An improved synthesis of indanocine and antiproliferative activity of 2-benzylindanocine via microtubule destabilization

Article abstract of DOI:10.1111/cbdd.13857

Indanocine, a potent anticancer investigational drug of National Cancer Institute-USA, has been much discussed in recent years. Present communication aimed at total synthesis of indanocine and its close analogues. Total synthesis was improved by double yields than previously reported yields. Some of the benzylidene and 2-benzyl derivatives with free rotation at C2 position exhibited potential cytotoxicities against various human cancer cell lines. Five such analogues exhibited potential antiproliferative effect against HCT-116 and MIA PACA-2 cell lines. Benzylindanocine 12i induced microtubule destabilization by occupying colchicine binding pocket of β-tubulin. It also exhibited anti-inflammatory activity by down-regulating IL-6 and TNF-α. In Ehrlich ascites carcinoma model, 12i reduced 78.4% of EAC tumour in Swiss albino mice at 90?mg/kg (i.p.) dose. Further, in in vivo safety studies, 12i was found to be safe to rodents up to 1,000?mg/kg dose. Concomitant anticancer and anti-inflammatory activity of benzylindanocine is distinctive, which suggests its further optimization for better efficacy and druggability.

Full text of DOI:10.1111/cbdd.13857

DR FEROZ KHAN (Orcid ID : 0000-0002-2704-5925)
Article type : Research Article
An improved synthesis of indanocine and antiproliferative activity of 2-benzyl-
indanocine via microtubule destabilization
Sadiya Khwaja,1,3 Kaneez Fatima,1,3 Divya Mishra, Vineet Babu, Yogesh Kumar, Sumera Banu Malik,
1
1
1
2
Misbah Tabassum, Suaib Luqman, Dnyaneshwar U. Bawankule,1,3 Debabrata Chanda,1,3 Feroz Khan,1,3
2
1,3
D. M. Mondhe,2,3 Arvind S. Negi1,3*
1
.
CSIR-Central Institute of Medicinal and Aromatic Plants (CSIR-CIMAP) P.O. CIMAP, Lucknow, 226015, India
2.CSIR-Indian Institute of Integrative Medicine (CSIR-IIIM), Canal Road, Jammu-180001, India.
3.Academy of Scientific and Innovative Research (AcSIR), Ghaziabad-201002, India
ABSTRACT
Indanocine, a potent anticancer investigational drug of National Cancer Institute-USA has been
much discussed in recent years. Present communication aimed at total synthesis of indanocine and
its close analogues. Total synthesis was improved by double yields than previously reported
yields. Some of the benzylidene and 2-benzyl derivatives with free rotation at C2 position
exhibited potential cytotoxicities against various human cancer cell lines. Five such analogues
exhibited potential antiproliferative effect against HCT-116 and MIA-PACA-2 cell lines.
Benzylindanocine 12i induced microtubule destabilization by occupying colchicine binding pocket
of β-tubulin. It also exhibited antiinflammatory activity by down-regulating IL-6 and TNF-α. In
Ehrlich ascites carcinoma model 12i reduced 78.4% of EAC tumour in Swiss albino mice at 90
mg/kg (i.p.) dose. Further, in in-vivo safety studies 12i was found to be safe to rodents up to 1000
mg/kg dose. Concomitant anticancer and antiinflammatory activity of benzylindanocine is
distinctive which suggests its further optimization for better efficacy and druggability.
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:
1
0.1111/CBDD.13857
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Keywords: Indanocine, Anticancer, antitubulin, Ehrlich ascites carcinoma, Acute oral toxicity
…
……………………………………….
*
2
Corresponding author: E-mail: arvindcimap@rediffmail.com ; ; Tel: +91-522- 2342676 x 583; Fax: +91-522-
718509;
CIMAP communication No. CIMAP/PUB/2020/Jun/43
1
. INTRODUCTION
Worldwide, cancer has caused 9.6 million human deaths which is about 17% of total human
deaths. In 2010, total annual economic cost of cancer was US$1.16 trillion [WHO Keyfacts
2
018]. Researchers are considerably exploring to identify diverse chemotherapeutics to tackle the
disease. Modulation of tubulin-microtubule dynamics is a distinctive capability of antimitotic
agents. Highly dynamic mitotic spindle microtubules are remarkable therapeutic targets for a
group of chemically diverse and clinically successful anticancer drugs. Even with the advent of
targeted therapies for cancer, antimitotic agents are some of the most effective drugs in the
treatment of solid tumours and hematological malignancies [Kaul et al. 2019]. A number of
antimitotic agents have successfully been developed as clinical anticancer drugs like paclitaxel,
docetaxel, cabazitaxel, ixabepilone, vinorelbine, vinflunine, eribulin mesylate (E7389), ENMD-
1
198 and STX-140 etc. and many more are under the development of various phases of clinical
trials namely epothilone derivatives patupilone (Phase-III), sagopilone (Phase-II), BMS310705,
larotaxel (Phase-III), tasetaxel (Phase-II), taxoprexin (Phase-III), xyotax (Phase-III),
combretastatin A4 and its phosphate salts (Various phases of clinical trials), dolastatin (Phase-II),
cryptophycin52 (Phase-II & Phase-III), halichondrins (phase-I), and noscapine (Phase-II) etc.
[Negi et al. 2015].
[Fig.1]
Indanocine (1), a synthetic indanone is a potent antiproliferative agent developed by NCI, USA. It
is a potent tubulin binding drug effective against a number of human cancer cell lines (mean
GL <20 nM) and against multidrug resistant cell lines [Leoni et al. 2000]. Studies demonstrated
5
0
that tumour cells over-expressing 170KD P-glycoprotein resistant to paclitaxel were very much
sensitive to indanocine. As a microtubule destabilizer, it binds to colchicine binding site of β-
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tubulin [Das et al. 2009]. Some other studies demonstrated that indanocine inhibited migration of
metastatic cancer cells [Kapoor and Panda 2012]. Owing to an important antimitotic anticancer
agent several analogues of indanocine have also been prepared in the recent past [Huang et al.
2
012; Tunbridge et al. 2013].
[Fig.2]
Present communication reports an improved synthesis of indanocine. The process is very efficient
and straightforward where new set of reagents have been used for better yields. The overall yield
of the process is 26.7% in three synthetic steps which is much better than the previously reported
methods [Shih et al. 2000; Coulup and George 2019]. Sixteen other analogues of indanocine
were also synthesized. Our main aim was to synthesize more flexible analogues of indanocine to
have better interaction with tubulin protein. So, we planned to have 2-benzyl analogues of
indanocine where 2-benzylidene ring was reduced for more flexibility. All these analogues were
evaluated against four human cancer cell lines. Benzylindanocine exhibited potent
antiproliferative activity against two human cancer cell lines. Both indanocine and
benzylindanocine were extensively evaluated for target studies, druggability, in-vivo efficacy and
benzylindanocine for safety in Swiss-albino mice.
2
2
. RESULTS
.1. Chemistry
Synthesis of indanocine
All the previous and present synthetic protocols have been depicted in Scheme 1. Previously, two
protocols were reported and both were from the same group of UCSD following identical route of
synthesis. Carson et al. mentioned only last two steps of protocol in this patent. In the first step 1N
o
NaOH in ethanol was used at 50 C to get nitroindanone derivative 11 in 96% yields from nitro
derivative 10. In the final step, nitro group of 11 was reduced with alkaline sodium bisulphite
solution to afford indanocine (1) in 18% yield. In this patent, preparation of compound 10 was not
mentioned. The overall yield in two steps was 17.3% only [Carson et al. 2000]. The second
communication by Shih et al., nitration of indanone 9 was done with sodium nitrite in TFA to get
nitroindanone 10. In the next step, 2-benzylidene derivative of nitroindanone (11) was prepared by
condensation of 3,5-dimethyl-4-hydroxybenzaldehyde using 5% methanesulphonic acid in acetic
acid. Reduction of nitro group of 11 was tried with two different reagents i.e. Sodium dithionate or
zinc dust in 2% acetic acid to get indanocine (1). However, yields of these steps were not
mentioned in this communication [Shih et al. 2000].
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In our strategy, we also followed the identical route of synthesis as done by UCSD group but,
using other set of reagents. In the first step, nitration of indanone 9 was done in cold acetic
anhydride-nitric acid mixture to get compound 10 in 74% yields. Compound 10 was condensed
with 3,5-dimethyl-4-hydroxybenzaldehyde in dioxane-conc. HCl system under reflux conditions
to get 2-benzylidenenitroindanone (11) in 82% yield. Finally, nitro group of 11i was reduced
under controlled conditions with 10% Pd-C in dry DMF to afford indanocine (1) in 44% yield.
The overall yield of our process was 26.7% in three steps.
