ChemMedChem
10.1002/cmdc.202100377
FULL PAPER
and H460 cells were screened and the structure-activity
relationships were investigated. The most potent anti-
proliferation effect on Hccc-9810 cells by using a falcarindiol
analogue (3R,8S)-2i was observed for the first time in a dose-
dependent manner. The falcarindiol analogue (3R,8S)-2i
induced obvious apoptosis preferentially in Hccc-9810 cells as
revealed by using Hoechst33258 staining and flow cytometry
analysis. Mechanistic investigations including LDH release, MDA
content and SOD activity suggest that the Hccc-9810 cell
apoptosis induced by the falcarindiol analogue (3R,8S)-2i could
involve intracellular free radical generation and oxidative stress.
These findings indicate that the falcarindiol analogue (3R,8S)-2i
could be considered as a promising therapeutic agent of human
cholangiocarcinoma (CCA) for further study.
addition of 1-decanal (0.5 mmol, 1equiv.). The reaction mixture was
stirred for 12 h, and then it was quenched with aqueous saturated NH
5 mL). The solution was extracted with CH Cl (4 × 50 mL), and the
combined organic phase was dried over Na SO filtered and
4
Cl
(
2
2
2
4
,
concentrated under reduce pressure. The crude residue was purified by
column chromatography on silica gel eluted with petroleum ether and
ethyl acetate to afford the corresponding compound (S)-or (R)-3.
The second asymmetric addition: under argon atmosphere, Zn powder
(3.06 mmol, 10.2 equiv.) and (R)- or (S)-BINOL (0.20 mmol, 0.68 equiv.)
were added to a 25 mL flask equipped with a mechanical stirrer. Then EtI
i
(
6.12 mmol, 20.4 equiv), Ti(OPr)
4
(0.51 mmol, 1.70 equiv.), and (S)-or
(
R)-5 (0.60 mmol, 2 equiv) were added dropwise by syringe. After 5 min,
THF (1 mL) was added, and the mixture was stirred at r.t. for 24 h. Then
diethyl ether (10 mL) was added, which was followed by the addition of
an aldehyde (0.3 mmol, 1 equiv) after one hour. The mixture was stirred
for 12 h, and then it was quenched with aqueous saturated NH
The solution was extracted with CH Cl (3 × 20 mL), and the combined
organic phase was dried (Na SO ), filtered and concentrated under
reduced pressure. The crude was purified by column chromatography on
silica gel eluted with petroleum ether and ethyl acetate to afford the
desired chiral falcarindiol analogues 2.
4
Cl (5 mL).
2
2
Experimental Section
2
4
General procedure and materials
All reactions were performed under inert gas conditions unless otherwise
specified. The chemicals were commercially available and were used
directly without purification. Methylene chloride, tetrahydrofuran, and
diethyl ether were purified by MBRAUN SPS-800-Systems, and other
solvents were dried by standard methods prior to use. The NMR data
were recorded with a Bruker TM400 NMR spectrometer. High-resolution
mass spectra (QTOF)were measured on SCIES-X500r. HPLC analyses
were performed with a Waters 1525 by using Diacel Chiralcel OD-H,
Chiracel AD-H, and Chiralcel AS-H columns, with detection at 254 or 220
nm by using a Waters 2487 instrument. Optical rotations were obtained
by using a Hanon P810/P850 automatic polarimeter with a sodium 589.3
nm filter.
Anticancer activities of the falcarindiol analogues 2
The viability of cells was measured by a colorimetric CCK8 assay.
Cultured cells were counted and suspended in DMEM and RPMI1640
medium. 96-well plates were seeded at concentrations of 7000 and 5000
cells/well (180 L medium) for Hccc-9810, HepG2, MDA-MB-231, HeLa,
MG-63 and H460 cell lines respectively and then preincubated for 24 h.
