5-Isopropylidene-3-ethyl rhodanine induce growth inhibition followed by apoptosis in leukemia cells

Article abstract of DOI:10.1016/j.ejmech.2010.02.054

5-Isopropylidene-3-ethyl rhodanine II was prepared by conventional and Microwave assisted synthesis. For the first time, we found that rhodanine II treatment led to cytotoxicity in leukemic cell line, CEM by inducing apoptosis.

Full text of DOI:10.1016/j.ejmech.2010.02.054

European Journal of Medicinal Chemistry 45 (2010) 2748e2752
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
5-Isopropylidene-3-ethyl rhodanine induce growth inhibition followed
by apoptosis in leukemia cells
,
b
a
a
b
Subban Ravi a , Kishore K. Chiruvella , K. Rajesh , V. Prabhu , Sathees C. Raghavan
*
a Department of Chemistry, Karpagam University, Coimbatore 21, Tamilnadu, India
b Indian Institute of Science, Bangalore, India
a r t i c l e i n f o
a b s t r a c t
Article history:
5-Isopropylidene-3-ethyl rhodanine II was prepared by conventional and Microwave assisted synthesis.
For the first time, we found that rhodanine II treatment led to cytotoxicity in leukemic cell line, CEM by
inducing apoptosis.
Received 13 November 2009
Received in revised form
19 February 2010
Accepted 19 February 2010
Available online 1 March 2010
Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords:
5-Isopropylidene-3-ethyl rhodanine
Synthesis
Cytotoxicity
Apoptosis
1. Introduction
discovery. Therefore, the synthesis of these compounds is of
considerable interest.
Rhodanine derivatives are attractive compounds due to their
biological activities. They are anticonvulsant, antibacterial, antiviral
and antidiabetic in nature [1]. Rhodanine derivatives have also
been reported as inhibitors of Hepatitis C Virus (HCV) protease [2],
The rhodanine moiety has been synthesized by various methods
such as addition of isothiocyanate to mercaptoacetic acid followed
by acid catalyzed cyclisation, or the reaction of ammonia or primary
amines with carbon disulfide and chloroacetic acid in the presence
of bases [17,18].
uridine diphospho-N-acetylmuramate/L-alanine ligase [3], bacterial
b
-lactamase and Mur ligases [4, 5]. Recently, substituted rhoda-
Arylidenerhodanines are frequently identified as potent hits in
high throughput screening against various prokaryotic and
eukaryotic targets. Condensation of aromatic aldehydes at the
nucleophilic C-5 active methylene has been performed using
piperidinium benzoate in refluxing toluene or sodium acetate in
refluxing glacial acetic acid [17,18]. Recently, Sim et al. [19] reported
the synthesis of 5-arylalkylidene rhodanines in 60e82% yields by
heating the reactants suspended in toluene at 110 ꢀC for 3 days.
Sing et al. [20] reported the condensation of rhodanine with an
aldehyde (0.1 mmol) by heating in anhydrous EtOH (200 mL) for
6 h at 80 ꢀC. Zhang Alloum et al. reported the successful synthesis of
some 5-arylalkylidene rhodanines on solid inorganic supports in
dry media under microwave irradiation [21,22]. Jian-Feng Zhou
et al. have reported the synthesis of bis(benzylidene)cyclo-
alkanones by the aldol condensation in an aqueous medium under
phase-transfer catalysis and microwave irradiation [23] and
synthesis of 5-arylalkylidene rhodanines by the aldol condensation
of aromatic aldehydes with rhodanine using tetrabutylammonium
bromide (TBAB) as phase-transfer catalyst in an aqueous medium
under microwave irradiation.
nines were investigated for tau aggregation inhibitor properties [6].
