In vivo efficacy studies of novel quinazoline derivatives as irreversible dual EGFR/HER2 inhibitors, in lung cancer xenografts (NCI-H1975) mice models

Article abstract of DOI:10.1016/j.bioorg.2020.103790

Lung cancer is the most common cancer and leading cause of cancer-related deaths worldwide. The first-generation reversible, ATP-competitive inhibitors gefetinib and elotinib showed good clinical responses in lung adenocarcinoma tumors (NSCLC). But almost all patients developed resistance to these inhibitors over time. Such resistance of EGFR inhibitors was frequently linked to the acquired L858R and T790M point mutations in the kinase domain of EGFR. To overcome these resistance problems, the second and the third generation inhibitors have been discovered. FDA approved afatinib, the second generation irreversible inhibitor and osimitinib, the third generation irreversible EGFR inhibitors for the treatments of NSCLC. We identified new covalent quinazoline inhibitors (E)-N-(4-(3-chloro-4-fluorophenylamino)-7-(2-ethoxyethoxy)quinazolin-6-yl)-4-(dimethylamino)but-2-enamide (6d) and (E)-N-(4-(3-chloro-4-(pyridin-2-ylmethoxy)phenylamino)-7-(2-ethoxyethoxy)quinazolin-6-yl)-4-(dimethyl-amino)but-2-enamide (6h) that exhibited potent EGFR kinase inhibitory activities on L858R and T790M mutations. The compound 6 h showed selectivity similar to AZD9291 (osimertinib) in mutated and wild type tumor cell lines. In vitro cell assay 6d and 6h were better than afatinib and osimertinib. In vivo antitumor efficacy studies of these compounds were done in NCI-H1975 mice xenografts.

Full text of DOI:10.1016/j.bioorg.2020.103790

Journal Pre-proofs
In vivo efficacy studies of novel quinazoline derivatives as irreversible dual
EGFR/HER2 inhibitors, in lung cancer xenografts (NCI-H1975) mice models
Debasis Das, Lingzhi Xie, Jingbing Wang, Jingli Shi, Jian Hong
PII:
S0045-2068(19)31735-3
DOI:
https://doi.org/10.1016/j.bioorg.2020.103790
YBIOO 103790
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Bioorganic Chemistry
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Revised Date:
Accepted Date:
15 October 2019
9 January 2020
21 March 2020
Please cite this article as: D. Das, L. Xie, J. Wang, J. Shi, J. Hong, In vivo efficacy studies of novel quinazoline
derivatives as irreversible dual EGFR/HER2 inhibitors, in lung cancer xenografts (NCI-H1975) mice models,
Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.103790
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In vivo efficacy studies of novel quinazoline derivatives as irreversible dual
EGFR/HER2 inhibitors, in lung cancer xenografts (NCI-H1975) mice
models
Debasis Das*, Lingzhi Xie, Jingbing Wang, Jingli Shi, Jian Hong*
Discovery Chemistry Research, Arromax Pharmatech Co. Ltd.
Sangtian Island Innovation Park, No. 1 Huayun Road, Suzhou 215123, P. R. China.
*
Corresponding authors:
Email: debasis.das@arromax.com (D. Das) & eric.hong@arromax.com (J. Hong).
Abstract
Lung cancer is the most common cancer and leading cause of cancer-related deaths
worldwide. The first-generation reversible, ATP-competitive inhibitors gefetinib and elotinib
showed good clinical responses in lung adenocarcinoma tumors (NSCLC). But almost all
patients developed resistance to these inhibitors over time. Such resistance of EGFR
inhibitors was frequently linked to the acquired L858R and T790M point mutations in the
kinase domain of EGFR. To overcome these resistance problems, the second and the third
generation inhibitors have been discovered. FDA approved afatinib, the second generation
irreversible inhibitor and osimitinib, the third generation irreversible EGFR inhibitors for the
treatments of NSCLC. We identified new covalent quinazoline inhibitors (E)-N-(4-(3-chloro-4-
fluorophenylamino)-7-(2-ethoxyethoxy)quinazolin-6-yl)-4-(dimethylamino)but-2-enamide (6d) and
(
E)-N-(4-(3-chloro-4-(pyridin-2-ylmethoxy)phenylamino)-7-(2-ethoxyethoxy)quinazolin-6-yl)-4-
dimethyl-amino)but-2-enamide (6h) that exhibited potent EGFR kinase inhibitory activities on L858R
(
and T790M mutations. The compound 6h showed selectivity similar to AZD9291 (osimertinib)
in mutated and wild type tumor cell lines. In vitro cell assay 6d and 6h were better than
afatinib and osimertinib. In vivo antitumor efficacy studies of these compounds were done in
NCI-H1975 mice xenografts.
Key Words: EGFR; HER2; Lung cancer; xenograft; NSCLC; NCI-H1975.
1

