Design, synthesis and biological evaluation of novel 1α,25- dihydroxyvitamin D3 analogues possessing aromatic ring on 2α-position

Article abstract of DOI:10.1016/j.tet.2005.08.116

In the present study, we describe the synthesis of new analogues of 1α,25-dihydroxyvitamin D₃ (1), which possess hydrophobic aromatic ring on the 2α position. Among these analogues, 2α-benzyl analogue showed the highest potency in the affinity

Full text of DOI:10.1016/j.tet.2005.08.116

Tetrahedron 61 (2005) 11253–11263
Design, synthesis and biological evaluation of novel
1a,25-dihydroxyvitamin D3 analogues possessing aromatic
ring on 2a-position
Shinobu Honzawa,a Koshiro Hirasaka,a Yasuhiro Yamamoto,a Sara Peleg,b Toshie Fujishima,a
Masaaki Kurihara,c Nozomi Saito,a Seishi Kishimoto,d Takayuki Sugiura,d Keizo Waku,d
Hiroaki Takayamaa and Atsushi Kittakaa,
*
aDepartment of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa 199-0195, Japan
bM. D. Anderson Cancer Center, The University of Texas, Houston, TX 77030, USA
cNational Institute of Health Sciences, Setagaya-ku, Tokyo 158-8501, Japan
dDepartment of Hygienic Chemistry, Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa 199-0195, Japan
Received 5 July 2005; accepted 29 August 2005
Available online 10 October 2005
Abstract—In the present study, we describe the synthesis of new analogues of 1a,25-dihydroxyvitamin D3 (1), which possess hydrophobic
aromatic ring on the 2a position. Among these analogues, 2a-benzyl analogue showed the highest potency in the affinity for the wild type
vitamin D receptor (VDR) and induction of HL-60 cell differentiation as well as transcriptional activity. Affinity for the mutant VDR related
to hereditary vitamin D-resistant rickets (R274L) was also examined.
q 2005 Elsevier Ltd. All rights reserved.
1. Introduction
1a,25-Dihydroxyvitamin D3 (1a,25-(OH)2D3, 1) is known
as the active metabolite of vitamin D3 and regulates a wide
variety of biological activities, such as intestinal calcium
absorption, bone resorption and mineralization.1 In addition,
1 has been found to induce cell differentiation and
proliferation in culture and in vivo.2 These biological
activities are mediated by ligand binding to the nuclear
vitamin D receptor (VDR), a ligand-dependent transcription
factor.3 In order to improve the biological profile of 1 for a
therapeutic application, numerous analogues of 1 have been
synthesized and developed for biological studies and
Figure 1. 1a,25-Dihydroxyvitamin D3 and its analogues.
clinical trials, most of which have modified side chain of 1.4
to binding affinity for the bovine thymus VDR, elevation of
serum calcium in rats, and induction of HL-60 cell
We have synthesized A-ring modified analogues of 1, in
differentiation. Among 2a-substituted analogues,
which the 2a-alkyl or the 2a-hydroxyalkyl group was
2a-methyl analogue 2 is worthy of comment, that is, only
introduced to 1, to study the A-ring conformation- and
structure-activity relationships (Fig. 1).5–8 The resulting
analogues exhibited interesting biological activities, in
a small structural modification on the 2a position leads to
4-fold increase in VDR affinity and 2-fold increase in
induction of HL-60 cell differentiation. Based on the X-ray
crystallographic analysis of the truncated VDR ligand
particular, 2a-methyl and 2a-(3-hydroxypropyl) analogues
(2 and 3) showed much higher potency than 1 with respect
binding domain (LBD) occupied by the natural hormone,9
we could see that the A-ring of 1 is surrounded by
Keywords: Vitamin D receptor; Aromatic ring; Ligand binding domain.
*
hydrophobic amino acid residues Tyr143, Tyr147, Phe150
and Tyr236 (Fig. 2). We considered that the affinity of the
Corresponding author. Tel.:/fax: C81 426 85 3713;
e-mail: akittaka@pharm.teikyo-u.ac.jp
0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2005.08.116

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S. Honzawa et al. / Tetrahedron 61 (2005) 11253–11263
analogues that have aromatic groups at the 2a-position.
That is, we prepared 2a-phenyl-(4), 2a-benzyl-(5) and
2a-phenethyl-1a,25-(OH)2D3 (6) and evaluated their
affinity for VDR and their ability to induce differentiation
of HL-60 cells (Fig. 3).
2. Synthesis
Retrosynthetic analysis of the analogues is shown in
Scheme 1. We adapted the convergent synthetic method
developed by Trost et al.10 This method utilized bromo-
olefin 710 and enyne 8 as reaction substrates and these were
coupled in the presence of a palladium catalyst. CD ring
bromoolefin 7 was synthesized from vitamin D3 and A-ring
enyne 8 was synthesized from sugar epoxide 11, easily
derived from D-glucose.11 2a-Substituents could be intro-
duced by the reaction of 11 with corresponding Grignard
reagents, which was reported from our laboratory quite
recently.12
Figure 2. X-ray structure of VDR ligand binding domain with 1 by Moras
et al.9
2a-methyl analogue (2) for the VDR should increase
because of hydrophobic interaction between 2a-methyl
group and this hydrophobic pocket. In the present study we
examined the hypothesis that the affinity for VDR would
increase if a larger hydrophobic group, for example, the
phenyl group, could interact with the hydrophobic pocket.
In order to elucidate this assumption, we synthesized novel
Summary of the synthesis of enynes 8a–c is shown in
Scheme 2. In short, the ring opened products 10a–c were
subjected to NBS in the presence of BaCO3 to give
Figure 3. Structures of 2a-aromatic substituted analogues.
Scheme 1. Retrosynthetic analysis of 4–6.

S. Honzawa et al. / Tetrahedron 61 (2005) 11253–11263
11255
Scheme 2. Synthetic route toward the A-ring precursors of 2a-aromatic analogues 4–6.
Scheme 3. Palladium-catalyzed coupling reaction.
bromides 12a–c, which upon reaction with activated zinc
powder and sodium cyanoborohydride gave diols 9a–c.
These diols were transformed to epoxides 13a–c via
monotosylates. Epoxides were converted to enynes 8a–c
through reaction with lithium acetylide followed by
treatment with K2CO3 and persilylation. Palladium cata-
lyzed coupling reaction of CD ring bromoolefin 7 with 8,
followed by deprotection gave the 2a-aromatic substituted
vitamin D derivatives 4–6 (Scheme 3). These derivatives
were purified for biological assays by recycling reverse-
phase HPLC.
6, respectively) showed 7, 15 and 1% affinities, respectively,
when compared with the natural hormone (Table 1). These
results would suggest that aromatic groups of 2a-sub-
stituents would be so large that these could not fit well to the
pocket around the A-ring of vitamin D in the LBD, contrary
to our hypothesis. In addition to the steric bulkiness of the
aromatic ring, tendencies of the aromatic substituents to
adopt the equatorial position14 would lower the affinities for
VDR due to the partial conformational changes in the
A-ring from the b-form, suitable for hydrogen bonding, to
the unsuitable a-form.6a Indeed, if the aromatic ring would
be forced to accommodate in the pocket, the analogue
molecules should shift their atomic positions largely in
order to form hydrogen bonds (see Figs. 4 and 5).
3. Results and discussions
Surprisingly, when HL-60 cell differentiating activities
were measured, the analogues were as potent or even higher
than the natural hormone. Above all, 2a-benzyl analogue 5
was 4.6-fold more potent than the natural hormone
(Table 1).
The new analogues were first examined for their affinity for
the bovine thymus VDR, by using competition assay.13 We
found that these analogues had lower affinity compared with
the natural hormone, 1a,25-(OH)2D3. In fact, 2a-phenyl,
2a-benzyl and 2a-phenethyl substituted analogues (4, 5 and
To further elucidate the origin of the discrepancies between
VDR binding profiles of the analogues and their cellular
responses, we considered the possibility that the bovine
VDR (calf thymus) and the human VDR (in HL-60 cells)
have different binding properties to these analogues.
Therefore, we used recombinant human VDR to perform
additional assays including a protease sensitivity assay and
transactivation assay.15 The protease sensitivity assay has
Table 1. Biological activities of analogues prepared
VDR affinitya,b
HL-60 cell differentiationa
2a-phenyl (4)
2a-benzyl (5)
2a-phenethyl (6)
7
15
1
220
460
140
a Activities of 1a,25-(OH)2D3 were normalized to 100.
b Bovine thymus.

