Synthesis of novel β-carboline based chalcones with high cytotoxic activity against breast cancer cells

Article abstract of DOI:10.1016/j.bmcl.2014.04.109

A series of novel β-carboline based chalcones was synthesized and evaluated for their cytotoxic activity against a panel of human cancer cell lines. Among them we found that two of the compounds 7c and 7d, showed marked anti-proliferative activity in a panel of solid tumor cell lines with highest effect in breast cancer. The compounds 7c and 7d showed an IC₅₀ of 2.25 and 3.29 μM, respectively against human breast cancer MCF-7 cell line. Further, the compound 7c markedly induced DNA fragmentation and apoptosis in breast cancer cells.

Full text of DOI:10.1016/j.bmcl.2014.04.109

Accepted Manuscript
Synthesis of novel β-carboline based chalcones with high cytotoxic activity
against breast cancer cells
Shikha S. Chauhan, Anup K. Singh, Sanjeev Meena, Minaxi Lohani, Akhilesh
Singh, Rakesh K. Arya, Srikanth H. Cheruvu, Jayanta Sarkar, Jiaur R. Gayen,
Dipak Datta, Prem M.S. Chauhan
PII:
S0960-894X(14)00466-1
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.04.109
BMCL 21602
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
26 February 2014
23 April 2014
26 April 2014
Please cite this article as: Chauhan, S.S., Singh, A.K., Meena, S., Lohani, M., Singh, A., Arya, R.K., Cheruvu, S.H.,
Sarkar, J., Gayen, J.R., Datta, D., Chauhan, P.M.S., Synthesis of novel β-carboline based chalcones with high
cytotoxic activity against breast cancer cells, Bioorganic & Medicinal Chemistry Letters (2014), doi: http://
dx.doi.org/10.1016/j.bmcl.2014.04.109
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Graphical Abstract
A novel series of β-carboline based chalcones was synthesized and evaluated for their cytotoxic
activity against a panel of human cancer cell lines. Among all, compound 7c was identified as
the most potent analogue that showed marked apoptotic activity in breast cancer cells.

Synthesis of novel β-carboline based chalcones with high cytotoxic activity
against breast cancer cells
Shikha S. Chauhan,a,c Anup K. Singh,b Sanjeev Meena,b Minaxi Lohani,c Akhilesh Singh,b
Rakesh K. Arya,b Srikanth H. Cheruvu,d Jayanta Sarkar,b Jiaur R. Gayen,d Dipak Datta,b and
Prem M. S. Chauhan,a,*
aMedicinal and Process Chemistry Division, CSIR-Central Drug Research Institute, Lucknow-
226031, India
bBiochemistry Division, CSIR-Central Drug Research Institute, Lucknow-226031, India.
cDepartment of Chemistry, Integral University, Lucknow-226 026, U.P., India.
dPharmacokinetics and Metabolism Division, CSIR-Central Drug Research Institute, Lucknow-
226031, India.
*Corresponding author. Tel.: +91 522 2771940; Fax: +91 522 2771941;
E-mail: prem_chauhan_2000@yahoo.com; premsc58@hotmail.com
Abstract:
A series of novel β-carboline based chalcones was synthesized and evaluated for their cytotoxic
activity against a panel of human cancer cell lines. Among them we found that two of the
compounds 7c and 7d, showed marked anti-proliferative activity in a panel of solid tumor cell
lines with highest effect in breast cancer. The compounds 7c and 7d showed an IC50 of 2.25 and
3.29 µM respectively against human breast cancer MCF-7 cell line. Further, the compound 7c
markedly induced DNA fragmentation and apoptosis in breast cancer cells.
Keywords: Anticancer, β-carboline, Chalcone, Cytotoxic.

