Synthesis of 25-hydroxy-19-norvitamin D3 analogs and their antiproliferative activities on prostate cells

Article abstract of DOI:10.3987/com-05-s(k)56

Synthesis of 25-hydroxy-19-norvitamin D₃ derivatives as prohormone type agents for anti-prostate diseases was accomplished utilizing Julia-type olefination. Synthesized compounds showed potent antiproliferative activity on an immortalized norma

Full text of DOI:10.3987/com-05-s(k)56

HETEROCYCLES, Vol. 66, 2005
469
HETEROCYCLES, Vol. 66, 2005, pp. 469 – 479. © The Japan Institute of Heterocyclic Chemistry
Received, 13th September, 2005, Accepted, 11th November, 2005, Published online, 15th November, 2005. COM-05-S(K)56
SYNTHESIS OF 25-HYDROXY-19-NORVITAMIN D3 ANALOGS AND
THEIR ANTIPROLIFERATIVE ACTIVITIES ON PROSTATE CELLS
Midori A. Arai,1 Ryuji Tsutsumi,1 Hideki Hara,1 Tai C. Chen,2 Toshiyuki
Sakaki,3 Naoko Urushino,4 Kuniyo Inouye,4 and Atsushi Kittaka*,1
1Faculty of Pharmaceutical Sciences, Teikyo University, Kanagawa 199-0195,
Japan 2Boston University School of Medicine, Boston, MA 02118, USA 3Faculty
of Engineering, Toyama Prefectural University, Toyama, Japan 4Graduate School
of Agriculture, Kyoto University, Kyoto 606-8502, Japan e-mail address:
akittaka@pharm.teikyo-u.ac.jp
Abstract – Synthesis of 25-hydroxy-19-norvitamin D3 derivatives as prohormone
type agents for anti-prostate diseases was accomplished utilizing Julia-type
olefination. Synthesized compounds showed potent antiproliferative activity on
an immortalized normal prostate cell line, PZ-HPV-7, which has high
1α-hydroxylase
activity.
Furthermore,
we
demonstrated
that
25-hydroxy-19-norvitamin D3 was hydroxylated at the 1α position to form
1α,25-dihydroxy-19-norvitamin D3 by a recombinant human 1α-hydroxylase
(CYP27B1) in a cell-free system.
INTRODUCTION
The biological active form of vitamin D, 1α,25-dihydroxyvitamin D3, 1α,25(OH)2D3 (1), plays a major
role in calcium-phosphorus homeostasis, cell differentiation and apoptosis.1 Synthetic analogs of 1 have
received much attention as potential drugs for the treatment of osteoporosis,2 psoriasis,3 hereditary
vitamin D-resistant rickets,4 and chemoprevention of cancer and cancer chemotherapy, especially prostate
cancer, colon cancer, and breast cancer.5 The natural hormone (1) is known to inhibit the proliferation and
invasiveness of prostate cancer cells. However, 1 can cause hypercalcemia; therefore, the analogs that are
less calcemic but exhibit potent antiproliferative activity would be attractive as therapeutic agents. It has
been shown that prostate cells possess 1α-hydroxylase, (1α(OH)ase), and can convert 25-hydroxyvitamin
D3, 25(OH)D3 (2) to 1 intracellularly to inhibit their proliferation.5 It is also known that a
This paper is dedicated to the memory of the Emeritus Professor Kenji Koga of the University of Tokyo.

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HETEROCYCLES, Vol. 66, 2005
19-demethylenated analog of 1, i.e., 1α,25-dihydroxy-19-norvitamin D2, possesses similar
pro-differentiation and antiproliferative activities as 1.6 Since 19-norvitamin D derivativesare known to
be less calcemic than 1 when administered systemically,7 we are interested in knowing whether
25-hydroxy-19-norvitamin D3, 25(OH)19-norVD3 (3) can exert potent antiproliferative activity toward
prostate cells which possess 1α(OH)ase activity and therefore can be used as chemopreventive agents
without causing hypercalcemic side effects. Herein, we report an improved synthesis of 3 and its
diastereoisomer (14) by using our new coupling method utilizing Julia-type olefination,8 and their
biological activities.