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S. Honzawa et al. / Tetrahedron 61 (2005) 11253–11263
Table 2. Biological activities of the analogues with recombinant human
VDR
Protease sensitivitya
Transcriptional activitya,b
2a-phenyl (4)
2a-benzyl (5)
2a-phenethyl (6)
0.02
20
0.2
25
250
77
a Activities of 1a,25-(OH)2D3 were normalized to 100.
b Co-transfection of wild type human VDR and osteocalcin VDRE-TK-GH
reporter gene.
been used to determine the effect of ligand on VDR’s
conformation, as reflected by the size of VDR fragments
that become resistant to trypsin digestion after ligand
binding. The assay, in some cases, also reflects transcrip-
tional potency of an analogue because ability to stabilize
VDR conformation in vitro may correlate with transcrip-
tional activity of the analogue in cultured cells.15 The
protease sensitivity assay was performed with in vitro
synthesized 35S-labeled VDR treated with different concen-
trations of the analogues and digested with trypsin. Then
protease-resistant fragments were analyzed by SDS-PAGE
and autoradiography of the dried gels.15 We found that the
three analogues stabilized a VDR conformation similar to
that stabilized by the natural hormone (data not shown).
However, we also found similar results (Table 2) to the
competitive assay with bovine thymus VDR (Table 1). That
is, aromatic ring-substituted analogues 4–6 had lower ability
to stabilize human VDR conformation against trypsin
digestion than the natural hormone 1, and 2a-benzyl
substituted analogue 5 had the highest potency among this
group of derivatives. In contrast, transcriptional activities of
the analogues were similar to that of the natural hormone
(Table 2), as in the case of HL-60 cell differentiation
(Table 1). The reason for restoring potency to induce cell
differentiation and transcriptional activity is not clear, but it
may suggest that cellular modifications of the analogue-
bound VDR or interaction of the analogue-bound VDR with
dimerization partners or coactivators of transcription change
the mode of interaction of these analogues with VDR in the
cells and lead to an increase in their transcriptional potency
and cellular activities which is not proportional to their
receptor binding properties in vitro. Our laboratory is now
developing such a high-throughput assay system that could
examine coactivator-dependent ligand binding with VDR.16
The origin of discrepancies shown here could be clarified
utilizing this assay system.
Figure 4. Hypothetical models for the structure of the VDR’s LBD bound
to 2a-phenyl analogue 4 (a) and 2a-benzyl analogue 5 (b). Hydrogen bonds
are drawn as dotted lines.
As the hydrophobic properties of the 2a-substituents of the
analogues 4–6 were considered, we anticipated that the
analogues 4–6 could be used to restore defective ligand
binding activity of the mutant VDR, Arg274Leu, which
causes hereditary vitamin D-resistant rickets (HVDRR).
HVDRR is caused by mutations to the VDR gene, which
lead to resistance to 1a,25-(OH)2D3 (1) under physiological
condition.17 Clinically, HVDRR is associated with hypo-
calcemia, secondary hyperparathyroidism, and relatively
high concentration of 1a,25-(OH)2D3 (1) in serum. Over 20
mutations that cause HVDRR have been reported. Most of
these mutations occur at the DNA binding domain, but a few
are localized at the LBD. We have focused our attention to
the mutated VDR whose Arg-274 is substituted to Leu,
Arg274Leu.18 The Arg-274 residue locates in the ligand
Figure 5. An overlay of the modeled VDR-2a-benzyl analogue 5 complex
with VDR-1 structure in the LBD. Ligands 1 and 5 are drawn in gray and
green, respectively.

S. Honzawa et al. / Tetrahedron 61 (2005) 11253–11263
11257
binding domain of the VDR and forms hydrogen bond with
the 1a-hydroxy group of 1 (Fig. 2). The mutation causes a
1000-fold decrease in the affinity for 1, thus the vitamin D
action should be disrupted. Vitamin D derivatives that bind
specifically to a mutated VDR of this type would be a
candidate for therapeutic agent against HVDRR.15 A few
research groups have reported such designs of ligand for the
mutant VDR. Peleg et al. have demonstrated the rationale
for using A ring-modified analogues to restore loss of
binding and transcriptional activity of the Arg274Leu
mutant.15 Koh et al. developed derivatives whose 1a-
hydroxy group is protected by a substituted benzyl group.19
On the other hand, we have approached to the issue by using
2a-hydroxypropylated 1 (3).20 In this case, we have shown
that 3 would form hydrogen bonding with Asp144 of
the mutant VDR through the terminal hydroxy group of
the 2a-substituent, thus could restore its complex formation
ability with the mutant VDR.
2a-benzyl analogue induced detectable stabilization of the
mutant VDR (Table 3).
In all cases where biological activities were assayed, the
2a-benzyl analogue (5) was the most potent in the aromatic
analogue series, that is, one methylene group appeared the
most appropriate to link the A-ring of the natural hormone
with the phenyl group.
In summary, we prepared novel analogues of 1 that possess
the aromatic ring on the 2a position. Data of biological
assays showed that these analogues induce HL-60 cell
differentiation and transcriptional activities as much potent
as 1, whereas the affinity for VDR was less than 10%
relative to the natural hormone. Further studies are now on
the way to elucidate the discrepancies between binding
affinity of the analogues for VDR in vitro and their potencies
in the cellular assays, including studies on metabolism by
CYP27A1 and CYP24A1.21
We hypothesized that analogues possessing hydrophobic
substituent at the 2a-position could fit the hydrophobic
pocket formed by the mutation and restore ligand-mediated
VDR action. Our modeling studies imply that the
hydrophobic pocket around the 2a-position of 1 would be
extended by the mutation of bulkier Arg to sterically less
demanding Leu. Because of this, the aromatic ring on the
2a-substituent could be accommodated more comfortably
in the pocket of the mutant receptor than that of the wild
type receptor, which would make the complex more stable
(Fig. 6). So, we decided to assay the analogues 4–6 for their
binding to the mutant VDR, Arg274Leu.
Transcriptional assay for the mutant VDR (Arg274Leu) is
now planning and the result will be reported elsewhere.
4. Experimental
4.1. General procedure
1H and 13C NMR spectra were recorded on a JEOL GSX-
400 or AL-400 NMR (400 MHz) or ECP-600 NMR
(600 MHz). 1H NMR spectra taken in CDCl3 were
referenced to tetramethylsilane (d 0.00) as an internal
standard. 13C NMR spectra taken in CDCl3 (d 77.0) were
referenced to the residual solvents. Melting points were
determined with Yanagimoto micro melting point apparatus
without correction. Optical rotations were measured on a
JASCO DIP-370 digital polarimeter. IR spectra were
measured on a JASCO FT/IR-8000 spectrophotometer.
Low- and high-resolution MS spectra were recorded on a
JEOL JMS-SX-102A spectrometer. Merck silica gel 60
(230–400 mesh) was employed for flash column chroma-
tography. The ratios of solvent mixtures for chromato-
graphy are shown in volume/volume.
Syntheses of 10a–c have been reported elsewhere.12
Figure 6. Superposition of modeled structures of 5 in the LBD of wild type
VDR (green) and mutant VDR (Arg274Leu, purple).
4.1.1. Methyl 4-O-Benzoyl-6-C-bromo-3,6-dideoxy-3-C-
phenyl-a-D-altropyranoside (12a). Under an Ar atmos-
phere, 10a (144.9 mg, 0.423 mmol) was dissolved in CCl4
(5 mL, passed through alumina pad before use), and to this
solution were added N-bromosuccinimide (90 mg,
0.51 mmol) and BaCO3 (46.7 mg, 0.24 mmol). After
refluxed for 40 min, the mixture was allowed to cool to
room temperature, diluted with AcOEt (10 mL) and washed
with saturated aqueous Na2SO3 (10 mL). Layers were
separated and organic layer was washed successively with
water (5 mL), saturated aqueous NaHCO3 solution (5 mL),
brine (5 mL), dried (MgSO4) and concentrated. Purification
by silica gel column chromatography (toluene–AcOEt (20/
1–18/1)) gave the product 12a (128 mg, 72%) as a white
foam.
Because competition assays cannot be used with this mutant
due to its low affinity for the natural hormone, we used
instead the protease sensitivity assay to assess binding of
the analogues to the human VDR, in vitro.15 Contrary to
our expectation, these analogues showed poor ability to
stabilize the conformation of the mutant VDR, and only the
Table 3. Binding affinity of the analogues with mutant VDR (Arg274Leu)
Protease sensitivitya
1a,25-(OH)2D3
2a-phenyl (4)
2a-benzyl (5)
2a-phenethyl (6)
0.1
NDb
0.1
NDb
a Affinity of 1a,25-(OH)2D3 to wild type VDR is normalized to 100.
1H NMR (400 MHz, CDCl3) d 1.97 (1H, d, JZ2.9 Hz), 3.32
b Not detectable.

