Highly potent cell differentiation-inducing analogues of 1α,25-dihydroxyvitamin D3: Synthesis and biological activity of 2-methyl-1,25-dihydroxyvitamin D3 with side-chain modifications

Article abstract of DOI:10.1016/S0968-0896(00)00267-4

Eight 2-methyl substituted analogues of 20-epi-22R-methyl-1α,25-dihydroxyvitamin D₃ (5) and 20-epi-24,26,27-trihomo-22-oxa-1α,25-dihydroxyvitamin D₃ (6: KH-1060) were convergently synthesized. Preparation of the CD-ring portions with modified side chains of 5 and 6, followed by palladium-catalyzed cross-coupling with the A-ring enyne synthons (20a-d), (3S,4S,5R)-, (3S,4R,5R)-, (3S,4S,5S)- and (3R,4R,5S)-3,5-bis[(tert-butyldimethylsilyl)oxy]-4-methyloct-1-en-7-yne, afforded two sets of four A-ring stereoisomers of 20-epi-2,22-dimethyl-1,25-dihydroxyvitamin D₃ (7a-d) and 20-epi-24,26,27-trihomo-2-methyl-22-oxa-1,25-dihydroxyvitamin D₃ (8a-d). The biological profiles of the hybrid analogues were assessed in terms of affinity for vitamin D receptor (VDR) and HL-60 cell differentiation-inducing activity in comparison with the natural hormone. The combined modifications of the A-ring at the 2-position and the side chain yielded analogues with high potency.

Full text of DOI:10.1016/S0968-0896(00)00267-4

Bioorganic & Medicinal Chemistry 9 (2001) 525±535
Highly Potent Cell Di€erentiation-Inducing Analogues of
1ꢀ,25-Dihydroxyvitamin D3: Synthesis and Biological
Activity of 2-Methyl-1,25-dihydroxyvitamin D3
with Side-Chain Modi®cations
Toshie Fujishima,a Liu Zhaopeng,a Katsuhiro Konno,a Kimie Nakagawa,b
Toshio Okano,b Kentaro Yamaguchic and Hiroaki Takayamaa,*
aFaculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa 199±0195, Japan
bDepartment of Hygienic Sciences, Kobe Pharmaceutical University, Kobe 658±8558, Japan
cChemical Analytical Center, Chiba University, Inage-ku, Chiba 263±8522, Japan
Received 23 August 2000; accepted 4 October 2000
AbstractÐEight 2-methyl substituted analogues of 20-epi-22R-methyl-1a,25-dihydroxyvitamin D3 (5) and 20-epi-24,26,27-trihomo-
22-oxa-1a,25-dihydroxyvitamin D3 (6: KH-1060) were convergently synthesized. Preparation of the CD-ring portions with modi®ed
side chains of 5 and 6, followed by palladium-catalyzed cross-coupling with the A-ring enyne synthons (20a±d), (3S,4S,5R)-,
(3S,4R,5R)-, (3S,4S,5S)- and (3R,4R,5S)-3,5-bis[(tert-butyldimethylsilyl)oxy]-4-methyloct-1-en-7-yne, a€orded two sets of four
A-ring stereoisomers of 20-epi-2,22-dimethyl-1,25-dihydroxyvitamin D3 (7a±d) and 20-epi-24,26,27-trihomo-2-methyl-22-oxa-1,25-
dihydroxyvitamin D3 (8a±d). The biological pro®les of the hybrid analogues were assessed in terms of anity for vitamin D receptor
(VDR) and HL-60 cell di€erentiation-inducing activity in comparison with the natural hormone. The combined modi®cations of the
A-ring at the 2-position and the side chain yielded analogues with high potency. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
D3 (3) and found that the potencies of the analogues
depend on the con®guration not only of the C-1 and C-3
hydroxyl groups, but also of the 2-methyl group.3 In
particular, 2a-methyl-1a,25-dihydroxyvitamin D3 (3a:
aab-isomer; the Greek letters denote the con®gurations
of C-1, C-2 and C-3, respectively, in the vitamin D
numbering system) exhibited 4-fold higher VDRbind-
ing potency than 1, while its 2-epimer, 2b-methyl-1a,25-
dihydroxyvitamin D3 (3b: abb-isomer), showed one-
eighth of the activity of 1. This simple A-ring modi®cation
yielded for the ®rst time an analogue (3a) with sig-
ni®cantly higher VDRbinding activity than the parent
hormone.
The hormonally active metabolite of vitamin D, 1a,25-
dihydroxyvitamin D3 (1), is now recognized as an impor-
tant cell-cycle regulator, which in¯uences cell prolifera-
tion, di€erentiation and apoptosis, in addition to its
classical role in calcium±phosphorus homeostasis.1 There-
fore, analogues of 1a,25-dihydroxyvitamin D3 should be
promising candidates to treat cancer, psoriasis and
numerous other diseases caused by cell-cycle disorders.
Most biological responses to 1a,25-dihydroxyvitamin
D3 are mediated by a ligand-inducible transcription
factor, vitamin D receptor (VDR), which is a member of
the nuclear receptor superfamily.2 Therefore, analogues
with high VDRanity are of great interest since the
whole sequence of actions is considered to be triggered
by the formation of the ligand±VDRcomplex.
20-epi-1a,25-Dihydroxyvitamin D3 (2) is noteworthy
due to its high activity in cell di€erentiation with rela-
tively low calcemic e€ect; its biological pro®le is clearly
distinct from that of the natural hormone.4 The VDR
binding potency of 2 relative to 1 (normalized to 100)
was estimated to be 4005 by us and 5004a by others
using bovine thymus VDR. We synthesized the above
2-methyl analogues with 20-epimerization, obtaining all
eight possible A-ring stereoisomers (4), and found that
2-methyl substitution and 20-epimerization had additive
In order to investigate the structure±function relationship
of the natural hormone, we synthesized all eight possible
A-ring diastereomers of 2-methyl-1,25-dihydroxyvitamin
*Corresponding author. Tel.: +81-426-85-3713; fax: +81-426-85-
3714; e-mail: hi-takay@pharm.teikyo-u.ac.jp
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00267-4

526
T. Fujishima et al. / Bioorg. Med. Chem. 9 (2001) 525±535
e€ects on the VDRbinding and cell di€erentiation-indu-
cing activities.6,7 Thus, 20-epi-2a-methyl-1a,25-dihydroxy-
vitamin D3 (4a: aab-isomer) exhibited 12-fold higher
anity to VDRand its 2-epimer, 20- epi-2b-methyl-
1a,25-dihydroxyvitamin D3 (4b: abb-isomer), showed
1.6-fold higher anity than 1. On the other hand, four
of the eight 20-epi analogues possessed higher cell dif-
ferentiation-inducing activity than 1, i.e., the aab-, abb-,
aaa- and bba-isomers, which correspond to 4a, 4b, 4c
and 4d, respectively, as illustrated in Chart 3.
Those remarkable e€ects of double modi®cation
prompted us, in the present work, to design 2-methyl
analogues modi®ed in the side chain, in order to study
further the structure±activity relationships of the hybrid
analogues. Four potent A-ring parts, aab-, abb-, aaa-
and bba-isomers, and two characteristic side chains
were selected. The ®rst side chain was that of 20-epi-
22R-methyl-1a,25-dihydroxyvitamin D3 (5), which has
11-fold higher anity to bovine thymus VDRand 20-
fold higher anity to porcine intestinal VDRthan 1,
Chart 1.
and is the most active side-chain analogue found to date
8
in terms of VDRbinding anity.
The second side
chain was that of 20-epi-24,26,27-trihomo-22-oxa-1a,25-
dihydroxyvitamin D3 (6: KH-1060), which is the most
potent known analogue in terms of cell di€erentiation and
immunosuppressive e€ects.4b In this paper, syntheses and
biological pro®les of the 2-methyl analogues of 20-epi-
22R-methyl-1,25-dihydroxyvitamin D3 (7a±d) and 20-epi-
24,26,27-trihomo-22-oxa-1,25-dihydroxyvitamin D3 (8a±
d) are disclosed.
Synthesis
Convergent synthesis can be more e€ective and ¯exible
for the synthesis of a variety of analogues than the
Chart 2.
Chart 3.

