Novel A-ring homodimeric C-3-carbamate analogues of 1α,25-dihydroxyvitamin D3: Synthesis and preliminary biological evaluation

Article abstract of DOI:10.1016/j.bmc.2006.07.021

The synthesis of a new class of vitamin D₃ analogues in which two units of 1α,25-dihydroxyvitamin D₃ are linked at the C-3 position by a dicarbamate functionality of variable length is described. The analogues demonstrated no affinit

Full text of DOI:10.1016/j.bmc.2006.07.021

Bioorganic & Medicinal Chemistry 14 (2006) 7512–7519
Novel A-ring homodimeric C-3-carbamate analogues
of 1a,25-dihydroxyvitamin D3: Synthesis and preliminary
biological evaluation
a
a
b
b
´
Daniel Oves, Susana Fernandez, Lieve Verlinden, Roger Bouillon,
Annemieke Verstuyf,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 24 May 2006; revised 30 June 2006; accepted 6 July 2006
Available online 1 August 2006
Abstract—The synthesis of a new class of vitamin D3 analogues in which two units of 1a,25-dihydroxyvitamin D3 are linked at the
C-3 position by a dicarbamate functionality of variable length is described. The analogues demonstrated no affinity for the vitamin
D receptor and possessed no antiproliferative or transactivating properties.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
25
R3
1a,25-Dihydroxyvitamin D3 [1a,25-(OH)2-D3, 1, Figure
1], the most active metabolite of vitamin D3 (2), plays
a major role in many biological processes including
calcium–phosphorus homeostasis, cell differentiation
and proliferation, and immune reactions.1 However,
the mechanisms for these differential actions have not
H
1, R1= R2= R3= OH, 1α,25-Dihydroxyvitamin D3
2, R1= R3= H; R2= OH, Vitamin D3
R2
R1
1
3
been clearly defined. In the last two decades, various
analogues2 of 1a,25-(OH)2-D3 have been developed to
improve the biological profile of the natural hormone
for a potential therapeutic application.3 Some of these
derivatives have similar or more potent antiproliferative,
yet reduced hypercalcemic actions, than the natural hor-
mone. An increasing number of synthetic vitamin D
derivatives are currently in use as drugs for treatment
of various human diseases and new candidates are in
human clinical trials.2a,4
Figure 1. 1a,25-Dihydroxyvitamin D3, the most active metabolite of
vitamin D3.
protein reveals distinct domains involved in nuclear
localization, DNA binding, ligand binding, receptor
dimerization, and gene transactivation.6 It is believed
that n-VDR-mediated gene expression is initiated by
binding of 1a,25-(OH)2-D3 to the ligand binding domain
of n-VDR which is assumed to cause conformational
changes in the receptor protein. Thus, a detailed under-
standing of the complementarity of the ligand shape
with that of the interior surface of the nuclear VDR
receptor ligand domain is the key to understand not
only the structural basis of receptor action, but also to
design new analogues of 1a,25-(OH)2-D3. The n-VDR
is a ligand-dependent transcription factor that regulates
target gene expression by interacting with response ele-
ments in gene promoters.
The genomic actions of 1a,25-(OH)2-D3 are mediated by
the vitamin D nuclear receptor (n-VDR), which is a
member of the nuclear steroid hormone receptor super-
family.5 Structure-function analysis of the n-VDR
Keywords: Vitamin D3 analogues; Enzymatic catalysis; Biological
evaluation.
*
Corresponding author. Tel./fax: +34 98 510 3448; e-mail: vgs@fq.
uniovi.es
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.07.021

D. Oves et al. / Bioorg. Med. Chem. 14 (2006) 7512–7519
7513
The vitamin D receptor binds to the vitamin D response
elements as a homodimer or as a heterodimer with reti-
noic acid receptors (RARs) or retinoic X receptors
(RXRs).7
a
b
O
HO
OH
O
O
OH
O
5
3
It is known that small differences in structure of ligand
may elicit different biological profiles. Crystal structure
of the VDR reveals that the relatively large ligand-bind-
ing cavity can accommodate much structural variation
in potential calcitriol analogue ligands.8 To the best of
our knowledge, there are only three research groups that
reported on the synthesis of vitamin D dimers. The first,
by Mourin˜o et al.9 describes the generation of dimeric
vitamin D3 and 1a,25-(OH)2-D3 comprising two units
linked through the C-11 position by a carbon side chain
of modulated size. In the second, Posner et al.10 report
the synthesis of a vitamin D dimeric analogue in which
the monomeric vitamin D units are linked via a side-
chain heteroaromatic ring. Finally, Fall et al.11 show
the preparation of a vitamin D dimer containing a disul-
fide bridge in the side-chain moiety.
4
5
+ 5
c
H2N
N
O
OH
n
H
6 a-c
O
O
RO
O
d
N
H
N
O
OR
n
H
7a-c, R= H
8a-c, R= TBDMS
a, n= 2; b, n= 4; c, n= 10
Scheme 1. Reagents and conditions: (a) CH2@CHOCO2N@CMe2,
CAL-B, toluene, 30 ꢁC, 4 h (quantitative); (b) NH2(CH2)nNH2, THF,
65 ꢁC, overnight (6a, 94%; 6b, 91%; and 6c, 93%); (c) 5, DMAP, 1,4-
dioxane, 85 ꢁC, 48 h (7a, 39%; 7b, 41%; and 7c, 40%); (d) TBDMSCl,
imidazol, DMAP, DMF, rt, 14 h (8a, 93%; 8b, 84%; and 8c, 79%).
Taking into account the efficient synthesis of A-ring car-
bamate precursors of 1a,25-(OH)2-D3 through a chemo-
enzymatic pathway,12 we describe here an efficient
convergent strategy to newly designed A-ring homodi-
meric derivatives of 1a,25-(OH)2-D3 (3, Fig. 2). In these
dimers two units of 1a,25-(OH)2-D3 are linked at the C-
3 position by a dicarbamate functionality of variable
length, while leaving the two hydroxyl groups at C-1
and C-25 unchanged. The crucial role of both groups
for the binding to VDR and DBP proteins has been estab-
lished.13 In addition, results of preliminary evaluation of
their biological properties are reported to investigate the
structure–activity relationships of the natural hormone.
1a,25-(OH)2-D3 A-ring fragment possesses two hydrox-
yl groups which can be selectively modified by means of
biocatalysts. The preparation of optically active com-
pounds via enzyme-mediated reactions has demonstrat-
ed the usefulness of these catalysts.15 Thus, Candida
antarctica lipase B (CAL-B) catalyzes the regioselective
alkoxycarbonylation at the C-5 hydroxyl group16 of diol
4.12b The reaction was carried out at 30 ꢁC in toluene
using acetone O-[(vinyloxy)carbonyl]oxime as acylating
agent. It is noteworthy that not C-3 alkoxycarbonyla-
tion product was observed being isolated exclusively,
after 4 h, vinyl carbonate 5 in quantitative yield.
