Novel quinazoline derivatives bearing various 6-benzamide moieties as highly selective and potent EGFR inhibitors

Article abstract of DOI:10.1016/j.bmc.2018.02.022

A series of novel quinazoline derivatives bearing various C-6 benzamide substituents were synthesized and evaluated as EGFR inhibitors, and most showed significant inhibitory potency against EGFR kinase. In particular, compound 6g possessed potent inhibitory activity against EGFR wild-type (IC₅₀ = 5 nM), and strong antiproliferative activity against HCC827 and Ba/F3 (L858R) cell lines. Kinase profiling against a panel of 365 kinases showed that 6g was highly selective for EGFR. Furthermore, 6g showed desirable properties in assays of liver microsome metabolic stability and cytochromes P450 inhibition and preliminary pharmacokinetic study. The overall attractive profile of 6g made it an interesting compound for further development.

Full text of DOI:10.1016/j.bmc.2018.02.022

Accepted Manuscript
Novel quinazoline derivatives bearing various 6-benzamide moieties as highly
selective and potent EGFR inhibitors
Weijie Hou, Yan Ren, Zhenhua Zhang, Huan Sun, Yongfen Ma, Bo Yan
PII:
S0968-0896(17)32202-2
https://doi.org/10.1016/j.bmc.2018.02.022
BMC 14208
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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Revised Date:
Accepted Date:
11 November 2017
11 February 2018
15 February 2018
Please cite this article as: Hou, W., Ren, Y., Zhang, Z., Sun, H., Ma, Y., Yan, B., Novel quinazoline derivatives
bearing various 6-benzamide moieties as highly selective and potent EGFR inhibitors, Bioorganic & Medicinal
Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.02.022
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Novel quinazoline derivatives bearing
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Weijie Hou*, Yan Ren, Zhenhua Zhang, Huan Sun, Yongfen Ma, Bo Yan
7 Science Park Rd. ZGC Life Science Park, Beijing

Bioorganic & Medicinal Chemistry
Novel quinazoline derivatives bearing various 6-benzamide moieties as highly
selective and potent EGFR inhibitors
Weijie Houa,b,, Yan Renb, Zhenhua Zhangc, Huan Sunb, Yongfen Mab, Bo Yanb
a College of Life Sciences, Beijing Normal University, Beijing 100875, China
b National Institute of Biological Sciences (NIBS), Beijing 102206, China
c Beijing Advanced Innovation Center for Food Nutrition and Human Health, Department of Applied Chemistry, China Agricultural University, Beijing 100193,
China
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A series of novel quinazoline derivatives bearing various C-6 benzamide substituents were
synthesized and evaluated as EGFR inhibitors, and most showed significant inhibitory potency
against EGFR kinase. In particular, compound 6g possessed potent inhibitory activity against
EGFR wild-type (IC50 = 5 nM), and strong antiproliferative activity against HCC827 and Ba/F3
(L858R) cell lines. Kinase profiling against a panel of 365 kinases showed that 6g was highly
selective for EGFR. Furthermore, 6g showed desirable properties in assays of liver microsome
metabolic stability and cytochromes P450 inhibition and preliminary pharmacokinetic study.
The overall attractive profile of 6g made it an interesting compound for further development.
Available online
Keywords:
EGFR
Kinase inhibitor
Quinazoline
High selectivity
Anti-cancer
2009 Elsevier Ltd. All rights reserved.
been approved, including afatinib,22 osimertinib,23 and
olmutinib.24
1. Introduction
Epidermal growth factor receptor (EGFR) is a receptor
tyrosine kinase (RTK) of the ErbB family.1 EGFR is known to be
a key regulator of many complex biological processes, including
cell motility, adhesion, and cell cycle regulation, as well as
angiogenesis, apoptosis, and metastasis.2, 3 Aberrant activity of
EGFR has been observed in a wide variety of human cancers,4
such as non-small cell lung cancer (NSCLC),5 head and neck
cancer,6 breast cancer,7 and colorectal cancer,8 and the
dysregulation of EGFR signaling is invariably associated with
poor clinical outcomes.2 Based on its critical role in the tumor
progression, many antibodies and small molecule inhibitors that
target EGFR have been developed over the last three decades.
Several of them (e.g., gefitinib9, 10 and erlotinib,11, 12) have been
approved by the US Food and Drug Administration (FDA).
Most irreversible EGFR inhibitors reported to date use
acrylamide fragment to react with the Cys797 within the EGFR
ATP-binding pocket.25, 26 It has been reported that the intrinsic
reactivity of acrylamide fragment can cause rapid metabolic
deactivation, toxicity or nonspecific reactions with off-target
biomolecules.26, 27 Some other cysteine-reactive warheads have
been explored in the course of covalent EGFR inhibitor
development efforts, (e.g., butynamide, vinylsulfonamide, α-
substituted acetamide, alkynyl thienopirimidine),25 but more
systematic studies about application of different covalent groups
in EGFR inhibitors are required to provide more information
about further optimization of the efficacy/toxicity ratio.
2. Compound design
However, the dramatic efficacy of gefitinib and erlotinib for
the treatment of NSCLC patients that harbor EGFR mutations,
L858R or delE746_A750,13 is threatened by drug resistance.14, 15,
Phenyl with leaving groups (LGs) and electron withdrawing
groups (EWGs) can be viewed as a potential cysteine-reactive
group. In 1945, Frederick Sanger used 1-fluoro-2,4-
dinitrobenzene(DNFB) to determine the identity of N-terminal
amino acid residues in polypeptide chains.28 In 2012, Merck
reported a novel class of HCV NS5B polymerase covalent
inhibitors that contained p-fluoro-nitrophenyl as the cysteine
acceptor: the fluoro atom is used as the LG and the nitro group is
used as the EWG.29 Thus, the reactivity of this type of functional
16
A secondary mutation T790M in the gatekeeper of EGFR
kinase domain accounts for about half of all known cases of
17
acquired resistance to gefitinib or erlotinib.15,
Irreversible
inhibitors of EGFR have been demonstrated as an effective tactic
for overcoming drug resistance that is driven by the T790M
mutation.18, 19, 20, 21 To date, several irreversible inhibitors have
———
 Corresponding author. Tel.: +86-010-80726688-8577; fax: +86-010-80705587; e-mail: houweijie@nibs.ac.cn

group can be finely adjusted by using different LGs and EWGs.
Importantly, LGs and EWGs can be placed at different phenyl
positions, offering considerable structural flexibility.
nitrobenzaldehyde. The synthetic route of analogs 9a-i is shown
in Scheme S1 (Supplementary data-1). Intermediate 3 was
reacted with different aliphatic alcohols, and then nitro group
reduction and amidation reactions according to Scheme 1 were
used to yield the title compounds. The synthesis of analogs 13a-j
are illustrated in Scheme S2 (Supplementary data-1).
Intermediate 2 was reacted with different aromatic aniline and
went through the next three steps according to Scheme 1 (steps c,
d, and e) to obtain the analogs 13a-j.
Here, we performed molecular docking simulation to help
better understand if these functional groups could be used to
develop EGFR inhibitors with a quinazoline scaffold. We
employed a known crystal structure of EGFR T790M (PDB ID:
4G5P)30 to model the binding mode of the quinazoline compound
with a benzamide moiety at the C-6 position (named as 6a below)
(Fig. 1). A highly conserved hydrogen bond was predicted
between the quinazoline ring N-1 and Met793 in the EGFR hinge
region, and the 4-aniline moiety protrudes into the hydrophobic
pocket of EGFR. The benzamide moiety occupies the sugar
region of the ATP-binding pocket. The carbon atoms at the
ortho- and meta-positions of benzamide could readily reach
(within 5Å) the sulfhydryl group of the Cys797 residue of EGFR
where they are ready for nucleophilic attack. Based on the
docking result, a series of novel quinazoline derivatives bearing
this new functional covalent group were synthesized.
3.2. Biological activity
3.2.1. Biological activity of 6a-p
Table 1.
In vitro EGFR kinase inhibition activities of 6a-p
EGFR WT
IC50(nM)
A431
No.
R1
IC50(nM)
Gefitinib
6a
—
H
0.5
27
768
134
6b
6c
2, 3-F, F
2-F-3-NO2
2-F-3-CF3
2-F-3-SO2Me
2-Cl-3-NO2
2-F-5-NO2
2-F-5-CN
2-F-5-SO2Me
2-Cl-5-NO2
5-F-2-NO2
2, 3, 5-F, F, F
2-F
29
220
13
1100
>10μM
189
6d
6e
30
24
6f
14
265
6g
5.0
1.3
57
2352
>50μM
396
6h
6i
6j
8.3
43
>50μM
887
Figure 1. Putative binding mode of 6a within the ATP-binding pocket of
6k
6l
EGFR T790M (PDB ID: 4G5P).
365
3
>10μM
146
3. Results and discussion
6m
6n
6o
5-NO2
50.9
4.3
242.4
107
3.1. Synthesis
—
66
6p
—
331
Table 2.
Cell antiproliferative activities of selected compounds
No.
