Synthesis of 24,24-ethanovitamin D3 lactones using ruthenium-catalyzed intermolecular enyne metathesis: Potent vitamin D receptor antagonists

Article abstract of DOI:10.1055/s-2005-872075

Novel vitamin D receptor antagonists, 24,24-ethanovitamin D ₃-26,23-lactones 6 and 7 and their 2α-functionalized analogues 6a-c and 7a-c were synthesized and their biological activities were evaluated. The triene structure of vitamin D₃

Full text of DOI:10.1055/s-2005-872075

PAPER
2533
Synthesis of 24,24-Ethanovitamin D3 Lactones Using Ruthenium-Catalyzed
Intermolecular Enyne Metathesis: Potent Vitamin D Receptor Antagonists
E
thanovitamin D3oLactones
w
it
z
hEnyne
M
o
etathesis mi Saito,a Manami Masuda,a Hiroshi Saito,b Kazuya Takenouchi,b Seiichi Ishizuka,b Jun-ichi Namekawa,b
Midori Takimoto-Kamimura,b Atsushi Kittaka*a
a
Faculty of Pharmaceutical Sciences, Teikyo University, Kanagawa 199-0195, Japan
Fax +81(426)853713; E-mail: akittaka@pharm.teikyo-u.ac.jp
Teijin Institute for Bio-Medical Research, Tokyo 191-8512, Japan
Received 25 March 2005; revised 19 April 2005
b
Abstract: Novel vitamin D receptor antagonists, 24,24-ethanovita-
min D3-26,23-lactones 6 and 7 and their 2a-functionalized ana-
logues 6a–c and 7a–c were synthesized and their biological
25
H
OH
activities were evaluated. The triene structure of vitamin D3 was
constructed using Pd-catalyzed alkenylative cyclization of A-ring
H
precursor enynes 12 and 12a–c with the CD-ring bromo-olefin
counterpart having 24,24-ethano-a-methylene-g-lactone on the side
1: R = H
3
1
chain (21 or 22). The CD-ring precursors 21 and 22 were efficiently
synthesized via Ru-catalyzed intermolecular enyne metathesis of 15
with ethylene to give dienone 17 followed by cyclopropanation.
The VDR antagonistic activity of the newly designed vitamin D3
lactones 6 and 7 increased to 2.8 times that of TEI-9647 (2) in a HL-
60 cell differentiation evaluating system. Moreover, introduction of
three substituents, that is, a methyl (6a and 7a), a 3-hydroxypropyl
1a: R = Me
2
HO
OH
1b: R = CH2CH2CH2OH
1c: R = OCH2CH2CH2OH
R
Figure 1 Structures of 1a,25-dihydroxyvitamin D3 (1) and its C2a-
modified analogues 1a–c
(6b and 7b), or a 3-hydroxypropoxyl group (6c and 7c) into the C2a In 1999, 25-dehydro-1a-hydroxyvitamin D3-26,23-lac-
tones, TEI-9647 (2) and TEI-9648 (3) in Figure 2,10 were
position of 6 and 7, resulted in marked enhancement, up to 19 times,
identified as the first VDR antagonists11 during the course
of the antagonistic activity toward VDR.
Key words: vitamins , antagonist, lactones, ruthenium, metathesis,
enynes
of experiments on the side chain modification of 1a,25-di-
hydroxyvitamin D3-26,23-lactone, which is the natural
metabolite of 1.12 Both 2 and 3 specifically antagonize the
VDR-mediated genomic action.13 Namely, 2 and 3 inhibit
1a,25-Dihydroxyvitamin D3 (1) (Figure 1) is the most the differentiation of human leukemia cells (HL-60 cells)
physiologically active metabolite of vitamin D3 and regu- induced by 1.10a Moreover, 2 suppresses the gene expres-
lates various biological events, including bone metabo- sion of 25-hydroxyvitamin D3-24-hydroxylase in human
lism as well as the proliferation and differentiation of osteosarcoma cells10b and in HL-60 cells.10d The unprece-
various types of tumor cells.1,2 In most cases, the biologi- dented biological profiles of 2 and 3 prompted us to inves-
cal responses of 1 are mediated via interaction with its tigate the structure–activity relationships of the vitamin
specific nuclear receptor, vitamin D receptor (VDR), D3 lactone derivatives, and we found some pertinent mod-
which is a member of the nuclear receptor superfamily ifications of 2 and 3 that resulted in a marked enhance-
and acts as a ligand-dependent gene transcription factor ment of their biological activities.14 That is, introduction
with co-activators.3,4 Recently, we have synthesized sev- of the above three motifs, i.e., the methyl, the 3-hydroxy-
eral 1a,25-dihydroxyvitamin D3 analogues, which sys- propyl or the 3-hydroxypropoxyl group, into the C2a po-
tematically introduced an alkyl, w-hydroxyalkyl, and w- sition of 2 and 3, increased the antagonistic activity up to
hydroxyalkoxyl group into the C2a position of 1.5–9 Some
30-fold in the case of 2b.14a On the other hand, it was
of these C2a-modified vitamin D3 analogues showed found that both the VDR-binding affinity and antagonistic
unique biological profiles. In particular, introduction of activity of 2 and 3 were affected by the orientation of the
the 2a-methyl (1a),5 2a-(3-hydroxypropyl) (1b),6 and 2a- substituents on the lactone ring.14b Interestingly, vitamin
(3-hydroxypropoxy) (1c)7 groups led to a 2- to 4-fold D3 lactones with dimethyl groups at position C24 on the
higher binding affinity for the bovine thymus VDR rela- lactone ring (especially 4) had 12 times the antagonistic
tive to the natural hormone 1 with potent agonistic activi- activity of 2.14c Furthermore, we demonstrated that double
ty.
functionalization of the C2a and C24 positions of 2 and 3
remarkably increased the antagonistic activity, that is, 4a
showed 89-fold stronger antagonism than 2, and 5a was
19-fold more potent than 3.
On the basis of the above results, we newly designed
24,24-ethanovitamin D3-26,23-lactones 6 and 7 to inves-
tigate further the structure–activity relationships on the
SYNTHESIS 2005, No. 15, pp 2533–2543
Advanced online publication: 20.07.2005
DOI: 10.1055/s-2005-872075; Art ID: F05705SS
© Georg Thieme Verlag Stuttgart · New York
x
x.
x
x
.
2
0
0
5

2534
N. Saito et al.
PAPER
R2
R2
R2
R2
24
10 was sulfonated, and the resulting monosufonate was
treated with lithium hexamethyldisilazide to give the ep-
oxide 11. Introduction of the TMS-ethynyl group into 11
followed by deprotection under basic conditions gave a
diol. The resulting diol was protected by the TBS groups
to provide the desired A-ring precursor 12 (Scheme 1).17
23R
23S
24
25
25
H
O
H
O
O
O
H
H
MeO
O
MeO
HO
O
2
2
O
1) LiAlH4
Br
OBz
HO
OH
HO
OH
R1
R1
2) NBS, BaCO3
O
Ph
O
2: R1 = R2 = H (TEI-9647)
2a: R1 = Me, R2 = H
2b: R1 = (CH2)3OH, R2 = H
3: R1 = R2 = H (TEI-9648)
3a: R1 = Me, R2 = H
3b: R1 = (CH2)3OH, R2 = H
8
9
1) 2-mesitylenesulfonyl
chloride
Zn/NaBH3CN
HO
2c: R1 = O(CH2)3OH, R2 = H 3c: R1 = O(CH2)3OH, R2 = H
1-PrOH–H2O
75%
OH OBz
2) LiHMDS
4: R1 = H, R2 = Me
4a: R1 = R2 = Me
5: R1 = H, R2 = Me
5a: R1 = R2 = Me
10
2 steps 66%
1)
TMS
BF3·OEt2
Li
Figure 2 Structures of 25-dehydro-1a-hydroxyvitamin D3-26,23-
lactones (TEI-9647: 2 and TEI-9648: 3), and their C2a-modified (2a–
c and 3a–c), 24-modified (4 and 5), and 2,24-double modified ana-
logues (4a and 5a)
O
OBz
11
TBSO
12
OTBS
2) K2CO3/MeOH
3) TBSOTf, 2,6-lutidine
3 steps 93%
Scheme 1 Improved synthesis of A-ring precursor 12
lactone ring core structure, and particularly, the effect of
the rigid spiro[2.4]ring system on the biological activities
(Figure 3). Furthermore, we expected the biological activ-
ities of the ethanolactone derivatives (6 and 7) to be en-
hanced by introducing three motifs, that is, the methyl (6a
and 7a), the 3-hydroxypropyl (6b and 7b) and the 3-hy-
droxypropoxyl group (6c and 7c) as in our previous stud-
ies.8,9,14 Here we report the synthesis and biological
evaluation of the novel potent VDR antagonists, 24,24-
ethano-1a-hydroxyvitmin D3 lactones and their C2a-
modified analogues.
Next, we synthesized the CD-ring counterpart having the
ethano-a-methylene-g-lactone unit on the side chain via
Mori’s Ru-catalyzed intermolecular enyne metathesis
with ethylene.18 The metathesis precursor 15 was pre-
pared by the addition of lithium siloxymethylacetylide
generated from 14 and BuLi to the aldehyde 1314a and the
following TPAP oxidation (Scheme 2).
25
CHO
24
OTBS
H
23
O
1)
(14)
OTBS
H
23S
O
23R
O
n-BuLi
2) TPAP, NMO
2 steps 77%
H
24
24
H
H
Br
H
13
O
O
15
Br
H
H
Scheme 2 Synthesis of alkynone 15
The Ru-catalyzed intermolecular enyne metathesis of 15
with ethylene was investigated for the introduction of a
methylene unit into positions C24 and C25 (based on the
steroidal numbering), respectively (Table 1). First of all,
according to Mori’s procedure, the alkyne 15 was treated
with the first generation Grubbs catalyst 16a under ethyl-
ene gas (run 1). However, no metathesis product was ob-
tained. The second generation Grubbs catalyst 16b19a was
not effective for the intermolecular enyne metathesis of
15, either, and only decomposition of 15 was observed
(runs 2 and 3). On the other hand, when the Hoveyda–
Grubbs catalyst 16c19b was used, the dienone 17 was ob-
tained in 29% (run 4). The same type of Ru catalyst 16d
reported by Blechert19c was found to be more effective for
the metathesis of 15 with ethylene, and 17 was produced
in 55% yield.19d Finally, we disclose that the alkynone–
ethylene metathesis proceeded smoothly in the presence
of 10 mol% of 16d at 0 °C to provide the desired dienone
derivative 17 in high yield (run 6).
