Synthesis and evaluation of a 3-position diastereomer of 1α,25-dihydroxy-2β-(3-hydroxypropoxy)vitamin D3 (ED-71)

Article abstract of DOI:10.1016/j.bmc.2006.07.039

A 3-position diastereomer of 1α,25-dihydroxy-2β-(3-hydroxypropoxy)vitamin D₃ (ED-71, 2), 3-epi-ED-71 (4), was synthesized by the convergent method coupling the A-ring fragment (5) with the C/D-ring fragment (6). As the results of preliminary in

Full text of DOI:10.1016/j.bmc.2006.07.039

Bioorganic & Medicinal Chemistry 14 (2006) 8050–8056
Synthesis and evaluation of a 3-position diastereomer
of 1a,25-dihydroxy-2b-(3-hydroxypropoxy)vitamin D3 (ED-71)q
Susumi Hatakeyama,a Satoshi Nagashima,a Naoko Imai,a Keisuke Takahashi,a
Jun Ishihara,a Atsuko Sugita,b Takeshi Nihei,b Hitoshi Saito,b
Fumiaki Takahashib and Noboru Kuboderab,*
aGraduate School of Biomedical Sciences, Nagasaki University, Nagasaki 852-8521, Japan
bChugai Pharmaceutical Co., Ltd., 2-1-1, Nihonbashi-Muromachi, Chuo-ku, Tokyo 103-8324, Japan
Received 27 June 2006; revised 20 July 2006; accepted 21 July 2006
Abstract—A 3-position diastereomer of 1a,25-dihydroxy-2b-(3-hydroxypropoxy)vitamin D3 (ED-71, 2), 3-epi-ED-71 (4), was syn-
thesized by the convergent method coupling the A-ring fragment (5) with the C/D-ring fragment (6). As the results of preliminary
in vitro biological evaluation of 3-epi-ED-71 (4), the inhibition of parathyroid hormone secretion in bovine parathyroid cells and
binding affinity to human recombinant vitamin D receptor and to human vitamin D binding protein in comparison with ED-71
(2), 1a,25-dihydroxyvitamin D3 (1,25(OH)2D3, 1), and 3-epi-1,25(OH)2D3 (3) are described.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
studies in Japan as a promising candidate for the treat-
ment of osteoporosis and bone fracture prevention.9
Active vitamin D3, 1a,25-dihydroxyvitamin D3
(1,25(OH)2D3, 1), is well recognized as a potent regula-
tor of cell proliferation and differentiation processes in
addition to possessing regulatory effects on calcium
and phosphorus metabolism.2 Various analogs of
1,25(OH)2D3 (1) have been investigated in attempts to
separate differentiation-induction and antiproliferation
activities from calcemic activity with the aim of obtain-
ing useful analogs for the medical treatment of psoriasis,
secondary hyperparathyroidism, cancer, etc.3 There is
also intense interest in obtaining analogs more potent
than 1,25(OH)2D3 (1) in terms of regulatory effects on
calcium and phosphorus metabolism with the objective
of treating bone diseases such as osteoporosis. 1a,25-Di-
hydroxy-2b-(3-hydroxypropoxy)vitamin D3 (ED-71, 2),
an analog of 1,25(OH)2D3 (1) from which a hydroxy-
propoxy substituent at the 2b-position is appended, is
such an analog that shows potent effects on bone thera-
py.4–8 ED-71 (2) is currently under phase III clinical
It is well known that the synthesis and secretion of
parathyroid hormone (PTH) is regulated by
1,25(OH)2D3 (1).10,11 Interestingly during our clinical
development of ED-71 (2), serum intact PTH in osteo-
porotic patients did not change significantly upon
treatment with 2, although the reason remains unclear,
however.12 Brown et al. reported that epimerization of
1,25(OH)2D3 (1) at the 3-position of the A-ring plays a
major role in hormone activation and inactivation,
especially in the case of parathyroid cells.13 It has been
also reported that 3-epi-1,25(OH)2D3 (3), an epimer of
1,25(OH)2D3 (1) at the 3-position, shows equipotent
and prolonged activity compared to 1 at suppressing
PTH secretion.14 Since ED-71 (2) has a bulky hydroxy-
propoxy substituent at the 2-position in the A-ring,
epimerization of 2 at the adjacent and sterically hin-
dered 3-position might be prevented. This could be
the reason why ED-71 (2) showed weak potency in
PTH suppression during clinical studies. We have sig-
nificant interest in ED-71 (2) epimerization at the 3-po-
sition and the biological potency of 3-epi-ED-71 (4) in
suppressing PTH production.
Keywords: 1a,25-Dihydroxyvitamin D3; 1a,25-Dihydroxy-2b-(3-hydr-
oxypropoxy)vitamin D3; ED-71; 3-epi-ED-71.
q
See Ref. 1.
In this paper, therefore, we describe the synthesis of
3-epi-ED-71 (4) and its in vitro suppression of PTH
*
Corresponding author. Tel.: +81 3 3273 8558; fax: +81 3 3281
2626; e-mail: kuboderanbr@chugai-pharm.co.jp
0968-0896/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.07.039

S. Hatakeyama et al. / Bioorg. Med. Chem. 14 (2006) 8050–8056
8051
OH
OH
OH
OH
HO
OH
HO
OH
HO
OH
HO
OH
O
OH
O
OH
1,25(OH)2D3 (1)
Figure 1. Structures of active vitamin D3 analogs.
