Versatile synthesis and biological evaluation of 1,3-diamino-substituted 1α,25-dihydroxyvitamin D3 analogues

Article abstract of DOI:10.1016/j.bmc.2005.09.009

A concise route to 1α,3β-diamino-25-hydroxy-3-deoxyvitamin D₃ (5) and 1β,3α-diamino-25-hydroxy-3-deoxyvitamin D ₃ (6) has been developed starting from (R)- or (S)-carvone for the construction of the modified A-ring fragments. The con

Full text of DOI:10.1016/j.bmc.2005.09.009

Bioorganic & Medicinal Chemistry 14 (2006) 928–937
Versatile synthesis and biological evaluation of
1,3-diamino-substituted 1a,25-dihydroxyvitamin D3 analogues
a
a
a
b
´
Daniel Oves, Susana Fernandez, Miguel Ferrero, Roger Bouillon,
Annemieke Verstuyfb and Vicente Gotora,*
a
´
´
´
´
Departamento de Quımica Organica e Inorganica, Facultad de Quımica, Universidad de Oviedo, 33071-Oviedo, Spain
bLaboratorium voor Experimentele Geneeskunde en Endocrinologie, Katholieke Universiteit Leuven, Gasthuisberg,
B-3000 Leuven, Belgium
Received 30 May 2005; revised 31 August 2005; accepted 6 September 2005
Available online 4 October 2005
Abstract—A concise route to 1a,3b-diamino-25-hydroxy-3-deoxyvitamin D3 (5) and 1b,3a-diamino-25-hydroxy-3-deoxyvitamin
D3 (6) has been developed starting from (R)- or (S)-carvone for the construction of the modified A-ring fragments. The con-
version of the hydroxyl group to amine function with complete inversion of the configuration was efficiently accomplished by
Mitsunobu reaction using phthalimide as nucleophile or activation of the hydroxyl group as mesylate followed by reaction
with NaN3. Diamino 5 and 6 as well as monoamino 3, 4, 30, and 31 vitamin D3 derivatives have shown poor binding to
VDR compared with 1a,25-dihydroxyvitamin D3. The most active compound in the inhibition of MCF-7 cell proliferation
and HL 60 cell differentiation was 1a-amino analogue 3. Also, very low in vivo calcemic effects of derivatives 3 and 4 were
found.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
25
R3
OH
1a,25-Dihydroxyvitamin D3 [1, 1a,25-(OH)2-D3, Fig. 1]
has been recognized as the hormonally active metabolite
of vitamin D3 (2) involved in intestinal calcium absorp-
tion, and bone resorption and mineralization. In addi-
tion, 1 regulates cell differentiation, cell proliferation,
and immune effects.1 Because of the biological profile
of 1a,25-(OH)2-D3, structurally modified compounds
have been prepared as sensitive molecular biology
probes and as potential new drugs of high efficacy and
low toxicity.2 Eight vitamin D analogues are currently
in use as drugs for chemotherapy of various human dis-
eases and four new derivatives in human clinical trials.2a
Of considerable interest is the development by Leo Phar-
maceutical of a compound called seocalcitol3 (Leo EB
1089), characterized by an altered side-chain structure
featuring 26,27-dimethyl groups and two double bonds.
Most of the analogues are altered in the side chain of the
CD-ring moiety, although modifications in the A-ring,
H
H
R2
R1
H2N
NH2
3
1
1, R1= R2= R3= OH
6
2, R1= R3= H; R2= OH
3, R1= NH2; R2= R3= OH
4, R1= R3= OH; R2= NH2
5, R1= R2= NH2; R3= OH
Figure 1.
less accessible synthetically, provide vitamin D deriva-
tives with an unique biological profile.4
1a-Fluoro-16,23-diene-20-epi derivative Ro 26-9228
(Hoffmann-La Roche)5 and 2b-(3-hydroxypropoxy)
analogue ED-71 (Chugai)6 are both used in human clin-
ical trials as promising candidates for the treatment of
osteoporosis. Another analogue with a substitution in
Keywords: Calcitriol; Monoamino and diamino A-ring; Novel
analogues.
*
Corresponding author. Tel./fax: +34 985 103 448; e-mail:
vgs@fq.uniovi.es
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.09.009

D. Oves et al. / Bioorg. Med. Chem. 14 (2006) 928–937
929
the A-ring on C-2 (2-methylene, 2MD) induces bone
formation in vitro and in vivo.7
For the synthesis of 1a,3b-A-ring precursor 13 (Scheme
1), we chose (R)-carvone as chiral template, which was
converted in a highly efficient synthesis to the monoace-
tate 8 by Okamura et al.13 We envisaged that the synthe-
sis of 13 could be achieved by Mitsunobu reaction14
using phthalimide as nucleophilic on the corresponding
diol 9. When 9 was allowed to react with phthalimide in
the presence of DEAD and Ph3P, diimide derivative 10
was obtained with complete inversion of the configura-
tion at both centers. However, simultaneous inversion
To investigate the structure–function relationships of
this hormone are of interest the synthesis and biological
evaluation of novel A-ring modified vitamin D ana-
logues. Recently, we reported the synthesis of 1a- and
3b-amino derivatives of 1a,25-(OH)2-D3 3 and 4, respec-
tively.8 Although hydroxyl groups at C-1 and C-25 posi-
tions are considered essential for binding to nVDR and
DBP proteins, various vitamin D derivatives with struc-
tural changes at the 1-position have showed significant
biological activities. Among them, the Ro 26-9228 deriv-
ative, above mentioned, and 1-hydroxyalkyl-25-hydrox-
yvitamin D3 analogues,9 which are known to retain
calcitriolÕs antiproliferative activity in murine keratino-
cytes even though these synthetic homologues are signif-
icantly less effective than calcitriol in binding to the
1a,25-(OH)2-D3 receptor. In addition, orientation of
the two hydroxyl groups on the A-ring is important in
the biological profile of such derivatives. Thus, the C-1
epimer of 1a,25-(OH)2-D3 was shown to be an antago-
nist of nongenomic, but not genomic, actions by Nor-
man and co-workers.10 Also, the C-3 epimer was
shown to be produced in the vitamin D metabolic path-
way in certain cell lines.11 Herein, we wish to describe
the synthesis of 1a,3b- and 1b,3a-diamino derivatives
of vitamin D3 5 and 6, respectively, and also the biolog-
ical significance of both the amino substitution and
stereochemistry at C-1 and/or C-3 position.
