Design, synthesis and biological evaluation of pyrimidine-based derivatives as VEGFR-2 tyrosine kinase inhibitors

Article abstract of DOI:10.1016/j.bioorg.2018.04.005

Vascular endothelial growth factor receptor-2 (VEGFR-2) plays a crucial role in tumor angiogenesis, and inhibition of the VEGFR-2 signaling pathway has already become an attractive approach for cancer therapy. In this study, a novel pyrimidine-based derivative 7j was designed as lead compound, and three series of potent VEGFR-2 inhibitors were synthesized and biologically evaluated against A549 and HepG2 cell lines. Compounds 7d, 9s and 13n exhibited superior inhibitory activities against A549 cell with IC₅₀ ranged from 9.19 to 13.17 μM and HepG2 cell with IC₅₀ ranged from 11.94 to 18.21 μM compared to those of Pazopanib (IC₅₀ = 21.18 and 36.66 μM). In addition, molecular docking study was performed to investigate the binding capacity and binding mode between target compounds and VEGFR-2.

Full text of DOI:10.1016/j.bioorg.2018.04.005

BioorganicChemistry78(2018)393–405
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design, synthesis and biological evaluation of pyrimidine-based derivatives
as VEGFR-2 tyrosine kinase inhibitors
Wuji Suna, Shengquan Hua, Shubiao Fangb, Hong Yana,⁎
a
Beijing Key Laboratory of Environmental and Viral Oncology, College of Life Science and Bio-engineering, Beijing University of Technology, Pingleyuan Street No. 100,
Chaoyang District, Beijing 100124, China
b
Tong Yi Tang Pharmaceutical Co., Ltd., Sui'an Industrial Park of Zhangpu County, Fujian Province 363200, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Vascular endothelial growth factor receptor-2 (VEGFR-2) plays a crucial role in tumor angiogenesis, and in-
hibition of the VEGFR-2 signaling pathway has already become an attractive approach for cancer therapy. In this
study, a novel pyrimidine-based derivative 7j was designed as lead compound, and three series of potent VEGFR-
2 inhibitors were synthesized and biologically evaluated against A549 and HepG2 cell lines. Compounds 7d, 9s
and 13n exhibited superior inhibitory activities against A549 cell with IC50 ranged from 9.19 to 13.17 μM and
HepG2 cell with IC50 ranged from 11.94 to 18.21 μM compared to those of Pazopanib (IC50 = 21.18 and
36.66 μM). In addition, molecular docking study was performed to investigate the binding capacity and binding
mode between target compounds and VEGFR-2.
VEGFR-2 inhibitors
Pyrimidine-based derivatives
Molecular docking
1. Introduction
Clinical experience demonstrates that these agents have the advantages
of broad spectrum anti-tumor potency and less prone to resistance.
Angiogenesis, the formation of new blood vessel from preexisting
vasculature, is a normal and vital process in growth and development,
as well as in wound healing, tissue repair and female reproductive cy-
cling. [1–3] Aberrant regulation of angiogenesis is associated with
major diseases including rheumatoid arthritis, diabetes, atherosclerosis,
retinopathies and cancer [4–6]. Tumor angiogenesis is a prerequisite
for tumor growth as it is a fundamental step in the growth and me-
tastasis of cancer [7]. Accordingly, the selective inhibition of tumor
angiogenesis may be a promising therapeutic strategy for inhibiting
tumor growth and metastasis. Tumor angiogenesis is regulated by a
number of proangiogenic and antiangiogenic factors, produced by both
host and tumor cells, including vascular endothelial growth factor
(VEGF), platelet-derived growth factor (PDGF), fibroblast growth factor
(FGF) and other cytokines [7]. Particularly, VEGF and its receptor
VEGFR-2 tyrosine kinase are very important in the direct regulation of
angiogenesis, and the overexpression of VEGFR-2 is implicated in the
angiogenesis of solid tumors [8–10]. Disruption of VEGF signaling
pathway by either neutralizing of circulating VEGF or inhibiting re-
ceptor tyrosine kinases with small molecules is found to effectively
inhibit angiogenesis, tumor progression and dissemination [11].
At present, many small molecule VEGFR-2 inhibitors have been
synthesized and approved to be potent anti-cancer agents, such as
Pazopanib [12], Sunitinib [13], Sorafenib [14] and Axitinib [15].
Nevertheless, the adverse effects that cause treatment discontinuations
and dose reductions may negate the life-prolonging effects of these
agents, indicating an unmet need for safer VEGFR-2 inhibitors with
high efficacy.
Pazopanib is a multi-target receptor tyrosine kinase inhibitor, tar-
geting the vascular endothelial growth factor receptor (VEGFR-1, -2,
and -3), platelet-derived growth factor receptor (PDGFR-α and -β) and
c-kit [16,17]. The chemical structure of Pazopanib is sectioned into two
main parts of indazole and anilinopyrimidine. The anilinopyrimidine
moiety, mimicking the behavior of the adenine ring of ATP, occupies
the adenine pocket and is closely related with the kinase inhibition
[12]. Sorafenib, a potent VEGFR-2 inhibitor, is approved by U.S. FDA
for the treatment of advanced renal cell carcinoma and hepatocellular
carcinoma [18]. Sorafenib presents a typical pharmacophore feature
where a central aryl ring, occupying the linker region, is bound to
pyridine core via an ether linker and the terminal aryl ring via a urea
group. More and more researchers have focused on the optimization of
Sorafenib due to its disadvantages, such as poor physicochemical
properties and intolerable adverse effects [19,20]. Axitinib is approved
for the treatment of advanced renal cell carcinoma [21,22]. It competes
with ATP for the ATP binding site, and subsequently inhibits activation
of VEGFR [21–23]. The molecule fits inside the tunnel of the catalytic
domain of VEGFR, and the ethenylpyridine segment is well embedded
⁎
Corresponding author.
E-mail address: hongyan@bjut.edu.cn (H. Yan).
https://doi.org/10.1016/j.bioorg.2018.04.005
Received 31 January 2018; Received in revised form 12 April 2018; Accepted 12 April 2018
Availableonline13April2018
0045-2068/©2018ElsevierInc.Allrightsreserved.

W. Sun et al.
BioorganicChemistry78(2018)393–405
Fig. 1. Design of pyrimidine-based derivatives.
into the hydrophobic pocket. Owing to its anti-cancer properties, sev-
eral researches are currently in progress to develop Axitinib as the
potential drug in the treatment of other cancers [24,25].
substituted by hydroxy group of 4a–4b to afford target compounds
9a–9t.
The synthesis of 13a–13n was shown in Scheme 3. The nucleophilic
substitution reactions of 4,6-dichloropyrimidine 10 with various
amines gave intermediates 11a–11d, which then could be reacted with
intermediates 4a–4b to give target compounds. Alternatively, 4,6-di-
chloropyrimidine 10 was reacted with 4a–4b to give intermediates
12a–12b, and subsequent nucleophilic substitution reaction of
12a–12b with various amines afforded target compounds.
Based on the structure-activity relationships (SARs) of above in-
hibitors, a hybrid scaffold of pyrimidine-based derivative 7j, containing
the anilinopyrimidine moiety of Pazopanib, the central aryl ring of
Sorafenib and the ethenylpyridine segment of Axitinib, was designed as
lead compound (Fig. 1). In an attempt to discover potential VEGFR-2
inhibitors, three series of pyrimidine-based derivatives (7, 9 and 13)
bearing the new scaffold were designed and synthesized as VEGFR-2
tyrosine kinase inhibitors. Biological evaluation against A549 and
HepG2 cell lines was carried out using Pazopanib as reference.
2.2. Biological evaluation
2.2.1. In vitro inhibitory activity in cancer cell lines
2. Results and discussions
In vitro inhibitory activity was conducted against A549 and HepG2
cell lines by CCK assay using Pazopanib as control. And both cell lines
were stimulated by VEGF and usually used as the tested cells in the
discovery for VEGFR-2 kinase inhibitors. The SARs of the compounds 7
with the varied substitution at the 2-position of the pyrimidine and the
side chain at the 4-position of the pyrimidine were first discussed
(Table 1). Among the derivatives with the 2-position substituted on the
terminal pyridine (7a–7j: X = N, Y = CH), the inhibitory potencies of
7d (R = 3-Cl-Ph, IC50 = 9.19 and 14.59 μM) and 7j (R = 4-Me-3-
SO2NH2-Ph, IC50 = 12.65 and 22.66 μM) were obviously better than
those of Pazopanib (IC50 = 21.18 and 36.66 μM) on A549 and HepG2
cell lines. Furthermore, benzyl and 3-sulfamoylphenyl derivatives (7e
and 7i) displayed potent inhibitions to A549 cell with IC50 = 19.24 μM
and 17.33 μM, and phenyl and 4-fluorobenzyl derivatives (7a and 7 g)
exhibited good inhibitions to HepG2 cell with IC50 = 29.47 and
31.89 μM. Among the derivatives with the 4-position substituted on the
terminal pyridine (7k-7q: X = CH, Y = N), benzyl derivative (7m) also
displayed better inhibitory potencies on both cell lines than Pazopanib.
Concurrent with SARs studies of 2,4-disubstitued pyrimidine deri-
vatives, we explored the substitution pattern of the pyrimidine
(Table 2). When the positions of the varied substitution and the side
chain were exchanged between the 2-position and 4-position of pyr-
imidine, a slight change in potencies on both cell lines was observed.
Among the derivatives with the 2-position substituted on the terminal
pyridine (9a–9g: X = N, Y = CH), substitution of 4-methoxyanilino in
2.1. Chemistry
To achieve a rapid SARs exploration, three efficient synthetic routes
adopted to obtain the different derivatives (7a-7q, 9–9t and 13a–13n)
were outlined in Schemes 1–3. The target compounds were obtained in
20–56% total yields and their structures were characterized by 1H
NMR, 13C NMR and HRMS.
The synthesis of 7a–7q was depicted in Scheme 1. Intermediates
4a–4b were prepared in 2 steps first by reacting picoline 1a–1b with 4-
hydroxybenzaldehyde 2 in refluxing acetic anhydride to furnish 3a–3b,
then hydrolysis of 3a–3b using potassium hydroxide. The reactions of
4a–4b with 2,4-dichloropyrimidine 5 proceeded via a nucleophilic
substitution reaction where hydroxy group of 4a–4b substituted with
the Cl at position 4 of 2,4-dichloropyrimidine 5 to produce 6a–6b. The
second Cl at position 2 of pyrimidine ring was substituted by amino
group of amine derivatives to yield target compounds 7a–7q.
The synthesis of 9a–9t was illustrated in Scheme 2. 2,4-Di-
chloropyrimidine 5 was chosen as a starting material. Intermediates
8a–8g were obtained by the nucleophilic substitution reaction of 2,4-
dichloropyrimidine 5 with amine derivatives at position 4 of 2,4-di-
chloropyrimidine 5, and intermediates 8h–8j were generated by the
palladium-catalyzed coupling of 2,4-dichloropyrimidine 5 with phe-
nylboronic acid derivatives. The Cl at position 2 of pyrimidine ring was
394

W. Sun et al.
BioorganicChemistry78(2018)393–405
Scheme 1. Synthesis of compounds 7a–7q.
9b (IC50 = 20.12 and 40.81 μM) and 4-chloroanilino in 9c
(IC50 = 26.85 and 35.02 μM) resulted in a significant rise in cellular
potencies on A549 and HepG2 cell lines compared to the counterparts
7b (IC50 > 100 and 100 μM) and 7c (IC50 = 50.55 and 63.71 μM).
Among the derivatives with the 4-position substituted on the terminal
pyridine (9k-9q: X = CH, Y = N), substitution of benzylamino in 9o
(IC50 = 66.18 and > 100 μM) led to a significant reduction in in-
hibitory potencies on both cell lines compared to the counterpart 7m
(IC50 = 17.76 and 31.47 μM). The aryl derivatives, especially with the
4-position substituted on the terminal pyridine (9r–9t: X = CH, Y = N),
led to a significant rise in potencies on both A549 and HepG2 cell lines.
The 4-methylphenyl and 4-chlorophenyl derivatives (9s and 9t) ex-
hibited more potent cellular potencies on A549 and HepG2 cell lines
(9s: IC50 = 9.95 and 18.21 μM; 9 t: IC50 = 15.78 and 21.95 μM), that is
comparable to the IC50 of Pazopanib (IC50 = 21.18 and 36.66 μM).
As an extension of the 2,4-disubstitued pyrimidine derivatives, 4,6-
disubstituted pyrimidine derivatives 13 were also explored (Table 3).