Gefitinib
6c
6g
6h
6j
6k
H1975 IC50(nM)
7184
2480 5340 >50μM >50μM 2675
We use an HTRF KinEASE-TK kit (Cat# 62TK0PEC) and a
Cell Titer-Glo Luminescent Cell Viability Assay kit (Cat# G7573)
to test, respectively, kinase inhibition activity and cell
antiproliferative activity; the assay results for analogs 6a-p are
summarized in Table 1 and Table 2. With benzamide at the C-6
position of quinazoline (6a), the EGFR kinase inhibitory potency
(IC50) was 27nM. A fluoro atom was introduced to the phenyl C-
2 position as the LG, and different EWGs (fluoro, nitro,
trifluoromethyl, and methyl sulfonyl) were introduced the ortho-
position of this fluoro atom (6b-6e). Compounds 6b, 6d, and 6e
showed the similar inhibitory activity against WT EGFR as did
6a, while 6c showed a 2-fold improvement. EWGs were also
introduced to the para-position of the fluoro atom (6g-6i), and as
compared with 6a, both 6g and 6h showed improved inhibitory
activity, 6i was 2-fold less potent than 6a, perhaps as a result of
steric hindrance by its methyl sulfonyl group. We also explored
Scheme 1. Synthesis of analogs 6a-o. Reagents and conditions: (a) SOCl2/cat.
DMF, reflux; (b) 3-chloro-4-fluoroaniline, i-PrOH, 90°C; (c) 3-
morpholinopropan-1-ol, t-BuOK, t-BuOH/THF, 0°C to rt; (d) Fe, NH4Cl,
EtOH /H2O, 80°C; (e) aromatic acid, oxalyl chloride/cat. DMF, DCM, 0°C to
rt; THF, TEA, 0°C.
The analogs 6a-o were prepared by using 7-fluoro-6-
nitroquinazolin-4(3H)-one (1) as the starting material (Scheme 1).
Briefly, 2 was obtained through chlorination with thionyl
chloride, and then reacted with 3-chloro-4-fluoroaniline to
produce 3. The C-7 position of quinazoline in 3 was substituted
with 3-morpholinopropan-1-ol to afford the intermediate 4, and
reduction of the nitro group in 4 with iron power/ammonium
chloride yielded the key intermediate 5. 5 was reacted with
different benzoic acids to obtain analogs 6a-o. Compound 6p was
prepared via reductive amination of
5 with 2-fluoro-5-

using a chloride atom as the LG (compounds 6f and 6j), but this
change had only very minor effects on inhibitory potency. We
examined the use of a fluoro atom as the EWG: we introduced
fluoro atom to both ortho- and para-positions of the LG (fluoro)
to obtain 6l, which was 13-fold less potent than 6a. When the
nitro group of 6g was removed, 6m had very similar potency;
when the fluoro of 6g atom was removed, 6n was about 10-fold
less potent than 6g. Replacement of phenyl ring of 6m with a
pyridine ring to obtain 6o, and 6o showed virtually equal potency
with 6m. When benzamide (6g) changed to benzylamine (6p), a
dramatic decrease (about 48-fold) in potency was observed.
Based on kinase inhibitory activity data, we selected five
compounds to test their antiproliferative activity against the
H1975 cell line, and found that compounds 6c, 6g, and 6k
showed more potent antiproliferative activity than gefitinib.
serine/threonine Kinase 10 (STK10) by erlotinib contributes to
severe skin disorders.36 Previously reported inhibition assays of
gefitinib and erlotinib (1µM) showed, respectively, 80.8 and
92.7% inhibition of STK10 activity.37 6g (1μM), however,
showed only 15.6% inhibition of STK10 activity (Table 2),
suggesting that 6g may be relatively safer and confer fewer
adverse effects. Our kinase profiling results demonstrate that
modification of the C-6 position of the quinazoline scaffold (the
sugar region of the ATP-binding pocket) can improve the kinase
selectivity of EGFR inhibitors based on quinazoline, a finding
that has been mentioned in several previous reports.38, 39
Table 3.
Percent inhibition of compound 6g at 1 μM against selected kinasesa
Kinase
Percent inhibition Kinase
Percent inhibition
3.2.2. Inhibitory effects of 6g on EGFR phosphorylation
DDR1
92.1%
35.2%
76.1%
56.9%
15.6%
EGFR
98.3%
46.7%
79.0%
52.8%
ERBB2/HER2
EPHA6
ERBB4/HER4
MEK5
c-Met
c-MER
LOK/STK10
Figure 2. (A) Chemical structure of CI-1033. (B) Inhibition of EGFR
autophosphorylation in A431 cells. The treatment concentration of
compounds: 10-fold of IC50.
a Compound was tested in single dose duplicate mode, at a concentration of 1
μM. Reactions were carried out at 1 μM ATP. This table includes all the six
kinases for which 6g showed an inhibitory percentage was above 50. Data for
the other kinases in the panel can be found in supplementary data-2.
Our bioactivity data did not enable us to determine if these
compounds inhibited EGFR in an irreversible manner, so
compound 6g was selected as the representative compound to
react with reduced glutathione (GSH)31 in vitro to confirm the
reactivity against thiol nucleophiles. The results demonstrated
that 6g could transform into the adduct in the presence of GSH
(Fig. S1, S2 and S3, Supplementary data-1). We also compared
6g with some 2nd (CI-1033 and Afatinib) and 3rd (WZ4002 and
Osimertinib) generation EGFR inhibitors against different
genotypes EGFR kinases, and 6g showed less potent kinase
activity than these four irreversible inhibitors (Table S5,
Supplementary data-1). 6g was also selected to perform the
wash/no wash assays. Western blotting revealed that CI-1033, the
positive control and a known irreversible inhibitor of EGFR,
inhibited the autophosphorylation of EGFR in both no wash and
wash out treatments, while gefitinib (negative control) and 6g
showed no effect in the wash-out treatment (Fig. 2). This result
established that 6g can inhibit the autophosphorylation of EGFR,
and does so in a reversible manner, and is thus similar to gefitinib.
One explanation for the non-irreversible inhibition that we
observed might be that the angle between the carbon atom
bearing the fluoro atom at the nitrophenyl and the sulfhydryl
group of Cys797 is not conducive for nucleophilic attack to form
the covalent bond.
3.2.4. Biological activity of 9a-i
Table 4.
In vitro EGFR kinase inhibitory activities (IC50: nM) of analogs 9a-i
EGFR EGFR
EGFR
No.
R2
WT
L858R d746-750
6g
5.0
2.7
1.3
9a
9b
F
44
68
17
74
11
35
OMe
9c
9d
9e
9f
2.6
6.6
21
1.9
3.8
14
0.6
3.9
5.3
1.7
3.2.3. Kinase profiling of 6g
Profiling of the activity of compound 6g against a panel of
365 kinases was conducted, and this analysis indicated that 6g is
highly selective as an EGFR inhibitor (partial results are shown
in Table 3 and Table S1, Supplementary data-1; complete results
can be found in Supplementary data-2). Using kinase activity
inhibition > 80% as the cutoff, 6g only showed activity against
two kinases in the panel: EGFR and DDR1. In clinical patients
13
12
9g
9h
50
9.2
6.3
18
3.8
3.8
9.2
3.1
treated with EGFR drugs, the most common adverse events are
33
skin disorders32,
and diarrhea.34 These side effects can
compromise the patients’ quality of life, and can even result in
dose-reduction or interruption of treatment.35 Naoko Yamamoto
et al. (2011) demonstrated that off-target inhibition of
9i
NDa
a ND: not determined.

On the basis of aforementioned results, we decided to pursue
the development of potent EGFR inhibitors with high selectivity
instead of developing irreversible inhibitors against drug
resistance. We conducted an investigation of C-7 substituents of
quinazoline (analogs 9a-i) to identify yet more potent EGFR
inhibitors (Table 4). When a fluoro atom was introduced to the
C-7 position of quinazoline (9a), the potency decreased by about
9-fold compared to 6g. Compound 9b, with a methoxy group at
the C-7 position, was 13-fold less potent than 6g in WT EGFR.
Several other substituents were also investigated, including
piperazine analogs (9c-f) and dimethylamine analogs (9g-i).
When the morpholine group of 6g was substituted with a
piperazine group (9c), these two compounds had almost the same
potency. When a methyl group was introduced to the 4-positon of
the piperazine (9d), the potency in WT EGFR decreased slightly.
Compound 9e has an acetyl group at the 4-positon of the
piperazine, and was 8-fold less potent than 9c. Compared to the
3-carbon chain of 9d, the shorter 2-carbon chain (9f) resulted in a
slightly decreased potency against WT EGFR. Compared with 9g,
extension of carbon chain, 9h (3-carbon chain) and 9i (4-carbon
chain) showed a 5-fold and 8-fold improvement, respectively,
against WT EGFR.
decreased by about 10 folds; a further 10-fold decrease in
potency resulted when the fluoro atom was removed (13b). The
introduction of a cyano group to the para-position of aniline (13c)
resulted in a dramatic drop (at least 485-fold) in potency
compared with 6g. A number of substituents, including small
aliphatic and large aromatic groups were investigated in
compounds 13d-13j. With the exception of 13i and 13j, these
compounds were less potent than 6g, with IC50 values above
150nM; these decreases in potency are likely attributable to
unfavorable interactions between these substituents and the
hydrophobic pocket of EGFR. Compounds 13i and 13j had IC50
values of 39nM and 28nM, respectively. Additionally, 14
compounds (Table S2, selected from among 9a-i and 13a-j) were
evaluated for their antiproliferative activity against the H1975
cell line; and 4 were found to be more potent than gefitinib.
3.2.6. Evaluation of 6g against various cell lines
Table 6.
