Design, synthesis and biological evaluation of 4-aniline quinazoline derivatives conjugated with hydrogen sulfide (H2S) donors as potent EGFR inhibitors against L858R resistance mutation

Article abstract of DOI:10.1016/j.ejmech.2020.112522

In this study, a series of 4-aniline quinazoline derivatives bearing hydrogen sulfide (H₂S) donors were designed, synthesized and evaluated for biological activities. The synthesized compounds were screened for the enzymatic activities against EGFR and EGFR mutants by kinase target-based cell screening method. The results demonstrate that most compounds exhibit selectively inhibitory activities against TEL-EGFR-L858R–BaF3, especially compound 9h with GI₅₀ = 0.008 μM (TEL-EGFR-L858R–BaF3), 0.0069 μM (TEL-EGFR-C797S–BaF3), >10 μM (BaF3), >10 μM (TEL-EGFR-BaF3) and 6.03 μM (TEL-EGFR-T790M-L858R–BaF3). The results from anti-proliferative assays in two NSCLC cell lines indicate that synthetic derivatives (9g, 9h, 15e and 15f) with H₂S donor ACS81 display greater anti-proliferative potency against NSCLC cell line H3255 bearing EGFR mutant (L858R) with GI₅₀ values ranging from 0.3486 to 1.348 μM. In addition, compound 9h exhibits weak anti-proliferative effects on other tumor cells (HepG2, MCF-7, HT-29 and A431) and has lower toxic effect on HUVEC cells than AZD9291 (positive control). Meanwhile, compound 9h inhibits the phosphorylation of EGFR in H3255 cells in a dose-dependent manner. Cell cycle analysis reveals that compound 9h suppresses the proliferation of cells by inducing cell cycle arrest in G0-G1 phase. The result of H₂S release evaluation suggests that the H₂S release of compound 9h is significantly more and faster than other compounds.

Full text of DOI:10.1016/j.ejmech.2020.112522

Journal Pre-proof
Design, Synthesis and Biological Evaluation of 4-Aniline Quinazoline Derivatives
Conjugated with Hydrogen Sulfide (H S) Donors as Potent EGFR Inhibitors against
2
L858R Resistance Mutation
You-Guang Zheng, Wu-Qi Zhang, Long Meng, Xiao-Qing Wu, Ling Zhang, Lin An,
Cheng-Lin Li, Cai-Yun Gao, Liang Xu, Yi Liu
PII:
S0223-5234(20)30494-3
DOI:
https://doi.org/10.1016/j.ejmech.2020.112522
EJMECH 112522
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 March 2020
Revised Date: 29 May 2020
Accepted Date: 1 June 2020
Please cite this article as: Y.-G. Zheng, W.-Q. Zhang, L. Meng, X.-Q. Wu, L. Zhang, L. An, C.-L. Li, C.-
Y. Gao, L. Xu, Y. Liu, Design, Synthesis and Biological Evaluation of 4-Aniline Quinazoline Derivatives
Conjugated with Hydrogen Sulfide (H S) Donors as Potent EGFR Inhibitors against L858R Resistance
2
Mutation, European Journal of Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.112522.
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition
of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of
record. This version will undergo additional copyediting, typesetting and review before it is published
in its final form, but we are providing this version to give early visibility of the article. Please note that,
during the production process, errors may be discovered which could affect the content, and all legal
disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier Masson SAS.

Design, Synthesis and Biological Evaluation of 4-Aniline
Quinazoline Derivatives Conjugated with Hydrogen Sulfide (H2S)
Donors as Potent EGFR Inhibitors against L858R Resistance
Mutation
You-Guang Zhenga, b*, Wu-Qi Zhanga, b, Long Menga, b, Xiao-Qing Wuc, Ling Zhanga,
b, Lin Ana, b, Cheng-Lin Lia, b, Cai-Yun Gaoa, b, Liang Xu c, d, Yi Liu a, b*.
aCollege of Pharmacy, Xuzhou Medical University, Xuzhou 221004, PR China;
bJiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Xuzhou
Medical University, Xuzhou 221004, PR China;
c Department of Molecular Biosciences, University of Kansas, Lawrence, KS, USA;
d
Department of Radiation Oncology, The University of Kansas Medical Center,
Kansas City, Kansas, USA.
*You-Guang Zheng and Yi Liu are corresponding authors.
ABSTRACT
In this study, a series of 4-aniline quinazoline derivatives bearing hydrogen sulfide
(H2S) donors were designed, synthesized and evaluated for biological activities. The
synthesized compounds were screened for the enzymatic activities against EGFR and
EGFR mutants by kinase target-based cell screening method. The results demonstrate
that most compounds exhibit selectively inhibitory activities against
TEL-EGFR-L858R-BaF3, especially compound 9h with GI50=0.008 μM
(TEL-EGFR-L858R-BaF3), 0.0069 μM (TEL-EGFR-C797S-BaF3), >10 μM
(BaF3),
>10
μM
(TEL-EGFR-BaF3)
and
6.03
μM
(TEL-EGFR-T790M-L858R-BaF3). The results from anti-proliferative assays in two
NSCLC cell lines indicate that synthetic derivatives (9g, 9h, 15e and 15f) with H2S
donor ACS81 display greater anti-proliferative potency against NSCLC cell line
H3255 bearing EGFR mutant (L858R) with GI50 values ranging from 0.3486 to 1.348
μM. In addition, compound 9h exhibits weak anti-proliferative effects on other tumor
cells (HepG2, MCF-7, HT-29 and A431) and has lower toxic effect on HUVEC cells
than AZD9291 (positive control). Meanwhile, compound 9h inhibits the

phosphorylation of EGFR in H3255 cells in a dose-dependent manner. Cell cycle
analysis reveals that compound 9h suppresses the proliferation of cells by inducing
cell cycle arrest in G0-G1 phase. The result of H2S release evaluation suggests that
the H2S release of compound 9h is significantly more and faster than other
compounds.
Keywords: EGFR, Conjugate, Hydrogen Sulfide, H3255 (L858R)
1. Introduction
Quinazoline alkaloids are nitrogen-containing heterocycles with a broad range of
biological properties, such as anticancer[1, 2], anticonvulsant[3], antifungal[4],
anti-inflammatory[5], antimalaria[6], antibacterial[7], antihypertensive[8], anti-HIV[9]
activities. Many quinazoline derivatives have been developed to bind to various
targets, including epidermal growth factor receptor (EGFR)[10-13], vascular
endothelial growth factor receptor (VEGFR)[14], janus kinase 2 (JAK2)[15], histone
deacetylase (HDAC)[16], Bromodomain-containing protein
4
(BRD4)[17],
cyclin-dependent kinases (CDK)[18], poly ADP-ribose polymerase (PARP)[19],
MEK5/ERK5 pathway[20], and so on. In our previous work, we have reported several
4-anilinoquinazoline derivatives, which show significant activity against cancer cells
[21-23]. On the basis of the literature and our preliminary studies, we used
4-anilinoquinazoline as the scaffold to design new antitumor agents in this study.
Hydrogen sulfide (H2S) was known as toxic pollutant for many years. However, it is
now widely recognized as an important biological and pharmacological molecule. In
the past decade, studies reveal that H2S plays critical role in physiology and
pathology[24]. In cancer, H2S has been showed to exhibit remarkable in vitro
antiproliferative activity against tumor cell lines, such as A549/DDP[25], HepG2[26],
and MCF-7[27]. Although H2S exerts crucial effects on antitumor, it cannot be used as
an anticancer agent due to the uncontrollability and toxicity[28]. Meanwhile, several
slow-releasing H2S donors were reported to display antitumor effect [26, 29], and
many H2S donors were discovered (Fig. 1). In addition, previous relevant studies also
demonstrate that the compounds bearing H2S donors have potent inhibitory activities
against tumor cells [28, 30-33].

