Synthesis of C-2 substituted vitamin D derivatives having ringed side chains and their biological evaluation, especially biological effect on bone by modification at the C-2 position

Article abstract of DOI:10.1039/c1ob05142c

In order to obtain vitamin D derivatives, which have strong activity for enhancing bone growth, we designed vitamin D derivatives with various substitutions at the C-2 position. Novel 2 α-substituted vitamin D derivatives were synthesized starting from d-

Full text of DOI:10.1039/c1ob05142c

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PAPER
Synthesis of C-2 substituted vitamin D derivatives having ringed side chains
and their biological evaluation, especially biological effect on bone by
modification at the C-2 position†
Hiroshi Saitoh,*a Takayuki Chida,a Kenichiro Takagi,a Kyohei Horie,a Yoshiyuki Sawai,a Yuko Nakamura,a
Yoshifumi Harada,a Kazuya Takenouchia and Atsushi Kittakab
Received 26th January 2011, Accepted 7th March 2011
DOI: 10.1039/c1ob05142c
In order to obtain vitamin D derivatives, which have strong activity for enhancing bone growth, we
designed vitamin D derivatives with various substitutions at the C-2 position. Novel 2 a-substituted
vitamin D derivatives were synthesized starting from D-glucose as a chiral template of the A-ring with a
CD-ring bromoolefin unit using the Trost coupling method. We evaluated these compounds by two in
vitro assays, affinity to VDR and transactivation assays, using human osteosarcoma (Hos) cells, and
demonstrated the SAR of the C-2 position of VD3. Furthermore, by using the OVX model, we found
that compound 5c, which has a hydroxypropoxy side chain at C-2 and 2,2-dimethyl cyclopentanone in
the CD-ring side chain, has a strong activity for enhancing bone growth, same as the reported
compound, 2a-(3-hydroxypropoxy)-1a,25-dihydroxyvitamin D3 1d, and this derivative shows a
possibility that calcemic activity is less than 1d in vivo.
such as the proliferation and differentiation of various types in
tumor cells, and the regulation of immune reactions,3,4 the most
1. Introduction
1a,25-Dihydroxyvitamin D3 (Fig. 1, 1a) shows biological activities
through the interaction with a vitamin D receptor (VDR),
which is a member of the nuclear hormone receptor superfamily,
and acts as a ligand-dependent gene transcription factor with
co-activators.1,2 Although the biological activities of 1a vary,
important physiological role of 1a is the regulation of calcium and
phosphorus metabolism as well as bone remodeling via its action in
the bone, intestine and kidney; therefore, 1a is used as a therapeutic
drug for osteoporosis, a systemic skeletal disease characterized by
low bone mineral density (BMD) leading to reduced bone strength
and fracture.
However, the use of vitamin D as a therapeutic agent is limited
because of its calcemic and phosphatemic activities; therefore, in
order to find new vitamin D analogs, which are more efficacious,
safer and more selective than natural 1a, numerous analogs of 1a
have been developed.4,5 Most of the analogs have a modified side
chain. As an example, 1a,25-dihydroxyvitamin D3-26,23-lactones
2, which was reported by Ishizuka et al., has a unique structure
aTeijin Institute for Bio-Medical Research, Asahigaoka, Hino, Tokyo,
191-8512, Japan. E-mail: hi.saitou@teijin.co.jp; Fax: +81-42-586-8293;
Tel: +81-42-586-8409
bFaculty of Pharmaceutical Sciences, Teikyo University, Sagamihara, Kana-
gawa, 252-5195, Japan
† Electronic supplementary information (ESI) available: Detailed experi-
mental procedures including spectral data of compounds 14, 15 and 5b–5h.
See DOI: 10.1039/c1ob05142c
Fig. 1 Structures of 1a,25-dihydroxyvitamin D3 (1) and its derivatives.
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with a lactone ring on the side chain.6 We therefore synthesized
and evaluated vitamin D derivatives with a ringed side chain in
their structures. In a series of derivatives, we report that compound
3, which has a cyclopentanone in the side chain, showed strong
affinity with VDR.7
On the other hand, Kittaka et al. developed the first systematic
synthesis of novel analogs of vitamin D3 based on the structural
modification of the A-ring in order to investigate A-ring structure–
activity relationships.8–13 During the course of their studies, they
found that the introduction of methyl (1b),8 3-hydroxypropyl (1c),9
and 3-hydroxypropoxy (1d) 10 groups at the C-2a position showed
2- to 4-fold higher binding affinity relative to the natural hormone
1a (Fig. 2). In addition, Saito et al. reported that 2a modification
of TEI-9647 (4a), which is the first vitamin D3 antagonist, increases
antagonistic activity significantly.14
Against this background, we modified 3 with 2a substitution
in order to obtain VD3 derivatives, which have strong activity for
enhancing bone growth. As mentioned above, the 2a-methyl group
(1b), 2a-3-hydroxypropyl group (1c), and 2a-3-hydroxypropoxy
group (1d) were appropriate substituted groups for the higher
affinity to VDR. Recent research about co-crystals of these ligands
and the ligand-binding domain (LBD) of VDR clarified the reason
for the higher activity of these compounds.15 Namely, the C-2a
methyl group of 1b interacts with amino acid residues Ser237,
Leu233, and Phe150 in the LBD, and each C-2a terminal hydroxy
group of 1c and 1d forms a new hydrogen bond between the
hydroxy group and Arg274; therefore, we were interested in the
effect of these substitutions for enhancing bone growth and we
designed 5a–5c (Fig. 3). In addition, we designed 5d–5h as new 2a
substituted groups. As stated above, the co-crystals of 1c and 1d
only showed an interaction between the terminal hydroxy group
and Arg274 of VDR, but there remained a cavity around the alkyl
side chain of 1c and 1d. We therefore designed 5d and 5e because
we expected that more occupation of the cavity around the C-2
position of the A-ring would increase the affinity to VDR and the
biological activities. Furthermore, we designed 5f–5h in order to
investigate the effects for the affinity to VDR and the biological
activities by the difference of the terminal group in 2a-substituted
groups.
2. Results
Our synthesis plan of 2a-substituted vitamin D3 derivatives is
shown in Scheme 1. The triene skeleton of the desired vitamin
D3 derivatives (5a–5h) was constructed by Trost’s Pd-catalyzed
alkenylative cyclization 16 of A-ring precursor enynes (7a–7h) with
the CD-ring bromoolefin counterpart (6).7 The A-ring precursor
enynes (7a–7h) could be synthesized from D-glucose derivatives on
the basis of the reported scheme.10
2.1. Synthesis
2.1.1. Preparation of A-ring precursors 7a–7h. We synthe-
sized the A-ring precursors 7d and 7g, in which the cyclopropyl
group is introduced into the center of the 2a side chain from the
D-glucose derivative (Scheme 2).