Cell antiproliferative activity of 6g against various cell lines
Cell Line
Ba/F3 (EGFR L858R)
Ba/F3 (EGFR Del E746
_A750)
IC50 (μM)
Cell Line
IC50 (μM)
0.182
SF295 (EGFR WT)
>10
3.2.5. Biological activity of 13a-j
0.178
SF539 (EGFR WT)
Ba/F3 (cat-EGFR)
>10
Table 5.
HCC827 (EGFR Del E7
46_A750)
In vitro EGFR kinase inhibitory activities (IC50: nM) of analogs 13a-j
0.098
2.35
5.34
>10
>10
>10
A431 (EGFR WT)
NCI-H1975 (EGFR L85
8R/T790M)
HEK293
Ba/F3 (Her2
negative)
Compound 6g was selected for further biological evaluation,
and was evaluated against various cell lines that harbored
different EGFR mutations and HEK293 (normal human
embryonic kidney cells). Compound 6g showed potent inhibition
of the growth of Ba/F3 (EGFR L858R), Ba/F3 (EGFR del
E746_A750), and HCC827 cell lines; it did not display obvious
antiproliferative activity against the cell lines with WT EGFR
(SF295, SF539, Ba/F3) (Table 6). It is known that in patients the
concurrent inhibition of WT EGFR by inhibitors can lead to
adverse effects, including skin rash and diarrhea.40, 41 The fact
that 6g has almost no antiproliferative activity against cell lines
with WT EGFR implies that it should be able to reduce these
adverse effects. The IC50 of compound 6g against HEK293
exceeded 10 μM, indicating that the cytotoxic effects of 6g
appear to be minimal.
EGFR EGFR
EGFR
No.
R3
R4
WT
L858R d746-750
6g
F
F
Cl
F
5.0
2.7
32
1.3
11
13a
13b
13c
53
F
H
Cl
722
427
2245
110
474
CN
2426
13d
13e
13f
Cl
Cl
Cl
172
503
374
735
463
270
71
210
128
3.2.7. Preliminary druggability evaluation of 6g.
Table 7.
Pharmacokinetic parameters of 6g in mousea
Cmax
Tmax
AUClast
AUCInf
T1/2
MRT
(hr)
F
13g
13h
13i
Cl
Cl
Cl
390
169
39
666
475
194
419
268
125
Route
(ng/mL) (hr) (hr*ng/mL) (hr*ng/mL) (hr)
(%)
Oral
44.2
2.00
563
669
8.77 12.47 57.1
a The pharmacokinetic parameters are determined after oral administration of
a single (10 mg/kg) dose.
We also evaluated the preliminary druggability of compound
6g. 6g was tested for its inhibition of human cytochrome P450
(CYP) enzymes (5 isoforms: 1A2, 2D6, 3A4, 2C9, 2C19) and the
IC50 values were all higher than 50 μM (Table S3, Supplementary
data-1). These results suggest that 6g has a low potential for
involvement in drug-drug interactions. Liver microsome
metabolic stability assays were also conducted, and the half-life
13j
Cl
28
>200
>200
Further exploration at the C-4 position of quinazoline was also
conducted to search for a group that was capable of establishing
additional interactions with the nearby hydrophobic pocket of
EGFR. When the chlorine atom at the meta-position of aniline
(6g) was replaced with a fluoro atom (13a), the potency

of 6g in human, rat, and mouse all exceeded 90min (Table S4,
Supplementary data-1), suggesting that 6g should have excellent
metabolic stability in the liver. A pharmacokinetic study of 6g
was performed in mouse (Table 7): 6g demonstrated fair oral
bioavailability (57.1%) and a desirable half-life time (8.77h). It is
known that nitro-anion radical or a nitroso group could form as a
result of the reduction of nitro group.29, 42 So we performed the
Salmonella/mammalian microsome Mini-AMES reverse
mutation assay43 to evaluate this potential safety issues of
compound 6g. No bacterial mutagenicity was observed under the
tested doses in TA98 strain with or without metabolic activation
(Table S6, Supplementary data-1); while 6g showed bactericidal
effect at highest dose in TA100 strain both with and without
metabolic activation; no bacterial mutagenicity was observed at
the rest doses in TA100 strain with or without metabolic
activation (Table S7, Supplementary data-1). The results above
demonstrated that little potential safety issues related to nitro
group of 6g has been found in Mini-AMES assay.
Compounds 1-4 were prepared following the Patent:
US2003/158408 A1, 2003. Compound 4 (414mg, 0.9mmol) was
dissolved in ethanol (4 mL) and water (2mL). Iron powder
(251mg, 4.5mmol) and ammonium chloride (240mg, 4.5mmol)
were then added, and the resulting mixture was heated to 80°C
for 3 hours. The reaction mixture was cooled to room
temperature and filtered through celite. The ethanol was removed
in vacuo, and the resulting residue was basified with saturated
sodium bicarbonate and extracted with ethyl acetate three times.
The organic layer was separated and dried using anhydrous
sodium sulfate, concentrated to afford 324mg of yellow solid
(84%).1H NMR (400 MHz, DMSO-d6
4H), 2.49 (t, J =8.4 Hz, 2H), 3.66 (m, 4H), 4.16 (t, J = 6.6Hz,
2H), 4.21 (m, 2H), 6.86 (s, 1H), 6.98 (m, 1H), 7.08 (t, J = 8.4Hz,
1H), 7.11 (s, 1H), 7.43 (m, 1H), 7.86 (dd, J = 2.4, 6.6Hz, 1H),
8.51 (s, 1H).
5.3. General method for preparing of analogs 6a-o
Analogs 6a-o were synthesized as outlined in Scheme 1: Acid
(1.2eq) was dissolved in anhydrous DCM, oxalyl chloride
(1.44eq) was added dropwise at 0°C and then one drop DMF was
added. The reaction mixture stirred at room temperature for 2
hours. The excess oxalyl chloride was removed under reduced
pressure, and the residue dissolved in THF for next
transformation.
4. Conclusion
In summary, a series of 4-amino quinazoline derivatives with
C-6 benzamide substituents were synthesized and evaluated as
EGFR inhibitors. The representative compound 6g strongly
inhibited the kinase activity of EGFR and displayed potent
cancer cell antiproliferative activity, while showing minimal
cytotoxicity to HEK293 cell line. Western blotting showed that
the phosphorylation of EGFR was inhibited by compound 6g,
and in a reversible manner. Kinase inhibition profiling (with a
panel of 365 kinases) for 6g demonstrated that this compound is
a highly selective EGFR inhibitor. Importantly, 6g performed
well in liver microsome metabolic stability and CYP inhibition
assays, and showed desirable pharmacokinetic properties. No
Intermediate 5 (1eq) and triethylamine (1.5eq) were added
sequent to a 25ml three-neck-bottom flask under a nitrogen
atmosphere. Acyl chloride in THF was added dropwise to flask at
0°C. The reaction mixture was stirred at 0°C. After pale yellow
solid appeared, the mixture reacted at room temperature until
TLC showed 5 disappeared. Ice water was added to reaction
mixture at 0°C, and stirred for another 30min until no insoluble
solid generated. The solid was filtered to get crude product. The
crude product further purified by medium pressure column
chromatography (C18 padding, ACN: H2O（containing 0.05%
TFA）=1: 99 – 99: 1) to get product as a solid.
bacterial
mutagenicity was
observed
with
6g in
Salmonella/mammalian microsome Mini-AMES reverse
mutation assay. In conclusion, we identified the new quinazoline
derivative 6g with a p-nitro-fluoro-benzamide moiety at the C-6
position and demonstrate that this new lead compound is a highly
selective inhibitor of EGFR. Further evaluation of this
compound’s druggability is currently in progress.
5.3.1. N-(4-((3-chloro-4-fluorophenyl) amino)-7-(3-morpholinop
ropoxy) quinazolin-6-yl) benzamide (6a)
Pale yellow solid; yield: 48%.1H NMR (400 MHz, DMSO-d6)
δ 2.22-2.27 (m, 2H), 2.98-3.05 (m, 2H), 3.32-3.36 (m, 2H), 3.43-
3.46 (m, 2H), 4.02 (m, 2H), 3.63 (t, J = 12.0Hz, 2H), 3.93-3.97
(m, 2H), 4.34 (t, J = 5.6Hz, 2H), 7.39 (s, 1H), 7.50 (t, J = 9.2Hz,
1H), 7.57-7.62 (m, 2H), 7.64-7.68 (m, 1H), 7.76-7.80 (m, 1H),
8.04-8.07 (m, 2H), 8.12 (dd, J = 2.8, 6.8Hz, 1H), 8.75 (s, 1H),
8.89 (s, 1H), 9.82(br, 1H), 10.00 (s, 1H). MS (ESI) m/z =
536.1879 (M+H+), calcd. for C28H27ClFN5O3 m/z = 536.1865.
