Fluorinated benzylidene indanone exhibits antiproliferative activity through modulation of microtubule dynamics and antiangiogenic activity

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Fluorinated benzylidene indanone exhibits antiproliferative activity

through modulation of microtubule dynamics and antiangiogenic

activity

Ankita Srivastava chemical synthesis ,

Kaneez Fatima Soft agar analysis; cell cycle; Antiangiogenesis; annexin V experiments ,

Eram Fatima chemical synthesis , Arjun Singh Acute oral toxicity ,

Aastha Singh chemical synthesis ,

Aparna Shukla Molecular docking studies ,

Suaib Luqman Soft agar analysis; cell cycle; Antiangiogenesis; annexin V experiments ,

Karuna Shanker Purity proﬁle ,

Debabrata Chanda Acute oral toxicity ,

Feroz Khan Molecular docking studies ,

Arvind S. Negi Design & synthesis; manuscript preparation; overall planning; responsibility etc

PII:

S0928-0987(20)30301-8

DOI:

Reference:

https://doi.org/10.1016/j.ejps.2020.105513

PHASCI 105513

To appear in:

European Journal of Pharmaceutical Sciences

Received date:

Revised date:

Accepted date:

3 May 2020

11 August 2020

Please cite this article as: Ankita Srivastava chemical synthesis , Kaneez Fatima Soft agar analysis; cell cycle; Anti

Eram Fatima chemical synthesis , Arjun Singh Acute oral toxicity , Aastha Singh chemical synthesis ,

Aparna Shukla Molecular docking studies , Suaib Luqman Soft agar analysis; cell cycle; Antiangiogenesis; annexin

Karuna Shanker Purity proﬁle , Debabrata Chanda Acute oral toxicity , Feroz Khan Molecular docking studies ,

Arvind S. Negi Design & synthesis; manuscript preparation; overall planning; responsibility etc , Flu-

orinated benzylidene indanone exhibits antiproliferative activity through modulation of microtubule

dynamics and antiangiogenic activity, European Journal of Pharmaceutical Sciences (2020), doi:

https://doi.org/10.1016/j.ejps.2020.105513

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Highlights

* An extensive cancer pharmacology of Fluorinated benzylidene indanone has been explored

* Induces late apoptosis in breast cancer cells (MCF-7 & MDA-MB-231)

* Occupies colchicine binding pocket of β-tubulin.

* Down-regulates tumour angiogenic factors; VEGF and HIF-α in MCF-7 cells.

* In-vivo efficacy: 48.2% tumour reduction at 120 mg/kg oral dose.

* Safety: in-vivo acute toxicity- safe up to 1000mg/kg dose

1

Fluorinated benzylidene indanone exhibits antiproliferative activity through

modulation of microtubule dynamics and antiangiogenic activity

Ankita Srivastava,^a,bKaneez Fatima,^a,bEram Fatima,^aArjun Singh,^aAastha Singh,^aAparna Shukla,^a

Suaib Luqman,^a,bKaruna Shanker,^a,bDebabrata Chanda,^a,bFeroz Khan,^a,bArvind S. Negi^a,b,*

^aCSIR-Central Institute of Medicinal and Aromatic Plants (CSIR-CIMAP), Kukrail Picnic Spot Road, P.O.

CIMAP, Lucknow-226015, India.

^bAcademy of Scientific and Innovative Research (AcSIR), Ghaziabad-201002, Uttar Pradesh, India.

Abstract

The application of fluorine in drug design has been understood significantly by the medicinal

chemists in recent years. Modulation of tubulin-microtubule dynamics is one of the most effective

targets for cancer chemotherapeutics. A logically designed and identified lead compound, fluorinated

benzylidene indanone 1 has been extensively evaluated for cancer pharmacology. It occupied

colchicine binding pocket acting as microtubule destabilizer and induced a G2/M phase arrest in

MCF-7 cells. Compound 1 exerted an antiangiogenic effect in MCF-7 cells by down-regulating

Vascular Endothelial Growth Factor (VEGF) and Hypoxia Inducible Factor-α (HIF-α). In in-vivo

efficacy in C3H/Jax mice mammary carcinoma model, benzylidene indanone 1 reduced tumour

volumes by 48.2%. Further in acute oral toxicity studies compound 1 was well tolerated and safe up

to 1000 mg/kg dose in Swiss albino mice. The fluorinated benzylidene indanone 1, a new chemical

entity (NCE) can further be optimized for better efficacy against breast adenocarcinoma.

Keywords: anticancer, breast cancer, benzylidene indanone, antitubulin, antiangiogenic, Ehrlich

ascites carcinoma, toxicity.

...........................................................................................................................................

*Corresponding author: E mail: arvindcimap@rediffmail.com (a.s. negi),

Tel: +91-522-2342676 x 583; Fax: +91-522-2342666;

2

CSIR-CIMAP Communication no. 2020-FEB/13

1. Introduction

Breast cancer is the second most common cancer in the world. It is the most frequent cancer among

women with an estimated 2.1 million new cancer cases diagnosed and 0.63 million deaths in 2018

[Cancer: WHO factsheet 2018]. It caused 0.57 million mortality in women which was more in less

developed countries. Over the past two decades the survival rate of breast cancer patients has been

enhanced significantly due to better diagnostic tools and more effective adjuvant therapies. In India,

breast cancer (14%) was the most prevalent among all types of cancers [Breast Globocon 2019].

There are sufficient evidences that endogenous estrogens play a critical role in the development of

breast cancer [Nowsheen et al. 2012; Samavat et al. 2015]. About two third of breast cancer cases

are hormone dependent breast cancer also known as estrogen positive (ER+ve) breast cancer. The ER

positive breast cancer is sensitive to hormone therapy and is commonly treated with estrogen

antagonists and/or aromatase inhibitors [Gupta et al. 2013]. Traditionally, breast cancer treatment

decisions are made based on tumour histology and status of three main biomarkers, ER (estrogen

receptor), PR (progesterone receptor) and HER2 (erb-B2 receptor tyrosine kinase 2) [Baselga et al.

2010]. There are several clinical drugs being used alone or in

combinations to tackle the disease. Raloxifene and tamoxifen as estrogen antagonists for preventive

medicines, paclitaxel, docetaxel, and abraxane, albumin bound paclitaxel nanoparticles as

microtubule stabilizers, anastrozole and exemestane as aromatase inhibitors, Doxorubicin as DNA

topo-II inhibitor, 5-fluorouracil as antimetabolites, fulvestrant as estrogen receptor down-regulator,

abemaciclib as CDK inhibitor, gefitinib as tyrosine kinase inhibitor, and several monoclonal

antibodies like herceptin etc. However, due to the high degree of heterogeneity of breast tumours,

multiple biomarkers for molecular profiling and diverse therapeutic treatment options are being

explored.

3

Figure 1: Some of the clinical drugs being used against breast cancer

Drugs that target tubulin and microtubule have been amongst the most successful modes of cancer

chemotherapy [Negi et al. 2015, Gan et al. 2007]. Tubulin is a globular protein which exists as α/β

dimeric form. Tubulin is polymerised to microtubule which plays an important role during mitosis in

cell division. A dynamic equilibrium exists between soluble tubulin and tubulin in microtubules. Any

interference in this process induces cell cycle arrest. Among the drugs that perturb microtubule

functionare microtubule stabilizers like paclitaxel and epothilones and microtubule destabilizers such

as colchicine, podophyllotoxin and combretastatins. There are several antitubulin drugs in clinical

use and many more are under various phases of clinical trials.