25
OH
OH
OH
H
H
H
1
HO
OH
HO
25(OH)D3 (2)
HO
1!,25(OH)2D3 (1)
25(OH)19-norVD3 (3)
OH
1!(OH)ase
1!,25(OH)219-norVD3 (4)
active form
3
inactive form
HO
OH
Prostate cell
Figure 1. 25-Hydroxy-19-norvitamin D3; Prohormone type anti-agent for prostate disease
RESULTS AND DISCUSSION
25-Hydroxy-19-nor-vitamin D3 (3) was synthesized according to our new method described previously,8,9
and its antiproliferative activity was evaluated in prostate cells. The A-ring precursor (12) for Julia-type
olefination coupling was synthesized from (-)-quinic acid (5) as an enantiomeric pure form as shown in
Scheme 1. After obtaining compound (6),8 the triol which was obtained by sodium borohydride reduction
was converted to benzyl ether (7). The isopropylidene group of 7 was removed under acidic conditions,
and the resulting cis-diol was treated with bis(imidazol-1-yl)thione to give cyclic carbonothioate (8). By
subsequent refluxing in P(OMe)3,10 the cis-diol unit of 8 was successfully converted to a C-C double bond
to give 9. Under hydrogenation conditions, the benzyl group was cleaved and the C-C-double bond was

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471
reduced simultaneously to give compound (10), then oxidative cleavage of vicinal diol and protection of
the secondary hydroxyl group by the tert-butyl dimethyl silyl group gave the A-ring precursor (12).
O
BnO
BnO
OBn
OH
BnO
BnO
OBn
O
HOOC
HO
OH
a
b, c
d, e
O
OH
O
S
O
O
O
O
OH
(-)-quinic acid
5
6
7
8
O
O
HO
OH
BnO
OBn
g
f
h
i
BnO
HO
HO
TBSO
9
10
11
12
Scheme 1. Reagents and conditions: (a) 2,2-dimethoxypropane, (+)-CSA, benzene, 80%; (b) NaBH4,
EtOH, quant.; (c) BnBr, NaH, THF, 91%; (d) PPTS, MeOH, 92%; (e) bis(imidazol-1-yl)thione, acetone,
quant.; (f) trimethyl phosphite, 86%; (g) 5% Pd-C, H2, THF, quant.; (h) NaIO4, CH2Cl2/H2O, 80%; (i)
TBSOTf, 2,6-lutidine, CH2Cl2, 62%.
The key step of Julia-type olefination proceeded with high yields between 12 and sulfone (13)8 using
LiHMDS in THF at -78˚C to give the protected 19-norvitamin D3 derivatives as a diastereomeric mixture.
After partial purification, mixture was treated with (+)-CSA in MeOH to yield deprotected compounds
(3) and 3-deoxy-1α,25(OH)2-19-norvitamin D3 (14) in the ration of 1 to 1 (Scheme 2). These isomers
1
were separated by preparative TLC followed by purification with reversed-phase HPLC. From H NMR
spectroscopic experiments including NOEs, the relative configuration was determined as shown in
Scheme 2.
9.5%
OMOM
OH
OH
6.1%
Ha
5.7%
Hb
O O
S
He
N
a, b
3.8 Hz
Hc
HO
12
S
7.6 Hz
6.9%
13
Hd
HO
OH
NOE
Coupling constant (J)
3
14
3
Scheme 2. Reagents and conditions: (a) LiHMDS, THF, -78C; (b) (+)-CSA, MeOH, 58% in two steps.

