Remarkable effect of 2[small alpha]-modification on the VDR antagonistic activity of 1small alpha-hydroxyvitamin D3-26,23-lactones.

Article abstract of DOI:10.1039/b311107e

Novel 2[small alpha]-methyl-, 2[small alpha]-(3-hydroxypropyl)- and 2[small alpha]-(3-hydroxypropoxy)-substituted 25-dehydro-1[small alpha]-hydroxyvitamin D-26,23-lactone derivatives were efficiently synthesized Reformatsky type allylation and palladium-c

Full text of DOI:10.1039/b311107e

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Remarkable effect of 2ꢀ-modification on the VDR antagonistic
activity of 1ꢀ-hydroxyvitamin D3-26,23-lactones
Nozomi Saito,a Toshihiro Matsunaga,a Toshie Fujishima,a Miyuki Anzai,b Hiroshi Saito,b
Kazuya Takenouchi,b Daishiro Miura,b Seiichi Ishizuka,b Hiroaki Takayamaa and
Atsushi Kittaka*a
a Faculty of Pharmaceutical Sciences, Teikyo University, Kanagawa 199-0195, Japan.
E-mail: akittaka@pharm.teikyo-u.ac.jp; Fax: ϩ81-426-85-3714; Tel: ϩ81-426-85-3715
b Teijin Institute for Bio-Medical Research, Tokyo 191-8512, Japan; Fax: ϩ81-42-586-8298;
Tel: ϩ81-42-586-8220
Received 12th September 2003, Accepted 17th October 2003
First published as an Advance Article on the web 6th November 2003
Novel 2α-methyl-, 2α-(3-hydroxypropyl)- and 2α-(3-hydroxypropoxy)-substituted 25-dehydro-1α-hydroxyvitamin
D3-26,23-lactone derivatives were efficiently synthesized via Reformatsky type allylation and palladium-catalyzed
alkenylative cyclization processes, and their biological activities were evaluated. Introducing functional groups into
the 2α-position of the vitamin D3-26,23-lactones resulted in remarkable enhancement of their antagonistic activity
on vitamin D receptor (VDR).
Introduction
1α,25-Dihydroxyvitamin D3 (1) is the most potent metabolite of
vitamin D3, and regulates various biological events, including
calcium and phosphorus homeostasis, cell proliferation and
differentiation, and immune reaction.1,2 Most of the biological
responses of 1 are mediated via interaction with its specific
receptor, vitamin D receptor (VDR), which is one of the
nuclear receptor superfamily and acts as a ligand-dependent
gene transcription factor with coactivators.3,4 Recently, we have
synthesized several 1α,25-dihydroxyvitamin D3 analogues,
which systematically introduced an alkyl, ω-hydroxyalkyl,
and ω-hydroxyalkoxyl group into the C2α position of 1, to
investigate A-ring conformation- and structure–activity
relationships.5–8 Some of these 2α-modified vitamin D3
analogues exhibited unique biological profiles with potent
agonistic activity. In particular, introduction of the 2α-methyl
Fig. 2 Structures of 25-dehydro-1α-hydroxyvitamin D3-26,23-lactones
(TEI-9647: 2 and TEI-9648: 3) and their 2α-modified analogues 2a–c
and 3a–c.
(1a),5 2α-(3-hydroxypropyl) (1b)6 and 2α-(3-hydroxypropoxy)
(1c)7 groups showed 2- to 4-fold higher binding affinity to the
bovine thymus VDR relative to the natural hormone 1 (Fig. 1).
leukemia cell (HL-60 cells) differentiation,10a as well as 25-
hydroxyvitamin D3-24-hydroxylase gene expression in human
osteosarcoma cells10b and in HL-60 cells10d induced by 1.
Furthermore, it is noteworthy that 2 shows antagonistic action
on the genomic-mediated calcium metabolism regulated by 1
in vivo in rats.10e The unique structures and interesting bio-
logical profiles of 2 and 3 prompted us to investigate the
structure–activity relationship of the vitamin D3 lactones 2 and
3 from the standpoint of the anti-D molecules. Particularly, we
focused on the introduction of the motifs described above into
the C2α position of lactone analogues 2 and 3. It was expected
that such modifications should increase their VDR binding
affinity and improve their antagonistic activity to VDR-
mediated biological actions. Here, we report the synthesis and
biological evaluation of the novel 2α-modified 25-dehydro-
1α-hydroxyvitamin D3-26,23-lactone analogues.
Fig. 1 Structures of 1α,25-dihydroxyvitamin D3 (1) and its 2α-
substitued analogues 1a–c.
In 1999, studies on the modification of the side-chain struc-
ture based on the 1α,25-dihydroxyvitamin D3-26,23-lactone
metabolite9 derived from active vitamin D3 (1) led to the
discovery of TEI-9647 (2) and TEI-9648 (3) (Fig. 2).10 Both
vitamin D3 analogues 2 and 3 have an α-methylene-γ-
butyrolactone moiety on the side-chain, and are the first specific
antagonists of the VDR-mediated genomic action of 1.11 That
is, vitamin D3-lactone derivatives 2 and 3 inhibit human
Results and discussion
Synthesis of the CD-ring part 6 is shown in Scheme 1.
Hydrindan derivative 4 was prepared from vitamin D2 by the
established method.12 Bromomethylenation of 4 gave 5 in 76%
yield. The substitution reaction of tosylate 5 with potassium
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 4 3 9 6 – 4 4 0 2
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 3
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Treatment of 8 with NaH in THF gave a CD-ring precursor
12 having a lactone moiety in quantitative yield. Similarly, the
lactone derivative 13 was also synthesized from γ-hydroxyester
9 in an excellent yield (Scheme 4).
Scheme 1
cyanide in DMSO followed by DIBAL-H reduction of the
resulting cyano group afforded aldehyde 6 in 83% yield (2 steps
from 5).
The zinc-mediated Reformatsky type allylation of aldehyde 6
with methyl bromomethylacrylate 7 proceeded smoothly to give
γ-hydroxyesters 8 and 9 in yields of 40% and 60%, respectively
(Scheme 2).
Scheme 4
The palladium-catalyzed coupling reaction14 of the above
bromoolefin 12 and enyne 14a, which was prepared by our
previously reported procedure,15 followed by deprotection of
silyl groups using HF/MeCN provided the desired 2α-methyl-
25-dehydro-1α-hydroxyvitamin D3-26,23-lactone 2a in good
yield (Scheme 5).
Scheme 2
The absolute configurations at the C23 position (based
on steroidal numbering) on the side chain of 8 and 9 were
determined using modified Mosher’s method13 (Scheme 3).
Thus, γ-hydroxyesters 8 and 9 were transformed into the corre-
Scheme 5
Encouraged by this result, we synthesized the other 2α-
modified vitamin D3-26,23-lactone derivatives (2b,c and 3a–c)
as shown in Scheme 6. Thus, the coupling reaction of bromo-
olefins 12 or 13 and enynes 14a, 14b,6 or 14c7 and subsequent
deprotection afforded the desired 2α-modified vitamin D3
analogues having a lactone side chain, i.e. 2α-methyl- (3a), 2α-
(3-hydroxypropyl)- (2b or 3b) and 2α-(3-hydroxypropoxy)-25-
dehydro-1α-hydroxyvitamin D3-26,23-lactones (2c or 3c), in
moderate to good yields.
sponding (S)- and (R)-MTPA esters 10 and 11, respectively.
1
The values of ∆δ = δ(R)-MTPA
Ϫ δ(S)-MTPA
in the H NMR
ester
ester
spectra of 10 and 11 were calculated and shown in Scheme 3.
These data were considered by applying a modified Mosher’s
method and the absolute configurations at the C23 position of
10 and 11 were determined to be S and R, respectively.
Scheme 6
Biological activities of vitamin D3-lactone analogues, thus
obtained 2a–c and 3a–c, were evaluated, and the results are
summarized in Table 1. Binding affinity to chick intestinal
VDR was examined as described previously,16 and TEI-9647 (2)
and TEI-9648 (3) showed 8 (12%) and 14 (7%) times weaker
potency than that of the natural hormone 1, respectively.
