Novel rhodanine derivatives induce growth inhibition followed by apoptosis

Article abstract of DOI:10.1016/j.bmcl.2010.08.084

We have designed and synthesized three novel compounds, 5-isopropylidiene derivatives of 3-dimethyl-2-thio-hydantoin (ITH-1), 3-ethyl-2-thio-2,4- oxazolidinedione (ITO-1), and 5-benzilidene-3-ethyl rhodanine (BTR-1), and have tested their chemotherapeutic properties. Our results showed that all three compounds induced cytotoxicity in a time- and concentration-dependent manner on leukemic cell line, CEM. Among the compounds tested, BTR-1 was 5- to 7-fold more potent than ITH-1 and ITO-1 when compared by trypan blue and MTT assays. IC₅₀ value of BTR-1 was estimated to be <10 μM. Both cell cycle analysis and tritiated thymidine assays revealed that BTR-1 affects DNA replication by inducing a block at S phase. BTR-1 treatment led to increased level of ROS production and DNA strand breaks suggesting activation of apoptosis for induction of cell death.

Full text of DOI:10.1016/j.bmcl.2010.08.084

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6297–6301
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel rhodanine derivatives induce growth inhibition followed by apoptosis
a
b,
⇑
a
a
b
a
Balaji T. Moorthy , Subban Ravi , Mrinal Srivastava , Kishore K. Chiruvella , H. Hemlal , Omana Joy ,
a,
⇑
Sathees C. Raghavan
Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India
Department of Chemistry, Karpagam University, Coimbatore 21, Tamilnadu, India
a
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 27 April 2010
Revised 27 July 2010
Accepted 18 August 2010
Available online 21 August 2010
We have designed and synthesized three novel compounds, 5-isopropylidiene derivatives of 3-dimethyl-
2-thio-hydantoin (ITH-1), 3-ethyl-2-thio-2,4-oxazolidinedione (ITO-1), and 5-benzilidene-3-ethyl rhoda-
nine (BTR-1), and have tested their chemotherapeutic properties. Our results showed that all three com-
pounds induced cytotoxicity in a time- and concentration-dependent manner on leukemic cell line, CEM.
Among the compounds tested, BTR-1 was 5- to 7-fold more potent than ITH-1 and ITO-1 when compared
by trypan blue and MTT assays. IC50 value of BTR-1 was estimated to be <10 lM. Both cell cycle analysis
Keywords:
and tritiated thymidine assays revealed that BTR-1 affects DNA replication by inducing a block at S phase.
BTR-1 treatment led to increased level of ROS production and DNA strand breaks suggesting activation of
apoptosis for induction of cell death.
Chemotherapy
Double-strand breaks
Cytotoxicity
DNA damage
Ó 2010 Elsevier Ltd. All rights reserved.
5
-Benzilidene-3-ethyl rhodanine
Cancer is one of the difficult diseases to be cured, and very few
of effective anti-cancer therapeutic agents with well-defined
pharmacokinetic properties is of importance.
Rhodanine derivatives have been proven to be attractive com-
pounds due to their outstanding biological activities and have
undergone rapid development as anticonvulsant, antibacterial,
effective drugs are available. The development of novel, efficient,
and less toxic anticancer agents remains an important and chal-
lenging goal in medicinal chemistry. Understanding the molecular
mechanism involved in cancers will lead to identification of novel
anticancer agents. Changes in DNA, RNA, and protein levels due to
mutations have been analyzed in many cancers, including leuke-
1
5,16
antiviral, and antidiabetic agents.
also been reported as hepatitis C virus (HCV) protease inhibitors17
and used as inhibitors of uridine diphospho-N-acetylmuramate/
At the same time, these have
1
–4
mia and lymphoma.
