Design, synthesis and biological evaluation of 7H-pyrrolo[2,3-d]pyrimidine derivatives containing 1,8-naphthyridine-4-one fragment

Article abstract of DOI:10.1016/j.ejmech.2021.113273

In this study, a series of pyrrolo [2,3-d]pyrimidine derivatives containing 1,8-naphthyridine-4-one fragment were synthesized and their biological activity were tested. Most of the target compounds displayed moderate to excellent activity against one or more cancer cell lines and low activity against human normal cell LO2 in vitro. The most promising compound 51, of which the IC₅₀ values were 0.66 μM, 0.38 μM and 0.44 μM against cell lines A549, Hela and MCF-7, shown more remarkable activity and better apoptosis effect than the positive control Cabozantinib. The structure-activity relationships (SARs) indicated that double-EWGs (such as R₃ = 2-Cl-4-CF₃) on the terminal phenyl rings was a key factor in improving the biological activity. In addition, the further research on compound 51 mainly included c-Met kinase activity and selectivity, concentration dependence, and molecular docking.

Full text of DOI:10.1016/j.ejmech.2021.113273

European Journal of Medicinal Chemistry 215 (2021) 113273
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Design, synthesis and biological evaluation of 7H-pyrrolo[2,3-d]
pyrimidine derivatives containing 1,8-naphthyridine-4-one fragment
,
b
,
,
Jianqing Zhang a 1, Pengqin Chen a 1, Yongli Duan c, Hehua Xiong d, Hongmin Li b,
Yao Zeng b, Guang Liang a, Qidong Tang a *, Di Wu a
,
b,
, **
a Chemical Biology Research Center, School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, 325035, PR China
b Jiangxi Provincial Key Laboratory of Drug Design and Evaluation, School of Pharmacy, Jiangxi Science & Technology Normal University, Nanchang, 330013,
PR China
c School of Optoelectronic Science and Engineering, University of Electronic Science and Technology of China (UESTC), Chengdu, 610054, PR China
d State Key Laboratory of Natural Medicines, Key Laboratory of Drug Quality Control and Pharmacovigilance, Department of Pharmaceutical Analysis, China
Pharmaceutical University, Nanjing, 210009, China
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, a series of pyrrolo [2,3-d]pyrimidine derivatives containing 1,8-naphthyridine-4-one
fragment were synthesized and their biological activity were tested. Most of the target compounds
displayed moderate to excellent activity against one or more cancer cell lines and low activity against
human normal cell LO2 in vitro. The most promising compound 51, of which the IC50 values were
Received 30 October 2020
Received in revised form
20 January 2021
Accepted 1 February 2021
Available online 9 February 2021
0.66 mM, 0.38 mM and 0.44 mM against cell lines A549, Hela and MCF-7, shown more remarkable activity
and better apoptosis effect than the positive control Cabozantinib. The structure-activity relationships
(SARs) indicated that double-EWGs (such as R3 ¼ 2-Cl-4-CF3) on the terminal phenyl rings was a key
factor in improving the biological activity. In addition, the further research on compound 51 mainly
included c-Met kinase activity and selectivity, concentration dependence, and molecular docking.
© 2021 Elsevier Masson SAS. All rights reserved.
Keywords:
7H-pyrrolo[2,3-d]pyrimidine
1,8-Naphthyridine-4-one
Inhibitors
Activity
Synthesis
SAR
1. Introduction
c-Met belongs to a subfamily of receptor tyrosine kinases (RTKs)
encoded by the c-Met proto-oncogene, which was discovered in
Cancer is a major public health problem worldwide [1e3].
Global demographic characteristics predict that cancer incidence
rate will continue to increase in the next few decades, with more
than 20 million cancer patients annually. Traditional treatment
methods (such as surgery, radiotherapy, chemotherapy) could in-
crease the survival rate of cancer patients, but they still have many
adverse reactions [4e6]. Thus, the search of novel treatment which
is efficacious and effective with low side effects is imminent. With
the continuous development of medical research, targeted therapy
has become a prevalent method in the treatment of cancer because
of its good efficacy and low side effects [7,8].
human osteosarcoma cell lines [9,10]. The binding of hepatocyte
growth factor (HGF) to c-Met induces a series of effects, including
proliferation, angiogenesis, invasion and migration, which are
essential in normal physiology [11,12]. However, the abnormal
activation of c-Met occurs in many types of human cancers, such as
breast, liver, gastric cancers and so on [13e15]. Therefore, c-Met has
garnered considerable attention for use as an anticancer drug
target, giving rise to numerous investigations pertaining to c-Met
kinase inhibitors with the goal of disrupting the abnormal activa-
tion of HGF/c-Met signaling pathway.
In view of how inhibitors and c-Met kinase domain bind, small-
molecular c-Met kinase inhibitors are categorized into either class I
or class II. Inhibitors of class I have the characteristics of small
binding region and easily produce drug resistance for patients due
to the mutation near the active site of c-Met [16]. In comparison,
inhibitors of class II are believed to be more effective for these
mutations as their binding interactions extend beyond the entry of
the c-Met active site [17]. Therefore, c-Met inhibitors of class II have
* Corresponding author. Chemical Biology Research Center, School of Pharma-
ceutical Sciences, Wenzhou Medical University, Wenzhou, 325035, PR China.
** Corresponding author.
E-mail addresses: tangqidongcn@126.com (Q. Tang), wudi2017@wmu.edu.cn
(D. Wu).
1
These authors contribute equally to this work.
https://doi.org/10.1016/j.ejmech.2021.113273
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

J. Zhang, P. Chen, Y. Duan et al.
European Journal of Medicinal Chemistry 215 (2021) 113273
been widely studied due to their decreased level of drug resistance
as well as their reduced toxicity and stronger efficacy. As shown in
Fig. 1, Cabozantinib (1) is the first small-molecular c-Met inhibitor
of class II approved by the FDA of the United States for the treat-
ment of late-stage non-small-cell lung cancer (NSCLC) and meta-
static medullary thyroid cancer [18e20]. In addition, many
Cabozantinib derivatives were reported, such as Foretinib (2),
BMS777607 (3) as well as compounds 4 and 5 [21,22]. Moreover,
compound 6 showed excellent efficacy with IC50 values in the
micromolar level, as demonstrated in our previous study [23].
The c-Met inhibitors of class II have similar structural character-
istics, which can be illustrated by the binding pattern of Cabozantinib
with c-Met protein (PDB code: 3LQ8), as shown in Fig. 2. First, moiety
A was a pyridine or its analogs, providing a nitrogen atom and mainly
forming hydrogen bond with Met1160. Subsequently, the central and
terminal benzene rings (moiety B) located at the hydrophobic region
forming weak interactions with the surrounding amino acid residues.
Inparticular, moietyC exhibitedanobviousstructuralcharacteristic, a
‘5-atom linker’, which means six chemical bonds distance existed
between the central and terminal benzene rings. The moiety C can be
modified with flexible or rigid chains to form hydrogen bond donors
or acceptors with the amino acid residues of c-Met (such as Lys1110
and Asp1222) in improving the activity of the compounds [15,21,23].
The space of c-Met protein around moieties A and C was large enough
for further optimization of the compounds in order to improve the
biological activity. While, only minor changes in the central and ter-
minal benzene rings were available.
In this study, the pyrrolo [2,3-d]pyrimidine fragment was
retained as moiety A based on compound 6 from our previous
work, which served as an important structure in maintaining bio-
logical activity. In this regard, different substituents were intro-
duced into moiety A so as to study the effect of hydrogen bond
formation on antiproliferative potency. As reported, various 1,8-
naphthyridine-4-one derivatives represented by vosaroxin (7)
(Fig. 3) have shown excellent anticancer effects in different types of
cancer. These derivatives possessed the characteristics of a high-
quality hydrogen bond receptor and gained considerable atten-
tion from researchers [24e26]. According to the 3D model of
Cabozantinib (Fig. 4), an intramolecular hydrogen bond
(distance ¼ 1.714 Å) between oxygen and hydrogen atoms was
formed in the ‘5-atom linker’. Moreover, inspired by the intra-
molecular hydrogen bond of Cabozantinib, 1,8-naphthyridine-4-
one was introduced into moiety C to further explore biological
activity while maintaining the possibility of forming intramolecular
hydrogen bonds between oxygen and hydrogen atoms. Finally,
various substituents such as F, Cl, CF3 and OCH3 were introduced to
the central and terminal benzene rings (moiety B) in order to
explore the influence of electron cloud accumulation in the hy-
drophobic region on biological activity (Fig. 4). Thus, a novel series
of pyrrolo [2,3-d]pyrimidine derivatives containing 1,8-
naphthyridine-4-one fragment were designed.
2. Results and discussion
2.1. Chemistry
2.1.1. Synthesis of 3-substituted-4-((7-substituted-7H-pyrrolo[2,3-
d]pyrimidin-4-yl)oxy)anilines (13a-d)
The key intermediates of 3-substituted-4-((7-substituted-7H-
pyrrolo [2,3-d]pyrimidin-4-yl)oxy)anilines 13a-d were acquired as
revealed in Scheme 1. Nucleophilic substitution reaction of
commercially available 4-chloro-7H-pyrrolo [2,3-d]pyrimidine 10a
or 4-chloro-7-methyl-7H-pyrrolo [2,3-d]pyrimidine 10b and p-
nitrophenol or 2-fluoro-4-nitrophenol (11a-d) gave intermediates
12a-d, which were then reduced to obtain intermediates 13a-
d [22,27].
Reagents and conditions: (i) p-nitrophenol or 2-fluoro-4-
nitrophenol, diphenyl oxide, 140 ꢀC, 8 h; (ii) Fe powder, NH4Cl
(cat.), EtOH/H2O, 85 ꢀC, 5 h.
