Design and synthesis of quinazolinones as EGFR inhibitors to overcome EGFR resistance obstacle

Article abstract of DOI:10.1016/j.bmc.2017.03.039

The epidermal growth factor receptor (EGFR) T790M mutant is found in about 50% of clinically acquired resistance to gefitinib among patients with non-small cell lung cancer (NSCLC). New derivatives of 4(3H)-quinazolinones were synthesized and evaluated for their inhibitory activity against NSCLC. The results of the study demonstrated that compound 79, 7-chloro-3-(5-(4-methoxyphenyl)-1,3,4-thiadiazol-2-yl)-2-phenylquinazolin-4(3H)-one was found to be the most potent compounds of the series with IC₅₀ value of 0.031?μM against mutant T790M/L858R EGFR. Compounds 15, 51, 73, 75, 78, 79 and 96 were less potent against A549 (WT EGFR and k-Ras mutation) and HT-29 (non-special gene type) cells, showing a high safety index. The obtained results showed that compounds 15, 51, 73, 75, 78, 79 and 96 could be the promising template to overcome drug resistance mediated by the EGFR T790 Mutant.

Full text of DOI:10.1016/j.bmc.2017.03.039

Accepted Manuscript
Design and Synthesis of Quinazolinones as EGFR inhibitors to Overcome
EGFR Resistance Obstacle
Harun M. Patel, Rahul Pawara, Azim Ansari, Malleshappa Noolvi, Sanjay
Surana
PII:
S0968-0896(17)30036-6
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.03.039
BMC 13637
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
9 January 2017
14 March 2017
16 March 2017
Please cite this article as: Patel, H.M., Pawara, R., Ansari, A., Noolvi, M., Surana, S., Design and Synthesis of
Quinazolinones as EGFR inhibitors to Overcome EGFR Resistance Obstacle, Bioorganic & Medicinal Chemistry
(
2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.03.039
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Design and Synthesis of Quinazolinones
as EGFR inhibitors to Overcome EGFR
Resistance Obstacle
a
a
a
b
a
Harun M. Patel *, Rahul Pawara , Azim Ansari , Malleshappa Noolvi , Sanjay Surana
a
Department of Pharmaceutical Chemistry, R. C. Patel Institute of Pharmaceutical Education and Research, Shirpur, District
Dhule-425 405, Maharashtra, India
b
Department of Pharmaceutical Chemistry, Shree Dhanvantary Pharmacy College, Kim (Surat)-3941110, Gujrat, India
Corresponding Author*
Tel: +91-8806621544; Email:hpatel_38@yahoo.com
Abstract
The epidermal growth factor receptor (EGFR) T790M mutant is found in about 50% of
clinically acquired resistance to gefitinib among patients with non-small cell lung cancer
(
NSCLC). New derivatives of 4(3H)-quinazolinones were synthesized and evaluated for their
inhibitory activity against NSCLC. The results of the study demonstrated that compound 79, 7-
chloro-3-(5-(4-methoxyphenyl)-1,3,4-thiadiazol-2-yl)-2-phenylquinazolin-4(3H)-one was found to
be the most potent compounds of the series with IC50 value of 0.031 µM against mutant
T790M/L858R EGFR. Compounds 15, 51, 73, 75, 78, 79 and 96 were less potent against A549
(
WT EGFR and k-Ras mutation) and HT-29 (non-special gene type) cells, showing a high safety
index. The obtained results showed that compounds 15, 51, 73, 75, 78, 79 and 96 could be the
promising template to overcome drug resistance mediated by the EGFR T790 Mutant.
Keywords: Quinazoline; Non Small Cell Lung Cancer, T790M EGFR
1

1
. Introduction
The role of the epidermal growth factor receptor (EGFR) in non small-cell lung cancer (NSCLC)
is well-known, and considerable therapeutic progress in the treatment of this disease has made
1
over thepast 10 years through the exploitation of this insight. The epidermal growth factor
,2
receptor (EGFR) has been established as one of the most important therapeutic targets for
3
NSCLC. Two small-molecule inhibitors of EGFR, gefitinib and erlotinib, have been approved
,4
5
-8
by the U.S. Food and Drug Administration (FDA) for the treatment of NSCLC.
Gefitinib and
erlotinib (Fig. 1), as the first generation ATP-competitive and reversible EGFR inhibitors,
were effective for NSCLC patients harbouring somatic EGFR mutations L858R and
9
delE746_A750 which account for 90% of all EGFR mutations in NSCLC. Unfortunately,
due to acquired secondary mutations such as T790M missense in EGFR leading to drug
resistance in roughly 50% NSCLC patients, the ability of these EGFR inhibitors to
1
0,11
effectively treat NSCLC patients is short-lived to later treatments.
EGFR T790M
mutation restores the affinity for ATP similar to that of wild type (WT) EGFR, and prevents the
1
2
reversible inhibitors from binding at higher ATP concentrations.
1
Second generation EGFR inhibitors such as canertinib , afatinib , neratinib , and
3
14
15
1
6
dacomitinib (Fig. 1) have been developed with intent to overcome the T790M mutation
1
7
related resistance. The molecules of these inhibitors are built based on a quinazoline or
quinoline core scaffold like gefitinib and erlotinib, but contain an electrophilic functionality that
can undergoes a Micheal addition reaction with a conserved cysteine residue present in EGFR
1
Cys797) to get occupancy greater than that of the reversible inhibitors. However, since the
7
(
ATP affinity of EGFR T790M is similar to that of WT EGFR, these EGFR inhibitors at
the concentrations required to inhibit EGFR T790M will also effectively inhibit WT EGFR,
leading to the dose-limiting toxicity such as diarrhoea and skin rash.
2

