2024 Volume 72 Issue 10 Pages 899-908
25-Hydroxyvitamin D3-23,26-lactone (1) and 1α,25-dihydroxyvitamin D3-23,26-lactone (2) have long been considered as among the end metabolites of vitamin D3. Recently, however, we found that these lactones exhibit biological activity related to the β-oxidation of fatty acids. We hypothesized that a metabolic pathway might exist to inactivate their physiological activity. Here, by means of metabolic experiments with a variety of cytochrome P450 (CYP) enzymes, we show that CYP3A4 metabolizes the lactones. The metabolites were presumed to be hydroxylated at C4 based on the previous reports showing that metabolism of 25-hydroxyvitamin D3 by CYP3A4 along with the current LC-MS analysis. To confirm this, we chemically synthesized 4α,25(OH)2D3-23,26-lactone (3), 4β,25(OH)2D3-23,26-lactone (4), 1α,4α,25(OH)3D3-23,26-lactone (5), and 1α,4β,25(OH)3D3-23,26-lactone (6). HPLC analysis using these authentic compounds as standards revealed that 1 was metabolized to 3 and 4, while 2 was metabolized exclusively to 6 by CYP3A4. Docking studies suggest that the hydroxyl group at C1 in 2 forms hydrogen bonds with Ser119 and Arg212 of CYP3A4, contributing to the fixation of C4β on heme iron in the CYP, thereby resulting in stereoselective hydroxylation at C4.
The metabolism of vitamin D3 has been extensively studied, and many metabolic pathways have been identified.1–4) Vitamin D3 is firstly metabolized to 25-hydroxyvitamin D3 (25(OH)D3) in the liver by CYP2R1 and CYP27A1, a member of the cytochrome P450 (CYP) superfamily (Chart 1). Then, 25(OH)D3 is hydroxylated at C1α by CYP27B1 in the kidney to produce the active form, 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). These metabolites of 25(OH)D3 and 1,25(OH)2D3 are further hydroxylated in their side chains by CYP24A1 to generate over 40 types of biologically inactive metabolites.5–7)
Among these metabolites, 25-hydroxyvitamin D3-23,26-lactone (25(OH)D3-23,26-lactone (1)) and 1α,25-dihydroxyvitamin D3-23,26-lactone (1α,25(OH)2D3-23,26-lactone (2)) were identified in 1979 and 1980 by the Jorgensen and Deluca groups, respectively,8,9) and they have been considered as among the inactive end metabolites of vitamin D3 for more than 40 years since their discovery. Recently, however, our group showed that 1 and 2 bind to HADHA (hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit alpha), which catalyzes the β-oxidation of fatty acids, and inhibit the biosynthesis of L-carnitine, thereby inhibiting the metabolism of fatty acids.10) These findings suggest that further metabolic pathways from 1 and 2 should exist in order to control these physiological activities. In the present study, we show that 1 and 2 are indeed further metabolized by CYP3A4. Potential metabolites were chemically synthesized and the structures of the metabolites of 1 and 2 were identified by HPLC comparison with the authentic samples.
Compounds 1 and 2 were each incubated with human liver microsomes to verify the further metabolism of 1 and 2. After incubation for 30 min, the medium was extracted with CHCl3/MeOH (3 : 1), and the extracts were analyzed by HPLC and compared to the extracts incubated for 0 min (Fig. 1).
In the case of 1, two new peaks, M1 and M2, were observed by HPLC analysis with a conversion ratio of 7.2% (Fig. 1A). In the case of 2, one major peak for M3 was detected by HPLC with a conversion ratio of 9.4% (Fig. 1B). The conversion ratios were calculated based on the peak area ratio of the starting materials 1 and 2 and the products M1–M3 in HPLC. These results support the idea that the vitamin D3 lactones 1 and 2 are further metabolized in the liver.
Next, 1 and 2 were incubated with each of the human drug-metabolizing CYPs: CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, and 3A4. The results indicated that 1 and 2 were metabolized exclusively by CYP3A4, which generated metabolites corresponding to M1, M2, and M3, respectively. The conversion ratios of 1 and 2 to their respective metabolites by CYP3A4 were 17.5 and 16.1%, respectively (Figs. 2A, 2B). The other CYPs examined were found to be ineffective.
LC-MS analysis of these metabolites revealed that M1–M3 have an additional hydroxyl group compared with vitamin D3 lactones 1 and 2, respectively (Supplementary Materials). Since CYP3A4 has been reported to hydroxylate C4 of 25(OH)D3,11) we hypothesized that the metabolites of 1 and 2 formed by CYP3A4 were 4α,25(OH)2D3-23,26-lactone (3) and 4β,25(OH)2D3-23,26-lactone (4), and 1α,4α,25(OH)3D3-23,26-lactone (5) or 1α,4β,25(OH)3D3-23,26-lactone (6), respectively. To confirm this, we synthesized the vitamin D3 lactones 3–6 hydroxylated at C4.
Synthesis of 4-hydoxylated Vitamin D3 Lactones 3–6We planned to synthesize lactones 3–6 by coupling bromoolefin 7 (CD-rings)12) with enynes 8 and 9 hydroxylated at C4 (A-ring synthons) using the palladium-catalyzed Heck-type reaction developed by Trost13) (Chart 2).
The enynes 8a and 8b were synthesized from 10, which was derived from L-(−)-malic acid as shown in Chart 3.14,15) The primary tert-butyldimethylsilyl (TBS) ether in 10 was selectively deprotected by treatment with Py-Br3 in MeOH at 0 °C to give the alcohol 11 in 91% yield.16) After oxidation of 11 under the Swern conditions, the resulting aldehyde was reacted with trimethylsilyl (TMS) acetylide prepared from TMS acetylene and n-butyllithium to give the alcohol 4β-12b stereoselectively in 90% yield. The stereochemistry of 4β-12b was determined by means of nuclear Overhauser effect spectroscopy (NOESY) experiments (Supplementary Materials). The diastereomer 4α-12a was obtained selectively from 4β-12b by Dess–Martin oxidation and subsequent reduction of the resulting ketone with L-Selectride in 94% yield. The secondary hydroxyl group of alcohol 12a was then protected as TBS ether in the presence of TBSOTf and 2,6-lutidine, and the benzoyl and TMS groups were removed with potassium carbonate to give alcohol 13a in 70% yield. The hydroxyl group of 13a was converted to iodide by reaction with iodine and triphenylphosphine, and the product was reacted with sodium cyanide to give nitrile 14a, which was further reduced with DIBAL-H followed by reaction with methylene Wittig reagent to give the A-ring synthon, enyne 8a, in 52% yield (4 steps). Similarly, 4β-12b was converted into the enyne 8b via 13b and 14b.
