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Definitions

• the natural hormone, l ,25-dihydroxyvitamin D 3 also referred to as l ,25- dihydroxycholecalciferol and calcitriol
• its analog in the ergosterol series i.e. la,25- dihydroxyvitamin D 2
• l ,25-dihydroxyvitamin D 3 also referred to as l ,25- dihydroxycholecalciferol and calcitriol
• la 25- dihydroxyvitamin D 2
• the present technology provides (20R)-2-methylene- 19-nor-22-dimethyl- l ,25-dihydroxyvitamin D 3 and (20S)- 2-methylene-19-nor-22-dimethyl-l ,25- dihydroxyvitamin D 3 and related compounds, pharmaceutical formulations that include (20S)-2-methylene-19-nor-22-dimethyl-la,25-dihydroxyvitamin D 3 or (20R)-2-methylene- 19-nor-22-dimethyl-l ,25-dihydroxyvitamin D 3 , methods of treating various disease states using this compound, and the use of this compound in the preparation of medicaments for treating various disease states.
• the present technology provides a compound having the formula I shown below:
• the carbon at position 20 has the S- configuration as shown in the compound of formula IA and in others it has the R- configuration as shown in the compound of formula IB:
• X , X and X are both hydroxy protecting groups such as silyl groups.
• X 1 and X 1 are both hydroxy protecting groups such as silyl groups.
• the compound has the formula II.
• the compound is (20S)-2-methylene-19-nor-22- dimethyl-l ,25-dihydroxyvitamin D 3 having the formula IIA as shown below or the compound is (20R)-2-methylene-19-nor-22-dimethyl-l ,25-dihydroxyvitamin D 3 having the formula IIB as shown below:
• the compound of formula IIA is a compound of formula IIC (MET-1), and in other embodiments, the compound of formula IIB is a compound of formula IID (MET-2) and have the structures shown below:
• Compounds of the present technology show a highly advantageous pattern of biological activity, including strong binding to the vitamin D receptor, strong cell differentiation and induction of 24-hydroxylase activity, yet low to very low calcemic activity.
• the present compounds may be used in methods of treating a subject suffering from certain biological conditions.
• the methods include administering an effective amount of a compound of the present technology to the subject, wherein the biological condition is selected from psoriasis; leukemia; colon cancer; breast cancer; prostate cancer; multiple sclerosis; lupus; diabetes mellitus; host versus graft reaction; rejection of organ transplants; an inflammatory disease selected from rheumatoid arthritis, asthma, or inflammatory bowel diseases; a skin condition selected from wrinkles, lack of adequate skin firmness, lack of adequate dermal hydration, or insufficient sebum secretion.
• the biological condition is selected from psoriasis; leukemia; colon cancer; breast cancer; prostate cancer; multiple sclerosis; lupus; diabetes mellitus; host versus graft reaction; rejection of organ transplants; an inflammatory disease selected from rheumatoid arthritis, asthma, or inflammatory bowel diseases; a skin condition selected from wrinkles, lack of adequate skin firmness, lack of adequate dermal hydration, or insufficient sebum
• a compound of the present technology may be present in a composition to treat the above-noted diseases and disorders in an effective amount and optionally including a pharmaceutically acceptable carrier.
• the amount of compound includes from about 0.01 ⁇ g per gram of composition to about 1 mg per gram of the composition, preferably from about 0.1 ⁇ g per gram to about 500 ⁇ g per gram of the composition, and may be administered topically, transdermally, orally, or parenterally in dosages of from about 0.01 ⁇ g per day to about 1 mg per day, preferably from about 0.1 ⁇ g per day to about 500 ⁇ g per day.
• Figures 1-5 illustrate various biological activities of (20S)-2-methylene-19- nor-22-dimethyl-l ,25-dihydroxyvitamin D 3 (referred to as “MET-1” in the figures) compared with those of the native hormone l ,25-dihydroxyvitamin D 3 (referred to as “l,25(OH) 2 D " in the figures).