T. Fujishima et al. / Bioorg. Med. Chem. 9 (2001) 525±535
527
classical steroidal approach.9 In particular, Trost's con-
vergent method10 using palladium-catalyzed coupling of
the A-ring enyne synthon with the CD-ring portion,
applied by us in the synthesis of A-ring analogues,3,5,6,11
seems most useful. The 2-methyl-A-ring synthons can be
easily obtained according to our procedure, a part of
which was reported in ref 3. Therefore, we focused on the
synthesis of CD-ring portions with modi®ed side chains.
con®guration was separated by chromatography as a
major product. The absolute con®guration at C-22 was
determined by X-ray crystallography of the diol 17
(Chart 4).14 Reductive elimination of a hydroxyl group
in 14 via the tosylate 15 produced the desired CD-ring
portion 16. Removal of both protecting groups in 16
with CSA a€orded the corresponding diol 17 in 95%
yield. The resulting secondary alcohol was oxidized with
PDC to give the ketone 18 in 99% yield. Finally,
bromomethylenation of 18 furnished the requisite 20-
epi-22R-methyl-CD-ring synthon 19. Each of the four
selected A-ring enyne synthons (20a, 20b, 20c and
20d),15 prepared separately as we previously reported,3
was coupled with the 20-epi-22R-methyl-CD-ring 19
using the palladium catalyst, followed by deprotection
to give the 2-methyl analogue (7a, 7b, 7c and 7d). In this
way, a set of four stereoisomers of 20-epi-2,22-dimethyl-
1,25-dihydroxyvitamin D3 was synthesized.
Scheme 1 outlines the synthesis of the 20-epi-22R-
methyl-CD-ring portion 1912 and the subsequent cou-
pling with an A-ring enyne 20a,3 as exempli®ed by the
aab-isomer, to produce the 2a-methyl analogue 7a. The
tosylate 95 of unnatural 20(S)-con®guration was con-
verted to the nitrile 10 in 99% yield using sodium cya-
nide. Condensation of 10 with the side-chain moiety
1113 using LDA as a base furnished 12 as an approxi-
mately 5:1 epimeric mixture at C-22 in 77% yield. Sub-
sequent reduction of this nitrile mixture 12 with
DIBAL-H, followed by NaBH4, a€orded the corre-
sponding C-22 epimeric alcohols in 79% yield (two
steps), from which the alcohol 14 of the desired 22(S)-
Scheme 2 shows the synthesis of the CD-ring portion of
KH-1060 (26), and the subsequent coupling with an
A-ring enyne 20a to a€ord the 2a-methyl substituted
Scheme 1. (a) NaCN/DMSO, 90 ꢀC, 99%; (b) LDA/THF, À78 to 0 ꢀC, 77%; (c) DIBAL-H/CH2Cl2, À10 ꢀC, 90%; (d) NaBH4/MeOH, rt, 74%;
(e) TsCl/pyridine, rt, 99%; (f) LiAlH4/ether, rt, 89%; (g) CSA/MeOH, rt, 95%; (h) PDC/CH2Cl2, rt, 99%; (i) Ph3P+CH2Brꢁ BrÀ, NaHMDS/THF,
50%; (j) Pd2(dba)3ꢁ CHCl3±PPh3±Et3N/toluene, re¯ux; (k) CSA/MeOH, rt.

528
T. Fujishima et al. / Bioorg. Med. Chem. 9 (2001) 525±535
KH-1060 analogue (8a). The synthetic approach to the
CD-ring ketone 25 was previously described in a com-
munication.16 Similar procedures and coupling condi-
tions were employed here. The alcohol 2117 was coupled
with the side-chain moiety 2218 using potassium hydride
as a base in the presence of 18-crown-6 ether to give the
protected diol 23 in 86% yield. Removal of both TBS
protecting groups in 23 by a quick treatment with TFA
furnished the diol 24 in 81% yield. Oxidation of 24 with
PDC, followed by bromomethylenation, a€orded the
requisite CD-ring portion 26. Finally, in the same man-
ner as described above, each of the four A-ring enyne
synthons (20a, 20b, 20c and 20d)15 was coupled with the
CD-ring 26 using the palladium catalyst, followed by
deprotection to give the 2-methyl substituted KH-1060
analogue (8a, 8b, 8c and 8d). Thus, syntheses of two sets
of four 2-methyl analogues of 20-epi-22R-methyl-1,25-
dihydroxyvitamin D3 (7a±d) and 20-epi-24,26,27-tri-
homo-22-oxa-1,25-dihydroxyvitamin D3 (8a±d) were
accomplished.
Biological Evaluation
The biological activities of the synthesized analogues
(7a±d, 8a±d) are summarized in Table 1 in comparison
with those of the natural hormone 1, together with the
2-methyl (3a±d)3,7 and 20-epi-2-methyl analogues (4a±
d).6,7 The activities were highly dependent not only on
the stereochemistry in the A-ring, but also on the side-
chain character. Thus, the double modi®cation yielded
analogues with unique activity pro®les.
In the vitamin D receptor binding assay using bovine
thymus VDR,19 7a and 7b, both of which have natural
hydroxyl con®gurations, exhibited 2-fold higher anity
than 1a,25-dihydroxyvitamin D3 irrespective of the
orientation of the 2-methyl group. The analogue 7c, the
3-epimer of 7a, showed rather high anity to VDR,
with half the potency of the natural hormone 1, whereas
7d exhibited low anity. The introduction of a 22R-
methyl group into 20-epi-1a,25-dihydroxyvitamin D3 (2)
produced 20-epi-22R-methyl-1a,25-dihydroxyvitamin D3
(5) with approximately 2.8-fold elevated VDRbinding
anity.8 Activation by 22R-methyl introduction was
also observed in the cases of 7b (versus 4b) and 7c (versus
Chart 4.
Scheme 2. (a) KH, 18-crown-6/THF, rt, 86%; (b) TFA/CH2Cl2, 0 ꢀC, 81%; (c) PDC/CH2Cl2, rt, 80%; (d) Ph3P+CH2Br ꢁ BrÀ, NaHMDS/THF,
33%; (e) Pd2(dba)3ꢁ CHCl3±PPh3±Et3N/toluene, re¯ux; (f) CSA/MeOH, rt.

T. Fujishima et al. / Bioorg. Med. Chem. 9 (2001) 525±535
529
Table 1. Biological activites of the 2-methyl analogues of 1a,25-
dihydroxyvitamin D3
di€erentiation-inducing activity in the case of the KH-
1060 analogues. The 2-epimerization of 8a reduced the
activity in cell di€erentiation by approximately 1/3,
while the 3-epimerization did so by 1/9. These results
revealed that the potency of the 2-methyl analogues
with KH-1060 side chains was a€ected by the A-ring
stereochemistry.
a
VDRb
HL-60c
VDRHL-60
400f
1
100
400d
13d
4d
100
258e
54e
22e
0e
2
3571f
3a
3b
3c
3d
(200)d 4a 1200g
9688e
1722e
705e
(59,000)g
(2600)g
(730)g
(10)d 4b
(13)d 4c
(3)d 4d
160g
17g
7g
0.8d
135e
(190)g
High potency of 5 in VDRbinding was explained by
anchoring of the 25-hydroxyl group to the right position
in the receptor due to the introduction of the 22R-
methyl group.8 The e€ect of the 22R-methyl substitu-
tion to restrict the side-chain conformation seems to be
independent of the A-ring or the CD-ring structure.
However, introduction of a 2a-methyl group into each
parent (1, 2, 5 and 6) a€orded analogues (3a, 4a and 8a)
with elevated VDRanity by 2- to 4-fold, except for
the case of the 20-epi-22R-methyl analogue (7a). The
combined modi®cation of 2a-methyl and 20-epi-22R-
methyl introduction resulted in an analogue with lower
anity than each parent, instead of a `super ligand' to
VDR. In addition, cell di€erentiation-inducing activities
of 7a±c suggested that the potency of the 20-epi-22R-
methyl analogues was barely a€ected by the orientation
of the 2-methyl or the 3-hydroxyl group, implying that
the double-modi®ed analogues would be dominated by
the side-chain character. These unique e€ects in hybrid
analogues might need to be explained by an alternative
concept. Further studies are required to elucidate the
structure±activity relationships of these compounds. On
the other hand, the cell di€erentiation-inducing activity
of KH-1060 (6) was elevated by the 2a-methyl substitu-
tion to yield 8a with 75-fold higher activity than 1. The
immunosuppresive e€ect of 8a, one of the characteristic
activities of KH-1060,4 would be of great interest.
5
1100h
200
200
50
20,000h
6190
5420
4330
58
6i
29
70
6430
7500
7a
7b
7c
7d
8a
8b
8c
8d
2.5 2610
0.5
<0.1
820
6
3
aThe potencies of 1a,25-dihydroxyvitamin D3 (1) are normalized to
100.
bBovine thymus.
cCell di€erentiation was assessed in terms of expression of CD11b.
Values in parentheses are results using NBT reductivity instead of
expression of CD11b.
dRef 3.
eRef 7.
fRef 5.
gRef 6.
hRef 8.
iFor comparison, 6 was synthesized and biologically evaluated in our
laboratory.
4c). As regards HL-60 cell di€erentiation-inducing
activities,20 7a and 7b showed 62- and 54-fold higher
potency than 1, respectively. Interestingly, 7c exhibited
43-fold higher activity than 1, having almost comparable
activity to 7a and 7b in potency. 3-Epimerization gen-
erally results in a marked reduction of potency and has
recently been reported to be involved in the metabolic
pathway not only of the natural hormone 1, but also of
several side-chain analogues.21 In the case of 7a, how-
ever, 3-epimerization would a€ord 7c with high cell-dif-
ferentiating activity, which might be advantageous for
in vivo administration. The cell-di€erentiation-inducing
activity of 7d was low but signi®cant, in spite of the 1b-
hydroxyl con®guration.
