Synthesis and in vitro evaluation of novel indole-based sigma receptors ligands

Article abstract of DOI:10.1111/j.1747-0285.2011.01215.x

To investigate the molecular features involved in sigma (σ) receptors binding, a series of compounds based on indole scaffolds were synthesized and their chemical structures were confirmed by ¹H-NMR, IR, and elemental analysis. Their affinity t

Full text of DOI:10.1111/j.1747-0285.2011.01215.x

ª 2011 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2011.01215.x
Chem Biol Drug Des 2011; 78: 869–875
Research Article
Synthesis and in vitro Evaluation of Novel
Indole-Based Sigma Receptors Ligands
Mine Yarim1, Meric Koksal1,
corresponding to r2 sites has not yet been cloned. In comparison
Dirk Schepmann2 and Bernard Wu¨ nsch2
with the r1 receptor, it appears to be slightly smaller in size (r1:
25–29 kDa, r2: 18–22 kDa) (9,10). Pharmacological experiments
reveal that r2 receptors may be lipid raft proteins that affect cal-
cium signaling via sphingolipid products. Unlike r1 receptors, r2
receptors do not appear to translocate. Both the subtypes of r
receptors are highly expressed on tumor cell lines from human
and rat cancer tissues. However, malignant tumor cells show a
higher expression of r2 receptors than quiescent tumor cells. The
overexpression of r2 receptors in human and murine tumors sug-
gests that r2 receptors may be a biomarker of tumor cell prolifer-
ation (11–13). Owing to the lack of availability of detailed protein
structural information and truly selective r2 ligands, the pharma-
cological characterization of the r2 subtype with regard to its
mechanism of action and biochemical role in various biological
effects has been very limited.
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
Yeditepe University, 34755 Kayisdagi, Istanbul, Turkey
2Institut fꢀr Pharmazeutische und Medizinische Chemie,
Hittorfstrasse 58-62, D-48149 Mꢀnster, Germany
*Corresponding author: Mine Yarim, myarim@yeditepe.edu.tr
To investigate the molecular features involved in
sigma (r) receptors binding, a series of compounds
based on indole scaffolds were synthesized and
their chemical structures were confirmed by
1H-NMR, IR, and elemental analysis. Their affinity
toward r1 and r2 receptor subtypes was evaluated.
1-{[4-(2-phenylethyl)piperazin-1-yl]methyl}-3-methyl
-1H-indole 3b had a high affinity to r1 receptors,
while three compounds, 1-{3-[4-(substitutedphe-
nyl)piperazin-1-yl]propyl}-1H-indole derivatives 4a–
c had shown high affinity and selectivity for r2
receptors. Cytotoxicity of the compounds was
demonstrated on cancer cell lines from liver
(HUH7), breast (MCF7), and colon (HCT-116) cancer
cell lines. Compound 1c (3-{[4-(3,4-dichloroben-
zyl)piperazin-1-yl]methyl}-1H-indole) showed signif-
icant cell growth inhibitory activity on the selected
cancer cell lines.
Therefore, ligands interacting with r receptors are of interest for
example as atypical antipsychotics (14,15), antidepressants (16),
anticocaine agents (17–19), and antitumor agents (20–23). Thus,
selective r1 and r2 agonists and antagonists may be potentially
useful drugs for treatment of several pathologic conditions such
as psychiatric disorders, cocaine abuse, memory and learning dis-
orders, dyskinesia and dystonic reactions induced by classical
antipsychotic drugs, cancer and tumor diagnosis. Several com-
pounds binding r1 receptors with high affinity and selectivity
have been discovered, whereas r2 receptor ligands generally
have poor selectivity over r1 receptors and new r2 ligands are
needed to define the structural features that may improve their
affinity and selectivity.
Key words: cytotoxicity assay, drug design, indole, piperazine, radio
ligand binding assay, radioligands, sigma ligands
Received 30 June 2011, revised 11 July 2011 and accepted for publica-
tion 10 August 2011
Glennon et al. (24) reported on the structure affinity relationships of
a series of phenylalkylamine derivatives with respect to their bind-
ing at r1 receptors and elaborated the features of these com-
pounds being important for high r1 receptor binding. According to
the proposed two-dimensional model, two hydrophobic substituents
in different distances from a basic nitrogen atom, which is sup-
posed to bind to a proton donor site (Asp 126 and ⁄ or Glu 172) of
the receptor, are required for a high r1 receptor affinity (Figure 1).
Additionally, 1,4-disubstituted piperazine derivatives are described
as high affinity r receptor ligands in the literature (25–28). Here,
we report the design of compounds according to the Glennon's
pharmacophore model as well as synthesis, their binding affinities
to the r1 and r2 receptors, and their effect on the inhibition of
cancer cell lines from liver (HUH7), breast (MCF7), and colon (HCT-
116) samples.
The class of r receptors is subdivided into at least two subtypes,
which are termed r1 and r2 receptors. To date, the r1 receptor
is pharmacologically well characterized because of the receptor
sequence information and availability of selective r1 ligands. The
223-amino acid r1 receptor with two transmembrane-spanning
regions (1,2) has been purified and cloned from several species,
including mouse, rat, guinea pig, and human (3–7). The r1 recep-
tors bind structurally diverse classes of compounds, including
diverse psychotherapeutic agents, drugs of abuse such as cocaine
and methamphetamine and steroid hormones such as progester-
one. The general pharmacophoric element appears to be an
N-alkyl, N,N-dialkyl, or N-arylalkyl amine (8). The protein
869

Yarim et al.