Reagents and conditions: (a) Py-Br3, MeOH, 0 °C, 29 h, 91%; (b) i) (COCl)2, dimethyl sulfoxide (DMSO), triethylamine (Et3N), dichloromethane (DCM), −78 °C, 1h, ii) n-butyllithium, trimethylsilylacetylene, tetrahydrofuran (THF), −78 °C, 15 min 90%; (c) i) DMP, pyridine, DCM, room temperature (r.t.), 30 min, ii) L-Selectride, THF, −78 °C, 94%; (d) i) TBSOTf, 2,6-lutidine, DCM, r.t., 30 min, ii) K2CO3, MeOH, .r.t, 1 h, 70% (13a) 76% (13b); I i) I2, PPh3, imidazole, THF, −20 °C, 30 min, ii) NaCN, N,N-dimethylformamide (DMF), 90 °C, 20 min, 83% (14a), 84% (14b); f) i) DIBAL-H, DCM, 0 °C, 30 min, ii) NaHMDS, methyltriphenylphosphonium iodide, THF, r.t., 1 h, 63% (8a) 70% (8b).
Enyne 9 was synthesized from triol 13 (Chart 4). The primary hydroxyl group of 13a was oxidized by IBX and the resulting aldehyde was reacted with vinyl Grignard reagent to give allyl alcohol 15 as a 1 : 2 diastereomeric mixture at C1 (15a:15a′) (Chart 4A). The stereochemistry at C1 in 15 was determined by the modified Mosher method; see Supplementary Materials.17,18) After separation of the diastereomers by silica gel column chromatography, 15a was reacted with TBSOTf and 2,6-lutidine to give the A-ring synthon 9a. Similarly, 4β-13b was converted into the enyne 9b via 15b (Chart 4B).
Reagents and conditions: (A) (a) i) IBX, DMSO, 50 °C, 3 h, ii) Vinylmagnesium bromide, THF, −78 °C, 2 h, iii) separation, 15a (23%) and 15a′ (42%); (b) TBSOTf, 2,6-lutidine, DCM, r.t., 1 h, 90% (9a). (B) (c) i) IBX, DMSO, 50 °C, 3 h, ii) Vinylmagnesium bromide, THF, −78 °C, 2 h, iii) separation, 15b (22%) and 15b′ (41%); (d) TBSOTf, 2,6-lutidine, DCM, r.t., 1 h, 98% (9b).
With the A ring synthons 8 and 9 in hand, the coupling reaction with CD ring synthon 7 was next carried out. The A ring synthon 8a and bromoolefin 7 were subjected to the coupling reaction in the presence of Pd(PPh3)4 and triethylamine (Et3N) in toluene followed by deprotection of the silyl ether of the coupling product with HF·Et3N to give 4α-hydroxyvitamin D3-lactone (3) in 65% yield (2 steps). Similarly, 8b, 9a, and 9b were reacted with bromoolefin 7 followed by deprotection of the silyl ether to give 4β,25(OH)2D3-23,26-lactone (4), and 1α,4α,25(OH)3D3-23,26-lactone (5) and 1α,4β,25(OH)3D3-23,26-lactone (6), respectively, in 59, 42, and 65% yield, respectively (Chart 5).
Reagents and conditions: (a) i) 7, Pd(PPh3)4, Et3N, toluene, 90 °C, 2 h; ii) HF·Et3N, THF, r.t., 6 d, 65% (3), 59% (4), 42% (5), 65% (6).
With the authentic samples of 4-hydroxyated vitamin D3 lactones 3–6 in hand, HPLC identification of the metabolites M1–M3 was carried out. As shown in Fig. 3A, the retention times of M1 and 4β-hydroxylated compound 4 were identical, while the retention times of M2 and 4α-hydroxylated compound 3 were also identical. Similarly, the retention times of M3 and the 1α,4β,25(OH)3D3-23,26-lactone (6) were identical (Fig. 3B). These results suggest that the 25(OH)2D3-23,26-lactone (1) is metabolized to 4α,25(OH)2D3-23,26-lactone (3) and 4β,25(OH)2D3-23,26-lactone (4), while 1,25(OH)2D3-23,26-lactone (2) is metabolized to 1α,4β,25(OH)3D3-23,26-lactone (6) by CYP3A4.
To get insight into the difference in stereoselectivity of the metabolites generated from 1 and 2 by CYP3A4, docking studies were performed. In the case of 2, the interaction of CYP3A4 and 2 is suggested to be stabilized by three hydrogen bonds between the carbonyl group of C26 and Arg106, the hydroxy group of C1 and Ser119, and Arg212 (docking score −7.18 kcal/mol), and oxidation by the heme iron proceeded stereoselectively at the β site to give 1α,4β,25(OH)3D3-23,26-lactone (6) (Fig. 4B). On the other hand, in the case of 1, the absence of the hydroxy group at C1 allows for greater conformational freedom and both the C4α and C4β sites can come into close proximity to heme iron, so that both 4α,25(OH)2D3-23,26-lactone (3) and 4β,25(OH)2D3-23,26-lactone (4) can be formed. Actually, from the docking experiments, only hydrogen bond formation between the carbonyl group of C26 and Arg106 was observed (docking score −6.18 kcal/mol) (Fig. 4A).
We found that 25(OH)D3-23,26-lactone (1) and 1α,25(OH)2D3-23,26-lactone (2), previously considered as end metabolites, are further metabolized by CYP3A4. In case of 1, hydroxylation proceeds at both the C4α and the C4β sites to afford 4α,25(OH)2D3-23,26-lactone (3) and 4β,25(OH)2D3-23,26-lactone (4). In the case of 2, the C4β site is selectively hydroxylated to generate exclusively 1α,4β,25(OH)3D3-23,26-lactone (6). Docking studies of CYP3A4 with 1 and 2 provided a rationale for these observations. Further research is ongoing about the significance of the metabolism of 1 and 2.19,20)
All reagents were supplied by commercial sources without further purification. All reactions involving air- or moisture-sensitive reagents were carried out in flame-dried glassware under argon atmosphere. Flash chromatography was performed using silica gel 60 (spherical, particle size 0.040 − 0.100 mm; Kanto Co., Inc., Tokyo, Japan), and preparative TLC (PLC) was performed using PLC silica gel 60 F254 (0.5 mm, Merck Ltd., Darmstadt, Germany). Optical rotations were measured on a JASCO P-2200 polarimeter (JASCO Co., Inc., Tokyo, Japan). 1H- and 13C-NMR spectra were recorded on JNM-AL300 (300 MHz, JEOL Ltd., Tokyo, Japan), JNM-ECX 400 (400 MHz, JEOL Ltd.), and JNMECA 500 (500 MHz, JEOL Ltd.) spectrometers. Chemical shift in CDCl3 was reported in the scale relative to CDCl3 (7.26 ppm) for 1H-NMR. For 13C-NMR, the chemical shift was reported in the scale relative to CDCl3 (77.0 ppm) and CD3OD (49.0 ppm) as an internal reference. High resolution (HR)MS (electrospray ionization (ESI)) measurements were performed on a JMS-T100LC spectrometer (JEOL Ltd.).
