Synthesis and biological characterization of 1α,24,25-trihydroxy-2β-(3-hydroxypropoxy)vitamin D3 (24-hydroxylated ED-71)

Article abstract of DOI:10.1016/S0039-128X(00)00149-5

24-Hydroxylated derivatives were synthesized in 24(R) and 24(S) forms by the convergent method as analogs related to 1α,25-dihydroxy-2β-(3-hydroxypropoxy)vitamin D₃. In the convergent synthesis, the A-ring fragment, synthesized from diethyl D-tartarate, and the C/D-ring fragments in 24(R) and 24(S) forms (vitamin D numbering), obtained from vitamin D₂ via the Inhoffen-Lythgoe diol, were coupled in moderate yields to give 1α,24(R),25-trihydroxy-2β-(3-hydroxypropoxy)vitamin D₃ and 1α,24(S),25-trihydroxy-2β-(3-hydroxypropoxy)vitamin D₃. In preliminary biological evaluations, 24-hydroxylation of 1α,25-dihydroxy-2β-(3-hydroxypropoxy)vitamin D₃ caused weakened affinity to vitamin D binding protein in vitro and less calcemic activity in vivo compared to the parent compound. While the affinity to vitamin D receptor in 24(R) epimer was sustained, the affinity in 24(S) epimer was less than that of the parent compound. Copyright

Full text of DOI:10.1016/S0039-128X(00)00149-5

Steroids 66 (2001) 267–276
Synthesis and biological characterization of 1␣,24,25-trihydroxy-2␤-(3-
hydroxypropoxy)vitamin D3 (24-hydroxylated ED-71)
Susumi Hatakeyamaa, Akira Kawaseb, Yasushi Uchiyamab, Junji Maeyamaa,
Yoshiharu Iwabuchia, Noboru Kuboderab,*
aFaculty of Pharmaceutical Sciences, Nagasaki University, Nagasaki 852-8521, Japan
bChugai Pharmaceutical Co., Ltd., 2–1-9, Kyobashi, Chuo-ku, Tokyo 104-8301, Japan
Abstract
24-Hydroxylated derivatives were synthesized in 24(R) and 24(S) forms by the convergent method as analogs related to 1␣,25-
dihydroxy-2␤-(3-hydroxypropoxy)vitamin D3. In the convergent synthesis, the A-ring fragment, synthesized from diethyl D-tartarate, and
the C/D-ring fragments in 24(R) and 24(S) forms (vitamin D numbering), obtained from vitamin D2 via the Inhoffen-Lythgoe diol, were
coupled in moderate yields to give 1␣,24(R),25-trihydroxy-2␤-(3-hydroxypropoxy)vitamin D3 and 1␣,24(S),25-trihydroxy-2␤-(3-hy-
droxypropoxy)vitamin D3. In preliminary biological evaluations, 24-hydroxylation of 1␣,25-dihydroxy-2␤-(3-hydroxypropoxy)vitamin D3
caused weakened affinity to vitamin D binding protein in vitro and less calcemic activity in vivo compared to the parent compound. While
the affinity to vitamin D receptor in 24(R) epimer was sustained, the affinity in 24(S) epimer was less than that of the parent compound.
© 2001 Elsevier Science Inc. All rights reserved.
Keywords: 1␣,25-Dihydroxyvitamin D3; 1␣,25-Dihydroxy-2␤-(3-hydroxypropoxy)vitamin D3; ED-71; 24-Hydroxylated ED-71; Sterol
1. Introduction
been conducted in Japan, based upon the preventive and ther-
apeutic effects on bone mineral loss in osteoporosis model rats
[4]. We recently reported that in the modification study of
ED-71 (3) at the 2␤-position, 3 was proved to be an optimized
analog possessing preventive activity in pre-osteoporosis
model rats, as well as 1␣,25-dihydroxy-2␤-(4-hydroxybutyl)
vitamin D3 (carbaED-71) (4) [5]. We also found that 1␣,25-
dihydroxy-26,27-dimethyl-2␤-(3-hydroxypropoxy)vitamin D3
(26,27-dimethyl ED-71) (5), obtained in our modification
study of ED-71 (3) at the side chain, showed a more potent
efficacy on spinal bone mineral density in ovariectomized rats
compared to 3 [6]. These results prompted us to investigate
further synthesis and biological evaluation of analogs of
ED-71 (3) especially modified at the side chain. We have
been also interested in the analogs of 3 having an oxidized
side chain, taking postulated metabolites of ED-71 (3)
into consideration. Thus, in this paper, we describe the
synthesis of 24-hydroxylated ED-71 in both 24(R) and
24(S) forms, 1␣,24(R),25-trihydroxy-2␤-(3-hydroxypro-
poxy)vitamin D3 [(24R)OH-ED-71] (6R) and 1␣,24(S),
25-trihydroxy-2␤-(3-hydroxypropoxy)vitamin D3
[(24S)OH-ED-71] (6S), respectively, as a continuation of
our modification study of ED-71 (3). Preliminary biolog-
ical evaluations of the synthesized 24-hydroxylated
Various analogs of 1␣,25-dihydroxyvitamin D3
[1␣,25(OH)2D3] (1), a hormonally active sterol of native vita-
min D3, have been investigated in attempts to separate differ-
entiation-induction and antiproliferation activities from calce-
mic activity, with the aim of obtaining useful analogs for the
medical treatment of psoriasis, secondary hyperparathyroid-
ism, cancer, etc [1]. There is also an intense interest in obtain-
ing analogs more potent than 1␣,25(OH)2D3 (1) or 1␣-hy-
droxyvitamin D3 (1␣OHD3) (2), a clinically important prodrug
of 1, in terms of regulatory effects in calcium and phosphorous
metabolism, with the aim of treating bone diseases such as
osteoporosis [2]. 1␣,25-Dihydroxy-2␤-(3-hydroxypropoxy)vi-
tamin D3 (ED-71) (3) is an analog of 1␣,25(OH)2D3 (1) bear-
ing a hydroxypropoxy substituent at the 2␤-position [2].
ED-71 (3) is characterized by high calcemic activity and long
half-life in plasma arising from the strong affinity to vitamin D
binding protein (DBP) [3]. Clinical trials of ED-71 (3) as a
promising candidate for the treatment of osteoporosis have
* Corresponding author. Tel.: ϩ81-3-3273-8558; fax: ϩ81-3-3281-
6675.
E-mail address: kuboderanbr@chugai-pharm.co.jp (N. Kubodera).
0039-128X/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved.
PII: S0039-128X(00)00149-5

268
S. Hatakeyama et al. / Steroids 66 (2001) 267–276
Fig. 1. Structures of active vitamin D3 analogs.
ED-71 (6R and 6S) in comparison with 1␣,25(OH)2D3
(1) and ED-71 (3) are also described (Fig. 1).
2.1.2. (2R,3S)-1,4-Dibenzyloxy-3-(3-pivaloyloxypropoxy)
butan-2-ol (13)
To a stirred solution of 12 (11.36 g, 0.031 mol) in
CH2Cl2 (500 ml), were added pyridine (20.42 ml, 0.252
mol) and pivaloyl chloride (15.48 ml, 0.126 mol). After
being stirred at room temperature for 4 h, the mixture was
extracted with CH2Cl2, washed with H2O and saturated
NaCl, dried, evaporated, and chromatographed on silica gel.
Elution with AcOEt-hexane (1:3) gave 13 (12.26 g, 88%) as
a colorless oil: IR (neat) 3487, 1725, 1478, 1455, 1364,
2. Experimental
2.1. General
All melting points were taken on Yanagimoto micro
melting point apparatus and are uncorrected. Optical rota-
tions were measured with JASCO DIP-140 polarimeter.
Infrared (IR) spectra were obtained using JASCO FT/IR-
5300, JEOL JIR-6000, and Hitachi 270–30 spectrophoto-
1
1285, 1162, 1106 cmϪ1; H NMR ␦: 1.85 (q, 2H, J ϭ 6.3
Hz), 2.64 (br d, 1H, J ϭ 5.2 Hz), 3.46–3.77 (m, 7H), 3.91
(br quint, 1H, J ϭ 5.2 Hz), 4.13 (dt, 2H, J ϭ 6.3, 1.6 Hz),
4.54 (s, 4H), 7.16–7.52 (m, 10H); HRMS calcd for
C26H36O6 (Mϩ): 444.2512; found: 444.2555; [␣]D ϩ6.55 (c
1.19, CHCl3).
