Efficient synthesis and biological evaluation of all A-ring diastereomers of 1α,25-dihydroxyvitamin D3 and its 20-epimer

Article abstract of DOI:10.1016/S0968-0896(99)00262-X

An improved synthesis of the diastereomers of 1α,25-dihydroxyvitamin D₃ (1) was accomplished utilizing our practical route to the A-ring synthon. We applied this procedure to synthesize for the first time all possible A- ring diastereomers of 20-epi-1α,25-dihydroxyvitamin D₃ (2). Ten-step conversion of 1-(4-methoxyphenoxy)but-3-ene (6), including enantiomeric introduction of the C-3 hydroxyl group to the olefin by the Sharpless asymmetric dihydroxylation, provided all four possible stereoisomers of A- ring enynes (3), i.e., (3R,5R)-, (3R,5S)-, (3S,5R)- and (3S,5S)-bis[(tert- butyldimethylsilyl)oxy]oct-1-en-7-yne, in good overall yield. Palladium- catalyzed cross-coupling of the A-ring synthon with the 20-epi CD-ring portion (5), (E)-(20S)-de-A,B-8-(bromomethylene)cholestan-25-ol, followed by deprotection, afforded the requisite diastereomers of 20-epi-1α,25- dihydroxyvitamin D₃ (2). The biological profiles of the synthesized stereoisomers were assessed in terms of affinities for vitamin D receptor (VDR) and vitamin D binding protein (DBP), HL-60 cell differentiation- inducing activity and in vivo calcium-regulating potency in comparison with the natural hormone. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(99)00262-X

Bioorganic & Medicinal Chemistry 8 (2000) 123±134
Ecient Synthesis and Biological Evaluation of All A-Ring
Diastereomers of 1ꢀ,25-Dihydroxyvitamin D3 and its 20-Epimer
Toshie Fujishima, a Katsuhiro Konno, a Kimie Nakagawa, b Mayuko Kurobe, b
Toshio Okano b and Hiroaki Takayama a,*
aFaculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa 199-0195, Japan
bDepartment of Hygienic Sciences, Kobe Pharmaceutical University, Kobe 658-8558, Japan
Received 30 July 1999; accepted 3 September 1999
AbstractÐAn improved synthesis of the diastereomers of 1a,25-dihydroxyvitamin D3 (1) was accomplished utilizing our practical
route to the A-ring synthon. We applied this procedure to synthesize for the ®rst time all possible A-ring diastereomers of 20-epi-
1a,25-dihydroxyvitamin D3 (2). Ten-step conversion of 1-(4-methoxyphenoxy)but-3-ene (6), including enantiomeric introduction of
the C-3 hydroxyl group to the ole®n by the Sharpless asymmetric dihydroxylation, provided all four possible stereoisomers of A-
ring enynes (3), i.e., (3R,5R)-, (3R,5S)-, (3S,5R)- and (3S,5S)-bis[(tert-butyldimethylsilyl)oxy]oct-1-en-7-yne, in good overall yield.
Palladium-catalyzed cross-coupling of the A-ring synthon with the 20-epi CD-ring portion (5), (E)-(20S)-de-A,B-8-(bromomethy-
lene)cholestan-25-ol, followed by deprotection, a€orded the requisite diastereomers of 20-epi-1a,25-dihydroxyvitamin D3 (2). The
biological pro®les of the synthesized stereoisomers were assessed in terms of anities for vitamin D receptor (VDR) and vitamin D
binding protein (DBP), HL-60 cell di€erentiation-inducing activity and in vivo calcium-regulating potency in comparison with the
natural hormone. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
synthesis. The orientations of the two hydroxyl groups
on the A-ring in the natural hormone are 1a and 3b (the
vitamin D numbering system was used). The other three
diastereomers on the A-ring moiety are depicted in
Chart 1. The 1-epimer of 1a,25-dihydroxyvitamin D3,
i.e., the (20R)-(1b,3b) isomer, was shown to be an
antagonist of nongenomic, but not genomic, actions by
Norman and co-workers.5 Most recently, the 3-epimer
of 1, i.e., the (20R)-(1a,3a) isomer, was shown to be
produced in the vitamin D metabolic pathway in certain
cell lines, though its functions remain unknown.6±8
Therefore, studies on A-ring diastereomers with all
possible hydroxyl con®gurations, as well as on 1a,25-
dihydroxyvitamin D3 itself have become of interest in
vitamin D research.
The biologically active form of vitamin D, 1a,25-dihy-
droxyvitamin D3 (1), plays a major role in calcium±
phosphorus homeostasis, and its functions, such as cell
di€erentiation-inducing and antiproliferative activities,
attracted us to explore analogues which might have
therapeutic potential for cancer, psoriasis and osteo-
porosis.1,2 In order to investigate the structure±function
relationships of this hormone, a number of analogues
have been synthesized and biologically evaluated. For
reasons of synthetic convenience and for metabolic stu-
dies of its inactivation process, most of the analogues
synthesized so far have been altered in the side chain.
Among them, 20-epi-1a,25-dihydroxyvitamin D3 (2) is
noteworthy due to its high activity in cell di€erentiation
with relatively low calcemic e€ects, having a clearly dif-
ferent biological pro®le from its parent hormone.3,4
Convergent synthesis can be more e€ective and ¯exible
for the synthesis of a variety of analogues than the
classical steroidal approach.9,10 In particular, Trost's
convergent method11 using palladium-catalyzed cou-
pling of the A-ring enyne synthon (A) with the CD-ring
portion (4) seems most useful (Scheme 1). We applied
this procedure to an improved synthesis of the dia-
stereomers of 1a,25-dihydroxyvitamin D3.12 We also
disclose herein the ®rst synthesis of all four possible
A-ring diastereomers of 20-epi-1a,25-dihydroxyvitamin D3
The A-ring moiety of 1, which bears two hydroxyl
groups on the six-membered ring, is of interest in con-
nection with metabolic studies as well as analogue
Keywords: vitamins; hormones; receptors; antiproliferative agents.
*Corresponding author. Tel.: +81-426-85-3713; fax: +81-426-85-
3714.
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00262-X

124
T. Fujishima et al. / Bioorg. Med. Chem. 8 (2000) 123±134
Scheme 1.
Chart 1.
(2) using the 20-epi CD-ring (5) instead of 4, and their
biological activities in comparison with those of the 20-
natural counterparts.
PHAL as a ligand instead resulted in comparable yield
with slightly better enantioselectivity.17 Thus, treatment
of p-anisyl ether 6 with AD-mix-a in t-BuOH:H2O (1:1)
at 0 ꢀC for 3 h a€orded the diol 7a of the desired 3(S)-
con®guration in 95% yield with 93% ee. This was
recrystallized from CH2Cl2-n-hexane to yield an enan-
tiomerically pure specimen. The enantiopurity and the
absolute con®guration were determined by 1H NMR
analyses of the MTPA esters of the mono-tosylate 8a
(Chart 2).18 The primary alcohol of 7a was converted in
94% yield to the tosylate 8a, which upon treatment with
lithium hexamethyldisilazide a€orded the epoxide 9a in
84% yield. The acetylene unit was introduced by the
reaction of 9a with lithium acetylide±ethylenediamine
complex in DMSO to give the alcohol 10a in 86% yield.
Protection of the resulting secondary alcohol of 10a
with a benzyloxymethyl (BOM) group a€orded 11a in
90% yield, and deprotection of the p-anisyl group with
cerium (IV) diammonium nitrate (CAN) furnished the
primary alcohol 12a in quantitative yield. Introduction
of a vinyl group into the aldehyde, obtained from 12a
with PDC in 96% yield, proceeded smoothly to give the
allyl alcohols 13aA and 13aS in 62% yield as an
approximately 1:1 mixture of the diastereomers. These
isomers were readily separable by silica gel column
Synthesis
The retrosynthetic analysis is shown in Scheme 1.
Introduction of an acetylene unit and a vinyl group into
the chiral epoxy-aldehyde precursor (B) would lead to
the desired A-ring enyne (A). Therefore, the 3-buten-1-ol
derivative (C) was chosen as a starting material. The
20-natural CD-ring portion (4) can be prepared from
vitamin D3 by known methods,11 while the 20-epi CD-
ring portion (5) can be obtained according to our pro-
cedure, a part of which was reported in ref 13.
The synthetic route to the A-ring enynes (3aA, 3aS) is
outlined in Scheme 2.14 Introduction of the C-3 hy-
droxyl group to 6 was accomplished by the Sharpless
asymmetric dihydroxylation (AD). Corey and co-workers
reported that, in the AD reaction of 3-buten-1-ol
derivatives, the combination of the chiral ligand
(DHQD)2PYDZ and the p-anisyl protecting group at
the primary alcohol gave high enantioselectivity (91%
ee).15,16 We found, however, that the use of (DHQ)2

T. Fujishima et al. / Bioorg. Med. Chem. 8 (2000) 123±134
125
Scheme 2. (a) AD-mix-a/ t-BuOH-H2O, 0 ꢀC, 95%; (b) TsCl/pyridine, 0 ꢀC, 94%; (c) LiHMDS/THF, 0 ꢀC, 84%; (d) Lithium acetylide-EDA/
DMSO, r.t., 86%; (e) BOMCl, DIPEA/CH2Cl2, r.t., 90%; (f) CAN/MeCN-H2O, 0 ꢀC, 99%; (g) PDC/CH2Cl2, r.t., 96%; (h) VinylMgBr/toluene,
78 ꢀC, 62%; (i) PhSH-BF3.Et2O/CH2Cl2, 0 ꢀC, 86%; (j) TBSOTf, 2,6-Lutidine/CH2Cl2, 0 ꢀC. 99%.
synthesized all four possible stereoisomers of A-ring
enyne synthon in good overall yield, 29% for 10 steps,
starting from 6.
The 20-epi CD-ring portion (5) was synthesized as illu-
strated in Scheme 4.13 The aldehyde 1520 was equili-
brated under basic conditions to give an approximately
Chart 2.
2:3 mixture of the aldehydes in favor of the 20-epimer.
chromatography and the C-1 con®guration of both iso-
mers was determined by 1H NMR analyses of their
Reduction of this aldehyde mixture with NaBH4 a€or-
ded the corresponding C-20 epimeric alcohols, which
were then separated by chromatography to obtain the
desired 20-epi alcohol 16 in 52% yield. Tosylation of the
primary alcohol 16 a€orded 17 in 98% yield, and
treatment with NaI gave the iodide 18 in 92% yield.
