Stereoselective Synthesis of 24-Fluoro-25-Hydroxyvitamin D3 Analogues and Their Stability to hCYP24A1-Dependent Catabolism

Two 24-fluoro-25-hydroxyvitamin D3 analogues (3,4) were synthesized in a convergent manner. The introduction of a stereocenter to the vitamin D3 side-chain C24 position was achieved via Sharpless dihydroxylation, and a deoxyfluorination reaction was utilized for the fluorination step. Comparison between (24R)- and (24S)-24-fluoro-25-hydroxyvitamin D3 revealed that the C24-R-configuration isomer 4 was more resistant to CYP24A1-dependent metabolism than its 24S-isomer 3. The new synthetic route of the CYP24A1 main metabolite (24R)-24,25-dihydroxyvitamin D3 (6) and its 24S-isomer (5) was also studied using synthetic intermediates (30,31) in parallel.

Synthesis of CD-ring fragments was achieved by side-chain elongation of Inhoffen-Lythgoe diol. Stereoselective introduction of the 24-hydroxy group was performed by Sharpless dihydroxylation reaction [17,18], and the fluorination step was achieved by deoxyfluorination reaction using N,N-diethylaminosulfur trifluoride (DAST).

Results and Discussion
For the synthesis of C24-substituted CD-ring fragments (13)(14)(15)(16), commercially available Inhoffen-Lythgoe diol was chosen as a starting material (Scheme 3). Iodination at C22-OH and hydroxy protection at the C8 position yielded iodide 18 [19]. After replacement of iodine with an allyl group utilizing allyl magnesium bromide, stereoselective dihydroxylation was achieved via Sharpless asymmetric dihydroxylation using AD-mix α and β to yield diols with 24S-OH (20) and 24R-OH (21), respectively. Protection of the C24 position with benzyl ether and two-step oxidation afforded carboxylic acids (28,29). These were treated with trimethylsilyl diazomethane in methanol to produce methyl esters (30,31), which were subsequently hydrogenated to afford 24-hydroxylated methyl esters (9,10). Next, introduction of a fluorine atom was achieved via deoxyfluorination reaction using DAST. The addition of an excess of methyl magnesium chloride to the resulting fluoro methyl esters (11,12) in THF, followed by desilylation at the C8 position in the presence of p-toluenesulfonic acid, yielded 24-fluorinated CD-ring fragments (15,16).

Experimental Section
1 H and 13 C NMR spectra were recorded on JEOL AL-400 NMR (400 MHz) and ECP-600 NMR (600 MHz) spectrometers (Tokyo, Japan). 1 H NMR spectra were referenced with (CH 3 ) 4 Si (δ 0.00 ppm) or CHCl 3 (δ 7.26 ppm) as internal standards. 13 C NMR spectra were referenced with deuterated solvent (δ 77.0 ppm for CDCl 3 ). IR spectra were recorded on a JASCO FT-IR-800 Fourier-transform infrared spectrophotometer (Tokyo, Japan). Highresolution mass spectra were obtained on a SHIMADZU LCMS-IT-TOF mass spectrometer (Kyoto, Japan) with an electrospray ionization (ESI) method or atmospheric-pressure chemical ionization (APCI). Optical rotations were measured on a JASCO DIP-370 digital polarimeter (Tokyo, Japan). Column chromatography was performed on silica gel 60N (40-50 µm, Kanto Chemical Co., Inc., Tokyo, Japan) or silica gel 60 (0.040-0.063 mm, Merck, Tokyo Japan). All experiments were performed under anhydrous conditions in an atmosphere of argon, unless otherwise stated. The supporting information of 1 H and 13 (20) A mixture of AD-mix α (4.01 g) in tBuOH (10 mL) and H 2 O (10 mL) was stirred at 0 • C for 25 min; 19 (303.5 mg, 0.255 mmol) was added to the mixture at 0 • C, and it was stirred at the same temperature for 5 h, and then at room temperature for 15 h under air. After the reaction was quenched with water, the mixture was extracted with EtOAc three times, dried over Na 2 SO 4 , filtered, and concentrated. The residue was purified via flash column chromatography on silica gel (hexane:EtOAc = 1:1) to obtain 20 (253.9 mg, 79%) as a colorless oil.  