[Scheme 1]
Synthesis of indanocine analogues
Synthetic strategy for preparation of indanocine analogues (11a-11m, 12a-12m & 13n-13p) was
as illustrated in Scheme 2. To get diverse 2-benzylidene derivatives, various substituted
benzaldehydes were condensed with 7-nitro-5,6,-dimethoxyindanone-1 (10) via Claisen Schmidt
reaction using different reaction conditions (One alkaline and two acidic conditions) to get 2-
benzylideneindanone derivatives (11a-11l) in 43-86% yields. Boc-piperazine derivative 11l was
treated with trifluoroacetic acid in dry DCM under cold conditions to afford 2-benzylidene
derivative 11m in 91% yield. All the 2-benzylidene-7-nitro indanone derivatives were reduced
with 10% Pd-C-dry DMF at RT to get fully reduced 2-benzyl-7-aminoindanone derivatives 12a-l
in 44-67% yields. But, compounds 12n-12p underwent partial reductions (Nitro group) only and
afforded 7-amino-2-benzylidenes (13n-13p). Nitration of 5,6-dimethoxyindanone (9) in NaNO2-
BF -etherate-MeOH system yielded nitration at 2-position and product obtained was rather 14 (aci
3
form) due to strong hydrogen bonding. All products were confirmed by spectroscopy.
[Scheme 2]
2
.2. Biological evaluation
Antiproliferative activity
All derivatives of indanocine were evaluated for antiproliferative activity by SRB assay against
four human cancer cell lines viz. HCT-116 (Colon), MIA PACA-2 (Pancreas), SW 620 (Colon)
and A549 (Lung). Initially, compounds were evaluated at two different concentrations of 10 µM
and 50 µM. Out of twenty nine analogues fifteen analogues exhibited significant cytotoxicity
(≥50% inhibition at 50 µM), While five analogues exhibited potent cytotoxicity (≥50% inhibition
at 10µM) (Table 1A). On further evaluation, indanocine (1) exhibited IC of 1.03 µM (HCT-116)
5
0
and 0.44 µM (MIA PACA-2) while benzylindanocine (12i) exhibited IC of 1.30 µM and 0.58
5
0
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µM against both the cell lines respectively (Table 1B). However, both the compounds were less
active than standard drug combretastatin A4 (IC <0.1 µM).
5
0
[Table 1A & 1B]
For structure activity relationship, we had mainly two series, Series-I as 2-benzylidene-7-
nitroindanones (11a-11m) and Series-II as 2-Benzyl-7-aminoindanones (12a-12m). Cytotoxicity
against MIA PACA-2 cell line (at 50 µM) was taken into consideration as most of the compounds
exhibited higher activity against this cell line. In general, Series-II compounds were more active
than the Series-I compounds. In Series-I, compound 11a was without any substitution,
introduction of a methoxy group (11c) marginally increased the cytotoxicity, cytotoxicity was
further increased on introducing second methoxy group (11f). But, on introducing third methoxy
group (11h), activity was decreased drastically. When a fluoro (11j) or hydroxy (11b) group was
introduced in 11a cytotoxicity was enhanced. Further, demethylation of any methoxy group of 11f,
resulted in decreased cytotoxicity (11d and 11e). Introduction of amino group in compounds (11k,
1
1l, 11m) possessed marginally higher activity. In Series-I, 4-hydroxybenzylidene derivative
(11b) possessed the highest cytotoxicity.
In Series-II (12a-12m), compound12a was 2-benzyl derivative with no substitution. Introduction
of one methoxy group (12c) significantly reduced cytotoxicity. But subsequent introduction as
dimethoxy (12f) and trimethoxy (12h) groups enhanced activity gradually. Both 4-hydroxy (12b)
and 4-fluoro (12j) derivatives showed less cytotoxicity than 12a. Subsequent introduction of two
methyl groups (12i) enhanced drastically, which was the best compound in this series. Both
piperazine based benzyl derivatives (12l and 12m) possessed higher activity than 12a.
Tubulin polymerisation inhibition activity
Tubulin targeting agents disrupt cellular process by modulating tubulin-microtubule dynamics
[Coulup and George 2019]. Effect of indanocine (1) and benzylindanocine (12i) on
polymerisation process of tubulin was determined by tubulin kinetics experiment. Paclitaxel was
used as standard stabilizer and podophyllotoxin as standard destabilizer. Three different
concentrations of each test compound were taken. Both the compounds exhibited moderate
destabilization of microtubules. Figures 3A and 3B show paclitaxel curves are above the GTP
protein clearly exhibiting stabilization effect. Podophyllotoxin treated curves are much below the
GTP protein showing strong destabilization effect. Both indanocine (1, Fig. 3A) and
benzylindanocine (12i, Fig. 3B) treated curves are also below the GTP protein curve, distinctly
exhibiting destabilization effects. But, their destabilization effects are lower than podophyllotoxin.
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Hence, both compound 1 and 12i exhibit moderate destabilization of tubulin polymerisation
process.
[Fig 3A & 3B]
Antiinflammatory activity of 2-benzylindanocine
Tumour microenvironment contains many different inflammatory cells and mediators (Cytokines,
chemokines, and prostaglandins) that are over-expressed during the pathogenesis of the disease.
Both TNF-α and IL-6 are well established prognostic markers in acute cancer inflammation
[Furman et al. 2019]. Interestingly, both indanocine (1) and benzylindanocine (12i) significantly
inhibited LPS-stimulated productions of pro-inflammatory cytokines TNF-α and IL-6, thus
exhibiting cancer related antiinflammatory activity as well (Table 2). Both indanocine (29.36%)
and benzylindanocine (28.38%) showed comparable activity against TNF-α. Benzylindanocine
(12i, 46.02%)) exhibited higher IL-6 inhibition activity as compared to indanocine (1, 41.41%)).
However, both the compounds had relatively lower activity than the standard anti-inflammatory
clinical drug dexamethasone.
[Table 2]
Molecular docking studies
a) Interaction with target protein β-tubulin
In a comparative molecular docking studies of indanocine (1), both 2α (2R) and 2β (2S)-benzyl
indanocine (12i), podophyllotoxin and colchicine were used as standard destabilizers of β-tubulin.
All five ligands occupied same binding pocket of β-tubulin (Table 3). There were six residual
amino acids common to all i.e. LEU B:248, ALA B:250, LEU B: 255, ASN B:258, ALA B316
and LYS B:352 indicating a common binding site. 2β-Benzyl derivative of indanocine showed
highest binding affinity (-8.1 Kcal/Mol) with β-tubulin. It showed marginally better affinity than
indanocine (-8 Kcal/Mol). While, 2α-benzylindanocine had less affinity (-7.9 Kcal/Mol) than the
indanocine. However, all these three ligands (1, 12i-α and 12i-β) possessed higher binding affinity
with β-tubulin than the both the standard inhibitors colchicine (-6.2 Kcal/Mol) and
podophyllotoxin -7 Kcal/Mol). Further, docking view showed a common site occupied by these
ligands. They also exhibited structural similarities as far as chemical space is concerned (Fig. 4).
[Table 3] [Fig. 4]
b) In-silico prediction of ADME properties
Physicochemical properties of drug candidate give an idea about the druggability of molecule.
Pharmacokinetic properties of both indanocine (1) and 2-benzylindanocine 12i were calculated
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using online SwissADME software. Physicochemical parameters, water solubility, lipophilicity,
pharmacokinetics, druglikeness and medicinal chemistry aspects were calculated. Both
compounds showed good druggability. All the required parameters were within desired limits.
Both did not possess PAINS (Pan-assay interference compounds) structure and did not violate
Lipinski’s rule. Overall, both compounds possessed satisfactory oral bioavailability and hence
druggability. Further, figure 5 exhibits bioavailability radar of both compounds within defined
limits of good bioavailability.
[Figure 5]
In-vivo antiproliferative efficacy by Ehrlich Ascites Carcinoma
Ehrlich ascites carcinoma is a spontaneous murine mammary adenocarcinoma which is well
established model in tumour biology for the study of tumour pathogenesis and development of
anticancer agents. Both indanocine (1) and 2-benzylindanocine (12i) were evaluated for their in-
vivo anticancer efficacy by EAC in Swiss albino mice. Both indanone derivatives 1 and 12i
exhibited significant antitumour activity (Tables 5a and 5b). However, efficacy of compound 1
was much better than compound 12i. Indanocine reduced 89.1% of EAC tumour at 30 mg/kg dose
(i.p.), while 2-benzylindanocine (12i) reduced 40.2%, 53.8%, and 78.4% of tumour at 10 mg/kg,
3
0 mg/kg and 90 mg/kg dose (i.p.) respectively. There was no mortality in any of group
throughout experimental period. Standard drug, 5-fluorouracil exhibited 97.2% tumour reduction
at 20 mg/kg i.p. dose.