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S. Honzawa et al. / Tetrahedron 61 (2005) 11253–11263
(1H, dd, JZ4.1, 11.5 Hz), 3.59 (3H, s), 3.72 (1H, dd, JZ
4.1, 11.0 Hz), 4.25 (1H, dt, JZ3.9, 7.3 Hz), 4.44 (1H, ddd,
JZ3.8, 5.4, 11.5 Hz), 4.77 (1H, d, JZ5.4 Hz), 5.37 (1H, t,
JZ3.8 Hz), 7.19 (1H, t, JZ7.7 Hz), 7.24–7.27 (2H, m),
7.34 (2H, d, JZ7.3 Hz), 7.42 (2H, t, JZ7.7 Hz), 7.56 (1H, t,
JZ7.3 Hz), 7.92 (2H, d, JZ7.3 Hz). 13C NMR (100 MHz,
CDCl3) d 32.0, 47.4, 56.4, 69.6, 73.2, 73.7, 103.4, 127.6,
128.3, 128.7, 129.0, 129.3, 129.5, 133.1, 135.1, 165.2. IR
(neat) 3470, 1719, 1453, 1316, 1269, 712 cmK1. LRMS (EI)
m/z: 389 (MC(79Br)–OCH3), 267, 209. HRMS (EI) calcd for
C19H1879BrO4; 389.0383. Found 389.0391. [a]2D1 K110.9 (c
1.1, CHCl3).
(10 mL), and layers were separated. Organic layer was
washed with water (10 mL), saturated aqueous NH4Cl
solution (10 mL), brine (10 mL), dried (MgSO4) and
concentrated. Purification by silica gel column chromato-
graphy (0.1% MeOH in CHCl3) gave the product 9a
(29.1 mg, 62%) as white foam.
1H NMR (400 MHz, CDCl3) d 2.99 (1H, dd, JZ3.7,
10.3 Hz), 3.21 (1H, dd, JZ8.1, 11.2 Hz), 3.42 (1H, dd, JZ
3.7, 11.2 Hz), 4.08 (1H, dd, JZ3.7, 8.1 Hz), 5.05 (1H, br d,
JZ10.6 Hz), 5.21 (1H, br d, JZ17.0 Hz), 5.67 (1H, ddd,
JZ6.1, 10.6, 17.0 Hz), 6.03 (1H, dd, JZ6.1, 10.3 Hz),
7.26–7.33 (5H, m), 7.45–7.49 (2H, m), 7.58–7.62 (1H, m),
8.09–8.12 (2H, m). 13C NMR (100 MHz, CDCl3) d 52.9,
65.2, 70.4, 75.0, 117.9, 127.3, 128.3, 128.4, 129.6, 129.7,
129.7, 133.3, 134.5, 136.2, 166.5. IR (neat) 3389, 1721,
1601, 1493, 1453, 712 cmK1. LRMS (EI) m/z: 312 (MC),
295, 281, 159, 105, 77. HRMS (EI) calcd for C19H20O4;
312.1362. Found 312.1361. [a]2D2 C66.6 (c 2.0, CHCl3).
12b and 12c could be synthesized substantially as the same
procedure.
4.1.2. Methyl 4-O-Benzoyl-3-C-benzyl-6-C-bromo-3,6-
dideoxy-a-D-altropyranoside (12b). A colorless foam
1
(85%). H NMR (600 MHz, CDCl3) d 2.20 (1H, d, JZ
4.6 Hz), 2.49 (1H, dddd, JZ4.6, 6.6, 7.7, 8.2 Hz), 2.78 (1H,
dd, JZ7.7, 13.7 Hz), 3.22 (1H, dd, JZ8.2, 13.7 Hz), 3.53
(1H, dd, JZ8.2, 11.1 Hz), 3.53 (3H, s), 3.57 (1H, dd, JZ
4.0, 11.1 Hz), 3.80 (1H, ddd, JZ3.8, 4.1, 6.6 Hz), 4.25 (1H,
ddd, JZ4.0, 6.3, 8.2 Hz), 4.62 (1H, d, JZ3.8 Hz), 5.14 (1H,
dd, JZ4.6, 6.3 Hz), 7.07–7.09 (2H, m), 7.12–7.15 (1H, m),
7.18–7.21 (2H, m), 7.44–7.47 (2H, m), 7.58–7.61 (1H, m),
8.04–8.06 (2H, m). 13C NMR (100 MHz, CDCl3) d 31.6,
31.8, 43.6, 56.2, 69.8, 70.4, 70.5, 102.1, 126.1, 128.3, 128.4,
128.9, 129.3, 129.6, 133.3, 138.9, 165.2. IR (neat) 3472,
9b and 9c were also synthesized substantially the same
manner.
4.1.5. (2S,3R,4R)-4-Benzoyloxy-3-benzylhex-5-ene-1,2-
diol (9b). A colorless oil (75%). 1H NMR (600 MHz,
CDCl3) d 2.21–2.24 (1H, m), 2.76 (1H, dd, JZ7.2,
14.5 Hz), 2.92 (1H, dd, JZ6.0, 14.5 Hz), 3.53 (1H, dd,
JZ3.8, 11.0 Hz), 3.63 (1H, dd, JZ8.5, 11.0 Hz), 4.04–4.05
(1H, m), 5.28 (1H, d, JZ10.7 Hz), 5.31 (1H, d, JZ
17.0 Hz), 5.59 (1H, apparent t, JZ5.5 Hz), 5.91 (1H, ddd,
JZ6.0, 10.7, 17.0 Hz), 7.16–7.18 (3H, m), 7.25–7.26 (2H,
m), 7.41–7.45 (2H, m), 7.56–7.58 (1H, m), 7.97–7.99 (2H,
m). 13C NMR (100 MHz, CDCl3) d 31.8, 46.6, 65.1, 71.5,
76.0, 118.1, 126.2, 128.5, 128.6, 129.0, 129.6, 129.8, 133.3,
134.9, 140.4, 165.8. IR (neat) 3424, 3063, 3028, 2928, 1718,
1603, 1495, 1452, 1315, 1113, 700 cmK1. LRMS (EI) m/z:
326 (MC), 308 (MCKH2O). HRMS (EI) calcd for
C20H22O4; 326.1521. Found 326.1521. [a]2D5 C0.2 (c 0.5,
CHCl3).
3063, 3028, 2934, 1716, 1603, 1495, 1452, 1267 cmK1
.
LRMS (EI) m/z: 434 (MC(79Br)). HRMS (EI) calcd for
C21H2379BrO5; 434.0729. Found 434.0742. [a]2D5 C4.1 (c
1.1, CHCl3).
4.1.3. Methyl 4-O-Benzoyl-6-C-bromo-3,6-dideoxy-3-C-
(2-phenylethyl)-a-D-altropyranoside (12c). A colorless oil
(85%). H NMR (400 MHz, CDCl3) d 1.72–1.84 (1H, m),
1
2.09–2.25 (3H, m), 2.58–2.62 (2H, m), 3.52 (3H, s), 3.54–
3.62 (2H, m), 3.83 (1H, m), 4.23 (1H, ddd, JZ5.3, 5.3,
7.1 Hz), 4.54 (1H, d, JZ4.4 Hz), 5.43 (1H, dd, JZ4.4,
5.3 Hz), 7.09–7.19 (3H, m), 7.20–7.25 (2H, m), 7.44–7.49
(2H, m), 7.60 (1H, m), 8.02–8.07 (2H, m). 13C NMR
(150 MHz, CDCl3) d 27.4, 31.3, 33.3, 40.2, 56.2, 70.2, 70.7,
71.8, 102.2, 126.0, 128.3, 128.4, 128.6, 129.4, 129.8, 133.5,
4.1.6. (2S,3R,4R)-4-Benzoyloxy-3-(2-phenylethyl)hex-5-
ene-1,2-diol (9c). A colorless oil (57%). 1H NMR
(400 MHz, CDCl3) d 1.68–1.92 (3H, m), 2.62 (1H, ddd,
JZ6.1, 10.1, 13.5 Hz), 2.71 (1H, br s), 2.78 (1H, ddd, JZ
5.9, 9.9, 13.5 Hz), 3.03 (1H, br s), 3.53 (1H, dd, JZ3.2,
10.9 Hz), 3.66 (1H, dd, JZ8.6, 10.9 Hz), 3.93 (1H, m), 5.29
(1H, d, JZ10.7 Hz), 5.29 (1H, d, JZ17.1 Hz), 5.70 (1H,
apparent t, JZ6.1 Hz), 5.92 (1H, ddd, JZ6.1, 10.7,
17.1 Hz), 7.10–7.19 (3H, m), 7.20–7.27 (2H, m), 7.38–
7.44 (2H, m), 7.51–7.57 (1H, m), 7.99–8.06 (2H, m). 13C
NMR (100 MHz, CDCl3) d 27.5, 34.4, 43.8, 65.0, 71.7,
76.0, 118.1, 125.8, 128.3, 128.3, 128.4, 129.5, 129.7, 133.1,
134.5, 141.8, 165.9. IR (neat) 3291, 2942, 1719, 1603, 1453,
1273, 1113, 1071, 1026, 940, 714 cmK1. LRMS (EI) m/z:
340 (MC), 322 (MCKH2O). HRMS (EI) calcd for
C21H24O4; 340.1675. Found 340.1670. [a]D C26.3 (c 1.0,
CHCl3).