3-Epi-1,25(OH)2D3 (3)
3-Epi-ED-71 (4)
ED-71 (2)
compared to ED-71 (2), 3-epi-1,25(OH)2D3 (3), and
1,25(OH)2D3 (1) (Fig. 1).
afford acetonide (12) in 88% yield. Swern oxidation of
12 followed by Grignard reaction of the resulting alde-
hyde with vinylmagnesium bromide produced alcohol
(13) as an epimeric mixture (S:R = 3:2, determined by
1H NMR) in 66% yield.19 To separate this epimeric mix-
ture, 13 was subjected to lipase-catalyzed acetylation
using vinyl acetate and Novozyme in tert-butyl methyl
ether.20 As a result, the R-epimer preferentially under-
went acetylation to give the acetate (14) and S-13
(R:S = 1:20) in 40% and 57% yields, respectively. Acidic
hydrolysis of 14 gave diol (15) in 90% yield, which, upon
Mitsunobu reaction using DEAD and triphenylphos-
phine in boiling toluene, afforded epoxide (16) in 75%
yield.21 Reaction of 16 with lithium trimethylsilylacety-
lide in the presence of BF3ÆOEt2 at ꢀ78 °C followed
by saponification provided eneyne (17) in 65% yield.22
Protection of 17 as its triethylsilyl ether produced A-ring
fragment (5) quantitatively.
2. Results and discussion
2.1. Synthesis of 3-epi-ED-71 (4)
The synthesis of 3-epi-ED-71 (4) was envisioned using
the convergent method via palladium-catalyzed
coupling of the A-ring fragment (5) prepared from the
C2 symmetrical epoxide (7) with known C/D-ring
fragment (6) obtained from the Inhoffen-Lythgoe diol
15,16
(8) (Fig. 2).
The synthesis of the A-ring fragment (5) began with
inversion of the C3 configuration of alcohol (9) which
was prepared from the C2 symmetrical epoxide (7)
according to our previously established procedure.17
Thus, reaction of 9 with p-nitrobenzoic acid in the pres-
ence of diethyl azodicarboxylate (DEAD) and triphenyl-
phosphine gave the p-nitrobenzoate (10) in 84% yield.18
Treatment of 10 with NaHCO3 in methanol allowed
selective methanolysis of the p-nitrobenzoate group to
give the inverted alcohol (11) in 86% yield. After
hydrogenolysis of the benzyl ether functionalities in
11, the resulting diol was protected as its acetonide to
Having secured A-ring fragment (5), we then performed
its coupling with C/D-ring fragment (6) using Trost’s
methodology.15,16 Thus, the A-ring fragment (5) was al-
lowed to react with C/D-ring fragment (6) in the pres-
ence of (Ph3P)4Pd and triethylamine in boiling toluene
to give the coupling product which was desilylated with
ammonium fluoride in boiling methanol to produce
3-epi-ED-71 (4) in 46% yield (Scheme 1).
O
TESO
OTES
BnO
OBn
O
OTES
OH
A-Ring fragment (5)
C Symmetrical epoxide (7)
2
HO
OH
O
OH
OH
OH
3-Epi-ED-71 (4)
H
H
OH
Br
C/D-Ring fragmenr (6)
Inhoffen-Lythgoe diol (8)
Figure 2. Retrosynthesis of 3-epi-ED-71 (4).

8052
S. Hatakeyama et al. / Bioorg. Med. Chem. 14 (2006) 8050–8056
BnO
BnO
O
Ref. 19
76%
BnO
a)
84%
OBn
HO
OBn
OPiv
RO
OBn
OPiv
O
(7)
O
(10): R=p-(NO2)PhCO
(9)
b) 86%
(11): R=H
O
O
O
O
O
c), d)
88%
e), f)
66%
g)
O
OAc
OPiv
OH
OH
O
O
OPiv
O
(12)
OPiv
(14) 40% (100% ee)
(13) S:R=3:2
+
S-(13) 57% (R:S=1:20)
HO
HO
O
m), n)
h)
90%
j), k)
65%
i)
75%
OAc
OAc
OPiv
(14)
RO
OR
(4)
O
OPiv
O
46%
O
OR
(15)
(16)
(17): R=H
l) 100%
(5): R=TES
Scheme 1. Synthesis of 3-epi-ED-71 (4). Reagents and conditions: (a) p-(NO2)PhCO2H, DEAD, Ph3P, toluene; (b) NaHCO3, MeOH; (c) Pd(OH)2,
H2, MeOH; (d) 2,2-dimethoxypropane, TsOH, acetone; (e) (COCl)2, DMSO, CH2Cl2, ꢀ78 °C, then Et3N; (f) CH2@CHMgBr, THF, ꢀ20 °C; (g)
Novozyme, CH2@CHOAc, t-BuOMe; (h) 60% AcOH; (i) DEAD, Ph3P, dioxane, reflux; (j) TMSC2H, n-BuLi, BF3ÆEt2O, THF, ꢀ78 °C; (k) 10 M
NaOH, MeOH; (l) TESOTf, NEt3, CH2Cl2, ꢀ40 °C; (m) 6, (Ph3P)4Pd, Et3N, toluene, reflux; (n) NH4F, MeOH, reflux.
2.2. Biological evaluation of 3-epi-ED-71 (4)
affinity to human recombinant VDR as shown in Ta-
ble 1. Regarding affinity to human DBP as reported
previously in the rat DBP case, ED-71 (2) showed
more potent affinity than 1,25(OH)2D3 (1). This in-
crease in DBP affinity is due to the existence of a
hydroxypropoxy substituent at the 2b-position and
was also observed in the 3-epi series: 3-epi-
1,25(OH)2D3 (3): 8.3 and 3-epi-ED-71 (4): 113.1, as
shown in Table 1.