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HETEROCYCLES, Vol. 66, 2005
The proposed mechanism of Julia olefination for the synthesis of 3 is shown in Scheme 3 on the basis of
a report by L. B. Baudin et al.11 The α-lithio derivative of 13 would react with the carbonyl group of 12 to
give intermediate (I) and lithium alkoxide. The alkoxide then reacts with the neighboring C=N group to
form spiro heterocyclic intermediate (II). A ring-opening of the spiro heterocycle followed by a
benzothiazole transfer from S to O will form the intermediate (III). Finally, the removal of sulfur
dioxide leads to diene compound (3) and benzothiazolone anion.
OMOM
OMOM
12 + 13
Li
O
S
O
H
N
O
O
H
N
S
H
S
LiO
S
H
O
OTBS
I
II
OTBS
Li
O
S
O
H
S
SO2
O
TBSO
3
S
N
OMOM
N
LiO
+
III
Scheme 3. Proposed mechanism of Julia-type olefination for synthesis of 3
Next, we evaluated the antiproliferative activities of 3 and 14 in an immortalized normal prostate cell line,
PZ-HPV-7, which has high 1α(OH)ase activity. The results are summarized in Figure 2. Interestingly,
both compounds showed high antiproliferative activities almost at the same level as that of the natural
hormone (1). In the case of 3, when the cells were treated with both 3 and the natural hormone (1),
synergistic effect was clearly observed (graph A at 10-6 M). It is interesting that both 3 and 4 showed such
high antiproliferative activities, even though the binding affinity of these two compounds to the vitamin D
receptor (VDR) is low.9 To evaluate a more precise molecular mechanism, we next studied the
metabolism of 3 by 1α(OH)ase (CYP27B1) in an in vitro system (Figure 3). After incubating 3 with the E.
coli extract containing the recombinant 1α(OH)ase (CYP27B1), the resulted mixture was analyzed by
HPLC. It was shown that 3 was hydroxylated at the 1α-position to form 1α,25(OH)2-19-norvitamin D3 (4),
which is known to be active. Thus, our data strongly suggest that the antiproliferative activity of 3 in
prostate cells is primarily the result of its hydroxylation by endogenous 1α(OH)ase (CYP27B1) at the
1α-position leading to the formation of the active 1α,25(OH)2-19-norvitamin D3 compound.

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Figure 2. Antiproliferative activity of 3 and 14 on immortalized normal prostate cells
Figure 3. Metabolic activation of 3 to 4 by 1α(OH)ase
CONCLUSION
We have synthesized the A-ring deoxy-19-norvitamin D3 derivatives, 25-hydroxy-19-norvitamin D3 (3)
and 3-deoxy-1α,25(OH)2-19-norvitamin D3 (14) by using Julia olefination, which we have reported as the
new coupling method for the synthesis of 19-norvitamin D derivatives.8 Both compounds exhibited high
antiproliferative activities in the immortalized normal prostate cells at the same level as that of the

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biological active form of vitamin D3 (1). We also evaluated the metabolic reaction of 3 by 1α(OH)ase
(CYP27B1) in cell-free system, and found that the 1α-hydroxylated 4 was produced. This is the first
report that A-ring deoxy-19-norvitamin D3 derivatives are highly active in inhibiting prostate cell growth.
Precise mechanistic studies are in progress.
EXPERIMENTAL
(3aS,4R,6R,7aR)-4,6-Bis(benzyloxy)-6-(benzyloxymethyl)hexahydro-2,2-dimethylbenzo[d][1,3]dio-
xole (7). To a solution of 6 (5.83 g, 27.2 mmol) in EtOH (120 mL) was added NaBH4 (3.09 g, 81.6 mmol)
at 0 ºC. The reaction mixture was stirred at the same temperature for 2.5 h. The reaction was quenched by
addition of acetone at 0 ºC. Evaporation of the solvent afforded a crude mixture, from which
(3aS,4R,6R,7aR)-hexahydro-6-hydroxymethyl-2,2-dimethyl-1,3-benzodioxole-4,6-diol8,12 (6.06 g) was
roughly separated by silica gel short pad column chromatography (MeOH : CHCl3 = 1 : 5) as a white
solid in quantitative yield.
