3-Arylindanones and related compounds as antiproliferative agents against colorectal cancer

Article abstract of DOI:10.1111/cbdd.13574

Diverse benzylidene indanones and their derivatives were synthesized as anticancer agents. Two of the analogues, that is 7 and 22, exhibited significant antiproliferative activity against several human cancer cell lines. Both the compounds possessed antimitotic activity and induced apoptosis in DLD1 colorectal adenocarcinoma cells through activation of caspase pathways. In cell cycle analysis, both the compounds induced predominantly G2/M phase arrest in DLD1 cells. Molecular docking studies revealed that compound 7 occupies colchicine binding pocket of β-tubulin. Both the compounds were safe in acute oral toxicity in rodents. Both the compounds are further being optimized for better efficacy.

Full text of DOI:10.1111/cbdd.13574

DR FEROZ KHAN (Orcid ID : 0000-0002-2704-5925)
DR ARVIND SINGH NEGI (Orcid ID : 0000-0002-1236-2493)
Article type : Research Article
3-Arylindanones and related compounds as antiproliferative agents against
colorectal cancer
Ankita Srivastava,#1,3 Kusumoori Ravi,#1 Kaneez Fatima,1,3 Mayank Maheshwari,2 Raghib
Ashraf2, Mohammad Hasanain2, Pankaj Yadav,1 Hina Iqbal,1 Yogesh Kumar,1 Suaib
Luqman,1,3 Debabrata Chanda,1,3 Feroz Khan,1,3 Karuna Shanker,1,3 Jayanta Sarkar,2,3 Arvind
Singh Negi*1,3
1CSIR_Central Institute of Medicinal and Aromatic Plants (CSIR-CIMAP), P.O. CIMAP, Kukrail Picnic Spot
Road, Lucknow-226 015, India.
2CSIR- CSIR-Central Drug Research Institute, Jankipuram Extension, Lucknow-226031, U.P. , INDIA.
3Academy of Scientific and Innovative Research (AcSIR), New Delhi-110001 (India).
#equal contribution
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.13574
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Abstract
Diverse benzylidene indanones and their derivatives were synthesized as anticancer agents.
Two of the analogues i.e. 7 and 22 exhibited significant antiproliferative activity against
several human cancer cell lines. Both the compounds possessed antimitotic activity and
induced apoptosis in DLD1 colorectal adenocarcinoma cells through activation of caspase
pathways. In cell cycle analysis, both the compounds induced predominantly G2/M phase
arrest in DLD1 cells. Molecular docking studies revealed that compound 7 occupies
colchicine binding pocket of β-tubulin. Both the compounds were safe in acute oral toxicity
in rodents. Both the compounds are further being optimized for better efficacy.
Keywords: cancer; apoptosis; caspase; antitubulin; acute oral toxicity.
CIMAP Communication No. CIMAP/PUB/2018/96
*Corresponding author: Dr. Arvind Singh Negi, Scientist, CSIR-CIMAP, P.O. CIMAP, Kukrail Picnic Spot
Road, Lucknow-226015, U.P., India; E-mail: arvindcimap@rediffmail.com ; Tel: +91-522- 2342676 x 327;
Fax: +91-522-2342666;
1. INTRODUCTION
Colorectal cancer is a multifaceted disease which is therapeutically challenged. Globally, it is
the third most common cancer after lung and breast cancers, which has caused about 1.80
million human deaths in 2018 (WHO Factsheet, 2018). It is more prevalent in developing
countries and less pervasive among women than men. It is estimated that there will be 60% of
rise in global colorectal cancer burden by 2030 (Farooqi et al., 2019). Most of the colorectal
cancers are due to old age, life style factors and only a few cases due to genetic disorders.
Some risk factors include diet, smoking, obesity, and lack of physical activity. The common
treatments include surgery, radiation, chemotherapy, and target therapy. Chemotherapy is an
integral part of treatment in advance stages of the disease which includes 5-fluorouracil,
irinotecan, oxaliplatin, and capecitabine etc. (Fig. 1). Available therapies for treatment show
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only limited efficacy and there are many complications with relapse in 30-50% of cases
(Thomas, 2016), which warrants for the development of new, efficient, and safer
chemotherapeutics.
Tubulin is a globular protein that plays an important role in cell growth and
development. Alpha-beta tubulin dimer polymerises to microtubules and afterwards
depolymerises also (Jordan & Wilson, 2004). Subsequently, microtubule bundles are formed
and lead to mitosis in cells during G2/M phase. This polymerisation/depolymerisation
process is in dynamic equilibrium. Any disturbance in this process leads to cell cycle arrest.
Owing to an important role in mitosis, tubulin has been an important target for cancer drug
development. Several successful drugs have been developed and being used as clinical drugs
notably paclitaxel, docetaxel, vincristine, vinblatine and many more are as clinical candidates
(Fig. 1) (Negi et al., 2015).
