Synthesis and evaluation of some novel N-substituted rhodanines for their anticancer activity

Article abstract of DOI:10.1007/s00044-016-1545-7

Novel N-substituted rhodanines 2a-g were synthesized by conventional and microwave-assisted methods and tested for their anticancer activity. Structure-activity relationship of the synthesized rhodanine 2a-g as antiproliferative agents was investigated. The results revealed that all the seven compounds showed potent antiproliferative activity in a concentration-dependent manner on leukemic cell line K562. Among the tested compounds, 2b was found to be more potent when compared by trypan blue and MTT assay. IC₅₀ values of 2b using trypan blue and MTT assay were found to be 11.1 and 20.3 μg/ml, respectively. A dose-dependent increase in the LDH release was also observed upon treatment with 2a-g. Cell cycle analysis revealed that 2b affects DNA replication and leads to accumulation of cells in G₀ and decline of G₂/M, G₁ and S phases which indicates apoptosis. The selective cytotoxic activity against human chronic myelogenous cell line (K562), via apoptosis, suggests that compound 2b is a promising scaffold for the development of novel anticancer drug.

Full text of DOI:10.1007/s00044-016-1545-7

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-016-1545-7
ORIGINAL RESEARCH
Synthesis and evaluation of some novel N-substituted rhodanines
for their anticancer activity
1
1
1
Sulaiman Ali Muhammad
•
Subban Ravi
•
Arumugam Thangamani
Received: 21 April 2015 / Accepted: 18 February 2016
Ó Springer Science+Business Media New York 2016
Abstract Novel N-substituted rhodanines 2a–g were
synthesized by conventional and microwave-assisted
methods and tested for their anticancer activity. Structure–
activity relationship of the synthesized rhodanine 2a–g as
antiproliferative agents was investigated. The results
revealed that all the seven compounds showed potent
antiproliferative activity in a concentration-dependent
manner on leukemic cell line K562. Among the tested
compounds, 2b was found to be more potent when com-
pared by trypan blue and MTT assay. IC50 values of 2b
using trypan blue and MTT assay were found to be 11.1
and 20.3 lg/ml, respectively. A dose-dependent increase in
the LDH release was also observed upon treatment with
Introduction
Cancer is one of the difficult diseases to be cured, and only
very few effective ways are available. The development of
novel, efficient and less toxic anticancer agents remains an
important and challenging goal in medicinal chemistry.
Understanding the molecular mechanism involved in can-
cers should lead to the identification of novel anticancer
agents. Leukemia is one of the major types of cancer
affecting a significant segment of the population. In fact,
leukemia is the most frequent childhood cancer with 26 %
of all cases and 30 % mortality (Brown et al., 1956; Lesyk
and Zimenkovsky, 2004; Moorthy et al., 2010; Ravi et al.,
2010). Although the incidence rate for this disease remains
relatively unchanged, some success has no doubt been
attained in its treatment. The current treatments, however,
have many limitations. This includes side effects and the
development of acquired drug resistance (Brown et al.,
1956; Moorthy et al., 2010). Therefore, there is a need for
the development of effective anticancer drugs with well-
defined pharmacokinetic properties.
2a–g. Cell cycle analysis revealed that 2b affects DNA
replication and leads to accumulation of cells in G and
0
decline of G /M, G and S phases which indicates apop-
1
2
tosis. The selective cytotoxic activity against human
chronic myelogenous cell line (K562), via apoptosis, sug-
gests that compound 2b is a promising scaffold for the
development of novel anticancer drug.
Keywords Rhodanine Á Microwave-assisted synthesis Á
Cytotoxicity Á Apoptosis
Rhodanine derivatives have been proven to be attractive
compounds due to their outstanding biological activities
and have undergone rapid development as anticonvulsant,
antibacterial, antiviral and antidiabetic agents (Brown and
Bradsher, 1951; Nitsche and Klein, 2012; Prashantha
Kumar et al., 2012; Kamila et al., 2012). At the same time,
these have also been reported as inhibitors of hepatitis C
virus (HCV) protease (Frankov et al., 1985), HIV-1 inte-
grase inhibitors (Kavya et al., 2010a, b) and also used as
inhibitors of uridinediphospho-N-acetylmuramate/L-alanine
ligase (Alizadeh et al., 2009). Recently, substituted rhoda-
nines were investigated for tau aggregation inhibitor prop-
erties (Jacobine and Posner, 2011). Rhodanines are
classified as nonmutagenic (Alizadeh and Zohreh, 2009),
&
Subban Ravi
ravisubban@rediffmail.com
1
Department of Chemistry, Karpagam Academy of Higher
Education, Coimbatore 641 021, Tamil Nadu, India
123

Med Chem Res
and a long-term study on the clinical effects of the rhoda-
nine-based Epalrestat as an antidiabetic has demonstrated
that it is well tolerated (Alizadeh et al., 2009). Additionally,
rhodanines have been designed as inhibitors of various
enzymes such as bacterial b-lactamase and Mur ligases
allyl residues. The N-substituted rhodanines 2a–g were
synthesized from amine/amino acid and carbon disulfide
adopting base-catalyzed process in the conventional
(Scheme 1) and microwave-assisted methods as shown in
Scheme 2. The addition of sodium hydroxide in the reac-
tion serves to support the nucleophilic attack of amine at
carbon disulfide. The hypothesis here is that the amino acid
moiety will change the polarity and acidity near the thia-
zolidine ring due to the freely exposed carboxyl group and
is also relatively safe with respect to its metabolism in the
body.
