BRPI0609666A2 - use of a vitamin d compound, method for preventing and / or treating endometriosis, pharmaceutical formulation, vitamin d compound, and kit - Google Patents

Abstract

USE OF A VITAMIN D COMPOUND, METHOD TO PREVENT AND / OR TREAT ENDOMETRIOSIS, PHARMACEUTICAL FORMULATION, VITAMIN D COMPOUND, E, KIT. The use of vitamin D compounds in the treatment or prevention of endometriosis, methods for the treatment or prevention of endometriosis by administering a vitamin D compound, and compounds for use therein.

Description

"USE OF A VITAMIN D COMPOUND, METHOD TO PREVENT AND / OR TREAT ENDOMETRIOSIS, PHARMACEUTICAL FORMULATION, VITAMIN D, E COMPOSITE"

This application claims the benefit of GB 0505955.5, filed March 23, 2005, and U.S. Provisional Application No. 60 / 667,367, filed March 31, 2006, the disclosures of any applications are incorporated herein by this reference.

The present invention relates to the use of vitamin D compounds in treating or preventing endometriosis, methods for treating or preventing endometriosis by administering a vitamin D compound, and compounds for use therein.

Endometriosis is a disease involving the growth of endometrium at ectopic sites that results in under-fertility, chronic pelvic pain and multiple surgeries. It affects approx. 10% of the female population at their reproductive age (Balweg, M. (2004) Best Practice Res. Cl. Ob. 18: 201 and Vigano, P. et al (2004) Best Practice Res. Cl. Ob. 18: 177). Stromal cell proliferation, vascular development, and inflammation are important factors in the pathogenesis of endometriosis (Kayama, C. M (2003) Reproductive Biology and Endocrinology 1: 123). Most current medical therapies involve inducing a hypoestrogenic state in patients. These treatments are associated with severe side effects and high relapse rates of the disease.

The present inventors have developed a novel method of treating endometriosis in order to mitigate or alleviate the aforementioned disadvantages.

The importance of vitamin D (cholecalciferol) in the biological systems of higher animals has been recognized since its discovery by Mellanby in 1920 (Mellanby, E. (1921) Spec. Rep. Ser. Med. Res. Council (GB) SRS 61: 4) . It was between 1920 and 1930 that vitamin D officially became classified as a "vitamin" that was essential for normal skeletal development and maintenance of calcium and phosphorus homeostasis.

Studies involving vitamin D3 metabolism were initiated with the discovery and chemical characterization of the plasma metabolite 25-hydroxyvitamin D3 [25 (OH) D3] (Blunt, JW et al. (1968) Biochemistry 6: 3317-3322) and the form hormonally active, 1-alpha, 25 (OH) 2D3 (Myrtle, JF et al (1970) J. Biol. Chem. 245: 1190-1196; Norman, AW et al. (1971) Science 173: 51-54; Lawson5 DEM et al (1971) Nature 230: 228-230; Holick 3 MF (1971) Proc. Natl. Acad. Sci. USA 68: 803-804). The formulation of the concept of an endocrine vitamin D system was dependent on both the evaluation of the key role of the kidney in producing 1-alpha, 25 (OH) 2D3 in a carefully regulated form (Fraser, DR and Kodicek5 E (1970) Nature 288: Wong5 RG et al (1972) J. Clin Invest 51: 1287-1291) and the discovery of a nuclear receptor for 1-alpha, 25 (OH) 2D3 (VD3R) in the intestine (Haussler, MR et al (1969) Exp. Cell Res. 58: 234242; Tsai5 HC and Norman5 AW (1972) J. Biol. Chem. 248: 5967-5975).

The operation of the endocrine vitamin D system depends on the following: first, in the presence of cytochrome P450 enzymes in the liver (Bergman, T. and Postlind5 H. (1991) Biochem. J. 276: 427-432; Ohyama5 Y and Okuda, K (1991) J. Biol. Chem. 266: 8690-8695) and kidney (Henry, HL and Norman, AW (1974) J. Biol. Chem. 249: 7529-7535; Gray5 RW and Ghazarian5 JG (1989) Biochem J. 259: 561-568), and in a variety of other tissues to effect the conversion of vitamin D3 to biologically active metabolites such as 1-alpha, 25 (OH) 2D3 and 24R, 25 (OH) 2D3; second, the existence of plasma vitamin D binding protein (DBP) to effect selective transport and release of these hydrophobic molecules to various tissue components of the endocrine vitamin D system (Van Baelen, Η et al (1988) Ann NY Acad Sci. 538: 60-68; Cooke, NE and Haddad, JG (1989) Endocr Rev. 10: 294-307; Bikle, DD et al (1986) J. Clin Endocrinol Metab 63: 954-959 ); and third, the existence of stereoselective receptors in a wide variety of target tissues that interact with the 1-alpha, 25 (OH) 2D3 agonist to generate the specific biological responses required for this dry steroid hormone (Pike, JW (1991) Annu. Rev Nutr 11: 189-216). To date, there is evidence that nuclear receptors for 1-alpha, 25 (OH) 2D3 (VD3R) exist in more than 30 cancer cell tissues and lineages (Reichel, H. and Norman, AW (1989) Annu. Rev. Med. 40: 71-78), including the normal bladder.

Vitamin D3 and its hormonally active forms are well-known regulators of calcium and phosphorus homeostasis. These compounds are known to stimulate at least one of intestinal calcium and phosphate absorption, bone mineral mobilization, and calcium retention in the kidney. In addition, the discovery of the presence of specific vitamin D receptors in more than 30 tissues has led to the identification of vitamin D3 as a pluripotent regulator outside its classic role in calcium / bone homeostasis. A role of paracrine for 1-alpha, 25 (OH) 2D3 has been suggested by the combined presence of enzymes capable of oxidizing vitamin D3 in their active forms, eg 25-OHD-1-alpha hydroxylase, and specific receptors in various tissues such as bone, keratinocytes, placenta, and immune cells. In addition, vitamin D3 hormone and active metabolites have been found to be able to regulate cell proliferation and differentiation of both normal and malignant cells (Reichel, H. et al (1989). Ann.Rev.Med. 40: 71-78) .

Given the activities of vitamin D3 and its metabolites, much attention has focused on the development of synthetic analogues of these compounds. A large number of these analogs involve structural modifications to ring A, ring B, rings C / D, and especially the side chain (Bouillon, R. et al. (1995) Endocrine Reviews 16 (2): 201-204). Although a vast majority of vitamin D3 analogs developed so far have involved structural changes in the side chain, few studies have reported the biological profile of ring A diastereomers (Norman, AW et al. (1993) J. Biol. Chem. 268 (27 ): 20022-20030). In addition, biological esterification of steroids has been studied (Hochberg, R.B., (1998) Endocr. Rev. 19 (3): 331-348), and vitamin D3 esters are known (WO 97/11053).

In addition, despite much effort to develop synthetic analogues, clinical applications of vitamin D and its structural analogues have been limited by the unwanted side effects evoked by these compounds following administration to a patient for known indications / applications of vitamin D compounds.

The activated form of vitamin D, vitamin D3, and some of its analogues have been described as potent regulators of cell growth and differentiation. It has previously been found that vitamin D3 as an analogue (analog V) inhibited BPH cell proliferation and neutralized the mitogenic activity of potent growth factors for BPH cells such as keratinocyte growth factor (KGF) and growth factor. similar to insulin (IGF1). In addition, the analog induced bcl-2 protein expression, intracellular calcium mobilization, and apoptosis in both unstimulated and KGF stimulated BPH cells.

Ailawadi et al FertiL SteriL 2004 81 (2): 290-296 describe the treatment of endometriosis and chronic pelvic pain with letrozole and norethindrone acetate. A range of additional medications including calcium and vitamin D supplements have been provided to reduce possible treatment-associated bone loss.

Shippen et al FertiL SteriL 2004 81 (5): 1395-1398 describe the treatment of severe endometriosis with an aromatase inhibitor. A range of additional medications have been provided, including calcitriol primarily to reduce the potential for bone loss.

US2005 / 0032741 discloses vitamin compositions containing calcium, vitamin D, folic acid, vitamin B12 and vitamin B6 for the treatment or prevention of conditions associated with hormonal changes in an individual. In one example, a patient suffering from endometriosis and osteoporosis while simultaneously receiving a gonadotropin releasing hormone antagonist, Leuprolide and Fosamax, showed a decrease in the rate of bone loss and endometriosis when the vitamin composition was administered. In light of the number of agents administered in the combination, there is no evidence that the reduction in endometriosis symptoms was a direct result of vitamin D administration.

US2002 / 0010163 discloses novel vitamin D compounds. Said compounds are intended to be of use as antiproliferative agents, for example in the treatment of hormone responsive tumors or hyperplasias (such as breast, prostate or ovarian cancers, fibroids or endometriosis) , or as suppressors of progesterone activity, for example in edema, acne, melasma or fertility control. No biological data is provided in the application for any of the stated indications.

Thus the invention provides vitamin D compounds, and novel treatment methods using such compounds, for the prevention or treatment of endometriosis, and associated symptoms for example chronic pelvic pain and / or sub-fertility. Treatment and / or prevention may include a reduction in the number and / or size of ectopic growths. In one embodiment the use and methods of the present invention may refer to adenomyosis (also known as internal endometriosis, uterine endometriosis or intrinsic endometriosis).

Suitably the methods of the present invention may be applied for the treatment of endometriosis. Alternatively, the methods of the present invention may be applied for the prevention of endometriosis.

Prior to another description of the present invention, and in order that the invention may be more readily understood, certain terms are first defined and collected herein for convenience.

The term "administering" or "administering" includes ways of introducing the vitamin D compound (s) to a patient to perform their intended function. Examples of routes of administration that may be used include injection (subcutaneously, intravenously, parenterally, intraperitoneally), oral, inhalation, rectal, transdermal or via bladder instillation. Pharmaceutical preparations are, of course, provided in forms suitable for each route of administration. For example, these preparations are administered in tablet or capsule form by injection, infusion, inhalation, lotion, ointment, suppository, etc. Oral administration is preferred. The injection may be bolus or it may be continuous infusion. Depending on the route of administration, the vitamin D compound may be coated with or disposed of a selected material to protect it from natural conditions that may adversely affect its ability to perform its intended function. The vitamin D compound may be administered alone, or in combination with another agent for use in the treatment of endometriosis, or a pharmaceutically acceptable carrier, or both. The vitamin D compound may be administered prior to administration of the other agent, simultaneously with the agent, or after administration of the agent. In addition, vitamin D compound can also be administered in a pro forma that is converted to its active metabolite, or more active metabolite in vivo.

The term "effective amount" includes an amount effective at the dosages and for periods of time necessary to obtain the desired result, that is, sufficient to treat and / or prevent endometriosis. An effective amount of vitamin D compound may vary depending on factors such as the patient's disease state, age and weight, and the ability of the vitamin D compound to elicit a desired response in the patient. Dosage regimens may be adjusted to provide the optimal therapeutic response. An effective amount is also one wherein any toxic or detrimental effects (e.g., side effects) of the vitamin D compound are prevailed by the therapeutically beneficial effects.

A therapeutically effective amount of the vitamin D compound (i.e. an effective dosage) may range from about 0.001 to 30 µg / kg body weight, preferably about 0.01 to 25 µg / kg body weight, more preferably about 0.1 to 20 μg / kg body weight, and even more preferably about 1 to 10 μg / kg, 2 to 9 μg / kg, 3 to 8 μg / kg, 4 to 7 μg / kg, or 5 to 6 μg / kg body weight. The skilled technician will appreciate that certain factors may influence the dosage required to effectively treat a patient, including but not limited to the severity of the disease or disorder, previous treatments, the patient's general health and / or age, and other present illnesses. In addition, the dose administered will also depend on the particular vitamin D compound used, the effective amount of each compound can be determined by titration methods known in the art. In addition, treating a patient with a therapeutically effective amount of a vitamin D compound may include a single treatment or, preferably, may include a series of treatments. In one example, a patient is treated with a vitamin D compound in the range of about 0.1 to 20 µg / kg body weight once daily for a duration of six months or more, depending on symptom management and of the evolution of the condition. Also, as with other chronic treatments an on-off or intermittent treatment regimen may be considered. It will also be appreciated that the effective dosage of a vitamin D compound used for treatment may increase or decrease during the course of a particular treatment. The term "alkyl" refers to the radical of saturated aliphatic groups, including straight chain alkyl groups, branched chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. Alkyl further includes alkyl groups, which may optionally further include (for example, in one embodiment alkyl groups do not include) oxygen, nitrogen, sulfur or phosphorus atoms substituting one or more carbons in the hydrocarbon backbone, e.g. oxygen, nitrogen, sulfur or phosphorus In preferred embodiments, a straight chain or branched alkyl The carbon atom has 30 or less carbon atoms in its main chain (e.g., C1-C30 for straight chain, C3-C30 for branched chain), preferably 26 or less, and more preferably or less, especially 6 or less. Likewise, preferred cycloalkyls have 310 carbon atoms in their ring structure, and more preferably have 3, 4, 5, 6 or 7 carbons in the ring structure.

Further, the term alkyl as used throughout the specification and claims is intended to include both "unsubstituted alkyl" and "substituted alkyls", the latter of which refers to alkyl moieties having substituents substituting one hydrogen on one or more hydrocarbon backbone carbons. Such substituents may include, for example, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxy, phosphate, phosphonate, cyanoamino, amino (dialk) , and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, sulfonate, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, heterocyclyl, cyano, alkylamino, an aromatic or heteroaromatic moiety. It will be understood by those skilled in the art that substituted portions in the hydrocarbon chain alone may be substituted, if appropriate. Cycloalkyl may be further substituted, for example, with the substituents described above. An "alkylaryl" moiety is an alkyl substituted with an aryl (e.g. phenylmethyl (benzyl)). Unsubstituted alkyl groups (including cycloalkyl) or halogen substituted groups, especially fluorine, are generally preferred over other substituted groups. The term "alkyl" also includes analogs of non-aliphatic groups saturated in length and possible substitution to the alkyls described above, but which contain at least one double or triple bond respectively.

Unless the number of carbons is otherwise specified, "lower alkyl" as used herein means an alkyl group as defined above, but having from one to ten carbons, more preferably from one to six, and most preferably from one to six carbons. to four carbon atoms in their main chain structure, which may be straight or branched chain. Examples of lower alkyl groups include methyl, ethyl, propyl (n-propyl and i-propyl), butyl (tert-butyl, n-butyl and sec-butyl), pentyl, hexyl, heptyl, octyl and so on. In a preferred embodiment, the term "lower alkyl" includes a straight chain alkyl having 4 or less carbon atoms in its main chain, for example C1-C4 alkyl.

Thus specific examples of alkyl include C1-6 alkyl or C1-6 alkyl (such as methyl or ethyl). Specific examples of hydroxyalkyl include C 1-6 hydroxyalkyl or C 1-6 hydroalkyl (such as hydroxymethyl).

The terms "alkoxyalkyl", "polyaminoalkyl" and "thioalkoxyalkyl" refer to alkyl groups as described above which further include oxygen, nitrogen or sulfur atoms substituting one or more carbons in the hydrocarbon backbone, e.g. oxygen, nitrogen or sulfur. The term "aryl" as used herein refers to the radical of aryl groups, including 5- and 6-membered single ring aromatic groups which may include from zero to four heteroatoms, for example benzene, pyrrol, furan, thiophene, imidazole benzoxazole, benzothiazole, triazole, tetrazol, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like. Aryl groups also include fused polycyclic aromatic groups such as naphthyl, quinolyl, indolyl, and the like. Those aryl groups having heteroatoms in the ring structure may also be referred to as "aryl heterocycles," "heteroaryls" or "heteroaromatics". The aromatic ring may be substituted at one or more ring positions with such substituents as described above, such as for example halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, phosphoryl, alkylthiocarbon phosphonate, phosphinate, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureide), amidino, imino, sulfhydryl, alkylthio, arylthio sulfate, thiocarboxylate, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. Aryl groups may also be fused or bridged with non-aromatic alicyclic or heterocyclic rings to form a polycyclic (e.g., tetraline).

The terms "alkenyl" and "alkynyl" refer to analogues of unsaturated aliphatic groups in length and possible substitution to the alkyls described above, but which contain at least one double or triple bond, respectively. For example, the invention considers cyano and propargyl groups.

The term "chiral" refers to molecules that have the non-overlapping property of the specular imaging partner, while the term "achiral" refers to molecules that are overlapping on their specular imaging partner.

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

The term "enantiomers" refers to two stereoisomers of a compound that are non-overlapping specular images. An equimolar mixture of two enantiomers is called a "racemic mixture" or a "racemate".

As used herein, the term "halogen" denotes -F, -Cl, -Br or -I; the term "sulfhydryl" or "thiol" means -SH; the term "hydroxyl" means -OH.

The term "haloalkyl" is intended to include alkyl groups as defined above which are mono-, di- or polysubstituted by halogen, for example haloC 1-6 or haloC 1-4 such as fluoromethyl and trifluoromethyl.

The term "heteroatom" as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, sulfur and phosphorus.

The terms "polycyclyl" or "polycyclic radical" refer to the radical of two or more cyclic rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and / or heterocyclyl) where two or more carbons are common to two adjacent rings for example, the rings are "fused rings". Rings that are joined through nonadjacent atoms are called "bridged" rings. Each of the polycyclic rings may be substituted with such substituents as described above, such as, for example, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate, alkoxy, phosphate cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and urea), amidino, imino, sulfldrila, alkylthio, arylthio, thiocarboxylate, sulfates, sulfonate, sulfonate, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkyl, alkylaryl, or an aromatic or heteroaromatic moiety.

The term "isomers" or "stereoisomers" refers to compounds that have identical chemical constitution but differ with respect to the arrangement of atoms or groups in space.

The terms "isolated" or "substantially purified" are used interchangeably herein and refer to vitamin D3 compounds in a non-naturally occurring state. The compounds may be substantially free of cell material or culture medium when naturally produced, or chemical precursors or other chemicals when chemically synthesized. In one embodiment of the invention an isolated vitamin D compound is at least 75% pure, especially at least 85% pure, in particular at least 95% pure and preferably at least 99% pure on a w / w basis. said purity being by reference to the compounds with which the vitamin D compound is naturally associated or chemically associated in the course of chemical synthesis. In certain preferred embodiments, the terms "isolated" or "substantially purified" also refer to preparations of a chiral compound that substantially lacks one of the enantiomers; that is, enanciomerically enriched or non-racemic preparations of a molecule. Similarly, the terms "isolated epimers" or "isolated diastereomers" refer to preparations of chiral compounds that are substantially free of other stereochemical forms. For example, isolated or substantially purified vitamin D3 compounds include synthetic or natural preparations of a stereoisomer-enriched vitamin D3 having a chiral carbon-linked substituent at the 3-position of ring A in an alpha configuration, and thus substantially lacking other isomers having a beta configuration. Unless otherwise specified, such terms refer to vitamin D3 compositions wherein the ratio of alpha to beta forms is greater than 1: 1 by weight. For example, an isolated preparation of an α-epimer means a preparation having greater than 50% by weight of the alpha epimer relative to the beta stereoisomer, more preferably at least 75% by weight, and even more preferably at least 85% by weight. Of course the enrichment may be much greater than 85%, providing "substantially epimer enriched" preparations, that is, preparations of a compound that have more than 90% of the alpha epimer relative to the beta stereoisomer, and even more preferably greater than 100%. that 95%. The term "substantially beta stereoisomer free" will be understood to have similar purity ranges.

As used herein, the term "vitamin D compound" includes any compound being a vitamin D analog that is capable of treating or preventing endometriosis. In general, compounds that are Vitamin D receptor ligands (VDR ligands) and are capable of treating or preventing endometriosis are considered to be within the scope of the invention. Vitamin D compounds are preferably vitamin D receptor agonists. Thus, vitamin D compounds are intended to include secoids. Examples of specific vitamin D compounds suitable for use in the methods of the present invention are further described herein. A vitamin D compound includes vitamin D2 compounds, vitamin D3 compounds, isomers thereof, or derivatives / analogs thereof. Preferred vitamin D compounds are vitamin D3 compounds that are vitamin D receptor ligands (more preferably agonists). Preferably the vitamin D compound (e.g., vitamin D3 compound) is a more potent vitamin D receptor agonist. vitamin D than the native ligand (ie vitamin D, for example vitamin D3). Vitamin D1 compounds, vitamin D2 compounds and vitamin D3 compounds include, respectively, vitamin D1, D2, D3 and the like. In certain embodiments, the vitamin D compound may be a steroid, such as a dry steroid, for example calciol, calcidiol or calcitriol. Non-limiting examples of vitamin D compounds according to the invention include those described in US Patent Nos. 6,017,908, 6,100,294, 6,030,962, 5,428,029 and 6,121,312, published international applications WO 98/51633, WO 01 / 40177A3. Other examples of vitamin D compounds include those described in US 6,492,353 and WO 2005/030222.

The term "dry steroid" is recognized in the art and includes compounds wherein one of the cyclopentanoperhydro-phenanthrene rings of the steroid ring structure is broken. For example, 1-alpha, 25 (OH) 2D3 and analogs thereof are hormonally active secoids. In the case of vitamin D3, the carbon-carbon bond 9-10 of ring B is broken, generating a dry-B-steroid. The official IUPAC name for vitamin D3 is 9,10-secocolesta- 5,7,10 (19) -trien-3B-ol. For convenience, a lalpha, 25 (OH) 2D3 6-s-trans conformation is illustrated herein having all carbon atoms numbered using standard steroid notation.

<formula> formula see original document page 15 </formula>

In the formulas presented herein, the various substituents on ring A are illustrated as attached to the steroid nucleus by one of these notations: a dashed line (-) indicating a substituent that is in beta orientation (i.e., above the plane of the ring), a cuneiform solid line () indicating a substituent that is in the alpha orientation (i.e. below the plane of the molecule), or a wavy line () indicating that a substituent may be above or below the ring plane. With reference to ring A, it should be understood that the stereochemical convention in the vitamin D field is opposite from the general chemical field, where a dashed line indicates a substituent on ring A which is in an alpha orientation (i.e., below the plane). of the molecule), and a solid cuneiform line indicates a substituent on ring A which is in beta orientation (ie above the plane of the ring).

In addition the indication of stereochemistry through a carbon-carbon double bond is also opposite from the general chemical field where "Z" refers to what is often referred to as an "eis" (same side) conformation whereas "E" refers to what is often referred to as a "trans" configuration (opposite side). Nevertheless, both cis / trans and / or Z / E configurations are considered for the compounds for use in the present invention.

As shown, the 1-alpha hormone ring A, 25 (OH) 2D3 contains two asymmetric centers at carbons 1 and 3, each containing a hydroxyl group in well-characterized configurations, that is, 1-alpha and 3-hydroxyl groups. beta. In other words, carbons 1 and 3 of ring A are said to be "chiral carbons" or "carbon centers".

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

Also, throughout the patent literature, ring A of a vitamin D compound is often described in generic formulas as any of the following structures: <formula> formula see original document page 17 </formula>

wherein X1 and X2 are defined as H or = CH2; or

<formula> formula see original document page 17 </formula>

wherein X1 and X2 are defined as H2 or CH2.

Although it does not appear to be any given convention, it is clear that a person of ordinary skill in the art understands formula (A) or (B) to represent an A ring where, for example, X1 is = CH2 and X2 is defined as H2, as follows:

<formula> formula see original document page 17 </formula>

For purposes of the present invention, formula (B) will be used in all generic structures.

Thus, in one aspect, the invention provides the use of a Vitamin D compound in the prevention or treatment of endometriosis. Also provided is a method of treating an endometriosis patient by administering an effective amount of a Vitamin D compound. Also provided is the use of a Vitamin D compound in the manufacture of a medicament for the prevention or treatment of endometriosis. Supplied further is a vitamin D compound for use in the prevention and / or treatment of endometriosis. Also provided is a kit containing a vitamin D compound together with instructions leading to administration of said compound to a patient in need of endometriosis treatment and / or prevention thereby to treat and / or prevent endometriosis in said patient. Endometriosis, for example, may be characterized by the presence of symptoms of chronic pelvic pain and / or under fertility.

Uses and methods are uses and methods in treating women, especially premenopausal women.

In one embodiment of the invention, the vitamin D compound is a compound of formula (I):

<formula> formula see original document page 18 </formula>

on what:

X is hydroxyl or fluorine;

Y is H2 or CH2;

Z1 and Z2 are H or a substituent represented by formula (II) as long as Z1 and Z2 are different (preferably both Z1 and Z2 do not represent formula (II)):

<formula> formula see original document page 18 </formula>

on what:

Z 3 represents formula (I) described above;

A is a single bond or a double bond; R 1, R 2, and Z 4 are all independently hydrogen, alkyl, or a saturated or unsaturated carbon chain represented by formula (III), as long as at least one of R 1, R 2, and Z 4 is saturated or carbon chain. unsaturated carbon represented by formula (III) and as long as all R1, R2, and Z4 are not saturated or unsaturated carbon chain represented by formula (III):

<formula> formula see original document page 19 </formula>

on what:

Z5 represents formula (II) described above;

A2 is a single bond, a double bond, or a triple bond; and

A3 is a single bond or a double bond; and

R3, and R4 are all independently hydrogen, alkyl, haloalkyl, hydroxyalkyl; and R5 is H2 or oxygen. R5 may also represent hydrogen or may be absent.

Thus, in the structure above formula (III) (and corresponding structures below), when A2 represents a triple bond R5 is absent. When A2 represents a double bond R5 represents hydrogen. When A2 represents a single bond R5 represents a carbonyl group or two hydrogen atoms.

In another embodiment of the invention, the vitamin D compound is a compound of formula (IV): wherein:

X1 and X2 are H2 or CH2, where X1 and X2 are not CH2 at the same time;

A is a single or double bond;

A2 is a single, double or triple bond;

A3 is a single or double bond;

R 1 and R 2 are hydrogen, C 1 -C 4 alkyl or 4-hydroxy-4-methylpentyl, wherein both R 1 and R 2 are not hydrogen;

R5 is H2 or oxygen, R5 may also represent hydrogen or may be absent;

R3 is C1-C4 alkyl, hydroxyalkyl or haloalkyl, for example fluoroalkyl, for example fluoromethyl and trifluoromethyl; and

R4 is C1 -C4 alkyl, hydroxyalkyl or haloalkyl, for example fluoroalkyl, for example fluoromethyl and trifluoromethyl.

In yet another embodiment of the invention, the vitamin D compound is a compound of formula (V):

<formula> formula see original document page 20 </formula>

on what:

X1 and X2 are H2 or CH2, where X1 and X2 are not CH2 at the same time;

A is a single or double bond;

A2 is a single, double or triple bond;

A3 is a single or double bond;

R1 and R2 are hydrogen, C1-C4 alkyl, wherein both R1 and R2 are not hydrogen;

Ro is H2 or oxygen, R5 may also represent hydrogen or may be absent;

R3 is C1-C4 alkyl, hydroxyalkyl or haloalkyl, for example fluoroalkyl, for example fluoromethyl and trifluoromethyl; and

R4 is C1 -C4 alkyl, hydroxyalkyl, haloalkyl, for example, or fluoroalkyl, for example, fluoromethyl and trifluoromethyl.

An example of the above structure of formula (V) is 1,25-dihydroxy-16-ene-23-one cholecalciferol.

In another embodiment, the vitamin D compound is a compound of formula (VII):

<formula> formula see original document page 21 </formula>

on what:

A is a single or double bond;

R 1 and R 2 are all independently hydrogen, alkyl (e.g. methyl);

R3, and R4, are all independently alkyl, and

X is hydroxyl or fluorine.

In another embodiment, the vitamin D compound is a compound having the formula (VIII): <formula> formula see original document page 22 </formula>

on what:

R1 and R2 are all independently hydrogen or alkyl, for example methyl;

R3 is alkyl, for example methyl,

R4 is alkyl, for example methyl; and

X is hydroxyl or fluorine.

In specific embodiments of the invention, the vitamin D compound is selected from the group consisting of:

<formula> formula see original document page 22 </formula> <formula> formula see original document page 23 </formula>

In other specific embodiments of the invention, the vitamin D compound is selected from the group consisting of:

<formula> formula see original document page 23 </formula>

In yet another embodiment, the vitamin D compound is a "geminal" compound of formula (VI):

<formula> formula see original document page 23 </formula>

wherein: X1 is H2 or CH2;

A2 is a single, a double bond or a triple bond;

R3 is C1-C4 alkyl, hydroxyalkyl, or haloalkyl, for example fluoroalkyl, for example fluoromethyl and trifluoromethyl;

R4 is C1 -C4 alkyl, hydroxyalkyl or haloalkyl, for example fluoroalkyl, for example fluoromethyl and trifluoromethyl;

and the setting at C20 is R or S.

Compounds of this type may be referred to as "twin" or "twin" vitamin D3 compounds due to the presence of two C20-6 alkyl chains.

An example of a geminal compound of formula (VI) is 1,25-dihydroxy-21- (3-hydroxy-3-methylbutyl) -19-nor-cholecalciferol (also referred to as Compound C here):

<formula> formula see original document page 24 </formula>

The synthesis of the above compound is described in WO 98/49138 and US6.030.962 which are incorporated herein in their entirety by reference.

In other specific embodiments of the invention, the vitamin D compound is selected from the group of twin compounds consisting of: <formula> formula see original document page 25 </formula>

In yet another aspect, the invention provides twin vitamin D3 compounds of formula (IX):

<formula> formula see original document page 25 </formula>

wherein: A1 is a single or double bond;

A2 is a single, double or triple bond;

R 1, R 2, R 3 and R 4 are all independently C 1 -C 4 alkyl, C 1 -C 4 deuteroalkyl, hydroxyalkyl, or haloalkyl;

R5, R6 and R7 are all independently hydroxyl, OC (O) C1 -C4 alkyl, OC (O) hydroxyalkyl, or OC (O) haloalkyl;

the setting at C20 is R or S;

X1 is H2 or CH2;

Z is hydrogen when at least one of R1 and R2 is C1-C4 deuteroalkyl and at least one of R3 and R4 is haloalkyl or when at least one of R1 and R2 is haloalkyl and at least one of R3 and R4 is C1-C4 deuteroalkyl ; or Z is -OH, = O, -SH, or -NH2;

and pharmaceutically acceptable esters, salts, and prodrugs thereof.

Various embodiments of this aspect of the invention include individual compounds of formula I wherein: A1 is a single bond; A2 is a single bond; A2 is a triple bond; R 1, R 2, R 3, and R 4 are all independently methyl or ethyl; R1, R2, R3, and R4 are all independently C1 -C4 deuteroalkyl or haloalkyl; R5 is hydroxyl; R6 and R7 are hydroxyl; R 6 and R 7 are all C (0) C 1 -C 4 alkyl; X1 is H2; X1 is CH2; Z is hydrogen; or Z is = O.

In certain embodiments, R 5, R 6 and R 7 are hydroxyl. In other embodiments, R 6 and R 7 are all acetyloxy.

In still other embodiments, Z is hydrogen when at least one of R1 and R2 is C1-C4 deuteroalkyl and at least one of R3 and R4 is haloalkyl or when at least one of R1 and R2 is haloalkyl and at least one of R3 and R4 is C1-C4 deuteroalkyl; Z is -OH, = O, -SH, or -NH2 when X1 is CH2; Z is -OH, = 0, -SH, or -NH2 when X1 is H2 and the setting at C20 is S; or Z is = O, -SH, or -NH2 when X1 is H2 and the setting at C20 is R. In one embodiment, Z is -OH.

Still other embodiments of this aspect of the invention include those wherein X1 is CH2; A2 is a single bond; R 1, R 2, R 3, and R 4 are all independently methyl or ethyl; and Z is -OH. In one embodiment, X1 is CH2; A2 is a single bond; R1, R2, R3, and R4 are all independently methyl or ethyl; and Z is = O. In one embodiment, X1 is H2; A2 is a single bond; R1, R2, R3, and R4 are all independently methyl or ethyl; the setting at C20 is S; and Z is -OH. In another embodiment, X1 is H2; A2 is a single bond; R1, R2, R3, and R4 are all independently methyl or ethyl; and Z is = 0. In these embodiments, R 1, R 2, R 3, and R 4 are all advantageously methyl. In certain embodiments, haloalkyl is fluoroalkyl. Advantageously, fluoroalkyl is fluoromethyl or trifluoromethyl.

Additional embodiments of this aspect of the invention include compounds X1 and H2; A2 is a triple bond; R1 and R2 are all C1-C4 deuteroalkyl; R3 and R4 are all haloalkyl; and Z is hydrogen. In other embodiments, X1 is CH2; A2 is a triple bond; R1 and R2 are all C1-C4 deuteroalkyl; R3 and R4 are all haloalkyl; and Z is hydrogen.

In these embodiments, R 1 and R 2 are all advantageously deuteromethyl and R 3 and R 4 are all advantageously trifluoromethyl.

In still other specific embodiments of the invention, the vitamin D compound is a geminal compound of formula (IX):

<formula> formula see original document page 27 </formula>

wherein: X1 is H2 or CH2;

A2 is a single, double or triple bond; R 1, R 2, R 3 and R 4 are all independently C 1 -C 4 alkyl, hydroxyalkyl, or haloalkyl, for example fluoroalkyl, for example, fluoromethyl and trifluoromethyl;

Z is -OH, Z may also be = O, -NH 2 or -SH; and the configuration at C20 and R or S;

and pharmaceutically acceptable esters, salts, and prodrugs of the

same.

In another embodiment, X1 is CH2. In another embodiment, A2 is a single bond. In another, R1, R2, R3, and R4 are all independently methyl or ethyl. In another embodiment, Z is -OH. In another, Xi is CH2; A2 is a single bond; R1, R2, R3, and R4 are all independently methyl or ethyl; and Z is -OH. In yet another embodiment, R 1, R 2, R 3, and R 4 are all methyl.