Present communication deals with an antitubulin lead compound 1 belonging to arylidene-

indanone class that was designed based on structure-activity relationship of several natural

antitubulins. Arylidene indanone scaffold possesses various biological activities specifically

anticancer activity [JCJMDS Menezes 2017]. Podophyllotoxin (PDT), colchicine and

combretastatin A4 (CA4) are important microtubule destabilizers possessing a 3,4,5-

trimethoxyphenyl fragment in their structures (Fig. 2). This fragment interacts with the β-tubulin and

is responsible for their effects on microtubules. Based on these facts, we designed a hexamethoxy-

2,3-diaryl-1-indanone pharmacophore containing ring A and this fragment as a possible antitubulin

agent. Compound 1 is a fluorinated derivative of hexamethoxy-2,3-diaryl-1-indanone moiety.

4

Attributed to the potent in-vitro anticancer activity of compound 1, its detailed pharmacology was

explored systematically. Various experiments were performed to establish its cytotoxicity,

mechanism of action and finally preclinical efficacy and toxicity in rodent models.

F

OH

O

H₃CO

NH

O

H₃CO

OH

H₃CO

OCH₃

H₃CO

OCH₃

H₃CO

OCH₃

O

OCH₃

Podophyllotoxin

Colchicine

Combretastatin A4

3,4,5-trimethoxyphenyl

motif

Fluorinated

benzylideneindanone 1

Fig. 2: Structures of naturally occurring microtubule destabilizers (PDT, colchicine and CA4)

possessing 3,4,5-trimethoxyphenyl fragment, designed pharmacophore and fluorinated lead

compound 1.

2. Materials and methods

2.1 General

Melting points were determined in open glass capillaries and were uncorrected. Gallic acid was

procured from S.d. Fine Chemicals India. Reagents and other chemicals were purchased either from

Sigma-Aldrich, USA or Avra Synthesis India and used without purification. Reactions were

monitored on Merck pre-coated silica gel TLC-GF₂₅₄aluminium sheets and visualization of

compounds was done under UV light (254 nm and 365 nm) and further charring with 2% ceric

sulphate in 10% sulphuric acid (aqueous). Purification of compound 1 was done on Flash

chromatography system (CombiFlash R_f200i, Teledyne-ISCO, USA) using glass column (13 cm

lengthx2 cm i.d.) and silica gel (230-400 mesh) using a UV detector (254 nm & 280 nm) and

1

characterised by H and ¹³C NMR, ESI-MS, and ESI-HRMS. NMR spectra were obtained with

Bruker Avance-500 MHz spectrometer. Chemical shifts are given in δ ppm values with respect to

1

tetramethylsilane (TMS) as an internal standard. H–¹H coupling constant (J) values are given in Hz.

ESI mass spectra were recorded using an APC3000 LC-MS-MS (Applied Biosystem) and High

Resolution Mass (HRMS) on Agilent 6520Q-TOF after dissolving the compounds in methanol.

DMEM (Dulbecco’s Modified Essential Eagle Medium) and FBS (Fetal Bovine Serum) were

purchased from Gibco (ThermoFisher, India). RNaseA, Crystal Violet Dye, HEPES, Trypsin-EDTA,

Antibiotic-Antimycotic (Ab-Am) Solution, Phosphate Buffer Saline (PBS), Citric Acid and Propidium

Iodide (PI) were acquired from Sigma-Aldrich, USA. Sodium bicarbonate (NaHCO₃), Agar, Sodium

Citrate and Di-sodium Hydrogen Phosphate were obtained from Himedia Laboratories, India. Solvents

including ethanol and isopropanol were procured from Merck, India Ltd.

5

2.2. Chemical synthesis

Compound 1 was synthesized from gallic acid as per our reported method [Saxena et al. 2008,

Prakasham 2012].

The selected physical data;

3-(3’,4’,5’-trimethoxyphenyl)-4,5,6-trimethoxy-2-(4″-fluorobenzylidene)-indan-1-one (1)

Overall

yield:14.29%

(Six

steps);

M.P.:151-153^oC

[156-158^oC,

11];

TLC

(Ethylacetate:Hexane=40:60): R_f=0.41; ¹H NMR (CDCl₃, 500MHz): δ 3.46 (s, 3H, OCH₃), 3.73 (s,

9H, 3xOCH₃), 3.91 (s, 3H, OCH₃), 3.94 (s, 3H, OCH₃), 5.26 (s, 1H, 3-CH), 6.43 (s, 2H, 2’ & 6’-CH

of 3-phenyl), 6.95-6.98 (t, 2H, 2” & 6”-CH of benzylidene ring, J=8.5Hz), 7.23 (s, 1H, 7-CH of

indanone ring), 7.47-7.51(bs, 2H, 3” & 5”-CH of benzylidene ring), 7.66 (s, 1H, benzylidene-CH).

¹³C NMR (CDCl₃, 75MHz): δ 46.15, 56.20, 56.31, 56.31, 60.29, 60.91, 60.94, 101.42, 105.89,

105.89, 115.44, 115.61, 130.56, 131.97, 133.03, 133.09, 133.37, 136.70, 136.81, 139.42, 139.44,

140.92, 148.78, 149.98, 153.04, 154.97, 162.21, 193.66. ESIMS (MeOH): 495 [M+H]⁺, 517

[M+Na]⁺; ESI-HRMS (CH₃CN): cacl for C₂₈H₂₈FO₇(M+H)+, 495.1819, found 495.1815.

2.3. UPLC analysis for purity profile

The purity profile was checked with a reverse phase Ultra Pressure Liquid Chromatography system

(Waters USA) with Empower® software using a C-18 column (BEH130 Å, 1.7X 50 mm, 1.7 µm,

Waters, USA, thermostated at 35±0.1 °C). Column analysis was performed with a using isocratic

elution of Mobile phase Water (0.1% acetic acid): Acetonitrile in 40:60 (v/v) at flow rate of 0.2

mL/min. The injection volume was 2 µL. The integration on the peak area was performed at λ_max

325 nm. While, 3D chromatographic data acquisition in the range of 210-400 nm was done to

monitor possible impurities under optimised analytical conditions. The purity was calculated by peak

area normalization. Under optimised chromatographic condition, compound 1 was eluted at 1.817

min without interference of any neighbouring co-eluting peak. As a reference method, the PDA

functions of UPLC software Empower® (Waters USA) were used to ensure the presence of a

spectrally homogeneous peak.

2.4. Antiproliferative activity by soft agar colony assay

Human breast cancer cells, MCF-7 and MDA-MB-231 were seeded (5x10⁴cells per mL) separately

in 24 well plates with or without compound 1 treatment for 24 h [Kakuguchi et al. 2010]. The

bottom jelly layer was made with 0.72% agar and 4 mL media in a 60 mm petridish followed by the

formation of the top layer consisting of cells and 3 mL media in 0.36% agar. Petridishes were

6

incubated in a CO₂incubator for 15-20 days until the appearance of colonies followed by staining

with Crystal Violet (0.04% in 2% ethanol) and incubated for 1h at RT. After the incubation period,

colonies were counted using an inverted microscope and pictures were captured accordingly.

