Synthesis and anticancer screening of triazine analogues

Article abstract of DOI:10.22159/ijpps.2019v11i4.28275

Objective: The study was aimed to investigate the cytotoxic effect of S-5H-[1,2,4]-triazino (5,6-b) indol-3-yl-3,4-phenylethane-thioate derivatives as epidermal growth factor Receptor (EGFR) inhibitors. Methods: In the present study 14 novel triazine analogues were synthesized and characterized using different spectroscopic techniques such as FTIR, NMR and Mass Spectroscopy. The anticancer activity was performed using MCF-7 (breast cancer) and K-562 (leukaemia) cell lines. Further, molecular docking was carried out using Vlife Molecular Docking Software (MDS) on crystal structure of epidermal growth factor receptor (EGFR) to identify the binding mode of interaction with an active site. Results: Compounds MA-7, MA-8, MA-12, MA-13 and MA-14 show potent activity against cancer cell lines in the range of<10 to 84.4 μg/ml. Further molecular docking on EGFR also supports that there is a strong correlation between in silico and in vitro biological activity. The results of this study may be further useful for lead optimization process. Conclusion: The results of this study indicates that the synthesized triazine analogues can give a potential lead as an anticancer agent.

Full text of DOI:10.22159/ijpps.2019v11i4.28275

International Journal of Pharmacy and Pharmaceutical Sciences
ISSN- 0975-1491
Vol 11, Issue 4, 2019
Original Article
SYNTHESIS AND ANTICANCER SCREENING OF TRIAZINE ANALOGUES
ANSHULY TIWARI, SIDDHARTH J. MODI, KAKASAHEB R. MAHADIK, MUGDHA R. SURYAWANSHI*
Department of Pharmaceutical Chemistry, Poona College of Pharmacy, Bharati Vidyapeeth (Deemed to be University), Pune, Maharashtra, India
Email: mugdha.rs@gmail.com
Received: 03 Jul 2018 Revised and Accepted: 06 Mar 2019
ABSTRACT
Objective: The study was aimed to investigate the cytotoxic effect of S-5H-[1,2,4]-triazino (5,6-b) indol-3-yl-3,4-phenylethane-thioate derivatives as
epidermal growth factor Receptor (EGFR) inhibitors.
Methods: In the present study 14 novel triazine analogues were synthesized and characterized using different spectroscopic techniques such as FT-
IR, NMR and Mass Spectroscopy. The anticancer activity was performed using MCF-7 (breast cancer) and K-562 (leukaemia) cell lines. Further,
molecular docking was carried out using Vlife Molecular Docking Software (MDS) on crystal structure of epidermal growth factor receptor (EGFR)
to identify the binding mode of interaction with an active site.
Results: Compounds MA-7, MA-8, MA-12, MA-13 and MA-14 show potent activity against cancer cell lines in the range of<10 to 84.4 µg/ml. Further
molecular docking on EGFR also supports that there is a strong correlation between in silico and in vitro biological activity. The results of this study
may be further useful for lead optimization process.
Conclusion: The results of this study indicates that the synthesized triazine analogues can give a potential lead as an anticancer agent.
Keywords: Cancer, Triazine Analogues, MCF-7, K-562, Anticancer activity, EGFR
© 2019 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open-access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
DOI: http://dx.doi.org/10.22159/ijpps.2019v11i4.28275
INTRODUCTION
(Neu, HER-2), ErbB-III (HER-3) and ErbB-IV (HER-4) [11]. RTK
family contains an extracellular ligand-binding domain that is a
hydrophobic transmembrane domain, and cytoplasmic tyrosine
kinase domain [12]. A knockout model for EGFR shows the lethality
of embryo and defects in tissues or organs. The abnormal activation
of EGFR is expressed in many types of specific diseases [13]. The
pleiotropic function of EGFR signaling helps to develop and regulate
various physiological functions of the body [14]. The incrimination
of various types of disorders has been defined by the exponential
expression of EGFR. There are several reports are available in the
literature suggesting that aromatic and heteroaromatic substitution
over chosen triazine ring moiety shows potent anticancer activity,
therefore, here in this study the substituted benzoic acids were used
and were studied as anticancer agents [15]. The main objective of
the study is to perform synthesis and anticancer screening of
synthesized compounds using various cell lines.
Cancer is a condition that refers to a state in which the cell present
in the body gone awry and reproduced in an uncontrollable manner
[1]. The healthy tissues and organs that are present in the body are
then proliferated or intrude get destroyed [2]. The growth of the
cancer begins with one part/specific organ of the body and further
spread into the entire body. Cancerous cells segregate and mimic
themselves and could be present in the form of a cluster of infected
cells that are known as malignancy (tumor) [3]. The symptom of
cancer originated from the coercing, pulverizing and destroying the
surrounding healthy cells and tissues [4]. The immune system has
crucial role in the management and elimination of early cancer or
premalignant cells because the immune system is associated with
increased incidences of a variety of cancer [5]. In women, breast
cancer is a more common while, colorectal, prostate and lung cancer
occurs most commonly in men [6]. Development of breast cancer is
seen most commonly in the cells present in the inner linings of the
milk ducts and the lobules, which are the major source for the
supply of the milk in the ducts [7]. Factors emerging risk of breast
cancer are obesity, decrease in physical exertion, alcohol intake,
hormone replacement therapy at the ongoing menopause, early age
menstruation, late pregnancy or no pregnancy at all. Approximately
5% of cases are occurring due to the inherited gene expression from
the patient’s parents. Risk factors for cancer also involve breast
cancer genes namely BRCA-1 and BRCA-2 among others [8]. The
other type of cancer we have focused is leukaemia which is known
as blood cancer and starts with the bone marrow and results into the
multiplication of white blood cells (WBCs) [9]. With the progression
of leukaemia, the infected cells crowed out to suppress the growth of
normal cells. Sometimes some hairy thin projections are seen in an
infrequent chronic leukaemia which is called as hairy cell leukaemia
(HCL) that starts with the bone marrow and the accumulation of
these hairy cells present in the bone marrow, liver and spleen.