Cancer has become a major cause of death in the world and posed a great challenge to the fields
of medicine and immunology. Approximately seven million people die from cancer every year
due to which this disease has become responsible for 12.5 % of death worldwide.1 Chemotherapy
has been widely employed for various cancer treatments. Most of the presently available
chemotherapeutic agents work through the induction of apoptosis.2 There are mainly two
pathways by which cells undergo apoptosis; one is extrinsic and another one is intrinsic. Cancer
cells are well known to aberrantly regulate either one or both of these pathways according to
their need for survival and become resistant against most of the common chemotherapeutic
drugs.3 Therefore, development of new anticancer drugs and more effective treatment strategies
for cancer are of utmost importance.
Natural anticancer pharmacophores play a major role in sensitizing the tumor cells
toward apoptosis.4 Among the natural products isolated from plants, chalcones, belonging to
flavonoid family, represent one of the most interesting classes of biologically active
compounds.5-7 Anticancer properties of naturally occurring and synthetic chalcones continue to
attract considerable interest.8-10 Chalcones isolated from various plants species like Angelica,
Glycyrrhiza, and Scutellaria, have been used in traditional medicine to treat various diseases
including cancer.11-14 Due to their high therapeutic index, ease of preparation, the potential for
oral administration and safety, chalcones have emerged as potential anticancer agents.15-17
Similarly, β-carboline alkaloids form a large group of natural and synthetic indole alkaloids.
Their presence in medicinal plants such as Peganum harmala and Eurycoma longifolia, have
drawn attention due to their antitumor activities.18,19 Harmine, a β-carboline alkaloid isolated
from the extracts of Peganum harmala, is highly cytotoxic and significantly inhibit tumor cell
growth with apoptotic effects.20 According to other reports, hyrtiocarboline, isolated from a

Papua New Guinea marine sponge, Hyrtios reticulates, have also been identified as an antitumor
22
agent.21 Moreover, our group has also synthesized some β-carboline
derivatives that have
shown good anticancer activity.23
Instigated with all the above facts and in continuation with our efforts towards natural
product inspired synthesis of novel compounds of medicinal value,24-26 we designed β-carboline
based chalcones where one phenyl ring (ring B) was replaced with the β-carboline
pharmacophore and discovered a new class of anticancer agents in this endeavor (Figure 1),
capable of targeting breast cancer cells.
[Insert Figure 1 here]
In the present investigation, designed chalcones were prepared by the classical method
that has been outlined in Scheme 1. The synthesis of chalcone involved Claisen−Schmidt
condensation of substituted 1-phenylethanone and β-carboline-1-carbaldehyde or N-alkylated β-
carboline-1-carbaldehyde in 10% aqueous solution of NaOH and methanol. Our synthetic
strategy started with the Pictet–Spengler reaction of tryptamine 1 with dimethoxyglyoxal, to
afford the tetrahydro-β-carboline derivative 2, which was oxidized with KMnO4 at room
temperature overnight, resulting in acetal 3. The acetal 3 was deprotected in aqueous acetic acid
to give the corresponding aldehyde 4. In order to obtain acetal 5a-j the intermediate 3 was
alkylated with methyl and ethyl iodide in DMF, after deprotonation with sodium hydride, to give
N-alkylated indole. Acetals 5a-j was further deprotected with the same methodology as followed
for acetal 3 to give corresponding aldehydes 6a-j.27 The aldehydes 4 and 6a-j were coupled with
substituted 1-phenylethanone to provide desired products 7a-h and 8a-h respectively. The
products were characterized by analysis and comparison of their spectral and physical data