5. Experiments
5.1. General
The reagents and solvents for reactions were reagent-grade
and used without further purification. Glass apparatus for
reaction dewater and deoxygenize under nitrogen atmospheric
and high temperature. Reactions sensitive to water and air
rigorously followed water-free and oxygen-free reaction
procedure, added reagents with injection syringe. LC-MS system:
5.3.2. N-(4-((3-chloro-4-fluorophenyl)amino)-7-(3-morpholinopr
opoxy)quinazolin-6-yl)-2,3-difluorobenzamide (6b)
1
Waters 2545 (XBndgeTM C18) / 3100 Mass Detector. H NMR
1
Pale yellow solid; yield: 35%. H NMR (400 MHz, DMSO-d6) δ
spectra were recorded using a Varian Mercury 400 spectrometer
under 400 MHz with tetramethylsilane (TMS) as an internal
standard. The NMR data are given as follows: chemical shift (δ)
in ppm, coupling constants (J) in Hz. 13C NMR were recorded
using a Varian Mercury 400 spectrometer under 100 MHz. Data
of 1H NMR are reported as follows: chemical shift, multiplicity (s
= singlet, br = broad, d = doublet, t = triplet, m = multiplet),
coupling constants and integration. High-resolution mass spectra
were obtained using Agilent Technolodies 6540 UHD Accurate-
Mass Q-TOF LC/MS. Thin-layer chromatography (TLC) was
carried out using HS-GF 254 silica plates, Chromatographic
purification was carried out using silica gel (Yan tai mou ping
yuan bo jing xi silica gel plant)
2.26-2.33 (m, 2H), 3.06-3.12 (m, 2H), 3.38-3.43 (m, 2H), 3.47-
3.52 (m, 2H), 3.69-3.76 (m, 2H), 3.96-4.03 (m, 2H), 4.38 (t, J =
5.6Hz, 2H), 7.40-7.45 (m, 2H), 7.51 (t, J = 9.2Hz, 1H), 7.64-7.77
(m, 3H), 8.05-8.08 (m, 1H), 8.78 (s, 1H), 9.10 (s, 1H), 10.12 (m,
1H), 10.75 (br, 1H). MS (ESI) m/z = 572.1673 (M+H+), calcd.
for C28H26ClF3N5O3 m/z = 572.1676.
5.3.3. N-(4-((3-chloro-4-fluorophenyl)amino)-7-(3-morpholinopr
opoxy)quinazolin-6-yl)-2-fluoro-3-nitrobenzamide (6c)
Pale yellow solid; yield: 31%. 1H NMR (400 MHz, DMSO-d6)
δ 2.23-2.28 (m, 2H), 3.04-3.13 (m, 4H), 3.64-3.70 (m, 2H), 3.99-
4.02 (m, 2H), 4.37 (t, J = 5.6Hz, 2H), 7.38 (s, 1H), 7.50 (t, J =
9.2Hz, 1H), 7.64 (t, J = 8.0Hz, 1H), 7.74-7.77 (m, 1H), 8.08-8.11
(m, 1H), 8.16 (t, J = 6.8Hz, 1H), 8.38 (t, J = 7.6Hz, 1H), 8.72 (s,
5.2. Preparation of compounds 1–5

1H), 9.02 (s, 1H), 9.80 (br, 1H), 10.28 (s, 1H). MS (ESI) m/z =
599.1618 (M+H+), calcd. for C28H26ClF2N6O5 m/z = 599.1621.
8.75 (s, 1H), 9.03 (s, 1H), 10.26 (s, 1H). MS (ESI) m/z =
632.1545 (M+H+), calcd. for C29H29ClF2N5O5S m/z = 632.1546.
5.3.4. N-(4-((3-chloro-4-fluorophenyl)amino)-7-(3-morpholinopr
opoxy)quinazolin-6-yl)-2-fluoro-3-(trifluoromethyl)benzamide (6
d)
5.3.10. N-(4-((3-chloro-4-fluorophenyl)amino)-7-(3-morpholinop
ropoxy)quinazolin-6-yl)-2-chloro-5-nitrobenzamide (6j)
Pale yellow solid; yield: 36%. 1H NMR (400 MHz, DMSO-d6)
δ 2.21-2.28 (m, 2H), 3.06 (br, 4H), 3.61-3.67 (m, 4H), 3.97-4.00
(m, 2H), 4.36 (t, J = 6.0Hz, 2H), 7.39 (s, 1H), 7.51 (t, J = 9.2Hz,
1H), 7.73-7.77 (m, 1H), 7.95 (d, J = 8.8Hz, 1H), 8.08 (dd, J = 2.8,
6.8Hz, 1H), 8.40 (dd, J = 2.8, 8.8Hz, 1H), 8.46 (d, J = 2.4Hz, 1H),
8.76 (s, 1H), 8.95 (s, 1H), 9.77 (br, 1H), 10.48 (s, 1H). MS (ESI)
m/z = 615.1326 (M+H+), calcd. for C28H26Cl2FN6O5 m/z =
615.1326.
Pale yellow solid; yield: 40%. 1H NMR (400 MHz, DMSO-d6)
δ 2.23-2.29 (m, 2H), 2.95-3.11 (m, 4H), 3.64-3.72 (m, 2H), 3.97-
4.01 (m, 2H), 4.37 (t, J = 5.6Hz, 2H), 7.37-7.39 (m, 1H), 7.45-
7.51 (m, 1H), 7.62 (t, J = 7.6Hz, 1H), 7.76-7.80 (m, 1H), 8.04 (t,
J = 7.6Hz, 1H), 8.09-8.13 (m, 1H), 8.68 (s, 1H), 8.97 (s, 1H),
10.23 (s, 1H). MS (ESI) m/z = 622.1650 (M+H+), calcd. for
C29H26ClF5N5O3 m/z = 622.1644.
5.3.5. N-(4-((3-chloro-4-fluorophenyl)amino)-7-(3-morpholinopr
opoxy)quinazolin-6-yl)-2-fluoro-3-(methylsulfonyl)benzamide (6e)
5.3.11. N-(4-((3-chloro-4-fluorophenyl)amino)-7-(3-morpholinop
ropoxy)quinazolin-6-yl)-5-fluoro-2-nitrobenzamide (6k)
Pale yellow solid; yield: 36%. 1H NMR (400 MHz, DMSO-d6)
δ 2.27-2.33 (m, 2H), 3.11 (bs, 2H), 3.38-3.43 (m, 4H), 3.44 (s,
3H), 3.68-3.74 (m, 2H), 3.99-4.03 (m, 2H), 4.38 (t, J = 5.6Hz,
2H), 7.43 (s, 1H), 7.50 (t, J = 9.2Hz, 1H), 7.66 (t, J = 8.0Hz, 1H),
7.74-7.78 (m, 1H), 8.07-8.12 (m, 2H), 8.14-8.18 (m, 1H), 8.76 (s,
1H), 9.06 (s, 1H), 10.10 (br, 1H), 10.31 (d, J = 3.2Hz, 1H). MS
(ESI) m/z = 632.1545 (M+H+), calcd. for C29H29ClF2N5O5S m/z
= 632.1546.
Pale yellow solid; yield: 28%. 1H NMR (400 MHz, DMSO-d6)
δ 1.93-2.00 (m, 2H), 2.35 (m, 4H), 2.45 (bs, 4H), 2.46 (t, J =
7.2Hz, 2H), 3.56 (t, J = 4.4Hz, 4H), 4.26 (t, J = 6.4Hz, 2H), 7.32
(s, 1H), 7.44 (t, J = 9.2Hz, 1H), 7.62-7.68 (m, 2H), 7.71-7.73 (m,
1H), 7.78-7.82 (m, 1H), 8.12 (dd, J = 2.4, 6.8Hz, 1H), 8.31 (dd, J
= 4.8, 8.8Hz, 1H), 8.55 (s, 1H), 8.82 (s, 1H), 9.96 (s, 1H), 10.30
(s, 1H). MS (ESI) m/z = 599.1627 (M+H+), calcd. for
C28H26ClF2N6O5 m/z = 599.1621.
5.3.6. N-(4-((3-chloro-4-fluorophenyl)amino)-7-(3-morpholinopr
opoxy)quinazolin-6-yl)-2-chloro-3-nitrobenzamide (6f)
5.3.12. N-(4-((3-chloro-4-fluorophenyl)amino)-7-(3-morpholinop
ropoxy)quinazolin-6-yl)-2,3,5-trifluorobenzamide (6l)
Pale yellow solid; yield: 35%. 1H NMR (400 MHz, DMSO-d6)
δ 2.23-2.29 (m, 2H), 3.02-3.09 (m, 2H), 3.34-3.38 (m, 2H), 3.44-
3.48 (m, 2H), 3.65-3.72 (m, 2H), 3.98-4.01 (m, 2H), 4.35 (t, J =
5.6Hz, 2H), 7.40 (s, 1H), 7.51 (t, J = 9.2Hz, 1H), 7.72-7.76 (m,
1H), 7.77 (t, J = 8.0Hz, 1H), 7.94 (dd, J = 2.0, 8.0Hz, 1H), 8.05-
8.07 (m, 1H), 8.20 (dd, J = 2.0, 8.4Hz, 1H), 8.77 (s, 1H), 8.98 (s,
1H), 10.09 (br, 1H), 10.47 (s, 1H). MS (ESI) m/z = 615.1332
(M+H+), calcd. for C28H26Cl2FN6O5 m/z = 615.1326.
Pale yellow solid; yield: 40%. 1H NMR (400 MHz, DMSO-d6)
δ 2.24-2.31 (m, 2H), 3.02-3.13 (m, 2H), 3.37-3.41 (m, 4H), 3.69-
3.73 (m, 2H), 3.96-4.02 (m, 2H), 4.37 (t, J = 5.6Hz, 2H), 7.40-
7.41 (m, 1H), 7.47-7.52 (m, 2H), 7.74-7.78 (m, 1H), 7.83-7.90
(m, 1H), 8.09 (dd, J = 2.4, 7.6Hz, 1H), 8.71 (s, 1H), 9.02 (s, 1H),
10.17-10.19 (m, 1H), 10.50 (s, 1H). MS (ESI) m/z= 590.1588
(M+H+), calcd. for C28H25ClF4N5O3 m/z = 590.1582.
