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  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61P19/00—Drugs for skeletal disorders
  • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
• • A—HUMAN NECESSITIES
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
  • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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  • A61P3/02—Nutrients, e.g. vitamins, minerals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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  • C07C35/00—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a ring other than a six-membered aromatic ring
  • C07C35/02—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a ring other than a six-membered aromatic ring monocyclic
  • C07C35/08—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a ring other than a six-membered aromatic ring monocyclic containing a six-membered rings
  • C07C35/18—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a ring other than a six-membered aromatic ring monocyclic containing a six-membered rings with unsaturation at least in the ring
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  • C07F7/00—Compounds containing elements of Groups 4 or 14 of the Periodic Table
  • C07F7/02—Silicon compounds
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  • C07F7/18—Compounds having one or more C—Si linkages as well as one or more C—O—Si linkages
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  • C07C2601/12—Systems containing only non-condensed rings with a six-membered ring
  • C07C2601/14—The ring being saturated
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  • C07—ORGANIC CHEMISTRY
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  • C07C2602/00—Systems containing two condensed rings
  • C07C2602/02—Systems containing two condensed rings the rings having only two atoms in common
  • C07C2602/14—All rings being cycloaliphatic
  • C07C2602/24—All rings being cycloaliphatic the ring system containing nine carbon atoms, e.g. perhydroindane
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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  • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
  • Y02P20/00—Technologies relating to chemical industry
  • Y02P20/50—Improvements relating to the production of bulk chemicals
  • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

• This invention relates to vitamin D compounds, and more particularly to 2-
• Methylene-Vitamin D analogs and their pharmaceutical uses and especially (20S)-25- hydroxy-2-methylene-vitamin D 3 , its biological activities, and its pharmaceutical uses, as well as (20R)-25-hydroxy-2-methylene-vitamin D 3 , its biological activities, and its pharmaceutical uses.
• This latter compound can also be named simply as 2-methylene-25- hydroxy-vitamin D 3 since the 20-methyl substituent is in its natural or "R" orientation.
• the natural hormone, l ,25-dihydroxyvitamin D3 and its analog in the ergosterol series, i.e. l a,25-dihydroxyvitamin D2 are known to be highly potent regulators of calcium homeostasis in animals and humans, and their activity in cellular differentiation has also been established, Ostrem et al, Proc. Natl. Acad. Sci. USA, 84, 2610 (1987). Many structural analogs of these metabolites have been prepared and tested, including loc- hydroxy vitamin D3, 1 a-hydroxy vitamin D2, various side chain homologated vitamins and fluorinated analogs. Some of these compounds exhibit an interesting separation of activities in cell differentiation and calcium regulation. This difference in activity may be useful in the treatment of a variety of diseases such as renal osteodystrophy, vitamin D-resistant rickets, osteoporosis, psoriasis, and certain malignancies.
• diseases such as renal osteodystrophy, vitamin D-resistant rickets, osteo
• 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 exocyclic 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-dihydroxy-l 9-nor-vitamin D3 revealed a selective activity profile with high potency in inducing cellular
• Patent 6,627,622 All three of these compounds have relatively high binding activity to vitamin D receptors and relatively high cell differentiation activity, but little if any calcemic activity as compared to la,25-dihydroxyvitamin D 3 . Their biological activities make these compounds excellent candidates for a variety of pharmaceutical uses, as set forth in the '352, '861 and '622 patents.
• the present invention is aimed at vitamin D compounds characterized by the presence of an A-ring exocyclic methylene group at carbon 2 (C-2) (e.g., 2-methylene-vitamin D analogs). Although these analogs lack la-OH, that is important for biological activity, such hydroxyl group can be potentially introduced enzymatically in the living organisms.
• the present invention is aimed at vitamin D compounds characterized by not only having the A-ring exocyclic methylene group at carbon 10 (C-10), but also by the presence of an additional exomethylene substituent at carbon 2 (C-2) (i.e., 2-methylene- vitamin D analogs). These analogs also lack a la-OH group, and thus the present invention is directed toward 2-methylene-vitamin D analogs, and their pharmaceutical uses, and more specifically toward (20S)-25-hydroxy-2-methylene-vitamin D 3 , its biological activity, and various pharmaceutical uses for this compound, as well as (20R)-25-hydroxy-2-methylene- vitamin D 3 , its biological activities, and its pharmaceutical uses.
