Total synthesis of biologically active 20S-hydroxyvitamin D3

Article abstract of DOI:10.1016/j.steroids.2015.09.009

A total synthetic strategy of 20S-hydroxyVitamin D3 [20S-(OH)D3] involving modified synthesis of key intermediates 7 and 12, Grignard reaction to stereoselectively generate 20S-OH and Wittig-Horner coupling to establish D3 framework, was completed in 16 steps with an overall yield of 0.4%. The synthetic 20S-(OH)D3 activated Vitamin D receptor (VDR) and initiated the expression of downstream genes. In addition, 20S-(OH)D3 showed similar inhibitory potency as calcitriol [1,25(OH)₂D3] on proliferation of melanoma cells.

Full text of DOI:10.1016/j.steroids.2015.09.009

Accepted Manuscript
Total synthesis of biologically active 20S-hydroxyvitamin D3
Qinghui Wang, Zongtao Lin, Tae-Kang Kim, Andrzej T. Slominski, Duane D.
Miller, Wei Li
PII:
S0039-128X(15)00248-2
http://dx.doi.org/10.1016/j.steroids.2015.09.009
STE 7843
DOI:
Reference:
Steroids
To appear in:
Received Date:
Revised Date:
Accepted Date:
2 June 2015
10 September 2015
28 September 2015
Please cite this article as: Wang, Q., Lin, Z., Kim, T-K., Slominski, A.T., Miller, D.D., Li, W., Total synthesis of
biologically active 20S-hydroxyvitamin D3, Steroids (2015), doi: http://dx.doi.org/10.1016/j.steroids.2015.09.009
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Total synthesis of biologically active 20S-hydroxyvitamin D3
Qinghui Wang 1, Zongtao Lin 1, Tae-Kang Kim 2, Andrzej T. Slominski 2,3, Duane D. Miller 1, Wei Li 1,*
1Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Science
Center, Memphis, TN 38163, United States.
2Departments of Dermatology and Pathology, University of Alabama at Birmingham, Birmingham, AL
35294, United States.
3Veteran Affairs Medical Center, Birmingham, AL 35294, United States.
Address for all correspondence and reprint requests to:
Wei Li, PhD
Department of Pharmaceutical Sciences
University of Tennessee Health Science Center
881 Madison Avenue, Room 561
Memphis, TN 38163
Tel: (901) 448-7532
Fax: (901) 448-6828
E-mail: wli@uthsc.edu
1

Abstract
A total synthetic strategy of 20S-hydroxyvitamin D3 [20S-(OH)D3] involving modified synthesis of key
intermediates 7 and 12, Grignard reaction to stereoseletively generate 20S-OH and Wittig-Horner
coupling to establish D3 framework, was completed in 16 steps with an overall yield of 0.4 %. The
synthetic 20S-(OH)D3 activated vitamin D receptor (VDR) and initiated the expression of downstream
genes. In addition, 20S-(OH)D3 showed similar inhibitory potency as calcitriol [1,25(OH)2D3] on
proliferation of melanoma cells.
Keywords
20S-hydroxyvitamin D3; total chemical synthesis; PCR; reporter assay; thymidine incorporation; VDR
activation.
Abbreviations
D3, Vitamin D3; 20S-(OH)D3, 20S-hydroxyvitamin D3; 1,25(OH)2D3, 1α,25-dihydroxyvitamin D3;
EOM, Ethoxymethyl; TBS, t-butyldimethysilyl.
2

1. Introduction
25-hydroxylase (CYP2R1 or CYP27A1) in the liver and 1α-hydroxylase (CYP27B1) in the kidney are
the enzymes responsible for sequential metabolism of vitamin D3 to produce bioactive 1α,25-
dihydroxyvitamin D3 [1,25(OH)2D3, calcitriol] [1, 2]. 1,25(OH)2D3 activates nuclear vitamin D receptor
(VDR), which is found in almost all tissues of the body, to trigger numerous cellular effects including but
not limited to stimulation of cell differentiation and/or apoptosis, inhibition of cell proliferation,
regulation of secretory and immune factors of many cells, and cell protective functions, in a context
dependent fashion [3-7]. Therefore, active forms of vitamin D can be used in therapy of cancer,
hyperproliferative, autoimmune and metabolic disorders [8-10]. However, these applications are limited
by hypercalcemic (toxic) effect of calcitriol at pharmacological concentrations [11]. This side effect has
led to the development of more than 3,000 synthetic vitamin D3 analogs showing low calcemic activity
[12]. Some of these analogs such as paricalcitol, oxacalcitriol, falecalcitriol, tacalcitol and doxercalciferol
were used to treat secondary hyperparathyroidism and psoriasis [8, 13]. Besides psoriasis, calcipotriol is
also a promising agent for the treatment of pancreatic cancer and is currently undergoing human clinical
trial [14] (Figure 1).
Mammalian cytochrome P450 side-chain cleavage enzyme (P450scc or CYP11A1) not only cleaves the
side chain of cholesterol to produce pregnenolone (precursor of all steroids) [15, 16] but also hydroxylates
vitamin D3 in a sequential fashion [16-20] starting from C20 to form 20S-hydroxyvitamin D3 [20S-
(OH)D3] (1, Figure 1), which is subsequently converted to di- and trihydroxy metabolites [17, 19-22].
Functional studies showed that 20S-(OH)D3 not only stimulated keratinocyte differentiation program but
also inhibited NF-κB activity in human keratinocytes [23]. In addition, it has shown anti-inflammatory
activities, strong anti-proliferative effects, anti-leukemia and tumorostatic effects [23-27], protective
effects against ultra-violet B (UVB) induced damage [28], as well as antifibrotic activity in vivo [29].
These activities are mediated either through activation of the VDR [23, 30] or inhibition of RORα and
RORγ transcriptional activities [31]. More importantly, while having comparable anti-proliferative
potency with 1,25(OH)2D3 which has strong hypercalcemic toxicity at a concentration of 1µg/kg, 20S-
(OH)D3 is not hypercalcemic at concentrations as high as 60 µg/kg [24, 26, 28, 32].
Unfortunately, in-depth evaluation of the biological activity for 20S-(OH)D3 or its analogs was
hampered by lack of an efficient synthetic method of its production [33] without using costly enzymatic
approaches [20, 21, 34]. 20S-(OH)D3 was firstly chemically synthesized from pregnenolone acetate via
Grignard reaction and low yielding UVB irradiation to afford low milligram scale of 20S-(OH)D3 stereo-
specifically. However, this method also generated structurally similar and physicochemically active by-
products including previtamin D3, lumisterol and tachysterol which presented a significant challenge in
the purification of 20S-(OH)D3 even through preparative HPLC [33]. Due to these disadvantages, scope
3