The results of preliminary in vitro biological evalua-
tion of 3-epi-ED-71 (4) in comparison with ED-71
(2), 1,25(OH)2D3 (1), and 3-epi-1,25(OH)2D3 (3) are
summarized in Table 1, which contains affinity to hu-
man vitamin D receptors (VDR) and human vitamin
D binding protein (DBP), and inhibition of PTH in
cultured bovine parathyroid cells. The dose-responsive
effects of analogs on PTH suppression are also shown
in Figure 3. 3-Epi-ED-71 (4) showed only slight inhi-
bition of PTH secretion in bovine parathyroid cells
compared to ED-71 (2). In our assay systems, 3-epi-
1,25(OH)2D3 (3) did not show greater activity than
1,25(OH)2D3 (1) in suppressing PTH secretion. The
inhibitory potency of vitamin D3 analogs was
1,25(OH)2D3 (1) >ED-71 (2) P3-epi-1,25(OH)2D3 (3)
ꢁ3-epi-ED-71 (4), and they were well responsive for
ED-71 (2) and its 3-position epimer, 3-epi-ED-71 (4), ap-
pear to be inherently weak agents toward PTH suppres-
sion. This should be examined further with in vivo
evaluation systems using renal insufficiency animal mod-
els such as 5/6 nephrectomized rats showing high level of
serum PTH.23 Nevertheless, the less potent activity of
ED-71 (2) toward native PTH suppression compared
to 1,25(OH)2D3 (1) might be a beneficial characteristic
of 2 for treating osteoporotic patients.
Table 1. Biological evaluation of 3-epi-ED-71 (4)
In our previous modification studies of 1,25(OH)2D3 (1)
and ED-71 (2) at the 1-position of the A-ring, 1-epi-
1,25(OH)2D3 and 1-epi-ED-71 showed enhanced affinity
to rat DBP compared to parent compounds, 1 and 2.24
The relative binding affinities of 1,25(OH)2D3, 1-epi-
1,25(OH)2D3, ED-71, and 1-epi-ED-71 to DBP were
100, 290, 410, and 670, respectively.24 Interestingly, epi-
merization of 1,25(OH)2D3 (1) and ED-71 (2) at the 3-
position in the present study did not result in enhanced
affinity to human DBP. At the present time, however,
the reasons for this remain unclear. Future studies along
these lines should provide clarification and will be
reported elsewhere.
VDR
100
DBP
PTH
100
1,25(OH)2D3 (1)
3-epi-1,25(OH)2D3 (3)
ED-71 (2)
100
9.62
44.6
0.02
8.3
421.9
113.1
1.25
3.54
0.11
3-epi-ED-71 (4)
VDR, relative affinity normalized by the potency of 1,25(OH)2D3
(=100) using human vitamin D receptors.
DBP, relative affinity normalized by the potency of 1,25(OH)2D3
(=100) using human vitamin D binding protein.
PTH, relative inhibitory activity of parathyroid hormone secretion
normalized by the potency of 1,25(OH)2D3 (=100) in cultured bovine
parathyroid cells.

S. Hatakeyama et al. / Bioorg. Med. Chem. 14 (2006) 8050–8056
8053
120
110
100
90
80
70
60
50
40
30
20
10
0
1,25(OH)2D3 (1)
3-Epi-1,25(OH)2D3 (3)
ED-71 (2)
3-Epi-ED-71 (4)
Vehicle 10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-5
concentration(mol/L)
Figure 3. Dose-responsive effects of active vitamin D3 analogs on PTH secretion in bovine parathyroid cells in vitro.
3. Experimental
3.1. General methods
7.45–7.15 (m, 10H), 5.51 (br q, J = 2.1 Hz), 4.53–4.45
(m, 4H), 4.09 (dt, 2H, J = 6.3, 1.6 Hz), 3.83 (br quint,
1H, J = 5.2 Hz), 3.78–3.45 (m, 7H), 2.64 (br d, 1H,
J = 4.8 Hz), 1.88 (quint, 2H, J = 6.3 Hz), 1.18 (s, 9H);
13C NMR (100 MHz, CDCl3) d 178.2, 163.9, 150.4,
137.6, 135.4, 129.5, 128.2, 127.5, 127.4, 127.3, 123.4,
74.1, 73.5, 73.2, 68.9, 67.9, 67.7, 61.2, 38.7, 29.4, 27.2;
HRMS (EI) m/z calcd for C33H39NO9 (M+) 593.2625,
found 593.2607.
Where appropriate, reactions were performed in flame-
dried glassware under argon atmosphere. All extracts
were dried over MgSO4 and concentrated by rotary
evaporation below 30 °C at 25 Toor. Anhydrous tetra-
hydrofuran (THF) was purchased from Kanto Chemical
Co., Inc., dichloromethane (CH2Cl2), triethylamine,
dimethylsulfoxide (DMSO), toluene, and acetonitrile
(MeCN) were distilled from CaH2. Methanol (MeOH)
was distilled from sodium. Thin-layer chromatography
was performed with Merck F-254 TLC plates. Column
chromatography was performed using Kanto Chemical
Co., Inc., silica gel 60 N (spherical, neutral). Infrared
spectra were measured on a JASCO FTIR-230 spec-
trometer. Optical rotations were recorded on a JASCO
3.3. (2S,3S)-3-(1,4-Bisbenzyloxy-3-hydroxybutan-2-
yloxy)propyl pivalate (11)
To a solution of 10 (22.3 g, 38.0 mmol) in MeOH
(380 mL) was added NaHCO3 (19.0 g, 22.6 mmol) at
0 °C, and the mixture was stirred at room temperature
for 8 h. The reaction mixture was diluted with CH2Cl2,
washed with H2O and brine, dried, concentrated, and
1
DIP-370 polarimeter at ambient temperature. H and
chromatographed (SiO2 400 g, hexane/AcOEt = 6:1) to
24
D
1.00, CHCl3); IR (neat) 3781, 3712, 2867, 1724, 1600,
13C NMR spectra were measured on a Varian Gemini
300, JEOL JNM-AL 400, or Varian Unity plus 500 spec-
trometer. For 1H NMR spectra, chemical shifts are
reported as d values in ppm downfield from tetrameth-
ylsilane. For 13C NMR spectra, chemical shifts are
reported as d values in ppm relative to chloroform or
methanol. EI Mass spectra were measured on a JEOL
JMS-700N. The melting point was measured on a
YANACO Melting Point Apparatus MP-83 and are
uncorrected.