1
meters. H and 13C NMR spectra were recorded on VAR-
IAN Gemini-300, JEOL FX-200, and JNM-270EX
spectrometers using CDCl3 as a solvent. Chemical shifts are
reported in parts per million (ppm) downfield from tetram-
ethylsilane or calibrated from CHCl3. Mass spectra (MS)
were measured with JEOL JMS-HX-100, Shimadzu GCMS
QP-1000, and Hitachi M1200H instruments. High resolu-
tion mass spectra (HRMS) were recorded on JEOL JMS-
AX-500 and VG Auto Spec Q instruments. Ultra violet
(UV) spectra were obtained with Shimadzu UV-240 spec-
trometer using EtOH as a solvent. All reactions were carried
out under an atmosphere of argon unless otherwise noted.
All extract were dried over MgSO4 and evaporated under
reduced pressure with a rotary evaporator. Chromatographic
purification was carried out with Merck silica gel 60 (col-
umn) or Merck silica gel 60 PF254 (thin layer).
2.1.3.
(2R,3S)-1,2-O-Isopropylidene-3-(3-pivaloyloxy-
propoxy)butane-1,2,4-triol (14)
To a stirred solution of 13 (12.26 g, 0.028 mol) in MeOH
(200 ml), was added palladium hydroxide (Pd(OH)2). The
resulting mixture was stirred at room temperature under
hydrogen atmosphere for 24 h. An insoluble material was
removed by Celite filtration and the filtrate was evaporated.
To a stirred solution of a colorless residue (7.29 g) in
acetone (20 ml), were added 2,2-dimethoxypropane (3.2 ml,
0.026 mol) and p-toluenesulfonic acid (TsOH) (8.2 mg,
0.043 mmol). The mixture was stirred at room temperature
for 4 h, extracted with AcOEt, washed with saturated NaCl,
dried, evaporated, and chromatographed on silica gel. Elu-
tion with AcOEt-hexane (1:2) gave 14 (4.79 g, 87%) as a
colorless oil: IR (neat) 3499, 1725, 1480, 1460, 1398, 1371,
2.1.1. (2R,3S)-1,4-Dibenzyloxy-3-(3-hydroxypropoxy)bu-
tan-2-ol (12)
1
1286, 1256, 1213, 1163, 1075 cmϪ1; H NMR ␦: 1.19 (s,
A mixture of 11 [7] (22.2 g, 0.078 mol) and potassium
tert-butoxide (t-BuOK) (29.82 g, 0.27 mol) in 1,3-pro-
panediol (60 ml) was stirred at 120°C for 3 h. Excess
1,3-propanediol was removed by distillation. The residue
was extracted with CH2Cl2. The extract was washed with
H2O and saturated NaCl, dried, evaporated, and chromato-
graphed on silica gel. Elution with AcOEt-hexane (1:1)
gave 12 (24.2 g, 86%) as a colorless oil: IR (neat) 3448,
1496, 1453, 1364, 1098 cmϪ1; 1H NMR ␦: 1.76 (q, 2H, J ϭ
5.7 Hz), 2.70 (br s, 2H), 3.51–3.84 (m, 9H), 3.89 (br q, 1H,
J ϭ 5.0 Hz), 4.54 (s, 1H), 7.23–7.41 (m, 10H); HRMS calcd
for C21H28O5 (Mϩ): 360.1937; found: 360.1937; [␣]D
ϩ9.32 (c 1.03, CHCl3).
3H), 1.34 (s, 3H), 1.41 (s, 3H), 1.89 (quint, 2H, J ϭ 6.3 Hz),
2.23 (br s, 1H), 3.34 (dt, 1H, J ϭ 6.2, 4.2 Hz), 3.53–3.72 (m,
3H), 3.79 (br d, 1H, J ϭ 12.0 Hz), 3.87 (dd, 1H, J ϭ 8.0, 5.4
Hz), 4.02–4.18 (m, 3H), 4.25 (dt, 1H, J ϭ 11.2, 6.1 Hz);
HRMS calcd for C15H28O6 (Mϩ): 304.1886; found:
304.1888; [␣]D ϩ9.51 (c 1.03, CHCl3).
2.1.4. (2R,3S,4RS)-1,2-O-Isopropylidene-4-pivaloyloxy-3-
(3-pivaloyloxypropoxy)-5-hexene-1,2-diol (15)
To a stirred solution of oxalyl chloride (5.85 ml, 0.067
mol) in CH2Cl2 (100 ml), was added dimethyl sulfoxide
(9.44 ml, 0.133 mol) in CH2Cl2 (100 ml) at –60°C. The

S. Hatakeyama et al. / Steroids 66 (2001) 267–276
269
mixture was stirred at –60°C for 15 min. To the stirred
mixture, was added 14 (10.02 g, 0.033 mol) in CH2Cl2 (200
ml) at –60°C. The mixture was stirred at –60°C for 30 min.
After addition of triethylamine (23.28 ml, 0.167 mol) at
–60°C, the resulting mixture was allowed to be room tem-
perature and the stirring was continued at room temperature
for 30 min. After addition of NH4Cl, the mixture was
extracted with AcOEt, washed with saturated NaCl, dried,
and evaporated to give a residue (10.71 g). To a stirred
solution of the residue (10.71 g) in THF (200 ml), was
added 0.97 M vinylmagnesium bromide in THF (102 ml,
0.099 mol) at –60°C. The mixture was stirred at –60°C for
2 h. After addition of NH4Cl, the mixture was extracted
with CH2Cl2, dried, and evaporated to give a residue (11.21
g). A mixture of the residue (11.21 g), triethylamine (18.54
ml, 0.133 mol), dimethylaminopyridine (10 mg, 0.825
mmol), pivaloyl chloride (8.25 ml, 0.067 mol) in CH2Cl2
(400 ml) was stirred at room temperature for 20 h. The
mixture was extracted with CH2Cl2, washed with saturated
NaCl, dried, evaporated, and chromatographed on silica gel.
Elution with AcOEt-hexane (1:10) gave 15 (7.89 g, 58%) as
a colorless oil: IR (neat) 1728, 1480, 1460, 1373, 1284,
was refluxed for 7 days, evaporated, and chromatographed
on silica gel. Elution with AcOEt-hexane (1:10) gave 17
(692 mg, 77%) as a colorless oil: IR (neat) 1730, 1480,
1
1397, 1365, 1282, 1156, 1066 cmϪ1; H NMR ␦: 1.20 (s,
9H), 1.23 (s, 9H), 1.85 (quint, 2H, J ϭ 6.0 Hz), 2.70–2.82
(m, 2H), 2.97–3.06 (m, 1H), 3.38 (t, 0.4H, J ϭ 4.2 Hz), 3.45
(t, 0.6H, J ϭ 4.7 Hz), 3.51–3.73 (m, 2H), 4.12 (m, 2H),
5.23–5.48 (m, 3H), 5.84–5.98 (m, 1H); HRMS calcd for
C19H32O6 (Mϩ): 356.2199; found: 356.2238.
2.1.7. (4R,5S,6R)-4,6-Di(tert-butyldimethylsilyloxy)-5-[3-(t-
butyldimethylsilyloxy)propoxy]-7-octen-2-yne (7) and
(4R,5S,6S)-4,6-Di(tert-butyldimethylsilyloxy)-5-[3-(tert-bu-
tyldimethylsilyloxy)propoxy]-7-octen-2-yne (18)
To a stirred solution of trimethylsilylacetylene (8.4 ml,
11.9 mmol), was added 1.59 M n-BuLi in hexane (35.2 ml,
56.0 mmol) at –78°C. The mixture was stirred at –78°C for
30 min. To the stirred mixture, was added borontrifluoride
etherate (BF3-OEt2) and the stirring was continued at –78°C
for 1 h. To the stirred mixture, was added 17 (4.24 g, 11.9
mmol) in THF (65 ml) and the stirring was continued at
–78°C for 2 h. The mixture was quenched by saturated
NaHCO3 and H2O, extracted with AcOEt, washed with
saturated NaCl, dried, and evaporated to give a yellow oil
(5.1 g), which was used without further purification. A
mixture of the yellow oil (5.1 g) and 10 N NaOH (35 ml) in
MeOH (80 ml) was stirred at room temperature for 1.5 h.
The mixture was neutralized by conc. HCl under ice-cool-
ing, azeotropically refluxed with toluene, extracted with
THF, dried, evaporated, and chromatographed on silica gel.