17
Treatment of the known epoxide 8 with 2,2-cyclopropane-
1,3-diol and KOtBu under heat conditions gave 3-(3-hydroxy-2,2-
ethanopropyl)oxyaltropyranoside 9. Protection of the primary
alcohol using pivaloyl chloride, followed by O-silylation, gave
Fig. 2 Structures of 1a,25-dihydroxyvitamin D3 (1a), 1a-hydroxyvitamin D3-26,23-lactones (TEI-9647:4a) and their representative C-2a-modified
analogs (1b–d, 4b–d).
Fig. 3 Design of derivatives of 3 (R2 = H).
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Scheme 1 Retrosynthesis.
Scheme 2 Synthetic route of 7d.
protected altropyranoside 10. Treatment with NBS, followed by
reduction using activated zinc powder and NaBH3CN gave alcohol
11. Tosylation and subsequent deprotection with TBAF provided
epoxide 12. Ethynylation using lithium TMS acetylide in the
presence of BF3-Et2O in THF and methanolysis gave triol 13.
Finally, protection of triol by TBS groups gave the desired enyne
A-ring precursor 7d.
For the A-ring precursor 7g, the end of 2a side chain of
7d was substituted with a cyano group and synthesized as
follows (Scheme 3). Protection of the primary alcohol of 13
with a pivaloyl group, followed by silylation and deprotection
of the pivaloyl group gave alcohol 14. Oxidation of alcohol
followed by conversion to oxime and dehydration provided nitrile
7g.
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Scheme 3 Synthetic route of 7g.
Scheme 4 Synthetic route of 7e, 7f and 7h.
For the A-ring precursor 7e, the primary alcohol of the 2a side
2.2. Biological evaluation
chain of 7c was changed to the tertiary alcohol, was synthesized as
follows (Scheme 4). The known enyne A-ring precursor 7c 10 was
treated with camphorsulfonic acid to give alcohol 15. Oxidation
of 15 with excess PDC (5 mol. equiv.) provided carboxylic
acid 16. Esterification followed by re-protection gave the A-ring
precursor 7h. Moreover, the desired A-ring precursor 7e was
provided by treatment of 7h with methyl magnesium bromide,
followed by protection. Furthermore, the A-ring precursor 7f
was obtained by tosylation of 15 and substitution to the cyano
group.
2.2.1. In vitro evaluation (affinity to VDR and transactivation
assays on Hos cells). The newly synthesized derivatives were
evaluated in vitro. We evaluated new derivatives by two in vitro
assays, affinity to VDR and transactivation assays, using human
osteosarcoma (Hos) cells. The evaluation results are shown in
Table 1.
As described above, it was reported that the methyl group,
hydroxypropyl group, and 3-hydroxypropoxy group at C-2 con-
tributed to increased affinity to VDR, but when the side chain
in the CD-ring was the ring, there was a difference in activity
between the above appropriate substitution groups. Namely,
modification by the 3-hydroxypropoxy group at C-2, compound
5c, increased the affinity to VDR, but 2a-Me modification, 5a, and
3-hydroxypropyl modification, 5b, did not contribute to improve
the affinity to VDR markedly. Additionally, new derivatives 5d
and 5e, which have branched 3-hydroxypropoxy side chains at
2.1.2. Synthesis of 2-substituted vitamin D3 derivatives. The
palladium-catalyzed coupling reaction of the above enynes 7a–
7h and bromoolefin 6, followed by deprotection of silyl groups
and purification by reversed-phase HPLC provided the desired
compounds 5a–5h, respectively (Scheme 5).
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Scheme 5 Coupling and deprotection of A-ring precursor and CD-bromoolefin.
Table 1 VDR binding affinity and osteocalcin promoter transactivation in Hos cells
compd
VDR affinity (%)a
Transactivation assay (EC50, pM)
compd
VDR affinity (%)
Transactivation assay (EC50, pM)
1
100
30
16.2
20
11.2
6.7
5d
5e
5f
5g
5h
5.5
2
10
18
1
36.9
630
59.0
105
3
22.7
11.6
32.3
58.8
5a
5b
5c
7460
a The potency of 1 is normalized to 100.
C-2, also decreased the affinity to VDR. In particular, 5e led to a
significantly decreased affinity. In the case of compounds 5f–5h, in
which terminal groups in the side chain are substituted from the
hydroxy group to other groups, the affinity to VDR of compounds
5f and 5g was moderate, but that of 5h was decreased.
With respect to trans activity assay on Hos cells, it was found that
there was a moderate correlation between activity of trans activity
assay and the affinity to VDR (Fig. 4). Especially, compound 5c
showed a stronger activity (6.9 pM) than that of natural hormone
1 and compound 5c.
week-old Sprague-Dawley female rats (Charles River, Japan) were
ovariectomized and fed a normal diet containing 1.0% Ca ad
libitum for 4 weeks. The rats were then administrated vitamin D3
derivatives at doses from 0.006 mg kg-1 to 1 mg kg-1, 5 times a week
for 4 weeks. The sham and OVX groups were administrated MCT
alone. Twenty-four hours after the final administration, blood was
collected under ether anesthesia, the rats were then euthanized,
and BMD of the spine (L4–L5) bone mass was measured by dual
X-ray absorptiometry. The results of BMD and serum Ca density
of 3 vitamin D3 derivatives are shown in Fig. 5, 6 and 7, respectively.
As shown in Fig. 5a and 5b, the starting compound 3 did
not show such a strong increase of BMD, although it showed
strong activity in vitro, and the high dose of 3 especially showed
hypercalcemia. In the case of 2a-hydroxypropoxy-1,25-(OH)2-
vitamin D3 1d (Fig. 6a and 6b), it showed an increase of BMD,
which was not linear. Furthermore, the calcemic activity was
observed from the lowest dose. It was found that by stepping
up the dose, the calcemic activity was stronger than the increase
of BMD.
On the other hand, newly synthesized derivative 5c (Fig. 7a and
7b) showed a significant increase of BMD at 0.025 mg kg-1, and
the significant difference of the calcemic activity was not observed
at this dose.
Fig. 4 Relationship between the affinity to VDR and the transactivation
assay on Hos cells of 1, 3 and 5a–5h.
2.2.2. In vivo evaluation: Evaluation of effects of derivatives
on bone mineral loss in ovariectomized rats. As described above,
we found that compound 5c had strong activity on Hos cells;
therefore, we evaluated the in vivo therapeutic effect of compound
5c using an ovariectomized rat therapeutic model (OVX). Twelve-
3. Discussion
In this research, we designed novel vitamin D derivatives with the
reported and new 2a side chains. We designed and synthesized
compounds 5d and 5e because we expected that the terminal
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Fig. 5 (a) Spiral bone mineral density (BMD) in the OVX rat therapeutic
model administrated compound 3 orally (b) Serum calcium level in the
OVX rat therapeutic model administrated compound 3 orally.