2. Results and discussion
Subsequent reaction of 5 with linear diamines of various
lengths between the two amino functionalities afforded
carbamates 6. Next, these derivatives were allowed to re-
act with vinyl carbonate 5 in 1,4-dioxane and DMAP as
catalyst. Conversion of the carbamates to the dimers
was relatively low and the reaction yielded 7 in only
moderate yields (39–41%). In addition, starting materi-
als were recovered.
The homodimeric derivatives 3 were synthesized accord-
ing to the enyne approach, originally developed by
Lythgoe.14 This method, based on the coupling between
an A-ring synthon enyne and an enol triflate of the CD-
ring/side-chain fragment, has become one of the most
convenient methods for the synthesis of 1a,25-(OH)2-
D3 analogues. For that, key A-ring precursors 8 are con-
structed as indicated in Scheme 1.
The resulting homodimeric A-ring synthons 7 were con-
veniently protected as silyl ethers with tert-butyldimeth-
ylsilyl chloride.
R=
R
R
OH
All attempts to obtain 8 directly from 5 were unsuccess-
ful. The reaction of 9, the protected silyl derivative of 5,
H
H
O
O
NH2(CH2)4NH2
O
HO
O
N
N
O
OH
n
LDA, THF, 0 C, 4 h
H
H
O
O
OΣ
HO
OΣ
quantitative
3a-c
a, n= 2; b, n= 4; c, n= 10
9
10
Σ= TBDMS
Scheme 2.
Figure 2. Homodimeric C-3-carbamate derivatives of 1a,25-
dihydroxyvitamin D3.

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D. Oves et al. / Bioorg. Med. Chem. 14 (2006) 7512–7519
with the lithium diamidate of butan-1,4-diamine affor-
ded alcohol 10 as sole product (Scheme 2). Unproduc-
tive results were also obtained in the treatment of silyl
ether of 6 with LDA and subsequent treatment with 9.
The synthesis of the homodimeric derivatives of 1a,25-
(OH)2-D3 starts with the coupling17 of A-ring precursors
8a–c and vinyl triflate 11,18 prepared according to the
published procedure, in the presence of catalytic amount
of bis(triphenylphosphine)palladium (II) acetate-copper
(I) iodide (Scheme 3). The reaction crude was subjected
to desilylation with tetrabutylammonium fluoride
(TBAF) to afford dienynes 12a–c in 70–89% yield (for
coupling and desilylation steps). Catalytic hydrogena-
tion of tetraols 12 in methanol, in the presence of Lind-
lar catalyst and quinoline poison, generated previtamins
13. A critical factor is the state of the catalyst, since it
should be previously predried at 50 ꢁC in vacuum. Ther-
molysis of the latter at 80 ꢁC for 4 h afforded the dimers
3a–c in 50–60% yield from 12.
Figure 3. Affinity of 1a,25-(OH)2-D3 and its analogues for pig vitamin
D receptor.
parent compound (Fig. 4). A 10ꢀ8 M concentration of
1a,25-(OH)2-D3 led to a half-maximal transactivation.
However, the compounds 3a, 3b, and 3c were almost un-
able to transactivate this reporter construct (a 1.5- to
1.9-fold induction at 10ꢀ6 M).
3. Biological evaluation
We evaluated the potencies of the C-3-carbamate vita-
min D3 analogs in terms of their ability to bind to the
pig intestinal vitamin D receptor in comparison to the
natural hormone. In addition, the transactivating capac-
ity of 1a,25-(OH)2-D3 and its analogues was measured
as well as their potency to inhibit MCF-7 breast cancer
cell proliferation.
These results were paralleled by the findings on cell pro-
liferation. The EC50 value of 1a,25-(OH)2-D3 to inhibit
the proliferation of MCF-7 cells was 7 · 108 Mꢀ1
(Fig. 5).
However, none of the three C-3-carbamate analogues
were able to inhibit the proliferation of MCF-7 breast
cancer cells.
The affinity of 1a,25-(OH)2-D3 for the pig duodenal
mucosa vitamin D receptor was 1.9 · 1010 Mꢀ1, whereas
the analogues possessed no affinity at all for the vitamin
D receptor (Fig. 3).
4. Conclusions
Next, the ability of the C-3-carbamate vitamin D3 ana-
logues to transactivate a reporter construct, which con-
tained a vitamin D responsive element, was determined
and compared to the transactivation potential of the
A facile synthetic strategy for the preparation of new
dimeric 1a,25-(OH)2-D3 derivatives was developed
through a chemoenzymatic pathway that allows the
selective functionalization of the C-3 position. Our ini-
R
R
H
H
OTMS
a, b
+
8a-c
H
11
TfO
O
O
HO
O
N
N
O
OH
R=
n
H
H
OH
12a-c
a, n=2; b, n=4; c, n=10
R
c
R
OH
HO
O
O
d
H
H
3a-c
O
N
N
O
n
H
H
13a-c
Scheme 3. Reagents and conditions: (a) Pd(PPh3)2(OAc)2, Cul, Et2NH, DMF, rt, 2 h; (b) Bu4NF, THF, rt, overnight (12a, 70%; 12b, 89%; and 12c,
72%, 2 steps); (c) H2, Lindlar cat., quinoline, MeOH, rt, 20 min; (d) acetone, 80 ꢁC, 4 h (3a, 52%; 3b, 50%; and 3c, 60%, 2 steps).

D. Oves et al. / Bioorg. Med. Chem. 14 (2006) 7512–7519
7515
100.61 MHz for 1H and 13C, respectively, was used
1
1
for the adquisition of H–1H and H–13C correlation
experiments. The chemical shifts are given in delta
(d) values and the coupling constants (J) in Hertz
(Hz). ESI+ and EI (70 eV) were used to record mass
spectra (MS). Microanalyses were performed on a
Perkin-Elmer model 2400 instrument. HPLC was per-
formed using UV detector and a Spherisorb W, 5lm
silica gel column, 250 · 10 mm. Compound 5 has been
previously synthesized.12b
5.2. Synthesis of 3,30-[Alkan(1,n-dicarbamoyloxy)]bis-
1a,25-dihydroxyvitamin D3 (3)
A flask containing Lindlar catalyst (88 mg) was exposed
to a positive pressure of hydrogen gas (balloon). Deoxy-
genated MeOH (3 mL) was added, and to this suspen-
sion quinoline (0.240 mL, 0.17 M in hexane) and 12
(0.0342 mmol) in MeOH (4 mL) were added. After
20 min, the mixture was filtered on Celite and concen-
trated to afford a crude, which was sufficiently pure
for the next step. A solution of the crude previtamin
13 (0.0237 mmol) in anhydrous acetone (4 mL) was
placed in a screw-capped vial and heated at 80 ꢁC for
4 h. The solvent was evaporated to leave a residue which
was purified by flash chromatography (gradient eluent
80–100% EtOAc/hexane). The compound was purified
further by HPLC (18% iPrOH/hexane, 2.5 mL/min,
Spherisorb W, 5l, 250· 10 mm).