Recently, extensive efforts were made to-
L-
1
8
wards characterization of the mechanism of chromosomal
alanine ligase. Recently, substituted rhodanines were investi-
gated for tau aggregation inhibitor properties. Rhodanines are
classified as nonmutagenic and a long-term study on the clinical
effects of the rhodanine-based Epalrestat as an anti-diabetic, dem-
5,6
19
translocations involved in many lymphoid cancers. The proteins
mostly responsible for leukemia and lymphoma in the recent past
2
0
are Recombination Activating Genes (RAGs, the enzyme responsi-
ble for antibody diversity)1
,7–9
and Activation Induced Deaminase
(
AID, an enzyme responsible for somatic hypermutation and class
2,10–12
switch recombination).
Leukemia is one of the major types of cancers affecting a
significant segment of the population. In fact, leukemia is the most
frequent childhood cancer, with 26% of all cases, and 30% mortality.
Although the incidence rate for this disease remains relatively
unchanged, some success has been attained in its treatment.
However, the current treatments have many limitations. This
X
13
HC
S
O
S
N
R
O
S
N
includes side effects induced by the drugs and the development
of acquired drug resistance.14 Thus, the need for the development
C2H5
BTR-1
ITO-1; R = C H , X = O
2
5
ITR-1; R = C H , X = S
2
5
⇑
Corresponding authors. Tel.: +91 9047174142; fax: +91 2611043 (S.R.); tel.: +91
80 2293 2674; fax: +91 080 2360 0814 (S.C.R.).
ITH-1; R = CH , X = CH3
3
0
E-mail addresses: ravisubban@rediffmail.com (S. Ravi), sathees@biochem.iisc.
ernet.in (S.C. Raghavan).
Figure 1. The five membered heterocyclic core; rhodanines (X = S), thiohydantoin
(X = NCH ), and thioxooxazolidine (X = O).
3
0
960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.08.084

6
298
B. T. Moorthy et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6297–6301
onstrated that it is well tolerated.21 Additionally, rhodanines have
been designed as inhibitors of various enzymes such as bacterial
b-lactamase and Mur ligases.22 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 (tetramethylthiu-
ram disulfide and the salts of dithiocarbamic acid), as well as a car-
bonyl group conjugated with an ethylenic linkage, found in
2
3
another class of fungicides. Due to various possibilities of chem-
ical derivatization of the rhodanine ring, rhodanine-based com-
CH3
N
pounds will probably remain
a privileged scaffold in drug
N
ammonium
malonate
discovery. Therefore, the synthesis of these compounds is of con-
siderable interest. However, the anticancer properties of rhodanine
derivatives were never investigated extensively.
O
O
3
CH COCH3
N
S
N
S
reflux, 20 h
Thus, in the present study we report synthesis of three novel
rhodanine derivatives, 5-isopropylidiene derivates of 3-methyl-2-
thio-hydantoin (ITH-1), 3-ethyl-2-thio-2,4-oxazolidinedione (ITO-
CH3
ITH-1
1
) and 5-benzilidene-3-ethyl rhodanine (BTR-1). The biological
S
ammonium
malonate
activity of the compounds was assayed using various cytotoxic
and cellular assays. Results showed that BTR-1 was many fold
more potent than ITH-1 and ITO-1 with an IC50 value around
10 lM. Further, we show that BTR-1 affects DNA replication by
inducing a block at S phase followed by induction of cell death
S
O
S
N
S
CH COCH3
O
3
N
reflux, 20 h
C2H5
C H
2
5
ITR-1
by apoptosis.
Chemical synthesis. In our previous study, ITR-1 showed encour-
aging results and was capable of inducing cytotoxic activity and
apoptosis in CEM with an IC50 of 40 lM, which makes it promis-
ing for further synthesis and optimization by QSAR studies. For
synthesis, we employed a very simple approach and focused on
the heterocycle as well as its substituents. Initially, we focused
on the central heterocycle itself, replacing the original rhodanine
core of ITR-1 with other heterocycles and in these experiments,
S
ammonium
malonate
HC
O
S
2
4
O
O
S
S
N
S
S
C6H5CHO
reflux, 20 h
N
C H
2
5
C H
2
5
BTR-1
O
ammonium
acetate, acetic acid
O
we prepared rhodanines (X = S), thiohydantoin (X = NCH
3
), and
thioxooxazolidine (X = O) (Fig. 1) as per the Scheme 1.
