GB2158072A

GB2158072A - 23,23-Difluoro steroids

Info

Publication number: GB2158072A
Application number: GB08513212A
Authority: GB
Inventors: Hector F Deluca; Yoko Tanaka; Nobuo Ikekawa; Yoshiro Kobayashi
Original assignee: Wisconsin Alumni Research Foundation
Current assignee: Wisconsin Alumni Research Foundation
Priority date: 1983-08-18
Filing date: 1985-05-24
Publication date: 1985-11-06
Also published as: GB8513212D0; GB8420957D0; GB2158072B; FR2550789A1; GB2145091B; GB2145091A; IT8422350A0; IT1175607B; DE3430390A1; FR2550789B1

Abstract

New 23,23-difluoro steroids of formulae I and II are used as intermediates in the synthesis of 23,23-difluoro-25-hydroxy- and 1 alpha ,25-dihydroxy vitamin D3 <IMAGE> R1 is H, acyl or tetrahydropyraryl; R2 and R3 are each CH3CH2OH or CO2 alkyl; X is halogen, OH or Oacyl.

Description

1

SPECIFICATION

23,23-Difluoro-25-hydroxy-and lct,25-dihydroxy- vitamin D.

GB 2 158 072 A 1 This invention relates to derivatives of vitamin 1), Vitamin D3 is a well-known agent for the control of calcium and phosphorus homeostatis. In the normal animal or human this compound is known to stimulate intestinal calcium transport and bone-calcium mobilization and is effective in preventing rickets.

It is also now well-known that to be effective, vitamin D, must be converted in vivo to its hydroxylated forms. For example, the vitamin is first hydroxylated in the liver to form 25-hydroxy-vitamin D, and is 10 further hydroxylated in the kidney to produce lo-,25- hydroxy-vitamin D, of 24,25-dihydroxy-vitamin D3. The lhydroxylated form of the vitamin is generally considered to be the physiologically active or hormonal form of the vitamin and to be responsible for what are termed the vitamin D-like activities, such as increasing intestinal absorption of calcium and phosphate, mobilizing bone mineral, and causing reab- sorption of calcium in the kidneys.

Since the discovery of biologically active metabolites of vitamin D there has been much interest in the preparation of structural analogs thereof because such compounds may represent useful therapeutic agents for the treatment of diseases resulting from calcium metabolism disorders. It is generally ac cepted that 1(x,25-dihydroxycholecalci- ferol is the circulating hormonal form of vitamin D.

New derivatives of vitamin D have now been found which are at least as potent as 25-hydroxyvitamin 20 D, as measured by their ability to stimulate calcium transport in the intestine or its ability to mobilize calcium from bone. These derivatives which form the subject of the present invention are 23,23-difluoro - 25-hydroxycholecalciferol (23,23-difluoro-25-hydroxy vitamin D, or 23,23-F,- 25(0H)D,,) and 23,23-difluoro- lu,25-dihydroxycholecalciferol (23,23-difluoro-1,25-dihydroxy vitamin D,, or 23,23-F,-1,25(0H),D,), and acylates thereof.

A major pathway for inactivation of vitamin D is believed to be 23Shydroxylation of 25-hydroxy vita min D, (see Biochemistry 20, 3875-3879, 1981) and its subsequent conversion to 25R-hydroxy-26,23S-lac- tone (see Proc. Nat'l. Acad. Sci. USA 78, 4805-4808, 1981). Accordingly it will appear that the derivatives 30 of the present invention, because of the fluorine substituents at C-23, would not be readily hydroxylated at that carbon and that, therefore, they would be characterized by prolonged vitamin D-like activity - an obvious advantage in many therapeutic applications.

23,23-Difluoro-25-hydroxyvitamin D can be obtained in accordance with the process hereinafter de scribed and shown in the accompanying schematic wherein like numbers refer to like compounds.

The steroid-l-ether (1) is oxidized to the C-22 aldehyde by pyridium chlorochromate or other suitable alcohol oxidizing reagent. This C-22 aldehyde is converted to a silyl ether carboxy ester (3) by a Wittig type condensation. Hydrolysis in acetic acid and TsOH afford the corresponding C-23ot -keto methyl ester and converts the 1 ether to the 3 acetoxy function (4). The ketone is fluorinated with DAST (diethyl amino sulphur trifluoride) to provide the C-23 difluoro carboxymethyl-3 acetate. Hydrolysis of the acetoxy group 40 and treatment with 2,3-dihydropyran and TsOH gave the 3-THP protected difluoro 24 ester. Reduction with lithium aluminium hydride produced the C-24 alcohol (7). Treatment of this alcohol with a mixture of trifluoromethanesulphonic anhydride and pyridine provides the trifluoromethanesulphonyl ester which undergoes a malonic ester condensation to yield the C-26,27 diethyl ester (9). Nchlorosuccinimide is used to convert (9) to the C-25 chloro derivative (10) which, upon reduction with lithium aluminium hy- 45 dride, affords the C-26,27 chlorodialcohol (11). Treatment of that chlorodiol with sodium hydride in dime thoxyethane yields the 25,26 epoxy 26-alcohol (12). Treatment with methanesulphonyl chloride and triethylamine provided the mesylate which upon reduction with lithium aluminium hydride yielded 23,23 difluoro, 25-hydroxyl - 31 -tetra hydropyranyl cholesterol (13) which was converted to the acetate (14). This compound was then converted to the 5,7- diene by the usual allylic bromination followed by dehydrobromination in collidine. This diene was then photolyzed to provide the corresponding previtamin which during the temperatures of work up results in the 23,23-difluoro-25- hydroxyvitamin D, (16).

