Design, synthesis and antitumor activity evaluation of trifluoromethyl-substituted pyrimidine derivatives

Article abstract of DOI:10.1016/j.bmcl.2021.128268

In order to find efficient new antitumor drugs, a series of novel trifluoromethyl-substituted pyrimidine derivatives were designed and synthesized, and the bioactivity against four human tumor cells (PC-3, MGC-803, MCF-7 and H1975) was evaluated by MTT assay. Compound 17v displayed potent anti-proliferative activity on H1975 (IC₅₀ = 2.27 μΜ), which was better than the positive control 5-FU (IC₅₀ = 9.37 μΜ). Further biological evaluation studies showed that compound 17v induced apoptosis of H1975 cells and arrested the cell cycle at G2/M phase. Furthermore, compound 17v induced H1975 cells apoptosis through increasing the expression of pro-apoptotic proteins Bax and p53 and down-regulating the anti-apoptotic protein Bcl-2. In addition, compound 17v was able to be tightly embedded in the active pocket of EGFR. In summary, these results demonstrated that compound 17v has a potential as a lead compound for further investigation.

Full text of DOI:10.1016/j.bmcl.2021.128268

Bioorg. Med. Chem. Lett. 51 (2021) 128268
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and antitumor activity evaluation of
trifluoromethyl-substituted pyrimidine derivatives
,
,
,
,
,
,b
Limin Liua b, Zhengjie Wanga b, Chao Gaoa b, Honglin Daia b, Xiaojie Sia b, Yang Zhanga
,
,
,
,
b
,
c
,
,
b
,
c,
,b,c,*
Yaqi Menga b, Jiaxin Zhenga c, Yu Kea
*, Hongmin Liua
*, Qiurong Zhanga
a School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001, China
b Collaborative Innovation Center of New Drug Research and Safety Evaluation, Zhengzhou 450001, China
c Key Laboratory of Advanced Drug Preparation Technologies, Ministry of Education of China, Zhengzhou 450001, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
In order to find efficient new antitumor drugs, a series of novel trifluoromethyl-substituted pyrimidine de-
rivatives were designed and synthesized, and the bioactivity against four human tumor cells (PC-3, MGC-803,
MCF-7 and H1975) was evaluated by MTT assay. Compound 17v displayed potent anti-proliferative activity
Pyrimidine derivatives
Synthesis
Antitumor activity
Trifluoromethyl moiety
on H1975 (IC50 = 2.27 μМ), which was better than the positive control 5-FU (IC50 = 9.37 μМ). Further biological
evaluation studies showed that compound 17v induced apoptosis of H1975 cells and arrested the cell cycle at
G2/M phase. Furthermore, compound 17v induced H1975 cells apoptosis through increasing the expression of
pro-apoptotic proteins Bax and p53 and down-regulating the anti-apoptotic protein Bcl-2. In addition, compound
17v was able to be tightly embedded in the active pocket of EGFR. In summary, these results demonstrated that
compound 17v has a potential as a lead compound for further investigation.
Although researchers have made a lot of efforts, cancer is still the
second leading cause of mortality after cardiovascular diseases1. As a
new cancer treatment modality, cancer immunotherapy has made
breakthrough progress in the treatment of numerous cancers2,3. Cancer
immunotherapy stimulates host immune responses to destroy local and
metastatic cancer cells4. Even though it brings promising results,
chemotherapy still plays an important role in the field. Chemotherapy,
one of three acknowledged cancer treatments, is frequently used in
clinical cancer therapy5. However, due to the existence of defects such as
poor selectivity and serious side effects, it is imperative to develop drugs
with better antitumor activity and lower toxicity6,7. Pyrimidine is a
nitrogen-containing heterocyclic compound with a wide range of
pharmacological effects, especially in antitumor applications.8,9. For
example, anticancer drugs such as the reversible ALK/EGFR inhibitor
Brigatinib (1)10 and the BTK inhibitor Vecabrutinib (2)11 (Fig. 1).
In recent years, N-(3-aminophenyl)acrylamide as an active fragment
has played a great role in antitumor activity. Compound 3 displayed
high activity against EGFRT790M/L858R kinase (IC50 = 0.71 nM) and
repressed H1975 cell replication harboring EGFRT790M mutations at a
concentration of 0.037 µM12. Li et al13 reported a series of 2,5-diamino-
pyrimidine derivatives, among them, the most potent compound (4)
showed potent anti-prolifera-
tive activities toward multiple B-cell lymphoma cell lines, and
significantly inhibited BTK enzyme with IC50 value of 5 nM. In addition,
introducing a trifluoromethyl group into an organic molecular structure
can greatly improve their physicochemical, and biological properties,
such as enhanced binding selectivity, higher lipophilicity, and increased
metabolic stability14. Compared with wild-type EGFR (IC50 = 465 nM),
compound 5 showed higher inhibitory selectivity to mutant EGFRL858R/
T790M (IC50 = 10.4 nM). Moreover, the potent antiproliferative activity
against H1975 cells (IC50 = 15 nM), suggested that compound 5 is a
potentially useful chemical entity for the treatment of NSCLC15. Wang et
al16 reported a series of 3,5-disubstituted and 3,5,7-trisubstituted
quinolines. Compound 6 showed the most potent enzyme activity with
IC50 of<1.0 nM. In addition, it markedly inhibited the proliferation in
both NIH-3 T3-TPR-Met and BaF3-TPR-Met cell models with IC50 of 2.0
μ
M and 0.39 μM.