Elution with AcOEt gave a colorless oil (1.73 g), which was
used without further purification. To a stirred solution of the
colorless oil (1.73 g) in CH2Cl2 (100 ml), were added
triethylamine (11.3 ml, 0.4 mol) and tert-butyldimethylsilyl
trifluoromethanesulfonate (TBSOTf) (10.7 ml, 40.4 mmol)
at 0°C. The stirring was continued at 0°C for 1.5 h. After the
addition of saturated NaHCO3 and H2O, the mixture was
extracted with CH2Cl2, washed with saturated NaCl, dried,
evaporated. Repetition of chromatography on silica gel
[AcOEt-hexane (1:50)] gave 7 (2.50 g, 36%) and 18 (1.58 g,
24%), respectively, as colorless oils. 7: IR (neat) 3314,
1
1214, 1160, 1058 cmϪ1; H NMR ␦: 1.20 (s, 3H), 1.34 (s,
3H), 1.42 (s, 3H x 0.6), 1.43 (s, 3H x 0.4), 1.88 (quint, 0.4H,
J ϭ 6.3 Hz), 1.89 (quint, 0.6H, J ϭ 6.3 Hz), 2.48 (d, 0.4H,
J ϭ 4.1 Hz), 2.65 (d, 0.6H, J ϭ 8.2 Hz), 3.41 (dd, 1H, J ϭ
6.4, 3.6 Hz), 3.45 (dd, 1H, J ϭ 5.5, 4.3 Hz), 3.70 (m, 2H),
3.89 (t, 0.4H, J ϭ 8.2 Hz), 3.90 (t, 0.6H, J ϭ 8.2 Hz), 4.04
(t, 0.4H, J ϭ 8.5 Hz), 4.05 (t, 0.6H, J ϭ 8.5 Hz), 4.08–4.34
(m, 3H), 5.23 (dt, 0.4H, J ϭ 3.0, 1.6 Hz), 5.25 (dt, 0.6H, J ϭ
3.0, 1.6 Hz), 5.35 (dt, 0.4H, J ϭ 8.9, 1.6 Hz), 5.38 (dt, 0.6H,
J ϭ 8.9, 1.6 Hz); HRMS calcd for C22H38O7 (Mϩ):
414.2618; found: 414.2618.
2.1.5.
(2R,3S,4RS)-4-Pivaloyloxy-3-(3-pivaloyloxyprop-
oxy)-5-hexene-1,2-diol (16)
A mixture of 15 (1.83 g, 4.4 mmol) and 1 M HCl (10 ml)
in MeOH (10 ml) was stirred at room temperature for 10 h.
The mixture was extracted with Et2O, washed with H2O and
saturated NaCl, dried, evaporated, and chromatographed on
silica gel. Elution with AcOEt-hexane (1:1) gave 16 (1.67 g,
100%) as a colorless oil: IR (neat) 3448, 1731, 1480, 1285,
1
1468, 1254 cmϪ1; H NMR ␦: 0.03 (s, 3H), 0.04 (s, 3H),
1
1170, 1108 cmϪ1; H NMR ␦: 1.20 (s, 9H), 1.23 (s, 9H),
0.06 (s, 3H), 0.08 (s, 3H), 0.10 (s, 3H), 0.89–0.90 (m, 2H),
1.78 (quint, 2H, J ϭ 6.0 Hz), 1.92 (t, 1H, J ϭ 2.7 Hz), 2.46
(ddd, 1H, J ϭ 3.0, 5.1, 17.1 Hz), 3.27 (dd, 1H, J ϭ 2.1, 6.6
Hz), 3.60–3.82 (m, 4H), 3.98 (ddd, 1H, J ϭ 1.8, 4.8, 7.2
Hz), 4.18 (t, 1H, J ϭ 6.3 Hz), 5.14 (dt, 1H, J ϭ 1.5, 10.5
Hz), 5.26 (dt, 1H, J ϭ 1.5, 17.1 Hz), 5.89 (ddd, 1H, J ϭ 6.0,
10.5, 17.4 Hz); 13C NMR ␦: 138.5, 116.0, 86.9, 83.2, 74.4,
72.1, 69.5, 60.5, 33.7, 26.1, 26.0, 23.3, 18.5, 18.4, Ϫ4.3,
Ϫ4.4, Ϫ4.5, Ϫ5.2; HRMS calcd for C28H57O4Si3 (Mϩ-Me):
1.89 (quint, 2H, J ϭ 6.0 Hz), 2.01 (br s, 0.6H), 2.50 (br s,
0.4H), 2.68 (br s, 0.4H), 2.95 (br s, 0.6H), 3.30–3.90 (m,
6H), 4.00–4.34 (m, 2H), 5.20–5.41 (m, 2H), 5.50 (m,
0.6H), 5.63 (m, 0.4H), 5.84–6.10 (m, 1H); HRMS calcd for
C19H34O7 (Mϩ): 374.2305; found: 374.2305.
2.1.6. (2R,3S,4RS)-1,2-Epoxy-4-pivaloyloxy-3-(3-pivaloy-
loxypropoxy)-5-hexene (17)
25
A solution of 16 (1.08 g, 2.9 mmol) in benzene (30 ml)
was azeotropically refluxed for 1 h. To the ice-cooled mix-
ture of 16 in benzene, were added triphenyl phosphine
(Ph3P) (3.04 g, 11.6 mmol) and diethyl azodicarboxylate
(DEAD) (1.83 ml, 11.6 mmol) [8]. The resulting mixture
541.3565; Found 541.3547; [␣]D ϩ15.4 (c 0.52, CHCl3).
1
18: IR (neat) 3314, 1468, 1254 cmϪ1; H NMR ␦: 0.03–
0.12 (m, 18H), 0.88 (s, 9H), 0.90 (s, 9H), 0.91 (s, 9H), 1.74
(quint, 2H, J ϭ 6.3Hz), 1.93 (t, 1H, J ϭ 2.7 Hz), 2.53 (ddd,
1H, J ϭ 2.7, 5.7, 17.4 Hz), 3.36 (dd, 1H, J ϭ 4.5, 5.7 Hz),

270
S. Hatakeyama et al. / Steroids 66 (2001) 267–276
1
3.67 (t, 2H, J ϭ 6.3 Hz), 3.74 (ddd, 2H, J ϭ 3.0, 6.3, 12.9
Hz), 3.85 (dt, 1H, J ϭ 4.2, 5.7 Hz), 4.27 (dd, 1H, J ϭ 4.5,
6.0 Hz), 5.14 (dt, 1H, J ϭ 1.5, 10.5 Hz), 5.26 (dt, 1H, J ϭ
1.5, 17.1 Hz), 5.88 (ddd, 1H, J ϭ 6.0, 10.2, 17.4 Hz); 13C
NMR ␦: 138.2, 116.1, 85.7, 82.3, 74.4, 71.0, 69.8, 69.7,
60.3, 33.7, 26.1, 23.3, 18.5, 18.2, Ϫ4.2, Ϫ4.3, Ϫ4.4, Ϫ5.2;
oil: IR (neat) 3460 (br), 2925, 1280, 1135, 1075 cmϪ1; H
NMR ␦: 0.69 (s, 3H), 1.05 (d, 3H, J ϭ 6.6 Hz, 3H), 1.20 (s,
3H), 1.25 (s, 3H), 3.35 (t, 1H, J ϭ 5.1 Hz), 3.45 (br s, 1H),
4.01 (br s, 1H), 7.53–7.94 (m, 5H); MS m/z: 439 (Mϩϩ1),
60 (100%).
25
[␣]D –2.3 (c 0.80, CHCl3).
2.1.11.
(1R,3aR,4S,7aR)-1-[(1R,4R)-4,5-Dihydroxy-1,5-
dimethylhexyl]-7a-methyl-octahydro-1H-inden-4-ol (24R)
Sodium amalgam (Na/Hg) (5%, 5.01 g, 10.9 mmol) was
added to a solution of 23R (104 mg, 0.237 mmol) in THF
(5.5 ml) and MeOH (3.5 ml). The mixture was stirred at
room temperature for 15 h. MeOH (5.4 ml) and H2O (5.4
ml) were added and the mixture was stirred for 30 min.
After removal of the excess Na/Hg by decantation, the
collected solution was poured into saturated NH4Cl and
extracted with AcOEt. The extract was washed with satu-
rated NaCl, dried, evaporated, and chromatographed on
silica gel. Elution with EtOH-CH2Cl2 (3:50) gave 24R (68
mg, 97%) as a colorless foam: IR (neat) 3400 (br), 2930
2.1.8. (1R,3aR,4S,7aR,1S)-4-Acetoxy-7a-methyl-1-[(S)-1-
methyl-2-(phenylsulphonyl)ethyl]octahydro-1H-indene (21)
To an ice-cooled solution of 20 [9,10] (334 mg, 0.994
mmol), 4-dimethylaminopyridine (18 mg, 0.148 mmol) and
pyridine (0.2 ml) in CH2Cl2 (20 ml), was added Ac2O
(0.188 ml, 1.99 mmol). The mixture was stirred at room
temperature for 16 h, poured into diluted HCl, and extracted
with CH2Cl2. The extract was washed with H2O and satu-
rated NaHCO3, dried, evaporated, and chromatographed on
silica gel. Elution with AcOEt-hexane (2:3) gave 21 (368
mg, 98%) as a colorless oil: IR (neat) 2945, 1735, 1310,
1250, 1150 cmϪ1; 1H NMR ␦: 0.86 (s, 3H), 1.20 (d, 3H, J ϭ
6.8 Hz), 2.03 (s, 3H), 2.84 (dd, 1H, J ϭ 14.1, 9.8 Hz), 3.11
(d, 1H, J ϭ 14.1 Hz), 5.12 (br s, 1H), 7.52–7.96 (m, 5H);
MS m/z: 336 (Mϩ–Ac), 135 (100%); UV ␭max 270, 263,
257, 217 nm.