Introduction of the A-ring motifs, i.e., 2α-methyl (2a), 2α-(3-
hydroxypropyl) (2b) and 2α-(3-hydroxypropoxy) (2c) groups,
Scheme 3
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 4 3 9 6 – 4 4 0 2
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Table 1 Biological activities of vitamin D3-lactone analogues
Compound
VDR binding affinitya
Relative antagonistic activityb
1
100
12
16
18
16
7
37
33
23
—
2 (TEI-9647)
100
1019
2989
1160
6
38
100
66
2a
2b
2c
3 (TEI-9648)
3a
3b
3c
a Chick intestinal VDR. The potency of 1 is normalized to 100. b Determined by the NBT-reduction method and relative antagonistic activity was
calculated at IC50. The potency of 2 is normalized to 100.
into the vitamin D3-lactone 2, little affected the binding affinity
to the VDR. On the other hand, the binding affinity of TEI-
9648 analogues (3a–c) to VDR was significantly increased to
3.3–5.3 times that of 3 by the introduction of C2α substituents.
Next, the antagonistic activities of 2a–c and 3a–c were assessed
in terms of inhibition of HL-60 cell differentiation induced by
treatment of the natural hormone 1, and their activities relative
to TEI-9647 (2) were calculated based on the IC50 values.
Interestingly, modification of the C2α position of the original
antagonist 2 resulted in marked enhancement of the antagon-
istic activities. The 2α-methyl analogue 2a showed ca. 10-fold
higher activity than 2. Surprisingly, the antagonistic activity of
2b, which possesses the ω-hydroxypropyl subsituent on the C2α
position, was 30 times stronger than that of 2. Furthermore,
introduction of the 2α-(3-hydroxypropoxy) group (2c) improved
the activity to ca. 12-fold higher than 2. Although 2α-modified
TEI-9648 (3) type analogues 3a–c generally showed weaker
antagonistic activities than 2, introduction of the three motifs
into 3 increased the activities compared to the original
compound 3 (6.3 to 16.7 times more potent than 3).
the mixture was stirred at the same temperature for 1 h. To
the mixture was added a solution of 4 (1.0 g, 2.7 mmol) in THF
(20 mL) at 0 ЊC, and the resulting mixture was stirred at the
same temperature for 2 h. To the mixture was added a saturated
NH4Cl aq. solution at 0 ЊC, and the aqueous layer was extracted
with AcOEt. The organic layer was washed with a saturated
NaCl aq. solution, dried over Na2SO4, and concentrated. The
residue was purified by flash column chromatography on silica
gel (hexane/AcOEt = 20 : 1) to give 5 (920 mg, 76%) as a color-
less oil. [α]2D6 ϩ72.4 (c 1.38, CHCl3); IR (neat) 1647, 1599, 1360,
1176 cmϪ1; 1H NMR (400 MHz, CDCl3) δ 0.53 (s, 3 H), 1.00 (d,
J = 6.6 Hz, 3 H), 1.05–1.81 (m, 10 H), 1.88–2.00 (m, 2 H), 2.45
(s, 3 H), 2.91 (m, 1 H), 3.82 (dd, J = 9.3, 6.1 Hz, 1 H), 3.96 (dd,
J = 9.3, 3.2 Hz, 1 H), 5.64 (s, 1 H), 7.34 (d, J = 8.1 Hz, 2 H), 7.78
(d, J = 8.1 Hz, 2 H); 13C NMR (150 MHz, CDCl3) δ 11.8, 17.0,
21.6, 22.0, 22.4, 36.8, 39.5, 45.4, 51.4, 55.4, 75.3, 97.3, 127.9,
129.8, 133.1, 144.5, 144.6; EI-LRMS m/z 440 (Mϩ), 361, 268,
227, 189, 172, 91; EI-HRMS calcd for C21H29O379Br 440.1021,
found 440.1023.
(1R,4E,3aR,7aR)-4-Bromomethylene-1-[(1R)-2-formyl-1-meth-
ylethyl]-7a-methylperhydroindene (6)
Conclusion
To a solution of 5 (1.2 g, 2.8 mmol) in DMSO (3 mL) was
added KCN (363 mg, 5.6 mmol), and the mixture was stirred at
70 ЊC for 1.5 h. The mixture was diluted with Et2O, and the
organic layer was washed with H2O and saturated NaCl aq.
solution, dried over Na2SO4 and concentrated. The residue was
dissolved in CH2Cl2 (5.5 mL). To the solution was added a
solution of DIBAL-H in toluene (1.0 M, 3 mL, 3.1 mmol) at
0 ЊC, and the mixture was stirred at the same temperature for
1.5 h. To the mixture was added, 10% potassium sodium
tartrate aq. solution, and the aqueous layer was extracted with
Et2O. The organic layer was washed with saturated NaCl aq.
solution, dried over Na2SO4 and concentrated. The residue was
purified by flash column chromatography on silica gel (hexane/
Et2O = 30 : 1) to give 6 (692 mg, 2.3 mmol in 2 steps) as a
colorless oil. [α]2D0 ϩ86.1 (c 1.08, CHCl3); IR (neat) 2950, 1725,
1381 cmϪ1; 1H NMR (400 MHz, CDCl3) δ 0.62 (s, 3 H), 1.04 (d,
J = 6.6 Hz, 3 H), 1.20–1.40 (m, 3 H), 1.45–1.75 (m, 5 H), 1.90
(m, 1 H), 1.95–2.15 (m, 3 H), 2.20 (ddd, J = 16.0, 9.3, 3.2 Hz,
1 H), 2.47 (dd, J = 16.0, 2.7 Hz, 1 H), 2.89 (m, 1 H), 5.67 (s,
1 H), 9.76 (dd, J = 3.2, 1.2 Hz, 1 H); 13C NMR (150 MHz,
CDCl3) δ 11.8, 20.0, 21.9, 22.4, 27.7, 30.9, 31.7, 39.6, 45.5, 50.7,
55.4, 55.7, 97.7, 144.6, 203.0; EI-LRMS m/z 298 (Mϩ) 254,
227, 148; EI-HRMS calcd for C15H23O79Br 298.0932, found
298.0934.
We introduced three motifs, 2α-methyl, 2α-(3-hydroxypropyl)
and 2α-(3-hydroxypropoxy) substituents, into 25-dehydro-1α-
hydroxyvitamin D3-26,23-lactone analogues, and investigated
their biological activities. It was found that the modification of
the C2α position of the vitamin D3-lactone skeleton remarkably
enhanced their antagonistic activities. The VDR antagonists are
expected to be potent therapeutic agents for some diseases
caused by hypersensitivity to 1α,25-dihydroxyvitamin D3, such
as Paget’s bone disease.17 We expect that these analogues with
potent anti-D activity would contribute to understanding the
mechanisms involved in the expression of antagonistic activity
on VDR as well as to finding the seeds of new medicines for
treating Paget’s bone disease.
Experimental
General
All manipulations were performed under an argon atmosphere
unless otherwise mentioned. All solvents and reagents were
purified when necessary using standard procedures. Column
chromatography was performed on silica gel 60 N (Kanto
Chemical CO., Inc., 100–210 µm), and flash column chromato-
graphy was performed on silica gel 60 (Merck, 0.040–0.063
mm).
