Synthesis and photochemical transformation of 3β,21-dihydroxypregna-5, 7-dien-20-one to novel secosteroids that show anti-melanoma activity

Article abstract of DOI:10.1016/j.steroids.2010.10.009

We have synthesized 3β,21-dihydroxypregna-5,7-dien-20-one (21(OH) 7DHP) and used UVB radiation to induce its photoconversion to analogues of vitamin D (pD), lumisterol (pL) and tachysterol (pT). The number and character of the products and the dynamics of the process were dependent on the UVB dose. The main products: pD and pT compounds were characterized by UV absorption, MS and NMR spectroscopy after RP-HPLC chromatography. In addition, formation of multiple oxidized derivatives of the primary products was detected and one of these derivatives was characterized as oxidized 21-hydroxyisotachysterol compound (21(OH)oxy-piT). These newly synthesized compounds inhibited growth of human melanoma cells in a dose dependent manner, with greater or equal potency to calcitriol. 3β,21-Dihydroxy-9β,10α-pregna-5,7-dien-20-one (21(OH)pL) and 21(OH)oxy-piT had higher potency against pigmented melanoma cells, while the EC₅₀ for compounds 21(OH)7DHP and (5Z,7E)-3β,21-dihydroxy-9,10-secopregna-5,7,10(19)-trien-20-one (21(OH)pD) were similar in both pigmented and non-pigmented cells. Moreover, 21(OH)7DHP and its derivatives inhibited proliferation of human epidermal HaCaT keratinocytes, albeit at a lower activity compared to melanoma cells. Importantly, 21(OH)7DHP derivatives strongly inhibited the colony formation of human melanoma cells with 21(OH)pD being the most potent. The potential mechanism of action of newly synthesized compounds was similar to that mediated by 1,25(OH)₂D ₃ and involved ligand-induced translocation of vitamin D receptor into the nucleus. In summary, we have characterized for the first time products of UVB-induced conversion of 21(OH)7DHP and documented that these compounds have selective, inhibitory effects on melanoma cells.

Full text of DOI:10.1016/j.steroids.2010.10.009

Steroids 76 (2011) 193–203
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis and photochemical transformation of
3␤,21-dihydroxypregna-5,7-dien-20-one to novel secosteroids that show
anti-melanoma activityଝ,ଝଝ
Michal A. Zmijewskia, Wei Lib, Jianjun Chenb, Tae-Kang Kimc, Jordan K. Zjawionyd,
Trevor W. Sweatmane, Duane D. Millerb, Andrzej T. Slominskic,∗
a Department of Histology, Medical University of Gdan´sk, Gdan´sk, Poland
b Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Science Center, Memphis, TN 38163, USA
c Department of Pathology and Laboratory Medicine, Center for Cancer Research, 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 38677-1848, USA
e 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:
We have synthesized 3␤,21-dihydroxypregna-5,7-dien-20-one (21(OH) 7DHP) and used UVB radiation
to induce its photoconversion to analogues of vitamin D (pD), lumisterol (pL) and tachysterol (pT).
The number and character of the products and the dynamics of the process were dependent on the
UVB dose. The main products: pD and pT compounds were characterized by UV absorption, MS and
NMR spectroscopy after RP-HPLC chromatography. In addition, formation of multiple oxidized deriva-
tives of the primary products was detected and one of these derivatives was characterized as oxidized
21-hydroxyisotachysterol compound (21(OH)oxy-piT). These newly synthesized compounds inhibited
growth of human melanoma cells in a dose dependent manner, with greater or equal potency to cal-
citriol. 3␤,21-Dihydroxy-9␤,10␣-pregna-5,7-dien-20-one (21(OH)pL) and 21(OH)oxy-piT had higher
potency against pigmented melanoma cells, while the EC50 for compounds 21(OH)7DHP and (5Z,7E)-
3␤,21-dihydroxy-9,10-secopregna-5,7,10(19)-trien-20-one (21(OH)pD) were similar in both pigmented
and non-pigmented cells. Moreover, 21(OH)7DHP and its derivatives inhibited proliferation of human
epidermal HaCaT keratinocytes, albeit at a lower activity compared to melanoma cells. Importantly,
21(OH)7DHP derivatives strongly inhibited the colony formation of human melanoma cells with
21(OH)pD being the most potent. The potential mechanism of action of newly synthesized compounds
was similar to that mediated by 1,25(OH)2D3 and involved ligand-induced translocation of vitamin D
receptor into the nucleus. In summary, we have characterized for the first time products of UVB-induced
conversion of 21(OH)7DHP and documented that these compounds have selective, inhibitory effects on
melanoma cells.
Received 28 May 2010
Received in revised form
27 September 2010
Accepted 30 October 2010
Available online 9 November 2010
Keywords:
Secosteroids
UV radiation
Vitamin D
SLOS
Skin
Melanoma
© 2010 Elsevier Inc. All rights reserved.
1. Introduction
5,7,10(19)-trien-3-ol) from 7-dehydrocholesterol (7DHC, cholesta-
5,7-dien-3␤-ol) [1–3]. This reaction is initiated by photolysis of
Human skin is the main organ for photo-induced synthe-
sis of vitamin D3 (cholecalciferol, 3␤,5Z,7E-9,10-secocholesta-
the unsaturated B ring of 7DHC, resulting in the formation of a
pre-D3 intermediate and subsequent isomerization to vitamin D3,
tachysterol (6E-9,10-secocholesta-5(10),6,8-trien-3␤-ol, T3) and
lumisterol3 (9␤,10␣-cholesta-5,7-diene-3␤-ol, L3). Depending on
strength and duration of ultraviolet B (UVB) exposure, tachysterol
might undergo further isomerization to isotachysterol, and subse-
quent oxidation of A, B or C rings [3–6]. The other factors influencing
the relative ratio and structure of the products of UV photolysis of
steroidal 5,7-dienes are temperature, wavelength, and the presence
of biological membranes or cellular compartments of the skin [4,6].
Production of 5,6-transvitamin D3, suprasterols I and II in the skin
in response to high doses of UVB has also been observed [4]. Irradia-
tion of 5,7-dienes also results in formation of 5,7,9(11)-trienes, with
ଝ
Supported by grant # AR052190 from NIH/NIAMS (ATS), Polish Ministry of Sci-
ence and Higher Education, project no. N405 623238 (MAZ), and 1R15CA125623
from NIH/NCI to WL.