HO
OH
HO
OH
R
R
6: R = H
7: R = H
6a: R = Me
6b: R = CH2CH2CH2OH
6c: R = OCH2CH2CH2OH
7a: R = Me
7b: R = CH2CH2CH2OH
7c: R = OCH2CH2CH2OH
Figure 3 Newly designed 24,24-ethanovitamin D3 lactones 6 and 7
and their 2a-modified analogues (6a–c and 7a–c)
Synthesis and Biological Evaluation of 24,24-Ethano-
vitamin D3-26,23-Lactones
First of all, A-ring precursor 12 was synthesized in an im-
proved manner from epoxide 8.15 That is, according to the
reported procedure,16 8 was reacted with LiAlH4 followed
by oxidative cleavage of the benzylidene acetal by NBS to
give the known compound 9. Pyranose ring-opening of 9
with activated zinc in the presence of NaBH3CN provided
10 in 75% yield. The primary hydroxyl group of the diol
Synthesis 2005, No. 15, 2533–2543 © Thieme Stuttgart · New York

PAPER
Ethanovitamin D3 Lactones with Enyne Metathesis
2535
Table 1 Ruthenium-Catalyzed Enyne Metathesis of 15 with Ethyl-
ene
23
24
Me3S(O)I, NaH
DMSO–THF
H
O
17
OTBS
24
25
18 (85%)
H
H
O
Ru catalyst (16)
OTBS
15
Br
1) DIBAL–H
2) TBAF
CH2Cl2
H2C=CH2 (1atm)
H
17
Br
23R
OH
23S
PCy3
MesN NMes
Cl
H
H
MesN NMes
Cl
Ph
OH
16c: R = H
16d: R = Ph
Cl
OH
OH
Ru
Ru
Cl
Cl
Ph
Ru
Cl
PCy3
O
H
H
PCy3
19 (2 steps 43%)
20 (2 steps 52%)
16a
16b
Br
Br
R
MnO2
MnO2
Run
1
‘Ru’ (mol%)
16a (5)
Temp (°C)
r.t.
Time (h)
Yielda (%)
0 (80)
0 (88)
0 (44)
29 (32)
55 (6)
92
16
24
4
H
O
H
O
2
16b (5)
r.t.
O
O
H
H
3
16b (10)b
16c (20)c
16d (20)d
16d (10)
reflux
r.t.
21 (97%)
22 (92%)
Br
Br
4
24
53
Scheme 3 Synthesis of CD-ring precursors 21 and 22
5
r.t.
6
0 °C
1.5
a The yield in parentheses is that of recovered 15.
b 5 mol% of 16b was used. After 2 h, 5 mol% of 16b was added.
c 5 mol% of 16c was used. Additional amounts of 16c were succes-
sively added (5 mol%, 5 h, 10 mol%, 19 h).
d 5 mol% of 16d was used. Additional amounts of 16d were succes-
sively added (5 mol%, 24 h, 10 mol%, 48 h).
Transformation of the dienone 17 into the CD-ring bro-
mo-olefin precursors 21 and 22 is shown in Scheme 3.
Cyclopropanation by the 1,4-addition of trimethylsulfox-
onium ylide to 17 provided the desired cyclopropyl ketone
derivative 18 in good yield. Treatment of 18 with DIBAL-
H followed by desilylation of the TBS group using TBAF
gave two diol derivatives 19 and 20, which were stereo-
isomers with respect to the position C23 on the side chain,
in 43% yield (2 steps) and in 53% yield (2 steps), respec-
tively. The crystal structure of 20 revealed the absolute
configuration of position C23 of 20 to be R (Figure 4).20
From this result, the absolute configuration of position
C23 of its stereoisomer 19 was determined to be S. Final-
ly, the diol 19 was converted into the desired CD-ring pre-
cursor 21 by oxidation using MnO2 in 97% yield.
Similarly, the lactone 22 was obtained from 20 by MnO2
oxidation in 92% yield.
Figure 4 Stereoscopic view of the crystal structure of compound
20. The thermal displacement parameters are drawn at 50%
23
*
H
O
O
21
1) 12, cat. Pd(PPh3)4
toluene/Et3N
H
or
2) HF/MeCN (5/95)
22
6 (62% from 21)
7 (76% from 22)
HO
OH
Scheme 4 Synthesis of 24,24-ethanovitamin D3-26,23-lactones 6
and 7
Biological activities of the synthesized 24,24-ethanovita-
min D3 lactones 6 and 7 were evaluated and the data are
shown in Table 2. We also show the data of 24,24-di-
methylvitamin D3 lactones 4 and 5 for comparison. The
binding affinity of 6 and 7 for the chick intestinal VDR
was examined as described previously.22 The binding af-
finity of (23S)-24,24-ethanovitamin D3 lactone (6) for the
VDR increased remarkably to 13.9 times that of 2 (1.7
times more potent than the natural hormone 1). In the case
of TEI-9648 type analogue 7, the binding affinity for the
VDR decreased to one tenth that of 3. The antagonistic ac-
tivities of 6 and 7 were assessed by the NBT-reduction
The construction of a vitamin D triene skeleton was
achieved by Trost’s alkenylative cyclization7,21 of the A-
ring precursor 12 with the CD-ring counterpart 21 or 22
(Scheme 4). That is, each CD-ring precursor 21 or 22 re-
acted with enyne 12 in the presence of Pd(0) catalyst, and
the protected vitamin D3 derivative was produced. Acid-
mediated deprotection gave the desired 24,24-ethanovita-
min D3 lactone derivatives 6 and 7, respectively.
Synthesis 2005, No. 15, 2533–2543 © Thieme Stuttgart · New York

2536
N. Saito et al.
PAPER
method23 in terms of inhibition of HL-60 cell differentia-
tion induced by 10 nM of the natural hormone 1. Although
the antagonistic activity of (23S)-24,24-ethanovitmain D3
lactone 6 was weaker than that of the corresponding
24,24-dimethly analogue 4, 6 was 2.8-fold more active
than the original 2. On the other hand, the (23R)-isomer 7
showed little antagonistic activity in contrast to (23R)-
24,24-dimethylvitamin D3 lactone 5 which was highly ac-
tive compared to the original 3.
1)
TBSO
OTBS
23
*
R
12a: R = Me
12b: R = (CH2)3OTBS
12c: R = O(CH2)3OTBS
H
O
O
H
21
or
cat. Pd(PPh3)4
6a (60%)
6b (62%)
6c (66%)
7a (49%)
7b (54%)
7c (56%)
2) HF/MeCN (5/95)
22
HO
OH
R
Table 2 Biological Activities of 24,24-Ethanovitamin D3 Lactones
6 and 7
Scheme 5 Synthesis of C2a-modified 24,24-ethanovitamin D3-
26,23-lactones 6a–c and 7a–c
Compounds
Binding affinity
for VDRa
Antagonistic activity
(IC50, nM)b
VDR antagonistic activity increased to 13–19 times that
of 2 (4.5–6.7 times stronger than 6). The antagonistic ac-
tivity of (23R)-lactone analogue 7 was strongly affected
by the substituents at position C2a (7a–c). That is, (23R)-
24,24-ethanovitamin D3 lactone having the 2a-methyl
group (7a) showed 2.4-times higher VDR antagonistic ac-
tivity than TEI-9648 (3). On the other hand, the antagonis-
tic activity of the vitamin D3 lactone derivatives having
the longer side-chain at the C2a position (7b and 7c) de-
creased to about half that of 8.
1
100
12
–
9.4
TEI-9647 (2)
4c
37
0.71
3.3
6
167
7
TEI-9648 (3)
134.4
51.5
>3000
5c
7
18
0.7
a Chick intestinal VDR. The potency of 1 is normalized to 100.
b Antagonistic activity was assessed in terms of IC50 for the differen-
tiation of HL-60 cells induced by 10 nM of 1.
c See ref.14c
Table 3 Biological Activities of 2a-Modified 24,24-Ethanovitamin
D3 Lactones 6a–c and 7a–c
Compounds
Binding affinity
for VDRa
Antagonistic activity
(IC50, nM)b
TEI-9647 (2)
12
111
83
59
7
9.4
0.49
0.74
0.55
134.4
55
Effect of C2a Modification of 24,24-Ethanovitamin
D3 Lactones
6a
6b
Next, we turned our attention to the C2a-funtionalization
of the 24,24-ethanovitmain D3 lactones 6 and 7. We dem-
onstrated that the C2a-modification of 24-methylvitamin
D3 lactones14b as well as 24,24-dimethyl derivatives14c
markedly enhanced their biological activities. From our
previous results, we expected a high increase in VDR-
binding affinity and marked enhancement of antagonistic
activity through the C2a-functionalization of 6 and 7. The
C2a-modified analogues were similarly synthesized by
the coupling reaction of the CD-ring precursors 21 and 22
with the A-ring precursor enynes 12a,24 12b14c and 12c,7
respectively (Scheme 5).
6c
TEI-9648 (3)
7a
7b
7c
15
29
13
240
220
a Chick intestinal VDR. The potency of 1 is normalized to 100.
b Antagonistic activity was assessed in terms of IC50 for the differen-
tiation of HL-60 cells induced by 10 nM of 1.
