Brief Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 6161
C-3 resulted in a decreased affinity for the VDR (4 vs 6,
Table 1). In the case of the epoxy analogues, derivative 10
retained a significant binding affinity (25% with respect to
the natural hormone), but the 1R,2R-epoxy analogue 9 was
devoid of any affinity for VDR. These results indicated that
vitamins with the axial orientation of the 1R-hydroxyl group
exhibited the most potent affinity to VDR.
2β-isomer 4. In vivo calcemic effects of synthesized analogues
were evaluated showing very low calcemic effect.
Experimental Section
Chemistry. Synthesis of 11b,7a 11c,7b 20,7a and 259 were pre-
viously reported. The purity of compounds was determined in all
cases as >99% by HPLC analysis.
When hDBP binding of the 2-hydroxy analogues were
compared, the 1R,2β,3R isomer 6 and the mixture 7 þ 8
proved to possess more affinity. Interestingly, introduction
of an epoxy function in the A-ring increased the binding
potency. Both oxirane derivatives, 9 and 10, showed 45%
and 38% binding afffinity for hDBP, respectively, relative to
the parent hormone. The lack of the C-1 hydroxyl group in 9
was not essential for binding. These epoxy analogues ex-
hibited large differences in hDBP binding compared with the
2-hydroxy derivatives with the same stereochemistry in the
A-ring (9 vs 3 and 10 vs 4, Table 1).
Next, the ability of these compounds to inhibit proliferation
was examined in MCF-7 breast cancer cells. The 2R-hydroxy
analogue 3 and the 2β,3β-epoxy derivative 10, which showed
the highest affinity to the VDR, displayed the highest potency
among the tested compounds. Thus, at a concentration of 10-6
M, compound 3 can inhibit cell proliferation by 60%, which is
comparable with 1R,25-(OH)2-D3 (p S7, Figure S7, SI). How-
ever, this derivative was 5 times less potent than 1R,25-(OH)2-
D3 at the EC50 concentration. Similar results were observed
with the epoxy analogue 10. In contrast, epoxy 9 was almost
unable to inhibit cell proliferation. In comparing antiproli-
ferative activity of the 2R- and 2β-hydroxy derivatives (3
and 4), the 2R-analogue 3 exhibited a 4-fold increase in
potency.
Comparison of the biological activity of these derivatives
with our previous results of the corresponding 6-s-cis
counterparts6,10 possessing the same functionalities and
stereochemistries showed that the 6-s-trans conformers
displayed markedly increased binding affinity to VDR
and hDBP and increased potency to inhibit MCF-7 cell
proliferation.
Besides the in vitro screening, the in vivo calcemic effects
of the compounds 3, 9, and 10 were evaluated. All tested
analogues were less calcemic than 1R,25-(OH)2-D3 (p S7,
Figure S8, SI). The tetraol analogue 3 demonstrated the
weakest calcemic activity in mice because a daily injection of
a dose exceeding 1000-fold the maximal tolerable dose of
1R,25-(OH)2-D3 (0.1 μg/kg/day) resulted in a smaller in-
crease (13%) in serum calcium levels than 1R,25-(OH)2-D3
(27%).
General Procedure for 3-8. To a solution of 19 (43 mg, 0.078
mmol) in anhyd THF (260 μL) at -50 °C was added dropwise
LHMDS (78 μL, 1.0 M in THF, 0.078 mmol) and the mixture
was stirred at the same temperature for 2 h. Next, a solution of
the corresponding ketone 14 (23 mg, 0.047 mmol) in anhyd THF
(310 μL) was added dropwise to the mixture. After being stirred
at -50 °C during 5 h, the reaction mixture was poured into a satd
aq solution of NH4Cl and extracted with Et2O. The combined
organic fractions were dried (Na2SO4), filtered, and concen-
trated to give a crude, which was dissolved in anhyd MeOH
(1.2 mL) and cooled down at 0 °C. (-)-CSA (50 mg, 0.220 mmol)
was then added, and the mixture was stirred a room temperature
overnight. The reaction was quenched by adding a satd aq
solution of NaHCO3. The aqueous layer was extracted with
EtOAc. The combined organic fractions were dried (Na2SO4),
filtered, and concentrated to give a crude, which was purified by
˚
column chromatography using silica gel 60 A (32-63 μm) pH 7
and EtOAc as eluent. The diastereoisomers were isolated
by reverse-phase preparative HPLC (ODS-A column, 5 μm,
250 mm ꢀ 10 mm). Conditions: 4.5 mL/min, CH3CN/H2O
(45:55) for 3 and 4; 4 mL/min, CH3CN/H2O (50:50) for 5 and
6. Retention times: tR (3) = 28.6 min; tR (4) = 24.1 min; tR (5) =
20.7 min; tR (6) = 18.9 min.
1r,2r-Epoxy-25-hydroxy-19-nor-vitamin D3 (9) and 2β,3β-
Epoxy-1R,25-dihydroxy-3-deoxy-19-nor-vitamin D3 (10). Cou-
pling of 19 (40 mg, 0.074 mmol) with 27 (20 mg, 0.044 mmol) and
subsequent desilylation was performed through the same pro-
cedure as described above for the synthesis of 3-8. To a solution
of the resulting crude in anhyd THF (500 μL) was added
dropwise DBU (10 μL, 0.066 mmol), and the reaction was
stirred at room temperature for 16 h. Solvent was concentrated,
and the crude was purified by column chromatography using
˚
silica gel 60 A (32-63 μm) pH 7 and 10% hexane/EtOAc as
eluent. The diastereoisomeric mixture was separated by reverse-
phase preparative HPLC (ODS-A column, 5 μm, 250 mm ꢀ 10
mm, 5 mL/min, CH3CN/H2O 65:35). Retention times: tR (9) =
20.4 min. tR (10) = 23.4 min.
Acknowledgment. Financial support by the Spanish Gov-
ernment (MEC-07-CTQ-61126) and the Fonds voor We-
tenschappelijk Onderzoek (FWO grant G.0553.06 and
G.0587.09) are gratefully acknowledged. L.S.-A. thanks MEC
for a predoctoral fellowship.
Supporting Information Available: Experimental procedures,
conformational analyses, energy-minimized conformations, in
vitro and in vivo assays, NMR spectra; HPLC data. This
material is available free of charge via the Internet at http://
pubs.acs.org.
Conclusions
We have described the synthesis and biological evaluation
of 1R,25-dihydroxy-19-nor-vitamin D3 analogues substituted
at C-2 with a hydroxy group or possessing an epoxy function-
ality in the A-ring. To investigate structure-activity relation-
ships, different stereochemistry at C-1, C-2, and C-3 were
investigated. The structures of the analogues were determined
by analysis of their coupling constants in addition to COSY
and ROESY experiments. Results on VDR binding affinity
and MCF-7 cell proliferation revealed that the axial orienta-
tion of the 1R hydroxyl group is necessary for biological
activity. Comparison of the 2R- and 2β-hydroxy derivatives
of 1R,25-dihydroxy-19-nor-vitamin D3 showed that the 2R-
isomer 3 has a higher VDR affinity than the corresponding
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