Design, synthesis, and biological evaluation of novel 2,4-thiazolidinedione derivatives as histone deacetylase inhibitors targeting liver cancer cell line

Article abstract of DOI:10.1007/s00044-011-9623-3

As a part of an ongoing effort to find alternate chemotherapeutic agents for hepatocellular carcinoma, we herein, report the design and synthesis of two novel compounds targeting histone deacetylase (HDAC) with 2,4-thiazolidinedione as zinc chelating group. Further, we demonstrate that these compounds show cytotoxicity that parallels their ability to inhibit HDACs activity in human liver cancer cell line HepG2. The findings obtained in this study indicate that 2,4-thiazolidinedione group may be utilized successfully to inhibit HDAC activity with future potential for lead optimization by chemical derivatization of active compound, N-(6-(2,4-dioxothiazolidin-3- yl)hexyl) benzenesulfonamide. Springer Science+Business Media, LLC 2011.

Full text of DOI:10.1007/s00044-011-9623-3

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:1156–1165
DOI 10.1007/s00044-011-9623-3
ORIGINAL RESEARCH
Design, synthesis, and biological evaluation of novel
2,4-thiazolidinedione derivatives as histone deacetylase
inhibitors targeting liver cancer cell line
•
•
•
Rhea Mohan Ajit K. Sharma Sanjay Gupta
C. S. Ramaa
Received: 4 January 2011 / Accepted: 5 March 2011 / Published online: 29 March 2011
Ó Springer Science+Business Media, LLC 2011
Abstract As a part of an ongoing effort to find alternate
chemotherapeutic agents for hepatocellular carcinoma,
we herein, report the design and synthesis of two novel
compounds targeting histone deacetylase (HDAC) with
2,4-thiazolidinedione as zinc chelating group. Further, we
demonstrate that these compounds show cytotoxicity that
parallels their ability to inhibit HDACs activity in human
liver cancer cell line HepG2. The findings obtained in this
study indicate that 2,4-thiazolidinedione group may be uti-
lized successfully to inhibit HDAC activity with future
potential for lead optimization by chemical derivatization of
active compound, N-(6-(2,4-dioxothiazolidin-3- yl)hexyl)
benzenesulfonamide.
(Marin et al., 2008). HCC is often diagnosed at an
advanced stage, when it is not amenable to curative ther-
apies. Treatment of liver cancer is primarily limited by its
refractivity to available chemotherapy and development of
resistance during treatment (Avila et al., 2006). Advances
in cancer biology suggest that a limited number of path-
ways are responsible for initiating and maintaining dys-
regulated cell proliferation, which is the major cellular
alteration responsible for the cancer phenotype (Roberts
and Gores, 2005). New treatments in development target
several of these critical pathways, one of them being the
epigenetic histone deacetylation pathway. Rikimaru et al.,
in their report, were the first to conclude that a high Histone
Deacetylase 1 (HDAC1) expression might play an impor-
tant role in the aggressiveness, cell dedifferentiation, and
poor outcome in patients with HCC. A high HDAC1
expression was found to be an independent poor prognostic
factor in patients with HCC. Normal hepatocytes did not
express HDAC1; therefore HDAC1 in HCC may be a good
molecular target for anticancer agents (Rikimaru et al.,
2007).
Keywords Hepatocellular carcinoma cell line Á
HDAC inhibitors Á 2,4-Thiazolidinedione Á Histones
Introduction
Primary liver tumors, mainly hepatocellular carcinoma
(HCC) and cholangiocarcinoma, constitute the fifth most
frequent types of cancer, whereas in the rank of mortality,
they are the third. The clinical relevance of liver tumor
increases further if the number of deaths due to liver
metastasis of cancers of extrahepatic origin is included
The last decade has seen an exponential growth in
research addressing HDACs and their inhibitors, and a
significant number of HDAC inhibitors have been identi-
fied and developed (Walkinshaw and Yang, 2008; Miller
et al., 2003; Paris et al., 2008). The mode of action of
HDAC inhibitors for anticancer activities is partially
through the transcriptional regulation of genes involved in
proliferation, differentiation, or apoptosis (Carew et al.,
2008). Normal cells are relatively resistant to HDAC
inhibitor induced cell death. It may be speculated that
cancer cells with multiple defects cannot reverse the crit-
ical effects of HDAC inhibitors as normal cells may do. It
was previously reported that Trichostatin A (TSA) induces
cell cycle arrest/apoptosis and hepatocyte differentiation in
R. Mohan Á C. S. Ramaa (&)
Bharati Vidyapeeth’s College of Pharmacy, Sector 8,
C.B.D. Belapur, Navi Mumbai 400614, Maharashtra, India
e-mail: sinharamaa@yahoo.in
A. K. Sharma Á S. Gupta (&)
Cancer Research Institute, Tata Memorial Centre, ACTREC,
Sector 22, Kharghar, Navi Mumbai 410210, Maharashtra, India
e-mail: sgupta@actrec.gov.in
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Med Chem Res (2012) 21:1156–1165
1157
human liver cancer cell lines (Yamashita et al., 2003). In
addition, HDAC inhibitors like suberoyl anilide hydroxa-
mic acid (SAHA) and sulfatase 1 enzyme (SULF1) were
seen to inhibit HCC tumor in in vivo animal models
(Venturelli et al., 2007; Lai et al., 2006). Thus, the current
results are considered to provide important evidence of the
future application of HDAC inhibitors in patients with
HCC.
