Design, synthesis, and biological action of 20 R -hydroxyvitamin D3

Article abstract of DOI:10.1021/jm201478e

The non-naturally occurring 20R epimer of 20-hydroxyvitamin D3 is synthesized based on chemical design and hypothesis. The 20R isomer is separated by semipreparative HPLC, and its structure is characterized. A comparison of 20R isomer to its 20S counterpa

Full text of DOI:10.1021/jm201478e

Brief Article
pubs.acs.org/jmc
Design, Synthesis, and Biological Action of 20R-Hydroxyvitamin D3
Yan Lu,† Jianjun Chen,† Zorica Janjetovic,‡ Phillip Michaels,‡ Edith K. Y. Tang,§ Jin Wang,†
Robert C. Tuckey,§ Andrzej T. Slominski,‡ Wei Li,*,† and Duane D. Miller*,†
†Department of Pharmaceutical Sciences, University of Tennessee Health Science Center, Memphis, Tennessee 38163, United States
‡Department of Pathology and Laboratory Medicine, Center for Cancer Research, and Department of Medicine, Division of
Dermatology, University of Tennessee Health Science Center, Memphis, Tennessee 38163, United States
§School of Biomedical, Biomolecular and Chemical Sciences, University of Western Australia, Crawley, WA, Australia
S
* Supporting Information
ABSTRACT: The non-naturally occurring 20R epimer of 20-hydroxyvitamin D3 is synthesized based on chemical design and
hypothesis. The 20R isomer is separated by semipreparative HPLC, and its structure is characterized. A comparison of 20R
isomer to its 20S counterpart in biological evaluation demonstrates that they have different behaviors in antiproliferative and
metabolic studies.
INTRODUCTION
■
Vitamin D hormone carries out essential biological functions
required for human health. Besides its critical role in regulating
bone formation and calcium homeostasis, vitamin D hormone
also regulates cell growth, differentiation, proliferation,
apoptosis, and immune responses through the activation of
the nuclear vitamin D receptor (VDR).1,2 Vitamin D3 is
Figure 1. Structures of calcitriol, vitamin D3, and new VD3 metabolite
20S(OH)D3.
derived from a cholesterol precursor in the skin, 7-
dehydrocholesterol (7-DHC). After absorption of ultraviolet
B (UVB) radiation (315−280 nm wavelength), 7-DHC is
converted to previtamin D3, which undergoes further thermally
induced transformation to vitamin D3 (cholecalciferol).
Vitamin D3 from the diet or produced in the epidermis is
biologically inactive and requires enzymatic conversion to 1,25-
dihydroxyvitamin D3 (1,25(OH)2D3, calcitriol), the active
form of vitamin D3. This activation involves sequential 25- and
1α-hydroxylation in the liver and kidney, respectively.3
Unfortunately, the toxicity (hypercalcemia) induced by high
levels of vitamin D largely prevents the clinical use of
pharmacological doses of 1,25(OH)2D3. More than 40
metabolites of vitamin D have been reported,4 and under-
standing the activity of these metabolites can assist in the
development of new vitamin D3 analogues with beneficial
actions. Searching for vitamin D analogues that retain biological
efficacy and display minimal or no hypercalcemia has been
pursued for several decades. As a result, several vitamin D
analogues that exhibit reduced hypercalcemia are entering
clinical trials and are showing great promise as potential
therapeutic agents for the treatment of cancer and other
diseases.5
ties similar to those of 20S(OH)D3 generated enzymatically
using P450scc. The S configuration of C-20 in chemically
synthesized 20S(OH)D3 was determined by NMR and HPLC
and was found to be identical to the enzymatically generated
metabolite. Many reported potent D3 analogues have 21β-Me
configuration which corresponds to the 20R(OH)D3 config-
uration.9 These analogues are very potent VDR activators with
very low hypercalcemic side effect.10 It will be very interesting
to see if 20R(OH)D3 has different activity compared with
20S(OH)D3. Hansen et al. have reported the preparation of
20R(OH)D3 analogues with 22-alkynyl side chains from
secosteroids.11 In this report, we designed an efficient synthetic
route to prepare 20R(OH)-7DHC from pregenolone, which
after UVB irradiation and HPLC purification yielded the
20R(OH)D3 epimer. Analysis of its NMR spectra and
comparison to that of 20S(OH)D3 confirmed the formation
of the 20R-epimer without detectable amounts of the other.
