Synthesis and biological evaluation of new 6-s-cis locked 1,2,25-trihydroxyprevitamin D3 analogues

Article abstract of DOI:10.1016/j.bmc.2007.03.058

An efficient synthesis of several diastereomers of 2-hydroxy substituted 1α,25-dihydroxyprevitamin D₃ derivatives was accomplished utilizing a practical route to the A-ring synthon. The biological activity of the analogues was evaluated in vitr

Full text of DOI:10.1016/j.bmc.2007.03.058

Bioorganic & Medicinal Chemistry 15 (2007) 4193–4202
Synthesis and biological evaluation of new 6-s-cis locked
1,2,25-trihydroxyprevitamin D3 analogues
a
a
b
´
´
Laura Sanchez-Abella, Susana Fernandez, Annemieke Verstuyf,
Lieve Verlinden,b Miguel Ferreroa,* and Vicente Gotora,*
aDepartamento de Quı´mica Orga´nica e Inorga´nica and Instituto Universitario de Biotecnologı´a de Asturias,
Universidad de Oviedo, 33006 Oviedo (Asturias), Spain
bLaboratorium voor Experimentele Geneeskunde en Endocrinologie, Katholieke Universiteit Leuven, Gasthuisberg,
B-3000 Leuven, Belgium
Received 15 February 2007; revised 15 March 2007; accepted 20 March 2007
Available online 24 March 2007
´
Dedicated to Professor Miguel Yus on the occasion of his 60th birthday.
Abstract—An efficient synthesis of several diastereomers of 2-hydroxy substituted 1a,25-dihydroxyprevitamin D3 derivatives was
accomplished utilizing a practical route to the A-ring synthon. The biological activity of the analogues was evaluated in vitro.
All the synthesized derivatives demonstrated low affinity for the vitamin D receptor and vitamin D-binding protein compared with
1a,25-dihydroxyvitamin D3, the natural hormone. 1a,2b,25-trihydroxy-19-nor-pre-D3 was the most potent of the analogues in inhib-
iting proliferation of MCF-7 cells but requires higher EC50 concentrations than 1a,25-dihydroxyvitamin D3.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
have been reported in a variety of systems including
stimulation of intestinal Ca2+ transport (transcaltachia),5
opening of chloride channels,6 and activation of PKC7
and MAP8 kinases.
1a,25-Dihydroxyvitamin D3 [1, 1a,25-(OH)2-D3], in
addition to its important role in calcium and phospho-
rus homeostasis and bone mineralization, exerts also cell
proliferation and differentiation, and serves as a modu-
lator of the immune system.1 The plethora of biological
actions that are attributable to 1a,25-(OH)2-D3 sug-
gested wide clinical applications of this metabolite or
modified compounds2 for the treatment of certain can-
cers, osteoporosis, inflammation, dermatological, and
autoimmune diseases.3
In comparison to other steroid hormones vitamin D3
and its metabolites are unusually conformationally flex-
ible.9 This includes the side chain, a seco B-ring, and a
conformationally active A-ring. These seco steroids
can undergo a rotation around the 6,7 carbon–carbon
single bond which generates a wide array of molecular
shapes extending from the 6-s-cis (steroid-like confor-
mation) to the more stable extended 6-s-trans conforma-
tion. Both conformers are present in slow chemical
equilibrium (5–10%) with 1a,25-dihydroxyprevitamin
D3 [2, 1a,25-(OH)2-pre-D3]. Pure pre-1a,25 standing in
solution rearranges to 1a,25 with a half-life of 13.5 h
at 37 ꢁC, whereas when the C-19 methyl group is deuter-
ated,10 rearrangement proceeds with a half-life of 81 h.
Due to the spontaneous isomerization of previtamin 2
via a [1,7]-sigmatropic hydrogen shift back to the ther-
mally more stable vitamin 1, biological evaluation of 2
is very difficult.
At the molecular level, 1a,25-(OH)2-D3 stimulates bio-
logical responses both via interaction with nuclear
receptors (VDRn) to regulate gene transcription (slow
genomic pathway) and via other membrane receptors
(VDRm), which generates rapid actions believed to be
independent of direct interaction with the genome.4
Non-genomic biological responses of 1a,25-(OH)2-D3
Keywords: Vitamin D3 analogues; Previtamin D3; Novel A-ring anal-
ogues; Biological evaluation; Calcitriol.
*
It has been observed that analogues that are either struc-
turally blocked in the previtamin form11 or that only
Corresponding authors. Tel./fax: +34 985 103 448; e-mail addresses:
vgs@fq.uniovi.es; MFerrero@fq.uniovi.es
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.03.058

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L. Sanchez-Abella et al. / Bioorg. Med. Chem. 15 (2007) 4193–4202
4194
R
R
25
HO
HO
OH
HO
HO
HO
HO
H
H
H
6
HO
19
OH
3
4
1α,2α,25-(OH)3-19-nor-pre-D3
1β,2α,25-(OH)3-19-nor-pre-D3
H
HO
3
1
OH
HO
1'
1
R
R
6-s
-
cis-1α,25-(OH)2-D3
6-s
-
trans-1α,25-(OH)2-D3
HO
HO
HO
HO
"1α,25"
H
H
HO
HO
5
6
HO
1α,2β,25-(OH)3-19-nor-pre-D3
1α,2α,25-(OH)3-3-epi-19-nor-pre-D3
OH
H
R=
HO
2
OH
1α,25-(OH)2-pre-D3
"Pre-1α,25"
Figure 1. Previtamin D3 analogues.
Scheme 1. Vitamin–previtamin equilibrium.
slowly isomerize to the vitamin form12 are able to fully
mimic the membrane actions of 1a,25-(OH)2-D3 but
have little action at the nuclear level with minimal effects
on cell proliferation and differentiation. These results
suggested that 1a,25-(OH)2-D3 produces biological
responses through two distinct receptors, which recognize
totally different ligand shapes. Thus, the genomic actions
occur as a consequence of the interaction of a 6-s-trans
shape of 1a,25-(OH)2-D3 with its VDRn; meanwhile,
the ability of 1a,25-(OH)2-D3 to generate rapid biologi-
cal effects requires a 6-s-cis shape, which is recognized
by a VDRm.4 In addition, a 9,19-methano-bridged ana-
coupling17 involving the A-ring enyne precursors
11a–d (Scheme 2) with an enol triflate of the CD-ring/
side chain fragment.
A-ring synthons 11a–d were obtained starting from
methyl shikimate and its epi-isomers. Methyl 3-epi,18
4-epi-,19 and 5-epi-shikimate20 derivatives 7b, c, and d
were synthesized as previously reported through simple
and efficient approaches. Methyl shikimate (7a) was pre-
pared from shikimic acid by treatment with HCl in
logue13 of 1a,25-(OH)2-D3 and A-ring diastereomers of
14
1a,25-(OH)2-19-nor-pre-D3
have been reported.
Recently, the first previtamin structure with genomic activi-
ties equivalent to 1a,25-(OH)2-D3 has been described.15
This analogue interacted as efficiently as the natural hor-
mone with the VDRn and uses the same contact points
within the receptor as did 1a,25-(OH)2-D3. An important
point in this research was the recent disclosure of the
detailed structure of the ligand binding of the VDRn.16
CO2Me
CO2Me
CH2OH
a
b
5
3
4
OH
OΣ
OΣ
HO
ΣO
ΣO
OH
OΣ
OΣ
7a-d
8a, 95%
8b, 95%
8c, 85%
8d, 75%
9a, 89%
9b, 72%
9c, 94%
9d, 85%
In order to further probe the less well investigated bio-
logical actions of the previtamin form, it is the purpose
of this article to describe the synthesis of a new class of
locked 1a,25-(OH)2-19-nor-previtamin D3 analogues
with a modified A-ring (3–6, Fig. 1), hence representing
a system in which the biological profile of a genuine pre-
vitamin form could be assessed. These derivatives are
unable to undergo rearrangement to the respective vita-
min D form by virtue of the absence of the C-19 methyl
group. In addition, results of preliminary biological
activities are reported to better understand their mode
of action Scheme 1.
a: (3
b: (3
c: (3
d: (3
R
S
R
,4
,4
,4
S
,5
R)
,5
R)
S,5
c
R
R
)
)
R,4S,5S
CHO
d
OΣ
OΣ
ΣO
ΣO
OΣ
OΣ
11a, 78%
11b, 66%
11c, 90%
11d, 60%
10a, 98%
10b, 96%
10c, 92%
10d, 95%
2. Results and discussion
Scheme 2. Reagents and conditions: (a) TBDMS-OTf, 2,6-lutidine,
CH2Cl2, 0 ꢁC ! rt, overnight; (b) DIBALH, toluene, ꢀ78 ꢁC, 4 h; (c)
PCC, CH2Cl2, rt, 5 h; (d) TMSCHN2, nBuLi, THF, ꢀ78 ꢁC ! 0 ꢁC, 4 h.
For synthesis of the target 2-substituted 19-nor-previta-
min D3 derivatives 3–6, we used standard Sonogashira

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L. Sanchez-Abella et al. / Bioorg. Med. Chem. 15 (2007) 4193–4202
4195
MeOH. Persilylation of 7a–d with tert-butyldimethylsi-
lyl trifuoromethanesulfonate (TBDMS-OTf) and 2,6-
lutidine afforded the desired protected silyl ethers 8a–d
in high yields (Scheme 2). Transformation of the ester
into the aldehyde was best carried out via a two-step se-
quence. Thus, reduction of 8a–d with DIBALH gave the
alcohols 9a–d, which upon oxidation with PCC yielded
the aldehydes 10a–d. Formation of the enynes 11a–d
was accomplished in good yields by reaction with
trimethylsilyldiazomethane.
readily available vitamin D3 following the general proce-
dure described previously.13,22 A-ring synthons 11a–d
were coupled to protected vinyl triflate 13 in the
presence of bis[triphenylphosphine]palladium (II)
acetate-copper (I) iodide catalyst and diethylamine in
DMF. The resulting silyloxy dienynes were deprotected
with TBAF to afford the tetrahydroxydienynes 14a–d in
60–94% yields. Careful catalytic hydrogenation of 14a–d
in the presence of Lindlar catalyst and quinoline
generated previtamins 3–6.
