CROSS-REFERENCE TO RELA TED APPLICATIONS

(0001 J The present application claims the benefit of priority to U.S. Provisional Patent

Application No. 61/656,157, filed on June 6, 2012, the content of which is incorporated herein by reference in its entirety.

BACKGROUND

|0002| The field of the invention relates to vitamin D compounds, and more particularly to 1 -nor- Vitamin D analogs and their pharmaceutical uses, and especially seco-A-2, 19-dinor- 1 ,25-dihydroxyvitamm D¾, its biological activities, and its pharmaceutical uses. This latter compound can also be abbreviated simply as "ΌΑ2ΗΕ."

[0003J The natural hormone, la,25-dihydroxyvitaniin D- ₅ and its analog in the ergosterol. series (i.e., 1 ,25-dihydroxyvitaniin D _: >) are known to be highly potent regulators of calcium homeostasis in animals and humans, and their activity in cellular differentiation has also been established. (See Ostrem et ai., Proc, Natl. Acad. Sci USA, 84, 2610 (J 987)). Many structural, analogs of these metabolites have been prepared and tested, including i -hydrox vitamin D la-hydroxy vitamin D , various side chain homologated vitamins and fluorinated analogs. Some of these compounds exhibit an interesting separation of activities in cell differentiation and calcium .regulation. These differences In activity may be useful in the treatmen of a variety of diseases such as renal osteodystrophy, vitamin D-resistant rickets, osteoporosis, psoriasis, and certain malignancies. ftl004| Another class of vitamin D analogs, i.e., the so called 19-nor- vitamin D compounds, is characterized by the replacement of the A-ring exocyciic methylene group (carbon 19), typical of the vitamin D system, by two hydrogen atoms. Biological testing of such 19-nor-analogs (e.g., l ,25-diiiydroxy- 19-nor~vitamin D _¾ ) revealed a selective activity profile with high potency in inducing cellular differentiation, and very low calcium mobilizing activity. Thus, these compounds are potentially useful as therapeutic agents for the treatment, of malignancies, or the treatment of various skin disorders. Two different methods of synthesis of such 1 -mir- itamin D analogs have been described, (See Perlmaa et ai, Tetrahedron Lett. 31. 1823 (1990); Perlman et al, Tetrahedron Lett. 32, 7663 (1991); and DeLuca ei ai, U.S. Pat. No. 5 ,086 J 91).

{00051 In U.S. Pat. No. 4,666,634, 2|¾-hydroxy and alkoxy (e.g., ED-71 ) analogs of l ,25-d.ibydtOxyvitainin have been described and examined by the Chugai group as potential dnrgs for osteoporosis and as antitumor agents. (See also Okano et a!.., Biochem. Biophys. Res, Conimim. 1 3. 1444 (1989)). Other 2-substituted [e.g., with, hydroxyalkyl, (ED-120) and fiuoroalkyl groups) A-ting analogs of ί a,25-dihydroxyvitami» D ₃ have also been prepared and tested.. (See Miyamoto et i, Chem. Pharm. Bull. 41 , 1111 (1993); Nishii et ai. Osteoporosis int. Sirppi. 1 , 190 (1993); and Posner et ai, i . Org. Chem. 59, 7855 (1994), and J. Org. Chem. 60, 4617 (1995)).

|0O96| 2-siibstititted analogs of 1 a,25-dihydroxy- 19-nor-vi tannn , have also been synthesized, for example compounds substituted at the 2-pcsitkra with hydroxy or alkoxy groups, (see DeLuca et /., U.S. Pat, No. 5,536,713), with 2-alkyl groups, (see DeLuca et l U.S. Patent No. 5,945,410), and with 2-alkylidene groups, (see DeLuca ei ai U.S. Patent No. 5,843,928), These 2-substituted analogs exhibit interesting and selective activity profiles. All these studies indicate that bindin sites on vitamin D receptors can accommodate different substituents at€-2 in the synthesized vitamin D analogs.

{0007J In a continuing effort to explore the 19-nor class of pharmacologically important vitamin D compounds, analogs which are characterized by the presence of a methylene substituenf at carbon 2 (C-2), a hydroxy!, group at carbon. (CM), and a shortened side chain attached at. carbon 20 (C-20) have also been synthesized and tested. 1.a-hydroxy-2-methylene- 19-nor-pregnacalciferol is described in U.S. Patent 6,566,352. I a-hydroxy-2 -methylene- 19-nor- ho opregnacalciferol is described i U.S. Patent 6,579,861, and la-hydroxy-2-methyIene-19- nor-bishomopregnacalciferol is described in U.S. Patent 6,627,622. All three of these compounds have relatively high binding activity to vitamin D receptors and relatively high cell differentiation activity. However, these compounds have little if any calcermc activity as- compared to 1 a,25^i hydroxyvitamin D3. Their biological activities make these compounds excellent candidates for a variety of pharmaceutical uses, as set forth in the '352, '861 and '622 patents,

|0008f Analogs of the natural hormone I a,25-dihydroxy vitamin D _? characterized by the transposition of the A-ring exocyclic methylene group from carbon 10 (€-10) to carbon 2 (C-2) (e.g., 1 o^S-d y&oxy^-methylene- 1 -nor- vitamin D analogs) have been synthesized and tested. (See Sicinski et aL, J. Med. Chem., 41. 4662 (1998); Sieinski et ί, Steroids 67, 247 (2002); and eLuca el al U.S. Pat Nos. 5,843,928; 5,936,133 and 6,382,071 ». hi these studies, it was found that 1 a _s 25-dihydroxy-2-methylene- 1 -nor-vitamin D analogs are characterized by significant biological potency. In addition, the biological potency of such analogs may be enhanced dramatically where "unnatural" (205 -configuration is present.