2.5. Cell cycle analysis

The effect of compound 1 on the cell division cycle of both MCF-7 and MDA-MB-231 cells under

two independent experiments was assessed by two independent flow cytometry analysis using

propidium iodide (PI) to stain cellular DNA [Riccardi et al. 2006]. Briefly, 2 x 10⁶cells per mL

were grown in a 6 well plate at 37^oC in 5% CO₂with or without compound 1 treatment for 24 h.

After incubation, cells were collected and washed with PBS. Following removal of the PBS cells

were fixed-in 70% ethanol added drop-wise while vortexing and left overnight at 4 ˚C. The fixed

cells were centrifuged at high rpm to remove ethanol followed by washing with PBS and addition of

DNA extraction buffer. The samples were incubated for 5 min at room temperature and centrifuged

again to get a pellet. The pellet thus obtained was dissolved in PI staining solution followed by

RNaseA (200 µg/mL) treatment and incubated for 30 min at RT. Finally, cell cycle analysis was

done with a BD Biosciences LSR II flow cytometer (San Jose, CA, USA) and the analysis was

performed using FACS Diva Software, version 6.1.3. Cell cycle experiments were performed in

duplicates and results were expressed as Mean ± 1SD.

2.6. Apoptosis Vs Necrosis induction by 6a by Annexin V-FITC assay

For Annexin V-FITC apoptosis assay manufacturer’s protocol was followed (BD Bioscience Kit).

Two independent experiments were done for each MCF-7 and MDA-MB-231 on Flow cytometry

[Looi et al. 2013]. 1 X 10⁶cells/mL cells (MCF-7 or MDA-MB-231) were seeded in 6-well plate

and left overnight before treatment with compound 1 at various concentrations (at half IC₅₀, IC₅₀and

2xIC₅₀) for 24 hours. All cells were collected and centrifuged at 5000 rpm for 5 min at 4 ºC and

washed twice with cold PBS. Pellets were dissolved in 100 µL 1x binding buffer and incubated with

5 µL FITC-annexin-V and 5 µL propidium iodide (PI) for 15 min. Samples were resuspended in 500

µL 1x binding buffer and stained cells were analysed by FACS Diva software of flow cytometer

within 1 h. The annexin V-FITC binds to phosphatidiylserine present on membrane surface in case

of apoptotic cells whereas PI labels-cellular DNA in necrotic cells. This combination allows the

differentiation among viable cells (annexin V negative, PI negative), early apoptotic cells (annexin V

positive, PI negative), late apoptotic cells (annexin V positive, PI positive), and necrotic cells

(annexin V positive, PI positive).

7

2.7. Molecular docking studies

The crystallographic structure of selected target β-Tubulin (PDB: 4O2B) was retrieved from protein

databank (http://www.pdb.org). The structure of protein β-Tubulin and ligands colchicine,

podophyllotoxin and compound 1 were prepared by using Autodock Tools v1.5.6 (MGL, La Jolla,

USA). The tool converts protein structure into AD4 type then adds atomic partial charges, polar

hydrogens and torsional degrees of freedom. The tool generates extended PDBQT file for protein and

ligands. The grid parameter file and docking parameter files were prepared by setting grid centre at

colchicine binding site with dimensions x= 13.686207, y= 8.663517 and z = -19.963483. The grid

dimension was taken as x= 64, y=64 and z=64. AutoDock docking software used free energy force

field and Lamarckian-Genetic algorithm to search stable conformation and binding free energy of

ligand. For docking protocol optimization, a co-crystallised ligand redocking study was performed

without randomizing ligand co-ordinates. Molsoft-chemist v3.8-6a 2018 was used for root mean

square deviation (RMSD) value between redocked and β-Tubulin co-crystallised colchicine pose.

Post docking analysis of protein-ligand complex was performed by using discovery studio

v17.2.0.16349 visualizer.

2.8. Prediction of in-silico oral bioavailability and toxicity assessment

Here, the parameters for oral bioavailability for studied compounds were assessed through standard

commercial and non-commercial tools namely TOPKAT (DS v3.5), Molsoft-chemist v3.8-6a (2018),

SwissADME (http://www.swissadme.ch). TOPKAT use their patented QSTR models to predict

ADME and toxicity for different endpoints e.g., hepatotoxicity, CYP2D6 interaction, plasma-protein

binding (PPB), blood-brain-barrier (BBB) penetration, carcinogenicity in rodents, Weight of

Evidence (WOE) prediction, Ames mutagenicity, developmental toxicity, skin irritancy, skin

sensitization, ocular irritancy, developmental toxicity and aerobic biodegradability etc. The

TOPKAT prediction reliability is assessed by the software based benchmarked QSTR model optimal

predictive space (OPS) applicability domain. Likewise, Molsoft and SwissADME used their

benchmarked models to predict different ADME parameters.

2.9. Antiangiogenesis activity: a) Hypoxia Inducible Factor-1α

Hypoxia-inducible factor 1-alpha, a constitutively expressed transcription factor includes promotion

of angiogenesis, responsible for tumor growth and its survival. The assay was carried out as per the

described protocol of HIF-1α human ELISA Kit from Abcam (Catalog no. ab171577) [Reen et al.

8

2016, Phelan et al. 2016]. Briefly, 50 µL of standards and samples were pipetted into the appropriate

well as described in the manual (cell lysate was prepared after giving 24 h treatment in MCF-7 cell

line) followed by the addition of 50 µL of antibody cocktail to each well. The plate was incubated for

an hour at room temperature on a shaker set to 400 rpm. After incubation the plate was washed with

buffer (300 µL) and the liquid was completely removed by aspiration or decantation. 100 µL

of tetramethylbenzidine (TMB) substrate solution was added to each well and the color was allowed

to develop in a proportion to the amount of bound HIF-1α. The stop solution (100 µL) changed the

color from blue to yellow and the intensity of the color was immediately measured at 450 nm. All

samples were assayed in replicate (duplicate). Doxorubicin (1 µg/mL) was used as a standard

inhibitor. The concentration of HIF-1α in the samples is then determined by comparing the optical

density of the samples to the standard curve and the total protein content of sample was estimated by

Nano Drop. The percent difference was calculated by comparing with control values.

b) Vascular Endothelial Growth Factor (VEGF)

Over-expression of vascular endothelial growth factor promotes tumor growth by formation of new

blood vessels, a process known as angiogenesis. The vascular endothelial growth factor (VEGF)

assay was carried out as per the described protocol in VEGF human ELISA kit from Abcam (Catalog

no. ab100663) [Petrica et al., 2017]. Briefly, this assay employs an antibody specific for human

VEGF coated on a 96 well plate. 100 µL of standards and samples were pipetted into the appropriate

wells as per the manual description (cell lysate was prepared after giving 24h treatment in MCF-7

cell line) and the samples having VEGF bound to immobilized antibody (Lihui et al. 2006). After

washing with buffer (300 µL) the liquids from the wells were completely removed by aspiration or

decantation. Then 100 µL of 1X biotinylated antibody specific to VEGF was added and the plate was

incubated for an hour followed by washing to remove unbound biotinylated antibody. 100 µL of

streptavidin conjugated horse radish peroxidase (HRP) was added into each well and allowed to

leave it for 45 min. The wells were again washed and 100 µL of tetramethylbenzidine (TMB)

substrate solution was added to the wells and the color developed is in proportion to the amount of

bound VEGF. The stop solution (50 µL) changed the color from blue to yellow and the intensity of

the color was measured at 450 nm immediately. All the samples were assayed in replicate.

