Journal of Natural Products
Article
4′-Acetoxy-6,7-(2,2-dimethylpyrano)-5-prenyloxyflavone (6).
Step i: A magnetically stirred solution of compound 5 (6.6 g, 19.6
mmol) in anhydrous CH2Cl2 (60 mL) maintained at 0 °C was treated
with pyridine (1.60 mL, 19.6 mmol) followed by Ac2O (1.86 mL, 19.6
mmol). The ensuing solution was warmed to 18 °C and stirred at this
temperature for 6 h before being filtered through a pad of TLC-grade
silica gel, and the filtrate concentrated under reduced pressure. The
resulting yellow solid was directly subjected to the next step of the
reaction sequence. Step ii: A magnetically stirred solution of the
acetate was dissolved in dry tetrahydrofuran (THF) (60 mL) and
cooled to 0 °C followed by the addition of Ph3P (7.7 g, 29.4 mmol),
3-methyl-2-buten-1-ol (3.0 mL, 29.4 mmol), and diethyl azodicarbox-
ylate (4.6 mL, 29.4 mmol). The ensuing mixture was warmed to 18
°C, stirred at this temperature for 6 h, then concentrated under
reduced pressure followed by flash chromatography (silica, 5:95 v/v
EtOAc/n-hexane elution). Concentration of the appropriate fractions
(Rf = 0.6 in 1:3 v/v EtOAc/n-hexane) afforded compound 6 (6.0 g,
(15); HRMS [M + Na]+ calcd for C25H24O523Na 427.1521, found
427.1506.
X-ray Crystallographic Studies. Crystallographic Data for
Compound 1. C25H24O5, M = 404.44, T = 150 K, monoclinic, space
group C2/c, Z = 8, a = 26.0009(6) Å, b = 9.6431(3) Å, c =
16.4553(4) Å, β = 94.559(2)°, V = 4112.77(19) Å3, Dx = 1.306 g
cm−3, 4326 unique data (2θmax = 56.8°), 2885 with I > 2.0σ(I); R =
0.052, Rw = 0.134, S = 1.03.
Crystallographic Data for Compound 7. C27H26O6, M = 446.48,
T = 100 K, monoclinic, space group P21/n, Z = 4, a = 18.695(2) Å, b
= 6.122(3) Å, c = 20.101(4) Å, β = 98.333(18)°, V = 2276.3(14) Å3,
Dx = 1.303 g.cm−3, 4646 unique data (2θmax = 52.8°), 3254 with I >
2.0σ(I); R = 0.0575, Rw = 0.1593, S = 1.046.
Structure Determination. Images for compound 1 were
measured on a Agilent SuperNova diffractometer (Mo Kα, graphite
monochromator, λ = 0.710 73 Å), and those for the acetate derivative
7 on the MX1 beamline at the Australian Synchrotron (Melbourne).
Data collection, cell refinement, and data reduction for compound 1
employed the CrysAlis PRO program,42 while SHELXT43 and
SHELXL44 were used for structure solution and refinement. Data
collection, cell refinement, and data reduction for compound 7 used
Blu-Ice45 and XDS,46 while the structure was solved with the ShelXT
2018/2 structure solution program47 using the dual solution method
and refined with SHELXL44 using Olex248 as the graphical interface.
Atomic coordinates, bond lengths and angles, and displacement
parameters have been deposited at the Cambridge Crystallographic
Data Centre (CCDC Deposition numbers 1888687 and 1888688).
contacting the Cambridge Crystallographic Data Centre, 12 Union
Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
Cell Cultures. The MDA-MB-231, MCF-7, HepG2, LNCaP, and
RAW 264.7 cell lines were cultured in DMEM, while the LO-2 and
HCT-116 cell lines were grown in RPMI-1640 medium. All media
were blended with 10% FBS, 86.0 μM streptomycin, 50 units/mL
penicillin, and 71.9 μM gentamycin, while cells were cultured under a
5% CO2 atmosphere at 37 °C.