In another embodiment of the invention, the vitamin D compound is a geminal compound of the formula:

<formula> formula see original document page 28 </formula>

The chemical names of the above-mentioned compounds 33 and 50 are 1,25-dihydroxy-21- (2R, 3-dihydroxy-3-methylbutyl) -20R-cholecalciferol and 1,25-dihydroxy-21 - (2R, 3-dihydroxy-3-methylbutyl) -20S-cholecalciferol respectively.

Additional embodiments of twin compounds include the following vitamin D compounds for use according to the invention:

<formula> formula see original document page 29 </formula>

(1,25-Dihydroxy-21- (2R, 3-dihydroxy-3-methylbutyl) -20S-19-norcolecalciferol),

<formula> formula see original document page 29 </formula>

(1,25-Dihydroxy-20S-21- (3-hydroxy-3-methyl-butyl) -24-keto-19-nor-checalciferol),

<formula> formula see original document page 29 </formula>

(1,25-Dihydroxy-20S-21- (3-hydroxy-3-methylbutyl) -24-keto-cholecalciferol),

<formula> formula see original document page 29 </formula>

(1,25-Dihydroxy-21 (3-hydroxy-3-trifluoromethyl-4-trifluoro-butynyl) -26,27-hexadeutero-19-nor-20S-cholecalciferol), and <formula> formula see original document page 30 </formula>

(1,25-Dihydroxy-21 (3-hydroxy-3-trifluoromethyl-4-trifluoro-butynyl) -26,27-hexadeutero-20S-cholecalciferol).

In other embodiments of the invention, the vitamin D compound is a compound of formula (X):

<formula> formula see original document page 30 </formula>

on what:

X1 and X1 are all independently H2 or = CH2, provided that both X1 and X1 are not = CH2; R 1 and R 2 are all independently hydroxyl, OC (O) C 1 -C 4 alkyl, OC (O) hydroxyalkyl, OC (O) Auroralkyl;

R 3 and R 4 are all independently hydrogen, C 1 -C 4 alkyl, hydroxyalkyl or haloalkyl, or R 3 and R 4 taken together with C 20 form C 3 -C 6 cycloalkyl; and

Rs and D are all independently C1-C4 alkyl and pharmaceutically acceptable esters, salts, and prodrugs of the

same.

Suitably R3 and R4 are all independently hydrogen, C1-C4 alkyl, or R3 and R4 taken together with C20 form C3-C6 cycloalkyl.

In an exemplary set of compounds R 5 and R 6 are all independently C 1 -C 4 alkyl.

In another exemplary set of compounds R5 and R6 are all independently haloalkyl for example fluoro C1-C4.

When R3 and R4 are taken together with C20 to form C3 -C6 cycloalkyl, an example is cyclopropyl.

In one embodiment, X1 and X1 are all H2. In another embodiment, R3 is hydrogen and R4 is C1-C4 alkyl. In a preferred embodiment R 4 is methyl.

In another embodiment, R 5 and are all independently methyl, ethyl, fluoromethyl or trifluoromethyl. In a preferred embodiment, R 5 and R 6 are all methyl.

In yet another embodiment, R 5 and R 1 are all independently hydroxyl or ° C (O) C 1 -C 4 alkyl. In a preferred embodiment, R 5 and R 1 are all ° C (O) C 1 -C 4 alkyl. In another preferred embodiment, R 1 and R 1 are all acetyloxy.

An example of such a compound is 1,3-0-diacetyl-1,25-dihydroxy-16-ene-24-keto-19-norcolecalciferol having the following structure:

<formula> formula see original document page 31 </formula>

In another embodiment of the invention the vitamin D compound for use according to the invention is 2-methylene-19-nor-20 (S) -1-alpha, 25-hydroxyvitamin D3: <formula> formula see original document page 32 </formula>

The synthesis of this and related compounds is described in WO 02/05823 and US5,536,713 which are incorporated herein in their entirety by reference.

In another embodiment of the invention, the vitamin D compound is a compound of formula (XII):

<formula> formula see original document page 32 </formula>

on what:

A1 is single or double bond; A2 is a single, double or triple bond; X1 and X2 are all independently H or = CH2, provided that both X1 and X2 are not = CH2;

R1 and R2 are all independently OC (O) C1-C4 alkyl (e.g. OAc), OC (O) hydroxyalkyl or OC (O) haloalkyl such as OC (O) C1-C4 alkyl or OC (O) hydroxyalkyl;

R1 and / or R2 may alternatively be OH;

R3, R4 and R5 are all independently hydrogen, alkyl

C 1 -C 4, hydroxyalkyl, or haloalkyl, or R 3 and R 4 taken together with C 20 form C 3 -C 6 cycloalkyl; and

R 6 and R 7 are all independently C 1-4 alkyl or haloalkyl; and

R 8 is H, -C 1 -C 4 -Coalkyl (e.g. Ac), -COhydroxyalkyl or -COhaloalkyl; and

pharmaceutically acceptable esters, salts, and prodrugs thereof.

When R3 and R4 are taken together with C20 to form C3 -C6 cycloalkyl an example is cyclopropyl.

Suitably R6 and R7 are all independently haloalkyl. R 8 may suitably represent H or Ac.

In one embodiment, A 1 is a single bond and A2 is a single bond, double bond E or Z, or a triple bond. In another embodiment, A 1 is a double bond and A2 is a single bond, double bond E or Z, or a triple bond. One of ordinary skill in the art will readily appreciate that when A2 is a triple bond, R5 is absent.

In one embodiment, X1 and X2 are all H. In another embodiment, Xj is CH2 and X2 is H2. In another embodiment, R3 is hydrogen and R4 is C1 -C4 alkyl. In a preferred embodiment R 4 is methyl.

In another exemplary set of compounds both R1 and R2 represent OAc.

In a set of example compounds R 6 and R 7 are all independently C 1-4 alkyl. In another set of example compounds R6 and R? they are all independently haloalkyl. In another embodiment, R 6 and R 7 are all independently methyl, ethyl or fluoroalkyl. In a preferred embodiment R 6 and R 8 are all trifluoroalkyl, for example trifluoromethyl. Suitably R5 represents hydrogen. Thus, in certain embodiments, vitamin D compounds for use according to the invention are represented by formula (XII):

<formula> formula see original document page 34 </formula>

(XII)

on what:

A1 is single or double bond;

A2 is a single, double or triple bond;

X1 and X2 are all independently H or = CH2, provided that both X1 and X2 are not = CH2;

R1 and R2 are all independently 0C (0) C1-C4 alkyl,

OC (0) hydroxyalkyl, or OC (0) haloalkyl;

R 3, R 4 and R 5 are all independently hydrogen, C 1 -C 4 alkyl, hydroxyalkyl, or haloalkyl, or R 3 and R 4 taken together with C 20 form C 3 -C 6 cycloalkyl;

R 6 and R 7 are all independently haloalkyl; R 6 and R 7 may alternatively be alkyl; and

R 8 is H, C (O) C 1 -C 4 alkyl, C (O) hydroxyalkyl, or C (O) haloalkyl; and

pharmaceutically acceptable esters, salts, and prodrugs thereof. In preferred embodiments, when A 1 is a single bond, R 3 is hydrogen and R 4 is methyl, then A2 is a double or triple bond.

An example compound of formula (XII) described above which is particularly preferred in the context of the present invention is 1,3-di-O-acetyl-1,25-dihydroxy-16,23Z-diene-26,27-hexafluoro - 19-nor-cholecalciferol:

<formula> formula see original document page 35 </formula>

In another preferred embodiment the compound is one of formula (XIII), wherein R 1 and R 2 are all OAc; Ai is a double bond; A2 is a triple bond; and Rg is H or Ac:

<formula> formula see original document page 35 </formula>

(XIII)

In certain embodiments of formula (XII) shown above, vitamin D compounds for use according to the invention are represented by formula (XIV):

<formula> formula see original document page 35 </formula>

(XIV)

In a preferred embodiment, X1 is = CH2 and X2 is H2. When A1 is a single bond, and A2 is a triple bond, it is preferred that R 8 is H or C (O) CH 3, and R 6 and R 7 are alkyl, preferably methyl. When Aj is a single bond, and A2 is a single bond, it is preferred that R 8 is H or C (O) CH 3, and R 6 and R 7 are alkyl, preferably methyl. When A1 is a double bond, and A2 is a single bond, it is preferable that R 8 is H or C (O) CH 3, and R 6 and R 7 are alkyl, preferably methyl.

In another preferred embodiment, X1 and X2 are all H2. When A 1 is a single bond, and A 2 is a triple bond, it is preferred that R 6 is H or C (O) CH 3, and R 6 and R 7 are alkyl or haloalkyl. It is preferred that the alkyl group is methyl, and the haloalkyl group is trifluoroalkyl, preferably trifluoromethyl. When A 1 is a single bond, and A 2 is a double bond, it is preferred that R 8 is H or C (O) CH 3, R 6 and R 7 are haloalkyl, preferably trifluoroalkyl, preferably trifluoromethyl. When A 1 is a double bond, and A 2 is a single bond, it is preferred that R 8 is H or C (O) CH 3, R 6 and R 7 are alkyl, preferably methyl.

Other exemplary compounds of the formula described above (XIV) include:

1,3-di-O-acetyl-1,25-dihydroxy-23-yna-cholecalciferol;

1,3-di-O-acetyl-1,25-dihydroxy-16-ene-23-yna-cholecalciferol;

1,3-di-O-acetyl-1,25-dihydroxy-16,23E-diene-cholecalciferol;

1,3-di-O-acetyl-1,25-dihydroxy-16-ene-cholecalciferol;

1,25-Tri-O-acetyl-1,25-dihydroxy-16-ene-23-one-26,27-hexafluoro-cholecalciferol:

1,3-di-O-acetyl-1,25-dihydroxy-16-ene-23-one-26,27-hexafluoro-cholecalciferol;

1,3-Di-O-acetyl-1,25-dihydroxy-16,23E-diene-25R-26-trifluoro-cholecalciferol;

1,3-Di-O-acetyl-1,25-dihydroxy-16-ene-23-one-26,27-hexafluoro-19-nor-cholecalciferol;

1,3,25-Tri-O-acetyl-1,25-dihydroxy-16-ene-23-yne-26,27-hexafluoro-19-norcecalciferol;

1,3-di-O-acetyl-1,25-dihydroxy-16-ene-19-nor-cholecalciferol;

1,3-Di-O-acetyl-1,25-dihydroxy-16-ene-23-one-19-nor-cholecalciferol;

1,3-Di-O-acetyl-1,25-dihydroxy-16-ene-23-one-26,27-bishomo-19-nor-cholecalciferol.

In certain other embodiments of formula (XII) represented above, vitamin D compounds for use according to the invention are represented by formula (XV):

<formula> formula see original document page 37 </formula>

Other exemplary compounds of formula (XV) described above include:

1,3-di-O-acetyl-1,25-dihydroxy-20-cyclopropyl-23-one-19-nor-cholecalciferol: 1,25-tri-O-acetyl-1,25-dihydro hydroxy-20-cyclopropyl-23-one-26,27-hexafluoro-19-norcolecalciferol; 1,3-di-O-acetyl-1,25-dihydroxy-20-cyclopropyl-23-one-26,27-hexafluoro-19-nor-cholecalciferol;

1,3-di-O-acetyl-1,25-dihydroxy-20-cyclopropyl-23-yna-cholecalciferol; 1,3-di-O-acetyl-1,25-dihydroxy-20-cyclopropyl-23Z-ene-26,27-hexafluoro-19-norcolecalciferol; 1,3-di-O-acetyl-1,25-dihydroxy-20-cyclopropyl-cholecalciferol;

1,3-di-O-acetyl-1,25-dihydroxy-16-ene-20-cyclopropyl-19-nor-cholecalciferol; and 1,3-di-O-acetyl-1,25-dihydroxy-16-ene-20-cyclopropyl-cholecalciferol.

A preferred compound of the formula (XV) is 1,3-di-O-acetyl-1,25-dihydroxy-20-cyclopropyl-23E-ene-26,27-hexafluoro-19-nor-cholecalciferol: <formula> formula see original document page 38 </formula>

An example of another preferred compound is 1,3-Di-O-acetyl-1,25-dihydroxy-20-cyclopropylcolecalciferol (referred to as "Compound D") having the formula:

<formula> formula see original document page 38 </formula>

Such compounds are described in WO2005 / 030222, the contents of which are incorporated herein by reference in their entirety. The invention also encompasses the use of esters and salts of Compound D. Esters include pharmaceutically acceptable unstable esters that can be hydrolyzed in the body to release Compound D. Salts of Compound D include adducts and complexes that can be formed with alkali metal ions and alkaline earth and metal ion salts such as sodium, potassium and calcium ions and salts thereof such as calcium chloride, calcium malonate and the like. However, although Compound D may be administered as a pharmaceutically acceptable salt or ester thereof, preferably Compound D is used as is, that is, it is not used as an ester or salt thereof.

Another compound is 1,25-dihydroxy-20,21,28-cyclopropyl-cholecalciferol having the formula: <formula> formula see original document page 39 </formula>

The compound is described in U.S. 6,492,353, the contents of which are incorporated herein by reference in their entirety.

The invention also encompasses the use of 1,25-dihydroxy-20,21,28-cyclopropylcolecalciferol esters and salts. The esters include pharmaceutically acceptable unstable esters which may be hydrolyzed in the body to release 1,25-dihydroxy-20,21,28-cyclopropyl-cholecalciferol. 1,25-Dihydroxy-20,21,28-cyclopropyl-cholecalciferol salts include adducts and complexes that can be formed with alkaline and alkaline earth metal ions and metal ion salts such as sodium, potassium and calcium ions. and salts thereof such as calcium chloride, calcium malonate and the like. However, although 1,25-dihydroxy-20,21,28-cyclopropyl-cholecalciferol may be administered as a pharmaceutically acceptable salt or ester thereof, preferably it is used as is ie it is not used as a an ester or a salt thereof.

Other preferred vitamin D compounds for use according to the invention included those having formula (XVII):

<formula> formula see original document page 39 </formula>

on what:

B is single, double or triple bond; X1 and X2 are all independently H2 or CH2, provided that both X1 and X2 are not CH2; and

R4 and R5 are all independently alkyl or haloalkyl.

The compounds of formula (XVII) including the following: 1,25-Dihydroxy-16-ene-23-yn-20-cyclopyl-cholecalciferol:

<formula> formula see original document page 40 </formula>

1,25-Dihydroxy-16-ene-23-one-20-cyclopropyl-19-nor-cholecalciferol:

<formula> formula see original document page 40 </formula>

1,25-Dihydroxy-16-ene-20-cyclopropyl-23-ya-26,27-hexafluoro-19-nor-cholecalciferol:

<formula> formula see original document page 40 </formula>

1,25-Dihydroxy-16-ene-20-cyclopropyl-23-ya-26,27-hexafluoro-cholecalciferol: <formula> formula see original document page 41 </formula>

1,25-Dihydroxy-16,23E-diene-20-cyclopropyl-26,27-hexafluoro-19-nor-cholecalciferol:

<formula> formula see original document page 41 </formula>

1,25-Dihydroxy-16,23E-diene-20-cyclopropyl-26,27-hexafluoro-cholecalciferol:

<formula> formula see original document page 41 </formula>

1,25-Dihydroxy-16,23Z-diene-20-cyclopropyl-26,27-hexafluoro-19-nor-5 cholecalciferol:

<formula> formula see original document page 41 </formula>

1,25-Dihydroxy-16,23Z-diene-20-cyclopropyl-26,27-hexafluoro-cholecalciferol: <formula> formula see original document page 42 </formula>

1,25-Dihydroxy-16-ene-20-cyclopropyl-19-nor-cholecalciferol:

<formula> formula see original document page 42 </formula>

1,25-Dihydroxy-16-ene-20-cyclopropyl-cholecalciferol:

<formula> formula see original document page 42 </formula>

In another embodiment, the vitamin D compounds for use in the invention are compounds of formula (XVI):

<formula> formula see original document page 42 </formula>

(XVI) where:

X is H2 or CH2

R1 is hydrogen, hydroxy or fluorine

R2 is hydrogen or methyl

R3 is hydrogen or methyl. When R2 or R3 is methyl, R3 or

R2 must be hydrogen.

R4 is methyl, ethyl or trifluoromethyl

R5 is methyl, ethyl or trifluoromethyl

A is a single or double bond

B is a single bond, double E, double Z or triple.

In preferred compounds each of R 4 and R 5 is methyl or ethyl, for example 1-alpha-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20- epi-cholecalciferol (referred to as "Compound A "in the examples, having the formula:

<formula> formula see original document page 43 </formula>

Such compounds are described in US 5,939,408 and EP808833, the contents of which are incorporated herein by reference in their entirety. The invention also encompasses the use of esters and salts of Compound A. Esters include pharmaceutically acceptable unstable esters which may be hydrolyzed in the body to release Compound A. Compound A salts include adducts and complexes which may be formed with alkali metal ions and alkaline earth and metal ion salts such as sodium, potassium and calcium ions and salts thereof such as calcium chloride, calcium malonate and the like. However, although Compound A may be administered as a pharmaceutically acceptable salt or ester thereof, preferably Compound A is used as is, that is, it is not used as an ester or salt thereof.

Another vitamin D compound of the invention is 1,25-dihydroxy-21- (3-hydroxy-3-trifluoromethyl-4-trifluoro-butynyl) -26,27-hexadeutero-19-nor-20S-cholecalciferol.

Still other preferred vitamin D compounds for use according to the invention include those having formula (XVIII):

<formula> formula see original document page 44 </formula>

In one embodiment, A 1 is a double bond, and X 1 is = CH 2 and X 2 is H 2. When A 2 is a triple bond, it is preferred that R 8 is H or C (O) CH 3, and R 6 and R 7 are alkyl or haloalkyl. It is preferred that the alkyl group is methyl and the haloalkyl group is trifluoroalkyl, preferably trifluoromethyl. When A2 is a double bond, it is preferred that R 6 is H or C (O) CH 3, and R 6 and R 7 are alkyl, preferably methyl. It is also preferred that R 6 and R 7 are independently alkyl and haloalkyl. When A2 is a single bond, it is preferred that R 8 is H or C (O) CH 3, and R 6 and R 7 are alkyl, preferably methyl.

In a preferred embodiment, Aj is a double bond, and X1 and X2 are all H2. When A 2 is a triple bond, it is preferred that R 8 is H or C (O) CH 3, and R 6 and R 7 are alkyl or haloalkyl. It is preferred that the alkyl group is methyl or ethyl and the haloalkyl group is trifluoroalkyl, preferably trifluoromethyl. Where A2 is a double bond, it is preferred that R 8 is H or C (O) CH 3, and R 6 and R 7 are haloalkyl, preferably trifluoroalkyl, preferably trifluoromethyl. When A2 is a single bond, it is preferred that R 8 is H or C (O) CH 3, and R 6 and R 7 are alkyl, preferably methyl.

In another embodiment of the invention of formula (XVIII), R 1 and R 2 are OC (O) CH 3, A 1 is a single bond, and A 2 is a single, double or triple bond, except that when R 3 is H and R 4 is methyl, A2 is a double or triple bond. In a preferred embodiment, R 3 is H, R 4 is methyl, R 5 is absent, R 6 is H or C (O) CH 3, and R 6 and R 7 are alkyl, preferably methyl.

Preferred compounds of the present include the following: 1,3-Di-O-acetyl-1,25-dihydroxy-16,23Z-diene-26,27-hexafluoro-19-norcecalciferol, 1,3-Di -O-Acetyl-1,25-Dihydroxy-16-ene-23-one-26,27-hexafluoro-19-nor-cholecalciferol, 1,2,2-Tri-O-acetyl-1,25-Di -hydroxy-16-eno-23-amino-26,27-hexafluoro-19-nor-cholecalciferol, 1,3-Di-O-acetyl-1,25-dihydroxy-16-eno-23-y-cholecalciferol 1,3-Di-O-acetyl-1,25-dihydroxy-16,23E-diene-cholecalciferol, 1,3-Di-O-acetyl-1,25-dihydroxy-16-ene-cholecalciferol 1,2,2-Tri-O-acetyl-1,25-dihydroxy-16-ene-23-one-26,27-hexafluoro-cholecalciferol, 1,3-Di-O-acetyl-1,25 -dihydroxy-16-eno-23-one-26,27-hexafluoro-cholecalciferol, 1,3-Di-O-acetyl-1,25-dihydroxy-16,23E-diene-25R, 26-trifluoro -colecalciferol, 1,3-Di-O-acetyl-1,25-dihydroxy-16-ene-19-nor-cholecalciferol, 1,3-Di-O-Acetyl-1,25-dihydroxy-16 -en-23-ya-19-nor-cholecalciferol, 1,3-Di-O-acetyl-1,25-dihydroxy-16-ene-23-ya-26,27-bishomo-19-nor-cholecalciferol el, 3-Di-O-acetyl-1,2 5-dihydroxy-23-amino-cholecalciferol. These compounds may be prepared, for example, as described in PCT Publication W02005030222. The use of compounds having the structures provided above is extended to pharmaceutically acceptable esters, salts, and prodrugs thereof.

Still other preferred vitamin D compounds for use according to the invention include those having the formula (XIX): <formula> formula see original document page 46 </formula>

on what:

A1 is single or double bond; A2 is a single, double or triple bond, X1 and X2 are all independently H2 or CH2, provided that both X1 and X2 are not CH2;

R 1 and R 2 are all independently OC (O) C 1 -C 4 alkyl, OC (O) hydroxyalkyl, or OC (O) haloalkyl;

R 3, R 4 and R 5 are all independently hydrogen, C 1 -C 4 alkyl, hydroxyalkyl, or haloalkyl, or R 3 and R 4 taken together with C 20 form C 3 -C 6 cycloalkyl;

R 6 and R 7 are all independently haloalkyl; and R 8 is H, C (C) C 1 -C 4 alkyl, OC (O) W droxyalkyl, or OC (O) haloalkyl; and

pharmaceutically acceptable esters, salts, and prodrugs thereof. In preferred embodiments, R 6 and R 7 are all independently trialoalkyl, especially trifluoromethyl.

These compounds may be prepared, for example, as described in PCT Publication W02005030222, the contents of which are incorporated herein by reference. The use of compounds having the structures provided above is extended to pharmaceutically acceptable esters, salts, and prodrugs thereof.

A vitamin D compound of particular interest is calcitriol (also referred to as Compound B herein. The use of compounds having the structures provided above is extended to pharmaceutically acceptable esters, salts, and prodrugs thereof.

Other exemplary compounds of use in the invention that are vitamin D receptor agonists include paricalcitol (ZEMPLAR®) (see US Patent 5,587,497), tacalcitol (BONALFA®) (see US Patent 4,022,891), doxercalciferol (HECTOROL). ®) (see Lam et al. (1974) Science 186, 1038), maxacalcitol (OXAROL®) (see US Patent 4,891,364), calcipotriol (DAIVONEX®) (see US Patent 4,866,048), and falecalcitriol ( FULSTAN®).

Other compounds include ecalcidene, calcitiazole and thisocalcitate.

Other compounds include atocalcitol, lexacalcitol and seocalcitol.

Another compound of possible interest is secalciferol ("OSTEO D").

Other non-limiting examples of vitamin D compounds which may be of use according to the invention include those described in published international applications: WO 01/40177, WOOO10548, WO0061776, WO0064869, WO0064870, WO00106589, WO011099, WO0130751, WO0140177 , WO0151464, WO0156982, WO0162723, WO0174765, WO0174766, WO0179166, WO0190061, WO0192221, WO0196293, WO02066424, WO0212182, WO0214268, WO03004036, WO03027065, WO03055854, WO03088977, WO04037781, WO04067504, WO8000339, WO8500819, WO8505622, WO8602078, WO8604333, WO8700834, WO8910351 , WO9009991, WO9009992, WO9010620, WO9100271, WO9100855, WO9109841, WO112239, WO9112240, WO9115475, WO9203414. WO9622973, WO9711053, WO9720811, WO9737972, WO9746522, WO9818759, WO9824762, WO9828266, WO9841500, WO9841501, WO9851663, WO9851678, WO990382 9, WO9912894, WO9915499, WO9918070, WO9943645, WO9952863, those described in US Pat. .

US6667298, US6683219, US6696431, US6774251, and those described in the published US Patent Applications: US2001007907, US2003083319, US2003125309, US2003130241, US2003171605, US2004167105. Additional vitamin D compounds of use in accordance with the present invention include those described in US4929609, US5393900, US5747478, WO2005 / 082375, WO2005 / 030223, WO2005 / 030222, WO2005 / 027923, WO2004 / 098522 and WO2004 / 098507.

It will be appreciated that the structures of some of the compounds of the invention include asymmetric carbon atoms. Accordingly, it should be understood that isomers that originate from such asymmetry (e.g., all enantiomers and diastereomers) are included within the scope of this invention unless otherwise indicated. Such isomers may be obtained in substantially pure form by classical separation techniques and / or by stereochemically controlled synthesis.

Naturally occurring or synthetic isomers may be separated in various ways known in the art. Methods for separating a racemic mixture of two enantiomers include chromatography using a chiral stationary phase (see, for example, "Chiral Liquid Chromatography," WJ. Lough, Ed. Chapman and Hall, New York (1989)). Enantiomers can also be separated by classical resolution techniques. For example, diastereomeric salt formation and fractional crystallization may be used to separate the enantiomers. For the separation of carboxylic acid enantiomers, diastereomeric salts may be formed by the addition of enanciomerically pure chiral bases such as brucine, quinine, ephedrine, strychnine, and the like. Alternatively, diastereomeric esters may be formed with enanciomerically pure chiral alcohols such as menthol, followed by separation of diastereomeric esters and hydrolysis to yield the free, enanciomerically enriched carboxylic acid. For the separation of optical isomers from amino compounds, the addition of chiral carboxylic or sulfonic acids such as camphorsulfonic acid, tartaric acid, mandelic acid, or lactic acid may result in the formation of diastereomeric salts.

The invention also provides a pharmaceutical composition comprising an effective amount of a vitamin D compound as described herein and a pharmaceutically acceptable carrier. In another embodiment, the effective amount is effective for treating endometriosis as previously described.

In one embodiment, the vitamin D compound is administered to the patient using a pharmaceutically acceptable formulation, for example, a pharmaceutically acceptable formulation that provides sustained release of the vitamin D compound to a patient for at least 12 hours, 24 hours, 36 hours. hours, 48 hours, one week, two weeks, three weeks, or four weeks after the pharmaceutically acceptable formulation is administered to the patient. In certain embodiments, these pharmaceutical compositions are suitable for topical or oral administration to a patient. In other embodiments, as described in detail below, the pharmaceutical compositions of the present invention may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, liquid medicaments ( aqueous or non-aqueous solutions or suspensions), tablets, cakes, powders, granules, pastes; (2) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection such as, for example, a sterile solution or suspension, (3) topical application, for example, as a cream, ointment or spray applied to the skin; (4) intravaginally or intraretally, for example, as a pessary, cream or foam; or (5) aerosol, for example as an aqueous aerosol, liposome preparation or solid particles containing the compound.

The phrase "pharmaceutically acceptable" refers to those vitamin D compounds of the present invention, compositions containing such compounds, and / or dosage forms that are, within the scope of legal medical judgment, suitable for use in contact with humans and animals without excessive toxicity, irritation, allergic response, or other commensurate problem or complication with a reasonable benefit / risk ratio.

The phrase "pharmaceutically acceptable carrier" includes pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in loading or transporting the chemical object of an organ, or body portion. , to another organ, or portion of the body. Each carrier must be "acceptable" in the sense that it is compatible with the other formulation ingredients and not deleterious to the patient. Some examples of materials that may serve as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose and sucrose; (2) starches, such as cornstarch and potato starch; (3) cellulose, and derivatives thereof, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerine, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen free water; (17) isotonic saline solution; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances used in pharmaceutical formulations.

Wetting agents, emulsifiers and lubricants such as sodium lauryl sulfate and magnesium stearate as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants may also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: (1) water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Compositions containing a vitamin D compound include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol and / or parenteral administration. The compositions may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient that may be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single overall dosage form will be that amount of the compound that produces a therapeutic effect. Generally, within one hundred percent, this amount will range from about 1 percent to about ninety-nine percent active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 100 percent. 10 percent to about 30 percent.

Methods of preparing these compositions include the step of bringing into association a vitamin D compound with the carrier and optionally one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately combining a vitamin D compound with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, forming the product.

Compositions of the invention suitable for oral administration may be in the form of capsules, tablets, pills, tablets, expectorant tablets (using a flavor base, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or suspension. in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as tablets (using an inert base such as gelatin and glycerin, or sucrose and acacia) and / or as mouthwash solutions and the like, each containing a predetermined amount of a vitamin D compound as an active ingredient. A compound may also be administered as a cake, electuary or paste.

In solid dosage forms of the invention for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers such as sodium citrate or dicalcium phosphate, and / or any of the following: (1) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and / or acacia; (3) humectants, such as glycerol; (4) disintegrating agents such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents such as paraffin; (6) absorption accelerators such as quaternary ammonium compounds; (7) wetting agents, such as, for example, acetyl alcohol and glycerol monostearate; (8) absorbents such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be used as fillers in soft and hard filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (e.g. gelatin or hydroxypropyl methylcellulose), lubricant, inert diluent, preservative, disintegrant (e.g. sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered active ingredient moistened with an inert liquid diluent.

Tablets, and other solid dosage forms of the pharmaceutical compositions of the present invention, such as tablets, capsules, pills and granules, may optionally be registered or prepared with coatings and shells, such as enteric coatings and other coatings well known in the formulation art. Pharmaceutical They may also be formulated to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropyl methylcellulose in varying proportions to provide the desired release profile, other polymeric matrices, liposomes and / or microspheres. They may be sterilized, for example, by filtration through a bacterial retention filter, or by incorporating sterilizing agents in the form of sterile solid compositions which may be dissolved in sterile water, or some other sterile injectable medium immediately prior to use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient (s) only or preferably into a certain portion of the gastrointestinal tract, optionally in a time-consuming manner. Examples of inclusion compositions that may be used include polymeric substances and waxes. The active ingredient may also be in microencapsulated form, if appropriate, with one or more of the excipients described above.

Liquid dosage forms for oral administration of the vitamin D compound (s) include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate. , benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular cottonseed, peanut, corn, germ, olive, castor oil and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and sorbitan fatty acid esters, and mixtures thereof.

In addition to inert diluents, oral compositions may include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preserving agents.

Suspensions, in addition to the active vitamin D compound (s) may contain suspending agents such as, for example, ethoxylated isoesteraryl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide, bentonite, agar and tragacanth, and mixtures thereof.

The pharmaceutical compositions of the invention for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more vitamin D compound (s) with one or more suitable non-irritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, which is solid at room temperature but liquid at body temperature and will therefore dissolve in the rectum or vaginal cavity and release the active agent.

Compositions of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.

Dosage forms for topical or transdermal administration of a vitamin D compound include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalers. The active vitamin D compound (s) may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that may be required.

Ointments, pastes, creams and gels may contain, in addition to the vitamin D compound (s) of the present invention, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

The powders and sprays may contain, in addition to a vitamin D compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures thereof. The sprays may additionally contain customary propellants such as chlorofluorocarbons and volatile unsubstituted hydrocarbons such as butane and propane.

The vitamin D compound (s) may alternatively be administered by aerosol. This is accomplished by preparing an aqueous aerosol, liposome preparation or solid particles containing the compound. A non-aqueous suspension (e.g. fluorocarbon propellant) may be used. Sonic nebulizers are preferred because they minimize exposure of the shear agent, which can result in compound degradation.

Generally, an aqueous aerosol is manufactured by formulating an aqueous solution or suspension of the agent together with conventional pharmaceutically acceptable carriers and stabilizers. Carriers and stabilizers vary with the needs of the particular compound, but typically include nonionic surfactants (Tweens, Pluronics, or polyethylene glycol), innocuous proteins such as serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars or sugar alcohols. Aerosols are generally prepared from isotonic solutions.

Transdermal patches have added the advantage of providing controlled release of a vitamin D compound to the body. Such dosage forms may be manufactured by dissolving or dispersing the agent in the medium itself. Absorption enhancers can also be used to increase the flow of the active ingredient through the skin. The rate of such flow can be controlled by providing a rate control membrane or dispersing the active ingredient in a polymer matrix or gel.

Pharmaceutical compositions of the invention suitable for parenteral administration comprise one or more vitamin D compounds in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted in solutions or sterile injectable dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes that render the formulation isotonic with the intended recipient's blood or suspending or thickening agents.

Examples of suitable aqueous and non-aqueous carriers which may be used in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils such as oil and organic injectable esters such as ethyl oleate. The flowability itself can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, sorbic acid phenol, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like in the compositions. Further, prolonged absorption of the injectable pharmaceutical form may be produced by the inclusion of absorption retarding agents such as aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirable to reduce the absorption of the drug from subcutaneous or intramuscular injection. This can be accomplished by using a liquid suspension of crystalline or amorphous material having insufficient water solubility. The absorption rate of the drug then depends on its rate of dissolution which, in turn, may depend on crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oily vehicle.

Injectable depot forms are fabricated by forming microencapsulation matrices of vitamin D compound (s) in biodegradable polymers such as polylactide-polyglycolide. Depending on the drug to polymer ratio, and the nature of the particular polymer used, the rate of drug release may be controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides). Depot injectable formulations are also prepared by capturing the drug in liposomes or microemulsions that are compatible with body tissue.

When the vitamin D compound (s) are administered as pharmaceuticals to humans and animals, they may be supplied by themselves or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.

Regardless of the route of administration selected, the vitamin D compound (s), which may be used in a suitable hydrated form, and / or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.

Actual dosage levels and time course of administration of the active ingredients in the pharmaceutical compositions of the invention may be varied to obtain an amount of the active ingredient that is effective in obtaining the desired therapeutic response for a particular patient, composition, and mode of administration. without being toxic to the patient. An exemplary dose range is 0.1 to 300 μg per day.

