Design, synthesis, antiproliferative activity and docking studies of quinazoline derivatives bearing oxazole or imidazole as potential EGFR inhibitors

Article abstract of DOI:10.1039/c8nj03594f

Six series of quinazoline derivatives bearing oxazole or imidazole (8a-f, 9a-f, 10a-d, 11a-f, 12a-d and 13a-i) were designed, synthesized and their IC₅₀ values evaluated against three cancer cell lines (A549, MCF-7 and PC-3). Most of the thirty-five target compounds showed excellent antiproliferative activity against one or several cancer cell lines. Compound 12a showed the best activity against A549, MCF-7 and PC-3 cancer cell lines, with IC₅₀ values of 1.90 ± 0.13 μM, 2.23 ± 0.28 μM and 2.03 ± 0.14 μM, respectively. Four selected compounds (8a, 9d, 10a and 12a) were further evaluated for the inhibitory activity against EGFR kinase. AnnexinV-FITC, propidium iodide (PI) double staining and acridine orange single staining results indicated that compound 12a could induce apoptosis of human lung cancer A549 cells.

Full text of DOI:10.1039/c8nj03594f

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Design, synthesis, antiproliferative activity and docking studies of quinazoline derivatives bearing oxazole or
imidazole as potential EGFR inhibitors
Yiqiang OuYang1, 2, †, Caolin Wang1,†, Bingbing Zhao1, Hehua Xiong1, Zhen Xiao1, Bingliang Zhang1, Pengwu Zheng1,
Jiayi Hu1, Yanli Gao1, Manli Zhang1, Wufu Zhu1,* , Shan Xu1,*
1 Jiangxi Provincial Key Laboratory of Drug Design and Evaluation, School of Pharmacy, Jiangxi Science & Technology
Normal University, Nanchang 330013
2 State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Drug Discovery for Metabolic Diseases, Center
of Drug Discovery, China Pharmaceutical University, Nanjing 210009, China
*Correspondence author.
Tel/Fax: +86 791 8380ꢀ2393; Eꢀmail address: shanxu9891@126.com (S. Xu), zhuwufuꢀ1122@163.com (W. Zhu)
Abstract: Six series of quinazoline derivatives bearing oxazole or imidazole (8a–f, 9a–f, 10a–d, 11a–f, 12a–d and 13a–i)
were designed, synthesized and evaluated for the IC50 values against three cancer cell lines (A549, MCFꢀ7 and PCꢀ3).
Most of the thirty five target compounds showed excellent antiproliferative activity against one or several cancer cell
lines. The compound 12a showed the best activity against A549, MCFꢀ7 and PCꢀ3 cancer cell lines, with the IC50 values
of 1.90 ± 0.13 ꢁM, 2.23 ± 0.28 ꢁM and 2.03 ± 0.14 ꢁM, respectively. Four selected compounds (8a, 9d, 10a and 12a)
were further evaluated for the inhibitory activity against EGFR kinase. AnnexinVꢀFITC, propidium iodide (PI) double
staining and acridine orange single staining results indicated that the compound 12a could induce apoptosis of human
lung cancer A549 cells.
Key words: Quinazoline derivatives; Synthesis; Antiproliferative activity; EGFR inhibitors
† These authors contribute equally to this work
1
Corresponding author. Tel./fax: +86 791 83802393.
Eꢀmail address: shanxu9891@126.com (S. Xu), zhuwf@jxstnu.edu.cn, zhuwufuꢀ1122@163.com (W. Zhu).

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1 Introduction
Malignant tumor is serious threat to human health, and the number of deaths due to cancer is increasing every year1.
The epidermal growth factor receptor (EGFR) is fundamentally important in cell proliferation, survival, migration,
adhesion, and differentiation, and closely related to the formation and deterioration of the tumor 2ꢀ5. Therefore, inhibition
of EGFR signaling pathway has become a hot spot for cancer prevention and treatment.
In recent years, many quinazoline derivatives were reported as EGFR signal transduction pathway inhibitors such as
6
7
8
gefitinib (1), erlotinib (2) and afatinib (3) (Fig. 1). The structureꢀactivity relationships studies of these compounds
revealed that the quinazoline skeleton plays a key role in their antitumor activities. Therefore, the quinazoline skeleton
was kept as a privileged scaffold. In our previous work, compound 4 showed the best activity against EGFR kinase, with
the IC50 value of 5 nM 9. Therefore, inspired by compound 4, several series of quinazoline derivatives bearing
2,3ꢀdihydroꢀindole or 1,2,3,4ꢀtetrahydroquinoline were designed. In addition, in order to investigate the effect of amide
bond at Cꢀ6 position of quinazoline on the antiproliferative activity, a fiveꢀatom ring structure between the Cꢀ6 and Cꢀ7
position of quinazoline were constructed, just as shown in Fig 2.
Furthermore, compound 5 10 was an excellent EGFR kinase inhibitor, and the IC50 value was 19 nM against EGFR
kinase. In order to investigate the effect to the activity of the aqueous solubility chains and inspired by compound 5, we
focused on the replacement of the oxazolidinꢀ2ꢀone group by oxazole moiety and introduced secondary amines group
11
into the target compounds to afford two series of compounds (8a–f and 9a–f). In addition, compound 6
bearing
imidazo[4,5ꢀg]ꢀquinazoline showed excellent activity and the IC50 value was 8 pM against EGFR kinase. Inspired by
compound 6, the oxazole moiety was substituted by imidazole moiety to obtain two series of compounds (10a–d and
11a–f). However, these compounds (10a–d and 11a–f) show unsatisfactory solubility which affects the pharmacokinetics
in vitro, the compounds were substituted with hydrogen group at R2 of imidazole ring have increased the antiproliferative
activity relative to substituted by methyl group. In order to increase the waterꢀsoluble, the secondary amine was added to
the end of the flexible chain on the imidazole ring. On the basis of the compounds (10aꢀb and 11bꢀc) to obtain the other
series of compounds (12a–d and 13a–i). The design strategy for all target compounds is shown in Fig. 2.
Hence we disclosed the design, synthesis and antiproliferative activity of novel quinazoline analogues against EGFR
overexpression cancer cells A549 (human lung cancer), MCFꢀ7 (human breast cancer) and PCꢀ3 (human prostate cancer)
12ꢀ14 and as well as the inhibitory on EGFR kinase. Moreover, flow cytometry, acridine orange single staining and docking
studies were presented in this paper as well.

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F
F
HN
N
Cl
HN
CN
HN
N
Cl
H
N
O
O
O
N
N
O
O
N
N
N
O
O
O
N
O
2, Erlotinib
1, Gefitinib
3, Afatinib
(IC50=2 nM)
(IC50=27 nM)
(IC50=0.5 nM)
O
O
Cl
N
N
O
O
HN
N
Br
H
N
O
HN
Cl
N
N
N
N
O
N
O
N
4
N
N
H
O
5
N
6
(IC50=8 pM)
(IC50=5 nM)
(IC50=19 nM)
O
n1
N
n
n
N
N
N
N
N
N
N
N
N
N
R1
N
N
N
O
N
R2
N
n2
Target Compounds
Target Compounds
Target Compounds
8aꢀf n=1
9aꢀf n=2
10aꢀd n=1
11aꢀf n=2
12aꢀd n1=1, n2=2 or 3
13aꢀi n1=2, n2=2 or 3
Fig. 1. Structures of smallꢀmolecule EGFR inhibitors based on the quinazoline scaffold and the target compounds 8a–f,
9a–f, 10a–d, 11a–f, 12a–d and 13a–i.
Fig. 2. Structures and design strategy for target compounds 8a–f, 9a–f, 10a–d, 11a–f, 12a–d and 13a–i.
2 Chemistry
The structures and preparation of target compounds 8a–f, 9a–f, 10a–d, 11a–f, 12a–d and 13a–i are described in
Schemes 1ꢀ2. These target compounds were synthesized from commercially available 2ꢀaminoꢀ4ꢀfluorobenzoic acid
through seven steps or eight steps, respectively. Cyclization reaction of 2ꢀaminoꢀ4ꢀfluorobenzoic acid in the present of

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formamidine acetate obtained compound 1 which was a white solid. Subsequently, compound 1 was nitrified with
concentrated sulfuric acid and fuming nitric acid refluxed for 2 h to obtain 2. Chlorination of compound 2 with SOCl2
and DMF for 4 h at 90 °C to obtain 3. Nucleophilic displacement of the compound 3 with 2,3ꢀdihydroꢀindole or
1,2,3,4ꢀtetrahydroquinoline gave access to intermediates 4a and 4b, respectively. Compound 4a or 4b was treated with
DMSO, 50% sodium hydroxide as base catalyst to furnish 5a and 5b, respectively. Then compounds 5a and 5b were
reduced by NH2NH2•H2O (80%), FeCl3 and activated carbon to generate key intermediates 6a and 6b, respectively.
Finally, intermediates 6a–d were cyclizing with CS2, 50% sodium hydroxide as base catalyst to get intermediates 7a and
7b, respectively. Finally, intermediates 7a and 7b were condensed with secondary amine (Dimethylamine, Diethylamine,
Pyrrolidine, Piperidine, Nꢀmethyl piperazine, Morpholine) to get target compounds 8a–f and 9a–f, respectively.
Scheme 1. Synthetic route of target compounds 8a–f and 9a–f.
Reagents and conditions: (a) ethanol, 24 h; (b) Con.H2SO4, fuming HNO3, 2 h; (c) SOCl2, DMF, 4 h; (d)
2,3ꢀdihydroindole or 1,2,3,4ꢀtetrahydroindole, isopropanol, triethylamine, 1.5 h; (e) 50%NaOH, DMSO, 3 h; (f) 80%
hydrazine monohydrate, FeCl3, activated carbon, ethanol, 1 h; (g) CS2, ethanol, 1 h; (h) K2CO3, 1,4ꢀdioxane, 4 h.
Compound 4a or 4b was treated with condensed with the corresponding amine (Methylamine, Ethanolamine,
Propanolamine) to furnish 5cꢀe, respectively. Then compounds 5cꢀe were reduced by NH2NH2•H2O (80%), FeCl3 and
activated carbon to generate key intermediates 6cꢀe, respectively. Finally, intermediates 6a–d were cyclizing with
(Formic acid or Triethyl orthoacetate) to get target compounds 10a–d and 11a–f, respectively.
Scheme 2. Synthetic route of target compounds 10a–d and 11a–f.
Reagents and conditions: (a) isopropanol, 2 h; (b) 80% hydrazine monohydrate, FeCl3, activated carbon, ethanol, 1 h; (c)
triethyl orthoacetate or formic acid, 4 h;