In an attempt to increase the yield of the 5-epi enyne
derivative 11d, ethynylation of 10d was carried out by
conversion to the vinyl dibromide 12 and subsequent
reaction with nBuLi.21 However, this alternative method
provides 11d in a similar overall yield (Scheme 3).
3. Biological evaluation
We examined the potencies of the previtamin D3 deriv-
atives 3–6 in terms of their ability to bind to the pig vita-
min D receptor (VDR) and human vitamin D-binding
protein (hDBP). The results of biological evaluation
are summarized in Table 1, in comparison with those
of 1a,25-(OH)2-D3. All the synthesized derivatives have
markedly reduced affinity for the VDR. Comparison of
the four isomers showed that compounds 3 and 5 pos-
sessing the configuration of the natural hormone at
C-1 and C-3 positions are more potent than correspond-
ing isomers 4 and 6. When comparing DBP binding,
previtamin analogues exhibited more than 10 times
lower affinity than 1a,25-(OH)2-D3.
Initially, the protection of 7a–d was performed with
trimethylsilyl chloride (TMSCl). However, the same se-
quence of reactions for A-ring preparation gave lower
yields due to the instability of the TMS group.
Four 6-s-cis locked previtamin D3 derivatives were suc-
cessfully synthesized as outlined in Scheme 4. CD-ring/
side chain fragment 13 was prepared starting from
Br
In addition, inhibition of MCF-7 breast cancer cell prolif-
eration was measured (Fig. 2). In this study, the rank or-
der of potency was parallel to that of VDR binding. The
2a- and 2b-hydroxy analogues 3 and 5 with 1a,3b config-
uration exhibited the most potent activity in this series.
Thus, 1a,2b,25-(OH)3-19-nor-pre-D3 (5) can inhibit the
cell proliferation by 70% at a concentration of 10ꢀ6 M
comparable with 1a,25-(OH)2-D3 but this compound
was 17 times less potent than 1a,25-(OH)2-D3 at the
EC50 concentration. The 1-epimer as well as 3-epimer
showed virtually no inhibition even at the concentration
of 10ꢀ6 M.
CHO
Br
n
Zn, Ph3P, CBr4
BuLi
11d
THF, -78 ˚C
Py, CH2Cl2
1.5 h1
ΣO
ΣO
OΣ
OΣ
h
OΣ
OΣ
93%
65%
10d
12
Scheme 3. Alternative synthesis of compound 11d.
The introduction of a 2b-OH group in the 1a,25-(OH)2-
19-nor-pre-D3 compound did not alter its biological
activity. The binding of compound 5 and 1a,25-(OH)2-
19-nor-pre-D3 to VDR (2% and 1%, respectively) and
OTMS
OH
H
13
TfO
H
a,b
+
Table 1. Biological activity of 2-hydroxy derivatives of 1a,25-(OH)2-
pre-D3
HO
OH
ΣO
OΣ
OH
OΣ
Compound
VDR
(%)
hDBP
(%)
MCF-7
(%)
(1α,2α,3β)-14a, 94%
(1β,2α,3β)-14b, 69%
(1α,2β,3β)-14c, 88%
(1α,2α,3α)-14d, 60%
11a-d
1a,25-(OH)2-D3
1a,25-(OH)2-19-nor-pre-D3
3
4
5
6
100
1a
100
5a
4
100
11a
2
0.2
<0.1
2
2
0
8
6
c
<0.1
7
0
Summary of the in vitro effects of 2-hydroxy analogues of 1a,25-
(OH)2-pre-D3 on receptor binding (VDR), human vitamin D-binding
protein (hDBP), and MCF-7 proliferation. The in vitro effect is
expressed as percentage activity at EC50 in comparison with 1a,25-
(OH)2-D3 (=100% activity).
3, 70%; 4, 63%
5, 66%; 6, 69%
Scheme 4. Reagents and conditions: (a) Pd(Ph3P)2(OAc)2, CuI,
Et2NH, DMF, rt, 5 h; (b) TBAF, THF, rt, overnight; (c) H2, Lindlar
cat., quinoline, MeOH, rt, 30 min.
a Data from Refs. 11c and 15.

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L. Sanchez-Abella et al. / Bioorg. Med. Chem. 15 (2007) 4193–4202
4196
5.2. Synthesis of 3–6
A flask containing Lindlar catalyst (45 mg) was exposed
to a positive pressure of hydrogen gas (balloon). A solu-
tion of 14 (15 mg, 0.036 mmol) in MeOH (1.8 mL) and
quinoline (130 lL of 0.17 M in hexane, 0.022 mmol)
were added. The reaction mixture was stirred vigorously
during 30 min. The mixture was filtered on Celite, con-
centrated, and the crude subjected to flash chromatogra-
˚
phy using silica 60 A (32–63 lm) pH 7 (gradient elution
with 10–30% acetone/CH2Cl2). Further purification by
HPLC (Dynamax column, 250 · 10 mm, 4 mL/min, hex-
ane/MeOH/EtOH, 92:4:4 for 3, 5, and 6; 5 mL/min, hex-
ane/MeOH/EtOH, 94:3:3 for 4) afforded 3–6.
5.2.1. 1a,2a,25-Trihydroxy-19-nor-previtamin D3 (3).
1
Yield 70%. H NMR (300.13 MHz, MeOH-d4): d 0.79
(s, 3H, Me18), 1.01 (s, 3H, Me21, JHH = 6.4 Hz), 1.11
Figure 2. In vitro antiproliferative effects of 2-hydroxy analogues of
1a,25-(OH)2-pre-D3 on breast cancer MCF-7 cells. 1a,25-(OH)2-D3
(d); 3 (s); 4 (j); 5 (e); 6 (h).
3
(m, 1H), 1.19 (s, 6H, Me26 + Me27), 1.2–1.5 (m, 14H),
1.68 (m, 1H, H14), 1.9–2.3 (m, 6H), 2.68 (dd, 1H, H4e,
2
3
j JHHj = 17.6, JHH = 5.4 Hz), 3.51 (m, 1H, H2), 3.86
3
(ddd, 1H, H3, JHH = 8.8, 8.8, 5.4 Hz), 4.26 (dd, 1H,
3
H1, JHH = 4.4, 4.4 Hz), 5.43 (sa, 1H, H9) 5.70 (br s,
hDBP (8% and 5%, respectively) was comparable.11c,15
Moreover, both compounds did inhibit the MCF-7 cell
proliferation more or less with the same potency (6% com-
pound 5 vs 11% 1a,25-(OH)2-19-nor-pre-D3).15 The intro-
duction of a 2a-OH group in 1a,25-(OH)2-19-nor-pre-D3
decreased the VDR binding affinity five (compound 3) to
ten (compounds 4 and 6) times as well as its capacity to in-
hibit the proliferation of MCF-7 cells.
3
1H, H10), 5.8 (d, 1H, H7, JHH = 12.2 Hz), and 5.88 (d,
3
1H, H6, JHH = 12.2 Hz) ppm; 13C NMR (50.5 MHz,
MeOH-d4): d 10.0 (C18), 17.9 (C21), 20.5 (CH2), 23.2
(CH2), 24.2 (CH2), 27.8 (CH2), 27.9 (CH3), 28.0
(CH3), 35.8 (CH2), 36.0 (CH2), 36.1 (CH), 36.4 (CH2),
41.9 (C), 43.9 (CH2), 50.8 (CH), 54.4 (CH), 66.9
(C3 + C1), 70.1 (C), 73.1 (C2), 125.2 (C9), 128.2 (C10),
129.1 (C6), 130.6 (C7), 136.5 (C), and 136.9 (C); (ESI+,
m/z) 441 [(M+Na)+, 100%], 442 [(M+H+Na)+, 25];
(EI+, m/z) 418 [M+, 30%], 400 [(MꢀH2O)+, 25], 382
(42), 193 (70), 157 (90), and 69 (100); HRMS (m/z) cal-
culated for C26H42O4 (M+): 418.3078. Found: 418.3072.
Such as for 1a,25-(OH)2-19-nor-pre-D3,15 the low bio-
logical profile of the studied 19-nor-previtamin D3 ana-
logues is probably due to weak interactions with
VDR, vitamin D responsive elements (VDRE), and
co-activators.
5.2.2. 1b,2a,25-Trihydroxy-19-nor-previtamin D3 (4).
Yield 63%.1H NMR (400.13 MHz, MeOH-d4): d 0.79
(s, 3H, Me18), 1.00 (d, 3H, Me21, JHH = 6.4 Hz), 1.09
3
4. Conclusions
(m, 1H), 1.18 (s, 6H, Me26 + Me27), 1.2–1.6 (m, 14H),
1.65 (dd, 1H, JHH = 19.2, 10 Hz), 1.9–2.3 (m, 6H),
3
We have described the synthesis and biological evalua-
tion of novel 2-hydroxy substituted 6-s-cis locked previ-
tamin D3 analogues. We investigated the potency in
inhibiting MCF-7 cell proliferation and found that
derivatives 3 and 5 possessing the natural C-1 and C-3
configuration in the A-ring showed inhibition effects,
while analogues 4 and 6 are inactive. These previtamin
D3 derivatives have shown poor binding to VDR and
DBP compared with 1a,25-dihydroxyvitamin D3. Fur-
ther studies are needed to elucidate fully the activity pro-
files and modes of action of these analogues.
