Synthesis of New 26,27-Difluoro- and 26,26,27,27-Tetrafluoro-25-hydroxyvitamin D₃: Effects of Terminal Fluorine Atoms on Biological Activity and Half-life

Abstract

As an extension of our research on providing a chemical library of side-chain fluorinated vitamin D₃ analogues, we newly designed and synthesized 26,27-difluoro-25-hydroxyvitamin D₃ (1) and 26,26,27,27-tetrafluoro-25-hydroxyvitamin D₃ (2) using a convergent method applying the Wittig–Horner coupling reaction between CD-ring ketones (13, 14) and A-ring phosphine oxide (5). The basic biological activities of analogues, 1, 2, and 26,26,26,27,27,27-hexafluoro-25-hydroxyvitamin D₃ [HF-25(OH)D₃] were examined. Although the tetrafluorinated new compound 2 exhibited higher binding affinity for vitamin D receptor (VDR) and resistance to CYP24A1-dependent metabolism compared with the difluorinated 1 and its non-fluorinated counterpart 25-hydroxyvitamin D₃ [25(OH)D₃], HF-25(OH)D₃ showed the highest activity among these compounds. Osteocalcin promoter transactivation activity of these fluorinated analogues was tested, and it decreased in the order of HF-25(OH)D₃, 2, 1, and 25(OH)D₃ in which HF-25(OH)D₃ showed 19-times greater activity than the natural 25(OH)D₃.

Introduction

The introduction of fluorine to the vitamin D₃ side-chain is a powerful tool for the application of vitamin D₃ to pharmaceuticals, since it significantly affects binding affinity to vitamin D receptor (VDR) and vitamin D binding protein (DBP), while it also confers resistance to CYP24A1 metabolism.^1–7) 26,26,26,27,27,27-Hexafluoro-1α,25-dihydroxyvitamin D₃ (falecalcitriol) is one such side-chain fluorinated analogue of active vitamin D₃, 1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃]. This fluorination was designed to resist CYP24A1 metabolism, and falecalcitriol has been clinically applied to treat secondary hyperparathyroidism^1,8–12) (Fig. 1).

Fig. 1. Structures of 25-Hydroxyvitamin D₃ [25(OH)D₃], 1α,25-Dihydroxyvitamin D₃ [1α,25(OH)₂D₃], 26,26,26,27,27,27-Hexafluoro-1α,25-dihydroxyvitamin D₃ (falecalcitriol), and Target Analogues of 26,27-Difluoro-25-hydroxyvitamin D₃ (1), 26,26,27,27-Tetrafluoro-25-hydroxyvitamin D₃ (2) as well as Known 26,26,26,27,27,27-Hexafluoro-25-hydroxyvitamin D₃ [HF-25(OH)D₃]⁸⁾

We have developed the efficient synthesis of side-chain fluorinated vitamin D₃ analogues to evaluate the effects of the fluorinated position, stereochemistry, and number of fluorine atoms at the side-chain on binding affinity to VDR, osteocalcin promoter transactivation activity, and metabolic resistance to CYP24A1.^4–7,13)

Here, we studied the effect of 26,27-difluoro- and 26,26,27,27-tetrafluoro-terminal dimethyl groups on vitamin D₃ activities compared with the 26,26,26,27,27,27-hexafluorodimethyl and natural dimethyl groups. In this paper, we describe the synthesis of novel C26- and C27-fluorinated analogues, 26,27-difluoro-25-hydroxyvitamin D₃ (1) and 26,26,27,27-tetrafluoro-25-hydroxyvitamin D₃ (2), along with their biological activity (Fig. 1).

Our synthetic plan for the C26- and C27-fluorinated analogues (1, 2) is outlined in Chart 1. We chose the convergent method, by which we could introduce various designed A-ring moieties (in this report, the A-ring structure was 5), and decided to synthesize two CD-ring fragments (3, 4) from Inhoffen–Lythgoe diol.

Chart 1. Convergent Synthesis of 1 and 2 via C26,27-Fluoro-CD-ring Fragments (3, 4)

Results and Discussion

The synthesis of 26,27-F₂-CD-ring (3) is shown in Chart 2. The aldehyde 6,¹⁴⁾ synthesized from Inhoffen–Lythgoe diol, was converted to an alkyne 8 by the Corey–Fuchs procedure.¹⁵⁾ The acetylide was produced in situ from 8,^16,17) followed by nucleophilic addition to 1,3-difluoroacetone to give 9. Reduction of the alkyne moiety of 9 by hydrogenation in the presence of a palladium catalyst, and subsequent desilylation of the C8-hydroxy group gave the target 26,27-F₂-CD-ring 3 in high yield.

Chart 2. Synthesis of 26,27-F₂-CD-ring (3) Using 1,3-Difluoroacetone as the Fluorine Source

Next, synthesis of the 26,26,27,27-F₄-CD-ring (4) is shown in Chart 3. First, the methyl ester 10¹⁸⁾ synthesized from Inhoffen–Lythgoe diol in 3 steps was converted to carboxylic acid 11 by hydrolysis. After preparing the acid chloride from 11, the tetrafluoro moiety was introduced to the side-chain terminal using a difluoromethylation reaction to obtain 12,¹⁹⁾ which was deprotected under acidic conditions to give 4.