1
cmϪ1; H NMR ␦: 0.91 (d, 3H, J ϭ 6.3 Hz), 0.94 (s, 3H),
1.21 (s, 3H), 1.24 (s, 3H), 3.33 (br t, 1H, J ϭ 5.4 Hz), 4.08
(br s, 1H); MS m/z: 298 (Mϩ), 135 (100%).
2.1.12.
(1R,3aR,4S,7aR)-1-[(1R,4S)-4,5-Dihydroxy-1,5-
dimethylhexyl]-7a-methyl-octahydro-1H-inden-4-ol (24S)
Na/Hg (5%, 12.4 g, 27.0 mmol) was added to a solution
of 23S (216 mg, 0.493 mmol) in THF (12 ml) and MeOH
(7.5 ml). The mixture was stirred at room temperature for
15 h. MeOH (5.4 ml) and H2O (5.4 ml) were added and the
mixture was stirred for 30 min. After removal of the excess
Na/Hg by decantation, the collected solution was poured
into saturated NH4Cl and extracted with AcOEt. The extract
was washed with saturated NaCl, dried, evaporated, and
chromatographed on silica gel. Elution with EtOH-CH2Cl2
(3:50) gave 24S (100 mg, 68%) as a colorless oil: IR (neat)
3400 (br), 2930 cmϪ1; 1H NMR ␦: 0.92 (d, 3H, J ϭ 6.6 Hz),
0.93 (s, 3H), 1.16 (s, 3H), 1.21 (s, 3H), 3.27 (dd, 1H, J ϭ
10.2, 2.0 Hz), 4.07 (br d, 1H, J ϭ 2.6Hz); MS m/z: 280
(Mϩ- H2O), 60 (100%).
2.1.9. (1R,3aR,4S,7aR)-7a-Methyl-1-[(1S,2RS,4R)-4,5-di-
hydroxy-1,5-dimethyl-2-(phenylsulphonyl)hexyl]octahydro-
1H-inden-4-ol (23R)
To a stirred solution of 21 (160 mg, 0.423 mmol) and
(R)-3-methyl-1-(p-toluenesulphonyl)oxybutane-2,3-diol
(22R) [11,12] (232 mg, 0.847 mmol) in THF (15 ml) at
–20°C, was added 1.63 M n-BuLi in hexane (2.3 ml, 3.75
mmol). After being stirred at –20°C for 2 h, the reaction
mixture was poured into saturated NH4Cl and extracted
with AcOEt. The extract was washed with saturated NaCl,
dried, evaporated, and chromatographed on silica gel. Elu-
tion with EtOH-CH2Cl2 (3:50) gave 23R (131 mg, 71%) as
a colorless foam: IR (neat) 3500 (br), 2935, 1300, 1280,
1
1135 cmϪ1; H NMR ␦: 0.67 (s, 3H), 0.96 (d, 3H, J ϭ 6.8
Hz), 1.20 (s, 3H), 1.26 (s, 3H), 3.51 (d, 1H, J ϭ 10.2 Hz),
3.81 (br d, 1H, J ϭ 11.7 Hz), 4.00 (br s, 1H), 7.52–7.94 (m,
5H); MS m/z: 439 (Mϩϩ1), 71 (100%); UV ␭max 271,
263, 257, 216 nm.
2.1.13. (1R,3aR,4S,7aR)-4-Acetoxy-7a-methyl-1-[(R)-1-
methyl-3-[(R)-2,2,4,4-tetramethyl-1,3-dioxolan-5-
yl]propyl]octahydro-1H-indene (25R)
To a solution of 24R (86 mg, 0.289 mmol) and 2,2-
dimethoxypropane (2.93 g, 28.1 mmol) in acetone (19.5
ml), was added TsOH (6.5 mg, 0.034 mmol). After being
stirred at room temperature for 15 h, the reaction mixture
was treated with NaHCO3 (10 mg) and evaporated. The
residue was extracted with CH2Cl2. The extract was washed
with saturated NaCl, dried, evaporated, and chromato-
graphed on silica gel. Elution with EtOH-CH2Cl2 (1:25)
gave a colorless residue (107 mg), which was used for the
next reaction without further purification. The crude residue
(107 mg) was dissolved in CH2Cl2 (10 ml) and pyridine
(128 ␮l), 4-dimethylaminopyridine (7 mg, 0.058 mmol) and
Ac2O (120 ␮l, 1.27 mmol) were added. After being stirred
2.1.10. (1R,3aR,4S,7aR)-7a-Methyl-1-[(1S,2RS,4S)-4,5-di-
hydroxy-1,5-dimethyl-2-(phenylsulphonyl)hexyl]octahydro-
1H-inden-4-ol (23S)
To a stirred solution of 21 (305 mg, 0.908 mmol) and
(S)-3-methyl-1-(p-toluenesulphonyl)oxybutane-2,3-diol
(22S) [12] (249 mg, 0.909 mmol) in THF (20 ml) at –20°C,
was added 1.69 M n-BuLi in hexane (4.3 ml, 7.27 mmol).
After being stirred at –20°C for 2 h, the reaction mixture
was poured into saturated NH4Cl and extracted with AcOEt.
The extract was washed with saturated NaCl, dried, evap-
orated, and chromatographed on silica gel. Elution with
AcOEt-hexane (4:1) gave 23S (110 mg, 28%) as a colorless

S. Hatakeyama et al. / Steroids 66 (2001) 267–276
271
at room temperature for 4 h, the reaction mixture was
poured into diluted HCl and extracted with CH2Cl2. The
extract was washed with saturated NaHCO3, dried, evapo-
rated, and chromatographed on silica gel. Elution with
AcOEt-hexane (3:17) gave 25R (91 mg, 83%) as a colorless
AcOEt. The extract was washed with H2O and saturated
NaCl, dried, evaporated, and chromatographed on silica gel.
Elution with AcOEt-hexane (2:3) gave 26S (25 mg, 83%) as
1
a colorless oil: IR (neat) 3460 (br), 2935, 1730 cmϪ1; H
NMR ␦: 0.88 (s, 3H), 0.93 (d, 3H, J ϭ 6.6 Hz), 1.16 (s, 3H),
1.22 (s, 3H), 2.04 (s, 3H), 3.23–3.32 (m, 1H)), 5.15 (br s,
1H); MS m/z: 340 (Mϩ), 135 (100%).
1
oil: IR (KBr) 3485 (br), 2920 cmϪ1; H NMR ␦: 0.93 (d,
3H, J ϭ 5.3 Hz), 0.95 (s, 3H), 1.10 (s, 3H), 1.25 (s, 3H),
1.33 (s, 3H), 1.42 (s, 3H), 3.64 (dd, 1H, J ϭ 8.5, 3.7 Hz),
4.08 (br d, 1H, J ϭ 2.4Hz); MS m/z: 338 (Mϩ), 323 (100%).
2.1.17. (1R,3aR,4S,7aR)-4-Acetoxy-1-[(1R,4R)-4,5-di(tert-
butyldimethylsilyloxy)-1,5-dimethylhexyl]-7a-methylocta-
hydro-1H-indene (27R)
2.1.14.
(1R,3aR,4S,7aR)-4-Acetoxy-7a-methyl-1-[(R)-1-
methyl-3-[(S)-2,2,4,4-tetramethyl-1,3-dioxolan-5-yl]pro-
pyl]octahydro-1H-indene (25S)
To an ice-cooled solution of 26R (46 mg, 0.135 mmol) in
CH2Cl2 (10 ml), were added 2,6-lutidine (355 ␮l, 3.03
mmol) and TBSOTf (466 ␮l ml, 2.03 mmol). After being
stirred at room temperature for 1.5 h, the reaction mixture
was poured into diluted HCl and extracted with CH2Cl2.