Fig. 1. Hydrogen sulfide (H2S) donors
In the light of above findings and our previous studies, we designed and synthesized
of a series novel 4-anilinoquinazoline derivatives linked with various H2S donors on
position 6 or 7 of quinazoline ring through different chains (Fig. 2).
Fig. 2. Design of novel 4-aniline quinazoline derivatives bearing hydrogen sulfide
(H2S) donors
2. Results and discussion
2.1. Chemistry
The overall synthetic route of compounds (9a-9h) was outlined in Scheme 1 starting
from vanillic acid. After the esterification, alkylation, nitration, reduction, cyclisation
and chlorination, compounds (8a-8f) were obtained. The target compounds (9a-9h)
were prepared by reacting compound (8) with two H2S donors.

Scheme 1 The synthesis of compounds 9a-9h. Reagents and conditions: (a)CH3OH,
98% sulfuric acid, reflux; (b)K2CO3, KI, 1-bromo-3-chloropropane, DMF, rt; (c) nitric
acid, acetic acid, acetic anhydride, 0-5 °C; (d) Pd/C, methanol, rt; (e) formamidine
acetate, alcohol, reflux; (f) SOCl2, DMF, reflux; (g) substituted aniline, isopropanol,
reflux; (h) K2CO3, KI, ADT-OH or ACS81, DMF rt.
As depicted in Scheme 2, 2-amino-5-nitrobenzoic acid (10) was converted by
cyclisation with formamidine acetate to obtain compound (11), which was chlorinated
by thionyl chloride to afford the chloride compound (12). Compound (12) was treated
with substituted aniline in isopropanol to give compounds (13a-13c). Then
compounds (13a-13c) were reduced to their amino derivatives (14a-14c) using iron
powder. The target compounds (15a-15f) were synthesized by reacting compounds
(14a-14c) with two H2S donors using HATU.

R2
R1
O
N
Cl
N
HN
N
O2N
O2N
COOH
NH2
O2N
NH
O2N
b
N
a
c
d
N
10
11
12
13a-13c
R2
R1
S
S
O
S
O
HN
N
OH
H
N
O
e
S
O
S
N
S
ACS60
15a-15c
R2
R1
15a R1=CF3 R2=H
15b R1=Br
15c R1=Cl
R2=H
R2=F
HN
N
H2N
R2
R1
N
HN
N
14a-14c
S
OH
H
S
S
N
e
S
N
O
O
ACS81
15d-15f
15d R1=CF3 R2=H
15e R1=Br
15f R1=Cl
R2=H
R2=F
Scheme 2 The synthesis of compounds 15a-15f. Reagents and conditions:
(a)formamidine acetate, 2-methoxyethanol, reflux; (b)SOCl2, DMF, reflux; (c)
substituted aniline, isopropanol, reflux; (d) iron powder, acetic acid/ethanol, refiux; (e)
HATU, triethylamine, ACS60 or ACS81, rt.
2.2. Biological activities
2.2.1. In vitro inhibition activities studies of kinases
Many novel 4-aniline quinazoline derivatives target EGFR, which is closely
associated with cancer[34-36]. Therefore, the target compounds were assayed with the
enzymatic activities against EGFR and EGFR mutants by kinase target-based cell
screening method [37, 38]. As showed in Table 1, most of the compounds (9a-9h)
displayed selective anti-proliferation efficacy against TEL-EGFR-L858R-BaF3
(GI50=0.008-0.85μM), while having poor activities against BaF3, TEL-EGFR-BaF3
and TEL-EGFR-T790M-L858R-BaF3, especially compound 9h with GI50=0.008 μM
(TEL-EGFR-L858R-BaF3), >10 μM (BaF3), >10 μM (TEL-EGFR-BaF3) and 6.03
μM (TEL-EGFR-T790M-L858R-BaF3). Compounds bearing H2S donor ACS81
showed better inhibiting activities (GI50=0.008-0.058μM) than others with ADT-OH
(GI50>0.10 μM). Meanwhile, compounds with m-Br in 4-aniline group exhibited
greater potency against TEL-EGFR-L858R-BaF3, such as compound 9h

(GI50=0.008μM) and 9d (GI50=0.12 μM). In addition, compound 9h was tested
against TEL-EGFR-C797S-BaF3 with GI50=0.0069μM, so there was no interaction
between the allyl-disulfanyl-propionic group and the C797 residue, and the
biochemical evaluation of the mechanism of the compound 9h is being further
investigated.
Table 1 The activities of the compounds (9a-9h) against EGFR and EGFR mutations
(GI50 μM)
TEL-EGFR-T
Com
TEL-EGFR-B TEL-EGFR-L
R1
Cl
H
R2
F
R3
BaF3
790M-L858R-
BaF3
pd
aF3
858R-BaF3
9a
>10
>10
0.27
0.25
>10
>10
9.23
9.56
9b
9c
9d
9e
9f
Br
NO2
H
3.79
9.23
3.79
2.65
4.08
3.06
1.98
3.39
0.60
0.12
0.38
0.83
>10
>10
H
Br
CF3
1.648
6.35
H
-OCH3 -OCH3
3.91
>10
2.6
2.34
>10
0.28
0.058
0.008
>10
6.03
0.06
9g
9h
H
Br
H
Br
AZD
9291
<0.003
Several of compounds 15a-15f were also tested the enzymatic activities against EGFR

and EGFR mutants. As showed in Table 2, the compounds with ACS81 also exhibited
stronger inhibition against TEL-EGFR-L858R-BaF3, such as compound 15e
(GI50=0.011μM) and compounds 15f (GI50=0.012μM).
Table 2 The activities of the compounds (15a-15f) against EGFR and EGFR
mutations (GI50 μM)
TEL-EGFR-T
Com
TEL-EGFR-B TEL-EGFR-L
R1
R2
R3
BaF3
790M-L858R-
BaF3
pd
aF3
858R-BaF3
15a
15b
15c
15d
15e
15f
CF3
Br
H
H
F
NT
NT
NT
NT
NT
NT
NT
NT
4.27
NT
2.49
NT
0.29
NT
3.16
NT
Cl
CF3
Br
H
H
F
＞10
＞10
2.6
＞10
＞10
0.28
＞10
＞10
0.06
0.011
0.012
<0.003
Cl
AZD
9291
2.2.2. Anti-proliferation assay
To explore the potential antitumor effect, the in vitro anti-proliferative activities of the
synthetic derivatives were evaluated against the NSCLC cell lines A549 (A549
harboring wide type EGFR) and H3255 (H3255 expressing EGFR-L858R).
AZD9291(a third-generation EGFR inhibitor) that shows inhibiting EGFR
phosphorylation in cells harboring sensitising EGFR mutants including H3255
(L858R), PC-9 (ex19del) and H1650 (ex19del) was choose as positive control. The
results were summarized in Table 3. All of the target compounds showed poor
anti-proliferative activities against A549 (GI50>10 μM), while most compounds
exhibited potent inhibitory activities against H3255 with the GI50 ranging from 0.3486
to 8.578 μM. Furthermore, compound 9g, 9h, 15e and 15f with H2S donor ACS81

showed greater anti-proliferative potency against H3255 with GI50 values at 1.348,
0.7873, 0.5195 and 0.3486 μM, respectively. It seems that the result of
anti-proliferative activities was consistent with their enzymatic inhibitory activities.
Then the H3255 cells were treated with compound 9h together with ZnCl2 (10μM
ZnCl2 to quench the released H2S), but the GI50 (GI50=0.3028μM) of which decreased
rather than increased compared with that of compound 9h only. We guess that it may
owe to the inhibitory activity of ZnCl2 against H3255 (Cell proliferation rate was only
54.02% at 10 μM of ZnCl2).
Table 3 In vitro anti-proliferative activities in two NSCLC cell lines of target
compounds.
compd
A549 (GI50 μM)
>10
H3255 (GI50 μM)
>10
9a
9b
9c
9d
9e
9f
9g
9h
15a
>10
>10
>10
>10
>10
>10
>10
>10
1.408
1.874
2.404
7.472
8.578
1.348
0.7873
NT
15b
>10
NT
15c
15d
>10
>10
4.355
NT
15e
15f
ZnCl2
>10
>10
NT
NT
0.5195
0.3486
54.02%a
0.3028
0.09
9h+ZnCl2(10μM)
AZD9291
NT
a Cell proliferation rate (10 μM)
In addition, in vitro anti-proliferative activities against other four tumor cells
(harboring wide type EGFR) of compound 9h were tested. As described in Table 4, 9h
exhibits weak anti-proliferative effects on HepG2, MCF-7, HT-29 and A431 cell lines
with IC50 value of >10, 7.309, >10 and 6.703 μM, respectively. To investigate the
tumor selectivity, the cytotoxicity of 9h on normal cells (HUVEC cells) was assayed.
The result showed that 9h has no toxic effect on HUVEC cells (IC50>10μM).