2 GB 2 158 072 A C"20H 2 CHO coo me c,'S --- 1 - > - c,'S ZEL3 > or-be 10 COOMie cHIR 15 0 > F F FF 10. C.0 5 3-pc- fl R: OH 20 C. 3-THP 8R = 050.2CF= a COOE-L r WEt cii N c: F il ---> a F FC0'1 F F 1 30 C-OCCo-.I 35 1 -F F ".r ab 0 m '^FXF OH rF on rt, ( ?12 40 12 1:5 3-THP 14 3 - A ci, Ii cn, ' ' so 1 1 95 & #h 55 60 65 3 GB 2 158 072 A 3 23,23-Difluoro-lot,25-dihydroxy-vitamin D, can be readily prepared from 23,23-difluoro-25-hydroxy- vitamin D, by in vitro enzymatic hydroxylation of the latter compound at carbon 1, for example by incubation with a homogenate prepared from kidney tissue of vitamin D-deficient chickens, as follows:

5 \YOH \OH F F 10 15 HO HO OH 20 The acylates i.e. where one or more of the hydroxy groups in the 1 (if present), 3 an 25 positions is 0 aliphatic acyl of 1 to 4 carbon atoms, such as 0- acetyl, 0-propionyl or O-butyryl, or 0-benzoyl, can readily be obtained from the free vitamins by treatment with the appropriate acid chloride or anhydride, typically in the presence of pyridine, from ambient temperature to reflux. For example, treatment of the free vita- 25 min (1 mg) with acetic anhydride (0.1 ml) in pyridine (0.1 ml) at ambient temperature for 1.5 hours yields the corresponding 1,3-diacetoxy derivative. The corresponding 1,3,25- triacetoxy derivative can be readily obtained by utilizing the same reagents at elevated temperatures, e.g. 75'C to 90'C. Similarly, the corre sponding benzoate compound can be prepared by reaction of the free vitamin with benzoyl chloride in pyridine at room temperature for 3 hours.

The present invention also provides the compounds of the formula:

F R 2 R 40 R 1 0 wherein R, represents acyl or tetra hyd ro-pyra nyl 45 X represents halogen or hydroxy and R, and R, independently represent COOR4, CH,OH or hydrogen where R4 is lower alkyl and of the formula R 2 50 F F X R 3 55 j ' 1 R 1 0 60 wherein R, represent hydrogen or acyl, X represents hydroxyl or 0-acyl and R, and R3 are as defined above.

The various physico-chemical characteristics of the compounds in the following synthesis were deter65 mined as follows:

4 GB 2 158 072 A 4 Melting points were determined on a hot stage microscope and were uncorrected. UV spectra were obtained in ethanol solution with a Shimadzu UV-200 double beam spectrophotometer. IR spectra were taken with a JEOL IRA-1 diffraction grating infrared s peGtro photometer. 1H-NMR spectra were recorded on a Varian EM-360L spectrometer in CDCI, unless otherwise stated, with tetra methylsil ane as an internal reference. 19F- NMR spectra were recorded on a Varian EM-360L spectrometer in CDCI,-solution, with benzotrifluoride as an internal reference (a plus means high field). Mass spectra were obtained with a HITACHI double focusing mass spectrometer RMU-7L. Column chromatography was effected with silican gel (Merck, 70-23 mesh). Preparative thin layer chromatography was carried out on precoated plates of silica gel (Merck, silica gel 60 F...). The "usual work-up" refers to dilution with water, extraction with an organic solvent, washing to neutrality, drying over magnesium sulfate, filtration, and removal of the sol- 10 vent under reduced pressure. The following abbreviations were used: THF- tetrahydrofuran; ether- diethyl ether; HMPA-hexamethylphosphoramide; TsOH- p-toluenesulfonic acid; THP-tetrahydropyranyl; s-singlet; d-doublet; t- triplet; q-quartet; m-multiplet; bs-broaden singlet.

