Combination of betulinic acid and its anticancer efficacy
magnetic resonance (NMR) and 13C NMR spectra were to afford product 10 (51%) as a white solid. 1H-NMR (CDCl3,
recorded with a Bruker AV-300 spectrometer in the indicated 300 MHz): δ 0.73, 0.84, 0.90, 0.97, 1.68 (each 3 H, s), 1.59
solvents (trimethylchlorosilane [TMS] as internal standard). (m, 4 H), 1.98 (2 H, m), 2.24 (2 H, m), 3.01 (1 H, m), 3.49
Mass spectra were obtained using FTMS-2000. The synthetic (4 H, t), 3.81, 4.01 (each 1 H, m), 4.59, 4.72 (each 1 H, m), 5.80
method and physicochemical date of compounds 6 and 9 have (2 H, s); 13C-NMR (CDCl3, 75 MHz): δ 15.9, 16.6, 18.1,
been disclosed in previous reports, the NMR spectrums of 19.3, 20.8, 15.4, 26.2, 29.7, 29.8, 30.6, 31.1, 32.3, 37.0, 38.3,
compounds 7, 8, and 10 are available in Figures S1–S3.15,16 38.5, 39.2, 40.6, 40.8, 42.1, 42.4, 45.1, 46.9, 49.2, 50.5, 56.3,
67.2, 72.1, 96.5, 109.6, 152.8, 173.4, 179.7; mass spectrom-
O2-(Methylthiomethyl)-1-(pyrrolidin-1-yl)
diazen-1-ium-1,2-diolate (7)
etry (MS) electrospray ionization (ESI) m/z [M+H]+ 642.4,
high resolution mass spectrometer (HRMS) (ESI) m/z calcu-
lated for C37H63N4O6 [M+NH4]+ 659.4742, found 659.4738.
The sodium NONOate 6 (6.88 g, 45.0 mmol) was added to
a suspension of sodium carbonate (4.78 g, 45.0 mmol) and
dimethylformamide (DMF) (100 mL) at room temperature,
and this mixture was stirred for 10 min. Chloromethyl methyl
sulfide (3.72 mL, 45.0 mmol) was added dropwise, and the
reaction was allowed to proceed at room temperature for 4 h
with stirring. Ethyl acetate (200 mL) was added to quench the
reaction, the solids were filtered off, the organic phase was
washed with water (5×60 mL), and dried with Na2SO4. The
solvent was removed in vacuo to give a liquid residue, which
was purified by silica gel column chromatography using
EtOAc/hexane (1:4, v/v) as the eluent. Compound 7 was
obtained as a canary yellow liquid (3.80 g, 45%): 1H NMR
(300 MHz, CDCl3): δ (ppm) 5.15 (s, 2 H), 3.53–3.48 (m, 4 H),
2.21 (s, 3 H), 1.91–1.86 (m, 4 H).
Determination of in vitro antiproliferative
activity
The viability of 5 human cancer cell lines (B16F10, MCF-7,
HCT-116, A549, and HepG2) treated with BA, BA-78, and
Cisplatin, was determined using the MTT assay. Briefly,
cells were seeded in 96-microwell plates, incubated at 37°C
in a humidified incubator with 5% CO2 for 24 h prior to the
experiments. After removing the medium, 100 μL of fresh
medium containing the test compounds at 5 concentra-
tions (ie, 10−4, 10−5, 10−6, 10−7, and 10−8 M) were added to
microtiter plates and cells were incubated with each com-
pound at 37°C for another 72 h. At the end of the incubation,
the medium was discarded, cells were washed with PBS and
100 μL of MTT buffer was added to each well. Cells were
incubated with MTT for at least 4 h at 37°C and the viabilities
were proportioned to the value of optical densities, which
was quantitatively measured by spectrophotometry (Biorad,
Nazareth, Belgium) at 570 nm. The dose–response curve (not
shown) was created by plotting the percent viability against
the log of concentrations of the corresponding compound for
each cell line. The molar concentrations that caused 50% cell
killing (IC50) were determined.
O2-chloromethyl-1-(pyrrolidin-1-yl)
diazen-1-ium-1,2-diolate (8)
A solution of compound 7 (273 mg, 1.44 mmol) in dry
dichloromethane (30 mL) was cooled to 0°C and sulfuryl
chloride (1.44 mL, 1.0 M solution in dichloromethane) was
added dropwise, the ice bath was removed and the reaction
mixture was stirred at room temperature for another 2 h.
The solvent was evaporated to afford compound 8 in nearly
quantitative yield, which was used without further purifica-
tion. 1HNMR (300 MHz, CDCl3): δ (ppm) 5.80 (s, 2 H), 3.63
(m, 4 H), 1.95 (m, 4 H).
Detection of nO production
NO was indirectly determined by measuring nitrite content
in the culture supernatant using Griess reagent. Briefly,
B16F10 cells (2×104 cells/mL) were seeded in a 96-well
Ba-78
To a solution of 0.1 g, 9 mmol in anhydrous DMF was added plate and incubated at 37°C for 24 h followed by incubation
Cs2CO3 (1 eq.) and compound 8 (1.2 eq.). The reaction was with 10 μM of BA-78 for different times (ie, 10, 30, 60 min,
stirred at room temperature for 6 h. The reaction mixture 3, 6, 12, 24, and 48 h). For each well, 50 μL of supernatants
was diluted with 20 mL of ethyl acetate, and the organic layer was collected in a tube, the collection was incubated with
was separated. The aqueous layer was extracted with ethyl 25 μL of sulfanilic acid for 10 min at room temperature
acetate (2×10 mL). The combined organic layer was dried in the dark. After the reaction, 25 μL of N-(1-naphthyl)
over anhydrous sodium sulfate, and the solvent was evapo- ethylenediamine was added into the tube and the plate was
rated under reduced pressure. The crude material was purified incubated for another 10 min at room temperature in the dark.
by flash column chromatography (1:1 hexane/ethyl acetate) The absorbance was read at 550 nm by a spectrophotometry
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