The results showed that substitution of 4-methoxyphenyl in 13b
(X = N, Y = CH) and 4-chlorophenyl in 13c (X = N, Y = CH) exhibited
a significant rise in inhibitory potencies on A549 and HepG2 cell lines
(13b: IC50 = 33.85 and 33.06 μM; 13c: IC50 = 22.36 and 31.21 μM)
compared to counterparts 7b (IC50 > 100 and 100 μM) and 7c
(IC50 = 50.55 and 63.71 μM), and equivalent inhibitions compared to
counterparts 9b (IC50 = 20.12 and 40.81 μM) and 9c (IC50 = 26.85 and
35.02 μM). Phenyl derivative 13 h (IC50 = 29.24 and 21.69 μM) dis-
played decreased potency on A549 cell and a moderate improvement
on HepG2 cell compared to counterparts 7 k (IC50 = 20.09 and
41.57 μM) and 9 k (IC50 = 22.36 and 32.04 μM). This trait is especially
obvious in other compounds. The 4,6-disubstituted pyrimidine deriva-
tives 13d (R = 3-Cl-Ph, X = N, Y = CH) and 13n (R = 4-Me-3-SO2NH2-
Ph, X = CH, Y = N) exhibited better potencies on both cell lines (13d:
IC50 = 16.65 and 21.67 μM; 13n: IC50 = 13.17 and 11.94 μM) than
Pazopanib. The sulfamoyl substituent (13e–13g and 13l–13n) dis-
played good inhibitory potencies, suggesting that sulfamoyl substitu-
tion was preferred in this series of compounds. In addition, most 4,6-
disubstituted pyrimidine derivatives showed more potent inhibitory
potencies on HepG2 cell when compared to the 2,4-disubstitued pyr-
imidine derivatives.
In summary, most derivatives exhibited considerable inhibitory
potencies on A549 and HepG2 cell lines. Among the derivatives with
the 2-position substituted on the terminal pyridine, 3-chloroanilino
derivative 7d displayed excellent inhibitory potencies on A549 and
HepG2 cell lines with IC50 = 9.19 and 14.59 μM. Among the derivatives
with the 4-position substituted on the terminal pyridine, 4-methyl-
phenyl derivative 9s exhibited potent cellular potencies on A549 and
HepG2 cell lines with IC50 = 9.95 and 18.21 μM, and 4-methyl-3-sul-
famoylanilino derivative 13n exhibited significant cellular potencies on
both cell lines with IC50 = 13.17 and 11.94 μM. Moreover, it was ob-
served that most 2,4-disubstitued derivatives 7a–7q and 9a–9t de-
monstrated better activity inhibitions in A549 cell while most 4,6-dis-
ubstitued derivatives 13a–13n exhibited more potent cellular potencies
on HepG2 cell.
2.2.2. Cytotoxicity test
In vitro inhibitory activity of target compounds is sometimes a re-
sult of their cytotoxicity effects, which might cause an erroneous
Scheme 2. Synthesis of compounds 9a–9 t.
395

W. Sun et al.
BioorganicChemistry78(2018)393–405
Scheme 3. Synthesis of compounds 13a–13n.
2.2.3. VEGFR-2 kinase assay
Table 1
The structures and IC50 values of the target compounds 7a–7q.
To assess the selectivity of target compounds, the most active
compounds 7d, 7j, 9s, 9t, 13d and 13n were also selected to further
evaluated for their inhibitory activity against VEGFR-2 kinase with
Pazopanib as positive control. The results were reported as summarized
in Table 4. These compounds displayed good inhibitory activity on
VEGFR-2 kinase with IC50 values ranging from 0.23 to 2.52 μM. Among
them, compound 13n potently inhibited VEGFR-2 at low IC50 values of
0.23 μM. Also, compounds 7d and 9t possessed IC50 values of 0.77 and
0.58 μM, respectively. The result of VEGFR-2 kinase assay was corre-
sponding with that of in vitro inhibitory activity on A549 and HepG2
cell lines, which might indicate some relative selectivity of these
compounds towards VEGFR-2 kinase.
Entry
Substituent
R
IC50 (μM)a
X
Y
A549
HepG2
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
Ph
4-OMe-Ph
4-Cl-Ph
3-Cl-Ph
Bn
4-OMe-Bn
4-F-Bn
4-SO2NH2-Ph
3-SO2NH2-Ph
4-Me-3-SO2NH2-Ph
Ph
4-Cl-Ph
Bn
4-F-Bn
4-SO2NH2-Ph
3-SO2NH2-Ph
4-Me-3-SO2NH2-Ph
–
N
N
N
N
N
N
N
N
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
N
N
N
N
N
N
–
19.32
> 100
50.55
9.19
29.47
> 100
63.71
14.59
35.84
40.02
31.89
38.71
37.73
22.66
41.57
42.13
31.47
> 100
81.54
44.37
38.55
36.66
2.3. Molecular docking study
19.24
25.15
20.53
30.73
17.33
12.65
20.09
27.35
17.76
39.33
27.49
24.48
22.71
21.18
In order to investigate the binding mode and evaluate the stability
of the docked poses, molecular docking study was performed by
AutoDock 4.2. [26] The VEGFR-2 protein (PDB ID: 4ASD) was obtained
from the Protein Data Bank. [27] Compound 7j was chosen as a re-
presentative compound and docked into the active site of VEGFR-2 to
identify the key interactions between this molecule and VEGFR-2. The
binding mode of 7j was consistent to our hypothesis: the three segments
of 7j interacted with the residues in a similar way to the known in-
hibitors (Fig. 2). The anilinopyrimidine moiety of 7j occupies the
adenine pocket in accordance with Pazopanib. It presents some hy-
drogen bonds to the key amino acid of the hinge region: the pyrimidine
N-2 and the anilino N-H form two hydrogen bonds with Asp1046 and
Glu885, respectively. The sulfamoyl N-H forms two hydrogen bonds
with Ile1044 in the hinge region. The O atom between pyrimidine core
and the central aryl ring provides an unexpected hydrogen bond in-
teraction with Lys868 and Van der Waals contact with Phe1047 com-
pared to Sorafenib. The central aryl ring of 7j is located in the linker
region and promotes hydrophobic interactions with Cys1045, Val848,
Ala866 and Leu1035, while Ala866 and Leu1035 involve some
N
N
CH
CH
CH
CH
CH
CH
CH
–
7q
Pazopanib
a
IC50 values are the mean of three separate experiments.
conclusion. The most active compounds 7d, 7j, 9s, 9t, 13d and 13n
were selected to evaluate their cytotoxicity. As summarized in Table 4,
they were tested in vitro for their cytotoxicity against human macro-
phage. The results showed that these compounds had quite a low cy-
totoxicity activity.
396

W. Sun et al.
BioorganicChemistry78(2018)393–405
Table 3
The structures and IC50 values of the target compounds 13a–13n.
Table 2
The structures and IC50 values of the target compounds 9a–9t.
Entry
Substituent
R
IC50 (μM)a
Entry
Substituent
R
IC50 (μM)a
X
Y
A549
HepG2
X
Y
A549
HepG2
9a
9b
9c
9d
N
N
N
N
CH
CH
CH
CH
21.68
20.12
26.85
22.73
35.40
40.81
35.02
39.21
13a
13b
13c
13d
13e
13f
13g
13h
13i
13j
13k
13l
Ph
4-OMe-Ph
4-Cl-Ph
N
N
N
N
N
N
N
CH
CH
CH
CH
CH
CH
CH
–
CH
CH
CH
CH
CH
CH
CH
N
N
N
N
N
39.71
33.85
22.36
16.65
24.30
28.95
20.10
29.24
59.03
66.67
23.58
27.70
23.33
13.17
21.18
23.25
33.06
31.21
21.67
24.32
29.01
23.19
21.69
45.38
52.05
32.83
33.84
24.34
11.94
36.66
3-Cl-Ph
4-SO2NH2-Ph
3-SO2NH2-Ph
4-Me-3-SO2NH2-Ph
Ph
4-OMe-Ph
4-Cl-Ph
9e
9f
N
N
N
CH
CH
CH
35.64
27.87
21.45
49.55
38.58
68.81
3-Cl-Ph
4-SO2NH2-Ph
3-SO2NH2-Ph
4-Me-3-SO2NH2-Ph
–
9g
13m
13n
Pazopanib
N
N
–
9h
9i
N
CH
CH
CH
N
25.82
16.39
18.06
22.36
29.96
23.59
20.33
37.42
26.09
32.68
32.04
50.44
43.36
30.90
N
a
IC50 values are the mean of three separate experiments.
9j
N
9k
9l
CH
CH
CH
CH
as shown in Fig. 4. The pyrimidine moiety occupies the adenine pocket,
also the central aryl ring and the ethenylpyridine segment interact with
the hydrophobic pocket. The O atom and the pyrimidine N-2 in both 7d
and 9s are engaged in two hydrogen bonds with Asp1046 and Lys868
while the other moieties are involved in Van der Waals contacts and
hydrophobic interactions with residues, which are considered a sig-
nificant opportunity for better binding capacities. Compound 13n
shows a nice fit in the binding sites and favorable binding modes with
VEGFR-2. The better binding capacity may be attributed to the direct
hydrogen bonds with Ile1044 and Asp1046. Backbone carbonyl oxygen
of Ile1044 makes two hydrogen bonds with the sulfamoyl N-H, and
another hydrogen bond is formed between the anilino N-H with
Asp1046. Moreover, the O atom and the terminal pyridine show Van
der Waals contacts with residues, and the other moieties promote hy-
drophobic interactions.
N
9m
9n
N
N
9o
9p
9q
CH
CH
CH
N
N
N
66.18
> 100
49.09
> 100
54.28
74.47
9r
CH
CH
CH
–
N
N
N
-
16.86
9.95
27.05
18.21
21.95
36.66
9s
9t
15.78
21.18
Pazopanib
–
3. Conclusions
a
IC50 values are the mean of three separate experiments.
In conclusion, a series of pyrimidine-based derivatives were de-
signed and synthesized as novel VEGFR-2 inhibitors. The derivatives
were obtained in good yields, and their structures were characterized
by 1H NMR, 13C NMR and HRMS. Biological evaluation revealed that
most derivatives exhibited better inhibitory potencies on A549 and
HepG2 cell lines compared to Pazopanib. Among these derivatives, 7d
(IC50 = 9.19 and 14.59 μM), 9s (IC50 = 9.95 and 18.21 μM) and 13n
(IC50 = 13.17 and 11.94 μM) displayed excellent cellular potencies on
both cell lines compared to Pazopanib. Molecular docking study was
performed to further analyze the SARs of the pyrimidine-based deri-
vatives. The docking results shown that 7d (ΔG = −10.54 kcal/mol,
Ki = 0.018 μM), 9s (ΔG = −10.60 kcal/mol, Ki = 0.016 μM) and 13n
(ΔG = −10.37 kcal/mol, Ki = 0.025 μM) displayed better binding ca-
pacities compared to Pazopanib, consistent with their excellent cellular
potencies. Furthermore, 7d, 9s and 13n had comparable docking
modes to that of lead compound 7j. The O atom and the pyrimidine N-2
in both 7d and 9s formed two hydrogen bonds with Asp1046 and
Lys868. The sulfamoyl N-H and the anilino N-H in 13n made the direct
hydrogen bonds with Ile1044 and Asp1046. And the other moieties of
these derivatives were involved in Van der Waals contacts and hydro-
phobic interactions with residues. The above results shown that these
interactions with the terminal pyridine ring. The ethenylpyridine seg-
ment of 7j binds the similar active site of VEGFR-2 to Axitinib, and the
pyridine ring makes contacts with Cys919, Phe918 and Leu840, re-
spectively. The comparison of 2D diagrams between 7j and the chosen
segments of known inhibitors for docking poses are presented in Fig. 3.
The stability of the docked poses is reported as the binding free energy
(ΔG) and inhibition constant (Ki). The docking results indicate that 7j
exhibits a better binding capacity with ΔG=-10.38 kcal/mol and
Ki = 0.024 μM, comparable to that of Pazopanib (ΔG = −10.28 kcal/
mol, Ki = 0.029 μM).
To further analyze the SARs of the pyrimidine-based derivatives, the
docking study of other target compounds with VEGFR-2 was in-
vestigated. Among them, 7d (ΔG = −10.54 kcal/mol, Ki = 0.018 μM),
9s
(ΔG = −10.60 kcal/mol,
Ki = 0.016 μM)
and
13n
(ΔG = −10.37 kcal/mol, Ki = 0.025 μM) exhibited significantly better
binding capacities compared to Pazopanib (ΔG = −10.28 kcal/mol,
Ki = 0.029 μM), which were consistent with their excellent inhibitory
potencies on A549 and HepG2 cell lines. A close look of their binding
modes provided some useful information. In general, the binding modes
of 7d, 9s and 13n with VEGFR-2 were similar with that of compound 7j
397

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BioorganicChemistry78(2018)393–405
Table 4
aqueous solution (80 mL) and distilled water (50 mL × 2), respectively.