Therefore, compound 9h selectively inhibits NSCLC cell line H3255 (expressing
EGFR-L858R) compared to other tumor cells and has lower toxic effect on HUVEC
cells (IC50>10μM) than AZD9291 (IC50=7.278μM).
Table 4 In vitro anti-proliferative activities against other tumor cells and normal cells
of compound 9h (IC50 μM)
Compound
9h
HepG2
MCF-7
7.309
2.54
HT-29
A431
6.703
2.823
HUVEC
＞10
＞10
＞10
AZD9291
2.239
2.834
7.278
2.2.3. Compound 9h inhibited EGFR phosphorylation in H3255 cells
In order to further verify the activities of compound 9h in cellular level, we examined
its effect on the phosphorylation of EGFR in the representative cancer cell line H3255,
which was treated with various concentrations of 9h for 4h. As shown in Fig. 3,
compound 9h inhibited the phosphorylation of EGFR in H3255 cells in a
dose-dependent manner.
Fig. 3. Compound 9h suppressed the phosphorylation of EGFR in H3255 cells
2.2.4. Cell cycle analysis
Previous studies reported that EGFR inhibitors can induce cell cycle arrest in tumor
cells [39-42]. To determine the effect of compound 9h on cell cycle, flow cytometry
was performed on H3255 cells treated with various concentrations of 9h for 72 h. As
described in Fig. 4, the percentage of H3255 cells in G0-G1 phase increased from
60.32% to 88.61% after treatment of compound 9h with 1, 5 and 10 μM, indicating
that compound 9h inhibits the proliferation of cells by inducing cell cycle arrest in

G0-G1 phase.
Fig. 4 9h induced G0-G1 phase cell cycle arrest on H 3255.
2.2.5 In vitro hydrogen sulfide release evaluation
Using the hydrogen sulfide probe NIR-HS) developed by our group[43], compound
(
9f, 9h and 15c bearing various H2S donors were tested their ability to release
hydrogen sulfide by the published method [44]. After incubating the compounds 9f,
9h and 15c (10, 50, 100, 200 μM) with the fluorescent probe NIR-HS (10 μM),
significant fluorescence signals were produced, and the fluorescence intensity
increased significantly compared with that without the compounds (*P < 0.05,**P <
0.01) (Fig. 5). This indicates that all compounds can release H2S in PBS buffer.

Fig. 5 Fluorescence emission spectra of NIR-HS (10μM) with varied concentrations
of 9f (A), 9h (B) and 15c (C) in PBS buffer (pH=7.4, 60% acetone) at 37 for 3 h.
Then quantitative determinations of H2S release from target compounds were
performed. As shown in Fig 6A and 6B, the standard curve between the concentration
of Na2S and fluorescence intensity of NIR-HS probe was established, which has
excellent linear relationship. Release of H2S by compound 9f, 9h and 15c were
quantitatively determined based on the standard curve. The results indicate that all the
compounds could release H2S, especially compound 9h with the ability of H2S
releasing significantly more than that of compound 9f and 15c. Within 1h, the release
rate of H2S of compound 9h was faster than other two compounds and tended to level
off after 2h (Fig 6C). This result is consistent with the in vitro inhibition activities of
kinases and cells.
Fig. 6 (A) Fluorescence emission spectra of NIR-HS (10μM) with varied
concentrations of Na2S (0, 2, 4, 6, 8μM) in PBS buffer (pH=7.4, 60% acetone) at 37

for 3h. (B) The fluorescence intensity (F/F0) of the reaction system at 723 nm against
the corresponding reagent blank (without Na2S), which is the calibration curve for this
study. (C) Time-dependent hydrogen sulfide-releasing derivatives of 9f, 9h and 15c.
3. Conclusions
In summary, we designed and synthesized a series of 4-aniline quinazoline derivatives
conjugated with hydrogen sulfide (H2S) donors and the target compounds were
assayed with the enzymatic activities against EGFR and EGFR mutations. Most
compounds displayed inhibitory activities against TEL-EGFR-L858R-BaF3 and
achieved selectivity over TEL-EGFR-BaF3 and TEL-EGFR-T790M-L858R-BaF3,
especially compound 9h with GI50=0.008 μM (TEL-EGFR-L858R-BaF3), >10 μM
(TEL-EGFR-BaF3) and 6.03 μM (TEL-EGFR-T790M-L858R-BaF3). Additionally,
anti-proliferation assays showed that compounds (9g, 9h, 15e and 15f) with H2S
donor ACS81 exhibited greater anti-proliferative potency against EGFR mutant
NSCLC cell line H3255 (L858R) with GI50 values at 1.348, 0.7873, 0.5195 and
0.3486 μM respectively, and it is consistent with their enzymatic inhibitory activities.
In addition, compound 9h selectively inhibits NSCLC cell line H3255 (expressing
EGFR-L858R) compared to other tumor cells (HepG2, MCF-7, HT-29 and A431 cell
lines) and has lower toxic effect on normal cells (HUVEC) than AZD9291.
Furthermore, compound 9h inhibited the phosphorylation of EGFR in H3255 cells in
a dose-dependent manner. Cell cycle analysis uncovered compound 9h inhibited the
proliferation of cells by inducing cell cycle arrest in G0-G1 phase. The result of
hydrogen sulfide release evaluation exhibited that all the compounds could release
H2S, especially compound 9h with the ability of H2S releasing significantly more and
faster than other compounds. Hence, compound 9h merits further exploration as a
potential candidate for the EGFR-L858R positive NSCLC.
4. Experimental
4.1. Chemistry
Unless otherwise noted, all reagents were purchased from commercial sources and

used without further purification. All compounds were routinely monitored by
thin-layer chromatography with silica gel GF-254 glass plates and viewed under UV
light at 254 nm. The 1H-NMR and 13C-NMR spectra were determined in DMSO-d6 on
a JNM-ECZ400s/L spectrometer. Chemical shifts (δ) were reported in parts per
million (ppm) relative to tetramethylsilane (TMS), which was used as an internal
standard. The mass spectra (MS) were obtained from Agilent 1100LC/MS
Spectrometry Services. HR-MS was obtained using a Q-tof high resolution mass
spectrometer.
4.1.1. Synthesis of compounds (2-8)
Compounds 2-8 were prepared according to a similar procedure in Refs[23].
4.1.2. General procedure for the preparation of the compounds 9
To a solution of compounds 8 (1mmol) in DMF (10 mL) was added K2CO3 (2mmol)
and catalytic amount of KI, stirred for 10mins, followed by corresponding H2S donors
o
(1mmol). The reaction mixture was stirred at 80 C and monitored by thin layer
chromatography. After completion of the reaction, the mixture was cooled to rt,
poured into ice water (100 mL), stirred for 30mins. The solid formed was filtered off
and purified by a silica gel column chromatography to afford compound 9.
4.1.2.1.
4-(4-(3-((4-((3-chloro-4-fluorophenyl)amino)-6-methoxyquinazolin-7-yl)oxy)propoxy)
phenyl)-3H-1,2-dithiole-3-thione (9a)
1
Orange solid, yield: 20%. H NMR (400 MHz, CDCl3) δ 8.66 (s, 1H), 7.91 (s, 1H),
7.64-7.59 (m, 2H), 7.31 (d, J = 16.0 Hz, 5H), 7.22-7.18 (m, 1H), 7.03 (d, J = 8.0 Hz,
2H), 4.41-4.31 (m, 4H), 4.04 (s, 3H), 2.46 (s, 2H); 13C NMR (100 MHz, d6-DMSO) δ
215.2, 174.2, 172.5, 162.4, 156.5, 154.1, 153.0, 152.5, 149.6, 134.6, 129.5, 124.2,
124.0, 122.9, 122.8, 119.4, 119.2, 117.1, 116.9, 116.0, 109.2, 108.1, 102.4, 65.6, 65.3,
56.8, 28.7; LC-MS m/z: 585.8 [M+H]+; MS (m/z): HRMS(ESI) Calcd for
C27H20ClFN3O3S3([M-H]-):584.0339
C27H20ClFN3O3S3([M-H]-):586.0310
(100%),
(32%),
found:584.0362;
found:586.0332;
Calcd
Calcd
for
for
C27H20ClFN3O3S3([M-H]-):585.0373 (29.2%), found:585.0380.

