Nicoletti et al.
for 12 h to give resin loaded with alcohol 12 (0.533 g, 0.800 mmol/
g, 97%). 13C NMR (63 MHz, CDCl3) δ 173.6, 145.3, 127.6, 125.6,
69.2, 66.0, 56.2, 52.2, 45.8, 44.0, 41.8, 40.3, 35.0, 33.5, 27.1, 22.5,
21.4, 18.5, 17.4, 13.5. IR (KBr) 3440 (OH), 2923, 2873, 2855, 1732
and then allowed to reach -40 °C. THF (5 mL) was added, and
the resin beads were filtered and washed successively with CH2Cl2
(3 × 3 mL), Et2O (3 × 3 mL), MeOH (3 × 3 mL), Et2O (3 × 3
mL), CH2Cl2 (3 × 3 mL), and Et2O (3 mL). The combined filtrate
was concentrated to recover the unreacted excess of phosphine oxide
13 (0.240 g, 75%, after flash chomatography). The remaining resin
was nitrogen-dried and then high-vacuum-dried to give protected
vitamin-loaded resin 14 (0.243 g). 13C NMR (63 MHz, CDCl3) δ
144.9, 127.7, 125.7, 121.4, 117.9, 111.9, 70.3, 66.0, 56.2, 47.0,
45.7, 40.4, 36.5, 35.9, 35.2, 32.6, 28.9, 27.7, 23.5, 22.3, 21.6, 18.7,
18.1, 12.4.
Polymer-Bound Vitamin 15. Hydrogen fluoride pyridine (HF-
Py, 0.35 mL) was added to a suspension of resin 14 (0.238 g, high-
vacuum-dried from P2O5 for 72 h) in dry CH2Cl2 (1.5 mL) and dry
CH3CN (1.5 mL). The resulting mixture was shaken at rt in the
dark for 48 h. The reaction was quenched by the adition of
diisopropylethylamine (DIPEA, 0.5 mL), and the resin was filtered
and washed successively with with CH2Cl2/DIPEA (1:1, 4 mL),
CH2Cl2 (4 mL), CH2Cl2/DIPEA (1:1, 4 mL), CH2Cl2 (3 × 4 mL),
DMF (2 mL), dioxane (6× 2 mL), DMF (2 mL), and CH2Cl2 (6×
2 mL). The resin was high-vacuum-dried to afford resin loaded
with vitamin D3 ester 15 (0.195 g), which was directly submitted
to next reactions.
25-Hydroxyvitamin D3 (1a). A solution of MeLi in Et2O (0.7
mL, 1.4 M, 1 mmol) was added to a suspension of resin 15 (0.101
g, high-vacuum-dried over P2O5 for 72 h) in dry THF (3 mL) at
-78 °C. The mixture was slowly warmed to -40 °C for 12 h in
the dark. The reaction was quenched at -78 °C by the addition of
a few drops of MeOH. The resin was filtered and washed with
Et2O (10 mL). The solution was washed with brine (5 mL), dried,
filtered, and concentrated. The residue was purified by flash
chromatography (8 × 1 cm, 40% Et2O/hexanes) to afford metabolite
1a. [20 mg, 0,05 mmol, 61% from resin 3, Rf ) 0.2 (30% EtOAc/
hexanes), 59% from resin 6]. Spectral data were identical to those
of an authentic sample.25
26,26,26,27,27,27-Hexadeutero-25-hydroxyvitamin D3 (1c). A
solution of CD3MgI in Et2O (0.9 mL, 1.0 M, 0.9 mmol) was added
to a suspension of resin 15 (94 mg, high-vacuum-dried over P2O5
for 72 h) in dry THF (3 mL) at -78 °C. The mixture was slowly
warmed to rt for 12 h in the dark. The reaction was quenched at 0
°C by the addition of a few drops of H2O. The resin was filtered
and washed with Et2O (10 mL). The solution was washed with
saturated NH4Cl (10 mL) and brine (5 mL), dried, filtered, and
concentrated. The residue was purified by flash chromatography
(8 × 1 cm, 40% Et2O/hexanes) to afford labeled metabolite 1c [19
mg, 0.047 mmol, 63% from resin 3, 61% from resin 6]. Spectral
data were identical to those of an authentic sample.25
(CO), 1491, 1159, 756, 698, 540 cm-1
.
Methyl 8ꢀ-[(tert-Butyldimethylsilyl)oxy]-de-A,B-cholane-24-car-
boxylate (5). An aqueous solution of LiOH (9 mL, 2 M, 18 mmol)
was added to a solution of ester 7 (0.60 g, 2.12 mmol) in dry THF
(36 mL). The reaction mixture was stirred at reflux for 5 h under
argon and then allowed to reach room temperature. An aqueous
solution of HCl (10%) was added until pH ) 2. The mixture was
stirred for 5 min and then extracted with Et2O (3 × 50 mL). The
combined organic phase was washed with brine (50 mL), dried,
filtered, and concentrated. The residue was purified by flash
chromatography (12 × 2 cm, 20% EtOAc /hexanes) to give 526 as
a white powder (0.53 g, 92%). Rf ) 0.2 (20% EtOAc/hexanes).
