A novel approach towards design, synthesis and evaluation of some Schiff base analogues of 2-aminopyridine and 2-aminobezothiazole against hepatocellular carcinoma

Biomedicine & Pharmacotherapy 89 (2017) 162 –1 76

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A novel approach towards design, synthesis and evaluation of some

Schiff base analogues of 2-aminopyridine and 2-aminobezothiazole

against hepatocellular carcinoma

*

Shinu Chacko , Subir Samanta

Division of Pharmaceutical Chemistry, Department of Pharmaceutical Sciences and Technology, Birla Institute of Technology, Mesra, Ranchi, Jharkhand 835215,

India

A R T I C L E I N F O

A B S T R A C T

Article history:

Hepatocellular carcinoma is the most common primary malignancy of the liver with poor prognosis. In

this study novel, Schiff’s bases of 2-aminopyridine (SSSC-26 to 31) and 2-aminobenzothiazole (SSSC-32

to 37) were designed, synthesised and evaluated for antioxidant potential using DPPH method, and anti-

hepatocellular carcinoma property using diethylnitrosamine (DEN) induced hepatocellular carcinoma rat

model. The in-silico pharmacokinetic, rule of ﬁve and toxicity studies reveals that all the leads have an

excellent intrinsic quality and sufﬁcient structural features necessary for an oral activity. Molecular

docking studies of all compounds into the ligand binding pocket of checkpoint kinase1 and vascular

endothelial growth factor receptor-2 was also performed using Schrodinger software suite v8.5, and

which have shown good Glide scores. Further compounds were synthesised based on the docking score

and ADMET proﬁle. The 1,1-diphenyl2-picrylhydrazil (DPPH) scavenging study was carried out, and

results showed that SSSC-29 (IC₅₀-63.60) and SSSC-33 (IC₅₀-60.32) were having good anti-oxidant

potential in comparison with ascorbic acid (IC₅₀-55.27). SSSC-33 further evaluated for anti-cancer

potential against diethylnitrosamine (200 mg/kg bw) induced hepatocellular carcinoma in rats. The

biochemical, histopathological and morphological data showed that SSSC-33 can reverse the changes

occurred in the cancerous liver signiﬁcantly. All these ﬁndings suggested that SSSC-33-((benzo[d]thiazol-

2-ylimino) methyl)phenol) could be a potential compound in combating the oxidative damage of hepatic

cells occurred due to the development of hepatocellular carcinoma induced by a chemical carcinogen,

DEN.

Received 1 December 2016

Received in revised form 8 January 2017

Accepted 17 January 2017

Keywords:

Hepatocellular carcinoma

Schiff’s base

Anti-oxidant

DPPH diethylnitrosamine

Aspartate transferase

Alanine transferase

Reactive oxygen species

Histopathology

1. Introduction

like surgical resection, locoregional ablation, cytotoxic chemo-

therapy (e.g., sorafenib) and liver transplantation but the later

Hepatocellular carcinoma (HCC), a primary malignancy of the

liver, is the ﬁfth most common cancer in men and seventh in

women. Poor prognosis is the main reason for decreased cure rate

of HCC and occurrence of approximately more than 50, 000 new

cases of HCC worldwide every year [1]. Globally, death rate from all

other common cancers (such as lung, breast, and prostate cancers)

is declining, whereas mortality rate from liver cancer are increased

by 2.8 and 3.4 percentage per year respectively in men and women

[2]. Most cases of HCC develop in the liver having chronic damage

with alcoholic liver disease, non-alcoholic fatty liver, hepatitis B

(accounts for most 50% of all cases of HCC worldwide) and hepatitis

C infection [3]. Multiple treatment options are available for HCC

diagnosis of HCC is the major limitation for the success. At an

advanced stage, sorafenib, a multi-kinase inhibitor, is the only US

FDA-approved therapy [4]. In the study conducted by Xiao-Ke Guo

et al., natural molecule like Gambogic acid (GA) and its derivatives

have been proved as apoptotic agent against HepG2 cell lines [5].

Reactive oxygen species (ROS) includes superoxide anion

radical (O₂^ꢀꢁ), singlet oxygen (O), hydrogen peroxide (H₂O), and

the highly reactive hydroxyl radical (^.OH) play a signiﬁcant role in

carcinogenesis [6]. Oxidative stress occurs due to excess ROS

production, depletion of antioxidants (like superoxide dismutase)

or both and subsequent imbalance in the average level of ROS and

antioxidants. Especially in HCC amount of ROS is more due to

permanent damage of liver, a major metabolising organ. Com-

pounds of having antioxidant property may have anti-HCC activity

due to its free radical scavenging activity [7].

* Corresponding author.

E-mail address: shinu10015@bitmesra.ac.in (S. Chacko).

http://dx.doi.org/10.1016/j.biopha.20 1 7 .01.108

S. Chacko, S. Samanta / Biomedicine & Pharmacotherapy 89 (2017) 162–176

163

Schiff bases or Azomethines, ﬁrst reported by Hugo Schiff in

1864, broadly display range of biological activities such as

anticancer, antifungal, antibacterial, antimalarial, antiviral, anti-

pyretic, anti-inﬂammatory characteristics [8]. Schiff’s bases with

CCl₄by CYP 450 causes lipid peroxidation and membrane damage

[17]. Phenobarbitone sodium (PB) (which increases the expression

of CYP 450 enzyme and leads to the enhanced effect of DEN and

CCl₄) was also given during induction period for the successful

hepatocarcinogenesis.

general formula RHC

¼NꢀꢀR’ (Imine group) are the class of

compounds formed by the condensation between the primary

amine and carbonyl compounds [9]. The lone pair of electrons

present in sp²hybridised orbital of the nitrogen atom in the imine

group responsible for its chemical and biological properties [10].

The chelating property of Schiff bases plays a signiﬁcant role in its

antioxidant activity, and this will be helpful in the development of

compounds with anti-hepatocellular carcinoma.

2. Material and methods

2.1. General information

Docking calculation was carried out on HP Intel¹Core 2 due ¹

processor E3-1200v2 family with 16GB RAMS, 1TB Hard disk,

NVIDIA Quadro 2000, Linux. Chem Ofﬁce (level: Ultra, version: 3.5)

was used for drawing and energy minimization of structures.

QikProp module of Schrödinger was employed for Absorption,

Distribution, Metabolism and Excretion (ADME) prediction and

docking studies. ProTox, a web server was the toxicity determining

tool used for Lethal Dose, 50% (LD50) calculation in the rat.

All the chemical used for the synthesis and in vivo study (DEN,

CCl₄, and phenobarbitone sod.) were of analytical grade purchased

from Sigma-Aldrich. The progress of the reaction was determined

by thin layer chromatography (TLC) on silica gel plates in various

In this work, some novel Schiff’s bases based on 2-amino-

pyridine and 2-aminobenzothiazole with various aldehydes are

designed and evaluated theirs in silico toxicity and absorption,

distribution, metabolism and excretion (ADME) properties using

Protox and QikProp software respectively. Further, we docked the

hits with checkpoint 1 (Chek1) and vascular endothelial growth

factor receptor (VEGFR) using Schrodinger software suite v8.5 [11].

The leads having good docking score and reasonable ADMET

property was selected for synthesis. The compounds were

characterised by ¹H NMR, ¹³C NMR, Mass spectral and elemental

analysis. Compounds were then screened for its anti-oxidant

property by 1,1-diphenyl-2-picrylhydrazil (DPPH) [12,13] method

using ascorbic acid as standard. Subsequently evaluation of anti-

HCC activity of the best compound by its anti-oxidant property and

docking score was performed in DEN-induced HCC rat model [1].

Diethylnitrosamine (DEN) is a chemical carcinogen and DEN-

induced HCC in the rat is considered an as well accepted model for

the development of drugs against hepatocarcinogenesis. DEN was

ﬁrst hydroxylated to alpha-hydroxy nitrosamine in the presence of

cytochrome P450- dependent enzymatic system and which is

capable of alkylating DNA structure (Fig. 1). ROS are also generated

during this enzymatic process and causes lipid peroxidation and

adds up to the hepatocarcinogenesis [1]. DEN-induced HCC rat

model to mimic the injury-ﬁbrosis-malignancy cycle. Here, DEN

was given to the rat in the period of hepatic cell proliferation,

initiated by partial hepatectomy (PH) [14], which followed a

necrotizing dose of carbon tetrachloride (CCl_4;hepatotoxin)

[15,16]. Trichloromethyl radicals formed by the metabolism of

solvents. UV-lamp (l= 254–365 nm) was used for visualisation of

TLC plate and iodine vapour or ninhydrin reagent as detecting

agent. Melting points were determined on OptiMelt (Stanford

Research Systems, California) and were uncorrected. ¹H NMR

(400 MHz) was performed on Bruker Advance 300 instrument

(Bruker Instruments Inc., USA) using DMSO-d₆as solvent and

tetramethylsilane (TMS) as an internal standard. Chemical shifts

were expressed in parts per million (ppm). Mass spectra were

recorded by WATERS-Q-T of Premier-HAB213 using the ESIMS

Electrospray Ionization technique.