Though hairy cell leukaemia strikes the WBC, the enlargement in the
lymph nodes doesn’t observed [10]. Although there are no clear
symptoms for the development of HCL in the early stage.
MATERIALS AND METHODS
Chemicals and reagents
Raw materials were purchased from Loba Chemie, Sigma Aldrich,
Merck (India), Sisco Research Laboratory SRL (India) and
Spectrochem Pvt. Ltd. and were of the synthetic grade. Melting point
detection was conducted by using Veego VMP-D digital apparatus.
Silica gel precoated F-254 TLC plates were used for monitoring the
progress of reaction; further detection of the retention factor is
obtained by either staining by iodine or visualizing under UV-light
chamber. Fourier transform infrared (FT-IR) spectra were obtained
by making sample cuvettes in anhydrous potassium bromide (KBr)
on “JASCO FTIR 4100” and are reported in cm-1. 1H-NMR spectra
were illustrated on “Bruker Avance having a frequency of 400 MHz
using DMSO-D6 in the presence of an internal standard
tetramethylsilane (TMS).
Step I: General procedure of synthesis of 1, 2, 4-triazino-(5, 6-
b)-indole-3-thione
To a mixture of Isatin (0.01 mol) and thiosemicarbazide (0.01 mol),
distilled water (300 ml) containing potassium carbonate (0.04 mol)
was taken in a round bottom flask. The reaction flask was refluxed
EGFR receptor is devided in to four receptors namely EGFR (ErbB-
I/HER-1{Human Epidermal Growth Factor Receptor-1}) ErbB-II

Suryawanshi et al.
Int J Pharm Pharm Sci, Vol 11, Issue 4, 114-121
for 4 h during which an initial intense red coloured solution turned
orange. Upon cooling at room temperature, the reaction mixture was
filtered and the residual filtrate obtained was acidified with glacial
acetic acid to precipitate the yellow coloured product. Precipitates
obtained by filtration were 3 to 4 times with water to obtain the
crude product. The crude product was then stirred with methanol
(25 ml) for 2 h, filtered, dried and subsequently re-crystallized to
furnish the final product (1.34 g, 66.3%) [16].
Scheme of synthesis
Step I
Step II
Step II General procedure of synthesis of S-5H-[1, 2, 4]-triazino-
(5,6-b)indol-3-yl-3,4-biphenylethanethioate (MA-1 to MA-14)
The grid map was streamlined at the residues namely ASP 212 (A),
LYS 179 (A), HIS 180(A), ARG 174(A), ASP 269(A), HIS 268 (A),
TYR59(A), GLN 63 (A), ASP 273 (A) respectively which were then
forecasted from the ligpot.
Substituted benzoic acid (0.01 mol) and 1-Ethyl-3-(3-dimethyl-
aminopropyl) carbodiimide (EDC. HCl) was taken in 100 ml round
bottom flask containing N-N dimethylformamide (DMF) as a solvent and
the reaction mixture was then agitated for 2 h at 0 °C. Later, on the
completion of 2 h the crude product (reaction step I (1)) was added (0.01
mol). The reaction mixture was then agitated at room temperature for
24 h. The reaction mixture was extracted with ethyl acetate (EtOAc), was
filtered, dried and recrystallized with chloroform.
Docking file generation
On the activation of the receptor EGFR (1M17) signalling pathways
are strongly activated for anticancer activity. Hence, EGFR-1M17
was selected as a biological target for the docking study. The
crystallized structure was retrieved from the Protein Data Bank
(PDB) {www. rcsb. org/pdb}. The receptor was optimized; saved as.
mol file and used for docking simulation. Two dimensional
structures of the synthesized compounds were drawn and then
transfigured into the 3D structures with Vlife MDS. Energy
minimization of the 3D structures was performed up to the rms
gradient of value 0.01 by the use of MMFF94. For all the conformers,
energy minimization method was performed. The selection of the
active site was done by picking the hollow having the utmost
hydrophobic surface area of the receptor. The GA docking method
was selected for the docking simulation. The virtual docking for all
the conformers was conducted at elucidated cavity of the receptor.
The fixed parameters of the docking simulation were the number of
placements: 30, rotation angles 30 ° comprehensive method, scoring
task: docking score. The rotation of the analogs to the divergent
postures was obtained by the rotation angle. Besides docking
simulation, the premier docked conformers of each ligand and
receptor was transpired and assembled by elucidating the results of
4Å. After optimization, complexes were examined for assorted
Molecular docking studies
Ligand preparation
4-biphenylethanethioate derivatives were drawn as the template for
building the compounds present in the dataset of V-Life molecular
docking software (MDS) version 3.5. The geometry of the ligands
was modified by the minimization of energy with the use of Merck
Molecular force field (MMFF94) and charges for atoms was applied
until a 0.001 kcal/mol/Å gradient was reached, perpetuating the
rigid structure template throughout the process of minimization of
energy [17].