Condensation of 18 with the side chain moiety 1921
using n-BuLi as a base in the presence of HMPA furn-
ished a mixture of C-23 epimeric sulfones 20 in 98%
yield. Desulfonylation with sodium amalgam in a buf-
fered mixture of methanol and THF22 produced the
desired CD-ring portion 21. Removal of both protecting
groups in 21 with TsOH a€orded the corresponding diol
22 in high yield. The resulting secondary alcohol was
oxidized with TPAP-NMO23 to give the ketone 23 in
96% yield. Finally, bromomethylenation of 23 furn-
ished the requisite 20-epi CD-ring synthon 5.
MTPA esters (Chart 3).18 Removal of the BOM pro-
19
.
tecting group by PhSH-BF3 OEt2 a€orded the desired
enynes 14aA and 14aS, from 13aA and 13aS, respec-
tively. Finally, the diols (14aA, 14aS) were protected
using TBSOTf in 99% yield to give the requisite A-ring
enyne synthons (3aA, 3aS). Oxidation of 6 using AD-
mix-b instead of AD-mix-a gave the diol 7b in 94%
yield with 93% ee, which led to the enantiomers 3bA
and 3bS, corresponding to 3aA and 3aS, respectively,
in the same way as above (Scheme 3). Thus, we have
Chart 3.
Scheme 3.

126
T. Fujishima et al. / Bioorg. Med. Chem. 8 (2000) 123±134
Scheme 4. (a) DBU/THF, re¯ux; (b) NaBH4/MeOH, 0 ꢀC, 52% (two steps); (c) TsCl/pyridine, r.t., 98%; (d) Nal/DMF, 50 ꢀC, 92%; (e) n-BuLi,
HMPA/THF, 78 ꢀC, 98%; (f) Na-Hg/Na2HPO4-MeOH-THF, r.t., 98%; (g) TsOH/MeOH, r.t., 85%; (h) TPAP-NMO-4A M.S./CH2Cl2, 96%; (i)
Ph3P+CH2Br Br , NaHMDS/THF, 57%.
.
The coupling reaction of the disilyl ether (3aA) with the
20-epi CD-ring portion (5) catalyzed by Pd2(dba)3-
PPh3-Et3N in toluene at 120 ꢀC for 2 h, followed by
deprotection with camphorsulfonic acid (CSA) in
MeOH gave the (20S)-(1a,3b) isomer (2) in 52% yield.11
The same treatment of 3aS, 3bS and 3bA a€orded the
other diastereomers, (20S)-(1b,3b), (20S)-(1a,3a) and
(20S)-(1b,3a), respectively. Use of the CD-ring part
(4)11 instead of 5 with the A-ring enyne (3) in the same
way gave 1a,25-dihydroxyvitamin D3 (1) and the other
three diastereomers, (20R)-(1b,3b), (20R)-(1a,3a) and
(20R)-(1b,3a).24 In this way, the two sets of all four
possible A-ring diastereomers of 1a,25-dihydroxy-
vitamin D3 (1) and its 20-epimer (2) were synthesized.
(VDR) binding assay using bovine thymus VDR,25
(20S)-(1a,3b) (2) exhibited 4-fold higher anity than
1a,25-dihydroxyvitamin D3. The VDR binding potency
of 2 relative to that of 1a,25-dihydroxyvitamin D3 (1)
(normalized to 100) was reported to be 120 for chick
intestinal VDR3 and 500 for bovine thymus VDR.4 Our
results were consistent with this estimation. The (20S)-
(1a,3a) isomer, the 3-epimer of 2, showed rather high
anity to VDR with half the potency of the natural
hormone 1. The (20S)-(1b,3b) and (20S)-(1b,3a) isomers
exhibited much lower anity for VDR, implying that
1b-hydroxyl orientation greatly decreases the potency
irrespective of the con®guration of the C-3 hydroxyl
group and the C-20 stereochemistry.
The anity of vitamin D binding protein (DBP) was
tested using rat serum DBP.26 Each 20-epi analogue
showed lower anity than its 20-natural counterpart.
The range of potencies among the isomers was narrower
Biological evaluation
The biological activities of the synthesized analogues are
summarized in Table 1. In the vitamin D receptor
Table 1. Biological activities of A-ring diastereomers of 1a,25-dihydroxyvitamin D3 (1) and 20-epi-1a,25-dihydroxyvitamin D3 (2)a
VDRb binding
DBPc binding
HL-60 celld
Ca mobilizatione
(20S)-(1a,3b)(2)
(20S)-(1a,3a)
(20S)-(1b,3b)
(20S)-(1b,3a)
(20R)-(1a,3b)(1)
(20R)-(1a,3a)
(20R)-(1b,3b)
(20R)-(1b,3a)
400
50
0.3
0.3
<0.1
3571
208
<10
<10
100
<10
<10
<10
360
110
NDf
ND
100
NTi
NT
NT
5
15
26
100
100
(24)g
(0.2)g
(0.8)g
45 (800)h
243 (450)h
410 (6570)h
aThe potencies of 1a,25-dihydroxyvitamin D3 (1) are taken as 100.
bBovine thymus.
cRat serum.
dCell di€erentiation was assessed in terms of expression of antigen CD11b.
eRat serum calcium level.
fNot detected.
gRef 5, chick intestinal.
hRef 27, human DBP.
iNot tested.

T. Fujishima et al. / Bioorg. Med. Chem. 8 (2000) 123±134
127
than the results using human DBP reported by Norman
et al.27 However, as they reported,27 the A-ring con®g-
uration of (1b,3a) was preferred, independent of the C-
20 stereochemistry.
VDR binding, HL-60 cell di€erentiation and in vivo
calcemic activity. Each 20-epi analogue with a 1b-hy-
droxyl group exhibited weak activity similar to that of
its 20-natural counterpart, implying that 1b-hydroxyl
orientation greatly decreases the potency irrespective of
the con®guration of the C-3 hydroxyl group and the
C-20 stereochemistry. In contrast, the DBP binding
potency of each analogue appeared to be reduced by the
20-epimerization independently of the A-ring stereo-
chemistry. These results shed some light on the struc-
ture±activity relationships of the A-ring and the side
chain. Some of these compounds would make useful
tools in research on the biology of vitamin D.
Cell di€erentiation-inducing activity of (20S)-(1a,3b) (2)
towards HL-60 cells28 was 36 times higher than that of
the natural hormone (1). The potency of 2 towards
U-937 cells was reported to be 27-fold higher than that
of 1,3 which is in reasonable agreement with our result
considering the di€erence in cell lines. The 3-epimer of
2, i.e., (20S)-(1a,3a), was approximately 2.1 times more
active than 1. Thus, the 3-epimerization of 2 resulted in
approximately 17-fold reduction in cell di€erentiation-
inducing activity, which is high compared with the
8-fold reduction in the VDR anity. Those results
might be explained by a di€erence in DBP binding,
which is believed to correlate to availability of the com-
pounds to target cells. The 1b-isomers, as well as (20R)-
(1a,3a) showed virtually no cell-di€erentiating activity
even at the concentration of 10 8 M.
Experimental
Melting points were determined by using a Yanagimoto
hot-stage melting point apparatus and are uncorrected.
NMR spectra were recorded on a JEOL GSX-400
spectrometer. Chemical shifts are expressed in ppm
relative to tetramethylsilane. Mass spectra and high-
resolution mass spectra (HRMS) were recorded on a
JMS-SX 102A. Fast atom bombardment mass spectra
(FAB±MS) were recorded with 3-nitrobenzyl alcohol
(NBA) as a matrix. Infrared spectra were recorded on a
Jasco FT/IR-8000 spectrometer and are expressed in
cm 1. Ultraviolet spectra were recorded with a Hitachi
200-10 spectrophotometer. Optical rotations were
determined by using a Jasco DIP-370 digital polari-
meter. Elemental analyses were carried out in the
Microanalytical Laboratory, Faculty of Pharmaceutical
Sciences, University of Tokyo, and were within 0.3% of
the theoretical values.
In vivo calcium-regulating activities of the diastereo-
mers were tested using normal SD male rats.29 The
(20S)-(1a,3b) (2) and (20S)-(1a,3a) compounds (single
dose) showed similar time courses with peak calcemic
activity at 48 h after administration, while the natural
hormone (1) showed the peak activity at 24 h under the
same experimental conditions (data not shown). The
potencies, based on the ability to elevate serum calcium
level by 1 mg/dL, were calculated at the time when the
e€ects were maximal. The e€ective concentrations of
(20S)-(1a,3b) (2), (20S)-(1a,3a) and (20R)-(1a,3b) (1)
were 0.8, 2.7 and 3.0 mg/kg, respectively, and Table 1
shows the data as relative potencies with respect to 1,
de®ned as 100. The 20-epi modi®cation of 1 increased
the calcemic activity concomitantly with the VDR
binding potency. However, in contrast to the 36-fold
di€erence of relative potency of 2 to 1 in cell di€er-
entiation, the 20-epimerization had much less e€ect on
the calcemic activity (3.6-fold). The (20S)-(1a,3a) com-
pound, the 3-epimer of 2, exhibited similar calcemic
activity to 1a,25-dihydroxyvitamin D3. Thus, alteration
in both C-3 and C-20 stereochemistry retained calcemic
and cell-di€erentiating activities similar to those of the
natural hormone. These results imply that the 3-epi-
merization of 2 would produce the (20S)-(1a,3a) com-
pound with relatively high calcium-regulating potency
but much less cell di€erentiation activity than the parent
compound 2. It is of interest to know whether 3-epi-
merization is involved in the metabolic pathway of 2 or
not.6±8 On the other hand, the 1b-isomers showed no
serum calcium-elevating activity under these conditions.
1-(4-Methoxyphenoxy)but-3-ene (6).15,16 1H NMR
(400 MHz, CDCl3) d 2.52 (2H, qt, J=6.7, 1.2 Hz), 3.77
(3H, s), 3.97 (2H, t, J=6.7 Hz), 5.10 (1H, dq, J=10.0,
1.2 Hz), 5.16 (1H, dq, J=17.0, 1.2 Hz), 5.99 (1H, ddt,
J=17.0, 10.0, 6.7 Hz), 6.83 (4H, m); 13C NMR
(100 MHz, CDCl3) d 33.7 (t), 55.7 (q), 68.0 (t), 114.7 (d),
115.7 (d), 116.9 (t), 134.7 (d), 153.2 (s), 153.9 (s); MS
178 [M]+, 124 [CH3OC6H4OH]+; HRMS calcd. for
[C21H24O4] 178.0994, found 178.0995.