afford 11 (14.6 g, 86%) as a colorless oil: ½aꢂ +8.3° (c
1529, 1481, 1351, 1272, 1105 cmꢀ1
;
1H NMR
(300 MHz, CDCl3) d 7.43–7.19 (m, 10H), 4.52 (s, 4H),
4.13 (dt, 2H, J = 6.0, 1.6 Hz), 3.91 (br quint, 1H,
J = 5.3 Hz), 3.80–3.46 (m, 6H), 2.62 (br d, 1H,
J = 4.8 Hz), 1.87 (quint, 2H, J = 6.0 Hz); 13C NMR
(100 MHz, CDCl3) d 178.3, 137.9, 128.3, 127.6, 127.5,
78.4, 73.4, 70.8, 70.7, 69.9, 67.5, 61.3, 38.7, 29.4, 27.2;
HRMS (EI) m/z calcd for C26H36O6 (M+) 444.2512,
found 444.2506.
3.2. (2S,3R)-3-[(4-Nitrobenzoyl)oxy-1,4-bisbenzyloxy-
butan-2-yloxy]propyl pivalate (10)
3.4. [(S)-2-Hydroxy-1-((S)-2,2-dimethyl-1,3-dioxolan-4-
yl)ethoxy]propyl pivalate (12)
To a mixture of triphenylphosphine (23.6 g, 90.0 mmol),
p-nitrobenzoic acid (15.0 g, 90.0 mmol), and 9 (20.0 g,
45.0 mmol) in toluene (600 mL) was added DEAD
(2.2 M in toluene, 40.9 mL, 90.0 mmol). After stirring
at room temperature for 20 h, most of the toluene was
evaporated. The residue was diluted with Et2O, washed
with H2O and brine, dried, concentrated, and chromato-
A mixture of 11 (12.3 g, 29.2 mmol) and Pd(OH)2
(1.30 g, 1.76 mmol) in MeOH (210 mL) was stirred un-
der H2 atmosphere at room temperature for 24 h. The
reaction mixture was filtered through Celite pad, and
the filtrate was concentrated to give the corresponding
triol (7.41 g) as a colorless oil. To a solution of the crude
triol (7.41 g) in acetone (31 mL) were added 2,2-dimeth-
oxypropane (4.37 g, 42.0 mmol) and p-toluenesulfonic
acid monohydrate (13.3 mg, 0.07 mmol), and the mix-
ture was stirred at room temperature for 4 h. The reac-
tion mixture was diluted with AcOEt, washed with
graphed (SiO2 500 g, hexane/AcOEt = 6:1) to give 10
24
D
CHCl3); IR (neat) 3808, 3739, 3455, 2863, 1718, 1465,
(22.4 g, 84%) as a colorless oil; ½aꢂ +5.9° (c 1.00,
1
1282, 1159, 1091 cmꢀ1; H NMR (300 MHz, CDCl3) d
8.26 (d, 2H, J = 8.7 Hz), 8.25 (d, 2H, J = 9.0 Hz),

8054
S. Hatakeyama et al. / Bioorg. Med. Chem. 14 (2006) 8050–8056
saturated NaHCO3 and brine, dried, concentrated, and
chromatographed (SiO2, 200 g, hexane/AcOEt = 4:1) to
afford 12 (7.78 g, 88%) as a colorless oil; ½aꢂ +8.30° (c
was added Novozyme (2.2 g), and the mixture was stir-
red at 30 °C for 7 d. The reaction mixture was filtered
through Celite pad, concentrated, and chromatographed
(SiO2 150 g, hexane/AcOEt = 10:1) to afford 14 (2.03 g,
24
D
1.00, CHCl3); IR (neat) 3500, 2973, 1720, 1473, 1375,
1288, 1216, 1164, 1052 cmꢀ1
;
1H NMR (400 MHz,
40%) and S-13 (R:S = 1:20, 2.50 g, 57%) each as a color-
26
D
CDCl3) d 4.25 (dt, 1H, J = 11.2, 6.1 Hz), 4.14 (quint,
1H, J = 5.6 Hz), 4.01 (dd, 1H, J = 7.6, 6.8 Hz), 3.78–
3.62 (m, 4H), 3.56 (quint, 1H, J = 6.0 Hz), 3.41 (dd,
1H, J = 8.1, 6.0 Hz), 2.32 (br t, 1H, J = 6.0 Hz), 1.93
(quint, 2H, J = 6.3 Hz), 1.43 (s, 3H), 1.36 (s, 3H), 1.20
(s, 9H); 13C NMR (100 MHz, CDCl3) d 178.7, 109.5,
80.7, 76.7, 67.5, 65.8, 61.9, 61.4, 36.0, 29.8, 27.5, 26.7,
25.6; HRMS (EI) m/z calcd for C15H28O6 (M+)
304.1886, found 304.1885.
less oil. Compound 14: ½aꢂ ꢀ9.1° (c 1.10, CHCl3); IR
(neat) 2985, 1752, 1644, 1481, 1378, 1251 cmꢀ1
;
1H
NMR (400 MHz, CDCl3) d 5.90 (ddd, 1H, J = 17.2,
10.4, 5.6 Hz), 5.35 (d, 1H, J = 17.2 Hz), 5.25 (d, 1H,
J = 10.8 Hz), 4.17 (quint, 3H, J = 6.0 Hz), 3.97 (t, 1H,
J = 8.5 Hz), 3.81–3.65 (m, 3H), 3.35 (dd, 1H, J = 5.2,
4.3 Hz), 2.10 (s, 3H), 1.92 (quint, 2H, J = 6.0 Hz), 1.43
(s, 3H), 1.36 (s, 3H), 1.20 (s, 9H); 13C NMR
(100 MHz, CDCl3) d 179.1, 170.4, 133.6, 109.9, 82.2,
77.0, 74.5, 69.8, 66.5, 62.0, 39.6, 30.2, 28.0, 27.4, 23.4,
21.9; HRMS (EI) m/z calcd for C19H33O7 (M+)
3.5. A 3:2 mixture of 3-[(1S,2R)-2-hydroxy-1-((S)-2,2-
dimethyl-1,3-dioxolan-4-yl)but-3-enyloxy]propyl pivalate
and 3-[(1S,2S)-2-hydroxy-1-((S)-2,2-dimethyl-1,3-dioxo-
lan-4-yl)but-3-enyloxy]propyl pivalate (13)
372.2148,
found
372.2137.
