23 _f 23-diflυo_-O-25-hyd_πxy-vitaπ n P., •and Process for Preparing Same

The invention described herein was made in the course of work under a grant or award frcm the Department of Health and Human Services.

The Government also has rights in this invention pursuant to U.S. Japan Cooperative Grant _Q7E ^, -76-05793 awarded by the National Science Foundation. Technical Field

This invention relates to a ccπpound which is characterized by vitamin D-like activity.

More specifically this invention relates to a derivative of vitamin D ₃ _

Vitamin D_ is a well-known agent for the control of calcium and phosphorous hαmeostasis. In the normal animal or human this c npound is known to stimulate intestinal calcium transport and bone-calcium mobilization and is ef ective 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-bydroxy-vitamin D_ and is further hydroxylated in the kidney to produce -_α,25-di___ydroxy vitamin D- or 24,25- dihydroxy vitamin D_. The 1-hydroxylated 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 i_ntestir_al absorption of calcium and phosphate, mobilizing bone mineral, and causing reabsorption of calcium in the kidneys.

Background Art

Since the discovery of biologically active metabolites of vitamin D there has been much interest in the preparation of st__uctur_il analogs of these metabolites, because such compounds may represent useful therapeutic agents for the treatment of diseases resulting frcm c_alcium metabolism disorders. A variety of vitamin D-like cαnpounds have been synthesized. See, for example, U.S. Pat. Nos. 3,741,996 directed to 3,907,843 directed to lα-hydι_O_ς?e__gocalciferol; 3,786,062 directed to 22-dehydro- 25-hydroxycholecalciferol; 4,069,321 directed to the preparation of various side chain-fluorinated vitamin D ₃ derivativ-es and side chain-fluorinated d-Lhydrotachysterol analogs; 4,201,881 directed to 24,24-difluoro-lα,25 ^**** dihydroxy- cholecalciferol and 4,196,133 directed to 24,24-difluoro- 25-hydrcκycfølecalciferol. Other metabolic alternatives are believed to be responsible for the metabolism and ultimate eliiriination of vitamin D cαtpounds frαn the body, with the generally accepted recognition that lα,25-dihyd__oxychole- calciferol (U.S. Patent 3,697,559) is the circulating hormonal form of vitamin D. Disclosure of Invention

A new derivative of vitamin D has new been found which is at least as potent as 25-h ^* yd__>xyvitamin D- (see U.S. Patent 3,565,924) .as measured by its ability to stimulate calcium transport in the intestine or its ability to mobilize calcium frcm bone. This derivative has been identified as 23,23-difl-ι__o__o-25-hyd-_Ojς^-cholecalciferol (23,23-difluoro-25- hydrαxy vitamin D or 23,23-F -25CH D .)

A major pathway for inactivation of vitamin D is 23S- hydroxylation of 25-hydroxy vitamin D_ (Tanaka et al. Biochemistry 20, 3875-3879, 1981) and its subsequent ■conversion to 25R-hyd__oxy-26,23S-lactone (Tanaka et al, Proc. Nat'l. Acad. Sci. USA 78, 4805-4808, 1981). In view of these

OMp

findings of Tanaka et al it would appear that the vitamin D derivative of the present invention, because of the fluorine substituents at C-23, would not be readily hydroxylated at that carbon and that, therefore, it would be characterized by prolonged vitamin D-like activity - a c-haracteristic which would be an obvious advantage in many therapeutic applications. Best Mode for C_arrying Out the Invention

23,23-difluoro-25-hydroxyvitamin D_ can be prepared in accordance with the process as hereinafter described and shown in the following schematic. In the schematic and the description like numbers identify like -compounds. Also, for those compounds in the schematic where a particul-ar steroid nucleus is not specifically depicted the designated cαnpoυnd has the same steroid nucleus shown for the compound from which it is most irrπediately derived. For example, compounds 2 and 3 have the steroid nucleus shown in compound 1, while coπpounds 5 through 14 have the steroid nucleus shown in C-cmpound 4.

_2_ 4 3-THP 8 R - OS0 ₂ CF ₃

so (1

In the following detailed description of the synthesis of 23,23-difluo__o-25-hydroxyvi1_amin D. the various physico- chemical cha3_acteristics of the -cαripoui-ds shown were determined utilizing the apparatus hereafter described and the various abbreviations and processes have the indicated definitions.