(400 MHz, CDCl3, ppm): 8.23 (bs,1H,indole N-H), 7.78 (d,1H,indole
H4), 7.33 (d,1H,indole H7), 7.24 (d, 2H, phenyl H3, H5), 7.21 (s,1H,
indole,H2), 6.96 (d, 2H, phenyl H2, H6), 6.87–6.84 (m, 2H, indole H5,
H6), 3.78 (s,2H, C-CH2-N), 3.11 (t,4H, piperazine H3, H5), 2.67 (t,4H,
piperazine H2, H6). 13C-NMR (400 MHz, DMSO, ppm): 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 (pipera-
zine C3, C5) 48.18 (piperazine C2, C6). Anal. Cacld. for C19H20FN3: C,
73.76; H, 6.52; N, 13.58. Found: C, 73.69; H, 6.50; N, 13.50.
A
N
B
3-{[4-(2,5-difluorobenzyl)piperazin-1-yl]methyl}-
1H-indole (1b)
Purified by column chromatography (SiO2, AcOEt ⁄ n-hexane 1:2). Yield:
23%; yellowish solid. Mp 126.7 ꢀC. IR (KBr, cm)1): 3057 (N-H), 2935–
1
2814 (C-H). H-NMR (400 MHz, CDCl3, ppm): 8.13 (bs,1H,indole N-H),
7.72 (d,1H,indole H4), 7.35 (dd,1H,indole H7), 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. Cacld. for C20H21F2N3: C,
70.36; H, 6.20; N, 12.31. Found: C, 70.27; H, 6.15; N, 12.26.
N
N
N
Figure 1: Pharmacophore model of r1 receptor ligands (24) and
hypothetical binding of the compound 3b to the r1 receptor.
3-{[4-(3,4-dichlorobenzyl)piperazin-1-yl]methyl}-
1H-indole (1c)
Materials and Methods
Purified by column chromatography (SiO2, AcOEt ⁄ n-hexane 1:2). Yield:
20%; yellowish solid. Mp 107 ꢀC. (KBr, cm)1): 3435 (N-H), 2933–2820
1
Chemistry
(C-H). H-NMR (400 MHz, CDCl3, ppm): 8.48 (bs,1H,indole N-H), 7.71
Melting points (ꢀC) were determined by using a Mettler Toledo
FP62 capillary melting point apparatus (Mettler-Toledo, Greifensee,
Switzerland) and are uncorrected. Infrared spectra were recorded
on a Perkin-Elmer Spectrum One series FTIR apparatus (Version
5.0.1) (Perkin Elmer, Norwalk, CT, USA), using potassium bromide
pellets; the frequencies are expressed in cm)1. The 1H NMR spec-
tra were recorded with a Varian Mercury-400 FT-NMR spectrometer
(Varian Inc., Palo Alto, CA, USA), using tetramethylsilane as the
internal reference, with chloroform-CDCl3 or dimethylsulphoxide-
DMSO-d6 as solvents, the chemical shifts are reported in parts per
million (ppm). Elemental analyses were performed on LECO 932
CHNS (Leco-932, St. Joseph, MI, USA) instrument and were within
€ 0.4% of the theoretical values.
(d,1H,indole H4), 7.39 (d,1H,indole H7), 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. Cacld. for C20H21Cl2N3: C,
64.18; H, 5.65; N, 11.23. Found: C, 64.08; H, 5.62; N, 11.20.
General procedure for the preparation of 1,3-di-
{[4-(substitutedphenyl)piperazin-1-yl]methyl}-1H-
indole (2a)
For disubstituted derivative, indole (2 mmol), formalin (6 mmol), and
4-fluorophenyl piperazine (4 mmol) were refluxed in ethanol for 4 h.
The precipitate was filtered, washed with cold ethanol, dried and
purified by recrystallization.
General procedure for the preparation of 3-{[4-
(substitutedphenyl ⁄ benzyl)piperazin-1-
yl]methyl}-1H-indole (1a–c)
ndole (2 mmol, 235 mg) was dissolved in 20-mL ethanol–water (1:1)
solution; formalin (3 mmol) and substituted phenyl piperazine
(2 mmol) were added. The mixture was stirred in room temperature
and the reaction monitored by TLC in benzene ⁄ methanol (9:1) and
toluene ⁄ ethyl acetate ⁄ DEA (75:25:1). At the end of the reaction,
the crude precipitate was filtered and purified by recrystallization or
column chromatography.
1,3-di-{[4-(4-fluorophenyl)piperazin-1-yl]methyl}-
1H-indole (2a)
Recrystallization from ethanol-water. Yield: 28.5%; white solid. Mp
1
192.8 ꢀC. (KBr, cm)1): 2938–2836 (C-H). H-NMR (400 MHz, CDCl3,
ppm): 7.76 (d,1H,indole H4), 7.47 (d,1H,indole H7), 7.26 (s,1H, indole,H2),
7.23–6.89 (m, 10H, indole H5, H6 + phenyl), 4.86 (s,2H, N-CH2-N), 3.78
(s,2H, C-CH2-N), 3.10–3.05 (m,8H, piperazine H3, H3¢, H5, H5¢), 3.58–
2.70 (m, 8H, piperazine H2, H2¢, H6, H6¢). Anal. Cacld. for C30H33F2N5: C,
71.83; H, 6.63; N, 13.96. Found: C, 71.75; H, 6.59; N, 13.94.
General procedure for the preparation of 1-{[4-
(substitutedphenyl ⁄ phenylethyl)piperazin-1-
yl]methyl}-3-methyl-1H-indole (3a–b)
3-{[4-(4-fluorophenyl)piperazin-1-yl]methyl}-1H-
indole (1a)a
Crystalized from ethanol-water. Yield: 63%; white solid. Mp
166.8 ꢀC. IR (KBr, cm)1): 3128 (N-H), 3094–2756 (C-H). 1H-NMR
To a solution of 3-methylindol (2.2 mmol, 300 mg) in ethanol
(20 mL), formalin (3 mmol) and substituted phenyl piperazine
870
Chem Biol Drug Des 2011; 78: 869–875

Indole-Based Sigma Receptors Ligands
(2.2 mmol) were added. The mixture was refluxed 4 h, and the
formed precipitate was filtered, dried and if necessary recrystalized
from appropriate solvent.