4.1.2.2.
4-(4-(3-((4-((4-bromophenyl)amino)-6-methoxyquinazolin-7-yl)oxy)propoxy)phenyl)-
3H-1,2-dithiole-3-thione (9b)
1
Orange solid, yield: 16%. H NMR (400 MHz, CDCl3) δ 8.66 (s, 1H), 7.63 (dd, J =
8.0, 12.0 Hz, 3H), 7.53 (d, J = 8.0 Hz, 2H), 7.39 (s, 1H), 7.29 (d, J = 12.0 Hz, 3H),
7.07-7.01 (m, 3H), 4.39-4.29 (m, 4H), 4.03 (s, 3H), 2.47-2.44 (m, 2H); 13C NMR (100
MHz, CDCl3) δ 195.7, 173.0, 169.7, 168.0, 162.2, 154.1, 149.7, 134.7, 132.0, 128.6,
124.3, 123.4, 121.8, 117.0, 116.2, 115.5, 100.7, 65.3, 64.7, 56.4, 29.7; LC-MS m/z:
611.8 [M+H]+; MS (m/z): HRMS(ESI) Calcd for C27H21BrN3O3S3 ([M-H]-):
609.9928(100%), found:609.9918, Calcd for C27H21BrN3O3S3 ([M-H]-):
611.9908(97.3%), found: 611.9930.
4.1.2.3.
4-(4-(3-((6-methoxy-4-((4-nitrophenyl)amino)quinazolin-7-yl)oxy)propoxy)phenyl)-3
H-1,2-dithiole-3-thione (9c)
1
Orange solid, yield: 19%. H NMR (400 MHz, d6-DMSO) δ 10.05 (s, 1H), 8.65 (s,
1H), 8.30 (d, J = 8.0 Hz, 2H), 8.20 (d, J = 12.0 Hz, 2H), 7.92-7.87 (m, 2H), 7.81-7.76
(m, 1H), 7.31 (s, 1H), 7.13 (dd, J = 4.0 Hz, 2H), 4.37-4.29 (m, 4H), 4.01 (s, 3H),
13
2.33-2.30 (m, 2H); C NMR (100 MHz, d6-DMSO) δ 215.3, 174.2, 162.4, 162.0,
156.2, 154.5, 152.6, 149.92, 142.1, 134.7, 129.5, 128.7, 125.1, 124.2, 121.2, 116.6,
116.0, 109.9, 108.0, 102.5, 65.7, 65.3, 56.9, 28.7; LC-MS m/z: 578.9 [M+H]+.
4.1.2.4.
4-(4-(3-((4-((3-bromophenyl)amino)-6-methoxyquinazolin-7-yl)oxy)propoxy)phenyl)-
3H-1,2-dithiole-3-thione (9d)
Orange solid, yield: 20%.1H NMR (400 MHz, d6-DMSO) δ 9.53 (s, 1H), 8.53 (s, 1H),
8.16 (s, 1H), 7.90-7.75 (m, 5H), 7.34 (d, J = 8.0 Hz, 1H), 7.27 (d, J = 16.0 Hz, 2H),
7.12 (d, J = 8.0 Hz, 2H), 4.33-4.28 (m, 4H), 3.98 (s, 3H), 2.30 (s, 2H); 13C NMR (100
MHz, d6-DMSO) δ 215.3, 174.2, 162.4, 156.5, 154.0, 153.1, 149.6, 147.3, 141.7,

134.6, 130.8, 129.5, 128.7, 126.1, 124.5, 124.2, 121.7, 121.1, 116.0, 115.8, 109.4,
108.3, 102.4, 65.6, 65.3, 56.8, 28.7; LC-MS m/z: 611.7 [M+H]+ (100%); MS (m/z):
HRMS(ESI) Calcd for C27H21BrN3O3S3([M-H]-): 611.9908(97.3%), found: 611.9960.
4.1.2.5.
4-(4-(3-((6-methoxy-4-((3-(trifluoromethyl)phenyl)amino)quinazolin-7-yl)oxy)propoxy
)phenyl)-3H-1,2-dithiole-3-thione (9e)
Orange solid, yield: 20%. 1H NMR (400 MHz, d6-DMSO) δ 9.67 (s, 1H), 8.54 (s, 1H),
8.23 (d, J = 8.0 Hz, 2H), 7.87 (d, J = 8.0 Hz, 3H), 7.75 (s, 1H), 7.63 (t, J = 8.0 Hz,
1H), 7.44 (d, J = 8.0 Hz, 1H), 7.26 (s, 1H), 7.13 (d, J = 12.0 Hz, 2H), 4.35-4.28 (m,
4H), 4.00 (s, 3H), 2.32-2.29 (m, 2H); 13C NMR (100 MHz, d6-DMSO) δ 215.2, 174.2,
170.8, 162.4, 156.5, 154.1, 153.0, 149.6, 147.4, 140.9, 134.6, 130.0, 129.5, 126.1,
125.9, 124.2, 123.4, 119.8, 118.3, 116.0, 109.5, 108.4, 102.4, 65.6, 65.3, 56.8, 28.7;
LC-MS m/z: 601.8 [M+H]+; MS (m/z): HRMS(ESI) Calcd for
C28H23F3N3O3S3([M+H]+): 602.0854, found:602.0830.
4.1.2.6.
4-(4-(3-((4-((3,4-dimethoxyphenyl)amino)-6-methoxyquinazolin-7-yl)oxy)propoxy)phe
nyl)-3H-1,2-dithiole-3-thione (9f)
Orange solid, yield: 17%.1H NMR (400 MHz, d6-DMSO) δ 9.39 (s, 1H), 8.42 (s, 1H),
7.90-7.83 (m, 3H), 7.75 (s, 1H), 7.38 (s, 1H), 7.32 (d, J = 8.0 Hz, 1H), 7.21 (s, 1H),
7.13 (d, J = 8.0 Hz, 2H), 6.99 (d, J = 12.0 Hz, 1H), 4.32-4.29 (m, 4H), 3.97 (s, 3H),
3.79 (d, J = 8.0 Hz, 6H), 2.32-2.29 (m, 2H); 13C NMR (100 MHz, d6-DMSO) δ 215.3,
174.2, 162.4, 157.1, 153.7, 149.3, 148.9, 145.88, 134.6, 133.0, 129.5, 128.7, 116.5,
116.0, 115.8, 115.6, 112.3, 109.2, 108.6, 108.1, 102.7, 65.5, 65.3, 56.8, 56.2, 56.1,
28.8; LC-MS m/z: 593.8[M+H]+; MS (m/z): HRMS(ESI) Calcd for C29H28N3O5S3
([M+H]+): 594.1191, found:594.1163; Calcd for C29H28N3O5S3 ([M+Na]+):616.1011,
found: 616.0981.
4.1.2.7.
3-((4-((4-bromophenyl)amino)-6-methoxyquinazolin-7-yl)oxy)propyl