2.1.2. Synthesis of the target compounds of 20e51
The target compounds 20e51 were prepared as illustrated in
Scheme 2. Here, 1-(2-chlorophenyl)ethan-1-one (14) and dimethyl
carbonate were refluxed in toluene at 85 ꢀC to afford intermediate
15 as an orange oily liquid [28]. The carbanion reaction of 15 with
N,N-dimethylformamide dimethyl acetal (DMF-DMA) in toluene
produced intermediate 16 as a light yellow liquid, which then
reacted with the corresponding anilines to obtain intermediates
17a-h. The corresponding acid chlorides 19a-h were then obtained
by hydrolysis and carboxylation of 17a-h, which reacted with so-
dium hydroxide (NaOH) and oxalyl chloride, respectively [29]. The
target compounds 20e51 were obtained at room temperature (r.t.),
and the reaction of the anilines 13a-d with the acid chlorides 19a-h
were facilitated by N,N-diisopropylethylamine (DIPEA) in
dichloromethane.
Reagents and conditions: (i) NaH, dimethyl carbonate, toluene,
85 ꢀC, 6 h; (ii) DMF-DMA, toluene, 100 ꢀC, 2 h; (iii) corresponding
anilines, Cs2CO3, toluene, 150 ꢀC, 6 h; (iv) NaOH, 1,4-dioxane, H2O,
100 ꢀC, 2 h; (v) oxalyl chloride, CH2Cl2, r. t., 0.5 h; (vi) corresponding
anilines, DIPEA, CH2Cl2, r. t., 1 h.
Fig. 1. The representative small-molecular c-Met kinase inhibitors from others work (1e5) and our previous work (6).
2

J. Zhang, P. Chen, Y. Duan et al.
European Journal of Medicinal Chemistry 215 (2021) 113273
Fig. 2. Analysis of the binding mode of Cabozantinib and c-Met kinase (PDB code: 3LQ8): (A) Cavity diagram of the docking results of Cabozantinib; (B) Chemical structure of
Cabozantinib and its depicted binding mode with c-Met kinase; (C) Ribbon map of the docking results of Cabozantinib. Hydrogen bonding interactions were indicated with yellow
dotted line.
Fig. 3. Anticancer agents with 1,8-naphthyridine-4-one fragment (7e9).
Fig. 4. The design strategy for the pyrrolo [2,3-d]pyrimidine derivatives with 1,8-naphthyridine-4-one fragment.
Scheme 1. Synthetic route of the key intermediates 13a-d.
2.2. Biological evaluation
MCF-7 (breast cancer cells). Cabozantinib was used as a positive
control, and the results were expressed as half-maximal inhibitory
concentration (IC50) values, as presented in Table 1.
2.2.1. In vitro cytotoxic activities and SARs
The synthesized compounds (20e51) were evaluated for cyto-
toxicity via MTT assay against three cancer cell lines including A549
(non-small cell lung cancer cells), Hela (cervical cancer cells) and
Among them, A549 and Hela are high and moderate expressing
cell lines of c-Met kinase, respectively, and were used to test the
cytotoxic activity in our study. Although there is no overexpression
3

J. Zhang, P. Chen, Y. Duan et al.
European Journal of Medicinal Chemistry 215 (2021) 113273
Scheme 2. Synthetic route of the target compounds.
Table 1
Structures and cytotoxic activities of the target compounds 20e51 against A549, Hela and MCF-7 cancer cell lines in vitro.
a
Compd.
R1
R2
R3
IC50
(
mM)
A549
Hela
MCF-7
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
F
F
F
F
F
F
F
F
F
F
F
F
4-H
2-F-4-Br
4-Br
4-Cl
4-F
3-Cl-4-F
4-OCH3
2-Cl-4-CF3
4-H
2-F-4-Br
4-Br
2.89 0.54
7.05 0.23
7.34 0.54
0.75 0.15
0.79 0.02
0.81 0.03
0.97 0.01
3.67 0.88
1.89 0.03
5.24 0.33
1.01 0.02
6.28 0.79
0.69 0.02
0.73 0.01
5.14 0.11
0.81 0.11
0.93 0.17
0.89 0.02
0.78 0.01
1.27 0.23
0.72 0.04
0.69 0.04
5.85 0.32
1.32 0.02
1.18 0.02
2.14 0.04
0.87 0.01
3.71 0.28
0.77 0.01
NA
6.31 0.82
1.46 0.29
3.46 0.53
3.12 0.65
NAb
2.18 0.43
9.38 0.25
NA
3.62 0.72
4.41 0.79
9.24 0.62
3.47 0.09
1.29 0.03
0.35 0.04
NA
4.27 0.81
11.09 1.23
6.49 1.17
9.24 0.33
2.12 0.16
0.46 0.01
NA
2.81 0.49
1.25 0.23
4.07 0.51
2.71 0.43
3.65 0.71
1.69 0.03
0.69 0.03
0.34 0.02
4.53 0.61
2.45 0.36
0.09 0.09
0.65 0.02
0.52 0.03
0.76 0.12
0.51 0.02
2.11 0.23
0.46 0.09
0.44 0.04
0.45 0.04
12.09 2.01
NA
3.16 0.43
1.23 0.33
1.54 0.25
0.34 0.03
1.43 0.54
3.25 0.13
1.26 0.29
3.12 0.63
1.29 0.03
0.30 0.02
0.33 0.01
NA
1.34 0.07
0.25 0.03
0.65 0.02
0.14 0.01
0.51 0.03
1.21 0.24
0.29 0.03
0.35 0.02
1.65 0.09
0.38 0.03
0.32 0.01
4-Cl
4-F
3-Cl-4-F
4-OCH3
2-Cl-4-CF3
4-H
2-F-4-Br
4-Br
4-Cl
4-F
F
F
F
3-Cl-4-F
4-OCH3
2-Cl-4-CF3
4-H
2-F-4-Br
4-Br
4-Cl
4-F
3-Cl-4-F
4-OCH3
2-Cl-4-CF3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
1.15 0.24
0.66 0.03
0.76 0.05
Cabozantinibc
a
Bold type showed that the IC50 value of the target compound was lower than that of the positive control.
NA: Not determined.
Used as the positive control.
b
c
of c-Met in MCF-7 cell line, we chose it to investigate whether these
compounds showed potent cytostatic activity against cells that c-
Met expressed lowly [30e32].
23 (IC50 ¼ 0.75
(IC50 ¼ 0.81
(IC50 ¼ 0.66
0.15
0.03
0.03
m
M), 24 (IC50 ¼ 0.79
0.02
0.01
m
M), 25
m
m
M), 48 (IC50 ¼ 0.77
mM), 51
M). The most promising compound 51
Overall, all tested compounds showed moderate to excellent
cytotoxic activity against different cancer cells. Fifteen of these
compounds were more potent than Cabozantinib against one or
more cancer cell lines, which further indicated that the introduc-
tion of the 1,8-naphthyridine-4-one fragment to the ‘5-atom linker’
(moiety C) was an effective optimization strategy. Notably, as
illustrated in Table 1, most target compounds were found to be
more potent against A549, such as the representative compounds
demonstrated excellent activity against A549, Hela and MCF-7 cell
lines with IC50 values of 0.66, 0.38 and 0.44 mM, respectively, which
was more active than that of the positive control Cabozantinib.
The data listed in Table 1 showed that compounds substituted
with a hydrogen atom (R1 group) on moiety A were more active
than those substituted with methyl. For instance, compound 46
(A549 IC50 ¼ 0.87
mM; R1 ¼ H, R2 ¼ H, R3 ¼ 4-Br) had superior
activity compared to compound 22 (A549 IC50 ¼ 7.34
mM; R1 ¼ CH3,
4

J. Zhang, P. Chen, Y. Duan et al.
European Journal of Medicinal Chemistry 215 (2021) 113273
R2 ¼ H, R3 ¼ 4-Br), and the same trend was observed in compounds
20/44, 24/48, 30/38, 31/39. Afterward, the anticancer activity of the
central benzene ring substituted by F/H atom of the linker con-
necting A and C was investigated, which showed that the intro-
duction of R2 on the central phenyl ring had no significant effect on
activity, such as compounds 42 and 50.
inhibited the growth of the three tumor cell lines in a dose-
dependent manner.
2.2.3. Morphological changes of A549 cells under an inverted
microscope and fluorescence microscope
Further analysis revealed that compounds with different sub-
stituents of R3 on the terminal phenyl ring (moiety B) showed
different cytotoxic activity. Among them, the activity of the com-
pounds was found to be significantly reduced when the 4-position
of the terminal benzene ring was replaced by a single electron-
donating group. For example, the IC50 value of compound 36 was
In order to further explore the apoptotic mechanism of com-
pound 51, the morphological changes of A549 cells were monitored
using an acridine orange (AO) assay. According to Fig. 6, cells in the
control group (Fig. 6A) were stained with AO, presenting as an oval
cell shape with a clear edge in a tight and orderly arrangement with
no abnormalities. However, when the cells were treated with
0.93
donating group (EDG) methoxy substituted had an IC50 value of
5.85 M. Compared to R3 substituted by EDG, compounds
mM against A549, while compound 42 with a single electron-
compound 51 at a concentration of 1.1 mM and AO staining (Fig. 6B),
the cells’ morphology was observed to be abnormal with evidence
of cell nucleus splitting and dispersion and the RNA fragments was
stained with Orange. The corresponding findings showed that
compound 51 could induce apoptosis in A549 cells.
m
substituted by electron-withdrawing groups (EWGs) exhibited
better activity than the lead compound (Cabozantinib), such as 23,
25, 26, and so forth. Interestingly, the introduction of double
electron-withdrawing groups (double-EWGs) to R3 on the terminal
phenyl ring demonstrated better activity than that of single-
electron-withdrawing groups (single-EWGs). For example, when
substituents were introduced to the C-2 and C-4 positions of the
terminal phenyl ring (moiety B), the cytotoxic activity of 46 (R3 ¼ 4-
2.2.4. Apoptosis results analysis
The mechanism of A549 apoptosis induced by compound 51 was
further revealed by annexin V/PI staining [23]. As shown in Fig. 7,
compared to the control group, compound 51 was seen to signifi-
cantly increase early apoptosis (20.05%), and its induction effect
was better than that of Cabozantinib (19.87%). In addition, the
advanced apoptosis rate of compound 51 was found to be higher
than that of Cabozantinib. The corresponding results showed that
target compound 51 could induce apoptosis of A549 cells, which
was observed to be superior to that of Cabozantinib.