The evaluation of the biological activities of 6a–c and 7a–
c demonstrated that the C2a-modification was effective in
improving the biological activities of 24,24-ethanovita-
min D3 lactones 6 and 7 (Table 3). Namely, the VDR-
binding affinity of (23S)-isomers 6a–c remained high. In
particular, the 2a-methyl analogue 6a showed 9.3 times
stronger VDR-binding affinity than the original TEI-9647
(2). On the other hand, the C2a-modification of TEI-9648
(3) type analogue 7 increased the low VDR affinity of 7 to
1.9–4.1 times higher than TEI-9648 (3) (7a–c). The antag-
onistic activity was also enhanced by the C2a-functional-
ization. In the case of TEI-9647 type analogues 6a–c, the
It is generally accepted that the first step in VDR-mediat-
ed transactivation is a ligand-binding process to the
ligand-binding domain (LBD) of the apo form of VDR.
Next, the ligand-VDR complex changes conformation
into a transcriptionally active holo form, which binds to
the co-activators to activate the target gene.25 During con-
formational change, helix 12, which is the most C-termi-
nal a-helix of VDR and has the site for interaction with
other proteins such as co-activators, is important and con-
trols whether the function of a ligand is agonism or antag-
onism.26 When the VDR antagonist TEI-9647 (2) binds to
the LBD of a VDR, the complex would change into an un-
Synthesis 2005, No. 15, 2533–2543 © Thieme Stuttgart · New York

PAPER
Ethanovitamin D3 Lactones with Enyne Metathesis
2537
usual transcriptionally inactive form.27 We speculate that
some amino acid residues in the LBD participate in the
conformational change of the VDR through the interac-
tion of the exo-methylene moiety on the lactone ring of 2.
Namely, there are two cysteine residues, Cys403 on helix
11 and Cys410 in the hinge region between helix 11 and
helix 12 in the LBD of the hVDR. Recently, it was re-
vealed that the two cysteines, Cys403 and Cys410, play an
important role in the VDR antagonism of TEI-9647
(2).28,29 Furthermore, the exo-methylene lactone structure
is dispensable for the antagonistic action of the vitamin D3
lactones.30 Based on the results, we consider that the nu-
cleophilic thiol groups of the cysteines could attack the a-
methylene-g-lactone of 2 via 1,4-addition to give the cor-
responding cysteine adduct.31 Such interaction between
the ligand and the LBD might prevent the positioning of
helix 12. As a result, the complex of VDR and antagonist
2 could not adopt the transcriptionally active conforma-
tion. Therefore, it is thought that the VDR antagonists
whose exo-methylene moiety is located at a more favor-
able position to interact with Cys403 and/or Cys410 show
stronger VDR antagonistic activity.
temperature for 40 min. After the mixture was filtered through a
Celite pad, the filtrate was concentrated. The residue was purified
by flash column chromatography (silica gel; hexane–EtOAc, 1:1) to
give 10.
Yield: 785 mg (75%); colorless oil; [a]D23 +10.9 (c 1.54, CHCl3).
IR (neat): 3381, 1716, 1604, 1275, 1026 cm–1.
1H NMR (400 MHz, CDCl3): d = 1.75–1.90 (m, 2 H), 2.09 (dd,
J = 7.3, 4.2 Hz, 1 H), 3.26 (d, J = 3.9 Hz, 1 H), 3.51 (ddd, J = 11.1,
6.9, 4.2 Hz, 1 H), 3.64 (ddd, J = 11.1, 7.3, 3.4 Hz, 1 H), 3.75 (m, 1
H), 5.24 (d, J = 10.4 Hz, 1 H), 5.79 (d, J = 17.3 Hz, 1 H), 5.78 (m,
1 H), 5.98 (ddd, J = 17.3, 10.4, 5.9 Hz, 1 H), 7.46 (dd, J = 7.8, 7.8
Hz, 2 H), 7.59 (dd, J = 7.8, 7.8 Hz, 1 H), 8.07 (d, J = 7.8 Hz, 2 H).
13C NMR (100 MHz, CDCl3): d = 38.1, 66.4, 68.1, 72.0, 116.3,
128.2 (2 C), 129.4 (2 C), 129.7, 133.0, 136.1, 166.3.
EI-LRMS: m/z = 236 (M+), 219, 105, 77.
EI-HRMS: m/z calcd for C13H16O4: 236.1049; found: 236.1054.
(3S)-3-Benzoyloxy-4-(S)-oxyranylbut-1-ene (11)
To a solution of 10 (560 mg, 2.4 mmol) in pyridine (2.4 mL) was
added 2-mesitylenesulfonyl chloride (596 mg, 2.7 mmol) at 0 °C,
and the mixture was stirred at r.t. for 16 h. To the mixture was added
H2O, and this was extracted with Et2O. The organic layer was
washed with sat. aq NaCl, dried (Na2SO4) and concentrated. The
residue was roughly purified by flash column chromatography (sil-
ica gel; hexane–AcOEt, 3:1) to give a crude sulfonate compound
(827 mg). To a solution of the crude sulfonate (827 mg) in THF (20
mL) was added a solution of LiHMDS in THF (1.0 M, 3.0 mL, 3.0
mmol) at –78 °C, and the mixture was warmed to 0 °C over 1 h. To
the mixture was added sat. aq NH4Cl, and the aq layer was extracted
with Et2O. The organic layer was washed with sat. aq NaCl, dried
(Na2SO4) and concentrated. The residue was purified by flash col-
umn chromatography (silica gel; hexane–EtOAc, 20:1) to give 11
The novel synthesized vitamin D3 lactones 6, 6a–c and
7a–c, which showed more potent antagonistic activity,
might be situated in a preferable position to interact with
the cysteine residues after the binding to the LBD of the
VDR. On the other hand, the exo-methylene moiety of the
weaker VDR antagonist (23R)-ethanovitamin D3 lactone
(7) is possibly located in an unfavorable position for inter-
action with the cysteines. Further investigation of the
mechanism of antagonistic action is currently in progress.
23
Yield: 342 mg (2 steps, 66%); colorless oil; [a]D +14.3 (c 1.12,
CHCl3).
We have succeeded in the development of novel potent vi-
tamin D receptor antagonists, 24,24-ethano-1a-hydroxy-
vitamin D3-26,23-lactones 6 and 7 and their C2a-
functionalized analogues 6a–c and 7a–c. The VDR antag-
onists are expected to be potent therapeutic agents for
some diseases caused by the hypersensitivity of the VDR
to 1a,25-dihydroxyvitamin D3 such as Paget’s disease of
bone.32 We expect these analogues with potent anti-vita-
min D activity to contribute to our understanding of the
mechanisms involved in the expression of antagonistic ac-
tivity toward VDR as well as to finding new medicines for
treating Paget’s disease of bone.
IR (neat): 1720, 1649, 1603, 1026 cm–1.
1H NMR (400 MHz, CDCl3): d = 1.94–2.05 (m, 2 H), 2.52 (dd,
J = 4.8, 2.8 Hz, 1 H), 2.77 (dd, J = 4.8, 4.8 Hz, 1 H), 3.07 (m, 1 H),
5.25 (ddd, J = 10.6, 1.2, 1.2 Hz, 1 H), 5.39 (ddd, J = 17.1, 1.2, 1.2
Hz, 1 H), 5.73 (ddt, J = 1.2, 6.1, 6.1 Hz, 1 H), 5.97 (ddd, J = 17.1,
10.6, 6.1 Hz, 1 H), 7.45 (dd, J = 7.1, 7.1 Hz, 2 H), 7.57 (dd, J = 7.1,
7.1 Hz, 1 H), 8.07 (d, J = 7.1 Hz, 2 H).
13C NMR (100 MHz, CDCl3): d = 37.9, 47.0, 48.9, 72.6, 116.9,
128.2 (2 C), 129.4 (2 C), 130.0, 132.9, 135.6, 165.3.
EI-LRMS: m/z = 218 (M+), 193, 105, 77.
EI-HRMS: m/z calcd for C13H14O3: 218.0943; found: 218.0948.
(3S,5R)-Bis(tert-butyldimethylsilyloxy)oct-1-en-7-yne (12)
To a solution of TMS acetylene (0.59 mL, 4.2 mmol) in THF (1.5
mL) was added a solution of BuLi in hexane (1.6 M, 2.2 mL, 3.5
mmol) at –78 °C, and the mixture was stirred at the same tempera-
ture for 30 min. To the mixture were added a solution of 11 (335 mg,
1.5 mmol) and BF3·OEt2 (0.23 mL, 1.8 mmol) at –78 °C, and the
mixture was warmed to r.t. over 2 h. To the mixture was added sat.
aq NH4Cl, and the aqueous layer was extracted with Et2O. The or-
ganic layer was washed with sat. aq NaCl, dried (Na2SO4) and con-
centrated. The residue was dissolved in MeOH (5.1 mL). To the
solution was added K2CO3 (635 mg, 4.6 mmol) at 0 °C, and the mix-
ture was stirred at r.t. for 1 h. The mixture was partitioned between
H2O and Et2O, and this was extracted with Et2O. The organic layer
was washed with sat. aq NaCl, dried (Na2SO4) and concentrated.
The residue was purified by flash column chromatography (silica
gel; hexane–EtOAc, 3:1–2:1) to give (3S,5R)-oct-1-en-7-yne-3,5-
diol.
All manipulations were performed under an argon atmosphere un-
less otherwise mentioned. All solvents and reagents were purified
when necessary using standard procedures. Ethylene gas was used
without purification. Column chromatography was performed on
silica gel 60 N (Kanto Chemical Co., Inc., 100–210 mm), and flash
column chromatography was performed on silica gel 60 (Merck,
40–63 mm). NMR spectra were measured on a JEOL AL-400 mag-
netic resonance spectrometer. IR spectra were recorded on a
JASCO FTIR-8000 spectrometer. MS were measured on a JEOL
JMX-SX 102 mass spectrometer. Specific optical rotations were
measured on JASCO DIP-370 digital polarimeter.
(2S,4S)-4-Benzoyloxy-5-hexene-1,2-diol (10)
To a solution of 9 (1.5 g, 4.4 mmol) in 1-propanol–H2O (9:1, 44
mL) were added activated Zn dust (13 g, 199 mmol) and NaBH3CN
(1.8 g, 29 mmol) at 95 °C, and the mixture was stirred at the same
Synthesis 2005, No. 15, 2533–2543 © Thieme Stuttgart · New York

2538
N. Saito et al.
PAPER
Yield: 208 mg (97%); colorless oil; [a]D23 –0.003 (c 1.16, CHCl3).
