Synthesis of novel hapten derivatives of 1α,25-dihydroxyvitamin D3 and its 20-epi analogue

Article abstract of DOI:10.1021/jo026061s

Hapten derivatives of 1α,25-dihydroxyvitamin D₃ and its 20-epimer were synthesized and conjugated to a carrier protein for raising polyclonal antibodies. The haptens were linked through spacers at C-16, thereby exposing both the A-ring and the side chain of the molecules, to maximize antibody specificity. The spacers were introduced via stereoselective hydroboration of 16-ene intermediates as the key step. In immunoassays, the antibodies raised toward the natural hormone were selective to this compound over derivatives with modifications in the A-ring or the side chain. The antibodies toward the 20-epimer, however, were unable to recognize modifications in the side chain.

Full text of DOI:10.1021/jo026061s

Syn th esis of Novel Ha p ten Der iva tives of 1r,25-Dih yd r oxy-
vita m in D3 a n d Its 20-Ep i An a logu e1
Lars K. A. Blæhr,*,† Fredrik Bjo¨rkling,† Martin J . Calverley,† Ernst Binderup,† and
Mikael Begtrup‡
Medicinal Chemistry Research, LEO Pharma, DK-2750 Ballerup, Denmark, and
Department of Medicinal Chemistry, Royal Danish School of Pharmacy, DK-2100 Copenhagen, Denmark
lars.blaehr@leo-pharma.com
Received J une 16, 2002
Hapten derivatives of 1R,25-dihydroxyvitamin D3 and its 20-epimer were synthesized and conjugated
to a carrier protein for raising polyclonal antibodies. The haptens were linked through spacers at
C-16, thereby exposing both the A-ring and the side chain of the molecules, to maximize antibody
specificity. The spacers were introduced via stereoselective hydroboration of 16-ene intermediates
as the key step. In immunoassays, the antibodies raised toward the natural hormone were selective
to this compound over derivatives with modifications in the A-ring or the side chain. The antibodies
toward the 20-epimer, however, were unable to recognize modifications in the side chain.
In tr od u ction
Vitamin D has been recognized as a pluripotent
hormone involved in a variety of biological processes,
including calcium homeostasis, cell differentiation and
proliferation, and immunology.2 The metabolite 1R,25-
dihydroxyvitamin D3 (1,25(OH)2D3; Figure 1) has been
identified as the hormonally most active form of vitamin
D. The detailed mechanism of action of vitamin D is yet
unclear, but it has been established that it involves
interaction with a specific vitamin D receptor (VDR)
which undergoes dimerization with other nuclear recep-
tors, in particular the retinoid X receptor. The receptors
then bind to response elements on DNA, causing stimu-
lation/inhibition of protein synthesis.3 For therapeutic
use, analogues of 1,25(OH)2D3 have been synthesized
with enhanced antiproliferative activity and with reduced
calcemic activity. Vitamin D analogues have been devel-
oped as antipsoriatic drugs and show promise in the
treatment of cancer and other proliferative disorders.4
Most analogues synthesized to date have been compounds
with modified C-20 side chains.5
F IGURE 1. The natural hormone 1,25(OH)2D3, its 20-epimer
(MC1288), and corresponding haptens 1a and 1b used for
raising antibodies.
One group of analogues that has attracted particular
attention is characterized by an inverted stereochemistry
at the C-20 position relative to that of the natural
hormone (Figure 1). These so-called “20-epi” analogues
are, in many cases, more potent in regulating cell growth
and differentiation than the corresponding compounds
with normal C-20 stereochemistry. In particular, 20-epi
analogues exhibit immunosuppressive properties.6
The purpose of the present study was to produce and
compare specific antibodies to 1,25(OH)2D3 and 20-epi-
1,25(OH)2D3. We were interested to see if the antibodies
could distinguish between the normal and C-20 epimeric
side chains. If this is the case, it would be possible to
raise specific antibodies to new 20-epi analogues selected
† LEO Pharma.
‡ Royal Danish School of Pharmacy.
(1) This work was taken in part from the doctoral thesis of Lars
Blæhr (Royal Danish School of Pharmacy, Copenhagen) and was
partially presented at the 10th Workshop on Vitamin D: Vitamin D.
Chemistry, Biology and Clinical Applications of the Steroid Hormone;
Norman, A. W., Bouillon, R., Thomasset, M., Eds.; Printing and
Reprographics, University of California: Riverside, CA, 1997; p 91.
(2) Bouillon, R.; Okamura, W. H.; Norman, A. W. Endocr. Rev. 1995,
16, 200.
(3) J ones, G.; Strugnell, S. A.; DeLuca, H. F. Physiol. Rev. 1998,
78, 1193.
(4) Verstuyf, A.; Segaert, S.; Verlinden, L.; Bouillon, R.; Mathieu,
C. Exp. Opin. Invest. Drugs 2000, 9, 443.
(5) Calverley, M. J .; J ones, G. In Antitumor Steroids; Blickenstaff,
R. T., Ed.; Academic Press: San Diego, 1992; p 193.
(6) Binderup, L.; Latini, S.; Binderup, E.; Bretting, C.; Calverley,
M.; Hansen, K. Biochem. Pharmacol. 1991, 42, 1569.
10.1021/jo026061s CCC: $25.00 © 2003 American Chemical Society
Published on Web 01/28/2003
J . Org. Chem. 2003, 68, 1367-1375
1367

Blæhr et al.
SCHEME 1
as drug candidates. Such antibodies would be useful in
clinical settings for analyzing serum concentrations of the
drugs in patients. In this study, we designed and
synthesized hapten derivatives of 1,25(OH)2D3 and 20-
epi-1,25(OH)2D3 (1a and 1b; Figure 1) for raising anti-
bodies. The haptens have a spacer for carrier conjugation
incorporated at the C-16R position to expose the A-ring
and the side chain, two structural elements that together
distinguish 1,25(OH)2D3 from a host of other natural
vitamin D metabolites. It has previously been shown that
exposing these elements in the hapten leads to high
antibody specificity.7 Thus, cross-reaction with related
metabolites, especially 25-hydroxyvitamin D3 (lacking the
1R-OH) present in high concentrations in serum, is
minimized.
SCHEME 2
We report herein the syntheses of the two haptens and
the preliminary examination of the properties of poly-
clonal antibodies raised toward bovine serum albumin
(BSA) conjugates of the haptens.
Resu lts a n d Discu ssion
There have been several reports in the literature on
the synthesis of haptens for raising polyclonal antibodies
to 1,25(OH)2D3. The first haptens reported were deriva-
tives of 1,25(OH)2D3 conjugated to a carrier protein
through spacers attached to one of the inherent hydroxyl
groups in the molecule.8 However, polyclonal antibodies
resulting from immunization with these conjugates were
not specific to 1,25(OH)2D3, probably because it is es-
sential to expose all three hydroxyl groups of 1,25(OH)2D3
in the hapten to obtain a high specificity. Later on it was
shown that the introduction of a fourth hydroxyl group
for conjugation improved the antibody specificity consider-
ably.7a Thus, we decided to follow this strategy for our
study and introduce a hydroxyl group at the C-16
position.
Syn th esis of 16-Glu ta r yloxy-1,25(OH)2D3. We de-
cided to introduce a 16,17 double bond as a starting point
for functionalizing the C-16 position. Subsequent hydro-
boration/oxidation of the double bond would be expected
to regiospecifically introduce a hydroxyl group at this
position, providing a chemical handle for attaching the
spacer.
(Scheme 1). Reduction of the ketone with NaBH4 gave
two epimeric alcohols11 in the ratio (R:S) 85:15 which
were easily separated by chromatography. The R-isomer
(3a ) was converted to the (Z)-ethylidene compound 4 by
E2 elimination of water with POCl3.12 Ene reaction with
paraformaldehyde under BF3‚OEt2 catalysis resulted in
highly stereoselective conversion to the homoallylic al-
cohol 5, the angular methyl group directing attack of
aldehyde solely from the R-face of the molecule.13
The 25-hydroxylated vitamin D3 side chain was as-
sembled by a modification of a previously described
procedure used to make 1,25(OH)2D3 (Scheme 2).14 Thus,
tosylation of the primary alcohol 5 to 6a followed by
coupling of the Grignard reagent of 715 in the presence
To obviate the problem of chemoselective hydroboration
of a 16-ene in the presence of the triene system of vitamin
D, we chose to work with CD-ring intermediates, defer-
ring assembly of the triene unit until the final steps of
the synthesis in a convergent approach.5
16
of Li2CuCl4 gave 8a .
Introduction of the 16,17 double bond9 involved ene
reaction of formaldehyde with the ethylidene compound
4, which was obtained from the known ketone 210
(11) (a) Posner, G. H.; White, M. C.; Dolan, P.; Kensler, T. W.;
Yukihiro, S.; Guggino, S. E. Bioorg. Med. Chem. Lett. 1994, 4, 2919.
For previous work on the reduction of steroidal 20-ketones, see: (b)
Midland, M. M.; Kwon, Y. C. J . Am. Chem. Soc. 1983, 105, 3725. (c)
Hansen, K.; Calverley, M. J .; Binderup, L. In Vitamin D: Gene
Regulation, Structure-Function Analysis and Clinical Application;
Norman, A. W., Bouillon, R., Thomasset, M., Eds.; Walter de Gruyter:
Berlin, 1991; p 161.
(12) Von Daehne, W.; Hansen, C. M.; Hansen, D.; Mathiasen, I. S.
In Vitamin D. Chemistry, Biology and Clinical Applications of the
Steroid Hormone; Norman, A. W., Bouillon, R., Thomasset, M., Eds.;
Printing and Reprographics, University of California: Riverside, CA,
1997; p 81.
