4-Amino-2-arylamino-5-indoloyl/cinnamoythiazoles, analogs of topsentin-class of marine alkaloids, induce apoptosis in HeLa cells

Article abstract of DOI:10.1016/j.ejmech.2013.02.032

Marine organisms provide several biologically active compounds that include alkaloids with high cytotoxic activity but only a few of them have so far reached clinical stage, due partly to their limited supply and complex structural features. In an attempt

Full text of DOI:10.1016/j.ejmech.2013.02.032

European Journal of Medicinal Chemistry 63 (2013) 474e483
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
4-Amino-2-arylamino-5-indoloyl/cinnamoythiazoles, analogs of
topsentin-class of marine alkaloids, induce apoptosis in HeLa cells
,
,
Manisha Juneja a 1, Uma Vanam a 1, Sripriya Paranthaman a, Asha Bharathan b,
Venugopal S. Keerthi b, Justus K. Reena b, Rama Rajaram c, Kallikat N. Rajasekharan b,
Devarajan Karunagaran a
,
*
a Department of Biotechnology, Indian Institute of Technology Madras, Chennai 600 036, India
b Department of Chemistry, University of Kerala, Trivandrum 695 581, India
c Biochemistry Laboratory, Central Leather Research Institute, Chennai 600 020, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Marine organisms provide several biologically active compounds that include alkaloids with high
cytotoxic activity but only a few of them have so far reached clinical stage, due partly to their limited
supply and complex structural features. In an attempt to develop novel anticancer compounds, we have
now synthesized diaminoindoloylthiazoles (4aec; DIT1e3) and diaminocinnamoylthiazoles (5a,b; DCT1
e2) as analogs based on a topsentin scaffold and investigated the cytotoxic and apoptotic activities of
these compounds in HeLa cells. The results suggest that diaminoindoloylthiazoles (DIT1e3) inhibit cell
Received 21 November 2012
Received in revised form
19 February 2013
Accepted 21 February 2013
Available online 1 March 2013
growth and among these, DIT3 is the most cytotoxic against HeLa cells (IC50
minocinnamoylthiazoles DCT1 and DCT2, which can be viewed as curcuminediaminothiazole hybrids,
also inhibited cell growth but at relatively higher concentrations with IC50 values of 60 and 30 M,
respectively. These compounds induced apoptosis through the intrinsic pathway by reducing the
mitochondrial membrane potential and activating caspases, 9 and 3, but not caspase 8. Among the
marine alkaloid analogs tested in this study, DIT1e3 are very effective in inducing apoptosis of HeLa cells
followed by DCT2 and DCT1. The treated cells were arrested in G2/M phase followed by accumulation of
the cells in the Sub G0 phase. The curcuminediaminothiazole hybrid DCT1 had the maximum effect in
1 mM). The dia-
Keywords:
Anticancer
Apoptosis
Cell-cycle
NF-kB
Topsentin
m
Diaminothiazoles
downregulating TNF-induced NF-
kB activation among the compounds tested in this study. Thus, we
demonstrate that diaminoindoloylthiazoles and diaminocinnamoylthiazoles induce apoptosis, regulate
cell cycle and NF-
investigation.
k
B signaling and thus show promising anticancer effects that warrant further
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
topsentins and nortopsentins, which possess excellent in vitro
cancer cell cytotoxicity, generally in the 1e10 mg/mL range [12e20].
Marine organisms are known to contain several pharmacologi-
cally active compounds [1e4]. Many marine natural products have
unique structural features and substitutions, such as the presence
of unusual heterocyclic ring systems or halogen substituents [5]. A
few marine natural products or their synthetic analogs are in
clinical use or at various stages of drug development [6e9]. Many
alkaloids are derived from marine organisms such as sponges,
coelenterates, tunicates (ascidians), bryozoans, red algae, acorn
worms and symbiotic bacteria [10]. Indole derivatives are quite
common among marine alkaloids [11] that include dragmacidins,
Dragmacidins (1), topsentins (2) and nortopsentins (3) are bis(in-
dolyl)piperazines, indoloylindolylimidazoles and bis(indolyl)imid-
azoles, respectively (Fig. 1).
Despite the discovery of several marine alkaloids with high
cytotoxic activity, not many have entered pre-clinical and clinical
trials, due to their scarcity and difficulties in total synthesis. In an
attempt to discover novel anticancer compounds, we now report
studies on thiazole analogs of the marine alkaloid topsentin. The
indoloyl group of topsentins has been retained in the first set of
analogs, whereas the indolylimidazole ring has been replaced with
a 2,4-diaminothiazole unit, leading to diaminoindoloylthiazoles
(DIT1e3; 4aec) (Fig. 2A). These compounds can also be viewed as
analogs of the marine alkaloid dendrodoine, which is 5-indol-3-
oyl-3-(N,N-dimethylamino)-1,2,4-thiadiazole, and these have
been reported earlier by us [21e24]. In a further modification, a
* Corresponding author. Tel.: þ91 44 22574126; fax: þ91 44 22574102.
E-mail addresses: karunagarand@gmail.com, karuna@iitm.ac.in (D. Karunagaran).
1
These authors contributed equally to this work.
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.02.032

M. Juneja et al. / European Journal of Medicinal Chemistry 63 (2013) 474e483
475
initiation and tumor promotion, induced by various carcinogens
[26e29]. Extensive research over the last 50 years established the
anti-carcinogenic activity of curcumin and found it to be safe in
human clinical trials [30,31]. We were further encouraged by the
cytotoxic activity of the marine indole alkaloid hemiasterlin A [7].
The present study was initiated to assess the cytotoxic effects of
three diaminoindoloylthiazoles (DIT1e3) as topsentin analogs,
and three diaminocinnamoylthiazoles (DCT1e2), as curcumine
diaminothiazole hybrids (Fig. 2), on a transformed cell line. In-
duction of apoptosis and modulation of NF-kB signaling in cancer
cells are considered as important underlying mechanisms by which
curcumin and many other anticancer agents act [30,31]. Hence, we
assessed the apoptotic potential of these marine alkaloid analogs
and studied their effects on cell cycle and NF-kB signaling.