Chart 3. Synthesis of 26,26,27,27-F₄-CD-ring (4) Using Difluoromethylation Reaction as a Key Step

The C8-OH groups of 3 and 4 were oxidized by tetrapropylammonium perruthenate (TPAP), and then C25-OH groups were protected to give ketones 13 and 14, respectively (Chart 4).

Chart 4. Synthesis of 8-Oxo-CD-rings 13 and 14

With the corresponding counterparts, the Wittig–Horner reaction was examined between the A-ring (5) and CD-rings (13, 14).²⁰⁾ In the presence of nBuLi, the coupling reaction proceeded smoothly to afford the desired products. Removal of the protective groups with tetrabutylammonium fluoride (TBAF) yielded 26,27-difluoro-25-hydroxyvitamin D₃ (1) and 26,26,27,27-tetrafluoro-25-hydroxyvitamin D₃ (2), respectively (Chart 5).

Chart 5. Coupling Reaction and Deprotection Steps for 1 and 2

The binding affinity for the human vitamin D receptor (hVDR) and human CYP24A1-dependent metabolism of the new synthetic analogues (1, 2) and HF-25(OH)D₃^8,21) were examined to compare with those of the natural 25(OH)D₃ (Tables 1, 2).

Table 1. Relative hVDR Binding Affinity of 26,27-Fluorinated 25(OH)D₃ Analogues

Compound	Relative hVDR binding affinity (%)
25(OH)D3	100
26,27-F2-25(OH)D3 (1)	95
26,26,27,27-F4-25(OH)D3 (2)	710
HF-25(OH)D3	5700

Table 2. Metabolic Activities of Human CYP24A1 toward 25(OH)D₃ and Its 26,27-Fluorinated Analogues

Substrate	(nmol/min/nmol-P450)
25(OH)D3	4.56 ± 0.38
26,27-F2-25(OH)D3 (1)	3.16 ± 0.15
26,26,27,27-F4-25(OH)D3 (2)	1.82 ± 0.14
HF-25(OH)D3	0.59 ± 0.10

Data were obtained at a substrate concentration of 5 µM. Each value represents the mean ± standard deviation (S.D.) of three separate experiments.

Although 26,27-difluorinated analogue 1 had similar properties to 25(OH)D₃, tetrafluoro-analogue 2 showed 7.1- and 2.5-times more potent VDR-binding and CYP24A1 resistance than 25(OH)D₃, respectively. However, HF-25(OH)D₃, which was synthesized by our group using the efficient two-step trifluoromethylation method,⁸⁾ exhibited the highest activities: 57- and 7.7-times greater activities than those of 25(OH)D₃, respectively.

As shown in Table 3, osteocalcin promoter transactivation activity of these analogues in human osteosarcoma (HOS) cells under serum-free conditions showed the same order of potency as above; HF-25(OH)D₃ had 19-times greater activity than 25(OH)D₃, and fluorine atoms at the C26- and C27-positions enhanced the biological activity of 25(OH)D₃.

Table 3. Osteocalcin Promoter Transactivation Activity of 25(OH)D₃ and Its 26,27-Fluorinated Analogues, 1, 2, and HF-25(OH)D₃

Compound	Osteocalcin transactivation activity in HOS/SF cells (EC50, nM)
25(OH)D3	319
26,27-F2-25(OH)D3 (1)	294
26,26,27,27-F4-25(OH)D3 (2)	179
HF-25(OH)D3	16.6

Through the above studies, it was clarified that the effect of fluorination on in vitro activities (VDR affinity and osteocalcin promoter transactivation activity) was enhanced in the order of the bismonofluoromethyl groups (1), bisdifluoromethyl groups (2), and bistrifluoromethyl groups [HF-25(OH)D₃].

As shown in Table 4, it was confirmed that pK_a at the 25-hydroxy group was decreased as the number of introduced fluorine atoms increased. We consider that the acidity of the 25-OH group affects the binding affinity for VDR directly, and consequently increases the biological activities.

Table 4. pK_a at the 25-Hydroxy Group of 25(OH)D₃ and Its 26,27-Fluorinated Analogues, 1, 2, and HF-25(OH)D₃

Compound	pKa at 25-hydroxy group
25(OH)D3	14.6
26,27-F2-25(OH)D3 (1)	12.9
26,26,27,27-F4-25(OH)D3 (2)	11.1
HF-25(OH)D3	9.3

pK_a of each compound was calculated by ChemDraw professional version 18.

Conclusion

In this study, we synthesized 26,27-difluoro-25-hydroxyvitamin D₃ (1) and 26,26,27,27-tetrafluoro-25-hydroxyvitamin D₃ (2) via a convergent coupling route. The key CD-ring fragments (3, 4) were prepared from Inhoffen-Lythgoe diol. Using the Wittig–Horner reaction between 8-oxo-CD-rings (13, 14) and A-ring phosphine oxide 5, we were able to synthesize the two target analogues 1 and 2. Compound 2 showed higher VDR binding affinity and resistance to CYP24A1-dependent metabolism than the natural 25(OH)D₃, while 1 had similar properties to 25(OH)D₃. 26,26,26,27,27,27-Hexafluoro-25-hydroxyvitamin D₃^8,21) showed even higher VDR binding affinity and resistance to CYP24A1 metabolism. The 26,27-fluorovitamin D₃ analogues showed greater osteocalcin promoter transactivation activity in HOS cells than 25(OH)D₃, and actually, HF-25(OH)D₃ exhibited more than 19-times stronger activity than 25(OH)D₃. Our results indicate that the addition of fluorine at positions 26 and 27 of 25(OH)D₃ had significantly favorable effects on biological activity.

