Anticancer activity of gallic acid template-based benzylidene indanone derivative as microtubule destabilizer

Article abstract of DOI:10.1111/cbdd.12805

Benzylidene indanones have been designed and synthesized from gallic acid, a plant phenolic acid as possible anticancer agent. The best analogue of the series, that is, 3-(3′,4′,5′-trimethoxyphenyl)-4,5,6-trimethoxy-2-(4?-nitrobenzylidene)-indan-1-one (8) exhibited potent cytotoxicity (IC₅₀=3–10?μm) against several human cancer cell lines through microtubule destabilization (IC₅₀=1.54?μm) after occupying colchicine-binding site of β-tubulin. In cell cycle analysis, compound 8 exerted G2/M phase arrest in both MCF-7 and MDA-MB-231 cells and induced apoptosis. It reduced 34.8% solid tumor in in vivo Ehrlich ascite carcinoma in Swiss albino mice at 30?mg/kg dose. In acute oral toxicity experiment, it was tolerable up to 300?mg/kg doses in Swiss albino mice. The lead compound 8 needs to be optimized for better activity.

Full text of DOI:10.1111/cbdd.12805

Received: 16 November 2015
DOI: 10.1111/cbdd.12805
Revised: 21 April 2016
Accepted: 2 June 2016
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R E S E A R C H A R T I C L E
Anticancer activity of gallic acid template-based benzylidene
indanone derivative as microtubule destabilizer
Aastha Singh1,*
Dev Priya2
D. M. Mondhe2
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Kaneez Fatima1,*
Arjun Singh1 Girish Mahajan2
Suaib Luqman1 Debabrata Chanda1
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Ankita Srivastava1
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Sadiya Khwaja1
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Sarfaraz Alam1
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Ajit K. Saxena3
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Feroz Khan1
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Arvind S. Negi1
1CSIR-Central Institute of Medicinal and
Aromatic Plants (CSIR-CIMAP), Lucknow,
Uttar Pradesh, India
2CSIR-Indian Institute of Integrative Medicine
(CSIR-IIIM), Jammu, Jammu and Kashmir,
India
Benzylidene indanones have been designed and synthesized from gallic acid, a
plant phenolic acid as possible anticancer agent. The best analogue of the series,
that is, 3-(3′,4′,5′-trimethoxyphenyl)-4,5,6-trimethoxy-2-(4˝-nitrobenzylidene)-
indan-1-one (8) exhibited potent cytotoxicity (IC50=3–10 μm) against several
human cancer cell lines through microtubule destabilization (IC50=1.54 μm) after
occupying colchicine-binding site of β-tubulin. In cell cycle analysis, compound 8
exerted G2/M phase arrest in both MCF-7 and MDA-MB-231 cells and induced
apoptosis. It reduced 34.8% solid tumor in in vivo Ehrlich ascite carcinoma in Swiss
albino mice at 30 mg/kg dose. In acute oral toxicity experiment, it was tolerable up
to 300 mg/kg doses in Swiss albino mice. The lead compound 8 needs to be
optimized for better activity.
3Amity University, Lucknow, Uttar Pradesh,
India
Correspondence
Arvind S. Negi
Email: arvindcimap@rediffmail.com
K E Y W O R D S
benzylidene indanone, cell cycle, cytotoxicity, Ehrlich ascite carcinoma, tubulin polymerization
inhibition
Cancer is a public health menace. The disease is of a great
concern to both developed and developing countries due to
its high morbidity and mortality. In many countries, it has
become second largest killer after cardiovascular disease.
In 2012, there were 14 million new cases added on and 8.2
million deaths were due to cancer.a Among men, lung can-
cer was the most predominant, while among women, it was
breast cancer. Africa, Asia, and Central and South America
diagnosed more than 60% cancer cases and 70% of total can-
cer deaths. There will be 24 million cancer cases annually
and 14.6 million annual deaths by the end of 2015.b These
troubling figures compel policy makers and the researchers
to combat this disease. Nevertheless, the complexity of the
disease is too much and still today, cancer is a challenge to
the researchers.
cylinders built by polymerization of tubulin dimers, a glob-
ular protein. Microtubules play a key role in various cellular
processes including mitosis. Some of the compounds known
as antitubulins interfere in microtubule dynamics either
through stabilizing or destabilizing (inhibiting) microtubule
assembly. In both the cases, the equilibrium of this process is
disturbed and this interference in microtubule dynamics has
a significant impact on cell division which ultimately causes
cell death.[3,4] Several natural molecules such as paclitaxel,
epothilones, laulimalide, peruloside, and discodermolide
are microtubule stabilizers while podophyllotoxin, combret-
astatins, vinca alkaloids, dolastatins etc. are microtubule
destabilizers.[5–7]
In this study, we modified gallic acid, a plant phe-
nolic acid, to a basic pharmacophore based on structure
activity relationship of some natural antitubulins such as
combretastatin A-4 (CA-4), colchicine, and podophyllo-
toxin (PDT; Figure 1).[8,9] In these natural antitubulins, a
3,4,5-trimethoxyphenyl unit is very crucial to interact with
Microtubules are considered as an attractive target in can-
cer chemotherapeutics.[1,2] Microtubules are rigid hollow
*Both authors contributed equally to this work.
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Chemical Biology & Drug Design 2016; 1–10
wileyonlinelibrary.com/journal/cbdd
© 2016 John Wiley & Sons A/S.