Cytotoxicity Analyses (Using a Methylene Blue Assay). Stock
solutions of isolaxifolin and apigenin were prepared in dimethyl
sulfoxide (DMSO) at a concentration of 100.0 mg/mL and later
diluted with fresh medium to achieve the required concentrations
(6.2, 17.3, 24.7, and 30.9 μM for isolaxifolin; 23.1, 46.3, 94.4, and
185.0 μM for apigenin). The final concentration of DMSO in each
culture was less than 0.1%. Evaluations of cytotoxicity properties were
performed using minor modifications of previously described49
protocols. Thus, cells were seeded at a density of 1.5 × 105 cell/
mL in 100 μL aliquots on a 96-well microplate and incubated
overnight (∼12 h). The supernatant liquid in each well was then
replaced with fresh medium (100 μL) containing isolaxifolin or
apigenin. Experiments were performed in triplicate for 48 h and then
stained with the methylene blue solution. The cell viability rate was
calculated using eq 1:
1
68% from 5) as an amorphous, pale-yellow powder: H NMR (600
MHz, DMSO-d6) δ 8.09 (d, J = 8.7 Hz, 2H), 7.34 (d, J = 8.7 Hz, 2H),
6.90 (s, 1H), 6.81 (s, 1H), 6.64 (d, J = 10.1 Hz, 1H), 5.90 (d, J = 10.1
Hz, 1H), 5.53 (m, 1H), 4.51 (d, J = 7.3 Hz, 2H), 2.32 (s, 3H), 1.72 (s,
3H), 1.61 (s, 3H), 1.44 (s, 6H); 13C NMR (150 MHz, DMSO-d6) δ
176.1, 169.4, 159.9, 158.5, 157.8, 153.6, 153.3, 138.3, 131.6, 128.8,
128.0, 123.1, 120.7, 116.4, 113.9, 112.8, 108.3, 101.1, 78.1, 71.7, 28.3,
26.0, 21.4, 18.2; IR (KBr) νmax 2974, 2931, 1756, 1638, 1599, 1505,
1445, 1361, 1291, 1191, 1164, 1119, 1086, 1019, 912 cm−1; MS (ESI,
+ve) m/z 469 ([M + Na]+, 100%), 447 ([M + H]+,10); HRMS [M +
Na]+ calcd for C27H26O623Na 469.1627, found 469.1628.
4′-Acetoxy-5-hydroxy-6,7-(2,2-dimethylpyrano)-8-prenylflavone
(7). A magnetically stirred solution of compound 6 (6.00 g, 13.4
mmol) in CHCl3 (60 mL) was treated with Eu(fod)3 (800 mg, 0.77
mmol), and the ensuing mixture was heated at 60 °C for 1 h before
being cooled and concentrated under reduced pressure. The yellow
solid obtained was subjected to flash chromatography (silica, 5:95 v/v
EtOAc/n-hexane elution), and concentration of the appropriate
fractions (Rf = 0.8 in 1:3 v/v EtOAc/n-hexane) afforded compound 7
1
(5.00 g, 83%) as yellow needles, mp = 132−134 °C: H NMR (400
MHz, CDCl3) δ 12.93 (s, 1H), 7.91 (d, J = 8.8 Hz, 2H), 7.28 (d, J =
8.8 Hz, 2H), 6.74 (d, J = 10.1 Hz, 1H), 6.62 (s, 1H), 5.63 (d, J = 10.1
Hz, 1H), 5.21 (m, 1H), 3.50 (d, J = 7.1 Hz, 2H), 2.35 (s, 3H), 1.83 (s,
3H), 1.70 (s, 3H), 1.47 (s, 6H); 13C NMR (100 MHz, CDCl3) δ
182.8, 169.1, 162.7, 157.1, 154.5, 153.2, 131.8, 129.3, 128.0, 127.6,
122.4, 122.2, 115.8, 107.6, 105.6, 105.5, 105.3, 77.9, 28.2, 25.8, 21.7,
21.2, 18.1; IR (KBr) νmax 2977, 2926, 1763, 1653, 1582, 1507, 1436,
1367, 1320, 1202, 1168, 1119, 1079, 910 cm−1; MS (ESI, +ve) m/z
469 ([M + Na]+, 100%), 443 (40), 427 (80), 405 (30); HRMS [M +
Na]+ calcd for C27H26O623Na 469.1627, found 469.1624.