(
Jacobine and Posner, 2011). Due to various possibilities of
chemical derivatization of the rhodanine ring, rhodanine-
based compounds will probably remain a privileged scaf-
fold in drug discovery. The synthesis of these compounds is
of considerable interest. The anticancer properties of rho-
danine derivatives have not been investigated extensively.
Earlier we have synthesized and evaluated 5-isopropy-
lidene-3-ethyl rhodanine (Ia) for its cytotoxicity (Brown
et al., 1956; Ravi et al., 2010; Nitsche and Klein, 2012) and
found that the compound shows cytotoxicity in leukemic
cell line, CEM and induces apoptosis. These encouraging
results make rhodanine a promising starting point for fur-
ther synthesis and optimization by structure–activity rela-
tionship studies. To compare them with their O and N
counterparts, we also synthesized and evaluated (Brown
et al., 1956; Ravi et al., 2010; Nitsche and Klein, 2012)
three novel compounds, mainly 5-isopropylidene deriva-
tives of 3-methyl-2-thio-hydantoin Ib, 3-ethyl-2-thio-2,4-
oxazolidinedione Ic and 5-benzylidene-3-ethyl rhodanine
Id (Fig. 1). The results revealed that Id was more potent
than Ia, Ib and Ic with an IC50 value around 10 lM.
Furthermore, we also showed that Id affects DNA repli-
cation by inducing a block at G /G followed by induction
It is known that reports with the straightforward syn-
thesis of the N-substituted rhodanine precursors with a
‘free’ 5-position are very scarce (Alizadeh and Zohreh,
2009). Only a small number of these N-substituted rhoda-
nine synthons are commercially available. There is a need
for a straightforward synthetic approach to make these key
building blocks. Reported protocols for N-substituted rho-
danines require long reaction time under aqueous condi-
tions (Vivek and Rajender, 2008). Considering these
aspects, we used an interesting approach for the synthesis
of rhodanine derivatives and report an aqueous, micro-
wave-assisted and one-pot protocol for the rapid and effi-
cient synthesis of N-substituted rhodanines using carbon
disulfide. This protocol enables the synthesis of N-substi-
tuted rhodanines from the corresponding amino acids under
environmentally friendly conditions. Our procedure
employed sodium hydroxide as the reagent in the first two
reaction steps and hydrochloric acid in the last step (Al-
izadeh et al., 2009). After some optimization using alanine
as a model substrate, we identified the conditions for a
0
1
by cell death by apoptosis. This showed the importance of
the presence of a benzylidene moiety in position C-5 of the
rhodanine nucleus. Now, in order to optimize the N-3
position, in the present study we have synthesized different
N-substituted rhodanines and tested them for their anti-
cancer properties.
1
S 2
S
1
2
.NaOH, CS2
. ClCH COONa
5
Chemistry
2
N3
RNH2
R
4
3. conc.HCl
O
We designed some novel rhodanines incorporated with
amino acid residues like glycine, alanine, phenylalanine,
valine and glutamic acid residues in addition to ethyl and
1a-g
r.t
2a-g
(each setps 3-4h)
R
a:-CH COOH
e:-CH-CH -Ph
2
2
S
COOH
S
b:-CH-COOH
O
X
O
S
2
-CH CH3
N
R
CH3
f:
N
C H
2
5
c:-CH-COOH
g: -CH CH=CH2
2
Ia R = C H X= S
;
,
2
5
CH(CH3)2
Ib R = C H X= NCH
;
;
,
2
5
,
5
3
d:-CH-COOH
Ic R = C H X= O
Id
2
CH -CH -COOH
2
2
Fig. 1 The five-membered heterocyclic core; rhodanine (X = S),
thiohydantoin (X = NCH ) and thioxooxazolidine (X = O)
3
Scheme 1 Conventional synthesis of 2a–g
1
23

Med Chem Res
O
O
Cl
CS (1 mol)
2
H
H
ONa
R-NH2
a-g
R N
S Na
R N
S
NaOH(22%),
O Na
o
(
1 mol), 100 C,
1
o
1
00 C, 80 W,
S
S
8
0 W, 5 min
5
min
Step 1
Step 2
o
1
10 C, 90 W,
conc.HCl
20-30 min
Step 3
R
1
a:-CH COOH
e:-CH-CH -Ph
S 2
2
2
S
5
COOH
N3
b:-CH-COOH
R
4
f: -CH
CH
O
CH3
2
3
2
a-g
c:-CH-COOH
g: -CH CH=CH2
2
CH(CH3)2
d:-CH-COOH
CH -CH -COOH
2
2
Scheme 2 Microwave-assisted synthesis of 2a–g
one-pot synthetic approach as shown in Table 1 as the
starting point for further investigations. Alanine and carbon
disulfide were allowed to react in water under basic con-
ditions in a microwave reactor for 5 min at 80 W and
other N-substituted rhodanines. For comparison purposes,
the compounds were also prepared by conventional
method.
1
00 °C, followed by the addition of chloroacetic acid and a
Synthesis of N-substituted rhodanine (2a–g)
second reaction for 5 min at 80 W and 100 °C. An excess
of aqueous hydrochloric acid was then added, and final
reaction step was executed at 80 W and 110 °C for 30 min.