1

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Singh et al.
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FIGURE 1 Natural microtubule
destabilizers, trimethoxyphenyl
unit, basic pharmacophore, and best
analogue of the series (compound 8)
tubulin. This motif is essentially required to induce antitu-
bulin effect in these compounds.[6,8–10] Thus, we designed
a benzylidene indanone unit with a 3,4,5-trimethoxyphenyl
unit in ring A in the pharmacophore. The mode of action of
these compounds was expected through tubulin polymeriza-
tion inhibition. Based on these facts, we synthesized several
benzylidene indanones with a 3,4,5-trimethoxyphenyl frag-
ment and restricted rotation around C–C bond as in case of
natural antitubulin combretastatin A4.[11] The compounds
have been evaluated against human cancer cell lines for cyto-
toxicity. The best analogue of the series has been evaluated
for extensive biology, that is, tubulin polymerization assay,
cell cycle analysis, in vivo anticancer activity, and in vivo
acute oral toxicity. Molecular docking studies revealed that it
occupies colchicine-binding pocket of β-tubulin.
18 mm, flow rate = 18 mL/min) using hexane-ethyl acetate
as eluents. The desired benzylidene indanones were obtained
in good yields (72–89%).
1.2.1
3-(3′,4′,5′-trimethoxyphenyl)-4,5,6-
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trimethoxy-2-(4″-nitrobenzylidene)-indan-1-
one (8)
Yield = 82%; m.p. = 162–64 °C; 1H NMR (300 MHz,
CDCl3):δ3.45(s, 3H, OCH3), 3.73(s, 9H, 3 × OCH3), 3.93(s,
6H, 2xOCH3), 5.28 (s, 1H, 3-CH), 6.38 (s, 2H, 2″ & 6″-CH of
3-phenyl), 7.15 (s, 1H, 7-CH), 7.24 (s, 1H, CH=benzylidene
double bond), 7.62 (d, 2H, 2′ & 6′-CH of 2-benzylidene ring,
J = 8.7 Hz), 8.12 (d, 2H, 3′ & 5′-CH of 2-benzylidene ring,
J = 7.2 Hz); 13C NMR (75 MHz, CDCl3): δ 46.5, 56.6, 56.7,
60.7, 61.3, 101.9, 106.3, 123.8, 131.6, 131.8, 132.0, 136.8,
137.5, 141.3, 143.9, 150.4, 153.6, 155.6, 192.6; HR-ESI-MS
(MeOH): for C28H28NO9 [M + H]+; calcd, 522.1764; found,
522.1755.
1
EXPERIMENTAL SECTION
Chemical synthesis
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1.1
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The synthesis of indanone 7 is previously reported.[12,13]
1.2.2
3-(3′,4′,5′-trimethoxyphenyl)-4,5,6-
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trimethoxy-2-(4″-benzyloxybenzylidene)-indan-
1-one (9)
1.2
GENERAL PROCEDURE
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FOR THE SYNTHESIS OF
BENZYLIDENE INDANONES 8, 9,
10, AND 12
Yield = 89%; m.p. = 190–92 °C; 1H NMR (300 MHz,
CDCl3): δ 3.56 (s, 3H, OCH3), 3.81 (s, 9H, 3 × OCH3),
3.98 (s, 6H, 2 × OCH3), 5.04 (s, 2H, Benzylic), 5.27 (s,
1H, 3-CH), 6.49 (s, 2H, 2″ & 6″-CH of 3-phenyl), 6.94 (d,
2H, 3′ & 5′-CH of 2-benzylidene ring, J = 8.7 Hz), 7.23 9s,
1H, 7-CH), 7.34 (bs, 5H, 5 × CH of benzyl ring), 7.51 (d,
2H, 2′ & 6′-CH of 2-benzylidene ring, J = 8.4 Hz), 7.66
(s, 1H, CH=benzylidene); 13C NMR (75 MHz, CDCl3): δ
46.7, 56.6, 56.7, 60.6, 61.2, 70.4, 101.8, 106.5, 115.2, 127.6,
127.8, 128.6, 129.0, 132.6, 133.6, 134.8, 136.8, 137.3,
137.8, 141.2, 148.9, 150.4, 153.4, 155.2, 160.4, 194.2; ESI-
MS (MeOH): 583.2 [M + H]+, 605.2 [M + Na]+, 621.2
[M + K]+.
To a stirred solution of hexamethoxyindanone 7 (200 mg,
0.51 mmol) in 3% methanolic KOH (6 mL) respective ben-
zaldehyde (0.60 mmol) was added. The reaction mixture
was stirred at room temperature for 2–4 h. The organic sol-
vent was evaporated and residue was taken in ethyl acetate
(20 mL) and acidified with dil. HCl (10%, 10 mL). Organic
layer was washed with water, dried over anhydrous sodium
sulfate, and evaporated in vacuo. The residue thus obtained
was purified through silica gel column (230–400 mesh) by
flash chromatography (glass column, length 130 mm, i.d.

Singh et al.
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residue was taken in ethyl acetate. It was acidified with dil.
HCl (5%, 10 mL). Organic phase was washed with water and
dried over anhydrous sodium sulfate. Organic solvents were
evaporated to give a residue which on recrystallization with
chloroform-hexane afforded compound 13.