(S)-3-((tert-Butyldimethylsilyl)oxy)-4-hydroxybutyl Benzoate (11)To a solution of 1016) (898 mg, 2.1 mmol) in MeOH (20 mL) was added pyridinium tribromide (65 mg, 0.21 mmol) at 0 °C. The reaction mixture was stirred at this temperature for 24 h, and pyridinium tribromide (65 mg, 0.21 mmol) was additionally added. After being stirred for 5 h, the reaction was quenched with sat. NaHCO3 aq. and the mixture was extracted with CH2Cl2 three times. The organic layer was washed with brine, dried over MgSO4, filtered and then concentrated in vacuo. The residue was purified by column chromatography on silica gel (ethyl acetate/n-hexane 1 : 10) to give 11 (608 mg, 91%) as a clear oil. αD25=−9.8 (c 0.53, CHCl3); 1H-NMR (300 MHz, CDCl3) δ: 8.03 (d, J = 7.2 Hz, 2H), 7.56 (t, J = 7.4 Hz, 1H), 7.44 (t, J = 7.7 Hz, 2H), 4.31–4.49 (m, 2H), 3.96–4.03 (m, 1H), 3.66 (q, J = 4.9 Hz, 1H), 3.55 (q, J = 5.3 Hz, 1H), 1.99 (q, J = 6.3 Hz, 2H), 0.91 (s, 9H), 0.10 (d, J = 2.1 Hz, 6H); 13C-NMR (75 MHz, CDCl3) δ: 166.5, 132.9, 130.2, 129.5, 128.4, 69.7, 66.4, 61.6, 32.9, 25.8, 18.0, −4.6, −4.8; HR-MS (ESI) m/z: [M + Na]+ Calcd for C17H28O4SiNa 347.1655. Found 347.1670.
(3S,4R)-3-((tert-Butyldimethylsilyl)oxy)-4-hydroxy-6-(trimethylsilyl)hex-5-yn-1-yl Benzoate (12b)To a solution of oxalic acid dichloride (0.48 mL, 5.6 mmol) in CH2Cl2 (7 mL) was added DMSO (1.0 mL, 14.0 mmol) at −78 °C. After being stirred for 10 min, a solution of 11 in CH2Cl2 (902 mg, 2.78 mmol in 7 mL) and triethylamine (3.9 mL, 27.8 mmol) were added dropwise. After being stirred for 1 h, the reaction was quenched with H2O and the mixture was extracted with ethyl acetate for three times. The organic layer was washed with brine, dried over MgSO4, filtered, and then concentrated in vacuo. The residue was filtered by column chromatography on silica gel (ethyl acetate/n-hexane 1 : 1) to give aldehyde. The crude residue was used in the subsequent step without purification. To a solution of trimethylsilylacetylene (1.0 mL, 7.2 mmol, 3.0 equivalent (equiv.)) in THF (12 mL) was added n-butyllithium (2.6 M in hexane; 2.3 mL, 6.0 mmol, 2.3 equiv.) at −78 °C. After being stirred for 30 min, a solution of crude residue in THF (crude product, 1.0 equiv. in 12 mL) was added dropwise. After being stirred for another 15 min, the reaction was quenched with sat. NH4Cl aq. and the mixture was extracted with ethyl acetate for three times. The organic layer was washed with brine, dried over MgSO4, filtered and then concentrated in vacuo. The residue was purified by column chromatography on silica gel (ethyl acetate/n-hexane 1 : 30) to give 12b (910 mg, 90%) as a clear oil. αD25=+1.5 (c 5.36, CHCl3); 1H-NMR (300 MHz, CDCl3) δ: 8.05–8.03 (m, 2H), 7.59–7.53 (m, 1H), 7.44 (t, J = 7.6 Hz, 2H), 4.56–4.26 (m, 3H), 4.04–3.99 (m, 1H), 2.33–1.87 (m, 3H), 0.92–0.88 (m, 9H), 0.16–0.08 (m, 15H); 13C-NMR (75 MHz, CDCl3) δ: 166.4, 132.9, 130.2, 129.5, 128.3, 102.9, 91.7, 71.9, 66.5, 61.6, 31.1, 25.7, 18.0, −0.3, −4.5, −4.7; HR-MS (ESI) m/z: [M + Na]+ Calcd. for C22H36O4Si2Na 443.2050. Found 443.2017.
(3S,4S)-3-((tert-Butyldimethylsilyl)oxy)-4-hydroxy-6-(trimethylsilyl)hex-5-yn-1-yl Benzoate (12a)To a solution of 12b (100 mg, 0.24 mmol) and pyridine (0.38 mL) in CH2Cl2 (19 mL) was added Dess–Martin periodinane (404 mg, 0.95 mmol) at 0 °C. The resulting mixture was allowed to warm to room temperature and stirred for 30 min. The reaction mixture was quenched with sat. Na2S2O3 aq. and the aqueous layer was extracted with CH2Cl2 for three times. The combined organic layer was washed with brine, dried over MgSO4, filtered, and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (n-hexane/ethyl acetate = 10 : 1) to give ketone (94 mg, 94%). To a solution of the ketone (94 mg, 0.22 mmol) in THF (11 mL) was added L-selectride (1.0 M in THF, 0.40 mL, 0.40 mmol) at −78 °C. The resulting mixture was stirred at the same temperature for 1 h. The reaction mixture was quenched with sat. NH4Cl aq. and the aqueous layer was extracted with ethyl acetate for three times. The combined organic layer was washed with brine, dried over MgSO4, filtered, and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (n-hexane/ethyl acetate = 10 : 1) to give 12a (94 mg, 99%) as a colorless oil. αD25=−9.1 (c 0.3, CHCl3); 1H-NMR (300 MHz, CDCl3) δ: 8.05 (d, J = 6.9 Hz, 2H), 7.56 (t, J = 7.4 Hz, 1H), 7.44 (t, J = 7.6 Hz, 2H), 4.32–4.51 (m, 3H), 3.99 (q, J = 5.4 Hz, 1H), 1.99–2.19 (m, 2H), 0.92 (s, 9H), 0.12–0.16 (m, 15H); 13C-NMR (75 MHz, CDCl3) δ: 166.4, 132.9, 130.1, 129.5, 128.3, 104.6, 90.6, 72.5, 65.8, 61.2, 32.7, 25.9, 18.1, −0.3, −4.3, −4.6; HRMS (ESI) m/z: [M + Na]+ Calcd for C22H36O4Si2Na 443.2050. Found 443.2054.
