Synthesis of 25-hydroxyvitamin D3 and 26,26,26,27,27,27- hexadeutero-25-hydroxyvitamin D3 on solid support

Article abstract of DOI:10.1021/jo900524u

(Chemical Equation Presented) A convenient five-step route to 25-hydroxylated vitamin D3 compounds on (hydroxymethyl)polystyrene support is reported. A CD-side chain fragment was anchored to the solid phase through an ester group at C25 and coupled to an

Full text of DOI:10.1021/jo900524u

Synthesis of 25-Hydroxyvitamin D3 and
26,26,26,27,27,27-Hexadeutero-25-hydroxyvitamin D3 on Solid
Support†
Daniel Nicoletti, Antonio Mourin˜o, and Mercedes Torneiro*
Departamento de Qu´ımica Orga´nica y Unidade Asociada al CSIC, UniVersidad de Santiago de
Compostela, 15782 Santiago de Compostela, Spain
mercedes.torneiro@usc.es
ReceiVed March 11, 2009
A convenient five-step route to 25-hydroxylated vitamin D3 compounds on (hydroxymethyl)polystyrene
support is reported. A CD-side chain fragment was anchored to the solid phase through an ester group
at C25 and coupled to an A ring building block to assemble the vitamin D triene system by the
Wittig-Horner approach. Deprotection of the hydroxy group was carried out on the support, prior to
functionalization at C25. The title compounds were released from the resin in excellent global yield by
nucleophilic attack on the ester carbonyl group using commercially available organometallic reagents.
This key last step offers an opportunity for the efficient generation of 26,27-labeled compounds and also
for diversification at the side chain without need for a pool of side chain fragments.
Introduction
hypercalcemic effects.2,6,7 Efforts aimed at developing vitamin
D analogues with strong cell-differentiating ability and low
calcemic action have led to the synthesis of more than 3000
vitamin D analogues.7,8 However, only a few of these have
successfully made it to the market,9 for example, calcipotriol
(MC903, Daivonex), which is currently used for the treatment
of psoriasis.10,11 Successful convergent synthetic strategies to
access these compounds have been reported.12-14 However,
aside from the pioneering work of Takahashi and co-workers,15,16
studies on the syntheses of new vitamin D analogues involving
Vitamin D3 undergoes two successive hydroxylations, first
in the liver at C25 to produce 25-hydroxyvitamin D3 (1a) and
then in the kidney at C1 to generate 1,25-dihydroxyvitamin D3
(1b), the active form of vitamin D3, which regulates a plethora
of biological functions including calcium homeostasis, bone
mineralization, proliferation and differentiation of various types
of cells, and immune modulation.1-3 The antiproliferative-
prodifferentiating activities of the secosteroid hormone 1b have
suggested its potential use in the treatment of various cancers
(breast, colon) and skin diseases.4,5 However, its therapeutic
application has been limited by the parallel induction of
(6) Ettinger, R. A.; DeLuca, H. F. AdV. Drug Res. 1996, 28, 269–312.
(7) Carlberg, C.; Mourin˜o, A. Expert Opin. Ther. Pat. 2003, 13, 761–772.
(8) Bouillon, R.; Okamura, W. H.; Norman, A. W. Endocr. ReV. 1995, 16,
200–256.
(9) Kabat, M. M.; Radinov, R. Curr. Opin. Drug DiscoVery DeV. 2001, 4,
† Dedicated to Prof. Josep Font on the occasion of his retirement.
(1) Bouillon, R.; Norman, A. W.; Pasqualini, J. R. Proceedings of the 13th
and 12th Workshops on Vitamin D. J. Steroid Biochem. Mol. Biol. 2007, 103,
201–822; 2004, 89-90, 1-633 and the previous 11 volumes in this series.
(2) Vitamin D, 2nd ed.; Feldman D., Pike, J. W., Glorieux, F. H., Eds.;
Academic Press: New York, 2005.
(3) Jones, G.; Strugnell, S. A.; DeLuca, H. F. Physiol. ReV. 1998, 78, 1193–
1231.