The separated organic layer was dried over anhydrous Na2SO4, filtered
and concentrated by a rotary evaporator. The crude product was dis-
solved in hot EtOH and crystallized to give 3a as a white solid (75%):
mp 109.5–110.4 °C; 1H NMR (400 MHz, CDCl3) δ 8.62 (d, J = 4.5 Hz,
1H), 7.69–7.59 (m, 4H), 7.38 (d, J = 7.4 Hz, 1H), 7.18–7.11 (m, 4H),
2.32 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 169.36, 155.42, 150.60,
149.66, 136.57, 134.45, 131.67, 128.11, 128.07, 122.18, 122.13,
121.87, 21.15; HRMS (ESI) m/z: calcd for C15H14NO2 [M + H]+
240.1019, found 240.1022.
The cytotoxicity on macrophage and VEGFR-2 inhibitory activity.
Entry
Macrophage CC50 (μM)a
VEGFR-2 IC50 (μM)b
7d
7j
9s
9t
228.12
347.20
267.56
231.42
298.56
306.13
256.78
0.77
1.38
0.81
0.58
2.52
0.23
1.04
13d
13n
Pazopanib
4-(2-(4-Pyridinyl)ethenyl)phenyl acetate (3b): The compound 3b
was prepared from 4-picoline 1b and 4-hydroxybenzaldehyde 2 in a
manner similar to that described for 3a to yield a yellow solid (72%):
a
CC50 values are the mean of three separate experiments.
IC50 values are the mean of three separate experiments.
b
mp 160.8–161.5 °C; 1H NMR (400 MHz, DMSO-d6)
δ 8.55 (d,
pyrimidine-based derivatives had potential to be the novel VEGFR-2
inhibitors.
J = 5.2 Hz, 2H), 7.70 (d, J = 8.5 Hz, 2H), 7.58–7.53 (m, 3H), 7.27–7.15
(m, 3H), 2.28 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ 169.57, 151.08,
150.49, 144.63, 134.31, 132.49, 128.56, 126.54, 122.72, 121.31,
21.33; HRMS (ESI) m/z: calcd for C15H14NO2 [M + H]+ 240.1019,
found 240.1020.
4. Experimental section
4-(2-(2-Pyridinyl)ethenyl)phenol (4a): To
a solution of 3a
4.1. Chemistry
(11.95 g, 50 mmol) in ethanol (80 mL) was added a 2 M KOH aqueous
solution (12 mL). The mixture was refluxed for 12 h. To the solution
was added 1 M HCl solution to acidify to pH 5–6. The precipitate was
filtered off and recrystallized from ethanol to give 4a as a white solid
(92%): mp 217.0–217.7 °C; 1H NMR (400 MHz, DMSO-d6) δ 9.73 (s,
1H), 8.53 (d, J = 4.5 Hz, 1H), 7.76–7.71 (m, 1H), 7.58 (d, J = 16.1 Hz,
1H), 7.50–7.46 (m, 3H), 7.21–7.17 (m, 1H), 7.08 (d, J = 16.1 Hz, 1H),
6.80 (d, J = 8.3 Hz, 2H); 13C NMR (100 MHz, DMSO-d6) δ 158.41,
155.95, 149.81, 137.13, 132.56, 129.04, 127.86, 125.19, 122.25,
122.19, 116.10; HRMS (ESI) m/z: calcd for C13H12NO [M + H]+
198.0913, found 198.0815.
All the materials we used were purchased from commercial sup-
pliers and used without further purification unless otherwise specified.
All of the solvents were reagent grade and flash chromatography was
performed using silica gel (200–300 mesh). Reactions were monitored
by thin layer chromatography on 0.25 mm silica gel plates (60GF-254).
The melting points were determined on an electrothermal melting point
apparatus and uncorrected. NMR spectra were recorded on a Bruker
ARX-400 MHz spectrometer for 1H NMR and 13C NMR. The chemical
shifts were reported as ppm down field using tetramethylsilane (TMS)
as an internal standard. High-resolution mass spectra (HRMS) were
determined on an Agilent G3250AA LC/MSD TOF mass spectrometer
and reported as m/z (relative intensity). The single crystal X-ray dif-
fraction was determined on a Rigaku RAXIS RAPID IP CCD area-de-
tector diffractometer.
4-(2-(2-Pyridinyl)ethenyl)phenyl acetate (3a): A mixture of 2-
picoline 1a (9.31 g, 100 mmol), 4-hydroxybenzaldehyde 2 (12.21 g,
100 mmol), acetic anhydride (20.42 g, 200 mmol) and acetic acid
(1.20 g, 20 mmol) in a round bottomed flask equipped with an anhy-
drous CaCl2 drying tube was heated under reflux for 24 h. Volatile
solvent was distilled out and the reaction residue was cooled. The re-
sidue was dissolved in CH2Cl2 (80 mL), washed with 20% NaHCO3
4-(2-(4-Pyridinyl)ethenyl)phenol (4b): The compound 4b was
prepared from 3b in a manner similar to that described for 4a to yield a
yellow solid (94%): mp 263.5–264.6 °C; 1H NMR (400 MHz, DMSO-d6)
δ 9.78 (s, 1H), 8.50 (d, J = 5.1 Hz, 2H), 7.50–7.48 (m, 4H), 7.43 (d,
J = 16.4 Hz, 1H), 7.00 (d, J = 16.4 Hz, 1H), 6.81 (d, J = 8.3 Hz, 2H);
13C NMR (100 MHz, DMSO-d6) δ 158.68, 150.34, 145.26, 133.51,
129.12, 127.65, 122.91, 120.94, 116.14; HRMS (ESI) m/z: calcd for
C
13H12NO [M + H]+ 198.0913, found 198.0818.
2-Chloro-4-(4-(2-(2-pyridinyl)ethenyl)phenoxy)pyrimidine
(6a): Compound 4a (1.97 g, 10 mmol) was dissolved in a solution of
sodium hydroxide (0.4 g, 10 mmol) in water (10 mL) and the mixture
Fig. 2. Binding modes of 7j in the active site of VEGFR-2.
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BioorganicChemistry78(2018)393–405
Fig. 3. 2D interactions diagram for docked compounds: (A) The interactions between anilinopyrimidine moiety of Pazopanib and VEGFR-2. (B) The interactions
between the central aryl ring of Sorafenib and VEGFR-2. (C) The interactions between ethenylpyridine segment of Axitinib and residues of VEGFR-2. (D) The
interactions between 7j and VEGFR-2.
Fig. 4. (A) Binding sites of 7d (magenta), 9s (blue) and 13n (purple) with VEGFR-2 compared to 7j (yellow). (B) Binding modes of 7d with VEGFR-2. (C) Binding
modes of 9s with VEGFR-2. (D) Binding modes of 13n with VEGFR-2. (For interpretation of the references to colour in this figure legend, the reader is referred to the
web version of this article.)
399

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BioorganicChemistry78(2018)393–405
was maintained at ice-bath temperature. To this was added, dropwise, a
solution of 2,4-dichloroprymidine 5 (1.49 g, 10 mmol) in acetone
(40 mL). The stirred mixture was refluxed for 5 h. The precipitate was
filtered off and recrystallized from ethanol to give 6a as a white solid
(77%): mp 157.5–158.3 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.64 (d,
J = 5.7 Hz, 1H), 8.59 (d, J = 3.9 Hz, 1H), 7.82–7.71 (m, 4H), 7.55 (d,
J = 7.8 Hz, 1H), 7.36–7.26 (m, 4H), 7.20 (d, J = 5.7 Hz, 1H); 13C NMR
122.85, 122.67, 121.37, 118.21, 117.47, 99.47; HRMS (ESI) m/z: calcd
for C23H18ClN4O2 [M + H]+ 401.1164, found 401.1168.
N-Benzyl-4-(4-(2-(2-pyridinyl)ethenyl)phenoxy)pyrimidin-2-
amine (7e): Yield 59%, white solid: mp 148.0–148.9 °C; 1H NMR
(400 MHz, DMSO-d6) δ: 8.58 (d, J = 4.1 Hz, 1H), 8.17 (d, J = 5.5 Hz,
1H), 7.81–7.70 (m, 5H), 7.55 (d, J = 7.8 Hz, 1H), 7.33–7.18 (m, 9H),
6.18 (s, 1H), 4.32 (d, J = 103.1 Hz, 2H); 13C NMR (100 MHz, DMSO-d6)
δ: 169.72, 162.71, 160.31, 155.41, 152.81, 149.94, 140.58, 137.28,
133.99, 131.62, 128.70, 128.51, 128.40, 127.73, 126.94, 122.88,
(100 MHz, DMSO-d6)
δ 170.47, 161.99, 159.44, 155.29, 151.93,
149.97, 137.32, 135.00, 131.35, 129.07, 129.00, 122.97, 122.95,
122.33, 108.16; HRMS (ESI) m/z: calcd for C17H13ClN3O [M + H]+
310.0742, found 310.0744.
122.83, 122.64, 44.47; HRMS (ESI) m/z: calcd for
C24H21N4O
[M + H]+ 381.1710, found 381.1715.
2-Chloro-4-(4-(2-(4-pyridinyl)ethenyl)phenoxy)pyrimidine
(6b): The compound 6b was prepared from 4b and 2,4-di-
chloroprymidine 5 in a manner similar to that described for 6a to yield
a yellow solid (70%): mp 162.3–163.2 °C; 1H NMR (400 MHz, DMSO-
d6) δ 8.64 (d, J = 5.7 Hz, 1H), 8.56 (d, J = 5.8 Hz, 2H), 7.77 (d,
J = 8.5 Hz, 2H), 7.61–7.56 (m, 3H), 7.34–7.25 (m, 3H), 7.20 (d,
J = 5.7 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ 170.43, 162.00,
159.43, 152.14, 150.50, 144.61, 134.76, 132.37, 129.10, 126.88,
122.39, 121.34, 108.18; HRMS (ESI) m/z: calcd for C17H13ClN3O
[M + H]+ 310.0742, found 310.0740.
N-(4-Methoxybenzyl)-4-(4-(2-(2-pyridinyl)ethenyl)phenoxy)
pyrimidin-2-amine (7f): Yield 58%, yellow solid: mp 144.8–145.5 °C;
1H NMR (400 MHz, DMSO-d6) δ: 8.58 (d, J = 4.0 Hz, 1H), 8.16 (d,
J = 5.5 Hz, 1H), 7.80–7.73 (m, 5H), 7.54 (d, J = 7.8 Hz, 1H), 7.34–7.19
(m, 5H), 6.95–6.80 (m, 3H), 6.17 (d, J = 5.5 Hz, 1H), 4.24 (d,
J = 110.0 Hz, 2H), 3.70 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ:
169.73, 162.67, 160.41, 158.46, 155.41, 152.86, 149.93, 137.26,
134.00, 132.51, 131.65, 129.17, 128.70, 128.40, 122.85, 122.81,
122.67, 113.89, 55.43, 43.91; HRMS (ESI) m/z: calcd for C25H23N4O2
[M + H]+ 411.1816, found 411.1817.
N-Phenyl-4-(4-(2-(2-pyridinyl)ethenyl)phenoxy)pyrimidin-2-
amine (7a): A mixture of 6a (309 mg, 1 mmol) and aniline (93 mg,
1 mmol) in DMF (5 mL) was heated in a microwave reactor at 130 °C
and 100 W for 30 min. The reaction mixture was diluted with ethyl
acetate (30 ml). The organic layer was washed with brine (25 mL × 3),
dried over anhydrous Na2SO4, filtered and concentrated. The residue
was purified by chromatography (ethyl acetate/petroleum ether) on
silica gel to give 7a as a white solid (70%): mp 170.3–171.1 °C; 1H NMR
(400 MHz, DMSO-d6) δ: 9.59 (s, 1H), 8.60 (d, J = 4.1 Hz, 1H), 8.37 (d,
J = 5.6 Hz, 1H), 7.82–7.74 (m, 4H), 7.57 (d, J = 7.8 Hz, 1H), 7.52 (d,
J = 8.0 Hz, 2H), 7.37–7.25 (m, 4H), 7.08 (t, J = 7.7 Hz, 2H), 6.84 (t,
J = 7.3 Hz, 1H), 6.48 (d, J = 5.6 Hz, 1H); 13C NMR (100 MHz, DMSO-
d6) δ: 169.81, 160.49, 160.09, 155.40, 152.93, 149.96, 140.56, 137.31,
134.33, 131.61, 128.84, 128.63, 128.50, 122.94, 122.91, 122.88,
N-(4-Fluorobenzyl)-4-(4-(2-(2-pyridinyl)ethenyl)phenoxy)pyr-
imidin-2-amine (7g): Yield 56%, yellow solid: mp 140.2–140.8 °C; 1H
NMR (400 MHz, DMSO-d6) δ: 8.58 (d, J = 4.1 Hz, 1H), 8.17 (d,
J = 5.3 Hz, 1H), 7.80–7.71 (m, 5H), 7.55 (d, J = 7.7 Hz, 1H), 7.34–7.05
(m, 8H), 6.19 (d, J = 5.3 Hz, 1H), 4.27 (d, J = 114.1 Hz, 2H); 13C NMR
(100 MHz, DMSO-d6) δ: 169.75, 162.67, 162.61, 160.40, 160.27,
155.41, 152.81, 149.93, 137.27, 136.70, 134.04, 131.63, 129.76,
128.69, 128.43, 122.86, 122.82, 122.68, 115.27, 115.06, 43.80; HRMS
(ESI) m/z: calcd for C24H20FN4O [M + H]+ 399.1616, found 399.1618.
