Design, synthesis and biological evaluation of 2,3-dihydro-[1,4]dioxino[2,3-f]quinazoline derivatives as EGFR inhibitors

Article abstract of DOI:10.1016/j.bioorg.2021.104743

Epidermal growth factor receptor (EGFR) is the most attractive target for drug research in non-small cell lung cancer (NSCLC). The first-generation EGFR tyrosine kinase inhibitors (TKIs) Gefetinib and Elotinib showed good clinical efficacy on lung adenocarcinoma tumors, but almost all patients developed resistance to these inhibitors over time. Quinazoline and quinoline derivatives are common targeted inhibitors of EGFR kinase, and their structural optimization is an important direction for the development of effective targeted anticancer drugs. Based on these facts, a series of heterocyclic 2,3-dihydro-[1,4]dioxino[2,3-f]quinazoline derivatives have been designed and synthesized and their structures were confirmed by spectral analyses. The cytotoxic activity of the newly synthesized compounds was evaluated against the human kidney epithelial T293 cell line, normal lung cell lines WI-38, non-small cell lung cancer A549 and NCI-H157 cell lines using MTT. The tested compounds showed an evident anticancer activity against the tested cell lines, especially compound 13c, which was the most potent anticancer agent with half maximal inhibitory concentrations (IC₅₀) between 8.82 and 10.24 μM. Docking study showed that compound 13b could be nicely bound to the ATP binding pocket of EGFR. In addition, the inhibitory activity of the target compounds against epidermal growth factor receptor tyrosine kinase (EGFR-TK) was evaluated. Results indicated the ability of the target compounds to inhibit EGFR-TK with half maximal inhibitory concentrations (IC₅₀) in the range of 10.29 nM to 652.3 nM. In view of the reported compound activity, the structure deserves further optimization as cancer treatment agents.

Full text of DOI:10.1016/j.bioorg.2021.104743

Bioorganic Chemistry 110 (2021) 104743
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design, synthesis and biological evaluation of 2,3-dihydro-[1,4]dioxino
[2,3-f]quinazoline derivatives as EGFR inhibitors
,
Xuemei Qina *, Leifu Yangb, Peng Liuc, Lifang Yangd, Linmeng Chend, Liming Hub,
,
*
Mingguo Jianga
a Guangxi Key Laboratory of Polysaccharide Materials and Modifications, School of Marine Sciences and Biotechnology, Guangxi University for Nationalities, Nanning
530008, China
b College of Life Science and Chemistry & Beijing Key Laboratory of Environmental and Oncology, Beijing University of Technology, Beijing 100124, China
c Guangzhou Institute of Biomedicine and Health, Chinese Academy of Science, Guangzhou 510530, China
d School of Chemistry and Chemical Engineering, Guangxi University for Nationalities, Guangxi Nanning 530008, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Epidermal growth factor receptor (EGFR) is the most attractive target for drug research in non-small cell lung
cancer (NSCLC). The first-generation EGFR tyrosine kinase inhibitors (TKIs) Gefetinib and Elotinib showed good
clinical efficacy on lung adenocarcinoma tumors, but almost all patients developed resistance to these inhibitors
over time. Quinazoline and quinoline derivatives are common targeted inhibitors of EGFR kinase, and their
structural optimization is an important direction for the development of effective targeted anticancer drugs.
Based on these facts, a series of heterocyclic 2,3-dihydro-[1,4]dioxino[2,3-f]quinazoline derivatives have been
designed and synthesized and their structures were confirmed by spectral analyses. The cytotoxic activity of the
newly synthesized compounds was evaluated against the human kidney epithelial T293 cell line, normal lung cell
lines WI-38, non-small cell lung cancer A549 and NCI-H157 cell lines using MTT. The tested compounds showed
an evident anticancer activity against the tested cell lines, especially compound 13c, which was the most potent
EGFR inhibitors
Quinazoline derivatives
Lung cancer
Docking
anticancer agent with half maximal inhibitory concentrations (IC50) between 8.82 and 10.24 μM. Docking study
showed that compound 13b could be nicely bound to the ATP binding pocket of EGFR. In addition, the inhibitory
activity of the target compounds against epidermal growth factor receptor tyrosine kinase (EGFR-TK) was
evaluated. Results indicated the ability of the target compounds to inhibit EGFR-TK with half maximal inhibitory
concentrations (IC50) in the range of 10.29 nM to 652.3 nM. In view of the reported compound activity, the
structure deserves further optimization as cancer treatment agents.
1. Introduction
resistance eventually. Especially a secondary point mutation (T790M) in
the EGFR active domain is responsible for approximately half of the
Non-small cell lung cancer (NSCLC) accounts for approximately 85%
of all lung cancer cases and is one of the leading causes of cancer death
worldwide [1]. Genetic aberrations in the tyrosine kinase domain of
EGFR have been confirmed as one of the key drivers of NSCLC pro-
gression [2]. Thus, EGFR has become the most effective and attractive
therapeutic targets for NSCLC [2,3].
clinically acquired drug resistance in NSCLC patients [7,8].
In order to address the drug resistance caused by T790M, several
second-generation irreversible EGFR inhibitors were developed such as
Afatinib and Canertinib (Fig. 1) [9–11]. Afatinib was approved by the
FDA for the treatment of late-stage NSCLC patients with actively
mutated EGFR in 2013 [12]. The inhibitor contains electrophilic
Michael-acceptor systems that can covalently alkylate the conserved
cysteine residue (Cys797) at the lip of the ATP binding cleft of EGFR.
However, all the second generation inhibitors lack significant selectivity
between EGFR T790M mutants and the wild-type EGFR kinase. The
inhibition against wild-type EGFR is thought to be responsible for the
Gefitinib and erlotinib (Fig. 1) [4], as first-generation ATP-compet-
itive and reversible EGFR inhibitors, have been approved for the treat-
ment of NSCLC patients whose lung tumors harbor somatic EGFR
mutations L858R and delE746_A750 [5,6]. Unfortunately, most patients
who initially respond to gefitinib or erlotinib develop acquired
* Corresponding authors.
E-mail addresses: qxmei313@126.com (X. Qin), mzxyjiang@163.com (M. Jiang).
https://doi.org/10.1016/j.bioorg.2021.104743
Received 2 August 2020; Received in revised form 15 December 2020; Accepted 9 February 2021
Available online 18 February 2021
0045-2068/© 2021 Published by Elsevier Inc.

X. Qin et al.
Bioorganic Chemistry 110 (2021) 104743
side-effect of skin and gastrointestinal toxicities, which will limit their
achievable activity against the T790M mutation in patients [13].
The third generation mutant-selective EGFR inhibitors such as Osi-
mertinib (AZD9291, Fig. 1) and rociletinib (CO-1686, Fig. 1) have
emerged as potential therapeutics to block the growth of EGFR T790M
positive tumors [14,15]. Unlike the first (gefitinib and erlotinib) and
second generation (afatinib) EGFR TKIs, both AZD9291 and CO-1686
have a significantly increased potency for EGFR mutants (including
T790M) than for EGFRwt [16]. Despite the high efficacy of the third-
generation EGFR TKIs, acquired resistance to CO-1686 and AZD9291
have emerged, and C797S point mutation is the leading mechanism of
resistance [17–19]. The tries for overcoming EGFR C797S mutation led
to the introduction of the fourth-generation inhibitors to the clinical
practice [20]. Nowadays, studies are still ongoing to find more efficient
EGFR inhibitors in light of the resistance to the current inhibitors
[21–23].
Fig. 2. Our previous compound.
In our previous research, we found that compound b1 (Fig. 2)
showed inhibitory activity against EGFRwt (IC50 = 2.0 nM) and
EGFRT790M/L858R (IC50 = 6.9 nM) [24], indicating that the core structure
of dihydro[1,4]dioxino [2,3-f]quinazoline has potential use for tyrosine
kinase inhibitors. Based on our previous researches and attractiveness of
tyrosine kinases as promising targets for the design of new cancer
agents, taking erlotinib as the leading compound, it was decided to
introduce new dihydro[1,4]dioxino [2,3-f]quinazoline derivatives by
keeping the core structure of dihydro[1,4]dioxino [2,3-f]quinazoline
remains unchanged. All the compounds were tested against EGFRWT
enzyme. The most active compounds were examined for their anti-
proliferative activities and toxicity against a number of cancer cells
lines and normal cell lines. A molecular docking of the most active
compound was carried out to investigate their binding patterns with the
EGFR active pocket.
1.1. Rational drug design
The ATP binding pocket of EGFR-TK consists of five main parts;
adenine region, hydrophilic ribose pocket, hydrophobic region I, hy-
drophobic region II and phosphate binding region (Fig. 3) [25–28].
Fig. 1. Basic pharmacophoric features of EGFR-TK inhibitors [4].
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Bioorganic Chemistry 110 (2021) 104743
2. Results and discussion
2.1. Chemistry
Compounds 13a-13k and 14a-14k were synthesized according to
Scheme 1. The synthesis started with the commercial available 2,3,4-tri-
hydroxybenzoic acid 1, esterfying with idomethane and KHCO3, to give
benzoate 2 in excellent yield. Then intermediate 2 were protected by
benzyl and the ortho-position benzyl of ester was deprotected selectively
by HAc/HCl (10:1) at 40 ◦C to give 3. The nucleophilic substitution of
compound 3 with epichloro-hydrin resulted in compound 4, which was
then catalyzed by ferric chloride to form compound 5 in methanol. The
key intermediate 7 was got by intramolecular cyclization after 6 being
chlorination, then reacted with different halogenoalkane to obtain 8.
The intermediate 9 was achieved by selective nitration with the mixture
of nitric acid-acetic acid solution. Then catalytic hydrogenation gave 10,
which was subjected to cyclization to generate intermediate 11, which
underwent a nucleophilic 13a-13k and 14a-14k.
Fig. 3. The ATP binding pocket of EGFR-TK [28].
