Structural elaboration of a natural product: Identification of 3,3′-diindolylmethane aminophosphonate and urea derivatives as potent anticancer agents

Article abstract of DOI:10.1002/cmdc.201300273

An approach involving rational structural elaboration of the biologically active natural product diindolylmethane (DIM) with the incorporation of aminophosphonate and urea moieties toward the discovery of potent anticancer agents was considered. A four-step approach for the synthesis of DIM aminophosphonate and urea derivatives was established. These novel compounds showed potent anticancer activities in two representative κidney and colon cancer cell lines, low toxicity to normal cells, higher potency than the parent natural product DIM and etoposide, and potent inhibition of cancer cell migration. Biophysical and immunological studies, including DAPI nuclear staining, western blot analysis with apoptotic protein marκers, flow cytometry, immunocytochemistry, and comet assays of the two most potent compounds revealed good efficacies in apoptosis and DNA damage. It was found that downregulation of nuclear factor κB (NF-κB p65) could be an important mode of action in apoptosis, and the two most potent derivatives were found to be more potent than parent compound DIM in the down-regulation of NF-κB. Our results show the importance of structural elaboration of DIM by rational incorporation of aminophosphonate and urea moieties to produce potent anticancer agents; they also suggest that this approach using other structurally simple bioactive natural products as scaffolds holds promise for future drug discovery and development.

Full text of DOI:10.1002/cmdc.201300273

CHEMMEDCHEM
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DOI: 10.1002/cmdc.201300273
Structural Elaboration of a Natural Product: Identification
of 3,3’-Diindolylmethane Aminophosphonate and Urea
Derivatives as Potent Anticancer Agents
[
a]
[b]
[a]
[a]
Somnath Kandekar, Ranjan Preet, Maneesh Kashyap, Renu Prasad M. U.,
[b]
[b]
[b]
[b]
Purusottam Mohapatra, Dipon Das, Shakti Ranjan Satapathy, Sumit Siddharth,
[a]
[b]
[b]
[a]
Vaibhav Jain, Maitrayee Choudhuri, Chanakya N. Kundu,* Sankar K. Guchhait,* and
[a]
Prasad V. Bharatam
An approach involving rational structural elaboration of the
biologically active natural product diindolylmethane (DIM) with
the incorporation of aminophosphonate and urea moieties
toward the discovery of potent anticancer agents was consid-
ered. A four-step approach for the synthesis of DIM amino-
phosphonate and urea derivatives was established. These
novel compounds showed potent anticancer activities in two
representative kidney and colon cancer cell lines, low toxicity
to normal cells, higher potency than the parent natural prod-
uct DIM and etoposide, and potent inhibition of cancer cell mi-
gration. Biophysical and immunological studies, including DAPI
nuclear staining, western blot analysis with apoptotic protein
markers, flow cytometry, immunocytochemistry, and comet
assays of the two most potent compounds revealed good effi-
cacies in apoptosis and DNA damage. It was found that down-
regulation of nuclear factor kB (NF-kB p65) could be an impor-
tant mode of action in apoptosis, and the two most potent de-
rivatives were found to be more potent than parent com-
pound DIM in the down-regulation of NF-kB. Our results show
the importance of structural elaboration of DIM by rational in-
corporation of aminophosphonate and urea moieties to pro-
duce potent anticancer agents; they also suggest that this ap-
proach using other structurally simple bioactive natural prod-
ucts as scaffolds holds promise for future drug discovery and
development.
Introduction
Natural products play a key role in healthcare. They continued
to be a major source of inspiration in drug discovery. About
therapeutic agents such as morphinanes, benzomorphanes,
[2]
and methadone. Another well-known example is nakijiqui-
[3]
40% of all marketed drugs are either natural products or their
none; the compounds of this class are inhibitors of Her2/Neu
receptor tyrosine kinases. During the study of these natural
products as inhibitors of a new set of receptor tyrosine kinases,
it was found that none of the natural nakijiquinones exhibited
significant inhibitory activity, though some of their analogues/
derivatives showed activity in the low micromolar range. Inves-
tigation of the podophyllotoxin class of natural products even-
tually led to the development of semisynthetic derivatives eto-
semisynthetic derivatives, while about 80% of all antibacterial
drugs and 60% of all anticancer drugs are these types of com-
[
1]
pounds. The use of herbal medicine in health care since an-
cient times has rooted the screening of natural extracts in the
discovery of natural bioactive agents. As an extension, the bio-
logical screening of analogues, structurally simplified com-
pounds inspired by natural products, has been found even
more effective than screening of natural products alone. A
popular example of this is morphine; its derivatization led to
the development of potent analgesics such as codeine and
etorphine, and structural simplification led to exploration of
[4]
poside and teniposide as efficient anticancer drugs. In light
of these examples, derivatization of bioactive complex natural
products isolated from natural sources or synthesized via mul-
tiple steps is often considered. However, the type of chemistry
that the natural products can undergo for derivatization or the
comprehensive multistep synthetic approach frequently limits
the preparation of analogues with relevant moieties incorpo-
rated or the generation of suitable libraries required for struc-
ture–activity relationship studies. Recently, an approach toward
structural simplification of bioactive natural products, which
+
[
a] S. Kandekar, M. Kashyap, R. P. M. U., V. Jain, Dr. S. K. Guchhait,
Prof. P. V. Bharatam
National Institute of Pharmaceutical Education & Research (NIPER)
Sector 67, S.A.S. Nagar (Mohali), Punjab 160062 (India)
E-mail: skguchhait@niper.ac.in
+
[
b] R. Preet, P. Mohapatra, D. Das, S. R. Satapathy, S. Siddharth, M. Choudhuri,
Dr. C. N. Kundu
School of Biotechnology
Kalinga Institute of Industrial Technology (KIIT) University
Campus-11, Patia, Bhubaneswar, Odisha 751024 (India)
E-mail: cnkundu@gmail.com
can be completed through simple multicomponent syntheses,
[4a,5]
has shown much promise.
Nevertheless, this approach may
completely change the pharmacodynamic as well as pharma-
cokinetic profiles, which are also important components of the
resulting class of compounds. In this regard, the pharmacologi-
+
[
] These authors contributed equally to this work.
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cally relevant elaboration of structurally simple bioactive natu-
tion of vinblastine with a urea moiety at the C-20 position was
[
6]
[17]
ral products can be an important strategy.
,3’-Diindolylmethane (DIM) is a biologically active and
abundant dietary compound found in Brassica genus vegeta-
found to enhance anticancer activity. The anticancer and cy-
3
toprotective activities of flavagline are known to be improved
[18]
by incorporation of an additional urea moiety.
Sorafenib,
[
7]
bles such as broccoli, cabbage, and brussels sprouts. It exhib-
used in the treatment of renal and hepatocellular carcinoma,
possesses a urea moiety which plays an important role in the
[8]
its remarkable antitumor activities against several tumors and
is presently in clinical trials for the treatment of prostate
[
19]
inhibition of Raf kinase.
The a-aminophosphonate deriva-
[
9]
[20]
cancer. The anticancer activity of DIM has been found to be
tives of carbazole also exhibit good anticancer activities.
[21]
associated with down-regulation of the nuclear factor kB
Aminophosphonate derivatives of pyridothiazole and thio-
[
10,11]
[22]
(
NF-kB) transcription factor.