5.3.13. N-(4-((3-chloro-4-fluorophenyl)amino)-7-(3-morpholinop
5.3.7. N-(4-((3-chloro-4-fluorophenyl)amino)-7-(3-morpholinopr
opoxy)quinazolin-6-yl)-2-fluoro-5-nitrobenzamide (6g)
ropoxy)quinazolin-6-yl)-2-fluorobenzamide (6m)
Pale yellow solid; yield: 30%. 1H NMR (400 MHz, DMSO-d6)
δ 2.27-2.32 (m, 2H), 3.05-3.12 (m, 4H), 3.64-3.72 (m, 4H), 4.00-
4.04 (m, 2H), 4.38 (t, J = 5.6Hz, 2H), 7.39 (s, 1H), 7.41-7.53 (m,
3H), 7.67-7.76 (m, 3H), 7.91 (t, J = 7.6Hz, 1H), 8.07 (dd, J = 2.4,
6.4Hz, 1H), 8.75 (s, 1H), 9.13 (s, 1H), 9.96 (d, J = 6.4Hz, 1H).
MS (ESI) m/z = 554.1766 (M+H+), calcd. for C28H27ClF2N5O3
m/z = 554.1770.
Pale yellow solid; yield: 32%. 1H NMR (400 MHz, DMSO-d6)
δ 1.98-2.02 (m, 2H), 2.37 (bs, 4H), 3.28-3.31 (m, 2H), 3.57 (bs,
4H), 4.29 (t, J = 5.6Hz, 2H), 7.34 (s, 1H), 7.44 (t, J = 9.2Hz, 1H),
7.75 (t, J = 9.2Hz, 1H), 7.80-7.84 (m, 1H), 8.15 (dd, J = 2.4,
6.8Hz, 1H), 8.52 (m, 1H), 8.58 (s, 1H), 8.60-8.64 (m, 1H), 8.91
(s, 1H), 9.90 (s, 1H), 10.17 (br, 1H). 13CNMR (100 MHz,
DMSO-d6) δ 25.57, 53.31, 54.59, 66.17, 67.08, 107.55, 108.74,
116.45, 117.31, 118.59, 122.48, 123.63, 124.29, 126.07, 126.39,
128.71, 136.70, 144.00, 149.80, 152.01, 154.36, 155.19, 156.92,
160.44, 161.43, 164.00. MS (ESI) m/z = 599.1627 (M+H+), calcd.
for C28H26ClF2N6O5 m/z = 599.1621.
5.3.14. N-(4-((3-chloro-4-fluorophenyl)amino)-7-(3-morpholinop
ropoxy)quinazolin-6-yl)-3-nitrobenzamide (6n)
Pale yellow solid; yield: 30%. 1H NMR (400 MHz, DMSO-d6)
-2.25 (m, 2H), 3.04 (bs, 4H), 3.55-3.65 (m, 4H), 3.94-3.97
δ (m, 2H), 4.35 (t, J = 5.6Hz, 2H), 7.39 (s, 1H), 7.49 (t, J = 9.2Hz,
1H), 7.77-7.81 (m, 1H), 7.91 (t, J = 8.0Hz, 1H), 8.15 (dd, J = 2.8,
6.8Hz, 1H), 8.46-8.53 (m, 2H), 8.72 (s, 1H), 8.81 (s, 1H), 8.88 (t,
J = 2.0Hz, 1H), 9.64 (br, 1H), 10.46 (s, 1H). MS (ESI) m/z =
581.1716 (M+H+), calcd. for C28H27ClFN6O5 m/z = 581.1715.
5.3.8. N-(4-((3-chloro-4-fluorophenyl)amino)-7-(3-morpholinopr
opoxy)quinazolin-6-yl)-2-fluoro-5-cyanobenzamide (6h)
Pale yellow solid; yield: 41%. 1H NMR (400 MHz, DMSO-d6)
δ 2.23-2.29 (m, 2H), 2.43-2.46 (m, 2H), 3.05-3.12 (m, 4H), 3.65-
3.71 (m, 2H), 3.99-4.03 (m, 2H), 4.37 (t, J = 5.6Hz, 2H), 7.39 (s,
1H), 7.50 (t, J = 9.2Hz, 1H), 7.68-7.73 (m, 1H), 7.74-7.78 (m,
1H), 8.08 (dd, J = 2.4, 6.4Hz, 1H), 8.18-8.22 (m, 1H), 8.30 (dd, J
= 2.4, 6.4Hz, 1H), 8.72 (s, 1H), 9.02 (s, 1H), 9.89 (br, 1H), 10.19
(d, J = 3.2Hz, 1H). MS (ESI) m/z = 579.1720 (M+H+), calcd. for
C29H26ClF2N6O3 m/z = 579.1723.
5.3.15. N-(4-((3-chloro-4-fluorophenyl)amino)-7-(3-morpholinop
ropoxy)quinazolin-6-yl)-2-fluoronicotinamide (6o)
Pale yellow solid; yield: 32%. 1H NMR (400 MHz, DMSO-d6)
δ 2.25-2.33 (m, 2H), 3.06-3.14 (m, 4H), 3.64-3.71 (m, 4H), 4.00-
4.05 (m, 2H), 4.38 (t, J = 5.6Hz, 2H), 7.39 (s, 1H), 7.50 (t, J =
9.2Hz, 1H), 7.59-7.63 (m, 1H), 7.73-7.77 (m, 1H), 8.07-8.09 (m,
1H), 8.38-8.43 (m, 1H), 8.48-8.49 (m, 1H), 8.74 (s, 1H), 9.10 (s,
1H), 9.86 (br, 1H), 10.14 (d, J = 4.8Hz, 1H). MS (ESI) m/z =
555.1718 (M+H+), calcd. for C27H26ClF2N6O3 m/z = 555.1723.
5.3.9. N-(4-((3-chloro-4-fluorophenyl)amino)-7-(3-morpholinopr
opoxy)quinazolin-6-yl)-2-fluoro-5-(methylsulfonyl)benzamide (6i)
Pale yellow solid; yield: 28%. 1H NMR (400 MHz, DMSO-d6)
δ 2.25-2.31 (m, 2H), 3.07-3.13 (m, 4H), 3.34 (s, 3H), 3.64-3.71
(m, 4H), 3.99-4.04 (m, 2H), 4.38 (t, J = 5.6Hz, 2H), 7.40 (s, 1H),
7.50 (t, J = 9.2Hz, 1H), 7.73-7.78 (m, 2H), 8.08 (dd, J = 2.8,
6.8Hz, 1H),, 8.22-8.26 (m, 1H), 8.34 (dd, J = 2.4, 6.4Hz, 1H),
5.3.16. Preparation of compound 6p
Intermediate
5
(22mg,
0.05mmol),
2-fluoro-5-
nitrobenzaldehyde (10mg, 0.06mmol) anhydrous methanol(5ml)
and acetic acid (2μl, 0.05mmol) were added sequent to 25ml a

dewatered three-neck-bottom flask under a nitrogen atmosphere.
The reaction mixture stirred at 60 ° C for 4 hours. Then
NaBH3CN(5mg，0.08mmol) was added to the mixture at 0°C.
The reaction was allowed to warm to room temperature and
stirred at room temperature for 2 hours. Saturated sodium
carbonate solution (3ml) was added to reaction mixture. The
solvent was removed under reduced pressure. The residue was
partitioned between dichloromethane and water. The organic
layer was washed with brine and dried over Na2SO4, the solid
was filtered off, and the filtrate was concentrated under reduced
pressure to get crude product. The crude product purified by
medium pressure column chromatography (C18 padding, ACN:
H2O (containing 0.05% TFA) =1: 99 – 99: 1) to get product as a
pale-yellow solid (12mg, yield: 41%).1H NMR (400 MHz,
DMSO-d6) δ 2.27-2.31 (m, 2H), 3.08-3.16 (m, 4H), 3.49-3.54 (m,
4H), 3.64-3.70 (m, 2H), 3.98-4.04 (m, 2H), 4.38 (t, J = 5.6Hz,
2H), 4.74 (d, J = 6.4Hz, 2H), 6.71 (s, 1H), 7.21 (s, 1H), 7.41 (s,
1H), 7.51 (t, J = 9.2Hz, 1H), 7.56-7.63 (m, 2H), 7.94-7.97 (m,
1H), 8.20 (dd, J = 2.8, 6.4Hz, 1H), 8.24-8.28 (m, 1H), 8.68 (s,
1H), 9.83 (br, 1H). MS (ESI) m/z = 585.1846 (M+H+), calcd. for
C28H28ClF2N6O4 m/z = 585.1829.
3.20-3.28 (m, 2H), 3.39-3.46 (m, 2H), 4.35 (t, J = 5.6Hz, 2H),
7.40-7.42 (m, 1H), 7.53 (t, J = 9.2Hz, 1H), 7.71-7.78 (m, 2H),
8.06 (dd, J = 2.4, 6.8Hz, 1H), 8.52-8.56 (m, 1H), 8.62 (dd, J =
3.2, 6.0Hz, 1H), 8.81 (s, 1H), 9.06 (s, 1H), 10.32 (d, J = 4.0Hz,
1H), 10.83 (s, 1H). MS (ESI) m/z = 612.1933 (M+H+), calcd. for
C29H29ClF2N7O4 m/z = 612.1938.