1
. Introduction
Lung cancer is one of the common deadly cancer worldwide and cause many deaths
globally every year. An estimated 228,150 new cases of lung cancer and approximately
42,670 associated deaths are predicted in the United States in 2019 [1]. Lung cancer is
1
prevalent in highly populated countries such as China [2] and India [3]. About 80-85% of
lung cancer patients are identified with non-small cell lung cancer (NSCLC) and the patients
have 5-year survival rate of less than 1% [4]. The small cell lung cancer (SCLC) accounts
1
0-15% of the patients. Overexpression of epidermal growth factor receptor (EGFR) is
frequently observed in various solid tumors [5]. EGFR regulates important tumorigenic
processes, including proliferation, apoptosis, angiogenesis, and invasion. EGFR has been
well-validated as one of key drivers of NSCLC progression [6], [7]. Now, EGFR tyrosine
kinase inhibitors (TKIs) are used as standard treatment in clinic for the patient with advanced
EGFR mutant NSCLC [8]. First generation EGFR TKIs, including gefitinib [9], erlotinib
[10], bound competitively and reversibly to the ATP-binding site of the EGFR tyrosine
kinase and give good response to NSCLC patients with EGFR mutations (L858R and Del19).
Despite of the initial response to the treatment with erlotinib or gefitinib, these EGFR TKIs
are limited by the up-regulation of bypass signalling pathways and acquired point mutations
,
[
11] [12]. After treatment 10–16 months, all the patients developed tumor resistance and
approximately 50% of the NSCLC patients gained an additional gate keeper mutation
T790M) [13].
(
F
Reversible TKIs
HN
HN
N
Cl
O
O
O
O
O
O
N
O
O
N
N
Gefitinib
Erlotinib
F
Irreversible TKIs
O
H
N
HN
N
Cl
N
H
N
N
N
N
N
N
O
O
N
O
NH
Afatinib
Osimertinib
(
AZD9291)
O
Figure 1: FDA approved EGFR TKIs for the treatment of NSCLC
2

The second generation irreversible EGFR TKIs have been developed to overcome
T790M mutation [14]. Irreversible inhibitors that target EGFR, form a covalent bond with
Cys-797 near to EGFR active site [15]. Afatinib was the first approved drug by the US FDA
for the treatment of late-stage (metastatic) NSCLC patients with L858R mutated EGFR [16].
The second generation irreversible EGFR TKIs have anti-T790M activities. But these
inhibitors have poor selectivity between EGFR T790M mutant and the wild-type kinasae
with dose-limiting toxicities in patients [17] [18]. Recently, third-generation covalent
inhibitor AZD9291 has been demonstrated selectivity for EGFRT790M mutants over wild-
wt
type EGFR (EGFR ) [19]. The third generation
EGFR TKIs target selectively and
irreversibly EGFR T790M and activating EGFR mutation with efficacy in NSCLC resistance
cases to the first and second generation EGFR TKIs. In April 2018, FDA approved,
osimertinib (AZD9291), the third generation EGFR TKI to treat metastatic EGFR T790M
NSCLC patients [20].
Towards identifying efficacious compounds for NSCLC, we reported new quinazoline
EGFR inhibitors [21], [22] and ALK inhibitors [23] recently. The biological evaluations of
selected compounds are discussed in this article including in vitro pharmacological profile,
pharmacokinetic and in vivo efficacy studies on lung cancer tumor xenograft mice models.
2
. Results and Discussion
The new 6- and 7-substituted amino-4-anilinequinazoline derivatives were prepared
following a general synthetic route, as shown in Scheme 1, In all covalent inhibitor, a
Michael acceptor was introduced at the 6-position of quinazoline ring. The Michael acceptor
form the covalent adduct between the inhibitor and the receptor’s cysteine nucleophile (Cys-
7
97 in EGFR and Cys-805 in HER2) expected to enhance the potency of ligands [24]. 4-
anilinoquinazoline nitro derivatives 3 could be made from 1 through one pot reaction via
intermediate 2.
3