H2, H6), 2.33 (t, 2H, CH2-CH2-CH2-Npiperazine), 2.03 (q, 2H, CH2-CH2-
CH2). 13C-NMR (400 MHz, DMSO, ppm): 158.79, 155.45, 148.62,
136.40, 129.31, 128.73, 121.57, 119.49, 117.70, 117.64, 116.01,
115.79, 110,43, 101.13 (aromatics), 55,13 (indoleN-CH2-CH2-CH2),
55.14 (piperazine C3, C5), 49.65 (piperazine C2, C6), 43.95 (CH2-CH2-
CH2-N piperazine), 27,67 (CH2-CH2-CH2). Anal. Cacld. for C21H24FN3: C,
74.75; H, 7.17; N, 12.45. Found: C, 74.71; H, 7.13; N, 12.46.
1-{[4-(4-fluorophenyl)piperazin-1-yl]methyl}-3-
methyl-1H-indole (3a)
Crystalized from ethanol-water. Yield: 50.6%; white solid. Mp
109.5 ꢀC. IR (KBr, cm)1): 3045–2788 (C-H). 1H-NMR (400 MHz,
CDCl3, ppm): 7.55 (d,1H,indole H4), 7.42 (d,1H,indole H7), 7.21 (t, 1H,
indol H6), 7.12 (t,1H, indole H5), 6.93 (d, 2H, phenyl H3, H5), 6.90
(s,1H, indole H2), 6.81 (dd, 2H, phenyl H2, H6), 4.80 (s,2H, N-CH2-N),
3.08 (t,4H, piperazine H3, H5), 2.69 (t,4H, piperazine H2, H6), 2.33 (s,
3H, -CH3). 13C-NMR (400 MHz, CDCl3, 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. Cacld. for C20H22FN3: C, 74.28;
H, 6.86; N, 12.99. Found: C, 74.25; H, 6.75; N, 12.96.
1-{3-[4-(2-fluorophenyl)piperazin-1-yl]propyl}-1H-
indole (4b)
Yellowish oily residue was purified by column chromatography
(SiO2, AcOEt ⁄ n-hexane – 1 ⁄ 2). Rf 0.21 (SiO2, AcOEt ⁄ n-hexane 1:1).
Yield: 15%. IR (KBr, cm)1): 3010–2755 (C-H), 1225 (C=C). 1H-NMR
(400 MHz, CDCl3, ppm): 6.84–7.65 (m, 10H, indole + phenyl), 4.22
(t,2H,indoleN-CH2-CH2-CH2), 3.10 (t,4H, piperazine H3, H3¢, H5, H5¢),
2.55 (t, 4H, piperazine H2, H2¢, H6, H6¢), 2.32 (t, 2H, CH2-CH2-CH2-
Npiperazine), 2.05 (q, 2H, CH2-CH2-CH2). Anal. Cacld. for C21H24FN3:
C, 74.75; H, 7.17; N, 12.45. Found: C, 74.70; H, 7.15; N, 12.26.
1-{[4-(2-phenylethyl)piperazin-1-yl]methyl}-3-
methyl-1H-indole (3b)
1-{3-[4-(phenyl)piperazin-1-yl]propyl}-1H-indole
(4c)b
Crystalized from ethanol-water. Yield: 39%; white solid. Mp
122.9 ꢀC. IR (KBr, cm)1): 3022–2763 (C-H). 1H-NMR (400 MHz,
CDCl3, ppm): 7.54 (d, 2H, indole H4), 7.40 (d, 1H, indole H7), 7.28–
7.08 (m, 7H, indol H5, H6 + phenyl), 6.92 (s,1H, indole H2), 4.76 s,
2H, N-CH2-N), 2.75. Anal. Cacld. for C22H27N3: C, 79.24; H, 8.16; N,
12.60. Found: C, 79.20; H, 8.15; N, 12.56.
Yellowish oily residue was purified by column chromatography
(SiO2, AcOEt ⁄ n-hexane – 1 ⁄ 2). Rf 0.20 (SiO2, AcOEt ⁄ n-hexane 1:1).
Yield: 17%. IR (KBr, cm)1): 3011–2788 (C-H), 1240 (C=C). 1H-NMR
(400 MHz, CDCl3, ppm): 7.00–7.64 (m, 11H, indole + phenyl), 4.20
(t,2H,indoleN-CH2-CH2-CH2), 3.11 (t,4H, piperazine H3, H3¢, H5, H5¢),
2.54 (t, 4H, piperazine H2, H2¢, H6, H6¢), 2.32 (t, 2H, CH2-CH2-CH2-
Npiperazine), 2.07 (q, 2H, CH2-CH2-CH2). Anal. Cacld. for C21H25N3:
C, 78.96; H, 7.89; N, 13.15. Found: C, 78.91; H, 7.83; N, 13.16.
General procedure for the preparation of 1-{3-
[4-(substitutedphenyl)piperazin-1-yl]_ propyl}-
1H-indole (4a–c)
To a solution of substituted phenyl piperazine (5 mmol) in 10 mL of
acetone was added 7.5 mL of a 25% solution sodium hydroxide.