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Using target compounds 10a, 10b, 11b and 11c as the key intermediates, then compounds 10a, 10b, 11b and 11c
with SOCl2 and DMF for 4 h at 90 °C to obtain 7cꢀf, respectively. Finally, intermediates 7cꢀf were condensed with
secondary amine (Diethylamine, Pyrrolidine, Piperidine, Nꢀmethyl piperazine, Morpholine) to get target compounds
12a–d and 13a–i, respectively.
Scheme 3. Synthetic route of target compounds 12a–d and 13a–i.
Reagents and conditions: (a) SOCl2, DMF, 4 h; (b) K2CO3, 1,4ꢀdioxane, 4 h;
3 Results and discussion
3.1 Biological evaluation
All the target compounds (8a–f, 9a–f, 10a–d, 11a–f, 12a–d and 13a–i) were tested for antiproliferative activity
against EGFR overexpression cancer cells A549, MCFꢀ7 and PCꢀ3 cancer cell lines through
3ꢀ(4,5ꢀdimethylthiazolylꢀ2)ꢀ2, 5ꢀdiphenyltetrazolium Bromide (MTT) assay, using EGFR inhibitor afatinib as positive
control. In addition, some selected compounds were evaluated for the IC50 values against EGFR kinase by the mobility
shift assay, together with reference compound afatinib. The results expressed as IC50 values were summarized in Tables
1–4 and the values are the average of at least two independent experiments.
Table 1. Structures and activity of target compounds 8a–f and 9a–f.
IC50 a (µM)
Compd.
n
A549
MCFꢀ7
PCꢀ3
1
1
1
8a
8b
8c
3.83 ± 0.27
3.85 ± 0.59
7.52 ± 0.88
4.21 ± 0.31
3.67 ± 0.56
4.34 ± 0.42
6.65 ± 0.69
2.56 ± 0.40 16.13 ± 1.21

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1
1
1
2
2
2
2
2
8d
8e
8f
11.23 ± 1.01 13.25 ± 1.15 15.58 ± 1.36
4.46 ± 0.53 4.98 ± 0.44 5.31 ± 0.47
24.36 ± 1.49 20.29 ± 1.89 18.58 ± 1.79
9a
9b
9c
9d
9e
5.25 ± 0.42
3.85 ± 0.40
5.83 ± 0.45
4.68 ± 0.41
5.15 ± 0.39
5.86 ± 0.51
14.82 ± 1.23 12.24 ± 1.29 13.59 ± 1.38
17.42 ± 1.08 14.37 ± 1.31 17.77 ± 1.81
13.62 ± 1.13
23.19 ± 1.56 23.01 ± 1.35 19.78 ± 1.39
1.07 ± 0.07 1.33 ± 0.16 2.68 ± 0.29
9.93 ± 0.88 11.14 ± 1.05
9f
2
ꢀ
Afatinibb
ꢀ
a The values are an average of two separate determinations
b Used as a positive controls
Table 2. Structures and activity of target compounds 10a–d and 11a–f.
IC50 a (µM)
MCFꢀ7
Compd.
n
R1
R2
A549
PCꢀ3
10a
10b
1
1
1
1
2
2
2
2
2
2
ꢀ
ꢀCH2CH2OH
ꢀCH2CH2CH2OH
ꢀCH2CH2OH
ꢀCH2CH2CH2OH
ꢀCH3
ꢀH
ꢀH
4.48 ± 0.39
5.11 ± 0.41
9.53 ± 0.75
8.25 ± 0.75
8.67 ± 0.76 10.34 ± 0.92
11.67 ± 1.06 13.89 ± 1.15
12.84 ± 0.99 9.02 ± 0.86
11.15 ± 1.08 14.37 ± 1.52
10c
ꢀCH3
ꢀCH3
ꢀH
10d
11a
12.49 ± 1.09 17.97 ± 1.25 12.55 ± 1.14
11b
ꢀCH2CH2OH
ꢀCH2CH2CH2OH
ꢀCH3
ꢀH
6.24 ± 0.49
8.88 ± 0.74
7.25 ± 0.62
9.22 ± 0.96
8.07 ± 0.69
9.46 ± 0.86
11c
ꢀH
11d
ꢀCH3
ꢀCH3
ꢀCH3
ꢀ
19.53 ± 1.75 24.84 ± 1.99 23.02 ± 1.86
11e
ꢀCH2CH2OH
ꢀCH2CH2CH2OH
ꢀ
5.49 ± 0.39
5.03 ± 0.40
1.07 ± 0.07
10.61 ± 1.04 8.47 ± 0.67
8.47 ± 0.74 10.88 ± 1.05
11f
Afatinibb
1.33 ± 0.16
2.68 ± 0.29
a The values are an average of two separate determinations
b Used as a positive controls

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Table 3. Structures and activity of target compounds 12a–d and 13a–i.
IC50 a (µM)
MCFꢀ7
Compd.
n1
n2
A549
PCꢀ3
12a
12b
12c
12d
13a
13b
13c
13d
13e
13f
1
1
1
1
2
2
2
2
2
2
2
2
2
3
3
3
2
2
2
2
3
3
3
3
1.90 ± 0.13
2.23 ± 0.28
4.55 ± 0.66
3.26 ± 0.23
6.08 ± 0.49
4.38 ± 0.32
6.42 ± 0.51
7.09 ± 0.59
2.03 ± 0.14
9.23 ± 0.88
3.39 ± 0.17
6.51 ± 0.51
5.58 ± 0.51
7.81 ± 0.64
6.89 ± 0.57
7.11 ± 0.59
5.62 ± 0.44
5.88 ± 0.44
4.32 ± 0.31
4.20 ± 0.32
6.48 ± 0.81
9.34 ± 0.74
5.33 ± 0.41
5.69 ± 0.48
5.52 ± 0.42
4.99 ± 0.43
9.28 ± 0.79 11.83 ± 0.83
4.28 ± 0.32
4.42 ± 0.45
6.08 ± 0.52
5.15 ± 0.45
4.87 ± 0.45
6.30 ± 0.55
5.78 ± 0.43
4.49 ± 0.38
13g
13h
2
ꢀ
3
ꢀ
13i
8.63 ± 0.78
1.07 ± 0.07
9.06 ± 0.77 10.76 ± 0.90
1.33 ± 0.16 2.68 ± 0.29
Afatinibb
ꢀ
The bold values showed excellent antiproliferative activity
a The values are an average of two separate determinations
b Used as a positive controls

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Fig. 3 Statistical results of the cell proliferation inhibition in A549, MCFꢀ7 and PCꢀ3 cells treated with different
concentrations of target compound 12a.
Table 4 EGFR kinase inhibitory activity of compounds 8a, 9d, 10a, 12a and afatinib.
IC50 (nM)
Compd.
EGFR
8a
9d
72
93
65
32
5
10a
12a
Afatiniba
a Used as a positive controls
As illustrated in Tables 1ꢀ3, most of the thirty five target compounds showed excellent antiproliferative activity
against the different tested cancer cells. In general, the antiproliferative activity of target compounds against A549 cell
line were more potent than against MCFꢀ7 and PCꢀ3 cell lines, It can be inferred that these compounds have a certain
selectivity for A549 cells. What’s more, we could easily see that the two series of compounds 12a–d and 13a–i against
the three cancer cell lines were much more active than the other series of compounds 8a–f, 9a–f, 10a–d and 11a–f. Four
selected compounds (8a, 9d, 10a and 12a) were further evaluated for the inhibitory activity against EGFR kinases and all
of them exhibited excellent activity with the IC50 values in nanomole level. On the whole, when the Cꢀ4 position of
quinazoline was substituted by 2,3ꢀdihydroindole or 1,2,3,4ꢀtetrahydroquinoline, there would be no significant effect on
antiproliferative activity.

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As illustrated in Table 1, it can be see that the compounds (8aꢀb and 9aꢀb) including dimethylamine or diethylamine
could improve the antiproliferative activity against A549, MCFꢀ7 and PCꢀ3 cell lines, which were more remarkable than
that of compounds (8cꢀf and 9cꢀf) contained other secondary amines. We hypothesized that dimethylamine or
diethylamine unit may contribute to high binding affinity. However, the antiproliferative activity of the compounds (8aꢀf
and 9aꢀf) was worse than the lead compound afatinib. It may be that the ability of the compound to bind to EGFR kinase
is reduced, after forms the oxazole ring at the Cꢀ6 and Cꢀ7 position of quinazoline.
In Table 2, the antiproliferative activity results indicated that replacement of the oxazole ring by imidazole ring
could maintained the antiproliferative activity. In addition, it can be seen that the compounds substituted with hydrogen
or methyl at R1 of imidazole ring showed slightly influence on the antiproliferative activity. However, the compounds
was substituted by alcoholic hydroxyl group at R2 of imidazole ring have significantly increased the antiproliferative
activity relative to substituted by methyl group. We speculated that the alcoholic hydroxyl group may increase the ability
to form hydrogen bonds with amino acid residues and advantageous for the pharmacokinetics of the compound in vitro.
In Table 3, the results suggested that the compounds (12aꢀd and 13aꢀi) substituted with secondary amines were
beneficial to the antiproliferative activity. We hypothesized that secondary amines may contribute to the increasing of
waterꢀsoluble and high binding affinity. On the whole, the length of the flexible chain at imidazole ring showed slightly
influence of the antiproliferative activity. What’s more, compound 12a showed the best antiproliferative activity against
the tested three human cancer cell lines. The results of the dosageꢀdependent effects on cell lines were showed in Figs. 3.
It was found that compound 12a exerted antiproliferative effects in a dosageꢀdependent manner and the IC50 values of
compound 12a were 1.90 µM, 2.23 µM and 2.03 µM against A549, MCFꢀ7 and PCꢀ3, which were equal to more active
than afatinib (1.07 µM, 1.33 µM and 2.68 µM), respectively.
Experiments against EGFR kinases of four selected compounds (8a, 9d, 10a and 12a) were carried out in this paper
for further study. In Table 4, obviously, it was found that the activity against EGFR kinase of compound 12a was better
than the other compounds, and the IC50 values of compound 12a was 32 nM against EGFR kinase. On the whole, it was
found that the overall activity against EGFR kinase of the selected compounds was a little bit lower than the reference
compound afatinib. In a word, the results prompted us that this series of compounds may be a kind of potential EGFR
kinase inhibitors.
3.2. Apoptosis result analyzing
To further explain the inhibition of cell growth, we first evaluated the effect of compound 12a induced apoptosis on
A549 cells using AnnexinVꢀFITC and propidium iodide (PI) double staining by flow cytometry, which stained