1
of both OH groups gives place to low yield (17%). H
NMR spectrum of the reaction crude showed the ab-
sence of starting material and traces of possible elimina-
tion products. The loss of crude weight can be explained
by the formation of 1-ethynyl-2-methylbenzene, which
was vaporized in the work-up. For this reason, sequen-
tial modification of C-3 and C-5 positions was tried.
Thus, allylic alcohol 8 was subjected to above-men-
tioned conditions being isolated compound 11 in 82%
yield. Saponification of the acetate ester and subsequent
treatment with phthalimide under Mitsunobu condi-
tions gives place to diimide 10. The reaction of 10 with
8 M MeNH2 in EtOH afforded the corresponding diami-
no, which is N-protected by adding di-tert-butyl-
dicarbonate to the reaction mixture. However, the
moderate yield of this step prompted us to search for
a more efficient route.
Treatment of 11 with MeNH2 in EtOH results in com-
plete deprotection of both the hydroxy and the amino
groups to give the corresponding aminoalcohol, which
is N-protected. The resulting derivative 14 (Scheme 2)
was treated with phthalimide to provide the required
stereoisomer 15 in a low yield of 30% due to the presence
of elimination products. The use of toluene instead of
THF, or replacement of Ph3P by Me3P did not have sig-
nificant impact on the yield.
2. Results and discussion
To synthesize the diamino analogues, we used a conver-
gent route12 based on the palladium-catalyzed coupling
reaction of the A-ring synthon with the CD-ring por-
tion, which are separately prepared. In this approach,
a dienyne is semihydrogenated to a previtamin structure
that undergoes rearrangement to the corresponding
vitamin D analogue.
The conversion of alcohol 14 to inverted amine was
best achieved by activation of the hydroxyl group as
mesylate followed by reaction with NaN3. This ap-
proach provides the azido-substituted compound 17
O
a
b
c
e
O
O
3 h, 17%
5 steps
N
NHBoc
AcO
OH
HO
OH
N
BocHN
8
9
13
O
O
7, (R)-(-)-carvone
10
c
4 h, 82%
d
c
O
N
O
N
2.5 h, 63%
AcO
HO
O
O
11
12
Scheme 1. (a) Ref. 13; (b) NaOMe, MeOH, 0 ꢁC, 2.5 h (96%); (c) phthalimide, Ph3P, DEAD, THF, rt; (d) K2CO3, MeOH, 0 ꢁC, 1.5 h (71%);
(e) i, MeNH2, EtOH, rt, 24 h, and then 65 ꢁC, 12 h. ii, (Boc)2O, NaHCO3 (aq), CHCl3, rt, 24 h (45%).

930
D. Oves et al. / Bioorg. Med. Chem. 14 (2006) 928–937
O
a
b
a
O
11
NHBoc
N
NHBoc
5 steps
HO
AcO
OH
14
19
O
18, (S)-(+)-carvone
15
c
b
d
e
d
c
13
O
NHBoc
NHBoc
N
MsO
3
NHBoc
AcO
N
HO
16
17
21
O
20
Scheme 2. (a) i, MeNH2, EtOH, rt, 24 h, and then 65 ꢁC, 12 h. ii,
(Boc)2O, NaHCO3 (aq), CHCl3, rt, 24 h (88%); (b) phthalimide, Ph3P,
DEAD, THF, rt, 3 h (30%); (c) MsCl, Et3N, CH2Cl2, 0 ꢁC, 3 h (94%);
(d) NaN3, DMF, 65 ꢁC, 6 h (79%); (e) Me3P, (Boc)2O, 1 M NaOH,
THF, 24 h (82%).
e
f
NHBoc
NHBoc
BocHN
NHBoc
MsO
N3
with inversion of the configuration at C-3 in good
yield. Transformation of azide to amine was accom-
plished by the Staudinger reaction.15 Thus, treatment
of 17 with Me3P in the presence of H2O and Boc2O
afforded the N-Boc derivative 13 in 53% yield after
flash chromatography. Recently, Vilarrasa et al.16 have
described that the presence of a basic medium, to avoid
22
23
24
Scheme 3. (a) Ref. 17; (b) phthalimide, Ph3P, DEAD, THF, rt, 4 h
(84%); (c) i, MeNH2, EtOH, rt, 24 h, and 65 ꢁC, 12 h. ii, (Boc)2O,
NaHCO3 (aq), CHCl3, rt, 24 h (77%); (d) MsCl, Et3N, CH2Cl2, 0 ꢁC,
3 h (81%); (e) NaN3, DMF, 65 ꢁC, 6 h (75%); (f) Me3P, (Boc)2O, 1 M
NaOH, THF, 24 h (79%).
t
t
the protonation of BuOCOOꢀ to BuOCOOH and/or
ꢀ
to remove CO2 as HCO3 or CO32ꢀ, is essential to
the success of the reaction. Thus, when 17 was treated
in degassed THF with Me3P and Boc2O in the presence
of degassed aqueous 1 M NaOH the yield of 13 was
increased up to 82%.
lation with tetrabutylammonium fluoride to afford
the most stable dienyne derivatives 26 and 27 in 82
and 89% yield, respectively (for coupling and desily-
lation steps). Subsequent catalytic hydrogenation in
the presence of Lindlar catalyst and quinoline poison
generated previtamins 28 and 29. One note concerns
the conditions used to reach high conversions in the
hydrogenation process, which was conducted in deox-
ygenated methanol using a vibromatic at 800 cycles/
min and with the catalyst pre-dried at 60 ꢁC in vac-
uum. The thermal isomerization of 28 and 29 (ace-
tone, 80 ꢁC, 4 h) reveals an interequilibrium between
the vitamin and the previtamin forms. 1H NMR
analysis of 30 and 31 indicated an equilibrium pro-
portion of the previtamin forms of 28 and 26%,
respectively. These minor constituents were readily
separable by HPLC being isolated derivatives 30
and 31 in 40–43% yield after purification (hydrogena-
tion and isomerization steps). Finally, deprotection of
the amino group with HCl and purification by semi-
Having established a route for the synthesis of 1a,3b-
A-ring precursor, we embarked on the preparation of
corresponding stereoisomer 1b,3a-A-ring synthon.