A mixture of AD-mix β (4.62 g) in tBuOH (15 mL) and H 2 O (15 mL) was stirred at 0 • C for 25 min; 19 (418.4 mg, 0.255 mmol) was added to the mixture at 0 • C, and it was stirred at the same temperature for 1 h 35 min under air. After the reaction was quenched with water, the mixture was extracted with EtOAc three times, dried over Na 2 SO 4 , filtered, and concentrated. The residue was purified via flash column chromatography on silica gel (hexane:EtOAc = 1:1) to obtain 21 (433.1 mg, 94%) as a colorless oil.  Benzaldehyde dimethyl acetal (374.4 mg, 369 µL, 2.46 mmol) and pyridinium ptoluenesulfonate (PPTS) (158.6 mg, 0.63 mmol) were added to a solution of 20 (472.0 mg, 1.23 mmol) in toluene (15 mL) at room temperature, and the mixture was stirred at the same temperature for 2 h. After the reaction was quenched with water and saturated aqueous NaHCO 3 , the mixture was extracted with EtOAc three times, dried over Na 2 SO 4 , filtered, and concentrated. The residue was purified via flash column chromatography on silica gel (hexane:EtOAc = 4:1) to obtain the crude acetal 22, which was used for the next reaction without further purification. To a solution of the above crude acetal 22 in CH 2 Cl 2 (15 mL), we added DIBAL-H (4.8 mL, 1.03 M in hexane solution, 4.92 mmol) at 0 • C, and the mixture was stirred at the same temperature for 20 min. After the reaction was quenched with MeOH at 0 • C, H 2 O and saturated aqueous potassium sodium tartrate were added at room temperature. The mixture was extracted with CH 2 Cl 2 four times, dried over Na 2 SO 4 , filtered, and concentrated. The residue was purified via flash column chromatography on silica gel (hexane:EtOAc = 4:1) to obtain 24 (501.1 mg, 86%) as a colorless oil.
Benzaldehyde dimethyl acetal (39.3 mg, 39 µL, 0.258 mmol) and pyridinium ptoluenesulfonate (PPTS) (4.5 mg, 0.018 mmol) were added to a solution of 21 (49.6 mg, 0.129 mmol) in toluene (0.7 mL) at room temperature, and the mixture was stirred at the same temperature for 2 h. After the reaction was quenched with water and saturated aqueous NaHCO 3 , the mixture was extracted with EtOAc three times, dried over Na 2 SO 4 , filtered, and concentrated. The residue was purified via flash column chromatography on silica gel (hexane:EtOAc = 5:1) to obtain the crude acetal 23, which was used for the next reaction without further purification. To a mixture of the above crude acetal 23 in CH 2 Cl 2 (2 mL), we added DIBAL-H (313 µL, 1.03 M in hexane solution, 0.322 mmol) at −40 • C, and the mixture was stirred at the same temperature for 1 h, and then at room temperature for 1 h. After the reaction was quenched with MeOH, H 2 O and saturated aqueous potassium sodium tartrate were added at room temperature. The mixture was extracted with CH 2 Cl 2 three times, dried over Na 2 SO 4 , filtered, and concentrated. The residue was purified via flash column chromatography on silica gel (hexane:EtOAc = 4:1) to obtain 25 (44.4 mg, 72%) as a colorless oil.   (28) Dess-Martin periodinane (1.42 g, 3.35 mmol) was added to a mixture of 24 (490.2 mg, 1.03 mmol) and 4Å molecular sieves (321.9 mg) in CH 2 Cl 2 (10 mL) at 0 • C, and the mixture was stirred at the same temperature for 2 h. After the reaction was quenched with water and saturated aqueous NaHCO 3 , the mixture was extracted with CH 2 Cl 2 three times, dried over Na 2 SO 4 , filtered, and concentrated. The residue was purified via flash column chromatography on silica gel (hexane:EtOAc = 10:1) to obtain the crude aldehyde 26, which was used for the next reaction without further purification. To a mixture of the above crude aldehyde 26 and NaH 2 PO 4 (1.216 g, 8.11 mmol) in H 2 O (9 mL) and t-BuOH (18 mL), NaClO 2 (575.9 mg, 6.37 mmol) was added at 0 • C under air and stirred at the same temperature for 30 min. After the reaction was quenched with aqueous saturated NH 4 Cl and aqueous saturated sodium thiosulfate, the mixture was extracted with EtOAc three times, washed with brine, dried over Na 2 SO 4 , filtered, and concentrated. The residue was purified via flash column chromatography on silica gel (hexane:EtOAc = 1:1) to obtain 28 (960.6 mg, 99%) as a colorless oil.   (29) Dess-Martin periodinane (2.76 g, 6.51 mmol) was added to a mixture of 25 (1.03 g, 2.17 mmol) and 4Å molecular sieves (600.0 mg) in CH 2 Cl 2 (10 mL) at 0 • C, and the mixture was stirred at the same temperature for 2 h. After the reaction was quenched with water and saturated aqueous NaHCO 3 , the mixture was extracted with CH 2 Cl 2 three times, dried over Na 2 SO 4 , filtered, and concentrated. The crude residue 27 was used for the next reaction without further purification. To a mixture of the above crude aldehyde 27 in H 2 O (3 mL) and t-BuOH (6 mL), NaH 2 PO 4 (134.8 mg, 0.898 mmol) and NaClO 2 (24.6 mg, 0.272 mmol) were added at 0 • C under air and stirred at the same temperature for 30 min. After the reaction was quenched with aqueous saturated NH 4 Cl and aqueous saturated sodium thiosulfate, the mixture was extracted with EtOAc three times, washed with brine, dried over Na 2 SO 4 , filtered, and concentrated. The residue was purified via flash column chromatography on silica gel (hexane:EtOAc = 1:1) to obtain 29 (125. 3 Trimethylsilyl diazomethane (1.1 mL, 2.0 M in diethyl ether, 2.16 mmol) was added to a solution of 28 (490.2 mg, 1.03 mmol) in MeOH (2 mL) and CH 2 Cl 2 (6 mL) at 0 • C, and the mixture was stirred at the same temperature for 17 min. After the reaction was quenched with acetic acid and saturated aqueous NaHCO 3 , the mixture was extracted with CH 2 Cl 2 three times, dried over Na 2 SO 4 , filtered, and concentrated. The residue was purified via flash column chromatography on silica gel (hexane:EtOAc = 4:1) to obtain 30 (387 mg, 100%) as a colorless oil.  5 mL) and CH 2 Cl 2 (4.5 mL) at 0 • C, and the mixture was stirred at the same temperature for 20 min. After the reaction was quenched with acetic acid and saturated aqueous NaHCO 3 , the mixture was extracted with CH 2 Cl 2 three times, dried over Na 2 SO 4 , filtered, and concentrated. The residue was purified via flash column chromatography on silica gel (hexane:EtOAc = 4:1) to obtain 31 (125.1 mg, 97%) as a colorless oil.   DAST (48.0 mg, 43 µL, 0.30 mmol) was added to a solution of 10 (20.5 mg, 0.05 mmol) in CH 2 Cl 2 (5 mL) at 0 • C, and the mixture was stirred at the same temperature for 90 min. After the reaction was quenched with MeOH, H 2 O, and saturated aqueous NaHCO 3 at 0 • C, the mixture was extracted with CH 2 Cl 2 three times, dried over Na 2 SO 4 , filtered, and concentrated. The residue was purified via flash column chromatography on silica gel (hexane:EtOAc = 8:1) to obtain 11 (15.5 mg, 75%) as a colorless oil.  (12) DAST (195.0 mg, 173 µL, 1.21 mmol) was added to a solution of 9 (99.7 mg, 0.24 mmol) in CH 2 Cl 2 (3 mL) at 0 • C, and the mixture was stirred at the same temperature for 2 h 15 min. After the reaction was quenched with MeOH, H 2 O, and saturated aqueous NaHCO 3 at 0 • C, the mixture was extracted with CH 2 Cl 2 three times, dried over Na 2 SO 4 , filtered, and concentrated. The residue was purified via flash column chromatography on silica gel (hexane:EtOAc = 8:1) to obtain 12 (31.0 mg, 31%) as a colorless oil.  