Experimental

General

¹H- and ¹³C-NMR spectra were recorded on JEOL AL-400 NMR (400 MHz) and ECP-600 NMR (600 MHz) spectrometers (Tokyo, Japan). ¹H-NMR spectra were referenced with (CH₃)₄Si (δ 0.00 ppm) or CHCl₃ (δ 7.26 ppm for CDCl₃), or with CH₃OD (δ 3.34 ppm for CD₃OD). ¹³C-NMR spectra were referenced with deuterated solvent (δ 77.0 ppm for CDCl₃) or CD₃OD (δ 49.3 ppm for CD₃OD). IR spectra were recorded on a JASCO FT-lR-800 Fourier transform IR spectrophotometer (Tokyo, Japan). High-resolution mass spectra (HR-MS) were obtained on a SHIMADZU LCMS-IT-TOF mass spectrometer (Kyoto, Japan) with an electrospray ionization (ESI) method. Optical rotations were measured on a JASCO DIP-370 digital polarimeter (Tokyo, Japan). Column chromatography was performed on silica gel 60N (Kanto Chemical Co., Inc., 40–50 µm, Tokyo, Japan) or silica gel 60 (Merck, 0.040-0.063 mm, Tokyo, Japan). Preparative TLC was performed on silica gel 60 F₂₅₄ (Merck, 0.5 mm, Tokyo, Japan). All experiments were performed under anhydrous conditions in an atmosphere of argon, unless otherwise stated.

tert-Butyl({(1R,3aR,4S,7aR)-1-[(2R)-5,5-dibromopent-4-en-2-yl]-7a-methyloctahydro-1H-inden-4-yl}oxy)dimethylsilane (7)

To a solution of aldehyde 6¹⁴⁾ (408 mg, 1.2 mmol) in CH₂Cl₂ (5 mL) was added triphenylphosphine (1.01 g, 3.85 mmol) and CBr₄ (611.5 mg, 1.84 mmol) at room temperature, and the mixture was stirred at the same temperature for 17 min. After the reaction was quenched with H₂O, the mixture was extracted with CH₂Cl₂ three times. The organic layer was dried over Na₂SO₄, filtered, and concentrated. The residue was purified by flash column chromatography on silica gel (hexane only) to obtain 7 (561 mg, 95%) as a colorless oil.

7: [α]_D²⁷ +33.1 (c 4.82, CHCl₃); IR (neat) 1456, 1379, 1248, 1162, 1085, 1023, 837, 768 cm⁻¹; ¹H-NMR (400 MHz, CDCl₃) δ: 0.00 (s, 3H), 0.01 (s, 3H), 0.89 (s, 9H), 0.92 (s, 3H), 0.94 (d, J = 6.9 Hz, 3H), 1.01–1.16 (m, 2H), 1.22–1.40 (m, 5H), 1.52–1.69 (m, 3H), 1.74–1.96 (m, 4H), 2.02 (ddd, J = 3.2, 6.4, 14.6 Hz, 1H), 3.99–4.01 (m, 1H), 6.39 (dd, J = 6.4, 7.8 Hz, 1H); ¹³C-NMR (100 MHz, CDCl₃) δ: −5.1, −4.8, 13.7, 17.6, 18.0, 23.0, 25.8, 27.4, 34.4, 35.2, 39.5, 40.6, 42.2, 53.0, 56.5, 69.4, 88.6, 137.8; HR-MS (ESI⁻) Calcd for C₂₁H₃₈OSiBr₂Cl [M + Cl]⁻ 527.0753, Found 527.0757.

tert-Butyldimethyl({(1R,3aR,4S,7aR)-7a-methyl-1-[(2R)-pent-4-yn-2-yl]octahydro-1H-inden-4-yl}oxy)silane (8)

To a solution of 7 (561 mg, 1.13 mmol) in tetrahydrofuran (THF) (5 mL) was added nBuLi (1.87 mL, 1.59 M hexane solution, 2.84 mmol) at −78 °C, and the mixture was stirred at −78 °C for 5 min. After the reaction was quenched with H₂O at −78 °C, the mixture was extracted with EtOAc three times. The organic layer was dried over Na₂SO₄, filtered, and concentrated. The residue was purified by flash column chromatography on silica gel (hexane only) to obtain 8 (355.6 mg, 94%) as a colorless oil.

8: [α]_D²⁷ +55.9 (c 5.46, CHCl₃); IR (neat) 3315, 2192, 1468, 1379, 1252, 1166, 1089, 1023, 837, 775, 632 cm⁻¹; ¹H-NMR (400 MHz, CDCl₃) δ: −0.01 (s, 3H), 0.01 (s, 3H), 0.89 (s, 9H), 0.92 (s, 3H), 1.06 (d, J = 6.6 Hz, 3H), 1.12–1.29 (m, 4H), 1.32–1.40 (m, 3H), 1.53–1.62 (m, 2H), 1.65–1.68 (m, 1H), 1.76–1.84 (m, 2H), 1.92–1.95 (m, 2H), 2.02 (ddd, J = 2.4, 7.8, 17.4 Hz, 1H), 2.23 (dt, J = 3.0, 16.2 Hz, 1H), 3.99–4.01 (m, 1H); ¹³C-NMR (150 MHz, CDCl₃) δ: −5.2, −4.8, 13.9, 17.6, 18.0, 18.9, 23.0, 25.5, 25.8, 27.2, 34.4, 34.8, 40.5, 42.1, 53.0, 55.6, 69.1, 69.4, 83.5; HR-MS (ESI⁻) Calcd for C₂₂H₃₉O₃Si [M + HCOO]⁻ 379.2674, Found 379.2675.