IR (neat): 3374, 3301, 2120, 1645, 1219, 1072 cm–1.
1H NMR (400 MHz, CDCl3): d = 1.74 (ddd, J = 14.5, 7.5, 2.9 Hz, 1
H), 1.83 (ddd, J = 14.5, 8.8, 3.3 Hz, 1 H), 2.05 (t, J = 2.5 Hz, 1 H),
2.35–2.50 (m, 3 H), 2.80 (br d, J = 3.2 Hz, 1 H), 4.11 (m, 1 H), 4.49
(m, 1 H), 5.16 (ddd, J = 10.4, 1.5, 1.5 Hz, 1 H), 5.29 (ddd, J = 17.0,
1.5, 1.5 Hz, 1 H), 5.92 (ddd, J = 17.0, 10.4, 5.8 Hz, 1 H).
13C NMR (100 MHz, CDCl3): d = 27.2, 41.2, 67.0, 69.8, 70.7, 80.7,
114.4, 140.2.
EI-LRMS: m/z = 122 [(M – H2O)]+, 101, 83, 66.
was stirred under ethylene (1 atm) at the same temperature for 1.5
h. After the mixture was concentrated, the residue was purified by
flash column chromatography (silica gel; hexane–EtOAc, 50:1) to
give 17.
Yield: 96 mg (92%); colorless oil; [a]D28 +64.3 (c 1.21, CHCl3).
IR (neat): 1686, 1632, 1095 cm–1.
1H NMR (400 MHz, CDCl3): d = 0.061 (s, 6 H), 0.60 (s, 3 H), 0.90
(s, 9 H), 0.94 (d, J = 6.6 Hz, 3 H), 1.23–1.39 (m, 3 H), 1.41–1.73
(m, 5 H), 1.80–2.13 (m, 4 H), 2.44 (dd, J = 16.1, 10.0 Hz, 1 H), 2.70
(dd, J = 16.1, 2.9 Hz, 1 H), 2.88 (m, 1 H), 4.26 (dd, J = 1.4, 1.4 Hz,
2 H), 5.11 (d, J = 1.4 Hz, 1 H), 5.33 (d, J = 1.4 Hz, 1 H), 5.65 (s, 1
H), 5.68 (s, 1 H), 5.76 (s, 1 H).
13C NMR (100 MHz, CDCl3): d = –5.3 (2 C), 11.9, 18.3, 19.9, 22.0,
22.5, 25.9 (3 C), 27.7, 31.0, 33.1, 39.8, 45.6, 46.4, 55.76, 55.84,
65.5, 97.5, 114.2, 122.2, 144.6, 145.4, 149.2, 202.4.
EI-HRMS: m/z calcd for C8H10O (M – H2O): 122.0732; found:
122.0739.
To a solution of the above diol (416 mg, 3.0 mmol) in CH2Cl2 (15
mL) were added TBSOTf (1.7 mL, 7.4 mmol) and 2,6-lutidine (1.0
mL, 8.6 mmol) at 0 °C, and the mixture was stirred at the same tem-
perature for 1 h. To the mixture was added sat. aq NH4Cl, and the
aq layer was extracted with Et2O. The organic layer was washed
with sat. aq NaCl, dried (Na2SO4) and concentrated. The residue
was purified by column chromatography (silica gel; hexane–
EtOAc, 50:1) to give 12, whose spectral data were identical with
those reported previously by us.17
EI-LRMS: m/z = 494 (M+), 479, 437, 415, 345, 253, 211, 183.
EI-HRMS: m/z calcd for C26H43O279BrSi: 494.2216; found:
494.2208.
(R)-6-[(1R,4E,3aR,7aR)-4-Bromomethylene-7a-methyloctahy-
droinden-1-yl)]-2-[(tert-butyldimethylsilyloxy)methyl]-3,3-eth-
anohept-1-en-4-one (18)
Yield:1.1 g (96%); colorless oil.
Trimethylsulfoxonium iodide (49 mg, 0.22 mmol) and NaH (60%
in mineral oil, 10.5 mg, 0.26 mmol) were weighed in a dry flask.
DMSO (1 mL) was poured into the flask at r.t. and the resulting het-
erogeneous mixture was stirred at the same temperature for 20 min.
To the mixture was added a solution of 17 (104 mg, 0.20 mmol) in
THF–DMSO (7:1; 2.3 mL) at 10 °C, and the resulting mixture was
stirred at r.t. for 30 min. To the mixture was added sat. aq NH4Cl at
10 °C and the mixture was extracted with Et2O. The organic layer
was washed with sat. aq NaCl, dried (Na2SO4) and concentrated.
The residue was purified by flash column chromatography (silica
gel; hexane–EtOAc, 100:1) to give 18.
Yield: 86 mg (85%); colorless oil; [a]D28 +70.1 (c 1.15, CHCl3).
IR (neat): 1695, 1651, 1632, 1103 cm–1.
1H NMR (400 MHz, CDCl3): d = 0.066 (s, 6 H), 0.58 (s, 3 H), 0.85–
0.93 (m, 2 H), 0.87 (d, J = 6.4 Hz, 3 H), 0.91 (s, 9 H), 1.20–1.33 (m,
5 H), 1.38–1.70 (m, 5 H), 1.82 (m, 1 H), 1.90–2.05 (m, 3 H), 2.35
(dd, J = 16.5, 10.0 Hz, 1 H), 2.51 (dd, J = 16.5, 3.1 Hz, 1 H), 2.86
(m, 1 H), 4.13 (dd, J = 1.6, 1.6 Hz, 2 H), 5.13 (d, J = 1.6 Hz, 1 H),
5.41 (d, J = 1.6 Hz, 1 H), 5.63 (br s, 1 H)
(R)-6-[(1R,4E,3aR,7aR)-4-Bromomethylene-7a-methyloctahy-
droinden-1-yl)]-1-(tert-butyldimethylsilyloxy)-hept-2-yn-4-one
(15)
To a solution of 1433 (692 mg, 4.1 mmol) in THF (3.8 mL) was
added a solution of BuLi in hexane (1.5 M, 2.7 mL, 4.1 mmol) at
–78 °C, and the mixture was stirred at the same temperature for 1 h.
To the mixture was added a solution of 13 (607 mg, 2.0 mmol) in
THF (3 mL) at –78 °C, and the mixture was stirred at the same tem-
perature for 1 h. To the mixture was added sat. aq NH4Cl at –78 °C,
and the resulting mixture was warmed to r.t. The mixture was ex-
tracted with Et2O. The organic layer was washed with sat. aq NaCl,
dried (Na2SO4) and concentrated. The residue was dissolved in
CH2Cl2 (6.8 mL). To the solution was added tetrapropylammonium
perruthenate (214 mg, 0.61 mmol) and N-methylmorpholine N-ox-
ide (357 mg, 3.0 mmol) at 0 °C, and the mixture was stirred at r.t.
for 30 min. After the mixture was diluted with Et2O and filtered
through a short column (silica gel), the filtrate was concentrated.
The residue was purified by flash column chromatography (silica
gel; hexane–EtOAc, 50:1) to give 15.
13C NMR (100 MHz, CDCl3): d = –5.40, –5.38, 11.9, 16.0, 16.2,
18.4, 19.9, 22.0, 22.5, 25.9 (3 C), 27.5, 31.0, 32.7, 35.6, 39.8, 45.5,
46.4, 55.7, 55.9, 65.3, 97.5, 114.5, 144.7, 147.5, 209.1.
EI-LRMS: m/z = 508 (M+), 451, 377, 297, 227.
EI-HRMS: m/z calcd for C27H45O279BrSi: 508.2372; found:
27
Yield: 733 mg (2 steps, 77%); colorless oil; [a]D +55.8 (c 1.49,
CHCl3).
IR (neat): 2212, 1676, 1632, 1254, 1103 cm–1.
1H NMR (400 MHz, CDCl3): d = 0.13 (s, 6 H), 0.61 (s, 3 H), 0.91
(s, 9 H), 0.99 (d, J = 6.3 Hz, 3 H), 1.25–1.38 (m, 3 H), 1.40–1.72
(m, 5 H), 1.89 (m, 1 H), 1.95–2.03 (m, 2 H), 2.10 (m, 1 H), 2.29 (dd,
J = 15.4, 10.1 Hz, 1 H), 2.64 (dd, J = 15.4, 3.4 Hz, 1 H), 2.88 (m, 1
H), 4.46 (s, 2 H), 5.65 (m, 1 H).
13C NMR (100 MHz, CDCl3): d = –5.2 (2 C), 11.9, 18.2, 19.7, 22.0,
22.4, 25.7 (3 C), 27.7, 30.9, 33.3, 39.6, 45.5, 51.5, 52.3, 55.5, 55.7,
84.1, 90.1, 97.6, 144.5, 187.2.
508.2366.
(4S,6R)-6-[(1R,4E,3aR,7aR)-4-Bromomethylene-7a-methyloc-
tahydroinden-1-yl]-3,3-ethano-2-methyleneheptane-1,4-diol
(19)
To a solution of 18 (86 mg, 0.17 mmol) in toluene (1.7 mL) was
added a solution of DIBAL-H in toluene (1.0 M, 0.25 mL, 0.25
mmol) at –78 °C, and the mixture was stirred at the same tempera-
ture for 1 h. To the mixture were added a few drops of MeOH and
sat. aq potassium sodium tartrate at –78 °C, and the mixture was
stirred at r.t. for 30 min. The aq layer was extracted with Et2O, and
the organic layer was washed with sat. aq NaCl, dried (Na2SO4) and
concentrated. The residue was purified by flash column chromatog-
raphy (silica gel; hexane–EtOAc, 30:1) to give 23S-alcohol (39 mg,
45%) and 23R-alcohol (48 mg, 55%), respectively.