The inhibitory activities of DIM
urea-containing pseudopeptide
possess potent antitumor
[12]
for angiogenesis and cancer invasion are also known, and
activities as well. Furthermore, the incorporation of urea and
a-aminophosphonates as additional moieties on scaffolds are
also known to improve the pharmacokinetic profiles of thera-
peutic agents. Based on all these aspects, DIM aminophospho-
nate and urea derivatives were evaluated as potential anticanc-
er agents that could possibly possess enhanced activity over
the parent compound, DIM.
[13]
DIM has been found to possess leishmanicidal activity. Diin-
dolylalkane and their derivatives are found in bioactive metab-
[
14]
olites of terrestrial and marine origin. Several members of
the indole class of anticancer natural products (Figure 1):
vibrindole A, bisindolylmaleimide (e.g., enzasturin), and indolo-
[
2,3-a]carbazole (e.g., rebeccamycin, arcyriaflavins A–D, and
[
15]
staurosporine) structurally resemble DIM.
In the present study, novel DIM aminophosphonate and
urea derivatives that possess potent anticancer activities in
kidney and colon cancer cells, low toxicity to normal cells, and
potent inhibition in the migration of cancer cells were ex-
plored. Two of these synthesized compounds were found to
be the most potent anticancer agents and exhibited higher ac-
tivities than parent compound DIM and the clinically used anti-
cancer drug etoposide. Biochem-
For possible enhancement of therapeutic activities, the in-
corporation of various pharmaceutically relevant moieties into
important scaffolds is common. In this context, the incorpora-
tion of aminophosphonate, especially its use as a bioisosteric
replacement for amino acids, and the inclusion of urea moiet-
[
16]
ies are valuable in drug discovery research. The derivatiza-
ical and immunological tech-
niques, including DAPI (4’-6-di-
amidino-2-phenylindole) nuclear
staining, expression of represen-
tative apoptotic protein markers
(
caspase-3, PARP cleavage, and
p53), caspase-3 staining (immu-
nocytochemistry), flow cytome-
try, and comet assays revealed
the activities of the investigated
compounds in apoptosis and
DNA damage. A luciferase-based
promoter assay indicated the
possible role of NF-kB in the
apoptotic, anticancer, and anti-
metastatic activities of these DIM
aminophosphonate and urea de-
rivatives.
Results and Discussion
Chemistry
For the synthesis of alkylamino-
phosphonate and alkylurea de-
rivatives of DIM,
method was
Scheme 1). This methodology
a
four-step
established
(
involved convenient preparation
of 2,2-bis(indol-3-yl)nitroethanes
and their required derivatization
toward the preparation of de-
Figure 1. Examples of bisindole-containing natural/synthetic anticancer agents and therapeutic agents/drugs that
possess aminophosphonate or urea as a pharmacologically important moiety. Based on these existing examples,
DIM aminophosphonate and urea derivatives were postulated as potential anticancer agents.
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Biological studies
Antiproliferative activities of DIM aminophosphonates (5a–e)
and ureas (6a–e)
To evaluate the effects of DIM aminophosphonate and urea
derivatives (5a–e and 6a–e) on anchorage-dependent cell via-
bility, an MTT assay was performed using representative kidney
cancer cell (HEK 293T) and normal kidney epithelial (Vero) cell
[6a]
lines. Cell viabilities were measured at different time periods
24, 48, and 72 h) after treating cells with the investigated
(
compounds at various concentrations (Figure 3a and Table 1).
Table 1. Cytotoxicities of investigated compounds in normal (Vero) and
cancer (HEK 293T) cells.
[
a]
[a]
Compd
LC50 [mm]
Compd
LC50 [mm]
Vero
HEK 293T
Vero
HEK 293T
5
5
5
5
5
a
b
c
d
e
58Æ2
61Æ2
66Æ2
56Æ3
55Æ2
61Æ2
22Æ2
19Æ3
23Æ1
18Æ2
13Æ2
44Æ2
6a
6b
6c
6d
6e
56Æ2
54Æ2
52Æ2
58Æ2
58Æ2
25Æ2
28Æ2
18Æ2
23Æ2
14Æ2
[
b]
DIM
Scheme 1. Synthesis of DIM aminophosphonate and urea derivatives 5a–
e and 6a–e. Reagents and conditions: a) NH
b) NBS (10 mol%), CH Cl , 408C, 16 h, 67%; c) Zn dust, 6n HCl, EtOH, RT, 1 h;
, Mg(ClO
(anhyd), RT, 1 h, 50–59%.
4
OAc, AcOH, 1108C, 4 h, 91%;
[a] Lethal concentrations required to kill 50% of cells in culture were de-
termined after incubation with test compounds for 48 h; data represent
the mean ÆSD of three independent experiments. [b] Diindolylmethane
(DIM) was used as a reference agent.
2
2
2
2
d) R CHO, H(O)P(OEt)
CH Cl
2
4 2
) (10 mol%), EtOH, 508C, 46–56%; e) R NCO,
2
2
signed compounds for investigation. 3-(2’-Nitrovinyl)indole (2),
a precursor for the synthesis of 2,2-bis(indol-3-yl)nitroethane
No significant cell death was observed after 48 h exposure
with tested compounds. DIM aminophosphonate (5e) and DIM
urea (6e) derivatives were found to be the most potent anti-
proliferative agents, while showing poor cytotoxicities in
normal cells after 48 h of treatment (Table 1). The cytotoxic ac-
tivities of these agents (5e and 6e), in comparison with parent
DIM, were studied. Accordingly, assays were performed after
exposure with varied concentrations of compounds 5e, 6e,
and DIM. Cell viability decreased in a dose-dependent manner.
In HEK 293T cells, the viability was significantly lower than in
Vero cells. Interestingly, DIM aminophosphonate (5e) and DIM
urea (6e) exhibited more potent antiproliferative activities,
with LC50 values of 13 and 14 mm, respectively, compared with
DIM (LC =44 mm). In addition, these DIM derivatives and DIM
(
3), was prepared by Henry aldol condensation of indole-3-car-
[
23]
boxaldehyde (1) with nitromethane.
Michael addition of
3-(2’-nitrovinyl)indole with NH-free/substituted indole by
N-bromosuccinamide (NBS) catalysis produced 2,2-bis(indol-3-
[
24]
yl)nitroethane (3) in good yields. The reduction of 2,2-bis(in-
dol-3-yl)nitroethane (3) to 2,2-di(indol-3-yl)ethanamine (4) was
found to be difficult. Various approaches were attempted for
this reduction, such as catalytic hydrogenation using hydrogen,
[
25]
[26]
transfer hydrogenation, use of hydride reducing agents,
[
27]
and Clemmensen reduction using zinc and HCl. Therefore,
the process of reduction of 2,2-bis(indol-3-yl)nitroethane (3) to
2,2-di(indol-3-yl)ethanamine (4) and its in situ derivatization
50
was considered. After several experiments, it was found that
Clemmensen reduction using zinc and HCl of compound 3 to
amine 4, followed by quick extraction using ethyl acetate with-
out chromatographic purification and Kabachnik–Fields reac-
itself exhibited similar cytotoxicities in normal Vero cells, and
no LC50 values were observed, even after treatment with com-
pounds at 50 mm concentrations.