In summary, we have synthesized a total of eight
2-methyl analogues with side-chain modi®cations (7a±d,
8a±d) by employing the convergent method using palla-
dium catalyst. The above procedure should be versatile
for the synthesis of a variety of analogues, in particular
those with A-ring and/or side-chain modi®cations. Bio-
logical evaluation revealed that further modi®cation in
the side chain di€erentially a€ected the potency of the
2-methyl analogues depending on the side-chain char-
acter. Thus, the 2-methyl analogues with 20-epi-22R-
methyl side chains exhibited a reduced dependency on
the A-ring stereochemistry. In contrast, the potency of
the corresponding KH-1060 analogues was a€ected by
the A-ring stereochemistry and the double modi®cation
resulted in synergistic e€ects on HL-60 cell di€erentia-
tion-inducing activity. These novel analogues with high
potency should be useful tools in research on the biology
of vitamin D. The results of broad-spectrum biological
screening of the analogues will be reported in due course.
In VDRbinding assay using bovine thymus, 8a showed
70% anity while 8b showed 2.5% anity, compared
with 1. The VDRbinding anity of KH-1060 ( 6) rela-
tive to 1, normalized to 100, was reported to be 120 for
chick intestinal VDR.4b Our estimate using bovine thy-
mus VDRwas 29, and the lower anity of 6 may be
explained by the di€erence of the VDRsource. Intro-
duction of a 2a-methyl group into 6 to produce the
analogue 8a increased the VDRbinding activity. The
analogue 8c, the 3-epimer of 8a, showed low anity to
VDRand 8d exhibited virtually no anity. The rank
order of potency to induce HL-60 cell di€erentiation
among 8a±d was almost parallel to that of VDRbinding
anity. Although the KH-1060 analogues (8a±c)
appeared to be relatively poor ligands for VDR, their
cell di€erentiation-inducing activities were high. In the
case of 8a, which exhibited 70% anity to VDRrelative
to 1, the potency in cell di€erentiation was 75-fold
higher than that of 1. In contrast, the corresponding 20-
epi-22R-methyl analogue 7a exhibited 62-fold higher
activity in cell di€erentiation, although its VDRanity
was 2-fold higher than that of 1. Thus, the double
modi®cation resulted in synergistic e€ects on HL-60 cell
Experimental
Melting points were determined by using a Yanagimoto
hot-stage melting point apparatus and are uncorrected.
NMRspectra were recorded on a JEOL GSX-400
spectrometer. Chemical shifts are expressed in ppm

530
T. Fujishima et al. / Bioorg. Med. Chem. 9 (2001) 525±535
relative to tetramethylsilane. Mass spectra (EI) and
high-resolution mass spectra (HRMS) were recorded on
a JMS-SX 102A. Infrared spectra were recorded on a
Jasco FT/IR-8000 spectrometer and are expressed in
cmÀ1. Ultraviolet spectra were recorded with a Hitachi
200-10 spectrophotometer. Optical rotations were deter-
mined by using a Jasco DIP-370 digital polarimeter. Ele-
mental analyses were carried out in the Microanalytical
Laboratory, Faculty of Pharmaceutical Sciences, Uni-
versity of Tokyo, and were within 0.3% of the theore-
tical values. Recycling preparative HPLC was performed
on a Waters LC equipped with a 510 HPLC pump and
484 tunable absorbance detector. Crystallographic data
were collected on a Rigaku AFC7S di€ractometer with
graphite-monochromated Cu-Ka radiation.
(1HÂ1/6, m), 2.89 (1HÂ5/6, ddd, J=8.9, 5.5, 3.4 Hz),
3.36 (3H, s), 4.01 (1H, m), 4.69 (2H, s); 13C NMR
(100MHz, CDCl3) (major) d À5.3 (q), À4.9 (q), 13.9 (q),
14.3 (q), 17.5 (t), 17.9 (s), 22.6 (t), 25.6 (t), 25.7 (q), 25.9
(q), 26.5 (q), 27.2 (t), 34.1 (t), 36.4 (d), 36.6 (d), 40.0 (t),
40.3 (t), 41.9 (s), 52.8 (d), 54.3 (d), 55.2 (q), 69.3 (d), 75.6
(s), 91.1 (t), 121.4 (s), (minor) d À5.3 (q), À4.9 (q), 14.0 (q),
14.5 (q), 17.0 (t), 17.9 (s), 20.7 (t), 22.9 (t), 25.6 (t), 26.0 (q),
26.1 (q), 26.3 (q), 26.5 (t), 34.2 (t), 36.1 (d), 36.2 (d), 39.7
(t), 40.0 (t), 42.2 (s), 52.2 (d), 54.4 (d), 55.1 (d), 55.0 (q),
69.1 (d), 75.6 (s), 91.0 (t), 122.7 (s); FTIR(neat) 3021,
2934, 2251, 1472, 1387, 1215, 926, 837, 760 cmÀ1; MS
465 [M]+, 450 [MÀMe]+, 408 [MÀt-Bu]+; HR MS
calcd for [C27H51NO3Si] 465.3638, found 465.3637.
(20S,22RS)-De-A,B-8ꢁ-[(tert-butyldimethylsilyl)oxy]-22-
formyl-25-[(methoxymethyl)oxy]cholestane (13). To a
solution of 12 (940 mg, 2.02 mmol) in CH2Cl2 (15 mL)
was added DIBAL-H solution (0.95 M in n-hexane,
2.34 mL, 2.22 mmol) with stirring at À10 ꢀC under an
argon atmosphere. After stirring for 1 h, the reaction
was quenched by adding 4% aqueous HCl solution. The
whole was extracted with ethyl acetate. The organic
solution was washed with brine, dried over magnesium
sulfate, ®ltered and concentrated. The crude product
was separated by silica gel column chromatography
(ethyl acetate:n-hexane=8:92) to give 13 (846 mg) in
90% yield as a colorless oil.
(20S)-De-A,B-8ꢁ-[(tert-butyldimethylsilyl)oxy]-20-(cya-
nomethyl)pregnane (10). To a solution of 9 (1.70 g,
3.54mmol) in DMSO (30 mL) was added NaCN (370 mg,
7.08mmol). The resulting mixture was stirred at 90ꢀC for
4 h. The reaction mixture was cooled, then water was
added. After extraction of the mixture with ethyl acetate,
the organic layer was washed with brine, dried over mag-
nesium sulfate and ®ltered. Evaporation of the ®ltrate
a€orded a crude mixture, from which 10 (1.12 g) was
separated by silica gel column chromatography (ethyl
acetate:n-hexane=5:95) in 99% yield as a colorless oil.
[a]D=+21.1 (c=3.0, CHCl3); 1H NMR(400 MHz,
CDCl3) d 0.00 (3H, s), 0.01 (3H, s), 0.89 (9H, s), 0.92
(3H, s), 1.06 (3H, d, J=6.7 Hz), 2.38 (1H, dd, J=16.8,
6.7 Hz), 2.45 (1H, dd, J=16.7, 4.0 Hz), 4.00 (1H, m);
1H NMR(400 MHz, CDCl 3) d 0.00 (3H, s), 0.01 (3H, s),
0.72 (3HÂ1/6, d, J=7.0 Hz), 0.89 (9H, s), 0.90 (3HÂ5/6,
d, J=6.7 Hz), 0.96 (3HÂ5/6, s), 1.00 (3HÂ1/6, s), 2.46
(1HÂ1/6, m), 2.59 (1HÂ5/6, m), 3.36 (3H, s), 4.00 (1H,
m), 4.69 (2H, s), 9.26 (1HÂ1/6, s), 9.81 (1HÂ5/6, s); 13C
13C NMR(100 MHz, CDCl ) d À5.3 (q), À4.9 (q), 13.9
3
(q), 17.4 (t), 17.9 (s), 19.5 (q), 22.6 (t), 23.9 (t), 25.7 (q),
26.9 (t), 31.7 (d), 34.1 (t), 40.1 (t), 41.9 (s), 52.7 (d), 55.0
(d), 69.2 (d), 118.9 (s); FTIR(neat) 2932, 2858, 2251,
1462, 1253, 1167, 1084, 1022, 837, 810, 775 cmÀ1; MS
335 [M]+, 320 [MÀMe]+, 278 [MÀt-Bu]+; HR MS
calcd for [C20H37NOSi] 335.2644, found 335.2652.