141.5, 165.7. IR (neat) 3443, 2930, 1719, 1453, 1269 cmK1
.
LRMS (EI) m/z: 448 (MC(79Br)), 416 (MC(79Br)–CH3OH),
294 (MC(79Br)–CH3OH–PhCOOH). HRMS (EI) calcd for
C22H2579BrO5; 448.0885. Found 448.0863. [a]22D C14.0 (c
0.6, CHCl3).
4.1.4. (2S,3R,4R)-4-Benzoyloxy-3-phenylhex-5-ene-1,2-
diol (9a). Under an Ar atmosphere, to a solution of bromide
(12a, 50.8 mg, 0.121 mmol) in 1-propanol (1.2 mL) was
added distilled water (120 mL) and the mixture was heated
at 95 8C. To the mixture were added Zn dust (activated by
washing with dil. HCl aq, then EtOH and Et2O, and dried in
vacuo, 239 mg, 3.63 mmol) and NaBH3CN (38 mg,
0.60 mmol) in one portion and stirred at the same
temperature for 20 min. Zn dust (160 mg, 2.42 mmol) and
NaBH3CN (23 mg, 0.36 mmol) were added and stirred
further 30 min. The mixture was cooled to room tempera-
ture, and insoluble materials were filtered off through Celite.
The filtrate was diluted with AcOEt (100 mL) and water
4.1.7. (3R,4R,5S)-3-Benzoyloxy-5,6-epoxy-4-phenylhex-
1-ene (13a). Synthesis of monotosylate: Under an Ar
atmosphere, to a solution of 9a (475 mg, 1.52 mmol) in
CH2Cl2 (15 mL) was added p-toluenesulfonyl chloride
(406 mg, 2.13 mmol), triethylamine (316 mL, 2.28 mmol)

S. Honzawa et al. / Tetrahedron 61 (2005) 11253–11263
11259
and 4-(N,N-dimethylamino)pyridine (1.14 mg, 9.3 mmol)
and stirred at room temperature for 4 h. The mixture was
diluted with AcOEt (100 mL) and washed with water
(10 mL!2), saturated aqueous NH4Cl (10 mL), brine
(10 mL), dried (MgSO4) and concentrated. Purification by
silica gel column chromatography (hexane–AcOEt (9/1–
6/1)) gave monotosylate (621 mg, 90%) as a colorless oil.
4.1.11. Base-promoted epoxide formation. Under an Ar
atmosphere, to a cooled (K78 8C) solution of phenyl-
substituted monotosylate (620 mg, 1.33 mmol) in THF
(13 mL) was added LiHMDS (1 M in hexane, 1.6 mL,
1.60 mmol) and stirred at K78 8C for 20 min. and at room
temperature for 45 min. The mixture was diluted with
AcOEt (200 mL) and washed with water (10 mL!2),
saturated aqueous NH4Cl solution (10 mL), brine (10 mL)
dried (MgSO4), and concentrated. Purification by silica gel
column chromatography (hexane–AcOEt (15/1)) gave the
product 13a (322.2 mg, 85%) as a colorless foam.
4.1.8. (2S,3R,4R)-4-Benzoyloxy-3-phenyl-1-(p-toluene-
sulfonyloxy)hex-5-en-2-ol. H NMR (400 MHz, CDCl3) d
1
2.40 (3H, s), 3.04 (2H, br dd, JZ3.7, 11.0 Hz), 3.70 (1H, d,
JZ6.6 Hz), 4.23–4.28 (1H, m), 5.03 (1H, d, JZ10.6 Hz),
5.19 (1H, d, JZ17.0 Hz), 5.63 (1H, ddd, JZ6.4, 10.6,
17.0 Hz), 6.00 (1H, dd, JZ6.4, 11.0 Hz), 7.24–7.26 (7H,
m), 7.48–7.51 (2H, m), 7.60–7.68 (3H, m), 8.09–8.11 (2H,
m). 13C NMR (100 MHz, CDCl3) d 21.7, 52.2, 67.5, 71.3,
74.7, 118.1, 127.5, 127.7, 128.3, 128.4, 129.4, 129.6, 129.7,
129.9, 132.4, 133.4, 134.3, 134.8, 144.7, 166.5. IR (neat)
3528, 3510, 1719, 1362, 1273, 1177 cmK1. LRMS (EI) m/z:
466 (MC), 344. HRMS (EI) calcd for C26H26O6S; 466.1364.
Found 466.1457. [a]1D7 C18.7 (c 1.0, CHCl3).
1H NMR (400 MHz, CDCl3) d 2.54 (1H, dd, JZ2.6,
5.0 Hz), 2.78 (1H, dd, JZ3.9, 4.9 Hz), 2.84 (1H, dd, JZ4.9,
7.6 Hz), 3.47 (1H, ddd, JZ2.6, 3.9, 7.6 Hz), 5.13 (1H, ddd,
JZ1.1, 1.1, 10.6 Hz), 5.26 (1H, ddd, JZ1.1, 1.1, 16.9 Hz),
5.76 (1H, ddd, JZ6.2, 10.6, 16.9 Hz), 5.92–5.96 (1H, m),
7.24–7.35 (5H, m), 7.45–7.48 (2H, m), 7.57–7.60 (1H, m),
8.07–8.09 (2H, m). 13C NMR (100 MHz, CDCl3) d 47.4,
52.8, 53.1, 75.8, 117.8, 127.3, 128.3, 128.4, 128.7, 129.4,
129.8, 133.0, 134.1, 136.8, 165.2. IR (neat) 1719, 1601,
1584, 1453 cmK1. LRMS (EI) m/z: 294 (MC), 172. HRMS
(EI) calcd for C19H18O3; 294.1256. Found 294.1257. [a]D19
K23.3 (c 0.8, CHCl3).
Benzyl- and 2-phenylethyl substituted compounds could
also be synthesized as substantially the same manner.
13b and 13c could also be synthesized as substantially the
same manner.
4.1.9. (2S,3R,4R)-4-Benzoyloxy-3-benzyl-1-(p-toluene-
sulfonyloxy)hex-5-en-2-ol. A colorless oil (82%). 1H
NMR (600 MHz, CDCl3) d 2.12–2.24 (1H, m), 2.42 (3H,
s), 2.71 (1H, dd, JZ7.1, 14.5 Hz), 2.81 (1H, dd, JZ7.1,
14.5 Hz), 3.81–3.89 (2H, m), 4.20–4.22 (1H, m), 5.25 (1H,
d, JZ10.6 Hz), 5.27 (1H, d, JZ16.7 Hz), 5.55 (1H,
apparent t, JZ5.5 Hz), 5.84 (1H, ddd, JZ6.0, 10.6,
16.7 Hz), 7.06–7.09 (2H, m), 7.16–7.19 (1H, m), 7.21–
7.22 (2H, m), 7.28–7.29 (2H, m), 7.43–7.46 (2H, m), 7.57–
7.59 (1H, m), 7.67–7.68 (2H, m), 7.97–7.98 (2H, m). 13C
NMR (150 MHz, CDCl3) d 21.7, 31.4, 46.4, 68.7, 72.3,
75.6, 118.3, 126.4, 128.0, 128.6, 128.6, 129.1, 129.6, 129.7,
129.9, 132.5, 133.4, 134.7, 139.6, 145.0, 165.5. IR (neat)
3526, 3065, 3028, 2922, 1718, 1601, 1495, 1452, 1361,
1271, 1176, 1111, 1097, 970 cmK1. LRMS (EI) m/z: 480
(MC). HRMS (EI) calcd for C27H28O6S; 480.1607. Found
480.1616. [a]2D5 C25.1 (c 0.3, CHCl3).