To further confirm the antiproliferative activity of the investi-
gated compounds in cancer cells, a clonogenic cell survival
assay was performed. This assay is a long-term cell viability
test that determines the ability of a single cell to proliferate in-
definitely, thereby retaining its reproductive ability to form
a colony or a clone. DIM, DIM aminophosphonate (5e), and
DIM urea (6e) showed decreased colony formation in a concen-
tration-dependent manner with LC50 (50% decrease in cultured
colonies) values of 31, 7, and 8 mm, respectively (Figure 3b).
With regard to the capacity to impede colony formation, the
enhanced potency of DIM derivatives relative to DIM was ob-
served and found to be consistent with MTT assay results. The
[
28]
tion provided DIM aminophosphonates (5) in good yields.
Likewise, Clemmensen reduction of compound 3, extraction,
and subsequent reaction of 4 with isocyanate led to the forma-
tion of DIM ureas (6) in good yields. Following the established
synthetic approach, various relevant substituted title com-
pounds (5a–e and 6a–e) were prepared (Figure 2). DIM, which
was used as a control in the bioevaluation of investigated
compounds, was prepared by a known reaction of indole with
paraformaldehyde.
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veals that DIM derivatives not only inhibit kidney
cancer cells but also cause cytotoxic effects in colon
cancer cells in a similar manner.
Anti-migratory activities in HEK 293T cells
The phenomena of cancer cell growth relates to its
rapid and uncontrolled proliferation and migration.
To examine the inhibitory effect of DIM amino-
phosphonate (5e) and DIM urea (6e) on anchorage-
dependent cell migration, a wound healing assay
using HEK 293T cells was performed. After making
a wound, cancer cells were treated with compounds
5
e and 6e at their LC50 concentrations, 13 mm and
14 mm, respectively. The wound was inspected micro-
scopically over time. In untreated cells, migration oc-
curred, and the wound healed in 48 h (Figure 5).
However, in treated cells, migration was inhibited,
and after prolonged time period of treatment, wide-
spread apparent cell death was observed (Figure 5).
Therefore, this assay, in conjunction with the assays
as discussed above (clonogenic and MTT cell survival),
demonstrates that DIM aminophosphonate (5e) and
DIM urea (6e) possess potent inhibitory activities
toward colony formation, viability, and migration of
cancer cells.
Apoptosis studies
To determine the effect of DIM aminophosphonate
(
5e) and DIM urea (6e) on apoptosis in cancer cells,
the formation of chromatin condensation and nuclear
fragmentation in HEK 293T cells was analyzed using
Figure 2. Synthesized DIM aminophosphonates 5a–e and urea derivatives 6a–e.
a DAPI nuclear staining experimental method (Fig-
ure 6a). Cells at 60–70% confluence were treated
with investigated compounds at their LC50 concentra-
results of MTT and clonogenic assays indicate that DIM amino-
phosphonate and urea derivatives possess higher cytotoxic ac-
tivities, relatively low toxicities toward normal cells, and higher
potencies than DIM.
tions for 48 h, followed by nuclear staining. Increased chroma-
tin condensation was observed, as well as nuclear fragmenta-
tion and shrunken (apoptotic) nuclei in treated HEK 293T cells
relative to untreated cells (Figure 6a). The relative number of
apoptotic/non-apoptotic nuclei are presented in a bar diagram
(
Figure 6b, *p<0.05). To confirm the apoptosis caused by
compounds 5e and 6e, fluorescence-activated cell sorting
FACS) analysis was performed. The kidney cancer cells were
Comparison of DIM derivatives and etoposide in colon cancer
cells
(
Once cytotoxic activities of DIM aminophosphonate and urea
derivatives were established in a kidney cancer cell line, their
cytotoxic potential in another cancer cell line, HCT-116 (colon
cancer cells), was evaluated. The activities of investigated com-
pounds were compared with that of etoposide, a clinically
used anticancer drug. Accordingly, an MTT assay using HCT-116
cells was performed (Figure 4) in which the cells were treated
with increasing concentrations of etoposide and the potent
compounds 5e and 6e. After 48 h of treatment, cell viabilities
were measured. The LC50 values for both 5e and 6e were
treated with the investigated compounds at their respective
LC50 concentrations for 48 h and then analyzed using FACS by
staining with propidium iodide (Figure 6c,d). DNA content of
the cells at various phases of the cell cycle was measured by
Cell Quest software (Becton and Dickinson, CA). A high in-
crease in apoptosis was observed for treated cells, with ~60
and 61% apoptosis observed for compounds 5e and 6e, re-
spectively, whereas, little (~1%) apoptosis was observed for
untreated cells (Figure 6c). A histogram of the cell cycle profile
is presented in Figure 6d. Therefore, these results indicate that
HEK 293T cells, after treatment with investigated DIM amino-
phosphonate and urea derivatives, undergo significant apopto-
sis.
1
5 mm, whereas etoposide showed an LC50 value of 22 mm
Figure 4). Thus, the results showed that DIM derivatives had
greater cytotoxicity in cancer cells than etoposide. This also re-
(
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product of poly(ADP-ribose)
polymerase (PARP), and down-
regulation of NF-kB expression
are the majorly observed phe-
nomena for the apoptotic activi-
[29]
ties of anticancer agents. The
expression of caspase-3 was de-
termined by immunocytochem-
istry in HEK 293T cells after expo-
sure to test compounds at their
respective LC50 concentrations
for 48 h. Increased accumulation
of caspase-3 expression was ob-
served in treated cells relative to
control (Figure 7). In comparison
with untreated cells, after expo-
sure to DIM derivatives in kidney
cancer cells, the expressions of
tumor suppressor gene p53 and
caspase-3 increased, cleaved
products of PARP appeared, and
the expression of NF-kB de-
creased (Figure 8a). Together,
these results reveal that investi-
gated DIM aminophosphonate
and urea derivatives possess
apoptotic activities in kidney
cancer cells.
Figure 3. Antiproliferative activities of DIM, DIM aminophosphonate (5e) and DIM urea (6e); the activities for the
two most potent compounds are presented. a) MTT cell viability assay: Cells (HEK 293T and Vero) were treated
with DIM and investigated compounds at various concentrations for 48 h as described in the Experimental Sec-
tion. b) Clonogenic cell survival assay: HEK 293T cells were treated with DIM and the investigated compounds 5e
and 6e, according to a protocol described in the Experimental Section. Data shown are the mean ÆSD of three
different experiments.
Potential role of NF-kB (p65)
transcription factor in anticanc-
er activities
The parent natural product, DIM,
is known to exhibit anticancer activity via NF-kB down-regula-
[30,31]
tion.
In our study of apoptosis using western blot analysis
with protein markers for DIM aminophosphonate and urea de-
rivatives, NF-kB was found to be expressed at relatively lower
levels (Figure 8a). Therefore, we speculated that NF-kB could
play an important role in anticancer activities exhibited by
these investigated compounds. Among all isoforms of NF-kB,
[32]
p65 plays crucial role in cancer.