(13) (a) Batcho, A. D.; Berger, D. E.; Davoust, S. G.; Wovkulich, P.
M.; Uskokovic, M. R. Helv. Chim. Acta 1981, 64, 1682. (b) Wovkulich,
P. M.; Barcelos, F.; Batcho, A. D.; Sereno, J . F.; Baggiolini, E. G.;
Hennessy, B. M.; Uskokovic, M. R. Tetrahedron 1984, 40, 2283.
(14) Andrews, D. R.; Barton, D. H. R.; Hesse, R. H.; Pechet, M. M.
J . Org. Chem. 1986, 51, 4819.
(7) (a) Kobayashi, N.; Kitahori, J .; Mano, H.; Shimada, K. Chem.
Pharm. Bull. 1993, 41, 1321. (b) Kobayashi, N.; Imazu, T.; Ebisawa,
A.; Shimada, K. J . Steroid Biochem. Mol. Biol. 1997, 63, 127. (c)
Kobayashi, N.; Higashi, T.; Saito, K.; Murayama, T.; Douya, R.;
Shimada, K. J . Steroid Biochem. Mol. Biol. 1997, 62, 79.
(8) (a) Bouillon, R.; De Moor, P.; Gabbiolini, E. G.; Uskokovic, M.
R. Clin. Chem. 1980, 26, 562. (b) Clemens, T. L.; Hendy, G. N.; Graham,
R. F.; Baggiolini, E. G.; Uskokovic, M. R.; O’Riordan, L. H. Clin. Sci.
Mol. Med. 1978, 54, 329. (c) Brown, W. B.; Peacock, M. Clin. Chim.
Acta 1986, 159, 111.
(9) The conventional steroid numbering system is used for the CD-
ring fragments.
(10) Ferna´ndez, B.; Pe´rez, J . A. M.; Granja, J . R.; Castedo, L.;
Mourin˜o, A. J . Org. Chem. 1992, 57, 3173.
1368 J . Org. Chem., Vol. 68, No. 4, 2003

Hapten Derivatives of 1,25(OH)2D3 and Its 20-Epimer
A-ring synthon 17 under standard conditions.20 The
acetate group was replaced with a hemiglutaroyl group,
and deprotection with TBAF generated the final hapten
1a .
SCHEME 3
Syn th esis of 20-Ep i-16-Glu ta r yloxy-1,25(OH)2D3.
The synthesis of the corresponding 1,25(OH)2D3 hapten
with 20-epi (S) configuration (1b) followed a similar
synthetic route. However, because the altered configu-
ration at C-20 had some unexpected influence on the
outcome of some reactions, a few modifications proved
to be necessary.
To establish the S-configuration at C-20, the E-isomer
of alkene 4 was required for the ene reaction with
formaldehyde (cf. Scheme 1). The E-isomer could be
formed by stereospecific dehydration of the alcohol 3b
obtained from reduction of ketone 2. However, since the
yield of this alcohol was poor, we looked at ways to
isomerize alkene 4 from the previous sequence, which
was available in larger amounts, to the corresponding
E-isomer.
Although a procedure has been described for the inter-
conversion of 17-ethylidene isomers (standard sequence
of epoxidation, ring-opening with lithium diphenylphos-
phide, and methylation),21 we decided on another ap-
proach, involving hydroboration of the double bond
followed by E2 elimination of water (Scheme 5). The
hydroboration of (Z)-17-ethylidene steroids has been
reported to occur predominantly by attack from the R-face
of the molecule, affording the (20S)-alcohol upon oxida-
tion.22 Thus, hydroboration/oxidation of the (Z)-ethylidene
compound 4 afforded the (S)-alcohol 3b in good yield, and
subsequent dehydration gave rise to the (E)-ethylidene
compound 21, which now could be prepared in adequate
amounts.
We then proceeded with ene reaction of 21 with
formaldehyde to produce the homoallylic alcohol 23.
Attack from the less hindered R-face would establish the
desired 20-epi configuration (cf. ref 12). Surprisingly,
when the (E)-alkene 21 was subjected to the same
reaction conditions used with the (Z)-alkene 4 in the
previous synthesis (Scheme 1), a compound was isolated
as the major product with the correct molecular mass
(determined by MS), but without the expected vinylic
signals in its 1H NMR and 13C NMR spectra. The
compound was identified as the oxetane 22, apparently
as a single diastereomer, which presumably is the
product of R-attack as shown (Scheme 6). TLC showed
that 23 was initially formed to some extent in the
reaction as a transient intermediate.
The generation of oxetanes in ene reactions is known.
It has been postulated that the mechanism of the ene
reaction in certain cases involves the formation of a 2 +
2 transition state complex (an oxetane) before the rate-
determining step to the ene product.23 Alternatively, the
oxetane could be formed via an intermediate character-
istic of the Prins reaction.24 Thus, instead of the concerted
It is known that hydrogenation of 16-ene steroids takes
place exclusively from the R-face due to steric hindrance
afforded by the angular C-13 methyl group.13 We antici-
pated that hydroboration of the double bond in 8a would
exhibit the same facial selectivity. Treating 8a with
diborane followed by oxidation with H2O2 resulted in two
isomeric alcohols (9a and 10a ) in the ratio 5:1. The
1
predominant isomer showed a signal in H NMR at 4.0
ppm (H-16), and this chemical shift corresponded with
spectra of 16R-hydroxy steroids found in the literature.17
The minor isomer displayed a signal at 4.3 ppm which
was in accordance with the literature data of 16â-hydroxy
steroids. Consequently, the major and minor isomers
were assigned the structures of 9a and 10a , respectively.
Since hydroboration is a syn-addition, the isomers should
also be 17-epimers. However, neither comparison of
chemical shifts nor 2D NOE experiments supplied con-
clusive evidence for assigning the absolute configuration
at C-17.
Therefore, we decided to confirm our assignments by
converting 9a to its corresponding 16-deoxy compound,
which is known.18 Thus, the alcohol was converted to the
mesylate, subsequently reduced with LAH, and depro-
tected (Scheme 3) to give 12a , which was identical (1H,
mp) with literature data. Evidently, hydroboration of a
16,17 double bond does preferentially take place from the
least hindered R-face. Bulkier borane reagents such as
9-BBN, which might have given improved selectivity,
were not effective at hydroborating the double bond.
The hydroboration/oxidation step resulted in adventi-
tious cleavage of the TMS group in the side chain. (In
some runs, the TMS intermediates were the isolated
products, from which the diols 9a and 10a could be
recovered after brief treatment with a catalytic amount
of PPTS in ethanol/THF.19) Fortunately, the unprotected
tertiary alcohol did not interfere in the following steps.
The secondary alcohol 9a was protected as an acetate to
give compound 13a (Scheme 4), and the TBS group was
removed with hydrofluoric acid, affording 14a . Oxidation
of the secondary alcohol with PDC and reprotection of
the tertiary alcohol afforded 16a , which was coupled with
(15) Calverley, M. J .; Binderup, E. T. International Patent WO
9,100,271-A:53 pp, 1991.
(16) Fouquet, G.; Schlosser, M. Angew. Chem. 1974, 86, 50.
(17) (a) Koreeda, M.; George, I. A. J . Am. Chem. Soc. 1986, 108,
8098. (b) Le thi Quyen; Ripperger, H.; Adam, G.; Schreiber, K. Liebigs
Ann. Chem. 1993, 167.
(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.
(21) de los Angeles Rey, M.; Martinez-Perez, J . A.; Fernandez-Gacio,
A.; Halkes, K.; Fall, Y.; Granja, J .; Mourino, A. J . Org. Chem. 1999,
64, 3196.
(18) Lythgoe, B.; Roberts, D. A.; Waterhouse, I. J . Chem. Soc., Perkin
Trans. 1 1977, 2608.
(19) Compound 10a was characterized as the 25-O-TMS ether.
(22) (a) Krubiner, A. M.; Oliveto, E. P. J . Org. Chem. 1966, 31, 24.
(b) Midland, M. M.; Kwon, Y. C. J . Org. Chem. 1981, 46, 229.
(23) Kwart, H.; Brechbiel, M. J . Org. Chem. 1982, 47, 5411.
J . Org. Chem, Vol. 68, No. 4, 2003 1369

Blæhr et al.
SCHEME 4
SCHEME 5
preparation of hapten 1a (Schemes 2 and 4). As before,
hydroboration of the 16-ene intermediate 8b with BH3‚
THF and subsequent oxidation with H2O2 afforded two
isomeric alcohols (9b and 10b) in the ratio 8.5:1. Again,
the hydrogen at the C-17 position of the major isomer
9b was established to be R-oriented (natural configura-
tion) by converting the isomer to the known 16-deoxy
compound 12b (Scheme 3) and correlating with literature
data.26 Due to the syn-addition mechanism of hydro-
boration, it then follows that the 16-OH of 9b is also
R-oriented. This configuration was also confirmed by an
NOE between 16-H and the angular C-13 methyl group.
In the case of the isomer 10b, no NOE was observed
between 16-H and the C-13 methyl, and this points to
the configuration as drawn. The spectrum of 10b also
revealed an NOE between 16-H and 17-H, which is
consistent with the trans-configuration of the protons in
a gauche-orientation.
SCHEME 6
Im m u n oa ssa ys
addition process typical of the ene reaction, the alkene
attacks the formaldehyde, thereby generating a tertiary
carbenium ion that collapses to the oxetane 22.
The haptens 1a and 1b were converted to their
corresponding N-hydroxysuccinimide esters and coupled
to BSA.27 Rabbits were immunized biweekly with the
resulting conjugates for 3 months. The polyclonal anti-
sera thus produced were examined for cross-reactivity
with a series of vitamin D metabolites in a standard
radioimmunoassay using radiolabeled 1,25(OH)2D3 as
ligand. The results are presented in Table 1.