Fragment Based Drug Discovery (FBDD) is one of the recent approaches in medicinal
chemistry to achieve bioactive compounds [Keseru et al. 2016]. It identifies low molecular
weight fragment or motif exhibiting quality interactions with biological targets to elicit a
particular pharmacological response. In the present study, a specific motif 3,4,5-
trimethoxyphenyl is placed at ring A of indanone pharmacophore. Some of the naturally
occurring antitubulin compounds like colchicine, podophyllotoxin, and combretastatin A4 etc
possess this fragment which interacts with the β-tubulin at α/β junctions to elicit antitubulin
effects (Negi et al., 2015). Further, we have diversified the indanone based anticancer
pharmacophore especially for nitrogen moiety. Twenty six diverse 3-arylindanone based
compounds were prepared and evaluated for biological activity against a penal of human
cancer cell lines. Two of the potential compounds were evaluated for detailed cancer
pharmacology. Both the potent compounds were also evaluated for in-vivo acute oral toxicity
in Swiss-albino mice.
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2 EXPERIMENTAL SECTION
2.1. Chemistry
The syntheses of potent compounds 7 and 22 are given in the manuscript, rest of the
compounds are given in Supplementary information (S1-S3).
2.1.1. Synthesis of compound 7
Indanone 1 (250mg, 0.64 mmol) was taken in dioxane (10mL). To this stirred reaction
mixture selenium dioxide (300mg, 2.72 mmol) was added in fractions and reaction mixture
was refluxed for 5 h. Dioxane was evaporated and residue was taken in ethyl acetate. It was
washed with water, dried over anhydrous sodium sulphate and evaporated. The crude mass
was purified through silica gel column to get compounds 6, 7 and 8.
2-Hydroxy, 3-(3´,4´,5´-trimethoxyphenyl)-4,5,6-trimethoxy-ind-2-en-1-one (7)
1
Yield:23%; m.p.=oil; H NMR (300MHz, CDCl3): δ3.32 (s, 3H, OCH3), 3.80 (s, 6H,
2xOCH3), 3.88 (s, 3H, OCH3), 3.96 (s, 6H, 2xOCH3), 6.65 (s, 2H, 2xCH of 3-phenyl ring),
13
6.97 (s, 1H, 7-CH); C NMR (75MHz, CDCl3): δ56.02, 56.22, 56.48, 60.86, 61.10, 61.45,
105.33, 105.93, 124.71, 127.68, 128.46, 129.10, 138.51, 147.51, 148.36, 152.34, 153.96,
160.40, 192.02; ESI mass for C21H22O8, 401 [M-H]+, 441 [M+K]+ , Negative mode: 401 [M-
H]-; HRMS (ESI-TOF) m/z [M+H] Calcd for C21H22O8 401.1236 , found 401.1235.
3-(3´,4´,5´-Trimethoxyphenyl)-4,5,6-trimethoxy--indan-1,2-dione (6)
1
Yield:16%; m.p.= 193-195oC ; H NMR (300MHz, CDCl3): δ3.61 (s, 3H, OCH3), 3.75 (s,
9H, 3xOCH3), 4.05 (s, 3H, OCH3), 4.08 (s, 3H, OCH3), 4.71 (s, 1H, 3-CH), 6.33 (s, 2H,
2xCH of 3-phenyl ring), 7.61 (s, 1H, 7-CH); 13C NMR (75MHz, CDCl3): δ49.87, 56.61,
56.82, 61.02, 61.24, 61.64, 102.42, 105.91, 133.16, 133.67, 137.98, 138.20, 150.99, 151.95,
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154.01, 155.68, 187.19, 198.44; ESI mass for C21H22O8, 403 [M+H]+, 425 [M+Na]+ , 441
[M+K]+ , Negative mode: 401 [M-H]-; HRMS (ESI-TOF) m/z [M+H] Calcd for C21H22O8
403.1392, found 403.1384.
1,2,3,5,6,7-Hexamethoxy-indeno[2,1b]-9-oxo-benzo[b]furan (8)
1
Yield:40%; m.p.=oil; H NMR (300MHz, CDCl3): δ3.90 (s, 3H, OCH3), 3.95 (s, 3H,
OCH3), 3.98 (s, 3H, OCH3), 4.00 (s, 3H, OCH3), 4.02 (s, 3H, OCH3), 4.07 (s, 3H, OCH3),
13
7.04 (s, 1H, 4-CH), 8.07 (s, 1H, 8-CH); C NMR (75MHz, CDCl3): δ56.14, 56.52, 60.88,
61.14, 61.27, 61.85, 105.23, 106.33, 126.62, 129.94, 132.65, 136.51, 141.19, 146.76, 146.91,
148.96, 153.42, 153.77, 156.09, 187.73; ESI mass for C21H20O8, 400 [M]+.
2.1.2. Synthesis of 3-(4'-N,N-Dimethylaminophenyl)-4,5,6-trimethoxyindan-1-one (22)
It was prepared as per our previously reported method (Saxena et al., 2008).