(6R)-6-{(1R,3aR,4S,7aR)-4-[(tert-Butyldimethylsilyl)oxy]-7a-methyloctahydro-1H-inden-1-yl}-1-fluoro-2-(fluoromethyl)-hept-3-yn-2-ol (9)

To a solution of 8 (148.8 mg, 0.45 mmol) in THF (1 mL) was added nBuLi (336 µL, 1.59 M hexane solution, 0.53 mmol) at −78 °C, and the mixture was stirred at −78 °C for 30 min. 1,3-Difluoroacetone (61 µL, 83.7 mg, 0.89 mmol) was added to the mixture at the same temperature. After being stirred at −78 °C for 10 min, the reaction was quenched with H₂O and saturated aqueous NH₄Cl at −78 °C. The mixture was extracted with EtOAc three times. The organic layer was dried over Na₂SO₄, filtered, and concentrated. The residue was purified by flash column chromatography on silica gel (hexane : EtOAc = 7 : 1) to obtain 9 (181.7 mg, 95%) as a colorless oil.

9: [α]_D²⁷ +46.5 (c 4.73, CHCl₃); IR (neat) 3408, 2253, 1464, 1371, 1255, 1162, 1085, 1035, 837, 775 cm⁻¹; ¹H-NMR (600 MHz, CDCl₃) δ: −0.01 (s, 3H), 0.00 (s, 3H), 0.88 (s, 9H), 0.91 (s, 3H), 1.03 (d, J = 6.6 Hz, 3H), 1.09–1.13 (m, 2H), 1.19–1.26 (m, 2H), 1.32–1.39 (m, 3H), 1.52–1.68 (m, 3H), 1.74–1.84 (m, 2H), 1.91–1.94 (m, 1H), 2.02 (dd, J = 8.1, 16.5 Hz, 1H), 2.28 (dd, J = 3.2, 16.8 Hz, 1H), 2.51 (br s, 1H), 3.99–4.00 (m, 1H), 4.40–4.51 (m, 4H); ¹³C-NMR (150 MHz, CDCl₃) δ: −5.2, −4.8, 13.8, 17.6, 18.0, 18.9, 23.0, 25.7, 25.8, 27.1, 34.3, 34.9, 40.5, 42.1, 53.0, 55.8, 68.9 (t, J = 20.9 Hz), 69.3, 76.6 (t, J = 5.8 Hz), 84.1 (dd, J = 2.9, 179.6 Hz), 88.1; HR-MS (ESI⁻) Calcd for C₂₄H₄₂O₂F₂SiCl [M + Cl]⁻ 463.2616, Found 463.2605.

(1R,3aR,4S,7aR)-1-[(2R)-7-Fluoro-6-(fluoromethyl)-6-hydroxyheptan-2-yl]-7a-methyloctahydro-1H-inden-4-ol (3)

To a solution of 9 (181.7 mg, 0.42 mmol) in MeOH (10 mL) was added 10% Pd/C catalyst (17.0 mg). The mixture was stirred at room temperature under 1 atm hydrogen atmosphere for 14 h. The reaction mixture was diluted with EtOAc, filtered through a Celite pad, and concentrated under reduced pressure to obtain crude alcohol, and this was used for the next reaction without further purification.

p-Toluenesulfonic acid monohydrate (482.9 mg, 2.54 mmol) was added to a solution of the above crude alcohol in MeOH (10 mL). The mixture was stirred at room temperature for 1 h under air. After the reaction was quenched with H₂O and saturated aqueous NaHCO₃ at room temperature, the mixture was extracted with EtOAc three times. The organic layer was dried over Na₂SO₄, filtered, and concentrated. The residue was purified by flash column chromatography on silica gel (hexane : EtOAc = 2 : 1) to obtain 3 (130.6 mg, 97%, in 2 steps) as a colorless oil.

3: [α]_D²⁷ +34.5 (c 1.64, CHCl₃); IR (neat) 3419, 1460, 1375, 1271, 1162, 1019, 941, 914, 736 cm⁻¹; ¹H-NMR (600 MHz, CDCl₃) δ: 0.90 (d, J = 6.6 Hz, 3H), 0.92 (s, 3H), 1.01–1.09 (m, 2H), 1.14 (dt, J = 3.6, 13.2 Hz, 1H), 1.22–1.57 (m, 13H), 1.77–1.86 (m, 3H), 1.98–2.00 (m, 1H), 2.26 (d, J = 7.2 Hz, 1H), 4.06–4.07 (m, 1H), 4.28–4.42 (m, 4H); ¹³C-NMR (150 MHz, CDCl₃) δ: 13.5, 17.4, 18.4, 18.7, 22.5, 27.1, 33.0, 33.5, 35.1, 36.1, 40.3, 41.8, 52.5, 56.5, 69.4, 72.6 (t, J = 17.3 Hz), 83.5–84.8 (m); HR-MS (ESI⁻) Calcd for C₁₈H₃₁O₂F₂ [M−H]⁻ 317.2298, Found 317.2285.