Fig. 1. First, second and third generation EGFR inhibitors.
3

Therefore, the clinical application of these inhibitors has been limited, especially in patients
with gefitinib or erlotinib-resistant NSCLC. Hence the EGFR NSCLC inhibitors developed
with less efficacy against WT EGFR has become a significant unmet need for the treatments of
NSCLC patients.
2
. Rationale
Third generation irreversible EGFR inhibitors containing a Michael acceptor functional group
have been developed to overcome the problem of second generation irreversible inhibitors with
minimal WT EGFR activity and enhanced ATP binding by irreversibly alkylating a cysteine
1
8-26
residue (C797) in the ATP binding site of the EGFR.
However, except for a few examples,
2
7
28
such as PF00299804, and WZ-4002, these irreversible inhibitors have thus far shown limited
clinical efficacy. Inherent weaknesses, including relatively high toxicity and a decreased binding
2
9-31
rate to the mutant kinase, may be responsible for the lack of clinical efficacy.
There has been
a recent report of C797S mutation or loss of the T790M mutation in cell-free plasma DNA
3
samples from patients who have developed resistance, and a second separate report of
2
33
C797S mutation in biopsy samples from a single patient . Additionally, studies with third-
generation resistant cell lines have shown that the allelic context of the activating
gatekeeper and C797S mutations affects the sensitivity to the three generations of inhibitors with
3
4
no EGFR TKIs alone or in combination able to suppress activity when the mutations are in cis.
These data suggest that there is a need for drugs which do not rely on covalent reaction
with Cys797 for potency or selectivity.
In view of this justification and in continuation of an ongoing program aiming at finding
3
5-38
new structure leads with potential chemotherapeutic activities
, in the present study new
series of quinazolinones have been synthesized, which comprises the 2,3-disubstituted
quinazoline pharmacophore (Fig. 2).
4

Fig. 2. Rationale for the designing of new scaffold.
5

Our strategy directs toward designing a variety of ligands with diverse chemical properties
hypothesizing that the potency of these molecules might be enhanced by adding alternative
binding group such as phenyl ring at place C-2, aniline with electron withdrawing groups, 2-
amino thiadiazoles and different primary amine containing motifs at position 3-of the
quinazoline ring. We intensely didn’t introduce Michel acceptor to avoid the covalent binding. In
this way, such substitution pattern could target different regions of the ATP-binding site of the
T790M EGFR protein kinase domain as compare to the existing first, second and third generation
agent to create differentially selective molecules.
3
. Chemistry
Anthranilic acid 1a, 1b reaction with benzoyl chloride 2, yielded 2-phenyl-4H-3,1-benzoxazin-4
one 3a and 7-chloro 2-phenyl-4H-3,1-benzoxazin-4 one 3b by N-acylation via dehydrative
3
9
cyclization mechanism as reported in our previous paper. The reaction of different primary
amines containing anilines, sulphonamides, thiadiazoles and hydrazides with 2-phenyl-4H-3,1-
benzoxazin-4 one 3a and 7-chloro 2-phenyl-4H-3,1-benzoxazin-4 one 3b in dry pyridine yielded
the corresponding quinazoline-4-one derivatives as shown in Scheme 1. It observed that while
synthesizing the compounds 16, 19, 20, 63-66, 70, 71, 74, 86, 92, 93, 95, 97-103 by fusion
reaction in an attempt to obtain 3-substituted-quinazolin-4-ones in different reaction conditions;
the reaction afforded the (ring open quinazolines) diamides (Scheme 1). It could have happend
because of ring opening tendancy of benzoxazinones with different nucleophiles, allowing
3
9
incorporation of substitution at the 3-position.
6

R
Compound Code
73
R’
R
Compound Code
86
R’
R
Cl
Cl
Cl
Cl
Cl
75
78
Cl
Cl
93
94
Cl
Cl
N
N
O
CH3
Cl
79
S
Cl
96
Cl

4
. Pharmacology
4
.1 In vitro cancer screen
The screening is a two-stage process, beginning with the evaluation of all compounds against the
-
5
6
0 cell lines at a single dose of 10 M.
The output from the single dose screen is reported as a mean graph and is available for analysis
by the COMPARE program. Compounds which exhibit significant growth inhibition are
evaluated against the 60 cell panel at five concentration levels. The human tumor cell lines of the
cancer screening panel are grown in RPMI 1640 medium containing 5% fetal bovine serum and 2
mM L-glutamine. For a typical screening experiment, cells are inoculated into 96 well microtiter
plates in 100 µL at plating densities ranging from 5,000 to 40,000 cells/well depending on the
doubling time of individual cell lines. After cell inoculation, the microtiter plates are incubated at
3
2
7°C, 5 % CO , 95 % air and 100 % relative humidity for 24 h prior to addition of experimental
drugs. After 24 h, two plates of each cell line are fixed in situ with TCA, to represent a
measurement of the cell population for each cell line at the time of drug addition (Tz).
Experimental drugs are solubilized in dimethyl sulfoxide at 400-fold the desired final maximum
test concentration and stored frozen prior to use. At the time of drug addition, an aliquot of frozen
concentrate is thawed and diluted to twice the desired final maximum test concentration with
complete medium containing 50 µg/ml gentamicin. Additional four, 10-fold or ½ log serial
dilutions are made to provide a total of five drug concentrations plus control. Aliquots of 100 µl
of these different drug dilutions are added to the appropriate microtiter wells already containing
1
00 µl of medium, resulting in the required final drug concentrations.
Following drug addition, the plates are incubated for an additional 48 h at 37°C, 5 % CO , 95 %
2
air, and 100 % relative humidity. For adherent cells, the assay is terminated by the addition of
8