The quinazoline derivatives with different 7-alkoxyalkoxide chain 4 were made from
. In presence of sodium hydride (NaH), alkoxy alkanols (ethylene / propelene glycol methyl
3
ether) was allowed to react with 3 at low temperature. The 6-nitro group of 4 was easily
reduced to give 7-alkoxy-6-amino quinazolines 5 by tin(II) chloride dehydrate and
o
o
hydrochloric acid or iron powder in acetic acid conditions at 40 C to 60 C temperature for
0min to 3hrs. Then the amine was coupled with (E)-4-(dimethylamino)but-2-enoyl chloride
or other acid chlorides to give the quinazolines derivatives 6 in good yield. The synthesis of
a-6g was reported earlier by us [22]. The synthesis of 6h was achieved similarly, following
3
6
the scheme 1. 3-chloro-4-(pyridin-2-ylmethoxy)aniline was synthesized in very good yield
starting from 2-chloromethyl pyridine hydrochloride, following a known method [25]. 3-
chloro-4-(pyridin-2-ylmethoxy)aniline was reacted chlorintermediate 2, generated in-situ
from 7-fluoro-6-nitro-4-hydroxyquinazoline (1), to give 3h in moderate yield (52%). In
presence of NaH, ethoxy ethanol was reacted with 3h at low temperature produced 4h in
good yield (71%). Using Fe/acetic acid condition, nitro group of 4h was reduced to give
aminoquinazoline 5h in very good yield 80%. (E)-4-(dimethylamino)but-2-enoyl chloride
4

o
was reacted with 5h at 0 C to give the desire product 6h in good yield (81%) after silica gel
chromatographic purification. The compounds were characterized by NMR.
The synthesized compounds were tested for their inhibitory effects on EGFR and HER2
enzymes and results of selected compounds are summarized in Table 1. We identified a
quinazoline series as dual EGFR / HER2 inhibitors [21] and their activities were reported
earlier [22]. The introduction of methoxyl group onto the 7-position of qunazoline ring
resulted in a significant improvement of the inhibitory activity for EGFR was reported [26].
Table 1. In vitro EGFR enzyme assay and anti-proliferation activities of
quinazoline derivatives on human cell lines
EGFR
NCI-H1975
HCC 827
A431
Compounds*
IC (nM)
IC (nM)
IC (nM)
IC (nM)
5
0
50
50
50
6
6
d
h
0.6
107.0/12.2**
60.6
0.2/0.3**
1.2
20.0/1.52**
288.3
1.01
AZD9291
Afatinib
Gefitinib
71.5
0.5
270.4
0.9
284.7/96.07** 1.3/0.43**
16870 0.4
105.0/10.21**
337.6
Notes: * In cell assay dimaleate salts of 6d, 6h, afatinib, AZD9291
mesylate, and gefeitbib hydrochoride were used; ** Reference [22]
Increasing the chain length from 2 to 6 atoms improved their inhibitory activities for both EGFR
and we found that most of the compounds showed significant EGFR activity. Better EGFR
inhibitory activities were reported when 3, 4-positon of the aryl group was substituted with
chloro- and fluoro- groups. In enzymatic assay, the length of the side chain at 7-position of
the quinazoline ring played a major role in EGFR activity. Using afatinib ((IC = 0.9 nM)
5
0
as the reference standard, the new compounds were evaluated. The longer chain length with
seven atoms, 6e and branched chain 6f showed less potent with IC = 2.84 nM and 1.54 nM
5
0
respectively. Chiral examples 6b & 6c of chain length five atoms showed poorer activity. 6b
was superior (IC = 0.9 nM) to 6c (IC = 2.7 nM) by three times due to match and mismatch
5
0
50
topology. The chain length of six-atoms showed good activities e.g., 6a (IC = 0.7 nM) 6d
5
0
(
IC = 0.6 nM). So, the six atoms chain length was optimum chain length at 7-position of the
50
5