5.4.5. N-(7-(3-(4-acetylpiperazin-1-yl)propoxy)-4-((3-chloro-4-fl
uorophenyl)amino)quinazolin-6-yl)-2-fluoro-5-nitrobenzamide (9
e)
Pale yellow solid; yield: 29%. 1H NMR (400 MHz, DMSO-d6)
δ 2.06 (s, 3H), 2.25-2.31 (m, 2H), 2.92-2.98 (m, 2H), 3.06-3.12
(m, 2H), 3.37-3.40 (m, 2H), 3.50-3.56 (m, 2H), 3.99-4.03 (m,
2H), 4.38 (t, J = 5.6Hz, 2H), 7.42 (s, 1H), 7.49 (t, J = 9.2Hz, 1H),
7.72-7.79 (m, 2H), 8.10 (dd, J = 2.8, 6.8Hz, 1H), 8.52-8.56 (m,
1H), 8.64 (dd, J = 3.2, 6.0Hz, 1H), 8.71 (s, 1H), 9.03 (s, 1H),
9.93 (br, 1H), 10.28 (d, J = 3.6Hz, 1H), 10.47 (br, 1H). MS (ESI)
m/z = 640.1900 (M+H+), calcd. for C30H29ClF2N7O5 m/z =
640.1887.
5.4.6. N-(4-((3-chloro-4-fluorophenyl)amino)-7-(2-(4-methylpipe
razin-1-yl)ethoxy)quinazolin-6-yl)-2-fluoro-5-nitrobenzamide (9f)
Pale yellow solid; yield: 30%. 1H NMR (400 MHz, DMSO-d6)
δ 2.77 (s, 3H), 3.06 (bs, 4H), 3.22-3.39 (m, 6H), 3.39 (bs, 2H),
4.43 (t, J = 4.4Hz, 2H), 7.44 (s, 1H), 7.52 (t, J = 9.2Hz, 1H),
7.71-7.78 (m, 2H), 8.07 (dd, J = 2.8, 7.2Hz, 1H), 8.53-8.57 (m,
1H), 8.62 (dd, J = 2.8, 5.6Hz, 1H), 8.78 (s, 1H), 9.05 (s, 1H),
10.33 (d, J = 3.2Hz, 1H), 10.74 (br, 1H). MS (ESI) m/z =
598.1791 (M+H+), calcd. for C28H27ClF2N7O4 m/z = 598.1781.
5.4. General method for preparing of analogs 9a-i
The analogs 9 was synthesized from intermediate 3 as shown
in scheme S1. In step a, different aliphatic alcohol was used to
replace 3-morpholinopropan-1-ol according to scheme 1 to
obtain intermediates 7. The next two steps were according to
analogs 6 to obtain analogs 9.
5.4.1. N-(4-((3-chloro-4-fluorophenyl)amino)-7-fluoroquinazolin
5.4.7. N-(4-((3-chloro-4-fluorophenyl)amino)-7-(2-(dimethylami
-6-yl)-2-fluoro-5-nitrobenzamide (9a)
no)ethoxy)quinazolin-6-yl)-2-fluoro-5-nitrobenzamide (9g)
Pale yellow solid; yield: 35%. 1H NMR (400 MHz, DMSO-d6)
δ 7.50 (t, J = 9.2Hz, 1H), 7.72-7.79 (m, 3H), 8.09 (dd, J = 2.8,
6.8Hz, 1H), 8.50-8.54 (m, 1H), 8.59 (dd, J = 2.8, 6.0Hz, 1H),
8.76 (s, 1H), 9.03 (d, J = 8.0Hz, 1H), 10.61 (br, 1H), 10.95 (s,
Pale yellow solid; yield: 30%. 1H NMR (400 MHz, DMSO-d6)
δ 2.94 (s, 3H), 2.95 (s, 3H), 3.67 (t, J = 4.4Hz, 2H), 4.64 (t, J =
4.8Hz, 2H), 7.44 (s, 1H), 7.47 (t, J = 9.2Hz, 1H), 7.70 (t, J =
9.2Hz, 1H), 7.76-7.80 (m, 1H), 8.11 (dd, J =2.8, 6.8Hz, 1H),
8.18-8.22 (m, 1H), 8.32 (dd, J = 2.4, 6.8Hz, 1H), 8.66 (s, 1H),
9.02 (s, 1H), 9.56 (br, 1H), 10.14 (s, 1H). LC-MS (ESI) m/z =
543.2 (M+H+), calcd. for C25H22ClF2N6O4 m/z = 543.1359.
1H). MS (ESI) m/z
=
474.0586 (M+H+), calcd. for
C21H12ClF3N5O3 m/z = 474.0581. 13C NMR (100 MHz, DMSO-
d6) δ 104.59, 111.58, 116.80, 118.34, 119.03, 120.62, 123.84,
124.48, 125.10, 125.47, 125.93, 128.74, 135.46, 143.87, 153.00,
153.94, 155.43, 157.65, 159.94, 161.39, 163.95.
5.4.8. N-(4-((3-chloro-4-fluorophenyl)amino)-7-(3-(dimethylami
no)propoxy)quinazolin-6-yl)-2-fluoro-5-nitrobenzamide (9h)
Pale yellow solid; yield: 36%. 1H NMR (400 MHz, DMSO-d6)
δ 2.20-2.27 (m, 2H), 2.83 (s, 3H), 2.84 (s, 3H), 3.30-3.35 (m, 2H),
4.36 (t, J = 5.6Hz, 2H), 7.38 (s, 1H), 7.48 (t, J = 9.2Hz, 1H),
7.73-7.79 (m, 2H), 8.12 (dd, J = 2.4, 6.8Hz, 1H), 8.52-8.56 (m,
1H), 8.63 (dd, J = 2.8, 6.0Hz, 1H), 8.68 (s, 1H), 8.99 (s, 1H),
9.50 (br, 1H), 10.26 (d, J = 3.2Hz, 1H). MS (ESI) m/z =
557.1510 (M+H+), calcd. for C26H24ClF2N6O4 m/z = 557.1516.
5.4.2. N-(4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazol
in-6-yl)-2-fluoro-5-nitrobenzamide (9b)
Pale yellow solid; yield: 32%. 1H NMR (400 MHz, DMSO-d6)
δ 4.04 (s, 3H), 7.36 (s, 1H), 7.44 (t, J = 9.2Hz, 1H), 7.72 (t, J =
9.2Hz, 1H), 7.79-7.83 (m, 1H), 8.14 (dd, J = 2.4, 6.8Hz, 1H),
8.48-8.52 (m, 1H), 8.57-8.59 (m, 2H), 8.95 (s, 1H), 9.95 (s, 1H),
10.28 (d, J = 3.2Hz, 1H). MS (ESI) m/z = 486.0797 (M+H+),
calcd. for C22H15ClF2N5O4 m/z = 486.0781.
5.4.9. N-(4-((3-chloro-4-fluorophenyl)amino)-7-(4-(dimethylami
no)butoxy)quinazolin-6-yl)-2-fluoro-5-nitrobenzamide (9i)
5.4.3. N-(4-((3-chloro-4-fluorophenyl)amino)-7-(3-(piperazin-1-y
Pale yellow solid; yield: 35%. 1H NMR (400 MHz, DMSO-d6)
δ 1.85-1.90 (m, 4H), 2.77 (s, 3H), 2.78 (s, 3H), 3.12-3.17 (m, 2H),
4.31 (t, J = 5.2Hz, 2H), 7.38 (s, 1H), 7.48 (t, J = 9.2Hz, 1H),
7.72-7.80 (m, 2H), 8.11 (dd, J = 2.4, 6.8Hz, 1H), 8.52-8.56 (m,
1H), 8.62 (dd, J = 3.2, 6.0Hz, 1H), 8.68 (s, 1H), 8.98 (s, 1H),
9.35 (br, 1H), 10.24 (d, J =4.0Hz, 1H). MS (ESI) m/z = 571.1693
(M+H+), calcd. for C27H26ClF2N6O4 m/z = 571.1672.
l)propoxy)quinazolin-6-yl)-2-fluoro-5-nitrobenzamide (9c)
Tert-butyl-4-(3-hydroxypropyl)piperazine-1-carboxylate was
used in step a and the intermediate got from step b and c went
through removal of t-butyloxy carbonyl by TFA to obtain the
1
compound 9c. HNMR (400 MHz, DMSO-d6) δ 2.11 (m, 2H),
2.92 (m, 4H), 3.23 (m, 4H), 4.34 (t, J = 5.6Hz, 2H), 7.38 (s, 1H),
7.50 (t, J = 9.2Hz, 1H), 7.73-7.78 (m, 2H), 8.08-8.10 (m, 1H),
8.53-8.56 (m, 1H), 8.61-8.63 (m, 1H), 8.73 (s, 1H), 9.02 (s, 1H),
10.29 (s, 1H), 10.54 (br, 1H). MS (ESI) m/z = 598.1802 (M+H+),
calcd. for C28H27ClF2N7O4 m/z = 598.1781.