SUMMARY jO009f Disclosed herein are vitamin D compounds characterized by lacking the A-ring exocyclic methylene group at carbon 10 (C~10), (i.e., a class of 1 -nor vitamin D compounds), and also characterized by the presence of a seco-A ring. These analogs also lack a carbon 2 (C- 2) group, and thus the present invention is directed toward 19~nor~ vitamin D analogs, and their pharmaceutical uses, and more specifically toward seeo-A-2, 1 -dinor- l.,2S->dihydroxyvitamin ¾ its biological activiiy, and various pharmaceutical uses for this compound.

1000] 0] Structurally these j 9-nor~viiamin D analogs are characterized by the general formula I shown below:

XO OX

!

where X. is selected from the group consisting of hydrogen and a hydroxy-protecting group. represents any of the typical side chains known for vitamin D type compounds or hydrogen, aikyl hydroxyalkyl, or iluoroalkyi group. For example, R may represent a side chain of the formula:

where the stereochemical center at carbon 20 may have the R or S configuration, and where Z in the above side chain structure is selected from Y, -OY, - CH^OY, -C≡CY and -CH=CHY, where the double bond in the side chain may have the cis (Z) or trans (E) geometry, and where Y is selected from hydrogen, methyl, -COR ⁵ and a radical of the structure:

where m mid % independently, represent the integers from 0 to 5, where ⁵ is selected from hydrogen, deuterium, .hydroxy, protected hydroxy, fluoro, trifluoromethy!, and Cj-s-alkyl, which may be straight chain or branched and, optionally, m y bear a hydroxy or protected-hydroxy substituent, and where each of R , R , and R ⁴ , independently, is selected from deuterium, deuteroaikyL hydrogen, fluoro, trifluoromethyl and Ct-s alkyl, which .may be straight-chain, or branched, and optionally, may bear a hydroxy or protected-hydroxy substituent, and where R ¹ and ^' R ^* , taken together, represent an oxo group, or an a!fcylkleoe group having a general formula fcBik" where k is an. integer, the group ^~ R¾ ^' ', or the gr up™(C¾) _j ,™, where p is an integer from 2 to 5, and where R'' and R ^' \ taken together, represent an oxo group, or the group -(<¾) _<} - where q is an integer from 2 to 5, and where R* represents hydrogen, hydroxy, protected hydroxy, or Cj.s alkyl and wherein any of the CH-groups at positions 20, 22, or 23 in the side chain may be replaced by a nitrogen atom, or where any of the groups ---CH(Cii¾) -(CH^,-, •C .] ·· ^■ or ^• -- ^• (Ci¾,) _» '" at positions 20, 22, and 23, respectively, may be replaced by an oxygen or sulfur atom.

(000111 Specific important examples of side chains are the structures represented by formulas (a), (b), (c), (d) and (e) below with natural 20R-eon†1guratioB, i.e., the side chain as it occurs in 25-hydrox vitamin ¾ (a); vitamin D-j (b); 25-hydroxyvitamm D2 (c); vitamin (d); and the C-24 epimer of 25-hydroxyvitamin D2 (e).

[00012] Additional important examples of side chains are the structures represented by formulas (a), (b), (c), (d) and (e) below having the 20-epi or 20S-configuration, i.e., the side chain as it occurs in (20S)-25-hydroxyvitamm D3 (a); (20S)-vitamm D3 (b); (20$)~25- hydroxyvitan in D? (c); (20S)- vitamin D2 (d); and the C-24 epimer of (20S)-25-bydroxyvttamtn

D ₂ (e).

[00013] The wavy line to the carbon 20 indicates thai carbon 20 may have either the R or S configuration.