Accordingly, a luciferase-
based promoter assay was performed using NF-kB (p65). The
direct transcription factor activity of NF-kB (p65) was measured
after treatment of HEK 293T cells with compounds 5e and 6e
[29]
for 48 h following the reported method. The parent com-
pound, DIM, was also used in the assay for comparison of its
activity with that of investigated DIM aminophosphonate and
urea derivatives. The results are presented in Figure 8b. Com-
pounds 5e and 6e were found to induce ~4.0- and 3.8-fold
decreases in luciferase activities, respectively, while DIM caused
a 2.0-fold decrease. These results signify that the structurally
elaborated DIM aminophosphonate and urea derivatives are
more potent NF-kB (p65) down-regulating agents than parent
DIM and, moreover, they decreased not only the expression of
NF-kB (p65) at transcriptional level (Figure 8b) but also at
translational level (Figure 8a). These observations indicate that
Figure 4. Effects of DIM aminophosphonate (5e) and DIM urea (6e) on cell
survival in an anchorage-dependent manner. Comparison of anti-cell viability
(HCT-116 cells) for potent investigated compounds with etoposide (Et). Data
are the mean ÆSD of three different experiments.
For further studies of apoptosis, immunocytochemistry and
western blot analyses using representative apoptosis markers
were also performed. Overexpression of caspase-3 and cleaved
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aminophosphonate and urea derivatives as potent apoptotic
anticancer agents. A four-step method was established for
their synthesis. DIM aminophosphonate (5e) and DIM urea
(
6e) were found to be the most potent anticancer agents and
exhibited higher anticancer activities than parent natural prod-
uct DIM and etoposide (an anticancer drug). These compounds
showed antiproliferative activities in kidney (HEK 293T) and
colon (HCT-116) cancer cells and anti-migratory activities in
kidney (HEK 293T) cancer cells while exhibiting low toxicity to
normal cells (Vero). Relevant biophysical and immunological
techniques, including DAPI nuclear staining, western blot anal-
ysis with apoptotic protein markers, flow cytometry, immuno-
cytochemistry, and comet assays revealed good efficiencies for
potent compounds 5e and 6e in apoptosis and DNA damage.
It was found that down-regulation of NF-kB could play an im-
portant role in apoptosis, and compounds 5e and 6e were
more potent than parent natural product DIM. The results of
the present study represent the importance of structural elabo-
ration of bioactive natural product DIM with rational incorpora-
tion of aminophosphonate and urea moieties toward the dis-
covery of potent NF-kB-down-regulating apoptotic, anticancer,
and anti-metastatic agents. This should motivate researchers
to consider DIM or any other structurally simple bioactive nat-
ural product as important natural scaffolds for rational structur-
al elaboration in drug discovery research.
Experimental Section
Figure 5. Anchorage-dependent cell migration of HEK 293T cells. After
wound formation, cells were treated with DIM aminophosphonate (5e) and
DIM urea (6e) at their LC50 concentrations. Micrographs were taken at inter-
vals of 0, 12, 24, 36, and 48 h to evaluate the healing of the wound at 40ꢁ
magnification. Images shown are representative of three independent ex-
periments.
Chemistry
General: All reactants and reagents were obtained from commer-
cial sources and used without further purification unless otherwise
noted. N-Bromosuccinimide was used after recrystallization. NMR
spectra were recorded on a Bruker Avance DPX 400 MHz spectrom-
eter in CDCl /[D ]DMSO/CD OD solvents using TMS as an internal
3
6
3
standard. J values are given in Hz. Mass spectra were recorded
using an LC–MS-Finnigan MAT-LCQ (for ESI and APCI), Bruker-Maxis
the down-regulation of NF-kB (p65) can be an important
mode of action for anticancer activities exhibited by the inves-
tigated DIM-aminophosphonate and urea derivatives.
(for HRMS) mass spectrometers. IR spectra were recorded on a Nico-
let FT-IR Impact 410 instrument as thin film (neat). The reactions
were monitored by TLC (Merck, silica gel 60 F254). The purity of the
synthesized compounds was analyzed by HPLC (Shimadzu LC-6AD
system), using a Phenomenex RP-C18 column (250ꢁ4.60 mm), par-
Induction of DNA damage by DIM derivatives
À1
ticle size 5 mm, flow rate 1 mLmin , using H O/CH CN (60:40) for
2
3
To further explore the possible involvement of DNA damage
repair as a mechanism of action for the anticancer activities of
DIM aminophosphonate and urea derivatives, a comet assay
was carried out. In HEK 293T cells treated with compounds 5e
and 6e, the average comet length was found to be greater (6-
to 8-fold) relative to untreated cells (Figure 9A). This result in-
dicates the DNA damaging efficacy of the investigated com-
pounds.
compounds 5a–e, and H O/CH CN (50:50) for compounds 6a–e de-
2 3
tection at 220 nm and 254 nm. The purity of tested compounds
was found to be >95%. The investigated compounds were syn-
thesized as racemic mixtures.
3-(2-Nitrovinyl)indole (2): Nitromethane (3 mmol, 0.16 mL) was
added dropwise to a solution of indole-3-carboxaldehyde (1)
(1 mmol, 145 mg) and CH COONH (1 mmol, 77 mg) in CH COOH
3 4 3
(0.75 mL). The mixture was stirred at reflux for 4 h and then cooled
to room temperature. The resulting mixture was extracted with
EtOAc (2ꢁ25 mL), and the solution was washed with H O (3ꢁ
2
2
0 mL) and dried with anhydrous Na SO . The solvent was evapo-
2 4
Conclusions
rated under vacuum to provide NMR-pure 3-(2-nitrovinyl)-indole in
91% yield (164 mg). The compound was used in the subsequent
reaction without further chromatographic purification.
In conclusion, rational structural elaboration of DIM, a biologi-
cally active natural product, led to the discovery of novel DIM
ꢀ
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Diethyl (2,2-di(indol-3-yl)ethyla-
minophenmethylphosphonate
(
5d): A mixture of 2,2-di(indol-3-
yl)ethylamine (0.5 mmol, 137.5 mg)
4a), benzaldehyde (0.5 mmol,
.05 mL), diethyl phosphite
(
0
(
(
0.6 mmol, 0.07 mL), and Mg(ClO4)2
10 mol%, 11 mg) in EtOH (2 mL)
was stirred at 508C. After comple-
tion of the reaction as indicated by
TLC, the resulting mixture was ex-
tracted with EtOAc (2ꢁ25 mL). The
combined organic solution was
washed with H O (2ꢁ10 mL) and
2
brine (10 mL), dried with anhy-
drous Na SO , and concentrated
2
4
under reduced pressure. Column
chromatographic purification of
crude mixture on silica gel, eluting
with EtOAc/hexane, provided 5d
in 52% isolated yield (260 mg).
DIM aminophosphonate deriva-
tives 5a, 5b, 5c, and 5e were also
prepared following this procedure.
Diethyl 2-(3-indolyl)-2-(1-methyl-
indol-3-yl)ethylaminophenme-
Figure 6. Apoptosis induced by DIM aminophosphonate (5e) and DIM urea (6e) in HEK 293T cells. a) DAPI nuclear
staining: HEK 293T cells were treated with investigated compounds, and DAPI nuclear staining was performed ac-
cording to the protocol described in the Experimental Section. Micrographs were taken at 40ꢁ magnification.
b) Bar diagram represents normal (black) and apoptotic (grey) cells. Data represent the best of three individual ex-
periments. Statistical significance was determined by Student’s paired t test; *p<0.05. c) Quantitative analysis of
percentages of DNA in each phase of the cell cycle was done using Cell Quest software (Becton-Dickinson). d) His-
thylphosphonate
(5a):
Light-
brown semisolid (288 mg, 56%):
Rf =0.45 (EtOAc/n-hexane, 75:25);
IR (neat): n˜ max =3269, 2981, 1613,
À1
1
1
(
228, 1026, 969 cm
400 MHz, CDCl3): d=8.19 (brs,
NH), 7.48–7.42 (m, 2H), 7.29–7.21
m, 7H), 7.18–7.12 (m, 2H) 7.00–
;
H NMR
tograms of HEK 293T cell populations were measured by FACS using Cell Quest software. Apoptosis, G
M phases are indicated; data are the mean ÆSD of three different experiments.