Thirty minutes later, 1-bromo-3-chloropropane (5.5 mmol) was added
carefully to minimize its mixing with aqueous layer. The mixture was
stirred slowly for 22 h with a magnetic stirrer. The organic phase
was then separated, and the solvent was removed under vacuum. A
mixture of indole (2.5 mmol) and 87% w ⁄ v solution KOH (7.5 mmol)
in DMSO (30 mL) was stirred at room temperature for 1 h. Reaction
mixture was cooled in ice-water bath to 0 ꢀC, and 1-(3-Chloropro-
pyl)-4-(substituted phenyl)piperazine in DMSO (10 mL) was added
dropwise. The stirring was continued at room temperature for 20–
30 h. After addition of water (50 mL) and extraction with Et2O, the
organic layer was washed with water and dried over anhydrous
Na2SO4. The solvent was evaporated and the oily residue was puri-
fied by column chromatography (SiO2, AcOEt ⁄ n-hexane 1:2) to give
1-{3-[4-(substituted phenyl)piperazin-1-yl]propyl}-1H-indole as an oil.
Receptor binding studies
Materials and general procedures
Guinea pig brains and rat livers were commercially available (Harlan-
Winkelmann, Germany). Homogenizer: Elvehjem Potter (B. Braun Bio-
tech International). Centrifuge: High-speed cooling centrifuge model
Sorvall RC-5C plus (Thermo Finnigan). Filter: Printed Filtermat Type B
(Perkin Elmer) presoaked in 0.5% aqueous polyethylenimine for 2 h at
room temperature before use. The filtration was carried out with a
MicroBeta FilterMate-96 Harvester (Perkin Elmer). The scintillation
analysis was performed using Meltilex (Type A) solid scintillator (Perkin
Elmer). The solid scintillator was melted on the filtermat at a tempera-
ture of 95 ꢀC for 5 min. After solidification of the scintillator at room
temperature, the scintillation was measured using a MicroBeta Trilux
scintillation analyzer (Perkin Elmer). The counting efficiency was 20%.
Membrane preparation for the r1 assay (29–32)
Five guinea pig brains were homogenized with the potter (500–
800 rpm, 10 up-and-down strokes) in six volumes of cold 0.32 M
sucrose. The suspension was centrifuged at 1200 · g for 10 min at
4 ꢀC. The supernatant was separated and centrifuged at 23 500 · g
for 20 min at 4 ꢀC. The pellet was resuspended in 5–6 volumes of buf-
fer (50 mM TRIS, pH 7.4) and centrifuged again at 23 500 · g
(20 min, 4 ꢀC). This procedure was repeated twice. The final pellet
1-{3-[4-(4-fluorophenyl)piperazin-1-yl]propyl}-1H-
indole (4a)
Yellowish oily residue was purified by column chromatography (SiO2,
AcOEt ⁄ n-hexane – 1 ⁄ 2). Rf 0.19 (SiO2, AcOEt ⁄ n-hexane 1:1). Yield:
18%. IR (KBr, cm)1): 3022–2763 (C-H), 1245 (C=C). 1H-NMR (400 MHz,
CDCl3, ppm) 6.86–7.64 (m, 10H, indole + phenyl), 4.24 (t,2H,indoleN-
CH2-CH2-CH2), 3.13 (t,4H, piperazine H3, H5), 2.57 (t, 4H, piperazine
Chem Biol Drug Des 2011; 78: 869–875
871

Yarim et al.
was resuspended in 5–6 volumes of buffer, the protein concentration
was determined according to the method of Bradford (33) using bovine
serum albumin as standard, and subsequently the preparation was fro-
zen ()80 ꢀC) in 1.5 mL portions containing about 1.5 mg protein ⁄ mL.
tion experiments with six concentrations of the test compounds and
were calculated with the program GraphPad Prismꢁ 3.0 (GraphPad
Software) by nonlinear regression analysis. The Ki-values were cal-
culated according to Cheng and Prusoff (34). The Ki-values are given
as mean values + SEM from three independent experiments.
Performing of the r1 assay (29–32)
The test was performed with the radioligand [3H]-(+)-pentazocine
(42.5 Ci ⁄ mmol; Perkin Elmer). The thawed membrane preparation
(about 75 lg of the protein) was incubated with various concentra-
tions of test compounds, 2 nM [3H]-(+)-pentazocine, and buffer
(50 mM TRIS, pH 7.4) in a total volume of 200 lL for 180 min at
37 ꢀC. The incubation was terminated by rapid filtration through the
presoaked filtermats by using the cell harvester. After washing each
well five times with 300 lL of water, the filtermats were dried at
95 ꢀC. Subsequently, the solid scintillator was placed on the filtermat
and melted at 95 ꢀC. After 5 min, the solid scintillator was allowed
to solidify at room temperature. The bound radioactivity trapped on
the filters was counted in the scintillation analyzer. The non-specific
binding was determined with 10 lM unlabeled (+)-pentazocine. The
Kd-value of the radioligand [3H]-(+)-pentazocine is 2.9 nM.
Cytotoxicity studies
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 and incubated in 37 ꢀC incubators containing 5% CO2
and 95% air.
