A synthetic 2,3-diarylindole induces cell death via apoptosis and autophagy in A549 lung cancer cells

Article abstract of DOI:10.1016/j.bmcl.2016.03.079

A series of 2,3-diarylindoles were synthesized via the Larock heteroannulation, and evaluated for their anticancer activity against A549 lung cancer cells. The most potent compound, PCNT13 with IC₅₀ = 5.17 μM, caused the induction of two modes of programmed cell death, apoptosis and autophagy.

Full text of DOI:10.1016/j.bmcl.2016.03.079

Accepted Manuscript
A Synthetic 2,3-Diarylindole Induces Cell Death via Apoptosis and Autophagy
in A549 Lung Cancer Cells
Thanya Rukkijakan, Lukana Ngiwsara, Kriengsak Lirdprapamongkol, Jisnuson
Svasti, Nared Phetrak, Pitak Chuawong
PII:
S0960-894X(16)30312-2
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.03.079
BMCL 23722
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
10 December 2015
20 March 2016
23 March 2016
Please cite this article as: Rukkijakan, T., Ngiwsara, L., Lirdprapamongkol, K., Svasti, J., Phetrak, N., Chuawong,
P., A Synthetic 2,3-Diarylindole Induces Cell Death via Apoptosis and Autophagy in A549 Lung Cancer Cells,
Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.03.079
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A Synthetic 2,3-Diarylindole Induces Cell Death via Apoptosis and Autophagy in
A549 Lung Cancer Cells
Thanya Rukkijakana, Lukana Ngiwsarab,*, Kriengsak Lirdprapamongkolb, Jisnuson Svastib, Nared
Phetraka, Pitak Chuawonga,*
aDepartment of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science,
and Special Research Unit for Advanced Magnetic Resonance (AMR), Kasetsart University, Bangkok
10900, Thailand
Tel +66 2 5625555 ext. 5161; Fax +66 2 5793955; E-mail address: Pitak.C@ku.ac.th
bLaboratory of Biochemistry, Chulabhorn Research Institute, Bangkok 10210, Thailand
Tel +66 2 5538555 ext. 8356; Fax +66 2 2596634; E-mail address: lukana@cri.or.th

Abstract
A series of 2,3-diarylindoles were synthesized via the Larock heteroannulation, and evaluated for their anticancer
activity against A549 lung cancer cells. The most potent compound, PCNT13 with IC50 = 5.17 M, caused the
induction of two modes of programmed cell death, apoptosis and autophagy.
Keywords
2,3-Diarylindole; Apoptosis; Autophagy; Lung Cancer

Lung cancer is one of the most common malignancies worldwide, and the long-term survival rate of lung
cancer patients is one of the lowest among cancers.1 Two major types of lung cancer have been characterized:
non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC).2 NSCLC is a more prevalent subtype
being relatively insensitive to chemotherapy and radiation therapy compared to SCLC. The high mortality in
NSCLC is due to the difficulty of early diagnosis and the high potential for local invasion and distant metastasis.
Although relatively effective chemotherapeutic agents have been developed, lung cancer still has a low cure rate.
Furthermore, the adverse effects of chemotherapeutic compounds often deteriorate the quality of life of lung
cancer patients. Therefore, the discovery of effective novel therapeutic compounds for NSCLC treatments is
urgently needed.
Indole alkaloids exhibit various biological activities including anti-inflammatory,3 anticancer,4 anti-HIV,5
and anti-depression.6 In particular, 2,3-disubstituted indole derivatives demonstrated inhibitory activity against
cyclooxygenase I and II (COX I and II), which play crucial roles in many physiological and pathological
processes including chronic inflammation, cancer, and neurodegenerative disorders.7 In addition, 2,3-
diarylindoles have been indicated to inhibit tubulin polymerization,8 a process that has been validated for
anticancer therapy.9 In this study, we explore the potential biological activity of synthetic 2,3-diarylindoles toward
a human NSCLC cell line, A549 lung adenocarcinoma.
We started our investigation with the synthesis of 2,3-diarylindoles. Several synthetic methods have been
reported for indole derivatives10 including palladium-catalyzed reactions,11 which have become very attractive due
to their high efficiency. Among them, the Larock heteroannulation is a palladium-catalyzed reaction utilizing 2-
iodoaniline and disubstituted alkynes as substrates.12 The reaction results in the formation of carbon-carbon and
carbon-nitrogen bond in a single operation. When unsymmetrical disubstituted alkynes are used, two
regioisomeric products can be obtained. The 2,3-diarylindoles bearing different substituents at the para position
of one of the phenyl moieties (PCNT01-13) were synthesized via the Larock heteroannulation according to our
previous report (Table 1).13

