Design and synthesis of VEGFR-2 tyrosine kinase inhibitors as potential anticancer agents by virtual based screening

Article abstract of DOI:10.1039/c5ra05277g

Vascular endothelial growth factor receptor-2 (VEGFR-2) plays a crucial role in cancer angiogenesis. A library of 6,7-dimethoxy quinazoline was prepared using a ligand based drug design approach and passed through different filters of virtual screening su

Full text of DOI:10.1039/c5ra05277g

View Article Online
View Journal
RSC Advances
This article can be cited before page numbers have been issued, to do this please use: H. Patel, P. Bari, R.
Karpoormath, M. noolvi, N. Thapliyal, S. Surana and P. Jain, RSC Adv., 2015, DOI: 10.1039/C5RA05277G.
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. This Accepted Manuscript will be replaced by the edited,
formatted and paginated article as soon as this is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the Ethical guidelines still
apply. In no event shall the Royal Society of Chemistry be held
responsible for any errors or omissions in this Accepted Manuscript
or any consequences arising from the use of any information it
contains.
www.rsc.org/advances

Page 1 of 71
Please Rd So Cn oA tda vd aj un sc te ms argins
View Article Online
DOI: 10.1039/C5RA05277G
RSC Advances
ARTICLE
Design and Synthesis of VEGFR-2 Tyrosine Kinase
Inhibitors as Potential Anticancer Agents by Virtual
Based Screening
Received 00th January 20xx,
Accepted 00th January 20xx
†
‡
†
§
DOI: 10.1039/x0xx00000x
Harun M. Patel , Pankaj Bari , Rajshekhar Karpoormath , Malleshappa Noolvi , Neeta
†
‡
‡, *
Thapliyal , Sanjay Surana , Pritam Jain
www.rsc.org/advances
Abstract
Vascular endothelial growth factor receptor-2 (VEGFR-2) plays a crucial role in cancer angiogenesis. A
library of 6,7-dimethoxy quinazoline was prepared using ligand based drug design approach and passed
through different filters of virtual screening such as docking study and Lipinski’s rule. Twenty virtually
screened compounds were synthesized and investigated against VEGFR-2 kinase and human umbilical vein
endothelial cells (HUVEC) in vitro. Virtually screened compound 47 having 4-chlorophenyl-1,3,4-thiadiazole
rd
substitution at 3 position of 6,7-dimethoxy-2-phenylquinazolin-4(3H)-one exhibited the most promising
activity, with IC50 value of 3.8 nm and 5.5 nm against VEGFR-2 tyrosine kinase and HUVEC cell line.
Docking simulation supported the initial pharmacophoric hypothesis and suggested a common mode of
interaction at the ATP-binding site of VEGFR-2 demonstrating that compound 47 was a potential agent for
cancer therapy that deserves further research.
Keywords: 6,7-dimethoxy quinazoline, virtual screening, pharmacophore mapping, docking
simulation,VEGFR-2.
Corrosponding Author: P. S. Jain, E-mail: pritash79@yahoo.com, Tel. No. : +91-2563-255189, Fax No.: +91-2563-251808
†
Dept. of Pharmaceutical Chemistry, University of KwaZulu-Natal (Westville
Campus), Private Bag X54001, Durban- 4000, South Africa.
‡
Department of Pharmaceutical Chemistry, R.C. Patel College of Pharmacy, Shirpur,
Dhule 425405, Maharashtra, India
§
Department of Pharmaceutical Chemistry, Shree Dhanvantary Pharmacy College,
Kim (Surat)-3941110, Gujrat, India
RSC Advances © The Royal Society of Chemistry 20xx
RSC Advances, 2015, 00, 1-3 | 1
Please do not adjust margins

Please Rd So Cn oA tda vd aj un sc te ms argins
Page 2 of 71
ARTICLE
. Introduction
RSC Advances
1
View Article Online
DOI: 10.1039/C5RA05277G
Cancer management is an extremely challenging field for medicinal chemists to discover effective yet safer chemotherapeutic agents
1
targeting various biochemical processes involved in progression of different kinds of cancers. Among these targets, angiogenesis is one of
the critical processes that affect growth and development of cancerous cells. Angiogenesis refers to generation of new blood vessels from the
existing vasculature. It is the key factor in advancement of various human diseases, including cancer, where it is essential for the growth,
2
spread and survival of tumors. Angiogenesis is a complex process regulated by multiple growth factors and cytokines. Among these
factors, vascular endothelial growth factor (VEGF) is one of the most potent angiogenic factors involved in tumor growth. It
stimulates endothelial cell proliferation, migration and tube formation by binding to its two main receptor tyrosine kinases (RTKs)
3
expressed on endothelial cells, VEGF receptor 1 (VEGFR-1) and VEGF receptor 2 (VEGFR-2). Current evidence suggests that the
interaction between VEGF and VEGFR-1 plays a minor role in angiogenesis, while VEGFR-2 mediates the major angiogenic
4
function of VEGF . Therefore, VEGF and VEGFR-2 have become therapeutic targets for the development of anticancer agents. Inhibition
of the VEGFR-2 signalling pathway has an important anti-angiogenic effect on human cancer, which is evident from the approval of the
4
5
6
small-molecule VEGFR-2 kinase inhibitors sorafenib (Bay 43-9006) [112], sunitinib (SU-11248), and pazopanib (GW786034) by the
7
Food and Drug Administration (FDA) for the treatment of advanced renal cell carcinoma. Presently, many anti-angiogenic and multikinase
8
9
agents are undergoing phase III clinical studies, including cediranib (AZD-2171) [16], brivanib (BMS-582664), axitinib (AG013736)
1
0
11
12
[
18] , tivozanib (KRN-951) [19] , and vandetanib (ZD-6474) [20].
During the course of pharmaceutical development of novel VEGFR2 inhibitors, pharmacophore and docking based in silico studies are
efficiently used to improve the discovery of lead identification and optimization, which is followed by the synthesis of lead compound
derivatives and their biological evaluation. The discovery of pazopanib is an example of virtual screening using homology models and
1
3
pharmacophore modeling.
Inspired by this, in the current study, a library of quinazoline analogues was prepared using ligand based approach and passed through the
different filters of virtual screening. Virtually screened hits were then synthesized and evaluated for their inhibitory activities against
VEGFR-2 tyrosine kinase and VEGF-stimulated proliferation of HUVEC.
2
. Virtual Screening Protocol
The virtual screening protocol used in this study is based on the application of sequential filters in order to select a restricted number of
compounds to be submitted for biological evaluation. In the present study, both ligand based and structure based virtual screening approaches
have been used. The workflow of the virtual screening campaign is outlined in Fig. 1. In detail, (i) a structure-based 3D pharmacophore
model was optimized; (ii) a library of quinazoline analogs was made based upon the optimized pharmacophore; (iii) a structure-based 3D
pharmacophore model was used as a search query on the quinazoline analogs (library), retaining the molecules that adhered to all the features
of the model; (iii) the binding mode of all retrieved compounds was evaluated by molecular docking, using the 3D structure of VEGFR-2
tyrosine kinase; (iv) The next filter was Lipinski’s rule of five to evaluate drug likeness, which becomes an essential tool to facilitate drug
discovery. Finally, the virtually screened hits were synthesized and evaluated for their inhibitory activities against VEGFR-2 kinase and
VEGF-stimulated proliferation of HUVEC.
2
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 3 of 71
Please Rd So Cn oA tda vd aj un sc te ms argins
Journal Name
.1 Software and Hardware
ARTICLE
2
View Article Online
DOI: 10.1039/C5RA05277G
Ligand based approach was carried out by the pharmacophore 3D-QSAR study using PHASE, version 3.0, Schrodinger, LLC, New York,
1
4
USA, 2008. PHASE supports various ligand-based drug design approaches like pharmacophore perception, structure alignment, 3D-QSAR
1
5
16
and database searching. Energy minimization of the dataset structures was accomplished using Macromodel with OPLS 2005 force field.
The minimized structures were imported in PHASE and appropriate protonation states were assigned to them at physiological pH 7.2 ± 2.0
1
7
by Ligprep. Different conformations were then generated using Confgen with OPLS 2001 force field using distance dependent dielectric
1
8
solvation treatment. Default pharmacophore features in PHASE include hydrogen bond acceptor (A), hydrogen bond donor (D),
hydrophobic (H), negative (N), positive (P) and aromatic ring (R). It determines how molecular structure affects drug activity by dividing
space into a fine cubic grid, encoding atom type occupation as numerical information, and performing a partial least-squares (PLS)
1
5
regression, resulting in prediction of a significant model. Structure based virtual screening was conducted using a graphical user interface
1
9
XP-docking mode of program Maestro 8. The protein structure of a complex VEGFR-2 was obtained from the RCSB Protein Data Bank
2
0
(
PDB) as entry 3B8Q (http://www.rcsb.org/pdb/explore/explore.3B8Q). The protein was optimized for docking from its raw state
2
1
employing protein preparation wizard with OPLS 2005 force field for minimization. Receptor gird generation was accomplished using
Glide. Further, we analyzed the compounds for Lipinski’s rule of five to evaluate drug likeness using QikProp.
2
.2 Ligand Based Drug Design
2
2
The novel 50 quinazoline derivatives were taken from literature to derive pharmacophore based 3D QSAR model. The IC50 values were
converted to pIC50 using the formula (pIC50 = -log IC ). Compounds that displayed insignificant or no inhibitions were excluded from the
50
present study. The structures of all the compounds along with their actual and predicted biological activities are presented in Table 1. Three-
dimensional (3D) conversion and minimization of 50 quinazoline ligands were performed using LigPrep (MMFFs force field) incorporated
in PHASE. Conformers were generated using a rapid torsion angle search approach followed by minimization of each generated structure
using the MMFFs force field, with an implicit GB/SA solvent model. A maximum of 1,000 conformers were generated per structure using a
pre-process minimization of 1,000 steps and post process minimization of 500 steps. Each minimized conformer was filtered through the
−1
23
−1
relative energy window of 50 kJ mol and the minimum atom deviation of 1.00 Å. This value (50 kJ mol ) sets an energy threshold with
respect to the lowest-energy conformer. Conformers having energy higher than the threshold are discarded. In order for two conformers to be
considered identical, the distance between pairs of corresponding heavy atoms must be below 1.00 Å. This rule is applied after the energy
−1
difference threshold, and only if the two conformers are within 1 kcal mol of each other. The total set of inhibitors was divided randomly
into a training set of 35 compounds for generation of 3D-QSAR models, and a test set of 15 compounds for validation of the developed
model. The training set molecules were selected in such a way that they contained information in terms of both their structural features and
2
4
biological activity range. The most active, moderately active and less active molecules were included, to spread out the range of activities.
Partial least-squares (PLS) regression analysis was applied to obtain the QSAR model. The maximum number of PLS factors were 5.
PHASE QSAR models do not use internal cross-validation techniques, but rather use distinct training and test sets. PHASE supports only
external validation, using an actual test set whose structures and activities are not considered when QSAR models are developed. Each of the
2
developed 3D-QSAR models was validated by predicting activities of 15 test set molecules (q ). The predictive ability of the models was
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
Please do not adjust margins

