Structure–activity relationship study of novel quinazoline-based 1,6-naphthyridinones as MET inhibitors with potent antitumor efficacy

Article abstract of DOI:10.1016/j.ejmech.2020.112785

As a privileged scaffold, the quinazoline ring is widely used in the development of EGFR inhibitors, while few quinazoline-based MET inhibitors are reported. In our ongoing efforts to develop new MET-targeted anticancer drug candidates, a series of quinaz

Full text of DOI:10.1016/j.ejmech.2020.112785

European Journal of Medicinal Chemistry 208 (2020) 112785
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Structureeactivity relationship study of novel quinazoline-based 1,6-
naphthyridinones as MET inhibitors with potent antitumor efficacy
,
,
,
Lin-Sheng Zhuo a 1, Feng-Xu Wu a 1, Ming-Shu Wang a 1, Hong-Chuang Xu a,
Fan-Peng Yang a, Yan-Guang Tian a, Xing-E. Zhao b, Zhi-Hui Ming b, Xiao-Lei Zhu a,
Ge-Fei Hao a **, Wei Huang a
,
, *
a Key Laboratory of Pesticide & Chemical Biology of Ministry of Education, International Joint Research Center for Intelligent Biosensor Technology and
Health, College of Chemistry, Central China Normal University, Wuhan, 430079, PR China
b Jiangsu Key Laboratory of Molecular Targeted Antitumor Drug Research, Jiangsu Simcere Pharmaceutical Co. Ltd, Nanjing, 210042, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
As a privileged scaffold, the quinazoline ring is widely used in the development of EGFR inhibitors, while
few quinazoline-based MET inhibitors are reported. In our ongoing efforts to develop new MET-targeted
Received 16 June 2020
Received in revised form
3 August 2020
Accepted 19 August 2020
Available online 31 August 2020
anticancer drug candidates,
a series of quinazoline-based 1,6-naphthyridinone derivatives were
designed, synthesized, and evaluated for their biological activities. The preliminary SARs studies indicate
that the quinazoline scaffold was also acceptable for the block A of class II MET inhibitors. The further
pharmacokinetic studies led to the identification of the most promising compound 22a with favorable
in vitro potency (MET, IC50 ¼ 9.0 nM), human microsomal metabolic stability (t1/2 ¼ 621.2 min) and oral
bioavailability (F ¼ 42%). Moreover, 22a displayed good in vivo antitumor efficacy (IR of 81% in 75 mg/kg)
in MET-positive human glioblastoma U-87 MG xenograft model. These positive results indicated that 22a
is a potential new MET-targeted antitumor drug lead, which is worthy of further development.
© 2020 Elsevier Masson SAS. All rights reserved.
Keywords:
MET kinase Inhibitor
Antitumor
1,6-naphthyridone
Quinazoline
Structure-activity relationship
1. Introduction
blocking MET activity is a promising approach to the treatment of
cancers where MET is activated [11e13].
Mesenchymal-epithelia transition factor (MET), a member of a
subfamily of heterodimeric receptor tyrosine kinases (RTKs), is a
proto-oncogene encoding the high affinity receptor for its natural
ligand-hepatocyte growth factor (HGF) [1]. The HGF/MET axis has
been identified to play a significant role in many normal physio-
logical processes, such as cell proliferation, motility, migration, and
survival, by triggering multiple downstream signaling pathways
including Ras/MEK/MAPK, PI3K-AKT-mTOR and Ras/RAC1/PAK
[2e5]. Abnormal activation of MET signaling has been reported in a
variety of human cancers, occurring as a consequence of activating
mutations, gene amplification, or protein overexpression [6e8].
Importantly, MET overexpression has been correlated with poor
prognosis, and MET amplification is one of the acquired mecha-
nisms of resistance to EGFR inhibitors [9,10]. Consequently,
To date, several MET-targeted therapies were or are under
development, including MET/HGF antibodies and small-molecule
MET inhibitors. With the failures of clinical trials of MET anti-
bodies [14], numerous small-molecule MET inhibitors have or are
being evaluated in different stages [15]. In general, the reported
MET inhibitors can be classified into two classes (class I and class II)
according to their chemotypes and binding modes with MET [16].
The prototype compound of class I inhibitor crizotinib (1, Fig. 1), a
multitargeted inhibitor of MET, ALK, ROS1, and RON, was approved
by FDA for the treatment of patients with ALK-positive advanced
non-small cell lung cancers (NSCLC, 2011) [17]. Capmatinib (2), a
highly potent and selective MET inhibitor, is the first and only
therapy recently approved by the FDA to specifically target meta-
static NSCLC with a mutation that leads to MET exon 14 skipping
(METex14) [18]. The representative class II inhibitor, cabozantinib
(3) had been marketed as multitargeted inhibitors of MET, VEGFR-2
and Ret for treatment of thyroid cancer (2012), renal cell carcinoma
(2016) and liver cancer (2018) [19]. Another good example of class II
inhibitor BMS-777607 (4), a pyridine-based selective MET inhibitor
which primarily targets MET and several MET family members,
* Corresponding author.
** Corresponding author.
E-mail addresses: gefei_hao@foxmail.com (G.-F. Hao), weihuangwuhan@126.
com (W. Huang).
1
L.Z., F.W. and M.W. contributed equally to this work.
https://doi.org/10.1016/j.ejmech.2020.112785
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.

2
L.-S. Zhuo et al. / European Journal of Medicinal Chemistry 208 (2020) 112785
Fig. 1. The structure of representative class I and class II MET inhibitors.
including AXL, RON, and Tyro 3, is currently undergoing phase II
clinical trials for various cancers [20]. Hence, the increasing FDA-
approved drugs and clinical trial drug candidates targeting MET
kinase push the development of small-molecule MET inhibitors to a
hotspot [21e31].
In our previous research, we reported the discovery of diverse
naphthyridine derivatives as kinase inhibitors [32e38]. Among
them, a 1,6-naphthyridinone-based MET inhibitor (5) was acquired
by the combination of strategy a (cyclization & bioisosterism) and b
(heteroatom migration) of block C in BMS-777607 (Fig. 2) [35].
However, compound 5 exhibited unfavorable pharmacokinetic (PK)
properties (CL ¼ 5.0 L/h/kg and AUC0-∞ ¼ 2.2
mg h/mL at oral dose of
10 mg/kg) and poor in vivo antitumor efficacy (Tumor growth in-
hibition/TGI of 15% at 12.5 mg/kg) [35]. Currently, among class II
MET inhibitors, the quinoline ring in the block A of cabozantinib
have been extensively studied. As a most common bioisotere of
quinoline, the quinazoline ring is frequently encountered in me-
dicinal chemistry [39,40], especially in FDA-approved EGFR in-
hibitors (Fig. 2, e.g. Gefitinib, Erlotinib, Vandetanib and Icotinib)
[41,42]. However, few quinazoline-based MET inhibitors are re-
ported [15].
In view of the above, we proposed a bioisosterism strategy c that
Fig. 2. Design of quinazoline-based 1,6-naphthyridinone derivatives as new MET inhibitors.

L.-S. Zhuo et al. / European Journal of Medicinal Chemistry 208 (2020) 112785
3
replaces the pyridine moiety in block A of lead compound 5 with
the privileged quinazoline scaffold from FDA-approved EGFR in-
hibitors. Furthermore, the extensive structural optimizations of
block A, B and C in quinazoline-based 1,6-naphthyridinone de-
rivatives 10 as MET inhibitors were carried out to improve its drug-
likeness.
structure, the compound 10l exists in an extended conformation
that is coincident with the binding conformation of class II in-
hibitors with MET kinase [48].
3. Results and discussion
3.1. The analyses of structure-activity relationships and microsomal
metabolic stability
2. Chemistry
The key 4-((quinazolin-4-yl)oxy)-aniline intermediates were
synthesized according to the pathways described in Schemes 1 and
2. According to the reported method [43], the key intermediate 15
was synthesized from readily available methyl vanillate 11a
(Scheme 1). The eOH group within commercially available methyl
3-hydroxy-4-methoxybenzoate 11a was protected with benzyl
bromide under basic condition to provide 11b. The intermediate 13
was prepared by nitration-reduction-cyclization-chlorination re-
actions of 11b. The resultant chlorinated quinazoline 13 was con-
verted to 14a by nucleophilic substitution with 2-fluoro-4-
nitrophenol under DIPEA in toluene, subsequent de-protection
and reduction of nitro easily afforded intermediate 14c. The key
intermediates 15a-h were successfully yielded by alkylation reac-
tion of 14c with various halides or 2,2-dimethyloxirane. Briefly, the
intermediates 17a-g were also obtained from methyl 3-hydroxy-4-
methoxybenzoate 16 according to synthetic route of compounds
15a-h.
As described in Scheme 2, the intermediates 19a-d were
generated by nucleophilic substitution reaction of commercially
available 18a-c with 2-fluoro-4-nitrophenol or 4-aminophenol
under DIPEA in toluene. Then the intermediates 20a-d were suc-
cessfully obtained by the reduction of nitro of 19a-d [44e46].
The synthesis of target compounds 10a-r and 22a-f were sum-
marized in Scheme 3. The diverse chlorinated 1,6-naphthyridone
21 could be easily obtained by our reported methods [45]. With
intermediate 15, 17 and 20 in hand, target compounds 10a-r and
22a-b were successfully synthesized through the direct reaction of
21 with 15 or 17 or 20 in the presence of hydrochloric acid or p-
toluenesulfonic acid. The 1-NH group of naphthyridine in 10l was
reacted with diverse halides to smoothly provide target compounds
22c-f. In addition, the crystal structure of 10l was further deter-
mined by X-ray diffraction analysis (Fig. 3) [47]. In the crystal
Newly synthesized compounds 10a-r were firstly evaluated for
their enzymatic activities against MET and VEGFR-2 by using the
protocol we previously reported [32,34], with the compound 3
(Cabozantinib) served as a positive control. The privileged quina-
zoline scaffolds (A-1 ~ A-4) from launched EGFR inhibitors were
firstly introduced into the block A to investigate their effects on
MET activity. As shown in Table 1, compounds 10a-d, to our sur-
prise, were found to be active against MET kinase with IC50 of
9.5e23.2 nM, among which compound 10d showed comparable
MET inhibitory activity (IC50 of 9.5 nM) to that of 3 and 5, indicating
that the quinazoline scaffold was also acceptable for block A of class
II MET inhibitors. Unfortunately, Compound 10d only showed
moderate half-life (t1/2 ¼ 49.8 min) from preliminary human
microsomal metabolic stability evaluation. To further improve the
drug-likeness, various substituents were installed on C-7 and C-6
positions of quinazoline moiety. Initially, the SAR of 7-R1 group and
metabolic stability studies were further investigated after fixed the
6-R2 group of quinazoline with methoxy group. The methox-
yethoxy substituted analogue 10e displayed moderate inhibition
against MET with IC50 of 42.0 nM. Interestingly, compound 10f,
substituted at the R1 position with hydroxyethoxy group, showed
comparable MET inhibitory activity (IC50 of 13.9 nM) to that of 3.
However, the introductions of methylene or di-methyl into
hydroxyethoxy group of 10f resulted in a 2.8e4.7-fold loss of ac-
tivity (10g and 10h, IC50 of 39.3 and 65.2 nM). Compared with the
MET inhibitory activity of 3, after the alicyclic amines were installed
on R1 position, the resulted compounds 10i and 10j exhibited about
2.7-fold lower potency, while compound 10k with (pyrrolidin-1-yl)
propoxy group exhibited comparable inhibitory activity (IC50 of
8.3 nM). Moreover, 10f showed 4.6-fold increase in the metabolic
stability (t1/2 ¼ 230.8 min) relative to 10d. Interestingly, compound
10l with prototypal methoxy group installed on the R1 position, not
Scheme 1. Reagent and Conditions: (i) BnBr, K2CO3, DMF, 100 ꢀC; (ii) AcOH, HNO3, 45 ꢀC; (iii) Fe, NH4Cl, EtOH/H2O, reflux; (iv) HCONH2, HCOONH4, 140 ꢀC; (v) POCl3, CHCl3, DMF,
reflux; (vi) 2-fluoro-4-nitrophenol, DIPEA, toluene, reflux; (vii) CF3COOH, reflux; (viii) R6X or R7X or 2,2-dimethyloxirane, K2CO3, CH3CN, reflux.