Doxorubicin (1 µg/mL) was used as a standard inhibitor. The concentration of VEGF in the samples

is then determined by comparing the optical density of the samples to the standard curve and the total

protein content of sample was estimated by Nano Drop. The percent difference was calculated and

compared with control values.

9

2.10. In vivo anticancer activity of compound 1 against mice mammary carcinoma

The in-vivo anticancer efficacy assay was performed against C3H/Jax mice mammary carcinoma

model at ACTREC, Mumbai, India. The experiment was approved by Institute’s Animal Ethical

Committee (IAEC) via approval No. 01-2015. Inbred C3H/Jax mice were obtained from Institute’s

(ACTREC Mumbai) animal colony. The mammary tumour incidence in virgin females is 6-70%.

Thirty six female mice of body weight 24±3 g each were used in the study. The temperature of the

animal colony was maintained at 21±1^oC and humidity at 50-60% and a 12 h light/dark cycle was

used. All the animals were fed ad libitum with standard laboratory diet. Five groups, each consisting

of six randomly chosen animals, were used in this assessment. Group I was kept as control group (no

treatment), Group II was for positive control (drug) Adriyamycin (Doxorubicin) at 2.5 mg/kg i.v. on

day 1, 5, 9. Group III-V were treatment groups with compound 1 at 30 mg/kg, 60 mg/kg and 120

mg/kg orally 5 days a week for four weeks respectively, Compound 1 was dissolved in 1% DMSO in

water and gavaged orally. This concentration of DMSO is not considered toxic to animals. The

control group mice received the only vehicle.

The body weight, tumour volume, % survival were observed. Relative tumour volume (RTV) and

T/C ratio were calculated;

Control groupmiceweregiveneither1%DMSO in PBS oral gavage or PBS intraperitoneal injection

alone

RTV=Tumour volume on day of measurement/Tumour volume on day 1

T/C (Activity criteria) = Relative tumour volume of mice receiving test compound/Relative tumour

volume of control animals

In-vivo efficacy of compound 1 was evaluated against C3H/Jax mice mammary tumour model taking

Nod/Scid mice strain. Five different groups were taken six mice in each group (n=6), one control,

one standard drug and three test groups.

A) Control

B) Positive control: Doxorubicin (Adriyamycin)-2.5mg/kg i.v. injection on 1, 5, and 9 days.

C) Compound 1: 20mg/kg oral gavaging 5days a weekx4 weeks

D) Compound 1: 40mg/kg oral gavaging 5days a weekx4 weeks

E) Compound 1: 80mg/kg oral gavaging 5days a weekx4 weeks

10

Animals were exposed to drug for four weeks and- were sacrificed on the 29^thday. Tumour volumes

were calculated by assuming that the shape of a tumour approximated that of a sphere: Volume of

sphere, V= (4/3) r³; average diameter d=(d₁+d₂)/2

V= (/6) d³; volume obtained was divided by 1000 to convert mm³to cm³.

Relative tumour volume of control (A) - Relative tumour volume of test

% tumour reduction =

-------------------------------------------------------------------------------------------------x100 ;

Relative tumour volume of control (A)

Relative tumour volume of test group

and T/C = --------------------------------------------------------

Relative tumour volume of control (A)

2.11. In-vivo safety studies by acute oral toxicity

In view of potent anti-cancer activity of compound 1 in in-vitro model, its safety profiling was done

via acute oral toxicity in Swiss albino mice. Experiment was conducted in accordance with the

Organization for Economic Co-operation and Development (OECD) test guideline No 423 (1987).

The study and number of animals used were approved by the Institutional Animal Ethics Committee

(IAEC) of CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow, India via

CIMAP/IAEC/2016-19/1 dated 09-02-2017.

For the acute oral toxicity experiment, 30 mice (15 male and 15 female) were taken and divided into

four groups comprising 3 male and 3 female mice in each group (body weight=20-25 g each). The

0

animals were maintained at 22±5 C with humidity control and also on an automatic dark and light

cycle of 12 hours. The animals were fed with the standard mice feed and provided ad libitum

drinking water. Mice of group 1 were kept as control and animals of groups 2, 3, 4, and 5 were kept

as experimental. The animals were acclimatized for 7 days in the experimental environment prior to

the actual experimentation. The test compound 1 was dissolved in a minimum volume of

dimethylsulphoxide, suspended in caboxymethylcellulose (0.7%) and was given at 5, 50, 300 and

1000 mg/kg body weight to animals of groups 2, 3, 4 and 5 respectively. Control animals received

only vehicle.

Observational, haematological, biochemical and gross pathological study

Animals were checked for mortality and any signs of ill health at hourly interval on the day of

administration of drug and thereafter a daily general case side clinical examination was carried out

including changes in skin, mucous membrane, and eyes, the occurrence of secretion and excretion

and also responses including lachrymation, pilo-erection and respiratory patterns. Changes in gait,

11

posture and response to handling were also recorded [Allan et al. 2007]. In addition to observational

study, body weights were recorded and blood and serum samples were collected from all the animals

on the 7^thday of the experiment to determine acute oral toxicity. The samples were analysed for total

RBC, WBC, differential leucocytes count, haemoglobin percentage and biochemical parameters like

ALKP, SGPT, SGOT, total cholesterol, triglycerides, creatinine, bilirubin, serum protein, tissue

protein, malonaldehyde and reduced GSH activity. The animals were then sacrificed and were

necropsied for any gross pathological changes. Weights of vital organs like liver, heart, kidney etc.

were recorded [Chanda et al. 2008].

2.12. Statistical analysis

Soft agar colony formation, cell cycle, and Annexin V FITC experiments were performed in

duplicates. In-vivo efficacy each group possessed six animal (n=6) and results are expressed as

Mean±SD. For multiple comparisons each value was compared by one-way ANOVA following

Dunnett test, Student t-test, and Tukey test in GraphPad Instat version 3.06. Comparisons are made

relative to the untreated controls. p value<0.05 were considered significant. The statistical analysis

was done using the Dunnett test. (**) indicates p values less than 0.01 and (*) denotes p values less

than 0.05.

3. Results

3.1 Chemistry

Synthesis of fluorinated 2,3diarylindanone (1)

The synthetic strategy was as shown in Scheme 1, also described in our previous paper [Saxena et

al. 2008, Prakasham et al. 2012]. It was little modified to get higher yields in several steps. Gallic

acid was used as the starting substrate. Overall yield of fluorinated benzylidene indanone 1 was

14.29% in six synthetic steps starting with gallic acid.

Briefly, gallic acid was modified to trimethoxybenzoic acid methyl ester (2, 74%) using dimethyl

sulphate in 40% aqueous alkali. It was reduced to 3,4,5-trimethoxybenzyl alcohol (3, 83%) by

treating with lithium borohydride in THF at RT. This benzyl alcohol was oxidised with pyridinium

chlorochromate in dry dichloromethane to get 3,4,5-trimethoxybenzaldehyde (4, 86%). Aldehyde 4

was treated with 3,4,5-trimethoxyacetophenone to get a hexamethoxychalcone (5, 95%) which

underwent Nazarov’s cyclization in trifluoroacetic acid in a sealed tube to get an indanone (6, 36%).