3-(allyldisulfaneyl)propanoate (9g)
1
Off-white solid, yield: 16%. H NMR (400 MHz, d6-DMSO) δ 9.53 (s, 1H), 8.49 (s,
1H), 7.84 (d, J = 12.0 Hz, 3H), 7.57 (d, J = 12.0 Hz, 2H), 7.22 (d, J = 8.0 Hz, 1H),
5.84-5.69 (m, 1H), 5.21-5.15 (m, 1H), 5.11-5.05 (m, 1H), 4.33-4.24 (m, 4H), 3.98 (s,
3H), 3.16 (d, J = 8.0 Hz, 2H), 2.67-2.61 (m, 2H), 2.51 (s, 2H), 2.20-2.14 (m, 2H); 13C
NMR (100 MHz, d6-DMSO) δ 172.0, 168.9, 156.5, 153.9, 153.2, 147.4, 139.5, 135.0,
131.7, 128.8, 124.4, 117.6, 115.3, 109.4, 102.5, 65.7, 61.7, 56.8, 34.5, 34.1, 28.3;
LC-MS m/z: 563.8[M+H]+; MS (m/z): HRMS(ESI) Calcd for C24H25BrN3O4S2
([M-H]-): 562.0470(100%), found:562.0589; Calcd for C24H25BrN3O4S2 ([M-H]-):
564.0449(97.3%), found:564.0569.
4.1.2.8.
3-((4-((4-bromophenyl)amino)-6-methoxyquinazolin-7-yl)oxy)propyl
3-(allyldisulfaneyl)propanoate (9h)
1
Off-white solid, yield: 20%. H NMR (400 MHz, d6-DMSO) δ 9.54 (s, 1H), 8.53 (s,
1H), 8.16 (s, 1H), 7.90 (d, J = 8.0 Hz, 1H), 7.84 (s, 1H), 7.36 (t, J = 8.0 Hz 1H), 7.29
(d, J = 8.0 Hz, 1H), 7.24-7.22 (m, 1H), 5.79-5.69 (m, 1H), 5.14-5.05 (m, 2H),
4.33-4.23 (m, 4H), 3.98 (s, 3H), 3.16 (d, J = 4.0 Hz, 2H), 2.63 (t, J = 4.0 Hz ,2H),
2.52-2.51 (m, 2H), 2.17-2.11 (m, 2H); 13C NMR (100 MHz, d6-DMSO) δ 172.0, 166.0,
156.4, 154.0, 153.1, 149.6, 147.5, 135.0, 130.8, 128.8, 126.0, 124.4, 121.6, 121.0,
117.6, 109.4, 102.4, 65.7, 61.5, 56.8, 34.5, 34.1, 28.1; LC-MS m/z: 564.1[M+H]+; MS
(m/z): HRMS(ESI) Calcd for ([M-H]-): 562.0470(100%), found:562.0494; Calcd for
C24H25BrN3O4S2 ([M-H]-): 564.0449(97.3%), found:564.0481.
4.1.3. Synthesis of compounds (11-14)
Compounds 11-14 were prepared according to a similar procedure in Refs[21].
4.1.4. General procedure for the preparation of the compounds 15.
To the corresponding H2S donors (1.1 mmol) in DMF (10 mL) was added HATU (1.1
mmol) and triethylamine (1.2 mmol) at room temperature and the reaction was run for
20 mins. Then compounds 14 (1.0 mmol) was added, and the reaction mixture was

stirred for 24 h at room temperature. After completion of the reaction, the mixture was
poured into ice water (100 mL), stirred for 30mins. The solid formed was filtered off
and purified by a silica gel column chromatography to afford compound 15.
4.1.4.1 2-(4-(3-thioxo-3H-1, 2-dithiol-5-yl)phenoxy)-N-(4-((3-(trifluoromethyl)phenyl)
amino)quinazolin-6-yl)acetamide (15a)
Yellow solid, yield: 16.6 %. 1H NMR (400 MHz, d6-DMSO) δ 10.52 (s, 1H), 10.05 (s,
1H), 8.79 (s, 1H), 8.60 (s, 1H), 8.27 (s, 1H), 8.20 (d, J = 8.0 Hz, 1H), 7.96-7.91 (m,
3H), 7.83 (d, J = 8.0 Hz, 1H), 7.76 (d, J = 4.0 Hz, 1H), 7.60 (t, J = 8.0 Hz, 1H), 7.43
13
(s, 1H), 7.20 (d, J = 8.0 Hz, 2H), 4.95 (s, 2H); C NMR (100 MHz, d6-DMSO) δ
215.4, 176.9, 166.8, 161.7, 157.9, 153.7, 147.4, 140.73, 136.5, 134.9, 129.9, 129.90,
129.6, 129.5, 129.0, 128.7, 128.1, 128.10, 126.2, 124.8, 124.6, 118.7, 116.3, 115.9,
67.5; LC-MS m/z: 571.1 [M+H]+; MS (m/z): HRMS(ESI) Calcd for C26H16F3N4O2S3
([M-H]-): 569.0387, found:569.0453.
4.1.4.2
N-(4-((3-bromophenyl)amino)quinazolin-6-yl)-2-(4-(3-thioxo-3H-1,2-dithiol-5-yl)phe
noxy)acetamide (15b)
Yellow solid, yield: 10.0 %. 1H NMR (400 MHz, d6-DMSO) δ 10.55 (s, 1H), 9.90 (s,
1H), 8.77 (s, 1H), 8.57 (s, 1H), 8.17 (s, 1H), 7.97 (d, J = 12.0 Hz, 1H), 7.87 (d, J = 8.0
Hz, 2H), 7.79 (d, J = 8.0 Hz, 2H), 7.72 (s, 1H), 7.30 (d, J = 8.0 Hz, 1H), 7.25 (s, 1H),
7.19 (d, J = 8.0 Hz, 2H), 4.93 (s, 2H); 13C NMR (100 MHz, d6-DMSO) δ 215.3, 173.9,
166.7, 161.7, 157.8, 153.7, 147.4, 141.6, 136.4, 134.8, 130.6, 129.4, 128.7, 126.3,
125.6, 124.8, 121.6, 121.3, 116.3, 116.1, 115.9, 67.5; LC-MS m/z: 581.0[M+H]+; MS
(m/z): HRMS(ESI) Calcd for C25H16BrN4O2S3 ([M-H]-): 578.9619(100%),
found:578.9648; Calcd for C25H16BrN4O2S3 ([M-H]-): 580.9598(97.3%),
found:580.9629.
4.1.4.3

N-(4-((3-chloro-4-fluorophenyl)amino)quinazolin-6-yl)-2-(4-(3-thioxo-3H-1,2-dithiol-
5-yl)phenoxy)acetamide (15c)
Yellow solid, yield: 15.0 %. 1H NMR (400 MHz, CDCl3) δ 8.83 (s, 1H), 8.76 (s, 1H),
8.53 (s, 1H), 7.98 (d, J = 12.0 Hz, 2H), 7.75-7.70 (m, 3H), 7.63 (d, J = 8.0 Hz, 1H),
7.57 (d, J = 8.0 Hz, 1H), 7.43 (s, 1H), 7.22 (d, J = 8.0 Hz, 1H), 7.19-7.16 (m, 2H),
13
4.79 (s, 2H); C NMR (100 MHz, d6-DMSO) δ 215.3, 172.4, 166.7, 161.6, 136.8,
134.8, 129.3, 128.6, 128.2, 124.9, 124.7, 119.5, 119.4, 116.9, 116.7, 116.2, 116.0,
113.1, 100.0, 67.5; LC-MS m/z: 555.0[M+H]+; MS(m/z):HRMS(ESI) Calcd for
C25H17ClFN4O2S3([M+H]+): 556.0654, found:556.0680.
4.1.4.4
3-(allyldisulfaneyl)-N-(4-((3-(trifluoromethyl)phenyl)amino)quinazolin-6-yl)propana
mide (15d)
Off-white solid, yield: 16.0 %. 1H NMR (400 MHz, d6-DMSO) δ 10.37 (s, 1H), 10.02
(s, 1H), 8.76 (s, 1H), 8.57 (s, 1H), 8.27-8.20 (m, 2H), 7.82 (dd, J = 8.0 Hz, 2H), 7.60
(d, J = 8.0 Hz, 1H), 7.41 (s, 1H), 5.90-5.80 (m, 1H), 5.19 (dd, J = 16.0 Hz, 2H), 3.43
(d, J = 8.0 Hz, 2H), 3.05 (m, 2H), 2.51-2.50 (m, 2H); 13C NMR (100 MHz, d6-DMSO)
δ 169.9, 157.8, 149.5, 147.1, 140.8, 137.3, 134.0, 129.9, 129.0, 128.0, 126.1, 126.1,
119.2, 118.4, 117.3, 116.0, 100.0, 41.5, 36.4, 33.9; LC-MS m/z: 465.1[M+H]+; MS
(m/z): HRMS(ESI) Calcd for C21H19F3N4NaOS2 ([M+H]+): 465.1031, found:
465.1007; Calcd for C21H19F3N4NaOS2 ([M+Na]+): 487.0850, found: 487.0836.
4.1.4.5
3-(allyldisulfaneyl)-N-(4-((3-bromophenyl)amino)quinazolin-6-yl)propanamide (15e)
Off-white solid, yield: 15.7 %. 1H NMR (400 MHz, d6-DMSO) δ 10.35 (s, 1H), 9.88
(s, 1H), 8.71 (s, 1H), 8.57 (d, J = 4.0 Hz, H), 8.17 (t, J = 4.0 Hz, 1H), 7.87 (d, J =8.0
Hz, 2H), 7.78 (dd, J = 4.0 Hz, 1H), 7.32-7.25 (m, 2H), 5.90-5.80 (m, 1H), 5.18 (dd, J
=16.0 Hz, 2H), 3.43 (d, J = 8.0 Hz, 2H), 3.04 (t, J = 8.0 Hz, 2H), 2.51-2.50 (m, 2H);