Yield:29%; m.p.=89-91oC; 1H NMR (300MHz, CDCl3): δ2.54 (dd, 1H, 2-CH2, J=17.1Hz and
2.4Hz), 3.10-3.19 (dd, 1H, 2-CH2, J=11.4Hz and 8.1Hz), 2.98 (s, 6H, N-(CH3)2), 3.36 (s, 3H,
OCH3), 3.89 (s, 6H, 2xOCH3), 4.48-4.52 (dd, 1H, 3-CH, J=2.4Hz and 5.4Hz), 6.63 (d, 2H,
2xCH of 3-Phenyl, J=8.7Hz), 6.94-6.97 (d, 2H, 2xCH of 3-Phenyl, J=8.7Hz), 7.07 (s, 1H, 7-
13
CH); C NMR (75MHz, CDCl3): δ41.14, 41.18, 47.80, 56.59, 60.54, 61.23, 100.67, 107.78,
113.39, 128.25, 132.49, 132.70, 145.69, 149.80, 150.85, 155.09, 206.35; ESI mass for
C27H27NO5, 342 [M+H]+, 364 [M+Na]+ ; ESI-HRMS for C27H28NO5 calc, 342.1705 ; found
342.1685.
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2.2 Biological evaluation
The details of all the biological assays are in Supplementary information (S4-S8).
2.2.1 Cytotoxicity evaluation by Sulphorhodamine assay
Cytotoxic activity of the compounds was assessed against human cancer cell lines viz. A549
(lung carcinoma), DU145 (Prostate carcinoma), FaDU (Hypopharyngeal carcinoma), DLD1
(colorectal adenocarcinoma), MCF-7 (Breast adenocarcinoma), and healthy/normal cell line
HEK-293 (Human embryonic kidney) by Sulphorhodamine B dye based plate assay as per
reported method (Adaramoye et al., 2011). Tamoxifen, doxorubicin, podophyllotoxin and
paclitaxel were used as positive controls.
2.2.2 Cell cycle analysis
The effect of potent compounds 7 and 22 on DLD1 cell division cycle was assessed by flow
cytometry with PI-stained cellular DNA (Sarkar et al., 2009).
2.2.3 Tubulin polymerisation assay
Tubulin polymerization assay experiment was performed using ‘assay kit’ procured from
Cytoskeleton, Inc. Denver, USA. The procedure was followed as per reported protocol (Lee
& Timassheff et al., 1997). The standard antimitotic agents nocodazole, podophyllotoxin (as
destabilizers) and paclitaxel (as stabilizer) were used as positive controls.
2.2.4 Apoptosis induction by caspase pathway
Lysate of DLD1 cells was prepared after treating with compounds 7 and 22 and further
processed as described in Supplementary information. Doxorubicin was used as positive
control.
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2.2.5 Molecular docking studies
Docking studies of compound 7 (Destabilizer) was determined at colchicine binding pocket
of β-tubulin (Prota et al., 2014), while both the enantiomers compound 22 (R) and 22 (S)
(Stabilizers) were assessed at paclitaxel binding pocket of tubulin (Atherton et al., 2017)
using docking software AutoDock Vina. Further, preliminary ‘Drug likeliness’ was also
assessed for both the potent compounds 7 and 22 as per ‘Lipinski’s rule of five’ using
Discovery Studio 3.5v software (Accelrys, USA).
2.2.6 Acute oral toxicity
Considering significant anti-cancer activity of compounds 7 and 22 in in-vitro models, two
different acute oral toxicity experiments were carried out in accordance with the Organization
for Economic Co-operation and Development (OECD) test guideline No 423 (1987) in Swiss
albino mice (Allan et al., 2007) after IAEC (Institutional Animal Ethical Committee)
approved protocols vide ref. No. CIMAP/IAEC/2016-19/01, dated 09/2/2017.
2.2.7 Statistical analysis
Statistical analysis for cytotoxicity was carried out in Microsoft Excel. The ANOVA
followed by Tukey’s multiple comparison test was used to assess the statistical significance
of vehicle verses treatment groups. Results are presented as the means ± SE. Differences with
a P value <0.05 were considered significant. In acute oral toxicity, Turkey’s multiple column
comparison method was used at 5% significant level (n=6).
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3 RESULTS
3.1 Chemical Syntheses
The synthetic strategy was as depicted in scheme 1. Gallic acid was starting substrate, which
was modified to indanones 1 as per our reported method (Saxena et al., 2008). Indanone 1
was transformed to various types of indanone derivatives. It underwent Claisen-Schmidt
reaction with various aromatic aldehydes in 7% alkali in methanol to give 2-benzylidene
indanones 2-5 in 79-91% yields. Indanone 1 was oxidised with selenium dioxide in dioxane
to give a 1,2-diketo derivative (6), keto-enol derivatives 7 and an indolobenzofuran
derivatives 8 as a condensed product of 7. Indanone 1 was treated with dimethylcarbonate in
THF using sodium hydride as a base to afford methyl-2-indolylcarboxylate (9). Ester 9 was
hydrolysed with 5% alkali to get corresponding acid 10. Indanone 1 was converted to its
keto-oxime 11 by treating it with hydroxylamine hydrochloride in ethanol. Oxime 11
underwent Beckmann rearrangement by treating it with p-toluene sulphonic acid (p-TSA) to
afford a lactam 12 (Gawley, 1988). Another indanone series was different than the previous
indanones 1. 3-(4-nitrophenyl)-4,5,6-trimethoxy-indanone-1 (13) was prepared in two steps.
