Design, synthesis, and biological evaluation of indole-based 1,4-disubstituted piperazines as cytotoxic agents

Article abstract of DOI:10.3906/kim-1111-5

A series of 3-[(4-substitutedpiperazin-1-yl)methyl]-1H -indole derivatives were synthesized, and their structures were confirmed by spectral analysis. All the compounds were tested for their cytotoxic activity in vitro against 3 human tumor cell lines: human liver (HUH7), breast (MCF7), and colon (HCT116). Among the designed derivatives, most of the compounds showed significant cytotoxicity against liver and colon cancer cell lines with lower IC50 concentrations than the standard drug 5-fluorouracil. Compound 3s, with 3,4-dichlorophenyl substituent on the piperazine ring, was the most active in suppressing the growth of all screened cancer cells. TUeBITAK.

Full text of DOI:10.3906/kim-1111-5

Turk J Chem
36 (2012) , 515 – 525.
¨
˙
c
ꢀ TUBITAK
doi:10.3906/kim-1111-5
Design, synthesis, and biological evaluation of
indole-based 1,4-disubstituted piperazines as cytotoxic
agents
1,∗
1
2
¨
¨
˙
Meri¸c KOKSAL AKKOC¸ , Mine YARIM YUKSEL , Irem DURMAZ ,
2
˙
Rengu¨l C¸ ETIN ATALAY
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Yeditepe University,
˙
34755, Kayı¸sda˘gı, Istanbul-TURKEY
e-mails: merickoksal@yeditepe.edu.tr, myarim@yeditepe.edu.tr
2Department of Molecular Biology and Genetics, Faculty of Science, Bilkent University,
06800, Bilkent, Ankara-TURKEY
e-mails: durmaz@bilkent.edu.tr, rengul@bilkent.edu.tr
Received: 02.11.2011
A series of 3-[(4-substitutedpiperazin-1-yl)methyl]-1H -indole derivatives were synthesized, and their
structures were confirmed by spectral analysis. All the compounds were tested for their cytotoxic activity in
vitro against 3 human tumor cell lines: human liver (HUH7), breast (MCF7), and colon (HCT116). Among
the designed derivatives, most of the compounds showed significant cytotoxicity against liver and colon
cancer cell lines with lower IC50 concentrations than the standard drug 5-fluorouracil. Compound 3s, with
3,4-dichlorophenyl substituent on the piperazine ring, was the most active in suppressing the growth of all
screened cancer cells.
Key Words: Anticancer activity, cytotoxicity, Mannich base, indole, 1,4-substituted piperazines
Introduction
Recent drug discovery studies have focused on the design and synthesis of small molecules that have an
indole nucleus as the core structure and that act as tubulin inhibitors.1 Drugs that bind to tubulin act by
interfering with the mitosis of cells during the M-phase, resulting in mitotic arrest and eventually leading to
∗Corresponding author
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Design, synthesis, and biological evaluation of..., M. KOKSAL AKKOC¸, et al.
apoptosis.2 Therefore, microtubules are a sensitive target for the development of anticancer drugs. Due to
the introduction of vinca alkaloids such as vincristine and vinblastine for the clinical therapy of cancer, indole-
carrying compounds have generated considerable interest.3−8 A large number of synthetic indole-containing
drugs and clinical candidates have been identified over the past few years (Figure).
O
NH
HO
N
N
H
HO
NH
O
N
H
Semaxanib
Dacinostat
N
O
NH
O
O
Cl
NH
N
H3CO
D-64131
D-24851
Figure. Structure comparison of small indole molecules as anticancer agents.
Chang and co-workers reported a large number of compounds with indole core structure. In addition to
the synthesis and evaluation of the anticancer activity of these compounds, they have revealed some SAR and
pharmacophore modeling data.4,5,9−13 Research on 1- and 3-aroylindoles9 showed that 3-substituted indole
derivatives exhibited significant activity compared with 1-aroylindoles and the electronic effects on the indole
ring were important for activity potency.11
Piperazines, especially disubstituted ones, exhibit a wide range of biological properties, as reported in the
literature. In the last decade, a number of piperazine derivatives have been synthesized and evaluated for their
cytotoxic activity.14−19 Additional preclinical/clinical drug development studies of the piperazine compounds
in small-animal models by the US National Cancer Institute (NCI) demonstrated that these targets had the
ability to suppress experimental tumors. As a result of the study for the lead compounds, it has been reported
that inhibitory action was observed against colon, prostate, breast, lung, and immune cell tumors in many
indole-carrying small anti-cancer molecules.20 Thus, based on these observations in the literature, the present
study was initiated with the aim of identifying the structural requirements of the indole-based piperazines in
terms of anticancer activity. The versatile utility of Mannich bases on the effectiveness and toxicity of the parent
compounds17 prompted us to prepare a series of piperazinomethyl derivatives of indole structure and evaluate
their cytotoxic activity against different cancer cell lines.