1.2.3
3-(3′,4′,5′-trimethoxyphenyl)-
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4,5,6-trimethoxy-2-(4″,4″-
dimethylaminobenzylidene)-indan-1-one (10)
Yield = 72%; m.p. = 182–85 °C; 1H NMR (300 MHz,
CDCl3): δ 2.98 (s, 6H, N-(CH3)2), 3.53 (s, 3H, OCH3), 3.75
(s, 9H, 3xOCH3), 3.96 (s, 6H, 2xOCH3), 5.24 (s, 1H, 3-CH),
6.53 (s, 2H, 2″ & 6″-CH of 3-phenyl), 6.63 (d, 2H, 3′ & 5′-CH
of 2-benzylidene ring, J = 8.4 Hz), 7.21 (s, 1H, 7-CH), 7.47
(d, 2H, 2′ & 6′-CH of 2-benzylidene ring, J = 8.4 Hz); 13C
NMR (75 MHz, CDCl3): δ 30.0, 40.9, 47.0, 56.6, 60.6, 61.2,
101.8, 106.5, 112.6, 133.0, 135.38, 135.7, 137.1, 137.5, 140.9,
148.5, 150.3, 150.8, 153.4, 155.1, 194.2; ESI-MS (MeOH):
520.4 [M + H]+, 542.4 [M + Na]+, 558.2 [M + K]+.
1.2.6
3-(3′,4′,5′-trimethoxyphenyl)-4,5,6-
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trimethoxy-2-(4″-carboxybenzylidene)-indan-1-
one (13)
Yield = 89%; m.p. = 230–31 °C; 1H NMR (300 MHz,
CDCl3): δ3.46 (s, 3H, OCH3), 3.74 (s, 9H, 3 × OCH3), 3.93
(s, 6H, 2 × OCH3), 5.33 (s, 1H, 3-CH), 6.41 (s, 2H, 2″ & 6″-
CH of 3-phenyl), 8.03 (d, 2H, 3′ & 5′-CH of 2-benzylidene
ring, J = 8.7 Hz), 7.27 (s, 1H, 7-CH), 7.59 (d, 2H, 2′ & 6′-CH
of 2-benzylidene ring, J = 8.4 Hz), 7.72 (s, 1H, CH= of ben-
zylidene); 13C NMR (75 MHz, CDCl3): δ 46.5, 56.6, 61.3,
61.4, 101.9, 106.35, 129.9, 130.4, 131.1, 132.2, 133.4, 137.1,
137.3, 140.0, 141.5, 142.7, 149.5, 150.3, 153.4, 155.5, 171.1,
193.9; ESI-MS (MeOH): 521.2 [M + H]+, 543.2 [M + Na]+,
559.1 [M + K]+.
1.2.4
3-(3′,4′,5′-trimethoxyphenyl)-4,5,6-
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trimethoxy-2-(4″-carboxymethylbenzylidene)-
indan-1-one (12)
Yield = 78%; m.p. = 160–62 °C; 1H NMR (300 MHz,
CDCl3): δ 3.44 (s, 6H, OCH3 and COOCH3), 3.72 (s, 9H,
3 × OCH3), 3.94 (s, 6H, 2 × OCH3), 5.30 (s, 1H, 3-CH),
6.39 (s, 2H, 2″ & 6″-CH of 3-phenyl), 7.23 (s, 1H, 7-CH),
7.54 (d, 2H, 2′ & 6′-CH of 2-benzylidene ring, J = 8.1 Hz),
7.69 (s, 1H, CH=benzylidene); 7.93 (d, 2H, 3′ & 5′-CH of
2-benzylidene ring, J = 8.1 Hz); 13C NMR (75 MHz, CDCl3):
δ 46.5, 52.6, 56.6, 56.7, 60.6, 61.2, 61.3, 101.8, 106.4, 129.8,
130.7, 131.1, 132.2, 133.4, 137.1, 137.3, 139.2, 141.4, 142.4,
149.5, 150.4, 153.3, 155.4, 166.8, 193.8; ESI-MS (MeOH):
535.1 [M + H]+, 557.1 [M + Na]+, 573.3 [M + K]+.
1.2.7
Synthesis of 3-(3′,4′,5′-
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trimethoxyphenyl)-4,5,6-trimethoxy-2-(4″-
carbamoylbenzylidene)-indan-1-one (14)
Ester 12 (100 mg, 0.19 mmol) was taken in methanol
(10 mL) and stirred with cooling (5–10 °C). Aqueous ammo-
nia (1 mL) was added to it and stirred for 2 h. Thereafter,
reaction mixture was stirred at room temperature overnight.
Reaction mixture was evaporated and residue was taken in
ethyl acetate. It was washed with water, and organic layer
was dried over anhydrous sodium sulfate and evaporated.
The crude mass was purified through column chromatogra-
phy over silica gel using ethyl acetate-hexane system to get
amide 14.
1.2.5
Synthesis of 3-(3′,4′,5′-
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trimethoxyphenyl)-4,5,6-trimethoxy-2-
(4″,4″,4″-trimethylaminium benzylidene)-
indan-1-one- iodide (11)
1
Compound 10 (100 mg, 0.19 mmol) was taken in dry ace-
tone (10 mL) and anhydrous potassium carbonate (300 mg)
and methyl iodide (0.2 mL, 3.21 mmol) were added and
reaction mixture was stirred at room temperature for 2 h.
Reaction mixture was filtered and washed with acetone. The
filtrate was evaporated and residue was recrystallized with
chloroform-acetone to get 11.