Fig. 7 (a) Spiral bone mineral density (BMD) in the OVX rat therapeutic
model administrated compound 5c orally (b) Serum calcium level in the
OVX rat therapeutic model administrated compound 5c orally.
terminal hydroxy groups of these compounds might move by
insertion of the substituted groups into the alkyl side chains. In
addition, we designed compounds 5f–5h, in which the terminal
groups were substituted. In these compounds, the affinity to VDR
of compounds 5f and 5g did not markedly decrease. In the previous
paper,9b,10c it was reported that VD3 derivatives, in which alkyl side
chains were introduced at C-2, markedly decreased the affinity to
VDR except for the 2a-methyl substituted VD3 derivative.9a From
the difference between the activity of the cyano side chains and
alkyl side chains, it was supposed that compounds 5f and 5g might
interact with amino acids in VDR.
About the activity for enhancing bone growth, we evaluated
newly synthesized derivatives by transactivation assays on Hos
cells. The activity showed a moderate correlation with the affinity
to VDR. So, we consider the activity for enhancing bone growth
was based on the affinity to VDR.
Furthermore, we evaluated 2a-substituted vitamin D3 deriva-
tives in vivo. It was confirmed that compound 1d has strong activity
for enhancing bone growth (Fig. 6a), but the calcemic activity was
also observed from the lowest dose (Fig. 6b), so we consider that
compound 1d has a strong calcemic activity. On the other hand,
compound 5c showed a strong activity for enhancing bone growth,
same as 1d (Fig. 7a), but the significant difference of the calcemic
activity was not observed at 0.025 mg kg-1 (Fig.7b). From these
results, we consider that compound 5c possibly has a calcemic
activity less than that of 1d. It was supposed that the structure
of the side chain in the CD-ring would contribute to the separate
activity for enhancing bone growth from calcemic activity.
Fig. 6 (a) Spiral bone mineral density (BMD) in the OVX rat therapeutic
model administrated compound 1d orally (b) Serum calcium level in the
OVX rat therapeutic model administrated compound 1d orally.
hydroxy group of these compounds would form hydrogen bonds
with Arg274 as reported with compound 1d; however, the affinity
to VDR of compounds 5d and 5e was decreased more than that of
compound 5c. From these results, we consider that the terminal
hydroxy group of 2a side chains in compounds 5d and 5e might
not be able to form a hydrogen bond with Arg274 because the
4. Conclusion
We designed and synthesized novel vitamin D3 derivatives with
various substituted side chains at the C-2 position in order to
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obtain vitamin D3 derivatives with strong activity for enhancing
bone growth. As the result of invitro experiments, we demonstrated
the SAR of the C-2 position of VD3. Furthermore, by using the
OVX model, we found that compound 5c, which has a hydrox-
ypropoxy side chain at C-2 and 2,2-dimethyl cyclopentanone in
the CD-ring side chain, has a strong activity for enhancing bone
growth, same as the reported compound, 2a-(3-hydroxypropoxy)-
1a,25-dihydroxyvitamin D3 1d, and this derivative shows a possi-
bility that the calcemic activity is less than that of 1d in vivo.
by filtration and concentration. The residue was dissolved in
dry CH2Cl2 (20 mL) and cooled to 0 ◦C. To the solution were
added 2,6-lutidine (1.3 mL, 11.6 mmol) and t-butyldimethylsilyl
trifluoromethanesulfonate (2.14 mL, 9.32 mmol), and the reaction
mixture was stirred at room temperature for 1 h. Dry MeOH
(5 mL) was added to the reaction mixture and stirred at room
temperature for 5 min. The reaction mixture was evaporated and
diluted with toluene. The solution was washed with brine, and the
organic layer was dried over MgSO4, followed by filtration and
concentration. Purification by flash silica gel chromatography
(n-hexane–EtOAc = 95/5, then 90/10) gave 10 (3.19 g, 5.61 mmol)
in 69% as a colorless syrup. [a]2D5 +36.71 (c 1.0, CHCl3); IR (film,
CHCl3) 3020, 1716, 1217, 1107, 1047 cm-1; 1H NMR (400 MHz,
CDCl3) d: 7.49–7.34 (5H, m), 5.56 (1H, s), 4.45 (1H, s), 4.29–4.25
(2H, m), 4.18 (1H, d, J = 11.2 Hz), 3.98–3.92 (3H, m), 3.75
(1H, t, J = 12.0 Hz), 3.65 (1H, t, J = 2.7 Hz), 3.60 (1H, d, J =
9.5 Hz), 3.52 (1H, d, J = 9.5 Hz), 3.35 (3H, s), 1.19 (9H, s), 0.91
(9H, s), 0.61–0.51 (4H, m), 0.10 (3H, s), 0.10 (3H, s); 13C NMR
(100 MHz, CDCl3) d: 178.5, 137.8, 129.0, 128.2, 126.2, 102.3,
102.2, 73.5, 70.0, 69.4, 67.6, 58.4, 55.3, 38.8, 27.2, 25.7, 25.5,
20.4, 18.0, 8.2, 8.1, -5.0; EI-LRMS m/z 587.2 (M+Na), 582.2
(M+H2O); EI-HRMS calcd for C30H48O8SiNa 587.3011 (M+Na),
found 587.3012.
5. Experimental section
5.1. Synthesis
5.1.1. General. NMR spectra were measured using a JEOL
AL-400 magnetic resonance spectrometer. Infrared spectra data
were recorded on a JASCO FTIR-5300 spectrometer. Mass spectra
were measured on a Shimadzu LC-MS-IT-TOF. Specific optical
rotations were measured using a JASCO P-1030 polarimeter.
Purification by flash column chromatography on silica gel was
carried out using a Biotage FLASH system. Preparative thin layer
chromatography was performed using Merck Kieselgel F254 plates.
Reversed-phase HPLC was carried out on a Shimadzu LC-2010
system.