The same conditions were used for the preparation of all
A mixture of amino acid, sodium hydroxide in water and
carbon disulfide was stirred for 4 h at room temperature,
followed by the addition of sodium chloroacetate and
Table 1 Evaluation of conditions for the synthesis of N-substituted rhodanine 2b by microwave-assisted synthesis
S. no
Solvent
Step 1
Time
Step 2
Time
Step 3
Time
Yield (%)
Conditions
Conditions
Conditions
o
Temp ( C)
o
Temp ( C)
o
Temp ( C)
MW (W)
MW (W)
MW (W)
1
2
3
4
5
6
7
8
9
–
100
100
100
100
100
100
100
100
100
100
100
100
80
80
80
60
80
100
80
80
80
80
80
80
10
10
10
10
10
10
5
100
100
100
100
100
100
100
100
100
100
100
100
80
80
80
60
80
100
80
80
80
80
80
80
10
10
10
10
10
10
5
100
100
100
100
100
100
100
100
100
100
100
100
80
80
80
60
80
100
80
80
80
80
80
80
10
10
10
10
10
10
5
55
61
69
48
74
72
76
71
74
72
79
84
Ethanol
Water
Water
Water
Water
Water
Water
Water
Water
Water
Water
10
15
5
10
15
5
10
15
10
20
30
1
1
1
0
1
2
5
10
15
5
123

Med Chem Res
further stirred for 3 h. Conc. HCl was then added, and the
product was separated and dried (Wang et al., 2012; Xing
et al., 2007; Kaminsky et al., 2012; Bernardo et al., 2009,
Table 2 Trypan blue exclusion test for compound 2a–g against the
human chronic myelogenous cell line K562
a,b
Compound
IC50 (lg/ml)
2
011) (Scheme 1).
2
2
2
2
2
2
2
a
b
c
d
e
f
14.60 ± 3.5
11.10 ± 2.0
19.98 ± 2.9
29.21 ± 3.8
36.52 ± 2.6
21.48 ± 2.4
23.74 ± 3.4
4.78 ± 3.2
In order to develop a greener synthesis, the above
mixture was treated in a microwave oven at 100 °C, 80 W
for 45 min using water as the solvent, and the product was
recovered as above. The yield was comparable to the
conventional method, but the reaction time was shorter
(
Scheme 2). The compounds were characterized by UV,
g
IR, NMR and elemental analysis.
Cisplatin
a
Determined by trypan blue exclusion method
Results and discussion
b
All experiments were conducted in triplicate
Compounds related to 2a have been reported to behave as
an imaging agent for neurofibrillary tangles (NFTs)
detection in Alzheimer’s disease and as GSK-3 inhibitors
-CH(CH(CH
)
)–COOH and -C
H
as N-substituents
3
2
2
5
showed good cytotoxic activity. In the other three mole-
cules 2d, 2e and 2g, the low activity is due to steric hin-
drance of the larger groups present as N-substituents. The
groups may have different orientation in space and may not
fit into the receptors.
(
Martinez et al., 2003; Anumala et al., 2013). Compounds
related to 2b have been tested and found to alter the life
span of eukaryotic organisms (David, 2009). Compounds
related of 2c have been reported as selective inhibitors for
antiapoptotic Bcl-2 proteins from BHI-1 by molecular
modeling studies (Xing et al., 2007). However, in none of
these studies cytotoxicity was studied in detail.
The antiproliferative effect induced by rhodanines on
proliferation of cancer cells was further verified using MTT
assay on K562 cancer cell line. The cancer cells treated
with the 6.25, 12.5, 25, 50 and 100 lg/ml concentrations of
the compounds 2a–g were harvested after 48 h and were
subjected to MTT assay. Results showed that cell viability
(Table 3) was affected upon treatment with 2a–g.
Antiproliferative activity in human chronic
myelogenous leukemia cell line in a dose-dependent
manner
Compounds 2a, 2b and 2f showed good antiproliferative
effect with an IC50 value of 27.73, 20.31 and 28.18 lg/ml,
respectively, and compounds 2c–e and 2 g also exhibited
cytotoxic effect against K562 cell line with an IC50 value
of 87.07, 51.35, 40.77 and 35.84 lg/ml, respectively.
Compounds 2a, 2b and 2f are seen to be twice potent than
2c–e and 2 g. The most potent compound was found to be
2b with an IC50 value of 20.31 lg/ml.
To further check the antiproliferative effect of 2a–g,
LDH release in the cell culture suspension was measured
using LDH assay (Fig. 3). The DMSO-treated cells were
used as vehicle control, and 2a–g-treated cells showed
LDH release suggesting antiproliferative effect on K562
cells. The membrane of K562 cells was disrupted due to
antiproliferative effect of 2a–g causing release of LDH
(lactate dehydrogenase) into the supernatant. Results
showed a dose-dependent increase in the LDH release upon
treatment of various concentrations of 2a–g further con-
firming the above results (Fig. 3).
In order to evaluate the antiproliferative activity of syn-
thesized rhodanines, 2a–g, we used trypan blue assay.
Cancer cell line K562 (human chronic myelogenous leu-
kemia) was treated with 6.25, 12.5, 25, 50 and 100 lg/ml
of compounds 2a–g (Table 2).
Since the compounds dissolved in 0.1 % DMSO, the
cells with DMSO were used as vehicle control. Following
the addition of compounds, cells were counted after 24 h
till the control cells attained stationary phase. Results
showed that cell growth was affected by the increase in
concentration. The effect was limited when 25 lg/ml of 2b
was used. However, concentrations of 50 and 100 lg/ml
resulted in increased cell death (Fig. 2). These results
suggest that 2b induces antiproliferative effect in human
cancer cell line K562.