(3S,4S)-3,4-bis((tert-butyldimethylsilyl)oxy)hex-5-yn-1-ol (13a)To a solution of 12a (115 mg, 0.27 mmol) in CH2Cl2 (3 mL) was added 2,6-lutidine (98 µL, 0.82 mmol) and tert-butyldimethylsilyl triflate (94 µL, 0.41 mmol) at 0 °C. The resulting mixture was allowed to warm to room temperature and stirred for 30 min. The reaction mixture was quenched with H2O, and the aqueous layer was extracted with CH2Cl2 for three times. The combined organic layer was washed with brine, dried over MgSO4HR-MS, filtered, and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (n-hexane/ethyl acetate = 40 : 1) to give silyl ether (143 mg, 98%). To a solution of the silyl ether (143 mg, 0.27 mmol) in MeOH (0.9 mL) was added K2CO3 (148 mg, 1.1 mmol) at room temperature. The resulting mixture was stirred at the same temperature for 1 h. The reaction mixture was quenched with H2O, and the aqueous layer was extracted with ethyl acetate for three times. The combined organic layer was washed with brine, dried over MgSO4, filtered, and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (n-hexane/ethyl acetate = 30 : 1) to give 13a (68 mg, 71%) as a colorless oil. αD25=−14.5 (c 0.3, CHCl3); 1H-NMR (300 MHz, CDCl3) δ: 4.38 (q, J = 2.3 Hz, 1H), 3.79 (tt, J = 8.1, 2.9 Hz, 3H), 2.38 (d, J = 2.1 Hz, 1H), 1.84–2.09 (m, 2H), 0.90 (d, J = 1.4 Hz, 18H), 0.09–0.15 (m, 12H); 13C-NMR (75 MHz, CDCl3) δ: 82.4, 74.0, 73.2, 66.9, 60.1, 34.8, 25.7, 18.1, 18.0, −4.6, −4.7, −4.8, −5.1; HR-MS (ESI) m/z: [M + Na]+ Calcd for C18H38O3Si2Na 381.2257. Found 338.2297.
(3S,4R)-3,4-Bis((tert-butyldimethylsilyl)oxy)hex-5-yn-1-ol (13b)To a solution of 12b (264 mg, 0.63 mmol) in CH2Cl2 (6 mL) were added 2,6-lutidine (0.22 mL, 1.9 mmol) and tert-butyldimethylsilyl triflate (0.22 mL, 0.94 mmol) at 0 °C. The mixture was allowed to warm to room temperature and stirred for 30 min. The reaction was quenched with H2O and the mixture was extracted with CH2Cl2 three times. The combined organic layer was washed with brine, dried over MgSO4, filtered, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (ethyl acetate/n-hexane 1 : 40) to give silyl ether (334 mg, 99%). To a solution of the silyl ether (206 mg, 0.39 mmol) in MeOH (1.3 mL) was added potassium carbonate (213 mg, 1.5 mmol) at room temperature. After being stirred for 1 h, the reaction was quenched with H2O and the mixture was extracted with ethyl acetate three times. The organic layer was washed with brine, dried over MgSO4, filtered, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (ethyl acetate/n-hexane 1 : 30) to give 13b (104 mg, 76%) as a clear oil. αD25=−22.3 (c 1.6, CHCl3); 1H-NMR (300 MHz, CDCl3) δ: 4.36 (q, J = 2.2 Hz, 1H), 3.94 (q, J = 4.8 Hz, 1H), 3.80–3.75 (m, 2H), 2.41 (d, J = 2.1 Hz, 1H), 2.15–1.82 (m, 3H), 0.90 (d, J = 1.7 Hz, 18H), 0.16–0.09 (m, 12H); 13C-NMR (75 MHz, CDCl3) δ: 83.4, 74.2, 74.1, 66.9, 59.0, 34.6, 25.9, 25.7, 18.2, 18.1, −4.3, −4.6, −4.8, −5.1; HR-MS (ESI) m/z: [M + Na]+ Calcd. for C18H38O3Si2Na 381.2257. Found 381.2263.
(4S,5S)-4,5-Bis((tert-butyldimethylsilyl)oxy)hept-6-ynenitrile (14a)To a solution of 13a (49 mg, 0.38 mmol) in THF (0.6 mL) was added triphenylphosphine (70 mg, 0.33 mmol), imidazole (28 mg, 0.41 mmol), and iodine (112 mg, 0.44 mmol) at −20 °C. The resulting mixture was stirred at the same temperature for 30 min. The resulting mixture was allowed to warm to room temperature and stirred for 20 min. The reaction mixture was quenched with sat. Na2S2O3 aq. and the aqueous layer was extracted with ethyl acetate for three times. The combined organic layer was washed with brine, dried over MgSO4, filtered, and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (n-hexane) to give iodide (54 mg, 83%). To a solution of the iodide (54 mg, 0.11 mmol) in DMF (0.3 mL) was added sodium cyanide (8.4 mg, 0.17 mmol) at room temperature. The resulting mixture was allowed to warm to 90 °C and stirred for 20 min. The reaction mixture was quenched with sat. NaHCO3 aq. and the aqueous layer was extracted with ethyl acetate for three times. The combined organic layer was washed with brine, dried over MgSO4, filtered, and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (n-hexane/ethyl acetate = 80 : 1) to give 14a (42 mg, 99%) as a yellow oil. αD25=−27.6 (c 0.3, CHCl3); 1H-NMR (300 MHz, CDCl3) δ: 4.38 (q, J = 2.3 Hz, 1H), 3.67–3.73 (m, 1H), 2.39–2.58 (m, 2H), 2.36 (d, J = 2.1 Hz, 1H), 2.04–2.16 (m, 1H), 1.90–2.02 (m, 1H), 0.90 (s, 18H), 0.11–0.14 (m, 12H); 13C-NMR (75 MHz, CDCl3) δ: 119.9, 82.0, 74.0, 72.3, 66.4, 31.1, 27.6, 25.7, 18.1, 18.0, −4.6, −4.8, −5.1; HR-MS (ESI) m/z: [M + Na]+ Calcd for C19H37NO2Si2Na 390.2261. Found 390.2290.
(4S,5R)-4,5-Bis((tert-butyldimethylsilyl)oxy)hept-6-ynenitrile (14b)To a solution of 13b (136 mg, 0.38 mmol) in THF (1.6 mL) was added triphenylphosphine (193 mg, 0.91 mmol), imidazole (77.2 mg, 1.1 mmol) and iodine (308 mg, 1.2 mmol) at −20 °C. After being stirred for 30 min, the mixture was allowed to warm to room temperature and stirred for another 20 min. The reaction was quenched with sat. Na2S2O3 aq. and the mixture was extracted with ethyl acetate for three times. The organic layer was washed with brine, dried over MgSO4, filtered, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (ethyl acetate/n-hexane 0 : 100) to give iodide (149 mg, 84%). To a solution of the iodide (133 mg, 0.28 mmol) in DMF (0.7 mL) was added sodium cyanide (21.0 mg, 0.43 mmol) at room temperature. The reaction mixture was allowed to warm to 90 °C. After stirred for 20 min, the reaction was quenched with sat. NaHCO3 aq. and the mixture was extracted with ethyl acetate for three times. The organic layer was washed with brine, dried over MgSO4, filtered, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (ethyl acetate/n-hexane 1 : 80) to give 14b (105 mg, 100%) as a colorless oil. αD25=−32.0 (c 2.1, CHCl3); 1H-NMR (300 MHz, CDCl3) δ: 4.27 (q, J = 2.1 Hz, 1H), 3.85–3.80 (m, 1H), 2.51–2.36 (m, 3H), 2.12–1.89 (m, 2H), 0.90 (s, 18H), 0.15–0.11 (m, 12H); 13C-NMR (75 MHz, CDCl3) δ: 120.1, 83.0, 74.4, 73.7, 66.7, 28.2, 25.8, 25.7, 18.1, 18.1, 13.0, −4.2, −4.6, −4.8, −5.2; HR-MS (ESI) m/z: [M + Na]+ Calcd. for C19H37NO2Si2Na 390.2261. Found 390.2249.