2
3
2.59 (dd, 1H, H4e, j JHHj = 17.2, JHH = 5.6 Hz), 3.36
3
(dd, 1H, H2, JHH = 10, 7.6 Hz), 3.56 (ddd, 1H, H3,
3JHH = 9.6, 9.6, 5.6 Hz), 4.07 (br s, 1H, H1), 5.42 (s,
1H, H9) 5.52 (s, 1H, H10), 5.79 (d, 1H, H7, JHH 12H,
3
3
and 5.87 (d, 1H, H6, JHH = 12 Hz); 13C NMR
(100.6 MHz, MeOH-d4): d 10.4 (C18), 17.9 (C21), 20.5
(CH2), 23.2 (CH2), 24.2 (CH2), 27.7 (CH3), 27.9
(CH3), 28.0 (CH2), 36.0 (CH2), 36.1 (CH), 36.4 (CH2),
41.9 (C), 43.9 (CH2), 50.7 (CH), 54.4 (CH), 69.7 (C3),
70.1 (C), 72.7 (C1), 77.8 (C2), 125.3 (C9), 128.5 (C6),
129.1 (C10), 130.4 (C7), 134.6 (C5), and 136.6 (C8);
(ESI+, m/z) 441 [(M+Na)+, 100%]; 436 [(M+H2O)+,
85%]; (EI+, m/z) 418 [M+, 15%], 400 [(MꢀH2O)+, 10],
382 [(Mꢀ2H2O)+, 20], 289 (30), 245 (55), 158 (95), 91
(98), and 69 (100). HRMS (m/z) calculated for
C26H42O4 (M+): 418.3078. Found: 418.3074.
5. Experimental
5.1. General
Synthesis of 7b,18 c,19 d,20 and vinyl triflate 1313,22 was
previously reported. Unless otherwise specified column
˚
chromatography was performed over silica 60 A (230–
400 mesh). HPLC was performed using UV detector
and a Varian Dynamax column (microsorb 100–5 Si,
250 · 10 mm).
5.2.3. 1a,2b,25-Trihydroxy-19-nor-previtamin D3 (5).
1
Yield 66%. H NMR (400.13 MHz, MeOH-d4): d 0.78
(s, 3H, Me18), 1.00 (d, 3H, Me21, JHH = 6.8 Hz), 1.09
3
(m, 1H), 1.18 (s, 6H, Me26 + Me27), 1.2–1.5 (m, 14H),
1.65 (m, 1H), 1.9–2.1 (m, 2H), 2.2–2.4 (m, 3H), 2.42

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L. Sanchez-Abella et al. / Bioorg. Med. Chem. 15 (2007) 4193–4202
4197
2
(dd, 1H, H4a, j JHHj = 16.8, 3JHH = 5.6 Hz), 2.51 (d, 1H,
ꢀ4.7 (SiMe), ꢀ4.4 (SiMe), ꢀ4.3 (SiMe), 17.9 (SiC),
18.1 (SiC), 18.5 (SiC), 25.7 (SiCMe3), 25.8 (SiCMe3),
26.2 (SiCMe3), 29.5 (C6), 51.7 (C8), 68.0 (C3), 69.8
(C5), 72.3 (C4), 126.9 (C1), 140.4 (C2), and 167.5 (C7);
(ESI+, m/z) 553 [(M+Na)+, 100%].
2
3
H4e, j JHHj = 16.8 Hz), 3.63 (dd, 1H, H2, JHH = 5.6,
3
2.4 Hz), 3.98 (ddd, 1H, H1, JHH = 6.4, 4.0, 2.4 Hz),
4.20 (m, 1H, H3), 5.45 (sa, 1H, H9) 5.61 (sa, 1H, H10),
3
5.77 (d, 1H, H7, JHH = 11.6 Hz), and 5.87 (d, 1H, H6,
3JHH = 12.4 Hz); 13C NMR (100.6 MHz, MeOH-d4): d
10.4 (C18), 17.9 (C21), 20.5 (CH2), 23.0 (CH2), 24.2
(CH2), 27.7 (CH3), 27.9 (CH3), 28.0 (CH2), 33.4
(CH2), 36.0 (CH2), 36.1 (CH), 36.4 (CH2), 41.9 (C),
43.9 (CH2), 50.9 (CH), 54.4 (CH), 67.8 (C1), 69.8 (C3),
70.1 (C), 73.9 (C2), 125.1 (C9), 127.5 (C10), 129.5 (C6),
130.2 (C7), 135.6 (C5), and 136.5 (C8); (APCIꢀ, m/z)
383 [(Mꢀ2H2O)+, 100%]; (EI+, m/z) 418 [M+, 30%],
400 [(MꢀH2O)+, 20], 382 [(Mꢀ2H2O)+, 37], 157 (55),
95 (61), 81 (76), and 69 (100); HRMS (m/z) calculated
for C26H42O4 (M+): 418.3078. Found: 418.3075.
5.3.2. Methyl (3S,4S,5R)-3,4,5-tri[(tert-butyldimethylsi-
lyl)oxy]cyclohex-1-enecarboxylate (8b). Yield 95%. IR
(NaCl):
t
2955, 2930, 2890, 2858, 1723, and
1
1655 cmꢀ1; H NMR (400.13 MHz, CDCl3): d 0.03 (s,
3H, SiMe), 0.06 (s, 3H, SiMe), 0.09 (s, 3H, SiMe),
0.10 (s, 3H, SiMe), 0.12 (s, 3H, SiMe), 0.13 (s, 3H,
SiMe), 0.87 (s, 9H, SiCMe3), 0.88 (s, 9H, SiCMe3),
0.92 (s, 9H, SiCMe3), 2.43 (m, 2H, 2H6), 3.74 (ddd,
3
4
1H, H4, JHH = 4, 1.8, j JHHj = 1.8 Hz), 3.77 (s, 3H,
3
H8), 3.91 (ddd, 1H, H5, JHH = 4, 4, 4 Hz), 4.06 (ddd,
3
4
1H, H3, JHH = 2, 2, j JHHj = 2 Hz), and 6.78 (ddd,
3
4
5.2.4. 1a,2a,25-Trihydroxy-3-epi-19-nor-previtamin D3
1H, H2, JHH = 1.2, j JHHj = 1.2, 1.2 Hz); 13C NMR
(100.6 MHz, CDCl3): d ꢀ4.4 (SiMe), ꢀ4.3 (SiMe),
ꢀ4.3 (SiMe), ꢀ2.9 (SiMe), 17.9 (SiC), 18.2 (SiC), 18.2
(SiC), 25.7 (SiCMe3), 25.8 (SiCMe3), 25.9 (SiCMe3),
28.8 (C6), 51.8 (C8), 68.2 (C5), 71.7 (C3), 74.8 (C4),
126.7 (C1), 137.7 (C2), and 167.7 (C7); (ESI+, m/z) 553
[(M+Na)+, 100%], 531 [(M+H2O)+, 70%].
1
(6). Yield 69%. H NMR (400.13 MHz, MeOH-d4): d
0.78 (s, 3H, Me18), 1.01 (d, 3H, Me21, JHH = 6.4 Hz),
3
1.09 (m, 1H), 1.19 (s, 6H, Me26 + Me27), 1.2–1.5 (m,
14H), 1.70 (ddd, 1H, JHH = 8.4, 8.4, 8.4 Hz), 1.9–2.1
(m, 2H), 2.2–2.3 (m, 3H), 2.33 (dd, 1H, H4e,
3
2
3
j JHHj = 16.8, JHH = 5.2 Hz), 2.51 (dd, 1H, H4a,
2
3
j JHHj = 16.8, JHH = 9.2 Hz), 3.78 (m, 1H, H3), 3.90
(s, 1H, H2), 4.23 (s, 1H, H1), 5.44 (s, 1H, H9) 5.54 (s,
1H, H10), 5.78 (d, 1H, H7, JHH = 12.4 Hz), and 5.86
5.3.3. Methyl (3R,4R,5R)-3,4,5-tri[(tert-butyldimethylsi-
lyl)oxy]cyclohex-1-enecarboxylate (8c). Yield 85%. IR
3
3
1
(d, 1H, H6, JHH = 12.4 Hz); 13C NMR (100.6 MHz,
(NaCl): t 2954, 2929, 2892, 2858, and 1725 cmꢀ1. H
MeOH-d4): d 10.4 (C18), 17.9 (C21), 21.9 (CH2), 24.3
(CH2), 25.6 (CH2), 29.1 (CH3), 29.2 (CH3), 29.4
(CH2), 33.4 (CH2), 37.4 (CH2), 37.6 (CH), 37.8 (CH2),
43.3 (C), 45.3 (CH2), 52.4 (CH), 55.8 (CH), 69.7 (C1),
70.4 (C3), 71.5 (C2), 72.2 (C), 126.6 (C9), 129.4 (C10),
130.8 (C6), 131.6 (C7), 136.7 (C5), and 137.7 (C8);
(ESI+, m/z) 441 [(M+Na)+, 100%]; (EI+, m/z) 418 [M+,
15%], 400 [(MꢀH2O)+, 25], 382 [(Mꢀ2H2O)+, 80], 69
(90), and 55 (100); HRMS (m/z) calculated for
C26H42O4 (M+): 418.3078. Found: 418.3076.
