Structure-guided design and development of novel N-phenylpyrimidin-2-amine derivatives as potential c-Met inhibitors

Article abstract of DOI:10.1016/j.ejmech.2021.113648

The HGF/Met signaling pathway is over-expressed in many types of cancers and closely related to oncogenesis and metastasis. Thus, we developed novel N-phenylpyrimidin-2-amine derivatives to test their inhibitory activities towards c-Met kinase, and most o

Full text of DOI:10.1016/j.ejmech.2021.113648

European Journal of Medicinal Chemistry 223 (2021) 113648
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Structure-guided design and development of novel N-
phenylpyrimidin-2-amine derivatives as potential c-Met inhibitors
,
,
,
,
, c
Daowei Huang a 1, Jixia Yang d 1, Qingwei Zhang b c, Guan Wang b c, Zixue Zhang b
,
Yue Zhang a **, Jianqi Li b
,
, c, *
a School of Chemical and Pharmaceutical Engineering, Hebei University of Science and Technology, Shijiazhuang, 050018, China
b Novel Technology Center of Pharmaceutical Chemistry, Shanghai Institute of Pharmaceutical Industry, Shanghai, 201203, China
c Shanghai Engineering Research Center of Pharmaceutical Process, Shanghai, 201203, China
d School of Pharmacy, Hebei University of Chinese Medicine, Shijiazhuang, 050018, China
a r t i c l e i n f o
a b s t r a c t
Article history:
The HGF/Met signaling pathway is over-expressed in many types of cancers and closely related to
oncogenesis and metastasis. Thus, we developed novel N-phenylpyrimidin-2-amine derivatives to test
their inhibitory activities towards c-Met kinase, and most of the compounds (15a-i, 15o-r, 20 and 34a-c)
could inhibit the target with IC50 values from 550.8 nM to 15.0 nM. Subsequently, compound 15b, 15d,
15f, 15i, 15o, 15r, 20, 34a and 34b also showed high antiproliferative activities in c-Met sensitive tumor
Received 20 February 2021
Received in revised form
10 June 2021
Accepted 11 June 2021
Available online 17 June 2021
cell lines (PC-3, Panc-1, HepG2, HCT116 and Caki-1) with IC50 values from 0.53 to 1.37 mM. The lead
compound 34a displayed outstanding c-Met inhibitory activity (IC50: 15.0 nM) and antiproliferative
activities. Furthermore, 34a also performed favorable pharmacokinetic properties in mice (F%: 59.3) and
an acceptable safety profile in preclinical studies. Further docking studies showed a common interaction
of 34a with c-Met at the ATP-binding site, which indicated that 34a could be a potential candidate for c-
Met inhibitors.
Keywords:
c-Met
Tyrosine kinase
N-Phenylpyrimidin-2-amine
Synthesis
Anti-cancer
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
Met kinase activity, besides antibodies and antagonists. Several
pipelines of small-molecule c-Met inhibitors with different
c-Mesenchymal epithelial transition factor (c-Met) is a receptor
tyrosine kinase (RTK) and also known as hepatocyte growth factor
receptor (HGFR) [1,2], which is critical in the progression of ma-
lignancy and essential for embryogenesis [3], wound healing [4]
and organ regeneration [5]. However, abnormally high levels of c-
Met kinase have been found to be implicated in a variety of solid
tumors (gastric cancer, thyroid cancer and lung cancer) [6] and they
are frequently associated with metastatic phenotypes and poor
prognosis [7]. Also, c-Met mutations might be one of the causes of
poor prognosis and drug-resistance in targeted therapies at clinical
studies [8]. Therefore, c-Met kinase and its ligand have become
desirable targets for cancer treatment.
binding modes and selectivity profiles have been launched or in
clinical trials. Type-I inhibitors, such as Crizotinib [9], JNJ-
38877605 [10], INC-280 [11] and MK-2461 [12], bind to c-Met in
a U shape conformation competed with ATP, and type-II in-
hibitors, such as Cabozantinib [13], Foretinib [14], MGCD265 [15],
Tepotinib [16], S-49076 [17] and Golvatinib [18], exhibit an
extended binding mode with c-Met other than the way of Type-I
inhibitors does (Fig. 1). Crizotinib (Xalkori) was approved by FDA
for the treatment of non-small cell lung cancer (NSCLC) in 2011,
and Cabozantinib (Cometriq) was first approved in 2012 for the
treatment of progressive metastatic medullary thyroid cancer, and
then in 2017 was approved in the USA for the treatment of
advanced renal cell carcinoma (RCC) in the patients who had
received prior antiangiogenic therapy. In 2020, Tepotinib devel-
oped by Merck was approved in Japan for the treatment of unre-
sectable non-small cell lung cancer with MET exon 14-skipping
mutations [19], which is the first oral c-Met inhibitor launched in
the world. Furthermore, new variations based on these molecules
have also been reported [20e26].
Small-molecule inhibitors are the main approach to inhibit c-
* Corresponding author. Novel Technology Center of Pharmaceutical Chemistry,
Shanghai Institute of Pharmaceutical Industry, Shanghai, 201203, China.
** Corresponding author.
E-mail addresses: yuezhang02@163.com (Y. Zhang), lijianqb@126.com (J. Li).
Authors of contributed equally to this paper.
1
https://doi.org/10.1016/j.ejmech.2021.113648
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

D. Huang, J. Yang, Q. Zhang et al.
European Journal of Medicinal Chemistry 223 (2021) 113648
Fig. 1. The representative small-molecule c-Met kinase inhibitors.
Generally, Type-II inhibitors show multi-target activities with
effective kinases inhibition, low toxicity and anti-drug resistance.
Cabozantinib is the first launched Type-II inhibitor and well inhibits
c-Met, VEGFR-2, c-Kit, Mer and other targets. The molecular
structure of Cabozantinib contains moiety A, moiety B, and moiety
C as three major moieties (Fig. 2). Binding mode (Fig. 3) indicates
that the quinoline group is competitively hydrogen-bonded to
Met1160, which results in the displacement of ATP at the binding
pocket to inhibit the kinase activity. Similarly, the two carbonyl
oxygens from moiety B form two hydrogen bonds with Lys1110 and
Asp1222, respectively, and moiety C is engaged in a p-p interaction
with Phe1223 to further improve c-Met inhibitory activity [27,28].
The reported structure-activity relationship may provide guidance
Fig. 2. The structure features of type II inhibitor Cabozantinib.
2

D. Huang, J. Yang, Q. Zhang et al.
European Journal of Medicinal Chemistry 223 (2021) 113648
inhibitory potency on c-Met kinase (Fig. 4). Twenty-seven new
compounds were designed, synthesized and evaluated by the
structure-activity relationship (SAR) studies to develop novel c-Met
inhibitors with high activity, low toxicity and favorable pharma-
cokinetic properties.
The binding model of the target compounds (Fig. 5) is similar to
that of Cabozantinib, which indicates that the N-phenylpyrimidin-
2-amine scaffold would be an important core structure for devel-
oping c-Met inhibitors. Compounds 15a-i, 15o-r, 20 and 34a-c
possess great inhibitory activities, and compound 15b, 15d, 15f, 15i,
15o, 15r, 20, 34a and 34b also perform great anti-proliferative ac-
tivities against five types of cancer cells (PC-3, Panc-1, HepG2,
HCT116 and Caki-1). In particular, compound 34a exhibits favorable
properties in pharmacokinetic study, and acceptable safety profiles
in MTD and hERG test, making it a promising compound for future
development.
2. Chemistry
A series of N-phenylpyrimidin-2-amine analogs and the related
intermediates were synthesized according to the pathways shown
in Schemes 1e5. These compounds were confirmed by MS, HRMS,
1H NMR, and 13C NMR spectroscopy.
Intermediates 13a-b were synthesized via peptide coupling be-
tween 1-((4-fluorophenyl)carbamoyl)cyclopropanecarboxylic acid
(11) and substituted 4-aminophenols (12) with EDCI, which were
then reacted with substituted 2,4-dichloropyrimidines or 2,4-
dichloropyridines to yield 14a through SNAr reactions. The prod-
ucts 15a-r were synthesized by nucleophilic substitution of 14a and
substituted anilines [40] in the catalyst of p-Toluenesulfonic acid
(PTSA). The method was shown in Scheme 1.
Intermediate 18 was obtained by SNAr substitution of 2,4-
dichloropyridine (16) by 4-aminophenol (17) in the presence of
potassium tert-butoxide as the base, which was found to enhance
the reactivity of 4-Cl. Then peptide coupling between intermediate
18 and 1-((4-fluorophenyl)carbamoyl)cyclopropanecarboxylic acid
yielded the key intermediate 19. Finally, 19 reacted with 3-
morpholinoaniline to give the desired compound 20 via Buch-
wald coupling. Noted that attempts failed to get compound 20 by
common SNAr conditions with either acid or base catalysis. The
method was shown in Scheme 2.
Fig. 3. The docking mode of Cabozantinib with c-Met protein (PDB: 3LQ8).
for designing Type-II inhibitors. Moiety B is commonly a linear or
cyclic group under “five-atom rule” [29e32], which contains at
least one amide bond. Many heterocycle scaffolds, such as
substituted quinolones [33], thienopyridines [34], pyrrolopyridines
[35], and pyrimidodiazepines [36,37] can be introduced to moiety
A, as their nitrogen atoms can facilitate the inhibitors to bind to the
target.
The N-phenylpyrimidin-2-amine fragment has been widely
used as a building block in the design of anticancer agents, which
contains a pyrimidine nitrogen that is capable of binding to the ATP
binding pocket [38,39]. In this work, the N-phenylpyrimidin-2-
amine fragment and different structures based on the “five-atom
rule” were introduced to moiety A and moiety B to achieve the
2-Bromobenzoic acid (21) was coupled with 4-fluoroaniline (22)
by Cu/Cu2O to yield the intermediate 23, which then reacted with
Fig. 4. Design strategy of the target compounds.
3

D. Huang, J. Yang, Q. Zhang et al.
European Journal of Medicinal Chemistry 223 (2021) 113648
Fig. 5. The docking mode of 34a with c-Met protein (PDB: 3LQ8).
Scheme 1. Reagents and conditions: (a) EDCI/DMF, rt, 4 h; (b) K2CO3/DMF, 80 ꢀC, 5 h; (c) PTSA/DMF, 90 ꢀC, 10 h.
4