(5R)-5-{(1R,3aR,4S,7aR)-4-[(tert-Butyldimethylsilyl)oxy]-7a-methyloctahydro-1H-inden-1-yl}hexanoic Acid (11)

Aqueous KOH (25 mL, 7.1 M solution) was added to a solution of methyl ester 10¹⁸⁾ (2.0 g, 5.05 mmol) in dioxane (50 mL), and the mixture was stirred at 60 °C for 19 h. After the reaction was quenched with H₂O and saturated aqueous NH₄Cl, the mixture was extracted with EtOAc three times. The organic layer was dried over Na₂SO₄, filtered, and concentrated. The residue was purified by flash column chromatography on silica gel (hexane : EtOAc = 3 : 1) to obtain 11 (1.95 g, 100%) as a colorless oil.

11: [α]_D²⁷ +48.8 (c 0.95, CHCl₃); IR (neat) 1706, 1458, 1409, 1232, 1164, 1082, 1017, 950, 724 cm⁻¹; ¹H-NMR (400 MHz, CDCl₃) δ: −0.01 (s, 3H), 0.00 (s, 3H), 0.88 (s, 9H), 0.90 (d, J = 6.4 Hz, 3H), 0.90 (s, 3H), 1.00–1.13 (m, 3H), 1.18–1.60 (m, 9H), 1.64–1.85 (m, 4H), 1.94 (dt, J = 2.7, 12.8 Hz, 1H), 2.24–2.38 (m, 2H), 3.98–4.00 (m, 1H); ¹³C-NMR (100 MHz, CDCl₃) δ: −5.2, −4.8, 13.7, 17.7, 18.0, 18.5, 21.3, 23.0, 25.8, 27.2, 34.5, 35.0, 35.2, 40.7, 42.1, 53.0, 56.5, 69.4, 179.8; HR-MS (ESI⁻) Calcd for C₂₂H₄₁O₃Si [M−H]⁻ 381.2830, Found 381.2824.

(1R,3aR,4S,7aR)-1-[(2R)-6-(Difluoromethyl)-7,7-difluoro-6-hydroxyheptan-2-yl]-7a-methyloctahydro-1H-inden-4-ol (4)

To a solution of 11 (604.6 mg, 1.58 mmol) in CH₂Cl₂ (5.0 mL) was added N,N-dimethylformamide (20 µL, 21.2 mg, 0.29 mmol) and oxalyl chloride (202 µL, 298.4 mg, 2.35 mmol) at room temperature, and the reaction mixture was stirred at room temperature for 1 h. The mixture was evaporated in vacuo, and the crude residue was used for the next reaction without further purification.

Ph₃P (1.90 g, 7.24 mmol) and N,N′-dimethylpropyleneurea (DMPU) (1.5 mL, 1.61 g, 12.6 mmol) were added to a solution of the above crude residue in CH₃CN (5 mL), and the mixture was cooled to 0 °C. TMSCF₂Br (1.2 mL, 1.59 g, 7.85 mmol) was added to the mixture at 0 °C. After the mixture was stirred at 0 °C for 30 min and at room temperature for 4 h, H₂O (2.5 mL) and pyridine (800 µL, 784.0 mg, 9.91 mmol) were added and stirred at 80 °C for 1.5 h. The reaction mixture was diluted with an excess amount of H₂O and extracted with EtOAc three times. The organic layer was dried over Na₂SO₄, filtered, and concentrated. The residue was purified by flash column chromatography on silica gel (hexane : EtOAc = 5 : 1) to obtain crude 12.

To the above crude 12 in MeOH (30 mL) was added p-toluenesulfonic acid monohydrate (3.18 g, 16.7 mmol), and the mixture was stirred at room temperature for 14 h under air. After the reaction was quenched with H₂O and saturated aqueous NaHCO₃ at room temperature, the mixture was extracted with CH₂Cl₂ three times. The organic layer was dried over Na₂SO₄, filtered, and concentrated. The residue was purified by flash column chromatography on silica gel (hexane : EtOAc = 3 : 1) to obtain 4 (286.5 mg, 51%, in 4 steps) as a white powder.

4: [α]_D²⁷ +34.7 (c 1.52, CHCl₃); IR (neat) 3417, 1465, 1379, 1116, 1070 cm⁻¹; ¹H-NMR (400 MHz, CDCl₃) δ: 0.91 (d, J = 6.4 Hz, 3H), 0.93 (s, 3H), 1.02–1.88 (m, 19H), 1.97–2.20 (m, 1H), 2.45 (br s, 1H), 4.07–4.08 (m, 1H), 5.67–6.00 (m, 2H); ¹³C-NMR (100 MHz, CDCl₃) δ: 13.5, 17.4, 18.4, 18.4, 22.5, 27.2, 30.0, 33.5, 35.1, 36.2, 40.3, 41.9, 52.6, 56.5, 69.4, 74.2 (quin, J = 20.0 Hz), 114.7 (t, J = 246.9 Hz); HR-MS (ESI⁻) Calcd for C₁₉H₃₁O₄F₄ [M + HCOO]⁻ 399.2164, Found 399.2155.