The synthesis started from key intermediate 19
(Scheme 3), which has been previously reported by
Okamura et al., using (S)-(+)-carvone.17 Substitution
of hydroxyl group in 19 by phthalimide and simulta-
neous inversion of the configuration was carried out
by the Mitsunobu method. Subsequent reaction of 20
with MeNH2 gives place to deprotection of both the
phthaloyl and acetyl groups yielding the appropriate
aminoalcohol which was N-protected to give 21. The
latter was transformed to the mesylate 22, which was
then converted to the azide 23 by the treatment with
NaN3. Finally, direct conversion of azido derivative
to N-Boc compound afforded the 1b,3a-A-ring precur-
sor 24 in high yield. We confirmed unambiguously the
stereochemistry of 13 and 24 by NOESY experiments
on compounds 14 and 21. In addition, comparison of
the optical rotation of derivatives obtained in Schemes
2 and 3 revealed the formation of corresponding
enantiomers.
preparative TLC [5% NH3(aq)/MeOH] yielded
and 6.
5
A-ring chair conformations of analogues 5 and 6 are
shown in Chart 1, as determined by H NMR analysis.
1
In both cases, the 1-amino group occupies the equatorial
position and the 3-amino group the axial position.
The B-seco steroidal structure was constructed12a,18
by standard Sonogashira coupling of the A-ring syn-
thons 13 and 24 with the CD-ring portion 25
(Scheme 4), prepared according to the published pro-
cedure.19 The reaction crude was subjected to desily-
3. Biological evaluation
We evaluated the potencies of the amino vitamin D3
derivatives 3, 4, 5, 6, 30, and 31 in terms of their ability

D. Oves et al. / Bioorg. Med. Chem. 14 (2006) 928–937
931
Table 1. Biological activity of amino derivatives of 1a,25-(OH)2-D3
Compound
VDR (%)
MCF-7 (%)
HL-60 (%)
1a,25-(OH)2-D3
3
4
100
5
100
30
2
100
20
2
0.4
0
5
0
0
6
0
0
0
30
31
0
0
0
0
0
0
Summary of the in vitro effects of amino analogues of 1a,25-(OH)2-D3
on receptor binding (VDR), MCF-7 proliferation, and HL 60 differ-
entiation. The in vitro effect is expressed as percentage activity at EC50
in comparison with 1a,25-(OH)2-D3 (= 100% activity).
bound very poorly to VDR when compared with 1a,25-
(OH)2-D3. As shown in Table 1, the 1a-amino derivative
3 exhibited 5% of affinity compared with the natural
hormone; meanwhile, the other analogues show no affin-
ity at all.
As a measure of cell proliferation, [3H]thymidine
incorporation of MCF-7 was determined after a 72 h
incubation period with various concentrations of 1a,
25-(OH)2-D3, analogues or ethanol. The most active
compound is the 1a-amino derivative 3, which can
inhibit the cell proliferation for 80% at a concentration
of 10ꢀ6 M comparable with 1a,25-(OH)2-D3 but this
compound was three times less potent than 1a,25-
(OH)2-D3 at the EC50 concentration (Fig. 2). Also
compound 3 was the most potent analogue to stimulate
the differentiation of
HL 60 cells (Fig. 3).
Scheme 4. (a) Pd(Ph3P)2(OAc)2, Cul, Et2NH, DMF, rt, 1 h; (b)
Bu4NF, THF, rt, 12 h (82% for 26 and 89% for 27, 2 steps); (c) H2,
Lindlar cat., quinoline, MeOH, rt, 30 min; (d) acetone, 80 ꢁC, 4 h (43%
for 30 and 40% for 31, 2 steps); (e) HCl(g), EtOH, rt, 30 min (60% for 5
and 55% for 6.
Besides the in vitro screening, the in vivo calcemic effects
of the compound 3 and 4 were evaluated. Both ana-
logues were much less calcemic than 1a,25-(OH)2-D3
even at 100-fold higher doses than 1a,25-(OH)2-D3
(0.1 lg/kg/d) (Fig. 4).
NH
2
H
H
NH
2
H
NH
H
2
NH
2
5
6
Chart 1.
to bind to the pig intestinal vitamin D receptor in com-
parison to the natural hormone. In addition, inhibition
of MCF-7 cell proliferation and the induction of HL
60 cell differentiation by 1a,25-(OH)2-D3 and its ana-
logues as well as their calcemic effects were measured.
The relative affinity of the analogues for the VDR was
calculated from their concentration required for 50%
displacement of [3H]1a,25-(OH)2-D3 from the receptor
protein compared with the activity of 1a,25-(OH)2-D3
(assigned a value of 100 by definition). These analogues
Figure 2. Antiproliferating effects of mono- and diamino 1a,25-(OH)2-
D3 analogues on breast cancer MCF-7 cells. 1a,25-(OH)2-D3 (d); 3
(s); 4 (j); 5 (h); 6 (m); 30 (n); 31 (.).

932
D. Oves et al. / Bioorg. Med. Chem. 14 (2006) 928–937
Figure 3. Prodifferentiating effects of mono- and diamino 1a,25-
(OH)2-D3 analogues on promyelocytic HL 60 leukemia cells. 1a,25-
(OH)2-D3 (d); 3 (s); 4 (j); 5 (h); 6 (m); 30 (n); 31 (.).
4. Conclusions
We have described the synthesis and biological evalua-
tion of novel 1-, 3-, and 1,3-diamino-substituted vitamin
D3 analogues. Key feature of these approaches is the
excellent yield and stereoselectivity of the Mitsunobu
reaction, which provide direct introduction of the amino
group at C-1 with total inversion of the configuration.
The conversion of alcohol at C-3 to inverted amine
was best achieved by activation of the hydroxyl group
as mesylate followed by reaction with NaN3. Biological
assays on diamino 5 and 6 as well as monoamino 3, 4,
30, and 31 vitamin D3 derivatives have shown poor
binding to VDR. The most active compound in the inhi-
bition of MCF-7 cell proliferation and HL 60 cell differ-
entiation was 1a-amino analogue 3. In vivo calcemic
effects of derivatives 3 and 4 were evaluated showing
very low calcemic effect.
Figure 4. In vivo biological activity of 1- and 3-monoamino 1a,25-
(OH)2-D3 analogues determined by body weight (A) as a marker of
toxicity and measuring serum (B) and urine (C) calcium levels in mice
after intraperitoneally injections after seven consecutive days. Mice
were injected with vehicle (arachis oil), 1a,25-(OH)2-D3 (0.1 lg/kg/d)
or analogues 3 and 4 (10 lg/kg/d).
5. Experimental section
5.1. General spectroscopic and experimental data
Melting points were taken on samples in open capillary
tubes and are uncorrected. IR spectra were recorded on
an Infrared Fourier Transform spectrophotometer using
KBr pellets. Flash chromatography was performed
detector and a Spherisorb W, 5 lm silica gel column,
250 · 10 mm.