Rhodanines are classified as nonmutagenic [7] and a long-term
study on the clinical effects of the rhodanine-based Epalrestat for
antidiabetic demonstrated that it is well tolerated [8]. The use of
the rodanine analogs as anti-leukemia agents has also been
reported in literatures [9e15]. Rhodanine derivatives were found to
have marked mildew-proofing activity. It is interesting to note that
the new mildew-proofing agents contain the structure
present in many plant fungicides (tetrame-
thylthiuram disulphide and the salts of dithiocarbamic acid), as
well as a carbonyl group conjugated with an ethylenic linkage,
found in another class of fungicides [16]. Due to various possibilities
of chemical derivatization of the rhodanine ring, rhodanine-based
compounds will probably remain a privileged scaffold in drug
* Corresponding author. Tel.: þ91 9047174142; fax þ91 2611043.
E-mail address: ravisubban@rediffmail.com (S. Ravi).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.02.054

S. Ravi et al. / European Journal of Medicinal Chemistry 45 (2010) 2748e2752
2749
Alternatively, the alkylidene rhodanines were prepared by the
condensation of 3-3-carboxyethylrhodanines with oxo-compounds
unrelated to aromatic aldehydes and tested for possible antimeta-
bolic activity [24]. Rhodanine, Ketone, and NH4OAc were refluxed in
toluene for 3 days to prepare the alkylidene rhodanin derivatives.
Obviously, these methods involve long reaction times, high
temperatures, use large quantities of organic solvents and some
give unsatisfactory yields. Due to these drawbacks the number of
available reports on the investigation of alkylidene rhodanines was
very few. This prompted us to synthesise few rhodanine derivatives
and their corresponding oxygen (2-thio-2,4-oxazolidine deriva-
tives) and nitrogen (thiohydantions) analogs. Besides we show that
the newly synthesized isopropylidene rhodanine derivatives
induced cytotoxicity in leukemic cells in a dose- and time-depen-
dent manner. The results are encouraging, which makes them
a promising starting point for further synthesis and optimization by
QSAR studies.
Fig. 1. Cytotoxicity of 5-isopropylidene-3-ethyl rhodanine in leukemic cells. Evaluation
of cell viability using trypan blue assay following 5-isopropylidene-3-ethyl rhodanine
treatment. CEM cells were treated with 10, 50, 100 and 250 mM of 5-isopropylidene-3-
ethyl rhodanine or CH3OH (vehicle control). Cells were harvested, trypan blue stained
and counted every 24 h until it reached stationary phase. The data represented is result
of three independent experiments.
2. Result and discussion
2.1. Synthesis of 5-isopropylidene-3-ethyl rhodanine II
To further check the cytotoxic effect of II, LDH release in the cell
culture suspension was measured using LDH assay (Fig. 3). The
methanol treated cells were used as vehicle control and II treated
cells showed LDH release suggesting cytotoxic effect on CEM cells.
The membrane of CEM cells are disrupted due to cytotoxic effect of
II causing release of LDH (Lactate dehydrogenase) into the super-
natant. Results showed time-dependent increase in the LDH release
upon treatment with the II, further confirming the above results
(Fig. 3).
A mixture of thioglycollic acid, ethyl isothiocyanate, methanol
and water were heated in an oil bath for four hours at 100 ꢀC to
yield N-ethyl rhodanine I (Scheme 1). A mixture of I, ammonium
malonate and acetone was refluxed in an oil bath at 90 ꢀC for 18 h to
yield 5-isopropylidene-3-ethyl rhodanine II. In order to develop
a greener synthesis the above mixture was treated in a microwave
oven at 300 W for 2 min. The yield was comparable to the
conventional method but the reaction time was shorter. The
compounds were characterized by elemental analysis, UV, IR, NMR
and MS data.
2.3. Cell cycle analysis by flow cytometry
We performed FACS analysis to determine the effect of II on cell
cycle progression. CEM cells were stained with ethidium bromide
after 72 h of treatment with II (10, 50, 100 and 250 mM) and sub-
2.2. II induces cytotoxicity in leukemic cells in a dose- and time-
dependent manner
jected to FACS. Histogram of control cells showed a standard cell
cycle pattern, which includes G1 and G2 separated by S phase
(Fig. 4). The subG1 phase (mostly dead cells) was not prominent.