D. Huang, J. Yang, Q. Zhang et al.
European Journal of Medicinal Chemistry 223 (2021) 113648
Scheme 2. Reagents and conditions: (a) Potassium tert-butylate/DMF, 100 ꢀC, 10 h; (b) EDCI/DMF, rt, 4 h; (c) Pd(OAc)2/BINAP/Cs2CO3/1,4-dioxane, reflux, 2 h.
Scheme 3. Reagents and conditions: (a) Cu/Cu2O/K2CO3/2-ethoxyethanol, 130 ꢀC, 24 h; (b) EDCI/DMF, rt, 4 h; (c) K2CO3/DMF, 80 ꢀC, 2 h; (d) PTSA/DMF, 90 ꢀC, 10 h.
Scheme 4. Reagents and conditions: (a) Cu/Cu2O/K2CO3/2-ethoxyethanol, 130 ꢀC, 24 h; (b) EDCI/DMF, rt, 4 h; (c) K2CO3/DMF, 80 ꢀC, 2 h; (d) PTSA/DMF, 90 ꢀC, 10 h.
5

D. Huang, J. Yang, Q. Zhang et al.
European Journal of Medicinal Chemistry 223 (2021) 113648
Scheme 5. Reagents and conditions: (a) EDCI/DMF, rt, 4 h; (b) K2CO3/DMF, 80 ꢀC, 5 h; (c) PTSA/DMF, 90 ꢀC, 10 h.
4-aminophenol to obtain intermediate 24. Intermediate 25 was
prepared from 2,4-dichloropyrimidine and intermediate 24 with a
similar method to intermediate 14. Compounds 26a-b were pre-
pared by nucleophilic substitution with substituted anilines [41].
The method was shown in Scheme 3.
Nine preferred compounds (15b, 15d, 15f, 15i, 15o, 15r, 20 and
34a-b) were selected to be further assayed for antiproliferative
activities in cancer cells (Table 3). All nine compounds have shown
significant antiproliferative activities with IC50 values ranging from
0.53 to 1.37
mM, which is comparable to Cabozantinib. More
Compounds 30a-b were prepared from 2-bromobenzoic acid
(21) and 4-aminophenol (17) with a similar method to Scheme 3,
which was shown in Scheme 4.
The general method to synthesize compounds 34a-c is shown in
Scheme 5 [42]. Intermediates 32a-b were prepared by peptide
coupling between 1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-
pyrazole-4-carboxylic acid (31) and substituted 4-aminophenols
(12) followed by SNAr reactions with 2,4-dichloropyrimidine to
yield 33a-b. The final products 34a-c were prepared from 33a-b
and substituted anilines with a similar method to compound 30.
specially, compound 34a displayed three to five-fold greater ac-
tivity than Cabozantinib. The results indicate the efficacy to apply
N-phenylpyrimidin-2-amine group in the development of novel c-
Met inhibitors.
The pharmacological activities indicate that the number and
position of substituents play critical roles in c-Met inhibitory ac-
tivities. For instance, the replacement of hydrogen with fluorine in
R1 position increased the c-Met activity (15o > 15f, 15r > 15q). The
presence of nitrogen in position “X” does not increase the inhibitory
activity, while affords the improved antiproliferative activity
(15f < 20), probably due to the better cell permeability. However,
the inhibitory activity decreases with nitrogen in position “Y”
(15q < 20), because this results in the change of molecular orien-
tation that breaks the hydrogen bonding.
3. Results and discussion
When R2 position is functionalized by aniline with an electron-
donating substitution, the activities of meta-substituted analogs
are better than the para-substituted analogs (15f > 15g, 15d > 15c,
15b > 15a), which could be attributed to different binding mode. In
addition, when R2 is a substituted aniline with carboxyl acid display
the superior activities compared to ester and amide
(15n > 15k > 15j > 15l > 15m).
According to Table 2, the lack of c-Met inhibitory activity in
compound 26a-b and 30a-b indicate that 2-((4-fluorophenyl)
amino)-N-phenylbenzamide and N-(4-fluorophenyl)-2-(phenyl-
amino)benzamide can't bind to c-Met kinase, so these two motifs
do not fit Moiety B. The better activities of 34a and 34b suggest the
corresponding moiety B form hydrogen bonds with the residues at
the binding pocket of c-Met kinase protein. Furthermore, the
replacement of hydrogen by fluorine would decrease the activity
3.1. In vitro activities and structureeactivity relationships
All the prepared compounds were treated by c-Met kinase-
based assay, then the lead compounds were selected to deter-
mine their antiproliferative activities against PC-3, Panc-1, HepG2,
HCT116 and Caki-1 cell lines. The assay procedures were the same
as reported in our previous work [36]. Cabozantinib was applied as
the reference compound. The IC50 values were summarized in
Table 1, as the mean values of experiments performed in triplicate.
Based on the results reported in Table 1 and Table 2, most com-
pounds (15a-i, 15o-r, 20 and 34a-c) have shown great inhibitory ac-
tivities against c-Met kinase (IC50: 550.8 nM ~ 15.0 nM). Particularly,
the activity of 34a (IC50: 15.0 nM) is better than that of Cabozantinib
(34.0 nM), and the activity of 15o (IC50: 34.9 nM) was comparable to
Cabozantinib, which further indicated that the designed compounds
are potent inhibitors of c-Met kinase.
6

D. Huang, J. Yang, Q. Zhang et al.
European Journal of Medicinal Chemistry 223 (2021) 113648
Table 1
Table 1 (continued )
Compd.
Structures and inhibitory activities against c-Met kinase of compound 15a-r, 20 and
Cabozantinib in vitro.
X
C
Y
R1
H
R2
IC50 (nM)①
15q
N
312.61 33.8
15r
20
C
C
N
C
F
110.2 8.4
59.4 6.9
H
Compd.
X
N
Y
C
R1
H
R2
IC50 (nM)①
15a
137.0 20.1
Cabozantinib
34.0 2.4
①Data are expressed as means SD of three experiments, confidence limits 95% (CL
95%).
15b
15c
15d
N
N
N
C
C
C
H
H
H
58.9 9.4
Table 2
Structures and inhibitory activities against c-Met kinase of compound 26a-b, 30a-b,
34a-c and Cabozantinib in vitro.
120.3 14.5
63.8 10.1
15e
15f
N
N
C
C
H
H
255.7 27.4
64.2 9.2
①
Compd.
R1
H
R2
moiety B
IC50 (nM)
26a
>1000
15g
15h
N
N
C
C
H
H
143.89 19.5
528.5 58.3
26b
30a
H
H
>1000
>1000
15i
15j
N
N
C
C
H
H
112.9 11.7
>2000
30b
34a
H
H
>1000
15k
15l
N
N
N
C
C
C
H
H
H
2182.7 149.6
>2000
15.0 1.6
34b
F
67.3 3.5
119.2 8.7
34.0 2.4
15m
>2000
15n
15o
N
N
C
C
H
F
1202.3 84.2
34.9 6.5
34c
H
Cabozantinib
15p
N
C
F
550.8 60.1
①Data are expressed as means SD of three experiments, confidence limits 95% (CL
95%).
(continued on next page)
(34b < 34a), which shows a different trend as compound 15f and
15o. However, the rationale for these results needs to be investi-
gated in further studies.
7

D. Huang, J. Yang, Q. Zhang et al.
European Journal of Medicinal Chemistry 223 (2021) 113648
Table 3
Cytotoxic activities of compounds cell lines in vitro.
①
IC50(mM) of 5 cell lines
Cpds
Panc-1
HepG2
PC-3
Caki-1
HCT116
15b
15d
15f
15i
15o
15r
20
34a
2.85 0.021
3.16 0.027
6.67 0.054
3.28 0.029
3.40 0.031
5.53 0.048
4.01 0.036
1.01 0.008
2.00 0.017
5.13 0.034
7.01 0.056
1.73 0.012
3.57 0.033
3.24 0.028
2.21 0.019
5.97 0.052
4.86 0.039
1.34 0.007
2.10 0.015
3.05 0.026
1.93 0.016
2.25 0.020
2.65 0.022
2.60 0.019
2.34 0.018
4.71 0.038
3.30 0.029
1.34 0.011
1.80 0.012
3.68 0.031
3.31 0.032
1.63 0.015
3.90 0.034
1.47 0.008
2.92 0.022
2.69 0.024
2.59 0.021
0.53 0.002
1.88 0.014
1.66 0.012
3.56 0.027
2.02 0.015
4.20 0.035
3.14 0.028
3.15 0.029
7.99 0.057
4.79 0.041
1.37 0.012
2.34 0.020
3.78 0.032
34b
Cabozantinib
①Data are expressed as means SD of three experiments, confidence limits 95% (CL 95%).
Table 5
Activity on hERG potassium currents of compound 34a.
3.2. Binding model analysis
To elucidate the binding mode of the compounds, a detailed
docking analysis was performed. In our study, the co-crystal
structure of Cabozantinib with c-Met was selected as the docking
mode. The inhibitor was docked using the GLIDE docking algorithm
in XP (extra-precision) mode [43]. Compound 34a shows great
inhibitory potency, which indicates that the N-phenylpyrimidin-2-
amine in moiety A and 1,5-dimethyl-2-phenyl-1H-pyrazol-3(2H)-
one in moiety B are effectively pharmacophores to inhibit the ac-
tivity of c-Met kinase. The potency of compound 34a may come
from the two hydrogen bonds by N-phenylpyrimidin-2-amine in
moiety A with MET (Fig. 5). In addition, the amide group forms a
hydrogen-bonding interaction with ASP1222, and the benzene
Compound
IC50 (mM)
34a
Cabozantinib
>40
>30
(Table 5). The detailed procedures are the same as our previous
work [36]. The corresponding IC50 values (>40 M) indicate the
inactivated status of hERG channels while c-Met kinase is inhibited,
m
so compound 34a has no risk of cardiotoxicity.
3.5. Pharmacokinetics study
The pharmacokinetics (PK) studies of 34a were conducted in
male SD rats at 20 mg/kg (PO). A Cmax of 3050.2 ng/mL was achieved
at 6.0 h. Compound 34a also was well cleared (CLz/F ¼ 0.66 L/h/kg)
in rats. The elimination half-life after administration was 9.04 h and
the oral bioavailability was 59.3%. In summary, compound 34a
displays favorable pharmacokinetics properties (Table 6).
group forms p-p interaction with residues. Further modification of
34a is currently in progress.
3.3. Acute toxicity test
The acute toxicity of compound 34a was tested to build up the
safety profile of this series of compounds with N-phenylpyrimidin-
2-amine and 1,5-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one scaf-
folds (Table 4). The ICR mice were treated with 34a orally at a single
dose of 600, 800, 1000, 1200, 1500 and 2000 mg/kg or vehicle
control (n ¼ 10), and then monitored for 14 days. The results
showed that the dosage of 1500 mg/kg killed 30% of the mice. On
the other hand, no abnormalities were observed in the dosage of
600e1200 mg/kg, which suggests that the maximum safety dosage
of 34a in mice should be 1500 mg/kg (PO).
4. Conclusions
In this study, a series of novel N-phenylpyrimidin-2-amine de-
rivatives were designed, synthesized and evaluated as c-Met kinase
inhibitors. SAR exploration led to the identification of a highly
potent c-Met inhibitor 34a with excellent activity (IC50: 15.0 nM). In
addition, its antiproliferative activities in multiple cancer cell lines
were three to five times better than Cabozantinib. Notably, as a
candidate, 34a has also shown good pharmacokinetic properties,
low body toxicity by acute toxicity tests, and negative cardiotoxicity
by hERG potassium channel tests. Further docking studies identi-
fied two hydron-bonding interactions between 34a and c-Met ki-
nase as the key structural rationale for the inhibitory activity. In
conclusion, our studies suggest that compound 34a is a promising
c-Met inhibitor worthy of further investigation.
3.4. Evaluation of activity on hERG potassium currents
The activation of hERG channels is related to cardiotoxicity, so it
is necessary to evaluate the activity on hERG potassium currents
5. Experimental
Table 4
Acute toxicity of compound 34a.
5.1. Chemistry
Dose (mg/Kg)
Mice (N)
Deaths (N)
Survival on day 14 (%)
All melting points were obtained on a BüchiMelting Point B-540
apparatus (BüchiLabortechnik) and were uncorrected. Mass spectra
(MS) were taken in ESI mode on Agilent 1100 LC-MS (Agilent, Palo
Alto, CA, USA). 1H NMR and 13C NMR spectra were generated on
Bruker AM-400 spectrometers (Bruker Bioscience, USA) with TMS
as the internal standard. All activities were tested at Shanghai Sudia
2000
1500
1200
1000
800
10
10
10
10
10
10
10
10
3
0
0
0
0
70
100
100
100
100
100
600
Vehicle
0
0
8

