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mixture of N-(3,4-difluorophenyl)chloroacetamide (14 mmol,
dropwise under N2. The mixture was stirred for 24 h, then
quenched with H2O (10 mL) and EtOAc (20 mL). A few drops of 2m
HCl were added to remove traces of LiAlH4, and the mixture was
then extracted with EtOAc (320 mL). The organic phase was
dried with anhydrous Na2SO4 and the solvent was removed in
vacuo. N-(3-Hydroxymethylphenyl)methanesulfonamide (21) was
obtained as an oil and was reacted in the next step without further
purification. 21 (1.1 mmol) was dissolved in CH2Cl2 (20 mL) fol-
lowed by the addition of pyridinium dichromate (3.5 equiv,
3.6 mmol). The reaction was stirred at room temperature (258C) for
1 h under N2. The brown crude mixture was filtered through a plug
of silica gel and washed with EtOAc. The solvent was removed
under reduced pressure to give a clear oil. Column chromatogra-
phy with EtOAc/hexane (2:3) gave 22 as a white crystalline solid.
2.8 g) and aluminum chloride (54 mmol, 7.2 g) was stirred and
heated at 200–2108C in a silicon oil bath for 4.5 h. On cooling, ice-
cold HCl (40 mL) was added, and the solid residue was removed
by vacuum filtration and purified by column chromatography with
hexane/EtOAc (1:1) as eluting solvents to give 5,6-difluoro-oxindole
as a beige solid: 970 mg, 41% yield; H NMR (300 MHz, [D6]DMSO):
d=10.46 (s, 1H, NH), 7.33 (t, J=8.7 Hz, 1H, Ar-H), 6.82 (q, J=
6.9 Hz, 1H, Ar-H), 3.47 ppm (s, 2H, ArCH2CO).
1
General procedure for the synthesis of benzylideneindolin-2-
ones (series I–VI): The method described by Zhang and Go was
followed.[3] Briefly, oxindole (1 mmol) and benzaldehyde (1 mmol)
were dissolved in EtOH (6 mL), a drop of piperidine (20 mL) was
added, and the reaction mixture was heated in a sealed 10-mL vial
on a microwave reactor for 15 min at 1408C. The mixture was
cooled to room temperature. If precipitation was observed, the
precipitate was removed by filtration under reduced pressure,
washed with cold EtOH, and recrystallized with MeCN to afford the
desired product. In the absence of precipitation, the mixture was
concentrated under reduced pressure, and the residue was purified
by flash column chromatography with hexane/EtOAc (1:1) as elut-
ing solvents.
Biological methods
Protocols for reagents for biological assays, cell lines, and cell culture:
3-(4,5)-Dimethylthiazol-2-yl)-2,5-diphenytetrazolium bromide (MTT)
was purchased from Duchefa Biochemie (Denmark). Penicillin G
and streptomycin were obtained from Sigma–Aldrich. Test com-
pounds were prepared in DMSO (ACS grade, NUS Laboratory
Supply) at 10 mm as stock solutions and stored at room tempera-
ture (258C). The following were purchased from Santa Cruz Bio-
technology (CA, USA): Rabbit monoclonal antibodies to PARP and
caspase 3, goat anti-rabbit horseradish peroxidase (HRP) conjugate,
and goat anti-mouse HRP; rabbit monoclonal antibodies to cleave
caspase 3, phospho-HER3 (Tyr1289), HER3, FGFR4, phospho-FRS2a
(Tyr436), phosphorylated Akt (Ser732), Akt mouse monoclonal anti-
bodies to phosphorylated tyrosine (p-Tyr100) were purchased from
Cell Signaling (MA, USA). Fetal bovine serum (FBS) and mouse
monoclonal antibodies to b-actin were purchased from Invitrogen
Life Technologies (CA, USA). Human phospho-RTK array kit (Cat.
No. ARY001B) was purchased from RnD Systems Inc. (Minneapolis,
MN, USA), and the immunoblot assay was from GE Healthcare
(Buckinghamshire, UK).