NCI-60 sulphorhodamine B assay
Cancer cells (range of 2000 cell ⁄ well to 5000 cell ⁄ well) were inoc-
ulated into 96-well plates in 200 lL of media and incubated in
37 ꢀC incubators containing 5% CO2 and 95% air. After a 24 h
incubation period, one plate for each cell line was fixed with
100 lL of 10% ice-cold trichloroacetic acid (TCA). This plate repre-
sents the behavior of the cells just prior to drug treatment and is
accepted as the time-zero plate. The compounds to be tested were
solubilized in dimethyl sulfoxide (DMSO) to a final concentration of
40 mM and stored at +4 ꢀC. While treating the cells with the com-
pounds, the corresponding volume of the compound was applied to
the cell to achieve the desired drug concentration and diluted
through serial dilution. After drug treatment, the cells were incu-
bated in 37 ꢀC incubators containing 5% CO2 and 95% air for
72 h. Following the termination of the incubation period after drug
treatment, the cells were fixed with 100 lL of 10% ice-cold TCA
and incubated in the dark at +4 ꢀC for 1 h. Then the TCA was
washed away with ddH2O five times, and the plates were left to
air dry. For the final step, the plates were stained with 100 lL of
0.4% Sulphorhodamine B (SRB) (cat. no. 86183–5 g from Sigma)
solution in 1% acetic acid solution. Following staining, the plates
were incubated in 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. To measure the absorbance results, the
bound stain was then solubilized using 200 lL of 10 mM Tris-Base.
The OD values were obtained at 515 nm.
Membrane preparation for the r2 assay (29–32)
Two rat livers were cut into small pieces and homogenized with a
potter (500–800 rpm, 10 up-and-down strokes) in six volumes of
cold 0.32 M sucrose. The suspension was centrifuged at 1200 · g
for 10 min at 4 ꢀC. The supernatant was separated and centrifuged
at 31 000 · g for 20 min at 4 ꢀC. The pellet was resuspended in
buffer (50 mM TRIS, pH 8.0) and incubated at room temperature for
30 min. After the incubation, the suspension was centrifuged again
at 31 000 · g for 20 min at 4 ꢀC. The final pellet was resuspended
in buffer, the protein concentration was determined according to
the method of Bradford (33) using bovine serum albumin as stan-
dard, and subsequently the preparation was frozen ()80 ꢀC) in
1.5 mL portions containing about 2-mg protein ⁄ mL.
Performing of the r2-assay (29–32)
The test was performed with the radioligand [3H]-di-o-tolylguanidine
(50 Ci ⁄ mmol; ARC). The thawed membrane preparation (about 100 lg
of the protein) was incubated with various concentrations of test com-
pounds, 3 nM [3H]-di-o-tolylguanidine, 500 nM (+)-pentazocine and buf-
fer (50 mM TRIS, pH 8.0) in a total volume of 200 lL for 180 min at
room temperature. The incubation was terminated by rapid filtration
through the presoaked filtermats using a cell harvester. After washing
each well five times with 300 lL of water, the filtermats were dried at
95 ꢀC. Subsequently, the solid scintillator was placed on the filtermat
and melted at 95 ꢀC. After 5 min, the solid scintillator was allowed to
solidify at room temperature. The bound radioactivity trapped on the
filters was counted in the scintillation analyzer. The non-specific bind-
ing was determined with 10 lM unlabeled ditolylguanidine. The
Kd-value of the radioligand [3H]-ditolylguanidine is 17.9 nM.
Results and Discussion
The new piperazine substituted indole derivatives have been
designed, according to the r1 receptorial model proposed by Glen-
non et al. (24,35,36), with the assumption that the indole moiety
may interact with a secondary hydrophobic site corresponding to
the hydrophobic ''A'' region, the basic piperazine N-atom linked by
the alkylene chain to the indole moiety may interact with a recep-
torial proton donor site and the substituted N-phenyl ⁄ benzyl moiety
may bind a primary hydrophobic region similar to the phenyl 'B'
region of the r1 receptorial model and modulate the binding affin-
ity of the compounds for r1 or r2 receptors. In Figure 1, the most
potent r1 ligand is compared with Glennon model. The distance
between the left basic N-atom and the terminal phenyl moiety is 5
Data analysis
All experiments were carried out in triplicate using standard 96-well
multiplates (Diagonal). The IC50-values were determined in competi-
872
Chem Biol Drug Des 2011; 78: 869–875

Indole-Based Sigma Receptors Ligands
atoms (6 bond lengths). This distance corresponds exactly with the
most potent compounds of Glennon with a 5-phenylpentyl residue
at the N-atom.
forming of the r2 assay in the presence of an excess of non-triti-
ated 1,3-di(o-tolyl)guanidine led to the non-specific binding of the
radioligand (29–32).
The preparation of the compounds is illustrated in
Scheme 1. The groups of 3-{[4-(substitutedphenyl ⁄ benzyl)piperazin-
1-yl]methyl}-1H-indole 1a–ca, 1,3-di-{[4-(4-fluorophenyl)piperazin-1-
yl]methyl}-1H-indole 2a and 1-{[4-(substitutedphenyl ⁄ phenyleth-
yl)piperazin-1-yl]methyl}-3-methyl-1H-indole 3a–b were prepared by
Mannich reaction of substituted piperazine and formaldehyde with
indole or 3-methylindole. The crude products were purified by
recrystallization or column chromatography.
Selectivity ratios between the r1 receptor and the r2 receptor were
also calculated to determine relative specificity and are summarized
in Table 1.
When structural modifications were examined (Table 1), the r recep-
tor affinities were low in 1-non-substitutedindol derivatives 1a–c.
Replacement of the phenyl ring by a benzyl moiety increased r affin-
ity of these compounds. Exchange of 2,5-difluoro for 3,4-dichloro sub-
stitution increased r1 and r2 affinity for the same group of
compounds. Only low r1 and r2 receptor affinities were determined
for 1,3-dipiperazinomethyl substituted indole 2a. 1-{[4-(2-Phenyleth-
yl)piperazin-1-yl]methyl}-3-methyl-1H-indole 3b shows very high
affinity for r1 (Ki = 14.2 nM) and r2 (Ki = 55.8 nM) receptors and,
therefore, can be regarded as unselective r ligand. Compound 3a
having 4-fluorophenyl subtitution showed 70 times lower affinity
than 3b for r1 receptor sites. A specific interaction of compound 3a
with r2 receptors could not be observed even at a concentration of
1 lM. A comparison of the r receptor affinities of 3a and 3b
showed that an increased distance between the piperazine ring and
the phenyl moiety resulted in enhanced selectivity for the r1 subtype.