Indanone 6 was treated with 4-fluorobenzaldehyde to get 2-benzylidene indanone 1 in 89% yield.

The overall yield of fluorobenzylidene indanone 1 was 14.29% in six synthetic steps.

12

Scheme 1

O

e

HO

COOH

a

H₃CO

COOCH₃

b

H₃CO

OCH₃

H₃CO

CH₂OH

c

H₃CO

CHO

d

5

H₃CO

OH

OCH₃

4

Gallic acid

2

3

O

H₃CO

F

H₃CO

f

OCH₃

6

H₃CO

Compound 1

Reagents and conditions: a) Me₂SO₄, 30% Aqueous KOH, reflux, 3 h, 74%; b) LiBH₄, THF, RT,

83%; c) PCC, dry DCM, 10 ^oC-RT, 2 h, 86%, d) 7% KOH in MeOH, 3,4,5-trimethoxyacetophenone,

RT, 6 h, 95%, e) TFA, 120 ^oC, 5 h, 36%, f) 3% KOH in MeOH, RT, 4 h, 89%.

3.2 Purity profile of compound 1 by UPLC

The purity of the fluorinated indanone 1, was checked in Waters reversed phase UPLC system (USA)

using PDA detector at 325 nm. The purity of the synthesized indanone 1 was found to be 99.70 %.

3.3 Cytotoxicity by Soft agar colony assay

To establish its in-vitro efficacy further, a soft agar colony assay was performed. The soft agar assay

is considered as a gold standard assay for the transformed cell phenotype [Rotem et al. 2015].

Compound 1 significantly inhibited the colony forming ability of both MCF-7 (IC₅₀=27.10 µM) and

MDA-MB-231 (IC₅₀=38.64 µM) cells (Table 1). This effect was more pronounced in MCF-7 cells as

compared to MDA-MB-231. However, the cytotoxicity of fluorinated benzylidene in MCF-7 cells

was lower than standard drug tamoxifen.

13

Table 1: Effect of compound 1 on percent colony formation in MCF-7 and MDA-MB-231 cells in

soft agar after 24 h incubation.

MCF-7 Cells^d

MDA-MB-231 cells^d

Compound

Conc.

µM

%

cells

Live

%

IC₅₀

Compound Conc.

µM

% Live cells

%

IC₅₀

µM

Dead µM

cells

Dead

cells

0

Control^a

100

0

---

Control^b

100

---

Tamoxifen

0

100

99.81

65.04

3.48

Tamoxifen

1.52±0.002

36.99±0.02

58.77±0.04

74.17±0.01

85.98±0.01

29.49±0.004

51. 83±0.007

61.92±0.01

79.88±0.02

93.02±0.003

98.48

63.01

41.23

25.83

14.02

60.51

48.17

38.08

20.12

6.98

36.04

135

54

27

13.5

4

135

54

27

13.5

4

0.19±0.02

4.96±0.03

19.42±0.13 80.58

42.13±0.61 57.87

Compound 1

2.85±0.05

30.95±0.2

97.15 27.10

69.05

Compound

38.64

101

40

20

10

3

101

40

20

10

3

1

65.53±0.72 34.47

85.89±0.15 14.11

99.27±0.03 0.73

^**p<0.01 (Dunnett test), ^atotal cells=41312; ^btotal cells=3708, ^dno. of seeded cells=5x10⁴cells/mL, area of 60 mm plate=2826 mm².

3.4 Cell cycle analysis

In cell cycle analysis in MCF-7 cells, compound 1 exhibited G2/M phase arrest as depicted in figure

2. Flow cytogram clearly showed induction of apoptosis at all experimental concentrations (1.5µM-

50µM). While in MDA-MB-231 cells, there was no significant change up to 10 µM concentrations.

However, there were S phase and G2/M phase arrest at higher concentrations (20 µM and 50 µM).

Here, apoptosis was induced only at 50 µM concentration.

14

MCF-7

MDA-MB-231

Figure 2: Cell cycle of compound 1 in MCF-7 and MDA-MB-231 cells at various concentrations.

3.5 Apoptosis induction by Annexin V-FITC

As shown in figure 3, compound1 induced early apoptosis at its IC₅₀concentration (0.68µM) in

MCF-7 cells (4.2%) and late apoptosis (5%). 19.8% of cells were necrotic at this concentration.

MDA-MB-231 cells treated with Compound 1 at its IC₅₀concentration also exhibited early apoptosis

(0.7%) , late apoptosis (12.1%) and 15.1% showed necrosis.

MCF-7: Control

½*IC₅₀(0.34 μM)

IC₅₀(0.68 μM)

2*IC₅₀(1.36 µM)

MDA-MB-231: Control 1/2IC₅₀(19.32 μM)

IC₅₀(38.64 μM)

2xIC₅₀(77.28 μM) Doxorubicin (1μg/mL)

Fig. 3: Effect of compound 1 on Apoptosis Vs necrosis in MCF-7 and MDA-MB-231 cells (Annexin V-FITC)

3.6 Molecular docking studies

The calculated RMSD value between superimposed structure of redocked and β-Tubulin co-

crystallised colchicine was found to be 0.63 which is well below cut off limit 2.0. A low RMSD

value standardized the docking protocol. Here, colchicine binding site was taken for receptor-ligand

interaction study. The binding site and its dimension are given under supplementary figure S1.

15

Colchicine redocking showed binding energy of -10.59 kcalmol^-1and form one hydrogen bond with

amino acid residue VAL-181 (Figure S2). Analysis of docking results indicate the studied compound

1 exhibit docking binding energy of -8.54 kcalmol^-1which is comparable to the binding energy of

positive control drug podophyllotoxin i.e., -8.69 kcalmol^-1. Here, a docked pose with lowest

estimated free energy of binding was selected as probable binding pose. Receptor-ligand complex

insight analysis indicate compound 1 form number of non-covalent interactions namely van der

Waals, halogen interaction, unfavourable acceptor-acceptor, pi-sigma, pi-sulfur and pi-alkyl (Figure

4A). Where binding pocket amino acid residues SER-178 and ALA-354 are involved in pi-sigma and

pi-alkyl bonding with compound 1 aromatic rings. A halogen based weak interaction and an

unfavourable acceptor-acceptor interaction was also observed between compound 1 amino acid

residues VAL-315 and ASN-101 respectively (Table 2). Positive control podophyllotoxin is making

four conventional hydrogen bonds with amino acid residues ASN-101, GLN-247, ASN-254 and

ASN-258. Binding pose analysis indicate compound 1 trimethoxy and fluoride containing aromatic

rings are shown to have similar orientation as that of colchicine aromatic rings with trimethoxy and

oxygen based functional groups (Figure 4B).

Table 2: Molecular docking based biding affinity results for positive control drug colchicine,

podophyllotoxin and Compound 1 against anticancer drug target tubulin (PDB: 4O2B).