2. Results and discussion
2.1. Diaminothiazoles exert cytotoxic effects on HeLa cells
Bisindole alkaloids, a class of deep-sea sponge metabolites, have
received much attention due to their potent antiviral, antimicrobial,
anti-inflammatoryand antitumor activities. Topsentins belong to the
bisindole class of marine alkaloids and are indoloylindolylimidazole
derivatives, often bearing halogen substitutions. Other topsentin-
like compounds, differing in the coupling pattern of the indole
units have also been found in several marine sponges andthese show
a wide spectrum of bioactivities [32]. The structurally related nor-
topsentins also exhibit diverse bioactivities including potent anti-
viral, antitumor, and anti-inflammatory activities. Coupled with this
wide-range of bioactivities, the lack of structural complexity has
made bisindole alkaloids attractive targets for investigations with
both natural and synthetic compounds [33]. In continuation to our
interest in the synthetic thiazole analogs of marine alkaloids and as
part of our effort in searching for novel antitumor compounds
modeled on marine alkaloids [34], we have now designed and syn-
thesized diaminothiazole analogs of topsentins and further elabo-
rated these to curcuminediaminothiazole hybrids.
Fig. 1. Chemical structure of dragmacidin (1), topsentin (2) and nortopsentin (3).
second set was selected in which the indoloyl unit in dia-
minoindoloylthiazoles (4) was further substituted with a cinna-
moyl group leading to diaminocinnamoylthiazoles (DCT1e2; 5a,b)
(Fig. 2B) and these compounds were obtained by a method devel-
oped by us earlier [25]. This substantial structural modification was
prompted by our interest in the diarylheptanoid curcumin (6)
(Fig. 2C). Curcumin, a biscinnamoylmethane, is the major active
component of turmeric (Curcuma longa) that inhibits both tumor
Cytotoxic activity was examined using alamar blue assay
after treating HeLa cells for 48 h with different concentrations of
diaminoindoloylthiazoles DIT1e3 or diaminocinnamoylthiazoles
DCT1e2 and these diaminothiazoles were cytotoxic in a concen-
tration dependent manner (Fig. 3). The IC50 values of these com-
pounds in HeLa cells are given in Table 1. Diaminothiazoles (DIT1e3)
inhibited cell growth and DIT3 was the most effective in its cyto-
toxicity against HeLa cells (IC50 1
hybrids (DCT1e2) also inhibited cell growth but at relatively
higher concentrations with IC50 values of 60 and 30 M, respec-
mM). Curcuminediaminothiazole
m
tively. Similar to the cytotoxic effects of diaminoindoloylthiazoles
DIT1e3 observed in the present study, several novel synthetic
2,5-bis(30-indolyl)furans and 3,5-bis(30-indolyl)isoxazoles have IC50
values in the micromolar range [35]. Cytotoxic effects were also
reported for a series of bis-indolylthiophenes, in which the imid-
azole moiety of the nortopsentin was replaced by a thiophene ring,
and a series of bisindolylpyrazoles, where a pyrazole central ring
replaced the imidazole ring of nortopsentin [36,37].
2.2. Diaminothiazoles induce apoptosis in HeLa cells
HeLa cells were treated with DIT1e3 or DCT1e2 at their
respective IC50 concentrations for 48 h and checked for morpho-
logical changes by ethidium bromide/acridine orange staining.
While the untreated cells were well spread with a flattened
morphology, many apoptotic bodies could be noticed in those
treated with DIT1e3 or DCT1e2 (Fig. 4). The apoptotic cells treated
with the compounds could be distinguished as they acquired a
Fig. 2. A. Diaminoindoloylthiazoles (DIT1e3; 4aec) synthesized as topsentin analogs;
B. Diaminocinnamoylthiazoles (DCT1e2; 5aeb) synthesized as curcuminedia-
minothiazole hybrids; C. Curcumin I (diferuloylmethane) from turmeric.

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M. Juneja et al. / European Journal of Medicinal Chemistry 63 (2013) 474e483
Fig. 3. Changes in viability of HeLa cells upon treatment with (A) DIT1 (B) DIT2 (C) DIT3 and (D) DCT1, DCT2 (5a,b). Cells grown in 96 well plates were treated with various
concentrations of DIT1e3 and DCT1e2 for 48 h. The mean of the percentage cell viability over control along with their standard deviation is indicated. All the experiments were
done three times with triplicate samples.
yellowish fluorescence whereas the untreated cells appeared to
have a greenish fluorescence. These results show that DIT1e3 and
DCT1e2 induce apoptosis in HeLa cells akin to curcumin that was
used as a positive control.
expressed in percentage. Table 2 shows the percentage distribution
of HeLa cells in various cell cycle stages upon treatment with DIT1e3
or DCT1e2 for 24 h. It can be seen that 81% of the untreated HeLa
cells (control) synchronized themselves at G0/G1 phase and 13% at
G2/M phase, 5% at S phase and only 1% in sub G0 phase that usually
contains the apoptotic fraction. DIT1 arrested 24% cells at sub G0
phase without significantly changing the number of cells at S (8%)
and G2/M (12%) phases but decreasing the percentage of cells at G0/
G1 (56%) phase. DIT2 arrested 13% cells at sub G0 phase and 10% at S
phase, without significantly changing the number of cells at G2/M
(18%) phases but decreasing the percentage of cells at G0/G1 (59%)
phase. DIT3 treatment arrested 35% cells at G2/M phase and 17% cells
were in sub G0/G1 phase without changing the number of cells at S
phase but decreasing the percentage of cells at G0/G1 phase. DCT1
arrested 27% cells at G2/M phase without significantly changing the
number of cells at S and sub G0 phases but decreasing the percentage
of cells at G0/G1 (65%) phase. In contrast DCT2 induced both cell cycle
arrest and apoptosis by driving 26% cells into sub G0 phase and also
arresting 51% cells at G2/M phase and by decreasing the percentage
of cells at G0/G1 (15%) phase.
2.3. Diaminothiazoles induce nuclear condensation
HeLa cells were treated with DIT1e3 or DCT1e2 at their
respective IC50 concentrations for 48 h, stained with DAPI and
checked for morphological changes using a fluorescent microscope.
Cells with homogenously glowing bright nuclei were considered to
have the non-apoptotic phenotype and apoptotic nuclei were
identified by the condensed chromatin or fragmented nuclear
bodies (Fig. 5). The nucleus in the treated cells had shrunken and
shifted to periphery and there was also a decrease in nucleus to
cytoplasmic ratio. The chromatin condensation was clearly visible
in cells treated with curcumin. Thus, HeLa cells treated with DIT1e3
or DCT1e2 formed apoptotic bodies and exhibited nuclear
condensation, characteristic features of apoptosis.