4-(4-(2-(2-Pyridinyl)ethenyl)phenoxy)-N-(4-sulfamoylphenyl)
pyrimidin-2-amine (7h): To a solution of 6a (309 mg, 1 mmol) and 4-
sulfamoylaniline (172 mg, 1 mmol) in isopropanol (10 mL) was added 5
drops of hydrochloric acid. The mixture was heated to reflux for 4 h and
then concentrated in vacuo. The residue was diluted with saturated
sodium bicarbonate solution (25 ml) and extracted with ethyl acetate
(30 mL). The extract was washed with brine (25 mL), dried over an-
hydrous Na2SO4, filtered and concentrated. The precipitate was filtered
off and recrystallized from ethyl acetate to give 7h as a white solid
(64%): mp > 250 °C; 1H NMR (400 MHz, DMSO-d6) δ: 10.00 (s, 1H),
8.59 (d, J = 4.1 Hz, 1H), 8.45 (d, J = 5.5 Hz, 1H), 7.82–7.70 (m, 6H),
7.56 (d, J = 8.3 Hz, 3H), 7.37–7.26 (m, 4H), 7.14 (s, 2H), 6.68 (d,
J = 5.5 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ: 169.81, 160.57,
159.73, 155.38, 152.79, 149.94, 143.71, 137.31, 136.68, 134.50,
131.55, 128.88, 128.72, 126.76, 122.98, 122.89, 122.79, 118.28,
99.94; HRMS (ESI) m/z: calcd for C23H20N5O3S [M + H]+ 446.1281,
found 446.1284.
121.83, 119.24, 98.71; HRMS (ESI) m/z: calcd for
C23H19N4O
[M + H]+ 367.1553, found 367.1555.
The following compounds 7b–7g were prepared from 6a using a
procedure similar to that described for 7a.
N-(4-Methoxyphenyl)-4-(4-(2-(2-pyridinyl)ethenyl)phenoxy)
pyrimidin-2-amine (7b): Yield 61%, purple solid: mp 206.4–207.3 °C;
1H NMR (400 MHz, DMSO-d6) δ: 9.40 (s, 1H), 8.59 (d, J = 4.1 Hz, 1H),
8.32 (d, J = 5.5 Hz, 1H), 7.82–7.74 (m, 4H), 7.56 (d, J = 7.8 Hz, 1H),
7.39–7.26 (m, 6H), 6.66 (d, J = 8.1 Hz, 2H), 6.41 (d, J = 5.5 Hz, 1H),
3.60 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ: 169.84, 160.46, 160.19,
155.42, 154.53, 153.01, 151.26, 149.96, 137.30, 134.31, 133.70,
131.65, 128.84, 128.53, 122.94, 122.86, 120.95, 113.82, 98.05, 55.43;
HRMS (ESI) m/z: calcd for C24H21N4O2 [M + H]+ 397.1659, found
397.1662.
The following compounds 7i and 7j were prepared from 6a using a
procedure similar to that described for 7h.
N-(4-Chlorophenyl)-4-(4-(2-(2-pyridinyl)ethenyl)phenoxy)pyr-
imidin-2-amine (7c): Yield 69%, white solid: mp 213.1–213.8 °C; 1H
NMR (400 MHz, DMSO-d6) δ: 9.75 (s, 1H), 8.59 (d, J = 4.2 Hz, 1H),
8.39 (d, J = 5.6 Hz, 1H), 7.81–7.75 (m, 4H), 7.57–7.55 (m, 3H),
7.37–7.25 (m, 4H), 7.12 (d, J = 8.6 Hz, 2H), 6.50 (d, J = 5.6 Hz, 1H);
13C NMR (100 MHz, DMSO-d6) δ: 169.84, 160.51, 159.84, 155.39,
152.87, 149.96, 139.61, 137.30, 134.43, 131.57, 128.88, 128.63,
128.45, 125.28, 122.93, 122.89, 120.64, 99.15; HRMS (ESI) m/z: calcd
for C23H18ClN4O2 [M + H]+ 401.1164, found 401.1166.
N-(3-Chlorophenyl)-4-(4-(2-(2-pyridinyl)ethenyl)phenoxy)pyr-
imidin-2-amine (7d): Yield 69%, white solid: mp 176.3–177.0 °C; 1H
NMR (400 MHz, DMSO-d6) δ: 9.82 (s, 1H), 8.59 (d, J = 4.2 Hz, 1H),
8.41 (d, J = 5.6 Hz, 1H), 7.81–7.71 (m, 5H), 7.55 (d, J = 7.8 Hz, 1H),
7.39 (d, J = 8.1 Hz, 1H), 7.33–7.25 (m, 4H), 7.10 (t, J = 8.1 Hz, 1H),
6.86 (d, J = 7.8 Hz, 1H), 6.55 (d, J = 5.6 Hz, 1H); 13C NMR (100 MHz,
DMSO-d6) δ: 169.82, 160.52, 159.79, 155.41, 152.73, 149.95, 142.09,
137.29, 134.48, 133.47, 131.70, 130.16, 129.02, 128.49, 122.86,
4-(4-(2-(2-Pyridinyl)ethenyl)phenoxy)-N-(3-sulfamoylphenyl)
pyrimidin-2-amine (7i): Yield 61%, yellow solid: mp 213.5–214.2 °C;
1H NMR (400 MHz, DMSO-d6) δ: 9.89 (s, 1H), 8.59 (d, J = 4.1 Hz, 1H),
8.41 (d, J = 5.6 Hz, 1H), 8.11 (s, 1H), 7.82–7.72 (m, 5H), 7.56 (d,
J = 7.8 Hz, 1H), 7.37–7.24 (m, 8H), 6.51 (d, J = 5.7 Hz, 1H); 13C NMR
(100 MHz, DMSO-d6) δ: 169.81, 160.49, 159.97, 155.39, 152.81,
149.95, 144.81, 141.01, 137.31, 134.40, 131.60, 129.25, 128.93,
128.61, 122.91, 122.88, 122.71, 122.19, 119.01, 116.41, 99.64; HRMS
(ESI) m/z: calcd for
446.1286.
C
23H20N5O3S [M + H]+ 446.1281, found
N-(4-Methyl-3-sulfamoylphenyl)-4-(4-(2-(2-pyridinyl)ethenyl)
phenoxy)pyrimidin-2-amine (7j): Yield 78%, white solid: mp
214.2–215.1 °C; 1H NMR (400 MHz, DMSO-d6) δ: 9.76 (s, 1H), 8.60 (d,
J = 3.9 Hz, 1H), 8.38 (d, J = 5.6 Hz, 1H), 8.15 (s, 1H), 7.82–7.70 (m,
5H), 7.57 (d, J = 7.8 Hz, 1H), 7.36–7.24 (m, 6H), 7.05 (d, J = 8.4 Hz,
1H), 6.47 (d, J = 5.6 Hz, 1H), 2.46 (s, 3H); 13C NMR (100 MHz, DMSO-
d6) δ: 169.78, 160.45, 160.09, 155.42, 152.85, 149.96, 142.34, 138.59,
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BioorganicChemistry78(2018)393–405
137.29, 134.38, 132.41, 131.61, 128.90, 128.64, 122.87, 122.71,
122.41, 118.59, 99.22, 19.56; HRMS (ESI) m/z: calcd for C24H22N5O3S
[M + H]+ 460.1438, found 460.1439.
The following compounds 7k–7n were prepared from 6b using a
procedure similar to that described for 7a.
(m, 5H), 7.03 (d, J = 8.2 Hz, 1H), 6.47 (d, J = 5.6 Hz, 1H), 2.45 (s, 3H);
13C NMR (100 MHz, DMSO-d6) δ: 169.76, 160.50, 160.07, 153.09,
150.49, 144.74, 142.33, 138.56, 134.16, 132.63, 132.41, 128.95,
128.64, 126.50, 122.76, 122.41, 121.33, 118.59, 99.22, 19.56; HRMS
(ESI) m/z: calcd for
460.1442.
C
24H22N5O3S [M + H]+ 460.1438, found
N-Phenyl-4-(4-(2-(4-pyridinyl)ethenyl)phenoxy)pyrimidin-2-
amine (7k): Yield 60%, yellow solid: mp > 250 °C; 1H NMR (400 MHz,
DMSO-d6) δ: 9.58 (s, 1H), 8.56 (d, J = 4.4 Hz, 2H), 8.37 (d, J = 5.5 Hz,
1H), 7.78 (d, J = 8.4 Hz, 2H), 7.61–7.59 (m, 3H), 7.50 (d, J = 7.6 Hz,
2H), 7.31–7.26 (m, 3H), 7.07 (t, J = 7.7 Hz, 2H), 6.84 (t, J = 7.1 Hz,
1H), 6.47 (d, J = 5.6 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ: 169.77,
160.52, 160.10, 153.17, 150.47, 144.74, 134.11, 132.63, 131.95,
129.17, 128.87, 128.63, 126.38, 122.91, 121.35, 119.25, 98.72; HRMS
(ESI) m/z: calcd for C23H19N4O [M + H]+ 367.1553, found 367.1557.
N-(4-Chlorophenyl)-4-(4-(2-(4-pyridinyl)ethenyl)phenoxy)pyr-
imidin-2-amine (7l): Yield 59%, yellow solid: mp 236.1–237.0 °C; 1H
NMR (400 MHz, DMSO-d6) δ: 9.74 (s, 1H), 8.56 (d, J = 5.7 Hz, 2H),
8.39 (d, J = 5.6 Hz, 1H), 7.78 (d, J = 8.6 Hz, 2H), 7.65–7.53 (m, 6H),
7.38–7.27 (m, 4H), 6.51 (d, J = 5.6 Hz, 1H); 13C NMR (100 MHz,
DMSO-d6) δ: 169.81, 160.54, 159.84, 153.12, 150.49, 144.72, 139.59,
134.22, 132.59, 129.21, 128.92, 128.44, 126.53, 122.93, 121.19,
120.65, 99.15; HRMS (ESI) m/z: calcd for C23H18ClN4O [M + H]+
401.1164, found 401.1166.
N-Benzyl-4-(4-(2-(4-pyridinyl)ethenyl)phenoxy)pyrimidin-2-
amine (7m): Yield 67%, white solid: mp 197.1–197.8 °C; 1H NMR
(400 MHz, DMSO-d6) δ: 8.55 (d, J = 5.2 Hz, 2H), 8.17 (d, J = 5.5 Hz,
1H), 7.91–7.71 (m, 3H), 7.61–7.56 (m, 3H), 7.27–7.02 (m, 8H), 6.18 (s,
1H), 4.30 (d, J = 112.2 Hz, 2H); 13C NMR (100 MHz, DMSO-d6) δ:
169.67, 162.71, 160.49, 153.05, 150.47, 144.73, 140.55, 133.77,
132.64, 128.73, 128.50, 127.82, 126.95, 126.27, 122.65, 121.30,
44.48; HRMS (ESI) m/z: calcd for C24H21N4O [M + H]+ 381.1710,
found 381.1712.
N-(4-Fluorobenzyl)-4-(4-(2-(4-pyridinyl)ethenyl)phenoxy)pyr-
imidin-2-amine (7n): Yield 70%, yellow solid: mp 201.9–202.5 °C; 1H
NMR (400 MHz, DMSO-d6) δ: 8.55 (d, J = 5.7 Hz, 2H), 8.17 (d,
J = 5.5 Hz, 1H), 7.87–7.72 (m, 3H), 7.61–7.56 (m, 3H), 7.27–7.04 (m,
7H), 6.19 (d, J = 5.5 Hz, 1H), 4.27 (d, J = 113.7 Hz, 2H); 13C NMR
(100 MHz, DMSO-d6) δ: 169.71, 162.67, 162.60, 160.26, 153.03,
150.47, 144.72, 136.71, 133.82, 132.63, 130.79, 129.88, 129.77,
128.73, 126.28, 122.72, 121.30, 115.28, 115.07, 43.79; HRMS (ESI) m/
z: calcd for C24H20FN4O [M + H]+ 399.1616, found 399.1619.
The following compounds 7o–7q were prepared from 6b using a
procedure similar to that described for 7h.