The extract was washed with saturated NaHCO3, dried,
evaporated, and chromatographed on silica gel. Elution with
AcOEt-hexane (3:47) gave 27R (71 mg, 92%) as a colorless
To a solution of 24S (100 mg, 0.336 mmol) and 2,2-
dimethoxypropane (3.80 g, 36.5 mmol) in acetone (23 ml),
was added TsOH (15 mg, 0.079 mmol). After being stirred
at room temperature for 15 h, the reaction mixture was
treated with NaHCO3 (10 mg) and evaporated. The residue
was extracted with CH2Cl2. The extract was washed with
saturated NaCl, dried, evaporated, and chromatographed on
silica gel. Elution with EtOH-CH2Cl2 (1:25) gave a color-
less residue (76 mg), which was used without further puri-
fication. The crude residue (76 mg) was dissolved in
CH2Cl2 (3 ml) and pyridine (500 ␮l), 4-dimethylaminopy-
ridine (7 mg, 0.058 mmol) and Ac2O (250 ␮l, 2.64 mmol)
were added. After being stirred at room temperature for 4 h,
the reaction mixture was poured into diluted HCl and ex-
tracted with CH2Cl2. The extract was washed with saturated
NaHCO3, dried, evaporated, and chromatographed on silica
gel. Elution with AcOEt-hexane (3:17) gave 25S (82 mg,
1
oil: IR (neat) 2945, 1745 cmϪ1; H NMR ␦: 0.04 (s, 3H),
0.06 (s, 3H), 0.07 (s, 3H), 0.08 (s, 3H), 0.86 (s, 9H), 0.89 (s,
9H), 1.11 (s, 3H), 1.19 (s, 3H), 2.04 (s, 3H), 3.22 (br s, 1H),
5.15 (br s, 1H); MS m/z: 553 (Mϩ–Me), 173 (100%).
2.1.18. (1R,3aR,4S,7aR)-4-Acetoxy-1-[(1R,4S)-4,5-di(tert-
butyldimethylsilyloxy)-1,5-dimethylhexyl]-7a-methylocta-
hydro-1H-indene (27S)
To an ice-cooled solution of 26S (31 mg, 0.091 mmol) in
CH2Cl2 (2 ml), were added 2,6-lutidine (64 ␮l, 0.55 mmol)
and TBSOTf (84 ␮l ml, 0.37 mmol). After being stirred at
room temperature for 1.5 h, the reaction mixture was poured
into diluted HCl and extracted with CH2Cl2. The extract
was washed with saturated NaHCO3, dried, evaporated, and
chromatographed on silica gel. Elution with AcOEt-hexane
(3:47) gave 27S (47 mg, 91%) as a colorless oil: IR (neat)
64%) as a pale yellow oil: IR (neat) 2930, 1735 cmϪ1; H
1
NMR ␦: 0.89 (s, 3H), 0.94 (d, 3H, J ϭ 6.6 Hz), 1.10 (s, 3H),
1.25 (s, 3H), 1.33 (s, 3H), 1.41 (s, 3H), 2.04 (s, 3H), 3.61 (t,
1H, J ϭ 6.3 Hz), 5.16 (br s, 1H); MS m/z: 365 (Mϩ– Me,
100%).
1
2945, 1735 cmϪ1; H NMR ␦: 0.04 (s, 3H), 0.06 (s, 3H),
2.1.15.
(1R,3aR,4S,7aR)-4-Acetoxy-1-[(1R,4R)-4,5-dihy-
0.07 (s, 3H), 0.08 (s, 3H), 0.85 (s, 9H), 0.88 (s, 9H), 1.10 (s,
3H), 1.19 (s, 3H), 2.04 (s, 3H), 3.18 (dd, 1H, J ϭ 7.4, 2.3
Hz), 5.15 (br s, 1H); MS m/z: 553 (Mϩ–Me), 173 (100%).
droxy-1,5-dimethylhexyl]-7a-methyloctahydro-1H-indene
(26R)
A mixture of 25R (46 mg, 0.121 mmol) and 1% I2-MeOH
(2 ml) was refluxed for 4.5 h. The reaction mixture was poured
into 10% Na2S2O3, evaporated, and extracted with AcOEt. The
extract was washed with H2O and saturated NaCl, dried, evap-
orated, and chromatographed on silica gel. Elution with
AcOEt-hexane (2:3) gave 26R (25 mg, 61%) as a colorless oil:
2.1.19.
(1R,3aR,4S,7aR)-1-[(1R,4R)-4,5-Di(tert-butyldi-
methylsilyloxy)-1,5-dimethylhexyl]-7a-methyloctahydro-
1H-inden-4-ol (28R)
To an ice-cooled solution of lithium aluminum hydride
(LiAlH4) (20 mg, 0.528 mmol) in THF (1 ml), was added
dropwise a solution of 27R (71 mg, 0.125 mmol) in THF (2
ml). After being stirred with cooling in an ice bath for 1.5 h,
the reaction mixture was quenched with 1M NaOH and
extracted with AcOEt. The extract was washed with satu-
rated NaCl, dried, evaporated, and chromatographed on
silica gel. Elution with AcOEt-hexane (3:22) gave 28R (63
1
IR (neat) 3455 (br), 2930, 1735 cmϪ1; H NMR ␦: 0.89 (s,
3H), 0.92 (d, 3H, J ϭ 6.6 Hz), 1.16 (s, 3H), 1.21 (s, 3H), 2.04
(s, 3H), 3.32 (br t, 1H, J ϭ 5.9 Hz), 5.14 (br s, 1H); MS m/z:
280 (Mϩ– Ac – H2O), 59 (100%).
2.1.16.
(1R,3aR,4S,7aR)-4-Acetoxy-1-[(1R,4S)-4,5-dihy-
droxy-1,5-dimethylhexyl]-7a-methyloctahydro-1H-indene
(26S)
A mixture of 25S (34 mg, 0.090 mmol) and 1% I2-MeOH
(2 ml) was refluxed for 4.5 h. The reaction mixture was
poured into 10% Na2S2O3, evaporated, and extracted with
mg, 96%) as a colorless oil: IR (neat) 3425 (br), 2945 cmϪ1
;
1H NMR ␦: 0.03 (s, 3H), 0.06 (s, 3H), 0.07 (s, 3H), 0.08 (s,
3H), 0.85 (s, 9H), 0.89 (s, 9H), 1.11 (s, 3H), 1.18 (s, 3H),
3.22 (br s, 1H), 4.08 (br s, 1H); MS m/z: 511 (Mϩ–Me), 173
(100%).

272
S. Hatakeyama et al. / Steroids 66 (2001) 267–276
2.1.20.
(1R,3aR,4S,7aR)-1-[(1R,4S)-4,5-Di(tert-butyldi-
62.1, 76.4, 81.2, 212.2; MS m/z: 509 (Mϩ–Me), 467 (Mϩ–57),
173 (100%); HRMS calcd for C29H57O3Si2 (Mϩ–Me):
509.3846, found: 509.3852; [␣]D24 –5.3° (c 1.185, CHCl3).
methylsilyloxy)-1,5-dimethylhexyl]-7a-methyloctahydro-
1H-inden-4-ol (28S)
To an ice-cooled solution of LiAlH4 (6.3 mg, 0.166
mmol) in THF (1 ml), was added dropwise a solution of 27S
(47 mg, 0.083 mmol) in THF (2 ml). After being stirred with
cooling in an ice bath for 1.5 h, the reaction mixture was
quenched with 1M NaOH and extracted with AcOEt. The
extract was washed with saturated NaCl, dried, evaporated,
and chromatographed on silica gel. Elution with AcOEt-
hexane (3:22) gave 28S (44 mg, 100%) as a colorless oil: IR
(neat) 3430 (br), 2920 cmϪ1; 1H NMR ␦: 0.04 (s, 3H), 0.06
(s, 3H), 0.07 (s, 3H), 0.08 (s, 3H), 0.85 (s, 9H), 0.89 (s, 9H),
0.92 (d, 3H, J ϭ 6.3 Hz), 1.11 (s, 3H), 1.19 (s, 3H), 3.18
(dd, 1H, J ϭ 7.6, 2.3 Hz), 4.00 (br s, 1H); MS m/z: 511
(Mϩ–Me), 173 (100%).