[Table 5A & 5B]
In-vivo safety studies of 12i
Acute oral toxicity specifies various adverse effects aroused on oral administration of single dose
of test compound to experimental animal. There were no observational changes throughout the
experimental period of 12i up to dose level of 1000 mg/kg body weight. There were no
detrimental effects in body weight of experimental mice. It did not induce any toxic effect in
haematological parameters, liver function parameters, kidney function parameters, and lipid
parameters of experimental animals (Table 6). However there was slight reduction in WBC count
which was statistically in significant. Gross pathological studies (Fig. 6A & 6B) showed no
significant changes in the absolute and relative organ weights of vital organs. Furthermore,
differential leukocyte count did not show any sign of toxic effects of compound 12i like invasion
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or inflammation (Fig. 7). Overall, 2-benzylindanocine 12i was safe and well tolerable up to dose
level of 1000 mg/kg body weight as a single acute oral dose in Swiss albino mice.
[Table 6 ]
[Figure 6A, 6B & Figure 7]
3
. DISCUSSION
Indanocine is a potent investigational anticancer drug. Our first objective was to develop an
improved protocol for its synthesis. We used same reaction route as previously done by Carson
group but a new set of reagents. The reagents were more efficient to improve yield of the process
by two fold. This group did not mention yield in the first step (Nitration of indanone) but in other
steps our overall yield was much better.
Colon and pancreatic cancers are among leading causes of cancer deaths. Globally, both colorectal
and pancreatic cancers caused more than 1 million deaths annually. Chemotherapy and surgery are
two most common medical remedies to tackle the disease. Chemotherapy is a preferred approach
with combination of various anticancer drugs with meager success rate of 64% for five years in
colon cancer and only 7% for five years in pancreatic cancer [Cancer facts and figures 2020].
2
-Benzylindanocine 12i exhibited significant antimitotic activity. We planned to synthesize more
flexible analogue of indanocine i.e. 2-benzylindanocine. In in-vitro studies, indanocine exhibited
better cytotoxicity than 2-benzylindanocine against colon cancer cells, while 2-benzylindanocine
showed more potent activity than indanocine against pancreatic cancer cells. Undoubtedly, both
possessed similar anticancer potential. However, reduction of 2-benzylidene to 2-benzyl group did
not enhance in-vivo efficacy. EAC is a general carcinoma model and not specific to colorectal or
pancreatic cancer. However, it is important to mention that amongst the five most potent
analogues of indanocine, four were 2-benzylindanocines i.e. 12e, 12g, 12i, and 12m indicating
higher prospects of success while dealing with these compounds.
Benzylindanocine exhibited tubulin polymerisation inhibition. Tubulin-microtubule dynamic
equilibrium is an important feature in cell growth and development. Any disturbance in this
dynamic equilibrium induces cell cycle arrest [Negi et al. 2015]. In molecular docking studies,
there were six residual amino acids in binding pocket common to all these ligands. Interestingly,
both indanocine and benzylindanocine showed interaction with β:316 and β:318, the crucial
residues interacting with target protein. Burns et al. (1992) revealed that ALA β:316 is an
important residue that is directly involved in the binding of trimethoxyphenyl unit of both
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colchicine and podophyllotoxin [Burns, 1992]. Although trimethoxyphenyl unit is important for
binding to β:316, but hydrophobicity around this residue also plays a role to some extent as it
specifies the rate of conformational changes of the ligand.
Deregulatory inflammation plays a pivotal role in the initiation, development and progression of
tumour [Chai et al. 2015]. The association between chronic inflammation and cancer has been
well perceived by epidemiologists since last two decades. both indanocine and benzylindanocine
exhibited moderate antiinflammatory activity by down-regulating production of pro-inflammatory
cytokines TNF-α and IL-6. Tumour necrosis factor-α (TNF-α) is a key multifunctional cytokine
with pleiotropic actions in the regulation of inflammation related immune responses. Being a
major cytokine, it regulates other cytokines and chemokines. Hence, TNF-α effectively influences
several hallmarks of cancer. Interleukin-6 has gained importance as a prime biomarker in the
prognosis of various types of cancer microenvironment. Now it is well established as therapeutic
target [Garbers et al. 2018]. Antiinflammatory effect of benzylindanocine 12i is a much desirable
attribute to be a broad spectrum anticancer agent.
ADME properties are very much important for a bioactive compound to become a successful drug
candidate. ADME properties predicted through SwissADME software were quite satisfactory for
both indanocine and 2-benzylindanocine. Water solubility prediction for indanocine was three fold
higher than indanocine which indicates that it may have better oral route administration. It did not
possess PAINS structure which otherwise might exhibit misleading activity due to artifact [Baell
and Walters 2014]. Inadequate ADME properties can be devastating to any good drug candidate
[Kenakin TP, 2017].
Both indanocine and benzylindanocine exhibited significant in-vivo efficacy. However, 2-
benzylindanocine displayed moderate activity against EAC carcinoma cells which are considered
difficult to break [Bean & Kassel 1968]. EAC is a general carcinoma and hence, the evaluation of
benzylindanocine against specific rodent models will be done in future.
Benzylindanocine 12i was safe and well tolerated by Swiss albino mice up to the 1000 mg/kg dose
in acute oral toxicity. However, sub-acute and chronic experiments of benzyl indanocine 12i will
be mandatory to be carried out to look for any adverse effect on its prolonged exposure. Safety is
an essential component in drug development process and nowadays referred as
‘Pharmacovigilance’. It thoroughly focuses on adverse drug reactions. According to WHO, it aims
to enhance safety and care of patient in relation to use of medicine and to support public health
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programmes by providing reliable, balanced information for effective assessment of risk-benefit
profile of medicines [WHO 2002].
4
. CONCLUSION
In conclusion, an efficient protocol has been developed for the synthesis of indanocine. It is a
simple, efficient and straight-forward method. Benzylindanocine 12i has been investigated as a
potent anticancer agent. It exhibited antiproliferative effect via microtubule destabilization by
occupying colchicine binding pocket of β-tubulin. It showed potential in-vivo efficacy and good
tolerability in rodent model. The concomitant antiinflammatory activity of benzylindanocine 12i is
paramount, which is very much required in anticancer drug development. Study also enlightens
that increase in flexibility at 2-benzylidene system does not necessarily enhance the tubulin
binding and hence the efficacy.
5
. EXPERIMENTAL
5
.1 Chemistry
Synthesis of indanocine
,3-Dihydro-5,6-dimethoxy-7-nitro-indan-1-one (10)
2
To a cold and stirred solution of acetic anhydride (6 mL, 648 mg, 6.4 mmol), nitric acid (3 mL)
o
was added dropwise and reaction mixture was kept in ice bath (3-5 C). After 10 min. 5,6-
o
dimethoxyindanone (192 mg, 1 mmol) was added in fractions and further stirred at 8-10 C for 2
h. On completion, it was poured into crushed ice, extracted with ethyl acetate (15 mLx3) and
washed with water. Combined organic layer was dehydrated over anhydrous Na SO and dried in
2
4
vacuo. The desired product was recrystallized with chloroform-hexane to get nitro derivative 10 as
amorphous solid.
o
1
Yield=74%; light yellowish brown solid, m.p.= 93-95 C; H NMR (500 MHz, CDCl ): δ 2.70-
3
2
.72 (t, 2H, 3-CH , J=6Hz), 3.08-3.10 (t, 2H, 2-CH , J=5.75Hz ), 3.89 (s, 3H, OCH ), 3.95 (s, 3H,
2 2 3
1
3
OCH ), 7.02 (s, 1H, 4-CH, aromatic); C NMR (125 MHz, CDCl ) : δ 25.78, 36.69, 56.77, 62.38,
3
3
1
2
10.09, 120.31, 139.77, 140.69, 152.83, 159.25, 200.34; ESI-MS for C H NO (MeOH):
11 11 5
+
+
+
38[M+H] , 260[M+Na] , 276[M+K] ; ESI-TOF (MeOH) for C H NO calculated 238.0715 for
1
1
11
5
+
[
M+H] , found; 238.0718.
E)-2-(4-hydroxy-3,5-dimethylbenzyliden)-2,3-dihydro-5,6-dimethoxy-7-nitroindan-1-one (11i)
To a stirred solution of dioxane (20 mL)-conc. HCl (12 N, 2 mL), 5,6-dimethoxy-7-nitroindanone
(
(10, 474 mg, 2 mmol) was added. After 10 min., 4-hydroxy-3,5-dimethylbenzaldehyde (328 mg,
o
2
.2 mmol) was added to it and refluxed at 110 C. After 3h, conc. HCl (2 mL) was added to
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reaction mixture and further stirred for 5 h. On completion, the reaction mixture was poured into
water (20 mL) and extracted with ethyl acetate (20 mLx2). Pooled organic layer was dehydrated
over anhydrous Na SO and evaporated to dryness. Residue thus obtained was charged on a silica
2
4
gel glass column and eluted with ethyl acetate-hexane to get the desired benzylidene derivative
1
1
6
1i. It was recrystallized from chloroform-pentane (1:3).