The antibodies to 1a (normal side chain configuration)
are clearly selective toward 1,25(OH)2D3 over its metabo-
lites and over 20-epi-1,25(OH)2D3. Removal of either the
25-hydroxy group (1-hydroxyvitamin D3) or the 1-hydroxy
group (25-hydroxyvitamin D3) resulted in a 100-fold
decrease in binding. Removal of both hydroxyl groups
(vitamin D3) resulted in a binding of less than 0.01%
On the basis of this mechanism, the formation of the
oxetane could presumably be minimized by using another
Lewis acid to alter the reactivity of the oxy anion. A
number of other Lewis acids were evaluated for the
reaction above, and it was found that substituting BF3‚
OEt2 with trimethylaluminum led to a good yield of the
desired homoallylic alcohol 23 (with the recovery of 17%
starting material). While only trace amounts of oxetane
were formed, a new byproduct (24) was produced, result-
ing from a Wagner-Meerwein rearrangement.25
The alcohol 23 was then converted to the hapten 1b
using a sequence similar to that described for the
(24) (a) Adams, D. R.; Bhatnagar, S. P. Synthesis 1977, 661. (b)
Meresz, O.; Leung, K. P.; Denes, A. S. Tetrahedron Lett. 1972, 27, 2797.
(25) The assigned structure of 24 is tentative on the basis of NMR
and MS data.
(26) Fujishima, T.; Konno, K.; Nakagawa, K.; Kurobe, M.; Okano,
T.; Takayama, H. Bioorg. Med. Chem. 2000, 8, 123.
(27) Hosoda, H.; Sakai, Y.; Yoshida, H.; Miyairi, S.; Ishii, K.;
Nambara, T. Chem. Pharm. Bull. 1979, 27, 742.
1370 J . Org. Chem., Vol. 68, No. 4, 2003

Hapten Derivatives of 1,25(OH)2D3 and Its 20-Epimer
as described previously28 using silica gel 60, 40-63 µm. NMR
TABLE 1. Sp ecificity of An tibod ies Der ived fr om
Im m u n iza tion in Ra bbits w ith BSA Con ju ga tes of 1a a n d
1b
spectra were obtained in CDCl3 unless otherwise stated. NMR
1
spectra were recorded at 300 MHz for H and 75 MHz for 13C.
Chemical shifts are reported in parts per million downfield
from that of internal trimethylsilane. For compounds contain-
percent cross-reactiona
1
metabolite
1,25(OH)2D3
20-epi-1,25(OH)2D3
25-hydroxyvitamin D3
1-hydroxyvitamin D3
vitamin D3
antibody to 1a
antibody to 1b
ing silicon, CHCl3 was used as reference (7.25 ppm in H NMR,
76.8 ppm in 13C NMR). All spectra were recorded at the
Department of Spectroscopy at LEO Pharma. Melting points
are uncorrected.
100
8
100
50
1.3
1.3
<0.01
1.5
(5Z,7E )-(1R ,3S )-9,10-Se coch ole st a -5,7,10(19)-t r ie n e -
1,3,25-tr iol-16r-yl Hyd r ogen Glu ta r a te (1a ). To a solution
of silyl ether 20a (32 mg, 0.04 mmol) in THF (4 mL) was added
TBAF (119 mg, 0.4 mmol), and the resulting solution was
heated to 60 °C for 5 h and then cooled to room temperature.
After standard workup, the residue was purified by flash
chromatography (CH2Cl2/methanol), affording 7 mg (33%) of
hemiglutarate 1a as a colorless oil. 1H NMR (500 MHz,
CD3OD): δ 0.63 (3H, s), 0.99 (3H, d, J ≈ 6.3 Hz), 1.16 (6H, s),
1.0-2.1 (series of m), 2.24 (2H, t), 2.33 (2H, t), 2.54 (2H, m),
2.88 (1H, br d), 4.12 (1H, m), 4.35 (1H, m), 4.89 (2H, m), 5.29
(1H, m), 6.01 (1H, d, J ≈ 11.2 Hz), 6.31 (1H, d, J ≈ 11.1 Hz).
MS: m/z 396 (M - glutaryl - H2O), 378 (M - glutaryl -
2H2O), 360 (M - glutaryl - 3H2O), 249. HRMS (EI): m/z calcd
for C27H36 ([M - HOOC(CH2)3COOH - 3H2O]+) 360.2817,
found 360.2820.
<0.01
<0.01
a
The percent cross-reaction of a metabolite is defined as (dose
of 1,25(OH)2D3 required to displace 50% of the bound radioligand)/
(dose of metabolite required to displace 50% of the bound radio-
ligand) × 100.
compared to that of 1,25(OH)2D3. These results indicate
that both the side chain and the A-ring are important
for binding, and an alteration in either moiety leads to a
reduction in binding affinity.
In contrast, the antibodies toward 1b (epimeric side
chain configuration) did not discriminate between the
natural hormone and its 20-epimer, but we detected a
large difference in affinity for 1-hydroxyvitamin D3 and
25-hydroxyvitamin D3. This result indicates that the
antibody has a stronger binding preference for the A-ring
moiety than for the side chain. Alterations in the side
chain do not seem to influence the binding affinity to a
large degree, while the antibody is much more sensitive
to alterations in the A-ring.
In conclusion, we have successfully prepared the pure
16R-glutaryloxy derivatives of 1R,25(OH)2D3 and 20-epi-
1R,25(OH)2D3. These derivatives were used as haptens
for the generation of antibodies toward the two vitamin
D compounds. The haptens were designed to present the
most characteristic parts of the vitamin D molecule, the
A-ring and the side chain, to maximize the specificity of
the antibodies. This was achieved to a satisfactory level
with the derivative of the natural hormone, but less so
for the corresponding epi compound. An explanation
could be that the side chain in the epi antigen interacts
with the carrier protein and thereby becomes less acces-
sible to the antibody-producing B-cells. Further work
concerning antibodies against epi compounds should
concentrate on other linker positions to obtain more
specific antibodies. Thus, this study has given us further
information on how to make useful haptens of specific
vitamin D derivatives.
(5Z,7E)-(1R,3S,20S)-9,10-Secoch olesta -5,7,10(19)-tr ien e-
1,3,25-tr iol-16r-yl Hyd r ogen Glu ta r a te (1b). Following the
procedure described for the preparation of 1a , 26 mg of 20b
was converted to 11 mg (59%) of 1b as a colorless oil. 1H NMR
(CD3OD): δ 0.62 (3H, s), 0.88 (3H, d, J ≈ 6.2 Hz), 1.17 (6H, s),
1.2-2.2 (series of m), 2.31 (1H, m), 2.33 (4H, m), 2.51 (1H,
dd), 2.90 (1H, m), 4.11 (1H, m), 4.34 (1H, m), 4.88 (1H, m),
4.96 (1H, m), 5.29 (1H, m), 6.02 (1H, d, J ≈ 11.1 Hz), 6.31
(1H, d, J ≈ 11.1 Hz). 13C NMR (CD3OD): δ 14.1, 18.8, 21.5,
22.0, 24.0, 29.3, 29.7, 33.7, 34.1, 34.4, 34.7, 37.9, 41.5, 43.7,
45.1, 46.2, 47.5, 48.2, 54.8, 62.7, 67.4, 71.5, 71.6, 79.7, 112.4,
119.5, 124.6, 136.5, 140.9, 149.8,174.7, 176.9. HRMS (EI): m/z
calcd for C32H46O5 [M - 2H2O]+ 510.3345, found 510.3358.
(20S)-Des-A,B-8â-[(ter t-bu tyldim eth ylsilyl)oxy]pr egn an -
20-ol (3b).11a To a solution of (Z)-ethylidene 4 (2.40 g, 8.40
mmol) in dry THF (80 mL) was added BH3‚THF (1.0 M in THF,
15 mL). The resulting mixture was stirred at room tempera-
ture for 2.5 h, and then water (60 mL) was slowly added. After
the mixture was cooled in an ice bath, H2O2 (30% aqueous, 40
mL) was added from a dropping funnel over a 10-15 min
period. After complete addition, the suspension was stirred
at 0 °C for 2 h and extracted twice with EtOAc. The combined
organic layers were washed with 10% NaHSO4, water, and
brine, dried, and evaporated. Flash chromatography (15%
1
EtOAc/pentanes) afforded 2.0 g (77%) of alcohol 3b. H NMR:
δ -0.01 (3H, s), 0.00 (3H, s), 0.87 (9H, s), 0.90 (3H, s), 1.19
(3H, d, J ≈ 6.3 Hz), 1.1-2.0 (series of m), 3.67 (1H, m), 4.01
(1H, m). 13C NMR: δ -5.4, -5.0, 14.2, 17.2, 17.8, 22.8, 23.2,
24.8, 25.6, 34.1, 39.6, 41.2, 52.7, 58.8, 69.0, 70.0. HRMS (EI):
m/z calcd for C18H34OSi [M - H2O]+ 294.2379, found 294.2389.