MeMgCl (264 µL, 3.0 M THF solution, 0.79 mmol) was added to the mixture at 0 • C and stirred at the same temperature for 5 min. After the reaction was quenched with H 2 O, the mixture was extracted with EtOAc three times, washed with saturated aqueous NH 4 Cl, dried over Na 2 SO 4 , filtered, and concentrated. The crude residue was used for the next reaction without further purification. To the above crude residue in MeOH (10 mL) and CH 2 Cl 2 (5 mL), we added p-toluenesulfonic acid monohydrate (399.2 mg, 2.10 mmol), and the mixture was stirred at room temperature for 24 h under air. After the reaction was quenched with H 2 O and saturated aqueous NaHCO 3 at room temperature, the mixture was extracted with CH 2 Cl 2 three times, dried over Na 2 SO 4 , filtered, and concentrated. The residue was purified via flash column chromatography on silica gel (hexane:EtOAc = 1:1) to obtain 15 (36.3 mg, 61%, in 2 steps) as a white powder.  MeMgCl (264 µL, 3.0 M THF solution, 0.79 mmol) was added to the mixture at 0 • C and further stirred for 10 min. After the reaction was quenched with H 2 O, the mixture was extracted with EtOAc three times, washed with saturated aqueous NH 4 Cl, dried over Na 2 SO 4 , filtered, and concentrated. The crude residue was used for the next reaction without further purification. To the above crude residue in MeOH (10 mL) and CH 2 Cl 2 (5 mL), we added p-toluenesulfonic acid monohydrate (380.7 mg, 2.0 mmol), and the mixture was stirred at room temperature for 24 h under air. After the reaction was quenched with H 2 O and saturated aqueous NaHCO 3 at room temperature, the mixture was extracted with CH 2 Cl 2 three times, dried over Na 2 SO 4 , filtered, and concentrated. The residue was purified via flash column chromatography on silica gel (hexane:EtOAc = 2:1) to obtain 16 (18.8  4-Methylmorpholine N-oxide (32.6 mg, 0.28 mmol) was added to a solution of 15 (22.2 mg, 0.074 mmol) in CH 2 Cl 2 (2 mL), and the mixture was cooled to 0 • C. Tetrapropylammonium perruthenate (TPAP, 15.2 mg, 0.043 mmol) was added to the mixture, and the mixture was stirred at room temperature for 1 h. The reaction was diluted with Et 2 O, and the mixture was directly purified via flash column chromatography on silica gel (Et 2 O only) to obtain the crude ketone, which was used for the next reaction without further purification.
nBuLi (191 µL, 1.55 M hexane solution, 0.30 mmol) was added to a solution of A-ring phosphine oxide [16] (132.6 mg, 0.29 mmol) in THF (1.5 mL) at −78 • C. After stirring for 15 min, the solution of crude 32 in THF (2 mL) was added, and the mixture was stirred at −78 • C for 15 min and 0 • C for 5 min. After the reaction was quenched with H 2 O and saturated aqueous NH 4 Cl at the same temperature, the mixture was extracted with EtOAc three times, washed with brine, dried over Na 2 SO 4 , filtered, and concentrated. The residue was purified via flash column chromatography on silica gel (hexane:EtOAc = 10:1) to obtain the crude coupling product (34.9 mg), which was used for the next reaction without further purification. Tetrabutylammonium fluoride (370 µL, 1 M THF solution, 0.37 mmol) was added to a solution of the crude coupling product (34.9 mg) in THF (2 mL), and the mixture was stirred at room temperature for 16 h. After the reaction was quenched with H 2 O at room temperature, the mixture was extracted with EtOAc three times, dried over Na 2 SO 4 , filtered, and concentrated. The residue was purified via flash column chromatography on silica gel (hexane:EtOAc = 1:1) to obtain 3 (15.6 mg, 50%, in 4 steps) as a white powder.  4-Methylmorpholine N-oxide (26.1 mg, 0.22 mmol) was added to a solution of 16 (18.8 mg, 0.063 mmol) in CH 2 Cl 2 (2 mL), and the mixture was cooled to 0 • C. TPAP (11.2 mg, 0.032 mmol) was added to the mixture, and the mixture was stirred at 0 • C for 10 min and room temperature for 20 min. The reaction was diluted with Et 2 O, and the mixture was directly purified via flash column chromatography on silica gel (Et 2 O only) to obtain the crude ketone, which was used for the next reaction without further purification.