• Figures 6-10 illustrate various biological activities of (20R)- 2-methylene-19-nor-22-dimethyl-l ,25-dihydroxyvitamin D 3 (referred to as "MET -2" in the figures) compared with those of the native hormone, l,25(OH) 2 D 3 .
• Fig. 1 shows a graph of competitive binding to the nuclear vitamin D hormone receptor between MET-1 and the native hormone, l,25(OH) 2 D 3 .
• MET-1 binds to the nuclear vitamin D hormone receptor with the same affinity as l,25(OH) 2 D 3 .
• Fig. 2 is a graph comparing the percent HL-60 cell differentiation as a function of the concentration of MET-1 with that of 1,25(0 H) 2 D 3 .
• MET-1 has the about four times the potency of l,25(OH) 2 D 3 in causing the differentiation of HL-60 cells into monocytes.
• Fig. 3 is a graph comparing the in vitro transcription activity of MET-1 with that of l,25(OH) 2 D 3 .
• MET-1 is about ten times more potent than l,25(OH) 2 D 3 in increasing transcription of the 24-hydroxylase gene.
• Fig. 4 is a bar graph comparing the bone calcium mobilization activity of
• MET-1 with that of l,25(OH) 2 D 3 in rat.
• MET-1 is approximately 20 times less potent than l,25(OH) 2 D 3 in releasing bone calcium stores.
• Fig. 5 is a bar graph comparing the intestinal calcium transport activity of
• Fig. 6 shows a graph of competitive binding to the nuclear vitamin D hormone receptor between MET-2 and the native hormone, l,25(OH) 2 D3.
• MET-2 binds to the nuclear vitamin D hormone receptor with the same affinity as l,25(OH) 2 D3.
• Fig. 7 is a graph comparing the percent HL-60 cell differentiation as a function of the concentration of MET-2 with that of 1,25(0 H) 2 D3.
• MET-2 has the about three times the potency of l,25(OH) 2 D3 in causing the differentiation of HL-60 cells into monocytes.
• Fig. 8 is a graph comparing the in vitro transcription activity of MET-2 with that of l,25(OH) 2 D3. MET-2 is about three times more potent than l,25(OH) 2 D3 in increasing transcription of the 24-hydroxylase gene.
• Fig. 9 is a bar graph comparing the bone calcium mobilization activity of
• Fig. 10 is a bar graph comparing the intestinal calcium transport activity of
• MET-2 with that of l,25(OH) 2 D 3 .
• MET-2 is less potent than l,25(OH) 2 D 3 in promoting active calcium transport in the rat gut.
• Yi and Y 2 are hydroxy-protecting groups such as silyl protecting groups.
• the t-butyldimethylsilyl (TBDMS) group is an example of a particularly useful hydroxy-protecting group.
• Phosphine oxide IV is a convenient reagent that may be prepared according to the procedures described by Sicinski et al, J. Med. Chem., 41, 4662 (1998), DeLuca et al., U.S. Patent No. 5,843,928; Perlman et al, Tetrahedron Lett. 32, 7663 (1991); and DeLuca et al, U.S. Patent No. 5,086,191.
• Scheme 1 shows the general procedure for synthesizing phosphine oxide IV as outlined in U.S. Patent No. 5,843,928 which is hereby incorporated by reference in its entirety as if fully set forth herein.
• hydroxy-protecting group signifies any group commonly used for the temporary protection of the hydroxy (-OH) functional group, such as, but not limited to, alkoxycarbonyl, acyl, alkylsilyl or alkylarylsilyl groups (hereinafter referred to simply as “silyl” groups), and alkoxyalkyl groups.
• Alkoxycarbonyl protecting groups are alkyl-O-CO- groups such as methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, isobutoxycarbonyl, tert-butoxycarbonyl,
• Preferred silyl-protecting groups are trimethylsilyl, triethylsilyl, t- butyldimethylsilyl, dibutylmethylsilyl, diphenylmethylsilyl, phenyldimethylsilyl, diphenyl-t- butylsilyl and analogous alkylated silyl radicals.