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Design, synthesis, and biological evaluation of..., M. KOKSAL AKKOC¸, et al.
Experimental
Chemistry
Chemicals and reagents used in the current study were of analytical grade. The reactions were monitored by thin
layer chromatography (TLC) on Merck pre-coated silica GF254 plates. Melting points were determined using
a Mettler Toledo FP62 capillary melting point apparatus (Mettler-Toledo, Greifensee, Switzerland) and were
uncorrected. Infrared spectra were recorded on a Perkin-Elmer 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 1 H- and 13 C-NMR spectra were recorded with a Varian Mercury-400 FT-NMR spectrometer
(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 analyses were performed on a LECO 932 CHNS instrument (Leco-932, St. Joseph, MI,
USA) and analyses for C, H, and N were within
0.4% of the theoretical values.
General procedure for the synthesis of compounds (3a-3s)
Indole (1) (2 mmol, 235 mg) was dissolved in 20 mL of ethanol-water (1:1) solution, and formaldehyde 37% (3
mmol) and substituted piperazine (2) (2 mmol) were added. The mixture was stirred at room temperature and
the reaction was controlled by TLC in benzene:methanol (9:1) and toluene:ethyl acetate:diethylamine (75:25:1).
At the end of the reaction, the precipitate was filtrated, dried, and recrystallized using an appropriate solvent.
3-[(4-Phenylpiperazin-1-yl)methyl]-1H-indole (3a)21−23
Yield: 45%: mp 179.7 ◦ C. IR (KBr) cm−1 : ν 3130 (N-H), 3095-2756 (C-H). 1 H-NMR (CDCl3): δ 8.10 (bs,
1H, indole N-H), 7.77 (d, 1H, indole H4 , J = 7.6), 7.36 (d, 1H, indole H7 , J = 8), 7.27-7.14 (m, 5H, phenyl),
6.92-6.82 (m, 3H, indole H2 , H5 , H6), 3.79 (s, 2H, C-CH2 -N), 3.20 (t, 4H, piperazine H3 , H5 , J = 4.8), 2.68
(t, 4H, piperazine H2 , H6 , J = 4.8). Anal. Calc. for C19 H21 N3 (291.39): C, 78.32; H, 7.26; N, 14.42%, found:
C, 78.18; H, 6.94; N, 14.25%.
3-{[4-(2-Fluorophenyl)piperazin-1-yl]methyl}-1H-indole (3b)
Yield: 58%: mp 164.6 ◦ C. IR (KBr) cm−1 : ν 3403 (N-H), 3068-2772 (C-H). 1 H-NMR (CDCl3): δ 8.15 (bs,
1H, indole N-H), 7.78 (d, 1H, indole H4 , J = 7.6), 7.36 (d, 1H, indole H7 , J = 7.6), 7.24 (s, 1H, indole, H2),
7.22-6.90 (m, 6H, indole H5 , H6 + phenyl), 3.80 (s, 2H, C-CH2 -N), 3.11 (t, 4H, piperazine H3 , H5 , J = 4.8),
2.71 (t, 4H, piperazine H2 , H6 , J = 4.8). Anal. Calc. for C19 H20 FN3 (309.38): C, 73.76; H, 6.52; N, 13.58;
F, 6.14%, found: C, 73.62; H, 6.21; N, 13.44; F, 6.12%.