Based on the studies of pyrimidine derivatives, we found that some
compounds with better bioactivity have a sulfhydryl group at the 2-po-
sition of the pyrimidine ring or a trifluoromethyl group at the 6-posi-
tion17–20. Therefore, the 2-mercapto-6-trifluoromethyl-pyrimidine was
selected as the scaffold to study. We used the principle of molecular
* Corresponding authors at: School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001, China.
E-mail addresses: ky@zzu.edu.cn (Y. Ke), liuhm@zzu.edu.cn (H. Liu), zqr409@yeah.net (Q. Zhang).
https://doi.org/10.1016/j.bmcl.2021.128268
Received 25 April 2021; Received in revised form 10 July 2021; Accepted 14 July 2021
Available online 21 July 2021
0960-894X/© 2021 Published by Elsevier Ltd.

L. Liu et al.
Bioorganic & Medicinal Chemistry Letters 51 (2021) 128268
hybridization to introduce acrylamide and trifluoromethyl group into
the pyrimidine skeleton to form a series of pyrimidine derivatives and
evaluate their bioactivity in vitro.
change in bioactivity. Particularly, compound 16d with 4-CF3 substi-
tution exhibited a better inhibitory effect than the positive control 5-Fu.
Next, through further structure ꢀ activity relationship (SAR) studies,
we found that different heterocyclics and anilines substitutions at R2
were also important for the bioactivity. Here, we replaced the N-(3-
aminophenyl)acrylamide of the best compound 16d in the previous
series with different heterocycles and anilines to synthesize the target
compounds 17a-z. The bioactivities of these compounds were shown in
Table 2. As compared to the compound 16d, the removal of the acryl-
amide group led to a decrease in the bioactivity of compound 17o.
Acrylamide can form a covalent bond with the cell target, which might
be the reason for the loss of activity after removal. Interestingly, a
similar phenomenon could be observed when acrylamide was
substituted by other substituents. As shown in Table 2, most heterocyclic
and piperazine substitutions attached to the 4-position of the pyrimidine
skeleton also decreased the bioactivity. Particularly, compound 17v
The synthetic route to 16a-z was depicted in Scheme 1. The key
intermediate 10 and 15a-z were synthesized according to previously
reported procedure21–23. In the presence of triethylamine,
3-nitroaniline (7) and acryloyl chloride (8) were reacted in aceto-
nitrile to form the intermediate 9, followed by a reduction reaction in
the presence of iron powder to give the key intermediate 10. Then, re-
action of ethyl 4,4,4-trifluoro-3-oxobutanoate (11) with thiourea (12) in
ethanol under reflux afforded pyrimidine 13. Compound 13 reacted
with diverse benzyl chlorides in the presence of KOH to offer compounds
14a-z, which were dissolved in 1,4-dioxane, and then heated with
phosphorus oxychloride to obtain the key intermediate 15a-z. The in-
termediates 15a-z and 10 were heated at 80℃ in N, N-dimethylforma-
mide (DMF) for 10 h to form the target products
16a-z. After that, 15d was reacted with heterocyclics and anilines to
obtain compounds 17a-z. The structures of target compounds 16a-z and
17a-z were confirmed by 1H NMR, 13C NMR and high-resolution mass
spectrometry (HR-MS).
exhibited the best bioactivity against H1975 (IC50 = 2.27
positive control 5-Fu (IC50 = 9.37 M) and compound 16d (IC50 = 4.77
M).
Compounds 16d and 17v were further examined for possible cyto-
μM) than the
μ
μ
The bioactivities of all the target compounds against PC-3 (human
prostate cancer cell line), MGC-803 (human gastric cancer cell line),
MCF-7 (human breast cancer cell line) and H1975 (non-small cell -
lung cancer cell line) were evaluated by MTT assay and 5-Fluorouracil
(5-FU) as the positive control. The results were summarized in Ta-
bles 1 ꢀ 2.
toxicity against GES-1 (normal human gastric epithelial cell line). As
shown in Table 3, compared with 5-FU, we found that com-
pounds 16d and 17v exhibited weak cytotoxicity against GES-1. The
results indicated that compounds 16d and 17v had higher selectivity
between the selected cancer cell line (H1975) and a normal cell line
(GES-1).
To investigate the effect of the acrylamide group on bioactivity,
compounds 16a-z were synthesized initially, and their bioactivities were
shown in Table 1. Compared with compound 16u, which had no sub-
stituent on benzene ring, the introduction of electron-donating groups at
R1 did not increase the bioactivity. Conversely, introducing an electron-
withdrawing group remarkably increased the bioactivity. It was worth
noting that increasing the electron withdrawing ability of substituent
significantly improved the bioactivity of most compounds. In addition,
the position of substituent also played a great role on the bioactivity,
para-substitution had better bioactivity than meta-substitution, while
ortho-substitution had worse bioactivity. Interestingly, replacement of
the benzene ring by thiazole or aliphatic ring (16v-z) led to no obvious
Based on the above findings, in order to investigate the mechanism of
these series of compounds, compound 17v and H1975 cell line were
selected for further study. Cancer cell colony formation can be used as an
indirect assessment method of tumor transformation24. Therefore, we
firstly explored the effect on cell colony formation. The results displayed
that 17v was able to inhibit the colony formation of H1975 cells dose-
dependently (Fig.2A) Moreover, as the concentration of 17v increased,
the colonies by the compound-added group became smaller and fewer,
compared with the control group. Obviously, the inhibition rate of
compound 17v was achieved 90% at 4 μM. Cell cycle arrest is a marker
closely related to cell proliferation inhibition25, the effect of compound
17v on cell cycle progression was investigated. As shown in Fig. 2B, with
Figure 1. Design strategy of trifluoromethyl-substituted pyrimidine derivatives.