D. Huang, J. Yang, Q. Zhang et al.
European Journal of Medicinal Chemistry 223 (2021) 113648
Table 6
Pharmacokinetic Parameters of compound 34a.
Dose (mg/Kg)
AUC(0-t) (ngh/mL)
CLZ/F (L/h/Kg)
T1/2Z (h)
Tmax (h)
Cmax (ng/mL)
F (%)
20 (po)
4 (iv)
18999.37
8224.8
0.66
0.41
9.04
8.9
6.0
3050.2
2469.0
59.3
Co., Ltd. All other chemicals were analytical grade and used without
further purification.
Toluenesulfonic acid monohydrate (7.60 g, 40.0 mmol). The mixture
was stirred at 90 ꢀC for 10 h. Then water (50 mL) was added to
precipitate white solid, which was filtered off, and recrystallized
from ethanol to afford 15.
5.2. Preparation of N-(4-fluorophenyl)-N-(3-substitued-4-
hydroxyphenyl) cyclopropane-1,1-dicarboxamide (13a-b)
5.4.1. N-(4-fluorophenyl)-N-(4-((2-((4-(methylcarbamoyl)phenyl)
amino)pyrimidin-4- yl)oxy)phenyl)cyclopropane-1,1-
dicarboxamide (15a)
To
a
solution
of
1-((4-fluorophenyl)carbamoyl)cyclo-
propanecarboxylic acid (1.0 g, 4.48 mmol) and 4-aminophenol or 4-
amino-2-fluorophenol (5.38 mmol) in DMF (15 mL) was added
EDCI.HCl (1.03 g, 5.38 mmol). The reaction solution was stirred at
room temperature for 2 h and monitored by thin-layer chromato-
graph (TLC). Water (30 mL) was added and adjusting pH 5.0 by 1 M
HCl, and the precipitate was filtered off, washed, dried in vacuum to
yield 13 as a white solid. The production was used for the next step
without further purification.
White solid; Yield: 70.0%. 1H NMR (400 MHz, DMSO‑d6)
d 10.16
(s, 1H, CONH), 10.10 (s, 1H, CONH), 9.84 (s, 1H, CONH), 8.39 (d,
J ¼ 5.6 Hz, 1H, ArH), 8.15 (d, J ¼ 4.4 Hz, 1H, ArH), 7.73e7.58 (m, 8H,
ArH), 7.22e7.13 (m, 4H, ArH), 6.48 (d, J ¼ 5.6 Hz, 1H, NH), 2.73 (d,
J ¼ 4.4 Hz, 3H, CH3), 1.53e1.47 (m, 4H, CH2). 13C NMR (100 MHz,
DMSO‑d6)
d 178.74, 168.49, 167.45, 167.02, 164.18, 162.73, 155.82,
147.31, 138.49, 136.13, 131.22, 127.28, 123.64, 121.54, 119.82, 117.74,
113.49, 96.42, 34.05, 29.76, 24.08. HRMS (ESI) m/z calcd. for
5.2.1. N-(4-fluorophenyl)-N-(4-hydroxyphenyl)cyclopropane-1,1-
dicarboxamide (13a)
C
29H25FN6O4 [MþH]þ 541.2214, found 541.2219. Purity: > 95%
(HPLC).
White solid; Yield: 87.9%; MS (ESI) m/z: 315.15 [MþH]þ. 1H NMR
(500 MHz, CDCl3)
d 10.72 (s, 1H), 7.53e7.49 (m, 2H), 7.03e6.96 (m,
5.4.2. N-(4-fluorophenyl)-N-(4-((2-((3-(methylcarbamoyl)phenyl)
amino)pyrimidin-4- yl)oxy)phenyl)cyclopropane-1,1-
dicarboxamide (15b)
2H), 6.85e6.81 (m, 2H), 6.71e6.66 (m, 2H), 1.97e1.92 (m, 4H).
White solid; Yield: 71.4%. 1H NMR (400 MHz, DMSO‑d6)
d 10.12
5.2.2. N-(3-fluoro-4-hydroxyphenyl)-N-(4-fluorophenyl)
cyclopropane-1,1-dicarbox- amide (13b)
(d, J ¼ 28.0 Hz, 2H, CONH), 9.68 (s,1H, CONH), 8.36 (d, J ¼ 5.6 Hz,1H,
ArH), 8.27 (d, J ¼ 4.4 Hz, 1H, ArH), 7.96 (s, 1H, ArH), 7.64e7.23 (m,
5H, ArH), 7.29 (d, J ¼ 7.2 Hz, 1H, ArH), 7.20e7.14 (m, 5H, ArH), 6.41
(d, J ¼ 5.6 Hz, 1H, NH), 2.76 (d, J ¼ 4.4 Hz, 3H, CH3), 1.48 (s, 4H, CH2).
White solid; Yield: 81.4%; MS (ESI) m/z: 333.12 [MþH]þ. 1H NMR
(400 MHz, DMSO‑d6) d 10.14 (s, 1H, CONH), 9.64 (s, 1H, CONH), 9.13
(s, 1H, OH), 6.61e7.82 (m, 7H, Ar-H), 1.44 (s, 4H, CH2CH2).
13C NMR (100 MHz, DMSO‑d6)
d 194.02, 180.67, 168.48, 167.23,
5.3. General procedure for preparation of intermediate 14
165.12, 162.41, 151.83, 145.96, 139.16, 136.42, 135.33, 134.51, 127.64,
126.65, 121.82, 119.74, 117.41, 115.68, 114.14, 96.45, 37.05, 29.31,
23.76. HRMS (ESI) m/z calcd. for C29H25FN6O4 [MþH]þ 541.5203,
found 541.5217. Purity: > 95% (HPLC).
To a solution of the intermediate 13 (10.0 mmol) and 2,4-
dichloropyrimidine or 4,6-dichloropyrimidine (5.38 mmol) in
DMF (15 mL) was added K2CO3 (1.52 g,11.0 mmol). The mixture was
stirred at 80 ꢀC for 5 h. Then water (50 mL) was added to precipitate
white solid, which was filtered off, washed and dried in vacuum to
afford intermediate 14.
5.4.3. N-(4-fluorophenyl)-N-(4-((2-((4-(4-methylpiperazin-1-yl)
phenyl)amino)pyrimidin-4-yl)oxy)phenyl)cyclopropane-1,1-
dicarboxamide (15c)
White solid; Yield: 74.2%. 1H NMR (400 MHz, DMSO‑d6)
d 10.13
5.3.1. N-(4-((2-chloropyrimidin-4-yl)oxy)phenyl)-N-(4-
fluorophenyl)cyclopropane-1,1 dicarboxamide (14a)
White solid; Yield: 87.4%; MS (ESI) m/z: 427.19 [MþH]þ.
(d, J ¼ 22.4 Hz, 2H, CONH), 9.32 (s, 1H, NH), 8.27 (d, J ¼ 5.6 Hz, 1H,
ArH), 7.70e7.64 (m, 4H, ArH), 7.29 (s, 2H, ArH), 7.16 (t, J ¼ 8.8 Hz, 4H,
ArH), 6.70 (d, J ¼ 8.0 Hz, 2H, ArH), 6.33 (d, J ¼ 5.6 Hz, 1H, ArH), 2.99
(s, 4H, CH2), 2.41 (s, 4H, CH2), 2.19 (s, 4H, CH2), 1.49 (m, J ¼ 6.8 Hz,
3H, CH2). 13C NMR (100 MHz, DMSO‑d6)
d 179.73, 168.42, 167.46,
5.3.2. N-(4-((2-chloropyrimidin-4-yl)oxy)-3-fluorophenyl)-N-(4-
fluorophenyl) cyclopropane-1,1-dicarboxamide (14b)
164.88, 162.61, 154.56, 141.65, 137.42, 135.11, 126.48, 124.61, 121.65,
119.76, 118.34, 116.16, 114.62, 94.37, 58.72, 54.03, 47.68, 32.01, 21.05.
HRMS (ESI) m/z calcd. for C32H32FN7O3 [MþH]þ 582.5422, found
582.5431. Purity: > 95% (HPLC).
White solid; Yield: 81.3%; MS (ESI) m/z: 445.12 [MþH]þ.
5.3.3. N-(4-((6-chloropyrimidin-4-yl)oxy)phenyl)-N-(4-
fluorophenyl)cyclopropane- 1,1-dicarboxamide (14c)
White solid; Yield: 82.4%; MS (ESI) m/z: 427.11 [MþH]þ.
5.4.4. N-(4-fluorophenyl)-N-(4-((2-((3-(4-methylpiperazin-1-yl)
phenyl)amino)pyrimidin-4-yl)oxy)phenyl)cyclopropane-1,1-
dicarboxamide (15d)
5.3.4. N-(4-((6-chloropyrimidin-4-yl)oxy)-3-fluorophenyl)-N-(4-
fluorophenyl) cyclopropane-1,1-dicarboxamide (14d)
White solid; Yield: 75.3%. 1H NMR (400 MHz, DMSO‑d6)
d 10.18
White solid; Yield: 84.6%; MS (ESI) m/z: 445.09 [MþH]þ.
(s, 1H, CONH), 10.07 (s, 1H, CONH), 9.36 (s, 1H, NH), 8.33 (d,
J ¼ 5.6 Hz, 1H, ArH), 7.71e7.63 (m, 4H, ArH), 7.18e7.14 (m, 5H, ArH),
7.03 (d, J ¼ 7.6 Hz, 1H, ArH), 6.93 (t, J ¼ 8.0 Hz, 1H, ArH), 6.44e6.38
(m, 2H, ArH), 2.94 (s, 4H, CH2), 2.40 (s, 4H, CH2), 2.20 (s, 3H, CH3),
5.4. General procedure for preparation of compound 15
To a solution of the intermediate 14 (11.0 mmol) and R2-sub-
stituedaniline (10.0 mmol) in DMF (20 mL) was added p-
1.48 (s, 4H, CH2). 13C NMR (100 MHz, DMSO‑d6)
d
178.75, 169.67,
164.84, 163.15, 162.16, 154.82, 149.37, 144.35, 137.41, 133.14, 130.92,
9

