A synthetic 2,3-diarylindole induces microtubule destabilization and G2/M cell cycle arrest in lung cancer cells

Article abstract of DOI:10.1016/j.bmcl.2019.126777

The anticancer potential of a synthetic 2,3-diarylindole (PCNT13) has been demonstrated in A549 lung cancer cells by inducing both apoptosis and autophagic cell death. In this report, we designed to connect a fluorophore to the compound via a hydrophilic linker for monitoring intracellular localization. The best position for linker attachment was identified from cytotoxicity and effect on cell morphology of newly synthesized PCNT13 derivatives bearing hydrophilic linker. Cytotoxicity and effect on cell morphology related to the parental compound were used to identify the optimum position for linker attachment in the PCNT13 chemical structure. The fluorophore-PCNT13 conjugate was found to localize in the cytoplasm. Microtubules were found to be one of the cytosolic target proteins of PCNT13, as the compound could inhibit tubulin polymerization in vitro. A molecular docking study revealed that PCNT13 binds at the colchicine binding site on the α/β-tubulin heterodimer. The effect of PCNT13 on microtubule dynamics caused cell cycle arrest in the G2/M phase as analyzed by flow cytometric analysis.

Full text of DOI:10.1016/j.bmcl.2019.126777

Journal Pre-proofs
A synthetic 2,3-diarylindole induces microtubule destabilization and G2/M cell
cycle arrest in lung cancer cells
Bongkotrat Thanaussavadate, Lukana Ngiwsara, Kriengsak Lirdprapamongkol,
Jisnuson Svasti, Pitak Chuawong
PII:
S0960-894X(19)30742-5
DOI:
https://doi.org/10.1016/j.bmcl.2019.126777
BMCL 126777
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
28 August 2019
18 October 2019
23 October 2019
Please cite this article as: Thanaussavadate, B., Ngiwsara, L., Lirdprapamongkol, K., Svasti, J., Chuawong, P., A
synthetic 2,3-diarylindole induces microtubule destabilization and G2/M cell cycle arrest in lung cancer cells,
Bioorganic & Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.126777
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A synthetic 2,3-diarylindole induces microtubule destabilization and G2/M cell
cycle arrest in lung cancer cells
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Bongkotrat Thanaussavadatea, Lukana Ngiwsarab,*, Kriengsak Lirdprapamongkolb, Jisnuson
Svastib, Pitak Chuawonga,*
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aDepartment of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of
Science, Kasetsart University, Bangkok 10900, Thailand
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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
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Abstract
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The anticancer potential of a synthetic 2,3-diarylindole (PCNT13) has been demonstrated in A549 lung
cancer cells by inducing both apoptosis and autophagic cell death. In this report, we designed to connect a
fluorophore to the compound via a hydrophilic linker for monitoring intracellular localization. The best
position for linker attachment was identified from cytotoxicity and effect on cell morphology of newly
synthesized PCNT13 derivatives bearing hydrophilic linker. Cytotoxicity and effect on cell morphology
related to the parental compound were used to identify the optimum position for linker attachment in the
PCNT13 chemical structure. The fluorophore-PCNT13 conjugate was found to localize in the cytoplasm.
Microtubules were found to be one of the cytosolic target proteins of PCNT13, as the compound could
inhibit tubulin polymerization in vitro. A molecular docking study revealed that PCNT13 binds at the
colchicine binding site on the tubulin heterodimer. The effect of PCNT13 on microtubule dynamics
caused cell cycle arrest in the G2/M phase as analyzed by flow cytometric analysis.
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Keywords
2,3-diarylindole; lung cancer; tubulin polymerization; cell cycle arrest