2

L. Liu et al.
Bioorganic & Medicinal Chemistry Letters 51 (2021) 128268
Scheme 1. Synthesis of the Target Pyrimidine-Based Derivatives. Reagents and conditions: (a) acetonitrile, Et3N, 0℃, 2 h; (b) absolute methanol, iron powder,
ammonium chloride, 65℃, 2 h; (c) absolute ethanol, KOH, reflux, 6 h; (d) Acetone-H2O, diverse benzyl chlorides, KOH, 80℃, 1.5 h; (e) 1,4-Dioxane, POCl3, 90℃, 1
h; (f) N-(3-aminophenyl)acrylamide, DMF, absolute K2CO3, 80℃, 10 h; (g) DMF, absolute K2CO3, 80℃, 6 h.
the increasing concentration of compound 17v (0, 1, 2, and 4
μ
M), the
compound 17v on H1975 cells, western blot analysis was used to
explore the expression of apoptosis-related proteins. As shown
in Fig. 3C, the expression of pro-apoptotic proteins Bax and p53 was
significantly increased, while anti-apoptotic protein Bcl-2 was down-
regulated. In summary, these results indicated that compound 17v
induced H1975 cells apoptosis through the activation of intrinsic
apoptotic pathway.
percentage of H1975 cells in G2/M phase increased dramatically (from
5.78% to 30.39%), which evidently indicated that compound 17v could
assert H1975 cell at G2/M phase.
Next, in consideration of the higher bioactivity of 17v against H1975
cells, we investigated whether compound 17v could effectively inhibit
the migration and invasion of H1975 cells. As shown in Fig. 2C, com-
pound 17v significantly and dose-dependently decreased the scratch-
repaired capacity of the cells, which indicated that compound 17v
inhibited the migration of H1975 cells in a time-dependent and
concentration-dependent manner.
At present, many third-generation EGFR inhibitors are based on
pyrimidine26,27, so we decided to insert the compounds 16d and 17v
into the EGFR pocket to explore the interaction between compounds and
EGFR. By using MOE 2020 for molecular docking, we found that com-
pounds 16d and 17v were able to be tightly embedded in the pocket of
EGFR (PDB code: 5GNK), which was retrieved from the protein database
(http://www.rcsb.org/pdb) for the docking calculations. As shown in
Fig. 4, as anticipated, compound 16d formed a covalent bond between
its acryloyl group and the Cys797 reside in the EGFR protein. Moreover,
compound 16d formed a hydrogen bond with residue Met793 (2.38 Å).
Interestingly, compound 17v used a completely different binding mode
To determine whether the inhibitory effect of 17v on cell pro-
liferation were accompanied by enhanced cancer cell apoptosis, DAPI
staining was performed after treating H1975 cells of compound 17v
with variable level (0, 2, and 4 μM) for 48 h. As shown in Fig. 3A, control
groups of H1975 cells showed intact nucleus; while after treatment of
compound 17v, the nucleus shrinked, dense particles appeared, the
nucleus were broken into fragments and the number of change in
apoptotic morphology increased significantly. These findings indicated
that compound 17v could induce apoptosis in H1975 cells. In addition,
Annexin V-FITC/PI double staining flow cytometry method was also
used. As shown in Fig. 3B, in H1975 cells, compound 17v increased the
percentage of apoptotic cells from 3.86% of the control to 5.04%,
11.13% and 16.68% (including the early and late apoptosis) at 1, 2 and
from 16d. Compound 17v interacted with Val726 (2.92 Å) by
π-H
interaction. At the same time, the 2-mercapto-1,3,4-thiadiazole group of
compound 17v formed hydrogen bonds with Arg841 (3.68 Å), Asn842
(3.32 Å), Phe723 (2.52 Å) and Gly724 (2.44 Å), respectively. These
findings indicated that compounds 16d and 17v may have inhibitory
effects on EGFR, while the detail mechanisms need to be further
examined in our future study.
4 μM for 48 h, respectively, while the percentage of apoptotic cells
increased to 7.70%, 17.31% and 29.08% at the same concentrations for
72 h. These results indicated that compound 17v could induce apoptosis
in H1975 cells. Finally, in order to further evaluate the mechanism of
In conclusion, a series of novel 2,4-substituted pyrimidine de-
rivatives were synthesized, and the anti-proliferative activities of the
target compounds against PC-3, MGC-803, MCF-7, and H1975 cells lines
3

L. Liu et al.
Bioorganic & Medicinal Chemistry Letters 51 (2021) 128268
Table 1
Table 2
Anti-proliferative activities [IC50/(
μ
mol•Lꢀ 1)] of 16a-z against four cancer cell
Anti-proliferative activities [IC50 / (
μ
mol•Lꢀ 1)] of 17a-z against four cancer cell
linesa.
Compounds
linesa.