Table 3 shows the percentage distribution of HeLa cells in
various cell cycle stages upon treatment with DIT1e3 or DCT1e2
for 48 h. Similar to the cells at 24 h, the control cells were mostly
in G0/G1 phase (73%) and 17% at G2/M phase, 7% at S phase and only
2% in sub G0/G1 phase. DIT1e2 induced apoptosis with 55% and 42%
of cells, respectively, at sub G0/G1 phase. DIT3 also dramatically
induced apoptosis with 49% cells at sub G0/G1 phase whereas DCT1
treatment induced 17% cells into this phase while DCT2 induced
35% cells into sub G0/G1 phase. While DIT2 (9.78%) and DIT3 (13%)
treatment had less number of cells at G2/M phase compared with
the control (17%), DIT1 induced 22% into G2/M, DCT1 induced 28%
cells and DCT2 could induce 36% cells into this phase of the cell
cycle. Thus upon treatment for 24 h, DIT3, DCT1 and DCT2 arrested
2.4. Diaminothiazoles induce cell cycle arrest and apoptosis
Cell cycle analysis was done by propidium iodide staining and the
cell distribution in the sub G0, G0/G1, S and G2/M phases was
Table 1
IC50 values of DIT1e3 and DCT1e2 in HeLa cells treated for 48 h.
S.no.
Compound
IC50 values (mM)
1.
2.
3.
4.
5.
DIT1
DIT2
DIT3
DCT1
DCT2
45
20
1
60
30

M. Juneja et al. / European Journal of Medicinal Chemistry 63 (2013) 474e483
477
Fig. 4. Apoptosis induction upon treatment with DIT1e3 and DCT1e2 in HeLa cells. HeLa cells were grown in 24 well plates and treated with or without the compounds for 48 h
and then stained with acridine orange/EtBr mixture, and viewed under fluorescent microscope. These results were confirmed in three independent experiments done in duplicates.
(A) Control, (B) curcumin, (C) DIT1, (D) DIT2, (E) DIT3, (F) DCT1 and (G) DCT2.
HeLa cells effectively at G2/M phase whereas DIT1 did not (12%)
while DIT2 was less effective (18%). In addition, DIT1e3 and DCT2
induced apoptosis but DCT1 was not effective in apoptosis induc-
tion at this time point. However, upon treatment for 48 h, all the
tested diaminothiazoles (DIT1e3 and DCT1e2) induced apoptosis
although DCT1 was relatively less effective. Interestingly, DIT1,
DCT1 and DCT2 effectively arrested cells at G2/M phase whereas
DIT2 and DIT3 did not do so at this time point. Among the five
marine alkaloid analogs, apoptosis was maximum in HeLa cells
treated with DIT1, followed by that with DIT3, DIT2 and DCT2.
In parallel with the results from the present study showing
cytotoxicity to HeLa cells upon treatment with DIT1e3 or DCT1e2
due to cell cycle arrest followed by the induction of apoptosis,
E7974, a synthetic analog of hemiasterlin, a marine sponge natural
product, induces G2/M arrest and marked disruption of mitotic
spindle formation [38]. Similarly, batzellines (pyrroloiminoqui-
nones alkaloids obtained from the deep-water Batzella sp of
Caribbean sponge) exhibit cytotoxicity in pancreatic cancer cells by
inducing cell cycle arrest mediated by their ability to intercalate
into DNA and/or inhibit topoisomerase II activity [39]. Derivatives
of pyridoacridine alkaloids (a group of marine sponge alkaloids)
induced G2/M arrest followed by apoptosis in Neuro 2A (murine
neuroblastoma) cells [40]. Granulatimide and isogranulatimide
(marine alkaloids) induced G2/M arrest, targeting checkpoint 1
kinase (Chk1) [41].
2.5. Diaminothiazoles reduce mitochondrial membrane potential
Induction of apoptosis by various chemotherapeutic drugs is
preceded by a reduction in the mitochondrial membrane potential.
Thus HeLa cells treated with DIT1e3 or DCT1e2 had lower mito-
chondrial membrane potential exhibiting primarily green fluores-
cence and were easily distinguished from control cells exhibiting
red fluorescence as shown (Fig. 6). As expected, curcumin-treated
HeLa cells also exhibited an increase in green fluorescence
compared to control.
2.6. Diaminothiazoles activate caspases 3 and caspase 9 but not
caspase 8
Cell extracts were analyzed for the changes in the activities of
caspases 3, 8 and 9 upon treatment with DIT1e3 or DCT1e2. Cas-
pase 3 activation was measured upon treatment with DIT1e3 or
DCT1e2 for 48 h and DIT1e2 treated cells had 2.5 and 2.4 fold
activation, respectively. In the case of DIT3 treatment, there was 2.8
fold activation while for DCT1e2 treated cells, there was 1.5 and 2.2

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M. Juneja et al. / European Journal of Medicinal Chemistry 63 (2013) 474e483
Fig. 5. Changes in nuclear morphology induced by DIT1e3 and DCT1e2 in HeLa cells using DAPI staining. Cells were grown in 24 well plates, with or without drug (48 h), stained
with DAPI, and viewed under fluorescent illumination. The results were confirmed in three independent experiments. (A) Control, (B) curcumin, (C) DIT1, (D) DIT2, (E) DIT3, (F)
DCT1 and (G) DCT2.
fold activation, respectively. Curcumin treatment showed 2.1 fold
activation (Fig. 7A). Caspase 9 activation was studied upon treat-
ment with DIT1e3 or DCT1e2 for 48 h. Upon DIT1e3 treatment for
48 h, there was 2.5, 4.4 and 1.6 fold activation, respectively, while
treatment with DCT1e2 for 48 h, there was 2.3 and 2.6 fold acti-
vation, respectively. Curcumin treatment activated caspase 9 by 1.7
fold (Fig. 7B). DIT2 activated caspase 8 by 1.54 fold and curcumin
activated it by 1.4 fold as shown. However, no significant activation
was observed upon treatment with DIT1, DIT3, DCT1, or DCT2 (0.96,
1.1, 0.9 and 1.2 fold, respectively) (Fig. 7C). Upon treating the cells
with DIT1e3 or DCT1e2, there was an upregulation of caspase 3
and caspase 9 while there was relatively no significant activation of
caspase 8. These results, together with the observation of a
reduction in the mitochondrial membrane potential induced by
diaminothiazoles suggest the involvement of the intrinsic pathway
of apoptosis but do not rule out the operation of extrinsic pathway.
Table 2
Table 3
Percentage distribution of HeLa cells after DIT1, 2 or 3 or DCT1 or 2 treatment during
various cell cycle stages at 24 h. Values represent mean ꢀ SD of three independent
experiments.