of synthesis through UVB irradiation of 5,7-dienal precursor, 20S-(OH)-7-dehydrocholesterol was
confined to very few derivatives, if not to almost exclusive production of 20S-(OH)D3. Therefore, an
approach that could easily generate large quantity of 20S-(OH)D3 with high quality ( > 98% purity)
would facilitate further modifications on the side chain to determine the structure-activity relationships of
this promising 20S-hydroxyl scaffold. Herein, we report an efficient total synthetic route for
stereospecific 20S-(OH)D3.
2. Experimental
2.1. General methods
Tetrahydrofuran was distilled from sodium-benzophenone. All other solvents and chemical reagents
were obtained from commercial sources and directly used without further purification. Ergocalciferol was
purchased from Chem Impex International Inc. Glassware was oven-dried before use. All reactions were
performed under an argon atmosphere. TLC was performed on silica gel 60 GF254 and monitored under
UV light or visualized using phosphomolybdic acid reagent. Flash chromatography was performed on
230-400 mesh silica gel (Fisher Scientific). Preparative TLC was performed on Analtech TLC Uniplates
(250 µm). Melting points were recorded on a MPA100 Automated Melting Point Apparatus. NMR
spectra were obtained on a Bruker Ascend 400 (Billerica, MA) spectrometer or a Varian Inova-500
spectrometer (Agilent Technologies, Santa Clara, CA). Chemical shifts are given in ppm with
tetramethylsilane (TMS) as an internal reference. All coupling constants (J) are given in Hertz (Hz).
2.2. Chemistry
2.2.1. (4R,7aR)-1-((2R,5R,E)-5,6-dimethylhept-3-en-2-yl)-4-((R,Z)-1-hydroxy-2-((S)-5-hydroxy-2-
methylenecyclohexylidene)ethyl)-7a-methyloctahydro-1H-inden-4-ol (2).
Following a reported procedure [35], to a solution of ergocalciferol (20 g, 50.5 mmol) in ethanol (2 L)
at -45 oC was added dropwise solution of potassium permanganate (9.0 g, 58.7 mmol) in water (300 mL),
o
the mixture was stirred for half an hour and further 1 hour at -15 C and then warmed to room
temperature. After 3 hours, the precipitate was filtered off and the solution was evaporated to give
yellowish crude oil. The crude oil was purified on silica gel column chromatography eluting with
hexane/ethyl acetate (3:1) to give pure triol compound 2 as white solid (10.5 g, 65%) and unreacted
starting material (5.1 g). Mp: 170-172 °C (MeOH). 1H NMR (400 MHz, CD3OD): δ 5.52 (dd, J = 9.9, 1.7
Hz, 1H, 6-H), 5.30 – 5.09 (m, 2H, 22/23-H), 5.02 – 4.98 (m, 2H, 7/19-H), 4.90 (d, J = 9.9 Hz, 1H, 19-H),
3.65 (m, 1H, 3-H), 2.55 (ddd, J = 12.4, 4.5, 1.9 Hz, 1H), 2.49 – 2.38 (m, 1H), 2.17 – 1.90 (m, 5H), 1.90 –
4

1.68 (m, 4H), 1.62 (m, 1H), 1.56 – 1.39 (m, 4H), 1.39 – 1.03 (m, 5H), 1.00 (d, J = 6.6 Hz, 3H, 21-H), 0.94
13
(d, J = 6.8 Hz, 3H, 28-H), 0.89 – 0.81 (t, J = 6.4 Hz, 9H, 18/26/27-H). C NMR (101 MHz, CD3OD): δ
147.35, 140.78, 137.06, 133.12, 126.72, 111.72, 76.25 (C7), 71.42 (C8 or C3), 71.39 (C3 or C8), 61.04,
58.79, 47.60, 45.14, 44.36, 41.70, 41.53, 39.23, 37.21, 34.50, 34.38, 29.13, 22.87, 21.76, 21.29, 20.52,
20.12, 18.27, 13.59. ESI-HRMS: calculated for C28H46O3Na [M+Na]+ 453.3345, found 453.3352.
2.2.2. (S,Z)-3-(2-hydroxyethylidene)-4-methylenecyclohexanol(3) and (4S,7aR)-1-((2R,5R,E)-5,6 -
dimethylhept-3-en-2-yl)-7a-methyloctahydro-1H-inden-4-ol (4).
To a suspension of 2 (6.0 g, 13.9 mmol) and sodium carbonate (14.6 g, 138 mmol) in dichloromethane
(50 mL) was added lead tetraacetate (6.7 g, 15.3 mmol) in portions at 0 °C. After 2 hours, the reaction
was quenched with ethylene glycol and the mixture was vigorously stirred at 0 °C for 10 minutes. Water
was then added and the mixture was extracted with dichloromethane for five times, washed with brine
and dried with Na2SO4. The combined extracts were evaporated to give crude oil mixture, which was used
for next step without further purification. To a solution of the above crude oily mixture in benzene (100
mL) at 0 °C under argon with stirring, Red-Al (28.7 mmol, 8.6 mL) was added dropwise and the mixture
was stirred for 2 hours. On completion reaction was quenched with water. The mixture was filtered off
and extracted with ethyl acetate, washed with brine and dried with anhydrous Na2SO4. Organic phase was
evaporated to generate crude oil mixture which was purified by flash chromatography on silica. Elution
with hexane/ethyl acetate (10:1) gave pure alcohol compound 4 as transparent oil (3.3 g, 86% for two
steps), further elution with hexane/ethyl acetate (1:2) gave pure allylic alcohol 3 as colorless oil (0.91 g,
43% for two steps). compound 3: 1H NMR (400 MHz, CDCl3): δ 5.45 (t, J = 6.6 Hz, 1H, 6-H), 4.97 (dd, J
= 2.4, 1.3 Hz, 1H, 19-H), 4.59 (d, J = 2.3 Hz, 1H, 19-H), 4.25 (ddd, J = 12.7, 7.0, 1.2 Hz, 1H, 7-H), 4.14
(ddd, J = 12.6, 6.2, 1.3 Hz, 1H, 7-H), 3.94 (m, 1H, 3-H), 2.56 – 2.33 (m, 2H), 2.24 (dd, J = 13.2, 7.1 Hz,
13
1H), 2.19 – 2.06 (m, 1H), 1.87 (m, 1H), 1.75 – 1.60 (m, 1H). C NMR (101 MHz, CDCl3): δ 144.51
(C10), 139.11 (C5), 125.95 (C6), 112.23 (C19), 68.64 (C3), 59.34 (C7), 44.82, 34.62, 31.45. ESI-HRMS:
1
calculated for C9H11, [M-2H2O+H]+ 119.0861, found 119.0861. compound 4: H NMR (400 MHz,
CDCl3): δ 5.25 – 5.02 (m, 2H, C22/23-H), 4.02 (d, J = 2.8 Hz, 1H, C8-H), 2.04 – 1.88 (m, 2H), 1.86 –
1.73 (m, 3H), 1.73 – 1.60 (m, 1H), 1.56 – 1.33 (m, 5H), 1.33 – 1.02 (m, 5H), 0.96 (d, J = 6.6 Hz, 3H, 22-
13
H), 0.92 (s, 3H, C18-H), 0.89 (d, J = 11.2 Hz, 3H, 23-H), 0.80 (t, J = 6.6 Hz, 6H, C26/27-H). C NMR
(101 MHz, CDCl3): δ 135.59, 131.70, 69.14 (C8), 56.48, 52.72, 42.78, 41.65, 40.32, 39.75, 33.53, 33.00,
27.57, 22.46, 20.71, 19.88, 19.55, 17.57, 17.40, 13.61. ESI-HRMS: calculated for C19H33 [M-H2O+H]+
261.2582, found 261.2582.
5