(S)-4-(4-Methoxyphenoxy)butane-1,2-diol (7a). A mix-
ture of AD-mix-a (Aldrich, 1.4 g) in t-BuOH (5 mL) and
H2O (5 mL) was cooled to 0 ꢀC. The resulting suspension
was treated with the ole®n 6 (178 mg, 1.0 mmol), then
stirred for 3 h, and quenched by addition of sodium
sul®te (1.5 g, 6.0 mmol). The mixture was stirred for
30 min at room temperature, and extracted with ethyl
acetate. The organic layer was washed with brine, dried
over magnesium sulfate and concentrated. The residue
was puri®ed by silica gel column chromatography (ethyl
acetate:n-hexane=4:1) to a€ord the desired diol
(202 mg) as a solid in 95% yield with 93% ee. It was
recrystallized from CH2Cl2:n-hexane to yield optically
pure 7a as colorless ®ne needles.
In summary, we have synthesized two sets of all four
possible A-ring diastereomers of 1a,25-dihydroxy-
vitamin D3 (1) and its 20-epimer (2) by employing the
convergent method using palladium catalyst. The above
procedure should be versatile for the synthesis of a
variety of A-ring analogues, as well as those with CD-
ring and/or side chain modi®cations. Biological evalua-
tion revealed that the 20-epimerization of the analogues
with a 1a-hydroxyl group increased the potencies of
Mp 64 ꢀC (recry. from CH2Cl2-n-hexane); [a]2d6 5.2
1
(c=1.35, CHCl3); H NMR (400 MHz, CDCl3) d 1.95
(2H, m), 2.61 (1H, d, J=4.7 Hz), 3.56 (1H, m), 3.72

128
T. Fujishima et al. / Bioorg. Med. Chem. 8 (2000) 123±134
(1H, ddd, J=11.0, 6.6, 3.7 Hz), 3.77 (3H, s), 4.01 (1H,
m), 4.12 (2H, m), 6.84 (4H, m); 13C NMR (100 MHz,
CDCl3) d 32.7 (t), 55.7 (q), 66.1 (t), 66.7 (t), 70.4 (d),
3.3 mmol) at 0 ꢀC under argon. The mixture was stirred
for 1 h at 0 ꢀC, then poured into satd NH4Cl aq and
extracted with ether. The organic layer was washed with
brine, dried over magnesium sulfate, ®ltered and con-
centrated. The crude mixture was puri®ed by silica gel
column chromatography (ethyl acetate:n-hexane=1:4)
to give 9a (443 mg) as a colorless oil in 84% yield.
114.7 (d), 115.5 (d), 152.6 (s), 154.1 (s); FTIR (Nujol)
1
3252, 1215, 1290, 1242, 1116, 1072, 1032, 993, 966 cm
;
MS 212 [M]+, 124 [CH3OC6H4OH]+; HRMS calcd. for
[C11H16O4] 212.1049, found 212.1049. Anal. calcd for
C11H16O4: C, 62.25; H, 7.60. Found: C, 61.97; H, 7.48.
[a]2d5 13.6 (c=1.65, CHCl3); 1H NMR (400 MHz,
CDCl3) d 1.92 (1H, dq, J=14.0, 5.8 Hz), 2.08 (1H, m),
2.57 (1H, dd, J=4.9, 2.8 Hz), 2.82 (1H, dd, J=4.9,
4.3 Hz), 3.15 (1H, m), 3.77 (3H, s), 4.07 (2H, m), 6.84
(4H, m); 13C NMR (100 MHz, CDCl3) d 32.4 (t), 47.1
(t), 49.8 (d), 55.7 (q), 65.3 (t), 114.7 (d), 115.5 (d), 152.9
(s), 154.0 (s); FTIR (neat) 1232 cm 1; MS 194 [M]+, 124
[CH3OC6H4OH]+; HRMS calcd for [C11H14O3]
194.0943, found 194.0941.
(R)-4-(4-Methoxyphenoxy)butane-1,2-diol (7b). This
compound was obtained by the same procedure as
described for 7a using AD-mix-b instead of AD-mix-a.
Mp 64 ꢀC (recry. from ethyl acetate:n-hexane); [a]2d6
+5.4 (c=1.15, CHCl3).
(S)-2-Hydroxy-4-(4-methoxyphenoxy)butan-1-ol p-toluene-
sulfonate (8a). To a solution of 7a (200 mg, 0.94 mmol)
in pyridine (3mL) was added TsCl (216mg, 1.1 mmol) at
0 ꢀC under argon. The reaction mixture was stirred at 0 ꢀC
for 22h, then poured into water and extracted with ether.
The organic layer was washed with 2 N HCl aq and brine,
dried over magnesium sulfate and concentrated. The
residue was puri®ed by silica gel column chromatography
(ethyl acetate:n-hexane=1:2) to give 8a (324 mg) in 94%
as a solid, which was recrystallized from ethyl acetate:n-
hexane to give analytically pure 8a as colorless needles.
(R)-4-(4-Methoxyphenoxy)-1,2-epoxybutane (9b). [a]d25
+15.6 (c=1.35, CHCl3).
(R)-1-(4-Methoxyphenoxy)hex-5-yn-3-ol (10a). To
a
solution of 9a (250 mg, 1.3 mmol) in DMSO (3 mL) was
added with stirring lithium acetylide ethylenediamine
complex (175 mg, 1.9 mmol) at room temperature. The
mixture was stirred for 30 min, then diluted with water
and extracted with ether. The organic layer was washed
with brine, and dried over magnesium sulfate and con-
centrated. The residue was puri®ed by silica gel column
chromatography (ethyl acetate:n-hexane=1:3) to give
10a (245 mg) in 86% as a solid, which was recrystallized
from CH2Cl2:n-hexane to give analytically pure 10a as
colorless ®ne needles.
Mp 70 ꢀC (recry. from ethyl acetate:n-hexane); [a]2d6
1
+2.4 (c=1.00, CHCl3); H NMR (400 MHz, CDCl3) d
1.91 (2H, m), 2.52 (1H, d, J=4.0 Hz), 3.77 (3H, s), 3.99±
4.13 (4H, m), 4.15 (1H, m), 6.80 (4H, m), 7.35 (2H, d,
J=8.5 Hz), 7.81 (2H, dt, J=8.5, 1.8 Hz); 13C NMR
(100 MHz, CDCl3) d 21.7 (q), 32.4 (t), 55.8 (q), 65.3 (t),
67.6 (d), 73.6 (t), 114.7 (d), 115.5 (d), 128.0 (d), 130.0
(d), 132.7 (s), 145.1 (s), 152.6 (s), 154.1 (s); FTIR
(Nujol) 3547, 2725, 1714, 1508, 1350, 1290, 1226, 1172,
1124, 1039, 962 cm 1; MS 366 [M]+; HRMS calcd. for
[C18H22O6S] 366.1137, found 366.1137. Anal. calcd for
C18H22O6S: C, 59.00; H, 6.05. Found: C, 58.71; H, 6.12.
Mp 45 ꢀC (recry. from CH2Cl2:n-hexane); [a]2d7 9.8
1
(c=1.18, CHCl3); H NMR (400 MHz, CDCl3) d 1.95±
2.10 (2H, m), 2.07 (1H, t, J=2.8 Hz), 2.44 (1H, ddd,
J=16.0, 6.1, 2.8 Hz), 2.50 (1H, ddd, J=16.0, 6.1,
2.8 Hz), 2.54 (1H, d, J=4.3 Hz), 3.77 (3H, s), 4.08 (2H,
m), 4.15 (1H, ddd, J=9.5, 7.3, 4.9 Hz), 6.84 (4H, m);
13C NMR (100 MHz, CDCl3) d 27.3 (t), 35.4 (t), 55.8
(q), 66.2 (t), 68.2 (d), 70.9 (d), 80.7 (s), 114.7 (d), 115.5
(d), 152.8 (s), 154.1 (s); FTIR (neat) 3919, 3422, 2932,
2118, 2056, 1508, 1290, 1230 cm 1; MS 220 [M]+, 124
[CH3OC6H4OH]+; HRMS calcd. for [C13H16O3]
220.1100, found 220.1099. Anal. calcd for C13H18O3: C,
70.89; H, 7.32. Found: C, 70.77; H, 7.32.
1
(S)-MTPA ester of 8a. H NMR (400 MHz, CDCl3) d
2.041 (2H, m), 2.440 (3H, s), 3.505 (3H, s), 3.631 (1H,
ddd, J=9.5, 8.2, 4.6 Hz), 3.773 (3H, s), 3.783 (1H, m),
4.176 (1H, dd, J=11.3, 6.1 Hz), 4.348 (1H, dd, J=11.3,
2.6 Hz), 5.525 (1H, m), 6.668 (2H, m), 6.800 (2H, m),
7.253±7.376 (5H, m), 7.461 (2H, d, J=7.9 Hz), 7.753
(2H, d, J=8.5 Hz).
1
(R)-MTPA ester of 8a. H NMR (400 MHz, CDCl3) d
(S)-1-(4-Methoxyphenoxy)hex-5-yn-3-ol (10b). Mp 44 ꢀC
(recry. from CH2Cl2:n-hexane; [a]2d6 +10.6 (c=0.97,
CHCl3).
2.132 (2H, m), 2.434 (3H, s), 3.411 (3H, s), 3.773 (3H, s),
3.863 (1H, ddd, J=9.5, 7.6, 5.2 Hz), 3.937 (1H, m),
4.146 (1H, dd, J=11.0, 5.8 Hz), 4.296 (1H, dd, J=11.0,
3.4 Hz), 5.526 (1H, m), 6.743 (2H, m), 6.818 (2H, m),
7.258±7.394 (5H, m), 7.432 (2H, d, J=8.1 Hz), 7.708
(2H, d, J=8.5 Hz).
(R)-3-[(Benzyloxymethyl)oxy]-1-(4-methoxyphenoxy)hex-
5-yne (11a). To a mixture of 10a (100 mg, 0.45 mmol) and
diisopropylethylamine (0.32 mL, 1.8 mmol) in CH2Cl2
(2 mL) was added with stirring BOMCl (213 mg,
1.4 mmol) under argon at room temperature. The reaction
mixture was stirred for 16 h, and diluted with CH2Cl2.
The organic layer was washed with 1 N HCl aq, dried
over magnesium sulfate and ®ltered. Evaporation of the
®ltrate a€orded a residue, from which 11a (138 mg) was
separated by silica gel column chromatography (ethyl
acetate:n-hexane=1:3) in 90% as a colorless oil.
(R)-2-Hydroxy-4-(4-methoxyphenoxy)butan-1-ol p-toluene-
sulfonate (8b). Mp 71 ꢀC (recry. from ethyl acetate:n-
hexane); [a]2d7 +2.1 (c=1.15, CHCl3).