Synthesis 6PMethyoxy-3a,5-cyclo-23,24-dinor-5a--cholan 22-al (2) 6p ethoxy-3ot-5-eyclo-23,24-dinor-5(x-cholan-22oI (1) (2.0 g, 15.8 mmol), which was prepared according to Helvetica Chimica Acta, 57, FASC 3, pp. 764- 771 (1974), was added to a suspension of pyridinium chlorochromate (3.8 g) and sodium acetate (1.4 g) in dichloromethane (40 m]); this mixture was stirred at room temperature for 2.5 hr.

Then, to this solution ether (100 mi) was added and the resultant precipitates were filtered off and washed with ether (100 mi). The combined filtrate was successively washed with 5% NaHCO, and brine, and dried over magnesium sulfate. After removal of the solvent in vacuo, the residue was applied to a column of silica gel (300 g). Elution with nhexane-ether (10:1) provided the aldehyde (2) (1.44 g, 73%), amorphous. WNMR 8: 0.76 (314, s, 18-H,), 1.30 (3H, d, J= 6Hz, 21-HJ, 1.17 (3H, s, 19-HA, 2.76 (1H, m, 6-H), 3.33 (3H, s, -OCHJ, 9.51 (1 H, d, J =3. 5Hz, -CHO). MS mlz: 344 (M-), 329, 312.

60-Methoxy-23-triethylsilyloxy-31,5-cyclo-5.- cholan-22-en-24-oic Acid Methyl Ester (3) To a solution of diisopropylamine (1.05 mi, 7.5 mmol) in THF (10 m]) n- butyllithium (6 mmol) was added at -78' C under argon atmosphere and this solution was stirred for 5 min. To this solution methyl 30 (x- triethylsi lyl oxy-u--d im ethyl p hosph o nbacetate (1.56 g, 5 mmol) in THF (10 mi) was added and this mix ture was stirred at room temperature for 15 min. Then, to the resulting solution the aldehyde (2) (491 mg, 1.43 mmol) in THF (10 mi) was added and this mixture was stirred at room temperature for 4 hr. The usual work-up (ether for extraction) gave a crude product, which was applied to a column of silica gel (150 g). Elution with n-hexane-ether (15: 1) provided the unsaturated ester (3) (615 mg, 81%), colorless 35 oil. WNMR 8: 3.30 (3H, s, -OCHJ, 3.73 (3H, s, -C02CHJ, 5.26 (1 H, d, J= 10Hz, 22-H). MS mIz: 530 (M,), 501,469.

3p-Acetoxy-23-oxochol5-en-24-oic Aci Methyl Ester (4) A solution of the unsaturated ester (3) (1.53 9, 2.9 mmol) in acetic acid (7 mi) was heated at 80-90'C for 40 6 hr. The usual work-up (ether for extraction) gave a crude product. This and a catalytic amount of TsOH in dioxane (10 mO and water (10 mi) were heated at 85-95'C for 7 hr. The usual work-up (ether for extrac tion) gave a crude product, which was applied to a column of silica gel (300 g). Elution with n- hexane ether (15: 1) provided the a-keto ester (4) (768 mg, 76%) mp, 146-147'C (n-hexane).]R yKBr cm-l: 1720, Max 1240. 'H-NMR 8: 0.73 (3H, s, 18-H,). 0.93 (3H, d, J=61-1z, 21-H,), 1.03 (3H s, 19-H,), 2.03 (3H, s, acetyl), 3.88 45 (3H S, -C02CHJ, 4.63 (1H, m, 3-H), 5.41 (1H, m, 6-H). MS mIz: 384 (M-CH. COOH), 369. Anal. Calcd for C,,H,,,0,: C, 72.92; H, 9.08. Found: C, 72.63; H, 9.13.

3p-Acetoxy-23,23-difluorochol-5-en24-oic Acid Methyl Ester (5) A mixture of a-ketoester (4) (400 mg, 0.9 mmol) and diethylaminosuifurtrifluoride (1.5 m]. 9.5 mmol) in 50 dichloromethane (15 mi) was stirred at room temperature for 16 hr. The usual work-up (ether for extrac tion) gave a crude product, which was applied to a column of silica gel (100 g). Elution was n-hexane ether (10: 1) provided the difluoroester (5) (312 mg, 74%), mp 132-132. 5'C (n-hexane). IR y KI3r CM-,:

max 1770,1730, 1255. WNMR 8: 0.70 (3H, s, 18-H,,), 1.0. (3H, d, J=6Hz, 21-1- 1), 2.03 (3H, s, acetyl), 3.87 (3H, s, -CO,CHJ, 4.60 (1H, m, 3-H), 5.38 (1H, m, 6-H). 9F-NMR: + 40.3. MS m/z:406 (M- -CH,COOH). Anal. 55 Calcd for CH27H... F2: C, 69.50; H, 8,64; F, 8.14. Found: C, 69.75; H, 8. 75; F, 8.26.