Percentage distribution of HeLa cells after DIT1, 2 or 3 or DCT1 or 2 treatment during
various cell cycle stages at 48 h. Values represent mean ꢀ SD of three independent
experiments.
Compound
Sub G0
G0/G1
S
G2/M
Compound
Sub G0
G0/G1
S
G2/M
Control
DIT1
DIT2
DIT3
DCT1
DCT2
1.2 ꢀ 0.29
80.7 ꢀ 0.47
56.34 ꢀ 0.52
59.13 ꢀ 0.92
43.2 ꢀ 1.7
4.5 ꢀ 0.2
13.2 ꢀ 0.48
11.8 ꢀ 0.56
17.51 ꢀ 0.83
35.3 ꢀ 1.5
Control
DIT1
DIT2
DIT3
DCT1
DCT2
2.3 ꢀ 0.37
73.1 ꢀ 1.02
13.75 ꢀ 0.82
38.53 ꢀ 0.66
24.0 ꢀ 0.16
38.0 ꢀ 1.4
7.2 ꢀ 0.77
16.6 ꢀ 0.16
21.56 ꢀ 0.42
9.78 ꢀ 0.65
12.5 ꢀ 3.5
23.74 ꢀ 0.25
13.17 ꢀ 0.8
16.7 ꢀ 0.9
3.4 ꢀ 0.64
25.8 ꢀ 2.0
8.12 ꢀ 0.4
55.33 ꢀ 0.55
42.37 ꢀ 0.64
49.0 ꢀ 4.1
8.95 ꢀ 0.84
7.69 ꢀ 0.55
7.8 ꢀ 1.2
10.19 ꢀ 0.38
4.2 ꢀ 0.18
4.4 ꢀ 0.02
6.8 ꢀ 0.38
64.7 ꢀ 4.0
27.2 ꢀ 3.4
16.7 ꢀ 0.91
35.0 ꢀ 6.2
14.2 ꢀ 0.01
9.0 ꢀ 0.86
28.2 ꢀ 0.26
35.8 ꢀ 12.7
15.37 ꢀ 0.77
51.4 ꢀ 2.57
15.0 ꢀ 1.6
Data represent mean ꢀ S.D.
Data represent mean ꢀ S.D.

M. Juneja et al. / European Journal of Medicinal Chemistry 63 (2013) 474e483
479
Fig. 6. Reduction in mitochondrial membrane potential induced by DIT1e3 and DCT1e2 in HeLa cells using JC1 dye. Cells were grown in 24 well plates, with or without drug (48 h),
stained with JC1 and viewed under fluorescent illumination. The results were confirmed in three independent experiments. (A) Control, (B) curcumin, (C) DIT1, (D) DIT2, (E) DIT3,
(F) DCT1 and (G) DCT2.
Another marine alkaloid, lamellarin D, initiated apoptosis by the
activation of Bax and decreased the expression levels of anti-
apoptotic proteins (Bcl-2 and cIAP2) in association with activation
of caspase 9 and caspase 3 in leukemic cells [42]. Lamellarin D
mediated apoptosis through the intrinsic pathway as it did not alter
the expression of Fas and Fas-L and in addition the leukemia cells
deficient in caspase 8 or Fas-associated death domain underwent
apoptosis through the typical mitochondrial apoptotic cascade [42].
Lamellarin D and its structural analogs induced apoptosis of cancer
cells by inducing reduction of mitochondrial membrane potential,
swelling and cytochrome c release [43]. Whereas Lamellarin D
promoted apoptosis in leukemia cells without prominent cell cycle
arrest, DIT3 and DCT1e2 used in our study caused cell cycle arrest
followed by the induction of apoptosis, thus highlighting the need
to understand the mechanisms by which different marine alkaloids
induce apoptosis [44]. E7974, the synthetic analog of hemiasterlin
was also shown to induce apoptosis via caspase-3 activation and
poly ADP ribose polymerase cleavage [38]. Another marine alkaloid,
naamidine A, induced apoptosis in cultured tumor cells by
disruption of the mitochondrial membrane potential, and cleavage
and activation of caspases 3, 8 and 9 [45]. Similarly, polycarpine
from the ascidian Polycarpa aurata and its synthetic derivative,
desmethoxyethoxypolycarpine (dimethylpolycarpine), were found
to induce apoptosis in JB6 cells through p53 and caspase 3
dependent pathways [46].
2.7. Diaminothiazoles inhibit TNF-a-induced NF-kB activation
HeLa cells were transiently transfected with NF-
gene luciferase construct and luciferase assay was performed in the
presence or absence of TNF- , as described in Experimental section.
kB reporter
a
Results shown in Fig. 8 indicate that diaminoindoloylthiazoles
(DIT1e3) used at their respective IC50 concentrations, decreased
NF-
cells activated by TNF-
2), used at their respective IC50 concentrations, showed NF-
downregulation by 87% and 56%, respectively, over the control cells
upon activation by TNF- . Curcumin at its IC50 concentration effi-
ciently downregulated NF- B by 79% over control, as expected.
Thus, the curcuminediaminothiazole hybrid DCT1 was found to be
k
B activity by 80%, 72% and 34%, respectively, over the control
a. The diaminocinnamoylthiazoles (DCT1e
kB
a
k
the most effective in downregulating TNF-induced NF-
activation.
kB
Curcumin (diferuloylmethane), a known anti-inflammatory and
anti-carcinogenic agent, is a potent inhibitor of NF- B activation
k

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M. Juneja et al. / European Journal of Medicinal Chemistry 63 (2013) 474e483
Fig. 7. Changes in caspase activity induced by DIT1e3 and DCT1e2. HeLa cells were grown in 60 mm petri plates and treated for 48 h with or without the compounds, lysed,
centrifuged and the supernatant was used for assaying caspase activity with specific caspase substrates for: (A) caspase 3; (B) caspase 9 and (C) caspase 8 activation. Results were
expressed as relative fluorescence units (RFU) over the control. The experiments were repeated thrice and values were expressed as mean ꢀ SD.
induced by TNF, phorbol ester and hydrogen peroxide through
quenching of ROI production or by inhibiting a protein kinase [47].
and the topsentin analog DIT1 needs further investigation.