2-Chloro-N-phenylpyrimidin-4-amine (8a): To a solution of 2,4-
dichloroprymidine 5 (1.49 g, 10 mmol) and triethylamine (1.52 g,
15 mmol) in ethanol (50 mL), aniline (0.93 g, 10 mmol) was added. The
reaction mixture was stirred overnight at room temperature. After
completion of the reaction, excess ethanol was evaporated. The crude
product was recrystallized from ethanol to give 8a as a white solid
(80%): mp 185.7–186.4 °C; 1H NMR (400 MHz, DMSO-d6) δ: 10.05 (s,
1H), 8.11 (d, J = 5.9 Hz, 1H), 7.55 (d, J = 7.9 Hz, 2H), 7.35–7.30 (m,
2H), 7.07–7.03 (m, 1H), 6.74 (d, J = 5.9 Hz, 1H).
The following compounds 8b–8g were prepared from 2,4-di-
chloroprymidine 5 using a procedure similar to that described for 8a.
2-Chloro-N-(4-methoxyphenyl)pyrimidin-4-amine (8b): Yield
77%, purple solid: mp 178.8–179.5 °C; 1H NMR (400 MHz, CDCl3) δ:
8.05 (d, J = 5.9 Hz, 1H), 7.28 (s, 1H), 7.21 (d, J = 8.8 Hz, 2H), 6.94 (d,
J = 8.8 Hz, 2H), 6.40 (d, J = 5.9 Hz, 1H), 3.83 (s, 3H).
2-Chloro-N-(4-chlorophenyl)pyrimidin-4-amine (8c): Yield 72%,
white solid: mp 198.9–199.6 °C; 1H NMR (400 MHz, CDCl3) δ: 8.15 (d,
J = 5.9 Hz, 1H), 7.38 (d, J = 8.1 Hz, 2H), 7.29 (d, J = 8.1 Hz, 2H), 6.97
(s, 1H), 6.55 (d, J = 5.9 Hz, 1H).
2-Chloro-N-(3-chlorophenyl)pyrimidin-4-amine (8d): Yield
70%, white solid: mp 168.4–169.9 °C; 1H NMR (400 MHz, DMSO-d6) δ:
10.17 (s, 1H), 8.21 (d, J = 5.8 Hz, 1H), 7.81 (s, 1H), 7.50 (d,
J = 8.2 Hz, 1H), 7.38 (t, J = 8.1 Hz, 1H), 7.13 (d, J = 7.9 Hz, 1H), 6.79
(d, J = 5.9 Hz, 1H).
N-Benzyl-2-chloropyrimidin-4-amine (8e): Yield 76%, white
solid: mp 130.7–131.3 °C; 1H NMR (400 MHz, DMSO-d6) δ: 8.38 (s, 1H),
7.93 (d, J = 5.9 Hz, 1H), 7.37–7.26 (m, 5H), 6.53 (d, J = 5.9 Hz, 1H),
4.51 (d, J = 4.7 Hz, 2H).
2-Chloro-N-(4-methoxybenzyl)pyrimidin-4-amine (8f): Yield
81%, yellow solid: mp 89.7–90.3 °C; 1H NMR (400 MHz, CDCl3) δ: 8.01
(d, J = 5.9 Hz, 1H), 7.25 (d, J = 8.3 Hz, 2H), 6.90 (d, J = 8.3 Hz, 2H),
6.24 (d, J = 5.9 Hz, 1H), 4.48 (s, 2H), 3.81 (s, 3H).
2-Chloro-N-(4-fluorobenzyl)pyrimidin-4-amine (8g): Yield 72%,
yellow solid: mp 150.9–151.6 °C; 1H NMR (400 MHz, CDCl3) δ: 7.99 (d,
J = 5.1 Hz, 1H), 7.32–7.28 (m, 2H), 7.06–7.02 (m, 2H), 6.27 (d,
J = 5.1 Hz, 1H), 4.69 (s, 1H), 4.54 (d, J = 4.1 Hz, 2H).
2-Chloro-4-phenylpyrimidine (8h):
A
mixture of 2,4-di-
4-(4-(2-(4-Pyridinyl)ethenyl)phenoxy)-N-(4-sulfamoylphenyl)
pyrimidin-2-amine (7o): Yield 64%, green solid: mp > 250 °C; 1H
NMR (400 MHz, DMSO-d6) δ: 9.98 (s, 1H), 8.56 (d, J = 5.9 Hz, 2H),
8.44 (d, J = 5.6 Hz, 1H), 7.79 (d, J = 8.5 Hz, 2H), 7.68–7.64 (m, 2H),
7.60–7.53 (m, 5H), 7.33–7.26 (m, 3H), 7.12 (s, 2H), 6.58 (d,
J = 5.6 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ: 169.80, 160.63,
159.69, 153.03, 150.44, 144.75, 143.67, 136.65, 134.29, 132.57,
128.94, 126.72, 126.65, 122.86, 121.40, 118.29, 99.92; HRMS (ESI) m/
z: calcd for C23H20N5O3S [M + H]+ 446.1281, found 446.1285.
4-(4-(2-(4-Pyridinyl)ethenyl)phenoxy)-N-(3-sulfamoylphenyl)
pyrimidin-2-amine (7p): Yield 62%, yellow solid: mp > 250 °C; 1H
NMR (400 MHz, DMSO-d6) δ: 9.88 (s, 1H), 8.56 (d, J = 5.1 Hz, 2H),
8.42 (d, J = 5.6 Hz, 1H), 8.11 (s, 1H), 7.82–7.76 (m, 3H), 7.63–7.57
(m, 3H), 7.37–7.24 (m, 7H), 6.51 (d, J = 5.6 Hz, 1H); 13C NMR
(100 MHz, DMSO-d6) δ: 169.77, 160.52, 159.97, 153.04, 150.49,
144.82, 144.72, 141.00, 134.19, 132.60, 129.25, 128.97, 126.50,
122.74, 122.17, 121.34, 119.02, 116.41, 99.64; HRMS (ESI) m/z: calcd
for C23H20N5O3S [M + H]+ 446.1281, found 446.1283.
chloroprymidine 5 (1.00 g, 6.7 mmol), phenylboronic acid (0.99 g,
8.1 mmol), (1,1′-bis(diphenylphosphino)ferrocene)dichloropalladium
(II) (0.51 g, 0.7 mmol) and sodium carbonate (2.13 g, 20.1 mmol) in
DMF/1,4-dioxane/water (4:2:1, 10 mL) was degassed with nitrogen and
stirred at 80 °C for 10 h. After completion of the reaction, the reaction
mixture was diluted with dichloromethane (50 ml), washed with brine
(30 mL × 3), dried over anhydrous Na2SO4, filtered and concentrated.
The residue was purified by chromatography (ethyl acetate/petroleum
ether) on silica gel to give 8h as a white solid (70%): mp 87.7–88.2 °C;
1H NMR (400 MHz, CDCl3) δ: 8.65 (d, J = 5.3 Hz, 1H), 8.11 (dd,
J = 8.1, 1.9 Hz, 2H), 7.66 (d, J = 5.3 Hz, 1H), 7.58–7.51 (m, 3H).
The following compounds 8i and 8j were prepared from 2,4-di-
chloroprymidine 5 using a procedure similar to that described for 8h.
2-Chloro-4-(4-methylphenyl)pyrimidine (8i): Yield 69%, white
solid: mp 104.5–105.3 °C; 1H NMR (400 MHz, CDCl3) δ: 8.61 (d,
J = 5.3 Hz, 1H), 8.01 (d, J = 8.2 Hz, 2H), 7.62 (d, J = 5.3 Hz, 1H), 7.33
(d, J = 8.2 Hz, 2H), 2.45 (s, 3H).
2-Chloro-4-(4-chlorophenyl)pyrimidine (8j): Yield 72%, white
solid: mp 129.6–130.1 °C; 1H NMR (400 MHz, CDCl3) δ: 8.65 (d,
J = 5.3 Hz, 1H), 8.05 (d, J = 8.6 Hz, 2H), 7.63 (d, J = 5.3 Hz, 1H), 7.49
(d, J = 8.6 Hz, 2H).
N-Phenyl-2-(4-(2-(2-pyridinyl)ethenyl)phenoxy)pyrimidin-4-
amine (9a): A mixture of 4a (197 mg, 1 mmol) and 8a (205 mg,
N-(4-Methyl-3-sulfamoylphenyl)-4-(4-(2-(4-pyridinyl)ethenyl)
phenoxy)pyrimidin-2-amine (7q): Yield 63%, green solid:
mp > 250 °C; 1H NMR (400 MHz, DMSO-d6) δ: 9.76 (s, 1H), 8.56 (d,
J = 5.1 Hz, 2H), 8.38 (d, J = 5.6 Hz, 1H), 8.13 (s, 1H), 7.77 (d,
J = 8.3 Hz, 2H), 7.70 (d, J = 8.2 Hz, 1H), 7.63–7.57 (m, 3H), 7.31–7.24
401

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BioorganicChemistry78(2018)393–405
1 mmol) was suspended in DMF (5 ml), then copper powder (6.4 mg,
0.1 mmol) and cesium carbonate (815 mg, 2.5 mmol) were added. The
reaction mixture was held at 110 °C under nitrogen for 6 h and then
diluted with ethyl acetate (30 ml). The organic layer was washed with
brine (25 mL × 3), dried over anhydrous Na2SO4, filtered and con-
centrated. The residue was purified by chromatography (ethyl acetate/
petroleum ether) on silica gel to give 9a as a yellow solid (68%): mp
192.1–193.1 °C; 1H NMR (400 MHz, DMSO-d6) δ: 9.74 (s, 1H), 8.59 (d,
J = 3.9 Hz, 1H), 8.11 (d, J = 5.8 Hz, 1H), 7.81–7.73 (m, 4H), 7.56 (d,
J = 7.8 Hz, 1H), 7.46 (d, J = 7.8 Hz, 2H), 7.34–7.22 (m, 4H), 7.14 (t,
J = 7.9 Hz, 2H), 6.93 (t, J = 7.3 Hz, 1H), 6.53 (d, J = 5.8 Hz, 1H); 13C
NMR (100 MHz, DMSO-d6) δ: 165.17, 162.19, 157.52, 155.48, 153.62,
149.94, 139.82, 137.28, 133.74, 131.79, 128.96, 128.61, 128.16,
122.96, 122.90, 122.85, 122.79, 120.08, 102.90; HRMS (ESI) m/z:
calcd for C23H19N4O [M + H]+ 367.1553, found 367.1557.
N-(4-Fluorobenzyl)-2-(4-(2-(2-pyridinyl)ethenyl)phenoxy)pyr-
imidin-4-amine (9g): Yield 60%, yellow solid: mp 174.5–175.2 °C; 1H
NMR (400 MHz, DMSO-d6) δ: 8.57 (d, J = 4.8 Hz, 1H), 8.12 (s, 1H),
7.87 (s, 1H), 7.80–7.68 (m, 4H), 7.54 (d, J = 7.8 Hz, 1H), 7.30–7.09
(m, 8H), 6.28 (d, J = 5.9 Hz, 1H), 4.36 (d, J = 5.9 Hz, 2H); 13C NMR
(100 MHz, DMSO-d6) δ: 165.11, 164.43, 162.86, 160.45, 156.05,
155.50, 153.66, 149.91, 137.24, 135.78, 133.34, 131.83, 129.98,
128.44, 128.03, 122.74, 122.59, 115.54, 115.33, 102.09, 43.06; HRMS
(ESI) m/z: calcd for C24H20FN4O [M + H]+ 399.1616, found 399.1620.
4-Phenyl-2-(4-(2-(2-pyridinyl)ethenyl)phenoxy)pyrimidine
(9h): Yield 63%, brown solid: mp 160.7–161.3 °C; 1H NMR (400 MHz,
CDCl3) δ: 8.63 (d, J = 4.8 Hz, 1H), 8.59 (d, J = 5.2 Hz, 1H), 8.07 (d,
J = 8.0 Hz, 2H), 7.71–7.66 (m, 4H), 7.52–7.47 (m, 4H), 7.40 (d,
J = 7.8 Hz, 1H), 7.30 (d, J = 8.6 Hz, 2H), 7.19–7.15 (m, 2H); 13C NMR
(100 MHz, CDCl3) δ: 167.01, 165.50, 160.01, 155.60, 153.09, 149.66,
136.55, 135.86, 133.85, 131.95, 131.45, 128.95, 128.24, 127.81,
127.29, 122.10, 122.05, 122.01, 111.79; HRMS (ESI) m/z: calcd for
The following compounds 9b-9j were prepared from 4a and 8b-8j
using a procedure similar to that described for 9a.