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phosphatidylserine residues and DNA, respectively. The results were shown in Fig. 4. As can be seen, compared with the
control group, the number of early apoptotic cells of afatinib and compound 12a was increased, the number of late
apoptotic and death cells was increased as well. The number of mechanical damage cells was within the error range,
which claimed that our data was credible. In addition, the number of early apoptotic cells of compound 12a was equal to
the number of afatinib. What’s more, the number of early apoptotic and late apoptotic and death cells (34.56%) was same
with the number of afatinib (33.32%). It could be concluded that the target compound 12a can induce apoptosis of A549
and it have the same effect as afatinib.
Fig. 4. Representative dot plot of Annexin V (xꢀaxis) versus PI (yꢀaxis) analyses. Q2ꢀ1: Mechanical damage; Q2ꢀ2: Late
apoptotic and death; Q2ꢀ3: Living cells; Q2ꢀ4: Early apoptotic.
3.3 Morphology of cells by fluorescence microscopy
To further confirm the induction of apoptosis, we monitored the morphological changes of A549 cells induced by
compound 12a by acridine orange single staining. After exposure to a concentration of 1.90 ꢁM of compound 12a for 12
h, cell morphology was investigated using fluorescence microscopy. As reflected in Fig. 5a, it can be observed that the
control cells showing the nucleus of A549 cells were stained into green from fluorescence microscopy, the cells grew
well, the edges were clear and the refraction was great in the control group. However, when the cells were treated with
compound 12a, it can be seen in 5b, the A549 cells showed significant evidence of apoptosis mediated cell death,
including that the nuclei were dyed red, and cells shrunk and chromatin condensed. It suggested that compound 12a
could induce the cellular apoptosis, which was consistent with the former result of antiproliferative activity assay.

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Fig. 5 Morphologic changes of A549 cells under inverted microscopy and fluorescence Microscopy. (5a) The control
group cell treated with nothing; (5b) Experimental group treated with 1.90 ꢁM concentration of compound 12a.
3.4 Molecular docking study
To explore the binding modes of target compounds with the active site of EGFR, molecular docking simulation
studies were carried out by using SURFLEXꢀDOCK module of SYBYL package version. Based on the inhibition results,
we selected 12a, our best EGFR inhibitor in this study, as ligand example, and the structure of EGFR was selected as the
docking model (PDB ID code :4G5P).
Fig. 6. Molecular docking analysis was carried out by the SYBYL to explore the binding model for the active site of
EGFR with its ligand. Docking simulations show that the compound 12a occupy the ATP site of EGFR kinase (Fig. 6a).
Binding model of afatinib and compound 12a with EGFR (Fig. 6b). The proteins were displayed by colors ribbon.
Compound 12a and afatinib were displayed by white and wine red sticks, respectively. Hꢀbonding interactions between
the 12a, afatinib and EGFR were indicated with dashed lines in yellow.
The binding modes of afatinib and compound 12a were depicted in Fig. 6. Visual inspection of the pose of the
compound 12a was tightly embedded into the active binding pocket in Fig. 6a. In Fig. 6b, compound 12a and afatinib

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can nearly overlap in the binding model. In addition, compound 12a and afatinib can formed hydrogen bonds with
residues LEUꢀ861 and LYSꢀ949. On the one hand, the oxygen atoms in the amide bond moiety of afatinib formed one
hydrogen bond with the amino hydrogen of LYSꢀ949. On the other hand, the nitrogen atoms in the tetrahydropyrrole
moiety of compound 12a formed one hydrogen bond with the amino hydrogen of LYSꢀ949. It is obviously that
compound 12a and afatinib have slightly different way of the active binding site of EGFR. It may be the reason that the
antiproliferative activity of the compounds (12aꢀd and 13aꢀi) was worse than the lead compound afatinib. In general, the
results suggested that compound 12a may be a potential inhibitor of EGFR. Analysis of the binding mode in the active
binding site demonstrated that the docking mode of the 12a was similar to the afatinib in the respect of binding site. The
Fig. 6b showed that compound 12a and afatinib can bind to the ATPꢀbinding pocket of the EGFR protein. Combining
with the antiproliferative activity results of compound 12a, whose antiproliferative activity against the tested three
human cancer cell lines was equal to or even higher than that of afatinib, the results suggested that the results of
molecular docking and antiꢀproliferative activity were consistent. The aboveꢀmentioned results of structureꢀactivity
relationships analysis and molecular docking study may allow the rational design of more potent EGFR inhibitors.
4. Conclusions
six series of quinazoline derivatives bearing oxazole or imidazole moiety (8a–f, 9a–f, 10a–d, 11a–f, 12a–d and
13a–i) were designed, synthesized and evaluated for the IC50 values against three cancer cell lines (A549, MCFꢀ7 and
PCꢀ3). Most of the thirty five target compounds showed excellent antiproliferative activity against the different cancer
cells. Notably, compound 12a showed the best activity against A549, MCFꢀ7 and PCꢀ3 cancer cell lines and exerted
antiproliferative effects in a dosageꢀdependent manner. The antiproliferative activity data showed that the six series of
compounds possessed more selectivity for A549 cell line than MCFꢀ7 and PCꢀ3 cell lines. That meant these compounds
had better treatment effect on lung cancer. On the whole, the compounds (12aꢀd and 13aꢀi) substituted with secondary
amines were beneficial to the antiproliferative activity. What’s more, the compound 12a could induce apoptosis of A549
cells and it’s binding model to the EGFR was similar to afatinib in the respect of Hꢀbond. Four selected compounds (8a,
9d, 10a and 12a) were evaluated for the inhibitory activity against EGFR kinases and all of them exhibited excellent
activity with the IC50 values in nanomole level. The results prompted us that compounds 8a, 9d, 10g and 12a may be
potential EGFR kinase inhibitors.
5 Experimental
5.1. Chemistry
All melting points were obtained on a Büchi Melting Point Bꢀ540 apparatus (Büchi Labortechnik, Flawil,
Switzerland) and were uncorrected. NMR spectra were performed using Bruker 400 MHz spectrometers (Bruker

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Bioscience, Billerica, MA, USA) with TMS as an internal standard. Mass spectra (MS) were taken in electrospray
ionization (ESI) mode on Agilent 1100 LC–MS (Agilent, Palo Alto, CA, USA). Thin layer chromatography (TLC)
analysis was carried out on silica gel plates GF254 (Qindao Haiyang Chemical, China). All the materials were obtained
from commercial suppliers and used without purification, unless otherwise specified. Yields were not optimized.
5.2 Preparation of 4ꢀchloroꢀ7ꢀfluoroꢀ6ꢀnitroquinazoline (3)
Compound 3 was synthesized according to the procedures in our previous research15.
5.3 Preparation of 4ꢀ(2,3ꢀdihydroꢀindolꢀ1ꢀyl)ꢀ7ꢀfluoroꢀ6ꢀnitroꢀquinazoline (4a)
To the solution of compound 3 (20.0 g, 0.088 mol), isopropanol (200 mL), triethylamine (14 mL),
2,3ꢀdihydroꢀindole(12.6 g, 0.106 mol) was added at room temperature. Then the reaction mixture was stirred at room
temperature for 1.5 h. After completion of reaction as indicated by TLC, the mixture was filtered, washed with
isopropanol and water, dried to compound 4a as orange solid (26.0 g, 95.4%). m.p. 261.4ꢀ262.5 °C. ESIꢀMS m/z:
[M+H]+311.2. 1H NMR (400 MHz, CDCl3) δ 9.02 (d, J = 8.1 Hz, 1H), 8.74 (s, 1H), 7.90 (d, J = 8.1 Hz, 1H), 7.85 (d, J =
12.5 Hz, 1H), 7.36 (d, J = 7.3 Hz, 1H), 7.20 (t, J = 7.8 Hz, 1H), 7.08 (t, J = 7.4 Hz, 1H), 4.59 (t, J = 7.8 Hz, 2H), 3.21 (t,
J = 7.7 Hz, 2H).
5.4 Preparation of 4ꢀ(3,4ꢀdihydroquinolinꢀ1(2H)ꢀyl)ꢀ7ꢀfluoroꢀ6ꢀnitroquinazoline (4b)
To the solution of compound 3 (20.0 g, 0.088 mol), isopropanol (160 mL), triethylamine (14 mL),
1,2,3,4ꢀtetrahydroquinoline (14.1 g, 0.106 mol) was added at room temperature. Then the reaction mixture was stirred at
room temperature for 1.5 h. After completion of reaction as indicated by TLC, the mixture was filtered, washed with
isopropanol and water, dried to compound 4b as orange solid (26.2 g, 92.1%). m.p. 252.4ꢀ254.6 °C. ESIꢀMS m/z:
[M+H]+324.3. 1H NMR (400 MHz, DMSOꢀd6) δ 9.04 (s, 1H), 7.98 (s, 1H), 7.39 (d, J = 7.4 Hz, 1H), 7.21 (t, J = 7.4 Hz,
1H), 7.05 (t, J = 7.6 Hz, 1H), 6.97 (s, 1H), 6.93 (d, J = 8.0 Hz, 1H), 4.15 (t, J = 6.3 Hz, 2H), 2.92 (t, J = 6.6 Hz, 2H), 2.09
(p, J = 6.5 Hz, 2H).
5.5 Preparation of 4ꢀ(indolinꢀ1ꢀyl)ꢀ6ꢀnitroquinazolinꢀ7ꢀol (5a)
To the mixture of compound 4a (5.0 g, 16.1 mmol), DMSO (100 mL) and 50% KOH (5 mL) was added at room
temperature. The reaction mixture was heated to 80 °C. Then the reaction mixture was refluxed for 3 h and monitored by
TLC. Then the mixture was poured into water and stirred for 30 minutes, yielding a precipitate. Then the mixture was
separated by filtration and washed with water, dried to afford compound 5a as yellow solid (4.1 g, 82.0%). m.p.
188.2ꢀ189.7 °C. ESIꢀMS m/z: [M+H]+309.1.