(5S,6S)-5-(But-3-en-1-yl)-6-ethynyl-2,2,3,3,8,8,9,9-octamethyl-4,7-dioxa-3,8-disiladecane (8a)To a solution of 14a (42 mg, 0.11 mmol) in CH2Cl2 (0.6 mL) was added diisobutylaluminium hydride (1.0 M in n-hexane; 0.14 mL, 0.14 mmol) at 0 °C. The resulting mixture was stirred at the same temperature for 30 min. To the reaction mixture was added 2-propanol (0.098 mL), silica gel (200 mg), and H2O (1 mL) at room temperature. The resulting mixture was stirred at the same temperature for 30 min. The reaction mixture was filtered through a pad of Celite® and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (n-hexane/ethyl acetate = 10 : 1) to give the aldehyde as a yellow oil. The crude residue was used in the subsequent step without purification. To a solution of methyltriphenylphosphonium iodide (240 mg, 0.39 mmol) in THF (0.6 mL) was added sodium bis(trimethylsilyl)amide (1.9 M in THF, 0.29 mL, 0.56 mmol) at 0 °C. The resulting mixture was stirred at the same temperature for 30 min, then a solution of the aldehyde in THF (0.2 M, 0.55 mL, 0.11 mmol) was added dropwise at the same temperature. The resulting mixture was allowed to warm to room temperature and stirred for 1 h. The reaction mixture was quenched with sat. NH4Cl aq. and the aqueous layer was extracted with n-hexane for three times. The combined organic layer was washed with brine, dried over MgSO4, filtered, and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (n-hexane) to give 8a (27 mg, 63%) as a colorless oil. αD25=−18.0 (c 0.2, CHCl3); 1H-NMR (400 MHz, CDCl3) δ: 5.78–5.88 (m, 1H), 4.94–5.06 (m, 2H), 4.34 (q, J = 2.3 Hz, 1H), 3.57–3.61 (m, 1H), 2.33 (d, J = 2.3 Hz, 1H), 2.18–2.27 (m, 1H), 2.02–2.11 (m, 1H), 1.78–1.86 (m, 1H), 1.67–1.75 (m, 1H), 0.90 (s, 18H), 0.07–0.14 (m, 12H); 13C-NMR (100 MHz, CDCl3) δ: 138.9, 114.3, 83.0, 74.0, 73.2, 66.8, 30.8, 29.6, 25.8, 25.8, 18.2, 18.1, −4.4, −4.5, −4.7, −5.0; HR-MS (ESI) m/z: [M + Na]+ Calcd for C20H40O2Si2Na 391.2465. Found 391.2465.
(5S,6R)-5-(But-3-en-1-yl)-6-ethynyl-2,2,3,3,8,8,9,9-octamethyl-4,7-dioxa-3,8-disiladecane (8b)To a solution of 14b (32.5 mg, 0.089 mmol) in CH2Cl2 (0.4 mL) was added diisobutylaluminium hydride (1.0 M in n-hexane; 0.11 mL, 0.11 mmol) at 0 °C. The resulting mixture was stirred at the same temperature for 30 min. To the reaction mixture was added 2-propanol (0.074 mL), silica gel (200 mg), and H2O (1 mL) at room temperature. The resulting mixture was stirred at the same temperature for 30 min. The reaction mixture was filtered through a pad of Celite® and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (n-hexane/ethyl acetate = 10 : 1) to give the aldehyde as a yellow oil. The crude residue was used in the subsequent step without purification. To a solution of methyltriphenylphosphonium iodide (189 mg, 0.47 mmol) in THF (0.5 mL) was added sodium bis(trimethylsilyl)amide (1.9 M in THF, 0.23 mL, 0.44 mmol) at 0 °C. The resulting mixture was stirred at the same temperature for 30 min, then a solution of the aldehyde in THF (0.2 M, 0.45 mL, 0.089 mmol) was added dropwise at the same temperature. The resulting mixture was allowed to warm to room temperature and stirred for 1 h. The reaction mixture was quenched with sat. NH4Cl aq. and the aqueous layer was extracted with n-hexane for three times. The combined organic layer was washed with brine, dried over MgSO4, filtered, and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (n-hexane) to give 8b (23 mg, 70%) as a colorless oil. αD25=−29.1 (c 3.3, CHCl3); 1H-NMR (300 MHz, CDCl3) δ: 5.76–5.89 (m, 1H), 4.93–5.05 (m, 2H), 4.23 (q, J = 2.3 Hz, 1H), 3.73 (q, J = 5.2 Hz, 1H), 2.35 (d, J = 2.4 Hz, 1H), 2.13 (q, J = 7.3 Hz, 2H), 1.59–1.82 (m, 2H), 0.91 (s, 18H), 0.08–0.15 (m, 12H); 13C-NMR (75 MHz, CDCl3) δ: 138.8, 114.3, 84.2, 75.2, 73.3, 66.7, 32.0, 28.9, 26.0, 25.8, 18.2, −4.1, −4.5, −4.5, −5.1; HR-MS (ESI) m/z: [M + Na]+ Calcd for C20H40O2Si2Na 391.2465. Found 391.2442.
