Chemical synthesis of 20S-hydroxyvitamin D3, which shows antiproliferative activity

Article abstract of DOI:10.1016/j.steroids.2010.05.021

20S-hydroxyvitamin D3 (20S-(OH)D3), an in vitro product of vitamin D3 metabolism by the cytochrome P450scc, was recently isolated, identified and shown to possess antiproliferative activity without inducing hypercalcemia. The enzymatic production of 20S-(OH)D3 is tedious, expensive, and cannot meet the requirements for extensive chemical and biological studies. Here we report for the first time the chemical synthesis of 20S-(OH)D3 which exhibited biological properties characteristic of the P450scc-generated compound. Specifically, it was hydroxylated to 20,23-dihydroxyvitamin D3 and 17,20-dihydroxyvitamin D3 by P450scc and was converted to 1α,20-dihydroxyvitamin D3 by CYP27B1. It inhibited proliferation of human epidermal keratinocytes with lower potency than 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) in normal epidermal human keratinocytes, but with equal potency in immortalized HaCaT keratinocytes. It also stimulated VDR gene expression with similar potency to 1,25(OH)2D3, and stimulated involucrin (a marker of differentiation) and CYP24 gene expression, showing a lower potency for the latter gene than 1,25(OH)2D3. Testing performed with hamster melanoma cells demonstrated a dose-dependent inhibition of cell proliferation and colony forming capabilities similar or more pronounced than those of 1,25(OH)2D3. Thus, we have developed a chemical method for the synthesis of 20S-(OH)D3, which will allow the preparation of a series of 20S-(OH)D3 analogs to study structure-activity relationships to further optimize this class of compound for therapeutic use.

Full text of DOI:10.1016/j.steroids.2010.05.021

Steroids 75 (2010) 926–935
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Chemical synthesis of 20S-hydroxyvitamin D3, which shows
antiproliferative activity
Wei Lia,∗, Jianjun Chena, Zorica Janjetovicb, Tae-Kang Kimb, Trevor Sweatmanc, Yan Lua,
Jordan Zjawionyd, Robert C. Tuckeye, Duane Millera, Andrzej Slominskib
a Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Science Center, Memphis, TN 38163, USA
b Department of Pathology and Laboratory Medicine, Center for Cancer Research, University of Tennessee Health Science Center, Memphis, TN 38163, USA
c Department of Pharmacology, University of Tennessee Health Science Center, Memphis, TN 38163, USA
d Department of Pharmacognosy and Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi, University, MS 38677, USA
e School of Biomedical, Biomolecular and Chemical Sciences, University of Western Australia, Crawley, WA, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
20S-hydroxyvitamin D3 (20S-(OH)D3), an in vitro product of vitamin D3 metabolism by the cytochrome
P450scc, was recently isolated, identified and shown to possess antiproliferative activity without inducing
hypercalcemia. The enzymatic production of 20S-(OH)D3 is tedious, expensive, and cannot meet the
requirements for extensive chemical and biological studies. Here we report for the first time the chemical
synthesis of 20S-(OH)D3 which exhibited biological properties characteristic of the P450scc-generated
compound. Specifically, it was hydroxylated to 20,23-dihydroxyvitamin D3 and 17,20-dihydroxyvitamin
D3 by P450scc and was converted to 1␣,20-dihydroxyvitamin D3 by CYP27B1. It inhibited proliferation
of human epidermal keratinocytes with lower potency than 1␣,25-dihydroxyvitamin D3 (1,25(OH)2D3)
in normal epidermal human keratinocytes, but with equal potency in immortalized HaCaT keratinocytes.
It also stimulated VDR gene expression with similar potency to 1,25(OH)2D3, and stimulated involucrin
(a marker of differentiation) and CYP24 gene expression, showing a lower potency for the latter gene
than 1,25(OH)2D3. Testing performed with hamster melanoma cells demonstrated a dose-dependent
inhibition of cell proliferation and colony forming capabilities similar or more pronounced than those
of 1,25(OH)2D3. Thus, we have developed a chemical method for the synthesis of 20S-(OH)D3, which
will allow the preparation of a series of 20S-(OH)D3 analogs to study structure–activity relationships to
further optimize this class of compound for therapeutic use.
Received 31 March 2010
Received in revised form 13 May 2010
Accepted 24 May 2010
Available online 11 June 2010
Keywords:
20S-Hydroxyvitamin D3
Melanoma
Chemical synthesis
Antiproliferative activity
© 2010 Elsevier Inc. All rights reserved.
1. Introduction
centration in serum leads to calcium deposition in many tissues,
including crucial organs such as heart or kidney. The search for vita-
Besides its critical role in regulating bone mineralization and cal-
cium homeostasis [1], it is well documented that the active form
of vitamin D3 also regulates cell growth, differentiation, prolif-
eration, apoptosis, and immune responses through the activation
of the nuclear vitamin D receptor (VDR) [2–4]. However, clinical
applications of pharmacological doses of its active form, 1␣,25-
dihydroxyvitamin D3 (1,25(OH)2D3), are hampered by its toxic
side effect of hypercalcemia [5–7]. Excessive elevated calcium con-
min D3 analogs displaying minimal or no hypercalcemic side effects
has been intense in the past decades. As a result, several vitamin
D analogs that exhibit reduced hypercalcemia are entering clini-
cal trials and are showing great promise as potential therapeutic
agents for the treatment of cancer and other diseases [2,4,8].
Recent studies have revealed that a crucial enzyme in steroido-
genesis, mammalian cytochrome P450scc, not only cleaves the
side chain of cholesterol but also metabolizes 7-dehydrocholesterol
(7DHC) to 7-dehydropregnenolone (7DHP) in vitro, in isolated
mitochondria and in incubated ex vivo adrenal glands through
sequential hydroxylation of carbon atoms C-20 and C-22 and subse-
quent cleavage of the side chain [9–11]. Importantly, P450scc also
hydroxylates the side chain of vitamin D3 to 20-hydroxyvitamin
D3 (20S-(OH)D3), with subsequent transformation to di-, and
tri-hydroxy metabolites [9,12–15]. Similarly, 20-hydroxyvitamin
D2 is a major product of P450scc action on vitamin D2, and
1␣,20-dihydroxyvitamin D3 is formed from 1␣-hydroxyvitamin D3
Abbreviations:
D3, vitamin D3; 20S-(OH)D3, 20S-hydroxyvitamin D3;
1,25(OH)2D3, 1␣,25-dihydroxyvitamin D3; ESI-MS, electrospray ionization Mass
Spectroscopy.
∗
Corresponding author at: Department of Pharmaceutical Sciences, University
of Tennessee Health Science Center, 847 Monroe Avenue, Room 327, Memphis, TN
38163, USA, Tel.: +1 901 448 7532; fax: +1 901 448 6828.