4
L.-S. Zhuo et al. / European Journal of Medicinal Chemistry 208 (2020) 112785
Scheme 2. Reagent and Conditions: (i) 4-aminophenol or 2-fluoro-4-nitrophenol, DIPEA, toluene, reflux; (ii) Fe, NH4Cl, EtOH/H2O, reflux.
Scheme 3. Reagent and Conditions: (i) a, HCl, isopropanol, 90 ꢀC; b, Et3N, DCM, r. t.; (ii) p-toluenesulfonic acid, isopropanol, 90 ꢀC; (iii) R4X or isobutylene oxide, K2CO3, DMF, r. t.
Fig. 3. X-ray crystal structure of 10l.
only performed comparable MET inhibitory activity (IC50 of 9.7 nM)
to that of 3, but also displayed 5.8-fold increase in the metabolic
activity (IC50 of 7.9e9.4 nM) to 3. Interestingly, compound 10n,
substituted at the R2 position with hydroxyethoxy group, showed a
2.4-fold increase in potency with IC50 of 3.5 nM relative to 3.
However, compounds 10n and 10o exhibited slightly higher clear-
ance rate (t1/2 ¼ 250.2e280.6 min) to that of compound 10l from
the analysis of metabolic stability in human liver microsomes.
Based on the understanding of the binding model of Type II
inhibitors with MET kinase, the block A (quinazoline bearing hy-
drophilic group) extending to solvent exposure area may provide
an opportunity for forming H-bonds with the Met1160 and Pro1158
residues as well as van der Waals interactions with MET backbone,
which may be one of the reasons why most of target compounds
showed moderate to significant potency against MET kinase.
stability (t1/2
¼
290.0 min) compared with compound 10d.
Furthermore, compound 10l displayed comparable cell-based MET
inhibitory activity (IC50 of 59.4 nM) to that of 3 (IC50 of 44.5 nM).
Next, 10l was chosen as an early lead compound and the re-
placements of methoxy group in C-6 position with a series of
substituents were performed to further investigate SAR and
metabolic stability. The installations of substituted-alkoxy or
alicyclic amine groups into 6-R2 position showed similar SAR with
that of 7-R1-substituted analogues 10e-k. The 2-hydroxy-2-
methylpropoxy, (diethylamino)propoxy and (pyrrolidin-1-yl)pro-
poxy analogues (10o-q) displayed comparable MET inhibitory

L.-S. Zhuo et al. / European Journal of Medicinal Chemistry 208 (2020) 112785
5
Table 1
Activities of 10a-r against MET and VEGFR-2 as well as microsomal metabolic stability (human).
Compd.
Block A
R1
IC50 (nM)
MET
Metabolic Stability (t1/2, min)
VEGFR-2
R2
enzyme-baseda
cell-basedb
enzyme-baseda
c
c
10a
10b
A-1
A-2
23.2
19.5
-
121.4
79.2
-
e
e
e
e
10c
10d
10e
A-3
A-4
15.4
9.5
33.0
47.6
139.9
e
49.8
OMe
OMe
OMe
OMe
42.0
e
10f
13.9
39.3
65.2
21.7
68.0
93.9
230.8
e
e
e
10g
10h
e
e
c
10i
10j
10k
OMe
OMe
OMe
OMe
24.5
21.7
8.3
-
e
e
e
e
226.7
127.9
e
130.0
10l
10m
OMe
OMe
9.7
36.0
59.4
267.3
97.3
290.0
e
e
10n
10o
OMe
OMe
3.5
7.9
12.3
52.9
280.6
250.2
e
e
10p
10q
OMe
OMe
9.4
8.1
396.8
e
163.5
145.4
e
e
10r
OMe
15.5
207.6
e
e
5 [35]
9.8
8.4
68
e
e
e
3 (cabozantinib)
44.5
7.0
a
In vitro kinase assays were performed with the indicated purified recombinant MET or VEGFR-2 kinase domains (nM).
IC50 values (nM) for HGF-mediated autophosphoylation in MKN-45 cell line.
No test.
b
c
However, the introduction of hydrophilic groups bearing carbon
Secondly, the introduction of a methyl group into the 2-position of
1,6-naphthyridinone (22b, IC50 of 222.7 nM) lead to a 23.4-fold
lower potency against MET compared to 10l. Interestingly, next,
the installation of a methyl group on R4 position in block C not only
showed comparable MET inhibitory activity (22c, IC50 of 7.1 nM) to
that of compound 10l but also resulted in a significant increase in
VEGFR-2 selectivity by > 422.5-fold. Meanwhile, compounds 20a
and 22c also exhibited comparable cell-based MET inhibitory ac-
tivity (IC50 of 86.5 nM and 82.9 nM, respectively) to that of com-
pounds 3 and 10l. After an ethyl group was introduced to R4
position, 3-fold lower potency against MET was observed relative to
10l (22d, IC50 of 28.8 nM, >275-fold improvement in VEGFR-2
selectivity). Compounds 22e-f exhibited 16.9-folde20.2-fold
lower potency (IC50 of 164.0e196.4 nM) against MET compared
with 10l, indicating that the R4 position can only tolerate less bulky
substituents to benefit the activity. The above results suggested that
steric clash of R4 and R5 group were sensitive to the MET kinase
inhibitory activities. More importantly, the further metabolic sta-
bility study of the potent compounds 22a and 22c indicated that
ether at R1/R2 positions of quinazoline made a negative influence
on metabolic stability of inhibitors. The pharmacological data
above indicated that the 6,7-dimethoxyquinazolin scaffold is the
superior one that can achieve a balance between potency and
metabolic stability.
Meanwhile, among all newly synthesized compounds, 10n with
6-hydroxyethoxy group exhibited comparable inhibitory activity
for VEGFR-2 (IC50 of 12.3 nM) to that of 3 (cabozantinib), and 10a-m
and 10o-r showed good to moderate inhibition against VEGFR-2
kinase with IC50 values ranging from 21.7 to 226.7 nM. It is worth
mentioning that compound 10l possessed moderate (27.6-fold)
selectivity to VEGFR-2.
Based on above description, the SARs of block B and C were
further investigated using the superior 6,7-dimethoxyquinazolin of
10l at the block A (Table 2). Firstly, the removal of F atom in block B
of compound 10l resulted in the maintenance of MET inhibitory
activity (22a, IC50 of 9.0 nM), suggesting that the electronic effect of
R3 group may have less influence on MET inhibitory activity.

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L.-S. Zhuo et al. / European Journal of Medicinal Chemistry 208 (2020) 112785
Table 2
Activities of 22a-f against MET and VEGFR-2 as well as microsomal metabolic stability (human).
Compd.
Block B
R3
Block C
R4
IC50 (nM)
MET
Metabolic Stability (t1/2, min)
VEGFR-2
R5
enzyme-baseda
cell-basedb
86.5
enzyme-baseda
22a
22b
22c
22d
H
F
F
H
H
Me
Et
H
Me
H
9.0
227.2
7.1
111.3
454.9
>3000
>3000
621.2
c
c
-
-
82.9
256.0
F
H
28.8
e
e
22e
F
H
164.0
>3000
e
e
22f
F
H
196.4
>3000
e
e
e
10l
F
H
H
9.7
59.4
267.3
a
In vitro kinase assays were performed with the indicated purified recombinant MET or VEGFR-2 kinase domains (nM).
IC50 values (nM) for HGF-mediated autophosphoylation in MKN-45 cell line.
No test.
b
c
the replacement of F atom with hydrogen atom in block B generated
compound 10l and 22a have the highly similar binding mode with
MET and the active site residues are conservative with little fluc-
tuation. Both of 10l and 22a can form hydrogen bonds (H-bonds)
with Asp1222, Glu1127, Met1160, and Tyr1159. It is reflected on the
binding free energy calculation that 10l and 22a have the approx-
1.1 fold improvement in metabolic stability (22a, t1/2 ¼ 621.2 min)
and the installation of substituents on R4 position led to compa-
rable clearance rate (22c, t1/2 ¼ 256.0 min) to compound 10l (t1/
¼ 290.0 min), respectively.
2
imative
DEele value (shown in Table 4). The highly similar binding
mode and interactions with MET make 10l (IC50 ¼ 9.7 nM) and 22a
(IC50 ¼ 9.0 nM) keep the enzyme activity at the same level.
As shown in Fig. 4B, the replacement of 6-OMe of 10l with
hydroxyethoxy group (10n, IC50 ¼ 3.5 nM) led to the formation of
H-bond between hydroxyl and Asp1164, which is not observed
between 10l and MET. Meanwhile, the two methylenes of
hydroxyethoxy group in 10n can form better hydrophobic in-
teractions with Phe1123 compared with 10l. As the hydroxyethoxy
group extends into solvent region of the active site, it may form the
H-bond network through the water molecules and other polar
groups to enhance the interaction with MET. Compound 10n has
3.2. Enzymatic selectivity assays
To examine the selectivity of compounds 10l and 22a on MET
kinase over other kinases, they were screened against 7 other
tyrosine kinases (Table 3). Consistent with their high potency
against MET (IC50 of 9.7 nM and 9.0 nM, respectively), both of 10l
and 22a also can strongly inhibit Flt-3 (IC50 of 10.2 nM and 16.9 nM,
respectively) and c-Kit (IC50 of 45.1 nM and 28.3 nM, respectively).
10l displayed moderate inhibitory activity against Ret (IC50 of
65.3 nM) and Ron (IC50 of 90.1 nM). Additionally, both of 10l and
22a showed good selectivity against PDGFR-b (IC50 of 1359 nM and
>2000 nM, respectively). These data suggested that compound 10l
and 22a are potential promising multitargeted inhibitors of kinases.
the more positive contribution to the binding with MET on
and EvdW than 10l (shown in Table 4), that proves our analysis
above.
DEele
D
As shown in Fig. 4C, after installed a methyl group on N (1) in
block C of 10l, the resulted compound 22c (IC50 ¼ 7.1 nM) led to the
rotation of Glu1127 sidechain and the disappearance of H-bond
between 22c and Glu1127. However, the methyl can form hydro-
phobic interactions with the sidechain of Met1131, which makes up
for the loss of binding energy caused by the loss of H-bond and
therefore make 10l and 22c keep the enzyme activity at the same
level. This was also can be observed from the free energy calcula-
3.3. Molecular docking studies
To further understand the SAR, molecular docking was per-
formed for the selected compounds. As shown in Fig. 4A,
Table 3
Kinase selectivity profile of compounds 10l and 22a.
tion result. Although 22c has less contribution on
DEele than 10l, it
kinase
Axl
10l (IC50, nM)
11.0
22a (IC50, nM)
has more contribution on EvdW (shown in Table 4).
D
As shown in Fig. 4D, after introduced a methoxyethyl group into
N (1) in block C of 10l, there is a steric clash between the resulted
compound 22e (IC50 ¼ 164.0 nM) with Glu1127. According to the
molecular docking and molecular dynamics (MD) simulation result,
although the sidechain of Glu1127 has a rotation, 22e still clash
with Glu1127 and makes the binding of the entire naphthyridine
ring (block C) in the active pocket slightly crowded. This is the
e
c-Kit
Flt-3
MET
45.1
10.2
9.7
28.3
16.9
9.0
PDGFR-
Ret
b
1359
65.3
>2000
e
Ron
90.1
e
VEGFR-2
267.3
111.3

L.-S. Zhuo et al. / European Journal of Medicinal Chemistry 208 (2020) 112785
7
Fig. 4. (A) The proposed binding mode overlay of 10l (gray) and 22a (purple) with MET (PDB: 3F82 [49]); (B) The proposed binding mode overlay of 10l (gray) and 10n (purple) with
MET; (C) The proposed binding mode overlay of 10l (gray) and 22c (purple) with MET; (D) The proposed binding mode overlay of 10l (gray) and 22e (purple) with MET. (For
interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Table 4
3.4. PK profile of compound 22a
Binding free energy calculation of compounds with MET.
On the basis of the favorable in vitro potency and prominent
metabolic stability in human liver microsomes, compound 22a was
selected to further evaluate in rat for the PK properties. As sum-
marized in Table 5, to our delight, oral dosing with compound 22a
at 10 mg/kg showed higher maximal plasma concentration
Compd.
Energy items (Kcal molꢁ1
)
DEele
D
EvdW
DGnp
D
GPB
D
He
-T
D
Sf
DGbind
a
b
c
d
g
10l
ꢁ16.81
ꢁ21.98
ꢁ17.45
ꢁ16.43
ꢁ17.92
ꢁ78.83
ꢁ88.54
ꢁ78.31
ꢁ80.79
ꢁ79.19
ꢁ6.15
ꢁ6.09
ꢁ6.01
ꢁ6.20
ꢁ6.58
36.36
42.91
36.57
36.26
39.87
ꢁ65.42
ꢁ73.69
ꢁ65.21
ꢁ67.15
ꢁ63.83
23.51
21.52
23.13
24.22
24.17
ꢁ41.91
ꢁ52.17
ꢁ42.08
ꢁ42.93
ꢁ39.65
10n
22a
22c
22e
(Cmax ¼ 25.1
m
g/mL), longer half-life (T1/2 ¼ 12.5 h), higher plasma
exposure (AUC0-∞
¼
427.0 h/mL), lower total clearance
mg
a
(CL ¼ 0.02 L/h/kg) and better oral bioavailability (F ¼ 42%) to that of
compound 5. These desirable PK properties strengthen our confi-
dence of selecting compound 22a to further in vivo efficacy studies.
D
D
D
D
D
Eele: electrostatic energies.
b
c
d
e
f
EvdW: van der Waals interaction.
Gnp: nonelectrostatic contribution.
GPB: electrostatic contribution.
H: enthalpy change, it is the sum of
D
Eele, DEvdW, DGnp, and DGPB.
3.5. In vivo antitumor activity of compound 22a
eT
D
S: entropic contribution.
g
DGbind: binding free energy.
The in vivo antitumor activity of compound 22a was evaluated in
the MET-positive human glioblastoma U-87 MG xenograft model
[51]. The compound was administered orally q. d. as tumors
reached ~150 mm3. As shown in Table 6 and Fig. 6, compound 22a
could suppress tumor growth in a dose-dependent manner, with
the minimum effective doses (MED/ED50, 50% inhibition of tumor
growth) of ~25 mg/kg. Moreover, 22a displayed good antitumor
efficacy (IR of 81%) at dose of 75 mg/kg. Additionally, the PK/PD
studies demonstrated that the dose-dependent enhancement in
suppression of tumor growth was proportional to the plasma
exposure of compound 22a.
reason why 22e has an activity reduction targeted on MET
compared with 10l.
As shown in Fig. 5A, the introduction of a C (3) methyl group in
block C of 10l resulted in the adjustment of molecular orientation in
binding pocket, which led to a disappearance of the H-bond
interaction between carbonyl group in block C of 22b and the
residue A1222. As a result, 22b showed 23.4-fold lower potency
compared with 10l. As shown in Fig. 5B, the methylation of N (1)
amino group in block C of 10l led to a disappearance of the H-bond
interaction with the residue Glu 885 obviously. Furthermore, longer
distance (3.0 Å) between the C (4) carboxyl group in block C of 22c
and Asp 1046 relative to 10l was observed, which led to the key H-
bond in binding model of type II inhibitors with VEGFR-2 [50]
became weak. Combination of the above led to 22c provided a
significant loss in potency against VEGFR-2 (IC50 > 3000 nM).
4. Conclusions
In the present work, enlightened by the quinazoline ring that is
wildly used in the development of EGFR inhibitors while barely