2.2. Biological activity
Nowadays, the EGFR-TKIs do not exploit the ribose and phosphate
binding site. Accordingly, the structure-activity relationships (SAR) of
majority EGFR-TKIs share four common pharmacophoric features [29],
including hydrophobic head, central hetero armotic system, NH spacer,
hydrophobic tail as shown in Figs. 1 and 2. The main target of this work
was synthesis of new dihydro[1,4]dioxino[2,3-f]quinazoline derivatives
having the same essential pharmacophoric features of the previous re-
searches and clinically used EGFR-TKIs erlotinib (Fig. 4). The core of our
molecular design rational comprised bioisosteric modification strategies
of EGFR-TKIs at four different positions.
2.2.1. Enzyme assay
The newly synthesized [1,4]dioxino[2,3-f]quinazoline derivatives
were evaluated for their ability to inhibit the autophosphorylation of
EGFR kinase using Kinase-Glo luminescent kinase assays. The results
were shown in Table 1. The studied compounds displayed inhibition to
EGFR kinase with IC50 values ranging from 652.3 nM to 10.29 nM. All
the compounds displayed significant EGFR inhibitory activities. Among
them, compound 13b showed the most potent inhibitory activity against
EGFR kinase (IC50 = 10.29 nM), which was comparable to the positive
The first position was the hetero aromatic ring system, where the
quinazoline was used the same as erlotinib. One consideration for using
the cyclization at 5- and 6-position of the quinazoline is that core
cyclization makes the molecule small, thus makes it easier to tolerate the
shift of the inhibitors inside the binding pocket due to the mutations,
such as T790M. The second position was the terminal hydrophobic head.
Diferent hydrophobic substituted phenyl including meta or para-posi-
tion halogen or alkyl substituted phenyl were selected. The third posi-
control erlotinib and gefitinib (IC50
respectively).
= 11.65 nM, 10.41 nM,
To explore the SARs of the novel scaffold, methoxyethyl was firstly
introduced as R1 (13a-13 k) and the inhibitory activities against EGFR
ranged from 10.29 nM to 208.6 nM. When R3 was hydrogen (13a, 13b,
13c), compounds substituted by Cl or Br (13b, IC50 = 10.29 nM; 13c,
IC50 = 15.13 nM) showed excellent potency against EGFR kinase and
displayed 6–10 fold increase in potency than substituted by F (13a, IC50
= 106.1 nM). The reason may be that small fluorine atom could be too
small to fill up well the hydrophobic pocket of EGFR kinase pocket. As
for bromine atom, its larger volume may not entry into the EGFR pocket
well. R2 or R3 of the target compounds substituted by methyl (13k, IC50
= 208.6 nM) or methoxy (13e, IC50 = 354.7 nM) show weaker activities
against EGFR kinase, indicating that grafting a large electron donating
group at the 4-positions of quinazoline scaffold is not beneficial for the
activity. By comparing 13a & 13h, 13b & 13i and 13c & 13j, it is easy to
find the compounds containing meta-substituted aniline showed much
more potency against EGFR than compounds containing para-
substituted aniline.
–
tion was the linker (spacer) region, where the linker was NH the same
as the lead compound erlotinib. The fourth position was the hydro-
phobic tail. Methoxyethyl (compound 13a-13k) and 3- morpholino-
propyl (14a-14k) was incorporated to occupy the front hydrophobic
region of ATP binding site. All modification pathways and molecular
design rationale were illustrated and summarized in Fig. 5.
To find a more potent EGFR inhibitor, R1 was substituted by 3-
morpholinopropyl in the subsequent synthesis (14a-14k) and the inhib-
itory activities against EGFR ranged from 13.59 nM to 653.2 nM. By
comparing target compounds 13 and 14, we could conclude that com-
pounds with R1 being mothoxyethyl showed almost the same trend
inhibitory efficiency against EGFR as compounds with R1 being 3-mor-
pholinopropyl do, indicating that the inhibitory activity of the synthe-
sized compounds depends mainly on the nature of the substituents of
aniline. With regard to the un-substituted derivative 14k (IC50 = 321.2
nM), had a lower activity than the substituted ones.
It is important to point ou that when 4-position of quinazoline scaffold
are substituted by 3-ethynylphenylamino (13d, 14d) or 3-chloro-4-fluo-
rophenylamino, compounds (13g, 14g) showed better inhibitory activity
against EGFR. As a whole, compounds substituted by halogens showed
better potency against EGFR. The different substituted halogen on aniline,
the inhibitory activity of compounds on EGFR was consistent with Cl > Br
> F (13b > 13c > 13a, 14b > 14c > 14a, 13i > 13j > 13 h, 14i > 14h).
Fig. 4. The basic structural requirements for erlotinib as reported EGFR-TK
inhibitor [28].
3

X. Qin et al.
Bioorganic Chemistry 110 (2021) 104743
Fig. 5. Rational of molecular design of the new proposed EGFR-TK inhibitors.
Scheme 1. Reagents and conditions: (a) CH3I, KHCO3, DMF, 40 ◦C; (b) BnCl, K2CO3, KI, DMF, 60 ◦C; (c) HCl/H2O, HAc, 40 ◦C; (d) epichlorohydrin, Cs2CO3, DMF,
70 ◦C; (e) CH3OH, FeCl3, rt; (f) Pd/C, H2, EtOH; (g) SOCl2, DMF, reflux; K2CO3, acetone, KI, reflux; (h) R1-Cl or R1-Br, K2CO3, DMF, 80 ◦C; (i) HNO3/HAc; (j) Pd/C,
H2, EtOH; (k) formamidine acetate, EtOH, reflux; (l) POCl3, reflux; (m) anilines, isopropanol, reflux.
2.2.2. Anti-proliferation assay
noticeable that almost all the tested compounds demonstrated good
activity in blocking tumor cell proliferation compared to the positive
control erlotinib.
Compounds with better inhibitory activities in enzyme assay were
selected to assess for anti-proliferative activity against two non-small
cell lung cancer A549 and NCI-H157 cell lines using MTT, using erloti-
nib as a positive control. The results were showed in Table 2. It is
4

X. Qin et al.
Bioorganic Chemistry 110 (2021) 104743
2.2.3. Cytotoxicity test
Table 1
In vitro enzymatic inhibitory activity of compounds 13a-13k and 14a-14k
As shown in Table 3, compounds with good inhibitory activities
against EGFR kinase were evaluated for their toxicity against the human
kidney epithelial T293 cell line and the normal lung cell lines WI-38
using the MTT, these compounds were tested at multiple doses to
study the viability of T293 cell and WI-38 cell. The median cytotoxic
concentration (CC50) showed that some of the tested compounds did not
display any significant cytotoxicity in vitro against T293 cells and WI-
38, especially compounds 13b, 13g and 14b.
against EGFR.a
Compound
R1
R2
R3
IC50 (nM)
13a
13b
13c
13d
13e
13f
13g
13h
13i
F
Cl
Br
ethynyl
MeO-
-CF3
Cl
H
H
H
H
H
H
H
H
F
106.1
10.29
15.13
39.62
354.7
92.89
23.1
164.2
86.79
136.3
208.6
115.9
2.2.4. Docking study
The most active compound 13b was docked into the three dimen-
sional EGFR active site (1 M17.pdb [30], EGFR/erlotinib crystalline
complex) using Autodock software package (version 4.0) with the help
of Autodock Tools. In the proposed binding mode, compound 13b was
nicely bound to the ATP binding pocket in a similar manner to erlotinib,
showed in Fig. 6A. The N-1 of the quinazoline forms an H-bond with the
Met769 backbone nitrogen. A water (HOH-10) molecule-mediated
hydrogen bonding interaction is observed between quinazoline nitro-
gen atom (N-3) and other amino acid. The 3-chloro-4-fluoro phenyl-
amino substituent extends into the hydrophobic pocket in the back of
the ATP-binding cleft. And compound 13b was overlaid well with the
binding conformation of erlotinib (gray) into the EGFR kinase domain
(Fig. 6B). The well binding of the inhibitor to the active sites of EGFR
may explain its excellent inhibitory activity.
F
Cl
Br
Me
H
13j
13k
14a
H
H
F
14b
14c
14d
14e
14f
Cl
H
13.59
15.84
37.36
281.4
100.9
17.6
Br
H
3. Conclusion
ethynyl
MeO-
-CF3
Cl
H
In summary, we identified a series of novel [1,4]dioxino[2,3-f]qui-
nazoline-based derivates as EGFR inhibitors. All of the compounds
showed good inhibitory activities against EGFR kinase (IC50 < 1 μM).
H
Among them, eight compounds (13b, 13c, 13d, 13g, 14b, 14c, 14d and
14g) showed excellent potency against EGFR kinase, as well as signifi-
cant anti-proliferative activities against A549 and NCI-H157 cells.
Docking study showed that compound 13b could be nicely bound to the
ATP binding pocket of EGFR. It is believed that this work would be
helpful for the further research of EGFR inhibitors and expanding the
types of compounds that act as EGFR inhibitors.
H
14g
14h
14i
F
H
F
132.8
90.7
H
Br
MeO-
H
4. Experiments
4.1. Materials and methods
14j
H
652.3
321.2
The reagents were purchased and used without further purification.
Melting points were determined on a MP120 melting point apparatus
(Hanon instruments Corp., Jinan, China) and are as read. The 1H NMR
and 13C NMR spectra were measured on a Bruker 400 MHz Avance or on
a Bruker 600 MHz Avance spectrometer with TMS and solvent signals
allotted as internal standards. The chemical shifts are reported in ppm
(δ). Splitting patterns are designed as s, singlet; d, doublet; t, triplet; m,
multiplet. ESI-MS spectra were obtained on an Esquire 6000 Mass
14k
H
gefitinib
erlotinib
10.41
11.65
a
Values are averages of two independent determinations.