Each sample of the falcarindiol analogues 2 was dissolved in DMSO to
produce a 0.2 mol/L stock solution. The falcarindiol 1 was selected as a
positive control. Serial dilutions were performed (with the addition of
DMEM and RPMI1640 medium for Hccc-9810, HepG2, MDA-MB-231,
HeLa, MG-63 and H460 cell lines respectively) to produce final ligand
concentrations ranging from 300-500 µmol/L. The testing sample was
added at 20 µL per well, followed by incubation for 48 h at 37 oC in a
For anticancer activities of falcarindiol analogues, DMSO, Penicillin,
Streptomycin was purchased from Sigma-Aldrich. DMEM medium with
high glucose and RPMI1640 medium were purchased from Gibco.
Human cholangiocarcinoma Hccc-9810, hepatocellular carcinoma
HepG2, breast cancer MDA-MB-231, cervical carcinoma HeLa,
osteosarcoma MG-63 and large cell lung cancer H460 cell lines were
purchased from the Institute of Cell Biology, Academic Sinica (Shanghai,
China). CCK-8 detection kit was obtained from Japanese colleagues.
Fetal bovine serum was purchased from Hangzhou Sijiqing company.
Trypsin was purchased from Bioder company. Malonaldehyde (MDA),
superoxide dismutase (SOD) and lactic dehydrogenase (LDH) kit were
purchased from Nanjing Jiancheng Institute of Biology. CKX53-SLP
inverted phase contrast microscope, fluorescence inverted microscope
2
humidified atmosphere of 5% CO . Then the medium was removed and
replaced with 100 µL 10% CCK8 (V/V) per well, followed by 30 min
incubation at 37 °C. The absorbance was recorded at 450 nm by using
microplate reader. The results were expressed as ratio of absorbance
between treatments and control cells (solvent vehicle set at 100%). Four
replicate wells were tested per assay which was repeated three times.
IC50 values (concentration resulting in 50% inhibition) were calculated
using GraphPad Prism (San Diego, CA).
Apoptosis morphology by Hoechst33258 staining
(
(
Olympus, Japan), Spectra Max M3 multi-function microplate reader
Molecular Devices, USA), TSX60086D refrigerator, HERACELL-150i
Hccc-9810 cells in the logarithmic growth phase were taken and
5
inoculated in a 6-well culture plate at a density of about 1 × 10
cells per
CO
2
incubator and 481HP constant temperature shaker (U.S. Thermo
well. After cultured overnight at 37 C and 5% CO incubator, the medium
o
2
Fisher Scientific company) were used.
was discarded, followed by treatment with 0.5 M, 1.0 M and 2.0 M of
the falcarindiol analogue (3R,8S)-2i (including control group). The cells
were washed twice with PBS and fixed in 0.5 mL methanol per well for 30
min. The fixed cells were incubated with 1 mL Hoechest33258 staining
Typical procedure for the asymmetric diyne addition to
aldehydes by using ligand (R)- and (S)-BINOL
o
solution for 10 min at 37 C in the dark. After removal of the staining
solution, the cells were washed twice with PBS, and then were observed
by using a fluorescence microscope (FL CD12-002, AMG, USA) with
excitation wavelength of 350 nm and emission wavelength of 461 nm.
For cell counts, 5 random fields (about 100 cells each field) were
observed per coverslip. Results were expressed as percentage of
Hoescht-positive nuclei (condensed or fragmented) relative to the total
number of nuclei counted per coverslip for each experimental condition.
The first asymmetric addition: under argon atmosphere, Zn powder (5.1
mmol, 10.2 equiv.) and (R)- or (S)-BINOL (0.34 mmol, 0.68 equiv.) were
added to a 25 mL flask equipped with a mechanical stirrer. Then EtI (10.2
i
mmol, 20.4 equiv.), Ti(OPr)
4
(0.85 mmol,1.70 equiv.) and buta-1,3-diynyl-
TIPS (1 mmol, 2 equiv.) were added dropwise via syringe. After 5 min,
THF (1 mL) was added and the mixture was stirred at room temperature
(r.t.) for 24 h. Then diethyl ether (15 mL) was added, followed by the
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