8
L.-S. Zhuo et al. / European Journal of Medicinal Chemistry 208 (2020) 112785
Fig. 5. (A) The proposed binding mode overlay of 10l (yellow) and 22b (gray) with MET; (B) The proposed binding mode overlay of 10l (yellow) and 22c (magenta) with VEGFR-2
(PDB: 3U6J [50]). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Table 5
In vivo PK profiles of compound 22a in rata,b
.
Compd.
route
Dose (mg/kg)
Cmax
(m
g/mL)
Tmax (h)
T1/2 (h)
AUC0-∞
(m
g* h/mL)
CL (L/h/kg)
Vz (L/kg)
F (%)
22a
p.o.
i.v.
10
5
25.1
44.0
3.50
0.03
12.5
14.0
427.0
509.7
0.02
0.01
0.4
0.2
42
e
5 [35]
p.o.
i.v.
10
2.5
0.6
16.4
2.70
1.0
0.5
2.2
4.5
5.0
0.6
7.5
10.4
12
e
e
a
Vehicle: 70% PEG400-30% water. Cmax, maximum concentration; Tmax, time of maximum concentration; T1/2, half-life; AUC0-∞, area under the plasma concentration time
curve; CL, clearance; VZ, volume of distribution; and F, oral bioavailability.
b
Data reported as the average of three animals.
Table 6
In vivo tumor growth inhibition activity.
c
Tumor model
U-87 MG
Compd.
Dose (mg/kg)a
IR (%)b
Cmax c
(m
g/mL)
AUC0-t (h*
m
g/mL)
22a
25
75
51
81
12.1
16.0
156.1
279.1
a
1q.d. ꢂ 21/70% PEG-400/H2O/p.o..
Inhibition rate value.
PK/PD studies.
b
c
involved in MET inhibitors, we described the design, synthesis, and
biological evaluation of series of quinazoline-based 1,6-
a
naphthyridinone derivatives as MET inhibitors. Most of target
compounds showed moderate to significant potency at the
enzyme-based and/or cell-based assay, which suggested the
introduction of quinazoline scaffold into the block A moiety
maintained the potent MET-inhibited activity. The SAR and PK
studies disclosed the most promising compound 22a with favorable
inhibitory activity against MET (IC50 ¼ 9.0 nM), human microsomal
metabolic stability (t1/2 ¼ 621.2 min), and oral bioavailability
(F ¼ 42%). Moreover compound 22a displayed good antitumor ef-
ficacy (IR of 81% in 75 mg/kg) with a dose-dependent manner in
MET-positive human glioblastoma U-87 MG xenograft model.
Collectively, the favorable drug-likeness of 22a proposed that the
quinazoline may be a valuable scaffold for the discovery of novel
MET inhibitors. The preliminary preclinical studies of 22a are
underway.
Fig. 6. Antitumor effects of 22a in the MET-positive human glioblastoma U-87 MG
xenograft model. Tumor-bearing nude mice were randomly divided into groups when
the tumor volume reached ~150 mm3 and given corresponding compounds p. o. at the
indicated dose levels or vehicle alone over the designated treatment schedule. Data are
presented as the mean ( SEM; n ¼ 6 mice per group).
5. Experimental
5.1. General methods
Unless otherwise noted, all chemical reagents were

L.-S. Zhuo et al. / European Journal of Medicinal Chemistry 208 (2020) 112785
9
commercially available and treated with standard methods. Silica
gel column chromatography (CC). Silica gel (200e400 Mesh;
Qingdao Makall Group Co., Ltd; Qingdao; China). Solvents were
dried in a routine way and redistilled. All reactions involving air- or
moisture-sensitive reagents were performed under a nitrogen or
argon atmosphere. Melting points of compounds were measured
on a Melt-Temp II apparatus and uncorrected. 1H NMR spectra (400
or 600 MHz) and 13C NMR (100 or 150 MHz) spectra were recorded
on a Bruker BioSpin AG (Ultrashield Plus AV 400) spectrometer as
deuterochloroform (CDCl3) or dimethyl sulfoxide-d6 (DMSO-d6)
solutions using tetramethylsilane (TMS) as an internal standard
5.2.5. 7-(benzyloxy)-4-chloro-6-methoxyquinazoline (13) [53]
A solution of 12 (15 g, 53 mmol) and DMF (2d) in POCl3 (50 mL)
was heated at 100 ꢀC for 1 h. The mixture was evaporated under
reduced pressure and the residue was added to ice water, and then
extracted with DCM. The organic layer was separated, washed with
saturated sodium bicarbonate and brine, dried over anhydrous
Na2SO4. The residue was purified by chromatography (PE/
EA ¼ 30:1) to yield 13 as a yellow solid (13.2 g, 83%). mp:
133e134 ꢀC (132e134 ꢀC, ref [53]); 1H NMR (600 MHz, DMSO‑d6)
d
8.88 (s, 1H), 7.57 (s, 1H), 7.55e7.51 (m, 2H), 7.47e7.42 (m, 2H),
7.40e7.36 (m, 2H), 5.37 (s, 2H), 4.00 (s, 3H).
(
d
¼ 0) unless noted otherwise. MS spectra were obtained on an
Agilent technologies 6120 quadrupole LC/MS (ESI). High-resolution
mass spectra (HR-MS) were obtained on an Agilent 6224 TOF LC/MS
(USA). All reactions were monitored using thin-layer chromatog-
raphy (TLC) on silica gel plates. Yields were of purified compounds
and were not optimized.
5.2.6. 7-(benzyloxy)-4-(2-fluoro-4-nitrophenoxy)-6-
methoxyquinazoline (14a) [55]
A solution of 13 (13 g, 43.3 mmol), 2-fluoro-4-nitrophenol
(65 mmol) and DIPEA (130 mmol) in toluene (150 mL) was
refluxed for 8 h, and then cooled to room temperature. The reaction
mixture was concentrated under reduced pressure, and purified by
chromatography (PE/EA ¼ 30:1) to yield 14a as a yellow solid
5.2. General procedures for the synthesis of intermediates
(15.8 g, 87%). mp: 177e178 ꢀC; 1H NMR (600 MHz, DMSO-d6)
d 8.57
5.2.1. Methyl 4-(benzyloxy)-3-methoxybenzoate (11b) [52]
(s, 1H), 8.41 (d, J ¼ 9.0 Hz, 1H), 8.24 (d, J ¼ 8.4 Hz, 1H), 7.85 (t,
J ¼ 8.4 Hz,1H), 7.61 (s,1H), 7.58e7.49 (m, 3H), 7.44 (t, J ¼ 7.2 Hz, 2H),
7.41e7.35 (m, 1H), 5.35 (s, 2H), 3.98 (s, 3H).
A mixture of methyl 4-hydroxy-3-methoxybenzoate 11a (20 g,
109 mmol), benzyl bromide (164 mmol) and K2CO3 (329 mmol) in
DMF (200 mL) was stirred at 100 ꢀC for 5 h. The reaction was cooled
to room temperature and poured into a mixture of ice and water
(1000 mL). The solid was collected by filtration, washed with water
and dried under vacuum to give the title compound. White solid
(28.9 g, 97%). mp: 69e70 ꢀC (70e72 ꢀC, ref [52]), 1H NMR (600 MHz,
5.2.7. 4-(2-fluoro-4-nitrophenoxy)-6-methoxyquinazolin-7-ol
(14b) [55]
A solution of 14a (15 g, 35.5 mmol) and TFA (355 mmol) in DCM
(150 mL) was stirred at room temperature for 30 min. The reaction
mixture was quenched with a saturated aqueous NaHCO3 solution.
The resulting suspension was filtered and the solid dried to afford
the title compound 14b (9.4 g, 80%). mp: 252e253 ꢀC; 1H NMR
DMSO-d6)
d
7.57 (d, J ¼ 8.4 Hz, 1H), 7.46 (d, J ¼ 9.6 Hz, 3H), 7.40 (t,
J ¼ 7.2 Hz, 2H), 7.38e7.31 (m,1H), 7.16 (d, J ¼ 8.4 Hz,1H), 5.16 (s, 2H),
3.81 (ovl, 6H).
(400 MHz, DMSO-d6) d 10.97 (s, 1H), 8.50 (s, 1H), 8.41e8.37 (m, 1H),
5.2.2. Methyl 4-(benzyloxy)-5-methoxy-2-nitrobenzoate (11c) [52]
In a 250 mL flask in an ice bath, 11b (28 g, 102.8 mmol), acetic
acid (80 mL) and HNO3 (10 mL) were charged and the mixture was
stirred for 1 h, subsequently stirred for 4 h at 100 ꢀC. After stirring,
the solid was precipitated with cold water 120 mL, suction filtration
was performed, the solid was washed with H2O, recrystallized with
EtOAc, dried under vacuum and the title compound was obtained
as a yellow solid (27.7 g, 85%). mp: 126e127 ꢀC (128e130 ꢀC, ref
8.23e8.16 (m, 1H), 7.83e7.75 (m, 1H), 7.57e7.49 (m, 1H), 7.27 (s,
1H), 4.00 (s, 3H).
5.2.8. 4-(4-amino-2-fluorophenoxy)-6-methoxyquinazolin-7-ol
(14c) [56]
Prepared according to the procedure for the preparation of 11d,
from 14b, to yield 14c as yellow solid (92%). mp: 245e246 ꢀC; 1H
NMR (400 MHz, CDCl3)
d 10.70 (s, 1H), 8.41 (s, 1H), 7.47e7.40 (m,
[52]); 1H NMR (600 MHz, DMSO-d6)
d
7.15 (s, 1H), 6.86e6.73 (m,
1H), 7.18e7.10 (m, 1H), 6.99 (s, 1H), 6.45e6.39 (m, 1H), 6.38e6.31
6H), 5.24 (s, 2H), 3.91 (s, 3H), 3.82 (s, 3H).
(m, 1H), 5.36 (s, 2H), 3.94 (s, 3H).
5.2.3. Methyl 2-amino-4-(benzyloxy)-5-methoxybenzoate (11d)
[53]
5.2.9. 3-Fluoro-4-{[6-methoxy-7-(substituted alkoxy)quinazolin-4-
yl]oxy}aniline (15)
A mixture of iron powder (595 mmol), ammonium chloride
(255 mmol), 11c (85 mmol, 27 g), ethanol (150 mL) and water
(50 ml) was refluxed for 3 h. The mixture was filtered through
Celite and washed with EtOAc. The organic layer was washed with
water and Sat. NaCl, dried over Na2SO4, and concentrated to afford
the product (22 g, 90%). mp: 131e132 ꢀC (131e133 ꢀC, ref [53]); 1H
A solution of 14c (200 mg, 0.66 mmol) and K2CO3 (1.99 mmol) in
CH3CN (15 mL) was stirred at room temperature for 30 min. The
various alkyl halides or 2,2-dimethyloxirane (0.99 mmol) was
added to the reaction mixture and subsequently refluxed for 4 h.
The reaction mixture was concentrated under reduced pressure,
and purified by chromatography (PE/EA ¼ 10:1) to yield the title
compound 15.
NMR (600 MHz, DMSO-d6)
J ¼ 7.2 Hz, 2H), 7.36 (d, J ¼ 6.6 Hz, 1H), 7.15 (s, 1H), 6.49e6.43 (m,
d
7.45 (d, J ¼ 7.2 Hz, 2H), 7.41 (t,
3-fluoro-4-{[6-methoxy-7-(2-methoxyethoxy)quinazolin-4-yl]
3H), 5.05 (s, 2H), 3.75 (s, 3H), 3.65 (s, 3H).
oxy}aniline (15a) [45]: yield: 88%; mp: 195e196 ꢀC; 1H NMR
(600 MHz, DMSO-d6)
d 8.54 (s, 1H), 7.53 (s, 1H), 7.39 (s, 1H), 7.05 (t,
5.2.4. 7-(benzyloxy)-6-methoxyquinazolin-4(3H)-one (12) [53,54]
A mixture of 11d (20 g, 69.6 mmol) and ammonium formate
(104.4 mmol) in formamide (100 mL) was stirred at 150 ꢀC for 3 h.
The reaction was cooled to room temperature and poured into a
mixture of ice and water (500 mL). The mixture was filtered,
washed with water, vacuum dried and the title compound was
obtained as a yellow solid. (15.9 g, 81%). mp: 254e255 ꢀC
J ¼ 8.8 Hz, 1H), 6.54e6.48 (m, 1H), 6.46e6.40 (m, 1H), 5.41 (s, 2H),
4.32 (t, J ¼ 4.2 Hz, 2H), 3.98 (s, 3H), 3.76 (t, J ¼ 4.2 Hz, 2H), 3.35 (s,
3H).
2-{[4-(4-amino-2-fluorophenoxy)-6-methoxyquinazolin-7-yl]
oxy}ethan-1-ol (15b): yield: 76%; mp: 179e180 ꢀC; 1H NMR
(600 MHz, DMSO-d6)
d 8.54 (s, 1H), 7.53 (s, 1H), 7.39 (s, 1H), 7.05 (t,
J ¼ 8.4 Hz, 1H), 6.50 (d, J ¼ 12.6 Hz, 1H), 6.43 (d, J ¼ 8.4 Hz, 1H), 5.41
(s, 2H), 4.99 (s, 1H), 4.22 (s, 2H), 3.98 (s, 3H), 3.82 (s, 2H).
3-{[4-(4-amino-2-fluorophenoxy)-6-methoxyquinazolin-7-yl]
oxy}propan-1-ol (15c): yield: 72%; mp: 190e191 ꢀC; 1H NMR
(266e267 ꢀC, ref [54]); 1H NMR (600 MHz, DMSO-d6)
d 12.05 (s,
1H), 7.97 (s, 1H), 7.52e7.31 (m, 6H), 7.13 (s, 1H), 5.19 (s, 2H), 3.90 (s,
3H).