5.2. Synthesis of 1a,3b-diamino-25-hydroxy-3-deoxyvita-
min D3 (5)
1
using silica gel 60 (230–400 mesh). H, 13C NMR, and
DEPT were obtained using AC-300 (1H, 300.13 MHz
and 13C, 75.5 MHz) or DPX 300 (1H, 300.13 MHz and
13C, 75.5 MHz) spectrometers for routine experiments.
AMX-400 spectrometer operating at 400.13 and
A solution of 30 (30.7 mg, 0.05 mmol) in 1.5 mL of eth-
anol was added to a solution of HCl/EtOH [15 mL, gen-
erated by bubbled HCl (g) in EtOH]. The solution was
stirred for 0.5 h, and then solvent was removed under re-
duced pressure. The residue was purified by semiprepar-
ative TLC [5% NH3(aq)/MeOH] to afford a white solid
1
100.61 MHz for H and 13C, respectively, was used for
1
1
the acquisition of H–1H homonuclear and H–13C het-
eronuclear correlation experiments. The chemical shifts
are given in delta (d) values and the coupling constants
(J) in Hertz (Hz). ES+ was used to record mass spectra
(MS). Microanalyses were performed on a Perkin-Elmer
model 2400 instrument. HPLC was performed using UV
1
(60% yield). H NMR (MeOH-d4, 300.13 MHz): d 0.75
(s, 3H, H18), 1.15 (d, 3H, H21, JHH 6.2 Hz), 1.36 (s,
3
6H, H26 + H27), 1.2–2.4 (m, 21H, 2H2 + 2H9 + 2H11
2H12 + H14 + 2H15 + 2H16 + H17 + H20 + 2H22 + 2H23
+
+

D. Oves et al. / Bioorg. Med. Chem. 14 (2006) 928–937
933
2
2H24), 2.73 (d, 1H, H4, JHH 13.3 Hz), 3.07 (d, 1H, H4,
5.6. Synthesis of (3S,5S)-5-acetoxy-1-ethynyl-2-methyl-3-
phthalimidocyclohex-1-ene (11)
2JHH 13.3 Hz), 3.40 (m, 1H, H3), 3.76 (m, 1H, H1), 5.41
(m, 1H, H19), 6.25 (d, 1H, H7, JHH 10.5 Hz) and 6.59
3
3
(d, 1H, H6, JHH 10.5 Hz); MS (ES+, m/z): 415
A similar procedure as that described for 10 afforded 11
as a white solid (82% yield). Mp: 105–107 ꢁC; IR (KBr):
t 3256, 2932, 2104, 1774, 1710, 1606, and 1468 cmꢀ1; 1H
NMR (CDCl3, 300.13 MHz): d 1.74 (s, 3H, H9), 2.04 (s,
3H, H11), 2.18 (m, 1H, H4), 2.42 (m, 2H, H4 + H6), 2.63
(d, 1H, H6, 2JHH 15.1 Hz), 3.14 (s, 1H, H8), 5.0 (m, 2H,
H3 + H5) and 7.80 (m, 4H, H14 + H15); 13C NMR
(CDCl3, 75.5 MHz): d 17.2 (C9), 21.0 (C11), 31.8, 34.8
(C4 + C6), 49.9 (C3), 67.6 (C5), 81.0 (C8), 82.4 (C7),
114.7 (C1), 123.3 (C14), 131.5 (C13), 134.1 (C15), 139.4
(C2), 167.4 (C12) y 170.1 (C10); MS (ES+, m/z): 346
[(M+H)+, 100%].
5.3. Synthesis of 1b,3a-diamino-25-hydroxy-3-deoxyvita-
min D3 (6)
A similar procedure as that described for 5 afforded 6 as
a white solid (55% yield). 1H NMR (MeOH-d4,
300.13 MHz): d 0.76 (s, 3H, H18), 1.15 (d, 3H, H21,
3JHH 6.0 Hz), 1.36 (s, 6H, H26 + H27), 1.2–2.4 (m,
21H,2H2 + 2H9 + 2H11 + 2H12 + H14 + 2H15 + 2H16 +
H17 + H20 + 2H22 + 2H23 + 2H24), 2.71 (dd, 1H, H4,
[(M+Na)+, 100%] and 362 [(M+K)+, 20%];
20
D
3
2
2JHH 13.1 Hz, JHH 4.3 Hz), 3.07 (dd, 1H, H4, JHH
13.1 Hz, JHH 4.3 Hz), 3.36 (m, 1H, H3), 3.76 (m, 1H,
½aꢁ = ꢀ102.3 (c 1.0, CHCl3); Anal. Calcd (%) for
3
C19H17NO4: C, 70.58; H, 5.30; N, 4.33. Found: C,
70.3; H, 5.5; N, 4.3.
3
H1), 5.40 (m, 1H, H19), 6.25 (d, 1H, H7, JHH 11.4 Hz)
and 6.58 (d, 1H, H6, JHH 11.4 Hz); MS (ES+, m/z):
3
415 [(M+H)+, 100%].
5.7. Synthesis of (3S,5S)-1-ethynyl-5-hydroxy-2-methyl-
3-phthalimidocyclohex-1-ene (12)
5.4. Synthesis of (3R,5S)-3,5-dihydroxy-1-ethynyl-2-
methylcyclohex-1-ene (9)
To a solution of 11 (200 mg, 0.619 mmol) in MeOH
(12 mL) K2CO3 (85 mg, 0.619 mmol) was added. The
reaction was stirred at 0 ꢁC for 1.5 h and then treated
with Dowex 50WX4-400 (200–400 mesh). Solution was
filtered, solvents were evaporated under reduced pres-
sure, and the residue was purified by flash chromatogra-
phy (30% EtOAc/hexane). White solid (71% yield). Mp:
178–180 ꢁC (decomp.). IR (KBr): t3490, 3268, 2922,
Acetate 8 (194 mg, 1 mmol) was treated with 0.2 M sodi-
um methoxide in methanol (10 mL). After stirring for
2.5 h at 0 ꢁC, the solution was acidified with Dowex
50X4-400 resin (200–400 mesh). After the removal of
the resin by filtration, the solution was evaporated,
and the residue was purified by flash chromatography
(EtOAc) to give the deprotected diol (96% yield). This
compound was previously reported.17a
2094, 1768, 1700, and 1460 cmꢀ1 1H NMR (CDCl3,
;
300.13 MHz): d 1.77 (s, 3H, H9), 2.1–2.65 (m, 4H,
H4 + H6), 3.17 (s, 1H, H8), 4.03 (m, 1H, H5), 4.96 (m,
1H, H3) and 7.8 (m, 4H, H12 + H13); 13C NMR (CDCl3,
75.5 MHz): d 17.6 (C9), 35.8, 38.4 (C4 + C6), 49.4 (C3),
64.9 (C5), 80.9 (C8), 82.9 (C7), 115.4 (C1), 123.4 (C12),
131.5 (C11), 134.2 (C13), 138.8 (C2) y 167.9 (C10); MS
5.5. Synthesis of (3S,5R)-1-ethynyl-2-methyl-3,5-
bis(phthalimido)cyclohex-1-ene (10)
5.5.1. From imide 9. Phthalimide (108 mg, 0.74 mmol),
Ph3P (193 mg, 0.74 mmol) and diethylazodicarboxilate
(0.115 mL, 0.74 mmol) were added to a stirred solution
of 9 (45 mg, 0.30 mmol) in THF (10 mL). The mixture
was stirred for 3 h at room temperature and then evap-
orated under reduced pressure to leave a residue which
was purified by flash chromatography (30% EtOAc/hex-
ane) to afford a white solid (17% yield).