New approaches to cancer therapy that inhibit nuclear trans-
Curcumin inhibits NF-
k
B activation pathway prior to I
kB
a
phos-
location of NF-kB are expected to be highly effective in the treat-
phorylation [48]. Since curcumin is known to inhibit NF-
k
B acti-
ment of tumors [49].
vation induced by TNF and other activators, the observation made
in the present study that the curcuminediaminothiazole hybrid
3. Conclusions
DCT1e2 could also inhibit NF-
kB is interesting. The mechanism of
NF- B inhibition by the curcuminediaminothiazole hybrid DCT1
k
We have shown that the diaminoindoloylthiazoles and dia-
minocinnamoylthiazoles induce apoptosis of HeLa cells by the
mitochondrial pathway. The present work has provided interesting
leads such as the observation of cell cycle arrest at G2/M phase upon
treatment with DIT3, DCT1 or DCT2 for 24 h (Table 2), suggesting
that these compounds may regulate certain target proteins of the
cell cycle. Similarly the significant downregulation of NF-kB activity
by the curcuminediaminothiazole hybrid, DCT1, and the topsentin
analogs (DIT1 or 2) suggests the need for further investigation of
the signaling pathways that are regulated by these compounds.
4. Experimental section
4.1. Cell culture and maintenance
HeLa (human cervical adenocarcinoma) cells were obtained
from the National Centre for Cell Science, Pune, India. The cells were
grown in Dulbecco’s modified Eagle’s medium (DMEM) (GIBCO,
Invitrogen Corporation) supplemented with 10% heat inactivated
fetal bovine serum (FBS) (GIBCO, Invitrogen Corporation) and an-
tibiotics (100 U/ml penicillin and 100
mg/mL streptomycin) in a
humidified atmosphere of 5% CO2 at 37 ꢁC. For sub culturing, the
monolayer was washed with PBS-EDTA at room temperature and
trypsinized for 3e4 min at 37 ꢁC. Complete medium was added to
neutralize trypsin and cells were pelleted at 2500 rpm, 5 min. The
cells were suspended in fresh medium and dispensed into fresh
tissue culture flasks for maintenance.
Fig. 8. Changes in NF-
transfected with NF- B luciferase and renilla luciferase constructs and treated for 24 h
with or without DIT1e3 or DCT1e2 at their respective IC50 concentrations, followed by
TNF- treatment (20 ng/ml) for 5 h. The levels of NF- B were determined by measuring
the luciferase activity by dual-luciferase assay kit (Promega) as per the manufacturer’s
protocol. The experiments were repeated thrice and NF- B levels were expressed as
relative luminescence units (RLU). Values represent mean ꢀ SD.
kB activity induced by DIT1e3 and DCT1e2. HeLa cells were co-
k
a
k
k

M. Juneja et al. / European Journal of Medicinal Chemistry 63 (2013) 474e483
481
4.2. Chemical synthesis
Chemicals used were of commercial grade. 3-Acetylindole was
purchased from Sigma Aldrich and copper (II) bromide was from
Aldrich (India). Silica gel G for TLC and as solid support in bromi-
nation was procured from Merck (India). Standard methods were
used for the synthesis of methyl styryl ketone [50] The spectra were
recorded on: Bruker or Varian NMR spectrometers, JEOL Mass
spectrometer (using Argon/Xenon, 6 KV, 10 mA as the FAB gas and
m-nitrobenzyl alcohol as the matrix) for FAB Mass spectra and
Nicolet 400D FTIR spectrometer. All compounds gave satisfactory C,
H and N analysis (Central Drug Research Institute (CDRI), Lucknow,
India and Sophisticated Analytical Instrumentation Facility (SAIF).
Kochi, India). The 1-aryl-3-(N,N0-diarylamidiono)thioureas and
1-aryl-3-(N-nitroamidino)thioureas were obtained by methods re-
ported earlier [51,52]. The bromination of 3-acetylindole (0.159 g,
1 mmol) was done by refluxing with silica gel supported copper (II)
bromide (1.3 g, 2 mmol) in chloroformeethyl acetate (1:1) solvent
system in the presence of HBr. Adopting a similar procedure, but
without HBr, methyl styryl ketones were also brominated and the
bromo compounds thus obtained were immediately used in the
next step of the synthesis of diaminothiazoles. Thus, the dia-
minoindoloylthiazoles DIT1 (4a) and DIT3 (4c) were obtained by the
reaction of 3-(2-bromoacetyl)indole (7) in N,N-dimethylformamide
with 1-(4-chlorophenyl)-3-(N-nitroamidino)thiourea (8a) or 1-(4-
methoxyphenyl)-3-(N-nitroamidino)thiourea (8c) respectively in
presence of triethylamine and these were found to be identical
with samples available from our earlier work [21,23]. The dia-
minoindoloylthiazole DIT2 (4b) was prepared from 3-(2-bro-
moacetyl)indole (7) and 1-[N,N0-di(4-chorophenyl)amidino]-3-(4-
methoxyphenyl)thiourea (8b) based on our earlier work [24]. The
diaminocinnamoylthiazoles DCT1e2 (5a,b) were prepared from
1-(4-chlorophenyl)-3-(N-nitroamidino)thiourea (8b) and bromo-
methyl styryl ketones (9a,b) by following a method reported earlier
with some modifications [25]. The synthesis (Scheme 1) and other
details including NMR spectra are given in the Supplementary
material.
Scheme 1. A. Reaction scheme for the synthesis of diaminoindoloylthiazoles (DIT1e3;
4aec). B. Reaction scheme for the synthesis of diaminocinnamoylthiazoles (DCT1e2;
5a,b). General reaction condition (i): reactants and reagent, 1 mmol each; N,N-
dimethylformamide as solvent; triethylamine as base, 60e80 ꢁC; 15 min.
4.5. Acridine orange/ethidium bromide staining
4.3. Reagents
Characteristic apoptotic morphological changes were assessed
by fluorescent microscopy using acridine orange/ethidium bromide
staining method. Briefly, cells were seeded in 24-well plates at
seeding densities of 75000 cells/well and then treated with the drug
at specified concentration for a definite time interval. After washing
The stock solutions of DIT1e3 and DCT1e2 were made in 100%
DMSO at a concentration of 10 mM and sub-stocks were prepared
freshly in growth medium prior to the experiments. Curcumin was
purchased from Sigma (catalog number C-7727). Alamar blue
(catalog number RM124-1G) was purchased from Himedia. Sub-
strate for caspase 8 (Ac-IETD-AFC) (catalog number 556552) was
bought from BD biosciences and the substrates for caspase 3 (Ac-
with PBS, cells were stained with 100
m
L of a mixture of acridine
orange (100 mg/mL) and ethidium bromide (100 mg/mL). The cells
were incubated for 30 min at 37 ꢁC and washed with PBS and finally
viewed under Nikon Eclipse TS100 inverted fluorescent microscope.