N
CH COCH , Benzene
O
ITH-1, ITO-1, and BTR-1 induces cytotoxicity in T-cell leukemic cell
line in a dose- and time-dependent manner. In order to evaluate the
cytotoxic effect of ITH-1, ITO-1, and BTR-1 on T-cell leukemic cell
line (CEM), we used trypan blue assay as described in Supplemen-
3
3
N
Dean Stark apparatus
reflux, 60 h
C2H5
C H
2
5
ITO-1
tary Methods. CEM cells were treated with 10, 50, 100, or 250
of ITH-1, ITO-1, and BTR-1. Since the compounds were dissolved in
lM
Scheme 1. Synthesis of the five member heterocyclic cores.
Figure 2. Cytotoxicity of different rhodanine derivatives on leukemia cell line. (A–C) Determination of cell viability on T-cell leukemia cell line, CEM following addition of
ITH-1 (A), ITO-1(B), and BTR-1 (C) (10, 50, 100, and 250 M) by trypan blue assay. DMSO was used as vehicle control. Live cells were counted at an interval of 24 h following
l
addition of compounds, till culture reached stationary phase and the data was represented as a graph. Error bars based on independent experiments are indicated. (D–F)
Determination of cell proliferation by MTT assay. CEM cells were cultured with ITH-1 (D), ITO-1 (E), and BTR-1 (F) as described in ‘panel A’, harvested after 24 and 48 h and
subjected to MTT assay and data is presented as bar diagram. The percentage of viable cells was calculated considering DMSO treated cells as 100% and plotted. In all the
cases, error bars indicate SD (standard deviation) from three independent experiments. p values (p <0.01, p <0.05) were calculated comparing control cells with treated cells.
p >0.05 are represented by asterisks (*).

B. T. Moorthy et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6297–6301
6299
DMSO, the cells with DMSO were used as vehicle control. Follow-
ing addition of the compound, cells were counted at intervals of
The effect of ITH-1, ITO-1, and BTR-1 on proliferation of T-cell
leukemic cells was further verified using MTT assay. CEM cells
2
4 h until the control cells attained stationary phase. Results
were treated with 10, 50, 100, and 250 lM of compound and were
showed that cell growth was affected with increase in duration
of treatment and concentration of the compound (Fig. 2A–C). The
subjected to MTT assay after 24 and 48 h (Fig. 2D–F). Results
showed that cell viability was affected at higher concentration in
case of ITH-1, ITO-1 while the effect was more pronounced in
the case of BTR-1. In case of BTR-1, cell viability was affected as
low as 10 lM. Interestingly, when normal T-lymphocyte cell line,
8E5, was used for the study, none of the compounds were cyto-
effect was limited in case of ITH-1, ITO-1 even when 100
was used, whereas in case of BTR-1, there was significant reduction
in the cell number even when 10 M was used for the study. How-
ever, concentration of 250 M resulted in cell death in all three
cases (Fig. 2A–C). The IC50 of ITH-1 was approximately 70 and
lM
l
l
toxic, except in the case of BTR-1, which showed a limited sensitiv-
4
7
6
1
0
l
M and ITO-1 was 80 and 50
2 h, respectively. Interestingly for BTR-1, IC50 value was 8 and
M at 48 and 72 h, respectively. These results suggest that ITH-
, ITO-1, and BTR-1 induce cytotoxicity in human leukemic cells
l
M, when estimated at 48 and
ity at the highest concentration (250 lM) and that could be due to
nonspecific interaction (Supplementary Fig. 1). Based on both try-
pan blue and MTT assays, we find that potency of BTR-1 (IC50 va-
lue <10 lM) is 5- and 7-fold higher than ITH-1 and ITO-1,
l
in a dose- and time-dependent manner.
respectively. Thus we have used BTR-1 for further studies.