(ES+, m/z): 304 [(M+Na)+, 100%] and 320 [(M+K)+,
20
D
25%]; ½aꢁ = ꢀ117.5 (c 0.9, CHCl3); Anal. Calcd (%)
for C17H15NO3: C, 72.58; H, 5.37; N, 4.98. Found: C,
72.7; H, 5.5; N, 5.2.
5.8. Synthesis of (3S,5R)-3,5-bis[(tert-butoxycarbon-
yl)amino]-1-ethynyl-2-methylcyclohex-1-ene (13)
5.5.2. From imide 12. A similar procedure as described
in Section 5.5.1 afforded 10 as a white solid (63%
yield). Mp: 199–201 ꢁC (decomp.); IR (KBr): t 3269,
5.8.1. From diimide 10. Compound 10 (205 mg,
0.5 mmol) was dissolved in 5 mL of a 8 M solution of
MeNH2 in ethanol under nitrogen. The mixture was stir-
red for 24 h at room temperature and then 12 h at 65 ꢁC.
Solvent was removed under reduced pressure to leave a
residue which was dissolved in CHCl3 (10 mL). Di-tert-
butyl-dicarbonate (350 mg, 1.6 mmol) and aqueous sat-
urated solution of NaHCO3 (0.5 mL) were added and
the mixture was stirred for 24 h at room temperature.
The mixture was extracted with CHCl3 (3· 10 mL)
and the combined organic layers were dried and evapo-
rated under reduced pressure. The crude was purified by
flash chromatography (25% EtOAc/hexane) to afford a
white solid (45% yield).
3064, 2916, 2088, 1744, 1711, 1612, and 1468 cmꢀ1
;
1H NMR (CDCl3, 300.13 MHz): d 1.88 (s, 3H, H9),
1.96 (m, 1H, H4), 2.58 (dd, 1H, H4, JHH 17.0, JHH
2
3
2
5.7 Hz), 3.0 (ddd, 1H, H6, JHH 13.5, JHH 13.5,
3
4JHH 6.9 Hz), 3.12 (m, 1H, H6), 3.18 (s, 1H, H8),
3
4.97 (ad, 1H, H3, JHH 6.3 Hz), 5.13 (m, 1H, H5) and
7.76 (m, 8H, H12 + H13 + H16 + H17); 13C NMR
(CDCl3, 75.5 MHz): d 18.7 (C9), 32.2, 32.5 (C4 + C6),
43.5 (C5), 49.5 (C3), 81.3 (C8), 82.5 (C7), 117.9 (C1),
123.1, 123.4 (C12 + C16), 131.5, 131.7 (C11 + C15),
133.9, 134.1 (C13 + C17), 136.5 (C2) and 168.1
(C10 + C14); MS (ES+, m/z): 433 [(M+Na)+, 45%] and
449 [(M+K)+, 100%]; Anal. Calcd (%) for
C25H18N2O4: C, 73.16; H, 4.42; N, 6.83. Found: C,
73.3; H, 4.6; N, 6.7.
5.8.2. From azide 17. A solution of NaOH (aq) (0.4 mL,
1 M, previously degassed), Me3P (0.543 mL, 1 M
in THF) and di-tert-butyl-dicarbonate (237 mg,

934
D. Oves et al. / Bioorg. Med. Chem. 14 (2006) 928–937
1.086 mmol) was added to a stirred solution of azide 17
(100 mg, 0.362 mmol) in THF (4 mL). The mixture was
stirred for 24 h at room temperature and then brine was
added. The mixture was extracted with CHCl3 and the
organic layers were dried and evaporated under reduced
pressure lo leave a residue which was purified by flash
chromatography (25% EtOAc/hexane) to afford a white
solid (82% yield). Mp: 208–210 ꢁC (decomp.). IR (KBr):
t3352, 3284, 2970, 2087, 1680, 1509, 1445, and
IR (KBr): t3367, 3282, 2986, 2944, 2098, 1682, 1527,
1
and 1420 cmꢀ1; H NMR (MeOH-d4, 300.13 MHz): d
1.64 (s, 9H, H13), 2.09 (s, 3H, H9), 2.15 (m, 1H, H4),
2.43 (m, 1H, H4), 2.65 (m, 1H, H6), 2.8 (m, 1H, H6),
3.3 (s, 3H, H10), 3.73 (s, 1H, H8), 4.47 (m, 1H, H3)
and 5.12 (m, 1H, H5); 13C NMR (MeOH-d4,
75.5 MHz):
d 18.2 (C9), 28.6 (C13), 36.1, 36.9
(C4 + C6), 38.3 (C10), 50.1 (C3), 76.3 (C5), 80.3 (C12),
82.4 (C8), 83.3 (C7), 114.0 (C1), 143.4 (C2) and 157.9
1391 cmꢀ1; H NMR (CDCl3, 300.13 MHz): d 1.45 (s,
(C11); MS (ES+, m/z): 352 [(M+Na)+, 100%] and 368
1
20
[(M+K)+, 55%]; ½aꢁ = ꢀ35.8 (c 1.06, EtOH); Anal.