1
including H NMR and HRMS given in the “Supporting Information” provided along with this
manuscript.
[Insert Scheme 1 here]
To study the in vitro anticancer activity of β-carboline based chalcones, we screened a
series of 18 compounds in eight different cancer cell lines representing eight cancer types.
Representative cell lines from different cancer types are DLD-1 (Colorectal adenocarcinoma),
MCF-7 (Breast adenocarcinoma), PLC/PRF/5 (Liver hepatoma), A549 (Lung carcinoma),
SKOV-3 (Ovarian adenocarcinoma), MiaPaca-2 (Pancreatic adenocarcinoma), DU145 (Prostate
carcinoma), A-172 (Brain glioblastoma). Detailed results of these investigations in terms of
percentage inhibitions of cell growth at 10 μM of each compound are given in Table 1. As part of
present investigation to determine the important features of β-carboline based chalcone
influencing their anticancer activity, we disclose here additional structural requirements for the
anti-proliferative activity of this newly designed chalcones. The substituents to be introduced on
the chalcone scaffold were chosen among those frequently found in naturally occurring
compounds, such as methoxy. Moreover, with the goal to explore the influence of electron
density on the activity of enone, we introduced substituent with varied electron density on the
phenyl ring of the chalcone counterpart. For that purpose, methyl, besides methoxy group was
used as electron donating groups and halogens were used as electron withdrawing groups. We
observed that compounds substituted with electron donating group at the phenyl ring of chalcone
(7b, 7c, 7d, 7e) had positive impact towards the inhibitory activity of the compounds in
comparison to the unsubstituted compounds (7a) and the compounds with electron withdrawing
groups (7f, 7g, 7h). Further, for the structural optimization of the compounds alkylation of the
indole NH was done with methyl (8a-e) and ethyl groups (8f-j) with the goal to determine

whether the modification at the indole NH can improve the efficacy of the compounds.
Unfortunately, a significant lowering was observed in the activity of the compounds in this case.
Although the ethyl substituted compounds were found to be more active than methyl substituted
compounds but overall these compounds were not as good as the parent compounds. These
results indicate the importance of free indole NH group in β-carboline moiety for its anticancer
activity in the compounds and can be correlated with the occurrence of natural β-carboline
alkaloid like harmine and hyrtiocarboline, with antitumor activities, having free indole NH.20,21
The primary screening of diversely substituted chalcones concluded that the OMe substituents
are quite promising in providing the most active tumor cell growth inhibitors and relevant
structure−activity relationship. Moreover, the activity of chalcones was found to be dependent on
the presence, the number, and the positions of the methoxy group. The compounds with 3',4'-
dimethoxy (7c) and 3',4',5'-trimethoxy (7d) groups were found to be the most active among all
the compounds screened against various cancer cell lines.
[Insert Table 1 here]
Therefore, based on the above observations compounds 7c and 7d were selected for
further analysis and their respective IC50 (Inhibitory Concentration 50) values were calculated in
six different cell lines by taking 4 different concentrations of 10, 3.16. 1 and 0.316 μM (half log
scale). IC50 values of compounds 7c and 7d in different cancer cell lines are shown in Figure 2;
Panel A. Consistent with our previous finding, here we observe that compounds 7c and 7d have
shown IC50 values less than 5 μM in multiple cancer cell lines. However, based on the minimal
IC50 values, we decided to perform rest of the experiments with compounds 7c and 7d on breast
cancer cell lines. In this endeavor, dose response curves were prepared for compounds 7c and 7d
at three different breast cancer cell lines MCF-7, MDA-MB 231 and 4T1. Dose response curve

clearly indicated that both compounds 7c and 7d were potentially active against all three breast
cancer cell lines MCF-7, MDA-MB 231 and 4T1 in a dose dependent manner. However, at a
same dose of compound 7c and 7d, we found that compound 7c was more effective in killing
three different types of breast cancer cells.
[Insert Figure 2 here]
To determine the gross morphology change after the exposure of compound 7c for 24 hours, we
examined treated and control cells under microscope and observed that there was change in
structured cell morphology as well as marked reduction of cell numbers in treated groups
compared to control. Reduction of cell numbers and gross phenotype alterations might be an
indication of cellular apoptosis. To further validate that whether compound 7c induces apoptosis
in MCF-7 cells, we performed 7-AAD/Annexin-V staining and cells were analyzed by flow
cytometry. Annexin-V binds to phosphatidylserine (PS) which is normally present in inner
membrane and translocate to outer membrane as an early event in apoptotic cells whereas 7-
AAD is a DNA labeling dye which does not enters the healthy cells as they are impermeable to it
but enters the cells and label their DNA once their permeability changes in advance stages of
apoptosis.
[Insert Figure 3 here]
As shown in the figure 4B, FACS-acquired dot plots of control and treated groups,
percentage of Annexin-V low/7-AAD low (non apoptotic healthy cells), Annexin-V high/7-AAD
low
high
(early apoptotic cells), and Annexin-V high/7-AAD
(late apoptotic cells) cells were
low
determined and found that after the treatment of compound 7c, Annexin V high/7-AAD