A preferred dose of the vitamin D compound for the present invention is the maximum that a patient can tolerate and not develop hypercalcemia. Preferably, the vitamin D compound of the present invention is administered at a concentration of about 0.001 μg to about 100 μg per kilogram body weight, about 0.001 to about 10 μg / kg or about 0.001 μg to about 100 μg / kg body weight. Intermediate ranges to the values reported above are also intended to be part of the invention.

The vitamin D compound may be administered separately, sequentially or simultaneously in separate pharmaceutical formulations or combined with a second medicament for the treatment of endometriosis.

Synthesis of the Invention Compounds

Various compounds of the present invention may be prepared by incubating vitamin D3 analogs in cells, for example, incubating vitamin D3 analogs in UMR 106 cells or Ros 17 / 2.8 cells results in the production of vitamin D3 compounds of the invention. . For example, incubation of 1,25-dihydroxy-16-ene-5,6-trans-calcitriol in UMR 106 cells results in the production of 1,25-dihydroxy-16-eno-24-oxo-5. , 6 trans-calcitriol.

In addition to the methods described herein, the compounds of the present invention may be prepared using a variety of synthetic methods. For example, one skilled in the art would be able to use methods for synthesizing existing vitamin D3 compounds to prepare the compounds of the invention (see, for example, Bouillon, R. et al, (1995) Endocrine Reviews 16 (2): 201- 204; Ikekawa N. (1987) Med. Res. Rev. 7: 333- 366; DeLuca HF and Ostrem VK (1988) Prog. Clin Biol. Res. 259: 41-55; Ikekawa N. and Ishizuka S. ( 1992) CRC Press 8: 293-316; Calverley MJ and Jones G. (1992) Academic Press 193-270; Pardo R. and Santelli M. (1985) Bull. Soc. Chim. Fr: 98-114; Bythgoe B. (1980) Chem. Soc. Rev. 449-475; Quinkert G. (1985) Synform 3: 41-122; Quinkert G. (1986) Synform 4: 131-225; Quinkert G. (1987) Synform 5: 1- 85 Mathieu C. et al (1994) Diabetologia 37: 552-558; Dai H. and Posner GH (1994) Synthesis 1383-1398); DeLuca et al., WO 97/11053.

Exemplary methods of synthesis include photochemical ring-opening of a modified 7-dehydrocholesterol 1-hydroxylated side chain derivative that initially produces a pre-vitamin that is easily thermolysed to vitamin D3 in a well-known form (Barton DHR et al ( J. Am. Chem. Soc. 95: 2748-2749; Barton DHR (1974) JCS Chem. Comm. 203-204); a phosphor oxide bonding method developed by (Lythgoe et al (1978) JCS Perkin Trans. 1: 590-595) which comprises attaching a phosphor oxide to a Grundmann ketone derivative to directly produce an l-alpha skeleton, 25 (OH) 2 D3 as described in Baggiolini EG, et al (1986) J. Org. Chem. 51: 3098-3108; DeSchrijver J. and DeClercq P.J. (1993) Tetrahed Lett 34: 4369-4372; Posner G.H and Kinter C.M. (1990) J. Org. Chem. 55: 3967-3969; dienine semihydrogenation to a pre-vitamin structure rearranged for the corresponding vitamin D3 analog as described by Harrison R.G. et al (1974) JCS Perkin Trans. 1: 2654-2657; Castedo L. et al (1988) Tetrahed Lett 29: 1203-1206; Mascarenas J.S. (1991) Tetrahedron 47: 3485-3498; Barrack S.A. et al. (1988) J. Org. Chem. 53: 1790-1796) and Okamura W.H. et al. (1989) J. Org. Chem. 54: 4072-4083; the vinylalene method involving intermediates which are subsequently arranged using heat or a combination of metal catalyzed isomerization followed by photosensitive isomerization (Okamura WH et al. (1989) J. Org. Chem. 54: 4072-4083; Van Alstyne EM et al. (1994) J. Am. Chem. Soc. 116: 6207-6210); the method described by Trost et al. B.M. et al. J. Am. Chem. Sound. 114: 9836-9845; Nagasawa K. et al. (1991) Tetrahed Lett 32: 4937-4940 involves an acyclic A-ring precursor that is intramolecular cross-linked to bromoenine leading directly to 1,25 (OH) 2 D3 skeleton formation; a tosylated derivative that is isomerized to the i-steroid that can be modified on carbon-1 and subsequently subsequently back-isomerized under solvolytic conditions to form 1-alpha, 25 (OH) 2D2 or analogs thereof (Sheves M. and Mazur Y. ( J. Am. Chem. Soc. 97: 6249-6250; Paaren HE et al (1980) J. Org. Chem. 45: 3253-3258; Kabat M. et al. (1991) Tetrahed Lett 32: 2343- 2346; Wilson SR et al. (1991) Tetrahed Lett 32: 2339-2342); direct modification of vitamin D derivatives to 1-oxygenated 5,6-trans vitamin D as described in (Andrews D.R. et al (1986) J. Org. Chem. 51: 1635-1637); Diels-Alders' pre-vitamin D3 cicloaduct method can be used to cyclo-reverse to 1-alpha, 25 (OH) 2D2 through the intermediate of a pre-vitamin form via thermal isomerization (Vanmaele L. et al (1985) Tetrahedron 41: 141-144); and, a final method requires the direct modification of 1-alpha, 25 (OH) 2D2 or an analog through the use of suitable protecting groups such as transition metal derivatives or other chemical transformations (Okarmura WH et al (1992)). J. Cell Biochem 49: 10-18). Additional methods for synthesizing vitamin D2 compounds are described, for example, in Japanese Patent Disclosures Nos. 62750/73, 26858/76, 26859/76, and 71456/77; Pat. No. 25 3,639,596; 3,715,374; 3,847,955 and 3,739,001.

Examples of compounds of this invention having a saturated side chain may be prepared according to the general procedure illustrated and described in U.S. Patent No. 4,927,815. Examples of compounds of the invention having an unsaturated side chain may be prepared according to the general procedure illustrated and described in U.S. Patent No. 4,847,012. Examples of compounds of the invention in which R groups together represent a cycloalkyl group may be prepared according to the general process illustrated and described in U.S. Patent No. 4,851,401.

Another synthetic strategy for the preparation of modified 1-alpha, 25-dihydroxyergocalciferol side chain analogs is disclosed in Kutner et al, The Journal of Organic Chemistry, 1988, 53: 3450-3457. In addition, the preparation of 24-homo and 26-homo vitamin D analogs is disclosed in U.S. Patent No. 4,717,721.

Enancioselective synthesis of chiral molecules is now state of the art. Through combinations of enancioselective synthesis and purification techniques, many chiral molecules can be synthesized as an enanciomerically enriched preparation. For example, methods have been reported for the enancioselective synthesis of 1-alpha, 25 (OH) 2D3 ring A diastereomers as described in Muralidharan et al (1993) J. Organic Chem. 58 (7): 1895-1899 and Norman et al (1993) J. BioL Chem. 268 (27): 20022-30. Other methods for the enanciomeric synthesis of various compounds known in the art include, inter alia, epoxides (see, for example, Johnson, RA; Sharpless, KB In Catalytic Asymmetric Synthesis; Ojima, I., Ed .: VCH: New York, 1993 ; Chapter 4.1 Jacobsen, EN Ibid. Chapter 4.2), diols (eg, by the Sharpless method, J. Org. Chem. (1992) 57: 2768), and alcohols (eg, by ketone reduction, EJ Corey et al, J. Am. Chem. Soc. (1987) 109: 5551). Other reactions useful for generating optically enriched products include hydrogenation of olefins (e.g., M. Kitamura et al, J. Org. Chem. (1988) 53: 708); DielsAlder reactions (e.g., K. Narasaka et al, J. Am. Chem. Soc. (1989) 111: 5340); aldol reactions and enolate alkylation (see, for example, DA Evans et al, J. Am. Chem. Soc. (1981) 103: 2127; DA Evans et al, J. Am. Chem. Soc. (1982) 104 : 1737); carbonyl additions (e.g., R. Noyori, Angew. Chem. Int. Ed. Eng. (1991) 30:49); and meso-epoxide ring opening (e.g., Martinez, L.E.; Leighton J.L., Carsten, D.H .; Jacobsen, E.N. J. Am. Chem. Soc. (1995) 117: 5897-5898). The use of enzymes to produce optically enriched products is also well known in the art (e.g., M.P. Scheider, ed. "Enzymes as Catalysts in Organic Synthesis", D. Reidel, Dordrecht (1986).

Chiral synthesis may result in high purity stereoisomer products. However, in some cases, the stereoisomer purity of the product is not high enough. The skilled artisan will appreciate that the separation methods described herein can be used to further enhance the stereoisomer purity of the vitamin D3 epimer obtained by chiral synthesis.

Compounds of formula (XVIII):

<formula> formula see original document page 63 </formula>

on what:

Χ1 and X1 are all independently H2 or = CH2, as long as both Xi and X1 are not = CH2;

R 1 and R 2 are all independently hydroxyl, C (O) C 1 -C 4 alkyl, OC (O) hydroxyalkyl, OC (O) Auroralkyl as long as both R 1 and R 2 are not hydroxyl;

R 3 and R 4 are all independently hydrogen, C 1 -C 4 alkyl, or R 3 and R 4 taken together with C 20 form C 3 -C 8 cycloalkyl; and

R 5 and R 6 are all independently C 1 -C 4 alkyl, hydroxyalkyl, or haloalkyl, for example fluoroalkyl, for example, fluoromethyl and trifluoromethyl;

and pharmaceutically acceptable esters, salts, and prodrugs thereof may be synthesized by methods described in this section, and in the chemical literature. In particular, the compounds of formula (XVIII) of the invention are prepared as shown in Scheme 1 below.

Accordingly, the compounds of formula (XVIII) are prepared by linking the compounds of formula (XIX) with the compounds of formula (XX) in tetrahydrofuran with n-butyllithium as a base to provide compounds of formula (XI). Subsequent removal of the silyl protecting groups (R1 = OSi (CH3) 2t.Bu) provides the 1,3 dihydroxy vitamin D3 compound of formula (XVIII) (Ri = OH, R2 = OH). Acylation at positions 1 and / or 3 is obtained using methods well known in the art. For example, preparation of the 1,3-diacetoxy compounds of formula IV (R 1 = R 2 = OAc) requires additional acetylation with acetic anhydride and pyridine as shown in Scheme 2 and described below.

Referring to Scheme 1, the compounds of formula (XX) are known compounds, and are prepared starting from the known epoxy ketone of formula (XII). The compound of formula (XII) is converted to the epoxy olefin of formula (XIII) by a Wittig reaction. Reduction with LiA1H4 to compound (XIV) and protection of the hydroxy group resulted in compound (XXV). Then, the reaction of eno of formula (XXV) with the known hydroxy conjugated ketone (XXVI) (R3 = Re = CH3) in tetrahydrofuran in the presence of Lewis acid (CH3) 2 Al Cl yields compound (XXVII) characterizing the C, D and total side chain rings of the target vitamin D analogs. Finally, removal of the silyl group and oxidation provide the key intermediate, the ketone of formula (XX). Scheme 1

<formula> formula see original document page 65 </formula>

Scheme 2 shows the binding of compound (XX) with a silylated phosphine oxide under Witting binding conditions. Removal of the silyl protecting group provides diols of formula (XVIII), where both R1 and R2 are hydroxyl. Scheme 2

<formula> formula see original document page 66 </formula>

wherein X1, X2, R3, R4, R5 and R4 are as defined above.

Scheme 3 demonstrates the acetylation of the vitamin D3 derivatives of formula (P) to the acetates of formula (Q). Scheme 3

<formula> formula see original document page 66 </formula>

Vitamin D3 compounds of the formula: <formula> formula see original document page 67 </formula>

on what:

A1 is single or double bond;

A2 is a single, double or triple bond;

X1 and X2 are all independently H or = CH2,

R 1 and R 2 are all independently C (0) C 1 -C 4 alkyl, OC (O) hydroxyalkyl, or OC (0) haloalkyl;

R 3, R 4 and R 5 are all independently hydrogen, C 1 -C 4 alkyl, hydroxyalkyl, or haloalkyl, or R 3 and R 4 taken together with C 20 form C 3 -C 6 cycloalkyl;

R 6 and R 7 are all independently haloalkyl; and

R 8 is H or C (O) C 1 -C 4 alkyl, C (O) hydroxyalkyl, or OC (O) haloalkyl; and pharmaceutically acceptable esters, salts, and prodrugs thereof.

can be prepared analogously to the synthesis of 1,3-Di-O-acetyl-1,25-dihydroxy-16,23Z-diene-26,27-hexafluoro-19-nor-cholecalciferol (1) which is carried out under Standard acetylation conditions of diol to corresponding diacetate:

<formula> formula see original document page 67 </formula> The present invention will now be described with reference to the following non-limiting examples, with reference to the figures, wherein:

Figure 1 shows the effect of treatment with a vitamin D compound (Compound A) on lesion weight in an in vivo endometriosis model. Panel A - pairs of treated and untreated patients receiving the same donor cells. Panel B - change in lesion weight for specific pairs. Panel C - Average lesion weight in treated and untreated patients.

Figure 2 shows the effect of treatment with a vitamin D compound (Compound A) on endometrial stromal cell proliferation. Panel A - Eutopic Cells, Panel B - Ectopic Cells.

Figure 3 shows the effect of treatment with a vitamin D compound (Compound A) on gene expression in cultured cells.

Figure 4 shows the effect of treatment with a vitamin D compound (Compound A) on lesion weight in an in vivo endometriosis model. Panel A - complete data set. Panel B - average lesion weight for treatment groups. Panel C - Relative reduction in lesion weight as a result of treatment.

Figure 5 shows the effect of treatment with a vitamin D compound (Compound B) on lesion weight in an in vivo endometriosis model. Panel A - complete data set. Panel B - average lesion weight for treatment groups. Panel C - Relative reduction in lesion weight as a result of treatment.

Figure 6 shows the effect of treatment with a vitamin D compound (Compound C) on lesion weight in an in vivo endometriosis model. Panel A - complete data set. Panel B - average lesion weight for treatment groups. Panel C - Relative reduction in lesion weight as a result of treatment.

Figure 7 illustrates the reduction in lesion weight as a function of different dosages of vitamin D compound Compound A.

Figure 8 illustrates the reduction in lesion weight that results from a range of different treatment regimens using vitamin D compound Compound A.

Figure 9 shows the effect of Compound A treatment on cell adhesion.

Figure 10 shows the effect of Compound A treatment on cell migration.

Figure 11 shows the effect of Compound A treatment on a range of inflammatory markers. SYNTHETIC EXAMPLES

All operations involving vitamin D3 analogs are conducted on amber colored glassware in a nitrogen atmosphere. Tetrahydrofuran was distilled from sodium cetyl benzophenone just prior to use and solute solutions were dried with sodium sulfate. Melting points were determined on a Thomas-Hoover capillary apparatus and are uncorrected. Optical rotations were measured at 25 ° C. 1H NMR Spectrum was recorded at 400 MHz in CDCl3 unless otherwise indicated. TLC was performed on silica gel plates (Merck PF-254) with short wavelength UV light visualization or by spraying the plates with 10% phosphomolybdic acid in methanol followed by heating. Flash chromatography was performed on 40-65 μιη mesh silica gel. Preparative HPLC was performed on a 5x50 cm column and 15-30 μηι mesh of silica gel at a flow rate of 100 ml / min.

Synthetic Example 1 - Synthesis of 1,3-Di-O-acetyl-1,25-dihydroxy-16,23Z-diene-26,27-hexafluoro-19-nor-cholecaciferol (1) <formula> formula see original document page 70 </formula>

1,25-Dihydroxy-16,23Z-diene-26,27-hexafluoro-19-nor-cholecaciferol starting material may be prepared as described in US Patent 5,428,029 to Doran et al. 25-dihydroxy-16,23Z-diene-26,27-hexafluoro-19-nor-cholecaciferol was dissolved in 0.8 ml pyridine, cooled to ice bath temperature and 0.2 ml acetic anhydride was added and kept at this temperature for 16 h. Then the reaction mixture was diluted with 1 ml water, stirred for 10 min in the ice bath and partitioned between 5 ml water and 20 ml ethyl acetate. The organic layer was washed with 3 x 5 ml of water, once with 5 ml of saturated sodium hydrogen carbonate, once with 3 ml of brine then dried (sodium sulfate) and evaporated. The oily residue was taken up in 1: 6 ethyl acetate - hexane and flash chromatographed using a 1: 6, 1: 4 and 1: 2 graded ethyl acetate - hexane gradient. Column chromatography was monitored by TLC (1: 4 ethyl acetate-hexane, viewing point with phosphomolybdic acid spray), appropriate fractions were mixed, evaporated, the residue taken up in methyl formate, filtered, then evaporated again to provide 23.8 mg of the title compound (1) as a colorless syrup; 400 MHz 1H NMR δ 0.66 (3Η, s), 0.90 (1H, m), 1.06 (3H, d, J = 7.2 Hz), 1.51 (1H, m), 1, 72 - 1.82 (3H, m), 1.9-2.1 (3H, m), 1.99 (3H, s) 2.04 (3H, s), 2.2 - 2.3 (3 m) 2.44 - 2.64 (6H, m), 2.78 (1H, m), 3.01 (1H, s), 5.10 (2H, m), 5.38 (1H, m) ), 5.43 (1H, d, J = 12 Hz), 5.85 (1H, d, J = 11.5 Hz), 5.97 (1H, dt, J = 12 and 7.3 Hz), 6.25 (1H, d, J = 11.5 Hz). Synthetic Example 2 - Synthesis of 1,3-Di-O-acetyl-1,25-Dihydroxy-16-eno-23-ya-26,27-hexafluoro-19-nor-cholecaciferol (2) and 1,3 , 25-Tri-O-acetyl-1,25-Dihydroxy-16-ene-23-one-26,27-hexafluoro-19-nor-cholecaciferol (3)

<formula> formula see original document page 71 </formula>

1,25-Dihydroxy-16-ene-23-one-26,27-hexafluoro-19-nor-cholecaciferol starting material may be prepared as described in US Patents 5,451,574 and 5,612,328 to Baggiolini et al. 314 mg (0.619 mmol) of 1,25-dihydroxy-16-ene-23-one-26,27-hexafluoro-19-nor-cholecaciferol was dissolved in 1.5 ml of pyridine, cooled to room temperature. ice bath, and 0.4 ml of acetic anhydride was added. The reaction mixture was kept at room temperature for 7 hours and then for 23 hours in a refrigerator. This was then diluted with 10 ml water and extracted with 30 ml ethyl acetate. The organic extract was washed with water and brine, dried over sodium sulfate and evaporated. The residue was flash chromatographed on a 10 x 140 mm column with 1: 6 and 1: 4 ethyl acetate - hexane as the mobile phase to afford 126 mg of 1,3-Di-O-acetyl-1, 25-Dihydroxy-16-ene-23-one-26,27-hexafluoro-19-nor-cholecaciferol (2), and 248 mg of 1,25-Tri-O-acetyl-1,25-Di -hydroxy-16-ene-23-one-26,27-hexafluoro-19-nor-cholecaciferol (3).

Synthetic Example 3 - Synthesis of 1,3-Di-O-acetyl-1,25-dihydroxy-16-ene-23-one-cholecaciferol (4)

<formula> formula see original document page 71 </formula> A 10 ml round bottom flask was charged with 40 mg of 1,25-dihydroxy-16-ene-23-yna-cholecaciferol. This material was dissolved in 1 ml of pyridine. This solution was cooled in an ice bath after 0.3 ml of acetic anhydride was added. The solution was stirred for 30 min, then refrigerated overnight, diluted with water and transferred to a separatory funnel with the addition of 10 mL of water and 40 mL of ethyl acetate. The organic layer was washed with 4 x 20 ml water, 10 ml brine passed through a sodium sulfate buffer and evaporated. The light brown oily residue was taken up in 1: 9 ethyl acetate - hexane then flash chromatographed on a 10 χ 130 mm column using 1: 9 ethyl acetate - hexane as the mobile phase for fractions 1 to 5. , 1: 6 for fractions 6 to 13 and ethyl acetate - hexane 1: 4 for fractions 14 to 20 (18 ml fractions). Fractions 14 to 19 contained the main range with Rf 0.15 (TLC 1: 4). These fractions were mixed and evaporated to a colorless oil, 0.044 g. The material was taken up in methyl formate, filtered and evaporated to afford a sticky, colorless foam, 0.0414 g of the title compound (4).

Synthetic Example 4 - Synthesis of 1,3-Di-O-acetyl-1,25-dihydroxy-16,23E-diene-cholecaciferol (5)

<formula> formula see original document page 72 </formula>

0.0468 g of 1,25-Dihydroxy-16,23E-diene-cholecaciferol was dissolved in 1.5 ml of pyridine. This solution was cooled in an ice bath then cooled overnight, diluted with 10 ml water while still immersed in the ice bath, stirred for 10 min and transferred to a separatory funnel with the aid of 10 ml water and 40 ml. of ethyl acetate. The organic layer was washed with 4 x 20 ml water, 10 ml brine, passed through a sodium sulfate buffer and evaporated. The light brown oily residue was taken up in 1: 9 ethyl acetate - hexane then flash chromatographed on a 10 χ 130 mm column using 1: 9 ethyl acetate - hexane as the mobile phase for fractions 1 to 3. (20 ml fractions), 1: 6 for fractions 6 to 8 and 1: 4 ethyl acetate - hexane for fractions of 9al7 (18ml each). Fractions 11 through 14 contained the main range with Rf 0.09 (TLC 1: 4). These fractions were mixed and evaporated to a colorless oil, 0.0153 g. This material was taken up in methyl formate, filtered and evaporated to afford 0.014 g of the title compound (5).

Synthetic Example 6 - Synthesis of 1,3-Di-O-acetyl-1,25-dihydroxy-16-eneolecalciferol (6)

<formula> formula see original document page 73 </formula>

0.0774 g of 1,25-Dihydroxy-16-ene-cholecaciferol was dissolved in 1.5 ml of pyridine. This solution was cooled in an ice bath after 0.3 ml of acetic anhydride was added. The solution was stirred, cooled overnight then diluted with 1 ml water, stirred for 1 h in the ice bath and diluted with 30 ml ethyl acetate and 15 ml water. The organic layer was washed with 4 x 15 ml of water, once with 5 ml of brine then dried (sodium sulfate) and evaporated. The light brown oily residue was taken up in 1: 9 ethyl acetate - hexane then flash chromatographed on a 10 x 130 mm column using 1: 9 ethyl acetate - hexane as the mobile phase for fraction 1 (fractions). 20 ml), 1: 6 for fractions 2 to 7 and ethyl acetate - hexane 1: 4 for fractions 8 to 13. Fractions 9 to 11 contained the main range with Rf 0.09 (TLC, ethyl acetate - hexane). hexane 1: 4). These fractions were mixed and evaporated to a colorless oil, 0.0354 g. This material was taken up in methyl formate, filtered and the solution evaporated, colorless film of 0.027 g, the title compound (6).

Synthetic Example 7 - Synthesis of 1,3,25-Tri-O-acetyl-1,25-dihydroxy-16-ene-23-one-26,27-hexafluoro-cholecaciferol (7) and 1,3-Di -0-acetyl-1,25-di-

<formula> formula see original document page 74 </formula>

0.0291 g of 1,25-dihydroxy-16-ene-23-one-26,27-hexafluorocholecaciferol was dissolved in 1.5 ml of pyridine. This solution was cooled in an ice bath after 0.25 ml of acetic anhydride was added. The solution was stirred for 20 min and kept in a refrigerator overnight. The cooled solution was diluted with 15 mL of water, stirred for 10 min, and diluted with 30 mL of ethyl acetate. The organic layer was washed with 4 x 15 ml of water, once with 5 ml of brine then dried (sodium sulfate) and evaporated. The light brown oily residue was taken up in 1: 6 ethyl acetate - hexane then flash chromatographed on a 10 x 110 mm column using 1: 6 ethyl acetate - hexane as the mobile phase. Fractions 2 through 3 give 72.3461 to 72.3285 = 0.0176 g. Evaporation of fractions 6 to 7 give 0.0055 g. The residue from fractions 2 to 3 was taken up in methyl formate, filtered and evaporated to afford 0.0107 g of the title triacetate (7). The residue from fractions 6 to 7 was taken up in methyl formate, filtered and evaporated to afford 0.0049 g of diacetate (8). Synthetic Example 8 - Synthesis of 1,3-Di-O-acetyl-1,25-dihydroxy-16,23E-diene-25R, 26-trifluoro-cholecaciferol (9)

<formula> formula see original document page 75 </formula>

1.5 ml of 1,25-dihydroxy-16,23E-diene-25R, 26-trifluorocholecaciferol was dissolved in 1.5 ml of pyridine, cooled to ice bath temperature and 0.4 ml of anhydride. Acetic acid has been added. The mixture was then refrigerated. After two days the mixture was diluted with 1 ml of water, stirred for 10 min in the ice bath then distributed between 10 ml of water and 30 ml of ethyl acetate. The organic layer was washed with 4 x 15 ml of water, once with 5 ml of brine then dried (sodium sulfate) and evaporated. The oily, light brown residue was taken up in 1: 6 ethyl acetate - hexane then flash chromatographed on a 10 x 130 mm column using 1: 6 ethyl acetate - hexane as the mobile phase. Fractions 4 to 6 (TLC, 1: 4) contained the main range (see TLC). These fractions were evaporated and provided 0.0726 g. This residue was taken up in methyl formate, filtered and evaporated to afford 0.0649 g of colorless foam, the title compound (9).

Synthetic Example 8 - Synthesis of 1,3-Di-O-acetyl-1,25-dihydroxy-16-ene-19-nor-cholecalciferol (10)

<formula> formula see original document page 75 </formula>

0.0535 g of 1,25-Dihydroxy-16-ene-19-nor-cholecaciferol was dissolved in 1.5 ml of pyridine, cooled to ice bath temperature and 0.3 ml of acetic anhydride was added. and the mixture was refrigerated overnight. The solution was diluted with 1 ml water, stirred for 10 min in the ice bath then distributed between 10 ml water and 30 ml ethyl acetate. The organic layer was washed with 4 x 15 ml of water, once with 5 ml of brine then dried (sodium sulfate) and evaporated. The almost colorless oily residue was taken up in 1: 6 ethyl acetate - hexane as the mobile phase for fractions 1 to 6 then 1: 4 ethyl acetate - hexane was used. Fractions 9 to 19 (TLC, 1: 4 ethyl acetate - hexane, Rf 0.09, see below) were mixed, evaporated to give 0.0306 g, which was taken up in methyl formate, filtered, then evaporated. These provided 0.0376 of the title compound (10).

Synthetic Example 9 - Synthesis of 1,3-Di-O-Acetyl-1,25-dihydroxy-16-en-23-one-19-nor-cholecaciferol (11)

<formula> formula see original document page 76 </formula>

50 mg of 1,25-dihydroxy-16-ene-23-one-19-nor-cholecaciferol was dissolved in 0.8 ml of pyridine, cooled to ice bath temperature and 0.2 ml of acetic anhydride. was added. The mixture was refrigerated for 3 days then diluted with 1 ml water, stirred for 10 min in the ice bath and partitioned between 5 ml water and 20 ml ethyl acetate. The organic layer was washed with 4 x 5 ml water, once with 3 ml brine then dried (sodium sulfate) and evaporated. The almost colorless oily residue was taken up in 1: 6 ethyl acetate - hexane then flash chromatographed on a 15 χ 120 mm column using 1: 6 ethyl acetate - hexane as the mobile phase for fractions 1 to 6. , 1: 4 for fractions 9-12, 1: 3 for fractions 13-15 and ethyl acetate - hexane 1: 2 for the remaining fractions. Fractions 11 to 16 (TLC, 1: 4 ethyl acetate-hexane, Rf 0.09, see below) were mixed, evaporated 76.1487 to 76.1260 = 0.0227 g, taken up in methyl formate, filtered, then evaporated. These provided 0.0186 g of the title compound (11).

Synthetic Example 10 - Synthesis of 1,3-Di-O-acetyl-1,25-dihydroxy-16-eno 23-one-26,27-bishomo-19-nor-cholecaciferol (12)

0.0726 g of 1,25-dihydroxy-16-ene-23-one-26,27-bishomo-19-nor-cholecaciferol was dissolved in 0.8 ml of pyridine, cooled to ice bath temperature. and 0.2 ml acetic anhydride was added. The solution was stirred in the ice bath then refrigerated overnight. The solution was then diluted with 1 ml water, stirred for 10 min in the ice bath and distributed between 10 ml water and 25 ml ethyl acetate. The organic layer was washed with 3 x 10 ml water, once with 5 ml saturated sodium hydrogen carbonate, once with 3 ml brine then dried and evaporated, 33,5512 to 33,4654 = 0.0858 g. a light brown oily residue that was flash chromatographed on a 15 χ 120 mm column using 1: 6 as the mobile phase. Fractions 7 to 11 (20 ml each) were mixed (TLC, 1: 4 ethyl acetate-hexane, Rf 0.14) and evaporated, 67.2834 to 67.2654 = 0.018 g. This residue was taken up in methyl formate, filtered and evaporated. This provided 0.0211 g of the title compound (12).

Synthetic Example 11 - Synthesis of 1,3-Di-O-acetyl-1,25-dihydroxy-20-cyclopropyl-23-one-19-nor-cholecaciferol (13) <formula> formula see original document page 78 < / formula>

0.282 g of 1,25-Dihydroxy-20-cyclopropyl-23-ya-19-nor-cholecaciferol was dissolved in 0.8 ml of pyridine, cooled to ice bath temperature and 0.2 ml of acetic anhydride. It was added and the mixture was refrigerated overnight, then diluted with 1 ml water, stirred for 10 min in the ice bath and partitioned between 5 ml water and 20 ml ethyl acetate. The organic layer was washed with 3 x 5 ml of water, once with 5 ml of saturated sodium hydrogen carbonate, once with 3 ml of brine then dried (sodium sulfate) and evaporated. The oily residue was taken up in 1: 6 ethyl acetate - hexane then flash chromatographed on a 15 χ 110 mm column using 1: 6 ethyl acetate - hexane as the mobile phase for fractions 1 to 4, 1: 4. for fractions 5 to 12, 1: 3 for fractions 13 to 15 ethyl acetate - hexane for the remaining fractions. Fractions 7 to 12 (TLC, 1: 4 ethyl acetate-hexane, Rf 0.13) were mixed, evaporated, the residue taken up in methyl formate, filtered, then evaporated to afford 0.023 g of the title compound (13). . Synthetic Example 12 - Synthesis of 1,3'-25-Tri-O-acetyl-1,25-dihydroxy-20-cyclopropyl-23-one-26,27-hexafluoro-19-nor-cholecaciferol (14) and 1 1,3-Di-O-acetyl-1,25-dihydroxy-20-cyclopropyl-23-one-26,27-hexafluoro-19-nor-cholecaciferol (15) 0.1503 g of 1,25-di- hydroxy-20-cyclopropyl-23-one-26,27-hexafluoro-19-nor-cholecaciferol was dissolved in 0.8 ml of pyridine, cooled to ice bath temperature and 0.2 ml of acetic anhydride was added. The mixture was refrigerated overnight, then diluted with 1 ml water, stirred for 10 min in the ice bath and distributed between 5 ml water and 20 ml ethyl acetate. The organic layer was washed with 3 x 5 ml of water, once with 5 ml of saturated sodium hydrogen carbonate, once with 3 ml of brine then dried (sodium sulfate) and evaporated. The oily residue was taken up in 1: 6 ethyl acetate - hexane then flash chromatographed on a 15 x 150 mm column using 1: 6 ethyl acetate - hexane as the mobile phase for fractions 1 to 5: 1: 4 for the remaining fractions. Fractions of 3a4e6a7 were mixed, evaporated, then taken up in methyl formate, filtered, and evaporated to afford 0.0476 g of the title triacetate (14) and 0.04670 g of the title diacetate (15).

Synthetic Example 13 - Synthesis of 1,3-Di-O-acetyl-1,25-dihydroxy-20-cyclopropyl-23-ya-cholecaciferol (16)

<formula> formula see original document page 79 </formula>

0.0369 g of 1,25-dihydroxy-20-cyclopropyl-23-inacolecaciferol was dissolved in 0.8 ml of pyridine, cooled to ice bath temperature and 0.2 ml of acetic anhydride was added. and the mixture was refrigerated overnight, then diluted with 1 ml water, stirred for 10 min in the ice bath and distributed between 5 ml water and 20 ml ethyl acetate. The organic layer was washed with 3 x 5 ml of water, once with 5 ml of saturated sodium hydrogen carbonate, once with 3 ml of brine then dried (sodium sulfate) and evaporated. The oily residue was taken up in 1: 6 ethyl acetate - hexane then flash chromatographed on a 13 χ 110 mm column using 1: 6 ethyl acetate - hexane as the mobile phase for fractions 1 to 7, ethyl acetate. ethyl - hexane 1: 4 for the remaining fractions. Fractions 9 to 11 (TLC, 1: 4 ethyl acetate - hexane) were mixed, evaporated, taken up in methyl formate, filtered, then evaporated to afford 0.0099 g of the title compound (16).

Synthetic Example 14 - Synthesis of 1,3-Di-O-acetyl-1,25-dihydroxy-20-cyclopropyl-23E-eno-26,27-hexafluoro-19-nor-cholecaciferol (17)

<formula> formula see original document page 80 </formula>

0.0328 g of 1,25-dihydroxy-20-cyclopropyl-23E-ene-26,27-hexafluoro-19-nor-coleeaeiferol was dissolved in 0.8 ml of pyridine, cooled to ice bath temperature. and 0.2 ml acetic anhydride was added. The solution was refrigerated overnight. The solution was then diluted with 1 ml water, stirred for 10 min in the ice bath and partitioned between 5 ml water and 20 ml ethyl acetate. (Extraction of the aqueous layer provided no detectable phosphomolybic acid material.) The organic layer was washed with 3 x 5 ml of water, once with 5 ml of saturated sodium hydrogen carbonate, once with 3 ml of brine then dried (sodium sulfate) and evaporated, the residue shows Rf 0.25 as The only point. The oily residue was taken up in 1: 6 ethyl acetate - hexane then flash chromatographed on a 13.5 χ 110 mm column using 1: 6 ethyl acetate - hexane as the mobile phase for fractions 1 to 10. Fractions 4 to 9 were mixed and evaporated, the residue taken up in methyl formate, filtered, then evaporated to afford 0.0316 g of the title compound (17).