(3S,5S,6S)-5,6-Bis((tert-butyldimethylsilyl)oxy)oct-1-en-7-yn-3-ol (15a)To a solution of 13a (37.7 mg, 0.11 mmol) in DMSO (0.23 mL) was added IBX (29 mg, 0.21 mmol) at room temperature. The resulting mixture was allowed to warm to 50 °C and stirred for 3 h. The reaction mixture was quenched with sat. Na2S2O3 aq. and the aqueous layer was extracted with ethyl acetate for three times. The combined organic layer was washed with brine, dried over MgSO4, filtered, and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (n-hexane/ethyl acetate = 40 : 1) to give aldehyde as a colorless oil. To a solution of the aldehyde (35.4 mg, 0.099 mmol) in THF (0.5 mL) was added vinylmagnesium bromide (1.0 M in THF; 0.3 mL, 0.3 mmol, 3 equiv.) at −78 °C. After being stirred for 20 min, the reaction was quenched with sat. NH4Cl aq. and the mixture was extracted with ethyl acetate for three times. The organic layer was washed with brine, dried over MgSO4, filtered, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (CH2Cl2/n-hexane 1 : 1) to give 15a (14.3 mg, 23%) and 15a′ (25.5 mg, 42%). Spectral data for 15a: αD25 = –14.3 (c 0.5, CHCl3); 1H-NMR (400 MHz, CDCl3) δ: 5.92 (dq, J = 17.2, 5.3 Hz, 1H), 5.27 (dt, J = 17.2, 1.6 Hz, 1H), 5.09 (dt, J = 10.5, 1.6 Hz, 1H), 4.42 (q, J = 2.4 Hz, 1H), 4.39 (dd, J = 6.0, 4.1 Hz, 1H), 3.91 (q, J = 5.5 Hz, 1H), 2.38 (d, J = 2.3 Hz, 1H), 1.89 (t, J = 6.0 Hz, 2H), 0.91 (d, J = 4.1 Hz, 18H), 0.16–0.07 (m, 12H) ; 13C-NMR (100 MHz, CDCl3) δ: 141.4, 113.7, 82.5, 73.9, 72.2, 69.6, 66.6, 39.0, 25.9, 25.8, 18.2, 18.0, −4.5, −4.7, −5.0; HR-MS (ESI) m/z: [M + Na]+ Calcd for C20H40O3Si2Na 407.24137. Found 407.24210. Spectral data for 15a′: 1H-NMR (400 MHz, CDCl3) δ: 5.88 (dq, J = 11.8, 5.5 Hz, 1H), 5.28 (d, J = 17.2 Hz, 1H), 5.11 (d, J = 10.3 Hz, 1H), 4.40 (q, J = 2.4 Hz, 1H), 4.35 (m, 1H), 3.84 (m, 1H), 2.38 (d, J = 2.0 Hz, 1H), 2.37 (m, 1H), 1.91 (m, 1H), 0.91 (d, J = 2.1 Hz, 18H), 0.16–0.11 (m, 12H).
(3S,5S,6R)-5,6-Bis((tert-butyldimethylsilyl)oxy)oct-1-en-7-yn-3-ol (15b)To a solution of 13b (97.8 mg, 0.27 mmol) in DMSO (2.7 mL) was added IBX (153 mg, 0.55 mmol) at room temperature. The resulting mixture was allowed to warm to 50 °C and stirred for 3 h. The reaction mixture was quenched with sat. Na2S2O3 aq. and the aqueous layer was extracted with ethyl acetate for three times. The combined organic layer was washed with brine, dried over MgSO4, filtered, and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (n-hexane/ethyl acetate = 4 : 1) to give the aldehyde as a colorless oil. To a solution of the aldehyde (60.7 mg, 0.17 mmol) in THF (0.9 mL) was added vinylmagnesium bromide (1.0 M in THF; 0.51 mL, 0.51 mmol, 3 equiv.) at −78 °C. After being stirred for 2 h, the reaction was quenched with sat. NH4Cl aq. and the mixture was extracted with ethyl acetate for three times. The organic layer was washed with brine, dried over MgSO4, filtered, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (CH2Cl2/n-hexane 1 : 1) to give 15b (14.3 mg, 21%) and 15b′ (28.5 mg, 41%). Spectral data for 15b: αD25=−59.3 (c 0.3, CHCl3); 1H-NMR (400 MHz, CDCl3) δ: 5.87 (dq, J = 17.1, 5.3 Hz, 1H), 5.28–5.23 (m, 1H), 5.10–5.06 (m, 1H), 4.42 (q, J = 2.4 Hz, 1H), 4.38 (q, J = 2.3 Hz, 1H), 4.02–4.00 (m, 1H), 2.39 (d, J = 2.3 Hz, 1H), 1.94–1.89 (m, 1H), 1.74–1.67 (m, 1H), 0.92–0.90 (m, 18H), 0.16–0.13 (m, 12H); 13C-NMR (100 MHz, CDCl3) δ: 141.2, 113.9, 83.7, 74.0, 69.4, 66.6, 39.0, 25.9, 25.8, 18.1, 18.1, −4.2, −4.4, −4.7, −5.1; HR-MS (ESI) m/z: [M + Na]+ Calcd for C20H40O3Si2Na 407.24137. Found 407.24247. Spectral data for 15b′: 1H-NMR (400 MHz, CDCl3) δ: 5.88 (dq, J = 9.9, 5.8 Hz, 1H), 5.28 (d, J = 17.2 Hz, 1H), 5.10 (d, J = 9.9 Hz, 1H), 4.35–4.43 (m, 2H), 3.95 (m, 1H), 2.44 (d, J = 2.1 Hz, 1H), 1.98 (m, 1H), 1.85 (dq, J = 3.3, 1.3 Hz, 1H), 0.91 (d, J = 3.1 Hz, 18H), 0.18–0.08 (m, 12H).
(5S,6S,8S)-6-((tert-Butyldimethylsilyl)oxy)-5-ethynyl-2,2,3,3,10,10,11,11-octamethyl-8-vinyl-4,9-dioxa-3,10-disiladodecane (9a)To a solution of 15a (10.3 mg, 0.027 mmol) in CH2Cl2 (0.3 mL) were added 2,6-lutidine (10 µL, 0.08 mmol) and tert-butyldimethylsilyl triflate (12 µL, 0.054 mmol) at 0 °C. The mixture was allowed to warm to room temperature and stirred for 2 h. The reaction was quenched with H2O and the mixture was extracted with CH2Cl2. The combined organic layer was washed with brine, dried over MgSO4, filtered, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (ethyl acetate/n-hexane 1 : 80) to give 9a (12.0 mg, 90%) as a clear oil. αD25=−11.3 (c 0.6, CHCl3); 1H-NMR (400 MHz, CDCl3) δ: 5.91–5.82 (m, 1H), 5.17–5.12 (m, 1H), 5.02 (d, J = 10.1 Hz, 1H), 4.37 (t, J = 2.3 Hz, 1H), 4.32–4.27 (m, 1H), 3.81–3.77 (m, 1H), 2.31 (d, J = 2.3 Hz, 1H), 2.08–2.02 (m, 1H), 1.62 (dq, J = 13.7, 4.1 Hz, 1H), 0.90–0.88 (m, 27H), 0.13–0.04 (m, 18H); 13C-NMR (100 MHz, CDCl3) δ: 142.4, 114.0, 73.3, 71.3, 67.0, 41.4, 25.9, 25.8, 25.7, 18.2, 18.1, 4.3, −3.5, −4.3, −4.4, −4.5, −4.7, −5.1; HR-MS (ESI) m/z: [M + Na]+ Calcd for C26H54O3Si3Na 521.32730. Found 521.32842.
