Photo-conversion of two epimers (20R and 20S) of pregna-5,7-diene-3β, 17α, 20-triol and their bioactivity in melanoma cells

Article abstract of DOI:10.1016/j.steroids.2008.10.017

Pregna-5,7-dienes and their hydroxylated derivatives can be formed in vivo when there is a deficiency in 7-dehydrocholesterol (7-DHC) Δ-reductase function, e.g., Smith-Lemli-Opitz syndrome (SLOS). Ultraviolet B (UVB) radiation induces photoconversion of 7-DHC to vitamin D₃, lumisterol₃ and tachysterol₃. Two epimers (20R and 20S) of pregna-5,7-diene-3β,17α,20-triol (4R and 4S, respectively) were synthesized and their UVB photo-conversion products identified as corresponding 9,10-secosteroids with vitamin D-like and tachysterol-like structures, and 5,7-dienes with inverted configuration at C-9 and C-10 (lumisterol-like). The number and character of the products and the dynamics of the process were dependent on the UVB dose. At high UVB doses, the formation of multiple oxidized derivatives of the primary products, and the formation of 5,7,9(11)-triene, were observed. The production of vitamin D-like, tachysterol-like and lumisterol-like derivatives was also observed in human skin treated with 4R and 4S, and subjected to UV irradiation, as shown by RP-HPLC. Newly synthesized compounds inhibited melanoma growth in dose dependent manner, and some of them showed equal or higher potency than 1,25(OH)₂D₃. In summary, we have characterized for the first time the products of UV induced conversion of pregna-5,7-diene-3β,17α,20-triols and documented that the newly synthesized compounds have antiproliferative properties against melanoma cells.

Full text of DOI:10.1016/j.steroids.2008.10.017

Steroids 74 (2009) 218–228
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Photo-conversion of two epimers (20R and 20S) of pregna-5,7-diene-3␤, 17␣,
20-triol and their bioactivity in melanoma cells
Michal A. Zmijewskia, Wei Lib, Jordan K. Zjawionyc, Trevor W. Sweatmand,
Jianjun Chenb, Duane D. Millerb, Andrzej T. Slominskia,∗
a Department of Pathology and Laboratory Medicine, Center for Cancer Research, University of Tennessee Health Science Center, Memphis, TN 38163, USA
b Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Science Center, Memphis, TN 38163, USA
c Department of Pharmacognosy and National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, School of Pharmacy,
University of Mississippi, MS 38677-1848, USA
d Department of Pharmacology and the Center for Anticancer Drug Research, University of Tennessee, Health Science Center, Memphis, TN 38163, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 July 2008
Received in revised form
22 September 2008
Accepted 29 October 2008
Available online 6 November 2008
Pregna-5,7-dienes and their hydroxylated derivatives can be formed in vivo when there is a deficiency in 7-
dehydrocholesterol (7-DHC) ꢀ-reductase function, e.g., Smith–Lemli–Opitz syndrome (SLOS). Ultraviolet
B (UVB) radiation induces photoconversion of 7-DHC to vitamin D3, lumisterol3 and tachysterol3. Two
epimers (20R and 20S) of pregna-5,7-diene-3␤,17␣,20-triol (4R and 4S, respectively) were synthesized and
their UVB photo-conversion products identified as corresponding 9,10-secosteroids with vitamin D-like
and tachysterol-like structures, and 5,7-dienes with inverted configuration at C-9 and C-10 (lumisterol-
like). The number and character of the products and the dynamics of the process were dependent on the
UVB dose. At high UVB doses, the formation of multiple oxidized derivatives of the primary products,
and the formation of 5,7,9(11)-triene, were observed. The production of vitamin D-like, tachysterol-like
and lumisterol-like derivatives was also observed in human skin treated with 4R and 4S, and subjected
to UV irradiation, as shown by RP-HPLC. Newly synthesized compounds inhibited melanoma growth
in dose dependent manner, and some of them showed equal or higher potency than 1,25(OH)2D3. In
summary, we have characterized for the first time the products of UV induced conversion of pregna-5,7-
diene-3␤,17␣,20-triols and documented that the newly synthesized compounds have antiproliferative
properties against melanoma cells.
Keywords:
Secosteroids
UV radiation
Lumisterol
Tachysterol
SLOS
Melanoma
© 2008 Elsevier Inc. All rights reserved.
1. Introduction
variables, including the ultraviolet B (UVB) dose (absorbed energy),
temperature, wavelength, the presence of biological membranes or
The UVB-driven isomerization of cholesta-5,7-dien-3␤-ol
(7-dehydrocholesterol, 7DHC) to (3␤,5Z,7E)-9,10-secocholesta-
5,7,10(19)-trien-3-ol (cholecalciferol, vitamin D3) is one of the
most fundamental reactions in the photobiology of the skin [1–3].
This conversion is initiated by photolysis of the unsaturated B
ring, with production of pre-D3 intermediate, and is followed
by its slow isomerization to three main products: vitamin D3,
tachysterol3 (6E-9,10-secocholesta-5(10),6,8-trien-3␤-ol, T3) and
lumisterol3 (9␤,10␣-cholesta-5,7-diene-3␤-ol, L3) (Scheme 1). The
relative ratio and the structure of products depend on a number of
when the reaction is carried out in the skin [3–5]. Higher doses of
UV or the presence of trace amounts of acid trigger further isomer-
ization of T3 to isotachysterol3 (isoT3) products, and is followed by
autoxidation [4,6]. Another pathway of degradation/isomerization
of vitamin D3 with production of 5,6-transvitamin D3, suprasterols I
and II in response to high doses of UVB was also demonstrated in the
skin [4]. It was shown that 7DHC in biological systems can also pro-
duce corresponding 5,7,9(11)-trienes. Although the detailed mech-
anism is not fully understood; it has been suggested that singlet
oxygen and photosensitizers may play a role in this reaction [7–11].
Accumulation of 5,7-dienes and 5,7,9(11)-trienes is impor-
tant in the etiology of a 7ꢀ-reductase deficiency syndrome
(Smith–Lemli–Opitz syndrome—SLOS) [9,10,12–15]. The lack of
the key enzyme converting 7DHC to cholesterol results in mul-
tiple metabolic defects with accumulation of 7DHC and its
5,7-diene steroidal derivatives, including cholesta-5,7,9(11)-trien-
3␤-ol (9DDHC) [10] and pregna-5,7-diene-3␤,17␣,20-triol [12–14].