Figure 4. Transactivating potency of 1a,25-(OH)2-D3 and its ana-
logues in COS-1 cells. Luciferase activity was normalized to b-
galactosidase activity and expressed relative to the normalized activity
of transfected, vehicle-stimulated cells.
5.2.1. 3,30-[Butan(1,4-dicarbamoyloxy)]bis-1a,25-dihydro-
xyvitamin D3 (3a). Yield 52%. Mp 96–98 ꢁC (decompose);
IR (KBr): m 3413, 2945, 2870, 1701, 1527 and 1377 cmꢀ1
;
1H NMR (CDCl3, 300 MHz): d 0.55 (s, 6H, 6H18), 0.94
(d, 6H, 6H21, JHH 6.3 Hz), 1.23 (s, 12H, 6H26 + 6H27),
3
1.2–2.6 (remaining ring and side-chain hydrogens, series
of m), 2.82 (d, 2H, JHH 9.4 Hz), 3.16 (m, 4H, 2CH2–
2
NH), 4.38 (m, 2H, 2H1), 4.80 (m, 2H, 2NH), 5.0 (s, 2H,
2H19), 5.12 (m, 2H, 2H3), 5.4 (s, 2H, 2H19), 6.04 (d, 2H,
Figure 5. In vitro antiproliferative effects of 1a,25-(OH)2-D3 and its
analogues in MCF-7 cells. [3H]thymidine incorporation was deter-
mined in cells which were incubated during a 72 h incubation period
with 1a,25-(OH)2-D3 or with the compounds 3a, 3b or 3c.
3
3
2H7, JHH 11.0 Hz) and 6.33 (d, 2H, 2H6, JHH
11.0 Hz);13C NMR (CDCl3, 75.5 MHz): d 11.9, 18.7,
20.7, 22.2, 23.6, 27.1, 27.6, 29.0, 29.1, 29.3, 36.0, 36.3,
40.2, 40.4, 41.7, 44.3, 45.9, 56.3, 56.4, 70.0, 71.1, 111.1,
117.0, 124.3, 132.9, 143.0, 147.5 and 156.0; (ESI+, m/z):
995 [(M+Na)+, 85%], 1011 [(M+K)+, 10%]; Anal. Calcd
(%) for C60H96N2O8: C, 74.03; H, 9.94; N, 2.88. Found:
C, 74.3; H, 10.1; N, 3.0.
tial aim was directed at the incorporation of linkers of
different length. Unfortunately the analogues were not
able to bind to the vitamin D receptor and demonstrated
no transactivating or antiproliferative activity.
5.2.2. 3,30-[Hexan(1,6-dicarbamoyloxy)]bis-1a,25-dihydro-
xyvitamin D3 (3b). Yield 50%. Mp 100–102 ꢁC (decom-
pose); IR (KBr): m 3414, 2941, 2867, 1701, 1618, 1527
5. Experimental
1
and 1377 cmꢀ1; H NMR (CDCl3, 300 MHz): d 0.47 (s,
3
6H, 6H18), 0.86 (d, 6H, 6H21, JHH 6.3 Hz), 1.14 (s,
5.1. General spectroscopic and experimental data
12H, 6H26 + 6H27), 1.0–2.5 (remaining ring and side-
chain hydrogens, series of m), 2.74 (d, 2H, JHH
2
Melting points were taken on samples in open capil-
lary tubes and are uncorrected. IR spectra were
recorded on an Infrared Fourier Transform spectro-
photometer using NaCl plates or KBr pellets. Flash
chromatography was performed using silica gel 60
(230–400 mesh). 1H, 13C NMR, and DEPT were ob-
tained using AC-300 (1H, 300.13 MHz and 13C,
75.5 MHz) spectrometer for routine experiments. An
AMX-400 spectrometer operating at 400.13 and
12.6 Hz), 3.06 (m, 4H, 2CH2–NH), 4.30 (m, 2H, 2H1),
4.67 (m, 2H, 2NH), 4.92 (s, 2H, 2H19), 5.04 (m, 2H,
3
2H3), 5.3 (s, 2H, 2H19), 5.97 (d, 2H, 2H7, JHH 11.0 Hz)
and 6.25 (d, 2H, 2H6, 3JHH 11.0 Hz); 13C NMR (CDCl3,
75.5 MHz): d 11.9, 18.7, 20.6, 22.2, 23.5, 26.0, 27.6, 29.1,
29.3, 29.6, 36.0, 36.3, 40.1, 40.4, 40.5, 41.7, 44.3, 45.9,
56.3, 56.4, 69.9, 71.1, 111.0, 117.0, 124.2, 133.0, 143.0,
147.5 and 155.9; (ESI+, m/z): 1023 [(M+Na)+, 100%];

7516
D. Oves et al. / Bioorg. Med. Chem. 14 (2006) 7512–7519
Anal. Calcd (%) for C62H100N2O8: C, 74.36; H, 10.06; N,
2.80. Found: C, 74.6; H, 10.3; N, 2.6.
(ESI+, m/z): 295 [(M+H)+, 100%]; Anal. Calcd (%) for
C16H26N2O3: C, 65.28; H, 8.90; N, 9.52. Found: C 65.1;
H, 9.1; N, 9.3.