To a suspension of NaH (60% in mineral oil, 2.26g, 56.6 mmol) in THF (7 mL) was added with stirring a
solution of the above triol (617 mg, 2.83 mmol) in THF (8 mL) at 0 ºC under argon. After stirring for 70
min at rt, to the reaction mixture was added benzyl bromide (5.05 mL, 42.5 mmol) at 0 ºC. The resulting
mixture was stirred at rt for 22 h, and then heated at 70 ºC for 25 h. After the addition of MeOH and water
to the reaction mixture at 0 ºC, the whole was extracted with ethyl acetate. The organic layer was washed
with brine, dried over magnesium sulfate and filtered. The solvent was removed under the reduced
pressure to give a residue, from which 7 (1.26 g) was purified by silica gel column chromatography (ethyl
acetate : n-hexane = 1 : 5) as a colorless oil in 91% yield. [α]2D5 = -21.1˚ (c = 0.94, CHCl3); 1H NMR (400
MHz, CDCl3) δ 1.34 (3H, s), 1.38 (3H, s), 1.51 (1H, dd, J = 13.7, 11.8 Hz), 1.89 (1H, dd, J = 15.6, 5.1
Hz), 2.15-2.02 (1H, m), 2.34-2.39 (1H, m), 3.45 (1H, d, J = 9.4 Hz), 3.49 (1 H, d, J = 9.4 Hz), 3.93 (1H,
ddd, J = 11.8, 7.0, 4.2 Hz), 4.06 (1H, dd, J = 7.0, 6.5 Hz), 4.34-4.39 (1H, m), 4.40 (1H, d, J = 11.0 Hz),
4.53 (2H, s), 4.56 (1H, d, J = 11.0 Hz), 4.62 (1H, d, J = 12.0 Hz), 4.74 (1H, d, J = 12.0 Hz), 7.21-7.36
13
(15H, m); C NMR (100 MHz, CDCl3) δ 25.9, 28.1, 31.0, 35.2, 64.6, 71.7, 73.4, 73.6, 75.1, 75.3, 76.2,
80.3, 108.6, 127.0, 127.3, 127.5, 127.6, 127.8, 128.0, 128.2, 128.3, 138.0, 138.7, 139.2; IR (film) 3031,
2984, 2932, 2865, 1497, 1455, 1366, 1240, 1053, 739, 698 cm-1; MS m/z 473 (M+ - Me); HRMS calcd for
C30H33O5 473.2328, found 473.2337.
(3aS,4R,6R,7aR)-4,6-Bis(benzyloxy)-6-(benzyloxymethyl)hexahydrobenzo[d][1,3]dioxole-2-thione
(8). A solution of 7 (1.24 g, 2.54 mmol) in MeOH (25 mL) was treated with PPTS (64 mg, 254 µmol) at
rt under argon. After refluxing at 70 ºC for 23 h, to the reaction mixture was added sat. aq. NaHCO3 and
evaporated. The whole was extracted with ethyl acetate, the organic layer was washed with brine, dried
over magnesium sulfate and filtered. Evaporation of the solvent afforded a crude mixture, from which

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(1R,2R,3R,5S)-3,5-bis(benzyloxy)-5-(benzyloxymethyl)cyclohexane-1,2-diol (1.05 g) was purified by