2.2.3. (S,Z)-tert-butyl(2-(5-((tert-butyldimethylsilyl)oxy)-2-
methylenecyclohexylidene)ethoxy)dimethylsilane (5).
Imidazole (1.4 g, 21.2 mmol) and tert-butyldimethylsilyl chloride (4.8 g, 31.6 mmol) was sequentially
added to a solution of compound 3 (0.82 g, 5.3 mmol) in dichloromethane (50 mL) in a round bottom
flask with stirring. The resulting suspension was stirred for 2 hours. Water was then added and the
mixture was extracted with dichloromethane, washed with brine and dried with anhydrous Na2SO4.
Evaporation under vacuum gave the oily residue which was subjected to flash chromatography on silica.
1
Elution with hexane/ethyl acetate (80:1) gave pure compound 5 as colorless oil (1.8 g, 88%). H NMR
(400 MHz, CDCl3): δ 5.38 (ddt, J = 6.8, 5.3, 1.3 Hz, 1H, 6-H), 4.95 (p, J = 1.1 Hz, 1H, 19-H), 4.57 (dd, J
= 2.4, 1.3 Hz, 1H, 19-H), 4.31 (ddt, J = 13.0, 7.2, 0.9 Hz, 1H, 7-H), 4.21 (ddd, J = 12.9, 5.3, 2.1 Hz, 1H,
7-H), 3.82 (tt, J = 8.9, 3.9 Hz, 1H, 3-H), 2.47 – 2.31 (m, 2H), 2.22 – 2.13 (m, 1H), 2.06 (m, 1H), 1.93 –
13
1.80 (m, 1H), 1.56 (m, 1H), 0.89 (d, J = 4.7 Hz, 18H, TBS), 0.08 – 0.03 (s, 12H, TBS). C NMR (101
MHz, CDCl3): δ 144.89 (C10), 138.24 (C5), 126.29 (C6), 111.95 (C19), 70.11 (C3), 60.59 (C7), 45.93,
36.03, 32.15, 26.01 (TBS), 25.86 (TBS), 18.43, 18.18, -4.65 (TBS), -4.69 (TBS), -4.99 (TBS), -5.05
(TBS). ESI-HRMS: calculated for C21H42O2Si2Na [M+Na]+ 405.2621, found 405.2629.
2.2.4. (S,Z)-2-(5-((tert-butyldimethylsilyl)oxy)-2-methylenecyclohexylidene)ethanol (6).
To a solution of compound 5 (1.7 g, 4.4 mmol) in anhydrous THF (20 mL) in a round bottom flask at -
5 °C under nitrogen, tetra-n-butylammonium fluoride (1.0 M in THF, 4.8 mmol) was added dropwise
with stirring. The resulting mixture was stirred for 3 hours, water was then added and the brown mixture
was extracted with dichloromethane, washed with brine and dried with anhydrous Na2SO4. The combined
extracts were evaporated under vacuum to give the oily residue which was purified with flash
chromatography on silica. Pure allylic alcohol 6 was eluted out with hexane/ethyl acetate (50:1) as
colorless oil (0.93 g, 78%). 1H NMR (400 MHz, CDCl3): δ 5.44 (ddt, J = 7.3, 5.9, 1.3 Hz, 1H, 6-H), 4.96
(d, J = 1.2 Hz, 1H, 19-H), 4.62 (t, J = 2.4 Hz, 1H, 19-H), 4.43 – 4.01 (m, 2H, 7-H), 3.84 (m, 1H, 3-H),
2.48 – 2.33 (m, 2H), 2.26 – 2.14 (m, 1H), 2.13 – 2.01 (m, 1H), 1.87 (m, 1H), 1.58 (m, 1H), 0.88 (s, 9H,
13
TBS), 0.06 (d, J = 2.9 Hz, 6H, TBS). C NMR (101 MHz, CDCl3): δ 144.83, 140.52, 124.88, 111.91,
70.11 (C3), 59.82 (C7), 46.02, 35.98, 32.26, 25.82 (TBS), 18.15, -4.67 (TBS), -4.71 (TBS). ESI-HRMS:
calculated for C15H28O2SiNa [M+Na]+ 291.1756, found 291.1755.
2.2.5. (S,Z)-(2-(5-((tert-butyldimethylsilyl)oxy)-2-methylenecyclohexylidene)ethyl)diphenylphosphine
oxide (7).
To a solution of the allylic alcohol 6 (0.81 g, 3.0 mmol) in anhydrous THF (20 mL) was added n-BuLi
(2.5 M in hexanes, 30.0 mmol) under argon at 0 °C with stirring. After 5 minutes, a solution of p-tosyl
6

chloride (598 mg, 3.15 mmol) dissolved in anhydrous THF (10 mL) was added to the allylic alcohol-BuLi
solution. On the other side, to a solution of diphenylphosphine (1.02 mL, 6.0 mmol) in anhydrous THF (5
mL) under argon at 0 °C with stirring n-BuLi (2.5 M in hexanes, 2.2 mL, 5.7 mmol) was added dropwise
to provide a red solution. After 10 minutes 4.1 mL of the red solution was then drawn to add to the
solution of tosylate. The mixture was stirred for 30 minutes at 0 °C and quenched with H2O (1 mL).
Evaporation of the solution under vacuum gave a phosphine residue. To the phosphine residue in
dichloromethane (15 mL) at 0 °C with stirring was added 10% H2O2 (1.1 mL). Reaction was quenched
with water, extracted with dichloromethane, washed with cold saturated sodium sulfite solution and brine
and dried with Na2SO4. Organic solvent was removed under vacuum to give crude residue which was
subjected to flash chromatography on silica for purification. Elution with hexane/ethyl acetate (3:1) gave
o
1
pure phosphine oxide compound 7 as white solid (800 mg, 59%). Mp: 94-96 C (EtOAc). H NMR (400
MHz, CDCl3): δ 7.71 (m, 4H, Ph), 7.56 – 7.36 (m, 6H, Ph), 5.35 (q, J = 7.2 Hz, 1H, 6-H), 4.92 (t, J = 1.9
Hz, 1H, 19-H), 4.68 (dd, J = 2.4, 1.3 Hz, 1H, 19-H), 3.52 (m, 1H, 3-H), 3.36 (m, 1H, 7-H), 3.20 (m, 1H,
7-H), 2.44 – 2.31 (m, 1H), 2.22 (dt, J = 13.3, 4.7 Hz, 1H), 2.15 – 2.05 (m, 1H), 1.81 – 1.71 (m, 1H), 1.66
(m, 1H), 1.47 (m, 1H), 0.83 (s, 9H, TBS), -0.00 (d, J = 2.0 Hz, 6H, TBS); 13C NMR (101 MHz, CDCl3): δ
145.03, 145.01, 142.62, 142.50, 133.49, 132.99, 132.52, 132.00, 131.75, 131.73, 131.61, 131.58, 131.20,
131.11, 130.95, 130.86, 128.59, 128.48, 128.44, 128.32, 113.19, 113.11, 111.60, 111.59, 70.46 (C3),
70.43, 46.68, 46.66, 36.21, 32.48, 32.46, 31.44, 30.74, 25.82 (TBS), 18.12, -4.69 (TBS), -4.75 (TBS).
ESI-HRMS: calculated for C27H37O2PSiNa [M+Na]+ 475.2198, found 475.2203.
2.2.6. (4S,7aR)-1-((2R,5R,E)-5,6-dimethylhept-3-en-2-yl)-7a-methyloctahydro-1H-inden-4-yl acetate (8).
Pyridine (1.3 mL, 16.1 mmol), 4-dimethylaminopyridine (352 mg, 2.88 mmol) and acetyl anhydride
(0.98 mL, 104 mmol) was sequentially added to a solution of compound 4 (3.2 g, 11.5 mmol) in
anhydrous dichloromethane (30 mL) in a round bottom flask under argon. The resulting mixture was
stirred for 3 hours. Water was then added and the mixture was extracted with dichloromethane, washed
with saturated NaHCO3, 0.1 M HCl and brine and then dried with Na2SO4. Evaporation under vacuum
gave the oily residue which was purified with flash chromatography on silica. Elution with hexane/ethyl
acetate (30:1) gave pure compound 8 as colorless oil (3.4 g, 92%). 1H NMR (400 MHz, CDCl3): δ 5.31 –
5.00 (m, 3H, 8/22/23-H), 2.12 – 1.92 (m, 5H), 1.92 – 1.53 (m, 4H), 1.53 – 1.03 (m, 10H), 0.99 (d, J = 2.4
13
Hz, 3H, 22-H), 0.94 – 0.85 (m, 6H), 0.82 (t, J = 6.8 Hz, 6H, 26/27-H). C NMR (101 MHz, CDCl3): δ
170.73 (acetyl), 135.47, 131.90, 71.35 (C8), 56.31, 51.41, 42.79, 41.82, 39.90, 39.84, 33.05, 30.50, 27.47,
22.61, 21.31, 20.77, 19.93, 19.60, 17.90, 17.60, 13.17. ESI-HRMS: calculated for C21H36O2Na [M+Na]+
343.2613, found 343.2614.
7