5.5. General method for preparing of analogs 13a-j
5.5.1. N-(4-((3,4-difluorophenyl)amino)-7-(3-morpholinopropoxy)
quinazolin-6-yl)-2-fluoro-5-nitrobenzamide (13a)
5.4.4. N-(4-((3-chloro-4-fluorophenyl)amino)-7-(3-(4-methylpipe
razin-1-yl)propoxy)quinazolin-6-yl)-2-fluoro-5-nitrobenzamide
(9d)
Pale yellow solid; yield: 31%. 1H NMR (400 MHz, DMSO-d6)
δ 2.08-2.14 (bs, 2H), 2.78 (s, 3H), 2.90 (bs, 2H), 3.08 (bs, 2H),
Pale yellow solid; yield: 32%. 1H NMR (400 MHz, DMSO-d6)
δ 2.24-2.30 (m, 2H), 3.07-3.13 (m, 2H), 3.37-3.41 (m, 2H), 3.48-
3.51 (m, 2H), 3.64-3.70 (m, 2H), 3.99-4.03 (m, 2H), 4.38 (t, J =
5.6Hz, 2H), 7.41 (s, 1H), 7.49-7.54 (m, 1H), 7.58-7.61 (m, 1H),
7.76 (t, J = 9.2Hz, 1H), 7.98-8.03 (m, 1H), 8.52-8.56 (m, 1H),

8.64 (dd, J = 3.2, 6.0Hz, 1H), 8.71 (s, 1H), 9.03 (m, 1H), 9.93 (br,
1H), 10.28 (d, J = 3.6Hz, 1H). MS (ESI) m/z = 583.1926 (M+H+),
calcd. for C28H25F3N6O5 m/z = 583.1917.
6.0Hz, 1H), 8.67 (s, 1H), 9.02 (s, 1H), 9.75 (br, 1H), 10.28 (d, J =
3.2Hz, 1H). MS (ESI) m/z = 668.2398 (M+H+), calcd. for
C32H36ClFN7O6 m/z = 668.2400.
5.5.2. 2-fluoro-N-(4-((4-fluorophenyl)amino)-7-(3-morpholinopr
opoxy)quinazolin-6-yl)-5-nitrobenzamide (13b)
5.5.7. 2-fluoro-N-(4-((3-chloro-4-((6-methylpyridin-3-yl)oxy)phe
nyl)amino)-7-(3-morpholinopropoxy)quinazolin-6-yl)-5-nitroben
zamide (13g)
Pale yellow solid; yield: 26%. 1H NMR (400 MHz, DMSO-d6)
δ 2.24-2.29 (m, 2H), 3.05-3.13 (m, 2H), 3.39 (m, 2H), 3.37-3.41
(m, 2H), 3.48-3.51 (m, 2H), 3.67 (t, J = 12.0Hz, 2H), 3.99-4.03
(m, 2H), 4.38 (t, J = 5.6Hz, 2H), 7.27-7.32 (m, 2H), 7.39 (s, 1H),
7.73-7.78 (m, 3H), 8.52-8.56 (m, 1H), 8.63 (dd, J = 3.2, 6.0Hz,
1H), 8.71 (s, 1H), 9.06 (s, 1H), 9.91 (br, 1H), 10.30 (d, J = 3.2Hz,
1H). MS (ESI) m/z = 565.2027 (M+H+), calcd. for C28H26F2N6O5
m/z = 565.2011.
Pale yellow solid; yield: 38%. 1H NMR (400 MHz, DMSO-d6)
δ 2.26-2.32 (m, 2H), 2.49 (s, 3H), 3.07-3.13 (m, 2H), 3.41 (t, J =
8.0Hz, 2H), 3.49-3.51 (m, 2H), 3.97-4.02 (m, 4H), 4.39 (t, J =
5.6Hz, 2H), 7.25 (d, J = 8.8Hz, 1H), 7.36-7.38 (m, 1H), 7.42-
7.46 (m, 2H), 7.73-7.78 (m, 2H), 8.11 (d, J = 2.4Hz, 1H), 8.31 (d,
J = 2.8Hz, 1H), 8.52-8.56 (m, 1H), 8.64 (dd, J = 2.8, 6.0Hz, 1H),
8.84 (s, 1H), 9.13 (s, 1H), 10.14 (br, 1H), 10.35 (d, J = 3.2Hz,
1H). MS (ESI) m/z 688.2094 (M+H+), calcd. for C34H32ClFN7O6
m/z = 688.2087.
5.5.3. N-(4-((3-chloro-4-cyanophenyl)amino)-7-(3-morpholinopr
opoxy)quinazolin-6-yl)-2-fluoro-5-nitrobenzamide (13c)
Pale yellow solid; yield: 29%. 1H NMR (400 MHz, DMSO-d6)
δ 1.98-2.02 (m, 2H), 2.37 (bs, 4H), 3.29 (m, 2H), 3.56-3.58 (m,
4H), 4.32 (t, J = 6.0Hz, 2H), 7.41 (s, 1H), 7.75 (t, J = 9.2Hz, 1H),
7.95 (d, J = 8.8Hz, 1H), 8.10 (dd, J = 2.0, 8.8Hz, 1H), 8.42 (d, J
= 2.0Hz, 1H), 8.50-8.53 (m, 1H), 8.61-8.64 (m, 1H), 8.72 (s, 1H),
8.98 (s, 1H), 10.19 (d, J =4.0 Hz, 1H), 10.24 (s, 1H). MS (ESI)
m/z 606.1674 (M+H+), calcd. for C29H26ClFN7O5 m/z = 606.1668.
5.5.8. N-(4-((3-chloro-4-(3-cyanophenoxy)phenyl)amino)-7-(3-m
orpholinopropoxy)quinazolin-6-yl)-2-fluoro-5-nitrobenzamide
(13h)
Pale yellow solid; yield: 34%. 1H NMR (400 MHz, DMSO-d6)
δ 2.26-2.30 (m, 2H), 3.06-3.13 (m, 4H), 3.65-3.72 (m, 4H), 3.99-
4.02(m, 2H), 4.39 (t, J = 6.0Hz, 2H), 7.29-7.33 (m, 1H), 7.35 (s,
1H), 7.41 (s, 1H), 7.48 (d, J = 2.4Hz, 1H), 7.60-7.62 (m, 2H),
7.76 (t, J = 9.2Hz, 1H), 7.85 (dd, J = 2.4, 9.2Hz, 1H), 8.19 (m,
1H), 8.52-8.56 (m, 1H), 8.64 (dd, J = 3.2, 6.0Hz, 1H), 8.75 (s,
1H), 9.06 (s, 1H), 9.96 (br, 1H), 10.29 (d, J = 3.6Hz, 1H). MS
(ESI) m/z = 698.1926 (M+H+), calcd. for C35H30FN7O6 m/z =
698.1930.
5.5.4. N-(4-((3-chloro-4-(cyclopropylmethoxy)phenyl)amino)-7-
(3-morpholinopropoxy)quinazolin-6-yl)-2-fluoro-5-nitrobenzami
de (13d)
Pale yellow solid; yield: 30%. 1H NMR (400 MHz, DMSO-d6)
δ 0.35-0.39 (m, 2H), 0.58-0.63 (m, 2H), 1.23-1.29 (m, 1H), 2.26
(m, 2H), 3.10 (m, 2H), 3.40 (d, J = 8.0Hz, 2H), 3.70 (m, 2H),
3.96 (d, J = 6.8Hz, 2H), 4.01 (m, 2H), 4.37 (t, J = 5.6Hz, 2H),
7.20 (d, J = 9.2Hz, 1H), 7.42 (s, 1H), 7.62 (dd, J = 2.8, 9.2Hz,
1H), 7.75 (t, J = 9.2Hz, 1H), 7.88 (d, J = 2.8Hz, 1H), 8.52-8.56
(m, 1H), 8.63 (dd, J = 2.8, 6.0Hz, 1H), 8.74 (s, 1H), 9.06 (s, 1H),
10.30 (d, J = 3.6Hz, 1H), 10.70 (s, 1H). MS (ESI) m/z =
5.5.9. N-(4-((3-chloro-4-(pyridin-2-ylmethoxy)phenyl)amino)-7-
(3-morpholinopropoxy)quinazolin-6-yl)-2-fluoro-5-nitrobenzami
de (13i)
Pale yellow solid; yield: 33%. 1H NMR (400 MHz, DMSO-d6)
δ 2.25-2.29 (m, 2H), 3.06-3.12 (m, 4H), 3.64-3.71 (m, 4H), 3.98-
4.03 (m, 2H), 4.38 (t, J = 5.6Hz, 2H), 5.33 (s, 2H), 7.33 (d, J =
8.8Hz, 1H), 7.38-7.41 (m, 2H), 7.59-7.64 (m, 2H), 7.76 (t, J =
9.2Hz, 1H), 7.88-7.93 (m, 2H), 8.52-8.56 (m, 1H), 8.60-8.65 (m,
2H), 8.78 (s, 1H), 9.07 (s, 1H), 9.96 (br, 1H), 10.32 (d, J = 3.2Hz,
651.2152 (M+H+), calcd. for C32H33ClFN6O6 m/z = 651.2134. 13
C
NMR (100 MHz, DMSO-d6) δ 3.12, 10.07, 22.80, 51.33, 53.34,
63.39, 66.23, 73.35, 107.83, 113.92, 117.84, 118.43, 120.86,
123.66, 124.30, 125.37, 126.08, 127.10, 128.80, 131.23, 144.00,
151.45, 152.29, 155.61, 158.00, 158.40, 160.64, 161.46, 164.04.
1H). MS (ESI) m/z
=
688.2089 (M+H+), calcd. for
C34H32ClFN7O6 m/z = 688.2087.