5.2.3. 3,30-[Dodecan(1,12-dicarbamoyloxy)]bis-1a, 25-di-
hydroxyvitamin D3 (3c). Yield 60%. Mp 103–105 ꢁC
(decompose); IR (KBr): m 3411, 2928, 2854, 1700, 1528,
5.3.3. (3S,5R)-5-[N-(12-Aminododecyl)carbamoyloxy]-1-
ethynyl-3-hydroxy-2-methylcyclohex-1-ene (6c). Yield
93%. IR (neat): m 3345, 3282, 2923, 2859, 2093, 1667,
1
1466 and 1377 cmꢀ1; H NMR (CDCl3, 300 MHz): d
0.56 (s, 6H, 6H18), 0.95 (d, 6H, 6H21, 3JHH 6.1 Hz), 1.23
(s, 12H, 6H26 + 6H27), 1.27 (s, 20H, 10CH2), 1.2–2.6
(remaining ring and side-chain hydrogens, series of m),
3.16 (m, 4H, 2CH2–NH), 4.38 (m, 2H, 2H1), 4.68 (m,
2H, 2NH), 5.03 (s, 2H, 2H19), 5.13 (m, 2H, 2H3), 5.37
1534 and 1468 cmꢀ1; H NMR (MeOH-d4, 300 MHz):
1
d 1.4–1.8 (m, 20H, 10 CH2), 2.17 (s, 3H, 3H9), 2.1–
2.17 (m, 2H, 2H4), 2.33 (dd, 1H, H6, JHH 17.1 Hz,
2
2
3JHH 6.8 Hz), 2.74 (d, 1H, H6, JHH 17.1 Hz), 2.96
(apparent t, 2H, CH2–NH2, JHH 7.4 Hz), 3.26 (appar-
3
3
3
ent t, 2H, CH2–NH, JHH 7.1 Hz), 3.66 (s, 1H, H8),
(s, 2H, 2H19), 6.05 (d, 2H, 2H7, JHH 11.2 Hz) and 6.33
(d, 2H, 2H6, JHH 11.2 Hz); 13C NMR (CDCl3,
3
4.38 (m, 1H, H3) and 5.17 (m, 1H, H5); 13C NMR
75.5 MHz): d 11.9, 18.7, 20.7, 22.2, 23.5, 26.7, 27.6, 29.0,
29.1, 29.2, 29.3, 29.4, 29.9, 36.0, 36.3, 40.2, 40.4, 40.9,
41.8, 44.3, 45.9, 56.3, 56.4, 67.0, 69.8, 70.1, 71.0, 111.0,
117.0, 124.3, 133.0, 143.0, 147.6 and 155.8; (ESI+, m/z):
1085 [(M+H)+, 10%], 1107 [(M+Na)+, 23%]; Anal. Calcd
(%) for C68H112N2O8: C, 75.23; H, 10.40; N, 2.58. Found:
C, 75.5; H, 10.4; N, 2.5.
(MeOH-d4, 75.5 MHz): d 18.5 (C9), 27.7, 27.7, 30.3,
30.6, 30.8, 31.0 (C12 + C13 + C14 + C15 + C16 + C17
+
C18 + C19 + C20 + C21), 36.5 (C6), 37.6 (C4), 41.5, 41.6
(C11+C22), 68.1 (C5), 68.6 (C3), 81.8 (C8), 83.8 (C7),
114.8 (C1), 144.1 (C2) and 158.4 (C@O); (ESI+, m/z):
379 [(M+H)+, 100%]; Anal. Calcd (%) for
C22H38N2O3: C, 69.80; H, 10.12; N, 7.40. Found: C,
69.6; H, 10.3; N, 7.5.
5.3. Synthesis of (3S,5R)-5-[N-(aminoalkyl)carbamoyl-
oxy]-1-ethynyl-3-hydroxy-2-methylcyclohex-1-ene (6)
5.4. Synthesis of (3S,5R,30S,50R)-1,10-Diethynyl-3,30-di-
hydroxy-2,20-dimethyl-5,50-[alkan(1,n-dicarbamoyl-
oxy)]dicyclohex-1-ene (7)
In a typical procedure, a solution of 5 (0.675 mmol) and
corresponding diamine (3 equiv of 1,4-butanodiamine,
1,6-hexanediamine or 1,12-dodecadiamine) in THF
(6 mL) was heated at 65 ꢁC for 16 h. Then, solvent
was evaporated under reduced pressure, and the residue
was subjected to flash chromatography (1% NH3/
MeOH) to afford a colorless oil.
A mixture of carbonate 5 (0.765 mmol), carbamate 6
(0.765 mmol), and DMAP (catalytic amount) in 1,4-di-
oxane (10 mL) was heated at 85 ꢁC for 60 h. After evap-
oration of the solvent, the residual mixture was purified
by flash chromatography (gradient eluent 70–100%
EtOAc/hexane) to give compound 7 as a white solid.
5.3.1. (3S,5R)-5-[N-(4-Aminobutyl)carbamoyloxy]-1-ethy-
nyl-3-hydroxy-2-methylcyclohex-1-ene (6a). Yield 94%. IR
5.4.1. (3S,5R,30S,50R)-1,10-Diethynyl-3,30-dihydroxy-2,20-
dimethyl-5,50-[butan(1,4-dicarbamoyloxy)]dicyclohex-1-ene
(7a). Yield 39%. Mp 206–208 ꢁC (decompose); IR
(KBr): m 3488, 3336, 2963, 2092, 1669, 1542 and
(neat): m 3288, 2933, 2855, 2091, 1711, 1552 and 1441 cmꢀ1
;
1H NMR (MeOH-d4, 300 MHz): d 1.69 (m, 4H, 2CH2),
2.17 (s, 3H, 3H9), 2.07–2.2 (m, 2H, 2H4), 2.33 (dd, 1H,
2
H6, JHH 17.1 Hz, JHH 6.5 Hz), 2.75 (d, 1H, H6, JHH
3
2
1
1451 cmꢀ1; H NMR (MeOH-d4, 300 MHz): d 1.67 (m,
3
17.1 Hz), 2.85 (apparent t, 2H, CH2–NH2, JHH 6.8 Hz),
3.29 (m, 2H, CH2–NH), 3.67 (s, 1H, H8), 4.38 (apparent
4H, 2CH2), 2.16 (s, 6H, 3H9 þ 3H09), 2.05–2.2 (m, 4H,
2H4 þ 2H0 ), 2.33 (dd, 2H, H6 þ H0 , 2JHH 16.7 Hz, 3JHH
4
6
t, 1H, H3, 3JHH 4.8 Hz) and 5.17 (m, 1H, H5); 13C NMR
4.9 Hz), 2.74 (d, 2H, H6 þ H06, JHH 16.7 Hz), 3.28 (m,
4H, 2CH2–NH), 3.65 (s, 2H, H8 þ H08), 4.37 (m, 2H,
H3 þ H0 ) and 5.17 (m, 2H, H5 þ H0 ); 13C NMR
2
(MeOH-d4, 75.5 MHz):
d
18.5 (C9), 28.1, 30.4
(C12 + C13), 36.5 (C6), 37.6 (C4), 41.3, 41.9 (C11 + C14),
68.1, 68.5 (C5 + C3), 81.9 (C8), 83.8 (C7), 114.7 (C1),
144.2 (C2) and 158.3 (C@O); MS (ESI+, m/z): 267
[(M+H)+, 100%], 289 [(M+Na)+, 90%]; Anal. Calcd (%)
for C14H22N2O3: C, 63.13; H, 8.33; N, 10.52. Found: C,
63.4; H, 8.5; N, 10.6.