3,4,5-Trimethoxyacetophenone was condensed with 4-nitrobenzaldehyde to give a chalcones
moiety i.e. 3-(4-nitrophenyl)-1-(3,4,5-trimethoxyphenyl)-2-propen-1-one which was further
transformed to indanones 13 by treating with trifluoroacetic acid. Several diverse analogues
were prepared onto indanones 13. It was treated with aromatic benzaldehydes to get 2-
benzylidenes (14-18). Pyridine-2-carboxaldehyde reacted with indanones 13 in an alkaline
medium to give 2-pyridinyl-methine indanones (17). Oxime of indanones 13 was prepared as
described previously for 12. Similarly, another indanones derivative 22 was prepared and
further 2-benzylideneindanones (23-26) were prepared as described earlier. All the
intermediates and final compounds were confirmed by spectroscopy [Supplementary
information].
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3.2 Purity profile of compounds 7 and 22
The purity of potential anticancer lead compounds 7 and 22 was assessed on Waters UPLC
system, using column C-18 (BEH 130Å, 1.7 50mm, 1.7µm, Waters, USA), PDA (190-400
nm) at max (282 nm and 263 nm). Under optimised chromatographic condition, compounds 7
and 22 were eluted at 3.01 min and 1.36 min without any interference of any neighbouring
peaks. The purity of the synthesized compounds 7 and 22 were 99% and 98.39%,
respectively.
3.3 Biological evaluation
3.3.1 Cytotoxicity evaluation by Sulphorhodamine assay
The cytotoxicity results of indanone derivatives exhibiting significant activity among 2-26
and standard drugs are depicted in Table 1. The preliminary evaluation of compounds was
done at 50µM concentration. Only six compounds exhibited significant cytotoxicity while
two of the compounds i.e. 7 and 22 showed potential cytotoxicity against most of the cell
lines. Compound 7 exhibited cytotoxicity IC50 in the range of 2.22-4.80 µM while compound
22 had IC50 in the range of 1.23-10.87 µM. Selectivity index (SI) was calculated using
healthy cell lines (HEK-293) to know the safety profile in-vitro. Compounds 7 and 22
exhibited SI >21 and 97 indicating quite safe (SI≥10), as compared to the clinical drugs
possessing very poor SI (tamoxifen, 1.29 and doxorubicin, 0.56).
3.2.2 Cell cycle analysis
Cell cycle has sequence of events which duplicates its genome and ultimately leads to cell
division into two daughter cells. There are several regulators at various phases of cell cycle
progression (Sherr, 2000). Compounds 7 and 22 both induced G2/M phase arrest in DLD1
colorectal cancer cell lines (Figure 2). This effect was more prominent at 24h but at 48h this
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effect was almost negligible. Both the compounds (7 and 22) also exerted S phase arrest in
DLD1 cells. However, G2/M phase arrest was comparatively more predominated over S
phase arrest.
3.2.3 Tubulin polymerisation assay
Compounds 7 and 22 exhibiting potential cytotoxicities were further evaluated for their effect
on tubulin polymerisation. As shown in Table 1, compound 7 inhibited tubulin
polymerisation strongly (IC50=0.57μM) thus acting as microtubule destabiliser quite
comparable to podophyllotoxin (IC50=0.72μM) and nocodazole (IC50=0.62μM). Conversely,
compound 22 exhibited stabilizing effect which was much weaker (EC50=1.8μM) than that of
paclitaxel (EC50=0.025μM) (Fig. 3). Overall, both the compounds exhibited antimitotic
effects.
3.2.4 Apoptosis induction by caspase pathway
In western blot experiments, compounds 7 and 22 cleaved PARP (Poly ADP-ribose
polymerase) enzyme at their IC50 values (Figure 4). Both the compounds cleaved PARP at
their IC50 concentrations (2.22μM and 1.23μM) to indicate apoptosis induction through
caspase cascade pathway. During apoptosis, PARP is cleaved by the activation of caspase
pathway. However, this effect was moderate and only up to 24h of exposure of compounds.
3.2.5 Molecular docking studies
In molecular docking studies, compound 7, podophyllotoxin, and colchicine all the ligands
bound to the colchicine binding pocket (PDB ID: 4O2B) and shared almost equal interacting
amino acids within the region of 4Å radius (Table S7A). There were seven common amino
acid residues (LEU β:248, , LYS β:254, LEU β:255, ASN β:258, , ALA B:316, LYS β:352,
ALA β:354) to all the three ligands, indicating the occupancy of same pocket. Compound 7
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showed docking energy of -6.1 kcal/mol which was comparable with the standard
destabilizers podophyllotoxin (-7.2 kcal/mol) and colchicine (-8.5 kcal/mol) (Fig. 5A).
