Journal of Natural Products
Article
(
12H, s), 0.14 (3H, s), 0.12 (3H, s); 13C NMR (50 MHz, CDCl ) δ
NE2, and the water molecule no. 35 was set to “toggle and spin”. This
means that the docking algorithm can choose to turn, keep, or delete
this water molecule depending on which setting gives the best docking
results. For the docking, both magnolol ligands were removed from
the binding site and used to define the binding site location. Docking
settings were validated by redocking of magnolol into the receptor.
Magnolol was docked at the location of either one or the other
cocrystallized magnolol binding sites with an average RMSD of 0.823
Å. The docking poses of compounds 3R,8S-1, 3R,8R-1, 3S,8S-1, and
3
1
2
3
36.8, 134.7, 127.9, 115.8, 79.5, 79.3, 69.4, 69.2, 64.2, 58.8, 31.9, 29.4,
9.3, 29.3, 27.8, 25.9, 22.8, 18.4, 14.2, −4.5, −4.8; HRMS m/z
97.2536 [M + Na] (calcd for C H O SiNa, 397.2539).
2
3
38
2
General Procedure for Steglich Esterification Using EDCI.
To a mixture of 3-methoxy-4-(tert-butyldimethylsilyl)oxycinnamic
acid (19) (52 mg, 0.17 mmol), DMAP (16 mg, 0.13 mmol), and 3-
(
(
(tert-butyldimethylsilyl)oxy)heptadeca-1,9-diene-4,6-diyn-8-ol (20)
50 mg, 0.13 mmol) in dry CH Cl (1.2 mL) was added EDCI·
2
2
HCl (33 mg, 0.17 mmol) under positive argon pressure at 0 °C. The
reaction vessel was sealed, and the mixture was allowed to warm to
room temperature. After the reaction was completed (TLC), the
mixture was cooled with an ice bath and 2 N HCl (1.2 mL) was
added dropwise. The mixture was extracted with CH Cl (3 × 2 mL),
the combined organic mixtures were washed with brine (5 mL) and
dried over sodium sulfate, and the solution was passed through a pad
of Celite. Volatiles were evaporated at reduced pressure at room
temperature. Products were purified by column chromatography using
silica gel and petroleum ether/EtOAc (95:5).
3
S,8R-1 were analyzed using LigandScout 4.2.1 (Inte:Ligand GmbH,
Vienna, Austria).
PPARγ Reporter Gene Transactivation. PPARγ luciferase
reporter gene transactivation experiments were performed using
HEK293 cells (ATCC, Manassas, VA, USA). HEK293 cells were
maintained in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% fetal bovine serum, 2 mM glutamine, 100
2
2
6
U/mL benzylpenicillin, and 100 μg/mL streptomycin. Then, 6 × 10
cells were seeded in 20 cm dishes, cultured for 18 h, and transfected
with 6 μg of a full-length human PPARγ expression plasmid, 6 μg of a
firefly luciferase reporter plasmid containing a PPAR response
element, and 3 μg of pEGFP-N1 (Clontech, Mountain View, CA,
USA) as internal control. After 6 h of transfection, cells were reseeded
(
E)-(3R,8S,Z)-3-((tert-Butyldimethylsilyl)oxy)hexadeca-1,9-
dien-4,6-diyn-8-yl-3-(4-((tert-butyldimethylsilyl)oxy)-3-
methoxyphenyl)acrylate (3R,8S-21). This was prepared according
to the general procedure described above and isolated as a colorless
4
in 96-well plates (4× 10 cells/well) in serum-free DMEM
oil in 81% (70 mg) yield: R 0.57 (petroleum ether/EtOAc, 9:1);
f
25
1
supplemented with 2 mM glutamine. Reseeded cells were treated
with 0.1, 0.3, 1, 3, 10, and 30 μM of each test compound dissolved in
DMSO and were then incubated for 18 h. After cell lysis, luciferase
activity and EGFP fluorescence were quantified on a GeniosPro plate
[
(
6
5
α] +91.8 (c 0.25, CHCl ); H NMR (200 MHz, CDCl ) δ 7.65
D
3
3
1H, d, J = 16.0 Hz), 7.03−7.00 (2H, m), 6.84 (1H, d, J = 8.8 Hz),
.33−6.25 (2H, m), 5.94−5.76 (1H, m), 5.72−5.50 (2H, m), 5.43−
.33 (1H, dt, J = 16.8 Hz, J = 1.2 Hz), 5.19−5.13 (1H, dt, J = 10.0 Hz,
J = 1.2 Hz), 4.93 (1H, d, J = 4.9 Hz), 3.83 (3H, s), 2.20 (2H, q, J =
reader (Tecan, Grodig, Austria). The ratio of luminescence units to
̈
6
0
.8 Hz), 1.39−1.26 (10H, m), 0.99 (9H, s), 0.91−0.83 (12H, m),
fluorescence units was calculated to account for differences in cell
number or transfection efficiency. Results are expressed as fold
induction compared to the solvent DMSO (0.1%). Pioglitazone (5
μM) was used as positive control.
1
3
.17 (6H, s), 0.14 (3H, s), 0.12 (3H, s); C NMR (100 MHz,
CDCl ) δ 165.8, 151.3, 147.9, 146.1, 136.8, 136.5, 128.3, 124.3, 122.6,
3
1
2
21.2, 115.8, 115.1, 111.0, 79.6, 76.3, 69.7, 69.4, 64.1, 60.2, 55.6, 32.0,
9.3, 29.3, 28.1, 25.9, 25.8, 22.8, 18.6, 18.4, 14.2, −4.8, −4.5; HRMS
Statistical analysis was performed using Prism software (ver. 4.03;
GraphPad Software Inc., San Diego, CA, USA). To calculate the EC50
values, data were curve fitted and nonlinear transformed using a
sigmoidal dose response with variable slope.
m/z 687.3893 [M + Na] (calcd for C H O Si Na, 687.3877).