• R may be an alkyl, hydrogen, hydroxyalkyl or fluoroalkyl group, or R may represent a side chain of the formula:
• stereochemical center at carbon 20 may have the R or S configuration
• Y is selected from hydrogen, methyl, -COR 5 and a radical of the structure: 1 ⁇ R 2 R 3
• R 1 is selected from hydrogen, deuterium, hydroxy, protected hydroxy, fluoro, trifluoromethyl, and Ci-5-alkyl, which may be straight chain or branched and, optionally, bear a hydroxy or protected-
• each of R , R , and R independently, is selected from deuterium, deuteroalkyl, hydrogen, fluoro, trifluoromethyl and C 1-5 alkyl, which may be straight-chain or branched, and optionally, bear a hydroxy or protected-hydroxy substituent,
• side chains are the structures represented by formulas (a), (b), (c), (d) and (e) below with natural 20R-configuration, i.e., the side chain as it occurs in 25 -hydroxy vitamin D 3 (a); vitamin D 3 (b); 25 -hydroxy vitamin D 2 (c); vitamin D 2 (d); and the C-24 epimer of 25 -hydroxy vitamin D 2 (e).
• 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-hydroxyvitamin D 3 (a); (20S)-vitamin D 3 (b); (20S)-25- hydroxyvitamin D 2 (c); (20S)-vitamin D 2 (d); and the C-24 epimer of (20S)-25- hydroxy vitamin D 2 (e).
• carbon 20 may have either the R or S configuration.
• Compound la may also be named "(20S)-2-methylene-25-hydroxy-vitamin
• Compound lb may also be named "2-methylene-25 -hydroxy- vitamin D 3 " herein.
• the above compounds of formula I exhibit a desired, and highly advantageous, pattern of biological activity. These compounds are characterized by relatively high binding to vitamin D receptors, i.e. they bind with about the same affinity as la.25-dihydroxyvitamin D They are only slightly less potent causing differentiation of HL-60 cells than l,25(OH) 2 D 3 . They also exhibit relatively high activity in their ability to mobilize calcium from bone, and in their ability to promote intestinal calcium transport, as compared to l ,25-dihydroxyvitamin D 3 .
• One or more of the compounds may be present in a composition to treat or prevent the above-noted diseases in an amount from about 0.0 ⁇ g/gm to about 1000 ⁇ g/gm of the composition, preferably from about 0. ⁇ g/gm to about 500 ⁇ g/gm of the composition, and may be administered topically, transdermally, orally, rectally, nasally, sublingually, or parenteraliy in dosages of from about 0.0 ⁇ g/day to about 1000 ⁇ g/day, preferably from about O. ⁇ ig/day to about 500 ⁇ g/day.
• Figures 1 -4 illustrate various biological activities of (20S)-25-hydroxy-2- methylene-vitamin D 3 , hereinafter referred to as "ID-QMS,” as compared to the native hormone l ,25-dihydroxyvitamin D 3 , hereinafter "l,25(OH) 2 D 3 .”
• Figure 1 is a graph illustrating the relative activity of ID-QMS and l ,25(OH) 2 D 3 to compete for binding with [ H]-l ,25-(OH) 2 D 3 to the full-length recombinant rat vitamin D receptor;
• Figure 2 is a graph illustrating the percent HL-60 cell differentiation as a function of the concentration of ID-QMS and l ,25(OH) 2 D 3 ;
• Figure 3 is a bar graph illustrating the bone calcium mobilization activity of l ,25(OH) 2 D 3 as compared to ID-QMS;
• Figures 4 is a bar graph illustrating the intestinal calcium transport activity of l ,25(OH) 2 D 3 as compared to ID-QMS.
• Figures 5-8 illustrate various biological activities of (20R)-25-hydroxy-2- methylene-vitamin D 3 , hereinafter referred to as " 1D-QM,” as compared to the native hormone la,25-dihydroxyvitamin D 3 , hereinafter " l ,25(OH) 2 D 3 .”