Yield = 42%; m.p. = oil; H NMR (300 MHz, CDCl3):
δ3.44 (s, 3H, OCH3), 3.76 (s, 9H, 3 × OCH3), 3.92 (s,
6H, 2 × OCH3), 5.31 (s, 1H, 3-CH), 6.40 (s, 2H, 2″ & 6″-
CH of 3-phenyl), 7.21 (s, 1H, 7-CH), 7.54 (d, 2H, 2′ &
6′-CH of 2-benzylidene ring, J = 8.1 Hz), 7.67 (s, 1H,
CH=benzylidene); 7.91 (d, 2H, 3′ & 5′-CH of 2-benzylidene
ring, J = 8.1 Hz); ESI-MS (MeOH): 520.2 [M + H]+.
Yield = 84%; m.p. = 269 °C (decompose); 1H NMR
(300 MHz,CDCl3):δ3.32(s,3H,OCH3),3.83(s,6H,N-(CH3)2),
3.92 (s, 9H, 3 × OCH3), 3.94 (s, 6H, 2 × OCH3), 5.29 (s, 1H,
3-CH), 6.36 (s, 2H, 2″ & 6″-CH of 3-phenyl), 7.22 (s, 1H, 7-CH),
7.68 (d, 2H, 3′ & 5′-CH of 2-benzylidene ring, J = 8.4 Hz), 7.78
(d, 2H, 2′ & 6′-CH of 2-benzylidene ring, J = 8.4 Hz); ESI-MS
(MeOH): 534.3 [M − I]+, 535.3 [M + H − I]+.
1.3
Biological evaluation
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The detail of biological assays is in Supporting information.
1.4
Antiproliferative activity by
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sulforhodamine assay
Synthesis of compound 13: Ester 12 (100 mg, 0.19 mmol)
was taken in 5% KOH (methanol:water = 9:1, 6 mL) and
heated at 50 °C for an hour. Solvent was evaporated and
Human cancer cell lines, THP-1 (leukemia), HCT-116
(colon), MCF-7 (breast), T-47D (breast), and A549 (lung),

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Singh et al.
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were originally obtained from American type of cell culture
collection (ATCC), USA, and assay was performed as per
reported method.[14]
1.11
Statistical analysis
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Data expressed as mean or representative of one of three simi-
lar experiments unless otherwise indicated. Both the soft agar
colony formation and cell cycle experiment were performed
in duplicates and results are expressed as Mean SD. For
multiple comparisons, each value was compared by one-way
anova following Dunnett’s test, Tukey’s test and Student’s t-
test in graphpad inStat version 3.06. Data with p value < 0.05
were considered as significant.
1.5
Soft agar colony assay
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Human breast cancer MCF-7 and MDA-MB-231 cells
(5 × 104/mL) were seeded separately in 24-well plates with
or without compound 8 treatment for 24 h.[15] Combretastatin
A-4 (CA-4) and tamoxifen (TAM) were used as standard
anticancer drugs (positive control) for cytotoxicity.
2
RESULTS AND DISCUSSION
Chemistry
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1.6
Tubulin polymerization assay
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2.1
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Tubulin polymerization experiment was carried out as per
reported protocol using ‘assay kit’ from Cytoskeleton,
USA.[16,17] Podophyllotoxin (PDT) was used as standard
inhibitor of tubulin polymerization.
The synthetic strategy was as depicted in Scheme 1. The start-
ing substrate gallic acid (1) was modified to ester 2 using dime-
thyl sulfate. Ester 2 was reduced with lithium borohydride to
get benzyl alcohol 3, which on oxidation with pyridinium chlo-
rochromate (PCC) yielded 3,4,5-trimethoxybenzaldehyde (4).
Aldehyde 4 was treated with 7% methanolic KOH to afford
hexamethoxy chalcone 6 through Claisen–Schmidt reaction.
On treatment with trifluoroacetic acid, chalcone 6 underwent
Nazarov’s cyclization reaction[12] to give a 3-(3,4,5-trime
thoxyphenyl)-hexamethoxy indanone (7). Indanone 7 was
treated with various aromatic aldehydes to get 2-benzylidene
indanones 8, 9, 10, 12, and 13. 2-(4-N,N-Dimethylamino)-b
enzylidene indanone (10) was transformed to its correspond-
ing quaternary ammonium salt (compound 11) by treating
it with methyl iodide in potassium carbonate-methyl iodide
system. 2-(4-Carboxymethyl)-benzylidene indanone (12) was
hydrolyzed with 5% KOH in aqueous methanol (1:9) to afford
corresponding carboxylic acid derivative 13. Benzylidene 13
on treatment with aqueous ammonia-THF system yielded the
corresponding amide derivative 14. All the derivatives were
as isomeric mixtures and were confirmed through spectros-
copy (Supporting Information).
1.7
Molecular docking studies
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In silico docking and visualization studies were completed
with Discovery Studio version 3.5 (Accelrys, USA, 2013), a
software for molecular modeling.[18] To prepare target pro-
tein, the structure of beta-tubulin (PDB ID 4O2B) has been
retrieved from RCSB PDB site (http://www.rcsb.org/pdb).
For pharmacokinetic, metabolism and toxicity (ADMET)
prediction, ADMET Predictor™ was used.