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Lung cancer is the leading cause of cancer-associated mortality worldwide. It is divided into two
types, according to histology, small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC).
NSCLC accounts for 85% of lung cancers1 and is insensitive to conventional treatments such as radiation
therapy and chemotherapy. Despite advances in diagnostics and therapeutics, the outcome for patients with
NSCLC remains poor. Therefore, the discoveries of effective new therapeutic drugs are still an urgent
requirement for this cancer. Previously, we have reported the potential anticancer activity of the synthetic
2,3-diarylindole (PCNT13), which induces two modes of programmed cell death, autophagy, and apoptosis,
toward a human NSCLC cell line, A549.1 In the present study, we further explored the mechanism
underlying the action of PCNT13 against A549 cells.
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We planned to investigate the cellular localization of PCNT13. It is worth mentioning that most of
the 2,3-diarylindoles possess fluorescent property, which facilitates monitoring the intracellular localization
of the compounds. However, the intrinsic fluorescent intensity of PCNT13 is rather weak, rendering the
compound impractical to be observed inside the cells under a fluorescence microscope. Therefore, the use
of fluorophore-PCNT13 conjugate may be an alternative approach. Since structural modifications of
biologically active compounds may interfere with non-covalent interactions with their biomolecular targets,
the position for the hydrophilic linker attachment needs to be optimized. As illustrated in Fig.1, four
different derivatives bearing a hydrophilic linker at different positions were considered. Derivatives 1 and
3 resemble PCNT13 with the linker on the indolic nitrogen and the ester moiety, respectively, while 2 and
4 resemble PCNT12, a regioisomer of PCNT13. PCNT12 was previously reported to have solubility
problems with unattainable cytotoxicity.2 Thus, introducing a hydrophilic linker might increase the
solubility of PCNT12 and allows us to evaluate the anticancer activity.

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Fig. 1. Four indole derivatives utilized in the optimization of linker installation.
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Syntheses of the hydrophilic linkers and PCNT13 derivatives were illustrated in Scheme 1 and 2,
respectively. One of the two hydroxyl groups of triethylene glycol 5 was activated using tosyl chloride to
afford 6 in 64% yield, followed by protection of the remaining hydroxyl group using dihydropyran under
the acidic conditions to afford 7 in 85% yield. Subsequently, 6 was subjected to azide substitution using
NaN3 in refluxing acetonitrile to provide 8 in 94% yield. The reaction between 7 and 8 in the presence of
NaH, followed by deprotection of the tetrahydropyranyl ether under the acidic conditions, provided the
hexaethylene glycol derivative 9 in 45% yield over two steps. A hydroxyl group of 5 was also protected as
the tetrahydropyranyl ether to afford 10 in 65% yield.

b
c
d
O
O
O
O
N3
N3
6 H
3 H
R
3H
R
3Ts
8
9
6: R = OH
5: R = OH
a
a
7: R = OTHP
10: R = OTHP
e
11: R = Br
f
I
H
N
3
H
O
O
3
12
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Scheme 1. Synthesis of hydrophilic linkers. Reagents and conditions: (a) DHP, conc. HCl, rt, 16 h; (b)
TsCl, Et3N, CH2Cl2, rt, 5 h; (c) NaN3, CH3CN, reflux, 16 h; (d) i) 7, NaH, THF, reflux, 26 h; ii) 1 M HCl,
MeOH, rt, 6 h; (e) PBr3, reflux, 4 h; (f) 2-iodoaniline, Na2CO3, CH2Cl2, rt, 3 h. (Note: DHP = 3,4-dihydro-
2H-pyran; THP = tetrahydro-2H-pyran)
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An N,N-disubstituted derivative of 2-iodoaniline (12) was prepared in 2 steps. First, a hydroxyl
group of 5 was converted to a bromide using PBr3 to provide 11 in 49% yield, followed by the N-alkylation
of 2-iodoaniline using 11 in the presence of Na2CO3 to provide 12 in 40%. Compound 12, along with the
synthesized hydrophilic linkers, were utilized in the synthesis of 2,3-diarylindole derivatives 1, 2, 3, and 4,
as described in Scheme 2.