2.2. Molecular design

2.2.1. ADME prediction

The prediction of ADME (Absorption, Distribution, Metabolism

and Excretion) properties are considered to be crucial in the

development of a new drug since lots of drugs are being withdrawn

from the market due to the inappropriate ADME properties. The

Fig. 1. Metabolism of DEN and CCl₄in the presence of CYP 450 enzymatic system.

164

S. Chacko, S. Samanta / Biomedicine & Pharmacotherapy 89 (2017) 162–176

Table 1

2.2.3.2. Protein preparation. The crystal structure of the target

Schiff’s bases of 2-aminopyridine.

protein downloaded from PDB was prepared by using the prep

wizard of Schrödinger, since it is not suitable for immediate use in

molecular modelling calculations. This protein preparation was

done by adding missing atoms, removing unwanted chains, water

molecules and heterocyclic atoms and set for receptor grid

generation. The procedure was completed with an energy

minimised protein-ligand complex, to which hydrogen was

added subjected to protonation states for ionizable residues,

modiﬁcation of tautomeric forms and the repositioning of the re-

orientable hydrogen.

Compound

SSSC-26

R

Molecular Formula

C₁₂H₁₀N₂

Mol. Wt.

182.22

2.2.3.3. Receptor grid generation. Blind docking is performed if

information and location about the binding site are unknown.

Here, a ligand with known binding modes and active site residues

are available with the downloaded crystal structure of protein. It is

utilised to create a speciﬁed area of the active site (grid) so that

search space can be reduced to focus on the region of interest. The

processed protein was used for receptor grid generation using

Glide 5.0 component of the Schrödinger. The grid deﬁnes the

binding site of the receptor protein to which ligand has to bind.

SSSC-27

SSSC-28

C₁₂H₁₀N₂O

198.22

227.22

C₁₂H₉N₃O₂

2.2.3.4. Ligand preparation. The proposed Schiff’s bases (Tables 1

and 2) were drawn in 2D and converted into 3D using Chem Ofﬁce

SSSC-29

C₁₄H₁₄N₂O₂

242.27

Table 2

Schiff’s bases of 2-Aminobenzothiazole.

SSSC-30

SSSC-31

C₁₁H₉N₃

183.21

282.34

C₂₀H₁₄N₂

Compound

SSSC-32

R₂

Molecular Formula

C₁₄H₁₀N₂S

Mol. Wt.

238.31

SSSC-33

SSSC-34

C₁₄H₁₀N₂OS

254.05

283.31

ADME properties of the proposed molecules were generated by the

QikProp, version 3.0, Schrödinger, LLC, New York, 2005 [18].

2.2.2. Acute rat toxicity prediction

C₁₄H₉N₃O₂S

A safer drug should have a high therapeutic index [ratio

between dose producing a therapeutic effect (effective dose, 50%,

ED₅₀) and dose producing toxic effects (LD₅₀). In silico prediction of

the LD₅₀in mg/kg in rodents after oral administration was done by

ProTox, a web server, for the prediction of oral toxicities of small

molecules. Additionally, it provides information about toxicity

class using similarity and fragment-based methods and possible

toxicity targets. It also suggests the mechanisms involved in

toxicity development [19].

SSSC-35

C₁₆H₁₄N₂O₂S

298.36

2.2.3. Docking studies of the designed molecules

SSSC-36

SSSC-37

C₁₃H₉N₃S

239.05

338.09

2.2.3.1. Target identiﬁcation. The crystal structure of the target

Proteins, Chk 1 (PDB entry: 1ZYS) [20] and VEGFR-2 (PDB entry:

2QU5) [21], were retrieved from Research Collaboratory for

C₂₂H₁₄N₂S

Structural Bioinformatics (RCSB) PDB,

a

crystallographic

database for the three-dimensional (3D) structural data of the

primary biological molecules like proteins and nucleic acids

(http://www.rcsb.org/pdb/home/home.do).

S. Chacko, S. Samanta / Biomedicine & Pharmacotherapy 89 (2017) 162–176

165

Scheme 1. Synthesis of Schiff’s Bases using 2-aminopyridine and 2-aminobenzo thiazole.

10.0. Preliminary energy minimization and molecular dynamics

study were done to identify low energy conformers which were

saved in mole format using the same software. These molecules

were imported into Schrödinger project table, and ligand

preparation was carried out using LigPrep 2.2. Fully customised

ligand libraries generated were further optimised for

computational analysis by including energy minimised

tautomeric, stereochemical and ionisation variations.

121.793-128.949 (C-phenyl), 121.490 (C-5 pyridine), 118.985 (C-3

pyridine).

2.3.3. 4-((pyridin-2-ylimino)methyl)phenol (SSSC-27)

SSSC-27 was prepared by the condensation of 2-aminopyridine

with p-hydroxy benzaldehyde in methanol using glacial acetic acid

as catalyst. Yield 80%, mp 117 ^ꢂC, ESI–MS (m/z): 198.0 [M + ]; ¹H

NMR (400 MHz, DMSOꢀꢀ d₆):

1H, NꢀꢀCH), 7.73 (t, 1H, CꢀꢀCH), 7.71 (s, 1H,OH), 7.3 (t, 1H,

NꢀꢀCꢀꢀCH), 7.40 (d, 1H, C CH) 6.8-7.5 (m, 4H, Ar-H-).

d

(ppm) 9.19 (s, 1H, N CH), 8.50 (d,

¼

2.2.3.5. Validation of docking protocol. Docking method was

validated by removing the inhibitor bound to the protein from

the active site and re-docked it [24] into the apo-form of the Chek 1

receptor and VEGFR-2 [25]. The conformation and orientations of

the ligand in the docked form were compared to that of the original

X-ray crystallographic structure downloaded from PDB.

¼

2.3.4. N-(2-nitrobenzylidene)pyridin-2-amine (SSSC-28)

SSSC-28 was prepared by the condensation of 2-aminopyridine

with 2-nitrobenzaldehyde in methanol using glacial acetic acid as

catalyst. Yield 82%, mp 128 ^ꢂC, ESI–MS (m/z): 228 [M + H]; ¹H NMR

(400 MHz, DMSOꢀꢀ d₆):

NꢀꢀCH), 8.22 (d, 1H, NO₂ꢀꢀCꢀꢀCH), 7.73 (t, 1H, CꢀꢀCH), 7.33 (t, 1H,

NꢀꢀC CH), 7.44 (d, 1H, C CH) 7.59-7.88 (m, 3H, Ar-H-).

d

(ppm) 8.97 (s, 1H, N CH), 8.51 (d, 1H,

¼

2.2.3.6. Molecular docking. The generated structures of various

conformations of designed molecule were docked into the

predicted binding site of the targeted receptor proteins (1ZYS

and 2QU5) using Glide, Version 5.0, Schrödinger, LLC, New York,

NY, 2008. Docking was done in standard precision (SP) mode.

Potential hits were found out based on Glide score [26,27].

¼

2.3.5. N-(2,3-dimethoxybenzylidene)pyridin-2-amine (SSSC-29)

SSSC-29 was prepared by the condensation of 2-aminopyridine

with 2,3-dimethoxybenzaldehyde in methanol using glacial acetic

acid as catalyst. Yield 79%, mp 232 ^ꢂC, ESI–MS (m/z): 242 [M + ]; ¹H

2.3. Synthesis of Schiff’s bases

NMR (400 MHz, DMSOꢀꢀ d₆):

1H, NꢀꢀCH), 7.77 (t, 1H, CꢀꢀCH), 7.28 (t, 1H, NꢀꢀC

CH) 6.89-6.99 (m, 3H, Ar-H-), 3.82 (s, 6H, OꢀꢀCH₃).

d

(ppm) 8.53 (s, 1H, N

¼

CH), 8.49 (d,

¼CH), 7.37 (d, 1H,

2.3.1. General procedure

C¼

Various Schiff bases of 2-aminopyridine (SSSC-26 to 31) and 2

ꢀamino benzothiazole (SSSC-32 to 37) were prepared as per the

Scheme 1.

2.3.6. N-(pyridin-3-ylmethylene)pyridin-2-amine (SSSC-30)

SSSC-30 was prepared by the condensation of 2-aminopyridine

with 3-pyridinecarboxaldehyde in methanol using glacial acetic

acid as catalyst. Yield 87%, mp 132 ^ꢂC, ESI–MS (m/z): 183 [M + ]; ¹H

A mixture of equimolar quantities of aryl aldehydes (0.01 mol)

and 2-aminopyridine/2-Aminobenzothiazole (0.01 mol) in etha-

nol/methanol (20 mL) was reﬂuxed on a water bath for 6–10 h in

the presence of a few drops of glacial acetic acid as catalyst. Thin

layer chromatography (TLC) monitored the progress of reaction at

an appropriate time interval. After completion of the reaction, the

solution was cooled, separated solid was ﬁltered and washed with

ice-cold water and dried. Finally, the product thus obtained was

recrystallized from ethyl acetate and ethanol in different

proportions depending on the nature of the compound [28].