NMR (400.13 MHz, CDCl3): d 0.07 (s, 3H, SiMe),
0.08 (s, 3H, SiMe), 0.09 (s, 3H, SiMe), 0.10 (s, 3H,
SiMe), 0.12 (s, 6H, SiMe), 0.86 (s, 9H, SiCMe3), 0.90
(s, 9H, SiCMe3), 0.91 (s, 9H, SiCMe3), 2.3–2.5 (m, 2H,
H6), 3.71 (br s, 1H, H4), 3.77 (s, 3H, H8), 3.99 (t, 1H,
3
H5, JHH = 6.8, 6.8 Hz), 4.10 (br s, 1H, H3), and 6.66
(s, 1H, H2); 13C NMR (100.6 MHz, CDCl3): d ꢀ4.9
(SiMe), ꢀ4.8 (SiMe), ꢀ4.7 (SiMe), ꢀ4.6 (SiMe), ꢀ4.5
(SiMe), ꢀ4.4 (SiMe), 18.0(SiC), 18.1 (SiC), 18.3 (SiC),
25.8 (SiCMe3), 26.0 (SiCMe3), 28.9 (br s C6),23 51.8
(C8), 67.4 (br s C5),23 71.0 (C3), 74.9 (C4), 130.5 (C1),23
135.8 (C2),23 and 167.4 (C7); (ESI+, m/z) 553
[(M+Na)+, 100%], 569 [(M+K)+, 5%].
5.3. Synthesis of 8a–d
2,6-Lutidine (2.6 mL, 22.3 mmol) was added to a sus-
pension of 7 (600 mg, 3.19 mmol) in anhydrous CH2Cl2
(3.2 mL). The solution was cooled at 0 ꢁC and TBDMS-
OTf (3.7 mL, 15.96 mmol) was added dropwise. The
reaction mixture was stirred at room temperature over-
night and then poured into water/CH2Cl2. The aqueous
layer was extracted with CH2Cl2, and the combined or-
ganic layers were dried over Na2SO4 and concentrated.
The crude product was subjected to flash chromatogra-
phy (2% EtOAc/hexane).
5.3.4. Methyl (3R,4S,5S)-3,4,5-tri[(tert-butyldimethylsi-
lyl)oxy]cyclohex-1-enecarboxylate (8d). Yield 75%. Mp
91–93 ꢁC; IR (KBr): t 2953, 2928, 2892, 2860, 1707,
and 1653 cmꢀ1 1H NMR (300.13 MHz, CDCl3): d
;
0.06 (s, 6H, 2SiMe), 0.07 (s, 3H, SiMe), 0.09 (s, 3H,
SiMe), 0.11 (s, 3H, SiMe), 0.12 (s, 3H, SiMe), 0.86 (s,
9H, SiCMe3), 0.90 (s, 9H, SiCMe3), 0.93 (s, 9H,
SiCMe3), 2.3–2.5 (m, 2H, 2H6), 3.74 (s, 4H, H5+3H8),
3.90 (br s, 1H, H4), 4.35 (br s, 1H, H3), and 6.53 (s,
1H, H2); 13C NMR (75.5 MHZ, CDCl3): d ꢀ4.7 (SiMe),
ꢀ4.6 (SiMe), ꢀ4.5 (SiMe), ꢀ4.4 (SiMe), ꢀ4.3 (SiMe),
18.3 (SiC), 18.5 (SiC), 18.6 (SiC), 25.9 (SiCMe3), 26.0
(SiCMe3), 26.1 (SiCMe3), 30.5 (C6), 51.8 (C8), 70.4
(C5), 71.4 (C3), 74.6 (C4), 128.5 (C1), 140.3 (C2), and
167.1 (C7); (ESI+, m/z) 553 [(M+Na)+, 100%], 531
[(M+H2O)+, 70%].
5.3.1. Methyl (3R,4S,5R)-3,4,5-tri[(tert-butyldimethylsi-
lyl)oxy]cyclohex-1-enecarboxylate (8a). Yield 95%. Mp
42–44 ꢁC; IR (KBr): t 2948, 2925, 2891, 2854, 1718,
and 1651 cmꢀ1 1H NMR (400.13 MHz, CDCl3): d
;
0.08 (s, 12H, 4 SiMe), 0.13 (s, 3H, SiMe), 0.14 (s, 3H,
SiMe), 0.86 (s, 9H, SiCMe3), 0.88 (s, 9H, SiCMe3),
2
0
0.96 (s, 9H, SiCMe3), 2.18 (dd, 1H, H6e , j JHHj = 18.2,
3
2
0
JHH = 1 Hz), 2.60 (dddd, 1H, H6a , j JHHj = 18.2,
5.4. Synthesis of 9a–d
3JHH = 3.6, 3, 3 Hz), 3.76 (m, 4H, H8+H4), 4.00 (ddd,
1H, H5, JHH = 4.6, 2, 2 Hz), 4.63 (m, 1H, H3), and
3
DIBAL-H (8.26 mL of 1.0 M in toluene, 8.26 mmol)
was added dropwise to a solution of 8 (1.46 g,
6.69 (s, 1H, H2); 13C NMR (75.5 MHz, CDCl3): d

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L. Sanchez-Abella et al. / Bioorg. Med. Chem. 15 (2007) 4193–4202
4198
2.75 mmol) in anhydrous toluene (23 mL) at ꢀ78 ꢁC,
and the reaction mixture was stirred for 4 h at the same
temperature. NH4Cl (aq.) was added and the mixture
was warmed to room temperature, diluted with Et2O,
and filtered through a short column of silica gel, using
additional Et2O to elute the column. The filtrate was
concentrated to give a crude sufficiently pure for the
next step.
(NaCl):
t
3346, 2951, 2929, 2895, 2857, and
1
1726 cmꢀ1; H NMR (400.13 MHz, CDCl3): d 0.06 (s,
3H, SiMe), 0.07 (s, 3H, SiMe), 0.08 (s, 3H, SiMe),
0.10 (s, 3H, SiMe), 0.10 (s, 3H, SiMe), 0.11 (s, 3H,
SiMe), 0.88 (s, 9H, SiCMe3), 0.91 (s, 9H, SiCMe3),
0
2
0.93 (s, 9H, SiCMe3), 1.99 (dd, 1H, H6a , j JHHj = 16.4,
3
0
JHH = 5.6 Hz), 2.33 (m, 1H, H6e ), 3.78 (ddd, 1H, H5,
3JHH = 6, 6, 6 Hz), 3.90 (br s, 1H, H4), 4.01 (br s, 2H,
2H7), 4.27 (br s, 1H, H3), and 5.34 (br s, 1H, H2); 13C
NMR (100.6 MHz, CDCl3): d ꢀ4.6 (SiMe), ꢀ4.4
(SiMe), ꢀ4.2 (SiMe), 18.3 (SiC), 18.6 (SiC), 18.7 (SiC),
26.0 (SiCMe3), 26.1 (SiCMe3), 26.3 (SiCMe3), 31.4
(C6), 66.1 (C7), 70.6 (C5), 71.2 (C3), 75.4 (C4), 124.1
(C2), and 136.6 (C1); (ESI+, m/z) 525 [(M+Na)+,
100%]; 541 [(M+K)+, 10%].
5.4.1. (3R,4S,5R)-3,4,5-Tri[(tert-butyldimethylsilyl)oxy]-
1-hydroxymethylcyclohex-1-ene (9a). Yield 89%. Mp 74–
76 ꢁC; IR (KBr): t 3430, 2953, 2928, 2894, 2856, and
1
1643 cmꢀ1; H NMR (400.13 MHz, CDCl3): d 0.07 (s,
6H, 2SiMe), 0.08 (s, 3H, SiMe), 0.09 (s, 3H, SiMe),
0.11 (s, 6H, 2 SiMe), 0.87 (s, 9H, SiCMe3), 0.88 (s,
9H, SiCMe3), 0.94 (s, 9H, SiCMe3), 1.85 (d, 1H, H6a,
2
2
j JHHj = 17.8 Hz), 2.44 (d, 1H, H6e, j JHHj = 18.1 Hz),
5.5. Synthesis of 10a–d
3
3.73 (dd, 1H, H4, JHH = 3.8, 3.8 Hz), 4.00 (m, 3H,
H5+2H7), 4.50 (br s, 1H, H3), and 5.5 (s, 1H, H2); 13C
NMR (75.5 MHz, CDCl3): d ꢀ4.8 (SiMe), ꢀ4.7 (SiMe),
ꢀ4.5 (SiMe), ꢀ4.3 (SiMe), ꢀ4.2 (SiMe), 17.9 (SiC), 18.2
(SiC), 18.5 (SiC), 25.8 (SiCMe3), 25.9 (SiCMe3), 26.2
(SiCMe3), 31.1 (C6), 66.7 (C7), 67.8 (C3), 70.0 (C5),
73.0 (C4), 123.9 (C2), and 135.3 (C1); (ESI+, m/z) 525
[(M+Na)+, 100%].
PCC (1.16 g, 5.38 mmol) was added to a solution of 9
(900 mg, 1.79 mmol) in anhydrous CH2Cl2 (18 mL).
The reaction mixture was stirred at room temperature
for 5 h. Et2O was added and the gummy residue was fil-
tered through a short column of fluorosil and washed
with EtOAc. The filtrate was concentrated to afford
10, which was sufficiently pure for direct use in the next
step. These aldehydes are instable and should be kept in
the refrigerator.