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5.6 Preparation of 4ꢀ(3,4ꢀdihydroquinolinꢀ1(2H)ꢀyl)ꢀ6ꢀnitroquinazolinꢀ7ꢀol (5b)
The synthetic method of compound 5b was the same to compound 5a. Compound 5a was obtained as yellow solid.
m.p. 182.4ꢀ184.5 °C. ESIꢀMS m/z: [M+H]+323.2.
5.7 Preparation of 2ꢀ((4ꢀ(indolinꢀ1ꢀyl)ꢀ6ꢀnitroquinazolinꢀ7ꢀyl)amino)ethanꢀ1ꢀol (5c)
To the mixture of compound 4a (5.0 g, 16.1 mmol), isopropanol (100 mL) and 40% methylamine (32.2 mmol) was
added at room temperature. The reaction mixture was heated to 80 °C and pressurized with nitrogen balloon. Then the
reaction mixture was refluxed for 2 h and monitored by TLC. The reaction solution was concentrated under reduced
pressure and poured into water, yielding a precipitate. Then the mixture was separated by filtration and washed with
water, dried to afford compound 5c as yellow solid (4.1 g, 72.4%). m.p. 202.8ꢀ204.6 °C. ESIꢀMS m/z: [M+H]+352.2.
5.8 Preparation of 3ꢀ((4ꢀ(indolinꢀ1ꢀyl)ꢀ6ꢀnitroquinazolinꢀ7ꢀyl)amino)propanꢀ1ꢀol (5d)
The synthetic method of compound 5d was the same to compound 5c. Compound 5d was obtained as yellow solid.
m.p. 194.1ꢀ195.4 °C. ESIꢀMS m/z: [M+H]+366.2.
5.9 Preparation of 4ꢀ(3,4ꢀdihydroquinolinꢀ1(2H)ꢀyl)ꢀNꢀmethylꢀ6ꢀnitroquinazolinꢀ7ꢀamine (5e)
The synthetic method of compound 5e was the same to compound 5c. Compound 5e was obtained as yellow solid.
m.p. 176.3ꢀ177.9 °C. ESIꢀMS m/z: [M+H]+335.4.
5.10 Preparation of 2ꢀ((4ꢀ(3,4ꢀdihydroquinolinꢀ1(2H)ꢀyl)ꢀ6ꢀnitroquinazolinꢀ7ꢀyl)amino)ethanꢀ1ꢀol (5f)
The synthetic method of compound 5f was the same to compound 5c. Compound 5f was obtained as yellow solid.
m.p. 173.6ꢀ175.2 °C. ESIꢀMS m/z: [M+H]+366.4.
5.11 Preparation of 3ꢀ((4ꢀ(3,4ꢀdihydroquinolinꢀ1(2H)ꢀyl)ꢀ6ꢀnitroquinazolinꢀ7ꢀyl)amino)propanꢀ1ꢀol (5g)
The synthetic method of compound 5g was the same to compound 5c. Compound 5g was obtained as yellow solid.
m.p. 178.4ꢀ179.9 °C. ESIꢀMS m/z: [M+H]+380.4.
5.12 Preparation of 6ꢀaminoꢀ4ꢀ(indolinꢀ1ꢀyl)quinazolinꢀ7ꢀol (6a)
The solution of compound 5a (4.1 g, 13.3 mmol) and ethanol (300 mL) was stirred at 60 °C, an appropriate amount
of activated carbon (1.5 g) and ferric chloride (0.5 g) were added at room temperature, the mixture was heated to 80 °C
and 80 percent hydrazine hydrate (3.5 mL) was added to the solution. The reaction mixture was refluxed for 1.5 h and
monitored by TLC. Then the mixture was filtered and the precipitate was washed with ethanol. The filtrate was

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concentrated under reduce pressure and the residue was poured into water with stirred for 30 min. The precipitate was
filtered and dried to obtain compound 6a as yellow solid (3.0 g, 81.1%). m.p. 175.6ꢀ178.1 °C. ESIꢀMS m/z: [M+H]+
279.2 . 1H NMR (400 MHz, DMSOꢀd6) δ 10.90 (s, 1H), 8.45 (s, 1H), 7.26 (d, J = 6.7 Hz, 1H), 7.08 – 7.01 (m, 2H), 6.97
(s, 1H), 6.85 (d, J = 7.1 Hz, 2H), 5.41 (s, 2H), 4.22 (t, J = 7.5 Hz, 2H), 3.14 (d, J = 7.4 Hz, 2H).
5.13 Preparation of compounds 6bꢀg
The synthetic method of compounds 6bꢀg was the same to compound 6a. Compounds 6bꢀg as light yellow solid.
5.14 Preparation of 8ꢀ(indolinꢀ1ꢀyl)oxazolo[4,5ꢀg]quinazolineꢀ2ꢀthiol (7a)
To the solution of 6a (3.1 g, 11.2 mmol) are placed in 100 ml of ethanol and combined with CS2 (1.6 g, 28.0 mmol)
and 50%KOH (1.3 mL) at room temperature. The reaction mixture was heated to 60 °C and pressurized with nitrogen
balloon. The reaction mixture was refluxed for 1 h and monitored by TLC. Then the mixture was concentrated under
reduce pressure and the residue was poured into water with stirred for 30 min. Then the reaction mixture was adjusted to
pH = 5−6 with 2 N HCl, yielding a precipitate. The precipitate was filtered and dried to obtain compound 7a as yellow
1
solid (2.1 g, 58.9%). m.p. 189.2ꢀ191.7 °C. ESIꢀMS m/z: [M+H]+321.1. H NMR (400 MHz, DMSOꢀd6) δ 14.43 – 13.82
(m, 1H), 8.74 (s, 1H), 7.91 (s, 1H), 7.63 (s, 1H), 7.33 (s, 1H), 7.17 – 7.06 (m, 2H), 6.98 (s, 1H), 4.38 (s, 2H), 3.19 (s,
2H).
5.15 Preparation of 8ꢀ(3,4ꢀdihydroquinolinꢀ1(2H)ꢀyl)oxazolo[4,5ꢀg]quinazolineꢀ2ꢀthiol (7b)
The synthetic method of compounds 7b was the same to compound 7a. Compounds 7b as light yellow solid. m.p.
179.5ꢀ181.1 °C. ESIꢀMS m/z: [M+H]+335.4.
5.16 General procedure for the preparation of compounds 8a–f and 9a–f
The corresponding secondary amine (Dimethylamine, Diethylamine, Pyrrolidine, Piperidine, Nꢀmethyl piperazine,
Morpholine) (1.0 mmol) was added to a stirred mixture of Intermediates 7aꢀb (0.4 mmol) and K2CO3 (0.4 mmol) in
1,4ꢀdioxane (10 mL) at room temperature. The reaction mixture was heated to 100 °C for 4 h and monitored by TLC.
Then the mixture was concentrated under reduce pressure and the residue was poured into water with stirred for 30 min,
yielding a precipitate. The precipitate was filtered and dried to obtain compounds 8a–f and 9a–f as white solid
.
5.17 General procedure for the preparation of compounds 10a–d and 11a–f
To the solution of 6cꢀg (0.6 mmol) are placed in 10 ml of formic acid at room temperature. The reaction mixture
was heated to 100 °C for 4 h and monitored by TLC. Then the mixture was concentrated under reduce pressure and the

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residue was poured into water with stirred for 30 min. The aqueous layers were adjusted to pH = 8 with 40% ammonia
water, yielding a precipitate. The precipitate was filtered and dried to obtain compounds 10a–b and 11a–c as white solid
.
To the solution of 6cꢀg (0.6 mmol) are placed in 10 ml of 1,4ꢀdioxane and combined with triethyl orthoacetate (1.2 mmol)
and acetic acid (2 d) at room temperature. The reaction mixture was heated to 100 °C for 4 h and monitored by TLC.
Then the mixture was concentrated under reduce pressure and the residue was poured into water with stirred for 30 min,
yielding a precipitate. The precipitate was filtered and dried to obtain compounds 10c–d and 11d–f as white solid
.
5.18 Preparation of compounds 7c–f
Using target compounds 10a, 10b, 11b and 11c as the key intermediates. To the solution of intermediates 10aꢀb and
11bꢀc (2.0 mmol) are placed in 10 ml of SOCl2 and combined with DMF (2 d) at room temperature. The reaction mixture
was heated to 90 °C for 4 h and monitored by TLC. Then the mixture was concentrated under reduce pressure and the
residue was poured into 10% K2CO3 (50 mL), the mixture was extracted with EtOAc (3 × 50 mL) three times and
saturated aqueous NaCl (50 mL × 1), and the organic phase was separated, dried, and evaporated to yield 7c–f
which were purified by dichloromethane.
5.19 General procedure for the preparation of compounds 12a–d and 13a–i
The corresponding secondary amine (Diethylamine, Pyrrolidine, Piperidine, Nꢀmethyl piperazine, Morpholine) (1.0
mmol) was added to a stirred mixture of intermediates 7c–f (0.4 mmol), KI (0.4 mmol) and K2CO3 (0.6 mmol) in
1,4ꢀdioxane (10 mL) at room temperature. The reaction mixture was heated to 100 °C for 4 h and monitored by TLC.
Then the mixture was concentrated under reduce pressure and the residue was poured into water with stirred for 30 min,
yielding a precipitate. The precipitate was filtered and dried to obtain compounds 12a–d and 13a–i as white solid
.
8-(indolin-1-yl)-N,N-dimethyloxazolo[4,5-g]quinazolin-2-amine (8a)
1
White solid in 62% yield. m.p. 180.3ꢀ182.7 °C. ESIꢀMS m/z: [M+H]+ 332.1. H NMR (400 MHz, CDCl3) δ 8.76
(s, 1H), 7.81 (s, 1H), 7.74 (s, 1H), 7.26 (d, J = 3.2 Hz, 1H), 7.09 (q, J = 8.0 Hz, 2H), 6.93 (td, J = 7.2, 1.4 Hz, 1H), 4.40 (t,
J = 7.9 Hz, 2H), 3.28 (s, 6H), 3.20 (t, J = 7.9 Hz, 2H).
8-(indolin-1-yl)-N,N-diethyloxazolo[4,5-g]quinazolin-2-amine (8b)
White solid in 68% yield. m.p. 187.1ꢀ189.5 °C. 1H NMR (400 MHz, DMSOꢀd6) δ 8.60 (s, 1H), 7.73 (s, 1H), 7.62 (s,
1H), 7.26 (s, 1H), 7.04 (d, J = 9.0 Hz, 2H), 6.89 (s, 1H), 4.33 (s, 2H), 3.53 (t, J = 16.9 Hz, 4H), 3.12 (s, 2H), 1.20 (s, 6H);
13C NMR (101 MHz, DMSOꢀd6) δ 163.79, 160.08, 153.58, 152.10, 148.49, 145.74, 143.91, 133.31, 126.77, 125.45,
122.38, 115.16, 114.98, 108.46, 105.83, 54.29, 43.43(2C), 29.10, 13.79(2C). HRMS (ESI): m/z: [M+H]+ calcd for
C21H21N5O, 360.1746; found 360.1758.