10
L.-S. Zhuo et al. / European Journal of Medicinal Chemistry 208 (2020) 112785
(600 MHz, DMSO-d6)
d
8.52 (s, 1H), 7.51 (s, 1H), 7.35 (s, 1H), 7.03 (s,
(600 MHz, DMSO-d6) d 8.53 (s, 1H), 7.52 (s, 1H), 7.37 (s, 1H), 7.04 (t,
1H), 6.47 (s, 1H), 6.40 (s, 1H), 5.38 (s, 2H), 4.60 (s, 1H), 4.25 (s, 2H),
3.96 (s, 3H), 3.59 (s, 2H), 1.95 (s, 2H).
J ¼ 8.9 Hz, 1H), 6.50 (d, J ¼ 12.7 Hz, 1H), 6.42 (d, J ¼ 8.9 Hz, 1H), 5.39
(s, 2H), 4.21 (t, J ¼ 6.6 Hz, 2H), 3.98 (s, 3H), 3.62e3.53 (m, 4H),
2.48e2.43 (m, 2H), 2.41e2.32 (m, 4H), 2.00e1.92 (m, 2H).
1-{[4-(4-amino-2-fluorophenoxy)-6-methoxyquinazolin-7-yl]
oxy}-2-methylpropan-2-ol (15d): yield: 77%; mp: 195e196 ꢀC; 1H
3-fluoro-4-({7-methoxy-6-[3-(pyrrolidin-1-yl)propoxy]quinazo-
NMR (600 MHz, DMSO-d6)
d 8.53 (s, 1H), 7.53 (s, 1H), 7.36 (s, 1H),
lin-4-yl}oxy)aniline (17f) [45]: yield: 86%; mp: 136e137 ꢀC; 1H NMR
7.04 (t, J ¼ 8.8 Hz, 1H), 6.50 (d, J ¼ 12.9 Hz, 1H), 6.45e6.39 (m, 1H),
5.49e5.32 (m, 2H), 4.76e4.68 (m, 1H), 3.98 (s, 3H), 3.94 (s, 2H), 1.26
(s, 6H).
(600 MHz, DMSO-d6) d 8.55 (s, 1H), 7.54 (s, 1H), 7.38 (s, 1H), 7.05 (t,
J ¼ 8.8 Hz,1H), 6.51 (dd, J ¼ 12.9, 2.5 Hz,1H), 6.44 (dd, J ¼ 8.8, 2.5 Hz,
1H), 5.41 (s, 2H), 4.23 (t, J ¼ 6.6 Hz, 2H), 4.00 (s, 3H), 2.62 (t,
J ¼ 6.6 Hz, 2H), 2.55e2.51 (m, 4H), 2.05e1.95 (m, 2H), 1.76e1.66 (m,
4H).
3-fluoro-4-{[6-methoxy-7-(3-morpholinopropoxy)quinazolin-4-
yl]oxy}aniline (15e) [45]: yield: 87%; mp: 135e136 ꢀC; 1H NMR
(600 MHz, DMSO-d6)
d
8.55 (s, 1H), 7.54 (s, 1H), 7.39 (s, 1H), 7.06 (t,
3-fluoro-4-({7-methoxy-6-[3-(piperidin-1-yl)propoxy]quinazolin-
J ¼ 8.9 Hz, 1H), 6.52 (d, J ¼ 12.8 Hz, 1H), 6.44 (d, J ¼ 8.8 Hz, 1H), 5.42
(s, 2H), 4.30e4.22 (m, 2H), 3.99 (s, 3H), 3.65e3.57 (m, 4H),
2.51e2.45 (m, 2H), 2.45e2.32 (m, 4H), 2.04e1.95 (m, 2H).
4-yl}oxy)aniline (17g) [45]: yield: 80%; mp: 133e134 ꢀC; 1H NMR
(600 MHz, DMSO-d6)
d 8.57 (s, 1H), 7.55 (s, 1H), 7.36 (s, 1H), 7.02 (t,
J ¼ 8.8 Hz, 1H), 6.52 (dd, J ¼ 12.9, 2.5 Hz, 1H), 6.44 (dd, J ¼ 8.8,
2.5 Hz, 1H), 5.42 (s, 2H), 4.22 (t, J ¼ 6.6 Hz, 2H), 4.01 (s, 3H), 2.63 (t,
J ¼ 6.6 Hz, 2H), 2.54e2.50 (m, 4H), 2.06e1.94 (m, 2H), 1.76e1.66 (m,
4H), 1.57e1.46 (m, 2H).
3-fluoro-4-({6-methoxy-7-[3-(pyrrolidin-1-yl)propoxy]quinazo-
lin-4-yl}oxy)aniline (15f) [45]: yield: 89%; mp: 162e163 ꢀC; 1H NMR
(600 MHz, DMSO-d6)
d 8.53 (s, 1H), 7.53 (s, 1H), 7.37 (s, 1H), 7.04 (t,
J ¼ 8.6 Hz, 1H), 6.51 (d, J ¼ 12.7 Hz, 1H), 6.43 (d, J ¼ 8.2 Hz, 1H), 5.38
(s, 2H), 4.33e4.15 (m, 2H), 3.98 (s, 3H), 2.73e2.59 (m, 2H),
2.59e2.52 (m, 4H), 2.09e1.92 (m, 2H), 1.81e1.61 (m, 4H).
5.2.11. 4-(2-substituted-4-nitrophenoxy)-6,7-bis(substituted
alkoxy)quinazoline (19)
3-fluoro-4-{[6-methoxy-7-(2-morpholinoethoxy)quinazolin-4-yl]
Prepared according to the procedure for the preparation of 14a,
from 4-chloro-6,7-bis(substituted alkoxy)quinazoline 18, to yield
the title compound 19.
oxy}aniline (15g) [45]: yield: 82%; mp: 177e178 ꢀC; 1H NMR
(600 MHz, DMSO-d6)
d 8.53 (s, 1H), 7.52 (s, 1H), 7.42 (s, 1H), 7.04 (t,
J ¼ 8.8 Hz,1H), 6.50 (dd, J ¼ 13.0, 2.6 Hz,1H), 6.42 (dd, J ¼ 8.7, 2.6 Hz,
1H), 5.40 (s, 2H), 4.32 (t, J ¼ 5.6 Hz, 2H), 3.97 (s, 3H), 3.66e3.54 (m,
4H), 2.79 (t, J ¼ 5.6 Hz, 2H), 2.58e2.51 (m, 4H).
6,7-dimethoxy-4-(4-nitrophenoxy)quinazoline (19a): yield: 89%;
mp: 186e187 ꢀC; MS (ESI). 328.1 [MþH]þ.
4-(2-fluoro-4-nitrophenoxy)-6,7-dimethoxyquinazoline
(19b)
3-fluoro-4-({6-methoxy-7-[(1-methylpiperidin-4-yl)methoxy]qui-
nazolin-4-yl}oxy)aniline (15h): yield: 86%; mp: 181e182 ꢀC; 1H
[56]: yield: 92%; mp: 188e189 ꢀC; MS (ESI). 346.1 [MþH]þ.
4-(2-fluoro-4-nitrophenoxy)-6,7-bis(2-methoxyethoxy)quinazo-
line (19c): yield: 83%; mp: 168e169 ꢀC; MS (ESI). 434.2 [MþH]þ.
4-(2-fluoro-4-nitrophenoxy)-7,8,10,11,13,14-hexahydro- [1,4,7,10]
tetraoxacyclododecino[2,3-g]quinazolþine (19d): yield: 88%; mp:
181e182 ꢀC; MS (ESI). 432.3 [MþH] .
NMR (600 MHz, DMSO-d6)
d 8.55 (s, 1H), 7.55 (s, 1H), 7.43 (s, 1H),
7.05 (t, J ¼ 8.8 Hz, 1H), 6.54 (d, J ¼ 12.8 Hz, 1H), 6.46 (d, J ¼ 8.7 Hz,
1H), 5.48 (s, 2H), 4.20e4.04 (m, 2H), 3.99 (s, 3H), 3.12e2.97 (m, 2H),
2.72 (s, 3H), 2.22e2.10 (m, 1H), 2.07e1.97 (m, 2H), 1.79e1.65 (m,
2H) (Remark: a signal peak of methylene overlaps with the peak of
water).
5.2.12. 4-{[6,7-bis(substituted alkoxy)quinazolin-4-yl]oxy}-3-
(substituted)aniline (20)
5.2.10. 3-Fluoro-4-{[7-methoxy-6-(substituted alkoxy)quinazolin-
4-yl]oxy}aniline (17)
Prepared according to the procedure for the preparation of 14c,
from 19, to yield the title compound 20.
The preparation of title compounds 17 were carried out from
methyl 3-hydroxy-4-methoxybenzoate according to synthetic
method of compounds 15.
4-[(6,7-dimethoxyquinazolin-4-yl)oxy]aniline (20a) [58]: yield:
80%; mp: 178e179 ꢀC; 1H NMR (400 MHz, DMSO-d6)
d 8.56 (s, 1H),
7.52 (s, 1H), 7.38 (s, 1H), 6.54e6.47 (m, 2H), 6.44e6.36 (m, 2H), 5.38
3-fluoro-4-{[7-methoxy-6-(2-methoxyethoxy)quinazolin-4-yl]
(s, 2H), 3.97 (vol, 6H).
oxy}aniline (17a) [57]: yield: 86%; mp: 181e182 ꢀC; 1H NMR
4-[(6,7-dimethoxyquinazolin-4-yl)oxy]-3-fluoroaniline
(20b)
(600 MHz, DMSO-d6)
d 8.53 (s, 1H), 7.55 (s, 1H), 7.38 (s, 1H), 7.03 (t,
[46,59]: yield: 87%; mp: 188e189 ꢀC; 1H NMR (400 MHz, DMSO-d6)
J ¼ 8.9 Hz, 1H), 6.52e6.47 (m, 1H), 6.44e6.39 (m, 1H), 5.39 (s, 2H),
4.37e4.23 (m, 2H), 3.99 (s, 3H), 3.74 (t, J ¼ 4.2 Hz, 2H), 3.34 (s, 3H).
2-{[4-(4-amino-2-fluorophenoxy)-7-methoxyquinazolin-6-yl]
oxy}ethan-1-ol (17b): yield: 79%; mp: 182e183 ꢀC; 1H NMR
d
8.55 (s, 1H), 7.53 (s, 1H), 7.38 (s, 1H), 7.05 (t, J ¼ 8.8 Hz, 1H), 6.51
(dd, J ¼ 13.0, 2.5 Hz, 1H), 6.44 (dd, J ¼ 8.7, 2.4 Hz, 1H), 5.40 (s, 2H),
3.98 (vol, 6H).
4-{[6,7-bis(2-methoxyethoxy)quinazolin-4-yl]oxy}-3-
fluoroaniline (20c) [45]: yield: 77%; mp: 185e186 ꢀC; 1H NMR
(600 MHz, DMSO-d6)
d 8.53 (s, 1H), 7.54 (s, 1H), 7.38 (s, 1H), 7.04 (t,
J ¼ 9.0 Hz, 1H), 6.49 (d, J ¼ 12.6 Hz, 1H), 6.42 (d, J ¼ 7.8 Hz, 1H), 5.39
(s, 2H), 4.94 (s, 1H), 4.19 (s, 2H), 3.99 (s, 3H), 3.80 (s, 2H).
3-{[4-(4-amino-2-fluorophenoxy)-7-methoxyquinazolin-6-yl]
oxy}propan-1-ol (17c): yield: 85%; mp: 182e183 ꢀC; 190e191 ꢀC; 1H
(400 MHz, DMSO-d6) d 8.57 (s, 1H), 7.54 (s, 1H), 7.39 (s, 1H), 7.04 (t,
J ¼ 8.8 Hz, 1H), 6.51e6.46 (m, 1H), 6.43e6.37 (m, 1H), 5.39 (s, 2H),
4.36e4.32 (m, 4H), 3.92e3.89 (m, 4H), 3.52 (s, 3H), 3.50 (s, 3H).
3-fluoro-4-[(7,8,10,11,13,14-hexahydro-
acyclododecino[2,3-g]quinazolin-4-yl)oxy]aniline (20d) [57]: yield:
79%; mp: 192e193 ꢀC; 1H NMR (400 MHz)
8.53 (s,1H), 7.76 (s, 1H),
[1,4,7,10]tetraox-
NMR (400 MHz, DMSO-d6)
d 8.50 (s, 1H), 7.51 (s, 1H), 7.35 (s, 1H),
7.02 (t, J ¼ 8.8 Hz, 1H), 6.48 (dd, J ¼ 12.9, 2.4 Hz, 1H), 6.43e6.37 (m,
1H), 5.36 (s, 2H), 4.57 (t, J ¼ 5.2 Hz, 1H), 4.22 (t, J ¼ 6.4 Hz, 2H), 3.98
(s, 3H), 3.59 (q, J ¼ 6.0 Hz, 2H), 2.11e1.80 (m, 2H).
d
7.47 (s, 1H), 7.02 (t, J ¼ 8.6 Hz, 2H), 6.48 (dd, J ¼ 12.8, 2.5 Hz, 1H),
6.40 (dd, J ¼ 8.6, 2.6 Hz, 1H), 5.41 (s, 2H), 4.39e4.27 (m, 4H),
3.87e3.77 (m, 2H), 3.77e3.70 (m, 2H), 3.69e3.59 (m, 4H).
4-({6-[3-(diethylamino)propoxy]-7-methoxyquinazolin-4-yl}
oxy)-3-fluoroaniline (17d) [45]: yield: 81%; mp: 131e132 ꢀC; 1H
NMR (600 MHz, DMSO-d6)
d
8.51 (s, 1H), 7.52 (s, 1H), 7.36 (s, 1H),
5.3. General procedures for the synthesis of targets 10a-r and 22a-f
7.03 (t, J ¼ 8.9 Hz, 1H), 6.51 (d, J ¼ 12.7 Hz, 1H), 6.40 (d, J ¼ 8.9 Hz,
1H), 5.38 (s, 2H), 4.23 (t, J ¼ 6.6 Hz, 2H), 3.96 (s, 3H), 2.43e2.38 (m,
2H), 2.35e2.26 (m, 4H), 1.98e1.90 (m, 2H), 1.06 (m, 6H).
General Procedure A:
To the solution of aromatic amine (0.7 mmol) and the corre-
sponding chlorinated 1,6-naphthyridine (0.7 mmol) in isopropanol
(10 mL), HCl (20 mmol%) was added drop-wise, and then heated to
3-fluoro-4-{[7-methoxy-6-(3-morpholinopropoxy)quinazolin-4-
yl]oxy}aniline (17e) [45]: yield: 79%; mp: 143e144 ꢀC; 1H NMR