1
, S, and
G
2
(
6
4
2
.93 (m, 3H), 6.81–6.75 (m, 1H),
.73–4.70 (m, 1H), 4.14–4.10 (m,
H), 3.95–3.88 (m, 2H), 3.81 (brs,
NH), 3.68–3.61 (m, 4H), 3.27–3.24 (m, 2H), 1.13 (t, J=8.0 Hz, 3H),
Representative experimental procedures
13
0
1
.98 ppm (t, J=6.9 Hz, 3H); C NMR (100 MHz, CDCl ): d=137.2,
36.6, 135.8, 128.7, 128.3 (2CH), 127.8, 127.2, 126.8 (2CH), 122.2,
3
2
,2-Di(indol-3-yl)-1-nitroethane (3a): A mixture of 3-(2-nitrovinyl)-
indole (2), (1 mmol, 188 mg), indole (1.3 equiv, 152 mg), and NBS
10 mol%, 17.7 mg) in CH Cl (2 mL) was stirred at 408C for 16 h.
121.8, 121.4, 119.6, 119.5, 119.0, 118.6, 117.2, 116.3, 115.8, 111.1,
109.1, 62.8, 62.7, 61.8 (J=152 Hz), 52.5, 34.1, 32.6, 16.3, 16.1 ppm;
+
+
(
MS (APCI): m/z (%): 516 (12) [M+H] ; HRMS-ESI: m/z [M+Na]
2
2
After completion of the reaction as indicated by TLC, the resulting
mixture was extracted with EtOAc (2ꢁ25 mL). The combined or-
calcd for C H N NaO P: 538.2230, found: 538.2234.
30
34
3
3
Diethyl 2-(3-indolyl)-2-(1-methylindol-3-yl)ethylamino-4-chloro-
phenmethylphosphonate (5b): Light-brown semisolid (263 mg,
ganic solution was washed with H O (2ꢁ10 mL) and brine (10 mL),
2
dried with anhydrous Na SO , and concentrated under reduced
2
4
4
2
8
8%): R =0.47 (EtOAc/n-hexane, 75:25); IR (neat): n˜ =3271,
f
max
pressure. Column chromatographic purification of the crude mix-
ture on silica gel, eluting with EtOAc/hexane provided 3a in 67%
isolated yield (203 mg). Compounds 3b and 3c were also prepared
following this procedure.
À1
1
982, 1614, 1228, 1023, 971 cm
;
H NMR (400 MHz, CDCl ): d=
3
.06–8.02 (m,1H), 7.61–7.01 (m, 12H), 6.84–6.78 (m, 1H), 4.71
(
1
s,1H), 4.06–3.84 (m, 4H), 3.69–3.66 (m, 4H), 3.24–3.22 (m, 2H),
.15 (s, 3H), 1.01 ppm (s, 3H); C NMR (100 MHz, CDCl ): d=137.3,
3
13
2
,2-Di(indol-3-yl)ethylamine (4a): 2,2-Di(indol-3-yl)-1-nitroethane
136.5, 133.4, 129.9, 129.8 (2CH), 128.4 (2CH), 126.8, 122.0, 121.9,
121.5, 119.5, 119.4, 119.2, 118.6, 117.7, 117.3, 116.0, 115.7, 111.1,
109.1, 62.9, 62.7, 61.4 (J=158 Hz), 52.9, 34.2, 32.7, 16.3, 16.1 ppm;
(3a) (1 mmol, 305 mg) was dissolved in EtOH (6 mL) to which 6n
HCl (3 mL) and Zn dust (10 equiv, 650 mg) were added. After stir-
ring for 1 h at room temperature (25–268C), the resulting mixture
was filtered. The filtrate was neutralized by dropwise addition of
saturated aqueous NaHCO3 solution and then extracted with
+
+
MS (APCI): m/z (%): 550 (3) [M+H] ; HRMS-ESI: m/z [M+Na]
calcd for C H ClN NaO P: 572.1840, found: 572.1841.
30
33
3
3
Diethyl 2-(3-indolyl)-2-(1-methylindol-3-yl)ethylamino-4-meth-
oxyphenmethylphosphonate (5c): Dark-brown semisolid (250 mg,
EtOAc (4ꢁ30 mL). The organic solution was washed with H O
2
(
10 mL) and dried with anhydrous Na SO . The evaporation of sol-
2 4
4
2
6
7
6%): R =0.42 (EtOAc/n-hexane, 75:20); IR (neat): n˜ =3245,
f
m
a
x
vent under reduced pressure provided the crude residue, which
was directly used in the next reaction without column chromatog-
raphy. Compounds 4b and 4c were also prepared following this
procedure.
À1
978, 1248, 1028, 969 cm ; 1H NMR (400 MHz, CDCl ): d=7.49–
3
.99 (m, 9H), 6.89 (d, J=8.4 Hz, 2H), 6.85–6.77 (m, 3H), 4.72 (t, J=
.4 Hz, 1H), 4.09–3.90 (m, 7H), 3.80 (s, 3H), 3.71–3.65 (m, 1H),
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(
100 MHz, CD OD): 137.0 (2C),
3
1
(
1
(
34.0, 130.3 (2CH), 128.7, 128.0
2CH), 126.7 (2C), 122.0 (2CH),
20.9 (2CH), 118.8 (2CH), 118.1
2CH), 116.4 (2C), 110.9 (2CH), 63.3,
6
3.1, 60.1 (d, J=153 Hz), 51.7, 34.0,
3
1.3, 15.4, 15.2 ppm; MS (APCI): m/
+
z (%): 536 (31) [M+H] ; HRMS-ESI:
m/z
+
[M+Na]
calcd
for
C H ClN NaO P: 558.1684, found:
29
31
3
3
5
58.1687.
1
-Benzyl-3-(2,2-di(indol-3-yl)-
ethyl)urea (6c): Benzyl isocyanate
0.5 mmol, 0.062 mL) was added to
(
a solution of 2,2-di(indol-3-yl)ethyl-
amine (4a) (0.5 mmol, 135.5 mg) in
anhydrous CH Cl (2 mL). The mix-
2
2
ture was stirred at 25–268C for 1 h.
After completion of the reaction as
indicated by TLC, the resulting
mixture was extracted with EtOAc
(2ꢁ25 mL). The combined organic
solution was washed with H O (2ꢁ
2
1
0 mL) and brine (10 mL), dried
with anhydrous Na SO , and con-
2
4
centrated under reduced pressure.
Column chromatographic purifica-
tion of the crude mixture on silica
gel, eluting with EtOAc/hexane,
provided 6c in 51% isolated yield
Figure 7. Caspase-3 expression: HEK 293T cells were treated with DIM aminophosphonate (5e) and DIM urea (6e),
and staining was performed with anti-caspase-3 antibody and DAPI as described in the Experimental Section. Mi-
crographs were taken at 40ꢁ magnification. The fields of DAPI-stained nuclei were merged with respective fields
of caspase-3 staining with the help of IMAGE J software. Micrographs shown are the best of three individual ex-
periments.