Isolaxifolin (1). A magnetically stirred solution of compound 7 (5.0
g, 11.2 mmol) in dry MeOH/THF (90 mL of a 2:1 v/v mixture) was
cooled to 0 °C and then treated with K2CO3 (1.50 g, 11.2 mmol).
The ensuing mixture was stirred for 4 h at 0 °C and concentrated
under reduced pressure, and the yellow oil obtained was then
subjected to flash chromatography (silica, 5:95 v/v EtOAc/n-hexane
elution). Concentration of the appropriate fractions (Rf = 0.5 in 1:3 v/
v EtOAc/n-hexane) and recrystallization of the resulting solid (from
CDCl3) afforded isolaxifolin 1 (3.20 g, 71%) as yellow crystals, mp =
120 °C [lit.2 mp = 259−260 °C (after recrystallization from EtOAc/
n-hexane)]; presumably, two distinct crystal morphologies have been
obtained from these two different solvent systems and, thus, the
melting points of the two samples are significantly different. 1H NMR
(400 MHz, CDCl3) δ 12.99 (s, 1H), 7.80 (d, J = 8.7 Hz, 2H), 6.97 (d,
J = 8.7 Hz, 2H), 6.74 (d, J = 10.0 Hz, 1H), 6.56 (s, 1H), 5.76 (br s,
1H), 5.62 (d, J = 10.0 Hz, 1H), 5.24 (m, 1H), 3.51 (d, J = 7.1 Hz,
2H), 1.83 (s, 3H), 1.69 (s, 3H), 1.47 (s, 6H); 13C NMR (100 MHz,
CDCl3) δ 182.9, 164.6, 160.4, 157.2, 154.4, 154.0, 131.8, 128.4, 128.1,
122.8, 122.1, 116.3, 115.7, 107.8, 105.6, 104.8, 102.9, 77.9, 28.2, 25.7,
21.6, 18.1; IR (KBr) νmax 3386, 2976, 2926, 1654, 1606, 1562, 1507,
1466, 1433, 1346, 1294, 1205, 1173, 1127, 835 cm−1; MS (ESI, +ve)
m/z 427 ([M + Na]+, 100%), 405 ([M + H]+,15), 245 (20), 217
Cell viability = (Absx/Abs0) × 100%
(1)
where Absx is the absorbance measured for cells treated with varying
concentrations of the test compounds and Abs0 is the absorbance
measured for the cells not exposed to the test substrates. Standard
deviations were calculated in triplicate.
Cell Cycle Analyses. Each phase of the cell cycle was evaluated
using the PI staining method. Thus, MDA-MB-231 cells were placed
in a six-well plate and incubated, under standard conditions, for 24 h.
The supernatant liquid was then replaced with fresh media containing
varying concentrations of isolaxifolin (8.6, 12.4, and 17.3 μM), and
incubation continued for a further 48 h. The treated MDA-MB-231
cells were pelleted and then fixed with 75% ethanol solution and
maintained at −20 °C overnight before being stained, in the dark at
ambient temperatures, with PI-containing RNase for 15 min. A ca.
10 000-cell sample from each well was then analyzed by flow
cytometry using a FACSCanto cell analyzer (Becton, Dickinson &
Co., USA).
G
J. Nat. Prod. XXXX, XXX, XXX−XXX