Another tubulin target PDB ID: 5M5C a tubulin-paclitaxel bound complex was taken for
docking studies of compound 22 and control drug paclitaxel (Table S7B). Both 22 (R) and 22
(S) enantiomers were used for docking studies. There were nine common residual amino
acids (VAL β:23, LEU β:230, ALA β:233, PHE β:272, LEU β:275, PRO β:360, ARG β:369,
LEU β:371, SER β:374) common in paclitaxel and compounds 22 (R) and 22 (S) indicating
that these ligands occupy almost the same binding pocket of tubulin. Compound 22 (R)
showed relatively low binding (-6.5 kcal/mol) as compared to isomer 22 (S) (-7.1 kcal/mol)
of whereas paclitaxel showed little higher docking energy of -8.2 kcal/mol (Fig. 5B). Both
the compounds 7 and 22 did not show any deviation as per Lipinski rule [Supplementary
information, Tables S7A, S7B, and S7C].
3.2.6 Acute oral toxicity
Owing to potential antiproliferative efficacy of compounds 7 and 22, both were evaluated for
safety aspects in rodent model by two independent experiments (Table 2 and Figure 6A/B;
Table 3 and Figure 7A/B). Four different oral doses (5 mg/kg, 50 mg/kg, 300 mg/kg, and
1000 mg/kg) of compounds 7 and 22 were given to Swiss-albino mice as single oral dose.
There was no morbidity or mortality in experimental animals treated with compound 7 up to
the dose level 1000 mg/kg in the acute oral toxicity test. Most of biochemical parameters
showed non-significant changes. However, SGOT and SGPT levels showed significant
increase in group of animals treated with the compound at 1000 mg/kg as single acute oral
dose. When an anticancer compound administered orally, it is quite possible. However, safety
index and therapeutic profile of the compound may be important to decide its dose and route
of administration to use this compound judiciously (Muller & Milton, 2012). Other
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parameters studied showed non-significant changes. It was absolutely safe up to 300 mg/kg
dose.
However, compound 22 was safer than 7 up to 1000 mg/kg oral dose. There was no
morbidity or mortality in the animals treated with compound 22 up to the dose level 1000
mg/kg in the acute oral toxicity. All the haematological, biochemical parameters and gross
pathological analysis showed non-significant changes.
DISCUSSION
Colorectal cancer is one of the most frequent malignancies in the world. Despite the
improved management in the chemotherapy, side effects and drug resistance have shown
limitations and led to the search for superior treatments. In our studies, both the compounds 7
and 22 exhibited potential cytotoxicities against colorectal adenocarcinoma cells. Hence,
detailed pharmacology of these compounds was explored against this cell line.
Tubulin-microtubule dynamic equilibrium is an important aspect in cell growth and
development. Antimitotic agents, both microtubule stabilizers like paclitaxel and microtubule
destabilizers like podophyllotoxin suppress spindle dynamics inducing mitotic arrest leading
to cell death in rapidly dividing cancer cells (Cao et al., 2018; Borisy et al., 2016). Both the
compounds (7 & 22) showed antimitotic activity. Highly dynamic spindle microtubules are
considered remarkable therapeutic targets for the development of cancer chemotherapeutics
(Brouhard & Rice, 2018). Further, in molecular docking studies, there were fourteen residual
amino acids in the binding pocket of compound 7 common to colchicine binding pocket. It
indicates almost same binding location of compound 7 to the β-tubulin. Burns et al. (1992)
reported that ALA β:316 is an important residue that is directly involved in the binding of
trimethoxyphenyl unit of both colchicine and podophyllotoxin (Burns, 1992). It was further
elaborated that although trimethoxyphenyl unit is important for binding to β:316, but
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hydrophobicity around this residue also plays a role to some extent as it specifies the rate of
conformational changes of the ligand. The designed compound 7, possesses similar
arrangements of trimethoxyphenyl unit in ring A and also exhibits similar effects of
microtubule destabilization as by colchicine and podophyllotoxin. On the other hand,
compound 22 exhibited weak effect of microtubule stabilization. Paclitaxel, a standard
microtubule stabilizer, binds to the luminal surface of β-tubulin in microtubules also known
as paclitaxel binding site. Nogales et al. (1998) found LEU β:275 as a crucial residue in M-
loop of binding pocket which is the main interaction point (Nogales et al., 1998). Further,
Field et al. (2013) revealed that in S9-S10 loop amino acid residues PRO β:360 and ARG
β:369 also play important roles (Field et al., 2013). All the three residues were present in the
binding pocket of ligands 22, and paclitaxel, which indicates the occupancy of similar
binding pocket by both the ligands. In docking studies with both the enantiomers 22(R) and
22 (S) independently, 22 (S) showed better binding affinity than the 22(R) isomer. However,
both the enantiomers occupied all the three crucial residues (LEU β:275, PRO β:360, and
ARG β:369) to exhibit microtubule stabilization effect.
Oncogenic processes mainly target on cell cycle regulators and abandon their control leading
to uncontrolled proliferation of cells which is the hallmark of cancer cells (Hanahan &
Weinberg, 2011). Compounds 7 and 22 exhibited G2/M phase arrest in DLD1 cells at 24 h.