5.1.4. (2R,3R,4R)-4-(Benzoyloxy)-2-(tert-butyldimethylsilo-
xy)-3-[3-(2,2-dimethylpropionyl)oxy-2,2-ethanopropyl]oxyhex-5-
en-1-ol (11). To a solution of 10 (3.17 g, 5.61 mmol) in
cyclohexane (63 mL) were added BaCO3 (775 mg, 3.92 mmol),
benzoyl peroxide (136 mg, 0.56 mmol), and N-bromo succinimide
(1.21 g, 6.73 mmol) at room temperature, and the reaction
mixture was refluxed for 1 h. The reaction mixture was cooled
to room temperature followed by filtration with celite. The residue
was washed with cylcohexane. The combined organic layer was
washed with brine and then dried over MgSO4, followed by
filtration and concentration. The residue (4.0 g) was dissolved
in a mixture of 1-PrOH (36 mL) and H2O (4 mL). To the
solution were added activated zinc powder (7.38 g, 112.2 mmol)
and NaBH3CN (1.42 g, 22.4 mmol), and the mixture was stirred
vigorously at 110 ◦C for 1 h. The reaction mixture was cooled to
room temperature, followed by filtration. Almost all 1-PrOH was
removed by evaporation and the residue was diluted with EtOAc,
washed with brine, and dried over MgSO4. After filtration and
concentration, purification by flash chromatography on silica gel
(n-hexane–EtOAc = 90/10 then 80/20) provided alcohol 11 (1.50 g,
2.80 mmol) in 50% yield (2 steps). [a]2D5 +31.21 (c 1.0, CHCl3); IR
(film, CHCl3) 3020, 1716, 1215 cm-1; 1H NMR (400 MHz, CDCl3)
d: 8.05–8.02 (2H, m), 7.59–7.43 (3H, m), 6.11 (1H, ddd, J = 11.0,
17.3, 6.0 Hz), 5.78–5.75 (1H, m), 5.41 (1H, dt, J = 17.3, 1.3 Hz),
5.30 (1H, dt, J = 10.4, 1.2 Hz), 4.17 (1H, d, J = 11.4 Hz), 3.96–3.93
(2H, m), 3.81 (1H, dd, J = 11.4, 5.1 Hz), 3.73–3.68 (2H, m), 3.64
(1H, d, J = 9.7 Hz), 3.50 (1H, d, J = 9.7 Hz), 1.18 (9H, s), 0.90 (9H,
s), 0.55 (4H, t, J = 1.9 Hz), 0.09 (3H, s), 0.07 (3H, s); 13C NMR
(100 MHz, CDCl3) d: 178.5, 165.3, 133.1, 133.0, 130.2, 129.5,
128.4, 118.9, 82.4, 75.1, 74.9, 72.6, 67.4, 63.9, 38.8, 27.1, 25.8,
20.7, 18.0, 8.6, -4.6; EI-LRMS m/z 557.2 (M+Na); EI-HRMS
calcd for C29H46O7SiNa 557.2905, found 557.2914.
5.1.2. Methyl 4,6-O-benzylidene-3-[(3-hydroxy-2,2-ethano)-
propyl]oxy-a-D-altoropyranoside (9). To a stirred solution of
epoxide 8 (6.0 g, 22.7 mmol) in N-methyl-pyrolidone (60 mL) was
added 2,2-cyclopropane-1,3-propanediol (15 g, 146.8 mmol) and
KOtBu (10.19 g, 90.8 mmol) at room temperature. The solution
was stirred at 130 ◦C for 8 h. The reaction mixture was cooled to
room temperature, the solution was diluted with H2O (240 mL),
and Dia-ion HP-20SS (Mitsubishi Chemical Industries, 30 g) was
added to the solution and stirred at room temperature overnight.
Filtration was followed by washing with saturated aq. NH4Cl,
H2O, and elution by acetone. The elution was evaporated, the
residue was diluted with EtOAc, and the solution was washed
with brine. The organic layer was dried over MgSO4, followed by
filtration and concentration. The residue was purified by flash
column chromatography on silica gel (n-hexane–EtOAc = 20/80)
to provide 2.97 g of 9 as a colorless syrup (8.1 mmol, 36%).
[a]2D5 +71.1 (c 1.0, CHCl3); IR (film, CHCl3) 3020, 1217, 1099,
1
1045 cm-1; H NMR (400 MHz, CDCl3) d: 7.51–7.36 (5H, m),
5.54 (1H, s), 4.61 (1H, s), 4.40–4.29 (2H, m), 4.08 (1H, t, J =
4.3 Hz), 4.01 (1H, dd, J = 9.3, 2.7 Hz), 3.93 (1H, br s), 3.83–3.75
(3H, m), 3.60–3.50 (3H, m), 3.41 (3H, s), 0.59–0.41 (3H, m); 13C
NMR (100 MHz, CDCl3) d: 137.6, 129.8, 129.2, 128.4, 126.3,
126.2, 102.6, 101.2, 80.1, 76.0, 70.4, 69.8, 69.3, 59.1, 55.5, 22.8,
9.2, 9.1; EI-LRMS m/z 389.1 (M+Na); EI-HRMS calcd for
C19H26O7Na 389.1571 (M+Na), found 389.1576.
5.1.3. Methyl
4,6-O-Benzylidene-2-(tert-butyldimethylsily-
loxy-3-[3-(2,2-dimethylpropionyl)oxy-2,2-ethanopropyl]oxy-a-
D-altoropyranoside (10). To a solution of 9 (2.97 g, 8.10 mmol)
in pyridine (30 mL), pivaloyl chloride (1.15 mL, 9.32 mmol)
was added at 0 ◦C and stirred at the same temperature for 1 h.
Dry MeOH (3 mL) was added to the reaction mixture and
stirred at room temperature for 5 min. The reaction mixture was
evaporated and diluted with toluene. The solution was washed
with brine, and the organic layer was dried over MgSO4, followed
5.1.5. (3R,4R,5R)-3-(Benzoyloxy)-4-[3-(2,2-dimethylpropion-
yl)oxy-2,2-ethanopropyl]oxy-5,6-epoxyhex-1-ene (12). To a solu-
tion of 11 (2.41 g, 4.5 mmol) in dry MeCN (25 mL) were added
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triethylamine (1.26 mL, 9 mmol), trimethylamine hydrochloride
salt (86 mg, 0.9 mmol), and p-toluenesulfonyl chloride (1.30 g,
6.8 mmol) at room temperature and stirred at the same temper-
ature for 1 h. The reaction mixture was quenched with saturated
aq.NaHCO3 followed by evaporation. The residue was diluted
with EtOAc and washed with brine. The organic layer was dried
over MgSO4, followed by filtration and concentration. The residue
(3.31 g) was diluted with THF (18 mL) and tetrabutyl ammonium
fluoride (1 M in THF, 13.5 mL, 13.5 mmol) was added to the
solution and refluxed for 1.5 h. The reaction mixture was cooled to
room temperature and diluted with EtOAc, washed with brine. The
organic layer was dried over MgSO4, filtrated and concentrated.
The residue was purified by flash column chromatography on silica
gel (hexane–EtOAc = 90/10) to give 12 (851 mg, 2.1 mmol) in 47%
yield. [a]2D5 +31.21 (c 1.0, CHCl3); IR (film, CHCl3) 3020, 1718,
263.1 (M+Na); EI-HRMS calcd for C13H20O4Na 263.1254, found
263.1244.
5.1.7. (3R,4S,5R)-3,5-Bis-[(tert-butyldimethylsilyl)oxy]-4-[3-
(tert-butyldimethylsilyl)oxy-2,2-ethanopropyl]oxyoct-1-en-7-yne
(7d). To a solution of 13 (257.0 mg, 1.07 mmol) in CH2Cl2
(5 mL) were added 2,6-lutidine (0.748 mL, 0.42 mmol)
and t-butyldimethylsilyl trifluoromethanesulfonate (1.11 mL,
4.82 mmol) at 0 ◦C and they were stirred at room temperature
for 1 h. Dry MeOH (2 mL) was added to the reaction mixture at
room temperature and stirred at the same temperature for 10 min.