E-mail address: wli@uthsc.edu (W. Li).
0039-128X/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2010.05.021

W. Li et al. / Steroids 75 (2010) 926–935
927
[12,16,17]. Thus, novel in vitro pathways have been identified that
can generate hydroxylated forms of vitamin D with 20-hydroxy
derivatives predominating.
rial was removed by filtration and the filtrate was concentrated
to yield a yellow-brown solid. To a solution of this yellow-brown
solid in tetrahydrofuran (50 ml) was added tetrabutylammonium
bromide (0.8 g, 2.5 mmol) and stirred for 75 min at room temper-
ature. To this reaction mixture was added tetrabutylammonium
fluoride (20 ml of 1.0 M solution in tetrahydrofuran, 20 mmol) and
the resulting solution was stirred for 50 min. Water was added
and the mixture was extracted by ethyl acetate. The organic layer
was dried and concentrated. The residual was subjected to flash
chromatography (eluted with hexane–ethyl acetate 2:1) to give
a white solid. Yield: 36%. 1H NMR (500 MHz, CDCl3): ı 5.60 (dd,
J = 12 Hz, 4.0 Hz, 1H), 5.44–5.56 (m, 1H), 4.73–4.75 (m, 1H), 2.66 (t,
J = 10 Hz, 1H), 2.53–2.55 (m, 1H), 2.38–2.41 (m, 1H), 2.23–2.26 (m,
1H), 2.17 (s, 3H), 2.13–2.16 (m, 1H), 2.06 (s, 3H), 1.72–1.96 (m, 8H),
1.52–1.62 (m, 3H), 1.40 (dt, J = 30 Hz, 5 Hz, 1H), 0.97 (s, 3H), 0.60 (s,
3H). ESI-MS: calculated for C23H32O3, 356.2, found 379.3 [M+Na]+.
Our recent studies have demonstrated that 20S-(OH)D3 acts as a
potent inducer of keratinocyte differentiation with strong antipro-
liferative effects and NF␬B inhibitory activity [18–20]. We have
also found that 20S-(OH)D3 has significant anti-cancer activities
in vitro, while being non-calcemic [20]. Collectively, these results
strongly indicate that 20S-(OH)D3 or its analogs can serve as a ther-
apeutic agent(s) with potentially lower toxicity in comparison to
many synthetic xenobiotics. To further assess its biological activ-
ity and efficacy in animal models larger quantities of 20S-(OH)D3
are required. The current enzymatic generation of 20S-(OH)D3 by
P450scc has substantial limitations with regard to scale-up, cost
and time needed for production of more than milligram quantities
of metabolites. The isolation and purification of metabolites is very
tedious and expensive. To provide larger quantities of 20S-(OH)D3
for in vivo biological studies and further chemical modifications,
we developed an efficient synthetic route for this compound. The
conformational analysis during the course of the nucleophilic addi-
tion of Grignard reagent to 7-dehydropregnenolone acetate and
analysis of NMR spectra indicated the formation of the 20S-epimer
of 20-(OH)D3 exclusively. Subsequent studies confirmed that 20S-
(OH)D3 can be enzymatically metabolized by P450scc and CYP27B1
to the expected dihydroxy products. These have potent antiprolif-
erative and pro-differentiation activities in cultured keratinocytes
and melanoma cells that are similar to those reported previously
for 20(OH)D3 generated through the action of P450scc [10,18].
2.4. Synthesis of compound 4
Compound 3 (1-bromo-4-methyl-pentane, 3.3 g, 20.0 mmol) in
dry THF (50 ml) was added dropwise to Mg (735 mg, 30.0 mmol,
1.5 equiv.) in an argon-purged flask and then stirred for 2 h at 45 ◦C.
The resulting solution was cooled to room temperature and used
for next step without further purification.
2.5. Synthesis of 20S-(OH)-7DHC
Compound 2 (712 mg, 2.0 mmol) was added to a solution of com-
pound 4 (excess, 20–30 equiv.) in dry THF at 0 ◦C under argon. The
solution was allowed to warm to room temperature and was stirred
overnight. The reaction mixture was quenched with aq. NH4Cl
(sat.), extracted with EtOAc, the organic layer was washed with
brine and water, dried by MgSO4 and concentrated. The crude mate-
rial was subject to column chromatography (hexane:ethyl acetate
10:1) to give a white solid. Yield: 75%. 1H NMR (500 MHz, CD3OD,
see Table 1 for full assignments): ␦ 5.54–5.57 (m, 1H), 5.39–5.42
(m, 1H), 3.49–3.58 (m, 1H), 2.39–2.45 (m, 1H), 2.24–2.30 (m, 1H),
2.17–2.24 (m, 1H), 1.29–2.00 (m, 18H), 1.27 (s, 3H), 1.14–1.23 (m,
3H), 0.96 (s, 3H), 0.91 (d, J = 6.5 Hz, 6H), 0.81 (s, 3H). ESI-MS: calcu-
lated for C27H44O3, 400.3, found 423.3 [M+Na]+.
2. Experimental
2.1. Generation, isolation and purification of biologically
generated 20(OH)D3
The enzymatic production of 20S-(OH)D3 has been reported
previously [12,13]. Briefly, cytochrome P450scc purified from
bovine adrenal glands was used to generate 20S-(OH)D3 from D3
followed by isolation with preparative thin layer chromatography
(TLC). The metabolite was further purified using a C18 column in
an RP-HPLC system equipped with a diode-array detector (Waters,
Milford, MA). Fractions with 20S-(OH)D3 of at least 99% purity were
combined, dried under nitrogen, and stored at −70 ◦C.
2.6. Synthesis of 20S-(OH)D3
2.2. Chemical synthesis
The UV conversion of 20S-(OH)-7DHC to 20S-(OH)D3 was
conducted using a UVB light source (4.8 0.2 mW cm−2) with a
maximum emission spectrum in the range of 280–320 nm, as
described previously [21]. Briefly, a solution of 20S-(OH)-7DHC
(10 mg, 1 mg/ml in methanol) was subjected to UV irradiation
for 5 min in a quartz cuvette, using a Biorad UV Transilluminator
2000 (Biorad, Hercules, CA). The reaction mixture was incubated
at room temperature (25 ◦C) for 3 days and the product was sepa-
rated by RP-HPLC chromatography using a Waters HPLC-system
equipped with a diode-array detector (Waters Associates, Mil-
ford, MA). The reaction mixture was injected by an autosampler
into an Atlantis C18 column (Waters, IL) with mobile phase as
70/30 methanol/water and a flow rate of 1.5 ml/min. Fractions were
collected every 15 s and were reanalyzed by RP-HPLC. Fraction
containing > 95% of pure compound was dried. The product was
identified based on the retention time, UV absorption spectra, MS
and NMR measurement (see Table 1 for full NMR assignments).