Melting points were detenπined 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 spectrophotometer. n-NMR spectra were recorded on a

Varian EMr360L specxctneter in CDC1- unless otherwise stated,

19 with tetrametliylsilane as an internal reference. F-NMR spectra were recorded on a Varian -ZM-360L spectrαneter in

CDCl, solution, with benzotrifluoride as an internal reference

(a plus means high field) . Mass spectra were obtained with a

HITACHI double focusing mass spectrαnenter EM -7L. Column c_h__ ^* c__ιatograρby was effected with silica gel (Merck, 70-23 meah) . Preparative thin lay-er c-hrαnatography was ca__ried out on precoated plates of silica gel (Merck, silica gel 60 _?t - .

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 solvent under reduced pressure. The following abbreviations were used? THF, tetrahydrofuran; ether, diethyl ether; HMPA, hexairethylphosphoramide; TsOH, p-toluenesulfonic acid; THP, tetrahydropyranyl; s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; bs, broaden singlet.

Synthesis

6β-Methoxy-3α,5-cyclo-23,24-dinor-5 ^α -cholan-22-al (2)

6β-Matho_^-__Λ,5-cyclo-23,24-dinor-5 ^α -chol-22ol (1) (2.0 g,

■311

15.8 πtπol) , which was prepared according to the literature itethod, was added to suspension of pyridinium chlorochromate (3.8 g) and sodium acetate (1.4 g) in dichloro- ethane (40 ml) , and this mixture was stirred at room temperature for 2.5

OMPI _

hr. Then, to this solution ether (100 ml) was added and the resultant precipitates were filtered off and washed with ether

(100 ml) . The ccπbined filtrate was succesively washed with

5%l ^* feHC0_ 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 n-hexane-ether ilO:l) provided the aldehyde (2) (1.44 g, 73%), amorphous. TI-NMR δ: 0.76 (3H, s, 18- -.) , 1.30 (3H, d _r J=6Hz, 21-H ₃ ) ,

1.17 (3H, s, 19-H ₃ ) , 2.76 (1H, m, 6-H) , 3.33 (3H _r s, -Cgy ,

9.51 (1H, d, J=3.5Hz, -CHO) . MS m/z: 344 (M ⁺ ) , 329, 312.

0L 0L

6β--Msthoxy--'23-triethylsilyloxy-3 ,5-cyclo-5 -cholan-22-en-24- oic Acid Methyl Ester (3)

To a solution of dii__op__opyla_T_ine (1.05 ml, 7.5 ttol) in THF (10 ml) n-butyllithium (6 nmol) was added at -78°C under argon atmosphere and this solution was stirred for 5 rain. To this solution methyl α-triethylsilylo_^-α-dime ^* t_hylphosphonoacetate (1.56 g, 5 nmol) in THF (10 ml) was added and this mixture was stirred at room taiperature for 15 min. Then, to the resulting solution the aldehyde (2) (491 mg, 1.43 nmol) in THF (10 ml) 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 oil. Tϊ-NMR δ : 3.30 (3H, s, -COL,) , 3.73 (3H, s, -• ^■ CO- _j CEy, 5.26 (1H, d, J=10Hz, 22-H) . MS m/z: 530 (M ⁺ ) , 501, 469.

3β-AoetQ∑ay-23-oxochol-5-en-24-oic Acid Methyl Ester (4) A solution of the unsaturated ester (3) (1.53 g, 2.9 nmol) in acetic acid (7 ml) was heated at 80-90°C for 6 hr. The usual work-up (ether for extraction) gave a crude product. This and a catalytic amount of TsCH in dioxane (10 ml) and water (10 ml) were heated at 85-95°C for 7 hr. The usual work-up (ether for extraction) gave a crude product, which was applied to a column of silica gel (300 g) . Elution with n-hexane-ether (15

: 1) provided the α-keto ester (4) (768 mg, 76%) , p 146-147°C

(n-hexane). IR γ ^KBr cm ^"1 : 1720, 1240. ^" hi-NMR 5: 0.73 (3H, s, 18-H ₃ ), 0.93 (3H, d, J=6Hz, 21-H ₃ ) , 1.03 (3H, s, 19-H. _j ) ,

Anal. Calcd for C^H^O^ C, 72.92; H, 9.08. Found: C, 72.63;

H, 9.13.