• aryl specifies a phenyl-, or an alkyl-, nitro- or halo-substituted phenyl group.
• An extensive list of protecting groups for the hydroxy functionality may be found in Protective Groups in Organic Synthesis, Greene, T.W.; Wuts, P. G. M., John Wiley & Sons, New York, NY, (3rd Edition, 1999) which can be added or removed using the procedures set forth therein and which is hereby incorporated by reference in its entirety and for all purposes as if fully set forth herein.
• 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 functional groups, e.g., the silyl, alkoxyalkyl, acyl or alkoxycarbonyl groups, as previously defined.
• these compounds are characterized by relatively high binding to vitamin D receptors (see Figs. 1 and 5) and high activity in HL-60 differentiation, but very low ability to mobilize calcium from bone (see Figs. 4 and 9) and relatively low intestinal calcium transport activity (see Figs. 5 and 10), as compared to that of l ,25-dihydroxyvitamin D 3 .
• these compounds can be characterized as having little, if any, calcemic activity at the dosages that l ,25-dihydroxyvitamin D 3 displays significant calcemic activity.
• the may be useful as a therapy for suppression of secondary hyperparathyroidism type renal osteodystrophy.
• the compounds of the present technology are also suited for treatment and prophylaxis of human disorders which are characterized by an imbalance in the immune system, e.g. in autoimmune diseases, including multiple sclerosis, lupus, diabetes mellitus, host versus graft reaction, and rejection of organ transplants; and additionally for the treatment of inflammatory diseases, such as rheumatoid arthritis, asthma, and inflammatory bowel diseases such as celiac disease, ulcerative colitis and Crohn's disease. Acne, alopecia and hypertension are other conditions which may be treated with the compounds of the present technology.
• autoimmune diseases including multiple sclerosis, lupus, diabetes mellitus, host versus graft reaction, and rejection of organ transplants
• inflammatory diseases such as rheumatoid arthritis, asthma, and inflammatory bowel diseases such as celiac disease, ulcerative colitis and Crohn's disease.
• Acne, alopecia and hypertension are other conditions which may be treated with the compounds of
• the above compounds are also characterized by cell differentiation activity either equal to or significantly higher than that of la,25(OH) 2 D 3 (see Figs. 2 and 7).
• this compound also provides a therapeutic agent for the treatment of psoriasis, or as an anticancer agent, especially against leukemia, colon cancer, breast cancer and prostate cancer.
• this compound due to its relatively high cell differentiation activity, provides a therapeutic agent for the treatment of various skin conditions including wrinkles, lack of adequate dermal hydration, i.e. dry skin, lack of adequate skin firmness, i.e. slack skin, and insufficient sebum secretion. Use of this compound thus not only results in moisturizing of skin but also improves the barrier function of skin.
• the compounds of the present technology may be used to prepare
• compositions or medicaments that include a compound of the present technology in combination with a pharmaceutically acceptable carrier.
• Such pharmaceutical formulations and medicaments may be used to treat various biological disorders such as those described herein.
• Methods for treating such disorders typically include administering an effective amount of the compound or an appropriate amount of a pharmaceutical formulation or a medicament that includes the compound to a subject suffering from the biological disorder.
• the subject is a mammal.
• the mammal is selected from a rodent, a primate, a bovine, an equine, a canine, a feline, an ursine, a porcine, a rabbit, or a guinea pig.
• the mammal is a rat or is a mouse.
• the subject is a primate such as, in some embodiments, a human.
• IIC may be formulated for 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 pharmaceutically acceptable and non-toxic excipients such as stabilizers, anti-oxidants, binders, coloring agents or emulsifying or taste-modifying agents. Pharmaceutically acceptable excipients and carriers are generally known to those skilled in the art and are thus included in the present technology. Such excipients and carriers are described, for example, in "Remingtons Pharmaceutical Sciences” Mack Pub. Co., New Jersey (1991), which is hereby incorporated by reference in its entirety and for all purposes as if fully set forth herein.