Grid generation
Using the Vlife molecular docking software (MDS) version 3.5 the grid
map was centralized at the active site of the protein.
115

Suryawanshi et al.
Int J Pharm Pharm Sci, Vol 11, Issue 4, 114-121
interSactions of the ligand with the receptor such as hydrogen
bonding, hydrophobic bonding and van der Waal’s interaction [18].
1665, 3069, 3305; 1H-NMR (δ ppm; CDCl3): 2.30 (s, 3H,-CH3), 4.27 (s,
2H,-CH2), 6.87-7.31 (m, 8H, biphenyl), 7.31-7.69 (m, 3H,-Ar).
In vitro anticancer evaluation
S-5H-[1,2,4]-triazino(5,6-b)indol-3-yl 2,4-dichlorobenzothioate
(MA-2)
The cell lines were grown on Roswell Park Memorial Institute 1640
(RPMI0-1640) media that contains fetal bovine serum (10%) and L-
glutamine (2 mmol). Ninety-six well microtiter plates in 100 µl at the
solidity of plates were used to inoculate the cells depending upon
the multiplication time of cell lines. After the cell inoculation
process, microtiter plates were incubated for 24h, the conditions
were sustained as 37 °C, 5% CO2, 95% air and 100% Relative
Humidity (Rh) before addition of synthesized compounds. The stock
solution of the synthesized analogs was prepared at 100 mg/kg and was
further diluted to 1 mg/ml with water and kept in chilled condition
before use. Further, one fraction of chilled concentrate (1 mg/ml) was
liquefied and was attenuated to 100, 200, 400 and 800 µg/ml
respectively with whole media comprising of standard drug. A portion of
10µg of dilutions containing diverse drugs was added to the suitable
wells previously having 90 µl media resulting in the ultimate drug
concentrations that are 10, 20, 40 and 80µg/ml respectively.
Molecular formula: C16H8Cl2N4OS (373.98); MP: 275 °C; yield: 80%;
Rf: 0.77 (toluene: EtOAc {4:1}); IR vmax (cm-1) (KBr): 1475.28,
1308.46, 1700.91, 599.62; 1H-NMR (δ ppm; DMSO): 7.38-7.85 (m,
3H,-Ar), 7.00-7.80 (m, 4H phenyl), ESI-MS: m/z 374.19 (M+H)+.
S-5H-[1, 2, 4]triazino[5,6-b]indol-3-yl 3-bromobenzothioate (MA-3)
Molecular formula: C16H9BrN4OS (385.24); MP: 276 °C; yield: 77%;
Rf: 0.68 (toluene: EtOAc {4:1}); IR vmax (cm-1) (KBr): 1479.81,
1311.36, 1698.34, 617.10; 1H-NMR (δ ppm; DMSO): 7.38-7.91
(m,4H,-Ar), 7.00-7.55 (m, 4H phenyl) ESI-MS: m/z 384.8 (M-H)+.
S-5H-[1,2,4]triazino[5,6-b]indol-3-yl 4-chlorobenzothioate (MA-4)
Molecular formula: C16H9ClN4OS (340.79); MP: 277 °C; yield: 88%;
Rf: 0.55(toluene: EtOAc {4:1}); IR vmax (cm-1) (KBr): 1478.33,
1312.06, 1698.34, 619.10; 1H-NMR (δ ppm; DMSO): 7.36-8.00 (m,
4H,-Ar), 7.05-7.43 (m, 5H phenyl) ESI-MS: m/z 340.02 (M+H)+.
After the addition of the compounds, plates were incubated in the
standard conditions for 48h and assay was discontinued after
inclusion of trichloroacetic acid (TCA). An in-situ condition was
maintained with mixing 50 µl of chilled TCA gently (10% of TCA)
followed by further incubation for 1h at the temperature of 4 °C.
Removal of resilient liquid was discarded; plates were washed with
water 4-6 times and were dried under air. Sulforhodamine B (SRB)
solution at the concentrations of 50 µl at 0.4 % (w/v) in 1% acetic
acid was appended to every well; 20 min. the incubation period of
these plates was obtained at room temperature. Once the dying
process is completed the portion of unbound dye was recuperated
and the unconsumed dye was omitted by washing 4-5 times with
acetic acid in the concentration of 1 % acetic acid. The plates were
dried under the influence of air. Calculation of the percentage
growth in plate sequence was compared to reference wells. Percent
development of cells was revealed in terms of the proportion (ratio)
of the mean absorbance of the well to the mean absorbance of the
reference well X 100. By applying measurements of 6 absorbance
[time 0], control growth (C), and the test in the presence of 4
concentration extents (Ti)], the % growth was calculated at the
concentration extent of every single drug [19-20].