23,23-Difluoro-3p-tetrahydropyranyloxychol-5- en-24oic Acid Methyl Ester (6) The difluoroester (5) (880 mg, 1.9 mmol) was treated with 2% KOH-MeOH (30 mi) at room temperature for 2 hr. The usual work-up (ether for extraction) gave a crude acid. This in ether (10 ml) was treated with 60 an ethereal solution of diazomethane until the gas evolution was ceased. This solution was concentrated under reduced pressure to leave the residue. This in dioxane (10 m]) was treated with 2,3-dihydropyran (516 K1) and TsOH (10 mg) at room temperature for 3 hr. The usual work-up (ether for extraction) gave a crude product, which was applied to a column of silica gel (200 g). Elution with n-hexane-ether (15: 1) provided the THP-ester (6) (907 mg, 95%), amorphous. H-NMR 8 0.70 (3H, s, 18-H,), 1.03 (3H, s, 19- H,), 65 GB 2 158 072 A 1.10 (3H, d, J =6Hz, 21 -HJ, 3.53 (2H, m, THP), 3.86 (3H, s, -CO,CH,), 3. 93 (1 H, m, 3- H), 4.73 (1 H, m, THP), 5.36 (1 H, m, 6-H), 19F- NIVIR 3 + 40.0 MS m/z: 424 (M- -DHP), 406, 391.

23,23-Difluorochol-5-ene-3p,24-dioI 3-THP Ether (7) To a suspension of lithium aluminium hydride (63 mg, 1.65 mmol) in ether (10 mO the difluoroester (6) 5 (1.40 g, 2.76 mmol) in ether (10 mi) was added and the mixture was stirred at O'C for 10 min and then stirred at room temperature for 10 min. The usual work-up (ether for extraction) gave a crude product, which was applied to a column of silica gel (100 g). Elution with nhexane-ether (5: 1) gave the alcohol (7) (1.139, 86%), viscous oil. 'H-NMR 3: 0.73 (3H, s, 18-1-1), 1.03 (3H, s, 19-HJ, 1,13 (3H, cl, J=61-1z, 21-H,), 3.33-4.10 (5H, m, 24-H2, 3-H and THP), 4.76 (1H, m, THP), 5.38 (1H, m, 6- H). 1917-NIVIR 8: + 43.3. MS m/z:

396 (M- -DHP), 378.

23,23-Difluoro-24trifluoromethanesulfonyloxychol- 5-en-3p-ol 3-THP Ester (8) The mixture of pyridine (124 IA) and trifluoromethanesulfonic anhydride (206 111) in dichloromethane (5 mO was stirred at -20'C under argon atmosphere for 5 min. To this solution the alcohol (7) (400 mg, 1.02 15 mmol) in dichloromethane (10 mi) was added and the mixture was stirred at room temperature for 40 min. The usual work-up (dichloromethane for extraction) gave the triflate (8) (612 mg), which was used in the next step without further purification. 'H-NMR 8: 0.73 (3H, s, 18-H), 1.00 (3H, s, 19-H,), 1.15 (3H, d, J=6Hz, 21-1-1j, 3.56 (2H, m, THP), 3.85 (1H, m, 3-H), 4.50 (2H, t, J12Hz, 24-H2), 4.70 (1H, m, THP), 5.37 (1H, m, 6-H). I9F-NIVIR: + 12.2 (317), + 41.3 (2F).

23,23-Difluoro-3p-tetrahydropyranyloxycholest-5- ene-26,27-didic Acid Diethyl Ester (9) A mixture of potassium tert-butoxide (1.1 g, 9.6 mmol) and diethyl malonate (3.8 g, 24 mmol) in THF (25 mi) and HMPA (8 mi) was stirred at room temperature under argon atmosphere for 1 hr. To this solution the triflate (8) (1.47 g, 2.4 mmol) in THF (20 mi) was added and the mixture was stirred at room 25 temperature for 26 hr. The usual work-up (ether for extraction) gave a crude product, which was applied to a column of silica gel (100 g). Elution with n-hexane-ether (5: 1) provided the diester (9) (1.20 g, 81%), mp 79-800C (ethanol). IR yKBr cm-1: 1750, 1740. H-NMR 8: 0.73 (3H, s, 18- H3), 1.00 (3H, s, 19-H3) max '1.10 (3H,d,J=6H,,H,) 1,27 (6H, t, J= 7Hz, -CO,CH2CH,), 3.46 (2H, m, THP), 3.62 (1H, t, J=6Hz, 25-H), 3.80 (11H, M, 3-H), 4.14 (4H, q, J=JHz, -COCH2CH,), 4.64 (1H, m, THP), 5.30 (1H, m, 6- H). MS m/z: 538 (M' -DHP), 520, 30 505. Anal. Calcd for C,^,0J2: C, 69.40; H, 9.06; F, 6.10. Found: C, 69.19; H, 9.11; F, 5.85.