EI-LRMS: m/z = 466 (M+), 409, 255, 175, 147.
EI-HRMS: m/z calcd for C24H39O279BrSi: 466.1902; found:
466.1889.
(R)-6-[(1R,4E,3aR,7aR)-4-Bromomethylene-7a-methyloctahy-
droinden-1-yl]-2-[(tert-butyldimethylsilyloxy)methyl]-3-meth-
ylenehept-1-en-4-one (17) (Table 1, Run 6)
To a solution of 15 (98 mg, 0.21 mmol) in CH2Cl2 (2.1 mL) was
added Ru catalyst 16d (15 mg, 0.021 mmol) at 0 °C, and the mixture
Synthesis 2005, No. 15, 2533–2543 © Thieme Stuttgart · New York

PAPER
Ethanovitamin D3 Lactones with Enyne Metathesis
2539
23S-Alcohol
[a]D27 +29.4 (c 0.83, CHCl3).
IR (neat): 3424, 1641, 1255, 1042 cm–1.
(4R,6R)-6-[(1R,4E,3aR,7aR)-4-Bromomethylene-7a-methyloc-
tahydroinden-1-yl]-3,3-ethano-2-methyleneheptane-1,4-diol
(20)
Similar to the synthesis of 19 from the 23S-alcohol, the crude prod-
uct, which was obtained from the above 23R-alcohol (269 mg, 0.53
mmol) and TBAF (1.0 M THF solution, 1.1 mL, 1.1 mmol) in THF
(5.3 mL) at r.t. for 15 min, was purified by flash column chromatog-
raphy (silica gel; hexane–EtOAc, 4:1) to give 20.
1H NMR (400 MHz, CDCl3): d = 0.11 (s, 6 H), 0.41 (ddd, J = 9.3,
5.4, 4.1 Hz, 1 H), 0.56 (m, 1 H), 0.57 (s, 3 H), 0.69 (ddd, J = 9.3,
5.4, 4.1 Hz, 1 H), 0.78 (ddd, J = 9.3, 5.4, 4.1 Hz, 1 H), 0.93 (s, 9 H),
0.97 (d, J = 6.6 Hz, 3 H), 1.18–2.05 (m, 15 H), 2.82–2.93 (m, 2 H),
4.13 (d, J = 12.3 Hz, 1 H), 4.23 (d, J = 12.3 Hz, 1 H), 5.01 (d,
J = 1.9 Hz, 1 H), 5.19 (s, 1 H), 5.63 (s, 1 H)
13C NMR (100 MHz, CDCl3): d = –5.2 (2 C), 10.4, 11.5, 11.9, 18.4,
19.8, 22.0, 22.6, 25.9 (3 C), 27.5, 30.3, 31.1, 33.8, 39.9, 42.5, 45.5,
55.8, 56.7, 67.0, 76.9, 97.3, 116.7, 145.0, 147.8.
Yield: 195 mg (93%); colorless solid; mp 167–170 °C (recrystal-
lized from THF–hexane); [a]D25 +142.9 (c 1.19, THF).
IR (KBr): 3318, 1634, 1219, 1014 cm–1.
1H NMR (400 MHz, CDCl3): d = 0.45–0.55 (m, 2 H), 0.55 (s, 3 H),
0.65–0.78 (m, 2 H), 0.92 (d, J = 6.6 Hz, 3 H), 1.09 (ddd, J = 14.1,
10.8, 1.5 Hz, 1 H), 1.20–1.75 (m, 10 H), 1.85–2.06 (m, 3 H), 2.83–
2.95 (m, 2 H), 3.13 (br s, 2 H), 4.08 (d, J = 12.2 Hz, 1 H), 4.12 (d,
J = 12.2 Hz, 1 H), 5.00 (d, J = 1.7 Hz, 1 H), 5.20 (s, 1 H), 5.64 (s, 1
H).
13C NMR (100 MHz, CDCl3): d = 11.4, 11.5, 12.0, 18.8, 22.1, 22.6,
27.9, 31.1, 31.2, 32.6, 39.9, 41.2, 45.6, 56.0, 56.3, 66.5, 75.8, 97.4,
117.7, 144.9, 148.2.
EI-LRMS: m/z = 510 (M+), 492, 453, 435, 361, 281, 241, 227, 147.
EI-HRMS: m/z calcd for C27H47O279BrSi: 510.2528; found:
510.2528.
23R-Alcohol
[a]D25 +113.7 (c 1.04, CHCl3).
IR (neat): 3382, 1634, 1256, 1026 cm–1.
1H NMR (400 MHz, CDCl3): d = 0.11 (s, 6 H), 0.43–0.53 (m, 2 H),
0.57 (s, 3 H), 0.64–0.78 (m, 2 H), 0.91 (d, J = 6.6 Hz, 3 H), 0.92 (s,
9 H), 1.04 (ddd, J = 13.8, 10.9, 1.7 Hz, 1 H), 1.18–1.72 (m, 11 H),
1.91 (m, 1 H), 1.96 (ddd, J = 12.5, 7.1, 1.5 Hz, 1 H), 2.01 (ddd,
J = 12.5, 2.3, 2.3 Hz, 1 H), 2.80–2.92 (m, 2 H), 4.11 (d, J = 12.5 Hz,
1 H), 4.21 (d, J = 12.5 Hz, 1 H), 4.99 (d, J = 1.7 Hz, 1 H), 5.18 (s, 1
H), 5.63 (s, 1 H).
13C NMR (100 MHz, CDCl3): d = –5.2 (2 C), 11.3, 11.9, 18.4, 18.7,
22.0, 22.6, 25.7, 26.0 (3 C), 27.8, 31.0, 31.1, 32.5, 39.9, 41.8, 45.6,
56.0, 56.5, 66.8, 74.8, 97.3, 116.7, 145.0, 147.9.
EI-LRMS: m/z = 492 (M – H2O)+, 453, 435, 361, 281, 241, 227,
147.
EI-HRMS: m/z calcd for C27H45O79BrSi (M – H2O): 492.2423;
found: 492.2423.
EI-LRMS: m/z = 396 (M+), 378, 299, 254, 225, 175, 127.
EI-HRMS: m/z calcd for C21H33O279Br: 396.1663; found: 396.1656.
Anal. Calcd for C21H33O2Br: C, 63.47; H, 8.37. Found: C, 63.73; H,
8.74.
(S)-4-{(R)-2-[(1R,4E,3aR,7aR)-4-Bromomethylene-7a-methyl-
octahydroinden-1-yl]propyl}-7-methylene-5-oxaspiro[2.4]hep-
tan-6-one (21)
To a solution of 19 (61 mg, 0.15 mmol) in CH2Cl2 (3.1 mL) was
added MnO2 (346 mg, 4.0 mmol) at r.t., and the mixture was stirred
at the same temperature for 30 h. After the mixture was filtered
through a short column (silica gel; Et2O), the filtrate was concen-
trated. The residue was purified by flash column chromatography
(silica gel; hexane–EtOAc, 30:1) to give 21 (59 mg, 97%) as an
amorphous solid.
To the 23S-alcohol (197 mg, 0.39 mmol) in THF (3.9 mL) was add-
ed a solution of TBAF in THF (1.0 M, 0.77 mL, 0.77 mmol) at 0 °C,
and the mixture was stirred at r.t. for 1.5 h. To the mixture was add-
ed sat. aq NH4Cl at 0 °C, and the mixture was extracted with Et2O.
The organic layer was washed with sat. aq NaCl, dried (Na2SO4)
and concentrated. The residue was purified by flash column chro-
matography (silica gel; hexane–EtOAc, 3:1) to give 19.
[a]D27 +27.5 (c 0.88, CHCl3).
IR (film, CHCl3): 1759, 1660, 1342, 1118 cm–1.
1H NMR (400 MHz, CDCl3): d = 0.56 (s, 3 H), 0.85 (ddd, J = 9.8,
7.2, 4.9 Hz, 1 H), 0.95 (ddd, J = 10.0, 7.2, 4.9 Hz, 1 H), 1.01 (ddd,
J = 9.8, 6.5, 4.9 Hz, 1 H), 1.06 (d, J = 6.6 Hz, 3 H), 1.21 (ddd,
J = 10.0, 6.5, 4.9 Hz, 1 H), 1.25–1.73 (m, 11 H), 1.88–2.05 (m, 3 H),
2.88 (m, 1 H), 4.49 (dd, J = 8.7, 4.0 Hz, 1 H), 5.02 (s, 1 H), 5.64 (s,
1 H), 5.93 (s, 1 H).
13C NMR (100 MHz, CDCl3): d = 11.8, 14.8, 16.5, 19.9, 22.1, 22.5,
27.0, 27.8, 31.0, 34.5, 39.4, 39.7, 45.6, 55.6, 55.8, 81.4, 97.5, 113.5,
140.9, 144.7, 169.7.
Yield: 146 mg (96%); colorless solid; mp 167 °C (recrystallized
from THF); [a]D24 +50.4 (c 0.80, THF).
IR (KBr): 3148, 1637, 1256, 1022 cm–1.
1H NMR (400 MHz, CDCl3): d = 0.47 (ddd, J = 9.3, 5.2, 4.0 Hz, 1
H), 0.58 (ddd, J = 9.3, 5.2, 4.0 Hz, 1 H), 0.59 (s, 3 H), 0.66 (ddd,
J = 9.3, 5.2, 4.0 Hz, 1 H), 0.85 (ddd, J = 9.3, 5.2, 4.0 Hz, 1 H), 0.98
(d, J = 6.6 Hz, 3 H), 1.20–1.70 (m, 11 H), 1.80 (ddd, J = 13.9, 7.1,
2.9 Hz, 1 H), 1.85–2.04 (m, 3 H), 2.88 (m, 1 H), 2.94 (br s, 1 H),
2.57 (t, J = 6.6 Hz, 1 H), 4.13 (s, 2 H), 5.01 (d, J = 1.7 Hz, 1 H), 5.22
(s, 1 H), 5.64 (s, 1 H).