silica gel column chromatography (ethyl acetate : n-hexane = 1 : 2) as a colorless oil in 92% yield. [α]2D6
= -1.0˚ (c = 1.03, CHCl3); 1H NMR (400 MHz, CDCl3) δ 1.42 (1H, dd, J = 14.1, 11.6 Hz), 1.64 (1H, dd, J
= 15.0, 3.4 Hz), 2.30 (1H, ddd, J = 15.0, 3.3, 3.2 Hz), 2.50 (1H, ddd, J = 14.1, 3.8, 3.8 Hz), 3.42 (1H, d, J
= 9.9 Hz), 3.46 (1H, d, J = 9.9 Hz), 3.52 (1H, dd, J = 9.3, 3.3 Hz), 3.72 (1H, ddd, J = 11.6, 9.3, 4.4 Hz),
4.03-4.04 (1H, m), 4.26 (1H, d, J = 10.6 Hz), 4.47 (1H, d, J = 10.6 Hz), 4.52 (2H, s), 4.72 (1H, d, J = 12.0
Hz), 4.77 (1H, d, J = 12.0 Hz), 7.14-7.16 (2H, m), 7.27-7.41 (13H, m); 13C NMR (100 MHz,
CDCl3) δ 33.7, 36.5, 64.9, 70.4, 72.3, 73.4, 74.1, 74.6, 76.3, 79.8, 127.5, 127.6, 127.7, 127.7, 127.9, 128.4,
128.4, 128.4, 137.6, 137.7, 138.5; IR (film) 3461, 3063, 3031, 2924, 2867, 1495, 1455, 1091, 737, 698
cm-1; MS m/z 357 (M+ - Bn); HRMS calcd for C21H25O5 357.1702, found 357.1697.
To a solution of the above diol (84 mg, 188 µmol) in acetone (2 mL) was added bis(imidazol-1-yl)thione
(168 mg, 940 µmol) at rt under argon. After refluxing at 60 ºC for 2 days, the reaction mixture was
evaporated. The whole was diluted with CH2Cl2, washed with 1 M aq. HCl, sat. aq. Na2CO3, and brine.
The organic layer was dried over magnesium sulfate and filtered. Removal of the solvent afforded a
residue, from which 8 (94 mg) was purified by silica gel column chromatography (ethyl acetate :
n-hexane = 1 : 3) as pale yellow oil in quantitative yield. [α]2D5 = -24.7˚ (c = 1.02, CHCl3); 1H NMR (400
MHz, CDCl3) δ 1.62 (1H, dd, J = 14.0, 11.9 Hz), 2.00 (1H, dd, J = 16.1, 5.0 Hz), 2.24 (1H, ddd, J = 14.0,
4.7, 2.1 Hz), 2.64 (1H, ddd, J = 16.1, 2.9, 2.6 Hz), 3.46 (1H, d, J = 9.3 Hz), 3.54 (1H, d, J = 9.3 Hz), 4.04
(1H, ddd, J = 11.9, 7.2, 4.7 Hz), 4.34 (1H, d, J = 10.8 Hz), 4.50 (1H, d, J = 10.8 Hz), 4.52 (2H, s), 4.57
(1H, d, J = 11.5 Hz), 4.70 (1H, d, J = 11.5 Hz), 4.83 (1H, dd, J = 7.6, 7.2 Hz), 5.11 (1H, ddd, J = 7.6, 5.0,
2.9 Hz), 7.23-7.38 (15H, m); 13C NMR (100 MHz, CDCl3) δ 29.9, 34.5, 64.9, 72.3, 73.5, 73.8, 74.2, 75.1,
80.5, 84.9, 127.4, 127.5, 127.6, 127.8, 127.8, 127.9, 128.3, 128.4, 128.4, 137.4, 137.4, 137.9, 190.9; IR
(film) 3063, 3031, 2932, 2863, 1497, 1455, 1273, 1121, 1096, 739, 698 cm-1; MS m/z 490 (M+), 399 (M+
- Bn); HRMS calcd for C29H30O5S 490.1814, found 490.1812.