NMR(100 MHz, CDCl ) (major) d À5.3 (q), À4.9 (q),
3
14.2 (q), 14.4 (q), 17.6 (t), 18.0 (s), 21.7 (t), 22.8 (t), 25.7
(q), 26.1 (q), 26.3 (q), 27.2 (t), 34.3 (t), 36.3 (d), 39.9 (t),
40.6 (t), 42.5 (s), 52.9 (d), 53.9 (d), 54.4 (d), 55.1 (q), 69.3
(d), 76.0 (s), 91.0 (t), 206.6 (d), (minor) d À5.3 (q), À4.9
(q), 14.3 (q), 14.7 (q), 17.0 (t), 18.0 (s), 21.1 (t), 22.9 (t),
26.0 (q), 26.2 (q), 26.5 (q), 27.0 (t), 34.4 (t), 36.0 (d), 40.0
(t), 40.2 (t), 42.3 (s), 52.7 (d), 52.8 (d), 53.0 (d), 55.0 (q),
69.4 (d), 76.1 (s), 90.9 (t), 205.3 (d); FTIR(neat) 3157,
2934, 1794, 1717, 1472, 1253, 1036, 909, 733 cmÀ1; MS 468
[M]+, 453 [MÀMe]+; HRMS calcd for [C27H52O4Si]
468.3634, found 468.3626.
(20S,22RS)-De-A,B-8ꢁ-[(tert-butyldimethylsilyl)oxy]-22-
cyano-25-[(methoxymethyl)oxy]cholestane (12). To
a
solution of diisopropylamine (0.92 mL, 6.56 mmol) in
THF (5 mL) was added n-BuLi solution (1.54 M in
n-hexane, 4.26 mL, 6.56 mmol) with stirring at 0 ꢀC
under an argon atmosphere. The resulting mixture was
stirred for 30 min at 0 ꢀC. The LDA solution was cooled
to À78 ꢀC, then a solution of 10 (1.10 g, 3.28 mmol) in
THF (7 mL) was added. After stirring for 30 min, a
solution of 11 (1.38 g, 6.56 mmol) in THF (10 mL) was
added, and the reaction mixture was stirred for 1 h at
À78 ꢀC. After an additional one hour at 0 ꢀC, the reac-
tion was quenched by adding saturated aqueous NH4Cl
solution. The whole was extracted with ethyl acetate.
The organic solution was washed with brine, dried over
magnesium sulfate, ®ltered and concentrated. The crude
product was separated by silica gel column chromato-
graphy (ethyl acetate:n-hexane=5:95) to give 12 (1.17 g)
in 77% yield as a 22-epimeric mixture.
(20S,22S)-De-A,B-8ꢁ-[(tert-butyldimethylsilyl)oxy]-22-
hydroxymethyl-25-[(methoxymethyl)oxy]cholestane (14).
A solution of 13 (810 mg, 1.73 mmol) in methanol
(10 mL) was treated with NaBH4 (134 mg, 3.46 mmol)
with stirring at 0 ꢀC. Stirring was continued for 2 h at
room temperature, then the reaction was quenched by
adding water. The whole was extracted with ethyl ace-
tate. The organic solution was washed with brine, dried
over magnesium sulfate, ®ltered and concentrated. The
crude product was separated by silica gel column chro-
matography (ethyl acetate:n-hexane=10:90) to give 14
(603 mg, 74%, more polar) and its 22-epimer (110 mg,
14%, less polar), both as colorless oils.
1H NMR(400 MHz, CDCl 3) d 0.00 (3H, s), 0.02 (3H, s),
0.89 (9H, s), 0.91 (3HÂ1/6, s), 0.93 (3HÂ5/6, s), 0.98
(3HÂ5/6, d, J=6.4 Hz), 1.03 (3HÂ1/6, d, J=7.0 Hz), 2.72
[a]D=+17.5 (c=0.92, CHCl3); 1H NMR(400 MHz,
CDCl3) d À0.01 (3H, s), 0.00 (3H, s), 0.73 (3H, d,

T. Fujishima et al. / Bioorg. Med. Chem. 9 (2001) 525±535
531
J=7.0 Hz), 0.88 (9H, s), 0.92 (3H, s), 1.22 (6H, s), 3.37
(3H, s), 3.41 (1H, dd, J=10.7, 8.2 Hz), 3.76 (1H, dd,
J=10.7, 3.7 Hz), 3.99 (1H, m), 4.71 (2H, q, J=7.3Hz);
J=6.7 Hz), 0.74 (3H, d, J=7.0 Hz), 0.89 (9H, s), 0.92
(3H, s), 1.21 (6H, s), 3.37 (3H, s), 4.00 (1H, m), 4.71
(2H, m); 13C NMR(100 MHz, CDCl ) d À5.3 (q), À4.9
3
13C NMR(100MHz, CDCl ) d À5.3 (q), À4.9 (q), 13.6
(q), 12.0 (q), 13.7 (q), 13.8 (q), 17.7 (t), 18.0 (s), 22.9 (t),
25.8 (q), 26.3 (q), 26.4 (q), 27.6 (t), 29.9 (t), 34.5 (t), 34.7
(d), 38.1 (d), 40.0 (t), 40.3 (t), 42.2 (s), 52.2 (d), 53.6 (d),
55.1 (q), 69.5 (d), 76.4 (s), 91.0 (t); FTIR(neat) 2934,
1381, 1253, 1165, 1096, 1038, 911, 733 cmÀ1; MS 439
[MÀMe]+, 393 [MÀOCH2OCH3]+; HRMS calcd for
[C26H51O3Si] 439.3608, found 439.3611.
3
(q), 13.8 (q), 17.6 (t), 18.0 (s), 22.9 (t), 24.4 (t), 25.8 (q),
26.2 (q), 26.5 (q), 27.5 (t), 34.4 (t), 35.8 (t), 39.8 (t), 40.3 (t),
42.4 (s), 43.2 (d), 53.1 (d), 53.6 (d), 54.1 (q), 63.6 (t), 69.4
(d), 76.5 (s), 91.0 (t); FTIR(neat) 3574, 3538, 3406, 3158,
2934, 1381, 1253, 1093, 909, 733 cmÀ1; MS 470 [M]+;
HRMS calcd for [C27H54O4Si] 470.3791, found 470.3798.
(22R)-Isomer: [a]D=+20.2 (c=1.11, CHCl3); 1H NMR
(400 MHz, CDCl3) d 0.00 (3H, s), 0.01 (3H, s), 0.75 (3H,
d, J=7.0 Hz), 0.89 (9H, s), 0.94 (3H, s), 1.16 (6H, s),
3.37 (3H, s), 3.51 (1H, dd, J=10.7, 7.9 Hz), 3.62 (1H,
dd, J=10.7, 4.9 Hz), 4.00 (1H, m), 4.72 (2H, q,
J=7.3 Hz); FTIR(neat) 3634, 3453, 2932, 2859, 1468,
(20S,22R)-De-A,B-22-methylcholestane-8ꢁ,25-diol (17).
A solution of 16 (300 mg, 0.66 mmol) in MeOH (5 mL)
was treated with CSA (306 mg, 1.32 mmol) at room
temperature. The reaction mixture was stirred at room
temperature for 2 days. After the reaction was com-
pleted, saturated aqueous NaHCO3 solution was added
to the mixture. The whole was extracted with ethyl ace-
tate. The organic layer was washed with brine, dried
over magnesium sulfate and ®ltered. Puri®cation by
silica gel column chromatography (ethyl acetate:n-hex-
ane=20:80) a€orded 17 (185 mg) in 95% yield as a
solid, which was recrystallized from ethyl acetate to give
analytically pure 17 as colorless prisms.
1381, 1038, 922, 872, 837 cmÀ1
.
(20S,22S)-De-A,B-8ꢁ-[(tert-butyldimethylsilyl)oxy]-25-
(methoxymethyl)oxy-22-[(p-tolylsulfonyl)oxymethyl]cho-
lestane (15). To a solution of 14 (780 mg, 1.66 mmol) in
pyridine (10 mL) was added p-tosyl chloride (475 mg,
2.49 mmol) with stirring at 0 ꢀC under an argon atmo-
sphere. The resulting mixture was stirred overnight at
room temperature. The reaction mixture was poured
into 4% aqueous HCl solution. After extraction with
ethyl acetate, the organic layer was washed with brine,
dried over magnesium sulfate and ®ltered. Evaporation
of the ®ltrate a€orded a crude mixture, from which 15
(1.02 g) was separated by silica gel column chromato-
graphy (ethyl acetate:n-hexane=10:90) in 99% yield as
a colorless oil.