(4) Abe, E.; Miyaura, C.; Sakagami, H.; Takeda, M.; Konno, K.; Yamazaki,
T.; Yoshiki, S.; Suda, T. Proc. Natl. Acad. Sci. U.S.A. 1981, 78, 4990–4994.
(5) Colston, K.; Colston, M. J.; Feldman, D. Endocrinology 1981, 108, 1083–
1086.
808–833.
(10) Jain, S.; Sehgal, V. N. Int. J. Dermatol. 1997, 36, 255–258.
(11) Calverley, M. J. Tetrahedron 1987, 43, 4609–4619.
(12) Dai, H.; Posner, G. H. Synthesis 1994, 1383–1398. Zhu, G.-D.; Okamura,
W. H. Chem. ReV. 1995, 95, 1877–1952.
(13) Posner, G. H.; Kahraman, M. Eur. J. Org. Chem. 2003, 3889–3895.
(14) Krause, S. Schmalz H.-G. In Organic Synthesis Highlights IV; Schmalz,
H.-G., Ed.; Wiley-VCH: Weinheim, 2000; pp 212-217.
(15) Doi, T.; Hijikuro, I.; Takahashi, T. J. Am. Chem. Soc. 1999, 121, 6749–
6750.
(16) Hijikuro, I.; Doi, T.; Takahashi, T. J. Am. Chem. Soc. 2001, 123, 3716–
3722.
4782 J. Org. Chem. 2009, 74, 4782–4786
10.1021/jo900524u CCC: $40.75  2009 American Chemical Society
Published on Web 06/03/2009

25-Hydroxylated Vitamin D3 Compounds
SCHEME 1. Takahashi’s Strategy to Vitamin D3 Analogues
materials, would be intriguing employing silylated linkers.
Another key step includes the formation of the vitamin D triene
system by Wittig-Horner coupling between phosphine anion
4 and polymer-linked ketone 3 because the resulting solid-
supported intermediate could serve as a handle for diversity-
oriented synthesis. At this point we were optimist that possible
basic species generated upon workup would not hydrolyze the
ester group. Access to ketone 3 would be carried out by selective
esterification of carboxylic acid 5 with simple commercial resins
containing a primary alcoholic hydroxy group. This approach
would also serve to access other known bioactive or antagonist
vitamin D3 analogues by simple adaptation of the corresponding
carboxylated side chain.
Results and Discussion
As representative targets we chose 25-hydroxyvitamin D3 (25-
OH-D3, 1a) and 26,26,26,27,27,27-hexadeutero-25-hydroxyvi-
tamin D3 (1c). 25-OH-D3 is the most abundant vitamin D
metabolite in circulation and serves as an indicator of nutritional
vitamin D status22 and in the treatment of several deseases.1
The hexadeuterated analogue 1c is used as an internal standard
in the quantification of circulating 25-OH-D3.23,24 Our prelimi-
nary studies on the formation of the ester linkage began with
hydroxyester 7 (Scheme 3), which was easily prepared from
commercial vitamin D2 in three steps.25,26 Silylation of the
secondary hydroxy group followed by hydrolysis of the ester
provided the desired carboxylic acid 9, which was anchored
directly to (hydroxymethyl)polystyrene resin (6) employing
1-(mesitylene-2-sulfonyl)-3-nitro-1H-1,2,4-triazole (MSNT) and
N-methylimidazole (MeIm) to activate the carboxylic group.27
The loading yield of the anchored CD-side chain fragment
10 was 100%,28 and the excess unreacted acid 9 (1 equiv) was
recovered by filtration. IR and 13C NMR spectra of resin 10
confirmed the presence of the ester linkage and the disappear-
ance of the free hydroxy groups by comparison with spectra of
unloaded resin 6 and partially loaded resin 10b obtained by
reaction of 6 with 0.6 equiv of 9. At this point we decided to
test the feasibility of the final releasing step of the synthesis
before building the vitamin D triene system. Addition of
methyllithium provided 11a in 93% yield, while addition of
trideutero-methylmagnesium iodide produced the tertiary alcohol
the construction of the labile vitamin D triene system on solid
support are unknown.17 Takahashi’s approach uses the Lythg-
oe’s powerful Wittig-Horner strategy18-20 to build the vitamin
D triene system by coupling a phosphine oxide anion (A-ring
fragment) with a solid supported ketone (CD-fragment), an-
chored to PS-DES resin via a sulfonated linker. Subsequently
a Cu(I)-catalyzed Grignard displacement of the resin can be used
to introduce the side chain fragment (Scheme 1). Final depro-
tection of analogues is accomplished in solution.16
As part of a broader research program to develop effective
synthetic pathways to bioactive vitamin D analogues, we
disclose here a convenient five-step method that involves the
construction of the vitamin D triene system employing Lythgoe’s
Wittig-Horner approach on solid phase to generate 25-
hydroxyvitamin D3 (1a) and their analogues functionalized at
C2521 (Scheme 2). It was assumed that the target compounds
could be released from the deprotected polymer-bound vitamin
D3 intermediate 2 by nucleophilic attack on the ester carbonyl
group using a variety of commercially available organometallic
reagents. Moreover, this key step offers an opportunity for the
generation of 26,27-labeled compounds (11C, 14C, 3H) for
2
biomedical studies or standards (13C, H) for quantification of
vitamin D3 metabolites by mass spectrometry.