NMR (400 MHz, DMSOꢀꢀ d₆):

(d, 1H, N

CH) 8.49 (d, 1H, NꢀꢀCH), 8.47 (s, 1H, N

CꢀꢀCH), 7.70 (t, 1H, CꢀꢀCH), 7.37 (d, 1H, C

CꢀꢀCH), 7.28 (t, 1H, NꢀꢀC CH).

d

(ppm) 8.79 (s, 1H, C

CH), 8.13 (d, 1H,

CH), 7.43 (t, 1H,

¼

CH-N), 8.68

¼

C

N

¼

2.3.7. N-(anthracen-9-ylmethylene)pyridin-2-amine (SSSC-31)

SSSC-31 was prepared by the condensation of 2-aminopyridine

with 9-anthraldehyde in ethanol using glacial acetic acid as

catalyst. Brown crystalline solid, yield 81%, mp 110 ^ꢂC, ESI–MS (m/

2.3.2. N-benzylidenepyridin-2-amine (SSSC-26)

z): 282 [M + ]; ¹H NMR (400 MHz, DMSOꢀꢀ d₆):

d

(ppm) 9.26 (s, 1H,

SSSC-26 was prepared by the condensation of 2-aminopyridine

with benzaldehyde in methanol using glacial acetic acid as catalyst.

Yield 78%, mp 120 ^ꢂC, ESI–MS (m/z): 183.0 [M + H]; ¹H NMR

N

¼CH) 8.57 (d, 1H, NꢀꢀCH), 8.13 (d, 1H, C¼CꢀꢀCH), 7.87-8.04 (m,

4H, anthracene), 7.38-7.39 (m, 4H, Phe), 7.75 (t, 1H, CꢀꢀCH), 7.41 (d,

1H, C CH).

¼

CH), 7.34 (t, 1H, NꢀꢀC

¼

(400 MHz, DMSOꢀꢀ d₆):

d (ppm) 10.89 (s, 1H, Ar-NH), 7.2-8.3 (m,

4H, Ar-H-), 4.3 (s, 2H, NH₂), 2.7 (s, 1H, ꢀC = S-NH-); ¹³CNMR

2.3.8. N-benzylidenebenzo[d]thiazol-2-amine (SSSC-32)

SSSC-32 was prepared by the condensation of 2-amino-

benzothiazole with benzaldehyde in ethanol using glacial acetic

(125 MHz, DMSO-d6):

d

(ppm) 165.040 (C2-pyridine), 152.580 (C-

Azomethine), 140.210 (C6-pyridine), 131.066 (C4-pyridine),

166

S. Chacko, S. Samanta / Biomedicine & Pharmacotherapy 89 (2017) 162–176

acid as catalyst. Yield 81%, mp 124 ^ꢂC, ESI–MS (m/z): 239 [M + H];

DPPH radicals by 50%. All tests were carried out in triplicates.

¹H NMR (400 MHz, DMSOꢀꢀ d₆):

d

(ppm) 8.24 (s, 1H, N CH) 8.01

¼

A_Cꢀ A_t

(dd, 2H, benzyl), 7.46 (dd, 2H, benzyl), 7.38-7.62 (m, 5H, phenyl).

DPPH Scaven ged ð%Þ ¼

ꢃ 100

A_C

2.3.9. 4-((benzo[d]thiazol-2-ylimino)methyl)phenol (SSSC-33)

SSSC-33 was prepared by the condensation of 2-amino-

benzothiazole with p-hydroxy benzaldehyde in ethanol using

glacial acetic acid as catalyst. Yield 81%, mp 130 ^ꢂC, ESI–MS (m/z):

where, A_cis the absorbance of the control (control containing all

reagents except the test compound), A_tis the absorbance of test

drugs (synthesised drugs) at different concentrations.

Concentration allowing 50% inhibition (IC₅₀) was calculated

from the graph plotted by considering inhibition percentage

against various concentrations and the linear regression analysis

equation was used to obtain the IC₅₀value.

255 [M + H]; ¹H NMR (400 MHz, DMSOꢀꢀ d₆):

d (ppm) 9.59 (s, 1H,

N CH) 7.47-8.10 (m, 4H, benzyl), 7.37 (s, 2H, phenyl), 6.8 (s, 2H,

¼

phenyl), 4.12 (s, 1H, ꢀOH); ¹³C NMR (125 MHz, DMSO-d₆):

d (ppm)

170.106 (C-OH), 156.059 (C2-benzothiazole), 151.085 (C-azome-

thine), 149.147 (C8-benzothiazole), 143.018 (C9-benzothiazole),

140.210 (C6-pyridine), 135.142 (C-phenyl-benzothiazole) 131.096

(2C-phenyl), 124.730-128.041 (C-phenyl- benzothiazole), 121.490

(C-5 pyridine), 116.267 (2C- phenyl).

2.4.2. Anti-HCC screening using DEN-induced HCC rat model

2.4.2.1. Experimental animals. Male Wistar rats weighing between

250 and 350 g were procured from the Animal House, Birla

Institute of Technology, Mesra, Ranchi, Jharkhand, India. The whole

study was performed here in controlled conditions of temperature

24 ꢄ 2 ^ꢂC, relative humidity 50–56% and photo schedule 12 h: 12 h

of light: dark. Animals were fed a standard pellet diet (Amrut

Feeds, Mumbai, India) and water ad libitum. The animals were

acclimatised to the laboratory conditions for one week before the

initiation of the experiment. The experiments were carried out in

accordance with the guidelines set by CPCSEA (Committee for the

purpose of control and supervision of experiments on animals),

India. The experimental design was approved by the institutional

animal ethics committee of Department of Pharmaceutical

Sciences and Technology, BIT, Mesra (approval No. BIT/PH/IAEC/

01/2015 dated 07/07/15).

2.3.10. N-(2-nitrobenzylidene)benzo[d]thiazol-2-amine (SSSC-34)

SSSC-34 was prepared by the condensation of 2-amino-

benzothiazole with 2-nitrobenzaldehyde in ethanol using glacial

acetic acid as catalyst. Yield 81%, mp 110 ^ꢂC, ESI–MS (m/z): 284

[M + H]; ¹H NMR (400 MHz, DMSOꢀꢀ d₆):

d (ppm) 8.24 (s, 1H,

N CH) 8.01 (dd, 2H, benzyl), 7.46 (dd, 2H, benzyl), 7.38-7.62 (m,

¼

5H, phenyl).

2.3.11. N-(2,3-dimethoxybenzylidene)benzo[d]thiazol-2-amine (SSSC-

35)

SSSC-35 was prepared by the condensation of 2-amino-

benzothiazole with 2,3-dimethoxybenzaldehyde in ethanol using

glacial acetic acid as catalyst. Yield 81%, mp 110 ^ꢂC, ESI–MS (m/z):

299 [M + H]; ¹H NMR (400 MHz, DMSOꢀꢀ d₆):

d

(ppm) 8.24 (s, 1H,

2.4.2.2.

Hepatocarcinogenesis

induction

and

treatment

N

¼

CH) 8.01 (dd, 2H, benzyl), 7.46 (dd, 2H, benzyl), 7.38-7.62 (m,

protocol. The experimental hepatocarcinogenesis was initiated

by DEN and promoted by CCl₄in rats as described by Farber et al.

[14], Pound et al. [16] and Yadav et al. [15] with some modiﬁcation.

Brieﬂy, rats (except control group I) were subjected to partial

hepatectomy (PH) and administered with DEN (200 mg/kg b.w., i.

p) after 24 h of stabilisation. Phenobarbital (PB, 0.05% w/v, P.O) was

given to the animals for up to four weeks through drinking water.

Rats were subjected to CCl₄administration (1 mL/kg b.w, s.c) twice

a week during the second and third week of pH for the promotion

of carcinogenesis and left for cancer development up to Week 6.

After six weeks, induced animals (six animals each) in Groups III to

VIII were treated with test compounds and group II with 0.5%

carboxymethylcellulose (CMC) for two weeks (Fig. 4).

5H, phenyl).

2.3.12. N-(pyridin-3-ylmethylene)benzo[d]thiazol-2-amine (SSSC-36)

SSSC-36 was prepared by the condensation of 2-amino-

benzothiazole with 3-pyridyl carboxaldehyde in ethanol using

glacial acetic acid as catalyst. White crystalline solid, yield 81%, mp

110 ^ꢂC, ESI–MS (m/z): 239 [M + ]; ¹H NMR (400 MHz, DMSOꢀꢀ d₆):

d

(ppm) 8.71 (s, 1H, N

¼CH), 8.47 (s, 1H, CꢀꢀCH-C in ring), 7.40-8.03

(m, 12H, 3 phe).

2.3.13. N-(anthracen-9-ylmethylene)benzo[d]thiazol-2-amine (SSSC-

37)

SSSC-36 was prepared by the condensation of 2-amino-

benzothiazole with 9-anthraldehyde in ethanol using glacial acetic

acid as catalyst. Yield 81%, mp 113 ^ꢂC, ESI–MS (m/z): 339 [M + H]; ¹H

2.4.2.3. Treatment regimen. Rats were randomly divided into four

groups (n=6). The entire grouping was as follows:

NMR (400 MHz, DMSOꢀꢀ d₆):

d

(ppm) 8.71 (s, 1H, N

¼CH), 8.47 (s,

Group I: Control; animals without any treatment or induction;

Group II: Induced control; animals induced with HCC by DEN

administration as described above;

1H, CꢀꢀCH-C-anthracene), 7.40-8.03 (m, 12H-phe).