Table 2
Antiproliferative assay in vitro.a
Table 3
Compound
A549 IC50 (nM)
NCI-H157 IC50 (nM)
The median cytotoxic concentration (CC50) data of texted compounds.a
13b
9.95 × 103
8.82 × 103
9.23 × 103
12.25 × 103
19.85 × 103
15.21 × 103
13.78 × 103
13.26 × 103
7.29 × 103
11.66 × 103
10.24 × 103
19.08 × 103
7.76 × 103
16.19 × 103
16.65 × 103
20.89 × 103
9.89 × 103
6.88 × 103
Compound
Cytotoxocity (CC50 in nM)
13c
T293
WI-38
13d
13g
13b
>100 × 103
40.10 × 103
69.21 × 103
82.38 × 103
61.50 × 103
22.35 × 103
38.15 × 103
59.16 × 103
65.56 × 103
90.55 × 103
70.60 × 103
80.64 × 103
94.35 × 103
100.56 × 103
55.37 × 103
93.45 × 103
45.68 × 103
89.38 × 103
14b
13c
14c
13d
14d
13g
14g
14b
erlotinib
14c
a
14d
Values are means of at least two experiment.
14g
erlotinib
a
Values are means of at least two experiment.
5

X. Qin et al.
Bioorganic Chemistry 110 (2021) 104743
mixture was allowed to cool to room temperature. Then the reaction
mixture was poured into 1000 mL ice water. The product precipitated as
a white solid. Filtration to get the solid and washed with water and
petroleum ether. Product was obtained after drying under vacuum.
Yield: 26 g, 95%. 1H NMR (400 MHz, DMSO‑d6) δ 7.28–7.56 (m, 16H),
7.08 (d, J = 8.8 Hz, 1H), 5.23 (d, 2H), 5.00 (d, 4H), 3.76 (s, 3H). ESI-MS
m/z: 455.1 [M + H]+.
4.2.3. Methyl 3,4-bis(benzyloxy)-2-hydroxybenzoate (3)
Methyl 2,3,4-tris(benzyloxy)benzoate 2 (10 g, 22 mmol) was dis-
solved in 550 mL HAc/HCl (V/V, 10:1). The mixture was heated in water
bath at 40 ◦C and monitored by TLC. After the conversion was
completed, the reaction mixture was poured into 800 mL ice water. The
product precipitated as a white solid. Filtration to get the solid and
washed with water. 1H NMR (400 MHz, DMSO‑d6) δ 7.57 (d, J = 9.2 Hz,
1H), 7.29–7.46 (m, 10H), 6.80 (d, J = 9.2 Hz, 1H), 5.20 (s, 2H), 4.97 (s,
2H), 3.87 (s, 3H). ESI-MS m/z: 363.0 [Mꢀ H]–.
4.2.4. Methyl 3,4-bis(benzyloxy)-2-(oxiran-2-ylmethoxy) benzoate (4)
Cesium carbonate (4.65 g, 14.3 mmol) and epichlorohydrin (1.4 mL,
17.85 mmol) were added to a solution of intermediate 3 (5 g, 11.9
mmol) in DMF. The mixture was heated at 70 ◦C for 8 h. The reaction
was cooled to room temperature, filtered, washed with ethyl acetate,
and concentrated, then diluted with water and extracted with ethyl
acetate. Organic layer was washed with water and concentrated to oil
and separated by chromatographic column on a silica gel to get the
product 3.8 g. White solid, yield 75%. 1H NMR (400 MHz, CDCl3) δ 7.64
(d, J = 8.9 Hz, 1H), 7.32–7.48 (m, 10H), 6.80 (d, J = 8.9 Hz, 1H), 5.15(s,
2H), 5.06 (q, J = 9.2 Hz, 2H), 4.28 (dd, J = 10.7, 3.6 Hz, 1H), 4.10–4.14
(m, 1H), 3.91 (s, 3H), 3.42 (dt, J = 7.9, 3.2 Hz, 1H), 2.84 (t, J = 4.6 Hz,
1H), 2.65 (dd, J = 5.0, 2.6 Hz, 1H). ESI-MS m/z: 443.2 [M + Na]+.
4.2.5. Methyl 4-(3-c methyl 3,4-bis(benzyloxy)-2-(2-hydroxy-3-methoxy-
propoxy)benzoate (5)
A mixture of intermediate 4 (2 g, 4.76 mmol) and anhydr. FeCl3
(0.15 g, 0.96 mmol) in MeOH (50 mL) was stirred at room temperature.
The reaction was monitored by TLC. The solvent was removed under
vacuum. Water (70 mL) was added and the mixture was extracted with
ethyl acetate (2 × 70 mL). The organic solution was dried (MgSO4).
Evaporation of the solvent followed by chromatography on a short
Fig. 6. (A) Binding modes of compound 13b with the active site of EGFR. (B)
Overview of compound 13b (grey) overlaid with the binding conformation of
erlotinib (yellow) into the EGFR active pocket.
column of silica gel gave the pure product, 1.4 g. Yellow oil, 65%. 1
H
NMR (400 MHz, CDCl3) δ 7.67 (d, J = 8.9 Hz, 1H), 7.47–7.35 (m, 7H),
7.35–7.30 (m, 3H), 6.76 (d, J = 8.9 Hz, 1H), 5.13 (d, J = 1.9 Hz, 2H),
5.03 (s, 2H), 4.83 (d, J = 4.0 Hz, 1H), 4.43 (dd, J = 10.2, 2.7 Hz, 1H),
4.22 (dd, J = 10.2, 6.9 Hz, 1H), 4.11 (d, J = 2.4 Hz, 1H), 3.87 (d, J = 1.9
Hz, 3H), 3.51 (d, J = 5.5 Hz, 2H), 3.37 (s, 3H). ESI-MS m/z: 475.3 [M +
Na]+.
Spectrometer. HRMS data were measured using a Bruker APEX IV
Fourier transform ion cyclotron resonance mass spectrometer.
4.2. Synthetic procedures
4.2.1. Methyl 2,3,4-trihydroxybenzoate (1)
4.2.6. Methyl 3,4-dihydroxy-2-(2-hydroxy-3-methoxypro-poxy)ben zoate
(6)
To a suspension of 2,3,4-trihydroxybenzoic acid (10.0 g, 58.82
mmol) in DMF (150 mL) was added KHCO3 (7.58 g, 70.58 mmol), and
the mixture was stirred at room temperature for 10 min, then added
CH3I (5.60 mL, 50.7 mmol). The mixture was heated in water bath at
40 ◦C and monitored by TLC. After the conversion was completed, the
mixture was allowed to cool to room temperature. Then the reaction
mixture was poured into 800 mL ice water. The product precipitated as a
white solid. Filtration to get the solid and washed with water (70 mL ×
4). Product was obtained after drying under vacuum. Yield: 10 g, 92%.
1H NMR (400 MHz, CDCl3) δ 11.01 (s, 1H), 7.38 (d, J = 8.8 Hz, 1H), 6.53
(d, J = 8.8 Hz, 1H), 5.80 (s, 1H), 5.47 (s, 1H), 3.94 (s, 3H). ESI-MS m/z:
183.1 [Mꢀ H]-.
Intermediate 5 (5.0 g, 11.0 mmol) was dissolved in ethanol (50 mL)
followed by the addition of 10% Pd/C (0.2 g). The air was expelled from
the vessel, and the mixture was stirred over an atmosphere of hydrogen
for 8 h at r.t. The solution was filtered over a bed of celite, and the
solvent was evaporated giving a white yellow oil. The product was used
for the next step without any purification. 1H NMR (400 MHz, DMSO‑d6)
δ 7.96 (s, 2H), 7.16 (d, J = 8.6 Hz, 1H), 6.62 (d, J = 8.6 Hz, 1H), 4.05
(dd, J = 10.1, 3.8 Hz, 1H), 4.01–3.92 (m, 1H), 3.84 (dd, J = 10.0, 6.5 Hz,
1H), 3.75 (s, 3H), 3.72–3.65 (m, 0.5H), 3.41 (ddd, J = 15.9, 10.0, 5.4 Hz,
2H), 3.28 (s, 3H), 3.22 (dd, J = 8.1, 5.5 Hz, 0.5H). ESI-MS m/z: 273.7 [M
+ H]+.
4.2.2. Methyl 2,3,4-tris(benzyloxy)benzoate (2)
4.2.7. Methyl 8-hydroxy-2-(methoxymethyl)-2,3-dihydrobenzo [b][1,4]
dioxine-5-carboxylate (7)
To a stirred solution of 1 (10 g, 54.30 mmol) in DMF (150 mL) were
added K2CO3 (37.47 g, 271.5 mmol), KI (0.9 g, 5.4 mmol), followed by
benzyl chloride (31.24 mL, 271.5 mmol) and the reaction mixture was
heated at 60 ◦C and monitored by TLC still the reaction completing. The
A suspension of intermediate 6 (2.9 g, 10.7 mmol) in SOCl2 (15 mL)
containing 3 drops of DMF was stirred at room temperature for 2 h and
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X. Qin et al.
Bioorganic Chemistry 110 (2021) 104743
then heated under reflux for 1 h. The SOCl2 was removed under reduced
pressure to give crude chloro-substitute. The crude product was dis-
solved in acetone (50 mL), and then added potassium carbonate (15 g,
107 mmol), KI (0.018 g, 0.11 mmol). The mixture was heated at reflux
and the reaction was monitored by TLC still the reaction completing.