Compounds
R1
IC50
(
μ
M)
R2
IC50
(μM)
PC-3
MGC-803
MCF-7
H1975
PC-3
MGC-
803
MCF-7
H1975
16a
16b
16c
16d
16e
16f
4-CH3-Bn
3-CH3-Bn
2-CH3-Bn
4-CF3-Bn
3-CF3-Bn
2-CF3-Bn
4-OCH3-Bn
3-OCH3-Bn
2-OCH3-Bn
4-F-Bn
17.81 ±
1.25
18.47 ±
1.26
25.71 ±
1.14
14.98 ±
0.77
17a
15.88
5.87 ±
10.73
6.21 ±
9.05 ±
0.95
12.56 ±
1.09
26.71 ±
1.42
13.23 ±
1.12
± 1.20
0.27
± 1.03
0.79
15.05 ±
1.17
18.19 ±
1.08
33.65 ±
1.52
14.63 ±
1.16
17b
17c
17d
40.84
>50
>50
>50
>50
>50
5.65 ±
0.98
9.80 ±
0.99
7.08 ±
0.85
4.77 ±
0.44
± 1.89
5.92 ±
0.77
18.68 ±
1.27
12.41 ±
1.09
5.07 ±
0.70
46.82
44.03
± 1.83
± 1.64
7.96 ±
0.91
26.61 ±
1.42
17.18 ±
1.23
6.55 ±
0.55
34.67
15.82
30.16
23.91 ±
16g
16h
16i
15.82 ±
0.76
15.07 ±
0.71
7.59 ±
0.88
10.24 ±
1.01
± 1.54
± 1.19
± 1.48
1.37
11.53 ±
1.06
32.44 ±
1.51
8.91 ±
0.95
9.45 ±
0.97
17e
17f
27.21
25.61
21.98
14.97 ±
9.96 ±
0.99
32.71 ±
1.51
22.03 ±
1.34
6.50 ±
0.81
± 1.43
± 1.25
± 1.34
1.17
16j
9.22 ±
0.96
9.25 ±
0.96
14.93 ±
0.69
10.14 ±
1.11
42.74
35.12
>50
>50
>50
16k
16l
3-F-Bn
8.57 ±
0.89
7.14 ±
0.85
23.03 ±
1.36
13.73 ±
1.03
± 1.91
± 1.34
2-F-Bn
8.67 ±
0.93
13.38 ±
1.12
31.14 ±
1.19
15.42 ±
0.73
16m
16n
16o
16p
16q
16r
16s
16t
4-Cl-Bn
9.99 ±
1.02
29.32 ±
1.46
15.77 ±
1.10
11.69 ±
0.43
17g
17h
40.61
22.15
>50
>50
3-Cl-Bn
10.52 ±
1.02
35.21 ±
1.54
10.13 ±
1.06
14.79 ±
0.57
± 1.60
± 1.87
2-Cl-Bn
13.56 ±
1.13
28.45 ±
1.45
11.81 ±
1.07
13.14 ±
0.71
>50
>50
23.54
17.38 ±
4-Br-Bn
6.32 ±
0.81
29.41 ±
1.46
11.70 ±
0.67
6.44 ±
0.92
± 1.02
0.86
3-Br-Bn
7.44 ±
0.87
22.81 ±
1.35
14.27 ±
1.15
7.44 ±
0.65
2-Br-Bn
8.17 ±
0.91
30.86 ±
1.49
20.15 ±
1.30
9.62 ±
0.75
17i
35.24
>50
18.34 ±
2.4-diCl-Bn
2.6-diCl-Bn
Bn
6.66 ±
0.82
18.58 ±
1.26
6.25 ±
0.79
15.31 ±
0.72
± 1.03
0.91
14.55 ±
1.16
34.68 ±
1.54
9.66 ±
0.98
9.03 ±
0.95
16u
16v
16w
16x
16y
16z
5-FUb
14.16 ±
1.15
22.29 ±
1.34
19.06 ±
1.09
21.94 ±
0.84
17j
14.13
19.23
>50
>50
17.45 ±
± 1.87
± 1.45
1.77
11.53 ±
1.06
38.32 ±
1.58
10.72 ±
1.03
10.89 ±
0.83
17k
17l
16.46
32.33
20.56 ±
12.48 ±
1.09
34.12 ±
1.53
11.24 ±
1.05
17.99 ±
0.90
± 1.98
± 1.47
1.51
8.96 ±
0.96
28.34 ±
1.45
17.67 ±
1.24
16.29 ±
0.51
35.34
8.70 ±
22.23
13.75 ±
± 1.54
0.94
± 1.34
1.13
12.87 ±
1.11
37.24 ±
1.57
18.32 ±
1.26
17.59 ±
0.74
17m
17n
34.97
41.44
>50
>50
22.09 ±
12.63 ±
1.10
38.66 ±
1.58
14.16 ±
1.15
19.18 ±
0.62
± 1.68
± 1.54
1.78
–
6.23 ±
0.67
9.05 ±
0.96
8.77 ±
0.56
9.37 ±
0.39
29.59
35.45
33.54 ±
± 1.45
± 1.55
1.76
a
Antitumor activity was assayed by exposure for 72 h to substances and
expressed as concentration required to inhibit tumor cell proliferation by 50%
(IC50). Data are presented as the mean ± SDs of three independent experiments
17o
17p
23.15
8.97 ±
13.54
23.56 ±
b
± 1.36
0.95
± 1.13
1.12
Positive control.