(3R,5R)-3,5-Bis(benzyloxy)-5-benzyloxymethylcyclohexene (9). Compound (8) (70 mg, 143 µmol) was
treated with trimethyl phosphite (2 mL) at rt under argon. After refluxing at 110 ºC for 2 days, the
reaction mixture was diluted with ether, washed with 10% aq. NaOH, water, and brine. The organic layer
was dried over sodium sulfate, filtered, and evaporated. The residue was diluted with toluene, and maleic
anhydride (240 mg) was added. The resulting mixture was refluxed at 110 ºC for 30 min, and then 10%
aq. NaOH was added at rt. After being stirred for 15 min, the reaction mixture was diluted with ether,
washed with 10% aq. NaOH, water, and brine. The organic layer was dried over sodium sulfate and
filtered. Removal of the solvent afforded a residue, from which 9 (51 mg) was purified by silica gel
column chromatography (ethyl acetate : n-hexane = 1 : 5) as a colorless oil in 86% yield. [α]2D5 = +66.3˚ (c
1
= 1.02, CHCl3); H NMR (400 MHz, CDCl3) δ 1.81 (1H, dd, J = 12.9, 8.8 Hz), 2.23-2.30 (1H, m),

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HETEROCYCLES, Vol. 66, 2005
2.33-2.38 (1H, m), 3.55 (2H, s), 4.26-4.32 (1H, m), 4.49 (2H, s), 4.54 (1H, s), 4.54 (1H, s), 4.58 (1H, s),
13
4.59 (1H, s), 5.69-5.74 (1H, m), 5.88 (1H, d, J = 10.3 Hz), 7.21-7.36 (15H, m); C NMR (100 MHz,
CDCl3) δ 31.8, 34.7, 64.5, 70.4, 72.4, 73.3, 74.5, 76.4, 125.8, 127.1, 127.2, 127.4, 127.5, 127.6, 128.1,
128.1, 128.2, 128.3, 138.2, 138.7, 139.3; IR (film) 3063, 3031, 2861, 1655, 1495, 1455, 1071, 912, 737,
693 cm-1; MS m/z 323 (M+ - Bn); HRMS calcd for C21H23O3 323.1647, found 323.1649.
(1S,3S)-1-Hydroxymethylcyclohexane-1,3-diol (10). A solution of 9 (511 mg, 1.23 mmol) in dry THF
(13 mL) was treated with Pd-C (5%, 787 mg) at rt under H2 atmosphere. The reaction mixture was stirred
for 5 days, diluted with MeOH, and filtered. Evaporation of the solvent afforded a residue, from which 10
(196 mg) was purified by silica gel column chromatography (MeOH : ethyl acetate = 1 : 10) as a white
solid in quantitative yield. mp 114.0-115.0 ˚C (AcOEt-Hexane); [α]2D1 = +7.4˚ (c = 0.93, EtOH); 1H NMR
(400 MHz, CDCl3) δ 1.18-1.28 (4H, m), 1.61-1.71 (5H, m), 1.98-2.02 (2H, m), 3.47 (2H, s), 4.00 (1H,
dddd, J = 8.8, 8.8, 4.4, 4.4 Hz); MS m/z 146 (M+), 115 (M+ - CH2OH), 97 (M+ - CH2OH - H2O); HRMS
calcd for C7H14O3 146.0943, found 146.0949; Anal. calcd for C7H14O3•1/7H2O: C, 56.52; H, 9.68, found:
C, 56.47; H, 9.95.
(S)-3-Hydroxycyclohexanone (11). To a solution of 10 (193 mg, 1.32 mmol) in CH2Cl2 (7 mL), NaIO4
(847 mg, 3.96 mmol) in water (7 mL) was added dropwise at 0 ºC. After stirring for 1.5 h at rt, the
mixture was extracted with CH2Cl2 and ethyl acetate. The organic layer was washed with brine, dried over
magnesium sulfate, and filtered. Evaporation of the solvent afforded a crude mixture, from which 11 (121
mg) was purified by silica gel column chromatography (ethyl acetate : n-hexane = 1 : 1) as a colorless oil
in 80% yield. 1H NMR (400 MHz, CDCl3) δ 1.66-1.82 (2H, m), 1.99-2.13 (2H, m), 2.33 (2H, dd, J = 7.1,
6.3 Hz), 2.41 (1H, dd, J = 14.1, 7.8 Hz), 2.67 (1H, dd, J = 14.1, 4.1 Hz), 4.20 (1H, dddd, J = 7.8, 7.8, 4.1,
3.9 Hz); C NMR (100 MHz, CDCl3) δ 20.7, 32.8, 40.9, 50.4, 69.7, 209.8; MS m/z 114 (M+), 97 (M+ -
13
OH); HRMS calcd for C6H10O2 114.0681, found 116.0681.