Synthetic Example 15 - Synthesis of 1,3-Di-O-acetyl-1,25-dihydroxy-20-cyclopropyl-23Z-ene-26,27-hexafluoro-19-nor-cholecaciferol (18)

<formula> formula see original document page 81 </formula>

0.0429 g of 1,25-dihydroxy-20-cyclopropyl-23Z-ene-26,27-hexafluoro-19-nor-cholecaciferol was dissolved in 0.8 ml of pyridine, cooled to ice bath temperature. and 0.2 ml acetic anhydride was added. The solution was refrigerated overnight. The solution was then diluted with 1 ml water, stirred for 10 min in the ice bath and partitioned between 7 ml water and 25 ml ethyl acetate. The organic layer was washed with 3 x 5 ml water, once with 5 ml saturated sodium hydrogen carbonate, once with 3 ml brine then dried (sodium sulfate, TLC (ethyl acetate - hexane 1: 4 shows evaporated, flash chromatographed on a 15 χ 120 mm column using 1: 6 as the mobile phase. Fractions of 3 to 6 (20 ml each) were mixed and evaporated. It is taken up in methyl formate, filtered and evaporated to give 0.0411 g of the title compound (18).

Synthetic Example 16 - Synthesis of 1,3-Di-O-acetyl-1,25-dihydroxy-20-cyclopropyl-cholecaciferol (19) <formula> formula see original document page 82 </formula>

0.0797 g of 1,25-dihydroxy-20-cyclopropyl-cholecaciferol was dissolved in 0.8 ml of pyridine, cooled to ice bath temperature and 0.2 ml of acetic anhydride was added. The solution was refrigerated overnight. The solution was then diluted with 1 ml water, stirred for 10 min in the ice bath and distributed between 10 ml water and 25 ml ethyl acetate. The organic layer was washed with 3 x 10 ml of water, once with 5 ml of saturated sodium hydrogen carbonate, once with 3 ml of brine then dried and evaporated to afford 0.1061 g of a light brown oily residue. it was flash chromatographed on a 15 x 120 mm column using 1: 6 as the mobile phase. Fractions 9 to 16 (20 ml each) were mixed (TLC, 1: 4 ethyl acetate-hexane, Rf 0.13) and evaporated. This residue was taken up in methyl formate, filtered and evaporated to afford 0.0581 g of the title compound (19).

Synthetic Example 17 - Synthesis of 1,3-Di-O-acetyl-1-alpha, 25-dihydroxy-16-ene-20-cyclopropyl-19-nor-cholecaciferol (20)

<formula> formula see original document page 82 </formula>

The solution of 1-alpha, 25-Dihydroxy-16-ene-20-cyclopropyl-19-nor-cholecaciferol (94 mg, 0.23 mmol) in pyridine (3 ml) at 0 ° C, acetic anhydride (0 0.5 mL, 5.3 mmol) was added. The mixture was stirred for 1h, refrigerated for 15h and then stirred for an additional 8h. Water (10 mL) was added and after stirring for 15 min the reaction mixture was extracted with 1: 1 EtOAc: Hexane (25 mL), washed with water (4 x 25 mL) and brine (20 mL), dried over Na2SO4. The residue (120 mg) after evaporation of the solvent was purified by FC (15 g, 30% EtOAc in hexane) to provide the title compound (20) (91 mg, 0.18 mmol, 80%). [α] 30D = +14.4 ° C and 0.34, EtOH; UV λ max (EtOH): 242 nm (ε 3434), 250 nm (ε 40458), 260 nm (ε 27545); 1H NMR (CDCl3): 6.25 (1H, d, J = 11.1 Hz), 5.83 (1H, d, J = 11.3 Hz), 5.35 (1H, m), 5.09 ( 2H, m), 2.82 - 1.98 (7H, m), 2.03 (3H, s), 1.98 (3H, s), 2.00 - 1.12 (15H, m), 1 , 18 (6H, s), 0.77 (3H, s), 0.80 - 0.36 (4H, m); 13 C NMR (CDCl 3): 170.73 (0), 170.65 (0), 157.27 (0), 142.55 (0), 130.01 (0), 125.06 (1), 123, 84 (1), 115.71 (1), 71.32 (0), 70.24 (1), 69.99 (1), 59.68 (1), 50.40 (0), 44.08 (2), 41.40 (2), 38.37 (2), 35.96 (2), 35.80 (2), 32.93 (2), 29.48 (3), 29.31 ( 2), 28.71 (2), 23.71 (2), 22.50 (2), 21.56 (3), 21.51 (0), 21.44 (3), 18.01 (3) ), 12.93 (2), 10.53 (2); MS HRES Calculated for C 31 H 46 O 5 M + Na 521.3237, Observed M + Na 521.3233

Synthetic Example 18 - Synthesis of 1,3-Di-0-α-yl-1-alpha, 25-hydroxy-16-ene-20-cyclopropyl-cholecaciferol (21)

<formula> formula see original document page 83 </formula>

To the solution of 1-alpha, 25-Dihydroxy-16-ene-20-cyclopropyl-cholecaciferol (100 mg, 0.23 mmol) in pyridine (3 mL) at 0 ° C, acetic anhydride (0.5 mL, 5%, 3 mmol) was added. The mixture was stirred for 2 h and then refrigerated for an additional 15 h. Water (10 ml) was added and after stirring for 15 min. The reaction mixture was extracted with 1: 1 EtOAc: Hexane (25 mL), washed with water (4 χ 25 mL), brine (20 mL) and dried over Na 2 SO 4. The residue (150 mg) after evaporation of the solvent was purified by FC (15 g, 30% EtOAc in hexane) to afford the title compound (21) (92 mg, 0.18 mmol, 78%). [α] D 30 -14.9 and 0.37, EtOH; Λ max UV (EtOH): 208 nm (ε 15949), 265 nm (ε 15745); 1H NMR (CDCl3): 6.34 (1H, d, J = 11.3 Hz), 5.99 (1H, d, J = 11.3 Hz), 5.47 (1H, m), 5.33 (1H, m), 5.31 (1H, s), 5.18 (1H, m), 5.04 (1H, s), 2.78 (1H, m), 2.64 (1H, m) 2.40 - 1.10 (18H, m), 2.05 (3H, s), 2.01 (3H, s), 1.18 (6H, s), 0.76 (3H, s), 0.66 - 0.24 (4H, m); 13 C NMR (CDCl 3): 170.76 (0), 170.22 (0), 157.18 (0), 143.02 (0), 142.40 (0), 131.94 (0), 125, 31 (1), 125.10 (1), 117.40 (1), 115.22 (2), 72.97 (1), 71.32 (0), 69.65 (1), 59.71 (1), 50.57 (0), 44.07 (2), 41.73 (2), 38.36 (2), 37.10 (2), 35.80 (2), 29.45 ( 3), 29.35 (2), 29.25 (3), 28.92 (2), 23.80 (2), 22.48 (2), 21.55 (3), 21.50 (3) ), 21.35 (0), 17.90 (3), 12.92 (2), 10.54 (2); MS HRES Calculated for C 32 H 46 O 5 M + Na 533.3237, Observed M + Na 533.3236

Synthetic Example 19 - Synthesis of 1,3-Di-O-acetyl-1,25-dihydroxy-23-inacolecalciferol (22)

<formula> formula see original document page 84 </formula>

0.2007 g (0.486 mmol) was dissolved in 2 ml of pyridine. This solution was cooled in an ice bath and 0.6 ml of acetic anhydride was added. The solution was kept in an ice bath for 45 h then diluted with 10 ml of water, stirred for 10 min and equilibrated with 10 ml of water and 40 ml of ethyl acetate. The organic layer was washed with 4 x 20 ml water, 10 ml brine, dried (sodium sulfate) and evaporated. The brown, oily residue was flash chromatographed using ethyl acetate - hexane 1:19, 1: 9, and 1: 4 as the step gradient. The main band with Rf 0.16 (TLC, acetate: hexane 1: 4) was evaporated to afford 1,3-di-O-acetyl-1,25-dihydroxy-23-yna-cholecaciferol (22) as a foam. colorless, 0.0939 g.

Synthetic Example 20 - Synthesis of (3aR, 4S, 7aR) -7a-Methyl-1- [1- (4-hydroxy-4-methyl-pent-2-ynyl) -cyclopropyl] -3a, 4,5,6, 7,7a-hexahydro-3H-inden-4-ol

<formula> formula see original document page 85 </formula>

To a stirred solution of (3aR, 4S, 7aR) -1- {1- [4- (tert-Butyl-dimethylsilanyloxy) -7a-methyl-3a, 4,5,6,7,7a-hexahydro-3H -inden-1-yl]) - cyclopropyl] ethynyl (1.0 g, 2.90 mmol) in tetrahydrofuran (15 mL) at -78 ° C was added n-BuLi (2.72 mL, 4.35 mmol, 1.6M in hexane). After stirring at -78 ° C for 1 h., Acetone (2.5 mL, 34.6 mmol) was added and stirring was continued for 2.5 h. NH 4 Clq was added (15 mL) and the mixture was stirred for 15 min at room temperature then extracted with EtOAc (2 x 50 mL). The combined extracts were washed with brine (50 mL) and dried over Na 2 SO 4. The residue after solvent evaporation (2.4 g) was purified by FC (50 g, 10% EtOAc in hexane) to provide (3aR, 4S, 7aR) -5- {1- [4- (tert-Butyl -dimethylsilanyloxy) -7α-methyl-3α, 4,5,6,7,7a-hexahydro-3 H -inden-1-yl] cyclopropyl} -2-methyl-pent-3-yn-2- ol (1.05 g, 2.61 mmol) which was treated with tetrabutylammonium fluoride (6 mL, 6 mmol, 1.0 M in THF) and stirred at 65 to 75 ° C for 48 h. The mixture was diluted with EtOAc (25 mL) and washed with water (5 x 25 mL), brine (25 mL).

The combined aqueous washes were extracted with EtOAc (25 mL) and the combined organic extracts were dried over Na 2 SO 4. The residue after evaporation of the solvent (1.1 g) was purified by FC (50 g, 20% EtOAc in hexane) to afford the title compound (0.75 g, 2.59 mmol, 90%).

[cc] 30d = +2.7 and 0.75, CHCl3. 1H NMR (CDCl3): 5.50 (1H, m), 4.18 (1H, m), 2.40 (2H, s), 2.35 - 1.16 (11H, m), 1.48 ( 6H, s), 1.20 (3H, s), 0.76 - 0.50 (4H,

<formula> formula see original document page 85 </formula> 85 m); 13 C NMR (CDCl 3): 156.39, 125.26, 86.39, 80.19, 69.21, 65.16, 55.14, 46.94, 35.79, 33.60, 31.67, 29.91, 27.22, 19.32, 19.19, 17.73, 10.94, 10.37; MS HREI Calculated for C 22 H 28 O 2 M + 288.2089, Observed M + 288.2091. Synthetic Example 21 - Synthesis of (3aR, 4S, 7aR) -7a-Methyl-1- [1- (4-hydroxy-4-methyl-pent-2Z-enyl) -cyclopropyl] -3a, 4,5,6, 7,7a-hexahydro-3H-inden-4-ol

The mixture of (3aR, 4S, 7aR) -7a-Methyl-1- [1- (-4-hydroxy-4-methylpent-2-ynyl) cyclopropyl] -3a, 4,5,6,7, 7a-hexahydro-3H-inden-4-ol (0.72 g, 2.50 mmol), ethyl acetate (10 mL), hexane (24 mL), absolute ethanol (0.9 mL), quinoline (47 L ) and Lindlar catalyst (156 mg, 5% Pd in CaCO3) was hydrogenated at room temperature for 2 h. The reaction mixture was filtered through a pad of celite and the pad was washed with EtOAc. The filtrates and washings were combined and washed with 1M HCl, NaHCO 3 and brine. After drying over Na 2 SO 4 the solvent was evaporated and the residue (0.79 g) was purified by FC (45 g, 20% EtOAc in hexane) to provide the title compound (640 mg, 2.2 mmol, 88%). ).

Synthetic Example 22 - Synthesis of (3aR, 4S, 7aR) -7a-Methyl-1- [1- (4-hydroxy-4-methyl-pentyl) -cyclopropyl] -3a, 4,5,6,7,7a- hexahydro-3H-inden-4-ol

(3aR, 4S, 7aR) -7a-Methyl-1- [1- (4-hydroxy-4-methyl-pent-2Z-enyl) -cyclopropyl] -3a, 4,5,6,7,7a -hexyhydro-3H-inden-4-ol (100 mg, 0.34 mmol), rhodium tetrafluoroborate 1,4-bis (diphenylphosphine) butane 1,5 cyclooctadiene (25 mg, 0.034 mmol), dichloromethane (5 mL) and one drop of mercury was hydrogenated using Paar apparatus at room temperature and 50 psi pressure for 3 h. The reaction mixture was filtered through a pad of Celite5 which was then washed with ethyl acetate. The combined filtrates and washings were evaporated to dryness (110 mg) and purified by FC (10 g, 20% EtOAc in hexane) to afford the title compound (75 mg, 0.26 mmol, 75%). [α] 30D = -8.5 and 0.65, CHCl3. 1H NMR (CDCl3): 5.37 (1H, m,), 4.14 (1H, m), 2.37 - 1.16 (17H, m), 1.19 (6H, s), 1.18 (3H, s), 0.66 - 0.24 (4H, m); MS HREI Calculated for C 19 H 32 O 2 M + H 292.2402, Observed M + H 292.2404.

Synthetic Example 23 - Synthesis of (3aR, 7aR) -7a-Methyl-1- [1- (4-methyl-4-trimethylsilanyloxy-pentyl) -cyclopropyl] -3a, 4,5,6,7,7a-hexahydro 3H-inden-4-one

<formula> formula see original document page 87 </formula>

To a stirred suspension of (3aR, 4S, 7aR) -7a-Methyl-1- [1- (4-hydroxy-4-methyl-pentenyl) -cyclopropyl] -3a, 4,5,6,7,7a-hexahydro -3H-inden-4-ol (440 mg, 1.50 mmol) and Celite (2.0 g) in dichloromethane (10 mL) at room temperature was added pyridinium dichromate (1.13 g, 3.0 15 mmol). ). The resulting mixture was stirred for 5 h, filtered through silica gel (10 g), and then silica gel pad was washed with 20% EtOAc in hexane. The combined filtrate and washings were evaporated to afford one (3 aR, 7aR) -7a-Methyl-1- [1- (4-hydroxy-4-methyl-pentenyl) cyclopropyl] -3a, 4,5,6, Crude 7,7a-hexahydro-3H-inden-4-one (426 mg, 1.47 mmol, 98%). To a stirred solution of (3aR, 7aR) -7a-Methyl-1- [1- (4-hydroxy-4-methyl-pentenyl) -cyclopropyl] -3a, 4,5,6,7,7a-hexahydro-3H -inden-4-one (424 mg, 1.47 mmol) in dichloromethane (10 mL) at room temperature was added trimethylsilyl imidazole (0.44 mL, 3.0 mmol). The resulting mixture was stirred for 1.0h filtered through silica gel (10g) and the silica gel pad was washed with 10% EtOAc in hexane. Combined filtrate and washings were evaporated to afford the title compound (460 mg, 1.27 mmol, 86%). [oc] 29D = -9.9 and 0.55, CHCl3.1H NMR (CDCl3): 5.33 (1 H, dd, J = 3.2, 1.5 Hz), 2.81 (1 H, dd , J = 10.7, 6.2 Hz), 2.44 (1Η, ddd, J = 15.6, 10.7, 1.5 Hz), 2.30 - 1.15 (13Η, m) overlap 2.03 (ddd, J = 15.8, 6.4, 3.2 Hz), 1.18 (6Η, s), 0.92 (3Η, s), 0.66 - 0.28 (4Η, m) 0.08 (9Η, s); 13 CRMN (CDCl 3): 211.08 (0), 155.32 (0), 124.77 (1), 73.98 (0), 64.32 (1), 53.91 (0), 44.70 (2), 40.45 (2), 38.12 (2), 34.70 (2), 29.86 (3), 29.80 (3), 26.80 (2), 24.07 ( 2), 22.28 (2), 21.24 (0), 18.35 (3), 12.60 (2), 10.64 (2), 2.63 (3); MS HRES Calculated for C 22 H 38 O 2 Si M + 362.2641. Observed M + 362.2648.

Synthetic Example 24 - Synthesis of (3aR, 7aR) -7a-Methyl-1- [1- (4-methyl-4-trimethylsilanyloxy-pent-2-ynyl) -cyclopropyl] -3a, 4,5,6,7, 7a-hexahydro-3H-inden-4-one

<formula> formula see original document page 88 </formula>

To a stirred suspension of (3aR, 4S, 7aR) -7a-Methyl-1- [1- (4-hydroxy-4-methyl-pent-2-ynyl) -cyclopropyl] -3a, 4,5,6,7 7a-hexahydro-3H-inden-4-ol (381 mg, 1.32 mmol) and Celite (2.0 g) in dichloromethane (10 ml) at room temperature were added pyridinium dichromate (1.0 g, 2.65 mmol). The resulting mixture was stirred for 1.5h filtered through silica gel (10g), and then silica gel pad was washed with 20% EtOAc in hexane. The filtrate and combined washings were evaporated to afford one (3aR, 7aR) -7a-Methyl-1- [1- (4-hydroxy-4-methyl-pent-2-ynyl) cyclopropyl] -3a, 4,5 Crude, 6,7,7a-hexahydro-3H-inden-4-one (360 mg, 1.26 mmol, 95%). To a stirred solution of (3aR, 7aR) -7a-Methyl-1- [1- (4-hydroxy-4-methyl-pent-2-ynyl) -cyclopropyl] -3a, 4,5,6,7,7a -hexyhydro-3H-inden-4-one (360 mg, 1.26 mmol) in dichloromethane (10 mL) at room temperature was added trimethylsilyl imidazole (0.25 mL, 1.7 mmol). The resulting mixture was stirred for 0.5h filtered through silica gel (10g) and the silica gel pad was washed with 5% EtOAc in hexane. Combined filtrate and washings were evaporated to afford the title compound (382 mg, 1.07 mmol, 81%).

Synthetic Example 25 - Synthesis of 1-alpha, 25-Dihydroxy-16-ene-20-cyclopropyl-23,24-α-cholecacifers (23)

<formula> formula see original document page 89 </formula>

To a stirred solution of a (1S, 5R) -1,5-bis - ((tert-butyldimethyl) silanyloxy) -3- [2- (diphenylphosphinoyl) -et- (Z) -ylidene] -2-methylene-cyclo hexane (513 mg, 0.88 mmol) in tetrahydrofuran (6 mL) at -78 ° C was added n-BuLi (0.55 mL, 0.88 mmol). The resulting mixture was stirred for 15 min and solution of (3aR, 7aR) -7a-Methyl-1- [1- (4-methyl-4-trimethylsilanyloxy-pent-2-ynyl) -cyclopropyl] -3a, 4,5 6.7,7a-hexahydro-3H-inden-4-one (179 mg, 0.50 mmol in tetrahydrofuran (2 mL) was added dropwise.) The reaction mixture was stirred at -72 ° C for 3 hours. 5 h diluted with hexane (25 mL) washed brine (30 mL) and dried over Na 2 SO 4 The residue (716 mg) after solvent evaporation was purified by FC (15 g, 5% EtOAc in hexane) to provide 1- alpha-3-beta-Di (tert-Butyl-dimethyl-silanyloxy) -25-trimethylsilanyloxy-16-ene-20-cyclopropyl-23,24-one-cholecaciferol (324 mg, 045 mmol). -beta-Di (tert-Butyl-dimethyl-silanyloxy) -25-trimethylsilanyloxy-16-ene-20-cyclopropyl-23,24-one-cholecaciferol (322 mg, 0.45 mmol) tetrabutylammonium fluoride (4 ml, 4 mmol, IM solution in THF) was added at room temperature.The mixture was stirred for 18 h diluted with EtOAc (25 mL) and washed with water (5 x 20 mL), brine (20 mL). ml) and dried over Na 2 SO 4. The residue (280 mg) after evaporation of the solvent was purified by FC (10 g, 50% EtOAc in hexane and EtOAc) to provide the title compound (23) (172 mg, 0.41 mmol, 82%). [α] 31D = +32.4 ° C and 0.50, MeOH. UV λ max (EtOH): 261 nm (ε 11930); 1H NMR (CDCl3): 6.36 (1H, d, J = 11.3 Hz), 6.09 (1H, d, J = 11.3 Hz), 5.45 (1H, m), 5.33 (1H, m), 5.01 (1H, m), 4.45 (1H, m), 4.22 (1H, m), 2.80 (1H, m), 2.60 (1H, m) 2.50 - 1.10 (16H, m), 1.45 (6H, s), 0.81 (3H, s), 0.72 - 0.50 (4H, m); MS HRES Calculated for C 28 H 38 O 3 M + 422.2821, Observed M + 422.2854.

Synthetic Example 26 - Synthesis of 1-alpha, 25-Dihydroxy-16-ene-20-cyclopropyl-23,24-one-19-nor-cholecaciferol (24)

<formula> formula see original document page 90 </formula>

To a stirred solution of (1R, 3R) -1,3-bis - ((tert-butyldimethyl) silanyloxy) -5- [2- (diphenylphosphinoyl) ethylidene] -cyclohexane (674 mg, 1.18 mmol) In tetrahydrofuran (8 mL) at -78 ° C was added n-BuLi (0.74 mL, 1.18 mmol). The resulting mixture was stirred for 15 min and solution of (3aR, 7aR) -7a-Methyl-1- [1- (4-methyl-4-trimethylsilanyloxy-pent-2-ynyl) cyclopropyl] -3a, 4,5 6.7,7a-hexahydro-3H-inden-4-one (235 mg, 0.66 mmol) in tetrahydrofuran (3 ml) was added dropwise.The reaction mixture was stirred at -72 ° C for 3, 5 h diluted with hexane (25 mL) washed brine (30 mL) and dried over Na 2 SO 4 The residue (850 mg) after solvent evaporation was purified by FC (15 g, 5% EtOAc in hexane) to provide 1- alpha, 3-beta-Di (tert-Butyl-dimethyl-silanyloxy) -25-trimethylsilanyloxy-16-ene-20-cyclopropyl-23,24-ya-19-nor-cholecaciferol (330 mg, 0.46 mmol). 1-alpha, 3-beta-Di (tert-Butyl-dimethylsilanyloxy) -25-trimethylsilanyloxy-16-ene-20-cyclopropyl-23,24-one-19-nor-cholecaciferol (328 mg, 0.46 mmol) tetrabutylammonium fluoride (5 mL, 5 mmol, 1M THF solution) was added at room temperature.The mixture was stirred for 62 h diluted with EtOAc (25 mL) and washed with water (5 x 20 m 1), brine (20 ml) and dried over Na 2 SO 4. The residue (410 mg) after evaporation of the solvent was purified by FC (10 g, 50% EtOAc in hexane and EtOAc) to provide the title compound (24) (183 mg, 0.45 mmol, 68%). [[oc] 29D = +72.1 and 0.58, MeOH. Amax UV (EtOH): 242 nm (ε 29286), 251 nm (ε 34518), 260 nm (ε 23875) 1H NMR (CDCl3): 6.30 (1H, d, J = 11.3 Hz), 5, 94 (1H, d, J = 11.3 Hz), 5.48 (1H, m), 4.14 (1H, m), 4.07 (1H, m), 2.78 (2H, m), 2.52 - 1.10 (18H, m), 1.49 (6H, s), 0.81 (3H, s), 0.72 - 0.50 (4H, m); MS HRES Calculated for C 27 H 38 O 3 M + 410.2821, Observed M + 410.2823.

Synthetic Example 27 - Synthesis of (3aR, 4S, 7aR) -7a-Methyl-1- [1- (5,5,5-trifluoro-4-hydroxy-4-trifluoromethyl-pent-2-ynyl) -cyclopropyl] - 3a, 4,5,6,7,7a-hexahydro-3H-inden-4-ol

<formula> formula see original document page 91 </formula>

To a stirred solution of (3aR, 4S, 7aR) -1- {1- [4- (tert-Butyl-dimethylsilanyloxy) -7a-methyl-3a, 4,5,6,7,7a-hexahydro-3H -inden-1-yl]) -cyclopropylethynyl (1.95 g, 5.66 mmol) in tetrahydroururane (35 mL) at -78 ° C was added n-BuLi (4.3 mL, 6.88 mmol, 1, 6M in hexane). After stirring at -78 ° C for 1 h., Hexafluoroacetone (six drops from the cooling probe) was added and stirring was continued for 1 h. NH 4 Cl 2 was added (10 mL) and the mixture was allowed to warm to room temperature. The reaction mixture was diluted with brine (100 mL) and extracted with hexane (2 x 125 mL). The combined extracts were dried over Na 2 SO 4. The residue after solvent evaporation (8.2 g) was purified by FC (150 g, 10% EtOAc in hexane) to provide (3aR, 4S, 7aR) -5- {1- [4- (tert-Butyl -dimethylsilanyloxy) -7a-methyl-3a, 4,5,6,7,7a-hexahydro-3H-inden-1-yl] cyclopropyl} -1,1,1-trifluoro-2-trifluoromethylpentyl 3-yn-2-ol (2.73 g, 5.35 mmol) which was treated with tetrabutylammonium fluoride (20 mL, 20 mmol, 1.0 M in THF) and stirred at 65 to 75 ° C for 30 h. The mixture was diluted with EtOAc (150 mL) and washed with water (5 x 150 mL), brine (150 mL). The combined aqueous washes were extracted with EtOAc (150 mL) and the combined organic extracts were dried over Na 2 SO 4. The residue after evaporation of the solvent (3.2 g) was purified by FC (150 g, 20% EtOAc in hexane) to afford the title compound (2.05 g, 5.17 mmol, 97%). [α] 28d = +6.0 c 0.47, CHCl3 ZH NMR (CDCl3): 5.50 (1H, br. s), 4.16 (1H, br. s), 3.91 (1H, s) 2.48 (1H, AB quartet part A, J = 17.5 Hz), 2.43 (1H, AB quartet part B, J = 17.5 Hz), 2.27 (1H, m), 2.00 - 1.40 (9H, m), 1.18 (3H, s), 0.8 - 0.5 (4H, m); NMR (CDCl3): 155.26 (0), 126.68 (1), 121.32 (0, q, J = 284 Hz), 90.24 (0), 71.44 (0, sep. J = 34 Hz), 70.54 (0), 69.57 (1), 55.17 (1), 47.17 (0), 36.05 (2), 33.63 (2), 30.10 ( 2), 27.94 (2), 19.50 (3), 19.27 (0), 17.90 (2), 11.56 (2), 11.21 (2); MS HREI Calculated for C 19 H 22 O 2 F 6 M + 396.1524, Observed M + 396.1513.

Synthetic Example 28 - Synthesis of (3aR, 7aR) -7a-Methyl-1- [1- (5,5,5-trifluoro-4-trifluoromethyl-4-hydroxy-pen-2-ynyl) -cyclopropyl] -3a, 4,5,6,7,7a-hexahydro-3H-inden-4-one

<formula> formula see original document page 92 </formula>

To a stirred suspension of (3aR, 4S, 7aR) -7a-Methyl-1- [1- (5,5,5-trifluoro-4-hydroxy-4-trifluoromethyl-pent-2-ynyl) -cyclopropyl] -3a 4,5,6,7,7a-hexahydro-3H-inden-4-ol (504 mg, 1.27 mmol) and Celite (1.5 g) in dichloromethane (12 ml) at room temperature were added. pyridinium (0.98 g, 2.6 mmol). The resulting mixture was stirred for 2.5 h filtered through silica gel (5 g), and then silica gel pad was washed with 20% EtOAc in hexane. The combined filtrate and washings were evaporated to afford a title compound (424 mg, 1.08 mmol, 85%). [α] 28d = +3.1 c 0.55, CHCl3.1H NMR (CDCl3): 5.46 (1H, br. s), 3.537 (1H, s), 2.81 (1H, dd, J = 10.7, 6.5 Hz), 2.49 - 1.76 (10H, m), 0.90 (3H, s), 0.77 - 0.53 (4, m); MS HREI Calculated for C 19 H 20 O 2 F 6 M + H 395.1440, Observed M + H 395.1443.

Synthetic Example 29 - Synthesis of 1-alpha, 25-Dihydroxy-16-ene-20-cyclopropyl-23,24-one-26,27-hexafluoro-19-nor-cholecaciferol (25)

To a stirred solution of one (1R, 3R) -1,3-bis - ((tert-butyldimethyl) silanyloxy) -5- [2- (diphenylphosphinoyl) ethylidene] cyclohexane (900 mg, 1.58 mmol) In tetrahydrofuran (8 mL) at -78 ° C n-BuLi (1.0 mL, 1.6 mmol) was added. The resulting mixture was stirred for 15 min and (3aR, 7aR) -7a-Methyl-1- [1- (5,5,5-trifluoro-4-trifluoromethyl-4-hydroxy-pen-2-ynyl) - cyclopropyl] -3a, 4,5,6,7,7a-hexahydro-3H-inden-4-one (200 mg, 0.51 mmol in tetrahydrofuran (3 mL) was added dropwise. The reaction mixture was stirred). at -12 ° C for 3.5 h diluted with hexane (25 mL) washed brine (30 mL) and dried over Na 2 SO 4 The residue (850 mg) after evaporation of the solvent was purified by FC (20 g, 10% AcOEt in hexane) to provide 1-alpha, 3-beta-Di (tert-Butyl-dimethyl-silanyloxy) -25-hydroxy-16-ene-20-cyclopropyl-23,24-one-26,27-hexafluoro- 19-nor-cholecaciferol (327 mg, 0.44 mmol, 86%) 1-alpha, 3-beta-Di (tert-Butyl-dimethyl-silanyloxy) -25-hydroxy-16-ene-20-cyclopropyl 23,24-Amino-26,27-hexafluoro-19-nor-cholecaciferol (327 mg, 0.44 mmol) Tetrabutylammonium fluoride (4 mL, 4 mmol, IM solution in 20 THF) was added at room temperature. The mixture was stirred for 24 h, diluted with EtOAc ( 25 ml) and washed with water (5 x 20 ml), brine (20 ml) and dried over Na 2 SO 4. The residue (250 mg) after evaporation of the solvent was purified by FC (10 g, 50% EtOAc in hexane and EtOAc) to provide the title compound (25) (183 mg, 0.45 mmol, 68%). [α] 30D = + 73.3 ° C and 0.51, EtOH. UV λ max (EtOH): 243 nm (ε 29384), 251 nm (ε 34973), 260 nm (ε 23924); NMR (CDCl 3): 6.29 (1, d, J = 11.1 Hz), 5.93 (1, d, J = 11.1 Hz), 5.50 (1, m), 4.12 ( 1H5 m), 4.05 (1H, m), 2.76 (2H, m), 2.55 - 1.52 (18H, m), 0.80 (3H, s), 0.80 - 0, 49 (4H, m); 13 C NMR (CDCl 3): 155.24 (0), 141.78 (0), 131.28 (0), 126.23 (1), 123.65 (1), 121.09 (O, q, J = 285 Hz), 115.67 (1), 89.63 (0), 70.42 (0), 67.48 (1), 67.29 (1), 59.19 (1), 49.87 (0), 44.49 (2), 41.98 (2), 37.14 (2), 35.76 (2), 29.22 (2), 28.47 (2), 27.57 ( 2), 23.46 (2), 19.32 (0), 17.97 (3), 11.89 (2), 10.18 (2); MS HRES Calculated for C 27 H 32 O 3 F 6 M + H 519.2329. Observed M + H 519.2325.

Synthetic Example 30 - Synthesis of 1-alpha, 25-Dihydroxy-16-ene-20-cyclopropyl-23,24-ya-26,27-hexafluoro-cholecaciferol (26)

<formula> formula see original document page 94 </formula>

To a stirred solution of a (1S, 5R) -1,5-bis - ((tert-butyldimethyl) silanyloxy) -3- [2- (diphenylphosphinoyl) -et- (Z) -ylidene] -2-methylene-cyclo hexane (921 mg, 1.58 mmol) in tetrahydrofuran (8 mL) at -78 ° C was added n-BuLi (1.0 mL, 1.6 mmol). The resulting mixture was stirred for 15 min and (3aR, 7aR) -7a-Methyl-1- [1- (5,5,5-trifluoro-4-trifluoromethyl-4-hydroxy-pen-2-ynyl) - cyclopropyl] -3a, 4,5,6,7,7a-hexahydro-3H-inden-4-one (197 mg, 0.50 mmol in tetrahydrofuran (2 mL) was added dropwise.) The reaction mixture was stirred at -12 ° C for 3.5 h diluted with hexane (25 mL) washed brine (30 mL) and dried over Na 2 SO 4. The residue (876 mg) after evaporation of the solvent was purified by FC (20 g, 105% of AcOEt in hexane) to provide 1-alpha, 3-beta-Di (tert-Butyl-dimethylsilanyloxy) -25-hydroxy-16-eno-20-cyclopropyl-23,24-one-26,27-hexafluoro-cholecaciferol (356 mg, 0.47 mmol) To 1-alpha, 3-beta-Di (tert-Butyl-dimethylsilanyloxy) -25-hydroxy-16-ene-20-cyclopropyl-23,24-one-26, 27-Hexafluoro-cholecaciferol (356 mg, 0.47 mmol) tetrabutylammonium fluoride (5 mL, 5 mmol, IM solution in THF) was added at room temperature.The mixture was stirred for 15 h, diluted with EtOAc (25 mL). ) and washed with water (5 χ 20 ml), brine (20 ml) and dried over Na 2 SO 4. The residue (270 mg) after evaporation of the solvent was purified by FC (20 g, 50% EtOAc in hexane and EtOAc) to provide the title compound (26) (216 mg, 0.41 mmol, 87%). [α] 30D = + 40.0 ° C and 0.53, EtOH. Λ max λ max (EtOH): 262 nm (ε 12919); 1H NMR (CDCl3): 6.38 (1H, d, J = 11.5 Hz), 6.10 (1H, d, J = 11.1 Hz), 5.49 (1H, m), 5.35 (1H, s), 5.02 (1H, s), 4.45 (1H, m), 4.25 (1H, m), 3.57 (1H, s), 2.83 - 1.45 ( 18H, m), 0.82 (3H, s), 0.80 - 0.51 (4H, m); MS HRES Calculated for C28H32O3F6M + H 531.2329. Observed M + H 531.2337.