Compounds 4a–c displayed very high affinity and selectivity for r2
receptors in vitro. Remarkably, the r2 affinity of the compounds hav-
ing a trimethylene spacer between indole and piperazine ring was
5–100 fold increased, whereas the r1 affinity was not changed. In
this series of compounds, phenylpiperazin-1-ylpropyl derivatives 4a–
c, without a substituent in 3-position of the indole system, exhibited
high affinity (Ki = 20, 9.9, and 75 nM) and selectivity for the r2
receptor. The highest r2 ⁄ r1 selectivity was found for the 2-fluor-
ophenyl derivative 4b having the highest r2 receptor affinity.
1-{3-[4-(substitutedphenyl)piperazin-1-yl]propyl}-1H-indole
4a–cb
were synthesized by the reaction of indole and 1-(3-chloropropyl)-4-
(substitutedphenyl)piperazine in presence of potassium hydroxide. To
obtain 1-(3-chloropropyl)-4-(substitutedphenyl)piperazine, substituted
phenyl piperazine was reacted with 1-bromo-3-chloropropane. Com-
pounds 4a–c were purified by column chromatography on silica gel
using ethyl acetate ⁄ n-hexane as
a mobile phase system
(Scheme 1).
The r receptor affinity of the compounds was evaluated with
receptor binding studies. The test compounds compete with tritium
labeled ligands for a limited number of receptors. Homogenates of
guinea-pig brain and rat liver were used as receptor material in the
r1 assay and the r2 assay, respectively. In the r1 assay, [3H]-(+)-
pentazocine was employed as radioligand, and the non-specific
binding was determined in the presence of a large excess of (+)-
pentazocine. As a r2 selective radioligand is not commercially avail-
able, the non-selective radioligand [3H]-ditolylguanidine was
employed in the presence of a large excess of non-radiolabeled (+)-
pentazocine (500 nM), which selectively occupies r1 receptors. Per-
R′
n
N
R′
N
N
N
N
n
N
H
i
3a–b
iii
R
1a–c
N
H
iv
ii
N
N
R′
R′
N
R′
N
N
N
N
N
4a–c
2a
1a
H
0
1b
H
1
1c
H
1
2a
H
-
3a
CH3
0
3b
CH3
2
4a
H
-
4b
H
-
4c
H
-
R
n
R′
4-F
2,5-diF
3,4-diCl 4-F
4-F
H
4-F
2-F
H
Scheme 1: Synthesis of compounds 1a–c, 2a, 3a–b, and 4a–c. Reagent and conditions: (i) HCHO, substituted piperazine, EtOH, room
temperature; (ii) HCHO, 4-F-phenylpiperazine, EtOH, reflux, 4 h; (iii) HCHO, substituted piperazine, EtOH, reflux, 4 h; and (iv) 87% KOH, DMSO,
room temperature, 1 h; 1-(3-chloropropyl)-4-(substitutedphenyl)piperazine, DMSO, 0 ꢀC, 20h.
Chem Biol Drug Des 2011; 78: 869–875
873

Yarim et al.
Table 1: Binding affinities of compounds at r1 and r2 receptors
Ki € SEM (nM)
the relative affinities and selectivities of ligand binding to r1 and
r2 receptor subtypes, we synthesized several modifications of the
indole derivatives. The findings from this study led to the conclu-
sion that an increase in the linker length between the indole and
piperazine rings to three methylene moieties results in compounds
with high affinity and selectivity for r2 receptors. Additionally, intro-
duction of two large substituents on indole ring was not tolerated
by sigma receptors.
a
b
Compounds
r1
r2
Selectivity ratio r1 ⁄ r2
1a
1b
1c
2a
3a
3b
4a
4b
4c
3640
660
5.5
0.5
1
2.7
–
0.3
22.8
26.7
19.9
216 € 98
126 € 23
2690
418 € 55
130 € 9
1000
1000
0%c
Currently, a large variety of chemotherapeutic drugs are used to
treat cancer. However, many compounds have limited efficacy due
to problems of delivery and penetration and a moderate degree of
selectivity for cancer cells. In this study, our results demonstrate
that some of the synthesized compounds exhibit a high cytotoxic
effect on growing cancer cells in vitro. This study identifies this
new series of agents for cancer therapy.
14.2 € 4
456 € 32
264 € 130
1492
55.8 € 1.6
20 € 3
9.9 € 2.7
75 € 7.7
Values are mean € SEM of three experiments performed in duplicate.
aDisplacement of [3H](+)pentazocine.
bDisplacement of [3H]-ditolylguanidine in the presence of (+)pentazocine.
cInhibition of radioligand [3H]ditolylguanidine at concentration of 1 lM.
The compounds were tested for their effect on cellular viability
against cancer cell lines from liver (HUH7), breast (MCF7), and colon
(HCT-116) samples. The results are given in Table 2.
Acknowledgments
This work is supported by The Scientific
& Technological
Research Council of Turkey (TUBITAK) (Project Number: 108S009).
We gratefully acknowledge Assoc. Prof. Dr. Rengul Atalay for the
cytotoxicity assays. The authors have declared no conflict of
interest.
The cytotoxic activity of the synthesized compounds was investi-
gated on liver (HUH7), breast (MCF7), and colon (HCT116) cancer
cell lines, by means of sulphorhodamine B (SRB) assays in triplicate.