D. Huang, J. Yang, Q. Zhang et al.
European Journal of Medicinal Chemistry 223 (2021) 113648
127.63, 121.67, 118.44, 115.84, 109.32, 106.31, 102.76, 94.32, 58.29,
54.02, 46.61, 34.11, 21.73. HRMS (ESI) m/z calcd. for C32H32FN7O3
[MþH]þ 582.5414, found 582.5423. Purity: > 95% (HPLC).
5.4.9. N-(4-fluorophenyl)-N-(4-((2-((2-oxo-2,3,4,5-tetrahydro-1H-
benzo[b]azepin-7-yl)amino)pyrimidin-4-yl)oxy)phenyl)
cyclopropane-1,1-dicarboxamide (15i)
White solid; Yield: 64.5%. 1H NMR (400 MHz, DMSO‑d6)
d 10.22
(s, 1H, NH), 10.01 (s, 1H, CONH), 9.80 (s, 1H, CONH), 9.28 (s, 1H,
CONH), 8.34 (d, J ¼ 4.0 Hz, 1H, ArH), 7.75 (d, J ¼ 8.0 Hz, 2H, ArH),
7.65e7.63 (m, 2H, ArH), 7.48 (s, 1H, ArH), 7.41 (s, 1H, ArH), 7.20 (d,
J ¼ 4.0 Hz, 2H, ArH), 7.17e7.11 (m, 3H, ArH), 6.51 (d, J ¼ 4.0 Hz, 1H,
ArH), 2.29 (s, 2H, CH2), 2.04 (t, J ¼ 4.0 Hz, 2H, CH2),1.95 (t, J ¼ 4.0 Hz,
2H, CH2), 1.46e1.52 (m, 4H, CH2). 13C NMR (100 MHz, DMSO‑d6)
5.4.5. N-(4-fluorophenyl)-N-(4-((2-((3-(morpholinomethyl)phenyl)
amino)pyrimidin-4-yl)oxy)phenyl)cyclopropane-1,1-dicarboxamide
(15e)
White solid; Yield: 75.1%. 1H NMR (400 MHz, DMSO‑d6)
d 10.18
(s, 1H, CONH), 10.04 (s, 1H, CONH), 9.62 (s, 1H, NH), 8.35 (d,
J ¼ 6.8 Hz, 1H, ArH), 7.75e7.64 (m, 5H, ArH), 7.47 (d, J ¼ 8.4 Hz, 1H,
ArH), 7.33e7.26 (m, 4H, ArH), 7.12e7.07 (m, 2H, ArH), 6.49 (d,
J ¼ 5.8 Hz, 1H, ArH), 5.13 (s, 2H, CH2), 3.67 (t, J ¼ 4.6 Hz, 4H, CH2),
2.95 (s, 4H, CH2), 1.46 (d, J ¼ 2.4 Hz, 4H, CH2). 13C NMR (100 MHz,
d
181.72,174.92,169.57,168.32,164.12,162.78,154.81,139.18,134.67,
134.22, 131.19, 126.56, 124.63, 122.61, 120.87, 119.73, 117.72, 115.86,
115.67, 93.57, 37.57, 35.61, 32.08, 23.24, 21.77. HRMS (ESI) m/z calcd.
for C31H27FN6O4 [MþH]þ 567.1749, found 566.1756. Purity: > 95%
(HPLC).
DMSO‑d6)
d 182.73, 169.65, 165.19, 163.46, 162.82, 154.81, 146.13,
138.36, 136.42, 134.17, 127.27, 124.64, 121.52, 119.59, 117.81, 116.73,
115.86, 115.26, 91.44, 68.78, 62.71, 58.74, 34.05, 23.72. HRMS (ESI)
m/z calcd. for C32H31FN6O4 [MþH]þ 583.2637, found 583.2654.
Purity: > 95% (HPLC).
5.4.10. Methyl 4-((4-(4-(1-((4-fluorophenyl)carbamoyl)
cyclopropanecarboxamido)phenoxy)pyrimidin-2-yl)amino)-3-
methoxybenzoate (15j)
White solid; Yield: 69.2%. 1H NMR (400 MHz, DMSO‑d6)
d 10.21
(s, 1H, CONH), 10.14 (s, 1H, CONH), 8.43 (s, 1H, NH), 8.11 (s, 1H, ArH),
8.02 (d, J ¼ 7.6 Hz, 1H, ArH), 7.76e7.66 (m, 4H, ArH), 7.46e7.38 (m,
2H, ArH), 7.23e7.16 (m, 4H, ArH), 6.58 (s, 1H, ArH), 3.90 (s, 3H, CH3),
3.80 (s, 3H, CH3), 1.53 (s, 4H, CH2). 13C NMR (100 MHz, DMSO‑d6)
5.4.6. N-(4-fluorophenyl)-N-(4-((2-((3-morpholinophenyl)amino)
pyrimidin-4-yl)oxy)phenyl)cyclopropane-1,1-
dicarboxamideCompound (15f)
White solid; Yield: 77.6%. 1H NMR (400 MHz, DMSO‑d6)
d
10.17
d 180.12, 169.45, 167.62, 165.56, 163.33, 162.71, 154.86, 149.32,
(s, 1H, CONH), 10.07 (s, 1H, CONH), 9.67 (s, 1H, NH), 8.34 (d,
J ¼ 6.0 Hz, 1H, ArH), 7.72e7.61 (m, 5H, ArH), 7.48 (d, J ¼ 8.0 Hz, 1H,
ArH), 7.31e7.21 (m, 4H, ArH), 7.09e7.01 (m, 2H, ArH), 6.47 (d,
J ¼ 5.6 Hz, 1H, ArH), 3.69 (t, J ¼ 4.0 Hz, 4H, CH2), 2.97 (s, 4H, CH2),
137.93, 134.28, 125.07, 123.68, 121.14, 120.92, 119.57, 117.74, 115.85,
114.93, 94.87, 56.81, 54.52, 34.02, 22.74. HRMS (ESI) m/z calcd. for
C
30H26FN5O6 [MþH]þ 567.3907, found 572.3916. Purity: > 95%
(HPLC).
1.47 (d, J ¼ 2.4 Hz, 4H, CH2). 13C NMR (100 MHz, DMSO‑d6)
d 179.78,
169.51, 166.17, 164.12, 162.34, 156.83, 150.18, 143.25, 137.43, 134.17,
128.46, 124.61, 121.64, 119.46, 116.78, 109.32, 105.38, 102.71, 96.42,
66.34, 57.39, 32.05, 27.72. HRMS (ESI) m/z calcd. for C31H29FN6O4
[MþH]þ 569.2744, found 569.2761. Purity: > 95% (HPLC).
5.4.11. 4-((4-(4-(1-((4-fluorophenyl)carbamoyl)