Br, IC50 ¼ 0.87
be 1.34 times to 1.16 times weaker than that of 51 (R3 ¼ 2-Cl-4-CF3,
M). Accordingly, the results showed that the cyto-
mM) and 48 (R3 ¼ 4-F, IC50 ¼ 0.77
mM) was found to
IC50 ¼ 0.66
m
toxicity of compounds with double-EWGs on the terminal benzene
ring was higher than that of other substituents.
As shown in Table 2, the promising compounds exhibited
excellent cytotoxicity against A549, Hela and MCF-7 cells were
screened for toxicity test on normal human liver cell line LO2 via
MTT assay. The results showed that the compounds had lower ac-
tivity against normal cell line LO2 than that of Cabozantinib, and far
lower than that of cancer cells, which indicated that the com-
pounds had certain selectivity on cancer cells. Then, combined with
the results of cytotoxicity test of Tables 1 and 2, we selected the
most promising compound 51 with the excellent activity against
cancer cells and low activity on normal cells to further study.
2.2.5. Tyrosine kinases assays
To examine the selectivity of compound 51 on c-Met over other
kinases, compound 51 was screened against c-Met and six other
tyrosine kinases (Table 3). At a concentration of 10
bition rate on c-Met of compound 51 reached 86.3%, while the in-
hibition rates on the six other enzymes, including PDGFR- , Flt-3,
mM, the inhi-
b
Ron, KDR, c-Kit and ALK, were all between 23.6 and 38.0%. The
results indicated that compound 51 exhibited selectivity on c-Met
over other kinases.
2.2.2. The relationship between the concentration of compound 51
and the cytotoxicity of three cancer cell lines
In order to effectively examine the relationship between inhi-
bition rate and concentration, the cells were treated with seven
different concentrations of compound 51 via MTT, as shown in
Fig. 5. Unsurprisingly, the results demonstrated that compound 51
Table 2
Cytotoxic activity of selected compounds against normal human liver cell line LO2
in vitro.
Compd.
R1
R2
R3
IC50
LO2
(mM)
23
24
25
33
38
39
CH3
CH3
CH3
CH3
H
H
H
H
F
F
F
4-Cl
4-F
3-Cl-4-F
3-Cl-4-F
4-Br
>100
>100
>100
>100
>100
>100
H
4-Cl
40
43
44
45
50
51
H
H
H
H
H
H
F
F
H
H
H
H
4-F
2-Cl-4-CF3
4-H
2-F-4-Br
4-OCH3
2-Cl-4-CF3
87.58 1.94
74.16 1.46
>100
>100
>100
67.43 1.25
54.97 0.39
Cabozantiniba
Fig. 5. The relationship between activity and concentration of selected compound 51
against our cancer cell lines.
a
Used as the positive control.
5

J. Zhang, P. Chen, Y. Duan et al.
European Journal of Medicinal Chemistry 215 (2021) 113273
Fig. 6. The morphological changes of A549 cells were observed under inverted microscope and fluorescence microscope. (A) Blank control group; (B) the experimental group was
treated with compound 51 with a concentration of 1.1 M.
m
Fig. 7. Representative dot plot of Annexin V (x-axis) versus PI (y-axis) analyses. Q2-1: Mechanical damage; Q2-2: Late apoptotic and death; Q2-3: Living cells; Q2-4: Early apoptotic.
Table 3
the molecular docking study showed that pyrrolo [2,3-d]pyrimi-
dine derivatives containing 1,8-naphthyridine-4-one fragment
could act synergistically to interact with the binding site of c-Met,
and 1,8-naphthyridine-4-one fragment may serve as an effective
scaffold in building a novel series of c-Met inhibitors.
Inhibition rates of tyrosine kinases by compound 51.
Kinases
Inhibition rates (%)
c-Met
PDGFR-
Flt-3
RON
KDR
c-Kit
ALK
86.3
38.0
36.0
30.7
30.4
29.7
23.6
b
3. Conclusions
In summary, pyrrolo [2,3-d]pyrimidine derivatives containing 1,8-
naphthyridine-4-one fragment were designed and synthesized, and
the cytotoxic potency against three cancer cell lines (A549, Hela and
MCF-7) were evaluated. Among them, most of target compounds
were more potent against one or more cancer cell lines and showed
lower activity against normal human liver cell line LO2 than Cabo-
zantinib. In particular, compound 51 showed remarkable cytotoxic
2.3. Binding model analysis
To further elucidate the binding modes of target compounds
with the active site of c-Met, molecular docking simulation studies
were carried out using AutoDock vina 1.1.2. Considerable research
has shown that c-Met inhibitors of class II penetrate the protein
cavity with a linear stretch configuration and form a primary
hydrogen bond with a pharmacodynamic effect through the aro-
matic ring of the hinge region and Met1160 [33,34]. The binding
mode of Cabozantinib with c-Met protein (PDB ID code: 3LQ8)
showed that a large enough cavity existed around the ‘5-atom
linker’ (moiety C) to accommodate the rigid segment (Fig. 8A).
Interestingly, the docking study demonstrated that the 1,8-
naphthyridine-4-one fragment in compound 51 could be well
accommodated to the cavity, as shown in Fig. 8B and C. In addition,
two nitrogen atoms of pyrrolo [2,3-d]pyrimidine formed a biden-
tate hydrogen bond with Met1160, and the amide and 1,8-
naphthyridine-4-one fragment of moiety C formed two hydrogen
bonds with residues Lys1110 and Asp1222, respectively. In general,
activities with IC50 values of 0.66 mM, 0.38 mM and 0.44 mM against
A549, Hela and MCF-7 cell lines, respectively. Notably, compound 51
had a better apoptosis effect than that of Cabozantinib against
A549 cells. The preliminary studies on enzymatic activity revealed
thatcompound 51 showed selectivityonc-Metoversixothertyrosine
kinases. Moreover, the SARs and docking studies indicated that
double-EWGs (such as R3 ¼ 2-Cl-4-CF3) on the terminal phenyl rings
was a key factor in improving the biological activity.
4. Experimental
4.1. General information
All melting points of the target compounds were obtained using
a Büchi melting point B-540 instrument (BüchiLabor technik, Flavil,
6

J. Zhang, P. Chen, Y. Duan et al.
European Journal of Medicinal Chemistry 215 (2021) 113273
Fig. 8. Docking diagram of Cabozantinib and compound 51 with c-Met protein (PDB ID code: 3LQ8). (A) docking cavity diagram and a partial enlarged drawing of Cabozantinib; (B)
3D interaction map between the 51 and c-Met; (C) 2D interaction map between the 51 and c-Met.
Switzerland) without correction. The structures of the target
compounds were determined using 1H NMR, 13C NMR and MS with
Bruker 400 MHz or 500 MHz spectrometers (Bruker Bioscience,
Billerica, MA, USA) in conjunction with tetramethylsilane (TMS) as
an internal standard. Unless otherwise stated, all materials were
purchased from commercial suppliers and used without purifica-
tion, and the yields were not optimized.
4.2.4. 3-fluoro-4-((7-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)
aniline (13d)
Light yellow solid; yield: 59.2%; m. p.: 185.1e187.2 ꢀC; 1H NMR
(400 MHz, DMSO‑d6)
1H), 7.23 (s, 1H), 6.99 (d, J ¼ 8.9 Hz, 1H), 6.47 (s, 1H), 5.47 (s, 2H),
d
8.32 (s, 1H), 7.50 (d, J ¼ 3.4 Hz, 1H), 7.35 (s,
þ
3.38 (s, 3H). ESI-MS: m/z 258.1 [MþH]
.
4.3. General procedure for the preparation of compounds 20e51
4.2. Synthesis of 3-substituted-4-((7-substituted-7H-pyrrolo[2,3-d]
O-chloroacetophenone 14 (0.16 mol) and NaH (0.54 mol) were
dissolved in toluene (50 mL) in an ice bath for 0.5 h. After dimethyl
carbonate (0.48 mol) was slowly added into the reaction mixture,
the reaction temperature was raised to 85 ꢀC and stirred for 6 h.
Following the reaction, the reaction system was cooled to r. t. and
adjusted to pH ¼ 7 with glacial acetic acid. Finally, the organic layer
was concentrated to obtain intermediate 15.
The key intermediate 15 (0.22 mol) and DMF-DMA (0.56 mol)
were sequentially dissolved to toluene (75 mL) and stirred at 100 ꢀC
for 2 h. The reaction mixture was then extracted with CH2Cl2. The
combined organic layers were washed with saturated sodium
chloride solution, dried over Na2SO4, filtered and concentrated
under reduced pressure to produce intermediate 16.
Afterward, intermediate 16 (0.14 mol), the appropriate anilines
(0.43 mol) and Cs2CO3 (0.1 mol) were dissolved in toluene (85 mL)
and stirred at 150 ꢀC for 6 h until TLC displayed completion of the
reaction. Finally, the precipitate was collected by filtration and
dried to give the corresponding intermediates 17a-h, which were
pale-yellow solids.
pyrimidin-4-yl)oxy)anilines (13a-d)
The preparation of the key intermediates 13a-d was illustrated
in detail in previous study [22,27], hence, the synthesis procedure
was omitted here.
