6-Aryl substituted 4-(4-cyanomethyl) phenylamino quinazolines as a new class of isoform-selective PI3K-alpha inhibitors

Article abstract of DOI:10.1016/j.ejmech.2016.07.006

Isoform-selective inhibition of PI3K-α has been identified as one of the important strategy to discover effective and safer anticancer agents. Herein, we report discovery of ‘quinazoline’ as a new chemotype for isoform-selective PI3K-α inhibitors. The indolyl substituted quinazoline 9u displayed selective inhibition of PI3K-α with IC₅₀value of 0.201?μM with >49.7 over PI3K-β, and δ-isoforms. Quinazoline 9u also inhibited PI3K-γ with IC₅₀value of 0.750 μM (3.7 fold selective for α-versus γ-isoform). The isoform-selective inhibition was also demonstrated at protein-expression level by western-blot analysis in MCF-7 and PC-3?cells. The isoform-selective inhibitor 9u also showed inhibition of phospho-Akt levels in these cells. Quinazoline 9u showed in-vitro cytotoxicity in MCF-7?cells with GI₅₀of 7?μM, which was highly selective for cancer cells, as it was non-toxic to normal cells fR2, HEK293 and hGF (GI₅₀?>?50?μM). Compound 9u at 25?mg/kg dose showed 62 and 37% TGI in Ehrlich Ascites Carcinoma and Ehrlich Solid Tumor mice models. In nutshell, our efforts to identify potent and efficacious PI3K inhibitors resulted in the discovery of a new class of isoform-selective PI3K-α inhibitors possessing promising in-vivo anticancer activity.

Full text of DOI:10.1016/j.ejmech.2016.07.006

Accepted Manuscript
6-Aryl substituted 4-(4-cyanomethyl) phenylamino quinazolines as a new class of
isoform-selective PI3K-alpha inhibitors
Rammohan R. Yadav, Santosh K. Guru, Prashant Joshi, Girish Mahajan, Mubashir
J. Mintoo, Vikas Kumar, Sonali S. Bharate, Dilip M. Mondhe, Ram A. Vishwakarma,
Shashi Bhushan, Sandip B. Bharate
PII:
S0223-5234(16)30553-0
10.1016/j.ejmech.2016.07.006
EJMECH 8724
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 28 April 2016
Revised Date: 4 July 2016
Accepted Date: 5 July 2016
Please cite this article as: R.R. Yadav, S.K. Guru, P. Joshi, G. Mahajan, M.J. Mintoo, V. Kumar,
S.S. Bharate, D.M. Mondhe, R.A. Vishwakarma, S. Bhushan, S.B. Bharate, 6-Aryl substituted 4-(4-
cyanomethyl) phenylamino quinazolines as a new class of isoform-selective PI3K-alpha inhibitors,
European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.07.006.
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ACCEPTED MANUSCRIPT
GRAPHICAL ABSTRACT
An isoform-selective quinazoline class of PI3K-alpha inhibitor possessing in-vitro and in-vivo
anticancer activity has been identified.

ACCEPTED MANUSCRIPT
6
-Aryl substituted 4-(4-cyanomethyl) phenylamino quinazolines as a
new class of isoform-selective PI3K-alpha inhibitors
a,b#
c#
a,b
b,c
Rammohan R. Yadav, Santosh K. Guru, Prashant Joshi, Girish Mahajan, Mubashir J.
b,c
b,d
d
b,c
a,b
Mintoo, Vikas Kumar, Sonali S. Bharate, Dilip M. Mondhe, Ram A. Vishwakarma,
b,c
a,b,
Shashi Bhushan* and Sandip B. Bharate *
a
Medicinal Chemistry Division, CSIR-Indian Institute of Integrative Medicine , Canal Road,
Jammu-180001, India.
b
Academy of Scientific & Innovative Research (AcSIR), CSIR-Indian Institute of Integrative
Medicine, Canal Road, Jammu-180001, India
c
Cancer Pharmacology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road,
Jammu-180001, India. *E-mail:
d
Preformulation Laboratory, PKPD Division, CSIR-Indian Institute of Integrative Medicine,
Canal Road, Jammu-180001, India
*
Corresponding authors. E-mail: sbharate@iiim.ac.in (SBB); sbhushan@iiim.ac.in (SB); Fax:
91-191-2585333; Tel: +91-191-2585006
+
ABSTRACT:
Isoform-selective inhibition of PI3K-α has been identified as one of the important strategy to
discover effective and safer anticancer agents. Herein, we report discovery of ‘quinazoline’ as a
new chemotype for isoform-selective PI3K-α inhibitors. The indolyl substituted quinazoline 9u

ACCEPTED MANUSCRIPT
displayed selective inhibition of PI3K-α with IC value of 0.201 µM with >49.7 over PI3K-β,
5
0
and δ-isoforms. Quinazoline 9u also inhibited PI3K-γ with IC value of 0.750 uM (3.7 fold
5
0
selective for α- versus γ-isoform). The isoform-selective inhibition was also demonstrated at
protein-expression level by western-blot analysis in MCF-7 and PC-3 cells. The isoform-
selective inhibitor 9u also showed inhibition of phospho-Akt levels in these cells. Quinazoline
9
u showed promising cytotoxicity in MCF-7 cells with GI of 7 µM, which was highly selective
50
for cancer cells, as it was non-toxic to normal cells fR2, HEK293 and hGF (GI > 50 µM).
5
0
Compound 9u at 25 mg/kg dose showed 62 and 37% TGI in Ehrlich Ascites Carcinoma and
Ehrlich Solid Tumor mice models. In nutshell, our efforts to identify potent and efficacious PI3K
inhibitors resulted in the discovery of a new class of isoform-selective PI3K-α inhibitors
possessing promising in-vivo anticancer activity.
KEYWORDS:
6
-aryl quinazolines, isoform-selective, PI3K-alpha, breast cancer, anticancer agents

ACCEPTED MANUSCRIPT
1
. INTRODUCTION
The PI3K signalling pathway coordinates large number of physiological functions in humans
including cell growth, proliferation, metabolism, survival and motility [1-4]. Biochemical studies
in large number of tumour tissues revealed that PI3K signalling cascade is dysregulated in breast,
colon, enodometrial, brain and prostate cancers. PI3K signalling genes PIK3CA, PIK3CB,
PIK3CD and PIK3CG encodes different PI3K isoforms (PI3K-α, β,
δ and γ). Several studies
have demonstrated that PI3K-α isoform plays crucial role in cancer progression, due to the
mutation or amplification of the PIK3CA gene or increased expression of P110α in most of the
tumor types, whereas PI3K-β isoform is involved in PTEN deficient tumors. However, both
PIK3CA mutation as well as PTEN deficiency is not common in PI3K-δ associated tumors [4-6].
The early discovery of pan-PI3K inhibitors wortmannin [7] and LY294002 [8] and GDC-0941
[
9, 10] were critical events that allowed rapid exploration of PI3K signalling, which led to their
extensive usage as a tool compounds against a variety human tumour histotypes in-vitro and in-
vivo. Later, with the discovery of mTOR as a molecular target of rapamycin, numerous dual
PI3K/mTOR inhibitors were discovered; and many of them reached to clinical stages, including
NVP-BEZ235 (1) [11-13]. The pan-PI3K inhibitors and dual PI3K/mTOR inhibitors faced
problems in clinical trials, with limited efficacies as a monotherapeutic agent as well as a
relatively high rate of side effects, because of their off-target activity [2, 6, 13-15]. Thus, the
discovery of isoform-selective PI3K kinase inhibitors has always remained a priority of
medicinal chemists in order to improve efficacy while minimizing undesirable side effects.
The success of PI3K isoform-selective inhibitors has been demonstrated by idelalisib
(CAL101, 2), a first-in-class PI3Kδ-selective small-molecule inhibitor that has been approved by
the FDA for the treatment of chronic lymphocytic leukemia, relapsed small lymphocytic

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lymphoma and B-cell non-Hodgkin's lymphoma [16]. Furthermore, more than a dozen isoform-
selective PI3K inhibitors are in various stages of clinical trials. Some of them includes NVP-
BYL719 (3) [17, 18], GDC-0032 (4) [19, 20] and INK1117 (5) [21] for PI3K-α, and AZD8186
(6) [22] and AZD-6482 (7) [23] for PI3K-β. The clinical success of isoform-selective PI3K
inhibitor idelalisib, a quinazoline class of compound has motivated us to look for isoform-
selective inhibitors from this scaffold. A pan-PI3K inhibitor BAY 80-6946 (8) [24] is reported
from this scaffold, however a PI3K-α selective inhibitor has never been reported.
Amongst all four isoforms of PI3K, the importance and high frequency of PIK3CA mutation
in most of the solid tumors has attracted attention towards the development of PI3Kα-selective
inhibitors. The aim of present work was to design, synthesize and optimize quinazoline scaffold
for selective binding to PI3K-α ATP binding site. A series of 6-aryl quinazolines 9a-x were
prepared and screened for PI3K-α/ mTOR inhibitory activity and cytotoxicity in a panel of
cancer cell lines; and the best compounds were investigated for their in-vivo anticancer activity
in murine tumor models.
<< Figure 1 >>
2
2
. RESULTS AND DISCUSSION
.1. Design. The close analysis of PI3K-α ATP binding site reveals the presence of a small
polypeptide chain (a hinge region) connecting the C-lobe and N-lobe of PI3K, which interacts
with adenine part of ATP. Literature precedence suggests that diverse fragments such as
quinazoline (idelalisib), quinoline (GSK2126458 [25] and PF-04979064 [26]), 2-aminothiazole
(NVP-BYL719 and PIK-93) [17] and morpholine group (PI-103 and GDC-0914) [9] interact
with the hinge region. Herein, we chose quinazoline as a key hinge binding pharmacophore in