quinazoline ring to get better results. The alkoxychain EtOCH CH O- was the substituent of
2
2
choice. SAR study was then focused on the substituents of aryl amino group on the 4-position of
quinazoline ring. The groups with chloro-, fluoro- and alkoxy- chains gave better results.
Compound 6g showed the lowest inhibitory activities for EGFR ((IC50 = 153.3 nM).
Interestingly, compound 6h with pyridylbenzyl showed better inhibitory effect (IC = 1.01
5
0
nM) in enzyme assay.
To evaluate the pharmacological activities of novel synthesized derivatives we investigated
their anti-proliferation activities on human lung cancer cell lines of L858R/T790M EGFR and
wt
EGFR . The cell lines were human non-small cell lung cancer cell lines NCI-H1975
(T790M), human non-small cell lung cancer cell line HCC 827 (L858R), human epithelial
wt
carcinoma cell lines A431 (EGFR ). The anti-proliferation activities of selected compounds
were compared against first generation, second generation and third generation irreversible
tyrosine kinase inhibitors- gefitinib, afatinib and AZD9291 respectively. Enzyme
inhibition results of 6a, 6d and 6h showed better than afatinib. We selected two compounds
(6d, 6h) for the anti-proliferation activities in four human lung cancer cell lines. Both
selected compounds 6d and 6h showed good anti-proliferative activities and promising
results. Both the compounds were more active than gefitinib. In vitro cell assay on NCI-
H1975 (mutation T790M) cell line, the compound 6h (IC = 60.6 nM) showed better result
5
0
than 6d (IC = 107.0 nM).
5
0
The activity of 6h was similar to AZD9291 (IC = 71. 5 nM) and many times better
5
0
than afatinib (IC = 284.7 nM) and gefitinib (IC = 16870 nM) respectively for NCI-H1975.
5
0
50
On HCC827 (L858R mutation) cell lines, 6d (IC = 0.23 nM) was better than 6h (IC =
5
0
50
1
1
.28 nM). The activity of 6d was better than AZD9291 (IC = 0.44 nM), afatinib (IC =
50 50
.39 nM) and gefitinib (IC = 0.42 nM). Compound 6h was similar to afatinib and inferior to
5
0
AZD9291. On wild type cell lines, A431, compound 6d (IC = 20.0 nM) was more potent
5
0
than 6h (IC50 = 288.3 nM). Compound 6h and AZD9291 showed better selectivity on mutant cell
lines than other. Compound 6h was approximately seven times more active than afatinib (IC50 =
2
5.9 nM) and AZD9291 (IC50 = 35.7 nM) respectively. The result confirmed that compound 6h was
a potent and selective L858 R/T790M mutant EGFR inhibitor. In vitro, compound 6h has
better inhibitory activity and selectivity towards mutant cell lines was better compared to
irreversible dual EGFR/HER2 inhibitor afatinib and is similar to third generation irreversible
EGFR inhibitor AZD9291.
6