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.
Routine TLC was performed on aluminum backed Uniplates (Anal-
tech, Newark, DE). NMR spectra were obtained on a Bruker
ARX-300 MHz (Billerica, MA) or a Varian Inova-500 MHz spectrom-
eter (Varian NMR Inc., Palo Alto, CA). Chemical shifts are reported as
parts per million (ppm) relative to the residue signals in methanol-
d4 (3.31 ppm for proton and 49.15 ppm for carbon). Temperature
was regulated with a general accuracy of 0.1 ◦C. Mass spectral
data were collected on a Bruker ESQUIRE-LC/MS system equipped
with an ESI source. The scheme for the synthesis of 20S-(OH)D3 is
shown in Fig. 1A.
2.3. Synthesis of compound 2
To
a
solution of compound
1
(3.58 g, 10.0 mmol) in
2.7. Molecular modeling
benzene–hexane (200 ml, 1:1 in volume) was added dibro-
mantin (1.72 g, 6.0 mmol) and 2,2ꢀ-azobisisobutyronitrile (68 mg,
0.4 mmol). The mixture was refluxed for 20 min in a preheated oil
bath (100 ◦C) and then placed in an ice bath to cool. Insoluble mate-
Molecular modeling studies were performed using Schrodinger
Molecular Modeling Suite 2009 (Schrodinger Inc., New York,
NY). Molecules were constructed and systematic conformation

928
W. Li et al. / Steroids 75 (2010) 926–935
Fig. 1. Synthesis and characterization of 20(OH)D3. (A) Reagents and conditions: (a) dibromantin, 2,2ꢀ-azobisisobutyronitrile, benzene/hexane (1:1), 100 ◦C, reflux; (b)
Bu4NBr, Bu4NF, THF, room temperature; (c) Mg, THF, 45 ◦C; (d) THF, 0 ◦C – RT; (e) UVB irradiation for 5 min followed by 3 days in solution at room temperature. Only major
products from the UVB irradiation are shown. (B) Monitoring reactions by NMR from the starting material 20S-(OH)-7DHC to 20S-(OH)D3 after preparative HPLC purifications,
only fingerprint regions are shown. (C) HPLC trace of product mixture detected at 280 nm of UV absorption, peak 3 is the desired 20(OH)D3 product. (D) Extracted UV spectra
from the diode-array detector of each HPLC peak. 20(OH)D3 is separated with high purity from this preparative HPLC operation, and the purified product is further confirmed
by 1H NMR and mass spectrometry.
search was performed using Macromodel and OPLS-2005 forcefield
within the software. Lowest energy conformers for molecules were
selected and their geometries were further optimized with density
functional theory (DFT) using the B3LYP hybrid functional [22,23]
together with Pople’s 6-31G(d,p) basis [24,25] for final calculations
(655 basis functions).
and grown in keratinocyte basal medium (KBM) supplemented
with keratinocytes growth factors (KGF) (Lonza) on collagen coated
plates [19]. For experiments, cells in their third passage were
used.
Hamster AbC1 melanoma cells were cultured in F10 media
(Sigma–Aldrich, St. Louis, MO), while immortalized human epider-
mal keratinocytes (HaCaT) were cultured in Dulbecco’s Modified
Eagle’s Medium (DMEM) supplemented with 5% fetal bovine serum
(FBS) and 1% penicillin/streptomycin/amphotericin antibiotic solu-
tion (Sigma–Aldrich, St. Louis, MO) [12]. Prior to treatment with
vitamin D3 metabolites, cells were serum deprived for 24 h and
further incubated in F10 medium containing 5% charcoal-treated
FBS (ctFBS) (HyClone, Logan, UT). All cultures were performed at
37 ◦C in 5% CO2.
2.8. Metabolism of 20S-(OH)D3 by cytochrome P450scc and
CYP27B1
Incorporation of 20S-(OH)D3 into phospholipid vesicles and
measurement of its metabolism by P450scc was carried out
as described previously [15]. The incubation mixture comprised
510 ␮M phospholipid vesicles containing 20S-(OH)D3 at a ratio to
phospholipid of 0.1 mol/mol, 2 ␮M bovine cytochrome P450scc,
15 ␮M adrenodoxin, 0.4 ␮M adrenodoxin reductase, 2 mM glu-
cose 6-phosphate, 2 U/ml glucose 6-phosphate dehydrogenase
and 50 ␮M NADPH. Samples were pre-incubated for 8 min, reac-
tions started by the addition of NADPH and incubations carried
out at 37 ◦C with shaking for 6 min. After extraction with
dichloromethane samples were analyzed by HPLC as described
2.10. Cell proliferation assays
2.10.1. MTS test
HEKn keratinocytes were plated in 96-well plates, 10,000
cells/well. After overnight incubation of cells, 1,25(OH)2D3 or
chemically synthesized 20S-(OH)D3, initially dissolved in ethanol
and then diluted in KGM medium containing 0.5% BSA, was added
to the medium to achieve final concentrations of 0.01 or 1 ␮M,
while in control cultures, the final concentration of ethanol vehi-
cle was 0.1%. After 20 h of incubation with these compounds, 20 ␮l
of MTS/PMS solution (Promega, Madison, WI) were added to the
cells. Four hours later, absorbance was recorded at 490 nm using
an ELISA plate reader. The number of viable cells was measured in
six replicates.
before using
a
C18 column (Grace 15 cm × 4.6 mm, particle
size 7 ␮m) [15]. Metabolism of 20S-(OH)D3 by CYP27B1 (25-
hydroxyvitamin D3 1␣-hydroxylase) was measured by a similar
procedure as described before [26].
2.9. Cell culture
Normal human keratinocytes (HEKn) were isolated from neona-
tal foreskin of African American donors using 2.5 UI/ml dispase

W. Li et al. / Steroids 75 (2010) 926–935
929
Table 1
Full structural assignment of D3, 20S-(OH)D3 and its precursor, 20S-(OH)-7DHC.