19.51
15.54
10.87
9.92 ±
± 1.29
± 1.19
± 1.03
0.99
were evaluated. Among them, compound 17v showed the best bioac-
tivity against H1975 cells. Further mechanism research showed that
compound 17v significantly inhibited the migration and colony forma-
tion of H1975 cells and induced cell cycle arrest at G2/M phase.
Moreover, DAPI staining, apoptosis and western blot experiments indi-
cated that compound 17v induced H1975 cells apoptosis through the
activation of intrinsic apoptotic pathway. In addition, molecular dock-
ing revealed that compound 17v could bind well to the pocket of EGFR.
Taken together, these results suggested that compound 17v might be a
valuable lead compound for antitumor agents.
17q
11.96
21.26
14.47
5.84 ±
± 1.07
± 1.32
± 1.16
0.76
17r
17s
17t
>50
28.58
>50
14.03 ±
± 1.45
1.14
25.21
>50
>50
43.22
23.94 ±
± 1.40
± 1.63
1.37
>50
>50
>50
(continued on next page)
4

L. Liu et al.
Bioorganic & Medicinal Chemistry Letters 51 (2021) 128268
General procedure for the synthesis of compound 10
Table 2 (continued)
Compounds
R2
IC50
(
μ
M)
Compound 10 was synthesized according to the literature21. Com-
pound 9 (5.20 mmol, 1.00 eq), iron powder (36.43 mmol, 7.00 eq) and
ammonium chloride (15.61 mmol, 3.00 eq) in 30 mL of absolute
methanol were heated at 65 ℃ for 2 h. The reaction mixture was filtered
under reduced pressure, water was added to the filtrate, and the mixture
was extracted with ethyl acetate, and then purified by column chro-
matography to get the target compound 10 (yellow solid, yield 63%).
PC-3
MGC-
803
MCF-7
H1975
17u
17v
45.26
12.41
39.48
24.32 ±
± 1.65
± 1.09
± 0.35
1.38
7.88 ±
10.85
8.81 ±
2.27 ±
General procedure for the synthesis of compound 13
1.03
± 1.03
1.27
0.35
17w
17x
17y
>50
11.44
>50
3.06 ±
Compound 13 was synthesized according to the literature22. Potas-
sium hydroxide (32.59 mmol, 1.50 eq) was dissolved in 50 mL absolute
ethanol, and then thiourea (26.07 mmol, 1.20 eq) and ethyl 4,4,4-tri-
fluoroacetoacetate (21.73 mmol, 1.00 eq) were added. The reaction
mixture was stirred and heated under reflux for 6 h. Upon completion,
the precipitated product was filtered off and washed with ethanol to
afford the product 13 (white solid, yield 79%).
± 1.05
0.48
40.59
24.47
44.93
24.17 ±
± 1.60
± 1.38
± 1.65
1.38
23.83
12.22
36.91
6.32 ±
± 1.37
± 0.08
± 1.56
0.80
17z
16d
7.35 ±
0.86
5.77 ±
0.76
10.09
± 1.04
7.08 ±
0.85
16.74 ±
1.22
5.65 ±
0.98
9.80 ±
0.99
4.77 ±
0.44
General procedure for the synthesis of compounds 14a-z
5-FUb
–
6.23 ±
9.05 ±
8.77 ±
9.37 ±
Compounds 14a-z were synthesized according to the literature23
.
0.67
0.96
0.56
0.39
Potassium hydroxide (7.11 mmol, 1.50 eq) and compound 13 (5.69
mmol, 1.20 eq) in 25 mL of water was stirred at room temperature for 5
min. Next, diverse benzyl chlorides (1.00 eq) were dissolved in 3 mL of
acetone and added to the above mixture. The reaction mixtures were
heated to 80 ◦C for 1.5 h. Upon completion, the precipitated products
were filtered off and washed with water to afford the products 14a-z
(yield 68–90%).
a
Antitumor activity was assayed by exposure for 72 h to substances and
expressed as concentration required to inhibit tumor cell proliferation by 50%
(IC50). Data are presented as the mean ± SDs of three independent experiments
b
Positive control
Table 3
Anti-proliferative activities [IC50 / (
μ
mol•Lꢀ 1)] of 16d and 17v against human
normal cell linea.
General procedure for the synthesis of compounds 15a-z
Compounds
IC50
(
μM)
SIc
Compounds 15a-z were synthesized according to the literature23
.
H1975
GES-1
Compounds 14a-z (2.10 mmol 1.00 eq) were dissolved in 15 mL 1,4-
Dioxane, then excessive POCl3 (2–3 mL) was slowly added. The reac-
tion mixtures were heated to 90 ◦C for 1 h, cooled to room temperature,
and then the mixtures were added to ice water (100 mL) while stirring.
The precipitates were filtered under reduced pressure and washed with
water to yield compounds 15a-z (yield 57–76%).
16d
4.77 ± 0.44
2.27 ± 0.35
9.37 ± 0.39
31.47 ± 0.79
37.42 ± 0.86
19.27 ± 0.58
6.60
16.48
2.06
17v
5-FUb
a
Cells were treated with different concentrations of the compounds for 72 h.