cyclopropanecarboxamido)phenoxy)pyrimidin-2-yl)amino)-3-
methoxybenzoic acid (15k)
White solid; Yield: 62.1%. 1H NMR (400 MHz, DMSO‑d6)
d 10.25
(s, 1H, CONH), 10.61 (s, 1H, CONH), 8.33 (d, J ¼ 5.6 Hz, 1H, NH), 7.83
(s, 1H, ArH), 7.77e7.67 (m, 5H, ArH), 7.47 (d, J ¼ 0.8 Hz, 1H, ArH),
7.27 (t, J ¼ 8.4 Hz, 1H, ArH), 7.17e7.09 (m, 4H, ArH), 6.42 (d,
J ¼ 5.6 Hz, 1H, ArH), 3.81 (s, 3H, CH3), 1.34 (s, 4H, CH3). 13C NMR
5.4.7. N-(4-fluorophenyl)-N-(4-((2-((4-morpholinophenyl)amino)
pyrimidin-4-yl)oxy)phenyl)cyclopropane-1,1-dicarboxamide (15g)
White solid; Yield: 73.2%. 1H NMR (400 MHz, DMSO‑d6)
d
10.16
(100 MHz, DMSO‑d6) d 171.04, 169.56, 169.17, 167.72, 164.18, 162.64,
(s, 1H, CONH), 10.09 (s, 1H, CONH), 9.34 (s, 1H, NH), 8.28 (d,
J ¼ 4.8 Hz,1H, ArH), 7.70e7.65 (m, 4H, ArH), 7.32 (s, 2H, ArH), 7.16 (t,
J ¼ 8.4 Hz, 4H, ArH), 6.71 (d, J ¼ 7.2 Hz, 2H, ArH), 6.34 (d, J ¼ 5.2 Hz,
1H, ArH), 3.68 (s, 4H, CH2), 2.96 (s, 4H, CH2), 1.49 (d, J ¼ 8.4 Hz, 4H,
154.82, 147.43, 138.83, 135.49, 134.54, 123.18, 122.79, 121.35, 120.71,
119.47, 117.83, 115.92, 115.63, 94.41, 56.82, 53.36, 19.46, 9.75. HRMS
(ESI) m/z calcd. for C29H24FN5O6 [MþH]þ 558.1962, found 558.1976.
Purity: > 95% (HPLC).
CH2). 13C NMR (100 MHz, DMSO‑d6)
d 181.24, 170.41, 167.43, 163.57,
162.93, 156.81, 138.65, 136.42, 135.17, 129.43, 124.65, 121.66, 119.78,
118.31, 116.74, 113.67, 97.41, 65.32, 54.31, 33.05, 24.74. HRMS (ESI)
m/z calcd. for C31H29FN6O4 [MþH]þ 569.4674, found 569.4692.
Purity: > 95% (HPLC).
5.4.12. N-(4-((2-((4-((cyclopropylmethyl)carbamoyl)-2-
methoxyphenyl)amino)pyrimidin-4-yl)oxy)phenyl)-N-(4-
fluorophenyl)cyclopropane-1,1-dicarboxamide (15l)
White solid; Yield: 59.3%. 1H NMR (400 MHz, DMSO‑d6)
d 10.16
(s, 1H, CONH), 10.09 (s, 1H, CONH), 8.39 (d, J ¼ 5.6 Hz, 1H, NH), 8.01
(s, 1H, ArH), 7.96 (s, 1H, ArH), 7.73e7.62 (m, 4H, ArH), 7.46 (d,
J ¼ 1.6 Hz, 1H, ArH), 7.22e7.13 (m, 5H, ArH), 6.50 (d, J ¼ 5.6 Hz, 1H,
ArH), 3.89 (s, 3H, CH3), 3.11 (t, J ¼ 6.0 Hz, 2H, CH2), 1.48 (t, J ¼ 5.6 Hz,
5.4.8. N-(4-fluorophenyl)-N-(4-((2-((5-oxo-5,6,7,8-
tetrahydronaphthalen-2-yl)amino)pyrimidin-4-yl)oxy)phenyl)
cyclopropane-1,1-dicarboxamide (15h)
4H, CH2), 1.35 (s, 2H, CH2), 1.23 (s, 2H, CH2), 1.04 (m, 1H, CH2). 13
C
White solid; Yield: 67.1%. 1H NMR (400 MHz, DMSO‑d6)
d
10.21
NMR (100 MHz, DMSO‑d6) d 180.46, 169.52, 167.63, 167.09, 164.18,
(s, 1H, NH), 10.04 (s, 1H, CONH), 10.00 (s, 1H, CONH), 8.42 (d,
J ¼ 4.8 Hz, 1H, ArH), 7.76 (d, J ¼ 8.0 Hz, 2H, ArH), 7.65e7.63 (m, 3H,
ArH), 7.51 (s, 1H, ArH), 7.31 (d, J ¼ 8.0 Hz, 1H, ArH), 7.21 (d,
J ¼ 8.0 Hz, 2H, ArH), 7.16 (t, J ¼ 4.0 Hz, 2H, ArH), 6.57 (d, J ¼ 4.0 Hz,
1H, ArH), 2.59 (t, J ¼ 4.0 Hz, 2H, CH2), 2.40 (t, J ¼ 4.0 Hz, 2H, CH2),
1.90e1.86 (m, 2H, CH2), 1.50e1.46 (m, 4H, CH2). 13C NMR (100 MHz,
162.83, 154.81, 149.57, 137.05, 134.81, 134.14, 127.39, 122.54, 122.02,
120.41, 119.79, 117.73, 115.83, 112.94, 94.46, 56.24, 47.56, 33.05,
21.74, 4.47. HRMS (ESI) m/z calcd. for C33H31FN6O5 [MþH]þ
611.12541, found 611.2569. Purity: > 95% (HPLC).
5.4.13. Methyl 6-((4-(4-(1-((4-fluorophenyl)carbamoyl)
cyclopropanecarboxamido)phenoxy)pyrimidin-2-yl)amino)
nicotinate (15m)
DMSO‑d6)
d 205.41, 180.63, 168.57, 167.85, 164.34, 162.18, 152.83,
147.71, 140.39, 137.42, 134.56, 129.72, 127.83, 124.79, 122.68, 119.49,
116.91, 115.62, 107.22, 95.47, 38.74, 34.02, 27.71, 17.62, 8.44. HRMS
(ESI) m/z calcd. for C31H26FN5O4 [MþH]þ 552.2021, found 552.2033.
Purity: > 95% (HPLC).
White solid; Yield: 34.2%. 1H NMR (400 MHz, DMSO‑d6)
d 10.18
(s, 1H, CONH), 10.07 (s, 1H, CONH), 8.24 (d, J ¼ 6.4 Hz, 1H, NH), 8.07
(s, 1H, ArH), 7.81 (s, 1H, ArH), 7.74e7.65 (m, 4H, ArH), 7.45 (d,
10