(S)-4-(4-Methoxyphenoxy)-1,2-epoxybutane (9a). A solu-
tion of 8a (1.00 g, 2.7 mmol) in dry THF (10 ml) was
treated with LiHMDS (1.0 M in THF, 3.3 mL,

T. Fujishima et al. / Bioorg. Med. Chem. 8 (2000) 123±134
129
[a]2d8 19.4 (c=1.04, CHCl3); 1H NMR (400 MHz,
CDCl3) d 2.04 (1H, t, J=2.7 Hz), 2.12 (2H, m), 2.51
(1H, ddd, J=16.8, 4.6, 2.7 Hz), 2.57 (1H, ddd, J=16.8,
6.1, 2.7 Hz), 3.76 (3H, s), 4.01±4.12 (3H, m), 4.58 (1H, d,
J=11.6 Hz), 4.66 (1H, d, J=11.6 Hz), 4.81 (1H, d,
J=7.0 Hz), 4.89 (1H, d, J=7.0 Hz), 6.81 (4H, m), 7.30
(5H, m); 13C NMR (100 MHz, CDCl3) d 24.7 (t), 34.0
(t), 55.7 (q), 64.7 (t), 69.8 (t), 70.5 (d), 72.6 (d), 80.6 (s),
94.0 (t), 114.7 (d), 115.4 (d), 127.8 (d), 128.4 (d), 137.7
(s), 153.0 (s), 153.8 (s); FTIR (neat) 3289, 2951, 2118,
1591, 1508, 1470, 1385, 1290, 1232 cm 1; MS 340 [M]+;
HRMS calcd. for [C21H24O4] 340.1675, found 340.1676.
mixture of 13aA and 13aS. This was separated by silica
gel column chromatography (ethyl acetate:n-hexane
=1:2) to give 13aA (9 mg) and 13aS (12 mg) both as
colorless oils in 62% total yield.
13aA. [a]2d6
33.2 (c=1.01, CHCl3); 1H NMR
(400 MHz, CDCl3) d 1.78 (1H, ddd, J=14.3, 9.5,
3.1 Hz), 1.93 (1H, ddd, J=14.3, 9.5, 3.1 Hz), 2.03 (1H, t,
J=2.7 Hz), 2.37 (1H, d, J=4.6 Hz), 2.50 (1H, ddd,
J=16.8, 4.6, 2.7 Hz), 2.57 (1H, ddd, J=16.8, 6.4,
2.7 Hz), 4.08 (1H, ddt, J=6.4, 4.6, 3.1 Hz), 4.41 (1H,
m), 4.66 (1H, d, J=11.6 Hz), 4.69 (1H, d, J=11.6 Hz),
4.85 (1H, d, J=7.0 Hz), 4.91 (1H, d, J=7.0 Hz), 5.12
(1H, dt, J=10.4, 1.5 Hz), 5.29 (1H, dt, J=17.4, 1.5 Hz),
5.91 (1H, ddd, J=17.4, 10.4, 5.5 Hz), 7.28±7.37 (5H, m);
13C NMR (100 MHz, CDCl3) d 24.8 (t), 41.0 (t), 69.1
(d), 70.1 (t), 70.5 (d), 73.5 (d), 80.5 (s), 94.5 (t), 114.3 (t),
127.9 (d), 128.0 (d), 128.6 (d), 137.5 (s), 140.8 (d); FTIR
(neat) 3448, 3289, 2918, 2120, 1647, 1496, 1456,
1419 cm 1; FAB±MS (NBA-NaI) 283 [M+Na]+.
(S)-3-[(Benzyloxymethyl)oxy]-1-(4-methoxyphenoxy)hex-
5-yne (11b). [a]d28 +23.0 (c=1.20, CHCl3).
(R)-3-[(Benzyloxymethyl)oxy]hex-5-yn-1-ol (12a). A solu-
tion of 11a (138 mg, 0.41 mmol) in CH3CN (4.8 mL) and
H2O (1.2 mL) was treated with diammonium cerium
nitrate (CAN) (553 mg, 0.98 mmol) with stirring at 0 ꢀC.
After 15 min, ethyl acetate (8 mL) and brine (8 mL) were
added to the solution and separated. The aqueous layer
was extracted with ethyl acetate. The combined organic
phases were washed with satd NaHCO3 aq, dried over
magnesium sulfate and ®ltered. Evaporation of the ®l-
trate a€orded a residue, from which 12a (95 mg) was
separated by silica gel column chromatography (ethyl
acetate:n-hexane=1:1) in 95% yield as a pale yellow oil.
1
(S)-MTPA ester of 13aA. H NMR (400 MHz, CDCl3)
d 2.000 (1H, t, J=2.4 Hz), 1.96±2.05 (2H, m), 2.392 (1H,
ddd, J=16.8, 3.7, 2.4 Hz), 2.540 (1H, ddd, J=16.8, 6.4,
2.4 Hz), 3.524 (3H, d, J=1.2 Hz), 3.596 (1H, m), 4.574
(1H, d, J=11.9 Hz), 4.675 (1H, d, J=7.3 Hz), 4.730
(1H, d, J=11.9 Hz), 4.818 (1H, d, J=7.3 Hz), 5.292
(1H, dt, J=10.4, 0.9 Hz), 5.414 (1H, dt, J=17.4,
0.9 Hz), 5.690 (1H, m), 5.869 (1H, ddd, J=17.4, 10.4,
7.3 Hz), 7.278±7.403 (8H, m), 7.516±7.548 (2H, m).
[a]2d7 50.3 (c=1.74, CHCl3); 1H NMR (400 MHz,
CDCl3) d 1.93 (2H, m), 2.02 (1H, t, J=2.4 Hz), 2.05
(1H, t, J=5.8 Hz), 2.50 (1H, ddd, J=17.1, 5.2, 2.4 Hz),
2.55 (1H, ddd, J=17.1, 6.1, 2.4 Hz), 3.74±3.88 (2H, m),
4.02 (1H, dq, J=8.2, 5.2 Hz), 4.67 (2H, s), 4.84 (1H, d,
J=7.0 Hz), 4.90 (1H, d, J=7.0 Hz), 7.28±7.37 (5H, m);
13C NMR (100 MHz, CDCl3) d 24.6 (t), 36.5 (t), 59.5 (t),
70.0 (t), 70.4 (d), 74.3 (d), 80.6 (s), 94.1 (t), 127.8 (d),
128.5 (d), 137.4 (s); FTIR (neat) 3383, 2964, 2872, 1456,
1377 cm 1; FAB±MS (NBA-NaI) 257 [M+Na]+.
1
(R)-MTPA ester of 13aA. H NMR (400 MHz, CDCl3)
d 1.998 (1H, ddd, J=14.7, 9.5, 3.4 Hz), 2.030 (1H, t,
J=2.4 Hz), 2.080 (1H, ddd, J=14.5, 9.5, 3.4 Hz), 2.479
(1H, ddd, J=16.8, 4.0, 2.4 Hz), 2.563 (1H, ddd, J=16.8,
6.4, 2.4 Hz), 3.538 (1H, d, J=1.2 Hz), 3.758 (1H, ddd,
J=6.4, 4.0, 3.4 Hz), 4.585 (1H, d, J=11.9 Hz), 4.716
(1H, d, J=11.9 Hz), 4.729 (1H, d, J=7.3 Hz), 4.860
(1H, d, J=7.3 Hz), 5.215 (1H, dt, J=10.4, 0.9 Hz),
5.308 (1H, dt, J=17.1, 0.9 Hz), 5.689 (1H, m), 5.776
(1H, ddd, J=17.1, 10.4, 7.0 Hz), 7.276±7.410 (8H, m),
7.511-7.534 (2H, m).
(S)-3-[(Benzyloxymethyl)oxy]hex-5-yn-1-ol (12b). [a]d27
+55.5 (c=1.54, CHCl3).
(3S,5R)-5-[(Benzyloxymethyl)oxy]oct-1-en-7-yn-3-ol (13aA)
and (3R,5R)-5-[(Benzyloxymethyl)oxy]oct-1-en-7-yn-3-ol
(13aS). A stirred mixture of 12a (73 mg, 0.31 mmol)
and powdered 4 A MS (50 mg) in CH2Cl2 (2 mL) was
treated with PDC (292 mg, 0.78 mmol) at room tem-
perature. The mixture was stirred at room temperature
for 1 day under argon, and separated by silica gel col-
umn chromatography (ethyl acetate:n-hexane=1:1) to
give the corresponding aldehyde (70 mg, 96%) as a col-
orless oil, which was used immediately in the following
step.
13aS. [a]2d7
31.5 (c=1.09, CHCl3); 1H NMR
(400 MHz, CDCl3) d 1.93 (2H, m), 2.03 (1H, t,
J=2.8 Hz), 2.53 (1H, ddd, J=16.8, 4.6, 2.8 Hz), 2.59
(1H, ddd, J=16.8, 6.1, 2.8 Hz), 4.00 (1H, m), 4.35 (1H,
m), 4.64 (1H, d, J=11.6 Hz), 4.70 (1H, d, J=11.6 Hz),
4.83 (1H, d, J=7.0 Hz), 4.91 (1H, d, J=7.0 Hz), 5.13
(1H, dt, J=10.4, 1.5 Hz), 5.28 (1H, dt, J=17.1, 1.5 Hz),
5.89 (1H, ddd, J=17.1, 10.4, 6.1 Hz), 7.28±7.37 (5H, m);
13C NMR (100 MHz, CDCl3) d 24.6 (t), 41.1 (t), 70.1 (t),
70.6 (d), 71.3 (d), 75.0 (d), 80.4 (s), 93.8 (t), 114.9 (t),
127.9 (d), 128.0 (d), 128.6 (d), 137.5 (s), 140.5 (d); FTIR
(neat) 3445, 3296, 2947, 2120, 1649, 1498, 1456,
1421 cm 1; FAB±MS (NBA-NaI) 283 [M+Na]+.
To a solution of the above aldehyde (30 mg, 0.13 mmol)
in dry toluene (3 mL) was added with stirring vinyl
magnesium bromide (1.0 M in THF, 390 mL, 0.39 mmol)
at 78 ꢀC under argon. The reaction mixture was stirred
for 40 min, and quenched by addition of satd NH4Cl aq
After extraction with ethyl acetate, the organic layer
was washed with brine, dried over magnesium sulfate
and ®ltered. Evaporation of the solvent a€orded a crude
1
(S)-MTPA ester of 13aS. H NMR (400 MHz, CDCl3)
d 2.030 (1H, t, J=2.4 Hz), 2.052 (1H, ddd, J=14.0, 7.6,
4.6 Hz), 2.150 (1H, ddd, J=14.0, 8.2, 5.8 Hz), 2.501
(1H, m), 2.555 (1H, m), 3.557 (3H, d, J=1.2 Hz), 3.777
(1H, m), 4.609 (1H, d, J=11.9 Hz), 4.693 (1H, d,
J=11.9 Hz), 4.747 (1H, d, J=7.3 Hz), 4.838 (1H, d,

130
T. Fujishima et al. / Bioorg. Med. Chem. 8 (2000) 123±134
J=7.3 Hz), 5.250 (1H, dt, J=10.7, 0.9 Hz), 5.310 (1H,
dt, J=17.8, 0.9 Hz), 5.647 (1H, m), 5.740 (1H, ddd,
J=17.8, 10.7, 7.0 Hz), 7.282±7.412 (8H, m), 7.503±7.526
(2H, m).