₀
1
1i: Yield= 82%; yellow solid; m.p.=220-222 C; H NMR (500 MHz, DMSOd ): δ 2.22 (s,
6
H,CH ), 3.85 (s, 3H, OCH ), 4.02 (s,3H, OCH ), 4.05 (s, 2H, 3-CH ), 7.31 (s, 2H, 2’& 6’-CH of
3
3
3
2
1
3
benzylidene ring), 7.35 ( s, 1H, 4-CH, aromatic), 7.56 (s, 1H, 2-benzylidene CH); C NMR (125
MHz, DMSOd ): δ 16.64, 16.64, 32.00, 57.21, 62.13, 111.57, 120.19, 124.90, 124.90, 125.66,
6
1
30.92, 131.80, 131.80, 134.00, 139.31, 139.45, 148.02, 155.94, 158.41, 187.95; ESI-MS for
+
+
-
C H NO (MeOH): Positive mode: 370 [M+H] , 408 [M+K] ; Negative mode: 368 [M-H] ; ESI-
2
0
19
6
+
TOF (MeOH) for C H NO calculated 370.1290 for [M+H] , found; 370.1289.
2
0
19
6
(E)-2-(4-hydroxy-3,5-dimethylbenzyliden)-2,3-dihydro-5,6-dimethoxy-7-aminoindan-1-one(1,
Indanocine)
Nitrobenzylidene derivative 11i (500 mg, 1.36 mmol) was taken in dry dimethylformamide (10
mL). To this stirred solution 10% palladium-charcoal (50 mg) was added and hydrogen gas was
supplied via balloon. Reaction mixture was stirred at RT for 30 min. On completion, reaction
mixture was carefully filtered through celite (filter aid), washed with dil. HCl (1N, 2 mL) and
extracted with ethyl acetate (20 mLx3), washed with water (20 mLx2). Combined organic fraction
was dehydrated over anhydrous Na SO and dried in vacuo. The crude mass was purified through
2
4
silica gel column eluting with ethyl acetate–hexane to get pure indanocine 1 as amorphous solid.
₀
₀
1
1
: Yield=44%, dark yellowish brown solid; m.p.=236-238 C [238-240 C]; H NMR (500 MHz ,
DMSOd ): δ 2.21 (s, 6H, 2xCH ), 3.64 (s, 3H, OCH ), 3.83 (s, 3H, OCH ), 3.85 (s,2H,3-CH ),
6
3
3
3
2
6
.28 (s, 2H, exchangeable, NH ), 6.50 (s, 1H, =CH of benzylidene ring), 7.14 (s, 1H, 4-CH of
2
benzylidene ring), 7.28 (s, 2H, 2’& 6’-CH, aromatic), 8.85 (s, 1H, exchangeable, OH, phenolic);
1
3
C NMR (125 MHz, DMSOd ): δ 16.72, 16.72, 31.88, 56.00, 59.45, 97.45, 97.45, 116.33, 124.80,
6
1
24.80, 126.42, 130.07, 131.10, 132.14, 133.19, 141.79, 146.88, 154.97, 158.30, 192.96; ESI-MS
+
+
+
for C H NO (MeOH): Positive mode: 340[M+H] , 362[M+Na] , 378[M+K] ; Negative mode:
2
0
21
4
-
+
3
38 [M-H] ; ESI-TOF (MeOH) for C H NO calculated, 340.1548 for [M+H] , found, 340.1545.
20 21 4
General procedure for synthesis of derivatives 11a, 11c, 11f-11h, and 11j-11l
E)-2-benzylidene-2,3-dihydro-5,6-dimethoxy-7-nitroindan-1-one (11a)
(
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To a stirred solution of 3% KOH in methanol (w/w, 10 mL), 5,6-dimethoxy-7-nitroindanone (300
mg, 1.3 mmol) was added. After 10 min, benzaldehyde (170 mg, 1.6 mmol) was added and
reaction mixture was further stirred at RT for 4 h. On completion, solvent was evaporated under
vacuum and reaction mixture was diluted with water (20 mL). It was extracted with ethyl acetate
(20 mLx3), washed with water (20 mLx2) and organic fraction was dehydrated over anhydrous
Na SO . It was dried in vacuo to get a crude mass which was purified through column
2
4
chromatography over silica gel by eluting with ethyl acetate-hexane to get the desired product 11a
as amorphous solid.
₀
1
1
1a: Yield=67%; light yellow solid; m.p.=207-209 C; H NMR (500 MHz, DMSOd6):δ 3.86 (s,
3
H, OCH ), 4.04 (s, 3H,OCH ), 4.12 (s, 2H, 3-CH ), 7.47-7.52 (bs, 5H, 5xCH of benzylidene
3
3
2
1
3
ring), 7.75 (bs, 2H, CH of benzylidene & 4-CH); C NMR (125 MHz, DMSOd6): δ 31.87, 57.25,
6
1
2.13, 111.50, 119.79, 129.05, 129.05, 130.09, 130.80, 130.80, 133.11, 134.51, 134.57, 139.44,
+
48.34, 158.82, 188.02; ESI-MS for C H NO (MeOH): Positive mode: 326[M+H] , 348
1
8
15
5
+
+
-
[
M+Na] , 364 [M+K] ; Negative mode: 324 [M-H] ; ESI-TOF (CH CN) for C H NO calculated
3
18 15
5
+
3
26.1028 for [M+H] , found; 326.1027.
Synthesis of (E)-2-(4-piperazin-1-yl)benzylidene)-2,3-dihydro-5,6-dimethoxy-7-nitro-indan-1-one
11m)
(
A stirred solution of Boc-piperazine deriv. 11l (200 mg, 0.39 mmol) in dry dichloromethane (8
o
mL) was cooled to 5-10 C. To this stirred solution, trifluoroacetic acid (2 mL) was added drop-
wise and stirred for an hour. On completion, solvent was evaporated without heating under
vacuum. Residue was diluted with water (10 mL), basified with aqueous ammonia (30%, 8-10
mL) and extracted with ethyl acetate (20 mLx2). Pooled organic fraction was washed with water
(
20 mLx2), dehydrated over anhydrous Na SO and dried in vacuo. The residue thus obtained was
2 4
recrystallized from chloroform-acetone to get 11m as amorphous solid.
₀
1
1
1m: Yield=91%; orange solid; m.p.=163-165 C; H NMR (500 MHz, CDCl +DMSOd ): δ 3.03
3 6
(
bs, 4H, N-(CH ) of piperazine ring), 3.55 (bs, 4H, N-(CH ) of piperazine ring), 3.92 (s, 3H,
2 2 2 2
OCH ), 3.98 (s, 3H, OCH ), 4.06 (s,2H, 3-CH ), 6.95 ( bs, 2H, 2’ &3’-CH of 2-benzylidene
3
3
2
phenyl ring), 7.45(bs, 2H, 5’& 6’-CH of benzylidene ring), 7.55 ( s, 1H, 4-CH, aromatic), 7.68 (s,
1
4
1
H, 2-benzylidene CH); 13C NMR (125 MHz, CDCl +DMSOd ): δ 31.37, 44.47, 44.47, 47.12,
3 6
7.12, 55.89, 61.25, 109.31, 113.61, 113.61, 120.40, 123.65, 128.87, 131.59, 131.67, 133.50,
33.58, 138.98, 139.17, 146.13, 151.34, 157.53, 187.18; ESI-TOF (MeOH) for C H N O
2
3
23
3
5
+
calculated, 410.1715 for [M+H] , found, 410.1715.
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General procedure for synthesis of 2-benzyl analogues of indanocine 12a-12l
-Nitro-2-benzylidene indanocine derivative (11a-11l, 1.5 mmol) was taken in dry
7
dimethylformamide (10 mL). To this stirred solution 10% palladium-charcoal (100 mg) was added
and hydrogen gas was supplied through a balloon via syringe. After 2 h, 50 mg palladium-charcoal
was again added to it and reaction was further stirred for 2-4 h at RT. On completion, it was
filtered carefully through celite (filter aid), washed with dil. HCl (5%, 5 mL) and extracted with
ethyl acetate (20 mLx2). Pooled organic fraction was washed with water, dried over anhydrous
Na SO and distilled off under vacuum to get a crude mass. It was charged on a silica gel column
2
4
to get desired 7-amino-2-benzyl indanocine derivative (12a-12l) and recrystallized from
chloroform-hexane.