18H, H12 + H15), 1.73 (m, 1H, H4), 1.94 (m, 5H,
H4 + H6 + H9), 2.62 (d, 1H, H6, JHH 16.4 Hz), 3.09 (s,
1H, H8), 3.72 (s, 1H, H5), 4.28 (m, 1H, H3), 4.49 (m,
D
2
Calcd (%) for C15H23NO5S: C, 54.69; H, 7.04; N, 4.25.
Found: C, 54.8; H, 7.3; N, 4.1.
3
1H, NH) and 4.65 (d, 1H, NH, JHH 8.9 Hz); 13C
NMR (CDCl3, 75.5 MHz):
d
18.8 (C9), 28.3
5.12. Synthesis of (3S,5R)-5-azido-3-[(tert-butoxycar-
bonyl)amino]-1-ethynyl-2-methylcyclohex-1-ene (17)
(C12 + C15), 35.7 (C4), 36.4 (C6), 42.7 (C5), 49.2 (C3),
79.5, 79.6 (C11 + C14), 80.6 (C8), 82.6 (C7), 115.5 (C1),
141.3 (C2) y 155.1, 155.3 (C10 + C13); MS (ES+, m/z):
NaN3 (228 mg, 3.5 mmol) was added to a stirred solu-
tion of 16 (385 mg, 1.17 mmol) in anhydrous DMF
(10 mL). The solution was stirred for 6 h at 65 ꢁC, and
then, the solvent was removed under reduced pressure
to leave a residue, which was purified by flash chroma-
tography (10% EtOAc/hexane). White solid (79% yield).
Mp: 89–91 ꢁC; IR (KBr): t3310, 2976, 2931, 2094, 1673,
1529, and 1453 cmꢀ1; 1H NMR (CDCl3, 300.13 MHz): d
1.43 (s, 9H, H12), 1.75 (m, 1H, H4), 1.92 (s, 3H, H9), 2.0
(m, 1H, H4), 2.15 (dd, 1H, H6, 2JHH 16.8, 3JHH 8.9 Hz),
20
373 [(M+Na)+, 100%]; ½aꢁ = ꢀ97.6 (c 0.85, CHCl3);
D
Anal. Calcd (%) for C19H30N2O4: C, 65.12; H, 8.63;
N, 7.99. Found: C, 65.3; H, 8.9; N, 7.7.
5.9. Synthesis of (3S,5S)-3-[(tert-butoxycarbonyl)amino]-
1-ethynyl-5-hydroxy-2-methylcyclohex-1-ene (14)
A similar procedure as that described in Section 5.5.1
afforded 14 as a white solid (88% yield). Mp: 188–
190 ꢁC (decomp.); IR (KBr): t3346, 2985, 2937, 2087,
2
3
2.49 (dd, 1H, H6, JHH 16.8, JHH 4.0 Hz), 3.11 (s, 1H,
H8), 3.6 (m, 1H, H5), 4.28 (m, 1H, H3) and 4.61 (d,
1682, 1519, 1458, and 1369 cmꢀ1; H NMR (MeOH-
1
3
d4, 300.13 MHz): d 1.64 (s, 9H, H12), 1.77 (m, 1H,
H4), 2.05 (s, 3H, H9), 2.25 (m, 2H, H4 + H6), 2.57 (d,
1H, NH, JHH 8.9 Hz); 13C NMR (CDCl3, 75.5 MHz):
d 18.7 (C9), 28.2 (C12), 34.5, 34.7 (C4 + C6), 49.0 (C3),
53.1 (C5), 79.7 (C11), 81.0 (C8), 82.2 (C7), 114.8 (C1),
2
1H, H6, JHH 16.5 Hz), 3.63 (s, 1H, H8), 4.07 (m, 1H,
H5) and 4.37 (m, 1H, H3); 13C NMR (MeOH-d4,
141.3 (C2) and 155.1 (C10); MS (ES+, m/z): 299
20
[(M+Na)+, 100%]; ½aꢁ = ꢀ127.5 (c 0.83, EtOH); Anal.
75.5 MHz):
(C4 + C6), 50.9 (C3), 65.8 (C5), 80.1 (C11), 81.5 (C8),
d
14.3 (C9), 28.7 (C12), 38.8, 39.5
D
Calcd (%) for C14H20N4O2: C, 60.85; H, 7.30; N,
20.28. Found: C, 60.6; H, 7.5; N, 20.5.
84.2 (C7), 115.0 (C1), 143.4 (C2) and 157.9 (C10); MS
(ES+, m/z): 274 [(M+Na)+, 100%] and 290 [(M+K)+,
20
22%]; ½aꢁ = ꢀ175.4 (c 0.73, CHCl3); Anal. Calcd (%)
5.13. Synthesis of (3R,5R)-5-acetoxy-1-ethynyl-2-methyl-
3-phthalimidocyclohex-1-ene (20)
D
for C14H21NO3: C, 66.91; H, 8.42; N, 5.57. Found: C,
67.1; H, 8.7; N, 5.6.
A similar procedure as that described for 11 afforded 20
20
D
5.10. Synthesis of (3S,5R)-3-[(tert-butoxycarbonyl)ami-
no]-1-ethynyl-2-methyl-5-phthalimidocyclohex-1-ene (15)
as a white solid (84% yield). ½aꢁ = +98.0 (c 0.8, CHCl3).
5.14. Synthesis of (3R,5R)-3-[(tert-butoxycarbonyl)ami-
no]-1-ethynyl-5-hydroxy-2-methylcyclohex-1-ene (21)
A similar procedure as that described in Section 5.5.1
afforded 15 as a white solid (30% yield). 1H NMR
(CDCl3, 300.13 MHz): d 1.41 (s, 9H, C Me3), 1.9
(m, 1H, H4), 1.97 (s, 3H, H9), 2.33 (m, 1H, H4), 2.72
(m, 1H, H6), 2.85 (m, 1H, H6), 3.08 (s, 1H, H8), 4.4
(m, 2H, H3 + H5), 4.78 (m, 1H, NH) and 7.7 (m, 4H,
H12 + H13).
A similar procedure as that described for 14 afforded 21 as
20
D
a white solid (77% yield). ½aꢁ = +177.7 (c 0.93, CHCl3).
5.15. Synthesis of (3R,5R)-3-[(tert-butoxycarbonyl)ami-
no]-1-ethynyl-5-metanesulfonyloxy-2-methylcyclohex-1-
ene (22)
5.11. Synthesis of (3S,5S)-3-[(tert-butoxycarbonyl)ami-
no]-1-ethynyl-5-metanesulfonyloxy-2- methylcyclohex-1-
ene (16)
A similar procedure as that described for 16 afforded 22
20
D
as a white solid (81% yield). ½aꢁ = +39.5 (c 0.97,
EtOH).