3-{[4-(4-Fluorophenyl)piperazin-1-yl]methyl}-1H-indole (3c)24,25
Yield: 63%: mp 166.8 ◦ C (lit. 164 ◦ C). IR (KBr) cm−1 : ν 3128 (N-H), 3094-2756 (C-H). 1 H-NMR (CDCl3):
δ 8.23 (bs, 1H, indole N-H), 7.76 (d, 1H, indole H4 , J = 7.6), 7.33 (d, 1H, indole H7 , J = 7.8), 7.24 (d, 2H,
phenyl H3 , H5 , J = 7.4), 7.21 (s, 1H, indole, H2), 6.96 (d, 2H, phenyl H2 , H6 , J = 7.4), 6.87-6.84 (m, 2H,
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indole H5 , H6), 3.78 (s, 2H, C-CH2 -N), 3.11 (t, 4H, piperazine H3 , H5 , J = 4.8), 2.67 (t, 4H, piperazine H2 ,
H6 , J = 4.8). 13 C-NMR (DMSO-d6): δ 150.99, 136.23, 128.74, 127.51, 124.53, 120.82, 118.97, 118.54, 118.31,
115.20, 111.23, 110.57 (aromatics), 53.09 (C-CH2 -N), 52.41 (piperazine C3 , C5), 48.18 (piperazine C2 , C6).
Anal. Calc. for C19 H20 FN3 (309.38): C, 73.76; H, 6.52; N, 13.58; F, 6.14%, found: C, 73.69; H, 6.50; N, 13.50;
F, 6.04%.
3-{[4-(2-Chlorophenyl)piperazin-1-yl]methyl}-1H-indole (3d)
Yield: 47%: mp 139.5 ◦ C. IR (KBr) cm−1 : ν 3409 (N-H), 3057-2767 (C-H). 1 H-NMR (CDCl3): δ 8.14
(bs, 1H, indole N-H), 7.78 (d, 1H, indole H4 , J = 8.0), 7.37 (d, 1H, indole H7 , J = 8.0), 7.33 (dd, 1H,
phenyl H3 , J = 8.0, Jꢀ = 1.2), 7.25 (s, 1H, indole, H2), 7.23-6.92 (m, 5H, indole H5 , H6 + phenyl H4 , H5 ,
H6), 3.81 (s, 2H, C-CH2 -N), 3.08 (bs, 4H, piperazine H3 , H5), 2.72 (bs, 4H, piperazine H2 , H6). 13 C-NMR
(CDCl3): δ 149.67, 136.45, 130.82, 128.97, 128.27, 127.77, 123.93, 123.74, 122.28, 120.622, 119.80, 119.77,
112.86, 111.27 (aromatics), 53.75 (C-CH2 -N), 53.48 (piperazine C3 , C5), 51.55 (piperazine C2 , C6). Anal.
Calc. for C19 H20 ClN3 (325.84): C, 70.04; H, 6.19; N, 12.90; Cl, 10.88%, found: C, 70.03; H, 6.28; N, 12.75;
Cl, 10.63%.
3-{[4-(3-Chlorophenyl)piperazin-1-yl]methyl}-1H-indole (3e)
Yield: 26%: mp 120.7 ◦ C. IR (KBr) cm−1 : ν 3247 (N-H), 2948-2779 (C-H). 1 H-NMR (CDCl3): δ 8.10 (bs,
1H, indole N-H), 7.77 (d, 1H, indole H4 , J = 7.6), 7.37 (d, 1H, indole H7 , J = 8.4), 7.26 (s, 1H, indole,
H2), 7.23-7.12 (m, 3H, indole H6 + phenyl H2 , H4), 6.86-6.74 (m, 3H, indole H5 + phenyl H5 , H6), 3.77
(s, 2H, C-CH2 -N), 3.19 (t, 4H, piperazine H3 , H5 , J = 4.8), 2.65 (t, 4H, piperazine H2 , H6 , J = 4.8).
13 C-NMR (CDCl3): δ 152.67, 136.48, 135.11, 130.19, 128.13, 123.85, 122.36, 119.84, 119.81, 119.27, 115.88,
114.02, 112.71, 111.29 (aromatics), 53.82 (C-CH2 -N), 53.08 (piperazine C3 , C5), 48.95 (piperazine C2 , C6).
Anal. Calc. for C19 H20 ClN3 (325.84): C, 70.04; H, 6.19; N, 12.90; Cl, 10.88%, found: C, 70.01; H, 6.08; N,
12.79; Cl, 10.67%.