(3S,5R)-Bis[(tert-butyldimethylsilyl)oxy]oct-1-en-7-yne
(3aA). To a stirred mixture of 14aA (30 mg, 0.21 mmol)
and 2,6-lutidine (73 mL, 0.63 mmol) in dry CH2Cl2 (2 mL)
was added TBSOTf (123 mL, 0.53 mmol) at 0 ꢀC under
argon. The resulting mixture was stirred at 0 ꢀC for
30 min, diluted with CH2Cl2 and washed with water.
The organic layer was dried over magnesium sulfate and
®ltered. Removal of the solvent a€orded a residue, from
which 3aA (76 mg) was separated by silica gel column
chromatography (ethyl acetate:n-hexane=1:8) as a col-
orless oil in 99% yield.
1
(R)-MTPA ester of 13aS. H NMR (400 MHz, CDCl3)
d 2.000 (1H, ddd, J=14.0, 7.3, 5.2 Hz), 2.015 (1H, t,
J=2.8 Hz), 2.082 (1H, ddd, J=14.0, 7.6, 6.4 Hz), 2.434
(1H, ddd, J=17.1, 4.6, 2.8 Hz), 2.504 (1H, ddd, J=17.1,
6.1, 2.8 Hz), 3.543 (3H, d, J=1.2 Hz), 3.671 (1H, m),
4.588 (1H, d, J=11.9 Hz), 4.675 (1H, d, J=11.9 Hz),
4.703 (1H, d, J=7.0 Hz), 4.793 (1H, d, J=7.0 Hz), 5.316
(1H, d, J=9.8 Hz), 5.405 (1H, dt, J=17.1, 0.9 Hz),
5.640 (1H, m), 5.864 (1H, ddd, J=17.1, 9.8, 7.6 Hz),
7.271±7.409 (8H, m), 7.510±7.533 (2H, m).
[a]2d7
7.5 (c=1.01, CHCl3); 1H NMR (400 MHz,
CDCl3) d 0.04 (3H, s), 0.07 (3H, s), 0.086 (3H, s), 0.093
(3H, s), 0.890 (9H, s), 0.894 (9H, s), 1.66 (1H, ddd,
J=13.7, 6.4, 4.9 Hz), 1.88 (1H, ddd, J=13.7, 7.6,
5.2 Hz), 1.97 (1H, t, J=2.8 Hz), 2.33 (1H, ddd, J=16.8,
5.2, 2.8 Hz), 2.38 (1H, ddd, J=16.8, 6.1, 2.8 Hz), 3.93
(1H, quintet, J=5.5 Hz), 4.23 (1H, m), 5.04 (1H, ddd,
J=10.1, 1.5, 0.5 Hz), 5.14 (1H, ddd, J=17.1, 1.5,
0.9 Hz), 5.82 (1H, ddd, J=17.1, 10.1, 7.0 Hz); 13C NMR
(100 MHz, CDCl3) d 4.6 (q), 4.6 (q), 4.4 (q), 3.8
(q), 18.1 (s), 18.2 (s), 25.86 (q), 25.93 (q), 28.0 (t), 45.9
(t), 68.1 (d), 70.1 (d), 71.7 (d), 81.5 (s), 114.3 (t), 141.9
(d); FTIR (neat) 3314, 2955, 2932, 2887, 2858, 1471,
1464, 1361, 1255, 1091 cm 1; MS 311 [M-tBu]+; HRMS
calcd. for [C16H31O2Si2] 311.1862, found 311.1857.
(3R,5S)-5-[(Benzyloxymethyl)oxy]oct-1-en-7-yn-3-ol
(13bA). [a]2d5 +33.7 (c=1.00, CHCl3).
(3S,5S)-5-[(Benzyloxymethyl)oxy]oct-1-en-7-yn-3-ol (13bS).
[a]2d6 +32.5 (c=1.08, CHCl3)
(3S,5R)-Oct-1-en-7-yn-3,5-diol (14aA). A solution of
.
BF3 OEt2 (420 mL, 3.4 mmol) in dry CH2Cl2 (2 mL) was
added slowly to a solution of 13aA (296 mg, 1.1 mmol)
and thiophenol (176 mL, 1.7 mmol) in dry CH2Cl2
(10 mL) with stirring at 0 ꢀC under argon. Stirring was
continued at 0 ꢀC for 15 min, then the reaction mixture
was diluted with phosphate bu€er and extracted with
ethyl acetate. The combined organic layer was washed
with brine, dried over magnesium sulfate, ®ltered and
concentrated. The residue was puri®ed by silica gel col-
umn chromatography (ethyl acetate:n-hexane=1:1) to
give 14aA (132 mg) as a colorless oil in 86% yield.
(3R,5S)-Bis[(tert-butyldimethylsilyl)oxy]oct-1-en-7-yne
(3bA). [a]2d5 +10.2 (c=0.55, CHCl3).
(3R,5R)-Bis[(tert-butyldimethylsilyl)oxy]oct-1-en-7-yne
10.9 (c=1.08, CHCl3); 1H NMR
(3aS). [a]2d5
(400 MHz, CDCl3) d 0.04 (3H, s), 0.06 (3H, s), 0.07
(3H, s), 0.09 (3H, s), 0.90 (18H, s), 1.80 (2H, t, J=
6.1 Hz), 1.97 (1H, t, J=2.8 Hz), 2.34 (1H, ddd, J=16.8,
5.5, 2.8 Hz), 2.40 (1H, ddd, J=16.8, 6.1, 2.8 Hz), 3.88
(1H, quintet, J=6.1 Hz), 4.25 (1H, m), 5.06 (1H, ddd,
J=10.4, 1.6, 1.2 Hz), 5.18 (1H, ddd, J=16.8, 1.8,
1.2 Hz), 5.81 (1H, ddd, J=16.8, 10.4, 6.4 Hz); 13C NMR
(100 MHz, CDCl3) d 4.8 (q), 4.6 (q), 4.3 (q), 4.2
(q), 18.1 (s), 18.2 (s), 25.86 (q), 25.90 (q), 27.6 (t), 45.2
(t), 68.0 (d), 70.0 (d), 71.1 (d), 81.5 (s), 114.3 (t), 141.2
(d); FTIR (neat) 3316, 2955, 2932, 2889, 2858, 1471,
1464, 1361, 1255, 1087 cm 1; MS 311 [M-tBu]+; HRMS
calcd for [C16H31O2Si2] 311.1862, found 311.1864.
[a]2d5
1.2 (c=1.25, CHCl3); 1H NMR (400 MHz,
CDCl3) d 1.76 (1H, ddd, J=14.7, 7.6, 3.1 Hz), 1.86 (1H,
ddd, J=14.7, 8.9, 3.1 Hz), 2.06 (1H, t, J=2.8 Hz), 2.43
(3H, m), 2.80 (1H, br.s), 4.10 (1H, m), 4.49 (1H, m),
5.17 (1H, d, J=10.4 Hz), 5.31 (1H, d, J=17.4 Hz), 5.94
(1H, ddd, J=17.4, 10.4, 5.5 Hz); 13C NMR (100 MHz,
CDCl3) d 27.3 (t), 41.2 (t), 67.2 (d), 70.3 (d), 70.9 (d),
80.6 (s), 114.7 (t), 140.4 (d); FTIR (neat) 3298, 2918,
2118, 1647, 1419, 1336, 1292, 1126 cm 1; FAB±MS
(NBA-NaI) 163 [M+Na]+.
(3R,5S)-Oct-1-en-7-yn-3,5-diol (14bA). [a]2d9 +1.3 (c=
1.58, CHCl3).
(3S,5S)-Bis[(tert-butyldimethylsilyl)oxy]oct-1-en-7-yne
(3bS). [a]2d5 +15.9 (c=0.41, CHCl3).
(3R,5R)-Oct-1-en-7-yn-3,5-diol (14aS). [a]2d8 10.3 (c=
1
0.70, CHCl3); H NMR (400 MHz, CDCl3) d 1.70 (1H,
(20S)-De-A,B-8ꢁ-[(tert-butyldimethylsilyl)oxy]-20-(hy-
droxymethyl)pregnane (16). A solution of 15 (6.15 g,
19 mmol) in THF (50 mL) was re¯uxed under an argon
atmosphere in the presence of DBU (3.4 mL, 23 mmol)
for 12 h. The reaction mixture was cooled and diluted
with ether (400 mL). The combined mixture was washed
with water, 1 N HCl aq, then brine, dried over magne-
sium sulfate and concentrated under reduced pressure to
a€ord a mixture of 20-epimeric aldehydes.
dt, J=14.7, 9.8 Hz), 1.84 (1H, dt, J=14.7, 3.1 Hz), 2.06
(1H, t, J=2.8 Hz), 2.41 (2H, m), 2.73 (1H, d,
J=3.1 Hz), 3.28 (1H, d, J=3.1 Hz), 4.05 (1H, m), 4.41
(1H, m), 5.13 (1H, dt, J=10.4, 1.2 Hz), 5.28 (1H, dt,
J=17.4, 1.2 Hz), 5.90 (1H, ddd, J=17.4, 10.4, 6.1 Hz);
13C NMR (100 MHz, CDCl3) d 27.6 (t), 41.9 (t), 70.3
(d), 70.9 (d), 73.3 (d), 80.5 (s), 114.9 (t), 140.4 (d); FTIR
1
(neat) 3298, 2916, 2120, 1649, 1417, 1331, 1126 cm
FAB±MS (NBA-NaI) 163 [M+Na]+.
;
The above residue was dissolved in MeOH (50 mL) and
treated with NaBH4 (3.57 g, 95 mmol) with stirring at
0 ꢀC. The reaction mixture was stirred for 1 h at 0 ꢀC,
(3S,5S)-Oct-1-en-7-yn-3,5-diol (14bS). [a]2d5 +9.2 (c=
0.52, CHCl3).