The pharmacokinetic profiles of 6d and 6h were studied. Pharmacokinetic parameters
of 6d oral (10mg/kg): oral Tmax = 4h, Cmax = 92.32μg/L and IV (5mg/kg): half life = 6.8h,
AUC0- = 1030.9g/L*h; oral bioavailability = 46.1%. 6h oral (10mg/kg): half life = 4.3h,
oral Tmax = 8h, Cmax = 39.4μg/L and IV (5mg/kg): half life = 4.9h, AUC0- = 780g/L*h;
oral bioavailability = 28.8%.
Table 2. In vivo anti-tumour effect on NCI-H1975 lung cancer xenografts tumour model
Groups
Animal
Administrati Mean tumour Tumour inhibition
Treatment
numbers
on mode
po, qd
weight (g)
1.01±0.44
0.049±0.05
0.294±0.21
0.45±0.29
0.161±0.13
0.347±0.18
0.537±0.25
rate (%)
-
1
2
3
4
5
6
7
6
6
6
6
6
6
6
Vehicle, 200 l/20g
6d (81.4 mg/kg),
6d (20.4 mg/kg),
6d (5.1 mg/kg),
6h (99.5 mg/kg),
6h (24.9 mg/kg),
6h (6.2 mg/kg),
po, qod
po, qod
po, qd
95.21%
71.01%
55.53%
84.11%
65.72%
47.00%
po, qod
po, qod
po, qd
Notes: po: oral doses, qd: administrated every day; qod; administrated every other day; The
th
administration period lasted for 25 days and sacrificed on 29 day
Based on its high in vitro activities and favourable in vivo pharmacokinetic properties,
compound 6d and 6h were selected for in vivo anti-tumor efficacy study. A NCI-H1975
tumor xenograft model was established in nude mice. When the tumor size reached a volume
3
of 140 mm , the tumor-bearing nude mice derived from NCI-H1975 cells were randomly
assigned into 7 groups (6 mice/ group), the drug dosing and administration period lasted for
th
2
5days and the rats were sacrificed on 29 day. The groups and dosages are summarized in
Table 2.
Previously, we studied the efficacy of 6d for three different doses and compared to
marketed drug afatinib 20 mg/Kg dose. 6d was demonstrated as better drug compared to afatinib
at 20 mg/Kg PO in NCI-H1975 xenograft rat model [22]. In PK analysis, oral bioavailability of 6h
(28.8%) was little weaker than 6d (46.1%). So, three doses of 6h (6.2, 24.9, 99.5 mg/Kg) were taken
higher than 6d (5.1, 20.4 and 81.4 mg/Kg). The control group was administrated with the same
amount of solvent (saline solution of 1% DMSO), during the period of administration and
7

recovery. Treatments with 6d and 6h demonstrated good dose-dependent inhibition on the
tumor. The results were represented in Figure 2. It was observed that all had good tumor
inhibition effect and the tumor inhibition rate of 6d and 6h which achieved to 95.1%, 84.0%
respectively. In vivo efficacy showed that the compound 6d had a better tumor inhibition
effect in NCI-H1975 constructed tumors model (Figure 2).
The anticancer effect on NCI-H1975xenograft tumor model in the Balb/c nude mice
2000
1800
1600
1400
1200
1000
800
600
400
200
0
0
5
10
15
20
25
30
Days after treatment
Figure 2
3
. Conclusion
In summary, new quinazoline derivatives were studied for their in vitro anti-
proliferation activities and in vivo antitumor efficacy in NCI-H1975 xenografts (supplement)
models. In mice median dosage, compound 6d (qod) and 6h (qod) were shown good efficacy.
Considering the cell based assay results, 6h was more selective to mutant variation (NCI-
H1975) than 6d which was more wild type (A431) inhibitor. The half life, P450 analysis and
tox data in rat were more promising for 6h. The selectivity wise, compound 6h showed more
8