3β-Acetoxy-23,23-diflυorochol-5--en-24-oic Acid Methyl

Ester (5)

A mixture of α-ketoester (4) (400 mg, 0.9 mmol) and diethylaminosulfurtriflxioride (1.5 ml, 9.5 mmol) in dichloromethane (15 ml) was stirred at room teiriperature for 16 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 (10 : 1) provided the difluoroester (5) (312 mg, 74%) , mp 132-132.5°C (n-hexane) .

IR γ ^KBr cm ^*1 : 1770, 1730, 1255. l-WR. δ: 0.70 (3H, s, max

18-H ₃ ),1.0. (3H, s, 19-H ₃ ), 1.10 (3H, d, J=6Hz, 21-H ₃ ) , 2.03 (3H, s, acetyl), 3.87 (3H, s, -C0CH^), 4.60 (lH, m, 3-H) , 5.38 (1H, m, 6-H). ^F-N R : + 40.3. MS m/z: 406 (M -CH ₃ C00H)o Anal. Calcd for CH ₂ ^ ₄₀ 0 ₄ F ₂ ^: C, 69.50; H, 8.64; F, 8.14. Found: C, 69.75; H,8.75; F, 8.26.

23,23-Difluoro-3β-tetrahydropyranyloxychol-5-en-24-oic Acid Methyl Ester (6)

The difluoroester (5) (880 mg, 1.9 irπol) was treated with 2% KOH-MεOH (30 ml) at room teπpe__ature for 2 hr. The usual work-up (ether for extraction) gave a crude acid. This in ether (10 ml) was treated with etheral solution of diazαnethane until the gas evolution was ceased. This solution was concentrated under reduced pressure to leave the residue. This in dioxane (10 ml) was treated with 2,3-dihydropyran (516 μl) 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 (2C0 g) . Elution with n-hexane-ether (15 : 1) provided the

WIPO

THP-ester (6) (907 mg, 95%) , amorphous. Tϊ-NMR δ: 0.70 (3H, s, 18-H ₃ ), 1.03 (3H, s, 19-H ₃ ), 1.10 (3H, d, J=6Hz, 21-H ₃ ), 3.53 (2H, m, THP), 3.86 (3H, s, (1H, m, 3-H) , 4.73 (1H, m, THP), 5.36 (1H, m, δ: + 40.0. MS m/z: 424 (M ⁺ - DHP) , 406, 391.

23,23-Difl\_orod-θl-5-ene-3β,24--diol 3-THP Ether (7) To a suspension of _Li-t ^** hium aluminium hydride (63 mg, I,65 nmol) in ether (10 ml) the difluoroester (6) (1.40 g, 2.76 mmol) in ether (10 ml) was added and the mixture was stirred at 0°C for 10 min and then stirred at room temperature for 10 min. Ηie 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) gave the alcohol (7) (1.13 g, 86%) , viscous oil. Tϊ-NMR δ: 0.73 (3H, s, 18-H ₃ ) , 1.03 (3H, s, 19-H ₃ ), 1.13 (3H, d, J=6Hz, 21-HJ, 3.33-4.10 (5H, m, 24rH ₂ , 3-H, .and THP) , 4.76 (1H, , THP) , 5.38 (1H, m, 6-H) . ¹⁹ F-NMR δ: + 43.3. MS m/z: 396 (M -DHP), 378. 23,23-Difluoro-24-trifluo_π3 ^* tethanesulfonyloxychol-5-en-3β-ol 3-THP Ether (8)

The mixture of pyridine (124 μl) and -_rifluo_x_rBthanesulfonic anhydride (206 μl) in dichlor nethane (5 ml) was stirred at -20°C under argon atmosphere for 5 min. To this solution the alcohol (7) (400 mg, 1.02 mmol) in dichlo-xmethane (10 ml) 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. TI-NMR δ: 0.73 (3H, s, 18-HJ , 1.00 (3H, s, 19-H ₃ ) , 1.15 (3H, d, J=6Hz, 21-H ₃ ) , 3.56 (2H, m, THP), 3.85 (1H, m, 3-H) , 4.50 (2H, t, J=12Hz, 24-H,,) , 4.70 (1H, m, THP), 5.37 (IH, m, 6-H) . ¹⁹ F-NMR δ: + 12.2 (3F) , + 41.3 (2F).

23,23-Difluoro-3β-tetrahydropyr.anyloxycholest-5-ene-26,27- di- oic Acid Diethyl Ester ,„.