Table 1. Synthesis of 2,3-diarylindoles and their IC50 values against A549 lung cancer adenocarcinoma cell line.
Compound
Structure
H
N
Compound
Structure
IC50 (M)
IC50 (M)
H
N
PCNT01
58.70
PCNT07
N/Aa
NHAc
H
N
H
N
NO2
CN
PCNT02
PCNT03
24.81
38.37
PCNT08
PCNT09
15.0
18.3
H
N
H
N
CN
NO2
H
N
H
N
NH2
OMe
PCNT04
PCNT05
54.70
83.38
PCNT10
PCNT11
288.31
30.0
H
N
H
N
OMe
NH2
H
N
H
N
NHAc
CO2Me
PCNT06
N/Aa
PCNT12
PCNT13
N/Aa
5.17
H
N
CO2Me
aCompounds become insoluble during the assay.

These compounds were first screened for their cytotoxic activity at 72 h using the MTT assay.14 Three
compounds (PCNT06, PCNT07, and PCNT12) had solubility problems hence their cytotoxic activity could not be
evaluated. Among all compounds tested, PCNT13 showed the highest cytotoxic activity with IC50 value of 5.17
μM (Table 1). Therefore, this compound was chosen for further study. Cell viability was reduced in a time- and
dose-dependent manner when treated with compound PCNT13 for 24, 48, and 72 h at a concentration range of
6.3-100 μM (Fig. 1).
Figure 1. Cytotoxic effect of PCNT13 against A549 cells. The viability of cells after exposed to PCNT13 at
various concentrations (6.3-100 M) for 24 h was determined by MTT assay. Data are reported as average values
from three independent experiments with standard deviations (SD).
After 24 h treatment, the morphology of A549 cells became more rounded in appearance, with more
vacuoles appearing in the cells when concentrations of PCNT13 were increased (10, 50, and 100 μM) (Fig. 2A).
The cell morphology began to change upon treatment of PCNT13 after 3 h (data not shown). We further
examined whether PCNT13 induced apoptosis in A549. Changes in apoptosis markers were analyzed by flow
cytometric technique with Muse Cell Analyzer using Annexin-V and Dead Cell assay kit.15 By increasing the
concentrations of PCNT13 for 24 h, treated cells showed increased Annexin-V and 7AAD-positive cell
population, indicating the activation of apoptosis (Fig. 2B). The highest proportion of late apoptosis cells showing
both Annexin-V and 7AAD positive was about 40% when cells were treated with PCNT13 at a concentration of
100 μM (Fig. 2C).

Figure 2. Effect of PCNT13 on cell morphology and apoptosis induction in A549 cells. Cells were treated with
PCNT13 at 10, 50, and 100 μM for 24 h and photographs were taken, after which cells were analyzed for
apoptosis using Muse Cell Analyzer with Annexin-V and Dead Cell assay kit. (A) Cell morphology changes,
original magnification of ×400. (B) Dot plots recording Annexin-V and 7-AAD positive cells in a representative
experiment. (C) Quantitative analysis of percent cell populations. Data are reported as average values from three
independent experiments with standard deviations (SD), ***p < 0.001 vs. control cells.