cold TCA. Cells are fixed in situ by the gentle addition of 50 µl of cold 50 % (w/v) TCA (final
concentration, 10 % TCA) and incubated for 60 minutes at 4°C. The supernatant is discarded, and
the plates are washed five times with tap water and air dried. Sulforhodamine B (SRB) solution
(
100 µl) at 0.4 % (w/v) in 1 % acetic acid is added to each well, and plates are incubated for 10
minutes at room temperature. After staining, unbound dye is removed by washing five times with
% acetic acid and the plates are air dried. Bound stain is subsequently solubilized with 10 mM
1
trizma base, and the absorbance is read on an automated plate reader at a wavelength of 515 nm.
For suspension cells, the methodology is the same except that the assay is terminated by fixing
settled cells at the bottom of the wells by gently adding 50 µl of 80 % TCA (final concentration,
1
6 % TCA). Using the seven absorbance measurements [time zero, (Tz), control growth, (C), and
test growth in the presence of drug at the five concentration levels (Ti)], the percentage growth is
calculated at each of the drug concentrations levels. Percentage growth inhibition is calculated as:
[
(Ti-Tz)/(C-Tz)] x 100 for concentrations for which Ti>/=Tz
(Ti-Tz)/Tz] x 100 for concentrations for which Ti<Tz.
Three dose response parameters are calculated for each experimental agent. Growth inhibition of
0 % (GI50) is calculated from [(Ti-Tz)/(C-Tz)] x 100 = 50, which is the drug concentration
[
5
resulting in a 50 % reduction in the net protein increase (as measured by SRB staining) in control
cells during the drug incubation. The drug concentration resulting in total growth inhibition (TGI)
is calculated from Ti = Tz. The LC50 (concentration of drug resulting in a 50 % reduction in the
measured protein at the end of the drug treatment as compared to that at the beginning) indicating
a net loss of cells following treatment is calculated from [(Ti-Tz)/Tz] x 100 = -50. Values are
calculated for each of these three parameters if the level of activity is reached; however, if the
9

effect is not reached or is exceeded, the value for that parameter is expressed as greater or less
4
0-43
than the maximum or minimum concentration tested.
-
5
4
.1.2 Primary single high dose (10 M) full NCI 60 cell panel in vitro assay
All the selected compounds submitted to National Cancer Institute (NCI) for in vitro anticancer
assay were evaluated for their anticancer activity. Primary in vitro one dose anticancer assay was
performed in full NCI 60 cell panel representing leukemia, melanoma and cancers of lung, colon
brain breast, ovary, kidney and prostate in accordance with the protocol of the NCI, USA. The
-
5
compounds were added at a single concentration (10 M) and the culture was incubated for 48 h.
End point determinations were made with a protein binding dye, Sulforhodamine B. Results for
each compound were reported as a mean graph of the percent growth of the treated cells when
compared to the untreated control cells. There after obtaining the results for one dose assay,
analysis of historical Development Therapeutics Programme (DTP) was performed and
compounds, which satisfied pre-determined threshold inhibition criteria was selected for NCI full
4
0-43
panel 5 dose assay.
4
.1.3 In vitro 5 dose full NCI 60 cell panel assay
All the cell lines (about 60), representing nine tumor subpanels, were incubated at five different
concentrations (0.01, 0.1, 1, 10 & 100 µM). The outcomes were used to create log concentration
Vs % growth inhibition curves and three response parameters (GI50, TGI and LC50) were
calculated for each cell line. The GI
concentration of the compound causing 50% decrease in net cell growth, the TGI value
cytostatic activity) is the concentration of the compound resulting in total growth inhibition and
LC50 value (cytotoxic activity) is the concentration of the compound causing net 50% loss of
5
value (growth inhibitory activity) corresponds to the
(
4
0-43
initial cells at the end of the incubation period of 48 h.
10

4
.2 EGFR Kinase Inhibitory assay
The assays were performed in vitro using Homogeneous timeresolved fluorescence (HTRF)
method (Cisbio). EGFR and EGFR T790M were purchased from Sigma. The kinases and
substrates were incubated first with synthesized analogues for 5 min in enzymatic buffer. Then
ATP (Sigma) was added into the reaction mixture to start the enzyme reaction. The ATP
concentrations used in each enzyme reaction were 1.65 µM for EGFR, equivalent to the Km of
ATP for the corresponding enzyme in this assay condition. The assays were conducted at room
temperature for 30 min and stopped by detection reagents which contain EDTA. The detection
4
4
step lasted for 1 h. The IC50 was calculated using GraphPad Prism 5.0.
. Docking Study
Docking study was carried out using a graphical user interface SP-docking mode of program
5
4
5-47
Maestro 8.
The protein structure of a complex T790M mutatd EGFR was obtained from the
4
8
RCSB Protein Data Bank (PDB) as entry 2JIU (http://www.rcsb.org/pdb/explore/explore.2jiu).
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 molecules in the
crystal structures were deleted, and hydrogens were added, wherever they were missing. The
4
9
energy of crystal structure was 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
5
0
that produces the low energy conformer of ligands using OPLS 2005 force 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.
11

6
. Results and Discussion
All the compounds were synthesized as depicted in Scheme 1. The structures of final compounds
1 13
4-105) were characterized based on their physicochemical and spectral (IR, H NMR, C NMR
(
and MS) analysis. The analytical data of all the newly synthesized compounds along with their
anticipated structures are summarized in supporting information.
The tumor growth inhibition properties of the 32 compounds selected among the all synthesized
compounds by National Cancer Institute (NCI), USA. Among the selected 32 compounds, 18
were further screened for 5-log dose molar range as they have shown prominent cell growth
-
5
inhibition at 10 M concentration.
To assess the potency for EGFR inhibitory activity, selected 18 compounds were tested against
gefitinib-resistant NSCLC cell H1975 possessing L858R/T790M mutant and EGFR human
epithelial carcinoma cell A431 overexpressing wild-type. The results of in vitro antiproliferation
demonstrate that compound 79, 7-chloro-3-(5-(4-methoxyphenyl)-1,3,4-thiadiazol-2-yl)-2-
phenylquinazolin-4(3H)-one was found to be the most active compound of the series with IC50
value of 0.031 µM, followed by compound 78 having IC50 value of 0.129 µM against H1975
NSCLC cell line. Similarly significant results were shown by compound 23 and 51 with IC50
value of 0.81 µM and 0.84 µM (Table 1).
Since EGFR is expressing in many tissues for normal cellular processes, the potential for the
toxicity from EGFR inhibitors is a concern. Therefore, the growth inhibitory of the compounds
against cancer cell lines with low level of EGFR expression was evaluated to assess the potential
non-specific toxicity. The cancer cell lines A549 express low levels of EGFR (WT EGFR and k-
Ras mutation) and therefore are good counter-screen cell lines for EGFR-targeting inhibitors.
12