4.2.1. 4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)aniline (13a)
Brown solid; yield: 55.6%; m. p.: 185.1e186.2 ꢀC; 1H NMR
(400 MHz, DMSO‑d6)
d
12.12 (s, 1H), 8.27 (s, 1H), 7.38 (d, J ¼ 3.1 Hz,
1H), 6.88 (d, J ¼ 8.6 Hz, 2H), 6.60 (d, J ¼ 8.5 Hz, 2H), 6.24 (d,
J ¼ 3.3 Hz, 1H), 5.07 (s, 2H). ESI-MS: m/z 226.1 [MþH] þ
.
4.2.2. 4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)-3-fluoroaniline
(13b)
Gray solid; yield: 56.1%; m. p.: 177.4e180.1 ꢀC; 1H NMR
(400 MHz, DMSO‑d6) d 12.17 (s,1H), 8.27 (s, 1H), 7.38 (s, 1H), 6.88 (d,
J ¼ 8.7 Hz, 2H), 6.60 (d, J ¼ 8.7 Hz, 2H), 6.24 (d, J ¼ 3.1 Hz, 1H), 5.07
þ
(s, 1H). ESI-MS: m/z 244.1 [MþH]
.
Key intermediates 17a-h (15 mol) and NaOH (35 mol) were then
added into the mixture solvent of 1,4-dioxane (35 mL) and water
(35 mL), and the reaction mixture was heated to 100 ꢀC. The
mixture was refluxed for 2 h, after which the solution was evapo-
rated and adjusted to pH ¼ 4 with the addition of 10% HCl aq. A
large amount of solid was aspirated, and the precipitate was
collected via filtration and then dried in order to obtain the
4.2.3. 4-((7-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)aniline
(13c)
White solid; yield: 56.8%; m. p.: 170.5e172.2 ꢀC; 1H NMR
(400 MHz, DMSO‑d6)
J ¼ 8.5 Hz, 2H), 6.77 (d, J ¼ 8.6 Hz, 3H), 6.31 (d, J ¼ 3.3 Hz, 2H), 3.36
d
8.32 (s, 1H), 7.46 (d, J ¼ 3.4 Hz, 1H), 6.97 (d,
(s, 3H). ESI-MS: m/z 240.1 [MþH] þ
.
7

J. Zhang, P. Chen, Y. Duan et al.
European Journal of Medicinal Chemistry 215 (2021) 113273
corresponding key intermediates 18a-h.
4.3.9. N-(4-((7-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)
phenyl)-4-oxo-1-phenyl-1,4-dihydro-1,8-naphthyridine-3-
carboxamide (20)
4.3.1. 4-oxo-1-phenyl-1,4-dihydro-1,8-naphthyridine-3-carboxylic
Yellow solid, yield: 67.8%; m. p.: 198.8e200.6 ꢀC; 1H NMR
acid (18a)
(400 MHz, DMSO‑d6)
d
12.16 (s, 1H), 8.89 (s, 1H), 8.83 (d, J ¼ 7.9 Hz,
Yellow solid, yield: 67.5%; 1H NMR (400 MHz, DMSO‑d6)
d 14.52
2H), 8.35 (s, 1H), 7.83 (d, J ¼ 8.5 Hz, 2H), 7.73 (br, 6H), 7.52 (s, 1H),
(s, 1H), 8.86 (s, 2H), 8.81 (d, J ¼ 8.0 Hz, 1H), 7.73 (dd, J ¼ 7.9, 4.7 Hz,
7.28 (d, J ¼ 8.5 Hz, 2H), 6.50 (s, 1H), 3.83 (s, 3H). 13C NMR (100 MHz,
1H), 7.66 (br, 5H).
DMSO‑d6)
d 178.37, 175.15, 163.79, 163.51, 155.75, 155.16, 154.05,
152.19,151.15,150.60,137.98,137.75, 131.52 (2C), 131.36,129.76 (2C),
124.53 (3C), 123.47, 123.00 (2C), 118.08, 114.05, 103.68, 102.17, 99.51,
4.3.2. 1-(4-bromo-2-fluorophenyl)-4-oxo-1,4-dihydro-1,8-
naphthyridine-3-carboxylic acid (18b)
33.27. Anal. Calcd. for C28H20N6O3 (%): C, 68.84; H, 4.13; N, 17.20;
þ
Brown solid, yield: 60.5%; 1H NMR (400 MHz, DMSO‑d6)
d
14.29
Found (%): C, 68.80; H, 4.10; N, 17.12. ESI-MS: m/z 488.2 [MþH]
.
(s, 1H), 9.11 (s, 1H), 8.89 (s, 1H), 8.81 (d, J ¼ 8.0 Hz, 1H), 7.92 (d,
J ¼ 9.3 Hz, 1H), 7.75 (d, J ¼ 7.1 Hz, 2H), 7.70 (d, J ¼ 8.6 Hz, 1H).
4.3.10. 1-(4-bromo-2-fluorophenyl)-N-(4-((7-methyl-7H-pyrrolo
[2,3-d]pyrimidin-4-yl)oxy)phenyl)-4-oxo-1,4-dihydro-1,8-
naphthyridine-3-carboxamide (21)
4.3.3. 1-(4-bromophenyl)-4-oxo-1,4-dihydro-1,8-naphthyridine-3-
carboxylic acid (18c)
Yellow solid, yield: 57.4%; m. p.: 233.8e234.6 ꢀC; 1H NMR
Brown solid, yield: 65.8%; 1H NMR (400 MHz, DMSO‑d6)
d
15.10
8.75 (t, J ¼ 9.7 Hz, 2H), 7.82 (d,
J ¼ 8.6 Hz, 2H), 7.61 (t, J ¼ 8.1 Hz, 2H).
(400 MHz, DMSO‑d6)
d 12.03 (s, 1H), 9.04 (s, 1H), 8.85 (s, 2H), 8.36
(s, 1H), 9.21 (s, 1H), 8.82 (s, 1H),
d
(s, 1H), 7.95 (s, 2H), 7.83 (d, J ¼ 8.1 Hz, 4H), 7.73 (s, 2H), 7.53 (s, 1H),
7.28 (d, J ¼ 8.5 Hz, 3H), 3.83 (s, 3H). Anal. Calcd. for C28H18BrFN6O3
(%): C, 57.45; H, 3.10; Br, 13.65; F, 3.25; N, 14.þ36; Found (%): C, 57.40;
4.3.4. 1-(4-chlorophenyl)-4-oxo-1,4-dihydro-1,8-naphthyridine-3-
H, 3.11; N, 14.31. ESI-MS: m/z 584.1 [MþH]
.
carboxylic acid (18d)
White solid, yield: 50.6%; 1H NMR (400 MHz, DMSO‑d6)
d 14.49
(s, 1H), 8.92 (s, 1H), 8.87 (s, 1H), 8.81 (d, J ¼ 8.0 Hz, 1H), 7.74 (dd,
4.3.11. 1-(4-bromophenyl)-N-(4-((7-methyl-7H-pyrrolo[2,3-d]
pyrimidin-4-yl)oxy)phenyl)-4-oxo-1,4-dihydro-1,8-naphthyridine-
3-carboxamide (22)
J ¼ 7.9, 4.5 Hz, 1H), 7.69 (br, 4H).
Yellow solid, yield: 71.8%; m. p.: 223.3e224.1 ꢀC; 1H NMR
4.3.5. 1-(4-fluorophenyl)-4-oxo-1,4-dihydro-1,8-naphthyridine-3-
carboxylic acid (18e)
(400 MHz, DMSO‑d6)
d
12.13 (s, 1H), 8.84 (dd, J ¼ 7.6, 6.5 Hz, 3H),
8.35 (s, 2H), 7.84 (d, J ¼ 5.1 Hz, 2H), 7.65 (d, J ¼ 7.9 Hz, 1H), 7.52 (s,
Yellow solid, yield: 70.9%; 1H NMR (400 MHz, DMSO‑d6)
d 14.52
2H), 7.27 (d, J ¼ 8.7 Hz, 3H), 6.50 (s, 1H), 3.83 (s, 3H). Anal. calcd. for
(s, 1H), 8.91 (s, 1H), 8.88 (d, J ¼ 3.5 Hz, 1H), 8.81 (d, J ¼ 7.9 Hz, 1H),
C
28H19BrN6O3 (%): C, 59.27; H, 3.38; Br,14.08; N,14.81; Found (%): C,
7.76 (br, 3H), 7.46 (t, J ¼ 8.6 Hz, 2H).
þ
59.26; H, 3.37; N, 14.80. ESI-MS: m/z 566.1 [MþH]
.
4.3.6. 1-(3-chloro-4-fluorophenyl)-4-oxo-1,4-dihydro-1,8-
naphthyridine-3-carboxylic acid (18f)
4.3.12. 1-(4-chlorophenyl)-N-(4-((7-methyl-7H-pyrrolo[2,3-d]
pyrimidin-4-yl)oxy)phenyl)-4-oxo-1,4-dihydro-1,8-naphthyridine-
3-carboxamide (23)
Yellow solid, yield: 55.6%; 1H NMR (400 MHz, DMSO‑d6)
d 14.46
(s, 1H), 9.01 (s, 1H), 8.89 (s, 1H), 8.82 (s, 1H), 8.03 (d, J ¼ 6.3 Hz, 1H),
7.77 (br, 3H).