S-5H-[1,2,4]triazino[5,6-b]indol-3-yl benzothioate (MA-5)
Molecular formula: C17H12N4OS (306.6); MP: >300 °C; yield: 77%; Rf =
0.55(toluene: EtOAc {4:1}); IR vmax (cm-1) (KBr):1475.28, 1306.46,
1
1700.91, 599.62; H-NMR (δ ppm; DMSO): 7.0-7.55 (m, 4H,-Indole),
7.5-7.97 (m, 5H,-Ar); m/z = 321.08 (M+H)+
S-5H-[1,2,4]triazino [5,6-b] indol-3-yl 4-methylbenzothioate (MA-6)
Molecular formula: C17H12N4OS (320.37); MP: >300 °C; yield: 90%; Rf
= 0.58(toluene: EtOAc {4:1}); IR vmax (cm-1) (KBr): 1500.35,
1
1281.47, 1714.41, 2988.05; H-NMR (δ ppm; DMSO): 2.35 (s,-CH3),
7.33-7.75 (m,-Ar), 7.00-7.85 (m, indole).
S-5H-[1,2,4]triazino [5,6-b] indol-3-yl 3-methylbenzothioate (MA-7)
Molecular formula: C17H12N4OS (320.37); MP: >300 °C; yield: 70%;
Rf: 0.62(toluene: EtOAc {4:1}); IR vmax (cm-1) (KBr): 1431.69,
1
1347.03, 1714.41, 2891.91, H-NMR (δ ppm; DMSO): 2.51 (s,-CH3),
7.25-7.85 (m,-Ar), 7.06-7.88 (m, indole).
S-5H-[1,2,4]triazino [5, 6-b] indol-3-yl 3,4,5-trihydroxybenzo-
thioate (MA-8)
ꢁꢂ
ꢀ
ꢄ ∗ 100%
ꢃ
Molecular formula: C16H10N4O4S (354.34); MP: >300 °C; yield: 70%;
Rf: 0.56 (toluene: EtOAc {4:1}); IR vmax (cm-1) (KBr): 1474.31,
1310.39, 1696.91, 3115.44, 1H-NMR (δ ppm; DMSO): 5.0 (m,-OH),
7.05-7.40 (m,-Ar), 7.0-7.85 (m, indole).
In silico ADMET study
For the identification and optimization of lead generation, in silico
ADMET (absorption, distribution, metabolism, excretion, and
toxicity) properties plays an essential role; ADMET SAR is open
source software available online and was employed to predict the
ADMET parameters. Here, the ADMET profile is estimated for the
different analogs using the online server (www. lmmd. ecust. edu. cn,
accessed on 5th March 2017) [20-21].
S-5H-[1, 2, 4]triazino[5,6-b]indol-3-yl 3-methoxybenzothioate
(MA-9)
Molecular formula: C16H10N4O4S (336.37); MP: >300 °C; yield: 70%;
Rf: 0.56 (toluene: EtOAc {4:1}); IR vmax (cm-1) (KBr): 1477.21,
1
1300.36, 1686.88, 3120.422, H-NMR (δ ppm; DMSO): 5.0 (m,-OH),
7.05-7.40 (m,-Ar), 7.0-7.85 (m, indole).
RESULTS AND DISCUSSION
Characterization study
S-5H-[1,2,4]triazino[5,6-b]indol-3-yl
(MA-10)
pyridine-3-carbothioate
Fourteen triazine derivatives MA-1 to MA-14 were synthesized. The
yield of final compounds were obtained in the range of 50.00% to
90.04%. Recrystallization was performed using ethanol. Structural
Molecular formula: C15H9N5OS (307.04); MP: >300 °C; yield: 88%; Rf:
0.68 (toluene: EtOAc {4:1}); IR vmax (cm-1) (KBr): 1475.28, 1306.46,
1700.91, 1H-NMR (δ ppm; DMSO): 7.00-7.85 (m, 4H, indole), 7.49-
7.64 (m,-Ar).
1
characterization of synthesized analogues was done by IR, H-NMR
and mass spectroscopy. The results of IR spectra confirm that all the
compounds shows stretching under respective frequency within the
range i. e; C-C Stretch (1500-1400 cm-1), C-N Stretch (1250-1335 cm-
S-5H-[1,2,4]triazino[5,6-b]indol-3-yl 3-aminobenzothioate (MA-
11)
1), S-C=O (1668-1705 cm-1) etc. H NMR spectra shows that protons
1
of S-5H-[1,2.4]triazino[5,6-b]indol-3 were observed in the aromatic
region under the range of 6.8-7.2 δ ppm. Further, Mass spectra of
compounds also confirms synthesized compounds.
Molecular formula: C16H11N5OS (321.37); MP: >300 °C; yield: 90%;
Rf: 0.68 (toluene: EtOAc {4:1}); IR vmax (cm-1) (KBr): 1499.05,
1
1384.43, 1718.15, 1600.65; H-NMR (δ ppm; DMSO): 7.06-7.89 (m,
4H, indole), 7.43-7.68 (m,-Ar) 4.32 (s, aromatic-NH).
S-5H-[1,2,4]-triazino
(5,6-b)
indol-3-yl-3,
4-biphenyl-
ethanethioate (MA-1)
S-5H-[1,2,4]triazino[5,6-b]indol-3-yl 5-aminobenzothioate (MA-12)
Molecular formula; C24H20N4OS (396.10), MP: 118-120ᵒC; yield
Molecular formula: C16H11N5OS (321.07); MP: >300 °C; yield: 93%;
74.04%; Rf = 0.69 (toluene: EtOAc {4:1}); IR vmax (cm-1) (KBr): 1297,
Rf: 0.68 (toluene: EtOAc {4:1}); IR vmax (cm-1) (KBr): 1500.35,
116

Suryawanshi et al.