Synthetic Example 31 - Synthesis of (3aR, 4S, 7aR) -7a-Methyl-1- [1- (5,5,5-trifluoro-4-hydroxy-4-trifluoromethyl-pent-2E-enyl) -cyclopropyl] - 3a, 4,5,6,7,7a-hexahydro-3H-inden-4-ol

<formula> formula see original document page 95 </formula>

To a lithium aluminum hydride (4.5 ml, 4.5 mmol, 15OM in THF) at 5 ° C was first added solid sodium methoxide (245 mg, 4.6 mmol) and then to the drops solution of (3aR, 4S, 7aR) -7a-Methyl-1- [1- (5,5,5-trifluoro-4-hydroxy-4-trifluoromethyl-pent-2-ynyl) -cyclopropyl] -3a, 4,5,6, 7,7a-hexahydro-3H-inden-4-ol (360 mg, 0.91 mmol) in tetrahydrofuran (5 mL). After the addition was complete the mixture was stirred at reflux for 2.5 h. It was then cooled in the ice bath and quenched with water (2.0 mL) and sodium hydroxide (2.0 mL, 2.0M water solution); Diluted with ether (50 mL) stirred for 30 min, MgSO 4 (5 g) was then added and stirring was continued for 30 min. The residue after evaporation of the filtrates (0.42 g) was purified by FC (20 g, 20% EtOAc in hexane) to afford the title compound (315 mg, 0.79 mmol, 87%). [α] 28 D = +2.0 c 0.41, CHCl3. 1H NMR (CDCl3): 6.24 (1H, dt, J = 15.7, 6.7 Hz), 5.60 (1H, d, J = 15.7 Hz), 5.38 (1H, br. s), 4.13 (1H, br. s), 3.27 (1H, s), 2.32 - 1.34 (12H, m), 1.15 (3H, s), 0.80 - 0 , 45 (4H, m); 13 C NMR (CDCl 3): 155.89 (0), 138.10 (1), 126.21 (1), 122.50 (0, q, J = 287 Hz), 119.15 (1), 76, 09 (0, sep J = 31 Hz), 69.57 (1), 55.33 (1), 47.30 (0), 40.31 (2), 36.05 (2), 33.71 (2), 30.10 (2), 20.36 (0), 19.46 (3), 17.94 (2), 11.96 (2), 11.46 (2); MS REI Calculated for C19H24O2F6 M + 398.1680. Observed M + 398.1675.

Synthetic Example 32 - Synthesis of (3aR, 7aR) -7a-Methyl-1- [1- (5,5,5-trifluoro-4-trifluoromethyl-4-trimethylsilanyloxy-pen-2E-enyl) -cyclopropyl] -3a, 4,5,6,7,7a-hexahydro-3H-inden-4-one

<formula> formula see original document page 96 </formula>

To a stirred suspension of (3aR, 4S, 7aR) -7a-Methyl-1- [1- (5,5,5-trifluoro-4-hydroxy-4-trifluoromethyl-pent-2E-enyl) -cyclopropyl] -3a , 4,5,6,7,7a-hexahydro-3H-inden-4 = oi (600 mg, 1.51 mmol) and Celite (2.0 g) in dichloromethane (10 ml) at room temperature was added. pyridinium (1.13 g, 3.0 mmol). The resulting mixture was stirred for 3.5h, filtered through silica gel (10g), and then silica gel pad was washed with 25% EtOAc in hexane. The combined filtrate and washings were evaporated to afford one (3aR, 7aR) -7a-Methyl-1- [1- (5,5,5-trifluoro-4-hydroxy-4-trifluoromethyl-pent-2E-enyl) - crude cyclopropyl] -3a, 4,5,6,7,7a-hexahydro-3H-inden-4-one (550 mg, 1.39 mmol, 92%). To a stirred solution of (3aR, 7aR) -7a-Methyl-1- [1- (5,5,5-trifluoro-4-hydroxy-4-trifluoromethyl-pent-2E-enyl) -cyclopropyl] -3a, 4,5,6,7,7a-hexahydro-3H-inden-4-one (550 mg, 1.39 mmol) in dichloromethane (15 mL) at room temperature was added trimethylsilylimidazole (1.76 mL, 12.0 mmol) ). The resulting mixture was stirred for 1.0h filtered through silica gel (10g) and the silica gel pad was washed with 10% EtOAc in hexane. Combined filtrate and washings were evaporated to afford the title compound (623 mg, 1.33 mmol, 88%). [α] 28D = -1.6 and 0.51, CHCl3. 1H NMR (CDCl3): 6.14 (1H, dt, J = 15.5, 6.7 Hz), 5.55 (1H, d, J = 15.5 Hz), 5.35 (1H, m) , 2.80 (1H, dd, J = 10.7, 6.4 Hz), 2.47 - 1.74 (10H, m), 0.90 (3H, s), 0.76 - 0.40 (4H, m) 0.2 (9H, s); 13 CRMN (CDCl 3): 210.99 (0), 154.28 (0), 137.41 (1), 126.26 (1), 122.59 (0, q, J = 289 Hz), 120.89 (1), 64.31 (1), 53.96 (0), 40.60 (2), 40.13 (2), 35.00 (2), 27.03 (2), 24.21 ( 2), 20.57 (0), 18.53 (3), 12.41 (2), 10.79 (2), 1.65 (3); MS HRES Calculated for C 22 H 30 O 2 F 6 Si M + H 469.1992. Observed M + H 469.1995.

Synthetic Example 33 - Synthesis of 1-alpha, 25-Dihydroxy-16-ene-20-cyclopropyl-23,24-E-eno-26,27-hexafluoro-19-nor-cholecaciferol (27)

<formula> formula see original document page 97 </formula>

To a stirred solution of one (1R, 3R) -1,3-bis - ((tert-butyldimethyl) silanyloxy) -542- (diphenylphosphinoyl) ethylidene] cyclohexane (514 mg, 0.90 mmol) in tetrahydrofuran ( 6 ml) at -78 ° C n-BuLi (0.57 ml, 0.91 mmol) was added. The resulting mixture was stirred for 15 min and (3 aR, 7aR) -7a-Methyl-1- [1- (5,5,5-trifluoro-4-trifluoromethyl-4-trimethylsilanyloxypent-2E-enyl) solution -cyclopropyl] -3a, 4,5,6,7,7a-hexahydro-3H-inden-4-one (200 mg, 0.43 mmol in tetrahydrofuran (2 mL) was added dropwise. The reaction mixture was stirred at -12 ° C for 3.5 h diluted with hexane (35 mL) washed brine (30 mL) and dried over Na 2 SO 4 The residue (750 mg) after evaporation of the solvent was purified by FC (15 g, 5% AcOEt in hexane) to provide a mixture of 1-alpha, 3-beta-Di (tert-Butyl-dimethyl-silanyloxy) -25-trimethylsilanyloxy-16-ene-20-cyclopropyl-23,24-E-ene-26 , 27-hexafluoro-19-nor-cholecaciferol and 1-alpha, 3-beta-Di (tert-Butyl-dimethyl-silanyloxy) -25-hydroxy-16-ene-20-cyclopropyl-23,24-E-ene 26,27-hexafluoro-19-nor-cholecaciferol (250 mg) The mixture of 1-alpha, 3-beta-Di (tert-Butyl-dimethylsilanyloxy) -25-trimethylsilanyloxy-16-ene-20-cyclopropyl 23,24-E-ene-26,27-hexafluoro-19-nor-colecac iferol and 1-alpha, 3-beta-Di (tert-Butyl-dimethyl-silanyloxy) -25-hydroxy-16-ene-20-cyclopropyl-23,24-E-ene-26,27-hexafluoro-19-nor -colecaciferol (250 mg) tetrabutylammonium fluoride (4 mL, 4 mmol, IM solution in THF) was added at room temperature. The mixture was stirred for 24 h. diluted with EtOAc (25 mL) and washed with water (5 x 20 mL), brine (20 mL) and dried over Na 2 SO 4. The residue (270 mg) after evaporation of the solvent was purified by FC (10 g, 50% EtOAc in hexane and EtOAc) to provide the title compound (27) (157 mg, 0.30 mmol, 70%). [α] 30D = +63.3 and 0.45, EtOH. UV λ max (EtOH): 243 nm (ε 30821), 251 nm (ε 36064), 260 nm (ε 24678); 1H NMR (CDCl3): 6.29 (1H, d, J = 11.3 Hz), 6.24 (1H, dt, J = 15.9, 6.4 Hz), 5.92 (1H, d, J = 11.1 Hz), 5.61 (1H, d, J = 15.7 Hz), 5.38 (1H, m), 4.13 (1H, m), 4.05 (1H, m) , 2.88 (1H, s), 2.82 - 1.34 (19H, m), 0.770 (3H, s), 0.80 - 0.36 (4H, m); MS HRES Calculated for C 27 H 34 O 3 F 6 M + H 521.2485. Observed M + H 521.2489.

Synthetic Example 34 - Synthesis of 1-alpha, 25-Dihydroxy-16-ene-20-cyclopropyl-23,24-E-eno-26,27-hexafluoro-cholecacifers (28)

<formula> formula see original document page 98 </formula>

To a stirred solution of a (1S, 5R) -1,5-bis - ((tert-butyldimethyl) silanyloxy) -3- [2- (diphenylphosphinoyl) -et- (Z) -ylidene] -2-methylene-cyclo hexane (525 mg, 0.90 mmol) in tetrahydrofuran (6 mL) at -78 ° C was added n-BuLi (0.57 mL, 0.91 mmol). The resulting mixture was stirred for 15 min and (3aR, 7aR) -7a-Methyl-1- [1- (5,5,5-trifluoro-4-trifluoromethyl-4-trimethylsilanyloxy-pent-2E-enyl) -solution. cyclopropyl] -3a, 4,5,6,7,7a-hexahydro-3H-inden-4-one (200 mg, 0.43 mmol in tetrahydrofuran (2 mL) was added dropwise.) The reaction mixture was stirred at -72 ° C for 2.5 h diluted with hexane (35 mL) washed brine (30 mL) and dried over Na 2 SO 4 The residue (760 mg) after solvent evaporation was purified by FC (15 g, 10% AcOEt in hexane) to provide a mixture of 1-alpha, 3-beta-Di (tert-Butyl-dimethylsilanyloxy) -25-trimethylsilanyloxy-16-ene-20-cyclopropyl-23,24-E-ene-26, 27-Hexafluoro-cholecaciferol and 1-alpha, 3-beta-Di (tert-Butyl-dimethyl-silanyloxy) -25-hydroxy-16-ene-20-cyclopropyl-23,24-E-ene-26,27-hexafluoro -colecaciferol (274 mg) The mixture of 1-alpha, 3-beta-Di (tert-Butyl-dimethylsilanyloxy) -25-trimethylsilanyloxy-16-ene-20-cyclopropyl-23,24-E-ene , 27-hexafluoro-cholecaciferol and 1-alpha, 3-beta-Di (tert -Butyl-dimethylsilanyloxy) -25-hydroxy-16-ene-20-cyclopropyl-23,24-E-ene-26,27-hexafluoro-cholecaciferol (274 mg) tetrabutylammonium fluoride (4 ml, 4 mmol, IM solution in TKF) was added at room temperature. The mixture was stirred for 15 h. diluted with EtOAc (25 mL) and washed with water (5 x 20 mL), brine (20 mL) and dried over Na 2 SO 4. The residue (280 mg) after evaporation of the solvent was purified by FC (15 g, 50% EtOAc in hexane and EtOAc) to provide the title compound (28) (167 mg, 0.31 mmol, 73%). [α] 30D = +18.3 and 0.41, EtOH. UV Aniax (EtOH): 207 nm (ε 17778), 264 nm (ε 15767); 1H NMR (CDCl3): 6.36 (1H, d, J = 11.1 Hz), 6.24 (1H, dt, J = 15.7, 6.7 Hz), 6.07 (1H, d, J = 11.3 Hz), 5.60 (1H, d, J = 15.5 Hz), 5.35 (1H, m), 5.33 (1H, s), 5.00 (1H, s), 4.44 (1H, m), 4.23 (1H, m), 3.14 (1H, s), 2.80 (1H, m), 2.60 (1H, m), 2.40 - 1 , 40 (15H, m), 0.77 (3H, s), 0.80 - 0.36 (4H, m); MS HRES Calculated for C 28 H 34 O 3 F 6 M + H 533.2485. Observed M + H 533.22483.

Synthetic Example 35 - Synthesis of (3aR, 4S, 7aR) -7a-Methyl-1- [1- (5,5,5-trifluoro-4-hydroxy-4-trifluoromethyl-pent-2Z-enyl) -cyclopropyl] - 3a, 4,5,6,7,7a-hexahydro-3H-inden-4-ol

<formula> formula see original document page 100 </formula>

(3aR, 4S, 7aR) -7a-Methyl-1- [1- (5,5,5-trifluoro-4-hydroxy-4-trifluoromethyl-pent-2-ynyl) -cyclopropyl] -3a, 4 , 5,6,7,7a-hexahydro-3H-inden-4-ol (300 mg, 0.76 mmol), ethyl acetate (5 mL), hexane (12 mL), absolute ethanol (0.5 mL) Quinoline (30 ml) and Lindlar catalyst (75 mg, 5% Pd in CaCO3) was hydrogenated at room temperature for 2 h. The reaction mixture was filtered through a pad of celite and the pad was washed with EtOAc. The solvent was evaporated to afford the title compound (257 mg, 0.65 mmol, 87%). [α] D 28 = +1.8 and 0.61, CHCl3 1H NMR (CDCl3): 6.08 (1H, dt, J = 12.3, 6.7 Hz), 5.47 (1H, m), 5.39 (1H, d, J = 12.1 Hz), 4.15 (1H, br. S), 3.28 (1H, s), 2.52 - 1.34 (12H, m), 1 , 16 (3H, s), 0.78 - 0.36 (4H, m); 13 C NMR (CDCl 3): 156.66 (0), 141.77 (1), 126.51 (1), 122.79 (0, q, J = 285 Hz), 115.77 (1), 69, 59 (1), 55.41 (1), 47.28 (0), 36.44 (2), 35.90 (2), 33.75 (2), 30.22 (2), 20.89 (0), 19.41 (3), 17.94 (2), 12.05 (2), 11.11 (2); MS HRES Calculated for C19H24O2F6 M + H 399.1753. Observed M + H 399.1757.

Synthetic Example 36 - Synthesis of (3aR, 7aR) -7a-Methyl-1- [1- (5,5,5-trifluoro-4-trifluoromethyl-4-trimethylsilanyloxy-pen-2Z-enyl) -cyclopropyl] -3a, 4,5,6,7,7a-hexahydro-3H-inden-4-one

<formula> formula see original document page 100 </formula>

To a stirred suspension of (3aR, 4S, 7aR) -7a-Methyl-1- [1- (5,5,5-trifluoro-4-hydroxy-4-trifluoromethyl-pent-2Z-enyl) -cyclopropyl] -3a 4,5,6,7,7a-hexahydro-3H-inden-4-ol (617 mg, 1.55 mmol) and Celite (2.0 g) in dichloromethane (10 ml) at room temperature were added. pyridinium (1.17 g, 3.1 mmol). The resulting mixture was stirred for 2.5 h filtered through silica gel (5 g), and then silica gel pad was washed with 20% EtOAc in hexane. The combined filtrate and washings were evaporated to afford one (3aR, 7aR) -7a-Methyl-1- [1- (5,5,5-trifluoro-4-hydroxy-4-trifluoromethyl-pentenyl) -cyclopropyl] -3a Crude, 4,5,6,7,7a-hexahydro-3H-inden-4-one (600 mg, 1.51 mmol, 98%). To a stirred solution of (3aR, 7aR) -7a-Methyl-1- [1- (5,5,5-trifluoro-4-hydroxy-4-trifluoromethyl-pent-2Z-enyl) -cyclopropyl] -3a, 4 , 5,6,7,7a-hexahydro-3H-inden-4-one (600 mg, 1.51 mmol) in dichloromethane (15 mL) at room temperature was added trimethylsilylimidazole (1.76 mL, 12.0 mmol) . The resulting mixture was stirred for 1.0h filtered through silica gel (10g) and the silica gel pad was washed with 10% EtOAc in hexane. Combined filtrate and washings were evaporated to afford the title compound (640 mg, 1.37 mmol, 88%). [α] 28D = -0.2 and 0.55, CHCl3. 1H NMR (CDCl3): 5.97 (1H, dt, J = 12.2, 6.2 Hz), 5.40 (1H, m), 5.38 (1H, d, J = 12.2 Hz) , 2.82 (1H, dd, J = 10.7, 6.6 Hz), 2.60 - 1.74 (10H, m), 0.89 (3H, s), 0.75 - 0.36 (4H, m), 0.21 (9H, s); 13 C NMR (CDCl 3): 210.56 (0), 154.30 (0), 139.28 (1), 125.81 (1), 122.52 (0, q, J = 289 Hz), 118, 17 (1), 64.11 (1), 53.69 (0), 40.43 (2), 35.51 (2), 34.85 (2), 26.94 (2), 24.07 (2), 20.89 (0), 18.39 (3), 12.26 (2), 10.61 (2), 1.43 (3); MS HRES Calculated for C 22 H 30 O 2 F 6 Si M + H 469.1992. Observed M + H 469.1992.

Synthetic Example 37 - Synthesis of 1-alpha, 25-Dihydroxy-16-ene-20-cyclopropyl-23,24-Z-eno-26,27-hexafluoro-19-nor-cholecaciferol (29)

<formula> formula see original document page 101 </formula> To a stirred solution of a (1R, 3R) -1,3-bis - ((tert-butyldimethyl) silanyloxy) -542- (diphenylphosphinoyl) ethylidene] -cyclo Hexane (514 mg, 0.90 mmol) in tetrahydrofuran (6 mL) at -78 ° C was added n-BuLi (0.57 mL, 0.91 mmol). The resulting mixture was stirred for 15 min and (3aR, 7aR) -7a-Methyl-1- [1- (5,5,54rifluoro-44-trifluoromethyl-4-trimethylsilanyloxy-pent-2Z-enyl) -cyclopropyl] -3a solution 4,5,6,7,7a-hexahydro-3H-inden-4-one (194 mg, 0.41 mmol in tetrahydrofuran (2 mL) was added dropwise.) The reaction mixture was stirred at -72 ° C. C for 3.0 h diluted with hexane (35 mL) washed brine (30 mL) and dried over Na 2 SO 4 The residue (750 mg) after solvent evaporation was purified by FC (15 g, 10% EtOAc in hexane) to provide a mixture of 1-alpha, 3-beta-Di (tert-Butyl-dimethyl-silanyloxy) -25-trimethylsilanyloxy-16-eno-20-cyclopropyl-23,24-Z-ene-26,27-hexafluoro- 19-nor-cholecaciferol and 1-alpha, 3-beta-Di (tert-Butyl-dimethyl-silanyloxy) -25-hydroxy-16-ene-20-cyclopropyl-23,24-Z-ene-26,27-hexafluoro -19-nor-cholecaciferol (230 mg) The mixture of 1-alpha, 3-beta-Di (tert-Butyl-dimethyl-silanyloxy) -25-trimethylsilanyloxy-16-ene-20-cyclopropyl-23,24-Z -ene-26,27-hexafluoro-19-nor-cholecaciferol and 1-alpha, 3 = beta = Di (tert-Butyl-dimethyl-silanyloxy) -25-hydroxy-16-ene-20-cyclopropyl-23,24-Z-ene-26,27-hexafluoro-19-nor-cholecaciferol (230 mg) Tetrabutylammonium fluoride (4 mL, 4 mmol, IM solution in THF) was added at room temperature. The mixture was stirred for 40 h. diluted with EtOAc (25 mL) and washed with water (5 x 20 mL), brine (20 mL) and dried over Na 2 SO 4. The residue (260 mg) after solvent evaporation was purified by FC (10 g, 50% EtOAc in hexane and EtOAc) to provide the title compound (29) (1327 mg, 0.25 mmol, 62%). [α] 28 D = + 53.6 c 0.33, EtOH. UV λ max (EtOH): 243 nm (ε 26982), 251 nm (ε 32081), 260 nm (ε 21689); 1H NMR (CDCl3): 6.29 (1H, d, J = 10.7 Hz), 6.08 (1H, dt, J = 12.5, 6.7 Hz), 5.93 (1H, d, J = 11.1 Hz), 5.46 (1H, m), 5.40 (1H, d, J = 12.7 Hz), 4.12 (1H, m), 4.05 (1H, m) 3.14 (1H, s), 2.80 - 1.40 (19H, m), 0.77 (3H, s), 0.80 - 0.36 (4H, m); MS HRES Calculated for C 2 H 34 O 3 F 6 M + H 521.2485. Observed M + H 521.2487.

Synthetic Example 38 - Synthesis of 1-alpha, 25-Dihydroxy-16-ene-20-cyclopropyl-23,24-Zreno-26,27-hexafluoro-cholecaciferol (30)

<formula> formula see original document page 103 </formula>

To a stirred solution of a (1S, 5R) -1,5-bis - ((tert-butyldimethyl) silanyloxy) -3- [2- (diphenylphosphmoyl) -et- (Z) -ylidene] -2-methylene-cyclo hexane (525 mg, 0.90 mmol) in tetrahydrofuran (6 mL) at -78 ° C was added n-BuLi (0.57 mL, 0.91 mmol). The resulting mixture was stirred for 15 min and (3aR, 7aR) -7a-Methyl-1- [1- (5,5,5-trifluoro-4-trifluoromethyl-4-trimethylsilanyloxy-pent-2Z-enyl) -solution. cyclopropyl] -3a, 4,5,6,7,7a-hexahydro-3H-inden-4-one (200 mg, 0.43 mmol in tetrahydrofuran (2 mL) was added dropwise.) The reaction mixture was stirred at -12 ° C for 2.5 h diluted with hexane (35 mL) washed brine (30 mL) and dried over Na 2 SO 4 The residue (680 mg) after evaporation of the solvent was purified by FC (15 g, 10% AcOEt in hexane) to provide a mixture of 1-alpha, 3-beta-Di (tert-Butyl-dimethylsilanyloxy) -25-trimethylsilanyloxy-16-ene-20-cyclopropyl-23,24-Z-ene-26, 27-hexafluoro-cholecaciferol and 1-alpha, 3-beta-Di (tert-Butyl-dimethyl-silanyloxy) -25-hydroxy-16-ene-20-cyclopropyl-23,24-Z-ene-26,27-hexafluoro -colecaciferol (310 mg) The mixture of 1-alpha, 3-beta-Di (tert-Butyl-dimethylsilanyloxy) -25-trimethylsilanyloxy-16-ene-20-cyclopropyl-23,24-Z-ene , 27-hexafluoro-cholecaciferol and 1-alpha, 3-beta-Di (ter c-Butyl-dimethylsilanyloxy) -25-hydroxy-16-ene-20-cyclopropyl-23,24-Z-eno-26,27-hexafluoro-cholecaciferol (310 mg) tetrabutylammonium fluoride (4 ml, 4 mmol, IM solution) in THF) was added at room temperature. The mixture was stirred for 15 h. diluted with EtOAc (25 mL) and washed with water (5 x 20 mL), brine (20 mL) and dried over Na 2 SO 4. The residue (370 mg) after evaporation of the solvent was purified by FC (10 g, 50% EtOAc in hexane and EtOAc) to provide the title compound (30) (195 mg, 0.37 mmol, 85%). [α] 30D = +9.4 and 0.49, EtOH. UV λη 4 χ (EtOH): 262 nm (ε 11846); 1H NMR (CDCl3): 6.36 (1H, d, J = 11.1 Hz), 6.08 (2H, m), 5.44 (1H, m), 5.40 (1H, d, J = 12.3 Hz), 5.32 (1H, s), 5.00 (1H, s), 4.43 (1H, m), 4.23 (1H, m), 3.08 (1H, s) , 2.80 (1H, m), 2.60 (1H, m), 2.55 - 1.40 (15H, m), 0.77 (3H, s), 0.80 - 0.34 (4H , m); MS HRES Calculated for C 28 H 34 O 3 F 6 M + H 533.2485. Observed M + H 533.2502.

Synthetic Example 39 - Synthesis of 1-alpha, 25-Dihydroxy-16-ene-20-cyclopropyl-19-nor-cholecaciferol (31)

<formula> formula see original document page 104 </formula>

To a stirred solution of one (1R, 3R) -1,3-bis - ((tert-butyldimethyl) silanyloxy) -542- (diphenylphosphinoyl) ethylidene] cyclohexane (697 mg, 1.22 mmol) in tetrahydrofuran ( 9 ml) at -78 ° C n-BuLi (0.77 ml, 1.23 mmol) was added. The resulting mixture was stirred for 15 min and solution of (3aR, 7aR) -7a-Methyl-1- [1- (4-methyl-4-trimethylsilanyloxy-pentyl) -cyclopropyl] -3a, 4,5,6, 7,7a-Hexahydro-3H-inden-4-one (220 mg, 0.61 mmol in tetrahydrofuran (2 mL) was added dropwise.) The reaction mixture was stirred at -12 ° C for 3.5 h diluted. Hexane (35 mL) Washed brine (30 mL) and dried over Na 2 SO 4 The residue (900 mg) after evaporation of the solvent was purified by FC (15 g, 10% EtOAc in hexane) to provide 1-alpha. - beta-Di (tert-Butyldimethylsilanyloxy) -25-trimethylsilanyloxy-16-ene-20-cyclopropyl-19-nor-cholecaciferol (421 mg, 0.59 mmol) To 1-alpha, 3-beta-Di ( t-butyl dimethyl silanyloxy) -25-trimethylsilanyloxy-16-ene-20-cyclopropyl-26,27-hexadeutero-19-nor-cholecaciferol (421 mg, 0.59 mmol) tetrabutylammonium fluoride (4 ml, 4 mmol (IM solution in THF) was added at room temperature The mixture was stirred for 40 h, diluted with EtOAc (25 mL) and washed with water (5 x 20 ml), brine (20 ml) and dried over Na 2 SO 4. The residue (450 mg) after evaporation of the solvent was purified by FC (15 g, 50% EtOAc in hexane and EtOAc) to provide the title compound (31) (225 mg, 0.54 mmol, 89%). [α] 29 D = +69.5 ° C and 0.37, EtOH. UV λ max (EtOH): 243 nm (ε 27946), 251 nm (ε 33039), 261 nm (ε 22701) 1H NMR (CDCl3): 6.30 (1 Η, d, J = 11.3 Hz), 5 , 93 (1H, d, J = 11.3 Hz), 5.36 (1H, m), 4.12 (1H, m), 4.04 (1H, m), 2.75 (2H, m) 2.52 - 1.04 (22H, m), 1.18 (6H, s), 0.79 (3H, s), 0.65 - 0.26 (4H, m); 13 C NMR (CDCl 3): 157.16 (0), 142.33 (0), 131.25 (0), 124.73 (1), 123.76 (1), 115.50 (1), 71, 10 (0), 67.39 (1), 67.19 (1), 59.47 (1), 50.12 (0), 44.60 (2), 43.84 (2), 42.15 (2), 38.12 (2), 37.18 (2), 35.57 (2), 29.26 (3), 29.11 (2), 29.08 (3), 28.48 ( 2), 23.46 (2), 22.26 (2), 21.27 (0), 17.94 (3), 12.70 (2), 10.27 (2); MS HRES Calculated for C27H42O3 M + H 415.3207. Observed M + H 415.3207.

Synthetic Example 40 - Synthesis of 1-alpha, 25-Dihydroxy-16-ene-20-cyclopropyl-cholecaciferol (32)

<formula> formula see original document page 105 </formula>

To a stirred solution of a (1S, 5R) -1,5-bis - ((tert-butyldimethyl) silanyloxy) -3- [2- (diphenylphosphinoyl) -et- (Z) -ylidene] -2-methylene-cyclo hexane (675 mg, 1.16 mmol) in tetrahydrofuran (8 mL) at -78 ° C was added n-BuLi (0.73 mL, 1.17 mmol). The resulting mixture was stirred for 15 min and solution of (3aR, 7aR) -7a-Methyl-1- [1- (4-methyl-4-trimethylsilanyloxy-pentyl) -cyclopropyl] -3a, 4,5,6,7 7α-hexahydro-3H-inden-4-one (210 mg, 0.58 mmol, in tetrahydrofuran (2 mL) was added dropwise.) The reaction mixture was stirred at -72 ° C for 3.5 h diluted with hexane (35 mL) washed brine (30 mL) and dried over Na 2 SO 4 The residue (850 mg) after evaporation of the solvent was purified by FC (15 g, 10% EtOAc in hexane) to provide 1-alpha, 3- beta-Di (tert-Butyl-dimethyl-silanyloxy) -25-trimethylsilanyloxy-16-ene-20-cyclopropyl-cholecaciferol (382 mg, 0.53 mmol) To 1-alpha, 3-beta-Di (tert-Butyldimethyl) -silanyloxy) -25-trimethylsilanyloxy-16-ene-20-cyclopropyl-cholecaciferol (382 mg, 0.53 mmol) tetrabutylammonium fluoride (4 mL, 4 mmol, 1M solution in THF) was added at room temperature. The mixture was stirred for 15 h, diluted with EtOAc (25 mL) and washed with water (5 x 20 mL), brine (20 mL) and dried over Na 2 SO 4. The residue (380 mg) after solvent evaporation was purified by FC (15 g, 50% EtOAc in hexane and EtOAc) to provide the title compound (32) (204 mg, 0.48 mmol, 83%). . [α] 29D = +16.1 and 0.36, EtOH. UV λmaχ (EtOH): 208 nm (ε 17024), 264 nm (ε 16028); 1H NMR (CDCl3): 6.37 (1H, d, J = 11.3 Hz), 6.09 (1H, d, J = 11.1 Hz), 5.33 (2H, m), 5.01 (1H, s), 4.44 (1H, m), 4.23 (1H, m), 2.80 (1H, m), 2.60 (1H, m), 2.38 - 1.08 ( 20H, m), 1.19 (6H, s), 0.79 (3H, s), 0.66 - 0.24 (4H, m); NMR (CDCl3): 157.07 (0), 147.62 (0), 142.49 (0), 133.00 (0), 124.90 (1), 124.73 (1), 117.19 (1), 111.64 (2), 71.10 (1), 70.70 (0) 66.88 (1), 59.53 (1), 50.28 (0), 45.19 (2) ), 43.85 (2), 42.86 (2), 38.13 (2), 35.59 (2), 29.27 (2), 29.14 (3) 28.65 (2), 23.57 (2), 22.62 (2), 21.29 (0), 17.84 (3), 12.74 (2), 10.30 (2); MS HRES Calculated for C 28 H 42 O 3 M + Na 449.3026.

Observed M + Na 449.3023.

Synthetic Example 41 - Synthesis of 1,25-Dihydroxy-21- (2R, 3-dihydroxy-3-methylbutyl) -20R-Cholecaciferol (33). <formula> formula see original document page 107 </formula>

[1R, 3aR, 4S, 7aR] -2 (R) - [4- (1,1-dimethylethyl) dimethylsilanyloxy) -7a-methyl octahydroinden-1-yl] -6-methylheptane-1,6 -thiol (34) and [1R, 3aR, 4S, 7aR] -2 (S) - [4- (1,1-dimethylethyl) dimethylsilanyloxy) -7a-methyl-octahydro-inden-1-yl] -6 - methylheptane-1,6-diol (35)

<formula> formula see original document page 107 </formula>

A solution of alkenol in tetrahydrofuran (9 ml) was cooled in an ice bath and an IM solution of borane-THF in tetrahydrofuran (17 ml) was added dropwise in an originally effervescent reaction. The solution was stirred overnight at room temperature, re-cooled in an ice water bath (17 mL) was added dropwise followed by sodium percarbonate (7.10 g, 68 mmol). The mixture was immersed in a 50 ° C bath and stirred for 70 min to produce a solution. The phase 2 system was allowed to cool then equilibrated with 1: 1 ethyl acetate - hexane (170 ml). The organic layer was washed with water (2 x 25 mL) then brine (20 mL), dried and evaporated to leave a colorless oil (2.76 g). This material was passed through a short scintillating column using 1: 1 ethyl acetate - hexane and silica gel. The effluent, obtained after exhaustive elution, was evaporated, taken up in ethyl acetate, filtered and chromatographed on the column. 2 χ 18 "silica YMC HPLC from 15 to 20 μ using 2: 1 ethyl acetate - hexane as the mobile phase and leading to 100 ml / min. Isomer 34 emerged in a maximum effluent of 2.9 L, colorless oil, 1.3114 g, [α] d + 45.2 ° (methanol, c 0.58; 1H NMR d -0.002 (3H, s), 0.011 (3H, s), 0.89 (9H, s), 0 , 93 (3H, s), 1.17 (1H, m), 1.22 (6H, s), 1.25 - 1.6 (16H, m), 1.68 (1H, m), 1, 80 (2H, m), 1.89 (1H, m), 3.66 (1H, dd, J = 4.8 and 11 Hz), 3.72 (1H, dd, J = 3.3 and 11 Hz), 4.00 (1H, m); LR-ES (-) m / z 412 (M), 411 (MH); HR-ES (+): calculated for (M + Na) 435.3265, found : 435,3269.