(1R,3aR,7aR)-1-{(2R)-7-Fluoro-6-(fluoromethyl)-6-[(trimethylsilyl)oxy]heptan-2-yl}-7a-methyloctahydro-4H-inden-4-one (13)

4-Methylmorpholine N-oxide (39.4 mg, 0.34 mmol) was added to a solution of 3 (21.3 mg, 0.07 mmol) in CH₂Cl₂ (1 mL), and the mixture was cooled to 0 °C. TPAP (14.2 mg, 0.04 mmol) was added to the mixture, and it was stirred at 0 °C for 1 h. The reaction was diluted with an excess amount of Et₂O. The mixture was directly purified by flash column chromatography on silica gel (Et₂O only) to obtain crude ketone, and this was used for the next reaction without further purification.

TESCl (70.7 mg, 79 µL, 0.47 mmol) was added to 0 °C cooled solution of the above crude ketone and imidazole (50.1 mg, 0.74 mmol) in CH₂Cl₂ (1 mL), and the mixture was stirred at room temperature for 24 h. After the reaction was quenched with H₂O, the mixture was extracted with CH₂Cl₂ three times. The organic layer was dried over Na₂SO₄, filtered, and concentrated. The residue was purified by flash column chromatography on silica gel (hexane : EtOAc = 20 : 1) to obtain 13 (23.0 mg, 80%, in 2 steps) as a colorless oil.

13: [α]_D²⁷ +2.71 (c 1.77, CHCl₃); IR (neat) 1712, 1464, 1379, 1235, 1170, 1035, 744 cm⁻¹; ¹H-NMR (400 MHz, CDCl₃) δ: 0.60 (q, J = 7.8 Hz, 6H), 0.64 (s, 3H), 0.94 (t, J = 7.8 Hz, 9H), 0.95 (d, J = 6.0 Hz, 3H), 1.02–1.09 (m, 1H), 1.23–1.60 (m, 10H), 1.69–1.76 (m, 1H), 1.85–1.94 (m, 2H), 1.98–2.04 (m, 1H), 2.10–2.13 (m, 1H), 2.19–2.29 (m, 2H), 2.44 (dd, J = 7.8, 11.8 Hz, 1H), 4.22–4.35 (m, 4H); ¹³C-NMR (150 MHz, CDCl₃) δ: 6.3 (3C), 6.9 (3C), 12.5, 18.6, 18.7, 19.0, 24.0, 27.5, 33.9, 35.4, 36.1, 39.0, 40.0, 49.9, 56.6, 62.0, 75.4 (t, J = 17.9 Hz), 83.3–84.7 (m, 2C), 212.0; HR-MS (ESI⁻) Calcd for C₂₄H₄₄O₂F₂SiCl [M + Cl]⁻ 465.2773, Found 465.2789.

(1R,3aR,7aR)-1-{(2R)-6-(Difluoromethyl)-7,7-difluoro-6-[(trimethylsilyl)oxy]heptan-2-yl}-7a-methyloctahydro-4H-inden-4-one (14)

4-Methylmorpholine N-oxide (33.6 mg, 0.29 mmol) was added to a solution of 4 (55.1 mg, 0.16 mmol) in CH₂Cl₂ (1 mL), and the mixture was cooled to 0 °C. TPAP (29.4 mg, 0.08 mmol) was added to the mixture, and it was stirred at 0 °C for 1 h. The reaction was diluted with an excess amount of Et₂O. The mixture was directly purified by flash column chromatography on silica gel (Et₂O only) to obtain the crude ketone, and this was used for the next reaction without further purification.

TMSCl (117.9 mg, 137 µL, 1.09 mmol) was added to the 0 °C cooled solution of the above crude ketone and imidazole (91.6 mg, 1.35 mmol) in CH₂Cl₂ (2 mL), and the mixture was stirred at 0 °C for 1 h. After the reaction was quenched with H₂O, the mixture was extracted with CH₂Cl₂ three times. The organic layer was dried over Na₂SO₄, filtered, and concentrated. The residue was purified by flash column chromatography on silica gel (hexane : EtOAc = 10 : 1) to obtain 14 (51.4 mg, 78%, in 2 steps) as a colorless oil.

14: [α]_D²⁷ +5.0 (c 3.95, CHCl₃); IR (neat) 1715, 1468, 1383, 1252, 1116, 1081, 853 cm⁻¹; ¹H-NMR (600 MHz, CDCl₃) δ: 0.16 (s, 9H), 0.63 (s, 3H), 0.96 (d, J = 6.0 Hz, 3H), 1.04–1.10 (m, 1H), 1.24–1.64 (m, 9H), 1.68–1.76 (m, 2H), 1.85–1.93 (m, 2H), 1.98–2.03 (m, 1H), 2.09–2.12 (m, 1H), 2.18–2.29 (m, 2H), 2.44 (dd, J = 7.8, 11.4 Hz, 1H), 5.73 (t, J = 54.9 Hz, 2H); ¹³C-NMR (150 MHz, CDCl₃) δ: 1.8, 12.4, 18.6, 18.7, 19.0, 24.0, 27.5, 29.9, 35.4, 36.3, 39.0, 40.9, 49.9, 56.6, 61.9, 77.2 (quin, J = 20.8 Hz), 114.9 (t, J = 250.7 Hz), 115.0 (t, J = 248.5 Hz), 211.9; HR-MS (ESI⁺) Calcd for C₂₁H₃₆O₂F₄SiNa [M + Na]⁺ 447.2313, Found 447.2318.