L.-S. Zhuo et al. / European Journal of Medicinal Chemistry 208 (2020) 112785
11
90 ꢀC under nitrogen for 2 h. The mixture was filtered, and the solid
651.6845 [MþH]þ.
was dissolved in ethyl acetate. The solution was stirred with K2CO3
(1 mmol) at r. t. for 1 h and filtered. The filtrate was concentrated in
vacuum and purified by flash chromatography (CH2Cl2/
MeOH ¼ 20:1) to yield the corresponding targets.
5.3.4. 5-({3-fluoro-4-[(7,8,10,11,13,14-hexahydro- [1,4,7,10]
tetraoxacyclododecino [2,3-g]quinazolin-4-yl)oxy]phenyl}amino)-
3-(4-fluorophenyl)-1,6-naphthyridin-4(1H)-one (10d)
General Procedure B:
Prepared according to the general procedure B. Yellow solid;
A solution of aromatic amine (0.7 mmol), the corresponding
chlorinated 1,6-naphthyridine (0.7 mmol) and p-toluenesulfonic
acid (0.5 mmol) in isopropanol (10 mL) was heated to 90 ꢀC for 2 h.
The mixture was filtered, and the solid was washed with ice-cold
isopropanol to yield the corresponding targets.
yield: 68%, mp: 224e225 ꢀC. 1H NMR (400 MHz)
d
13.37 (s, 1H),
13.29 (br, 1H), 8.65 (s, 1H), 8.27 (d, J ¼ 5.7 Hz, 1H), 8.09e7.91 (m,
2H), 7.84 (s, 1H), 7.77e7.64 (m, 2H), 7.60e7.39 (m, 3H), 7.26 (t,
J ¼ 8.3 Hz, 2H), 7.04 (d, J ¼ 6.7 Hz,1H), 4.43e4.25 (m, 4H), 3.88e3.69
(m, 4H), 3.69e3.55 (m, 4H). 13C NMR (150 MHz, DMSO-d6)
d 176.69,
General Procedure C:
163.23, 161.59, 159.36, 157.69, 155.78, 154.32, 152.40, 151.18, 150.83,
147.63, 147.31, 145.33, 139.18, 138.01, 131.25, 131.19, 130.03, 128.25,
125.51, 125.34, 124.25, 115.23, 115.14, 109.12, 106.23, 105.21, 103.24,
101.20, 73.42, 71.41, 70.81, 70.43, 69.11, 68.79. MS (ESI). 640.1
[MþH]þ. HRMS (EI): calcd for C36H32F2N6O4 639.1929; found:
640.1926 [MþH]þ.
A solution of 10l (200 mg, 0.36 mmol) and K2CO3 (0.72 mmol) in
anhydrous DMF (5 mL) was stirred at 25 ꢀC under nitrogen for 0.5 h.
Then diverse halides or isobutylene oxide (0.72 mmol) was added.
The resulted mixture was stirred at 25 ꢀC for 2 h. Ice-cold water was
added, and the mixture was extracted with dichloromethane. The
organic layer was dried and concentrated. The residue was purified
by chromatography (CH2Cl2/MeOH ¼ 100:1) to yield corresponding
targets.
5.3.5. 5-[(3-fluoro-4-{[6-methoxy-7-(2-methoxyethoxy)
quinazolin-4-yl]oxy} phenyl)amino]-3-(4-fluorophenyl)-1,6-
naphthyridin-4(1H)-one (10e)
5.3.1. 5-[(3-fluoro-4-{[7-methoxy-6-(3-morpholinopropoxy)
quinazolin-4-yl]oxy}phenyl) amino]-3-(4-fluorophenyl)-1,6-
naphthyridin-4(1H)-one (10a)
Prepared according to the general procedure A. Yellow solid;
yield: 80%, mp: 236e237 ꢀC. 1H NMR (400 M, DMSO-d6)
d 13.37 (s,
1H), 13.22 (br, 1H), 8.68 (s, 1H), 8.28 (d, J ¼ 5.6 Hz, 1H), 8.12 (d,
J ¼ 12.0 Hz, 1H), 8.06 (d, J ¼ 6.4 Hz, 1H), 7.73e7.77 (m, 2H), 7.62 (s,
1H), 7.48e7.57 (m, 3H), 7.29 (t, J ¼ 8.8 Hz, 2H), 7.05 (d, J ¼ 6.4 Hz,
Prepared according to the general procedure A. Yellow solid;
yield: 68%; m. p.: 220e221 ꢀC; 1H NMR (400 M, DMSO-d6)
d 13.31
(s, 1H), 12.93 (br, 1H), 8.64 (s, 1H), 8.27 (d, J ¼ 5.6 Hz, 1H), 8.21e8.10
(m, 2H), 7.77e7.73 (m, 2H), 7.65 (s, 1H), 7.51e7.48 (m, 3H), 7.29 (t,
J ¼ 8.8 Hz, 2H), 6.99 (d, J ¼ 6.0 Hz,1H), 4.34 (t, J ¼ 6.4 Hz, 2H), 4.21 (s,
3H), 4.03e3.79 (m, 4H), 3.53e3.50 (m, 2H), 3.16e3.09 (m, 4H),
1H), 4.35 (t, J ¼ 4.4 Hz, 2H), 4.01 (s, 3H), 3.78 (t, J ¼ 4.4 Hz, 2H); 13
C
NMR (100 M, DMSO-d6)
d 177.5, 164.6, 163.1, 162.9, 160.6, 156.2,
155.6, 155.0, 152.8, 152.6, 150.8, 149.3, 149.0, 146.5, 139.6, 138.7,
133.7, 131.5, 131.2, 124.7, 123.3, 116.3, 115.3, 115.1, 109.7, 108.3, 108.1,
107.8, 103.6, 101.2, 70.4, 68.7, 58.7, 56.5. MS (ESI). 598.2 [MþH]þ.
HR-MS (EI) m/z calcd for C32H25F2N5O5 597.1824; found 598.1922
[MþH]þ.
2.34e2.31 (m, 2H); 13C NMR (100 M, DMSO-d6)
d 176.6, 164.7, 162.7,
160.1, 156.5, 155.2, 152.7, 151.3, 149.7, 146.8, 138.8, 131.3124.9, 123.7,
118.9, 115.9, 115.1, 110.0, 106.6, 105.4, 103.9, 102.3, 66.9, 63.8, 56.8,
54.2, 51.7, 23.4. MS (ESI). 667.2 [MþH]þ. HR-MS (EI) m/z calcd for
C
36H32F2N6O5 666.2402; found 667.2481 [MþH]þ.
5.3.6. 5-[(3-fluoro-4-{[7-(2-hydroxyethoxy)-6-methoxyquinazolin-
4-yl]oxy}phenyl) amino]-3-(4-fluorophenyl)-1,6-naphthyridin-
4(1H)-one 4-methylbenzenesulfonate (10f)
5.3.2. 5-[(4-{[6,7-bis(2-methoxyethoxy)quinazolin-4-yl]oxy}-3-
fluorophenyl)amino]-3- (4-fluorophenyl)-1,6-naphthyridin-4(1H)-
one (10b)
Prepared according to the general procedure B. White solid;
yield: 58%, mp: 256e257 ꢀC. 1H NMR (600 MHz, DMSO-d6)
d 13.43
Prepared according to the general procedure A. Yellow solid;
(s, 1H), 13.11 (br, 1H), 8.69 (s, 1H), 8.32 (d, J ¼ 5.6 Hz, 1H), 8.09e7.97
(m, 2H), 7.80e7.71 (m, 2H), 7.65e7.55 (m, 2H), 7.54e7.44 (m, 4H),
7.30 (t, J ¼ 8.7 Hz, 2H), 7.15e7.09 (m, 2H), 7.02 (d, J ¼ 6.6 Hz, 1H),
4.25 (t, J ¼ 4.8 Hz, 2H), 4.02 (s, 3H), 3.84 (t, J ¼ 4.7 Hz, 2H), 2.29 (s,
yield: 72%, mp: 236e237 ꢀC. 1H NMR (400 M, DMSO-d6)
d 13.40 (s,
1H), 13.16 (br, 1H), 8.67 (s, 1H), 8.30 (d, J ¼ 5.6 Hz, 1H), 8.07 (d,
J ¼ 12.0 Hz, 1H), 8.03 (d, J ¼ 6.4 Hz, 1H), 7.73e7.77 (m, 2H), 7.66 (s,
1H), 7.48e7.59 (m, 3H), 7.30 (t, J ¼ 8.8 Hz, 2H), 7.04 (d, J ¼ 6.4 Hz,
1H), 4.37 (t, J ¼ 4.4 Hz, 4H), 3.66e3.80 (m, 10H); 13C NMR (100 M,
3H). 13C NMR (101 MHz, DMSO-d6)
d 176.29, 164.80, 162.89, 160.50,
156.44, 154.89, 154.59, 152.52, 151.25, 151.02, 147.23, 145.89, 145.39,
144.62, 137.78, 130.89, 130.81, 129.91, 128.16, 125.49, 124.90, 123.42,
115.09, 114.89, 109.09, 107.41, 105.02, 102.22, 101.01, 66.71, 57.85,
56.75, 21.19. MS (ESI). 584.1 [MþH]þ. HR-MS (EI) m/z calcd for
DMSO-d6)
d 176.8, 164.2, 155.6, 154.5, 151.9, 149.7, 148.0, 146.8,
138.5,130.9, 130.8, 124.8, 123.7, 114.9, 114.7, 109.2,107.1, 106.7, 103.6,
102.0, 70.1, 68.5, 58.3. MS (ESI). 642.2 [MþH]þ. HR-MS (EI) m/z calcd
for C34H29F2N5O6 641.2086; found 642.2175 [MþH]þ.
C
31H23F2N5O5 583.1667; found 584.1615 [MþH]þ.
5.3.3. 5-{[3-fluoro-4-({6-methoxy-7-[(1-methylpiperidin-4-yl)
methoxy]quinazolin-4-yl} oxy)phenyl]amino}-3-(4-fluorophenyl)-
1,6-naphthyridin-4(1H)-one (10c)
5.3.7. 5-[(3-fluoro-4-{[7-(3-hydroxypropoxy)-6-
methoxyquinazolin-4-yl]oxy}phenyl)amino]-3-(4-fluorophenyl)-
1,6-naphthyridin-4(1H)-one 4-methylbenzenesulfonate (10g)
Prepared according to the general procedure B. White solid;
Prepared according to the general procedure A. Yellow solid;
yield: 75%, mp: 211e212 ꢀC. 1H NMR (600 MHz, DMSO-d6)
d
13.22
yield: 61%; m. p. 229e230 ꢀC; 1H NMR (600 MHz, DMSO-d6)
d 13.39
(s,1H),12.39 (br,1H), 8.57 (s,1H), 8.34 (d, J ¼ 13.3 Hz,1H), 8.26e8.13
(m, 2H), 7.80e7.67 (m, 2H), 7.58 (s, 1H), 7.53e7.46 (m, 1H),
7.45e7.35 (m, 2H), 7.27 (t, J ¼ 8.7 Hz, 2H), 6.86 (d, J ¼ 5.9 Hz, 1H),
4.07 (d, J ¼ 5.9 Hz, 2H), 4.00 (s, 3H), 2.87e2.75 (m, 2H), 2.18 (s, 3H),
1.98e1.86 (m, 2H), 1.86e1.71 (m, 3H), 1.44e1.30 (m, 2H). 13C NMR
(s, 1H), 12.96 (br, 1H), 8.67 (s, 1H), 8.31 (d, J ¼ 5.5 Hz, 1H), 8.14e7.94
(m, 2H), 7.81e7.68 (m, 2H), 7.65e7.53 (m, 2H), 7.53e7.37 (m, 4H),
7.35e7.22 (m, 2H), 7.17e7.05 (m, 2H), 6.98 (d, J ¼ 6.5 Hz,1H), 4.29 (t,
J ¼ 6.0 Hz, 2H), 4.00 (s, 3H), 3.62 (t, J ¼ 6.0 Hz, 2H), 2.28 (s, 3H),
2.05e1.89 (m, 2H). 13C NMR (101 MHz, DMSO-d6)
d 176.81, 164.31,
(100 MHz, DMSO-d6)
d
176.96, 164.17, 162.56, 160.14, 155.75, 155.27,
162.88, 160.44, 155.94, 155.07, 153.96, 152.62, 151.63, 150.74, 147.27,
147.04, 145.43, 138.78, 137.83, 131.00, 130.83, 130.22, 128.13, 125.51,
125.07, 124.13, 115.02, 114.81, 109.01, 106.64, 105.98, 103.82, 100.77,
66.19, 57.16, 56.21, 31.70, 20.79. MS (ESI). 598.1 [MþH]þ. HRMS
(ESI): calcd for C32H25F2N5O5 597.1824, found: 598.17464 [MþH]þ.
152.45, 152.18, 150.38, 148.92, 138.71, 133.31, 133.19, 131.25, 130.85,
122.75, 115.72, 114.82, 114.61, 109.06, 107.81, 107.34, 107.01, 103.42,
100.65, 73.06, 56.13, 54.78, 46.10, 34.48, 28.37. MS (ESI). 651.6
[MþH]þ. HRMS (EI): calcd for C36H32F2N6O4 650.6868; found:

12
L.-S. Zhuo et al. / European Journal of Medicinal Chemistry 208 (2020) 112785
5.3.8. 5-[(3-fluoro-4-{[7-(2-hydroxy-2-methylpropoxy)-6-
methoxyquinazolin-4-yl]oxy}phenyl)amino]-3-(4-fluorophenyl)-
1,6-naphthyridin-4(1H)-one 4-methylbenzenesulfonate (10h)
Prepared according to the general procedure B. White solid;
yield: 85%; m. p. 280e281 ꢀC; 1H NMR (400 M, DMSO-d6)
d 13.41 (s,
1H), 13.23 (br, 1H), 8.68 (s, 1H), 8.31 (d, J ¼ 5.6 Hz, 1H), 8.08 (d,
J ¼ 12.0 Hz, 1H), 8.03 (d, J ¼ 5.6 Hz, 1H), 7.75 (t, J ¼ 8.8 Hz, 2H), 7.61
(s,1H), 7.55e7.61 (m, 2H), 7.46 (s,1H), 7.29 (t, J ¼ 9.2 Hz, 2H), 7.04 (d,
yield: 86%; m. p. 212e213 ꢀC; 1H NMR (600 MHz, DMSO-d6)
d
13.53
J ¼ 6.8 Hz, 1H), 4.01 (vol, 6H); 13C NMR (100 M, DMSO-d6)
d 176.9,
(s, 1H), 13.23 (br, 1H), 8.74 (s, 1H), 8.33 (d, J ¼ 5.4 Hz, 1H), 8.06e7.93
(m, 2H), 7.70 (d, J ¼ 7.8 Hz, 2H), 7.66e7.60 (m, 2H), 7.55e7.44 (m,
5H), 7.39 (t, J ¼ 7.2 Hz, 1H), 7.12 (d, J ¼ 7.8 Hz, 2H), 7.05 (d, J ¼ 5.4 Hz,
1H), 4.03 (s, 3H), 3.99 (s, 2H), 2.29 (s, 3H), 1.28 (s, 6H). MS (ESI).
612.2 [MþH]þ. HRMS (ESI): calcd for C33H27F2N5O5 611.1983, found:
612.1942 [MþH]þ.
164.2, 160.6, 156.3, 154.2, 152.6, 151.9, 150.5, 148.1, 146.8, 144.9,
138.6, 136.2, 130.9, 130.5, 124.8, 123.5, 114.9, 114.7, 109.1, 108.0,
106.7, 106.1, 104.0, 100.6, 59.3, 56.1. MS (ESI). 554.1 [MþH]þ. HR-MS
(EI) m/z calcd for C30H21F2N5O4, 553.1562; found 554.1659 [MþH]þ.
5.3.13. 5-[(3-fluoro-4-{[7-methoxy-6-(2-methoxyethoxy)
quinazolin-4-yl]oxy}phenyl) amino]-3-(4-fluorophenyl)-1,6-
naphthyridin-4(1H)-one (10m)
5.3.9. 5-[(3-fluoro-4-{[6-methoxy-7-(2-morpholinoethoxy)
quinazolin-4-yl]oxy}phenyl) amino]-3-(4-fluorophenyl)-1,6-
naphthyridin-4(1H)-one (10i)
Prepared according to the general procedure A. Yellow solid;
yield: 67%; m. p. 188e189 ꢀC; 1H NMR (400 M, DMSO-d6)
d 13.42 (s,
Prepared according to the general procedure A. Yellow solid;
1H),13.26 (br,1H), 8.69 (s,1H), 8.31 (d, J ¼ 5.6 Hz,1H), 8.01e8.07 (m,
2H), 7.74e7.78 (m, 2H), 7.65 (s, 1H), 7.48e7.60 (m, 3H), 7.30 (t,
J ¼ 8.8 Hz, 2H), 7.06 (d, J ¼ 6.8 Hz,1H), 4.35 (t, J ¼ 4.0 Hz, 2H), 4.03 (s,
3H), 3.77 (t, J ¼ 4.0 Hz, 2H), 3.36 (s, 3H); 13C NMR (100 M, DMSO-d6)
yield: 76%; m. p. 254e255 ꢀC; 1H NMR (400 M, DMSO-d6)
d 13.35 (s,
1H), 13.09 (br, 1H), 8.61 (s, 1H), 8.28 (d, J ¼ 5.6 Hz, 1H), 8.14 (d,
J ¼ 12.8 Hz, 1H), 8.08 (d, J ¼ 6.4 Hz, 1H), 7.73e7.77 (m, 2H), 7.66 (s,
1H), 7.51e7.56 (m, 3H), 7.29 (t, J ¼ 8.8 Hz, 2H), 7.02 (d, J ¼ 6.4 Hz,
1H), 4.74 (t, J ¼ 6.0 Hz, 2H), 3.99e4.03 (m, 5H), 3.83e3.89 (m, 2H),
3.65e3.70 (m, 2H), 3.58e3.62 (m, 2H), 3.25e3.31 (m, 2H); 13C NMR
d
176.8, 164.4, 162.0, 160.4, 156.7, 154.0, 152.6, 151.5, 149.9, 147.1,
146.9, 138.6, 130.8125.1, 124.2, 115.0, 114.7, 109.1, 106.6, 105.4, 103.8,
101.6, 70.0, 68.3, 58.2, 56.4. MS (ESI). 597.9 [M]þ. HR-MS (EI) m/z
calcd for C32H25F2N5O5, 597.1824; found 598.1902 [MþH]þ.
(100 M, DMSO-d6)
d 176.9, 164.2, 154.2, 152.5, 152.3, 150.3, 148.3,
146.0, 142.9, 137.9, 130.9, 130.7, 125.4, 114.9, 114.7, 110.2, 109.7, 107.9,
106.7, 103.5, 101.1, 63.8, 63.2, 56.3, 54.5, 51.9. MS (ESI). 653.2
[MþH]þ. HR-MS (EI) m/z calcd for C35H30F2N6O5 652.2246; found
653.2323 [MþH]þ.
5.3.14. 5-[(3-fluoro-4-{[6-(2-hydroxyethoxy)-7-
methoxyquinazolin-4-yl]oxy}phenyl) amino]-3-(4-fluorophenyl)-
1,6-naphthyridin-4(1H)-one 4-methylbenzenesulfonate (10n)
Prepared according to the general procedure B. white solid;
5.3.10. 5-[(3-fluoro-4-{[6-methoxy-7-(3-morpholinopropoxy)
quinazolin-4-yl]oxy} phenyl)amino]-3-(4-fluorophenyl)-1,6-
naphthyridin-4(1H)-one (10j)
yield: 57%; m. p. 256e257 ꢀC; 1H NMR (600 MHz, DMSO-d6)
d 13.47
(s, 1H), 13.34 (br, 1H), 8.73 (s, 1H), 8.33 (d, J ¼ 5.4 Hz, 1H), 8.04e7.93
(m, 2H), 7.79e7.70 (m, 2H), 7.68e7.56 (m, 2H), 7.55e7.38 (m, 4H),
7.30 (t, J ¼ 8.4 Hz, 2H), 7.18e6.96 (m, 3H), 4.24 (t, J ¼ 4.8 Hz, 2H),
4.03 (s, 3H), 3.83 (t, J ¼ 4.8 Hz, 2H), 2.29 (s, 3H). 13C NMR (101 MHz,
Prepared according to the general procedure A. Yellow solid;
yield: 67%; m. p. 197e198 ꢀC; 1H NMR (400 M, DMSO-d6)
d 13.30 (s,
1H), 12.91 (br, 1H), 8.63 (s, 1H), 8.26 (d, J ¼ 5.6 Hz, 1H), 8.20 (d,
J ¼ 13.6 Hz, 1H), 8.11 (d, J ¼ 6.0 Hz, 1H), 7.73e7.77 (m, 2H), 7.63 (s,
1H), 7.48e7.53 (m, 3H), 7.29 (t, J ¼ 8.8 Hz, 2H), 6.98 (d, J ¼ 6.0 Hz,
1H), 4.36 (t, J ¼ 5.6 Hz, 2H), 3.99e4.02 (m, 5H), 3.83 (t, J ¼ 11.2 Hz,
2H), 3.52 (d, J ¼ 12.0 Hz, 2H), 3.28e3.33 (m, 2H), 3.11e3.16 (m, 2H),
DMSO-d6) d 177.18, 164.83, 163.35, 160.78, 157.15, 155.51, 154.22,
153.14, 151.89, 150.51, 147.59, 147.29, 145.90, 139.21, 138.23, 131.29,
131.24, 130.55, 128.53, 125.90, 125.55, 124.67, 115.45, 115.24, 109.60,
107.01, 105.89, 104.29, 101.75, 69.81, 60.94, 56.11, 21.19. MS (ESI).
584.1 [MþH]þ. HR-MS (EI) m/z calcd for C31H23F2N5O5, 583.1667;
found 584.1658 [MþH]þ.
2.31e2.38 (m, 2H); 13C NMR (100 M, DMSO-d6)
d 177.2, 162.9, 160.8,
160.5, 154.3, 154.0, 152.5, 150.2, 149.2, 147.8, 144.8, 143.9, 138.9,
131.3, 130.6, 126.0, 125.4, 118.9, 116.0, 115.4, 114.5, 110.8, 106.8, 105.7,
103.8, 66.7, 63.8, 56.3, 54.3, 51.8, 23.3. MS (ESI). 665.2 [M ꢁ H]þ. HR-
MS (EI) m/z calcd for C36H32F2N6O5, 666.2402; found 689.2300
[MþNa]þ.
5.3.15. 5-[(3-fluoro-4-{[6-(2-hydroxy-2-methylpropoxy)-7-
methoxyquinazolin-4-yl]oxy}phenyl)amino]-3-(4-fluorophenyl)-
1,6-naphthyridin-4(1H)-one (10o)
Prepared according to the general procedure A. Yellow solid;
yield: 75%; m. p. 226e227 ꢀC; 1H NMR (600 MHz, DMSO-d6)
d 8.72
5.3.11. 5-{[3-fluoro-4-({6-methoxy-7-[3-(pyrrolidin-1-yl)propoxy]
quinazolin-4-yl} oxy)phenyl]amino}-3-(4-fluorophenyl)-1,6-
naphthyridin-4(1H)-one (10k)
(s, 1H), 8.39e8.28 (m, 2H), 8.09e7.93 (m, 1H), 7.64 (s, 1H), 7.59 (t,
J ¼ 8.7 Hz, 1H), 7.53e7.47 (m, 2H), 7.33e7.26 (m, 3H), 7.12e7.09 (m,
1H), 4.04 (s, 3H), 3.97 (s, 2H), 1.27 (s, 6H). MS (ESI). 612.2 [MþH]þ.
HR-MS (EI) m/z calcd for C33H27F2N5O5, 611.1980; found 612.1965
[MþH]þ.
Prepared according to the general procedure A. Yellow solid;
yield: 77%; m. p. 194e195 ꢀC; 1H NMR (400 M, DMSO-d6)
d 13.30 (s,
1H), 13.02 (br, 1H), 8.63 (s, 1H), 8.24 (d, J ¼ 6.0 Hz, 1H), 8.11 (d,
J ¼ 12.8 Hz, 1H), 8.11 (d, J ¼ 6.0 Hz, 1H), 7.73e7.77 (m, 2H), 7.63 (s,
1H), 7.47e7.51 (m, 3H), 7.29 (t, J ¼ 8.8 Hz, 2H), 7.00 (d, J ¼ 6.0 Hz,
1H), 4.36 (t, J ¼ 6.0 Hz, 2H), 4.02 (s, 3H), 3.57e3.62 (m, 2H),
3.29e3.33 (m, 2H), 3.01e3.06 (m, 2H), 2.28e2.32 (m, 2H),
2.00e2.05 (m, 2H), 1.89e1.94 (m, 2H); 13C NMR (100 M, DMSO-d6)
5.3.16. 5-{[4-({6-[3-(diethylamino)propoxy]-7-methoxyquinazolin-
4-yl}oxy)-3-fluorophenyl]amino}-3-(4-fluorophenyl)-1,6-
naphthyridin-4(1H)-one (10p)
Prepared according to the general procedure A. Yellow solid;
yield: 65%; m. p. 136e137 ꢀC; 1H NMR (400 M, DMSO-d6)
d 13.31 (s,
d
177.2, 161.0, 160.8, 154.5, 154.0, 152.6, 150.2, 149.2, 147.7, 144.8,
1H), 12.94 (br, 1H), 10.21 (s, 1H), 8.64 (s, 1H), 8.26 (d, J ¼ 6.4 Hz, 1H),
8.20 (d, J ¼ 12.8 Hz, 1H), 8.11 (d, J ¼ 6.0 Hz, 1H), 7.73e7.77 (m, 2H),
7.65 (s, 1H), 7.47e7.53 (m, 3H), 7.29 (t, J ¼ 8.8 Hz, 2H), 6.99 (d,
J ¼ 6.0 Hz, 1H), 4.34 (t, J ¼ 6.0 Hz, 2H), 4.03 (s, 3H), 3.14e3.27 (m,
6H), 2.23e2.27 (m, 2H), 1.26 (t, J ¼ 7.2 Hz, 6H); 13C NMR (100 M,
143.9, 139.0, 131.3, 130.5, 125.1, 119.0, 116.1, 115.5, 115.1, 108.6, 106.8,
105.7, 103.7, 66.6, 56.7, 53.6, 51.8, 25.5, 23.3. MS (ESI). 649.2
[M ꢁ H]þ. HR-MS (EI) m/z calcd for C36H32F2N6O4, 650.2453; found
651.2542 [MþH]þ.
DMSO-d6)
d 177.4, 163.4, 160.9, 160.4, 155.2, 154.7, 152.6, 150.2,
5.3.12. 5-({4-[(6,7-dimethoxyquinazolin-4-yl)oxy]-3-fluorophenyl}
amino)-3- (4-fluorophenyl)-1,6-naphthyridin-4(1H)-one (10l)
Prepared according to the general procedure A. Yellow solid;
148.0,147.7,144.8,144.6,138.9,131.4,131.2,130.5,127.4,124.9, 121.3,
118.9, 115.9, 115.5, 115.3, 113.3, 108.1, 106.9, 103.6, 66.3, 56.5, 48.2,
46.9, 23.5, 9.0. MS (ESI). 653.2 [MþH]þ. HR-MS (EI) m/z calcd for