DEVD-AFC) and caspase
9 (Ac-LEHD-AFC) (catalog numbers
264151-1 MG and 218765-1 MG, respectively) were purchased from
Calbiochem Inc., USA. Lipofectamine was purchased from Invi-
trogen. Dual luciferase assay kit was procured from Promega.
4.6. DAPI staining
Briefly, cells were seeded at a density of 75000 cells/well and
allowed to grow for 24 h. Subsequently the cells were incubated
with desired concentration of the tested compound for specific time
period. The medium was removed and cells were washed with PBS.
4.4. Cell viability assay
Cell viability assays were carried out using alamar blue (resa-
zurin), a nontoxic, cell permeable nonfluorescent compound that is
blue in color. Viable cells reduce resazurin to resorufin, which pro-
duces very bright red fluorescence that is used for a quantitative
measure of viability. Briefly cells were seeded at a density of
5000 cells/well into 96 well plates. After 24 h, drugs were added to
Then 300
m
l of DAPI stain (1
m
g/mL) in methanol was added and kept
L of PBS was
at 37 ꢁC for 10 min. The stain was removed and 200
m
added and morphology was observed using fluorescent microscopy.
the medium at various concentrations and incubated for 48 h. 10
mL
4.7. JC1 staining
of 0.2 mg/mL alamar blue was added into each well followed by
incubation for 2e4 h. The absorbance of alamar blue was read using
the Biorad microplate reader (model no. 680) at 570 and 595 nm. Cell
survival was expressed as percentage over the untreated control.
JC-1 (5,50,6,60-tetrachloro-1,10,3,30-tetraethylbenzimidazolcarbo-
cyanine iodide) is a lipophilic fluorescent cationic dye. Intact living
cells stained with JC-1 exhibit a pronounced orange fluorescence

482
M. Juneja et al. / European Journal of Medicinal Chemistry 63 (2013) 474e483
whereas apoptosis results in a loss of mitochondrial membrane
potential and a subsequent green fluorescence. JC-1 was diluted in
medium to a final concentration of 5 mg/mL and the JC-1 containing
medium was added to the treated cells, followed by incubation for
10 min at 37 ꢁC (or RT for 15 min). Subsequently the cells were
washed twice with PBS and analyzed under Nikon Eclipse TS100
inverted fluorescent microscope.
Technology (a), Government of India. Manisha Juneja was a
recipient of MHRD fellowship from IIT Madras. Uma Vanam and
Sripriya Paranthaman were recipients of post-doctoral fellow-
ships from the Department of Biotechnology, Government of India.
Asha Bharathan was a recipient of fellowship from University of
Kerala. Justus K. Reena was a recipient of University Grants Com-
mission (UGC) fellowship. Venugopal S. Keerthi obtained her
research fellowship from Kerala State Council for Science, Tech-
nology and Environment. K.N. Rajasekharan is a recipient of UGC
BSR Faculty Fellowship.
4.8. Flow cytometry
Cells were treated with DIT1e3 or DCT1e2 for 24 h and 48 h. The
cells were then harvested, washed in PBS-EDTA and fixed overnight
in cold 70% ethanol at 4 ꢁC. The cells were then pelleted by
centrifugation, washed twice with PBS-1% FBS, and were resus-
Appendix A. Supplementary data
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.ejmech.2013.02.032.
pended in 300
ml of PBS. 2.5
m
l RNase A (20 mg/mL) was added to it
and incubated at 37 ꢁC for 1 h. This is followed by addition of 3
mL PI
(1 mg/mL) and then cells were analyzed through Cell lab QuantaÔ
SC flow cytometer from Beckman Coulter.
References
[1] T.E. Adrian, Novel marine-derived anti-cancer agents, Curr. Pharm. Des. 13
(2007) 3417e3426.
[2] G. Schwartsmann, Marine organisms and other novel natural sources of new
cancer drugs, Ann. Oncol. 11 (Suppl. 3) (2000) 235e243.
[3] B. Haefner, Drugs from the deep: marine natural products as drug candidates,
Drug Discov. Today 8 (2003) 536e544.
[4] G.M. Cragg, D.J. Newman, K.M. Snader, Natural products in drug discovery and
development, J. Nat. Prod. 60 (1997) 52e60.
4.9. Caspase assays
Activation of Caspases 3, 8 and 9 was assessed by using a fluoro-
genic substrate assay.1 ꢂ106 cells were seeded in a 60 mm Petri dish
and treated with DIT1e3 or DCT1e2 for desired timings. Cells were
lysed in lysis buffer (10 mM TriseHCl, 10 mM NaH2PO4/Na2HPO4, pH
7.5, 130 mM NaCl, 1% Triton-X-100, 10 mM NaPPi). For each reaction
[5] S. Urban, S.J.H. Hickford, J.W. Blunt, M.H.G. Munro, Bioactive marine alkaloids,
Curr. Org. Chem. (2000) 765e807.
[6] K.B. Glaser, A.M. Mayer, A renaissance in marine pharmacology: from pre-
clinical curiosity to clinical reality, Biochem. Pharmacol. 78 (2009) 440e448.
[7] T.F. Molinski, D.S. Dalisay, S.L. Lievens, J.P. Saludes, Drug development from
marine natural products, Nat. Rev. Drug Discov. 8 (2009) 69e85.
[8] G. Schwartsmann, A.B. Da Rocha, J. Mattei, R. Lopes, Marine-derived
anticancer agents in clinical trials, Exp. Opin. Investig. Drugs 12 (2003)
1367e1383.
[9] R. Singh, M. Sharma, P. Joshi, D.S. Rawat, Clinical status of anti-cancer agents
derived from marine sources, Anticancer Agents Med. Chem. 8 (2008) 603e617.
[10] J.W. Blunt, B.R. Copp, M.H.G. Munro, P.T. Northcote, M.R. Prinsep, Marine
natural products, Nat. Prod. Rep. 23 (2006) 26e78.
or time point, 5
well containing 50
10 mMDTT,1 mM EDTA, 0.1%CHAPS,10%sucrose, pH7.2). 50
m
l reconstituted caspase substrate was added to each
L of 2ꢂ assay buffer (20 mM PIPES,100 mM NaCl,
L ofcell
m
m
lysate was added to each well/reaction and incubated for 1 h at 37 ꢁC.
The amount of AFC liberated was read using a plate reader (Spec-
traMax) with excitation wavelength of 400 nm and an emission
wavelength of 480e520 nm. Caspase activation was expressed in
relative fluorescence units in folds over the untreated control.
[11] U. Pindur, T. Lemster, Advances in marine natural products of the indole and
annelated indole series: chemical and biological aspects, Curr. Med. Chem. 8
(2001) 1681e1698.