26,27-Difluoro-25-hydroxyvitamin D₃ (1)

nBuLi (133 µL, 1.59 M hexane solution, 0.21 mmol) was added to a solution of A-ring phosphine oxide (5) (103.9 mg, 0.23 mmol) in THF (1 mL) at −78 °C. After stirring for 20 min, a solution of 13 (23.0 mg, 0.053 mmol) in THF (1 mL) was added to the reaction mixture, and it was stirred at −78 °C for 4 h. After the reaction was quenched with H₂O at the same temperature, the mixture was extracted with EtOAc three times. The organic layer was dried over Na₂SO₄, filtered, and concentrated. The residue was purified by flash column chromatography on silica gel (hexane : EtOAc = 10 : 1) to obtain the crude coupling product (30.2 mg), and it was used for the next reaction without further purification.

Tetrabutylammonium fluoride (424 µL, 1 M THF solution, 0.42 mmol) was added to a solution of the above crude coupling product (30.2 mg) in THF (3 mL), and the mixture was stirred at room temperature for 14 h. After the reaction was quenched with H₂O and aqueous saturated NH₄Cl at room temperature, the mixture was extracted with EtOAc three times. The organic layer was dried over Na₂SO₄, filtered, and concentrated. The residue was purified by flash column chromatography on silica gel (hexane : EtOAc = 2 : 1) to afford 1 (18.3 mg, 79%, in 2 steps) as a white powder.

1: [α]_D²⁷ +42.9 (c 1.41, EtOH); IR (neat) 3381, 1441, 1371, 1259, 1023, 899 cm⁻¹; ¹H-NMR (600 MHz, CD₃OD) δ: 0.59 (s, 3H), 1.00 (d, J = 6.0 Hz, 3H), 1.09–1.15 (m, 1H), 1.29–1.62 (m, 13H), 1.70–1.75 (m, 2H), 1.91–2.08 (m, 4H), 2.12–2.17 (m, 1H), 2.22 (dd, J = 9.6, 12.6 Hz, 1H), 2.44 (dt, J = 4.8, 13.8 Hz, 1H), 2.57 (dd, J = 4.2, 12.6 Hz, 1H), 2.87–2.90 (m, 1H), 3.77–3.81 (m, 1H), 4.29–4.41 (m, 4H), 4.78 (d, J = 1.8 Hz, 1H), 5.07 (br s, 1H), 6.07 (d, J = 10.8 Hz, 1H), 6.25 (d, J = 10.8 Hz, 1H); ¹³C-NMR (150 MHz, CD₃OD) δ: 12.7, 19.6, 20.2, 23.6, 24.9, 29.0, 30.2, 33.9, 34.6, 36.9, 37.7, 38.0, 42.2, 47.2, 47.3, 57.8, 58.2, 70.9, 73.8 (t, 17.9 Hz), 85.0–86.3 (m), 112.9, 119.3, 122.9, 137.6, 142.8, 147.3; HR-MS (ESI⁻) Calcd for C₂₇H₄₁O₂F₂ [M−H]⁻ 435.3080, Found 435.3063.

26,26,27,27-Tetrafluoro-25-hydroxyvitamin D₃ (2)

nBuLi (228 µL, 1.59 M hexane solution, 0.36 mmol) was added to a solution of A-ring phosphine oxide (5) (176.2 mg, 0.39 mmol) in THF (1 mL) at −78 °C. After stirring for 30 min, a solution of 14 (51.4 mg, 0.12 mmol) in THF (1 mL) was added to the reaction mixture, and it was stirred at −78 °C for 3 h 20 min. After the reaction was quenched with H₂O at the same temperature, the mixture was extracted with EtOAc three times. The organic layer was dried over Na₂SO₄, filtered, and concentrated. The residue was purified by flash column chromatography on silica gel (hexane : EtOAc = 10 : 1) to obtain the crude coupling product (61.3 mg), and it was used for the next reaction without further purification.

Tetrabutylammonium fluoride (500 µL, 1 M THF solution, 0.5 mmol) was added to a solution of the above crude coupling product (61.3 mg) in THF (5 mL), and the mixture was stirred at room temperature for 17 h. After the reaction was quenched with H₂O and brine at room temperature, the mixture was extracted with EtOAc three times. The organic layer was dried over Na₂SO₄, filtered, and concentrated. The residue was purified by flash column chromatography on silica gel (hexane : EtOAc = 2 : 1) to afford 2 (25.2 mg, 44%, in 2 steps) as a white powder.

2: [α]_D²⁷ +74.5 (c 1.94, EtOH); IR (neat) 3361, 1441, 1375, 1112, 1069, 957, 895, 741 cm⁻¹; ¹H-NMR (600 MHz, CD₃OD) δ: 0.60 (s, 3H), 1.00 (d, J = 6.0 Hz, 3H), 1.10–1.16 (m, 1H), 1.30–1.75 (m, 15H), 1.91–2.08 (m, 4H), 2.12–2.17 (m, 1H), 2.22 (dd, J = 9.0, 12.0 Hz, 1H), 2.44 (dt, J = 5.4, 13.8 Hz, 1H), 2.56 (dd, J = 2.4, 12.6 Hz, 1H), 2.87–2.90 (m, 1H), 3.77–3.81 (m, 1H), 4.78 (d, J = 1.2 Hz, 1H), 5.07 (br s, 1H), 5.88 (t, J = 54.9 Hz, 2H), 6.07 (d, J = 11.4 Hz, 1H), 6.25 (d, J = 11.4 Hz, 1H); ¹³C-NMR (150 MHz, CD₃OD) δ: 12.7, 19.6, 20.2, 23.6, 24.9, 29.0, 30.2, 33.9, 34.6, 36.9, 37.7, 38.0, 42.2, 47.2, 47.3, 57.8, 58.2, 70.9, 73.8 (t, 17.9 Hz), 85.0–86.3 (m), 112.9, 119.3, 122.9, 137.6, 142.8, 147.3; HR-MS (ESI⁻) Calcd for C₂₇H₃₉O₂F₄ [M−H]⁻ 471.2892, Found 471.2908.