ଝଝ
Its contents are solely the responsibility of the authors and do not necessarily
represent the official views of the NIH.
∗
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 4486979.
E-mail address: aslominski@uthsc.edu (A.T. Slominski).
0039-128X/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2010.10.009

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M.A. Zmijewski et al. / Steroids 76 (2011) 193–203
the probable involvement of singlet oxygen and photosensitizers
[7–11]. Detection of non-classical products of UV mediated photol-
ysis of 5,7-dienes might represent a naturally occurring mechanism
of regulation of vitamin D3 synthesis and response to UV.
2.3. 3ˇ,21-Dihydroxypregna-5,7-dien-20-one diacetate (3)
Compound 3 was synthesized according to a known pro-
cedure [35]. To a solution of compound 2 (4.16 g, 10.0 mmol)
in benzene–hexane (250 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 under argon for 25 min in
a preheated oil bath (100 ◦C) and then placed in an ice bath to
cool. The insoluble material was filtered off. The filtrate was con-
centrated to yield a yellow-brown solid. To a solution of this
yellow-brown material in tetrahydrofuran (60 ml) was added tetra-
butylammonium bromide (0.8 g, 2.5 mmol) and stirred for 75 min at
room temperature. To this reaction mixture was added tetrabuty-
lammonium 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 over anhydrous Na2SO4 and concentrated
to yield crude compound 3. The crude compound 3 was subjected
to flash chromatography (eluted with hexane–ethyl acetate 2:1) to
produce a white solid. Yield: 41%. 1H NMR (500 MHz, CDCl3): ı 5.60
(dd, J = 9.6 Hz, 2.8 Hz, 1 H), 5.44–5.47 (m, 1 H), 4.78 (d, J = 16.0 Hz,
1 H), 4.70–4.76 (m, 1 H), 4.58 (d, J = 16.0 Hz, 1 H), 2.64 (t, J = 9.6 Hz,
1 H), 2.52–2.56 (m, 1 H), 2.39 (t, J = 14.8 Hz, 1 H), 2.25–2.32 (m, 1
H), 2.20 (s, 3 H), 2.12–2.15 (m, 1 H), 2.08 (s, 3 H), 2.04–2.10 (m,
2 H), 1.50–1.96 (m, 8H), 1.50 (dt, J = 14.8 Hz, 8.0 Hz, 1 H),1.40 (dt,
J = 14.0 Hz, 5.0 Hz, 1 H), 0.96 (s, 3 H), 0.65 (s, 3 H). ESI-MS: calculated
for C25H34O5, 414.2, found 437.3 [M+Na]+.
A deficiency of 7DHC ꢀ-reductase can lead to formation
of pregna-5,7-dienes in vivo [12–16]. Similar to 7DHC, such
compounds [9,10,13,17–19], are sensitive to UVB-induced pho-
toconversion to vitamin D, lumisterol and tachysterol analogues
[17–19]. Recently, it was shown that cytochrome P450scc is capa-
ble of the oxidation and cleavage of the side chain of 7-DHC to
produce 7-dehydropregnenolone (7DHP) [17,20]. 7DHP can serve
as a substrate for further metabolism by classical enzymes of the
steroidogenic pathway [17,20], which raises the possibility of gen-
eration of a new class of vitamin D3 derivatives after UVB irradiation
[18,19]. Moreover, cytochrome P450scc was shown to hydroxy-
late vitamins D3 and D2 and ergosterol without the subsequent
side chain cleavage [21–25], broadening the spectrum of possible
secosteroidal products of CYP11A1 activity.
Vitamin D3 is known as a master regulator of body calcium
level [2,26–28]. Furthermore, vitamin D3 affects proliferation, dif-
ferentiation and apoptosis of normal and malignant cells [2,26].
However, its use as an antiproliferative agent is largely limited due
to toxicity (hypercalcemia) at high doses [1,2]. It has been shown
that one way to significantly reduce this effect is by shortening or
removal of the side chain of 7DHC [29,30]. Since UVB can photolyze
pregna-5,7-dienes and their hydroxylated derivatives into vitamin
D analogs with short side chains, it is logical to examine their poten-
tial anticancer properties [31]. Indeed, some of these compounds
have already demonstrated antiproliferative activity against skin
cells [19,20] and leukemia [32].
2.4. 3ˇ,21-Dihydroxypregna-5,7-dien-20-one (4, 21(OH)7DHP)
The aim of this study was to generate new vitamin
D
Compound 4 was synthesized according to a reported procedure
[37]. To a solution of compound 3 (414 mg, 1 mmol) in THF:MeOH
(60 ml, 1:2 in volume) was added potassium carbonate (276 mg,
2 mmol) and stirred for 10 h at room temperature. Argon bubbling
into the reaction mixture was applied to exclude oxygen since the
presence of oxygen initiates oxidative cleavage of 21-CH2 leading
to the formation of 17-carboxylic acid [38]. Water was added and
the mixture was extracted with ethyl acetate. The organic layer was
dried over anhydrous Na2SO4 and concentrated. The residue was
purified by flash chromatography (eluent: hexane–ethyl acetate
1:1) to produce a white solid. Yield: 66%. 1H NMR (500 MHz, CDCl3):
ı 5.51 (dd, J = 8.0 Hz, 2.8 Hz, 1 H), 5.36–5.38 (m, 1 H), 4.10–4.20 (m,
2 H), 3.57–3.60 (m, 1 H), 3.17–3.20 (m, 1 H), 2.5 (t, J = 8.0 Hz, 1 H),
2.40–2.44 (m, 1 H), 2.18–2.24 (m, 2 H), 1.91–1.98 (m, 3 H), 1.76–1.84
(m, 4 H), 1.52–1.66 (m, 2 H), 1.36–1.44 (m, 2 H), 1.18–1.26 (m, 1 H),
0.86 (s, 3 H), 0.54 (s, 3 H). ESI-MS: calculated for C21H30O3, 330.3,
found 353.3 [M+Na]+.
derivatives with low calcemic effects and good antiprolifer-
ative properties. Here we report on the UVB photolysis of
3␤,21-dihydroxypregna-5,7-dien-20-one [33] synthesized from 3-
acetylated 5-diene precursor [34,35]. The photolytic reaction led to
synthesis of novel 9,10-secosteroids with vitamin D and lumisterol
backbone structures, and oxidized derivatives. The biological activ-
ity of these derivatives was determined against human melanoma
cells since this cancer is poorly responsive to current treatment
regimens, especially once metastasis has occurred.