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the designed compounds, wherein the “N=CH-N” fragment interacts with the Val851 of hinge
region (Figure 1B).
The chemical association of hinge binding functionality with the aromatic and heteroaromatic
rings increases the potency drastically as seen in the case of NVP-BEZ235, NVP-BYL719,
GSK2126458 and PF-04979064. Therefore, C6-substitution on quinazoline with heteroaromatic
rings was selected in order to maintain interactions in the affinity pocket. Another common
feature in pan-PI3K inhibitors and isoform-selective PI3K-α inhibitors is their ATP-competitive
inhibition via blockage of substrate lipid phosphorylation, however, interactions with the un-
conserved residues in ATP binding site and solvent exposed area, has been reported to provide
isoform-selectivity. Docking studies of NVP-BEZ235 and NVP-BYL719 indicated that
introduction of 4-(4-cyanomethyl)phenylamino functionality at C-4 position led to interactions of
designed analogs with un-conserved residues. Therefore, this functionality was incorporated in
designed compounds (as shown in Figure 1B).
2.2. Synthesis. The synthetic scheme for preparation of quinazolines 9a-x is depicted in Scheme
1. The first step involves treatment of anthranilic acid (10) with bromine in acetic acid to produce
2-amino-5-bromobenzoic acid 11 in 55% yield [27]. Further, the treatment of 2-amino-5-
bromobenzoic acid (11) with formamide resulted in formation of 4-hydroxy quinazoline (12) in
2% yield. Treatment of 4-hydroxy quinazoline (12) with phosphorous oxychloride resulted in
7
formation of 4-chloro 6-bromoquinazoline 13 in 92% yield [28]. The intermediate 13 was then
treated with 4-amino benzylcyanide (14) to get a key intermediate (15) required for Suzuki
coupling reaction [29]. Finally, Suzuki-Miyaura coupling of intermediate 15 with various aryl
and heteroaryl boronic acids using tetrakis(triphenylphosphine)palladium catalyst produced 6-
aryl substituted 4-(4-cyanomethyl) phenylamino quinazolines 9a-x in 43-81% yield. The 6,7-

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dimethoxy 4-(4-cyanomethyl) phenylamino quinazoline 19 was also synthesized using similar
synthetic strategy as depicted in Scheme 1.
<
< Scheme 1 >>
.3. In-vitro screening for PI3K inhibition and cytotoxicity and isoform-selectivity. All
synthesized 6-substituted quinazolines were first tested for inhibition of PI3K-α and mTOR at
.5-1 µM. Amongst all tested compounds, analogs 9e, 9p, 9u, 9w and 9x were the potent
2
0
inhibitors of PI3K-α showing 70, 48.6, 47.5, 45.6 and 49% inhibition at 0.5 µM. Other analogs
with >30% inhibition of PI3K-α were 9a, 9d, 9f, 9h, and 9o. Further, the IC was determined for
50
best 10 analogs for PI3K-α inhibition and results are shown in Table 1. Analogs 9e, 9u and 9x
were found to inhibit PI3K-α with IC values of 115, 201 and 150 nM, respectively. None of the
5
0
compound showed inhibition of mTOR at a tested concentration (1 µM).
< Table 1 >>
<
The analysis of PI3K-α crystal structures and function reveals that all known PI3K inhibitors
interacts with the common hinge binding region Val851 residue by H-bonding and mimic the
adenine part of ATP, which consequently inhibit substrate phosphorylation. However, further
extension and interaction of inhibitor structural features in the affinity pocket, and solvent
exposed area defines the enzyme inhibition potency. As anticipated, molecular docking of 9u
and 9x with PI3K-α ATP binding site (using PDB: 4L23) considering PI-103 as centroid of
docking grid reveals that quinazoline core interacts with the Val851 residue backbone in hinge
pocket by H-bonding and mimic the adenine ring of ATP. The bisaromatic heterocyclic core
interacts with the Tyr836 residue of ribose pocket by aromatic π-π stacking. H-bonding and π-π
stacking at hinge pocket stabilize the conformation of ligands in such a way that it extends its 6-
heteroaryl ring i.e. indolyl and quinolinyl group towards the affinity pocket Lys802 and Asp933

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through bypassing the gatekeeper Ile848 residue which physically block entry of donor substrate
ATP as displayed in Figure 2.
<< Figure 2 >>
Next, we studied selected 10 best compounds for their isoform-selectivity against other PI3K
isoforms. Amongst tested compounds, indolyl substituted analog 9u displayed promising
selectivity towards PI3K-α isoform over β- and δ-isoforms (>49 fold selectivity). The
isoquinolinyl substituted analog 9x displayed high selectivity towards PI3K-α isoform over β-
and γ-isoform (≥56-fold selectivity). In particular, the compound 9x did not inhibit (0%
inhibition) PI3K-β up to 20 µM. Thus, the compound 9u is a selective dual alpha/gamma
inhibitor with a slight preference for alpha; whereas compound 9x is a selective dual alpha/delta
inhibitor with a slight preference for alpha.
4
-Acetylphenyl substituted analog 9d was found to be an inhibitor of PI3K-α and γ isoforms with
IC values of 270 and 150 nM, respectively. Interestingly, 4-vinylphenyl substituted analog 9h
5
0
was found to be a selective inhibitor of PI3K-γ, and with no inhibition of PI3K-α, β and δ
isoforms (IC50 >10 µM). PI3K family member kinases share close homology in structural
sequence, function and topology and therefore, targeting of cancer cells by isoform-selective
PI3K inhibitors is challenging due to conserved kinase residues (PI3K-α and γ share ~35%
overall homology and ~43.5% kinase domain) [30] in ATP binding site.
Experimentally, it was observed that 9u inhibit both α and γ isoforms with moderate selectivity
(
4-fold selective towards PI3K-α), however, 9x selectively inhibit PI3K-α enzyme over PI3K-γ
>50-fold selective). Comparison of interactions of 9x with PI3K-α (PDB: 4L23) and PI3K-γ
(
isoform (PDB: 3IBE) reveals that at selectivity pocket/activation loop of enzymes, benzyl

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cyanide group adopts different conformations due to difference in topology of PI3K-α and PI3K-
γ isoform. Furthermore, in PI3K-γ solvent exposed area, neutral Gln859, Ser854, and Ser 919
residues of PI3K-α were replaced by the charged Lys890, Ala885, and Asp950 residues, which
leads to loss of vander waal interactions as shown in supporting information (Figure S1).
All compounds were then screened for cytotoxicity in a panel of cancer lines including HL-60
(human leukemia cells), A-375 (human malignant melanoma), MCF-7 (human breast
adenocarcinoma cell line), Panc-1 (human pancreatic cancer), and PC-3 (human prostate cancer)
using 48 h as incubation time. Results are depicted in Table 2. The 6-isoquinolinyl derivative 9x
displayed significant cytotoxicity in all tested cancer lines with GI values in the range of 9-12
5
0
µM. Compound 9x displayed time-dependent cytotoxicity in PC-3 cells, possessing GI of 567,
5
0
1
89, 28 and 9 µM at 6, 12, 24 and 48 h, respectively. The 6-indolyl substituted derivative 9u
showed promising in-vitro cytotoxicity in breast cancer MCF-7 cells with GI value of 7 µM.
5
0
The 6-(2,4-difluorophenyl) substituted analog 9b showed significant cytotoxicity in HL-60 and
A375 cells with GI values of 7 and 9 µM, respectively. Two best compounds 9u and 9x were
5
0
then screened for in-vitro cytotoxicity in 3 normal cell lines (Table 3). Results indicated that
these compound are non-toxic to normals cells (GI >50 µM). In order to address the cell death
5
0
caused by compounds 9u and 9x, the extent of apoptotic death in MCF-7 and PC-3 cell lines was
assessed using flow cytometry and by analyzing effect on mitochondrial membrane potential
(
MMP) loss. Both compounds exhibited a dose-dependent increase in sub-G1 population
apoptosis), and also showed mitochondrial membrane potential (MMP) loss in MCF-7 and PC-3
(
cells (see, Figure S2 and S3 of supporting information).
<
< Table 2 >>
< Table 3 >>
<

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To assess the obtained isoform-selectivity results of enzyme assays, the effect of compounds 9u
and 9x on the expression of various isoforms of PI3K (α, β, γ and δ) was investigated in MCF-7
and PC-3 cells by western-blot analysis. As seen in Figure 3a, compound 9u showed 44, 4, 16
and 30% inhibition of PI3K-α, β, γ and δ isoforms at 30 µM in MCF-7 cells, indicating
selectivity towards α-isoform. Similarly, another compound 9x also displayed higher selectivity
towards α-isoform over other three isoforms. As seen in Figure 3b, compound 9x showed 100,
4
2, 20 an 32% inhibition of PI3K-α, β, γ and δ isoforms at 30 µM in PC-3 cells, respectively
(Figure 3a-d).
The PI3K activation phosphorylates and activates Akt in the plasma membrane. Activated Akt
mediates downstream responses, including cell survival, growth, proliferation, cell migration and
angiogenesis, by phosphorylating a range of intracellular proteins [31-33]. Therefore, the
downstream effect of PI3K inhibition was assessed by investigating effect of these compounds
on levels of phospho-Akt. Compounds 9u and 9x displayed 80% and 50% inhibition of phospho-
Akt levels at 30 µM, in MCF-7 and PC-3 cells, respectively (Figure 3a-d).
<< Figure 3 >>
2
.4. Solubility, Caco-2 permeability, CYL liability and in-vivo anticancer activity. The
solubility of most potent compounds 9e, 9u and 9x was determined in water, PBS, SGF and SIF.
All three compounds displayed good solubility in SGF (80-125 µg/ml), whereas low solubility in
water, PBS and SIF (1-10 µg/ml) (Table S1 of supporting information). Compound 9x does not
have CYP liability against major metabolizing enzymes CYP3A4 and CYP2D6 (27 and 0%
inhibition at 10 µM). Similarly, another compound 9u also do not have CYP liability (51 and 29
inhibition at 10 µM) for these two major CYPs. Further, in Caco-2 permeability assay, the efflux