Please Rd So Cn oA tda vd aj un sc te ms argins
Page 4 of 71
ARTICLE
RSC Advances
measured by Pearson-R value. To overcome the over-fitting problem, the run was performed using 1-5 PLS factors, in whicVhietwhAertsictlaenOdnalirnde
DOI: 10.1039/C5RA05277G
deviation of regression was approximately equal to the experimental error. The stability value was used to check the strength of the resulting
3
D QSAR model and compare models from the different hypotheses. The training set was used to identify the common pharmacophore
hypothesis (CPH) by following tree-based partition algorithms. For finding the CPH, the dataset was divided into active and inactive sets
depending upon the observed activity; active ligands are those with IC50 below 1.7 nm and inactive above 1.7 nm. Based on sites, maximum
five features were allowed to develop hypotheses and a number of CPHs were reported that were common in all 50 molecules. Among the
1
47 hypotheses developed, five of them were selected for molecular alignment based upon the survival score. PLS analysis was conducted
using five factors with a grid spacing of 1 Å and five regression models were derived. The pharmacophore hypothesis labelled as Models 1-
2
2
5
, together with their statistical scores, is listed in Table 2. Based on r , q , SD and RMSE, as well as on the highest value on the Pearson-R,
2
2
Model-I was found to be the best model (r = 0.9687, q = 0.7106, F =148.6). The graph of observed versus predicted biological activity of
training and test sets are shown in Fig. 2. The pharmacophore model hypothesis distances and angles are depicted in Fig. 3 and 4,
respectively. Table 3 displayed the alignment of active compounds in accordance with the hypothesis. For Model-I, the training set
2
correlation is characterized by PLS factors (r = 0.9687, SD = 0.1969, F = 148.6, P = 0.8709). The test set correlation is characterized by PLS
2
factors (q = 0.7106, RMSE = 0.4487, Pearson-R = 0.8709). The contribution maps obtained from Model-I AAARR.8 show how 3D-QSAR
methods can identify features important for the interaction between ligands and their target protein.
2
.3 Designing of the Library based upon Ligand based Strategy
The library was designed based on the developed 3D-QSAR models and earlier reported work on quinazoline based inhibitors of VEGFR-2.
2
Among them, Model-I was found to be significantly more accurate, characterized by PLS factors (r = 0.9687, SD = 0.1969, F = 148.6, P =
2
0
.8709) and the test set correlation is characterized by PLS factors (q = 0.7106, RMSE = 0.4487, Pearson-R = 0.8709). The resulting 3D-
QSAR contour maps provide useful insights in active-structure relationship, allowing a discussion in terms of drug design. N-1, 6 and 7
alkoxy oxygen of quinazoline ring served as hydrogen bond acceptor (A1, A3 and A4 respectively) in drug receptor interaction. Ring
residue, R13, in this model occupies much of the favourable blue cubes due to the presence of hydrophobic quinazoline ring. Similarly, ring
residues, R15, suggest that bulky substituent is essential for producing VEGFR-2 kinase inhibition as shown in Fig. 3 and 4.
Our strategy is directed towards designing a variety of ligands with diverse chemical properties as per the developed 3D QSAR
pharmacophore model hypothesizing that the potency of these molecules might be enhanced by adding an alternative binding group
nd
rd
such as phenyl at 2 position, and substituted thiadiazoles, oxadiazoles, different amines and substituted hydrazides at 3 position of the
,7-dimethoxy quinazoline ring. In this way, such a substitution pattern could target various regions of the ATP-binding site of the
6
protein kinase domain to create differentially selective molecules. Based upon pharmacophore based 3D-QSAR and literature survey of
VEGFR-2 inhibitors, we designed the library of 75 compounds as shown in Fig. 5, 6(A), 6(B) and 6(C).
2
.4 Structure Based Drug Design
2
5
The molecular docking tool, GLIDE was used for ligand docking studies into the VEGFR-2 tyrosine kinase receptor binding pocket. The
crystal structure of VEGFR-2 tyrosine kinase was obtained from the protein data bank, PDB ID: 3B8Q. The protein preparation was carried
out using ‘protein preparation wizard’ in Maestro 8.0 in two steps, preparation and refinement. After ensuring chemical correctness, water
4
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 5 of 71
Please Rd So Cn oA tda vd aj un sc te ms argins
Journal Name
ARTICLE
molecules in the crystal structures were deleted, and hydrogens were added, wherever they were missing. The energy of crystalVisetwruAcrtticulereOnwlinaes
DOI: 10.1039/C5RA05277G
2
6
minimized. Grids were defined centering them on the ligand in the crystal structure using the default box size. The ligands were developed
using maestro build panel and prepared by Ligprep 2.2 module that produces the low energy conformer of ligands using OPLS 2005 force
2
7
field. The low energy conformation of the ligands was selected and docked into the grid generated from protein structures using standard
precision (SP) docking mode. The final evaluation was done with glide score (docking score), and a single best pose is generated as the
output for a particular ligand. 6,7-dimethoxy quinazoline analogues were modeled by positioning them in the co-crystallized ligand’s binding
site. The entire complex was subjected to alternate cycles of minimization and dynamics. The co-crystallized ligand was re-docked into the
active site of the enzyme and then replaced with 6,7-dimethoxy quinazoline derivatives in order to compare the binding mode of both co-
crystallized ligand and the compounds under investigation.
2
.5 Lipinski’s Rule for Drug Likeliness
Pharmacokinetic property optimization is a rather complex undertaking that is likely to require changes in those molecular determinants that
are responsible for binding affinity and specificity like hydrogen bonds. It is well known that numerous drug candidates have failed during
clinical tests because of problems related to ADME (absorption, distribution, metabolism and excretion) properties. We analyzed physically
significant descriptors and pharmaceutically relevant properties such as molecular weight, log p, H-bond donors and H-bond acceptors of all
the synthesized compounds, according to Lipinski’s rule of five. Hydrogen bond acceptor (HBA) and hydrogen bond donor (HBD) groups in
the compound optimize the drug receptor interaction. Lipinski’s rule of five is 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 most likely make it an orally active drug
in humans. The rule describes delicate balance between the molecular properties of a compound, which directly influence its
pharmacodynamics and pharmacokinetics, and ultimately affect their ADME in the human body like a drug. In general, these parameters
allow to ascertain poor oral absorption or membrane permeability that occurs when the evaluated molecules present values higher than five
2
8
H-bond donors (HBD), 10 H-bond acceptors (HBA), molecular weight (MW) > 500 Da and LogP (cLogP) > 5 (Lipinski’s ‘rule-of-five’).
We also evaluated the number of violations of Lipinski’s rule of five. Compounds that satisfy these rules are considered as drug like.
Compounds with fewer (and preferably no) violations of these rules are more likely to be orally available.
3
. Chemistry
The synthesis of virtually screened 6,7-dimethoxy quinazoline analogues was achieved through an efficient and versatile synthetic route, as
illustrated in Scheme 1 and 2. It is quite clear that unique final steps were involved in the synthesis of target compounds, having structural
rd
variations at the 3 position of 6,7-dimethoxy quinazoline ring. Reaction of equimolar quantity of 4,5-dimethoxy anthranilic acid (A) with
benzoyl chloride (B) in dry pyridine yielded 6,7-dimethoxy-2-phenyl-4H-benzo[d][1,3]oxazin-4-one(C) by N-benzoylation via dehydrative
cyclization mechanism. Subsequently, 6,7-dimethoxy-2-phenyl-4H-benzo[d][1,3]oxazin-4-one(C) reacted with different substituted primary
amines containing oxadiazole/ thiadiazoles/ anilines in dry pyridine to obtain the virtually screened compounds as shown in Scheme 1 and
rd
2
. Benzoxazinones undergo ring opening with different nucleophiles, allowing incorporation of substitution at the 3 position. Hence, the use
of dry pyridine and maintenance of anhydrous condition is mandatory, while synthesizing the above-mentioned analogues. It is also reported
2
9
that the benzoxazinones are liable to hydrolysis by water. The actual extent to which this hydrolysis occurs varies greatly across a range of
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 5
Please do not adjust margins