A noticeable phenomenon was reported in the preparation of
20S(OH)-7DHC that only 20S epimer was produced from the
Grignard reaction, while theoretically 20R- and 20S(OH)-
7DHC should be obtained. This result suggested that the attack
of Grignard agent is highly stereoselective at the 20-cabonyl
position (Figure 2). A similar phenomenon was also reported in
the synthesis of 20S-hydroxycholesterol and other 20-
hydroxysteroids.12 Theoretical calculation and NMR spectra
characterization6b of synthesized 20S(OH)-7DHC clearly
20S-Hydroxyvitamin D3 (20S(OH)D3, Figure 1) is a newly
isolated metabolite of vitamin D3 produced by the action of the
enzyme cytochrome P450scc (CYP11A1).6,7 It inhibits cell
proliferation and differentiation without inducing hyper-
calcemia at a dose of 30 μg/kg on mice (unpublished results),
stimulates VDR gene expression, and inhibits NFκß activity
with similar potency to 1,25(OH)2D3.8 We have chemically
synthesized 20S(OH)D3,6b which exhibited biological proper-
Received: November 11, 2011
Published: March 9, 2012
© 2012 American Chemical Society
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Journal of Medicinal Chemistry
Brief Article
to introduce Weinreb amide, but the major product separated
was the intermediate active ester of benzotriazolyl carboxylate,
which is close to the desired 2 on the TLC plate but showed
UV absorption. We therefore changed coupling conditions to
HBTU/DIPEA/HNMeOMe·HCl salt which was reacted with
17α carboxylic acid to yield Weinreb amide in 75.9% yield. We
chose introducing silicon ether protection of the 3β-hydroxyl in
the next step to reduce the consumption of (4-methylpentyl)
magnesium bromide 4, which was prepared from 1-bromo-4-
methylpentane and magnesium turnings in anhydrous THF.
Three silyl chlorides (TMSCl, TBDMSCl, and TBDPSCl) were
used in the presence of various bases such as triethylamine,
imidazole, and N-methylimidazole. After comparing and
examining the above conditions, we chose TBDPSCl as the
silylation agent for introducing chromophore to 3 and using N-
methylimidazole as the base with iodine as the catalyst to
accelerate the reaction (89.1% yield).14 Grignard reaction of 3
and (4-methylpentyl)magnesium bromide yielded the bulky
side chain ketone 5 with 47.6% yield.
We designed the synthesis route by introducing a 5,7-diene
to 5 under 1,3-dibromo-5,5-dimethylhydantoin (dibromantin)/
AIBN/TBAB/TBAF conditions; thus, the deprotection of
silicon ether can be used in the same step under TBAF
treatment. However, we found from TLC analysis that there
was a more complex set of reaction products with siliyl
protected substrate compared with a similar reaction from
acetyl protected substrate in the preparation of 20S(OH)-
7DHC. Neither the diabromantin condition nor the NBS/γ-
collidine reaction afforded satisfactorily pure 5,7-diene product.
Moreover, 5,7-diene structures are known to be unstable under
light and acidic conditions. Thus, we chose to postpone the
formation of the 5,7-diene and to replace TBDPS protection
with acetyl protection. The deprotection of silicon ether with
TBAF afforded the 3β-hydroxyl 6 in satisfactory yield
(quantitative). Introducing the acetyl protecting group at 3β-
OH with acetyl chloride and pyridine gave 7 in an 88.7% yield.