Name

Docking

binding

energy

Binding site residues within 4Å region

Key amino acid

residues

kcal/mol

Colchicine (co-

crystallised

ligand)

Chain C: SER178, THR179, ALA180, VAL181

Chain D: CYS241, LEU242, LEU248, ALA250, ASP251,

LYS254, LEU255, ASN258, MET259, THR314, VAL315,

ALA316, ILE318, ASN350, LYS352, ALA354

-10.59

-8.69

VAL181

Podophyllotoxin

Chain C: GLN11, ASN101, SER178, THR179, ALA180,

VAL181, TYR224

Chain D: GLN247, LEU248, ASN249, ALA250, LYS254, ASN258, GLN247

LEU255, ASN258, LYS352, THR353

ASN101, LYS254,

Compound 1

Chain C: GLN11, ASN101, SER178, THR179, ALA180,

VAL181, TYR224

-8.54

Chain D: GLN-247, LEU248, ALA250, LEU 255, ASN258, SER178, ALA354

MET259, VAL315, ALA316, ALA317, LYS352, THR353,

ALA354,

16

(A) Colchicine

(B) Podophyllotoxin

(C) Compound 1

Fig. 4A: The 2D visualization of binding interactions between (A) colchicine, (B) podophyllotoxin

and (C) Compound 1 and tubulin inhibitor binding pocket residues. The important interactions are

highlighted with different colour dashed lines. The legend for colour code is given in the bottom.

17

Figure 4B: A 3D representation of docked pose of compound 1 (orange) at β-Tubulin (PDB: 4O2B)

colchicine binding site. Where ligands are shown in ‘ball and stick’ form and binding pocket residues are

shown in ‘stick’ form with element based color coding (carbon; grey, oxygen; red, nitrogen; blue, sulphur;

yellow and fluoride; sky blue). Compound 1 is shown in orange and colchicine is in blue colors ball and stick

form.

3.7 In silico prediction of ADME properties

In in-vivo system, a drug candidate should reach to biological target in a sufficient concentration to

exhibit desired pharmacological effect. There are several physicochemical parameters which play a

crucial role in defining its success inside the body. A few physicochemical parameters like

lipophilicity, water solubility, pharmacokinetics, Lipinski parameters, and PAINS (Pan-assay

interference compounds) structure were predicted through SwissADME software (Table 3).

Compound 1 exhibited satisfactory scores for various parameters. Figure 5 shows two dimensional

bioavailability within the good bioavailability confined limits.

Table 3: Various druggability parameters of Podophyllotoxin Compound 1.

Parameter Podophyllotoxin

Compd. 1

Acceptable

range

Parameter

Log P_o/w

Podophyllotoxin Compd. Acceptable

1

range

Physicochemical properties

Lipophilicity

Molecular

formulae

M. Wt.

Rotable

bonds

C22H23O8

C28H27FO7

---

2.99

4.22

≤5

414.41

4

494.51

8

≤500

≤10

Pharmacokinetics

GI absorption

High

---

H-bond

acceptors

H-bond

donors

8

1

8

0

≤10

≤5

BBB

permeability

P-gp substrate

No

Yes

No

Molar

Refractivity

10.3.85

0.41

132.50

0.25

40-130

CYP1A2

inhibition

CYP2C9/19

inhibition

No

Sp3

Not less

than 0.25

No

Yes

hybridisation

fraction

TPSA

92.68Å²

72.45Å²

20 Å²to

130 Å²

Drug likeness

Lipinski rule Yes, no violation Yes, no

violation

1 violation

moderate

Water solubility

Water

solubility

Solubility

class

81.2 μg/mL

Moderate

-3.71

0.49 μg/mL

Bioavailability

score

0.55

Medicinal chemistry

No No

0.55

Poorly

soluble

-6.01

acceptable

>-4

Log S

PAINS

18

Podophyllotoxin

Compound 1

Fig. 5: Two dimensional bioavailability radars of podophyllotoxin and compound 1

(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 and + 5.0, size: MW between 150 and 500 g/mol, polarity: TPSA between 20 Å²and 130 Å², 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, the compound is predicted not orally bioavailable,

because too flexible and too polar.)

3.8 Antiangiogenic activity of benzylidene indanone 1 via VEGF and HIF-α inhibition

Compound 1 exhibited moderate antiangiogenic activity in MCF-7 cells (Figure 6). Compound 1

inhibited VEGF by 15-22% in concentration dependent manner. At its IC₅₀VEGF was inhibited by

17.2% in MCF-7 cells while doxorubicin induced 19-33% inhibition at its various concentrations. At

its IC₅₀doxorubicin inhibited 33% of VEGF. Hypoxia inducible factor-α was inhibited by 28-40% by

compound 1 in a dose-dependent manner. It inhibited 34.6% HIF-α at its IC₅₀(0.68 µM) while

doxorubicin inhibited the same by 43.3% at its IC₅₀(5.83 μM).

19

Fig. 6: Effect of Compound 1 on angiogenesis in MCF-7 cells VEGF and HIF at its various

concentrations [Half IC₅₀(0.34 µM), IC₅₀(0.68 µM), and double IC₅₀(1.36 µM)] (Mean±SD; n=2,

n*p<0.05 compared to control, ** p<0.01 compared to control)

3.9 In-vivo efficacy studies

In in-vivo efficacy experiment compound 1 showed moderate efficacy against C3H/Jax mice at 30

mg/kg oral dose. The results are presented in tables 4, 5 and 6. It reduced 48% tumour volume at 30

mg/kg oral dose. There was no significant improvement on further increasing doses to 60 and 120

mg/kg of compound 1. There was no mortality and loss in weight of experimental animals

throughout the experimental period. However, the T/C ratio was moderate i.e. 0.52.

Table 4: Average body weight (g) of mice during the experimental period (n=6)

Treatment

Dose

Body weight (g)***

Day 1

Day9

Day 18

Day 29

Control

Compd. 1

Doxorubicin

---

23.2±1.05

24.6±2.27

26.1±1.35

26.6±1.46

24.7±2.15

23.5±0.98

24.8±2.18

26.2±1.37

26.3±1.44

23.9±2.13

24.5±1.11

25.0±2.47

26.8±1.22

26.7±1.78

24.9±1.98

26.5±1.63

27.4±2.72

28.1±4.21

27.9±1.65

25.7±2.74

30 mg/kg, oral

60 mg/kg, oral

120 mg/kg, oral

2.5 mg/kg, i.v.

Mean±SD; ***p<0.01 compared to control

Table 5. Relative tumour volume (RTV) in various groups of experimental mice on treatment with

compounds (n=6)

Treatment

Dose

Relative Tumour Volume (RTV)*

% Tumour

reduction

%

Survival

Day 1

1.00

Day9

5.98±1.63

2.13±0.22

2.97±0.33

2.70±0.38

1.92±0.62

Day 18

17.32±4.14

9.47±3.38

12.40±5.43

9.3±1.63

Day 29

43.83±14.35

22.71±7.16

Control

---

100

Compd. 1

30 mg/kg, oral

60 mg/kg, oral

120 mg/kg, oral

1.00

48.19±8.17

34.50±18.51 21.29±11.21 100

24.00±4.78

8.87±3.01

45.24±4.12

79.76±4.17

100

Doxorubicin 2.5 mg/kg, i.v.