25-Chloro-23,23-difluoro-3p-tetrahydropyranyloxycholest- 5-ene-26, 27dioic Acid Diethy Ester (10) The diester (9) (700 mg, 1.125 mmol) was treated with sodium hydride (39 mg, 1.625 mmol) in dime- thoxyethane, (20 ml) at room temperature under argon atmosphere for 1 hr. Then, to this solution N chlorosuccinimide (180 mg, 1.35 mmol) was added and the mixture was stirred at room temperature for 1 hr. The usual work-up (ether for extraction) gave a crude product, which was applied to a column of silica gel (20 g). Elution with n-hexane- ether (10: 1) provided the chlorodiester (10) (730 mg, 99%), glass.

H-NMR: 0.72 (3H, s, 18-H3), 1.02 (3H, s, 19-HJ, 1.10 (3H, cl, J=6Hz, 21-11j, 1.30 (6H, t, J=7.5Hz, - C02CH2CH.), 2.95 (2H, t, J= 15Hz, 24-H2), 3.52 (2H, m, THP), 3.88 (1 H, m, 3-H), 4.32 (4H, q, J=7.5Hz, - 40 C02CO,CH,), 4.72 (1 H, m, THP), 5.38 (1 H, m, 6-H). MS mlz: 554,520.

25-Chloro-23,23-difluorocholest-5-ene-3p, 26,27- triol 3-THP Ether (11) To a solution of the chlorodiester (10) (730 gm, 1.1 mmol) in ether (15 mi) lithium aluminium hydride (48 mg) was added and the mixture was stirred at O'C for 1 hr. and then stirred at room temperature for 45 2 hr. The usual work-up (ether for extraction) gave a crude product, which was applied to a column of silica gel (50 g). Elution with dichloromethane provided the chlorodiol (11) (250 mg, 39%) mp 152-153'C (n-hexane- ether). 1WNIVIR 8 (CDCI, - acetone d, - DIVISO dj: 0.77 (3H, s, 18-HJ, 1.00 (3H, s, 19-H,), 1.10 (3H, d, J=6Hz, 21 -HJ, 3.50-4.50 (7H, m, 3- H, 26-H2, 27-H, and THP), 4. 77 (3H, m, 26-0H, and THP), 5,38 (1 H, m, 6-H); S(MC13 - acetone d,, DIVISO d,-D20); 3.60 (2H, m, THP), 3. 77 (4H, s, 26- H2 and 27-HJ, 4.77 (1 H, m, THP). MS m/z: 434, 416, 404. Anal. Calcd for C.2H,04CIF2: C, 67. 05; H, 8.97; Cl, 6.19; F, 6.63.

Found: C, 67.08; H, 8.89, Cl, 5.99; F, 6.53.

25E)25,26-Epoxy-23,23-difluorocholest5-ene-3p, 27-diol 3- THP Ether (12) The chlorodiol (11) (183rng, 0.32 mmol) was treated with sodium hydride (18 mg, 0.75 mmol) in dime thoxyethane (18 m]) at room temperature for 6 days. The usual work- up (ether for extraction) gave a crude product, which was applied to a column of silica gel (100 g). Elution with dichoromethane provided the epoxyalcohol (12) (56mg, 32%), glass. 'H-NMR 8: 2.92 (2H, m, 26-1-1j, 3.67-4.16(3H, m, 3-H and 27-H,,).

MS mIz: 434 (M- -THP OH), 416, 404, and the recovery of chlorodiol 11 (92 mg, 50%).

23,23-Difuorocholest-5-ene-3p,25-dioI 3- THP Ether (13) The epoxyalcohol (12) (55 mg, 0.103 mmol) was treated with methanesulfonyl chloride (20 K]) and trie- thylamine (30 KI) in dichloromethane (10 mi) at room temperature for 13 hr. The usual work-up (ether for extraction) gave the crude mesyiate (69 mg). This mesylate was treated with lithium aluminum hydride (5 mg) in ether (10 mi) at O'C for 1.5 hr. The usual work-up (ether for extraction) gave a crude product, 65 6 GB 2 158 072 A which was applied to a column of silica gel (20 g). Elution with n-hexane- ether (5: 2) provided the 25-ol (13) (43.3 mg, 80%), mp 148-149'C (nhexane-eyclohexane). H-NMR 8: 0.72 (3H, s, 18-H,), 1.01 (3H; s, 19H, 1. 10 (3H, d, J =6Hz, 21 -H), 1.35 (6H, s, 26-H3 and 27-H3), 3.53 (2H, m, THP), 3.87 (1 H, rn, 3-H), 4.71 (1 H, m, THP), 5.37 (1 H, m, 6-H). MS mIz: 420 (M' - TEPOH), 405. High resolution MS Calcd for C27H42F20 (M- 5 THPOH): 420, 3214. Found: 420. 3208.