5.4.2. (3S,4S,5R)-3,4,5-Tri[(tert-butyldimethylsilyl)oxy]-
1-hydroxymethylcyclohex-1-ene (9b). Yield 72%. Mp
80–81 ꢁC; IR (KBr): t 3417, 2952, 2928, 2892, 2857,
5.5.1. (3R,4S,5R)-3,4,5-Tri[(tert-butyldimethylsilyl)oxy]-
cyclohex-1-enecarbaldehyde (10a). Yield 98%. Mp 47–
48 ꢁC; IR (KBr): t 2953, 2931, 2889, 2852, and
and 1471 cmꢀ1 1H NMR (400.13 MHz, CDCl3): d
;
0.08 (s, 3H, SiMe), 0.10 (s, 3H, SiMe), 0.11 (s, 6H,
2SiMe), 0.11 (s, 6H, 2SiMe), 0.89 (s, 9H, SiCMe3),
0.90 (s, 9H, SiCMe3), 0.92 (s, 9H, SiCMe3), 2.00 (dd,
1
1690 cmꢀ1; H NMR (400.13 MHz, CDCl3): d 0.06 (s,
3H, SiMe), 0.07 (s, 3H, SiMe), 0.08 (s, 3H, SiMe),
0.09 (s, 3H, SiMe), 0.15 (s, 3H, SiMe), 0.16 (s, 3H,
SiMe), 0.84 (s, 9H, SiCMe3), 0.87 (s, 9H, SiCMe3),
0
0.98 (s, 9H, SiCMe3), 2.17 (d, 1H, H6a ,
2
3
0
1H, H6a , j JHHj = 16.8, JHH = 4.8 Hz), 2.33 (dd, 1H,
2
3
0
H6e , j JHHj = 16.4, JHH = 3.6 Hz), 3.70 (dd, 1H, H4,
3JHH = 5.2, 2.4 Hz), 3.86 (ddd, 1H, H5, JHH = 4.4,
3
2
2
0
4.4, 4.4 Hz), 4.03 (s, 4H, H3+2H7), and 5.58 (s, 1H,
H2); 13C NMR (100.6 MHz, CDCl3): d ꢀ4.8 (SiMe),
ꢀ4.2 (SiMe), ꢀ4.2 (SiMe), ꢀ4.1 (SiMe), ꢀ4.0 (SiMe),
ꢀ4.0 (SiMe), 18.3 (SiC), 17.9 (SiC), 26.0 (SiCMe3),
26.1 (SiCMe3), 26.2 (SiCMe3), 31.3 (C6), 66.8 (C7),
69.1 (C5), 72.1 (C3), 75.8 (C4), 122.9 (C2), and 135.0
(C1); (ESI+, m/z) 525 [(M+Na)+, 100%].
j JHHj = 18.2 Hz), 2.48 (dddd, 1H, H6e , j JHHj = 18.2,
3JHH = 3, j JHHj = 3, 3 Hz), 3.84 (m, 1H, H4), 4.04
4
3
(dd, 1H, H5, JHH = 3, 3 Hz), 4.78 (sa, 1H, H3), 6.5 (s,
1H, H2), and 9.47 (s, 1H, H7); 13C NMR (75.5 MHz,
CDCl3): d ꢀ4.9 (SiMe), ꢀ4.8 (SiMe), ꢀ4.4 (SiMe),
17.9 (SiC), 18.0 (SiC), 18.5 (SiC), 25.6 (SiCMe3), 25.7
(SiCMe3), 26.1 (SiCMe3), 26.7 (C6), 68.4 (C3), 69.5
(C5), 73.0 (C4), 138.0 (C1), 151.1 (C2), and 194.2 (C7);
(ESI+, m/z) 523 [(M+Na)+, 100%].
5.4.3. (3R,4R,5R)-3,4,5-Tri[(tert-butyldimethylsilyl)oxy]-
1-hydroxymethylcyclohex-1-ene (9c). Yield 94%. Mp 68–
70 ꢁC; IR (KBr): t 3425, 2954, 2929, 2891, 2857, and
5.5.2. (3S,4S,5R)-3,4,5-Tri[(tert-butyldimethylsilyl)oxy]-
cyclohex-1-enecarbaldehyde (10b). Yield 96%. Mp 75–
77 ꢁC; IR (KBr): t 2948, 2925, 2889, 2856, and
1
1471 cmꢀ1; H NMR (400.13 MHz, CDCl3): d 0.07 (s,
3H, 2 SiMe), 0.08 (s, 3H, SiMe), 0.09 (s, 3H, SiMe),
0.10 (s, 9H, 3SiMe), 0.87 (s, 9H, SiCMe3), 0.91 (s, 9H,
1683 cmꢀ1 1H NMR (400.13 MHz, CDCl3): d 0.02
;
0
SiCMe3), 0.91 (s, 9H, SiCMe3), 1.98 (dd, 1H, H6e ,
(s, 6H, 2SiMe), 0.08 (s, 3H, SiMe), 0.10 (s, 3H,
SiMe), 0.13 (s, 6H, SiMe), 0.82 (s, 9H, SiCMe3),
2
3
0
j JHHj = 16.4, JHH = 5.2 Hz), 2.27 (dd, 1H, H6a
,
2
3
j JHHj = 16.4, JHH = 9.6 Hz), 3.70 (s, 1H, H4), 4.0–4.1
(m, 4H, H3+H5+2H7), and 5.52 (br s, 1H, H2); 13C
NMR (100 MHZ, CDCl3): d ꢀ4.8 (SiMe), ꢀ4.7 (SiMe),
ꢀ4.6 (SiMe), ꢀ4.5 (SiMe), ꢀ4.4 (SiMe), ꢀ4.3 (SiMe),
18.2 (SiC), 18.3 (SiC), 25.8 (SiCMe3), 25.9 (SiCMe3),
26.1 (SiCMe3), 30.8 (C6),23 66.4 (C7), 67.9 (C5),23 71.5
(C3), 75.5 (C4), 120.8 (C2),23 and 139.8 (C1)23; (ESI+,
m/z) 525 [(M+Na)+, 95%]; 541 [(M+K)+, 30%].
0.85 (s, 9H, SiCMe3), 0.91 (s, 9H, SiCMe3), 2.27
0
2
0
(d, 1H, H6a , j JHHj = 17.2 Hz), 2.38 (d, 1H, H6e ,
2
j JHHj = 17.2), 3.81 (s, 1H, H4), 3.94 (s, 1H, H5),
4.14 (s, 1H, H3), 6.53 (s, 1H, H2), and 9.50 (s, 1H,
H7); 13C NMR (100.6 MHZ, CDCl3): d ꢀ4.9 (SiMe),
ꢀ4.6 (SiMe), ꢀ4.5 (SiMe), ꢀ4.4 (SiMe), 17.9 (SiC),
18.1 (SiC), 18.2 (SiC), 25.4 (C6), 25.8 (SiCMe3),
25.9 (SiCMe3), 25.9 (SiCMe3), 67.4 (C5), 71.7 (C3),
75.3 (C4), 136.9 (C1), 147.9 (C2), and 194.4 (C7);
(ESI+, m/z) 523 [(M+Na)+, 100%]; 501 [(M+H)+,
70%].
5.4.4. (3R,4S,5S)-3,4,5-Tri[(tert-butyldimethylsilyl)oxy]-
1-hydroxymethylcyclohex-1-ene (9d). Yield 85%. IR

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L. Sanchez-Abella et al. / Bioorg. Med. Chem. 15 (2007) 4193–4202
4199
5.5.3. (3R,4R,5R)-3,4,5-Tri[(tert-butyldimethylsilyl)oxy]-
cyclohex-1-enecarbaldehyde (10c). Yield 92%. IR
(100.6 MHZ, CDCl3): d ꢀ4.8 (SiMe), ꢀ4.7 (SiMe),
ꢀ4.4 (SiMe), ꢀ4.3 (SiMe), 17.9 (SiC), 18.1 (SiC), 18.4
(SiC), 25.7 (SiCMe3), 25.8 (SiCMe3), 26.1 (SiCMe3),
34.2 (C6), 67.6 (C3), 69.8 (C5), 72.3 (C4), 74.9 (C8),
84.7 (C7), 117.0 (C1), and 137.4 (C2); (ESI+, m/z) 519.2
[(M+Na)+, 90%].
1
(NaCl): t 2952, 2930, 2858, and 1694 cmꢀ1; H NMR
(400.13 MHz, CDCl3): d 0.07 (s, 3H, SiMe), 0.08 (s,
6H, 2SiMe), 0.10 (s, 3H, SiMe), 0.14 (s, 6H, 2SiMe),
0.85 (s, 9H, SiCMe3), 0.90 (s, 9H, SiCMe3), 0.92 (s,
2
0
9H, SiCMe3), 2.29 (d, 1H, H6a
,
j JHHj = 17.6,
3
2
0
JHH = 8.8 Hz), 2.40 (dd, 1H, H6e , j JHHj = 17.6,
3JHH = 5.3 Hz), 3.77 (s, 1H, H4), 4.00 (dd, 1H, H5,
3JHH = 6.4, 5.8 Hz), 4.24 (br s, 1H, H3), 6.45 (s, 1H,
H2), and 9.51 (s, 1H, H7); 13C NMR (100.6 MHz,
CDCl3): d ꢀ4.9 (SiMe), ꢀ4.7 (SiMe), ꢀ4.6 (SiMe),
ꢀ4.5 (SiMe), ꢀ4.4 (SiMe), 18.0 (SiC), 18.1 (SiC), 18.3
(SiC), 25.9 (SiCMe3), 26.0 (SiCMe3), 26.1 (SiCMe3),
26.9 (C6), 67.5 (C5),23 70.9 (C3), 75.6 (C4), 141.7
(C1),23 145.0 (C2),23 and 194.1 (C7)23; (ESI+, m/z) 523
[(M+Na)+, 13%]; 539 [(M+Na)+, 4%].