1.8
Cell cycle analysis
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The effect of benzylidene indanone 8 on cell division cycle
of MCF-7 and MDA-MB-231 cells was assessed by flow
cytometry with PI-stained cellular DNA.[19]
1.9
In vivo anticancer activity of 8 against
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Ehrlich ascites tumor (solid)
Ehrlich ascites carcinoma (EAC) experiment was per-
formed[20] on Swiss albino mice using compound 8 at 30 mg/
kg given intraperitoneal (i.p.) after dissolving in carboxym-
ethylcellulose (CMC) from day 1 to 9. 5-Fluorouracil (22 mg/
kg) was used as positive control.
2.2
Biological evaluation
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The benzylidene indanones (8–14) were evaluated for cytotox-
icity against a panel of human cancer cell lines by sulforho-
damine assay.[14] Paclitaxel (taxol) and 5-fluorouracil (5-FU)
were used as standard anticancer drugs (positive control) for
cytotoxicity. Compounds exhibiting IC50 > 50 μm were con-
sidered as inactive. For structure and activity relationship,
mainly the IC50 data in MCF-7 were taken in to considera-
tion. In the series, presence of an electron-withdrawing group
at 2-benzylidene ring exhibited good cytotoxicity. Compound
8 with 4-nitro group (IC50 = 4 μm) and compound 12 with
4-carboxylic ester (IC50 = 24 μm) possessed good cytotoxic-
ity. However, compound 13 with a 4-carboxylic acid group
was weak cytotoxic (IC50 = 48 μm). Compounds 8, 10, 12,
1.10
Acute oral toxicity of benzylidene
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indanone 8
The acute oral toxicity compound 8 was evaluated in accord-
ance with the Organization for Economic Co-operation and
Development (OECD) test guideline No 423 (1987) in Swiss
albino mice.[21,22] Compound 8 was suspended in double-
distilled water and given through oral route at various doses, that
is, 5, 50, 300, and 1000 mg/kg (groups II–IV) on body weight.

Singh et al.
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SCHEME 1 Reagents and conditions: (i) Me2SO4, 30% aqueous KOH, 0 °C,1 h, reflux, 4 h, 76%; (ii) THF, LiBH4, 82%; (iii) DCM, PCC, RT, 79%;
(iv) 7% KOH in MeOH, RT, 91%; (v) TFA, 150 °C, 5 h, 22%; (vi) 3% KOH in MeOH, RT, 72–89%; (vii) Anhyd. K2CO3, dry acetone, MeI, RT, 2 h, 83%;
(viii) 5% KOH in MeOH:H2O (9:1), 50 °C, 1 h, 84%; (ix) THF, aqueous NH3, ice for 2 h then RT for 14 h, 46%
TABLE 1 In vitro cytotoxicity of benzylidene indanones and standard drugs against various human cancer cell lines after 48-h incubation and tubulin
polymerization inhibition
Human cancer cell lines*
Compound
No.
Tubulin polymerization
THP-1, IC50 (μm)
0.2
HCT-116, IC50 (μm)
MCF-7, IC50 (μm)
T-47D, IC50 (μm)
A549, IC50 (μm)
inhibition, IC50 (μm)
8
9
3
7
0.4
4.0 0.3
42.0 2.4
44 1.8
44 2.2
24 1.4
48 2.2
>50
5.0 0.3
50 3.4
40 2.3
50 3.6
10 0.6
>50
1.54 0.12
n.d.
50 1.8
4.0 0.3
12 0.7
18 0.6
30 1.4
>50
>50
10
11
12
13
14
15.0 0.6
20 0.5
40 1.2
12.5 0.2
>50
4.0 0.23
>50
2.08 0.16
n.d.
4
0.2
30 1.6
1.55 0.09
n.d.
32 1.2
>50
8
0.5
>50
n.d.
PDT
CA4
—
—
—
—
3.5 0.6
0.01 0.003
—
—
<1.3
<1.87
0.62 0.12
1.20 0.26
Human cancer cell lines
Standard
drugs
Concentration, μm
THP-1, % inhibition
HCT-116, % inhibition
MCF-7, % inhibition
T-47D, % inhibition
A549, % inhibition
Taxol
5-FU
1.0
5.0
60
90
3
7
45
88
2
3
77
50
2
2
78
60
5
3
88
54
4
2
*IC50 > 50 μm considered inactive.
and 13 exhibited IC50 < 10 μm against one or two cell lines,
which is considered as good activity. Compound 8, the best
compound of the series, exhibiting good activity against all
the cell lines (IC50 = 3–10 μm) was extensively evaluated fur-
ther. However, the cytotoxicity (MCF-7) of compound 8 was
comparable to podophyllotoxin but less active than combret-
astatin A4, paclitaxel, and 5-fluorouracil. Compounds 8, 10,
12, and podophyllotoxin were evaluated for interaction with
tubulin polymerization process (Cytoskeleton, USA).[16,17] The
cytotoxicity and antitubulin data are represented in Table 1.

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Singh et al.