The IC50 of values obtained for 2a–g after 48 h of
treatment is shown in Table 2. The data revealed that
compounds 2a–c and 2f are more potent with an IC50 of
1
4.60, 11.10, 19.98, 21.48 lg/ml, respectively, and are
twice or three times more potent than compounds 2d, 2e
and 2g with an IC50 of 29.23, 36.52 and 23.72 lg/ml,
respectively. The compounds 2a–c and 2f with small
groups such as -CH COOH, -CH–(CH )–COOH,
Cell cycle analysis by flow cytometry
We performed fluorescence-activated cell sorting (FACS)
analysis to determine the effect of the most potent
2
3
1
23

Med Chem Res
Fig. 2 Evaluation of cell
viability using trypan blue assay
following 2a–g treatment. K562
cells were treated with 6.25,
1
2.5, 25, 50 and 100 lg/ml. The
data presented is result of three
independent experiments and
error bar are indicated
compound 2b on cell cycle progression. K562 cells were
stained with propidium iodide after 72 h of treatment with
2b (25 lg/ml) and subjected to FACS. Histogram of con-
trol cells showed a standard cell cycle pattern, which
includes G and G separated by S phase (Fig. 4). The G
0
Table 3 MTT assay for compound 2a–g against human chronic
myelogenous cell line K562
a,b
Compound
IC50 (lg/ml)
2
2
2
2
2
2
2
a
b
c
d
e
f
27.73 ± 4.7
20.31 ± 5.3
87.07 ± 6.4
51.35 ± 3.2
40.77 ± 4.3
28.18 ± 5.9
35.84 ± 5.1
5.947 ± 2.3
1
2
phase (mostly dead cells) was not prominent. Interestingly,
upon addition of 2b, a concentration of 25 lg/ml change
was observed in the cell cycle pattern (Table 4) leading to
the accumulation of cells in G and decline of G /M, G
0
2
1
and S phases indicating apoptosis.
g
These results of the antiproliferative effect of rhodanine
2b on cancer cells were recognized mainly due to the
induction of apoptosis with less or no contribution from
cell cycle arrest. The results obtained thus confirm our
Cisplatin
a
Determined by MTT assay
b
All experiments were conducted in triplicate
Fig. 3 Measurement of LDH
release following treatment with
2a–g. After the contact of K562
with 2a–g at different
concentrations (6.25, 12.5, 50
and 100 lg/ml) for 24 h, the
release of LDH was measured at
4
90 nm. The data presented is
result of three independent
experiments and error bar are
indicated
123

Med Chem Res
Fig. 4 Cell cycle analysis of K562 cells (A is control, and B is 25 lg of 2b treated) following 2b treatment. K562 cells were treated with 25 lg/
ml of 2b for 24 h, harvested and stained with propidium iodide and quantified by flow cytometry. The histograms show the percentage of cells in
the G /G , G , S and G /M phase of the cell cycle, and 10,000 cells were used for sorting
0 1 1 2
earlier observation of rhodanine 2b-induced cytotoxicity
Table 5).
ethyl and allyl group, respectively, in the N-3 position, and
the compound 2d has two hydrophilic carboxylic acid
groups. By the introduction of larger groups, the steric
effect increases.
(
Structure–activity relationship
The compound 2a with a hydrophilic group in the N-3
position showed a marked cytotoxic activity with an IC50
value of 27.3 lg/ml in the MTT assay. By the introduction
of hydrophobic methyl, isopropyl and benzyl groups into
2a, to form compounds 2b, 2c and 2e, respectively, the
activity increased in 2b (IC50 value of 20.3 lg/ml) but
decreased in 2c (IC50 value of 51.35 lg/ml) and 2e (IC50
value of 87.07 lg/ml). The activity decreases as the
The molecules were designed in such a way that they have
different moieties in the N-3 position of the rhodanine
nucleus. The compound 2a possesses a hydrophilic moiety
(
-CH COOH), whereas the compounds 2b, 2c and 2e have
2
a hydrophobic methyl, isopropyl and benzyl moieties,
respectively, along with a hydrophilic carboxylic acid
group. The compounds 2f and 2g have only a hydrophobic
1
23

Med Chem Res
Table 4 LDH release of human chronic myelogenous cell line K562 against 2a–g
Concentration (lg/ml) Enzyme units (U/ml)
a
Control
2a
2b
2c
2d
2e
2f
2g
0
.0096 ± 0.0010
6
1
2
5
1
a
.25
2.5
5
0.106 ± 0.028 0.0149 ± 0.0023 0.014 ± 0.0016 0.173 ± 0.024 0.154 ± 0.011 0.103 ± 0.014 0.053 ± 0.025
0.27 ± 0.031 0.227 ± 0.022 0.033 ± 0.024 0.193 ± 0.021 0.178 ± 0.016 0.24 ± 0.026 0.077 ± 0.024
0.357 ± 0.022 0.454 ± 0.014 0.053 ± 0.016 0.391 ± 0.018 0.193 ± 0.021 0.33 ± 0.044 0.14 ± 0.031
0.376 ± 0.019 0.811 ± 0.023 0.082 ± 0.013 0.463 ± 0.027 0.468 ± 0.022 0.343 ± 0.024 0.169 ± 0.028
0.42 ± 0.028 0.908 ± 0.023 0.106 ± 0.025 0.647 ± 0.018 0.507 ± 0.028 0.398 ± 0.026 0.294 ± 0.034
0
00
Control use as cells without drug
Table 5 FACS analysis to determine the effect of 2b on cell cycle progression
a,b
Compound
Cell cycle
/G
G
0
1
S
2
G /M
Control
64.7
74.0
18.7
16.4
12.6
7.2
2
a
b
Determined by flow cytometry
b
Experiments were conducted in 25 lg/ml concentration
bulkiness of the group increases in the N-3 position from
c to 2e. Compared to 2b, the cytotoxic activity was less in
compounds which possess only hydrophobic groups as in
N-(4-chlorophenyl)-2-{5-[(5-nitro-2-furyl)methylene]-rho-
danine-3-amino-2-thioxoacetamide and N-(4-methox-
yphenyl)-2-{5-[(5-nitro-2-furyl)methylene]-rhodanine-3-
amino-2-thioxoacetamide were found to possess anticancer
activity against colon cancer cell line (HCT 116) and a
pancreatic cell line (Panc-1) with an GI50 value of less than
10 lM concentration (Kavya et al., 2010a, b). All these
results indicate that if the rhodanines are substituted in the
fifth position, the anticancer activity of the molecule
increases. Potential anticancer activity of the above rhoda-
nines prepared from amino acids and their amenability to
structural modification in the fifth position will help in
designing novel anticancer agents with N-a-carboxyethyl
rhodanine nucleus.