2.4. Pharmacological screening

Group III: SSSC-33 LD (70 mg/kg bw, i.p); Induced animals

treated with low dose (LD) of SSSC-33 from Weeks 7 to 8;

Group IV: SSSC-33 HD (140 mg/kg bw, i.p); Induced animals

treated with high dose (HD) of SSSC-33 from Weeks 7 to 8.

Rats in all groups were starved overnight, anaesthetized and

killed by cervical decapitation eight weeks after pH for histological

and biochemical analyses. Serum was separated from the blood

and used for the analysis of biochemical parameters. The liver

tissue was excised immediately, rinsed in ice-cold saline and stored

the frozen tissue for further histological and molecular level

investigations.

2.4.1. Antioxidant study by DPPH method

The free radical scavenging activity was measured by 1, 1-

diphenyl-2-picryl-hydrazyl (DPPH) assay using the method

described by Blois [12,13].

About 0.3 mM DPPH solution was prepared and added to 1 mL

of the DPPH solution, 3 mL of the test drug solutions were added,

which were dissolved in 100% methanol at different concen-

trations (50–250 mg/ml). The mixture was vigorously shaken and

kept at room temperature in the dark for 20 min, and the

absorbance was measured at 517 nm using a UV spectrophotome-

ter. The antioxidant activity of the synthesised drugs was

expressed as IC₅₀values. The IC₅₀value was deﬁned as the

2.4.2.4. Sample preparations. The liver tissue was excised

immediately after killing, rinsed in ice-cold phosphate buffer

saline (PBS), weighed and observed morphologically and divided

concentration (mg/ml) of extracts that inhibits the formation of

S. Chacko, S. Samanta / Biomedicine & Pharmacotherapy 89 (2017) 162–176

167

into two portions. One portion was immediately ﬁxed in 10%

formalin for histopathological examination. The remaining part of

the liver was homogenised (at 20,000 rpm for 10 min) in cold PBS

(pH 7.4) by using glass homogenizer tube to get tissue homogenate.

Collected blood samples were centrifuged (at 3000 rpm for 15 min)

to obtain serum. The serum and tissue homogenate were used for

the biochemical assays.

Kumar and co-workers [38]. Total cholesterol (TC) was determined

according to the method given by Parekh and Jung [39]. The level of

High Density Lipoprotein Cholesterol (HDL-c), Low Density

Lipoprotein Cholesterol (LDL-c), and Very Low Density

Lipoprotein Cholesterol (VLDL-c) was determined through the

colorimetric enzymatic (at 505 nm) method given by Sujatha and

Sachdanandam [40] using HDL-c kit (Span Diagnostics Ltd., Surat,

India).

2.4.2.5. Biochemical examination

2.4.2.5.1. Hepatic markers of liver damage and cancer. Hepatic

markers enzyme activity was measured both in serum and liver

tissue homogenate. Activities of aspartate aminotransferase (AST),

alanine aminotransferase (ALT), alkaline phosphatase (ALP) and

gamma glutamyl transpeptidase (GGT) were measured

2.4.2.6. Histopathological assays. The buffered formalin (10% v/v

formaldehyde in PBS, for 24 h) ﬁxed portion of the liver tissues

followed through the process of dehydration using 70% v/v

isopropyl alcohol (IPA). After proper dehydration, conﬁrmed by

using Xylene (clearing), tissues were impregnated in parafﬁn wax

for 1 h at 58–60 ^ꢂC (temperature should not be increased more than

65 ^ꢂC) in parafﬁn bath and prepared parafﬁn blocks. The blocks

spectrophotometrically

spectrophotometer (UV–vis 1800). The ALT and AST were

estimated colorimetrically as moles of pyruvate liberated/min/

mg of protein by the method described by Huang et al. [29], ALP

was measured as moles of phenol liberated/min/mg of protein by

the method illustrated by King and Armstrong [30] and GGT was

measured as moles of p-nitroaniline was formed/min/mg of

using

Shimadzu

recording

m

were then sectioned in pieces of 4–6 mm size using rotary

microtome. The sections were mounted on the slide,

deparafﬁnized, rehydrated and stained using Hematoxylin and

Eosin (H&E) stain. Finally, slides were dehydrated, put a cover slip

and observed under LEICA DME microscope with Zoom Browser EX

remote shooting software with appropriate magniﬁcation as

required (4ꢃ, 10ꢃ, 40ꢃ and 100ꢃ).

m

protein by the method given by King [31].

Alpha-fetoprotein (AFP) is produced by the fetal liver and is

used as a marker for hepatic carcinoma. AFP was determined in

serum by enzyme immunoassay method using a commercial kit

(Calbiotech) and following their protocol [32].

2.4.2.7. Data analysis and statistics. Statistical analysis was carried

out using Graph Pad Prism v6.0 (Graph Pad Software, San Diego,

CA, USA). All the in vitro DPPH assay have been conducted in

triplicate and expressed as mean ꢄ standard error of the mean

(SEM). All the in-vivo values were expressed as mean ꢄ SEM of data

obtained from six animals per group and were statistically

analysed by one-way analysis of variance (ANOVA) followed by

Bonferroni’s multiple comparison tests with equal sample size. The

difference was considered statistically signiﬁcant when p value is

less than (<) 0.05.

2.4.2.5.2.

Enzymatic

and

non-enzymatic

antioxidant

activity. Enzymatic antioxidants like Superoxide Dismutase

(SOD) and Catalase (CT) and non-enzymatic antioxidants like

Glutathione (GSH) & Glutathione S-transferase (GST) levels were

determined to conﬁrm the antioxidant potential of the compound.

SOD enzymic activity determination was based on the principle

of nitroblue tetrazolium (NBT) reduction described by Rukmini

et al. [33]. The optical density of blue coloured formazan was taken

at 560 nm by UV–vis spectrophotometer (Shimadzu UV–vis 1800).

Tubes containing all reaction mixture except tissue sample serve as

blank. Each unit of enzyme activity deﬁned as the amount of

enzyme required to inhibit the 50% reduction of NBT under

speciﬁed condition.

3. Results and discussion

3.1. Molecular design, and ADMET prediction

Early in-silico ADME screening and pharmacokinetic (PK)

proﬁling provide a basis for choosing new molecular entities

and lead compounds that have desirable drug metabolism, PK or

safety proﬁle, necessary selection of drug candidates and late-

stage preclinical and clinical development. The molecular design-

ing approach in early drug development process helps to select

ligand molecules having good ADME property from a large lot and

reduces unnecessary cost on the irrelevant ligands. In the past,

many ligands were discarded in the later stage of drug discovery

due to inappropriate PK and pharmacodynamics (PD) properties

[41]. So it is very necessary to consider PK and PD parameters in the

early stage of drug development [42]. Molecular modelling

supports drug discovery process by producing ligands with

satisfactory PK and PD parameters so that the resulted molecules

will probably give good activity regarding disease concerned with

good PK and PD properties.

The primary aim of any new drug discovery is to develop orally

active drugs. Lipinski’s Rule of 5 (Ro5) [43] states that orally active

molecules have a molecular weight (MW) below 500D, hydrogen

bond acceptor (HBA) groups below 10, hydrogen bond donor (HBD)

groups below 5 and oil/water partition coefﬁcient (logPo/w) below

5. We utilised Rule of ﬁve (RO5) as a rule of thumb to evaluate drug-

likeness or determine if a chemical compound with a certain

pharmacological or biological activity has properties that would

make it a likely orally active drug in humans. All the designed

molecules obeyed Ro5 parameters (Table 3), indicates that all

CT activity was analysed by utilising the method described by

Sinha et al. [34]. In this process, the level of chromic acetate formed

from the reduction of dichromate in acetic acid in the presence of

hydrogen peroxide (H₂O₂) determined calorimetrically at 570 nm.

Catalase endogenously converts two H₂O₂molecules to two water

and one oxygen molecule. So the amount of chromic acetate

indirectly reﬂects the amount of catalase in the sample.

GSH content in the sample was determined using the method of

Van Dooran et al. [35]. The principle behind the GSH determination

method is the reaction of Ellman’s reagent (5,5⁰bis-[2-nitrobenzoic

acid]) with thiol groups of reduced GSH at pH 8.0 to produce the

yellow 5-thiol-2-nitrobenzoate anion (determined calorimetrical-

ly at 412 nm). The precipitate formed by the addition of 1.0 mL of

TCA to 1.0 mL of the sample was centrifuged at 1200g for 20 min to

convert all the GSH to reduced form. The supernatant thus

produced contains reduced GSH and was used for the analysis.

GST activity was determined according to the method of Habig

et al. [36]. In this assay, GST catalyses the conjugation of GSH with

1-chloro-2,4-dinitrobenzene, producing

a

chromophore at

340 nm. The activity of GST was expressed as moles of 1-chloro-

2,4-dinitrobenzene-glutathione conjugate formed min/mg of

protein.