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8-(indolin-1-yl)-2-(pyrrolidin-1-yl)oxazolo[4,5-g]quinazoline (8c)
1
White solid in 63% yield. m.p. 201.2ꢀ202.5 °C. ESIꢀMS m/z: [M+H]+ 358.2. H NMR (400 MHz, DMSOꢀd6) δ
8.76 (s, 1H), 7.81 (s, 1H), 7.73 (s, 1H), 7.24 (s, 1H), 7.08 (dd, J = 19.2, 7.8 Hz, 2H), 6.92 (t, J = 7.1 Hz, 1H), 4.39 (t, J =
7.7 Hz, 2H), 3.72 (s, 4H), 3.20 (t, J = 7.7 Hz, 2H), 2.08 (s, 4H).
8-(indolin-1-yl)-2-(piperidin-1-yl)oxazolo[4,5-g]quinazoline (8d)
1
White solid in 68% yield. m.p. 179.9ꢀ182.1 °C. ESIꢀMS m/z: [M+H]+ 372.1. H NMR (400 MHz, CDCl3) δ 8.75
(s, 1H), 7.79 (s, 1H), 7.74 (s, 1H), 7.27 (d, J = 3.0 Hz, 1H), 7.15 – 7.06 (m, 2H), 6.94 (t, J = 7.3 Hz, 1H), 4.40 (t, J = 7.9
Hz, 2H), 3.74 (s, 4H), 3.20 (t, J = 7.8 Hz, 2H), 1.73 (s, 6H).
8-(indolin-1-yl)-2-(4-methylpiperazin-1-yl)oxazolo[4,5-g]quinazoline (8e)
1
White solid in 67% yield. m.p. 185.6ꢀ187.3 °C, ESIꢀMS m/z: [M+H]+ 387.1. H NMR (400 MHz, DMSOꢀd6) δ
8.67 (s, 1H), 7.72 (s, 1H), 7.64 (s, 1H), 7.16 (s, 1H), 6.98 (d, J = 6.0 Hz, 2H), 6.85 – 6.81 (m, 1H), 4.30 (t, J = 7.9 Hz,
2H), 3.73 – 3.69 (m, 4H), 3.10 (t, J = 7.9 Hz, 2H), 2.48 – 2.45 (m, 4H), 2.27 (s, 3H).
8-(indolin-1-yl)-2-morpholinooxazolo[4,5-g]quinazoline (8f)
1
White solid in 62% yield. m.p. 188.5ꢀ189.8 °C, ESIꢀMS m/z: [M+H]+ 374.1. H NMR (400 MHz, CDCl3) δ 8.75
(s, 1H), 7.83 (s, 1H), 7.76 (s, 1H), 7.24 (s, 1H), 7.07 (q, J = 8.1 Hz, 2H), 6.93 (t, J = 7.1 Hz, 1H), 4.39 (t, J = 7.9 Hz, 2H),
3.85 – 3.82 (m, 4H), 3.79 – 3.75 (m, 4H), 3.19 (t, J = 7.9 Hz, 2H).
8-(3,4-dihydroquinolin-1(2H)-yl)-N,N-dimethyloxazolo[4,5-g]quinazolin-2-amine (9a)
1
White solid in 69% yield. m.p. 178.1ꢀ180.3 °C, ESIꢀMS m/z: [M+H]+ 346.1. H NMR (400 MHz, CDCl3) δ 8.86
(s, 1H), 7.71 (s, 1H), 7.42 (s, 1H), 7.17 (d, J = 7.5 Hz, 1H), 6.95 – 6.87 (m, 2H), 6.56 (d, J = 7.7 Hz, 1H), 4.01 (t, J = 6.2
Hz, 2H), 3.70 (s, 2H), 3.25 (d, J = 15.3 Hz, 6H), 2.92 (t, J = 6.7 Hz, 2H), 2.13 (dt, J = 13.1, 6.6 Hz, 2H).
8-(3,4-dihydroquinolin-1(2H)-yl)-N,N-diethyloxazolo[4,5-g]quinazolin-2-amine (9b)
1
White solid in 63% yield. m.p. 193.4ꢀ195.5 °C. H NMR (400 MHz, DMSOꢀd6) δ 8.74 (s, 1H), 7.75 (s, 1H), 7.21
(d, J = 6.1 Hz, 1H), 7.10 (s, 1H), 6.89 (dd, J = 16.5, 6.2 Hz, 2H), 6.42 (d, J = 6.6 Hz, 1H), 3.90 (s, 2H), 3.53 (s, 4H), 2.87
(s, 2H), 2.02 (s, 2H), 1.17 (s, 6H); 13C NMR (101 MHz, DMSOꢀd6) δ 163.83, 162.18, 153.41, 152.71, 148.40, 144.17,
143.17, 129.48(2C), 126.53, 122.80, 120.11, 116.34, 108.66, 106.05, 48.31, 43.41(2C), 26.93, 24.02, 13.78(2C). HRMS
(ESI): m/z: [M+H]+ calcd for C22H23N5O, 374.1903; found 374.1912.
8-(3,4-dihydroquinolin-1(2H)-yl)-2-(pyrrolidin-1-yl)oxazolo[4,5-g]quinazoline (9c)
1
White solid in 62% yield. m.p. 191.2ꢀ192.5 °C, ESIꢀMS m/z: [M+H]+ 372.1. H NMR (400 MHz, CDCl3) δ 8.85
(s, 1H), 7.71 (s, 1H), 7.43 (s, 1H), 7.17 (d, J = 6.8 Hz, 1H), 6.95 – 6.86 (m, 2H), 6.56 (d, J = 7.6 Hz, 1H), 4.01 (t, J = 6.2
Hz, 2H), 3.68 (s, 4H), 2.92 (t, J = 6.6 Hz, 2H), 2.13 (dt, J = 13.1, 6.5 Hz, 2H), 2.06 (t, J = 6.5 Hz, 4H).
8-(3,4-dihydroquinolin-1(2H)-yl)-2-(piperidin-1-yl)oxazolo[4,5-g]quinazoline (9d)
1
White solid in 67% yield. m.p. 194.1ꢀ195.8 °C, ESIꢀMS m/z: [M+H]+ 386.1. H NMR (400 MHz, CDCl3) δ 8.85
(s, 1H), 7.69 (s, 1H), 7.40 (s, 1H), 7.17 (d, J = 7.3 Hz, 1H), 6.95 – 6.87 (m, 2H), 6.57 (d, J = 7.5 Hz, 1H), 4.01 (t, J = 6.2
Hz, 2H), 3.70 (s, 4H), 2.92 (t, J = 6.6 Hz, 2H), 2.17 – 2.08 (m, 2H), 1.70 (s, 6H).
8-(3,4-dihydroquinolin-1(2H)-yl)-2-(4-methylpiperazin-1-yl)oxazolo[4,5-g]quinazoline (9e)

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1
White solid in 63% yield. m.p. 200.2ꢀ202.8 °C, ESIꢀMS m/z: [M+H]+ 401.1. H NMR (400 MHz, DMSOꢀd6) δ
8.86 (s, 1H), 7.71 (s, 1H), 7.42 (s, 1H), 7.17 (d, J = 7.4 Hz, 1H), 6.96 – 6.86 (m, 2H), 6.57 (d, J = 7.8 Hz, 1H), 4.01 (t, J
= 6.2 Hz, 2H), 3.79 – 3.74 (m, 4H), 2.92 (t, J = 6.7 Hz, 2H), 2.55 – 2.51 (m, 4H), 2.35 (s, 3H), 2.12 (dd, J = 12.9, 6.4 Hz,
2H).
8-(3,4-dihydroquinolin-1(2H)-yl)-2-morpholinooxazolo[4,5-g]quinazoline (9f)
1
White solid in 65% yield. m.p. 203.5ꢀ204.8 °C, ESIꢀMS m/z: [M+H]+ 388.1. H NMR (400 MHz, DMSOꢀd6) δ
8.78 (s, 1H), 7.82 (s, 1H), 7.25 (d, J = 7.6 Hz, 1H), 7.15 (s, 1H), 6.94 (d, J = 7.2 Hz, 1H), 6.89 (d, J = 7.3 Hz, 1H), 6.45
(d, J = 7.8 Hz, 1H), 3.94 (d, J = 5.9 Hz, 2H), 3.71 (s, 4H), 3.64 (s, 4H), 2.90 (d, J = 5.9 Hz, 2H), 2.06 (d, J = 6.2 Hz, 2H).
2-(8-(indolin-1-yl)-3H-imidazo[4,5-g]quinazolin-3-yl)ethan-1-ol (10a)
1
White solid in 60% yield. m.p. 167.1ꢀ169.4 °C, ESIꢀMS m/z: [M+H]+ 332.1. H NMR (400 MHz, DMSOꢀd6) δ
8.69 (s, 1H), 8.59 (s, 1H), 8.41 (s, 1H), 8.19 (s, 1H), 7.34 (d, J = 7.2 Hz, 1H), 7.26 (d, J = 8.1 Hz, 1H), 7.10 (t, J = 7.8 Hz,
1H), 6.97 (t, J = 7.3 Hz, 1H), 4.71 (t, J = 4.7 Hz, 2H), 4.55 (t, J = 4.7 Hz, 2H), 4.49 (t, J = 7.8 Hz, 2H), 3.20 (t, J = 7.8 Hz,
2H).
3-(8-(indolin-1-yl)-3H-imidazo[4,5-g]quinazolin-3-yl)propan-1-ol (10b)
1
White solid in 55% yield. m.p. 181.3ꢀ182.5 °C, ESIꢀMS m/z: [M+H]+ 346.1. H NMR (400 MHz, DMSOꢀd6) δ
8.71 (s, 1H), 8.60 (s, 1H), 8.44 (s, 1H), 8.08 (s, 1H), 7.35 (t, J = 7.0 Hz, 2H), 7.12 (t, J = 7.7 Hz, 1H), 7.00 (t, J = 7.5 Hz,
1H), 4.79 (s, 1H), 4.53 (t, J = 7.9 Hz, 2H), 4.45 (t, J = 6.9 Hz, 2H), 3.45 (t, J = 6.0 Hz, 2H), 3.20 (t, J = 7.8 Hz, 2H), 2.02
(p, J = 6.4 Hz, 2H).
2-(8-(indolin-1-yl)-2-methyl-3H-imidazo[4,5-g]quinazolin-3-yl)ethan-1-ol (10c)
1
White solid in 62% yield. m.p. 180.2ꢀ181.8 °C, ESIꢀMS m/z: [M+H]+ 346.1. H NMR (400 MHz, DMSOꢀd6) δ
8.48 (s, 1H), 7.23 (d, J = 7.2 Hz, 1H), 7.01 (s, 1H), 6.96 (s, 1H), 6.87 – 6.82 (m, 1H), 6.77 (s, 1H), 5.62 (t, J = 5.5 Hz,
1H), 4.88 (s, 1H), 4.24 – 4.22 (m, 2H), 3.63 (t, J = 5.3 Hz, 2H), 3.29 (dd, J = 11.4, 5.7 Hz, 2H), 3.10 (t, J = 7.9 Hz, 2H),
1.88 (s, 3H); 13C NMR (101 MHz, DMSOꢀd6) δ 163.88, 158.75, 153.28, 151.95, 147.12, 146.31, 136.38, 132.97, 126.74,
125.38, 121.79, 114.07, 113.66, 108.95, 102.02, 62.22, 59.43, 53.75, 16.94, 14.57.
3-(8-(indolin-1-yl)-2-methyl-3H-imidazo[4,5-g]quinazolin-3-yl)propan-1-ol (10d)
1
White solid in 64% yield. m.p. 172.3ꢀ174.0 °C, ESIꢀMS m/z: [M+H]+ 360.1. H NMR (400 MHz, CDCl3) δ 8.73
(s, 1H), 8.35 (s, 1H), 7.96 (s, 1H), 7.30 – 7.24 (m, 2H), 7.07 (t, J = 7.7 Hz, 1H), 6.95 (t, J = 7.4 Hz, 1H), 4.47 (t, J = 7.9
Hz, 2H), 4.41 (t, J = 6.7 Hz, 2H), 4.23 (s, 1H), 3.71 (t, J = 5.4 Hz, 2H), 3.22 (t, J = 7.8 Hz, 2H), 2.72 (s, 3H), 2.14 (d, J =
6.1 Hz, 2H); 13C NMR (101 MHz, DMSOꢀd6) δ 160.84, 157.19, 151.96, 146.55, 144.60, 141.05, 140.07, 132.42, 126.22,
124.52, 122.14, 114.42, 113.91, 112.76, 104.91, 57.84, 53.99, 40.29, 31.29, 28.65, 13.80.
8-(3,4-dihydroquinolin-1(2H)-yl)-3-methyl-3H-imidazo[4,5-g]quinazoline (11a)
1
White solid in 63% yield. m.p. 188.2ꢀ190.5 °C. ESIꢀMS m/z: [M+H]+ 316.1. H NMR (400 MHz, DMSOꢀd6) δ
8.81 (d, J = 9.9 Hz, 1H), 8.43 (s, 1H), 8.01 (s, 1H), 7.78 (d, J = 10.3 Hz, 1H), 7.25 (d, J = 7.2 Hz, 1H), 6.94 (t, J = 7.2 Hz,
1H), 6.83 (t, J = 7.5 Hz, 1H), 6.45 (d, J = 8.0 Hz, 1H), 4.02 – 3.94 (m, 2H), 3.90 (d, J = 9.7 Hz, 3H), 2.90 (t, J = 6.4 Hz,
2H), 2.10 – 2.01 (m, 2H).