3
5
(MeOH-d4, 75.5 MHz):
d
19.3 ðC9 þ C0 Þ, 28.9
9
ðC12 þ C0 Þ, 37.4, 38.5 ðC4 þ C0 þ C6 þ C0 Þ, 42.1
12
4
6
(2CH2–NH), 69.1, 69.5 ðC3 þ C0 þ C5 þ C0 Þ, 82.7
3
5
ðC8 þ C0 Þ, 84.7 ðC7 þ C0 Þ, 115.7 ðC1 þ C0 Þ, 145.0
8
7
1
ðC2 þ C0 Þ and 159.3 (2C@O); (ESI+, m/z): 467
2
[(M+Na)+, 100%]; Anal. Calcd (%) for C24H32N2O6: C,
64.85; H, 7.26; N, 6.30. Found: C, 65.0; H, 7.2; N, 6.4.
5.3.2. (3S,5R)-5-[N-(6-Aminohexyl)carbamoyloxy]-1-ethy-
nyl-3-hydroxy-2-methylcyclohex-1-ene (6b). Yield 91%. IR
(neat): m 3287, 2921, 2091, 1699, 1565, 1443 and
5.4.2.
(3S,5R,30S,50R)-1,10-Diethynyl-3,30-dihydroxy-
1
1359 cmꢀ1; H NMR (MeOH-d4, 200 MHz): d 1.4–1.8
2,20-dimethyl-5,50-[hexan(1,6-dicarbamoyloxy)]dicyclo-
hex-1-ene (7b). Yield 41%. Mp 170–172 ꢁC (decompose);
IR (KBr): m 3459, 3346, 3294, 2934, 2092, 1667, 1543,
(m, 8H, 4CH2), 2.08 (m, 2H, 2H4), 2.15 (s, 3H, 3H9),
2.28 (dd, 1H, H6, JHH 18 Hz, JHH 6.8 Hz), 2.71 (dd,
2
3
2
1H, H6, JHH 18 Hz), 2.87 (apparent t, 2H, CH2–NH2,
1
1455 and 1370 cmꢀ1; H NMR (MeOH-d4, 300 MHz):
3
3JHH 6.8 Hz), 3.24 (apparent t, 2H, CH2–NH, JHH
d 1.52 (m, 4H, 2CH2), 1.67 (m, 4H, 2CH2), 2.12 (m,
6.6 Hz), 3.64 (s, 1H, H8), 4.34 (m, 1H, H3) and 5.2 (m,
1H, H5); 13C NMR (MeOH-d4, 50.3 MHz): d 18.5 (C9),
27.4, 30.6, 31.8 (C12 + C13 + C14 + C15), 36.5 (C6), 37.5
(C4), 41.4, 41.7 (C11 + C16), 68.1 (C5), 68.5 (C3), 81.9
(C8), 83.8 (C7), 114.8 (C1), 144.1 (C2) and 158.3 (C@O);
4H, 2H4 þ 2H04), 2.16 (s, 6H, 3H9 þ 3H09), 2.33 (dd,
2
3
2H, H6 þ H06, JHH 17.1 Hz, JHH 6.3 Hz), 2.74 (d, 2H,
2
H6 þ H06, JHH 17.1 Hz), 3.27 (apparent t, 4H, 2CH2–
3
NH, JHH 6.8 Hz), 3.66 (s, 2H, H8 þ H08), 4.38 (m, 2H,
H3 þ H0 ) and 5.17 (m, 2H, H5 þ H0 ); 13C NMR
3
5

D. Oves et al. / Bioorg. Med. Chem. 14 (2006) 7512–7519
7517
(MeOH-d , 75.5 MHz): d 18.5 ðC9 þ C0 Þ, 27.3, 30.7
NMR (CDCl3, 300 MHz): d 0.13 (s, 12H, 2SiMe2), 0.93
(s, 18H, 2CMe3), 1.36 (m, 4H, 2CH2), 1.53 (m, 4H,
2CH2), 1.98 (s, 6H, 3H9 þ 3H09), 1.8–2.0 (m, 4H,
2H4 þ 2H0 ), 2.22 (d, 2H, H6 þ H0 , 2JHH 18 Hz), 2.63 (d,
ðC12 þ C0 4þ C13 þ C0 Þ, 36.5, 37.5 ðC4 þ C0 þ C6 þ
9
12
13
4
C0 Þ, 41.4 (2CH2–NH), 68.1, 68.5 ðC3 þ C0 þ C5 þ C0 Þ,
6
3
5
81.8 ðC8 þ C0 Þ, 83.8 ðC7 þ C0 Þ, 114.8 ðC1 þ C0 Þ, 141.1
8
7
1
4
6
ðC2 þ C0 Þ and 158.3 (2C@O); (ESI+, m/z): 495
2H, H6 þ H06, JHH 18 Hz), 3.09 (s, 2H, H8 þ H08), 3.19
(m, 4H, 2CH2-NH), 4.27 (m, 2H, H3 þ H03), 4.68 (m,
2H, 2NH) and 5.09 (m, 2H, H5 þ H05Þ; 13C NMR (CDCl3,
75.5 MHz): d -4.9 (2SiMe), -4.4 (2SiMe), 17.9 (2SiCMe3),
18.5 ðC9 þ C0 Þ, 25.7 (2CMe3), 26.2, 29.8 ðC12 þ C0 þ
2
2
[(M+Na)+, 100%]; Anal. Calcd (%) for C26H36N2O6: C,
66.08; H, 7.68; N, 5.93. Found: C, 66.2; H, 7.8; N, 6.0.
5.4.3. (3S,5R,30S,50R)-1,10-Diethynyl-3,30-dihydroxy-2,20-
dimethyl-5,50-[dodecan(1,12-dicarbamoyloxy)]dicyclohex-
1-ene (7c). Yield 40%. Mp 123–125 ꢁC (decompose); IR
(KBr) m 3347, 2925, 2093, 1686, 1534 and 1466 cmꢀ1; 1H
NMR (MeOH-d4, 300 MHz): d 1.49 (m, 16H, 8CH2),
1.66 (m, 4H, 2CH2), 2.17 (s, 6H, 3H9 þ 3H09), 2.05–2.2
9
12
C13 þ C0 Þ, 35.2, 36.8 ðC4 þ C0 þ C6 þ C0 Þ, 40.7 (2CH2-
13
4
6
NH), 67.3, 68.4 ðC3 þ C0 þ C5 þ C0 Þ, 79.8 ðC8 þ C0 Þ,
3
5
8
83.2 ðC7 þ C0 Þ, 112.8 ðC1 þ C0 Þ, 144.1 ðC2 þ C0 Þ and
7
1
2
155.8 (2C@O); (ESI+, m/z): 723 [(M+Na)+, 100%]; Anal.