2.4.2.5.3. Determination of lipids and lipoprotein level. The level of

triglycerides (TG) in plasma and hepatic tissues were estimated by

the method of Van Handel [37] with small modiﬁcations done by

168

S. Chacko, S. Samanta / Biomedicine & Pharmacotherapy 89 (2017) 162–176

Table 3

coefﬁcient (PlogBB). The data justiﬁes that none of the molecules

can cross the blood-brain barrier to producing CNS toxicity.

Lipinski Rule of Five analyses of the compounds.

Serial No.

Comp.

“Rule of Five”

3.2. Molecular docking studies

Mol.Wt^a

HBA^b

HBD^c

QPlogPo/w^d

1

2

3

4

5

6

7

8

SSSC-26

SSSC-27

SSSC-28

SSSC-29

SSSC-30

SSSC-31

SSSC-32

SSSC-33

SSSC-34

SSSC-35

SSSC-36

SSSC-37

Sorafenib

168.21

225.26

282.32

360.45

239.29

296.34

324.40

271.35

360.45

328.40

443.49

464.82

2

3

4

2

3

4

3

6

0

1

0

1

0

3

3.091

2.41

2.48

3.34

2.03

4.93

3.53

3.08

3.13

3.94

2.69

5.58

4.09

The main aim of docking study was to establish the multi-

receptor interaction of the designed ligands to counter the drug

resistance. As in the case of Sorafenib, a multikinase inhibitor, the

development of drug resistance can be overcome through the

discovery of medicines having multiple receptor activities. In the

case of HCC chek1 and EGFR receptors are overexpressed and so

utilised in this study to carry out docking simulation using Glide

v5.0 of Schrodinger software suite v8.5.

Chk1 is responsible for directing cellular mechanism to repair

damaged DNA and which is the only repairing mechanism

available for HCC cells since all the other mechanisms were

mutated during the HCC formation. Through the inhibition of Chk1

receptor, the entire damaged DNA repairing mechanism will be

stopped and damaged cells undergoes apoptosis. Similarly, VEGFR

is responsible for the angiogenesis in HCC and is overexpressed.

Continued tumour expansion in HCC occurs with the production of

new blood vessels (angiogenesis). Anti-angiogenic effect of the

compounds and thus anti-HCC activity can be established by the

EGFR inhibition.

Docking studies were started with the ligand preparation

utilising LigPrep, protein preparation employing Prep Wizard and

receptor grid generation. All these processes are necessary to meet

minimum requirements for molecular docking calculation. In

ligand preparation, optimisation and energy minimization of

designed ligands were carried out. After ligand preparation, the

downloaded receptors (Chk1, 1ZYS; VEGFR, 2QU5) from PDB

(Figs. 2 and 3) were reﬁned (protein preparation) to include

missing side chain, remove water molecules which are not having

interaction, adjust formal charges of the atoms of the protein and

metal ion associated and to correct the ionisation and tautome-

rization state of the protein.

All the 12 designed molecules (SSSC-26 to 37) and sorafenib

were docked into the active site of Chk1 and VEGFR-2 using Glide

v5.0. The Glide score (G-score) thus obtained were tabulated in

Table 5. The validation of docking program was done by re-docking

method. The low root mean square deviation (RMSD) value

indicates the docking method has better ability to reproduce the X-

ray bound conﬁrmation for the receptors, Chk1 and VEGFT-2.

The G-scores of the test compounds against Chk1 were found in

between ꢀ5.13 and ꢀ6.44 Kcal/mol and against VEGFR-2 were

9

10

11

12

13

a

Molecular weight.

Hydrogen bond acceptor.

Hydrogen bond donor.

b

c

d

Predicted octanol/water partition coefﬁcient.

compounds are more apt to be membrane permeable and easily

absorbed by the body [44].

In this study, all the designed molecules were analysed in-silico

using QikProp module of Schrodinger software suite v8.5 to

establish their PK parameters (Table 4). The descriptors like

QPPCaco, QPlogS, the percentage of oral absorption, dipole

moment, PlogBB, SASA and QplogKhsa were analysed [45]. Cell

permeability of the designed molecules was determined regarding

QPPCaco. The results depicted that molecules SSSC-26 to SSSC-37

have QPPCaco values greater than 500 nm/s and which shows that

these molecules have better cell permeability than sorafenib (a

multi-kinase inhibitor approved for HCC treatment). The sufﬁcient

number of hydrophilic-lipophilic groups may be the reason for

their high cell permeability. The percentage of oral absorption of all

the molecules are 100. The predicted aqueous solubility (QPlogS)

and solvent-accessible surface area (SASA) of the compounds were

in the accepted range of ꢀ6.235 and ꢀ2. 036 and 300–1000

respectively, which are sufﬁciently useful for the oral activity.

The fraction bound to human serum albumin acts as reservoir

and releases as the concentration in the plasma reduces through

elimination. Prediction of binding to human serum albumin

(QPlogKhsa) was shown that SSSC-31 to 35 and SSSC-37 have

higher values than others. So these molecules can be considered for

extended release of action. The central nervous system (CNS)

toxicity proﬁle was determined by predicting brain/blood partition

Table 4

Predicted pharmacokinetic properties using QikProp module of Schrodinger software suite v8.5.

Serial No.

Comp.

QPP Caco^a

QPlogS^b

% of Oral Abso.

Dipole Moment

PlogBB^c

SASA^d

QPlog Khsa^e

**

1

2

3

4

5

6

7

8

SSSC-26

SSSC-27

SSSC-28

SSSC-29

SSSC-30

SSSC-31

SSSC-32

SSSC-33

SSSC-34

SSSC-35

SSSC-36

SSSC-37

Sorafenib

4704.2

1425.3

927.8

6239.5

2548.3

5071.3

3920.5

1376.8

878.9

5720.7

2542.9

4864.1

333.48

ꢀ2.958

ꢀ2.847

ꢀ2.864

ꢀ3.172

ꢀ2.036

ꢀ5.210

ꢀ3.599

ꢀ3.892

ꢀ3.900

ꢀ4.089

ꢀ3.038

ꢀ6.235

ꢀ6.900

100

96.06

0.083

436.724

449.111

468.033

506.684

429.532

555.609

476.481

507.627

527.506

559.856

487.451

615.789

759.72

ꢀ0.021

ꢀ0.118

ꢀ0.133

ꢀ0.068

ꢀ0.462

0.727

0.125

0.089

0.063

0.115

ꢀ0.469

ꢀ0.645

0.069

ꢀ0.161

0.096

0.093

ꢀ0.429

ꢀ0.627

0.120

9

10

11

12

13

ꢀ0.096

ꢀ0.270

0.143

0.927

6.0

ꢀ0.929

0.324

a

Predicted apparent Caco-2 cell (the model for the gut blood brain barrier) permeability in nm/s (<25 poor > 500 great).

b

c

Predicted aqueous solubility (ꢀ6.5–0.5); S in moldm^ꢀ3

Predicted brain/blood partition coefﬁcient (ꢀ3–1.2).

.

d

e

Total solvent accessible surface area in square angstrom (300–1000).

Prediction of binding to human serum albumin (ꢀ1.5–1.5).

Data not available.

**

S. Chacko, S. Samanta / Biomedicine & Pharmacotherapy 89 (2017) 162–176

169

Fig. 2. (A): Co-crystal structure of Checkpoint Kinase 1 (Chk1) (PDB: 1ZYS) with a pyrrolo-pyridine inhibitor. (B): 2D Pose view of N-[5-[4-(4-Methylpiperazin-1-yl)phenyl]-

1h- pyrrolo[2,3-b]pyridin-3-yl] nicotinamide in IZYS. Retrieved from protein data bank (PDB) site [20].

Fig. 3. (A): Crystal structure of the VEGFR2 kinase (PDB: 2QU5) domain in complex with a benzimidazole inhibitor. (B): 2D Pose view of 4-[[2-[[4-chloro-3-(triﬂuoromethyl)

phenyl]amino]-3H-benzimidazol-5-yl]oxy]-N-methyl-pyridine-2-carboxamide in 2QU5. Retrieved from PDB site [22,23].

found in between ꢀ6.47 and ꢀ8.37 Kcal/mol. SSSC-37 and SSSC-32

were shown higher G-score towards Chk1 and VEGFR-2 protein

respectively. Whereas, G-score of sorafenib was found to be ꢀ3.68

Kcal/mol against Chk1 and ꢀ10.6 Kcal/mol against VEGFR-2. The

higher scoring function directly relates to the higher binding

afﬁnities. The structural features that provide hydrophobic

interaction, Van Der Waals (vdW) forces, dispersion interaction,

hydrogen bonding, and other electrostatic interactions between

amino acids of the protein and ligand and solvation effect

determine the strength of the ligand-protein binding. The 3D

images of receptor-ligand interaction (Fig. 5) after docking show a

sufﬁcient number of hydrogen bonding and hydrophobic inter-

actions to establish the inhibitory effect of the ligands on both the

receptors.

sufﬁciently large. Many drugs were called back from the market

and failed in the later stage of drug development due to the severe

toxicity [46]. So early assessment of toxicity is necessary for the

successful approval and marketing of new drugs.