Et3N (0.420 mL, 3 mmol) and MsCl (0.230 mL, 3 mmol)
were added to a stirred solution of 14 (375 mg,
1.5 mmol) in anhydrous CH2Cl2 (20 mL). The solution
was stirred for 3 h at 0 ꢁC. Then, the solvent was re-
moved under reduced pressure to leave a residue, which
was purified by flash chromatography (25% EtOAc/hex-
ane) to afford a white solid (94% yield). Mp: 135–137 ꢁC;
5.16. Synthesis of (3R,5S)-5-azido-3-[(tert-butoxycar-
bonyl)amino]-1-ethynyl-2-methylcyclohex-1-ene (23)
A similar procedure as that described for 17 afforded 23
20
D
as a white solid (75% yield). ½aꢁ = +139.7 (c 1.09,
EtOH).

D. Oves et al. / Bioorg. Med. Chem. 14 (2006) 928–937
935
5.17. Synthesis of (3R,5S)-3,5-bis[(tert-butoxycarbon-
yl)amino]-1-ethynyl-2-methylcyclohex-1-ene (24)
635 [(M+Na)+, 100%]; Anal. Calcd (%) for
C37H60N2O5: C, 72.51; H, 9.87; N, 4.57. Found: C,
72.2; H, 9.6; N, 4.7.
A similar procedure as that described for 13 afforded 24
20
D
as a white solid (79% yield). ½aꢁ = +92.2 (c 0.5, CHCl3).
5.20. Synthesis of 1a,3b-bis[(tert-butoxycarbonyl)amino]-
25-hydroxy-3-deoxyprevitamin D3 (28)
5.18. Synthesis of 1a,3b-bis[(tert-butoxycarbonyl)amino]-
25-hydroxy-6,7-didehydro-3-deoxyprevitamin D3 (26)
A flask containing Lindlar catalyst (74 mg) was exposed
to a positive pressure of hydrogen gas (balloon). Deox-
ygenated MeOH (1 mL) was added, and to this suspen-
sion of quinoline (0.205 mL, 0.17 M in hexane) and 26
(35 mg, 0.058 mmol) in MeOH (4 mL) was 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.
CuI (2.8 mg, 0.015 mmol), Pd(PPh3)2(OAc)2 (3.4 mg,
0.067 mmol), and Et2NH (1.2 mL) were added to a stir-
red solution of 25 (73 mg, 0.149 mmol) and 13 (57.4 mg,
0.164 mmol) in DMF (1.2 mL). The reaction mixture
was stirred at room temperature for 1 h under nitrogen
and then poured into water and extracted with diethyl
ether (3· 10 mL). The combined ether layers were dried
and concentrated to give a crude, which was sufficiently
pure for the next step. Although it was possible to purify
this compound by flash chromatography, it decomposed
in a few hours. TBAF (0.3 mL, 1 M in THF) was added
to a solution of the crude in THF (3 mL) at 0 ꢁC, and
the reaction was stirred for 12 h at room temperature.
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 and evaporated under reduced pressure.
The crude was purified by flash chromatography to af-
ford a white solid (82% yield). Mp: 104–106 ꢁC; IR
(KBr): t3451, 3366, 2971, 1700, 1513, 1359, and
5.21. Synthesis of 1b,3a-bis[(tert-butoxycarbonyl)amino]-
25-hydroxy-3-deoxyprevitamin D3 (29)
A similar procedure as that described for 28 afforded 29.
5.22. Synthesis of 1a,3b-bis[(tert-butoxycarbonyl)amino]-
25-hydroxy-3-deoxyvitamin D3 (30)
A solution of the crude previtamin 28 (0.058 mmol) in
anhydrous acetone (4 mL) was placed in a screw-capped
vial and heated at 80 ꢁC for 4 h. Then, solvent was evap-
orated under reduced pressure lo leave a residue, which
was purified by flash chromatography (20% EtOAc/hex-
ane). The compound was purified further by HPLC
1
1239 cmꢀ1; H NMR (CDCl3, 300.13 MHz): d 0.67 (s,
3
3H, H18), 0.93 (d, 3H, H21, JHH 6.3 Hz), 1.20 (s, 6H,
H26 + H27), 1.42 (s, 18H, 2 Me3CO), 1.89 (s, 3H, H19),
i
(2.7% PrOH/hexane, 3 mL/min, Spherisorb W, 5 lm,
250 · 10 mm) to afford a white solid (43% yield). Mp:
1.0–2.3 (m, 21H, 2H2 + H4 + 2H11 + 2H12 + H14
2H15 + 2H16 + H17 + H20 + 2H22 + 2H23 + 2H24 + OH),
+
105–107 ꢁC; IR (KBr): t3446, 2953, 1692, 1502, 1363
and 1243 cmꢀ1
0.54 (s, 3H, H18), 0.92 (d, 3H, H21, JHH 6.3 Hz), 1.20
(s, 6H, H26 + H27), 1.44 (s, 18H, 2 Me3CO), 1.0-2.2
;
1H NMR (CDCl33, 300.13 MHz): d
2
2.56 (d, 1H, H4, JHH 14.4 Hz), 3.73 (m, 1H, H3), 4.26
3
(m, 1H, H1), 4.61 (d, 1H, NH, JHH 7.5 Hz), 4.78 (d,
3
3
1H, NH, JHH 9.1 Hz) and 5.94 (d, 1H, H9, JHH
2.6 Hz); 13C NMR (CDCl3, 75.5 MHz): d 10.9, 18.5,
18.8, 20.7, 24.0, 25.0, 27.8, 28.2, 28.3, 29.0, 29.2, 35.7,
36.0, 36.2, 36.7, 41.7, 42.7, 44.2, 49.2, 49.9, 54.5, 70.8,
79.3, 87.0, 93.2, 116.7, 122.2, 133.6, 138.1, 155.1 and
155.3; MS (ES+, m/z): 635 [(M+Na)+, 100%]; Anal.
Calcd (%) for C37H60N2O5: C, 72.51; H, 9.87; N, 4.57.
Found: C, 72.6; H, 10.0; N, 4.7.