(1R,4E,3aR,7aR)-4-Bromomethylene-1-[(1R,3S )-3-hydroxy-5-
methoxycarbonyl-1-methyl-5-hexenyl]-7a-methylperhydroindene
(8) and (1R,4E,3aR,7aR)-4-Bromomethylene-1-[(1R,3R)-3-
hydroxy-5-methoxycarbonyl-1-methyl-5-hexenyl]-7a-methyl-
perhydroindene (9)
(1R,4E,3aR,7aR)-4-Bromomethylene-7a-methyl-1-[(1S )-
methyl-2-p-toluenesulfonyloxyethyl]perhydroindene (5)
To
a
suspension of bromomethyltriphenylphosphonium
bromide (5.9 g, 14 mmol) in THF (20 mL) was added a solution
of NaHMDS in THF (1.0 M, 14 mL, 14 mmol) at 0 ЊC and
To a solution of 6 (250 mg, 0.84 mmol) in saturated NH4Cl aq.
solution/THF (5 : 1, 4.3 mL) were added 7 (0.21 mL, 1.7 mmol)
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 4 3 9 6 – 4 4 0 2
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and activated zinc dust (219 mg, 3.3 mmol) at 0 ЊC, and the
mixture was stirred at the same temperature for 1.5 h. The
mixture was diluted with AcOEt. The organic layer was washed
with saturated NaCl aq. solution, dried over Na2SO4, and
concentrated. The residue was purified by flash column
chromatography on silica gel (hexane/AcOEt = 8 : 1) to give
8 (129 mg, 40%) and 9 (198 mg, 60%) as colorless oils, respect-
ively. 8: [α]2D0 ϩ108.3 (c 0.23, CHCl3); IR (neat) 1719, 1632, 1439,
To a solution of the above pivaloyl ester (17 mg, 37 µmol) in
pyridine (0.37 mL) were added (R)-MTPACl (14 µL, 74 µmol)
and DMAP (4.6 mg, 37 µmol) at 0 ЊC, and the mixture was
stirred at room temperature for 4 h. To the mixture was added
water, and the aqueous layer was washed with saturated NaCl
aq. solution, dried over Na2SO4, and concentrated. The residue
was purified by preparative thin-layer chromatography (hexane/
AcOEt = 15 : 1) on silica gel to give the corresponding (S)-
MTPA ester 10 (20 mg, 80%) as a colorless oil. [α]2D0 ϩ21.5 (c
1.15, CHCl3); IR (neat) 1736, 1456, 1269, 1157 cmϪ1; 1H NMR
(600 MHz, CDCl3) δ 0.53 (s, 3 H), 0.91 (m, 1 H), 0.97 (d, J = 5.8
Hz, 3 H), 1.22 (s, 9 H), 1.26 (m, 1 H), 1.32–1.46 (m, 4 H), 1.48–
1.53 (m, 2 H), 1.62–1.67 (m, 3 H), 1.86–1.91 (m, 2 H), 1.95 (br
d, J = 12.6 Hz, 1 H), 2.34 (dd, J = 15.0, 8.7 Hz, 1H), 2.43 (dd, J =
15.0, 3.8 Hz, 1 H), 2.87 (m, 1 H), 3.52 (s, 3 H), 4.51 (d, J = 13.6
Hz, 1 H), 4.55 (d, J = 13.6 Hz, 1 H), 5.00 (s, 1 H), 5.15 (s, 1 H),
5.32 (dddd, J = 8.4, 8.4, 4.2, 3.8 Hz, 1 H), 5.63 (s, 1 H), 7.37–
7.40 (m, 3 H), 7.52–7.54 (m, 2 H); 13C NMR (150 MHz, CDCl3)
δ 11.8, 19.2, 21.9, 22.5, 27.2, 27.7, 30.9, 33.9, 37.7, 38.8, 39.7,
40.2, 45.5, 55.5, 55.7, 56.0, 66.3, 74.2, 84.5 (q, 2JC–F = 27.3 Hz),
1
1203 cmϪ1; H NMR (400 MHz, CDCl3) δ 0.58 (s, 3 H), 0.86
(m, 1 H), 1.02 (d, J = 6.3 Hz, 3 H), 1.25–1.34 (m, 4 H), 1.41–1.69
(m, 7 H), 1.90–2.03 (m, 3 H), 2.18 (dd, J = 9.0, 13.9 Hz, 1 H),
2.68 (dd, J = 1.7, 13.9 Hz, 1 H), 2.87 (m, 1 H), 3.77 (s, 3 H), 3.85
(m, 1 H), 5.64 (s, 1 H), 5.68 (s, 1 H), 6.27 (s, 1 H); 13C NMR (100
MHz, CDCl3) δ 12.3, 19.8, 22.5, 23.0, 28.2, 31.5, 34.5, 40.3,
40.5, 44.2, 45.9, 52.5, 56.2, 56.8, 69.5, 97.7, 128.1, 137.6, 145.1,
168.1; EI-LRMS m/z 398 (Mϩ), 287, 256, 229; EI-HRMS calcd
for C20H3179BrO3 398.1457, found 398.1460. 9: [α]2D0 ϩ93.1
1
(c 0.38, CHCl3); IR (neat) 1719, 1630, 1439, 1203 cmϪ1; H
NMR (400 MHz, CDCl3) δ 0.59 (s, 3 H), 0.97 (d, J = 6.3 Hz,
3 H,), 1.07 (m, 1 H), 1.21–1.37 (m, 4 H), 1.40–1.69 (m, 7 H),
1.86–2.04 (m, 3 H), 2.86 (dd, J = 13.9, 8.2Hz, 1H), 2.51 (dd,
J = 13.9, 3.7 Hz, 1 H), 2.87 (m, 1 H), 3.77 (s, 3 H), 3.85 (m, 1 H),
5.64 (s, 1 H), 5.66 (s, 1 H), 6.25 (s, 1 H); 13C NMR (100 MHz,
CDCl3) δ 12.4, 19.1, 22.5, 23.0, 28.2, 31.5, 33.2, 40.3, 42.0, 44.1,
46.0, 52.5, 56.3, 56.8, 67.9, 97.7, 127.9, 137.6, 145.2, 168.2;
EI-LRMS m/z 398 (Mϩ), 366, 287, 256, 229; EI-HRMS calcd
for C20H3179BrO3 398.1457, found 398.1452.
1
97.6, 115.8, 123.3 (q, JC–F = 289.1 Hz), 127.4, 128.3, 129.5,
132.1, 139.7, 144.8, 166.1, 177.9; EI-LRMS m/z 670 (Mϩ), 591,
489, 436, 357, 255; EI-HRMS calcd for C34H46O579BrF3
670.2481, found 670.2485. <Synthesis of (R)-MTPA ester 10>
In a similar manner to that for the synthesis of (S)-MTPA ester,
a crude product, which was obtained from the above pivaloyl
ester (13 mg, 29 µmol), (S)-MTPACl (11 µL, 58 µmol) and
DMAP (3.5 mg, 29 µmol) in pyridine (0.29 mL), was purified by
preparative thin-layer chromatography on silica gel (hexane/
AcOEt = 15 : 1) to give the corresponding (R)-MTPA ester 10
(18 mg, 94%) as a colorless oil. [α]1D8 ϩ55.0 (c 1.23, CHCl3); IR
Transformation of 8 into the corresponding (S )- and (R)-MTPA
ester 10
To a solution of 8 (58 mg, 0.15 mmol) in toluene (3 mL) was
added a solution of DIBAL-H in toluene (1.04 M, 0.63 mL,
0.65 mmol) at 0 ЊC, and the mixture was stirred at the same
temperature for 1 h. To the mixture was added 10% potassium
sodium tartrate aq. solution, and the aqueous layer was
extracted with Et2O. The organic layers were combined and
washed with saturated NaCl aq. solution, dried over Na2SO4,
and concentrated. The residue was purified by flash column
chromatography on silica gel (hexane/AcOEt = 2 : 1) to give the
corresponding diol (52 mg, 96%) as a colorless oil. [α]1D8 Ϫ5.6
(neat) 1742, 1458, 1277, 1167 cmϪ1 1H NMR (600 MHz,
;
CDCl3) δ 0.55 (s, 3 H), 1.04 (d, J = 6.0 Hz, 3 H), 1.21 (s, 9 H),
1.25–1.32 (m, 3 H), 1.41–1.49 (m, 2 H), 1.51–1.59 (m, 3 H),
1.64–1.68 (m, 2 H), 1.74 (dd, J = 10.2, 9.5 Hz, 1 H), 1.95 (m, 3
H), 2.26 (dd, J = 14.8, 8.9 Hz, 1 H), 2.40 (dd, J = 14.8, 3.5 Hz,
1 H), 2.88 (m, 1 H), 3.52 (s, 3 H), 4.41 (d, J = 14.3 Hz, 1 H), 4.44
(d, J = 14.3 Hz, 1 H), 4.84 (s, 1H), 4.97 (s, 1 H), 5.31 (dddd, J =
9.5, 8.9, 4.1, 3.5, 1 H), 5.65 (s, 1 H), 7.36–7.69 (m, 3 H), 7.51 (br
d, J = 6.6 Hz, 2 H); 13C NMR (150 MHz, CDCl3) δ 11.8, 19.1,
22.0, 22.5, 27.2, 27.8, 31.0, 33.9, 37.5, 38.8, 39.8, 40.5, 45.5,
55.4, 55.8, 56.2, 66.2, 74.1, 84.3 (q, 2JC–F = 27.6 Hz), 97.6, 115.9,
1
(c 1.54, CHCl3); IR (neat) 3478, 1653 cmϪ1; H NMR (400
1
MHz, CDCl3) δ 0.58 (s, 3 H), 1.00 (d, J = 6.3 Hz, 3 H), 1.20–1.35
(m, 4 H), 1.41–1.71 (m, 7 H), 1.90–2.08 (m, 4 H), 2.43 (dd,
J = 1.7, 4.2 Hz, 1 H), 2.51 (br s, 2 H), 2.88 (m, 1 H), 3.84 (m,
123.3 (q, JC–F = 288.7 Hz), 127.3, 128.3, 129.5, 132.2, 139.2,
144.8, 166.0, 177.9; EI-LRMS m/z 670 (Mϩ), 591, 489, 436, 357,
255; EI-HRMS calcd for C34H46O579BrF3 670.2481, found
670.2483.