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ratio of 1.2 and 1.3, indicated that these compounds are not substrates of efflux transporter
pumps. Next, in order to demonstrate the anticancer activity in in-vivo models, these two
compounds were tested for tumor growth inhibition in Ehrlich Ascites Carcinoma (EAC) model.
EAC has undifferentiated cells and has a rapid growth rate and thus it resembles human tumors
which are most sensitive to chemotherapy. The effect of compound treatment on the body weight
of mice is shown in Table S2, which indicated that both these compounds does not led to change
in body weight of animals. The tumor growth inhibition results of EAC cells are shown in Table
4
. Compound 9x showed 25.8% inhibition in the growth of EAC cells in the peritoneal cavity of
experimental animals at a dose of 70 mg/kg of body weight. While, compound 9u displayed
promising tumor growth inhibition (62%) at 25 mg/kg dose. Based on the results obtained in
EAC study, compound 9u was further studied for its effect in Ehrlich Solid Tumor (EST) model,
wherein it showed 36.5% TGI at 25 mg/kg dose without causing any mortality and change in
body weight.
<< Table 4 >>
3
. CONCLUSION
In summary, a series of 6-aryl substituted 4-(4-cyanomethyl)phenylamino quinazolines have
been identified as a new class of potent and selective PI3K-α inhibitors. The 6-indolyl substituted
quinazoline 9u showed promising PI3K-α inhibition activity with excellent fold selectivity
towards α-isoform with respect to PI3Kβ and PI3K-δ isoforms and moderate selectivity over
PI3K-γ isoforms in both cell-free enzymatic and cell-based gene expression assay. The drug-like
properties (no CYP/ efflux pump liability) and promising in-vivo tumor growth inhibition in

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solid tumor model, indicated athat compound 9u has a promise for development as an anticancer
agent.
4
. EXPERIMENTAL SECTION
4
.1. General. All chemicals were obtained from Sigma-Aldrich Company and used as received.
1
13
H, C and DEPT NMR spectra were recorded on Brucker-Avance DPX FT-NMR 500 and 400
MHz instruments. Chemical data for protons are reported in parts per million (ppm) downfield
from tetramethylsilane and are referenced to the residual proton in the NMR solvent (CDCl3,
7
.26 ppm; CD OD, 3.31 ppm; DMSO-d , 2.50 ppm). The carbon nuclear magnetic resonance
3 6
1
3
spectra ( C NMR) were recorded at 125 MHz or 100 MHz: chemical data for carbons are
reported in parts per million (ppm, δ scale) downfield from tetramethylsilane and are referenced
to the carbon resonance of the solvent (CDCl , 77.16 ppm; CD OD, 49.0 ppm; DMSO-d , 39.52
3
3
6
ppm). HR-ESIMS spectra were recorded on Agilent HR-ESIMS-6540-UHD machine. IR spectra
were recorded on Perkin-Elmer IR spectrophotometer. Melting points were recorded on digital
melting point apparatus. HPLC analysis was done on Shimadzu HPLC system (model:
Shimadzu-LC 10AT) equipped with a PDA detector using Inertsil RP-18 (E-Merck, 5 µM, 4.0 x
2
0
50 mm) column and mobile phase used was acetonitrile and water as gradient at the flow rate of
.8 ml/min. All kinase screening was done at International Center for Kinase profiling (ICKP),
University of Dundee UK. The animals used in the study were breed in-house and used. The
animal facility is registered with CPCSEA vide registration no. 67/99/CPCSEA. All of the
biologically tested compounds have met the purity requirement.
4
.2. Synthesis of 2-amino-5-bromobenzoic acid (11). The solution of anthranilic acid (10, 1
g, 7.3 mmol) in glacial acetic acid (10 mL) was cooled to 15 °C. Bromine solution (0.45 mL,
.76 mmol) was added dropwise to the reaction mixture, which resulted in thick mass of white
8

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glistening crystals consisting of the hydrobromides of the mono and dibromo anthranilic acid.
The product was filtered off, washed with benzene and dried. It was then refluxed with dilute
hydrochloric acid (20 mL) and filtered while hot under suction. The insoluble residue was
extracted twice with boiling water (500 ml). The filtrate, upon cooling yielded an abundant
precipitate of 2-amino-5-bromobenzoic acid (11). Yield: 55%; light brown solid; m.p. 209-211
1
°
C; H NMR (CD OD, 500 MHz): δ 7.87 (t, 1H, J = 5.2 Hz), 7.31-7.28 (m, 1H), 6.67 (dd, 1H, J
3
=
8.8, 5.2 Hz); IR (CHCl ): ν 3474, 3360, 2919, 1674, 1608, 1581, 1548, 1481, 1421, 1311,
3 max
-1
+
1
293, 1168, 1127, 1089, 1019 cm ; HR-ESIMS: m/z 215.9654 calcd for C H BrNO +H
7 6 2
(215.9655).
4
.3. Synthesis of 6-bromoquinazolin-4-ol (12) and 6,7-dimethoxyquinazolin-4-ol (17). To
the solution of 2-amino-5-bromo-benzoic acid (11, 1 g, 4.63 mmol) or 2-amino-4,5-
dimethoxybenzoic acid (16, 1 g, 5.08 mmol), formamide (0.74 mL, 18.52 mmol for 11 and 0.8
mL, 20.3 mmol for 16) was added and the resultant mixture was allowed to reflux at 150 °C for 6
h. After completion of reaction, the reaction mixture was filtered through Whatmann filter paper
and dried under vacuum to get desired products 12 or 17.
1
4
.3.1. 6-Bromoquinazolin-4-ol (12): Yield: 70%; white solid; m.p. 273-275 °C; H NMR
(
DMSO-d , 500 MHz): δ 8.20-8.16 (m, 1H), 7.98-7.95 (m, 1H), 7.63 (d, 1H, J = 8.7 Hz); IR
6
(
CHCl ): ν 3442, 2922, 2852, 1726, 1697, 1605, 1465, 1389, 1318, 1272, 1239, 1220, 1178,
3
max
-1
81
+
1
019 cm ; HR-ESIMS: m/z 226.9633 calcd for C H BrN O+H (226.9658).
8 5 2
1
4
.3.2. 6,7-Dimethoxyquinazolin-4-ol (17): Yield: 76%; white solid; m.p. 296-298 °C; H
NMR (DMSO-d , 400 MHz): δ 7.99 (s, 1H), 7.44 (s, 1H), 7.13 (s, 1H), 3.90 (s, 3H), 3.87 (s, 3H);
6
IR (CHCl ): ν 3416, 2919, 1649, 1606, 1505, 1438, 1402, 1356, 1307, 1287, 1270, 1247,
3
max
-1
+
1
212, 1125, 1074, 1025 cm ; HR-ESIMS: m/z 207.0766 calcd for C H N O +H (207.0764).
10 10 2 3

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4
.4.
Synthesis
of
6-bromo-4-chloroquinazoline
(13)
and
4-chloro-6,7-
dimethoxyquinazoline (18). The solution of 6-bromoquinazolin-4-ol (12, 1 g, 4.44 mmol) or
,7-dimethoxyquinazolin-4-ol (17, 1 g, 4.13 mmol) in phosphorous oxychloride (5 mL) was
6
refluxed for 6 h at 120 °C. The mixture was cooled to room temperature and poured into ice-
water containing sodium bicarbonate to quench phosphorous oxychloride. The mixture was
extracted with dichloromethane (3 × 100 ml) and the solvent was evaporated to get chlorinated
products 13 or 18.
4
.4.1. 6-Bromo-4-chloroquinazoline (13): Yield: 74%; light yellow solid; m.p. 161-163 °C;
1
H NMR (CDCl , 500 MHz): δ 9.07 (s, 1H), 8.44 (d, 1H, J = 2.0 Hz), 8.05 (dd, 1H, J = 8.9, 2.2
3
Hz), 7.96 (d, 1H, J = 8.9 Hz); IR (CHCl ): ν 2922, 1632, 1559, 1546, 1474, 1461, 1388, 1360,
3
max
-1
+
1
241, 1160, 1019 cm ; HR-ESIMS: m/z 242.9326 calcd for C H BrClN +H (242.9319).
8 4 2
4
.4.2. 4-Chloro-6,7-dimethoxyquinazoline (18): Yield: 82%; light yellow solid; m.p. 184-186
1
°
C; H NMR (CDCl , 400 MHz): δ 8.88 (s, 1H), 7.40 (s, 1H), 7.34 (s, 1H), 4.09 (s, 6H); IR
3
(
CHCl ): ν 1674, 1614, 1557, 1510, 1473, 1455, 1442, 1435, 1413, 1357, 1349, 1281, 1259,
3 max
-1
+
1
204, 1164, 1124, 1071, 1029, 1013 cm ; HR-ESIMS: m/z 225.0428 calcd for C H ClN O +H
10 9 2 2
(225.0425).
4
.5. Synthesis of 6-bromo-4-(4-cyanomethyl)phenylamino quinazoline (15) and 6,7-
dimethoxy-4-(4-cyanomethyl)phenylamino quinazoline (19). The mixture of 6-bromo-4-
chloroquinazoline (13, 0.2 g, 0.83 mmol) or 4-chloro-6,7-dimethoxyquinazoline (18, 0.2 g, 0.89
mmol) and 4-amino benzyl cyanide (14, 0.142 g, 1.07 mmol for 13 and 0.153 g, 1.16 mmol for
1
8) in isopropanol (5 mL) was allowed to stir for 3 h under reflux condition. After completion of
reaction, the mixture was filtered through Whatman filter paper and dried under vacuum to get
the desired products 15 or 19.