The reaction was cooled to room temperature, neutralized with HCl at
4.2.10.1. Methyl 6-amino-8-(2-methoxyethoxy)-2-(methoxy- methyl)-2,3-
dihydrobenzo[b][1,4]dioxine-5-carboxylate (10a). Brown oily liquid,
90%. 1H NMR (400 MHz, CDCl3) δ 5.76 (s, 1H), 4.27 (dd, J = 11.2, 1.9
Hz, 1H), 4.21–4.13 (m, 1H), 3.96 (dt, J = 11.3, 6.1 Hz, 3H), 3.76 (s, 3H),
3.68–3.62 (m, 2H), 3.58 (dd, J = 10.3, 4.8 Hz, 1H), 3.52 (dd, J = 10.4,
5.9 Hz, 1H), 3.33 (s, 3H), 3.31 (s, 3H).
0
◦C and extracted with ethyl acetate. The organic solution was dried
(MgSO4). Evaporation of the solvent followed by chromatography on a
short column of silica gel gave the pure product, 1.77 g. White solid,
65%. 1H NMR (400 MHz, CDCl3) δ 7.45 (d, J = 8.7 Hz, 1H), 6.55 (d, J =
8.7 Hz, 1H), 4.47–4.38 (m, 2H), 4.16–4.09 (m, 1H), 3.85 (s, 3H), 3.67
(ddd, J = 14.9, 10.5, 5.0 Hz, 2H), 3.43 (s, 3H), 2.14 (s, 1H). ESI-MS m/z:
277.1 [M + Na]+.
4.2.10.2. Methyl 6-amino-2-(methoxymethyl)-8-(3-morpholino propoxy)-
2,3-dihydrobenzo[b][1,4]-dioxine-5-carboxylate
(10b). Brown
oily
liquid, 90%. ESI-MS m/z: 397.4 [M + H]+.
4.2.11. General synthesis method of compounds 11
A mixture of 10 (20 mmol) and formamidine acetate (24 mmol) in
ethanol (60 mL) was heated at reflux for 5 h. The mixture was cooled and
filtered. The precipitate was washed with ethanol and dried to afford the
product.
4.2.8. General procedure for the preparation of compounds 8a and 8b
Intermediate 7 (9.6 mmol), corresponding halogenoalkane (12.5
mmol), and potassium carbonate (28.9 mmol) were stirred in 200 mL
acetone at 80 ◦C for 8 h. The reaction was cooled to room temperature,
filtered, concentrated, and purified by silica gel chromatography to
afford the product.
4.2.11.1. 5-(2-methoxyethoxy)-3-(methoxymethyl)-2,3-dihydro-[1,4]
dioxino[2,3-f]quinazolin-10(9H)-one (11a). White solid; Yield: 69%. 1H
NMR (400 MHz, CDCl3) δ 11.80 (s, 1H), 7.99 (s, 1H), 6.85 (s, 1H), 4.60
(dd, J = 11.4, 2.2 Hz, 1H), 4.47–4.37 (m, 1H), 4.34–4.20 (m, 3H), 3.84
(dd, J = 7.1, 4.7 Hz, 2H), 3.74 (qd, J = 10.5, 5.3 Hz, 2H), 3.47 (s, 3H),
3.44 (s, 3H). ESI-MS m/z: 320.8 [M + H]+.
4.2.8.1. Methyl
8-(2-methoxyethoxy)-2-(methoxymethyl)-2,3-dihy-
drobenzo[b][1,4]dioxine-5-carboxylate (8a). Yellow liquid, yield 88%.
1H NMR (400 MHz, CDCl3) δ 7.46 (d, J = 8.8 Hz, 1H), 6.54 (d, J = 8.9
Hz, 1H), 4.46 (dd, J = 11.4, 1.9 Hz, 1H), 4.39 (dd, J = 11.8, 5.9 Hz, 1H),
4.21 (t, J = 4.9 Hz, 2H), 4.15 (dd, J = 11.3, 7.1 Hz, 1H), 3.87 (s, 3H),
3.80 (t, J = 4.7 Hz, 2H), 3.73 (dd, J = 10.4, 4.8 Hz, 1H), 3.66 (dd, J =
10.4, 6.1 Hz, 1H), 3.46 (s, 3H), 3.43 (s, 3H). ESI-MS m/z: 335.1 [M +
Na]+.
4.2.11.2. 3-(methoxymethyl)-5-(3-morpholino-propoxy)-2,3-dihydro[1,4]
dioxino[2,3-f]quinazolin-10(9H)-one (11b). White solid; Yield: 70%. 1H
NMR (400 MHz, DMSO‑d6) δ 11.78 (s, 1H), 7.85 (s, 1H), 6.76 (s, 1H),
4.44–4.32 (m, 2H), 4.14 (t, J = 6.4 Hz, 2H), 4.04 (dd, J = 11.2, 7.0 Hz,
1H), 3.64–3.55 (m, 6H), 2.40 (dd, J = 18.0, 10.9 Hz, 6H), 1.91 (p, J =
6.6 Hz, 2H).
4.2.8.2. Methyl 2-(methoxymethyl)-8-(3-morpholinopropoxy)-2,3-dihy-
drobenzo[b][1,4]-dioxine-5-carboxylate (8b). Yellow liquid, yield 88%.
ESI-MS m/z: 382.3 [M + H]+.
4.2.12. General synthesis method of compounds 12
A mixture of intermediate 11 (7.0 mmol) and POCl3 (15 mL) was
heated at reflux temperature for 3 h. The solvent were removed under
reduced pressure. The residue was dissolved in CH2Cl2 (50 mL) and the
organic layer was washed with cold aqueous NaHCO3 solution and
brine, and dried over MgSO4, filtered and evaporated to give the
product.
4.2.9. General procedure for the preparation of compounds 9a and 9b
To a suspension of intermediate 8 (7 mmol) dissolved in AcOH (16
mL) at 0 ◦C was slowly added fumic HNO3/HAc (4 mL/12 mL). Then stir
at room temperature still the reaction completing and poured into ice
water (500 mL). After stirring for 30 min, the resulting precipitate was
filtered off, washed with ice water and dried to obtain the nitro com-
pound 9 as a yellow solid.
4.2.12.1. 10-chloro-5-(2-methoxyethoxy)-3-(methoxymethyl)-2,3-dihy-
dro-[1,4]dioxino[2,3-f]quinazoline (12a). White solid; Yield: 72%. 1H
NMR (400 MHz, CDCl3) δ 8.78 (s, 1H), 7.06 (s, 1H), 4.55 (dd, J = 11.4,
2.3 Hz, 1H), 4.52–4.45 (m, 1H), 4.34 (t, J = 4.1 Hz, 2H), 4.25 (dd, J =
11.4, 6.8 Hz, 1H), 3.92–3.85 (m, 2H), 3.76 (ddd, J = 16.5, 10.4, 5.4 Hz,
2H), 3.48 (d, J = 7.9 Hz, 6H). ESI-MS m/z: 341.3 [M + H]+.
4.2.9.1. Methyl
8-(2-methoxyethoxy)-2-(methoxymethyl)-6-nitro-2,3-
dihydrobenzo[b][1,4]-dioxine-5-carboxylate (9a). Yellow solid, 83%. 1H
NMR (400 MHz, CDCl3) δ 7.43 (s, 1H), 4.42 (t, J = 9.6 Hz, 2H), 4.24 (t, J
= 4.5 Hz, 2H), 4.15 (dd, J = 11.5, 7.1 Hz, 1H), 3.95 (s, 3H), 3.80 (d, J =
2.0 Hz, 2H), 3.74 (dd, J = 10.4, 4.6 Hz, 1H), 3.67 (dd, J = 10.4, 6.1 Hz,
1H), 3.45 (s, 3H), 3.42 (s, 3H). ESI-MS m/z: 380.2 [M + Na]+.
4.2.12.2. 10-chloro-3-(methoxymethyl)-5-(3-morpholinopropoxy) ꢀ 2,3-
dihydro-[1,4]dioxino[2,3-f]quinazoline (12b). White solid; Yield:75%.
ESI-MS m/z: 410.3 [M + H]+.
4.2.9.2. Methyl 2-(methoxymethyl)-8-(3-morpholinopropoxy)-6-nitro-2,3-
dihydrobenzo[b][1,4]dioxine-5-carboxylate (9b). Yellow solid, 83%. 1H
NMR (400 MHz, CDCl3) δ 7.40 (s, 1H), 4.40 (dd, J = 8.5, 3.7 Hz, 2H),
4.16 (dd, J = 12.9, 6.8 Hz, 3H), 3.95 (s, 3H), 3.76–3.68 (m, 6H), 3.42 (s,
3H), 2.53 (t, J = 7.0 Hz, 2H), 2.48 (s, 4H), 2.08–1.99 (m, 2H). ESI-MS m/
z: 427.2 [M + H]+.
4.2.13. General synthesis method of compounds 13 & 14
A mixture of 4-chloroquinazolines derivatives 12 (5 mmol) and
substituted anilines (6 mmol) in isopropanol (25 mL) was stirred at
reflux for 3 h. The reaction mixture was cooled to room temperature and
the resultant precipitate was collected by filtration. The solid was
further dried in vacuum to give the product.
4.2.10. General synthesis method of compounds 10
To a stirred suspension of intermediate 9 (4.7 mmol) in acetic acid
(15 mL) was added zinc powder (1.2 g, 18.8 mmol) and the mixture was
heated at 60 ◦C for 3 h. The reation was cooled to room temperature and
then filtered through a celite bed. The filtrate was concentrated and the
residue was diluted with EtOAc and washed with water (2 × 100 mL)
and dried over anhydrous magnesium sulfate. The solvent was evapo-
rated to give the product as brown oil.