• Figure 5 is a graph illustrating the relative activity of 1D-QM and l ,25(OH) 2 D 3 to compete for binding with [ 3 H]-l ,25-(OH)2-D 3 to the full-length recombinant rat vitamin D receptor;
• Figure 6 is a graph illustrating the percent HL-60 cell differentiation as a function of the concentration of 1D-QM and l ,25(OH) 2 D 3 ;
• Figure 7 is a bar graph illustrating the bone calcium mobilization activity of l ,25(OH) 2 D 3 as compared to 1D-QM;
• Figure 8 is a bar graph illustrating the intestinal calcium transport activity of l ,25(OH) 2 D 3 as compared to 1D-QM.
• hydroxy-protecting group signifies any group commonly used for the temporary protection of hydroxy functions, such as for example, alkoxycarbonyl, acyl, alkylsilyl or alkylarylsilyl groups (hereinafter referred to simply as “silyl” groups), and alkoxyalkyl groups.
• Alkoxycarbonyl protecting groups are alkyl-O-CO- groupings such as methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, isobutoxycarbonyl, tert- butoxycarbonyl, benzyloxycarbonyl or allyloxycarbonyl.
• acyl signifies an alkanoyl group of 1 to 6 carbons, in all of its isomeric forms, or a carboxyalkanoyl group of 1 to 6 carbons, such as an oxalyl, malonyl, succinyl, glutaryl group, or an aromatic acyl group such as benzoyl, or a halo, nitro or alkyl substituted benzoyl group.
• alkyl as used in the description or the claims, denotes a straight-chain or branched alkyl radical of 1 to 10 carbons, in all its isomeric forms.
• Alkoxy refers to any alkyl radical which is attached by oxygen, i.e.
• Alkoxyalkyl protecting groups are groupings such as methoxymethyl, ethoxymethyl, methoxyethoxymethyl, or tetrahydrofuranyl and tetrahydropyranyl.
• 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.
• a "protected hydroxy” group is a hydroxy group derivatised 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.
• hydroxyalkyl deuteroalkyl
• fluoroalkyl refer to an alkyl radical substituted by one or more hydroxy, deuterium or fluoro groups respectively.
• An “alkylidene” refers to a radical having the general formula C k H 2k - where k is an integer.
• group X represents a leaving group selected from halogen (iodine, bromine or chlorine) and alkyl- or aryl-sulphonyloxy such as mesyloxy, tosyloxy or - most preferably - trifloxy.
• group Y and R represent groups defined above; Y being preferably hydroxy-protecting group, it being also understood that any functionalities in R that might be sensitive, or that interfere with the coupling reaction, be suitable protected as is well-known in the art.
• Bicyclic compounds of the general structure II are known, or can be easily prepared by known methods from the corresponding Windaus-Grundmann type ketones.
• Specific important examples of such known bicyclic ketones are the structures with the side chains (h), (i), (j), (k), (1), (m), and (n) below described above, i.e., 25 -hydroxy Grundmann's ketone (h) [Baggiolini et al., J. Org. Chem., 51 , 3098 (1986)] ; Grundmann's ketone (i) [Inhoffen et al., Chem.
• the introduced secondary hydroxyl in the product 6a was silylated and the protected compound 7 was subjected to the Wittig reaction with an ylide generated from methyltriphenylphosphonium bromide and /7-butyllithium.
• the pivaloyl protecting group in the formed olefin 8 was removed by treatment with DIBAL and the obtained cyclohexanol derivative 9 was oxidized to the ketone 10.
• Its reaction with lithium acetylide provided tertiary alcohol 11 which was dehydrated in two-step process (mesylation and reduction of the mesylate).
• the desired A-ring fragment 13 was prepared.
• SCHEME II shows the subsequent Sonogashira coupling of the obtained A- ring dienyne 13 with an enol inflate 14 [ Sanchez- Abella et al., Bioorg. Med. Chem. 16, 10244 (2008)], representing C,D-fragment derived from the protected 25-hydroxy Grundmann's ketone.
• the reaction should be preferentially carried out in the presence of bis(triphenylphosphine)palladium (II) acetate-copper (I) iodide catalyst and diethylamine.
• the coupling resulted in formation of the trienyne 15 which was further hydrogenated in the presence of Lindlar catalyst and quinoline.
• the protected vitamin D compound 17 was isolated by HPLC, and after hydroxyls deprotection with tetrabutylammonium fluoride provided the desired 25-hydroxy-2-methylene-vitamin D 3 (18). This synthetic path is described in EXAMPLE I herein.