But, this effect was negligible at 48 h. It might be due to faster metabolism of both the
compounds by cancer cells. In general, antimitotic agents induce G2/M phase arrest due to
interference in mitotic phase (Negi et al., 2015). Triggering of cell cycle arrest in cancer cells
comprises one of the most prevalent strategies to halt or restrict cancer spreading (Hartwell &
Weinert, 1989). As a result of cell cycle arrest compound 22 exhibited inductions of
apoptosis. But, it was not visible in case of compound 7. Many compounds induce apoptosis
as a result of cell cycle interruption.
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Apoptosis is a controlled and energy dependent cellular process which leads to programmed
cell death (Susan, 2007). During apoptosis PARP is cleaved by caspases and inactivated
(Chiarugi & Moskowitz, 2002). PARP cleavage in DLD1 cells by both the compounds
clearly indicates activation of caspase pathway to trigger apoptosis. However, this effect was
moderate in both the cases and only up to 24h. There were similar indications in cell cycle
analysis where both the compounds exhibited prominent effects only up to 24h. It indicates
that the antiproliferative effects of both the compounds are early on DLD1 cells. Both
apoptotic signalling pathways induce cell death by activating protease enzymes i.e. caspase.
Caspases 8, 9, and 10 are initiators while caspases 3, 6, and 7 are effector caspases (Degterev
et al., 2003). Cancer cells circumvent apoptosis and continue proliferating cells. Induction of
apoptosis by drug entities is preferred in cancer drug discovery (Olsson & Zhivotovsky,
2014).
There was no deviation in any of the parameters of compounds 7 and 22 specified in Lipinski
rule, which indicates good druggability of both the compounds. Drug candidates complying
rule of five tend to have lower attrition during clinical trials and are more successful in
reaching to market (Leeson & Springthorpe, 2007).
Assessment and optimization of safety of drug candidates is an important aspect in drug
discovery and development to recognise the molecules with balanced safety-efficacy profile
via therapeutic window. Acute oral toxicity is used to assess the adverse effects of
investigational drug within a short time after single oral dose or multiple doses given within
24h (IUPAC Gold Book, 2004).
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Both the compounds were safe and well tolerated by experimental animals in acute oral
toxicity. However, experiments with prolonged exposure of drug candidate will be required
during future development through sub-acute, chronic and sub-chronic toxicity experiments.
Nowadays, safety has become most important concern and a key criterion for successful new
drugs due to regulatory, societal and legal concerns (Ray, 2009).
4. CONCLUSION
In summary, diverse analogues of 1-indanone were prepared as possible anticancer agents.
Two of the analogues exhibited potential antiproliferative activity against colorectal
adenocarcinoma. These analogues showed antimitotic effects and induced G2/M phase arrest
in cell cycle analysis. Both the potential lead compounds moderately induced apoptosis in
DLD1 cells through induction of caspase pathway. Both the lead compounds showed good
druggability under Lipinski rule and were found to be safe in rodent models. Thus, both the
indanone based lead compounds 7 and 22 are potential anticancer agents. The optimization of
these lead compounds is underway to get more efficacious compounds from this series.
Acknowledgements
Ms. Ankita Srivastava extends her thanks to Department of Science and Technology, Govt. of
India for financial support for this work under WOS-A from KIRAN Division (GAP-407).
Ms. Kaneez Fatima acknowledges CSIR for her Senior Research Fellowship. Chemical
Central Facility of CSIR-CIMAP is duly acknowledged for Sophisticated Instrument support.
CONFLICT OF INTEREST
Authors declare no conflict of interest.
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Supplementary information
13
1H NMR, C NMR and Mass analysis data of compounds, detailed biological protocols and
scanned spectra are available online version.
Figure legends:
Figure 1. Chemical structure of some clinical drugs for colorectal cancer, microtubule stabilizers (Paclitaxel,
docetaxel), destabilizers (Colchicine, podophyllotoxin, combretastatin A4), and indanone core.
Figure 2: Cell cycle analysis of compounds 7 and 22 in DLD1 colorectal cancer cells.
Figure 3. Effects of compound 7 and compound 22 on tubulin kinetics (Positive controls, PAC=Paclitaxel as
microtubule stabilizer, PDT=podophyllotoxin as microtubule destabilizer)
Figure 4. Cleavage of PARP (Poly ADP-ribose polymerase) by compounds 7 and 22 at their IC50(s)
Figure 5A: 2D docking poses of compound 7, colchicine, and paclitaxel with PDB ID: 4O2B (Tubulin-
colchicine complex).
Figure 5B: 2D docking poses of isomeric compounds 22 (R), 22 (S) and paclitaxel with PDB ID: 5M5C
(Microtubule-paclitaxel bound complex)
Figure 6 A. Effect of 7 as a single acute oral dose at 5, 50, 300 and 1000 mg/kg on absolute and relative organ
weights in Swiss albino mice (Mean±SE; n=6).
Figure 6B. Effect of 7 as a single acute oral dose at 5, 50, 300 and 1000 mg/kg body weight on differential
leucocytes counts in Swiss albino mice (Mean±SE; n=6).