H
H
O
O
3
3
CO2Me
CO2H
CO2Me
CO2Me
N
a
d
N
c
b
+
CO2Me
H2N
H2N
I
14
15
13
16
CO2Me
e
2
1
H
H
H
N
N
N
f
g
OH
3
O
CO2Me
CO2H
O
PCNT13
17
3
+
H
H
N
H
N
O
N
h
CO2H
CO2Me
f
O
OH
3
18
PCNT12
4
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Scheme 2. Synthesis of PCNT13 derivatives bearing hydrophilic linker. Reagents and conditions: (a)
º
MeOH, H2SO4, reflux, 5 h; (b) H2SO4, NaNO2, 0 C, 5 h, then KI/H2O, rt, 16 h; (c) Phenyl acetylene,
Pd(OAc)2, PPh3, CuI, Et3N, THF, rt, 4 h; (d) 12, Pd(OAc)2, PPh3, n-Bu4NCl, Na2CO3, DMF, 80 ºC, 16 h;
(e) 2-iodoaniline, Pd(OAc)2, PPh3, n-Bu4NCl, Na2CO3, DMF, 80 ºC, 16 h; (f) EtOH, sat. KOH, reflux, 3 h;
(g) 5, PPh3, DIAD, THF, rt, 3 h; (h) i) 10, PPh3, DIAD, THF, rt, 3 h; ii) 5 M HCl, MeOH, rt, 30 min.
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The synthesis of 1, 2, 3, and 4 started with esterification of 13 to afford methyl ester 14 in 98%
yield (Scheme 2). Subsequently, the Sandmeyer reaction was utilized to convert arylamine 14 to
iodobenzene derivative 15 in 66% yield.3 Sonogashira coupling reaction between 15 and phenylacetylene
provided diarylacetylene derivative 16 in 85% yield.4 Then, the Larock heteroannulation between 16 and
12 afforded the regioisomeric products 1 and 2 in 15% and 47% yields, respectively.5, 6 It is worth
mentioning that one of the two alkyl groups on the nitrogen atom of 12 was removed during the catalytic
cycle as previously reported.7 Compound 16 was also subjected to the Larock heteroannulation using 2-
iodoaniline to afford PCNT13 and PCNT12 in 37% and 47% yields, respectively. The methyl ester in
PCNT13 and PCNT12 was hydrolyzed under basic conditions using saturated KOH in ethanol to afford
carboxylic acids 17 and 18 in 99% yields. Esterification of 17 and 18 using carbodiimide reagents was

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unsuccessful. We then turned to Mitsunobu esterification utilizing PPh3, diisopropyl azodicarboxylate
(DIAD) in THF.8 Gratifyingly, 3 was obtained in 50% yield using triethylene glycol 5 as a nucleophile.
Interestingly, Mitsunobu esterification of 18 using 5 as alcohol provided an inseparable mixture. To
overcome this problem, 10 was used instead of 5, and the reaction proceeded to give the ester intermediate,
followed by deprotection of the tetrahydropyranyl ether to afford 4 in 23% yield over two steps. (See
synthetic details and spectroscopic data of synthesized compounds in the Supporting Information.)
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The derivative with an optimum position for linker attachment should display cytotoxicity similar
or relatively close to the parent compound, to ensure a similar cytotoxic mechanism. With four indole
derivatives in hand, we proceeded to evaluate cytotoxicity against A549 lung cancer cells. As illustrated in
Table 1, all four compounds exhibited cytotoxic activity, with 3 showing the lowest IC50 value of 10.2±1.04
M, closet to the 5.17 M of the parent compound PCNT13.2 In addition, the changes in cell morphology
observed after 48 h treatment with 3 were similar to those treated with PCNT13.2 Therefore, an ester
functional group at the 4-position of the aromatic ring of PCNT13 was the best site for hydrophilic linker
attachment.
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Table 1. IC50 values of compound 1-4 against A549 cells and their effect on cell morphology
Compound
IC50 (M)
Cell morphology (48 h)
No
-
compound