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Further, in soft agar colonization assay,[15] compound 8
of apoptosis. Cell cycle is controlled by a complex series of sig-
naling pathways by which cell grows, replicates its DNA, and
divides. Cell cycle regulation ensures the fidelity of genomic
replication and cell division.[24] Cell cycle arrest is prevention
of normal progression of the cell cycle. It is induced via vari-
ous mechanisms. Some compounds interfere with CDK/cyclin
complexes leaving cells stuck at the G2/M phase border, while
others affect CaMKII phosphorylation, inducing G1 phase
arrest. Among several other mechanisms, interference with
RNA function and inhibition of protein synthesis are import-
ant. As a result of cell cycle interruption, many compounds
ultimately induce apoptosis.[25] In cell cycle analysis such as
other antitubulins, compound 8 induced mainly G2/M phase
arrest in both MCF-7 and MDA-MB-231 cells (8). Induction
of cell cycle arrest in cancer cells constitutes one of the most
prevalent strategies to stop or limit cancer spreading.[26,27]
In tubulin polymerization assay, compound 8 inhibited
polymerization (IC50 = 1.54 μm), while the standard antitu-
bulin podophyllotoxin showed higher effect (IC50 = 0.62 μm).
Microtubules are tube-like hollow filamentous structures
suppressed the growth of MCF-7 cells by 10–96% at 1.5–
50 μg/mL concentrations (IC50 = 8.35 μg/mL) and in MDA-
MB-231 cells by 28–90% (IC50 = 9.68 μg/mL; Table 2).
Combretastatin A4 exhibited 23–96% and 25–97% growth
inhibition in MCF-7 and MDA-MB-231, respectively, under
the same conditions. Tamoxifen exerted 62–100% growth
reduction at 1.5–50 μg/mL concentration in MCF-7 cells.
These results indicate that the cytotoxicity of compound 8
is quite significant and concentration dependent. Soft agar
colonization assay is used to confirm cellular anchorage-
independent growth in vitro and it is an astringent method
to detect the tumorigenic potential of transformed cells and
tumor suppressive effects on transformed cells.[23]
In cell cycle analysis,[19] compound 8 exerted cell cycle
arrest and induced apoptosis in both cells lines, that is, MCF-7
and MDA-MB-231 (Table 3). In both the cancer cell lines, cell
cycle was arrested at G2/M phase (Figure 2A,B). Thus, com-
pound 8 exhibits significant increase in number of subdiploid
cells at all concentrations in both cell lines, indicating induction
TABLE 2 Effect of compound 8 on percent colony formation in MCF-7 and MDA-MB 231 cells in soft agar after 24-h incubation
Condition
Concentration, μg/mL
MCF-7, % Live cells
MCF-7, IC50 (μg/mL)
MDA-MB-231, % Live cells
MDA-MB-231, IC50 (μg/mL)
Control
—
100
100
Compound 8
50
20
10
5
3.62 0.23**
9.69 0.15**
44.25 0.58**
80.76 0.66**
90.43 0.18**
8.35 (16 μm)
10.19 0.87**
19.22 0.45**
52.36 1.43**
66.12 1.09**
72.45 0.35**
9.68 (18.57 μm)
1.5
CA-4
Tam
50
20
10
5
3.72 0.24**
4.81 0.43**
16.27 0.16**
49.48 8.14**
76.75 4.11**
4.32 (10 μm)
0.82 (2.2 μm)
3.14 0.08**
22.76 1.61**
34.4 5.55**
53.54 2.34**
76.67 1.05**
5.37 (12.43 μm)
1.5
50
20
10
5
0
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
1.25 0.25**
5.03 0.35**
19.73 0.71**
38.56 0.17**
1.5
n.d., means not determined.
**p < 0.01 (Dunnett’s test).
TABLE 3 Effect of compound 8 on different phases of cell cycle of MCF-7 cells and MDA-MB-231 after 24-h incubation
MCF-7
% of Cells
G0/G1
MDA-MB-231
% of Cells
G0/G1
Concentration
of 8 (μg/mL)
S
G2/M
12 2.83
Apoptosis
S
G2/M
Apoptosis
Control
50
76.25 4.03
61.6 0.14
64.8 8.77
74.45 4.31
57.45 13.22
52.3 10.61
8.85 1.20
2.9 0.00
7.05 0.21
5.45 3.46
4.8 2.26
5.95 2.19
9.1 3.11
74.8 0.14
43.45 0.92
78.25 0.07
83.9 0.00
78.1 0.28
78.25 2.19
9.3 0.00
11.95 0.21
43.25 0.21
12.2 0.00
8.3 0.14
3.2 0.42
4.85 0.92
3.85 0.07
3.1 0.28
2.75 0.07
2.65 0.07
13.9 1.70
14.45 9.26
12.2 2.69
13.05 5.16
14.15 8.41
17.35 1.77
14.2 5.52
10.55 0.64
14.2 3.11
13.9 2.69
8.35 0.21
5.6 0.28
4.65 0.07
9.35 0.92
7.85 2.33
20
10
5
9.7 1.13
1.5
11.15 0.21

Singh et al.
7
|
A
a
b
c
d
e
f
B
a
b
c
d
e
f
FIGURE 2 Effect of compound 8 on different phases of cell cycle of (A) MCF-7 and (B) MDA-MB 231: (a) control, (b) 50 μg/mL, (c) 20 μg/mL, (d)
10 μg/mL, (e) 5 μg/mL, (f) 1.5 μg/mL
formed on association of α/β-tubulin heterodimers. They
serve as cytoskeletons of the cells and form framework for
structures such as spindle apparatus useful in chromosome
segregation during cell division. Microtubules play diverse
roles in cellular structure and function.[28] Thus, their crucial
role in mitotic process makes them an attractive target, and

8
Singh et al.
|
the drugs that affect microtubule assembly by disturbing the
machinery are useful especially in the treatment of cancer.