ACCEPTED MANUSCRIPT
4
.5.1. 6-Bromo-4-(4-cyanomethyl)phenylamino quinazoline (15): Yield: 82%; brown solid;
1
m.p. 275-277 ºC; H NMR (DMSO-d , 400 MHz): δ 9.20 (s, 1H), 8.93 (s, 1H), 8.30-8.23 (m,
6
1
3
1
H), 7.96-7.89 (m, 1H), 7.83-7.76 (m, 2H), 7.48 (d, 2H, J = 7.2 Hz), 4.10 (s, 2H); C NMR
(
DMSO-d , 100 MHz): δ 158.71, 151.16, 138.71, 138.18, 135.91, 129.56, 128.43, 127.17,
6
1
1
24.96, 122.15, 120.86, 119.05, 115.01, 21.98; IR (CHCl ): νmax 3437, 2922, 1649, 1621, 1594,
3
-1
+
545, 1464, 1406, 1273, 1098, 1018 cm ; HR-ESIMS: m/z 339.0243 calcd for C H BrN +H
1
6
11
4
(339.0240).
4
.5.2. 6,7-Dimethoxy-4-(4-cyanomethyl)phenylamino quinazoline (19): Yield: 72%; brown
1
solid; m.p. 268-270 °C; HPLC purity: 99% (t = 24.58 min); H NMR (DMSO-d , 400 MHz): δ
R
6
1
1.40 (s, 1H), 8.82 (s, 1H), 8.31 (s, 1H), 7.72 (d, 2H, J = 8.4 Hz), 7.47 (d, 2H, J = 8.4 Hz), 7.35
1
3
(
s, 1H), 4.10 (s, 2H), 4.02 (s, 3H), 4.0 (s, 3H); C NMR (Pyridine-d + D O, 100 MHz): δ
5 2
1
5
1
59.45, 157.24, 155.19, 151.90, 148.72, 141.53, 130.70, 128.24, 121.57, 111.88, 108.97, 104.32,
8.68, 58.25, 24.69; IR (CHCl ): ν 3369, 2953, 2921, 2852, 1738, 1628, 1508, 1575, 1453,
3
max
-1
+
402, 1363, 1320, 1152, 1065, 1018 cm ; HR-ESIMS: m/z 321.1348 calcd for C H N O +H
1
8
16
4
2
(
321.1346).
.6. Procedure for Suzuki coupling: Synthesis of compounds 9a-x. A solution of 2M
K CO solution (3 ml) in dioxane (3 ml) was taken in round bottom flask and was purged with
4
2
3
nitrogen balloon for 5 min at room temperature. A mixture of boronic acid (1.2 mmol) and
compound 15 (0.1 g, 1 mmol) was added to this reaction mixture, and it was again purged with
nitrogen for 5 min. Pd(PPh ) (0.05 mmol) was then added, followed by purging and allowed the
3
4
reaction mixture to stir at 90 °C for overnight. After completion of the reaction, product was
extracted with ethyl acetate (2 × 50 ml). The combined organic layer was concentrated in vacuo

ACCEPTED MANUSCRIPT
and crude reaction mixture was purified by silica gel (#100-200) column chromatography using
EtOAc: hexane as eluent to get desired products 9a-x.
4
.6.1. 6-Phenyl-4-(4-cyanomethyl)phenylamino quinazoline (9a). Yield: 81%, brick red solid;
1
m.p. 253-255 °C; HPLC purity: 96% (t = 33.92 min); H NMR (DMSO-d 400 MHz): δ 10.01
R
6,
(s, 1H), 8.85 (s, 1H), 8.60 (s, 1H), 8.21 (d, 1H, J = 7.0 Hz), 7.91-7.87 (m, 4H), 7.62-7.58 (m,
1
3
4
1
1
1
H), 7.57-7.39 (m, 2H), 4.04 (s, 2H); C NMR (DMSO-d 125 MHz): δ 157.83, 154.42, 149.01,
6,
39.12, 138.49, 138.07, 131.79, 131.50, 131.42, 129.02, 128.78, 128.69, 128.37, 128.22, 127.92,
27.14, 126.39, 122.95, 120.45, 119.40, 115.33, 21.91; IR (CHCl ): ν 3400, 2924, 2853, 1609,
3
max
-1
+
437, 1192, 1119 cm ; HR-ESIMS: m/z 337.1452 calcd for C H N +H (337.1448).
2
2
16
4
4
.6.2. 6-(2,4-Difluorophenyl)-4-(4-cyanomethyl)phenylamino quinazoline (9b). Yield: 45%;
1
yellow solid; m.p. 186-188 °C; HPLC purity: 98% (t = 30.25 min); H NMR (CDCl , 400
R
3
MHz): δ 8.74 (s, 1H), 8.15 (s, 1H), 7.98-7.92 (m, 2H), 7.80 (d, 1H, J = 8.8 Hz), 7.55-7.46 (m,
1
9
2
H), 7.38 (d, 2H, J = 8.4 Hz), 7.06-6.97 (m, 2H), 3.78 (s, 2H); F NMR (DMSO-d , 376 MHz):
6
1
3
δ -110.14 to -110.23 (m, 1F), -113.34 to -113.41 (m, 1F); C NMR (CDCl + CD OD, 100
3
3
1
1
MHz): δ 162.53 (d, J = 237 Hz), 159.62 (d, J = 238 Hz), 154.55, 148.49, 138.13, 133.96,
CF
CF
1
1
1
33.44, 131.60-131.46 (m), 128.23, 127.45, 125.79, 123.14, 121.90, 117.86, 115.31, 111.79-
2
11.54 (m), 104.26 (t, J = 26 Hz), 22.72; IR (CHCl ): ν 3391, 2955, 2923, 2854, 1606,
CF
3
max
-1
574, 1532, 1515, 1495, 1424, 1401, 1269, 1142, 1101, 1020 cm ; HR-ESIMS: m/z 373.1258
+
calcd for C H F N +H (373.1259).
2
2
14
2
4
4
.6.3. 6-(2-Formylphenyl)-4-(4-cyanomethyl)phenylamino quinazoline (9c). Yield: 67%; brick
1
red solid; m.p. 177-179 °C; HPLC purity: 93% (t = 28.7 min); H NMR (CDCl + CD OD 400
R
3
3
,
MHz): δ 9.99 (s, 1H), 8.80 (s, 1H), 8.32 (s, 1H), 8.12 (d, 1H, J = 1.6 Hz), 7.99 (t, 2H, J = 8.8
Hz), 7.84-7.76 (m, 2H), 7.71-7.62 (m, 1H), 7.57-7.48 (m, 2H), 7.43-7.39 (m, 1H), 7.32-7.30 (m,

ACCEPTED MANUSCRIPT
1
3
1
1
1
1
1
H), 3.74 (s, 2H); C NMR (CDCl + CD OD, 100 MHz): δ 192.32, 154.88, 148.58, 144.54,
3 3
36.48, 134.82, 134.00, 133.56, 131.95, 131.85, 131.14, 128.73, 128.60, 128.45, 128.42, 128.22,
27.45, 123.72, 123.41, 118.03, 115.22, 22.88; IR (CHCl ): ν 3369, 2956, 2924, 2854, 2250,
3
max
689, 1626, 1596, 1571, 1529, 1515, 1479, 1423, 1402, 1360, 1307, 1252, 1194, 1173, 1120,
-1
+
070, 1021 cm ; HR-ESIMS: m/z 365.1397 calcd for C H N O+H (365.1397).
2
3
16
4
4
.6.4. 6-(4-Acetylphenyl)-4-(4-cyanomethyl)phenylamino quinazoline (9d). Yield: 45%; pale
1
yellow solid; m.p. 157-159 °C; HPLC purity: 93% (t = 31.16 min); H NMR (CDCl + CD OD,
R
3
3
4
00 MHz): δ 8.65 (d, 1H, J = 9.0 Hz), 8.12 (t, 2H, J = 8.5 Hz), 7.95-7.91 (m, 2H), 7.83-7.82 (m,
1
3
1
H), 7.42 (s, 2H), 7.41 (s, 1H), 7.40 (s, 3H), 3.84 ( s, 2H), 2.70 (s, 3H); C NMR (DMSO-d ,125
6
MHz): δ 197.59, 157.93, 154.87, 149.53, 143.38, 138.36, 136.63, 135.90, 132.05, 131.50,
1
2
1
31.42, 128.95, 128.80, 128.71, 128.58, 128.26, 127.27, 126.57, 123.08, 121.20, 119.41, 115.33,
6.85, 21.90; IR (CHCl ): ν 3369, 2953, 2924, 2855, 2250, 1738, 1678, 1603, 1573, 1532,
3
max
-1
+
515, 1424, 1362, 1265, 1020 cm ; HR-ESIMS: m/z 379.1555 calcd for C H N O+H
2
4
18
4
(379.1553).
4
.6.5. 6-(3-Hydroxyphenyl)-4-(4-cyanomethyl)phenylamino quinazoline (9e). Yield: 62%;
1
brown solid; m.p. 222-224 °C; HPLC purity: 98% (t = 32.75 min); H NMR (DMSO-d , 400
R
6
MHz): δ 10.01 (s, 1H), 9.65 (s, 1H), 8.81 (d, 1H, J = 1.6 Hz), 8.59 (s, 1H), 8.13 (dd, 1H , J = 8.4,
2
2
1
2
1
.0 Hz), 7.90-7.84 (m, 2H), 7.65-7.55 (m, 4H), 7.41-7.26 (m, 2H), 6.87-6.85 (m, 1H), 4.05 (s,
13
H); C NMR (DMSO-d 100 MHz): δ 157.89, 157.85, 154.34, 149.01, 140.59, 138.53, 138.28,
6
,
31.76, 129.99, 128.29, 128.19, 126.36, 122.96, 120.33, 119.35, 117.95, 115.30, 114.87, 114.05,
1.91; IR (CHCl ): ν 3400, 3055, 2955, 2924, 2854, 1731, 1591, 1484, 1437, 1400, 1275,
3
max
-1
+
219, 1189, 1119, 1072, 1020 cm ; HR-ESIMS: m/z 353.1402 calcd for C H N O+H
2
2
16
4
(353.1397).