• SCHEME III and SCHEME IV show a different synthetic sequence leading to the building block 26 and to the final vitamin 32.
• the hydroxy ester 20 easily prepared from the commercially available (-)-quinic acid (19) by the method elaborated by Sibilska et al. [J. Steroid Biochem. Mol. Biol., 121, 51 (2010)], was used as a starting compound and the synthetic strategy was based on the work of Desmaele and Tanier [Tetrahedron Lett., 26, 4941 (1985)].
• the tertiary alcohol was dehydrated and the formed ⁇ , ⁇ -unsaturated ester 21 subjected to reaction with diazomethane.
• Such stereospecific 1 ,3-dipolar cycloaddition provided the expected bicyclic product 22.
• the subsequent thermolysis of the adduct 22 lead to efficient extrusion of nitrogen and formation of unsaturated ester 23.
• Its reduction with DIBALH furnished the allylic alcohol 24 that was oxidized with PDC to the ⁇ , ⁇ -unsaturated aldehyde 25.
• SCHEME IV shows a preparation of the enol trifiate 28, representing a C,D- fragment, from the protected (20S)-25-hydroxy Grundmann's ketone 27 [Sicinski et al., J. Med. Chem., 4J_, 4662 (1998)].
• the subsequent Sonogashira coupling of the obtained A-ring dienyne 26 with an enol triflate 28 resulted in formation of the trienyne 29 which was further hydrogenated in the presence of Lindlar catalyst and quinoline.
• previtamin D compound 30 The expected product of such catalytic hydrogenation, previtamin D compound 30, was purified by preparative TLC and subjected to the thermal reaction in hexane.
• the protected vitamin D compound 31 was isolated by HPLC, and after hydroxyls deprotection with tetrabutylammonium fluoride provided the desired (20S)-25-hydroxy-2-methylene- vitamin D 3 (32). This synthetic path is described in EXAMPLE II herein.
• Butyldiphenylsilyl)oxy]-5-methyl-4-pivaloyloxy-cyclohexanone (7) was added to a solution of a-hydroxy ketone 6a (765 mg, 3.6 mmol) and silver nitrate (1.72 g, 10.14 mmol) in anhydrous DMF (16 mL) under argon at room temperature; white precipitate formed immediately. Reaction was stirred for 17 h and then it was quenched by the addition of water. The mixture was extracted with hexane, dried over MgS0 4 , and concentrated. Purification by column chromatography on silica (1% ⁇ 4% diethyl ether/hexane) gave protected ⁇ -hydroxy ketone 7 (1.2 g, 97%).
• methyltriphenylphosphonium bromide 122 mg, 342 ⁇
• o-BuLi 1.6 M in hexanes
• 431 ⁇ iL, 689.5 ⁇ After 15 min another portion of phosphonium salt (122 mg, 342 ⁇ ) was added, and the solution was stirred at 0 °C for 10 min, and at room temperature for 20 min.
• a solution of 77-BuLi (1.6 M in hexanes, 326 ⁇ ,, 522 ⁇ ) was added dropwise to a solution of trimethylsilylacetylene (76 ⁇ ,, 537 ⁇ ) in anhydrous THF (2 mL) under argon at 0 °C. The solution was stirred for 30 min and cooled to -78 °C, then precooled (-78 °C) solution of ketone 10 (158 mg, 417.3 ⁇ ) in dry THF (2 mL) was slowly added.
• Butyldiphenylsilyl)oxy]-2-methylene-25-[(triethylsilyl)oxy]-9,10-secocholesta-5(10),8-dien- 6-yne (15).
• acetylene 13 47 mg, 121.6 ⁇ ⁇ ⁇
• triflate 14 50 mg, 95 ⁇
• Cul 2.7 mg, 14.2 ⁇
• Pd(OAc) 2 2.0 mg, 2.7 ⁇
• Et 2 NH 945 ⁇
• Silylated previtamin was then dissolved in anhydrous hexane (6 mL) and stirred at 65 °C for 5 h and at 40 °C overnight under argon. Solvent was evaporated and residue was purified by HPLC (9.4 mm x 25 cm Zorbax-Sil column, 4 mL/min) using hexane/ethyl acetate (99: 1) solvent system. Pure protected vitamin 17 (9.7 mg, 76%) was eluted at Rv 15.5 mL.