Mp 139±141 ꢀC (recryst. from ethyl acetate); [a]D=+
1
36.7 (c=0.91, CHCl3); H NMR(400 MHz, CDCl ) d
3
0.69 (3H, d, J=6.7 Hz), 0.76 (3H, d, J=6.7 Hz), 0.93
(3H, s), 1.21 (6H, s), 1.96 (1H, m), 4.08 (1H, m); 13C
NMR(100 MHz, CDCl ) d 12.0 (q), 13.5 (q), 13.7 (q),
3
17.4 (t), 22.3 (t), 27.3 (t), 29.1 (q), 29.3 (q), 30.2 (t),
33.6 (t), 34.7 (d), 38.0 (d), 40.0 (t), 41.9 (s), 42.1 (t), 52.7
(d), 53.4 (d), 69.4 (d), 71.1 (s); FTIR(neat) 3339, 2923,
1456, 1375, 1266, 1161, 1113, 1069, 1034, 987, 906,
729 cmÀ1; MS 278 [MÀH2O]+, 260 [MÀ2H2O]+;
HRMS calcd for [C19H34O] 278.2610, found 278.2611.
Anal. calcd for C19H36O2: C, 76.97; H, 12.24; found: C,
77.06; H, 12.01.
[a]D=+3.53 (c=0.85, CHCl3); 1H NMR(400 MHz,
CDCl3) d À0.02 (3H, s), 0.00 (3H, s), 0.67 (3H, d,
J=7.0 Hz), 0.86 (3H, s), 0.87 (9H, s), 1.18 (6H, s), 1.91
(1H, m), 2.44 (3H, s), 3.35 (3H, s), 3.82 (1H, t,
J=8.9 Hz), 3.95 (1H, m), 4.07 (1H, dd, J=9.2, 3.7 Hz),
4.67 (2H, m), 7.34 (2H, d, J=7.9 Hz), 7.79 (2H, d,
(20S,22R)-De-A,B-25-hydroxy-22-methylcholestan-8-one
(18). Pyridinium dichromate (PDC, 1.04 g, 2.76 mmol)
was added to a stirred mixture of 17 (205 mg, 0.69
mmol) in dry CH2Cl2 (10 mL) at room temperature
under argon. The mixture was stirred at room tempera-
ture for 4 h, and separated by silica gel column chro-
matography (ethyl acetate:n-hexane=5:95) to give the
corresponding ketone 18 (200 mg) as a colorless oil in
99% yield.
J=7.9 Hz); 13C NMR(100 MHz, CDCl ) d À5.3 (q),
3
À4.9 (q), 13.2 (q), 13.6 (q), 17.5 (t), 17.9 (s), 21.5 (q),
22.7 (t), 24.0 (t), 25.7 (q), 26.2 (q), 26.5 (q), 27.6 (t), 34.3
(t), 35.1 (d), 39.8 (t), 39.4 (t), 39.7 (d), 39.9 (t), 42.2 (s),
52.9 (d), 53.4 (d), 55.1 (d), 69.3 (d), 71.8 (t), 76.0 (s), 91.0
(t), 128.1 (d), 129.8 (d), 133.2 (s), 144.6 (s); FTIR(neat)
2932, 1531, 1468, 1176, 1096, 1038, 910, 733 cmÀ1
.
(20S,22R)-De-A,B-8ꢁ-[tert-butyldimethylsilyl)oxy]-25-
[(methoxymethyl)oxy]-22-methylcholestane (16). A solu-
tion of 15 (420 mg, 0.67 mmol) in dry ether (10 mL) was
treated with LiAlH4 (250 mg, 6.7 mmol) with stirring at
0 ꢀC. The resulting suspension was allowed to stand
overnight with stirring at room temperature. The mix-
ture was diluted with ether and the whole was ®ltered
through a small pad of silica gel. Evaporation of the
®ltrate a€orded a residue, from which 16 (271 mg, 89%)
and 14 (30 mg, 9.5%) were separated by silica gel col-
umn chromatography (ethyl acetate:n-hexane=5:95),
both as colorless oils.
[a]D=À7.9 (c=1.77, CHCl3); 1H NMR(400 MHz,
CDCl3) d 0.63 (3H, s), 0.73 (3H, d, J=6.7 Hz), 0.81 (3H, d,
J=7.0 Hz), 1.22 (6H, s), 2.23 (2H, m), 2.46 (1H, dd,
J=11.3, 7.6 Hz); 13C NMR(100 MHz, CDCl 3) d 12.0 (q),
12.3 (q), 13.8 (q), 18.8 (t), 23.9 (t), 27.6 (t), 29.1 (q), 29.3 (q),
30.1 (t), 35.0 (d), 38.2 (d), 38.6 (t), 40.9 (t), 42.0 (t), 49.9 (s),
53.6 (d), 62.0 (d), 71.0 (s), 212.2 (s); FTIR(neat) 3158,
2967, 2878, 1705, 1469, 1385, 1219, 1098, 908, 740 cmÀ1
;
MS 294 [M]+, 276 [M±H2O]+, 261 [M±H2OÀMe]+;
HRMS calcd for [C19H34O2] 294.2559, found 294.2555.
(E)-(20S,22R)-De-A,B-8-bromomethylene-22-methylcho-
lestan-25-ol (19). Sodium hexamethyldisilazide (NaH-
MDS) (1.0 M in THF, 3.40 mL, 3.40 mmol) was
[a]D=+17.9 (c=1.54, CHCl3); 1H NMR(400 MHz,
CDCl3) d 0.00 (3H, s), 0.01 (3H, s), 0.68 (3H, d,

532
T. Fujishima et al. / Bioorg. Med. Chem. 9 (2001) 525±535
added to (bromomethyl)triphenylphosphonium bromide
(1.55 g, 3.55 mmol) in THF (1.5 mL) at À60 ꢀC under
argon. After 1 h, a solution of 18 (210 mg, 0.71 mmol) in
THF (5 mL) was added. After an additional one hour at
room temperature, the reaction mixture was diluted
with n-hexane and the whole was ®ltered over Celite1.
The ®ltrate was concentrated, then puri®ed by silica
gel column chromatography (ethyl acetate:n-hex-
ane=10:90) to give 19 (132 mg) as a colorless oil in
50% yield.
0.54 (3H, s), 0.71 (3H, d, J=6.1 Hz), 0.78 (3H, d,
J=6.7 Hz), 1.15 (3H, d, J=6.7 Hz), 1.21 (6H, s), 2.42
(1H, dd, J=13.7, 4.9 Hz), 2.52 (1H, d, J=13.7 Hz), 2.83
(1H, m), 4.03 (2H, m), 5.02 (1H, s), 5.37 (1H, s), 6.04
(1H, d, J=11.0 Hz), 6.35 (1H, d, J=11.0 Hz); MS 444
[M]+, 426 [MÀH2O]+, 408 [MÀ2H2O]+, 393
[MÀ2H2OÀMe]+, 375 [MÀ3H2O-Me]+; HRMS calcd
for [C29 H48O3] 444.3603, found 444.3606.
(5Z,7E)-(1S,2S,3S,20S,22R)-2,22-Dimethyl-9,10-seco-
5,7,10(19)-cholestatriene-1,3,25-triol (7c). This com-
pound was obtained by the same procedure as described
for 7a using 20c instead of 20a, in 31% yield.
[a]D=+87.4 (c=0.54, CHCl3); 1H NMR(400 MHz,
CDCl3) d 0.55 (3H, s), 0.71 (3H, d, J=6.4 Hz), 0.78 (3H,
d, J=6.7 Hz), 1.21 (6H, s), 2.87 (1H, m), 5.65 (1H, s);
13C NMR(100 MHz, CDCl ) d 11.7 (q), 12.1 (q), 13.8
[a]D=+86.8 (c=0.04, CHCl3); UV (EtOH) lmax
264 nm, lmin 228 nm; 1H NMR(400 MHz, CDCl 3) d
0.53 (3H, s), 0.71 (3H, d, J=5.8 Hz), 0.78 (3H, d,
J=6.7 Hz), 1.21 (6H, s), 2.49 (1H, d, J=13.7 Hz), 2.58
(1H, dd, J=13.7, 4.0 Hz), 2.83 (1H, m), 3.84 (1H, m),
4.18 (1H, m), 4.98 (1H, d, J=1.8 Hz), 5.23 (1H, d,
J=1.8 Hz), 6.04 (1H, d, J=11.0 Hz), 6.48 (1H, d,
J=11.0 Hz); MS 444 [M]+, 426 [MÀH2O]+, 408
[MÀ2H2O]+, 393 [MÀ2H2OÀMe]+; HRMS calcd for
[C29H48O3] 444.3603, found 444.3603.
3
(q), 21.8 (t), 22.5 (t), 27.7 (t), 29.1 (q), 29.3 (q), 30.2 (t),
31.0 (t), 35.0 (d), 38.7 (d), 39.5 (t), 42.1 (t), 45.5 (s), 52.7
(d), 55.9 (d), 71.1 (s), 97.5 (d), 145.2 (s); FTIR(neat)
3381, 2964, 2872, 1632, 1466, 1377, 1088 cmÀ1; MS 370
[M]+; HRMS calcd for [C20H35O79Br] 370.1870, found
370.1871.