SRR1 (Andrew’s pKi: 2.052, Predicted pKi: 5.765) and
N-(6-(2,4-dioxothiazolidin-3-yl)hexyl) benzenesulfonamide
SRR2 (Andrew’s pKi: 0.9527, Predicted pKi: 6.602), both
having 2,4-thiazolidinedione as the enzyme inhibiting
group, showed better affinity for the receptor as compared to
other molecules. SAHA was re-docked into active site of
HDLP. The root mean square deviation (RMSD) between
the heavy atom positions of bound and MOE conformation
˚
was 1.19 A. Both conformations were quite well overlaid.
Herein we report the results of design, synthesis, anti-
proliferative and HDAC inhibitory activity of two novel
compounds with 2,4-thiazolidinedione as zinc chelating
group, which may be used as a lead for the development of
new antitumor agents targeting liver cancer.
Figure 2 depicts the docked poses of SRR1 and SRR2 with
HDLP. These molecules also show high metal ligation val-
ues (SRR1: 0.206, SRR2: 0.275). The considerable differ-
ence in their values for Andrew’s pKi and predicted pKi
indicate that these molecules maybe specific to the receptor.
These compounds were therefore selected for synthesis and
detailed biological evaluation with respect to their antican-
cer and HDAC inhibiting potential.
Results and discussions
Designing
Chemistry
The HDAC catalytic domain consists of a narrow, tube like
pocket of a length equivalent to that of a 4–6 carbon
straight chain. A zinc cation is positioned near the bottom
of this enzyme pocket, which in co-operation with two
His-Asp charge relay system facilitates the deacetylation
catalysis (Finnin et al., 1999).
Compounds SRR1 and SRR2 were synthesized by follow-
ing the route outlined in Scheme 1. 1,6-Dibromohexane was
refluxed with 2,4-thiazolidinedione in the presence of tri-
ethylamine (TEA) to obtain 3-(6-bromohexyl)-2,4-thiazo-
lidinedione (a) (Aoki et al., 2003). This intermediate was then
heated under reflux in a solution of hexamine in chloroform
which resulted in formation of a white precipitate of 1-[N-(6-
(2,4-dioxothiazolidin-3-yl)hexyl)]-1-azonia-3,5,7-triazatri-
cyclo decane bromide (b). This precipitate was filtered under
vacuum and subjected to overnight hydrolysis in acidic
ethanol at RT to obtain [N-(6-(2,4-dioxothiazolidin-3-
yl)hexyl)] ammonium chloride (c) (Warmus et al., 1993).
This reaction gave a product which was highly unstable
making its isolation difficult. Therefore, the final step
was carried out in the same assembly without isolation of
[N-(6-(2,4-dioxothiazolidin-3-yl)hexyl)] ammonium chlo-
ride using dichloromethane (DCM) as the solvent and TEA
as base. The resultant final products were isolated by column
chromatography and their structures were confirmed by
various spectral analyses.
Mai et al. have elaborated a structural model for Class
I/II HDAC inhibition. All known natural or synthetic
inhibitors possess an extremely variable cap group. This
moiety contacts residues on the rim of the catalytic pocket
and is connected to an electronegative group (X), that is
able to interact by hydrogen bond with other residues. This
in turn is bound to a hydrocarbon linker interacting with
channel residues of the active site of the enzyme and fin-
ishing with the enzyme inhibiting group (EIG), that in many
cases chelate the zinc cation near the bottom of the catalytic
pocket (Mai et al., 2005). Using this structural model as a
scaffold, we designed and docked several molecules having
the general structure shown in Fig. 1 into the active pocket
of histone deacetylase-like protein (HDLP).
Based on the scoring values, it was observed that com-
pounds N-(6-(2,4-dioxothiazolidin-3-yl)hexyl)benzamide
Biological evaluation
The biological activity of newly synthesized HDAC
inhibitors having 2,4-thiazolidinedione as enzyme inhibit-
ing group was evaluated using both cell proliferation assay
and HDAC enzyme assay.