Please Rd So Cn oA tda vd aj un sc te ms argins
Page 6 of 71
ARTICLE
RSC Advances
molecules. A molecule of water opens the benzoxazinone ring by attacking the intracyclic carbonyl and effectively hydrolyzViinewgAtrhtieclecOyncllinice
DOI: 10.1039/C5RA05277G
2
9
ester (Fig. 7). Hence, the pyridine should be completely dry.
It is also suggested that the nature of the substitution on the benzoxazinone can modulate the reactivity of the carbonyl, such as the electron
3
0
donating groups cause the carbonyl to be less electrophilic and reduce the reactivity of the benzoxazinone carbonyl to nucleophillic attack.
This is desirable in terms of stability on storage, or if they are to be the final molecules themselves, it also being a factor when these
molecules are altered for further analogue production.
An alkyl chain act as an electron donor (+I effect), suggesting that the longer the chain gets, the reaction and insertion of the group that
rd
constitute the substitution at the 3 position of the corresponding 6,7-dimethoxy quinazoline becomes more difficult (Fig. 8).
All the newly synthesized compounds exhibited acceptable analysis of their anticipated structures, which have been summarized in
experimental section. In general, the IR spectrum of all virtually screened compounds revealed typical absorption bands around 1640-1685
-
1
th
-1
cm for C=O (of 6,7-dimethoxy quinazoline at 4 position) and 1531-1563 cm for C=N, which confirms the formation of 6,7-dimethoxy
1
quinazoline ring. H NMR spectrum of virtually screened compounds exposed the characteristic two methoxy peak at 3.91-3.92 δ ppm and
1
3
3
5
4
.93-3.98 δ ppm, which is magnetically different. This was further confirmed from C NMR spectrum that exhibited two methoxy peaks at
6.12δ ppm and 56.41δ ppm. The mass spectra of these compounds additionally confirmed the assigned structures.
. Results and Discussion
In the beginning, a reported series of 6,7-dimethoxy quinazoline derivatives with VEGFR-2 tyrosine kinase inhibitory activity was subjected
to a 3D-QSAR study. All the developed 3D-QSAR models showed good predictabilities and statistical validation. Model-I was significantly
2
more accurate than other models and was characterized by PLS factors (r = 0.9687, SD = 0.1969, F = 148.6, P = 0.8709). The test set
2
correlation was characterized by the PLS factors (q = 0.7106, RMSE = 0.4487, Pearson-R = 0.8709). The resulting 3D-QSAR contour maps
provide useful insights in active-structure relationship, allowing a discussion in terms of drug design. Nitrogen atom of 6,7-dimethoxy
quinazoline ring served as hydrogen bond acceptor (A1) in drug receptor interaction. Ring residue (R13) in this model occupies much of the
favourable blue cubes due to the presence of hydrophobic 6,7-dimethoxy quinazoline ring. Similarly ring residue (R15) suggests that bulky
substituent is essential for producing VEGFR-2 kinase inhibition. It is also inferred from the docking results that the bulky moiety is located
in a deep hydrophobic pocket. Hydrogen bond acceptor (A3 and A4) at 6th and 7th position of quinazoline ring indicates that electron
donating substituent methoxy is favourable for VEGFR-2 inhibition activity, as shown in Fig. 3 and 4.
As per the pharmacophore based 3D-QSAR and literature survey of VEGFR-2 inhibitors, we designed the library of 75 compounds.
Designed library was passed through the developed 3D-QSAR model to validate the designing. Among the 75 compounds, 51 compounds
showing significant predicted activity (IC50 below 5.17) were further selected for next filter of docking study, as shown in Table 4.
Docking studies revealed that the quinazoline ring binds to a narrow hydrophobic pocket in the N-terminal domain of VEGFR-2 tyrosine
kinase where N-1 of the quinazoline ring interacts with H-atom of amino acid backbone of CYS-919 via a hydrogen bond. These interactions
underscore the importance of both nitrogen atoms for binding and the subsequent inhibitory capacity. 6,7-dimethoxy quinazoline ring is
surrounded by the hydrophobic residue such as Val848, Lys868, Thr916, indicating its role in hydrophobic interaction, with the ring residue
(R13) in 3D QSAR model also suggesting the same. The thiadiazoles/ oxadiazole/ aniline moiety at C-3 position of 6,7-dimethoxy
6
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 7 of 71
Please Rd So Cn oA tda vd aj un sc te ms argins
Journal Name
ARTICLE
quinazoline is observed to be inserted deeply in the cavity, interacting with Leu840, Ala866, Phe918 and Phe1047 throughViehwyAdrtrioclpehOonbliniec
DOI: 10.1039/C5RA05277G
interaction, as shown in Fig. 9, 10 and 11 . The ring residue (R15) also confirms the hydrophobic interaction of thiadiazoles/ oxadiazole/
aniline moiety in the 3D-QSAR study. This deep cavity is very well conserved in all tyrosine kinase iso forms and coincides with the ATP-
binding site region. As a measure of docking reliability, the docking results are evaluated in terms of glide dock score values by comparison
of the docked poses of the co-crystallized ligand. Compounds having docking score above -5.77 are allowed for the next filter of lipnski’s
rule. Out of 51 compounds, 27 compounds show good docking score and glide energy, as depicted in Fig. 9, 10, 11 and Table 5.We further
analyzed virtually filtered 27 compounds for Lipinski’s rule of five. The rule describes molecular properties important for a drug’s
pharmacokinetics in the human body, including its ADME. Although the cytotoxic effects of lead compounds are thought to be primarily due
to their ability to modulate cell death, other factors such as solubility, stability and/ or efflux properties within the cell may also contribute.
The QikProp 3.2 is used to analyze drug likeness (Lipinski’s Rule of Five), and the results are given in Table 6. It was found that all the
synthesized compounds comply with these rules, except for 28, 31, 32, 44, 61, 70 and 72, which did not comply for molecular weight and
QPlogP O/W, showing violation of Lipinski’s Rule of Five.
Finally, 20 virtually screened compounds are further synthesized, as shown in Scheme 1 and 2. The reaction of 4,5-dimethoxy anthranilic
acid (A) and benzoyl chloride (B) in dry pyridine afforded 6,7-dimethoxy-2-phenyl-4H-benzo[d][1,3]oxazin-4-one (C) (Scheme 1 and 2). C
was refluxed with different substituted thiadiazoles, oxadiazoles and anilines containing primary amine to obtain the desired compounds.
While synthesizing virtually screened compounds, the quinazoline ring opened and two products, a 90% closed ring and a 10% open ring
product, i.e. diamides, were obtained, as shown in Scheme 3.
Following are some of the predictions and the reasons that probably caused this opening and closing of the ring. The substituents have a
dominant bearing on the reactivity of the benzoxazinone carbonyl and the electronic contribution, in particular, of the ring substituents seems
to dominate. The order of reactivity was observed to follow the trend of the electronic contribution of the substituents through either
induction, resonance or a combination of the two. Conversely, if there is an N-N type molecule acting as the nucleophile, the factor that
determines which nitrogen attacks is most probably the steric effect, rather than nucleophilicity. The least hindered nitrogen, rather than the
most nucleophiles, attacks the carbonyl. This information is valuable if a range of nitrogen nucleophiles are used in the formation of
quinazolines and may explain differing yields of final product. Thus, the benzoxazinone molecule was useful as there was potential to
introduce diversity into the system at specific points by simply changing the nature of the starting materials. It is shown that the molecule
undergoes ring opening with different nucleophiles allowing incorporation of substitution at the 3rd position. This in turn generates a number
of molecules for the SAR study. One of the main reasons that these molecules did not easily purify was due to their potential to degrade
when exposed to water. It is known that the benzoxazinones are liable to hydrolysis by water and the increase in stability of 2-
alkylbenzoxazinones is related to the length of the side chain. The actual extent to which this hydrolysis occurs varies greatly across a range
of molecules. A molecule of water opens the benzoxazinone ring by attacking the intracyclic carbonyl and effectively hydrolyzes the cyclic
ester as shown in Fig. 7.
All the virtually synthesized compounds were tested for VEGFR-2 kinase activity using homogeneous time resolved fluorescence (HTRF)
3
1
method. The catalytic activity of kinases was measured by phosphorylated biotin-peptide conjugate using streptavidin linked-APC and
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 7
Please do not adjust margins

Please Rd So Cn oA tda vd aj un sc te ms argins
Page 8 of 71
ARTICLE
RSC Advances
europium-labelled anti-phosphotyrosine antibody. Subsequently, a cell proliferation assay was performed to find the potent VEVGiewFRAr-ti2clekOinnalisnee
DOI: 10.1039/C5RA05277G
inhibitors among these compounds for their ability to inhibit VEGF-stimulated proliferation of HUVEC 39, with the results being shown in
Table 7. Overall, considerable relationships between their structures and inhibitory activities were observed.
With respect to the VEGFR-2 inhibition assay, all compounds elicited moderate to significant kinase inhibition activity with IC50
values in the range of 3.8-124 nM. Regarding the SAR findings of 6,7-dimethoxy quinazoline analogues, it is observed that thiadiazole
derivatives are more potent as compared to the oxadiazole and aniline at 3rd position of 6,7-dimethoxy-2-phenylquinazolin-4(3H)-one.
Among the virtually screened compounds, compound 47 with 4-chlorophenyl-1,3,4-thiadiazole substitution at 3rd position of 6,7-dimethoxy-
2
-phenylquinazolin-4(3H)-one exhibited most promising activity, with IC50 value of 3.8 nM against VEGFR-2 tyrosine kinase and 5.5 nm
against the HUVEC cell line. Less bulky hydrophobic substitution on 5th position of 1,3,4-thiadiazole and 1,3,4-oxadiazole decreases the
activity (compound 21, 46), indicating that optimum hydrophobicity is required at 5th position to have VEGFR-2 inhibition activity.
Within set of 1,3,4-oxadiazole substituted quinazoline derivatives, it was found that presence of electron withdrawing group on phenyl ring
at 5th position of 1,3,4-oxadiazole increases the activity and vice versa, which is obvious by observing the VEGFR-2 inhibition of compound
2
2 (IC =12 nM, 4-Cl substituted) and 26 (IC =107 nM, 3-methyl substituted). By comparing the VEGFR-2 inhibition of compound 23
50 50
(
IC =15 nM) and 40 (IC =13.5 nM), it is concluded that para-methoxy is more active than ortho on phenyl ring at 5th position of 1,3,4-
50 50
oxadiazole and same thing is also observed with aniline series, where para-methoxy aniline (compound 10, IC =13.5 nM) is more active
5
0
than ortho-methoxy aniline (compound 2, IC =13.5 nM).
5
0
5
. Conclusion
In conclusion, virtually screened twenty 6,7-dimethoxy quinazoline derivatives were synthesized by ligand based and structured based drug
design approach. With respect to the VEGFR-2 inhibition assay, all compounds elicited moderate to significant kinase inhibition
activity with IC50 values in the range of 3.8-124 nM. Among the virtually screened compounds, compound 47 with 4-chlorophenyl-1,3,4-
thiadiazole substitution at 3rd position of 6,7-dimethoxy-2-phenylquinazolin-4(3H)-one exhibited most promising activity with IC50 value of
3
.8 nM against VEGFR-2 tyrosine kinase and 5.5 nm against the HUVEC cell line. Docking studies revealed that the quinazoline ring binds
to a narrow hydrophobic pocket in the N-terminal domain of VEGFR-2 tyrosine kinase where N-1 of the quinazoline ring interacts with H-
atom of amino acid backbone of CYS-919 via a hydrogen bond. These interactions underscore the importance of both nitrogen atoms for
binding and the subsequent inhibitory capacity. Regarding the SAR findings of 6,7-dimethoxy quinazoline analogues, it is seen that
thiadiazole derivatives are more potent as compared to the oxadiazole and aniline at 3rd position of 6,7-dimethoxy-2-phenylquinazolin-
4
(3H)-one. The overall outcome of this model revealed that optimum hydrophobicity is required at 5th position to have VEGFR-2 inhibitory
activity. Within set of 1,3,4-oxadiazole substituted quinazoline derivatives, it was found that presence of electron withdrawing group on
phenyl ring at 5th position of 1,3,4-oxadiazole increases the activity and vice versa. These encouraging results of biological screening of the
tested compounds offer an excellent framework in this field that may lead to discovery of potent VEGFR-2 inhibitors. Finally, it is
conceivable that further derivatization of such compounds will be of interest with the hope to get more selective and potentVEGFR-2
inhibitors.
8
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 9 of 71
Please Rd So Cn oA tda vd aj un sc te ms argins
Journal Name
. Experimental
ARTICLE
6
View Article Online
DOI: 10.1039/C5RA05277G
All the chemicals and solvents were supplied by Sigma-Aldrich and Spectrochem Pvt. Ltd. Solvents were distilled and dried before use as
required. The reactions were monitored with the help of thin-layer chromatography using pre-coated aluminium sheets with GF254 silica gel,
0
.2 mm layer thickness (Merck) by using solvent systems benzene : acetone (7:3 and 9:1) and toluene: ethyl acetate: formic acid (5:4:1). The
spots were visualized under UV lamp. Melting points of the synthesized compounds were determined and are uncorrected using one end
open capillary tubes on a scientific melting point apparatus, Analab Scintific Instruments. FTIR spectrum was recorded using KBr on FTIR-
1
8
400S Shimadzu spectrometer. HNMR (DMSO) spectra of the synthesized compounds were performed with BrukerAvance-II 400 NMR
spectrometer operating at 400 MHz in SAIF, Punjab University, Chandigarh. Chemical shifts were measured relative to internal standard
TMS and are reported in δ ppm. Mass spectra of the synthesized compounds were recorded at MAT 120 in SAIF, Punjab University.
6
.1 Synthesis of 6,7-dimethoxy-2-phenyl-4H-benzo[d][1,3]oxazin-4-one (C)
An equimolar quantity of 4,5-dimethoxy anthranilic acid (A) and benzoyl chloride (B) was stirred in dry pyridine at 0-5°C for 1 hr, to get the
solid product. Obtained solid was washed with dil. HCl to remove excess pyridine and with sodium bicarbonate solution to remove excess
3
2
benzoyl chloride. It was further recrystallized from ethanol to get the pure compound C.
.2 General procedure for the synthesis of virtually screened compounds
6
Equimolar quantity of 6,7-dimethoxy-2-phenyl-4H-benzo[d][1,3]oxazin-4-one(C) and different substituted primary amines containing
oxadiazole/ thiadiazoles/ anilines in dry pyridine was added slowly and reaction mixture was refluxed for 24 hrs. The mixture so obtained
was added to crushed ice and the separated precipitate was filtered off and washed with dil. HCl. Recrystallization was carried out from
ethanol. All the compounds were further purified by column chromatography using Benzene: Acetone (7:3) as eluant.
6
.2.1 6,7-dimethoxy-3-(2-methoxyphenyl)-2-phenylquinazolin-4(3H)-one (2)
-
1
This compound was prepared and purified as per the above mentioned procedure: yield 62 %; mp 288-291 °C. IR (KBr, ѵmax, cm ):
1
2
957.97 (CH), 1640.54 (C=O), 1545.12 (C=N); H NMR (DMSO-d6) δ ppm: 3.91-3.92 (s, magnetically different, 3H, OCH ), 3.93-3.98 (s,
3
1
3
magnetically different 3H, OCH ), 3.83 (s, 3H, OCH ), 6.98-8.09 (m, 11H, Ar-H); C NMR (DMSO-d6) δ ppm: 55.21, 56.12, 56.41,
3
3
1
1
6
06.43, 108.48, 114.36, 115.37, 116.38, 118.38, 125.29, 126.23, 128.67, 128.87, 128.92, 132.47, 143.56, 155.57, 156.47, 154.38, 156.58,
63.77; HRMS (EI) m/z calcd for C H N O : 388.1423; found: 388.1428.
2
3
20
2
4
.2.2 3-(4-chlorophenyl)-6,7-dimethoxy-2-phenylquinazolin-4(3H)-one (3)
-
This compound was prepared and purified as per the above mentioned procedure: yield 62 %; mp 288-291 °C. IR (KBr, ѵmax, cm
1
1
)
:2924.18(CH), 1673.30 (C=O), 1538.23 (C=N), 768.66 (C-Cl); H NMR (DMSO-d6) δ ppm: 3.91-3.92 (s, magnetically different, 3H,
1
3
OCH3), 3.93-3.98 (s, magnetically different 3H, OCH ) 7.02-8.09 (m, 11H, Ar-H); C NMR (DMSO-d6) δ ppm: 56.12, 56.41, 108.73,
3
1
09.47, 116.47, 125.13, 128.53, 128.90, 129.45, 130.14, 130.03, 131.00, 133.56, 146.24, 152.17, 153.56, 158.58, 163.26; HRMS (EI) m/z
calcd for C H ClN O : 392.0928; found: 392.0932
2
2
17
2
3
6
.2.3 6,7-dimethoxy-3-(4-methoxyphenyl)-2-phenylquinazolin-4(3H)-one (10)
-
1
This compound was prepared and purified as per the above mentioned procedure: yield 62 %; mp 288-291 °C. IR (KBr, ѵmax, cm ):2923.22
1
(
CH), 1672.34 (C=O), 1531.34 (C=N); H NMR (DMSO-d6) δ ppm: 3.91-3.92 (s, magnetically different, 3H, OCH ), 3.93-3.98 (s,
3
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 9
Please do not adjust margins