∗
Corresponding author at: Department of Pathology and Laboratory Medicine,
Center for Cancer Research, University of Tennessee Health Science Center, 930
Madison Avenue, RM525, Memphis, TN 38163, USA. Tel.: +1 901 448 3741;
fax: +1 901 448 6979.
E-mail address: aslominski@utmem.edu (A.T. Slominski).
0039-128X/$ – see front matter © 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2008.10.017

M.A. Zmijewski et al. / Steroids 74 (2009) 218–228
219
Recently, it was proposed that oxidation of 7DHC and 9DDHC leads
to severe photosensitivity in SLOS patients [7,9].
(m, 1H), 2.59–2.63 (m, 1H), 2.49–2.54 (m, 1H), 2.36 (t, J = 15 Hz, 1H),
2.11 (s, 3H), 2.08 (s, 3H), 2.05 (s, 3H), 2.02–2.04 (m, 1H), 1.82–1.94
(m, 4H), 1.56–1.73 (m, 6H), 1.38 (dt, J = 15 Hz, 5 Hz, 1H), 0.95 (s, 3H),
0.57 (s, 3H). ESI-MS: calculated for C25H34O5, 414.24, found 437.3
[M+Na]+.
Vitamin D3 is not only a master regulator of calcium metabolism
but also a powerful oncostatic agent affecting proliferation, dif-
ferentiation and apoptosis of normal and malignant cells [2,16].
Although its potential therapeutic use against cancer or hyperpro-
liferative disorders is largely limited due to toxicity (hypercalcemia)
at pharmacological concentrations, this adversity can be reduced
or attenuated by shortening or removal of the side chain [17,18].
Thus, the generation of vitamin D-like compounds with pregnane
side chains may have a potential utility in treatment of cancers or
other pathological disorders.
Here were report on the UVB photolysis of two epimers (20R and
20S) of pregna-5,7-diene-3␤,17␣,20-triols synthesized from series
of 3-acetylated 5-diene precursors [13,19]. The dynamics of the
photolytic reactions and identification of novel 9,10-secosteroids
with D- T- and L-like structures, and corresponding 5,7,9(11)-
trienes. In addition, we studied the formation of secosteroidal
derivatives in human skin exposed to UVB and tested their bio-
logical activity on the human melanoma SKMEL-188 cell line.
2.4. General procedure for the synthesis of 4 (4R and 4S)
Compounds 4 (4R and 4S) were synthesized according to
a known procedure [13]. Reaction resulted in a crude, white,
solid mixture of compounds 4R and 4S: 40–50% yield. The mix-
ture was subjected to flash chromatography (column eluted with
hexane–ethyl acetate 20:1, 10:1, 5:1, 1:1 in order), but only the
isomer with a shorter retention time (Peak 1, 4R), was purified in
this manner. The separation of the second isomer (Peak 2, 4S) was
accomplished using RP-HPLC.
1H NMR (500 MHz, CD3OD) for compound 4S: ı 5.54 (dd,
J = 7.5 Hz, 3.2 Hz, 1H), 5.38–5.40 (m, 1H), 3.94 (q, J = 18 Hz, 1H),
3.47–3.55 (m, 1H), 2.57 (t, J = 10 Hz, 1H), 2.38–2.43 (m, 1H), 2.2–2.28
(m, 1H), 1.88–1.98 (m, 3H), 1.62–1.88 (m, 8H), 1.42–1.58 (m, 3H),
1.26–1.34 (m, 2H), 1.15 (d, J = 6 Hz, 3H), 0.96 (s, 3H), 0.77 (s, 3H). ESI-
MS: calculated for C21H32O3, 332.24, found 355.3 [M+Na]+; 4R: ı
5.54 (dd, J = 8.3 Hz, 2.5 Hz, 1H), 5.41–5.44 (m, 1H), 3.79 (q, J = 18 Hz,
1H), 3.47–3.56 (m, 1H), 2.60 (t, J = 10 Hz, 1H), 2.38–2.43 (m, 1H),
2.20–2.28 (m, 1H), 2.07–2.14 (m, 1H), 1.89–1.97 (m, 2H), 1.62–1.89
(m, 8H), 1.42–1.53 (m, 2H), 1.26–1.34 (m, 2H), 1.12 (d, J = 6 Hz, 3H),
0.95 (s, 3H), 0.7 (s, 3H). ESI-MS: calculated for C21H32O3, 332.24;
found 355.3 [M+Na]+.
2. Experimental
2.1. Chemical synthesis
The synthesis of compounds 4R and 4S is shown in Scheme 1.
2.2. Synthesis of 2
The acetylation of steroid 1 was carried out following a known
procedure [20]. Yield: 95%. 1H NMR (500 MHz, CDCl3) for com-
pound 2a: ı 5.39 (d, J = 5 Hz, 1H), 4.58–4.64 (m, 1H), 2.92–2.96
(m, 1H), 2.30–2.36 (m, 2H), 2.12 (s, 3H), 2.04 (s, 3H), 2.05 (s, 3H),
1.98–2.02 (m, 2H), 1.86–1.90 (m, 2H), 1.46–1.80 (m, 9H), 1.26–1.32
(m, 1H), 1.14–1.18 (m, 1H), 1.06–1.08 (m, 1H), 1.03 (s, 3H), 0.64
(s, 3H). ESI-MS: calculated for C25H36O5, 416.26; found: 439.3
[M+Na]+.