Antiproliferative testing
The antiproliferative activity was assessed on human liver
cell lines, transformed (HepG2) and untransformed embry-
onic (WRL68) cell lines using dose-dependent and time-
Fig. 1 General structure of designed histone deacetylase inhibitors
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Med Chem Res (2012) 21:1156–1165
Fig. 2 Docked SRR1 (a) and SRR2 (b) showing zinc chelation (dotted lines) with HDLP
dependent MTT assay (spectrophotometric quantification of
viable cells by cleavage of tetrazolium salt) (Mosmann,
1983). WRL68 cell line was used to determine the effect of
test compounds on normal untransformed cells. The results
obtained have been depicted in Figs. 3 and 4. In addition,
cells treated with compounds SRR1 and SRR2 exhibited a
more elongated shape than that of control HepG2 cells; this
is similar to the morphological changes seen in sodium
butyrate (SB) or other known HDAC inhibitor-treated cells
(data not shown).
death, respectively, at the end of 48 h. The same dose for
same time period caused 34.66% (SRR1) (Fig. 3a) and
37.5% (SRR2) (Fig. 3b) cell death in WRL68 cells. The
dose-dependent studies indicate that test compounds are
cytotoxic in both HepG2 as well as WRL68 cell lines
although more selective toward transformed HepG2 cells.
Also, the cellular effect of test compounds was further
assessed for time-dependent differential growth inhibition in
HepG2 and WRL 68 cell lines by MTT assay. The growth
inhibition or cellular proliferation was measured at 48, 72,
96, and 120 h time points. The time-dependent studies
indicate both the compounds retain their cytotoxicity at the
end of 120 h causing 82.98% (SRR1) (Fig. 4a) and 69.45%
(SRR2) (Fig. 4b) cell death in HepG2 cell line. The pattern
Treatment of cells with test compounds resulted in cell
line specific growth inhibition. Treatment of HepG2 cell line
with 100 lM doses of test compounds SRR1 and SRR2
brought about 42.31% (Fig. 3a) and 57.27% (Fig. 3b) cell
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Med Chem Res (2012) 21:1156–1165
1159
O
O
TEA
Br
+
S
NH
Br
S
N
Br
reflux
1,6-dibromohexane
O
O
thiazolidine-2,4-dione
3-(6-bromohexyl)thiazolidine-2,4-dione
hexamine
reflux
N
O
N
N
N
N
Br
S
O
1-[N-(6-(2,4-dioxothiazolidin-3-yl)hexyl)]-1-azonia-3,5,7-triazatricyclo decane bromide
ethanol
conc HCl
O
N
S
ClH3N
O
[N-(6-(2,4-dioxothiazolidin-3-yl)hexyl)] ammonium chloride
benzoyl chloride/ benzene sulfonyl
chloride
TEA
O
X
N
S
HN
O
Scheme 1 Synthesis of 2,4-thiazolidinedione derivatives as HDAC inhibitors
of cell death in WRL 68 cell line shows a different pattern
from that of HepG2. Even though cytotoxicity is seen, WRL
68 cells treated with compound SRR2 recover since there is
significant difference in the percent cell death in these cells
(55.6%) as compared to the cell death in HepG2 cell line at
the end of 120 h (Fig. 4b). Compound SRR1 shows better
cytotoxicity but it suffers from the drawback that it is equally
active against transformed (82.98% cell death) and
untransformed (92.34% cell death) cells (Fig. 4a).
of treated and untreated HepG2 cells were analyzed for
their HDAC activity at different time intervals ranging
from 2 to 24 h. Maximum HDAC inhibition was seen in
the 8th hour time interval (Fig. 5) wherein compounds
SRR1 and SRR2 showed 42.11 and 56.85% HDAC inhi-
bition as compared to positive controls SB (52.32% HDAC
inhibition) and SAHA (57.89% HDAC inhibition).
Hyperacetylation of histones
In vitro HDAC assay
Many studies have demonstrated that inhibition of HDAC
activity by specific inhibitors induces hyperacetylation of
histones in drug-treated samples. To investigate whether
newly synthesized compounds inhibits HDAC activity in
The HDAC inhibitory potential of synthesized molecules
was assessed using HDAC enzyme assay. Nuclear extracts
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Fig. 3 Dose-dependent response curves of HDAC inhibitors. WRL
68 and HepG2 cells were treated with various concentrations of SRR1
(a) and SRR2 (b) as indicated, for 48 h. Cell viability was determined
by MTT assay. Results are expressed as % of cell death as compared
with control. Data represents mean values of at least three separate
experiments
Fig. 4 Time-dependent response curves of HDAC inhibitors. WRL
68 and HepG2 cells were treated with SRR1 (a) and SRR2 (b) at
various time intervals as indicated. Cell viability was determined by
MTT assay. Results are expressed as % of cell death compared with
control. Data represents mean values of at least three separate
experiments
cells, the levels of post-translational modifications (PTMs)
in histone was visualized using triton acid urea polyacryl-
amide gel electrophoresis (TAU PAGE) and antibodies
specific for acetylated H3-histone (Abcam ab47915).