(5Z,7E)-(1S,2S,3R,20S,22R)-2,22-Dimethyl-9,10-seco-
5,7,10(19)-cholestatriene-1,3,25-triol (7a). A mixture of
ꢁ
(dba)3Pd2 CHCl3 (4.0 mg, 0.0039 mmol), Ph3P (9.0 mg,
0.034 mmol) and triethylamine (1 mL) in toluene (1 mL)
was stirred for 10 min at room temperature, then a
solution of the A-ring moiety 20a3 (15 mg, 0.039 mmol)
and the CD-ring moiety 19 (22 mg, 0.059 mmol) in tol-
uene (0.5 mL) was added. After having been heated at
re¯ux for 6 h and diluted with pentane, the reaction
mixture was ®ltered through a pad of silica gel with
ether. After evaporation of the solvent, the crude
mixture was dissolved in methanol (1.5 mL) and trea-
ted with CSA (9.0 mg, 0.039 mmol) at room tempera-
ture for 2 days. After removal of the solvent, the
residue was subjected to silica gel column chromato-
graphy (ethyl acetate:n-hexane=50:50) to give 7a
(5.4 mg) as a colorless oil in 30% yield. Further pur-
i®cation for biological evaluation was conducted by
using reversed-phase recycle HPLC (YMC-Pack ODS
column, 20 mmÂ150 mm, 9.0 ml/min, acetonitrile:
water=85:15).
(5Z,7E)-(1R,2R,3S,20S,22R)-2,22-Dimethyl-9,10-seco-
5,7,10(19)-cholestatriene-1,3,25-triol (7d). This com-
pound was obtained by the same procedure as described
for 7a using 20d instead of 20a, in 46% yield.
[a]D=À56.5 (c=0.23, CHCl3); UV (EtOH) lmax
265 nm, lmin 226 nm; 1H NMR(400 MHz, CDCl 3) d
0.54 (3H, s), 0.71 (3H, d, J=5.8 Hz), 0.78 (3H, d,
J=6.7 Hz), 1.11 (3H, d, J=6.7 Hz), 1.21 (6H, s), 2.24
(1H, m), 2.66 (1H, dd, J=13.1, 4.0 Hz), 2.82 (1H, dd,
J=12.5, 4.0 Hz), 3.82 (1H, m), 4.27 (1H, m), 5.02 (1H,
d, J=1.5 Hz), 5.28 (1H, d, J=1.5 Hz), 6.02 (1H, d,
J=11.3 Hz), 6.39 (1H, d, J=11.3 Hz); MS 444 [M]+,
426 [MÀH2O]+, 408 [MÀ2H2O]+, 393 [MÀ2H2O±
Me]+, 375 [MÀ3H2O±Me]+; HRMS calcd for
[C29H48O3] 444.3603, found 444.3606.
(20R)-De-A,B-8ꢁ-[(tert-butyldimethylsilyl)oxy]pregnan-
20-ol (21).17 1H NMR(400 MHz, CDCl ) d À0.01 (3H,
3
[a]D=+10.0 (c=0.11, CHCl3); UV (EtOH) lmax 265
s), 0.01 (3H, s), 0.89 (9H, s), 1.00 (3H, s), 1.12 (3H, d,
J=6.1 Hz), 3.74 (1H, m), 4.01 (1H, d, J=2.4 Hz); MS
312 [M]+, 297 [MÀMe]+; HRMS calcd for
[C18H36O2Si] 312.2485, found 312.2465.
1
nm, lmin 228 nm; H NMR(400 MHz, CDCl ) d 0.53
3
(3H, s), 0.71 (3H, d, J=5.8 Hz), 0.78 (3H, d, J=6.7 Hz),
1.08 (3H, d, J=7.0 Hz), 1.21 (6H, s), 2.23 (1H, dd,
J=13.1, 7.9 Hz), 2.67 (1H, dd, J=13.1, 4.0 Hz), 2.83
(1H, m), 3.85 (1H, m), 4.31 (1H, m), 5.01 (1H, d,
J=1.5 Hz), 5.28 (1H, d, J=1.5 Hz), 6.02 (1H, d,
J=11.3 Hz), 6.39 (1H, d, J=11.3 Hz); MS 444 [M]+,
426 [MÀH2O]+, 408 [MÀ2H2O]+, 393 [MÀ
2H2OÀMe]+, 375 [MÀ3H2OÀMe]+; HRMS calcd for
[C29H48O3] 444.3603, found 444.3603.
1
(20S)-Isomer. H NMR(400 MHz, CDCl ) d 0.01 (3H,
3
s), 0.02 (3H, s), 0.88 (9H, s), 0.97 (3H, s), 1.20 (3H, d,
J=6.1 Hz), 3.68 (1H, m), 3.99 (1H, d, J=2.7 Hz); MS
312 [M]+, 297 [MÀMe]+; HRMS calcd for [C18H36O2Si]
312.2485, found 312.2483.
(20R)-De-A,B-8ꢁ,25-bis[(tert-butyldimethylsilyl)oxy]-
24,26,27-trihomo-22-oxacholestane (23). To a stirred
suspension of potassium hydride (30%, 200 mg,
3.30 mmol) and 18-crown-6 ether (698 mg, 2.64 mmol)
in THF (5 mL) was added 21 (688 mg, 2.20 mmol) under
an argon atmosphere. The mixture was stirred for
15 min at room temperature, then a solution of side-
chain moiety 22 (1.30 g, 3.52 mmol) in THF (5 mL) was
(5Z,7E)-(1S,2R,3R,20S,22R)-2,22-Dimethyl-9,10-seco-
5,7,10(19)-cholestatriene-1,3,25-triol (7b). This com-
pound was obtained by the same procedure as described
for 7a using 20b instead of 20a, in 23% yield.
[a]D=À79.1 (c=0.12, CHCl3); UV (EtOH) lmax
263 nm, lmin 227 nm; 1H NMR(400 MHz, CDCl 3) d

T. Fujishima et al. / Bioorg. Med. Chem. 9 (2001) 525±535
533
added dropwise to it. The resulting mixture was stirred
overnight at room temperature, then cooled and water
was added to destroy the excess reagent. After extrac-
tion with ethyl acetate, the organic phase was washed
with brine, dried over magnesium sulfate and ®ltered.
Evaporation of the ®ltrate gave a residue, from which
23 (1.03 g) was separated by silica gel column chroma-
tography (ethyl acetate:n-hexane=1:4) in 86% yield as
a colorless oil.
(1.0 M in THF, 1.6 mL, 1.6 mmol) at À60 ꢀC under
argon. The mixture was stirred at À60 ꢀC for 1 h, then a
solution of 25 (107 mg, 0.33 mmol) in THF (2 mL) was
added to it. The resulting solution was stirred at À60 ꢀC
for 70 min, then at room temperature for 2 h. The reac-
tion was quenched by addition of a small amount of
water. After extraction 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:hexane
=1:5) to a€ord the desired ole®n 26 (44 mg) as a pale
yellow oil in 33% yield.
1H NMR(400 MHz, CDCl ) d À0.01 (3H, s), 0.01 (3H,
3
s), 0.06 (6H, s), 0.815 (3H, t, J=7.6 Hz), 0.823 (3H, t,
J=7.6 Hz), 0.86 (9H, s), 0.89 (9H, s), 0.94 (3H, s), 1.04
(3H, d, J=5.8 Hz), 3.12 (1H, m), 3.25 (1H, dq, J=9.8,
6.1 Hz), 3.56 (1H, m), 4.00 (1H, m); MS 525 [MÀEt]+;
HRMS calcd for [C30H61O3Si2] 525.4159, found
525.4155.
1H NMR(400 MHz, CDCl ) d 0.57 (3H, s), 0.84 (3H, t,
3
J=7.6 Hz), 0.85 (3H, t, J=7.3 Hz), 1.09 (3H, d,
J=5.8 Hz), 1.46 (4H, m), 2.00 (1H, ddd, J=12.2, 6.7,
1.5 Hz), 2.16 (1H, m), 2.88 (1H, m), 3.22 (1H, dt, J=8.9,
6.1 Hz), 3.27 (1H, dq, J=10.1, 5.8 Hz), 3.56 (1H, dt,
J=8.9, 6.1 Hz), 5.63 (1H, t, J=1.5 Hz); 13C NMR
(100 MHz, CDCl3) d 7.8 (q), 7.9 (q), 12.5 (q), 18.2 (q),
22.2 (t), 22.5 (t), 24.2 (t), 25.0 (t), 30.9 (t), 31.1 (t), 35.6
(t), 39.7 (t), 45.5 (s), 55.4 (d), 56.1 (d), 68.8 (t), 74.1 (s),
78.1 (d), 97.2 (d), 145.2 (s); FTIR(neat) 3447, 2964,
2876, 2363, 1456, 1371, 1336, 1130, 1105, 949 cmÀ1; MS
371 [MÀEt]+; HRMS calcd for [C19H32O279Br]
371.1586, found 371.1585.