6 23,23-Difluorocholest-5-ene-3p,25-dio1 3-Acetate (14) The THP-ether (13) (26 mg, 0.0498 mmol) in methanol (4 mi) and THP (4 mi) was treated with a catalytic amount of TsOH at room temperature for 1 hr. The usual work-up (ethyl acetate for extraction) gave the crude diol (21.4 mg). This diol was treated with acetic anhydride (1 mi) and pyridine (1 mi) at room 10 temperature for 14 hr. The usual work-up (ethyl acetate for extraction) gave a crude product, which was applied to a column of silica gel (5 g). Elution with benzene-ethyl acetate (10: 1) provided the acetate (14) (23. 0 mg, 96%); mp 168-170'C (methanol). H-NMR 8: 0.82 (2H, s, 18-H,), 1.02 (3H, s, 19-H,), 1.07 (3H, d, J =6Hz, 21 -H,), 1.35 (6H, s, 26-H, and 27-H, ), 2.03 (3H, s, acetyl), 4.55 (1 H, rn, 3-H), 5.36 (1 H, m, 6-H). High resolution MS Caled for C271-1J,0 (M-pi- CH,COOH): 420, 3202. Found: 420, 3206.

23,23-Difluorocholesta-5,7-diene-3,25-dio1 (15) To a solution of the acetate (14) (19 mg, 0.0396 mmol) in ca rbo ntetrach lo ride (2 m]), N-bromosuccinim- ide (10 mg, 0.0571 mmol) was added and this mixture was refluxed under argon atmosphere for 20 min.

After cooling to O'C, the resulting precipitate was filtered off. The filtrate was concentrated below 400C to 20 leave the residue. This residue in xylene (2 mi) was added dropwise to a refluxing solution of S-collidine (0.5), and xylene (1.5 mi) and refluxing was continued for 20 min. The usual work-up (ethyl acetate for extraction) gave the crude diene. This diene in acetone (10 mi) was treated with a catalytic amount of TsOH at room temperature under argon atmosphere in the dark for 14 hr. The usual work-up (ethyl ace tate for extraction) gave the crude 5,7-diene acetate. This acetate in THF (5 mi) was treated with 5% KOH- 2.5 MeOH (1.0 mi) at room temperature under argon atmosphere in the dark for 30 min. The usual work-up (ethyl acetate for extraction) gave a crude product, which was submitted to preparative TLC (benzene ethyl acetate 2A, developed twice). The band of Rf value 0.47 was scraped off and eluted with ethyl acetate. Removal of the solvent provided the 5,7-diene (15) (3.75 mg, 21. 7%). LIV -y.,,mm: 294, 282, 272.

23,23-Difluoro-25-hydroxyvitamin D, (16) A solution of the 5,7-diene (15) (3.75 mg, 8.60limol) in benzene (90 mi) and ethanol (40 mi) was irradi ated with a medium pressure mercury lamp through a Vycor filter with ice cooling under argon atmos phere for 2.5 min. Removal of the solvent under reduced pressure gave a crude product, which was submitted to preparative TLC (benzene-ethyl acetate 2A, developed twice). The band of Rf value 0.59 was 35 scraped off and eluted with ethyl acetate. Removal of the solvent provided the vitamin D,, derivative (16) (0.96 mg, 25.6%). This was further purified by high performance liquid chromatography on a Zorbax SIL normal phase column (4.6 mm(D X 15 cm) at a flow rate of 2 milmin with hexane-dichloromethane (1: 2) as an eluent. The retention time of (16) was 7.4 min. LIVy_nrn: 265, -y,,, nm:228. H-NMR 6: 0.58 (3H, s, 18-1-13), 1.07 (3H, d, J=6.11-1z, 21-1-13) 1.34 (6H, s, 26-1-1, and 27-H, ), 3.95 (1H, rn, 3-H), 4.81 (1H, bs, 19-H), 40 5.04 (1H, bs, 19-H), 6.03 (1H, d, J=10.71-1z, 7-H), 6.23 (11-1, cl, J=10. 71-1z, 6-H). MS mIz: 436 (M-pi), 418, 403, 398, 380, 378, 300, 271, 265, 145, 118. High resolution MS caicd for C2,1- 142F,02: 436, 3150. Found: 436, 3155.

It will be apparent that other reactants may be utilized to provide equivalent substituents at various places in the compounds. For example, in compound 4 the acetoxy shown in the 3-position in the mole- 45 cule could readily be some other acyloxy group where the acyl group contains from about 1 to 4 carbon atoms or tetra hydro pyra nyi. Also the ethyl ester shown in the 26 and 27 positions in compounds 9 and 10 can readily be another alkyl ester where the alkyl group is a lower alkyl group containing from about 1 to about 4 carbon atoms. Likewise other halogen atoms can be introduced for the chlorine in compounds 10 and 11.