13C NMR (100 MHz, CDCl3): d = 10.6, 11.4, 11.9, 19.8, 22.1, 22.6,
27.6, 30.4, 31.1, 33.9, 39.9, 41.9, 45.6, 55.8, 56.6, 66.7, 77.8, 97.4,
117.5, 145.0, 148.1.
EI-LRMS: m/z = 392 (M+), 377, 313, 255, 227, 147.
EI-HRMS: m/z calcd for C21H29O279BrSi: 392.1351; found:
392.1368.
(R)-4-{(R)-2-[(1R,4E,3aR,7aR)-4-Bromomethylene-7a-methyl-
octahydroinden-1-yl]propyl}-7-methylene-5-oxaspiro[2.4]hep-
tan-6-one (22)
Similar to the synthesis of 21 from 19, a crude product, which was
obtained from 20 (42 mg, 0.10 mmol) and MnO2 (272 mg, 3.1
mmol) in CH2Cl2 (3.1 mL) at r.t. for 45 h, was purified by flash col-
umn chromatography (silica gel; hexane–EtOAc, 20:1) to give 22.
EI-LRMS: m/z = 396 (M+), 378, 299, 254, 225, 175, 127.
EI-HRMS: m/z calcd for C21H33O279Br: 396.1664; found: 396.1679.
Anal. Calcd for C21H33O2Br: C, 63.47; H, 8.37. Found: C, 63.55; H,
8.67.
26
Yield: 38 mg (92%); amorphous solid; [a]D +192.5 (c 0.68,
CHCl3).
IR (film, CHCl3): 1765, 1657, 1630, 1379, 1117 cm–1.
Synthesis 2005, No. 15, 2533–2543 © Thieme Stuttgart · New York

2540
N. Saito et al.
PAPER
1H NMR (400 MHz, CDCl3): d = 0.59 (s, 3 H), 0.87 (m, 1 H), 0.91–
1.06 (m, 3 H), 0.98 (d, J = 6.6 Hz, 3 H), 1.15 (ddd, J = 10.1, 6.4, 4.8
Hz, 1 H), 1.20–1.38 (m, 3 H), 1.40–1.73 (m, 6 H), 1.74–1.92 (m, 2
H), 1.97 (ddd, J = 12.8, 6.8, 1.6 Hz, 1 H), 2.01 (ddd, J = 12.8, 2.7,
2.7 Hz, 1 H), 2.88 (m, 1 H), 4.50 (dd, J = 11.7, 2.0 Hz, 1 H), 5.02
(s, 1 H), 5.65 (br s, 1 H), 5.94 (s, 1 H).
13C NMR (100 MHz, CDCl3): d = 11.9, 14.8, 16.9, 18.4, 22.0, 22.5,
26.1, 27.6, 31.0, 32.5, 39.8, 39.9, 45.6, 55.8, 56.2, 79.4, 97.5, 113.6,
141.0, 144.6, 169.6.
Yield: 25 mg (2 steps, 76%); amorphous solid; [a]D27 +92.0 (c 1.76,
CHCl3).
IR (film, CHCl3): 3428, 1755, 1657, 1607, 1342, 1215, 1051 cm–1.
1H NMR (400 MHz, CDCl3): d = 0.57 (s, 3 H), 0.82–1.05 (m, 4 H),
0.97 (d, J = 6.6 Hz, 3 H), 1.15 (ddd, J = 10.1, 6.4, 4.9 Hz, 1 H),
1.20–2.08 (m, 17 H), 2.31 (dd, J = 13.4, 6.6 Hz, 1 H), 2.60 (dd,
J = 13.4, 3.2 Hz, 1 H), 2.82 (m, 1 H), 4.23 (br s, 1 H), 4.42 (br s, 1
H), 4.51 (dd, J = 11.6, 1.8 Hz, 1 H), 5.00 (s, 1 H), 5.02 (s, 1 H), 5.33
(s, 1 H), 5.94 (s, 1 H), 6.01 (d, J = 11.2 Hz, 1 H), 6.46 (d, J = 11.2
Hz, 1 H).
EI-LRMS: m/z = 392 (M+), 377, 313, 255, 227, 147.
EI-HRMS: m/z calcd for C21H29O279BrSi: 392.1351; found:
392.1341.
13C NMR (100 MHz, CDCl3): d = 12.0, 14.8, 16.8, 18.4, 22.2, 23.5,
26.4, 27.6, 29.0, 32.5, 39.8, 40.5, 42.8, 45.2, 46.0, 56.3, 57.0, 66.8,
70.8, 79.6, 111.8, 113.7, 117.2, 124.8, 133.1, 141.2, 142.6, 147.6,
169.9.
Vitamin D3 Lactones; General Procedure
To a solution of an A-ring precursor (1.5 equiv to a CD-ring precur-
sor), and the CD-ring precursor in toluene were added Et3N and
Pd(PPh3)4 (30 mol% to the CD-ring precursor) and the mixture was
stirred at 110 °C for 1–1.5 h. After the mixture was filtered through
a silica gel pad, the filtrate was concentrated. The crude product was
dissolved in MeCN (2 mL). To the solution was added a solution of
concd HF (10%) in MeCN (2 mL) at 0 °C, and the mixture was
stirred at r.t. Sat. aq NaHCO3 was added, and the mixture was ex-
tracted with EtOAc. The organic layer was washed with sat. aq
NaCl, dried (Na2SO4), and concentrated. The residue was purified
by TLC (silica gel) to give the vitamin D3 derivative. Further puri-
fication for biological assays was conducted by reversed-phase re-
cycle HPLC (YMC-Pack ODS column, 20 × 150 mm, 9.9 mL/min,
eluent: MeCN–H2O, 90:10).
EI-LRMS: m/z = 452 (M+), 434, 416, 311, 285, 134, 105.
EI-HRMS: m/z calcd for C29H40O4: 452.2927; found: 452.2930.
(23S)-25-Dehydro-24,24-ethano-2a-methyl-1a-hydroxyvitamin
D3-26,23-Lactone (6a)
According to the General Procedure, a crude product, which was
obtained from 21 (25 mg, 62 mmol), 12a (36 mg, 94 mmol), Et3N
(1.5 mL) and Pd(PPh3)4 (22 mg, 19 mmol) in toluene (1.5 mL) at
110 °C for 1.5 h, was treated with concd HF in MeCN for 2.5 h. Af-
ter the usual work up, the crude product was purified by preparative
TLC (silica gel; hexane–EtOAc, 1:1) to give 6a.
Yield: 17 mg (2 steps, 60%); amorphous solid; [a]D24 +0.30 (c 1.34,
CHCl3).
IR (film, CHCl3): 3422, 1757, 1651, 1628, 1608, 1342, 1198, 1028
cm–1.
(23S)-25-Dehydro-24,24-ethano-1a-hydroxyvitamin D3-26,23-
Lactone (6)
1H NMR (400 MHz, CDCl3): d = 0.54 (s, 3 H), 0.84 (ddd, J = 9.9,
7.1, 5.0 Hz, 1 H), 0.91–1.05 (m, 2 H), 1.05 (d, J = 6.6 Hz, 3 H), 1.08
(d, J = 6.8 Hz, 3 H), 1.15–1.75 (m, 14 H), 1.85–2.05 (m, 4 H), 2.23
(dd, J = 13.6, 7.7 Hz, 1 H), 2.67 (dd, J = 13.6, 4.2 Hz, 1 H), 2.82 (m,
1 H), 3.84 (ddd, J = 7.7, 7.7, 4.2 Hz, 1 H), 4.31 (br d, J = 2.4 Hz, 1
H), 4.49 (dd, J = 8.7, 3.8 Hz, 1 H), 5.00 (br d, J = 2.0 Hz, 1 H), 5.01
(br s, 1 H), 5.28 (s, 1 H), 5.93 (s, 1 H), 6.00 (d, J = 11.2 Hz, 1 H),
6.38 (d, J = 11.2 Hz, 1 H).
13C NMR (100 MHz, CDCl3): d = 12.0, 12.6, 14.8, 16.5, 20.0, 22.3,
23.5, 27.1, 27.8, 29.0, 34.6, 39.5, 40.4, 43.4, 44.2, 46.0, 56.1, 56.6,
71.6, 75.3, 81.9, 113.1, 113.4, 117.0, 124.6, 133.1, 141.0, 142.7,
146.4, 169.8.
According to the General Procedure, a crude product, which was
obtained from 21 (25 mg, 64 mmol), 12 (35 mg, 95 mmol), Et3N (1.5
mL) and Pd(PPh3)4 (22 mg, 19 mmol) in toluene (1.5 mL) at 110 °C
for 1.5 h, was treated with concd HF in MeCN for 1 h. After the usu-
al work-up, the crude product was purified by preparative TLC (sil-
ica gel; hexane–EtOAc, 2:3) to give 6.
Yield: 19 mg (2 steps, 62%); amorphous solid; [a]D27 –17.0 (c 1.18,
CHCl3).
IR (film, CHCl3): 3387, 1759, 1657, 1611, 1342, 1055 cm–1.
1H NMR (400 MHz, CDCl3): d = 0.55 (s, 3 H), 0.83 (ddd, J = 9.9,
7.1, 5.1 Hz, 1 H), 0.95 (ddd, J = 9.9, 7.1, 4.7 Hz, 1 H), 1.01 (ddd,
J = 9.9, 6.5, 4.7 Hz, 1 H), 1.05 (d, J = 6.6 Hz, 3 H), 1.16–1.75 (m,
14 H), 1.85–2.08 (m, 5 H), 2.31 (dd, J = 13.5, 6.5 Hz, 1 H), 2.59 (dd,
J = 13.5, 3.4 Hz, 1 H), 2.82 (m, 1 H), 4.90 (m, 1 H), 4.43 (m, 1 H),
4.49 (dd, J = 8.8, 3.9 Hz, 1 H), 4.99 (s, 1 H), 5.01 (s, 1 H), 5.33 (s,
1 H), 5.93 (s, 1 H), 6.01 (d, J = 11.2 Hz, 1 H), 6.37 (d, J = 11.2 Hz,
1 H).
EI-LRMS: m/z = 466 (M+), 448, 430, 265, 166.