2.5. General procedure for UVB irradiation of 4R and 4S
The UVB irradiation was performed as described previously [21]
with some modifications. Briefly, a methanol solution of 4R or 4S
at 1 mg/mL was subjected to UV irradiation for various times in a
quartz cuvette (or glass HPLC insert) using Biorad UV Transillumi-
nator 2000 (Biorad, Hercules, CA). Spectral characteristics of the
UVB (280–320 nm) source were published previously [22] and its
strength (4.8 0.2 mW cm−2) was routinely measured with digital
UVB Meter Model 6.0 (Solartech Inc., Harrison Twp, MI). Irradiation
was followed by 14 h incubation at room temperature and selected
products were purified by RP-HPLC chromatography as described
[21]. The major products were identified on the basis of their reten-
tion time and characteristic UV absorption. Initial identification was
2.3. General procedure for the synthesis 3
Compound 3 was synthesized according to a known procedure
[13]. Yield: 40–50%. 1H NMR (500 MHz, CDCl3) for compound 3: ı
5.57–5.59 (m, 1H), 5.44–5.46 (m, 1H), 4.68–4.74 (m, 1H), 2.96–2.90
Scheme 1. Photolysis of cholesta-5,7-dien-3␤-ol.

220
M.A. Zmijewski et al. / Steroids 74 (2009) 218–228
Scheme 2. Synthesis of 4R and 4S (reagents and conditions: (a) Ac2O, microwave, p-toluenesulfonic acid monohydrate; (b) dibromantin, 2,2ꢀ-azobisisobutyronitrile, ben-
zene/hexane (1:1), 100 ◦C, reflux; (c) Bu4NBr, Bu4NF, THF, room temperature; (d) LiAlH4, THF, 0 ◦C).
Table 1
UV and MS spectra data date for pregna-5,7-diene-3␤, 17␣, 20-triols and its derivatives.
No.
Parental compound
Structure type
UV max (nm)
Predicted MW
MS+
4R
4R1
1
5,7-diene
preD-like
D-like
L-like
T-like
isoT-like (oxide)
5,7,9(11) triene
5,7-diene
preD-like
D-like
L-like
T-like
isoT-like
isoT-like (oxide)
5,7,9(11) triene
261, 270, 281, 290
260
265
262, 271, 281
270, 279, 291
240, 248, 257
312, 323, 340
263, 271, 282, 292
260
332.25
332.25
332.25
332.25
332.25
364.25
330.22
332.25
332.25
332.25
332.25
332.25
332.25
332.25
330.22
355.25 [M+Na]+
ND
4R
4R
4R
4R
4R
4R
1
4S
4S
4S
4S
4S
4S
4S
4R-D
4R-L
4R-T
4R-iT
4R7
355.25 [M+Na]+
355.25 [M+Na]+
355.25 [M+Na]+
387.15 [M+Na]+
ND
4S
4S1
355.25 [M+Na]+
ND
4S-D
4S-L
4S-T
4S-iT1
4S-iT2
4S7
265
355.25 [M+Na]+
355.25 [M+Na]+
355.25 [M+Na]+ND
355.25 [M+Na]+
387.15 [M+Na]+
ND
262, 271, 282
272, 282, 291
241, 248, 257
241, 248, 257
312, 323, 340
Bold: purified and characterized (NMR); italics: characterized by UV spectra; ND: not determined.
Table 2
1H NMR chemical shifts of pregna-5,7-diene-3␤,17␣,20-triols, D-like, L-like and 5,7,9(11)-triene.
4R
4S
4R-D
4S-D
4R-L
4S-L
5S
1 CH2
2 CH2
␣ 1.31
␤ 1.90
␣ 1.29
␤ 1.90
␣ 2.12
␤ 2.42
␣2.12
␤2.41
˛ 1.31
ˇ 1.90
˛ 1.31
ˇ 1.90
1.48
1.70
␣ 1.92
␤ 1.45
␣ 1.93
␤ 1.54
␣ 1.97
␤ 1.68
␣1.97
ˇ 1.68
˛ 1.92
ˇ 1.54
˛ 1.92
ˇ 1.54
1.89
1.68
3 CH
3.51
3.51
3.76
3.76
4.03
4.03
3.47
4 CH2
␣ 2.41
␤ 2.23
␣ 2.40
␤ 2.23
␣ 2.55
␤ 2.19
␣ 2.54
␤ 2.19
˛ 2.41
ˇ 2.29
˛ 2.44
ˇ 2.35
2.34
2.42
6 CH
7 CH
5.54
5.42
5.54
5.39
6.23
6.09
6.24
6.09
5.59
5.47
5.54
5.39
5.65
5.40
9 CH
2.11
2.02
2.85
1.54
2.85
1.56
2.02
2.02
11 CH2
12 CH2
␣ 1.67
␤ 1.75
␣ 1.6–1.8
␤ 1.6–1.8
␣ 1.77
␤ 1.77
˛ 1.77
ˇ 1.77
␣ 1.7–1.8
␤ 1.7–1.8
˛ 1.7–1.8
ˇ 1.7–1.8
5.61
␣ 1.49
␤ 1.80
˛ 1.49
ˇ 2.12
␣ 1.54
␤ 2.05
˛ 1.56
␤ 2.04
˛ 1.49
ˇ 2.18
˛ 1.49
ˇ 2.18
2.61
2.14
14 CH
2.62
2.57
2.12
2.12
2.05
2.05
2.78
15 CH2
␣ 1.83
␤ 1.63
˛ 1.84
ˇ 1.56
␣ 1.54
␤ 2.53
˛ 1.61
ˇ 1.61
˛ 1.82
ˇ 1.51
˛ 1.82
ˇ 1.51
1.85
1.50
16 CH2
␣ 1.56
␤ 1.74
␣ 1.7–1.8?
␤ 1.9?
␣ 1.87
␤ 2.7
˛ 1.7
␤ 2.66
˛ 1.76
ˇ 2.22
˛ 1.76
ˇ 2.22
1.60
1.75
18 CH3
19 CH3
0.7
0.77
0.95
0.6
0.67
0.76
0.67
0.76
0.75
0.67
1.24
0.94
␣ 4.76
␤ 5.05
␣ 4.74
␤ 5.05
20 C
21 CH3
3.78 CH
1.19
3.94 CH
1.15
3.76
1.18
3.61
1.14
3.72 CH
1.14
3.65 CH
1.15
3.9
1.16
Italics: not fully determined presumably similar to parental compound.

M.A. Zmijewski et al. / Steroids 74 (2009) 218–228
221
Scheme 3. Photolysis of pregna-5,7-diene-3␤,17␣,20-triols.