ACCEPTED MANUSCRIPT
4
.6.6. 6-(3-Nitrophenyl)-4-(4-cyanomethyl)phenylamino quinazoline (9f). Yield: 57%; pale
1
yellow solid; m.p. 216-218 °C; HPLC purity: 95% (t = 33.91 min); H NMR ( CDCl + CD OD,
R
3
3
4
1
2
1
1
1
00 MHz): δ 8.69 (d, 2H, J = 8.4 Hz), 8.29 (d, 1H, J = 8.0 Hz), 8.14 ( d, 1H, J = 7.2 Hz), 7.97 (d,
H, J = 8.8 Hz), 7.83 (d, 2H, J = 8.0 Hz), 7.73 (t, 1H, J = 8.0 Hz), 7.65-7.58 (m, 2H), 7.51 (s,
1
3
H), 3.84 (s, 2H); C NMR (CDCl + CD OD 125 MHz): δ 158.67, 154.75, 148.69, 141.30,
3
3
,
36.79, 133.06, 132.30, 131.87, 131.77, 131.69, 129.94, 128.59, 128.49, 128.29, 127.99, 126.09,
23.36, 122.37, 121.85, 120.86, 117.96, 22.63; IR (CHCl ): ν 3401, 2955, 2923, 2853, 1733,
3
max
-1
+
607, 1536, 1423, 1384, 1157, 1021 cm ; HR-ESIMS: m/z 382.1302 calcd for C H N O +H
2
2
15
5
2
(382.1299).
4
.6.7. 6-(2-Methylphenyl)-4-(4-cyanomethyl)phenylamino quinazoline (9g). Yield: 57%;
1
orange solid; m.p. 173-175 °C; HPLC purity: 95% (t = 33.79 min); H NMR (CDCl 400
R
3,
MHz): δ 8.83-8.81 (m, 1H), 8.03-8.00 (m, 1H), 7.98-7.96 (m, 4H), 7.55 (s, 1H), 7.43-7.27 (m,
1
3
6
1
1
1
3
H), 3.78 (s, 2H), 2.32 (s, 3H); C NMR (CDCl 100 MHz): δ 157.67, 154.73, 148.95, 140.83,
3,
40.54, 138.33, 135.42, 134.63, 130.60, 129.79, 128.64, 128.37, 128.09, 126.06, 125.66, 122.52,
20.74, 117.97, 115.07, 23.14, 20.47; IR (CHCl ): ν 3368, 2955, 2924, 2853, 2252, 1626,
3
max
-1
604, 1572, 1527, 1515, 1486, 1421, 1403, 1360, 1307, 1242, 1190, 1020 cm ; HR-ESIMS: m/z
+
51.1602 calcd for C H N +H (351.1604).
2
3
18
4
4
.6.8. 6-(4-Vinylphenyl)-4-(4-cyanomethyl)phenylamino quinazoline (9h). Yield: 71%; pale
1
yellow solid; m.p. 216-218 °C; HPLC purity: 93% (t = 35.16 min); H NMR (CDCl + CD OD
R
3
3
,
4
00 MHz): δ 8.58 (d, 1H, J = 17.2 Hz), 8.10 (d, 1H, J = 8.4 Hz), 7.89 (d, 1H, J = 8.4 Hz), 7.82-
.75 (m, 4H), 7.60-7.51 (m, 3H), 7.44-7.39 (m, 2H), 6.83-6.76 (m, 1H), 5.85 (d, 1H, J = 17.6
7
1
3
Hz), 5.33 (d, 1H, J = 10.8 Hz), 3.84 (s, 2H); C NMR (DMSO-d 125 MHz): δ 158.30, 154.92,
6
,
1
49.57, 138.96, 138.87, 137.93, 137.19, 136.56, 132.05, 131.97, 128.89, 128.70, 127.72, 127.27,

ACCEPTED MANUSCRIPT
1
26.90, 123.48, 120.64, 119.87, 115.83, 115.30, 22.39; IR (CHCl ): ν 3369, 2951, 2923, 2857,
3
max
-1
2
248, 1741, 1623, 1603, 1571, 1514, 1497, 1423, 1360, 1021 cm ; HR-ESIMS: m/z 363.1605
+
calcd for C H N +H (363.1604).
2
4
18
4
4
.6.9. 6-(4-Fluorobenzyloxyphen-4-yl)-4-(4-cyanomethyl)phenylamino quinazoline (9i).
1
Yield: 58%; pale yellow solid; m.p. 230-232 °C; HPLC purity: 96% (t = 33.92 min); H NMR
R
(
CDCl + CD OD 500 MHz): δ 8.61 (s, 1H), 8.45 (s, 1H), 8.04 (d, 1H, J = 2.0 Hz), 7.88 (d, 1H,
3 3 ,
J = 8.8 Hz), 7.81 (d, 2H, J = 8.4 Hz), 7.73-7.70 (m, 2H), 7.47 (d, 2H, J = 5.4 Hz), 7.45-7.39 (m,
1
3
3
1
1
H), 7.13-7.11 (m, 3H), 5.12 (s, 2H), 3.83 (s, 2H); C NMR (CDCl + CD OD 125 MHz): δ
3 3 ,
2
58.42, 153.75, 147.84, 139.15, 132.26 (d, J = 37.5 Hz), 129.20, 129.13, 128.17, 127.26,
CF
19
25.84, 123.26, 119.11, 117.95, 115.59, 115.21, 115.08, 115.04, 69.15, 22.50; F NMR
(
DMSO-d , 376 MHz): δ -114.38 to -114.45 (m, 1F); IR (CHCl ): ν 3400, 2955, 2923, 2854,
6 3 max
-1
1
605, 1573, 1498, 1514, 1423, 1401, 1384, 1225, 1157, 1020 cm ; HR-ESIMS: m/z 461.1777
+
calcd for C H FN O+H (461.1772).
2
9
21
4
4
.6.10. 6-(3-Acetylaminophenyl)-4-(4-cyanomethyl)phenylamino quinazoline (9j). Yield: 61%;
1
pale yellow solid; m.p. 195-197 °C; HPLC purity: 92% (t = 34.26 min); H NMR (DMSO-d
R
6,
4
00 MHz): δ 10.10 (s, 1H), 10.01 (s, 1H), 8.80 (d, 1H, J = 1.2 Hz), 8.60 (s, 1H), 8.09 (dd, 1H, J
=
8.8, 1.6 Hz), 8.00 (s, 1H), 7.89 (dd, 3H, J = 8.8, 1.6 Hz), 7.66-7.46 (m, 3H), 7.39 (d, 2H, J =
1
3
8
1
1
3
.4 Hz), 4.04 (s, 2H), 2.09 (s, 3H); C NMR (DMSO-d 125 MHz): δ 168.40, 157.79, 154.38,
6,
48.98, 139.85, 139.80, 138.43, 138.23, 131.42, 131.34, 129.30, 128.71, 128.61, 128.38, 128.14,
26.32, 122.87, 122.05, 120.57, 119.31, 118.50, 117.79, 115.26, 23.95, 21.83; IR (CHCl ): ν
3
max
-1
368, 2921, 1676, 1608, 1534, 1515, 1480, 1426, 1119 cm ; HR-ESIMS: m/z 394.1667 calcd for
+
C H N O+H (394.1662).
24
19
5