Atom
20S-(OH)D3
20S-(OH)-7DHC
D3
1H
1H
13C
1H
13C
13C
1
2
3
4
5
6
7
8
2.11␣, 2.40␤
1.97␣, 1.53␤
3.76
2.53␣, 2.19␤
NA
6.21
6.02
NA
1.68␣, 2.85␤
NA
1.56␣, 1.66␤
1.37␣, 2.07␤
NA
2.00
1.51
1.69␣, 1.78␤
1.67
0.69
4.74, 5.04
NA
5.49
1.23
1.32, 1.46
1.33
1.16
1.55
0.89
32.2
35.2
69.2
45.6
136.3
121.2
118.0
141.0
28.4
145.6
23.1
40.9
45.6
56.4
21.7
21.7
58.5
12.8
111.3
74.5
NA
1.30␣, 1.89␤
1.88␣, 1.48␤
3.64
2.47␣, 2.27␤
NA
5.57
5.40
NA
1.96
38.5
32.0
70.5
40.8
141.4
119.6
116.7
140.4
46.1
37.1
21.1
39.6
43.3
54.7
22.4
22.6
57.9
13.7
16.4
75.4
NA
2.12␣, 2.40␤
1.98␣, 1.54␤
3.76
2.53␣, 2.19␤
NA
6.22
6.04
NA
1.69␣, 2.86␤
NA
1.55␣, 1.67␤
1.33␣, 2.03␤
NA
2.02
1.46
1.31␣, 1.91␤
1.30
0.56
33.8
36.8
70.7
47.2
137.5
122.8
119.1
142.7
30.1
147.1
24.7
42.1
47.1
57.7
23.4
28.9
58.1
12.5
112.8
37.6
NA
9
10
11
12
13
14
15
16
17
18
19
20
20-OH
21
22
23
24
25
26, 27
NA
1.61␣, 1.74␤
1.28␣, 2.16␤
NA
1.88
1.75␣, 1.25␤
1.45␣,1.75␤
1.58
0.79
0.94
4.75, 5.04
1.40
NA
NA
a
24.8
43.9
21.7
39.6
27.8
21.7
1.29
1.32, 1.44
1.28
1.15
1.54
26.6
43.9
22.0
39.8
28.0
22.7
0.95
19.6
37.5
25.1
40.8
29.3
23.2
1.04, 1.39
1.19, 1.39
1.16
1.54
0.89
0.87
NMR chemical shift assignments for 20S-(OH)-7DHC, 20S-(OH)D3, and D3 by analysis of their 2D NMR. (Solvent: CD3OD for D3 and 20S-(OH)D3; CDCl3 for 20S-(OH)-7DHC.)
CDCl3 was used for 20S-(OH)-7DHC in order to compare its proton chemical shifts with reported values for 20R- and 20S-(OH)-cholesterol and to assign the absolute
configuration at C20. The S-configuration at C20 is established based on the large downfield shift for 21-Me based on well-established literature. 1D and 2D spectra for
20S-(OH)-7DHC and 20S-(OH)D3 are shown in the supplementary materials. NA – not applicable (ternary carbons).
a
Chemical shift was not observable at this position.
2.10.2. DNA synthesis
2.10.3. Colony forming assay
Testing of DNA synthesis was carried out as described pre-
viously [12,21]. Cells were inoculated into 24-well plates at
5000–25,000 cells/well, depending on cell type. After overnight
incubation at 37 ◦C, the cultures were placed in serum free media
to synchronize cells at the G0/G1 phase of the cell cycle. After
24 h 20S-(OH)D3 was added with fresh media containing growth
supplements and incubated for additional 72 h. After a defined
period of time, [3H]-thymidine (specific activity 88.0 Ci/mmol;
Amersham Biosciences, Piscataway, NY, USA) was added to a
final concentration of 0.5 ␮Ci/ml in medium. After 4 h of incu-
bation at 37 ◦C, media were discarded, cells precipitated in
10% TCA in PBS (phosphate-buffered saline) for 30 min, washed
twice with 1 ml PBS and then incubated with 1 N NaOH/ 1%
SDS (250 ␮l/well) for 30 min at 37 ◦C. The extracts were col-
lected in scintillation vials and 5 ml of scintillation cocktail was
added to each. 3H-radioactivity incorporated into DNA was mea-
sured with a beta counter (Direct Beta-Counter Matrix 9600;
Packard).
The assay followed our standard methodology, as described pre-
viously [12,21]. Briefly, cells were plated in six-well plates at a
density of 192 cells/well in medium containing 5% ctFBS (charcoal-
treated fetal bovine serum), 1% antibiotic solution and 20S-(OH)D3
at graded concentrations or ethanol (vehicle control). Cells were
cultured at 37 ◦C for 7 days with media being changed every 3
days. At the end of incubation, the colonies were fixed with 4%
paraformaldehyde in PBS overnight at 4 ◦C, washed, stained with
5% crystal violet in PBS for 30 min, rinsed, and air-dried. The num-
ber and size of the colonies were measured using an ARTEK counter
880 (Dynex Technologies Inc., Chantilly, VA). Colony forming units
were calculated by dividing the number of colonies by the number
of cells plated and then multiplying by 100.
2.10.4. ApoTox-Glo triplex assay
Melanoma cells were plated in 96-well plates, 10,000 cells/well.
After overnight incubation of cells, 1,25(OH)2D3 or 20S-(OH)D3
was added to the medium as described above. After 24 h of

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W. Li et al. / Steroids 75 (2010) 926–935
Table 2
P450scc-generated metabolite that shows very promising biolog-
Primers used for RT-PCR DNA amplification.
ical activities. The isolated 20S-(OH)D3 was dried under a gentle
stream of nitrogen, and the structure was confirmed by analysis of
its NMR spectra (Fig. 1B) and mass spectrometry. 20S-(OH)D3 with
at least 97% purity was stored at −70 ◦C until further use.
Gene
Primer sequence (left and right)
Cyclophilin B1
L 5ꢀ-TGTGGTGTTTGGCAAAGTTC-3ꢀ
R 5ꢀ-GTTTATCCCGGCTGTCTGTC-3ꢀ
L 5ꢀ-CTTACCTGCCCCCTGCTC-3ꢀ
VDR
Interestingly, while in theory both 20R-(OH)-7DHC and 20S-
(OH)-7DHC should be produced from the Grignard reaction (after
step d in Fig. 1A), only one epimer of 20-(OH)-7DHC and hence only
the corresponding single epimer of 20-(OH)D3 was obtained. This
strongly suggested that even though the acetyl side chain of com-
pound 2 is not very bulky, the attack of Grignard agent is highly
stereo-selective at the side chain. The same course of the reaction
was observed in the synthesis of 20S-hydroxycholesterol and other
20-hydroxysteroids [28–30].