(Z)-Des-A,B-8â-[(ter t-b u t yld im et h ylsilyl)oxy]p r egn -
17(20)-en e (4). A solution of alcohol 3a 11a (2.0 g, 6.4 mmol)
in dry pyridine (50 mL) was cooled to 0-5 °C, and POCl3 (6
mL) was added slowly over 20 min. The mixture was stirred
overnight in the slowly melting ice bath and then poured into
175 mL of ice-cooled ethyl acetate. Cold water (75 mL) was
added and the mixture acidified with HCl (aqueous, 4 M) to
pH 3.0 (approximately 130 mL). The water phase was sepa-
rated and extracted with ethyl acetate. The combined organic
layers were washed with 1 M HCl (40 mL), water, NaHCO3,
water, and brine, then dried, and concentrated to 1.76 g. The
oil was chromatographed (2% ethyl acetate/pentanes) to yield
1.7 g (68%) of 4 as a colorless oil. 1H NMR: δ 0.00 (6H, s), 0.88
(9H, s), 1.10 (3H, s), 1.3-2.4 (series of m), 1.63 (3H, m), 4.06
(1H, m), 5.02 (1H, m). 13C NMR: δ -5.3, -5.0, 12.8, 17.7, 17.8,
Exp er im en ta l Section
Gen er a l P r oced u r es. THF was dried by distillation under
argon from sodium benzophenone ketyl immediately prior to
use. DMSO was dried by distillation under argon from CaH2
and then stored over 4 Å molecular sieves. All other solvents
(CH2Cl2, Et2O, DMF, pyridine) were dried by storage over 4 Å
molecular sieves. Other commercial reagents were used as
received. Water-sensitive reactions were performed in oven-
dried (130 °C) or flame-dried glassware under argon. Thin-
layer chromatography was carried out on precoated silica gel
60 F254 glass plates. Plates were visualized using UV radiation
(254 nm) or with 4 M H2SO4 containing 1.5% cerium sulfate
and 1.0% molybdic acid followed by heating to 150 °C.
Standard workup means extracting 2-3 times with an organic
solvent (ethyl acetate unless otherwise stated), washing with
water and brine, drying over MgSO4 and removing the solvent
under reduced pressure. Flash chromatography was performed
(28) Still, W. C.; Kahn, M.; Mitra, A. J . Org. Chem. 1978, 43, 2923.
J . Org. Chem, Vol. 68, No. 4, 2003 1371

Blæhr et al.
19.3, 23.6, 25.6, 30.2, 34.2, 37.9, 44.0, 52.5, 69.5, 112.1, 150.6.
Anal. Calcd for C18H34OSi: C, 73.40; H, 11.64. Found: C, 73.36;
H, 11.56.
poured into ice-water. After standard workup with ether,
flash chromatography (ether/pentanes, 0.5% f 1.0%) afforded
956 mg (66%) of crude 8a as an oil. H NMR: δ 0.02 (6H, s),
1
0.08 (9H, s), 0.88 (9H, s), 0.97 (3H, d, J ≈ 6.6 Hz), 1.01 (3H,
s), 1.18 (6H, 2 s), 1.2-2.1 (series of m), 2.23 (1H, m), 4.08 (1H,
m), 5.25 (1H, m). 13C NMR: δ -5.4, -5.1, 2.4, 17.8, 17.9, 18.8,
22.1, 22.2, 25.6, 29.6, 29.9, 30.6, 31.5, 34.6, 35.6, 37.0, 44.8,
46.5, 54.9, 69.0, 73.9, 119.2, 160.5. HRMS (EI): m/z calcd for
Des-A,B-23,24-d in or -8â-[(ter t-bu tyld im eth ylsilyl)oxy]-
ch ol-16-en -22-ol (5). A suspension of alkene 4 (760 mg, 2.58
mmol) and paraformaldehyde (387 mg, 12.9 mmol) in 80 mL
of dry CH2Cl2 was treated with 4 drops of BF3‚OEt2 and stirred
for 8 min at room temperature. The purple suspension was
poured into water, and after standard workup with CH2Cl2,
the residue (a yellow oil) was purified by flash chromatography
C
26H51O2Si2 [M - CH3]+ 451.3428, found 451.3430. Anal. Calcd
for C27H54O2Si2: C, 69.46; H, 11.66. Found: C, 69.58; H, 11.69.
(20S)-Des-A,B-8â-[(ter t-bu tyldim eth ylsilyl)oxy]-25-[(tr i-
m eth ylsilyl)oxy]ch olest-16-en e (8b). Following the proce-
dure described for the preparation of 8a , 1.9 g of tosylate 6b
(4.0 mmol) was converted to 1.67 g (90%) of 8b (colorless oil).
1H NMR: δ 0.01 (6H, s), 0.08 (9H, s), 0.88 (9H, s), 1.02 (3H, s),
1.04 (3H, d, J ≈ 6.6 Hz), 1.18 (6H, s), 1.2-2.1 (series of m),
1.86 (1H, ddd, J ≈ 14.4, 6.2, 3.2 Hz), 2.21 (1H, ddt, m), 4.08
(1H, m), 5.28 (1H, m). 13C NMR: δ -5.4, -5.0, 2.4, 17.8, 17.9,
19.2, 21.1, 22.1, 25.6, 29.7, 30.6, 30.9, 34.6, 35.5, 38.0, 44.7,
46.8, 54.6, 69.0, 73.9, 119.8, 161.0. HRMS (EI): m/z calcd for
1
to yield 497 mg (59%) of 5 as a colorless oil. H NMR: δ 0.02
(6H, s), 0.88 (9H, s), 1.11 (3H, d, J ≈ 6.9 Hz), 1.03 (3H, s),
1.2-2.0 (series of m), 2.23 (1H, m), 2.38 (1H, m), 3.46 (2H,
m), 4.09 (1H, m), 5.44 (1H, m). 13C NMR: δ -5.4, -5.0, 17.8,
18.2, 18.9, 25.6, 30.7, 34.4, 34.5, 35.3, 46.5, 54.8, 66.4, 68.7,
121.6, 157.4. HRMS (EI): m/z calcd for C19H36O2Si (M+)
324.2484, found 324.2498.
Des-A,B-23,24-d in or -8â-[(ter t-bu tyld im eth ylsilyl)oxy]-
22-[(p-t olylsu lfon yl)oxy]ch ol-16-en e (6a ). Alcohol 5 (497
mg, 1.53 mmol) and pyridine (1.82 g, 23.0 mmol) were
dissolved in dry CH2Cl2, and the solution was cooled to 0 °C.
p-Toluenesulfonyl chloride (1.46 g, 7.66 mmol) was added, and
the reaction mixture was stirred at 0 °C for 30 min and then
at room temperature overnight. Methanol (5 mL) was added
and the mixture stirred for an additional 45 min and then
poured into water (50 mL). The mixture was extracted twice
with ether, and the combined ether layers were washed with
water (twice), 1 M HCl, water, and brine. The organic phase
was dried and concentrated. Purification by flash chromatog-
C
27H54O2Si2 (M+) 466.3662, found 466.3648.
Des-A,B-8â-[(ter t-b u t yld im et h ylsilyl)oxy]ch olest a n e-
16r,25-diol (9a) an d (17S)-Des-A,B-8â-[(ter t-bu tyldim eth yl-
silyl)oxy]ch olesta n e-16â,25-d iol (10a ). A solution of 8a (420
mg, 0.90 mmol) in dry THF (10 mL) was treated with BH3‚THF
(7.7 mL, 1.0 M in hexane) at 0 °C and stirred in the melting
ice bath overnight. The reaction mixture was cooled in the ice
bath, and water (3 mL), aqueous NaOH (3 N, 10 mL), and
aqueous H2O2 (30%, 5 mL) were slowly added while the
temperature was maintained below 6 °C. After being stirred
at 0 °C for 2 h, the mixture was acidified to pH 7.5 with HCl
(4 M). Standard workup with ether afforded 817 mg of crude
product as an oil, which was purified by flash chromatography
(ethyl acetate/petroleum ether) to give 254 mg of deprotected
alcohol 9a (68%) and 53 mg of the isomer 10a (14%), both as
colorless oils.
1
raphy afforded 6a (699 mg, 95%) as a colorless oil. H NMR:
δ 0.00 (6H, s), 0.86 (9H, s), 0.91 (3H, s), 0.99 (3H, d, J ≈ 6.9
Hz), 1.2-1.9 (series of m), 2.13 (1H, dd), 2.3-2.5 (1H, m), 2.43
(3H, s), 3.85 (1H, dd, J ≈ 8.8 Hz), 4.00 (1H, dd, J ≈ 9.4, 5.6
Hz), 4.05 (1H, m), 5.21 (1H, m), 7.32 (2H, d, J ≈ 8.1 Hz), 7.77
(2H, dd, J ≈ 8.1, 1.8 Hz). 13C NMR: δ -5.4, -5.1, 17.7, 17.8,
18.3, 18.7, 21.4, 25.5, 30.7, 31.0, 34.3, 35.1, 46.6, 54.4, 68.6,
74.0, 122.3, 127.7, 129.5, 133.1, 144.3, 155.2. Anal. Calcd for
1
Da ta for Isom er 9a . H NMR: δ -0.03 (3H, s), -0.02 (3H,
s), 0.85 (9H, s), 0.91 (3H, d), 0.92 (3H, s), 0.98 (1H, dd, J ≈
10.2, 5.4 Hz), 1.18 (6H, s), 1.2-2.0 (series of m), 2.05 (1H, dt),
3.99 (1H, m), 4.02 (1H, m). 13C NMR: δ -5.4, -5.0, 15.1, 17.1,
17.8, 18.7, 20.7, 25.6, 28.8, 29.3, 33.3, 34.1, 35.9, 36.0, 40.5,
43.8, 43.9, 49.8, 67.0, 68.8, 70.9, 75.9. HRMS (EI): m/z calcd
for C24H46O2Si [M - H2O]+ 394.3267, found 394.3255. Anal.
Calcd for C27H56O3Si2 (as 25-O-TMS ether): C, 66.88; H, 11.64.
Found: C, 66.97; H, 11.59.
C
26H42O4SSi: C, 65.23; H, 8.84; S, 6.70. Found: C, 65.09; H,
8.86; S, 6.92.