Yellow solid, yield: 76.7%; m. p.: 233.3e234.1 ꢀC; 1H NMR
(400 MHz, DMSO‑d6) d 12.14 (s, 1H), 8.91 (s, 1H), 8.82 (s, 1H), 8.36 (s,
1H), 7.83 (d, J ¼ 8.6 Hz, 3H), 7.72 (br, 5H), 7.52 (s, 1H), 7.28 (d,
4.3.7. 1-(4-methoxyphenyl)-4-oxo-1,4-dihydro-1,8-naphthyridine-
J ¼ 8.8 Hz, 2H), 6.51 (s, 1H), 3.83 (s, 3H). 13C NMR (100 MHz,
3-carboxylic acid (18g)
DMSO‑d6)
d 176.90, 161.70, 161.60, 153.63, 152.50, 150.10, 149.82,
White solid, yield: 58.4%; 1H NMR (400 MHz, DMSO‑d6)
d 14.57
149.03, 148.49, 139.22, 135.91, 135.65, 133.89, 129.73 (2C), 129.37
(2C), 129.27, 122.44 (2C), 122.05, 120.88 (2C), 112.12, 104.73, 97.41,
31.18. Anal. calcd. for C28H19ClN6O3 (%): C, 64.31; H, 3.66; Cl, 6.78; N,
16.07; Found (%): C, 64.30; H, 3.65; N, 16.08. ESI-MS: m/z 522.1
(s, 1H), 8.88 (s, 1H), 8.85 (br, 2H), 7.73 (dd, J ¼ 7.4, 4.2 Hz, 1H), 7.55
(d, J ¼ 8.1 Hz, 2H), 7.14 (d, J ¼ 8.2 Hz, 2H), 3.86 (s, 3H).
4.3.8. 1-(2-chloro-4-(trifluoromethyl)phenyl)-4-oxo-1,4-dihydro-
1,8-naphthyridine-3-carboxylic acid (18h)
[MþH] þ
.
Yellow solid; yield: 60.5%; 1H NMR (400 MHz, DMSO‑d6)
d 14.67
(s, 1H), 9.01 (s, 1H), 8.60 (s, 1H), 8.55 (d, J ¼ 7.8 Hz, 1H), 8.05 (d,
J ¼ 8.3 Hz, 1H), 7.78 (d, J ¼ 8.5 Hz, 2H), 7.48 (d, J ¼ 7.7 Hz, 1H).
The reaction of oxalyl chloride and key intermediates 18a-h
(4 mol) was carried out to obtain the corresponding intermediates
19a-h. The appropriate anilines 13a-d (2 mol) and DIPEA (6 mol)
were dissolved in dichloromethane (25 mL) to form system I. Sec-
ondly, 19a-h obtained by acylation of 18a-h with oxalyl chloride as
acylating agent, were dissolved in anhydrous dichloromethane
(20 mL) to form system II. Finally, the mixed solution of system II
was dropped into system I, and stirred in an ice bath for 1 h. Finally,
the mixture was removed under reduced pressure to get a crude
product which was purified by column chromatography on silica
gel using CH2Cl2: MeOH 15:1 to give the target compounds 20e51
as a pale yellow solid.
4.3.13. 1-(4-fluorophenyl)-N-(4-((7-methyl-7H-pyrrolo[2,3-d]
pyrimidin-4-yl)oxy)phenyl)-4-oxo-1,4-dihydro-1,8-naphthyridine-
3-carboxamide (24)
Yellow solid, yield: 66.5%; m. p.: 254.3e256.1 ꢀC; 1H NMR
(400 MHz, DMSO‑d6) d 12.15 (s, 1H), 8.90 (s, 1H), 8.84 (s, 1H), 8.82 (s,
1H), 8.36 (s, 2H), 7.83 (d, J ¼ 8.7 Hz, 2H), 7.74 (d, J ¼ 8.5 Hz, 3H), 7.53
(br, 1H), 7.48 (s, 1H), 7.28 (d, J ¼ 7.9 Hz, 2H), 6.51 (s, 1H), 3.83 (s, 3H).
13C NMR (100 MHz, DMSO‑d6)
d 176.88, 163.28, 161.69, 161.64,
159.66 (d, J ¼ 226.0 Hz), 153.62, 152.52, 150.09, 149.16, 148.52,
136.68, 135.87, 135.66, 134.92, 130.08, 129.99, 129.25, 122.40, 122.11,
121.38, 120.89, 120.28, 116.27 (d, J ¼ 13 Hz), 116.04 (d, J ¼ 12 Hz),
112.03, 104.75, 97.40, 31.17.19F NMR (471 MHz, DMSO‑d6)
d
ꢁ112.05.
Anal. calcd. for C28H19FN6O3 (%): C, 66.40; H, 3.78; F, 3.75; N, 16.59;
Found (%): C, 66.42; H, 3.77; N, 16.58. ESI-MS: m/z 506.2 [MþH] þ
.
8

J. Zhang, P. Chen, Y. Duan et al.
European Journal of Medicinal Chemistry 215 (2021) 113273
4.3.14. 1-(3-chloro-4-fluorophenyl)-N-(4-((7-methyl-7H-pyrrolo
[2,3-d]pyrimidin-4-yl)oxy)phenyl)-4-oxo-1,4-dihydro-1,8-
naphthyridine-3-carboxamide (25)
1H), 8.40 (s, 1H), 8.35 (d, J ¼ 8.7 Hz, 1H), 7.84 (d, J ¼ 8.5 Hz, 2H), 7.65
(d, J ¼ 8.6 Hz, 3H), 7.60 (s, 1H), 7.59 (s, 1H), 7.57 (d, J ¼ 6.4 Hz, 2H),
7.53 (d, J ¼ 5.1 Hz, 2H), 3.85 (s, 3H). Anal. calcd. for C28H18BrFN6O3
(%): C, 57.45; H, 3.10; Br, 13.65; F, 3.25; N, 14.36; Found (%): C, 57.44;
Yellow solid, yield: 56.5%; m. p.: 234.3e236.1 ꢀC; 1H NMR
(400 MHz, DMSO‑d6)
J ¼ 7.4 Hz, 2H), 7.94 (s, 1H), 7.82 (s, 3H), 7.53 (s, 2H), 7.27 (s, 3H), 7.15
(s, 1H), 3.83 (s, 3H). 13C NMR (100 MHz, DMSO‑d6)
177.34, 162.20,
d
12.13 (s,1H), 8.96 (s,1H), 8.85 (s,1H), 8.35 (d,
H, 3.11; N, 14.35. ESI-MS: m/z 584.1 [MþH] þ
.
d
4.3.20. 1-(4-chlorophenyl)-N-(3-fluoro-4-((7-methyl-7H-pyrrolo
[2,3-d]pyrimidin-4-yl)oxy)phenyl)-4-oxo-1,4-dihydro-1,8-
naphthyridine-3-carboxamide (31)
162.06, 159.04, 154.18, 153.00, 152.62, 150.73, 149.80 (d, J ¼ 160 Hz),
148.40,148.15, 131.03, 129.78, 122.95, 122.65, 122.56, 122.35, 122.24,
121.79, 121.39, 120.92, 120.70, 117.91 (d, J ¼ 32 Hz), 114.77, 112.62,
Yellow solid, yield: 55.6%; m. p.: 245.3e246.1 ꢀC; 1H NMR
105.23, 97.90, 31.68.19F NMR (471 MHz, DMSO‑d6)
calcd. for C28H18ClFN6O3 (%): C, 62.17; H, 3.35; Cl, 6.55; F, 3.51; N,
d
ꢁ116.09. Anal.
(400 MHz, DMSO‑d6) d 12.26 (s,1H), 8.91 (s,1H), 8.84 (s,1H), 8.82 (s,
1H), 8.36 (s, 1H), 7.73 (br, 4H), 7.57 (s, 2H), 7.47 (s, 2H), 7.46 (s, 1H),
6.64 (s, 1H), 3.85 (s, 3H). Anal. calcd. for C28H18ClFN6O3 (%): C, 62.17;
15.54; Found (%): C, 62.16; H, 3.36; N, 15.55. ESI-MS: m/z 540.1
þ
[MþH]
.
H, 3.35; Cl, 6.55; F, 3.51; N, 15.54; Found (%): C, 67.16; H, 3.34; N,
þ
15.55. ESI-MS: m/z 540.1 [MþH]
.
4.3.15. 1-(4-methoxyphenyl)-N-(4-((7-methyl-7H-pyrrolo[2,3-d]
pyrimidin-4-yl)oxy)phenyl)-4-oxo-1,4-dihydro-1,8-naphthyridine-
3-carboxamide (26)
4.3.21. N-(3-fluoro-4-((7-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)
oxy)phenyl)-1-(4-fluorophenyl)-4-oxo-1,4-dihydro-1,8-
naphthyridine-3-carboxamide (32)
Yellow solid, yield: 50.5%; m. p.: 245.3e246.1 ꢀC; 1H NMR
(400 MHz, DMSO‑d6)
d
12.18 (s, 1H), 8.87 (s, 1H), 8.82 (d, J ¼ 6.1 Hz,
Yellow solid, yield: 70.6%; m. p.: 225.3e226.1 ꢀC; 1H NMR
2H), 8.36 (s,1H), 7.83 (d, J ¼ 8.7 Hz, 2H), 7.59 (s,1H), 7.57 (s,1H), 7.53
(s, 2H), 7.28 (d, J ¼ 8.2 Hz, 2H), 7.15 (d, J ¼ 8.6 Hz, 2H), 6.50 (s, 1H),
(400 MHz, DMSO‑d6) d 12.25 (s, 1H), 8.92 (s, 1H), 8.82 (s, 1H), 8.39
(d, J ¼ 7.4 Hz, 1H), 8.36 (s, 1H), 7.71 (s, 3H), 7.69 (s, 1H), 7.60 (s, 2H),
3.83 (s, 3H). 13C NMR (100 MHz, DMSO‑d6)
d
176.78, 161.72, 159.56,
7.58 (s, 1H), 7.52 (s, 1H), 6.65 (s, 1H), 3.86 (s, 3H). Anal. calcd. for
153.64, 152.46, 150.09, 149.37, 148.41, 135.83, 135.69, 133.24, 129.28,
128.83 (2C), 122.47 (2C), 122.19, 121.91, 121.40, 120.84 (2C), 114.47
(2C), 111.76, 104.69, 97.40, 55.55, 31.19. Anal. calcd. for C29H22N6O4
(%): C, 67.17; H, 4.28; N, 16.þ21; Found (%): C, 67.16; H, 4.27; N, 16.20.
C28H18F2N6O3 (%): C, 64.12; H, 3.46; F, 7.24; N, 16.02; Found (%): C,
64.11; H, 3.45; N, 16.01. ESI-MS: m/z 524.1 [MþH] þ
.