2.1.23. (1R,3aR,7aR)-4-[(E)-Bromomethylene]-1-[(1R,4R)-
4,5-di(tert-butyldimethylsilyloxy)-1,5-dimethylhexyl]-7a-
methyloctahydro-4H-inden (9R)
To a stirred solution of (bromomethylene)triphenylphos-
phonium bromide (Ph3PCH2Br2) (130.5 mg, 0.30 mmol) in
THF (1 ml), was added 1 M sodium hexamethyldisilazide
(NaN(TMS)2) in THF (290 ␮l, 0.29 mmol) at –60°C. The
resulting mixture was stirred at –60°C for 1 h. After addition
of 29R (19.6 mg, 0.037 mmol) in THF (0.3 ml) at –60°C, the
mixture was stirred at room temperature for 1 h. The mixture
was diluted with hexane and filtered with silica gel. The filtrate
was evaporated. The residue was purified by preparative TLC
developed with hexane to give 9R (11.0 mg, 49%) as an
1
2.1.21. (1R,3aR,7aR)-1-[(1R,4R)-4,5-Di(tert-butyldimethyl-
silyloxy)-1,5-dimethylhexyl]-7a-methyloctahydro-4H-in-
den-4-one (29R)
yellow oil: IR (neat) 1467, 1253 cmϪ1; H NMR ␦: 0.04 (s,
3H), 0.06 (s, 3H), 0.07 (s, 3H), 0.08 (s, 3H), 0.56 (s, 3H), 0.85
(s, 9H), 0.89 (s, 9H), 0.92 (d, 3H, J ϭ 6.0 Hz), 1.11 (s, 3H),
1.18 (s, 3H), 1.20–2.04 (m, 16H), 2.82–2.91 (m, 1H), 3.18–
3.24 (m, 1H), 5.64 (s, 1H); 13C NMR ␦: Ϫ3.9, –3.1, –1.9, –1.8,
18.2, 18.3, 19.0, 22.2, 22.7, 23.6, 26.0, 26.2, 27.7, 29.1, 29.6,
31.2, 33.9, 36.8, 40.0, 45.6, 55.9, 56.0, 76.4, 81.1, 97.4, 145.3;
MS m/z: 585 (Mϩ–Me), 543 (Mϩ–57), 73; HRMS calcd for
A mixture of 28R (15.4 mg, 0.029 mmol), N-methylmor-
pholine N-oxide (NMO) (7.0 mg, 0.059 mmol), and 4A mo-
lecular sieves (14.0 mg) in CH2Cl2 (2 ml) was stirred at room
temperature for 1 h and then tetrapropylammonium perruthe-
nate (n-Pr4NRuO4) (0.8 mg, 0.0023 mmol) was added. After
being stirred at room temperature for 2.5 h, the mixture was
filtered through Celite, evaporated, and chromatographed on
silica gel. Elution with AcOEt-hexane (1:5) gave 29R (14.8
21
C30H58O2Si2Br (Mϩ–Me): 585.3150, found: 585.3134; [␣]D
–106.18° (c 0.55, CHCl3).
mg, 96%) as a colorless oil: IR (neat) 1716, 1460, 1252 cmϪ1
;
2.1.24. (1R,3aR,7aR)-4-[(E)-Bromomethylene]-1-[(1R,4S)-
4,5-di(tert-butyldimethylsilyloxy)-1,5-dimethylhexyl]-7a-
methyloctahydro-4H-inden (9S)
1H NMR ␦: 0.04 (s, 3H), 0.06 (s, 3H), 0.07 (s, 3H), 0.08 (s,
3H), 0.63 (s, 3H), 0.85 (s, 9H), 0.89 (s, 9H), 0.94 (d, 3H, J ϭ
5.7 Hz), 1.11 (s, 3H), 1.18 (s, 3H), 1.20–2.34 (m, 16H), 2.45
(dd, 1H, J ϭ 7.5, 11.1 Hz), 3.18–3.24 (m, 1H); 13C NMR ␦:
–3.1, –1.9, –1.8, 12.6, 18.2, 18.3, 18.9, 19.2, 23.6, 24.2, 25.9,
26.2, 27.6, 29.0, 29.6, 33.8, 36.3, 39.1, 41.1, 50.0, 56.7, 62.1,
76.4, 81.0, 212.2; MS m/z: 509 (Mϩ–Me), 173 (100%);
HRMS calcd for C29H57O3Si2 (Mϩ–Me): 509.3846, found:
509.3845; [␣]D23 ϩ17.0° (c 0.83, CHCl3).
To a stirred solution of Ph3PCH2Br2 (159.3 mg, 0.37 mmol)
in THF (0.9 ml), was added 1 M NaN(TMS)2 in THF (355 ␮l,
0.36 mmol) at –60°C. The resulting mixture was stirred at
–60°C for 1 h. After addition of 29S (23.9 mg, 0.046 mmol) in
THF (0.3 ml) at –60°C, the mixture was stirred at room
temperature for 1 h. The mixture was diluted with hexane and
filtered with silica gel. The filtrate was evaporated. The residue
was purified by preparative TLC developed with hexane to
give 9S (15.6 mg, 57%) as an yellow oil: IR (neat) 1466, 1252
cmϪ1; 1H NMR ␦: 0.04 (s, 3H), 0.06 (s, 3H), 0.07 (s, 3H), 0.08
(s, 3H), 0.56 (s, 3H), 0.85 (s, 9H), 0.88 (s, 9H), 0.92 (d, 3H,
J ϭ 6.0 Hz), 1.10 (s, 3H), 1.19 (s, 3H), 1.00–2.16 (m, 16H),
2.82–2.91 (m, 1H), 3.18 (dd, 1H, J ϭ 7.5, 2.1 Hz), 5.64 (s,
1H); 13C NMR ␦: Ϫ3.7, –3.1, –1.9, –1.8, 11.9, 18.2, 18.3, 18.9,
22.1, 22.7, 23.2, 25.9, 26.2, 27.9, 29.0, 29.7, 31.2, 34.2, 36.7,
40.0, 45.6, 55.9, 56.0, 76.4, 81.2, 97.4, 145.3; MS m/z: 585
(Mϩ–Me), 543 (Mϩ–57), 73; HRMS calcd for C27H52O2Si2Br
(Mϩ–57): 543.2690, found: 543.2692; [␣]D23 ϩ47.31° (c 0.78,
CHCl3).
2.1.22. (1R,3aR,7aR)-1-[(1R,4S)-4,5-Di(tert-butyldimethyl-
silyloxy)-1,5-dimethylhexyl]-7a-methyloctahydro-4H-in-
den-4-one (29S)
A mixture of 28S (23.7 mg, 0.044 mmol), NMO (7.8 mg,
0.066 mmol), and 4A molecular sieves (19.0 mg) in CH2Cl2 (3
ml) was stirred at room temperature for 1 h and then
n-Pr4NRuO4 (0.8 mg, 0.0023 mmol) was added. After being
stirred at room temperature for 5 h, the mixture was filtered
through Celite, evaporated, and chromatographed on silica gel.
Elution with AcOEt-hexane (1:5) gave 29S (23.7 mg, 100%)
as a colorless oil: IR (neat) 1716, 1467, 1252 cmϪ1; 1H NMR
␦: 0.04 (s, 3H), 0.06 (s, 3H), 0.07 (s, 3H), 0.08 (s, 3H), 0.63 (s,
3H), 0.85 (s, 9H), 0.88 (s, 9H), 0.94 (d, 3H, J ϭ 6.3 Hz), 1.10
(s, 3H), 1.19 (s, 3H), 1.20–2.28 (m, 16H), 2.43 (dd, 1H, J ϭ
7.5, 11.1 Hz), 3.18 (dd, 1H, J ϭ 2.4, 7.5 Hz); 13C NMR ␦:
Ϫ3.7, –3.1, –1.9, –1.8, 12.5, 18.2, 18.3, 18.8, 19.2, 23.2, 24.2,
26.0, 26.2, 27.8, 29.0, 29.7, 34.1, 36.2, 39.1, 41.1, 50.0, 56.8,
2.1.25.