Figure 7A. Effect of 22 as a single acute oral dose at 5, 50, 300 and 1000 mg/kg on absolute and relative organ
weights in Swiss albino mice (Mean±SE; n=6).
Figure 7B. Effect of 22 as a single acute oral dose at 5, 50, 300 and 1000 mg/kg body weight on differential
leucocytes counts in Swiss albino mice (Mean±SE; n=6).
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Table 1: In-vitro Cytotoxicity of indanones and related derivatives against various human cancer cells by
sulphorhodamine assay (IC50 in µM).
S. no.
Cytotoxicity IC50 in µM
Cytotoxic
ity CC50
in μM
Selectivity
Index (SI)**
Antitubulin
A549
DU145
FaDu
DLD1
MCF-7
HEK-93
IC50(DLD1)/
CC50
IC50
(Inhibition
EC50
(Stabilization)
)
4.80
>40
2.89
>40
2.40
>40
3.63
>40
47.15
ND
----
---
---
---
1.8
---
---
---
---
---
7
2.22
7.02
21.24
---
0.57
---
---
15
>40
>40
>40
>40
>40
ND
---
17
18
>40
>40
>40
>40
>40
ND
---
---
2.23
2.47
10.87
>40
>40
119.44
ND
---
22
1.23
97.11
---
20.73±1.99
15.68
0.63
24.40±1.94
19.82
0.30
24.91±1.18
16.34
2.19±0.34
---
>40
---
25
Tamoxifen
Doxorubicin
Nocodazole
Podophyllotoxin
8.29
<1.25
---
8.61
21.0
3.90
---
1.29
0.56
---
---
5.83±0.32
---
---
---
---
0.62±
0.74±0.12
<1.25
<1.25
0.104±0.01
<1.25
---
---
3
Paclitaxel
ND
ND
ND
ND
1.02±1.7
>25
---
---
0.025
* IC50>40μM was considered as inactive; **SI=IC50/CC50 ; IC50 against DLD1 cells, CC50 against HEK-93 cells
Table 2. Effect of compound 7 as a single acute oral dose at 5, 50, 300, and 1000 mg/kg on body weight,
haematological and serum biochemical parameters in Swiss albino mice (Mean±SE; n=6; *significant compared
to control, P<0.05).
Parameters
Dose of compound 7 at mg/kg body weight as single oral dose
Control
5 mg/kg
50 mg/kg
300 mg/kg
1000 mg/kg
33.51±1.04
31.03±2.20
29.30±1.01
28.81±0.60
29.38±1.21
Body weight
Body weight (g)
13.96±0.21
9.36±0.50
6.56±0.12
44.71±6.44
45.16±3.59
14.66±0.41
9.07±0.68
7.56±1.56
46.14±2.48
43.24±19.75
15.55±0.61
9.79±0.75
10.33±1.84
47.82±5.71
53.67±9.07
14.30±0.57
8.86±0.46
9.05±1.68
57.47±6.46
53.03±3.81
12.81±0.68
8.57±0.41
10.39±0.60
49.39±6.71
103.91±15.33*
Haematological
parameters
Haemoglobin (g/dL)
RBC (million/mm3)
WBC(thousands/mm3)
ALP (U/L)
Liver Function
Test
SGOT (U/L)
SGPT (U/L)
26.64±1.90
2.66±0.10
27.49±1.59
2.73±0.15
22.49±1.37
2.56±0.15
23.40±2.10
2.89±0.07
49.73±3.92*
2.42±0.10
Albumin(g/dL)
Protein(mg/ml)
2.82±0.08
0.51±0.02
0.76±0.10
3.24±0.03
0.56±0.02
0.42±0.03
2.79±0.07
0.55±0.04
1.03±0.21
2.64±0.04
0.54±0.07
0.86±0.20
2.91±0.12
0.50±0.02
0.78±0.21
Total Bilirubin(mg/dL)
Creatinine (mg/dL)
Kidney Function
Test
Lipid Profile
Cholesterol (mg/dL)
Triglycerides (mg/dL)
177.91±18.68
141.33±22.51
193.29±18.65
114.67±11.58
171.03±15.64
113.21±21.38
150.90±8.79
123.63±16.75
162.08±23.25
95.41±2.82
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Table 3. Effect of compound 22 as a single acute oral dose at 5, 50, 300, and 1000 mg/kg on body weight,
haematological and serum biochemical parameters in Swiss albino mice (Mean±SE; n=6).