2. Experimental
2.1. Chemical synthesis
The synthesis of compounds 4 is shown in Scheme 1.
2.5. General procedure for UVB irradiation of 21(OH)7DHP
2.2. 3ˇ,21-Dihydroxypregn-5-en-20-one diacetate (2)
The procedure of UVB irradiation was performed essentially as
described previously [18] with some minor modifications. Briefly,
a methanol solution of 4 at 1 mg/ml concentration was subjected
to UVB irradiation for 5–15 min in a 5 mm quartz NMR tube using
a Biorad UV Transilluminator 2000 (Biorad, Hercules, CA). Spec-
tral characteristics of the UVB (280–320 nm) source were published
previously [39] and its strength (3.8 0.2 mW cm−2) was routinely
measured with a digital UVB Meter Model 6.0 (Solartech Inc., Har-
rison Twp, MI). Irradiation was followed by rapid separation of
the products by RP-HPLC chromatography, as described previously
[18]. The major products were identified on the basis of their reten-
tion time and characteristic UV absorption. Fractions containing
21(OH)pre-pD (ꢁmax at 260 nm) were re-purified after effective
conversion to 21(OH)pD (at least 90% of D, ꢁmax at 265 nm). Ini-
tial identification was confirmed by MS, and NMR spectroscopic
measurements, as described below.
Compound 1 (3␤,21-dihydroxypregn-5-en-20-one acetate) was
acetylated following a known procedure [36]. A mixture of com-
pound 1 (7.48 g, 20 mmol) and pTSA·H2O (76 mg, 0.4 mol) in acetic
anhydride (40 ml) was placed in an open glass tube. This mixture
was irradiated in the microwave-assisted synthesizer for 10 min.
Ice-cold water was added and stirred until solid product precipi-
tated out. The solid was collected by filtration followed by washing
with saturated sodium bicarbonate solution and water. The dried
material was used for next step without further purification. Yield:
95%. 1H NMR (500 MHz, CDCl3): ı 5.38–5.41 (m, 1 H), 4.75 (d,
J = 18.0 Hz, 1 H), 4.60–4.66 (m, 1 H), 4.56 (d, J = 18.0 Hz, 1 H), 2.54 (t,
J = 9.8 Hz, 1 H), 2.33–2.36 (m, 2 H), 2.22–2.26 (m, 1 H), 2.18 (s, 3 H),
2.06 (s, 3 H), 2.01–2.08 (m, 2 H), 1.87–1.90 (m, 2 H), 1.40–1.76 (m,
10H), 1.28–1.34 (m, 1 H), 1.14–1.22 (m, 1 H), 1.04 (s, 3 H), 0.70 (s, 3
H). ESI-MS: calculated for C25H36O5, 416.3, found 439.3 [M+Na]+.

M.A. Zmijewski et al. / Steroids 76 (2011) 193–203
195
Scheme 1. Synthesis of 3␤,21-dihydroxypregna-5,7-dien-20-one (4) and its UVB-driven photolysis to vitamin D, tachysterol and lumisterol analogues. Reagents and condi-
tions: (a) Ac2O, microwave, p-toluenesulfonic acid monohydrate; (b) dibromantin, 2,2ꢀ-azobisisobutyronitrile, benzene/hexane (1:1), 100 ◦C, reflux; (c) Bu4NBr, Bu4NF, THF,
room temperature; (d) K2CO3, MeOH–THF, H2O, argon, room temperature.
2.5.1. 21(OH)pD (5Z,7E)-3ˇ,21-dihydroxy-9,10-secopregna-
5,7,10(19)-trien-20-one
dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide; 5 mg/ml
in PBS; Sigma, St. Louis, MI) was added and the plates were incu-
bated at 37 ◦C for 4 h in the presence of 5% CO2. Media was discarded
and 100 ␮L of acidified isopropanol (contained 0.1 M hydrochlo-
ric acid) was added prior to spectroscopic measurement of optical
density at 570 nm, with a 96-well plate reader.
1H NMR (500 MHz, CD3OD): ı 6.24 (d, J = 11.0 Hz, 1 H), 6.09 (d,
J = 11.0 Hz, 1 H), 5.06–5.08 (m, 1 H), 4.76–4.78 (m, 1 H), 4.17–4.26
(m, 2 H), 2.77–2.81 (m, 1 H), 2.90–2.93 (m, 1 H), 2.78 (t, J = 9.0 Hz, 1
H), 2.56 (dd, J = 13.0 Hz, 4.0 Hz, 1 H), 2.41–2.45 (m, 1 H), 1.32–2.24
(m, 14 H), 0.52 (s, 3 H). ESI-MS: calculated for C21H30O3, 330.3,
found 353.3 [M+Na]+.
2.7. [3H]-thymidine incorporation
2.5.2. 21(OH)pL
3ˇ,21-dihydroxy-9ˇ,10˛-pregna-5,7-dien-20-one
The cells were grown in Ham’s F-10 media containing 5% char-
coal treated FBS. After synchronization at G0/G1 phase of the cell
cycle by incubation in serum free media, the chemicals were added
with fresh media with 5% charcoal treated FBS and incubated for
72 h. After a defined period of time, [3H]-thymidine (specific activ-
ity 88.0 Ci/mmol; Amersham Biosciences, Piscataway, NY, USA) was
added to a final concentration of 0.5 ␮Ci/ml in medium, and after
4 h of incubation cells were precipitated with 10% TCA, processed as
described previously, and subjected to liquid scintillation counting
using a beta counter (Direct Beta-Counter Matrix 9600; Packard)
[38].