• the compounds may be administered orally, topically, parenterally, or transdermally.
• the compounds are 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.
• doses of from about 0.001 ⁇ g to about 1 mg per day of the compound are appropriate for treatment purposes.
• an appropriate and effective dose may range from about 0.01 ⁇ g to about 1 mg per day of the compound.
• an appropriate and effective dose may range from about 0.1 ⁇ g to about 500 ⁇ g per day of the compound.
• Such doses will be adjusted according to the type of disease or condition to be treated, the severity of the disease or condition, and the response of the subject as is well understood in the art.
• the compound may be suitably administered alone, or together with another active vitamin D compound.
• compositions for use in the present technology include an effective amount of
• an effective amount of a compound herein for use in accordance with some embodiments of the present technology will generally be a dosage amount such as those described herein, and may be administered topically, transdermally, orally, nasally, rectally, or parenterally.
• the compounds of formula IIA, IIB, IIC and IID 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.
• the compounds may be formulated as creams, lotions, ointments, aerosols, suppositories, topical patches, pills, capsules or tablets, 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, emulsifiers, coloring agents, binders or taste-modifying agents.
• other pharmaceutically innocuous or beneficial components such as stabilizers, antioxidants, emulsifiers, coloring agents, binders or taste-modifying agents.
• reaction mixture was stirred at -78 °C 1 h and then at room temperature for 1 h. It was quenched with saturated aqueous NH 4 C1 solution and extracted with ethyl acetate. Combined organic phases were washed with brine, dried (Na 2 S0 4 ) and concentrated. The residue was purified by column chromatography on silica gel (5%, then 10% ethyl acetate/hexane) to give the product 20 (0.197 g, 56% yield).
• Triethylsilyl trifluoromethanesulfonate (30 ⁇ , 35.1 mg, 0.133 mmol) was added dropwise to a solution of the ketone (29.2 mg, 0.095 mmol) and 2,6-lutidine (40 ⁇ , 37 mg, 0.343 mmol) in dichloromethane (1.6 mL) at -50 °C.
• the reaction mixture was stirred at -40 °C for 15 min, then it was diluted with dichloromethane and washed with water.
• the organic layer was dried (Na 2 S0 4 ) and concentrated. The residue was applied to a Waters silica Sep-Pak cartridge (5 g). Elution with hexane/ethyl acetate (1%, then 2%) gave the protected ketone 25 (34.4 mg, 86% yield).
• Phenyllithium (1.70 M in di-n-buthylether, 105 ⁇ , 0.178 mmol) was added to a stirred solution of the phosphine oxide H (73.6 mg, 0.126 mmol) in anhydrous THF (500 ⁇ ) at -30 °C. After 30 min the mixture was cooled to -78 °C and a precooled (-78 °C) solution of the ketone 25 (35 mg, 82.9 ⁇ ) in anhydrous THF (400 + 300 ⁇ ) was added. The reaction mixture was stirred under argon at -78 °C for 4 hours and then at +4 °C for 19 h.
• the protected compound 26 (50.94 mg, 64.8 mol) was dissolved in THF (5 mL) and acetonitrile (3 mL). A solution of aqueous 48% HF in acetonitrile (1 :9 ratio, 4 mL) was added at 0 °C and the resulting mixture was stirred at room temperature for 2 h. Saturated aqueous NaHC0 3 solution was added and the reaction mixture was extracted with dichloromethane. The combined organic phases were dried (Na 2 S0 4 ) and concentrated under reduced pressure. The residue was diluted with 2 mL of hexane/ethyl acetate (7:3) and applied to a Waters silica Sep-Pak cartridge (5 g).
• Codon Plus RIL cells and purified to homogeneity using two different column
• the first system was a nickel affinity resin that utilizes the C- terminal histidine tag on this protein.
• the protein that was eluted 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.