2
-(3-Hydroxy-4-methoxybenzyl)-7-amino-5,6-dimethoxy-2,3-dihydroindan-1-one (12e)
₀
1
Yield=46%, brown solid; m.p.=110-114 C; H NMR (500 MHz, CDCl ): δ 2.48-2.70 (bm, 2H, 3-
3
CH ), 2.81-2.99 (bm, 2H, CH benzylic), 3.22-3.26 (bm, 1H, 2-CH), 3.78 (s,3H, OCH ), 3.89
2
2
3
(
s,3H, OCH ), 3.91(s, 3H, OCH ), 5.61 (s, 2H, exchangeable, NH ), 6.20 (s, 1H, 4-CH, aromatic),
3 3 2
6
.69-6.70 ( d, 1H, 5’-CH, aromatic, J=8Hz), 6.76-6.78 (d, 1H, 6’-CH, aromatic, J=8Hz), 6.82 (s
1
3
,
1H, 2’-CH, aromatic); C NMR (125MHz, CDCl ): δ 32.10, 36.79, 49.36, 55.92, 55.99, 59.87,
3
9
2
7.59, 110.72, 115.10, 115.38, 120.32, 132.55, 133.27, 141.04, 145.09, 145.50, 151.25, 158.68,
+
06.98; ESI-MS for C H NO (MeOH): 344[M+H] ; ESI-TOF (MeOH) for C H NO
1
9
21
5
19 21
5
+
calculated, 344.1497 for [M+H] , found, 344.1493;
-(3,4-Methylenedioxybenzyl)-7-amino-5,6-dimethoxy-2,3-dihydroindan-1-one (12g)
2
₀
1
Yield=47%, brown solid; m.p.=131-133 C; H NMR (500 MHz, CDCl ): δ2.51-2.56 (bm, 2H,
3
phenyl ring CH ), 2.64-2.68 (bm, 2H, 3-CH ), 3.21 (m, 1H, 2-CH), 5.61 (s, 2H, exchangeable,
2
2
NH ), 5.91 (s, 2H, -O-CH -O-), 6.20 (s,1H, 2’-CH, aromatic), 6.65-6.72 (bs, 3H, 5’& 6’-CH and
2
2
1
3
4
1
-CH, aromatic); C NMR (125 MHz, CDCl ): δ 31.95, 36.50, 49.41, 55.93, 59.87, 97.57, 100.84,
3
08.18, 109.26, 115.34,121.82, 132.54, 133.70, 141.05, 145.95, 151.16, 158.70, 162.58, 206.79;
+
+
ESI-MS for C H NO (MeOH): 364[M+Na] , 380[M+K] ; ESI-TOF (MeOH) for C H NO
5
1
9
19
5
19 19
+
calculated, 342.1341 for [M+H] , found, 342.1336.
-(3,5-Dimethyl-4-hydroxybenzyl)-7-amino-5,6-dimethoxy-2,3-dihydroindan-1-one (12i)
2
₀
1
Yield=47.6%, light yellow solid; m.p.=121-123 C; H NMR (500 MHz, CDCl ): δ2.22 (s, 6H,
3
2
3
xCH ), 2.31-2.40 (bm, 1H, CH benzylic), 2.69-2.70 (bm, 1H, CH ,benzylic), 2.87-2.99 (bm, 2H,
3 2 2
-CH ), 3.20-3.23 (bm, 1H, 2-CH), 3.81 (s, 3H, OCH ), 3.93 (s, 3H, OCH ), 5.62 (s, 1H, 2’-CH,
2
3
3
aromatic), 6.21 (s, 1H, 6’-CH, aromatic), 6.83 (s, 1H, 4-CH, aromatic); 13C NMR (125MHz,
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CDCl ): δ15.98, 15.98, 32.23, 36.71, 49.61, 55.92, 59.87, 97.59, 115.40, 123.04, 123.04, 129.00,
3
1
3
29.00, 131.52, 132.54, 141.04, 150.64, 151.34, 158.67, 207.23; ESI-MS for C H NO (MeOH):
20 23 4
+
+
+
42[M+H] , 364 [M+Na] , 380 [M+K] ; ESI-TOF (MeOH) for C H NO calculated 342.1705
2
0
23
4
+
for [M+H] , found; 342.1699.
-[4-(4-Piperazin-1-yl) benzyl]-7-amino-5,6-dimethoxy-2,3-dihydroindan-1-one (12m)
N-Boc-piperazinyl-2-benzyl indanocine derivative 11m was used as starting substrate.
2
₀
1
Yield=46%, yellow solid; m.p.=137-139 C; H NMR (500MHz, CDCl ): δ2.63-2.67 (bm,2H,
3
CH , benzylic), 2.95-3.00 (bm, 2H, 3-CH ), 3.23-3.26 (t, 1H, 2-CH, J=9Hz ), 3.33-3.38 (t, N-
2
2
(
CH ) of piperazine ring, J=12.5Hz ), 3.85 (s, 3H, OCH ), 3.87 (s, 3H, OCH ), 5.62 (s,2H,
2
2
3
3
exchangeable, NH ), 6.20 ( s,1H, 4-CH, aromatic), 6.84-86 (d, 2H, 2’ & 3’-CH, aromatic,
2
J=8.5Hz), 7.15-7.16 (d, 2H, 5’& 6’-CH, aromatic, J=8.5Hz ); 13C NMR (125MHz, CDCl ): δ
3
3
1
2.06, 36.49, 43.49, 43.49, 47.23, 47.23, 49.27, 55.94, 59.89, 97.57, 115.35, 117.47, 117.47,
29.90, 129.90, 132.53, 133.30, 141.04, 148.44, 151.18, 158.71, 206.92; ESI-TOF (MeOH) for C
+
H N O calculated, 382.2130 for [M+H] , found, 382.2128.
2
2
27
3
3
General procedure for synthesis of 2-benzylidene-7-amino-indanocine analogues (13n-13p)
-Nitro-2-benzylidene indanocine analogue (11e/11g/11p, 0.6 mmol) was stirred in dry
7
dimethylformamide (10 mL) at RT. To this 10% palladium-charcoal (50 mg) was added and
hydrogen was supplied via balloon. Reaction mixture was stirred for 4 h. On completion, reaction
mixture was carefully filtered through celite, washed with dil. HCl (3 mL), extracted with ethyl
acetate and washed with water (20 mLx2). Combined organic layer was dehydrated over
anhydrous Na SO and evaporated in-vacuo to get a residue which was purified through silica gel
2
4
column eluting with ethyl acetate-hexane to afford desired 7-amino-2-benzylidene indanocine
derivatives (13n-13p).
(E)-2-(4-Hydroxy-3-methoxybenzylidene)-2,3-dihydro-5,6-dimethoxy-7-amino-indan-1-one (13n)
₀
1
Yield=46%, brownish yellow solid; m.p.=90-92 C; H NMR (500 MHz, CDCl ): δ 3.81 (s, 3H,
3
OCH ), 3.85 (s, 2H, 3-CH ), 3.94 (s, 3H, OCH ), 3.96 (s,3H, OCH ), 5.75 (s,2H, exchangeable,
3
2
3
3
NH ), 6.38 (bs, 1H, 2’- CH, aromatic), 6.97-6.99 (d, 1H, 5’-CH, aromatic, J=8Hz), 7.11-7.11 (d,
2
1
H, 6’-CH, aromatic, J=1.5Hz), 7.26 (s, 1H, 4-CH, aromatic), 7.40 (s, 1H, =CH, 2-benzyidene);
1
3
C NMR (125 MHz, CDCl ): δ 32.35, 55.98, 56.01, 59.94, 97.37, 112.96, 114.89, 117.42, 124.31,
3
1
28.38, 130.85, 132.84, 133.88, 141.66, 146.65, 146.76, 146.90, 158.42, 193.85; ESI-MS for
+
+
+
C H NO (MeOH): Positive mode: 342[M+H] , 364[M+Na] , 380[M+K] ; Negative mode:
1
9
19
5
-
+
3
40[M-H] ; ESI-TOF (MeOH) for C H NO calculated 342.1341 for [M+H] , found, 342.1334.
19 19 5
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Synthesis of 5,6-dimethoxy-1-oxo-indan-2-yl-2-acinitro-methanium (14)
To a cold stirred solution of sodium nitrite (50 mg) in dry methanol (6 mL), boron trifluoride-
etherate (0.5 mL) was added dropwise. To this reaction mixture, a solution 5,6-
dimethoxyindanone (9, 50 mg, 0.26 mmol) pre-dissolved in methanol (1 mL) was added dropwise
to it. The reaction mixture was further stirred on ice-bath for 2 h followed by at RT for an hour.