(20R)-Des-A,B-8â-[(ter t-bu tyld im eth ylsilyl)oxy]-22-[(p-
tolylsu lfon yl)oxym eth yl]p r egn -16-en e (6b). Following the
procedure described for the preparation of 6a , alcohol 23 (1.75
g, 5.39 mmol) was converted to 2.14 g of tosylate 6b (83%) as
a white solid. Mp: 59.0-60.5 °C (recrystallized from n-hexane).
1H NMR: δ -0.01 (3H, s), 0.00 (3H, s), 0.86 (9H, s), 0.95 (3H,
s), 1.06 (3H, d, J ≈ 7.0 Hz), 1.06 (1H, m), 1.3-1.9 (series of
m), 2.17 (1H, ddd, J ≈ 11.7, 1.5 Hz), 2.4 (1H, m), 2.43 (3H, s),
3.63 (1H, dd, J ≈ 9.6, 8.5 Hz), 3.97 (1H, J ≈ 9.6, 4.4 Hz), 4.03
(1H, m), 5.35 (1H, br t, J ≈ 1.5 Hz), 7.33 (2H, d, J ≈ 7.7 Hz),
7.76 (2H, dt, J ≈ 8.2, 1.8 Hz). 13C NMR: δ -5.4, -5.1, 17.7,
17.8, 18.8, 21.4, 25.5, 30.7, 30.9, 34.3, 35.1, 46.6, 54.2, 68.6,
74.1, 124.0, 127.8, 129.5, 133.1, 144.3, 154.5. HRMS (EI): m/z
calcd for C25H39O4SSi [M - CH3]+ 463.2338, found 463.2309.
Anal. Calcd for C26H42O4SSi: C, 65.23; H, 8.84; S, 6.70.
Found: C, 65.40; H, 8.77; S, 6.58.
Des-A,B-8â-[(ter t-bu tyld im eth ylsilyl)oxy]-25-[(tr im eth -
ylsilyl)oxy]ch olest-16-en e (8a ). Magnesium turnings (910
mg, 37.4 mmol) and freshly distilled THF (20 mL) were placed
in a dry two-necked flask equipped with a condenser and
an addition funnel. 4-Bromo-2-methyl-2-(trimethylsilyloxy)-
butane15 (7) (8.21 g, 34.3 mmol) was placed in the funnel, and
approximately 1/10 was added to the Mg suspension. By
heating to reflux, the reaction started, and the remaining
bromide was added so that a steady reflux was maintained.
After complete addition, the reaction mixture was cooled to
room temperature. Meanwhile, LiCl (132 mg, 3.12 mmol) and
CuCl2 (210 mg, 1.56 mmol) were dissolved in dry THF (10 mL)
by sonication and added to the reaction mixture. After being
stirred for 10 min, the mixture was cooled in an ice bath, and
a solution of tosylate 6a (1.49 mg, 3.12 mmol) in THF (10 mL)
was added. Stirring was continued for 60 min at 0 °C, after
which ether (50 mL) was added and the mixture was then
1
Da ta for Isom er 10a (a s 25-O-TMS Eth er ). H NMR: δ
0.01 (6H, s), 0.10 (9H, s), 0.8-2.1 (series of m), 3.98 (1H, m),
4.31 (1H, m). 13C NMR: δ -5.3, -5.0, 2.4, 14.9, 17.2, 17.8, 17.9,
20.9, 25.6, 29.4, 29.6, 29.9, 34.2, 35.5, 36.1, 40.5, 41.8, 44.8,
50.7, 61.8, 68.7, 71.6, 73.9. HRMS (ES): m/z calcd for C27H57
-
O3Si2 [M + H]+ 485.3846, found 485.3834.
(20S)-Des-A,B-8â-[(ter t-bu tyld im eth ylsilyl)oxy]ch oles-
t a n e-16r,25-d iol (9b ) a n d (17S,20S)-Des-A,B-8â-[(ter t-
bu tyld im eth ylsilyl)oxy]ch olesta n e-16â,25-d iol (10b). Us-
ing the procedure described for the preparation of 9a , 1.5 g of
alkene 8b (3.2 mmol) was converted to 840 mg of deprotected
alcohol 9b (64%) as a white solid, mp 104-106 °C (recrystal-
lized from n-hexane), and 98 mg (7.4%) of isomer 10b as a
colorless oil.
1
Da ta for Isom er 9b. H NMR: δ -0.01 (3H, s), 0.00 (3H,
s), 0.87 (9H, s), 0.91 (3H, s), 1.01 (3H, d, J ≈ 6.9 Hz), 1.01
(1H, dd), 1.1-1.8 (series of m), 1.20 (6H, s), 1.86 (1H, m), 2.06
(1H, dt, J ≈ 13.5, 8.3 Hz), 4.02 (1H, m), 4.04 (1H, m). 13C NMR
(125 MHz): δ -5.4, -5.0, 15.5, 17.2, 17.8, 18.5, 20.6, 25.6, 29.0,
32.3, 34.1, 35.5, 36.2, 40.3, 43.9, 44.0, 49.8, 66.3, 68.8, 70.9,
75.6. MS: m/z 412 (M+), 394 (M - H2O), 379 (M - H2O - Me),
337 (M - H2O - t-Bu), 279 (M - H2O - TBS), 263 (M - H2O
- OTBS), 95. HRMS (EI): m/z calcd for C24H46O2Si [M - H2O]+
394.3267, found 394.3264. Anal. Calcd for C24H48O3Si: C,
69.84; H, 11.72. Found: C, 69.85; H, 11.63.
1
Data for Isomer 10b. H NMR: δ 0.01 (6H, s), 0.88 (9H, s),
0.97 (1H, m), 0.97 (3H, d, J ≈ 6.5 Hz), 1.08 (3H, s), 1.20 (6H,
1372 J . Org. Chem., Vol. 68, No. 4, 2003

Hapten Derivatives of 1,25(OH)2D3 and Its 20-Epimer
s), 1.1-2.1 (series of m), 3.97 (1H, m), 4.30 (1H, m). 13C NMR:
δ -5.3, -5.0, 15.1, 17.3, 17.8, 18.4, 20.6, 25.6, 29.0, 29.2, 34.1,
34.6, 35.6, 40.6, 41.9, 44.0, 50.9, 61.7, 68.7, 70.9, 72.0. HRMS
-5.0, 15.0, 17.1, 17.8, 18.6, 20.3, 21.3, 25.6, 29.0, 29.2, 33.0,
33.3, 34.0, 35.4, 40.4, 43.0, 44.1, 50.1, 61.8, 68.5, 70.8, 78.8,
170.7. HRMS (EI): m/z calcd for C26H50O4Si (M+) 454.3478,
found 454.3486. Anal. Calcd for C26H50O4Si: C, 68.67; H, 11.08.
Found: C, 68.16; H, 11.07.
-
(ES): m/z calcd for C24H47O2Si [MH+ H2O] 395.3345, found
395.3449.
(20S)-Des-A,B-8â-[(ter t-bu tyld im eth ylsilyl)oxy]ch oles-
ta n -25-ol-16r-yl Aceta te (13b). Following the procedure
described for the preparation of 13a , 395 mg (0.96 mmol) of
9b was converted quantitatively to 13b (colorless oil). 1H NMR:
δ -0.03 (3H, s), -0.01 (3H, s), 0.82 (3H, d, J ≈ 6.9 Hz), 0.86
(9H, s), 0.93 (3H, s), 1.20 (6H, s), 1.1-1.8 (series of m), 1.88
(1H, m), 2.00 (3H, s), 2.13 (1H, dt, J ≈ 13.7, 8.4 Hz), 3.99 (1H,
m), 4.95 (1H, br t, J ≈ 6.9 Hz). 13C NMR: δ -5.4, -5.0, 15.4,
17.1, 17.8, 17.9, 20.5, 21.3, 25.6, 29.0, 29.1, 32.1, 33.0, 34.1,
36.1, 40.1, 43.0, 44.0, 50.1, 61.2, 68.4, 70.8, 78.3, 170.7. HRMS
(ES): m/z calcd for C26H50O4SiNa [M + Na]+ 477.3376, found
477.3374.
Des-A,B-8â-[(ter t-bu tyld im eth ylsilyl)oxy]-16r-[(m eth -
ylsu lfon yl)oxy]-25-[(tr im eth ylsily)oxy]ch olesta n e (11a ).
To a solution of 125 mg (0.30 mmol) of 9a in pyridine (5 mL)
was added methanesulfonyl chloride (50 µL) at 0 °C. The
solution was stirred at 0-5 °C for 3 h, then TMSCl (50 µL)
was added, and stirring was continued for another 45 min.
After addition of water (0.5 mL), the mixture was poured into
ice/water (25 mL) and extracted with ether. The combined
ether layers were washed with water, aqueous CuSO4 (0.2 M),
and brine, dried, and evaported. Flash chromatography af-
forded 146 mg (87%) of 11a as a colorless oil. 1H NMR: δ -
0.02 (3H, s), -0.00 (3H, s), 0.08 (9H, s), 0.86 (9H, s), 0.92 (3H,
s), 0.95 (3H, overlapping d), 1.1-1.8 (series of m), 1.19 (6H,
s), 1.94 (1H, br d), 2.09 (1H, dt, J ≈ 14.1, 6.4 Hz), 2.97 (3H, s),
4.03 (1H, m), 4.95 (1H, m).