5.6.2. (3S,4S,5R)-3,4,5-Tri[(tert-butyldimethylsilyl)oxy]-
1-ethynylcyclohex-1-ene (11b). Yield 66%. IR (NaCl): t
20
3317, 2956, 2930, 2889, and 2858 cmꢀ1. ½aꢁ þ 2 (c
Na
0.9, CHCl3); 1H NMR (400.13 MHz, CDCl3): d 0.07
(s, 3H, SiMe), 0.08 (s, 3H, SiMe), 0.09 (s, 3H, SiMe),
0.10 (s, 3H, SiMe), 0.11 (s, 3H, SiMe), 0.11 (s, 3H,
SiMe), 0.89 (s, 9H, SiCMe3), 0.90 (s, 9H, SiCMe3),
0
2
0.92 (s, 9H, SiCMe3), 2.11 (dd, 1H, H6a , j JHHj = 16.8,
3
2
0
JHH = 4 Hz), 2.47 (dddd, 1H, H6e , j JHHj = 16.8,
3JHH = 4, j JHHj = 2, 2 Hz), 2.86 (s, 1H, H8), 3.69 (dd,
4
3
1H, H4, JHH = 5.2, 2.8 Hz), 3.84 (ddd, 1H, H5,
5.5.4. (3R,4S,5S)-3,4,5-Tri[(tert-butyldimethylsilyl)oxy]-
cyclohex-1-enecarbaldehyde (10d). Yield 95%. Mp 92–
94 ꢁC; IR (KBr): t 2951, 2929, 2889, 2857, and
3JHH = 4.4, 4.4, 4.4 Hz), 4.00 (br s, 1H, H3), and 6.03
(s, 1H, H2); 13C NMR (100.6 MHz, CDCl3): d ꢀ4.9
(SiMe), ꢀ4.3 (SiMe), ꢀ4.2 (SiMe), ꢀ4.1 (SiMe), 17.9
(SiC), 18.2 (SiC), 25.9 (SiCMe3), 26.0 (SiCMe3), 26.0
(SiCMe3), 34.2 (C6), 68.3 (C5), 71.6 (C3), 74.8 (C4),
75.8 (C8), 84.9 (C7), 117.1 (C1), and 135.5 (C2); (ESI+,
m/z) 519 [(M+Na)+, 90%].
1
1689 cmꢀ1; H NMR (400.13 MHz, CDCl3): d 0.08 (s,
6H, 2SiMe), 0.09 (s, 3H, SiMe), 0.11 (s, 3H, SiMe),
0.15 (s, 3H, SiMe), 0.16 (s, 3H, SiMe), 0.85 (s, 9H,
SiCMe3), 0.91 (s, 9H, SiCMe3), 0.96 (s, 9H, SiCMe3),
2
3
0
2.31 (dddd, 1H, H6a
,
j JHHj = 16.8, JHH = 10,
4
2
0
j JHHj = 3.2, 3.2 Hz), 2.41 (dd, 1H, H6e , j JHHj = 17.2,
5.6.3. (3R,4R,5R)-3,4,5-Tri[(tert-butyldimethylsilyl)oxy]-
1-ethynylcyclohex-1-ene (11c). Yield 90%. IR (NaCl): t
3JHH = 6 Hz), 3.79 (s, 1H, H5, JHH = 10, 6, 1.2 Hz),
3
3.97 (s, 1H, H4), 4.46 (br s, 1H, H3), 6.34 (s, 1H, H2),
and 9.47 (s, 1H, H7); 13C NMR (100.6 MHZ, CDCl3):
d ꢀ4.8 (SiMe), ꢀ4.7 (SiMe), ꢀ4.6 (SiMe), ꢀ4.5 (SiMe),
ꢀ4.4 (SiMe), ꢀ4.3 (SiMe), 18.3 (SiC), 18.5 (SiC), 18.6
(SiC), 25.9 (SiCMe3), 26.0 (SiCMe3), 26.1 (SiCMe3),
27.2 (C6), 70.3, (C5), 71.8 (C3), 75.4 (C4), 139.4 (C1),
150.8 (C2), and 193.2 (C7); (ESI+, m/z) 523 [(M+Na)+,
100%]; 539 [(M+K)+, 10%].
3316, 2955, 2929, 2887, 2858, 2090, and 1630 cmꢀ1
;
20
Na
½aꢁ ꢀ 70 (c 0.9, CHCl3); 1H NMR (400.13 MHz,
CDCl3): d 0.08 (s, 3H, SiMe), 0.09 (s, 3H, SiMe),
0.09 (s, 3H, SiMe), 0.10 (s, 3H, SiMe), 0.10 (s, 3H,
SiMe), 0.11 (s, 3H, SiMe), 0.88 (s, 9H, SiCMe3),
0.91 (s, 9H, SiCMe3), 0.92 (s, 9H, SiCMe3), 2.13
2
3
0
(dd, 1H, H6e , j JHHj = 16.4, JHH = 4.7 Hz), 2.42 (dd,
2
0
1H, H6a , j JHHj = 16.4, 3JHH = 9.4 Hz), 2.86 (s, 1H,
H8), 3.68 (br s, 1H, H4), 4.02 (m, 2H, H3+H5), and
5.94 (br s, 1H, H2); 13C NMR (100.6 MHZ, CDCl3):
d ꢀ4.8 (SiMe), ꢀ4.7 (SiMe), ꢀ4.7 (SiMe), ꢀ4.6
(SiMe), ꢀ4.4 (SiMe), 18.0 (SiC), 18.1 (SiC), 18.3
(SiC), 25.8 (SiCMe3), 26.0 (SiCMe3), 34.1 (C6), 67.1
(C5), 70.0 (C3), 71.2 (C4), 74.8 (C8), 84.4 (C7), 121.2
(C1), and 133.6 (C2); (ESI+, m/z) 519 [(M+Na)+,
30%]; 535 [(M+K)+, 5%].
5.6. Synthesis of 11a–d
nBuLi (0.38 mL of 1.6 M in hexane, 0.60 mmol) was
added to a solution of TMSCHN2 (0.29 mL of
2.0 M in hexane, 0.57 mmol) at ꢀ78 ꢁC. To this solu-
tion was added 10 (255 mg, 0.51 mmol) in THF
(2 mL). The mixture was stirred and allowed to reach
room temperature during 4 h. The reaction mixture
was poured into water/Et2O and the aqueous layer
extracted with Et2O. The combined organic fractions
were dried (Na2SO4) and concentrated, and the resi-
due purified by flash chromatography using silica
5.6.4. (3R,4S,5S)-3,4,5-Tri[(tert-butyldimethylsilyl)oxy]-
1-ethynylcyclohex-1-ene (11d). Yield 60%. Mp 93–
20
95 ꢁC. ½aꢁ ꢀ 39 (c 0.7, CHCl3); IR: 3307, 2953, 2928,
Na
2289, and 2857 cmꢀ1; H NMR (400.13 MHz, CDCl3):
d 0.07 (s, 3H, SiMe), 0.08 (s, 6H, 2SiMe), 0.10 (s, 3H,
SiMe), 0.10 (s, 3H, SiMe), 0.11 (s, 3H, SiMe), 0.89 (s,
9H, SiCMe3), 0.91 (s, 9H, SiCMe3), 0.93 (s, 9H,
1
˚
60 A (32–63 lm) pH 7 (gradient elution with hexane-
1% Et2O/hexane).
2
3
0
5.6.1. (3R,4S,5R)-3,4,5-Tri[(tert-butyldimethylsilyl)oxy]-
1-ethynylcyclohex-1-ene (11a). Yield 78%. IR (NaCl): t
SiCMe3), 2.13 (dd, 1H, H6e , j JHHj = 16, JHH 5.6 Hz),
2
3
0
2.47 (dddd, 1H, H6a
,
j JHHj = 16.4, JHH = 10,
3316, 2955, 2930, 2887, 2858, 2099, and 1633 cmꢀ1
;
j JHH3j = 3.2, 3.2 Hz), 2.86 (s, 1H, H8), 3.76 (ddd, 1H,
H5, JHH = 10, 5.6, 1.2 Hz), 3.88 (br s, 1H, H4), 4.28
(br s, 1H, H3), and 5.81 (s, 1H, H2); 13C NMR
(100.6 MHz, CDCl3): d ꢀ4.7 (SiMe), ꢀ4.6 (SiMe),
ꢀ4.6 (SiMe), ꢀ4.5 (SiMe), ꢀ4.4 (SiMe), 18.3 (SiC),
18.5 (SiC), 18.6 (SiC), 25.9 (SiCMe3), 26.2 (SiCMe3),
34.5 (C6), 70.0 (C5), 71.4 (C3), 74.5 (C4), 75.8 (C8),
83.9 (C7), 118.4 (C1), and 137.5 (C2); (ESI+, m/z) 519
[(M+Na)+, 100%].