T. Fujishima et al. / Bioorg. Med. Chem. 8 (2000) 123±134
131
then water was added to destroy the excess reagent. To
the resulting mixture was added brine (50 mL), followed
by extraction with ethyl acetate. The organic layer was
dried over magnesium sulfate, ®ltered and concentrated.
Puri®cation by silica gel column chromatography (ethyl
acetate:n-hexane=1:8) a€orded the desired alcohol 16
(3.14 g, 52%) and the more polar epimer (2.49 g, 39%),
both as colorless oils.
(t), 42.0 (s), 52.7 (d), 55.2 (d), 69.3 (d); FTIR (neat)
2930, 2856, 1730 cm 1; MS 436 [M]+; HRMS calcd for
[C19H37IOSi] 436.1658, found 436.1658.
The 23-epimeric sulfones (20). To a solution of 19
(1.50 g, 5.5 mmol) in dry THF (2 mL) and HMPA
(2.2 mL, 13 mmol), n-BuLi solution (1.6 M in n-hexane,
3.4 mL, 5.5 mmol) was added dropwise with stirring at
78 ꢀC. The resulting mixture was stirred for 10 min,
then a solution of 18 (800 mg, 1.8 mmol) in dry THF
(5 mL) was added. The mixture was stirred at 78ꢀC for
40 min, then the reaction was quenched by adding satu-
rated aqueous NH4Cl solution. The whole was extracted
with ethyl acetate. The organic solution was dried over
magnesium sulfate, ®ltered and concentrated. The crude
product was separated by silica gel column chromato-
graphy (ethyl acetate:n-hexane=1:4) to give a mixture
of 23-epimeric sulfones 20 (1.04 g) in 98% yield.
1H NMR (400 MHz, CDCl3) d 0.00 (3H, s), 0.01 (3H, s),
0.89 (9H, s), 0.93 (3H, s), 0.94 (3H, d, J=6.7 Hz), 3.45
(1H, dd, J=10.6, 7.0 Hz), 3.71 (1H, dd, J=10.6, 3.7 Hz),
4.00 (1H, m); 13C NMR (100 MHz, CDCl3) d 5.1 (q),
4.8 (q), 14.1 (q), 16.6 (q), 17.7 (t), 18.0 (s), 22.9 (t), 25.9
(q), 26.7 (t), 34.4 (t), 37.5 (d), 40.2 (t), 42.0 (s), 53.0 (d),
53.1 (d), 66.9 (t), 69.4 (d); FTIR (neat) 3333, 2930, 2858,
1471, 1375, 1253, 1167 cm 1; MS 326 [M]+; HRMS
calcd for [C19H38O2Si] 326.2641, found 326.2642.
(20S)-De-A,B-8ꢁ-[(tert-butyldimethylsilyl)oxy]-20-[(p-to-
lylsulfonyl)oxymethyl]pregnane (17). To a solution of 16
(3.14 g, 9.6 mmol) in pyridine (20 mL) was added p-tosyl
chloride (2.20 g, 12 mmol) with stirring at 0 ꢀC under an
argon atmosphere. The resulting mixture was stirred for
13 h at room temperature. The reaction mixture was
cooled in an ice bath, then water was added to the mix-
ture. After extraction with ether, the organic layer was
washed with 1 N HCl aq and brine, dried over magne-
sium sulfate and ®ltered. Evaporation of the ®ltrate
a€orded a crude mixture, from which 17 (4.53 g) was
separated by silica gel column chromatography (ethyl
acetate:n-hexane=1:4) in 98% yield as a colorless oil.
23-Epimeric mixture: 1H NMR (400 MHz, CDCl3) d
0.02 (3H, s), 0.00 (3H, s), 0.66 (3H, d, J=6.4 Hz), 0.85
and 0.88 (9H, s), 1.23 and 1.27 (6H, s), 2.32 (1H, dd,
J=15.3, 4.3 Hz), 3.26 (1H, m), 3.30 (3H, s), 3.96 (1H,
m), 4.57 (1H, d, J=7.3 Hz), 4.67 (1H, d, J=7.3 Hz),
7.55 (2H, t, J=6.3 Hz), 7.63 (1H, t, J=6.3 Hz), 7.88
(2H, t, J=6.3 Hz); 13C NMR (100 MHz, CDCl3) d 5.3
(q), 4.9 (q), 14.2 (q), 17.5 (t), 17.8 (q), 17.9 (s), 22.7 (t),
24.9 (q), 25.7 (q), 26.7 (t), 27.7 (q), 30.8 (d), 34.3 (t), 38.0
(t), 40.8 (t), 42.1 (t), 52.9 (d), 55.1 (q), 56.9 (d), 58.9 (d),
69.3 (d), 75.1 (s), 90.9 (t), 129.0 (d), 129.1 (d), 133.4 (d),
138.4 (s); FTIR (neat) 2932, 2856, 2361, 1469, 1448,
1375, 1304, 1145 cm 1; MS 580 [M]+; HRMS calcd for
[C32H56O5SSi] 580.3618, found 580.3618.
1H NMR (400 MHz, CDCl3) d 0.02 (3H, s), 0.00 (3H,
s), 0.81 (3H, s), 0.87 (9H, s), 0.88 (3H, d, J=6.7 Hz),
2.45 (3H, s), 3.78 (1H, dd, J=9.2, 7.2 Hz), 3.97 (1H, m),
4.11 (1H, dd, J=9.2, 3.4 Hz), 7.34 (2H, d, J=8.2 Hz),
7.78 (2H, d, J=8.2 Hz); 13C NMR (100 MHz, CDCl3) d
5.2 (q), 4.8 (q), 14.1 (q), 16.7 (q), 17.6 (t), 18.0 (s),
21.6 (q), 22.7 (t), 25.8 (q), 26.6 (t), 34.2 (t), 34.7 (d), 39.9
(t), 41.8 (s), 52.7 (d), 52.8 (d), 69.2 (d), 74.3 (t), 128.0 (d),
129.7 (d), 133.3 (s), 144.6 (s); FTIR (neat) 2930, 2856,
2361, 1473, 1458, 1361, 1251, 1176 cm 1; MS 465 [M-
Me]+, 423 [M-tBu]+; HRMS calcd for [C25H41O4SSi]
465.2495, found 465.2493.
(20S)-De-A,B-8ꢁ-[(tert-butyldimethylsilyl)oxy]-25-[(meth-
oxymethyl)oxy]cholestane (21). A saturated solution of
Na2HPO4 (20 g) in MeOH (30 mL) was added to a stir-
red solution of 20 (1.50 g, 2.6 mmol) in THF (30 mL).
To this mixture was added 5% sodium amalgam (35 g)
at 0 ꢀC. The resulting suspension was stirred under
argon for 1 h at room temperature. The mixture was
diluted with ether and the whole was ®ltered over
Celite1 and concentrated. The residue was puri®ed by
silica gel column chromatography (ethyl acetate:n-hex-
ane=1:8) to give 21 (1.10 g) as a colorless oil in 98%
yield.
(20S)-De-A,B-8ꢁ-[(tert-butyldimethylsilyl)oxy]-20-(iodo-
methyl)pregnane (18). To a solution of 17 (127 mg,
0.26 mmol) in DMF (3 mL) was added NaI (126 mg,
0.84 mmol) and the mixture was stirred at 85 ꢀC for 3 h.
It was then cooled, and brine was added. After extrac-
tion with ether, the organic layer was dried over mag-
nesium sulfate, ®ltered and concentrated. Puri®cation
by silica gel column chromatography (ethyl acetate:n-
hexane=1:4) a€orded the iodide 18 (104 mg) as a col-
orless oil in 92% yield.
1H NMR (400 MHz, CDCl3) d 0.01 (3H, s), 0.01 (3H,
s), 0.81 (3H, d, J=6.7 Hz), 0.89 (9H, s), 0.91 (3H, s),
1.21 (6H, s), 3.36 (3H, s), 3.99 (1H, m), 4.70 (2H, s); 13C
NMR (100 MHz, CDCl3) d 5.1 (q), 4.8 (q), 14.0 (q),
17.8 (t), 18.1 (s), 18.6 (t), 20.6 (t), 23.0 (t), 25.8 (q), 26.4
(q), 27.2 (t), 34.5 (t), 34.8 (d), 35.8 (t), 40.7 (t), 42.1 (t),
42.2 (s), 53.2 (d), 55.1 (q), 56.5 (d), 69.5 (d), 76.4 (s),
91.0 (t); FTIR (neat) 2932, 2858, 1469, 1377, 1251, 1165,
1145, 1086, 1041, 922 cm 1; MS 440 [M]+, 425 [M-
Me]+; HRMS calcd for [C26H52O3Si] 440.3686, found
440.3687.
1H NMR (400 MHz, CDCl3) d 0.01 (3H, s), 0.01 (3H,
s), 0.88 (9H, s), 0.92 (3H, s), 0.95 (3H, d, J=6.4 Hz),
3.18 (1H, dd, J=9.4, 6.4 Hz), 3.46 (1H, dd, J=9.4,
3.1 Hz), 4.00 (1H, m); 13C NMR (100 MHz, CDCl3) d
5.2 (q), 4.8 (q), 14.1 (q), 17.6 (t), 18.0 (s), 19.5 (t),
21.4 (q), 22.8 (t), 25.8 (q), 26.9 (t), 34.2 (t), 36.2 (d), 40.5
(20S)-De-A,B-cholestane-8ꢁ,25-diol (22). A solution of
21 (80 mg, 0.18 mmol) in MeOH (3 mL) was treated
.
with p-TsOH H2O (174 mg, 0.91 mmol) at room tem-
perature. The reaction mixture was stirred at room

132
T. Fujishima et al. / Bioorg. Med. Chem. 8 (2000) 123±134
temperature for 1 day. After the reaction was com-
pleted, the mixture was concentrated and puri®ed by
silica gel column chromatography (ethyl acetate:n-
hexane=1:2) to give 22 (43 mg) as a colorless oil in 85%
yield.
and triethylamine (0.5 mL) in toluene (0.5 mL) was stir-
red for 10 min at room temperature, then a solution of
the A-ring moiety 3aA (21 mg, 0.057 mmol) and the 20-
epi CD-ring 5 (34 mg, 0.095 mmol) in toluene (0.5 mL)
was added. After having been heated at re¯ux for 2 h
and diluted with pentane, the reaction mixture was ®l-
tered through a pad of silica gel with ether. After eva-
poration of the solvent, the crude mixture was dissolved
in methanol (2 mL) and treated with CSA (15 mg,
0.065 mmol) at room temperature for 12 h. After
removal of the solvent, the residue was subjected to silica
gel column chromatography (ethyl acetate:n-hexane=
1:1) to give the vitamin 2 (12 mg) as a white solid in
52% yield. Further puri®cation for biological evalua-
tion was conducted by using reversed-phase recycle
HPLC (YMC-Pack ODS column, 20Â150 mm, 9.0 mL/
min, acetonitrile:water=9:1).