similar to AZD9291. Thus, compound 6h can be a suitable candidate for further preclinical
investigation in mutant lung cancer therapy.
4
4
. Experimental
.1. Chemistry
All reagents were purchased from commercial vendors and were used without further
purification. All oxygen-sensitive or moisture-sensitive reactions were run under nitrogen
1
atmosphere. H NMR spectra were recorded on a BRUKER AVIII 400 MHz spectrometer
with tetramethylsilane (TMS) as the internal standard, the values of the chemical shifts ()
are given in ppm, and coupling constants (J) are given in Hz. Mass spectra (ESIMS) were
performed on Agilent 6120. All reactions were monitored using thin-layer chromatography
(TLC) on silica gel plates at 254 nm under a UV lamp. Column chromatography separations
were performed on normal phase silica gel (100-200 mesh) and the melting points are
uncorrected.
N-(4-(3-chloro-4-(pyridin-2-ylmethoxy)phenylamino)-7-fluoro-6-nitro-4-quinazolinamine
(3h). To phosphorus oxychloride (44.6 mL, 478 mmol), taken in a 100 mL three-neck flask,
was added 7-fluoro-6-nitro-4-hydroxyquinazoline (1, 5 g, 24 mmol) slowly at room
o
temperature. The mixture was heated to reflux at 145-150 C for 5 h. Then the reaction
mixture was cooldown to room temperature, phosphorus oxychloride was removed
completely under reduced pressure, The residue was diluted with anhydrous dichloromethane
and evaporated again. The procedure was repeated for 3 times and diluted with acetonitrile
(100 mL), then 3-chloro-4-(pyridin-2-ylmethoxy)aniline (3.5g, 15 mmol) was added. After
heating overnight the desired compounds 3h was separated out as a yellow solid. The solid
filtrated and it was pure enough to proceed for the next step of the synthesis. Yield: 3.4 g,
1
5
2%. H NMR (400 MHz, DMSO-d6, δ ppm): 9.64-9.65 (m, 1 H), 8.78 (s, 1 H), 8.01-8.03
(m, 2 H), 7.85-7.88 (m, 1 H), 7.67-7.72 (m, 2 H), 7.47-7.49 (m, 2 H), 7.33-7.35 (m, 1 H),
5
.37 (s, 2 H). m/z: 426 (M+1).
N-(4-(3-chloro-4-(pyridin-2-ylmethoxy)phenylamino)-7-(2-ethoxyethoxy)-6-nitro-4-
quinazolinamine (4h)
Under nitrogen gas atmosphere, 60 wt.% NaH (500 mg, 12.5 mmol,) was added into a three-
o
neck flask containing anhydrous tetrahydrofuran (5 mL), cooled to 0 C, into which a solution
9

of propylene glycol methyl ether in anhydrous tetrahydrofuran (5 mL) was added drop wise
and effervescence observed. The reaction mixture was stirred for 2 h at room temperature and
o
cooled to –15 C, then added into a suspension of compound 3h (500 mg, 1.18 mmol) in THF
(5 mL) and DMF (1 mL), during the dripping the system turned to blood red. The reaction
o
mixture was reacted at –15 C for 1.5 and allowed to stir at room temperature for 1 h. The
reaction solution was diluted with a lot of ice-water, extracted with ethyl acetate (5 mL × 3),
the organic phases were combined, dried, and filtrated, The filtrate was concentrated by
evaporation to give 4h as a yellow solid. The compound was pure enough use for the next
step without further purification. Yield: 420 mg, 71%; 1H NMR (400 MHz, DMSO-d6, δ
ppm): 10.07 (s, 1 H), 9.19 (s, 1 H), 8.61 (m, 2 H), 8.00-8.01 (m, 1 H), 7.89-7.91 (m, 1 H),
7
1
1
.86-7.88 (m, 1 H), 7.67-7.69 (m, 1 H), 7.67 (s, 1 H), 7.48-7.57 (m, 1 H), 7.37 (d, J = 8.0 Hz,
H), 5.29 (s, 2 H), 4.41 (t, J = 4.4 Hz, 2 H), 3.76 (t, J = 4.4 Hz, 2 H), 3.52-3.54 (m, 2 H),
.12 (t, J = 6.8 Hz, 3 H); m/z: 496 (M+1).
N-(4-(3-chloro-4-(pyridin-2-ylmethoxy)phenylamino)-7-(2-ethoxyethoxy)-6-amino-4-
quinazolineamine (5h)
To a mixture of 4h (500 mg, 1.0 mmol) and acetic acid (6 mL) in a a 100 mL one-neck flask
was added iron powder (0.26 g, 4.63 mmol) portion wise and reacted for 1.5 h at room
temperature. The reaction was monitored by TLC (using ethyl acetae and hexanes system).
After completion of the reaction, the mixture was filtrated. The pH value of the filtrate was
adjusted to 11 with 10% NaOH aqueous solution. The product was precipitated out. The
product was filtrated and washed with water to give the desired product 5h. Yield: 375mg,
1
8
0%; mp. 175-178; H NMR (400 MHz, DMSO-d6, δ ppm): 9.34 (br, 1 H), 8.59-8.60 (m, 1
H), 8.36 (s, 1 H), 8.05 (s, 1 H), 7.89-7.86 (m, 1 H), 7.70-7.72 (m, 1 H), 7.58-7.60 (m, 1 H),
.44 (s, 1 H), 7.34-7.37 (m, 1 H), 7.22 (d, J = 8.0 Hz, 1 H), 7.11 (s, 1 H), 5.25 (s, 2 H), 5.23
s, 2 H), 4.27 (t, J = 4.4 Hz, 2 H), 3.87 (t, J = 4.4 Hz, 2 H), 3.52-3.54 (m, 2 H), 1.14 (t, J = 6.8
Hz, 3 H). m/z: 466 (M +1).
7
(
(E)-N-(4-(3-chloro-4-(pyridin-2-ylmethoxy)phenylamino)-7-(2-ethoxyethoxy)quinazolin-6-
yl)-4-(dimethylamino)but-2-enamide (6h)
To a solution of 5h（1g, 2.1 mmol）in anhydrous N-methyl-2-pyrrolidone (10 mL) in a
three-neck flast, was added (E)-4-(dimethylamino)but-2-enoyl chloride hydrochloride (1 g,
o
6
1
.1 mmol) in anhydrous THF (20 mL) drop wise at 0 C. The reaction mixture was stirred for
h. Then water (15 mL) was added, and pH was adjusted to 11 with 10% NaOH aqueous
1
0