1H NMR (500 MHz, CD3OD): ı 5.61 (dd, J = 5.0 Hz, 2.5 Hz, 1 H),
5.49–5.51 (m, 1 H), 4.20–4.29 (m, 2 H), 4.05–4.07 (m, 1 H), 2.96 (t,
J = 8.5 Hz, 1 H), 2.74–2.76 (m, 1 H), 2.46 (d, J = 16.0 Hz, 1 H), 2.34–2.36
(m, 1 H), 2.20–2.35 (m, 4 H), 1.25–1.91 (m, 9 H), 0.80 (s, 3 H), 0.59 (s,
3 H). ESI-MS: calculated for C21H30O3, 330.3, found 353.3 [M+Na]+.
2.5.3. 21(OH)oxy-piT
(6E)-3ˇ,21-dihydroxy-9,10-secopregna-5(10),6,8-triene-20-one
derivative (oxidized compound)
1H NMR (500 MHz, CD3OD): ı 6.59 (d, J = 11.5 Hz, 1 H), 6.11 (d,
J = 9.5 Hz, 1 H), 4.19–4.28 (m, 2 H), 3.99–4.00 (m, 1 H), 3.11 (s, 3
H), 2.79–2.85 (m, 2 H), 2.67 (dd, J = 13.5 Hz, 4.0 Hz, 1 H), 2.35 (d,
J = 13.5 Hz, 1 H), 1.33–2.25 (m, 13 H), 1.36 (s, 3 H), 0.55 (s, 3 H).
ESI-MS: calculated for C21H30O5, 362.3, found 401.3 [M+K]+.
2.8. Colony forming assay
The inhibition of melanoma SKMEL-188 colony formation by
derivatives of 21(OH)7DHP (21(OH)pD and 21(OH)pL) was per-
formed as described [19,20]. Briefly, melanoma SKMEL-188 cells
were plated at density of 500 cells per well in 12 well plates and
grown in F10 medium (GIBCO, Invitrogen Corp., Carlsbad, CA) sup-
plemented 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. Cells were
treated with 21(OH)7DHP, 21(OH)pD, 21(OH)pL, 21(OH)oxy-piT
and 1,25-(OH)2 D3 as a positive control at various concentrations
(0.1, 10 and 1000 nM). A corresponding dilution of ethanol was
used as a negative (vehicle) control. Cells were grown for 6–10
days and colonies were stained with 0.1% crystalline blue, pho-
tographed and the number and the size of colonies were measured
by ImageJ software (NIH, Bethesda, MD). Colony forming efficiency
2.6. MTT assay
The SKMEL-188 cells are amelanotic when grown in the Ham’s
F10 and start to produce melanin pigment after the switch to Ham’s
F10:DMEM (50:50, v/v) [40,41]. SKMEL-188 cells were seeded at
a density of 5000 cells per well into 96-well plates in Hams’
F10 or Hams’ F10:DMEM (50:50, v/v) medium (GIBCO, Invitro-
gen Corp., Carlsbad, CA) supplemented with charcoal-stripped fetal
bovine serum (HyClone, Logan, UT) and an antibiotic–antimycotic
solution (Sigma, St. Louis, MO). After 24 h, stock media was
exchanged with media containing serial dilutions of 21(OH)7DHP
and its derivatives. After incubation for 48 h, 20 ␮l of MTT ((3-(4,5-

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M.A. Zmijewski et al. / Steroids 76 (2011) 193–203
(CFU) was determined by dividing the number of colonies formed
by the number of cells plated, and multiplied by 100.
2.9. Generation of cell lines expressing VDR-EGFP fusion protein
In order to construct pLenti-CMV-VDR-EGFP-pgk-puro,
EGFP cDNA was amplified by PCR from pLenti-CMV-VDR-
p2a-EGFP-pgk-puro (submitted for publication) using primers
5ꢀ-GGACCTCTCGAGATGGTGAGCAAGGGCGAGGAG-3ꢀ
(forward);
5ꢀ-GCGAATTCCTACTTGTACAGCTCGTCCATGCC-3ꢀ (reverse), then
digested with XhoI and EcoRI and subcloned into pLenti-CMV-
VDR-p2a-EGFP-pgk-puro in which EGFP including p2a gene was
removed. The VDR in pLenti-CMV-VDR-EGFP was expressed with
EGFP together as a fusion protein. Lentivirus was produced in
293FT cells by UTHSC viral vector core using a method described
previously [42]. 10 MOI of lentivirus (construct pLenti-CMV-VDR-
EGFP-pgk-puro) was used to transduce SKMEL-188 melanoma
in the presence of 6 ␮g/ml polybrene in the corresponding
culture media (see above). The transduction efficiency was deter-
mined using a Nikon Eclipse TE300 microscope (Japan) by green
fluorescence.
Fig. 1. UVB-driven photolysis of 21(OH)7DHP. Representative UV spectra of
irradiated sample of 21(OH)7DHP. Peaks were assigned as follows: dotted
line—21(OH)7DHP (substrate), solid line—21(OH)pre-pD, dashed line—21(OH)pD,
dash double dot line—iso-pT or oxidized iso-pT. The UV spectra of fractions collected
from RP-HPLC separation of UVB-irradiation products were acquired by Nanodrop.
the known procedure [35]. Physico-chemical properties of 3␤,21-
dihydroxypregna-5,7-diene-20-one as well as byproducts (2 and
3) were monitored by means of MS and NMR spectroscopy and
their correctness confirmed (Section 2).
2.10. VDR translocation
Using SKMEL-188 transduced by pLenti-CMV-VDR-EGFP-pgk-
puro (VDR and EGFP expressed as fusion protein), VDR transloca-
tion was determined by counting cells with a fluorescent nucleus.
The transduced cells were incubated with drugs for 90 min and then
fixed with 4% PFA (paraformaldehyde). Fixed cells were mounted
with fluorescent mounting media (Dako, Denmark) and analyzed
with a fluorescent microscope. At least 10 pictures were taken from
different fields and the cells containing fluorescent nuclei were
counted. Data are presented as a percentage of total cell numbers.
