7294 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Roth et al.
using 4-dimethylaminomethyl-phenylamine18 and (E)-3-(hydroxy-
phenyl-methylene)-2-oxo-2,3-dihydro-1H-indole-6-carboxylic acid
ethylamide 11. Yield: 77% of 47i. 1H NMR: δ 1.06 (t, 3H), 2.04 (s,
6H), 3.18 (m, 2H) superimposed on 3.20 (s, 2H), 5.71 (d, 1H), 6.77
(d, 2H), 7.04 (m, 3H), 7.32 (s, 1H), 7.46 (m, 2H), 7.54 (m, 3H), 8.13
(t, 1H), 10.84 (s, 1H), 12.09 (s, 1H). MS: m/z 441 [M þ H]þ. HRMS
(ESþ) calcd for C27H28N4O2 [M þ H]þ m/e 441.2285, found m/e
441.2277.
(Z)-3-[(4-Ethylaminomethyl-aminophenyl)-phenyl-methylene]-
2-oxo-2,3-dihydro-1H-indole-6-carboxylic Acid N-Methyl-Ethy-
lamide Trifluoroacetate (47l). (Z)-Ethyl-[4-({[6-(ethyl-methyl-
carbamoyl)-2-oxo-1,2-dihydro-indol-3-ylidene]-phenyl-methyl}-
amino)-benzyl]-carbamic acid tert-butyl ester 50 (170 mg, 0.306
mmol) was dissolved in methylene chloride (20 mL) and trifluoro-
acetic acid (0.5 mL, 6.5 mmol) was added at 0 ꢀC. The mixture
was stirred for 1 h at 0 ꢀC and for 12 h at ambient temperature.
After that time, the organic phase was washed with water (2ꢀ)
and dried over sodium sulfate. The solvent was removed by
evaporation togive 170 mg (98%) of 47l. 1H NMR:δ 1.04(t, 3H),
1.16 (t, 3H), 2.85 (s, 3H), 2.91 (q, 2H), 3.25 (q, 2H, superimposed
on H2O), 4.00 (s, 2H), 5.79 (d, 1H), 6.57 (d, 1H), 6.85 (d, 2H)
superimposed on 6.88 (s, 1H), 7.25 (d, 2H), 7.51 (m, 2H), 7.59
(m, 3H), 8.60 (br, 2H), 10.83 (s, 1H), 12.10 (s, 1H). MS: m/z 455
[M þ H]þ. HRMS (ESþ) calcd for C28H30N4O2 [M þ H]þ m/e
455.2441, found m/e 455.2436.
(Cell Signaling, Danvers, MA, USA) supplemented with 1 mM
PMSF (Sigma-Aldrich, St. Louis, MO, USA).
96-Well plates (Nunc MaxiSor, Rochester, NY, USA) were
coated with anti-Smad2/3 monoclonal antibody (1 μg/mL; BD
Bioscience, San Jose, CA, USA) for 24 h at 4 ꢀC. To reduce
unspecific binding, the wells were rinsed with PBS þ 0.1%
Tween 20 and blocked with PBS þ 2% BSA for 2 h at room
temperature. After washing three times with PBS þ 0.1% Tween
20, the protein lysate was added and incubated for 2 h at room
temperature. Wells were washed three times and incubated with
an antiphoshpo-Smad2/3 rabbit antisera (Eurogentec, Liege,
Belgium) diluted in PBS þ 0.2% BSA þ 0.02% Tween 20, and
incubated for 2 h at room temperature. An AP-conjugated mAb
mouse anti rabbit IgG (Sigma-Aldrich, St. Louis, MO, USA)
was added and incubated for 2 h at room temperature. Finally,
after washing, pNPP liquid substrate system (Sigma-Aldrich, St.
Louis, USA) was added and developed in the dark at 37 ꢀC for
2 h before the absorbance was measured at 405 nm in a Synergy
HT plate reader (BioTek, Winooski, VT, USA).
Cytotoxicity Assay. The high-content cytotoxicity assay (kit
I) was performed according to the manufacturer’s instructions
(Cellomics, ThermoFisher, Waltham, MA, USA). HaCaT cells
were cultured overnight in black 96-well plates, incubated for
24 h with each compound at different concentrations, and
stained with cytotoxicity cocktail. Cells were fixed, washed,
and scanned on the Cellomics ArrayScan II platform. Images
were analyzed with the Cell Health image analysis algorithm.