The reaction mixture was diluted with hexane, washed with H2O
and dried over MgSO4, followed by filtration and evaporation. The
residue was purified by flash column chromatography on silica gel
(hexane–EtOAc = 96/4) to give 7d (571 mg, 0.979 mmol) in 91%
yield. [a]2D5 +4.62 (c 0.5, CHCl3); IR (film, CHCl3) 3020, 1253,
1217, 1086 cm-1; 1H-NMR (400 MHz, CDCl3) d: 6.01–5.92 (1H,
m), 5.20 (1H, d, J = 17.3 Hz), 5.11 (1H, d, J = 12.2 Hz), 4.32 (1H,
dd, J = 7.2, 3.8 Hz), 3.96 (1H, dd, J = 11.4, 5.4 Hz), 3.62 (2H, dd,
J = 14.4, 10.0 Hz), 3.49 (2H, dd, J = 9.9, 7.4 Hz), 3.36 (1H, t, J =
4.3 Hz), 2.53 (1H, ddd, J = 16.8, 6.6, 2.7 Hz), 2.36 (1H, ddd, J =
16.8, 6.6, 2.7 Hz), 1.94 (1H, t, J = 2.7 Hz), 0.90 (9H, s), 0.88 (9H, s),
0.88 (9H, s), 0.40 (4H, d, J = 7.3 Hz), 0.12 (3H, s), 0.09 (3H, s), 0.06
(3H, s), 0.03 (3H, s), 0.02 (3H, s). 13C-NMR (100 MHz, CDCl3)
d: 139.1, 116.0, 84.9, 82.5, 75.7, 75.5, 74.5, 71.7, 69.6, 65.5, 26.0,
25.8, 25.7, 23.8, 22.8, 18.4, 18.2, 18.1, 7.8, 7.4, -4.0, -4.1, -4.2,
-4.7, -5.3, -5.3; EI-LRMS m/z 605.7 (M+Na); EI-HRMS calcd
for C31H62O4Si3Na 605.3848, found 605.3862.
1
1271, 1217, 1165, 1111 cm-1; H NMR (400 MHz, CDCl3) d:
8.05–8.02 (2H, m), 7.59–7.43 (3H, m), 6.11 (1H, ddd, J = 11.0,
17.3, 6.0 Hz), 5.78–5.75 (1H, m), 5.41 (1H, dt, J = 17.3, 1.3 Hz),
5.30 (1H, dt, J = 10.4, 1.2 Hz), 4.17 (1H, d, J = 11.4 Hz), 3.96–
3.93 (2H, m), 3.81 (1H, dd, J = 11.4, 5.1 Hz), 3.73–3.68 (2H, m),
3.64 (1H, d, J = 9.7 Hz), 3.50 (1H, d, J = 9.7 Hz), 1.18 (9H,
s), 0.90 (9H, s), 0.55 (4H, t, J = 1.9 Hz), 0.09 (3H, s), 0.07 (3H,
s); 13C NMR (100 MHz, CDCl3) d: 178.5, 165.3, 133.1, 133.0,
130.2, 129.5, 128.4, 118.9, 82.4, 75.1, 74.9, 72.6, 67.4, 63.9, 38.8,
27.1, 25.8, 20.7, 18.0, 8.6, -4.6; EI-LRMS m/z 425.1 (M+Na),
420.2 (M+H2O), 403.1 (M+H); EI-HRMS calcd for C23H30O6Na
425.1935, found 425.1929
5.1.8. (3R,4S,5R)-3,5-Bis-[(tert-butyldimethylsilyl)oxy]-4-[2-
(cyano)-2,2-ethanopropyl]oxyoct-1-en-7-yne (7g). To a solution
of 14 (155 mg, 0.33 mmol) in CH2Cl2 (3 mL) were added Dess–
Martin reagent (212 mg, 0.5 mmol) at 0 ◦C and it was stirred
at the same temperature for 1.5 h. The reaction mixture was
diluted with EtOAc and washed with aq. Na2S2O3, aq. NaHCO3
successively. The organic layer was dried over MgSO4, filtered
and evaporated. The residue (251.3 mg) was dissolved in EtOH
(4 mL), and hydroxylamine hydrochloride (46 mg, 0.66 mmol)
and sodium acetate (82 mg, 1.0 mmol) were added to the solution.
The reaction mixture was stirred at room temperature for 1.5 h.
The reaction mixture was diluted with EtOAc and washed with
brine. The organic layer was dried over MgSO4, followed by
filtration and evaporation. The residue (290 mg) was dissolved
in pyridine (6 mL), and cyanuric chloride (243 mg, 1.32 mmol)
was added to the solution. The reaction mixture was stirred
at 60 ◦C overnight. The reaction mixture was cooled to room
temperature and diluted with toluene. The solution was washed
with H2O, brine successively. The organic layer was dried over
MgSO4, filtered and evaporated. The residue was purified by flash
column chromatography on silica gel (hexane–EtOAc = 95/5) to
give 7g (107.2 mg, 0.23 mmol) in 70% yield. [a]2D5 -3.3 (c 1.0,
CHCl3); IR (film, CHCl3) 3314, 2930, 1637, 1253, 1095 cm-1; 1H-
NMR (400 MHz, CDCl3) d: 5.98 (1H, ddd, J = 17.0, 10.0, 6.0 Hz),
5.24 (1H, ddd, J = 17.2, 1.5, 1.0 Hz), 5.18 (1H, ddd, J = 10.0, 1.5,
1.0 Hz), 4.35–4.32 (1H, m), 3.86 (1H, q, J = 5.5 Hz), 3.74 (1H,
d, J = 10. 0 Hz), 3.65 (1H, d, J = 10.0 Hz), 3.48 (1H, dd, J =
5.7, 3.3 Hz), 2.51 (1H, dq, J = 16.9, 2.8 Hz), 2.37 (1H, dq, J =
17.1, 2.6 Hz), 1.98 (1H, t, J = 2.7 Hz), 1.23–1.20 (2H, m), 1.02–
0.98 (2H, m), 0.91 (9H, s), 0.89 (9H, s), 0.12 (3H, s), 0.12 (3H, s),
0.08 (3H, s), 0.04 (3H, s); 13C-NMR (100 MHz, CDCl3) d: 137.9,
5.1.6. (3R,4S,5R)-4-[3-Hydroxy-2,2-ethanopropyl]oxyoct-
1en-7yne-3,5-diol (13). A solution of trimethylsilyl acetylene
(1.62 mL, 11.5 mmol) in THF (3 mL) under N2 gas atmosphere
was cooled in dry ice–acetone. To the solution was added n-butyl
lithium in hexane (2.64 M, 3.97 mL, 10.5 mmol) and the solution
was stirred at the same temperature for 45 min. To the solution
were added a solution of 12 (846 mg, 2.1 mmol) in THF (6 mL)
and BF3-diethylether complex (0.343 mL, 2.73 mmol) successively
and the reaction mixture was stirred at the same temperature for
2 h, and then at 0 ◦C for 1 h. Saturated aq. NH4Cl was added
to the solution and the reaction mixture was warmed to room
temperature and extracted with EtOAc. The organic layer was
washed with saturated aq. NaHCO3, brine, dried over MgSO4, and
evaporated. The residue was dissolved in dry MeOH (10 mL) and
sodium methoxide (870 mg, 6.3◦mmol) was added to the solution.