(5Z,7E)-(1R,2R,3R,24R)-1,3,24-Tris(tert-butyldi-
methylsilyloxy)-2-(3-tert-butyldimethylsilyloxypropoxy)-9,
10-secocholesta-5,7,10(19)-triene (30R)
A mixture of triphenyphosphine (PPh3) (2.0 mg, 7.6
␮mol), tris(dibenzylideneacetonedipalladium (0)-chloro-

S. Hatakeyama et al. / Steroids 66 (2001) 267–276
273
form adduct ((dba)3Pd2-CHCl3) (1.8 mg, 1.7 ␮mol), and
triethylamine (0.3 ml) in toluene (0.2 ml) was stirred at
room temperature for 10 min. To the stirred mixture, were
added 7 (7.9 mg, 0.014 mmol) and 9R (11.0 mg, 0.018
mmol) in toluene (0.2 ml) at room temperature. The result-
ing mixture was refluxed for 6h, diluted with hexane, fil-
tered with silica gel, and evaporated. The residue was pu-
rified by preparative TLC developed with hexane-benzene
(2:1) to give 30R (5.5 mg, 36%) as a colorless oil: IR (neat)
1467, 1252 cmϪ1; 1H NMR ␦: 0.02–0.10 (m, 30H), 0.52 (s,
3H), 0.82–0.94 (m, 48H), 1.18 (s, 3H), 1.20–2.05 (m, 18H),
2.23 (br dd, 1H, J ϭ 12.6, 3.3 Hz), 2.46 (br dd, 1H, J ϭ
12.9, 7.8 Hz), 2.81 (br d 1H, J ϭ 12.3 Hz), 3.22 (m, 2H),
3.58–3.76 (m, 4H), 4.16–4.26 (m, 2H), 4.98 (br s, 1H), 5.26
(br s, 1H), 6.00 (d, 1H, J ϭ 10.8 Hz), 6.22 (d, 1H, J ϭ
A mixture of 30S (5.1 mg, 4.73 ␮mol) and 1 M TBAF in
THF (237 ␮l, 237 ␮mol) in toluene (0.5 ml) was stirred at
105°C for 2 h. The mixture was extracted with AcOEt, washed
with H2O and saturated NaCl, dried and evaporated. The res-
idue was purified by preparative TLC developed with CH2Cl2-
EtOH (5:1) to give 6S (1.45 mg, 60%) as a colorless oil: IR
1
(neat) 3400, 2947, 2929, 2873, 1378, 1107, 1072 cmϪ1; H
NMR ␦: 0.55 (s, 3H), 0.95 (d, 3H, J ϭ 6.3 Hz), 1.16 (s, 3H),
1.22 (s, 3H), 2.42 (br d, 1H, J ϭ 13.9 Hz), 2.55 (dd, 1H, J ϭ
14.5, 4.0 Hz), 2.75–2.87 (m, 1H), 3.20–3.37 (m, 2H), 3.68–
3.78 (m, 1H), 3.80–3.99 (m, 3H), 4.20–4.36 (m, 2H), 5.08 (br
s, 1H), 5.50 (br s, 1H), 6.04 (d, 1H, J ϭ 11.2 Hz), 6.36 (d, 1H,
J ϭ 11.2 Hz); UV ␭max 264 nm; [␣]D24 Ϫ49° (c 0.07, EtOH).
2.1.29. Vitamin D receptor (VDR) binding assay [13]
Chick embryonic intestinal 1␣,25(OH)2D3 receptor (Ya-
masa Shoyu Co., Tokyo, Japan) was incubated at 4°C for 3
h with 4.67 nCi of [3H]1␣,25(OH)2D3 and various concen-
tration of analogs (1, 3, 6R, and 6S). Bound and free forms
of [3H]1␣,25(OH)2D3 were separated by addition of dext-
ran-charcoal and centrifugation. The radioactivity of the
receptor-bound [3H]1␣,25(OH)2D3 was measured with a
PACKARD TRI-CARB 2700TR.
23
10.5Hz); [␣]D ϩ25.8° (c 0.28, CHCl3).
2.1.26. (5Z,7E)-(1R,2R,3R,24S)-1,3,24-Tris(tert-butyldimeth-
ylsilyloxy)-2-(3-tert-butyldimethylsilyloxypropoxy)-9,10-seco-
cholesta-5,7,10(19)-triene (30S)
A mixture of PPh3 (2.4 mg, 9.2 ␮mol), (dba)3Pd2-CHCl3
(1.7 mg, 1.6 ␮mol), and triethylamine (0.3 ml) in toluene (0.2
ml) was stirred at room temperature for 10 min. To the stirred
mixture, were added 7 (9.6 mg, 0.017 mmol) and 9S (15.6 mg,
0.026 mmol) in toluene (0.2 ml) at room temperature. The
resulting mixture was refluxed for 4 h, diluted with hexane,
filtered with silica gel, and evaporated. The residue was puri-
fied by preparative TLC developed with hexane-benzene (2:1)
to give 30S (6,2 mg, 34%) as a colorless oil: IR (neat) 1467,
2.1.30. Vitamin D binding protein (DBP) binding assay
[14,15]
The binding affinity of analogs (1, 3, 6R, and 6S), with
rat DBP was performed according to Revelle et al. [14,15].
1
1252 cmϪ1; H NMR ␦: 0.02–0.10 (m, 30H), 0.52 (s, 3H),
2.1.31. Determination of ionized plasma calcium levels
Mice (BALB/c, male, 9 weeks of age) were i.v. given
vehicle (EtOH/saline ϭ 1/99) and 10 ␮g/kg of analogs (1, 3,
6R, and 6S). Their ionized plasma calcium levels at 24 h
and 48 h after a single i.v. injection were determined by
automatic Ca2ϩ/pH analyzer (The 634 Ca2ϩ/pH Analyzer,
CIBA-CORNING, Tokyo, Japan). The results (mmol/l) are
expressed as the mean Ϯ S.D. The statistical significance of
the differences between the vehicle and the experimental
groups was analyzed by the use of Student’s t test.
0.82–0.94 (m, 48H), 1.11 (s, 3H), 1.19 (s, 3H), 1.20–2.05 (m,
18H), 2.22 (br dd, 1H, J ϭ 12.6, 3.8 Hz), 2.46 (br dd, 1H, J ϭ
11.6, 8.1 Hz), 2.81 (br d, 1H, J ϭ 13.5 Hz), 3.16–3.24 (m, 2H),
3.58–3.76 (m, 4H), 4.16–4.26 (m, 2H), 4.98 (br s, 1H), 5.26
(br s, 1H), 6.00 (d, 1H, J ϭ 10.8 Hz), 6.22 (d, 1H, J ϭ 10.5
Hz); [␣]D23 ϩ10.7° (c 0.31, CHCl3).
2.1.27.
(5Z,7E)-(1R,2R,3R,24R)-2-(3-Hydroxypropoxy)-
9,10-secocholesta-5,7,10(19)-triene-1,3,24.25-tetraol (6R)
A mixture of 30R (3.3 mg, 3.06 ␮mol) and 1 M tetrabu-
tylammonium fluoride (TBAF) in THF (153 ␮l, 153 ␮mol) in
toluene (0.5 ml) was stirred at 105°C for 2 h. The mixture was
extracted with AcOEt, washed with H2O and saturated NaCl,
dried and evaporated. The residue was purified by preparative
TLC developed with CH2Cl2-EtOH (5:1) to give 6R (1.02 mg,
66%) as a colorless oil: IR (neat) 3400, 2947, 2927, 2871,
1377, 1105, 1070 cmϪ1; 1H NMR ␦: 0.56 (s, 3H), 0.94 (d, 3H,
J ϭ 5.9 Hz), 1.16 (s, 3H), 1.22 (s, 3H), 2.42 (br d, 1H, J ϭ 14.5
Hz), 2.55 (dd, 1H, J ϭ 14.5, 4.0 Hz), 2.76–2.87 (m, 1H), 3.27
(dd, 1H, J ϭ 8.8, 2.8 Hz), 3.30–3.37 (m, 1H), 3.68–3.78 (m,
1H), 3.80–4.00 (m, 3H), 4.21–4.36 (m, 2H), 5.08 (br s, 1H),
5.50 (br s, 1H), 6.04 (d, 1H, J ϭ 11.2 Hz), 6.36 (d, 1H, J ϭ
11.2 Hz); UV ␭max 264 nm; [␣]D24 Ϫ23° (c 0.07, EtOH).
3. Results
3.1. Synthesis
To synthesize 24-hydroxylated ED-71 (6), we adopted
the convergent method, in which the A-ring fragment (7)
and the C/D-ring fragments (9) in 24(R) and 24(S) forms
(vitamin D numbering) are coupled to produce a triene
system of vitamin D3 structure [16]. While the A-ring frag-
ment (7) was synthesized from diethyl D-tartarate (8), the
C/D-ring fragments (9) were prepared from vitamin D2 (10)
in 24(R) and 24(S) forms, respectively, as shown in the
retrosynthetic pathway in Fig. 2.
2.1.28.
(5Z,7E)-(1R,2R,3R,24S)-2-(3-Hydroxypropoxy)-
First, we undertook the synthesis of the A-ring fragment (7).
The epoxide (11), obtained from diethyl D-tartarate (8) by a
9,10-secocholesta-5,7,10(19)-triene-1,3,24.25-tetraol (6S)

274
S. Hatakeyama et al. / Steroids 66 (2001) 267–276
Fig. 2. Retrosynthesis of 24-hydroxylated ED-71.
known 5-step sequence [7], was cleaved by 1,3-propandiol in
the presence of potassium t-BuOK at 120°C to give the alcohol
(12) in 86% yield. After the protection of the introduced
primary hydroxy part as the pivalate (13) in 88% yield, the
cleavage of the benzyl ether in 13 and the subsequent protec-
tion of the resulting 1,2-diol moiety as the acetonide gave the
alcohol (14) in 87% overall yield. The Swern oxidation of 14
and the Grignard reaction toward the resulting aldehyde af-
forded the dipivalate (15) as an epimeric mixture (R/S ϭ 3/2)
after the protection of the hydroxy part as the pivalate, al-
though the stereoselectivity in the Grignard reaction was not
able to be realized. Without separation of the epimeric mixture
at this stage, acetonide moiety in 15 was cleaved quantitatively
to give the diol (16). By the Mitsunobu reaction [8], 16 was
converted to the epoxide (17) in 77% yield. The subsequent
introduction of the acetylene unit was successfully carried out
based upon the regioselective epoxide cleavage in 17 to pro-
vide the A-ring fragment (7) in 36% yield after separation of
the accompanied (S)-epimer (18) (24%) by column chroma-
tography (Fig. 3).