4.10. Transient transfection and luciferase assay
[12] J. Kobayashi, T. Murayama, M. Ishibashi, S. Kosuge, M. Takamatsu, Y. Ohizumi,
H. Kobayashi, T. Ohta, S. Nozoe, T. Sasaki, Hyrtiosins A and B, new indole al-
kaloids from the Okinawan marine sponge Hyrtios erecta, Tetrahedron 46
(1990) 7699e7702.
[13] S. Kohmoto, Y. Kashman, O.J. McConnell, K.L. Rinehart, A. .Wright, F. .Koehn,
Dragmacidin, a new cytotoxic bis(indole) alkaloid from a deep water marine
sponge, Dragmacidon sp, J. Org. Chem. 53 (1988) 3116e3118.
[14] A.E. Wright, S.A. Pomponi, S.S. Cross, P. McCarthy, A new bis(indole) alkaloid
from a deep-water marine sponge of the genus Spongosorites, J. Org. Chem.
57 (1992) 4772e4775.
HeLa cells were seeded at 60% confluency per well in 96 well
plates. After overnight culture, the cells in each well were trans-
fected with 0.2
mg DNA consisting of NF-kB-luciferase reporter
plasmid and renilla luciferase plasmid as internal control (pGL4,
Promega) using lipofectamine reagent (Invitrogen). After 24 h of
transfection, the cells were incubated in medium containing DIT1e
3 or DCT1e2 or DMSO (control) for 24 h and then treated with TNF-
a
(20 ng/ml) for 5 h. The cells were harvested and luciferase assay
[15] S. Tsujii, K.L. Rinehart, S.P. Gunasekera, Y. Kashman, S.S. Cross, M.S. Lui,
S.A.
Pomponi,
M.C.
Diaz,
Topsentin,
bromotopsentin,
and
was performed using dual-luciferase assay kit (Promega), according
to the manufacturer’s protocol. The luminescence was measured
using Spectramax-L luminometer, Molecular Devices, USA. Results
were normalized with internal control plasmid and expressed as
relative luminescence units in fold over control.
dihydrodeoxybromo-topsentin: antiviral and antitumor bis-(indolyl)imidaz-
oles from Caribbean deep-sea sponges of the family halichondriidae, J. Org.
Chem. 53 (1988) 5446e5453.
[16] K. Bartik, J.C. Braekman, D. Daloze, C. Stoller, J. Huysecom, G. Vandevyver,
R. Ottinger, Topsentins, new toxic bisindole alkaloids from the marine sponge
Topsentia genitrix, Can. J. Chem. 65 (1987) 2118e2121.
[17] S. Sakemi, H.H. Sun, Nortopsentins A, B, and C. Cytotoxic and antifungal
imidazolediylbis[indoles] from the sponge Spongosorites ruetzleri, J. Org.
Chem. 56 (1991) 4304e4307.
4.11. Statistical significance
[18] B. Jiang, X.H. Gu, Syntheses and cytotoxicity evaluation of bis(indolyl)thiazole,
bis(indolyl)pyrazinone and bis(indolyl)pyrazine: analogues of cytotoxic ma-
rine bis(indole) alkaloid, Bioorg. Med. Chem. 2 (2000) 363e371.
[19] X.H. Gu, X.Z. Wan, B. Jiang, Syntheses and biological activities of bis(3-indolyl)
thiazoles, analogues of marine bis(indole)alkaloid nortopsentins, Bioorg. Med.
Chem. Lett. 9 (1999) 569e572.
[20] W.N. Xiong, C.G. Yang, B. Jiang, Synthesis of novel analogues of marine indole
alkaloids: mono(indolyl)-4-trifluoromethylpyridines and bis(indolyl)-4-
trifluoromethylpyridines as potential anticancer agents, Bioorg. Med. Chem.
9 (2001) 1773e1780.
[21] S.K.C. Devi, Chemistry and Bioactivity of Compounds from Marine Organisms
and Their Analogs, Ph.D. dissertation, Univ. of Kerala, India, Nov 1999.
[22] T.F.A.F. Reji, K.N. Rajasekharan, Synthesis of 3-(2,4-diaminothiazol-5-oyl)in-
doles, J. Saudi Chem. Soc. 12 (2008) 269e274.
[23] K.G. Sreejalekshmi, S.K.C. Devi, K.N. Rajasekharan, An efficient protocol for
solid phase aminothiazole synthesis, Tetrahedron Lett. 47 (2006) 6179e6182.
Statistical significance was evaluated using Graphpad Prism
software. All the data were expressed as mean ꢀ SD and P value
<0.05 was considered significant.
Conflicts of interest
The authors declare that there are no conflicts of interest.
Acknowledgment
The authors are thankful to the financial support by the
Department of Biotechnology and Department of Science and

M. Juneja et al. / European Journal of Medicinal Chemistry 63 (2013) 474e483
483
[24] T.F.A.F. Reji, S.L. Manju, K.N. Rajasekharan, Synthesis of bis(arylamino)thai-
zoloylindoles as novel analogs of dendrodoine, Indian J. Chem. 49B (2010)
323e326.
[25] R. Binu, S. Deepa, K.N. Rajasekharan, Synthesis of 2,4-diamino-5-
cinnamoylthiazoles and their attempted cyclization, Synth. Commun. 28
(1998) 3625e3632.
antimitotic mechanism of E7974, a novel analogue of the marine sponge
natural product hemiasterlin, Mol. Cancer Ther. 8 (2009) 2852e2860.
[39] E.A. Guzman, J.D. Johnson, M.K. Carrier, C.I. Meyer, T.P. Pitts, S.P. Gunasekera,
A.E. Wright, Selective cytotoxic activity of the marine-derived batzelline
compounds against pancreatic cancer cell lines, Anticancer Drugs 20 (2009)
149e155.
[26] M.T. Huang, Z.Y. Wang, C.A. Georgiadis, J.D. Laskin, A.H. Conney, Inhibitory
effects of curcumin on tumor initiation by benzo[a]pyrene and 7,12-
dimethylbenz[a]anthracene, Carcinogenesis 13 (1992) 2183e2186.
[27] M.A. Azuine, S.V. Bhide, Chemopreventive effect of turmeric against stomach
and skin tumors induced by chemical carcinogens in Swiss mice, Nutr. Cancer
17 (1992) 77e83.
[28] M.A. Azuine, S.V. Bhide, Protective single/combined treatment with betel leaf
and turmeric against methyl (acetoxymethyl) nitrosamine-induced hamster
oral carcinogenesis, Int. J. Cancer 51 (1992) 412e415.