D. Huang, J. Yang, Q. Zhang et al.
European Journal of Medicinal Chemistry 223 (2021) 113648
J ¼ 2.4 Hz, 1H, ArH), 7.26e7.11 (m, 5H, ArH), 6.74 (d, J ¼ 6.4 Hz, 1H,
ArH), 3.88 (s, 3H, CH3), 1.48 (t, J ¼ 5.6 Hz, 4H, CH2). 13C NMR
5.4.18. N-(3-fluoro-4-((6-((3-morpholinophenyl)amino)pyrimidin-
4-yl)oxy)phenyl)-N- (4-fluorophenyl)cyclopropane-1,1-
dicarboxamide (15r)
(100 MHz, DMSO‑d6) d 180.12, 169.32, 167.67, 165.01, 163.45, 162.61,
157.32, 152.83, 149.65, 139.77, 134.75, 134.34, 122.56, 121.43, 119.52,
116.79, 115.62, 109.23, 93.67, 57.52, 34.02, 24.74. HRMS (ESI) m/z
calcd. for C28H23FN6O5 [MþH]þ 543.2043, found 543.2059. Purity:
> 95% (HPLC).
White solid; Yield: 72.7%. 1H NMR (400 MHz, DMSO‑d6)
d 10.32
(s,1H, CONH),10.02 (s,1H, CONH), 9.53 (s,1H, NH), 8.30 (s,1H, ArH),
6.79 (dd, 1H, J ¼ 16.0 Hz, 4.0 Hz, ArH), 7.66e7.62 (m, 2H, ArH), 7.42
(d, J ¼ 8.0 Hz,1H, ArH), 7.29 (t, J ¼ 8.0 Hz,1H, CH2), 7.19e7.13 (m, 4H,
ArH), 7.04 (d, J ¼ 8.0 Hz, 1H, ArH), 6.64 (dd, 1H, J ¼ 8.0 Hz, 4.0 Hz,
ArH), 6.20 (s,1H, ArH), 3.74 (t, J ¼ 4.0 Hz, 4H, CH2), 3.07 (t, J ¼ 4.0 Hz,
4H, CH2), 1.46 (d, J ¼ 4.0 Hz, 4H, CH2). 13C NMR (100 MHz, DMSO‑d6)
5.4.14. 3-((4-(4-(1-((4-fluorophenyl)carbamoyl)
cyclopropanecarboxamido)phenoxy)pyrimidin-2-yl)amino)benzoic
acid (15n)
d
180.92, 172.41, 171.67, 164.92, 162.57, 159.75, 147.13, 144.39, 137.92,
White solid; Yield: 69.3%. 1H NMR (400 MHz, DMSO‑d6)
d
12.85
136.67, 134.24, 131.45, 127.16, 120.62, 118.84, 116.71, 114.19, 108.37,
104.74, 101.77, 90.36, 66.72, 54.21, 31.57, 21.74. HRMS (ESI) m/z
calcd. for C31H28F2N6O4 [MþH]þ 586.2159, found 586.2167. Purity:
> 95% (HPLC).
(s, 1H, COOH), 10.17 (s, 1H, CONH), 10.10 (s, 1H, CONH), 9.76 (s, 1H,
NH), 8.38 (d, J ¼ 5.6 Hz,1H, ArH), 8.17 (s, 1H, ArH), 7.87 (d, J ¼ 8.4 Hz,
1H, ArH), 7.72e7.65 (m, 4H, ArH), 7.46 (d, J ¼ 7.6 Hz, 1H, ArH),
7.26e7.14 (m, 5H, ArH), 6.43 (d, J ¼ 5.6 Hz, 1H, ArH), 1.49 (s, 4H,
CH2). 13C NMR (100 MHz, DMSO‑d6)
d
180.19, 169.73, 167.92, 166.58,
5.5. Preparation of intermediate 4-((2-chloropyridin-4-yl)oxy)
164.17, 162.43, 152.81, 146.32, 135.42, 134.15, 133.22, 129.06,
124.18 122.63, 121.92, 120.77, 119.79, 117.71, 112.68, 94.43, 33.09,
23.83. HRMS (ESI) m/z calcd. for C28H22FN6O5 [MþH]þ 528.1703,
found 528.1729. Purity: > 95% (HPLC).
aniline (18)
To a solution of 2,4-dichloropyridine (1.47 g, 0.01 mol) and KOBu-
t (1.34 g, 12.0 mmol) in DMF (10 mL) was added 4-aminophenol
(1.09 g, 0.01 mol). The mixture then was stirred at 100 ꢀC for 10 h.
The reaction solution was cooled to room temperature and ice water
was added, then viscous solid was precipitated, filtered off to obtain
crude product which was purified by silica gel chromatography us-
ing a mixture of PE/EA (5:1) to afford the intermediate 18 as a white
solid (1.57 g, 71%). ESI-MS(m/z): 221.07 [MþH]þ.
5.4.15. N-(3-fluoro-4-((2-((3-morpholinophenyl)amino)pyrimidin-
4-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-
dicarboxamide (15o)
White solid; Yield: 51.6%. 1H NMR (400 MHz, DMSO‑d6)
d 10.35
(s, 1H, CONH), 10.05 (d, J ¼ 7.6 Hz, 1H, CONH), 9.43 (s, 1H, NH), 8.28
(d, J ¼ 4.8 Hz, 1H, ArH), 7.70e7.65 (m, 3H, ArH), 7.32 (s, 2H, ArH),
7.16 (t, J ¼ 8.4 Hz, 4H, ArH), 6.71 (d, J ¼ 7.2 Hz, 2H, ArH), 6.34 (d,
J ¼ 5.2 Hz, 1H, ArH), 3.68 (s, 4H, CH2), 2.96 (s, 4H, CH2), 1.49 (d,
5.6. Preparation of N-(4-((2-chloropyridin-4-yl)oxy)phenyl)-N-(4-
fluorophenyl)cyclopropane-1,1-dicarboxamide (19)
J ¼ 8.4 Hz, 4H, CH2). 13C NMR (100 MHz, DMSO‑d6)
d 180.52, 169.62,
167.74, 164.17, 162.79, 159.76, 152.18, 143.86, 138.91, 137.19, 134.55,
132.46, 125.14, 121.63, 119.28, 116.74, 112.17, 107.3, 104.71, 102.03,
96.49, 68.32, 54.39, 32.05, 21.74. HRMS (ESI) m/z calcd. for
Intermediate 18 (1.0 g, 4.54 mmol), 1-((4-fluorophenyl)carba-
moyl)cyclopro panecarboxylic acid (1.01 g, 4.54 mmol) and EDCI
(3.48 g, 18.16 mmol) in DMF (15 mL). The solution was stirred at
room temperature for 4 h. Then water (20 mL) was added, extracted
with ethyl acetate (10 mL ꢁ 3), washed by saturated brine
(10 mL ꢁ 3), and then dried by anhydrous Na2SO4, filtered and
concentrated to yield the crude product, which was purified by
stirring it with ethanol (40 mL) for 30 min to yield the intermediate
19 as a white solid (1.60 g, 83%). ESI-MS(m/z): 426.10 [MþH]þ.
C
31H28F2N6O4 [MþH]þ 587.1713, found 587.1721. Purity: > 95%
(HPLC).
5.4.16. N-(3-fluoro-4-((2-((2-oxo-2,3,4,5-tetrahydro-1H-benzo[b]
azepin-7-yl)amino)pyrimidin-4-yl)oxy)phenyl)-N-(4-fluorophenyl)
cyclopropane-1,1-dicarboxamide (15p)
White solid; Yield: 70.2%. 1H NMR (400 MHz, DMSO‑d6)
d 10.16
(s, 1H, CONH), 10.07 (s, 1H, CONH), 9.91 (s, 1H, CONH), 9.74 (s, 1H,
NH), 8.37 (s, 1H, ArH), 6.74 (d, J ¼ 16.0 Hz, 2H, ArH), 7.41 (d,
J ¼ 8.0 Hz, 2H, ArH), 7.27 (t, J ¼ 8.0 Hz, 1H, CH2), 7.17e7.11 (m, 3H,
ArH), 7.05 (d, J ¼ 8.0 Hz, 1H, ArH), 6.61 (t, J ¼ 8.0 Hz, 1H, ArH), 6.21
(s, 1H, ArH), 3.23 (t, J ¼ 4.0 Hz, 2H, CH2), 3.17 (t, J ¼ 4.6 Hz, 2H, CH2),
2.74e2.56 (m, 2H, CH2), 1.46 (d, J ¼ 4.0 Hz, 4H, CH2). 13C NMR
5.7. Preparation of N-(4-fluorophenyl)-N-(4-((2-((3-
morpholinophenyl)amino)pyridine-4-yl)oxy)phenyl)cyclopropane-
1,1-dicarboxamide (20)
To a solution of intermediate 19 (0.50 mmol, 1.18 mmol), 3-
morpholinoaniline (0.23 g, 1.29 mmol) and BINAP (37 mg,
0.06 mmol) in 1,4-dioxane (15 mL) was added Cs2CO3 (0.57 g,
1.76 mmol). The mixture then was stirred at reflux for 2 h. The
reaction solution was cooled to room temperature and filtered
through celite, then concentrated to obtain crude product which
was purified by silica gel chromatography using a mixture of DCM/
MeOH (40:1) to afford the title compounds 20.
(100 MHz, DMSO‑d6)
d 181.78, 174.41, 169.57, 167.83, 164.19, 162.85,
160.33, 138.64, 136.89, 136.34, 134.75, 131.39, 128.47, 124.12, 122.61,
120.94, 118.73, 116.49, 115.13, 115.09, 111.12, 94.41, 38.59, 35.72,
33.01, 23.24, 21.75. HRMS (ESI) m/z calcd. for C31H26F2N6O4 [MþH]þ
585.1249, found 585.1261. Purity: > 95% (HPLC).
5.4.17. N-(4-fluorophenyl)-N-(4-((6-((3-morpholinophenyl)amino)
pyrimidin-4-yl)oxy)phenyl)cyclopropane-1,1-dicarboxamide (15q)
White solid; Yield: 67.0%. 1H NMR (400 MHz, DMSO‑d6)
d 10.17
(s, 1H, CONH), 10.04 (s, 1H, CONH), 8.85 (s, 1H, NH), 8.03 (d,
J ¼ 4.0 Hz, 1H, ArH), 7.72 (d, J ¼ 8.0 Hz, 2H, ArH), 7.65e7.62 (m, 2H,
ArH), 7.19e7.13 (m, 5H, ArH), 7.04 (d, J ¼ 8.0 Hz, 2H, ArH), 6.49e6.46
(m, 1H, ArH), 6.39 (dd, J1 ¼ 1.6 Hz, J2 ¼ 4.0 Hz, 1H, ArH), 6.16 (d,
J ¼ 4.0 Hz, 1H, ArH), 3.72 (t, J ¼ 4.0 Hz, 4H, CH2), 3.02 (t, J ¼ 4.0 Hz,
4H, CH2), 1.46 (d, J ¼ 4.0 Hz, 4H, CH2). 13C NMR (100 MHz, DMSO‑d6)
White solid; Yield: 71.4%. 1H NMR (400 MHz, DMSO‑d6)
d 10.15
(s,1H, CONH),10.06 (s,1H, CONH), 9.45 (s,1H, NH), 8.34 (s,1H, ArH),
7.62e7.69 (m, 5H, ArH), 7.17e7.13 (m, 5H, ArH), 7.02 (d, J ¼ 8.0 Hz,
1H, ArH), 6.62 (dd, J ¼ 8.0 Hz, 1.6 Hz,1H, ArH), 6.04 (s, 1H, ArH), 2.73
(t, J ¼ 4.0 Hz, 4H, CH2), 3.05 (t, J ¼ 4.0 Hz, 4H, CH2), 1.46 (s, 4H, CH2).
13C NMR (100 MHz, DMSO‑d6)
d
180.42, 172.59, 171.46, 162.77,
d 180.42, 166.61, 163.63, 155.27, 149.82, 148.55, 146.01, 143.74,
161.81, 154.81, 147.12, 144.37, 135.46, 134.32, 130.93, 124.67, 121.64,
119.75, 116.71, 108.63, 104.87, 102.76, 91.82, 66.72, 54.31, 31.73,
22.47. HRMS (ESI) m/z calcd. for C31H29FN6O4 [MþH]þ 569.1775,
found 569.1782. Purity: > 95% (HPLC).
134.25, 134.64, 131.48, 126.61, 122.69, 119.93, 117.78, 109.33, 105.32,
101.87, 100.46, 95.17, 68.38, 54.35, 39.02, 23.74. HRMS (ESI) m/z
calcd. for C32H30FN5O4 [MþH]þ 568.1419, found 568.1426. Purity: >
95% (HPLC).
11