4.1.12. (3R,4R,5S)-3-Benzoyloxy-4-benzyl-5,6-epoxyhex-
1-ene (13b). A colorless oil (86%). H NMR (600 MHz,
1
CDCl3) d 1.80 (1H, dddd, JZ4.4, 5.4, 8.4, 8.4 Hz), 2.65
(1H, dd, JZ2.8, 4.9 Hz), 2.84 (1H, dd, JZ3.9, 4.9 Hz), 2.90
(1H, dd, JZ8.4, 13.8 Hz), 3.00 (1H, dd, JZ5.4, 13.8 Hz),
3.12 (1H, ddd, JZ2.8, 3.9, 8.4 Hz), 5.26 (1H, ddd, JZ1.4,
1.4, 10.4 Hz), 5.30 (1H, ddd, JZ1.4, 1.4, 17.2 Hz), 5.83
(1H, dddd, JZ1.4, 1.4, 4.4, 6.1 Hz), 5.89 (1H, ddd, JZ6.1,
10.4, 17.2 Hz), 7.16–7.22 (3H, m), 7.25–7.29 (2H, m), 7.45–
7.48 (2H, m), 7.57–7.61 (1H, m), 8.01–8.04 (2H, m). 13C
NMR (100 MHz, CDCl3) d 35.0, 47.9, 48.4, 52.5, 74.5,
117.7, 126.4, 128.5, 128.5, 129.3, 129.6, 130.0, 133.2,
134.5, 138.8, 165.3. IR (neat) 3065, 3030, 2928, 1718, 1603,
1495, 1452, 1273, 1133, 1070, 1026 cmK1. LRMS (EI) m/z:
308 (MC). HRMS (EI) calcd for C20H20O3; 308.1412.
Found 308.1410. [a]2D5 K3.3 (c 0.7, CHCl3).
4.1.10. (2S,3R,4R)-4-Benzoyloxy-3-(2-phenylethyl)-1-(p-
toluenesulfonyloxy)hex-5-en-2-ol. A colorless oil (88%).
1H NMR (400 MHz, CDCl3) d 1.62–1.82 (2H, m), 1.89 (1H,
dddd, JZ2.8, 4.4, 6.2, 6.2 Hz), 2.42 (3H, s), 2.53 (1H, br d,
JZ3.6 Hz), 2.56 (1H, ddd, JZ6.8, 10.0, 13.6 Hz), 2.75 (1H,
ddd, JZ6.0, 10.0, 13.6 Hz), 3.99 (1H, dd, JZ4.8, 10.0 Hz),
4.05 (1H, dd, JZ7.6, 10.0 Hz), 4.11–4.18 (1H, m), 5.29
(1H, ddd, JZ1.2, 1.2, 10.5 Hz), 5.35 (1H, ddd, JZ1.2, 1.2,
17.1 Hz), 5.69 (1H, dddd, JZ1.2, 1.2, 6.2, 6.2 Hz), 5.88
(1H, ddd, JZ6.2, 10.5, 17.1 Hz), 7.07–7.13 (2H, m), 7.14–
7.20 (1H, m), 7.22–7.28 (2H, m), 7.28–7.34 (2H, m), 7.42–
7.48 (2H, m), 7.58–7.62 (1H, m), 7.75–7.80 (2H, m), 7.99–
8.03 (2H, m). 13C NMR (100 MHz, CDCl3) d 21.7, 27.1,
34.1, 43.4, 68.7, 71.8, 75.6, 118.4, 125.9, 127.9, 128.3,
128.3, 128.5, 129.5, 129.8, 132.5, 133.3, 134.5, 141.4,
145.0, 165.7. IR (neat) 3499, 3065, 3029, 2938, 1719, 1599,
1453, 1362, 1271, 1177, 1098, 972 cmK1. LRMS (EI) m/z:
494 (MC), 476 (MCKH2O). HRMS (EI) calcd for
C28H30O6S; 494.1763. Found 494.1759. [a]2D0 C23.4 (c
0.8, CHCl3).
4.1.13. (3R,4R,5S)-3-Benzoyloxy-5,6-epoxy-4-(2-phenyl-
ethyl)hex-1-ene (13c). A colorless oil (82%). 1H NMR
(400 MHz, CDCl3) d 1.54 (1H, m) 1.89–2.00 (2H, m), 2.60
(1H, dd, JZ2.6, 5.0 Hz), 2.80 (1H, ddd, JZ7.2, 8.8,
14.2 Hz), 2.85 (2H, ddd, JZ7.2, 9.2, 14.2 Hz), 2.79 (1H, dd,
JZ4.0, 5.0 Hz), 3.00 (1H, ddd, JZ2.6, 4.0, 8.4 Hz), 5.28
(1H, ddd, JZ1.2, 1.2, 10.8 Hz), 5.37 (1H, ddd, JZ1.2, 1.2,
17.2 Hz), 5.68 (1H, dddd, JZ1.2, 1.2, 5.3, 6.5 Hz), 5.90
(1H, ddd, JZ6.5, 10.8, 17.2 Hz), 7.14–7.23 (3H, m), 7.25–
7.29 (2H, m), 7.45–7.48 (2H, m), 7.57–7.61 (1H, m), 8.01–
8.04 (2H, m). 13C NMR (100 MHz, CDCl3) d 31.4, 33.2,
45.8, 46.8, 53.2, 75.5, 117.9, 125.7, 128.2, 128.3, 128.3,
129.4, 129.9, 133.0, 134.2, 141.7, 165.2. IR (neat) 3063,
3029, 2994, 2926, 2867, 1721, 1603, 1495, 1453, 1271,
1111, 712 cmK1. LRMS (EI) m/z: 322 (MC), 218 (MCK
PhCOCH). HRMS (EI) calcd for C21H22O3; 322.1569.
Found 322.1572. [a]2D1 C22.2 (c 1.2, CHCl3).

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S. Honzawa et al. / Tetrahedron 61 (2005) 11253–11263
4.1.14. (3R,4S,5R)-3-Benzoyloxy-4-phenyl-8-(trimethyl-
silyl)oct-1-en-7-yn-5-ol (14a). Under an Ar atmosphere,
to a cold (K78 8C) solution of ethynyltrimethylsilane
(440 mL, 3.12 mmol) in THF (3 mL) was added n-BuLi
(1.6 M in hexane, 1.6 mL, 2.5 mmol) and stirred at the same
temperature for 10 min. To the mixture were added epoxide
13a (369 mg, 1.25 mmol) in THF (2 mL) and BF3$OEt2
(174 mL, 1.37 mmol), and stirred at the same temperature
for 2 h. The reaction was quenched by adding water (10 mL)
and the mixture was extracted with AcOEt (150 mL).
Organic layer was washed with water (10 mL), saturated
aqueous NH4Cl solution (10 mL), brine (10 mL), dried
(MgSO4) and concentrated. Purification by silica gel
column chromatography (hexane–AcOEt (40/1–30/1))
gave the product 14a (427 mg, 87%) as a colorless foam.
7.56–7.62 (1H, m), 8.03–8.07 (2H, m). 13C NMR
(100 MHz, CDCl3) d 0.2, 26.6, 34.5, 44.9, 69.5, 76.1,
87.5, 103.1, 117.8, 125.8, 128.2, 128.4, 129.5, 129.8, 133.1,
134.8, 141.7, 165.6. IR (neat) 3497, 3023, 2961, 2176, 1721,
1603, 1453, 1273, 1113, 1026, 845, 758, 712 cmK1. LRMS
(EI) m/z: 420 (MC), 405 (MCKCH3). HRMS (EI) calcd
for C26H32O3Si; 420.2121. Found 420.2117. [a]1D9 C19.7
(c 0.8, CHCl3).
4.1.17. (3R,4S,5R)-3,5-Bis-(tert-butyldimethylsilyloxy)-4-
phenyloct-1-en-7-yne (8a). Deprotection: To a solution of
enyne 14a (387 mg, 0.986 mmol) in MeOH (6.5 mL) was
added K2CO3 (409 mg, 2.96 mmol) and stirred at room
temperature for 4.5 h. The mixture was diluted with AcOEt
(200 mL) and washed with water (20 mL!2), saturated
aqueous NH4Cl solution (20 mL), brine (20 mL), dried
(MgSO4) and concentrated. The residue was used for the
next reaction without further purification, but, analytical
sample could be obtained by purification by silica gel
column chromatography (PhMe–AcOEt (9/1)) as a colorless
foam.
1H NMR (400 MHz, CDCl3) d 0.16 (9H, s), 2.07 (1H, dd,
JZ7.2, 16.7 Hz), 2.24 (1H, dd, JZ7.2, 16.7 Hz), 2.74 (1H,
d, JZ4.9 Hz), 3.16 (1H, dd, JZ2.7, 10.5 Hz), 4.20 (1H, ddt,
JZ2.7, 4.9, 7.9 Hz), 5.05 (1H, ddd, JZ1.2, 1.2, 11.2 Hz),
5.22 (1H, ddd, JZ1.2, 1.2, 17.8 Hz), 5.68 (1H, ddd, JZ6.3,
11.2, 17.8 Hz), 6.07 (1H, dddd, JZ1.2, 1.2, 6.3, 10.5 Hz),
7.28–7.38 (5H, m), 7.48–7.52 (2H, m), 7.60–7.64 (1H, m),
8.13–8.16 (2H, m). 13C NMR (100 MHz, CDCl3) d 0.2,
26.6, 54.0, 68.4, 75.1, 87.4, 103.4, 117.7, 127.3, 128.2,
128.4, 129.7, 129.8, 130.4, 133.2, 134.9, 135.6, 166.3. IR
(neat) 2176, 1703 cmK1. LRMS (EI) m/z: 392 (MC), 377
(MCKCH3). HRMS (EI) calcd for C24H28O3Si; 392.1815.