Polymer-Bound Alcohol 12 from Acid 5. A mixture of acid 5
(0.088 g, 0.33 mmol), MeIm (65 µL, 0.66 mmol), and MSNT (0.085
g, 0.33 mmol) in dry CH2Cl2 (3 mL) was stirred until an
homogeneous solution was obtained. Dry THF (3 mL) was added.
The resulting solution was added in two portions via syringe to
hydroxymethylpolystyrene resin (6, 0.105 g, 1.0 mmol/g, 0.105
mmol, previously dried for 12 h under high vacuum). The mixture
was shaken for 90 min under argon atmosphere. The resin was
filtered and washed with CH2Cl2 (20 mL). The filtrate was
concentrated, and the residue was purified by flash chromatography
(12 × 1 cm, 10-30% EtOAc/hexanes) to recover the unreacted
excess of 5 (58 mg, 97%). The resin was washed successively with
CH2Cl2 (6 × 2 mL), DMF (2 mL), MeOH (2 × 2 mL), dioxane (3
× 2 mL), DMF (2 mL), MeOH (2 × 2 mL), and CH2Cl2 (6 × 2
mL). The resin was air-dried and high-vacuum-dried for 12 h to
give resin loaded with alcohol 12 (0.131 g, 0.800 mmol/g, 100%).
The 13C NMR spectrum was identical to the spectrum of the resin
12 obtained from 10.
Polymer-Bound Ketone 3. Method A: TEMPO (13.1 mg, 0.084
mmol) and DAIB (0.300 g, 0.93 mmol) were successively added
to a suspension of resin loaded with alcohol 12 (0.168 g, 0.800
mmol/g, 0.134 mmol) in dry CH2Cl2 (5 mL). After shaking in the
darkness for 18 h at room temperature, the resin was filtered and
washed successively with CH2Cl2 (2 × 4 mL), MeOH (4 mL), aq
KHCO3 (3 × 4 mL), H2O (3 × 4 mL), MeOH (4 × 4 mL), and
CH2Cl2 (3× 4 mL). The resin was nitrogen-dried and high-vacuum-
dried for 12 h to give the solid-supported ketone 3 (0.163 g, 0.801
mmol/g, 97%). 13C NMR (63 MHz, CDCl3) δ 211.8, 145.1, 127.7,
125.6, 66.0, 61.9, 56.3, 49.8, 45.8, 44.0, 40.3, 38.9, 35.2, 34.6, 30.6,
27.5, 24.0, 21.4, 19.0, 18.7, 12.5. IR (KBr) 3025, 2923, 1734 (CO-
25), 1714 (CO-8), 1601, 1311, 1450, 1097, 757, 698, 540 cm-1
.
Method B: Dess-Martin periodinane (0.40 g, 0.94 mmol) was
added to a suspension of resin 12 (0.200 g, 0.800 mmol/g, 0.16
mmol) in dry CH2Cl2 (6 mL) and dry pyridine (0.2 mL). The
reaction mixture was shaken in the dark at rt for 18 h. The resin
was filtered and washed successively with THF/
(NaHCO3-Na2S2O3)aq (3:1, 2 × 4 mL), THF/H2O (3:1, 2 × 4
mL), MeOH (2× 4 mL), THF/H2O (3:1, 2 × 4 mL), MeOH (2×
4 mL), and Et2O (3× 4 mL). The resin was nitrogen-dried and
high-vacuum-dried for 12 h to give 3 (0.194 g, 0.801 mmol/g, 97%),
whose 13C NMR spectrum was identical to the spectrum of the
resin prepared by method A.
Polymer-Bound Protected Vitamin 14. A solution of phosphine
oxide 1326 (0.400 g, 0.809 mmol, high-vacuum-dried over P2O5
for 72 h) in dry THF (3 mL) at -78 °C was treated under Ar with
a solution of n-BuLi in hexanes (0.31 mL, 2.5 M, 0.78 mmol).
After stirring for 60 min the deep red solution of the anion of 13
was added to a suspension of resin 3 (0.200 g, 0.801 mmol/g, 0.160
mmol, high-vacuum-dried over P2O5 for 72 h) in dry THF (3 mL)
at -78 °C. The reaction mixture was shaken in the dark for 6 h
Acknowledgment. We thank the Spanish Ministry of Educa-
tion and Science (projects SAF2001-3187 and SAF2004-1885)
and Xunta de Galicia (INCITE08PXIB-209130PR and ACEUIC-
2006/XA050) for financial support, Zaida Rodr´ıguez for pre-
liminary experimentation, and Solvay Pharmaceuticals BV
(Weesp, The Netherlands) for the gift of vitamin D2. D. N.
thanks the Argentine National Council for Scientific Research
(CONICET) for a postdoctoral fellowship.
Supporting Information Available: General experimental
procedures. Experimental description for labeled compound 11b.
Copies of 13C NMR spectra for solid-supported compounds 3,
1
10, 10b, 12, and 14. Copies of H and 13C NMR spectra for
compounds 8, 9, 11a, 11b, 5, 1a and 1c. This material is
JO900524U
4786 J. Org. Chem. Vol. 74, No. 13, 2009