[00014] A preferred analog as disclosed herein is seco-A-2,19-dinor-l ,25- dihydroxwitamin D¾ (which is referred to herein as ' Α2ϊ-!Ε") which has the following formula la;

fflO0J5| The above compounds of formula Ϊ, especially formula la, exhibit a desired, and highly advantageous, pattern of biological activity. These compounds are characterized by relatively low binding to vitamin D receptors as compared to 1 ,25(OH)2D3, These compounds also exhibit markedly lower in vitro transcription activity. as well as significantly less potency for causing differentiation of HL-60 cells than l ,25(QH ¾. These compounds also exhibit relatively low activity in their ability to mobilize calcium from bone, but relativeiy high ability to promote intestinal calcium transport, as compared to 1 ,25-dihydroxyviuwnin ¾,

£00016] These compounds exhibit significant biological activity profiles- in vivo, specifically in intestinal tissue. This is likely due to these compounds maintaining two hydroxy! groups in the approximate same location as the natural hormone l a,25~diliydroxy itamin D3. Accordingly, these analogs may serve as important therapies for metabolic bone diseases where intestinal calcium transport activity is desirable, such as bone diseases like senile osteoporosis, postmenopausal osteoporosis, steroid-induced osteoporosis, low bone turnover osteoporosis, osteomalacia, and renal osteodystrophy,

[00017) Because DA2HE showed surprisingly high biological activity in vivo, specifically in intestinal tissue, but relativeiy low activities in promyelocyte leukemia cells and osteosarcoma ceils, there is potential for this compound and the compounds of formula, I to ha ve a strong selectivity for some ceil types. Such cell selectsviiy could make DA2HE and the compounds of formula Ϊ an important therapeutic choice for treatment or prevention of some types of cancers, such as colon cancer, or polyps, as well as hyperplastic intestinal disorders, such as Crohn's disease, ulcerative colitis and celiac disease.

(00018) One or more of the compounds may be present in a pharmaceutical composition to treat and/or prevent the above-noted diseases m an amount from about Q.Olpg/gni to about 1000 pg/gm of the composition, preferably from about OJ pg/gni to about 5<X g/gm of the composition. The pharmaceutical compositions typically may be administered topically, transdermal! , orally, rectally, nasally, sublingually, or parenierally in. dosages of from, abou 0,01 ^ig day to about l OOOpg/day, preferably from about 0.1 pg/da to about 500 pg day.

BRIEF DESCRIPTION OF THE DRAWINGS

(00019) Figures 1-4 illustrate various biological activities of seco-A-2, 1 -dinor- 1 ,25diirydroxy vitamin D¾, herein referred to as "DA2HE," as compared to the native hormone i a,25-dihydrox viiamin Dj, herein "1 ,25 ΟΗ)2θ3."

(00020} Figure 1 is a graph illustrating the relative activity of DA2HE and 1 _s 25(OH)2D3 to compete for binding with (¾]-! , 25-(OH)2D3 to the fall-length recombinant rat vitamin D receptor.

(000211 Figure 2 is a graph illustrating the percent HL-60 cell differentiation as a function of the concentration of DA2HE and 1 ,25(GH) ₂ ¾,

(00022) Figure 3 is a graph illustrating the in vitro transcriptio activity of DA2HE and 5(OH} ₂ D _s ,

(00023) Figure 4 is a bar graph illustrating the bone calcium mobilization activity of DA2HE and l ₅ 2S(OH) ₂ D ₃ .

(00024] Figure 5 is a ba graph illustrating the intestinal calcium transport activity of .1 ,25(OH) ₂ D _? as compared to DA2HE. DETAILED DESCRIPTION OF THE INVENTION

(000251 Disclosed are vitamin D analogs, and more particularly 19-nor-V.itaniin D analogs and their pharmaceutical uses, and especially seco-A-2,19-dinor-l ,25-dihydroxyvitamiii D-$, its biological activities, and its pharmaceutical uses.

{00026! Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. to which this invention belongs. All publications and patents specifically mentioned herein are incorporated by reference in their entirety for all purposes including describing and disclosing the chemicals, instruments, statistical analyses and methodologies which are reported in the publications which might be used in connection with the invention. If a definition in this specification, is contrary to a definition in a referenced publication or patent, the definition in this specification should be utilized to construe the meaning of the claims and the disclosure. All references cited in this specification are to be taken as indicati ve of the level of skill in the art. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue or prior invention.

[00027| in the specification and in the claims, the terms ''including" and "comprising" are open-ended terms and should be interpreted to mean "including, but not limited to," These terms encompass the more restrictive terms "consisting essentially of and "consisting of," It is also to be noted that the terms "comprising," "including," "characterized by" and "having ^"' ca be used interchangeably.

{00028] As used herein and in the appended claims, the singular forms "a " "an," and "the" include plural reference unless the context clearly dictates otherwise. As well, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein .

{00029) Where a range of values is provided, it is understood that each intervening value, and any combination or subcombination of intervening values, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the range of values recited. (00030} Certain terras or ranges may be presented herein with numerical values being preceded by the term "about" The term "about" is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately tie number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating itnrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number, and thus will typically refer to a number or value that is 10% below or above the specifically recited number or value.