4.84±3.06

20

Mean±SD; *p<0.05 compared to control

Table 6: T/C ratios for compound 1 at various doses and control drug doxorubicin (n=6)

Treatment

Dose

T/C^{#, **}

Day 18

Day 1

1.00

Day9

---

Day 29

---

Control

Compd. 1

Doxorubicin

---

30 mg/kg, oral

60 mg/kg, oral

120 mg/kg, oral

2.5 mg/kg, i.v.

0.36±.04

0.50±0.10

0.46±0.06

0.32±0.11

0.55±0.17

0.72±0.46

0.53±0.09

0.28±0.18

0.52±0.16

0.79±0.41

0.55±0.09

0.20±0.07

^#Mean tumour volume of drug-treated mice/ mean tumour volume of control mice; Maen±SD; **p<0.01 compared to control

3.10 Safety studies by acute oral toxicity

There were no significant observational changes in, morbidity and mortality throughout the

experimental period up to the dose level of 1000 mg/kg body weight of test compound 1.

Blood and serum samples showed non-significant changes in all the parameters studied including

total haemoglobin level, RBC count, WBC count, differential leucocyte-count, SGPT, ALKP,

creatinine, triglycerides, albumin, serum protein, tissue protein, hepatic MDA and hepatic GSH

activity (Table 7, 8 and Figure 8). However, SGOT was significantly increased in animals treated

with the test drug at 1000 mg/kg body weight as compared to untreated control animals. Serum total

cholesterol was significantly decreased in groups of animals treated with compound 1 at 5, 50 and

1000 mg/kg body weight as compared to the control group. A gross pathological study of drug-

treated animals showed no changes in any of the organs studied including their absolute and relative

weight (Figure 7). Therefore, the experiment showed that compound 1 is well tolerated by the Swiss

albino mice up to the dose level of 1000 mg/kg body weight as a single acute oral dose. Overall,

there were no observed adverse effect level (NOAEL) on experimental mice. Long term experiments

need to be carried out to determine if any adverse effects are associated with -repeated exposure to

compound 1 [M.N. Ghosh 1984].

21

Table 7. Effect of Compound 1 as a single acute oral dose at 5, 50, 300 and 1000 mg/kg body weight

on body weight, haemogram and serum biochemical parameters in Swiss albino mice (Mean±SD; n=6).

Fig. 7. Effect of Compound 1 as a single acute oral dose at 5, 50, 300 and 1000 mg/kg on absolute

and relative organ weight in Swiss albino mice. (Mean±SD; n=6, Non significant changes were found

compared to control).

Dose of indanone 1 at mg/kg body weight as a single oral dose

Parameters

Control

20.63±3.08

9.92±2.68

5 mg/kg

17.42±1.45

9.30±8.73

50 mg/kg

21.09±1.13

7.62±3.17

300 mg/kg

21.68±2.65

8.95±6.59

1000 mg/kg

19.12±3.20

7.19±0.08

Body weight (g)

SGPT (U/L)

SGOT (U/L)

ALKP (U/L)

15.99±7.78

258.24±48.21

11.41±2.30

88.45±26.79

60.61±16.58

0.40±0.13

17.21±3.67

261.57±65.31

11.95±1.02

56.33±7.23

67.24±13.68

0.51±0.09

15.87±8.17

267.65±33.11

11.45±1.62

68.81±12.41

45.26±22.99

0.50±0.07

33.39±6.34

257.24±88.01

12.11±1.78

57.98±11.21

55.46±19.43

0.51±0.10

24.07±1.71

246.52±121.87

11.49±0.15

44.83±13.30

43.94±2.95

0.51±0.09

Haemoglobin (g/dL)

Cholesterol (mg/dL)

Triglycerides (mg/dL)

Creatinine (mg/dL)

Albumin (mg/dL)

3.85±0.48

3.55±0.30

3.72±0.22

3.79±0.30

3.57±0.49

Serum protein (mg/mL)

Hepatic Tissue Protein

(mg/ml; 10% w/v)

Hepatic MDA (nM/ mg protein)

0.15±0.01

0.035±0.00

0.12±0.02

0.040±0.00

0.13±0.03

0.036±0.00

0.12±0.02

0.037±0.01

0.14±0.01

0.040±0.01

0.031±0.009

0.21±0.025

0.026±0.009

0.20±0.023

0.027±0.008

0.21±0.022

0.035±0.020

0.22±0.043

0.045±0.018

0.18±0.052

Hepatic GSH (µM/mg protein)

22

Fig. 8. Effect of Compound 1as a single acute oral dose at 5, 50, 300 and 1000 mg/kg body weight on

differential leucocytes counts in Swiss albino mice.(n=6, mean±SD, Non significant changes were

found compared to control).

Table 8: The differential leucocytes count at different acute oral doses (n=6, percentage; mean±SD; Non

significant changes were found compared to control).

Neutrophils Lymphocytes Monocytes Eosinophils Basophils

Control

44.00±10.32 38.50±10.30 11.20±1.10 5.40±1.14 0.80±0.45

5mg/kg

50mg/kg

300mg/kg

49.00±4.69

45.83±6.27

44.83±15.60 40.50±13.69 9.00±2.00

36.50±2.12 8.50±0.71

35.80±4.21 10.80±1.30 3.40±1.14 1.00±0.71

39.67±7.28

8.83±1.72

4.33±1.03 1.33±0.82

4.33±1.86 1.33±1.37

5.00±0.00 0.50±0.71

1000mg/kg 50.00±1.41

4. Discussion

Breast cancer is a multifarious disease. Numerous biological targets have been established which has

helped in developing newer cancer chemotherapeutics. The disease has been tackled to some extent

with the help of the target specific anticancer drugs. However, there is still high morbidity and

mortality due to this disease [Nuzzolese et al. 2020]. Affordability is one of the major concerns with

the disease and researchers need to explore efficient, safer, and affordable cancer chemotherapeutics.

Over the years the unique attributes of fluorine has been well understood which yielded

several useful fluorinated motifs [N.A. Meanwell 2018]. The judicious use of this element in a

pharmacophore can affect the physical, biochemical, pharmacological, and pharmacokinetic

properties beneficially [Park et al., 2001; Shan & Westwell 2007]. Its strong electronegativity,

small size, and modest lypophilicity prefers it in drug design in recent years [Jodnson et al. 2020].

Benzylidene indanone 1 exhibited potent antiproliferative activity against hormone dependent

breast cancer cell line MCF-7 with IC₅₀at 0.68 μM and microtubule destabilization with IC₅₀at

0.73µM [Prakasham et al. 2012]. Further, in soft agar colonisation assay it reduced the colony of

MCF-7 cells with IC₅₀at 27.10 μM and MDA-MB-231 (IC₅₀=38.6 µM) cells. Soft agar colonisation

23

assay is used to confirm in-vitro cellular anchorage-independent growth. It is a well established assay

to detect the tumorigenic potential of transformed cells and tumour suppressive effects on

transformed cells [Shin et al. 1975]. Fluorinated indanone 1 significantly inhibited the proliferation

of MCF-7 and MDA-MB-231 cells. Thus, compound 1, may be effective against both hormone

dependent (luminal) and hormone independent (basal) breast tumour cells i.e. a broad spectrum of

breast tumour cells.

‘Cell division cycle’ deals with a series of coordinated events to replicate DNA in G1/S and

to separate chromosomes to two daughter cells following mitotic phase [Collins 1997]. Cell cycle

checkpoints ensure the accuracy of DNA replication and division [Malumbres and Baracid 2009].