ACCEPTED MANUSCRIPT
4
.6.11. 6-(4-Phenylphenyl)-4-(4-cyanomethyl)phenylamino quinazoline (9k). Yield: 54%;
1
yellow solid; m.p. 214-216 °C; HPLC purity: 99% (t = 34.25 min); H NMR (DMSO-d , 400
R
6
MHz): δ 10.03 (s, 1H), 8.92 (d, 1H, J = 1.6 Hz), 8.61 (s, 1H), 8.29-8.27 (m, 1H), 8.03 (d, 2H, J =
8
4
1
1
1
.4 Hz), 7.92-7.87 (m, 5H), 7.80 (d, 2H , J = 1.2 Hz), 7.52 (t, 2H, J = 7.6 Hz), 7.43-7.40 (m, 3H),
1
3
.01 (s, 2H); C NMR (DMSO-d , 125 MHz): δ 157.84, 154.47, 149.11, 139.59, 139.41, 138.46,
6
38.01, 137.44, 131.64, 129.02, 128.47, 128.25, 127.69, 127.62, 127.25, 126.65, 123.03, 120.27,
19.42, 115.38, 21.90; IR (CHCl ): ν 3401, 2953, 2927, 1604, 1567, 1515, 1486, 1423, 1358,
3
max
-1
+
021 cm ; HR-ESIMS: m/z 413.1769 calcd for C H N +H (413.1761).
2
8
20
4
4
.6.12. 6-(4-(4-Ethoxyphenyl)phenyl)-4-(4-cyanomethyl)phenylamino quinazoline (9l). Yield:
1
4
4
8
8
7%; brown solid; m.p. 195-197 °C; HPLC purity: 99% (t = 37.33 min); H NMR (DMSO-d ,
R
6
00 MHz): δ 10.04 (s, 1H), 8.91 (s, 1H), 8.61 (s, 1H), 8.27 (d, 1H, J = 8.8 Hz), 7.99 (d, 2H, J =
.8 Hz), 7.91-7.88 (m, 3H), 7.83 (d, 2H, J = 8.4 Hz), 7.72 (d, 2H, J = 8.4 Hz), 7.41 (d, 2H, J =
13
.4 Hz), 7.06 (d, 2H, J = 8.8 Hz), 4.12-4.07 (m, 2H), 4.06 (s, 2H), 1.37 (t, 3H, J = 6.8 Hz); C
NMR (DMSO-d , 125 MHz): δ 158.37, 157.85, 154.41, 149.03, 139.31, 138.48, 137.56, 137.22,
6
1
31.58, 128.43, 128.24, 127.73, 127.54, 126.63, 123.02, 120.08, 114.90, 63.12, 21.91, 14.64; IR
(
CHCl ): ν 3306, 2956, 2925, 2855, 1729, 1604, 1568, 1515, 1494, 1424, 1401, 1360, 1252,
3
max
-1
+
1
190, 1082, 1019 cm ; HR-ESIMS: m/z 457.2012 calcd for C H N O+H (457.2023).
30 24 4
4
.6.13. 6-(4-Phenyl-2-fluorophenyl)-4-(4-cyanomethyl)phenylamino quinazoline (9m). Yield:
1
5
7%; pale yellow solid; m.p. 229-231 °C; HPLC purity: 98% (t = 34.64 min); H NMR (CDCl
R
3
+
CD OD, 400 MHz): δ 8.64-8.61 (m, 2H), 8.13 (dd, 1H, J = 8.4, 2.0 Hz), 7.93 (d, 1H, J = 8.8
3
Hz), 7.83 (d, 2H, J = 8.4 Hz), 7.68-7.61 (m, 4H), 7.51-7.47 (m, 2H), 7.43-7.38 (m, 4H), 3.85 (s,
1
9
13
2
H); F NMR (DMSO-d , 376 MHz): δ -117.72 (t, 1F, J = 11.3 Hz); C NMR (CDCl +
6 3
CD OD, 125 MHz): δ 162.53, 160.56, 160.21, 155.89, 150.11, 142.10, 139.31, 136.60, 133.53,
3

ACCEPTED MANUSCRIPT
1
1
1
32.63, 132.60, 130.25, 130.22, 129.86, 129.83, 129.23, 129.15, 127.63, 124.94, 124.43, 121.75,
2
19.58, 117.2, 116.07 (d, J = 23.7 Hz), 24.13; IR (CHCl ): ν 3392, 2951, 2924, 2853, 2250,
CF
3
max
-1
+
604, 1573, 1515, 1483, 1424, 1021 cm ; HR-ESIMS: m/z 431.1666 calcd for C H FN +H
2
8
19
4
(431.1667).
4
.6.14. 6-(Naphthalen-2-yl)-4-(4-cyanomethyl)phenylamino quinazoline (9n). Yield: 57%;
1
pale yellow solid; m.p. 204-206 °C; HPLC purity: 91% (t = 36.18 min); H NMR (DMSO-d ,
R
6
4
8
7
1
1
00 MHz): δ 9.98 (s, 1H), 8.93 (d, 1H, J = 1.6 Hz), 8.55 (s, 1H), 8.37 (s, 1H), 8.30 (dd, 1H, J =
.8, 2.0 Hz), 8.04 (s, 2H), 7.98 (d, 1H, J = 7.2 Hz), 7.93 (d, 1H, J = 6.8 Hz), 7.87-7.83 (m, 2H),
1
3
.54-7.50 (m, 3H), 7.34 (d, 2H, J = 8.4 Hz), 3.98 (s, 2H); C NMR (DMSO-d , 125 MHz): δ
6
57.85, 154.48, 149.12, 136.42, 133.24, 132.34, 132.00, 131.46, 131.38, 128.75, 128.66, 128.56,
28.46, 128.20, 127.55, 126.59, 126.42, 126.39, 125.78, 125.28, 123.03, 120.69, 115.40, 21.89;
IR (CHCl ): ν 3369, 3053, 2955, 2924, 2854, 2250, 1733, 1603, 1572, 1529, 1515, 1468,
3
max
-1
1
422, 1404, 1385, 1361, 1245, 1175, 1119, 1070, 1020 cm ; HR-ESIMS: m/z 387.1611 calcd for
+
C H N +H (387.1604).
26
18
4
4
.6.15. 6-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-4-(4-cyanomethyl)phenylamino quinazoline
1
(
9o). Yield: 69%; pale yellow solid; m.p. 207-209 °C; HPLC purity: 96% (t = 30.66 min); H
R
NMR (DMSO-d , 400 MHz): δ 9.95 (s, 1H), 8.76 (d, 1H, J = 1.6 Hz), 8.57 (s, 1H), 8.15 (dd, 1H,
6
J = 8.8, 2.0 Hz), 7.89 (d, 2H, J = 8.4 Hz), 7.82 (d, 1H, J = 8.8 Hz), 7.65-7.53 (m, 1H), 7.46 (d,
1
3
1
H, J = 2.0 Hz), 7.41-7.37 (m, 2H), 7.03 (d, 1H, J = 8.4 Hz), 4.32 (s, 4H), 4.05 (s, 2H); C NMR
(
CDCl + CD OD, 125 MHz): δ 158.43, 153.87, 148.00, 143.73, 143.59, 139.00, 133.02, 132.12,
3
3
1
1
1
31.78, 131.70, 128.59, 128.49, 128.24, 127.37, 125.78, 123.26, 120.13, 119.17, 118.00, 117.58,
15.79, 64.32, 64.26, 22.65; IR (CHCl ): ν 3400, 2922, 2853, 1602, 1514, 1495, 1422, 1307,
3
max
-1
+
249, 1068, 1021 cm ; HR-ESIMS: m/z 395.1508 calcd for C H N O +H (395.1503).
2
4
18
4
2

ACCEPTED MANUSCRIPT
4
.6.16. 6-(Benzo[d][1,3]dioxol-5-yl)-4-(4-cyanomethyl)phenylamino quinazoline (9p). Yield:
1
5
4
7%; yellow solid; m.p. 241-243 °C; HPLC purity: 91% (t = 29.68 min); H NMR (DMSO-d ,
R
6
00 MHz): δ 9.90 (s, 1H), 8.76 (d, 1H, J = 1.6 Hz), 8.58 (s, 1H), 8.23-8.15 (m, 1H), 7.99-7.81
(m, 3H), 7.53 (d, 1H, J = 1.6 Hz), 7.41-7.39 (m, 2H), 7.12 (d, 1H, J = 4.8 Hz), 6.12 (s, 2H), 4.05
1
3
(
s, 2H); C NMR (DMSO-d , 100 MHz): δ 157.74, 154.21, 148.78, 148.13, 147.26, 138.52,
6
1
1
37.73, 133.28, 131.58, 128.25, 128.20, 126.38, 122.95, 120.85, 119.71, 119.33, 115.29, 108.74,
-1
07.42, 101.32, 21.91; IR (CHCl ): ν 3400, 2923, 1603, 1514, 1419, 1220, 1039 cm ; HR-
3
max
+
ESIMS: m/z 381.1317 calcd for C H N O +H (381.1346).
2
3
16
4
2
4
.6.17. 6-(Benzofuran-2-yl)-4-(4-cyanomethyl)phenylamino quinazoline (9q). Yield: 62%;
1
pale yellow solid; m.p. 245-247 °C; HPLC purity: 92% (t = 32.17 min); H NMR (DMSO-d ,
R
6
4
00 MHz): δ 10.14 (s, 1H), 9.09 (d, 1H, J = 1.6 Hz), 8.61 (s, 1H), 8.41-8.38 (m, 1H), 7.93-7.88
(m, 2H), 7.75 (d, 1H, J = 7.2 Hz), 7.71-7.69 (m, 1H), 7.65-7.60 (m, 2H), 7.58-7.53 (m, 1H),
1
3
7
1
1
2
.42-7.39 (m, 2H), 7.32-7.30 (m, 1H), 4.06 (s, 2H); C NMR (DMSO-d 125 MHz): δ 157.84,
6,
54.88, 154.57, 154.46, 149.84, 138.42, 131.50, 131.42, 128.79, 128.70, 128.65, 128.23, 125.09,
23.46, 123.06, 121.45, 119.42, 118.65, 115.39, 111.15, 103.34, 21.91; IR (CHCl ): ν 3400,
3
max
-1
955, 2923, 2853, 1605, 1572, 1515, 1422, 1385, 1020 cm ; HR-ESIMS: m/z 377.1399 calcd for
+
C H N O+H (377.1397).
24
16
4
4
.6.18. 6-(Benzo[b]thiophen-2-yl)-4-(4-cyanomethyl)phenylamino quinazoline (9r). Yield:
1
6
4
5%; yellow solid; m.p. 263-265 °C; HPLC purity: 98% (t = 32.98 min); H NMR (DMSO-d ,
R
6
00 MHz): δ 10.04 (s, 1H), 8.91 (s, 1H), 8.58 (s, 1H), 8.24 (dd, 1H, J = 8.4, 2.0 Hz), 8.05-8.02
1
3
(
m, 2H), 7.91-7.84 (m, 4H), 7.44-7.38 (m, 4H), 4.02 (s, 2H); C NMR (DMSO-d 100 MHz): δ
6,
1
57.69, 154.65, 149.52, 142.35, 140.22, 138.89, 138.30, 131.42, 130.88, 128.63, 128.14, 126.51,
1
24.90, 124.86, 123.76, 123.09, 122.45, 121.09, 119.89, 119.25, 115.36, 21.85; IR (CHCl ): ν
3
max