Interestingly, upon addition of II, a concentration dependent
change was observed in the cell cycle pattern (Fig. 4) leading to
accumulation of cells in subG1 and decline of G2/M, G1 and S
phases indicating apoptosis.
Results showed dose-dependent accumulation of subG1 pop-
ulation of cells, which is a hall mark of apoptosis (Fig. 4). These
findings indicate that at the ranges of concentration studied, the
anti-proliferative effect of II on leukemic cells could be attributed
primarily to the induction of apoptosis, with less or no contribution
In order to evaluate the cytotoxic effect of 5-isopropylidene
rhodanine on growth of leukemic cells, we used trypan blue assay.
Leukemic cell line, CEM (T-cell leukemia) was treated with 10, 50,
100 and 250 mM of II. Since the compound was dissolved in
methanol, the cells with methanol were used as vehicle control.
Following addition of compound, cells were counted at intervals of
24 h till the control cells attained stationary phase. Results showed
that cell growth was affected with increase in time (Fig. 1). The
effect was limited when 10
trations of 100 and 250 M resulted in increased cell death (Fig. 1).
The IC50 of II was approximately 40 M, at 48 h of treatment. These
mM of II was used. However, concen-
m
m
results suggest that II induces cytotoxicity in human leukemic cells,
in a dose- and time-dependent manner.
The cytotoxicity induced by II on proliferation of leukemic cells
was further verified using MTT assay. CEM cells treated with 10,
50,100 and 250
were subjected to MTT assay (Fig. 2). Results showed that cell
viability was affected upon treatment with II at 50,100 and 250 M,
mM of II were harvested after 24, 48 or 72 hrs and
m
especially after 48 h. These results further suggest that 5-iso-
propylidene rhodanine induces the cytotoxicity.
S
S
(ii)
(i)
+
HS
COOH
NCS
O
S
O
S
N
N
C2H5
C2H5
Fig. 2. Determination of % cell viability by MTT assay in CEM cells. Cells were cultured
with 10, 50, 100 and 250 mM of 5-isopropylidene-3-ethyl rhodanine or vehicle control
I
II
for 24, 48 and 72 h. The percentage of cell viability was calculated considering
methanol treated (control) cells as 100% and plotted with representation of error bars.
Scheme 1. (i) CH3OH, H2O, 100 ꢀC, 4 h; (ii) ammonium malonate, acetone, 90 ꢀC, 18 h.

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S. Ravi et al. / European Journal of Medicinal Chemistry 45 (2010) 2748e2752
previous reports showed that similar doses were also used for other
compounds by Roy et al. [27] and Kumar et al. [28]. IC50 values
suggest that II exhibited good cytotoxicity, showed remarkable
suppression on the CEM cell growth as the concentration and time
increases in dose dependent manner. Hence, our results showed
that II affected growth of leukemic cells in a time- and dose-
dependent manner. Generally, cell death could be either through
necrosis or apoptosis. The former process is associated with rela-
tively large damage to the surrounding tissue. The latter is associ-
ated with controlled elimination of cancer cells. Thus for the
possible treatment of cancer, cytotoxic compounds should prefer-
entially act via apoptosis. To address the question whether II
induces cell death via apoptosis, we performed FACS analysis to
determine the effect on cell cycle progression. Appearance of Sub-
G1 peak accumulation in present study as indicated by FACS anal-
ysis is a hall mark of apoptosis (Fig. 4) and characteristic of
apoptotic cell behaviour, which is not for the case of necrosis [29].
However, we could not find any evidence for cell cycle arrest. FACS
analysis showed that II interferes with cell division leading to
inhibition of cell proliferation ultimately leading to cell death.
These findings indicate that at the ranges of concentration studied,
the anti-proliferative effect of II on leukemic cells could be attrib-
uted primarily to the induction of apoptosis, with less or no
contribution from cell cycle arrest.