Transformation of 7 into the 5,7-diene 9 was carried out by
diabromantin/AIBN employed in the synthesis of 20S(OH)-
7DHC using hexane and benzene as the solvent in the
bromination step. Treatment of TBAB/TBAF in THF yielded
the diene via dehydrobromination. The purification of the
product 9 (34% overall yield) was carried out through silver
nitrate impregnated silica gel chromatography to remove other
impurities such as the 4,6-diene byproduct. Grignard reaction
of 5,7-diene substrate 9 with CH3MgI afforded the precursor
20R(OH)-7DHC 10 (yield 85.6%).
Figure 2. Hypothesis to obtain 20R-epimer. Molecular models were
constructed using the MM2 energy minimization algorithm in
ChemBioOffice 2010 Chem 3D Pro (version 12.0). (A) It was
proved that “bottom” side attack by the bulky Grignard reagent to
form the 20S epimer is favored while attack from the “top” is
prohibited because of the steric hindrance from 18-CH3 and the
steroid scaffold. (B) We hypothesize that “bottom” side attack of the
small sized CH3MgI is favored by the pre-existing bulky side chain
while it is not inclined to attack from “top” side to form the 20R
epimer.
indicate that there is a preference for the orientation of the 17-
acetyl group. It is conceivable that this conformational
preference dictated the outcome of the bulky side chain
addition to form 20S epimer. As shown in Figure 2A, the attack
of the nucleophilic Grignard reagent takes place predominantly
from the less hindered “bottom” side of the carbonyl group to
form 20S(OH)-7DHC while the attack from the “top” side to
form 20R(OH)-7DHC is sterically prohibited.
The above observations encourage us to design a synthesis
approach to exclusively produce 20R(OH)-7DHC. We predict
that if the bulky side chain at the 20-cabonyl position is
introduced in earlier synthesis steps, followed by introducing
the 21-methyl group with smaller Grignard reagent CH3MgI,
the 20R conformation should be obtained as the only epimer
product (Figure 2B). To validate our hypothesis, we employed
chemical synthesis to access material for biological evaluation.
The synthesis route of 20R(OH)D3 is shown in Scheme 1.
RESULTS AND DISCUSSION
■
Chemistry. The synthesis of 20R(OH)-7DHC started with
high yield (>95%) conversion of pregnenolone acetate to 3β-
acetoxyetientic acid 1 under oxidation with in situ prepared
NaOBr from bromine and sodium hydroxide solution.13 We
tried the coupling conditions employing EDCI/HOBt/NMM
Scheme 1. Chemical Synthesis of 20R(OH)D3
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For B-ring-opening, 20R(OH)-7DHC 10 was subjected to
UVB irradiation for 20 min in a quartz tube at 65 °C to achieve
the maximum conversion to pre-20R(OH)D3 (11). The
mixture was incubated at room temperature for 1 week,
which produced a mixture of 20R(OH)D3 (12), 20R(OH)-
tachysterol (13), 20R(OH)-lumisterol (14), and other minor
20R(OH) products. The photolysis reaction and subsequent
time-dependent conversion to 20R(OH)D3 were analyzed by
HPLC. The final 20R(OH)D3 was purified by a Gilson
semipreparative HPLC system.
with the highest potency at >10−10 M.6b,8b,15 In the present
study we compared the antiproliferative activity of 20S(OH)D3
with its epimer 20R(OH)D3. Figure 4 shows that 20S(OH)D3
Different epimers of both 20(OH)-7DHC precursors and
final 20-hydroxyvitamin D3 analogues have characteristic
HPLC retention times (tR). The tR of precursor 20R(OH)-
7DHC appeared at 16.0 min, and its 20S counterpart could be
eluted earlier at 13.8 min under the same HPLC conditions.
The final 20R(OH)D3 showed a slight difference as compared
to the 20S(OH)D3 isomer peak at tR of 12.3 min vs 12.2 min,
respectively (see Supporting Information).