2
2
f (IC50 value of 28.18 lg/ml) and 2 g (IC50 value of
5.84 lg/ml). The cytotoxic activity of compound 2d with
3
two hydrophilic groups was less than 2f and 2 g with
hydrophobic groups. It clearly indicates that the compound
with hydrophilic (-COOH) and a small hydrophobic
(
-CH ) groups is required at N-3 position of the rhodanine
3
nucleus to exhibit a good cytotoxic activity. Further, it
indicates that irrespective of the hydrophilic or hydropho-
bic nature of the groups, the activity decreases with the
increase in size.
The same trend was observed in the trypan blue assay.
The IC50 values are marginally different in this assay. The
trypan blue assay required evaluation using a microscope
and a human observer, while the MTT assay is a colori-
metric assay performed by a spectrophotometer. This
explains, at least partly, why the IC50 values assessed by
trypan blue and MTT are so different.
Conclusion
We have described the synthesis, antiproliferative activity
and apoptosis of novel rhodanine derivatives. Synthesis
was conducted by both conventional and microwave-as-
sisted methods. Microwave synthesis gets completed in
shorter reaction time when compared with conventional
method and also results in good yield. We also report the
antiproliferative activity of the synthesized rhodanines
against human cancerous cell line K562. From the results,
we have obtained a simplified analogue 2b with a consid-
erable cytotoxicity for the further work. FACS analysis
confirmed the effect of 2b in the change of cell cycle
profile. The structure–activity relationship of 2a–g as
Further, it was reported in the literature that 5-(5-phenyl-
furan-2-ylmethylene)-substituted 3-a-carboxy ethyl rhoda-
nine having the same rhodanine nucleus as in 2b has shown
a good in vitro ASK1 inhibitory activity with an IC50 value
of 0.65 lM (Volynets et al., 2013). 5-Aryloxypyrazole
derivatives bearing a rhodanine-3-(3-phenyl propanoic
acid) and 3-a-carboxy isobutyl rhodanine derivatives
bearing a quinoline moiety in the fifth position exhibited
cytotoxicity against HeLa cell lines with an IC50 value
of 8.84 and 9.83 lg/mL, respectively (Guo et al., 2013).
123

Med Chem Res
antiproliferative agents against human chronic myeloge-
nous leukemia cell (K562) has been investigated.
-5 mg/ml dissolved in PBS). It was then incubated at
37 °C for 3 h. MTT was removed by washing with 19
PBS, and 200 ll of DMSO was added to the culture.
Incubation was carried out at room temperature for 30 min
until the cell got lysed and color was obtained. The solution
was transferred to centrifuge tubes and centrifuged at top
speed for 2 min to precipitate cell debris. Optical density
was measured at 570 nm using DMSO as blank in an
ELISA reader (LISASCAN, Erba). All the experiments
were performed in triplicate. IC50 value calculated using
GraphPad Prism software 6.01 version.
Experimental
Cell culture
Human chronic myelogenous leukemia cell line (K562)
purchased from NCCS, Pune, was maintained in Dul-
becco’s modified Eagle’s medium (Sigma) supplemented
with 10 % FBS (Invitrogen) and grown to confluency at
3
7 °C in 5 % CO (NBS, Eppendorf, Germany) in a 90 %
Lactate dehydrogenase (LDH) release assay
2
air in a CO incubator. The cells were trypsinized (500 ll
2
of 0.025 % trypsin in PBS/0.5 mM EDTA solution
Lactate dehydrogenase (LDH) release is an indicator of
membrane breaking, thus resulting in cell injury. LDH
assay was performed to evaluate the LDH release to the
media following treatment with the 2a–g (6.25, 12.5, 25, 50
and 100 lg) on K562 for 24 h and measured using standard
protocols (Korzeniewski and Callewaert, 1983). The
intracellular LDH was determined after lysing the cells by
rapid freezing and thawing in liquid nitrogen. The LDH
release was measured at an absorbance of 490 nm. The
percentage of LDH release was calculated as: (LDH
activity in media)/(LDH activity in media/intracellular
LDH activity) 9 100 %. Results are presented as per-
centage of LDH release subtracting the control values from
treated ones. All the experiments were performed in trip-
licate (Korzeniewski and Callewaert, 1983).
(
HiMedia)) for 2 min and passaged to T flasks in complete
aseptic conditions.
Cell viability by trypan blue exclusion
The effect of compounds 2a–g on the viability of cancer
cells (K562) was determined by trypan blue dye exclusion
assay (Arung et al., 2009; Shahabuddin et al., 2009).