R 5ꢀ-AGGGTCAGGCAGGGAAGT-3ꢀ
L 5ꢀ-TGCCTCAGCCTTACTGTGAGT-3ꢀ
R 5ꢀ-TCATTTGCTCCTGATGGGTA-3ꢀ
L 5ꢀ-CATCATGGCCATCAAAACAAT-3ꢀ
R 5ꢀ-GCAGCTCGACTGGAGTGAC-3ꢀ
Involucrin
CYP24
incubation with these compounds, 20 ␮l of Viability/Cytotoxicity
Reagent containing both GF-AFC Substrate and bis-AAF-R110 Sub-
strate (Promega, Madison, WI) were added to the cells. After
30 min of incubation at 37 ◦C, fluorescence was recorded at
400 nm excitation/505 nm emission for viability and 485 nm exci-
tation/520 nm emission for cytotoxicity using a microplate plate
reader for fluorescence and luminescence (Turner). One hundred
␮l of Caspase-Glo 3/7 Reagent (Promega) was further added to the
cells, and after 30 min of incubation at room temperature, lumi-
nescence was recorded. Numbers of viable, cytotoxic and apoptotic
cells were measured in four replicates.
Extensive efforts have been made to determine the absolute
configuration of the 20(OH)-7DHC epimer in order to unambigu-
ously assign the absolute configuration of the resultant 20(OH)D3.
However, both Mosher’s method (tertiary alcohol, failed to form
Mosher’s ester) and crystallization for X-ray crystallography were
not successful. NMR and HPLC analyses have clearly indicated that
both 20(OH)-7DHC and the resultant 20(OH)D3 are pure com-
pounds, not a mixture of epimers.
Since the goal of this work was to synthesize 20S-(OH)D3 that
would be identical with the biologically potent one produced by
P450scc, we compared the NMR spectra and HPLC retention times
of the biologically generated 20S-(OH)D3 and the synthetic product
(Fig. 2). To our satisfaction, both NMR spectra and HPLC retention
time in different solvent compositions were identical for the two
compounds, confirming that the chemically synthesized 20(OH)D3
isomer is the same as the enzymatically generated one and we
currently propose it to be the S-epimer, 20S-(OH)D3. Complete
1H and 13C assignment of 20S-(OH)D3 is shown in Table 1. The
S-configuration is confirmed based on well-established literature
[28,29,31,32]. First, condensation of pregnenolone acetate and the
Grignard reagent (isohexylmagnesium bromide) resulted exclu-
sively in 20S-OH-cholesterol [28,31], very similar to the current
study (Fig. 1A, the only difference being the presence of an extra
double bond in the B-ring). Second and most importantly, it is
well established that in the pregnane series, the 1H NMR chem-
ical shift of 21-Me in 20S-OH isomers was downfield relative to
20R-OH isomers [28]. They have distinct values and are the bases
used to assign the absolute configuration at C20 of these two
epimers [28–32]. The 1H NMR chemical shift for 21-Me in 20S-
OH-cholesterol is 1.17–1.28 ppm [30], and 20R-OH-cholesterol is
1.00–1.12 ppm, depending on NMR solvents [30]. The 1H NMR
chemical shift for 21-Me in synthesized 20-(OH)-7DHC is 1.29 ppm
which is almost identical to that in 20S-OH-cholesterol. Since the
side chain and the nearby rings have exactly the same structure, the
absolute configuration most likely is 20S-(OH)-7DHC. Subsequent
photolysis will not affect the side chain. Consistent with this analy-
sis, the 1H NMR chemical shifts for 21-Me are 1.23 ppm for both
enzymatically generated and chemically synthesized 20(OH)D3
(Table 1). Collectively, these results strongly suggest that our com-
pound is 20S-(OH)D3, confirming previous analysis [13]. We are
currently in the process of making different derivatives in order to
obtain satisfactory crystals to further confirm this result. For com-
parison, complete assignments for D3 and 20S-(OH)-7DHC are also
shown in this table.
2.11. Real-time RT PCR
The RNA from HEKn keratinocytes treated with 20S-(OH)D3 or
1,25(OH)2D3 was isolated using the Absolutely RNA Miniprep Kit
(Stratagen). Reverse transcription (1 ␮g RNA/reaction) was per-
formed using the Transcriptor First Strand cDNA Synthesis Kit
(Roche). Real-time PCR was performed using cDNA diluted 5-fold
in sterile water and a TaqMan PCR Master Mix. Reactions (in tripli-
cate) were performed at 95 ◦C for 5 min and then 45 cycles of 95 ◦C
for 10 s, 60 ◦C for 30 s and 72 ◦C for 30 s. The primers and probes
were designed with the universal probe library (Roche). Data were
collected on a Roche Light Cycler 480. The amount of amplified
product for each gene was compared to that for Cyclophilin B using
a comparative CT method. A list of the primers used for RT-PCR DNA
amplification is shown in Table 2.
2.12. Statistical analyses
Data are presented as mean SEM or SD as indicated in the
figure legends and were analyzed with a Student’s t-test (for two
groups) or ANOVA using Prism 4.00 (GraphPad, San Diego, CA).
3. Results
3.1. Synthesis of 20S-hydroxyvitamin D3 (20S-(OH)D3)
As shown in Fig. 1A, pregnenolone acetate 1 was bromi-
nated at the C-7 followed by dehydrobromination to generate
7-dehydropregnenolone acetate 2 [27]. Compound 2 was then
reacted with freshly prepared 4-methylpentylmagnesium bromide
4 generated from 4-methylpentyl bromide 3 to afford 20S-(OH)-
7DHC [28–30]. UVB irradiation of 20S-(OH)-7DHC followed by
3–4 days incubation at room temperature in methanol solution
produced a mixture of 20S-(OH)D3, 20S-(OH)-tachysterol, 20S-
(OH)-lumisterol, and other minor 20S-(OH)-steroids (Fig. 1A).
Reaction progress was readily monitored by 1H NMR based on
characteristic peaks of each product (Fig. 1B). The reaction mix-
ture was subsequently separated by preparative HPLC (Fig. 1C) and
each product was identified by its distinct UV absorption profile
(Fig. 1D) using a diode-array detector (Waters Corporation, Milford,
MA). Although we could isolate different 20S-hydroxy products, at
this stage we focused our attention on 20S-(OH)D3, since this is the
To understand the possible reason why only 20S-(OH)-7DHC
was formed synthetically, we performed quantum mechanical cal-
culations employing density functional theory (DFT) using the
Jaguar module (version 7.6) in Schrodinger Molecular Modeling
Suite 2009. The geometrically optimized structure for the precur-
sor to 20(OH)-7DHC (compound 2 in Fig. 1) is shown in Fig. 3. The
calculation results clearly indicate that there is a preference for the
orientation of the acetyl group for compound 2. It is conceivable

W. Li et al. / Steroids 75 (2010) 926–935
931
Fig. 2. The chemically synthesized compound (20S(OH)D3-chem) is identical to the enzymatically generated metabolite (20S(OH)D3-bio), confirmed by both NMR (A) and
HPLC (B). The S-configuration is established based on the chemical shift of 21-Me for similar structures and the stereospecificity of enzymatic reactions. “s” denotes residue
methanol solvent peaks, and the peak at 4.81 ppm (HDO) was suppressed with pre-saturation.
that this conformational preference dictated the outcome of the
bulky side chain addition to form 20-(OH)-7DHC (Fig. 1A). As shown
in Fig. 3, the attack of the nucleophilic Grignard reagent takes place
predominantly from the less hindered “back” side of the carbonyl
group to form 20S-(OH)-7DHC, while the attack from the “front”
side to form 20R-(OH)-7DHC is sterically prohibited.