population was increased. This indicates that treatment of compound 7c enabled induction of
apoptosis in MCF-7 cells.
To further confirm the apoptotic process in compound 7c exposed cells, we assessed
DNA fragmentation which is an essential step in the cells undergoing apoptosis. We performed
APO-BrdU TUNNEL assay to detect DNA fragmentation as an indication of apoptosis in MCF-
7 cells after 7c treatment. This assay act on the basis that DNA breaks generated during
apoptosis will expose a larger fraction of 3’-hydroxyl ends which can serve as starting point of
terminal deoxynucleotidyl transferase (TdT) to label these breaks with deoxythymidine analog 5-
bromo-2´-deoxyuridine 5´-triphosphate (BrdUTP) which is finally detected using anti-BrdU
antibody using immouno-fluorescence techniques. Results of this experiment as shown in the
figure 4C clearly indicate that the number of MCF-7 cells showing enhanced green fluorescence
in response to compound 7c treatment compared to vehicle (DMSO) treated cells, which further
proves its capability to induce apoptosis in breast cancer cells.
In conclusion, the present investigation led to the identification of newly designed β-
carboline based chalcones with potential cytotoxic activities and brings into account the new
structural elements that will aid in the development of more active chalcones. The compound 7c
demonstrated strong inhibition of the cellular proliferation of several cancer cell lines and found
to be the highly effective in inducing apoptosis in breast cancer cells. Further, the easy and
economical preparation opens a new avenue for the development of this new class of compounds
as novel anticancer agents.
Acknowledgement:
SSC and AKS are grateful to Council of Scientific and Industrial Research (CSIR) for the
financial support in the form of fellowship. We also thank SAIF Division of CDRI, Lucknow, for

providing the spectroscopic data. Further, we gratefully acknowledge the excellent technical help
of Mr. A. L. Vishwakarma of SAIF for the Flow Cytometry studies. Research in the authors’
laboratory is also supported by CSIR funds BSC106, and MOES fund GAP0118; the University
Grants Commission fellowship grants (to RKA).
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Table 1: Percentage Inhibition of compounds in various cancerous cell lines
MiaPaCa-2
(Pancreas)
21.74±2.71
33.68±2.40
87.53±7.14
84.75±9.11
2.10±5.01
PLC/PRF/5
(Liver)
SKOV-3
(Ovarian)
0.91±2.01
DU-145
A-172
A549
MCF7
DLD-1
(Colon)
Comp no.
(Prostate)
(Brain)
(Lung)
(Breast)
7a
7b
7c
7d
7e
7f
76.65±5.11
70.78±4.54
78.29±5.44
73.93±7.32
70.54±5.14
26.45±3.55
80.54±5.44
49.85±1.55
64.9±4.12
16.93±1.34
42.44±6.54
40.09±6.50
32.21±4.42
20.12±3.22 78.44±10.50 11.74±6.22 32.87±5.34 70.08±5.55
29.2±0.80 37.17±3.11 74.19±8.11
33.85±2.71 61.86±2.11 77.18±6.20
41.57±4.45 71.91±11.05 74.32±4.32 84.67±2.44 94.55±5.14 88.10±9.70
36.65±6.50 76.09±9.01 64.96±4.24 73.44±6.24 86.79±10.14 80.58±4.54
5.10±3.11
-2.43±3.14
0.20±5.04
8.15±2.11
11.37±4.09 60.47±8.22
9.91±5.21
9.19±3.32
4.68±2.34
6.73±4.33
20.92±4.24 42.42±2.44
-4.72±3.00
-13.08±3.37
-4.04±3.01
25.49±1.29
-0.03±4.12
7.71±5.34
-0.24±3.22
7.63±2.04
-5.2±3.22
5.41±2.10
75.78±7.03
16.25±2.87
7.15±2.85
15±3.12
14.88±2.22
41.16±5.02
25.64±4.12
7g
7h
8a
8b
8c
8d
8e
9.92±1.97
28.63±5.52 53.25±4.31 75.46±4.24 19.29±3.11 27.24±2.26 67.46±6.62
37.43±4.12
9.1±3.25
0.18±4.14
-3.88±5.09
19.6±6.68
8.18±3.24
28.25±5.44
68.67±10.35 34.13±4.45 15.54±3.34
12.94±3.57 45.29±4.45
19.04±8.93
13.72±5.35
37.35±9.21 59.49±8.53 38.75±3.59 33.84±7.44 28.77±5.53 49.45±.7.64
46.16±11.34 45.69±5.65 19.19±5.35 13.21±7.46 20.79±4.34 35.79±5.67