Parameters
Dose of compound 22 at mg/kg body weight as single oral dose
Control
28.48±1.00
10.84±0.50
9.85±0.57
5 mg/kg
27.17±0.64
9.96±0.71
8.90±0.59
50 mg/kg
31.47±0.88
10.91±0.59
7.63±0.41
300 mg/kg
24.10±0.43
9.66±0.63
7.51±1.43
1000 mg/kg
26.05±0.53
12.89±0.83
8.98±0.90
Body weight
Body weight (gm)
Haemoglobin (gm/dL)
RBC (million/mm3)
WBC(thousands/mm3)
Haematological
parameters
5.80±0.58
7.80±1.06
9.79±0.86
10.28±0.83
8.09±0.77
98.03±15.84
48.55±4.94
23.06±1.33
1.86±0.16
2.71±0.13
0.41±0.02
100.76±10.57
30.83±1.59
20.27±3.14
1.81±0.16
2.86±0.12
0.46±0.05
105.63±10.50
36.99±4.15
24.42±1.77
2.81±0.05
2.81±0.17
0.43±0.06
91.52±7.60
48.22±6.07
22.34±1.19
1.26±0.16
3.08±0.21
0.57±0.07
90.89±6.99
41.47±4.78
21.57±2.85
2.47±0.10
2.69±0.12
0.39±0.03
Liver Function Test
ALP (U/L)
SGOT (U/L)
SGPT (U/L)
Albumin(g/dL)
Serum Protein(mg/ml)
Bilirubin(mg/dL)
Creatinine (mg/dL)
Kidney Function
Test
0.60±0.06
108.63±7.46
80.67±9.76
0.54±0.05
130.07±8.73
85.85±11.08
0.87±0.20
0.78±0.18
169.27±19.21
96.33±12.49
0.49±0.02
214.71±25.45
90.09±13.77
Lipid profile
Cholesterol (mg/dL)
Triglycerides (mg/dL)
177.98±17.68
104.70±16.38
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Scheme 1
O
O
H3CO
H3CO
O
H3CO
H3CO
HO
HO
COOH
H3CO
H3CO
CHO
H3CO
OCH3
OCH3
OCH3
TFA
A.
110oC
7% KOH in MeOH
RT, 2h
H3CO
H3CO
OH
OCH3
OCH3
OCH3
OCH3
OCH3
H3CO
1
O
R2
i
H3CO
H3CO
2: R1=4-hydroxyphenyl;
3: R1=4-(ethoxycarboxymethoxyphenyl);
4: R1=3-(1-anisyl)-propenyl;
ii
H3CO
H3CO
O
5: R1=3-(2,3,5-trimethoxyphenyl)-propenyl
OCH3
OCH3
OCH3
OCH3
O
O
H3CO
iii
H3CO
H3CO
O
H3CO
H3CO
O
+
OH
+
H3CO
H3CO
OCH3
O
OCH3
H3CO
H3CO
B.
6
8
OCH3
OCH3
OCH3
OCH3
H3CO
7
H3CO
H3CO
H3CO
H3CO
O
O
H3CO
H3CO
O
C
COOCH3
OCH3
OH
H3CO
iv
OCH3
H3CO
H3CO
v
H3CO
1
OCH3
9
OCH3
OCH3
10
H3CO
OCH3
OH
H3CO
O
H
N
N
H3CO
H3CO
H3CO
H3CO
vii
vi
H3CO
H3CO
OCH3
OCH3
11
H3CO
12
OCH3
OCH3
H3CO
C.
O
O
O
H3CO
H3CO
H3CO
CHO
H3CO
H3CO
viii
ix
NO2
+
H3CO
O2N
OCH3
OCH3
13
OCH3
NO2
D.
O
14: R4=2-pyridyl;
15: R4=3-pyridyl;
16: R4=2-pyrolyl;
17: R4=4-(3,4,-methylenedioxyphenyl);
18: R4=4-hydroxyphenyl
H3CO
R4
x
H3CO
OCH3
O
H3CO
H3CO
NO2
OCH3
13
OR5
N
OH
N
H3CO
H3CO
NO2
H3CO
xii
xi
H3CO
OCH3
OCH3
19
NO2
NO2
20: R5=-CH2-COOC2H5;
21: R5=-CH2-CH=CH-COOC2H5
R6
O
O
O
O
H3CO
H3CO
H3CO
H3CO
H3CO
TFA
110oC
N
xiii
H3CO
H3CO
E.
+
H3CO
OHC
N
OCH3
22
OCH3
OCH3
OCH3
N
N
23: R6=4-hydroxy;
24: R6=3-hydroxy,4-methoxy;
25: R6=,3,5-dimethyl-4-hydroxy;
26 R6=3,4-methylenedioxy;
Reagents and conditions: i) 3% MeOH-KOH, RT, 3-5h, 64-91%; ii) Anhyd. K2CO3, dry acetone, for 3 and 20
ethylbromoacetate/ for 21 ethylbromocrotonate, reflux, 2-3h, 73-84%; iii) Dioxane, SeO2, reflux, 5h, 6:16%, 7:
23% and 8: 40%; iv) Me2CO3, NaH, dry toluene, reflux, 6h, 91%; v) 5% KOH, MeOH:H2O (9:1), 50oC, 1h,
87%;
vi) EtOH, NH2OH.HCl, reflux, 3-5h, 11: 72% and 19: 79%; vii) p-TSA, dry THF, reflux, 3h, 39%;
viii) 7% KOH, MeOH, RT, 2h, 93%; ix) TFA, 110oC, 6h, 41%; x) Piperidine, dry THF, RT, 54-63% for 17 &
18.
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