A mixture potassium tert-butoxide (1.1 g, 9.6 mmol) and diethyl malonate (3.8 g, 24 mmol) in THF (25 ml) and HMPA (8 ml) was stirred at rocm tenperature under argon atmosphere for 1 hr. To this solution the triflate (8) (1.47 g, 2.4 mmol) in THF (20 ml) was added and the mixture was stirred at room tenperature 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 9-80°C 0.73 (3H, s, 18-H ₃ ), 1.00 (3H, s, 19-H ₃ ), 1.10 (3H, d, J=6Hz, 21-H ₃ ) , 1.27 (6H, t, J=7Hz, -00 ₂ CH ₂ CH ₃ ) , 3.46 (2H, m, THP), 3.62 (1H, t, J=6Hz, 25-H), 3.80 (lH,m, 3-H) , 4.14 (4H,q,J=0Hz, -C0CH ₂ CH ₃ ),4.64 (lH,m,THP) , 5.30 (1H m, 6-H) . MS m/z: 538 (M ⁺ -DHP) , 520, 505. Anal. Calcd for C^H^O^: C, 69.40; H, 9.06; F, 6.10. Found: C, 69.19; H, 9.11; P, 5.85. 25-Chloro-23,23-diflι_oro-3β-tetrahydropyranyloxycholest-5 -ene- -26,27-dioic Acid Diethyl Ester (10)

The diester (9) (700 mg, 1.125 mmol) was treated with sodium hydride (39 mg, 1.625 nmol) in dimethoxyethane (20 ml) at rocm 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 rocm 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. ³ H- ^* N : 0.72 (3H, s, 18-H ₃ ) , 1.02 (3H, s, 19-H ₃ ), 1.10 (3H, d, J=6Hz, 21-R..) , 1.30 (6H, t, J=7.5Hz, 2.95 (2H, t, J=15Hz, 24-H ₂ ) , 3.52 (2H, , THP), 3.88 (1H, m, 3-H) , 4.32 (4H, q, J=7.5Hz, -CO CO CH ) , 4.72 (1H, m. THP), 5.38 (1H, m, 6-H) . MS m/z: 554, 520.

25-Chloro-23,23-difluorocholest-5-ene-3β, 26,27-triol 3-THP Ether (11)

To a solution of the chlorodiester (10) (730 mg, 1.1 mmol) in ether (15 ml) lithium aluminium hydride (48 mg) was added and the mixture was stirred at 0°C for 1 hr. and then stirred at room tatperature for 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%) p 152-153°C (n-hexane-ether). ^" TI-NMR δtCDCU - acetone d _g - DMS0 d _g ) : 0.77 (3H, s, 18-H ₃ ) , 1.00 (3H, s, 19-H ₃ ) , 1.10 (3H, d, J=6Hz _r 21-H ₃ ), 3.50-4.50 (7H, m, 3-H, 26-H ₂ , 27-H ₂ , -and THP) , 4.77 (3H, m, 26-0H, 27-€H, and THP), 5.38 (1H, m, 6-H); δ(CDCl ₃ - acetone d.-DMSOd^-D-O) : 3.60 (2H, , THP), 3.77 (4H, s, 26-H_ and 27-H ₂ ) , 4.77 (1H, , THP) . MS m/z: 434, 416, 404. Anal. Calcd for C H _χ 0 CJLF : 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.

25ε)-25,26-Epoxy-23,23-dif^l ^* uorocholest-5-erιe-3β, 27-diol 3-IHP Ether (12)

The -chlorodiol (11) (183 mg, 0.32 mmol) was treated with sodium hydride (18 g, 0.75 mmol) in dimet±iojQrethaie (18 ml) at rocm tenperature 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 dichorαnethane P-Tovided the epoxy-alcohol (12) (56 mg, 32%) , glass. ^" TI-NMr δ: 2.92 (2H, m, 26-H,,) , 3.67-4.16 (3H, m, 3-H and 27-H.,) . MS m/z: 434 (M ⁺ -THP OH) , 416, 404, and the recovery of chlorodiol 11 (92 mg, 50%) .