Antitubulins have been one of the most successful approach-
es in cancer chemotherapeutics (4), and many drug candi-
dates are under various phases of clinical trials (8).
In silico Pharmacokinetic studies showed that compound
8 slightly violates Lipinski rule for one parameter (molecular
weight = 521 > 500). It is lipophilic in nature, lacks water
solubility, and has poor oral absorption. It has high tendency
to cross blood brain barrier. Its various PK parameters vol-
ume of distribution (0.88–1.56 L/kg), blood-to-plasma con-
centration (0.64), and clearance rate (7.47 mL/min) etc. have
been determined. All these parameters seem to be in nor-
mal range. Five probable metabolites (M1–M5) are mostly
demethylated phenolic derivatives. CYP 2C8 and CYP 3A4
are mainly involved in the metabolism of indanone 8. The
details have been provided in Supporting information.
Ehrlich ascites carcinoma is a spontaneous murine mam-
mary adenocarcinoma adapted to ascites form and carried out
by serial intraperitoneal passages in outbred mice.[31] From
day 1 to 9, first treatment group was treated with benzylidene
indanone 8 (30 mg/kg i.p.). The second treatment group was
treated with 5-fluorouracil, a standard drug of solid tumors
(22 mg/kg, i.p.) from day 1 to 9. The control group was
administered normal saline (0.2 mL, i.p.) from day 1 to 9.
Compound 8 exhibited only 34.8% tumor reduction, while
5-fluorouracil exerted 53.5% tumor reduction (Table 5).
Molecular docking studies (Table 4) revealed that com-
pound 8 occupies colchicine-binding pocket of β-tubulin. It
has LibDock score of 83.66 comparable to colchicine (92.22),
but lower than podophyllotoxin (120.80). The residual amino
acids confirmed the same binding site of compound 8 like
colchicine. The docking pose in Figure 3 shows that amino
acid residue of β-tubulin is close proximity of compound 8.
Inourpharmacophoredesigning,a3,4,5-trimethoxyphenyl
motif has been placed in ring A. This motif is supposed to
interact with tubulin. Based on calculations of ligand–tubu-
lin association constants, it was reported that residue β:316
(ALA316) of tubulin is directly involved in binding the com-
mon trimethoxyphenyl unit of ring A of colchicine.[29] Later,
it was reported that residue β:318 (VAL318) is more crucial
and has strong binding with the middle methoxy group of tri-
methoxyphenyl unit of colchicine with various isotypes (βIV,
βII, and βIII) of β-tubulin.[30]
TABLE 4 Comparison of docking score and amino acid residues of β-tubulin (PDB ID 4O2B) in the binding site pocket of colchicine
Binding pocket amino acid residues (bold represents the similar amino acid residues with
colchicine)
Compound
LibDock score
H-bond
Colchicine
92.22
No
VAL238, CYS241, LEU242, LEU248, ALA250, ASP251, LYS254, LEU255, ASN258, MET259,
THR314, VAL315, ALA316, ALA317, ILE318, ASN350, VAL351, LYS352, ALA354
Podophyllotoxin
120.80
83.66
No
No
CYS241, LEU248, ALA250, LYS254, LEU255, ASN258, MET259, THR314, VAL315,
ALA316, ALA317, ILE318, ASN350, VAL351, LYS352, THR353, ALA354
VAL238, CYS241, LEU242, LEU248, ALA250, ASP251, LYS254, LEU255, ASN258,
MET259, THR314, VAL315, ALA316, ILE318, ASN349, ASN350, LYS352, THR353, ALA354
Compound 8
FIGURE 3 Representing the best
conformation of colchicine (A) and
superimposition of colchicine (yellow
color) and Podophyllotoxin (B) and
compound 8 and colchicine (yellow
color) (C) in the binding site pocket
of β-tubulin. Also showing the amino
acid residues of β-tubulin (PDB ID
4O2B) in close proximity of colchicine
TABLE 5 Effect of compound 8 on body weight of mice and against Ehrlich tumor (solid)
Av. Body weights (g) of animals on days
Day 13
% Tumor
growth
inhibition
Av. Body
weights (g)
Av. Tumor weights
(mg)
Treatment groups
1
5
9
Mortality
Compound 8 (30 mg/kg i.p.)
Fluorouracil (22 mg/kg i.p.)
20.71
20.71
21.5
21.85
21.57
22.9
22.14
20.28
23.4
21.85
20.0
23.6
1006.71 100.75
718.71 67.11
1545.5 105.25
34.80
53.49
–
0/7
0/7
Normal control
NS (0.2 mL i.p.)
0/10

Singh et al.
9
|
There was no mortality throughout the experimental period.
Hence, the anticancer effect of compound 8 was moderate
as compared to clinical drug, 5-fluorouracil. EAC is referred
to as an undifferentiated carcinoma, originally hyperdiploid,
high transplantable capability, no-regression, rapid prolifera-
tion, short life span, 100% malignancy.[32] However, Ehrlich
ascites tumor cells are much more difficult to break than most
non-malignant somatic cells.[33]
and albumin levels (Table 6) and also differential leukocyte
count (Figure 5). Benzylidene indanone 8 was well tolera-
ble up to 300 mg/kg dose in Swiss albino mice. However,
there was some indication of higher levels of albumin, creat-
inine, triglycerides, and total cholesterol at 1000 mg/kg dose
which were statistically insignificant. It indicated caution for
safety at higher dosage of compound 8 and suggests evaluat-
ing it for longer duration toxicity experiments.