[000311 As used in the description and in the claims, the term "hydroxy-protectmg group" signifies any group commonly used for the temporary protection of hydroxy functions, such as for example, alkoxycarbonyl, acyl, alkylsilyl or alky!aryisHyl groups (hereinafter referred to simply as "silyl" groups), and alkoxyalkyl groups. Alkoxycarbonyl protecting groups are alkyl- O-CO- groupings such as methoxycarbonyl, ethoxycarbonyi, propo ycarbonyl, isopropoxycarbonyb butoxycarbonyl, isobutoxyearbonyl, tert-butoxycarbonyl, benzyloxycarbonyl or allyloxycarbonyl. The term "acyl" signifies an aikanoyt group of 1 to 6 carbons, in all of its isomeric forms, or a earboxyalkaooyl group of 1 to 6 carbons, such as an oxalyl, malonyl. suceinyb glutaryl group, or an aromatic acyl group such, as benzoyl, or a halo, nitro or alkyS substituted benzoyl group. The word ^v alkyl ^M as used in the description or the claims, denotes a straight-chain or branched alky! radical of I to 10 carbons, in all its isomeric forms. "Alkoxy" refers to any alkyl radical which is attached by oxygen, i.e. a group represented by "alkyl-O " Alkoxyalkyl protecting groups are groupings such as methoxymethyi, ethoxymethyl, methoxyethoxymethyl, or tetrahydrofuranyl and tetrahydropyranyl. Preferred silyl-protecting groups are rrimethylsilyl, triethylsilyl, f-buty dimethySsilyl, dibutyimetiiyis iyl; diphenylinethy!si!yl, phenyldimethylsilyl, di henyl-t-butyl silyl and analogous alkylated silyl radicals. The term "aryl" specifies a phenyl-, or an alkyl-, nitro- or halo-substituted phenyl group. j00u32J A "protected hydroxy" group is a hydroxy group derivatized or protected by any of the above groups commonly used for the temporary or permanent protection of hydroxy functions, e.g. the silyl alkoxyalkyl, acyl or alkoxycarbonyl groups, as previously defined. The

to terras "hydroxyalkyl", "deuteroalkyl" and "fluoroalkyl" refer to an alkyl radical substituted by one or more hydroxy, deuterium or ffuoro groups respectively. An "alky!idene" refers to a radical having the general formula C^th^-whe k is an integer. 00033] The preparation of 1 -nor- vitamin D analogs of the basic structure I can be accomplished by a common general method, i.e., the Julia olefmation involving a coupling of an unsaturated sulfone IV (easily prepared from a bicyclic Wmdaus-Grundmann. type ketone III) with the ketone V;

[00034] i the structures III, IV and V groups Y and R represent groups defined above whereas Ar represents phenyl, substituted phenyl, benzothiazol-2-yl (preferably) and other aromatic groups that can be suitable for the Julia olefmation process, it being also understood that any functionalities in Ar that might be sensitive, or that interfere with the condensation reaction, should be avoided. The process shown above represents an application of the convergent synthesis concept, which has been applied effectively for the preparation of vitamin compounds. (See, e.g., ittaka el /,, Synlett 8, 1 175 (2003), and J. Org. Chem., 68, 7407 (2003).

[00035] Bicyclic hydrindanone compounds of the general structure II . are known, or can be easily prepared by known methods from the corresponding Windaus-Grundmann type ketcmes. Specific important examples of such known bicyclic ketones are the strtictures with the side chains (h), (i), (j), (k), (I), (m and («) below described above, i.e., 25-hydroxy Grandraann's ketone ( ) (see Baggiolmi et at, J. Org. Chem., 51 , 3098 ( 1986)): Gruadmann's ketone (i) (see Inhoffen el αί , Chem. Ber., 90, 664 (.1957)); 25-hydroxy Windaus ketone (j) (see BaggioHni ei aL, J. Org. Chem., 5i, 3098 (1986)); VVindaus ketone (k) (see VVindaus ei aL, Ann., 524, 297 (.1 36)); <20S)-25~hydroxy Grundmaiii's ketone (!) (see Sk nskt ei aL, J. Med. Chem., 41 , 4662 ( 1998)); (20S)-Griradniafrn's ketone (m) (see Grzywacz ei a , 3. Steroid Biochem. Moi Biol., 89-90, 13 (2004)); and (20S)-25-methyl Grundmartn's ketone (n) (see Grzywacz ei aL, J. Steroid Biochem, Moi. Biol., 89-90, 13 (2004)):

[00036| The preparation of the reqivired ketone of structure V was accomplished starting from the acetic acid S-acetoxy-S^ ^' r-batyl-diinethyl-silanyloxj'^pentyt ester (1), that was prepared from penia-1 ,4-dien-3-of, according to a known procedure. (See A. Scoriente ei aL , Tetrahedron* Asymmetry, 6, 4, 859 (1995)). Transfor ation, of I into the desired ketone of the general structure V is shown in SCHEME L Thus, the secondary /-buiyldimethylsilyl. group in 1 was hydrolyzed under TBAF condition leading to the hydroxyl compound 2, which was next oxidized with pyridimura chlorochromate to the desired ketone 3. The latter compound was then subjected to modified Julia Olefination with the anion generated with lithium bis(irimemylsilyl)amide from benzothiazolyl sulfone 4, prepared form the corresponding Grundmann ketone according to the know procedure. (See Glebocka et ai, J, Med. Chem., 49, 2909 (2006)). Removal of th protecting groups in the obtained bis-seco-steroid gave the expected vitamin D derivative 5 which was purified by HPLC

[00037} Several, other vitamin D compounds characterized by the A-seco structure may be synthesized by the method disclosed herein using the ketone 3 and the appropriate CD- fragments derived from the Windaus-Grandmann ketones having the desired side chain structure.