On completion, solvent was evaporated under vacuum and residue was taken in ethyl acetate (20
mL), organic layer was washed with water and dehydrated over anhydrous Na SO . It was
2
4
evaporated under vacuum to get a residue which was recrystallized from acetone-chloroform to
get compound 14 as amorphous solid.
1
4: Yield=61%, yellowish brown solid; m.p.=204-206 ₀C; 1H NMR (500 MHz,
CDCl +DMSOd6): δ3.89 (s, 3H, OCH ), 4.38 (s, 3H, OCH ), 4.48 (s, 2H, 3-CH ), 7.48 (s, 1H,4-
3
3
3
2
1
3
CH, aromatic), 7.71 (s, 1H, 7-CH), 8.21 (s,1H, OH); C NMR (125MHz, CDCl +DMSOd6): δ
3
2
6.86, 49.32, 54.93, 55.26, 103.64, 106.89, 130.03, 141.42, 148.45, 153.56, 155.11, 187.23; ESI-
+
TOF (MeOH) for C H NO calculated, 238.0715 for [M+H] , found, 238.0852.
1
1
11
5,
5
.2. Biological evaluations
Detailed protocols are in Supplementary information.
Antiproliferative activity by Sulphorhodamine assay
Antiproliferative assay was performed as per reported method [Akindele et al. 2015].
Combretastatin A4 and paclitaxel were used as standard drugs for comparison.
Tubulin polymerisation inhibition assay
Tubulin polymerization assay was performed using ‘assay kit-BK-006P’ from Cytoskeleton, USA,
as per Manufacturer’s protocol [Lee et al. 1997]. Paclitaxel was used as a standard stabilizer of
tubulin polymerase and podophyllotoxin (PDT) as standard inhibitor.
Anti-inflammatory activity against LPS-induced inflammation
Antiinflammatory assay was performed on primary macrophages cells which were isolated from
peritoneal cavities of Swiss albino mice as per our reported method [Pathak et al. 2019]. TNF-α
and IL-6 released in the cell culture supernatant were determined with ELISA kits.
Molecular docking studies
Docking studies were performed to know the binding conformation of ligands in active site of
protein by AutoDockVina 1.1.2 version by keeping all the docking parameters on default settings
[Trott and Olson 2010]. The bound protein 3D crystallographic structure of Tubulin-colchicine
complex PDB ID:4O2B was used to know the binding of enantiomers (2R/S)
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benzylindanocine12i, indanocine along with colchicine and podophyllotoxin as control drugs.
Whole protein was selected with maximum grid size for docking. All ligands and protein
structures were prepared for docking studies by protonating them and removing water molecules
from protein. After docking top 10 ligand protein interaction poses were selected and visualize for
their 2D and 3D poses using Discovery Studio visualizer. Further, ADME properties were
predicted at free online software provided by Swiss Institute of Bioinformatics, Switzerland dated
2
6/04/2020 [www.swissadme.ch 2020].
Ehrlich Ascites carcinoma
In vivo anticancer activity of benzylindanocine (12i) was evaluated against EAC mice model.
Study and number of animals used were approved by Institutional Animal Ethics Committee of
CSIR-CIMAP, Lucknow, India via CIMAP/IAEC/2016-19/32 dated 09-02-2017.
Acute oral toxicity in Swiss albino mice
Safety evaluation of compound 12i was done as per reported method [Chanda et al. 2009] at
four different single acute oral doses at 5 mg/kg, 50 mg/kg, 300 mg/kg, and 1000 mg/kg. Study
and number of animals used were approved via CIMAP/IAEC/2016-19/01 dated 09-02-2017 by
Institutional Animal Ethics Committee of CSIR-CIMAP, Lucknow, India.
4
.3.7. Statistical analysis
All data have been expressed as mean±SD and calculated using MS-Excel. Statistical analysis
of differences was carried out by ANOVA followed by Tukey’s multiple comparison test.
Comparisons were made relative to the untreated controls. Differences with p value<0.05 were
considered significant.
Declaration of competing interest
Authors declare that there is no conflict of interest.
Acknowledgement
The study was financially supported from CSIR CIMAP under MLP-10. Ms Sadiya Khwaja and
Ms. Kaneez Fatima acknowledge to CSIR for Senior Research Fellowships. Biological Central
Facility and Chemical Central Facility, CSIR-CIMAP are duly acknowledged for Sophisticated
Instruments support.
Supplementary information
Additional information may be found online in the Supporting Information section
REFERENCES
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Akindele, A. J., Wani, Z., Mahajan, G., Sharma, S., Aigbe, F. R., Satti, N., Adeyemi, O. O., &
Mondhe, D. M. (2015). Anticancer activity of Aristolochia ringens Vahl. (Aristolochiaceae).
J. Trad. Compl. Med., 5, 35-41.
Baell, J., & Walters, M. A. (2014). Chemistry: Chemical con artists foil drug discovery. Nature,
5
13, 481-483.
Beams, H. W., & Kessel, R. G. (1968). Properties of the Ehrlich Ascites tumor cell as determined
by electron microscopy, ultracentrifugation, and hydrostatic pressure. Cancer Res. 28 ,
1
944–1951.
Burns R. G. (1992). Analysis of the colchicine-binding site of beta-tubulin. FEBS J, 297, 205-208.
Cancer facts and figures (2020), American Cancer Society, Atlanta, GA 30303-1002, USA.
Cancer Keyfacts (2018). WHO, 12 September 2018.
Carson, D. A., Shih, C. H., Cottam, H. B., & Leoni, L. (2000). Indanone tetralone compounds for
inhibiting cell proliferation. US Pat., 6,162,810.
Chai, E. Z. P., Siveen, K. S., Shanmugam, M. K., Arfuso, F., & Sethi, G. (2015). Analysis of the
intricate relationship between chronic inflammation and cancer. Biochem. J., 468, 1–15.
Chanda, D., Shanker, K., Pal, A., Luqman, S., Bawankule, D. U., Mani, D. N., & Darokar, M.
P.(2009). Safety evaluation of Trikatu, a generic Ayurvedic medicine in Charles Foster rats.
J. Toxicol. Sci., 34, 99-108.
Coulup, S. K., & George, G. I. (2019). Revisiting microtubule targeting agents: α-tubulin and the
pironetin binding site as unexplored targets for cancer therapeutics. Bioorg. Med. Chem.
Lett., 29, 1865-1873.
Das, L., Gupta, S., Dasgupta, D., Poddar, A., Janik, M. E., & Bhattacharyya, B. (2009). Binding of
indanocine to the colchicine site on tubulin promotes fluorescence, and its binding
parameters resemble those of the colchicine analogue AC. Biochemistry, 48, 1628–1635.
Furman, D., Campisi, J., Verdin, E., Carrera-Bastos, P., Targ, S., franceschi, C., Ferrucci, L.,
Gilroy, D.W., Fasano, A., Miller, G. W., Miller, A. H., Mantovani, A., Weyand, C. M.,
Barzilai, N., Goronzy, J. J., Rando, T. A., Effros, R. B., Lucia, A., Kleinstreuer, N., &
Slavich, G. M. (2019). Chronic inflammation in the etiology of disease across the life span.
Nat. Med., 25, 1822-1832.
Garbers, C., Heink, S., Korn, T., & Rose-John, S. (2018). Interleukin-6: designing specific
therapeutics for the complex cytokine. Nat. Rev. Drug Discov., 17, 395-412.
This article is protected by copyright. All rights reserved

Giannakakou, P., Sackett, D., & Fijo, T. (2000). Tubulin/microtubule: still a promising target for
new chemotherapeutic agents. J. Natl. Cancer Inst., 92, 182-183.
http://www.swissadme.ch/, Swiss Institute of Bioinformatics 2019, access date 26/04/2020.
Huang, L., Lu, C., Sun, Y., Mao, F., Luo, Z., Su, T., Jiang, H., Shan, W., & Li, X. (2012).
Multitarget-directed benzylideneindanone derivatives: anti-β-amyloid (Aβ) aggregation,
antioxidant, metal chelation, and monoamine oxidase B (MAO-B) inhibition properties
against Alzheimer's disease. J. Med. Chem., 55, 8483–8492.
Kapoor, S., & Panda, D. (2012). Kinetic stabilization of microtubule dynamics by indanocine
perturbs EB1 localization, induces defects in cell polarity and inhibits migration of MDA-
MB-231 cells. Biochem. Pharmacol., 83, 1495–1506.
Kaul, R., Risinger, A. L., & Mooberry, S. L. (2019). Microtubule-targeting drugs: More than
antimitotics. J. Nat. Prod. , 82, 680-685.
Kenakin T. P. (2017). In Book: Pharmacology in drug discovery and development. Chapter 7:
Pharmacokinetics I: Permeation and metabolism”, Second edition, Academic Press, Elsevier
Inc., pp.157-191.