(20S)-Des-A,B-8â-[(ter t-b u t yld im et h ylsilyl)oxy]-16r-
[(m e t h ylsu lfon yl)oxy]-25-[(t r im e t h ylsily)oxy]ch ole s-
ta n e (11b). Using the procedure described for the preparation
of 11a , 60 mg of alcohol 9b (0.15 mmol) was converted to 69
mg of mesylate 11b (85%) as a colorless oil. 1H NMR: δ -0.01
(3H, s), 0.00 (3H, s), 0.09 (9H, s), 0.86 (9H, s), 0.91 (3H, s),
0.98 (3H, d, J ≈ 6.5 Hz), 1.19 (6H, s), 1.1-2.0 (series of m),
2.11 (1H, dt, J ≈ 14.1, 7.9 Hz), 2.98 (3H, s), 4.03 (1H, m), 5.00
(1H, t, J ≈ 7.1 Hz). 13C NMR: δ -5.4, -5.0, 2.4, 15.5, 17.0,
17.8, 18.1, 20.7, 25.6, 29.6, 29.7, 31.6, 32.7, 34.0, 36.5, 39.0,
39.7, 42.8, 44.8, 49.8, 62.2, 68.2, 73.8, 85.9. HRMS (ES): m/z
calcd for C28H59O5SSi2 [M + H]+ 563.3622, found 563.3635.
Des-A,B-ch olesta n e-8â,25-d iol (12a ). LiAlH4 (118 mg,
3.11 mmol) was added to a solution of mesylate 11a in ether
(10 mL). The mixture was heated to reflux for 3.5 h, at which
point the reaction was complete. Ethyl acetate was added until
gas evolution ceased. After standard workup with ether, the
residue was dissolved in THF and acetonitrile (each 1 mL),
treated with aqueous HF (48%, 0.5 mL), and stirred overnight
at room temperature. Water (1 mL) was added, and after being
stirred for 45 min, the mixture was extracted three times with
CH2Cl2. The combined extracts were then washed with 2 M
KHCO3, water, and brine, dried (Na2SO4), and concentrated
under reduced pressure. The residue was purified by flash
chromatography to afford 21 mg (68%) of 12a as a white solid,
which was recrystallized in hexane/dichloromethane for analy-
sis. Mp: 91-91.5 °C (lit.18 mp: 91-92 °C). 1H NMR: δ 0.91
(3H, s, J ≈ 6.9 Hz), 0.93 (3H, s), 1.21 (6H, s), 1.0-1.9 (series
of m), 1.95-2.05 (1H, m), 4.07 (1H, m). 13C NMR: δ 13.5, 17.5,
18.5, 20.8, 22.5, 27.2, 29.2, 29.4, 33.6, 35.3, 36.3, 40.4, 41.9,
44.4, 52.6, 56.7, 69.5, 71.1.
(20S)-Des-A,B-ch olesta n e-8â,25-d iol (12b).26 Using the
procedure described for the preparation of 12a , 66 mg of
mesylate 11b (0.12 mmol) was converted to 15 mg of diol 12b
(44%) as a white solid. Mp: 79.5-81 °C. 1H NMR: δ 0.84 (3H,
d, J ≈ 6.5 Hz), 0.93 (3H, s), 1.21 (6H, s), 1.0-2.1 (series of m),
4.07 (1H, m). 13C NMR: δ 13.8, 17.5, 18.5, 20.9, 22.4, 27.1,
29.2, 29.3, 33.6, 34.7, 35.7, 40.4, 41.9, 44.3, 52.7, 56.3, 69.4,
71.1. HRMS (EI): m/z calcd for C18H32O [M - H2O]+ 264.2453,
found 264.2466.
Des-A,B-ch olesta n e-8â,25-d iol-16r-yl Aceta te (14a ). To
a solution of 13a (210 mg, 0.462 mmol) in CH3CN (7 mL) was
added aqueous HF (40%, 230 µL), and the resulting mixture
was stirred at room temperature for 8 h. The reaction mixture
was neutralized with saturated NaHCO3 and concentrated by
evaporation. After standard workup, the residual oil was
purified by flash chromatography (ethyl acetate/pentanes),
affording 101 mg of 14a (64%) as a colorless oil. 1H NMR: δ
0.92 (3H, d, J ≈ 6.6 Hz), 0.95 (3H, s), 1.18 (6H, s),1.0-2.0
(series of m), 2.00 (3H, s), 2.18 (1H, dt, J ≈ 13.9, 8.2 Hz), 4.07
(1H, m), 4.90 (1H, t, J ≈ 6.9 Hz). 13C NMR: δ 14.7, 16.9, 18.6,
20.3, 21.3, 29.0, 29.2, 32.9, 33.1, 33.4, 35.3, 40.2, 42.7, 44.1,
49.6, 61.6, 68.2, 70.8, 78.5, 170.6. HRMS (EI): m/z calcd for
C
20H34O3 [M - H2O]+ 322.2509, found 322.2482.
(20S)-Des-A,B-ch olestan e-8â,25-diol-16r-yl Acetate (14b).
Following the procedure described for the preparation of 14a ,
444 mg (0.96 mmol) of 13b was converted to 257 mg of 14b
1
(79%) as a colorless oil. H NMR: δ 0.85 (3H, d, J ≈ 6.7 Hz),
0.97 (3H, s), 1.22 (6H, s), 1.1-2.0 (series of m), 2.02 (3H, s),
2.19 (1H, dt, J ≈ 13.9, 8.4 Hz), 4.09 (1H, m), 4.98 (1H, br t, J
≈ 7.1 Hz). 13C NMR: δ 15.3, 17.2, 18.1, 20.8, 21.5, 29.27, 29.34,
32.4, 32.8, 33.7, 36.3, 40.1, 43.0, 44.2, 49.9, 61.3, 68.4, 71.0,
78.2, 170.9. MS: m/z 322 (M - H2O), 280 (M - HOAc), 262
(M - H2O - HOAc), 247 (M - H2O - CH3 HOAc). HRMS
-
(EI): m/z calcd for C20H34O3 [M - H2O]+ 322.2509, found
322.2509.
Des-A,B-25-h ydr oxych olestan -8-on -16r-yl Acetate (15a).
PDC (219 mg, 0.583 mmol) was added to an ice/water-cooled
solution of 14a (131 mg, 0.385 mmol) in dry CH2Cl2 (20 mL).
The resulting suspension was then stirred at room tempera-
ture for 6 h, filtered through Celite, and evaporated. The
residue was redissolved in ethyl acetate, washed twice with
saturated NaHCO3, dried, and evaporated. The resulting crude
material was purified by flash chromatography (ethyl ether/
petroleum ether, 3:1), affording 108 mg (83%) of 15a as an
oil. 1H NMR: δ 0.68 (3H, s), 0.99 (3H, d, J ≈ 6.4 Hz), 1.21 (6H,
s), 1.0-3.4 (series of m), 2.01 (3H, s), 2.72 (1H, dd, J ≈ 12.6,
6.8 Hz), 4.95 (1H, dd, J ≈ 7.2, 6.1 Hz). 13C NMR: δ 14.0, 18.9,
20.5, 21.4, 23.5, 29.3, 29.5, 30.0, 33.6, 35.5, 38.9, 40.8, 44.2,
50.1, 59.3, 61.7, 70.9, 78.3, 170.5, 210.4. HRMS (EI): m/z calcd
for C20H34O4 (M+) 338.2457, found 338.2480.
(20S)-Des-A,B-25-h yd r oxych olest a n -8-on -16r-yl Ace-
ta te (15b). Following the procedure described for the prepara-
tion of 15a , 228 mg of 14b was converted to 216 mg of 15b
Des-A,B-8â-[(ter t-bu tyld im eth ylsilyl)oxy]ch olesta n -25-
ol-16r-yl Aceta te (13a ). Alcohol 9a (293 mg, 0.710 mmol)
dissolved in pyridine (5 mL) was treated with acetic anhydride
(1.10 mL) at 0 °C under argon. After being stirred at room
temperature for 3 h, the reaction mixture was poured into
water (20 mL), cooled in an ice bath, and acidified to pH 2.5
with aqueous HCl (4 M). After standard workup using ether,
the residue was purified by flash chromatography to afford
240 mg of 13a (74%) as an oil. 1H NMR: δ -0.04 (3H, s), -0.02
(3H, s), 0.85 (9H, s), 0.91 (3H, d, J ≈ 6.6 Hz), 0.93 (3H, s),
1.18 (6H, s), 1.0-2.0 (series of m), 1.99 (3H, s), 2.12 (1H, dt, J
≈ 13.9, 8.2 Hz), 3.98 (1H, m), 4.88 (1H, m). 13C NMR: δ -5.4,
1
(95%, colorless oil). H NMR: δ 0.67 (3H, s), 0.89 (3H, d, J ≈
6.5 Hz), 1.23 (6H, s), 1.1-2.1 (series of m), 2.01 (3H, s), 2.29
(3H, m), 2.73 (1H, dd, J ≈ 12.2, 6.9), 5.00 (1H, m). 13C NMR:
δ 14.2, 18.0, 20.8, 21.4, 23.5, 29.3, 29.4, 29.7, 32.7, 36.4, 38.6,
40.8, 44.1, 50.1, 59.2, 61.1, 70.9, 77.5, 170.5, 210.3. HRMS
(ES): m/z calcd for C20H34O4Na [M + Na]+ 361.2355, found
361.2337.
Des-A,B-25-[(t r im et h ylsilyl)oxy]ch olest a n -8-on -16r-
yl Aceta te (16a ). To a solution of 15a (108 mg, 0.319 mmol)
in dry CH2Cl2 (10 mL) were added DMAP (1 mg, 0.010 mmol),
Et3N (0.16 mL, 1.16 mmol), and TMSCl (0.16 mL, 1.28 mmol)
J . Org. Chem, Vol. 68, No. 4, 2003 1373

Blæhr et al.
at 0 °C under argon. The resulting mixture was warmed to
room temperature and stirred for 3 h. The reaction mixture
was quenched with water and extracted twice with CH2Cl2 (2
× 15 mL). The combined organic extracts were washed with
water and concentrated under reduced pressure. The residue
was taken up in ether, washed twice with water and brine,
dried, and evaporated to dryness. Flash chromatography in
ethyl acetate/pentanes afforded 118 mg (90%) of 16a as an oil.