4.3.22. 1-(3-chloro-4-fluorophenyl)-N-(3-fluoro-4-((7-methyl-7H-
pyrrolo[2,3-d]pyrimidin-4-yl)oxy)phenyl)-4-oxo-1,4-dihydro-1,8-
naphthyridine-3-carboxamide (33)
ESI-MS: m/z 518.2 [MþH]
.
4.3.16. 1-(2-chloro-4-(trifluoromethyl)phenyl)-N-(4-((7-methyl-
7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)phenyl)-4-oxo-1,4-dihydro-
1,8-naphthyridine-3-carboxamide (27)
Yellow solid, yield: 60.7%; m. p.: 215.3e216.1 ꢀC; 1H NMR
(400 MHz, DMSO‑d6)
d
12.23 (s, 1H), 8.97 (s, 1H), 8.83 (d, J ¼ 8.0 Hz,
1H), 8.35 (s, 1H), 8.07 (d, J ¼ 7.5 Hz, 1H), 7.99 (d, J ¼ 7.8 Hz, 1H), 7.79
(br, 4H), 7.55 (dd, J ¼ 7.8, 6.0 Hz, 3H), 7.48 (s, 1H), 6.64 (d, J ¼ 7.5 Hz,
1H), 3.85 (s, 3H). Anal. calcd. for C28H17ClF2N6O3 (%): C, 60.17; H,
3.07; Cl, 6.34; F, 6.80; N, 15.þ04; Found (%): C, 60.16; H, 3.06; N, 15.03.
Yellow solid, yield: 56.1%; m. p.: 234.3e236.1 ꢀC; 1H NMR
(400 MHz, DMSO‑d6) d 12.03 (s,1H), 9.04 (s,1H), 8.85 (s,1H), 8.36 (s,
2H), 7.97 (s, 2H), 7.83 (d, J ¼ 8.9 Hz, 3H), 7.74 (s,1H), 7.53 (s,1H), 7.29
(s, 2H), 6.51 (s, 1H), 3.83 (s, 3H). Anal. calcd. for C29H18ClF3N6O3 (%):
C, 58.94; H, 3.07; Cl, 6.00; F, 9.64; N, 14.2þ2; Found (%): C, 58.83; H,
ESI-MS: m/z 558.1 [MþH]
.
3.08; N, 14.21. ESI-MS: m/z 590.1 [MþH]
.
4.3.23. N-(3-fluoro-4-((7-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)
oxy)phenyl)-1-(4-methoxyphenyl)-4-oxo-1,4-dihydro-1,8-
naphthyridine-3-carboxamide (34)
4.3.17. N-(3-fluoro-4-((7-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)
oxy)phenyl)-4-oxo-1-phenyl-1,4-dihydro-1,8-naphthyridine-3-
carboxamide (28)
Yellow solid, yield: 63.2%; m. p.: 205.3e206.1 ꢀC; 1H NMR
(400 MHz, DMSO‑d6) d 12.30 (s,1H), 8.87 (s,1H), 8.83 (s,1H), 8.39 (s,
Yellow solid, yield: 65.1%; m. p.: 234.3e235.1 ꢀC; 1H NMR
1H), 8.36 (s, 1H), 8.01 (s, 1H), 7.98 (s, 1H), 7.79 (s, 1H), 7.61 (s, 2H),
7.57 (s, 2H), 7.17 (s, 1H), 7.14 (s, 1H), 6.69 (d, J ¼ 7.9 Hz, 1H), 6.65 (s,
1H), 3.86 (s, 3H), 3.84 (s, 3H). Anal. calcd. for C29H21FN6O4 (%): C,
64.92; H, 3.95; F, 3.54; N, 15.66;þFound (%): C, 64.91; H, 3.94; N,
(400 MHz, DMSO‑d6) d 12.28 (s,1H), 8.90 (s,1H), 8.84 (s,1H), 8.83 (s,
1H), 8.36 (s,1H), 8.02 (s,1H), 7.98 (d, J ¼ 7.3 Hz,1H), 7.66 (s,1H), 7.64
(s, 1H), 7.62 (s, 1H), 7.58 (d, J ¼ 7.3 Hz, 2H), 7.53 (s, 1H), 7.45 (s, 1H),
6.65 (t, J ¼ 8.1 Hz, 2H), 3.85 (s, 3H). Anal. calcd. for C28H19FN6O3 (%):
C, 66.40; H, 3.78; F, 3.75; N, 16.59; Found (%): C, 66.41; H, 3.73; N,
15.65. ESI-MS: m/z 536.2 [MþH]
.
16.58. ESI-MS: m/z 506.2 [MþH] þ
.
4.3.24. 1-(2-chloro-4-(trifluoromethyl)phenyl)-N-(3-fluoro-4-((7-
methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)phenyl)-4-oxo-1,4-
dihydro-1,8-naphthyridine-3-carboxamide (35)
4.3.18. 1-(4-bromo-2-fluorophenyl)-N-(3-fluoro-4-((7-methyl-7H-
pyrrolo[2,3-d]pyrimidin-4-yl)oxy)phenyl)-4-oxo-1,4-dihydro-1,8-
naphthyridine-3-carboxamide (29)
Yellow solid, yield: 75.2%; m. p.: 203.3e206.1 ꢀC; 1H NMR
(400 MHz, DMSO‑d6) d 12.13 (s, 1H), 9.05 (s, 1H), 8.84 (s, 1H), 8.36 (s,
Yellow solid, yield: 70.4%; m. p.: 205.3e207.1 ꢀC; 1H NMR
1H), 8.01 (s, 1H), 7.98 (s, 1H), 7.94 (s, 1H), 7.92 (s, 1H), 7.79 (d,
J ¼ 7.3 Hz, 1H), 7.73 (s, 2H), 7.57 (s, 2H), 6.64 (s, 1H), 3.85 (s, 3H).
Anal. calcd. for C29H17ClF4N6O3 (%): C, 57.20; H, 2.81; Cl, 5.82; F,
12.48; N, 13.8þ0; Found (%): C, 57.21; H, 2.82; N, 13.82. ESI-MS: m/z
(400 MHz, DMSO‑d6) d 12.14 (s, 1H), 9.06 (s, 1H), 8.84 (s, 1H), 8.36 (s,
2H), 7.97 (d, J ¼ 7.0 Hz, 2H), 7.80 (s, 2H), 7.73 (s,1H), 7.58 (s, 2H), 6.65
(s, 2H), 3.85 (s, 3H). Anal. calcd. for C28H17BrF2N6O3 (%): C, 55.74; H,
2.84; Br, 13.24; F, 6.30; N, 13.93; Found (%): C, 55.73; H, 2.83; N,
608.1 [MþH]
.
13.92. ESI-MS: m/z 602.1 [MþH] þ
.
4.3.25. N-(4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)-3-
fluorophenyl)-4-oxo-1-phenyl-1,4-dihydro-1,8-naphthyridine-3-
carboxamide (36)
4.3.19. 1-(4-bromophenyl)-N-(3-fluoro-4-((7-methyl-7H-pyrrolo
[2,3-d]pyrimidin-4-yl)oxy)phenyl)-4-oxo-1,4-dihydro-1,8-
naphthyridine-3-carboxamide (30)
Yellow solid, yield: 44.5%; m. p.: 225.3e226.1 ꢀC; 1H NMR
Yellow solid, yield: 60.5%; m. p.: 245.3e247.1 ꢀC; 1H NMR
(400 MHz, DMSO‑d6)
d 12.27 (s, 1H), 8.90 (s, 1H), 8.84 (s, 2H), 8.29
(400 MHz, DMSO‑d6)
d
12.26 (s, 1H), 8.92 (s, 1H), 8.83 (d, J ¼ 6.1 Hz,
(s, 1H), 8.01 (s, 1H), 7.98 (s, 1H), 7.70 (br, 6H), 7.52 (s, 3H), 6.60 (s,
9

J. Zhang, P. Chen, Y. Duan et al.
European Journal of Medicinal Chemistry 215 (2021) 113273
1H). Anal. calcd. for C27H17FN6O3 (%): C, 65.85; H, 3.48; F, 3.86; N,
calcd. for C27H15ClF2N6O3 (%): C, 59.51; H, 2.77; Cl, 6.51; F, 6.97; N,
17.07; Found (%): C, 65.84; H, 3.47; N, 17.06. ESI-MS: m/z 492.1
15.42; Found (%): C, 59.50; H, 2.76; N, 15,43. ESI-MS: m/z 544.1
þ
[MþH]
.
[MþH] þ
.