Compound
S-13:
25
D
½aꢂ ꢀ 21:7ꢃ (c 0.42, CHCl3); IR (neat) 3487, 2976,
1722, 1471, 1375, 1286, 1216, 1163, 1065 cmꢀ1
;
1H
NMR (300 MHz, CDCl3) d 5.93 (ddd, 1H, J = 17.4,
10.7, 6.2 Hz), 5.34(dt, 1H, J = 17.4, 1.5 Hz), 5.23 (dt,
1H, J = 10.2, 1.5 Hz), 4.29–4.09 (m, 4H), 4.00 (dd, 1H,
J = 8.4, 6.3 Hz), 3.79–3.65 (m, 3H), 3.30 (dd, 2H,
J = 6.0, 5.0 Hz), 2.89 (d, 1H, J = 6.3 Hz), 1.92 (quint,
2H, J = 6.3 Hz), 1.42 (s, 3H), 1.36 (s, 3H), 1.20 (s,
9H); 13C NMR (100 MHz, CDCl3) d 178.2, 137.1,
116.4, 108.9, 82.4, 72.5, 67.9, 65.8, 61.0, 38.5, 29.2,
27.0, 26.2, 25.4, 20.8, 14.0; HRMS (EI) m/z calcd for
C17H30O6 (M+) 330.2048, found 330.2039.
To a solution of oxalyl chloride (624 mg, 4.92 mmol) in
CH2Cl2 (10 mL) was added DMSO (768 mg, 9.84 mmol)
at ꢀ78 °C. After stirring for 15 min at ꢀ78 °C, a solu-
tion of 12 (500 mg, 1.64 mmol) in CH2Cl2 (7 mL) was
added and stirring was continued at ꢀ78 °C for
30 min. Triethylamine (1510 mg, 14.8 mmol) was added,
and the mixture was allowed to warm to 0 °C and stirred
at 0 °C for 1 h. The reaction mixture was diluted with
AcOEt, washed with saturated NH4Cl (10 mL), H2O,
and brine, dried, concentrated, and chromatographed
(SiO2 10 g, hexane/AcOEt = 1:1) affording the corre-
sponding aldehyde (580 mg) which was used for the next
reaction without purification. To a solution of the crude
aldehyde (580 mg) in THF (17 mL) was added vinyl-
magnesium bromide (1 M in THF, 5.05 mL, 5.05 mmol)
at ꢀ40 °C, and the mixture was stirred at ꢀ40 °C for
3 h. The reaction was quenched with saturated NH4Cl
and the reaction mixture was extracted with AcOEt.
The extract was washed with H2O and brine, dried, con-
centrated, and chromatographed (SiO2 20 g, hexane/
AcOEt = 6:1) to give 13 (330 mg, 66%), a colorless oil,
as a diastereomer mixture (R:S = 2:3); IR (neat) 3488,
3.7. 3-[(2S,3S,4R)-4-Acetoxy-1,2-dihydroxyhex-5-en-3-
yloxy]propyl pivalate (15)
A solution of 14 (300 mg, 0.805 mmol) in 60% aqueous
AcOH (2 mL) was stirred at room temperature for 22 h.
The reaction mixture was carefully basified by the addi-
tion of NaHCO3, diluted with CH2Cl2, washed with
H2O and brine, dried, and concentrated. Purification
of the residue by chromatography (SiO2 20 g, hexane/
AcOEt = 2:1) gave 15 (240 mg, 90%) as a colorless oil;
26
D
½aꢂ +18.5° (c 1.00, CHCl3); IR (neat) 3478, 2969,
1729, 1481, 1371, 1286, 1238, 1162, 1079 cmꢀ1
;
1H
2981, 1729, 1481, 1371, 1286, 1168 cmꢀ1
;
1H NMR
NMR (400 MHz, CDCl3) d 5.90 (ddd, 1H, J = 17.2,
10.4, 5.6 Hz), 5.39 (t, 1H, J = 6.0 Hz), 5.34 (d, 1H,
J = 17.2 Hz), 5.27 (d, 1H, J = 10.4 Hz), 4.28 (dt, 1H,
J = 10.8, 6.4 Hz), 4.12 (dt, 1H, J = 11.2, 6.0 Hz), 3.81
(dt, 1H, J = 8.0, 4.8 Hz), 3.80–3.55 (m, 3H), 3.42 (t,
1H, J = 5.2 Hz), 2.68 (d, 1H, J = 4.0 Hz), 2.10 (s, 4H),
1.90 (quint, 2H, J = 6.0 Hz), 1.43 (s, 3H), 1.20 (s, 9H);
13C NMR (100 MHz, CDCl3) d 178.5, 169.7, 132.4,
118.4, 80.0, 73.7, 70.9, 68.8, 63.5, 60.9, 38.7, 29.5, 27.2,
21.1; HRMS (EI) m/z calcd for C16H29O7 [(M+H)+]
333.1913, found 333.1910.