TAU PAGE is a commonly used method to separate
histone and their post-translationally modified forms. The
TAU PAGE separates proteins on the basis of mass, charge
and hydrophobicity. The hyperacetylation induced by
newly synthesized compounds peaks at 8th hour time point
as shown by HDAC assay. Therefore, after 8 h, histones
were extracted and subjected to TAU gel electrophoresis to
determine changes, if any, in the PTM status of the various
histones. TAU gel shows the presence of linker histone H1.
Preliminary identification of core histones can be obtained
in this gel system because core histones show histone-
specific differences in their affinity for triton and thus a
histone-specific reduction in gel mobility. Affinity for tri-
ton typically increases from low for histone H4, to mod-
erate for H2B, to intermediate or high for H3, and to high
or very high for H2A.
TAU gel electrophoresis of total histones purified from
HepG2 cells, treated with SB, SAHA or newly synthesized
compounds SRR1 and SRR2 shows modified forms of his-
tone H3 and H4 and low levels of similar modifications in
histone H2A and H2B in comparison to untreated cells
(Fig. 6).
Western blotting
To further investigate whether newly synthesized com-
pounds, SRR1 and SRR2 inhibits HDAC activity in
HepG2 cells, the levels of hyperacetylated histone H3 was
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Med Chem Res (2012) 21:1156–1165
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Fig. 5 HDAC activity of test compounds on HepG2 cell line. Eight
hours post-treatment with sodium butyrate (SB), SAHA, SRR1, and
SRR2 cells were harvested for HDAC assay. SB and SAHA were
used as positive control for the experiment. % HDAC activity is
expressed on basis of OD at 400 nm. Data represents mean values of
at least three separate experiments
studied by western blotting (Dickinson and Fowler, 2002).
As shown in Fig. 7, test compounds increase the acetyla-
tion of lysine residue in the N-terminal tail of H3 histone.
Fig. 6 AUT gel analysis of total nuclear histones extracted from
HepG2 cells with or without (control) treatment with sodium butyrate,
SAHA, SRR1, SRR2. 5 lg of histones were resolved on TAU PAGE.
Gel was stained using ammoniacal silver nitrate staining method.
Mobility of different histones are marked in the figure. Charge
modified forms are marked as PTM
Experimental
Designing
All computations for designing were carried out on a
Pentium 2.67 GHz workstation, 256 MB memory with
Windows operating system and Molecular Operating
Environment (MOE 2006.08) as computational software.
The crystal structure of an archbacterial HDAC homologue
(HDAC like protein- HDLP) complexed with SAHA [1c3s]
was obtained from Protein Data Bank library of MOE and
used for docking studies.
All the designed molecules were built, energy minimized
and used as ligands for the docking studies using MOE
software. The protein was prepared for docking by addi-
tion of hydrogen atoms and energy minimization using
AMBER99 forcefield. The active site was generated using
atom selector wherein atoms involved in binding the inhib-
itor, SAHA, to HDLP were taken into consideration. These
atoms were assigned as ‘‘binding pocket’’. The inhibitor was
then deleted from the crystal structure.
The energy minimized ligands were then docked into the
active site of HDLP. Docking was performed using Alpha
triangle placement method with London dG scoring based on
the free energy of binding of a ligand from a given pose. The
alpha triangle placement method generates different ligand
orientations by superposing ligand and receptor triplet points.
These are the alpha spheres that represent tight packing of
residues in the active site. The London dG scoring method
evaluates the binding free energy for the poses generated.
Fig. 7 Western blot analysis for hyperacetylation of histone H3.
HepG2 cells were treated with compounds sodium butyrate, SAHA,
SRR1, and SRR2 for 8 h. Cells were harvested and histones were acid
extracted. a Histogram plotted after densitometer scanning, b immu-
noblot with antibody specific for acetylated H3 histones, and c silver-
stained core histones to confirm the equal loading on the blot
Poses were further filtered using a pharmacophore
query. Metal ligation was the most critical parameter that
determined the inhibitory effect of designed molecule. It
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was therefore decided to give prominence to this feature.
Hydrophobicity of the linker chain was also included in the
pharmacophore query. The entire system was further
energy minimized.
in the previous step. This mixture was heated under reflux
for 4 h, after which reaction mixture was cooled and fil-
tered under vacuum to give 1-[N-(6-(2,4-dioxothiazolidin-
3-yl)hexyl)]-1-azonia-3,5,7-triazatricyclo decane bromide
as a white precipitate. The yield was found to be 74%.