[40] V.M. Dembitsky, T.A. Gloriozova, V.V. Poroikov, Novel antitumor agents:
marine sponge alkaloids, their synthetic analogs and derivatives, Mini Rev.
Med. Chem. 5 (2005) 319e336.
[41] S. Deslandes, S. Chassaing, E. Delfourne, Marine pyrrolocarbazoles and ana-
logues: synthesis and kinase inhibition, Mar. Drugs 7 (2009) 754e786.
[42] J. Kluza, M.A. Gallego, A. Loyens, J.C. Beauvillain, J.M. Sousa-Faro, C. Cuevas,
P. Marchetti, C. Bailly, Cancer cell mitochondria are direct proapoptotic targets
for the marine antitumor drug lamellarin D, Cancer Res. 66 (2006) 3177e3187.
[43] C. Ballot, J. Kluza, S. Lancel, A. Martoriati, S.M. Hassoun, L. Mortier, J.C. Vienne,
G. Briand, P. Formstecher, C. Bailly, R. Neviere, P. Marchetti, Inhibition of
mitochondrial respiration mediates apoptosis induced by the anti-tumoral
alkaloid lamellarin D, Apoptosis 15 (2010) 769e781.
[44] C. Ballot, J. Kluza, A. Martoriati, U. Nyman, P. Formstecher, B. Joseph, C. Bailly,
P. Marchetti, Essential role of mitochondria in apoptosis of cancer cells
induced by the marine alkaloid lamellarin D, Mol. Cancer Ther. 8 (2009)
3307e3317.
[29] M. Nagbhusan, S.V. Bhide, Curcumin as an inhibitor of cancer, J. Am. Coll. Nutr.
11 (1992) 192e198.
[30] B.B. Aggarwal, A. Kumar, A.C. Bharti, Anticancer potential of curcumin: pre-
clinical and clinical studies, Anticancer Res. 23 (2003) 363e398.
[31] G. Bar-Sela, R. Epelbaum, M. Schaffer, Curcumin as an anti-cancer agent: re-
view of the gap between basic and clinical applications, Curr. Med. Chem. 17
(2010) 190e197.
[32] A. Casapullo, G. Bifulco, I. Bruno, R. Riccio, New bisindole alkaloids of the
topsentin and hamacanthin classes from the Mediterranean marine sponge
Rhaphisia lacazei, J. Nat. Prod. 63 (2000) 447e451.
[33] J. Shin, Y. Seo, K.W. Cho, J.R. Rho, C.J. Sim, New bis(indole) alkaloids of the
topsentin class from the sponge Spongosorites genitrix, J. Nat. Prod. 62 (1999)
647e649.
[34] T.F.A.F. Reji, S.K.C. Devi, K.G. Sreejalekshmi, S.L. Manju, A. Bharathan,
M. Francis, S.K. Philip, K.N. Rajasekharan, Synthesis and cytotoxicity studies of
thiazole analogs of the anticancer marine alkaloid dendrodoine, Indian J.
Chem. 47B (2008) 1145e1150.
[35] P. Diana, A. Carbone, P. Barraja, G. Kelter, H.H. Fiebig, G. Cirrincione, Synthesis
and antitumor activity of 2,5-bis(30-indolyl)furans and 3,5-bis(30-indolyl)iso-
xazoles, nortopsentin analogues, Bioorg. Med. Chem. 18 (2010) 4524e4529.
[36] P. Diana, A. Carbone, P. Barraja, A. Montalbano, A. Martorana, G. Dattolo,
O. Gia, L. Dalla Via, G. Cirrincione, Synthesis and antitumor properties of 2,5-
bis(30-indolyl)thiophenes: analogues of marine alkaloid nortopsentin, Bioorg.
Med. Chem. Lett. 17 (2007) 2342e2346.
[45] D.V. LaBarbera, K. Modzelewska, A.I. Glazar, P.D. Gray, M. Kaur, T. Liu,
D. Grossman, M.K. Harper, S.K. Kuwada, N. Moghal, C.M. Ireland, The marine
alkaloid naamidine A promotes caspase-dependent apoptosis in tumor cells,
Anticancer Drugs 20 (2009) 425e436.
[46] S.N. Fedorov, A.M. Bode, V.A. Stonik, I.A. Gorshkova, P.C. Schmid,
O.S. Radchenko, E.V. Berdyshev, Z. Dong, Marine alkaloid polycarpine and its
synthetic derivative dimethylpolycarpine induce apoptosis in JB6 cells
through p53- and caspase 3-dependent pathways, Pharm. Res. 21 (2004)
2307e2319.
[47] S. Reddy, B.B. Aggarwal, Curcumin is a non-competitive and selective inhibitor
of phosphorylase kinase, FEBS Lett. 341 (1994) 19e22.
[48] S. Singh, B.B. Aggarwal, Activation of transcription factor NF-kappa B is sup-
pressed by curcumin (diferuloylmethane), J. Biol. Chem. 270 (1995) 24995e
25000.
[49] C.Y. Wang, M.W. Mayo, A.S. Baldwin Jr., TNF- and cancer therapy-induced
apoptosis: potentiation by inhibition of NF-kappaB, Science 274 (1996)
784e787.
[37] P. Diana, A. Carbone, P. Barraja, A. Martorana, O. Gia, L. DallaVia,
G. Cirrincione, 3,5-Bis(30-indolyl)pyrazoles, analogues of marine alkaloid
nortopsentin: synthesis and antitumor properties, Bioorg. Med. Chem. Lett.
17 (2007) 6134e6137.
[50] A.I. Vogel, A Text Book of Practical Organic Chemistry, English Language Book
Society and Longman, London, 1978. p. 794.
[51] T.F.A.F. Reji, Studies on Bioactive Natural Products Analogs, Ph.D. dissertation,
Univ. of Kerala, India, Jun. 2004.
[38] G. Kuznetsov, K. TenDyke, M.J. Towle, H. Cheng, J. Liu, J.P. Marsh, S.E. Schiller,
M.R. Spyvee, H. Yang, B.M. Seletsky, C.J. Shaffer, V. Marceau, Y. Yao, E.M. Suh,
S. Campagna, F.G. Fang, J.J. Kowalczyk, B.A. Littlefield, Tubulin-based
[52] R. Binu, K.K. Thomas, G.C. Jenardanan, K.N. Rajasekharan, Synthesis and
cyclization of 1-(N-nitroamidino)thioureas to 2,4-diaminothiazoles, Org. Prep.
Procedures Int. 30 (1998) 93e96.