D. Huang, J. Yang, Q. Zhang et al.
European Journal of Medicinal Chemistry 223 (2021) 113648
5.8. Preparation of the intermediate 2-((4-fluorophenyl)amino)
benzoic acid (23)
5.11.2. 2-((4-fluorophenyl)amino)-N-(4-((2-((3-
(morpholinomethyl)phenyl)amino)pyridin-4-yl)oxy)phenyl)
benzamide (26b)
A
mixture of 2-bromobenzoic acid (2.0 g,0.01 mol), 4-
White solid, Yield: 79.4%. 1H NMR (400 MHz, DMSO‑d6)
d 10.49
fluoroaniline (1.17 g,10.5 mmol), Cu (power, 0.06 g,0.9 mmol),
Cu2O (64 mg, 0.45 mmol) and K2CO3 (1.38 g,0.01 mmol) in 2-
ethoxyethanol (10 mL) was heated at 130 ꢀC for 24 h under nitro-
gen atmosphere. Upon cooling to room temperature, H2O (20 mL)
was added to, and filtered through celite. The filtrate was adjusted
to pH 7.0 to precipitate solid, and then filtered and dried to yield the
intermediate 23 (1.51 g, 65.3%). MS (ESI) m/z: 232.08 [MþH]þ
(s, 1H, CONH), 9.59 (s, 1H, NH), 9.11 (s, 1H, NH), 8.35 (d, 1H,
J ¼ 4.0 Hz, ArH), 7.80e7.86 (m, 3H, ArH), 7.46 (s, 1H, ArH), 7.33e7.41
(m, 2H, ArH), 7.14e7.25 (m, 7H, ArH), 7.07 (t, 1H, J ¼ 8.0 Hz, ArH),
6.94 (t, 1H, J ¼ 8.0 Hz, ArH), 6.79 (d, 1H, J ¼ 8.0 Hz, ArH), 6.45 (t, 1H,
J ¼ 8.0 Hz, ArH), 3.48 (s, 4H, CH2), 3.19 (s, 2H, CH2), 2.24 (s, 4H, CH2).
13C NMR (100 MHz, DMSO‑d6)
d 169.31, 167.47, 167.21, 163.67,
158.81, 152.33, 149.42, 143.11, 138.32, 136.89, 133.87, 133.36, 130.28,
128.27, 126.41, 123.87, 121.29, 119.73, 119.54, 118.71, 118.13, 116.77,
116.62, 115.52, 94.41, 67.12, 65.92, 56.37. HRMS (ESI) m/z calcd. for
5.9. Preparation of 2-((4-fluorophenyl)amino)-N-(4-
hydroxyphenyl)benzamide (24)
C
34H31FN6O3 [MþH]þ 591.4007, found 591.4022. Purity: > 95%
(HPLC).
A solution of 23 (2.31 g, 0.01 mol), 4-aminophenol (1.09 g,
0.01 mmol) and EDCI (5.75 g, 0.03 mmol) in DMF (20 mL) was
stirred at room temperature for 4 h. Then water (40 mL) was added,
extracted with ethyl acetate (15 mL ꢁ 3), washed by saturated brine
(10 mL ꢁ 3), and then dried by anhydrous Na2SO4, filtered and
concentrated to yield the crude product, which was purified by
silica gel chromatography using a mixture of petroleum ether/ethyl
acetate (40:1) to yield the intermediate 24 as a white solid (2.89 g,
5.12. Preparation of the intermediate 2-((4-hydroxyphenyl)amino)
benzoic acid (27)
A
mixture of 2-bromobenzoic acid (2.0 g,0.01 mol), 4-
aminophenol (1.15 g,10.5 mmol), Cu (power, 0.06 g,0.9 mmol),
Cu2O (64 mg, 0.45 mmol) and K2CO3 (1.38 g,0.01 mmol) in 2-
ethoxyethanol (10 mL) was stirred at 130 ꢀC for 24 h under nitro-
gen atmosphere. Upon cooling to room temperature, H2O (20 mL)
was added and filtered through celite. The filtrate was adjusted to
pH 7.0 to precipitate solid, and then filtered and dried to yield the
intermediate 27 (1.53 g, 67.0%). MS (ESI) m/z: 230.09 [MþH]þ
89.6%). 1H NMR (500 MHz, DMSO‑d6)
d 10.10 (s, 1H), 9.27 (s, 1H),
9.17 (s, 1H), 7.78e7.68 (m, 1H), 7.47 (d, J ¼ 8.8 Hz, 2H), 7.38e7.29 (m,
1H), 7.21e7.09 (m, 5H), 6.88 (t, J ¼ 7.8 Hz, 1H), 6.79e6.68 (m, 2H).
ESI-MS(m/z): 323.13[MþH]þ.
5.10. Preparation of the intermediate N-(4-((2-chloropyrimidin-4-
yl)oxy)phenyl)-2- ((4-fluorophenyl)amino)benzamide (25)
5.13. Preparation of N-(4-fluorophenyl)-2-((4-hydroxyphenyl)
amino)benzamide (28)
To a solution of intermediate 24 (3.22 g, 0.01 mol) and 2,4-
dichloropyrimidine (1.48 g, 0.01 mmol) in DMF (15 mL) was
added K2CO3 (1.09 g, 0.01 mol). The mixture then was stirred at
80 ꢀC for 2 h. The reaction mixture was cooled to room temperature
and ice water was added, then viscous solid was precipitated,
filtered off to obtain intermediate 25 as a pale white solid (3.45 g,
A solution of 27 (2.29 g, 0.01 mol), 4-fluoroaniline (1.11 g,
0.01 mmol) and EDCI (5.75 g, 0.03 mmol) in DMF (20 mL) was
stirred at room temperature for 4 h. Then water (40 mL) was added,
extracted with ethyl acetate (15 mL ꢁ 3), washed by saturated brine
(10 mL ꢁ 3), and then dried by anhydrous Na2SO4, filtered and
concentrated to yield the crude product, which was purified by
silica gel chromatography using a mixture of petroleum ether/ethyl
acetate (20:1) to yield the intermediate 28 as a white solid (3.05 g,
94.6%). ESI-MS(m/z): 323.12[MþH]þ.
79.4%). 1H NMR (500 MHz, DMSO‑d6)
d 10.45 (s, 1H), 9.04 (s, 1H),
8.62 (d, J ¼ 5.7 Hz, 1H), 7.81 (d, J ¼ 9.0 Hz, 2H), 7.77 (d, J ¼ 7.1 Hz,
1H), 7.37 (t, J ¼ 7.7 Hz,1H), 7.25 (d, J ¼ 8.9 Hz, 2H), 7.23e7.17 (m, 3H),
7.17e7.11 (m, 3H), 6.92 (t, J ¼ 7.5 Hz, 1H). ESI-MS(m/z): 435.11
[MþH]þ.
5.14. Preparation of the intermediate 2-((4-((2-chloropyrimidin-4-
yl)oxy)phenyl) amino)-N-(4-fluorophenyl)benzamide (29)
5.11. Preparation of the title compounds 26a-b
To a solution of intermediate 28 (3.22 g, 0.01 mol) and 2,4-
dichloropyrimidine (1.48 g, 0.01 mmol) in DMF (15 mL) was
added K2CO3 (1.52 g, 0.01 mol). The mixture then was stirred at
80 ꢀC for 2 h. The reaction solution was cooled to room temperature
and ice water was added, then viscous solid was precipitated,
filtered off to obtain intermediate 29 as a pale white solid (3.70 g,
85.2%). ESI-MS(m/z): 435.11[MþH]þ.
To a solution of the intermediate 25 (4.77 g, 11.0 mmol) and R2-
substituedaniline (10.0 mmol) in DMF (20 mL) was added p-Tol-
uenesulfonic acid monohydrate (7.60 g, 40.0 mmol). The solution
was stirred at 90 ꢀC for 10 h. Then water (50 mL) was added to
precipitate white solid. The white solid was filtered and recrystal-
lized from ethanol to afford 26.
5.11.1. 2-((4-fluorophenyl)amino)-N-(4-((2-((3-morpholinophenyl)
amino)pyridine-4-yl)oxy)phenyl)benzamide (26a)
5.15. Preparation of the title compounds 30a-b
White solid, Yield: 62.8%. 1H NMR (400 MHz, DMSO‑d6)
d
10.44
To a solution of the intermediate 29 (4.77 g, 11.0 mmol) and R2-
substituedaniline (10.0 mmol) in DMF (20 mL) was added p-Tol-
uenesulfonic acid monohydrate (7.60 g, 40.0 mmol). The solution
was stirred at 90 ꢀC for 10 h. Then water (50 mL) was added to
precipitate white solid. The white solid was filtered and recrystal-
lized from ethanol to afford 30.
(s, 1H, CONH), 9.67 (s, 1H, NH), 9.15 (s, 1H, NH), 8.34 (d, J ¼ 4.4 Hz,
1H, ArH), 7.87e7.82 (m, 3H, ArH), 7.41 (s, 1H, ArH), 7.39e7.33 (m,
2H, ArH), 7.24e7.12 (m, 7H, ArH), 7.03 (t, J ¼ 8.6 Hz,1H, ArH), 6.98 (t,
J ¼ 6.8 Hz, 1H, ArH), 6.77 (d, J ¼ 8.4 Hz, 1H, ArH), 6.34 (t, J ¼ 8.0 Hz,
1H, ArH), 3.46 (s, 4H, CH2), 2.27 (s, 4H, CH2). 13C NMR (100 MHz,
DMSO‑d6)
d 169.56, 167.43, 167.09, 164.25, 157.72, 152.73, 150.36,
148.61, 145.72, 138.88, 134.51, 134.63, 131.73, 129.93, 127.43, 124.61,
121.69, 119.21, 119.33, 118.25, 117.38, 109.33, 107.28, 103.71, 94.42,
65.39, 54.34. HRMS (ESI) m/z calcd. for C33H29FN6O3 [MþH]þ
577.2457; found 577.2462. Purity: > 95% (HPLC).
5.15.1. N-(4-fluorophenyl)-2-((4-((2-((3-morpholinophenyl)amino)
pyridin-4-yl)oxy)phenyl)amino)benzamide (30a)
White solid, Yield: 63.7%. 1H NMR (400 MHz, DMSO‑d6)
d
10.45
(s, 1H, CONH), 9.62 (s, 1H, NH), 9.15 (s, 1H, NH), 8.34 (d, 1H,
12