(
208 mg). DIM urea derivatives 6a,
b, 6d, and 6e were also pre-
pared following this procedure.
6
1
-(2-(Indol-3-yl)-2-(1-methylindol-
3
3
.34–3.23 (m, 2H), 1.28 (t, J=7.0 Hz, 3H), 1.22 ppm (t, J=7.0 Hz,
1
3
3-yl)ethyl)-3-benzylurea (6a): Brown semisolid (248 mg, 59%):
R =0.45 (EtOAc/n-hexane, 60:40); IR (neat): n˜ =3401, 2976, 1628,
H NMR (400 MHz, CDCl ): d=8.32 (brs, NH),
.57–7.50 (m, 2H), 7.21–7.16 (m, 4H), 7.08–6.96 (m, 7H), 6.82 (s,
1H), 6.65 (s, 1H), 4.73 (brs, NH), 4.65–4.61 (m, 1NH+ 1CH), 4.08 (s,
H), 3.85–3.81 (m, 2H), 3.53 ppm (s, 3H); C NMR (100 MHz,
CDCl ): d=157.6, 139.0, 137.2, 136.5, 128.5 (2CH), 128.1, 127.3
2CH), 127.2, 126.9, 126.5, 122.1, 122.0, 121.6, 119.8, 119.6, 119.3,
H); C NMR (100 MHz, CDCl ): d=158.6, 138.7, 137.2, 136.6, 128.7
3
(
1
2CH), 128.4, 127.8, 127.2, 126.9, 123.0, 121.8, 121.4, 119.7, 119.5,
19.2, 117.2, 116.3, 115.8, 113.8 (2CH), 109.2, 62.8, 62.6, 61.8 (J=
f
max
À1
1
1
7
275, 1051 cm
;
3
1
5
54 Hz), 54.7, 52.8, 34.1, 32.7, 16.4, 16.4 ppm; MS (APCI): m/z (%):
+
+
46 (2) [M+H] ; HRMS-ESI: m/z [M+Na]
calcd for
13
2
C H N NaO P: 568.2336, found: 568.2339.
3
1
36
3
4
3
Diethyl
2,2-di(3-indolyl)ethylaminophenmethylphosphonate
(
(5d): Light-brown semisolid (260 mg, 52%): R =0.50 (EtOAc/
f
118.8, 117.0, 115.4, 111.2, 109.2, 44.9, 44.5, 34.6, 32.6 ppm; MS
n-hexane, 80:20); IR (neat): n˜ =3228, 2926, 1634, 1238, 1024,
+
+
max
(APCI): m/z (%): 423 (2) [M+H] ; HRMS-ESI: m/z [M+Na] calcd
À1
1
9
7
7
2
6
69 cm ; H NMR (400 MHz, CD OD): d=7.32–7.30 (m, 1H), 7.26–
3
for C H N NaO: 445.1999, found: 445.1999.
27
26
4
.19 (m, 8H), 6.98–6.88 (m, 4H), 6.80–6.77 (m, 2H), 4.60 (t, J=
.2 Hz, 1H), 4.09–4.04 (m, 1H), 3.79–3.75 (m, 2H), 3.68- 3.64 (m,
H), 3.14 (d, J=7.2 Hz, 2H), 1.00 (t, J=7.0 Hz, 3H), 0.88 ppm (t, J=
1
-(2-(Indol-3-yl)-2-(1-methylindol-3-yl)ethyl)-3-(4-methoxyphe-
nyl)urea (6b): Pale-brown semisolid; (236 mg, 54%): R =0.42
(EtOAc/n-hexane, 60:40); IR (neat): n˜ max =3435, 2913, 1632,
212 cm ; H NMR (400 MHz, CDCl ): d=8.35 (brs, NH), 7.57–7.54
3
m, 2H), 7.25–6.62 (m, 12H), 6.25 (brs, NH), 4.84 (brs, NH), 4.68 (m,
H), 3.95–3.93 (m, 2H), 3.70 (s, 3H), 3.60 ppm (s, 3H); C NMR
(100 MHz, CDCl ): d=156.6, 154.9, 137.2, 136.5, 130.8, 127.3, 126.9,
3
24.2, 122.1, 121.6, 120.3, 119.6, 119.3 (2CH), 119.2, 118.9, 117.0,
f
1
3
.9 Hz, 3H); C NMR (100 MHz, CD OD): d=137.0 (2C), 134.9, 128.6
3
À1
1
1
(
1
(
(
2CH), 128.1 (2CH), 127.7, 126.8 (2C), 121.9 (2CH), 120.8 (2CH), 118.6
2CH), 118.0 (2CH), 116.5 (2C), 110.8 (2CH), 62.9, 62.7, 60.5 (J=
1
3
1
53 Hz), 51.7, 33.7, 15.1, 15.0 ppm; MS (APCI): m/z (%): 502 (30)
+
+
[M+H] ; HRMS-ESI: m/z [M+Na] calcd for C H N NaO P:
29
32
3
3
1
1
5
24.2073, found: 524.2077.
2,2-di(3-indolyl)ethylamino-4-chlorophenmethylphos-
15.8, 115.5, 114.3 (2CH), 111.2, 109.2, 55.4, 44.7, 34.4, 32.6 ppm;
MS (APCI): m/z (%): 439 (1) [M+H] ; HRMS-ESI: m/z [M+Na]
calcd for C H N NaO : 461.1948, found: 461.1948.
Diethyl
phonate (5e): Light-brown semisolid (272 mg, 51%): R =0.52
+
+
f
27
26
4
2
(
EtOAc/n-hexane, 80:20); IR (neat): n˜ =3243, 2956, 1625, 1238,
max
À1
1
1-Benzyl-3-(2,2-di(indol-3-yl)ethyl)urea (6c): Brown semisolid;
208 mg, 51%): R =0.52 (EtOAc/n-hexane, 70:30); IR (neat): n˜
403, 2927, 1642, 1548, 1241, 1093 cm
CD OD): d=7.54 (d, J=7.7 Hz, 2H), 7.32–7.02 (m, 11H), 6.91 (t, J=
9
7
1
2
1
72 cm ; H NMR (400 MHz, CD OD): d=7.38 (t, J=7.7 Hz, 2H),
3
(
3
=
.30–7.28 (m, 2H), 7.22–7.14 (m, 4H), 7.07–7.01 (m, 3H), 6.98 (s,
H), 6.78 (t, J=7.6 Hz, 2H), 4.68 (t, J=7.2 Hz, 1H), 4.09–4.00 (m,
H), 3.93–3.86 (m, 2H), 3.72–3.68 (m, 1H), 3.23 (d, J=7.2 Hz, 2H),
f
max
À1
1
;
H NMR (400 MHz,
3
13
7.0 Hz, 2H), 4.70 (t, J=6.9 Hz, 1H), 4.24 (s, 2H), 3.91 ppm (d, J=
.08 (t, J=7.0 Hz, 3H), 0.98 ppm (t, J=7.0 Hz, 3H); C NMR
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1
26.7 (2CH), 126.5 (2CH), 122.7, 121.9, 120.8, 118.8, 118.0, 116.4,
1
16.2, 111.4, 111.1(2C), 110.8, 54.8, 44.5, 43.2, 34.7 ppm; MS (APCI):
+
+
m/z (%): 439 (2) [M+H] ; HRMS-ESI: m/z [M+Na] calcd for
C H N NaO : 461.1948, found: 461.1948.