PCNT13
5.17±1.61
30.8±2.56
41.3±2.08
1
2
3
4
10.2±1.04
29.5±1.50
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Fluorescein isothiocyanate (FITC), a commonly used fluorophore, was chosen to connect to
PCNT13 through a hydrophilic linker. Synthesis of the fluorophore-PCNT13 conjugate (FL-PCNT13) was
illustrated in Scheme 3. The synthesis started from Mitsunobu esterification of 17 using a hydrophilic linker
9 to afford 19 in 75%.8 A longer linker (9, 12 carbon atoms) was used to provide ample space between the

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PCNT13 indole moiety and the fluorescein to minimize any steric hindrance upon target binding.
Subsequently, the azido group of 19 was reduced via catalytic hydrogenation, followed by a coupling
reaction with FITC to afford FL-PCNT13 in 19% over two steps.
H
H
H
N
N
N
a
b
S
N3
HN NH
CO2H
17
O
O
6
6
O
O
CO2H
19
O
O
OH
FL-PCNT13
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Scheme 3. Synthesis of FL-PCNT13. Reagents and conditions: (a) 9, PPh3, DIAD, THF, rt, 3 h; (b) i) Pd/C,
MeOH, H2, rt, 3 h, ii) FITC, Et3N, MeOH, rt, 8 h in the dark.
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The FITC moiety allows observing the intracellular localization of FL-PCNT13 by confocal laser
microscopy. The A549 cells were incubated with 250 μM FL-PCNT13 for 3 h, then stained with a nuclear
probe Hoechst 33342 (0.1 g/mL) for 5 min before subjecting to live-cell imaging under a confocal laser
microscope. As shown in Fig. 2, the observed fluorescent signal of FL-PCNT13 was distributed
throughout the cytoplasm while the fluorescent signal in the nucleus was faintly detected, suggesting that
PCNT13 and its primary target proteins are localized in the cytoplasm of the cells.
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Fig. 2. Intracellular localization of FL-PCNT13 in living A549 cells. Confocal fluorescent images were
taken after the cells were incubated with 250 μM FL-PCNT13 (Green) for 3 h, followed by 5-min staining
with 0.1 g/mL Hoechst 33342 nuclear dye (Blue).
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Since the chemical structure of PCNT13 was similar to arylindoles, which have been reported as
tubulin polymerization inhibitors,9-20 we examined the effect of PCNT13 on tubulin polymerization by
using the Tubulin Polymerization Assay Kit (Cytoskeleton, Inc.). Tubulins purified from the porcine brain
were allowed to polymerize in a buffer containing 80 mM PIPES pH 6.9, 0.6 mM EGTA, 2 mM MgCl2,
and 1 mM GTP at 37 °C. Polymerization was monitored by recording the absorbance at 340 nm as a result
of microtubule formation. As shown in Fig. 3, an anticancer drug Taxol (10 M), which is known to be a
microtubule-stabilizing agent, enhanced tubulin polymerization. In contrast, the polymerization was
drastically inhibited in the presence of 10 M PCNT13, indicating that PCNT13 might act as a microtubule-
destabilizing agent. Since PCNT13 was found to localize in the cytoplasm of cells where the microtubules
are formed, we suggested that the cytotoxicity of PCNT13 might result from inhibiting microtubule
formation, and tubulin is one of the target proteins of this compound.
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Fig. 3. Effect of PCNT13 on microtubule formation in vitro. Tubulins were allowed to polymerize in the
presence of 10 M PCNT13 or 10 M Taxol. The microtubule formation was monitored by measuring
absorbance at 340 nm.
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Molecular docking simulation was employed to reveal binding mode between PCNT13 and
tubulins. The three-dimensional structure of PCNT13 was obtained from DFT calculations using the
B3LYP method with 6-31G(d,p) basis set implemented in Gaussian03 package.21 Crystal structures of
tubulin  and  were obtained from the Protein Data Bank (PDB ID: 1SA0).22 Molecular docking was
performed using AutoDock 4.0.23 Ligands and protein structures were prepared using AutoDockTools
1.5.6.24 Grid box was assigned to adequately cover the tubulin  and  subunits and the interface between
them. Docking analysis reveals that PCNT13 binds to the colchicine binding site located at the interface
between  and  subunits of tubulin as illustrated in Fig. 4. (See detailed analysis of the binding site in the
Supporting Information.) Colchicine is known to destabilize microtubules. The docking results are
consistent with the microtubule-destabilizing activity of PCNT13 observed in vitro.
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Fig. 4. The best-docked conformation of PCNT13 on the heterodimer of tubulins. (A) PCNT13
(Magenta) binds to the interface of tubulin  (Light brown) and  (Light blue). (B) Comparison of PCNT13