(300 MHz, CDCl3) d 5.91 (ddd, 1H, J = 16.2, 10.5,
5.7 Hz), 5.42 (dd, 1H, J = 15.6, 3.3 Hz), 5.23 (dd, 1H,
J = 12.0, 3.6 Hz), 4.29–4.15 (m, 4H), 4.05 (t, 1H,
J = 8.5 Hz), 3.65 (m, 2H), 3.30 (dd, 1H, J = 5.2,
4.3 Hz), 3.25 (dd, 1H, J = 6.4, 3.6 Hz), 2.83 (d, 0.6H,
J = 3.6 Hz), 2.55 (d, 0.4H, J = 3.6 Hz), 1.92 (quint, 2H,
J = 6.3 Hz), 1.40 (s, 3H), 1.34 (s, 3H), 1.20 (s, 9H); 13C
NMR (100 MHz, CDCl3) d 178.6, 178.5, 137.9, 137.1,
128.4, 116.6, 115.0, 109.4, 109.3, 82.5, 82.2, 77.6, 77.1,
76.8, 72.9, 72.9, 69.5, 68.2, 66.3, 66.1, 61.4, 61.3, 39.0,
39.0, 29.7, 29.7, 27.6, 26.8, 26.6, 25.8; HRMS (EI) m/z
calcd for C17H30O6 (M+) 330.2642, found 330.2036.
3.8. 3-[(1S,2R)-2-Acetoxy-1-((S)-oxiran-2-yl)but-3-enyl-
oxy]propyl pivalate (16)
3.6. 3-[(1S,2R)-2-Acetoxy-1-((S)-2,2-dimethyl-1,3-dioxo-
lan-4-yl)but-3-enyloxy]propyl pivalate (14) and 3-
[(1S,2S)-2-hydroxy-1-((S)-2,2-dimethyl-1,3-dioxolan-4-
yl)but-3-enyloxy]propyl pivalate (S-13)
To a solution of 15 (1.80 g, 5.12 mmol) in dioxane
(50 mL) were added triphenylphosphine (2.01 g,
7.57 mmol) and DEAD (2.2 M in toluene, 3.49 mL,
7.67 mmol), and the mixture was refluxed for 17 h.
The reaction mixture was concentrated and chromato-
graphed (SiO2 150 g, hexane/AcOEt = 10:1) to give 16
A solution of vinyl acetate (2.3 g, 26.7 mmol) and 13
(4.40 g, 13.3 mmol) in tert-butyl methyl ether (133 mL)

S. Hatakeyama et al. / Bioorg. Med. Chem. 14 (2006) 8050–8056
8055
24
(1.20 g, 75%) as a colorless oil; ½aꢂ +11.3° (c 1.00,
J = 6.0, 5.2 Hz), 2.60 (ddd, 1H, J = 15.6, 6.0, 2.4 Hz),
2.32 (ddd, 1H, J = 15.6, 6.0, 2.4 Hz), 2.16 (s, 1H), 1.92
(quint, 2H, 8.0 Hz), 0.94–0.91 (m, 30H), 0.62–0.55 (m,
21H); 13C NMR (100 MHz, CDCl3) d 138.7, 115.6,
84.6, 82.6, 74.0, 71.7, 70.1, 70.1, 34.0, 24.3, 6.7, 6.6,
5.7, 5.4, 4.8; HRMS (EI) m/z calcd for C29H60O4Si3
(M+) 556.3799, found 556.3798.
D
CHCl3); IR (neat) 2969, 1735, 1473, 1371, 1284, 1236,
1
1162, 1108, 1029 cmꢀ1; H NMR (400 MHz, CDCl3) d
5.90 (ddd, 1H, J = 17.2, 10.6, 6.4 Hz), 5.39 (t, 1H,
J = 6.0 Hz), 5.32 (d, 1H,, J = 17.6 Hz), 5.27 (d, 1H,
J = 10.8 Hz), 4.17 (dt, 3H, J = 6.4, 1.2 Hz), 3.84 (dt,
1H, J = 9.6, 6.0 Hz), 3.60 (dt, 1H, J = 10.0, 6.0 Hz),
3.05–3.00 (m, 2H), 2.77 (t, 1H, J = 2.0 Hz), 2.55 (quant,
1H, J = 4.4 Hz), 2.11 (s, 3H), 1.90 (quint, 2H,
J = 3.6 Hz); 13C NMR (100 MHz, CDCl3) d 179.0,
170.3, 133.1, 119.1, 82.7, 75.0, 68.0, 61.9, 52.8, 44.3,
29.9, 28.0, 21.8; HRMS (EI) m/z calcd for C14H23O5
[(MꢀAc)+] 271.1546, found 271.1541.
3.11. (5Z,7E)-(1R,2R,3S)-2-(3-Hydroxypropoxy)-9,10-
secocholesta-5,7,10(19)-triene-1,3,25-triol (4)
To a solution of 5 (144.3 mg, 0.26 mmol) and 6
(134.6 mg, 0.38 mmol) in degassed toluene (6.2 mL) were
added triethylamine (3.7 mL, 26.5 mmol) and (Ph3P)4Pd
(87.9 mg, 0.076 mmol). After being refluxed for 2 h, the
reaction mixture was diluted with Et2O, filtered through
Celite pad, concentrated, and chromatographed (SiO2
18 g, hexane/AcOEt = 10:1) to give a mixture of the cou-
pling product and 6 (183.2 mg) as an yellow oil. The mix-
ture (183.2 mg) thus obtained was dissolved in MeOH
(16 ml) and NH4F (55.9 mg, 1.51 mmol) was added.