Calcd (%) for C38H64N2O6Si2: C, 65.10; H, 9.20; N,
4.00. Found: C, 65.3; H, 9.6; N, 3.8.
2
(m, 4H, 2H4 þ 2H04), 2.33 (dd, 2H, H6 þ H06, JHH 16.5
3
2
Hz, JHH 6 Hz), 2.73 (d, 2H, H6 þ H06, JHH 16.5 Hz),
2.25 (m, 4H, 2CH2–NH), 3.66 (s, 2H, H8 þ H08), 4.37 (m,
5.5.3.
(3S,5R,30S,50R)-3,30-Di[(tert-butyldimethylsilyl)-
2H, H3 þ H0 ) and 5.16 (m, 2H, H5 þ H0 ); 13C NMR
oxy]-1,10-diethynyl-2,20-dimethyl-5,50-[dodecan(1,12-dicar-
(MeOH-d4, 75.5 MHz): d 18.4 ðC9 þ C0 5Þ, 27.7, 30.3,
bamoyloxy)]dicyclohex-1ene (8c). Yield 79%. IR (neat) m
3
9
ðC12 þ C0 þ C13 þ C0 þ C14 þ C0
þ
3317, 2932, 2095, 1701, 1528, 1460 and 1360 cmꢀ1; H
1
30.6,
30.8
12
13
14
C15 þ C0 þ C16 þ C0 Þ, 36.5, 37.6 ðC4 þ C0 þ C6 þ C0 Þ,
NMR (CDCl3, 300 MHz): d 0.13 (s, 12H, 2CMe3), 0.93
(s, 18H, 2CMe3), 1.29 (m, 16H, 8CH2), 1.51 (m, 4H,
2CH2), 1.98 (s, 6H, 3H9 þ 3H09), 1.85-2.05 (m, 4H,
15
16
4
6
41.6 (2CH2-NH), 68.1, 68.6 ðC3 þ C0 þ C5 þ C0 Þ, 81.8
3
5
ðC8 þ C0 Þ, 83.8 ðC7 þ C0 Þ, 114.8 ðC1 þ C0 Þ, 144.1
8
7
1
ðC2 þ C0 Þ and 158.4 (2C@O); (ESI+, m/z): 579 [(M+
2H4 þ 2H04), 2.23 (d, 2H, H6 þ H60 , JHH 17.3 Hz), 2.63
2
2
Na)+, 100%]; Anal. Calcd (%) for C32H48N2O6: C,
69.04; H, 8.69; N, 5.03. Found: C, 69.2; H, 8.8; N, 4.9.
(d, 2H, H6 þ H60 , JHH 17.3 Hz), 3.09 (s, H, H8 þ H08),
2
3.19 (m, 4H, 2CH2-NH), 4.27 (m, 2H, H3 þ H03), 4.66 (m,
2H, 2NH), and 5.10 (m, 2H, H5 þ H0 ); 13C NMR (CDCl3,
5
5.5. Synthesis of (3S,5R,30S,50R)-3,30-Di[(tert-butyl-
dimethylsilyl)oxy]-1,10-diethynyl-2,20-dimethyl-5,50-
[alcan(1,n-dicarbamoyloxy)]dicyclohex-1ene (8)
75.5 MHz):
d
ꢀ4.9 (2SiMe), ꢀ4.4 (2SiMe), 17.9
(2SiCMe3), 18.5 ðC9 þ C0 Þ, 25.7 (2CMe3), 26.7, 29.2, 29.4,
9
29.9
ðC12 þ C0 þ C13 þ C0 þ C14 þ C0 þ C15 þ C0 þ
12
13
14
15
C16 þ C0 Þ, 35.6, 36.8 ðC4 þ C0 þ C6 þ C0 Þ, 40.9 (2CH2-
16
4
6
Imidazol (93 mg, 1.404 mmol), DMAP (catalytic
amount), and tert-butyldimethylsilyl chloride (140 mg,
0.936 mmol) were added to a stirred solution of dimer
7 (0.234 mmol) in DMF (3 mL) under nitrogen. The
solution was stirred at room temperature for 12 h, and
then evaporated under reduced pressure to leave a resi-
due which was purified by flash chromatography (30%
EtOAc/hexane). Colorless oil.
NH), 67.2, 68.4 ðC3 þ C0 þ C5 þ C0 Þ, 79.8 ðC8 þ C0 Þ,
3
5
8
83.2 ðC7 þ C0 Þ, 112.8 ðC1 þ C0 Þ, 144.1 ðC2 þ C0 Þ and
7
1
2
155.7 (2C@O); (ESI+, m/z): 807 [(M+Na)+, 100%]; Anal.
Calcd (%) for C44H76N2O6Si2: C, 67.30; H, 9.76; N, 3.57.
Found: C, 67.2; H, 9.7; N, 3.3.
5.6. Synthesis of (3S,5R)-3-[(tert-Butyldimethylsilyl)oxy]-
1-ethynyl-2-methyl-5-[(vinyloxy)carbonyloxy]cyclohex-1-
ene (9)
5.5.1.