LDH release assay was performed to test the cell membrane
damage induced by BTR-1. For this, CEM cells were cultured with
1
0, 50, 100, and 250 lM of BTR-1 and LDH released was measured
at 24, 48, and 72 h. Consistent with above results, we observed a
dose- and time-dependent increase in LDH release, further con-
firming the cytotoxic potential of BTR-1 (Fig. 3).
Compounds ITH-1, ITO-1, and BTR-1 were employed for induction
of cytotoxic activity and apoptosis in CEM cells. The rhodanine
heterocycle BTR-1 appeared to be most potent followed by
thiohydantoin ITH-1 and oxazolidinedione ITO-1. The thioxo group
in rhodanines is a carboxylic acid bioisoster by size has low
electronegativity and has ability to build hydrogen bonds, which
2
5
could be attributed for the activity. The activity of BTR-1 was
2
4
found to be higher than ITR-1 which was tested earlier and this
may be due to the increase in the delocalization of the electrons. It
further indicates that the activity is in the order of electronegativity
values of the atom X. As the electronegativity values increase from S
to O through N, the activity decreases in the same order. When the
electronegativity increases probably it will attract more electrons
and disturb the delocalisation of electrons between the double bond
and the carbonyl group of the heterocyclic ring.
Figure 3. Detection of LDH release induced by BTR-1. After exposure of CEM cells
with BTR-1 (10, 50, 100, and 250 lM for 24, 48, and 72 h), LDH release was
measured at 490 nm. Results are presented as percentage of LDH release. Error bars
based on independent experiments are indicated. Error bars indicate SD (standard
deviation) from three independent experiments. p values (p <0.05) were calculated
comparing control cells with treated cells. p >0.05 are represented by asterisks (*).
3
3
Figure 4. Determination of effect of BTR-1 on cell division by [ H]-thymidine incorporation assay and cell cycle analysis. (A) [ H]-thymidine assay to determine effect of BTR-
1
3
treatment on CEM cells. [ H]-thymidine was added to BTR-1 treated cells (10, 50, 100, or 250
bar diagram. The data presented is derived from independent experiments and error bars are indicated. (B and C) Flowcytometric analysis of CEM cells following BTR-1
treatment. BTR-1 treated cells (10, 20, 50, 100, and 250 M) or vehicle control were harvested after 48 h. The cells were fixed and stained with propidium iodide and
quantified by flow cytometry and presented as histogram (B). For each sample, 10,000 cells were used for sorting. Bar diagram showing quantification of percentage of cells in
the G /G , G1, S, and G /M phase of the cell cycle (C). Error bars indicate SD (standard deviation) from three independent experiments. p values (p <0.05) were calculated
comparing control cells with treated cells and p >0.05 are represented by asterisks (*).
lM) and radioactive counts per minute (cpm) were calculated and plotted as a
l
0
1
2

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B. T. Moorthy et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6297–6301
BTR-1 affects the proliferation of CEM cells. The potential of BTR-1
cation. However, it is also possible that, in addition to its effect on
cell division, it could directly induce apoptosis.
FACS analysis was performed to determine whether BTR-1 has
any effect on cell cycle progression. CEM cells were stained with
propidium iodide after 48 h of treatment (10, 20, 50, 100, and
to inhibit cell division was tested by tritiated thymidine incorpora-
tion assay. To test this, cultured CEM cells were incubated in the
3
presence of [ H] thymidine following addition of BTR-1. Results
showed that BTR-1-treatment led to the reduction in incorporation
3
of [ H] thymidine, in all concentrations tested especially after 48 h
250 lM) and subjected to FACS as described in Supplementary
(
Fig. 4A). These results suggest that BTR-1 affects the cell viability
Methods. Histogram of vehicle (DMSO) treated cells showed a
standard cell cycle pattern, which includes G1 and G2 separated
by S phase (Fig. 4B and C). The subG1 phase (mostly dead cells)
was not prominent. Interestingly, upon addition of BTR-1, a con-
centration dependent change was observed in the cell cycle pattern
(Fig. 4B and C) leading to accumulation of cells in subG1 and de-
cline of G2/M and G1. Importantly results showed a transient cell
cycle arrest at S phase in low doses of treatment (10, 20, and
by inhibiting cell division probably by interfering with DNA repli-
Time points (h) C 12 24 36 48 72 M
50 lM) (Fig. 4C). Hence, these results indicate that BTR-1 may
interfere with cell division by inducing S phase arrest followed
by apoptosis. However, more studies are required to understand
the mechanism of cell cycle arrest.