(5R,6S,8S)-6-((tert-Butyldimethylsilyl)oxy)-5-ethynyl-2,2,3,3,10,10,11,11-octamethyl-8-vinyl-4,9-dioxa-3,10-disiladodecane (9b)To a solution of 15b (14.3 mg, 0.037 mmol) in CH2Cl2 (0.4 mL) were added 2,6-lutidine (13 µL, 0.11 mmol) and tert-butyldimethylsilyl triflate (17 µL, 0.074 mmol) at 0 °C. The mixture was allowed to warm to room temperature and stirred for 1 h. The reaction was quenched with H2O and the mixture was extracted with CH2Cl2 for three times. The combined organic layer was washed with brine, dried over MgSO4, filtered, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (ethyl acetate/n-hexane 1 : 80) to give 9b (18.1 mg, 98%) as a clear oil. αD25 = –14.2 (c 0.7, CHCl3); 1H-NMR (400 MHz, CDCl3) δ: 5.87–5.78 (m, 1H), 5.14 (dt, J = 17.2, 1.4 Hz, 1H), 5.05–5.02 (m, 1H), 4.31–4.26 (m, 2H), 3.91–3.88 (m, 1H), 2.34 (d, J = 2.3 Hz, 1H), 1.84 (dq, J = 14.0, 4.1 Hz, 1H), 1.61 (dq, J = 7.0, 4.6 Hz, 1H), 0.93–0.84 (m, 27H), 0.13–0.03 (m, 18H); 13C-NMR (100 MHz, CDCl3) δ: 142.1, 114.2, 85.9, 83.1, 73.5, 73.1, 71.5, 68.0, 42.8, 26.0, 25.9, 25.9, 18.3, 18.3, 18.2, −3.6, −3.7, −4.5, −5.0; HR-MS (ESI) m/z: [M + Na]+ Calcd for C26H54O3Si3Na 521.32730. Found 521.32712.
4α,25(OH)2D3-23S,26R-lactone (3)To a solution of 8a (13.3 mg, 0.036 mmol) and CD rings 7 (15.0 mg, 0.030 mmol) in triethylamine (0.21 mL) and toluene (0.21 mL) were added tetrakis(triphenylphosphine)palladium (3.5 mg, 0.0030 mmol) at room temperature. The mixture was allowed to warm to 90 °C and stirred for 2 h. The reaction mixture was concentrated in vacuo. The residue was filtered by column chromatography on silica gel (ethyl acetate/n-hexane 0 : 100) to give coupling product (15.3 mg, 65%) as a pale-yellow oil. The crude residue was used in the subsequent step without purification. To a solution of the coupling product (8.1 mg, 0.0103 mmol) in THF (0.5 mL) was added triethylamine trihydrofluoride (0.26 mL) at room temperature. After being stirred for 6 d, the reaction was quenched with sat. NaHCO3 aq. and the mixture was extracted with ethyl acetate for three times. The organic layer was washed with brine, dried over MgSO4, filtered, and concentrated in vacuo. The residue was purified by a preparative silica gel TLC plate (methanol/chloroform 1 : 20) to give 3 (8.1 mg, 99%) as a colorless amorphous. αD25=+40.7 (c 0.3, CHCl3); 1H-NMR (400 MHz, CDCl3) δ: 6.58 (d, J = 11.4 Hz, 1H), 6.04 (d, J = 11.4 Hz, 1H), 5.12 (s, 1H), 4.87 (s, 1H), 4.44 (d, J = 9.2 Hz, 1H), 3.96 (d, J = 7.8 Hz, 1H), 3.56 (s, 1H), 2.90 (d, J = 13.3 Hz, 1H), 2.69 (s, 1H), 2.41–2.34 (m, 2H), 2.22–1.25 (m, 17H), 1.02 (d, J = 6.4 Hz, 3H), 0.91 (d, J = 29.3 Hz, 3H), 0.55 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ: 179.4, 143.5, 137.6, 118.9, 117.3, 114.7, 78.3, 74.6, 73.4, 56.5, 56.3, 45.9, 43.2, 41.7, 40.4, 33.8, 32.2, 31.4, 29.7, 29.0, 28.0, 24.5, 23.4, 22.1, 19.3, 12.0, 1.0; HR-MS (ESI) m/z: [M + Na]+ Calcd for C27H40O5Na 467.27734. Found 467.27702.
4β,25(OH)2D3-23S,26R-lactone (4)To a solution of 8b (10.0 mg, 0.027 mmol) and CD rings 7 (16.3 mg, 0.033 mmol) in triethylamine (0.19 mL) and toluene (0.19 mL) were added tetrakis(triphenylphosphine)palladium (3.1 mg, 0.0027 mmol) at room temperature. The mixture was allowed to warm to 90 °C and stirred for 2 h. The reaction mixture was concentrated in vacuo. The residue was filtered by column chromatography on silica gel (ethyl acetate/n-hexane 0 : 100) to give coupling product (12.6 mg, 59%) as a pale-yellow oil. The crude residue was used in the subsequent step without purification. To a solution of the coupling product (4.0 mg, 0.0051 mmol) in THF (0.25 mL) was added triethylamine trihydrofluoride (0.13 mL) at room temperature. After being stirred for 6 d, the reaction was quenched with sat. NaHCO3 aq. and the mixture was extracted with ethyl acetate for three times. The organic layer was washed with brine, dried over MgSO4, filtered, and concentrated in vacuo. The residue was purified by a preparative silica gel TLC plate (methanol/chloroform 1 : 20) to give 4 (2.8 mg, 99%) as a colorless amorphous. αD25=+61.1 (c 0.2, CHCl3); 1H-NMR (400 MHz, CDCl3) δ: 6.50 (d, J = 11.4 Hz, 1H), 6.04 (d, J = 10.5 Hz, 1H), 5.17 (s, 1H), 4.92 (s, 1H), 4.45–4.41 (m, 1H), 4.22 (d, J = 3.2 Hz, 1H), 3.86 (s, 1H), 2.86 (d, J = 12.8 Hz, 1H), 2.61 (s, 1H), 2.38 (q, J = 6.0 Hz, 2H), 2.17–1.25 (m, 17H), 1.02 (d, J = 6.4 Hz, 3H), 0.95–0.88 (m, 3H), 0.55 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ: 179.4, 144.7, 142.5, 137.4, 123.3, 117.2, 115.4, 73.3, 71.8, 56.5, 56.3, 46.0, 43.2, 41.7, 40.3, 33.8, 31.9, 30.0, 29.7, 29.1, 27.9, 24.5, 23.4, 22.1, 19.2, 12.0, 1.0; HR-MS (ESI) m/z: [M + Na]+ Calcd for C27H40O5Na 467.27734. Found 467.27652.