ACCEPTED MANUSCRIPT
-1
3
3
392, 2951, 2922, 2852, 1603, 1572, 1514, 1419, 1403, 1361, 1157, 1020 cm ; HR-ESIMS: m/z
+
93.1163 calcd for C H N S+H (393.1168).
2
4
16
4
4
.6.19. 6-(Dibenzo(b,d)furan-4-yl)-4-(4-cyanomethyl)phenylamino quinazoline (9s). Yield:
1
4
2%; pale yellow solid; m.p. 207-209 °C; HPLC purity: 93% (t = 37.87 min); H NMR
R
(
DMSO-d , 400 MHz) : δ 10.03 (s, 1H), 9.02 (d, 1H, J = 1.6 Hz), 8.66 (s, 1H), 8.46 (dd, 1H, J =
6
8
.8, 2.0 Hz), 8.26-8.23 (m, 2H), 7.99 (d, 1H , J = 8.8 Hz), 7.91 (d, 3H, J = 8.0 Hz), 7.79 (d, 1H, J
1
3
=
8.0 Hz), 7.65-7.55 (m, 3H), 7.48-7.39 (m, 2H), 4.05 (s, 2H); C NMR (CDCl + CD OD, 125
3 3
MHz): δ 156.04, 154.55, 153.18, 148.63, 134.29, 131.94, 131.86, 128.70, 128.60, 128.44,
1
2
27.50, 127.02, 123.39, 123.31, 123.04, 120.76, 120.44, 111.73, 22.93; IR (CHCl ): νmax 3392,
3
-1
955, 2923, 2853, 1604, 1573, 1530, 1515, 1490, 1451, 1402, 1362, 1189, 1120, 1020 cm ; HR-
+
ESIMS: m/z 427.1553 calcd for C H N O+H (427.1553).
2
8
18
4
4
.6.20. 6-(Dibenzo(b,d)thiophene-4-yl)-4-(4-cyanomethyl)phenylamino quinazoline (9t).
1
Yield: 45%; pale yellow solid; m.p. 199-201 °C; HPLC purity: 91% (t = 34.88 min); H NMR
R
(
CDCl + CD OD, 400 MHz): δ 8.57 (s, 1H), 8.17-8.15 (m, 2H), 7.89 (d, 1H, J = 8.8 Hz), 7.78-
3 3
1
3
7
.70 (m, 2H), 7.56-7.51 (m, 5H), 7.44-7.39 (m, 3H), 7.30 (d, 1H, J = 8.8 Hz), 3.75 (s, 2H); C
NMR (CDCl + CD OD, 125 MHz): δ 148.58, 139.38, 138.51, 135.59, 133.53, 132.46, 132.43,
3
3
1
1
1
31.94, 131.86, 128.75, 128.66, 128.42, 127.22, 127.04, 126.17, 125.26, 124.61, 123.53, 122.59,
22.01, 121.80, 121.07, 118.12, 22.79; IR (CHCl ): ν 3392, 2922, 2853, 1605, 1571, 1537,
3
max
-1
+
514, 1421, 1026 cm ; HR-ESIMS: m/z 443.1320 calcd for C H N S+H (443.1325).
2
8
18
4
4
.6.21. 6-(1H-Indol-5-yl)-4-(4-cyanomethyl)phenylamino quinazoline (9u). Yield: 63%;
1
brown solid; m.p. 267-269 °C; HPLC purity: 95% (t = 29.14 min); H NMR (DMSO-d 400
R
6,
MHz): δ 11.23 (s, 1H), 9.99 (s, 1H), 8.85 (d, 1H, J = 1.2 Hz), 8.58 (s, 1H), 8.23 (dd, 1H, J = 8.8,
.6 Hz), 8.08 (s, 1H), 7.93-7.84 (m, 3H), 7.66-7.53 (m, 3H), 7.44-7.39 (m, 2H), 6.55 (s, 1H),
1

ACCEPTED MANUSCRIPT
1
3
4
1
1
1
.05 (s, 2H); C NMR (DMSO-d 125 MHz): δ 157.70, 153.86, 148.37, 139.94, 138.63, 135.70,
6,
31.50, 131.42, 130.21, 128.79, 128.70, 128.27, 128.19, 126.39, 126.25, 122.94, 120.72, 119.57,
19.43, 118.79, 111.91, 101.61, 21.90; IR (CHCl ): ν 3212, 2923, 2853, 1603, 1572, 1529,
3
max
-1
514, 1437, 1421, 1309, 1175, 1119, 1070 cm ; HR-ESIMS: m/z 376.1564 calcd for
+
C H N +H (376.1557).
24
17
5
4
.6.22. 6-(Quinolin-3-yl)-4-(4-cyanomethyl)phenylamino quinazoline (9v). Yield: 49%; off-
1
white solid; m.p. 225-227 °C; HPLC purity: 99% (t = 31.57 min); H NMR (DMSO-d , 400
R
6
MHz): δ 10.06 (s, 1H), 9.52 (d, 1H, J = 2.0 Hz), 9.10 (d, 1H, J = 1.6 Hz), 8.86 (d, 1H, J = 2.0
Hz), 8.64 (s, 1H), 8.45-8.42 (m, 1H), 8.12 (d, 2H, J = 8.4 Hz), 7.97 (d, 3H, J = 8.4 Hz), 7.91 (d,
1
3
1
H, J = 8.4 Hz), 7.83 (d, 1H, J = 1.2 Hz), 7.42 (d, 2H, J = 8.8 Hz), 4.05 (s, 2H); C NMR
(
DMSO-d , 125 MHz): δ 157.87, 154.84, 149.70, 149.42, 146.93, 138.39, 134.89, 133.29,
6
1
1
31.95, 131.82, 129.86, 128.74, 128.45, 128.27, 127.58, 127.27, 126.55, 123.01, 121.18, 119.38,
-1
15.44, 21.92; IR (CHCl ): ν 3400, 2922, 1617, 1423, 1130 cm ; HR-ESIMS: m/z 388.1559
3
max
+
calcd for C H N +H (388.1557).
2
5
17
5
4
.6.23. 6-(Pyridin-4-yl)-4-(4-cyanomethyl)phenylamino quinazoline (9w). Yield: 61%; pale
1
yellow solid; m.p. 234-236 °C; HPLC purity: 99% (t = 25.18 min); H NMR (CDCl + CD OD
R
3
3
,
4
00 MHz): δ 8.69-8.62 (m, 3H), 8.09 (t, 1H, J = 1.6 Hz), 7.97 (d, 1H, J = 8.4 Hz), 7.83 (d, 4H, J
1
3
=
8.4 Hz), 7.41-7.32 (m, 3H), 3.81 (s, 2H). C NMR (CDCl + CD OD, 125 MHz): δ 158.51,
3 3
1
1
1
54.91, 149.30, 147.56, 137.93, 135.52, 131.31, 128.13, 127.95, 125.94, 123.18, 121.70, 120.98,
17.80, 115.54, 22.50; IR (CHCl ): ν 3392, 2957, 2923, 2850, 1606, 1573, 1532, 1493, 1425,
3
max
-1
+
021 cm ; HR-ESIMS: m/z 338.1365 calcd for C H N +H (338.1400).
2
1
15
5
4
.6.24. 6-(Isoquinolin-4-yl)-4-(4-cyanomethyl)phenylamino quinazoline (9x). Yield: 37%;
1
pale yellow solid; m.p. 237-239 °C; HPLC purity: >99% (t = 32.66 min); H NMR (DMSO-d ,
R
6

ACCEPTED MANUSCRIPT
4
00 MHz): δ 9.90 (s, 1H), 9.45 (s, 1H), 8.81 (s, 1H), 8.69 (s, 1H), 8.63 (s, 1H), 8.30 (d, 1H, J =
.0 Hz), 8.06 (dd, 1H, J = 8.8, 1.6 Hz), 7.98 (d, 1H, J = 8.8 Hz), 7.92-7.77 (m, 4H), 7.37 (d, 2H,
8
13
J = 8.8 Hz), 4.03 (s, 2H); C NMR (DMSO-d , 125 MHz): δ 157.76, 154.84, 152.44, 149.23,
6
1
1
1
42.99, 138.46, 134.75, 134.55, 133.22, 131.71, 131.45, 128.20, 128.04, 127.97, 127.73, 126.36,
24.17, 124.10, 122.72, 119.35, 115.26, 21.89; IR (CHCl ): ν 3400, 2923, 2853, 1624, 1423,
3
max
-1
+
042 cm ; HR-ESIMS: m/z 388.1564 calcd for C H N +H (388.1557).
2
5
17
5
4
.7. PI3K-α assay. PI3K-α (diluted in 12.5 mM Glycine-NaOH (pH 8.5), 50 mM KCl, 2.5
mM MgCl , 1 mM DTT, 0.05% CHAPS) is assayed in total volume of 20 µL containing 12.5
2
mM glycine-NaOH (pH 8.5), 50 mM KCl, 2.5 mM MgCl , 1 mM DTT, 0.05% CHAPS, 0.01
2
mM ATP and 0.05 mM diC8 PIP2. The enzyme is assayed for 80 min after which 20 µL of
ADP-Glo reagent is added. After a further incubation of 40 min, 40 µL of kinase detection
buffer is added. The assays are incubated for 40 min and then read on PerkinElmer Envision for
1
sec/well.
4
.8. PI3K-β assay. PI3K beta (diluted in 12.5 mM glycine-NaOH (pH 8.5), 50 mM KCl,
2
.5mM MgCl , 1 mM DTT, 0.05% CHAPS) is assayed in total volume of 20 µL containing 12.5
2
mM Glycine-NaOH (pH 8.5), 50 mM KCl, 2.5 mM MgCl , 1 mM DTT, 0.05% CHAPS, 0.01
2
mM ATP and 0.05 mM diC8 PIP2. The enzyme is assayed for 60 min after which 20 µL of
ADP-Glo reagent is added. After a further incubation of 40 min, 40 µL of kinase detection
buffer is added. The assays are incubated for 40 min and then read on PerkinElmer Envision for
1
sec/ well.
.9. PI3K-δ assay. PI3K delta (diluted in 12.5 mM glycine-NaOH (pH 8.5), 50 mM KCl, 2.5
4
mM MgCl , 1 mM DTT, 0.05% CHAPS) is assayed in total volume of 20 µL containing 12.5
2
mM Glycine-NaOH (pH 8.5), 50 mM KCl, 2.5 mM MgCl , 1 mM DTT, 0.05% CHAPS, 0.01
2