Measurement of hVDR Binding Affinity of 25(OH)D₃, 1, 2, and HF-25(OH)D₃

Binding affinity of each analogue for hVDR was evaluated using the in vitro system based on the split-luciferase technique described in our previous study.²²⁾ Briefly, 50 µL of cell lysate prepared from recombinant Escherichia (E.) coli expressing split-luciferase vitamin D biosensor protein 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 of MgSO₄, 2 mM of D-luciferin, and 4 mM of adenosine triphosphate in 25 mM Tris–HCl (pH 7.4) was injected into each well and incubated for 30 min at room temperature. The luminescence (photon counts) was measured using a luminometer (Infinite 200 Pro 96-microplate luminometer, Tecan). Relative hVDR binding affinity of each analogue was evaluated based on the concentration at which the luminescence showed 50% of the maximum value.

Metabolism of 25(OH)D₃, 1, 2, and HF-25(OH)D₃ by Recombinant hCYP24A1

The metabolism of 25(OH)D₃ and its analogues (1, 2) and HF-25(OH)D₃ by CYP24A1 was analyzed using a membrane fraction prepared from recombinant Escherichia coli cells expressing human CYP24A1, as described in our previous study.²³⁾ Briefly, the reaction mixture containing 0.02 µM of human CYP24A1, 2.0 µM of bovine adrenodoxin (ADX), 0.2 µM of bovine reduced nicotinamide adenine dinucleotide phosphate (NADPH)-adrenodoxin reductase (ADR), 1 mM of ethylenediaminetetraacetic acid (EDTA), 1 mM of NADPH, and 5.0 µM of each substrate in 100 mM Tris–HCl (pH 7.4) was incubated at 37 °C for 0, 5, or 15 min. The metabolites were extracted with 4 volumes of CHCl₃-CH₃OH (3 : 1) and analyzed by HPLC under the following conditions: column, CAPCELL PAK C18 UG120 (5 µm) (4.6 × 250 mm) (SHISEIDO, Tokyo, Japan); UV detection, 265 nm; flow-rate, 1.0 mL min⁻¹; column temperature, 40 °C; mobile phase, CH₃CN: a linear gradient of 20–100% CH₃CN aqueous solution per 25 min and 100% CH₃CN for 10 min.

Transactivation Assay of Human Osteocalcin Promoter

A mixed solution of expression plasmids, pGL4.26 DR3 (×4) and pcDNA3-humanVDR (full length) at a ratio of 5 : 1, and the control plasmid pGL4-CMV-Rluc (Promega, WI, U.S.A.) for correcting transfection efficiency were simultaneously used to transfect human-derived osteoblast-like cells (HOS cells, ATCC) using the gene introduction device MaxCyte STX (MaxCyte), so that the final amount of DNA was 300 µg/mL. The transfected cells were incubated in a humid environment of 5% CO₂ at 37 °C for 20 min. The incubated cells were cryopreserved using a cell cryopreservation reagent cell banker (Nippon Zenyaku Kogyo Co., Ltd.). The above VDR-transfected HOS cells were thawed and suspended in Dulbecco’s modified Eagle’s medium (DMEM) containing 5% inactivated and charcoal-treated fetal bovine serum. The suspended expressing cells were seeded on a 384-well low-volume white plate (Aurora, Cat.#AUR-30411) at 4000 cells/10 µL/well and incubated at 37 °C for 4 h in a humid environment of 5% CO₂. Dimethyl sulfoxide (DMSO) was used to dissolve the test compound, and the test compound diluted over the test concentration range was added to a final DMSO concentration of 0.1% using Echo^® Liquid Handler (Labcyte) and incubated for 20 h. Luciferase activities of test compounds were detected using the Dual-Glo luciferase assay system (Promega). Using a multi-label counter, EnVison (PerkinElmer, Inc., MA, U.S.A.), the effect of the test compound on activation of firefly luciferase transcription via VDR was measured under the condition of an integration time of 0.1 s. Renilla luciferase activity was also measured at the same time and used to correct for transfection efficiency. Firefly luciferase activity was corrected for Renilla luciferase activity, and the activity increase rate of the test compound was calculated with the DMSO control group as 100%, and Emax and EC₅₀ were calculated from the fit#208 formula of XLfit (manufactured by IDBS).

Acknowledgments

This work was supported in part by Grants-in-Aid from the Japan Society for the Promotion of Science (No. 22K14688 to F.K., Nos. 18K06556 and 23K06029 to A.K., and Nos. 19H02889 and 22H02263 to T.S.).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials of ¹H- and ¹³C-NMR spectra of all new compounds 1–4, 7–9, 11, 13, and 14.

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