Serial dilutions from 40 to 2.5 lM were used, and Camptothecin
was the positive control for the cytotoxic effect (Table 2). As seen
in Table 2, especially, 1c and 1b showed high cytotoxicity levels
on the selected cancer cell lines. 1c had lower IC50 values when
compared with 5-FU. A 50% growth inhibition of the cancer cell
lines was observed in micromolar concentrations. Among com-
pounds, the best inhibitory activity against HUH7 (IC50 = 3.42 lM)
was exhibited by compound 1c (3-{[4-(3,4-dichlorobenzyl)piperazin-
1-yl]methyl}-1H-indole) (Table 2). For MCF7 (IC50 = 2.92 lM),
HCT116 (IC50 = 9.19 lM) cell lines, compound 1c and compound
1b showed the lowest IC50 values, respectively.
References
1. Aydar E., Palmer C.P., Klyachko V.A., Jackson M.B. (2002) The
sigma receptor as a ligand-regulated auxiliary potassium channel
subunit. Neuron;34:399–410.
2. Jbilo O., Vidal H., Paul R., De Nys N., Bensaid M., Silve S., Cara-
yon P. et al. (1997) Purification and characterization of the human
SR 31747A-binding protein – a nuclear membrane protein related
to yeast sterol isomerase. J Biol Chem;272:27107–27115.
3. Hanner M., Moebius F.F., Flandorfer A., Knaus H.G., Striessnig
J., Kempner E., Glossmann H. (1996) Purification, molecular clon-
ing, and expression of the mammalian sigma(1)-binding site.
Proc Natl Acad Sci USA;93:8072–8077.
4. Mei J.F., Pasternak G.W. (2001) Molecular cloning and pharma-
cological characterization of the rat sigma(1) receptor. Biochem
Pharmacol;62:349–355.
5. Pan Y.X., Mei J., Xu J., Wan B.L., Zuckerman A., Pasternak G.W.
(1998) Cloning and characterization of a mouse sigma(1) recep-
tor. J Neurochem;70:2279–2285.
6. Seth P., Leibach F.H., Ganapathy V. (1997) Cloning and structural
analysis of the cDNA and the gene encoding the murine type 1
sigma receptor. Biochem Biophys Res Commun;241:535–540.
7. Seth P., Fei Y.J., Li H.W., Huang W., Leibach F.H., Ganapathy V.
(1998) Cloning and functional characterization of a sigma recep-
tor from rat brain. J Neurochem;70:922–931.
8. Ablordeppey S.Y., Fischer J.B., Glennon R.A. (2000) Is a nitrogen
atom an important pharmacophoric element in sigma ligand
binding? Bioorg Med Chem;8:2105–2111.
Conclusion and Future Directions
In conclusion, four different series of r receptor ligands were syn-
thesized and evaluated for their r receptor affinities. To modulate
Table 2: IC50(lM) of the compounds for liver (HUH7), breast
(MCF7), and colon (HCT116) carcinoma cell lines
Compounds
HUH7a
MCF7a
HCT116a
1a
1b
1c
14.63
13.87
9.32
5.47
11.65
9.19
3.42
2.92
9.33
b
b
b
2a
3a
3b
4a
4b
4c
NI
NI
17.67
NI
NI
40.95
26.23
21.66
23.95
NI
NI
23.86
20.45
30.24
NI
5-FU
30.66
3.5
18.7
aAll the experiments were conducted in triplicate (1 < R2 < 0.8). NI: no inhi-
9. Hellewell S.B., Bowen W.D. (1990) A Sigma-like binding-site in
rat pheochromacytoma (PC12) cells – Decreased affinity for (+)-
bition.
bInsoluble.
874
Chem Biol Drug Des 2011; 78: 869–875

Indole-Based Sigma Receptors Ligands
Benzomorphans and lower molecular-weight suggest a different
sigma receptor form from that of guinea-pig brain. Brain
Res;527:244–253.
ligand as a potential breast tumor imaging agent. J Med
Chem;41:2445–2450.
24. Glennon R.A., Ablordeppey S.Y., Ismaiel A.M., El-Ashmawy M.B.,
Fischer J.B., Howie B.K. (1994) Structural features important
sigma-1 receptor binding. J Med Chem;37:1214–1219.
25. de Costa R.B., He X.-S., Linder J.T.M., Dominguez C., Gu Z.Q.,
Williams W., Bowen W.D. (1993) Synthesis and evaluation of
conformationally restricted N-[2-(3,4-dichlorophenyl)ethyl]-N-
methyl-2-(1-pyrrolidinyl)ethylamines at sigma receptors. 2. Piper-
azines, bicyclic amines, bridged bicyclic amines, and miscella-
neous compounds. J Med Chem;36:2311–2320.
26. Younes S., Labssita Y., Baziard-Mouysset G., Payard M., Rettori
M.-C., Renard P., Pfeiffer B., Caignard D.-H. (2000) Synthesis and
structure–activity relationships of novel arylalkyl 4-benzyl pipera-
zine derivatives as r site selective ligands. Eur J Med
Chem;35:107–121.
27. Bedꢀrftig S., Wꢀnsch B. (2004). Chiral, nonracemic (piperazin-2-
yl)methanol derivatives with sigma-receptor affinity. Bioorg Med
Chem;12:3299–3311.
28. Weigl M., Wꢀnsch B. (2007) Chiral, nonracemic (piperazin-2-
yl)methanol derivatives with sigma-receptor affinity. Eur J Med
Chem;42:1247–1262.
10. Hellewell S.B., Bruce A., Feinstein G., Orringer J., Williams W.,
Bowen W.D. (1994) Rat-liver and kidney contain high-densities
of sigma (1) and sigma (2) receptors – Characterization by
ligand-binding and photoaffinity-labeling. Eur
col;268:9–18.