Please Rd So Cn oA tda vd aj un sc te ms argins
Page 10 of 71
ARTICLE
RSC Advances
1
3
magnetically different 3H, OCH ), 3.89 (s, 3H, OCH ), 6.70-7.88 (m, 11H, Ar-H); C NMR (DMSO-d6) δ ppm: 55.81, 56.12, 56.41,
3
3
View Article Online
DOI: 10.1039/C5RA05277G
1
08.45, 109.57, 115.47, 116.12, 126.48, 127.24, 128.62, 128.82, 128.90, 132.37, 146.46, 154.16, 156.11, 157.47, 158.53, 163.91; HRMS
(
EI) m/z calcd for C H N O : 388.1423; found: 388.1419
2
3
20
2
4
6
.2.4 6,7-dimethoxy-2,3-diphenylquinazolin-4(3H)-one (14)
-
1
This compound was prepared and purified as per the above mentioned procedure: yield 62 %; mp 288-291 °C. IR (KBr, ѵmax, cm ):2924.18
1
(
CH), 1663.78 (C=O), 1552.12 (C=N); H NMR (DMSO-d6) δ ppm: 3.91-3.92 (s, magnetically different, 3H, OCH ), 3.93-3.98 (s,
3
1
3
magnetically different 3H, OCH ) 7.14-8.28 (m, 12H, Ar-H); C NMR (DMSO-d6) δ ppm: 56.12, 56.41, 107.62 108.45, 116.47, 124.67,
3
1
3
6
25.35, 126.35, 127.16, 128.34, 128.48, 131.47, 132.46, 146.24, 154.08, 155.12, 156.46, 163.48; HRMS (EI) m/z calcd for C H N O :
22 18 2 3
58.1317; found: 358.1322.
.2.5 6,7-dimethoxy-3-(5-methyl-1,3,4-oxadiazol-2-yl)-2-phenylquinazolin-4(3H)-one (21)
-
1
This compound was prepared and purified as per the above mentioned procedure: yield 62 %; mp 288-291 °C. IR (KBr, ѵmax, cm ):2978.63
1
(
Aro-CH), 2924.18 (Ali-CH), 1685.69 (C=O), 1561.24 (C=N); H NMR (DMSO-d6) δ ppm: 2.61 (s, 3H, CH ), 3.91-3.92 (s, magnetically
3
1
3
different, 3H, OCH ), 3.93-3.98 (s, magnetically different 3H, OCH ) 7.12-8.24 (m, 11H, Ar-H); C NMR (DMSO-d6) δ ppm: 20.23, 56.12,
3
3
5
6.41, 109.57, 110.58, 116.58, 128.21, 128.45, 128.72, 130.56, 146.45, 154.18, 155.42, 156.43, 163.56, 164.87, 170.64; HRMS (EI) m/z
calcd for C H N O : 364.1172; found: 364.1176
1
9
16
4
4
6
.2.6 3-(5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)-6,7-dimethoxy-2-phenylquinazolin-4(3H)-one (22)
-
1
This compound was prepared and purified as per the above mentioned procedure: yield 62 %; mp 288-291 °C. IR (KBr, ѵmax, cm ):
1
2
990.73 (CH), 1668.66 (C=O), 1541.78 (C=N), 762.12 (C-Cl); H NMR (DMSO-d6) δ ppm: 3.91-3.92 (s, magnetically different, 3H,
1
3
OCH ), 3.93-3.98 (s, magnetically different 3H, OCH ) 7.18-8.42 (m, 11H, Ar-H); C NMR (DMSO-d6) δ ppm: 56.12, 56.41, 108.34,
3
3
1
10.56, 114.56, 125.78, 126.34, 127.12, 128.00, 128.31, 129.72, 130.46, 134.46, 146.46, 154.27, 155.13, 156.28, 163.72, 164.37, 170.18;
HRMS (EI) m/z calcd for C H ClN O : 460.0938; found: 462.0933.
2
4
17
4
4
6
.2.7 6,7-dimethoxy-3-(5-(2-methoxyphenyl)-1,3,4-oxadiazol-2-yl)-2-phenylquinazolin-4(3H)-one (23)
-
1
This compound was prepared and purified as per the above mentioned procedure: yield 62 %; mp 288-291 °C. IR (KBr, ѵmax, cm ):2989.76
1
(
CH), 1679.81 (C=O), 1556.24 (C=N);. H NMR (DMSO-d6) δ ppm: 3.91-3.92 (s, magnetically different, 3H, OCH ), 3.93-3.98 (s,
3
1
3
magnetically different 3H, OCH ), 3.89 (s, 3H, OCH ), 7.02-8.09 (m, 11H, Ar-H); C NMR (DMSO-d6) δ ppm: 55.62, 56.12,56.41, 108.37,
3
3
1
1
6
09.28, 112.38, 115.12, 118.13, 121.28, 124.12, 128.38, 128.67, 129.38, 131.28, 133.88, 144.37, 153.12, 154.45, 156.45, 157.23, 163.12,
64.47, 169.72; HRMS (EI) m/z calcd for C H N O : 456.1434; found: 456.1438.
2
5
20
4
5
.2.8 3-(5-cyclopropyl-1,3,4-oxadiazol-2-yl)-6,7-dimethoxy-2-phenylquinazolin-4(3H)-one (24)
-
1
This compound was prepared and purified as per the above mentioned procedure: yield 62 %; mp 288-291 °C. IR (KBr, ѵmax, cm ):
1
2
940.58 (CH), 1650.24 (C=O), 1538.24 (C=N). H NMR (DMSO-d6) δ ppm: 1.14-2.50 (m, 5H, cylopropyl), 3.91-3.92 (s, magnetically
1
3
different, 3H, OCH ), 3.93-3.98 (s, magnetically different 3H, OCH ) 7.02-8.09 (m, 11H, Ar-H); C NMR (DMSO-d6) δ ppm: 8.21, 9.25,
3
3
5
6.12, 56.41, 108.52, 109.87, 116.12, 128.17, 128.56, 128.74, 131.98, 145.45, 153.86, 154.45, 158.34, 163.87, 165.34, 170.87; HRMS (EI)
m/z calcd for C H N O : 390.1328; found: 390.1333.
2
1
18
4
4
1
0 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 11 of 71
Please Rd So Cn oA tda vd aj un sc te ms argins
Journal Name
ARTICLE
6
.2.9 6,7-dimethoxy-2-phenyl-3-(5-m-tolyl-1,3,4-oxadiazol-2-yl)quinazolin-4(3H)-one (26)
View Article Online
DOI: 10.1039/C5RA05277G
-
1
This compound was prepared and purified as per the above mentioned procedure: yield 62 %; mp 288-291 °C. IR (KBr, ѵmax, cm ):
1
2
924.18 (Ar-CH), 2901.45 (Ali-CH), 1662.34 (C=O), 1539.12 (C=N); H NMR (DMSO-d6) δ ppm: 2.34 (s, 3H, CH3), 3.91-3.92 (s,
1
3
magnetically different, 3H, OCH ), 3.93-3.98 (s, magnetically different 3H, OCH ), 7.11-8.31 (s, 11H, Ar-H); C NMR (DMSO-d6) δ ppm:
3
3
2
1
6
2.62, 56.12, 56.41, 109.87, 110.67, 115.45, 125.83, 126.32, 128.29, 128.64, 128.75, 129.41, 130.68, 132.43, 138.12, 140.45, 146.89,
53.42, 154.87, 156.55, 163.12, 165.78, 169.76; HRMS (EI) m/z calcd for C H N O : 440.1485; found: 441.1489.
2
5
20
4
4
.2.10 3-(5-benzyl-1,3,4-oxadiazol-2-yl)-6,7-dimethoxy-2-phenylquinazolin-4(3H)-one (29)
-
1
This compound was prepared and purified as per the above mentioned procedure: yield 62 %; mp 288-291 °C. IR (KBr, ѵmax, cm ):2982.23
1
(
Ar-CH), 2918.28 (Ali-CH) 1644.88 (C=O), 1561.23 (C=N); H NMR (DMSO-d6) δ ppm: 2.75 (s, 2H, CH2), 3.91-3.92 (s, magnetically
1
3
different, 3H, OCH ), 3.93-3.98 (s, magnetically different 3H, OCH ) 7.09-8.19 (s, 12H, Ar-H). C NMR (DMSO-d6) δ ppm: 30.51, 56.12,
3
3
5
1
6
6.41, 108.87, 109.46, 116.76, 126.13, 128.47, 128.67, 128.82, 129.97, 130.46, 132.47, 136.53, 145.73, 153.24, 155.46, 158.26, 163.32,
67.42, 168.12; HRMS (EI) m/z calcd for C H N O : 440.1485; found: 440.1481.
2
5
20
4
4
.2.11 6,7-dimethoxy-2-phenyl-3-(5-(pyridin-3-yl)-1,3,4-oxadiazol-2-yl)quinazolin-4(3H)-one (34)
-
1
This compound was prepared and purified as per the above mentioned procedure: yield 62 %; mp 288-291 °C. IR (KBr, ѵmax, cm ):2924.18
1
(
CH), 1641.12 (C=O), 1542.87 (C=N); H NMR (DMSO-d6) δ ppm: 3.91-3.92 (s, magnetically different, 3H, OCH ), 3.93-3.98 (s,
3
1
3
magnetically different 3H, OCH ) 7.14-8.23 (m, 11H, Ar-H); C NMR (DMSO-d6) δ ppm: 56.12, 56.41, 108.74, 110.13, 115.54, 124.78,
3
1
25.46, 128.63, 128.78, 128.84, 130.64, 134.56, 144.56, 148.45, 153.43, 154.13, 155.62, 156.76, 163.53, 164.94, 168.73; HRMS (EI) m/z
calcd for C H N O : 427.1281; found: 427.1285.
2
3
17
5
4
6
.2.12 6,7-dimethoxy-3-(5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-yl)-2-phenylquinazolin-4(3H)-one (40)
-
1
This compound was prepared and purified as per the above mentioned procedure: yield 62 %; mp 288-291 °C. IR (KBr, ѵmax, cm ):
1
2
920.32 (CH), 1647.26 (C=O), 1534.78 (C=N); H NMR (DMSO-d6) δ ppm: 3.91-3.92 (s, magnetically different, 3H, OCH ),3.93-3.98 (s,
3
1
3
magnetically different 3H, OCH ), 3.89 (s, 3H, OCH ), 6.84-8.09 (m, 11H, Ar-H); C NMR (DMSO-d6) δ ppm: 55.62, 56.12, 56.41,
3
3
1
1
6
08.56, 109.83, 116.65, 117.74, 117.94, 118.42, 128.22, 128.46, 128.78, 132.41, 145.67, 153.11, 154.34, 158.27, 163.76, 164.76, 166.35,
68.13; HRMS (EI) m/z calcd for C H N O : 456.1434; found: 456.1438.
2
5
20
4
5
.2.13 3-(5-(4-chlorobenzyl)-1,3,4-oxadiazol-2-yl)-6,7-dimethoxy-2-phenylquinazolin-4(3H)-one (41)
-
1
This compound was prepared and purified as per the above mentioned procedure: yield 62 %; mp288-291 °C. IR (KBr, ѵmax, cm ):2991.69
1
(
Ar-CH), 2865.45 (Ali-CH), 1642.74 (C=O), 1541.86 (C=N), 768.66 (C-Cl); H NMR (DMSO-d6) δ ppm: 2.94 (s,2H, CH ), 3.91-3.92 (s,
2
1
3
magnetically different, 3H, OCH ),3.93-3.98 (s, magnetically different 3H, OCH ) 7.04-8.29 (m, 11H, Ar-H); C NMR (DMSO-d6) δ ppm:
3
3
3
1
0.45, 56.12, 56.41, 108.57, 109.77, 116.31, 126.27, 128.36, 128.67, 128.82, 130.81, 131.43, 132.49, 134.53, 148.67, 153.24, 154.54,
56.52, 163.56, 166.64, 168.33; HRMS (EI) m/z calcd for C H ClN O : 474.1095; found: 474.1091.
2
5
19
4
4
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 11
Please do not adjust margins