The final models were evaluated for binding orienta-
tions using scoring svl functionality available in MOE
which is used to visualize intermolecular contacts like
direct hydrogen bonds, water-mediated hydrogen bonds,
transition metal interactions, and hydrophobic interactions
(Joshi et al., 2006). It also computes predicted pKi of the
ligand. Predicted pKi is an estimate of the affinity of a
ligand for a specific receptor.
Synthesis of [N-(6-(2,4-dioxothiazolidin-3-yl)hexyl)]
ammonium chloride
In a flask were mixed 4 ml of 95% ethanol and 0.7 ml conc.
HCl. To the still warm solution was added 0.7 g (0.0023
mol) of 1-[N-(6-(2,4-dioxothiazolidin-3-yl)hexyl)]-1-azo-
nia-3,5,7-triazatricyclo decane bromide in one portion. The
reaction mixture was allowed to stir at RT for 18 h, cooled
to 0°C and filtered to remove the precipitated ammonium
chloride. Filtrate was concentrated on rotary evaporator to
obtain the product as a yellow solid which was immediately
taken to the next step, i.e., benzoylation.
Chemistry
IR spectra were recorded as KBr discs, using Shimadzu
1
8400S FTIR spectrophotometer. H NMR spectrums and
13C NMR spectrums were recorded on Varian Mercury Plus
spectrometer at 300 and 75 MHz, respectively. The syn-
thesized compounds were dissolved in deuterated chloro-
form, CDCl3, for this purpose and trimethylsilane (TMS)
was used as an internal standard. Chemical shift values have
been reported in ppm (d). The elemental analysis (C, H, N,
S, O) were performed using the Thermo Fisher FLASH EA
1112 analyzer and were within 0.4% of theoretical values.
All reactions as well as column chromatography were fol-
lowed by TLC using Merck pre-coated silica gel 60 F254
plates and spots were visualized by observing in UV cabinet
under short UV. All reagents were used as received unless
otherwise stated.
Synthesis of N-(6-(2,4-dioxothiazolidin-3-yl)
hexyl)benzamide (SRR1)
[N-(6-(2,4-Dioxothiazolidin-3-yl)hexyl)] ammonium chlo-
ride obtained in the previous step (0.0016 mol, 0.3 g) was
dissolved in dichloromethane (10 ml). To this was added
0.6 ml (0.00417 mol) of TEA. This mixture was cooled to
2–5°C and then 0.2 ml (0.0016 mol) of benzoyl chloride
was added dropwise. This mixture was stirred at RT for one
and a half hours.
At the end of this time period, the mixture was given
DCM-water (3 9 30 ml) washings. The organic layer was
then subjected to dilute hydrochloric acid (HCl) washing to
remove excess of TEA. The organic layer was again
washed with water and dried over anhydrous sodium sul-
fate. This was then concentrated. The residue was given
ether washings (2 9 20 ml). The precipitate was collected
and subjected to column chromatography using chloro-
form: ethyl acetate (3:2) as the eluent.
Synthesis of 3-(6-bromohexyl)-2,4-thiazolidinedione
36.6 g (22.8 ml, 0.15 mol) of 1,6-dibromohexane, 5.9 g
(0.063 mol) of 2,4 thiazolidinedione, and 7.1 g of TEA
(9.8 ml, 0.07 mol) were added to 125 ml of acetonitrile
(ACN) and mixture was heated with stirring under reflux
for 8 h. After cooling to room temperature (RT), solvent
was evaporated under reduced pressure and the residue was
poured into water. Mixture was extracted with DCM–water
and organic layer was washed with saturated brine and
dried over sodium sulfate. After solvent was evaporated
under reduced pressure, residue was purified by column
chromatography using chloroform as eluent to obtain title
compound as pale yellow oil. This reaction gave a good
yield of 92% of the title compound.
Yield: 47%; Rf: 0.71 (4:1, chloroform/ethyl acetate); IR
(KBr, cm-1): 3311.55 (NH stretching vibration of amide),
1751.24 and 1668.31 (C=O stretching vibrations of cyclic
imides), 1633.59 (C=O stretching vibration of amide),
1257.50 (C–N stretching vibration of amide); 1H NMR
(300 MHz, CDCl3) d (ppm): 1.5 (m, 8H, aliphatic protons),
3.4 (q, 2H, –CH2–NH), 3.6 (t, 2H, –CH2 adjacent to thia-
zolidinedione ring), 4.0 (s, 2H, thiazolidinedione ring
protons), 6.3 (bs, 1H, NH), 7.7 (m, 5H, aromatic protons).