1H NMR (400 MHz, CDCl3) d 0.84 (3H, d, J=6.7 Hz),
0.93 (3H, s), 1.21 (6H, s), 4.07 (1H, m); 13C NMR
(100 MHz, CDCl3) d 13.8 (q), 17.5 (t), 18.5 (q), 20.9 (t),
22.4 (t), 27.1 (t), 29.2 (q), 29.3 (q), 33.6 (t), 34.7 (d), 35.7
(t), 40.4 (t), 41.9 (s), 44.3 (t), 52.7 (d), 56.3 (d), 69.4 (d),
71.1 (s); FTIR (neat) 3385, 2932, 2870, 1468, 1375, 1265,
1165, 1082, 1068, 989, 947 cm 1; MS 264 [M-H2O]+,
246 [M-2H2O]+; HRMS calcd for [C18H32O] 264.2453,
found 264.2453.
(20S)-De-A,B-25-hydroxycholestan-8-one (23). Solid
tetrapropylammonium perruthenate (TPAP, 135 mg,
0.38 mmol) was added to a stirred mixture of 22
(186 mg, 0.66 mmol), 4-methylmorpholine N-oxide
(NMO, 198 mg, 1.7 mmol), and 4 A MS (50 mg) in dry
CH2Cl2 (10 mL) at room temperature under argon.
Upon completion of the reaction, the mixture was
directly puri®ed by silica gel column chromatography
(ethyl acetate:n-hexane=1:3) to give the ketone 23
(178 mg) as a colorless oil in 96% yield.
UV (EtOH) lmax 263 nm, lmin 226 nm; 1H NMR
(400 MHz, CDCl3) d 0.54 (3H, s), 0.85 (3H, d, J=
6.4 Hz), 1.21 (6H, s), 2.31 (1H, dd, J=13.4, 6.4 Hz), 2.60
(1H, dd, J=13.4, 3.1 Hz), 2.83 (1H, dd, J=12.5, 4.3 Hz),
4.23 (1H, m), 4.43 (1H, m), 5.01 (1H, s), 5.33 (1H, s),
6.02 (1H, d, J=11.3 Hz), 6.38 (1H, d, J=11.3 Hz); MS
416 [M]+, 398 [M-H2O]+, 380 [M-2H2O]+; HRMS
calcd for [C27H44O3] 416.3291, found 416.3286.
1H NMR (400 MHz, CDCl3) d 0.64 (3H, s), 0.87 (3H, d,
J=6.1 Hz), 1.22 (6H, s), 2.45 (1H, dd, J=11.6, 7.3 Hz),
4.07 (1H, m); 13C NMR (100 MHz, CDCl3) d 12.8 (q),
18.5 (q), 19.0 (t), 20.9 (t), 24.1 (t), 29.3 (q), 29.4 (d), 34.9
(t), 36.0 (t), 41.0 (t), 44.2 (t), 50.0 (s), 56.3 (d), 62.0 (d),
71.0 (s), 212.0 (s); FTIR (neat) 3445, 2964, 2876, 1711,
1468, 1427, 1305, 1269, 1224, 1157 cm 1; MS 280 [M]+,
262 [M-H2O]+; HRMS calcd. for [C18H32O2] 280.2402,
found 280.2401.
(5Z,7E)-(1R,3R,20S)-9,10-Seco-5,7,10(19)-cholestatriene-
1,3,25-triol ((20S)-(1ꢁ,3ꢁ)). UV (EtOH) lmax 261 nm,
lmin 225 nm; H NMR (400 MHz, CDCl3) d 0.55 (3H,
1
s), 0.85 (3H, d, J=6.7 Hz), 1.21 (6H, s), 2.47 (1H, dd,
J=13.4, 4.3 Hz), 2.56 (1H, d, J=14.0 Hz), 2.85 (1H, dd,
J=12.5, 4.3 Hz), 4.11 (1H, m), 4.36 (1H, m), 5.01 (1H,
d, J=1.8 Hz), 5.30 (1H, s), 6.06 (1H, d, J=11.3 Hz),
6.45 (1H, d, J=11.3 Hz); 13C NMR (100 MHz, CDCl3)
d 12.1 (q), 18.5 (q), 20.8 (t), 22.1 (t), 23.6 (t), 27.2 (t),
29.0 (t), 29.2 (q), 35.4 (d), 36.0 (t), 40.0 (t), 40.3 (t), 44.2
(t), 45.4 (t), 45.9 (s), 56.1 (d), 56.4 (d), 68.2 (d), 71.1 (s),
73.6 (d), 113.4 (t), 117.0 (d), 125.8 (d), 131.7 (s), 143.2
(s), 147.3 (s); FTIR (neat) 3356, 2943, 2870, 1437, 1377,
1064, 1016, 908, 725 cm 1; MS 416 [M]+, 398 [M-
H2O]+, 380 [M-2H2O]+; HRMS calcd for [C27H44O3]
416.3291, found 416.3292.
(E)-(20S)-De-A,B-8-(bromomethylene)cholestan-25-ol
(5). Sodium hexamethyldisilazide (NaHMDS) (1.0 M
in THF, 0.86 mL, 0.86 mmol) was added to (bromo-
methylene)triphenylphosphonium bromide (389 mg,
0.90 mmol) in THF (1.5 mL) at 60 ꢀC under argon.
After 1 h, a solution of 23 (50 mg, 0.18 mmol) in THF
(1.5 mL) was added. After an additional 1 h at room
temperature, the reaction mixture was diluted with n-
hexane and the whole was ®ltered over Celite1. The ®l-
trate was concentrated, then puri®ed by silica gel column
chromatography (ethyl acetate:n-hexane=1:8, then 1:3)
to give 5 (36 mg) as a colorless oil in 56% yield.
(5Z,7E)-(1S,3S,20S)-9,10-Seco-5,7,10(19)-cholestatriene-
1,3,25-triol ((20S)-(1ꢀ,3ꢀ)). UV (EtOH) lmax 261 nm,
1
lmin 225 nm; H NMR (400 MHz, CDCl3) d 0.54 (3H,
s), 0.85 (3H, d, J=6.4 Hz), 1.22 (6H, s), 2.44 (1H, dd,
J=13.4, 5.5 Hz), 2.56 (1H, d, J=13.4 Hz), 2.84 (1H, dd,
J=11.6, 4.3 Hz), 4.06 (1H, m), 4.31 (1H, m), 5.00 (1H,
d, J=1.5 Hz), 5.30 (1H, s), 6.03 (1H, d, J=11.3 Hz),
6.44 (1H, d, J=11.3 Hz); 13C NMR (100 MHz, CDCl3)
d 12.2 (q), 18.5 (q), 20.8 (t), 22.1 (t), 23.4 (t), 27.2 (t),
29.0 (t), 29.2 (q), 35.4 (d), 36.0 (t), 40.4 (t), 40.7 (t), 44.3
(t), 45.5 (t), 45.9 (s), 56.1 (d), 56.4 (d), 68.2 (d), 71.1 (s),
73.1 (d), 112.9 (t), 117.1 (d), 125.6 (d), 131.7 (s), 143.2
(s), 147.3 (s); FTIR (neat) 3356, 2941, 2872, 1456, 1377,
1064, 1016, 908, 733 cm 1; MS 416 [M]+, 398 [M-
H2O]+, 380 [M-2H2O]+; HRMS calcd for [C27H44O3]
416.3291, found 416.3283.
1H NMR (400 MHz, CDCl3) d 0.56 (3H, s), 0.85 (3H, d,
J=6.4 Hz), 1.22 (6H, s), 2.88 (1H, m), 5.64 (1H, d,
J=1.5 Hz); 13C NMR (100 MHz, CDCl3) d 12.1 (q),
18.5 (q), 19.0 (t), 20.9 (t), 21.9 (t), 22.6 (t), 27.3 (t), 29.2
(q), 29.3 (q), 31.1 (t), 35.3 (d), 36.0 (t), 39.8 (t), 44.3 (t),
45.6 (s), 55.4 (d), 55.9 (d), 71.1 (s), 97.4 (d), 145.1 (s);
1
FTIR (neat) 3383, 2964, 2872, 1631, 1466, 1377 cm
;
MS 356 and 358 [M]+, 338 and 340 [M-H2O]+; HRMS
calcd. for [C19H33 79BrO] 356.1716, found 356.1715.
(5Z,7E)-(1S,3R,20S)-9,10-Seco-5,7,10(19)-cholestatriene-
.
1,3,25-triol (2: (20S)-(1ꢀ,3ꢁ)). A mixture of (dba)3Pd2
CHCl3 (2.0 mg, 0.0013mmol), Ph3P (5.0 mg, 0.019 mmol)
(5Z,7E)-(1R,3S,20S)-9,10-Seco-5,7,10(19)-cholestatriene-
1,3,25-triol ((20S)-(1ꢁ,3ꢀ)). UV (EtOH) lmax 263 nm,

T. Fujishima et al. / Bioorg. Med. Chem. 8 (2000) 123±134
133
1
lmin 225 nm; H NMR (400 MHz, CDCl3) d 0.54 (3H,
were recorded as the mean ¯uorescence index, which is
the product of the % ¯uorescence and the mean ¯uo-
rescence intensity, with 104 cells being counted per
treatment.
s), 0.85 (3H, d, J=6.7 Hz), 1.22 (6H, s), 2.30 (1H, dd,
J=13.1, 7.6 Hz), 2.62 (1H, dd, J=13.1, 3.7 Hz), 2.83
(1H, dd, J=12.5, 4.3 Hz), 4.22 (1H, m), 4.44 (1H, m),
5.01 (1H, s), 5.32 (1H, s), 6.01 (1H, d, J=11.3 Hz), 6.39
(1H, d, J=11.3 Hz); 13C NMR (100 MHz, CDCl3) d
12.2 (q), 18.5 (q), 20.8 (t), 22.1 (t), 23.5 (t), 27.2 (t), 29.0
(t), 29.2 (q), 35.4 (d), 36.0 (t), 40.3 (t), 42.8 (t), 44.2 (t),
45.4 (t), 45.9 (s), 56.1 (d), 56.3 (d), 66.8 (d), 71.1 (s), 71.4
(d), 112.6 (t), 117.1 (d), 125.1 (d), 132.8 (s), 143.3 (s),
147.4 (s); FTIR (neat) 3358, 2945, 2872, 1456, 1375,
1057, 910, 733 cm 1; MS 416 [M]+, 398 [M-H2O]+, 380
[M-2H2O]+; HRMS calcd for [C27H44O3] 416.3291,
found 416.3291.