3.2. UVB irradiation of 3ˇ,21-dihydroxypregna-5,7-dien-20-one
(21(OH)7DHP) and identification of products by combination of
RP-HPLC, UV, mass and NMR spectrometry
The UV conversion of 21(OH)7DHP was performed using a
UVB light source [39,43] (3.8 0.2 mW cm−2) with maximum emis-
sion spectrum in a range of 290–320 nm, as described previously
[18,19]. In order to optimize conditions for UVB-driven photol-
ysis of 21(OH)7DHP, the conversion was routinely monitored
by HPLC separation, and resulting products were characterized
by UV spectrometry [18,20,44]. Similar to cholesta-5,7-dien-3␤-
ol (7DHC) [44], four main products (Scheme 1) were formed in
a time-dependent fashion, namely 21(OH)pre-pD, 21(OH)pL and
21(OH)pD (Fig. 1). In addition, substantial production of com-
pounds with a characteristic shift of UV absorption to shorter
wave-lengths was also observed (ꢁmax = 240 3, 249 3, 260 3).
Those compounds are presumably isotachysterols (isotachysterol
and its oxidized derivatives), as described previously [6,18,20].
Due to the rapid and time-dependent rearrangement towards
isotachysterol derivatives, the reaction mixture was separated
immediately after irradiation. The photo-conversion and subse-
quent time-dependent structural rearrangements were monitored
by an HPLC equipped with a diode array that enabled fast detection
of products by characteristic UV spectra [18,20,44]. All products
were separated by HPLC and identified based on their unique UV
absorption spectra (Fig. 1).
Initial characterization of main products as pre-D (ꢁmax at
260 nm; 21(OH)pre-pD), D (ꢁmax at 265 nm; 21(OH)pD) and L
((ꢁmax at 262, 274, 283; 21(OH)pL) analogues was confirmed by
MS and NMR (see Section 2 for details). Surprisingly, the forma-
tion of T-derivative of 21(OH)7DHP, with predicted ꢁmax at 274,
281, 290 nm, was not detected. Instead, the rapid accumulation of
compounds with ꢁmax = 240 3, 249 3, 260 3 nm ( 2 nm) was
observed (several peaks on chromatogram; not shown). Detec-
tion of iso-T products indicates further metabolism of 21(OH)pT
(Scheme 1), with the potential formation of suprasterols [4], with
a ꢁmax below 250 nm. The pre-vitamin D derivative (ꢁmax = 260;
21(OH)pre-pD) underwent subsequent slow isomerization into
vitamin D compounds (21(OH)pD), with maximum UV absorption
at ꢁmax = 265 nm.
2.11. Statistical analyses
Data were tested for distribution and homogeneity of variances,
and for statistical significance with one-way analysis of variance
(Anova) with Tuckey post hoc test (for more than two groups) using
Prism 4.00 (GraphPad Software, San Diego, CA). Data are presented
as the mean SEM; for n = 4–6. *P < 0.05, **P < 0.01 and ***P < 0.005.
2.12. Other procedures
All RP-HPLC purification and analyses were performed on an
Atlantis C18 column using a Waters HPLC-system equipped with
a diode-array detector, an auto-sampler and a fraction collector
(Waters Associates, Milford, MA) as described elsewhere [18–20].
Mass spectra were recorded using a Bruker Esquire-LC/MS Spec-
trometer equipped with an electrospray ionization (ESI) source, as
described previously [18,19].
NMR measurements were performed on a Varian Unity Inova-
500 MHz spectrometer (Varian NMR Inc., Palo Alto, CA) using either
a 3 mm or a 5 mm probe, as described before [18,19].
3. Results
3.1. Synthesis of 3ˇ,21-dihydroxypregna-5,7-dien-20-one
(21(OH)7DHP)
The synthesis of 21(OH)7DHP following Scheme 1 was per-
formed as described in Section 2 [18,19].
The
synthesis
of
3␤,21-dihydroxypregna-5,7-dien-20-
one (21(OH)7DHP) was carried out from 21-acetylated
5-en precursor (1) by acetylation in position 3␤,
bromination–dehydrobromination and reduction, following

M.A. Zmijewski et al. / Steroids 76 (2011) 193–203
197
Table 1
UV and MS spectra data for 21(OH)7DHP and its derivatives.
Name
Parental compound
Structure type
UV max (nm)
Formula
Predicted MW
Found MW
21(OH)7DHP
21(OH)pre-pD
21(OH)pD
21(OH)pL
21(OH)-piT
21(OH)oxy-piT
21(OH)oxy-piT1
21(OH)oxy-piT2
21-OHprega
21(OH)7DHP
21(OH)7DHP
21(OH)7DHP
21(OH)7DHP
21(OH)7DHP
21(OH)7DHP
21(OH)7DHP
5,7-Diene
preD-like
D-like
L-like
260, 268, 280, 292
260
265
260, 271, 283
238, 247, 257
241, 250, 259
241, 250, 259
241, 250, 259
C21H30O3
C21H30O3
C21H30O3
330.22
330.22
330.22
330.22
330.22
362.21b
362.21b
362.21b
353.3 [M+Na]+
353.3 [M+Na]+
353.3 [M+Na]+
353.3 [M+Na]+
353.3 [M+Na]+
401.3 [M+K]+
401.3 [M+K]+
401.3 [M+K]+
C21H30O3
iT-like
C21H30O3
C21H30O5
C21H30O5
C21H30O5
b
b
b
isoT-like (oxide)
isoT-like (oxide)
isoT-like (oxide)
Bold—purified and characterized (NMR).
a
3␤,21-Dihydroxypregn-5-en-20-one acetate.
b
Alternative calculation base on predicted formula C21H30O6 and found 401.3 [M+Na]+.
Further identification was performed after purification by RP-
HPLC equipped with a fraction collector (see Section 2 for details)
and selected fractions were subsequently analyzed by MS. As
expected, all pD, pL and pT products had identical mass (m/z = 353.3
[M+Na]+) with the parental compound (Table 1). In addition to the
predicted (Scheme 1) products with molecular ion at m/z = 353.3
[M+Na]+, several products initially identified as iso-tachysterols
(by UV spectra with ꢁmax below 250 nm) showed a main ion at
m/z = 401.3 (Fig. 2B). This indicates either the addition of 2 (or
3) oxygen atoms with formation of peroxide or hydroperoxide
derivatives. Similar process, such as autooxidation of isotachys-
terol (6E-9,10-secocholesta-5(10),6,8(14)-trien-3␤-ol) has been
reported previously [6]. The oxidation of compound 21(OH)7DHP
without photolysis of the B ring resulting in production of endoper-
oxide and hydroperoxide, cannot be excluded, such process has
been shown for 7DHC [8,10].