2.2.7. (4S,7aR)-1-((2S,3R,4R,5R)-3,4-dihydroxy-5,6-dimethylheptan-2-yl)-7a-methyloctahydro-1H-inden-
4-yl acetate (9).
To a solution of the compound 8 (3.3 g, 10.3 mmol) in acetone (40 mL) was added N-
methylmorpholine N-oxide (6.0 g, 51.5 mmol) and 2.5% osmium tetroxide in tert-butanol (6.5 mL, 0.52
mmol) at room temperature with stirring. After 48 hours, water and ethyl acetate was added and the
organic phase was separated, washed with brine and dried with Na2SO4. Evaporation under vacuum gave
the oily residue which was purified with flash chromatography on silica. Elution with hexane/ethyl
acetate (10:1) gave pure compound 9 as colorless oil (2.44 g, 67%). 1H NMR (400 MHz, CDCl3): δ 5.14
(d, J = 2.6 Hz, 1H, 8-H), 3.71 (dd, J = 2.4, 4.4 Hz, 1H, 22-H), 3.59 (t, J = 2.8 Hz, 1H, 23-H), 2.39 (s, 1H),
2.04 (s, 4H), 1.94 – 1.79 (m, 2H), 1.70 (m, 5H), 1.55 – 1.09 (m, 9H), 1.00 (d, J = 6.9 Hz, 3H, 21-H), 0.96
13
(d, J = 6.7 Hz, 3H, 28-H), 0.92 – 0.84 (m, 9H, 18/26/27-H). C NMR (101 MHz, CDCl3): δ 170.80
(acetyl), 73.26 (C23), 71.19 (C8), 70.26 (C22), 53.02, 51.04, 44.11, 43.93, 42.45, 41.65, 40.00, 30.49,
29.89, 29.75, 26.91, 22.82, 21.44, 21.34, 18.94, 17.88, 13.91, 13.52, 12.83, 9.91. ESI-HRMS: calculated
for C21H38O4Na [M+Na]+ 377.2668, found 377.2668.
2.2.8. (4S,7aR)-7a-methyl-1-((S)-1-oxopropan-2-yl)octahydro-1H-inden-4-yl acetate (10).
To a suspension of compound 9 (2.2 g, 6.2 mmol) in THF-H2O (4:1, 10 mL), sodium periodate (1.45 g,
6.82 mmol) was added at 0 °C in portions with stirring. After 1 hour, the reaction was quenched with
ethylene glycol and the mixture was stirred at 0 °C for 10 minutes. Water was then added and the mixture
was extracted with ethyl acetate for three times, washed with brine and dried with Na2SO4. The combined
extracts were evaporated to give crude oil, which was purified with flash chromatography on silica.
Elution with hexane/ethyl acetate (5:1) gave pure compound 10 as transparent oil (1.03 g, 66%). 1H NMR
(400 MHz, CDCl3): δ 9.57 (d, J = 3.2 Hz, 1H, CHO ), 5.17 (d, J = 2.0 Hz, 1H, 8-H), 2.36 (m, 1H, 20-H),
2.05 (s, 3H), 1.97 (dt, J = 12.3, 3.3 Hz, 1H), 1.89 – 1.71 (m, 3H), 1.59 – 1.24 (m, 8H), 1.11 (d, J = 6.8 Hz,
13
3H, 21-H), 0.93 (s, 3H, 18-H). C NMR (101 MHz, CDCl3): δ 204.78 (CHO), 170.70 (acetyl), 70.89
(C8), 51.37, 50.74, 49.17, 42.47, 39.75, 30.47, 26.00, 22.95, 21.32, 17.83, 13.42, 13.35. ESI-HRMS:
calculated for C15H23O2 [M-H2O+H]+ 235.1698, found 235.1696.
2.2.9. (1S,4S,7aS)-1-acetyl-7a-methyloctahydro-1H-inden-4-yl acetate (12).
To a stirred solution of compound 10 (1.0 g, 3.9 mmol) in benzene (8 mL) was added morpholine
(0.524 mL, 4.8 mmol) and p-toluenesulfonic acid (34mg, 0.195 mmol). The mixture was refluxed over a
Dean-Stark water separator overnight. During the course of the reaction the theoretical amount of water
(~70 mg) was separated. Upon completion as indicated by mass spectrum, the reaction mixture was
8