Found 392.1808. [a]1D8 K3.7 (c 1.0, CHCl3).
4.1.18. (3R,4S,5R)-4-phenyloct-1-en-7-yne-3,5-diol. 1H
NMR (400 MHz, CDCl3) d 2.04 (1H, t, JZ2.7 Hz), 2.11
(1H, ddd, JZ2.7, 8.2, 16.5 Hz), 2.24 (1H, ddd, JZ2.7, 7.4,
16.5 Hz), 2.43 (1H, br s), 2.70 (1H, br s), 2.88 (1H, dd, JZ
2.9, 7.4 Hz), 4.49 (1H, ddt, JZ2.7, 7.4, 8.2 Hz), 4.67 (1H,
dd, JZ5.7, 7.6 Hz), 5.09 (1H, ddd, JZ1.4, 1.4, 10.5 Hz),
5.25 (1H, ddd, JZ1.4, 1.4, 17.0 Hz), 5.83 (1H, ddd, JZ5.7,
10.5, 17.0 Hz), 7.26–7.36 (5H, m). 13C NMR (100 MHz,
CDCl3) d 25.3, 54.4, 69.6, 70.6, 74.3, 81.1, 115.6, 127.1,
128.2, 129.9, 137.3, 139.3. IR (neat) 3443, 3283, 2119, 1239
704, 630 cmK1. LRMS (EI) m/z: 216 (MC), 198 (MCK
H2O). HRMS (EI) calcd for C14H16O2; 216.1152. Found
216.1150. [a]1D8 K17.0 (c 1.0, CHCl3).
14b and 14c could also be prepared as substantially the same
manner.
4.1.15. (3R,4S,5R)-3-Benzoyloxy-4-benzyl-8-(trimethyl-
silyl)oct-1-en-7-yn-5-ol (14b). A colorless oil (78%). H
1
NMR (600 MHz, CDCl3) d 0.17 (9H, s), 2.36–2.39 (2H, m),
2.46 (1H, dd, JZ7.7, 16.9 Hz), 2.55 (1H, dd, JZ6.9,
16.9 Hz), 2.66 (1H, dd, JZ9.0, 14.5 Hz), 2.98 (1H, dd, JZ
4.6, 14.5 Hz), 4.25 (1H, t, JZ7.1 Hz), 5.22 (1H, ddd, JZ
1.2, 1.2, 5.0 Hz), 5.25 (1H, ddd, JZ1.2, 1.2, 11.2 Hz), 5.64
(1H, dddd, JZ1.2, 1.2, 4.2, 5.5 Hz), 5.86 (1H, ddd, JZ5.5,
10.7, 16.2 Hz), 7.18–7.30 (5H, m), 7.31–7.49 (2H, m), 7.58–
7.62 (1H, m), 7.96–8.07 (2H, m). 13C NMR (150 MHz,
CDCl3) d 0.2, 26.7, 30.8, 47.6, 69.3, 75.9, 87.7, 103.0,
117.2, 126.0, 128.4, 128.5, 129.1, 129.4, 129.7, 133.1,
134.8, 140.2, 165.3. IR (neat) 3505, 2959, 2928, 2175, 1722,
1645, 1603, 1585, 1495, 1452, 1412, 1271, 1251, 1113,
1026 cmK1. LRMS (EI) m/z: 406 (MC), 388 (MCKH2O).
HRMS (EI) calcd for C25H30O3Si; 406.1964. Found
406.1963. [a]2D5 K6.9 (c 0.6, CHCl3).
Benzyl- and 2-phenylethyl substituted compounds could
also be prepared as substantially the same manner.
4.1.19. (3R,4S,5R)-4-Benzyloct-1-en-7-yne-3,5-diol. 1H
NMR (600 MHz, CDCl3) d 2.02 (1H, dddd, JZ1.7, 3.4,
4.9, 10.4 Hz), 2.07 (1H, t, JZ2.7 Hz), 2.43 (1H, ddd, JZ
2.7, 7.1, 16.8 Hz), 2.52 (1H, d, JZ3.3 Hz), 2.57 (1H, ddd,
JZ2.7, 7.4, 16.8 Hz), 2.83 (1H, dd, JZ4.9, 14.3 Hz), 2.91
(1H, dd, JZ10.4, 14.3 Hz), 3.14 (1H, br d, JZ1.4 Hz), 4.21
(1H, br s), 4.26 (1H, apparent t, JZ7.1 Hz), 5.22 (1H, ddd,
JZ1.7, 1.7, 10.7 Hz), 5.32 (1H, ddd, JZ1.7, 1.7, 17.0 Hz),
5.84 (1H, ddd, JZ4.4, 10.7, 17.0 Hz), 7.20–7.31 (5H, m).
13C NMR (150 MHz, CDCl3) d 24.6, 30.4, 46.9, 69.6, 70.6,
73.0, 80.9, 115.1, 126.0, 128.3, 129.2, 139.6, 140.1. IR
(neat) 3302, 3026, 2922, 2120, 1495, 1454, 1321 cmK1
.
LRMS (EI) m/z: 230 (MC). HRMS (EI) calcd for C15H18O2;
230.1307. Found 230.1296. [a]2D5 K18.2 (c 0.9, CHCl3).
4.1.16. (3R,4S,5R)-3-Benzoyloxy-4-(2-phenylethyl)-8-
(trimethylsilyl)oct-1-en-7-yn-5-ol (14c). A colorless oil
(80%). H NMR (400 MHz, CDCl3) d 0.15 (9H, s), 1.74
1
(1H, dddd, JZ5.6, 7.4, 9.9, 14.5 Hz) 1.91 (1H, dddd, JZ
4.0, 6.5, 10.3, 14.5 Hz), 2.01 (1H, dddd, JZ2.7, 4.0, 5.3,
7.4 Hz), 2.36 (1H, br d, JZ2.4 Hz), 2.43 (1H, dd, JZ6.8,
16.8 Hz), 2.55 (1H, dd, JZ7.2, 16.8 Hz), 2.68 (2H, ddd, JZ
6.5, 9.9, 13.7 Hz), 2.85 (1H, ddd, JZ5.6, 10.3, 13.7 Hz),
4.07–4.13 (1H, m), 5.31 (1H, ddd, JZ1.2, 1.2, 10.6 Hz),
5.38 (1H, ddd, JZ1.2, 1.2, 16.8 Hz), 5.77 (1H, apparent ddt,
JZ1.2, 1.2, 6.0 Hz), 5.93 (1H, ddd, JZ6.3, 10.6, 16.8 Hz),
7.15–7.20 (3H, m), 7.24–7.29 (2H, m), 7.44–7.50 (2H, m),
4.1.20. (3R,4S,5R)-4-(2-Phenylethyl)oct-1-en-7-yne-3,5-
diol. H NMR (400 MHz, CDCl3) d 1.70 (1H, dddd, JZ
1
2.6, 4.3, 4.3, 8.6 Hz) 1.75 (1H, dddd, JZ4.3, 6.4, 9.4,
13.8 Hz), 1.89–2.00 (1H, m), 2.00 (1H, t, JZ2.7 Hz), 2.29
(1H, ddd, JZ2.7, 6.2, 16.7 Hz), 2.47 (1H, ddd, JZ2.7, 7.9,
16.7 Hz), 2.63 (1H, ddd, JZ6.4, 9.7, 14.0 Hz), 2.63 (1H, br
s), 2.83 (1H, ddd, JZ5.2, 9.4, 14.0 Hz), 2.93 (1H, br s), 4.20
(apparent t, JZ7.2 Hz), 4.43 (1H, br s), 5.25 (1H, ddd, JZ
1.5, 1.5, 10.6 Hz), 5.36 (1H, ddd, JZ1.5, 1.5, 17.4 Hz), 5.90

S. Honzawa et al. / Tetrahedron 61 (2005) 11253–11263
11261
(1H, ddd, JZ5.0, 10.6, 17.4 Hz), 7.16–7.22 (3H, m), 7.26–
7.31 (2H, m). 13C NMR (100 MHz, CDCl3) d 24.4, 26.1,
33.9, 44.6, 70.2, 70.5, 74.0, 81.0, 115.5, 125.8, 128.3, 128.3,
139.8, 141.8. IR (neat) 3303, 2934, 2122, 1647, 1605, 1123,
1082, 1040, 924, 750, 700 cmK1. LRMS (EI) m/z: 244
(MC), 226 (MCKH2O). HRMS (EI) calcd for C16H20O2;