1
11b in 96% yield. Both alcohols were analytically pure by H
We hoped that the desilylation step could be performed after
formation of the triene system without problem. This operation,
which is of particular importance in the pathway to labeled
and 13C NMR spectral analysis. Desilylation of 10 with aqueous
hydrogen fluoride provided the polymer-bound alcohol 12 in
97% yield.29
At this stage and as discussed on the retrosynthetic plan, we
considered the possibility of preparing hydroxyester 12 directly
from acid 5, thus avoiding the protection-deprotection steps.
(17) For the synthesis of vitamin D-related compounds on solid support not
involving the construction of the vitamin D triene system, see: Hanazawa, T.;
Wada, T.; Masuda, T.; Okamoto, S.; Sato, F. Org. Lett. 2001, 3, 3975–3977.
D’herde, J. N.; De Clercq, P. J. Molecules 2006, 11, 655–660.
(18) Lythgoe, B.; Roberts, D. A.; Waterhouse, I. J. Chem. Soc., Perkin Trans.
1 1977, 2608–2612.
(22) Hollis, B. W.; Wagner, C. L.; Drezner, M. K.; Binkley, N. C. J. Steroid
Biochem. Mol. Biol. 2007, 103, 631–634.
(23) Maunsell, Z.; Wright, D. J.; Rainbow, S. J. Clin. Chem. 2005, 51, 1683–
1690.
(24) Tsugawa, N.; Suhara, Y.; Kamao, M.; Okano, T. Anal. Chem. 2005,
77, 3001–3007.
(25) Mascaren˜as, J. L.; Pe´rez-Sestelo, J.; Mourin˜o, A. Tetrahedron Lett. 1991,
32, 2813–2816.
(19) Lythgoe, B. Chem. Soc. ReV. 1980, 9, 449–475.
(20) Baggiolini, E. G.; Iacobelli, J. A.; Hennessy, B. M.; Batcho, A. D.;
Sereno, J. F.; Uskokovic, M. R. J. Org. Chem. 1986, 51, 3098–3108.
(21) Elongation of the side chain at positions C26 and C27 increases the
biological activity of the vitamin D hormone: Honda, A.; Mori, Y.; Otomo, S.;
Ishizuka, S.; Ikekawa, N. Steroids 1991, 56, 142–147. Miyahara, T.; Harada,
M.; Miyata, M.; Sugure, A.; Ikemoto, Y.; Takamura, T.; Higuchi, S.; Otomo,
S.; Kozuka, H.; Ikekawa, N. Calcif. Tissue Int. 1992, 51, 218–222. For recent
examples of active analogues modified at C25, see: Shimizu, M.; Miyamoto,
Y.; Takaku, H.; Matsuo, M.; Nakabayashi, M.; Masuno, H.; Udagawa, N.;
DeLuca, H. F.; Ikura, T.; Ito, N. Bioorg. Med. Chem. 2008, 16, 6949–6964, and
references therein. Yoon, J. S.; Kim, J. Y.; Park, H. K.; Kim, E. S.; Ahn, K. S.;
Yoon, S. S.; Cho, C. G.; Kim, B. K.; Lee, Y. Y. Int. J. Oncol. 2008, 32, 387–
396. Brown, A. J.; Slatopolsky, E. Mol. Aspects Med. 2008, 29, 433–452.
Sundaram, S.; Beckman, M. J.; Bajwa, A.; Wei, J.; Smith, K. M.; Posner, G. H.;
Gewirtz, D. A. Mol. Cancer Ther. 2006, 5, 2806–2814.