IC50 values are the Mean ± SD of three separate experiments.
b
Positive control. SIc = IC50(GES-1)/IC50(H1975)
General procedure for the synthesis of compounds 16a-z
Experimental
Compound 10 (787.62 μmol, 1.20 eq) and absolute K2CO3 (984.53
General procedure
μ
mol, 1.50 eq) were dissolved in 5 mL DMF and then compounds 15a-z
(1.00 eq) were added, next, the reaction mixtures were heated to 80 ◦C
for 10 h. When the reactions were completed, extracted with water and
ethyl acetate, and then purified by column chromatography to get the
target compounds 16a-z. All of the target compounds have never been
reported in published articles.
Reagents and solvents were purchased from commercial sources and
were used without further purification. All reactions were monitored by
thin-layer chromatography (TLC) with detection by UV light (254 nm).
1H and 13C NMR spectra were respectively determined with a 400 and
101 MHz spectrometer. High-resolution mass spectra of target com-
pounds were recorded on a Waters Q-T Micromass spectrometer using
electrospray ionization. The melting points were measured on an X-5
micromelting apparatus.
N-(3-((2-((4-methylbenzyl)thio)-6-(trifluoromethyl)pyrimidin-4-yl)
amino)phenyl)acrylamide (16a) white solid, yield 61%, mp
176.4–177.8℃. 1H NMR (400 MHz, DMSO‑d6) δ 10.24 (s, 1H), 10.21 (s,
1H), 8.13 (s, 1H), 7.42 (d, J = 6.6 Hz, 1H), 7.36–7.28 (m, 2H), 7.25 (d, J
= 8.0 Hz, 2H), 7.07 (d, J = 7.9 Hz, 2H), 6.87 (s, 1H), 6.44 (dd, J = 17.0,
10.1 Hz, 1H), 6.26 (dd, J = 17.0, 1.9 Hz, 1H), 5.76 (dd, J = 10.1, 1.9 Hz,
1H), 4.35 (s, 2H), 2.25 (s, 3H). 13C NMR (101 MHz, DMSO‑d6) δ 171.71,
163.17, 160.25, 151.91, 139.47, 138.62, 136.19, 134.31, 131.76,
129.14, 128.86, 126.98, 124.71, 121.98, 119.25, 115.98, 114.93,
111.80, 34.01, 20.61. HR-MS (ESI): Calcd for C22H20F3N4OS [M + H]+:
445.1310, found: 445.1307.
General procedure for the synthesis of compound 9
Compound 9 was synthesized according to the literature21. 3-nitroa-
niline (7.24 mmol, 1.00 eq) was dissolved in 20 mL acetonitrile, then
triethylamine (10.86 mmol, 1.50 eq) and acryloyl chloride (10.86 mmol,
1.50 eq) were slowly added in an ice bath. The reaction mixture was
stirred at room temperature for 2 h, and then it was added to ice water
(100 mL) with constant stirring. After 5 min, the precipitate was filtered
under reduced pressure and washed with water to yield product 9
(brown solid, yield 82%).
N-(3-((2-((3-methylbenzyl)thio)-6-(trifluoromethyl)pyrimidin-4-yl)
amino)phenyl)acrylamide (16b) white solid, yield 57%, mp
164.6–165.8℃. 1H NMR (400 MHz, DMSO‑d6) δ 10.24 (s, 1H), 10.21 (s,
1H), 8.15 (s, 1H), 7.42 (d, J = 6.6 Hz, 1H), 7.34–7.28 (m, 2H), 7.16 (t, J
= 6.7 Hz, 3H), 7.03 (d, J = 4.0 Hz, 1H), 6.87 (s, 1H), 6.45 (dd, J = 17.0,
5

L. Liu et al.
Bioorganic & Medicinal Chemistry Letters 51 (2021) 128268
Figure 2. Compound 17v inhibited the proliferation of H1975 cells. (A) H1975 cell colonies treated with compound 17v at different concentrations for 9 days, and
the quantitative analysis of colony formation inhibition rate. (B) Effect of compound 17v on cell cycle in H1975 cells, and the quantitative analysis of cell cycle
distribution of H1975 cells for 24 h. (C) Scratch assay of H1975 cells with different concentrations of compound 17v. The dates were represented as the Mean ± SD.
All experiments were performed at least three times.
10.1 Hz, 1H), 6.27 (dd, J = 17.0, 2.0 Hz, 1H), 5.76 (dd, J = 10.1, 2.0 Hz,
1H), 4.35 (s, 2H), 2.21 (s, 3H). 13C NMR (101 MHz, DMSO‑d6) δ 171.65,
163.18, 160.24, 152.26, 139.49, 138.60, 137.39, 137.32, 131.77,
129.70, 129.12, 128.18, 127.62, 126.95, 125.97, 121.98, 119.26,
116.03, 114.92, 111.84, 34.20, 20.81. HR-MS (ESI): Calcd for
N-(3-((6-(trifluoromethyl)-2-((3-(trifluoromethyl)benzyl)thio)
pyrimidin-4-yl)amino)phenyl)acrylamide (16e) white solid, yield 59%,
mp 179.1–180.2℃. 1H NMR (400 MHz, DMSO‑d6) δ 10.26 (s, 1H), 10.21
(s, 1H), 8.15 (s, 1H), 7.78 (s, 1H), 7.69 (d, J = 7.6 Hz, 1H), 7.58 (d, J =
7.8 Hz, 1H), 7.50 (t, J = 7.7 Hz, 1H), 7.39 (s, 1H), 7.35–7.26 (m, 2H),
6.88 (s, 1H), 6.45 (dd, J = 17.0, 10.1 Hz, 1H), 6.26 (dd, J = 17.0, 1.9 Hz,
1H), 5.76 (dd, J = 10.1, 2.0 Hz, 1H), 4.49 (s, 2H). 13C NMR (101 MHz,
DMSO‑d6) δ 171.18, 163.18, 160.26, 152.20, 139.72, 139.49, 138.54,
133.04, 131.76, 129.27, 129.12, 128.77, 126.91, 125.58, 123.68,
122.69, 121.95, 119.23, 115.94, 114.95, 111.74, 33.51. Calcd for
C
22H20F3N4OS [M + H]+: 445.1310, found: 445.1311.