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2-(8-(3,4-dihydroquinolin-1(2H)-yl)-3H-imidazo[4,5-g]quinazolin-3-yl)ethan-1-ol (11b)
1
White solid in 60% yield. m.p. 166.7ꢀ168.3 °C, ESIꢀMS m/z: [M+H]+ 346.1. H NMR (400 MHz, DMSOꢀd6) δ
8.82 (s, 1H), 8.44 (s, 1H), 8.10 (s, 1H), 7.78 (s, 1H), 7.28 (d, J = 7.3 Hz, 1H), 6.97 (t, J = 7.3 Hz, 1H), 6.87 (t, J = 7.5 Hz,
1H), 6.50 (d, J = 8.0 Hz, 1H), 5.03 (s, 1H), 4.38 (s, 2H), 4.00 (t, J = 6.1 Hz, 2H), 3.79 (s, 2H), 2.93 (t, J = 6.3 Hz, 2H),
2.11 – 2.03 (m, 2H).
3-(8-(3,4-dihydroquinolin-1(2H)-yl)-3H-imidazo[4,5-g]quinazolin-3-yl)propan-1-ol (11c)
1
White solid in 64% yield. m.p. 186.2ꢀ188.4 °C, ESIꢀMS m/z: [M+H]+ 360.1. H NMR (400 MHz, DMSOꢀd6) δ
8.80 (s, 1H), 8.50 (s, 1H), 8.20 (s, 1H), 8.08 (s, 1H), 7.79 (s, 1H), 7.24 (d, J = 7.4 Hz, 1H), 6.94 (t, J = 7.4 Hz, 1H), 6.84
(t, J = 7.6 Hz, 1H), 6.50 (d, J = 8.0 Hz, 1H), 4.41 (t, J = 7.0 Hz, 2H), 4.12 (t, J = 6.2 Hz, 2H), 3.96 (t, J = 6.3 Hz, 2H),
2.89 (t, J = 6.6 Hz, 2H), 2.19 (p, J = 6.6 Hz, 2H), 2.04 (p, J = 6.5 Hz, 2H); 13C NMR (101 MHz, DMSOꢀd63) δ 162.67,
153.31, 150.39, 147.74, 143.46, 143.24, 139.21, 130.31, 129.63, 126.66, 123.34, 120.85, 116.10, 114.11, 107.33, 61.38,
48.54, 42.06, 28.87, 27.05, 24.23.
8-(3,4-dihydroquinolin-1(2H)-yl)-2,3-dimethyl-3H-imidazo[4,5-g]quinazoline (11d)
1
White solid in 67% yield. m.p. 192.4ꢀ194.6 °C, ESIꢀMS m/z: [M+H]+ 330.1. H NMR (400 MHz, DMSOꢀd6) δ
8.80 (s, 1H), 7.95 (s, 1H), 7.62 (s, 1H), 7.27 (d, J = 7.3 Hz, 1H), 6.95 (t, J = 7.3 Hz, 1H), 6.84 (t, J = 7.5 Hz, 1H), 6.43 (d,
J = 8.4 Hz, 1H), 3.98 (t, J = 6.3 Hz, 2H), 3.81 (s, 3H), 2.92 (t, J = 6.6 Hz, 2H), 2.55 (s, 3H), 2.07 (p, J = 6.5 Hz, 2H).
2-(8-(3,4-dihydroquinolin-1(2H)-yl)-2-methyl-3H-imidazo[4,5-g]quinazolin-3-yl)ethan-1-ol (11e)
1
White solid in 61% yield. m.p. 185.2ꢀ186.8 °C. H NMR (400 MHz, DMSOꢀd6) δ 8.82 (s, 1H), 7.96 (s, 1H), 7.60
(s, 1H), 7.28 (d, J = 7.4 Hz, 1H), 6.98 (t, J = 7.3 Hz, 1H), 6.87 (t, J = 7.5 Hz, 1H), 6.50 (d, J = 8.0 Hz, 1H), 5.01 (s, 1H),
4.33 (s, 2H), 4.00 (t, J = 5.8 Hz, 2H), 3.76 (s, 2H), 2.92 (t, J = 6.1 Hz, 2H), 2.58 (s, 3H), 2.11 – 2.04 (m, 2H); 13C NMR
(101 MHz, DMSOꢀd6) δ 162.30, 158.43, 151.46, 145.47, 142.15, 141.11, 139.93, 129.29, 128.41, 125.48, 122.32, 119.67,
113.02, 112.43, 105.11, 58.84, 47.33, 45.79, 25.84, 23.06, 13.64. HRMS (ESI): m/z: [M+H]+ calcd for C21H21N5O,
360.1780; found 360.1774.
3-(8-(3,4-dihydroquinolin-1(2H)-yl)-2-methyl-3H-imidazo[4,5-g]quinazolin-3-yl)propan-1-ol (11f)
1
White solid in 58% yield. m.p. 184.0ꢀ186.3 °C, ESIꢀMS m/z: [M+H]+ 374.1. H NMR (400 MHz, DMSOꢀd6) δ
8.81 (s, 1H), 7.97 (s, 1H), 7.63 (s, 1H), 7.28 (d, J = 7.4 Hz, 1H), 6.97 (t, J = 7.3 Hz, 1H), 6.87 (t, J = 7.5 Hz, 1H), 6.49 (d,
J = 8.0 Hz, 1H), 4.76 (s, 1H), 4.33 (t, J = 6.9 Hz, 2H), 3.98 (t, J = 6.3 Hz, 2H), 3.43 (d, J = 5.0 Hz, 2H), 2.92 (t, J = 6.5
Hz, 2H), 2.59 (s, 3H), 2.11 – 2.03 (m, 2H), 1.96 – 1.89 (m, 2H); 13C NMR (101 MHz, DMSOꢀd6) δ 162.28, 157.73,
151.76, 146.30, 142.28, 141.15, 139.66, 128.97, 128.37, 125.44, 121.98, 119.51, 113.01, 112.71, 108.28, 105.34, 56.96,
47.25, 31.22, 25.86, 23.04, 13.26.
8-(indolin-1-yl)-3-(2-(pyrrolidin-1-yl)ethyl)-3H-imidazo[4,5-g]quinazoline (12a)
1
White solid in 54% yield. m.p. 183.2ꢀ184.7 °C. H NMR (400 MHz, CDCl3) δ 8.74 (s, 1H), 8.49 (s, 1H), 8.24 (d,
J = 3.6 Hz, 1H), 7.91 (s, 1H), 7.24 (s, 1H), 7.23 – 7.19 (m, 1H), 7.06 (t, J = 7.7 Hz, 1H), 6.93 (t, J = 7.4 Hz, 1H), 4.46 (t,
J = 8.0 Hz, 2H), 4.39 (t, J = 6.5 Hz, 2H), 3.20 (t, J = 7.5 Hz, 2H), 3.02 (t, J = 6.5 Hz, 2H), 2.60 (s, 4H), 1.78 (s, 4H); 13
NMR (101 MHz, CDCl3) δ 161.40, 152.90, 148.40, 147.57, 145.04, 142.41, 138.69, 132.85, 126.65, 124.96, 122.63,
C