Lethal Dose 50% (LD₅₀₎, the dose at which 50% of the tested

animal die upon administration of the compound, gives insight to

the level of acute toxicity. The LD₅₀values were predicted after oral

administration in rodents using ProTox and are tabulated in

Table 6. All the compounds showed LD₅₀values between 300 <

LD₅₀ꢅ2000 mg/kg and so came under the class 4 according to the

Globally Harmonized System of Classiﬁcation of Labeling of

Chemicals (GHS), United Nations guidelines. This clearly indicates

that all these compounds are only harmful if swallowed and are

safe for the development of a new drug.

3.3. In-silico acute toxicity study using ProTox

3.4. Chemistry

The safety of the drugs developed is one of the primary

requirement for regulatory authorities for the approval for clinical

use. Acute toxicity studies are performed to prove the safety of the

new drug. The gap between minimum effective concentration and

minimum toxic concentration (therapeutic window) should be

The strategy followed in the synthesis of proposed Schiff bases

(SSSC-26 to SSSC-37) are outlined in the Scheme 1. The

condensation between primary amines (2-aminopyridine and 2-

aminobenzothaizole) and aldehydes (benzaldehyde, p-hydroxy-

benzaldehyde, 2-nitrobenzaldehyde, 2,3-dimethoxybenzaldehyde,

170

S. Chacko, S. Samanta / Biomedicine & Pharmacotherapy 89 (2017) 162–176

Fig. 4. Treatment protocol of the study. CCl₄-carbontetrachloride, DEN-diethylnitrosamine, i.p- intraperitoneal, PB-phenobarbitone sodium, PH-partial hepatectomy, P.O-

peroral.

Table 5

Docking score generated by Glide 5.0.

Entry

Compound

Glide 5.0 Score

CHK1^areceptor (PDB Id-1ZYS)

VEGFRꢀ2^bKinase PDB Idꢀ2QU5

1

2

3

4

5

6

7

8

SSSC-26

SSSC-27

SSSC-28

SSSC-29

SSSC-30

SSSC-31

SSSC-32

SSSC-33

SSSC-34

SSSC-35

SSSC-36

SSSC-37

Sorafenib (std)

ꢀ6.29

ꢀ5.44

ꢀ5.24

ꢀ5.67

ꢀ6.24

ꢀ5.13

ꢀ5.55

ꢀ5.58

ꢀ5.38

ꢀ5.21

ꢀ6.44

ꢀ3.68

ꢀ7.04

ꢀ6.84

ꢀ7.15

ꢀ7.08

ꢀ7.29

ꢀ6.47

ꢀ8.37

ꢀ7.87

ꢀ6.98

ꢀ7.62

ꢀ7.55

ꢀ7.32

ꢀ10.60

9

10

11

12

13

a

Checkpoint kinase1.

Vascular endothelial growth factor receptor-2.

b

Fig. 5. (A) SSSC-33 in the active site of Chk 1 receptor (PDB ID: 1ZYS), G-score:-5.55; (B) SSSC-33 in the active site of VEGFR-2 (PDB ID: 2QU5), G-score:-7.87. The image was

generated by Glide 5.0. Protein is depicted in the ribbon model and ligand in ball and stick model in which grey colour represents carbon (C); red, oxygen (O); blue, nitrogen

v

(N); yellow, Sulphur (S) and white, hydrogen (H).

3-pyridylcarboxaldehyde and 9-anthraldehyde) in the presence of

glacial acetic acid as catalyst methanol or ethanol as solvent yield

12 different Schiff bases in good yield. The formation of Schiff’s

base occurs in two separate steps; formation of carbinolamine and

formation of imine (Schiff’s base). The carbinolamine was

converted to imine in the presence of glacial acetic acid [47].

The recrystallization of the compounds was done using a mixture

of ethyl acetate and ethanol.

spectroscopic methods. The mass spectrum supports the proposed

empirical formula of the compound. It reveals the molecular ion

peak m/z at consistent with the molecular weight of the ligand. The

NMR Spectral data showed characteristic peaks relevant to the

synthesised compounds and depicted in Section 2.3.

3.5. Free radical scavenging activity by DPPH method

Characterization was carried out by elemental analysis and

MASS, ¹H NMR, and ¹³C NMR (SSSC-26 and SSSC-33; one each from

2-aminopyridine and 2-aminobenzo thiazole series respectively)

All the synthesised compounds were subjected to free radical

scavenging assay through DPPH method to ﬁnd out its anti-oxidant

potential. The DPPH assay is a reliable method for the evaluation of

S. Chacko, S. Samanta / Biomedicine & Pharmacotherapy 89 (2017) 162–176

171

Table 6

Predicted LD₅₀of and toxicity class of designed compounds.

SL. No.

Compound

LD₅₀mg/Kg

Toxicity class

1

2

3

4

5

6

7

8

SSSC-26

SSSC-27

SSSC-28

SSSC-29

SSSC-30

SSSC-31

SSSC-32

SSSC-33

SSSC-34

SSSC-35

SSSC-36

SSSC-37

Sorafenib

2000

1500

2000

1000

562

Class IV

9

10

11

12

13

1000

800

Class I: fatal if swallowed (LD50 ꢅ 5 mg/kg), Class II: fatal if swallowed (5 < LD50

ꢅ 50 mg/kg), Class III: toxic if swallowed (50 < LD50 ꢅ 300 mg/kg), Class IV: harmful

if swallowed (300 < LD50 ꢅ 2000 mg/kg), Class V: may be harmful if swallowed

(2000 < LD50 ꢅ 5000 mg/kg), Class VI: non-toxic (LD50 > 5000 mg/kg).

antioxidant property because of its stability in the radical form and

simplicity of the test [48]. The method is based on the reduction of

DPPH (purple in colour) to corresponding hydrazine (yellow in

colour) by receiving hydrogen atom from the anti-oxidant. The

colour changes can be measured quantitatively by spectropho-

tometer absorbance at 517 nm [49].

Fig. 6. The concentration versus percentage inhibition graph with linear regression

line of ascorbic acid and test compounds.

The percentage of inhibition at 50, 100, 150, 200 and 250 mg/ml

concentration and IC₅₀(concentration at which 50% DPPH activity

inhibited) values of the synthesised compound were determined

and reported (Table 7). The data was compared with the ascorbic

acid (standard), a known anti-oxidant (Figs. 6 and 7). Out of 12

synthesised compounds SSSC-29 and 33 showed IC₅₀values of

63.60

m

g/ml and 60.32

m

g/ml respectively and which was compa-

g/ml). The Higher

rable with that of ascorbic acid (IC₅₀ꢀ 55.27

m

antioxidant potential is associated with the lower IC₅₀value. So,

SSSC-33 has higher DPPH scavenging property thus anti-oxidant

potential among other test compounds.

3.6. Anti-HCC screening

On the basis of anti-oxidant property and docking score SSSC-

33 was selected for the anti-HCC screening using DEN-induced

hepatocellular carcinoma rat model. The activity was determined

by comparing with the normal control (without induction and

treatment) and induced control (HCC induced but not treated).

Fig. 7. Comparison of IC₅₀values of test compounds with ascorbic acid.

3.6.1. Restoration of liver function

The effect of SSSC-33 in HCC induced hepatic damage was

evaluated by estimating activities of hepatic marker enzymes such

as AST, ALT, ALP, and GGT both in serum and liver homogenate and

level of AFP in serum. Serum activities of hepatic markers AST, ALT,

Table 7

The percentage of scavenging of DPPH radical and IC₅₀values of standard (ascorbic acid) and test compounds (SSSC-26 to SSSC-37).

Sl.No.

Compound

% Scavenging of DPPH radical

IC₅₀(mg/ml)

50

100

150

200

250

1

2

3

4

5

6

7

8

Ascorbic Acid (Std)

SSSC-26

SSSC-27

SSSC-28

SSSC-29

SSSC-30

SSSC-31

SSSC-32

SSSC-33

SSSC-34

SSSC-35

SSSC-36

SSSC-37

47.23 ꢄ 0.36

24.51 ꢄ 0.26

36.51 ꢄ 0.15

37.03 ꢄ 0.51

44.73 ꢄ 0.32

30.52 ꢄ 0.44

40.41 ꢄ 0.25

43.91 ꢄ 0.21

44.24 ꢄ 0.13

26.61 ꢄ 0.71

25.69 ꢄ 0.58

30.55 ꢄ 0.26

43.85 ꢄ 0.65

60.27 ꢄ 0.39

29.32 ꢄ 0.31

43.71 ꢄ 0.26

42.51 ꢄ 0.12

58.33 ꢄ 0.32

44.79 ꢄ 0.22

47.37 ꢄ 0.37

48.31 ꢄ 0.31

60.99 ꢄ 0.31

30.51 ꢄ 0.17

30.77 ꢄ 0.29

38.51 ꢄ 0.45

48.01 ꢄ 0.41

79.29 ꢄ 0.45

37.32 ꢄ 0.24

48.31 ꢄ 0.38

48.52 ꢄ 0.14

67.01 ꢄ 0.35

49.03 ꢄ 0.28

57.66 ꢄ 0.28

53.66 ꢄ 0.25

69.46 ꢄ 0.41

47.37 ꢄ 0.22

36.47 ꢄ 0.36

45.77 ꢄ 0.31

53.61 ꢄ 0.24

90.63 ꢄ 0.28

45.21 ꢄ 0.34

54.71 ꢄ 0.18

55.73 ꢄ 0.31

77.29 ꢄ 0.31

54.71 ꢄ 0.25

63.74 ꢄ 0.22

58.31 ꢄ 0.15

78.23 ꢄ 0.36

51.61 ꢄ 0.09

45.55 ꢄ 0.19

51.89 ꢄ 0.34

59.41 ꢄ 0.15

98.54 ꢄ 0.25

53.71 ꢄ 0.41

58.44 ꢄ 0.16

61.52 ꢄ 0.24

80.03 ꢄ 0.15

60.21 ꢄ 0.19

68.31 ꢄ 0.06

64.61 ꢄ 0.35

84.21 ꢄ 0.33

58.88 ꢄ 0.24

51.34 ꢄ 0.31

60.44 ꢄ 0.33

63.71 ꢄ 0.14

55.27

230.64

165.20

157.52

63.60

165.50

111.94

113.42

60.32

190.87

241.00

181.24

113.68

9

10

11

12

13

Each value represents mean ꢄ SEM, (n = 3); SEM-standard error of the mean.