cooled to room temperature and solvent was removed under vacuum to afford yellowish oil which was
immediately used for next step.
To a stirred solution of the above crude mixture in acetone (20 mL) was added alumina (481 mg, 4.72
mmol) supported potassium permanganate (616 mg, 3.9 mmol) in portions. The reaction mixture was
filtered after 2 hours and washed with ethyl acetate. Solvents were removed under vacuum to afford crude
oily product which was purified with flash chromatography on silica. Elution with hexane/ethyl acetate
(6:1) gave pure compound 12 as colorless oil (659 mg, 70% for two steps). 1H NMR (400 MHz, CDCl3):
δ 5.16 (d, J = 2.8 Hz, 1H, 8-H), 2.48 (dd, J = 10.0, 8.0 Hz, 1H, 17-H), 2.20 – 1.99 (m, 8H), 1.92 – 1.82
13
(m, 1H), 1.70 – 1.39 (m, 8H), 0.81 (s, 3H, 18-H). C NMR (101 MHz, CDCl3): δ 208.52 (C20), 170.26
(acetyl), 70.27 (C8), 63.54, 51.15, 43.06, 38.85, 31.26, 30.08, 22.54, 21.46, 20.98, 17.64, 14.51 (C18).
ESI-HRMS: calculated for C14H22O3Na [M+Na]+ 261.1467, found 261.1467.
2.2.10. (1S,4S,7aS)-1-((S)-2-hydroxy-6-methylheptan-2-yl)-7a-methyloctahydro-1H-inden-4-ol (14).
To a stirred suspension of magnesium (181 mg, 7.5 mmol) in anhydrous THF (10 mL) was added 1-
bromo-4-methyl-pentane ( 0.733 mL, 5.0 mmol ) under argon at room temperature. The mixture was then
refluxed for 2 hours and cooled down in ice bath to give Grignard reagent 13 which was used without
further purification. Compound 12 (100 mg, 0.42 mmol) in 2 mL THF was subsequently added to the
reaction mixture which was warmed to room temperature after 30 minutes and stirred overnight. Cooled
saturated NH4Cl solution was added to quench the reaction and the mixture was extracted with ethyl
acetate for three times, washed with brine and dried with Na2SO4. The combined extracts were evaporated
to give crude oil, which was purified with flash chromatography on silica. Elution with hexane/ethyl
1
acetate (3:1) afforded pure compound 14 as colorless oil (94 mg, 80%). H NMR (400 MHz, CDCl3): δ
4.09 (d, J = 2.8 Hz, 1H, 8-H), 2.14 – 2.02 (m, 1H), 1.91 – 1.74 (m, 3H), 1.74 – 1.37 (m, 9H), 1.37 – 1.08
13
(m, 14H), 0.87 (dd, J = 6.7, 0.8 Hz, 6H, 26/27-H). C NMR (101 MHz, CDCl3): δ 75.14 (C20), 69.41
(C8), 57.82, 52.55, 44.10, 42.52, 40.77, 39.56, 33.42, 27.92, 26.29, 22.71, 22.55, 22.21, 21.94, 21.30,
17.39, 15.44. ESI-HRMS: calculated for C18H31 [M-2H2O+H]+ 247.2426, found 247.2430.
2.2.11. (1S,7aR)-1-((S)-2-hydroxy-6-methylheptan-2-yl)-7a-methylhexahydro-1H-inden-4(2H)-one (15).
To a stirred solution of compound 14 (84 mg, 0.3 mmol) in dichloromethane (10 mL) was added
pyridinium dichromate (225 mg, 0.6 mmol) at room temperature. The reaction mixture was stirred for 1
hour and filtered through celite. The celite was washed with dichloromethane and the combined solvent
was removed under vacuum to give crude oily product which was purified with flash chromatography on
1
silica. Elution with hexane/ethyl acetate (8:1) gave pure compound 15 as colorless oil (74 mg, 89%). H
NMR (400 MHz, CDCl3): δ 2.43 (dd, J = 10.5, 6.9 Hz, 1H, 9-H), 2.33 – 2.13 (m, 3H, 9/12/14-H), 2.07 –
9

1.83 (m, 2H), 1.82 – 1.66 (m, 4H), 1.66 – 1.40 (m, 4H), 1.35 – 1.10 (m, 9H), 0.87 (d, J = 6.6 Hz, 6H,
13
26/27-H), 0.80 (s, 3H, 18-H)). C NMR (101 MHz, CDCl3): δ 211.99 (C8), 74.63 (C20), 62.10, 58.04,
49.84, 44.07, 40.80, 39.47, 39.26, 27.89, 26.53, 23.90, 22.67, 22.52, 21.95, 21.77, 18.73, 14.37. ESI-
HRMS: calculated for C18H31O [M-H2O+H]+ 263.2375, found 263.2377.
2.2.12. (1S,7aR)-1-((S)-2-(ethoxymethoxy)-6-methylheptan-2-yl)-7a-methylhexahydro-1H-inden-4(2H)-
one (16).
To a stirred solution of compound 15 (67 mg, 0.24 mmol), N,N-diisopropylethylamine (83 µL, 0.48
mmol) and 4-dimethylaminopyridine (12 mg, 0.096 mmol) in anhydrous dichloromethane (3 mL) was
added chloromethyl ethyl ether (45 µL, 0.48 mmol) dropwise at ice temperature. The resulting mixture
was stirred for overnight at room temperature. Water was then added and the reaction mixture was
extracted with dichloromethane, washed with brine and dried with Na2SO4. The combined extracts were
evaporated under vacuum to give the oily residue which was separated with flash chromatography on
silica. Elution with hexane/ethyl acetate (30:1) gave pure compound 16 as colorless oil (73 mg, 90%). 1H
NMR (400 MHz, CDCl3): δ 4.81 – 4.64 (m, 2H, EOM), 3.69 – 3.45 (m, 2H, EOM), 2.41 (dd, J = 11.5, 7.0
Hz, 1H), 2.32 – 2.06 (m, 3H), 2.03 – 1.43 (m, 11H), 1.31 (s, 3H, 21-H), 1.26 – 1.09 (m, 7H), 0.86 (dd, J =
6.6, 0.9 Hz, 6H, 26/27-H), 0.76 (s, 3H, 18-H). 13C NMR (101 MHz, CDCl3): δ 212.17 (C8), 88.76
(EOM), 79.75 (C20), 63.20 (EOM), 62.19, 57.31, 49.72, 40.83, 40.26, 39.70, 39.40, 27.87, 23.92, 23.21,
22.70, 22.57, 22.50, 21.70, 18.74, 15.14, 14.66. ESI-HRMS: calculated for C21H38O3Na [M+Na]+
361.2719, found 361.2719.
2.2.13. tert-butyl(((1S,Z)-3-((E)-2-((1S,7aS)-1-((S)-2-(ethoxymethoxy)-6-methylheptan-2-yl)-7a-
methylhexahydro-1H-inden-4(2H)-ylidene)ethylidene)-4-methylenecyclohexyl)oxy)dimethylsilane (17).
To a stirred solution of compound 7 (51 mg, 0.15 mmol) in anhydrous THF (3 mL) was added
phenyllithium (1.8 M in hexanes, 0.37 mmol) under argon at -78 oC. The mixture was stirred for another
30 minutes at this temperature and was added precooled compound 16 (167 mg, 0.37 mmol) in 100µL
anhydrous THF. Reaction was kept stirring at this temperature for 3 hours and further 12 hours at room
temperature. Water was then added and the reaction mixture was extracted with ethyl acetate, washed
with brine and dried with Na2SO4. The combined extracts were evaporated under vacuum to give the oily
residue which was purified with flash chromatography on silica. Elution with hexane/ethyl acetate (30:1)
1
gave pure compound 17 as colorless oil (45 mg, 52%). H NMR (400 MHz, CDCl3): δ 6.16 (d, J = 11.2
Hz, 1H, 6-H), 6.01 (d, J = 11.2 Hz, 1H, 7-H), 5.02 (s, 1H, 19-H), 4.85 – 4.64 (m, 3H, 19-H and EOM),
3.81 (m, 1H, H-3), 3.71 – 3.50 (m, 2H, EOM), 2.82 (d, J = 12.4 Hz, 1H, 9-H), 2.44 (dd, J = 13.0, 4.2 Hz,
1H), 2.36 (dt, J = 13.5, 4.8 Hz, 1H), 2.28 – 2.17 (m, 1H), 2.16 – 1.93 (m, 3H), 1.93 – 1.79 (m, 2H), 1.77 –
10