The 21(OH)pD and 21(OH)pL irradiation products of
21(OH)7DHP, with defined UV and mass spectra, were sub-
jected to NMR analysis. Elucidation of the structures was based
on 1H 1D and 1H–1H 2D correlation spectroscopy (COSY) experi-
ments. The 21(OH)pD), and 21(OH)pL compounds were assigned
based on expected chemical shifts for vinylic and methyl protons
with the characteristic pattern we have previously described in
detail [18,19]. The main difference between the NMR spectra
for 21(OH)pL and its parental compound is a downfield shift
(∼0.17 ppm) of 19-methyl protons of the parent compound and
a upfield shift of 3-H (from 3.58 ppm to 4.06 ppm) (Fig. 3A and
B).
3.3. Detection and characterization of novel oxidized product of
UVB irradiation of 21(OH)7DHP
In addition to well-characterized products of 5,7-diene irra-
diation (21(OH)pD, 21(OH)pL, and 21(OH)piT compounds), we
detected products with UV absorption ꢁmax = 241, 249, and 262 nm
(Fig. 1) and the molecular ion at m/z = 401.3 (Fig. 2). The unexpected
mass could be calculated for C21H30O5, 362.3 as [M+K]+ or for
C
21H30O6, 378.3 as [M+Na]+. It is worthwhile to note that UV irra-
diation of 21(OH)7DHP resulted in formation of at least 3 oxidized
derivatives with ꢁmax below 250 nm and found m/z = 401.3.
The proton NMR (Fig. 3D) study revealed only two double
bond protons assigned to 6-H (6.59 ppm) and 7-H (6.11 ppm).
This strongly suggested an iso-tachysterol arrangement of double
bonds of compound 21(OH)oxy-piT had predictably occurred, in
agreement with the UV spectrum (ꢁmax 250 nm, characteristic for
conjugated double bond). In contrast to 21(OH)pD, this compound
lacks the two double bond protons from 19-CH2 with chemical
shifts around 5.07 ppm and 4.77 ppm, instead it has an additional
(when compared to 21(OH)pD) methyl group with a chemical shift
at 1.33 ppm for 19-CH3. Surprisingly, the chemical shift for 19-CH3
at 1.33 ppm suggests that this methyl is not in the proximity of
the double bond between C10 and C5. The expected chemical shift
for 19-CH3 should be at 1.7–1.8 ppm if it is adjacent to a double
bond [6]. MS data suggested the presence of 2 (or 3) additional
oxygen atoms in the structure. The COSY spectrum showed clearly
all expected correlations for 3-H group (4␣-H, 4␤-H, 2␣-H, 2␤-H,
1␣-H). In addition, 6-H has a long-range correlation to 4␤-H, and
7-H with proton assign as 9-H, which strongly suggested that the
double bond between 6 and 7-position remains intact. The chemical
shifts for 18-CH3 and 21-CH2 are similar to its parental compound
21(OH)7DHP, thus rings C, D and carbonyl group (20-C O) are
Although we were able to detect 21(OH)pT and 21(OH)isopT
derivatives of compound 21(OH)7DHP by means of characteristic
UV spectrum, these compounds were not stable, which prevented
their in-depth characterization.
Fig. 2. Identification of oxidized derivatives of 21(OH)7DHP by mass spectrometry. Sample were purified after UV treatment using RP-HPLC and analyzed with LC–MS.
Representative spectrum of potentially oxidized derivative of 21(OH)7DHP is shown on panel B in comparison to starting material (A). The compound 21(OH)piT shows
m/z = 401.3. The parental compound (C21H30O3, mass = 330.25) gave m/z = 353.25 and compound 21(OH)oxy-piT could be calculated as C21H30O5, 362.3 [M+K]+ or as C21H30O6,
378.3 [M+Na]+.

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Table 2
Inhibition of growth of SKMEL-188 melanoma cells. EC50 values were calculated as
described in Section 2 and Fig. 4.
SKMEL-188
HaCaT (nM)
Amelanotic (nM)
190
91
3.4
Melanotic (nM)
1,25(OH)2D3
21(OH)7DHP
21(OH)pD
12.4
118
2.5
180
>1000
40
21(OH)pL
21(OH)oxy-piT
130
40
0.1
0.8
>1000
>1000
melanogenic activity can affect the behavior of melanoma cells
[41,46,47] we tested the anti-melanoma properties of 21(OH)7DHP
and its derivatives under amelanotic and melanotic phenotype.
We have found that 21(OH)7DHP, 21(OH)pD, 21(OH)pL and
21(OH)oxy-piT inhibited growth of both amelanotic and melan-
otic melanoma cells with an effect modified by the presence
or absence of melanin pigment (Fig. 4). The strongest inhibi-
tion (EC50 < 1 nM) was observed for compounds 21(OH)pL and
21(OH)oxy-piT against melanizing SKMEL-188 cells (Table 2). The
EC50 values for 21(OH)pL and 21(OH)oxy-piT were 40–1300-fold
lower for pigmented versus non-pigmented cells (Table 2). By con-
trast, 21(OH)pD and 21(OH)7DHP showed comparable EC50 values
for both pigmented and non-pigmented cells (2.5 nM versus 3.4 nM
and 91 nM and 118 nM, respectively).
Next, we tested the effect of vitamin D analogs against non-
malignant human epidermal HaCaT keratinocytes. As expected,
1,25(OH)2D3 inhibited proliferation of HaCaT keratinocytes with
similar EC50 to non-pigmented SKMEL-188 melanoma cells
(180 nM versus 190 nM, respectively) (Table 2). Interestingly,
21(OH)7DHP and its derivatives had no effect on proliferation
of HaCaT cells, with exception of 21(OH)pD. The potency (EC50
)
of 21(OH)pD towards keratinocytes was about 10 times higher
when compared to melanoma cells (EC50 of 40 nM versus 3.4 nM).