• Butyldimethylsilyl)oxy]-4-methylene-cyclohex-l-enecarboxylic acid methyl ester (21)
• solution of alcohol 20 (162.5 mg, 540.8 ⁇ ) in anhydrous carbon tetrachloride (5.1 mL) at room temperature under argon was added solution of [ ⁇ , ⁇ - bis(trifluoromethyl)benzenemethanolato]diphenylsulfur (545.6 mg, 81 1 .2 ⁇ ) in anhydrous carbon tetrachloride (1.8 mL). Reaction was stirred for 2 h and water was added. The mixture was extracted with methylene chloride, dried over Na 2 S0 4 and concentrated.
• Solution of diazomethane in diethyl ether [5.5 mL; prepared according to the procedure of Arndt, Org. Synth., 15, 3 and 48 (1935)] was added to the solution of unsaturated ester 21 (375 mg, 1.327 mmol) in anhydrous diethyl ether (2 mL) at room temperature. Reaction mixture was protected from light and stirred overnight.
• Butyldimethylsilyl)oxy]- l -ethynyl-2-methyl-4-methylene-cyclohexene (26). «-BuLi (1.6 M in hexanes; 101 iL, 161.71 ⁇ ) was added to a solution of (trimethylsilyl)diazomethane (2.0 M in hexane, 76 151.52 ⁇ ) in anhydrous THF (150 ⁇ ,) at -78 °C under argon, and a solution of aldehyde 25 (33.5 mg, 125.73 ⁇ ) in dry THF (100 +100 ⁇ ,) was added via cannula. After 1 h the cooling bath was removed and stirring was continued at room temperature overnight.
• Butyldimethylsilyl)oxy]-2-methylene-25-[(triethylsilyl)oxy]-9,10-secocholesta-5(10),8-dien- 6-yne 29.
• dienyne 26 6.1 mg, 22.9 ⁇
• triflate 28 8.9 mg, 16.89 ⁇
• Cul 0.507 mg, 2.66 ⁇
• Pd(OAc) 2 0.38 mg, 0.507 ⁇
• Et?NH 185 ⁇ ,
• FIG. 4 shows that ID-QMS has relatively high activity as compared to that of 1 , 25 -dihydroxy vitamin D 3 (l ,25(OH) 2 D 3 ), the natural hormone, in stimulating intestinal calcium transport.
• ID-QMS is about equivalent to l ,25(OH) 2 D 3 in promoting active calcium transport across the gut.
• Figure 3 demonstrates that ID-QMS has relatively high bone calcium mobilization activity, as compared to l ,25(OH) 2 D3. ID-QMS has about the same potency as the native hormone in releasing bone calcium stores as similar activity is observed when 780 pmol/rat is administered.
• Figures 3-4 thus illustrate that ID-QMS may be characterized as having relatively high calcemic activity.
• Figure 2 illustrates that ID-QMS is almost as potent as l ,25(OH) 2 D 3 on HL- 60 cell differentiation, making it an excellent candidate for the treatment of a cancer, especially for the prevention or treatment of osteosarcoma, leukemia, colon cancer, breast cancer, skin cancer and prostate cancer.
• Codon Plus RIL cells and purified to homogeneity using two different column chromatography systems.
• 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
• the protein was diluted in TEDK 50 (50 mM Tris, 1.5 mM EDTA, pH7.4, 5 niM DTT, 150 mM KCI) 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 mcl 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 mcl of hydroxylapatite 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.
• 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 3 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., 1979; J. Exp. Med. 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. Verification of differentiation to monocytic cells was determined by measuring phagocytic activity (data not shown). In vitro Transcription Assay
• ID-QMS will have significant activity as an anti-cancer agent, especially for preventing or treating osteosarcoma, leukemia, colon cancer, breast cancer, skin cancer and prostate cancer because it has direct cellular activity in causing cell differentiation and in suppressing cell growth.
• ID-QMS D-deficient animals. Using vitamin D-deficient rats on a low calcium diet (0.02%), the activities of ID-QMS and l ,25(OH) 2 D 3 in intestine and bone were tested. As expected, the native hormone (l ,25(OH) 2 D 3 ) increased serum calcium levels at the dosages tested ( Figure 3). Figure 3 also shows that ID-QMS has about the same activity in mobilizing calcium from bone as l ,25(OH) 2 D 3 . Administration of ID-QMS at 780 pmol/day for 4 consecutive days resulted in significant mobilization of bone calcium. ID-QMS is equivalent to the native hormone in releasing bone calcium stores as significant increases in serum calcium are observed at 780 pmol when both ID-QMS and the native hormone are given.