Cytotoxicity indices were calculated for each of the four param-
eters (cellular membrane integrity, nuclear fragmentation and
density, and lysosomal mass) to indicate the percentage of cells
outside of the normal range which was defined using a vehicle-
treated reference cell population.
(Z)-3-{[4-(2-Dimethylaminoethyl)-aminophenyl]-phenyl-meth-
ylene}-2-oxo-2,3-dihydro-1H-indole-6-carboxylic Acid N-Meth-
yl-Ethylamide (47p). Synthesized according to the general pro-
cedure using 4-(2-dimethylamino-ethyl)-phenylamine30 and
(E)-3-(hydroxy-phenyl-methylene)-2-oxo-2,3-dihydro-1H-indole-
6-carboxylic acid N-methyl-ethylamide 10. Yield: 180 mg
1
(50%) of 47p. H NMR: δ 1.04 (t, 3H), 2.11 (s, 6H), 2.38 (t,
2H), 2.56 (t, 2H), 2.85 (s, 3H), 3.25 (q, 2H, superimposed on
H2O), 5.76 (d, 1H), 6.56 (d, 1H), 6.73 (d, 2H), 6.85 (s, 1H), 7.00
(d, 2H), 7.47 (m, 2H), 7.56 (m, 3H), 10.79 (s, 1H), 12.09 (s, 1H).
MS: m/z 469 [M þ H]þ. HRMS (ESþ) calcd for C29H32N4O2
[M þ H]þ m/e 469.2598, found m/e 469.2590.
Acknowledgment. We thank Stephanie Isambert, Simone
Linke, and Josef Zeiler for their excellent technical expertise in
the synthesis of the compounds.
(Z)-3-[(4-Piperidin-1-ylmethyl-phenylamino)-methylene]-2-
oxo-2,3-dihydro-1H-indole-6-carboxylic Acid Ethylamide (47y).
Synthesized according to the general procedure using 4-piperi-
din-1-ylmethyl-phenylamine18 and (E)-3-hydroxymethylene-2-
oxo-2,3-dihydro-1H-indole-6-carboxylic acid ethylamide 15.
Yield: 26% of 47y. 1H NMR: δ 1.11 (t, 3H), 2.30-2.50 (m, 6H),
2.31 (m, 4H), 3.24 (m, 2H, superimposed on H2O), 3.39 (s, 2H), 7.30
(d, 2H), 7.36 (m, 3H), 7.47 (d, 1H), 7.62 (d, 1H), 8.28 (t, 1H), 8.67
(d, 1H), 10.63 (s, 1H), 10.82 (d, 1H). MS: m/z 405 [M þ H]þ.
HRMS (ESþ) calcd for C24H28N4O2 [M þ H]þ m/e 405.2285,
found m/e 405.2282.
Supporting Information Available: Kinase selectivity patterns
for compounds 5 and 47i, the description of the X-ray structure
of compound 5 soaked into the TGFβRI kinase domain (shown
in Figure 1), as well as additional experimental and spectro-
scopic data. This material is available free of charge via the
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TGFβRI- and PDGFRr IC50 Determinations. The inhibition
of the kinase activity of TGFβRI was determined using the
Promega Kinase-Glo kit (Promega, Mannheim, Germany)
according to the manufacturer’s protocol in the presence of
600 nM ATP. N-terminally his-tagged human TGFβRI (aa
162-end) expressed in baculovirus and purified using nickel
affinity chromatography was used at a final concentration of
0.03 μg/mL. The inhibition of the kinase activity of PDGFRR
was determined using the Z0-LYTE assay technology from
Invitrogen (Invitrogen Corporation, Carlsbad, CA, USA)
according to the manufacturer’s protocol. Full-length human
PDGFRR (5.6 nM per assay) and the Tyr4 peptide (2 μM per
assay) were obtained from Invitrogen, too. IC50 values were
determined by the use of the Graph PadPrism software.
Phospho-Smad ELISA Assay. HaCaT cells were cultured in
DMEM (Gibco, Invitrogen Corporation, Carlsbad, CA, USA)
containing 5% FCS. Cells starved for 3 h in DMEM containing
no FCS were preincubated with increasing compound concen-
trations (3.2 nM to 50 μM) or vehicle (DMSO) for 15 min and
subsequently stimulated with 5 ng/mL of TGF-βI (R&D Sys-
tem, Minneapolis, MN, USA). After 60 min, cells were washed
with ice cold PBS and lysed with 100 μL of cell lysis buffer