These results indicate selective effects of new compounds towards
melanoma cells in comparison to non-malignant epidermal ker-
atinocytes (Table 2).
The process of DNA synthesis, required for proper growth and
cell division, was measured by [3H]-thymidine incorporation assay.
The inhibition of DNA synthesis in melanoma SKMEL-188 cells
is shown in Fig. 5. 21(OH)pL had the highest inhibitory potency,
although other 21-OH derivatives (21(OH)-7DHP and 21(OH)pD)
also inhibited DNA synthesis in SKMEL-188 cells. Insert in Fig. 5C
showed that 21(OH)pL efficiently inhibited DNA synthesis in AbC1
cell line derived from Bomirski hamster melanoma [48]. These com-
pounds were non-toxic as the viability of the cells cultured in their
presence was >95% as measured by trypan exclusion method (not
shown).
3.5. Inhibition of melanoma SKMEL-188 colony formation by
3ˇ21-dihydroxypregna-5,7-diene-20-one derivatives
Fig. 3. Proton NMR spectra of 21(OH)7DHP (A), 21(OH)pL (B), 21(OH)pD (C), and
oxidized derivative—21(OH)oxy-piT (D) analogs as well as their predicted structures
(as insert for each panel).
All of the tested compounds inhibited the formation of
melanoma colonies in a dose dependent manner (Fig. 6). The best
inhibition was observed for 21(OH)pD in a 10–1000 nM concentra-
tion range (Fig. 6B). A less potent inhibitory effect was also seen
with 21(OH)pL and 21(OH)oxy-piT (p < 0.05) at 1000 nM concen-
tration (Fig. 6C and D). Ethanol (solvent control) had no effect on
colony formation (Fig. 6A).
intact. Insufficient amount of 21(OH)oxy-piT prevented us from
collection of 13C NMR data or 1H–13C HMBC which could otherwise
provide an unambiguous assignment for this structure.
3.4. The compound 21(OH)7DHP and its derivatives inhibit
growth of human SKMEL-188 melanoma cells
3.6. Translocation of vitamin D receptor by 21(OH)7DHP and its
derivatives
The phenotype of human SKMEL-188 melanoma cells and the
ability to produce melanin pigment are dependent on concentra-
tion of melanogenic precursors in the culture media [40,45]. Since
Ligand induced translocation of VDR receptor into the nucleus
was studied using SKMEL-188 cells stably transduced with pLenti-

M.A. Zmijewski et al. / Steroids 76 (2011) 193–203
199
Fig. 4. Inhibition of growth of SKMEL-188 melanoma cells by 21(OH)7DHP and its derivatives. SKMEL-188 were seeded into 96-well plates and incubated in Ham’s F10
medium containing low tyrosine levels, 10 ␮M (amelanotic phenotype) or in DMEM:Ham’s F10 (50:50, v:v) media containing high tyrosine levels, 200 ␮M (melanotic
phenotype). The media were supplemented with serial dilutions of compound 21(OH)7DHP and its derivative 21(OH)pD, 21(OH)pL and 21(OH)oxy-piT. After 48 h the
cells were submitted to an MTT test. The experiment was repeated with similar results. Examples of amelanotic and melanotic cells are shown as inserts for each graph.
1,25(OH)2D3 was used as positive control. Data is presented as means SEM for 6 independent measurements. *p < 0.05, **p < 0.005, ***p < 0.0005 versus control.

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M.A. Zmijewski et al. / Steroids 76 (2011) 193–203
Fig. 5. 21(OH)7DHP (A), 21(OH)pD (B) and 21(OH)pL (C) inhibit DNA synthesis in SKMEL-188. (D) 1,25(OH)2D3 was used as a positive control. The cells were incubated with
compounds for 72 h in Ham’s F10 media containing 5% charcoal treated FBS. [3H]-thymidine was added for last 4 h of incubation. DNA synthesis was measured by counting
the radioactivity incorporated into TCA precipitable material. Data are presented as means SD (n = 4). The dose dependent inhibition was analyzed by one-way ANOVA with
#P < 0.05 and ##P < 0.01. The differences between control and the chemicals were analyzed with Student’s t-test; *P < 0.05, **P < 0.01 and ***P < 0.001.
Fig. 6. Inhibition of colony formation of melanoma SKMEL-188 treated with photo derivatives of 21(OH)7DHP. Ethanol-solvent control (A); 21(OH)pD (B); 21(OH)pL (C);
21(OH)oxy-piT (D). The inhibition of melanoma SKMEL-188 colony formation was used to assay biological activity of newly synthesized compounds at 0.1, 10 and 1000 nM
concentrations. Ethanol was used as a solvent control. The average value (n = 6) for vehicle-treated cells is presented as control for drug treated cells. The total number of
colonies was expressed in colony forming units (CFU). Data are presented as means SEM (n = 3 or 4); *P < 0.05, **P < 0.005, and ***P < 0.001.

M.A. Zmijewski et al. / Steroids 76 (2011) 193–203
201
ously studied hydroxylated derivatives of 7DHP [18,19], production
of pT compound was not observed by means of UV spectroscopy.
This was probably due to rapid transformation of pT product to its
iso-pT derivative under UVB, followed by its oxidation. The autoox-
idation of isotachysterol was described in detail by Jin et al. [6]. At
least three potentially oxidized derivatives of 21(OH)7DHP were
characterized by combination of UV and MS spectra with ꢁmax
below 250 nm and ESI-MS found 401.3 (Figs. 1 and 2 and Table 1).
The ꢁmax below 250 nm suggested the presence of conjugated dou-
ble bonds, and an m/z = 401.3 could be explained by the addition
of two or three oxygens (C21H30O5, 362.3 [M+K]+ or C21H30O6,
378.3 [M+Na]+). Low stability and small quantity prevented further
characterization of those derivatives, with exception of product
21(OH)oxy-piT, which was additionally analyzed by NMR. Open
ring B structure was confirmed by the presence of the double
bond protons of 6-C and 7-C (Fig. 3). These protons in the double
bond region of the spectra (7–4.5 ppm) suggested an isotachysterol
type of structure. Comparison of NMR spectra for 21(OH)oxy-piT
to 21(OH)pD and starting compound revealed that correlations
between protons of ring A were intact (as judged by COSY experi-
ment) and a similar chemical shift for 18-CH3 suggested only slight
changes in arrangement of C and D rings. Two hydroxyl groups were
also present thus, it is most likely that two additional oxygens are
present in A ring, replacing double bond (5-C, 10-C), as proposed
by Jin et al. (compound 9) [6].