4
20
Na
½aꢁ ꢀ 52 (c 0.5, CHCl3); 1H NMR (400.13 MHz,
CDCl3): d 0.08 (s, 6H, 2 SiMe), 0.09 (s, 3H, SiMe),
0.09 (s, 3H, SiMe), 0.11 (s, 6H, 2 SiMe), 0.88 (s, 9H,
SiCMe3), 0.89 (s, 9H, SiCMe3), 0.94 (s, 9H, SiCMe3),
2
1.95 (d, 1H, H6a, j JHHj = 17.6 Hz), 2.55 (dddd, 1H,
2
3
4
H6e, j JHHj = 14.7, JHH = 3.4, j JHHj = 3.4, 2.9 Hz),
2.82 (s, 1H, H8), 3.72 (m, 1H, H4), 3.94 (m, 1H, H5),
4.54 (sa, 1H, H3), and 5.95 (s, 1H, H2); 13C NMR

´
L. Sanchez-Abella et al. / Bioorg. Med. Chem. 15 (2007) 4193–4202
4200
5.7. Synthesis of 11d from 12
5.9.1. 1a,2a,25-Trihydroxy-6,7-dehydro-19-nor-previta-
min D3 (14a). Yield 94%; 1H NMR (300.13 MHz,
MeOH-d4): d 0.75 (s, 3H, Me18), 1.02 (d, 3H,Me21,
3JHH = 6.4 Hz), 1.12 (m, 1H), 1.20 (s, 6H, Me26 + Me27),
1.1–1.6 (m, 14H), 1.82 (m, 1H), 1.9–2.3 (m, 6H), 2.58
nBuLi (1 mL of 1.6 M in hexane, 1.6 mmol) was added to
a solution of 12 (363 mg, 0.55 mmol) in THF (5.5 mL) at
ꢀ78 ꢁC, and the resulting mixture was stirred during 1 h
at the same temperature. The reaction mixture was al-
lowed to reach room temperature and then quenched with
NH4Cl (aqueous). The moisture was extracted with Et2O,
and the combined organic fractions dried (Na2SO4) and
concentrated. Flash chromatography of the residue using
2
3
0
(dd, 1H, H4e , j JHHj = 17.4, JHH = 5.5 Hz), 3.59 (dd,
3
1H, H2, JHH = 8.6, 4.0 Hz), 3.95 (ddd, 1H, H3,
3JHH = 7.4, 7.4, 7.4 Hz), 4.29 (dd, 1H, H1, JHH = 4.3,
3
4.3 Hz), and 5.94 (s, 2H, H10 + H9). 13C NMR
(75.5 MHz, MeOH-d4): d 10.0 (C18), 17.8 (C21), 20.5
(CH2), 23.8 (CH2), 24.6 (CH2), 27.6 (CH2), 27.7
(CH3), 27.9 (CH3), 35.8 (CH2), 36.1 (CH), 36.3 (CH2),
36.4 (CH2), 41.6 (C), 43.9 (CH2), 49.9 (CH), 54.7
(CH), 66.4 (C1 y C3), 70.1 (C), 72.1 (C2), 87.7 (C6),
88.7 (C7), 121.7 (C5), 122.8 (C2), 131.4 (C9), and 133.4
(C10). (ESI+, m/z) 439 [(M+Na)+, 100%], 440
[(M+H+Na)+, 35], 455 [(M+K)+, 15], and 417
[(M+H)+, 13]. (EI+, m/z): 416 [(M+), 25%], 398
[(M+ꢀH2O), 20], 380 [(M+ꢀ2H2O), 35], 179 (40), 165
(50), 105 (65), 91 (95), 69 (98), and 55 (100). HMRS
Calcd for C26H40O4 (M+) 416.2921. Found 416.2926.
˚
silica 60 A (32–63 lm) pH 7 (gradient elution with hex-
ane-1% Et2O/hexane) afforded 11d in 65% yield.
5.8. Synthesis of (3R,4S,5S)-3,4,5-tri[(tert-butyldimeth-
ylsilyl)oxy]-1-(2,2-dibromoethenyl)cyclohex-1-ene (12)
CBr4 (370 mg, 1.11 mmol) was added to a solution of zinc
(73 mg, 1.11 mmol) and Ph3P (280 mg, 1.07 mmol) in
CH2Cl2 (5.6 mL). The resulting suspension was stirred
for 30 min at room temperature. Pyridine (178 lL) and
a solution of compound 10d (89 mg, 0.18 mmol) in
CH2Cl2 (1.8 mL) were added stepwise to the reaction mix-
ture and stirring was continued for 1 h additional. Work-
up was accomplished by dilution of the mixture with Et2O
followed by filtration through a pad of silica gel. The fil-
trate was concentrated and the crude residue purified by
flash chromatography (hexane) to afford 12 in 93% yield.
Mp 73–75 ꢁC; IR (KBr): t 2953, 2921, 2889, and
5.9.2. 1b,2a,25-Trihydroxy-6,7-dehydro-19-nor-previta-
min D3 (14b). Yield 69%. 1H NMR (400.13 MHz,
MeOH-d4): d 0.73 (s, 3H, Me18), 0.99 (d, 3H,Me21,
3JHH = 6.8 Hz), 1.11 (m, 1H), 1.18 (s, 6H, Me26 + Me27),
1.2–1.5 (m, 14H), 1.78 (ddd, 1H, 3JHH = 6.8, 6.8, 6.8 Hz),
1.9–2.1 (m, 2H), 2.2–2.3 (m, 4H), 2.47 (dd, 1H, H4e,
1
2
3
2857 cmꢀ1; H NMR (400.13 MHz, CDCl3): d 0.03 (s,
j JHHj = 16.4, JHH = 5.6 Hz), 3.35 (dd, 1H, H2,
3
3H, SiMe), 0.02 (s, 3H, SiMe), 0.02 (s, 3H, SiMe), ꢀ0.01
3JHH = 9.6, 7.6 Hz), 3.64 (ddd, 1H, H3, JHH = 10, 10,
(s, 3H, SiMe), 0.00 (s, 3H, SiMe), 0.01 (s, 3H, SiMe),
0.78 (s, 9H, SiCMe3), 0.81 (s, 9H, SiCMe3), 0.83 (s, 9H,
6 Hz), 4.07 (br s, 1H, H1), 5.74 (dd, 1H, H10, 3JHH = 2.4,
4
j JHHj = 2.4 Hz), and 5.93 (s, 1H, H9); 13C NMR
SiCMe3), 2.19 (dd, 1H, H6e , j J j = 11.2, 3JHH = 4 Hz),
(100.6 MHz, MeOH-d4): d 11.4 (C18), 19.2 (C21), 21.9
(CH2), 25.1 (CH2), 26.0 (CH2), 29.0 (CH2), 29.1 (CH3),
29.3 (CH3), 37.2 (CH2), 37.5 (CH), 37.7 (CH2), 38.7
(CH2), 42.9 (C), 45.3 (CH2), 51.3 (CH), 56.1 (CH), 70.2
(C3), 71.5 (C), 73.7 (C1), 78.5 (C2), 88.5 (C6), 90.1 (C7),
120.9 (C5), 123.6 (C8), and 134.8 (C9 + C10); (APCIꢀ, m/
z) 399 [(MꢀOH)+, 100%]; (EI+, m/z): 416 [(M+), 30%],
398 [(MꢀH2O)+, 35], 380 [(Mꢀ2H2O)+, 40], 165 (55),
129 (65), 91 (80), 69 (90), and 55 (100); HMRS Calcd
for C26H40O4 (M+) 416.2921. Found 416.2918.
2
0
j JHHHHj = 10.8, JHH = 6.8,
2
3
0
2.45 (dddd, 1H, H6a
,
4
3
j JHHj = 2, 2 Hz), 3.65 (ddd, 1H, H5, JHH = 6.8, 4,
1.2 Hz), 3.78 (s, 1H, H4), 4.14 (s, 1H, H3), 5.48 (s, 1H,
H2), and 6.77 (s, 1H, H7); 13C NMR (100.6 MHz, CDCl3):
d ꢀ4.7 (SiMe), ꢀ4.6 (SiMe), ꢀ4.6 (SiMe), ꢀ4.4 (SiMe),
ꢀ4.4 (SiMe), ꢀ4.3 (SiMe), 18.3 (SiC), 18.6 (SiC), 18.7
(SiC), 26.0 (SiCMe3), 26.2 (SiCMe3), 33.2 (C6), 70.4
(C5), 71.4 (C3), 74.5 (C4), 87.5 (C8), 132.9 (C1), 133.2
(C2), and 138.2(C7); (ESI+, m/z) 679 [(M79Br81Br+Na)+,
45%]; 681 [(M81Br81Br+Na)+, 35%], and 677
[(M79Br79Br+Na)+, 22%].
5.9.3. 1a,2b,25-Trihydroxy-6,7-dehydro-19-nor-previta-
min D3 (14c). Yield 88%. 1H NMR (400.13 MHz,
MeOH-d4): d 0.77 (s, 3H, Me18), 1.03 (d, 3H,Me21,
3JHH = 8.9 Hz), 1.12 (m, 1H), 1.22 (s, 6H, Me26 + Me27),
1.3–1.6 (m, 14H), 1.82 (ddd, 1H, 3JHH = 9, 9, 9 Hz), 1.9–
5.9. Synthesis of 14a–d
CuI (3.5 mg, 0.02 mmol), (PPh3)2Pd(OAc)2 (4 mg,
0.005 mmol), and Et2NH (1.4 mL) were added to a solu-
tion of 11 (97 mg, 0.20 mmol) and 13 (73 mg,
0.18 mmol) in DMF (1.4 mL). The reaction was moni-
tored by TLC (hexane). After 5 h, the mixture was
poured into water/Et2O and the aqueous layer extracted
with Et2O. The combined organic fractions were dried
(Na2SO4) and concentrated. TBAF (1.8 mL of 1.0 M
in THF, 1.8 mmol) was added dropwise to a solution
of this crude in THF (3.6 mL) at 0 ꢁC and the reaction
mixture was stirred overnight at room temperature.
The crude residue was poured into water/EtOAc and
the aqueous layer extracted with EtOAc. The combined
organic fractions were concentrated and subjected to
0
2.1 (m, 2H), 2.2–2.3 (m, 3H), 2.35 (dd, 1H, H4a
0
,
2
3
j JHHj = 17.3, JHH = 5.6 Hz), 2.45 (m, 1H, H4e ), 3.65
(dd, 1H, H2, 3JHH = 5.6, 2 Hz), 4.04 (br s, 1H, H3), 4.24
(br s, 1H, H1), 5.87 (s, 1H, H10), and 5.95 (s, H, H9); 13
C
NMR (100.6 MHz, MeOH-d4): d 11.4 (C18), 19.2 (C21),
21.9 (CH2), 25.2 (CH2), 26.0 (CH2), 29.0 (CH2), 29.1
(CH3), 29.3 (CH3), 36.7 (CH2), 37.2 (CH2), 37.5 (CH),
37.7 (CH2), 42.9 (C), 45.2 (CH2), 51.3 (CH), 56.0 (CH),
68.6 (C3), 70.6 (C1) 71.4 (C), 74.8 (C2), 89.2 (C), 89.8
(C), 122.0 (C), 123.6 (C), 133.1 (C10), and 134.6 (C9);
(ESI+, m/z) 439 [(M+Na)+, 95%], 855 [(2M+Na)+,
100%]; (EI+, m/z): 416 [(M+), 10%], 398 [(M+ꢀH2O),
75], 380 [(M+ꢀ2H2O), 60], 165 (70), 129 (75), 91 (93), 69
(96), and 59 (100); HMRS Calcd for C26H40O4 (M+)
416.2921. Found 416.2917.
˚
flash chromatography using silica 60 A (32–63 lm) pH
7 (gradient elution with 30–50% acetone/CH2Cl2).