3-{[4-(4-Chlorophenyl)piperazin-1-yl]methyl}-1H-indole (3f)26−29
Yield: 21%: mp 174.3 ◦ C. IR (KBr) cm−1 : ν 3402 (N-H), 3079-2778 (C-H). 1 H-NMR (CDCl3): δ 8.09 (bs,
1H, indole N-H), 7.77 (d, 1H, indole H4 , J = 7.6), 7.38 (d, 1H, indole H7 , J = 8.4), 7.23-7.12 (m, 5H, indole
H2 , H5 , H6 + phenyl H2 , H6), 6.82 (dd, 2H, phenyl H3 , H5 , J = 6.8, Jꢀ = 2.0), 3.78 (s, 2H, C-CH2 -N), 3.16
(t, 4H, piperazine H3 , H5 , J = 5.2), 2.66 (t, 4H, piperazine H2 , H6 , J = 5.2). Anal. Calc. for C19 H20 ClN3
(325.84): C, 70.04; H, 6.19; N, 12.90; Cl, 10.88%, found: C, 70.06; H, 6.19; N, 12.84; 10.52%.
3-{[4-(3-Methoxyphenyl)piperazin-1-yl]methyl}-1H-indole (3g)
Yield: 37%: mp 129.2 ◦ C. IR (KBr) cm−1 : ν 3412 (N-H), 3005-2772 (C-H). 1 H-NMR (CDCl3): δ 8.13 (bs,
1H, indole N-H), 7.77 (d, 1H, indole H4 , J = 7.6), 7.37 (d, 1H, indole H7 , J = 7.6), 7.25 (s, 1H, indole, H2),
7.22-7.13 (m, 3H, indole H5 , H6 + phenyl H2), 6.54-6.38 (m, 3H, phenyl H4 , H5 , H6), 4.98 (s, 5H, C-CH2 -N
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and O-CH3), 3.19 (t, 4H, piperazine H3 , H5 , J = 4.8), 2.66 (t, 4H, piperazine H2 , H6 , J = 4.8). Anal. Calc.
for C20 H23 N3 O (321.42): C, 74.74; H, 7.21; N, 13.07%, found: C, 74.73; H, 7.10; N, 13.16%.
3-{[4-(4-Methoxyphenyl)piperazin-1-yl]methyl}-1H-indole (3h)
Yield: 22%: mp 157.2 ◦ C. IR (KBr) cm−1 : ν 3435 (N-H), 3098-2808 (C-H). 1 H-NMR (CDCl3): δ 8.11 (bs,
1H, indole N-H), 7.77 (d, 1H, indole H4 , J = 7.6), 7.37 (d, 1H, indole H7 , J = 8.4), 7.25 (s, 1H, indole, H2),
7.23-7.12 (m, 2H, indole H5 , H6), 6.90-6.81 (m, 2H, phenyl), 3.78 (s, 2H, C-CH2 -N), 3.76 (s, 3H, -OCH3), 3.09
(t, 4H, piperazine H3 , H5 , J = 4.8), 2.69 (t, 4H, piperazine H2 , H6 , J = 4.8). Anal. Calc. for C20 H23 N3 O
(321.42): C, 74.74; H, 7.21; N, 13.07%, found: C, 74.50; H, 7.11; 13.25%.
3-{[4-(2-Methylphenyl)piperazin-1-yl]methyl}-1H-indole (3i)
Yield: 68%: mp 89.9 ◦ C. IR (KBr) cm−1 : ν 3433 (N-H), 3013-2809 (C-H). 1 H-NMR (CDCl3): δ 8.20 (bs, 1H,
indole N-H), 7.79 (d, 1H, indole H4 , J = 8.0), 7.37 (d, 1H, indole H7 , J = 8.8), 7.26-6.95 (m, 7H, indole H2 ,
H5 , H6 + phenyl), 3.82 (s, 2H, C-CH2 -N), 2.95 (t, 4H, piperazine H3 , H5 , J = 4.4), 2.70 (bs, 4H, piperazine
H2 , H6), 2.30 (s, 3H, -CH3). Anal. Calc. for C20 H23 N3 (305.42): C, 78.65; H, 7.59; N, 13.76%, found: C,
78.63; H, 7.38; N 13.98%.