Please Rd So Cn oA tda vd aj un sc te ms argins
Page 12 of 71
ARTICLE
.2.14 6,7-dimethoxy-2-phenyl-3-(5-phenyl-1,3,4-thiadiazol-2-yl)quinazolin-4(3H)-one (45)
RSC Advances
6
View Article Online
DOI: 10.1039/C5RA05277G
-
1
This compound was prepared and purified as per the above mentioned procedure: yield 62 %; mp 288-291 °C. IR (KBr, ѵmax, cm ):
1
2
924.18 (CH), 1639.55 (C=O), 1552.34 (C=N); H NMR (DMSO-d6) δ ppm: 3.91-3.92 (s, magnetically different, 3H, OCH ), 3.93-3.98 (s,
3
1
3
magnetically different 3H, OCH ) 7.21-8.44 (m, 12H, Ar-H); C NMR (DMSO-d6) δ ppm: 56.12, 56.41, 108.67, 110.47, 116.41, 124.25,
3
1
26.46, 128.76, 128.88, 130.42, 132.71, 134.79, 136.55, 144.75, 153.26, 154.47, 156.29, 163.76, 172.78, 174.13; HRMS (EI) m/z calcd for
C H N O S: 442.1100; found: 443.1104.
2
4
18
4
3
6
.2.15 6,7-dimethoxy-3-(5-methyl-1,3,4-thiadiazol-2-yl)-2-phenylquinazolin-4(3H)-one (46)
-
1
This compound was prepared and purified as per the above mentioned procedure: yield 62 %; mp 288-291 °C. IR (KBr, ѵmax, cm ):
1
2
924.35 (Ar-CH), 2835.09 (Ali-CH), 1677.82 (C=O), 1541.32 (C=N); H NMR (DMSO-d6) δ ppm: 2.54 (s, 3H, CH3), 3.91-3.92 (s,
1
3
magnetically different, 3H, OCH ),3.93-3.98 (s, magnetically different 3H, OCH ) 7.02-8.09 (m, 11H, Ar-H). C NMR (DMSO-d6) δ ppm:
3
3
1
9.78, 56.12, 56.41, 108.75, 109.75, 116.31, 128.22, 128.46, 128.58, 130.31, 140.27, 145.57, 153.21, 154.54, 156.72, 163.76, 172.47; HRMS
(
EI) m/z calcd for C H N O S: 380.0943; found: 380.0948.
1
9
16
4
3
6
.2.16 3-(5-(4-chlorophenyl)-1,3,4-thiadiazol-2-yl)-6,7-dimethoxy-2-phenylquinazolin-4(3H)-one (47)
-
1
This compound was prepared and purified as per the above mentioned procedure: yield 62 %; mp 288-291 °C. IR (KBr, ѵmax, cm ):
1
2
924.18 (CH), 1673.30 (C=O), 1538.23 (C=N), 766.83 (C-Cl). H NMR (DMSO-d6) δ ppm: 3.91-3.92 (s, magnetically different, 3H,
1
3
OCH ), 3.93-3.98 (s, magnetically different 3H, OCH ) 7.18-8.42 (m, 11H, Ar-H); C NMR (DMSO-d6) δ ppm:56.12,56.41, 108.87,
3
3
1
10.37, 116.14, 126.24, 128.36, 128.58, 128.69, 129.73, 130.81, 132.86, 136.83, 144.87, 153.21, 153.54, 156.92, 160.36, 173.74, 174.23;
HRMS (EI) m/z calcd for C H ClN O S: 476.0710; found: 478.0715.
2
4
17
4
3
6
.2.17 3-(5-cyclopropyl-1,3,4-thiadiazol-2-yl)-6,7-dimethoxy-2-phenylquinazolin-4(3H)-one (50)
-
1
This compound was prepared and purified as per the above mentioned procedure: yield 62 %; mp 288-291 °C. IR (KBr, ѵmax, cm ):
1
2
3
925.99 (Ar-CH), 2851.55 (Ali-CH), 1641.73 (C=O), 1532.34 (C=N); H NMR (DMSO-d6) δ ppm: 1.50-2.43 (m, 5H, cylopropyl), 3.91-
1
3
.92 (s, magnetically different, 3H, OCH ),3.93-3.98 (s, magnetically different 3H, OCH ) 6.75-8.01 (m, 11H, Ar-H); C NMR (DMSO-d6)
3
3
δ ppm: 8.14, 9.35, 56.12, 56.41, 108.75, 110.73, 116.31, 128.38, 128.55, 128.69, 130.51, 144.57, 153.61, 154.78, 156.42, 163.76, 168.84,
1
72.48; HRMS (EI) m/z calcd for C H N O S: 406.1100; found: 406.1106.
21 18 4 3
6
.2.18 N-(5-(6,7-dimethoxy-4-oxo-2-phenylquinazolin-3(4H)-yl)-1,3,4-thiadiazol-2-yl) benzamide (51)
-
1
This compound was prepared and purified as per the above mentioned procedure: yield 62 %; mp 288-291 °C. IR (KBr, ѵmax, cm ):
1
3
212.34 (NH), 2918.18 (CH), 1678.34 (C=O), 1563.32 (C=N); H NMR (DMSO-d6) δ ppm: 3.91-3.92 (s, magnetically different, 3H,
1
3
OCH ), 3.93-3.98 (s, magnetically different 3H, OCH ) 6.65-7.88 (m, 12H, Ar-H), 9.12 (s,1H,NH); C NMR (DMSO-d6) δ ppm:
3
3
5
1
6
6.12,56.41, 108.47, 109.75, 116.15, 126.59, 128.26, 128.56, 128.74, 129.84, 130.14, 132.81, 134.42, 144.73, 150.83, 152.13, 153.44,
56.92, 163.64, 165.87, 172.64; HRMS (EI) m/z calcd for C H N O S: 485.1158; found: 485.1162.
2
5
19
5
4
.2.19 3-(5-benzyl-1,3,4-thiadiazol-2-yl)-6,7-dimethoxy-2-phenylquinazolin-4(3H)-one (55)
-
1
This compound was prepared and purified as per the above mentioned procedure: yield 62 %; mp 288-291 °C. IR (KBr, ѵmax, cm ):
1
2
925.15 (Ar-CH), 2901.24 (Ali-CH), 1652.48 (C=O), 1563.46 (C=N); H NMR (DMSO-d6) δ ppm: 2.53 (s, 3H, CH ), 3.91-3.92 (s,
2
1
2 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 13 of 71
Please Rd So Cn oA tda vd aj un sc te ms argins
Journal Name
ARTICLE
1
3
magnetically different, 3H, OCH ), 3.93-3.98 (s, magnetically different 3H, OCH ) 7.21-8.41 (s, 12H, Ar-H); C NMR (DMSO-d6) δ ppm:
3
3
View Article Online
DOI: 10.1039/C5RA05277G
3
1
6
3.62, 56.12, 56.41, 108.57, 109.37, 116.51, 124.67, 128.46, 128.55, 128.77, 128.82, 129.34, 130.15, 134.52, 142.57, 153.23, 154.44,
58.23, 163.45, 164.23, 172.43; HRMS (EI) m/z calcd for C H N O S: 456.1256; found: 458.1260.
2
5
20
4
3
.2.20 6,7-dimethoxy-2-phenyl-3-(5-(pyridin-3-yl)-1,3,4-thiadiazol-2-yl)quinazolin-4(3H)-one (63)
-
1
This compound was prepared and purified as per the above mentioned procedure: yield 62 %; mp 288-291 °C. IR (KBr, ѵmax, cm ):
1
2
996.16 (CH), 1645.09 (C=O), 1561.34 (C=N); H NMR (DMSO-d6) δ ppm: 3.91-3.92 (s, magnetically different, 3H, OCH ),3.93-3.98 (s,
3
1
3
magnetically different 3H, OCH ) 7.01-8.19 (m, 11H, Ar-H); C NMR (DMSO-d6) δ ppm: 56.12, 56.41, 108.47, 110.75, 116.61, 126.23,
3
1
28.33, 128.45, 128.78, 130.51, 132.54, 134.53, 146.57, 148.59, 150.92, 153.25, 154.54, 156.52, 163.56, 173.34, 174.51; HRMS (EI) m/z
calcd for C H N O S: 443.1052; found: 443.1056.
2
3
17
5
3
6
.2.21 6,7-dimethoxy-3-(5-(4-methoxyphenyl)-1,3,4-thiadiazol-2-yl)-2-phenylquinazolin-4(3H)-one (68)
-
1):
This compound was prepared and purified as per the above mentioned procedure: yield 62 %; mp 288-291 °C. IR (KBr, ѵmax, cm 2930.57
1
(
CH), 1666.48 (C=O), 1564.78 (C=N); H NMR (DMSO-d6) δ ppm: 3.91-3.92 (s, magnetically different, 3H, OCH ), 3.93-3.98 (s,
3
1
3
magnetically different 3H, OCH ), 3.89 (s, 3H, OCH ), 6.78-8.12 (m, 11H, Ar-H); C NMR (DMSO-d6) δ ppm:55.08, 56.12,56.41, 108.13,
3
3
1
10.13, 116.42, 118.31, 122.13, 128.23, 128.42, 128.67, 129.57, 130.15, 146.27, 152.15, 153.37, 154.54, 156.62, 162.26, 164.18, 172.37;
HRMS (EI) m/z calcd for C H N O S: 472.1205; found: 472.1209
2
5
20
4
4
6
.2.22 3-(5-(4-chlorobenzyl)-1,3,4-thiadiazol-2-yl)-6,7-dimethoxy-2-phenylquinazolin-4(3H)-one (69)
-
1
This compound was prepared and purified as per the above mentioned procedure: yield 62 %; mp 288-291 °C. IR (KBr, ѵmax, cm ):2918.29
1
(
Ar-CH), 2910.24 (Ali-CH), 1664.14 (C=O), 1534.24 (C=N), 752.68 (C-Cl). H NMR (DMSO-d6) δ ppm: 2.91 (s, 2H, CH ), 3.91-3.92 (s,
2
1
3
magnetically different, 3H, OCH ), 3.93-3.98 (s, magnetically different 3H, OCH3) 7.02-8.28 (m, 11H, Ar-H); C NMR (DMSO-d6) δ
3
ppm: 34.26, 56.12, 56.41, 108.37, 110.74, 116.31, 128.23, 128.46, 128.67, 128.83, 130.41, 132.82, 133.59, 134.35, 146.73, 153.41, 154.54,
1
56.25, 163.56, 164.34, 172.23; HRMS (EI) m/z calcd for C H ClN O S: 490.0866; found: 490.0871.
25 19 4 3
3
1
6
.3 In vitro VEGFR-2 kinase assay and HUVEC Cell Assay
HTRF assays are homogeneous time-resolved assays that generate a signal by FRET between donor and acceptor molecules. When formatted
for kinase assays, the Eu-cryptate is usually conjugated to a phospho-specific antibody and is presented upon binding of the antibody to the
phosphorylated product, while the streptavidin-conjugated allophycocyanin binds to the biotin on the substrate to complete the detection
complex. When the two entities get close proximity, energy transfer occurs upon excitation and APC emits a specific long-lived fluorescence
at 665 nm. The kinases were purified as the intracellular domain of human VEGFR-2 fused by GST. The catalytic activity of the kinase was
detected by using a biotinylated synthetic peptide as a substrate, biotin-aminohexyl-EEEEYFELVAKKKK-NH2, for VEGFR-2.
Phosphorylated substrate is measured by streptavidin linked-APC and europium-labeled anti-phosphorylated tyrosine antibody. Briefly, the
assay method is as follows: the working solution (100 nM TK-substrate, 3 µg/ml GSTVEGFR-2, 100 nM ATP, 1 mM DTT, 1 mM MnCl , 5
2
mM MgCl and 20 nM SEB in 10 µl reaction volume) is incubated at 37 ºC for 30 min; the detection solution (6.25 nM Streptavidin-XL665,
2
5
µl/well TK antibody-cryptate) is added to stop the reaction, and placed at room temperature for 30 min for determination (excitation at 314
nm, emission at 665 nm/ 620 nm).
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 13
Please do not adjust margins