N-(3-((2-((2-methylbenzyl)thio)-6-(trifluoromethyl)pyrimidin-4-yl)
amino)phenyl)acrylamide (16c) off-white solid, yield 62%, mp
162.2–163.3℃. 1H NMR (400 MHz, DMSO‑d6) δ 10.26 (s, 1H), 10.21 (s,
1H), 8.16 (s, 1H), 7.42 (d, J = 7.2 Hz, 1H), 7.37–7.31 (m, 1H), 7.28 (dd,
J = 12.5, 7.9 Hz, 2H), 7.21–7.13 (m, 2H), 7.09 (td, J = 7.2, 1.6 Hz, 1H),
6.89 (s, 1H), 6.44 (dd, J = 17.0, 10.1 Hz, 1H), 6.24 (dd, J = 17.0, 2.0 Hz,
1H), 5.74 (dd, J = 10.1, 2.0 Hz, 1H), 4.42 (s, 2H), 2.36 (s, 3H). 13C NMR
(101 MHz, DMSO‑d6) δ 171.72, 163.17, 160.26, 152.26, 139.47, 138.64,
136.57, 134.72, 131.74, 130.16, 129.74, 129.12, 127.42, 126.93,
125.89, 121.98, 119.25, 115.91, 114.91, 111.78, 32.67, 18.78. HR-MS
(ESI): Calcd for C22H20F3N4OS [M + H]+: 445.1310, found: 445.1310.
N-(3-((6-(trifluoromethyl)-2-((4-(trifluoromethyl)benzyl)thio)
C
22H17F6N4OS [M + H]+: 499.1027, found: 499.1048.
N-(3-((6-(trifluoromethyl)-2-((2-(trifluoromethyl)benzyl)thio)
pyrimidin-4-yl)amino)phenyl)acrylamide (16f) white solid, yield 54%,
mp174.6–175.1℃. 1H NMR (400 MHz, DMSO‑d6) δ 10.31 (s, 1H), 10.19
(s, 1H), 8.12 (s, 1H), 7.73 (t, J = 8.3 Hz, 2H), 7.61 (t, J = 7.5 Hz, 1H),
7.49 (t, J = 7.6 Hz, 1H), 7.43 (s, 1H), 7.30–7.24 (m, 2H), 6.92 (s, 1H),
6.43 (dd, J = 17.0, 10.1 Hz, 1H), 6.22 (dd, J = 17.0, 1.9 Hz, 1H), 5.73
(dd, J = 10.1, 1.9 Hz, 1H), 4.61 (s, 2H). 13C NMR (101 MHz, DMSO‑d6) δ
171.04, 163.16, 160.35, 152.21, 139.51, 138.56, 135.57, 132.76,
131.73, 129.08, 127.96, 127.32, 126.87, 126.09, 125.68, 122.95,
121.93, 119.20, 115.90, 115.00, 111.79, 31.02. Calcd for C22H17F6N4OS
[M + H]+: 499.1027, found: 499.1098.
pyrimidin-4-yl)amino)phenyl)acrylamide (16d) white solid, yield 52%,
mp 166.1–167.3℃. 1H NMR (400 MHz, DMSO‑d6) δ 10.26 (s, 1H), 10.22
(s, 1H), 8.13 (s, 1H), 7.60 (q, J = 8.5 Hz, 4H), 7.38 (s, 1H), 7.35–7.26 (m,
2H), 6.88 (s, 1H), 6.46 (dd, J = 17.0, 10.1 Hz, 1H), 6.27 (dd, J = 17.0,
2.0 Hz, 1H), 5.76 (dd, J = 10.1, 2.0 Hz, 1H), 4.48 (s, 2H). 13C NMR (101
MHz, DMSO‑d6) δ 171.14, 163.20, 160.35, 152.25, 142.91, 139.50,
138.52, 131.74, 129.15, 127.73, 127.41, 126.97, 125.04, 122.83,
121.94, 119.21, 116.18, 115.09, 112.01, 33.56. Calcd for C22H17F6N4OS
[M + H]+: 499.1027, found: 499.1028.
N-(3-((2-((4-methoxybenzyl)thio)-6-(trifluoromethyl)pyrimidin-4-yl)
amino)phenyl)acrylamide (16 g) light yellow solid, yield 70%, mp
132.6–133.4℃. 1H NMR (400 MHz, DMSO‑d6) δ 10.24 (s, 1H), 10.21 (s,
1H), 8.13 (s, 1H), 7.44 (d, J = 6.3 Hz, 1H), 7.34–7.27 (m, 4H), 6.87 (s,
1H), 6.83 (d, J = 8.7 Hz, 2H), 6.45 (dd, J = 17.0, 10.1 Hz, 1H), 6.27 (dd,
6

L. Liu et al.
Bioorganic & Medicinal Chemistry Letters 51 (2021) 128268
Figure 3. Compound 17v induced apoptosis in H1975 cells and possible mechanisms. (A) Analysis of apoptosis of H1975 Cells with DAPI staining. Yellow arrow
indicates nuclear lysis. (B) Apoptotic H1975 cells were analyzed with Annexin V-FITC/PI double staining. Quantitative analysis of apoptosis rates by flow cyometry.