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116.26, 114.89, 113.45, 105.91, 54.58, 54.46, 54.18(2C), 44.28, 29.08, 23.53(2C). HRMS (ESI): m/z: [M+H]+ calcd for
C23H24N6, 385.2096; found 385.2090.
8-(indolin-1-yl)-3-(3-(pyrrolidin-1-yl)propyl)-3H-imidazo[4,5-g]quinazoline (12b)
1
White solid in 59% yield. m.p. 172.4ꢀ173.9 °C, ESIꢀMS m/z: [M+H]+ 399.1. H NMR (400 MHz, CDCl3) δ 8.68
(s, 1H), 8.58 (s, 1H), 8.39 (s, 1H), 8.11 (s, 1H), 7.34 (d, J = 7.2 Hz, 1H), 7.24 (d, J = 8.0 Hz, 1H), 7.09 (t, J = 7.7 Hz, 1H),
6.96 (t, J = 7.4 Hz, 1H), 4.48 (t, J = 7.9 Hz, 2H), 4.43 (t, J = 6.8 Hz, 2H), 3.20 (t, J = 8.2 Hz, 2H), 2.50 (s, 2H), 2.48 –
2.42 (m, 4H), 2.09 – 2.01 (m, 2H), 1.71 (s, 4H).
8-(indolin-1-yl)-3-(3-(piperidin-1-yl)propyl)-3H-imidazo[4,5-g]quinazoline (12c)
1
White solid in 51% yield. m.p. 168.2ꢀ170.2 °C, ESIꢀMS m/z: [M+H]+ 413.1. H NMR (400 MHz, DMSOꢀd6) δ
8.67 (s, 1H), 8.55 (s, 1H), 8.37 (s, 1H), 8.08 (s, 1H), 7.31 (d, J = 7.3 Hz, 1H), 7.20 (d, J = 7.9 Hz, 1H), 7.06 (t, J = 7.5 Hz,
1H), 6.94 (t, J = 7.1 Hz, 1H), 4.45 (t, J = 7.7 Hz, 2H), 4.39 (t, J = 6.4 Hz, 2H), 3.21 – 3.16 (m, 2H), 2.96 (d, J = 5.0 Hz,
2H), 2.32 (s, 4H), 2.03 (d, J = 6.1 Hz, 2H), 1.65 (s, 4H), 1.35 (s, 2H); 13C NMR (101 MHz, DMSOꢀd6) δ 160.31, 151.53,
149.20, 146.67, 144.62, 141.91, 138.40, 132.40, 125.64, 124.40, 121.58, 114.29(2C), 111.98, 105.92, 54.31, 53.46, 53.14,
43.04, 28.05, 25.35, 24.60, 23.24, 21.59, 21.15.
8-(indolin-1-yl)-3-(3-(4-methylpiperazin-1-yl)propyl)-3H-imidazo[4,5-g]quinazoline (12d)
1
White solid in 61% yield. m.p. 171.4ꢀ173.1 °C, ESIꢀMS m/z: [M+H]+ 428.1. H NMR (400 MHz, DMSOꢀd6) δ
8.67 (s, 1H), 8.57 (s, 1H), 8.37 (s, 1H), 8.09 (s, 1H), 7.32 (d, J = 7.4 Hz, 1H), 7.22 (d, J = 7.8 Hz, 1H), 7.10 – 7.04 (m,
1H), 6.94 (t, J = 7.2 Hz, 1H), 4.46 (t, J = 7.8 Hz, 2H), 4.40 (d, J = 5.9 Hz, 2H), 3.21 – 3.15 (m, 2H), 3.03 (s, 2H), 2.33
(dd, J = 19.8, 13.7 Hz, 8H), 2.20 (s, 3H), 2.00 (dd, J = 11.9, 5.6 Hz, 2H); 13C NMR (101 MHz, DMSOꢀd6) δ 161.45,
152.70, 150.39, 147.81, 145.75, 143.01, 139.55, 133.57, 126.81, 125.57, 122.77, 115.50(2C), 113.12, 107.07, 54.97,
54.77, 52.67, 52.11, 46.05, 45.86, 43.62, 42.97, 29.22, 26.72.
8-(3,4-dihydroquinolin-1(2H)-yl)-3-(2-(pyrrolidin-1-yl)ethyl)-3H-imidazo[4,5-g]quinazoline (13a)
1
White solid in 50% yield. m.p. 180.6ꢀ181.9 °C, ESIꢀMS m/z: [M+H]+ 399.1. H NMR (400 MHz, DMSOꢀd6) δ
8.81 (s, 1H), 8.50 (s, 1H), 8.09 (s, 1H), 7.79 (s, 1H), 7.27 (d, J = 5.6 Hz, 1H), 6.95 (d, J = 7.0 Hz, 1H), 6.87 (s, 1H), 6.51
(d, J = 6.9 Hz, 1H), 4.44 (s, 2H), 3.98 (s, 2H), 2.88 (t, J = 12.7 Hz, 6H), 2.72 (s, 2H), 2.06 (s, 2H), 1.63 (s, 4H); 13C NMR
(101 MHz, DMSOꢀd6) δ 162.84, 153.01, 150.52, 147.45, 143.22, 139.07, 130.02, 129.40, 128.94, 126.43, 126.21, 123.05,
120.56, 115.65, 107.18, 54.81, 53.93(2C), 48.28, 43.94, 26.81, 23.99, 23.57(2C).
8-(3,4-dihydroquinolin-1(2H)-yl)-3-(2-(piperidin-1-yl)ethyl)-3H-imidazo[4,5-g]quinazoline (13b)
1
White solid in 59% yield. m.p. 183.5ꢀ185.3 °C, ESIꢀMS m/z: [M+H]+ 413.1. H NMR (400 MHz, DMSOꢀd6) δ
8.79 (s, 1H), 8.44 (s, 1H), 8.07 (s, 1H), 7.76 (s, 1H), 7.23 (d, J = 6.8 Hz, 1H), 6.92 (d, J = 7.1 Hz, 1H), 6.89 – 6.82 (m,
1H), 6.48 (d, J = 8.1 Hz, 1H), 4.39 (d, J = 4.8 Hz, 2H), 3.94 (s, 2H), 2.88 (s, 2H), 2.63 (s, 2H), 2.34 (s, 4H), 2.02 (s, 2H),
1.38 (s, 4H), 1.30 (s, 2H); 13C NMR (101 MHz, DMSOꢀd6) δ 163.56, 153.07, 150.59, 147.54, 143.33, 139.26, 130.03,
129.48, 126.48, 126.27, 123.11, 120.61, 115.66, 113.89, 107.31, 57.81, 54.49(2C), 48.39, 42.45, 26.90, 26.09(2C), 24.43,
24.06. HRMS (ESI): m/z: [M+H]+ calcd for C25H28N6, 413.2409; found 413.2416.
8-(3,4-dihydroquinolin-1(2H)-yl)-3-(2-(4-methylpiperazin-1-yl)ethyl)-3H-imidazo[4,5-g]quinazoline (13c)

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1
White solid in 62% yield. m.p. 181.4ꢀ182.8 °C, ESIꢀMS m/z: [M+H]+ 428.1. H NMR (400 MHz, CDCl3) δ 8.82
(s, 1H), 8.47 (s, 1H), 8.11 (s, 1H), 7.80 (s, 1H), 7.26 (d, J = 7.1 Hz, 1H), 6.96 (t, J = 7.2 Hz, 1H), 6.87 (t, J = 7.7 Hz, 1H),
6.52 (d, J = 7.6 Hz, 1H), 4.43 (t, J = 9.5 Hz, 2H), 3.98 (s, 2H), 2.91 (d, J = 6.0 Hz, 2H), 2.72 (d, J = 5.4 Hz, 2H), 2.46 (s,
2H), 2.25 (d, J = 24.9 Hz, 4H), 2.12 (s, 3H), 2.09 – 2.00 (m, 4H); 13C NMR (101 MHz, DMSOꢀd6) δ 163.47, 153.00,
150.52, 147.49, 143.26, 139.14, 129.95, 129.39, 128.96, 126.40, 126.19, 123.01, 120.54, 115.58, 107.23, 56.87,
55.06(4C), 52.87, 48.29, 46.00, 26.82, 23.99.
4-(2-(8-(3,4-dihydroquinolin-1(2H)-yl)-3H-imidazo[4,5-g]quinazolin-3-yl)ethyl)morpholine (13d)
1
White solid in 56% yield. m.p. 187.0ꢀ188.6 °C, ESIꢀMS m/z: [M+H]+ 415.1. H NMR (400 MHz, CDCl3) δ 8.82
(s, 1H), 8.51 (s, 1H), 8.12 (s, 1H), 7.80 (s, 1H), 7.27 (d, J = 7.2 Hz, 1H), 6.97 (t, J = 7.3 Hz, 1H), 6.87 (t, J = 7.6 Hz, 1H),
6.51 (dd, J = 8.5, 3.4 Hz, 1H), 4.46 (t, J = 5.8 Hz, 2H), 3.99 (d, J = 6.0 Hz, 2H), 3.51 (s, 4H), 2.91 (d, J = 6.4 Hz, 2H),
2.73 (s, 2H), 2.45 (s, 4H), 2.09 – 2.04 (m, 2H); 13C NMR (101 MHz, CDCl3) δ 163.56, 153.11, 150.70, 147.56, 143.01,
139.23, 130.11, 129.51, 129.06, 126.52, 126.31, 123.16, 120.67, 115.70, 107.36, 66.73(2C), 57.44, 53.66(2C), 48.40,
41.89, 26.91, 24.09.
3-(8-(3,4-dihydroquinolin-1(2H)-yl)-3H-imidazo[4,5-g]quinazolin-3-yl)-N,N-diethylpropan-1-amine (13e)
1
White solid in 58% yield. m.p. 179.2ꢀ181.6 °C, ESIꢀMS m/z: [M+H]+ 415.1. H NMR (400 MHz, CDCl3) δ 8.81
(s, 1H), 8.51 (s, 1H), 8.07 (s, 1H), 7.80 (s, 1H), 7.26 (d, J = 7.2 Hz, 1H), 6.95 (dd, J = 15.5, 8.2 Hz, 1H), 6.87 (d, J = 7.3
Hz, 1H), 6.51 (d, J = 6.2 Hz, 1H), 4.38 (dd, J = 26.9, 6.0 Hz, 2H), 3.98 (s, 2H), 2.90 (d, J = 9.9 Hz, 4H), 2.43 (dd, J =
32.9, 26.2 Hz, 4H), 2.05 (d, J = 6.2 Hz, 2H), 2.01 – 1.93 (m, 2H), 0.89 (t, J = 6.4 Hz, 6H); 13C NMR (101 MHz, CDCl3)
δ 163.54, 153.13, 150.41, 147.57, 143.33, 143.15, 139.18, 130.10, 129.47, 126.51, 123.13, 120.68, 115.83, 113.88,
107.26, 58.07, 49.62, 48.36, 46.54(2C), 32.68, 26.91, 24.09, 11.94(2C).
8-(3,4-dihydroquinolin-1(2H)-yl)-3-(3-(pyrrolidin-1-yl)propyl)-3H-imidazo[4,5-g]quinazoline (13f)
1
White solid in 60% yield. m.p. 178.5ꢀ179.9 °C, ESIꢀMS m/z: [M+H]+ 413.1. H NMR (400 MHz, DMSOꢀd6) δ
8.81 (s, 1H), 8.51 (s, 1H), 8.07 (s, 1H), 7.79 (s, 1H), 7.35 – 7.24 (m, 1H), 6.95 (dd, J = 14.8, 7.4 Hz, 1H), 6.87 (d, J = 8.1
Hz, 1H), 6.51 (d, J = 7.4 Hz, 1H), 4.40 (dd, J = 13.5, 6.4 Hz, 2H), 3.98 (s, 2H), 2.97 – 2.71 (m, 4H), 2.42 – 2.22 (m, 4H),
2.05 (d, J = 6.2 Hz, 2H), 2.03 – 1.87 (m, 4H), 1.66 (s, 2H); 13C NMR (101 MHz, DMSOꢀd6) δ 163.63, 153.20, 150.50,
147.64, 143.40, 130.14, 129.55, 129.11, 126.57, 123.20, 120.73, 115.90, 113.97, 107.33, 58.16, 53.98, 52.79, 48.44,
32.74, 28.91, 26.98, 24.16, 23.74(2C).
8-(3,4-dihydroquinolin-1(2H)-yl)-3-(3-(piperidin-1-yl)propyl)-3H-imidazo[4,5-g]quinazoline (13g)
1
White solid in 64% yield. m.p. 167.9ꢀ169.3 °C, ESIꢀMS m/z: [M+H]+ 427.1. H NMR (400 MHz, CDCl3) δ 8.81
(s, 1H), 8.51 (s, 1H), 8.08 (s, 1H), 7.80 (s, 1H), 7.27 (d, J = 7.3 Hz, 1H), 6.96 (t, J = 7.3 Hz, 1H), 6.88 (dd, J = 11.9, 5.9
Hz, 1H), 6.52 (d, J = 4.9 Hz, 1H), 4.43 – 4.34 (m, 2H), 3.99 (s, 2H), 2.91 (dd, J = 12.6, 5.7 Hz, 4H), 2.29 – 2.09 (m, 4H),
2.09 – 2.04 (m, 2H), 2.01 – 1.96 (m, 2H), 1.48 (t, J = 61.6 Hz, 6H); 13C NMR (101 MHz, CDCl3) δ 163.55, 153.13,
150.49, 150.43, 143.35, 143.15, 139.31, 129.49(2C), 126.52, 123.13, 120.69, 115.84, 113.89, 107.47, 107.28, 58.08,
55.60, 54.44(2C), 48.38, 32.68, 26.92, 26.06(2C), 24.67, 24.10.
8-(3,4-dihydroquinolin-1(2H)-yl)-3-(3-(4-methylpiperazin-1-yl)propyl)-3H-imidazo[4,5-g]quinazoline (13h)