Please Rd So Cn oA tda vd aj un sc te ms argins
Page 14 of 71
ARTICLE
RSC Advances
-
1
Cell assay: HUVEC were developed in M199 containing 10% FBS and kanamycin (50 µml ) in a humidified 5% CO incubator at 37 ºC.
2
View Article Online
DOI: 10.1039/C5RA05277G
After the cells had grown to confluence, they were disaggregated in trypsin solution, washed with M199 containing 10% FBS, centrifuged at
1
25 g for 5 min, re-suspended and then subcultured in accordance to standard protocols. Cells from passages 4-8 were used. HUVEC
proliferation in the presence of growth factors was evaluated using Sulforhodamine B (SRB) assay. In brief, HUVEC were plated in 96-well
plates (1000 cells/well) and dosed with tested compound + VEGF (15 ng/ml). The cultures were incubated for 48 h (37 ºC; 5% CO2)
followed by addition of SRB and finally, reincubated for 15 min. Cells were harvested and assayed using a ELISA counter with reading at
4
90 nm. IC50 data was interpolated as described above.
Acknowledgement
The authors would like to thank National Research Foundation, South Africa and College of Health Sciences, University of KwaZulu-Natal,
South Africa for funding this project.
1
4 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 15 of 71
Please Rd So Cn oA tda vd aj un sc te ms argins
Journal Name
References
ARTICLE
View Article Online
1
Yang, Y.; Shi, L.; Zhou, Y.; Li, H.-Q.; Zhu, Z.-W.; Zhu, H. L.Design, synthesis and biological evaluatDioOnI: 1o0f.1q0u3i9n/oCl5iRnAe0a5m27i7dGe
derivatives as novel VEGFR-2 inhibitorsBioorg. Med. Chem. Lett.2010, 20, 6653-6656
2
(a) Harris, P. A.; Boloor, A.; Cheung, M.; Kumar, R.; Crosby, R. M.; Davis-Ward, R. G.; Epperly, A. H.; Hinkle, K. W.; Hunter,
R. N.; Johnson, J. H.; Knick, V. B.; Laudeman, C. P.; Luttrell, D. K.; Mook, R. A.; Nolte, R. T.; Rudolph, S. K.; Szewczyk, J. R.;
Truesdale, A. T.; Veal, J. M.; Wang, L. P.; Stafford, J. A.Discovery of 5-[[4-[(2,3-dimethyl-2H-indazol-6-yl)methylamino]-2-
pyrimidinyl]amino]-2-methyl-benzenesulfonamide (pazopanib), a novel and potent vascular endothelial growth factor receptor
inhibitor J. Med. Chem.2008, 51, 4632-4640. (b) Rizvi, S.U.F.; Siddiqui, H.L.; Nisar, M.; Khan, N.; Khan, I. Discovery and
molecular docking of quinolyl-thienyl chalcones as anti-angigeonic agents targeting VEGFR-2 tyrosine kinase. Bioorg. Med.
Chem. Lett.2012, 22, 942-944.
3
(a) Ferrara, N. VEGF and the quest for tumour angiogenesis factors. Nat. Rev. Cancer2002, 2,795-803. (b) Ferrara, N.; Gerber, H.
P.; LeCouter, J. The biology of VEGF and its receptors. Nat. Med. 2003, 9, 669-676.
4
5
6
7
Ferrara, N. Vascular endothelial growth factor: basic science and clinical progress. Endocr. Rev.2004, 25, 581-611.
Cook, K. M.;Figg, W. D. Angiogenesis inhibitors: current strategies and future prospects. Cancer J. Clin.2010,60, 222-243.
Weis, S. M.; Cheresh, D. A. Tumor angiogenesis: molecular pathways and therapeutic targets. Nat. Med.2011,17, 1359-1370.
Qi, F.; Li, A.; Inagaki, Y.; Kokudo, N.; Tamura, S.; Nakata, M. Antitumor activity of extracts and compounds from the
skin of the toad Bufo bufo gargarizans Cantor. Int. Immunopharmacol.2011,11,342-349.
8
9
1
1
1
1
Cruz, J. S.; Matsuda, H. Depressive effects of arenobufagin on the delayed rectifier K+ current of guinea-pig cardiac
myocytes. Eur. J. Pharmacol.1994,266, 317-325.
Cruz, J. S.; Matsuda, H. Arenobufagin, a compound in toad venom, blocks Na+,-K+ pump current in cardiac myocytes.
Eur. J. Pharmacol.1993, 239, 223-226.
0
1
2
3
Cao, H.; Zhang, D.; Liu, J.; Hou, C.; Kurihara, H.; Ye, W. Inhibitory effect of arenobufagin on the adhesion, invasion
and migration of human hepatoma carcinoma cells. Chin. Pharm. Bull.2011, 27, 19-23.
Baudin, B.; Bruneel, A.; Bosselut, N.; Vaubourdolle, M. A protocol for isolation and culture of human umbilical vein
endothelial cells. Nat. Protoc.2007,2, 481-485.
Corrigan, C. J.; Wang, W.; Meng, Q.; Fang, C.; Wu, H.; Reay, V. T-helper cell type 2 (Th2) memory T cell-potentiating
cytokine IL-25 has the potential to promote angiogenesis in asthma. Proc. Natl. Acad. Sci. USA2011,108,1579–1584.
Harris, P.A.; Harris P. A.; Cheung, M.; Brown, M. L.; Veal, J. M.; Nolte, R.T.; Wang, L.; Liu, W.; Crosby, R. M.; Johnson, J. H.;
Epperly, A. H.; Kumar, R.; Luttrell, D. K.; Stafford, J. A. Discovery and evaluation of 2-anilino-5-aryloxazoles as a novel class
of VEGFR2 kinase inhibitors.J. Med. Chem.2005, 48, 1610–1619.
1
1
4
5
Phase, version 3.1, Schrödinger, LLC, New York, NY, 2009, 7.
Dixon, S. L.; Smondyrev, A. M.; Knoll, E. H.; Rao, S. N.; Shaw, D. E.; Friesner, R. A. PHASE: a new engine for pharmacophore
perception, 3D QSAR model development, and 3D database screening: 1. Methodology and preliminary results. J. Comput. Aided.
Mol. Des.2006, 20, 647-671.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 15
Please do not adjust margins