Table 1. Antiproliferative activity of compounds against cells harboring a different status of EGFR.
NCI-H1975
(EGFR L858R/T790M)
A549/ATCC
(WT EGFR)
(IC50 µM)
HT 29
(Non-Special Gene Type)
(IC50 µM)
Compound
Code
Selectivity Ratio
NCI-H1975/A549
Structure
(
IC50 µM)
5
.23 ± 0.42
100 ± 0.51
32.5 ± 0.13
0.052
15
17
23
4.89 ± 0.31
0.81 ± 0.08
5.42 ± 0.23
6.12 ± 0.16
1.15 ± 0.14
0.90
1.54
0.523 ± 0.05
3
4
1
8
2.38 ± 0.12
4.87 ± 0.15
3.02 ± 0.34
18.3 ± 0.45
3.46 ± 0.18
50 ± 0.32
0.78
0.26
5
1
0.846 ± 0.05
50 ± 0.10
50 ± 0.52
0.016
5
5
0
2.45 ± 0.43
2.88 ± 0.34
3.33 ± 0.23
2.87 ± 0.34
3.80 ± 0.28
3.92 ± 0.26
0.73
1.00
6
7
7
3
5
0.76 ± 0.05
3.79 ± 0.11
2.09 ± 0.33
0.717 ± 0.05
21.2 ± 0.24
2.72 ± 0.21
0.732 ± 0.06
0.20
0.101
0.17
0.213 ± 0.05
0.129 ± 0.05
7
8
13

Table 1. Continue...
NCI-H1975
A549/ATCC
(WT EGFR)
(IC50 µM)
HT 29
(Non-Special Gene Type)
(IC50 µM)
Compound
Code
Selectivity Ratio
Structure
(EGFR L858R/T790M)
NCI-H1975/A549
(
IC50 µM)
7
9
0.031 ± 0.02
14.41 ± 0.34
0.390 ± 0.05
0.388 ± 0.04
0.079
86
17.4 ± 0.28
3.36 ± 0.21
0.82
93
1.67 ± 0.07
1.57 ± 0.08
4.80 ± 0.24
1.06
9
4
2.45 ± 0.12
5.30 ± 0.28
2.45 ± 0.10
4.50 ± 0.32
0.46
96
0.378 ± 0.05
0.428 ± 0.08
0.154
8
.71 ± 0.22
9.86 ± 0.18
1.87 ± 0.08
8.19 ± 0.32
2.95 ± 0.08
0.88
Gefitinib
WZ4002
0.058 ± 0.01
0.031
*
Lesser the selectivity ratio towards zero more is selectivity (safety Index)
14

Table 2. Selectivity profile of compound 79 against panel of kinases
Kinase
EGFR
VEGFR-1
VEGFR-2
Abl
% inhibition at 3 µM
78
34
42
0
AMPKα1
urora-1
Blk
0
0
0
CDK1
cKIT
0
0
FAK
0
FGFR1
Flt1
0
0
Flt3
0
IGF-1R
IR
10
0
JAK-2
LKB-1
MEK-1
Met
0
0
0
0
Syk
0
This test was conducted by Millipore Kinase Profiling Service.
15

Table 3. In vitro inhibition of EGFR tyrosine kinase activity
Compound Structure EGFR Inhibition IC50 (µM)
T790M EGFR
WT EGFR
1
2
5
5
3
1
7.23 ± 0.10
0.78 ± 0.05
0.72 ± 0.05
78.23 ± 0.12
0
.42 ± 0.02
34.32 ± 0.21
1
2.12 ±0.10
3.21 ± 0.21
7
7
3
5
1.12 ± 0.08
0.102 ± 0.01
0.021 ± 0.001
1.68 ± 0.09
0.53 ± 0.01
0.32 ± 0.02
7
7
8
9
9
6
0.287 ± 0.05
0.173 ± 0.06
2.11 ± 0.08
Gefitinib
0.076 ± 0.001
EGFR activity assays were performed using HTRF method. The data reported are the mean values
from three independent experiments
16

These compounds are also evaluated against HT-29 colon cancer cells, which expressed a non-
special gene type, to test their toxic effects. As shown in Table 1 compounds 15, 51, 73, 75, 78,
7
9 and 96 were less potent against A549 (WT EGFR and k-Ras mutation) and HT-29 (non-
special gene type) cells, showing a high safety index. Therefore, compounds 15, 51, 73, 75, 78,
9 and 96 might be promising candidates to overcome drug resistance mediated by the EGFR
7
T790Mutant.
The inhibitory activity of 79 against various kinases is summarized in Table 2 and results
confirmed that 79 is a selective EGFR inhibitor. The inhibitory activities of the selected
compounds against different types of kinases (EGFR T790M, EGFR WT) were evaluated using
Homogeneous time-resolved fluorescence (HTRF) method, and gefitinib was employed as
positive controls. Notably compound 79 and 78 shows significant T790M inhibitory activity of
IC50 value 0.021 µM and 0.102 µM. Inhibition of WT EGFR leads to the dose-limiting toxicity
such as diarrhoea and skin rash. Therefore, the clinical application of these inhibitors has been
limited, especially in patients with gefitinib or erlotinib-resistant NSCLC. As shown in Table
3, all the tested compounds displayed weaker inhibitory WT EGFR. The results indicate that the
non-specific cytotoxic effects of this hybrid are minimal
Lung cancers caused by activating mutations in the epidermal growth factor receptor (EGFR) are
initially responsive to small molecule tyrosine kinase inhibitors (TKIs), but the efficacy of these
agents is often limited because of the emergence of drug resistance conferred by a second
mutation, T790M. Threonine 790 is the ‘‘gatekeeper’’ residue, an important determinant of
inhibitor specificity in the ATP binding pocket. The T790M mutation has been thought to cause
resistance by sterically blocking binding of TKIs such as gefitinib and erlotinib.
17