3-{[4-(4-Methylphenyl)piperazin-1-yl]methyl}-1H-indole (3j)30,31
Yield: 36%: mp 168.2 ◦ C. IR (KBr) cm−1 : ν 3133 (N-H), 3099-2813 (C-H). 1 H-NMR (CDCl3): δ 8.10 (bs,
1H, indole N-H), 7.77 (d, 1H, indole H4 , J = 7.6), 7.37 (d, 1H, indole H7 , J = 8.0), 7.25 (s, 1H, indole, H2),
7.23-6.82 (m, 6H, indole H5 , H6 + phenyl H2 , H3 , H5 , H6), 3.78 (s, 2H, C-CH2 -N), 3.15 (t, 4H, piperazine
H3 , H5 , J = 5.2), 2.68 (t, 4H, piperazine H2 , H6 , J = 5.2), 2.27 (s, 3H, -CH3). Anal. Calc. for C20 H23 N3
(305.42): C, 78.65; H, 7.59; N, 13.76%, found: C, 78.45; H, 7.53; N 13.95%.
3-{[4-(2,3-Dimethylphenyl)piperazin-1-yl]methyl}-1H-indole (3k)
Yield: 75%: mp. 173.8 ◦ C. IR (KBr) cm−1 : ν 3435 (N-H), 3080-2810 (C-H). 1 H-NMR (CDCl3): δ 8.16 (bs,
1H, indole N-H), 7.78 (d, 1H, indole H4 , J = 8.0), 7.38 (d, 1H, indole H7 , J = 8.0), 7.25 (s, 1H, indole H2),
7.23-6.87 (m, 5H, indole H5 , H6 + phenyl), 3.81 (s, 2H, C-CH2 -N), 2.91 (t, 4H, piperazine H3 , H5 , J = 4.8),
2.70 (bs, 4H, piperazine H2 , H6), 2.25 (s, 3H, -CH3), 2.20 (s, 3H, -CH3). Anal. Calc. for C21 H25 N3 (319.44):
C, 78.96; H, 7.89; N, 13.15%, found: C, 78.47; H, 7.78; 12.84%.
3-{[4-(2-Cyanophenyl)piperazin-1-yl]methyl}-1H-indole (3l)
Yield: 48%: mp 121.9 ◦ C. IR (KBr) cm−1 : ν 3420 (N-H), 3113-2808 (C-H), 2219 (C≡N). 1 H-NMR (CDCl3):
δ 8.17 (bs, 1H, indole N-H), 7.78 (d, 1H, indole H4 , J = 7.6), 7.54 (dd, 1H, indole H7 , J = 8.0, Jꢀ = 1.6),
7.48-7.36 (m, 2H, phenyl H3 , H4), 7.25 (s, 1H, indole, H2), 7.23-7.12 (m, 2H, indole H5 , H6), 6.99-6.96 (m,
2H, phenyl H5 , H6), 3.80 (s, 2H, C-CH2 -N), 3.23 (t, 4H, piperazine H3 , H5 , J = 4.8), 2.73 (t, 4H, piperazine
H2 , H6 , J = 4.8). Anal. Calc. for C20 H20 N4 (316.40): C, 75.92; H, 6.37; N 17.71%, found: C, 75.78; H, 6.43;
16.82%.
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3-{[4-(4-Nitrophenyl)piperazin-1-yl]methyl}-1H-indole (3m)
Yield: 52%: mp 186.1 ◦ C. IR (KBr) cm−1 : ν 3435 (N-H), 3000-2828 (C-H). 1 H-NMR (CDCl3): δ 8.13 (bs,
1H, indole N-H), 8.10 (d, 2H, phenyl H3 , H5 , J = 6.8), 7.76 (d, 1H, indole H4 , J = 7.6), 7.39 (d, 1H, indole
H7 , J = 8.4), 7.26 (s, 1H, indole H2), 7.24-7.12 (m, 2H, indole H5 , H6), 6.78 (d, 2H, phenyl H2 , H6 , J =
6.8), 3.81 (s, 2H, C-CH2 -N), 3.42 (t, 4H, piperazine H3 , H5 , J = 5.2), 2.64 (t, 4H, piperazine H2 , H6 , J =
5.2), 2.25 (s, 3H, -CH3), 2.20 (s, 3H, -CH3). Anal. Calc. for C19 H20 N4 O2 (336.39): C, 67.84; H, 5.99; N,
16.66%, found: 67.74; H, 5.93; N, 16.41%.