13C NMR: (75 MHz, CDCl3) d (ppm): 26.148, 26.239,
27.392, 29.415, 33.765, 39.763, 41.796, 126.899, 128.528,
131.330, 134.759, 167.554 (C=O), 171.560 (C=O),
171.954 (C=O); Anal. Calc. for C16H20N2O3S, %: C,
59.98; H, 6.29; N, 8.74; S, 10.01; O, 14.98. Found, %: C,
59.76; H, 6.32; N, 8.72; S, 9.87; O, 15.21.
Synthesis of 1-[N-(6-(2,4-dioxothiazolidin-3-yl)hexyl)]-1-
azonia-3,5,7-triazatricyclo decane bromide
To a solution of 1 g hexamethylene tetramine/hexamine
(0.0072 mol) in 15 ml CHCl3, was added 2 g of 3-(6-
bromohexyl)-2,4-thiazolidinedione (0.0072 mol) obtained
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Med Chem Res (2012) 21:1156–1165
1163
Synthesis of N-(6-(2,4-dioxothiazolidin-3-yl)
hexyl)benzenesulfonamide (SRR2)
CO2/95% air atmosphere with 95% humidity. For testing,
cells were harvested by trypsinization and plated (5,000
cells/well) in 96 well plates and incubated for 24 h at 37°C
in the incubator.
The procedure followed for the synthesis of N-(6-(2,4-di-
oxothiazolidin-3-yl)hexyl)benzenesulfonamide was the
same as described for synthesis of SRR1, wherein the
benzoyl chloride was substituted with benzene sulfonyl
chloride (0.0016 mol).
Dose-dependent MTT assay
After incubating the plates for 24 h, the cytotoxic effects of
synthesized compounds were tested in vitro against the cell
lines at tenfold dilutions of four concentrations ranging
from 10-3 to 10-6 M. The percentage growth was evalu-
ated spectrophotometrically versus negative control not
treated with test agents and positive control, treated with
SB (1 mM), a proven HDAC inhibitor. At the end of 48 h,
the medium from each well was removed by aspiration and
the cells washed with Dulbecco’s phosphate buffered saline
(PBS). 20 ll of MTT solution (stock 5 mg/ml) was added
to each well and plate was incubated at 37°C for 4 h. The
media was then removed from each well and 100 ll of
DMSO was added. Plates were then transferred to plate
reader and absorbance was measured at 540 nm with ref-
erence absorbance at 690 nm (Mosmann, 1983).
Yield: 43%; Rf: 0.68 (4:1, chloroform/ethyl acetate); IR
(KBr, cm-1): 3288.40 (NH stretching vibration of sulfon-
amide), 1751.24 and 1664.10 (C=O stretching vibrations of
cyclic imides), 1325.01 and 1159.14 (C=O stretching
1
vibrations of sulfonamide); H NMR (300 MHz, CDCl3) d
(ppm): 1.5 (m, 8H, aliphatic protons), 2.9 (q, 2H, –CH2–
NH), 3.6 (t, 2H, –CH2 adjacent to thiazolidinedione ring),
4.0 (s, 2H, thiazolidinedione ring protons), 5.0 (t, 1H, NH),
7.8 (m, 5H, aromatic protons). 13C NMR: (75 MHz, CDCl3)
d (ppm): 25.834, 25.986, 27.331, 29.354, 33.795, 39.763,
41.786, 43.000, 127.071, 129.155, 132.645, 140.050,
171.590 (C=O), 171.924 (C=O); Anal. Calc. for C15H20N2
O4S2, %: C, 50.54; H, 5.66; N, 7.86; S, 17.99; O, 17.95.
Found, %: C, 50.36; H, 5.24; N, 8.14; S, 17.65; O, 18.13.
Biological evaluation
Time-dependent MTT assay
Materials and methods
For the time-dependent assay, the growth inhibition or
cellular proliferation was measured at 48, 72, 96, and 120 h
time points. The dose of synthesized molecules selected for
this assay was 100 lM as determined from the results
obtained for the dose-dependent assay. The procedure
followed was the same as reported in dose-dependent
assay.
The HDAC inhibitor, SB and the tetrazolium salt, MTT
were procured from Sigma-Aldrich. Compounds SRR1 and
SRR3 were synthesized in our laboratory with purities
exceeding 99%, as is evident from NMR. For in vitro
studies, stock solutions of inhibitors were prepared in
dimethyl sulfoxide (DMSO) and stored frozen prior to use.