1
0 kb
BTR-1 induces DNA fragmentation. FACS analysis is also indica-
tive of the integrity of genome as propidium iodide staining was
performed. The observed accumulation of cells at G0 phase indi-
cated fragmentation of the DNA. In order to test the extent of
3
.0
.0
2
DNA breaks induced by BTR-1, CEM cells treated with 20 lM of
BTR-1 were harvested after 12, 24, 36, 48, and 72 h and used for
gel electrophoresis. The results showed fragmentation of the chro-
mosomal DNA leading to a smear in the lanes representative of
cells treated with BTR-1 (Fig. 5). Such breakage was extensive
when cells were incubated for 48 and 72 h with BTR-1. These re-
sults further confirm the activation of cell death pathways upon
treatment with BTR-1.
1
.0
.5
0
ROS production by BTR-1. We examined whether BTR-1 treat-
ment (20
Supplementary Methods. Results showed that there was ROS pro-
2 2
duction at 2 and 4 h of treatment with BTR-1 (Fig. 6C–E). The H O
induced ROS production was used as a positive control (Fig. 6F).
However, ROS production was weaker when the incubation period
lM) led to ROS production in CEM cells as detailed in
Figure 5. Agarose gel profile showing DNA fragmentation. The chromosomal DNA
was extracted from CEM cells, following treatment with 20 M BTR-1. The purified
DNA was then resolved on a 1% agarose gel. Lane C, DMSO; lanes 2–6, 12–72 h,
l
respectively. ‘M’ is molecular weight ladder (2 log DNA ladder).
Figure 6. Effect of BTR-1 on ROS formation. CEM cells were treated with BTR-1 (20
l
M) for 1 (C), 2 (D), and 4 h (E). ROS production was quantified by flow cytometry and
2 2
presented. Cells treated with (B) and without (A) dye are also shown. H O was used as a positive control (F).

B. T. Moorthy et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6297–6301
6301
was more than 4 h (data not shown). Hence, our results suggest
that ROS generation could be an intermediate step in BTR-1 in-
duced cytotoxicity.
Thus, in the present study, we have designed and synthesized
three novel derivatives of 5-isopropylidiene, ITH-1, ITO-1, and
BTR-1. We find that among the compounds tested, BTR-1 was
many fold potent in inducing cytotoxicity than ITH-1 and ITO-1
3. Aplan, P. D. Trends Genet. 2005, 22, 46.
4.
5.
6.
Rowley, J. D. Nat. Rev. Cancer 2001, 1, 245.
Raghavan, S. C.; Lieber, M. R. Bioessays 2006, 28, 480.
Raghavan, S. C.; Swanson, P. C.; Wu, X.; Hsieh, C. L.; Lieber, M. R. Nature 2004,
428, 88.
7.
8.
9.
Raghavan, S. C.; Lieber, M. R. Cell Cycle 2004, 3, 762.
Raghavan, S. C.; Kirsch, I. R.; Lieber, M. R. J. Biol. Chem. 2001, 276, 29126.
Marculescu, R.; Le, T.; Simon, P.; Jaeger, U.; Nadel, B. J. Exp. Med. 2002, 195, 85.