Fig. 1. UVB-driven photolysis of 4S (as an example). (A) RP-HPLC separation of 4S irradiation products after treatment with UVB for 15 min. (B) Representative UV spectra
of irradiated samples. Peak number (Panel A) corresponds to predicted structure based on UV spectra (Panel B). Peaks were assigned as follows—1–4, 10, 11: isoT-like and
oxidized isoT-like; 5: 5S; 6: 4S-L; 7: 4S-D; 8: 4S; 9: 4S-T; 12: 4S-pD. The UV spectra for RP-HPLC (Panel A) were measured at 280 nm. Representative UV spectra of irradiated
samples of 4R (c) and 4S (d). 1: 5c; 2: 5cD; 3: unknown product; 4: 5cT; 5: 5c1; 6: unknown product.

222
M.A. Zmijewski et al. / Steroids 74 (2009) 218–228
confirmed by means of MS and NMR measurements. The quanti-
ties of products varied and were predominantly dependent on the
UVB radiation dose. Fifteen minutes reaction resulted in 30–35% of
pre-D-like, 20% T-like, 10% of substrate and lower quantity of other
products.
2.7. Colony-forming assay
To measure colony formation and growth of melanoma
SKMEL-188, cells were plated at a colony-forming density (200
cells per well on six well plates). Cells were grown in F10
medium (GIBCO, Invitrogen Corp., Carlsbad, CA) supplemented
with charcoal-stripped fetal bovine serum (HyClone, Logan, UT)
and an antibiotic–antimycotic solution (Sigma, St. Louis, MO) at
37 ◦C in an atmosphere of 95% air and 5% CO2 [23]. Cells were
treated with compounds: 4R, 4R-pD, 4R-pL, 4S, 4S-pD, 4S-pL and
1,25-dihydroxyvitamin D3 as a positive control at different concen-
trations (0.1, 10 and 100 nM). Compounds were added to the media
from 1 mM ethanol stock solutions, and corresponding dilution of
methanol was used as negative (vehicle) control. Cells were grown
for 6–10 days and colonies were fixed with 4% paraformaldehyde in
PBS at 4 ◦C. The fixed cells were washed with distilled water, stained
with 0.1% crystalline blue for 30 min and rinsed with distilled water
to remove excess of the dye. Plates were photographed and the
number and the size of colonies were measured by ImageJ software
(NIH, Bethesda, MD). Colony-forming efficiency (CFU) was deter-
mined by dividing the number of colonies formed by the number
of cells plated, and multiplied by 100.
2.6. UVB irradiation of 4R and 4S incorporated into human skin
Human neonatal foreskin from African-American donors was
collected after routine circumcision, fat was removed, and the
skin was washed with DMEM medium and cut into approxi-
mately 5 mm × 5 mm pieces. Skin fragments (epidermis facing
up), were placed into 24 well plates containing 100 ␮L of DMEM
medium, leaving the epidermis above the surface of the medium.
The compounds 4R or 4S (2 ␮L of 10 mM solution) were dropped
on the epidermis and the samples were incubated for 1–2 h in
DMEM with dermis down (liquid phase) and epidermis exposed
to air a room temperature. Next, the samples were washed
two times for 5 min with PBS to remove not absorbed steroids.
Plates containing skin fragments were placed upside down on
Biorad UV Transilluminator 2000 (Biorad, Hercules, CA) and irra-
diated with 100, 500, 1000 and 2500 mJ/cm2. After irradiation
tissue was shredded and steroids were extracted with excess of
methylene chloride (twice). The samples were dried under nitro-
gen and analyzed by RP-HPLC. Untreated skin (irradiated and
non-irradiated), skin treated with 4R or 4S without irradiation
and compounds 4R or 4S irradiated in ethanol served as con-
trols.
2.8. Statistical analyses
Data were tested for distribution and homogeneity of vari-
ances, and for statistical significance with one-way analysis of
variance (Anova) with Tuckey post hoc test (for more than two
Fig. 2. Dose (time of irradiation) dependence of pregna-5,7-diene-3␤,17␣,20-triols (4R and 4S) photo-conversion. The compound 4R (Panels A, C, E and G) or 4S (Panels B,
D, F and H) was irradiated for 5 min (A and B), 15 min (C and D), 30 min (E and F) or 60 min (G and H) and products were separated by using RP-HPLC. The absorption profiles
of products were recorded by using diode-array detector. The samples were separated after incubation for 2 h at room temperature.

M.A. Zmijewski et al. / Steroids 74 (2009) 218–228
223
groups) using Prism 4.00 (GraphPad Software, San Diego, CA). Data
is presented as the mean S.E.M.; for n = 4–6. *P < 0.05, **P < 0.01
and ***P < 0.001.
ation (5 min; Fig. 2A, B) of both 4R and 4S resulted in the formation
of pre-D-like (ꢁmax at 260 nm; 4R-pD and 4S-pD), T-like (ꢁmax at
274, 281, 290 nm 4R-T and 4S-T) and L-like (262, 271, 281; 4R-L and
4S-L) products. The products 4R-pD and 4S-pD underwent sub-
sequent slow isomerization into vitamin D-like compounds (4R-D
and 4S-D) with maximum UV absorption at 265 nm. Longer UVB
irradiation (15 min; Fig. 2C and D) resulted in conversion of T-like
products to isoT-like compounds with characteristic UV absorption
for a conjugated double bond (ꢁmax at 248 nm). Irradiation for 30
and 60 min (Fig. 2E–H) resulted in further conversion into numer-
ous isoT-like products with a corresponding decrease in pre-D-like
and T-like products, indicating further metabolism (Scheme 2)
with the potential formation of suprasterols [4] with a ꢁmax below
250 nm.
Further identification was performed after purification by RP-
HPLC (see Materials and Methods for details) and the corresponding
fractions were analyzed by MS. As expected, all D-like, L-like and
T-like products had identical mass (m/z = 355.25 [M+Na]+) with
the parental compounds (Table 1). In addition to a molecular
ion at m/z = 355.25 [M+Na]+ the majority of isoT-like products
of irradiation had an additional ion at m/z = 387.15 (Fig. 3). This
indicates either the addition of O2 with formation of perox-
ide or hydroxyperoxide derivatives similar to those shown for
isoT3 (6E-9,10-secocholesta-5(10),6,8(14)-trien-3␤-ol) [6], or oxi-
dation of 4S and 4R without photolysis of the B ring, with
production of endoperoxide and hydroperoxide, as was shown
for 7-DHC [8,10]. The small scale of reaction and instability of
isoT-like compounds prevented their more complete characteriza-
tion.