TMSCl (68.4 mg, 80 µL, 0.63 mmol) was added to the 0 • C cooled solution of crude ketone and imidazole (43.7 mg, 0.64 mmol) in CH 2 Cl 2 (2 mL), and the mixture was stirred for 7 min at room temperature. After the reaction was quenched with H 2 O and saturated aqueous NH 4 Cl, the mixture was extracted with CH 2 Cl 2 three times, dried over Na 2 SO 4 , filtered, and concentrated. The residue was purified via flash column chromatography on silica gel (hexane:EtOAc = 10:1) to obtain crude 33.
nBuLi (163 µL, 1.55 M hexane solution, 0.25 mmol) was added to a solution of A-ring phosphine oxide [16] (117.4 mg, 0.26 mmol) in THF (1.5 mL) at −78 • C. After stirring for 15 min, a solution of crude 33 in THF (2 mL) was added, and the mixture was stirred at −78 • C for 15 min and 0 • C for 5 min. After the reaction was quenched with H 2 O at the same temperature, the mixture was extracted with EtOAc three times, washed with brine, dried over Na 2 SO 4 , filtered, and concentrated. The residue was purified via flash column chromatography on silica gel (hexane:EtOAc = 10:1) to obtain the crude coupling product (24.7 mg), and it was used for the next reaction without further purification. Tetrabutylammonium fluoride (315 µL, 1 M THF solution, 0.32 mmol) was added to a solution of the crude coupling product (24.7 mg) in THF (2 mL), and the mixture was stirred at room temperature for 16 h. After the reaction was quenched with H 2 O at room temperature, the mixture was extracted with EtOAc three times, dried over MgSO 4 , filtered, and concentrated. The residue was purified via flash column chromatography on silica gel (hexane:EtOAc = 1:1) to obtain 4 (16.0 mg, 61%, in 4 steps) as a white powder.  MeMgCl (0.53 mL, 3.0 M THF solution, 1.59 mmol) was added to a solution of 30 (133.4 mg, 0.265 mmol) in THF (4 mL) at 0 • C, and the mixture was stirred at 0 • C for 11 min. After the reaction was quenched with H 2 O, the mixture was extracted with EtOAc three times, washed with saturated aqueous NH 4 Cl, dried over Na 2 SO 4 , filtered, and concentrated. The residue was purified via flash column chromatography on silica gel (hexane:EtOAc = 4:1) to obtain crude 34 (130.0 mg), and it was used for the next reaction without further purification.
To a solution of crude 34 (130.0 mg) in MeOH (4 mL), we added 10% Pd/C catalyst (20.0 mg). The mixture was stirred for 6 days at room temperature under a hydrogen atmosphere. The reaction mixture was diluted with EtOAc, filtered through a Celite pad, and concentrated under reduced pressure. Purification via flash column chromatography on silica gel (hexane:EtOAc = 2:1) yielded 36 (87.2 mg, 82%) as a colorless oil [25].  MeMgCl (415 µL, 3.0 M THF solution, 1.25 mmol) was added to a solution of 31 (125.1 mg, 0.249 mmol) in THF (3 mL) at 0 • C, and the mixture was stirred at 0 • C for 7 min. After the reaction was quenched with H 2 O, the mixture was extracted with EtOAc three times, washed with saturated aqueous NH 4 Cl, dried over Na 2 SO 4 , filtered, and concentrated. The residue was purified via flash column chromatography on silica gel (hexane:EtOAc = 4:1) to obtain crude 35, which was used for the next reaction without further purification.