It is now well known that the reduction in tumor size caused by chemotherapy is a function of apoptotic
cell death (type I programmed cell death). However, apoptosis is suppressed in certain tumors that progress to
stages with high-grade malignancy and resistance to therapy.16 Recent evidence has pointed to the existence of
autophagy, or type II programmed cell death, which is normally activated in response to nutrient deprivation, but
is also activated by exposure to anticancer agents.17 Autophagy is characterized by the formation of
autophagosomes in the cytoplasm, which fuse with lysosomes, organelles, so that cytoplasmic macromolecules
are sequestered and ultimately degraded within the vesicle by lysosomal enzymes.18 Autophagy in response to
cellular stress serves as a potent death signal, as in the case of chemotherapy. Since the morphology of PCNT13
treated cells exhibited vacuole containing cells as a hallmark of autophagy (Fig. 2A), we assessed whether
PCNT13 also induces autophagy in A549 cells. After treatment for 24 h, PCNT13 treated cells displayed positive
results for staining with monodansylcadaverin (MDC), an autophagic vacuole-specific fluorescent probe (Fig. 3A).
Moreover, PCNT13 treatment induced MDC accumulation in A549 cells in a dose-dependent manner (Fig. 3B).
The results suggest that PCNT13 induces both autophagy and apoptosis in A549 cells.
Figure 3. Induction of autophagy by PCNT13 in A549 cells. Cells were treated with PCNT13 at different
concentrations (10, 50, and 100 μM) for 24 h. (A) Fluorescent imaging of the treated cells after staining with an

autophagic vacuole-specific fluorescent probe, monodansylcadaverin (MDC). (B) The relative fluorescent
intensity of MDC staining, Ex/Em at 335/512 nm, in treated cells after exposure to PCNT13 at various
concentrations (6.2-100 μM) for 6 h. Data are reported as average values from three independent experiments
with standard deviations (SD).
We further confirmed the occurrence of both apoptosis and autophagy processes by monitoring the
alteration of key proteins required for those two specific events. During the execution phase of apoptosis, PARP is
specifically cleaved by caspase-3. Autophagy is initiated by conjugation of Agt12, Atg5, and Atg8/microtubule-
associated protein light chain 3 (LC3) to the nascent autophagosome membrane. The increase of LC3-II levels has
been used to represent the autophagy extent.19 After 24 h treatment with PCNT13, A549 cells were harvested and
subjected to Western blot analysis. As shown in Figure 4, PCNT13 effectively enhanced PARP cleavage in a
dose-dependent manner, and this corresponds to the elevated conversion of LC3-I to LC3-II.
Figure 4. Western blot analysis of apoptosis and autophagy progression in PCNT13-treated A549 cells. Cells
were exposed to PCNT13 at different concentrations (10, 50, and 100 μM) for 24 h, and then harvested and
subjected to Western blot analysis. Results shown are representative of at least three independent experiments.
PARP cleavage and LC3 conversion are the hallmark of apoptosis and autophagy, respectively. Tubulin is used as
a loading control.
Apoptosis and autophagy are considered to be two different, but closely related programmed cell death
mechanisms. Apoptotic signaling can regulate autophagy, and conversely autophagy can regulate apoptosis.
However, the molecular connections between autophagy and cell death are complicated and, in different contexts,

autophagy may either promote or inhibit cell death.20 We used an autophagy inhibitor, Bafilomycin A1 (Baf), to
explore the role of autophagy in the growth inhibition and apoptosis induced by PCNT13. Baf prevents
maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes.21 Our results
showed that Baf co-treatment was able to block PCNT13-promoted autophagy in A549 cells, as shown by a lower
MDC fluorescent intensity (Fig. 5A). Interestingly, Baf co-treatment partially reversed the apoptosis-inductive
effect of PCNT13 (Fig. 5B), implying that autophagy contributes to PCNT13-induced apoptosis in A549 cells.
Figure 5. Effect of autophagy inhibition on apoptosis rate in PCNT13 treated A549 cells. The cells were exposed
to PCNT13 for 24 h, at various concentrations (10, 50, and 100 μM) with or without 10 nM Bafilomycin A1 (Baf).
(A) Quantitative analysis of autophagy in PCNT13 treated A549 cells using MDC staining. (B) The rate of
apoptosis was analyzed using Muse Cell Analyzer with Annexin-V and Dead Cell assay kit. Data are reported as