2.9. Other procedures
All RP-HPLC analyses were performed using a Waters’ HPLC-
system equipped with a diode-array detector, auto-sampler and
fraction collector (Waters Associates, Milford, MA) as was described
elsewhere [21]. Samples were injected into an Atlantis C18
column running on 70/30 methanol/water at a flow rate of
1.5 mL/min. Collected fractions (15 s) containing above 95% of
pure compound (for 240 nm and 280 nm spectra) were pooled
together and used for further characterization. The procedure was
optimized to achieve the best separation of each selected prod-
uct.
Mass spectra were recorded using a Bruker Esquire-LC/MS Spec-
trometer equipped with an electrospray ionization (ESI) source, as
described previously [21].
NMR measurements were performed on a Varian Unity Inova-
500 MHz spectrometer (Variannmr Inc., Palo Alto, CA) using either
a 3 mm probe or a 5 mm probe. Deuterated methanol was used
instead of CDCl3 to avoid observed decomposition due to traces of
acid that are present in commercial chloroform.
3. Results
3.1. Synthesis of pregna-5,7-diene-3ˇ,17˛,20-triols
The synthesis was performed essentially as described previously
[21] (see Scheme 2).
The synthesis of pregna-5,7-diene-3␤,17␣,20-triols (4) was
carried out from 17␣-acetylated 5-en precursor (1) by acetyla-
tion, bromination–dehydrobromination and reduction, following
the known procedure [13]. Deprotection was performed simul-
taneously with reduction of the carbonyl group. The procedure
resulted in formation of two diastereomers: 4R and 4S (20R
and 20S) with a ratio of 50:50. The mixture was effectively
separated by reverse phase HPLC (RP-HPLC). The presence of 4,6-
dienes was not detected, as was reported during the synthesis
of 3␤-hydroxypregna-5,7-dien-20-one and androsta-5,7-diene-
3␤,17␤-diol [21].
Physico-chemical properties of pregna-5,7-diene-3␤,17␣,20-
triols are summarized in Table 1. The detailed NMR data are
presented in Table 2. NMR chemical shifts for 4R and 4S are in agree-
ment with those presented previously [13], with small differences
related to a solvent effect due to the use of CD3OD instead of CDCl3
for NMR experiments.
UVB irradiation of pregna-5,7-diene-3␤,17␣,20-triols (4) and
identification of products by combination of RP-HPLC, UV and mass
spectrometry.
The UV conversion of 4R and 4S were performed using a UVB
light source (4.8 0.2 mW cm−2) with maximum emission spec-
trum in a range of 290–320 nm [22] as described previously [21].
The photo-conversion and subsequent time dependent structural
rearrangements were monitored by an HPLC equipped with a diode
array that enabled fast detection of products by characteristic UV
spectra [24]. Similarly to cholesta-5,7-dien-3␤-ol (7DHC) [24], four
main products (Scheme 3) were formed in time-dependent fashion,
namely pro-D-like, T-like, L-like and D-like. All products were sep-
arated by HPLC and identified based on their unique UV absorption
spectra (Fig. 1).
The irradiation of 4R and 4S with increasing UV doses resulted
in the formation of diverse products, as shown in Fig. 2. Short irradi-
Fig. 3. Identity of oxidized derivatives of 4R (A) and 4S by mass spectrometry. Sam-
ple were purified after UV treatment using RP-HPLC and analyzed with LC-MS.

224
M.A. Zmijewski et al. / Steroids 74 (2009) 218–228
Fig. 4. Identification of pregna-5,7,9(11)-triene-3␤,17␣,20S-triol (A) 500 MHz proton NMR (B) COSY showing correlation from 11-CH to 12a-CH2, 20-H to 21-CH3 and 3-CH
to 4-CH2 and 2CH2. Additionally, correlation from 7-CH to C-14 and from 14-CH to 15CH2. Impurities are marked with asterisk.
3.2. Identification of novel L-like, D-like, and T-like compounds
by NMR
other products with UV absorption (ꢁmax) at 312, 332 and 340 nm.
This shift in UV absorption suggests the presence of a triene
system, presumable similar to cholesta-5,7,9(11)-trien-3␤-ol (9-
DDHC), with reported ꢁmax at 324 nm [9,10]. Although irradiation
of both 4R and 4S resulted in the formation of compounds with
ꢁmax above 300 nm, the process was more efficient from 4S precur-
sor (Fig. 2). NMR analysis of this product confirmed the presence of
5,7,9(11)-triene system, and the product was identified as pregna-
5,7,9(11)-triene-3␤,17␣,20S-triol (Fig. 4), a compound that was
previously reported.13 The chemical shifts we observe for 5S
(Table 2) are essentially identical (with small changes due to differ-
ent solvent) to these earlier data.
The D-, L- or T-like irradiation products of 4R and 4S of defined
UV and mass spectra were subjected to NMR analysis. Elucida-
tion of the structures was based on 1H NMR data and selected
2D experiments (COSY, TOCSY). The detailed list of chemical shifts
with an assignment of signals is shown in Table 2. The D-like (4R-D
and 4S-D), and L-like (4R-L and 4S-L) compounds were assigned
based on expected chemical shifts for vinylic and methyl protons
with the characteristic pattern. The main difference between NMR
data for L-like compounds (4R-L and 4S-L) and their respective
parental compounds is a downfield shift of the methyl group at
C19 (∼0.20 ppm).
3.4. Production of pregna-5,7-diene-3ˇ,17˛,20-triols (4)
derivatives in human skin subjected to UV radiation (UVR)
Although we were able to detect and initially characterize T-like
and isoT-like compounds derived from 4R and 4S, these were not
stable, which prevented their in-depth characterization.