• ID-QMS is an excellent candidate for numerous human therapies as described herein.
• ID-QMS is an excellent candidate for treating a cancer because: (1) it has significant VD binding, transcription activity and cellular differentiation activity; and (2) it is easily synthesized. BIOLOGICAL ACTIVITY OF
• Figure 8 shows that ID-QM has relatively high activity as compared to that of 1 ,25 -dihydroxy vitamin D 3 (l ,25(OH) 2 D 3 ), the natural hormone, in stimulating intestinal calcium transport.
• ID-QM is about equivalent to l,25(OH) 2 D 3 in promoting active calcium transport across the gut.
• Figure 7 demonstrates that ID-QM has relatively low bone calcium mobilization activity, as compared to l ,25(OH) 2 D 3 .
• ID-QM is less potent than the native hormone in releasing bone calcium stores as little to no activity is observed until 780 pmol/rat is administered; whereas, significant increases in serum calcium are observed at 87 pmol when the native hormone is given.
• Figure 6 illustrates that ID-QM is almost as potent as l ,25(OH) 2 D 3 on HL-60 cell differentiation, making it an excellent candidate for the treatment of a cancer, especially for the prevention or treatment of osteosarcoma, leukemia, colon cancer, breast cancer, skin cancer and prostate cancer.
• 1D-QM will have significant activity as an anti-cancer agent, especially for preventing or treating osteosarcoma, leukemia, colon cancer, breast cancer, skin cancer and prostate cancer because it has direct cellular activity in causing cell differentiation and in suppressing cell growth.
• the compounds of this invention defined by formula I, la and lb 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.
• the compounds of formula I and particularly ID-QMS of formula la, and 1D-QM of formula lb may be administered orally, topically, parenterally, rectally, 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 0.01 ⁇ g to 1000 ⁇ g per day of the compounds I, particularly ID-QMS, and 1D-QM, preferably from about 0.1 ⁇ ig to about 500 ⁇ ig 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. 1 a-hydroxyvitamin D2 or D3, or la,25-dihydroxy vitamin D3 — in situations where different degrees of bone mineral mobilization and calcium transport stimulation is found to be advantageous.
• another active vitamin D compound ⁇ e.g. 1 a-hydroxyvitamin D2 or D3, or la,25-dihydroxy vitamin D3 — in situations where different degrees of bone mineral mobilization and calcium transport stimulation is found to be advantageous.
• compositions for use in the above-mentioned treatments comprise an effective amount of the compounds I, particularly ID-QMS and 1D-QM, as defined by the above formula I, la and lb 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 ⁇ g to about 1000 ⁇ g per gm of composition, preferably from about 0.1 ⁇ g to about 500 ⁇ g per gram of composition, and may be administered topically, transdermally, orally, rectally, nasally, sublingually or parenterally in dosages of from about 0.0 ⁇ g/day to about 1000 ⁇ g /day, and preferably from about 0.1 ⁇ g/day to about 500 ⁇ g/day.
• the compounds I 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 imiocuous 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.
• the compounds I 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 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.
• 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 solution or a suspension in an aqueous liquid or non-aqueous liquid; or in the form of an oil-in-water emulsion or a water-in-oil emulsion.
• 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.
• 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.
• Formulations suitable for topical administration include liquid or semi-liquid preparations such as liniments, lotions, applicants, oil-in-water or water-in-oil emulsions such as creams, ointments or pastes; or solutions or suspensions such as drops; or as sprays.
• a nebulizer or an atomizer can be used for nasal administration, inhalation of powder, self-propelling or spray formulations.
• a nebulizer or an atomizer can be used for nasal administration, inhalation of powder, self-propelling or spray formulations.
• the formulations, when dispensed, preferably have a particle size in the range of 10 to 100 ⁇ .
• formulations may conveniently be presented in dosage unit form and may be prepared by any of the methods well known in the art of pharmacy.
• dosage unit is meant a unitary, i.e. a single dose which is capable of being

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• 2012
  • 2012-10-09 JP JP2014537107A patent/JP6072811B2/ja not_active Expired - Fee Related
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