4.2.13.1. N-(3-fluorophenyl)-5-(2-methoxyethoxy)-3-(methoxy-methyl)-
2,3-dihydro-[1,4]dioxino[2,3-f]quinazolin-10-amine (13a). White solid;
Yield: 58%; mp: 231.6–232.2 ◦C. 1H NMR (500 MHz, CD3OD + CDCl3) δ
8.58 (s, 1H), 7.63 (dt, J = 10.3, 2.2 Hz, 1H), 7.48–7.37 (m, 2H), 7.11 (s,
1H), 7.09–7.01 (m, 1H), 4.83 (dd, J = 11.4, 2.2 Hz, 1H), 4.55–4.50 (m,
1H), 4.44 (dd, J = 11.4, 7.5 Hz, 1H), 4.38 (t, J = 4.4 Hz, 2H), 3.88 (dd, J
= 7.2, 3.7 Hz, 2H), 3.82 (qd, J = 10.7, 4.9 Hz, 2H), 3.47 (d, J = 1.5 Hz,
6H). 13C NMR (125 MHz, CD3OD + CDCl3) δ 162.67, 158.18, 156.41,
7

X. Qin et al.
Bioorganic Chemistry 110 (2021) 104743
148.41, 139.59, 137.34, 134.53, 133.55, 130.20, 119.49, 113.67,
111.41, 99.30, 94.35, 72.00, 70.23, 70.17, 69.50, 66.87, 59.19, 58.75.
HRMS (ESI) m/z: 416.1622 calcd for C21H22FN3O5, [M + H]+, found
416.1624. HPLC purity: 100%.
156.54, 148.42, 139.67, 136.60, 134.51, 133.58, 131.21, 129.65,
127.73, 123.61, 123.44, 121.15, 99.29, 94.25, 72.04, 70.25, 70.17,
69.53, 66.85, 59.19, 58.75. HRMS (ESI) m/z: 466.1590 calcd for
C22H22F3N3O5 [M + H]+, found 466.1596. HPLC purity: 100%.
4.2.13.2. N-(3-chlorophenyl)-5-(2-methoxyethoxy)-3-(methoxy-methyl)-
2,3-dihydro-[1,4]dioxino[2,3-f]quinazolin-10-amine (13b). White solid;
Yield: 62%; mp: 233.3–234.7 ◦C. 1H NMR (500 MHz, CD3OD + CDCl3) δ
8.59 (s, 1H), 7.82 (s, 1H), 7.56 (d, J = 8.0 Hz, 1H), 7.43 (t, J = 8.1 Hz,
1H), 7.33 (d, J = 8.0 Hz, 1H), 7.14 (s, 1H), 4.84 (dd, J = 11.3, 1.7 Hz,
1H), 4.54 (d, [1]J = 5.0 Hz, 1H), 4.45 (dd, J = 11.3, 7.5 Hz, 1H), 4.40 (t,
J = 4.3 Hz, 2H), 3.92–3.87 (m, 2H), 3.83 (qd, J = 10.8, 4.8 Hz, 2H), 3.49
(d, J = 0.9 Hz, 6H). 13C NMR (125 MHz, CD3OD + CDCl3) δ 158.25,
156.45, 148.37, 139.62, 137.08, 134.47, 134.42, 133.54, 130.02,
126.96, 124.22, 122.33, 99.25, 94.26, 72.02, 70.25, 70.17, 69.52,
66.87, 59.19, 58.75. HRMS (ESI) m/z: 432.1326 calcd for C21H22ClN3O5
[M + H]+, found 432.1329. HPLC purity: 100%.
4.2.13.7. N-(4-chloro-3-fluorophenyl)-5-(2-methoxyethoxy)-3-(methox-
ymethyl)-2,3-dihydro-[1,4]dioxino[2,3-f]quinazolin-10-amine
(13g).
Light yellow solid; Yield: 60%; mp: 248.2–250.5 ◦C. 1H NMR (500 MHz,
CD3OD + CDCl3) δ 8.57 (s, 1H), 7.86 (dd, J = 6.5, 2.6 Hz, 1H), 7.58–7.54
(m, 1H), 7.29 (t, J = 8.7 Hz, 1H), 7.13 (s, 1H), 4.83 (dd, J = 11.4, 2.1 Hz,
1H), 4.56–4.51 (m, 1H), 4.44 (dd, J = 11.4, 7.5 Hz, 1H), 4.40 (t, J = 4.3
Hz, 2H), 3.89 (dd, J = 7.2, 3.7 Hz, 2H), 3.83 (qd, J = 10.8, 4.9 Hz, 2H),
3.49 (d, J = 1.9 Hz, 6H). 13C NMR (125 MHz, CD3OD + CDCl3) δ 158.40,
156.53, 155.53, 148.38, 139.66, 134.44, 133.52, 132.57, 126.78,
124.57, 121.08, 116.68, 99.17, 94.26, 72.00, 70.24, 70.17, 69.52,
66.82, 59.21, 58.77. HRMS (ESI) m/z: 450.1232 calcd for
C
21H21ClFN3O5 [M + H]+, found 450.1235. HPLC purity: 95.33%.
4.2.13.3. N-(3-bromophenyl)-5-(2-methoxyethoxy)-3-(methoxy-methyl)-
2,3-dihydro-[1,4]dioxino[2,3-f]quinazolin-10-amine (13c). White solid;
Yield: 70%; mp: 239.1–241.7 ◦C. 1H NMR (500 MHz, CD3OD + CDCl3) δ
8.59 (s, 1H), 7.95 (t, J = 1.8 Hz, 1H), 7.63–7.58 (m, 1H), 7.48 (dd, J =
8.0, 0.8 Hz, 1H), 7.38 (t, J = 8.1 Hz, 1H), 7.13 (s, 1H), 4.84 (dd, J = 11.4,
2.1 Hz, 1H), 4.57–4.52 (m, 1H), 4.45 (dd, J = 11.4, 7.5 Hz, 1H), 4.40 (t,
J = 4.3 Hz, 2H), 3.89 (dd, J = 6.9, 3.7 Hz, 2H), 3.83 (qd, J = 10.7, 4.8
Hz, 2H), 3.49 (d, J = 0.8 Hz, 6H). 13C NMR (125 MHz, CD3OD + CDCl3)
δ 158.26, 156.46, 148.37, 139.62, 137.17, 134.42, 133.55, 130.27,
129.94, 127.10, 122.83, 122.23, 99.24, 94.25, 72.01, 70.24, 70.17,
69.52, 66.86, 59.20, 58.76. HRMS (ESI) m/z: 476.0816 calcd for
4.2.13.8. N-(4-fluorophenyl)-5-(2-methoxyethoxy)-3-(methoxy-methyl)-
2,3-dihydro-[1,4]dioxino[2,3-f]quinazolin-10-amine (13h). Light yellow
solid; Yield: 55%; mp: 239.3–240.9 ◦C. 1H NMR (500 MHz, CD3OD +
CDCl3) δ 8.51 (s, 1H), 7.67–7.61 (m, 2H), 7.22–7.16 (m, 2H), 7.10 (s,
1H), 4.82 (dd, J = 11.4, 2.2 Hz, 1H), 4.56–4.51 (m, 1H), 4.44 (dd, J =
11.4, 7.5 Hz, 1H), 4.40 (t, J = 4.4 Hz, 2H), 3.89 (dd, J = 7.2, 3.7 Hz, 2H),
3.83 (qd, J = 10.8, 4.9 Hz, 2H), 3.49 (d, J = 2.4 Hz, 6H). 13C NMR (125
MHz, CD3OD + CDCl3) δ 162.17, 158.41, 156.26, 148.41, 139.75,
134.42, 133.38, 131.84, 126.41, 115.84, 115.66, 99.12, 94.29, 71.96,
70.26, 70.18, 70.26, 69.45, 66.80, 59.20, 58.76. HRMS (ESI) m/z:
416.1622 calcd for C21H22FN3O5 [M + H]+, found 416.1625. HPLC
purity: 100%.
C
21H22BrN3O5 [M + H]+, found 476.0819. HPLC purity: 100%.
4.2.13.4. N-(3-ethynylphenyl)-5-(2-methoxyethoxy)-3-(methoxy-methyl)-
2,3-dihydro-[1,4]dioxino[2,3-f]quinazolin-10-amine (13d). White solid;
Yield: 50%; mp: 238.1–239.3 ◦C. 1H NMR (500 MHz, CD3OD + CDCl3) δ
8.56 (s, 1H), 7.82 (d, J = 0.9 Hz, 1H), 7.69–7.64 (m, 1H), 7.46–7.41 (m,
2H), 7.14 (s, 1H), 4.84 (dd, J = 11.4, 2.1 Hz, 1H), 4.57–4.53 (m, 1H),
4.45 (dd, J = 11.4, 7.5 Hz, 1H), 4.40 (t, J = 4.3 Hz, 2H), 3.89 (dd, J =
6.9, 3.6 Hz, 2H), 3.83 (qd, J = 10.8, 4.9 Hz, 2H), 3.49 (s, 6H), 3.38 (s,
1H). 13C NMR (125 MHz, CD3OD + CDCl3) δ 158.26, 156.36, 148.34,
139.64, 136.00, 134.37, 133.48, 130.48, 129.04, 127.47, 124.58,
123.25, 99.19, 94.26, 82.23, 78.48, 72.01, 70.25, 70.16, 69.50, 66.85,
59.20, 58.75. HRMS (ESI) m/z: 422.1716 calcd for C23H23N3O5 [M +
H]+, found 422.1720. HPLC purity: 99.27%.
4.2.13.9. N-(4-chlorophenyl)-5-(2-methoxyethoxy)-3-(methoxy-methyl)-
2,3-dihydro-[1,4]dioxino[2,3-f]quinazolin-10-amine (13i). White solid;
Yield: 61%; mp: 247.1–247.8 ◦C. 1H NMR (500 MHz, CD3OD + CDCl3) δ
8.55 (s, 1H), 7.68–7.63 (m, 2H), 7.48–7.43 (m, 2H), 7.12 (s, 1H), 4.83
(dd, J = 11.4, 2.2 Hz, 1H), 4.56–4.51 (m, 1H), 4.45 (dd, J = 11.4, 7.5 Hz,
1H), 4.40 (t, J = 4.4 Hz, 2H), 3.89 (dd, J = 7.3, 3.7 Hz, 2H), 3.83 (qd, J
= 10.7, 4.8 Hz, 2H), 3.49 (d, J = 2.1 Hz, 6H). 13C NMR (125 MHz,
CD3OD + CDCl3) δ 158.22, 156.37, 148.33, 139.66, 134.49, 134.36,
133.48, 132.45, 129.02, 125.58, 99.22, 94.24, 71.99, 70.24, 70.17,
69.49, 66.84, 59.20, 58.76. HRMS (ESI) m/z: 432.1326 calcd for
C
21H22ClN3O5 [M + H]+, found 432.1330. HPLC purity: 100%.