EI-HRMS: m/z calcd for C30H42O4: 466.3083; found: 466.3087.
(23R)-25-Dehydro-24,24-ethano-2a-methyl-1a-hydroxyvita-
min D3-26,23-Lactone (7a)
According to the General Procedure, a crude product, which was
obtained from 22 (21 mg, 52 mmol), 12a (30 mg, 79 mmol), Et3N
(1.5 mL) and Pd(PPh3)4 (18 mg, 16 mmol) in toluene (1.5 mL) at
110 °C for 1 h, was treated with concd HF in MeCN for 2.5 h. After
the usual work up, the crude product was purified by preparative
TLC (silica gel; hexane–EtOAc, 1:1) to give 7a.
13C NMR (100 MHz, CDCl3): d = 12.0, 14.8, 16.5, 20.0, 22.3, 23.6,
27.1, 27.8, 29.1, 34.6, 39.5, 40.4, 42.9, 45.3, 46.0, 56.1, 56.7, 66.8,
70.8, 81.6, 111.7, 113.5, 117.1, 124.8, 133.0, 141.0, 142.7, 147.6,
170.0.
EI-LRMS: m/z = 452 (M+), 434, 416, 311, 285, 134, 105.
25
EI-HRMS: m/z calcd for C29H40O4: 452.2926; found: 452.2925.
Yield: 12 mg (2 steps, 49%); amorphous solid; [a]D +118.9 (c
0.85, CHCl3).
(23R)-25-Dehydro-24,24-ethano-1a-hydroxyvitamin D3-26,23-
Lactone (7)
IR (film, CHCl3): 3441, 1754, 1657, 1604, 1344, 1215, 1055 cm–1.
1H NMR (400 MHz, CDCl3): d = 0.56 (s, 3 H), 0.83–1.05 (m, 4 H),
0.97 (d, J = 6.3 Hz, 3 H), 1.08 (d, J = 6.8 Hz, 3 H), 1.15 (ddd,
J = 10.3, 6.4, 4.8 Hz, 1 H), 1.20–1.33 (m, 4 H), 1.40–2.05 (m, 12 H),
2.23 (dd, J = 13.5, 7.8 Hz, 1 H), 2.67 (dd, J = 13.5, 4.1 Hz, 1 H),
2.82 (m, 1 H), 3.85 (ddd, J = 7.8, 7.8, 4.1 Hz, 1 H), 4.31 (br d,
J = 3.4 Hz, 1 H), 4.51 (dd, J = 11.5, 1.7 Hz, 1 H), 5.00 (br d, J = 2.0
According to the General Procedure, a crude product, which was
obtained from 22 (28 mg, 71 mmol), 12 (39 mg, 107 mmol), Et3N
(1.5 mL) and Pd(PPh3)4 (25 mg, 21 mmol) in toluene (1.5 mL) at
110 °C for 1.5 h, was treated with concd HF in MeCN for 1 h. After
the usual work up, the crude product was purified by preparative
TLC (silica gel; hexane–EtOAc, 2:3) to give 7.
Synthesis 2005, No. 15, 2533–2543 © Thieme Stuttgart · New York

PAPER
Ethanovitamin D3 Lactones with Enyne Metathesis
2541
Hz, 1 H), 5.02 (s, 1 H), 5.28 (s, 1 H), 5.94 (s, 1 H), 6.00 (d, J = 11.2
Hz, 1 H), 6.38 (d, J = 11.2 Hz, 1 H).
(23S)-25-Dehydro-24,24-ethano-2a-(3-hydroxypropoxyl)-1a-
hydroxyvitamin D3-26,23-Lactone (6c)
According to the General Procedure, a crude product, which was
obtained from 21 (24 mg, 62 mmol), 12c (52 mg, 93 mmol), Et3N
(1.5 mL) and Pd(PPh3)4 (21 mg, 19 mmol) in toluene (1.5 mL) at
110 °C for 1.5 h, was treated with concd HF in MeCN for 2.5 h. Af-
ter the usual work-up, the crude product was purified by preparative
TLC (silica gel; EtOAc) to give 6c.
13C NMR (100 MHz, CDCl3): d = 12.2, 12.6, 14.9, 16.9, 18.4, 22.3,
23.5, 26.5, 27.6, 29.1, 32.6, 39.8, 40.6, 43.5, 44.2, 46.0, 56.4, 57.0,
71.7, 75.3, 79.5, 113.1, 113.6, 117.1, 124.6, 133.2, 141.1, 142.6,
146.5, 169.7.
EI-LRMS: m/z = 466 (M+), 448, 430, 265, 166.
Yield: 21 mg (2 steps, 66%); amorphous solid; [a]D23 +17.2 (c 1.65,
EI-HRMS: m/z calcd for C30H42O4: 466.3083; found: 466.3075.
CHCl3).
(23S)-25-Dehydro-24,24-ethano-2a-(3-hydroxypropyl)-1a-hy-
droxyvitamin D3-26,23-Lactone (6b)
IR (film, CHCl3): 3395, 1759, 1659, 1342, 1059 cm–1.
According to the General Procedure, a crude product, which was
obtained from 21 (29 mg, 74 mmol), 12b (60 mg, 111 mmol), Et3N
(1.5 mL) and Pd(PPh3)4 (26 mg, 22 mmol) in toluene (1.5 mL) at
110 °C for 1.5 h, was treated with concd HF in MeCN for 2.5 h. Af-
ter the usual work-up, the crude product was purified by preparative
TLC (silica gel; EtOAc) to give 6b.
Yield: 23 mg (2 steps 62%); amorphous solid; [a]D24 +20.1 (c 1.78,
CHCl3).
IR (film, CHCl3): 3351, 1759, 1655, 1343, 1198, 1055 cm–1.
1H NMR (400 MHz, CDCl3): d = 0.53 (s, 3 H), 0.84 (ddd, J = 9.9,
7.0, 5.0 Hz, 1 H), 0.95 (ddd, J = 9.9, 7.0, 4.6 Hz, 1 H), 1.01 (ddd,
J = 9.9, 6.4, 4.6 Hz, 1 H), 1.05 (d, J = 6.6 Hz, 3 H), 1.15–1.75 (m,
17 H), 1.85–2.05 (m, 3 H), 2.13 (br s 1 H), 2.24 (dd, J = 13.2, 8.3
Hz, 1 H), 2.60 (br s, 2 H), 2.65 (dd, J = 13.2, 4.3 Hz, 1 H), 2.82 (m,
1 H), 3.60–3.73 (m, 2 H), 3.87 (ddd, J = 8.3, 8.3, 4.3 Hz, 1 H), 4.36
(br d, J = 2.9 Hz, 1 H), 4.49 (dd, J = 8.7, 3.8 Hz, 1 H), 4.98 (d,
J = 2.0 Hz, 1 H), 5.01 (s, 1 H), 5.26 (br s, 1 H), 5.93 (s, 1 H), 5.99
(d, J = 11.2 Hz, 1 H), 6.38 (d, J = 11.2 Hz, 1 H).
1H NMR (400 MHz, CDCl3): d = 0.54 (s, 3 H), 0.84 (ddd, J = 9.8,
7.1, 4.7 Hz, 1 H), 0.95 (ddd, J = 9.8, 6.7, 4.7 Hz, 1 H), 1.01 (ddd,
J = 9.8, 6.7, 4.7 Hz, 1 H), 1.05 (d, J = 6.6 Hz, 3 H), 1.15–2.05 (m,
17 H), 2.24 (dd, J = 13.5, 8.7 Hz, 1 H), 2.67 (dd, J = 13.5, 4.5 Hz, 1
H), 2.74 (br s, 3 H), 2.82 (m, 1 H), 3.37 (dd, J = 7.6, 3.2 Hz, 1 H),
3.70–3.93 (m, 4 H), 4.05 (ddd, J = 8.7, 7.6, 4.5 Hz, 1 H), 4.44 (d,
J = 3.2 Hz, 1 H), 4.49 (dd, J = 8.7, 3.8 Hz, 1 H), 5.01 (s, 1 H), 5.08
(d, J = 2.0 Hz, 1 H), 5.38 (d, J = 1.0 Hz, 1 H), 5.93 (s, 1 H), 6.00 (d,
J = 11.1 Hz, 1 H), 6.40 (d, J = 11.1 Hz, 1 H).
13C NMR (100 MHz, CDCl3): d = 12.0, 14.8, 16.5, 20.0, 22.3, 23.5,
27.1, 27.8, 29.1, 31.9, 34.6, 39.4, 40.3, 41.0, 46.0, 56.1, 56.6, 61.1,
68.3, 68.5, 71.9, 81.6, 84.5, 113.4, 116.1, 117.2, 125.3, 131.7,
141.0, 143.0, 144.1, 169.8.
EI-LRMS: m/z = 526 (M+), 508, 490, 464, 432, 265, 171.
EI-HRMS: m/z calcd for C32H46O6: 526.3295; found: 526.3297.
(23R)-25-Dehydro-24,24-ethano-2a-(3-hydroxypropoxyl)-1a-
hydroxyvitamin D3-26,23-Lactone (7c)
According to the General Procedure, a crude product, which was
obtained from 22 (30 mg, 77 mmol), 12c (64 mg, 115 mmol), Et3N
(1.5 mL) and Pd(PPh3)4 (27 mg, 23 mmol) in toluene (1.5 mL) at
110 °C for 1.5 h, was treated with concd HF in MeCN for 2.5 h. Af-
ter the usual work-up, the crude product was purified by preparative
TLC (silica gel; EtOAc) to give 7c.
13C NMR (100 MHz, CDCl3): d = 12.0, 14.8, 16.5, 20.0, 22.3, 22.8,
23.5, 27.1, 27.8, 29.0, 30.1, 34.6, 39.5, 40.4, 44.1, 46.0, 49.0, 56.1,
56.6, 62.6, 70.2, 73.5, 81.6, 113.4, 113.5, 117.1, 124.5, 133.0,
141.0, 142.7, 146.4, 169.8.