Theoretically detailed quantum mechanics calculations of var-
ious transition states from compound 2 to the two epimers
of 20-(OH)-7DHC may be more suitable to explain the prod-
uct selectivity, but such calculations, especially involving the
metal containing Grignard reagents, are beyond the scope of
the present study. While such calculations for the transition
state energies are difficult and may not be reliable, compar-
ing energies of the starting compound 2 and the products may
provide some insight into the product selectivity. At the DFT/6-
31G** level and in aqueous solution, the energy for the preferred
orientation (Fig. 3, energy = −1211.32927 hartree) of compound
2, which will result in the preferred formation of 20S-(OH)-
7DHC, is 3.44 kJ/mol lower than the alternative conformation
(energy = −1121.32796 hartree) that exposes the other face of
the carbonyl group to attack by the Grignard reagent. Hence,
the preferred conformation dominates compound 2. In addi-
tion, the product 20S-(OH)-7DHC (energy = −1205.86798 hartree)
is slightly more stable (ꢀE = 0.97 kJ/mol) than 20R-OH-7DHC
(energy = −1205.86761 hartree). Thermodynamically, the product
formation of 20S-(OH)-7DHC is also expected to be favored. Thus

932
W. Li et al. / Steroids 75 (2010) 926–935
Fig. 3. Preferred conformation of the precursor (compound 2) to 20(OH)-7DHC calculated with density function theory with 6-31G** baseset. “Front” side attack by the bulky
Grignard agent to form 20R isomer is prohibited due to the steric hindrance from the steroid core, while the “back” side attack to form 20S isomer is strongly favored.
these theoretical calculations support our designation of the prod-
uct as the S-epimer.
3.2. Chemically synthesized 20-S(OH)D3 is metabolized
byP450scc and CYP27B1
To test enzymatic metabolism, chemically synthesized 20S-
(OH)D3 was incorporated into phospholipid vesicles prepared by
sonication and incubated with bovine P450scc or mouse CYP27B1,
plus adrenodoxin and adrenodoxin reductase. The 20S-(OH)D3 was
hydroxylated by P450scc identically to the enzymatically gener-
ated compound [15] with the expected 20,23-dihydroxyvitamin
D3 and 17,20-dihydroxyvitamin D3 appearing as products (Fig. 4A).
The chemically synthesized 20S-(OH)D3 was 1␣-hydroxylated by
CYP27B1 with greater than 90% of it being converted to 1␣,20-
dihydroxyvitamin D3 during the incubation (Fig. 4B), in agreement
with the reported in vitro metabolism of enzymatically synthesized
compound [26].
3.3. Chemically synthesized 20S-(OH)D3 is biologically active
The biological activity of 20S-(OH)D3, measured in human nor-
mal epidermal keratinocytes, immortalized HaCaT keratinocytes
and hamster AbC1 melanoma cells, was similar to that reported
for the P450scc-generated compound [18,19].
First, we tested the effect on keratinocytes proliferation and
found that treatment of normal human epidermal keratinocytes
with 20S-(OH)D3 and 1,25(OH)2D3 at concentrations of 10−8 and
10−6 M for 24 h led to the inhibition of cell proliferation when
compared to control (vehicle treated) cells (Fig. 5). Similarly, 20S-
(OH)D3 inhibited proliferation (as measured by 3H-thymidine
incorporation into DNA) of HaCaT immortalized epidermal ker-
atinocytes in a dose-dependent manner (Fig. 6). The inhibitory
effect was similar to that exerted by 1,25(OH)2D3, being seen at
a concentration as low as 10−11 M (Fig. 6). Interestingly, the pre-
cursor compound, 20S-(OH)-7DHC, also inhibited DNA synthesis,
albeit at higher concentrations (≥10−10 M).
Next we tested the effect of 20S-(OH)D3 on the vitamin D recep-
tor (VDR), involucrin and CYP24 gene expression and found that
20S-(OH)D3 stimulated expression of VDR at comparable levels to
1,25(OH)2D3 (Fig. 7A). However, it was less potent in the stim-
ulation of the expression of involucrin (2- vs 5-fold) and CYP24
(60- vs 10,000-fold) in comparison to 1,25(OH)2D3 (Fig. 7B and
7C). The more than 100 times difference between 1,25(OH)2D3
Fig. 4. Chromatograms showing metabolism of chemically synthesized 20S-
(OH)D3 by P450scc and CYP27B1. 20S-(OH)D3 in phospholipid vesicles was
incubated with P450scc for 6 min at 37 ◦C (Fig. 4A) or with CYP27B1 for 1 h at
25 ◦C (Fig. 4B) and products analyzed by reverse phase HPLC. Products 20,23-
dihydroxyvitamin D3 (20,23(OH)2D3), 17,20-dihydroxyvitamin D3 (17,20(OH)2D3)
and 1␣,20-dihydroxyvitamin D3 (1,20(OH)2D3) were identified from their identi-
cal retention times to authentic standards. Control chromatograms (not shown)
confirmed that no products were present in the absence of enzyme.

W. Li et al. / Steroids 75 (2010) 926–935
933
is connected with slowed cell cycling as described previously for
biochemically generated 20S-(OH)D3 [18,20].
4. Discussion
The ability to chemically synthesize highly active and non-
hypercalcemic 20S-(OH)D3 is very important, because it enables
large scale production of this compound as well as serving
as the basis for further structural modification and extensive
structure–activity relationship studies for this novel class of vita-
min D derivatives. Interestingly, the synthetic method shown in
Fig. 1 stereo-specifically produces only 20S-(OH)D3, instead of
both epimers. This is most likely due to the preferred conforma-
tion of the reactant and the associated strong steric preference for
the formation of this isomer. While we have not been successful
in obtaining good crystals for X-ray crystallography determina-
tion, detailed spectral comparison of the chemically synthesized
compound with the enzymatically generated metabolite revealed
identical structures. The stereospecificity of enzymatic metabolism
by P450scc at C20 and the NMR chemical shifts for 21-Me confirms
the 20S-configuration in the chemically synthesized compound.