Fig. 3a. Docking interaction of the active compounds with T790M EGFR-tyrosine kinase PDB: 2JIU.
18

Fig. 3b. Docking interaction of the active compounds with T790M EGFR-tyrosine kinase PDB: 2JIU.
19

Fig. 4. Structure activity relationship (NCI-H1975)
20

Docking studies revealed that the synthesized compounds binds differently to the ATP-binding
site of the T790M EGFR tyrosine kinase domain as compare to the existing first, second and third
rd
generation agent. The compounds having hydrogen bond donor at 3 position of quinazoline
show hydrogen bond interaction with the Arg 841 amino acid of T790M EGFR rather than
interacting with Met793 ( compounds 48,55 and 96) as shown in Fig. 3a and 3b. Such kind of
allosteric site binding of the compounds to the T790M EGFR could resolve the problem of
resistance. Since the predominant strategy to combat the resistance is the use of covalent
inhibitors, increasing target residence time and providing strong potency. For this reason, second
and third generation EGFR inhibitors have been designed to form a covalent bond with Cys797
showing promising efficacy. On the other hands thiadiazoles (C-3 position of quinazoline) and
ring open quinazolines interact via Met-793 reversibly except compound 51, which shows
reversible hydrogen bonding interaction with that of Cys-797 as shown in Fig. 3a and 3b. These
findings indicate that drugs owning excellent potency on EGFR without relying on the covalent
modification of C797 are in demand. Herein, our work indicates alternative noncovalent strategy
that provides new inhibitors with the potency to overcome the problem of gefitinib-resistance. It
will also provide chemical starting points for optimization of the efficacy/toxicity ratio.
7
. Structure Activity Relationship
Structure-activity correlation of the synthesized compounds against NCI-H1975 revealed that,
th
quinazolines having chloro substituent at 7 position; is more active as compared to the
unsubstituted quinazoline derivatives (compound 78 and 31), which could contribute in
rd
enhancement of lipid solubility compounds. Presence of bulkier group at 3 position of the
quinazolines gave significant results. It is also observed that ring opened quinazolines (diamides)
are not as active as that of the ring closed quinazolines (compound 86 and 93) and confirms that
21

the quinazoline ring is satisfactory backbone for anti-tumor activity. 2-amino-1,3,4-thiadiazoles
th
having chloro substitution at 7 position are more potent as compared to the anilines at third
position of quinazolines. 4-methoxy phenyl is more potent on 1,3,4-thiadiazoles as compare to
the other substitution (compound 79). Compound 15 containing 4-chlorophenyl on 1,3,4-
thiadiazoles was less potent against A549 (WT EGFR and k-Ras mutation) and HT-29 (non-
special gene type) cells, showing a high safety index. It could be crucial chemical starting points
for the optimization of the efficacy/toxicity ratio, which is major problem of second and third
rd
generation EGFR inhibitors. Among the anilines at 3 position of quinazolines; 3-chloro 4-fluro
aniline has come out with significant results as compared to other anilines (compounds 17) (Fig.
4
).
. Conclusion
8
New derivatives of 4(3H)-quinazolinones were synthesized and evaluated for their inhibitory
activity against NSCLC (Non Small Cell Lung Cancer cell lines). The results of the study
demonstrated that compound 79, 7-chloro-3-(5-(4-methoxyphenyl)-1,3,4-thiadiazol-2-yl)-2-
phenylquinazolin-4(3H)-one was found to be the most potent compounds of the series with IC50
value of 0.031 µM. Compounds 15, 51, 73, 75, 78, 79 and 96 were less potent against A549 (WT
EGFR and k-Ras mutation) and HT-29 (non-special gene type) cells, showing a high safety
index. The obtained results showed that compounds 15, 48, 51, 78, 86 could be useful as a
template for future design, optimization, and investigation to construct more active analogs to
overcome drug resistance mediated by the EGFR T790M mutant. The inhibitory effect of
selected potent compounds on T790M EGFR tyrosine kinase activity was determined and
Compound 78 (0.102 µM) and 79 (0.021 µM) exhibited significant inhibition with subnanomolar
IC50 values as compare to the gefitinib. Docking studies further revealed that the quinazolinones
22

(
48, 55 and 96) allosterically binds to the Arg 841 amino acid of T790M EGFR rather than
rd
interacting with Met793 via substitution at 3 position of quinazoline. Herein, our work indicates
alternative noncovalent strategy that provides new inhibitors with the potency to overcome the
problem of gefitinib-resistance. It will also provide chemical starting points for optimization of
the efficacy/toxicity ratio. Structure-activity correlation of the synthesized compounds revealed
th
that, quinazolines having chloro substituent at 7 position is more active as compared to the
rd
unsubstituted quinazoline derivatives. Presence of bulkier group at 3 position of the
quinazolines gave significant results. Finally, it is conceivable that further derivatization of such
compounds will be of interest with the hope to get more selective and potent T790M EGFR
inhibitors, which could overcome EGFR resistance obstacle.
3
9
9
. Experimental
All the chemicals and solvents were supplied by Sigma-Aldrich and Spectrochem Pvt Ltd.
Solvents were distilled and dried before use if required. The reactions were monitored with the
help of thin-layer chromatography using pre-coated aluminum sheets with GF254 silica gel, 0.2
mm layer thickness (Merck) and the solvent systems of Benzene : Acetone (7:3 and 9:1),
Toluene: Ethyl acetate: Formic acid (5:4:1) and Chloroform: Methanol (9:1) were used. The
spots were visualized under UV lamp. Melting points of the synthesized compounds were
determined using one end open capillary tubes on a liquid paraffin bath and are uncorrected. IR
1 13
spectrum was acquired on Perkin Elmer FT-IR spectrometer. Both H-NMR (DMSO) and C
NMR (DMSO) spectra of the synthesized compounds were performed with Bruker Avance-II
4
00 NMR Spectrometer operating at 400 MHz and 100 MHz respectively in SAIF, Punjab
University (Chandigarh) and the chemical shifts are reported in ppm units with respect to TMS
23