10. Dorsett, Y.; McBride, K. M.; Jankovic, M.; Gazumyan, A.; Thai, T. H.; Robbiani, D.
F.; Di Virgilio, M.; San-Martin, B. R.; Heidkamp, G.; Schwickert, T. A.; Eisenreich,
T.; Rajewsky, K.; Nussenzweig, M. C. Immunity 2008, 28, 630.
with an IC50 value of <10 lM. It affected cell division by inducing
a block at S phase, which finally led to the activation of apoptosis.
Conflict of interest. Authors disclose that there is no conflict of
interest.
11. Okazaki, I. M.; Kotani, A.; Honjo, T. Adv. Immunol. 2007, 94, 245.
12. Lieber, M. R.; Yu, K.; Raghavan, S. C. DNA Repair (Amst.) 2006, 5, 1234.
1
3. Kavitha, C. V.; Nambiar, M.; Ananda Kumar, C. S.; Choudhary, B.; Muniyappa,
K.; Rangappa, K. S.; Raghavan, S. C. Biochem. Pharmacol. 2009, 77, 348.
4. Robert, J.; Jarry, C. J. Med. Chem. 2003, 46, 4805.
5. Ohishi, Y.; Mukai, T.; Nagahara, M.; Yajima, M.; Kajikawa, N.; Miyahara, K.;
Takano, T. Chem. Pharm. Bull. (Tokyo) 1990, 38, 1911.
1
1
Acknowledgments
1
6. Momose, Y.; Meguro, K.; Ikeda, H.; Hatanaka, C.; Oi, S.; Sohda, T. Chem. Pharm.
Bull. (Tokyo) 1991, 39, 1440.
We thank Ms. Mridula Nambiar, Ms. M. Nishana and other
members of SCR laboratory for discussions and help. This work
was supported by Lady Tata Memorial Trust international award
for leukemia research. K.K.C. acknowledges Postdoctoral fellow-
ship from DBT, India.
17. Sudo, K.; Matsumoto, Y.; Matsushima, M.; Fujiwara, M.; Konno, K.;
Shimotohno, K.; Shigeta, S.; Yokota, T. Biochem. Biophys. Res. Commun. 1997,
2
38, 643.
1
1
2
2
8. Sim, M. M.; Ng, S. B.; Buss, A. D.; Crasta, S. C.; Goh, K. L.; Lee, S. K. Bioorg. Med.
Chem. Lett. 2002, 12, 697.
9. Bulic, B.; Pickhardt, M.; Khilstunova, I.; Biernat, J.; Mandelkow, E. M.;
Mandelkow, E.; Waldmann, H. Angew Chem. Int. Ed. Engl. 2007, 46, 9215.
0. Zeiger, E.; Anderson, B.; Haworth, S.; Lawlor, T.; Mortelmans, K.; Speck, W.
Environ. Mutagen. 1987, 9, 1.
1. Hotta, N.; Akanuma, Y.; Kawamori, R.; Matsuoka, K.; Oka, Y.; Shichiri, M.;
Toyota, T.; Nakashima, M.; Yoshimura, I.; Sakamoto, N.; Shigeta, Y. Diabetes
Care 2006, 29, 1538.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.08.084.
2
2. Frlan, R.; Kovac, A.; Blanot, D.; Gobec, S.; Pecar, S.; Obreza, A. Molecules 2008,
References and notes
1
3, 11.
23. Krishman, P. S. Nature 1951, 168, 171.
1
2
.
.
Nambiar, M.; Kari, V.; Raghavan, S. C. Biochim. Biophys. Acta 2008, 1786, 139.
Nickoloff, J. A.; De Haro, L. P.; Wray, J.; Hromas, R. Curr. Opin. Hematol. 2008, 15,
24. Ravi, S.; Chiruvella, K. K.; Rajesh, K.; Prabhu, V.; Raghavan, S. C. Eur. J. Med.
Chem. 2010, 45, 2748.
3
38.
25. Patani, G. A.; LaVoie, E. J. Chem. Rev. 1996, 96, 3147.