The solution was stirred at 50 C for 1 h. The reaction mixture
was cooled to room temperature and evaporated. The residue was
diluted with EtOAc and the solution was washed with brine, dried
over MgSO4, and evaporated. The residue was purified by flash
column chromatography on silica gel (hexane–EtOAc = 60/40,
then 50/50 then 35/65) to give 13 (311.5 mg, 1.30 mmol) in 62%
yield.
[a]2D5 -15.06 (c 0.5, CHCl3); IR (film, CHCl3) 3020, 1217,
1
1041 cm-1; H NMR (400 MHz, CD3OD) d: 5.57 (1H, ddd, J =
17.0, 11.0, 6.0 Hz), 4.88 (1H, dt, J = 17.0, 1.7 Hz), 4.73 (1H, dt,
J = 11.0, 1.7 Hz), 3.85–3.81 (1H, m), 3.51 (1H, ddd, J = 8.4, 5.7,
2.1 Hz), 3.16 (1H, d, J = 9.5 Hz), 3.05 (1H, d, J = 9.5 Hz), 2.85 (2H,
dd, J = 4.6, 2.2 Hz), 2.12–1.92 (2H, m), 1.85 (1H, t, J = 2.7 Hz);
13C NMR (100 MHz, CD3OD) d: 139.95, 116.22, 83.51, 81.92,
77.77, 71.46, 71.07, 67.23, 24.31, 23.77, 9.14, 9.02; EI-LRMS m/z
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122.6, 116.7, 85.1, 81.2, 74.8, 74.2, 71.1, 70.3, 31.5, 25.9, 25.8, 24.1,
22,7, 18.1, 18.0, 14.1, 12.3, 11.8, 10.6, -4.2, -4.4, -4.8; EI-LRMS
m/z 557.2 (M+Na); EI-HRMS calcd for C25H45NO3Si2 464.3011,
found 464.3021.
5.1.11. (3R,4S,5R)-3,5-Bis-[(tert-butyldimethylsilyl)oxy]-4-[2-
(ethoxycarbonyl)ethyl]oxyoct-1-en-7-yne (7h). To a solution of 16
(180 mg, 0.394 mmol) in EtOH (2 mL) was added sulfonic acid
(44 mL, 0.79 mmol) at 0 ◦C and it was refluxed for overnight.
The reaction mixture was cooled to room temperature and
quenched with saturated aq. NaHCO3. After evaporation of
the reaction mixture, the residue was diluted with EtOAc and
washed with brine. The organic layer was dried over MgSO4,
filtered and evaporated. The residue (88 mg) was dissolved in
dry CH2Cl2 (2 mL) and cooled to 0 ◦C. 2,6-Lutidine (177 mL,
1.576 mmol) and t-butyldimethylsilyl trifluoromethanesulfonate
(272 mL, 1.182 mmol) were added to the solution and stirred at
room temperature for 1 h. The reaction mixture was quenched with
dry MeOH (2 mL), diluted with EtOAc and washed with H2O. The
organic layer was dried over MgSO4, filtrated and evaporated. The
residue was purified by flash column chromatography on silica gel
(hexane–EtOAc = 95/5) to give 7h (92.7 mg, 0.191 mmol) in 48%
yield. [a]2D5 +0.58 (c 0.5, CHCl3); IR (film, CHCl3) 3020, 1215 cm-1;
1H-NMR (400 MHz, CDCl3) d: 5.99–5.91 (1H, m), 5.21 (1H, ddd,
J = 17.3, 1.1, 2.0 Hz), 5.14 (1H, ddd, J = 10.4, 1.0, 2.0 Hz),
4.32–4.29 (1H, m), 4.13 (2H, q, J = 7.2 Hz), 4.02–3.95 (1H, m),
3.89–3.81 (2H, m), 3.40 (1H, dd, J = 5.5, 3.5 Hz), 2.56 (2H, t,
J = 6.7 Hz), 2.48 (1H, ddd, J = 17.0, 3.0, 6.0 Hz), 2.35 (1H, ddd,
J = 17.0, 3.0, 6.0 Hz), 1.96 (1H, t, J = 2.7 Hz), 1.26 (3H, t, J =
7.1 Hz), 0.90 (9H, s), 0.89 (9H, s), 0.09 (3H, s), 0.08 (3H, s),
0.07 (3H, s), 0.03 (3H, s). 13C-NMR (100 MHz, CDCl3) d: 171.7,
138.6, 116.1, 85.4, 81.9, 74.7, 71.4, 69.9, 67.9, 60.3, 35.6, 25.9, 25.8,
24.0, 18.2, 18.1, 14.2, -4.1, -4.2, -4.4, -4.8; EI-LRMS m/z 507.2
(M+Na). EI-HRMS calcd for C25H48O5Si2Na 507.2933, found
507.2919.
5.1.9. (3R,4S,5R)-3,5-Bis-[(tert-butyldimethylsilyl)oxy]-5-(3-
cyanopropyl)oxyoct-1-en-7-yne (7f). To a solution of 15 (1.64 g,
3.70 mmol) in CH2Cl2 (15 mL) were added triethylamine
(1.03 mL, 7.40 mmol) and trimethylamine hydrochloride (35.4 mg,
0.370 mmol) and it was stirred under an argon atmosphere at
0 ◦C. p-Toluenesulfonyl chloride (1.06 g, 5.56 mmol) was added to
the reaction mixture, which was warmed to room temperature
overnight. H2O was added to the reaction mixture at room
temperature, it was stirred for 30 min, and CH2Cl2 was removed
by evaporation. The residue was diluted with EtOAc, and washed
with H2O, saturated aq. NaHCO3 and brine. The organic layer was
dried over Na2SO4, followed by filtration and evaporation. The
residue was purified by flash column chromatography on silica gel
(hexane–EtOAc = 95/5) to give a tosylate (1.85 g, 3.10 mmol).