D. Huang, J. Yang, Q. Zhang et al.
European Journal of Medicinal Chemistry 223 (2021) 113648
J ¼ 4.4 Hz, ArH), 7.87e7.82 (m, 3H, ArH), 7.41 (s, 1H, ArH), 7.39e7.33
(m, 2H, ArH), 7.24e7.12 (m, 7H, ArH), 7.03 (t, 1H, J ¼ 8.6 Hz, ArH),
6.98 (t, 1H, J ¼ 6.8 Hz, ArH), 6.77 (d, 1H, J ¼ 8.4 Hz, ArH), 6.34 (t, 1H,
J ¼ 8.0 Hz, ArH), 3.46 (s, 4H, CH2), 2.27 (s, 4H, CH2). 13C NMR
5.17.2. N-(4-((2-chloropyrimidin-4-yl)oxy)-3-fluorophenyl)-1,5-
dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazole-4-carboxamide
(33b)
Pale white solid, Yield: 84.27%, ESI-MS(m/z): 454.11 [MþH]þ.
(100 MHz, DMSO‑d6) d 169.51, 167.68, 167.27, 164.85, 162.76, 149.47,
148.15, 144.74, 143.84, 139.31, 135.13, 133.62, 130.79, 129.43, 126.38,
125.67, 122.56, 122.84, 119.76, 118.55, 116.13, 109.34, 105.63, 101.45,
94.02, 64.28, 54.43. HRMS (ESI) m/z calcd. for C33H29FN6O3 [MþH]þ
577.2527, found 577.2542; Purity: > 95% (HPLC).
5.18. Preparation of the title compounds 34a-c
To a solution of the intermediate 33 (11.0 mmol) and R2-sub-
stituedaniline (10.0 mmol) in DMF (20 mL) was added p-toluene-
sulfonic acid monohydrate (7.60 g, 40.0 mmol). The solution was
stirred at 90 ꢀC for 10 h. Then water (50 mL) was added to pre-
cipitate white solid. The white solid was filtered and recrystallized
from ethanol to afford 34.
5.15.2. N-(4-fluorophenyl)-2-((4-((2-((3-(morpholinomethyl)
phenyl)amino)pyridin-4-yl)oxy)phenyl)amino)benzamide (30b)
White solid, Yield: 59.4%. 1H NMR (400 MHz, DMSO‑d6)
dppm:
10.47 (s, 1H, CONH), 9.64 (s, 1H, NH), 9.17 (s, 1H, NH), 8.35 (d,
J ¼ 4.0 Hz, 1H, ArH), 7.86e7.80 (m, 3H, ArH), 7.46 (s, 1H, ArH),
7.41e7.33 (m, 2H, ArH), 7.25e7.14 (m, 7H, ArH), 7.01 (t, J ¼ 8.0 Hz,
1H, ArH), 6.91 (t, J ¼ 8.0 Hz, 1H, ArH), 6.76 (d, J ¼ 8.0 Hz, 1H, ArH),
6.42 (t, J ¼ 8.6 Hz, 1H, ArH), 3.46 (s, 4H, CH2), 3.22 (s, 2H, CH2), 2.15
5.18.1. 1,5-dimethyl-N-(4-((2-((3-morpholinophenyl)amino)
pyrimidin-4-yl)oxy)phenyl)-3-oxo-2-phenyl-2,3-dihydro-1H-
pyrazole-4-carboxamide (34a)
(s, 4H, CH2). 13C NMR (101 MHz, DMSO‑d6)
d
169.54, 167.61, 167.02,
White solid, Yield: 63.1%. 1H NMR (400 MHz, DMSO‑d6)
d 10.80
163.95, 162.85, 150.32, 145.31, 142.71, 140.23, 138.12, 134.42, 133.21,
130.25, 128.74, 126.89, 124.72, 121.55, 121.67, 119.36, 118.81, 118.45,
117.92, 116.54, 115.62, 94.41, 67.54, 65.37, 53.65. HRMS (ESI) m/z
calcd. for C33H29FN6O3 [MþH]þ 591.2657, found 591.2672; Purity: >
95% (HPLC).
(s, 1H, CONH), 9.35 (s, 1H, NH), 8.33 (d, J ¼ 5.6 Hz, 1H, ArH), 7.66 (d,
J ¼ 8.9 Hz, 2H, ArH), 7.59 (t, J ¼ 7.6 Hz, 2H, ArH), 7.51 (t, J ¼ 7.4 Hz,
1H, ArH), 7.44 (d, J ¼ 7.1 Hz, 2H, ArH), 7.20e7.15 (m, 3H, ArH), 7.08
(d, J ¼ 8.3 Hz, 1H, ArH), 6.95 (t, J ¼ 8.1 Hz, 1H, ArH), 6.48 (dd, J ¼ 8.2,
2.3 Hz, 1H, ArH), 6.39 (d, J ¼ 5.5 Hz, 1H, ArH), 3.67 (t, J ¼ 4.0 Hz, 4H,
CH2), 3.36 (s, 3H, CH3), 2.92 (t, J ¼ 4.7 Hz, 4H, CH2), 2.71 (s, 3H, CH3).
13C NMR (100 MHz, DMSO‑d6)
d 169.81, 163.56, 161.62, 160.32,
5.16. Preparation of N-(3-substituted-4-hydroxyphenyl)-1,5-
dimethyl-3-oxo-2-phenyl- 2,3-dihydro-1H-pyrazole-4-carboxamide
(32a-b)
154.33, 151.84, 148.11, 141.31, 136.59, 133.55, 129.93, 129.29, 129.08,
127.58, 122.50, 120.59, 110.91, 109.42, 106.36, 98.73, 97.62, 66.53,
49.04, 33.79, 11.94. HRMS Calcd for C32H31N7O4 [MþH]þ, 578.2524;
found, 578.2523. Purity: > 95%.
A
mixture of 1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-
pyrazole-4- carboxylic acid (2.32 g, 0.01 mol), 2-substituted-4-
aminophenol (0.01 mmol) and EDCI (5.75 g, 0.03 mmol) in DMF
(15 mL) was heated at room temperature for 4 h. Then water
(40 mL) was added, extracted with ethyl acetate (15 mL ꢁ 3),
washed by saturated brine (10 mL ꢁ 3), and then dried by anhy-
drous Na2SO4, filtered and concentrated to yield the intermediate
32 as a white solid.
5.18.2. N-(3-fluoro-4-((2-((3-morpholinophenyl)amino)pyrimidin-
4-yl)oxy)phenyl)-1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-
pyrazole-4-carboxamide (34b)
White solid, Yield: 71.4%. 1H NMR (400 MHz, DMSO‑d6)
d 10.74
(s, 1H, CONH), 9.23 (s, 1H, NH), 8.13 (d, J ¼ 5.6 Hz, 1H, ArH), 7.59 (d,
J ¼ 8.0 Hz, 2H, ArH), 7.47 (t, J ¼ 8.0 Hz, 2H, ArH), 7.24 (t, J ¼ 8.0 Hz,
1H, ArH), 7.37 (d, J ¼ 8.0 Hz, 2H, ArH), 7.18 (d, J ¼ 8.0 Hz, 2H, ArH),
7.07 (d, J ¼ 8.0 Hz, 1H, ArH), 6.67 (t, J ¼ 8.0 Hz, 1H, ArH), 6.38 (d,
J ¼ 8.0 Hz, 1H, ArH), 6.27 (d, J ¼ 8.0 Hz, 1H, ArH), 3.47 (t, J ¼ 4.0 Hz,
4H, CH2), 2.75 (t, J ¼ 4.0 Hz, 4H, CH2), 2.64 (s, 3H, CH3), 1.27 (d,
5.16.1. Intermediate N-(4-hydroxyphenyl)-1,5-dimethyl-3-oxo-2-
phenyl-2,3-dihydro-1H-pyrazole-4-carboxamide (32a)
White solid, Yield: 94.0%, ESI-MS(m/z): 324.14 [MþH]þ.
J ¼ 4.0 Hz, 3H, CH3). 13C NMR (100 MHz, DMSO‑d6)
d 169.72, 166.54,
164.16, 163.76, 162.53, 160.37, 158.48, 147.79, 145.51, 137.01, 134.14,
133.96, 131.18, 129.42, 125.19, 123.58, 122.41, 117.24, 111.37, 108.32,
106.36, 103.95, 101.71, 94.48, 67.39, 55.30, 34.23, 12.06. HRMS (ESI)
m/z calcd. for C32H30FN7O4 [MþH]þ 596.2759, found 596.2771;
Purity: > 95% (HPLC).
5.16.2. Intermediate N-(3-fluoro-4-hydroxyphenyl)-1,5-dimethyl-
3-oxo-2-phenyl-2,3-dihydro-1H-pyrazole-4-carboxamide (32b)
White solid, Yield: 92.4%, ESI-MS(m/z): 342.13 [MþH]þ.
5.17. Preparation of the intermediate 33
5.18.3. 1,5-dimethyl-N-(4-((2-((3-(morpholinomethyl)phenyl)
amino)pyrimidin-4-yl)oxy)phenyl)-3-oxo-2-phenyl-2,3-dihydro-
1H-pyrazole-4-carboxamide (34c)
To
a solution of intermediate 32 (0.01 mol) and 2,4-
dichloropyrimidine (1.48 g, 0.01 mmol) in DMF (15 mL) was
added K2CO3 (1.52 g, 0.01 mol). The mixture then was stirred at
80 ꢀC for 2 h. The reaction mixture was cooled to room temperature
and ice water was added, then viscous solid was precipitated,
filtered off to obtain intermediate 33 as a pale white solid.
White solid, Yield: 67.3%. 1H NMR (400 MHz, DMSO‑d6)
d 10.87
(s, 1H, CONH), 9.58 (s, 1H, NH), 8.34 (d, J ¼ 8.0 Hz, 1H, ArH), 7.70 (d,
J ¼ 8.0 Hz, 2H, ArH), 7.60 (t, J ¼ 8.0 Hz, 2H, ArH), 7.51 (t, J ¼ 8.0 Hz,
1H, ArH), 7.44 (d, J ¼ 4.0 Hz, 3H, ArH), 7.30 (d, J ¼ 4.0 Hz, 1H, ArH),
7.20 (d, J ¼ 8.0 Hz, 2H, ArH), 7.04 (t, J ¼ 8.0 Hz, 1H, ArH), 6.83 (d,
J ¼ 8.0 Hz, 1H, ArH), 6.44 (d, J ¼ 4.0 Hz, 1H, ArH), 3.49 (s, 4H, CH2),
3.36 (s, 3H, CH3), 3.17 (s, 2H, CH2), 2.72 (s, 3H, CH3), 2.24 (s, 4H,
5.17.1. N-(4-((2-chloropyrimidin-4-yl)oxy)phenyl)-1,5-dimethyl-3-
oxo-2-phenyl-2,3-dihydro-1H-pyrazole-4-carboxamide (33a)
Pale white solid, Yield: 86.4%. 1H NMR (500 MHz, CDCl3)
d
10.79
CH2); 13C NMR (100 MHz, DMSO‑d6)
d 169.49, 166.13, 164.41, 163.15,
(s, 1H), 8.40 (d, J ¼ 5.7 Hz, 1H), 7.75e7.72 (m, 2H), 7.55 (t, J ¼ 7.8 Hz,
2H), 7.49e7.44 (m, 1H), 7.39e7.34 (m, 2H), 7.10e7.07 (m, 2H), 6.74
(d, J ¼ 5.6 Hz, 1H), 3.35 (s, 3H), 2.78 (s, 3H). ESI-MS(m/z): 436.12
[MþH]þ.
162.37, 158.26, 151.83, 147.35, 137.74, 134.59, 134.18, 130.42, 126.63,
124.76, 124.44, 119.14, 118.27, 116.81, 115.23, 104.91, 94.44, 67.79,
64.25, 55.70, 34.64, 12.09. HRMS (ESI) m/z calcd. for C33H33N7O4
[MþH]þ 592.4143, found 592.4168; Purity: > 95% (HPLC).
13

D. Huang, J. Yang, Q. Zhang et al.
European Journal of Medicinal Chemistry 223 (2021) 113648
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Declaration of competing interest
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The authors declare that they have no known competing
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Acknowledgments
[34] K.S. Kim, L.P. Zhang, R. Schmidt, Z.W. Cai, D. Wei, D.K. Williams, L.J. Lombardo,
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This work was financially supported by the National Natural
Science Foundation of China (No.82003601), and Biomedical co-
funding of Natural Science Foundation of Hebei Province
(No.H2020208008).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
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