Lee, J. C., & Timassheff, S. N. (1997). In vitro reconstitution of calf brain microtubules: effects of
solution variables. Biochemistry, 16, 1754-1764.
Leoni, L. M., Hamel, E., Genini, D., Shih, H., Carrera, C. J., Cottam, H. B., & Carson, D. A.
(2000). Indanocine, a microtubule-binding indanone and a selective inducer of apoptosis in
multidrug-resistant cancer cells. J. Natl. Cancer Inst., 92, 217–224.
Negi, A. S., Gautam, Y., Alam, S., Chanda , D., Luqman, S., Sarkar, J., Khan, F., & Konwar, R.
(2015). Natural antitubulin agents: importance of 3,4,5-trimethoxyphenyl fragment. Bioorg.
Med. Chem., 23, 373-389.
Pathak, N., Fatima, K., Singh, S., Mishra, D., Gupta, A. C., Kumar, Y., Chanda, D., Bawankule,
D. U.,
Shanker, K., Khan, F., Gupta, A., Luqman, S., & Negi, A. S. (2019). Bivalent furostene
carbamates as antiproliferative and antiinflammatory agents. J. Steroid Biochem. Mol. Biol.,
1
94 Article no. 105457.
Shih, H. C., Deng, L., Carrera, C. J., Adachi, S., Cottam, H. B., & Carson, D. A. (2000). Rational
design, synthesis and structure-activity relationships of antitumore (E)-2-benzylidene-1-
tetralones and (E)-2-benzylidene-1-indanones. Bioorg. Med. Chem. Lett., 10, 487–490.
This article is protected by copyright. All rights reserved

Trott, O., & Olson, A. J. (2010). AutoDock Vina: Improving the speed and accuracy of docking
with a new scoring function, efficient optimization, and multithreading. J. Comput. Chem.,
3
1, 455-461.
Tunbridge, G. A., Oram, J., & Caggiano, L. (2013). Design, synthesis and antiproliferative activity
of indole analogues of indanocine. Med. Chem. Commun., 4, 1452-1456.
WHO (2002). The Importance of Pharmacovigilance, safety monitoring of medicinal products.
World Health Organization.
…
…………………….
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Table 1A: Antiproliferative activity of indanocine derivatives (% Inhibition).
Cytotoxicity (% inhibition)a,*
Conc.
Sample code
HCT-116
SW 620
A549
(μM)
MIA PACA-2
1
5
1
5
1
5
1
5
1
5
1
5
1
5
1
5
1
5
1
5
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
---
3.14± 0.077
55.95± 0.043
---
14.94± 0.027
51.39± 0.031
42.14± 0.331
51.32± 0.048
41.85± 0.052
53.98± 0.036
19.67± 0 .021
30.61± 0.059
22.29± 0.049
33.88± 0.110
17.65± 0.169
39.15± 0.053
27.6± 0.212
45.41± 0.067
42.06± 0.056
54.3± 0.070
20.03± 0.163
41.13± 0.176
16± 0.033
---
1
1b
---
---
23.27 ± 0.064
50.81 ±0.102
16.42 ±0.157
49.43 ± 0.075
11.8±0.056
---
1
1e
36.01± 0.093
31.63± 0.019
41.54± 0.034
27.47± 0.024
42.86± 0.035
17.71± 0.127
50.19± 0.050
30.83± 0.070
48.31± 0.049
42.26± 0.024
60.38± 0.013
42.95± 0.059
52.33± 0.037
39.49± 0.049
53.41± 0.033
40.25± 0.092
55.63± 0.031
63.48± 0.014
66.94± 0.015
44.04± 0.128
64.89± 0.005
43.15± 0.094
65.15± 0.046
28.69± 0.069
50.72± 0.115
59.59± 0.069
62.51± 0.047
63.31± 0.019
68.8± 0.011
28.99± 0.039
20.57± 0.085
20.26± 0.152
26.1± 0.130
24.58± 0.088
31.78± 0.067
5.53± 0.042
9.68± 0.036
19.9± 0.036
20.79± 0.028
23.01± 0.036
28.24± 0.023
16.26± 0.125
21.82± 0.174
14.01± 0.023
22.94± 0.033
21.57± 0.014
30.01± 0.044
6.5± 0.122
1
1f
1
1k
56.8±0.060
9.94 ± 0.160
36.97± 0.102
49.35± 0.051
57.24± 0.008
51.84± 0.015
56.52± 0.050
30.46± 0.027
61.26± 0.076
50.42± 0.042
53.81± 0.035
35.51± 0.129
55.65± 0.087
57.06± 0.056
61.91± 0.017
24.25± 0.029
26.11± 0.085
55.51± 0.029
67.99± 0.003
22.4±0.072
1
2a
1
2d
1
2e
1
2f
1
2g
1
2h
48.02± 0.006
34.89± 0.140
44.8± 0.183
34.31± 0.041
47.47± 0.058
31.36± 0.053
37.86± 0.091
36.99± 0.087
42.8± 0.152
28.82± 0.042
47.85± 0.007
39.45± 0.027
40.91± 0.060
28.35± 0.119
1
2i
10
(
Benzylindanocine)
50
9.99± 0.157
18.26± 0.016
37.93± 0.038
19.09± 0.012
50.83± 0.019
19.2± 0.071
19.23± 0.070
7.02± 0.024
12.84± 0.023
30.65± 0.016
35.85± 0.025
27.45± 0.056
1
5
1
5
1
5
0
0
0
0
0
0
1
2l
1
2m
1
3o
38.6±0.077
1
3p
10
66.13±0.007
68.13 ± 0.013
61.93± 0.012
67.51± 0.013
48.87± 0.045
(
1:Indanocine)
50
1
0
CA4
5
0
Paclitaxel
10
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5
0
53.64± 0.023
29.37± 0.043
51.32± 0.044
36.39± 0.059
*
%inhibition<50% at 50 µM was considered inactive, *p<0.05 as compared to control
Table 1B: Cytotoxicity (IC ) of indanocine (1) and benzylindanocine (12i)
5
0
Compound
Cytotoxicity IC50 in µM
HCT- 116
MIA PACA-2
0.438 ±0.190
0.577 ±0.208
<0.1
Indanocine (1)
Benzylindanocine (12i)
Combretastatin A4
1.032 ±0.142
1.295 ±0.232
<0.1
Table 2. Effect of indanocine (1) and benzylindanocine (12i) on the pro-inflammatory cytokine TNF-α and
IL-6 levels (mean±SEM: *P<0.05; Vehicle vs treatment; #Vehicle vs normal; n=3)
Compound
LPS
Dose
TNFα (pg/mL)
TNF-α %
inhibition
NA
IL-6 (pg/mL)
IL-6 %
inhibition
NA
(
0.1µg/mL)
(µg/mL)
Normal
Vehicle
-
-
-
348.89±92.74
1049.62±19.59#
993.41±78.08
751.73±76.98*
839.51±28.62
741.36±30.55*
654.2±42.82*
1222±201.34
8505±400.02#
5285±506.44*
4591±526.04*
5360±355.16*
4982±418.39*
2582±114.29*






---
---
Benzylindanocine (12i)
1
5.35±7.43
28.38±7.33
20.01±2.72
29.36±2.91
37.67±4.08
37.85±5.95
46.02±6.18
36.97±4.17
41.41±4.91
69.63±1.34
10
1
Indanocine (1)
10
1
Dexamethasone
Table 3: Interaction of indanocine and both enantiomers of benzylindanocine with β-tubulin (PDB
ID:402B)
S.
Compounds
Docking
Energy
Interacting amino acids
Docking 2D poses
No.
Kcal/Mol
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1
(R)-
-7.9
VAL B:238, CYS B:241,
LEU B:242, LEU B:248,
ALA B:250, ASP B:251,
LEU B:255, ASN B:258,
THR B:314, VAL B:315,
ALA B:316, ALA B:317,
ILE B:318, ILE B:347,
ASN B:349, ASN B:350,
LYS B:352, ALA B:354,
ILE B:378
Benzylindanocine
(
12i-2α-isomer)
2
(S)-
-8.1
TYR B:202, VAL B:238,
CYS B:241, LEU B:242,
GLN B:247, LEU B:248,
ALA B:250, ASP B:251,
LEU B:252, LYS B:254,
LEU B:255, ASN B:258,
ALA B:316, ALA B:317,
ILE B:318, LYS B:352,
THR B:353, ALA B:354,
ILE B:378
Benzylindanocine
12i-2β-isomer)
(
3
Indanocine (1)
-8
TYR B:202, VAL B:238,
CYS B:241, LEU B:242,
LEU B:248, ALA B:250,
ASP B:251, LEU B:252,
LYS B:254, LEU B:255,
ASN B:258, ALA B:316,
ILE B:318, LYS B:352,
ILE B: 378
4
Colchicine
-6.2
GLN B:247, LEU B:248,
ASN B:249, ALA B:250,
LEU B:255, LYS B:254,
ASN B:258, MET B:259,
THR B:314, ALA B:316,
LYS B:352, THR B:353,
ALA B:354
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5
Podophyllotoxin
-7
CYS B:241, LEU B:248,
ALA B:250, LYS B:254,
LEU B:255, MET B:259,
ASN B:258, THR B:314,
VAL B:315, ALA B:316,
ALA B:317, ILE B:318,
ASN B:349, ASN B:350,
LYS B:352, THR B:353,
ALA B:354
Table 4 : Various druggability parameters of indanocine (1) and 2-benzylindanocine (12i)
Parameter
1
12i
Acceptable
range
Parameter
1
12i
Acceptable
range
Physicochemical properties
Molecular formulae C20H21NO4 C20H23NO4
Lipophilicity
---
≤500
≤10
Log Po/w
3.29
3.23
≤5
M.Wt.