3
9
60
5
2
General Procedure for Global Deprotection. To a solution of
1 (50 mg, 0.075 mmol) in THF (0.4 mL) was added dropwise HF·
2
pyridine (70:30 mixture, 97.5 μL, 50 equiv of HF) at 0 °C. The
mixture was stirred at room temperature. After the reaction was
completed (TLC), the mixture was cooled with an ice bath and a
saturated aqueous NaHCO3 solution (∼1 mL) was added. The
mixture was extracted with ether (3 × 2 mL), and the combined
organic layers were washed with brine, dried over sodium sulfate, and
filtered. Volatiles were evaporated under reduced pressure at room
temperature. The product was purified with column chromatography
with silica gel and petroleum ether/EtOAc (4:1).
Resazurin Conversion Assay. Cell viability was detected by
employing a resazurin conversion assay. This method is based on the
reduction of resazurin into resorufin by redox equivalents resulting
from cellular metabolism. The fluorescence signal of resorufin
produced in viable cells is proportional to the number of cells and
can be used as a measure for cell viability or cytotoxicity. HEK293
4
cells were seeded in 96-well plates at a density of 4 × 10 cells/well
and grown overnight. Cells were then treated with each test
compound (0.3, 1, 3, 10, and 30 μM) for 24 h and incubated for 4
h with resazurin solution (10 μg/mL in PBS) at 37 °C. Fluorescence
was quantified in a plate reader using a 535 nm excitation/590 nm
emission filter set.
(3R,8S)-Notoincisol A (3R,8S-1). This was prepared according to
the general procedure and isolated as a colorless oil in 83% (29 mg)
2
0
yield: R = 0.57 (petroleum ether/EtOAc, 9:1); [α] +87.7 (c 0.09,
f
D
1
MeOH); H NMR (400 MHz, CDCl ) δ 7.66−7.63 (1H, d, J = 15.9
3
GABA Electrophysiology. All four notoincisol A isomers were
A
Hz), 7.08−7.06 (1H, dd, J = 8.2 Hz, J = 1.8 Hz), 7.03−7.02 (1H, d, J
investigated on receptor subtypes α1β3 and α1β2γ2. Oocytes were
=
1.8 Hz), 6.92−6.91 (1H, d, J = 8.2 Hz), 6.29−6.27 (2H, m), 5.96−
prepared and injected with mRNA mixtures of the subunits, and
5
5
5
1
.90 (1H, ddd, J = 17.1, Hz, J = 10.2 Hz, J = 5.1 Hz), 5.89 (1H, s),
.72−5.68 (1H, dt, J = 10.7, Hz, J = 7.5 Hz), 5.54 (1H, t, J = 9.7 Hz),
.49−5.46 (1H, d, J = 17.1 Hz), 5.27−5.25 (1H, dt, J = 10.2 Hz, J =
.1 Hz), 3.92 (3H, s), 4.94−4.93 (1H, t, J = 5.1 Hz), 2.21−2.17 (2H,
18
receptor expressing oocytes were evaluated as described previously.
Initial preliminary screenings of all four compounds at different
GABA concentrations (EC3-5, EC20, EC50, and EC80) and at
compound concentrations of 1 and 10 μM diluted into measurement
buffer were performed. Moderate enhancement of GABA-elicited
currents was observed for α1β3 as well as for α1β2γ2. This effect was
strongest at EC3-5, consistent with an allosteric modulatory action of
the compounds. Further measurements were thus performed at EC3-5
by coapplying GABA+ compound. In α1β3 receptors the resulting
currents did not reach maximum strength even at longer (120 s)
recording times. α1β3-specific kinetic properties could be a reason for
this phenomenon. In an effort to obtain the maximum enhancement
of the GABA EC3-5 current, further measurements with preapplica-
tion of the compound for up to 105 s were performed until saturation
was reached. Direct effects were tested by applying compound
without GABA. Data were recorded and evaluated using standard
q, J = 7.5 Hz), 1.97−1.96 (1H, d, J = 6.4 Hz), 1.43−1.35 (2H, m),
1
3
1
.29−1.25 (8H, s), 0.87−0.85 (3H, t, J = 7.1 Hz,); C NMR (50
MHz, CDCl ) δ 165.8, 148.3, 146.9, 146.2, 136.7, 135.8, 126.9, 124.0,
3
1
2
23.5, 114.8, 114.6, 109.4, 78.5, 77.0, 70.4, 69.3, 63.6, 60.1, 56.1, 32.0,
9.3, 28.1, 22.8, 14.3; HRMS m/z 459.2150 [M + Na] (calcd for
C H O SiNa, 459.2147).
27
32
5
Molecular Docking. The ligands were constructed using
ChemDraw 15 Professional (PerkinElmer, Inc.) and a Pipeline Pilot
8
(Dassault Systemes, Inc.) protocol translating .cdx into .sd files.
4
0−42
Docking was performed with GOLD version 5.2
employing the
ChemPLP scoring function. The X-ray crystal structure of human
33
PPARγ bound to two molecules of magnolol (PDB entry 3r5n),
a
natural product and partial agonist of the receptor, was selected for
the docking calculations. The protonation state of His323 was set to
18
procedures as described previously.
H
J. Nat. Prod. XXXX, XXX, XXX−XXX