Please Rd So Cn oA tda vd aj un sc te ms argins
Page 16 of 71
ARTICLE
RSC Advances
1
1
1
6
7
8
MacroModel, version 9.7, Schrödinger, LLC, New York, NY, 2009,11.
LigPrep, version 2.3, Schrödinger, LLC, New York, NY, 2009,12.
View Article Online
DOI: 10.1039/C5RA05277G
(a) Regina, G. L.; Silvestri, R.; Artico, M.; Lavecchia, A.; Novellino, E.; Befani, O.; Turini, P. New pyrrole inhibitors of
monoamine oxidase: synthesis, biological evaluation, and structural determinants of MAO-A and MAO-B selectivity. J. Med.
Chem.2007, 50, 922-931.(b) Shivakumar, D.; Williams, J.; Wu, Y. Prediction of absolute solvation free energies using molecular
dynamics free energy perturbation and the OPLS force field. J. Chem. Theory Comput. 2010, 6, 1509-1519. (c) Jorgensen, W. L.;
Maxwell, D. S.; Tirado-Rives, J., et al., Development and testing of the OPLS all-atom force field on conformational energetics
and properties of organic liquids. J. Am. Chem. Soc. 1996, 118, 11225-11236. (d) Jorgensen, W. L.; Tirado-Rives, J. The OPLS
[optimized potentials for liquid simulations] potential functions for proteins, energy minimizations for crystals of cyclic peptides
and crambin. J. Am. Chem. Soc. 1988, 110, 1657-1666.
1
9
(a) Halgren, T. A.; Murphy, R. B.; Friesner, R. A.; Beard, H. S.; Frye, L. L.; Pollard, W. T.; Banks, J. L. Glide: a new approach for
rapid, accurate docking and scoring. 2. Enrichment factors in database screening.J. Med. Chem.2004, 47, 1750–1759. (b) Friesner,
R. A.; Banks, J. L.; Murphy, R. B.; Halgren, T. A.; Klicic, J. J.; Mainz, D. T.; Repasky, M. P.; Knoll, E. H.;Shelley, M.; Perry, J.
K.; Shaw, D. E.; Francis, P.; P.S. Shenkin, Glide: A new approach for rapid, accurate docking and scoring. 1. method and
assessment of docking accuracy. J. Med. Chem.2004, 47, 1739–1749. (c) Sherman, W.; Day, T.; Jacobson, M. P.; Friesner, R. A.;
Farid, R.Novel Procedure for Modeling Ligand/Receptor Induced Fit Effects. J. Med. Chem.2006, 49, 534–553.
2
2
0
1
http://www.rcsb.org/pdb/explore/explore.3B8Q.
Schrödinger Suite 2009 Protein Preparation Wizard; Epik version 2.0; Impact version 5.5. Schrödinger, LLC, New York, NY,
2
009, 14.
2
2
2
3
Antonio, G.; Amaury, F.; Severine R.; Amelie, L.; Perrine, S.; Philippe, C.; Laurence, G.; Patrick D. Synthesis and Structure-
Activity Relationships of (Aryloxy) quinazoline Ureas as Novel, Potent, and Selective Vascular Endothelial Growth Factor
Receptor-2 Inhibitors. J. of Med. Chem.2012, 55, 1189-1204.
(a) Friesner, R.A.; Banks, J.L.; Murphy, R.B.; Halgren, T.A.; Klicic, J.J.; Mainz, D.T.; Repasky, M.P.; Knoll, E.H.; Shelley, M.;
Perry, J.K.; Shaw, D.E.; Francis, P.; Shenkin, P.S. Glide: A New Approach for Rapid, Accurate Docking and Scoring. 1. Method
and Assessment of Docking Accuracy. J. Med. Chem.2004, 47, 1739-1749. (b) Sherman, W.; Day, T.; Jacobson, M.P.; Friesner,
R.A.; Farid, R. Novel Procedure for Modeling Ligand/Receptor Induced Fit Effects. J. Med. Chem.2006, 49, 534–553. (c)
Narkhede, S.S.; Degani, M.S. Pharmacophore Refinement and 3D-QSAR Studies of Histamine H3 Antagonists. QSAR Comb.
Sci.2007, 26, 744–753. (d) Tawari, N.R.; Bag, S.; Degani, M.S. Pharmacophore mapping of a series of pyrrolopyrimidines,
indolopyrimidines and their congeners as multidrug resistance-associated protein (MRP1) modulators. J. Mol. Model.2008, 14,
9
11–921. (e) Golbraikh, A.; Shen, M.; Xiao, Z.; Xiao, Y.D.; Lee, K.H.; Tropsha, A. Rational selection of training and test sets for
the development of validated QSAR models. J. Comput. Aided Mol. Des.2003, 17, 241–253.
Zhong, H.; Tran, L.M.; Jenna, L. Induced-fit docking studies of the active and inactive states of protein tyrosine kinases. J. Mol.
Graph. Mod.2009, 28, 336-346.
2
4
1
6 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 17 of 71
Please Rd So Cn oA tda vd aj un sc te ms argins
Journal Name
Combiglide 2.5 (2009) User manual. Schrodinger, New York.
ARTICLE
2
5
View Article Online
DOI: 10.1039/C5RA05277G
2
6
QikProp, version 9.0, Schrodinger, LLC, New York, NY, 2010.
Ligprep, version 9.0, Schrodinger, LLC, New York, NY, 2010.
2
2
7
8
Lipinski, C.A.; Lombardo, F.; Dominy, B.W.; Feeney, P.J. Experimental and computational approaches to estimate solubility and
permeability in drug discovery and development settings. Adv. Drug Deliv. Rev.2001, 23, 3-26.
2
3
3
3
9
0
1
2
Krantz, A.; Spencer, R. W.; Tam, T.F.; Liak, T. J.; Copp, L. J. Design and synthesis of 4H-3,1-benzoxazin-4-ones as potent
alternate substrate inhibitors of human leukocyte elastase. J.Med. Chem. 1990, 33,464-479.
Zentmyer, D. T.; Wagner, E. C. The so-called acylanthranils (3,1,4-benzoxazones). I. Preparation; reactions with water, ammonia,
and aniline; structure. J. Org. Chem.1949,14,967-981.
Yu, B.; Tang, L.; Li, Y.; Song, S. H.; Ji, X. L.; Lin, M. S.; Wu, C. F. Design, synthesis and antitumor activity of 4-
aminoquinazoline derivatives targeting VEGFR-2 tyrosine kinase. Bioorg. Med. Chem. Lett. 2012, 22, 110-114.
Noolvi, M. N.; Patel, H. M. Synthesis and in vitro anti-tumor activity of substituted quinazoline and quinoxaline derivatives: search
for anti-cancer agent. Eur. J. Med. Chem. 2011, 46, 2327-46.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 17
Please do not adjust margins

Please Rd So Cn oA tda vd aj un sc te ms argins
Page 18 of 71
ARTICLE
RSC Advances
Table 1 Experimental and predicted activity of quinazoline derivatives used in training and test set for VEGFR-2 inhibition uVsieiwngArMticloedOenlli-nIe
DOI: 10.1039/C5RA05277G
pIC50
Compounds
Residual
IC50 (nm)
Sr. No
Exp.
Pred.
H
HN
N
T
1
7.00
0.845
0.967
-0.122
O
O
N
Br
HN
N
T
2
0.80
0.097
0.108
-0.011
O
O
N
Cl
F
T
HN
N
3
5.30
0.724
0.784
-0.060
O
O
N
F
Br
HN
N
4
1.65
7.30
0.217
0.863
0.315
0.881
-0.098
-0.018
O
O
N
H
N
O
T
HN
N
5
O
O
N
H
N
CH2 2CH3
(
)
O
HN
6
4.0
0.845
0.967
-0.122
O
O
N
N
1
8 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 19 of 71
Please Rd So Cn oA tda vd aj un sc te ms argins
Journal Name
Table 1 Continue
ARTICLE
View Article Online
DOI: 10.1039/C5RA05277G
pIC50
Compounds
Residual
IC50 (nm)
Sr. No
Exp.
Pred.
NH
CH2 3CH3
( )
O
HN
T
7
6.80
5.80
0.833
0.848
-0.015
O
O
N
N
NH OCH3
O
HN
N
8
9
0.763
0.748
0.810
0.795
-0.047
-0.047
O
O
N
NH OCH CH3
2
O
HN
5.60
5.00
O
N
O
N
NH O CH 2CH3
(
2
)
T
O
1
0
0.699
0.781
-0.082
HN
O
N
O
N
NH O CH 3CH3
(
2
)
O
HN
T
1
1
6.80
5.05
5.05
0.833
0.703
0.703
0.894
0.835
0.721
-0.061
-0.132
-0.018
O
N
O
N
NH OCH CH
3
)
2
2
(
O
HN
N
1
2
O
O
N
Cl
NH OCH3
O
1
3
HN
O
N
O
N
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 19
Please do not adjust margins

Please Rd So Cn oA tda vd aj un sc te ms argins
Page 20 of 71
ARTICLE
RSC Advances
Table 1 Continue
View Article Online
DOI: 10.1039/C5RA05277G
pIC50
Compounds
Residual
IC50 (nm)
Sr. No
Exp.
Pred.
Cl
NH OCH CH3
2
O
T
1
4
HN
0.50
0.301
0.541
-0.240
O
O
N
N
H
N
O CH 2CH3
(
2
)
O
HN
Cl
T
1
5
3.30
0.519
0.712
-0.193
O
O
N
N
H
N
O CH 3CH3
(
2
)
O
HN
N
Cl
T
1
6
4.90
0.69
0.78
-0.009
O
O
N
NH OCH3
O
HN
N
T
1
7
0.85
0.65
0.85
0.071
0.187
0.071
0.084
0.301
0.097
-0.013
-0.118
-0.026
O
O
N
NH OCH CH
2
3
O
HN
1
8
O
N
O
N
NH O CH 2CH3
(
2
)
O
HN
T
1
9
O
N
O
N
2
0 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 21 of 71
Please Rd So Cn oA tda vd aj un sc te ms argins
Journal Name
ARTICLE
View Article Online
Table 1 Continue
DOI: 10.1039/C5RA05277G
pIC50
Compounds
Residual
IC50 (nm)
Sr. No
Exp.
Pred.
NH O CH 3CH3
(
2
)
O
HN
N
T
2
2
0
1
5.20
5.25
0.716
0.803
-0.087
O
O
N
NH OCH CH
3
( )
2
O
HN
N
T
0.72
0.75
-0.003
-0.096
-0.069
-0.107
-0.144
-0.160
O
N
O
Cl
O
HN
N
NH OCH CH
T
2
3
2
2
5.55
7.80
6.50
4.65
5.80
0.744
0.892
0.813
0.667
0.763
0.840
0.961
0.920
0.811
0.923
O
O
N
O
HN
N
NH OCH3
2
3
O
O
N
O
HN
NH OCH CH3
2
T
2
4
O
N
O
N
H
N
H
N
O
HN
2
5
O
N
O
N
H
H
N
N
CH2 3CH
3
(
)
O
T
HN
N
2
6
O
O
N
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 21
Please do not adjust margins