{00038} This invention is described by the following illustrative examples, in these examples specific products identified by Arabic numerals (e.g. 1, 2, 3, etc.) refer to the specific structures so identified in the preceding description and in the SCHEME i.

EXAMPLES

[00039} The following examples are illustrative and should not be construed to limit the scope of the claims.

[00Θ40] Chemistry. Ultraviolet (UV) absorption spectra were recorded with a Perkm- Elmer Lambda 3B UV-VIS spectrophotometer in ethanol. ^! H nuclear magnetic resonance (NMR) spectra were recorded in deuterioch!oroforra at 400 and 500 MHz with a Braker DMX- 400 and Braker DM -500 spectrometers, respectively. * ^" 'C nuclear magnetic resonance (NMR) spectra were recorded at 100 and 125 Hz with the same spectrometers in deuieriochlorofbr . Chemical shifts (5) were reported downfieid from internal Me^Si (5 0.00). Electron impact (El) mass spectra were obtained with a Mkroraass AutoSpec (Beverly, MA) instrument High- performance .liquid chromatography (HPLC) was performed on a Waters Associated liquid chromatograph equipped with a Model 6000A solvent delivery system, a Model U6 Universal injector, and a Model 486 tunable absorbance detector. THF was freshly distilled before use from sodium benzophenone kety! under argon.

1000411 Example I - Preparation of vitamin D analog $ 100042} Hydrolysis of /-bufyldimethyisilyl group 1 (SCHEME 1 ) and preparation of acetic acid S-a.cetoxy-3~hydroxy~penryj ester ( 2 ). The starting bicyclic compound 1 was obtained according to the described procedure. (See Armesto et aL, Tetrahedron Letters 4T, 8759 (2000)}. Compound 1 (70mg, 0.220 mmol) was dissolved in anhydrous THF (5 mL) and treated with TBAF (1M in THF; 350 μηιοΐ). The solution was stirred at room temperature under argon for 2 h, poured into brine, and extracted with ethyl acetate. The extract was washed with brine, dried (Na^SO _* }) and evaporated. The obtained product was purified by silica gel chromatography. Elution wit hexane/ethyl acetate (1 : 1 ) gave pure, colorless oily hydroxy compound 2 (32 rag, 71%).

100043) 2: 1H NMR (400 MHz, CDC¾) δ 1.73 and 1.82 (2 x 3H, each m, 2~H ₂ and 4-¾), 2.07 (6H, S, COCffc), 3,77 (IH ₅ m _s 3-H), 4.16 and 4.33 (2 x 2H _S each m, l -¾ and 5-¾); °C NMR δ 21 ,0 (COCHs), 36.3 (C2 and C4), 61.5 (CI and C5), 65.4 (C3), 171.5 (COCf L); ESI MS for C ₉ H| 0 ₅ Na (M + Na) ⁺ 227.2.

{00044J Oxid rio hydKml g

pentyl ester (3). A suspension of 2 (31 mg, 0.152 mmol) and pyridinium c iorochroraate (65 mg, 0.30 mmol) in anhydrous CI¾C¾ (2 ml..) was stirred at roorn temperature for 5 h, then filtered through a pad a silica gel The pad was washed with ethyl acetate to give ketone 3 (28 mg, 90%). 00045] 3: ¹ E NMR (400 MHz CDC ) S 2.79 (4H, t, J - 6:2, 2-H ₂ and 4-H ₂ ), 2.04 (6H, s, COCJ ), 4.35 (4H, T, J - 6.2, 1 -H ₂ and 5-H ₂ ); ^{f 3} C NMR 6 20.8 (CO£H ₃ ), 42.7 (C2 and C4), 59 (CI and C5), 171.5 iCOCH ); 204.7 (C3), ESI MS for C ₉ H _i 0sNa (M + Naf 225.2.

|00046] ijjl cQ pliiA^

o f 1 a.25 - dihy drox y vi tarn i C JS) . To a solution of sulfbne 4 (54.0 mg, 89 umol) in dry THF (700 μΕ), LiHMDS (I M in THF; 90 μϊ,, 90 μηιοΙ) was added at -78 "C under argon. The solution turned deep red. The mixture was stirred at -78 °C for 5 rain, and a solution of ketone 3 (9 mg, 44.5 μηιο!) in THF (200 + J 00 pL) was added. The reaction mixture was stirred at -78 C for 2,5 h, then brine containing 1% HCI was added, and the mixture was poured into brine and extracted with ethyl acetate. The extract was washed with brine, dried (NasSO-j). and evaporated. The yellow oily residue was applied on a silica column, tinted with hexane/ethyl acetate (9:1) and concentrated under vacuum. The oily product was dissolved in anhydrous dr MeOB (1.2 mL) and treated with (+)-camphofsulfonic acid (20 mg, 86 μη οΐ). The solution was stirred at room temperature under argon for 24 h, poured into brine, and extracted with ethyl acetate. The extract was washed with, brine, dried (N sSQ*) and evaporated. The obtained product was purified by straight-phase HPLC (9.4 ram x 25 cm Zorbax Rx-Sil column, 4 raL/min) using hexane/lPA (8:2) solvent system; 3,5~Seco~2,1.9-dino.r analog of Ia,25-(QH ₂ D3 5 (4.5 mg, 26%) was collected at Ry 23 mL. Additional purification was accomplished by reversed-phase HPLC (9,4 mm x 25 cm Zorbax XDB, C-18 column, 4 niL/min) using MeOH/iTO (9:1) solvent system; the analog 5 (4.5 mg, 26%) was collected at Rv 23 mL.