8f
25.01±5.59
9.18±4.53
75.13±5.24
61.61±4.30
56.2±9.58
33.54±11.24 54.23±7.56 80.55±8.58 12.54±3.45 39.28±7.59 77.57±8.34
43.74±5.40 52.52±8.93 39.24±9.58 21.25±7.49 27.24±5.66 55.5±8.24
57.58±4.59 56.83±4.38 70.09±2.35 35.63±3.59 29.56±2.59 66.04±6.35
68.54±5.49 60.15±7.39 81.12±6.23 44.4±2.21 32.7±3.69 73.38±5.24
8g
8h
15.35±8.36
24.73±9.48
11.93±3.29
85.13±4.54
8i
8j
70.66±4.91
34.09±7.29
90.66±5.64
47.70±9.69 35.03±7.47 26.57±6.35 16.43±4.39 30.01±6.36 44.14±6.49
69.20±5.45 73.92±7.12 67.63±8.58 69.05±9.57 56.93±6.48 73.22±7.74
Doxorubicin
Scheme 1:
Reagents and conditions: (a) OHCCH(OMe)2, 5% TFA, CH2Cl2, rt, 17 h. (b) KMnO4, THF, rt, 17h. (c) AcOH/H2O
(2:3 ratio), 120°C, 1 h. (d) NaOH (10% in H2O): MeOH, rt, 4h (e) RI, NaH, DMF, rt, 4 h

Figure 1: Designing of β-carboline based chalcones.

Figure 2: Selected β-carboline based chalcones (compounds 7c and 7d) inhibit cellular proliferation
of different human carcinoma cells with varying potential and were found to be highly effective on
breast cancer cells. Different human cancer cells (A172, MCF7, A549, PC3, SKOV-3 and normal Vero
cells) were treated with varying concentrations of compounds for 48 hours and the half maximal
proliferation inhibitory concentration (IC50) values were determined (Panel A) by SRB assay. Dose
response histograms were shown for human breast cancer MCF-7 and MDA-MB-231 (Panel B and Panel
C respectively) and mouse breast cancer 4T1 (Panel C) cell lines. Results are representative of three
independent experiments. Columns, average of triplicate readings of samples; error bars, ± S.D. *, p <
0.01, compared with vehicle treated cells.

A
Comp' 7c- Treated
Vehicle Control
B
Comp' 7c- Treated
Apo- BrdU
Vehicle Control
Bright Field
C
Vehicle
Control
Comp'7c
Treated
Figure 3: Effect of β-carboline based chalcone (compound 7c) on breast cancer cell apoptosis. (A)
Photo micrographs of confluency of MCF7 cells in vehicle treated and 24 hours treatment of compound
7c. (B) Flow cytometric analysis of MCF7 cells undergoing apoptosis after the treatment with compound
7c at the indicated concentration for 24 hours. The cells were harvested and labeled with Annexin-V-PE
and 7-AAD and analyzed by Flow cytometry. (C) Apoptosis indicator DNA fragmentation was
determined by Apo-BrdU TUNNEL assay. Bright Field (Left panels) and fluorescent photo (right panels)
micrographs of Alexa Flour 488-labeled anti-BrdU antibody for vehicle and compound 7c treated MCF7
cells.