4.3.26. N-(4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)-3-
fluorophenyl)-1-(4-bromo-2-fluorophenyl)-4-oxo-1,4-dihydro-1,8-
naphthyridine-3-carboxamide (37)
4.3.31. N-(4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)-3-
fluorophenyl)-1-(4-methoxyphenyl)-4-oxo-1,4-dihydro-1,8-
naphthyridine-3-carboxamide (42)
Yellow solid, yield: 57.5%; m. p.: 225.3e226.1 ꢀC; 1H NMR
Yellow solid, yield: 65.5%; m. p.: 212.3e215.6 ꢀC; 1H NMR
(400 MHz, DMSO‑d6)
d
12.32 (s, 1H), 12.14 (s, 1H), 9.06 (s, 1H), 8.83
(400 MHz, DMSO‑d6) d 12.32 (s, 1H), 12.29 (s, 1H), 8.87 (s, 1H), 8.84
(d, J ¼ 7.9 Hz, 2H), 8.31 (s, 1H), 7.94 (dd, J ¼ 7.6, 8.8 Hz, 2H), 7.81 (d,
(s, 2H), 8.31 (s, 1H), 7.98 (s, 1H), 7.57 (s, 2H), 7.52 (s, 3H), 7.45 (s, 1H),
J ¼ 7.8 Hz, 1H), 7.73 (s, 2H), 7.52 (s, 2H), 7.45 (d, J ¼ 7.8 Hz, 1H), 6.62
7.16 (d, J ¼ 8.2 Hz, 2H), 6.62 (s, 1H), 3.87 (s, 3H). 13C NMR (100 MHz,
(s, 1H). 13C NMR (100 MHz, DMSO‑d6)
d
176.97, 161.65, 160.88,
DMSO‑d6) d 176.94, 161.64, 160.85, 156.05, 154.00, 153.54, 149.98,
158.38, 154.96 (d, J ¼ 179 Hz), 154.71 (d, J ¼ 228 Hz), 152.59, 149.98,
149.41, 149.23, 136.82 (d, J ¼ 10 Hz), 136.07, 135.27 (d, J ¼ 13 Hz),
131.24, 128.61, 127.13 (d, J ¼ 13 Hz), 125.54, 124.66, 123.31 (d,
J ¼ 9 Hz), 122.41, 121.11, 120.01, 119.78, 116.16, 112.55, 108.27 (d,
J ¼ 14 Hz), 104.07, 97.79. Anal. calcd. for C27H15BrF2N6O3 (%): C,
55.03; H, 2.57; Br, 13.56; F, 6.45; N, 14.26; Found (%): C, 55.02; H,
149.46, 149.20, 136.84, 136.79, 136.02, 135.13, 131.23, 128.61, 127.07,
127.02, 125.57, 124.64, 122.40, 121.07, 119.79, 116.13, 112.47, 108.30,
104.02, 97.73, 58.98. Anal. calcd. for C28H19FN6O4 (%): C, 64.37; H,
3.67; F, 3.64; N,16.þ08; Found (%): C, 64.36; H, 3.68; N,16.09. ESI-MS:
m/z 522.1 [MþH]
.
2.56; N, 14.27. ESI-MS: m/z 588.0 [MþH] þ
.
4.3.32. N-(4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)-3-
fluorophenyl)-1-(2-chloro-4-(trifluoromethyl)phenyl)-4-oxo-1,4-
dihydro-1,8-naphthyridine-3-carboxamide (43)
4.3.27. N-(4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)-3-
fluorophenyl)-1-(4-bromophenyl)-4-oxo-1,4-dihydro-1,8-
naphthyridine-3-carboxamide (38)
Yellow solid, yield: 55.7%; m. p.: 211.1e213.4 ꢀC; 1H NMR
(400 MHz, DMSO‑d6)
d 12.31 (s, 1H), 12.13 (s, 1H), 9.06 (s, 1H), 8.83
Yellow solid, yield: 56.5%; m. p.: 225.3e226.1 ꢀC; 1H NMR
(d, J ¼ 7.9 Hz, 2H), 8.31 (s, 1H), 8.01 (br, 2H), 7.83 (br, 3H), 7.52 (s,
(400 MHz, DMSO‑d6) d 12.25 (s,1H), 8.92 (s,1H), 8.84 (s,1H), 8.82 (s,
2H), 7.45 (t, J ¼ 8.8 Hz, 1H), 6.62 (s, 1H). 13C NMR (100 MHz,
1H), 8.29 (s,1H), 8.00 (d, J ¼ 7.8 Hz,1H), 7.85 (s,1H), 7.83 (s,1H), 7.65
DMSO‑d6)
d
177.48, 162.17, 161.37, 158.89, 155.21 (d, J ¼ 229 Hz),
(d, J ¼ 8.3 Hz, 3H), 7.52 (s, 3H), 6.61 (s, 1H). Anal. calcd. for
154.58, 153.10, 150.49, 149.96, 149.75, 137.34 (d, J ¼ 10 Hz), 136.59,
135.76 (d, J ¼ 12 Hz), 131.76, 128.35 (d, J ¼ 154 Hz), 126.06, 125.19,
123.86, 122.94, 121.62, 120.51, 120.28, 116.68, 113.05, 108.94, 108.71,
104.55, 98.30. Anal. calcd. for C28H15ClF4N6O3 (%): C, 56.53; H, 2.54;
Cl, 5.96; F, 12.77; N, 14.13; Found (%): C, 56.53; H, 2.55; N, 14.14. ESI-
C
27H16BrFN6O3 (%): C, 56.76; H, 2.82; Br, 13.98; F, 3.33; N, 14.71;
þ
Found (%): C, 56.75; H, 2.81; N, 14.70. ESI-MS: m/z 570.0 [MþH]
.
4.3.28. N-(4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)-3-
fluorophenyl)-1-(4-chlorophenyl)-4-oxo-1,4-dihydro-1,8-
naphthyridine-3-carboxamide (39)
MS: m/z 594.1 [MþH] þ
.
Yellow solid, yield: 55.2%; m. p.: 204.3e207.1 ꢀC; 1H NMR
4.3.33. N-(4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)phenyl)-4-
oxo-1-phenyl-1,4-dihydro-1,8-naphthyridine-3-carboxamide (44)
Yellow solid, yield: 64.2%; m. p.: 198.1e201.5 ꢀC; 1H NMR
(400 MHz, DMSO‑d6)
(s, 2H), 8.31 (s, 1H), 7.99 (s, 2H), 7.72 (br, 5H), 7.53 (s, 2H), 6.62 (s,
1H). 13C NMR (100 MHz, DMSO‑d6)
177.38, 162.46, 161.38, 154.19,
d 12.31 (s, 1H), 12.25 (s, 1H), 8.92 (s, 1H), 8.83
d
(400 MHz, DMSO‑d6)
d
12.22 (s, 1H), 12.16 (s, 1H), 8.86 (d, J ¼ 8.6 Hz,
154.08, 151.79 (d, J ¼ 268 Hz), 150.34, 149.71, 139.67, 137.41 (d,
J ¼ 10 Hz), 136.40, 135.71 (d, J ¼ 13 Hz), 134.42, 130.23 (2C), 129.87
(2C), 126.07, 125.20, 122.63, 121.87, 116.59, 112.30, 108.87, 108.64,
104.56, 98.30. Anal. calcd. for C27H16ClFN6O3 (%): C, 61.55; H, 3.06;
Cl, 6.73; F, 3.61; N, 15.95; Found (%): C, 61.54; H, 3.05; N, 15.96. ESI-
4H), 8.31 (s, 1H), 7.83 (d, J ¼ 8.9 Hz, 2H), 7.67 (s, 2H), 7.64 (s, 1H),
7.63 (s, 2H), 7.47 (s, 1H), 7.28 (s, 2H), 6.48 (s, 1H). Anal. calcd. for
C
27H18N6O3 (%): C, 68.35; H, 3.82; N, 17.7þ1; Found (%): C, 68.34; H,
3.81; N, 17.70. ESI-MS: m/z 474.1 [MþH]
.
MS: m/z 526.1 [MþH] þ
.
4.3.34. N-(4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)phenyl)-1-(4-
bromo-2-fluorophenyl)-4-oxo-1,4-dihydro-1,8-naphthyridine-3-
carboxamide (45)
4.3.29. N-(4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)-3-
fluorophenyl)-1-(4-fluorophenyl)-4-oxo-1,4-dihydro-1,8-
naphthyridine-3-carboxamide (40)
Yellow solid, yield: 51.5%; m. p.: 223.3e224.6 ꢀC; 1H NMR
(400 MHz, DMSO‑d6) d 12.02 (s,1H), 9.04 (s,1H), 8.85 (s,1H), 8.64 (s,
2H), 8.45 (s, 1H), 8.29 (s, 1H), 7.94 (s, 2H), 7.81 (s, 2H), 7.73 (s, 1H),
Yellow solid, yield: 62.1%; m. p.: 220.3e221.1 ꢀC; 1H NMR
(400 MHz, DMSO‑d6)
(s, 2H), 8.31 (s, 1H), 7.98 (s, 1H), 7.73 (s, 3H), 7.55 (br, 5H), 6.62 (s,
1H). 13C NMR (100 MHz, DMSO‑d6)
176.86, 163.30, 161.98, 160.89,
d
12.31 (s, 1H), 12.26 (s, 1H), 8.91 (s, 1H), 8.84
7.66 (s, 1H), 7.47 (s, 1H), 7.28 (d, J ¼ 8.6 Hz, 2H). Anal. calcd. for
C
27H16BrFN6O3 (%): C, 56.76; H, 2.82; Br, 13.98; F, 3.33; N, 14.71;
d
Found (%): C, 56.75; H, 2.81; N, 14.70. ESI-MS: m/z 570.0 [MþH] þ
.
154.37 (d, J ¼ 221 Hz), 154.02 (d, J ¼ 203 Hz), 152.61, 149.98 (2C),
149.38, 136.96, 136.65, 135.86, 130.09 (2C), 130.00, 125.55, 124.68,
122.06, 121.38, 116.34, 116.11 (2C), 111.70, 108.26 (d, J ¼ 23 Hz),
4.3.35. N-(4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)phenyl)-1-(4-
bromophenyl)-4-oxo-1,4-dihydro-1,8-naphthyridine-3-
carboxamide(46)
104.07, 97.80.19F NMR (471 MHz, DMSO‑d6)
Anal. calcd. for C27H16F2N6O3 (%): C, 63.53; H, 3.16; F, 7.44; N, 16.46;
Found (%): C, 63.52; H, 3.15; N, 16.45. ESI-MS: m/z 510.1 [MþH]
d
ꢁ104.52, ꢁ113.10.
Yellow solid, yield: 50.6%; m. p.: 245.3e246.1 ꢀC; 1H NMR
þ
.
