D. Sawada et al. / Tetrahedron 72 (2016) 2838e2848
2847
was extracted with AcOEt, washed with brine, dried over Na2SO4,
filtered and concentrated. The residue was purified by flash col-
umn chromatography on silica gel (AcOEt/hexane¼1:3) to give
alcohols (430 mg, 84%) as an inseparable mixture. The above ob-
tained alcohols were dissolved in DMF (4 mL), and to the solution
3H), 1.22e1.30 (m, 2H), 1.33e1.64 (m, 12H), 1.82e1.91 (m, 3H),
1.99e2.05 (m, 1H), 2.18 (dd, J¼7.6, 12.9 Hz, 1H), 2.22e2.29 (m, 1H),
2.32 (dd, J¼7.6, 12.9 Hz, 1H), 2.48 (dd, J¼3.7, 13.7 Hz, 1H), 2.62 (dd,
J¼3.7, 13.7 Hz, 1H), 2.68 (dd, J¼7.3, 11.5 Hz, 1H), 4.05e4.11 (m, 2H),
6.09 (d, J¼11.2 Hz, 1H), 6.21 (d, J¼11.2 Hz, 1H); 13C NMR (150 MHz,
were added imidazole (180 mg, 2.65 mmol) and TESCl (222
mL,
CDCl3) d 20.0, 21.3, 22.0, 23.5, 27.4, 29.3, 29.4, 29.6, 32.8, 33.9, 34.5,
1.32 mmol) at 0 ꢀC and the whole mixture was stirred at room
temperature for 5 h. After the reaction was quenched by saturated
aqueous NaHCO3 at 0 ꢀC, the mixture was extracted with AcOEt,
washed with brine, dried over Na2SO4, filtered and concentrated.
The residue was purified by flash column chromatography on
silica gel (hexane:AcOEt¼30:1) to give compound 17 (24 mg, 33%
in five steps) and the isomer (7,8-trans-14-epi, 40 mg, 56% in five
36.9, 38.6, 42.2, 44.3, 44.9, 45.0, 49.3, 56.0, 67.3, 67.5, 71.1, 119.6,
123.6, 131.4, 143.4; HRFABMS calcd for C26H44NaO3 427.3183,
found 427.3182.
4.6. Human VDR binding assay
steps).
Binding affinity to hVDR was evaluated using a 1a,25(OH)2D3
27
Compound 17: [
a
]
D
þ33.7 (c 1.0, CHCl3); IR (neat) 2952, 2873,
assay kit (Polarscreen Vitamin D Receptor Competitor Assay, RED,
Cat. No. PV4569) purchased from Invitrogen. The solution of test
compound (1 mM in EtOH) was diluted to 10 times with DMSO.
The solution was diluted to 50 times with the assay buffer in-
cluded in the kit further. The solution was defined as the com-
pound solution. On the other hand, VDR/Fluoromone and VDR
RED, both of which are included in the kit, were diluted with the
assay buffer included in the kit so that the concentration of VDR/
Fluoromone was 2.8 nM, and that of VDR RED was 2 nM in the
mixture. The solution was defined as the VDR/Fluoromone and
VDR RED complex. To a 384 well Black plate (Coring, #3677) was
1652, 1472, 1380, 1333, 1236, 1153, 1041, 727 cmꢁ1
;
1H NMR
0.55 (q, J¼7.8 Hz, 6H), 0.68 (s, 3H), 0.82 (d,
(400 MHz, CDCl3)
d
J¼6.3 Hz, 3H), 0.93 (t, J¼7.8 Hz, 9H), 0.96e1.03 (m, 1H), 1.17e1.51
(m, 14H), 1.19 (s, 6H), 1.73e1.81 (m, 1H), 2.01e2.05 (m, 1H),
2.11e2.20 (m, 1H), 2.36 (m, 1H), 4.21 (dd, J¼7.8, 17.1 Hz, 1H), 4.25
(dd, J¼8.1, 17.1 Hz, 1H), 5.30 (dd, J¼7.8, 8.1 Hz, 1H), 7.53e7.62 (m,
2H), 7.96e7.99 (m, 1H), 8.18e8.21 (m, 1H); 13C NMR (100 MHz,
CD3OD)
d 6.8. 7.1, 19.8, 20.9, 21.8, 23.0, 27.4, 29.6, 29.8, 30.1, 32.4,
33.7, 34.3, 38.1, 45.1, 45.4, 49.4, 53.8, 55.7, 73.3, 107.7, 122.1, 125.2,
127.4, 127.7, 136.8, 152.1, 152.5, 165.7; HRFABMS calcd for
C
26H44NaNO3SiS2, 626.3128, found 626.3131.
added the compound solution (10
mL), and the VDR/Fluoromone
24
The isomer (7,8-trans-14-epi): [
(neat) 2956, 2873, 1653, 1472, 1380, 1332, 1236, 1151, 1041, 910,
729 cmꢁ1 1H NMR (400 MHz, CDCl3)
a]
þ31.5 (c 1.0, CHCl3); IR
and VDR RED complex (10 L) was added to each wells. The
m
D
mixture was incubated under 20e25 ꢀC for 2 h. The polarized
fluoresce in each wells was measured (384 nm, emission: 595 nm,
excitation: 535 nm, time: 250 ms/well). All compounds were
evaluated with N¼2 within the range from 10ꢁ6 M to 10ꢁ10 M. IC50
values were calculated by using the average of the measured
value. The activities of each compound were shown as relative
value, in which the activity of the natural hormone 1 was nor-
malized to 100%.