Fragments of neonatal human skin (approximately,
5 mm × 5 mm) were treated with 4R and 4S, incubated in culture
medium for 1–2 h, washed and subjected to UV-irradiation as
described in Section 2. The products of irradiation were separated
using RP-HPLC (Fig. 5). Previously identified products of UV radi-
ation of 4R and 4S in methanol were used as standards. Based on
identical retention times we detected UVR induced production of
3.3. Detection and characterization of novel triene-like products
of UVB irradiation of 4R and 4S
In addition to well-characterized products of 5,7-diene irradi-
ation (D-like, L-like, T-like and isoT-like compounds), we detected

M.A. Zmijewski et al. / Steroids 74 (2009) 218–228
225
Fig. 5. Ex vivo production of pregna-5,7-diene-3␤,17␣,20-triols (4R and 4S) derivatives in human neonatal skin. Skin fragments were treated with 2 ␮L of 10 mM of 4R (Panel
A) or 4S (Panel B) and subjected to UV irradiation. The products of photolysis in skin were subjected to RP-HPLC chromatography (chromatograms 2–7) and their retention
times were compared with previously identified products of UV irradiation of 4R and 4S in methanol (chromatogram 1). Different UV doses were used to show dynamics of
the process. The UVB doses per cm2 were as follows—chromatograms 1 and 7: 2500 mJ/cm2; 3: 0 mJ/cm2; 4: 100 mJ/cm2; 5: 500 mJ/cm2; 6: 1000 mJ/cm2. Chromatograms 2
show normal profile of non-treated and non-irradiated skin.
L-like, D-like, T-like and preD-like compounds in human skin from
4R (Fig. 5A) and 4S (Fig. 5B). The formation of 4R and 4S secoderiva-
tives was UVR dose dependent and the maximum production was
observed at 2500 mJ/cm2 (Fig. 5; chromatograms 7 on panels A
and B). These secosteroids were absent in the untreated human
skin (Fig. 5, chromatograms 2) or human skin exposed only to
pregna-5,7-diene-3␤,17␣,20-triols without following UVR (Fig. 5,
chromatograms 3 on panels A and B). We also noted appearance of
products corresponding to isoT-like derivatives with characteristic
UV absorption (ꢁmax at 250 nm) and retention time around 2 min
(not shown). Further identification of these products was not
possible due to limited absorption of steroidal precursors by skin
and subsequently relatively low production of secosteroids.
3.5. Inhibition of melanoma SKMEL-188 colony formation by
pregna-5,7-diene-3ˇ,17˛,20-triols (4R and 4S) and their D-like
and L-like derivatives
The inhibition of melanoma SKMEL-188 colony formation by
4R or 4S and their D-like and L-like derivatives was used to assay
their biological activity as described in Section 2. All studied com-
pounds had antiproliferative properties as showed by inhibition of
Fig. 6. Inhibition of colony formation of melanoma SKMEL-188 treated with pregna-5,7-diene-3␤,17␣,20-triols 4R (B) and 4S (E) and their D-like and L-like derivatives: 4R-D
(C), 4R-L (D), 4S-D (F) and 4S-L (G). An active form of vitamin D3 1,25(OH)2D3 was used as positive control (A). The inhibition of melanoma SKMEL-188 colony formation
was used to assay biological activity of newly synthesized compounds at 0.1, 10 and 100 nM concentrations. The average value (n = 6) for ethanol treated cells was used as a
0 nM on all graphs. The total number of colonies and their diameters with cutoff below and above 20 nm was measured and expressed in colony-forming units (CFU). Data
are presented as mean S.E.M. for colony-forming assay (n = 4–6); *P < 0.05, **P < 0.005, and ***P < 0.001. Representative pictures of SKMEL treated with studied compounds
and the vehicle control are shown (H).

226
M.A. Zmijewski et al. / Steroids 74 (2009) 218–228

M.A. Zmijewski et al. / Steroids 74 (2009) 218–228
227
Scheme 4. Putative model of UVR induced generation of pregna-5,7,9(11)-triene-3␤,17␣,20S-triol. (1) Singlet oxygen initiates photooxidation of 4S or 4R, with formation of
endoperoxide and hydroperoxide. (2) UV stimulates H2O2 and triene formation. (3) Triene acts as a photosensitizer for singlet oxygen generation. (4) The source of initial
singlet oxygen is uncertain, but possibly it is formed from O2 during irradiation.
colony formation (Fig. 6). When all colonies were counted, the aver-
age inhibition for the highest concentrations of tested compounds
(100 nM) was 50–70%. The inhibitory effect was relatively lower
(≤50%) for small colonies with diameter below 20 nm and higher
for colonies above 20 nm (50–80%) at 100 nM concentrations. Inter-
estingly, the pregna-5,7-diene-3␤,17␣,20-triols precursors (4R and
4S) were more efficient in growth inhibition than their photoderiva-
tive products (Fig. 6B and E). When comparing to 1,25(OH)2D3,
(Fig. 6A) we found that the precursor 4S (Fig. 6E) was the most
potent compound, while L-like compound 4R-L showed the lowest
activity (Fig. 6D). Furthermore, other compounds including 4S-D,
4S-L and 4R-D had similar effects as 1,25-(OH)2D3.
nature, and could be triggered by singlet oxygen in the presence
of photosensitizing agents [8,9,11]. The 9-DDHC can also act as
chromatophore, stimulating the production of singlet oxygen and
subsequent oxidation of 7DHC to 7-hydroxy-5,8(9)-cholestatriene,
which undergoes decomposition to give 9-DDHC and hydrogen
peroxide [9]. Such a self-regenerating system producing singlet
oxygen was also shown for ergosterol and its triene derivative [25].
Our data not only support such a model but also indicate for the
first time that UV irradiation is sufficient for the auto-catalytic
conversion of pregna-5,7-diene-3␤,17␣,20S-triol (4S) to pregna-
5,7,9(11)-triene-3␤,17␣,20S-triol (5S), as is presented in Scheme 3;
the presence of oxidized derivatives was confirmed by MS (Fig. 3).
Interestingly, formation of triene for 7DHC was observed only in
the presence of photosensitizers [8–11,25] and extensive irradiation
of 3␤-hydroxypregna-5,7-dien-20-one resulted in the production
of oxidized iT-like compounds [21]. This suggests a correlation
between the presence of two hydroxyls at C17 and C20 and UVR
induced formation of the triene Scheme 4.