100047] 5: UV (in ΕΐΟΗ) X _Ttmx 250.5 nm; 1H NMR (400 MHz, CDC¾) δ 0.544 (3H, s, 18- H ₅ ), 0-940 (3H, d, J - 6.3 Hz, t~¾), 1.21 (6H, s, 26- and 27-H ₃ ), 2.38 and 2.49 (2 x 2H, each J - 6.3 Hz, 4-and 10-¾), 2.81 (Hi, br d, J - 12.2 Hz, 9β-Ή), 3.75 (411, t, J - 6.3 Hz, 1 - and 3- ¾ _. ), 5.85 and 6.38 (1H and I H, each d, J = 1 1.1 Hz, 7- and 6~H); HRMS (ESI) exact mass calculated for C ₂ sH 4(¾ a (M + Na)+ 415.6174, measured 415.6179.

SCHEME L

(00048} Example 2 ----- Biological Activity of seco-A-2J9-di,oor-l ,25-dib.yciroxyvitai¾io. Di (DA2IIE)

[000491 Elimination of the A-ring of 1 a 25-dihy roxyviramni D3 by removing carbon 2 (C~2) while maintaining two hydroxy! groups in the approximate same location as the natural vitamin D hormone had significant effect on binding to the full length recombinant rat vitamin D receptor (VDR), as compared to J a,25~dihydrox vitamin D3. The compound DA2HE bound to the VDR with about one log less activity as compared to the standard la,25~(OH)JD (Figure 1).

This result might suggest that compound DA2HB would have little or insignificant biological activity. As shown in Figure 2, DA2HE is 100 times less potent than l ₅ 25(OH) D? on HL-60 cell differentiation, and as shown in Figure 3, DA2HE is 500 times less potent than the native hormone in stimulation of gene transcription. 000S0| However, DA2HE was observed to have a unique biological activity with respect to caleemic activity. As shown in Figure 4, DA2HE lacks any bone calcium mobilization activity, as compared to 1.25(ΟΗ)2θ¾, even after up to 35100 pmol was administered intraperitonealiy one time daily for four consecutive days. Surprisingly, however, Figure 5 shows that DA2HE has relaiively high activity as compared to that of 1 a,25-dthydroxyvitamin 3 ί1 α,25{ΌΗ) ₂ ¾}, the natural hormone, in stimulating intestinal calcium transport, DA2HE is only about 10 times less potent than J a,25(OH)-?D _. :¾ in promoting active calcium transport across the gut. Figures 4-5 thus illustrate that DA2HE may be characterized as having relaiively high intestinal calcium activity, but no bone calcium mobilization activity.

EXPERIMENTAL METHODS

[Θ005Ί] The compounds of the invention were prepared and studied using the following methods.

[00052] Vitamin Receptor Binding

[00053] Protein Source. Full-length recombinant rat receptor was expressed in E. colt BL21 (DE3) Codon Plus R1L cells and purified to homogeneity using two different column chromatography systems. The first system was a nickel affinity resin that utilizes- the C-teraiina! histidine tag on this protein. The protein that was elated from this resin was further purified using ion exchange chromatography (S-Sepharose Fast Flow). Aliquots of the purified protein were quick frozen in liquid nitrogen and stored at -80°C until use. For use in binding assays, the protein was diluted in TEDK.se (50 mM Tris, 1.5 mM EDTA, pH7.4, 5 mM DTT ₅ 150 m CI) with 0.1% Chaps detergent. The receptor protein and ligan concentration was optimized such that no more than 20% of the added radiolabeled ligand was bound to the receptor.

(00054) Study Drugs. Unlabeled ligands were dissolved in ethanoi and the concentrations determined using UV spectrophotometry ( 1 ,25(QH)2.D _. 3: molar extinction coefficient - 18,200 and jaas ^:::: 265 nm; Analogs: molar extinction coefficient ^:::: 42,000 and λ„ _1¾ί ^:::: 252 nm). Radiolabeled ligand ( ^' Ή- l ,25(OH)2¾ -159 Ci/ mole) was added in ethanoi at a final concentration of 1 nM.