13C NMR (100 MHz, d6-DMSO) δ 169.9, 157.8, 153.5, 147.1, 141.6, 137.3, 134.0,
130.7, 128.9, 126.4, 124.8, 121.6, 121.4, 119.2, 115.9, 100.0, 41.5, 36.4, 33.9; LC-MS
m/z: 475.0[M+H]+; MS (m/z): HRMS(ESI) Calcd for C20H18BrN4OS2 ([M-H]-):
473.0105(100%), found:473.0166; Calcd for C20H18BrN4OS2 ([M-H]-):
475.0085(97.3%), found:475.0153.
4.1.4.6
3-(allyldisulfaneyl)-N-(4-((3-chloro-4-fluorophenyl)amino)quinazolin-6-yl)propanami
de (15f)
Off-white solid, yield: 17.0 %. 1H NMR (400 MHz, d6-DMSO) δ 10.35 (s, 1H), 9.90
(s, 1H), 8.71 (s, 1H), 8.54 (s, 1H), 8.13 (dd, J =4.0 Hz, 1H), 7.86-7.75 (m, 3H), 7.38
(s, 1H), 5.90-5.80 (m, 1H), 5.18 (dd, J = 16.0 Hz, 2H), 3.43 (d, J = 8.0 Hz, 2H), 3.04
13
(t, J = 8.0 Hz, 2H), 2.51-2.50 (m, 2H); C NMR (100 MHz, d6-DMSO) δ 169.9,
157.8, 153.4, 152.6, 147.0, 137.3, 137.1, 134.0, 128.9, 124.3, 123.1, 119.3, 119.1,
117.0, 115.8, 112.0, 41.5, 36.4, 34.0; LC-MS m/z: 449.1[M+H]+; MS (m/z):
HRMS(ESI) Calcd for C20H17ClFN4OS2 ([M-H]-):447.0516(100.0%), found:447.0608;
Calcd for C20H17ClFN4OS2 ([M-H]-):448.0550(21.6%), found:448.0573; Calcd for
C20H17ClFN4OS2 ([M-H]-):449.0487(32.0%), found:449.0534.
4.2. General Procedure for Anti-proliferation Assays
Cells were seeded in 96-well culture plates (2500−3000/well). The compounds of
various concentrations were added to the plates. Cell proliferation was determined
after treatment with compounds for 72 h. Cell viability was measured using the Cell
Titer-Glo assay (Promega, USA) according to the manufacturer’s instructions, and
luminescence was measured in a multi-label reader (Envision, PerkinElmer, USA).
Data were normalized to control groups (DMSO) and represented by the mean of
three independent measurements with standard error of <20%. GI50 values were
calculated using Prism 5.0 (GraphPad Software, San Diego, CA).

4.3. Western blotting analysis
H3255 cells were treated with compounds at different concentrations. Cells were then
harvested, washed with ice-cold PBS and lysed in cold RIPA buffer (containing 1%
PMSF). The cells were centrifuged at 16200 rpm at 4 °C for 10 min and the
supernatant was collected as whole cell lysate. Protein concentration was determined
by BCA method. 1 x loading buffer was added and samples were heated to 100 °C for
10mins. Proteins were separated by SDS/PAGE electrophoresis and transferred to
PVDF membranes. After blocking the membranes with 5% fat-free milk, the target
protein was proved with anti-phospho specific EGFR (Py1068) antibodies. Blots were
developed by enhanced chemiluminescence (BD FACSCalibur).
4.4. Cell cycle analysis
H3255 and Pffeifer cells in 10% FBS/RPMI were treated with DMSO or compound
9h (0-10μM) for 72 hours and washed with cold 1×PBS buffer. The cells were then
fixed in 70% cold ethanol and incubated at -20 °C overnight. On the day of flow
cytometry, cells were spun down, washed with 1×PBS buffer and stained in PI/RNase
Staining Buffer (BD Bioscience) for 15 min at room temperature and moved to 4 °C
until the time of analysis. Flow cytometry was performed using a FACS Calibur (BD),
and results were analyzed by ModFit software.
4.5 hydrogen sulfide release evaluation
The H2S fluorescent probe NIR-HS (10μM) and the target compounds (0μM, 5μM,
10μM, 50μM, 100μM and 200μM) were incubated in PBS buffer (PBS: acetone =2:3)
for 3h (37°C). Subsequently, the fluorescence emission spectra (λex=670nm,
λem=723nm) were determined by an F4600 fluorescence spectrophotometer.
The H2S fluorescent probe NIR-HS (10μM) and Na2S (0μM, 2μM, 4μM, 6μM and
8μM) were incubated in PBS buffer (PBS: acetone =2:3) for 3h (37°C). Then the
fluorescence emission spectra (λex=670nm, λem=723nm) were determined by an
F4600 fluorescence spectrophotometer, and the standard curve was established

according to the fluorescence intensity.
The H2S fluorescent probe NIR-HS (10μM) and target compounds (20μM) were
incubated in PBS buffer solution (PBS: acetone =2:3) for 0min, 5min, 30min, 60min,
90min, 120min, 150min, 180min (37°C), respectively. After that, fluorescence
emission spectra (λex=670nm, λem=723nm) were determined. According to Na2S
standard curve, the amount of H2S released by compounds was converted.
Acknowledgment
The authors are grateful for the financial support by Science and technology project of
Xuzhou City (KC16SG255). Thanks to PreceDo pharmaceuticals Co., Ltd. for kinase
target-based cell screening and biological evaluation.
References
[1] G.I. Solyanik, Quinazoline compounds for antitumor treatment, Experimental oncology, 41 (2019)
3-6.
[2] E.-d. Li, Q. Lin, Y.-q. Meng, L.-y. Zhang, P.-p. Song, N. Li, J.-c. Xin, P. Yang, C.-n. Bao, D.-q.
Zhang, Y. Zhang, J.-k. Wang, Q.-r. Zhang, H.-m. Liu, 2,4-Disubstituted quinazolines targeting breast
cancer cells via EGFR-PI3K, European Journal of Medicinal Chemistry, 172 (2019) 36-47.
[3] H.S.A. Al-Salem, G.H. Hegazy, K.E.H. El-Taher, S.M. El-Messery, A.M. Al-Obaid, H.I.
El-Subbagh, Synthesis, anticonvulsant activity and molecular modeling study of some new
hydrazinecarbothioamide, benzenesulfonohydrazide, and phenacylacetohydrazide analogues of
4(3H)-quinazolinone, Bioorganic & Medicinal Chemistry Letters, 25 (2015) 1490-1499.
[4] M.A. Pradeep, N.R. Kumar, D.K. Swaroop, N.S. Reddy, K. Sirisha, C.G. Kumar, N.J. Babu, T.
Ganapathi, B. Narsaiah, Design and Synthesis of Novel Pyrimidine/Hexahydroquinazoline-Fused
Pyrazolo 3,4-b Pyridine Derivatives, Their Biological Evaluation and Docking Studies(#),
Chemistryselect, 4 (2019) 138-144.
[5] M. Yu V, A. O M, N. I S, B. G G, K. S I, Directed Search of Anti-inflammatory Agents among
(3H-Quinazoline-4-ylidene)hydrazides of N-protected Amino acids and their Heterocyclization
Products, Anti-inflammatory & anti-allergy agents in medicinal chemistry, (2019).
[6] R. Neelarapu, J.R. Maignan, C.L. Lichorowic, A. Monastyrskyi, T.S. Mutka, A.N. LaCrue, L.D.
Blake, D. Casandra, S. Mashkouri, J.N. Burrows, P.A. Willis, D.E. Kyle, R. Manetsch, Design and
Synthesis of Orally Bioavailable Piperazine Substituted 4(1H)-Quinolones with Potent Antimalarial
Activity: Structure-Activity and Structure-Property Relationship Studies, Journal of Medicinal
Chemistry, 61 (2018) 1450-1473.
[7] L.M. Antypenko, S.I. Kovalenko, O.V. Karpenko, A.M. Katsev, V.P. Novikov, N.S. Fedyunina,
1-R-2-( 1,2,4 Triazolo 1,5-c quinazoline-2-ylthio) etanon(ol) s: Synthesis, Bioluminescence Inhibition,
Molecular Docking Studies, Antibacterial and Antifungal Activities, Current Computer-Aided Drug