Since 4S and 4R and other androsta- and pregna-5,7-dienes
derivatives were detected in living organisms under physiological
and pathological conditions [10,13,14,26–30] our current data sug-
gest (by inference) that they may be converted into corresponding
secosteroids in the skin if exposed to solar radiation [31]. To test
this hypothesis we performed ex vivo experiments with photolytic
reactions conducted in human skin. Thus, using a physiologically
relevant topical application of pregna-5,7-diene-3␤,17␣,20-triols
with following ex-vivo incubation, washing of unincorporated
products and UVR exposure, we were able to detect peaks with
retention times corresponding to L-like, D-like, T-like and preD-
like standards that were absent in negative controls. This indicates
that under physiological conditions (skin) steroidal dienes without
the side chain (pregna-5,7-diene-3␤,17␣,20-triols) undergo similar
pathway of photochemical transformations to their corresponding
secosteroidal products as in the model UVR induced transforma-
4. Discussion
We are reporting for the first time an efficient, reproducible and
detailed method for the UV-driven derivatization of two epimers
(20R and 20S) of pregna-5,7-diene-3␤,17␣,20-triol (4R and 4S). To
our knowledge, this is the first description of vitamin D-like (4S-
D, 4R-D), L-like (4S-L and 4R-L), T-like (4S-T and 4R-T) derivatives
of 4R and 4S. Careful observation of the dynamics of conversion
reveal a rapid, UVB dose-dependent conversion of T-like com-
pounds to isoT, and their further oxidation. Longer UVB irradiation
results in the synthesis of pregna-5,7,9(11)-triene-3␤,17␣,20S-triol
(5S), its structure being confirmed by NMR. Although, chemical
synthesis of 5S was previously reported [13], the role of UV in
its formation has not been described. Interestingly, it was postu-
lated that pregna-5,7,9(11)-triene-3␤,17␣,20S-triol is produced in
humans because 21-deoxypregnane is almost exclusively reduced
to 20S-hydroxyls [14]. In our hands, compound 4S was more effi-
ciently converted to triene by UVB irradiation, despite the reduction
of compound 3 resulting in an almost 50–50 mixture of 4R and
4S. It was also shown that the synthesis of steroidal trienes has
two potential intermediates of endoperoxide and hydroperoxide

228
M.A. Zmijewski et al. / Steroids 74 (2009) 218–228
tion of 7-dehydrocholestrol to secosteroids containing the full side
chain (vitamin D3, tachysterol3 and luminosterol3) described by
the Holick group [1,3]. These findings also substantiate future effort
to study photolytic transformation of pregna-5,7-diene-3␤,17␣,20-
triols in SLOS patients, since these were detected in body fluids of
those patients [13,14].
syndrome indicates formation of sterol hydroperoxide. J Lipid Res 1996;37:
2280–7.
[11] Albro PW, Corbett JT, Schroeder JL. Doubly allylic hydroperoxide formed in the
reaction between sterol 5,7-dienes and singlet oxygen. Photochem Photobiol
1994;60:310–5.
[12] Ruan B, Wilson WK, Pang J, Gerst N, Pinkerton FD, Tsai J, et al. Sterols in blood
of normal and Smith–Lemli–Opitz subjects. J Lipid Res 2001;42:799–812.
[13] 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;68:31–42.
[14] Shackleton CH, Roitman E, Kelley R. Neonatal urinary steroids in Smith–
Lemli–Opitz syndrome associated with 7-dehydrocholesterol reductase defi-
ciency. Steroids 1999;64:481–90.
[15] Smith DW, Lemli L, Opitz JM. A newly recognized syndrome of multiple con-
genital anomalies. J Pediatr 1964;64:210–7.
[16] DeLuca HF. Overview of general physiologic features and functions of vitamin
D. Am J Clin Nutr 2004;80:1689S–96S.
[17] Plum LA, Prahl JM, Ma X, Sicinski RR, Gowlugari S, Clagett-Dame M, et al. Bio-
logically active noncalcemic analogs of 1alpha. 25-dihydroxyvitamin D with
an abbreviated side chain containing no hydroxyl. Proc Natl Acad Sci USA
2004;101:6900–4.
[18] Murari MP, Londowski JM, Bollman S, Kumar R. Synthesis and biological activ-
ity of 3 beta-hydroxy-9,10-secopregna-5,7, 10[19]-triene-20-one: a side chain
analogue of vitamin D3. J Steroid Biochem 1982;17:615–9.
[19] Bridgewater AJ, Cheung HTA, Vadasz A, Watson TR. Ring C aromatic steroids.
Part 2. Rearrangement of 16␣,17␣-epoxy- and 17␣-hydroxy-5,7-dienes. J Chem
Soc, Perkin Trans 1 Org Bio-Org Chem 1980;2:556–62.
[20] Marwah P, Marwah A, Lardy HA. Microwave induced selective enolization of
steroidal ketones efficient acetylation of sterols in semisolid state. Tetrahedron
2003;59:2273–87.
[21] Zmijewski MA, Li W, Zjawiony JK, Sweatman TW, Chen J, Miller DD, et al. Syn-
thesis and photo-conversion of androsta- and pregna-5,7-dienes to vitamin
D3-like derivatives. Photochem Photobiol Sci 2008, doi:10.1039/b809005j.
[22] Fischer TW, Sweatman TW, Semak I, Sayre RM, Wortsman J, Slominski A. Consti-
tutive and UV-induced metabolism of melatonin in keratinocytes and cell-free
systems. FASEB J 2006;20:1564–6.
[23] Slominski A, Semak I, Pisarchik A, Sweatman T, Szczesniewski A, Wortsman J.
Conversion of l-tryptophan to serotonin and melatonin in human melanoma
cells. FEBS Lett 2002;511:102–6.
[24] MacLaughlin JA, Anderson RR, Holick MF. Spectral character of sunlight mod-
ulates photosynthesis of previtamin D3 and its photoisomers in human skin.
Science 1982;216:1001–3.