Int J Pharm Pharm Sci, Vol 11, Issue 4, 114-121
1381.28, 1714.41, 1599.66; 1H-NMR (δ ppm; DMSO): 7.00-7.85(m,
4H, indole), 7.49-7.64 (m,-Ar) 4.0 (s, aromatic-NH).
1468.53, 1699.94; 1H-NMR (δ ppm; DMSO): 7.00-7.85(m, 4H, indole),
6.92-7.48 (m,-Ar).
S-5H-[1,2,4]triazino[5,6-b]indol-3-yl 2-hydroxybenzthioate (MA-13)
Molecular docking
Molecular formula: C16H10N4O2S (322.35); MP: >300 °C; yield: 83%;
Rf: 0.50 (toluene: EtOAc {4:1}); IR vmax (cm-1) (KBr): 1459.85,
1297.86, 1699.34, 3233.88; 1H-NMR (δ ppm; DMSO): 7.00-7.85(m,
4H, indole), 6.92-7.48 (m,-Ar), 5.0 (s, Ar-OH).
To investigate the interaction between epidermal growth factor
receptor (EGFR) and synthesized derivatives; molecular docking
study was carried out. Active compound MA-8 binds with ASP813A
and VAL741A; MA-12 binds with ASP8134A, HIS811A and PHE832A;
MA-13 binds with ASP8134A and PHE832A; MA-14 binds with
ASP813A and LYS721A amino acids. Interaction of compounds with
the crystal structure of epidermal growth factor receptor (EGFR) is
shown in the fig. 1.
S-5H-[1,2.4]triazino[5,6-b]indol-3-yl 3-nitrobenzthioate (MA-14)
Molecular formula: C16H10N4O2S (322.35); MP: >300 °C; yield: 83%;
Rf: 0.50 (toluene: EtOAc {4:1}); IR vmax (cm-1) (KBr): 1259.29,
Fig. 1: Docking interaction of compounds with 1M17
Fig. 2: Phase contrast images of MCF-7 cells showing morphological changes following the treatment with synthesized compounds
117

Suryawanshi et al.
Int J Pharm Pharm Sci, Vol 11, Issue 4, 114-121
Fig. 3: Phase contrast images of K-562 cells showing morphological changes following the treatment with synthesized compounds
Fig. 4: Growth Curve: Human Breast Cancer Cell Line (MCF-7)
Fig. 5: Growth Curve: Human Leukaemia Cell Line (K-562)
118

Suryawanshi et al.
Int J Pharm Pharm Sci, Vol 11, Issue 4, 114-121
Table 1: Cytotoxicity on human breast cancer cell line MCF-7
Human breast cancer cell line MCF-7
% Control growth
Drug concentrations (µg/ml)
Compound
code
Experiment No. 1
Experiment No. 1
Experiment No. 1
Average values
10
20
40
80
10
20
40
80
10
20
40
80
10
20
40
80
MA-1
80.4
87.7
80.5
45.9
75.8
76.6
86.9
78.2
69.7
77.0
87.2
92.4
85.3
89.3
-
79.0
86.8
73.3
6.9
79.7
77.3
81.8
68.4
56.4
71.0
81.4
91.2
93.9
75.5
64.1
75.0
56.3
1.4
74.0
81.0
71.2
8.8
37.8
67.3
74.5
78.5
91.8
53.4
58.4
65.4
40.2
7.6
72.7
79.8
58.3
11.8
34.6
86.0
65.6
83.2
72.9
42.0
75.3
82.8
80.8
39.7
78.2
76.7
77.7
80.8
72.7
79.7
88.0
95.0
92.2
89.6
76.4
83.5
75.4
7.7
79.1
80.8
79.2
76.9
61.9
74.2
84.9
89.1
90.8
86.1
69.5
72.4
55.4
2.9
70.6
81.3
73.2
14.2
41.8
73.8
77.9
80.0
88.0
69.2
53.0
62.2
42.4
11.2
68.7
78.5
60.4
11.8
39.9
89.4
72.0
92.2
88.5
57.7
91.6
93.1
95.2
105.5
116.3
101.5
53.1
79.2
79.0
75.5
98.8
-46.3
-46.3
-46.5
-43.4
91.0
82.6
95.6
110.9
114.2
103.2
67.3
64.9
62.7
62.6
102.8
-48.3
-46.5
-29.1
-47.3
27.8
58.8
84.6
94.8
109.2
107.4
49.7
33.8
31.6
35.4
93.1
-49.3
-53.2
-56.0
-50.2
30.1
57.3
98.7
75.9
103.5
87.1
34.9
26.7
23.4
28.2
109.7
-15.2
-10.5
-18.4
-33.7
82.5
87.9
85.5
63.7
90.1
84.9
72.6
79.4
73.8
77.4
91.3
47.0
43.8
44.1
82.1
84.3
81.4
41.8
91.0
87.1
76.1
70.1
60.3
69.3
89.7
44.0
46.1
44.2
53.8