As expected, the chemically synthesized 20S-(OH)D3 under-
goes enzymatic reactions similar to that of biochemically generated
metabolite and is biologically active. Its activity is similar to that
of 1,25(OH)2D3 in immortalized keratinocytes and melanoma, but
has lower potency in primary cultures of normal keratinocytes.
The differences in activity against immortalized and normal ker-
atinocytes may suggest a certain degree of selectivity against
immortalized or malignant cell lines. In addition, 20S-(OH)D3
has lower potency compared to 1,25(OH)2D3 in activating CYP24
which catalyses the major bio-deactivation pathway for vitamin
D analogs [18–20]. This may provide an advantage in using 20S-
(OH)D3 analogs as therapeutic agents because their deactivation
would be slow, which could effectively increase their lifetime dur-
ing circulation for potential reduced dose intervals.
Fig. 5. 20S-(OH)D3 inhibits growth of normal epidermal keratinocytes. Ker-
atinocytes were treated with 10−8 M (black bar) or 10−6 M (open bar) of chemically
synthesized 20S-(OH)D3 or 1␣,25(OH)2D3 (1,25(OH)2D3). Corresponding concen-
trations of 0.001 and 0.1% ethanol were used as negative (vehicle) controls. Relative
number of MTS positive cells is presented as a change in absorbance at 490 nm.
The experiment was repeated twice. Data are presented as mean SD (*p < 0.05,
**p < 0.01, ***p < 0.001).
and 20S-(OH)D3 on the induction of CYP 24 expression is similar to
that reported previously for biochemically synthesized 20S-(OH)D3
[18–20], and suggests that 20S-(OH)D3 will have only a minor
influence on the inactivation (24 hydroxylation) of active forms of
vitamin D3.
We also tested the effect of chemically synthesized 20S-(OH)D3
on cultured hamster AbC1 melanoma cells. As shown in Fig. 8,
20S-(OH)D3 inhibited cell proliferation in a dose-dependent man-
ner with an IC50 value of 1.2 × 10−10 M. Importantly, 20S-(OH)D3
also inhibited colony formation being more potent than its precur-
sor 20-(OH)-7DHC (Fig. 8) in inhibition of all (>0.2 mm) or larger
(>1 mm) colonies. Interestingly, 1,25(OH)2D3 had either minimal
or no effect on colony formation (data not shown). Moreover, test-
ing using ApoTox-Glo Triplex Assay showed lack of measureable
pro-apoptotic or cytotoxic effects of the vitamin D hydroxyl deriva-
tives against melanoma cells in comparison to ethanol controls
(data not shown). This indicates that the antiproliferative effect
In conclusion, we have chemically synthesized 20S-(OH)D3 that
has biological activity similar to that of 20S-(OH)D3 generated
Fig. 6. 20S-(OH)D3 inhibits DNA synthesis in HaCaT keratinocytes. HaCaT cells were incubated with drugs for 72 h. [3H]-thymidine was added for last 4 h of incubation. DNA
synthesis was measured by counting the radioactivity incorporated into TCA precipitable material. The experiment was repeated twice. Data are presented as means SEM.
The dose-dependent inhibition was analyzed by one-way ANOVA with #p < 0.05 and ##p < 0.01. The differences between control and treatments were also analyzed with
Student’s t-test where *p < 0.05; **p < 0.01; ***p < 0.001.
Fig. 7. 20S-(OH)D3 stimulates expression of VDR (A), involucrin (B), and CYP24 (C) genes in cultured human epidermal keratinocytes. Keratinocytes were treated with
10−7 M of chemically synthesized 20S-(OH)D3, 1␣,25(OH)2D3, or 0.01% ethanol (vehicle control) for 24 h. mRNA was isolated and subjected to RTPCR. Data are presented as
mean SD from 3 assays (*p < 0.05, **p < 0.01, ***p < 0.001).

934
W. Li et al. / Steroids 75 (2010) 926–935
Fig. 8. 20S-(OH)D3 inhibits DNA synthesis and colony formation in hamster AbC1 melanoma cells. For colony forming assays (left panel), 4 independent plates per each
concentration were incubated with drugs for 7 days and colonies stained with crystal violet. The number of colony units (CFU) formed was counted for all colonies (>0.2 mm)
and large colonies (>1 mm). To measure DNA synthesis (right panel), AbC1 cells plated into 4 independent cultures were incubated with drugs for 72 h, and [3H]-thymidine
was added for the last 4 h of incubation. DNA synthesis was measured by counting the radioactivity incorporated into TCA precipitable material. Data are presented as
means SEM for DNA synthesis and the colony forming assay (n = 4). *p < 0.05; **p < 0.01; ***p < 0.001 in Student’s t-test with the differences between control and treatments.
The dose-dependent inhibition was analyzed by one-way ANOVA with #p < 0.05 and ##p < 0.01. Lower panel shows representative plates with AbC1 colony formations that
were treated with vehicle (control) or test compound at a concentration of 10−9 M.
through the enzymatic action of P450scc on vitamin D3 [18–20].
Analytical procedures have confirmed that both compounds have
identical structures and stereochemistry. Synthesis of larger quan-
tities of material for further extensive in vivo biological studies
can now take place. Furthermore, the developed synthetic method
will provide a means for chemical modifications of 20S-(OH)D3
to generate the structure–activity relationship data necessary to
deliver the most promising therapeutic agents for treating hyper-
proliferative disorders.
References
[1] Holick MF. Vitamin D deficiency. N Engl J Med 2007;357(3):266–81.
[2] Pinette KV, Yee YK, Amegadzie BY, Nagpal S. Vitamin D receptor as a drug
discovery target. Mini Rev Med Chem 2003;3(3):193–204.
[3] Masuda S, Jones G. Promise of vitamin D analogues in the treatment of hyper-
proliferative conditions. Mol Cancer Ther 2006;5(4):797–808.
[4] Takahashi T, Morikawa K. Vitamin D receptor agonists: opportunities and chal-
lenges in drug discovery. Curr Top Med Chem 2006;6(12):1303–16.
[5] Beer TM, Myrthue A. Calcitriol in the treatment of prostate cancer. Anticancer
Res 2006;26(4A):2647–51.
[6] Eelen G, Gysemans C, Verlinden L, Vanoirbeek E, De Clercq P, Van Haver D, et
al. Mechanism and potential of the growth-inhibitory actions of vitamin D and
ana-logs. Curr Med Chem 2007;14(17):1893–910.
