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(M+H)+, 344 (M+Na)+; Anal. Calcd for C20H19NO3: C, 74.75; H, 5.96;
N, 4.36. Found: C, 74.70; H, 5.93; N, 4.27.
7H), 7.57 (dd, J1 = 3 Hz, J2 = 6.5 Hz, 1H), 7.85–7.91 (m, 3H), 8.60
(s, 1H); 13C NMR (CDCl3): d 42.3, 42.8, 110.0, 110.1, 111.4, 111.7,
112.0, 118.0, 119.2, 120.9, 121.5, 123.2, 127.2, 128.0, 128.9,
135.1, 135.6, 136.9, 138.2, 138.8, 191.9. MS (ESI) m/z 315
(M+H)+; Anal. Calcd for C21H18N2O: C, 80.23; H, 5.77; N, 8.91.
Found: C, 80.17; H, 5.72; N, 8.90.
4.1.2.8. 3-(2,6-Dimethoxy-phenyl)-1-(1-methyl-1H-indol-3-yl)-
propenone (8). Mp: 161–163 °C; 1H NMR (CDCl3): d 3.85 (s,
3H), 3.93 (s, 6H), 6.59 (d, J = 6 Hz, 1H), 7.25–7.34 (m, 5H), 7.80 (s,
1H), 7.85 (d, J = 15.2 Hz, 1H), 8.26 (d, J = 15.2 Hz, 1H), 8.54 (br s,
1H); 13C NMR (CDCl3): d 41.2, 56.0, 106.5, 109.1, 111.2, 111.7,
119.0, 120.8, 122.0, 122.5, 125.9, 130.3, 134.8, 136.9, 145.7,
159.7, 191.1. MS (ESI) m/z 322 (M+H)+, 344 (M+Na)+; Anal. Calcd
for C20H19NO3: C, 74.75; H, 5.96; N, 4.36. Found: C, 74.72; H,
5.91; N, 4.32.
4.1.2.15. 3-(1-Methyl-1H-indol-5-yl)-1-(2,4,6-trimethoxy-phe-
nyl)-propenone (15). Mp: 159–161 °C; 1H NMR (CDCl3): d 3.78
(s, 6H), 3.80 (s, 3H), 3.95 (s, 3H), 6.18 (s, 2H), 6.30 (br s, 1H), 6.97
(d, J = 15.7 Hz, 1H), 7.07 (br s, 1H), 7.29 (d, J = 15.6 Hz, 7.48 (d,
J = 15.7 Hz, 1H), 7.76 (s, 1H); 13C NMR (CDCl3): d 42.4, 55.7, 56.1,
93.7, 100.6, 103.7, 110.4, 111.2, 118.9, 122.4, 128.0, 129.3, 136.1,
138.2, 145.5, 163.0, 168.2, 189.2. MS (ESI) m/z 352 (M+H)+, 374
(M+Na)+; Anal. Calcd for C21H21NO4: C, 71.78; H, 6.02; N, 3.99.
Found: C, 71.72; H, 5.97; N, 3.95.
4.1.2.9.
1-(1-Methyl-1H-indol-3-yl)-3-(2,4,6-trimethoxy-phe-
nyl)-propenone (9). Mp: 183–185 °C; 1H NMR (CDCl3): d 3.85
(s, 3H), 3.86 (s, 3H), 3.92 (s, 6H), 6.15 (s, 2H), 7.31–7.37 (m, 3H),
7.80 (s, 1H), 7.74 (s, 1H), 7.78 (d, J = 15.1 Hz, 1H), 8.25 (d,
J = 15.1 Hz, 1H), 8.53 (br s, 1H); 13C NMR (CDCl3): d 42.2, 55.9,
56.6, 93.1, 102.4, 110.2, 111.1, 118.1, 120.5, 121.7, 122.4, 126.4,
135.2, 137.2, 160.7, 162.2, 190.8. MS (ESI) m/z 352 (M+H)+, 374
(M+Na)+; Anal. Calcd for C21H21NO4: C, 71.78; H, 6.02; N, 3.99.
Found: C, 71.75; H, 5.99; N, 3.92.
4.1.3. Preparation of arylindolylpropenones solutions
Compounds were dissolved in DMSO to a final concentration of
10ꢀ2 M. These solutions were then directly diluted into culture
medium. In order to test its innocuousness, DMSO alone was used
at the same dilution than the other compounds.
4.1.2.10. 1-(1-Methyl-1H-indol-3-yl)-3-(3,4,5-trimethoxy-phe-
nyl)-propenone (10). Mp: 201–203 °C; 1H NMR (CDCl3): d 3.89
(s, 3H), 3.91 (s, 3H), 3.94 (s, 6H), 6.87 (s, 2H), 7.24–7.38 (m, 6H),
7.80 (s, 1H), 7.73 (d, J = 14.9 Hz, 1H), 7.89 (s, 1H), 8.52 (br s, 1H);
13C NMR (CDCl3): d 43.0, 56.0, 56.3, 103.6, 110.8, 111.3, 119.6,
120.5, 122.0, 122.9, 126.1, 130.2, 134.2, 136.6, 139.3, 151.7,
191.8. MS (ESI) m/z 352 (M+H)+, 374 (M+Na)+; Anal. Calcd for
4.2. Cell culture
Human bladder cancer cell line RT112 was obtained from Cell
Lines Service (Eppelheim, Germany). RT112 cells were cultured in
RPMI 1640 medium supplemented with 10% (v/v) fetal calf serum
(FCS) and 2 mM glutamine (Invitrogen Life Technologies, Paisley,
UK). Cells were maintained at 37 °C in a 5% CO2-humidified
atmosphere and tested to ensure freedom from mycoplasma
contamination.
C21H21NO4: C, 71.78; H, 6.02; N, 3.99. Found: C, 71.73; H, 6.00; N,
3.95.
4.1.2.11.
1-(5-Methoxy-1-methyl-1H-indol-3-yl)-3-(2,4,6-tri-
methoxy-phenyl)-propenone (11). Mp: 206–208 °C; 1H NMR
(CDCl3): d 3.39 (s, 3H), 3.86 (s, 3H), 3.41 (s, 3H), 3.46 (s, 9H), 5.70
(s, 2H), 6.50 (d, J = 7 Hz, 1H), 6.78–6.85 (m, 2H), 7.26 (d,
J = 15 Hz, 1H), 7.30 (s, 1H), 7.65 (s, 1H), 7.79 (d, J = 15.1 Hz, 1H);
13C NMR (CDCl3): d 41.4, 55.9, 56.2, 56.6, 93.0, 101.6, 102.3,,
109.2, 110.2, 112.4, 121.4, 127.6, 129.3, 135.4, 145.0, 155.1,
159.2, 162.3, 190.1. MS (ESI) m/z 382 (M+H)+, 404 (M+Na)+; Anal.
Calcd for C22H23NO5: C, 69.28; H, 6.08; N, 3.67. Found: C, 69.24;
H, 6.04; N, 3.62.
4.3. Cell proliferation assay
Inhibition of cell proliferation by arylindolylpropenones deriva-
tives was measured by a MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) assay. RT112 cells were seeded into
96-well plates (3 ꢁ 103 cells/well) in 100
ll of culture medium.
After 24 h, cells were treated with cisplatin (Sigma-Aldrich, Lyon
France) or arylindolylpropenones compounds at various concen-
trations. In parallel, a control with DMS0 (vehicle alone) at the
same dilutions was done. Following incubation for 48 h, 10 ll of
a MTT (Euromedex, Mundolsheim, France) stock solution in PBS
at 5 mg/ml was added in each well and the plates were incubated
at 37 °C for 3 h. Plates were then centrifugated 5 min at 1500 rpm
before the medium was discarded and replaced with DMSO
4.1.2.12. 1-Methyl-3-[3-(2,4,6-trimethoxy-phenyl)-acryloyl]-1H-
indole-5-carboxylic acid (12). Mp: 205–207 °C; 1H NMR
(CD3OD): d 3.91 (s, 3H), 3.94 (s, 3H), 4.01 (s, 6H), 3.46 (s, 9H),
6.29 (s, 2H), 7.45 (d, J = 6.5 Hz, 1H), 7.93 (d, J = 15.6 Hz, 1H), 8.03
(d, J = 6.5 Hz, 1H), 8.14 (s, 1H), 8.23 (d, J = 15.5 Hz, 1H), 9.10 (s,
1H); 13C NMR (CD3OD): d 43.3, 55.9, 56.7, 93.2, 103.2, 111.6,
112.4, 115.0, 122.0, 122.9, 126.9, 135.5, 143.6, 146.7, 160.1,
162.2, 169.1, 192.1. MS (ESI) m/z 396 (M+H)+; Anal. Calcd for
(100 ll/well) to solubilize water-insoluble purple formazan crys-
tals. After 15 min under shaking, absorbance was measured on
an ELISA reader (Tecan, Männedorf, Switzerland) at a test wave-
length of 570 nm and a reference wavelength of 650 nm. Absor-
bance obtained by cells treated with the same dilution of the
vehicle alone (DMSO) was rated as 100% of cell survival. Each data
point is the average triplicates of three independent experiments.
C22H21NO6: C, 66.83; H, 5.35; N, 3.54. Found: C, 66.79; H, 5.28;
N, 3.52.
4.1.2.13. 1,3-Bis-(1-methyl-1H-indol-3-yl)-propenone (13). Mp:
238–240 °C; 1H NMR (CD3OD): d 3.86 (s, 6H), 7.25–7.50 (m, 8H),
7.68–7.71 (m, 2H), 8.31–8.38 (m, 2H); 13C NMR (CD3OD): d 42.0,
42.5, 110.4, 111.0, 111.2, 111.9, 112.4, 118.1, 119.2, 120.1, 120.5,
122.3, 123.5, 126.7, 126.9, 134.4, 135.5, 136.8, 137.3, 145.0,
192.2. MS (ESI) m/z 315 (M+H)+; Anal. Calcd for C21H18N2O: C,
80.23; H, 5.77; N, 8.91. Found: C, 80.20; H, 5.79; N, 8.89.
4.4. Cell cycle analysis
RT112 cells were cultured in 6-wells plates (2 ꢁ 105 cells/well)
for 24 h before treatment with DMSO or compounds 9, 11 and 14
for 24 or 48 h. Trypsinized and floating cells were then pooled,
fixed with 70% ethanol, washed with PBS and stained with 20 lg/
ml propidium iodide (PI) in the presence of 0.5 mg/ml of RNAse
(Sigma-Aldrich, Lyon France). Data acquisitions were performed
4.1.2.14. 3-(1-Methyl-1H-indol-5-yl)-1-(1-methyl-1H-indol-3-
yl)-propenone (14). Mp: 202–204 °C; 1H NMR (CDCl3): d 3.80 (s,
3H), 3.85 (s, 3H), 6.54 (d, J = 3 Hz), 7.07(d, J = 3 Hz), 7.28–7.39 (m,
with
a FACScan (Becton Dickinson France, Le Pont-De-Claix,
France) equipped with a 488 nm laser Argon and PI fluorescence