1H NMR: δ 0.07 (9H, s), 0.66 (3H, s), 0.96 (3H, d, J ≈ 6.4 Hz),
1.17 (6H, s), 1.0-2.4 (series of m), 1.98 (3H, s), 2.70 (1H, dd,
J ≈ 12.6, 6.8 Hz), 4.90 (1H, dd, J ≈ 7.2, 6.2 Hz), 13C NMR: δ
2.4, 13.7, 18.6, 20.3, 21.1, 23.3, 29.6, 29.7, 29.8, 33.4, 35.2, 38.7,
40.6, 45.0, 49.9, 59.1, 61.6, 73.7, 78.0, 170.2, 210.1. MS: m/z
322 (M - H2O), 262 (M - H2O - CH3COOH). HRMS (EI): m/z
calcd for C22H39O4Si [M - CH3]+ 395.2618, found 395.2600.
- TBSOH), 513 (M - 2TBSOH), 131. HRMS (EI): m/z calcd
for C44H82O5Si3 (M+) 774.5470, found 774.5467.
(5Z,7E)-(1R,3S)-1,3-Bis[(ter t-b u t yld im et h ylsilyl)oxy]-
25-[(tr im eth ylsilyl)oxy]-9,10-secoch olesta-5,7,10(19)-tr ien -
16r-ol (19a ). Acetate 18a (135 mg, 0.174 mmol) was added
to a solution of KOH in methanol (0.4 M, 20 mL), and the
resulting mixture was stirred at 40 °C for 6 h. The mixture
was concentrated to approximately half the initial volume and
diluted with ethyl acetate. After standard workup, the residue
was purified by flash chromatography (ethyl acetate/pentanes)
1
to give 70 mg (55%) of 19a as a colorless oil. H NMR: δ 0.05
(12H, s), 0.10 (9H, s), 0.56 (3H, s), 0.86 (18H, s), 0.95 (1H, dd,
J ≈ 7.4, 3.8 Hz), 1.20 (6H, s), 1.1-2.5 (series of m), 2.82 (1H,
m), 4.00 (1H, m), 4.18 (1H, m), 4.36 (1H, m), 4.85 (1H, m),
5.17 (1H, m), 5.95 (1H, d, J ≈ 11.2 Hz), 6.21 (1H, d, J ≈ 11.1
Hz). 13C NMR: δ -5.2, -5.0, -4.9, -4.8, 2.5, 13.3, 17.95, 18.0,
18.7, 21.3, 22.8, 25.6, 25.7, 28.5, 29.6, 29.7, 30.1, 34.2, 34.9,
36.0, 40.6, 44.6, 44.9, 45.8, 46.9, 53.3, 67.3, 67.6, 71.9, 73.9,
111.1, 118.0, 122.8, 135.2, 139.7, 148.1. MS: m/z 732 (M), 600
(M - TBSOH), 248. HRMS (EI): m/z calcd for C42H80O4Si3 (M+)
732.5364, found 732.5331.
(20S)-Des-A,B-25-[(tr im eth ylsilyl)oxy]ch olesta n -8-on -
16r-yl Aceta te (16b). Following the procedure described for
the preparation of 16a , 15b was converted to 16b (79% from
1
14b) as a colorless oil. H NMR: δ 0.09 (9H, s), 0.65 (3H, s),
0.86 (3H, d, J ≈ 6.5), 1.20 (6H, s), 1.1-2.1 (series of m), 2.00
(3H, s), 2.27 (3H, m), 2.71 (1H, dd, J ≈ 6.9, 12.2 Hz), 4.98 (1H,
m). 13C NMR: δ 2.4, 14.0, 17.8, 20.5, 21.2, 23.3, 29.5, 29.6, 29.7,
32.5, 36.2, 38.4, 40.6, 44.8, 49.9, 59.0, 61.0, 73.7, 77.4, 170.3,
210.1. MS: m/z 395 (M - CH3), 335 (M - HOAc - CH3), 131.
HRMS (EI): m/z calcd for C22H39O4Si [M - CH3]+ 395.2618,
found 395.2614.
(5Z,7E)-(1R,3S,20S)-1,3-Bis[(ter t-b u t yld im et h ylsilyl)-
oxy]-9,10-secoch olesta -5,7,10(19)-tr ien e-16r,25-d iol (19b).
Following the procedure described for the preparation of 19a ,
18b was converted quantitatively to 19b (colorless oil). 1H
NMR: δ 0.04-0.05 (12H, singlets), 0.54 (3H, s), 0.86 (18H, s),
1.03 (3H, d, J ≈ 6.9 Hz), 1.20 (6H, s), 1.2-2.0 (series of m),
2.20 (1H, dd, J ≈ 13.0, 7.6 Hz), 2.36 (1H, overlaid m), 2.43
(1H, dd, J ≈ 13.0, 3.8 Hz), 2.82 (1H, br d, J ≈ 13.40 Hz), 4.04
(1H, m), 4.17 (1H, m), 4.36 (1H, dd, J ≈ 6.5, 3.8 Hz), 4.84 (1H,
m), 5.16 (1H, m), 5.94 (1H, d, J ≈ 11.1 Hz), 6.21 (1H, d, J ≈
11.1 Hz). 13C NMR: δ -5.3, -5.0, -4.89, -4.86, 13.5, 17.95,
18.0, 18.4, 20.6, 22.7, 25.61, 25.65, 28.4, 29.1, 33.2, 34.6, 36.1,
40.3, 44.0, 44.6, 45.8, 46.8, 53.2, 66.3, 67.3, 70.9, 71.9, 75.9,
111.1, 118.0, 122.8, 135.3, 139.5, 148.1. HRMS (EI): m/z calcd
for C39H72O4Si2 (M+) 660.4969, found 660.4952.
(5Z,7E)-(1R,3S)-1,3-Bis[(ter t-b u t yld im et h ylsilyl)oxy]-
25-[(tr im eth ylsilyl)oxy]-9,10-secoch olesta-5,7,10(19)-tr ien -
16r-yl Hyd r ogen Glu ta r a te (20a ). To a solution of 19a (70
mg, 0.096 mmol) in CH2Cl2 (5 mL) were added DMAP (70 mg,
0.573 mmol) and glutaric anhydride (54 mg, 0.477 mmol). The
resulting solution was stirred at 4 °C for 3 days. Standard
workup with CH2Cl2 followed by flash chromatography (30%
EtOAc/pentanes) afforded 32 mg of 20a (39%) as a colorless
oil. 1H NMR: δ 0.04 and 0.05 (12H, singlets), 0.08 (9H, s), 0.56
(3H, s), 0.85 (9H, s), 0.87 (9H, s), 0.93 (3H, d, J ≈ 5.7 Hz),
1.18 (6H, s), 1.2-2.7 (series of m), 2.83 (1H, m), 4.18 (1H, m),
4.35 (1H, m), 4.82 (1H, m), 4.93 (1H, m), 5.14 (1H, m), 5.92
(1H, d, J ≈ 11.1 Hz), 6.21 (1H, d, J ≈ 11.2 Hz). 13C NMR: δ
-5.2, -5.0, -4.9, -4.7, 2.5, 13.2, 17.9, 18.7, 20.1, 20.8, 22.8,
25.6, 25.7, 28.6, 29.6, 29.7, 32.4, 33.6, 33.8, 35.4, 40.4, 44.6,
44.9, 46.1, 46.2, 53.5, 62.3, 67.2, 72.2, 73.9, 78.9, 111.7, 118.0,
122.7, 135.3, 139.1, 147.6, 172.8. MS: m/z 846 (M), 582 (M -
2TBSOH), 75. HRMS (EI): m/z calcd for C47H86O7Si3 (M+)
846.5681, found 846.5635.
(5Z,7E)-(1R,3S)-1,3-Bis[(ter t-b u t yld im et h ylsilyl)oxy]-
25-[(tr im eth ylsilyl)oxy]-9,10-secoch olesta-5,7,10(19)-tr ien -
16r-yl Aceta te (18a ). Ketone 16a and phosphine oxide 1720
were dried thoroughly with an oil pump. A solution of 17 (248
mg, 0.425 mmol) in dry THF (10 mL) was treated with n-BuLi
(1.56 M, 275 µL) at -78 °C under argon. The resulting orange
solution was stirred for 25 min at -78 °C, upon which a
solution of 16a (95 mg, 0.231 mmol) in THF (2 mL) was added
over 5 min. The reaction mixture was stirred at -78 °C for 2
h, warmed slowly to room temperature, where stirring was
continued for 3 h, and finally quenched with a few drops of
water. The solvent was removed in vacuo. The residue was
dissolved in a 1:1 mixture of ethyl acetate and petroleum ether,
washed with saturated NaHCO3 and brine, and dried. Filtra-
tion and evaporation afforded 381 mg of residue that was
chromatographed on silica gel (2.5% ethyl acetate/petroleum
ether) to give 160 mg (89%) of 18a as a colorless oil. 1H NMR:
δ 0.05 (12H, s), 0.08 (9H, s), 0.57 (3H, s), 0.86 (18H, s), 0.94
(3H, d, J ≈ 5.9 Hz), 1.18 (6H, s), 1.0-2.1 (series of m), 2.00
(3H, s), 2.23 (2H, m), 2.42 (1H, dd, J ≈ 13.1, 3.4 Hz), 2.83 (1H,
m), 4.18 (1H, m), 4.35 (1H, m), 4.81 (1H, d, J ≈ 2.3 Hz), 4.90
(1H, m), 5.15 (1H, J ≈ 1.6 Hz), 5.93 (1H, d, J ≈ 11.2 Hz), 6.20
(1H, d, J ≈ 11.2 Hz). 13C NMR: δ -5.2-5.0, -4.9, -4.7, 2.4,
13.1, 17.9, 18.0, 18.7, 20.4, 21.3, 22.8, 25.6, 25.7, 28.5, 29.6,
29.8, 32.4, 33.9, 35.4, 40.4, 44.6, 45.1, 45.9, 46.1, 53.5, 61.9,
67.3, 71.9, 73.8, 78.9, 111.3, 118.1, 122.6, 135.4, 139.1, 147.9,
170.6. HRMS (EI): m/z calcd for C44H82O5Si3 (M+) 774.5470,
found 774.5467.