At the time of compound addition, an aliquot of frozen
concentrate was thawed and diluted to the desired final test
concentration in 10% serum-containing medium for treat-
ment of cells (final concentration of DMSO, \0.1%). The
transformed liver cancer cell line HepG2 and untrans-
formed normal embryonic liver cell line WRL68 were
purchased from National Centre for Cell Science (NCCS),
Pune. Culture media, RPMI 1640 and Dulbecco’s Modified
Eagle’s Medium, DMEM, were obtained from Sigma-
Aldrich. HDAC assay kit was purchased from Biovision.
PVDF membrane and chemiluminescence kit were pur-
chased from Millipore. Acetylated histone H3 antibody
was procured from Abcam.
In vitro HDAC assay
HepG2 cells were plated and treated with synthesized
compounds (100 lM), SB (1 mM) and SAHA (250 nm)
for different time periods ranging from 2 to 24 h. HDAC
activity was assayed using 10 lg of nuclear extract pre-
pared from control and drug-treated samples. Commer-
cially available colorimetric HDAC assay kit (Biovision
K331-100) was used to determine the HDAC inhibitory
potential of synthesized compounds. The HDAC assay was
carried out in accordance to the instructions provided by
the manufacturer along with the kit. This method required
two steps, both performed on the same microtiter plate.
First, the HDAC colorimetric substrate, which comprises of
an acetylated lysine side chain, was incubated with nuclear
extract. Deacetylation of the substrate sensitized the sub-
strate, so that, in the second step, treatment with lysine
developer produced a chromophore. The chromophore was
analysed using an ELISA plate reader at 400 nm.
Antiproliferative testing
HepG2 cells were grown in monolayer cultures in RPMI
1640 medium and WRL68 cells in DMEM medium, both
supplemented with 10% heat-inactivated fetal bovine
serum and 1% antibiotics and maintained at 37°C in a 5%
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Med Chem Res (2012) 21:1156–1165
Acknowledgments Dr C. S. Ramaa thanks Board of Research in
Nuclear Sciences (BRNS), BARC, Mumbai, India (Sanction Number:
2006/37/41/BRNS) for financial support. Ms. Rhea Mohan is grate-
ful to Board of Research in Nuclear Sciences (BRNS) for provid-
ing fellowship. We thank Dr. V. J. Kadam, Principal, Bharati
Vidyapeeth’s College of Pharmacy, Navi Mumbai for providing
support for the ongoing project. The authors would also like to
express their gratitude to Mr. Bharat S. Khade, Scientific Assistant
‘D’, ACTREC, for technical support during the tenure of this work.
Hyperacetylation of histones
TAU PAGE HepG2 cell line cells were plated and treated
with test compounds, SRR1 and SRR2 (100 lM), SB
(1 mM), and SAHA (250 nM). At the end of 8 h incuba-
tion, histones were isolated from these cells. Total histones
were resolved on TAU PAGE according to method
described by Waterborg (Waterborg, 2002). Five micro-
gram histones were loaded in each well of the vertical slab
gel (Macrokin, Technosource). Histone samples were
electrophoresed at constant voltage of 250 V till methylene
blue dye front reached bottom of gel. Amido black-silver
staining was carried out as described by Mold et al. (1983).
After electrophoresis, gel was immersed in 10 volumes of
1% amido black in 40% methanol and 7% acetic acid for
3 h at room temperature on shaker. Gel was destained
overnight in excess of 32% ethanol and 8% acetic acid.
After destaining, gel was stained by ammoniacal silver
method (Khare et al., 2009).
Conflict of interest Authors have no conflict of interest regarding
the work reported in this manuscript.
References
Aoki K, Aikawa K, Kawakami M (2003) Benzimidazole compounds and
drugs containing the same. US Patent US6593323B1, July 15, 2003
Avila MA, Berasain C, Sangro B, Prieto J (2006) New therapies for
hepatocellular carcinoma. Oncogene 25:3866–3884
Carew JS, Giles FJ, Nawrocki ST (2008) Histone deacetylase
inhibitors: mechanisms of cell death and promise in combination
cancer therapy. Cancer Lett 269:7–17
Dickinson J, Fowler SJ (2002) Quantification of proteins on western
blots using ECL. In: Walker JM (ed) The protein protocols
handbook, 2nd edn. Humana Press, Totowa, NJ, pp 429–438
Finnin MS, Donigian JR, Cohen A, Richon VM, Rifkind RA, Marks
PA, Breslow R, Pavletich NP (1999) Structures of histone
deacetylase homologue bound to the TSA and SAHA inhibitors.
Nature 401:188–193
Joshi UJ, Shah FH, Tikhele SH (2006) Homology model of the human
5-HT1A receptor using the crystal structure of bovine rhodopsin.
Internet Electron J Mol Des 5:403–415
Khare SP, Khade B, Gupta S (2009) Mass spectrometry-compatible
silver staining of histones resolved on acid-urea-triton PAGE.
