P. Pathak et al.
capillaries occurs throughout the surroundings and acceler-
ate the growth of tumor beyond 2–3 mm size. In this process,
vascular endothelial growth factor (VEGF) pathways play an
important role in their growth and metastatic potential (Folk-
man 2006; Yang et al. 2013). Thus, its inhibition is a prime
target for various agents (Bjerkvig et al. 2009). In cancer
biology, clinical applications of novel angiogenic inhibitors
are limited due to modest eꢁciency and resistance (Bergers
and Benjamin 2003; Folkman 2006; Nishida et al. 2006).
Biological applications of quinazoline and its derivatives
identiꢀed through large no. of scientiꢀc communication dur-
ing the second half of the twentieth century. Whereas second
heterocycle 1,3,5-triazine and its derivatives reported as a
potent skeleton for various therapeutic targets. Mono-, di- or
tri-amino substituted 1,3,5-triazine conjugate such as treta-
mine, furazil and dioxadet have been reported as anticancer
agents (Inomata et al. 2004). These therapeutic applications
give us the brief idea that both of the nuclei can be utilized
nicely towards designing of the novel anticancer agent. Thus,
we adopted these as a signiꢀcant factor and attempted to
design, synthesis, docking, in vitro anticancer and antian-
giogenic evaluation on cancer-induced chick embryo via the
modiꢀcation of quinazoline nucleus with 1,3,5-triazine. In
the present work, designing of derivatives done on the basis
of molecular ꢀeld mapping, alignment and further compared
with the standard angiogenic inhibitor vandetanib. The
designed quinazoline clubbed 1,3,5-triazine (QCT) deriva-
tives synthesized through cost-eꢂective methodology and
docking calculation is done on VGFR2 protein model. The
biological evaluation performed using MTT assay followed
by cancer-induced angiogenesis inhibition in chick chorioal-
lantoic membrane model (CAM).
In vitro anticancer assay (cytotoxic study)
The QCT derivatives were the screen for their anticancer activity
on four diꢂerent cancer cell line HeLa (human cervical cancer),
MCF-7 (human breast cancer cell), HL-60 (human promyelo-
cytic leukemia cell), HepG2 (human hepatocellular carcinoma
cell), and one normal cell line HFF (human foreskin ꢀbroblasts).
Cell culture
HeLa, MCF-7, HL-60, HepG2, and HFF cell lines were main-
tained in monolayer cultures in supplemented Dulbecco’s
modiꢀed Eagle’s medium (DMEM) with 10% heat-inactivated
fetal bovine serum (FBS), 1% l-glutamine, and 50 µg/ml of
gentamycin sulfate, at 37 °C, in CO incubator in an atmos-
2
phere of humidiꢀed 5% CO and 95% air (Wang et al. 2011).
2
IC value determination
5
0
Cells were seeded into 96-well microtiter plates. Each well was
5
ꢀlled with 200 ll of cell suspension (10 cells/well) and cultured
in DMEM, 10% FBS, 1% l-glutamine, and 50 µg/ml gentamycin
sulfate. The cells were incubated for 12 and 72 h with diꢂerent
concentrations of reference drug vandetanib and synthesized
QCT derivatives. Untreated control cells were also included for
each sample, which was maintained in DMEM and 5% FBS.
The ꢀnal concentration of DMSO used to solubilize synthe-
sized derivatives and 0.1%, similar amount added to the control
cells. The ꢀnal concentration of ethanol was used to dissolve
standard and 0.01%; a similar amount was added to the control
cells. Cytotoxicity calculation of all derivatives and standard
were determined using MTT (3-[4,5-dimethylthiazol-2-y1]-
2
,5-diphenyltetrazolium\ bromide) (Sigma) assay. The assay for
selected concentration of sample (10–100 µg/ml for initial trial;
10–30 µg/ml in the second trial; 1–30 µg/ml in the third trial) was
performed in triplicate, and the culture plates were kept in 5%
Experimental
Commercially available analytical grade solvents and rea-
gents were used for the experiment without further puriꢀca-
tion. Melting points were determined by Veego, MPI melt-
(v/v) CO humidiꢀed incubator at 37 °C for 24 and 72 h. Follow-
2
ing that, 20 µl of 0.5% w/v MTT, dissolved in phosphate-buꢂered
(PBS) saline, was added to each well and further incubated for
4 h with 100 µl of DMSO was added to each well and mixed
vigorously to dissolve the formazan crystals. Absorbance values
were determined using MRX II ELISA reader at 540 nm. All
calculations were carried out in triplicates (Ahmadian et al. 2009;
Patel and Patel 2011).
−
1
ing point apparatus and uncorrected. FTIR (2.0 cm , ꢃat,
smooth, abex) were recorded on Perkin Elmer RX-I Spectro-
1
13
photometer. H-NMR and C-NMR spectra were recorded
in DMSO-d using Bruker Avance II 400 and Bruker Avance
6
II 100 NMR spectrometer, respectively, using TMS as an
internal standard. Mass spectra were obtained on VG-
AUTOSPEC spectrometer equipped with electrospray ioni-
zation (ESI) sources. Elemental analysis was carried out on
Vario EL-III CHNOS elemental analyzer.
In ovo antiangiogenic activity
on cancer‑induced CAM (Grodzik et al. 2011)
Preparation of egg for xenograft
Chemistry
Fertilized eggs (204 nos.) from a local commercial
hatchery guaranteeing 95% fertilization of the eggs were
The whole synthesis presented in the supplementary ꢀle.
1
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Pathak, Prateek