23,23-Difluoro-la,25-dihydroxyvitamin D, One day-old leghorn chickens were fed a vitamin D- defficient diet containing 1% calcium for two weeks (Omdahl et al, Biochemistry, 10, 2935-2940 (1971)). They were then killed, their kidneys were re moved, and a 20% (W/V) homogenate was prepared in ice-cold 0.19M sucrose solution containing 15mM. 55 Tris-acetate (tri hyd roxym ethyl am i noetha n eacetate) (pH 7.4 at room temperature) and 1.9 mM magnesium acetate (Tanaka, Y. et al, Arch. Biochem. Biophys, 171, 521-526 (1975)). The incubation involved the addi tion of 911 of 23,23difluora-25-hydroxyvitamin D, dissolved in 100 [LI of 95% of ethanol to a 125 ml Er lenmeyer flask which contained 1 g of kidney tissue, 0.19 M sucrose, 1.5 mM Trisacetate, 1.9 mM magnesium acetate and 25 mN succinate in a final volume of 7.5 mi. After shaking the mixture at 37'C 60 for 2 hrs.,the reaction was stopped with 15 mi of MeOh and 7.5ml of CH2C'2. After another 7.5 mi of CH2Cl2 was added to the organic phase, the resulting mixture was separated and evaporated under vac uum. The residue containing the desired 23,23- difluro-1,25- dihydroxyvitamin D,, was then subjected to chromatographic purification by high pressure liquid chromatography using a model ALC/GPC 204 high pressure liquid chromatograph (Waters Associates, Medford, Mass.) equipped with an ultraviolet detector 65 7 GB 2 158 072 A 7 operating at 254 nm. The residue, dissolved in 100 RI of 10% 2- propanol in hexane, was injected onto a silica gel column (Zorbax-SIL, 0.46 x 25 cm, Dupont, Inc.) operating under a pressure of 1000 psi which produced a flow rate of 2 ml/min. Using a solvent system containing 10% 2-propanol in hexane, the sample was purified twice through this column and then collected. Putative 23,23- difluoro-1,25-dihydroxyvi5 tamin D, was further purified on a reverse-phase column (Lichrosorb RP-15, 0.46 x 25 cm, E. Merck, Darmstadt, Federal Republic of Germany) using the same high pressure liquid chromatograph operating at a pressure of 2000 psi. The product was eluted with a solvent mixture of H20/MeOH (1/4) and collected. The residue was rechromatographed on the Zorbax SIL column using conditions exactly as described above.

The identity of the product as 23,23-difluoro-1,25- clihydroxy vitamin D, was confirmed by its spectro- 10 scopic properties. The compound showed the typical vitamin D-like ultraviolet absorption with a maximum at 264 nm. The mass spectrum of the product contained a molecular ion at m/e 452 as required for a 23,23- difluoro-1,25- dihydroxyvitamin D, Fragments at m/e 434 and 416 represent elimination of one and two molecules of H20. Loss of entire side chain results in the fragment of m/e 287 which, by elimina- tion of one and two molecules of H,O, gives rise to peaks at m/e 269 and 251. In addition, the spectrum 15 shows prominent peaks at m/e 152 and m/e 134 (elimination of one molecule of H2H) which represent ring A fragments and are diagnostic for lot,3- dihydroxy vitamin D3 compounds.

23,23-Difluoro-25-hydroxy- and 1,25- dihydroxy-vitamin D, can be obtained in crystalline form if desired by recrystallization from appropriate hydrocarbon solvents, or combinations of such solvents with alco- holic solvents, e.g. a combination of hexane and methanol, as is well- known.

The desired compounds can be obtained in crystalline form if desired by recrystallization from appropriate hydrocarbon solvents, or combinations of such solvents with alcoholic solvents, e.g. a combination of hexane and methanol, as is well-known in the organic chemical art.

Biological Activity The biological activity of the new analogs is evidenced by appropriate in vivo assays in the rat.

23,23-Difluoro-25-hydroxyvitamin D, Male weanling rats (Holtzman Company, Madison, Wis.) were fed a low calcium vitamin D-cleficient diet (0.02% calcium, 0.3% phosphorus - J. Nutr. 100, 1045-1052 (1970) for 3 weeks. They are then divided 30 into three groups of 6 rats each. Rats in the control group were given 0. 05 ml of 95% ethanol by intraju gular injection. Rats in the second group were administered, in same manner, a dose of 650 omole of 25 hydroxyvitamin D, (25-OHDJ dissolved in 0.05 ml ethanol, while rats in the third group were injected with a dose of 650 omole of 23,23-difluoro-25-hydroxyvitamin D3 (23,23- F, -25-OHD3) dissolved in 0.05 ml ethanol for comparative purposes. Twenty four hours after dosing, the effect of the test compounds on intestinal calcium transport and on bone calcium mobilization measured as by the serum calcium con centration were determined by the assay methods of Martin and DeLuca (Am. J. Physiol. 216, 1351-1359 (1969)) and of Tanaka et a[ (Biochemistry, 14, 3293-3296 (1975)) respectively. Results are shown in Table 1.