(20R)-De-A,B-24,26,27-trihomo-22-oxacholestane-8ꢁ,
25-diol (24). A solution of 23 (240 mg, 0.44 mmol) in
CH2Cl2 (4 mL) was treated with TFA (1.5 mL) at 0 ꢀC
with stirring. The mixture was stirred for 45 min, then
diluted with water and extracted with ethyl acetate. The
organic layer was washed with brine, and dried over
magnesium sulfate. Evaporation of the ®ltrate a€orded
a residue, from which 24 (116 mg) was separated by
silica gel column chromatography (ethyl acetate:hex-
ane=1:4) in 81% as a colorless oil.
(5Z,7E)-(1S,2S,3R,20R)-24,26,27-Trihomo-2-methyl-22-
oxa-9,10-seco-5,7,10(19)-cholestatriene-1,3,25-triol (8a).
1H NMR(400 MHz, CDCl 3) d 0.871 (3H, t, J=7.6 Hz),
0.874 (3H, t, J=7.6 Hz), 0.95 (3H, s), 1.06 (3H, d,
J=5.8 Hz), 1.92 (2H, q, J=7.6 Hz), 1.94 (2H, q,
J=7.6 Hz), 2.10 (1H, m), 3.16 (1H, dt, J=8.9, 6.4 Hz),
3.28 (1H, dq, J=9.8, 5.8 Hz), 3.57 (1H, dt, J=8.9,
6.4 Hz), 4.10 (1H, m); MS 308 [MÀH2O]+; HR MS
calcd for [C20H36O2] 308.2715, found 308.2716.
A mixture of (dba)3Pd2 CHCl3 (6.0 mg, 0.0062 mmol),
ꢁ
Ph3P (15 mg, 0.056 mmol) and triethylamine (1 mL) in
toluene (0.5 mL) was stirred for 10 min at rt, then a
solution of the A-ring moiety 20a (30 mg, 0.076 mmol)
and the CD-ring moiety 26 (25 mg, 0.062 mmol) in tol-
uene (0.5 mL) was added. After having been heated at
re¯ux for 6 h and diluted with pentane, the reaction mix-
ture was ®ltered through a pad of silica gel with ether.
After evaporation of the solvent, the crude mixture was
dissolved in methanol (2 mL) and treated with CSA
(6.0 mg, 0.026 mmol) at room temperature overnight.
After removal of the solvent, the residue was subjected to
silica gel column chromatography (ethyl acetate:n-hex-
ane=2:3) to give 8a (1.7 mg) as a colorless oil in 6%
yield. Further puri®cation for biological evaluation was
conducted by using reversed-phase recycle HPLC
(YMC-Pack ODS column, 20 mmÂ150 mm, 9.0 mL/
min, acetonitrile:water= 80:20).
(20R)-De-A,B-24,26,27-trihomo-25-hydroxy-22-oxacho-
lestan-8-one (25). A stirred mixture of 24 (164 mg,
0.50 mmol) and powdered 4 A MS (60 mg) in CH2Cl2
(4 mL) was treated with PDC (472 mg, 1.25 mmol) at
room temperature. The mixture was stirred at room
temperature for 1 h under argon, and separated by silica
gel column chromatography (ethyl acetate:hexane=1:6)
to give the corresponding ketone 25 (130 mg, 80%) as a
colorless oil.
1H NMR(400 MHz, CDCl ) d 0.55 (3H, s), 0.777 (3H,
3
t, J=7.6 Hz), 0.781 (3H, t, J=7.6 Hz), 0.90 (3H, d,
J=5.8 Hz), 1.83 (4H, q, J=7.6 Hz), 2.69 (1H, dd,
J=11.0, 7.3 Hz), 3.07 (1H, dt, J=9.2, 6.2 Hz), 3.17 (1H,
m), 3.49 (1H, dt, J=9.2, 6.2 Hz); 13C NMR(100 MHz,
CDCl3) d 7.4 (q), 7.5 (q), 12.9 (q), 18.2 (q), 19.4 (t), 24.0
(t), 24.1 (t), 25.1 (t), 27.2 (t), 31.4 (t), 39.0 (t), 41.2 (t),
49.9 (s), 56.8 (d), 61.5 (d), 67.8 (t), 77.6 (d), 94.8 (s),
212.0 (s); FTIR(neat) 3750, 2968, 2880, 1776, 1714,
1458, 1373, 1336, 1217, 1169 cmÀ1; MS 306 [MÀH2O]+;
HRMS calcd for [C20H34O2] 306.2559, found 306.2559.
[a]2D2=+253 (c=0.00316, EtOH); UV (EtOH) lmax
265 nm, lmin 226 nm; 1H NMR(400 MHz, CDCl 3) d
0.55 (3H, s), 0.848 (3H, t, J=7.6 Hz), 0.853 (3H, t,
J=7.3 Hz), 1.08 (6H, d, J=6.4 Hz), 1.46 (4H, m), 1.91
(1H, ddq, J=3.4, 7.3, 6.4 Hz), 2.01 (1H, t, J=9.8 Hz),
2.14 (1H, d, J=12.2 Hz), 2.23 (1H, dd, J=13.4, 7.9 Hz),
2.67 (1H, dd, J=13.4, 4.6 Hz), 2.83 (1H, dd, J=12.5,
4.0 Hz), 3.22 (1H, dt, J=8.9, 5.8 Hz), 3.26 (1H, dq,
J=9.8, 6.4 Hz), 3.57 (1H, dt, J=8.9, 6.1 Hz), 3.84 (1 H,
m), 4.29 (1H, m), 5.01 (1H, d, J=1.8 Hz), 5.27 (1H, d,
J=0.9 Hz), 5.99 (1H, d, J=11.3 Hz), 6.40 (1H, d,
J=11.3 Hz); FTIR(neat) 3387, 2964, 2876, 1454, 1373,
1332, 1103, 949 cmÀ1; MS 474 [M]+, 456 [MÀH2O]+,
438 [MÀ2H2O]+, 420 [MÀ3H2O]+; HRMS calcd for
[C30H50O4] 474.3709, found 474.3718.
(20R)-De-A,B-8-bromomethylene-24,26,27-trihomo-22-
oxacholestan-25-ol (26). A stirred suspension of (bro-
momethyl)triphenylphosphonium bromide (719 mg,
1.65 mmol) in THF (4 mL) was treated with NaHMDS

534
T. Fujishima et al. / Bioorg. Med. Chem. 9 (2001) 525±535
(5Z,7E)-(1S,2R,3R,20R)-24,26,27-Trihomo-2-methyl-22-
oxa-9,10-seco-5,7,10(19)-cholestatriene-1,3,25-triol (8b).
This compound was obtained by the same procedure as
described for 8a using 20b instead of 20a, in 38% yield.
J=11.3 Hz); FTIR(neat) 3406, 2964, 2876, 1649, 1454,
1371, 1332, 1103, 972, 949 cmÀ1; MS 474 [M]+, 456
[MÀ H2O]+, 438 [MÀ2H2O]+; HRMS calcd for
[C30H50O4] 474.3709, found 474.3711.
[a]2D4=+434 (c=0.00115, EtOH); UV (EtOH) lmax
263 nm, lmin 227 nm; 1H NMR(400 MHz, CDCl 3) d
0.56 (3H, s), 0.848 (3H, t, J=7.6 Hz), 0.853 (3H, t,
J=7.6 Hz), 1.09 (3H, d, J=5.8 Hz), 1.15 (3H, d,
J=7.0 Hz), 1.46 (4H, m), 1.79 (1H, ddq, J=2.6, 9.2,
7.0 Hz), 2.00 (1H, t, J=9.9 Hz), 2.16 (1H, d, J=
12.5 Hz), 2.42 (1H, dd, J=13.4, 5.2 Hz), 2.52 (1H, dd,
J=13.4 Hz), 2.83 (1H, dd, J=12.2, 4.0 Hz), 3.22 (1H,
dt, J=8.9, 5.6 Hz), 3.27 (1H, dq, J=9.8, 6.1 Hz), 3.56
(1H, dt, J=8.9, 6.1 Hz), 4.00 (1H, m), 4.02 (1H, m), 5.02
(1H, t, J=1.8 Hz), 5.37 (1H, t, J=1.8 Hz), 6.01 (1H, d,
J=11.3 Hz), 6.35 (1H, d, J=11.3 Hz); FTIR(neat)
3406, 2963, 2930, 2876, 2235, 1639, 1456, 1373, 1332,
1217, 1105, 993, 949 cmÀ1; MS 474 [M]+, 456 [MÀ
H2O]+, 438 [MÀ2H2O]+, 420 [MÀ3H2O]+; HR MS
calcd for [C30H50O4] 474.3709, found 474.3710.