23,23-Difluorocholest-5-ene-3β,25-diol 3-1_HP Ether (13) The epoxyalcohol (12) (55 mg, 0.103 mmol) was treated with πethanesulfonyl chloride (20 μl) and triethylamine (30 μl) in dichlor iethane (10 ml) at room temperature for 13 hr. The usual work-up (ether for extraction) gave the crude mesylate (69 mg) . This mesylate was treated with lithium aluminum hydride (5 mg) in ether (10 ml) at 0°C for 1.5 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 (5 : 2) provided the 25-ol (13) (43.3 mg, 80%) , mp 148-149°C (n-hexane-cyclchexane) . TI-NMR δ: 0.72 (3H, s, 18-H ₃ ), 1.01 (3H; s,19-H ₃ , 1.10(3H,d,J=6Hz,21-H) , 1.35 (6H,s,26-H ₃ and 27-H ₃ ) , 3.53 (2H, m, THP), 3.87 (IH, m, 3-H) , 4.71 (IH, m, ΪHP), 5.37 ( H, m, 6-H) . MS m/z: 20 (M ⁺ - TEPOH) , 405. High resolution MS Calcd for (M ⁺ - THPOH) : 420, 3214. Found: 420, 3208.

23,23-Difluo__ocholest-5-ene-3β,25-diol 3-Acetate (14) The THP-ether (13) (26 mg, 0.0498 nmol) in nethanol (4 ml) and THP (9) (4 ml) 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 ml) and pyridine (1 ml) at room temperature for 14 hr. The usual work-up (ethyl acetate for ertraction) 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%); πp 168-170°C (methanol). ^" ϊ-NMR δ: 0.82 (3H, s, 18-H ₃ ) , 1.02 (3H, s, 19-H ₃ ), 1.07 (3H, d, J=6Hz, 21-H ₃ ) , 1.35 (6H, s, 26-H ₃ and 27-H ₃ ) , 2.03 (3H, s, acetyl) , 4.55 (IH, m, 3-H) , 5.36 (IH, m, 6-H) . High resolution MS Calcd for C ₂₇ H ₄₂ F ₂ 0 (M ⁺ -CH ₃ C00H): 420, 3202. Found: 420, 3206.

23,23-Difluorocholesta-5,7-diene-3,25-diol (15) To a solution of the acetate (14) (19 mg, 0.0396 nmol) in •carbontetrachloride (2 ml) N-b-xπosuccinimide (10 mg, 0.0571 mmol) was added and this mixture was refluxed under argon atmosphere for 20 min. After cooling to 0°C, the resulting precipitate was filtered off. The filtrate was concentrated below 40°C to leave the residue. This residue in xylene (2 ml) was added dropwise to a refluxing solution of S-collidine (0.5) and xylene (1.5 ml) and refluxing was continued for 20 min. The usual work-up (ethyl acetate for extraction) gave the crude diene. This diene in acetone (10 ml) was treated

OMPI VIPO &?iVAT10

with a catalytic amount of TsOH at room tenperature under argon atmosphere in the dark for 14 hr. The usual work-up

(ethyl acetate for extraction) gave the crude 5,7-diene acetate. This acetate in THF (5 ml) was treated with 5% KOH-MeOH (1.0 ml) at roo tenperature 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 TDC (benzene-ethyl acetate 2:1, developed twice) ♦ The band of Rf value 0.47 was strapped off and eluted with ethyl acetate. Removal of the solvent provided the 5,7-diene (15) (3.75 mg, 21.7%). UV irm : 294, 282, 272.

23,23-Dif^l-uoro-25-hydroxyvitamin D-, (16) A solution of the 5,7-diene (15) (3.75 mg, 8.60 μmol) in benzene (90 ml) and ethanol (40 ml) was irradiated with a medium pressure mercury lamp through a Vycor filter with ice cooling under argon atmosphere for 2.5 min. Removal of the solvent under reduced pressure gave a crude product, which was submitted to preparative T C (benzene-ethyl acetate 2:1, develcped twice) . The band of Rf value 0.59 was strapped 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 chrcmatography -on a Zorbax SIL normal phase column (4.6 mm§ x 15 cm) at a flow rate of 2 ml/min with hex__ιιe-diciιlo_x--ιethane (1 : 2) as an eluent. The retention time of (16) was 7.4 min. ^ϋV ''max ^{11111 : 265} ' ^λ min ^{nm : 228* 1]a""NMR δ :} °- ⁵⁸ * ^3H ' ^s ' 18-H ₃ ), 1.07 (3H, d, J=6.1Hz, 21-H ₃ ) 1.34 (6H, s, 26-H-, and

27-H ₃ ), 3.95 (IH, m, 3-H) , 4.81 (IH, bs, 19-H) , 5.04 (IH, bs, 19-H), 6.03 (H, d, J=10.7Hz, 7-H) , 6.23 (IH, d, J=10.7Hz, 6-H). MS m/z: 436 (M ⁺ ) , 418, 403, 398, 380, 378, 300, 271, 265, 145, 118. High resolution MS calcd for ₂ 7 ^H 42 ^F 2°2 ^{: 436} 3150. Found: 436, 3155.