as internal standard. Mass spectra of the synthesized compounds were recorded at MAT 120 in
SAIF, Punjab University.
9
.1 2-phenyl-4H-benzo[d][1,3]oxazin-4-one (3a and 3b)
39
It is synthesized as per the reported method.
9
4
.2 General procedure for the synthesis of title compounds 3-substituted-2-phenylquinazolin-
(3H)-ones
Equimolar quantity of 2-phenyl-4H-benzo[d][1,3]oxazin-4-one 3a or 7-chloro-2-phenyl-4H-
benzo[d][1,3]oxazin-4-one 3b and different primary amino group containing moieties were
°
fused together at 200-250 C in an oil bath for 30 minutes, in such way that reaction mixture
should not degraded due to high heating. The mixture was cooled and ethanol was added to the
mixture. The separated solid was collected by filtration, washed with ethanol, dried and
recrystallized multiple times with ethanol to get the pure product.
9
.2.1 3-(5-(2-chlorophenyl)-1,3,4-thiadiazol-2-yl)-2-phenylquinazolin-4(3H)-one (15)
°
Yield 69 %; mp above 250 C; IR (KBr) ѵmax 3098.12 (Arom.CH strech), 2918.68 (Aliph.CH
-1 1
strech), 1671.12 (C=O), 1561.11 (C=C), 1465.48 (CH bend), 731.12 (C-Cl) cm ; H NMR
1
3
(
DMSO-d
6
) δ 7.11-8.14 (m, 12H, Ar-H), 2.45 (s, 3H, CH
70.15, 164.18, 158.28, 150.15, 140.16, 135.67, 134.16, 132.89, 132.67, 131.65, 130.86, 129.68,
28.50, 128.00, 127.66, 127.00, 126.86, 126.00, 122.14, 24.36. HRMS (ESI) m/z calcd. for
OS: 416.0499; found: 416.0492.
3 6
); C NMR (DMSO-d ) δ 175.14,
1
1
22 4
C H13ClN
9
.2.2 3-(3-chloro-4-fluorophenyl)-2-phenylquinazolin-4(3H)-one (17)
°
Yield 68 %; mp 168-170 C; IR (KBr) ѵmax 3060.36 (CH strech), 1680.53 (C=O), 1604.96
-
1 1
(
(
C=C), 1445.27 (CH bend), 1084.23 (C-F), 770.71 (C-Cl) cm ; H NMR (DMSO-d
6
) δ 6.92-8.23
1
m, 12H, Ar-H); C NMR (DMSO-d
3
6
) δ 162.06, 158.88, 156.38, 154.59, 134.92, 134.09,
24

1
34.05, 131.52, 129.15, 129.08, 128.88, 128.35, 127.86, 127.69, 121.65, 120.64, 116.92; HRMS
(
ESI) m/z calcd. for C20H12ClFN O: 350.0622; found: 350.0629.
2
9
.2.3 4-bromo-N-(4-oxo-2-phenylquinazolin-3(4H)-yl) benzamide (23)
°
Yield 41 %; mp 221-224 C; IR (KBr) ѵmax 3282.34 (NH strech), 3083.26 (CH strech), 1728.72
[
C=O (ketone)], 1672.38 [C=O (amide)], 1626.84 (NH bend), 1568.26 (C=C), 1452.97 (CH
-
1 1
bend), 601.83 (C-Br) cm ; H NMR (DMSO-d
6
) δ 11.31 (s, 1H, NH), 6.82-7.98 (m, 13H, Ar-H);
) δ 166.37, 162.15, 158.36, 152.32, 135.98, 132.84, 131.86, 130.68, 129.65,
28.94, 128.47, 128.00, 127.80, 126.5, 126.37, 126.00, 120.90. HRMS (ESI) m/z calcd. for
: 419.0269; found: 419.0274.
1
3
C NMR (DMSO-d
6
1
21 3 2
C H14BrN O
9
.2.4 3-(5-cyclopropyl-1,3,4-thiadiazol-2-yl)-2-phenylquinazolin-4(3H)-one (31)
°
Yield 81 %; mp 224-228 C; IR (KBr) ѵmax 3068.74 (Arom.CH strech), 2973.01 (Aliph.CH
-
1 1
strech), 1677.32 (C=O), 1560.25 (C=C), 1468.23 (CH bend) cm ; H NMR (DMSO-d
6
) δ 7.17-
) δ 175.41, 170.90,
59.15, 146.90, 140.84, 134.81, 134.45, 131.99, 129.45, 128.83, 127.42, 123.22, 121.44, 117.58,
1.09, 10.65; HRMS (ESI) m/z calcd. for C19 OS: 347.0888; found: 347.0882 [M+1].
1
3
8
1
1
9
.88 (m, 9H, Ar-H), 1.08-2.34 (m, 5H, cyclopropyl); C NMR (DMSO-d
6
14 4
H N
.2.5 3-(2-aminophenyl)-7-chloro-2-phenylquinazolin-4(3H)-one (48)
°
Yield 62 %; mp above 250 C; IR (KBr) ѵmax 3171.26 and 3131.98 (NH
2
), 3065.33 (CH strech),
-
1 1
1
671.48 (C=O), 1602.48 (NH bend), 1569.04 (C=C), 1444.24 (CH bend), 693.14 (C-Cl) cm ; H
1
3
NMR (DMSO-d
6
2 6
) δ 7.14-8.21 (m, 12H, Ar-H), 6.65 (s, 2H, NH ); C NMR (DMSO-d ) δ
1
1
3
9
61.67, 151.26, 149.21, 143.67, 134.91, 130.08, 129.49, 128.55, 126.35, 122.27, 121.47, 121.23,
18.63, 118.09, 114.92, 114.64, 111.08, 110.55; HRMS (ESI) m/z calcd. for C20
48.0825; found: 348.0831 [M+1].
3
H14ClN O:
.2.6 3-(2-acetylphenyl)-7-chloro-2-phenylquinazolin-4(3H)-one (51)
25