4.2.13.10. N-(4-bromophenyl)-5-(2-methoxyethoxy)-3-(methoxy-
methyl)-2,3-dihydro-[1,4]dioxino[2,3-f]quinazolin-10-amine
4.2.13.5. 5-(2-methoxyethoxy)-3-(methoxymethyl)-N-(3-methoxy-
(13j).
phenyl)-2,3-dihydro-[1,4]dioxino-[2,3-f]quinazolin-10-amine
(13e).
White solid; Yield: 74%; mp: 240.4–242.4 ◦C. 1H NMR (500 MHz,
CD3OD + CDCl3) δ 8.55 (s, 1H), 7.60 (s, 4H), 7.11 (s, 1H), 4.83 (dd, J =
11.4, 2.2 Hz, 1H), 4.56–4.51 (m, 1H), 4.45 (dd, J = 11.4, 7.5 Hz, 1H),
4.40 (t, J = 4.4 Hz, 2H), 3.89 (dd, J = 7.3, 3.7 Hz, 2H), 3.83 (qd, J =
10.7, 4.8 Hz, 2H), 3.49 (d, J = 1.9 Hz, 6H). 13C NMR (125 MHz, CD3OD
+ CDCl3) δ 158.15, 156.36, 148.35, 139.64, 135.02, 134.43, 133.49,
132.01, 125.81, 120.21, 99.26, 94.28, 71.99, 70.24, 70.17, 69.49,
66.84, 59.20, 58.76. HRMS (ESI) m/z: 476.0821 calcd for C21H22BrN3O5
[M + H]+, found 476.0821. HPLC purity: 100%.
White solid; Yield: 85%; mp: 233.0–233.7 ◦C. 1H NMR (500 MHz,
CD3OD + CDCl3) δ 8.54 (s, 1H), 7.39 (t, J = 8.2 Hz, 1H), 7.31 (t, J = 2.2
Hz, 1H), 7.28 (s, 1H), 7.17 (dd, J = 8.0, 1.3 Hz, 1H), 6.91 (dd, J = 8.1,
2.1 Hz, 1H), 4.80 (dd, J = 11.3, 2.1 Hz, 1H), 4.53 (dt, J = 6.7, 3.6 Hz,
1H), 4.45 (dd, J = 11.3, 7.4 Hz, 1H), 4.41 (t, J = 4.3 Hz, 2H), 3.90–3.78
(m, 7H), 3.49 (s, 6H). 13C NMR (125 MHz, CD3OD + CDCl3) δ 160.15,
158.13, 156.21, 148.28, 139.33, 136.70, 134.47, 133.41, 129.91,
116.08, 112.43, 110.14, 99.15, 94.69, 71.75, 70.16, 70.12, 69.52,
66.93, 59.43, 58.92, 55.30. HRMS (ESI) m/z: 428.1822 calcd for
C
22H25N3O6 [M + H]+, found 428.1830. HPLC purity: 100%.
4.2.13.11. 5-(2-methoxyethoxy)-3-(methoxymethyl)-N-p-tolyl-2,3-dihy-
dro-[1,4]dioxino[2,3-f]quinazolin-10-amine (13k). White solid; Yield:
85%; mp: 233.0–233.7 ◦C. 1H NMR (500 MHz, CD3OD + CDCl3) δ 8.54
(s, 1H), 7.39 (t, J = 8.2 Hz, 1H), 7.31 (t, J = 2.2 Hz, 1H), 7.28 (s, 1H),
7.17 (dd, J = 8.0, 1.3 Hz, 1H), 6.91 (dd, J = 8.1, 2.1 Hz, 1H), 4.80 (dd, J
= 11.3, 2.1 Hz, 1H), 4.53 (dt, J = 6.7, 3.6 Hz, 1H), 4.45 (dd, J = 11.3,
4.2.13.6. 5-(2-methoxyethoxy)-3-(methoxymethyl)-N-(3-(trifluoro
methyl)phenyl)-2,3-dihydro-[1,4]dioxino[2,3-f]quinazolin-10-amine
(13f). White solid; Yield: 45%; mp: 232.3–233.0 ◦C. 1H NMR (500 MHz,
CD3OD + CDCl3) δ 8.60 (s, 1H), 8.05 (s, 1H), 7.89 (d, J = 7.8 Hz, 1H),
7.63 (dt, J = 24.5, 8.7 Hz, 2H), 7.14 (s, 1H), 4.85 (dd, J = 11.4, 2.2 Hz,
1H), 4.58–4.53 (m, 1H), 4.46 (dd, J = 11.4, 7.5 Hz, 1H), 4.41 (t, J = 4.4
Hz, 2H), 3.90 (dd, J = 7.2, 3.7 Hz, 2H), 3.84 (qd, J = 10.8, 4.9 Hz, 2H),
3.49 (d, J = 1.5 Hz, 6H). 13C NMR (125 MHz, CD3OD + CDCl3) δ 158.45,
7.4 Hz, 1H), 4.41 (t, J = 4.3 Hz, 2H), 3.90–3.78 (m, 7H), 3.49 (s, 6H). 13
C
NMR (125 MHz, CD3OD + CDCl3) δ 160.15, 158.13, 156.21, 148.28,
139.33, 136.70, 134.47, 133.41, 129.91, 116.08, 112.43, 110.14, 99.15,
94.69, 71.75, 70.16, 70.12, 69.52, 66.93, 59.43, 58.92, 55.30, 0.75.
8

X. Qin et al.
Bioorganic Chemistry 110 (2021) 104743
HRMS (ESI) m/z: 428.1872 calcd for C22H25N3O6 [M + H]+, found
4.37 (dd, J = 11.1, 6.8 Hz, 1H), 4.23 (t, J = 6.5 Hz, 2H), 3.87 (s, 3H),
3.85–3.71 (m, 6H), 3.49 (s, 3H), 2.55 (dd, J = 21.4, 14.2 Hz, 6H),
2.19–2.04 (m, 2H). 13C NMR (125 MHz, CDCl3 + CD3OD) δ 160.13,
156.70, 154.08, 153.16, 146.71, 140.02, 137.87, 131.17, 129.50,
114.03, 109.22, 107.89, 102.37, 101.51, 71.32, 70.46, 67.36, 66.98,
66.25, 59.65, 55.32, 55.31, 53.70, 25.97. HRMS (ESI) m/z: 497.2400
calcd for C26H32N4O6 [M + H]+, found 497.2349. HPLC purity: 99.64%.
428.1879. HPLC purity: 100%.
4.2.13.12. N-(3-fluorophenyl)-3-(methoxymethyl)-5-(3-morpholino
propoxy)-2,3-dihydro-[1,4]dioxino[2,3-f] quinazolin-10-amine (14a).
White solid; Yield: 65%; mp: 125.0–125.9 ◦C. 1H NMR (400 MHz, CDCl3
+ CD3OD) δ 9.44 (s, 1H), 8.58 (s, 1H), 7.86 (d, J = 11.3 Hz, 1H), 7.34 (q,
J = 7.5 Hz, 2H), 6.97 (d, J = 9.2 Hz, 1H), 6.83 (t, J = 7.5 Hz, 1H), 4.65
(d, J = 11.0 Hz, 1H), 4.56–4.45 (m, 1H), 4.38 (dd, J = 11.1, 6.9 Hz, 1H),
4.23 (t, J = 6.4 Hz, 2H), 3.82 (dd, J = 10.3, 4.6 Hz, 1H), 3.75 (dd, J =
8.6, 5.2 Hz, 5H), 2.55 (dd, J = 21.4, 14.2 Hz, 6H), 2.10 (p, J = 6.7 Hz,
2H). 13C NMR (125 MHz, CDCl3 + CD3OD) δ 163.09, 156.49, 153.91,
153.38, 146.84, 140.58, 137.76, 131.40, 129.83, 116.63, 110.25,
108.82, 102.52, 101.51, 71.41, 70.50, 67.46, 67.04, 66.38, 59.72,
55.35, 53.77, 26.02. HRMS (ESI) m/z: 485.2200 calcd for C25H29FN4O5
[M + H]+, found 485 0.2193. HPLC purity: 100%.
4.2.13.17. 3-(methoxymethyl)-5-(3-morpholino-propoxy)-N-(3-(tri-
fluoromethyl)phenyl)-2,3-dihydro-[1,4]dioxino[2,3-f]quinazolin-10-amine
(14f). White solid; Yield: 52%; mp: 130.6–134.4 ◦C. 1H NMR (400 MHz,
CDCl3 + CD3OD) δ 9.47 (s, 1H), 8.58 (s, 1H), 8.03 (d, J = 8.4 Hz, 2H),
7.51 (t, J = 7.7 Hz, 1H), 7.38 (d, J = 7.6 Hz, 1H), 6.99 (d, J = 9.6 Hz,
1H), 4.68 (d, J = 11.0 Hz, 1H), 4.51 (d, J = 6.0 Hz, 1H), 4.40 (dd, J =
11.0, 6.9 Hz, 1H), 4.24 (t, J = 6.4 Hz, 2H), 3.83 (dd, J = 10.2, 4.6 Hz,
1H), 3.75 (dd, J = 10.6, 6.3 Hz, 5H), 3.49 (s, 3H), 2.57 (t, J = 7.1 Hz,
2H), 2.50 (s, 4H), 2.12 (dd, J = 13.6, 6.8 Hz, 2H). 13C NMR (125 MHz,
CDCl3 + CD3OD) δ 156.52, 153.88, 153.48, 146.90, 139.54, 137.74,
131.49, 131.31, 129.37, 124.66, 123.30, 120.13, 118.15, 102.55,
101.47, 71.45, 70.52, 67.48, 67.04, 66.45, 59.72, 55.35, 53.77, 26.03.