68.7
65.4
33.0
84.6
89.9
64.7
18.9
37.1
58.8
81.8
36.4
42.2
22.2
-50.2
47.2
61.7
60.4
31.5
81.6
81.8
51.2
16.7
32.7
67.9
82.4
53.4
50.3
27.1
-33.7
MA-2
MA-3
MA-4
MA-5
MA-6
MA-7
MA-8
MA-9
MA-10
MA-11
MA-12
MA-13
MA-14
Adriamycin
-47.3 -50.2 -33.7
-43.4 -47.3
-50.2 -33.7
-43.4 -47.3
43.4
Table 2: Cytotoxicity on human breast cancer cell line K-562
Human breast cancer cell line K-562
% Control growth
Drug concentrations (µg/ml)
Compound
code
Experiment No. 1
Experiment No. 1
Experiment No. 1
Average values
10
20
40
80
10
100.9
94.7
101.6
91.5
100.0
96.1
96.4
101.9
102.7
102.1
103.5
100.0
99.1
20
40
80
10
98.3
20
40
80
10
20
40
80
MA-1
91.8
98.6
98.2
83.8
91.7
90.9
92.6
84.7
96.5
96.7
94.5
98.4
97.2
90.5
12.4
96.3
98.3
97.3
8.1
96.1
102.0
95.0
9.8
91.7
89.5
47.6
13.5
85.0
72.2
87.6
22.6
95.9
84.8
92.8
97.7
83.3
28.3
-
100.4
101.6
99.2
7.1
99.2
100.7
96.9
0.4
99.0
97.9
67.8
9.2
98.7
99.7
98.0
15.0
100.9
101.8
99.1
96.2
101.6
88.5
101.4
106.4
103.0
82.5
13.5
101.9
104.0
101.8
18.4
103.2
100.0
104.2
59.1
111.1
87.5
107.0
103.9
98.8
89.5
89.9
65.5
12.0
96.7
99.5
97.5
-10.2
83.9
32.0
90.3
100.9
81.5
27.1
-34.0
97.0
98.4
98.9
89.2
96.7
95.0
92.3
96.6
101.0
102.4
101.6
100.7
100.0
94.0
11.2
98.4
99.9
98.2
10.0
99.3
98.8
97.2
92.9
94.3
86.2
99.3
101.4
99.3
87.3
9.1
99.0
102.2
97.9
9.5
93.4
92.5
60.3
11.6
93.8
90.5
94.4
8.5
MA-2
101.9
96.9
MA-3
MA-4
92.4
MA-5
96.5
96.6
94.2
86.8
82.7
73.4
99.3
96.7
93.1
82.8
7.5
95.8
91.7
92.5
67.0
105.7
96.8
105.5
101.2
99.7
69.0
-20.5
100.5
98.0
98.2
95.5
98.6
96.6
97.3
101.2
101.9
96.5
6.4
95.4
93.7
96.3
61.8
102.1
82.1
99.6
99.5
98.7
82.4
-29.0
99.6
99.9
98.2
13.0
89.8
84.2
95.4
99.1
92.5
47.1
-
98.4
98.1
95.1
97.7
62.6
106.3
88.8
104.0
101.5
99.1
73.1
-24.6
MA-6
98.0
MA-7
88.0
MA-8
103.1
103.9
108.6
106.9
103.8
103.8
94.4
MA-9
89.9
67.0
92.8
99.2
85.8
34.1
-
MA-10
MA-11
MA-12
MA-13
MA-14
Adriamycin
97.1
7.5
67.9
-24.2
13.6
30.8
26.0
30.3
Table 3: GI50 values (µg/ml) of standard and test compounds
Growth inhibition 50% (GI50)values (µg/ml)
S. No.
Compound code
MCF-7
67.6
NE
NE
17.2
NE
K-562
1.
2.
3.
4.
5.
6.
7.
8.
MA-1
MA-2
MA-3
MA-4
MA-5
MA-6
MA-7
MA-8
>80
>80
>80
10.4
>80
>80
NE
49.1
>80
>80
>80
>80
63.2
72.3
<10
NE
84.4
33.5
39.3
>80
>80
<10
<10
<10
<10
9.
MA-9
10.
11.
12.
13.
14.
STD
MA-10
MA-11
MA-12
MA-13
MA-14
Adriamycin
represented in table 1and table 2. GI50 values for standard and test
compounds are depicted in the table 3. Compounds MA-12 (GI50
=<10 µg/ml), MA-13 (GI50 =<10 µg/ml) and MA-14 (GI50 =<10
µg/ml) were found to be the most active compounds against the
breast cancer cell line and compound MA-4 (GI50 = 10.4 µg/ml)
showed the mild anticancer activity against the Adriamycin
(positive control) as shown in fig. 2 and fig. 3.
In vitro anticancer evaluation
The newly synthesized conjugates bearing various substituents on
the triazine ring were screened for their cytotoxicity on two cell
lines namely breast cancer (MCF-7) and leukaemia (K-562) cell
lines using SRB assay; Adriamycin was used as the positive
control. The results of cytotoxic activity against cell lines are
119

Suryawanshi et al.