It has to be noted that the compound with characteristic UV
spectra (ꢁmax at 320 nm) for a conjugated triene system (most
probably 5,7,9(11)-triene) was also detected by relatively low effi-
ciency of conversion (below 1%); this prevented us from further
identification (not shown). Thus, observed UVB-driven formation
of triene derivatives for several 5,7-dienes such as 7DHC or 17,20-
diOH derivatives of 7DHP [19] is a common reaction with potential
implications for photosensitivity of the skin [9].
The derivatives of vitamin D have a broad spectrum of bio-
logical functions [2,26,49] including anti-carcinogenic properties
in the skin (reviewed in [27,50,28]) and melanomas (reviewed in
[51]). Our focus has been on analogs of vitamin D3 without the
cholesterol-type side-chain with potentially low calcemic activ-
ity [18,19,30–32,38]. In the present studies we have shown that
3␤,21-dihydroxypregna-5,7-dien-20-one and its secosteroidal and
oxidized derivatives had an inhibitory effect on growth (Fig. 4
and Table 2), DNA synthesis (Fig. 5) and colony formation of
human melanoma cells (Fig. 6). Interestingly, the presence or
absence of melanin pigment influenced the sensitivity of the cells
to pL- and pT-photoderivatives of 21(OH)7DHP. Thus, production
of melanin pigment sensitized melanoma cells towards 21(OH)pL
and 21(OH)oxy-piT. This effect was absent in the case of 21(OH)pD.
It might be suggested, that active melanogenesis positively affects
the anti-melanoma activity of the above compounds. Although the
mechanism for this action would require future extensive stud-
ies, it must noted that intermediates of melanogenesis are highly
toxic towards melanoma cells [46,52]. Importantly, our initial study
showed that 21(OH)7DHP, 21(OH)pL and 21(OH)piT had no effect
on proliferation of HaCaT keratinocytes and the inhibitory effect
of 21(OH)pD was at least 10 times lower then for non-pigmented
SKMEL-188 cells. This suggests at least partial selectivity of 21-
OH photoderivatives towards melanoma cells. The antiproliferative
action of newly synthesized 21-OH7DHP and its photoderivatives
were additionally confirmed using radiolabeled thymidine incor-
poration into DNA assay. All tested compounds (21(OH)7DHP,
21(OH)pD and 21(OH)pL) significantly inhibited DNA synthesis in
SKMEL-188 cells with 21(OH)pL being the most potent compound
(Fig. 5). Inhibition of DNA synthesis was also observed for hamster
AbC1 melanoma, indicating that the observed effect is not limited
to human SKMEL-188 melanoma cells, and identifying an attractive
animal model of melanoma [53] for future in vivo testing.
Fig. 7. 21(OH)7DHP, 21(OH)pD and 21(OH)pL (10 nM) induced translocation of VDR
from the cytoplasm to the nucleus. (A) Data are presented as mean SD (n ≥ 10). The
differences were analyzed with Student’s t-test. ***P < 0.001: differences between
control and chemicals. #P < 0.05: differences between chemicals. (B) Representative
epifluorescence microphotographs of cells expressing VDR-EGFP fusion protein.
CMV-VDR-EGFP lentiviral construct (see Section 2). The addition of
21(OH)pD or 21(OH)pL resulted in efficient translocation of VDR-
GFP fusion protein into the nucleus (Fig. 7). The effect of 21(OH)pD
was similar to that of 1,25(OH)2D3 (positive control). 21(OH)pL
also translocated VDR-GFP to the nucleus, however, the effect was
significantly lower, when compared to 21(OH)pD or 1,25(OH)2D3.
These results document that novel secosteroidal derivatives of
21(OH)7DHP can bind to VDR and induce its translocation to the
nucleus in a similar manner as 1,25(OH)2D3.
4. Discussion
We are reporting for the first time an efficient, reproducible and
detailed method for the UVB-driven photolysis of 21-OH deriva-
tives of 3␤-hydroxypregna-5,7-dien-20-one (21(OH)7DHP). To our
knowledge, this is the first description of secosteroidal 21(OH)pD,
(21(OH)pL) and stable oxidized iso-pT derivatives of 21(OH)7DHP.
Further analysis of 21(OH)pD and 21(OH)pL confirmed proper
identification by MS and NMR spectroscopy. In contrast to previ-

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In order to gain some information on the mechanism of action
[17] Slominski A, Zjawiony J, Wortsman J, Semak I, Stewart J, Pisarchik A, et al.
novel pathway for sequential transformation of 7-dehydrocholesterol
A
of test compounds their effect on translocation of VDR from
cytoplasm to the nucleus was studied using EGFP labeled VDR
and epifluorescence microscopy (Fig. 7). Treatment of SKMEL-
188 resulted in efficient translocation of VDR into the nucleus
(Fig. 7B) indicating involvement of VDR in the observed pheno-
typic effects. Interestingly, the most potent compound—21(OH)pL
showed lower capacity to translocate VDR into nucleus in com-
parison to 21(OH)pD or 1,25(OH)2D3 (Fig. 7A). This suggests that
21(OH)pL, in addition to VDR, might possibly interact with other
proteins. For example, 1,25D3-MARRS (membrane associated rapid
response to steroids, ERp57) receptor protein [54] is one of the
possible candidates.
The present data may help to identify more efficient drugs
for adjuvant melanoma therapy, including the described ana-
logues of vitamin D (21(OH)pD) and, interestingly, its precursor
(21(OH)7DHP) and isomer (21(OH)pL). These compounds may
have different mechanisms of action from 1,25(OH)2D3, the sub-
ject of future laboratory studies. Synthesis and biological activity
of oxidized isotachysterol derivatives may also suggest a recep-
tor independent mechanism of action, e.g., intoxication of the
cells already stressed by cytotoxic intermediates of melanogene-
sis. These areas may represent an exciting and fruitful line of future
investigations.
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