(3S,5R,30S,50R)-3,30-Di[(tert-butyldimethylsilyl)-
oxy]-1,10-diethynyl-2,20-dimethyl-5,50-[butan(1,4-dicarba-
To a solution of 10 (1.73 mmol) in CH2Cl2 (10 mL) and
pyridine (0.4 mL) at 0 ꢁC was added dropwise vinyl chlo-
roformate (4.33 mmol). The progress of the reaction was
followed by TLC (40% EtOAc/hexane). After consump-
tion of the starting material (4 h), the reaction mixture
was extracted with CH2Cl2 and brine. The organic layer
was dried, evaporated under reduced pressure, and the
residue was subjected to flash chromatography (15%
EtOAc/hexane). White solid. Yield 87%. mp 63–65 ꢁC;
IR (KBr) m 3500, 2956, 2096, 1748, 1648, 1470 and
moyloxy)]dicyclohex-1ene (8a). Yield 93%. IR (neat) m
1
3500, 2956, 2940, 1700, 1549 and 1521 cmꢀ1; H NMR
(CDCl3, 300 MHz): d 0.11 (s, 12H, 2SiMe2), 0.91 (s, 18H,
2CMe3), 1.53 (m, 4H, 2CH2), 1.95 (s, 6H, 3H9 þ 3H09),
1.8–2.0 (m, 4H, 2H4 þ 2H0 ), 2.18 (d, 2H, H6 þ H0 , 2JHH
4
6
2
15 Hz), 2.58 (d, 2H, H6 þ H06, JHH 15 Hz), 3.06 (s, 2H,
H8 þ H08), 3.18 (m, 4H, 2CH2–NH), 4.24 (m, 2H,
H3 þ H03), 4.71 (m, 2H, 2NH) and 5.06 (m, 2H,
H5 þ H0 ); 13C NMR (CDCl3, 75.5 MHz): d -4.9 (2SiMe),
5
ꢀ4.4 (2SiMe), 18.0 (2SiCMe3), 18.5 ðC9 þ C0 Þ, 25.7
1364 cmꢀ1; H NMR (CDCl3, 200 MHz): d 0.12 (s, 6H,
1
(2CMe3), 27.2 ðC12 þ C0 Þ, 35.2, 36.8 ðC4 þ C09þ C6 þ
2MeSi), 0.91 (s, 9H, SiCMe3), 1.96 (s, 3H, 3H9), 1.95-2.1
12
4
C0 Þ, 40.4 (2CH2-NH), 67.4, 68.4 ðC3 þ C0 þ C5 þ C0 Þ,
(m, 2H, 2H4), 2.30 (dd, 1H, H6, 2JHH16.4, 3JHH 6.9 Hz),
2.68 (dd, 1H, H6, JHH16.4, JHH 3.8 Hz), 3.08 (s, 1H,
6
3
5
79.8 ðC8 þ C0 Þ, 83.2 ðC7 þ C0 Þ, 112.8 ðC1 þ C0 Þ, 144.1
2
3
8
7
1
ðC2 þ C0 Þ and 155.8 (2C@O); (ESI+, m/z): 695
3
H8), 4.27 (apparent t, JHH4.6 Hz), 4.58 (dd, 1H,
2
[(M+Na)+, 100%]; Anal. Calcd (%) for C36H60N2O6Si2:
C, 64.24; H, 8.99, N, 4.16. Found: C, 64.0; H, 9.2; N, 3.9.
H2C@cis, 3JHH6.4, 2JHH1.8 Hz), 4.93 (dd, 1H,
3
2
H2C@trans, JHH13.8, JHH3 1.8 Hz), 5.10 (m, 1H, H5)
3
and 7.10 (dd, 1H, @CH, JHH13.8, JHH6.4 Hz); 13C
5.5.2.
(3S,5R,30S,50R)-3,30-Di[(tert-butyldimethylsilyl)-
NMR (CDCl3, 50 MHz): d ꢀ4.9 (SiMe), ꢀ4.5 (SiMe),
17.9 (SiCMe3), 18.7 (C9), 25.6 (CMe3), 34.7, 36.6
(C4 + C6), 68.4, 71.7 (C3 + C5), 80.2 (C8), 82.8 (C7), 97.6
(CH2@), 112.5 (C1), 142.5, 143.8 (C2 + CH@) and 151.9
oxy]-1,10-diethynyl-2,20-dimethyl-5,50-[hexan(1,6-dicarba-
moyloxy)]dicyclohex-1ene (8b). Yield 84%. IR (neat) m
1
3313, 2931, 2094, 1709, 1529, 1472 and 1362 cmꢀ1; H

7518
D. Oves et al. / Bioorg. Med. Chem. 14 (2006) 7512–7519
(C@O); (EI, m/z):336(M+, 2%), 279 (M-tBu, 20), 249 (45),
248 (40), 233 (14), 192 (16), 145 (91), 117 (59), 101 (53), 75
(100); Anal. Calcd (%) for C18H28O4Si: C, 64.25; H, 8.39.
Found: C, 64.1; H, 8.6.
1007 [(M+K)+, 15%]; Anal. Calcd (%) for
C60H92N2O8: C, 74.34; H, 9.57; N, 2.89. Found: C,
74.2; H, 9.7; N, 2.6.
5.8.2. 3,30-[Hexan(1,6-dicarbamoyloxy)]bis-1a,25-dihy-
droxy-6,7-dehydroprevitamin D3 (12b). Yield 89%. Mp
105–107 ꢁC; IR (KBr): m 3413, 2948, 2874, 1701, 1528
5.7. Synthesis of (3S,5R)-3[(tert-Butyldimethylsilyl)oxy]-
1-ethynyl-5-hydroxy-2-methylcyclohex-1-ene (10)
1
and 1466 cmꢀ1; H NMR (MeOH-d4, 300 MHz): d 0.92
3
(s, 6H, 6H18), 1.18 (d, 6H, 6H21, JHH 6.3 Hz), 1.37 (s,
To a solution of 1,4-butanodiamine (0.044 mmol) in
THF (3 mL) at ꢀ78 ꢁC was added dropwise LDA
(0.45 mmol). The reaction mixture was stirred at 0 ꢁC
for 15 min and then cooled to ꢀ78 ꢁC. Compound 9
was added (0.089 mmol) and the reaction was followed
by TLC. After 4 h, the reaction mixture was extracted
with Et2O/brine, the organic layer was dried and evapo-
rated under reduced pressure, and the crude was subject-
ed to flash chromatography to obtain 10 in quantitative
yield. This compound has been previously described.19
1H NMR (CDCl3, 300 MHz): d 0.10 (s, 6H, 2MeSi),
12H, 6H26 + 6H27), 1.2–2.5 (remaining ring and side-
chain hydrogens, series of m), 2.14 (s, 6H, 6H19), 2.73
(d, 2H, JHH 13.9 Hz), 3.28 (t, 4H, 2CH2NH, JHH
2
3
6.6 Hz), 4.38 (m, 2H, 2H1), 5.15 (m, 2H, 2H3) and 6.10
3
(d, 2H, 2H9, JHH 2.8 Hz); 13C NMR (MeOH-d4,
75.5 MHz): d 11.4, 18.8, 19.2, 21.8, 25.3, 26.0, 27.3, 29.0,
29.1, 29.2, 30.7, 36.9, 37.1, 37.4, 37.6, 37.7, 41.4, 42.9,
45.1, 51.3, 56.0, 68.3, 68.8, 71.3, 88.4, 94.0, 116.1, 123.7,
134.1, 141.2 and 158.4; (ESI+, m/z): 1019 [(M+Na)+,
60%]; Anal. Calcd (%) for C62H96N2O8: C, 74.66; H,
9.70; N, 2.81. Found: C, 74.5; H, 9.9; N, 2.9.
2
0.89 (s, 9H, Me3CSi), 1.75 (ddd, 1H, H4, JHH13.4,
3
3JHH9.8, JHH4.6 Hz), 1.85–2.0 (m, 3H9 + H4), 2.0 (dd,
1H, H6, JHH16.5, JHH7.8 Hz), 2.55 (m, 1H, H6), 3.06
(s, 1H, H8), 4.15 (m, 1H, H5) and 4.24 (m, 1H, H3).