• the protein was diluted in TEDK 50 (50 mM Tris, 1.5 mM EDTA, pH 7.4, 5 mM DTT, 150 mM KC1) with 0.1% Chaps detergent.
• the receptor protein and ligand concentration was optimized such that no more than 20% of the added radiolabeled ligand was bound to the receptor.
• Radiolabeled and unlabeled ligands were added to 100 of the diluted protein at a final ethanol concentration of ⁇ 10%, mixed and incubated overnight on ice to reach binding equilibrium. The following day, 100 of hydroxyl apatite slurry (50%>) was added to each tube and mixed at 10-minute intervals for 30 minutes. The hydroxylapaptite was collected by centrifugation 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.
• Tris-EDTA buffer 50 mM Tris, 1.5 mM EDTA, pH 7.4
• the pellets were transferred to scintillation vials containing 4 mL of Biosafe II scintillation cocktail, mixed and placed in a scintillation counter. Total binding was determined from the tubes containing only radiolabeled ligand.
• the study drugs were dissolved in ethanol 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 ethanol ( ⁇ 0.2%) present in the cell cultures.
• HL60 Human promyelocytic leukemia (HL60) cells were grown in RPMI-1640 medium containing 10% fetal bovine serum. The cells were incubated at 37°C in the presence of 5%> C0 2 .
• HL60 cells were plated at 1.2 x 10 5 cells/mL. Eighteen hours after plating, cells in duplicate were treated with drug. Four days later, the cells were harvested and a nitro blue tetrazolium reduction assay was performed (Collins et al., J. Exp. Med. (1979) 149:969- 974). The percentage of differentiated cells was determined by counting a total of 200 cells and recording the number that contained intracellular black-blue formazan deposits.
• (20S)-2-methylene-19-nor-22-dimethyl-la,25-dihydroxyvitamin D 3 (MET-1) is approximately equally effective as l,25-(OH) 2 D 3 in binding to the recombinant vitamin D receptor as shown in Fig. 1 (K; of 4 x 10 "U M and 5 x 10 "11 M respectively).
• MET- 1 is 4 times more potent than l,25-(OH) 2 D 3 at inducing the differentiation of HL-60 cells in culture as shown in Fig. 2 (7 x 10 "10 M and 3 x 10 "9 M respectively).
• MET-1 is 10 times as potent in stimulating 24-OHase gene expression in bone cells than l,25-(OH) 2 D as shown in Fig.
• (20R)-2-methylene- 19-nor-22-dimethyl- 1 a,25-dihydroxyvitamin D 3 (MET-2) is equally effective as l,25-(OH) 2 D 3 in binding to the recombinant vitamin D receptor as shown in Fig. 6 (both display a K; of 5 x 10 "U M).
• MET-2 is 3 times more potent than 1,25- (OH) 2 D3 at inducing the differentiation of HL-60 cells in culture as shown in Fig. 7 (7 x 10 " 10 M and 3 x 10 "9 M respectively).
• MET-2 is about 3 times less potent in stimulating 24-OHase gene expression in bone cells than l,25-(OH) 2 D as shown in Fig.
• MET-2 is 16 times less active than l,25-(OH) 2 D3 on bone calcium mobilization (Fig. 4), and noticeably less active than l,25-(OH) 2 D3 in causing intestinal calcium transport (Fig. 5).
• Comparative Example Table 1 shows biological data for 2-methylene- 19- nor-l ,25-dihydroxyvitamin D 3 and its 20R isomer in comparison to compounds from the present disclosure (MET-1 and MET-2).
• the former compounds differ from the latter in that they have two methyl groups attached to the position 21 carbon rather than two hydrogens.
• binding of the vitamin D receptor and HL-50 differentiation is generally within an order of magnitude for each compound, the present compounds demonstrate surprising and unexpected effects on calcemic activity.
• the 2MD compounds show extremely potent net bone calcium mobilization activity ranging from 4.5 mg/dL in the 20R isomer to 9.3 mg/dL in the 20S isomer
• the present MET compounds show virtually no calcemic activity.

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