172

S. Chacko, S. Samanta / Biomedicine & Pharmacotherapy 89 (2017) 162–176

with HCC by DEN injection. These results coincide with the

observations obtained by Jahan et al. [50].

Assessment of hepatic function is essential to monitor HCC

progression and prognosis and the effect of anti-cancer drugs.

Hepatic marker level signiﬁcantly alters in the case of cancer

induction. Increase transaminase activities in HCC have been

reported by Rochi et al. [51] ALP and AST are considered as a most

sensitive tumour marker for malignant diseases. The increased

serum activity of AST and ALT in HCC induced group was due to the

presence of high cancerous cells and thereby high liver marker

enzyme synthesis [52]. ALP is involved in the transport of

metabolites across the cell membrane, protein synthesis, secretory

activities and glycogen metabolism. The rise in an activity of ALP in

DEN-induced HCC animals may be due to the disturbance in the

secretory activity and altered synthesis of the enzyme [53]. An

increase in GGT activity paralleled with an increase in ALP activity

is very common in HCC [54].

a-feto protein (AFP) is a serum

protein is detected in elevated concentration in conditions like

HCC. It is size, structure and composition are similar to serum

albumin, but is detectable in minute quantities in normal

condition. Elevated serum concentration is speciﬁcally observed

in HCC [50]. So its serum concentration can be used to conﬁrm HCC

and for the diagnosis of tumour response therapy. Here in this

study, the elevated level of AFP in DEN-treated group (Group II)

was signiﬁcantly restored towards normal level in Group IV (SSSC-

33 HD treated), which indicates clearly suggests the anti-HCC

potential of SSSC-33.

Fig. 8. Effect of SSSC-33 on hepatic markers in serum of rat with HCC. Values are

expressed as mean ꢄ SEM of six rats per group; where, a- Group II, III, IV compared

with Group I; b- Group III & IV compared to Group II; ***p < 0.001; **p < 0.01;

*p < 0.05; ^nsp > 0.05; Group I- Normal Control; Group II- Induced Control; Group III-

treated with SSSC-33 LD; Group IV- treated SSSC-33 HD. AST-aspartate

aminotransferase, ALT-alanine transferase, ALP-alkaline phosphatase, GGT-gamma

glutamyl transferase, AFP-alpha fetoprotein.

ALP, and GGT and level of AFP were increased signiﬁcantly

(p < 0.001) in Group II as compared to control rats (Group I), thus

indicating liver damage (Fig. 8). On the other hand, the activity of

AST and ALT was signiﬁcantly (p < 0.001) decreased, and the

activity of ALP and GGT was signiﬁcantly (p < 0.001) increased in

the liver tissue of animals from Group II as compared with Group I

(Fig. 9). The low dose of SSSC-33 was restored hepatic marker

enzymes activity less signiﬁcantly whereas the high dose restored

the activity of AST (p < 0.01), ALT (p < 0.01), ALP (p < 0.001), GGT

(p < 0.001), and AFP (p < 0.001) in serum on comparison with

induced control (Group II) signiﬁcantly. In the case of liver

homogenate of rats from Group IV (rats treated with SSSC-33 HD)

the activity of AST (p < 0.001), and ALT (p < 0.001) was increased

signiﬁcantly, and the activity of ALP (p < 0.001), and GGT

(p < 0.001) was decreased signiﬁcantly compared with rats from

the induced control group. This indicates that SSSC-33 HD can

revoke the changes happened in the liver functions of rats induced

3.6.2. Effect of SSSC-33 on the level of enzymic and non-enzymic

antioxidants

The in vitro antioxidant property of SSSC-33 was detected by the

DPPH method. Further, it was conﬁrmed by measuring levels of

enzymic antioxidants in both liver homogenate and serum and

non-enzymic antioxidants in serum and is summarised in Tables 8

and 9. Group II (induced control) animals exhibited signiﬁcant

increase (P < 0.001) in GSH level and GST activity and a decrease in

SOD and CAT activities compared to Group I (normal control).

These ﬁndings are consistent with the hepatic oxidative stress and

damage caused by DEN. The high dose of SSSC-33 (p < 0.001)

increases the level of SOD and CAT and decreases the level of GSH

and activity of GST towards normal in comparison with Group II.

Endogenously, a dynamic equilibrium between generated free

radicals and antioxidant defence system exists that protects from

deleterious effects of oxidative damage. During oxidative stress,

like in cancer, this equilibrium disrupts, and the level of enzymic

antioxidants like SOD and CAT decreases due to the over usage to

act against free radicals. GSH, a tripeptide, act as intracellular anti-

oxidant and protects from free radical induced cellular damage and

produce a substrate for GST and other anti-oxidant enzymes. The

increased level of GSH and increased GSTactivity in Group II clearly

indicates the over production of these enzymes due to increased

level of ROS occurred in hepatocarcinogenesis. So, the anti-cancer

potential of any drug can be assessed by screening the level of

these enzymic and non-enzymic antioxidants. The anti-cancer

potential of SSSC-33 established through the reversal of SOD, CAT

and GST towards normal level (normal control).

3.6.3. Lipids and lipoprotein evaluation in plasma

The level of lipids and lipoprotein were evaluated as described

to get additional information for a complete assessment and

monitoring of the liver function with HCC and results obtained are

depicted in Fig. 10. The data showed that the level of TC, TG, LDL,

and VLDL was signiﬁcantly (p < 0.001) increased and the level of

HDL (p < 0.001) signiﬁcantly decreased in the plasma sample of

rats from Group II as compared with Group I. This clearly indicates

the development of cancerous tissue in response with DEN. SSSC-

Fig. 9. Effect of SSSC-33 on hepatic markers in liver homogenate of rats induced

with HCC. Values are expressed as mean ꢄ SEM of six rats per group; where, a-

Group II, III, IV compared with Group I; b- Group III & IV compared to Group II;

***p < 0.001; **p < 0.01; *p < 0.05; ^nsp > 0.05; Group I- Normal Control; Group II-

Induced Control; Group III- treated with SSSC-33 LD; Group IV- treated SSSC-33 HD.

AST-aspartate aminotransferase, ALT-alanine aminotransferase, ALP-alkaline phos-

phatase, GGT-gamma-glutamyltransferase.

S. Chacko, S. Samanta / Biomedicine & Pharmacotherapy 89 (2017) 162–176

173

Table 8

Effect of SSSC-33 on the level of enzymic antioxidants in HCC induced animals.

SOD (units/mg of protein)

CAT (

mmol of H₂O₂consumed/min/mg of protein)

Serum

Liver

Serum

Liver

Group I

8.77 ꢄ 0.153

5.05 ꢄ 0.16

61.76 ꢄ 0.25

31.32 ꢄ 0.29

Group II

Group III

Group IV

4.56 ꢄ 0.22a^***

5.69 ꢄ 0.213a^***b^ns

6.91 ꢄ 0.33a^***b^***

2.07 ꢄ 0.05 a^***

3.40 ꢄ 0.03 a^***b^***

4.55 ꢄ 0.20 a^*b^***

40.24 ꢄ 0.21 a^***

44.39 ꢄ 0.21 a^***b^*

46.95 ꢄ 0.22 a^***b^***

19.19 ꢄ 0.19 a^***

21.24 ꢄ 0.92 a^***b^ns

22.26 ꢄ 0.61 a^***b^***

Values are expressed as mean ꢄ SEM of six rats per group; where, a- Group II, III, IV compared with Group I; b- Group III & IV compared to Group II; Group I- Normal Control;

Group II- Induced Control; Group III- treated with SSSC-33 LD; Group IV- treated with SSSC-33 HD. SOD-superoxide dismutase, CAT-catalase.

^**p < 0.01.

*

p < 0.05.

p < 0.001.

p > 0.05.

***

ns

3.6.4. Effect of SSSC-33 on liver weight and histopathological

investigation of liver sections

Table 9

Effect of SSSC-33 on the level of non-enzymic antioxidants in liver of HCC induced

animals.

On completion of eight weeks, all the animals were sacriﬁced

and examined the liver weight (Table 10). It was observed from the

data that cancer induction causes an overall increase in liver

weight due to the presence of a tumour. Liver regains its normal

weight after the treatment with SSSC-33 HD (p < 0.01) in

comparison with induced control (Fig. 11).