(5Z,7E)-(1R,3S,20S)-1,3-Bis[(ter t-b u t yld im et h ylsilyl)-
oxy]-9,10-secoch olesta - 5,7,10(19)-tr ien -25-ol-16r-yl Ace-
ta te (18b). Following the procedure described for the prepara-
tion of 18a , 98 mg of 16b was converted to 103 mg (61%) of
18b (TMS deprotected) and 60 mg (32%) of TMS-protected
product, both as colorless oils. Spectral data of the TMS-
protected product were recorded. 1H NMR: δ 0.04 (6H, s), 0.05
(6H, s), 0.09 (9H, s), 0.56 (3H, s), 0.86 (18H, s), 1.19 (6H, s),
1.1-2.1 (series of m), 2.01 (3H, s), 2.23 (2H, m), 2.43 (1H, dd,
J ≈ 13.4, 3.8 Hz), 2.84 (1H, br d, J ≈ 12.6 Hz), 4.18 (1H, septet,
J ≈ 3.4 Hz), 4.35 (1H, dd, J ≈ 6.5, 3.8 Hz), 4.81 (1H, br d, J ≈
2.3 Hz), 4.96 (1H, dd, J ≈ 7.2, 5.7 Hz), 5.16 (1H, br d, J ≈ 1.5
Hz), 5.93 (1H, d, J ≈ 11.4 Hz), 6.20 (1H, d, J ≈ 11.4 Hz). 13C
NMR: δ -5.3, -5.0, -4.9, -4.7, 2.4, 13.3, 17.90, 17.93, 18,0,
20.5, 21.3, 22.8, 25.59, 25.65, 28.5, 29.6, 29.7, 32.2, 33.0, 36.0,
40.2, 44.5, 44.9, 45.9, 46.1, 53.4, 61.3, 67.3, 71.9, 73.8, 78.4,
111.3, 118.0, 122.6, 135.4, 139.1, 147.9, 170.7. MS: m/z 774
(M), 714 (M - HOAc), 642 (70, M - HOTBS), 582 (M - HOAc
(5Z,7E)-(1R,3S,20S)-1,3-Bis[(ter t-b u t yld im et h ylsilyl)-
oxy]-9,10-secoch olest a -5,7,10(19)-t r ien -25-ol-16r-yl Hy-
d r ogen Glu ta r a te (20b). Following the procedure described
for the preparation of 20a , 44 mg of 19b was converted to 40
1
mg (77%) of 20b as a colorless oil. H NMR: δ 0.04, 0.05, and
0.05 (12H, 3 s), 0.55 (3H, s), 0.86 (18H, s), 0.85 (3H, overlaid),
1.21 (6H, s), 1.3-2.5 (series of m), 2.33 and 2.38 (4H, over-
lapping t), 2.84 (1H, m), 4.18 (1H, m), 4.35 (1H, m), 4.82 (1H,
m), 5.00 (1H, m), 5.16 (1H, m), 5.93 (1H, d, J ≈ 11.2 Hz), 6.20
(1H, d, J ≈ 11.1 Hz). 13C NMR: δ -5.2, -5.0, -4.9, -4.7, 13.5,
17.9, 19.9, 20.6, 22.7, 25.6, 25.7, 28.5, 29.0, 29.1, 32.2, 32.6,
33.0, 33.6, 36.4, 40.0, 43.9, 44.5, 46.0, 46.1, 53.4, 61.1, 67.2,
71.1, 72.0, 78.0, 111.4, 118.0, 122.6, 135.5, 139.0, 147.8, 172.4,
177.9. HRMS: (EI) m/z calcd for C44H78O7Si2 (M+) 774.5286,
found 774.5278.
(E)-Des-A,B-8â-[(ter t-b u t yld im et h ylsilyl)oxy]-17(20)-
p r egn en e (21). Following the procedure described for the
1374 J . Org. Chem., Vol. 68, No. 4, 2003

Hapten Derivatives of 1,25(OH)2D3 and Its 20-Epimer
preparation of 7, 2.36 g of alcohol 3b (7.55 mmol) was
converted to 1.88 g of alkene 21 (85%) as a colorless oil. 1H
NMR: δ 0.01 (6H, s), 0.88 (9H, s), 0.97 (3H, s), 1.1-1.9 (series
of m), 1.52 (3H, dt, J ≈ 6.6, 1.5 Hz), 2.10-2.35 (2H, m), 4.07
(1H, q, J ≈ 2.6 Hz), 4.97 (1H, tq, J ≈ 6.4, 2.5 Hz). 13C NMR:
δ -5.3, -5.0, 13.4, 17.5, 17.8, 21.6, 23.3, 25.3, 25.6, 34.6, 36.7,
2 h, methanol was carefully added until the reaction subsided.
This was followed by the addition of water to fully quench the
reaction. While being cooled in an ice bath, the mixture was
acidified to pH 3.0 with aqueous HCl. After standard workup
with CH2Cl2, the residue was purified by flash chromatography
(12% EtOAc/pentanes) to afford 1.22 g of alcohol 23 (colorless
oil, 53%) and 358 mg (17%) of starting material 21. In some
runs, the byproduct 24 was produced in <10% yield as a
43.1, 51.4, 69.2, 108.8, 153.2. HRMS (EI): m/z calcd for C18H34
-
OSi (M+) 294.2379, found 294.2391.
1
Des-A,B-8â-[(ter t-bu tyld im eth ylsilyl)oxy]-17,20-ep oxy-
m eth a n op r egn a n e (22). To a solution of (E)-alkene 21 (468
mg, 1.59 mmol) in dry CH2Cl2 (50 mL) were added paraform-
aldehyde (239 mg, 7.94 mmol) and BF3‚OEt2 (3 drops, ∼23 mg,
0.16 mmol) under argon. The resulting red-brown suspension
was stirred for 10 min at room temperature and then poured
into water (70 mL). After standard workup, the crude product
was purified by flash chromatography (10% EtOAc/pentanes)
to afford 354 mg of oxetane 22 (69%) as a colorless oil. 1H NMR
(500 MHz, d6-DMSO): δ 0.02 (3H, s), 0.03 (3H, s), 0.86 (3H,
d, J ≈ 6.9 Hz), 0.87 (9H, s), 0.95 (3H, s), 1.2-1.9 (series of m),
3.14 (1H, dd, J ≈ 11.1, 8.5 Hz), 3.69 (1H, dd, J ≈ 8.2, 7.3 Hz),
3.93 (1H, m). 13C NMR: δ -5.0, -4.8, 10.0, 18.0, 18.1, 21.8,
24.9, 25.7, 30.0, 30.0, 32.1, 43.9, 52.3, 52.5, 69.9, 70.8, 93.4.
colorless oil. H NMR: δ 0.01 (6H, 2 s), 0.87 (9H, s), 1.03 (3H,
s), 1.10 (3H, d, J ≈ 7.0 Hz), 1.2-2.0 (series of m), 2.25 (1H,
m), 2.37 (1H, m), 3.46 (2H, m), 4.08 (1H, m), 5.44 (1H, br t, J
≈ 1.5 Hz). 13C NMR: δ -5.4, -5.1, 17.8, 17.9, 18.9, 25.6, 30.8,
33.9, 34.4, 35.4, 46.8, 54.5, 66.4, 68.7, 123.3, 156.7. HRMS
(EI): m/z calcd for C19H36O2Si (M+) 324.2485, found 324.2487.
The NMR spectra of the byproduct 24 are available in the
Supporting Information.
Ack n ow led gm en t. This study was generously sup-
ported by the Academy of Technical Sciences, Denmark.
We thank Mr. Niels Rastrup-Andersen and Dr. Simon
Bolvig for their help with the interpretation of the
NMR spectra and Mrs. Priscila Madsen for experimen-
tal assistance. In addition, we thank Dr. Sven Erik
Godtfredsen and Dr. Peter Kaastrup for facilitating the
production of antisera at DAKO A/S.
IR (CHCl3): 835, 1035, 1090, 1255, 1460, 2800-3000 (s) cm-1
.
MS: m/z 324 (M+), 267 (M - t-Bu), 193 (M - OTBS), 175,
135, 75.
(20R)-Des-A,B-8â-[(ter t-bu tyld im eth ylsilyl)oxy]-20-h y-
d r oxym eth ylp r egn -16-en e (23). To a solution of alkene 21
(2.1 g, 7.1 mmol) in CH2Cl2 (150 mL) was added paraform-
aldehyde (1.07 g, 35.7 mmol), and the resulting suspension
was cooled in an ice bath. Trimethylaluminum (2 M in
heptanes, 9 mL) was added using a syringe, and the reaction
mixture was stirred for 4.5 h at 0 °C. Then, additional Me3Al
(9 mL) was added. After the mixture was stirred for another
Su p p or t in g In for m a t ion Ava ila b le: 1H and 13C NMR
spectra for new compounds and experimental details of the
radioimmunoassay. This material is available free of charge
via the Internet at http://pubs.acs.org.
J O026061S
J . Org. Chem, Vol. 68, No. 4, 2003 1375