Figure 4. 20(OH)D3 isomers inhibit growth of human keratinocytes.
HEKn cells were treated for 24 h with 1,25(OH)2D3 or R or S epimers
of 20(OH)D3 at the concentrations listed. The rate of H-thymidine
incorporation into DNA served as a measure of proliferative activity.
Data are presented as the mean SD, n = 4. Incorporation into DNA
is shown as percent (%) of control (ethanol treated cells). Statistical
significance was measured using Student t test (∗) and one-way
ANOVA (red asterisk) presented as (∗∗) p < 0.05, (∗∗∗) p < 0.01,
and (∗∗∗∗) p < 0.001.
3
1H NMR comparisons of both 20(OH)-7DHC epimer
precursors and final 20(OH)D3 products are effective methods
to identify 20R and 20S isomers of vitamin D3 analogues. It is
1
established that in the pregnane compounds, the H NMR
and 1,25(OH)2D3 (positive control) exhibited similar dose-
dependent inhibition of proliferation, as expected, with an EC50
of 7.28 × 10−10 and 4.54 × 10−10 M, respectively. In contrast,
20R(OH)D3 had a biphasic effect, slightly stimulating DNA
synthesis at 0.1 nM, with significant (p < 0.001) inhibition of
cell proliferation at ≥10 nM. This inhibitory effect was similar
to that of 20S(OH)D3 (<50% of control), however, with higher
EC50 of 4.09 × 10−9 M. These data show a clear difference
between the R and S isomers at low concentrations of the
ligand (0.1 nM) and similar effects at the higher concentrations,
however, with lower potency for the R form. In a separate
experiment we demonstrated that 20S(OH)D3 and 1,25-
(OH)2D3 inhibit colony formation by HaCaT epidermal
keratinocytes with similar potency while 25(OH)D3 has no
or a small effect (Supporting Information).
chemical shifts for the 21-Me in the 20S-OH isomers are
downfield relative to 20R-OH isomers.12a They have distinct
values and are the basis used to assign the absolute
configuration at C20 of these two epimers. The R-configuration
at C20 can be deduced from comparisons of 1H NMR spectra of
21-methyls in precursor 20R/S(OH)-7DHC and final 20R/
S(OH)D3. An upfield chemical shift (1.16 ppm) was obtained
for the 21-methyl in 20R(OH)-7DHC, while in 20S(OH)-
7DHC a downfield chemical shift at 1.27 ppm was observed.
Similarly, we found that the 21-Me showed a chemical shift at
1.13 ppm in 20R-isomer of 20(OH)D3 while a downfield
chemical shift of 1.24 ppm was observed for 21-Me in
1
20S(OH)D3 (as shown in Figure 3, H NMR spectra of 20R/
S(OH)-7DHC are shown in Supporting Information).
Metabolism of 20(OH)D3 by CYP11A1 and CYP27B1.
Since biologically generated 20S(OH)D3 is derived from the
action of P450scc on vitamin D3 and can be further
metabolized to dihydroxy (e.g., 17,20(OH)2D3, 20,22-
(OH)2D3, 20,23(OH)2D3) and trihydroxy (e.g., 17,20,23-
(OH)3, 20,22,X(OH)3D3) metabolites by this enzyme,6a,7,16
we compared the ability of P450scc to metabolize 20S(OH)D3
and 20R(OH)D3 (Figure 5A). Because of the formation of
many products, metabolites were characterized in the case of
the 20S-isomer16 but not for the 20R-isomer; data are
presented as the amount of substrate metabolized.