HRMS (ESI) m/z: 535.2168 calcd for C26H29F3N4O5 [M + H]+, found
535.2170. HPLC purity: 96.48%.
4.2.13.13. N-(3-chlorophenyl)-3-(methoxymethyl)-5-(3-morpholinopro-
poxy)-2,3-dihydro-[1,4]dioxino[2,3-f] quinazolin-10-amine (14b). White
solid; Yield: 78%; mp: 149.0–150.8. 1H NMR (400 MHz, CDCl3
+
CD3OD) δ 9.38 (s, 1H), 8.57 (s, 1H), 7.93 (s, 1H), 7.61 (d, J = 8.2 Hz,
1H), 7.37–7.22 (m, 1H), 7.09 (d, J = 7.3 Hz, 1H), 6.96 (d, J = 8.4 Hz,
1H), 4.64 (d, J = 10.9 Hz, 1H), 4.54–4.44 (m, 1H), 4.38 (dd, J = 11.1,
6.9 Hz, 1H), 4.22 (t, J = 6.5 Hz, 2H), 3.78 (ddd, J = 13.9, 9.0, 4.4 Hz,
6H), 3.48 (s, 3H), 2.54 (dd, J = 21.3, 14.1 Hz, 6H), 2.10 (p, J = 6.7 Hz,
2H). 13C NMR (125 MHz, CDCl3 + CD3OD) δ 156.41, 153.84, 153.33,
146.77, 140.13, 137.68, 134.43, 131.35, 129.76, 123.54, 121.39,
119.41, 102.44, 101.42, 71.35, 70.45, 67.39, 66.98, 66.32, 59.65,
55.29, 53.71, 25.96. HRMS (ESI) m/z: 501.1905 calcd for C25H29ClN4O5
[M + H]+, found 501.1892. HPLC purity: 95.63%.
4.2.13.18. N-(3-chloro-4-fluorophenyl)-3-(methoxymethyl)-5-(3-morpho-
linopropoxy)-2,3-dihydro-[1,4]dioxino[2,3-f]
quinazolin-10-amine
(14g). White solid; Yield: 59%; mp: 158.7–160.8 ◦C. 1H NMR (400
MHz, CDCl3 + CD3OD) δ 9.28 (s, 1H), 8.55 (s, 1H), 7.95 (d, J = 4.3 Hz,
1H), 7.60–7.53 (m, 1H), 7.16 (t, J = 8.8 Hz, 1H), 6.97 (d, J = 9.5 Hz,
1H), 4.65 (d, J = 11.2 Hz, 1H), 4.50 (d, J = 5.0 Hz, 1H), 4.38 (dd, J =
11.0, 6.9 Hz, 1H), 4.23 (t, J = 6.4 Hz, 2H), 3.82 (dd, J = 10.2, 4.6 Hz,
1H), 3.75(dd, J = 8.9, 5.2 Hz, 5H), 3.49 (s, 3H), 2.57 (t, J = 7.1 Hz, 2H),
2.50 (s, 4H), 2.17–2.05 (m, 2H). 13C NMR (125 MHz, CDCl3 + CD3OD) δ
156.55, 153.90, 153.50, 146.81, 137.76, 135.55, 131.42, 123.87,
121.47, 120.91, 116.56, 116.38, 102.52, 101.33, 71.41, 70.49, 67.48,
67.04, 66.41, 59.72, 55.35, 53.77, 26.02. HRMS (ESI) m/z: 519.1811
calcd for C25H28ClFN4O5 [M + H]+, found 519.1812. HPLC purity:
95.80%.
4.2.13.14. N-(3-bromophenyl)-3-(methoxymethyl)-5-(3-morpho-
propoxy)-2,3-dihydro-[1,4]dioxino-[2,3-f]quinazolin-10-amine
lino
(14c).
White solid; Yield: 80%; mp: 139.4–141.2 ◦C. 1H NMR (400 MHz, CDCl3
+ CD3OD) δ 9.37 (s, 1H), 8.57 (s, 1H), 8.04 (s, 1H), 7.70 (d, J = 1.1 Hz,
1H), 7.25 (d, J = 4.6 Hz, 2H), 6.96 (d, J = 8.8 Hz, 1H), 4.65 (d, J = 11.0
Hz, 1H), 4.55–4.45 (m, 1H), 4.38 (dd, J = 11.1, 6.9 Hz, 1H), 4.23 (t, J =
6.5 Hz, 2H), 3.78 (ddd, J = 13.8, 8.9, 4.4 Hz, 6H), 3.49 (s, 3H), 2.55 (dd,
J = 21.3, 14.1 Hz, 6H), 2.10 (p, J = 6.7 Hz, 2H). 13C NMR (125 MHz,
CDCl3 + CD3OD) δ 156.40, 153.86, 153.34, 146.77, 140.27, 137.68,
131.35, 130.08, 126.49, 124.24, 122.46, 119.95, 102.44, 101.42, 71.36,
70.44, 67.40, 66.97, 66.33, 59.66, 55.29, 53.70, 25.96. HRMS (ESI) m/z:
545.1400 calcd for C25H29BrN4O5 [M + H]+, found 545.1403. HPLC
purity: 100%.
4.2.13.19. N-(4-fluorophenyl)-3-(methoxymethyl)-5-(3-morpholino pro-
poxy)-2,3-dihydro-[1,4]dioxino[2,3-f]quinazolin-10-amine (14h). White
solid; Yield: 63%; mp: 122.3–123.4 ◦C. 1H NMR (400 MHz, CDCl3
+
CD3OD) δ 9.24 (s, 1H), 8.51 (s, 1H), 7.68 (dd, J = 8.7, 4.8 Hz, 2H), 7.10
(t, J = 8.6 Hz, 2H), 6.95 (d, J = 9.4 Hz, 1H), 4.63 (d, J = 11.0 Hz, 1H),
4.50 (d, J = 6.0 Hz, 1H), 4.37 (dd, J = 11.1, 6.8 Hz, 1H), 4.23 (t, J = 6.5
Hz, 2H), 3.81 (dd, J = 10.2, 4.6 Hz, 1H), 3.75 (t, J = 6.6 Hz, 5H), 3.49 (s,
3H), 2.57 (t, J = 7.1 Hz, 2H), 2.49 (s, 4H), 2.17–2.04 (m, 2H). 13C NMR
(125 MHz, CDCl3 + CD3OD) δ 156.86, 154.26, 154.07, 153.23, 146.69,
137.91, 134.69, 131.18, 123.91, 123.85, 115.64, 115.46, 102.37,
101.31, 71.32, 70.45, 67.38, 66.98, 66.28, 59.65, 55.30, 53.71, 25.97.
HRMS (ESI) m/z: 485.2200 calcd for C25H29FN4O5 [M + H]+, found
485.2200. HPLC purity: 100%.
4.2.13.15. N-(3-ethynylphenyl)-3-(methoxymethyl)-5-(3-morpho-
linopropoxy)-2,3-dihydro-[1,4]dioxino[2,3-f]
quinazolin-10-amine
(14d). White solid; Yield: 59%; mp: 112.1–113.7 ◦C. 1H NMR (400
MHz, CDCl3 + CD3OD) δ 9.35 (s, 1H), 8.55 (s, 1H), 7.84 (d, J = 9.9 Hz,
2H), 7.34 (t, J = 7.8 Hz, 1H), 7.24 (s, 1H), 6.95 (d, J = 9.4 Hz, 1H), 4.63
(d, J = 11.2 Hz, 1H), 4.49 (d, J = 5.6 Hz, 1H), 4.37 (dd, J = 11.1, 6.8 Hz,
1H), 4.21 (t, J = 6.4 Hz, 2H), 4.02 (d, J = 5.5 Hz, 1H), 3.84–3.66 (m,
6H), 3.47 (s, 3H), 2.53 (dd, J = 21.4, 14.2 Hz, 6H), 2.17–2.00 (m, 2H).
13C NMR (125 MHz, CDCl3 + CD3OD) δ 155.59, 152.99, 152.31, 145.78,
137.98, 136.80, 130.31, 127.93, 126.45, 123.94, 121.68, 121.25,
101.44, 100.47, 82.52, 70.38, 69.47, 66.42, 66.01, 65.31, 58.69, 54.33,
52.74, 24.99. HRMS (ESI) m/z: 491.2294 calcd for C27H30N4O5 [M +
H]+, found 491.2293. HPLC purity: 100%.
4.2.13.20. N-(4-bromophenyl)-3-(methoxymethyl)-5-(3-morpholino pro-
poxy)-2,3-dihydro-[1,4]dioxino[2,3-f]quinazolin-10-amine (14i). White
solid; Yield: 85%; mp: 169.8–172.3 ◦C. 1H NMR (400 MHz, CDCl3
+
CD3OD) δ 9.29 (d, J = 32.7 Hz, 1H), 8.54 (s, 1H), 7.67 (d, J = 8.6 Hz,
2H), 7.50 (d, J = 8.7 Hz, 2H), 6.96 (d, J = 9.1 Hz, 1H), 4.64 (d, J = 10.9
Hz, 1H), 4.55–4.45 (m, 1H), 4.37 (dd, J = 11.1, 6.9 Hz, 1H), 4.23 (t, J =
6.5 Hz, 2H), 3.81 (dd, J = 10.2, 4.6 Hz, 1H), 3.74 (t, J = 6.5 Hz, 5H),
3.49 (s, 3H), 2.55 (dd, J = 21.4, 14.2 Hz, 6H), 2.10 (p, J = 6.8 Hz, 2H).