(5Z,7E)-(1S,3R,20R)-24,26,27-Trihomo-22-oxa-9,10-
seco-5,7,10(19)-cholestatriene-1,3,25-triol (6).4 For com-
parison, KH-1060 (6) was synthesized by the same pro-
cedure as described for 8a using (3S,5R)-3,5-bis[(tert-
butyldimethylsilyl)oxy]oct-1-en-7-yne5 instead of 20a, in
30% yield.
UV (EtOH) lmax 262 nm, lmin 226 nm; 1H NMR
(400 MHz, CDCl3) d 0.56 (3H, s), 0.848 (3H, t, J=
7.3 Hz), 0.852 (3H, t, J=7.6 Hz), 1.08 (6H, d, J=
6.1 Hz), 1.46 (4H, m), 2.31 (1H, dd, J=13.1, 6.4 Hz),
2.60 (1H, dd, J=13.1, 3.7 Hz), 2.83 (1H, dd, J=11.9,
4.0 Hz), 3.22 (1H, dt, J=8.9, 6.1 Hz), 3.28 (1H, dq,
J=9.8, 5.8 Hz), 3.60 (1H, dt, J=8.9, 6.1 Hz), 4.23 (1H,
m), 4.43 (1H, m), 5.00 (1H, m), 5.32 (1H, t, J=1.5 Hz),
5.99 (1H, d, J=11.0 Hz), 6.39 (1H, d, J=11.0 Hz).
(5Z,7E)-(1S,2S,3S,20R)-24,26,27-Trihomo-2-methyl-22-
oxa-9,10-seco-5,7,10(19)-cholestatriene-1,3,25-triol (8c).
This compound was obtained by the same procedure
as described for 8a using 20c, instead of 20a in 40%
yield.
X-ray crystallographic analysis of 17. A colorless pris-
matic crystal with dimensions of 0.30 Â 0.10 Â 0.40mm
was obtained by recrystallization from ethyl acetate. The
observed cell parameters were as follows: C19H36O2,
Mr=296.49, orthorhombic, P21212, a=9.911(14) A,
b=19.205(10) A, c=9.825(15) A, V=1870(6) A3, Z=4,
Dx=1.053g/cm3, l(CuKa)=1.54178 A, m(CuKa)=
5.01cmÀ1, F(000)=664.00, 23 ꢀC. The structure was
solved by direct methods and expanded using Fourier
techniques. The non-hydrogen atoms were re®ned ani-
sotropically by full matrix least-squares calculations.
Hydrogen atoms were included but not re®ned.
R=0.079, Rw=0.097 for 1136 re¯ections (I>2.00 s(I)).
[a]2D2=+534 (c=0.00187, EtOH); UV (EtOH) lmax
265 nm, lmin 226 nm; 1H NMR(400 MHz, CDCl 3) d
0.55 (3H, s), 0.849 (3H, t, J=7.6 Hz), 0.855 (3H, t,
J=7.3 Hz), 1.09 (3H, d, J=5.8 Hz), 1.22 (3H, d,
J=7.3 Hz), 1.47 (4H, m), 1.92 (1H, tq, J=2.7, 7.3 Hz),
2.01 (1H, t, J=9.5 Hz), 2.16 (1H, d, J=8.9 Hz), 2.50
(1H, d, J=13.7 Hz), 2.58 (1H, dd, J=13.7, 4.0 Hz), 2.86
(1H, dd, J=12.5, 4.0 Hz), 3.22 (1H, dt, J=8.9, 5.8 Hz),
3.27 (1H, dq, J=9.8, 5.8 Hz), 3.56 (1H, dt, J=8.9,
6.4 Hz), 3.91 (1H, m), 4.17 (1H, m), 4.97 (1H, d,
J=2.1 Hz), 5.23 (1H, d J=1.8 Hz), 6.01 (1H, d,
J=11.3 Hz), 6.48 (1H, d, J=11.3 Hz); FTIR(neat)
3383, 2964, 2876, 2365, 2239, 1736, 1649, 1456, 1373,
1334, 1271, 1217, 1062, 1030, 970, 949 cmÀ1; MS 474
[M]+, 456 [MÀH2O]+, 438 [MÀ2H2O]+, 420 [MÀ
3H2O]+; HRMS calcd for [C30H50O4] 474.3709, found
474.3711.
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,
England) was then counted.
(5Z,7E)-(1R,2R,3S,20R)-24,26,27-Trihomo-2-methyl-22-
oxa-9,10-seco-5,7,10(19)-cholestatriene-1,3,25-triol (8d).
This compound was obtained by the same procedure as
described for 8a using 20d instead of 20a, in 10% yield.
[a]2D1=+114 (c=0.00262, EtOH); UV (EtOH) lmax
264 nm, lmin 226 nm; 1H NMR(400 MHz, CDCl 3) d
0.56 (3H, s), 0.848 (3H, t, J=7.6 Hz), 0.853 (3H, t,
J=7.6 Hz), 1.09 (3H, d, J=6.1 Hz), 1.10 (3H, d,
J=7.3 Hz), 1.46 (4H, m), 1.86 (1H, ddq, J=3.4, 8.2,
7.3 Hz), 2.00 (1H, t, J=9.1 Hz), 2.15 (1H, d, J=
12.4 Hz), 2.24 (1H, dd, J=13.4, 8.2 Hz), 2.67 (1H, dd,
J=13.4, 4.3 Hz), 2.83 (1H, dd, J=12.2, 4.0 Hz), 3.22
(1H, dt, J=8.5, 6.1 Hz), 3.27 (1H, dq, J=9.8, 5.8 Hz),
3.56 (1H, dt, J=8.5, 6.1 Hz), 3.82 (1H, dt, J=8.2,
4.3 Hz), 4.27 (1H, m), 5.01 (1H, d, J=2.1 Hz), 5.27 (1H,
d J=1.8 Hz), 6.00 (1H, d, J=11.3 Hz), 6.40 (1H, d,
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 10À12 and 10À7 M 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-buf-
fered 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

T. Fujishima et al. / Bioorg. Med. Chem. 9 (2001) 525±535
535
monoclonal
¯uorescein
isothiocyanate-conjugated
Konno, K.; Nakagawa, K.; Okano, T.; Takayama, H. Bioorg.
Med. Chem. Lett. 2000, 10, 1129.
Â
12. Vitale, C.; Fall, Y.; Carril, A. G.; Rey, M. A.; Moman, E.;
CD11b antibody for 30 min at room temperature in the
dark. The cells were washed once with Diluent solution
and then ®xed in 500 mL of PBS containing 2% para-
formaldehyde. Fluorescence was read on a Becton
Dickinson FACSanTM at an excitation wavelength of
490 nm and emission wavelength of 520 nm. Results
were recorded as the mean ¯uorescence index, which
is the product of the % ¯uorescence and the mean
¯uorescence intensity, with 104 cells being counted per
treatment.
Mourino, A. In Vitamin D: Chemistry, Biology and Clinical
Ä
Applications of the Steroid Hormone: Proceedings of the Tenth
Workshop on Vitamin D; Norman, A. W.; Bouillon, R.; Tho-
masset, M., Eds.; University of California, Printing and
Reprographics: Riverside, 1997; p 34.
13. The side-chain moiety 11, 4-bromo-2-methylbutan-2-ol
MOM-ether, was synthesized via two-step conversion [(a)
MeMgBr/THF, 0 ꢀC, 1 h, 66%. (b) MOMCl/ iPr2NEt, rt,
13.5 h, 77%] of commercially available ethyl 3-bromopropio-
nate. 11: 1H NMR(400 MHz, CDCl 3) d 1.24 (6H, s), 2.11 (2H,
m), 3.36 (3H, s), 3.46 (2H, m), 4.69 (2H, s); MS 195 [MÀ
Me]+.
Acknowledgements
14. Introduction of a 22R-methyl group into 20-epi-1a,25-
dihydroxyvitamin D3 was predicted by computational meth-
ods to restrict the dihedral angle at C(17-20-22-23) to anti in
ref 8a. Our experimental data show that the dihedral angle at
C(17-20-22-23) in 17 is 169.2(7)ꢀ, suggesting that the anti con-
former also dominates in the case of 17 in the solid state.
15. The A-ring synthons, 20a, 20b, 20c and 20d, correspond to
(3S,4S,5R)-, (3S,4R,5R)-, (3S,4S,5S)- and (3R,4R,5S)-3,5-
bis[(tert-butyldimethylsilyl)oxy]-4-methyloct-1-en-7-yne.
16. Wilson, S. R.; Zhao, H.; Dewan, J. Bioorg. Med. Chem.
Lett. 1993, 3, 341: although we tried their procedure ®rst, we
found that it was practically dicult to obtain the side-chain
moiety, 6-bromo-3-ethylhexan-3-ol TMS ether. The TMS
protective group in the side-chain moiety was also problem-
atic, being lost under the coupling conditions to a€ord a
complex mixture.
This work was supported by a Grant-in-Aid from the
Ministry of Education, Science and Culture of Japan,
and a Grant from the Hayashi Memorial Foundation
for Female Natural Scientists.
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