Int J Pharm Pharm Sci, Vol 11, Issue 4, 114-121
Table 4: Predicted ADMET-SAR properties of synthesized compounds
Probability
0.7701
0.7670
0.7616
0.7088
0.7597
0.7597
0.6947
0.6981
0.6377
0.6899
0.6572
0.6516
0.7382
0.6941
0.8551
0.8950
0.6342
0.9826
0.8474
0.8982
0.9332
0.7045
0.6488
0.6601
0.5904
0.5498
0.5498
0.5292
0.9135
0.9018
0.9331
0.9003
0.9266
0.9266
0.9169
0.8556
0.8979
0.9105
0.8952
0.9223
0.9173
0.9214
0.6213
0.5761
0.6050
0.6185
0.6320
0.6778
0.6211
0.5884
0.6252
0.5773
0.6042
0.5720
0.5089
0.5279
2.3435
2.4510
2.4631
2.4294
2.3361
2.2955
2.4114
0.8682
1.1856
1.0988
1.0799
0.8416
0.7848
0.7633
MA-1
MA-2
MA-3
MA-4
MA-5
MA-6
MA-7
0.9744
0.9493
0.9638
0.9646
0.9738
0.9676
0.9566
1.0000
1.0000
1.0000
1.0000
1.0000
1.0000
1.0000
0.5511
0.5171
0.5311
0.5130
0.5444
0.5509
0.5000
0.7590
0.7313
0.7328
0.7314
0.7492
0.7640
0.7659
0.5571
0.5784
0.5258
0.5455
0.5577
0.5474
0.5313
0.9967
1.0000
1.0000
1.0000
0.9882
0.9927
0.9927
0.6908
0.5704
0.6670
0.6325
0.6316
0.6887
0.9118
0.7687
0.8622
0.5814
0.6950
0.5875
0.9178
0.9696
0.8585
0.8879
0.9343
0.9223
0.5480
0.5187
0.6320
0.5859
0.4758
0.5720
2.3483
2.3532
2.3361
0.7288
0.7727
0.8416
MA-8
MA-9
0.6011
0.9559
0.9837
1.0000
0.6440
0.5000
0.8815
0.6978
0.5577
0.5437
0.9792
0.9605
MA-10
0.9738
1.0000
0.5444
0.7492
0.5577
0.9882
0.7597
0.9597
0.9597
0.7382
0.6155
0.6155
0.8982
0.9135
0.9135
0.5498
0.7022
0.7022
0.9266
0.9403
0.9403
0.8684
0.8684
0.8906
0.6683
0.6683
0.5402
0.4508
0.4508
0.5525
2.3803
2.3803
2.4074
0.6039
0.6039
0.9266
MA-11
MA-12
MA-13
0.9621
0.9621
0.9134
0.9954
0.9954
1.000
0.5137
0.5137
0.5641
0.7974
0.7974
0.7801
0.6170
0.6170
0.5360
1.0000
1.0000
0.9806
0.6884
0.6462
0.6942
0.7118
0.9098
0.8074
0.5853
0.4511
0.6728
0.8016
MA-14
0.9141
0.9875
0.5385
0.8366
0.8212
0.9843
120

Suryawanshi et al.
Int J Pharm Pharm Sci, Vol 11, Issue 4, 114-121
Further, only active compounds show significant interaction at the
active site of receptor via hydrogen bonding to some important amino
acids such as ASP, VAL, MET, LYS etc. Combination of different
interactions leads to significant penetration of compounds at the active
site of the receptor with good docking score. Hence, it is observed that
molecular docking and anticancer activity are correlated with each
other. The results of anticancer activity are depicted in fig. 2 and fig. 3
respectively.
2. McBee Jr WC, Gardiner AS, Edwards RP, Lesnock JL, Bhargava
R. MicroRNA analysis in human papillomavirus (HPV)-
associated cervical neoplasia and cancer. J Carcinog
Mutagen 2000;1. ).
3. Augustin HG. Antiangiogenic tumour therapy: will it
work? Trends Pharm Sci 1998;19:216-22.
4. Hinck L, Näthke I. Changes in cell and tissue organization in
cancer of the breast and colon. Curr Opi Cell Bio 2000;26:87-95.
5. Grivennikov SI, Greten FR, Karin M. Immunity, inflammation,
and cancer. Cell 2010;140:883-99.
In silico ADMET SAR study
6. Racil Z, Razga F, Drapalova J, Buresova L, Zackova D, Palackova
M, et al. Mechanism of impaired glucose metabolism during
nilotinib therapy in patients with chronic myelogenous
leukemia. Haematol Haematol 2013;98:e124-6.
7. Sharma GN, Dave R, Sanadya J, Sharma P, Sharma KK. Various
types and management of breast cancer: an overview. J Adv
Pharm Tech Res 2010;1:109.
8. Perou, Charles M, Therese Sørlie, Michael B Eisen, Matt Van De
Rijn, Stefanie S Jeffrey, Christian A Rees, et al. Molecular
portraits of human breast tumours. Nature 2000;406:747.
9. Hallek M, Cheson BD, Catovsky D, Caligaris Cappio F, Dighiero
G, Dohner H, et al. Guidelines for the diagnosis and treatment of
chronic lymphocytic leukemia: a report from the international
workshop on chronic lymphocytic leukemia updating the
Pharmacokinetic and pharmacodynamic profiling of the drug is one
of the most important and critical Parameters; beginning with the
site of administration, absorption into the systemic circulation,
gesticulation in blood, elimination through urine. ADMET profiling of
the synthesized triazine derivatives has been calculated with the
help of online software database namely admet SAR. There are
diverse parameters for determining the permeability of the drugs
through various body barriers such as blood-brain barrier (BBB),
human intestinal absorption, Caco-2 cell permeation, absorption in
the intestine, renal organic cation transport and AMES toxicity test.
P-glycoprotein is the membrane efflux transporter which plays a
critical role in the determination of pharmacokinetic [ADMEandT]
profile of some therapeutic agents and in the multidrug resistance
(MDR) phenomena. Recognition of the P-glycoprotein substrates is
an important process in the early stage of the drug discovery.