(26) Gregorio, C.; Eduardo, S.; Rodrigues, L. C.; Fraga, R.; Riveiros, R.;
Maestro, M.; Mourin˜o, A. J. Steroid Biochem. Mol. Biol. 2007, 103, 227–230.
(27) Blankemeyer-Menge, B.; Nimtz, M.; Frank, R. Tetrahedron Lett. 1990,
31, 1701–1704.
(28) The solid-phase reactions could be conveniently monitored by 13C NMR
spectrometry of suspended resins (CDCl3). See Supporting Information. The
yields of solid-phase reactions were determined by gravimetric analysis of the
supported product.
(29) We attribute the 3% wastage of this step to manipulative loss of resin.
J. Org. Chem. Vol. 74, No. 13, 2009 4783

Nicoletti et al.
SCHEME 2. Retrosynthetic Analysis of Vitamin D3 Metabolites and Analogues on Solid Support
SCHEME 3. Synthesis of the Solid-Supported CD-Side Chain Upper Fragment 12
DAIB (diacetoxyiodobenzene)30 or with Dess-Martin perio-
dinane31 delivered the desired polymer-supported ketone 3 in
high yield (89% overall yield from 7, three steps).29
SCHEME 4. Shorter Route to 12 and Oxidation
With the upper-keto fragment 3 in hand, we were ready to
install the vitamin D triene system. Toward this end, the
Wittig-Horner reaction proceeded stereoselectively to join
ketone 3 (Scheme 5) to the anion of phosphine oxide 1326 (5
equiv) to produce the desired immobilized vitamin 14. The
excess of phosphine oxide was recovered by filtration. Desily-
lation of 14 with hydrogen fluoride pyridine complex in CH2Cl2/
CH3CN gave the target alcohol 15. The ester linkage allows
for an early deprotection in the solid phase that avoids the
practical difficulties associated with a solution-phase deprotec-
tion after cleavage from the resin in the case of diversity-oriented
synthesis16 and prevents the contamination of the final com-
pounds with silyl species, which is of primary importance in
the case of labeled analogues.
Finally, exposure of 15 to methyllithium in THF delivered,
after simple purification, the desired 25-hydroxyvitamin D3
metabolite (1a) in 61% yield from the solid-supported CD-
moiety 3. Alternatively, treatment of 15 with CD3MgI in THF
produced the hexadeuterated analogue 1c in 63% yield (3 steps).
In summary, a practical five-step solid-phase synthetic method
for the preparation of 25-hydroxyvitamin D3 metabolite and
The hydroxyacid 5 was prepared by saponification of ester 7
as previously described (Scheme 4)26 and then was treated with
resin 6 under the above reaction conditions to afford polymer-
supported 12 (92% yield for the two steps). Importantly, the
1H and 13C NMR spectra of the product were identical to those
of the same compound obtained by the longer synthetic route.
The excess of hydroxyacid 5 (2 equiv) was recovered by
filtration. Attempts to prepare the solid-supported ketone 3,
required for the Wittig-Horner coupling, by oxidation of 12
with pyridinium dichromate resulted in a dark-colored material.
The IR spectrum shows the presence of carbonyl groups;
however, no 13C NMR spectrum could be obtained, probably
because of contamination with chromium species. Fortunately,
oxidation of 12 with a catalytic amount of TEMPO (2,2,6,6-
tetramethyl-1-piperidinyloxy free radical) in the presence of
(30) De Mico, A.; Margarita, R.; Parlanti, L.; Vescovi, A.; Piancatelli, G. J.
Org. Chem. 1997, 62, 6974–6977.