Finally, compound 8 was evaluated for acute oral toxic-
ity in Swiss albino mice in accordance with the OECD test
guideline No 423 (1987).[21,22] Acute oral toxicity is used to
assess the ability of a substance to cause adverse effects with-
in a short period of time following dosing or exposure. When
in vivo testing is performed, estimation of rodent acute tox-
icity is an important task in safety assessment of drug candi-
dates. Compound 8 was suspended in double-distilled water
and given through oral route at various doses, that is, 5, 50,
300, and 1000 mg/kg (groups II–IV) on body weight. Control
animals (group I) received only vehicle.
A
There were no observational changes, morbidity, and
mortality throughout the experimental period in all the
groups of experimental animal up to the dose levels of
300 mg/kg including body weight. However, at 1000 mg/kg
dose, there was loss in body weight. However, animals on
gross pathological study showed insignificant changes in any
of the organs studied including their absolute and relative
weight (Figure 4A,B). In most of the cases, blood and serum
samples upon analysis showed non-significant changes in
all the blood and liver parameters studied such as red blood
cells (RBC) count, white blood cells (WBC) count, serum
glutamate-pyruvate transaminase (SGPT), serum glutam-
ic oxaloacetic transaminase (SGOT), alkaline phosphatase
(ALKP) activity, and creatinine, triglycerides, cholesterol,
B
FIGURE 4 Effect of compound 8 as a single acute oral dose at 5, 50,
300, and 1000 mg/kg on absolute and relative organ weight in Swiss albino
mice (n = 6, non-significant changes were found compared to control)
TABLE 6 Effect of compound 8 as a single acute oral dose at 5, 50, 300, and 1000 mg/kg on body weight, hematological, and serum biochemical
parameters in Swiss albino mice (Mean SE; n = 6)
Dose of compound 8 at mg/kg body weight as a single oral dose
Parameters
Control
5 mg/kg
50 mg/kg
300 mg/kg
1000 mg/kg
Body wt. 7th day (g)
Hemoglobin (g/dL)
RBC (millions/mm3)
WBC (thousands/mm3)
ALKP (U/L)
21.74 0.53
17.75 0.17
6.73 0.28
21.85 0.77
18.25 0.30
6.60 0.31
22.60 0.50
17.19 0.46
6.72 0.04
21.92 0.84
16.93 0.11
6.34 0.34
19.25 1.40
16.01 0.56*
5.92 0.37
12.06 0.66
225.10 20.32
25.09 1.86
26.78 2.25
2.77 0.19
13.36 1.14
225.26 13.82
27.84 1.22
25.64 0.95
2.84 0.20
11.60 0.37
202.41 31.04
28.21 1.08
28.86 1.32
3.00 0.13
12.11 0.69
200.59 19.19
31.28 1.05
28.42 1.60
3.25 0.13
12.50 0.42
190.29 22.47
28.06 2.40
29.92 1.00
3.20 0.13
SGOT (U/L)
SGPT (U/L)
Albumin (g/dL)
Creatinine (mg/dL)
Triglycerides (mg/dL)
Serum protein (mg/mL)
Total cholesterol (mg/dL)
0.11 0.03
0.18 0.07
0.21 0.04
0.20 0.05
0.24 0.05
156.5 5.28
6.18 0.07
179.0 4.69
6.74 0.11
197.2 24.73
6.22 0.13
199.6 9.92
6.87 0.15*
110.42 6.05
196.7 17.87
6.89 0.21*
118.47 5.87
101.94 3.27
104.06 5.41
112.83 7.03
*p < 0.05 compared to control.

10
Singh et al.
|
FIGURE 5 Effect of compound 8 as a single acute
oral dose at 5, 50, 300, and 1000 mg/kg body weight
on differential leukocytes counts in Swiss albino mice
(Mean SE; n = 6; *p < 0.05 compared to control)
[6] A. S. Negi, Y. Gautam, S. Alam, D. Chanda, S. Luqman, J. Sarkar, F. Khan,
R. Konwar, Bioorg. Med. Chem. 2015, 23, 373.
3
CONCLUSION
|
[7] G. C. Tron, T. Pirali, G. Sorba, F. Pagliai, S. Busacca, A. A. Genazzani,
J. Med. Chem. 2006, 49, 3033.
In conclusion, benzylidene indanones are potential anticancer
agents. The representative compound 8 of this series exhib-
its potent in vitro anticancer activity against various human
cancer cell lines. Its anticancer activity is attributed through
microtubule destabilization. Compound 8 shows moderate in
vivo anticancer activity against solid tumors at 30 mg/kg, and
in toxicity profile, it seems tolerable up to 300 mg/kg dose.
It can be optimized for better activity, and soft drug design
approach may also be used in future to optimize it for safer
anticancer lead molecule.
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ACKNOWLEDGMENTS
The financial support from SERB-DST India is duly
acknowledged.
[17] J. C. Lee, S. N. Timassheff, Biochemistry 1997, 16, 1754.
[18] S. Alam, F. Khan, Drug Des. Dev. Ther. 2014, 8, 183.
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CONFLICT OF INTEREST
Authors declare no conflict of interest.
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Chem. Toxicol. 2007, 45, 1928.
NOTES
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