Original Article 391
3.80 (s, 3H, O-CH3), 3.05 (bs, 4H, piperazine H3, H5), 2.74 (t, 4H,
piperazine H2, H6, J=4.8), 2.32 (s, 3H, -CH3). Anal. calcd. for
C21 H25N3O (335.44): C, 75.19; H, 7.51; N, 12.53. Found: C, 75.12;
H, 7.40; N, 12.43.
(s, 1H, indole H2), 6.90 (d, 2H, phenyl H4, H6, J=8.8), 4.84 (s, 2H,
N-CH2-N), 2.88 (t, 4H, piperazine H3, H5, J=4.4), 2.72 (bs, 4H,
piperazine H2, H6), 2.34 (s, 3H, -CH3), 2.23 (s, 3H, -CH3), 2.14
(s, 3H, -CH3). Anal. calcd. for C22H27N3 (333.47): C, 79.24; H,
8.16; N, 12.60. Found: C, 79.10; H, 8.11; N, 12.52.
1-{[4-(3-Methoxyphenyl)piperazin-1-yl]methyl}-3-
methyl-1H-indole (3g)
3-Methyl-1-{[4-(2-phenylethyl)piperazin-1-yl]methyl}-
1H-indole (3l) [20]
Yield: 57%; m.p.: 111.6°C. IR (KBr) ν (cm−1): 3050–2842 (C-H).
1H NMR (CDCl3, 400MHz) δ (ppm): 7.56 (d, 1H, indole H4, J=8.0),
7.42 (d, 1H, indole H7, J=8.4), 7.24–7.10 (m, 3H, indole H5,
H6,+phenyl H2), 6.94 (s, 1H, indole H2), 6.49–6.38 (m, 3H, phe-
nyl), 4.80 (s, 2H, N-CH2-N), 3.76 (s, 3H, O-CH3), 3.16 (t, 4H, pip-
erazine H3, H5, J=5.2), 2.67 (t, 4H, piperazine H2, H6, J=4.8), 2.32
(s, 3H, -CH3). Anal. calcd. for C21 H25N3O (335.44): C, 75.19; H,
7.51; N, 12.53. Found: 75.18; H, 7.53; N, 12.45.
Yield: 39%; m.p.: 122.9°C. IR (KBr) ν (cm−1): 3022–2763 (C-H).
1H NMR (CDCl3, 400MHz) δ (ppm): 7.54 (d, 1H, indole H4, J=8.0),
7.41(d, 1H, indole H7, J=8.0), 7.28–7.16 (m, 6H, indol
H5+phenyl), 7.11 (t, 1H, indole H6, J=7.2), 6.92 (s, 1H, indole H2),
4.76 (s, 2H, N-CH2-N), 2.75 (m, 2H, -CH2-C6H5), 2.60-2.54 (m,
10H, -CH2-N), 2.31 (s, 3H, -CH3). Anal. calcd. for C28H31N3
(409.57): C, 79.24; H, 8.16; N, 12.56. Found: C, 79.20; H, 8.15; N,
12.56.
1-{[4-(4-Cyanophenyl)piperazin-1-yl]methyl}-3-methyl-
1H-indole (3h)
Cytotoxicity studies
Yield: 48%; m.p.: 139.5°C. IR (KBr) ν (cm−1): 2938–2832 (C-H),
2211 (C≡N). 1H NMR (CDCl3, 400MHz) δ (ppm): 7.56 (d, 1H,
indole H4, J=7.6), 7.44–7.39 (m, 3H, indole H7+phenyl H3, H5),
7.21 (t, 1H, indole H5, J=7.2), 7.12 (t, 1H, indole H6, J=7.6), 6.91
(s, 1H, indole H2), 6.74 (d, 2H, phenyl H2, H6, J=8.8), 4.78 (s, 2H,
N-CH2-N), 3.26 (t, 4H, piperazine H3, H5, J=4.8), 2.63 (t, 4H, pip-
erazine H2, H6, J=4.8), 2.32 (s, 3H, -CH3). 13C NMR (CDCl3,
400MHz) δ (ppm): 153.44, 137.44, 133.71, 129.20, 126.32,
122.08, 120.30 (aromatics), 119.32 (-CN), 114.47, 111.30, 109.98,
100.49 (aromatics), 67.74 (C-CH2-N), 50.21 (piperazine C3, C5),
47.21 (piperazine C2, C6), 9.87 (-CH3). Anal. calcd. for C21 H22N4
(330.43): C, 76.33; H, 6.71; N, 16.96. Found: C, 76.29; H, 6.46; N.
16.93.
Cell culture
The human cancer cell lines were grown in Dulbecco’s Modified
Eagle’s Medium (DMEM), with 10% fetal bovine serum (FBS) and
1% penicillin. They were incubated in 37 °C incubators contain-
ing 5% CO2 and 95% air.
NCI-60 Sulphorhodamine B (SRB) assay
Cancer cells (range of 2000 cells/well to 5000 cells/well) were
inoculated into 96-well plates in 200μL of media and incubated
in 37 °C incubators containing 5% CO2 and 95% air. After a 24h
incubation period, one plate for each cell line was fixed with
100μL of 10% ice-cold trichloroacetic acid (TCA). This plate rep-
resents the behavior of the cells just prior to compound treat-
ment and is accepted as the time-zero plate. The compounds to
be tested were solubilized in dimethyl sulfoxide (DMSO) to a
final concentration of 40mM and stored at +4 °C. While treating
the cells with the compounds, the corresponding volume of the
compound was applied to the cell to achieve the desired drug
concentration and diluted through serial dilution (40, 20, 10, 5,
2.5μM). After drug treatment, the cells were incubated in 37 °C
incubators containing 5% CO2 and 95% air for 72h. Following the
termination of the incubation period after drug treatment, the
cells were fixed with 100μL 10% ice-cold TCA and incubated in
the dark at +4°C for 1h. Then the TCA was washed away with
ddH2O 5 times and the plates were left to air dry. In the final
step, the plates were stained with 100μL of 0.4% SRB
(cat.86183-5g from Sigma) solution in 1% acetic acid solution.
Following staining, the plates were incubated in dark for 10min
at room temperature. The unbound dye was washed away using
1% acetic acid and the plates were left to air dry. To measure the
absorbance results, the bound stain was then solubilized using
200μL of 10mM Tris-Base. Camptothecin was the positive con-
trol and 5-Fluorouracil (5-FU) was standard drug for the cyto-
toxic effect. The OD values were obtained at 515nm.
3-Methyl-1-{[4-(4-nitrophenyl)piperazin-1-yl]methyl}-
1H-indole (3i)
Yield: 48%; m.p.: 134°C. IR (KBr) ν (cm−1): 3042–2852 (C-H),
1332 (NO2). 1H NMR (CDCl3, 400MHz) δ (ppm): 8.08 (d, 2H,
phenyl H3, H5, J=9.6 ), 7.56 (d, 1H, indole H4, J=7.6), 7.42 (d, 1H,
indole H7, J=8.4 ), 7.22 (t, 1H, indole H5, J=7.2), 7.15 (t, 1H,
indole H6, J=7.6), 6.93 (s, 1H, indole H2), 6.74 (d, 2H, phenyl H2,
H6, J=9.6), 4.82 (s, 2H, N-CH2-N), 3.40 (t, 4H, piperazine H3, H5,
J=4.8), 2.68 (t, 4H, piperazine H2, H6, J=4.8), 2.33 (s, 3H, -CH3).
Anal. calcd. for C20H22N4O2 (350.41): C, 68.55; H, 6.33; N, 15.99.
Found: 68.71; H, 6.27; 15.94.
3-Methyl-1-{[4-(4-methylphenyl)piperazin-1-yl]
methyl}-1H-indole (3j)
Yield: 45%; m.p.: 117.3°C. IR (KBr) ν (cm−1): 3048–2789 (C-H).
1H NMR (CDCl3, 400MHz) δ (ppm): 7.55 (d, 1H, indole H4, J=8.0),
7.42 (d, 1H, indole H7, J=8.4), 7.21 (t, 1H, indole H5, J=7.6), 7.11
(t, 1H, indole H6, J=6.8), 7.04 (d, 2H, phenyl H3, H5, J=8.4), 6.94
(s, 1H, indole H2), 6.78 (d, 2H, phenyl H2, H6, J=8.4), 4.80 (s, 2H,
N-CH2-N), 3.11 (t, 4H, piperazine H3, H5, J=4.8), 2.68 (t, 4H, pip-
erazine H2, H6, J=5.2), 2.32 (s, 3H, -CH3), 2.25 (s, 3H, -CH3). Anal.
calcd. for C21 H25N3 (319.44): C, 78.96; H, 7.89; N, 13.15. Found:
C, 79.28; H, 7.85; N, 12.97.
Hoechst staining
Apoptotic morphological alterations were visualized by Hoechst
33258 staining under fluorescent microscope. Cells were inocu-
lated into 6-well plates (60000cell/well) and incubated for 24h.
Then, they were treated with solvent DMSO, 3h or paclitaxel and
incubated for 72h. Fixation of the cells were accomplished by
methanol followed by Hoechst 33258 staining. Finally, cells were
destained with ddH2O and observed under fluorescent micro-
scope.
3-Methyl-1-{[4-(2,3-dimethylphenyl)piperazin-1-yl]
methyl}-1H-indole (3k)
Yield: 43%; m.p.: 108.7°C. IR (KBr) ν (cm−1): 3053–2785 (C-H).
1H NMR (CDCl3, 400MHz) δ (ppm): 7.57 (d, 1H, indole H4, J=7.6),
7.45 (d, 1H, indole H7, J=8.0), 7.22 (t, 1H, indole H5, J=7.2), 7.15
(t, 1H, indole H6, J=7.6), 7.05 (t, 1H, phenyl H5, J=6.8), 6.96
Koksal M et al. Cytotoxic Activity of Indole Derivatives… Arzneimittelforschung 2012; 62: 389–394