°
Yield 32 %; mp 192-194 C; IR (KBr) ѵmax 3111.20 (Arom.CH strech), 2924.66 (Aliph.CH
strech), 1681.55 [C=O (ketone)], 1645.32 [C=O (amide)], 1606.71 (C=C), 1421.30 (CH bend),
-
1
1
59.92 (C-Cl) cm ; H NMR (DMSO-d
13
3
); C
7
6
) δ 7.23-8.38 (m, 12H, Ar-H), 2.74 (s, 3H, CH
NMR (DMSO-d
6
) δ 199.14, 164.94, 160.01, 141.35, 139.60, 138.65, 134.81, 133.88, 131.72,
1
31.66, 128.39, 128.33, 126.82, 123.09, 122.79, 122.03, 120.58, 120.47, 118.13, 28.16; HRMS
ESI) m/z calcd. for C22 : 375.0822; found: 375.0828[M+1].
.2.7 7-chloro-2-phenyl-3-(pyridin-3-yl) quinazolin-4(3H)-one (55)
(
2 2
H15ClN O
9
°
Yield 81 %; mp 230-235 C; IR (KBr) ѵmax 3063.52 (CH strech), 1684.71 (C=O), 1598.68
-
1 1
(
C=C), 1421.88 (CH bend), 702.79 (C-Cl) cm ; H NMR (DMSO-d
6
) δ 7.25-8.50 (m, 12H, Ar-
) δ 161.49, 155.82, 149.65, 149.34, 148.30, 141.35, 136.48, 134.42,
34.34, 130.01, 129.07, 128.67, 128.44, 128.21, 127.49, 123.51, 119.06; HRMS (ESI) m/z calcd.
O: 334.0669; found: 334.0671[M+1].
.2.8 7-chloro-3-(4-methoxyphenyl)-2-phenylquinazolin-4(3H)-one (60)
1
3
H); C NMR (DMSO-d
6
1
for C19H12ClN
3
9
°
Yield 62 %; mp 200-204 C; IR (KBr) ѵmax 3056.62 (CH strech), 1711.81 (C=O), 1595.94
-
1 1
(
C=C), 1446.28 (CH bend), 771.14 (C-Cl) cm ; H NMR (DMSO-d
6
) δ 6.68-7.52 (m, 12H, Ar-
1
); C NMR (DMSO-d
3
H), 3.78 (s, 3H, OCH
3
6
) δ 65.89, 160.23, 156.12, 150.04, 138.41, 134.13,
1
32.29, 130.12, 129.94, 128.92, 127.95, 127.46, 123.05, 121.70, 119.34, 113.16, 55.44; HRMS
ESI) m/z calcd. for C21 : 363.0822; found: 363.0825 [M+1].
.2.9 7-chloro-3-(5-methyl-1,3,4-thiadiazol-2-yl)-2-phenylquinazolin-4(3H)-one (73)
(
2 2
H15ClN O
9
°
Yield 68 %; mp 243-247 C; IR (KBr) ѵmax 3064.98 (Arom.CH strech), 2923.34 (Aliph.CH
-1 1
strech), 1686.93 (C=O), 1590.32 (C=C), 1462.36 (CH bend), 724.38 (C-Cl) cm ; H NMR
1
3
(
DMSO-d
6
3 6
) δ 6.74-7.81 (m, 8H, Ar-H), 2.81 (s, 3H, CH ); C NMR (DMSO-d ) δ 171.31,
26

1
1
9
63.43, 158.44, 154.35, 146.32, 138.43, 132.62, 130.65, 129.62, 129.20, 129.01, 128.60, 124.62,
20.33, 22.30. HRMS (ESI) m/z calcd. for C17 OS: 354.0342; found: 354.0347.
H11ClN
4
.7.10 7-chloro-2-phenyl-3-(5-phenyl-1,3,4-thiadiazol-2-yl)quinazolin-4(3H)-one (75)
°
Yield 45 %; mp above 250 C; IR (KBr) ѵmax 3023.98 (CH strech), 1668.84 (C=O), 1568.47
-
1 1
(
C=C), 1472.62 (CH bend), 710.23 (C-Cl) cm ; H NMR (DMSO-d
6
) δ 6.89-8.34 (m, 13H, Ar-
) δ 172.35, 170.14, 162.34, 158.35, 156.35, 139.43, 135.65, 132.64,
32.15, 130.25, 129.63, 128.62, 128.43, 126.45, 126.14, 124.35, 122.34, 120.35. HRMS (ESI)
m/z calcd. for C22 OS: 416.0499; found: 416.0493.
.2.11 7-chloro-3-(5-cyclopropyl-1,3,4-thiadiazol-2-yl)-2-phenylquinazolin-4(3H)-one (78)
1
3
H); C NMR (DMSO-d
6
1
H13ClN
4
9
°
Yield 68 %; mp 222-226 C; IR (KBr) ѵmax 3123.35 (Arom.CH strech), 2956.32 (Aliph.CH
-1 1
strech), 1665.87 (C=O), 1543.76 (C=C), 1446.87 (CH bend), 678.92 (C-Cl) cm ; H NMR
1
3
(
DMSO-d
6
) δ 7.31-8.21 (m, 8H, Ar-H), 1.10-2.61 (m, 5H, cyclopropyl); C NMR (DMSO-d
6
) δ
1
1
3
9
71.36, 166.37, 162.35, 158.37, 154.48, 139.45, 132.66, 132.37, 130.89, 130.67, 130.25, 128.65,
24.66, 120.37, 11.25, 10.86. HRMS (ESI) m/z calcd. for C19
80.0493.
4
H13ClN OS: 380.0499; found:
.2.12 7-chloro-3-(5-(4-methoxyphenyl)-1,3,4-thiadiazol-2-yl)-2-phenylquinazolin-4(3H)-one
79)
Yield 63 %; mp above 250 C; IR (KBr) ѵmax 3046.38 (CH strech), 1658.24 (C=O), 1572.82
(
°
-
1 1
(
C=C), 1462.83 (CH bend), 756.32 (C-Cl) cm ; H NMR (DMSO-d
6
) δ 6.81-8.12 (m, 12H, Ar-
1
); C NMR (DMSO-d
3
H), 3.85 (s, 3H, OCH
3
6
) δ 172.67, 169.45, 162.36, 160.37, 158.85, 156.36,
27