1 H), 4.11 (s, 2 H), 4.98 (s, 1 H), 5.15 (s, 1 H), 5.64 (s, 1 H); 13
C
NMR (100 MHz, CDCl3) δ 12.0, 19.6, 22.2, 22.6, 27.9, 31.1,
34.2, 39.9, 41.7, 43.8, 45.6, 55.9, 56.5, 66.5, 69.3, 97.4, 114.4,
144.8, 145.8; EI-LRMS m/z 370 (Mϩ), 352, 337, 298, 256, 227;
EI-HRMS calcd for C19H31O279Br 370.1507, found 370.1507.
To a solution of the above diol (50 mg, 0.14 mmol) in CH2Cl2
(1.3 mL) were added pyridine (43 µL, 0.54 mmol) and pivaloyl
chloride (20 µL, 0.16 mmol) at 0 ЊC, and the mixture was stirred
at room temperature for 4 h. To the mixture was added water,
and the aqueous layer was extracted with Et2O. The organic
layer was washed with saturated NaCl aq. solution, dried over
Na2SO4, and concentrated. The residue was purified by column
chromatography on silica gel (hexane/AcOEt = 9 : 1) to give the
corresponding pivaloyl ester (30 mg, 48%) as a colorless oil. [α]1D7
Transformation of 9 into the corresponding (S )- and (R)-MTPA
ester 11
To a solution of 9 (65 mg, 0.16 mmol) in toluene (3 mL) was
added a solution of DIBAL-H in toluene (1.04 M, 0.63 mL,
0.65 mmol) at 0 ЊC, and the mixture was stirred at the same
temperature for 1.2 h. To the mixture was added 10% potassium
sodium tartrate aq. solution, and the aqueous layer was
extracted with Et2O. The organic layer was washed with satur-
ated NaCl aq. solution, dried over Na2SO4, and concentrated.
The residue was purified by flash column chromatography on
silica gel (hexane/AcOEt = 2 : 1) to give the corresponding diol
(61 mg, quant.) as a colorless oil. [α]1D8 ϩ90.9 (c 1.46, CHCl3); IR
(neat) 3320, 1630, 909 cmϪ1; 1H NMR (400 MHz, CDCl3) δ 0.59
(s, 3 H), 0.98 (d, J = 6.3 Hz, 3 H), 1.06 (m, 1 H), 1.21–1.36 (m, 3
H), 1.40–1.69 (m, 7 H), 1.87–2.04 (m, 3 H), 2.18 (dd, J = 14.2,
8.7 Hz, 1 H), 2.29 (dd, J = 14.2, 3.1 Hz, 1 H), 2.41 (br s, 2 H),
2.87 (m, 1 H), 3.85 (m, 1 H), 4.10 (s, 2 H), 4.98 (s, 1 H), 5.14 (s, 1
H), 5.64 (s, 1 H); 13C NMR (100 MHz, CDCl3) δ 12.1, 18.8,
22.2, 22.7, 27.9, 31.1, 32.9, 40.0, 43.3, 43.8, 45.7, 56.0, 56.5,
66.4, 67.6, 97.4, 114.2, 144.8, 145.8; EI-LRMS m/z 370 (Mϩ),
352, 337, 298, 256, 227; EI-HRMS calcd for C19H31O279Br
370.1507, found 370.1501. To a solution of the above diol (63
1
ϩ51.2 (c 1.54, CHCl3); IR (neat) 1730, 1287, 1152 cmϪ1; H
NMR (400 MHz, CDCl3) δ 0.57 (s, 3 H), 1.01 (d, J = 6.3 Hz,
3 H), 1.23 (s, 9 H), 1.25–1.39 (m, 4 H), 1.41–1.47 (m, 2 H), 1.50–
1.61 (m, 3 H), 1.63–1.70 (m, 2 H), 1.91–2.05 (m, 5 H), 2.36 (d,
J = 14.2 Hz, 1 H), 2.88 (m, 1 H), 3.85 (m, 1 H), 4.52 (d, J = 13.7
Hz, 1 H), 4.58 (d, J = 13.7 Hz, 1 H), 5.05 (s, 1 H), 5.17 (s, 1 H),
5.65 (s, 1 H); 13C NMR (100 MHz, CDCl3) δ 12.0, 19.7, 22.2,
22.7, 27.3, 28.0, 31.1, 34.3, 39.9, 41.8, 43.7, 45.6, 55.9, 56.4,
66.4, 68.1, 97.4, 114.8, 141.2, 144.8, 177.9; EI-LRMS m/z 454
(Mϩ), 375, 357, 299; EI-HRMS calcd for C24H39O379Br
454.2083, found 454.2086. <Synthesis of (S)-MTPA Ester 10>
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 4 3 9 6 – 4 4 0 2
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mg, 0.17 mmol) in CH2Cl2 (0.6 mL) were added pyridine (54
µL, 0.68 mmol) and pivaloyl chloride (36 µL, 0.32 mmol) at
0 ЊC, and the mixture was stirred at room temperature for 19 h.
To the mixture was added water, and the aqueous layer was
extracted with Et2O. The organic layer was washed with satur-
ated NaCl aq. solution, dried over Na2SO4, and concentrated.