Isomer 35 was eluted in a maximum effluent of 4.9 L, colorless oil, 0.8562 g which crystallized on prolonged standing: mp 102-3 °, [α] D + 25.2 ° (methanol, c 0.49); 1H NMR d - 0.005 (3H, s), 0.009 (3H, s), 0.89 (9H, s), 0.93 (3H, s), 1.16 (1H, m), 1.22 (6H , s), 1.3-1.5, (14H, m), 1.57 (2H, m), 1.67 (1H, m), 1.80 (2H, m), 1.91 (1H m) 3.54 (1H, dd, J = 4.8 and 11 Hz), 3.72 (1H, dd, J = 2.9 and 11 Hz), 4.00 (1H, m); ); LR-ES (-) mlz 412 (M), 411 (M-H). Anal. Calcd for C24 H48 O3 Si: C, 69.84, H, 11.72; Found: C, 69.91; H, 11.76.

[1R, 3aR, 4S, 7aR] -6 (R) - [4- (tert-Butyl-dimethyl-silanyloxy) -7a-methyl-octahydro-inden-1-yl] -7-iodo-2-methyl-heptan -2-ol (36)

<formula> formula see original document page 108 </formula>

A stirred mixture of triphenylphosphine (0.333 g, 1.27 mmol) and imidazole (0.255 g, 3 mmol) in dichloromethane (3 mL) was cooled in an ice bath and iodine (0.305 g, 1.20 mmol) was added. This mixture was stirred for 10 min then a solution of 34 (0.4537 g, 1.10 mmol) in dichloromethane (3 mL) was added dropwise over a period of 10 min. The mixture was stirred in the ice bath for 30 min then at room temperature for 2 h. TLC (1: 1 ethyl acetate - hexane) confirmed absence of extract. A solution of sodium thiosulfate (0.1 g) in water (5 ml) was added, the mixture equilibrated and the organic phase washed with 0.1 N sulfuric acid (10 ml) containing a few drops of brine then water-brine. 1: 1 (2 x 10 ml) once with brine (10 ml) then dried and evaporated. The residue was purified by flash chromatography using 1: 9 ethyl acetate - hexane as the mobile phase to afford 36 as a colorless syrup, 0.5637 g, 98%: 1H NMR δ -0.005 (3H, s), 0.010 ( 3H, s), 0.89 (9H, s), 0.92 (3H, s), 1.23 (6H, s), 1.1-1.6 (16H, m), 1.68 (1H , m), 1.79 (2H, m), 1.84 (1H, m), 3.37 (1H, dd, J = 4 and 10 Hz), 3.47 (1H, dd, J = 3 and 10 Hz), 4.00 (1H, m); LR-EI (+) mlz 522 (M), 465 (M-C 4 H 9), 477 (M-C 4 H 9 -H 2 O); HR-EI (+): calculated for C24H47IO2Si: 522.2390, found: 522.2394.

[1R, 3aR, 4S, 7aR] -6 (S) - [4- (tert-Butyl-dimethyl-silanyloxy) -7a-methyl-octahydro-inden-1-yl] -2-methyl-non-8-in -2-ol (37)

<formula> formula see original document page 109 </formula>

Lithium acetylide DMA complex (0.110 g, 1.19 mmol) was added to a solution of 36 (0.2018 g (0.386 mmol) in dimethyl sulfoxide (1.5 mL) and tetrahydrofuran (0.15 mL) The mixture was stirred overnight TLC (1: 4 ethyl acetate - hexane) showed a two point mixture passing very close together (Rf 0.52 and 0.46). containing pure alkenol, which is the elimination product of 36, and was produced as the main product. Fractions at the end of the elution range, however, were also homogeneous and provided the desired acetylene 37 on evaporation. his 6-epimer that served as identification were previously reported.

[1R, 3aR, 4S, 7aR] -7-Benzenesulfonyl-6 (S) - [4- (tert-butyl-dimethylsilanyloxy) -7a-methyloctahydro-inden-1-yl] -2-methyl-heptan-2 -ol (38).

<formula> formula see original document page 109 </formula>

A mixture of 37b (0.94 g, 1.8 mmol), sodium benzenesulfinate (2.18 g, 13 mmol) and N, N-dimethylformamide (31.8 g) was stirred at room temperature for 12h, then in a bath at 40 ° C for about 6 h until all extracts are converted as shown by TLC (1: 4 ethyl acetate - hexane). The solution was equilibrated with 1: 1 ethyl acetate - hexane (120 mL) and 1: 1 brine - water (45 mL). The organic layer was washed with water (4 x 25 mL) brine (10 mL), then dried and evaporated to leave a colorless oil, 1.0317 g. This material was flash chromatographed using a stepped gradient (1: 9, 1: 6, 1: 3 ethyl acetate - hexane) to provide a colorless oil, 0.930 g, 96%: 300 MHz 1H NMR δ -0 .02 (3H, s), 0.00 (3H, s), 0.87 (9H, s), 0.88 (3H, s), 1.12 (1H, m), 1.20 (6H, s), 1.2 - 1.8 (18H, m), 1.81 (1H, m), 3.09 (2H, m), 3.97 (1H, brs), 7.59 (3H, m) ), 7.91 (2H, m). [1R, 3aR, 4S, 7aR] -1- (1 (S) -Benzenesulfonylmethyl-5-methyl-5-trimethylsilanyloxyhexyl) -4- (tert-butyl-dimethylsilanyloxy) -7a-methyl-octahydro-indene (39).

<formula> formula see original document page 110 </formula>

1- (Trimethylsilyl) imidazole (1 ml) was added to a solution of 38 (0.8 g) in cyclohexane (10 ml) and stirred overnight then flash chromatographed using a staggered hexane gradient, ethyl acetate - hexane 1:39 and 1:19. Elution was monitored by TLC (1: 4 ethyl acetate - hexane) leading to 39 as a colorless syrup, 0.7915 g: 300 MHz 1H NMR δ 0.00 (3H, s), 0.02 (3H, s ), 0.12 (9H, s), 0.90 (12H, s, t-butyl + 7a-Me), 1.16 (1H, m), 1.20 (6H, s), 1.2 - 1.6 (15H, m), 1.66 - 1.86 (3H, m), 3.10 (2H, m), 4.00 (1H, brs), 7.56 - 7.70 (3H, m m), 7.93 (2H, m).

[1R, 3aR, 4S, 7aR] -6 (R) - [4- (tert-Butyl-dimethyl-silanyloxy) -7a-methyl-octahydro-inden-1-yl] -2,10-dimethyl-undecane-2 0.3 (R), 10-triol (40). <formula> formula see original document page 111 </formula>

A solution of 39 (0.7513 g, 1.23 mmol) and diol (0.508 g, 1.85 mmol) in tetrahydrofiirane (28 ml) was cooled to -35 ° C then 2.5 M butyl lithium in hexane (2, 75 ml) was added dropwise. The temperature was allowed to rise to -20 ° C and held at this temperature for 6 h or until the extract was consumed. The progress of the reaction was monitored by TLC (1: 4 ethyl acetate - hexane) showing the extract (Rf 0.71) and the two epimeric diols (Rf 0.09 and 0.12). Towards the end of the reaction period the temperature was briefly raised to 0 ° C, decreased again to -10 ° C, then saturated ammonium chloride (25 mL) was added followed by ethyl acetate (50 mL) and sufficient water to dissolve the salts. precipitated. The resulting aqueous phase was extracted with ethyl acetate (15 mL). The combined extracts were washed with brine (15 mL), dried and evaporated. The resulting syrup was flash chromatographed using a 1: 9, 1: 6, 1: 4 and 1: 1 stepped gradient of ethyl acetate - hexane to afford 39a as a colorless syrup, 0.8586 g. This material was dissolved in a mixture of tetrahydrofuran (30 mL) and methanol (18 mL), then 5% sodium amalgam (20 g) was added. Reductive desulfonylation was complete after stirring the mixture for 14 h. The progress of the reaction was monitored by TLC (1: 1 ethyl acetate - hexane) which showed the disappearance of the epimeric diols (Rf 0.63 and 0.74) and the generation of 40a (Rf 0.79) and the analog partially desilylated 40 (Rf 0.16). The mixture was diluted with methanol (20 mL), stirred for 3 min, then ice (20 g) was added, stirred for 2 min and the supernatant decanted into a mixture containing saturated ammonium chloride (50 mL). The residue was repeatedly washed with small amounts of tetrahydrofuran which was also added to the salt solution, which was then equilibrated with ethyl acetate (80 ml). The aqueous layer was re-extracted once with ethyl acetate (20 mL), the combined extracts were washed with brine (10 mL) then dried and evaporated. The resulting colorless oil containing both 40a and 40 was dissolved in 10 ml of a 1 N oxalic acid in methanol solution (prepared from the dihydrate) performing selective hydrolysis of trimethylsilyl ether within minutes. Calcium carbonate (1 g) was added and the suspension stirred overnight, then filtered. The solution was evaporated and the resulting residue was flash chromatographed using a 1: 4, 1: 2, 1: 2, 1: 2 and 1: 2 step gradient of ethyl acetate - hexane providing a triol 40 residue that crystallized on needles. very thin derivation of acetonitrile, 0.45 g: mp 94 at 95 ° C, [α] D + 44.1 ° (methanol, c 0.37); 400 MHz 1H NMR δ -0.005 (3H, s), 0.007 (3H, s), 0.89 (9H, s), 0.92 (3H, s), 1.15 (1H, m), 1.16 (3H, s), 1.21 (9H, s), 1.2 - 1.6 (19H, m), 1.67 (1H, m), 1.79 (2H, m), 1.90 ( 2H, m), 2.06 (1H, m), 3.31 (1H, brd, J = 10 Hz), 4.00 (1H, brs), LR-ES (-) m / z: 533 (M + Cl) 497 (MH); HR-ES (+): Calculated for C 29 H 58 O 4 Si + Na: 521.3996, found: 521.4003. Anal. Calcd for C 29 H 58 O 4 Si: C, 69.82, H, 11.72; Found: C, 69.97; H, 11.65. [1R, 3aR, 4S, 7aR] -6 (R) - (4-Hydroxy-7a-methyl-octahydro-inden-1-yl) -2,10-dimethyl-undecane-2,3 (R), 10- triol (41).

<formula> formula see original document page 112 </formula>

A stirred solution of triol 40 (0.4626 g, 0.927 mmol) in acetonitrile (10 mL) and dioxane (0.7 mL) was cooled to 10 ° C and a solution of fluorosilicic acid (2 mL) was added dropwise. The cooling bath was removed, the phase 2 system further diluted with acetonitrile (2 ml) then stirred at room temperature for 374 h. The disappearance of the extract was monitored by TLC (ethyl acetate). The mixture was equilibrated with water (10 mL) and ethyl acetate (30 mL). The aqueous phase was re-extracted with ethyl acetate (2 x 20 mL), the combined extracts were washed with water (5 mL) and brine (10 mL), then 1: 1 saturated brine-hydrogen hydrogen carbonate solution and dry. The residue was purified by flash chromatography using a stepped gradient of 1: 1 to 2: 1 ethyl acetate - hexane and pure ethyl acetate to give a residue which was taken up in dichloromethane - hexane 1: 1, filtered and evaporated to provide amorphous solids, 0.3039 g (85%): [α] D + 42.6 ° (methanol, c 0.48); 1H NMR (DMSO-d6): δ 0.87 (3H, s), 0.97 (3H, s), 1.02 (3H, s), 1.04 (6H, s), 1.1 - 1 , 4 (18H, m), 1.5 - 1.8 (4H, m), 1.84 (1H, m), 2.99 (1H, dd, J = 6 and 10 Hz), 3.87 ( 1H, brs), 4.02 (1H, s, OH), 4.05 (1H, s, OH), 4.16 (1H, d, OH, J = 3.6 Hz), 4.20 (1H d, OH, J = 6.4 Hz); LR-ES (+): m / z 384 (M), 383 (M-H); HR-ES (+): Calculated for (M + Na) 407.3132, found: 407.3134. [1R, 3aR, 4S, 7aR] -1- {5-Hydroxy-5-methyl-1 (R) - [2- (2,2,5,5-tetramethyl- [1,3] dioxolan-4 ( R) -yl) -ethyl] -hexyl} -7a-methyl-octahydro-inden-4-yl (42)

<formula> formula see original document page 113 </formula>

A solution of tetraol 40 (0.2966 g, 0.771 mmol) and pyridinium tosylate (100 mg) in acetone (8 mL) and 2,2-dimethoxypropane (8 mL) was kept at room temperature for 12 h. TLC analysis (ethyl acetate) showed the absence of extract (Rf 0.21) and two new points with Rf 0.82 and 0.71, the first expected 42 and the last considered to be methylacetal.

The reaction mixture was diluted with water (5 mL) and stirred for 10 min. At this time only the point with higher Rf value was observed. The mixture was neutralized with sodium hydrogen carbonate (0.5 g) then equilibrated with ethyl acetate (50 ml) and brine (5 ml). The organic layer was washed with water (5 ml) and brine (5 ml) then dried and evaporated to leave a sticky residue (0.324 g) which was directly used in the next step: 300 MHz 1 H NMR: δ 0.94 (3H, s), 1.10 (3H, s), 1.20 (1H, m), 1.22 (6H, s), 1.25 (3H, s), 1.34 (3H, s), 1, 41 (3H, s), 1.2 - 1.65 (20H, m), 1.78 - 1.86 (3H, m), 1.93 (1H, m), 3.62 (1H, dd, J = 4.6 and 8.3 Hz), 4.08 (1H, brs).

1- {5-Hydroxy-5-methyl-1 (R) - [2- (2,2,5,5-tetramethyl [1,3] dioxolan-4 (R) -yl) -ethyl] -hexyl} ester [1R, 3aR, 4S, 7aR] acetic acid-7α-methyl-octahydro-inden-4-yl (43).

<formula> formula see original document page 114 </formula>

The residue obtained above was dissolved in pyridine (6.9 g) and further diluted with acetic anhydride (3.41 g). The mixture was allowed to stand at room temperature for 24h, then in a 35 ° C bath for about 10h until the extract was no longer detectable (TLC, ethyl acetate). The mixture was diluted with toluene and evaporated. The residue was purified by flash chromatography (1: 4 ethyl acetate - hexane) to afford 43 as colorless syrup, 0.3452 g, 97%: 1H NMR: δ 0.89 (3H, s), 1.10 (3H, s), 1.20 (1H, m), 1.22 (6H, s), 1.25 (3H, s), 1.33 (3H, s), 1.41 (3H, s) 1.25 - 1.6 (19H, m), 1.72 (1H, m), 1.82 (2H, m), 1.95 (1H, m), 2.05 (3H, s), 3.63 (1H, dd, J = 4.4 and 8.4 Hz); 5.15 (1H, brs); LR-F AB (+) mlz 467 (M + H), 465 (M-H), 451 (M-Me).

1- [4 (R), 5-Dihydroxy-1 (R) - (4-hydroxy-4-methyl-pentyl) -5-methylhexyl] -7α-methyl-octahydro-inden-4-yl ester of [1R, 3aR, 4S, 7aR] -acetic acid (44).

<formula> formula see original document page 114 </formula>

A solution of 43 (0.334 g, 0.716 mmol) in 80% acetic acid (2 mL) was kept in a 68 ° C bath. TLC (ethyl acetate, Rf 0.33) monitored the progress of hydrolysis. The extract was no longer detectable after 2.5 h. The mixture was evaporated then coevaporated with a small amount of toluene to leave a colorless film (0.303 g) which was directly used in the next step: 300 MHz 1 H NMR: δ 0.89 (3H, s), 1.17 ( 3H, s), 1.22 (6H, s), 1.56 (3H, s), 1.1-1.6 (21H, m), 1.6-2.0 (5H, m), 2 .04 (3H, s), 3.32 (1H, brd, J = 10 Hz), 5.15 (1H, brs).

1- [4 (R) - [Dimethyl- (1,1,2-trimethyl-propyl) -silanyloxy] -5-hydroxy-1 (R) - (4-hydroxy-4-methyl-pentyl) -5-ester [1R, 3aR, 4S, 7aR] -acetic acid methylhexyl] -7a-methyl-octahydro-inden-4-yl (45)

<formula> formula see original document page 115 </formula>

A solution of triol 44 (0.30 g), imidazole (0.68 g, 10 mmol) and dimethyltexylsilyl chloride (1.34 g, 7.5 mmol) in N, N-dimethylformamide (6 g) was kept at room temperature. room temperature. After 48 h 4- (N, N-dimethylamino) pyridine (15 mg) was added and the mixture stirred for an additional 24 h. The progress of the reaction was monitored by TLC (ethyl acetate; 24, Rf 0.83; 25a, Rf 0.38). The mixture was diluted with water (2 mL), stirred for 10 min then partitioned between ethyl acetate (45 mL) and water (20 mL). The aqueous layer was extracted once with ethyl acetate (10 mL). The combined organic phases were washed with water (4 x 12 ml) and brine (8 ml) then dried and evaporated. The residual oil was purified by flash flash chromatography using a step gradient of 1: 9 and 1: 4 ethyl acetate - hexane to give 45 as a colorless syrup. A small amount of unreacted extract (80 mg) was eluted with ethyl acetate. Syrup 45 was directly used in the following step: 400 MHz 1H NMR: δ 0.13 (3H, s), 0.14 (3H, s), 0.87 (6H, s), 0.91 (9H, m ), 1.10 (1Η, m), 1.14 (3Η, s), 1.15 (3Η, s), 1.21 (6Η, s), 1.1 - 1.6 (19Η, m) , 1.6 - 1.9 (5Η, m), 1.94 (1Η, brd, J = 12.8 Hz), 2.05 (3Η, s), 3.38 (1Η, brs), 5, 15 (1Η, brs).

1- [4 (R) - [Dimethyl- (2,1,2-trimethyl-propyl) -silanyloxy] -5-methyl-1 (R) - (4-methyl-4-trimethylsilanyloxy-pentyl) -5-ester [1R, 3aR, 4S, 7aR] -acetic acid trimoctahydro-inden-4-yl (46).

<formula> formula see original document page 116 </formula>

1- (Trimethylsilyl) imidazole (0.90 mL, 6.1 mmol) was added to a solution of 45 (0.2929 mg) in cyclohexane (6 mL) and stirred for 12 h, then subjected to spray chromatography. (ethyl acetate - hexane 1:79) to yield 46 as colorless syrup (0.3372 g). Elution was monitored by TLC (1: 4 ethyl acetate - hexane) leading to 46 as a colorless syrup, 0.7915 g: 1 H NMR δ: 0.074 (3H, s), 0.096 (3H, s), 0.103 (9H , s), 0.106 (9H, s), 0.82 (1H, m), 0.83 (6H, s), 0.88 (9H, m), 1.32 (3H, s), 1.20 (9H, s), 1.15 - 1.6 (17H, m), 1.6-1.9 (5H, m), 1.97 (1H, brd, J = 12.8 Hz), 2, Δ (3H, s), 3.27 (1H, m), 5.15 (1H, brs); LR-FAB (+) mlz: 712 (M), 711 (M-H), 697 (M-Me), 653 (M-AcO) δ 627 (M-C6H13). [1R, 3aR, 4S, 7al2] -1- [4 (R) - [Dimethyl- (1,1,2-trimethyl-propyl) -silanyloxy] -5-methyl-1 (R) - (4-methyl-methoxy) 4-trimethylsilanyloxy-pentyl) -5-trimethylsilanyloxy-7α-methyl-octahydroinden-4-ol (47)

<formula> formula see original document page 116 </formula>

A stirred solution of 46 (0.335 mg, 0.47 mmol) in tetrahydrofuran (15 mL) was cooled in an ice bath and an IM solution of lithium aluminum hydride in tetrahydrofuran (2 mL) was added dropwise. TLC (1: 9 ethyl acetate - hexane) showed complete conversion 25b (Rf 0.61) to 26 (Rf 0.29) after 1.5 h. A solution of 2M sodium hydroxide (14 drops) was added, followed by water (0.5 mL) and ethyl acetate (30 mL). A small amount of Celite was added and, after stirring for 15 min, the liquid layer was separated by filtration. The solid residue was repeatedly rinsed with ethyl acetate and the combined liquid phases evaporated to leave a colorless syrup, which was taken up in hexane, filtered and evaporated to yield 26 (0.335 g) which was used without further purification: 1 H NMR δ: 0.075 (3H, s), 0.10 (21H, brs), 0.82 (1H, m), 0.84 (6H, s), 0.89 (6H, m), 0.93 (3H, s) 1.13 (3H, s), 1.20 (9H, s), 1.2-1.6 (16H, m), 1.6-1.7 (2H, m), 1.82 (3H m) 1.95 (1H, brd, J = 12.4 Hz), 3.27 (1H, m), 4.08 (1H, brs); LR-FAB (+) mlz: 585 (M-C 6 Hn), 481 (M-TMSO); HR-ES (+) m / z: Calculated for C37H78O4Si3 + Na: 693.5100 found: 693.55100.

[1R, 3aR, 7aR] -1- [4 (R) - [Dimethyl- (1,1,2-trimethyl-propyl) -silanyloxy] -5-methyl-1 (R) - (4-methyl-4- trimethylsilanyloxy-pentyl) -5-trimethylsilanyloxy-hexyl] -7α-methyl-octahydroinden-4-one (48)

<formula> formula see original document page 117 </formula>

Celite (0.6 g) was added to a stirred solution of 47 (0.310 g, 0.462 mmol) in dichloromethane (14 mL) followed by pyridinium dichromate (0.700 g, 1.86 mmol). The conversion of 47 (Rf 0.54) to ketone 27 (Rf 0.76) was followed by TLC (1: 4 ethyl acetate - hexane). The mixture was diluted with cyclohexane after 4.5 h then filtered through a silica gel layer. Filtrate and ether washes were combined and evaporated. The residue was flash chromatographed (1:39 ethyl acetate - hexane) to afford 27 as a colorless syrup, 0.2988 g, 96.6%: 1 H NMR δ: 0.078 (3H, s), 0.097 ( 3H, s), 0.107 (18H, s), 0.64 (3H, s), 0.81 (1H, m), 0.84 (6H, s), 0.89 (6H, m), 1.134 ( 3H, s), 1.201 (3H, s), 1.207 (3H, s), 1.211 (3H, s), 1.3-1.6 (14H, m), 1.6-1.7 (3H, m) ), 1.88 (1H, m), 2.04 (2H, m), 2.2 - 2.32 (2H, m), 2.46 (1H, dd, J = 7.5 and 11.5 Hz), 3.28 (1H, m); LR-FAB (+) mlz: 583 (M-C 6 H 13), 479 (M-OTMS); HR-ES (+) m / z: Calculated for C 37 H 76 O 4 Si 3 + Na: 691.4943, found: 691.4949.

[1R, 3aR, 7aR, 4E] -4- {2 (Z) - [3 (S), 5 (R) -Bis {tert-Butyl-dimethylsilanyloxy) -2-methylene-cyclohexylidene] -ethylidene} -7α-methyl-1- [5-methyl-1 (R) - (4-methyl-4-trimethylsilanyloxy-pentyl) -4 (R) - [dimethyl- (1,1,2-trimethyl-propyl) -silanyloxy ] -5-trimethylsilanyloxyhexyl] octahydroindene (49)

<formula> formula see original document page 118 </formula>

A solution of 2.5 M butyl lithium in hexane (0.17 mL) was added to a solution of 28 in tetrahydrofuran (2 mL) at -70 ° C to produce a deep cherry red color of the product. After 10 min a solution of ketone 27 (0.1415 g, 0.211 mmol) in tetrahydrofuran (2 mL) was added dropwise over a period of 15 min. The reaction was quenched after 4 h by the addition of pH 7 phosphate buffer (2 ml). The temperature was allowed to rise to 0 ° C then hexane (30 mL) was added. The aqueous layer was reextracted with hexane (15 mL). The combined extracts were washed with brine (5 ml), dried and evaporated to afford a colorless oil which was purified by flash chromatography (1: 100 ethyl acetate - hexane) to yield 49 as colorless syrup, 0.155 g, 71%. 1 H NMR δ: 0.068 (15H, m), 0.103 (12H, s), 0.107 (9H, s), 0.53 (3H, s), 0.82 (1H, m), 0.84 (6H, s), 0.88 (18H, m), 0.89 (6H, m), 1.14 (3H, m), 1.20 (9H, s), 12-1.9 (22H, m), 1.97 (2H, m), 2.22 (1H, dd, J = 7.5 at 13 Hz), 2.45 (1H, brd, J = 13 Hz), 2.83 (1H, brd, J = 13 Hz), 3.28 (1H, m), 4.20 (1H, m), 4.38 (1H, m), 4.87 (1H, d, J = 2 Hz), 5.18 ( 1H, d, J = 2 Hz); 6.02 (1H, d, J = 11.4 Hz); 6.24 (1H, d, J = 11.4 Hz); LR-FAB (+) mlz 1033 (M + H), 1032 (M), 1031 (M-H), 901 (M-TBDMS).

Synthesis of 1,25-Dihydroxy-21- (2R, 3-dihydroxy-3-methylbutyl) -20R-Cholecaciferol (33)

<formula> formula see original document page 119 </formula>

The 49 residue (0.153 g, 0.148 mmol) as obtained in the previous experiment was dissolved in an IM tetrabutylammonium fluoride solution (3.5 mL). TLC (ethyl acetate) monitored the progress of the reaction. Thus, the solution was diluted with brine (5 mL) after 24 h, stirred for 5 min then equilibrated with ethyl acetate (35 mL) and water (15 mL). The aqueous layer was reextracted once with ethyl acetate (15 mL). The combined organic layers were washed with water (5 x 10 mL), once with brine (5 mL) then dried and evaporated. The residue was purified by flash chromatography using a step gradient of 1: 100 ethyl acetate and methanol - ethyl acetate providing 33 as colorless, methyl pentane formate microcrystalline material, 70 mg, 91%: [α] D + 34.3 ° (methanol, c 0.51); 1H NMR (DMSCM6) δ: 0.051 (3Ηs), 0.98 (3Hs), 1.03 (3Hs), 1.05 (6Hs), 1.0 - 1.6 (17Hs) , m), 1.64 (3Η, m), 1.80 (2Η, m), 1.90 (1Η, d, J = 11.7 Hz), 1.97 (1H, dd, J = 9, 8 Hz), 2.16 (1H, dd, J = 5.9 and J = 13.7 Hz), 2.36 (1H, brd), 2.79 (1H, brd), 3.00 (1H, dd, J = 5 and 10 Hz), 3.99 (1H, brs), 4.01 (1H, s, OH), 4.04 (1H, s, OH), 4.54 (1H, OH, d , J = 3.9 Hz), 4.76 (1H, brs), 4.87 (1H, OH, d, J = 4.9 Hz), 5.22 (1H, brs), 5.99 (1H , d, J = 10.7 Hz); 6.19 (1H, d, J = 10.7 Hz); LR-ES (+) mlz: 519 (M + H), 518 (M), 517 (M-H), 501 (M-OH); HR-ES (+) calcd for C 32 H 54 O 5 + Na: 541.3863; found 541.3870; Λ max (ε): 213 (13554), 241sh (12801), 265 (16029) nm.

Synthetic Example 42 - Synthesis of 1,25-Dihydroxy-21 (2R, 3-dihydroxy-3-methylbutyl) -20S-CoIecaciferol (50).

<formula> formula see original document page 120 </formula>

[1R, 3aR, 4S, 7aR] -7-Benzenesulfonyl-6 (R) - [4- (tert-butyl-dimethyl-silyanoxy) -7a-methylthihydro-inden-1-yl] -2-methyl-heptan-2 -ol (51).

<formula> formula see original document page 120 </formula>

A solution of 36 and sodium benzenesulfinate (0.263 g, 1.6 mmol) in β, β-dimethyl formamide (5 ml) was stirred in a bath at 77 ° C for 3h. The solution was equilibrated with 1: 1 ethyl acetate - hexane (25 mL) and the organic layer washed with water (5x10 mL), dried and evaporated. The residue was flash chromatographed with a 1: 9, 1: 4, and 1: 3 stepped gradient of ethyl acetate - hexane to provide the sulfone as a colorless syrup: 1H NMR δ -0.02 (3H, s), 0.005 (3H, s), 0.79 (3H, s), 0.87 (9H, s), 1.12 (1H, m), 1.19 (6H, s), 1.12 ( 1H, m), 1.20 (6H, s), 1.2 - 1.8 (18H, m), 2.08 (1H, m), 3.09 (1H, dd, J = 9.3 and 14.5 Hz), 3.31 (1Η, dd, J = 3 and 14.5 Hz), 3.97 (1Η, brs), 7.58 (3Η, m), 7.66 (1Η, m) 7.91 2Η, m); LR-ES (+) mlz: 600 (M + Na + MeCN), 559 (M + Na); LR-ES (-) mlz: 536 (M), 535 (M-H); HR-ES (+): Calculated for C 30 H 52 O 4 SSi + Na 559.3248; found 559.3253.

[1R, 3aR, 4S, 7aR] -1- (1 (R) -Benzenesulfonylmethyl-5-methyl-5-trimethylsilanyloxyhexyl) -4- (tert-butyl-4-indene (52)).

<formula> formula see original document page 121 </formula>

1- (Trimethylsilyl) imidazole (0.146 mL) was added to a solution of 51 (0.145 g, 0.27 mmol) in cyclohexane (2 mL). After 17 h the product was purified by flash chromatography using a stepped gradient of 1:79 and 1:39 ethyl acetate - hexane to give 52 as a colorless residue, 0.157 g 0.258 mmol, TLC (ethyl acetate - hexane 1: 9) Rf 0.14. 300 MHz 1H NMR: δ -0.02 (3H, s), 0.00 (3H, s), 0.87 (12H, s), 1.12 (1H, m), 1.17 (6H, s) ), 1.2 - 1.6 (15H, m), 1.6 - 1.9 (3H, m), 3.08 (2H, m), 3.97 (1H, brs), 7.53 - 7.70 (3H, m); 7.90 (2H, d, J = 7Hz). [1R, 3aR, 4S, 7aR] -5 (R, S) -Benzenesulfonyl-6 (R) - [4- (tert-butyl-dimethylsilanyloxy) -7a-methyl-octahydro-inden-1-yl] - 240-dimethyl-10-trimethylsilanyloxy-undecane-2,3 (R) -diol (53)

<formula> formula see original document page 121 </formula>

A solution of 52 (0.2589, 0.425 mmol) and diol (0.176 g, 0.638 mmol) in tetrahydrofuran (9 mL) was cooled to -25 ° C and 1.6 M butyl lithium in hexane (1.4 mL) was added. . The temperature was raised to -20 ° C and held for 3 h then at -10 ° C for 2.5 h and 0 ° C for 10 min. The mixture was recooled to -10 ° C, saturated ammonium chloride solution (5 mL) was added, then equilibrated with ethyl acetate (50 mL) and sufficient water to dissolve precipitated salts. The aqueous layer was extracted with ethyl acetate (15 ml), the combined extracts were dried and evaporated and the residue purified by flash chromatography using a staggered 1: 6, 1: 4 ethyl acetate - hexane gradient. and 1: 1 to yield 53 as a colorless syrup, 0.212 g, 70%: 300 MHz 1 H NMR: δ 0.00 (3H, s), 0.017 (3H, s), 0.12 (9H, s), 0 , 81 (3H, s), 0.89 (9H, s), 1.16 (1H, m), 1.19 (12H, m), 1.1 - 1.6 (20H, m), 1, 6 - 1.8 (2H, m), 3.10 (1H, dd, J = 8.4 and 14.7 Hz), 3.30 (1H, m), 3.99 (1H, brs), 7 , 61 (2H, m), 7.67 (1H, m), 7.93 (2H, m).

[1R, 3aR, 4S, 7aR] -6 (S) - [4- (tert-Butyl-dimethylsilanyloxy) -7a-methyl-octahydro-inden-1-yl] -2,10-dimethyl-10-trimethylsilanyloxy -undecane-2,3 (R) -diol (54).

<formula> formula see original document page 122 </formula>

Compound 53 (0.186 mg, 0.262 mmol) was dissolved in 0.5M oxalic acid dihydrate in methanol (2.5 mL). The solution was stirred for 15 min then calcium carbonate (0.5 g) was added and the suspension stirred overnight then filtered. The filtrate was evaporated to afford 54 as a white foam, 0.188 g, 98%: TLC (1: 1 ethyl acetate-hexane) Rf 0.06. This material was used in the next step without further purification.

[1R, 3aR, 4S, 7aR] -6 (S) - [4- (tert-Butyl-dimethylsilanyloxy) -7a-methyl-octahydro-inden-1-yl] -2,10-dimethyl-undecane-2 0.3 (R), 10-triol (triol 55).

<formula> formula see original document page 122 </formula> Sodium amalgam (5% sodium, 10.8 g) was added to a vigorously stirred solution of 54 (0.426 g, 0.667 mmol) in a mixture of tetrahydrofuran (15 ml) and methanol (9 ml). The suspension was stirred for 24 h and the reaction monitored by TLC (1: 1 ethyl acetate - hexane) to observe 55 (Rf 0.17) yield. The mixture was diluted with methanol (3 mL), stirred for 5 min then further diluted with water (10 mL), stirred for 2 min and decanted into saturated ammonium chloride solution (25 mL). The aqueous layer was extracted with ethyl acetate (2 x 20 mL). The combined extracts were washed with pH 7 phosphate buffer (5 mL) then brine (10 mL), dried and evaporated. The residue was purified by flash chromatography using a staggered 1: 1 and 2: 1 ethyl acetate - hexane gradient to give 55 as a colorless syrup, 0.244 g, 73%: 1H NMR: δ -0.006 (3H, s ), 0.006 (3H, s), 0.86 (9H, s), 0.92 (3H, s), 1.11 (1H, m), 1.15 (3H, s), 1.21 (9H , s), 1.2-1.75 (21H, m), 1.7-1.85 (3H, m), 1.90 (1H, m), 3.29 (1H, brd), 3, 99 (1H, brs); LR-ES (+) mlz: 521 (M + Na), 481 (M-OH); LR-ES (-): m / z 544: (M + CH 2 O 2), 543 (M-H + CH 2 O 2), 533 (M-Cl); HR-ES (+) m / z: Calculated for C 29 H 58 O 4 Si + Na: 521.3996, found 521.3999. [1R, 3aR, 4S, 7aR] -6 (S) - (4-Hydroxy-7a-methyl-octahydro-inden-1-yl) -2,10-dimethyl-undecane-2,3 (R)> 10- triol (56).