(S)-3-(tert-Butyldimethylsiloxy)cyclohexanone (12). To a stirred mixture of 11 (115 mg, 1.01 mmol)
and 2,6-lutidine (410 µL, 3.54 mmol) in dry CH2Cl2 (5 mL) was added TBSOTf (487 µL, 2.12 mmol) at 0
ºC under argon. The resulting mixture was stirred for 4 h, and the reaction was quenched by addition of
water. After extraction with CH2Cl2, the organic layer was washed with brine, dried over magnesium
sulfate, and filtered. Removal of the solvent afforded a residue, from which 12 (144 mg) was purified by
silica gel column chromatography (ethyl acetate : n-hexane = 1 : 9) as a colorless oil in 62% yield.
[α]2D5 = -5.6˚ (c = 1.02, CHCl3); 1H NMR (400 MHz, CDCl3) δ 0.05 (6H, s), 0.87 (9H, s), 1.63 - 1.78 (2H,
m), 1.86-1.92 (1H, m), 2.03-2.12 (1H, m), 2.27-2.32 (2H, m), 2.38 (1H, dd, J = 13.9, 6.8 Hz), 2.53 (1H,
dddd, J = 13.9, 3.7, 1.1, 1.1 Hz), 4.17 (1H, dddd, J = 6.8, 6.8, 3.7, 3.4 Hz); 13C NMR (100 MHz, CDCl3)
δ -4.8, -4.8, 18.0, 20.5, 25.7, 33.4, 41.1, 50.8, 70.2, 209.7; IR (film) 2955, 2857, 1715, 1462, 1362, 1254,

HETEROCYCLES, Vol. 66, 2005
477
t
1034, 835, 777 cm-1; MS m/z 228 (M+), 215 (M+ - Me), 171 (M+ - Bu); HRMS calcd for C12H24O2Si
228.1545, found 228.1546.
(1S,5E,7E)-19-Nor-9,10-secocholesta-5,7-diene-1,25-diol
(4) and
(1S,5Z,7E)-19-Nor-9,10-
secocholesta-5,7-diene-1,25-diol (13). To a solution of 12 (172 mg, 323 µmol) in dry THF (800 µL) was
added LiHMDS (1 M solution in THF, 303 µL, 303 µmol) at -78 ºC under argon. After stirring at the
same temperature for 65 min, a solution of 13 (46 mg, 202 µmol) in dry THF (800 µL) was added
dropwise to the mixture. After stirring for 3 h, the reaction mixture was quenched by addition of sat. aq.
NH4Cl, and the aqueous layer was extracted with ether. The organic layer was washed with brine, dried
over sodium sulfate, and filtered. After evaporation of the solvent, the crude mixture was applied on silica
gel column chromatography (ethyl acetate: n-hexane = 1 : 30) to give the crude protected vitamin D
mixture (75 mg in total). To a solution of the protected vitamin D mixture (75 mg) in dry MeOH (1.5 mL)
was added (+)-CSA (64 mg, 274 µmol) at 0 ºC under argon. After stirring at rt for 20 h, the reaction
mixture was diluted with ethyl acetate. The resulting mixture was washed with sat. aq. NaHCO3 and brine,
dried over magnesium sulfate, and evaporated. Purification by silica gel column chromatography (ethyl
acetate : n-hexane = 1 : 1) gave a mixture of 3 and 14 (45 mg, 58% in two steps) as white solid.