L.-S. Zhuo et al. / European Journal of Medicinal Chemistry 208 (2020) 112785
13
C36H34F2N6O4, 652.2610; found 653.2687 [MþH]þ.
7.48e7.58 (m, 3H), 7.46 (s, 1H), 7.29 (t, J ¼ 8.8 Hz, 2H), 6.97 (d,
J ¼ 6.4 Hz, 1H), 4.00 (s, 6H), 3.86 (s, 3H); 13C NMR (100 M, DMSO-d6)
5.3.17. 5-((3-fluoro-4-({7-methoxy-6-[3-(pyrrolidin-1-yl)propoxy)
quinazolin-4-yl)oxy)phenyl)amino)-3-(4-fluorophenyl)-1,6-
naphthyridin-4(1H)-one (10q)
d 176.3, 164.1, 162.6, 160.2, 156.0, 155.9, 152.3, 152.1, 150.2, 149.3,
148.9, 146.6, 143.9, 138.9, 133.4, 130.8, 130.7, 124.2, 122.5, 115.9,
114.8, 114.6, 109.1, 108.0, 107.1, 106.7, 101.3, 100.5, 56.2, 56.0, 40.6.
MS (ESI). 568.1 [MþH]þ. HR-MS (EI) m/z calcd for C31H23F2N5O4,
567.1718; found 568.1797 [MþH]þ.
Prepared according to the general procedure A. Yellow solid;
yield: 79%; m. p. 172e173 ꢀC; 1H NMR (400 M, DMSO-d6)
d 13.43 (s,
1H), 13.26 (br, 1H), 8.73 (s, 1H), 8.61 (s, 1H), 8.43 (d, J ¼ 5.6 Hz, 1H),
7.90 (s, 1H), 7.58 (s, 1H), 7.29e7.05 (m, 5H), 6.96e6.90 (m, 1H), 6.52
(d, J ¼ 5.6 Hz, 1H), 6.30e6.22 (m, 1H), 4.05 (t, J ¼ 7.2 Hz, 2H), 3.96 (s,
3H), 3.81e3.70 (m, 4H), 2.52 (t, J ¼ 7.2 Hz, 2H), 1.90e1.77 (m,
5.3.22. 5-({4-[(6,7-dimethoxyquinazolin-4-yl)oxy]-3-fluorophenyl}
amino)-1-ethyl-3-(4-fluorophenyl)-1,6-naphthyridin-4(1H)-one
(22d)
6H)。13C NMR (100 M, DMSO-d6)
d
177.3, 161.1, 160.8, 154.3, 154.0,
Prepared according to the general procedure C. Yellow solid;
152.4,150.2,149.1,147.4, 144.8,143.9,139.0, 131.3, 130.5,125.1,119.0,
116.1, 115.5, 115.1, 108.6, 106.8, 105.7, 103.7, 66.6, 56.7, 53.6, 51.9,
25.6, 23.5. MS (ESI). 651.2 [MþH]þ. HR-MS (EI) m/z calcd for
yield: 85%; m. p. 169e170 ꢀC; 1H NMR (400 M, DMSO-d6)
d 13.48 (s,
1H), 8.59 (s, 1H), 8.38 (s, 1H), 8.29e8.35 (m, 2H), 7.74e7.78 (m, 2H),
7.60 (s, 1H), 7.40e7.51 (m, 3H), 7.29 (t, J ¼ 8.8 Hz, 2H), 7.07 (d,
J ¼ 6.0 Hz, 1H), 4.32e4.37 (m, 2H), 4.02 (s, 3H), 4.00 (s, 3H), 1.04 (t,
C
36H32F2N6O4, 650.2453; found 651.2487 [MþH]þ.
J ¼ 6.8 Hz, 2H); 13C NMR (100 M, DMSO-d6)
d 176.4, 164.2, 160.2,
5.3.18. 5-{[3-fluoro-4-({7-methoxy-6-[3-(piperidin-1-yl)propoxy]
quinazolin-4-yl}oxy) phenyl]amino}-3-(4-fluorophenyl)-1,6-
naphthyridin-4(1H)-one (10r)
156.4, 155.9, 152.2, 150.3, 149.3, 148.9, 145.7, 142.9, 138.9, 133.5,
133.3, 130.9, 130.8, 130.7, 124.2, 123.0, 116.0, 114.9, 114.6, 109.2,
107.4, 106.8, 101.0, 100.6, 56.2, 48.6, 14.2. MS (ESI). 582.1 [MþH]þ.
HR-MS (EI) m/z calcd for C32H25F2N5O4, 581.1875; found 582.1964
[MþH]þ.
Prepared according to the general procedure A. Yellow solid;
yield: 77%; m. p. 162e163 ꢀC; 1H NMR (400 M, DMSO-d6)
d 13.17 (s,
1H), 12.84 (br, 1H), 8.59 (s, 1H), 8.34 (d, J ¼ 13.2 Hz, 1H), 8.19 (d,
J ¼ 6.8 Hz, 1H), 7.72e7.76 (m, 2H), 7.61 (s, 1H), 7.38e7.52 (m, 3H),
7.27 (t, J ¼ 9.2 Hz, 2H), 6.96 (d, J ¼ 5.2 Hz,1H), 4.35 (t, J ¼ 6.0 Hz, 2H),
4.01 (s, 3H), 2.82e2.9 (m, 6H), 2.19e2.23 (m, 2H), 1.69e1.74 (m, 4H),
5.3.23. 5-({4-[(6,7-dimethoxyquinazolin-4-yl)oxy]-3-fluorophenyl}
amino)-3-(4-fluorophenyl)-1-(2-methoxyethyl)-1,6-naphthyridin-
4(1H)-one (22e)
1.47e1.52 (m, 2H); 13C NMR (100 M, DMSO-d6)
d
177.5, 164.7, 163.2,
Prepared according to the general procedure C. Yellow solid;
160.3, 156.4, 156.1, 154.9, 152.7, 149.7, 149.4, 148.9, 146.6, 139.6,
138.8, 133.8, 131.3, 131.2, 124.7, 123.3, 116.3, 115.3, 115.1, 109.6, 108.3,
108.1, 107.4, 103.6, 102.1, 67.2, 63.8, 56.8, 54.4, 53.2, 24.5, 23.8, 22.6.
MS (ESI). 665.2 [MþH]þ. HR-MS (EI) m/z calcd for C37H34F2N6O4,
664.2610; found 665.2681 [MþH]þ.
yield: 72%; m. p. 237e238 ꢀC; 1H NMR (400 M, DMSO-d6)
d 13.44 (s,
1H), 8.58 (s, 1H), 8.24e8.34 (m, 3H), 7.71e7.74 (m, 2H), 7.58 (s, 1H),
7.39e7.49 (m, 3H), 7.30 (t, J ¼ 8.8 Hz, 2H), 7.08 (d, J ¼ 6.4 Hz, 1H),
4.50 (t, J ¼ 4.8 Hz, 2H), 4.00 (s, 3H), 3.71 (t, J ¼ 4.8 Hz, 2H), 3.36 (s,
3H); 13C NMR (100 M, DMSO-d6)
d 176.5, 163.9, 162.7, 160.2, 156.3,
155.9, 154.7, 152.3, 152.1, 150.2, 149.2, 148.9, 146.2, 143.8, 139.0,
133.5, 133.3,130.8,130.7,124.2, 122.3,116.0, 114.9,114.7, 109.2, 108.1,
107.9, 106.7, 101.2, 100.6, 69.4, 58.4, 56.2, 56.0, 51.9. MS (ESI). 612.2
[MþH]þ. HR-MS (EI) m/z calcd for C33H27F2N5O5, 611.1980; found
612.2068 [MþH]þ.
5.3.19. 5-({4-[(6,7-dimethoxyquinazolin-4-yl)oxy]phenyl}amino)-
3-(4-fluorophenyl)-1,6-naphthyridin-4(1H)-one (22a)
Prepared according to the general procedure A. Yellow solid;
yield: 83%; m. p. 284e285 ꢀC; 1H NMR (400 M, DMSO-d6)
d 13.39
(vol, 2H), 8.68 (s, 1H), 8.29 (d, J ¼ 3.4 Hz, 1H), 7.93 (d, J ¼ 6.4 Hz, 1H),
7.74e7.78 (m, 4H), 7.60 (s, 1H), 7.44e7.48 (m, 3H), 7.46 (s, 1H), 7.29
(t, J ¼ 9.2 Hz, 2H), 7.04 (d, J ¼ 6.4 Hz, 1H), 4.01 (vol, 6H); 13C NMR
5.3.24. 5-({4-[(6,7-dimethoxyquinazolin-4-yl)oxy]-3-fluorophenyl}
amino)-3-(4-fluorophenyl)-1-(2-oxo-2-phenylethyl)-1,6-
naphthyridin-4(1H)-one (22f)
(100 M, DMSO-d6)
d 176.9, 164.2, 160.6, 156.3, 154.2, 152.6, 151.9,
150.5, 148.1, 146.8, 144.9, 138.6, 136.2, 130.9, 130.5, 124.8, 123.5,
114.9, 114.7, 109.1, 108.0, 106.7, 106.1, 104.0, 100.6, 59.3, 56.1. MS
(ESI). 536.1 [MþH]þ. HR-MS (EI) m/z calcd for C30H22FN5O4,
535.1656; found 536.1632 [MþH]þ.
Prepared according to the general procedure C. Yellow solid;
yield: 76%; m. p. 280e281 ꢀC; 1H NMR (400 M, DMSO-d6)
d 13.46 (s,
1H), 8.66 (s, 1H), 8.41e8.44 (m, 2H), 8.27 (d, J ¼ 6.4 Hz, 1H), 8.18 (d,
J ¼ 7.6 Hz,1H), 7.79e7.86 (m, 3H), 7.72 (t, J ¼ 7.6 Hz, 2H), 7.66 (s, 1H),
7.59 (d, J ¼ 9.6 Hz,1H), 7.49 (t, J ¼ 6.8 Hz, 2H), 6.93 (d, J ¼ 6.4 Hz,1H),
5.3.20. 5-({4-[(6,7-dimethoxyquinazolin-4-yl)oxy]-3-fluorophenyl}
amino)-3-(4-fluorophenyl)-2-methyl-1,6-naphthyridin-4(1H)-one
(22b)
6.17 (s, 2H), 4.06 (s, 6H); 13C NMR (100 M, DMSO-d6)
d 192.6, 176.7,
164.2, 160.3, 156.1, 155.9, 152.3, 152.2, 150.3, 149.3, 148.9, 147.2,
144.0, 139.0, 134.3, 134.2, 133.4, 130.7, 128.9, 128.3, 124.2, 122.8,
116.1, 115.0, 114.7, 109.2, 108.2, 108.0, 107.1, 106.8, 101.5, 100.6, 58.6,
56.2, 56.0. MS (ESI). 673.2 [M]þ. MS (ESI). 672.2 [MþH]þ. HR-MS (EI)
m/z calcd for C33H27F2N5O5, 672.2058; found 672.2961 [MþH]þ.
Prepared according to the general procedure A. Yellow solid;
yield: 65%; m. p. 282e283 ꢀC; 1H NMR (400 M, DMSO-d6)
d 13.34 (s,
1H), 12.77 (br, 1H), 8.69 (s, 1H), 8.11e8.18 (m, 2H), 7.65 (s, 1H),
7.49e7.57 (m, 3H), 7.32e7.41 (m, 4H), 7.04 (d, J ¼ 6.0 Hz,1H), 4.07 (s,
6H), 2.32 (s, 3H); 13C NMR (100 M, DMSO-d6)
d
177.3, 163.2, 160.8,
5.4. In silico studies
155.1, 154.2, 152.6, 150.2, 149.2, 148.7, 147.5, 144.5, 140.0, 133.0,
132.9, 130.2, 127.1, 125.3, 121.4, 119.0, 115.7, 115.4, 113.5, 107.8, 105.4,
103.9, 103.4, 56.4, 56.2, 18.8. MS (ESI). 568.1 [MþH]þ. HR-MS (EI) m/
z calcd for C31H23F2N5O4, 567.1718; found 568.1733 [MþH]þ.
5.4.1. Molecular docking
The three dimensional (3D) structure of the MET kinase com-
plex (PDB code: 3F82) and VEGFR-2 kinase (PDB code: 3U6J)
complex was obtained from PDB database. All water molecules and
ligand were removed from the complex structure and hydrogen
atoms were added with pH equaling to 7.0 using Sybyl-X. The
AutoDock 4.2 [60] program was applied to docking compound 10l,
10n, 22a, 22b, 22c and 22e into the binding site of MET kinase or
VEGFR-2 kinase. The Gasteiger charges were used for these in-
hibitors. In the docking process, a conformational search was
5.3.21. 5-({4-[(6,7-dimethoxyquinazolin-4-yl)oxy]-3-fluorophenyl}
amino)-3-(4-fluorophenyl)-1-methyl-1,6-naphthyridin-4(1H)-one
(22c)
Prepared according to the general procedure C. Yellow solid;
yield: 89%; m. p. 176e177 ꢀC; 1H NMR (400 M, DMSO-d6)
d
13.41 (s,
1H), 8.58 (s, 1H), 8.31e8.36 (m, 3H), 7.71e7.75 (m, 2H), 7.59 (s, 1H),