(31) Dess, D. B.; Martin, J. C. J. Am. Chem. Soc. 1991, 113, 7277–7287.
4784 J. Org. Chem. Vol. 74, No. 13, 2009

25-Hydroxylated Vitamin D3 Compounds
SCHEME 5. Synthesis of 25-Hydroxyvitamins 1a and 1c
The residue was purified by flash chromatography (12 × 2 cm,
20% EtOAc/hexanes) to give acid 932 as a white powder (0.477 g,
99%). Rf ) 0.2 (20% EtOAc/hexanes).
Polymer-Bound (tert-Buthyldimethylsilyl) Ether 10. A mixture
of acid 9 (0.343 g, 0.896 mmol), MeIm (0.17 mL, 1.80 mmol),
and MSNT (0.267 g, 0.901 mmol) in dry CH2Cl2 (3 mL) was stirred
until a homogeneous solution was obtained, and then dry THF (3
mL) was added. The resulting solution was added in two portions
via syringe over hydroxymethylpolystyrene resin (6, 0.450 g, 1.0
mmol/g loading, 0.450 mmol, previously dried for 12 h under high
vacuum) in a reaction vessel, and the mixture was shaken for 2 h
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 × 2 cm, 20% EtOAc/hexanes)
to recover unreacted excess 9 (161 mg, 94%). The resin was
successively washed with CH2Cl2 (6 × 2 mL), DMF (2 mL), MeOH
(4 × 2 mL), dioxane (3 × 2 mL), DMF (2 mL), MeOH (3 × 2
mL), and CH2Cl2 (6 × 2 mL). The loaded resin was air-dried and
high-vacuum-dried for 12 h to give 10 (0.613 g, 0.733 mmol/g,
0.450 mmol, 100%). 13C NMR (63 MHz, CDCl3) δ 173.6, 144.9,
138.7, 127.7, 125.7, 69.4, 66.0, 56.4, 53.0, 45.8, 44.0, 42.1, 40.6,
35.0, 34.5, 27.3, 25.8, 23.5, 21.5, 18.6, 17.7, 13.8, -4.7, -5.1. IR
(KBr) 3026, 2927, 2853, 1737 (CO), 1451, 1251, 1163, 1025, 835,
758, 698, 539 cm-1
.
Partially loaded resin 10b was prepared to corroborate the
absence of free hydroxyl groups by the disappearance of the signals
of CH2OH moieties in the 13C NMR spectrum of the above loaded
resin 10.
26,26,26,27,27,27-d6-25-hydroxyvitamin D3 has been developed.
Key steps of the synthesis include (1) the use of inexpensive
(hydroxymethyl)polystyrene resin; (2) the immobilization of the
CD-side chain fragment through an ester function at C25; (3)
the construction of the vitamin D triene system on solid support;
and (4) the possibility of diversification of the side chain at
C25, including the preparation of labeled material, in the last
step of the synthesis, with concomitant release from the resin.
The strategy is amenable to diversity-oriented synthesis by
combining different CD-system and A-ring building blocks.
Diversification at the side chain can be carried out using
commercial organometallic reagents during the release step,
avoiding the preparation of a pool of different side chain
fragments. This report expands the generality of Lythgoe’s
Wittig-Horner approach to prepare vitamin D metabolites and
analogues on solid support.
Partially Loaded Resin 10b. A mixture of acid 9 (25 mg, 0.065
mmol, 0.64 equiv), MeIm (10 µL, 0.12 mmol, 1,2 equiv), and
MSNT (19 mg, 0.065 mmol, 0.64 equiv) in dry CH2Cl2 (3 mL)
was stirred until a homogeneous solution was obtained. Dry THF
(3 mL) was added. The resulting solution was added in two portions
via syringe over hydroxymethylpolystyrene resin 6 (102 mg, 1.0
mmol/g loading, 0.102 mmol, previously dried for 12 h under high
vacuum) in a reaction vessel. The mixture was shaken for 2 h under
argon atmosphere. The resin was filtered and washed successively
with CH2Cl2 (6 × 2 mL), DMF (2 mL), MeOH (4 × 2 mL), dioxane
(3 × 2 mL), DMF (1 × 2 mL), MeOH (3 × 2 mL), and CH2Cl2 (6
× 2 mL). The washed resin was air-dried and high-vacuum-dried
for 12 h to give partially loaded resin 10b (∼50%). 13C NMR (63
MHz, CDCl3) δ 173.6, 144.9, 138.7, 127.7, 125.7, 69.4, 66.0
(RCOOCH2-resin-loaded), 65.1 (HOCH2-resin-unloaded), 56.4,
53.0, 45.8, 44.0, 42.1, 40.6, 35.0, 34.5, 27.3, 25.8, 23.5, 21.5, 18.6,
17.7, 13.8, -4.7, -5.1.