In vivo bone mineral mobilization assay. Six-week-old
normal SD male rats were divided into several groups of
®ve rats each receiving either an iv injection of 1.0,
10.0 or 100.0 mg/kg of 1a,25-dihydroxyvitamin D3 or an
analogue in 2 mL/kg of 0.1% Triton X-100 solution.
The resulting increases in serum calcium were measured
at 8, 24, 48 and 72 h after administration by the o-cre-
solphthalein complexone method. Relative activity of
each analogue with respect to 1a,25-dihydroxyvitamin
D3 was evaluated as follows: dose response e€ect of
each analogue was calculated at the time when the e€ect
was maximum and expressed as the dosage required to
elevate the serum calcium level by 1 mg/dL.
Binding to vitamin D receptor (VDR). Bovine thymus
1a,25-dihydroxyvitamin D3 receptor was obtained from
Yamasa Biochemical (Chiba, Japan) and dissolved in
0.05 M phosphate bu€er (pH 7.4) containing 0.3 M KCl
and 5 mM dithiothreitol just before use. The receptor
solution (500 mL, 0.23 mg protein) was pre-incubated
with 50 mL of ethanol solution of 1a,25-dihydroxy-
vitamin D3 or an analogue at various concentrations for
60 min at 25 ꢀC. Then, the receptor mixture was left to
stand overnight with 0.1 nM [3H]-1a,25-dihydroxy-
vitamin D3 at 4 ꢀC. The bound and free [3H]-1a,25-
dihydroxyvitamin D3 were separated by treatment with
dextran-coated charcoal for 30 min at 4 ꢀC and cen-
trifuged at 3000 rpm for 10 min. The radioactivity of the
supernatant (500 mL) with ACS-II (9.5 mL) (Amersham,
UK) was then counted.
Acknowledgements
This work was supported by a Grant-in-Aid (No.
10771257) from the Ministry of Education, Science and
Culture of Japan, and a Grant from Hayashi Memorial
Foundation for Female Natural Scientist.
References and Notes
1. Bouillon, R.; Okamura, W. H.; Norman, A. W. Endocrine
Rev. 1995, 16, 200.
2. Ettinger, R. A.; DeLuca, H. F. Adv. Drug Res. 1996, 28,
269.
3. Binderup, L.; Latini, S.; Binderup, E.; Bretting, C.; Calver-
ley, M. J.; Hansen, K. Biochem. Pharmacol. 1991, 42, 1569.
4. Dilworth, F. J.; Calverley, M. J.; Makin, H. L. J.; Jones, G.
Biochem. Pharmacol. 1994, 47, 987.
5. Norman, A. W.; Bouillon, R.; Farach-Carson, M. C;
Bishop, J. E.; Zhou, L.-X.; Nemere, I.; Zhao, J.; Mur-
alidharan, K. R.; Okamura, W. H. J. Biol. Chem. 1993, 268,
20022.
6. Bischof, M. G.; Siu-Caldera, M.-L.; Weiskopf, A.; Vouros,
P.; Cross, H. S.; Peterlik, M.; Reddy, G. S. Exp. Cell. Res.
1998, 241, 194.
7. Reddy, G. S.; Siu-Caldera, M.-L.; Schuster, I.; Astecker, N.;
Tserng, K.-Y.; Muralidharan, K. R.; Okamura, W. H.;
McLane, J. A.; Uskokovic, M. R. In Vitamin D: Chemistry,
Biology and Clinical Applications of the Steroid Hormone:
Proceedings of the Tenth Workshop on Vitamin D, Norman, A.
W.; Bouillon, R.; Thomasset, M., Eds., University of Cali-
fornia, Printing and Reprographics: Riverside, 1997; p 139.
8. Masuda, S.; Okano, T.; Kamao, M.; Kobayashi, T. In
Vitamin D: Chemistry, Biology and Clinical Applications of the
Steroid Hormone: Proceedings of the Tenth Workshop on
Vitamin D, Norman, A. W.; Bouillon, R.; Thomasset, M.,
Eds., University of California, Printing and Reprographics:
Riverside, 1997; p 159.
9. Dai, H.; Posner, G. H. Synthesis 1994, 1383.
10. Zhu, G.-D.; Okamura, W. H. Chem. Rev. 1995, 95, 1877.
11. Trost, B. M.; Dumas, J.; Villa, M. J. Am. Chem. Soc. 1992,
114, 9836.
12. Muralidharan, K. R.; de Lera, A. R.; Isae€, S. D.; Nor-
man, A. W.; Okamura, W. H. J. Org. Chem. 1993, 58, 1895.
13. Fujishima, T.; Liu, Z.-P.; Miura, D.; Chokki, M.; Ishi-
zuka, S.; Konno, K.; Takayama, H. Bioorg. Med. Chem. Lett.
1998, 8, 2145.
Binding to vitamin D binding protein (DBP). Serum
from vitamin D-de®cient Wistar male rats was diluted
(Â70,000) with 3.5 mM barbiturate bu€er (pH 8.6) con-
taining 0.13 M NaCl and 0.1% (w/v) bovine serum
albumin (BSA) and used as a source of DBP. The dilu-
ted serum (1 mL) was incubated with 0.1 nM [3H]-25-
hydroxyvitamin D3 and 100 mL of ethanol solution of
1a,25-dihydroxyvitamin D3 or an analogue at various
concentrations for 60 min at 4 ꢀC. Unbound [3H]-25-
hydroxyvitamin D3 was removed by treatment with
dextran-coated charcoal for 10 min at 4 ꢀC and cen-
trifuged at 3000 rpm for 10 min. The radioactivity of the
supernatant (1 mL) was measured.
Cell surface antigen expression analysis. HL-60 cells
were seeded at 105 cells/well in 24-well plates, and incu-
bated for 72 h with between 10 12 and 10 7 (or 10 8) M
of 1a,25-dihydroxyvitamin D3 or an analogue at 37 ꢀC
in a humidi®ed atmosphere of 5% carbon dioxide in air.
The cells were then washed with PBS and adjusted to
2Â106 cells/100 mL of Diluent solution [phosphate-
bu€ered saline (minus Mg2+, minus Ca2+) containing
1% BSA and 1% NaN3]. Aliquots of cell suspension
(100 mL) were incubated with 10 mL of the human
monoclonal
¯uorescein
isothiocyanate-conjugated
CD11b antibody for 30 min at room temperature with-
out light. The cells were washed once with Diluent
solution and then ®xed in 500 mL of PBS containing 2%
paraformaldehyde. Fluorescence was read on a Becton
Dickinson FACSanTM at an excitation wavelength of
490 nm and emission wavelength of 520 nm. Results

134
T. Fujishima et al. / Bioorg. Med. Chem. 8 (2000) 123±134
14. Konno, K.; Maki, S.; Fujishima, T.; Liu, Z.-P.; Miura, D.;
Chokki, M.; Takayama, H. Bioorg. Med. Chem. Lett. 1998, 8,
151.
15. Corey, E. J.; Guzman-Perez, A.; Noe, M. C. Tetrahedron
Lett. 1995, 36, 3481.
butan-2-ol MOM-ether, was synthesized via three-step con-
version (a. MeOH/H2SO4, rt, quant., b. MeMgBr/THF, 0 ꢀC,
96%, c. MOMCl/iPr2NEt, rt, 83%) of commercially available
3-(phenylsulfonyl)propionic acid. 19: 1H NMR (400 MHz,
CDCl3) d 1.20 (6H, s), 1.88 (2H, m), 3.22 (2H, m), 3.28 (3H, s),
4.59 (2H, s), 7.58 (2H, tt, J=7.6, 1.2 Hz), 7.67 (1H, tt, J=7.6,
1.2 Hz), 7.92 (2H, dt, J=7.6, 1.2 Hz), MS 257 [M-Me]+,
HRMS calcd for [C12H17O4S] 257.0847, found 257.0848.
22. Kutner, A.; Perman, K. L.; Lago, A.; Sicinski, R. R.;
Schnoes, H. K.; DeLuca, H. F. J. Org. Chem. 1988, 53, 3450.
23. Grith, W. P.; Ley, S. V.; Whitcombe, G. P.; White, A. D.
J. C. S. Chem. Commun. 1987, 1625.
24. All spectral data of these compounds were identical with
those previously reported in ref 12.
25. Imae, Y.; Manaka, A.; Yoshida, N.; Ishimi, Y.; Shinki, T.;
Abe, E.; Suda, T.; Konno, K.; Takayama, H.; Yamada, S.
Biochim. Biophys. Acta 1994, 1213, 302.
16. Corey, E. J.; Guzman-Perez, A.; Noe, M. C. J. Am. Chem.
Soc. 1995, 117, 10805.
17. We examined a variety of 3-buten-1-ol derivatives with
di€erent protecting groups, for example TBDMS, MEM,
benzyl, benzoyl etc., in the Sharpless AD reaction using
(DHQD)2PHAL or (DHQD)2PYR. The combination of the
benzoyl protecting group and (DHQD)2PYR resulted in the
second best enantioselectivity of only 66% e.e. For details of
the chiral ligand (DHQD)2PYR, which was reported as a good
ligand for terminal ole®ns, see: Crispino, G. A.; Jeong, K.-S.;
Kolb, H. C.; Wang, Z.-M.; Xu, D.; Sharpless, K. B., J. Org.
Chem., 1993, 58, 3785.
18. Ád values obtained were expressed in ppm: Ohtani, I.;
Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem. Soc.
1991, 113, 4092.
19. Suzuki, K.; Tomooka, K.; Kitayama, E.; Matsumoto, T.;
Tsuchihashi, G.-P. C. J. Am. Chem. Soc. 1986, 108, 5221.
20. Fernandez, B.; Perez, J. A. M.; Granja, J. R.; Castedo, L.;
Mourino, A. J. Org. Chem. 1992, 57, 3173.
26. Honda, A.; Mori, Y.; Otomo, S.; Ishizuka, S.; Ikekawa, N.
Steroids 1991, 56, 142.
27. Bishop, J. E.; Collins, E. D.; Okamura, W. H.; Norman,
A. W. J. Bone Mine. Res. 1994, 9, 1277.
28. Collins, S. J.; Ruscetti, F. W.; Gallagher, R. E.; Gallo, R.
C. J. Exp. Med. 1979, 149, 969.
29. Ishizuka, S.; Norman, A. W. J. Steroid Biochem. 1986, 25,
505.
21. The side chain moiety 19, 2-methyl-4-(phenylsulfonyl)-