1.44 (m, 13H), 1.39 – 1.08 (m, 12H), 0.87 (d, J = 7.7 Hz, 15H, 26/27-H and TBS), 0.67 (s, 3H, 18-H),
13
0.06 (d, J = 2.2 Hz, 6H, TBS); C NMR (101 MHz, CDCl3): δ 145.34 (C10), 141.18 (C8), 136.36 (C5),
121.32 (C6), 118.21 (C7), 112.13 (C19), 88.80 (EOM), 80.42 (C20), 70.54 (C3), 63.14 (EOM), 57.38,
56.63, 46.84, 45.73, 40.98, 40.30, 39.79, 36.34, 32.73, 28.80, 27.94, 25.99, 25.96, 25.91, 25.88, 23.32,
22.97, 22.77, 22.55, 21.98, 21.91, 18.18, 15.19, 14.15, -4.59 (TBS), -4.63 (TBS). ESI-HRMS: calculated
for C36H64O3SiNa [M+Na]+ 595.4522, found 595.4509.
2.2.14. (1S,Z)-3-((E)-2-((1S,7aS)-1-((S)-2-hydroxy-6-methylheptan-2-yl)-7a-methylhexahydro-1H-inden-
4(2H)-ylidene)ethylidene)-4-methylenecyclohexanol (1).
To a stirred solution of compound 17 (10 mg, 0.017 mmol) in MeOH-DCM (5:1, 3 mL) was added
camphorsulfonic acid (4.3 mg, 0.020 mmol) at room temperature. The mixture was stirred for 3 hours and
saturated NaHCO3 was added. The mixture was extracted with dichloromethane, washed with brine and
dried with Na2SO4. The combined extracts were evaporated under vacuo to give the oily residue which
was purified with preparative TLC (hexane/ethyl acetate = 5:1) to afford pure compound 1 as colorless
solid (2.6 mg, 37%). Mp: 91-93 oC (MeOH). 1H NMR (400 MHz, CD3OD): δ 6.22 (d, J = 11.2 Hz, 1H, 6-
H), 6.02 (d, J = 11.2 Hz, 1H, 7-H), 5.08 – 5.00 (s, 1H, 19-H), 4.74 (d, J = 1.6 Hz, 1H, 19-H), 3.76 (m, 1H,
3-H), 2.85 (dd, J = 11.9, 4.0 Hz, 1H, 9-H), 2.58 – 2.48 (dd, 13.0, 4.2, 1H, 4-H), 2.40 (dt, J = 13.6, 4.9 Hz,
1H, 1-H), 2.25 – 1.91 (m, 5H), 1.81 – 1.62 (m, 5H), 1.61 – 1.43 (m, 6H), 1.43 – 1.09 (m, 11H), 0.89 (d, J
= 6.6 Hz, 6H, 26/27-H), 0.69 (s, 3H, 18-H). 13C NMR (101 MHz, CD3OD): δ 147.00 (C10), 142.30 (C8),
137.34 (C5), 122.61 (C6), 119.37 (C7), 112.65 (C19) , 75.99 (C20), 70.55 (C3), 59.89, 57.80, 47.02,
45.11, 42.25, 40.95, 36.60, 33.59, 29.84, 29.17, 26.15, 24.44, 23.16, 23.06, 23.02, 22.97, 14.14. ESI-
HRMS: calculated for C27H44O2Na [M+Na]+ 423.3239, found 423.3235.
2.3. VDRE luciferase reporter assay
Dulbecco’s Modified Eagle Medium (DMEM) supplemented with glucose, L-gultamin, pyridoxine
hydrochloride, together with 5% fetal bovine serum (FBS) and 1% penicillin/streptomycin/amphotericin
antibiotic solution (Ab) (Sigma-Aldrich, St. Louis, MO) was used to culture immortalized human
keratinocytes (HaCaT). Jurkat cells were cultured in RPMI 1640 medium supplemented with 10% FBS
and 1% Ab. All cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2. Jurkat and
HaCaT cells were transduced with lentiviral VDRE luciferase using Cignal Lenti VDRE Reporter (luc)
Kit according to manufacturer’s protocol (QIAGEN, Valencia, CA). After one week selection by
11

puromycin (1 µg/mL), transduced HaCaT, Jurkat and Caco-2 cells were seeded in 96-well plate (1000
cells/well) using partial media (without FBS) for 24 h incubation. DMSO solutions of test compounds
were added to cells, which were then incubated for another 24 h and measured for luciferase signal
following the manufacturer’s procedure of ONE-GloTM Luciferase Assay System (Promega, Madison,
WI). The concentrations of DMSO were maintained consistently at 0.1% for compound treatment, and
0.1% DMSO were used as negative control. Concentration (100 nM) of 20S-(OH)D3 and 1,25(OH)2D3
was used and tested in triplicates.
2.4. PCR
HaCaT cells were cultured as described above. The RNA from HaCaT keratinocytes treated with 20S-
(OH)D3, 1,25(OH)2D3 or DMSO, was isolated using the Absolutely RNA Miniprep Kit (Stratagen).
Reverse transcription (100 ng RNA/reaction) was performed using the Transcriptor First Strand cDNA
Synthesis Kit (Roche Inc, Mannheim, Germany). Real-time PCR was performed using cDNA diluted 10-
fold in sterile water and a SYBR Green PCR Master Mix. Reactions (in triplicate) were performed at 50
ºC for 2 min, 95 ºC for 10 min and then 40 cycles of 95 ºC for 15 sec, 60 ºC for 30 sec and 72 ºC for 30
sec. Data were collected on a Roche Light Cycler 480. The amount of amplified product for each gene
was compared to that of β-actin as a housekeeping gene using a comparative Ct method [25].
2.5. Anti-proliferation assay
To test anti-proliferative effect of 20S-(OH)D3 in comparison to 1,25(OH)2D3 we measured [3H]-
thymidine (Moravek Biochemicals Inc. Brea, CA) incorporation into the DNA of human SKEML-188
melanoma line as described previously[30]. Briefly, the cells were grown in 24-well plates using Ham’s
F10 media (Mediatech, Inc. Manassas, VA, USA) containing 5% charcoal treated FBS (Atlanta
Biologicals, Lawrenceville, GA, USA) at 37˚C in 5% CO2 until 30% confluence. The cells were
synchronized in serum free medium for 1 day and then grown in the media containing serum and 10-8 to
10-10 M of the compounds or vehicle for 72 hours. For the final 4 hours, the cells were incubated with
12

[3H]-thymidine (final concentration of 0.5 µCi/mL). Afterwards media were replaced with 10% TCA in
PBS (phosphate-buffered saline) and plates were washed twice with 1 mL PBS. The cells were lysed with
1 N NaOH/1% SDS, the extracts mixed with scintillation cocktail, and radioactivity was counted using
Packard Matrix 9600 direct beta-counter (Packard, Meridan, CT, USA) as described previously [33].
3. Results
3.1. Chemistry
A retrosynthetic strategy was proposed as Scheme 1. The vitamin D3 scaffold could be established by
Wittig-Horner coupling reaction between phosphine oxide 7 and C/D-ring fragment 16. Due to steric
effect of 18-methyl, stereoselective generation of 20S-hydroxy of 16 could be achieved using Grignard
reagent 13 to attack 12. In addition, introducing a relatively bulky protecting group on 8S-hydroxy was
envisioned to assist the stereoselectivity of Grignard reaction. Conversion of VD2 to 7 and 12 could be
carried out through oxidative cleavages.
The synthesis of phosphine oxide 7 and protected C/D-ring ketone 12 were performed following
literature reports [35, 36]. To prepare 7 and 12 in large scale so as to facilitate future structure-activity
relationship studies, we modified the synthesis which were outlined as Scheme 2 and Scheme 3. Initially,
commercially available ergocalciferol was regioselectively dihydroxylated on the triene linkage to
provide triol 2 with a yield of 65% when treating with potassium permanganate in ethanol at low
temperature. Due to steric effect of 18-methyl, single 7R,8R-diostereoisomer was exclusively isolated
over chromatography [35]. It is worth mentioning that when 3-OH was protected with TBS,
dihydroxylation of the triene did not proceed. Subsequent treatment of vicinol 2 with lead tetraacetate in
dichloromethane in the presence of sodium carbonate led to formation of corresponding C/D-ring ketone
and A-ring allylic aldehyde. Although complete consumption of 2 was indicated on TLC, after workup
procedures and purification using flash chromatography, A-ring allylic aldehyde ended up with poor yield
(<20%) while C/D-ring ketone was obtained in 94% yield. We found that A-ring allylic aldehyde was
unstable when exposed to air and moisture as clearly indicated by its spot on TLC and it should be used
immediately. Thus, after quenching the oxidation reaction with ethylene glycol followed by simple
13