To the tosylate (1.85 g, 3.10 mmol) in N,N-dimethylformamide
(15 mL) were added potassium cyanide (404 mg, 6.20 mmol) and
18-crown-6-ether (78.7 mg, 0.31 mmol), and it was stirred at 50 ◦C
overnight. The reaction mixture was cooled to room temperature
and diluted with H2O. The mixture was extracted with EtOAc, and
the organic layer was washed with brine and dried over Na2SO4,
followed by filtration and evaporation. The residue was purified
by flash column chromatography on silica gel (hexane–EtOAc =
95/5 then 90/10) to give 7f (1.31 g, 2.88 mmol) in 78%. [a]D25
-5.37 (c 1.0, CHCl3); IR (film)) 3314, 2930, 1095 cm-1; 1H-NMR
(400 MHz, CDCl3) d: 5.97–5.89 (1H, m), 5.23–5.18 (2H, m), 4.30
(1H, dd, J = 7.0, 3.0 Hz), 3.84–3.68 (3H, m), 3.44 (1H, dd, J =
5.6, 3.2 Hz), 2.53–2.39 (3H, m), 2.35 (1H, dq, J = 16.9, 2.6 Hz),
1.99 (1H, t, J = 2.7 Hz), 1.92–1.85 (2H, m), 0.91 (9H, s), 0.90 (9H,
s), 0.10 (3H, s), 0.08 (6H, s), 0.05 (3H, s); 13C-NMR (100 MHz,
CDCl3) d:138.0, 119.9, 116.6, 85.4, 81.2, 74.6, 71.3, 70.3, 70.1,
26.5, 25.9, 25.8, 24.3, 18.2, 18.1, 14.2, -4.1, -4.3, -4.4, -4.7; EI-
LRMS m/z 474.2 (M+Na), EI-HRMS calcd for C24H45O3Si2Na
474.2830, found 474.2832.
5.1.12. (3R,4S,5R)-3,5-Bis-[(tert-butyldimethylsilyl)oxy]-5-
[[3-methyl-3-(trimethylsilyl)oxy]butyloxy]oct-1-en-7-yne (7e). To
a solution of 7h (68 mg, 0.14 mmol) in THF (2.1 mL) was added
MeMgBr (1 M in THF, 0.58 mL, 0.58 mmol) at room temperature
and it was stirred at the same temperature for 2h. The reaction
mixture was quenched with saturated aq. NH4Cl, diluted with
EtOAc and washed with brine. The organic layer was dried over
MgSO4, followed by filtration and evaporation. To a solution of
crude product in DMF (1 mL) were added imidazole (29 mg,
0.42 mmol) and trimethylsilyl chloride (26.6 mL, 0.21 mmol) at
0 ◦C and it was stirred at room temperature for 1 h. The reaction
was quenched with dry MeOH (1 mL). The reaction mixture was
diluted with EtOAc, washed with brine and dried over MgSO4.
After filtration and evaporation, the residue was purified by flash
column chromatography on silica gel (hexane–EtOAc = 98/2) to
give 7e (49 mg, 0.09 mmol) in 64% yield. [a]2D5 +4.62 (c 0.5, CHCl3);
5.1.10. 3-{[(3R,4S,5R)-3,5-Bis-(t-butyldimethylsilyl)oxyoct-
1-en-7-yn-4-yl]oxy}propanoic acid (16). To a solution of 15
(540.5 mg, 1.22 mmol) in DMF (5 mL) was added PDC (2.25 g,
6.10 mmol) at room temperature and it was stirred at the same
temperature overnight. Water was added to the reaction mixture
and extracted twice with EtOAc. The combined organic layer
was washed with brine and dried over MgSO4, followed by
filtration and evaporation. The residue was purified by flash
column chromatography on silica gel (hexane–EtOAc = 91/9 then
85/15 then 67/33) to give 16 (335.3 mg, 0.73 mmol) in 60% yield.
[a]D25 +0.58 (c 0.5, CHCl3); IR (film, CHCl3) 3020, 1215 cm-1; 1H-
NMR (400 MHz, CDCl3) d: 5.91 (1H, ddd, J = 11.0, 17.0, 6.0 Hz),
5.30–5.18 (2H, m), 4.32 (1H, dd, J = 6.8, 3.4 Hz), 3.98–3.93 (2H,
m), 3.82 (1H, q, J = 5.5 Hz), 3.52 (1H, dd, J = 6.1, 3.7 Hz), 2.64–
2.61 (2H, m), 2.46 (1H, ddd, J = 17.1, 2.8, 6.0 Hz), 2.36 (1H,
ddd, J = 17.2, 2.5, 6.0 Hz), 2.01 (1H, t, J = 2.7 Hz), 0.91 (18H,
s), 0.10 (3H, s), 0.09 (3H, s), 0.08 (3H, s), 0.07 (3H, s). 13C-NMR
(100 MHz, CDCl3) d: 174.4, 137.1, 117.4, 85.7, 80.7, 74.3, 70.9,
70.7, 68.0, 35.3, 29.7, 25.9, 25.8, 24.3, 18.2, 18.1, 13.0, -4.2, -4.4,
-4.5, -4.8; EI-LRMS m/z 479.1 (M+Na). EI-HRMS calcd for
C25H45NO3Si2Na 479.2644, found 479.2640.
1
IR (film, CHCl3) 3020, 1215 cm-1; H-NMR (400 MHz, CDCl3)
d: 6.00–5.92 (1H, m), 5.23–5.11 (2H, m), 4.33–4.29 (1H, m), 3.87–
3.60 (3H, m), 3.37 (1H, dd, J = 5.6, 3.4 Hz), 2.52–2.32 (2H, m), 1.96
(1H, t, J = 2.3 Hz), 1.75 (2H, t, J = 7.6 Hz), 1.23 (3H, s), 1.22 (3H,
s), 0.91 (9H, s), 0.89 (9H, s), 0.13 (3H, s), 0.10 (3H, s), 0.10 (9H,
d, J = 1.7 Hz), 0.07 (3H, s), 0.03 (3H, s). 13C-NMR (100 MHz,
CDCl3) d: 138.8, 115.9, 85.3, 82.1, 74.7, 73.0, 71.6, 69.8, 69.4,
30.4, 30.2, 25.9, 25.8, 14.1, 18.2, 18.1, 2.6, -4.1, -4.2, -4.4, -4.7;
EI-LRMS m/z 507.2 (M-TMS+2H2O), 493.2 (M-TMS+Na); EI-
HRMS calcd for C25H50O4Si2Na (M-TMS+Na) 493.3140, found
493.3136.
3962 | Org. Biomol. Chem., 2011, 9, 3954–3964
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5.1.13. (5Z,7E)-(1S,2S,3R)-2-Methyl-20-[(2,2-dimethylcyclo-
pentanone-(5E)-ylidene)] methyl-9,10-seco-5,7,10(19)-pregnatri-
ene-1,3-diol (5a). Under N2 atmosphere, a solution of 6 (40 mg,
0.105 mmol), 7a (52 mg, 0.148 mmol) and Pd(PPh3)4 (20 mg,
0.019 mmol) in toluene (1 mL) and Et3N (1 mL) was stirred at
110 ◦C for 2 h. The reaction mixture was evaporated and purified
with PTLC (Merck Kiseigel plate Art. 113794 1 mm, the eluent
was hexane–EtOAc = 95/5) to give a crude product (32.4 mg),
which was dissolved in dry MeCN (1 mL) and CH2Cl2 (1 mL). To
the solution was added LiBF4 (20 mg, 0.21 mmol) and 1 M H2SO4
in MeCN (20 mL, 0.02 mmol) at 0 ◦C and it was stirred at the
same temperature for 1.5 h. After the work up, the crude product
(6.0 mg) was obtained with preparative TLC (Merck Kiseigel
plate Art. 113794 1 mm, the eluent was hexane–EtOAc = 50/50).