27
26
4
2
Biology
Maintenance and treatment of cell lines: HEK 293T (kidney), HCT-116
(colon), and Vero (normal) cell lines were cultured in monolayers
and maintained in DMEM (Dulbecco’s modified eagle medium)
À1
with 10% fetal bovine serum (FBS), 100 UmL penicillin–strepto-
[30]
mycin and 1.5 mm glutamine.
All other cell culture reagents
were purchased from HIMEDIA (India). Cells were allowed to grow
in a humidified atmosphere of 5% CO at 378C. After reaching 70–
2
8
0% confluence, cells were treated with the indicated concentra-
tions of different compounds dissolved in sterile DMSO. MTT and
etoposide were purchased from Sigma–Aldrich (St. Louis, MO,
USA). Cells were treated at various time intervals (24, 48, and 72 h)
with the addition of freshly prepared working concentrations of
compounds as shown.
MTT assay: To investigate the cytotoxicity of investigated com-
pounds in kidney cancer (HEK 293T), colon cancer (HCT-116), and
normal kidney (Vero) cells, an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-
[33]
diphenyltetrazolium bromide] assay was performed. This is a col-
orimetric assay that measures the reduction of MTT (yellow) by mi-
tochondrial succinate dehydrogenase. The MTT enters the cells and
passes into the mitochondria, where it is reduced to an insoluble,
dark purple formazan product. The cells are then solubilized with
an organic solvent (e.g., isopropanol) and the released, solubilized
formazan reagent is measured spectrophotometrically. As reduc-
tion of MTT can only occur in metabolically active cells, the level of
activity is thus a measure of the viability of the cells. For our study,
cells were seeded in triplicate for different time periods (24, 48,
and 72 h) and then treated with investigated compounds at con-
centrations ranging from 0 mm to 100 mm. After treatment, MTT
was added, and cells were incubated for 4–6 h for the formation of
formazan crystals. The resulting formazan crystals were then dis-
solved in organic solvent, leading to formation of purple color,
which was measured spectrophotometrically at a wavelength of
Figure 8. a) Western blot analysis of HEK 293T whole-cell lysate after treat-
ment with DIM aminophosphonate (5e) and DIM urea (6e). Cells were treat-
ed with compounds at their LC50 concentrations (5e: 13 mm and 6e: 14 mm)
for 48 h prior to western blot analysis. GAPDH served as a protein loading
control. Images shown are representative of three independent experiments.
b) Relative luciferase activity of NF-kB (p65; p5XIP10B plasmid-containing
NF-kB (p65) constructs) measured in HEK 293T cells is plotted as the mean
ÆSD of three different experiments. Statistical significance was determined
by the paired t test, *p<0.05.
1
3
6
.9 Hz, 2H); C NMR (100 MHz, CD OD): d=159.8, 139.8, 136.9,
3
1
28.1 (2CH), 127.0 (2C), 126.8 (2CH), 126.5 (2C), 122.1 (2CH), 120.8
(
2
2CH), 118.7 (2CH), 118.1 (2CH), 116.4 (2C), 110.8 (2CH), 44.68, 43.27,
5
70 nm using an ELISA reader (Tristar, Berthold Technologies, Ger-
+
9.29 ppm; MS (APCI): m/z (%): 409 (2) [M+H] ; HRMS-ESI: m/z
many). This experiment was performed in triplicate.
+
[M+Na] calcd for C H N NaO: 431.1842, found: 431.1842.
26 24 4
Clonogenic assay: The clonogenic cell survival assay is a long-term
cell viability assay that determines the ability of a single cell to pro-
liferate indefinitely, thereby retaining its reproductive ability to
form a colony or a clone. This assay was performed to examine the
anti-colony-forming potential of investigated DIM derivatives in
1
-(2,2-Di(indol-3-yl)ethyl)-3-(4-methoxyphenyl)urea (6d): White
semisolid; (233 mg, 55%): R =0.48 (EtOAc/n-hexane, 70:30); IR
f
À1
1
(
neat): n˜ max =3410, 2920, 1632, 1450, 1275 cm ; H NMR (400 MHz,
[
D ]DMSO): d=8.27 (brs, NH), 7.56 (d, J=7.6 Hz, 2H), 7.32 (d, J=
6
7
6
3
1
.8 Hz, 2H), 7.25–7.21 (m, 4H), 7.02 (t, J=6.7 Hz, 2H), 6.90 (t, J=
.9 Hz, 2H), 6.78 (d, J=7.7 Hz, 2H), 5.95 (brs, NH), 4.59 (m, 1H),
.86 (m, 2H), 3.67 ppm (s, 3H); C NMR (100 MHz, [D ]DMSO): d=
55.9, 154.2, 136.9 (2CH), 134.1, 127.1 (2CH), 122.9 (2CH), 121.2
kidney cancer (HEK 293T) cell lines, according to the protocol de-
[29]
scribed previously.
Approximately 2000–2500 HEK 293T cells
13
6
were seeded in 24-well plates, allowed to grow for 24 h, and then
treated with various investigated compounds at their LC50 concen-
trations for the next 48 h. The media was then aspirated, and cells
were allowed to grow in fresh medium for another five to six dou-
blings; afterward, media was removed from the wells, and the
plate was air-dried and stained with 0.2% crystal violet. The wells
were washed twice with distilled water, and colonies were counted
using a gel documentation system (UVP, Germany).
(
1
2C), 119.5 (2CH), 119.4 (2C), 118.5 (2CH), 116.7 (2CH), 114.3 (2CH),
11.8 (2C), 55.5, 44.1, 35.1 ppm; MS (APCI): m/z (%): 425 (5) [M+
+
+
H] ; HRMS-ESI: m/z [M+Na] calcd for C H N NaO : 447.1791,
found: 447.1791.
26
24
4
2
1
-(2-(Indol-3-yl)-2-(5-methoxyindol-3-yl)ethyl)-3-benzylurea (6e):
Light-brown semisolid; (219 mg, 50%): R =0.46 (EtOAc/n-hexane,
f
À1
1
7
0:30); IR (neat): n˜ max =3432, 2917, 1630, 1441, 1275 cm ; H NMR
DAPI nuclear staining: Chromatin condensation, nuclear fragmenta-
tion, and bubble-shaped nuclei are the major phenotypically ob-
served phenomena for apoptotic cells. Apoptosis can be detected
by staining the cells with 4,6-diamidino-2-phenylindole (DAPI), ac-
(
400 MHz, CD OD): d=7.57 (d, J=7.9 Hz, 1H), 7.34 (d, J=8.1 Hz,
3
1
6
3
H) 7.27–7.17 (m, 6H), 7.08–7.04 (m, 4H), 6.94 (t, J=7.5 Hz, 1H),
.45 (dd, J=8.7, 2.3 Hz, 1H), 4.68 (t, J=7.4 Hz, 1H), 4.27 (s, 2H),
.92 (d, J=7.4 Hz, 2H), 3.71 ppm (s, 3H); C NMR (100 MHz,
13
[34]
cording to the protocol described previously. Briefly, HEK 293T
CD OD): d=159.8, 153.2, 136.9, 132.1, 128.0 (2CH), 127.3, 127.2,
3
cells were treated after reaching 70–80% confluence with LC50 con-
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(Nikon, Japan) at 10ꢁ magnifica-
tion to observe the migration of
fragmented DNA. Tail lengths of
the comets were measured using
commercially available TriTek Com-
etScore software.