average values from three independent experiments with standard deviations (SD). **p < 0.01 and ***p <
0.001 vs. control cells, and significance between PCNT13 and PCNT13 + Baf are indicated by #, p < 0.05.
In conclusion, this study sheds light on the anticancer potential of 2,3-diarylindole derivatives against
human NSCLC cells via promoting autophagy and apoptosis induction. Further studies are in progress on the
exact cytotoxic mechanism(s) exerted by this class of compounds, and the results should expand the scope for
further investigation on target-based anticancer drug design derived from two modes of programmed cell death.
Acknowledgments
This work was supported by the Kasetsart University Research and Development Institute (KURDI) and
the Faculty of Science, Kasetsart University, and a research grant from the Chulabhorn Research Institute.
Financial support from the Center of Excellence for Innovation in Chemistry (PERCH-CIC), the Office of the
Higher Education Commission, Ministry of Education is also gratefully acknowledged.
References
1.
(a) Jemal, A.; Bray, F.; Center, M. M.; Ferlay, J.; Ward, E.; Forman, D. CA: Cancer J. Clin. 2011, 61, 69;
(b) Wang, H.; Rayburn, E. R.; Wang, W.; Kandimalla, E. R.; Agrawal, S.; Zhang, R. W. Mol. Cancer Ther. 2006,
5, 1585; (c) Lokshin, A.; Levitt, M. L. J. Cell. Biochem. 1996, 24, 186.
2.
3.
Linnoila, R. I. J. Cell. Biochem. 1996, 24, 92.
(a) Sharma, V.; Kumar, P.; Pathak, D. J. Heterocycl. Chem. 2010, 47, 491; (b) Rodrigues de Sa Alves, F.;
Barreiro, E. J.; Fraga, C. A. M. Mini-Rev. Med. Chem. 2009, 9, 782; (c) Gul, W.; Hamann, M. T. Life Sci. 2005,
78, 442.
4.
(a) Li, X.; Li, J. R.; Chen, K.; Zhu, H. L. Curr. Med. Chem. 2013, 20, 3903; (b) Ahmad, A.; Biersack, B.;
Li, Y.; Kong, D.; Bao, B.; Schobert, R.; Padhye, S. B.; Sarkar, F. H. Anticancer Agents Med. Chem. 2013, 13,

1002; (c) Patil, S. A.; Patil, R.; Miller, D. D. Future Med. Chem. 2012, 4, 2085; (d) Biersack, B.; Schobert, R.
Curr. Drug Targets 2012, 13, 1705.
5.
(a) Olgen, S. Mini-Rev. Med. Chem. 2013, 13, 1700; (b) Xu, H.; Lv, M. Curr. Pharm. Des. 2009, 15,
2120.
6.
Kochanowska-Karamyan, A. J.; Hamann, M. T. Chem. Rev. 2010, 110, 4489.
7.
(a) Laube, M.; Tondera, C.; Sharma, S. K.; Bechmann, N.; Pietzsch, F.-J.; Pigorsch, A.; Koeckerling, M.;
Wuest, F.; Pietzsch, J.; Kniess, T. RSC Adv. 2014, 4, 38726; (b) Prasanna, S.; Manivannan, E.; Chaturvedi, S. C. J.
Enzyme Inhib. Med. Chem. 2005, 20, 455; (c) Hu, W.; Guo, Z.; Yi, X.; Guo, C.; Chu, F.; Cheng, G. Bioorg. Med.
Chem. 2003, 11, 5539; (d) Hu, W.; Guo, Z.; Chu, F.; Bai, A.; Yi, X.; Cheng, G.; Li, J. Bioorg. Med. Chem. 2003,
11, 1153; (e) Bailey, D. M.; DeGrazia, G. D.; Alexander, E. J.; Powles, R. G.; Johnson, R. E.; Patrick, R. A.;
Heerdt, B. G.; Fairbain, M. E.; Pruden, D. J. J. Med. Chem. 1985, 28, 160.
8.
(a) De Martino, G.; La Regina, G.; Coluccia, A.; Edler, M. C.; Barbera, M. C.; Brancale, A.; Wilcox, E.;
Hamel, E.; Artico, M.; Silvestri, R. J. Med. Chem. 2004, 47, 6120; (b) Flynn, B. L.; Hamel, E.; Jung, M. K. J.
Med. Chem. 2002, 45, 2670.
9.
(a) Antonarakis, E. S.; Heath, E. I.; Posadas, E. M.; Yu, E. Y.; Harrison, M. R.; Bruce, J. Y.; Cho, S. Y.;
Wilding, G. E.; Fetterly, G. J.; Hangauer, D. G.; Kwan, M. F. R.; Dyster, L. M.; Carducci, M. A. Cancer
Chemother. Pharmacol. 2013, 71, 883; (b) Antonarakis, E. S.; Heath, E. I.; Posadas, E. M.; Harrison, M. R.;
Bruce, J. Y.; Yu, E. Y.; Cho, S. Y.; Wilding, G. E.; Fetterly, G. J.; Hangauer, D. G.; Kwan, M. F. R.; Dyster, L.
M.; Carducci, M. A.; Cons, P. C. C. T. J. Clin. Oncol. 2012, 30; (c) Beckers, T.; Mahboobi, S. Drugs Future 2003,
28, 767; (d) Li, Q.; Sham, H. L. Expert Opin. Ther. Pat. 2002, 12, 1663.
10.
(a) Taber, D. F.; Tirunahari, P. K. Tetrahedron 2011, 67, 7195; (b) Humphrey, G. R.; Kuethe, J. T. Chem.
Rev. 2006, 106, 2875.
11.
12.
Cacchi, S.; Fabrizi, G. Chem. Rev. 2011, 111, Pr215.
Larock, R. C.; Yum, E. K. J. Am. Chem. Soc. 1991, 113, 6689.
13.
Phetrak, N.; Rukkijakan, T.; Sirijaraensre, J.; Prabpai, S.; Kongsaeree, P.; Klinchan, C.; Chuawong, P. J.
Org. Chem. 2013, 78, 12703.