339.39
341.40
Pharmacokinetics
Retable bonds
H-bond acceptors
H-bond donors
Molar Refractivity
3
4
4
4
GI absorption
BBBpermeability
P-gp substrate
CYP1A2
High
No
High
---
---
---
---
≤10
No
No
2
2
≤5
No
98.44
98.13
40-130
Yes
Yes
inhibition
Sp3 hybridisation
fraction
0.25
0.35
Not less
CYP2C9/19
inhibition
Yes
Yes
---
than 0.25
81.78Å2
81.78Å2
20 Å to
2
Drug likeness
TPSA
1
30 Å2
Water solubility
Lipinski rule
Yes, no
Yes, no
Up to 1
violation
moderate
violation violation
Water solubility
3.85 μg/mL
11.6 μg/mL
Bioavailability
score
0.55
0.55
Solubility class
Log S
Moderate
-4.61
Moderate
-4.47
acceptable
>-4
Medicinal chemistry
No No
PAINS
No
Table 5a. Effect of indanocine (1) and 2-benzylindanocine (12i) on body weight of mice bearing EAC
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Treatment
Control
Dose
Body weight (g)
Day 1
Day 5
Day 9
Day 12
Normal saline
26.60±0.75
27.28±0.86
25.24±1.89
25.875±0.62
26.66±2.07
33.32±1.31
34.30±1.28
(
0.2 mL i.p.)
Benzylindanocine
10 mg/kg i.p.
30 mg/kg i.p
90 mg/ kg i.p.
25.4±1.77
23.75±1.77
25.4±1.77
27.964±1.63
22.655±1.97
31.476±1.65
28.6±1.76
24.4±2.23
32.56±1.66
(
12i)
Benzylindanocine
12i)
Benzylindanocine
12i)
Indanocine (1)
-Fluorouracil
(
(
30 mg/kg i.p.
20 mg/kg i.p.
25±1.22
25.88±1.14
27.24±0.49
28.386±1.19
28.582±0.45
28.32±1.22
29±0.45
5
27.6±0.24
Table 5b. In vivo efficacy of indanocine (1) and 2-benzylindanocine (12i) against EAC
Treatment
Dose
Day 12
Av. Volume Av. Weight
Av. No. of
tumor cells
(1×107)
% Tumor Mortality
growth
of ascitic
fluid (mL)
4.28±0.37
of ascitic
fluid (g)
inhibition
Control
Normal saline
3.94±0.37
92.60±20.63
---
0
(0.2 mL i.p.)
Benzylindanocine (12i)
Benzylindanocine (12i)
Benzylindanocine (12i)
Indanocine (1)
10 mg/kg i.p.
30 mg/kg i.p.
90 mg/kg i.p.
30 mg/kg i.p.
20 mg/kg i.p.
3.52±0.13
3.075±0.13
2.54±0.43
0.76±0.05
0.58±0.09
3.28±0.09
3.175±0.07
2.42±0.49
0.532±0.07
0.2±0.04
55.4±2.06
42.75±5.15
20±1.48
40.2
53.8
78.4
89.1
97.2
0
0
0
0
0
10.1±1.27
2.55±0.42
5
-Fluorouracil
*
**p<0.001 (Dunnett test);
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Dose of 2-benzylindanocine (12i) at mg/kg body weight as a single oral
dose
Parameters
Control
5 mg/kg
21.09±0.28
13.27±0.27
5.97±0.29
4.80±0.46
75.97±2.46
30.69±3.06
14.21±0.71
2.67±0.06
0.48±0.03
3.42±0.04
50 mg/kg
22.30±0.58
15.30±0.59
6.06±0.53
4.24±0.67
77.56±8.42
27.32±1.45
14.72±0.39
2.76±0.03
0.58±0.04
3.43±0.05
300 mg/kg
21.30±0.65
14.16±0.64
7.05±0.59
3.91±0.34*
60.05±4.19
25.27±2.17
13.98±0.56
2.55±0.05
0.46±0.05
3.14±0.24
1000 mg/kg
21.29±0.63
14.56±0.82
5.70±0.67
3.33±0.32*
73.11±3.82
25.54±2.01
11.60±0.93
2.62±0.05
0.56±0.05
3.39±0.03
Body weight (gm)
24.19±1.19
Haematological Haemoglobin (gm/dL) 13.81±0.95
RBC (million/mm3)
WBC (1000*/mm3)
ALKP (U/L)
6.48±0.74
6.52±0.83
80.35±6.36
33.52±2.61
15.92±0.90
2.70±0.13
0.47±0.04
3.48±0.06
profile
SGOT (U/L)
SGPT (U/L)
Liver Function
Test
Albumin (g/dL)
Bilirubin (mg/dL)
Serum Protein
(mg/ml)
Kidney Function
Test
Creatinine (mg/dL)
0.37±0.02
0.35±0.03
0.32±0.02
0.36±0.04
0.30±0.03
Lipid Profile
Triglycerides (mg/dL)
87.52±6.76
74.18±4.64
85.21±3.50
81.85±5.33
93.94±7.80
Table 6. Effect of 2-benzylindanocine (12i) as a single acute oral dose in Swiss albino mice (Mean±SE;
n=6; *, significant compared to control P<0.05).
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Figure legends:
Figure 1. Structures of some of the important antimitotic anticancer drugs
Figure 2: Structure and antiproliferative activity of indanocine and some previously reported derivatives
Figure 3A: Effect of indanocine (1) (5, 10 and 20 µM) on tubulin polymerisation
Figure 3B: Effect of 2-benzylindanocine (12i) (5, 10 and 20 µM) on tubulin polymerisation
Figure 4. Molecular docking representation of compounds binding within the same binding pocket. The
compounds were represented in color as 2α-benzylindanone (12i)- (cyn), 2β-benzylindanone (12i) (yellow),
indanocine (green), colchicine (red), and podophyllotoxin (blue)
Figure 5: Two dimensional bioavailability radars of Indanocine (1) and 2-benzylindanocine (12i)
(
Six physicochemical properties are taken into account: lipophilicity, size, polarity, solubility, flexibility and
saturation. The pink area represents the optimal range for each properties (lipophilicity: XLOGP3 between − 0.7
2
and + 5.0, size:MW between 150 and 500 g/mol, polarity: TPSA between 20 and 130 A , solubility: log S not
higher than 6, saturation: fraction of carbons in the sp3 hybridization not less than 0.25, and flexibility: no more
than 9 rotatable bonds. In this example, both the compounds are predicted to be moderate orally bioavailable)
Figures 6 A & 6B: Effect of benzylindanocine 12i on absolute and relative organ weight in Swiss albino mice
(
Mean±SE; n=6).
Figure 7: Effect of benzylindanocine 12i on differential leucocytes counts 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).
Scheme 2:
o
o
Reagents and conditions: iv) Ac
2
O-HNO
3
, 5-10 C, 3h, 74%; v) NaNO
2
-dry MeOH, BF
3
-Et O, 10 C-
2
RT, 2h, 61%; vi) For 11a, 11c, 11f-11h &11j-11l: substituted benzaldehyde, 3% KOH in MeOH, 3-
6
h, 59-86% 11a,67%; 11c,59%; 11f, 86%; 11g, 61%; 11h,65%;&11j, 56%, 11k, 69%; 11l, 76%; For
o
11b, 11d, 11e, &11i: substituted benzaldehyde, dioxane-HCl, 110 C, 7-8h, 11b, 69%; 11d, 83%;
o
&
11e, 64%;11i, 82%. EtOH-SOCl
2
, RT, 20h, %; For 11m: 11l, dry DCM-TFA, 0 C,1h, 91%; vii)
1
0%Pd-C, H , RT, 2-4h, 12a-12m: 44-67%; 13n-13p: 44-46%.
2

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