The residue was purified by column chromatography on silica
gel (hexane/AcOEt = 9 : 1) to give the corresponding pivaloyl
ester (56 mg, 72%) as a colorless oil. [α]1D7 ϩ64.4 (c 1.54, CHCl3);
IR (neat) 1732, 1285, 1154 cmϪ1; 1H NMR (400 MHz, CDCl3)
δ 0.59 (s, 3 H), 0.98 (d, J = 6.3 Hz, 3 H), 1.23 (s, 9 H), 1.26–1.40
(m, 4 H), 1.43–1.74 (m, 7 H), 1.87–2.04 (m, 4 H), 2.11–2.22 (m,
2 H), 2.88 (m, 1 H), 3.87 (m, 1 H), 4.54 (s, 2 H), 5.03 (s, 1 H),
5.14 (s, 1 H), 5.64 (s, 1 H); 13C NMR (100 MHz, CDCl3) δ 12.1,
14.3, 18.9, 22.2, 22.7, 27.3, 27.9, 31.1, 32.9, 40.0, 43.2, 43.7,
45.7, 56.0, 56.4, 66.4, 97.4, 114.5, 141.2, 144.8, 178.0; EI-LRMS
m/z 454 (Mϩ), 375, 357, 299; EI-HRMS calcd for C24H39O379Br
454.2083, found 454.2086. <Synthesis of (S)-MTPA ester 11>
To a solution of the above pivaloyl ester (20 mg, 44 µmol) in
pyridine (0.44 mL) were added (R)-MTPACl (73 µL, 0.38
mmol) and DMAP (24 mg, 0.20 mmol) at 0 ЊC, and the mixtre
was stirred at room temperature for 2 days. To the mixture was
added water, and the aqueous layer was washed with saturated
NaCl aq. solution, dried over Na2SO4, and concentrated. The
residue was purified by preparative thin-layer chromatography
(hexane/AcOEt = 15 : 1) on silica gel to give the corresponding
(S)-MTPA ester 11 (28 mg, 95%) as a colorless oil. [α]1D9 ϩ38.0 (c
0.31, CHCl3); IR (neat) 1742, 1458, 1277, 1167 cmϪ1; 1H NMR
(600 MHz, CDCl3) δ 0.48 (s, 3 H), 0.98 (d, J = 6.3 Hz, 3 H), 1.22
(s, 9 H), 1.12–1.30 (m, 3 H), 1.36–1.44 (m, 2 H), 1.50–1.58 (m,
3 H), 1.63–1.68 (m, 2 H), 1.79 (dd, J = 12.6, 11.5 Hz, 1 H), 1.88
(m, 1 H), 1.94–1.99 (m, 2 H), 2.23 (dd, J = 14.4, 5.8 Hz, 1 H),
2.45 (dd, J = 14.4, 6.2 Hz, 1 H), 2.87 (m, 1 H), 3.49 (s, 3 H), 4.49
(d, J = 13.7 Hz, 1 H), 4.53 (d, J = 13.7 Hz, 1 H), 4.94 (s, 1 H),
5.06 (s, 1 H), 5.38 (ddd, J = 11.5, 6.2, 5.8 Hz, 1 H), 5.64 (s, 1 H),
7.36–7.40 (m, 3 H), 7.52 (br d, J = 7.1 Hz, 2 H); 13C NMR (150
MHz, CDCl3) δ 11.7, 18.6, 22.0, 22.5, 27.2, 27.6, 30.9, 32.8,
38.8, 39.1, 39.8, 40.5, 45.5, 55.2, 55.8, 56.0, 66.2, 72.7, 84.6 (q,
2JC–F = 27.6 Hz), 97.6, 115.7, 123.3 (q, 1JC–F = 289.0 Hz), 127.5,
128.4, 129.6, 132.0, 139.3, 144.8, 166.2, 177.9; EI-LRMS m/z
591 (M–Br)ϩ, 489, 436, 357, 255; EI-HRMS calcd for C34H46-
O5F3 (M–Br) 591.3297, found 591.3286. <Synthesis of (R)-
MTPA ester 11> In a similar manner to that for the synthesis of
(S)-MTPA ester, a crude product, which was obtained from the
above pivaloyl ester (30 mg, 66 µmol), (S)-MTPACl (40 µL,
0.21 mmol) and DMAP (24 mg, 0.20 mmol) in pyridine (0.66
mL), was purified by preparative thin-layer chromatography on
silica gel (hexane/AcOEt = 15 : 1) to give the corresponding (R)-
MTPA ester 11 (38 mg, 86%) as a colorless oil. [α]1D8 ϩ63.3 (c
0.23, CHCl3); IR (neat) 1740, 1453, 1273, 1165 cmϪ1; 1H NMR
(600 MHz, CDCl3) δ 0.40 (s, 3 H), 0.93 (d, J = 6.0 Hz, 3 H), 1.04
(m, 1 H), 1.13–1.25 (m, 4 H), 1.22 (s, 9 H), 1.34 (m, 1 H), 1.47
(m, 1 H), 1.54 (m, 1 H), 1.61–1.65 (m, 2 H), 1.73 (dd, J = 12.2
11.3 Hz, 1 H), 1.81 (m, 1 H), 1.90–1.95 (m, 2 H), 2.29 (dd, J =
14.3, 6.0 Hz, 1 H), 2.51 (dd, J = 14.3, 6.1 Hz, 1 H), 2.85 (m, 1
H), 3.53 (s, 3 H), 4.52 (d, J = 13.6 Hz, 1 H), 4.57 (d, J = 13.6 Hz,
1 H), 4.99 (s, 1 H), 5.12 (s, 1 H), 5.40 (ddd, J = 11.3, 6.1, 6.0 Hz,
1 H), 5.62 (s, 1 H), 7.37–7.38 (m, 3 H), 7.53 (br d, J = 6.3 Hz, 2
H); 13C NMR (150 MHz, CDCl3) δ 11.7, 18.5, 22.0, 22.5, 27.2,
27.4, 30.9, 32.5, 38.8, 39.3, 39.8, 40.5, 45.5, 55.4, 55.8, 55.9,
mmol) in THF (3.0 mL) at 0 ЊC, and the mixture was stirred at
the same temperature for 30 min. To the mixture was added
saturated NH4Cl aq. solution, and the aqueous layer was
extracted with Et2O. The organic layers were combined and
washed with saturated NaCl aq. solution, dried over Na2SO4,
and concentrated. The residue was purified by flash column
chromatography on silica gel (hexane/AcOEt = 8 : 1) to give 12
(80 mg, quant.) as a colorless oil. [α]1D9 ϩ64.5 (c 0.19, CHCl3); IR
(neat) 1763, 1671, 1437, 1140 cmϪ1 1H NMR (400 MHz,
;
CDCl3) δ 0.58 (s, 3 H), 1.04 (d, J = 6.3 Hz, 3 H), 1.19–1.70 (m,
11 H), 1.90–2.01 (m, 3 H), 2.54 (dddd, J = 16.8, 6.5, 2.9, 2.9 Hz,
1 H), 2.87 (m, 1 H), 3.05 (m, 1 H), 4.61 (ddt, J = 6.5, 6.9, 6.9 Hz,
1 H), 5.62 (s, 1 H), 5.65 (s, 1 H), 6.22 (br s, 1 H); 13C NMR (100
MHz, CDCl3) δ 12.0, 19.4, 22.1, 22.6, 28.0, 31.1, 33.7, 34.2,
39.9, 42.4, 45.6, 55.8, 76.5, 97.6, 121.8, 134.5, 144.6, 170.0;
EI-LRMS m/z 366 (Mϩ), 287, 229, 201; EI-HRMS calcd for
C19H27O279Br 366.1194, found 366.1204.
(R)-5-{(R)-2-[(1R,4E,3aR,7aR)-4-Bromomethylene-7a-methyl-
perhydroinden-1-yl]propyl}-3-methylenedihydrofuran-2-one (13)
In a similar manner to that for the synthesis of 12 from 8, a
crude product, which was obtained from 9 (112 mg, 0.28
mmol), NaH (60% oil dispersion, 12.3 mg, 0.31 mmol) in THF
(5.6 mL), was purified by (hexane/AcOEt = 8 : 1) to give 13 (104
mg, quant.) as a colorless oil. [α]1D9 ϩ127.6 (c 0.46, CHCl3); IR
(neat) 1763, 1667, 1437, 1127 cmϪ1 1H NMR (400 MHz,
;
CDCl3) δ 0.58 (s, 3 H), 1.02 (d, J = 6.6 Hz, 3 H), 1.20–1.34 (m, 5
H), 1.44–1.69 (m, 4 H), 1.75–1.89 (m, 3 H), 1.95–2.03 (m, 2 H),
2.52 (dddd, J = 16.9, 5.9, 2.9, 2.9 Hz, 1 H), 2.87 (m, 1 H), 3.07
(dddd, J = 16.9, 7.3, 2.3, 2.3 Hz, 1 H), 4.64 (m, 1 H), 5.62 (br s,
1 H), 5.65 (s, 1 H), 6.22 (dd, J = 2.7, 2.3 Hz, 1 H); 13C NMR
(100 MHz, CDCl3) δ 12.4, 19.1, 22.5, 22.9, 28.0, 31.4, 33.4, 34.9,
40.3, 43.8, 46.0, 56.2, 56.5, 75.4, 97.9, 122.2, 134.9, 144.9, 170.4;
EI-LRMS m/z 366 (Mϩ), 287, 229, 201; EI-HRMS calcd for
C19H27O279Br 366.1194, found 366.1190.