244.1463. Found 244.1463. [a]1D6 K12.7 (c 0.8, CHCl3).
ddd, JZ6.5, 10.3, 13.9 Hz), 2.73 (1H, ddd, JZ6.5, 10.3,
13.9 Hz), 4.02 (1H, dt, JZ4.2, 6.3 Hz), 4.19 (1H, dd, JZ
5.6, 7.2 Hz), 5.12 (1H, m), 5.19 (1H, m), 5.86 (1H, ddd, JZ
7.2, 10.0, 17.2 Hz), 7.17 (3H, m), 7.26 (2H, m). 13C NMR
(100 MHz, CDCl3) d K4.4, K4.2, K4.0, K3.5, 18.3, 18.3,
26.0, 26.1, 26.3, 28.0, 35.7, 49.2, 70.1, 71.5, 75.6, 81.9,
115.6, 125.4, 128.1, 128.2, 139.8, 143.0. IR (neat) 3314,
2955, 2930, 2859, 2122, 1472, 1462, 1254, 1076, 837,
776 cmK1. LRMS (EI) m/z: 472 (MC), 457 (MCKCH3),
415 (MCKt-Bu), 340 (MCKTBSOH). HRMS (EI) calcd
for C28H48O2Si2; 472.3193. Found 472.3187. [a]2D3 C4.4
(c 0.7, CHCl3).
4.1.21. Protection of hydroxy group. Under an Ar
atmosphere, to a cooled (0 8C) solution of crude phenyl-
substituted diol and 2,6-lutidine (459 mL, 3.94 mmol) in
CH2Cl2 (14 mL) was added TBSOTf (1.5 mL, 6.41 mmol)
and stirred at the same temperature for 40 min. The mixture
was diluted with AcOEt (200 mL) and washed with water
(20 mL!2), saturated aqueous NH4Cl solution (20 mL),
brine (20 mL), dried (MgSO4) and concentrated. Purifi-
cation by silica gel column chromatography (hexane–
AcOEt (50/1)) gave the product 8a (434 mg, 98% for 2
steps) as a colorless oil.
4.1.24. 1a,25-Dihydroxy-2a-phenylvitamin D3 (4).
A solution of enyne 8a (50 mg, 0.112 mmol), bromoolefin
710 (66 mg, 0.185 mmol), Pd(PPh3)4 (64 mg, 0.056 mmol)
in toluene (1 mL) and triethylamine (3 mL) was stirred
under an Ar atmosphere at reflux for 2 h. After cooled to
room temperature, the mixture was diluted with Et2O and
filtered. The filtrate was diluted with AcOEt (50 mL),
washed with water (5 mL!2), brine (10 mL), dried
(MgSO4) and concentrated. The residue was partially
purified by passing through silica gel pad (elution;
hexane–AcOEt (30/1)). The crude bisTBS ether was treated
with TBAF (1 M in THF, 2.2 mL, 2.2 mmol) and stirred at
room temperature for 48 h, and then 60 8C for 30 min. The
mixture was diluted with AcOEt (50 mL), washed with
water (5 mL!2), brine (5 mL) and concentrated. The
residue was purified by silica gel column chromatography
(hexane–AcOEt (2/1)) to give the product 4 (7 mg, 13% for
2 steps) as a white amorphous solid. This sample was further
purified for biological studies by recycled reverse phase
HPLC (YMC-Pack ODS column, 20!150 mm, 9.9 mL/
min, CH3CN–H2O (9/1)).
1H NMR (400 MHz, CDCl3) d 0.03 (3H, s), 0.05 (3H, s),
0.10 (3H, s), 0.10 (3H, s), 0.85 (9H, s), 0.91 (9H, s), 1.96
(1H, ddd, JZ2.7, 9.8, 16.4 Hz), 2.06 (1H, t, JZ2.7 Hz),
2.24 (1H, ddd, JZ2.7, 4.4, 16.4 Hz), 3.04 (1H, dd, JZ2.6,
9.2 Hz), 4.49–4.53 (2H, m), 4.86 (1H, dd, JZ1.2, 9.7 Hz),
4.92 (1H, dd, JZ1.2, 17.5 Hz), 5.54 (1H, ddd, JZ8.1, 9.7,
17.5 Hz), 7.15–7.19 (5H, m). 13C NMR (100 MHz, CDCl3)
d K4.1, K4.0, K3.9, K2.4, 18.3, 18.4, 25.8, 26.0, 26.1,
26.2, 56.4, 69.6, 70.6, 75.2, 81.3, 116.9, 126.2, 127.1, 131.1,
137.4, 140.2. IR (neat) 3314, 2965, 2897, 2858, 2122, 1495,
1472, 1464, 1428, 1091, 700, 640 cmK1. LRMS (EI) m/z:
444 (MC), 312 (MCKTBSOH). HRMS (EI) calcd for
C26H44O2Si2; 444.2875. Found 444.2880. [a]1D8 K3.0 (c 2.5,
CHCl3).
1H NMR (400 MHz, CDCl3) d 0.54 (3H, s), 0.94 (3H, d, JZ
6.3 Hz), 1.21 (6H, s), 1.00–1.64 (16H, m), 1.64–1.78 (2H,
m), 1.82–1.95 (1H, m), 1.98–2.03 (2H, m), 2.40 (1H, br t,
JZ11.9 Hz), 2.81–2.91 (3H, m), 4.31 (1H, br s), 4.50 (1H,
dd, JZ4.6, 10.2, 10.2 Hz), 5.07 (1H, d, JZ1.8 Hz), 5.31
(1H, d, JZ1.8 Hz), 6.04 (1H, d, JZ11.2 Hz), 6.53 (1H, d,
JZ11.2 Hz), 7.29–7.32 (1H, m), 7.36–7.44 (4H, m). 13C
NMR (100 MHz, CDCl3) d 12.3, 18.9, 21.0, 22.4, 23.6,
27.8, 29.2, 29.3, 29.4, 36.2, 36.5, 40.6, 44.3, 44.5, 46.0,
56.4, 56.6, 57.6, 68.3, 71.1, 115.1, 116.8, 125.4, 127.3,
128.7, 129.2, 132.0, 138.8, 143.5, 146.0. IR (film) 3405,
2944, 2870, 1647, 1603, 1495, 1377, 735 cmK1. LRMS (EI)
m/z: 492 (MC), 456 (MCK2!H2O). HRMS (EI) calcd for
C33H48O3; 492.3571. Found 492.3601. [a]1D7 C80.9 (c 0.05,
CHCl3).
8b and 8c could also be prepared as substantially the same
manner.
4.1.22. (3R,4S,5R)-4-Benzyl-3,5-bis-(tert-butyldimethyl-
silyloxy)oct-1-en-7-yne (8b). A colorless oil (88% for 2
steps). 1H NMR (600 MHz, CDCl3) d 0.01 (3H, s), 0.02 (3H,
s), 0.03 (3H, s), 0.05 (3H, s), 0.89 (9H, s), 0.91 (9H, s), 1.94
(1H, t, JZ2.7 Hz), 2.20 (1H, ddd, JZ2.7, 7.1, 16.8 Hz),
2.26 (1H, dddd, JZ2.7, 6.0, 6.0, 8.2 Hz), 2.30 (1H, ddd, JZ
2.7, 6.0, 14.3 Hz), 2.62 (1H, dd, JZ6.0, 14.3 Hz), 2.70 (1H,
dd, JZ8.2, 14.3 Hz), 4.07 (1H, ddd, JZ2.7, 6.0, 7.2 Hz),
4.18 (1H, dd, JZ6.0, 7.3 Hz), 5.08 (1H, ddd, JZ1.1, 1.1,
10.4 Hz), 5.17 (1H, ddd, JZ1.1, 1.1, 17.3 Hz), 5.83 (1H,
ddd, JZ7.3, 10.4, 17.3 Hz), 7.14–7.26 (5H, m). 13C NMR
(100 MHz, CDCl3) d K4.5, K4.3, K3.9, K3.3, 18.3, 18.3,
25.9, 26.0, 26.1, 32.2, 51.4, 69.9, 70.9, 75.8, 82.2, 115.7,
125.4, 127.9, 129.2, 140.1, 142.0. IR (neat) 3314, 2955,
2930, 2858, 2122, 1471, 1361, 1253, 1084, 1064, 924, 837,
775 cmK1. LRMS (EI) m/z: 458 (MC). HRMS (EI) calcd
for C27H46O2Si2; 458.3036. Found 458.3034. [a]2D5 C12.1
(c 0.9, CHCl3).
5 and 6 were also synthesized as substantially the same
manner.