14.
Lirdprapamongkol, K.; Kramb, J. P.; Suthiphongchai, T.; Surarit, R.; Srisomsap, C.; Dannhardt, G.;
Svasti, J. J. Agric. Food Chem. 2009, 57, 3055.
15.
16.
17.
Khan, A.; Gillis, K.; Clor, J.; Tyagarajan, K. Postepy Biochem. 2012, 58, 492.
Gozuacik, D.; Kimchi, A. Curr. Top. Dev. Biol. 2007, 78, 217.
(a) Adams, J. M.; Cory, S. Oncogene 2007, 26, 1324; (b) Tsujimoto, Y.; Shimizu, S. Cell Death Differ.
2005, 12 Suppl 2, 1528.
18.
(a) Todde, V.; Veenhuis, M.; van der Klei, I. J. Biochim. Biophys. Acta 2009, 1792, 3; (b) Kelekar, A.
Autophagy 2008, 4, 555; (c) Cecconi, F.; Levine, B. Dev. Cell 2008, 15, 344.
19.
Yoshimori, T. EMBO J. 2000, 19, 5720.
20. (a) Carew, J. S.; Espitia, C. M.; Esquivel, J. A., 2nd; Mahalingam, D.; Kelly, K. R.; Reddy, G.; Giles, F.
Kabeya, Y.; Mizushima, N.; Ueno, T.; Yamamoto, A.; Kirisako, T.; Noda, T.; Kominami, E.; Ohsumi, Y.;
J.; Nawrocki, S. T. J. Biol. Chem. 2011, 286, 6602; (b) Wu, Y. C.; Wu, W. K.; Li, Y.; Yu, L.; Li, Z. J.; Wong, C.
C.; Li, H. T.; Sung, J. J.; Cho, C. H. Biochem. Biophys. Res. Commun. 2009, 382, 451; (c) Boya, P.; Gonzalez-
Polo, R. A.; Casares, N.; Perfettini, J. L.; Dessen, P.; Larochette, N.; Metivier, D.; Meley, D.; Souquere, S.;
Yoshimori, T.; Pierron, G.; Codogno, P.; Kroemer, G. Mol. Cell. Biol. 2005, 25, 1025.
21.
Yamamoto, A.; Tagawa, Y.; Yoshimori, T.; Moriyama, Y.; Masaki, R.; Tashiro, Y. Cell Struct. Funct.
1998, 23, 33.

Graphical Abstract