Further purification with reversed-phase HPLC (YMC-Pack
ODS column, 30–250 mm, 10 mL min-1, eluent A: MeCN–H2O =
5/95, eluent B: MeCN–MeOH–H2O = 59.5/40/0.5, eluent A/B =
15/85) gave 5a (4.0 mg, 0.008 mmol, 8% yield). [a]2D5 +128.8 (c
0.1, CHCl3); IR (film, CHCl3) 3348, 2974, 2885, 1454, 1381, 1089,
5.2.2. General procedure for transactivation assay of human
osteocalcin promoter. The human osteocalcin gene promoter
fragment -838/+10 was cloned into the reporter plasmid pGL3
(Promega) as reported. Human VDR and RXR genes were cloned
into expression vector pcDNA3 (Invitrogen). Hos cells were
maintained in phenol red-free DMEM (Invitrogen) containing
10% FCS (Invitrogen). Prior to transfection, the cells were plated
in a 96-well plate at a density of 400,000 cells per well in
Opti-MEM (Invitrogen). The cells were transfected with human
osteocalcin reporter vector (pGL3-hOc: 100 ng/well), human
VDR and RXR expression vector (pcDNA-hVDR, pcDNA-
hRXR: 10 ng/well) and phRL-TK (Promega: 25 ng/well) using
0.45 mL Lipofectamine 2000 reagent (Invitrogen). After incubation
at 37 ◦C for 3 h, the culture media were replaced with phenol red-
free DMEM containing 10% FCS. The cells were treated with
ethanol vehicle or various concentrations of compounds (from 0.1
pM to 100 nM). After incubation at 37 ◦C for 24 h, the luciferase
activity of the cells was quantitated by a luminometer (Berthold)
using the Dual-Glo luciferase assay system (Promega).
1
1051 cm-1; H-NMR (400 MHz, CDCl3) d: 6.43–6.37 (2H, m),
5.2.3. Evaluation of derivatives on bone mineral loss in
ovariectomized rats. Twelve-week-old Sprague-Dawley female
rats (Charles River, Japan) were ovariectomized and fed ad libitum
with a normal diet containing 1.0% Ca for 4 weeks. The rats were
orally administrated vitamin D derivatives at various doses in
MCT as the vehicle 5 times a week for 4 weeks. The sham and
OVX groups were administrated MCT alone. Twenty-four hours
after the final administration, blood was collected under ether
anesthesia, and the rats were euthanized. The BMD of spine (L4–
L5) bone mass was measured by a dual X-ray absorptiometer
(QDR-2000; HOLOGIC, USA). The results are expressed as the
mean standard error of the mean. The statistical significance
of differences between the OVX and experimental groups was
analyzed by Student’s t-test.
6.00 (1H, d, J = 11.2 Hz), 5.27 (1H, s), 4.99 (1H, d, J = 1.9 Hz),
4.30 (1H, br s), 3.84 (1H, br s), 2.84 (1H, dd, J = 12.2, 3.9 Hz),
2.67 (1H, dd, J = 13.4, 4.2 Hz), 2.53–2.33 (3H, m), 2.23 (1H,
dd, J = 13.4, 8.1 Hz), 2.03–1.90 (3H, m), 1.76–1.65 (5H, m),
1.54–1.34 (7H, m), 1.17–1.11 (1H, m), 1.08 (3H, d, J = 6.8 Hz),
1.07 (3H, s), 1.05 (3H, s), 1.04 (3H, d, J = 6.0 Hz), 0.57 (3.0H,
s); 13C-NMR (100 MHz, CDCl3) d: 211.2, 146.5, 142.6, 152.5,
133.7, 133.2, 124.7, 117.1, 113.2, 75,4, 71.7, 56.5, 55.9, 46.0, 45.6,
44.2, 43.5, 40.3, 37.2, 35.4, 29.0, 26.8, 23.8, 23.5, 23.3, 22.2, 19.2,
12.5, 12.4; EI-LRMS m/z 475.2 (M+Na) 435.2 (M-H2O+H),
417.2 (M-2H2O+H); EI-HRMS calcd for C30H44O3Na (M+Na)
475.3183, found 475.3171.
5.2. Biology
Acknowledgements
5.2.1. Evaluation of affinity to vitamin D receptor (VDR).
The binding affinity to VDR was evaluated using a 1a,25-
(OH)2VD3 assay kit (POLARSCREEN VITAMIN D RECEP-
TOR COMPETITOR ASSAY, RED, Cat.No.PV4569; Invitro-
gen). The solution of test compound (1 mM in EtOH) was diluted
10 times with DMSO. The solution was diluted 50 times with
the assay buffer included in the kit. The solution was defined as
the compound solution. On the other hand, VDR/Fluoromone
and VDR RED, both of which are included in the kit, were
diluted with the assay buffer included in the kit so that the
concentration of VDR/Fluoromone was 2.8 nM, and that of
VDR RED was 2 nM in the mixture. The solution was defined
as the VDR/Fluoromone and VDR RED complex. To a 384-well
Black plate (Coring, #3677) was added the compound solution
(10 mL), and the VDR/Fluoromone and VDR RED complex
(10 mL) was added to each well. The mixture was incubated at
20–25 ◦C for 2 h. The polarized fluorescence in each well was
measured (384 nm, emission: 595 nm, excitation: 535 nm, time:
250 ms/well). All compounds were evaluated with N = 2 within
the range from 10-6 M to 10-10 M. IC50 values were calculated using
the average measured value. The activities of each compound were
shown as a relative value in which the activity of natural hormone
1 was normalized to 100%.
We gratefully thank Mrs. Ayako Mori-Ishii and Mrs. Bingbing
Song (Teijin Pharma Limited) for synthetic support, and Mr.
Takahiro Takeuchi, Ms. Saori Masuda and Mr. Yuki Kobayashi
(Teijin Pharma Limited) for the spectroscopic measurements.
We are grateful to Mr. Eiji Ochiai, Ms. Mariko Fujita and
Mr. Yasunori Nakayama (Teijin Pharma Limited) for in vitro
and in vivo evaluations. We also thank Dr Keiichiro Imaizumi
(Teijin Pharma Limited) for PK evaluation. We also wish to
thank Dr Seiichi Ishizuka, Dr Midori Takimoto-Kamimura, Dr
Tomohiro Ohta, Dr Yosiaki Azuma, Dr Hiroshi Kawabata, and
Dr Takashi Kamimura (Teijin Pharma Limited) for their advice
and encouragement.
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