Fig. 3. Measurement of LDH release following treatment with 5-isopropylidene-3-
ethyl rhodanine. After the exposure of CEM cells with 5-isopropylidene-3-ethyl rho-
danine at different concentrations (10, 50, 100 and 250 mM) for 24, 48 and 72 h, the
release of LDH was measured at 490 nm. Results are presented as percentage of LDH
release by subtracting the control values from treated ones. The data presented is
result of three independent experiments and error bars are indicated.
from cell cycle arrest. Thus, the results obtained, confirmed our
earlier observation of II induced cytotoxicity.
2.4. Discussion
Leukemia, being one of the most threatening hematological
malignant cancers, has been found to be very sensitive to anti-
cancer chemotherapeutic agents, which either interfere with cell
cycle or cause apoptosis [25]. This factor intrigues scientists to look
for more specific and effective chemical drugs against it. Leukemic
cells lines serve as useful tools to study factors and processes
associated with their differentiation and apoptosis pathways [26].
Hence, cytotoxic effects of II on CEM cells (a T-cell leukemic cell line
derived from T-cell leukemia patient) is studied.
3. Experimental protocols
3.1. General procedures
All compounds prepared were characterized by 1H NMR, IR and
elemental analyses and are described in the experimental section.
Melting points were determined in a XT-5 digital melting point
instrument and are uncorrected. IR spectra were recorded on
a Nicolet Avatar 360 FT-IR spectrometer. 1H NMR spectra were
measured at 400 MHz on a Bruker-400 spectrometer using TMS as
internal standard and CDCl3 as solvent. MS spectra were obtained
on a Shimadzu LCMS instrument. Elemental analyses were deter-
mined using a PerkineElmer 240C Elemental Analyzer.
In the present study, we find that II is capable of inducing
cytotoxic activity and induction of apoptosis in CEM. Although II
induced cytotoxicity in these cells, the IC50 value was about 40
mM
after 72 h, which is higher than many synthetic compounds. In fact,
Fig. 4. Cell cycle analysis of leukemic cells following 5-isopropylidene-3-ethyl rhodanine treatment. CEM cells were treated with 5-isopropylidene-3-ethyl rhodanine (10, 50, 100
and 250 M) for 72 h, harvested and stained with ethidium bromide and quantified by flow cytometry. In all cases the histograms show the percentage of cells in the G0/G1, G1, S
and G2/M phase of the cell cycle. For each sample 10,000 cells were used for sorting.
m

S. Ravi et al. / European Journal of Medicinal Chemistry 45 (2010) 2748e2752
2751
3.1.1. Preparation of N-ethyl rhodanine I
yl)-2, 5-diphenyltetrazolium bromide) assay [31]. The cleavage and
conversion of the soluble yellowish MTT to the insoluble purple
formazan by active mitochondrial dehydrogenase of living cells has
been used to develop an assay system alternative to other assays for
measurement of cell proliferation. Harvested cells were seeded in to
96-well plate (1 ꢂ105 cell/ml) with varying concentrations of the
A mixture of thioglycollic acid (9.2 g, 0.1 mol), ethyl iso-
thiocyanate (10.45 g, 0.12 mol), methanol (80 ml) and water
(1000 ml) were taken in a 3 lit capacity flask and was heated in an
oil bath for 4 h at 100 ꢀC. After 4 h, the reaction mixture was cooled
and lower oily layer, which was slight yellow in colour, was sepa-
rated by decantation and cooled strongly in a freezing mixture. The
oily layer got solidified. The solid was extracted with ether, dried
and distilled out at low pressure to yield I. Yield 72%, mp 38 ꢀC. IR
compound II (10, 50, 100 and 250
the cells were collected and MTT assay was performed. Four hours to
the end of incubation, 10 l of MTT solution (5 mg/ml in PBS) was
mM) and incubated. For every 24 h,
m
(KBr,
n
, cmꢁ1): 1544 (CeN), 1720. MS: APCI (Positive mode) m/z:
added to each well containing fresh and cultured medium. At the
end, the insoluble formazan produced was dissolved in solution
containing 10% SDS and 50% DMF (left for 2 h at 37 ꢀC in dark
conditions) and optical density (OD) was read against reagent blank
with multi well scanning spectrophotometer (ELISA reader, Model
Expert 96, Asys Hitchech, Austria) at a wavelength of 570 nm.