14
L.-S. Zhuo et al. / European Journal of Medicinal Chemistry 208 (2020) 112785
performed for the ligand using the Solis and Wets local search
method, and the Lamarckian genetic algorithm (LGA) [61,62] was
applied for the conformational search of the binding complex of
ligand with two kinases. Among a series of docking parameters, the
grid size was set to be 70 ꢂ 70 ꢂ 80 in 3F82, 46 ꢂ 40 ꢂ 60 in 3U6J,
and the used grid space was the default value of 0.375 Å. Finally,100
conformations were generated and top ranked conformation was
selected as the binding conformation.
MM_PBSA module in amber16 program.
solvent accessible surface area [61,78].
D
Gnp is determined by the
DGsol
¼
DGPB
þ
DGnp
(4)
For the entropic contribution, an empirical method [79,80] was
used, it consists two subitems, the solvation entropy change ( Ssol
and conformational entropy change ( Sconf):
D
)
D
DS ¼
DSsol
þ
D
Sconf
(5)
5.4.2. Molecular dynamics simulation
Molecular dynamics (MD) simulation were performed on the
selected inhibitor-receptor complex structures to obtain stable
binding mode. First, the Antechamber module in Amber16 program
was used to assign BCC charges on the ligands [63e65]. Then, the
complex coordinate and topology parameters were constructed by
using Leap module in Amber16 packages. We used AMBER ff14SB as
the force filed for the amino acids and general AMBER force field
(GAFF) for the ligands. Clꢁ or Naþ was added to the system to
neutralize the system [66e70]. The complex was finally solvated
with the TIP3P waters [71].
The
D
Ssol is obtained by the tendency of water molecules to
minimize their contacts with hydrophobic groups in protein, Sconf
D
is related to the change of the number of rotatable bonds during the
binding process. The entropic contribution is evaluated by using the
procedure developed by Dr. Zhan [79,80], and the conformational
entropy change is proportional to the number (
rotatable bonds during the binding:
D
Nrot) of the lost
eT
DSconf ¼ w(
DNrot
)
(6)
The complex was subjected to a three-step energy minimization
using the Sander module of Amber16 before the MD simulation.
First, only waters, ions and hydrogens were allowed to move, and
in which w is a scaling factor which was set to be 1 kcal/mol for the
binding energy calculation. Thus, Eq. (1) can be written as:
the solute was kept fixed with a constraint of 100 kcal molꢁ1 Åꢁ2
.
D
Gbind
¼
DEMM
þ
DGPB
þ
DGnp e T
DSsol þ w(DNrot
)
(7)
Second, we fixed the backbone atoms of the protein and allowed
other atoms to move. Third, all the atoms of the system were free to
move. The 2000-step steepest descent method and the 2000-step
conjugated gradient method were used in the three-step minimi-
zation processes on the ligand-receptor complexes. The MD simu-
lation was performed employing the periodic boundary condition
with the NPT ensemble. First, a 10-ps simulation was performed on
the solvent molecules and ions to obtain an equilibrated solvent
environment. Second, the system temperature was gradually
heated from 0 to 298 K over 50 ps? The systems were finally
maintained at 298 K with a constant pressure until the MD reached
6 ns? The SHAKE algorithm [72] was employed to constrain all
bonds involving hydrogens. The particle mesh Ewald (PME) algo-
rithm [73e76] was used to deal with long-range electrostatic in-
teractions and van der Waals (vdW) energy terms with a cutoff
distance of 10 Å. The time step was 2.0 fs, and the coordinates were
collected every 1 ps during the MD simulation.
all other parameters in the energy calculation are the standard
parameters or the default values of the Amber16 program. 50
snapshots were extracted from the last 1ns of the MD trajectory
with a time step 20ps. The final
compound was evaluated by using the average
D
Gbind of the receptor with every
Gbind value of the
D
50 snapshots. 5.5. In vivo Antitumor Activity Assay.
Female nude mice (4e6 weeks old) were housed and main-
tained under specific-pathogen free conditions. Animal experi-
ments were performed according to institutional ethical guidelines
of animal care. Tumor cells were inoculated into the flanks of
athymic nude mice (2 ꢂ 106 cells/mouse). When the tumor volume
reached about 150 mm3, the mice were randomly assigned into
control and treatment groups. Control groups were given vehicle
alone, and treatment groups received synthesized compounds as
indicated doses via p. o. administration 7 days per week for 3
weeks. The sizes of the tumors were measured three times per
week using micro caliper. The tumor weight (TW) was measured on
the final day of study. Percent (%) inhibition values (IR) were
calculated as 100% ꢂ (TW/controlfinal day eTW/treatedfinal day)/TW/
5.4.3. Free energy calculation
The MM-PBSA method was used to calculate the binding free
energy (DGbind) between the receptor and every ligand [77]. This
value was obtained by calculating the differences in free energies
between the ligand-receptor complex (Gcpx) and the unbound re-
ceptor (Grec) and ligand (Glig) as follows:
controlfinal day
.
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.
D
Gbind ¼ Gcpx e (Grec þ Glig
)
(1)
The Gbind consists of the molecular mechanical (MM) gas-
D
phase binding energy (
entropic contribution (eT
D
D
EMM), solvation free energy (
S):
DGsol) and
Acknowledgement
We thank the National Key Research and Development Program
of China (No. 2017YFA0505200), the National Natural Science
Foundation of China (No. 201502063), the Science and Technology
Program of Wuhan, China (No. 2019020701011460) for financial
support.
D
Gbind
¼
D
EMM
þ
D
G
sol e T
D
S
(2)
The
D
EMM includes two parts, the electrostatic energies (
EvdW):
DEele)
and van der Waals interaction (
D
DEMM
¼
D
Eele
þ
DEvdW
(3)
Appendix A. Supplementary data
The
nonelectrostatic contribution (
GPB is calculated by the Poisson-Boltzman (PB) method using
D
Gsol is made up of electrostatic contribution (
DGPB) and
D
Gnp) to the solvation free energy.
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2020.112785.
D

L.-S. Zhuo et al. / European Journal of Medicinal Chemistry 208 (2020) 112785
[18] http://www.globenewswire.com/news-release/2020/05/06/2028748/0/en/
15
Abbreviations used
Novartis-announces-FDA-approval-of-MET-inhibitor-Tabrecta-for-
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HGF
mesenchymalꢁepithelial transition factor
hepatocyte growth factor
EGFR
MEK
MAPK
PI3K
AKT
mTOR
RAC1
PAK
epidermal growth factor receptor
mitogen-activated protein kinase
mitogen-activated protein kinase
phosphatidylinositol 3-kinase
protein kinase B
mammalian target of rapamycin
Ras-related C3 botulinum toxin substrate 1
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ALK
anaplastic lymphoma kinase
c-ros Oncogene 1 kinase
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