Synthesis of 3-sulfamoylbenzoic acid (6) and N-substituted sul-
famoylbenzoic acids (7–12): The method of Shen et al.[16] was fol-
lowed with modifications. To a saturated solution of 3- or 4-chloro-
sulfonylbenzoic acid (8 mmol, 1.6 g) in EtOAc (4 mL) was added
a concentrated NH3 solution (30% w/w) or a solution of alkylamine
(40% w/w aqueous methylamine or dimethylamine, or 1m ethyla-
mine in MeOH) or pure alkylamine (propylamine or isopropyla-
mine). After stirring for 30 min at 08C, the mixture was neutralized
with HCl in dioxane (4m) and extracted with EtOAc. The organic
phase was concentrated under reduced pressure to give a solid
which was recrystallized from MeCN to give the desired com-
pound.
Synthesis of formyl benzenesulfonamides (13–19): To a solution
of the sulfamoylbenzoic acid (6–12; 2 mmol) in anhydrous THF
(6 mL) was added borane/THF complex (1m, 6 mL). After stirring
for 15 h at 258C the mixture was diluted with 10 mL brine and
5 mL H2O. The organic phase was separated, dried with anhydrous
Na2SO4, and concentrated under reduced pressure to give the de-
sired alcohol which was not purified. The crude alcohol (2 mmol)
was suspended in anhydrous THF (12 mL) and stirred. Activated
4 molecular sieves (4 g) and pyridinium dichromate (5 mmol)
was added, and the mixture was stirred for 3 h at 258C. Another
portion of pyridinium dichromate (5 mmol) was added, and the
mixture was stirred for 3 h. The mixture was filtered through silica
gel, and the filtrate was concentrated under reduced pressure to
give the product, which was purified by column chromatography
with hexane/EtOAc (1:1) as eluting solvents.
Determination of growth inhibitory IC50 by MTT assay: HuH7 cells
were purchased from the Japanese Collection of Research Biore-
sources Cell Bank (Japan). Hep3B was a gift from Dr. Ho Han Kiat,
Department of Pharmacy, NUS. Cells were cultured in Dulbecco’s
modified Eagle’s medium (DMEM, high glucose, with HEPES) with
10% v/v FBS, 100 mgLÀ1 penicillin G, and 100 mgLÀ1 streptomycin.
They were sub-cultured when the following densities in a T75 mL
flask were reached: HuH7: 60–80105 cells, Hep3B: 35–40105
cells. Cells were maintained within 2–10 passages for experiments.
An aliquot of cells in media (200 mL, 3104 cellsmLÀ1 HuH7, 2.5
104 cellsmLÀ1 Hep3B) were added to each well in a 96-well micro-
titer plate and incubated for 24 h at 378C under 5% CO2. The
media was removed, and fresh media (200 mL) containing test
compound at a known concentration was added and incubated
for 72 h. Final DMSO concentration per well was 0.5% v/v. The
media were then removed and replaced with FBS-free media
(200 mL) and MTT (50 mL of 2 mgmLÀ1 solution in phosphate-buf-
fered saline (PBS), pH 7.4). After incubation for 3 h at 378C under
5% CO2, the supernatant was removed, and a solution of 200 mL
DMSO and 25 mL Sorenson buffer (0.1m glycine, 0.1m NaCl, adjust-
ed to pH 10.5 with 0.1m NaOH) was added to dissolve the forma-
zan crystals. Absorbance was measured at l 570 nm on a micro-
plate reader (Tecan Infinite 200). Cell viability was determined from
readings of treated wells relative to control wells (absence of test
compound) with correction of background absorbance. IC50 values
(concentration required to decrease cell viability to 50% of con-
trol/untreated cells) were determined in triplicates on separate oc-
Synthesis of N-(3-formylphenyl)methanesulfonamide (22): 3-Ami-
nobenzoic acid methyl ester (3 mmol, 0.5 g) was dissolved in anhy-
drous CH2Cl2 (30 mL) in an ice bath, followed by the addition of
pyridine (0.5 mL) and methanesulfonyl chloride (0.5 mL,
6.58 mmol). The reaction was stirred at room temperature (258C)
for 17 h. H2O (20 mL) and CH2Cl2 (20 mL) were added, the aqueous
layer was extracted with CH2Cl2 (230 mL), after which it was
washed with 1n HCl (225 mL) to remove excess pyridine. The or-
ganic layer was concentrated under vacuum to give 3-methanesul-
fonylaminobenzoic acid methyl ester 20 as a white solid.
Compound 20 (2 mmol, 0.5 g) was dissolved in anhydrous THF
(20 mL) to which LiAlH4 (1m in THF, 9 mL, 9 mmol) was added
ChemMedChem 2015, 10, 1548 – 1558
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