[000551 Assay Conditions. Radiolabeled and unlabeled ligands were added to 100 mcl of the diluted protein at a final ethanoi concentration of <10%, mixed and incubated overnight on. ice to reach binding equilibrium. The following day, 100 mcl of hydroxyiapatite slurry (50%) was added to each tube and mixed at 10~minute intervals for 30 minutes. The hydroxy! paptiie was collected by centrifugatton and then washed three times with Tris-EDTA buffer (50 mM Tris, 1.5 mM EDTA, pH 7.4) containing 0.5% Titron X-100. After the final wash, the pellets were transferred to scintillation vials containing 4 ml of Biosafe 11 scintillation cocktail mixed and placed in a scintillation counter. Total binding was determined from the tubes containing only radiolabeled ligand.

100056 j HL-60 Differentiation

|00057i Study Drugs. The study drugs were dissolved in ethanoi and the concentrations determined using UV spectrophotometry. Serial dilutions were prepared so that a range of drug concentrations could be tested without changing the final concentration of ethanoi (< 0.2%) present in the cell cultures. (00058} Cells. Human promyelocyte leukemia. (HL60) cells were grown in RPMl-3640 medium containing 10% fetal bovine serum. The cells were incubated at 37°C in the presence of 5% C<¾.

{000591 Assay Condttioris. HL60 cells were plated at 1.2 x I C cells/ml. Eighteen hours after plating, cells in duplicate were treated with drag. Four days later, the cells were harvested and a nitro blue tetrazolium reduction assay was performed. (See Collies et ai, 1979; j. Exp. Med. 149:969-974), The percentage of differentiated cells was determined by counting a total of 200 ceils and recording the number that contained intracellular black-blue formazan deposits. Verification of differentiation to monocytic cells was determined by measuring phagocytic activity.

[OO06 | in vitro Transcription Assay. Transcription activity was measured in. ROS 1.7/2.8 (bone) cells that were stably transfected with a 24 -hydroxylase (240hase) gene promoter upstream of a luciferase reporter gene. (See Arbour et αί., 1998). Ceils were given a range of doses. Sixteen hours after dosing the cells were harvested and luciferase activities were measured using a lu inoraeter. RLU - relative luciferase units.

[00061] Intestinal Calcium Transport and Bone Calcium Mobilization

|00062| Male, weanling Sprague-Dawley rats were placed on Diet 1 1 (see Siida et /., J, Nutr. 100:1049, 1970) (0.47% Ca) + vitamins AEK for one week followed by Diet 11 (0.02% Ca) + vitamins AEK for 3 weeks. The rats were then switched to the same diet containing 0.47% Ca for one week followed by two weeks on the same diet containing 0.02% Ca. Dose administration began during the last week on 0.02% calcium diet. Four consecutive ip doses were given approximately 24 hours apart. Twenty-four hours after the last dose, blood was collected from the severed neck and the concentration of serum calcium determined by atomic absorption spectrometry as a measure of bone calcium mobilization. The first 10 cm of the intestine was also collected for intestinal calcium transport analysis using the everted got sac method.

INTERPRETATION OF DATA 100063} V R binding. HL60 cell differentiation, and transcription activity. DA2HE (Kf ^: 4xl 0 ^"w M) has about 10 times less activity as the natural hormone 10L25-dihydroxyvitai«m Dj ( ^4χ10 ^"Η Μ) in its ability to compete with [ ³ H]-l,25(OH)2D.} for binding to the full-length recombinant rat vitamin D receptor { Figure 1). DA2HE is also about 100 times less potent (ECso ^: ~3xl0" ) in its ability {efficacy or potency) to promote HL60 differentiation as compared to I a,25-dihydrox itamin ¾ (ECso-S l O^M) (See Figure 2). Also, DA2BE is about 500 times less potent (EC so - 4x10 ^' M) than 1 a,25-dihydroxyvitamin D (ECso-S l 0 ^' M) in stimulation of gene transcription in bone cells (Figure 3).

100064 j Calcium mobilization from bone and intestinal calcium absorption in vitamin D- defieient animals. Using vitamin D-deficient rats on a low calcium diet (0.02%), the activities of DA2B E and 1 ,25(OH)2Di in intestine and bone were tested. As expected, the native hormone (1.25(ϋΗ)2Γ> ) increased serum calcium levels at the dosages tested {Figure 4). Figure 4 also shows that DA2HE lacks an activity in mobilizing calcium from bone at the tested doses. Administration of DA2HE at 35100 pmoJ/day for 4 consecutive days resulted in no mobilization of bone calcium.

|¾O065] Intestinal calcium transport wa evaluated in the same groups of animals using the everted gut sac method (Figure 5). These results show that the compound DA2HE only has slightly less ability to promote intestinal calcium transport activity when administered at the recommended lower dosages, as compared to i ,25(OH) ₂ D;H. Thus, it may be concluded that DA2HE has relatively high intestinal calcium transport activity which increases in a dose dependent manner.