Cell cycle analysis: To investigate
cell cycle regulation and to under-
stand the mechanism of action of
DIM derivatives on apoptosis of
kidney cancer cells, FACS analysis
was used; 70–80% confluent HEK
2
93T cells were treated by DIM de-
rivatives at LC50 concentrations for
8 h and incubated. Cells were
then harvested according to the
4
[29]
protocol described previously.
For cell cycle analysis, signals of
0000 cells per sample were con-
1
sidered. Evaluation of DNA content
in cells was carried out using FACS
(Becton-Dickinson, CA) analysis.
Calculation of the population of
cells in different phases of the cell
cycle was determined using Cell
Quest Software (Becton-Dickinson).
Data shown here is the best of
three independent experiments.
Figure 9. Comet assay: a) HEK 293T cells were treated with DIM aminophosphonates (5e) and DIM urea (6e), and
a comet assay was performed as described in the Experimental Section. Images were captured using a fluores-
cence microscope (Nikon, Japan) at 10ꢁ magnification and represent one of three different experiments. b) The
diagram shows average tail length of comet formation taken from images in panel a). The tail lengths of the
comets were measured using commercially available TriTek CometScore software. Data are the mean ÆSD of
three different experiments; *p<0.05.
Western blot analysis: HEK 293T
cells were seeded in 100 mm
tissue culture plates and incubated
at 378C in a CO incubator. At 80%
2
confluence, cells were treated by
DIM derivatives at their LC50 con-
centrations for 48 h. Cells were then harvested, and the lysate was
prepared by adding RIPA lysis buffer containing protease inhibi-
tors. Protein concentrations were estimated using the Bradford
method. Proteins (40–50 mg) were separated by 10% SDS-PAGE.
For immunoblotting, antibodies for anti-caspase-3, anti-NF-kB
centrations of DIM derivatives for 48 h. Cells were washed with ice-
cold 1ꢁ PBS and fixed in an ice-cold acetone/MeOH (1:1) mixture
for 10 min at 48C in the dark. Cells were then washed twice with
ice-cold 1ꢁ PBS and stained with DAPI. The excess DAPI stain was
removed with repeated washings with ice-cold 1ꢁ PBS, apoptotic
cells were examined, and micrographs were taken at 40ꢁ magnifi-
cation using a fluorescence microscope (Nikon, Japan).
[34]
(p65), anti-PARP, anti-p53, and GAPDH (loading control) were used.
All antibodies were procured from Cell Signaling Technology (CA,
USA).
[29]
Wound healing assay: A wound healing assay was carried out to
assess the anti-migratory activity of cancerous cells in the presence
of investigated compounds. For this, HEK 293T cells were seeded in
Immunocytochemistry analysis of caspase-3 expression: HEK 293T
cells were seeded in a 96-well plate, and the plate was kept inside
a CO incubator for 24 h. After confluence, the cells were treated at
2
3
5 mm tissue culture plates. After reaching 90% confluence,
the LC50 value of the investigated compounds and were incubated
for 24 h. After incubation, the media was removed and washed
with 1ꢁ PBS. The cells were fixed by adding acetone/MeOH (1:1),
and the plate was incubated for 20 min at À208C. The fixer was
then removed, and the plate was blocked for 30 min by adding
a sharp wound was made on the monolayer by scratching cells at
an angle of 378 from the plane by using a sterile 200 mL microtip.
Cells were treated with DIM derivatives at their LC50 concentrations
and allowed to proliferate for 48 h with respect to the control. Mi-
crographs were taken at different time intervals (0, 12, 24, 36, and
2
% BSA and triton X-100 in 1ꢁ PBS. After blocking, primary anti-
4
8 h) at 40ꢁ magnification with an inverted microscope (Nikon,
body against caspase-3 (cat. #9662, Cell Signaling) was added, and
the plate was incubated for 2 h at 378C. The plate was then
washed with PBS, and secondary antibody conjugated with FITC
was added before incubating for 1 h at 378C. Finally, the plate was
stained with DAPI nuclear stain, and the cells were observed under
a fluorescent microscope (Nikon, Japan). Micrographs were taken
at 40ꢁ magnification.
Japan) to determine the healing of the wound. Data presented
here is the best of three independent experiments.
Comet assay: For evaluation of DNA damage capacity of the cells
after treatment with DIM derivatives, a single cell gel electrophore-
sis (comet assay) was performed according to the protocol de-
[35]
scribed earlier. Briefly, HEK 293T cells were treated with DIM de-
rivatives at their LC50 concentrations for 48 h. After treatment, cells
were harvested by trypsinization, and slides were prepared in 0.5%
low melting agarose and then stained with SYBR green in the dark
at 48C. Images were captured using a fluorescence microscope
Luciferase assay: To measure the activity of transcription factor pro-
moters, a luciferase-based assay was performed. According to the
principle, the relative luciferase activity is proportional to the pro-
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[
29]
moter activity of the cells. After reaching 70–80% confluence,
the two plasmid constructs, NF-kB (p65) (2.0 mg) and b-galactosi-
dase (1.0 mg), were transfected in HEK 293T cells and incubated for
shyap, S. Kandekar, A. T. Baviskar, D. Das, R. Preet, P. Mohapatra, S. R. Sa-
tapathy, S. Sidhharth, S. K. Guchhait, C. N. Kundu, U. C. Banerjee, Bioorg.
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6
–8 h. The NF-kB luciferase construct p5XIP10B (containing five
(
NIPER, S.A.S. Nagar, India), Indian Pat. Appl. 91/DEL/2011, 2011.
tandem copies of the NF-kB site from the IP10 gene) and b-galac-
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After incubation, existing media was replaced with fresh media
and incubated again for the next 12 h. The cells were then treated
with compounds according to their respective LC50 concentrations.
Transfection efficiency was obtained by measuring the activity of
b-galactosidase. Luciferase activity was measured using a micro-
plate reader (Multimode ELISA reader, Tristar, Berthold Technolo-
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3
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Acknowledgements
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The authors thank Dr. George R. Stark (Lerner Research Institute,
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The authors also gratefully acknowledge financial support of this
investigation from the Indian Council of Scientific & Industrial Re-
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agents · cancer · indole alkaloids · NF-kB
· apoptosis · antitumor
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Received: June 19, 2013
Revised: July 23, 2013
Published online on && &&, 0000
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FULL PAPERS
DIM the lights: A rational approach
toward structural elaboration of a bio-
active natural product, diindolylmeth-
ane (DIM), led to our exploration of
novel DIM aminophosphonate and urea
derivatives as potent NF-kB p65-down-
regulating apoptotic anticancer agents
that possess enhanced activities over
the parent DIM.
S. Kandekar, R. Preet, M. Kashyap,
R. P. M. U., P. Mohapatra, D. Das,
S. R. Satapathy, S. Siddharth, V. Jain,
M. Choudhuri, C. N. Kundu,*
S. K. Guchhait,* P. V. Bharatam
&& – &&
Structural Elaboration of a Natural
Product: Identification of 3,3’-Di-
indolylmethane Aminophosphonate
and Urea Derivatives as Potent
Anticancer Agents
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