Proteomics 9(9):2589–2592
Lai JP, Yu C, Moser CD, Adreca I, Han T, Garvey TD, Murphy LM,
Garrity-Park MM, Shridhar V, Adjei AA, Roberts LR (2006)
SULF1 inhibits tumor growth and potentiates the effects of
histone deacetylase inhibitors in hepatocellular carcinoma.
Gastroenterology 130:2130–2144
Western blotting HepG2 cell line cells were cultured and
treated with test compounds, SRR1 and SRR2 (100 lM),
SB (1 mM) and SAHA (250 nM) for 8 h. After treatment,
cells were cultured and harvested by centrifugation. His-
tones were extracted and isolated histones were subjected
to 18% sodium dodecyl sulfate-polyacrylamide gel elec-
trophoresis (SDS-PAGE) and transferred to a PVDF
membrane. Membrane was immunoblotted with antibodies
against acetylated H3 histones. Acetylated histones were
determined using chemiluminescence kit (Dickinson and
Fowler, 2002).
Mai A, Silvio M, Rotili D, Cerbara H, Valente S, Pezzi R, Simeoni S,
Ragno R (2005) Histone deacetylation in epigenetics: an attractive
target for anticancer therapy. Med Res Rev 25(3):261–309
Marin JGJ, Castano B, Martinez-Becerra P, Rosales R, Monte MJ
(2008) Chemotherapy in the treatment of primary liver tumors.
Cancer Therapy 6:711–728
Miller TA, Witter DJ, Belvedere S (2003) Histone deacetylase
inhibitors. J Med Chem 46(24):5097–5116
Mold DE, Weingart J, Assaraf J, Lubahn DB, Kelner DN, Shaw BR,
McCarthy KS Sr (1983) Silver staining of histones in triton-acid-
urea gels. Anal Biochem 135:44–47
Mosmann T (1983) Rapid colorimetric assay for cellular growth and
survival: application to proliferation and cytotoxicity assays.
J Immunol Methods 65:55–63
Paris M, Porcelloni M, Binaschi M, Fattori D (2008) Histone
deacetylase inhibitors: from bench to clinic. J Med Chem 51(6):
1505–1529
Rikimaru T, Taketomi A, Yamashita Y, Shirabe K, Hamatsu T,
Shimada M, Maehara Y (2007) Clinical significance of histone
deacetylase 1 expression in patients with hepatocellular carci-
noma. Oncology 72:69–74
Roberts LR, Gores GJ (2005) Hepatocellular carcinoma: molecular
pathways and new therapeutic targets. Semin Liver Dis 25(2):
212–225
Conclusion
In summary, we have successfully performed docking
studies of molecules with alternate zinc-binding heterocy-
clic groups to determine their effectiveness as HDAC
inhibitors. Based on their scoring, two novel compounds
containing 2,4-thiazolidinedione as enzyme inhibiting
group were synthesized and subjected to antiproliferative
and HDAC inhibition studies on liver cancer cell line
HepG2. Although both the compounds showed good
cytotoxicity and HDAC inhibition as confirmed by H3
acetylation status, compound SRR2 proved to be the better
candidate for future consideration on account of relative
selectivity toward transformed cells. Thus, the experi-
mental validation of 2,4-thiazolidinedione as a hitherto
unexplored enzyme inhibiting group for HDAC will
advance further efforts for lead optimization by chemical
derivatization of newly synthesized active compound
SRR2.
123

Med Chem Res (2012) 21:1156–1165
1165
Venturelli S, Armeanu S, Pathil A, Hsieh CJ, Weiss TS, Vonthen R,
Wehrmann M, Gregor M, Lauer UM, Bitzer M (2007) Epigenetic
combination therapy as a tumor-selective treatment approach for
hepatocellular cancer. Cancer 109(10):2132–2141
Walkinshaw DR, Yang XJ (2008) Histone deacetylase inhibitors as
novel anticancer agents. Curr Oncol 15(5):70–76
Warmus JS, Dilley GJ, Meyers AI (1993) A modified procedure for
the preparation of 2,5-dihydropyrrole (3-pyrroline). J Org Chem
58:270–271
Waterborg JH (2002) Acid-urea-triton polyacrylamide gel electro-
phoresis of histones. In: Walker JM (ed) The protein protocols
handbook, 2nd edn. Humana Press, Totowa, NJ, pp 113–124
Yamashita Y, Shimada M, Harimoto N, Rikimaru T, Shirabe K,
Tanaka S, Sugimachi K (2003) Histone deacetylase inhibitor
trichostatin A induces cell-cycle arrest/apoptosis and hepatocyte
differentiation in human hepatoma cells. Int J Cancer 103:
572–576
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