2
3
5.8.3. 3,30-[Dodecan(1,12-dicarbamoyloxy)]bis-1a,25-di-
hydroxy-6,7-dehydroprevitamin D3 (12c). Yield 72 %.
Mp 120–122 ꢁC; IR (KBr): m 3416, 2928, 1700, 1541,
1
5.8. Synthesis of 3,30-[Alkan(1,n-dicarbamoyloxy)]bis-
1a,25-dihydroxy-6,7-dehydroprevitamin D3 (12)
1466 and 1381 cmꢀ1; H NMR (MeOH-d4, 300 MHz):
3
d 0.92 (s, 6H, 6H18), 1.17 (d, 6H, 6H21, JHH 6.0 Hz),
1.37 (s, 12H, 6H26 + 6H27), 1.49 (s, 20H, 10CH2), 1.4–
CuI (5 mg, 0.026 mmol), Pd(PPh3)2(OAc)2 (7 mg,
0.009 mmol), and Et2NH (2.5 mL) were added to a solu-
tion of 8 (0.148 mmol) and 11 (137 mg, 0.282 mmol) in
DMF (2.5 mL). The reaction mixture was stirred at
room temperature for 2 h under nitrogen and then
poured into water and extracted with diethyl ether (3·
10 mL). The combined ether fractions were dried over
Na2SO4, filtered, and concentrated to give a crude,
which was sufficiently pure for the next step. Although
it was possible to purify this compound by flash chroma-
tography, it decomposed in a few hours.
2.5 (remaining ring and side-chain hydrogens), 2.14 (s,
6H, 6H19), 2.73 (d, 2H, JHH 16.0 Hz), 3.25 (m, 4H,
2CH2NH), 4.39 (m, 2H, 2H1), 5.16 (m, 2H, 2H3) and
2
6.10 (d, 2H, 2H9, JHH 3.0 Hz); 13C NMR (MeOH-d4,
3
75.5 MHz): d 11.3, 18.7, 19.1, 21.8, 25.3, 25.9, 27.5,
27.7, 29.0, 29.2, 30.3, 30.5, 30.8, 36.9, 37.1, 37.4, 37.6,
37.7, 41.5, 42.9, 45.1, 51.3, 56.0, 68.3, 68.8, 71.3, 88.4,
93.9, 116.1, 123.7, 134.1, 141.2 and 158.4; (ESI+, m/z):
1103 [(M+Na)+, 5%]; Anal. Calcd (%) for
C68H108N2O8: C, 75.51; H, 10.06; N, 2.59. Found: C,
75.8; H, 10.3; N, 2.8.
TBAF (5 mL of 1.0 M in THF, 5 mmol) was added to a
solution of the crude in THF (10 mL) at 0 ꢁC and the
reaction mixture was stirred at room temperature for
18 h. THF was evaporated and the crude residue was
poured into water/EtOAc. The aqueous layer was
extracted with EtOAc (3· 10 mL) and the combined
organic layers were dried over Na2SO4 and evaporated
in a vacuum. The crude was purified by flash chroma-
tography (EtOAc) to afford 12 as a white solid.
5.9. In vitro biological evaluation
5.9.1. Affinity for VDR. The affinity of 1a,25-(OH)2-D3
and its analogues to the vitamin D receptor was evaluat-
ed 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 de-
scribed previously. The relative affinity of the analogues
was calculated from their concentration needed to dis-
place 50% of [3H]1a,25-(OH)2-D3 from its receptor com-
pared with the activity of 1a,25-(OH)2-D3 (assigned a
100% value).20
5.8.1.
3,30-[Butan(1,4-dicarbamoyloxy)]bis-1a,25-dihy-
droxy-6,7-dehydroprevitamin D3 (12a). Yield 70%. Mp
99–101 ꢁC; IR (KBr): m 3393, 2951, 1699, 1541, 1458
and 1377 cmꢀ1
;
1H NMR (CDCl3, 300 MHz): d 0.68
5.9.2. Transactivation assay. COS-1 cells (American
Type Culture Collection, Manassas, VA) (30,000) were
seeded in 24-well dishes. The next day, cells were trans-
fected with 100 ng of a luciferase reporter construct,
which contained three copies of the mouse osteopontin
3
(s, 6H, 6H18), 0.94 (d, 6H, 6H21, JHH 6.5 Hz), 1.20 (s,
12H, 6H26+6H27), 1.97 (s, 6H, 6H19), 1.0–2.3 (remaining
ring and side-chain hydrogens, series of m), 2.57 (d, 2H,
2JHH 13.4 Hz), 3.15 (m, 6H, 2CH2–NH+2OH), 4.20 (m,
2H, 2H1), 5.01 (br s, 2H, 2NH), 5.14 (m, 2H, 2H3) and
vitamin
D responsive element, 50 ng pSG5-based
3
5.94 (d, 2H, 2H9, JHH 3.1 Hz); 13C NMR (CDCl3,
expression vectors for VDR and RXR, and 20 ng
pcDNA3.1(-)/Myc-His/lacZ (Invitrogen, Merelbeke,
Belgium). Cells were transfected with FuGENE 6
(Roche, Mannheim, Germany). The day after transfec-
tion, cells were stimulated with vehicle (ethanol) or dilu-
75.5 MHz): d 10.9, 18.4, 18.6, 20.7, 24.0, 25.1, 27.0,
27.9, 29.0, 29.2, 35.7, 36.0, 36.2, 40.3, 41.7, 44.2, 49.9,
54.5, 67.1, 68.2, 71.0, 87.3, 93.1, 114.9, 122.3, 133.5,
140.2, and 156.1; (ESI+, m/z): 991 [(M+Na)+, 100%],

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Acknowledgments
We thank Novo Nordisk Co. for the generous gift of the
lipase CAL-B. Financial support to this work by the
´
Spanish Ministerio de Educacion y Ciencia (MEC,
Project CTQ-2004-04185), Principado de Asturias
(FICYT, Project IB05-109), and the Fonds voor
8. Rochel, N.; Wurtz, J. M.; Mitschler, A.; Klaholz, B.;
Moras, D. Mol. Cell 2000, 5, 173–179.
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Wetenschappelijk
Onderzoek
(FWO)
(Grants
G.0508.05 and G.0553.06) is gratefully acknowledged.
´
S. F. thanks MEC for a personal grant (Ramon y Cajal
Program). D.O. thanks FICYT for a predoctoral fellow-
ship. L.V. is post-doctoral researcher from the FWO.
10. Suh, B.-C.; Jeon, H.; Posner, G. H.; Silverman, S. M.
Tetrahedron Lett. 2004, 45, 4623–4625.
´
11. Fall, Y.; Fernandez, C.; Gomez, G.; Bolan˜o, T.; Alonso,
D.; Perez, M. Abstracts of the 12th Workshop on Vitamin
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