solution. The reaction mixture was extracted with EtOAc (20 ml × 3), dried, filtrated,
evaporated. The crude product was purified by silica gel column chromatography using ethyl
acetate and hexanes as eluting mixture to give the pure product 6h as light yellow solid.
o
1
Yield: 1g, 81%; mp: 187-190 C; H NMR (400 MHz, DMSO-d6, δ ppm): 9.98 (s, 1 H),
9
1
1
.85 (s, 1 H), 8.9 (s, 1 H), 8.60-8.59 (m, 1 H), 8.56 (s, 1 H), 7.94-7.90 (m, 1 H), 7.89-7.86 (m,
H), 7.67-7.64 (m, 1 H), 7.59-7.57 (m, 1 H), 7.39-7.37 (m, 1 H), 7.33 (s, 1 H), 7.28-7.25 (m,
H), 6.80-6.76 (m, 1 H), 6.55 (d, J = 16 Hz, 1 H), 5.3 (s, 2 H), 4.37 (t, J = 4.4 Hz, 2 H), 3.97
(t, J = 4.4 Hz, 2 H), 3.56-3.53 (m, 2 H), 3.13 (d, J = 6.8 Hz, 2 H), 2.84 (s, 6H), 1.12 (t, J = 6.8
Hz, 3 H); m/z: 577 (M+1).
4
.2. In vitro cytotoxicity assay
The cytotoxicity of the compounds was evaluated by cell counting kit 8 (CCK8) assay against
NCI-H1975, HCC 827, A 431 cell lines , using gefetinib, afatinib and AZD9291 as the
reference standard. Cell culture: Logarithmic growth phase cells were collected and
counted, the cells were suspended again with complete medium. The cell concentration was
adjusted to an appropriate concentration, inoculated to 96 holes, each of which with 100 L
o
cells suspension. The cells were incubated in a 37 C at 100% relative humidity and 5% CO
2
incubator for 24 h. Relative inhibition rate test: The test compounds were diluted to the
corresponding concentration stated by culture medium, and the cells were inoculated as
2
5L/hole. The final concentration of the compound was from 10 M to 0 M, with a
o
gradient of 4 times, and 10 concentration points totally. The cells were incubated in a 37 C,
1
00% relative humidity, 5% CO incubator for 72 h. Absorbingmthe culture medium, the
2
o
complete medium containing 10% CCK-8 was added and incubated in the incubator of 37 C
for 2-4 h. After a slight shake, the absorbance at 450 nm was measured by SpectraMax M5
Microplate Reader,taking the absorbance at 650 nm as a reference, the inhibition rate was
calculated. Data processing and results: The inhibition rate of drugs on tumor cell growth
was calculated by the following formula: the growth inhibition rate of tumor cell % = [(Ac-
A )/(A -A )](Afol. A : OA of samples (cell +CCK-8+ the test compounds). A : OA of
s
c
b
s
c
negative control (cell +CCK-8+DMSO). A : OA of positive control (culture medium +CCK-
b
8
+DMSO). The IC curve fitting was performed by using the software Graphpad Prism 5
50
and the calculation formula log (inhibitor) vs. response to calculate the IC value.
5
0
4
.3. In vivo pharmacodynamics evaluation
BALB/c nude mice, female, 6 to 7 weeks of age, weight 20 g  2 g, purchased from Shanghai
1
1