1
5
9
39.47, 132.66, 132.17, 130.67, 130.35, 128.52, 128.40, 127.64, 126.35, 124.68, 120.66, 118.33,
5.67. HRMS (ESI) m/z calcd. for C23 S: 446.0604; found: 446.0609.
4 2
H15ClN O
.2.13 2-benzamido-4-chloro-N-(4-fluorophenyl) benzamide (86)
°
Yield 73 %; mp 245-248 C; IR (KBr) ѵmax 3264.62 (NH strech), 3150.42 (CH strech), 1651.20
(
C=O), 1601.11 (NH bend), 1550.26 (C=C), 1408.83 (C-H bend), 1223.84 (C-F), 702.43 (C-Cl)
-
1 1
cm ; H NMR (DMSO-d
6
) δ 12.03 (s, 1H, NHCO), 10.71(s, 1H, CONH), 6.83-8.69 (m, 12H,
Ar-H); C NMR (DMSO-d ) δ 166.76, 164.01, 159.28, 140.56, 139.04, 134.03, 132.41, 128.97,
28.08, 127.42, 123.07, 122.79, 122.71, 121.67, 119.28, 116.04, 115.82; HRMS (ESI) m/z calcd.
: 391.0728; found: 391.0735 [M+Na].
.2.14 2-benzamido-N-benzyl-4-chlorobenzamide (93)
1
3
6
1
2 2
for C20H14ClFN O
9
°
Yield 68 %; mp 200-205 C; IR (KBr) ѵmax 3257.31 (NH strech), 3111.10 (CH strech), 1652.02
-
1 1
(
(
C=O), 1600.85 (NH bend), 1561.87 (C=C), 1413.34 (C-H bend), 705.37 (C-Cl) cm ; H NMR
DMSO-d ) δ 11.57 (s, 1H, NHCO), 10.14 (s, 1H, CONH), 6.91-8.84 ((m, 13H, Ar-H), 4.63 (s,
6
1
3
2
1
H, CH
28.92, 128.89, 127.87, 127.80, 127.42, 122.86, 121.33, 118.37, 44.16; HRMS (ESI) m/z calcd.
: 364.0979; found: 364.0973.
.2.15 7-chloro-3-(3-chlorophenyl)-2-phenylquinazolin-4(3H)-one (94)
2 6
); C NMR (DMSO-d ) δ 167.43, 165.78, 140.93, 138.81, 137.36, 134.30, 132.19,
2 2
for C21H17ClN O
9
°
Yield 71 %; mp 194-200 C; IR (KBr) ѵmax 3069.99 (CH strech), 1686.80 (C=O), 1580.47
-
1 1
(
C=C), 1447.27 (CH bend), 701.77 (C-Cl) cm ; H NMR (DMSO-d
6
) δ 6.77-8.70 (m, 12H, Ar-
1
H); C NMR (DMSO-d
3
6
) δ 161.48, 155.96, 141.21, 138.41, 134.67, 134.65, 129.94, 129.88,
28

1
29.42, 128.97, 128.93, 128.89, 128.67, 128.26, 128.10, 127.41, 127.38, 119.20; HRMS (ESI)
m/z calcd. for C20 O: 367.0327; found: 367.0321 [M+1].
.2.16 3-(2-aminoethyl)-7-chloro-2-phenylquinazolin-4(3H)-one (96)
2 2
H12Cl N
9
°
Yield 89 %; mp 223-228 C; IR (KBr) ѵmax 3268.12 and 3214.78 (NH
2
), 3087.21 (Arom.CH
strech), 2912.98 (Aliph.CH strech), 1656.12 (C=O), 1601.78 (NH bend), 1491.23 (C=C),
-
1 1
1
438.12 (CH bend), 712.98 (C-Cl) cm ; H NMR (DMSO-d
6
) δ 6.98-7.97 (m, 8H, Ar-H), 6.11
) δ 160.11, 158.25, 154.34, 139.30,
32.26, 132.05, 130.87, 130.24, 130.00, 129.45, 124.84, 120.87, 50.14, 36.39. HRMS (ESI) m/z
O: 299.0825; found: 299.0829.
1
3
(
2 2 6
s, 2H, NH ,), 2.91 (t, 2H, CH ); C NMR (DMSO-d
1
calcd. for C16H14ClN
3
Acknowledgement
The authors would like to thank “Science and Engineering Research Board (SERB) of
Department of Science and Technology (DST) Govt. of India” (Grant No.YSS/2015/002017) for
funding the project.
29