<formula> formula see original document page 123 </formula>

A solution of aqueous fluorosilicic acid (3 mL) was added to a stirred solution of 55 (0.240 g, 0.481 mmol) in acetonitrile (12 mL). TLC (ethyl acetate) monitored the reaction. After 2.5 h Compound 56 (Rf 0.37) was the predominant species, produced at the expense of the less polar 55. The mixture was equilibrated with ethyl acetate and water (10 mL), the aqueous layer was re-extracted with water (2 x 10 mL) and the combined extracts were washed with water (6 mL) and brine (2 x 10 mL). then dried and evaporated. The colorless residue was flash chromatographed using a stepped gradient of 1: 2, 1: 1 and 2: 1 ethyl acetate - hexane to elute some unreacted 55, followed by 56, obtained as colorless syrup, 0.147 g, 79%: 1H NMR: 0.94 (3H, s), 1.12 (1H, m), 1.15 (3H, s), 1.21 (9H, s), 1.15 - 1.7 ( 20H, m), 1.7 - 1.9 (5H, m), 1.96 (1H, brd), 3.29 (1H, d, J = 9.6 Hz), 4.08 (1H, brs) ); LR-ES (+): m / z 448: (M + Na + MeCN), 407 (M + Na); LR-ES (-): m / z 419 (M + Cl); HR-ES (+) m / z: Calculated for C 23 H 44 O 4 + Na: 407.3132, found 407.3135. [1R, 3aR, 4S, 7aR] -1- (5-Hydroxy-1 (S) - {2- [2- (4-methoxy-phenyl) -5,5-dimethyl- [1,3] dioxolan-4 (R) -yl] -ethyl} -5-methylhexyl) -7α-methyl-octahydro-inden-4-ol (57).

<formula> formula see original document page 124 </formula>

4-Methoxybenzaldehyde dimethyl acetal (60 ml, 0.35 mmol) was added to a solution of 56 (81.2 mg, 0.211 mmol) in dichloromethane (2 ml), followed by a solution (0.2 ml) containing tosylate pyridinium (200 mg) in dichloromethane (10 ml). The reaction progress was followed by TLC (1: 2 ethyl acetate - hexane) which showed 4-methoxybenzaldehyde dimethyl acetal (Rf 0.80), 4-methoxybenzaldehyde (Rf 0.65), extract 56 (Rf 0.42) and product 57 (Rf 0.26). After 5 h the mixture was stirred for 15 min with saturated sodium hydrogen carbonate solution (5 mL) then equilibrated with ethyl acetate (25 mL). The organic layer was washed with brine (5 mL), dried and evaporated. The residue was flash chromatographed using a stepped gradient of 1: 3 and 1: 2 ethyl acetate - hexane to yield 57 as colorless syrup, 0.106 mg (100%): 1H NMR: 0.94 (3Η, s ), 1.19, 1.21 (6Η, unit s, Me 2 COH), 1.23, 1.35 and 1.24, 1.37 (6H, unit s, major and minor 5,5-dimethyloxolane diastereomer) 1.1-1.7 (18H, m), 1.7-1.9 (5H, m), 1.9-2.0 (2H, m), 3.65 (1H, m), 3 , 81 (3H, s), 4.08 (1H, brs), 5.78 and 5.96 (1H, unit s, major and minor acetal diastereomers), 6.89 (2H, m), 7.41 (2H, m). [1R, 3aR, 7aR] -1- (5-Hydroxy-1 (S) - {2- [2- (4-methoxy-phenyl) -5,5-dimethyl- [1,3] dioxolan-4 ( R) -yl] -ethyl} -5-methylhexyl) -7a-methyl-octahydro-inden-4-one (58)

<formula> formula see original document page 125 </formula>

Pyridinium dichromate (230 mg, 0.61 mmol) was added to a stirred mixture containing 57 (0.0838, 0.167 mmol), Celite (185 mg), and dichloromethane (4 mL). The conversion of 57 (Rf 0.31) to 58 (Rf 0.42) was monitored by TLC (1:25 methanol-chloroform). The mixture was diluted with dichloromethane (10 mL) after 2.5 h, then filtered through a silica gel layer. Filtrate and washings (dichloromethane - ethyl acetate 1: 1) were evaporated and the residue chromatographed (ethyl acetate - hexane 1: 4) to afford ketone 58, 0.0763 g, 91%: 1H NMR: 0.63 (3H, s), 1.19, 1.21 and 1.23 (6H, s unit, Me 2 COH), 1.25, 1.36, 1.38 (6H, m, s, s, diastereomer of 5, 5-dimethyloxolane), 1.1 - 1.9 (18H, m), 1.9 - 2.1 (3H, m), 2.1 - 2.4 (2H, m), 2.45 (1H, m), 3.66 (1 H, m), 3.802 and 3.805 (3H, unit s), 5.78 and 5.95 (1 H, unit s, major and minor acetal diastereomer), 6.89 (2H m, 7.39 (2H, m).

[1R, 3aR, 7aR] -1- [4 (R), 5-Dihydroxy-1 (S) - (4-hydroxy-4-methylpentyl) -5-methylhexyl] -7a-methylmethyl octaidro-inden-4-one (59) <formula> formula see original document page 126 </formula>

Ketone 58 was stirred in a solution of 1 N oxalic acid in 90% methanol. The mixture became homogeneous after a few min. TLC (ethyl acetate) suggested complete reaction after 75 min (Rf 0.24 to 59). Thus, calcium carbonate (0.60 g) was added and the suspension stirred overnight, then filtered. The filtrate was evaporated and flash chromatographed using a stepped gradient of 4: 1: 5 dichloromethane - ethyl acetate - hexane, 1: 1 ethyl acetate - hexane, and pure ethyl acetate yields 59 as a colorless residue, 0.060 mg, 94%: 1H NMR: 0.5 (3H, s), 1.17 (3H, s), 1.22 (6H, s), 1.23 (3H, s), 1.2 - 1 , 21 (23H, m), 2.15 - 2.35 (2H, m), 2.45 (1H, dd, J = 7 and 11 Hz), 3.30, 1 H, brd). [1R, 3aR, 7aR] -7a-Methyl-1- [5-methyl-1 (S) - (4-methyl-4-triethylsilanyloxy-pentyl) -4 (R), 5-bistriethylsilyloxy-hexyl] -octahydro- inden-4-one (60)

<formula> formula see original document page 126 </formula>

A mixture of 59 (0.055 g, 0.143 mmol), imidazole, (14.9 mg, 1.69 mmol), N, N-dimethylpyridine (6 mg), triethylchlorosilane (0.168 mL, 1 mmol) and N, N-dimethylformamide HCl (1.5 mL) was stirred for 17 h. The reaction was followed by TLC (1: 4 ethyl acetate - hexane) and showed rapid conversion to the disilyl intermediate (Rf 0.47). Another reaction proceeded gently overnight to provide the completely silylated 60 (Rf 0.90). The solution was equilibrated with water (3 mL), equilibrated with ethyl acetate (20 mL), the ethyl acetate layer was washed with water (3 x 4 mL), dried and evaporated. The residue was flash chromatographed using a 1: 100 stepped gradient of hexane and ethyl acetate - hexane to yield 60 as a colorless syrup, 0.0813 g, 78.4%: 1H NMR δ 0.55 - 0 , 64 (21H, m), 0.92 - 0.97 (27H, m), 1.12 (3H, s), 1.18 (3H, s), 1.19 (3H, s), 1, 21 (3H, s), 1.1 - 1.7 (18H, m), 1.9 - 2.15 (2H, m), 2.15 - 2.35 (2H, m), 2.43 ( 1H, dd, J = 7.7 and 11 Hz), 3.30 (1H, dd, J = 3 and 8.4 Hz). [1R, 3aR, 7aR, 4E] -4- [2 (Z) 13 (S), 5 (R) -Bis- (tert-Butyl-dimethylsilanyloxy) -2-methylene-cyclohexylidene] -ethylidene} -7α-methyl-1- [5-methyl-1 (S) - (4-methyl-4-triethylsilanyloxy-pentyl) -4 (TR), 5-bis-tritylsylanoxy-hexyl] -octahydro-indene (61)

<formula> formula see original document page 127 </formula>

A solution of 1.6M butyl lithium in hexane (0.14 mL) was added to a solution of phosphine (0.1308 g, 0.224 mmol) in tetrahydrofuran (1.5 mL) at -70 ° C. After 10 min a solution of ketone 60 (0.0813 g, 0.112 mmol) in tetrahydrofuran (1.5 mL) was added dropwise over a period of 15 min. The color of the ilid was faded after 3 h so that the pH 7 phosphate buffer (2 ml) was added and the temperature allowed to rise to 0 ° C. The mixture was equilibrated with hexane (30 mL), the organic layer was washed with brine (5 mL), dried and evaporated to afford a colorless oil which was purified by flash chromatography (1: 100 ethyl acetate - hexane). Only the range with Rf 0.33 (TLC, 1:39 ethyl acetate - hexane) was collected. Evaporation of these fractions provided 61 as colorless syrup, 0.070 g, 57%: 1 H NMR δ 0.06 (12H, brs), 0.53-0.64 (21H, m), 0.88 (18H, s), 0.92 - 0.97 (27H, m), 1.11 (3H, s), 1.177 (3H, s), 1.184 (3H, s), 1.195 (3H, s), 1-1.9 (22H , m), 1.98 (2Η, m), 2.22 (1Η, m), 2.45 (1Η, m), 2.83 (1Η, brd, J = 13 Hz, 3.27 (1Η, d, J = 6 Hz), 4.19 (1H, m), 4.38 (1H, m), 4.87 (1H, brs), 5.18 (1H, brs), 6.02 (1H, d, J = 11 Hz), 6.24 (1H, d, J = 11 Hz).

Synthesis of 1,25-Dihydroxy-21 (2R, 3-dihydroxy-3-methylbutyl) -20S-Cholecaciferol (50).

<formula> formula see original document page 128 </formula>

The deprotection reaction of 61 (0.068 g, 0.06238 mmol) in solution of tetrabutyl ammonium fluoride IM in tetrahydrofuran, followed by TLC (ethyl acetate), proceeded gradually to give 50 (Rf 0.19). The mixture was diluted with brine (5 mL) after 25 h, stirred for 5 min and equilibrated with ethyl acetate (35 mL) and water (15 mL). The aqueous layer was back extracted once with ethyl acetate (35 mL), the combined extracts were washed with water (5 x 10 mL) and brine (5 mL) then dried and evaporated. The residue was flash chromatographed using a linear gradient of 1: 1 and 2: 1 ethyl acetate - hexane and 2:98 methanol - ethyl acetate to give a residue which was taken up in methyl formate and evaporated to a white foam, 30 mg, 93%: [α] D + 29.3 ° (methanol, c 0.34); 1 H NMR δ: 0.55 (3H, s), 1.16 (3H, s), 1.21 (9H, s), 1.1 - 1.75 (22H, m), 1.80 (2H , m), 1.9-2.1 (5H, m), 2.31 (1H, dd, J = 7 and 13 Hz), 2.60 (1H, brd), 284 (1H, m), 3 , 29 (1H, d, J = 9.5 Hz), 4.22 (1H, m), 4.43 (1H, m), 5.00 (1H, s), 5.33 (1H, s) 6.02 (1H, d, J = 11 Hz); 6.02 (1H, d, J = 11 Hz); LR-ES (-) m / z: 564 (M + H 2 CO 2), 563 M-H + H 2 CO 2); HR-ES (+) calcd for C 32 H 54 O 5 + Na: 541.3863; found 541.3854; UV max (ε): 211 (15017), 265 (15850), 204 sh (14127), 245 sh (13747) nm. Synthetic Example 43 - Synthesis of 1,25-Dihydroxy-21- (2R, 3-dihydroxy-3-methylbutyl) -20S-19-nor-cholecaciferol (62)

<formula> formula see original document page 129 </formula>

[1R, 3aR, 7aR, 4E] -4- {2 (Z) -3 (S), 5 (R) -Bis- (tert-Butyl-dimethylsilanyloxy) -cyclohexylidene] -ethylidene} -7a- methyl-1- [5-methyl-1 (S) - (4-methyl-4-triethylsilanyloxy-pentyl) -4 (R), 5-bistriethylsilanoxy-hexyl] -octahydro-indene (63)

<formula> formula see original document page 129 </formula>

A solution of 1.6M butyl lithium in hexane was added to a solution of phosphine in tetrahydrofuran at -70 ° C. After 10 min a solution of ketone 60 of Example 2 in tetrahydrofuran was added dropwise over a period of 15 min. After the color of the ilid was faded, phosphate buffer at pH 7 was added and the temperature allowed to rise to 0 ° C. The mixture was equilibrated with hexane, the organic layer was washed with brine, dried and evaporated to afford a colorless oil which was purified by flash chromatography (1: 100 ethyl acetate - hexane) which provided 63. 1,25-Di -hydroxy-21- (2R, 3-dihydroxy-3-methylbutyl) -20S-19-nor-cholecaciferol (62) <formula> formula see original document page 130 </formula>

The deprotection reaction of 63 was performed in solution of tetrabutyl ammonium fluoride IM in tetrahydrofuran to provide 62. The mixture was diluted with brine after 25 h, stirred for 5 min and then equilibrated with ethyl acetate and water. The aqueous layer was extracted once with ethyl acetate, the combined extracts were washed with water and brine, then dried and evaporated. The residue was flash chromatographed to give a residue which was taken up in methyl formate and evaporated to yield 62. Synthetic Example 44 - Synthesis of 1,25-dihydroxy-20S-21 (3-hydroxy-3- methyl butyl) -24-keto-19-nor-cholecaciferol (64)

<formula> formula see original document page 130 </formula>

(R) -6 - [(1R, 3aR, 4S, 7aR) -4- (tert-Butyl-dimethylsilanyloxy) -7a-methyl-octahydro-inden-1-yl] -2-methyl-7-phenylsulfanyl-1-one heptan-2-ol (65)

<formula> formula see original document page 130 </formula> The above reaction was performed as described in Tet. Lett. 1975, 17: 1409-12. Specifically, a 50 ml round bottom flask was charged with 1.54 g (3.73 mmol) of (R) -2 - [(1R, 3aR, 4S, 7aR) -4- (tert-Butyldimethylsilanyloxy) -7a -methyloctahydroinden-1-yl] -6-methylheptane-1,6-diol (1) (Eur. J. Org. Chem. 2004, 1703-1713) and 2.45 g (11.2 mmol) of diphenylsulfide. The mixture was dissolved in 5 mL of pyridine and 2.27 g (11.2 mmol, 2.80 mL) of tributylphosphine was added. The mixture was stirred overnight and then diluted with 20 mL of toluene and evaporated. The residue was again taken up in toluene and evaporated, the remaining liquid chromatographed on silica gel using stepped gradients of hexane, ethyl acetate - hexane 1:39, 1:19 and 1: 9 to provide the title compound 65 as one syrup, 1.95 g.

(R) -7-Benzenesulfonyl-6 - [(1R, 3aR, 4S, 7aR) -4- (tert-Butyl-dimethylsilanyloxy) -7a-methyloctahydro-inden-1-yl] -2-methyl-heptan-2-one 2-ol (67) and (1R, 3aR, 4S, 7aR) -1 - ((R) -1-Benzenesulfonylmethyl-5-methyl-5-triethylsilanyloxyhexyl) -4- (tert-butyl-dimethylsilanyloxy) -7a methyl octahydro indene (68)

<formula> formula see original document page 131 </formula>

A 500 ml round bottom flask containing 1.95 g (3.9 mmol) of crude sulfide 65 was mixed with 84 g of dichloromethane (63 mL). The solution was stirred in an ice bath, then 2.77 g (11 mmol) of meta-chloroperbenzoic acid was added in one portion. The suspension was stirred in the ice bath for 40 min then at room temperature for 2 h. The reaction was monitored by TLC (1:19 methanol - dichloromethane). At the end of the reaction period, only one point at Rf 0.45 observed. Then 5.68 g (20 mmol) of solid sodium hydrogen carbonate was added to the suspension, the suspension was stirred for 10 min, then 30 mL of water was added portionwise and continued vigorous stirring for 5 min to dissolve all solids. The mixture was further diluted with 40 ml hexane, stirred for 30 min, transferred to a separatory funnel with 41.6 g hexane. The lower layer was discarded and the upper unit was washed with 25 mL of saturated sodium hydrogen carbonate solution, dried (sodium sulfate) and evaporated to afford 3.48 g of 67. This material was triturated with hexane, filtered, and evaporated to leave 67 as a cloudy syrup (2.81 g) which was directly used in the next step.

A 100 ml round bottom flask containing 2.81 g of 67 obtained above was charged with 30 ml of N, N-dimethylformamide 1.43 g of imidazole (21 mmol) and 1.75 ml of (10 mmol) of triethylsilyl chloride. The mixture was stirred for 17 h then diluted with 50 g of ice-water, stirred 15 for 10 min, further diluted with 5 mL brine and 60 mL hexane. The aqueous layer was back extracted with 20 ml hexane, both extracts were combined, washed with 2 x 30 ml water, dried, evaporated. This material contained a larger point with Rf 0.12 (ethyl acetate - hexane 1:39) and a smaller point with Rf 0.06. This material was chromatographed on silica gel using hexane, ethyl acetate - hexane 1: 100, 1:79, 1:39 and 1:19 as step gradients. The larger band was eluted with 1:39 and 1:19 ethyl acetate - hexane to yield 1.83 g of 68. (R) -5-Benzenesulfonyl-6 - [(1R, 3aR, 48.7aR) -4- (tert-Butyl-dimethylsilanyloxy) -7α-methyloctahydro-inden-1-yl] -10-methyl-2- (R) -methyl-10-triethylsilanyloxy-undecane-2,3-dioI (69)

<formula> formula see original document page 132 </formula> A 100 ml 3-neck round-bottom flask equipped with magnetic stirrer, thermometer and rubber-septum Claisen adapter was loaded with 1.7636 g of sulfone 68 (2.708 mmol), 1.114 g (4.062 mmol) tosylate, and 50 ml of freshly distilled tetrahydrofuran from cetyl benzophenone. This solution was cooled to -20 ° C and 9.31 ml of a 1.6M butyl lithium solution in hexane was added dropwise at -20 ° C. The temperature range between -10 and -20 ° C was maintained for 5 h. The cooling bath was removed and 50 ml of saturated ammonium chloride solution added followed by 75 ml of ethyl acetate and sufficient water to dissolve all salts. The organic layer was washed with 15 ml brine, dried, and evaporated to a colorless oil. This residue was chromatographed on silica gel using hexane, ethyl acetate - hexane 1: 9, 1: 6, 1: 4 and 1: 3 as staggered gradients. The main strip was eluted with 1: 4 and 1: 3 ethyl acetate - hexane to afford 1.6872 g of compound 69 as colorless syrup.

(S) -6 - [(1R, 3aR, 4S, 7aR) -4- (tert-Butyl-dimethylsilanyloxy) -7a-methyl-octahydro-inden-1-yl] -10-methyl-2- (R ) -methyl-10-triethylsilanyloxy-undecane-2,3-diol (70)

<formula> formula see original document page 133 </formula>

A 25 ml 2-neck round bottom flask equipped with magnetic stirrer, thermometer and rubber-septum Claisen adapter with nitrogen sweep was charged with 1.6872 g (2.238 mmol) of sulfone 69 and 40 mL of methanol. Then 1.25 g (51.4 mmol) of magnesium was added to the stirred solution in two equal portions over 30 min. The suspension was stirred for 70 min then another 0.17 g of magnesium and 5 ml of methanol was added and continued stirring 1 h. The mixture was then diluted with 100 mL hexane and 50 mL 1M sulfuric acid was added dropwise to provide two liquid phases. The aqueous layer was neutral. The aqueous layer was reextracted once with 25 ml of 1: 1 dichloromethane - hexane. The organic layers were combined then washed once with 15 ml brine, dried and evaporated. The resulting material was chromatographed on silica gel using hexane, ethyl acetate - hexane 1:39, 1:19 and 1: 9 as step gradients. The main strip was eluted with 1: 9 ethyl acetate - hexane to provide 1.2611 g of 70 as a colorless syrup. (S) -6 - [(1R, 3aR, 4S, 7aR) -4- (tert-Butyl-dimethylsilanyloxy) -7a-methyl-octahydro-inden-1-yl] -2,10-dihydroxy- 2,10-dimethyl-undecan-3-one (71)

<formula> formula see original document page 134 </formula>

A 25 ml round bottom flask equipped with magnetic stirrer, thermometer, nitrogen-scanned Claisen adapter and rubber septum was charged with 518 mg (3.88 mmol) of N-chlorosuccinamide and 11 ml of toluene. Stir for 5 min (not all dissolved) after cooling to 0 ° C and add 2.4 ml (4.8 mmol) of a solution of 2M dimethyl sulfide in toluene. The mixture was stirred from 5 min then cooled to -30 ° C and a solution of 0.7143 g (1.165 mmol) of diol 70 in 4 x 1.5 mL of toluene was added dropwise at -30 ° C. Stirring was continued at this temperature for 1 h. The mixture was then allowed to warm to -10 ° C over a period of 2 h then cooled to -17 ° C and 3.20 ml (6.4 mmol) of 2M triethylamine in toluene added dropwise. The mixture was stirred at -17 to -20 ° C for 10 min then allowed to slowly warm to room temperature. The mixture was chromatographed on a silica gel column using hexane, ethyl acetate - hexane 1:79, 1:39, 1:19, 1: 9, 1: 4, and 1: 1 as staggered gradients. The larger band was eluted with 1: 1 ethyl acetate - hexane providing 0.3428 g of compound 71 as solids.

(S) -2,10-Dihydroxy-6 - ((1R, 3aR, 4S, 7aR) -4-hydroxy-7a-methyl-octahydro-inden-1-yl) -2,10-dimethyl-undecan-2-one 3-one (72)

<formula> formula see original document page 135 </formula>

A 25 ml round bottom flask equipped with magnetic stirrer was charged with 0.3428 g (0.69 mmol) of diol 71, dissolved in 5 ml of acetonitrile then 1.25 ml of fluorosilicic acid solution. After 3 h, the mixture was partitioned between 35 mL of ethyl acetate and 10 mL of water, the aqueous layer was re-extracted with 10 mL of ethyl acetate, the combined organic layers washed with 2 x 5 mL of water. , once with 5 ml brine - 1: 1 saturated sodium hydrogen carbonate solution, dried and evaporated. This material was subjected to silica gel chromatography using 1: 4, 1: 3, 1: 2, and 1: 1 as stepped gradients providing 0.2085g of the title compound 72.

(1R, 3aR, 7aR) -1 - [(S) -5-Hydroxy-1- (4-hydroxy-4-methyl-pentyl) -5-methyl-4-oxohexy;] -7a-methyloctahydro -inden-4-one (73)

<formula> formula see original document page 135 </formula>

A 25 mL round bottom flask was charged with 0.2153 g (0.56 mmol) of 72.5 mL dichloromethane, and 0.20 g Celite. To this stirred suspension was added 1.00 g (2.66 mmol) of pyridinium dichromate. The reaction was stirred for 3 h and progress was monitored by TLC (1: 1 ethyl acetate - hexane). The reaction mixture was diluted with 5 ml of cyclohexane then filtered through silica gel G. The column was eluted with dichloromethane followed by 1: 1 ethyl acetate-hexane until no solute was detectable in the effluent. The effluent was evaporated and the oil colorless. This oil was then chromatographed on a silica gel using 1: 4, 1: 3, 1: 2, 1: 1 and 2: 1 ethyl acetate - hexane as staggered gradients to provide 0.2077 g of diketone 73.

(1R, 3aR, 7aR) -7a-Methyl-1 - [(S) -5-methyl-1- (4-methyl-4-trimethylsilanyloxypentyl) -4-oxo-5-trimethylsilyloxyhexyl] -octahydro-1-one inden-4-one (74)

<formula> formula see original document page 136 </formula>

A 25 mL round bottom flask was charged with 0.2077 g (0.545 mmol) of diketone 73. This material was dissolved in a mixture of 0.5 mL tetrahydrofuran and 3 mL cyclohexane. To the resulting mixture was added 0.30 ml (2.0 mmol) of TMS-imidazole. The reaction mixture was diluted with 3 ml hexane after 10 h then concentrated and subjected to silica gel chromatography using hexane, and 1:79, 1:39, 1:19 ethyl acetate - hexane as stepped gradients to provide 0.2381 g of 74 as a colorless oil.

(S) -6 - ((1R, 3aS, 7aR) -4- {2 - [(R) -34 (R) -tert-Butyldimethylsilanyloxy) -5- (tert-butyldimethylsilanyloxy) -cyclohexylidene] ethylidene} -7α-methyloctahydroinden- 1-i]) - 2,10-dimethyl-2,10-bis-trimethylsilaniyloxyundecan-3-one (75)

<formula> formula see original document page 136 </formula>

A 15-ml 3-neck peripheral flask, equipped with a magnetic stirrer, thermometer and a Claisen adapter containing a nitrogen sweep and rubber septum, was charged with 0.2722 g (0.4768 mmol) of [2- [2] oxide. (3R, 5R) -3,5-bis (tert-butyldimethylsilanyloxy) cyclohexylidene] ethyl] diphenylphosphine and 2 ml of tetrahydrofuran. The solution was cooled to -70 ° C and 0.30 ml of 1.6M butyl lithium in hexane was added. The deep red solution was stirred at this temperature for 10 min then 0.1261 g (0.240 mmol) of diketone 74 dissolved in 2 ml of tetrahydrofuran was added via syringe to the drops over a period of 10 min. After 3 h and 15 min, 5 mL of saturated ammonium chloride solution was added at -65 ° C, the mixture allowed to warm to 10 ° C then distributed between 35 mL of hexane and 10 mL of water. The aqueous layer was re-extracted once with 10 mL hexane, the combined layers washed with 5 mL brine containing 2 mL pH 7 buffer, then dried and evaporated. This material was chromatographed on a 15 χ 150 mm scintillating column using hexane and 1: 100 ethyl acetate - hexane as stepped gradients to yield 0.1572 g of the title compound 75 as a colorless syrup.

1,25-Dihydroxy-20S-21 (3-hydroxy-3-methylbutyl) -24-keto-19-nor-cholecaciferol (64)

<formula> formula see original document page 137 </formula>

A 15 ml 3-neck round bottom flask equipped with magnetic stirrer was charged with 155 mg (0.17 mmol) of tetrasilyl ether 75. This colorless residue was dissolved is 2 ml of a solution of tetrabutylammonium fluoride IM in tetrahydrofuran. After 43 h an additional 0.5 ml of tetrabutylammonium solution fluoride IM solution was added and stirring continued for 5 h. The light brown solution was diluted with 5 mL of brine, stirred for 5 min and transferred to a separatory funnel with 50 mL of ethyl acetate and 5 mL of water then reextracted with 5 mL of ethyl acetate. The organic layers were combined, washed with 5 x 10 ml water, 10 ml brine, dried and evaporated. The resulting residue was chromatographed on a 15 χ 123 mm column using 2: 3, 1: 1, 2: 1 ethyl acetate - hexane, and ethyl acetate as stepped gradients to provide 64 as a white solid (TLC ethyl acetate, Rf 0.23) which was taken up in methyl formate, filtered and evaporated affording 0.0753 g of the title compound 64 as a solid substance.

Synthetic Example 45 - Synthesis of 1,25-dihydroxy-20S-21 (3-hydroxy-3-methylbutyl) -24-keto-cholecaciferol (76)

<formula> formula see original document page 138 </formula>

(S) -6 - {(1R, 3aS, 7aR) -442 - [(R) -3- (tert-Butyl-dimethylsilanyloxy) -54 (S) -tert-butyl-dimethylsilanoyl) -2-methylene-2 cyclohexylidene] -et- (E) -ylidene] -7a-methyl-octahydro-inden-1-yl} -2,10-dimethyl-2,10-bis-trimethylsilanyloxy-undecan-3-one (77)

Compound 77 was prepared as described for 75 in Example 4 but reacting 74 with [(2Z) -2 - [(3S, 5R) -3,5-bis (tert-butyldimethylsilanyloxy) methylene cyclohexylidene] ethyl] oxide diphenylphosphine.

1,25-Dihydroxy-20S-21 (3-hydroxy-3-methylbutyl) -24-keto-cholecaciferol (76)

Compound 76 was prepared from 77 by deprotecting 77 as described in Example 44 to 64.

Synthetic Example 46 - Synthesis of 1α, 25-Dihydroxy-16-ene-20-cyclopropylcolecalciferol (78)

Compound (78) was synthesized according to the following synthetic procedure.

To a stirred solution of (3aR, 4S, 7aR) -1- {1- [4- (tert-Butyl-dimethylsilanyloxy) -7α-methyl-3a, 4,5,6,7,7a-hexahydro-3H -inden-1-yl]) - cyclopropyl] ethynyl (1.0 g, 2.90 mmol) in tetrahydrofuran (15 mL) at -78 ° C was added n-BuLi (2.72 mL, 4.35 mmol, 1.6M in hexane). After stirring at -78 ° C for 1 h., Acetone (2.5 mL, 34.6 mmol) was added and stirring was continued for 2.5 h. NH 4 Clq was added (15 mL) and the mixture was stirred for 15 min at room temperature then extracted with EtOAc (2 x 50 mL). The combined extracts were washed with brine (50 mL) and dried over Na 2 SO 4. The residue after solvent evaporation (2.4 g) was purified by FC (50 g, 10% EtOAc in hexane) to provide (3aR, 4S, 7aR) -5- {1- [4- (tert-Butyl -dimethylsilanyloxy) -7a-methyl-3α, 4,5,6,7,7a-hexahydro-3H-inden-1-yl] cyclopropyl} -2-methyl-pent-3-yn-2-ol (1.05 g, 2.61 mmol) which was treated with tetrabutylammonium fluoride (6 mL, 6 mmol, 1.0 M in THF) and stirred at 65-75 ° C for 48 h. The mixture was diluted with EtOAc (25 mL) and washed with water (5 x 25 mL), brine (25 mL). The combined aqueous washes were extracted with EtOAc (25 mL) and the combined organic extracts were dried over Na 2 SO 4. The residue after evaporation of the solvent (1.1 g) was purified by FC (50 g, 20% EtOAc in hexane) to afford the title compound (0.75 g, 2.59 mmol, 90%). [α] 30D = +2.7 and 0.75, CHCl3. 1H NMR (CDCl3): 5.50 (1H, m), 4.18 (1H, m), 2.40 (2H, s), 2.35 - 1.16 (11H, m), 1.48 ( 6H, s), 1.20 (3H, s), 0.76 - 0.50 (4H, m); 13 C NMR (CDCl 3): 156.39, 125.26, 86.39, 80.19, 69.21, 65.16, 55.14, 46.94, 35.79, 33.60, 31.67, 29.91, 27.22, 19.32, 19.19, 17.73, 10.94, 10.37; MS HREI Calculated for C 22 H 28 O 2 M + 288.2089 Observed M + 288.2091.

<formula> formula see original document page 140 </formula>

The mixture of (3aR, 4S, 7aR) -7a-Methyl-1- [1- (-4-hydroxy-4-methylpent-2-ynyl) cyclopropyl] -3a, 4,5,6,7, 7a-hexahydro-3H-inden-4-ol (0.72 g, 2.50 mmol), ethyl acetate (10 mL), hexane (24 mL), absolute ethanol (0.9 mL), quinoline (47 μ ) and Lindlar catalyst (156 mg, 5% Pd in CaCO3) was hydrogenated at room temperature for 2 h. The reaction mixture was filtered through a pad of celite and the pad was washed with EtOAc. The filtrates and washings were combined and washed with 1M HCl, NaHCO 3 and brine. After drying over Na 2 SO 4 the solvent was evaporated and the residue (0.79 g) was purified by FC (45 g, 20% EtOAc in hexane) to provide the title compound (640 mg, 2.2 mmol, 88%). ).

<formula> formula see original document page 140 </formula>

(3aR, 4S, 7aR) -7a-Methyl-1- [1- (4-hydroxy-4-methyl-pent-2Z-enyl) -cyclopropyl] -3a, 4,5,6,7,7a -hexhydro-3H-inden-4-ol (100 mg, 0.34 mmol), 1,4-bis (diphenylphospho) butane 1,5-cyclooctadiene rhodium tetrafluoroborate (25 mg, 0.034 mmol), dichloromethane (5 mL) and one drop of mercury was hydrogenated using Paar apparatus at room temperature and 50 psi pressure for 3 h. The reaction mixture was filtered through a pad of Celite, which was then washed with ethyl acetate. The combined filtrates and washings were evaporated to dryness (110 mg) and purified by FC (10 g, 20% EtOAc in hexane) to afford the title compound (75 mg, 0.26 mmol, 75%). [α] 30D = -8.5 and 0.65, CHCl3. 1H NMR (CDCl3): 5.37 (1Η, m), 4.14 (1Η, m), 2.37 - 1.16 (17Η, m), 1.19 (6Η, s), 1.18 (3Η, s), 0.66 - 0.24 (4Η, m); MS HREI Calculated for C 19 H 32 O 2 M + H 292.2402. Observed M + H 292.2404.