3-[(4-Benzoylpiperazin-1-yl)methyl]-1H-indole (3n)32
Yield: 55%: mp 159.2 ◦ C. IR (KBr) cm−1 : ν 3255 (N-H), 3116-2768 (C-H), 1708 (C=O). 1 H-NMR (CDCl3):
δ 8.41 (bs, 1H, indole N-H), 7.72 (d, 1H, indole H4 , J = 7.6), 7.38-7.07 (m, 9H, indole H2 , H5 , H6 , H7 and
ꢀ
phenyl), 3.79 (bs, 2H, piperazine H3), 3.74 (s, 2H, C-CH2 -N), 3.40 (bs, 2H, piperazine H3 ), 2.59 (bs, 2H,
ꢀ
piperazine H2), 2.42 (bs, 2H, piperazine H2 ). Anal. Calc. for C20 H21 N3 O (319.40): C, 75.21; H, 6.63; N,
13.16%, found: C, 75.18; H, 6.62; N, 13.14%.
3-[(4-Acetylpiperazin-1-yl)methyl]-1H-indole (3o)
Yield: 72%: mp 197.3 ◦ C. IR (KBr) cm−1 : ν 3148 (N-H), 3101-2847 (C-H), 1706 (C=O). 1 H-NMR (CDCl3):
δ 8.23 (bs, 1H, indole N-H), 7.75 (d, 1H, indole H4 , J = 7.6), 7.37 (dd, 1H, indole H7 , J = 8.4, Jꢀ = 0.8), 7.25
(s, 1H, indole, H2), 7.21 (t, 1H, indole H6 , J = 7.6), 7.13 (t, 1H, indole H5 , J = 7.6), 3.74 (s, 2H, C-CH2 -N),
ꢀ
3.60 (t, 2H, piperazine H3 , J = 4.8), 3.44 (t, 4H, piperazine H3 , J = 4.8), 2.48 (t, 4H, piperazine H2 , H6 ,
J = 4.8), 2.07 (s, 3H, -COCH3). Anal. Calc. for C15 H19 N3 O (257.33): C, 70.01; H, 7.44; N, 16.33%, found:
C, 69.78; H, 7.26; N, 16.20%.
3-{[4-(Pyrimidin-2-yl)piperazin-1-yl]methyl}-1H-indole (3p)
Yield: 25%: mp 200.0 ◦ C. IR (KBr) cm−1 : ν 3429 (N-H), 2990-2764 (C-H). 1 H-NMR (CDCl3): δ 8.28 (d,
2H, pyrimidinyl H3 , H5 , J = 4.8), 8.14 (bs, 1H, indole N-H), 7.77 (d, 1H, indole H4 , J = 7.6), 7.37 (d, 1H,
indole H7 , J = 8.0), 7.26 (s, 1H, indole H2), 7.23-7.12 (m, 2H, indole H5 , H6), 6.45 (t, 1H, pyrimidinyl H4 ,
J = 4.8), 3.83 (t, 4H, piperazine H3 , H5 , J = 4.8), 3.77 (s, 2H, C-CH2 -N), 2.57 (bs, 4H, piperazine H2 , H6).
Anal. Calc. for C17 H19 N5 (293.37): C, 69.60; H, 6.53; N, 23.87%, found: C, 69.45; H, 6.52; N, 23.84%.
3-{[4-(2,5-Difluorobenzyl)piperazin-1-yl]methyl}-1H-indole (3r)33
Yield: 23%: mp 126.7 ◦ C. IR (KBr) cm−1 : ν 3057 (N-H), 2935-2814 (C-H). 1 H-NMR (CDCl3): δ 8.13 (bs,
1H, indole N-H), 7.72 (d, 1H, indole H4 , J = 8.0), 7.35 (d, 1H, indole H7 , J = 7.6), 7.26 (s, 1H, indole,
H2), 7.21-6.89 (m, 5H, indole H5 , H6 , phenyl), 3.74 (s, 2H, C-CH2 -N), 3.54 (s, 2H, N-CH2 -Ph), 2.53 (bs, 8H,
piperazine). Anal. Calc. for C20 H21 F2 N3 (341.40): C, 70.36; H, 6.20; N, 12.31; F, 11.13%, found: C, 70.27;
H, 6.15; N, 12.26; F: 10.90%.