J
Pharma-
11. Bem W.T., Thomas G.E., Mamone J.Y., Homan S.M., Levy B.K.,
Johnson F.E., Coscia C. (1991) Overexpression of delta-receptors
in nonneuronal human tumors. Cancer Res;51:6558–6562.
12. Vilner B.J., Bowen W.D. (1992) Characterization of sigma-like
binding sites of NB41A3, S-20Y, and N1E-115 neuroblastomas,
C6 glioma, and NG108–15 neuroblastoma-glioma hybrid cells:
further evidence for sigma-2 receptors. In: Kamenka J.-M.,
Domino E.F., editors. Multiple Sigma and PCR Receptor Ligands
Mechanisms for Neuromodulation and Neuroprotection? Ann
Arbor: NPP Books; p. 341–353.
13. Mach R.H., Smith C.R., Al-Nabulsi I., Whirrett B.R., Childers
S.R., Wheeler K.T. (1997) Receptors as potential biomarkers of
proliferation in breast cancer. Cancer Res;57:156–161.
14. Abou-Gharbia M., Ablordeppey S.Y., Glennon R. (1993) Sigma
receptors and their ligands: The Sigma Enigma. In: Bristol J.A.,
editor. Annual Reports in Medicinal Chemistry. San Diego: Aca-
demic; 28, p. 1.
29. Maier C.A., Wꢀnsch B. (2002) Novel spiropiperidines as highly
potent and subtype selective r-selective ligands.
J Med
Chem;45:438–448.
15. Hayashi T., Su T.-P. (2004) r-1 receptor ligands: potential in the
treatment of neuropsychiatric disorders. CNS Drugs;18:269–284.
16. Sorbera L.A., Silvestre J., Castaner J. Monograph igmesine
hydrochloride (1999) Drugs Fut; 24: 133–140.
30. Oberdorf C., Schepmann D., Vela J.M., Diaz J.L., Holenz J.,
Wꢀnsch B. (2008) Thiophene bioisosteres of spirocyclic r recep-
tor ligands. 1. N-substituted spiro[piperidine-4,4¢-thieno[3,2-c]py-
rans]. J Med Chem;51:6531–6537.
17. Foster A., Wu H., Chen W., Williams W., Bowen W.D., Matsum-
oto R.R., Coop A. (2003) 1,4-dibenzylpiperazines possess antico-
caine activity. Bioorg Med Chem Lett;13:749–751.
18. Matsumoto R.R., Liu Y., Lerner M., Howard E.W., Bracket D.J.
(2003) r receptors: potential medications development target for
anti-cocaine agents. Eur J Pharmacol;469:1–12.
19. Matsumoto R.R., McCracken K.A., Pouw B., Miller J., Bowen
W.D., Williams W., deCosta B.R. (2001) N-alkyl substituted ana-
logs of the r receptor ligand BD1008 and traditional r receptor
ligands affect cocaine-induced convulsions and lethality in mice.
Eur J Pharmacol;411:261–273.
20. Crawford K.W., Bowen W.D. (2002) Sigma-2 receptor agonists
activate a novel apoptotic pathway and potentiate antineoplastic
drugs in breast tumor cell lines. Cancer Res;62:313–322.
21. Spruce B.A., Campbell L.A., McTavish N., Cooper M.A., Apple-
yard V.L., O'Neill M., Howie J. et al. (2004) Small molecule
antagonists of the sigma-1 receptor cause selective release
of the death program in tumor and self-reliant cells and inhi-
bit tumor growth in vitro and in vivo. Cancer Res;64:4875–
4886.
31. Geiger C., Zelenka C., Weigl M., Frçhlich R., Wibbeling B., Le-
hmkuhl K., Schepmann D., Grꢀnert R., Bednarski P.J., Wꢀnsch B.
(2007) Synthesis of bicyclic r receptor ligands with cytotoxic
activity. J Med Chem;50:6144–6153.
32. Holl R., Schepmann D., Frçhlich R., Grꢀnert R., Bednarski P.J.,
Wꢀnsch B. (2009) Dancing of the second aromatic residue
around the 6,8-diazabicyclo[3.2.2]nonane framework: influence
on sigma receptor affinity and cytotoxicity.
Chem;52:2126–2137.
J
Med
33. Bradford M.M. (1976) A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing the prin-
ciple of protein-dye binding. Anal Biochem;72:248–254.
34. Cheng Y., Prusoff W.H. (1973) Relationship between the inhibi-
tion constant, Ki, and the concentration of inhibitor which
causes 50 per cent inhibition (I50) of an enzymatic reaction. Bio-
chem Pharmacol;22:3099–3108.
35. Ablordeppey S.Y., Fischer J.B., Law H., Glennon R.A. (2002) Prob-
ing the proposed phenyl-a region of the sigma-1 receptor. Bioorg
Med Chem;10:2759–2765.
36. Glennon R.A. (2005) Pharmacophore identification for sigma1
receptor binding. Mini Rev Med Chem;5:927–940.
22. Choi S.-R., Yang B., Plossl K., Chumpradit S., Wey S.P., Acton
P.D., Wheeler K., Mach R.H., Kung H.F. (2001) Development of a
Tc-99m labeled sigma-2 receptor-specific ligand as a potential
breast tumor imaging agent. Nucl Med Biol;28:657–666.
23. John C.S., Lim B.B., Vilner B.J., Geyer B.C., Bowen W.D. (1998)
Development of a Tc-99m labeled sigma-2 receptor-specific
Notes
aMauvernay R., Busch N. (1969) US 3453366.
bSydney A., Bentlehem N.Y. (1964) US 3135794.
Chem Biol Drug Des 2011; 78: 869–875
875