Design, 12 (2016) 29-41.
[8] M.U. Rahman, A. Rathore, A.A. Siddiqui, G. Parveen, M.S. Yar, Synthesis and characterization of
quinazoline derivatives: search for hybrid molecule as diuretic and antihypertensive agents, Journal of
Enzyme Inhibition and Medicinal Chemistry, 29 (2014) 733-743.
[9] R.P. Modh, E. De Clercq, C. Pannecouque, K.H. Chikhalia, Design, synthesis, antimicrobial activity
and anti-HIV activity evaluation of novel hybrid quinazoline-triazine derivatives, Journal of Enzyme
Inhibition and Medicinal Chemistry, 29 (2014) 100-108.
[10] D. Das, L. Xie, J. Wang, X. Xu, Z. Zhang, J. Shi, X. Le, J. Hong, Discovery of new quinazoline
derivatives as irreversible dual EGFR/HER2 inhibitors and their anticancer activities - Part 1,
Bioorganic & Medicinal Chemistry Letters, 29 (2019) 591-596.
[11] S. Lee, J. Kim, K.B. Duggirala, A. Go, I. Shin, B.C. Cho, G. Choi, C.H. Chae, K. Lee, Allosteric
Inhibitor TREA-0236 Containing Non-hydrolysable Quinazoline-4-one for EGFR T790M/C797S
Mutants Inhibition, Bulletin of the Korean Chemical Society, 39 (2018) 895-898.
[12] M.M. Ghorab, M.S. Alsaid, A.M. Soliman, Dual EGFR/HER2 inhibitors and apoptosis inducers:
New benzo g quinazoline derivatives bearing benzenesulfonamide as anticancer and radiosensitizers,
Bioorganic Chemistry, 80 (2018) 611-620.
[13] C. Wang, S. Xu, L. Peng, B. Zhang, H. Zhang, Y. Hu, P. Zheng, W. Zhu, Design, synthesis and
biological evaluation of novel 4-anlinoquinazoline derivatives as EGFR inhibitors with the potential to
inhibit the gefitinib-resistant nonsmall cell lung cancers, Journal of Enzyme Inhibition and Medicinal
Chemistry, 34 (2019) 204-218.
[14] P. Pathak, P.K. Shukla, V. Kumar, A. Kumar, A. Verma, Quinazoline clubbed 1,3,5-triazine
derivatives as VEGFR2 kinase inhibitors: design, synthesis, docking, in vitro cytotoxicity and in ovo
antiangiogenic activity, Inflammopharmacology, 26 (2018) 1441-1453.
[15] S.J. Buggana, M.C. Paturi, H. Perka, G.D. Reddy, V.V.S.R. Prasad, Novel 2,4-disubstituted
quinazolines as cytotoxic agents and JAK2 inhibitors: Synthesis, in vitro evaluation and molecular
dynamics studies, Computational Biology and Chemistry, 79 (2019) 110-118.
[16] J. Chen, Z. Sang, Y. Jiang, C. Yang, L. He, Design, synthesis, and biological evaluation of
quinazoline derivatives as dual HDAC1 and HDAC6 inhibitors for the treatment of cancer, Chemical
Biology & Drug Design, 93 (2019) 232-241.
[17] S.-M. Yang, D.J. Urban, M. Yoshioka, J.W. Strovel, S. Fletcher, A.Q. Wang, X. Xu, P. Shah, X. Hu,
M.D. Hall, A. Jadhav, D.J. Maloney, Discovery and lead identification of quinazoline-based BRD4
inhibitors, Bioorganic & Medicinal Chemistry Letters, 28 (2018) 3483-3488.
[18] X. Hu, H. Zhao, Y. Wang, Z. Liu, B. Feng, C. Tang, Synthesis and biological evaluation of novel
5,6-dihydropyrimido 4,5-f quinazoline derivatives as potent CDK2 inhibitors, Bioorganic & Medicinal
Chemistry Letters, 28 (2018) 3385-3390.
[19] J. Zhou, M. Ji, H. Yao, R. Cao, H. Zhao, X. Wang, X. Chen, B. Xu, Discovery of
quinazoline-2,4(1H,3H)-dione derivatives as novel PARP-1/2 inhibitors: design, synthesis and their
antitumor activity, Organic & Biomolecular Chemistry, 16 (2018) 3189-3202.
[20] S. Patel, A. Motta, P. Flaherty, A. Bhatt, T. Wright, J. Cavanaugh, 4,6-Disubstituted quinazoline
derivatives as inhibitors of the MEK5/ERK5 pathway, Abstracts of Papers of the American Chemical
Society, 256 (2018).
[21] Y.-G. Zheng, J. Su, C.-Y. Gao, P. Jiang, L. An, Y.-S. Xue, J. Gao, Y. Liu, Design, synthesis, and
biological evaluation of novel 4-anilinoquinazoline derivatives bearing amino acid moiety as potential
EGFR kinase inhibitors, European Journal of Medicinal Chemistry, 130 (2017) 393-405.