(400 MHz, DMSO‑d6) d 12.13 (s, 1H), 8.91 (s, 1H), 8.81 (s, 1H), 8.30 (s,
1H), 7.84 (d, J ¼ 6.8 Hz, 5H), 7.67 (t, J ¼ 8.3 Hz, 4H), 7.47 (s, 1H), 7.28
4.3.30. N-(4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)-3-
fluorophenyl)-1-(3-chloro-4-fluorophenyl)-4-oxo-1,4-dihydro-1,8-
naphthyridine-3-carboxamide (41)
(d, J ¼ 8.4 Hz, 2H), 6.48 (s, 1H). 13C NMR (100 MHz, DMSO‑d6)
d
176.86, 170.03, 161.67, 161.56, 153.62, 153.51, 150.08, 149.72,
148.94, 148.42, 139.63, 135.89, 135.59, 132.32 (2C), 130.02 (2C),
125.24,122.53 (2C),122.04,121.31,120.83 (2C),112.03,104.57, 97.96.
Anal. calcd. for C27H17BrN6O3 (%): C, 58.60; H, 3.10; Br, 14.44; N,
15.19; Found (%): C, 58.61; H, 3.11; N, 15.18. ESI-MS: m/z 552.1
Yellow solid, yield: 61.5%; m. p.: 214.3e215.1 ꢀC; 1H NMR
(400 MHz, DMSO‑d6)
d 12.22 (s, 1H), 12.13 (s, 1H), 8.91 (s, 1H), 8.83
(d, J ¼ 7.8 Hz, 2H), 8.31 (s, 1H), 7.83 (d, J ¼ 8.7 Hz, 2H), 7.70 (d,
J ¼ 8.5 Hz, 5H), 7.47 (s, 1H), 7.28 (d, J ¼ 8.4 Hz, 2H), 6.49 (s, 1H). Anal.
[MþH] þ
.
10

J. Zhang, P. Chen, Y. Duan et al.
European Journal of Medicinal Chemistry 215 (2021) 113273
4.3.36. N-(4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)phenyl)-1-(4-
chlorophenyl)-4-oxo-1,4-dihydro-1,8-naphthyridine-3-
carboxamide (47)
incubated in 5% CO2 at 37 ꢀC for 24 h. Next, the cancer cell lines
adding test compounds at concentrations of 1.234, 3.704, 11.11,
33.33 and 100 m
mol/L were continued for 72 h in 5% CO2 at 37 ꢀC.
Yellow solid, yield: 43.5%; m. p.: 213.4e215.5 ꢀC; 1H NMR
Afterward, fresh MTT was added to each well at a terminal con-
centration of 5 g/mL and was incubated with cells at 37 ꢀC for 4 h.
Finally, after the removal of MTT methoxypyrimidine, the cells
(400 MHz, DMSO‑d6)
d 12.22 (s, 1H), 12.13 (s, 1H), 8.91 (s, 1H), 8.83
(d, J ¼ 7.8 Hz, 2H), 8.31 (s, 1H), 7.83 (d, J ¼ 8.7 Hz, 2H), 7.70 (d,
J ¼ 7.5 Hz, 5H), 7.47 (s, 1H), 7.28 (d, J ¼ 8.4 Hz, 2H), 6.49 (s, 1H). Anal.
were dissolved in 100 mL dimethyl sulfoxide, and the data were
calcd. for C27H17ClN6O3 (%): C, 63.72; H, 3.37; Cl, 6.97; N, 16.51;
recorded under an absorbance of 492 nm. All compounds were
tested three times under the same conditions, and the test results
were expressed by IC50 with the Bacus Laboratories Incorporated
Slide Scanner (Bliss) software.
þ
Found (%): C, 63.71; H, 3.38; N, 16.53. ESI-MS: m/z 508.1 [MþH]
.
4.3.37. N-(4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)phenyl)-1-(4-
fluorophenyl)-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxamide
(48)
4.5. Cell apoptosis assay by flow cytometry
Yellow solid, yield: 45.1%; m. p.: 220.3e221.3 ꢀC; 1H NMR
(400 MHz, DMSO‑d6)
d 12.22 (s, 1H), 12.15 (s, 1H), 8.90 (s, 1H), 8.82
A549 cells were seeded in 6-well plates at a density of
1 ꢂ 106 cells/well in RPMI 1640 medium and were incubated for
(s, 1H), 8.49 (s, 1H), 8.31 (s, 1H), 7.83 (d, J ¼ 9.0 Hz, 2H), 7.75 (s, 2H),
7.47 (br, 4H), 7.28 (d, J ¼ 8.5 Hz, 2H). Anal. calcd. for C27H17FN6O3
24 h. The cancer cell lines were then treated with 0.5 mM Cabo-
zantinib and compound 51 for 48 h at 37 ꢀC in 5% CO2. Cultured cells
stained with Annexin V-FITC and propidium iodide (PI) at 4 ꢀC for
0.5 h were tested using the FACS Calibur flow cytometer (Becton
Dickinson, San Jose, CA) by the Cell Quest software in the dark.
(%): C, 65.85; H, 3.48; F, 3.86; N, 17.07; Found (%): C, 65.84; H, 3.47;
þ
N, 17.06. ESI-MS: m/z 492.1 [MþH]
.
4.3.38. N-(4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)phenyl)-1-(3-
chloro-4-fluorophenyl)-4-oxo-1,4-dihydro-1,8-naphthyridine-3-
carboxamide (49)
4.6. Tyrosine kinases assays
Yellow solid, yield: 44.8%; m. p.: 204.3e206.9 ꢀC; 1H NMR
(400 MHz, DMSO‑d6) d 12.22 (s, 1H), d 12.11 (s, 1H), 8.96 (s, 1H), 8.84
First, the kinase buffer base was prepared with configured
0.0015% Brij-35 at 50 mM HEPES (pH ¼ 7.5). Additionally, all
compounds were diluted to five concentrations using 100% DMSO.
A new solution consisting of the above dissolved compound along
with the kinase buffer group was added to the 96-well plate and
shaken under r. t. for 10 min. The mixture in the 96-well plates was
(s, 1H), 8.76 (s, 1H), 8.65 (s, 1H), 8.31 (s, 2H), 7.84 (s, 3H), 7.74 (br,
2H), 7.66 (s, 1H), 7.30 (s, 2H). Anal. calcd. for C27H16ClFN6O3 (%): C,
61.55; H, 3.06; Cl, 6.73; F, 3.61; N,15.95; Found (%): C, 61.54; H, 3.07;
N, 15.96. ESI-MS: m/z 526.1 [MþH] þ
.
4.3.39. N-(4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)phenyl)-1-(4-
methoxyphenyl)-4-oxo-1,4-dihydro-1,8-naphthyridine-3-
carboxamide (50)
then transferred in duplicate to 384-well plates, and 10
enzyme solution was added to each 384-well assay plate. After
incubating at r. t. for 10 min, the 2.5x peptide solution (10 L)
mL of
m
Yellow solid, yield: 46.3%; m. p.: 222.8e225.6 ꢀC; 1H NMR
formed by adding FAM-labeled peptide and ATP to the kinase base
buffer was added to each well on the plates. Stop buffer was added
to stop the reaction after incubating for a certain period at 28 ꢀC.
The final result was obtained by the caliper collecting the data and
copying the conversion data, after which it further converted the
data to the inhibition value. The correspondingly obtained formula
was: percent inhibition ¼ (max-conversion)/(max-min)*100. Here,
“max” stands for DMSO control, while “min” stands for low control
[35,36].
(400 MHz, DMSO‑d6)
d 12.24 (s, 1H), 12.17 (s, 1H), 8.87 (s, 1H), 8.82
(s, 2H), 8.31 (s, 1H), 7.82 (d, J ¼ 9.0 Hz, 2H), 7.58 (d, J ¼ 9.1 Hz, 3H),
7.48 (s, 1H), 7.28 (d, J ¼ 9.0 Hz, 2H), 7.16 (d, J ¼ 9.0 Hz, 2H), 3.87 (s,
3H). 13C NMR (100 MHz, DMSO‑d6)
d 177.29, 162.22, 160.07, 154.14,
154.03, 150.89, 150.62, 149.88, 148.98, 136.34, 136.15, 133.76, 129.33
(2C), 125.66, 123.01 (2C), 122.41, 121.91, 121.42, 121.35 (2C), 114.98
(2C), 112.29, 105.09, 98.49, 56.06. Anal. calcd. for C28H20N6O4 (%): C,
66.66; H, 4.00; N, 16.66; Found (%): C, 66.67; H, 4.02; N, 16.68. ESI-
þ
MS: m/z 504.2 [MþH]
.
Declaration of competing interest
4.3.40. N-(4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)phenyl)-1-(2-
chloro-4-(trifluoromethyl)phenyl)-4-oxo-1,4-dihydro-1,8-
naphthyridine-3-carboxamide (51)
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.
Yellow solid, yield: 43.9%; m. p.: 212.6e215.9 ꢀC; 1H NMR
(400 MHz, DMSO‑d6)
d 12.22 (s, 1H), 12.02 (s, 1H), 9.04 (s, 1H), 8.84
(s, 2H), 8.31 (s, 1H), 7.94 (s, 1H), 7.82 (d, J ¼ 8.7 Hz, 3H), 7.72 (s, 2H),
Acknowledgments
7.47 (s, 2H), 7.28 (d, J ¼ 8.7 Hz, 2H). 13C NMR (100 MHz, DMSO‑d6)
d
177.48, 162.16, 161.38, 158.89, 156.36, 155.54, 154.56, 154.09,
We gratefully acknowledge the generous support provided by
The National Natural Science Foundation of China (No. 81660572),
and Natural Science Foundation of Jiangxi, China (No.
20192ACBL21009).
150.49, 149.94, 136.58, 131.76, 128.34 (d, J ¼ 153 Hz), 126.04, 125.18,
123.76, 122.91 (2C), 121.62, 120.51, 116.66 (2C), 113.07 (2C), 108.94,
108.71, 104.57. Anal. calcd. for C28H16ClF3N6O3 (%): C, 58.29; H, 2.80;
Cl, 6.14; F, 9.88; N, 14.5þ7; Found (%): C, 58.28; H, 2.81; N, 14.56. ESI-
MS: m/z 576.1 [MþH]
.
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