£000661 In vivo distinct activity profiles emerge most likely due to these compounds maintaining two hydroxy! groups in the approximate same location as the natural hormone 1 a,25-dihydroxyvitamm D;¾ These analogs may serve as important therapies for metabolic bone diseases where intestinal calcium transport activity is desirable, such as hone diseases like senile osteoporosis, postmenopausal osteoporosis, steroid-induced osteoporosis, low bone turnover osteoporosis, osteomal cia, and renal osteodystrophy. |OO067] Because DA2HE showed surprisingly high activity in vivo, specifically in intestinal tissue, but relatively low activities in promyelocytic leukemia cells and osteosarcoma cells, there is potential for this compound and the compounds of formula I to have a strong selectivity for some cell types. Such cell selectivity could make, DA2HE and the compounds for formula I an important therapeutic choice for treatment or prevention of some types of cancers such as colon cancer, or polyps, as well as hyperplastic intestinal disorders, such as Crohn's disease, ulcerative colitis and celiac disease.

[00068| For prevention and/or treatment purposes, the compounds of this invention defined by formula I, and la may be formulated tor pharmaceutical applications as a solution in innocuous solvents, or as an emulsion, suspension or dispersion in suitable solvents or carriers, or as pills, tablets or capsules, together with solid carriers, according to conventional methods known in the art. Any such formulations may also contain other pharmacentically-acceptable and non-toxic excipients such as stabilisers, anti -oxidants, binders, coloring agents or emulsifying or taste-modifying agents.

|OO06 j The compounds of formula I and particularly DA2HE of formula la. may be administered orally, topically, parenterally, rectaily, nasally, sublingually, or transdermally. The compound is advantageously administered by injection or by intravenous infusion or suitable sterile solutions, or in the form of liquid or solid doses via the alimentary canal, or in the form of creams, ointments, patches, or similar vehicles suitable for transdermal applications. A dose of from 0.0 lpg to lOOOpg per day of the compounds I, particularly DA2HE, preferably from about 0.l|ig to about 500 μg per day, is appropriate for prevention and or treatment purposes, such dose being adjusted according to the disease to be treated, its severity and the response of the subject as is well understood in the art Since the compound exhibits specificity of action, each may be suitably administered alone, or together with graded doses of another active vitamin D compound— e.g. l.a~hydroxyvitamht D„ or D , or I a,25-dihydroxyvitamra D„— in situations where different degrees of bone mineral mobilization and calcium transport stimulation is found to be advantageous. (00070} Compositions for use in the above-mentioned treatments comprise an effective amount of the compounds L particularly DA2HE, as defined by the above formula I, and l» as the active ingredient, and a suitable carrier. An effective amount of such compound for use in accordance with this invention is from about 0.01 μ§ to about 1000 μ§ per gm of composition, preferably from about 0.1 μ$ to about 500 μ per gram of composition, and may be administered topically, transdermal ly, orally, rectally, nasally, sublingually or parenterally in dosages of from about 0.01 g da to about 1000 μ« /day, and preferably from about 0.1 pg day to about 500

{Ό007Ϊ] The compounds Ϊ, particularly DA2HE, may be formulated as creams, lotions, ointments, topical patches, pills, capsules or tablets, suppositories, aerosols, or in liquid form as solutions, emulsions, dispersions, or suspensions in pharmaceutically innocuous and acceptable solvent or oils, and such preparations may contain in addition other pharmaceutically innocuous or beneficial components, such as stabilizers, antioxidants, emulsiflers, coloring agents, binders or taste-modtfying agents.

|OO0?2| The compounds I, particularly ΌΑ2ΗΕ, may be advantageously administered in amounts sufficient to effect the differentiation of promyelocytes to normal macrophages. Dosages as described above are suitable, it being understood that the amounts given are to be adjusted in accordance with the severity of the disease, and the condition and response of the subject as is well understood in the art.

{00073 J The formulations of the present invention comprise an active ingredient in. association with a pharmaceutically acceptable carrier therefore and optionally other therapeutic ingredients. The carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulations and not deleterious to the recipient thereof.

{00074] Formulations of the present invention suitable for oral administration may be in the form of discrete units as capsules, sachets, tablets or lozenges, each containing a predetermined amount of the active ingredient:; in the form of a powder or granules; in the form of a solutioa or a suspension in an aqueous liquid or non-aqueous liquid; or in the form of an oil- in-water emulsion or a waier-m-oil emulsion.

{00075J Formulations for rectal administration may be in the form of a suppository incorporating the active ingredient and carrier such as cocoa butter, or in the form of an enema.

{OO076j Formulations suitable for parenteral administration conveniently comprise a sterile oily or aqueous preparation of the active ingredient which is preferably isotonic with the blood of the recipient.

[00077 { Formulations suitable for topical administration include liquid or semi-liquid preparations such as liniments, lotions, applicants, oii-in-water or water-in-oil emulsions such as creams, ointments or pastes; or solutions or suspensions such as drops; or as sprays.

{00078J For nasal administration, inhalation of powder, self-propelling or spray formulations, dispensed with a spray can, a nebulizer or an atomizer can be used. The formulations, when dispensed, preferably have a particle size in the range of 10 to 100μ.

{0007 1 The formulations ma conveniently be presented in dosage unit form and may be prepared by any of the methods well known in the art of pharmacy. By the term "dosage unit" is meant a unitary, i.e. a single dose which is capable of being administered to a patient as a physically and chemically stable unit dose comprising either the active ingredient as such or a mixture of it with solid or liquid pharmaceutical diluents or carriers.