After being refluxed for 4 h, the reaction mixture was
concentrated, extracted with AcOEt, washed with H2O
and saturated NaHCO3, dried, and concentrated. The
residue was purified by preparative TLC (AcOEt) fol-
lowed by HPLC (ODS-M80) (MeCN/H2O = 1:1) to af-
ford 4 (58.4 mg, 46%) as a colorless oil. This material
3.9. (3R,4R,5S)-4-(3-Hydroxypropoxy)oct-1-en-7-yne-
3,5-diol (17)
To
a
solution of trimethylsilylacetylene (1.72 g,
17.5 mmol) in THF (20 mL) was added n-BuLi
(1.45 M in THF, 11.3 mL, 17.5 mmol) at ꢀ78 °C, and
the mixture was stirred at ꢀ78 °C for 30 min. BF3ÆEt2O
(2.49 g, 17.5 mmol) and a solution of 16 (1.11 g,
3.53 mmol) in THF (50 mL) were added and stirring
was continued at ꢀ78 °C for 1 h. After the mixture
was allowed to warm to room temperature over 1 h,
the reaction was quenched with saturated NaHCO3.
The reaction mixture was extracted with CH2Cl2,
washed with H2O and brine, dried, and concentrated.
The residue was dissolved in MeOH (8 mL) and 10 M
aqueous NaOH (8 mL) was added. After being stirred
at room temperature for 6 h, the reaction mixture was
concentrated and extracted with AcOEt. The aqueous
layer was concentrated and extracted with THF. Com-
bined organic extracts were concentrated and chromato-
was crystallized from a small amount of MeCN to form
26
D
MeOH); IR (KBr) 3332, 2939, 1641, 1442, 1375,
colorless crystals; mp 126–128°; ½aꢂ ꢀ61.6° (c 0.39,
1082 cmꢀ1 1H NMR (500 MHz, CD3OD) d 6.32 (d,
;
1H, J = 9.8 Hz), 5.99 (d, 1H, J = 11.2 Hz), 5.13 (dt,
2H, J = 24.3, 2.4 Hz), 3.96–3.87 (m, 2H), 3.79 (dt, 1H,
J = 8.9, 2.2 Hz), 3.71 (t, 2H, J = 6.1 Hz), 3.51–3.46 (m,
1H), 2.97 (t, 1H, J = 8.9 Hz), 2.84 (dd, 1H, J = 5.0,
11.2 Hz), 2.50 (dd, 1H, J = 12.8, 5.2 Hz), 2.17 (t, 1H,
J = 11.1), 2.04–1.99 (m, 2H), 1.99–1.87 (m, 1H), 1.82
(quint, 2H, J = 6.1 Hz), 1.70–1.66 (m, 2H), 1.58–1.40
(m, 7H), 1.37–1.28 (m, 4H), 1.26–1.22 (m, 1H), 1.16 (s,
6H), 1.10–1.03 (m, 1H), 0.96 (d, 3H, J = 6.4 Hz), 0.58
(s, 3H); 13C NMR (100 MHz, CD3OH) d 147.3, 143.6,
134.0, 124.5, 118.7, 111.8, 90.2, 75.3, 73.0, 71.5, 71.4,
60.5, 58.0, 57.6, 47.1, 45.3, 43.6, 41.8, 37.7, 33.7, 30.0,
29.3, 29.1, 28.7, 24.8, 23.4, 21.9, 19.4, 12.3; HRMS
(EI) m/z calcd for C30H50O5 (M+) 490.3659, found
490.3658.
graphed (SiO2, 35 g, hexane/AcOEt = 1:1) to give 17
24
D
CHCl3); IR (neat) 3754, 3417, 2884, 2235, 2117, 1643,
(503 mg, 65%) as a colorless oil; ½aꢂ +13.3° (c 0.50,
1
1423, 1072 cmꢀ1; H NMR (300 MHz, CDCl3) d 5.90
(ddd, 1H, J = 17.2, 10.6, 6.4 Hz), 5.41 (d, 1H,
J = 17.6 Hz), 5.25 (d, 1H, J = 10.5 Hz), 4.31 (dd, 1H,
J = 9.0, 5.2 Hz), 3.93–3.80 (m, 5H), 3.42 (dd, 1H, 6.3,
3.0 Hz), 2.52 (m, 2H), 2.06 (s, 1H), 1.85 (quant,
J = 5.2 Hz); 13C NMR (100 MHz, CDCl3) d 137.2,
116.9, 82.7, 80.3, 73.7, 72.0, 70.7, 70.4, 60.5, 31.9, 24.0;
HRMS (EI) m/z calcd for C14H23O5 [(M+H)+]
215.1283, found 215.1274.
3.10. (3R,4R,5S)-4-(3-(Triethylsilyloxy)propoxy)-3,5-
di(triethylsilyloxy)oct-1-en-7-yne (5)
3.12. Inhibition of PTH secretion in cultured bovine
parathyroid cells
To a solution of triethylamine (1.52 g, 14.9 mmol) and
17 (400 mg, 1.87 mmol) in CH2Cl2 (20 mL) was added
triethylsilyl triflate (2.47 g, 9.33 mmol) at ꢀ40 °C, and
stirring was continued at ꢀ40 °C for 2 h. The reaction
mixture was diluted with CH2Cl2, washed with H2O
and brine, dried, concentrated, and chromatographed
The inhibitory activity of analogs (1–4) in cultured
bovine parathyroid cells was analyzed according to
Brown et al.25
3.13. VDR binding assay
(SiO2 40 g, hexane) to afford 5 (1.10 g, 100%) as a color-
The binding affinity of analogs (1–4) with human recom-
binant VDR was analyzed according to Wecksler et al.26
27
D
less oil; ½aꢂ +16.8° (c 0.98, CHCl3); IR (neat) 3313,
2954, 2877, 1459, 1415, 1240, 1095, 1006 cmꢀ1
;
1H
NMR (300 MHz, CDCl3) d 5.94 (ddd, 1H, J = 17.0,
10.8, 6.4 Hz), 5.24 (d, 1H, J = 17.2 Hz), 5.10 (d, 1H,
J = 10.4 Hz), 4.34 (dd, 1H, J = 12.0, 6.8 Hz), 3.85 (dd,
1H, J = 10.8 8.0 Hz), 3.74–3.64 (m, 4H), 3.20 (dd, 1H,
3.14. DBP binding assay
The binding affinity of analogs (1–4) with human DBP
was analyzed according to Preece et al.27

8056
S. Hatakeyama et al. / Bioorg. Med. Chem. 14 (2006) 8050–8056
11. Horst, R. L.; Reinhardt, T. A.; Reddy, G. S. In Vitamin
Acknowledgment
D; Feldman, D., Pike, J. W., Glorieux, F. H., Eds.,
2nd ed.; Elsevier Academic Press: Burlington, 2005; pp
24–25.