To a solution of crude 35 in MeOH (4 mL), we added 10% Pd/C catalyst (20.0 mg). The mixture was stirred for 68 h at room temperature under a hydrogen atmosphere. The reaction mixture was diluted with EtOAc, filtered through a Celite pad, and concentrated under reduced pressure. Purification via flash column chromatography on silica gel (hexane:EtOAc = 2:1) yielded 37 (24.1 mg, 23%, 35 recovery 54%) as a colorless oil [25].  p-Toluenesulfonic acid monohydrate (199.1 mg, 1.01 mmol) was added to a solution of 36 (46.5 mg, 0.11 mmol) in MeOH (4 mL) and CH 2 Cl 2 (4 mL), and the mixture was stirred at room temperature for 45 h under air. After the reaction was quenched with H 2 O and saturated aqueous NaHCO 3 at room temperature, the mixture was extracted with CH 2 Cl 2 three times, washed with brine, dried over Na 2 SO 4 , filtered, and concentrated. The residue was purified via flash column chromatography on silica gel (EtOAc only) to obtain 13 (30.4 mg, 90%) as a colorless oil. The spectral data of the product matched those reported in the literature [25]. p-Toluenesulfonic acid monohydrate (192.9 mg, 1.01 mmol) was added to a solution of 37 (49.2 mg, 0.12 mmol) in MeOH (5 mL) and CH 2 Cl 2 (5 mL), and the mixture was stirred at room temperature for 53 h under air. After the reaction was quenched with H 2 O and saturated aqueous NaHCO 3 at room temperature, the mixture was extracted with CH 2 Cl 2 three times, washed with brine, dried over Na 2 SO 4 , filtered, and concentrated. The residue was purified via flash column chromatography on silica gel (EtOAc only) to obtain 14 (30.4 mg, 85%) as a colorless oil. The spectral data of the product matched those reported in the literature [25].  PPTS (19.4 mg, 0.08 mmol) was added to a solution of 14 (30.4 mg, 0.10 mmol) in acetone (1 mL) and 2,2-dimethoxypropane (1 mL), and the mixture was stirred at room temperature for 4 h under air. After the reaction was quenched with H 2 O and saturated aqueous NaHCO 3 at room temperature, the mixture was extracted with EtOAc three times, dried over Na 2 SO 4 , filtered, and concentrated. The residue was purified via flash column chromatography on silica gel (hexane:EtOAc = 1:1) to obtain 39 (28.6 mg, 83%) as a colorless oil. The spectral data of the product matched those reported in the literature [25].  4-Methylmorpholine N-oxide (28.8 mg, 0.25 mmol) was added to a solution of 39 (28.6 mg, 0.085 mmol) in CH 2 Cl 2 (1 mL), and the mixture was cooled to 0 • C. TPAP (13.9 mg, 0.04 mmol) was added to the mixture, and the mixture was stirred at 0 • C for 40 min. The reaction was diluted with Et 2 O, and the mixture was directly purified via flash column chromatography on silica gel (Et 2 O only), followed by purification via flash column chromatography on silica gel (hexane:EtOAc = 4:1), to obtain 41 (28.2 mg, 99%) as a colorless oil. The spectral data of the product matched those reported in the literature [25].  5 mL) was added, and the mixture was stirred at −78 • C for 2 h. After the reaction was quenched with H 2 O at the same temperature, the mixture was extracted with EtOAc three times, washed with brine, dried over Na 2 SO 4 , filtered, and concentrated. The residue was purified via flash column chromatography on silica gel (hexane:EtOAc = 10:1) to obtain the crude coupling product (39.4 mg), which was used for the next reaction without further purification. Tetrabutylammonium fluoride (414 µL, 1 M THF solution, 0.414 mmol) was added to the solution of the crude coupling product (39.4 mg) in THF (3 mL), and the mixture was stirred at room temperature for 15 h. After the reaction was quenched with H 2 O and aqueous saturated NH 4 Cl at room temperature, the mixture was extracted with EtOAc three times, dried over Na 2 SO 4 , filtered, and concentrated. The residue was purified via flash column chromatography on silica gel (hexane:EtOAc = 1:2) to obtain the crude product, which was used for the next reaction without further purification.