N-(4-Methoxyphenyl)-2-(4-(2-(2-pyridinyl)ethenyl)phenoxy)
pyrimidin-4-amine (9b): Yield 64%, yellow solid: mp 177.0–177.6 °C;
1H NMR (400 MHz, DMSO-d6) δ: 9.61 (s, 1H), 8.58 (d, J = 4.3 Hz, 1H),
8.06 (d, J = 5.8 Hz, 1H), 7.78–7.73 (m, 4H), 7.53 (d, J = 7.8 Hz, 1H),
7.37–7.30 (m, 3H), 7.25–7.21 (m, 3H), 6.74 (d, J = 8.7 Hz, 2H), 6.45
(d, J = 5.8 Hz, 1H), 3.62 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ:
165.30, 162.22, 157.18, 155.47, 155.35, 153.74, 149.93, 137.22,
133.68, 132.77, 131.85, 128.61, 128.15, 122.97, 122.76, 122.08,
122.04, 114.16, 102.26, 55.45; HRMS (ESI) m/z: calcd for C24H21N4O2
[M + H]+ 397.1659, found 397.1661.
C
23H18N3O [M + H]+ 352.1444, found 352.1446.
4-(4-Methylphenyl)-2-(4-(2-(2-pyridinyl)ethenyl)phenoxy)pyr-
imidine (9i): Yield 57%, brown solid: mp 201.7–202.6 °C; 1H NMR
(400 MHz, CDCl3) δ: 8.63 (d, J = 4.8 Hz, 1H), 8.55 (d, J = 5.2 Hz, 1H),
7.98 (d, J = 8.0 Hz, 2H), 7.70–7.65 (m, 4H), 7.44 (d, J = 5.3 Hz, 1H),
7.40 (d, J = 7.8 Hz, 1H), 7.30–7.28 (m, 4H), 7.18–7.14 (m, 2H), 2.42
(s, 3H); 13C NMR (100 MHz, CDCl3) δ: 167.00, 165.46, 159.79, 155.62,
153.14, 149.66, 141.99, 136.54, 133.79, 133.08, 131.97, 129.68,
128.22, 127.77, 127.23, 122.08, 122.01, 111.47, 21.47; HRMS (ESI) m/
z: calcd for C24H20N3O [M + H]+ 366.1601, found 366.1605.
4-(4-Chlorophenyl)-2-(4-(2-(2-pyridinyl)ethenyl)phenoxy)pyr-
imidine (9j): Yield 65%, white solid: mp 206.9–207.8 °C; 1H NMR
(400 MHz, CDCl3) δ: 8.63 (d, J = 4.8 Hz, 1H), 8.59 (d, J = 5.3 Hz, 1H),
8.01 (d, J = 8.5 Hz, 2H), 7.70–7.66 (m, 4H), 7.47–7.39 (m, 4H), 7.28
(d, J = 8.4 Hz, 2H), 7.18–7.14 (m, 2H); 13C NMR (100 MHz, CDCl3) δ:
165.76, 165.52, 160.25, 155.56, 152.97, 149.68, 137.73, 136.54,
134.30, 133.97, 131.86, 129.23, 128.57, 128.25, 127.91, 122.12,
122.07, 121.97, 111.52; HRMS (ESI) m/z: calcd for C23H17ClN3O
[M + H]+ 386.1055, found 386.1058.
N-(4-Chlorophenyl)-2-(4-(2-(2-pyridinyl)ethenyl)phenoxy)pyr-
imidin-4-amine (9c): Yield 61%, yellow solid: mp 214.9–215.7 °C; 1H
NMR (400 MHz, DMSO-d6) δ: 9.90 (s, 1H), 8.58 (d, J = 4.1 Hz, 1H),
8.14 (d, J = 5.8 Hz, 1H), 7.79–7.74 (m, 4H), 7.55–7.49 (m, 3H),
7.34–7.15 (m, 6H), 6.54 (d, J = 5.8 Hz, 1H); 13C NMR (100 MHz,
DMSO-d6) δ: 165.13, 161.95, 157.70, 155.45, 153.56, 149.93, 138.87,
137.23, 133.84, 131.75, 128.75, 128.64, 128.27, 126.34, 122.99,
122.83, 122.76, 121.44, 103.12; HRMS (ESI) m/z: calcd for
C
23H18ClN4O [M + H]+ 401.1164, found 401.1167.
N-(3-Chlorophenyl)-2-(4-(2-(2-pyridinyl)ethenyl)phenoxy)pyr-
The following compounds 9k–9t were prepared from 4b and 8a-8j
using a procedure similar to that described for 9a.
imidin-4-amine (9d): Yield 60%, white solid: mp 181.3–182.0 °C; 1H
NMR (400 MHz, DMSO-d6) δ: 9.92 (s, 1H), 8.58 (d, J = 4.0 Hz, 1H),
8.18 (d, J = 5.8 Hz, 1H), 7.80–7.70 (m, 5H), 7.55 (d, J = 7.8 Hz, 1H),
7.31–7.15 (m, 6H), 6.94 (d, J = 7.9 Hz, 1H), 6.56 (d, J = 5.8 Hz, 1H);
13C NMR (100 MHz, DMSO-d6) δ: 165.06, 161.94, 157.95, 155.50,
153.38, 149.93, 141.36, 137.25, 133.95, 133.67, 131.89, 130.46,
128.84, 128.16, 122.78, 122.75, 122.71, 122.35, 119.11, 118.08,
103.35; HRMS (ESI) m/z: calcd for C23H18ClN4O [M + H]+ 401.1164,
found 401.1165.
N-Phenyl-2-(4-(2-(4-pyridinyl)ethenyl)phenoxy)pyrimidin-4-
amine (9k): Yield 64%, orange solid: mp 234.0–234.9 °C; 1H NMR
(400 MHz, DMSO-d6) δ: 9.76 (s, 1H), 8.56 (d, J = 5.3 Hz, 2H), 8.11 (d,
J = 5.8 Hz, 1H), 7.74 (d, J = 8.6 Hz, 2H), 7.64–7.57 (m, 3H), 7.46 (d,
J = 8.0 Hz, 2H), 7.28–7.24 (m, 3H), 7.14 (t, J = 7.8 Hz, 2H), 6.93 (t,
J = 7.3 Hz, 1H), 6.53 (d, J = 5.8 Hz, 1H); 13C NMR (100 MHz, DMSO-
d6) δ: 165.12, 162.21, 157.52, 153.85, 150.47, 144.79, 139.81, 133.52,
132.78, 128.98, 128.66, 126.02, 122.96, 122.92, 121.30, 120.09,
102.94; HRMS (ESI) m/z: calcd for C23H19N4O [M + H]+ 367.1553,
found 367.1555.
N-(4-Methoxyphenyl)-2-(4-(2-(4-pyridinyl)ethenyl)phenoxy)
pyrimidin-4-amine (9l): Yield 61%, yellow solid: mp 218.5–218.4 °C;
1H NMR (400 MHz, DMSO-d6) δ: 9.63 (s, 1H), 8.55 (d, J = 5.1 Hz, 2H),
8.05 (d, J = 5.8 Hz, 1H), 7.72 (d, J = 8.2 Hz, 2H), 7.62–7.55 (m, 3H),
7.34 (d, J = 8.5 Hz, 2H), 7.27–7.21 (m, 3H), 6.72 (d, J = 8.5 Hz, 2H),
6.44 (d, J = 5.9 Hz, 1H), 3.62 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ:
165.25, 162.17, 157.19, 155.31, 153.94, 150.45, 144.77, 133.44,
132.79, 128.67, 125.97, 123.01, 122.05, 122.00, 121.25, 114.14,
102.37, 55.44; HRMS (ESI) m/z: calcd for C24H21N4O2 [M + H]+
397.1659, found 397.1662.
N-Benzyl-2-(4-(2-(2-pyridinyl)ethenyl)phenoxy)pyrimidin-4-
amine (9e): Yield 64%, yellow solid: mp 164.1–164.7 °C; 1H NMR
(400 MHz, DMSO-d6) δ: 8.57 (d, J = 4.1 Hz, 1H), 8.14 (d, J = 5.9 Hz,
1H), 7.87 (s, 1H), 7.79–7.67 (m, 4H), 7.54 (d, J = 7.8 Hz, 1H),
7.32–7.22 (m, 7H), 7.15 (d, J = 8.4 Hz, 2H), 6.31 (d, J = 5.9 Hz, 1H),
4.41 (d, J = 4.6 Hz, 2H); 13C NMR (100 MHz, DMSO-d6) δ: 165.12,
164.52, 155.96, 155.50, 153.68, 149.91, 139.58, 137.23, 133.30,
131.85, 128.76, 128.44, 128.08, 128.00, 127.32, 122.75, 122.72,
122.56, 102.13, 43.77; HRMS (ESI) m/z: calcd for
C24H21N4O
[M + H]+ 381.1710, found 381.1711.
N-(4-Methoxybenzyl)-2-(4-(2-(2-pyridinyl)ethenyl)phenoxy)
pyrimidin-4-amine (9f): Yield 59%, yellow solid: mp 165.6–166.3 °C;
1H NMR (400 MHz, DMSO-d6) δ: 8.57 (d, J = 3.8 Hz, 1H), 8.06 (s, 1H),
7.85 (s, 1H), 7.80–7.69 (m, 4H), 7.54 (d, J = 7.8 Hz, 1H), 7.30–7.12
(m, 6H), 6.86 (d, J = 8.1 Hz, 2H), 6.27 (d, J = 5.7 Hz, 1H), 4.31 (d,
J = 5.8 Hz, 2H), 3.72 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ: 165.08,
164.40, 158.74, 155.78, 155.48, 153.70, 149.89, 137.27, 133.30,
131.86, 131.47, 129.42, 128.45, 127.97, 122.75, 122.60, 114.16,
N-(4-Chlorophenyl)-2-(4-(2-(4-pyridinyl)ethenyl)phenoxy)pyr-
imidin-4-amine (9m): Yield 67%, yellow solid: mp > 250 °C; 1H NMR
(400 MHz, DMSO-d6) δ: 9.89 (s, 1H), 8.57 (s, 2H), 8.13 (d, J = 5.7 Hz,
1H), 7.75 (d, J = 8.5 Hz, 2H), 7.65–7.58 (m, 3H), 7.48 (d, J = 8.8 Hz,
2H), 7.28–7.24 (m, 3H), 7.16 (d, J = 8.8 Hz, 2H), 6.53 (d, J = 5.7 Hz,
1H); 13C NMR (100 MHz, DMSO-d6) δ: 165.09, 161.94, 157.76, 153.79,
150.49, 144.76, 138.85, 133.65, 132.75, 128.76, 128.70, 126.32,
126.18, 123.01, 121.45, 103.13; HRMS (ESI) m/z: calcd for
102.10, 55.50, 43.21; HRMS (ESI) m/z: calcd for
C25H23N4O2
[M + H]+ 411.1816, found 411.1818.
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BioorganicChemistry78(2018)393–405
C23H18ClN4O [M + H]+ 401.1164, found 401.1168.
6-Chloro-N-phenylpyrimidin-4-amine (11a): Yield 72%, yellow
solid: mp 119.9–120.7 °C; 1H NMR (400 MHz, CDCl3) δ: 8.46 (s, 1H),
7.65 (s, 1H), 7.43 (d, J = 8.0 Hz, 2H), 7.33–7.25 (m, 3H), 6.72 (s, 1H).
6-Chloro-N-(4-methoxyphenyl)pyrimidin-4-amine (11b): Yield
81%, gray solid: mp 135.5–136.1 °C; 1H NMR (400 MHz, DMSO-d6) δ:
9.80 (s, 1H), 8.35 (s, 1H), 7.44 (d, J = 8.8 Hz, 2H), 6.89 (d, J = 8.8 Hz,
2H), 6.68 (s, 1H), 3.70 (s, 3H).
6-Chloro-N-(4-chlorophenyl)pyrimidin-4-amine (11c): Yield
75%, yellow solid: mp 179.4–180.2 °C; 1H NMR (400 MHz, CDCl3) δ:
8.46–8.45 (m, 2H), 7.53 (d, J = 8.8 Hz, 2H), 7.31 (d, J = 8.8 Hz, 2H),
6.89 (s, 1H).
6-Chloro-N-(3-chlorophenyl)pyrimidin-4-amine (11d): Yield
72%, yellow solid: mp 170.2–170.8 °C; 1H NMR (400 MHz, DMSO-d6) δ:
10.02 (s, 1H), 8.54 (s, 1H), 7.91 (s, 1H), 7.48 (d, J = 8.2 Hz, 1H), 7.36
(t, J = 8.1 Hz, 1H), 7.10 (d, J = 8.1 Hz, 1H), 6.83 (s, 1H).
The following compounds 12a and 12b were prepared from 4a–4b
and 4,6-dichloroprymidine 10 using a procedure similar to that de-
scribed for 6a.