EI-LRMS: m/z = 510 (M+), 492, 474, 415, 327, 309.
23
EI-HRMS: m/z calcd for C32H46O5: 510.3345; found: 510.3342.
Yield: 22 mg (2 steps, 56%); amorphous solid; [a]D +107.1 (c
1.72, CHCl3).
(23R)-25-Dehydro-24,24-ethano-2a-(3-hydroxypropyl)-1a-hy-
droxyvitamin D3-26,23-Lactone (7b)
IR (film, CHCl3): 3382, 1759, 1651, 1628, 1343, 1057 cm–1.
According to the General Procedure, a crude product, which was
obtained from 22 (29 mg, 74 mmol), 12b (60 mg, 111 mmol), Et3N
(1.5 mL) and Pd(PPh3)4 (26 mg, 22 mmol) in toluene (1.5 mL) at
110 °C for 1.5 h, was treated with concd HF in MeCN for 2.5 h. Af-
ter the usual work-up, the crude product was purified by preparative
TLC (silica gel; EtOAc) to give 7b.
Yield: 20 mg (2 steps 54%); amorphous solid; [a]D24 +123.4 (c 1.57,
CHCl3).
IR (film, CHCl3): 3358, 1757, 1651, 1343, 1196, 1051 cm–1.
1H NMR (400 MHz, CDCl3): d = 0.56 (s, 3 H), 0.82–1.05 (m, 4 H),
0.97 (d, J = 6.6 Hz, 3 H), 1.15 (m, 1 H), 1.20–2.05 (m, 18 H), 2.19
(br s, 1 H), 2.24 (dd, J = 13.2, 8.6 Hz, 1 H), 2.60 (br s, 2 H), 2.65
(dd, J = 13.2, 4.3 Hz, 1 H), 2.83 (m, 1 H), 3.60–3.75 (m, 2 H), 3.88
(ddd, J = 8.6, 8.6, 4.3 Hz, 1 H), 4.36 (d, J = 2.7 Hz, 1 H), 4.50 (dd,
J = 11.5, 1.5 Hz, 1 H), 4.97 (d, J = 1.7 Hz, 1 H), 5.02 (s, 1 H), 5.27
(d, J = 1.7 Hz, 1 H), 5.94 (s, 1 H), 6.00 (d, J = 11.4 Hz, 1 H), 6.37
(d, J = 11.4 Hz, 1 H).
13C NMR (100 MHz, CDCl3): d = 12.2, 14.9, 16.9, 18.4, 22.3, 22.8,
23.5, 26.4, 27.7, 29.0, 30.1, 32.6, 39.8, 40.6, 44.3, 46.0, 49.0, 56.3,
56.9, 62.6, 70.2, 73.5, 79.5, 113.5, 113.7, 117.2, 124.4, 133.1,
141.1, 142.4, 146.4, 169.8.
1H NMR (400 MHz, CDCl3): d = 0.56 (s, 3 H), 0.82–1.05 (m, 4 H),
0.97 (d, J = 6.4 Hz, 3 H), 1.15 (m, 1 H), 1.20–1.35 (m, 3 H), 1.40–
2.06 (m, 12 H), 2.23 (dd, J = 13.2, 8.7 Hz, 1 H), 2.67 (dd, J = 13.2,
4.5 Hz, 1 H), 2.81 (br s, 3 H), 2.84 (m, 1 H), 3.37 (dd, J = 7.5, 2.9
Hz, 1 H), 3.73–3.92 (m, 4 H), 4.06 (ddd, J = 8.7, 7.5, 4.5 Hz, 1 H),
4.45 (d, J = 2.9 Hz, 1 H), 4.51 (br d, J = 10.7 Hz, 1 H), 5.02 (s, 1
H), 5.08 (s, 1 H), 5.38 (s, 1 H), 5.94 (s, 1 H), 6.01 (d, J = 11.2 Hz, 1
H), 6.40 (d, J = 11.2 Hz, 1 H).
13C NMR (100 MHz, CDCl3): d = 12.2, 14.9, 16.9, 18.4, 22.2, 23.5,
26.5, 27.6, 29.0, 31.9, 32.6, 39.8, 40.5, 41.0, 46.0, 56.3, 57.0, 61.0,
68.3, 68.4, 71.8, 79.5, 84.4, 113.7, 116.1, 117.3, 125.2, 131.8,
141.1, 142.8, 144.2, 169.8.
EI-LRMS: m/z = 526 (M+), 508, 490, 464, 432, 265, 171.
EI-HRMS: m/z calcd for C32H46O6: 526.3294; found: 526.3291.
Vitamin D Receptor (VDR) Binding Assay
[26,27-Methyl-3H]-1a,25-dihydroxyvitamin D3 (specific activity
6.623 TBq/mmol, 15,000 dpm, 15.7 pg) and various amounts of
1a,25-dihydroxyvitamin D3 and the analogue to be tested were dis-
solved in absolute EtOH (50 mL) in 12 × 75-mm polypropylene
tubes. The chick intestinal VDR (0.2 mg) and gelatin (1 mg) in aq
phosphate buffer solution (1 mL; 25 nM KH2PO4, 0.1 M KCl, and
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 °C and then chilled in an ice bath. Polypropylene glycol 6000
EI-LRMS: m/z = 510 (M+), 492, 474, 415, 327, 309.
EI-HRMS: m/z calcd for C32H46O5: 510.3345; found: 510.3344.
Synthesis 2005, No. 15, 2533–2543 © Thieme Stuttgart · New York

2542
N. Saito et al.
PAPER
(6) (a) Suhara, Y.; Nihei, K.; Tanigawa, H.; Fujishima, T.;
(40%; 1 mL) in distilled H2O was added to each tube, which was
mixed vigorously and centrifuged (2260 × g) for 60 min at 4 °C. Af-
ter the supernatant was decanted, the bottom of the tube containing
the pellet was cut off into a scintillation vial containing 10 mL of di-
oxane-based scintillation fluid and the radioactivity was measured
with a Beckman liquid scintillation counter (Model LS6500). The
relative potency of the analogue was calculated from the concentra-
tion needed to displace 50% of [26,27-methyl-3H]-1a,25-dihydroxy-
vitamin D3 from the receptor compared with the activity of 1a,25-
dihydroxyvitamin D3 (assigned a 100% value).
Konno, K.; Nakagawa, K.; Okano, T.; Takayama, H. Bioorg.
Med. Chem. Lett. 2000, 10, 1129. (b) Suhara, Y.; Nihei, K.;
Kurihara, M.; Kittaka, A.; Yamaguchi, K.; Fujishima, T.;
Konno, K.; Miyata, N.; Takayama, H. J. Org. Chem. 2001,
66, 8760.
(7) (a) Kittaka, A.; Suhara, Y.; Takayanagi, H.; Fujishima, T.;
Kurihara, M.; Takayama, H. Org. Lett. 2000, 2, 2619.
(b) Saito, N.; Suhara, Y.; Kurihara, M.; Fujishima, T.;
Honzawa, S.; Takayanagi, H.; Kozono, T.; Matsumoto, M.;
Ohmori, M.; Miyata, N.; Takayama, H.; Kittaka, A. J. Org.
Chem. 2004, 69, 7463.
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. Exponen-
tially proliferating cells were collected, suspended in fresh medium
and seeded in culture plates (Falcon, Becton Dickinson and Compa-
ny, Franklin Lakes, NJ). The cell concentration at seeding was ad-
justed to 2 × 104 cells/mL and the seeding volume was 1 mL/well.
An EtOH solution of 1a,25-dihydroxyvitamin D3 (final concentra-
tion: 10–8 M) and an analogue (final concentration: 10–11 to 10–6 M)
was added to the culture medium at 0.1% volume and culture was
continued for 96 h at 37 °C in a humidified atmosphere of 5% CO2–
air without a change of medium. The same amount of vehicle was
added to the control culture. The NBT-reducing assay was per-
formed according to the method of Collins.23 Briefly, cells were col-
lected, washed with PBS, and suspended in serum-free medium.
NBT–TPA solution (dissolved in PBS) was added. Final concentra-
tions of NBT and TPA were 0.1% and 100 ng/mL, respectively.
Then, the cell suspensions were incubated at 37 °C 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.
(8) For our account, see: Takayama, H.; Kittaka, A.; Fujishima,
T.; Suhara, Y. Vitamin D Analogs in Cancer Prevention and
Therapy, Recent Results in Cancer Research, Vol. 164;
Reichrath, J.; Friedrich, M.; Tilgen, W., Eds.; Springer-
Verlag: Berlin, 2003, 289.
(9) This concept was also good for 1a,25-dihydroxy-19-
norvitamin D3, see: (a) Ono, K.; Yoshida, A.; Saito, N.;
Fujishima, T.; Honzawa, S.; Suhara, Y.; Kishimoto, S.;
Sugiura, T.; Waku, K.; Takayama, H.; Kittaka, A. J. Org.
Chem. 2003, 68, 7407. (b) Yoshida, A.; Ono, K.; Suhara, Y.;
Saito, N.; Takayama, H.; Kittaka, A. Synlett 2003, 1175.
(10) (a) Miura, D.; Manabe, K.; Ozono, K.; Saito, M.; Gao, Q.;
Norman, A. W.; Ishizuka, S. J. Biol. Chem. 1999, 274,
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Vitamin D: Molecular, Cellular and Chemical
Acknowledgment
N.S. acknowledges the Takeda Science Foundation for financial
support. The authors thank Miss J. Shimode and Miss A. Tonoki
(Teikyo University) for spectroscopic measurements. This study
was supported by Grants-in-aid from the Ministry of Education,
Culture, Sports, Science and Technology, Japan, Japan Society of
Promotion of Science, and by Uehara Memorial Foundation.
Endocrinology; Norman, A. W.; Schaefer, K.; Grigoleit, H.
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143.
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A.; Gonzalez, M. M.; Ruf, D.; Steinmeyer, A.; Ishizuka, S.;
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Ethanovitamin D3 Lactones with Enyne Metathesis
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Synthesis 2005, No. 15, 2533–2543 © Thieme Stuttgart · New York