Cytochrome-P enzyme family (CYPs) is the essential enzyme which
plays a critical role in detoxification of drugs and the biosynthesis of
endogenous molecules. The direct or indirect correlation of the
carcinogenicity of therapeutic agents with the properties of the
molecule is an important feature in the pharmacokinetic profiling of
the drugs. The results of carcinogenicity, mutagenicity from the
admetSAR software have been listed in table 4. The triazine
derivatives might be able to pass through the human intestinal
barrier and could be absorbed from the intestine. Out of all the
synthesized triazine derivatives, majority of the compounds do not
show any kind of toxicity or mutagenicity.
national
cancer
institute–working
group
1996
guidelines. Blood 2008;111:5446-56.
10. Else M, Ruchlemer R, Osuji N, Del Giudice I, Matutes E,
Woodman A, et al. Long remissions in hairy cell leukemia with
purine analogs: a report of 219 patients with a median
follow-up of 12.5 y. Cancer Interdisciplinary Int
Soc 2005;104:2442-8.
J Am
11. Wood ER, Truesdale AT, McDonald OB, Yuan D, Hassell A,
Dickerson SH, et al. A unique structure for epidermal growth
factor receptor bound to GW572016 (Lapatinib): relationships
among protein conformation, inhibitor off-rate, and receptor
activity in tumor cells. Can Res 2004;64:6652-9.
12. Campbell P, Morton PE, Takeichi T, Salam A, Roberts N,
Proudfoot LE, et al. Epithelial inflammation resulting from an
inherited loss-of-function mutation in EGFR. J Inves Derm
2014;134:2570-8.
13. Yun UJ, Sung JY, Park SY, Ye SK, Shim J, Lee JS, et al. Oncogenic
role of rab escort protein 1 through EGFR and STAT3
pathway. Cell Death Dis 2017;8:e2621.
14. Zwick E, Hackel PO, Prenzel N, Ullrich A. The EGF receptor as
central transducer of heterologous signalling systems. Trends
Pharm Sci 1999;20:408-12.
15. Aswar UM, Kalshetti PP, Shelke SM, Bhosale SH, Bodhankar SL.
Effect of newly synthesized 1, 2, 4-triazino [5, 6-b] indole-3-
thione derivatives on olfactory bulbectomy induced depression
in rats. Asi Pac J Trop Biomed 2012;2:992-8.
CONCLUSION
A series of novel triazine derivatives were synthesized and chemical
structure of all analogs was confirmed by IR, 1H-NMR and mass
spectroscopy. The compounds were evaluated for anticancer activity
using MCF-7 and K-562 cell lines. The compound MA-7, MA-8, MA-
12, MA-13 and MA-14 were found most active from the series
against different cell lines. Further, synthesized compounds were
docked on the crystal structure of EGFR receptor to identify the
binding mode of interaction. Docking interaction and score supports
triazine derivative as potent anticancer agents. Moreover, all
compounds were subjected to in silico ADMET prediction using
admetSAR online tool which suggest that all molecules from the
series have appropriate ADMET properties to become a potential
drug candidate. The results of a computational study and in vitro
study strongly reveals that the synthesized compounds may be used
for lead optimization for development of novel anticancer agents
with a high degree of selectivity and specificity towards cancer cells.
16. Babu TMC, Rajesh SS, Bhaskar BV, Devi S, Rammohan A,
Sivaraman T, et al. Molecular docking, molecular dynamics
simulation, biological evaluation and 2D QSAR analysis of
flavonoids from syzygium alternifolium as potent anti-
helicobacter pylori agents. RSC Adv 2017;7:18277-92.
17. Noolvi MN, Patel HM. A comparative QSAR analysis and
molecular docking studies of quinazoline derivatives as
tyrosine kinase (EGFR) inhibitors: A rational approach to
anticancer drug design. J Saudi Chem Soc 2013;17:361-79.
18. Vichai V, Kirtikara K. Sulforhodamine B colorimetric assay for
cytotoxicity screening. Nat Protoc 2006;1:1112.
ACKNOWLEDGMENT
We Thank ‘Advanced Centre for Treatment, Research and Education in
Cancer (ACTREC), Mumbai’ for assistance with the cell line studies,
Centre of Food Testing, Bharati Vidyapeeth (Deemed to Be University),
Pune for providing LC-MS Services.
19. Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica
D, et al. New colorimetric cytotoxicity assay for anticancer-drug
screening. JNCI: J Nat Can Ins 1990;82:1107-12.
20. Variya B, Modi S, Savjani J, Patel S. In silico molecular docking
and pharmacokinetic prediction of gallic acid derivatives as
PPAR-γ agonists. Int J Pharm Pharml Sci 2017;9:102-7.
21. Shinde MG, Modi SJ, Kulkarni VM. Synthesis, pharmacological
evaluation, molecular docking and in silico ADMET prediction
of nitric oxide releasing biphenyls as anti-inflammatory
agents. J Appl Pharm Sci 2017;7:37-47.
CONTRIBUTIONS
All the authors have contributed equally.
CONFLICT OF INTERESTS
There is no conflict of interest pertaining to this manuscript
REFERENCES
1. Subrahmanyam V, Vanita P, Jhansi K. A short note on cancer. J
Carcinog Mutagen 2011;2:128.
121