4.1.25. 2a-Benzyl-1a,25-dihydroxyvitamin D3 (5).
A white amorphous solid (32% for 2 steps). 1H NMR
(600 MHz, CDCl3) d 0.53 (3H, s), 0.93 (3H, d, JZ6.6 Hz),
1.21 (9H, s), 1.00–1.72 (16H, m), 1.83–2.05 (6H, m), 2.27
(1H, dd, JZ9.3, 12.6 Hz), 2.73 (1H, dd, JZ4.4, 13.2 Hz),
2.82–2.86 (2H, m), 2.99 (1H, dd, JZ5.5, 13.7 Hz), 3.93
(1H, ddd, JZ4.4, 8.8, 8.8 Hz), 4.12 (1H, d, JZ2.8 Hz), 4.94
(1H, d, JZ2.0 Hz), 5.14 (1H, d, JZ2.0 Hz), 5.97 (1H, d,
JZ11.0 Hz), 6.42 (1H, d, JZ11.0 Hz), 7.21–7.31 (5H, m).
4.1.23. (3R,4S,5R)-3,5-Bis-(tert-butyldimethylsilyloxy)-4-
(2-phenylethyl)oct-1-en-7-yne (8c). A colorless oil (74%
for 2 steps). 1H NMR (400 MHz, CDCl3) d K0.08 (3H, s),
0.05 (3H, s), 0.06 (3H, s), 0.09 (3H, s), 0.89 (18H, s), 1.59–
1.73 (2H, m), 1.88 (1H, dddd, JZ4.2, 5.6, 5.6, 5.6 Hz), 1.97
(1H, t, JZ2.5 Hz), 2.41 (2H, dd, JZ2.5, 6.3 Hz), 2.66 (1H,

11262
S. Honzawa et al. / Tetrahedron 61 (2005) 11253–11263
13C NMR (100 MHz, CDCl3) d 12.0, 18.8, 20.8, 22.2, 23.5,
27.6, 29.1, 29.2, 29.3, 33.1, 36.1, 36.4, 40.5, 44.4, 44.6,
45.9, 51.6, 56.3, 56.5, 70.1, 71.1, 72.9, 113.9, 116.9, 124.9,
125.9, 128.4, 129.3, 132.7, 140.1, 143.4, 146.4. LRMS (EI)
m/z: 506 (MC). HRMS (EI) calcd for C34H50O3; 506.3760.
Found 506.3373. [a]2D5 C22.1 (c 0.07, CHCl3).
Laboratories, Kumamoto, Japan). After pre-incubation at
37 8C for 7 min, phorbol 12-myristate 13-acetate (4 mM,
Sigma, St. Louis, MO) was added to the cell suspension and
the suspension was incubated for 30 min at 37 8C. EDTA
(10 mM) was added to stop the reaction, and then the
percentage of positive cells (blue-stained cells) was
determined using a hemocytometer. EC50 value was
estimated from the obtained dose-response curve. Relative
differentiation activity was calculated according to the
following formula: Relative differentiation activityZEC50
of 1a,25-dihydroxyvitamin D3/EC50 of the analogue!100.
4.1.26. 1a,25-Dihydroxy-2a-(2-phenylethyl)vitamin D3
(6). A white amorphous solid (15% for 2 steps). H NMR
1
(400 MHz, CDCl3) d 0.53 (3H, s), 0.93 (3H, d, JZ6.4 Hz),
1.02–1.10 (2H, m), 1.22 (6H, s), 1.18–1.61 (13H, m), 1.62–
1.78 (4H, m), 1.82–1.95 (3H, m), 1.95–2.04 (2H, m), 2.24
(1H, dd, JZ8.5, 13.0 Hz), 2.62–2.72 (2H, m), 2.78–2.88
(2H, m), 3.92 (1H, ddd, JZ4.3, 8.5, 8.5 Hz), 4.43 (1H, d,
JZ3.2 Hz), 4.99 (1H, d, JZ1.6 Hz), 5.27 (1H, d, JZ
1.6 Hz), 6.00 (1H, d, JZ11.2 Hz), 6.40 (1H, d, JZ11.2 Hz),
7.15–7.31 (5H, m). 13C NMR (100 MHz, CDCl3) d 12.0,
18.8, 20.8, 22.2, 23.5, 27.6, 28.4, 29.0, 29.1, 29.3, 33.4,
36.0, 36.3, 40.4, 44.2, 44.3, 45.8, 48.9, 56.2, 56.4, 70.1,
70.9, 73.2, 113.2, 116.5, 124.5, 125.4, 128.0, 128.1, 132.3,
142.1, 143.0, 146.2. IR (film) 3357, 2934, 2870, 1456, 1377,
1063, 1040, 910, 733 cmK1. HRMS (EI) calcd for
C35H52O3; 520.3916. Found 520.3920. [a]1D8 C53.0 (c 0.5,
CHCl3).
4.4. Transfections and transcriptional assays
CV-1 monkey kidney cells were plated in 35-mm dishes at
a density of 105 cells/dish. Cells were transfected by the
DEAE-dextran method with 2 mg/dish of WT or mutant
VDR expression vectors and a reporter construct containing
the human osteocalcin VDRE (ocVDRE) linked to the
thymidine kinase promoter and the growth hormone
reporter gene (ocVDRE/TK-GH).15 The medium was
collected 2days after transfection, and growth hormone
was measured using a radioimmunoassay as described by
the manufacturer (Nichols Institute, San Juan Capistrano,
CA).
4.2. Vitamin D receptor binding assay
4.5. Protease sensitivity assays
Bovine thymus VDR was obtained from Yamasa Bio-
chemical (Chiba, Japan) and dissolved in 0.05 M phosphate
buffer (pH 7.4) containing 0.3 M KCl and 5 mM dithio-
threitol just before use. The receptor solution (500 mL) was
pre-incubated with 50 mL of ethanol solution of 1a,25-
dihydroxyvitamin D3 or an analogue at various concen-
trations for 60 min at 25 8C. Then, the receptor mixture was
left to stand overnight with 0.1 nM [3H]-1a,25-dihydroxy-
vitamin D3 at 4 8C. The bound and free [3H]-1a,25-
dihydroxyvitamin D3 were separated by treatment with
dextran-coated charcoal for 30 min at 4 8C and centrifuged
at 3000 rpm for 10 min. The supernatant (500 mL) was
mixed with ACS-II (9.5 mL) (Amersham, UK) and the
radioactivity was counted. The relative potency of the
analogues was calculated from their concentration needed to
displace 50% of [3H]-1a,25-dihydroxyvitamin D3 from the
receptor compared with the activity of 1a,25-dihydroxy-
vitamin D3 (assigned a 100% value).
WT and mutant VDRs were synthesized and labeled in vitro
with [35S]methionine (1000 Ci/mmol) using the TNT
coupled transcription/translation system (Promega). The
translated receptor preparations were incubated with 1a,25-
dihydroxyvitamin D3 or analogues for 10 min at ambient
temperature. Next, trypsin was added to a concentration of
20 mg/mL, and the mixtures were incubated for 10 min. The
digestion products were then separated by SDS polyacryl-
amide gel electrophoresis and detected by autoradiography.
Acknowledgements
We are grateful to Miss Junko Shimode, Miss Maroka
Kitsukawa and Miss Akiko Tonoki (Teikyo University) for
the spectroscopic measurements. This work has been
supported by a Grant-in-Aid for Young Scientists (B)
from the Ministry of Education, Culture, Sports, Science
and Technology, Japan, (No. 17799095 to S.H.) and in part
by Grants-in-Aid for Scientific Research (C) from Japan
Society for the Promotion of Science (No. 15590021 and
17590012 to A.K.) and an NIH grant DK-50583 (to S.P.).
A.K. also thanks Uehara Memorial Foundation for financial
support.
4.3. HL-60 cell differentiation assay
Activities of these analogues on differentiation of HL-60
cells were estimated by nitroblue tetrazolium (NBT)
reduction assay22 with some modifications. Human pro-
myelocytic leukemia HL-60 cells were grown at 37 8C in
RPMI 1640 medium (Asahi Technoglass Co., Chiba, Japan)
supplemented with 10% heat-inactivated fetal bovine serum
(Sigma, St. Louis, MO) in an atmosphere of 95% air and
5% CO2. Cells were collected, suspended in 1.5 mL of
the culture medium containing various concentrations
(0, 10K10–10K6 M) of the analogues at a density of ca.
5!105 cells/mL, and then cultivated for 5 days. After the
treatment, cells were harvested and suspended in Tyrode’s
solution (140 mM NaCl, 2.7 mM KCl, 1.1 mM MgCl2,
1 mM CaCl2, 0.45 mM NaH2PO4, 5.6 mM D-glucose, pH
7.4) containing 25 mM HEPES and 0.05% NBT (Dojindo
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