20R(OH)D3 proved to be a much better substrate than
20S(OH)D3 for P450scc with 58% being consumed in the first
2 min of incubation for the R-isomer but only 13% for the S-
isomer. While the structures of the products from 20R(OH)D3
remain to be elucidated, HPLC retention times are consistent
with formation of di- and trihydroxy derivatives analogous to
those produced from the 20S-isomer.16
Biological Evaluations. Previously we documented that
20S(OH)D3 and related 20S(OH)D2 inhibit proliferation and
stimulate differentiation of human epidermal keratinocytes
(HEKn) in a dose dependent manner similar to 1,25(OH)2D3
CYP27B1 catalyzes the 1α-hydroxylation of a range of
vitamin D derivatives including 20(OH)D3 and 20(OH)-
D2.15,17 In the case of 20S(OH)D3, the 1α,20S-dihydrox-
yvitamin D3 product exhibits calcemic activity which is in
contrast to the parent 20S(OH)D3 which lacks this activity.8a
We therefore compared the ability of CYP27B1 to metabolize
20S(OH)D3 and 20R(OH)D3 (Figure 5B). Each isomer was
metabolized to a single product at comparable initial rates, but
subsequently the rate declined more rapidly for the R-isomer
1
Figure 3. Comparison of H NMR chemical shifts for 21-Me in 20R
and 20S(OH)D3. 21-Me showed a chemical shift at 1.13 ppm in
20R(OH)D3, while the downfield chemical shift of 1.24 ppm was
observed for 21-Me in 20S(OH)D3.
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Waters Xevo G2 QTOF LCMS using ESI. The purity of the final
compounds was analyzed by an Agilent 1100 HPLC system (Santa
Clara, CA). Purities of the compounds were established by careful
integration of areas for all peaks detected and ≥95%.
(1S,Z)-3-((E)-2-((7αS)-1-((R)-2-Hydroxy-6-methylheptan-2-yl)-
7α-methylhexahydro-1H-inden-4(2H)-ylidene)ethylidene)-4-
methylenecyclohexanol (20R(OH)D3, 12). A methanol solution of
20R(OH)-7DHC (10) (5 mg, 2 mg/mL) was subjected to UVB
irradiation for 20 min in a quartz tube at 65 °C, using a Rayonet RPR-
100 photochemical reactor (Branford, CT). The mixture was
incubated at room temperature for 1 week to allow the conversion
from pre-20R(OH)D3 (11) to 20R(OH)D3 (12). The mixture was
analyzed using an Agilent 1100 HPLC system (Santa Clara, CA) to
confirm the production of 20R(OH)D3 and to optimize the
conditions for the separation using a semipreparative HPLC system.
The reaction mixture (5 μL) of irradiated 20R(OH)-7DHC was
injected by an autosampler onto a 5 μm Phenomenex Luna-PFP
column (250 mm × 4.6 mm) (Torrance, CA) with mobile phase of
85% methanol−water at a flow rate of 1.0 mL/min. The separation of
the mixture was conducted using a Gilson semipreparative HPLC
system with a 5 μm Phenomenex Luna-PFP semipreparative column
(250 mm × 10 mm) and a mobile phase as 85% methanol−water at a
flow rate of 6.0 mL/min. Fractions were collected based on a presetup
UV threshold and were reanalyzed by RP-HPLC. Fractions containing
above 95% of pure 12 (for 240 and 265 nm spectra) were pooled,
freeze-dried, and characterized by NMR and MS. 1H NMR (500 MHz,
MeOH-d4): δ 6.22 (d, 1 H, J = 10.0 Hz), 6.02 (d, 1 H, J = 10.0 Hz),
5.04 (s, 1 H), 4.74 (s, 1 H), 3.78−3.74 (m, 1 H), 2.87−2.84 (m, 1 H),
2.54−2.52 (m, 1 H), 2.42−2.39 (m, 1 H), 2.21−2.09 (m, 2 H), 2.01−
1.96 (m, 1 H), 1.74−1.14 (m, 26 H), 1.13 (s, 3 H), 0.91 (d, 6 H, J =
5.0 Hz), 0.71 (s, 3 H). MS (ESI) m/z 423.4 [M + Na]+. HPLC purity
100%. HRMS calculated for C27H45O2 [M + H]+ 401.3420. Found
401.3423.