Separation of 3 and 14 was conducted by using a silica gel preparative TLC (ethyl acetate : n-hexane = 1 :
3) to yield 3 and 14 as white powders, respectively.
(1S,5E,7E)-19-Nor-9,10-secocholesta-5,7-diene-1,25-diol (3). [α]2D1 = +61.4˚ (c = 0.70, CHCl ); UV
3
1
(EtOH) λmax 243, 252, 261 nm; H NMR (600 MHz, CDCl3) δ 0.55 (3H, s), 0.94 (3H, d, J = 6.3 Hz),
1.03-1.09 (1H, m), 1.18-1.66 (19H, m), 1.22 (6H, s), 1.71-1.77 (1H, m), 1.85-1.91 (2H, m), 1.98-2.01 (2H,
m), 2.11-2.17 (2H, m), 2.35 (1H, ddd, J = 13.5, 6.9, 4.5 Hz), 2.50 (1H, dd, J = 12.9, 3.8 Hz), 3.80 (1H,
dddd, J = 7.6, 7.6, 3.8, 3.8 Hz), 5.82 (1H, d, J = 11.2 Hz), 6.13 (1H, d, J = 11.2 Hz); 13C NMR (150 MHz,
CDCl3) δ 12.1, 18.8, 20.8, 22.3, 23.1, 23.5, 27.7, 27.9, 28.8, 29.2, 29.4, 34.7, 36.1, 36.4, 40.5, 44.4, 45.7,
46.0, 56.3, 56.5, 70.1, 71.1, 115.6, 120.6, 135.7, 141.7; IR (film) 3389, 2942, 2870, 1618, 1468, 1377,
1055, 758 cm-1; MS m/z 388 (M+), 370 (M+ - H2O); HRMS calcd for C26H44O2 338.3341, found 338.3333.
(1S,5Z,7E)-19-Nor-9,10-secocholesta-5,7-diene-1,25-diol (14). [α]2D7 = +73.8˚ (c = 0.84, CHCl ); UV
3
1
(EtOH) λmax 243, 251, 261 nm; H NMR (400 MHz, CDCl3) δ 0.55 (3H, s), 0.94 (3H, d, J = 6.6 Hz),
1.02-1.10 (1H, m), 1.20-1.67 (18H, m), 1.22 (6H, s), 1.75-1.82 (1H, m), 1.88-1.95 (2H, m), 1.98-2.03 (2H,
m), 2.07-2.10 (2H, m), 2.13-2.18 (1H, m), 2.78-2.88 (2H, m), 3.69 (1H, dddd, J = 8.6, 8.6, 4.3, 4.3 Hz),
5.85 (1H, d, J = 11.2 Hz), 6.21 (1H, d, J = 11.2 Hz); 13C NMR (150 MHz, CDCl3) δ 12.0, 18.8, 20.8, 22.3,
23.5, 24.3, 27.7, 28.8, 29.2, 29.4, 35.3, 36.1, 36.3, 36.4, 37.9, 40.5, 44.4, 45.7, 56.3, 56.5, 70.3, 71.1,
115.4, 120.3, 135.9, 141.9; MS m/z 388 (M+), 370 (M+ - H2O); HRMS calcd for C26H44O2 338.3341,
found 338.3332.

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ACKNOWLEDGEMENTS
We thank Ms. Junko Shimode and Ms. Akiko Tonoki (Teikyo University) for spectroscopic
measurements. This study was supported by a Grant-in-Aid for Young Scientists (B) from the Ministry
of Education, Culture, Sports, Science and Technology, Japan, (No. 16790027 to M.A.A.) and in part by
Grants-in-Aid for Scientific Research (C) from Japan Society for the Promotion of Science (No.
15590021 and 17590012 to A.K.). A.K. also gratefully acknowledges Uehara Memorial Foundation for
financial support.
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