5.5.10. N-(4-((3-chloro-4-((3-fluorobenzyl)oxy)phenyl)amino)-7-
(3-morpholinopropoxy)quinazolin-6-yl)-2-fluoro-5-nitrobenzami
de (13j)
5.5.5. N-(4-((3-chloro-4-(2-methoxyethoxy)phenyl)amino)-7-(3-m
orpholinopropoxy)quinazolin-6-yl)-2-fluoro-5-nitrobenzamide (1
3e)
Pale yellow solid; yield: 32%. 1H NMR (400 MHz, DMSO-d6)
δ 2.23-2.29 (m, 2H), 3.06-3.12 (m, 4H), 3.64-3.71 (m, 4H), 3.98-
4.04 (m, 2H), 4.37 (t, J = 5.6Hz, 2H), 5.28 (s, 2H), 7.16-7.22 (m,
1H), 7.29-7.35 (m, 3H), 7.37 (s, 1H), 7.45-7.51 (m, 1H), 7.65 (dd,
J = 2.4, 8.8Hz, 1H), 7.76 (t, J = 9.2Hz, 1H), 7.93 (d, J = 2.8Hz,
1H), 8.52-8.56 (m, 1H), 8.63 (dd, J = 2.8, 6.0Hz, 1H), 8.70 (s,
1H), 9.02 (s, 1H), 9.85 (br, 1H), 10.28 (s, 1H). MS (ESI) m/z =
705.2069 (M+H+), calcd. for C35H32ClF2N6O6 m/z = 705.2040.
Pale yellow solid; yield: 33%. 1H NMR (400 MHz, DMSO-d6)
δ 2.28 (m, 2H), 3.10 (m, 2H), 3.36 (s, 3H), 3.41 (m, 2H), 3.50 (m,
2H), 3.72 (m, 4H), 4.00 (m, 2H), 4.23 (m, 2H), 4.38 (t, J = 5.6Hz,
2H), 7.26 (d, J = 9.2Hz, 1H), 7.47 (s, 1H), 7.62 (dd, J = 2.8,
8.8Hz, 1H), 7.75 (t, J = 9.2Hz, 1H), 7.85 (d, J = 2.4Hz, 1H),
8.51-8.56 (m, 1H), 8.64 (dd, J = 2.8, 6.0Hz, 1H), 8.83 (s, 1H),
9.13 (s, 1H), 10.35 (d, J = 3.6Hz, 1H), 11.05 (s, 1H). MS (ESI)
m/z = 655.2081 (M+H+), calcd. for C31H33ClFN6O7 m/z =
655.2083. 13C NMR (100 MHz, DMSO-d6) δ 22.76, 51.31, 53.30,
58.39, 63.36, 66.40, 68.54, 70.25, 107.44, 113.73, 117.88, 118.42,
120.82, 124.18, 124.33, 125.91, 126.08, 127.54, 128.83, 130.81,
144.00, 151.48, 151.80, 155.98, 158.37, 158.39, 160.69, 161.47,
164.05.
5.6. EGFR kinase inhibition assay
The cell culture reagents RPMI1640, DMEM, FBS were
purchased from Invitrogen (life technologies). The EGFR
proteins were purchased from Carna Bioscience. The HTRF
KinEASE-TK kit (Cat# 62TK0PEC) was from Cisbio. The Cell
Titer-Glo Luminescent Cell Viability Assay kit (Cat# G7573)
was purchased from Promega. All the other chemicals were from
Sigma. The assay and cell culture plates were purchased from
Corning.
5.5.6. N-(4-((3-chloro-4-(2-(dimethylamino)ethoxy)phenyl)amino)
-7-(3-morpholinopropoxy) quinazolin-6-yl)-2-fluoro-5-nitrobenz
amide (13f)
Pale yellow solid; yield: 31%. 1H NMR (400 MHz, DMSO-d6)
δ 2.24-2.33 (m, 2H), 2.94 (s, 3H), 2.95 (s, 3H), 3.07-3.12 (m, 2H),
3.36-3.39 (m, 4H), 3.60-3.67 (m, 4H), 4.00-4.03 (m, 2H), 4.37 (t,
J = 5.6Hz, 2H), 4.44 (t, J = 4.8Hz, 2H), 7.30 (d, J = 9.2Hz, 1H),
7.39 (s, 1H), 7.71 (dd, J = 2.4, 9.2Hz, 1H), 7.76 (t, J = 9.2Hz,
1H), 7.97 (d, J = 2.4Hz, 1H), 8.52-8.56 (m, 1H), 8.63 (dd, J = 3.2,
The EGFR kinase assays were performed in 384-well plate
(Corning 3676, low volume, black, NBS), using Cisbio HTRF
KinEASE-TK kit. The assay buffer contained 50mM HEPES
(pH7.0), 0.02% NaN3, 0.01% BSA, 0.1mM Orthovanadate,

5.11. Molecular docking
10mM MgCl2, 2.5mM DTT and 6.25nM SEB. EGFR kinases
was first incubated with compounds for 2h respectively, then
ATP/Peptide substrate mixture was added to initiate the reaction.
The final concentration EGFR WT was 0.1nM, EGFR d746-750
and EGFR L858R and EGFR L858R/T790M were 0.06nM. The
final concentration of biotin-labeled peptide substrate was 1.4μM,
and ATP was 20μM for EGFR WT, 100μM for EGFR d746-750
and EGFR L858R. After 30min reaction at room temperature, the
detection reagents were added. The TR-FRET signal was
measured on PerkinElmer Envision using excitation 320nm and
emission 615nm/665nm. The data was analyzed using Graphpad
Prism (GraphPad Software; www.graphpad.com). The curves
were fitted using a non-linear regression model with a sigmoidal
dose response.
The molecular docking was performed using MOE 2015.10
software. The co-crystal structure of EGFR T790M in complex
with BIBW2992 (PDB ID:4G5P) was used for docking studies.
The optimized structure of 6g was generated by MOE 2015.10.
Before docking simulation, ligands and protein were prepared
with the standard protocol using MOE 2015.10. 6g was inserted
into the ATP-binding pocket of EGFR T790M to replace
BIBW2992. The complex of 6g-EGFR was used for geometrical
optimization. All docking calculations were performed using
default settings.
Acknowledgments
We thank Dr. Liang Chen (Jinan University) for the Ba/F3 cell
lines. We thank Mr. Yupeng Wang for his help of several
compounds synthesis. This work was supported by the National
High Technology Project 973 (2011CB504300).
5.7. Cell culture and cell survival assay
Ba/F3 (EGFR L858R), Ba/F3 (EGFR Del E746_A750), Ba/F3
(cat-EGFR), and Ba/F3 (Her2 negative) stable cell lines were
kindly provided by Dr. Chen Liang from Jinan University.
Cancer cell lines A431, HCC827 and NCI-H1975 were
purchased from ATCC. All cells were cultured in RPMI-1640
medium (Hyclone, Logan, UT), supplemented with 10% FBS
(Invitrogen) and 100 units/ml penicillin/streptomycin (Hyclone).
Cells were maintained at 37°C in humidified atmosphere of 5%
CO2.
References and notes
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Cancer cells or Ba/F3 cells were plated in 384-well cell
culture plate (Corning 3570), and exposed to compounds
treatment for 72 hours. Cell survival analysis was performed
using the Cell Titer-Glo Luminescent Cell Viability Assay kit
(Promega) following manufactory instruction with minor
modification. In brief, 20 μl of Cell Titer-glo reagent was added
to the 30μl cell culture medium. Cell culture plates were place on
a shaker for 10 min and were then incubated at room temperature
for an additional 10 min. Luminescent reading was carried on
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5.8. Western blotting
9. Barker, A. J.; Gibson, K. H.; Grundy, W.; Godfrey, A. A.; Barlow, J. J.;
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A431 cells were seeded to 6-well plate on day1 and on day2
replace with new medium. The cells were treated with CI-1033,
gefitinib, or 6g respectively for 1 hour, then either immediately
treated with EGF for 15min or thoroughly washed with fresh
medium 3 times for 8h before EGF treatment. Cells was collected
and re-suspended with lysis buffer (20 mM Tris-HCl, pH 7.4,
150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM Na3VO4,
25 mM ß-glycerol-phosphate, 0.1mM PMSF, complete protease
inhibitor cocktail phosphatase inhibitor cocktail(Roche)). The re-
suspended cell pellet was incubated on ice for 30 min and
centrifuged at 15,000 g for 10 min. The supernatants were
collected for western-blot analysis.
5.9. Compounds stability test in liver microsome assays
Compound 6g were incubated with human, rat or mouse liver
microsomes, and reactions were initiated by the addition of
NADPH in 0.05 M Phosphate buffer (pH=7.4) at 37°C for 0-60
min. The reaction was quenched, and the amount of the
remaining compound was analyzed using LC-MS/MS.
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Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Non–Small
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15. Pao, W.; Miller, V. A.; Politi, K. A.; Riely, G. J.; Somwar, R.; Zakowski,
M. F.; Kris, M. G.; Varmus, H. Acquired Resistance of Lung
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Following oral administration (PO) of 6g to C57BL/6 mice (n
= 3), blood sample was collected at various time points. The
concentrations of the compound in plasma samples were
measured by LC-MS/MS. Pharmacokinetic parameters were
determined using noncompartmental analysis in phoenix 64
(winNonlin 6.3).

17. Engelman, J. A.; Jänne, P. A. Mechanisms of Acquired Resistance to
Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Non–Small
Cell Lung Cancer. Clin. Cancer. Res. 2008, 14: 2895.
36. Yamamoto, N.; Honma, M.; Suzuki, H. Off-Target Serine/Threonine
Kinase 10 Inhibition by Erlotinib Enhances Lymphocytic Activity Leading to
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