Metabolic Studies of 20(OH)D3. To test enzymatic metabolism,
the 20S(OH)D3 and 20R(OH)D3 substrates were incorporated into
phospholipid vesicles prepared from dioleoylphosphatidylcholine and
cardiolipin as before,19 with the ratio of substrate to phospholipid
being 0.025 mol/mol phospholipid. Vesicles were incubated with 2
μM bovine CYP11A1 or 0.06 μM human CYP27B1 at 37 °C for up to
10 min. Products and remaining substrate were extracted with
dichloromethane and measured by reverse phase HPLC as described
before.16,17b
Inhibition of Proliferation by Different Epimers of 20(OH)D3
in Comparison to Calcitriol. Neonatal human epidermal
keratinocytes (HEKn) were isolated from neonatal foreskin of
African-American donors and grown in HKM (Lonza) medium
supplemented with HKGF (Lonza) as described previously.8b,c For the
cell proliferation assay the cells from a third passage were seeded into
24-well plates (TPP, Switzerland) and grown to ∼80% confluence.
Secosteroids were dissolved in ethanol and then diluted in keratinocyte
medium containing 0.1% BSA (Sigma). Cells were incubated for 24 h.
Then 1 μCi/ml [3H]thymidine (Moravek Biochemicals Inc., Brea,
CA) was added and cells were incubated for a further 4 h. Excess of
unbound thymidine was removed by washing cells with PBS. Cells
were precipitated with 10% trichloroacetic acid (TCA) (Sigma) and
then the precipitate was dissolved with 1 N NaOH. The solution was
collected in vials, and thymidine incorporation was determined using a
liquid scintillation counter (Beckman LS 6000, Santa Clara, CA).
Figure 5. Metabolism of 20(OH)D3 isomers by CYP11A1 and
CYP27B1. Substrates were incorporated into phospholipid vesicle and
incubated with CY11A1 (A) or CYP27B1 (B) and then substrate and
products extracted with dichloromethane and analyzed by reverse
phase HPLC.
than the S-isomer. This phenomenon could suggest that the
20R(OH)D3 isomer could show less calcemic toxicity than
20S(OH)D3 because of its lower efficiency of 1α-hydrox-
ylation. The hypercalcemic effects of the 1α-hydroxy
metabolites will be examined in future studies.
CONCLUSION
■
We chemically synthesized the R epimer of 20(OH)D3 based
on the preferred conformation of the reactant and the
associated strong steric preference for the formation of this
isomer. NMR characterization of the chemically synthesized
compound and comparisons with 20S(OH)-7DHC and
20S(OH)D3 confirmed the R chirality at the C20 position.
Biological studies demonstrated the antiproliferative activity of
R-epimer on keratinocytes, similar to that of the S-epimer and
1,25(OH)2D3 at 10 and 100 nM, with a noted difference
(opposite effect) at lower concentrations. This overlapping but
different behavior was further demonstrated by the ability of
P450scc and CYP27B1 to metabolize the R and S epimers but
with the different efficiencies. 20R(OH)D3 was a better
substrate for P450scc than 20S(OH)D3, while 20S(OH)D3
was a better substrate for CYP27B1 than for 20R(OH)D3. The
latter could explain the higher potency of 20S(OH)D3 in the
inhibition of HEKn cell proliferation. Alternatively, P450scc,
which is present in keratinocytes,18 may cause a more rapid
inactivation of the R-epimer than the S-epimer; however it
remains to be established if the products of P450scc action on
20R(OH)D3 are more or less potent than the parent
compound. In depth investigations of the biological activity
of 20R/S(OH)D3 epimers for anti-inflammatory and hyper-
calcemic effects, the nature of signal transduction pathways
induced by these compounds, and the potential role of 1-
hydroxylation in these processes will be carried out in the
future.