13C NMR (125 MHz, CDCl3 + CD3OD) δ 156.53, 153.94, 153.36, 146.81,
138.02, 137.79, 131.85, 131.36, 123.33, 116.30, 102.49, 101.49, 71.39,
70.51, 67.45, 67.04, 66.38, 59.72, 55.35, 53.77, 26.03. HRMS (ESI) m/z:
547.1379 calcd for C25H29BrN4O5 [M + H]+, found 547.1366. HPLC
purity: 100%.
4.2.13.16. 3-(methoxymethyl)-N-(3-methoxyphenyl)-5-(3-mor-
pholinopropoxy)-2,3-dihydro-[1,4]dioxino-[2,3-f]
quinazolin-10-amine
(14e). White solid; Yield: 67%; mp: 127.6–128.1 ◦C. 1H NMR (400
MHz, CDCl3 + CD3OD) δ 9.36 (s, 1H), 8.55 (s, 1H), 7.57 (s, 1H), 7.31 (d,
J = 8.1 Hz, 1H), 7.21 (d, J = 7.9 Hz, 1H), 6.96 (d, J = 9.2 Hz, 1H), 6.71
(d, J = 7.0 Hz, 1H), 4.63 (d, J = 10.5 Hz, 1H), 4.50 (d, J = 6.4 Hz, 1H),
9

X. Qin et al.
Bioorganic Chemistry 110 (2021) 104743
4.2.13.21. 3-(methoxymethyl)-N-(4-methoxy
phenyl)-5-(3-morpholin
(14j).
MTT assay (Dojindo, Japan). Cells lines were seeded at a density of 3000
cells/well in 96-well microtiter plates and were incubated at 37 ◦C
overnight in a humidified incubator containing 5% CO2. Cells were
dosed with compounds at final concentrations ranging from 0.025 to 80
opropoxy)-2,3-dihydro-[1,4]dioxino[2,3-f]quinazolin-10-amine
White solid; Yield: 65%; mp: 129.8–131.3 ◦C. 1H NMR (400 MHz, CDCl3
+ CD3OD) δ 9.14 (s, 1H), 8.48 (s, 1H), 7.58 (d, J = 8.6 Hz, 2H),
7.02–6.88 (m, 3H), 4.61 (d, J = 10.8 Hz, 1H), 4.49 (d, J = 6.4 Hz, 1H),
4.36 (dd, J = 11.1, 6.8 Hz, 1H), 4.22 (t, J = 6.4 Hz, 2H), 3.88–3.77 (m,
4H), 3.75 (d, J = 3.9 Hz, 5H), 3.48 (s, 3H), 2.54 (dd, J = 21.3, 14.2 Hz,
6H), 2.22–2.02 (m, 2H). 13C NMR (125 MHz, CDCl3 + CD3OD) δ 157.17,
156.53, 154.33, 153.06, 146.61, 138.08, 131.59, 130.99, 124.22,
114.21, 102.29, 101.33, 71.31, 70.48, 67.34, 66.98, 66.21, 59.64,
55.50, 55.31, 53.70, 25.98. HRMS (ESI) m/z: 497.2400 calcd for
μ
M in each well of the plate. After 48 h, 50 µL of the MTT solution was
added and incubated for 4 h. Cell survival was determined by measuring
the absorbance at 492 nm using a microplate reader. A calibration curve
was prepared using the data obtained from the wells that contain known
numbers of viable cells to determine the IC50 of the target compounds.
26H32N4O6 [M + H]+, found 497.2391. HPLC purity: 95.84%.
4.5. Docking study
C
The molecular docking of compound 13b into the three-dimensional
EGFR complex structure (PDB code: 1 M17.pdb, downloaded from the
PDB) was performed using CDocker. Unwanted water and ligands were
removed by the DS4.0. The structures of the molecules were drawn by
Gaussian 03 software, then optimized the molecules to the minimum
energy conformation used the semi-empirical AM1 method. Docking
procedure was performed by AutoDock 4.0 software with the help of
Autodock Tools. Tutorials may be found at: http://autodock.scripps.
edu/faqs-help/tutorial.
4.2.13.22. 3-(methoxymethyl)-5-(3-morpholino-propoxy)-N-phenyl-2,3-
dihydro-[1,4]dioxino[2,3-f]quinazolin-10-amine (14k). White solid;
Yield: 78%; mp: 128.0–129.7 ◦C. 1H NMR (400 MHz, CDCl3 + CD3OD) δ
9.35 (s, 1H), 8.54 (s, 1H), 7.75 (d, J = 7.9 Hz, 2H), 7.41 (t, J = 7.7 Hz,
2H), 7.15 (t, J = 7.2 Hz, 1H), 6.96 (d, J = 9.2 Hz, 1H), 4.63 (d, J = 10.8
Hz, 1H), 4.50 (d, J = 5.4 Hz, 1H), 4.37 (dd, J = 11.1, 6.8 Hz, 1H), 4.23 (t,
J = 6.4 Hz, 2H), 3.87–3.68 (m, 6H), 3.49 (s, 3H), 2.55 (dd, J = 21.5,
14.3 Hz, 6H), 2.10 (p, J = 6.7 Hz, 2H). 13C NMR (125 MHz, CDCl3
+
CD3OD) δ 156.83, 154.19, 153.22, 146.79, 138.84, 137.99, 131.21,
128.97, 124.00, 121.93, 102.44, 101.54, 71.39, 70.53, 67.42, 67.05,
66.31, 59.71, 55.37, 53.77, 26.04. HRMS (ESI) m/z: 467.2294 calcd for
4.5.1. EGFR binding pocket model building
C
25H30N4O6 [M + H]+, found 467.2293. HPLC purity: 98.78%.
The EGFR binding pocket model was generated by Discovery Studio
4.0 (DS4.0) based on the crystal structure of EGFR/erlotinib. Unwanted
water and ligands were removed by the DS4.0 to build the initial re-
ceptor structure for docking.
4.3. EGFR inhibitory assay
Kinase-Glo luminescent kinase assay is
a homogeneous non-
4.5.2. Small molecular preparation
radioactive method for determining the activity of purified kinases by
quantifying the amount of ATP remaining in solution following a kinase
reaction. Target compounds and positive compound gefitinib were dis-
solved in DMSO as 10 mM stock solution, then diluted it to 100 µM with
DMSO and transferred to the dose plate. The compounds was serially
diluted with DMSO in 5-fold. Then each concentration was diluted 10-
fold with reaction buffer (containing 25 mM HEPES, 10 mM MgCl2,
100 µg/mL BSA, 0.01% TritonX-100, 2.5 mM DTT and adjusted pH to
7.4) to obtain a 10 × final concentration. Transferred compounds with
its concentration ranging from 10 µM to 0.0006 µM to assay plate for
EGFR activity test with a volume of 1 µL/well. For HPE (hundred percent
effect: No kinase and no compound, but containing ATP, substrate and
1% DMSO) and ZPE (zero percent effect: No compound but containing
kinase, ATP, substrate and 1% DMSO) well, diluted 2 µL DMSO 10-fold
with reaction buffer to obtain 10% DMSO solution. Then transferred it to
the assay plat, 1 µL/well. Procedure for kinase reaction is: 1) Add 10 ×
compound to the assay plate in a 384-well plate layout, 1 µL/well. For
the HPE and ZPE wells, equal volume (1 µL/well) of 10% DMSO was
added to the 384-well assay plate; 2) Added 2.5 × kinase EGFR into the
assay plate as 384-well plate layout, 4 µL/well. For HPE wells, an equal
volume (4 µL/well) of assay buffer was added to the 384-well assay
plate; 3) Centrifuged the assay plate with 1000 rpm for 1 min to mix
them; 4) Pre-incubated the assay plate at 30℃ for 30 min; 5) Mixed
equal volume of 4 × ATP and 4 × substrate to obtain 2 × ATP-substrate
mixtures; 6) Added 2 × ATP-substrate mixture to the assay plate, 5 µL/
well; 7) Centrifuged the assay plate at 1000 rpm for 1 min to mix; 8)
Incubated the plate for one hour at 30 ◦C; 9) Kinase glo plus was added
The small molecular structure of 13b was drawn by Gaussian03
software, then optimized the molecules to the minimum energy
conformation used the semi-empirical AM1 method.
4.5.3. Docking protocol
Docking procedure was performed by AutoDock 4 software with the
help of Autodock Tools. The grid maps were calculated using AutoGrid4
for three dockings, a grid map with 60 × 70 × 60 points and a grid-point
spacing of 0.375 Å was applied. In order to fully explore the possible
binding conformations, 100 conformations were generated using the
Lamarckian Genetic Algorithm (LGA). For other docking parameters,
standard values were used as software default. Cluster analysis was
performed on the results using a root mean square (RMS) tolerance of
2.0 Å. The conformations and binding energy for further analysis were
obtained from the average of the biggest cluster.
Declaration of Competing Interest
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.
Acknowledgments
The authors would like to acknowledge financial support from Sci-
entific Research Project of Guangxi University for Nationalities
(2019KJQN005), the Middle-aged and Young Teachers’ Basic Ability
Promotion Project of Guangxi (2017KY0165), the Talent Introduction
Project of Guangxi University for Nationalities (2016MDQD008), and
the Science and Technology Major Project of Guangxi (AA18242026).
to each well (10 μL/well), and then incubated the assay plate for 20 min
at 27 ◦C; 10) Read luminescence signal with Envision. The raw data were
analysed by Prism 5.0 and the inhibitory rate was calculated by the
following formula: Compound inhibitory rate = (“compound” reading-
ZPE)/(HPE-ZPE) × 100%.
Appendix A. Supplementary material
4.4. The anti-proliferative and cytotoxicity activity
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.104743.
The anti-proliferative and cytotoxicity activity was determined using
10

X. Qin et al.
Bioorganic Chemistry 110 (2021) 104743
T. Klinowska, G.H. Richmond, M. Cantarini, D.W. Kim, M.R. Ranson, W. Pao,
Cancer Discoverv 4 (9) (2014) 1046–1061.
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