Analogs of vitamin D3 without cholesterol-type side-chain have
low calcemic activity [18,32] and vitamin D3 and its derivatives
have broad spectrum of biological functions [2,16,33], including
anti-carcinogenic properties in the skin (reviewed in [34,35]) and
melanomas (reviewed in [36]). In the present studies we have
shown that pregna-5,7-diene-3␤,17␣,20-triols and its D-like and L-
like derivatives have clear tumorostatic activity as defined by their
inhibition of colonies formation by human melanoma cells. Inter-
estingly, 5,7-diene-3␤,17␣,20-triols precursors showed the highest
activity with 4S being the most active, even more active than
1,25(OH)2D3, while L-like compound 4R-L was the least potent.
The other compounds showed similar activity to 1,25(OH)2D3.
Thus, these studies may help to identify efficient drugs for adju-
vant melanoma therapy including the described secosteroids
and, interestingly, their precursor 5,7-diene-3␤,17␣,20-triols. Thus,
newly generated dihydroxy derivatives, as described in this paper,
may serve as promising candidates for the treatment of various
pathological processes, including malignant melanoma, which is
resistant to any type of therapy once metastatic process has started
[37].
In conclusion, we have characterized in details an in vitro UVB
induced conversion of two epimers (20R and 20S) of pregna-5,7-
diene-3␤,17␣,20-triol, identified and defined the structures of new
products of this process, demonstrated that similar transformation
can occur in the skin and demonstrated anti-melanoma activity of
the substrates and secosteroidal products of the photoconversion.
[25] Albro PW, Bilski P, Corbett JT, Schroeder JL, Chignell CF. Photochemical reac-
tions and phototoxicity of sterols: novel self-perpetuating mechanisms for lipid
photooxidation. Photochem Photobiol 1997;66:316–25.
Acknowledgement
[26] Tint GS, Irons M, Elias ER, Batta AK, Frieden R, Chen TS, et al. Defective choles-
terol biosynthesis associated with the Smith–Lemli–Opitz syndrome. N Engl J
Med 1994;330:107–13.
Supported by grant # AR052190 from NIH/NIAMS to AS.
[27] Nowaczyk MJ, Waye JS. The Smith–Lemli–Opitz syndrome: a novel metabolic
way of understanding developmental biology, embryogenesis, and dysmor-
phology. Clin Genet 2001;59:375–86.
[28] Tait AD, Hodge LC, Allen WR. Biosynthesis of 3 beta-hydroxy-5,7-pregnadien-
20-one by the horse fetal gonad. FEBS Lett 1983;153:161–4.
References
[1] Holick MF, Clark MB. The photobiogenesis and metabolism of vitamin D. Fed
Proc 1978;37:2567–74.
[2] Holick MF. Vitamin D: a millenium perspective. J Cell Biochem 2003;88:
296–307.
[3] Holick MF, Tian XQ, Allen M. Evolutionary importance for the membrane
enhancement of the production of vitamin D3 in the skin of poikilothermic
animals. Proc Natl Acad Sci USA 1995;92:3124–6.
[4] Webb AR, DeCosta BR, Holick MF. Sunlight regulates the cutaneous produc-
tion of vitamin D3 by causing its photodegradation. J Clin Endocrinol Metab
1989;68:882–7.
[5] Tian XQ, Holick MF. A liposomal model that mimics the cutaneous production
of vitamin D3. Studies of the mechanism of the membrane-enhanced thermal
isomerization of previtamin D3 to vitamin D3. J Biol Chem 1999;274:4174–9.
[6] Jin X, Yang X, Yang L, Liu Z-L, Zhang F. Autoxidation of isotachysterol Tetrahedron
2004;60:2881–8.
[7] Valencia A, Kochevar IE. Ultraviolet A induces apoptosis via reactive oxygen
species in a model for Smith–Lemli–Opitz syndrome. Free Radic Biol Med
2006;40:641–50.
[8] Feng K, Zhang RY, Wu LZ, Tu B, Peng ML, Zhang LP, et al. Photooxidation of olefins
under oxygen in platinum(II) complex-loaded mesoporous molecular sieves. J
Am Chem Soc 2006;128:14685–90.
[9] Chignell CF, Kukielczak BM, Sik RH, Bilski PJ, He YY. Ultraviolet A sensitivity
in Smith–Lemli–Opitz syndrome: possible involvement of cholesta-5,7, 9(11)-
trien-3 beta-ol. Free Radic Biol Med 2006;41:339–46.
[29] Tait AD, Hodge LC, Allen WR. The biosynthesis of
androstadien-17-one by the horse fetal gonad. FEBS Lett 1985;182:107–10.
[30] Tait AD, Santikarn S, Allen WR. Identification of beta-hydroxy-5,7-
3 beta-hydroxy-5,7-
3
pregnadien-20-one and 3 beta-hydroxy-5, 7-androstadien-17-one as endoge-
nous steroids in the fetal horse gonad. J Endocrinol 1983;99:87–92.
[31] 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;271:4178–88.
[32] Holick MF, Garabedian M, Schnoes HK, DeLuca HF. Relationship of 25-
hydroxyvitamin D3 side chain structure to biological activity. J Biol Chem
1975;250:226–30.
[33] Bikle DD. Vitamin D regulated keratinocyte differentiation. J Cell Biochem
2004;92:436–44.
[34] Kamradt J, Rafi L, Mitschele T, Meineke V, Gartner BC, Wolfgang T, et al. Analysis
of the vitamin D system in cutaneous malignancies. Recent Results Cancer Res
2003;164:259–69.
[35] Bikle DD, Oda Y, Xie Z. Vitamin D and skin cancer: a problem in gene regulation.
J Steroid Biochem Mol Biol 2005;97:83–91.
[36] Brozyna A, Zbytek B, Granese J, Ross J, Carlson JA, Slominski A. Mechanism
of UV-related carcinogenesis and its contribution to nevi/melanoma. Exp Rev
Dermatol 2007;2:451–69.
[37] Carlson JA, Ross JS, Slominski A, Linette G, Mysliborski J, Hill J, et al. Molecular
diagnostics in melanoma. J Am Acad Dermatol 2005;52:775–8, 743–75 (quiz).
[10] De Fabiani E, Caruso D, Cavaleri M, Galli Kienle M, Galli G. Cholesta-5,7,
9(11)-trien-3 beta-ol found in plasma of patients with Smith–Lemli–Opitz