Lythgoe diol (19), prepared from vitamin D2 (10) by the
ozonolysis [17], was converted to the sulfone (21) via 20 by
known methodology [9,10]. The sulfone (21) was alkylated by
both (R)- and (S)-2,3-dihydroxy-3-methylbutyl p-toluenenesul-
fonate (22R and 22S) [13,14] to afford the alcohol (23R) in
71% yield and the alcohol (23S) in 28% yield, respectively.
The (R)- and (S)-alcohol (23R and 23S) were converted to the
C/D ring fragments (9R and 9S) as follows;
1. Desulfurization of 23R and 23S with Na/Hg giving
24R and 24S in 97% and 68% yields, respectively
2. Acetonide formation of 24R and 24S with 2,2-dime-
thoxypropane and the subsequent acetylation giving
25R and 25S in 83% and 64% yields, respectively
3. Deprotection of acetonide parts in 25R and 25S with
iodine in methanol giving 26R and 26S in 61% and
83% yields, respectively
4. Silylation of 26R and 26S with TBSOTf giving 27R
and 27S in 92% and 91% yields, respectively
5. Reductive cleavage of the acetyl parts in 27R and 27S
with LiAlH4 giving 28R and 28S in 96% and 100%
yields, respectively
Next, we performed the synthesis of the C/D-ring fragments
in both 24(R) and 24(S) series, respectively. The Inhoffen-
Fig. 3. Synthesis of the A-ring part. a) HO(CH2)3OH/t-BuOK; b) t-BuCOCl/pyridine; c) 1) H2/Pd(OH)2, 2) Me2C(OMe)2/TsOH, d); 1) Swern oxidation, 2)
CH2 ϭ CHMgBr, 3) t-BuCOCl/Et3N; e) 1M HCl; f) Ph3P/DEAD; g) 1) LiC ϵ CTMS/BF3-OEt2, 2) 10N NaOH, 3) TBSOTf/Et3N, 4) separation.

S. Hatakeyama et al. / Steroids 66 (2001) 267–276
275
Fig. 4. Synthesis of the C/D-ring parts. a) Ac2O/pyridine; b)n-BuLi; c) Na/Hg; d) 1) Me2C(OMe)2/TsOH, 2) Ac2O/pyridine; e) I2/MeOH; f) TBSOTf/lutidine;
g) LiAlH4; h) TPAP/NMO; i) Ph3PCH2Br2/NaN(TMS)2.
6. Oxidation of the hydroxy parts in 28R and 28S with
NMO and tetra n-Pr4NRuO4 giving 29R and 29S in
96% and 100% yields, respectively
7. Wittig reaction of 29R and 29S with Ph3PCH2Br2 in
the presence of NaN(TMS)2 giving 9R and 9S in 49%
and 57% yields, respectively (Fig. 4).
analog, plasma ionized calcium levels in BALB/c mice in-
creased significantly at 24 and 48 h after i.v. administration
compared to vehicle group. ED-71 (3) showed the most potent
increasing activity of iozined calcium among analogs tested
(vehicle: 1.32 Ϯ 0.02 vs. ED-71 (3): 1.63 Ϯ 0.02 mmol/l; P Ͻ
0.001, vehicle: 1.31 Ϯ 0.03 vs. ED-71 (3): 2.23 Ϯ 0.11
mmol/l; P Ͻ 0.001). Although 1␣,25(OH)2D3 (1), (24R)OH-
ED-71 (6R), and (24S)OH-ED-71 (6S) showed tendency of
increase in plasma calcium levels compared to vehicle, the
changes were less than those of ED-71
Having the A-ring fragment (7) and the C/D-ring frag-
ments (9R and 9S), we performed the coupling reaction in
the presence of PPh3 and (dba)3Pd2-CHCl3 following the
Trost methodology [16], to give 30R and 30S in 34% and
36% yields, respectively, which were deprotected by TBAF
to (24R)OH-ED-71 (6R) and (24S)OH-ED-71 (6S) in 60%
and 66% yields, respectively (Fig. 5).
4. Discussion
As analogs related to ED-71 (3), (24R)OH-ED-71 (6R) and
(24S)OH-ED-71 (6S) were synthesized by the convergent
method based upon the reaction between the A-ring fragment
(7) obtained from diethyl D-tartarate (8) and the C/D-ring
fragments (9R and 9S) prepared from vitamin D2 (10) via the
Inhoffen-Lythgoe diol (19). 24-Hydroxylation of ED-71 (3)
caused weakened affinity to DBP in vitro and less calcemic
activity in vivo compared to 3. The weakened calcemic char-
acters of 24-hydroxylated analogs (6R and 6S) might derive
from the weak affinity to DBP, taking the high calcemic
character of ED-71 (3) based on its strong affinity to DBP into
3.2. Biological evaluation
The results of preliminary biological evaluations of synthe-
sized (24R)OH-ED-71 (6R) and (24S)OH-ED-71 (6S) in com-
parison with 1␣,25(OH)2D3 (1) and ED-71 (3) are summarized
in Table 1. The binding potency to rat DBP [14,15] in both
(24R)OH-ED-71 (6R) and (24S)OH-ED-71 (6S) was less than
that of ED-71 (3). The binding affinity to chick VDR [13] in
(24R)OH-ED-71 (6R) was comparable to ED-71 (3). On the
other hand, that of (24S)OH-ED-71 (6S) showed 1/10 affinity
compared to ED-71 (3). At the dose of 10 ␮g/kg of each
Fig. 5. Coupling the A-ring part with C/D-ring parts. a) (dba)3Pd2CHCl3/PPh3; b) TBAF.

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S. Hatakeyama et al. / Steroids 66 (2001) 267–276
Table 1
Biological characters of 24-hydroxylated ED-71
VDR
DBP
Ca (24 h)
Ca (48 h)
Vehicle
1␣,25(OH)2D3 (1)
ED-71 (3)
(24R)OH-ED-71 (6R)
(24S)OH-ED-71 (6S)
1.32 Ϯ 0.02 (n ϭ 4)
1.59 Ϯ 0.04 (n ϭ 4)*
1.63 Ϯ 0.02 (n ϭ 5)*
1.48 Ϯ 0.04 (n ϭ 4)*
1.49 Ϯ 0.05 (n ϭ 4)**
1.31 Ϯ 0.03 (n ϭ 5)
1.46 Ϯ 0.05 (n ϭ 4)*
2.23 Ϯ 0.11 (n ϭ 5)*
1.45 Ϯ 0.03 (n ϭ 4)*
1.44 Ϯ 0.06 (n ϭ 4)**
1.00
0.13
0.10
0.01
1.00
2.7
0.3
0.1
VDR: Affinity to chick intestinal vitamin D receptor.
DBP: Affinity to rat vitamin D binding protein.
Ca: Ionized calcium levels (mmol/l) after 10 ␮g/kg of analogs intravenous administration to BALB/c mice. * P Ͻ 0.001 vs. vehicle group; ** P Ͻ 0.01
vs. vehicle group.
stituted at the 2␤-position and their preventive effects on bone mineral
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consideration. While the affinity to VDR in (24R)OH-ED-71
(6R) was sustained, the affinity in (24S)OH-ED-71 (6S) was
less than ED-71 (3). The observed effect of (24R)- and (24S)-
hydroxylation of ED-71 (3) on the affinity to VDR corresponds
well to the reported cases of the 24-hydroxylation of
1␣,25(OH)2D3 (1) [18,19] and 1␣OHD3 (2) [13]. The preven-
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(24S)OH-ED-71 (6S) on bone mineral loss in ovariectomized
rats are highly interested and under investigation. When ED-71
(3) was orally administered to rats, (24R)OH-ED-71 (6R) and
(24S)OH-ED-71 (6S) were found in rat plasma as metabolites
of 3 with accompanying several other metabolites. The de-
tailed metabolic pathway and metabolites of ED-71 (3) will be
reported elsewhere.
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We are grateful to Drs. Hiroyoshi Watanabe, Kazumi
Morikawa, and Naoki Kubota, Chugai Pharmaceutical Co.
Ltd., for their assistance and to Associate Professor David
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