To a stirred suspension of (3aR, 4S, 7aR) -7a-Methyl-1- [1- (4-hydroxy-4-methyl-pentenyl) -cyclopropyl] -3a, 4,5,6,7,7a-hexahydro -3H-inden-4-ol (440 mg, 1.50 mmol) and Celite (2.0 g) in dichloromethane (10 mL) at room temperature was added pyridinium dichromate (1.13 g, 3.0 mmol) . The resulting mixture was stirred for 5 h, filtered through silica gel (10 g), and then silica gel pad was washed with 20% EtOAc in hexane. The combined filtrate and washings were evaporated to afford one (3 aR, 7aR) -7a-Methyl-1- [1- (4-hydroxy-4-methyl-pentenyl) cyclopropyl] -3a, 4,5,6, Crude 7,7a-hexahydro-3H-inden-4-one (426 mg, 1.47 mmol, 98%). To a stirred solution of (3aR, 7aR) -7a-Methyl-1- [1- (4-hydroxy-4-methyl-pentenyl) -cyclopropyl] -3a, 4,5,6,7,7a-hexahydro-3H -inden-4-one (424 mg, 1.47 mmol) in dichloromethane (10 mL) at room temperature was added trimethylsilyl imidazole (0.44 mL, 3.0 mmol). The resulting mixture was stirred for 1.0h filtered through silica gel (10g) and the silica gel pad was washed with 10% EtOAc in hexane. Combined filtrate and washings were evaporated to afford the title compound (460 mg, 1.27 mmol, 86%). [a] 29D = -9.9 and 0.55, CHCl3, 1H NMR (CDCl3): 5.33 (1H, dd, J = 3.2, 1.5 Hz), 2.81 (1H, dd, J = 10.7, 6.2 Hz), 2.44 (1H, ddd, J = 15.6, 10.7, 1.5 Hz), 2.30 - 1.15 (13H, m) overlap 2 .03 (ddd, J = 15.8, 6.4, 3.2 Hz), 1.18 (6H, s), 0.92 (3H, s), 0.66 - 0.28 (4H, m ), 0.08 (9H, s); 13 CRMN (CDCl 3): 211.08 (0), 155.32 (0), 124.77 (1), 73.98 (0), 64.32 (1), 53.91 (0), 44.70 (2), 40.45 (2), 38.12 (2), 34.70 (2), 29.86 (3), 29.80 (3), 26.80 (2), 24.07 ( 2), 22.28 (2), 21.24 (0), 18.35 (3), 12.60 (2), 10.64 (2), 2.63 (3); MS HRES Calculated for C 22 H 38 O 2 Si M + 362.2641. Observed M + 362.2648.

<formula> formula see original document page 142 </formula>

To a stirred solution of (1S, 5R) -1,5-bis - ((tert-butyldimethyl) silanyloxy) -3- [2- (diphenylphosphinoyl) -et- (Z) -ylidene] -2-methylene-cyclo hexane (675 mg, 1.16 mmol) in tetrahydrofuran (8 mL) at -78 ° C was added n-BuLi (0.73 mL, 1.17 mmol). The resulting mixture was stirred for 15 min and solution of (3aR, 7aR) -7a-Methyl-1- [1- (4-methyl-4-trimethylsilanyloxy-pentyl) -cyclopropyl] -3a, 4,5,6,7 7α-hexahydro-3H-inden-4-one (210 mg, 0.58 mmol, in tetrahydrofuran (2 mL) was added dropwise.) The reaction mixture was stirred at -720 ° C for 3.5 h diluted with hexane ( 35 ml) washed brine (30 ml) and dried over Na 2 SO 4 The residue (850 mg) after evaporation of the solvent was purified by FC (15 g, 10% EtOAc in hexane) to provide 1α, 3 P-Di ( tert-Butyl-dimethyl-silanyloxy-25-trimethylsilanyloxy-16-ene-20-cyclopropyl-cholecaciferol (382 mg, 0.53 mmol) To 1α, 33-Di (tert-Butyl-dimethyl-silanyloxy) -25- trimethylsilanyloxy-16-ene-20-cyclopropyl-cholecaciferol (382 mg, 0.53 mmol) tetrabutylammonium fluoride (4 mL, 4 mmol, IM solution in THF) was added at room temperature.The mixture was stirred for 15 h. diluted with EtOAc (25 mL) and washed with water (5 x 20 mL), brine (20 mL) and dried over Na 2 SO 4. mg) after evaporation of the solvent was purified by FC (15 g, 50% EtOAc in hexane and EtOAc) to provide the title compound (78) (204 mg, 0.48 mmol, 83%). [α] D 29 = +16.1 and 0.36, EtOH. UV λ max (EtOH): 208 nm (ε 17024), 264 nm (ε 16028); 1H NMR (CDCl3): 6.37 (1H, d, J = 11.3 Hz), 6.09 (1H, d, J = 11.1 Hz), 5.33 (2H, m), 5.01 (1H, s), 4.44 (1H, m), 4.23 (1H, m), 2.80 (1Η, m), 2.60 (1Η, m), 2.38 - 1.08 ( 20Η, m), 1.19 (6Η, s), 0.79 (3Η, s), 0.66 - 0.24 (4Η, m); 13 C NMR (CDCl 3): 157.07 (0), 147.62 (0), 142.49 (0), 133.00 (0), 124.90 (1), 124.73 (1), 117, 19 (1), 111.64 (2), 71.10 (1), 70.70 (0), 66.88 (1), 59.53 (1), 50.28 (0), 45.19 (2), 43.85 (2), 42.86 (2), 38.13 (2), 35.59 (2), 29.27 (2), 29.14 (3), 28.65 ( 2), 23.57 (2), 22.62 (2), 21.29 (0), 17.84 (3), 12.74 (2), 10.30 (2); MS HRES Calculated for C 28 H 42 O 3 M + Na 449.3026. Observed M + Na 449.3023.

Synthetic Example 47 - Synthesis of 1-alpha-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epi-cholecaciferol (79) (Compound A)

Compound (79) is synthesized according to the following synthetic procedure.

To a stirred suspension of 11- (5-Hydroxy-1,5-dimethyl-hex-3-enyl) -7a-methyl-3a, 4,5,6,7,7a-hexahydro-3H-inden-4-ol ) and Celite in dichloromethane (10 ml) at room temperature is added pyridinium dichromate. The resulting mixture is stirred for 5 h filtered through silica gel, and then silica gel pad is washed with 20% EtOAc in hexane. The combined filtrate and washings are evaporated to afford a ketone. To a stirred solution of ketone in dichloromethane at room temperature is added trimethylsilyl imidazole. The resulting mixture is stirred for 1.0h filtered through silica gel and the silica gel pad is washed with 10% EtOAc in hexane. Combined filtrate and washings are evaporated to afford the title compound. <formula> formula see original document page 144 </formula>

To a stirred solution of a tert-Butyl- {3- [2- (diphenylphosphinoyl) ethylidene] -5-fluoro-4-methylene cyclohexyloxy} dimethyl silanein tetrahydrofuran at -7 ° C is added -BuLi. The resulting mixture is stirred for 15 min and 1- (5-Ethyl-1-methyl-5-trimethylsilanyloxy-hept-3-enyl) -7a-methyl-3,3a, 5,6,7,7a-hexahydro solution -inden-4-one in tetrahydrofuran is added dropwise. The reaction mixture is stirred at -78 ° C for 3.5 h diluted with hexane, brine washed and dried over Na 2 SO 4. The residue after evaporation of the solvent was purified by FC (15 g, 10% EtOAc in hexane) to afford the silylated compound. To the silylated compound, tetrabutylammonium fluoride is added at room temperature. The mixture is stirred for 15 h. diluted with EtOAc (25 mL) and washed with water (5 x 20 mL), brine (20 mL) and dried over Na 2 SO 4. The residue (380 mg) after solvent evaporation is purified by FC (15 g, 50% EtOAc in hexane and EtOAc) to provide the title compound (79). mm3) and incubated at 37 ° C for 1 h with 0.1% type A collagenase. At the end of incubation, single stromal cells were separated from large pieces of epithelium for a period of 10 min. of differential sedimentation in gravity in unit. The top 8 ml of medium containing predominantly stromal cells were then slowly removed and the cells collected by centrifugation. The stromal enriched fraction was

BIOLOGICAL EXAMPLES

EXAMPLE 1

Materials and methods

Stromal Cell Preparation

Tissue was gently cut into small pieces (1-2 washed twice in culture medium and allowed to selectively adhere to tissue culture plates for 15 min. After that, unbound epithelial cells still present were removed and a purified stromal preparation was obtained on the surface of the culture plates.

Total RNA Extraction

Cells were incubated at 37 ° C in DMEM containing 3% FBS (without Compound A, with Compound A at 1 μΜ concentration or with Compound A at 0.1 μΜ concentration) or DMEM containing 10% FBS (without Compound A A, with Compound A at 1 μΜ concentration or with Compound A at 0.1 μ concentração concentration). After 4 or 8 hours of incubation the cells were trypsinized and collected as a cell pellet.

For total RNA extraction, the RNeasy QIAGEN Mini Kit (cat. No. 74106) briefly described below was used.

The cells were disrupted by addition of RLT Buffer and the lysate was loaded onto a QIAshredder spin column (QIAGEN cat. # 79656) placed in a 2 ml collection tube and centrifuged for 2 min at full speed. A 70% volume of ethanol was added to the homogenized lysate. The sample was loaded onto an RNeasy mini column placed in a 2 ml collection tube and centrifuged for 15 s at> 10,000 rpm. Column-bound RNA was digested with a DNase treatment. The column was washed with Buffer RW1 and centrifuged for 15 s at> 10,000 rpm. The sample was incubated with DNase I (RNase-Free Dnase Set QIAGEN cat. # 79254) mixture at room temperature for 15 min. The RNeasy mini column was washed with RWl Buffer and transferred into a new 2 ml collection tube. The column was washed twice with RPE Buffer and centrifuged for 15 s at> 10,000 rpm; RNase-free water was loaded onto the column and RNA was eluted after centrifugation for 1 min at> 10,000 rpm. RNA concentration was assessed by NanoDrop Spectrophotometer cDNA Synthesis CDNA synthesis was performed using the Applied Biosystems TaqMan Reverse Transcription Reagents kit (Applied Biosystems cat. No. 8080234).

1µg / mL total RNA was retrotranscribed in an RT mixture containing RT IX Buffer, 5.5 mM MgCl2, 500 μΜ dNTPs, 2.5 μΜ Random Hexamers, 40 U RNase Inhibitor, and Multiscribe 125 U Reverse Transcription at a final volume of 100 ml. The mixture was incubated at room temperature for 10 min followed by 30 min at 48 ° C; cDNA concentration obtained was 10µg / mL.

Real-Time PCR for Gene Expression Quantification

Real-time PCR was performed using the ABI PRISM 7000 Sequence Detection System (Applied Biosystems). 30 ng cDNA were amplified in a 25 ml volume containing TaqMan Universal PCR Master Mix IX (Applied Biosystems cat. No. 4304437) and Assay Mix target gene IX (Applied Biosystems). Genes analyzed included Vitamin D Receptor (VDR), Cytochrome P450 (CYP24), Vascular Endothelial Growth Factor (VEGF), Estrogen Receptor Alpha (ERa), Estrogen Receptor Beta (ERP), Progesterone Receptor (PR), Aromatase (CYP19), Cyclooxygenase Type 2 (COX-2), Interleukin-8 (IL-8), Alpha Factor

Tumor Necrosis (TNF a), Caspase-3 (CASP3), Caspase-6 (CASP6), Ki-67 Nuclear Antigen (Ki-67).

Samples were incubated 2 min at 50 ° C, 10 min at 95 ° C and extended to 40 cycles at 950 ° C for 15 s (denaturation) and at 60 ° C for 1 min (annealing / extension). The amount of gene expression was normalized to rRNA 18S gene expression and comparative CT methods (User Bulletin # 2 ABI PRISM 7000 Sequence Detection System) were used for relative quantitation. In vitro endometrial stromal cell proliferation

Cells were maintained in DMEM supplemented with 10% Fetal Bovine Serum (SIGMA) and 100 U / ml penicillin, 100 μ & πιΐ streptomycin (GIBCO cat. No. 15140-122).

When stromal cells were developed at confluence, they were washed in PBS and then trypsinized using IX trypsin / EDTA solution (PromoCell cat. No. C-41002). Cells were seeded at 1 χ 10 3 cells / ml in a 96 well flat bottomed plate in DMEM, 5% fetal bovine serum and VDR ligand (Compound A) at different concentrations (1 μΜ to 0.1 nM). After 48 to 96 hours, supernatants were harvested and the plates were stored at -80 ° C during proliferation determination. Proliferation was determined with CyQuant Cell Proliferation Assay (Molecular Probe cat. No. C7026). The plates were thawed at room temperature, and 200 ml of CyQUANT GR dye / cell lysis buffer were added to each sample well.

The plates were incubated for 2 to 5 minutes at room temperature, protected from light.

Fluorescence was determined using a fluorescence microplate reader with appropriate filters for ~ 480 nm excitation and ~ 520 nm emission.

Hu-IL-8 in ELISA

IL-8 was detected with Human IL-8 ELISA Set (BD OptEIA BD Biosciences cat. No. 555244)

The plate was coated with 100 ml 1: 250 diluted capture human anti-IL-8 in Coating Buffer (0.1 M Sodium Carbonate, pH 9.5) and incubated overnight at 4 ° C. After washing, plates were blocked by adding 200 ml Assay Diluent (PBS with 10% FBS, pH 7.0) for 1 to 2 hours at room temperature. The supernatant was discarded and 100 ml of standard (200 pg / ml recombinant human IL-8 at 3.1 pg / ml) or 1: 2 diluted sample in Assay Diluent was added. The plates were incubated for 2 hours at room temperature. After washing, 100 ml Detection Antibody (1: 250 Detection Antibody + 1: 250 SAv-HRP Reagent) was added and incubated 1 hour at room temperature.

The plates were washed and 100 ml of Substrate Solution was added to each well. The colorimetric reaction was blocked with Stop Solution (H2SO4 1 Μ). Optical density was determined at 405 nm using microtiter plate reader.

In vivo model of endometriosis

Balb / c donor mice were injected with estrogen (Estradiol AMSA; 3 μg / mouse) and one week later were sacrificed and the uterus was removed, their horns isolated and reduced to small fragments. Fragments derived from isolated uterine horns were resuspended in saline with ampicillin (1 mg / ml) and then injected into the peritoneum of two previously anesthetized Balb / c receptor mice through a 0.5 cm incision in the abdominal wall. . Estrogen was injected subcutaneously once a week for two weeks to sustain endometrial growth. Antibiotic (ampicillin 1 mg / ml) was administered on the day of surgery and on the day after. Four hours after surgery, one mouse in each pair was injected with the test compound (100 μg / kg) and the other with control, ip once a day, 5 days a week for two weeks. After two weeks, the mice were given a lethal dose of anesthetic and their abdomen was opened to check for injury. Lesions can be identified as isolated or clustered translucent cysts mainly found on the abdominal wall, pancreas, and around the uterus. In some cases the lesions are necrotic. The lesions were carefully removed and placed on a glass slide to dry for 48 hours, and then weighed. In other experiments the lesions are transferred to a lysis solution and the isolated mRNA for gene expression analysis. For immunohistochemical analysis, the lesions were frozen immediately after isolation.

Results

Injury weight

Figure 1 illustrates the effect of treatment using 1-alpha-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epi-cholecalciferol (Compound A) versus treatment using control (vehicle only). Figure 1A presents the entire dataset for 17 mouse pairs. Figure IB presents data as the percentage inhibition of lesion growth in treated mice relative to their control partner. Figure 1C shows the mean for the treated and control groups. Statistical analysis shows a significant reduction in lesion weight for those mice receiving treatment with a vitamin D compound (t-test p = 0.0034 for correlated and p = 0.020 for uncorrelated).

In vitro endometrial stem cell proliferation

Figure 2 shows the observed cell proliferation levels for treatment with different concentrations of vitamin D compound (Panel A - Eutopic Endometrium, Panel B - Ectopic Endometrium). Although there is a degree of variation in results due to the small data set used, treatment with Compound A generally leads to a reduction in cell proliferation for Eutopic (Figure 2A) and Ectopic (Figure 2B) endometrium. Figure 2B suggests that this effect may occur in a dose dependent manner.

Ideally, treatment with a vitamin D compound leads to a preferential reduction in ectopic cell proliferation over reduction in eutopic cell proliferation.

Gene Expression Quantification

Figure 3 shows the expression levels of VDR (Panel A), VEGF (Panel Β), Cyp24 (Panel C) and Cyp 19 (Panel D) for untreated groups treated with 1 μΜ Compound A and treated with 0, 1 μΜ of Compound A.

A striking up-regulation of Cyp24 expression can be seen in Figure 3C. Little or no change in VDR, VEGF, or Cyp 19 expression is observed.

Effect of Vitamin D Compounds

It can be clearly seen that in an in vivo model of endometriosis the vitamin D compound tested significantly reduced the total lesion weight.

The data therefore demonstrate the potential for the use of vitamin D compounds in the prevention and treatment of endometriosis.

EXAMPLE 2

Materials and Methods In vivo model of endometriosis

Balb / c donor mice were injected with estrogen (Estradiol AMSA; 3 μg / mouse) and one week later were sacrificed and the uterus was removed, both horns isolated and reduced to small fragments. Fragments derived from isolated uterine horns were resuspended in saline with ampicillin (1 mg / ml) and then injected into the peritoneum of two previously anesthetized Balb / c receptor mice through a 0.5 cm incision in the abdominal wall. . Antibiotic (ampicillin 1 mg / ml) was administered on the day of surgery and on the day after. Starting four hours after surgery, one mouse in each pair was injected with the test compound and the other with control ip once a day, 5 days a week for two weeks. Dosage levels of the test compounds were at most tolerated levels for the subject compound, i.e. 1-alpha-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epi-cholecalciferol (Compound A) 100 μg / kg, calcitriol (Compound Β) 0.3 μg / kg and 1,25-dihydroxy-21- (3-hydroxy-3-methyl butyl) -19-norcolecalciferol (Compound C) 3 μg / kg

After two weeks, the mice were given a lethal dose of anesthetic and their abdomen was opened to check for injury. Lesions can be identified as isolated or clustered translucent cysts mainly found on the abdominal wall, pancreas, and around the uterus. In some cases the lesions are necrotic. The lesions were carefully removed and placed on a glass slide to dry for 48 hours, and then weighed.

Results

Figure 4 illustrates the effect of treatment using 1-alpha-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epi-cholecalciferol (Compound A) versus treatment using control (vehicle only). Figure 4A presents the entire data set for 24 mice in each group. Figure 4B presents data as the mean lesion weight in treated and untreated mice (mean and standard error are shown). Figure 4C shows the relative reduction in lesion weight between treated and control groups (mean and standard error are shown). The reduction in lesion weight between paired animals was calculated: Compound A at 100 μg / kg is capable of reducing lesion weight by 51 ± 11% (mean ± standard error) when provided for two weeks after uterus transfer. (mean lesion weight: 8.452 ± 1.039 mg vs 3.527 ± 0.5400 mg in animals treated with migliol and Compound A respectively).

Statistical analysis shows a significant reduction in lesion weight for those mice receiving treatment with the vitamin D analog Compound A (t-test p = 0.0001 for uncorrelated, P = 0.0001 for correlated).

Figure 5 illustrates the effect of treatment using calcitriol (Compound B) versus treatment using control (vehicle only). Figure 5Α presents the entire dataset for 7 mice in each group. Figure 5B presents data as the mean lesion weight in treated and untreated mice (mean and standard error are shown). Figure 5C shows the relative reduction in lesion weight between treated and control groups (mean and standard error are shown). Statistical analysis again shows a significant reduction in lesion weight for mice receiving treatment with a vitamin D analog, in this case Compound B (test t p = 0.0207 for uncorrelated, test t p = 0.0252 for correlated).

Figure 6 illustrates the effect of treatment using 1,25-dihydroxy-2- 1- (3-hydroxy-3-methylbutyl) -19-nor-cholecalciferol (Compound C) versus treatment using the control (vehicle only). Figure 6A presents the entire dataset for 9 mice in each group. Figure 6B presents data as the mean lesion weight in treated and untreated mice (mean and standard error are shown). Figure 6C shows the relative reduction in lesion weight between treated and control groups (mean and standard error are shown). Statistical analysis shows no significant reduction in lesion weight for those mice receiving treatment with Compound C vitamin D analogue (t-test p = 0.112 for uncorrelated, t-test p = 0.0781 for correlated).

Effect of Vitamin D Compounds

Example 2 demonstrates that a range of vitamin D compounds may be used in the present invention. Each of the three test compounds leads to a reduction in lesion weight, although this is most pronounced following treatment with Compound A (which may be administered at a higher dosage level than the other compounds tested due to the lower associated toxicity).

EXAMPLE 3

Materials and Methods Dose / Response Analysis

Balb / c donor mice were injected with estrogen (Estradiol AMSA; 3 μg / mouse) and one week later were sacrificed and the uterus was removed, both horns isolated and reduced to small fragments. Fragments derived from isolated uterine horns were resuspended in saline with ampicillin (1 mg / ml) and then injected into the peritoneum of previously anesthetized Balb / c receptor mice through a 0.5 cm incision in the abdominal wall. Antibiotic (ampicillin 1 mg / ml) was administered on the day of surgery and on the day after. Starting four hours after surgery, each mouse was injected with a specific dose of Compound A or with control, ip once a day, 5 days a week for two weeks.

After two weeks, the mice were given a lethal dose of anesthetic and their abdomen was opened to check for injury. Lesions can be identified as isolated or clustered translucent cysts mainly found on the abdominal wall, the pancreas, and around the uterus. In some cases the lesions are necrotic. The lesions were carefully removed and placed on a glass slide to dry for 48 hours, and then weighed. At least 10 test animals were used in each group.

Treatment Regimens

Other experiments were performed using Compound A at 100 μg / kg but varying the time at which administration of vitamin D compound was initiated and the point at which time administration was discontinued. Specifically: (i) administration for 1 week prior to injection of uterine fragments (ii) administration for 2 weeks subsequent to injection of uterine fragments (iii) administration for 1 week prior to and 2 weeks following injection of uterine fragments (iv) administration during 2 weeks, started two days after injection of uterine fragments (ν) administration for 2 weeks, started two weeks after injection of uterine fragments. In these experiments, patients were sacrificed within the last two weeks post injection or at the end of the treatment period as appropriate.

Results

The effect of four different doses of Compound A up to the maximum tolerated dose of 100 μg / kg is shown in Figure 7. Mean and standard error are indicated. Results follow a typical dose / response profile, with greater reduction in lesion weight resulting from higher doses of test compound. Of note is the fact that the weight of the lesion is reduced at dose levels well below the maximum tolerated dose (ie approximately 20% to 1/10 MTD and approximately 35% around 1/3 MTD).

Figure 8 illustrates the effect of different treatment times on reduction in lesion weight. Advances in treatment with Compound A, group (i), led to a 40% injury weight after two weeks. Treatment with Compound A for two weeks starting at the time of uterus transfer, group (ii), demonstrated a 48% reduction in lesion weight. The maximum effect was obtained by treating the animals for three weeks, one week before and two weeks after uterus transfer (group (iii)), leading to a 73% reduction in lesion weight. Compound A is still effective when animal treatment is started 2 days (group (iv), 35% reduction) or 2 weeks (group (v), 34% reduction) after uterine transfer when endometriotic cysts are well settled down.

Effect of Vitamin D Compounds

Compound A is effective in treating endometriosis in a mouse model, even at dosages well below the maximum tolerated dose (above which the compound becomes hypercalcemic). In addition, Compound A can be expected to be of use in both treatment and prevention of the disorder, based on the fact that both pretreatment and posttreatment lead to the lowest lesion weight, with the largest reduction observed where Pre and post treatment with Compound A are provided.

EXAMPLE 4

Materials and methods

Cell adhesion

Paired animals were treated with Compound A (100 orally once daily for two days. The animals were then sacrificed and the uterus horns removed. The myometrium was removed by scraping with a scalpel blade and tissue The remaining endometrial tissue was reduced to small fragments with scissors.

Tissue was cut into small pieces (1 to 2 mm) and incubated at 37 ° C for 1 h with 0.1% type A collagenase. At the end of incubation, single stromal cells were separated from large pieces of epithelium by a 10 min period of differential sedimentation in gravity in unit. The top 8 ml of the medium containing predominantly stromal cells was then slowly removed and the cells were collected by centrifugation. The enriched stromal fraction was washed twice in culture medium and allowed to selectively adhere to tissue culture plates for 15 min. Thereafter, unbound epithelial cells still present were removed and a purified stromal preparation was obtained on the surface of the culture plates.

Polystyrene 96 well plates (Costar) were coated with 50 ml / 8 mg / ml extracellular matrix (ECM) well (Sigma, USA), and left uncapped in a laminar flow hood overnight to allow evaporation. The plates were then rinsed with PBS and used for binding assays. Cells were washed three times with PBS, trypsinized and seeded into 200 ml of cells at a density of 2 x 10 ^ 5 / ml in ECM. After 1 to 2 h incubation at 37 ° C, the wells were gently rinsed three times with PBS to remove unbound cells. Remaining cells in 96 well plates were tested with the CyQuant cell proliferation kit (Molecular Probes). The fluorescence of the sample in each reservoir was measured using a fluorescence microplate reader with appropriate filters for maximum 480 nm excitation and 520 nm emission. Results were expressed as the percentage of total cells assuming that cell adhesion in the control was 100%. Adhesion percentage was determined using the formula: (Abs after rinsing with PBS / Abs without rinse) χ 100%. The experiments were performed in triplicate.

Cell chemotaxis assay

Human stromal cell preparation: The tissue was gently cut into small pieces (1 to 2 mm3) and incubated at 37 ° C for 1 h with 0.1% type A collagenase. At the end of incubation, single stromal cells were separated. of large pieces of epithelium for a period of 10 min. of differential sedimentation in gravity in unit. The top 8 ml of medium containing predominantly stromal cells were then slowly removed and the cells collected by centrifugation. The enriched stromal fraction was washed twice in culture medium and allowed to selectively adhere to tissue culture plates for 15 min. Thereafter, unbound epithelial cells still present were removed and a purified stromal preparation was obtained on the surface of the culture plates.

Endometrial stromal cell migration was evaluated by chemotaxis experiments in a modified Boyden chamber of 48 reservoirs. With the migration assay, the ability of cells to migrate toward a chemoattractant on a two-dimensional substrate (in our case, type IV collagen) was evaluated. Briefly, chemotaxis experiments were performed using 8 μm of Nuclepore polyvinylpyrrolidine-free polycarbonate filters coated with 10 μg / ml type IV collagen and placed in a bottom chamber containing 20 ng / ml PDGF and / or 1 μΜ estrogen. as the chemo-attractive factor. Serum-free medium was used as a negative control. Suspended in D-MEM medium containing 0.1% fatty acid free bovine serum albumin, ESC cells were pretreated for 30 min with Compound A at 1 μΜ and then cells were treated with β-Estradiol for 24 minutes. H. After treatment the cells were added to the upper chamber at a density of 4 x 10 4 cells / well. After six hours incubation at 37 ° C, unmigrated cells on the upper surface of the filter were scraped off. Cells migrating to the lower side of the filter were stained with Diff-Quick stain (VWR Scientific Products, Bridgeport, NJ), and 5 to 8 unit fields per filter were counted at 160x magnification using a Zeiss microscope. Assays were conducted in triplicate.

Quantitation of cytokine ELISA produced by peritoneal macrophages

Peritoneal cells were recovered two weeks after uterus transfer in cold PBS, 2 mM EDTA, by peritoneal lavage of treated (Compound A at 100 μg / kg) and untreated (vehicle only) animals prepared according to the procedure described. previously in Examples 1 to 3 (mixture of 5 mice per group). Peritoneal macrophages were counted directly after collection using washed Turk reagent and cultured with RPM 1 / glutamax 5% FC I, pen / strep, pyruvate Na. After 2 h at 37 ° C non-adherent cells were removed and macrophages were cultured for an additional 48 h. Supernatant was harvested and cytokines (TNF-alpha, IL1-alpha, IL1-beta, IL6, MIP2 and VEGF) were quantified using a specific ELISA (R&D System DuoSet). All ELISA determinations were performed in duplicate on the undiluted sample. Total plated cell number was evaluated by the CyQuant test and protein production values normalized to cell number.

Results

Cell adhesion

Compound A is capable of dramatically reducing endometriotic cell adhesion to collagen, as shown in Figure 9 (mean and standard error are shown for a total of 5 patients per group).

Cell chemotaxis assay

Figure 10 demonstrates that Compound A is capable of reducing estrogen-induced chemotaxis in human stromal endometrial cells. No effect of Compound A is evident at baseline migration compared to approximately 50% reduction in migration observed with estrogen stimulation.

ELISA Quantification

Figure 11 shows that inflammatory cytokine and VEGF production is dramatically reduced by Compound A, suggesting that an anti-inflammatory mechanism contributes to this endometriosis mouse model.

Effect of Vitamin D Compounds

Among the different possible mechanisms of action Compound

In endometriotic lesions there is a direct effect on endometrial cell adhesion and chemotactic sensitivity. Compound A is capable of reducing both the number of adherent cells and may decrease chemotactic migration of endometrial cells in response to estrogen.

Other possible mechanisms of action for vitamin D compounds include inhibition of inflammation. The inflammatory response of peritoneal macrophages is well documented to support the progression of endometriosis in humans. We therefore tested the hypothesis that vitamin D compounds such as Compound A may modulate peritoneal inflammation in the endometriosis mouse model and we have shown that the production of inflammatory cytokine and VEGF is dramatically reduced by Compound A (Figure 11). However the same macrophages are still capable of producing the same cytokines if reactivated in vitro with an unrelated stimulus such as LPS (data not shown).

FORMULATION EXAMPLES

Formulation Example 1: Oral Dosage Form Soft Gelatin Capsule An oral administration capsule is formulated under amber nitrogen: 150 mg of Compound A in 150 mg of fractionated coconut oil (Miglyol 812) with 0.015 mg of hydroxytoluene butylated (BHT) and 0.015 mg of butylated hydroxyanisole (BHA), filled into a soft gelatin capsule.

Formulation Example 3: Oral Dosage Form Soft Gelatin Capsule

One capsule for oral administration is formulated under amber nitrogen: 75 μg Compound A in 150 mg fractionated coconut oil (Miglyol 812), with 0.015 mg butylated hydroxytoluene (BHT) and 0.015 mg butylated hydroxyanisole (BHA) , filled in a soft gelatin capsule.

Throughout the specification and the claims that follow, unless the context otherwise requires, the word "comprise", and variations such as "comprise" and "comprising" shall be understood to indicate the inclusion of an integer, step, whole number group or step group established but not to the exclusion of any other integer, step, whole number group or step group

Incorporation by reference

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

Equivalents

Those skilled in the art will recognize, or be able to verify using no more than routine experimentation, many equivalents of the specific embodiments of the invention described herein.

Such equivalents are intended to be encompassed by the following claims.

Claims (12)

1. Use of a vitamin D compound, characterized in that it is for prevention or treatment of endometriosis. Use of a vitamin D compound according to claim 1, characterized in that it is for the manufacture of a medicament for the prevention or treatment of endometriosis. A method for preventing and / or treating endometriosis, which comprises administering an effective amount of a vitamin D compound. Pharmaceutical formulation, characterized in that it comprises a vitamin D compound and a pharmaceutically acceptable carrier for use in the prevention and / or treatment of endometriosis. Pharmaceutical formulation, characterized in that it comprises a vitamin D compound and a pharmaceutically acceptable carrier packaged with instructions for use in the prevention and / or treatment of endometriosis. 6. Vitamin D compound, characterized by the fact that it is for use in the prevention and / or treatment of endometriosis. Kit, characterized in that it contains a vitamin D compound together with instructions leading the administration of said compound to a patient in need of treatment and / or prevention of endometriosis in this manner to treat and / or prevent endometriosis in said patient. Use, method, formulation, compound or kit according to any one of claims 1 to 7, characterized in that (a) the vitamin D compound is administered separately, sequentially or simultaneously in separate pharmaceutical formulations or combined with a second drug for the treatment of endometriosis. Use, method, formulation, compound or kit according to any one of claims 1 to 8, characterized in that said vitamin D compound is a compound of the formula: <formula> formula see original document where: A1 is single or double bond; A2 is a single, double or triple bond; X1 and X2 are all independently H or = CH2, provided that both X1 and X2 are not = CH2; R 1 and R 2 are all independently C (0) C 1 -C 4 alkyl, OC (0) hydroxyalkyl or OC (0) haloalkyl; R1 and / or R2 may alternatively be OH; R 3, R 4 and R 5 are all independently hydrogen, C 1 -C 4 alkyl, hydroxyalkyl, or haloalkyl, or R 3 and R 4 taken together with C 20 form C 3 -C 6 cycloalkyl; and R 6 and R 7 are all independently C 1-4 alkyl or haloalkyl; and R 8 is H, -C 1 -C 4 -alkyl, -COhydroxyalkyl or -COhaloalkyl; and pharmaceutically acceptable esters, salts, and prodrugs thereof. Use, method, formulation, compound or kit according to any one of claims 1 to 8, characterized in that said vitamin D compound is a compound of the formula: <formula> formula see original document where: X is H2 or CH2 R1 is hydrogen, hydroxy or fluorine R2 is hydrogen or methyl R3 is hydrogen or methyl provided that when R2 or R3 are methyl, R3 or R2 must be hydrogen R4 is methyl, ethyl or trifluoromethyl R5 is methyl, ethyl or trifluoromethyl A is a single or double bond B is a single, double E, double Z or triple bond Use, method, formulation, compound or kit according to claim 10, characterized in that (a) each R 4 and R 5 is methyl or ethyl. Use, method, formulation, compound or kit according to any one of claims 1 to 8, characterized in that said vitamin D compound is 1,25-dihydroxy-21- (3-hydroxy) -3-methylbutyl) -19-nor-cholecalciferol having the formula: