Synthesis and cytotoxic activity of novel 3-methyl-1-[(4- substitutedpiperazin-1-yl)methyl]-1H-indole derivatives

Article abstract of DOI:10.1055/s-0032-1314868

A series of novel 3-methyl-1-[(4-substitutedpiperazin-1-yl)methyl]-1H- indoles (3a-l) were synthesized and their cytotoxicities were analyzed against 3 different human cell lines, including liver (HUH7), breast (MCF7) and colon (HCT116). The Mannich react

Full text of DOI:10.1055/s-0032-1314868

Original Article 389
Synthesis and Cytotoxic Activity of Novel 3-methyl-
1-[(4-substitutedpiperazin-1-yl)methyl]-1H-indole
Derivatives
Authors
M. Koksal1, M. Yarim1, I. Durmaz2, R. Cetin-Atalay2
Affiliations
1 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Yeditepe University, Kayisdagi, Istanbul, Turkey
2 Department of Molecular Biology and Genetics, Faculty of Science, Bilkent University, Bilkent, Ankara, Turkey
Key words
Abstract
revealed that the tested compounds showed com-
parable activity to the reference drug 5-fluorour-
acil and compounds 3g, 3h, 3i and 3k, had lower
50% inhibition (IC50) concentration than refer-
ence drug. Moreover, the cytotoxic effect of the
most potent compound 3h on HUH7 and MCF7
cells through apoptosis was visualized by Hoechst
staining and compared with paclitaxel, which is a
mitotic inhibitor acting on microtubules. The mor-
phological features of apoptosis were observed as
condensed and fragmented nuclei that are similar
to paclitaxel.
▶
anticancer
apoptosis
indole
●
▼
▶
●
A series of novel 3-methyl-1-[(4-substitutedpi-
perazin-1-yl)methyl]-1H-indoles (3a–l) were syn-
thesized and their cytotoxicities were analyzed
against 3 different human cell lines, including
liver (HUH7), breast (MCF7) and colon (HCT116).
The Mannich reaction of 3-methylindole (1) with
4-substitutedpiperazines (2) and formaldehyde
resulted to the 3-methyl-1-[(4-substitutedpiper-
azin-1-yl)methyl]-1H-indoles (3a–l) in 38–69%
yields. The investigation of anticancer screening
▶
●
▶
Mannich base
1,4-disubstitutedpiperazines
●
▶
●
Introduction
potent anticancer agents have been reported
[7,9–11]. A series of small indole containing
▼
▶
Cancer treatment has been a major attempt of
research in academia and pharmaceutical indus-
try for many years as it is one of the leading
causes of death [1,2]. Recent drug discovery
efforts are highly focused towards design and
synthesis of small molecules as anticancer agents
due to the advantages of easier synthesis and
lower cost. A wide variety of heterocyclic sys-
tems have been explored for the development of
novel chemical entities as a lead molecule in
anticancer drug discovery [3–5].
drugs and clinical candidates are given in Fig. 1.
●
Despite the fact that indole is core structure for
inhibition of tubulin polymerization, numerous
papers have also shown that Mannich base ana-
logs of heterocyclic rings exhibited potent cyto-
toxicity against several human tumor cell lines
[12–18]. Dimmock and Kumar reviewed antican-
cer and cytotoxic properties of Mannich bases
and outlined the effects of these compounds on
anticancer activity [19].
Based on these prior observations, we designed
new Mannich base analogues of 3-methylindole
and aimed to evaluate their in vitro anticancer
screening data against different cancer cell lines.
received 16.03.2012
accepted 22.05.2012
Bibliography
DOI http://dx.doi.org/
10.1055/s-0032-1314868
Published online:
June 29, 2012
Arzneimittelforschung 2012;
62: 389–394
© Georg Thieme Verlag KG
Stuttgart · New York
ISSN 0004-4172
Microtubules, one of the basic components of cell
structure, are involved in a wide number of vital
cellular functions, such as motility, division, shape
maintenance and cellular transport [6]. The
research for novel drugs that can modulate the
microtubule assembly either by inhibition of
tubulin polymerization or by blocking microtu-
bule disassembly are of great interest in antican-
cer therapy. Vincristine and vinblastine are among
the earliest antitumor agents recognized as tubu-
lin polymerization inhibitors since 1965 [7].
The indole ring is represented as the core nucleus
of several tubulin polymerization inhibitors
[7,8]. In the last decade, an increasing number of
small synthetic molecules with indole ring as
Correspondence
M. Koksal
Department of Pharmaceutical
Chemistry
Faculty of Pharmacy
Yeditepe University
26 Agustos Campus
34755 Kayisdagı
Istanbul
T u r k e y
Tel.: +90/216/578 00 66
Fax: +90/216/578 00 68
merickoksal@yeditepe.edu.tr
Materials and Methods
▼
Chemistry
All chemicals and reagents used in current study
were analytical grade. The reactions were moni-
tored by thin layer chromatography (TLC) on
Merck pre-coated silica GF254 plates. Melting
points were determined by using a Mettler
Toledo FP62 capillary melting point apparatus
(Mettler-Toledo, Greifensee, Switzerland) and are
Koksal M et al. Cytotoxic Activity of Indole Derivatives… Arzneimittelforschung 2012; 62: 389–394

390 Original Article
Fig. 1 Structure comparison of small indole
molecules as anticancer agents.
uncorrected. Infrared spectra were recorded on a Perkin-Elmer
1-{[4-(4-Fluorophenyl)piperazin-1-yl]methyl}-3-
methyl-1H-indole (3c) [20]
Spectrum One series FT-IR apparatus (Version 5.0.1) (Perkin
Elmer, Norwalk, CT, USA), using potassium bromide pellets, the
frequencies were expressed in cm−1. The 1H- and 13C-NMR spec-
tra were recorded with a Varian Mercury-400 FT-NMR spec-
trometer (Varian, Palo Alto, CA, USA), using tetramethylsilane as
the internal reference, with chloroform-CDCl3 as solvent, the
chemical shifts were reported in parts per million (ppm) and
coupling constants (J) were given in hertz (Hz). Elemental analy-
ses were performed on LECO 932 CHNS instrument (Leco-932,
St. Joseph, MI, USA) and were within±0.4% of the theoretical
values.
Yield: 51%; m.p.: 109.5°C. IR (KBr) ν (cm−1): 3046–2788 (C-H).
1H NMR (CDCl3, 400MHz) δ (ppm): 7.55 (d, 1H, indole H4, J=8),
7.43 (d, 1H, indole H7, J=8.8), 7.21 (t, 1H, indole H5, J=6.8), 7.12
(t, 1H, indole H6, J=6.8), 6.93 (dd, 2H, phenyl H3, H5, J=6.0,
J’=2.8), 6.90 (s, 1H, indole H2), 6.81 (dd, 2H, phenyl H2, H6, J=9.4,
J’=4.4), 4.80 (s, 2H, N-CH2-N), 3.08 (t, 4H, piperazine H3, H5,
J=5.2), 2.69 (t, 4H, piperazine H2, H6, J=5.2), 2.33 (s, 3H, -CH3).
13C NMR (CDCl3, 400MHz) δ (ppm): 158.65, 156.27, 148.14,
137.48, 129.17, 126.33, 121.92, 119.20, 118.25, 115.72, 111.15,
110.01 (aromatics), 67.86 (C-CH2-N), 50.72 (piperazine C3, C5),
50.33 (piperazine C2, C6), 9.83 (-CH3). Anal. calcd. for C20H22FN3
(323.41): C, 74.28; H, 6.86; N, 12.99. Found: C, 74.25; H, 6.75; N,
12.96.
General procedure for the synthesis of 3-methyl-1-[(4-
substitutedpiperazin-1-yl)methyl]-1H-indoles (3a–3l)
A mixture of 3-methylindole (1) (0.39g, 0.003mol), the appro-
priate N-substituted amine (2) (0.003mol) and 37% formalde-
hyde solution (1mL) in ethanol (15mL), was refluxed for 3–5h.
The crude products were either precipitated or it was necessary
to add water in case not precipitated. The crude products were
filtered, washed with water, dried and crystallized from appro-
priate solvents.
1-{[4-(3-Chlorophenyl)piperazin-1-yl]methyl}-3-
methyl-1H-indole (3d)
Yield: 42%; m.p.: 95.4°C. IR (KBr) ν (cm−1): 3043–2718 (C-H).
1H NMR (CDCl3, 400MHz) δ (ppm): 7.55 (d, 1H, indole H4, J=8.0),
7.41 (d, 1H, indole H7, J=8.4), 7.21 (t, 1H, indole H5, J=8.0), 7.14–
7.09 (m, 2H, indole H6+phenyl H2), 6.93 (s, 1H, indole H2), 6.81–
6.69 (m, 3H, phenyl), 4.78 (s, 2H, N-CH2-N), 3.14 (t, 4H,
piperazine H3, H5, J=5.2), 2.65 (t, 4H, piperazine H2, H6, J=5.2),
2.32 (s, 3H, -CH3). Anal. calcd. for C20H22ClN3 (339.86): C, 70.68;
H, 6.52; N, 10.43. Found: C, 70.41; H, 6.65; N, 10.38.
3-Methyl-1-[(4-phenylpiperazin-1-yl)methyl]-1H-indole
(3a)
Yield: 69%; m.p.: 160.2°C. IR (KBr) ν (cm−1): 3054–2797 (C-H).
1H NMR (CDCl3, 400MHz) δ (ppm): 7.56 (d, 1H, indole H4,
J=11.6), 7.43 (d, 1H, indole H7, J=8), 7.25-7.20 (m, 3H, indole
H5+phenyl H2, H6), 7.12 (t, 1H, indole H6, J=8), 6.95 (s, 1H, indole
H2), 6.88-6.82 (m, 3H, phenyl H3, H4, H5), 4.81 (s, 2H, N-CH2-N),
3.17 (t, 4H, piperazine H3, H5, J=5.2), 2.69 (t, 4H, piperazine H2,
H6, J=5.2), 2.33 (s, 3H, -CH3). Anal. calcd. for C20H23N3 (305.42):
C, 78.65; H, 7.59; N, 13.76. Found: C, 78.57; H, 7.56; N, 13.60.
1-{[4-(4-Chlorophenyl)piperazin-1-yl]methyl}-3-
methyl-1H-indole (3e)
Yield: 58%; m.p.: 144.1°C. IR (KBr) ν (cm−1): 3041–2794 (C-H).
1H NMR (CDCl3, 400MHz) δ (ppm): 7.56 (d, 1H, indole H4, J=7.6),
7.41 (d, 1H, indole H7, J=8.4), 7.24–7.10 (m, 4H, indole H5,
H6+phenyl H3, H5), 6.93 (s, 1H, indole H2), 6.76 (d, 2H, phenyl H2,
H6, J=9.2), 4.79 (s, 2H, N-CH2-N), 3.11 (t, 4H, piperazine H3, H5,
J=5.2), 2.67 (t, 4H, piperazine H2, H6, J=5.2), 2.32 (s, 3H, -CH3).
Anal. calcd. for C20H22ClN3 (339.86): C, 70.68; H, 6.52; N, 10.43.
Found: C, 70.56; H, 6.56; N, 10.39.
1-{[4-(2-Fluorophenyl)piperazin-1-yl]methyl}-3-methyl-
1H-indole (3b)
Yield: 60%; m.p.: 120.2°C. IR (KBr) ν (cm−1): 3045–2788 (C-H).
1H NMR (CDCl3, 400MHz) δ (ppm): 7.56 (d, 1H, indole H4, J=7.6),
7.43 (d, 1H, indole H7, J=8.4), 7.22 (t, 1H, indole H5, J=8.0), 7.12
(t, 1H, indole H6, J=6.8), 7.05-6.88 (m, 5H, indole H2+phenyl),
4.81 (s, 2H, N-CH2-N), 3.08 (t, 4H, piperazine H3, H5, J=4.8), 2.72
(t, 4H, piperazine H2, H6, J=4.8), 2.33 (s, 3H, -CH3). Anal. calcd.
for C20H22FN3 (323.41): C, 74.28; H, 6.86; N, 12.99. Found: C,
74.24; H, 6.89; N, 12.98.
1-{[4-(2-Methoxyphenyl)piperazin-1-yl]methyl}-3-
methyl-1H-indole (3f)
Yield: 53%; m.p.: 114.5°C. IR (KBr) ν (cm−1): 3046–2788 (C-H).
1H NMR (CDCl3, 400MHz) δ (ppm): 7.56 (d, 1H, indole H4, J=8.0),
7.43 (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), 7.00-6.89 (m, 4H, indole H2+phenyl H3,
H4, H5), 6.82 (d, 1H, phenyl H6, J=7.2), 4.82 (s, 2H, N-CH2-N),
Koksal M et al. Cytotoxic Activity of Indole Derivatives… Arzneimittelforschung 2012; 62: 389–394

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

392 Original Article
Fig. 2 Synthesis of compounds 3a–3l.
Results and Discussion
Table 1 Cytotoxic activity data for compounds 3a-l.
▼
Chemistry
The target 3-methyl-1-[(4-substitutedpiperazin-1-yl)methyl]-
1H-indole derivatives (3a–l) have been prepared by Mannich
N
▶
reaction between indole and appropriate piperazines ( Fig. 2).
●
N
Although 2 of compounds were registered in literature by our
lab group for sigma receptor binding studies [20], for the entirety
of the 3-methyl-1-substitutedindole group, data for them are
given again.
N
R
Compound
R
IC50 (μM)*
The prepared Mannich bases showed IR bands at 3054–2832
cm−1 (C-H) for all derivatives and a typical nitrile band at
2211cm−1 for Compound 3h. In the 1 H NMR spectra, the signals
of the respective protons of the prepared compounds 3a–l were
verified on the basis of their chemical shifts, multiplicities and
coupling constants. General characteristic peaks of 3-methylin-
dole ring were observed at about δ 7.55 (d, 1H, indole H4), 7.40
(d, 1H, indole H7), 7.22 (t, 1H, indole H5), 7.20 (t, 1H, indole H6),
6.90 (s, 1H, indole H2), and 2.30 (s, 3H, -CH3). The 1H NMR spec-
tra of compounds 3a–l showed a singlet with 2H intensity each,
in the range 4.84–4.76ppm assigned to the methylene protons,
confirming the Mannich condensation. The chemical shift of the
aliphatic protons of the piperazine ring were observed in the
range 3.40–3.08 (t, 4H, piperazine H3, H5) and 2.72–2.63ppm (t,
4H, piperazine H2, H6). In 13C-NMR of 3c and 3h significant
peaks at δ 67.74 (C-CH2-N), 50.21 (piperazine C3, C5), 47.21 (pip-
erazine C2, C6), 9.87 (-CH3) were observed.
HUH7
MCF7
HCT116
3a
Phenyl
NI
NI
NI
3b
3c
2-Fluorophenyl
4-Fluorophenyl
3-Chlorophenyl
4-Chlorophenyl
NI
23.30
40.95
68.81
64.73
41.29
13.69
23.48
NI
14.48
NI
NI
3d
3e
3f
NI
63.30
64.55
26.88
19.60
8.75
NI
2-Methoxyphenyl
3-Methoxyphenyl
4-Cyanophenyl
4-Nitrophenyl
NI
3g
3h
3i
24.76
14.38
25.01
NI
15.91
28.15
16.62
23.86
>0.01
18.78
3j
4-Methylphenyl
2,3-Dimethylphenyl
2-Phenylethyl
NI
3k
3l
22.20
NI
22.89
26.23
>0.01
3.50
CPT
0.15
5-FLU
30.70
*All the experiments were conducted in triplicate (1< R2 <0.8);
NI: no inhibition at a concentration lower than 100μM;
CPT: Camptothecin; 5-FLU: 5-fluorouracil
Biological activity
HUH7 and MCF7 cells through apoptosis and compared with
paclitaxel, which is a mitotic inhibitor acting on microtubules.
Apoptotic morphological alterations were visualized by Hoechst
33258 staining under fluorescent microscope. The morphologi-
cal features of apoptosis, i.e., condensation of chromatin and
fragmentation of the nucleus, were examined. DMSO treated
control cells showed round and homogeneous nuclei, whereas
The cytotoxic activity of the synthesized compounds 3a–3l was
investigated on liver (HUH7), breast (MCF7) and colon (HCT116)
cancer cell lines, by means of sulphorhodamine B (SRB) assays in
triplicate.
▶
▶
●
As shown in Table 1 and Fig. 3, all tested compounds were
●
screened on 3 different human cell lines with mean 50% inhibi-
tion (IC50) in micromolar concentration range. For liver cell line,
HUH7, most of the compounds showed no activity at a concen-
tration higher than 100μM. However, it is interesting that com-
pounds with cytotoxic activity (3g, 3h, 3i and 3k) exhibited
better cell growth inhibition than standard drug 5-fluorouracil
(5-FU) with IC50 values of 24.76, 14.38, 25.01 and 22.20μM,
respectively. The cytotoxic effects were not impressive against
MCF7 breast cancer cells, all of the compounds showed cell via-
bility with IC50 values ranging from 13.69–68.81μM concentra-
tions. It was noteworthy that the cytotoxic effects were more
pronounced against colon carcinoma cell line, HCT116. Similar to
HUH7 cell line, compounds 3h (IC50 =8.75μM), 3i (IC50 =15.91μM)
and 3k (IC50 =16.62μM) have better IC50 values than 5-FLU
(IC50 =18.78μM) and also compound 3h possessed 8.75μM value,
which represents good druggable cytotoxic activity.
3h and paclitaxel treated-cells showed condensed and frag-
▶
mented nuclei ( Fig. 4) .
●
In order to study the structure-activity relationship (SAR), these
results of activity screening are not clear enough, but on the
other hand, some observations can be stated. Compound 3a
with non-substituted phenyl indicated no cytotoxic activity
against all 3 cell lines. Interestingly, the SAR study reveals that
substitution on the phenyl ring plays an important role. Among
the compounds, 3g, 3h, 3i and 3k which have 3-OCH3, 4-CN,
4-NO2 and 2,3-diCH3 substituents on phenyl ring showed better
activity than 5-FLU. The activity results of these 4 compounds
are almost parallel for both liver HUH7 and colon HCT116 cells.
Generally, the derivatives carrying halogen on different posi-
tions of phenyl exhibited moderate or low activity results; the
only exception is compound 3b carrying o- fluoro substituent on
phenyl ring for HCT116 cell line. The findings for MCF7 repre-
Considering the indole core structure of the compounds, we
showed the cytotoxic effect of the most active compound 3h on
Koksal M et al. Cytotoxic Activity of Indole Derivatives… Arzneimittelforschung 2012; 62: 389–394

Original Article 393
Fig. 3 Inhibitory effects of compounds on HUH7,
MCF7 and HCT116 cell growth.
Fig. 4 The apoptotic effect of inhibitors on the
morphology of the nuclear chromatin in HUH7
(upper panel) and MCF7 (lower panel) cells.
sented that the compounds were inactive for breast carcinoma.
Especially, in HUH7 liver cell line, although halogen substituted
compounds had no cytotoxic activity, introduction of an elec-
tron rich substituent such as cyano, nitro to phenyl group have
produced compounds with significant IC50 values.
Conclusion
▼
In conclusion, the present study showed that the synthesized
compounds could be used as a part of a template for future
development through modification and derivatization to design
Koksal M et al. Cytotoxic Activity of Indole Derivatives… Arzneimittelforschung 2012; 62: 389–394

394 Original Article
7 Brancale A, Silvestri R. Indole, a core nucleus for potent inhibitors of
tubulin polymerization. Med Res Rev 2007; 27: 209–238
8 Duflos A, Kruczynski A, Barret JM. Novel aspects of natural and modified
vinca alkaloids. Curr Med Chem Anticancer Agents 2002; 2: 55–70
9 Kumar D, Sundaree S, Johnson EO et al. A n e fficient synthesis and bio-
logical study of novel indolyl-1,3,4-oxadiazoles as potent anticancer
agents. Bioorg Med Chem Letters 2009; 19: 4492–4494
10 Marchand P, Antoine M, Le Baut G et al. Synthesis and structure-
activity relationships of N-aryl(indol-3-yl)glyoxamides as antitumor
agents. Bioorg Med Chem 2009; 17: 6715–6727
more potent anticancer compounds that carry indole core struc-
ture. As a preliminary work, this group representing 1-substi-
tuted indoles can build our research interest and experience in
the discovery of novel promising antitumor agents. With combi-
nation of these and future indole-based series, a QSAR study can
be valuable and these results may be helpful for extensive func-
tionalization of the substituent of the indole scaffold on a lead
compound in future.
11 Ziedan NI, Stefanelli F, Fogli S et al. Design, synthesis and pro-apoptotic
antitumour properties of indole-based 3,5-disubstituted oxadiazoles.
Eur J Med Chem 2010; 45: 4523–4530
Acknowledgement
12 Dimmock JR, Kandepu NM, Hetherington M et al. Cytotoxic Activities
of Mannich Bases of Chalcones and Related Compounds. J Med Chem
1998; 41: 1014–1026
▼
The chemistry part of this work was supported by a grant from
The Scientific & Technological Research Council of Turkey
(TUBITAK) (Project No. 108S009).
13 Aboraia AS, Abdel-Rahman HM, Mahfouz NM
et al. N o v e l
5-(2-hydroxyphenyl)-3-substituted-2,3-dihydro-1,3,4-oxadiazole-2-
thione derivatives: Promising anticancer agents. Bioorg Med Chem
2006; 14: 1236–1246
14 Ivanova Y, Momekov G, Petrov O et al. Cytotoxic Mannich bases of
6-(3-aryl-2-propenoyl)-2(3H)-benzoxazolones. Eur
2007; 42: 1382–1387
J Med Chem
Conflict of Interest
15 Reddy MVB, Su CR, Chiou WF et al. Design, synthesis, and biological
evaluation of Mannich bases of heterocyclic chalcone analogs as cyto-
toxic agents. Bioorg Med Chem 2008; 16: 7358–7370
▼
The authors have declared no conflict of interest.
16 Solomon VR, Hu C, Lee H. Hybrid pharmacophore design and synthesis
of isatin-benzothiazole analogs for their anti-breast cancer activity.
Bioorg Med Chem 2009; 17: 7585–7592
17 Chen J, Lou J, Liu T et al. Synthesis and in-vitro antitumor activities of
some Mannich bases of 9-alkyl-1,2,3,4-tetrahydrocarbazole-1-ones.
Arch Pharm Chem Life Sci 2009; 342: 165–172
18 Shaw AY, Chang CY, Hsu MY et al. Synthesis and structure-activity
relationship study of 8-hydroxyquinoline-derived Mannich bases as
anticancer agents. Eur J Med Chem 2010; 45: 2860–2867
19 Dimmock JR, Kumar P. Anticancer and Cytotoxic properties of Mannich
bases. Curr Med Chem 1997; 4: 1–22
20 Yarim M, Koksal M, Schepmann D et al. Synthesis and in vitro evalu-
ation of novel indole-based sigma receptors ligands. Chem Biol Drug
Des 2011; 78: 869–875
References
1 Waring MJ. The Search for New Anticancer Drugs. Dordrecht, The
Netherlands: Kluwer Academic Publishers, 1992; 1–18
2 Jemal A, Siegel R, Ward E et al. Cancer statistics, 2008. CA Cancer J
Clin 2008; 58: 71–96
3 Bacher G, Beckers T, Emig P et al. New small-molecule tubulin inhibi-
tors. Pure Appl Chem 2001; 73: 1459–1464
4 Buolamwini JK, Addo J, Kamath S et al. Small molecule antagonists
of the MDM2 oncoprotein as anticancer agents. Curr Cancer Drug
Targets 2005; 5: 57–68
5 Dass CR, Choong PFM. Targeting of small molecule anticancer drugs to
the tumour and its vasculature using cationic liposomes: lessons from
gene therapy. Cancer Cell International 2006; 6: 17
6 Downing KH, Nogales E. Tubulin and microtubule structure. Curr Opin
Cell Biol 1998; 10: 16–22
Koksal M et al. Cytotoxic Activity of Indole Derivatives… Arzneimittelforschung 2012; 62: 389–394