5
Briefly, the cells were plated at a density of 1 9 10 in six-
well plates followed by the addition of different concen-
trations of compound (6.25, 12.5, 50 and 100 lg in 1 ml of
DMSO) or untreated cell. After incubation for every 24 h,
cells were collected and diluted in equal volume of media
and mixed with 20 ml of trypan blue. Cells were counted
under the microscope using hemocytometer. All the
experiments were performed in triplicate. IC50 value cal-
culated using GraphPad Prism software 6.01 version.
Cell cycle analysis by fluorescence-activated cell
sorting (FACS) method
Cell proliferation by MTT assay
K562 cell was cultured and treated with 25 lg of com-
pound 2b, and the treated cells were incubated for 24 h.
After overnight incubation cells were trypsinized by adding
100 ll of 0.25 % trypsin (Invitrogen) and allowed the cells
to detach. The detached cells were pipetted out and spun at
3009g (RPM of rotor 9 gravity constant) for 5 min and
washed once with 19 PBS. The cells were fixed with 1 ml
of ice-cold ethanol and incubated at -20 °C for overnight.
The ethanol fixed cells were washed once with PBS fol-
lowed by 200 ll of Muse cell cycle reagent. The tubes
were incubated for 30 min at dark and analyzed on Muse
flow cytometer (Millipore, USA, 2012).
Extracts were added to grown cells at concentrations of
6
.25, 12.5, 25, 50 and 100 lg from a stock of 1 mg/ml
0
.1 % DMSO and incubated for 24 h. The % difference in
viability was determined by standard MTT assay after 24 h
of incubation (Mosmann, 1983; Arung et al., 2009).
MTT is a colorimetric assay that measures the reduction
of yellow 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetra-
zolium bromide (MTT) by mitochondrial succinate dehy-
drogenase. The MTT enters the cells and passes into the
mitochondria where it is reduced to an insoluble, colored
(
dark purple) formazan product. The cells are then solu-
bilized with an organic solvent, dimethyl sulfoxide (Sigma-
Aldrich), and the released, solubilized formazan product
was measured at 570 nm. Since reduction of MTT can only
occur in metabolically active cells, the level of activity is a
measure of the viability of the cells.
Chemistry
1
All compounds synthesized were characterized by IR, H
1
3
NMR and C NMR elemental analyses and are described
in experimental section. Melting points were determined in
a BUNA melting point instrument and are uncorrected. IR
spectra were recorded on a Shimadzu Affinity I FT-IR
The cell culture suspension was washed with 1x PBS
and then added 30 ll of MTT solution to the culture (MTT
1
23

Med Chem Res
spectrometer. NMR spectra were measured at 400 MHz on
a Bruker 400 spectrometer using TMS as internal standard
reacted in a microwave reactor (CEM Discover, Bench-
mate, USA) for 5 min at 100 °C at 80 W. After automated
cooling to 40 °C, sodium chloroacetate (1 mol) was added
and the mixture was reacted again at 100 °C for 5 min.
After cooling (40 °C), conc. HCl (3 ml) was added and the
reaction was completed at 110 °C for 20–30 min. The
crude product was extracted with ethyl acetate and purified
(Nitsche and Klein, 2012).
and DMSO-d as solvent. Elemental analyses were deter-
6
mined using a PerkinElmer 240c elemental analyzer.
Microwave reaction was carried out using CEM Discover,
Benchmate, USA.
General procedure for the synthesis of 2a, 2b, 2f and 2 g
2
-(4-Oxo-2-thioxothiazolidin-3-yl)acetic acid (2a) Yield;
In a 22 % aqueous NaOH, 1 mol of glycine/dl-alanine/
ethyl amine or allyl amine was dissolved. The solution was
cooled, and 1 mol of carbon disulfide was added with
stirring which was continued for 3 h. To this, a solution of
82.5 %; mp 245–247 °C; UV kmax 266, 215 nm; IR(KBr)
-
1
1
mmax 3439, 1663, 1512, 1321, 896 cm
;
H NMR
(
(
(
DMSO-d , 400 MHz): d = 4.56 (2H, s, N–CH ), 4.41
2H, s, H-5); C NMR (DMSO-d , 100 MHz): d = 202.80
6
2
1
3
6
1
mol of sodium chloroacetate was added and stirring was
C = S, C-2), 173.72 (C = O, COOH), 167.29 (C = O,
continued for another 3 h. The mixture was then made
acidic with conc. HCl and kept overnight. Then the mixture
was heated on a water bath for 90 min till the product
separated as oil which was solidified on cooling. It was
collected on a filter, washed with water and dried (Brooker
et al., 1950; Bernardo et al., 2009, 2011).
C-4), 44.77 (CH , N–CH ), 35.94 (CH , C-5); Anal.calcd.
2
2
2
for C H NO S : C, 31.40; H, 2.64; N, 7.32. Found: C,
5
5
3 2
31.55; H, 2.53; N, 7.22.
2
-(4-Oxo-2-thioxothiazolidin-3-yl)propanoic acid (2b)
Yield; 81.2 %; mp 113–114 °C; UV kmax 285, 261 nm; IR
-
1 1
(KBr) mmax 3452, 1675, 1458, 1355, 844 cm ; H NMR
General procedure for the synthesis of (2d)
(
DMSO-d , 400 MHz): d = 3.5 (2H, s, H-5), 5.27 (1H, m,
6
1
N–CH), 1.43 (3H, d, J = 7.2 H, CH–CH3); C NMR
3
In a 22 % aqueous NaOH, 1 mol of L-glutamic acid
hydrochloride was dissolved. The solution was cooled, and
(
(
DMSO-d , 100 MHz): d = 203.01 (C = S, C-2), 173.75
C = O, COOH), 168.60 (C = O, C-4), 54.31 (CH, N–
6
1
mol of carbon disulfide was then added with stirring
CH), 34.71 (CH , C-5), 13.73 (CH , CH–CH ); Anal.calcd.