ACCEPTED MANUSCRIPT
mM ATP and 0.05 mM diC8 PIP2. The enzyme is assayed for 120 min after which 20 µL of
ADP-Glo reagent is added. After a further incubation of 40 min, 40 µL of kinase detection
buffer is added. The assays are incubated for 40 min and then read on PerkinElmer Envision for
1
sec/well.
4
.10. PI3K-γ assay. PI3K gamma (diluted in 12.5 mM Glycine-NaOH (pH 8.5), 50 mM KCl,
2
.5 mM MgCl , 1 mM DTT, 0.05% CHAPS) is assayed in total volume of 20 µL containing 12.5
2
mM glycine-NaOH (pH 8.5), 50 mM KCl, 2.5 mM MgCl , 1 mM DTT, 0.05% CHAPS, 0.01
2
mM ATP and 0.05 mM diC8 PIP2. The enzyme is assayed for 75 min after which 20 µL of
ADP-Glo reagent is added. After a further incubation of 40 min, 40 µL of kinase detection buffer
is added. The assays are incubated for 40 min and then read on PerkinElmer Envision for 1sec/
well. All PI3K assays were carried out at International center for kinase profiling, University of
Dundee UK on commercial basis.
4
.11. mTOR assay. mTOR assay was performed at Reaction Biology Corporation using the
HotSpot’ assay platform. Kinase assay protocol; reaction buffer: base reaction buffer; 20 mM
Hepes (pH 7.5), 10 mM MgCl , 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM Na VO ,
‘
2
3
4
2
mM DTT, 1% DMSO. Required cofactors were added individually to kinase reaction. Reaction
procedure: To a freshly prepared buffer solution was added required cofactor for the enzymatic
reaction, followed by the addition of kinase at a concentration of 1 µM. The contents were mixed
gently, then the compounds under test dissolved in DMSO was added to the reaction mixture in
3
3
the appropriate concentration. P-ATP (specific activity 500 µCi/µL) was added to the mixture
in order to initiate the reaction, and the mixture was incubated at room temperature for 2 h. All
compounds were tested by single dose duplicate made at a concentration of 1 µM. Reaction was

ACCEPTED MANUSCRIPT
carried out at 10 µM ATP concentration. Reaction was spotted onto P81 ion exchange paper and
detected the kinase activity by filter-binding method.
4
.12. In-vitro cytotoxicity. In-vitro cytotoxicity of all compounds was determined in five
cancer cell lines viz. Panc-1 (pancreatic cancer), MCF-7 (breast cancer), PC-3 (prostate), HL-60
3
(leukemia) and A-375 (melanoma) using MTT assay. In each well of a 96-well plate, 3 x 10
cells were grown in 100 ꢀL of medium. After 24 h, each test molecules were added to achieve a
final concentration of 10 to 0.01 ꢀmol/L, respectively. After 48 h of treatment, 20 ꢀL of 2.5
mg/mL MTT (Organics Research, Inc.) solution in phosphate buffer saline was added to each
well. After 48h, supernatant was removed and formazan crystals were dissolved in 200 ꢀL of
DMSO. Absorbance was then measured at 570 nm using an absorbance plate reader (Bio-Rad
Microplate Reader). Data are expressed as the percentage of viable cells in treated relative to
non-treated conditions. Each experiment was repeated thrice and data was expressed as mean ±
SD of three independent experiments [34].
4
.13. Preparation of total cell lysates for immunoblotting. MCF-7 and PC-3 Cells (1 ×
6
1
0 ) were collected, washed with cold PBS, and incubated with cold lysis buffer (50 mM Tris pH
.0, 150 mM NaCl, 5 mM EDTA, 1% v/v Nonidet P-40, 1 mM PMSF, and 1% v/v eukaryotic
8
protease inhibitor cocktail) for 30 min on ice. Cells were centrifuged at 12,000 g for 10 min at 4
C, and the supernatant was collected as whole cell lysates for Western blot analysis of various
proteins.
°
4
.14. Western-blot analysis. The protein lysates prepared were subjected to discontinuous
SDS-PAGE analysis. Proteins aliquots (50 ꢀg) were resolved on SDS−PAGE and then electro
transferred to PVDF membrane overnight at 4 °C at 30 V. Nonspecific binding was blocked by
incubation with 5% nonfat milk in tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h

ACCEPTED MANUSCRIPT
at room temperature. The blots were probed with respective primary antibodies for 2 h and
washed three times with TBST. The blots were then incubated with horseradish peroxidase
conjugated mouse or rabbit secondary antibodies for 1 h, washed again three times with TBST,
and signals detected using ECL plus Chemiluminescence’s kit on X-ray film. Protein was
measured employing the Bio-Rad protein assay kit using bovine serum albumin as the standard
[
35].
.15. Cell cycle analysis by flow cytometry. The effect of compounds 9u and 9x on the DNA
4
content was analysed by cell cycle phase distribution analysis in MCF-7 and PC-3 cells,
respectively. Cells were incubated with 9u and 9x at indicated concentrations for 24 h time
period. Cells were collected at 400µg, washed with ice-cold PBS and fixed with ice-cold 70%
ethanol for overnight at 4 °C. Next day cells were incubated with RNase at concentration of 0.2
mg/mL at 37 °C for 1 h time period and stained with propidium iodide (10 ꢀg/mL) for 30 min in
dark. Cells were analysed on flow-cytometer (FACS Calibur, Becton Dickinson) and data were
collected in list mode on 10,000 events for FL2-A vs. FL2-W. Resulting DNA distributions were
analyzed by Modfit (Verity Software House Inc., Topsham, ME) for the proportions of cells in
apoptosis, G -phase, S- phase, and G -M phases of the cell cycle.
1
2
4
.16. Flow cytometric determination of mitochondrial membrane potential. Change in
mitochondrial transmembrane potential (∆Ψm) due to mitochondrial perturbation was measured
after staining with Rhodamine-123. MCF-7 and PC-3 cells were incubated with the indicated
doses of compounds 9u and 9x, respectively for 24 h. Rhodamine- 123 (5 ꢀM) was added 1 h
before the termination of the experiment and cells were collected, washed in PBS. The
fluorescence intensity of 10,000 events was analyzed in FL-1 channel on a BD FACS Calibur

ACCEPTED MANUSCRIPT
(Becton Dickinson, USA) flow cytometer. The decrease in fluorescence intensity caused by loss
of mitochondrial membrane potential was analyzed in FL-1 channel.
.17. Determination of thermodynamic equilibrium solubility by 96-well plate-based
4
assay. The compound was dissolved in methanol to get 2000 µg/mL stock solution. The stock
solution was introduced into 96-well plates and allowed to evaporate at room temperature to
ensure that the compound (1, 2, 4, 8, 16, 25, 40, 80, 160 and 300 µg) is in solid form in the
beginning of the experiment. Thereafter, 200 µl of the dissolution medium (water, PBS, SGF,
and SIF) was added to the wells and plates were shaken horizontally at 300 rpm (Eppendorf
Thermoblock Adapter, North America) for 4 h at room temperature (25 ± 1 °C). The plates were
covered with aluminium foil and were kept overnight for equilibration. Later, the plates were
centrifuged at 3000 rpm for 15 min (Jouan centrifuge BR4i). Samples of 50 µl was withdrawn
into UV 96-well plates (Corning® 96 Well Clear Flat Bottom UV-Transparent Microplate) for
analyses with plate reader at corresponding λmax of the sample (SpectraMax Plus384). The
analysis was performed in triplicate for each compound. The solubility curve of concentration
(
µg/mL) vs absorbance was plotted to find out saturation point and the corresponding
concentration was noted [36].
.18. hrCYP P450 isoenzyme assay. hrCYP P450 isoenzyme were aliquoted as per the total
4
concentration required to conduct the study and stored at -70 °C until use. Total assay volume
was adjusted to 200 ꢀL and consists of three components: cofactors, inhibitor/vehicle, and
enzyme-substrate (ES) mix. The 50 ꢀL of working cofactor stock solution was dispensed to all
the specified wells in a black colored nunc microtiter polypropylene plate. The 50 ꢀL of diluted
working concentrations of test compounds/positive control/vehicle were dispensed in triplicate to
the specified wells as per the plate map design. Reaction plate with cofactor and test item was