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(Magenta) binding mode with that of colchicine (Green), a ligand co-crystallized with tubulin  and 
subunits in 1SA0 crystal structure.
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Since microtubule formation plays a critical role in the mitotic (M) phase of cell division,
therefore, we further determined the effect of PCNT13 on A549 cell cycle distribution by flow cytometric
technique using Muse Cell Analyzer (Merck Millipore) and Muse Cell Cycle Assay kit. After 24 h
treatment, the A549 cells treated with PCNT13 (2.5 and 5 M) exhibited a dose-dependent increase in the
proportion of cells in the G2/M phase as 42.8±1.77% and 70.0±1.0%, respectively, compared with
24.0±3.44% of the untreated cells (Fig. 5). The results indicated that the mitosis phase of A549 cell
division was disrupted by the microtubule-destabilizing activity of PCNT13, resulting in the cell cycle
arrest in G2/M phase.
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Fig. 5. Effect of PCNT13 on cell cycle distribution of A549 cells. The cells were treated with vehicle (A)
or 2.5 and 5 M PCNT13 (B and C) for 24 h. The percentages of cell populations in G0/G1, S, and G2/M
phases were determined by using Muse Cell Analyzer and Muse Cell Cycle kit. DNA content histograms
from a representative experiment were shown. Similar results were obtained in three independent
experiments.
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The effect of FL-PCNT13 on microtubule dynamics was similar to that of PCNT13 treatment as
G2/M cell cycle arrest was observed when the cells were treated with 200 M FL-PCNT13 for 24 h, by

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53.3% increase in G2/M phase compared with 16.2% of untreated cells. The fluorophore FITC has been
shown by other groups to localize in cytosol, and when tagged with compounds of interest, the FITC
exhibited minimal interference to the binding of the compounds to the molecular targets.25, 26 These
results are consistent with our finding that the FL-PCNT13 conjugate was found to localize mainly in the
cytoplasm, where microtubules, one of the cytosolic target proteins, were located. Nonetheless, FL-
PCNT13 exhibited less cytotoxic potency (IC50 = 238+7.63 M) compared with the parent compound
PCNT13 (IC50 = 5.17+1.61 M), presumably due to steric hindrance of the FITC moiety.
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In conclusion, we have elucidated a mechanism underlying the apoptosis-inducing activity of 2,3-
diarylindole PCNT13 using the FITC fluorophore connected to the optimum position of the compound
through a hydrophilic linker. This study demonstrated a correlation between cytoplasmic localization,
inhibition of tubulin polymerization, and G2/M arrest effect of PCNT13 in A549 cells. Our results are
consistent with the reported biological effects of other microtubule inhibitors that induce G2/M arrest and
subsequently lead to apoptosis in cancer cells.27 Hence, we conclude that the microtubule destabilization
is the mechanism underlying G2/M cell cycle arrest and apoptosis induction by PCNT13 in A549 lung
cancer cells.
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Acknowledgments
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This project was supported by the Thailand Research Fund (MRG5180073), by the Kasetsart
University Research and Development Institute (KURDI), by the Faculty of Science, Kasetsart University,
and a research grant from the Chulabhorn Research Institute. BT is supported by the Development and
Promotion of Science and Technology Talents Project (DPST). Financial support from the Center of
Excellence for Innovation in Chemistry (PERCH-CIC), Ministry of Higher Education, Science, Research
and Innovation is gratefully acknowledged.
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Appendix A. Supplementary Data
Supplementary data to this article can be found online at XXX.
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Declaration of interests
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☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
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☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests:
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