(m, 22H, 2H2 + 2H9 + 2H11 + 2H12 + H14 + 2H15
2H16 + H17 + H20 + 2H22 + 2H23 + 2H24 + OH), 2.59
+
2
2
(d, 1H, H4, JHH 12.5 Hz), 2.79 (d, 1H, H4, JHH
12.5 Hz) 3.88 (m, 1H, H3), 4.30 (m, 1H, H1), 4.60 (m,
2H, 2NH), 4.92 (m, 1H, H19), 5.23 (m, 1H, H19) 5.94
3
3
(d, 1H, H7, JHH 11.1 Hz) and 6.34 (d, 1H, H6, JHH
11.1 Hz); 13C NMR (CDCl3, 75.5 MHz): d 11.9, 18.7,
20.7, 22.1, 23.5, 27.5, 28.3, 29.0, 29.1, 29.3, 36.0, 36.3,
38.8, 40.4, 43.0, 44.3, 45.9, 46.2, 51.6, 56.3, 56.4, 71.0,
79.4, 113.2, 116.9, 124.9, 132.4, 143.4, 144.4, 154.9 and
155.0; MS (ES+, m/z): 637 [(M+Na)+, 100%] and 653
[(M+K)+, 5%]; Anal. Calcd (%) for C37H62N2O5: C,
72.27; H, 10.16; N, 4.56. Found: C, 72.4; H, 10.4; N, 4.3.
5.19. Synthesis of 1b,3a-bis[(tert-butoxycarbonyl)amino]-
25-hydroxy-6,7-didehydro-3-deoxyprevitamin D3 (27)
A similar procedure as that described for 26 afforded 27
as a white solid (89% yield). Mp: 112–114 ꢁC; IR (KBr):
t3344, 2972, 1709, 1691, 1529, 1501, and 1359 cmꢀ1; 1H
NMR (CDCl3, 300.13 MHz): d 0.68 (s, 3H, H18), 0.93
5.23. Synthesis of 1b,3a-bis[(tert-butoxycarbonyl)amino]-
25-hydroxy-3-deoxyvitamin D3 (31)
3
(d, 3H, H21, JHH 6.4 Hz), 1.20 (s, 6H, H26 + H27),
1.43 (s, 18H, 2 Me3CO), 1.89 (s, 3H, H19), 1.0–2.3 (m,
21H,2H2 + H4 + 2H11 + 2H12 + H14 + 2H15 + 2H16 + H
17 + H20 + 2H22 + 2H23 + 2H24 + OH), 2.58 (d, 1H, H4,
2JHH 14.4 Hz), 3.71 (m, 1H, H3), 4.25 (m, 1H, H1),
A similar procedure as that described for 30 afforded 31
as a white solid (40% yield). Mp: 112–114 ꢁC; IR (KBr):
1
3344, 2972, 1709, 1691, 1529, 1501, and 1359 cmꢀ1; H
NMR (CDCl3, 300.13 MHz): d 0.68 (s, 3H, H18), 0.93
(d, 3H, H21, JHH 6.4 Hz), 1.20 (s, 6H, H26 + H27),
3
3
3
4.52 (d, 1H, NH, JHH 6.7 Hz), 4.67 (d, 1H, NH, JHH
3
7.6 Hz) and 5.94 (d, 1H, H9, JHH 2.8 Hz); 13C NMR
1.43 (s, 18H, 2 Me3CO), 1.89 (s, 3H, H19), 1.0–2.3 (m,
21H,2H2 + H4 + 2H11 + 2H12 + H14 + 2H15 + 2H16 + H
17 + H20 + 2H22 + 2H23 + 2H24 + OH), 2.58 (d, 1H, H4,
2JHH 14.4 Hz), 3.71 (m, 1H, H3), 4.25 (m, 1H, H1),
(CDCl3, 75.5 MHz): d 10.9, 18.6, 18.8, 20.7, 24.1, 25.1,
27.9, 28.3, 29.1, 29.2, 35.8, 36.0, 36.3, 36.8, 41.7, 42.9,
44.2, 49.3, 49.9, 54.6, 70.9, 79.4, 87.0, 93.3, 116.8,
122.2, 133.7, 138.1, 155.1, and 155.3; MS (ES+, m/z):
3
3
4.52 (d, 1H, NH, JHH 6.7 Hz), 4.67 (d, 1H, NH, JHH

936
D. Oves et al. / Bioorg. Med. Chem. 14 (2006) 928–937
3
7.6 Hz) and 5.94 (d, 1H, H9, JHH 2.8 Hz); 13C NMR
(CDCl3, 75.5 MHz): d 10.9, 18.6, 18.8, 20.7, 24.1, 25.1,
27.9, 28.3, 29.1, 29.2, 35.8, 36.0, 36.3, 36.8, 41.7, 42.9,
44.2, 49.3, 49.9, 54.6, 70.9, 79.4, 87.0, 93.3, 116.8,
122.2, 133.7, 138.1, 155.1 y 155.3; MS (ES+, m/z): 635
[(M+Na)+, 100%]; Anal. Calcd (%) for C37H62N2O5:
C, 72.27; H, 10.16; N, 4.56. Found: C, 72.5; H, 10.3;
N, 4.4.
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6. In vitro and in vivo biological evaluation
6.1. Cell proliferation assays
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, analogues or
vehicle as described previously.20
6.2. Cell differentiation assays
Differentiation of promyeolocytic HL 60 leukemia cells
(ATCC) was measured by the nitro blue tetrazolium
(NBT) reduction assay after a 72 h incubation period
in the presence of 1a,25-(OH)2-D3, analogues or
vehicle.20
6.3. In vivo calcemic activity
NMRI mice were obtained from the Proefdierencentrum
of Leuven (Belgium) and fed with a vitamin D-replete
diet (1% calcium, 1% phosphate, and 2500 U vitamin
D/kg; Hope Farms, Woerden, The Netherlands). The
calcemic effects of the analogues were tested by daily
injections intraperitoneally of 1a,25-(OH)2-D3 (0.1 lg/
kg/d), analogues (10 lg/kg/d) or vehicle (arachis oil)
for seven consecutive days. Serum and urinary calcium
were measured as calcemic parameters using commer-
cially available kit (Sigma Diagnostics).
´
8. Oves, D.; Ferrero, M.; Fernandez, S.; Gotor, V. J. Org.
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Acknowledgments
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This work has been supported by the Spanish Ministerio
´
de Educacion y Ciencia (MEC) (Project CTQ-2004-
04185). S.F. thanks MEC for a personal grant (Ramon
´
y Cajal Program).
Supplementary data
Supplementary data associated with this article can be
found in the online version at doi:10.1016/
j.bmc.2005.09.009.
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