ASSOCIATED CONTENT
■
S
* Supporting Information
EXPERIMENTAL PROCEDURES
Preparations and spectra of intermediates. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
All reagents for the synthesis were purchased from commercial sources
and were used without further purification. Moisture-sensitive
reactions were carried out under an argon atmosphere. NMR spectra
were obtained on a Bruker ARX 300 MHz (Billerica, MA) or an
Agilent Inova 500 MHz spectrometer (Santa Clara, CA). Mass spectral
data were collected on a Bruker ESQUIRE-LC/MS system equipped
with an ESI source. High resolution mass spectra were recorded on a
AUTHOR INFORMATION
■
Corresponding Author
*For W.L.: phone, (901) 448-7532; e-mail, wli@uthsc.edu. For
D.D.M.: phone, (901) 448-6026; e-mail, dmiller@uthsc.edu.
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Journal of Medicinal Chemistry
Brief Article
Nguyen, M. N.; Pfeffer, L. M.; Slominski, A. T. 20-Hydroxychole-
calciferol, product of vitamin D3 hydroxylation by P450scc, decreases
NF-kappaB activity by increasing IkappaB alpha levels in human
keratinocytes. PloS One 2009, 4 (6), e5988.
(9) Tocchini-Valentini, G.; Rochel, N.; Wurtz, J. M.; Mitschler, A.;
Moras, D. Crystal structures of the vitamin D receptor complexed to
superagonist 20-epi ligands. Proc. Natl. Acad. Sci. U.S.A. 2001, 98 (10),
5491−5496.
(10) (a) Liu, Y. Y.; Collins, E. D.; Norman, A. W.; Peleg, S.
Differential interaction of 1alpha,25-dihydroxyvitamin D3 analogues
and their 20-epi homologues with the vitamin D receptor. J. Biol.
Chem. 1997, 272 (6), 3336−3345. (b) Yang, W.; Freedman, L. P. 20-
Epi analogues of 1,25-dihydroxyvitamin D3 are highly potent inducers
of DRIP coactivator complex binding to the vitamin D3 receptor. J.
Biol. Chem. 1999, 274 (24), 16838−16845.
(11) Hansen, K. B.; Claus Aage, S. Vitamin D Analogues Containing
a Hydroxy Group in the 20-Position. U.S. Patent US 5589471, 1996.
(12) (a) Mijares, A.; Cargill, D. I.; Glasel, J. A.; Lieberman, S. Studies
on the C-20 epimers of 20-hydroxycholesterol. J. Org. Chem. 1967, 32
(3), 810−812. (b) Nes, W. R.; Varkey, T. E. Conformational analysis
of the 17(20) bond of 20-keto steroids. J. Org. Chem. 1976, 41 (9),
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank College of Pharmacy of UTHSC; the Van
Vleet Endowed Professorship, UT Research Foundation,
University of Western Australia; and NIH/NIAMS Grants
R01AR052190 and 1R01AR056666-01A2 to A.T.S. for financial
support. We thank Jerrod Scarborough for performing HRMS
experiments.
ABBREVIATIONS USED
■
D3, vitamin D3; 7-DHC, 7-dehydrocholesterol; 1,25(OH)2D3,
1α,25-dihydroxyvitamin D3; VDR, vitamin D receptor;
P450scc, CYP11A1; HBTU, O-benzotriazole-N,N,N′,N′-tetra-
methyluronium hexafluorophosphate; TMSCl, trimethylsilyl
chloride; DIPEA, N,N-diisopropylethylamine; TBDMSCl, tert-
butyldimethylsilyl chloride; AIBN, 2-2′-azobisisobutyronitrile;
TBAF, tetrabutylammonium fluoride; TBDPSCl, tert-butyl-
chlorodiphenylsilane
(13) Djerassi, C.; Staunton, J. Optical Rotatory Dispersion Studies.
XLI. α-Haloketones (Part 9). Bromination of Optically Active cis-1-
Decalone. Demonstration of Conformational Mobility by Rotatory
Dispersion. J. Am. Chem. Soc. 1961, 83, 736−743.
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