Absorbance is a function of concentration of converted dye. The OD
value of study groups was divided by the OD value of untreated
control and presented as percentage of control (as 100%).
161.99 [M þ H]þ. 1H NMR (CDCl3, 400 MHz)
: 1.21 (t, 3H, J ¼ 7.1 Hz),
d
3.76 (m, 2H), 3.95 (q, 2H, J ¼ 7.0 Hz). Anal. Calcd. for C5H7NOS2: C,
37.24; H, 4.38; N, 8.69. Found: C, 37.32; H, 4.43; N, 8.58.
3.1.2. Preparation of 5-isopropylidene-3-ethyl rhodanine II
A mixture of N-ethyl rhodanine (2.4 g, 0.15 mol), ammonium
malonate (4.4 g, 0.035 mol) and acetone (13.8 ml, 0.25 mol) was
refluxed in an oil bath at 90 ꢀC for 18 h. After reflux it was cooled
and the separated solid was filtered. It was recrystallised from
methanol to yield pale yellow needles II. Yield 62%, m.p. 54 ꢀC. lmax
3.1.8. LDH release assay
(MeOH) 342 nm. IR (KBr,
n
, cmꢁ1): 1600 (C]C), 1720 (C]O). MS:
Release of lactate dehydrogenase (LDH) is an indicator of
membrane integrity and hence cell injury. LDH assay was per-
formed to assess the LDH release to the media following treatment
with the compound 5-isopropylidene rhodanine (10, 50,100 and
APCI (Positive mode) m/z: 202.02 [M þ H]þ. 1H NMR (CDCl3,
400 MHz)
d
: 1.23 (t, 3H, J ¼ 7.1 Hz), 2.03 (s, 3H), 2.46 (s, 3H), 4.15 (q,
2H, J ¼ 7.0 Hz). 13C NMR (CDCl3)
d: 11.7, 17.0, 17.2, 37.6, 116, 147, 164
and 192. Anal. Calcd. for C8H11NOS2: C, 47.73; H, 5.51; N, 6.96.
Found: C, 47.71; H, 5.48; N, 6.99.
250 mM) on CEM cells for 24, 48 and 72 h and it was measured using
standard protocols [32,33]. 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 þ intracellular LDH
activity) X100%. Results are presented as percentage of LDH release
substracting the control values from treated ones.
3.1.3. Microwave assisted synthesis
A mixture of N-ethyl rhodanine (2.4 g, 0.15 mole), ammonium
malonate (4.4 g, 0.035 mol) and acetone (13.8 ml, 0.25 mol) was
heated to 160 ꢀC in a microwave oven at 300 W for 2 min. After
heating it was cooled and the separated solid was recrystallised
from methanol to yield pale yellow needles II. The physical data
were identical.
3.1.9. Cell cycle analysis
CEM cells were cultured and treated with different concentra-
tions of compound II. Cells were harvested after 72 h. The cells
were processed, stained with ethidium bromide (Sigma, USA) and
subjected to flow cytometry (FACScan, BD Biosciences, USA) using
CellQuest Pro software using excitation 488 nm laser and emission
at 560/670 nm. A minimum of 10,000 cells were acquired per
sample and histograms were analyzed using WinMDI 2.8 software.
3.1.4. Cell culture
Human leukemia cell line CEM was purchased from National
Center for Cell Science, Pune, India. Cells were grown in RPMI 1640
medium (Gibco) supplemented with 10% heat-inactivated fetal
bovine serum (FBS; Sigma) and 100 mg/L pencillin-streptomycin at
37 ꢀC in a humidified atmosphere with 5% CO2. Cells were diluted at
a ratio of 1:5 every 2e3 days.
3.1.5. Compound
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used as vehicle control. The final concentration of MeOH used in
control was 0.8% which was equivalent to the maximum concen-
tration of methanol used in the experiments. In all the experiments
described herein compound was added after 24 h of cell culture.
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