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1
White solid in 57% yield. m.p. 174.3ꢀ176.3 °C, ESIꢀMS m/z: [M+H]+ 442.1. H NMR (400 MHz, CDCl3) δ 8.87
(s, 1H), 8.59 (s, 1H), 8.14 (s, 1H), 7.86 (s, 1H), 7.33 (d, J = 8.0 Hz, 1H), 7.02 (t, J = 7.2 Hz, 1H), 6.92 (s, 1H), 6.56 (d, J
= 8.1 Hz, 1H), 4.04 (s, 2H), 2.98 (s, 2H), 2.94 (s, 2H), 2.77 (d, J = 11.2 Hz, 2H), 2.26 (d, J = 21.4 Hz, 8H), 2.17 (d, J =
4.6 Hz, 3H), 2.11 (d, J = 6.2 Hz, 2H), 2.03 (s, 2H).
4-(3-(8-(3,4-dihydroquinolin-1(2H)-yl)-3H-imidazo[4,5-g]quinazolin-3-yl)propyl)morpholine (13i)
1
White solid in 54% yield. m.p. 184.7ꢀ186.5 °C, ESIꢀMS m/z: [M+H]+ 429.1. H NMR (400 MHz, DMSOꢀd6) δ
8.81 (s, 1H), 8.53 (s, 1H), 8.08 (s, 1H), 7.80 (s, 1H), 7.35 – 7.25 (m, 1H), 6.96 (t, J = 7.2 Hz, 1H), 6.93 – 6.83 (m, 1H),
6.51 (t, J = 6.3 Hz, 1H), 4.40 (dd, J = 12.9, 6.2 Hz, 2H), 3.98 (s, 2H), 3.52 (dd, J = 34.5, 12.8 Hz, 4H), 2.90 (d, J = 11.0
Hz, 4H), 2.73 (s, 2H), 2.23 (d, J = 6.4 Hz, 2H), 2.08 – 1.97 (m, 4H); 13C NMR (101 MHz, DMSOꢀd6) δ 162.93, 153.19,
150.49, 147.63, 143.41, 131.82, 130.17, 129.55, 129.11, 126.58, 123.21, 120.74, 115.90, 113.98, 107.32, 66.70, 58.14,
53.74, 48.44, 36.39, 32.74, 31.38, 26.98, 24.16, 23.58.
5.23 antiproliferative assay
The cytotoxic activities of target compounds (8a–f, 9a–f, 10a–d, 11a–f, 12a–d and 13a–i) were evaluated with
A549, MCFꢀ7 and PCꢀ3 cancer cell lines by the standard MTT assay, with compounds EGFR inhibitors afatinib as
positive control. The cancer cell lines were cultured in minimum essential medium (MEM) supplement with 10% fetal
bovine serum (FBS). Approximately 4×103 cells, suspended in MEM medium, were plated onto each well of a 96ꢀwell
plate and incubated in 5% CO2 at 37 °C for 24 h. The test compounds at indicated final concentrations were added to the
culture medium and the cell cultures were continued for 72 h. Fresh MTT was added to each well at a terminal
concentration of 5ꢁg/mL and incubated with cells at 37 °C for 4 h. The formazan crystals were dissolved in 100 ꢁL
DMSO each well, and the absorbency at 492 nm (for absorbance of MTT formazan) and 630 nm (for the reference
wavelength) was measured with the ELISA reader. All of the compounds were tested three times in each of the cell lines.
The results expressed as inhibition rates or IC50 (halfꢀmaximal inhibitory concentration) were the averages of two
determinations and calculated by using the Bacus Laboratories Incorporated Slide Scanner (Bliss) software.
5.24 EGFR kinase assay
The target compounds 8a, 9d, 10a and 12a are tested for their activity against EGFR kinase through the mobility
shift assay. All kinases assays were performed in 96ꢀwell plates in a 50 ꢁL reaction volume. The kinase buffer contains
50 mM HEPES, pH 7.5, 10 mM MgCl2, 0.0015% Brijꢀ35 and 2 mM DTT. The stop buffer contains 100 mM HEPES, pH
7.5, 0.015% Brijꢀ35, 0.2% Coating Reagent #3 and 50 mM EDTA. Dilute the compounds to 500 ꢁM by 100% DMSO,
then transfer 10 ꢁL of compound to a new 96ꢀwell plate as the intermediate plate, add 90 ꢁL kinase buffer to each well.
Transfer 5 ꢁL of each well of the intermediate plate to 384ꢀwell plates. The following amounts of enzyme and substrate
were used per well: kinase base buffer, FAMꢀlabeled peptide, ATP and enzyme solution. Wells containing the substrate,

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DOI: 10.1039/C8NJ03594F
enzyme, DMSO without compound were used as DMSO control. Wells containing just the substrate without enzyme
were used as low control. Incubate at room temperature for 10 min. Add 10 ꢁL peptide solution to each well. Incubate at
28 °C for specified period of time and stop reaction by 25 ꢁL stop buffer. At last collect data on Caliper program and
convert conversion values to inhibition values. Percent inhibition = (max − conversion)/(max − min) × 100. “max” stands
for DMSO control; “min” stands for low control.
5. 25 Cell apoptosis assay by flow cytometry
A549 cells were seeded in 16ꢀwell plates at a density of 1 × 106 cells/well in RPMI 1640 medium and grown for 24
h at 37 °C in 5% CO2. Then treated with 1 ꢁM afatinib or compound 12a for 48 h. Cultured cells were stained with
Annexin VꢀFITC and propidium iodide (PI) in the dark at 4 °C for 30 min and analyzed by FACS Calibur flow cytometer
(Becton Dickinson, San Jose, CA) using Cell Quest software.
5.26 Acridine orange single staining
The cancer cell apoptotic of target compounds 12a were evaluated with A549 cancer cell lines by acridine orange
single staining. The cancer cell lines were cultured in minimum essential medium (MEM) supplement with 10% fetal
bovine serum (FBS). Approximately 2×103 cells, suspended in MEM medium, were plated onto each well of a 24ꢀwell
plate and incubated in 5% CO2 at 37 °C for 24 h. The test compounds at indicated final concentrations were added to the
culture medium and the cell cultures were continued for 12 h. Fresh acridine orange was added to each well at a terminal
concentration of 10ꢁg/mL and keep in dark place to stained for 15 min. Then, the staining cells washed three times with
PBS and placed under fluorescence microscopy to observe cell morphology.
5.27 Docking studies
For docking purposes, the threeꢀdimensional structure of the EGFR (PDB ID code :4G5P) were obtained from
RCSB Protein Data Bank. Hydrogen atoms were added to the structure allowing for appropriate ionization at
physiological pH. The protonated state of several important residues, such as LEUꢀ861 was adjusted by using
SYBYL6.9.1 (Tripos, St. Louis, USA) in favor of forming reasonable hydrogen bond with the ligand. Molecular docking
analysis was carried out by the SURFLEXꢀDOCK module of SYBYL 6.9.1 package to explore the binding model for the
active site of EGFR with its ligand. All atoms located within the range of 5.0 Å from any atom of the cofactor were
selected into the active site, and the corresponding amino acid residue was, therefore, involved into the active site if only
one of its atoms was selected. Other default parameters were adopted in the SURFLEXꢀDOCK calculations. All
calculations were performed on Silicon Graphics workstation.
Conflicts of interest

New Journal of Chemistry
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DOI: 10.1039/C8NJ03594F
There are no conflicts to declare.
Acknowledgments
We gratefully acknowledge the generous support provided by The National Natural Science Funds of China (No.
21662014), Outstanding Youth Foundation of Jiangxi, Natural Science Foundation of Jiangxi, China
(20171BCB23078), Natural Science Foundation of Jiangxi, China (20171ACB21052 & 20171BAB215073 &
20171BAB205101), Innovative Research Team of Jiangxi Science & Technology Normal University
(2017CXTD002).
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