It will be apparent that in the foregoing other reactants may be utilized which will provide equivalent substituents at various places in the compounds depicted in the abreviated

schematic. For example, in compound 4 the acetoxy shown in the 3-position in the molecule could readily be seme other acyloxy group where the acyl group contains from about 1 to 4 carbon atoms. Also the ethyl ester shown in the 26 and 27 positions in cαπpounds 9 and 10 can as readily be another alkyl ester where the alkyl group is a lower alkyl group conteining from about 1 to about 4 carbon atoms.

Tήe desired compound, 23,23 ^* ^ifluoro-25-hydroxyvitemiιι D , can be obtained in crystalline form if desired by recrystallization frcm appropriate hydrocarbon solvents, or cαnbinations of such solvents with alcoholic solvents, e.g. a coπbination of hexane and methanol, as is well known in the organic chemical art.

Biological Activity

The biological activity of the new analog is evidenced by appropriate in vivo assays in the rat.

Male weanling rats (Holtzman Company, Madison, Wis.) were fed a low calcium vitamin D-deficient diet. (J. Nutr. 100, 1045-1052 (1970)) for 3 weeks. They are then divided into three groups of 6 rats each. Rats in the control group were given 0.05 ml of 95% ethanol by intrajugular injection. Rats in the second group were administered, in same manner, a dose of 650 p.mole of 25-hyα-roxyvitaιτιin D (25-0HD dissolved in 0.05 ml ethanol, while rats in the third group were injected with a dose of 650 pmole of 23,23-difluoro-25~hydroxyvitemin D ₃ (23,23-F ₂ -25-OHD ₃ ) dissolved in 0.05 ml ethanol for corrparative purposes. Twenty four hours after dosing, the effect of the test cempounds on intestinal calcium transport and on bone calcium mobilization measured as by the serum calcium concentration were de-termin-ed by the assay methods of Martin and DeLuca (Am. J. Physiol. 216, 1351-1359 (1969)) and of Tanaka et al (Biochemistry, 14, 3293-3296 (1975)) respectively. Results -are shown in Table 1.

Table 1

Cαcpound given -Intestinal Calcium Serum transport calcium

(Ca serosal/Ca muco.sal) (mg/lCO ml)

Vehicle (ethanol 2.8 ±0.4 ⁾ * 2.8 i 0.1 ^d)

25-0HD ₂ 5.5 ± 0.7 ^b) 3.5 - 0.05 ^eϊ

23,23-F ₂ -25-0HD ₃ 5.0 ± 1.4 ^C) 3.4 i 0.2 ^f

Significance of b) & c) frcm a) e) & f) from d) Difference: p<0.005 p O.001 b) from c) e) frcm f)

N.S. N.S.

*Standard deviation of the mean

The foregoing data indicate that 23,23-F ₂ -25-0HD ₃ 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-hyd_.Oxyvit.amin D.., strongly suggesting its use as a substitute for that vitamin D derivative or for vitamin D.

The 23,23-difluo__o-25-dihyd__t_xycholecal compound of this invention may be.readily administered as sterile pjarenteral solutions by injection or intravenously or by alimentary canal in the form of oral dosages, or by suppository. Doses of frcm about 1 μg to about 25 μg per day are effective in obtaining the physiological calcium balance responses described and which are characteristic of vitamin D-like activity, with maintenance doses of about 5 μg being suitable.

Dosage form of the c rpounds can be prepared by c_orribining them with a non-toxic pharmaceutically acceptable carrier as is well kncwn in the art. Such carriers may be either solid

or liquid such as, for example, corn starch, lactose, sucrose, peanut oil, olive oil, sesame oil and water. If a solid carrier is used the dosage forms of the -compounds of the invention may be tablets, capsules, powders, troches or lozenges. If a liquid carrier is used, soft gelatin capsules, or syrup or liquid suspension, emulsions or solutions may be the dosage form. The dosage forms may also conta n adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution prcmoters, etc. 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 in a particular case, as well as other factors known to those skilled in the art in the therapeutic use of such medicinal agents.

OMPI