6-Chloro-4-(4-(2-(2-pyridinyl)ethenyl)phenoxy)pyrimidine
(12a): Yield 55%, yellow solid: mp 162.5–163.1 °C; 1H NMR (400 MHz,
DMSO-d6) δ: 8.66 (s, 1H), 8.58 (d, J = 4.6 Hz, 1H), 7.82–7.69 (m, 4H),
7.55 (d, J = 7.8 Hz, 1H), 7.43 (s, 1H), 7.34–7.25 (m, 4H); 13C NMR
(100 MHz, DMSO-d6) δ: 170.32, 161.41, 158.99, 155.29, 152.24,
149.94, 137.32, 134.79, 131.42, 128.96, 128.85, 122.93, 122.91,
122.40, 108.68; HRMS (ESI) m/z: calcd for C17H13ClN3O [M + H]+
310.0742, found 310.0745.
6-Chloro-4-(4-(2-(4-pyridinyl)ethenyl)phenoxy)pyrimidine
(12b): Yield 51%, brown solid: mp 173.7–174.5 °C; 1H NMR (400 MHz,
DMSO-d6) δ: 8.67 (s, 1H), 8.55 (d, J = 5.6 Hz, 2H), 7.75 (d, J = 8.3 Hz,
2H), 7.60–7.55 (m, 3H), 7.41 (s, 1H), 7.31–7.23 (m, 3H); 13C NMR
(100 MHz, DMSO-d6) δ: 170.29, 161.43, 159.02, 152.47, 150.49,
144.62, 134.58, 132.42, 128.99, 126.75, 122.44, 121.32, 108.73;
HRMS (ESI) m/z: calcd for C17H13ClN3O [M + H]+ 310.0742, found
310.0743.
The following compounds 13a–13d were prepared from 4a and
11a–11d using a procedure similar to that described for 9a.
N-Phenyl-6-(4-(2-(2-pyridinyl)ethenyl)phenoxy)pyrimidin-4-
amine (13a): Yield 70%, white solid: mp 177.6–178.3 °C; 1H NMR
(400 MHz, DMSO-d6) δ: 9.60 (s, 1H), 8.58 (d, J = 4.1 Hz, 1H), 8.38 (s,
1H), 7.81–7.60 (m, 4H), 7.62 (d, J = 7.9 Hz, 2H), 7.55 (d, J = 7.8 Hz,
1H), 7.34–7.23 (m, 6H), 7.02 (t, J = 7.3 Hz, 1H), 6.16 (s, 1H); 13C NMR
(100 MHz, DMSO-d6) δ: 169.65, 163.01, 158.63, 155.38, 153.09,
149.95, 140.18, 137.28, 134.17, 131.53, 129.24, 128.97, 128.53,
122.98, 122.89, 122.85, 122.39, 120.40, 89.44; HRMS (ESI) m/z: calcd
for C23H19N4O [M + H]+ 367.1553, found 367.1558.
N-(4-Methoxyphenyl)-6-(4-(2-(2-pyridinyl)ethenyl)phenoxy)
pyrimidin-4-amine (13b): Yield 63%, yellow solid: mp
132.4–133.1 °C; 1H NMR (400 MHz, DMSO-d6) δ: 9.43 (s, 1H), 8.58 (d,
J = 4.4 Hz, 1H), 8.30 (s, 1H), 7.82–7.70 (m, 4H), 7.56 (d, J = 7.7 Hz,
1H), 7.46 (d, J = 8.3 Hz, 2H), 7.32–7.21 (m, 4H), 6.91 (d, J = 8.6 Hz,
2H), 6.05 (s, 1H), 3.72 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ:
169.52, 163.25, 158.62, 155.65, 155.28, 153.19, 149.79, 137.45,
134.01, 132.92, 131.70, 128.94, 128.28, 122.90, 122.72, 122.68,
122.35, 114.50, 88.53, 55.65; HRMS (ESI) m/z: calcd for C24H21N4O2
[M + H]+ 397.1659, found 397.1661.
N-(3-Chlorophenyl)-2-(4-(2-(4-pyridinyl)ethenyl)phenoxy)pyr-
imidin-4-amine (9n): Yield 70%, yellow solid: mp 217.7–218.4 °C; 1H
NMR (400 MHz, DMSO-d6) δ: 9.92 (s, 1H), 8.61(s, 2H), 8.18 (d,
J = 5.2 Hz, 1H), 7.75–7.57 (m, 6H), 7.29–7.15 (m, 5H), 6.94 (d,
J = 7.8 Hz, 1H), 6.56 (d, J = 5.2 Hz, 1H); 13C NMR (100 MHz, DMSO-
d6) δ: 165.02, 161.95, 157.95, 153.61, 150.49, 144.73, 141.35, 133.74,
133.66, 132.86, 130.47, 128.89, 126.04, 122.73, 122.37, 119.11,
118.09, 103.39; HRMS (ESI) m/z: calcd for C23H18ClN4O [M + H]+
401.1164, found 401.1169.
N-Benzyl-2-(4-(2-(4-pyridinyl)ethenyl)phenoxy)pyrimidin-4-
amine (9o): Yield 60%, yellow solid: mp 197.0–197.6 °C; 1H NMR
(400 MHz, DMSO-d6) δ: 8.54 (d, J = 5.5 Hz, 2H), 8.13 (d, J = 6.0 Hz,
1H), 7.87 (s, 1H), 7.67 (d, J = 8.5 Hz, 2H), 7.58–7.54 (m, 3H),
7.32–7.16 (m, 8H), 6.30 (d, J = 6.0 Hz, 1H), 4.40 (d, J = 5.5 Hz, 2H);
13C NMR (100 MHz, DMSO-d6) δ: 165.07, 164.50, 155.97, 153.91,
150.45, 144.80, 139.58, 133.06, 132.82, 128.76, 128.49, 128.02,
127.33, 125.84, 122.57, 121.26, 102.16, 43.74; HRMS (ESI) m/z: calcd
for C24H21N4O1 [M + H]+ 381.1710, found 381.1715.
N-(4-Methoxybenzyl)-2-(4-(2-(4-pyridinyl)ethenyl)phenoxy)
pyrimidin-4-amine (9p): Yield 56%, white solid: mp 197.5–198.2 °C;
1H NMR (400 MHz, DMSO-d6) δ: 8.54 (d, J = 5.1 Hz, 2H), 8.05 (s, 1H),
7.85 (s, 1H), 7.69 (d, J = 8.4 Hz, 2H), 7.59–7.55 (m, 3H), 7.23–7.11
(m, 5H), 6.85 (d, J = 8.0 Hz, 2H), 6.27 (d, J = 5.7 Hz, 1H), 4.31 (d,
J = 5.8 Hz, 2H), 3.72 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ: 165.08,
164.34, 158.74, 155.85, 153.94, 150.44, 144.81, 133.07, 132.83,
131.48, 129.42, 128.50, 125.82, 122.61, 121.26, 114.16, 102.12,
55.50, 43.20; HRMS (ESI) m/z: calcd for C25H23N4O2 [M + H]+
411.1816, found 411.1817.
N-(4-Fluorobenzyl)-2-(4-(2-(4-pyridinyl)ethenyl)phenoxy)pyr-
imidin-4-amine (9q): Yield 64%, yellow solid: mp 226.1–226.8 °C; 1H
NMR (400 MHz, DMSO-d6) δ: 8.54 (d, J = 5.0 Hz, 2H), 8.12 (d,
J = 6.9 Hz, 1H), 7.87 (s, 1H), 7.68 (d, J = 8.2 Hz, 2H), 7.58–7.54 (m,
3H), 7.23–7.09 (m, 7H), 6.29 (d, J = 6.9 Hz, 1H), 4.36 (d, J = 5.9 Hz,
2H); 13C NMR (100 MHz, DMSO-d6) δ: 165.07, 164.43, 162.87, 160.46,
156.06, 153.89, 150.44, 144.80, 135.78, 133.11, 132.80, 129.98,
128.50, 125.86, 122.61, 121.30, 121.26, 115.55, 115.33, 102.13,
43.03; HRMS (ESI) m/z: calcd for C24H20FN4O [M + H]+ 399.1616,
found 399.1619.
4-Phenyl-2-(4-(2-(4-pyridinyl)ethenyl)phenoxy)pyrimidine
(9r): Yield 60%, brown solid: mp 190.5–191.1 °C; 1H NMR (400 MHz,
CDCl3) δ: 8.66–8.58 (m, 3H), 8.07 (d, J = 6.2 Hz, 2H), 7.62 (d,
J = 8.5 Hz, 2H), 7.52–7.48 (m, 4H), 7.40–7.28 (m, 5H), 7.00 (d,
J = 16.3 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ: 167.05, 165.41,
160.03, 153.41, 150.17, 144.54, 135.83, 133.30, 132.37, 131.49,
128.96, 128.17, 127.29, 125.88, 122.13, 111.89; HRMS (ESI) m/z:
calcd for C23H18N3O [M + H]+ 352.1444, found 352.1449.
4-(4-Methylphenyl)-2-(4-(2-(4-pyridinyl)ethenyl)phenoxy)pyr-
imidine (9s): Yield 54%, yellow solid: mp 223.9–224.8 °C; 1H NMR
(400 MHz, CDCl3) δ: 8.59 (d, J = 5.2 Hz, 2H), 8.55 (d, J = 5.3 Hz, 1H),
7.98 (d, J = 8.0 Hz, 2H), 7.62 (d, J = 8.5 Hz, 2H), 7.45 (d, J = 5.3 Hz,
1H), 7.46–7.28 (m, 7H), 7.00 (d, J = 16.3 Hz, 1H), 2.42 (s, 3H); 13C
NMR (100 MHz, CDCl3) δ: 167.03, 165.37, 159.80, 153.47, 150.03,
144.73, 142.05, 133.21, 133.04, 132.49, 129.70, 128.17, 127.23,
125.75, 122.14, 120.86, 111.56, 21.48; HRMS (ESI) m/z: calcd for
C
24H20N3O [M + H]+ 366.1601, found 366.1602.
N-(4-Chlorophenyl)-6-(4-(2-(2-pyridinyl)ethenyl)phenoxy)pyr-
imidin-4-amine (13c): Yield 63%, yellow solid: mp 190.6–191.4 °C; 1H
NMR (400 MHz, DMSO-d6) δ: 9.73 (s, 1H), 8.58 (d, J = 4.1 Hz, 1H),
8.41 (s, 1H), 7.76–7.68 (m, 6H), 7.52 (d, J = 7.8 Hz, 1H), 7.36–7.27
(m, 3H), 7.25–7.22 (m, 3H), 6.18 (s, 1H); 13C NMR (100 MHz, DMSO-
d6) δ: 169.65, 162.72, 158.58, 155.36, 153.04, 149.92, 139.31, 137.20,
134.23, 131.52, 129.04, 128.97, 128.55, 126.35, 122.86, 122.81,
122.37, 121.57, 89.95; HRMS (ESI) m/z: calcd for C23H18ClN4O
[M + H]+ 401.1164, found 401.1168.
4-(4-Chlorophenyl)-2-(4-(2-(4-pyridinyl)ethenyl)phenoxy)pyr-
imidine (9t): Yield 66%, yellow solid: mp 192.2–192.9 °C; 1H NMR
(400 MHz, CDCl3) δ: 8.73–8.59 (m, 3H), 8.00 (d, J = 8.6 Hz, 2H), 7.62
(d, J = 8.2 Hz, 2H), 7.47–7.28 (m, 8H), 6.99 (d, J = 16.3 Hz, 1H); 13C
NMR (100 MHz, CDCl3) δ: 165.79, 165.42, 160.26, 153.29, 150.04,
149.12, 137.79, 134.26, 133.42, 132.31, 129.24, 128.56, 128.19,
126.00, 122.11, 111.62; HRMS (ESI) m/z: calcd for C23H17ClN3O
[M + H]+ 386.1055, found 386.1059.
The following compounds 11a–11d were prepared from 4,6-di-
chloroprymidine 10 using a procedure similar to that described for 8a.
N-(3-Chlorophenyl)-6-(4-(2-(2-pyridinyl)ethenyl)phenoxy)pyr-
imidin-4-amine (13d): Yield 61%, white solid: mp 166.8–167.4 °C; 1
H
403

W. Sun et al.
BioorganicChemistry78(2018)393–405
NMR (400 MHz, DMSO-d6) δ: 9.78 (s, 1H), 8.58 (d, J = 4.3 Hz, 1H),
8.44 (s, 1H), 7.95 (s, 1H), 7.80–7.70 (m, 4H), 7.55 (d, J = 7.8 Hz, 1H),
7.46 (d, J = 8.2 Hz, 1H), 7.34–7.24 (m, 5H), 7.04 (d, J = 7.7 Hz, 1H),
6.18 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ: 169.70, 162.66, 158.64,
155.36, 152.97, 149.94, 141.87, 137.27, 134.30, 133.59, 131.50,
130.80, 129.01, 128.60, 122.89, 122.86, 122.41, 122.25, 119.30,
118.28, 90.22; HRMS (ESI) m/z: calcd for C23H18ClN4O [M + H]+
401.1164, found 401.1165.