[22] M. Zheng, Y. Zheng, Y. Xue, Y. Liu, L. An, L. Zhang, M. Ji, B. Xue, X. Wu, X. Gong, N. Gu, X.
Zhan, Synthesis and Quantum Chemical Studies of New 4-aminoquinazoline Derivatives as Aurora
A/B Kinase Inhibitors, Chemical Biology & Drug Design, 81 (2013) 399-407.
[23] X. Wu, M. Li, Y. Qu, W. Tang, Y. Zheng, J. Lian, M. Ji, L. Xu, Design and synthesis of novel
Gefitinib analogues with improved anti-tumor activity, Bioorganic & Medicinal Chemistry, 18 (2010)
3812-3822.
[24] C.-M. Park, Y. Zhao, Z. Zhu, A. Pacheco, B. Peng, N.O. Devarie-Baez, P. Bagdon, H. Zhang, M.
Xian, Synthesis and evaluation of phosphorodithioate-based hydrogen sulfide donors, Molecular
Biosystems, 9 (2013) 2430-2434.
[25] Y. Ma, Z. Yan, X. Deng, J. Guo, J. Hu, Y. Yu, F. Jiao, Anticancer effect of exogenous hydrogen
sulfide in cisplatin-resistant A549/DDP cells, Oncology Reports, 39 (2018) 2969-2977.
[26] S. Lu, Y. Gao, X. Huang, X. Wang, GYY4137, a hydrogen sulfide ( H2S) donor, shows potent
anti- hepatocellular carcinoma activity through blocking the STAT3 pathway, International Journal of
Oncology, 44 (2014) 1259-1267.
[27] M. Ciocci, E. Iorio, F. Carotenuto, H.A. Khashoggi, F. Nanni, S. Melino, H2S-releasing
nanoemulsions: a new formulation to inhibit tumor cells proliferation and improve tissue repair,
Oncotarget, 7 (2016) 84338-84358.
[28] S. Pu, X. Wu, X. Yang, Y. Zhang, Y. Dai, Y. Zhang, X. Wu, Y. Liu, X. Cui, H. Jin, J. Cao, R. Li, J.
Cai, Q. Cao, L. Hu, Y. Gao, The Therapeutic Role of Xenobiotic Nuclear Receptors Against Metabolic
Syndrome, Curr Drug Metab, 20 (2019) 15-22.
[29] Z.W. Lee, J. Zhou, C.-S. Chen, Y. Zhao, C.-H. Tan, L. Li, P.K. Moore, L.-W. Deng, The
Slow-Releasing Hydrogen Sulfide Donor, GYY4137, Exhibits Novel Anti-Cancer Effects In Vitro and
In Vivo, Plos One, 6 (2011).
[30] X. Hu, R. Jiao, H. Li, X. Wang, H. Lyu, X. Gao, F. Xu, Z. Li, H. Hua, D. Li, Antiproliferative
hydrogen sulfide releasing evodiamine derivatives and their apoptosis inducing properties, European
Journal of Medicinal Chemistry, 151 (2018) 376-388.
[31] F. Xu, X. Gao, H. Li, S. Xu, X. Li, X. Hu, Z. Li, J. Xu, H. Hua, D. Li, Hydrogen sulfide releasing
enmein-type diterpenoid derivatives as apoptosis inducers through mitochondria-related pathways,
Bioorganic Chemistry, 82 (2019) 192-203.
[32] R. Kodela, M. Chattopadhyay, K. Kashfi, Synthesis and biological activity of NOSH-naproxen
(AVT-219) and NOSH-sulindac (AVT-18A) as potent anti-inflammatory agents with chemotherapeutic
potential, Medchemcomm, 4 (2013) 1472-1481.
[33] R. Kodela, M. Chattopadhyay, K. Kashfi, NOSH-Aspirin: A Novel Nitric Oxide-Hydrogen
Sulfide-Releasing Hybrid: A New Class of Anti-inflammatory Pharmaceuticals, Acs Medicinal
Chemistry Letters, 3 (2012) 257-262.
[34] S. Wu, J. Fu, Y. Dong, Q. Yi, D. Lu, W. Wang, Y. Qi, R. Yu, X. Zhou, GOLPH3 promotes glioma
progression via facilitating JAK2-STAT3 pathway activation, Journal of neuro-oncology, 139 (2018)
269-279.
[35] X.P. Zhou, S. Xie, S.S. Wu, Y.H. Qi, Z.H. Wang, H. Zhang, D. Lu, X. Wang, Y. Dong, G.Z. Liu,
D.X. Yang, Q. Shi, W.B. Bian, R.T. Yu, Golgi phosphoprotein 3 promotes glioma progression via
inhibiting Rab5-mediated endocytosis and degradation of epidermal growth factor receptor,
Neuro-Oncology, 19 (2017) 1628-1639.
[36] J.G. Paez, P.A. Janne, J.C. Lee, S. Tracy, H. Greulich, S. Gabriel, P. Herman, F.J. Kaye, N.
Lindeman, T.J. Boggon, K. Naoki, H. Sasaki, Y. Fujii, M.J. Eck, W.R. Sellers, B.E. Johnson, M.

Meyerson, EGFR mutations in lung cancer: Correlation with clinical response to gefitinib therapy,
Science, 304 (2004) 1497-1500.
[37] X.X. Li, A.L. Wang, K.L. Yu, Z.P. Qi, C. Chen, W.C. Wang, C. Hu, H. Wu, J.X. Wu, Z. Zhao, J.
Liu, F.M. Zou, L. Wang, B.L. Wang, W. Wang, S.C. Zhang, J. Liu, Q.S. Liu, Discovery of
(R)-1-(3-(4-Amino-3-(4-phenoxyphenyl)-1H-pyrazolo
3,4-d
pyrimidin-1-yl)p
iperidin-1-yl)-2-(dimethylamino)ethanone (CHMFL-FLT3-122) as a Potent and Orally Available FLT3
Kinase Inhibitor for FLT3-ITD Positive Acute Myeloid Leukemia, Journal of Medicinal Chemistry, 58
(2015) 9625-9638.
[38] J.S. Melnick, J. Janes, S. Kim, J.Y. Chang, D.G. Sipes, D. Gunderson, L. Jarnes, J.T. Matzen, M.E.
Garcia, T.L. Hood, R. Beigi, G. Xia, R.A. Harig, H. Asatryan, S.F. Yan, Y.Y. Zhou, X.J. Gu, A. Saadat,
V. Zhou, F.J. King, C.M. Shaw, A.I. Su, R. Downs, N.S. Gray, P.G. Schultz, M. Warmuth, J.S. Caldwell,
An efficient rapid system for profiling the cellular activities of molecular libraries, Proceedings of the
National Academy of Sciences of the United States of America, 103 (2006) 3153-3158.
[39] S. La Monica, D. Cretella, M. Bonelli, C. Fumarola, A. Cavazzoni, G. Digiacomo, L. Flammini, E.
Barocelli, R. Minari, N. Naldi, P.G. Petronini, M. Tiseo, R. Alfieri, Trastuzumab emtansine delays and
overcomes resistance to the third-generation EGFR-TKI osimertinib in NSCLC EGFR mutated cell
lines, Journal of Experimental & Clinical Cancer Research, 36 (2017).
[40] N.J. Zhao, P.C. Liang, X.Y. Zhuo, C.L. Su, X.M. Zong, B.N. Guo, D. Han, X.L. Yan, S.Q. Hu,
Q.G. Zhang, X. Tie, After Treatment with Methylene Blue is Effective against Delayed Encephalopathy
after Acute Carbon Monoxide Poisoning, Basic & clinical pharmacology & toxicology, 122 (2018)
470-480.
[41] C. Pham-Danis, S. Gehrke, E. Danis, A.I. Rozhok, M.W. Daniels, D.X. Gao, C. Collins, J.T. Di
Paola, A. D'Alessandro, J. DeGregori, Urea Cycle Sustains Cellular Energetics upon EGFR Inhibition
in EGFR-Mutant NSCLC, Molecular Cancer Research, 17 (2019) 1351-1364.
[42] Y.L. Zhang, L. Chen, H.J. Xu, X.B. Li, L.J. Zhao, W. Wang, B.L. Li, X.Q. Zhang,
6,7-Dimorpholinoalkoxy quinazoline derivatives as potent EGFR inhibitors with enhanced
antiproliferative activities against tumor cells, European Journal of Medicinal Chemistry, 147 (2018)
77-89.
[43] L. Zhang, X.E. Zheng, F. Zou, Y.G. Shang, W.Q. Meng, E. Lai, Z.C. Xu, Y. Liu, J. Zhao, A highly
selective and sensitive near-infrared fluorescent probe for imaging of hydrogen sulphide in living cells
and mice, Scientific Reports, 6 (2016).
[44] N. Lougiakis, A. Papapetropoulos, E. Gikas, S. Toumpas, P. Efentakis, R. Wedmann, A. Zoga, Z.M.
Zhou, E.K. Iliodromitis, A.L. Skaltsounis, M.R. Filipovic, N. Pouli, P. Marakos, I. Andreadou,
Synthesis and Pharmacological Evaluation of Novel Adenine-Hydrogen Sulfide Slow Release Hybrids
Designed as Multitarget Cardioprotective Agents, Journal of Medicinal Chemistry, 59 (2016)
1776-1790.

Highlights
ò We devised and synthesized a series of quinazoline derivatives bearing H2S
donors.
ò The
target
compounds
exhibits
inhibiting
activities
against
TEL-EGFR-L858R-BaF3.
ò Most compounds displayed anti-proliferative activities against H3255 cells.
ò Compound 9h exhibits lower toxic effect on other tumor cells and HUVEC cells.
ò
Compound 9h inhibits the phosphorylation of EGFR in H3255 cells.
ò compound 9h induces cell cycle arrest in G0-G1 phase.
ò The H2S release of compound 9h is more and faster than other compounds.

Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests:
You-Guang Zheng, Wu-Qi Zhang, Long Meng, Xiao-Qing Wu, Ling Zhang, Lin An, Cheng-Lin Li, Cai-Yun Gao,
Liang Xu, Yi Liu.