workup procedure the crude oily mixture was directly used without purification to the following reduction
o
step using excess equivalent of sodium bisaluminumhydride (65% in toluene) in benzene at 0 C.
Gratifyingly, these modified procedures afforded pure A-ring allylic alcohol 3 and C/D-ring alcohol 4
with yield of 43% and 86% for two steps after chromatography, respectively. It is worth noting that after
TLC indicated completion of the oxidation reaction, quenching the reaction by adding ethylene glycol
was crucial to prevent further oxidation of the aldehyde to carboxylic acid. In addition, compared with
lithium aluminum hydride (LAH) and diisobutylaluminium hydride (DIBAL-H in toluene or
tetrahydrofuran), Red-Al (>65% in toluene) was more efficient and regioselective toreduce only aldehyde
rather than conjugated diene to form3. Compound 3 was subsequently protected with TBS to generate 5,
which underwent selective removal of TBS on the allylic moiety to produce alcohol 6 in 71% yield for
two steps when stirring with tetra-n-butylammonium fluoride (TBAF) in THF under low temperature (-5
oC to ice temperature). The final A-ring intermediate phosphine oxide 7 was prepared following literature
procedures in three steps with 59% overall yield [36].
Acetate 8 was produced when secondary alcohol 4 was treated with acetic anhydride and pyridine.
Compound 8 was subjected to Upjohn reaction stirring with osmium tetroxide (0.5% wt in t-butanol) and
N-methylmorpholine N-oxide in acetone at ambient temperature for 48 hr to form the major
diastereoisomer 9 due to strong steric effect of 21- and 24-methyl groups. While TLC indicated existence
of the other diastereomer as minor product, 9 was isolated in 67% yield. Cleavages of vicinol 9 in the
presence of sodium periodate and sodium carbonate in H2O-THF (1:4) provided aldehyde 10 in 66%
yield. Aldehyde 10 was then refluxed in a Dean-Stark apparatus stirring with morpholine and catalytic
amount of p-toluenesulfonic acid in benzene to form crude enamine 11 as, after dried on vacuum, slightly
yellowish oil. Due to its instability and sensitivity to moisture, the crude enamine mixture was used for
next step immediately without further purification. While the removal of C-22 on the chiron could be
accomplished when treating 11 with singlet oxygen [36], this photo-oxygenation procedure was rather
tedious. By referring to an alternative strategy [37], the yellowish oil was dissolved in acetone and treated
14

with alumina supported potassium permanganate that was made prior to use, to provide the other key
intermediate 12 in 70% for two steps after chromatography.
As shown in Scheme 4, Grignard reagent 13 was readily accessible by refluxing commercially
available 1-bromo-4-methyl-pentane with magnesium in anhydrous THF. After 2 hours, 13 was
immediately subjected to Grignard reaction to form the diol 14 with 80% yield. Similar to our previous
studies [33], only the 20S-epimer was isolated over chromatography due to the steric effect of both 18-
methyl and 8-acetyl. This stereo-specificity is further supported by quantum mechanical calculations
employing density functional theory (DFT) using Schrodinger Molecular Modeling Suite 2015. Figure 2
shows the thermodynamically most stable and the preferred orientation of the acetyl group for the
intermediate 12. This conformational preference dictated the strongly favored back-side attack by the
Grignard reagent 13 to form 14 with 20S-OH configuration. The attack from the front side to form 20R-
OH configuration is sterically prohibited, consistent with what we found previously [33, 38]. Oxidation of
diol 14 using Cornforth reagent pyridinium dichloromate (PDC) gave 20S-hydroxy Grundmann`s ketone
15 in 89% yield. Compound 15 was subsequently introduced protecting group EOM on its tertiary alcohol
to form ketone 16, the other key intermediate for the final coupling step. With both C/D-ring fragment 16
and phosphine oxide 7 in hand, construction of the 20S-(OH)D3 framework was successfully
accomplished by employing Wittig-Horner reaction in the presence of phenyllithium (0.18 M, diluted
from commercial source) in THF to form 17 in 52% yield. Simultaneously removal of EOM and TBS was
achieved using camphorsulfonic acid (CSA) to produce the target compound 20S-(OH)D3 in 37% yield.
3.2. Biological assays
3.2.1. 20S-(OH)D3 activates VDR
To confirm that the synthetic 20S-(OH)D3 can activate VDR, two cell lines (Jurkat and HaCaT) were
transduced by VDRE luciferase vector to establish corresponding reporter assays. We compared the VDR
activation activity of 20S-(OH)D3 with that of 1,25(OH)2D3 as shown in Figure 3. At a concentration of
100 nM, both 20S-(OH)D3 and 1,25(OH)2D3 can activate VDR as compared with control in both cell
15

lines, however, 1,25(OH)2D3 showed much stronger activity than 20S-(OH)D3 suggesting deficiency in
coupling of 20S-(OH)D3 with activation of genes having VDRE consensus in their promoter. These
results are consistent with results obtained using enzymatically generated 20S-(OH)D3 [25] indicating
that 20S-(OH)D3 acts as a biased agonist on the VDR [23].
3.2.2. 20S-(OH)D3 stimulates the mRNA levels of VDR and CYP24A1
Since the CYP24A1 gene is an important target of 1,25(OH)2D3 activation, we compared the action of
20S-(OH)D3 with that of 1,25(OH)2D3 on the expression of VDR itself and CYP24A1 genes in HaCaT
cells at different time points as shown in Figure 3. After 6 hrs of treatment with 100 nM of the compound,
cells exposed to 1,25(OH)2D3 showed 350 fold increased expression of CYP24A1 and only 2- to 3-fold
after treatment with 20S-(OH)D3, which is consistent with previously reported data [23]. Interestingly,
20S-(OH)D3 is much stronger inducer of VDR gene expression than 1,25(OH)2D3 (Fig. 3B).
3.2.3. Anti-proliferation activity of 20S-(OH)D3
As shown in Figure 4, 20S-(OH)D3 showed potent anti-proliferative and dose-dependent effect on
SKMEL-188 melanoma cells comparable to classical active form of vitamin D3, 1,25(OH)2D3.
20S(OH)D3 gave an IC50 of 2.09 × 10-10 M while the IC50 of 1,25(OH)2D3 was 2.78 × 10-9 M. This result
is consistent with previous studies showing that 20S-(OH)D3 shows equal or higher potency for inhibition
of melanoma cell proliferation in comparison to 1,25(OH)2D3 [27, 39].
4. Discussion
20S-(OH)D3, a noncalcemic vitamin D3 analog, was enzymatically converted from vitamin D3 by
P450scc and was first chemically synthesized via UVB irradiation [33]. However, this photochemical
method also produces structurally similar and physicochemically active by-products that present a
significant challenge for purification and further SAR studies for the promising 20S-hydroxyl scaffold.
Therefore, we seek to develop an alternative synthetic method. The total synthetic strategy described in
16