N-(3-Chlorophenyl)-6-(4-(2-(4-pyridinyl)ethenyl)phenoxy)pyr-
imidin-4-amine (13k): Yield 59%, yellow solid: mp 215.1–215.9 °C; 1H
NMR (400 MHz, DMSO-d6) δ: 9.78 (s, 1H), 8.55 (d, J = 4.2 Hz, 2H),
8.43 (s, 1H), 7.94 (s, 1H), 7.75 (d, J = 8.4 Hz, 2H), 7.61–7.56 (m, 3H),
7.46 (d, J = 8.0 Hz, 1H), 7.34–7.23 (m, 4H), 7.04 (d, J = 8.0 Hz, 1H),
6.18 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ: 169.64, 162.68, 158.63,
153.22, 150.46, 144.70, 141.85, 134.05, 133.59, 132.51, 130.82,
129.05, 126.47, 122.42, 122.29, 121.32, 119.32, 118.31, 90.30; HRMS
The following compounds 13e–13g were prepared from 12a using a
procedure similar to that described for 7h.
(ESI) m/z: calcd for
401.1168.
C
23H18ClN4O [M + H]+ 401.1164, found
6-(4-(2-(2-Pyridinyl)ethenyl)phenoxy)-N-(4-sulfamoylphenyl)
pyrimidin-4-amine (13e): Yield 58%, white solid: mp > 250 °C; 1H
NMR (400 MHz, DMSO-d6) δ: 9.96 (s, 1H), 8.58 (d, J = 4.1 Hz, 1H),
8.46 (s, 1H), 7.84–7.70 (m, 8H), 7.55 (d, J = 7.8 Hz, 1H), 7.34–7.23
(m, 6H), 6.25 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ: 169.81, 162.59,
158.63, 155.37, 152.96, 149.95, 143.41, 137.53, 137.30, 134.35,
131.50, 129.03, 128.64, 127.22, 122.89, 122.42, 119.16, 90.66; HRMS
The following compounds 13l–13n were prepared from 12b using a
procedure similar to that described for 7h.
6-(4-(2-(4-Pyridinyl)ethenyl)phenoxy)-N-(4-sulfamoylphenyl)
pyrimidin-4-amine (13l): Yield 55%, yellow solid: mp > 250 °C; 1H
NMR (400 MHz, DMSO-d6) δ: 9.95 (s, 1H), 8.56 (d, J = 5.2 Hz, 2H),
8.46 (s, 1H), 7.83–7.75 (m, 6H), 7.61–7.57 (m, 3H), 7.28–7.22 (m, 5H),
6.26 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ: 169.73, 162.60, 158.62,
153.21, 150.48, 144.70, 143.39, 137.56, 134.09, 132.52, 129.06,
127.22, 126.50, 122.43, 121.33, 119.17, 90.74; HRMS (ESI) m/z: calcd
for C23H20N5O3S [M + H]+ 446.1281, found 446.1286.
6-(4-(2-(4-Pyridinyl)ethenyl)phenoxy)-N-(3-sulfamoylphenyl)
pyrimidin-4-amine (13m): Yield 53%, yellow solid: mp
232.6–233.2 °C; 1H NMR (400 MHz, DMSO-d6) δ: 9.92 (s, 1H), 8.56 (d,
J = 4.4 Hz, 2H), 8.42 (s, 1H), 8.19 (s, 1H), 7.84 (d, J = 7.9 Hz, 1H),
7.76 (d, J = 8.5 Hz, 2H), 7.61–7.46 (m, 5H), 7.39 (s, 2H), 7.28–7.23
(m, 3H), 6.22 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ: 169.65, 162.76,
158.62, 153.24, 150.47, 145.15, 144.71, 140.71, 134.04, 132.54,
129.94, 129.04, 126.46, 122.86, 122.43, 121.33, 119.75, 116.82,
90.26; HRMS (ESI) m/z: calcd for C23H20N5O3S [M + H]+ 446.1281,
found 446.1282.
N-(4-Methyl-3-sulfamoylphenyl)-6-(4-(2-(4-pyridinyl)ethenyl)
phenoxy)pyrimidin-4-amine (13n): Yield 60%, yellow solid:
mp > 250 °C; 1H NMR (400 MHz, DMSO-d6) δ: 9.80 (s, 1H), 8.56 (d,
J = 5.1 Hz, 2H), 8.38 (s, 1H), 8.13 (d, J = 1.7 Hz, 1H), 7.83 (d,
J = 8.1 Hz, 1H), 7.75 (d, J = 8.4 Hz, 2H), 7.61–7.57 (m, 3H), 7.37–7.23
(m, 6H), 6.18 (s, 1H), 2.53 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ:
169.57, 162.85, 158.61, 153.29, 150.49, 144.70, 142.71, 138.23,
133.98, 133.03, 132.54, 129.67, 129.00, 126.44, 123.11, 122.41,
121.31, 118.93, 89.90, 19.59; HRMS (ESI) m/z: calcd for C24H22N5O3S
[M + H]+ 460.1438, found 460.14342.
(ESI) m/z: calcd for
446.1283.
C
23H20N5O3S [M + H]+ 446.1281, found
6-(4-(2-(2-Pyridinyl)ethenyl)phenoxy)-N-(3-sulfamoylphenyl)
pyrimidin-4-amine (13f): Yield 60%, white solid: mp 215.7–216.6 °C;
1H NMR (400 MHz, DMSO-d6) δ: 9.90 (s, 1H), 8.58 (d, J = 4.1 Hz, 1H),
8.43 (s, 1H), 8.20 (s, 1H), 7.86–7.70 (m, 5H), 7.56–7.47 (m, 3H),
7.38–7.25 (m, 6H), 6.23 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ:
169.72, 162.76, 158.63, 155.37, 152.99, 149.94, 145.17, 140.75,
137.28, 134.29, 131.52, 129.93, 129.00, 128.61, 122.88, 122.87,
122.42, 119.73, 116.82, 90.19; HRMS (ESI) m/z: calcd for
C
23H20N5O3S [M + H]+ 446.1281, found 446.1282.
N-(4-Methyl-3-sulfamoylphenyl)-6-(4-(2-(2-pyridinyl)ethenyl)
phenoxy)pyrimidin-4-amine (13g): Yield 55%, white solid: mp
148.1–148.8 °C; 1H NMR (400 MHz, DMSO-d6) δ: 9.80 (s, 1H), 8.58 (d,
J = 4.1 Hz, 1H), 8.39 (s, 1H), 8.13 (d, J = 1.8 Hz, 1H), 7.84–7.70 (m,
5H), 7.55 (d, J = 7.8 Hz, 1H), 7.36–7.23 (m, 7H), 6.18 (s, 1H), 2.54 (s,
3H); 13C NMR (100 MHz, DMSO-d6) δ: 169.64, 162.85, 158.61, 155.39,
153.05, 149.95, 142.71, 138.25, 137.28, 134.23, 133.03, 131.53,
129.65, 128.97, 128.58, 123.10, 122.86, 122.39, 118.92, 89.83, 19.59;
HRMS (ESI) m/z: calcd for C24H22N5O3S [M + H]+ 460.1438, found
460.1439.
The following compounds 13h–13k were prepared from 4b and
11a-11d using a procedure similar to that described for 9a.
N-Phenyl-6-(4-(2-(4-pyridinyl)ethenyl)phenoxy)pyrimidin-4-
amine (13h): Yield 60%, brown solid: mp 234.8–235.7 °C; 1H NMR
(400 MHz, DMSO-d6) δ: 9.62 (s, 1H), 8.58 (s, 2H), 8.37 (s, 1H), 7.74 (d,
J = 8.4 Hz, 2H), 7.63–7.56 (m, 5H), 7.34–7.22 (m, 5H), 7.02 (t,
J = 7.3 Hz, 1H), 6.18 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ: 169.56,
163.03, 158.62, 153.36, 150.47, 144.67, 140.17, 133.92, 132.54,
129.24, 128.99, 126.41, 123.00, 122.38, 121.43, 120.43, 89.54; HRMS
(ESI) m/z: calcd for C23H19N4O [M + H]+ 367.1553, found 367.1556.
N-(4-Methoxyphenyl)-6-(4-(2-(4-pyridinyl)ethenyl)phenoxy)
pyrimidin-4-amine (13i): Yield 62%, yellow solid: mp 209.5–210.3 °C;
1H NMR (400 MHz, DMSO-d6) δ: 9.70 (s, 1H), 8.55 (s, 2H), 8.28 (s, 1H),
7.73 (d, J = 8.3 Hz, 2H), 7.60–7.49 (m, 5H), 7.26–7.21 (m, 3H), 6.90
(d, J = 8.7 Hz, 2H), 6.17 (s, 1H), 3.72 (s, 3H); 13C NMR (100 MHz,
DMSO-d6) δ: 169.26, 163.26, 158.55, 155.53, 153.47, 150.46, 144.72,
133.73, 133.12, 132.59, 128.93, 126.28, 122.55, 122.28, 121.36,
4.2. Biological evaluation
4.2.1. In vitro inhibitory activity in cancer cell lines
In vitro inhibitory activity was tested using a CCK-8 assay on A549
and HepG2 cell lines [28,29]. Briefly, cells (5 × 103 per well in 96-well
plates) were incubated overnight at 37 °C in a 5% CO2 incubator and
treated with target compounds at different concentrations for 48 h.
Then 10% CCK-8 solution was added into each well, and the cells were
further incubated at 37 °C and with 5% CO2 for another 2 h. The ab-
sorbance value was measured using a microplate reader (PerkinElmer,
USA). Half-maximal inhibitory concentration (IC50) was calculated
from a log plot of percent of control versus concentration [30,31].
4.2.2. Cytotoxicity test
114.44, 88.90, 55.67; HRMS (ESI) m/z: calcd for
C
24H21N4O2
The cytotoxicity test of target compounds was measured by CCK-8
assay. Human macrophage cells were incubated in a 96-well plate at a
density of 2 × 104 cells per well. Then, target compounds at different
concentrations were added to each well respectively. Cells without
treatment by target compounds were used as control. After incubation
for 48 h at 37 °C and with 5% CO2, 10% CCK-8 solution was added to
each well and cells were further incubated for 2 h at 37 °C and with 5%
CO2. The absorbance value was determined by a microplate reader
(PerkinElmer, USA), and the concentration of target compounds that
killed 50% of cells (CC50) was calculated by comparison with control
wells [32].
[M + H]+ 397.1659, found 397.1660.
N-(4-Chlorophenyl)-6-(4-(2-(4-pyridinyl)ethenyl)phenoxy)pyr-
imidin-4-amine (13j): Yield 57%, yellow solid: mp 216.8–217.4 °C; 1H
NMR (400 MHz, DMSO-d6) δ: 9.72 (s, 1H), 8.56 (s, 2H), 8.39 (s, 1H),
7.74 (d, J = 8.3 Hz, 2H), 7.67 (d, J = 8.9 Hz, 2H), 7.60–7.55 (m, 3H),
7.35 (d, J = 8.8 Hz, 2H), 7.26–7.22 (m, 3H), 6.17 (s, 1H); 13C NMR
(100 MHz, DMSO-d6) δ: 169.59, 162.72, 158.60, 153.26, 150.46,
144.68, 139.26, 133.99, 132.51, 129.07, 129.02, 126.43, 126.35,
122.41, 121.60, 121.35, 89.98; HRMS (ESI) m/z: calcd for
C
23H18ClN4O [M + H]+ 401.1164, found 401.1165.
404

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BioorganicChemistry78(2018)393–405
4.2.3. VEGFR-2 kinase assay
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The kinase activity of VEGFR-2 was measured by use of the ADP-
GloTM kinase assay with Pazopanib as positive control. It measured
kinase activity by quantitating the amount of ATP remaining in solution
after a kinase reaction. The general procedure was as the following:
Kinase was incubated with substrate, ATP and variable concentrations
of target compounds in a buffer containing 40 mM Tris, 10 mM MgCl2,
0.1 mg/mL BSA, 1 mM DTT in a 384-well plate. The kinase reaction was
allowed to proceed at 30 °C for 1 h, and then cooled for 5 min at room
temperature. ADP-Glo reagent was added to stop the reaction and
consumed the remaining ADP for 40 min, followed by another 30 min
incubation with kinase detection reagent. The luminescence signal was
measured by a VICTOR multilabel plate reader (PerkinElmer, USA). The
signal was correlated with the amount of ATP present in the reaction
and was inversely correlated with the kinase activity. The IC50 value
was determined as the concentration causing half-maximal percent
activity [33,34].
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with a grid spacing of 0.375 Å was used to define the binding site. The
binding affinity was reported as the binding free energy (ΔG) and in-
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This work was financially supported by the Key Projects in the
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