[7] Vieth R. Vitamin D and cancer mini-symposium: the risk of additional vitamin
D. Ann Epidemiol 2009;19(7):441–5.
Acknowledgements
[8] Schwartz GG. Vitamin D and intervention trials in prostate cancer: from theory
to therapy. Ann Epidemiol 2009;19(2):96–102.
[9] Guryev O, Carvalho RA, Usanov S, Gilep A, Estabrook RW. A pathway for the
metabolism of vitamin D3: unique hydroxylated metabolites formed dur-
ing catalysis with cytochrome P450scc (CYP11A1). Proc Natl Acad Sci USA
2003;100(25):14754–9.
[10] Slominski A, Zjawiony J, Wortsman J, Semak I, Stewart J, Pisarchik A, et
al. A novel pathway for sequential transformation of 7-dehydrocholesterol
and expression of the P450scc system in mammalian skin. Eur J Biochem
2004;271(21):4178–88.
The project described was supported by Grant Number
AR052190 from NIH/NIAMS to AS and 1R15CA125623 from
NIH/NCI to WL. Its contents are solely the responsibility of the
authors and do not necessarily represent the official views of the
NIH. We thank Edith Tang for performing the activity assay with
CYP27B1.
[11] Slominski AT, Zmijewski MA, Semak I, Sweatman T, Janjetovic Z, Li W, et
al. Sequential metabolism of 7-dehydrocholesterol to steroidal 5,7-dienes
in adrenal glands and its biological implication in the skin. PLoS One
2009;4(2):e4309.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.steroids.2010.05.021.

W. Li et al. / Steroids 75 (2010) 926–935
935
[12] Slominski A, Semak I, Wortsman J, Zjawiony J, Li W, Zbytek B, et al.
An alternative pathway of vitamin metabolism. Cytochrome P450scc
[21] Kim TK, Chen J, Li W, Zjawiony J, Miller D, Janjetovic Z, et al. A new steroidal 5,7-
diene derivative, 3beta-hydroxyandrosta-5,7-diene-17beta-carboxylic acid,
shows potent anti-proliferative activity. Steroids 2010;75(3):230–9.
[22] Becke AD. Density-functional thermochemistry. III. The role of exact exchange.
J Chem Phys 1993;98(7):5648–52.
[23] Lee C, Yang W, Parr RG. Development of the Colle-Salvetti correlation-energy
formula into a functional of the electron density. Phys Rev B 1988;37(2):785–9.
[24] Francl MM, Pietro WJ, Hehre WJ, Binkley JS, Gordon MS, DeFrees DJ, et al. Self-
consistent molecular orbital methods. XXIII. A polarization-type basis set for
second-row elements. J Chem Phys 1982;77(7):3654–65.
D
(CYP11A1)-mediated conversion to 20-hydroxyvitamin D2 and 17,20-
dihydroxyvitamin D2. FEBS J 2006;273(13):2891–901.
[13] Slominski A, Semak I, Zjawiony J, Wortsman J, Li W, Szczesniewski A, et al.
The cytochrome P450scc system opens an alternate pathway of vitamin D3
metabolism. FEBS J 2005;272(16):4080–90.
[14] Tuckey RC, Janjetovic Z, Li W, Nguyen MN, Zmijewski MA, Zjawiony JK, et
al. Metabolism of 1alfa-hydroxyvitamin D3 by cytochrome P450scc to bio-
logically active 1alfa,20-dihydroxyvitamin D3. J Steroid Biochem Mol Biol
2008;112:213–9.
[15] Tuckey RC, Li W, Zjawiony JK, Zmijewski MA, Nguyen MN, Sweatman T, et
al. Pathways and products for the metabolism of vitamin D3 by cytochrome
P450scc. FEBS J 2008;275(10):2585–96.
[25] Hariharan PC, Pople JA. Influence of polarization functions on MO hydrogena-
tion energies. Theor Chim Acta 1973;28(3):213–22.
[26] Tang EKY, Voo KJQ, Nguyen MN, Tuckey RC. Metabolism of substrates
incorporated into phospholipid vesicles by mouse 25-hydroxyvitamin
[16] Nguyen MN, Slominski A, Li W, Ng YR, Tuckey RC. Metabolism of vitamin D2
to 17,20,24-trihydroxyvitamin D2 by cytochrome P450scc (CYP11A1). Drug
Metab Dispos 2009;37:761–7.
[17] Tuckey RC, Nguyen MN, Slominski A. Kinetics of vitamin D3 metabolism by
cytochrome P450scc (CYP11A1) in phospholipid vesicles and cyclodextrin. Int
J Biochem Cell Biol 2008;40(11):2619–26.
D3 1alfa-hydroxylase (CYP27B1).
171–9.
[27] Guo LW, Wilson WK, Pang J, Shackleton CH. Chemical synthesis of
7- and 8-dehydro derivatives of pregnane-3,17alpha,20-triols, potential
steroid metabolites in Smith–Lemli–Opitz syndrome. Steroids 2003;68(1):
31–42.
J Steroid Biochem Mol Biol 2010;119:
[18] Zbytek B, Janjetovic Z, Tuckey RC, Zmijewski MA, Sweatman TW, Jones E,
[28] Mijares A, Cargill DI, Glasel JA, Lieberman S. Studies on the C-20 epimers of
20-hydroxycholesterol. J Org Chem 1967;32(3):810–2.
et al. 20-Hydroxyvitamin D3,
a product of vitamin D3 hydroxylation by
cytochrome P450scc, stimulates keratinocyte differentiation. J Invest Dermatol
2008;128(9):2271–80.
[29] Nes WR, Varkey TE. Conformational analysis of the 17(20) bond of 20-keto
steroids. J Org Chem 1976;41(9):1652–3.
[19] Janjetovic Z, Zmijewski MA, Tuckey RC, DeLeon DA, Nguyen MN, Pfeffer LM, et
al. 20-Hydroxycholecalciferol, 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.
[20] Slominski AT, Janjetovic Z, Fuller BE, Zmijewski MA, Tuckey RC, Nguyen MN, et
al. Products of vitamin D3 or 7-dehydrocholesterol metabolism by cytochrome
P450scc show anti-leukemia effects, having low or absent calcemic activity.
PLoS One 2010;5(3):e9907.
[30] Honda M, Komori T. Structures of thornasterols A and B (biologi-
cally active glycosides from asteroidia, XI). Tetrahedron Lett 1986;27(29):
3369–72.
[31] Burstein SH, Peron FG, Williamson E. Reactions of 20-hydroxylated steroids
with bovine adrenal tissue preparations. Steroids 1969;13(3):399–412.
[32] Corey EJ, Virgil SC, Sarshar S. New mechanistic and stereochemical insights
on the biosynthesis of sterols from 2,3-oxidosqualene.
1991;113(21):8171–2.
J Am Chem Soc