this report involves Wittig-Horner coupling and Grignard reaction and is successful in generating 20S-
(OH)D3 in 0.4% overall yield in 16 steps. A distinct advantage of this method over our previously
reported method is that after we generate the two key intermediates phosphine oxide 7 and C/D-ring
ketone 12 on gram scale from a common starting material vitamin D2, we can efficiently make side chain
derivatives for in-depth SAR studies of this 20S-hydroxyl scaffold.
As the native ligand of VDR, 1,25(OH)2D3 activated VDR (Figure 5) and initiated the rapid
transcriptional activity inside the nucleus. This included highly enhanced expression of CYP24A1 (~ 350-
fold) after 6 h treatment. In contrast, 20S-(OH)D3 showed weak stimulation of CYP24A1 gene expression
consistent with our previous data identifying 20S-(OH)D3 as the biased agonist for the VDR [23].
Enhanced expression of VDR by treatment with 20S-(OH)D3 with higher potency than that of
1,25(OH)2D3 further emphasized certain differences with this classical agonist, and suggested
amplification of VDR mediated effects, a subject warranting further studies.
In conclusion, we have established an efficient total synthesis of biologically active 20S-(OH)D3,
which involved divergently generation of key intermediates 7 and 12 from a common starting material
VD2 via modified methods, stereoselective formation of the 20S-hydroxyl scaffold through Grignard
reaction and simultaneously removal of EOM and TBS using CSA. Results from various biological
assays demonstrated the similarity of 20S-(OH)D3 produced through this total synthesis method with the
authentic compound generated enzymatically. Further modifications on the side chain to generate 20S-
(OH)D3 analogs for SAR study will be carried out via nucleophilic attack on intermediate ketone 12
before it will be coupled to the other key intermediate 7.
Acknowledgements
This work was supported by NIH grants 1R21AR063242-01A1 (W.L. and D.D.M.), 1S10OD010678-
01 (W.L.), 1S10RR026377-01 (W.L.), 2R01AR052190-(A.S.) and R21 AR066505-01A1 (A.S.). The
content is solely the responsibility of the authors and does not necessarily represent the official views of
the National Institutes of Health.
17

Supplementary data
Supplementary data associated with this article can be found, in the online version, at doi:
Figure Captions:
Fig. 1. Marketed vitamin D analogs and noncalcemic 20S-(OH)D3
Fig. 2. Preferred conformation of the intermediate (compound 12) calculated with density function theory
with 6-31G** baseset. “Front” side attack by the bulky Grignard agent 13 to form 20R isomer of 14 is
prohibited due to the steric hindrance from the 18-methyl and other moieties in compound 12, while the
“back” side attack to form 20S isomer is strongly favored.
Fig. 3. 20S-(OH)D3 stimulates mRNA levels of VDR and CYP24A1 at 6 h (A) and 24 h (B). HaCaT
cells were treated for 6 h with 100 nM of 20S-(OH)D3, 1,25(OH)2D3 or DMSO only (solvent) as a
control. The mRNA was isolated and qPCR performed using specific primers for VDR and CYP24A1
genes. Data are presented as mean SE (n = 3, * p < 0.05, *** p < 0.005, **** p < 0.0001).
Fig. 4. Anti-proliferative effects of 20S-(OH)D3 and 1,25(OH)2D3 on human SKEML-188 melanoma
cells. Cells were treated with 20S-(OH)D3, 1,25(OH)2D3 or DMSO (solvent) as a control to assess their
inhibitory effects on DNA synthesis. Data are presented as mean SE (n = 3, * p < 0.05, ** p < 0.01 ,
*** p < 0.005).
Fig. 5. 20S-(OH)D3 activates luciferase activityin VDRE-LUC transduced Jurkat and HaCaT cells. Cells
were treated for 24 h with 100 nM 20S-(OH)D3, 1,25(OH)2D3 or DMSO as a control. Data are presented
as mean SE (n = 3, * p < 0.05, ** p < 0.01).
Scheme 1. Retrosynthetic analysis of 20S-(OH)D3
Scheme 2. Synthesis of intermediate 7 Reagents: (a): KMnO4, EtOH-H2O; 65%; (b): i. Pb(OAc)4,
Na2CO3, DCM; ii. Red-Al, benzene; 43% for 3 and 86% for 4 in two steps; (c): TBSCl, Imidazole, DCM;
18

88%; (d): TBAF, THF; 78%; (e): i. n-BuLi, p-TsCl, THF; ii. n-BuLi, Ph2PH, THF; iii. H2O2, H2O, DCM;
59% in three steps.
Scheme 3. Synthesis of intermediate 12 Reagents: (a): Ac2O, Pyr, DMAP, DCM; 92%; (b): OsO4, NMO,
Acetone/t-BuOH; 67%; (c): NaIO4, H2O-THF; 66% (d): Morpholine, p-TsOH, PhH, reflux; (e):
KMnO4/Al2O3, Acetone; 70% in two steps.
Scheme 4. Synthesis of 20S-(OH)D3 Reagents: (a): Mg. THF; (b): THF;80% (c): PDC, DCM; 89%; (d):
EOMCl, DMAP, TEA, DCM; 90%; (e): 7, PhLi, THF; 53%; (f): CSA, DCM-MeOH; 37%.
19

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22

Fig. 1. Marketed vitamin D analogs and noncalcemic 20S-(OH)D3

Fig. 2. Preferred conformation of the intermediate (compound 12)
calculated with density function theory with 6-31G** baseset. “Front”
side attack by the bulky Grignard agent 13 to form 20R isomer of 14 is
prohibited due to the steric hindrance from the 18-methyl and other
moieties in compound 12, while the “back” side attack to form 20S
isomer is strongly favored.

****
A
B
***
*
****
****
***
6 h
24 h
VDR
CYP24A1
VDR
CYP24A1
Fig. 3. 20S-(OH)D3 stimulates mRNA levels of VDR and CYP24A1 at 6 h (A) and 24 h (B). HaCaT cells
were treated for 6 h with 100 nM of 20S-(OH)D3, 1,25(OH)2D3 or DMSO only (solvent) as a control. The
mRNA was isolated and qPCR performed using specific primers for VDR and CYP24A1 genes. Data are
presented as mean ± SE (n = 3, * p < 0.05, *** p < 0.005, **** p < 0.0001).

Fig. 4. Anti-proliferative effects of 20S-(OH)D3 and 1,25(OH)2D3 in human
SKEML-188 melanoma cells. Cells were treated with 20S-(OH)D3, 1,25(OH)2D3 or
DMSO (solvent) as a control to assess their inhibitory effects on DNA synthesis. Data
are presented as mean ± SE (n = 3, * p < 0.05, ** p < 0.01 , *** p < 0.005).

**
*
*
*
Jurkat
HaCaT
Fig. 5. 20S-(OH)D3 activates luciferase activity in VDRE-LUC transduced Jurkat and
HaCaT cells. Cells were treated for 24 h with 100 nM 20S-(OH)D3, 1,25(OH)2D3 or
DMSO as a control. Data are presented as mean ± SE (n = 3, * p < 0.05, ** p < 0.01).

Scheme 1. Retrosynthetic analysis of 20S-(OH)D3

Scheme 2. Synthesis of intermediate 7 Reagents: (a): KMnO4, EtOH-H2O; 65%; (b): i. Pb(OAc)4, Na2CO3, DCM;
ii. Red-Al, benzene; 43% for 3 and 86% for 4 in two steps; (c): TBSCl, Imidazole, DCM; 88%; (d): TBAF, THF;
78%; (e): i. n-BuLi, p-TsCl, THF; ii. n-BuLi, Ph2PH, THF; iii. H2O2, H2O, DCM; 59% in three steps.