2
3
3
which was continued for 4 h. To this, a solution of 1 mol of
sodium chloroacetate was added and stirring was continued
another 3 h. The mixture was kept overnight and then made
for C H NO S : C, 35.11; H, 3.44; N, 6.82. Found: C,
3 2
6
7
35.22; H, 3.48; N, 6.73.
acidic with dil. H SO . The mixture was heated on a water
2
4
3-Methyl-2-(4-oxo-2-thioxothiazolidin-3-yl)butanoic acid
2c) Yield; 79.0 %; mp 115–117 °C; UV kmax 298,
bath for 2 h till the product separated as an oil which
solidified on cooling. It was collected on a filter, washed
with water and dried, and the crystallized product was
collected (Brooker et al., 1950).
(
2
8
6
59 nm; IR (KBr) m
85 cm ; H NMR (DMSO-d , 400 MHz): d = 0.89 (m,
3428, 1667, 1502, 1413, 1317,
max
-
1 1
6
H, CH(CH ) ), 2.75 (1H, m, N–CH–CH(CH ) ), 3.56
3 2
3
2
1
3
(
(
(
2H, m, H-5), 4.20 (1H, m, N–CH–CH(CH ) ); C NMR
3 2
General procedure for the synthesis of 2c and 2e
DMSO-d , 100 MHz): d = 203.30 (C = S, C-2), 174.41
6
C = O, COOH), 168.06 (C = O, C-4), 57.59 (CH, N–
In a 22 % aqueous NaOH, 1 mol of dl-valine or L-pheny-
lalanine was dissolved. The solution was cooled, and 1 mol
of carbon disulfide was then added with stirring which was
continued for 6 h. To this, a solution of 1 mol of sodium
chloroacetate was added and stirring was continued another
CH), 35.76 (CH , C-5), 19.06 (CH , CH–CH ), 17.86
2
3
3
(CH , CH–CH ); Anal.calcd. for C H NO S : C, 41.18;
3 3 8 11 3 2
H, 4.75; N, 6.00. Found: C, 41.24; H, 4.80; N, 6.11.
2-(4-Oxo-2-thioxothiazolidin-3-yl)pentanedioic acid (2d)
Yield; 89 %; mp 123–127 °C; UV kmax 302, 289 nm; IR
3
h. The mixture was kept overnight and then made acidic
-
1
with conc. HCl. The mixture was refluxed for 8 h. The
product separated as oil which was solidified on cooling. It
was collected on a filter by column chromatography
(KBr) mmax 3417, 1716, 1676, 1508, 1425, 1271, 819 cm ;
1
H NMR (DMSO-d
CH –CH –), 2.36 (2H, m, N–CH–CH
m, H-5), 4.30 (1H, t, J = 10.4 Hz, N–CH-); C NMR
DMSO-d , 100 MHz,): d = 201.50 (C = S, C-2), 173.90
, 400 MHz): d = 2.23 (2H, m, N–CH–
6
–CH –), 4.20 (2H,
2
2
2
2
1
3
(
Bernardo et al., 2009, 2011).
(
6
General procedure for the microwave-assisted synthesis
(C = O, CH-COOH), 173.67 (C = O, CH
168.60 (C = O, C-4), 56.47 (CH, N–CH), 41.38 (CH ,
2
2
-COOH),
of N-substituted rhodanines (2a–g)
C-5), 30.03 (CH , N–CH–CH –CH –), 22.58 (CH , N–
2
2
2
2
Mixture of amine or amino acid (1 mol), sodium hydroxide
CH–CH
2
–CH
2
–); Anal.calcd. for C
8
H
9
NO
5
S
2
: C, 36.49; H,
(
22 %) and carbon disulfide (1 mol) in 3 ml water was
3.45; N, 5.32. Found: C, 36.56; H, 3. 57; N, 5.44.
123

Med Chem Res
2
-(4-Oxo-2-thioxothiazolidin-3-yl)-3-phenylpropanoic acid
2e) Yield; 84 %; mp 20–23 °C; UV kmax 285, 261 nm;
IR (KBr) m 3449, 1691, 1558, 1402, 1313, 1149,
Bernardo PH, Xu J, Wan KF, Sivaraman T, Krishnamurthy J, Mok
HYK, Yu VC, Chai CLL (2009) Rhodanine-based Pan-Bcl-2
inhibitors and Mcl-1-specific inhibitors as anti-cancer com-
pounds (WO 2010024783). International Patent no (PCT/
SG2009/000301)
(
max
-
1 1
8
35 cm ; H NMR (DMSO-d , 400 MHz): d = 3.76 (2H,
6
m, H-5), 4.54 (1H, m, N-CH), 3.44, (1H, dd, J = 11 &
Bernardo PH, Sivaraman T, Wan K, Xu J, Krishnamurthy J, Song
CM, Liming T, Chin JSF, Lim DSW, Mok HYK, Yu VC, Tong
JC, Chai CLL (2011) Synthesis of a rhodanine-based compound
library targeting Bcl-XL and Mcl-1. Pure Appl Chem
83:723–731
7
.0 Hz CH–CH ) 3.30 (1H, dd, J = 11.4 & 7.2 Hz CH–
2
1
3
CH ), 7.04-7.29 (5H, m, Ar–H); C NMR (100 MHz,
2
DMSO-d ): d = 202.82 (C = S), 173.99 (C = O, COOH),
6
1
69.7 (C = O), 129.50, 128.9, 128.3, 128.1, 126.4 (C, Ar–
Brooker IAS, Keyes GHF, Roch N (1950) Acid merocyanine dyes.