The above crude residue was dissolved in MeOH (10 mL), and AG 50W-X4 resin (177.2 mg) was added. The mixture was then stirred for 26 h, and the solids were filtered off, washed with MeOH, and the solution was concentrated in vacuo. The residue was purified via flash column chromatography (hexane:EtOAc = 1:2) to obtain 5 (20.7 mg, 66%) as a white powder. The spectral data of the product matched those reported in the literature [25].

(24R)-24,25-Dihydroxyvitamin D 3 (6)
nBuLi (163 µL, 1.55 M hexane solution, 0.252 mmol) was added to a solution of Aring phosphine oxide [16] (110.7 mg, 0.24 mmol) in THF (1 mL) at −78 • C. After stirring for 20 min, a solution of 41 (28.2 mg, 0.084 mmol) in THF (1 mL) was added, and the mixture was stirred at −78 • C for 2 h 30 min. After the reaction was quenched with H 2 O at the same temperature, the mixture was extracted with EtOAc three times, washed with brine, dried over Na 2 SO 4 , filtered, and concentrated. The residue was purified via flash column chromatography on silica gel (hexane:EtOAc = 10:1) to obtain the crude coupling product (43.3 mg), which was used for the next reaction without further purification. Tetrabutylammonium fluoride (420 µL, 1 M THF solution, 0.42 mmol) was added to the solution of the crude coupling product (43.3 mg) in THF (3 mL), and the mixture was stirred at room temperature for 17 h. After the reaction was quenched with H 2 O and aqueous saturated NH 4 Cl at room temperature, the mixture was extracted with EtOAc three times, dried over Na 2 SO 4 , filtered, and concentrated. The residue was purified via flash column chromatography on silica gel (hexane:EtOAc = 1:1) to obtain the crude product, which was used for the next reaction without further purification.
The above crude residue was dissolved in MeOH (5 mL), and AG 50W-X4 resin (167.5 mg) was added. The mixture was stirred for 24 h, and the solids were filtered off, washed with MeOH, and the solution was concentrated in vacuo. The residue was purified via flash column chromatography (hexane:EtOAc = 1:2) to obtain 6 (26.6 mg, 76%, in 3 steps) as a white powder. The spectral data of the product matched those reported in the literature [25].

Measurement of the hVDR Binding Affinity of 3, 4, and 24,24-Difluoro-25(OH)D 3
The binding affinity of each analogue for hVDR was evaluated using an in vitro system based on the split-luciferase technique described in our previous study [29]. Briefly, 50 µL of cell lysate prepared from recombinant Escherichia coli expressing split-luciferase vitamin D biosensor protein [29] was added to each well of a 96-well plate, and left for 10 min at room temperature. Then, 50 µL of the luciferin solution containing 20 mM MgSO 4 , 2 mM D-luciferin, and 4 mM adenosine triphosphate in 25 mM Tris-HCl (pH 7.4) was injected into each well and incubated for 15 min at room temperature. The luminescence (photon counts) was measured using a luminometer. The relative hVDR binding affinity of each analogue was evaluated based on the concentration at which the luminescence showed 50% of the maximum value.

Conclusions
In summary, in this paper we described novel stereoselective syntheses of 24-fluoro-25hydroxyvitamin D 3 (3 and 4) and 24,25-dihydroxyvitamin D 3 (5 and 6). To our knowledge, this is the first reported study to synthesize both 24R-and 24S-24-fluorinated vitamin D 3 analogues. This approach also provides a practical synthetic route to one of the main natural metabolites of 25(OH)D 3 by hCYP24A1-(24R)-24,25-dihydroxyvitamin D 3 (6). This synthetic method paves the way for efficient access to 24-substituted vitamin D 3 analogues. Synthesis of new 24-substituted vitamin D 3 analogues utilizing this method, along with evaluation of their biological activities, is in progress.
Funding: This work was supported in part by Grants-in-Aid from the Japan Society for the Promotion of Science (No. 18K06556 to A.K. and No. 19H02889 to T.S.).