The general procedure for the synthesis of 2ꢀ-substituted
25-dehydro-1ꢀ-hydroxyvitamin D3-26,23-lactones
To a solution of bromoolefin and enyne (1.5 eq. to bromoolefin)
in toluene-Et3N (1 : 3) and Pd(PPh3)4 (30 mol% to bromoolefin),
and the mixture was stirred at 110 ЊC for 2 h. The mixture was
filtered through a silica gel pad, and the filtrate was con-
centrated. The residue was dissolved in MeCN (1 mL). To the
solution was added HF/MeCN (1 : 9, 1 mL) at 0 ЊC, and the
mixture was stirred at room temperature for 2 h. To the mixture
was added saturated NaHCO3 aq. solution, and the aqueous
layer was extracted with AcOEt. The organic layer was washed
with saturated NaCl aq. solution, dried over Na2SO4, and con-
centrated. The residue was purified by flash column chromato-
graphy on silica gel to give the vitamin D3-lactone analogues.
(23S )-25-Dehydro-2ꢀ-methyl-1ꢀ-hydroxyvitamin D3-26,23-
lactone (2a)
A crude product, which was obtained from 12 (13 mg, 35 µmol),
14a (20 mg, 53 µmol) and Pd(PPh3)4 (13 mg, 11 µmol), was
treated with HF/MeCN. After usual workup, the crude product
was purified by column chromatography on silica gel (hexane/
AcOEt = 1 : 1) to give 2a (10 mg, 64% in 2 steps) as an amorph-
ous solid. [α]1D9 ϩ72.2 (c 0.15, CHCl3); IR (neat) 3345, 2926,
66.2, 72.4, 84.4 (q, 2JC–F = 27.6 Hz), 97.5, 115.6, 123.4 (q, 1JC–F
=
1
288.7 Hz), 127.3, 128.3, 129.5, 132.2, 139.5, 144.9, 166.2, 177.9;
EI-LRMS m/z 670 (Mϩ), 591, 489, 436, 357, 255; EI-HRMS
calcd for C34H46O579BrF3 670.2481, found 670.2459.
1752, 1277 cmϪ1; H NMR (400 MHz, CDCl3) δ 0.55 (s, 3 H),
1.03 (d, J = 6.3 Hz, 3 H), 1.08 (d, J = 6.8 Hz, 3 H), 1.19–2.04 (m,
17 H), 2.23 (dd, J = 13.2, 8.1 Hz, 1 H), 2.54 (dddd, J = 16.8, 6.6,
3.0, 3.0 Hz, 1 H), 2.67 (dd, J = 13.2, 3.9 Hz, 1 H), 2.83 (m, 1 H),
3.05 (dd, J = 16.8, 7.2 Hz, 1 H), 3.84 (m, 1 H), 4.30 (br s, 1 H),
4.59 (ddt, J = 6.6, 7.2, 7.0 Hz, 1 H), 5.00 (s, 1 H), 5.28 (s, 1 H),
5.62 (br s, 1 H), 6.00 (d, J = 11.2 Hz, 1 H), 6.22 (br s, 1 H), 6.39
(d, J = 11.2 Hz, 1 H); 13C NMR (150 MHz, CDCl3) δ 12.1, 12.5,
18.6, 22.2, 23.5, 27.6, 29.0, 33.0, 34.5, 40.5, 43.4, 44.2, 46.0,
(S )-5-{(R)-2-[(1R,4E,3aR,7aR)-4-Bromomethylene-7a-methyl-
perhydroinden-1-yl]propyl}-3-methylenedihydrofuran-2-one (12)
To a suspension of NaH (60% oil dispersion, 9.6 mg, 0.24
mmol) of THF (1.4 mL) was added a solution of 8 (87 mg, 0.22
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 4 3 9 6 – 4 4 0 2
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56.3, 56.9, 71.7, 75.1, 75.4, 113.2, 117.1, 121.9, 124.7, 133.2,
134.8, 142.7, 146.5, 170.4; EI-LRMS m/z 440 (Mϩ) 422, 404,
394, 378, 283; EI-HRMS calcd for C28H40O4 440.2927, found
440.2920.
MHz, CDCl3) δ 12.2 (2 C), 12.7, 18.8, 22.4, 23.6, 27.7, 29.1,
33.1, 34.6, 40.6, 43.5, 44.3, 46.1, 56.4, 56.9, 71.7, 75.2, 75.4,
113.1, 117.0, 121.8, 124.5, 133.1, 134.7, 142.5, 146.4, 170.1;
EI-LRMS m/z 440 (Mϩ) 422, 404, 394, 378, 283; EI-HRMS
calcd for C28H40O4 440.2927, found 440.2932.
(23S )-25-Dehydro-2ꢀ-(3-hydroxypropyl)-1ꢀ-hydroxyvitamin
D3-26,23-lactone (2b)
(23R)-25-Dehydro-2ꢀ-(3-hydroxypropyl)-1ꢀ-hydroxyvitamin
D3-26,23-lactone (3b)
A crude product, which was obtained from 12 (14 mg, 38 µmol),
14b (31 mg, 57 µmol) and Pd(PPh3)4 (13 mg, 11 µmol), was
treated with HF/MeCN. After usual workup, the crude product
was purified by flash column chromatography on silica gel
(AcOEt) to give 2b (10 mg, 56% in 2 steps) as an amorphous
solid. [α]2D1 ϩ47.5 (c 0.31, CHCl3); IR (neat) 3382, 2944, 1757,
A crude product, which was obtained from 13 (12 mg, 33 µmol),
14b (27 mg, 49 µmol), Pd(PPh3)4 (11 mg, 9.8 µmol), was treated
with HF/MeCN. After usual workup, the crude product was
purified by flash column chromatography on silica gel (AcOEt)
to give 3b (7 mg, 45% in 2 steps) as an amorphous solid. [α]1D9
ϩ93.6 (c 0.31, CHCl3); IR (neat) 3382, 2926, 1752, 1645, 1442
1
1667, 1437 cmϪ1; H NMR (400 MHz, CDCl3) δ 0.55 (s, 3 H),
1
1.03 (d, J = 6.3 Hz, 3 H), 1.22–1.73 (m, 19 H), 1.88–2.04 (m, 3
H), 2.25 (dd, J = 13.0, 8.8 Hz, 1 H), 2.54 (ddt, J = 16.8, 6.3, 3.1
Hz, 1 H), 2.66 (dd, J = 13.0, 4.4 Hz, 1 H), 2.82 (m, 1 H), 3.05
(ddt, J = 16.8, 7.4, 2.3 Hz, 1 H), 3.70 (t, J = 5.0 Hz, 2 H), 3.89
(ddd, J = 8.3, 8.3, 4.3 Hz, 1 H), 4.38 (d, J = 2.4 Hz, 1 H), 4.59
(dddd, J = 7.4, 7.0, 7.0, 6.3 Hz, 1 H), 4.99 (d, J = 2.0 Hz, 1 H),
5.28 (m, 1 H), 5.62 (dd, J = 2.4, 2.4 Hz, 1 H), 5.99 (d, J = 11.2
Hz, 1 H), 6.22 (dd, J = 2.8, 2.8 Hz, 1 H), 6.40 (d, J = 11.5 Hz, 1
H); 13C NMR (150 MHz, CDCl3) δ 12.0, 19.2, 22.2, 22.8, 23.4,
28.0, 29.0, 30.2, 33.7, 34.1, 40.4, 42.3, 45.9, 49.1, 56.2, 56.5,
62.9, 70.5, 73.6, 76.6, 113.7, 117.1, 121.9, 124.7, 132.9, 134.7,
142.9, 146.5, 170.3; EI-LRMS m/z 484 (Mϩ) 466, 448, 438, 389,
338, 309, 253; EI-HRMS calcd for C30H44O5 484.3189, found
484.3176.