silaike experimental animal Co. Ltd., SPF environment. Amplify and implant (S.C.) the
6
cancer cells (NCI-H1975) into 5 nude mice (right dorsal)with 5.0×10 cells in 0.1 ml PBS,
when the tumor size reached 300~500mm3, the tumor were removed and cutted into
1
~2mm3 pieces in PBS solution, and then implanted into right dorsal area of nude mice(1~2
3
pieces/mice). When the tumor size reaches a volume of 140 mm , the tumor-bearing nude
mice derived from NCI-H1975 cells were randomly assigned into 7 groups (6 mice/group),
The drug dosing was conducted by two persons or double checked to avoid any mistake and
th
error. The administration period lasted for 25days and sacrificed on 29 day. The tumor
volume and relative tumor volume were calculated according to the measured data, and the
2
calculation formula of the tumor volume (TV) was TV=1/2×a×b , of which a and b represent
the tumor length and diameter respectively. The relative tumor volume (RTV) was calculated
according to the measured results, and the calculation formula was: RTV=V /V , wherein V
0
t
0
represents the tumor volume at the beginning of the experiment, V represents the volume of
t
the tumor each measured. The evaluation index of anti-tumor activity was relative tumor
growth rate (T/C%), the calculation formula was as follows: T/C (%)=TRTV/CRTV×100%,
TRTV represents the RTV of the treatment group; CRTV represents the RTV of the negative
control group; the relative tumor growth inhibition rate (tumor inhibition rate) = (1-
T/C)×100%, the results were shown in Table 2.
5
. Acknowledgments
The authors would like to acknowledge financial support from Gusu City Scientific
Leadership Program and Jiangsu Province Social development program, P. R. China. The
authors are thankful to Shanghai Medicilon Inc. and the center for new drug safety evaluation
and research center, China Pharmaceutical University for providing biological assay services.
6
. Conflict of Interest.
No
Supplementary data
Rat tumor autopsy results are available.
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Conflict of interest
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1
6

Graphical Abstract
Synthesis of irreversible, dual EGFR/HER2 inhibitors
In vitro cell assay
HCC-827, NCI-H1975, A431
F
O
N
O
O
N
HN
N
Cl
N
HN
Cl
HN
O
HN
N
N
O
O
O
N
6d
6
h
HCC-827 cell: IC50 = 0.2 nM
NCI-H1975 cell: IC50 = 107 nM
IC50 = 1.2 nM
IC50 = 60 nM
In vivo efficiacy study on lung cancner xenograft (H1975) mice model
1
7

Highlights

New quinazoline derivatives as dual EGFR/HER-2 inhibitors.

In vitro anti-proliferation studied on NCI H1975, HCC827, A431 cell lines.

In vitro efficacy 6d and 6h were compared with afatinib, gefitinib,
osimertinib.

In vivo efficacies of 6d and 6h were demonstrated in lung cancer NCI H1975
xenograft.
1
8