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3-{[4-(3,4-Dichlorobenzyl)piperazin-1-yl]methyl}-1H-indole (3s)33
Yield: 20%. Yellow liquid. IR (KBr) cm−1 : ν 3435 (N-H), 2933-2820 (C-H). 1 H-NMR (CDCl3): δ 8.48 (bs,
1H, indole N-H), 7.71 (d, 1H, indole H4 , J = 8.4), 7.39 (d, 1H, indole H7 , J = 2.0), 7.34 (s, 1H, indole,
H2), 7.32-7.05 (m, 5H, indole H5 , H6 , phenyl), 3.74 (s, 2H, C-CH2 -N), 3.41 (s, 2H, N-CH2 -Ph), 2.45 (bs, 8H,
piperazine). Anal. Calc. for C20 H21 Cl2 N3 (374.31): C, 64.18; H, 5.65; N, 11.23; Cl, 18.94%, found: C, 64.08;
H, 5.62; N, 11.20; Cl, 18.77%.
Cytotoxicity
Cell culture
Human cancer cell lines (all except KATO-3 and MFE-296) were grown in Dulbecco’s Modified Eagle Medium
(DMEM), with 10% fetal bovine serum (FBS) and 1% penicillin, and incubated in 37 ◦ C incubators containing
5% CO2 and 95% air. KATO-3 gastric cancer cell lines were grown in high glucose DMEM (4.5 g/L glucose)
with 10% FBS, 1% penicillin, 1% L-glutamine, and 1% non-essential amino acid. MFE-296 endometrial cancer
cell lines were grown in medium containing 40% RPMI 1640, 40% minimum essential medium (MEM) (with
Earle’s salts), 20% FBS, 2 mM L-glutamine, and 1× insulin-transferrin-sodium selenite.
NCI-60 sulforhodamine B (SRB) assay
Cancer cells (range of 2000 cells/well to 5000 cells/well) were inoculated into 96-well plates in 200 μL of media
and incubated in 37 ◦ C incubators containing CO2 (5%) and air (95%). After a 24 h incubation period,
one plate for each cell line was fixed with 100 μL of ice-cold trichloroacetic acid (TCA) (10%). This plate
represents the behavior of the cells just prior to drug treatment and can be regarded as the time-zero plate. The
compounds to be tested were solubilized in dimethyl sulfoxide (DMSO) to the final concentration of 40 mM and
stored at +4 ◦ C. While treating the cells with the compounds, the corresponding volume of the compound was
applied to the cell in order to achieve the desired drug concentration and diluted by serial dilution. After drug
treatment, the cells were incubated in 37 ◦ C incubators containing 5% CO2 and 95% air for 72 h. Following
the termination of the incubation period after drug treatment, cells were fixed with 100 μL of 10% ice-cold
TCA and incubated in the dark at +4 ◦ C for 1 h. Then TCA was washed away with ddH2 O 5 times, and the
plates were left to air dry. In the final step, the plates were stained with 100 μL of 0.4% sulforhodamine B
(SRB) solution in 1% acetic acid solution. Following staining, the plates were incubated in the dark for 10 min
at room temperature. The unbound dye was washed away using 1% acetic acid and the plates were left to air
dry. In order to measure the absorbance results, the bound stain was then solubilized using 200 μL of 10 mM
Tris-Base. The OD values were obtained at 515 nm.
Results and discussion
Chemistry
The target 3-(piperazinomethylsubstituted)indole compounds (3a-3s) were prepared by Mannich reaction be-
tween indole (1) and appropriate piperazines (2) at room temperature (Scheme). Although a group of com-
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Design, synthesis, and biological evaluation of..., M. KOKSAL AKKOC¸, et al.
pounds were reported in the literature,21−33 all the compounds of the series were synthesized with a view to
structural elucidation, first time anticancer screening, and evaluation of the structure activity relationship.
R
N
N
HCHO
HN
N R
+
N
H
C2H5OH
N
H
3a-3s
1
2
Scheme. Synthesis of compounds 3a-3s.
The prepared Mannich bases showed IR bands at 3469-3130 cm−1 (N-H), representing substitution of
indole on the third position. Other strong absorption bands for characteristics of the functional groups were
displayed at 3116-2714 cm−1 (C-H) and 2219 cm−1 (C≡N) for compound 3l and approximately 1700 cm−1
(C=O) for ketonic compounds 3n and 3o. The 1 H-NMR spectrum was more informative: typical characteristic
peaks for the compounds were observed at approximately δ 8.10 (bs, 1H, indole N-H) and 3.80 (s, 2H, C-CH2 -
N). The other protons were seen at the expected chemical shifts.
Biological studies
In vitro evaluation of the target compounds 3a-3s for their cytotoxic properties was performed by means of
SRB assays in triplicate using 3 human cancer cell lines: liver (HUH7), breast (MCF7), and colon (HCT116).