1α,4α,25(OH)3D3-23S,26R-lactone (5)To a solution of 9a (8.9 mg, 0.018 mmol) and CD rings 7 (9.8 mg, 0.020 mmol) in triethylamine (0.13 mL) and toluene (0.13 mL) were added tetrakis(triphenylphosphine)palladium (4.2 mg, 0.0036 mmol) at room temperature. The mixture was allowed to warm to 90 °C and stirred for 2 h. The reaction mixture was concentrated in vacuo. The residue was filtered a pad of silica gel (ethyl acetate/n-hexane 0 : 100) to give coupling product (9.7 mg, 59%) as a pale-yellow oil. The crude residue was used in the subsequent step without purification. To a solution of the coupling product (9.7 mg, 0.0106 mmol) in THF (0.41 mL) was added triethylamine trihydrofluoride (0.12 mL) at room temperature. After being stirred for 3 d, the reaction was quenched with sat. NaHCO3 aq. and the mixture was extracted with ethyl acetate for three times. The organic layer was washed with brine, dried over MgSO4, filtered, and concentrated in vacuo. The residue was purified by a preparative silica gel TLC plate (ethyl acetate/chloroform 4 : 1) to give 5 (3.5 mg, 72%) as a colorless amorphous. αD25=+15.2 (c 0.3, MeOH); 1H-NMR (400 MHz, CDCl3) δ: 6.76 (d, J = 11.4 Hz, 1H), 6.02 (d, J = 11.4 Hz, 1H), 5.38 (d, J = 1.4 Hz, 1H), 5.06 (d, J = 1.8 Hz, 1H), 4.47–4.40 (m, 2H), 4.01–3.94 (m, 2H), 2.93–2.88 (m, 1H), 2.39 (dd, J = 12.8, 5.5 Hz, 1H), 2.25 (td, J = 9.2, 4.4 Hz, 1H), 2.06–1.99 (m, 2H), 1.96–1.25 (m, 14H), 1.02 (d, J = 6.4 Hz, 3H), 0.89–0.81 (m, 3H), 0.54 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ: 179.3, 145.3, 144.9, 134.7, 122.9, 116.8, 116.0, 78.0, 73.3, 71.6, 71.2, 56.4, 56.3, 46.0, 43.2, 41.7, 40.4, 38.4, 33.8, 29.7, 29.1, 27.9, 24.5, 23.4, 22.2, 19.2, 12.0; HR-MS (ESI) m/z: [M + Na]+ Calcd for C27H40O6Na483.27226. Found 483.27113.
1α,4β,25(OH)3D3-23S,26R-lactone (6)To a solution of 9b (9.0 mg, 0.018 mmol) and CD rings 7 (9.9 mg, 0.020 mmol) in triethylamine (0.13 mL) and toluene (013 mL) were added tetrakis(triphenylphosphine)palladium (4.2 mg, 0.0036 mmol) at room temperature. The mixture was allowed to warm to 90 °C and stirred for 2 h. The reaction mixture was concentrated in vacuo. The residue was filtered through a pad of silica gel (ethyl acetate/n-hexane 0 : 100) to give coupling product (14.7 mg, 89%) as a pale-yellow oil. The crude residue was used in the next step without purification. To a solution of the coupling product (14.7 mg, 0.016 mmol) in THF (0.61 mL) was added triethylamine trihydrofluoride (0.19 mL) at room temperature. After being stirred for 3 d, the reaction was quenched with sat. NaHCO3 aq. and the mixture was extracted with ethyl acetate for three times. The organic layer was washed with brine, dried over MgSO4, filtered, and concentrated in vacuo. The residue was purified by a preparative silica gel TLC plate (ethyl acetate/chloroform 4 : 1) to give 6 (5.4 mg, 73%) as a colorless amorphous. αD25=+22.3 (c 0.4, MeOH); 1H-NMR (400 MHz, CDCl3) δ: 6.66 (d, J = 11.4 Hz, 1H), 6.03 (d, J = 11.4 Hz, 1H), 5.41 (s, 1H), 5.09 (d, J = 1.8 Hz, 1H), 4.47–4.41 (m, 2H), 4.28 (d, J = 2.7 Hz, 1H), 4.21–4.17 (m, 1H), 2.89–2.86 (m, 1H), 2.38 (dd, J = 12.8, 5.5 Hz, 1H), 2.16–2.10 (m, 1H), 2.06–1.98 (m, 2H), 1.96–1.90 (m, 1H), 1.75–1.24 (m, 13H), 1.02 (t, J = 6.4 Hz, 3H), 0.92–0.79 (m, 3H), 0.55 (d, J = 5.0 Hz, 3H); 13C-NMR (100 MHz, CDCl3) δ: 179.4, 145.6, 144.6, 134.7, 125.6, 116.7, 115.6, 76.4, 76.1, 73.3, 71.3, 68.8, 56.4, 56.3, 46.0, 43.2, 41.7, 40.3, 37.4, 33.8, 29.1, 27.9, 24.5, 23.5, 22.2, 19.2, 12.0; HR-MS (ESI) m/z: [M + Na]+ Calcd for C27H40O6Na 483.27226. Found 483.27139.
Metabolism StudyMetabolism of 1 and 2 by human liver microsomes: A mixture of 0.5 mg/mL human liver microsomes, 5 µM substrate, and 1 mM reduced nicotinamide adenine dinucleotide phosphate (NADPH) in 100 mM potassium phosphate (pH 7.4) was incubated at 37 °C for 30 min. Metabolites were extracted with 4 volumes of CHCl3/MeOH (3 : 1) and analyzed by reversed-phase HPLC under the same conditions as in our previous study.21)
Metabolism of 1 and 2 by recombinant human drug-metabolizing CYPs: The metabolism of 1 and 2 by human CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 was examined as follows using commercially available recombinant microsomes prepared from baculovirus-infected insect cells. A mixture containing 10 nM CYP (Corning Inc., NY, U.S.A.), 5 µM substrate, and 1 mM NADPH in 100 mM potassium phosphate (pH 7.4) was incubated at 37 °C for 30 min. Metabolites were extracted with 4 volumes of CHCl3/MeOH (3 : 1) and analyzed by reversed phase HPLC under the same conditions as in our previous study.21)
LC/MS analysis: LC/MS analysis was performed using HPLC Agilent 1260-Infinity system (Agilent, CA, U.S.A.) connected to API-3200 QTrap (AB Sciex, MA, U.S.A.). The LC condition is same with our previous paper.22) The mass spectrometer operated in positive ion mode using APCI. The full scan range was from m/z 100 to 600.
Docking StudyThe initial structure of the human CYP3A4 was obtained from the Protein Data Bank (5A1R) and refined for docking simulations using the Protein Preparation Wizard script within Maestro (Schrödinger, LLC, New York, NY, U.S.A.). For all molecules, ionization and energy minimization were performed using the OPLS3e force field in the LigPrep script in Maestro. These minimized structures were used as input structures for the docking simulations using the Glide SP docking23,24) program (Schrödinger, LLC), with a grid box defined by a potential binding site position from SiteMap25,26) (Schrödinger, LLC). After the docking simulations were completed, the shortest distances between the Fe atom of the HEM molecule and C4 of the docked compounds from 100 poses on a binding site were selected.
This work was supported by JSPS KAKENHI Grant Numbers 22K19101 (KN) and 23KJ0861 (RS). This work was inspired by the international and interdisciplinary environment of the JSPS Asian CORE Program of ACBI (Asian Chemical Biology Initiative).
The authors declare no conflict of interest.
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