8ꢀ-[(tert-Butyldimethylsilyl)oxy]-de-A,B-cholestan-25-ol (11a). A
solution of MeLi in Et2O (0.40 mL, 1.6 M, 0.64 mmol) was added
to a suspension of resin 10 (100 mg, 0.733 mmol/g, 0.0733 mmol)
in dry THF (3 mL) at -78 °C in a cylindrical flask. The mixture
was shaken for 12 h with slow warming to -40 °C. The reaction
was quenched at -78 °C by the addition of a few drops of H2O.
The mixture was filtered, and the solids were washed with CH2Cl2
(5 × 2 mL) and Et2O (5 × 2 mL). The filtrate was washed with
brine (10 mL), and the organic layer was dried, filtered and
concentrated in vacuum to give alcohol 11a33 (27 mg, 93%) as a
colorless oil.
Polymer-Bound Alcohol 12 from Resin 10. Resin 10 (0.600
g, 0.733 mmol/g, 0.440 mmol) was placed into a HF-resistant vial
and suspended in dry CH2Cl2 (3 mL) and dry CH3CN (3 mL). After
30 min, aqueous HF (0.30 mL, 48-50%) was added, and the
mixture was shaken for 72 h. Unreacted HF was quenched with
DIPEA (0.5 mL). After shaking for 5 min the resin was filtered
and washed successively with CH2Cl2/DIPEA (1:1, 4 mL), CH2Cl2
(14 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 successively air-dried and high-vacuum-dried
Experimental Section
Methyl 8ꢀ-[(tert-Butyldimethylsilyl)oxy]-de-A,B-cholane-24-car-
boxylate (8). Imidazole (1.24 g, 18.2 mmol) and TBSCl (2.20 g,
14.6 mmol) were consecutively added to a solution of ester 725,26
(1.03 g, 3.65 mmol) in dry DMF (12 mL). The reaction mixture
was stirred at 60 °C for 48 h. The reaction was quenched with
brine (70 mL), and the aqueous phase was extracted successively
with hexanes (3 × 60 mL) and Et2O (1 × 80 mL). The combined
organic phase was washed with H2O (3 × 30 mL), dried, filtered,
and concentrated. The residue was purified by flash chromatography
(12 × 3.5 cm, 0-5% EtOAc/hexanes) to give ester 832 (1.42 g,
98%) as a white solid. Rf ) 0.5 (5% EtOAc/hexanes).
8ꢀ-[(tert-Butyldimethylsilyl)oxy]-de-A,B-cholane-24-carboxylic
Acid (9). An aqueous solution of LiOH (5 mL, 2 M, 10 mmol)
was added to a solution of ester 8 (0.500 g, 1.26 mmol) in THF
(30 mL), and the reaction mixture was stirred at reflux for 5 h. To
the mixture at room temperature was added aqueous HCl (10%)
until pH ) 2. After stirring for 5 min, the mixture was extracted
with Et2O (3 × 50 mL), and the combined organic phase was
washed with brine (1 × 50 mL), dried, filtered, and concentrated.
(32) Nemoto, H.; Ando, M.; Fukumoto, K. Tetrahedron Lett. 1990, 31, 6205–
6208.
(33) Fall, Y.; Vitale, C.; Mourin˜o, A. Tetrahedron Lett. 2000, 41, 7337–
7340.
J. Org. Chem. Vol. 74, No. 13, 2009 4785

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
available free of charge via the Internet at http://pubs.acs.org.
JO900524U
4786 J. Org. Chem. Vol. 74, No. 13, 2009