TABLE 1

Compound given Intestinal Calcium Serum transport calcium (Ca serosa#Ca mucosal) (Mgliooml) 45 Vehicle (ethanol 2.8 0.4-) 2.8 0.1d) 25-OHD2 5.5-tO.7b) 3.5 0.05-) 23,23-F,-25-OHD3 5.0:LlA) 3.4--0.21) Significance of b) & c) from a) e) & f) from d) 50 Difference: p<0.005 p<0.001 b) from c) e) from f) N.S. N.S.

Standard deviation of the mean The foregoing data indicated that 23,23F2-25-OHD, is active in both intestine and bone and that the compound exhibits vitamin D-like activity at least as great as that exhibited by 25-hydroxyvitamin D, strongly suggesting its use as a substitute for that vitamin D derivative or for vitamin D.

23,23-Difluoro- 1a,25-dihydroxyvitamin D, Male weanling rats (Holtzman Company, Madison, Wis.) were fed the low calcium vitamin D-cleficient diet for two weeks. They were divided into three groups of 6- 7 rats each. Rats in the control group were given 0.05 ml of 95% ethanol by intrajugular injection. Rats in the other two groups were each adminis tered, in the same manner, a dose, respectively, of 100 p moles of 1,25dihydroxyvitamin D, (1,25 6E (OH),D,) in 0.05 ml of ethanol or23,23-difluoro-lo-,25- dihydroxyvitamin D, (23,23-F2-1,2540HOD, in 0.05 65 8 GB 2 158 072 A 8 ml ethanol. 96 Hours after dosing the effect of the compounds on intestinal calcium transport was determined by the method of Martin and DeLuca. Results are shown in the Table below.

TABLE 1

Compound Given None (vehicle only) 1,25-(OH),D:, 23,23-F,-1,25-(OH)2D3 Significance of difference: b) & c) from a) p<0.001 Intestinal Calcium Transport (Ca serosollCa mucosal) (A vg. t SEM) 2.6 0.1.) 4.6-tO.4b, 4.5-hO.31) The foregoing data indicate that 23,25-F,.-1,25- (OH)2D, is as active in promoting intestinal calcium transport as 1,25-(OH),D,, strongly suggesting its use as a substitute for the hormonal form of the vitamin where pharmacologically increased intestinal calcium transport is indicated.

The compounds of this invention may typically be readily administered as sterile parenteral solutions by injection or intravenously or by alimentary canal in the form of oral dosages, or by suppository. Doses 20 of from about 0.1 Rg to about 10 Lg per day of 23,23-F,.- 1ot,25-(OH2)-D, and from 1 jig to about 25 [tg per day of 23,23-F2-25-OH-D3 are generally effective on obtaining the physiological calcium balance responses described (which are characteristic of vitamin D-like activity) with maintenance doses of about 0.25 [Lg of 23,23-F2_1a,25-(OH2)-D, and about 5 I.Lg of 23,23-F2-25-OH-D, being suitable.

Dosage forms can be prepared by combining the compound with a non-toxic pharmaceutically accept- 25 able carrier as is well-known in the art. Such carriers may be either solid or liquid such as corn starch, lactose, sucrose, peanut oil, olive oil, sesame oil and water. If a solid carrier is used the dosage forms include tablets, capsules, powders, troches or lozenges. If a liquid carrier is used, soft gelatin capsules, or syrup or liquid suspensions, emulsions or solutions may be the dosage form.

The dosage forms may also contain adjuvants, such as preserving, stabilizing, wetting or emulsifying 30 agents or solution promoters. They may also contain other therapeutically valuable substances.

It should be understood that although dosage ranges are given, the particular dose to be administered to a host will depend upon the specific disease state being treated, the end results being sought, as well as other factors known to those skilled in the therapeutic use of such medicinal agents.

Claims

1. 23,23-Difluoro-cholesta-5,7-diene.

2. A compound having the formula:

R 2 40 3 45 R6 50 1 wherein R represents acyl or tetra hyd ro-pyra nyl X represents halogen or hydroxy and R, and R, independently represent COOR,, CH20H or hydrogen where R4 is lower alky].

3. A compound according to Claim 2 wherein R, is acetyl and R, and R3 are COOCA.

4. A compound according to Claim 2 wherein R, is acetyl, X is chlorine and R, and R3 are CO0C^.

5. A compound according to Claim 2 wherein R, is acetyl, X is hydroxy and R2 and R3 are hydrogen.

6. A compound having the formula:

9 GB 2 158 072 A 9 R 2 X R3 RIO R 3 wherein R, represents hydrogen, acyl or tetra hydropyranyl, X represents hydroxyl or 0-acyl and R2 and R, are as defined in Claim 2.

7. A compound according to Claim 6 wherein R, represents hydrogen and X represents hydroxyl.

Printed in the UK for HMSO, D8818935, 9185, 7102. Published by The Patent Office, 25 Southampton Buildings, London, WC2A lAY, from which copies may be obtained.