cmϪ1; H NMR (400 MHz, CDCl3) δ 0.55 (s, 3 H), 1.02 (d,
J = 6.3 Hz, 3 H), 1.26–1.82 (m, 19 H), 1.96–2.04 (m, 3 H), 2.25
(dd, J = 13.0, 8.3 Hz, 1 H), 2.52 (m, 1 H), 2.67 (dd, J = 13.2, 4.0
Hz, 1 H), 2.84 (m, 1H), 3.04–3.10 (m, 1 H), 3.71 (t, J = 5.3 Hz,
2 H), 3.90 (ddd, J = 8.3, 8.3, 4.5 Hz, 1 H), 4.38 (d, J = 2.0 Hz,
1 H), 4.63–4.65 (m, 1 H), 4.99 (s, 1 H), 5.28 (s, 1 H), 5.62 (br s,
1 H), 6.00 (d, J = 11.2 Hz, 1 H), 6.23 (br s, 1 H), 6.39 (d, J = 11.2
Hz, 1 H); 13C NMR (100 MHz, CDCl3) δ 11.9, 18.4, 22.0, 22.6,
23.2, 27.4, 28.8, 30.0, 32.8, 34.3, 40.3, 43.2, 44.1, 45.8, 48.9,
56.1, 56.7, 62.7, 70.2, 73.4, 74.9, 113.4, 116.9, 121.7, 124.5,
132.7, 134.6, 142.6, 146.4, 170.2; EI-LRMS m/z 484 (Mϩ) 466,
448, 438, 389, 338, 309, 253; EI-HRMS calcd for C30H44O5
484.3189, found 484.3174.
(23R)-25-Dehydro-2ꢀ-(3-hydroxypropoxy)-1ꢀ-hydroxyvitamin
D3-26,23-lactone (3c)
(23S )-25-Dehydro-2ꢀ-(3-hydroxypropoxy)-1ꢀ-hydroxyvitamin
D3-26,23-lactone (2c)
A crude product, which was obtained from 13 (16 mg, 44 µmol),
14c (36 mg, 65 µmol) and Pd(PPh3)4 (15 mg, 13 µmol), was
treated with HF/MeCN. After usual workup, the crude product
was purified by flash column chromatography on silica gel
(AcOEt) to give 3c (10 mg, 46% in 2 steps) as an amorphous
solid. IR (neat) 3393, 2946, 1759, 1278 cmϪ1; [α]1D8 ϩ71.0 (c 0.37,
A crude product, which was obtained from 12 (11 mg, 29 µmol),
14c (25 mg, 45 µmol) and Pd(PPh3)4 (10 mg, 9 µmol), was
treated with HF/MeCN. After usual workup, the crude product
was purified by flash column chromatography on silica gel
(AcOEt) to give 2c (11 mg, 73% in 2 steps) as a colorless oil. [α]1D9
ϩ41.4 (c 0.21, CHCl3); IR (neat) 3379, 2946, 1761, 1279 cmϪ1
;
CHCl3); H NMR (400 MHz, CDCl3) δ 0.56 (s, 3 H), 1.01 (d,
1
1H NMR (400 MHz, CDCl3) δ 0.55 (s, 3 H), 1.02 (d, J = 6.4 Hz,
3 H), 1.21–2.01 (m, 16 H), 2.17 (t, J = 5.0 Hz, 1 H), 2.24 (dd, J =
13.0, 9.3 Hz, 1 H), 2.47 (d, J = 3.4 Hz, 1 H), 2.54 (d, J = 4.4 Hz,
1 H), 2.56 (m, 1 H), 2.69 (dd, J = 13.0, 4.6 Hz, 1 H), 2.82 (m, 1
H), 3.05 (dddd, J = 16.9, 7.4, 2.6, 2.3 Hz, 1 H), 3.38 (dd, J = 7.5,
3.5 Hz, 1 H), 3.75–3.90 (m, 4 H), 4.06 (m, 1 H), 4.45 (dd, J = 3.5,
3.5 Hz, 1 H), 4.59 (dddd, J = 7.4, 7.0, 7.0, 7.0 Hz, 1 H), 5.09 (br
s, 1 H), 5.39 (br s, 1 H), 5.62 (dd, J = 2.3, 2.3 Hz, 1 H), 6.01 (d,
J = 11.4 Hz, 1 H), 6.22 (dd, J = 2.7, 2.6 Hz, 1 H), 6.42 (d,
J = 11.4 Hz, 1 H); 13C NMR (150 MHz, CDCl3) δ 12.1, 19.2,
22.2, 23.4, 28.0, 29.0, 31.9, 33.7, 34.1, 40.4, 40.9, 42.3, 45.9,
56.2, 56.5, 61.3, 68.4, 68.5, 71.9, 76.8, 84.5, 116.2, 117.3, 121.9,
125.4, 131.7, 134.7, 143.1, 144.2, 170.4; EI-LRMS m/z 500 (Mϩ)
482, 464, 406, 390, 352; EI-HRMS calcd for C30H44O6 500.3138,
found 500.3133.
J = 6.3 Hz, 3 H), 1.26–2.03 (m, 16 H), 2.23 (dd, J = 13.2, 9.0 Hz,
1 H), 2.35 (br s, 1 H), 2.54 (m, 2 H), 2.61 (d, J = 3.9 Hz, 1 H),
2.68 (dd, J = 4.2 Hz, 1 H), 2.83 (m, 1 H), 3.06 (dd, J = 17.3, 7.6
Hz, 1 H), 3.37 (dd, J = 7.2, 3.1 Hz, 1 H), 3.76–3.90 (m, 4 H),
4.06 (m, 1 H), 4.44 (br s, 1 H), 4.63 (m, 1 H), 5.01 (br s, 1 H),
5.39 (br s, 1 H), 5.61 (br s, 1 H), 6.01 (d, J = 11.0 Hz, 1 H), 6.22
(br s, 1 H), 6.41 (d, J = 11.0 Hz, 1 H); 13C NMR (150 MHz,
CDCl3) δ 12.1, 18.6, 22.2, 23.4, 27.6, 29.0, 31.9, 33.0, 34.5, 40.5,
40.9, 43.4, 46.0, 56.3, 56.9, 61.2, 68.4, 68.5, 71.8, 75.2, 84.4,
116.1, 117.3, 125.4, 131.8, 134.8, 143.1, 144.3, 170.4; EI-LRMS
m/z 500 (Mϩ) 482, 464, 406, 390, 352; EI-HRMS calcd for
C30H44O6 500.3138, found 500.3134.