We are grateful to Professor David Horne of Oregon
State University for helpful comments and English
editing.
12. Matsumoto, T.; Miki, T.; Hagino, H.; Sugimoto, T.;
Okamoto, S.; Hirota, T.; Tanigawara, Y.; Hayashi, Y.;
Fukunaga, M.; Shiraki, M.; Nakamura, T. J. Clin.
Endocrinol. Metab. 2005, 90, 5031.
13. Brown, A. J.; Ritter, C.; Weiskopf, A. S.; Vouros, P.;
Sasso, G. J.; Uskokovic, M. R.; Wang, G.; Reddy, G. S.
J. Cell. Biochem. 2005, 96, 569.
14. Brown, A. J.; Ritter, C. J.; Slatopolsky, E.; Muralidharan,
K. R.; Okamura, W. H.; Reddy, G. S. J. Cell. Biochem.
1999, 73, 106.
15. Trost, B. M.; Dumas, J. J. Am. Chem. Soc. 1992, 114,
1924.
References and notes
1. A part of this work has been reported in proceedings of
the13th Workshop on Vitamin D (Victoria, Canada, 8–12
April, 2006). Hatakeyama, S.; Nagashima, S.; Imai, N.;
Takahashi, K.; Ishihara, J.; Sugita, A.; Nihei, T.; Saito,
H.; Takahashi, F.; Kubodera, N. J. Steroids Biochem.
Molecular Biol., in press.
2. Bouillon, R.; Okamura, W. H.; Norman, A. W. Endocr.
Rev. 1995, 16, 200.
16. Trost, B. M.; Dumas, J.; Villa, M. J. Am. Chem. Soc. 1992,
114, 9836.
3. Posner, G. H.; Kahraman, M. In Vitamin D; Feldman, D.,
Pike, J. W., Glorieux, F. H., Eds., 2nd ed.; Elsevier
Academic Press: Burlington, 2005; pp 1405–1422.
4. Miyamoto, K.; Murayama, E.; Ochi, K.; Watanabe, H.;
Kubodera, N. Chem. Pharm. Bull. 1993, 41, 1111.
5. Ono, Y.; Watanabe, H.; Shiraishi, A.; Takeda, S.; Higu-
chi, Y.; Sato, K.; Tsugawa, N.; Okano, T.; Kobayashi, T.;
Kubodera, N. Chem. Pharm. Bull. 1997, 45, 1626.
6. Ono, Y.; Kawase, A.; Watanabe, H.; Shiraishi, A.;
Takeda, S.; Higuchi, Y.; Sato, K.; Yamauchi, T.; Mikami,
T.; Kato, M.; Tsugawa, N.; Okano, T.; Kubodera, N.
Bioorg. Med. Chem. 1998, 6, 2517.
7. Okano, T.; Tsugawa, N.; Masuda, S.; Takeuchi, A.;
Kobayashi, T.; Takita, Y.; Nishii, Y. Biochem. Biophys.
Res. Commun. 1989, 163, 1444.
8. Kobayashi, T.; Okano, T.; Tsugawa, N.; Murano, M.;
Masuda, S.; Takeuchi, A.; Sato, K.; Nishii, Y. Bioorg.
Med. Chem. Lett. 1993, 3, 1815.
17. Hatakeyama, S.; Kawase, A.; Uchiyama, Y.; Maeyama,
J.; Iwabuchi, Y.; Kubodera, N. Steroids 2001, 66, 267.
18. Martin, S. F.; Dodge, J. A. Tetrahedron Lett. 1991, 32,
3017.
19. Although a ratio of R and S was reported as R/S = 3/2 in a
previous report (Ref. 1), a correct ratio should be R/S =
2/3.
20. Nakamura, K.; Hirose, Y. J. Synth. Org. Chem. Jpn. 1995,
53, 668.
21. Mitsunobu, O. Synthesis 1981, 1.
22. Yamaguchi, M.; Hirao, I. Tetrahedron Lett. 1983, 24, 391.
23. Hirata, M.; Endo, K.; Katsumata, K.; Ichikawa, F.;
Kubodera, N.; Fukagawa, M. Nephrol. Dial. Transplant.
2002, 17, 41.
24. Ono, Y.; Watanabe, H.; Kawase, A.; Kubodera, N.
Bioorg. Med. Chem. Lett. 1994, 4, 1523.
25. Brown, E.; Hurwitz, S.; Aurbach, G. Endocrinology 1976,
99, 1582.
26. Wecksler, W. R.; Norman, A. W. Anal. Biochem. 1979, 92,
314.
9. Kubodera, N.; Tsuji, N.; Uchiyama, Y.; Endo, K. J. Cell.
Biochem. 2003, 88, 286.
10. Silver, J.; Naveh-Many, T. In Vitamin D; Feldman, D.,
Pike, J. W., Glorieux, F. H., Eds., 2nd ed.; Elsevier
Academic Press: Burlington, 2005; pp 537–549.
27. Preece, M. A.; O’Riordan, J. L.; Lawson, D. E.; Kodicek,
E. Clin. Chim. Acta 1974, 54, 2352.