Please Rd So Cn oA tda vd aj un sc te ms argins
Page 22 of 71
ARTICLE
RSC Advances
View Article Online
DOI: 10.1039/C5RA05277G
Table 1 Continue
pIC50
Sr. No
Compounds
Residual
IC50 (nm)
Exp.
Pred.
H
N
H
N
O
T
HN
2
7
5.10
0.708
0.792
-0.084
O
O
N
N
H
N
H
N
O
O
HN
N
2
8
6.20
0.792
0.857
-0.065
O
O
N
H
N
H
N
Cl
O
T
HN
N
F
2
9
4.30
0.633
0.766
-0.133
O
N
O
Cl
F
3
0
HN
6.90
0.839
0.965
-0.126
O
O
N
N
N
F
Br
HN
N
T
3
3
3
1
2
3
1.00
6.45
5.20
0
0.019
-0.019
-0.06
O
N
N
O
Br
T
HN
0.81
0.94
O
N
N
O
N
Cl
NH OCH3
O
T
HN
N
0.716
0.779
-0.063
O
O
N
N
2
2 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 23 of 71
Please Rd So Cn oA tda vd aj un sc te ms argins
Journal Name
Table 1 Continue
ARTICLE
View Article Online
DOI: 10.1039/C5RA05277G
pIC50
Sr. No
Compounds
Residual
IC50 (nm)
Exp.
Pred.
Cl
NH OCH CH
2
3
O
HN
N
3
3
4
5
3.35
6.95
0.525
0.697
-0.172
O
O
N
N
NH OCH3
O
HN
N
0.842
0.568
0.82
0.732
0.631
0.90
-0.090
-0.063
-0.06
O
O
N
N
NH OCH CH3
2
O
T
HN
N
3
6
7
3.70
6.60
O
O
N
N
O
HN
N
H
T
3
3
HN
O
N
N
O
N
F
Br
HN
N
T
8
7.45
7.25
5.30
0.872
0.952
-0.080
-0.22
O
N
N
O
NH OCH CH3
2
O
HN
N
3
9
0.86
1.08
O
N
N
O
F
Br
T
HN
4
0
0.724
0.820
-0.096
O
O
N
N
N
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 23
Please do not adjust margins

Please Rd So Cn oA tda vd aj un sc te ms argins
Page 24 of 71
ARTICLE
Table 1 Continue
RSC Advances
View Article Online
DOI: 10.1039/C5RA05277G
pIC50
IC50
Compounds
Residual
Sr. No
(nm)
Exp.
Pred.
Cl
H
N
OCH3
O
T
HN
N
4
1
7.40
0.869
1.197
-0.328
O
O
N
N
Cl
O
O
H
N
T
4
2
HN
N
4.80
0.681
0.817
-0.138
O
O
N
N
O
O
H
N
T
T
T
T
HN
N
4
4
4
4
3
4
5
6
9.40
0.973
0.756
1.398
2.222
1.14
0.856
1.464
2.291
-0.167
-0.100
-0.066
-0.069
O
N
N
O
O
O
H
N
HN
N
5.70
O
N
N
O
H
H
N
N
O
O
N
0.040
O
O
N
O
H
H
N
N
Br
O
O
N
0.006
O
O
N
2
4 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 25 of 71
Please Rd So Cn oA tda vd aj un sc te ms argins
Journal Name
ARTICLE
View Article Online
DOI: 10.1039/C5RA05277G
Table 1 Continue
pIC50
IC50
Compounds
Residual
Sr. No
(nm)
Exp.
Pred.
H
N
H
N
O
T
O
N
Br
4
7
0.040
1.398
1.571
-0.173
O
O
N
H
N
H
N
Br
O
O
N
4
8
0.004
2.398
2.535
-0.137
O
N
O
H
N
H
N
O
T
O
N
F
4
5
9
0
0.006
2.222
2.420
-0.198
O
Cl
N
O
H
H
N
N
O
T
O
0.002
2.699
2.918
-0.219
O
O
N
N
Expt. = Experimental activity, Pred. = Predicted activity, T = Training Set
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 25
Please do not adjust margins

Please Rd So Cn oA tda vd aj un sc te ms argins
Page 26 of 71
ARTICLE
RSC Advances
View Article Online
DOI: 10.1039/C5RA05277G
Table 2 Statistical data for QSAR Models by PLS method for quinazoline derivatives.
2
2
Model
Hypothesis
Factor
SD
r
F
Stability
RMSE
q
Pearson R
Model-I
AAARR.8
5
0.1969
0.9687
148.6
0.8945
0.4487
0.7106
0.6867
0.6634
0.6432
0.6023
0.5723
0.5423
0.5213
0.5129
0.4987
0.8709
0.8423
0.8276
0.7876
0.7654
0.7123
0.6452
0.6128
0.5987
0.5862
Model-II
Model-III
Model-IV
Model-V
AAAHR.3
AAHHR.7
AADHR.9
AAADR.41
AAAAD.3
AADRR.10
AADHR.16
AAADH.5
ADHRR.34
5
5
5
5
5
5
5
5
5
0.2156
0.2648
0.3585
0.5881
0.6445
0.6865
0.7234
0.7457
0.8045
0.9212
0.9045
0.8534
0.8278
0.8097
0.7856
0.6434
0.5213
0.5045
139.8
129.9
121.3
102.8
92.2
0.7723
0.7123
0.6987
0.6824
0.6542
0.5284
0.2789
0.2553
0.2325
0.4823
0.5234
0.5678
0.5876
0.6234
0.6532
0.6856
0.7234
0.7324
Model-VI
Model-VII
Model-VIII
Model-IX
Model-X
90.6
79.4
70.2
48.1
2
Factor = Number of factors in the partial least squares regression model; SD = Standard deviation of the regression; r = coefficient of
determination; F = F test score; Stability = Stability of the model predictions to changes in the training set composition, maximum value
2
is 1, this statistic can be used to compare models from different hypotheses; RMSE = Root-mean-square error; q2 = cross validated r ;
Pearson R = correlation between experimental and predicted activity for the test set.
2
6 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 27 of 71
Please Rd So Cn oA tda vd aj un sc te ms argins
Journal Name
ARTICLE
View Article Online
Table 3 Distances and angles of pharmacophore hypothesis by Model-I
DOI: 10.1039/C5RA05277G
Site
2
Distance
(Å)
Site
1
Site
2
Site
3
Site
1
Site
2
Site
3
Site
1
Site
2
Site
3
Hypothesis
Site 1
Hypothesis
Angle
Hypothesis
Angle
Hypothesis
Angle
AAARR.8
AAARR.8
AAARR.8
AAARR.8
AAARR.8
AAARR.8
AAARR.8
AAARR.8
AAARR.8
AAARR.8
A1
A1
A1
A1
A3
A3
A3
A4
A4
R15
A3
A4
50574
4.856
2.775
6.425
2.667
2.800
5.489
2.798
7.462
5.24
AAARR.8
AAARR.8
AAARR.8
AAARR.8
AAARR.8
AAARR.8
AAARR.8
AAARR.8
AAARR.8
AAARR.8
A3
A3
A3
A1
A1
R13
A4
A4
A4
A1
A4
A4
A4
A4
A4
A4
A3
A3
A3
A3
A1
90.8
61.6
34.8
29.2
58.4
30.3
60.6
61.5
129.1
1
AAARR.8
AAARR.8
AAARR.8
AAARR.8
AAARR.8
AAARR.8
AAARR.8
AAARR.8
AAARR.8
AAARR.8
A1
R13
A4
A4
A4
A3
A3
R13
A4
A4
A3
A3
A1
A1
A1
A1
A1
A1
R13
R13
R15
R15
A3
71
70
AAARR.8
AAARR.8
AAARR.8
AAARR.8
AAARR.8
AAARR.8
AAARR.8
AAARR.8
AAARR.8
AAARR.8
A4
A3
A3
A1
A4
A4
A4
A3
A3
A1
R13
R13
R13
R13
R15
R15
R15
R15
R15
R15
R15
A1
134.1
178
R13
R15
R13
R15
R15
A1
R13
R15
A4
28.6
29.5
81.5
1
R15
R15
A3
79.8
102.1
16.1
40.1
15.6
55.1
30.1
25
R13
R15
R13
R15
R15
A3
R13
R15
R13
R15
R13
A1
53.9
52.9
56.9
121.2
R13
A1
R13
R15
R13
R13
R13
A1
Distances and angles between the pharmacophoric points (Site 1, 2 and 3) of hypothesis AAARR8 (refer Fig. 3)
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 27
Please do not adjust margins

Please Rd So Cn oA tda vd aj un sc te ms argins
Page 28 of 71
ARTICLE
Table 4 Pharmacophoric alignment and predicted activity of designed compounds
RSC Advances
View Article Online
DOI: 10.1039/C5RA05277G
Predicted Activity
Code
Compounds
Pharmacophoric Fitness
(
IC )
50
O N
2
O
N
N
H CO
3
1
8.28
H CO
3
O
O
H CO
3
2
3
4
N
N
1.923
H CO
3
Cl
O
N
N
H CO
3
1.201
H CO
3
F
O
H CO
3
N
N
6.23
H CO
3
Br
O
N
N
H CO
3
9.076
5
H CO
3
NO2
O
N
N
H CO
3
6
6.101
H CO
3
2
8 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 29 of 71
Please Rd So Cn oA tda vd aj un sc te ms argins
Journal Name
ARTICLE
Table 4 Continue
View Article Online
DOI: 10.1039/C5RA05277G
Predicted Activity
Code
Compounds
Pharmacophoric Fitness
(
IC )
50
F
O
N
N
H CO
3
7
8.115
H CO
3
NO2
O
N
N
H CO
3
8
9.124
H CO
3
Cl
O
H CO
3
N
N
9
15.101
H CO
3
O
O
N
N
H CO
3
1
1
1
0
1
2
0.795
H CO
3
O
N
N
H CO
3
7
.066
H CO
3
O
N
N
H CO
3
8.089
H CO
3
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 29
Please do not adjust margins