GSH (

m

g/mg of protein)

GST (unit/mg protein)

Group I

2.85 ꢄ 0.04

391.58 ꢄ 1.12

Group II

Group III

Group IV

3.27 ꢄ 0.02 a^***

3.11 ꢄ 0.04 a^***b^**

3.08 ꢄ 0.05 a^***b^***

786.52 ꢄ 0.80 a^***

774.02 ꢄ 3.21 a^***b^**

612.45 ꢄ 2.58 a^***b^***

The histological examination of the liver tissue of all groups by

hematoxylin and eosin staining is shown in Figs. Fig. 12C&D and

Fig. 13C&D. In the normal control group the section showed cells

with intact architecture with normal nuclei, but the liver sections

of HCC rats (Group II), showed loss of architecture, the presence of

binucleate, enlarged, polygonal hepatocytes with acidophilus

staining cytoplasm. The DEN-treated groups also showed irregular

sinusoids and degenerated tumour cells. Some cells in DEN-treated

liver tissue exhibited multiple proliferating nucleoli, and some

cells possess intranuclear vacuole or cytoplasmic vacuoles. A

massive area of vacuolated hepatocytes, cellular inﬁltration,

pyknotic nuclei, and Numerous Kupffer cells was found.

The histopathological investigation of the rats treated with

SSSC-33 (Group III&IV) showed cells with architecture more or less

like the control one. The sinusoids were regularised in greater

extent in Group IV compared with Group III.

Values are expressed as mean ꢄ SEM of six rats per group; where, a- Group II, III, IV

compared with Group I; b- Group III & IV compared to Group II; ***p < 0.001;

**p < 0.01; *p < 0.05; ^nsp > 0.05; Group I- Normal Control; Group II- Induced

Control; Group III- treated SSSC-33 LD; Group IV- treated with SSSC-33 HD. GSH-

glutathione, GST-glutathione-S-transferase.

33 treated groups showed a decrease in TC, TG, LDL and VLDL and

increased in HDL towards normal control group. The SSSC-33 HD

had given signiﬁcance difference from induced control to normal

control values. The reestablishment of lipids and lipoprotein level

in the plasma with the treatment with SSSC-33 indicates its anti-

cancer effect in HCC, but the effect signiﬁcantly differed from the

control group. As the liver plays a crucial role in lipid and

lipoprotein metabolism, the signiﬁcant impairment of the hepatic

function occurring during chronic liver diseases, such as HCC, can

inﬂuence plasma lipoprotein proﬁles. There is always a major

change found in the levels of lipoproteins along with TC and TG

with patients with HCC [55].

4. Concluding remarks

In this study we were tried to develop new azomethine based

lead compound having multi-receptor action on HCC like sorafenib

through molecular modelling, synthesis and pharmacological

screening. Twelve different azomethine compounds based on 2-

aminopyridine (SSSC-26 to 31) and 2-aminobenzothiazole (SSSC-

32 to 37) were designed. The pharmacokinetic evaluation using

QikProp showed that all compounds have sufﬁcient structural

features to develop as an oral drug. Further, Ro5 analysis proved

that compounds were having all the parameter required for the

oral activity. Acute toxicity studies using ProTox web server

revealed LD₅₀values (500–2000 mg/kg, b.w) of each compound

and showed that these molecules are safe enough to apply as a

drug. The docking score of the compounds was satisfactory enough

to explain the ability of the compounds to bind with VEGFR, and

Chk1 receptor to elicit anti-HCC action. After molecular modelling

Table 10

The effect of SSSC-33 on liver weight of rats induced with HCC.

Fig. 10. Effect of SSSC-33 on lipids and lipoprotein in plasma of rat induced with

HCC. Values are expressed as mean ꢄ SEM of six rats per group; where, a- Group II,

III, IV compared with Group I; b- Group III & IV compared to Group II; ***p < 0.001;

**p < 0.01; *p < 0.05; ^nsp > 0.05; Group I- Normal Control; Group II- Induced

Control; Group III- treated with SSSC-33 LD; Group IV- treated SSSC-33 HD. LC-total

cholesterol, TG-triglycerides, HDL-high density lipoprotein, LDL- low-density

lipoprotein, VLDL-very low-density lipoprotein.

Groups

Liver weight in Grams

Group I (Normal control)

7.54 ꢄ 0.15

Group II (Induced control

Group III (SSSC-33 LD treated)

Group IV (SSSC-33 HD treated)

9.67 ꢄ 0.22a^***

9.02 ꢄ 0.28a^***b^ns

8.37 ꢄ 0.14a^nsb^**

174

S. Chacko, S. Samanta / Biomedicine & Pharmacotherapy 89 (2017) 162–176

histopathological slides of liver and compared statistically with

the respective data of induced control and normal control.

The elevation of hepatic markers like AST, ALT, ALP and GGT in

serum was observed in cancer-induced groups as previously

reported due to the overproduction and membrane damage and

conﬁrms the HCC induction. On the contrary, SSSC-33 HD

treatment in Group IV resulted in a signiﬁcant reversal of altered

level of hepatic markers to control values which are a sign of the

liver improvement to a normal state. The antioxidative defence

system of the body scavenges ROS that helps in the initiation of

lipid peroxidation and, therefore, plays a protective role in cancer

development. This protection system operates through enzymatic

(SOD and CAT), and non-enzymatic (GST and GSH) components. In

the present study, the decreased activity of SOD, CAT, GSH and GST

in liver found in HCC rats was restored SSSC-33 HD towards normal

value. The elevation in the level of enzymatic and nonenzymatic

antioxidants may be due to the ability of SSSC-33 to prevent ROS

formation. Finally, comparison of histopathological changes and

liver weight variation of animals in Group IV with Group II further

conﬁrms the anti-HCC potential of SSSC-33.

Fig. 11. The effect of SSSC-33 on the liver weight of rats induced with HCC. Values

are expressed as mean ꢄ SEM of six rats per group; where, a- Group II, III, IV

compared with Group I; b- Group III & IV compared to Group II; ***p < 0.001;

**p < 0.01; *p < 0.05; ^nsp > 0.05; Group I- Normal Control; Group II- Induced

Control; Group III- treated with SSSC-33 LD; Group IV- treated SSSC-33 HD.

Thus

SSSC-33-((benzo[d]thiazol-2-ylimino)methyl)phenol),

leads were synthesised and characterised by different spectro-

scopic methods. The results thus obtained proved the structural

integrity of the compounds.

We evaluated DPPH scavenging property of all compounds,

SSSC-29 and SSSC-33 showed lowest IC₅₀values of 63.60 and 60.32

respectively and showed signiﬁcant antioxidant potential in

comparison with ascorbic acid (IC₅₀- 55.27), a known antioxidant.

The anti-HCC evaluation of SSSC-33 in DEN (200 mg/kg, bw)

induced rat model was carried out and determined level of hepatic

markers, enzymic and non-enzymic antioxidants, examined

appeared to be effective free radical scavenger with antioxidant

activities. Also, it could protect rat liver from DEN-induced altered

hepatic functioning, and prevent further progression through the

inhibition of Chk1and VEGFR-2. Furthermore, from all the above

mentioned overwhelming results it can be concluded that Schiff's’

base prepared from 2-aminopyridine like SSSC-33 is a potential

anti-cancer therapeutic agent and an appropriate candidate for

further development as an anti-HCC agent. Also, it is necessary to

improve the anti-HCC property of SSSC-33 without hampering the

pharmacokinetic and toxicological proﬁle.

Fig 12. A&B morphological views of normal liver (Group I) and HCC induced liver (Group II) respectively; C&D Hematoxylin and Eosin stained liver tissue of normal liver and

HCC induced liver respectively observed under LEICA DME microscope (40ꢃ). T-tumour tissue.

S. Chacko, S. Samanta / Biomedicine & Pharmacotherapy 89 (2017) 162–176

175

Fig.13. A&B morphological views of SSSC-33 LD treated liver (Group III), and SSSC-33 HD treated liver (Group IV) respectively; C&D Hematoxylin and Eosin stained liver tissue

of SSSC-33 LD treated, and SSSC-33 HD treated respectively observed under LEICA DME microscope (40ꢃ). N- non-tumorous tissue T-tumour tissue adjacent to the tumorous

fabric.

Conﬂict of interest

[2] D. Hashim, P. Boffetta, C. La Vecchia, M. Rota, P. Bertuccio, M. Malvezzi, E. Negri,

The global decrease in cancer mortality: trends and disparities, Ann. Oncol. 27

(5) (2016) 926 –933.

[3] H.B. El-Serag, Hepatocellular carcinoma, New Engl. J. Med. 365 (12) (20 1 1 )

111 8 –1 127.

The authors conﬁrm that this article content has no conﬂict of

interest.

[4] A. Raza, Hepatocellular carcinoma review: current treatment, and evidence-

based medicine, World J. Gastroenterol. 20 (15) (2014) 4 1 15.

[5] X.K. Guo, H.P. Sun, S. Shen, Y. Sun, F.L. Xie, L. Tao, Q.L. Guo, C. Jiang, Q.D. You,