;
d
0.54 (q, J¼7.8 Hz, 6H),
0.70 (s, 3H), 0.74 (d, J¼6.6 Hz, 3H), 0.92 (t, J¼7.8 Hz, 9H),
0.91e0.95 (m, 1H), 1.15 (s, 6H), 1.16e1.46 (m, 14H), 1.68e1.72 (m,
1H), 1.76e1.85 (m, 1H), 2.04 (dd, J¼8.5, 8.8 Hz, 1H), 2.08e2.15 (m,
1H), 4.20 (dd, J¼7.8, 14.4 Hz, 1H), 4.27 (dd, J¼8.1, 14.4 Hz, 1H),
5.20 (dd, J¼7.8, 8.1 Hz, 1H), 7.52e7.61 (m, 2H), 7.92e7.97 (m, 1H),
8.15e8.19 (m, 1H); 13C NMR (100 MHz, CD3OD)
d 6.8. 7.1, 19.5,
21.3, 21.8, 21.9, 24.9, 26.7, 29.0, 29.7, 29.9, 33.9, 34.5, 37.1, 45.2,
45.3, 53.9, 54.1, 57.1, 73.2, 107.0, 122.0, 125.1, 127.4, 127.7, 136.8,
152.2, 152.6, 165.6; HRFABMS calcd for
626.3128, found 626.3127.
C33H53NaNO3SiS2,
4.7. X-ray co-crystallographic studies
The human VDR LBD protein was the same construct and was
purified and complexed by the same protocol as described in the
literature.26 Crystallization experiments were performed using the
hanging-drop vapor diffusion method. The ligand 3 or 4 was added
to aliquots of the purified protein in a 5-fold molar excess. Crys-
tallization conditions were similar conditions for the VDR LBD-
4.5.3. 7,8-cis-14-epi-1a,25-Dihydroxy-19-norvitamin D3 (4). To
a solution of 17 (25 mg, 0.041 mmol) in THF (0.4 mL) was added
LiHMDS (46
m
L, 1.0 M THF solution, 0.46 mmol) at ꢁ78 ꢀC. After
the mixture was stirred at the same temperature for 20 min, 14
(15 mg, 0.0414 mmol) in THF (0.15 mL) was added, and the whole
mixture was gradually warmed up to ꢁ40 ꢀC for 4 h. After the
reaction was quenched by saturated aqueous NH4Cl, the mixture
was extracted with AcOEt, washed with brine, dried over Na2SO4,
filtered and concentrated. The residue was purified by flash col-
umn chromatography on neutral silica gel (hexane:AcOEt¼50:1).
To a solution of the coupling product obtained above in THF
(1.2 mL) was added TBAF (0.42 mL, 1.0 M THF solution, 0.42 mmol)
at 0 ꢀC and stirred at room temperature for 16 h. After the reaction
was quenched by saturated aqueous NH4Cl at 0 ꢀC, the mixture
was extracted with AcOEt, washed with brine, dried over Na2SO4,
filtered and concentrated. The residue was purified by flash col-
umn chromatography on silica gel (AcOEt-AcOEt/MeOH¼20:1)
followed by purification on preparative silica gel TLC plate (AcOEt)
to give compound 4 as an inseparable mixture, which was further
purified by reverse-phase HPLC (YMC-Pack ODS column) using
MeOH/H2O (9/1) as a solvent system to give compound 4 (9.0 mg,
1a,25(OH)2D3 complex crystals by mixing 1 mL of protein solution
(10 mg/mL) with an equal volume of the reservoir solution, which
contained 1.4 M ammonium sulfate with 0.1 M MES, pH 6.5 and
equilibrating against 1 mL reservoir solution. Single crystals grew to
suitable dimensions in 2e4 days. hVDR complex crystals with 3 or 4
were cryo-protected in 30% glycerol and cooled at 79 K, and X-ray
data were collected using beamline MX2 at the Australian Syn-
chrotron and beamline NW12 at the Photon Factory (PF), re-
spectively. The data for the complex with compound 3 was
processed using the XDS software package28 and the complex with
compound 4 was processed the HKL2000 software package.29 We
carried out molecular replacement using MOLREP30 from CCP4
(Collaborative Computational Project, Number 4, 1994) with the
coordinates of the VDR LBD-1
1DB1; the solvent molecules and 1
a
,25(OH)2D3 complex (PDB code
,25(OH)2D3 were removed) as
a
the initial model. Refinement was carried out using the programs
REFMAC.31 A sample containing a random 5% of the total reflections
in the data set was excluded for Rfree calculations. After rigid-body
refinement, electron density for ligand 3 or 4 was clearly con-
structed using COOT.32 Statistics of the data collection and final
54% in two steps) as a colorless oil: [
a
]
25 þ71.9 (c 0.23, CHCl3); UV
D
(EtOH) lmax 261.5, 252.5, 244.0 nm, lmin 258.5, 247.5 nm; IR (neat)
3362, 2952, 2858, 1602 cmꢁ1; 1H NMR (400 MHz, CDCl3)
J¼6.3 Hz, 3H), 0.93 (s, 3H), 1.02e1.09 (m, 1H), 1.23 (s, 3H), 1.23 (s,
d 0.89 (d,