US Patented (US2493747 A). 605:472
Brown FC, Bradsher CK (1951) Mildew-preventing activity of
rhodanine derivatives. Nature 168:171–172
Brown FC, Bradsher CK, Morgan EC, Tetenbaum M, WilderJr P
C), 59.01 (CH, N–CH), 37.44 (CH , C-5), 34.34 (CH ,
2
2
CH–CH ); Anal.calcd. for C H NO S : C, 51.23; H,
2
12 11
3 2
3
.94; N, 4.98. Found: C, 51.34; H, 3.83; N, 4.94; O, 17.10.
(
7
1956) Some 3-substituted rhodanines.
8:384–388
J Am Chem Soc
3
1
1
4
-Ethyl-2-thioxothiazolidin-4-one (2f) Yield; 84 %; mp
11–113 °C; UV kmax 265, 211 nm; IR (KBr) m 3407,
687, 1500, 1400, 1149, 827 cm ; H NMR (DMSO-d ,
max
David SG (2009) Method using lifespan-altering compounds for
altering the lifespan of eukaryotic organisms, and screening for
such compounds (US 20090163545 A1 20090625). US Patent
App Pub USA
Frankov IA, Kirillov MV, Sokolova TN, Skupskaya RV, Kharitono-
vich AN, Chizhevskaya II (1985) Synthesis and pharmacoloical
properties of 3-carboxyalkylrhodanines containing alkylating
moieties. Pharm Chem J 19:544–547
-
1 1
6
0
00 MHz): d = 4.06 (2H, m, H-5), 3.95 (2H, s, H-2 ), 1.21
0
3H, t, J = 6.0 Hz, CH , H-1 ); C NMR (DMSO-
3
13
(
d6,100 MHz): d = 200.9 (C = S, C-2). 173.6 (C = O,
C-4), 39.9 (CH , C-5), 35.4(CH , N-CH ), 12.04 (CH
3,
2
2
2
CH -CH ); Anal.calcd. for C H NOS : C, 37.24; H, 4.38;
2
3
5
7
2
Guo M, Zheng CJ, Song MX, Wu Y, Sun LP, Li YJ, Liu Y, Piao HR
(
N, 8.69. Found: C, 37.32; H, 4.51; N, 8.69.
2013) Synthesis and biological evaluation of rhodanine deriva-
tives bearing a quinoline moiety as potent antimicrobial agents.
Bioorg Med Chem Lett 23:4358–4361
3
1
1
4
5
-Allyl-2-thioxothiazolidin-4-one ( 2g) Yield; 82 %; mp
41–143 °C; UV kmax 275, 223 nm; IR (KBr) m 3458,
max
Jacobine AM, Posner GH (2011) Three-component, one-flask
synthesis of rhodanine (thiazolidinone) derivatives. J Org Chem
-
1
1
674, 1423, 1298, 823 cm
;
H NMR (DMSO-d6,
7
6:8121–8125
00 MHz): d = 3.82 (2H, m, H-5), 4.63 (2H, s, N-CH ),
2
Kamila S, Ankati H, Harry E, Edward RB (2012) A facile synthesis of
novel 3-(aryl/alkyl-2-ylmethyl)-2-thioxothiazolidin-4-ones using
microwave heating. Tetrahedron Lett 53:2195–2198
1
3
.32 (3H, m, CH = CH ), 5.63 (1H, m, CH = CH );
2
C
2
NMR (DMSO-d ,100 MHz): d = 202.9 (C = S, C-2),
6
1
72.6 (C = O), 131.78 (CH, CH = CH ), 116.72(CH ,
2
Kaminsky D, Bednarczyk-Cwynar B, Vasylenko O, Kazakova O,
Zimenkovsky B, Zaprutko L, Lesyk R (2012) Synthesis of new
potential anticancer agents based on 4-thiazolidinone and
oleanane scaffolds. Med Chem Res 21:3568–3580
2
CH = CH ), 43.92 (CH , N-CH ), 39.68 (CH , C-5);
2
2
2
2
Anal.calcd. for C H NOS : C, 41.59; H, 4.07; N, 8.08.
6
7
2
Found: C, 41.78; H, 4.03; N, 8.38.
Kavya R, Vladimir NY, Alexandra SN, Igor VZ, Mikhail MK, Leonid
VK, Adrian E, Srinivas O, Nouri N (2010a) Design, synthesis
and structure-activity studies of rhodanine derivatives as HIV-1
integrase inhibitors. Molecules 15:3958–3992
Acknowledgments The authors thank Science and Engineering
Research Board (SERB), Department of Science and Technology
(
2
DST), Government of India, for funding the project (No. SR/S1/OC-
8/2011). The authors thank SAIF-STIC, Cochin, for NMR facility.
Kavya R, Vladimir NY, Alexandra SN, Igor VZ, Mikhail MK, Leonid
VK, Adrian E, Srinivas O, Nouri N (2010b) Design, synthesis
and structure-activity studies of rhodanine derivatives as HIV-1
integrase inhibitors. Molecules 15:3958–3992
Korzeniewski C, Callewaert DM (1983) An enzyme-release assay for
natural cytotoxicity. J Immunol Methods 64:313–320
Lesyk RB, Zimenkovsky BS (2004) 4-Thiazolidones: centenarian
history, current status and perspectives for modern organic and
medicinal chemistry. Curr Org Chem 8:1547–1577
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