Serial dilutions from 40 μM to 2.5 μM were used; camptothecin was the positive control and 5-flourouracil
(5-FU) was the standard drug for the cytotoxic effect. The biological activity data are presented in the Table.
Some of the compounds exhibited enhanced antitumor potency compared to the standard drug 5-
fluorouracil against one or more cell lines in the tested micromolar range. The most promising compound
of the series, compound 3s, showed better cell growth inhibition than 5-FU, with IC50 values of 3.42, 2.92, and
9.33 μM for HUH7, MCF7, and HCT116 cell lines, respectively. A receptor binding study of the compound
has also been reported by Yarim et al.33 Generally, most of the compounds displayed significant cytotoxicity
towards HUH7 and HCT116 cancer cell lines with lower IC50 values than standard drug in the range 3.42-16.48
μM and 6.38-17.48 μM. In addition, IC50 values were parallel for liver and colon cancer cell lines. The only
compound that had a lower IC50 value (2.92 μM) than 5-FU (3.50 μM) against the breast cancer cell line was
compound 3s. Among the derivatives, compounds 3m, 3n, and 3o, carrying electron withdrawing groups by
resonance effect, showed no activity against any cancer cell lines.
The preliminary structure-activity relationships (SARs) revealed that substitution of phenyl on the fourth
position of piperazine plays an important role in activity. In particular, chloro substituted derivatives showed
the best activity values for each cell line.
Although the introduction of other substituents such as methoxy, methyl, and cyano onto the phenyl
ring caused fluctuations in the activity score, it was noteworthy that carbonyl substitution on phenyl decreased
potency.
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Design, synthesis, and biological evaluation of..., M. KOKSAL AKKOC¸, et al.
Table. IC50 value for tested compounds 3a-3s against cancer cell lines.
R
N
N
N
H
Compounds
R
HUH7a MCF7a HCT116a
3a
Phenyl
38.15
10.72
14.63
6.41
9.31
5.04
10.44
16.48
7.14
NI
7.97
8.50
9.32
8.72
6.14
9.70
10.01
18.51
13.37
NI
11.14
6.38
11.65
6.86
7.73
7.71
10.06
17.48
8.48
NI
3b
2-Fluorophenyl
4-Fluorophenyl
2-Chlorophenyl
3-Chlorophenyl
4-Chlorophenyl
3-Methoxyphenyl
4-Methoxyphenyl
2-Methylphenyl
4-Methylphenyl
3c
3d
3e
3f
3g
3h
3i
3j
3k
2,3-Dimethylphenyl
2-Cyanophenyl
4-Nitrophenyl
4-Benzoyl
5.14
12.25
NI
8.92
19.48
NI
7.96
12.31
NI
3l
3m
3n
NI
NI
NI
3o
4-Acetyl
NI
NI
NI
3p
3r
2-Pyrimidinyl
2,5-Difluoro
3,4-Dichlorobenzyl
-
15.50
13.87
3.42
0.15
30.70
12.46
5.47
2.92
>0.01
3.50
16.47
9.19
9.33
>0.01
18.78
3s
Camptothecin
5-Flourouracil
-
a All the experiments were conducted in triplicate (1 < R2 < 0.8). NI: no inhibition.
Conclusion
In summary, a series of 3-[(4-substitutedpiperazin-1-yl)methyl]-1H -indole derivatives were synthesized via the
Mannich reaction. The cytotoxicity of compounds 3a-3s on 3 cell lines was studied and showed a variable
extent of IC50 values. The cytotoxicity data of compounds demonstrate the importance of substitution at the
N-4 position of piperazine. Compound 3s is the most potent compound of the series against all cell lines. Other
promising compounds 3b-3f, 3i, and 3k have an IC50 of less than 10 μM, which indicates significant cytotoxic
activity. Further structure-activity studies are also required to clearly elucidate the role of the substitution on
the indole ring. The possible improvement of the anticancer properties of indole-based series through a QSAR
study and mechanistic determination on activity of lead compound will be the focus of our further investigation.
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Design, synthesis, and biological evaluation of..., M. KOKSAL AKKOC¸, et al.
Acknowledgement
The synthetic part of this work was supported by a grant from the Scientific and Technological Research Council
¨
˙
of Turkey (TUBITAK) (Project number: 108S009)
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