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L. Sanchez-Abella et al. / Bioorg. Med. Chem. 15 (2007) 4193–4202
4201
5.9.4.
1a,2a,25-Trihydroxy-6,7-dehydro-3-epi-19-nor-
References and notes
previtamin D3 (14d). Yield 60%. 1H NMR
(400.13 MHz, MeOH-d4): d 0.73 (s, 3H, Me18), 0.99 (d,
3H,Me21, JHH = 6.8 Hz), 1.09 (m, 1H), 1.19 (s, 6H,
1. (a) Feldman, D.; Pike, J. W.; Glorieux, F. H., Eds.;
Vitamin D, 2nd ed.; Elsevier: New York, 2005; (b)
Ettinger, R. A.; DeLuca, H. F. Adv. Drug Res. 1996, 28,
269–312; (c) Christakos, S.; Raval-Pandya, M.; Wernyj, R.
P.; Yang, W. Biochem. J. 1996, 316, 361–371; (d) Bouillon,
R.; Okamura, W. H.; Norman, A. W. Endocrinol. Rev.
1995, 16, 200–257.
3
3
Me26 + Me27), 1.2–1.6 (m, 14H), 1.78 (dd, 1H, JHH
=
17.6, 11.2 Hz), 1.9–2.3 (m, 6H), 2.40 (dddd, 1H, H4a,
2
3
4
j JHHj = 16.3, JHH = 8.8, j JHHj = 2.4, 2.4 Hz), 3.84
3
(ddd, 1H, H3, JHH = 8.4, 5.6, 1.6 Hz), 3.90 (br s, 1H,
H2), 4.24 (br s, 1H, H1), 5.76 (br s, 1H, H10), and 5.92
(m, 1H, H9); 13C NMR (100.6 MHz, MeOH-d4): d 11.4
(C18), 19.2 (C21), 21.9 (CH2), 25.2 (CH2), 26.0 (CH2),
29.0 (CH2), 29.1 (CH3), 29.3 (CH3), 35.0 (CH2), 37.2
(CH2), 37.5 (CH), 37.7 (CH2), 42.9 (C), 45.2 (CH2), 51.3
(CH), 56.1 (CH), 69.3 (C3), 69.6 (C1), 71.5 (C), 72.0
(C2), 89.0 (C6), 89.9 (C7), 121.7 (C5), 123.7 (C8), 133.8
(C10), and 134.5 (C9); (APCIꢀ, m/z) 448 [(M+MeOH)+,
100%]; 415 [(MꢀH)+, 65%]; (EI+, m/z): 416 [(M+), 30%],
398 [(MꢀH2O)+, 40], 380 [(Mꢀ2H2O)+, 65], 165 (73), 91
(98), and 59 (100); HMRS Calcd for C26H40O4 (M+)
416.2921. Found 416.2917.
2. (a) Saito, N.; Kittaka, A. Chembiochem 2006, 7, 1478–
1490; (b) Posner, G. H.; Kahraman, M. Eur. J. Org.
Chem. 2003, 3889–3895; (c) Takayama, H.; Kittaka, A.;
Fujishima, T.; Suhara, Y.. In Recent Results in Cancer
Research; Reichrath, J., Friedrich, M., Tilgen, W., Eds.;
Springer-Verlag: Berlin-Heidelberg, 2003; Vol. 164, pp
289–317; (d) Zhu, G.-H.; Okamura, W. H. Chem. Rev.
1995, 95, 1877–1952; (e) Posner, G. H.; Dai, H. Synthesis
1994, 1383–1398.
3. (a) Proceedings of the 12th workshop on vitamin D, J.
Steroid Biochem. Mol. Biol.-Special Issue 2004, 89–90,
1–603; (b) Norman, A. W.; Mizwicki, M. T.; Okamura,
W. H.. In Recent Results in Cancer Research; Reich-
rath, J., Friedrich, M., Tilgen, W., Eds.; Springer-
Verlag: Berlin-Heidelberg, 2003; Vol. 164, pp 55–82; (c)
Thacher, S. M.; Vasudevan, J.; Tsang, K.-Y.; Nagpal,
S.; Chandraratna, R. A. S. J. Med. Chem. 2001, 44,
281–297; (d) Norman, A. W.; Bouillon, R.; Thomasset,
6. In vitro biological evaluation
6.1. Cell proliferation assay
M., Eds.; Vitamin
D Endocrine System: Structural,
As a measure of cell proliferation, [3H]thymidine incor-
poration of breast cancer MCF-7 (ATCC, Rockville,
MD) was determined after a 72-h incubation period
with various concentrations of 1a,25-(OH)2-D3, ana-
logues or vehicle as described previously.24
Biological, Genetic and Clinical Aspects; University of
California Riverside: Riverside, CA, 2000; (e) Tsugawa,
N.; Nakagawa, K.; Furobe, M.; Ono, Y.; Kubodera,
N.; Ozono, K.; Okano, T. Biol. Pharm. Bull. 2000, 23,
66–71.
4. Norman, A. W.; Henry, H. L.; Bishop, J. E.; Song, X.-D.;
Bula, C.; Okamura, W. H. Steroids 2001, 66, 147–158, and
references cited therein.
6.2. Binding studies
5. (a) Zhou, L.-X.; Nemere, I.; Norman, A. W. J. Bone
Miner. Res. 1992, 7, 457–463; (b) De Boland, A. R.;
Norman, A. W. Endocrinology 1990, 127, 2475–2480; (c)
Nemere, I.; Yoshimoto, Y.; Norman, A. W. Endocrinology
1984, 115, 1476–1483.
The affinity of 1a,25-(OH)2-D3 and its analogues to the
vitamin D receptor was evaluated by their ability to
compete with [3H]-1a,25-(OH)2-D3 for binding to high
speed supernatant from intestinal mucosa homogenates
obtained from normal pigs as described previously.24
The relative affinity of the analogues was calculated
from their concentration needed to displace 50% of
[3H]-1a,25-(OH)2-D3 from its receptor compared with
the activity of 1a,25-(OH)2-D3 (assigned a 100% value).
6. Zanello, L. P.; Norman, A. W. Biochem. Biophys. Res.
Commun. 1996, 225, 551–556.
7. (a) Schmitz, J. P.; Schwartz, Z.; Sylvia, V. L.; Dean, D. D.;
Calderon, F.; Boyan, B. D. J. Cell Physiol. 1996, 168, 570–
579; (b) Slater, S. J.; Kelly, M. B.; Taddeo, F. J.; Larkin, J.
D.; Yeager, M. D.; McLane, J. A.; Ho, C.; Stubbs, C. D.
J. Biol. Chem. 1995, 270, 6639–6643; (c) Bissonnette, M.;
Tien, X.-Y.; Niedziela, S. M.; Hartmann, S. C.; Frawley,
B. P., Jr.; Roy, H. K.; Sitrin, M. D.; Perlman, R. L.;
Brasitus, T. A. Am. J. Physiol. 1994, 267, G465–G475.
8. (a) Song, X.; Bishop, J. E.; Okamura, W. H.; Norman, A.
W. Endocrinology 1998, 139, 457–465; (b) De Boland, A.
R.; Norman, A. W. J. Cell. Biochem. 1998, 69, 470–482;
(c) Beno, D. W. A.; Brady, L. M.; Bissonnette, M.; Davis,
B. H. J. Biol. Chem. 1995, 270, 3642–3647.
9. Okamura, W. H.; Midland, M. M.; Hammond, M. W.;
Rahman, N. A.; Dormanen, M. C.; Nemere, I.; Norman,
A. W. J. Steroid Biochem. Mol. Biol. 1995, 53, 603–613.
10. Curtin, M. L.; Okamura, W. H. J. Am. Chem. Soc. 1991,
113, 6958–6966.
11. (a) Norman, A. W.; Okamura, W. H.; Hammond, M. W.;
Bishop, J. E.; Dormanen, M. C.; Bouillon, R.; van Baelen,
H.; Ridall, A. L.; Daane, E.; Khoury, R.; Farach-Carson,
M. C. Mol. Endocrinol. 1997, 11, 1518–1531; (b) Okamura,
W. H.; Midland, M. M.; Norman, A. W.; Hammond, M.
W.; Dormanen, M. C.; Nemere, I. Ann. NY. Acad. Sci.
1995, 761, 344–348; (c) Bouillon, R.; Sarandeses, L. A.;
Allewaert, K.; Zhao, J.; Mascaren˜as, J. L.; Mourin˜o, A.;
Binding of vitamin D analogues to the human vitamin D
binding protein (hDBP) was performed at 4 ꢁC as de-
scribed previously.25
Acknowledgments
Financial support of this work by the Spanish Ministe-
´
rio de Educacion y Ciencia (MEC, Project CTQ-2004-
04185) and Principado de Asturias (FICYT, Project
IB05-109) is gratefully acknowledged. S.F. thanks
´
MEC for a personal grant (Ramon y Cajal Program).
L.S.-A. thanks MEC for a pre-doctoral fellowship.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmc.
2007.03.058.

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L. Sanchez-Abella et al. / Bioorg. Med. Chem. 15 (2007) 4193–4202
4202
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18. Armesto, N.; Ferrero, M.; Fernandez, S.; Gotor, V.
Tetrahedron Lett. 2000, 41, 8759–8762.
´
´
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