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

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^,³Kaneez Fatima,¹^,³Divya Mishra, Vineet Babu, Yogesh Kumar, Sumera Banu Malik,

1

2

Misbah Tabassum, Suaib Luqman, Dnyaneshwar U. Bawankule,¹^,³Debabrata Chanda,¹^,³Feroz Khan,¹^,³

2

1,3

D. M. Mondhe,²^,³Arvind 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

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-

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

. 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].

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

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.

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

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

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

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

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

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

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

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

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

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

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

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)

(

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

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

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

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

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.

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

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

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

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

aromatic), 6.21 (s, 1H, 6’-CH, aromatic), 6.83 (s, 1H, 4-CH, aromatic); ¹³C NMR (125MHz,

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

(

CH ) of piperazine ring, J=12.5Hz ), 3.85 (s, 3H, OCH ), 3.87 (s, 3H, OCH ), 5.62 (s,2H,

2

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

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

27

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

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

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

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

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)

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

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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

---

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

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

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

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

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

GI absorption

BBBpermeability

P-gp substrate

CYP1A2

High

No

High

---

≤10

No

2

≤5

No

98.44

98.13

40-130

Yes

inhibition

Sp3 hybridisation

fraction

0.25

0.35

Not less

CYP2C9/19

inhibition

Yes

---

than 0.25

81.78Å²

20 Å to

2

Drug likeness

TPSA

1

30 Å²

Water solubility

Lipinski rule

Yes, no

Up to 1

violation

moderate

violation violation

Water solubility

3.85 μg/mL

11.6 μg/mL

Bioavailability

score

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

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)

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

10.1±1.27

2.55±0.42

5

-Fluorouracil

*

**p<0.001 (Dunnett test);

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).

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

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

Article Doi

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

DOI: 10.1111/cbdd.13857

Source and publish data:

Authors:

Article abstract of DOI:10.1111/cbdd.13857

Full text of DOI:10.1111/cbdd.13857

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