The prime factor of cancer cells is to deregulate the cell cycle control [Sher and Bartek 2017].

Fluorinated indanone 1 effectively induced G2/M phase arrest in MCF-7 cells. However, in MDA-

MB-231 cells G2/M phase arrest was effective only at higher concentration i.e. at 50µM.

Microtubule dynamics modulators interfere during mitosis and induce cell cycle arrest at G2/M

phase. Induction of cell cycle arrest in cancer cell lines constitutes one of the most prevalent

strategies to stop or limit cancer spreading [Hartwell and Weinert 1989, C.J. Sher 1996].

The fluorinated indanone derivative 1 has exhibited antimitotic effect by destabilizing

polymerisation of α/β-tubulin to microtubule polymer. Microtubules play an essential role in various

cellular functions. Several studies indicate that their cellular actions are much more complex via

protein-protein interactions and need to be understood [Kaul et al. 2019]. Docking studies

demonstrated that it occupied colchicine binding pocket of β-tubulin. Both colchicine and compound

1 interacted with amino acid residue ALA316 which is considered crucial in inducing antimitotic

effect. From competitive binding experiments, Burns et al. observed that residue β316 is closely

involved in the hydrophobic interactions with 3,4,5-trimethoxy group of colchicine. Therefore, 3,4,5-

trimethoxyphenyl is considered a crucial fragment in inducing antitubulin effect in the molecule

[Negi et al. 2015].

Apoptosis and necrosis are two different processes causing cell death. Necrosis is caused by

external factors to the cell and is considered to be a toxic process. Both these are natural barriers to

restrict malignant cells from surviving and disseminating [Su et al. 2015]. Benzylidene indanone 1

induced apoptosis in the both MCF-7 and MDA-MB-231 cells. By induction of early and late

apoptosis in cancer cells better therapeutic strategies can be developed for cancer treatment [Cong et

al. 2019] .

Angiogenesis, a physiological process for the formation of new blood vessels plays a pivotal

role in both normal embryonic and adult development [Musumeci et al. 2012, Zhang et al. 2018]. It

24

is considered as one of the six hallmarks of cancer [Hanahan and Weinberg 2011]. Uncontrolled

proliferation of tumour cells requires high levels of nutrients and oxygen for rapid growth and

development. These needs are met by tumour-induced angiogenesis which surprisingly is induced

early in the multistage development of invasive cancers both in animal models and in humans [Raica

et al. 2009]. The US FDA has approved several antiangiogenic drugs for clinical use in various types

of cancer i.e. metastatic colorectal cancer, non-small lung cancer, metastatic breast cancer and

metastatic renal cell carcinoma [Carmeliet and Jain 2011]. The fluorinated indanone 1 has

exhibited moderate antiangiogenic activity by inhibiting VEGF and HIF-α significantly. VEGF plays

a pivotal role in the process of angiogenesis for the formation of new blood vessels by modulating

vascular network growth, vascular permeability and vessel cell survival [Niu and Chen 2010]. An

imbalance in angiogenesis contributes to numerous malignant, inflammatory, ischaemic, infectious

and immune disorders [P. Carmeliet 2005]. Therefore, the modulation of the VEGF-mediated

angiogenic pathway is considered as an important target in anticancer drug development [Niu and

Chen 2010]. Angiogenic inhibitors are relatively new class of anticancer drugs differing from

classical cytotoxic drugs [Kerbel and Folkman 2002].

Based on our studies, it is evident that the antiproliferative effect of fluorinated indanone 1, is

through multitarget effect. Due to complexity of multifactorial diseases like triple negative breast

cancer, single target drugs do not always exhibit satisfactory efficacy [Zhou et al. 2019]. However,

pharmacokinetic effects and safety aspects should also be taken into consideration.

Fluorobenzylidene indanone 1 exhibited significant antitumour activity against EAC model.

However, the effect on tumour reduction was not concentration dependent. The antitumour effect

was evident from the doses i.e. 30 mg/kg (48% tumour reduction) and 120 mg/kg (45% tumour

reduction). It seems that the dose response was saturated at 30 mg/kg and reaching pleatue as after

that there was no improvement in the efficacy. In 60 mg/kg experimental error was more which

exhibited poor activity. It is worth to mention that Ehrlich ascites carcinoma cells are considered

difficult to break [Beans and Kessel 1968]. It is appraised as one of the most reliable models to

assess preclinical efficacy of anticancer agents.

In safety studies benzylidene indanone 1 was well tolerated by the experimental mice and

found to be safe up to 1000 mg/kg oral dose. Acute oral toxicity is a preliminary assessment of a lead

compound after single or multiple exposures. It indicates adverse effects of the drug candidate in

animal. There was some elevated level of SGOT at higher doses of benzylidene indanone 1. It is

quite possible with an anticancer compound when orally ingested. However, it was statistically non-

25

significant. Drug safety assessment is also termed as ‘Pharmacovigilance’ to focus on adverse drug

reactions [WHO 2002]. It also gives as idea about risk-benefit profile of investigational drugs.

5. Conclusion

The fluorinated benzylidene indanone 1 exhibited potential antiproliferative activity through

modulation of microtubule dynamics by occupying colchicine binding pocket. It also exhibited

antiangiogenic effect by inhibiting VEGF and HIF-1α pathway. It was non-toxic and safe at much

higher concentration than its efficacy dose. Overall, it shows moderate anticancer efficacy against

breast cancer in rodent model. This investigational lead is under optimization for better efficacy.

6. Abbreviations

ER, Estrogen Receptor; ERα, Estrogen Receptor α-subtype; NCE, New Chemical Entity; HER-2,

Human Epidermal Growth Factor Receptor-2; CDK, Cyclin Dependent Kinase; ESI-HRMS,

Electrospray Ionisation-High Resolution Mass Spectrometry; UPLC, Ultra Pressure Liquid

Chromatography; EAC, Ehrlich Ascites Carcinoma; US-FDA, United States Food and Drug

Administration; VEGF, Vascular Endothelial Growth Factor; PAINS, Pan Assay Interference

Compounds; IAEC, Institute Animal Ethical Committee; OECD, Organization for Economic Co-

operation and Development ; SGOT, Serum Glutamate Oxaloacetic Transaminase; SGPT, Serum

Glutamic Pyruvic Transaminase; ALKP, Alkaline Phosphatase

7. Declaration of competing interest

The authors declare no competing financial interest.

8. Acknowledgement

The study was financially supported from Department of Health Research, Govt. of India under

GAP-432. Ms. Ankita Srivastava acknowledges to Department of Science and Technology, Govt. of

India for her fellowship under WOS-A from KIRAN Division (GAP-407). Ms. Kaneez Fatima

acknowledges CSIR for her Senior Research Fellowship. Chemical Central Facility of CSIR-CIMAP

is duly acknowledged for Sophisticated Instrument support.

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Article Doi

Fluorinated benzylidene indanone exhibits antiproliferative activity through modulation of microtubule dynamics and antiangiogenic activity

DOI: 10.1016/j.ejps.2020.105513

Source and publish data:

Authors:

Article abstract of DOI:10.1016/j.ejps.2020.105513

Full text of DOI:10.1016/j.ejps.2020.105513

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