Vitamin D receptor (VDR) binding assay
[26,27-methyl-3H]-1α,25-dihydroxyvitamin D3 (specific activity
6.623TBq mmolϪ1, 15,000 dpm, 15.7 pg) and various amounts
of 1α,25-dihydroxyvitamin D3 and an analogue to be tested
were dissolved in 50 µl of absolute ethanol in 12 × 75 mm
polypropylene tubes. 0.2 mg of the chick intestinal VDR and
1 mg of gelatin in 1 ml of phosphate buffer solution (25 nM
KH2PO4, 0.1 M KCl, 1 mM dithiothreitol, pH 7.4) were added
to each tube in an ice bath. The assay tubes were incubated in a
shaking water bath for 1 h at 25 ЊC and then chilled in an ice
bath. 1 ml of 40% polypropylene glycol 6000 in distilled water
was added to each tube, which was then mixed vigorously and
centrifuged at 2260 × g for 60 min at 4 ЊC. After the supernatant
was decanted, the bottom of the tube containing the pellet was
cut off into a scintillation vial containing 10 ml of dioxane-
based scintillation fluid and the radioactivity was counted with
a Beckman liquid scintillation counter (Model LS6500). The
relative potency of the analogues were calculated from their
(23R)-25-Dehydro-2ꢀ-methyl-1ꢀ-hydroxyvitamin D3-26,23-
lactone (3a)
A crude product, which was obtained from 13 (16 mg, 44 µmol),
14a (25 mg, 65 µmol), Pd(PPh3)4 (15 mg, 13 µmol), was treated
with HF/MeCN. After usual workup, the crude product was
purified by flash column chromatography on silica gel (hexane/
AcOEt = 3 : 2) to give 3a (9 mg, 47% in 2 steps) as an amorph-
ous solid. [α]1D9 ϩ94.1 (c 0.48, CHCl3); IR (neat) 3387, 2936,
1
1748, 1275 cmϪ1; H NMR (400 MHz, CDCl3) δ 0.56 (s, 3 H),
1.01 (d, J = 6.3 Hz, 3 H), 1.08 (d, J = 6.8 Hz, 3 H), 1.23–2.03 (m,
17 H), 2.23 (dd, J = 13.3, 7.8 Hz, 1 H), 2.52 (br d, J = 16.9, 1 H),
2.67 (dd, J = 13.3, 3.5 Hz, 1 H), 2.83 (m, 1 H), 3.06 (br d,
J = 16.9 Hz, 1 H), 3.85 (m, 1 H), 4.31(br s, 1 H), 4.64 (m, 1 H),
5.00 (s, 1 H), 5.28 (s, 1 H), 5.62 (br s, 1 H), 6.00 (d, J = 11.2 Hz, 1
H), 6.22 (br s, 1 H), 6.38 (d, J = 11.2 Hz, 1 H); 13C NMR (150
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 4 3 9 6 – 4 4 0 2
4401

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concentration needed to displace 50% of [26,27-methyl-3H]-
1α,25-dihydroxyvitamin D3 from the receptor compared with
the activity of 1α,25-dihydroxyvitamin D3 (assigned a 100%
value).
S. Ishizuka, K. Yamaguchi, Y. Kan, M. Kurihara, N. Miyata,
C. Smith, H. F. DeLuca and H. Takayama, J. Med. Chem., 2000, 43,
4247.
6 (a) Y. Suhara, K.-i. Nihei, H. Tanigawa, T. Fujishima, K. Konno,
K. Nakagawa, T. Okano and H. Takayama, Bioorg. Med. Chem.
Lett., 2000, 10, 1129; (b) Y. Suhara, K.-i. Nihei, M. Kurihara,
A. Kittaka, K. Yamaguchi, T. Fujishima, K. Konno, N. Miyata and
H. Takayama, J. Org. Chem., 2001, 66, 8760.
Assay for HL-60 cell differentiation
Nitro blue tetrazolium (NBT)-reducing activity was used as a
cell differentiation marker. HL-60 cells were cultured in RPMI-
1640 medium supplemented with 10% heat-inactivated FCS.
Exponentially proliferating cells were collected, suspended in
fresh medium and seeded in culture plates (Falcon, Becton
Dickinson and Company, Franklin Lakes, NJ). Cell concen-
tration at seeding was adjusted to 2 × 104 cells mLϪ1 and the
seeding volume was 1 mL/well. An ethanol solution of 1α,25-
dihydroxyvitamin D3 (final concentration: 10Ϫ8 M) and an
analogue (final concentration: 10Ϫ11 to 10Ϫ6 M) was added to
the culture medium at 0.1% volume and culture was continued
for 96 h at 37 ЊC in a humidified atmosphere of 5% CO2/air
without medium change. The same amount of vehicle was
added to the control culture. NBT-reducing assay was per-
formed according to the method of Collins et al.18 Briefly, cells
were collected, washed with PBS and suspended in serum-free
medium. NBT/TPA solution (dissolved in PBS) was added.
Final concentrations of NBT and TPA were 0.1% and 100 ng
mLϪ1, respectively. Then, the cell suspensions were incubated at
37 ЊC for 25 min. After incubation, cells were collected by
centrifugation and resuspended in FCS. Cytospin smears were
prepared, and the counter-staining of nuclei was done with
Kemechrot solution. At least 500 cells per preparation were
observed. The relative potency of the analogues were calculated
from the concentration needed to inhibit 50% of cell differen-
tiation by 10Ϫ8 M 1α,25-dihydroxyvitamin D3 (IC50) compared
with the activity of TEI-9647 (2) (assigned a 100% value).
7 A. Kittaka, Y. Suhara, H. Takayanagi, T. Fujishima, M. Kurihara
and H. Takayama, Org. Lett., 2000, 2, 2619.
8 H. Takayama, A. Kittaka, T. Fujishima and Y. Suhara, in Vitamin D
Analogues in Cancer Prevention and Therapy, eds. J. Reichrath,
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289–317.
9 (a) S. Ishizuka, S. Ishimoto and A. W. Norman, Biochemistry, 1984,
23, 1473; (b) S. Ishizuka, T. Ohba and A. W. Norman, in Vitamin D:
Molecular, Cellular and Chemical Endocrinology, eds. A. W.
Norman, K. Schaefer, H. G. Grigoleit and D. von Herrath, Walter
de Gruyter, Berlin, 1988, pp. 143–144.
10 (a) D. Miura, K. Manabe, K. Ozono, M. Saito, Q. Gao, A. W.
Norman and S. Ishizuka, J. Biol. Chem., 1999, 274, 16392; (b)
K. Ozono, M. Saito, D. Miura, T. Michigami, S. Nakajima and
S. Ishizuka, J. Biol. Chem., 1999, 274, 32376; (c) D. Miura,
K. Manabe, Q. Gao, A. W. Norman and S. Ishizuka, FEBS Lett.,
1999, 460, 297; (d ) S. Ishizuka, D. Miura, H. Eguchi, K. Ozono,
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M. Chokki and A. W. Norman, Steroids, 2001, 66, 227.
11 To date, only two types of VDR antagonists are known. For the
other type of VDR antagonist of 25-carboxylic esters ZK159222
and its analogue ZK168281, see: (a) M. Herdick, A. Steinmeyer and
C. Carlberg, J. Biol. Chem., 2000, 275, 16506; (b) Y. Bury,
A. Steinmeyer and C. Carlberg, Mol. Pharmacol., 2000, 58, 1067;
(c) M. Herdick, A. Steinmeyer and C. Carlberg, Chem. Biol., 2000,
7, 885; (d ) A. Toell, M. M. Gonzalez, D. Ruf, A. Steinmeyer,
S. Ishizuka and C. Carlberg, Mol. Pharmacol., 2001, 59, 1478.
12 I. Hijikuro, T. Doi and T. Takahashi, J. Am. Chem. Soc., 2001, 123,
3716 and references cited therein.
13 I. Ohtani, T. Kusumi, Y. Kashman and H. Kakisawa, J. Am. Chem.
Soc., 1991, 113, 4092.
14 B. M. Trost, J. Dumas and M. Villa, J. Am. Chem. Soc., 1992, 114,
9836.
15 Y. Suhara, A. Kittaka, S. Kishimoto, M. J. Calverly, T. Fujishima,
N. Saito, T. Sugiura, K. Waku and H. Takayama, Bioorg. Med.
Chem. Lett., 2002, 12, 3255.
16 (a) J. A. Eisman, A. J. Hamstra, B. E. Kream and H. F. DeLuca,
Arch. Biochem. Biophys., 1976, 176, 235; (b) M. Inaba and
H. F. DeLuca, Biochim. Biophys. Acta, 1989, 1010, 20.
17 Recent reports on Paget’s bone disease, see: (a) C. Menaa,
J. Barsony, S. V. Reddy, J. Cornish, T. Cundy and G. D. Roodman,
J. Bone Miner. Res., 2000, 15, 228; (b) N. Kurihara, S. V. Reddy,
C. Menaa, D. Anderson and G. D. Roodman, J. Clin. Invest., 2000,
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Acknowledgements
The authors thank Ms J. Shimode and Ms M. Kitsukawa
(Teikyo University) for spectroscopic measurements. This
study was supported by Grants-in-Aid from the Ministry of
Education, Culture, Sports, Science and Technology, Japan.
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