(C) Expression of apoptosis-related proteins including Bax, Bcl-2 and P53. The GAPDH was used as a control. The dates were represented as the Mean ± SD. All
experiments were performed at least three times. *p < 0.5, **p < 0.01, ***p < 0.001 compared with the control.
Figure 4. The 2D and 3D representation of the predicted binding model of compounds 16d (A) and 17v (B) with EGFR (PDB code: 5GNK).
J = 17.0, 2.0 Hz, 1H), 5.76 (dd, J = 10.1, 2.0 Hz, 1H), 4.35 (s, 2H), 3.71
(s, 3H). 13C NMR (101 MHz, DMSO‑d6) δ 171.79, 163.18, 160.26,
158.32, 152.27, 139.48, 138.64, 131.76, 130.10, 129.14, 126.98,
124.53, 121.99, 119.26, 116.02, 114.94, 113.71, 111.82, 54.98, 33.77.
Calcd for C22H20F3N4O2S [M + H]+: 461.1259, found: 461.1258.
N-(3-((2-((3-methoxybenzyl)thio)-6-(trifluoromethyl)pyrimidin-4-yl)
7

L. Liu et al.
Bioorganic & Medicinal Chemistry Letters 51 (2021) 128268
amino)phenyl)acrylamide (16 h) white solid, yield 72%, mp
151.7–152.4℃. 1H NMR (400 MHz, DMSO‑d6) δ 10.26 (s, 1H), 10.21 (s,
1H), 8.16 (s, 1H), 7.44 (d, J = 6.3 Hz, 1H), 7.34–7.26 (m, 2H), 7.19 (t, J
= 7.9 Hz, 1H), 6.95 (d, J = 7.1 Hz, 2H), 6.88 (s, 1H), 6.81–6.76 (m, 1H),
6.45 (dd, J = 17.0, 10.1 Hz, 1H), 6.27 (dd, J = 17.0, 2.0 Hz, 1H), 5.76
(dd, J = 10.1, 2.0 Hz, 1H), 4.39 (s, 2H), 3.66 (s, 3H). 13C NMR (101 MHz,
DMSO‑d6) δ 171.62, 163.18, 160.22, 159.14, 152.25, 139.50, 139.20,
138.63, 131.76, 129.36, 129.14, 126.97, 121.97, 121.12, 119.25,
115.88, 114.88, 114.31, 112.73, 111.67, 54.80, 34.17. Calcd for
5H), 6.88 (s, 1H), 6.45 (dd, J = 17.0, 10.1 Hz, 1H), 6.27 (dd, J = 17.0,
2.0 Hz, 1H), 5.76 (dd, J = 10.1, 2.0 Hz, 1H), 4.40 (s, 2H). 13C NMR (101
MHz, DMSO‑d6) δ 171.25, 163.18, 160.28, 152.22, 140.54, 139.50,
138.53, 132.74, 131.76, 130.06, 129.14, 128.83, 127.58, 126.97,
126.92, 121.96, 119.23, 116.05, 114.99, 111.84, 33.48. Calcd for
C
21H17ClF3N4OS[M + H]+: 465.0764, found: 465.0765.
N-(3-((2-((2-chlorobenzyl)thio)-6-(trifluoromethyl)pyrimidin-4-yl)
amino)phenyl)acrylamide (16o) white solid, yield 73%, mp
173.2–173.9℃. 1H NMR (400 MHz, DMSO‑d6) δ 10.28 (s, 1H), 10.21 (s,
1H), 8.10 (s, 1H), 7.54–7.46 (m, 2H), 7.41 (s, 1H), 7.34–7.27 (m, 3H),
7.25–7.20 (m, 1H), 6.89 (s, 1H), 6.44 (dd, J = 17.0, 10.1 Hz, 1H), 6.25
(dd, J = 17.0, 1.9 Hz, 1H), 5.75 (dd, J = 10.1, 2.0 Hz, 1H), 4.49 (s, 2H).
13C NMR (101 MHz, DMSO‑d6) δ 171.21, 163.18, 160.32, 152.23,
139.51, 138.56, 134.86, 133.35, 131.75, 131.19, 129.38, 129.17,
128.62, 127.13, 126.97, 121.96, 119.23, 116.01, 115.02, 111.84, 32.35.
Calcd for C21H17ClF3N4OS[M + H]+: 465.0764, found: 465.0765.
N-(3-((2-((4-bromobenzyl)thio)-6-(trifluoromethyl)pyrimidin-4-yl)
C
22H20F3N4O2S [M + H]+: 461.1259, found:461.1260.
N-(3-((2-((2-methoxybenzyl)thio)-6-(trifluoromethyl)pyrimidin-4-yl)
amino)phenyl)acrylamide (16i) yellow solid, yield 57%, mp
151.2–152.6℃. 1H NMR (400 MHz, DMSO‑d6) δ 10.23 (s, 1H), 10.21 (s,
1H), 8.07 (s, 1H), 7.46 (s, 1H), 7.33–7.28 (m, 3H), 7.27–7.22 (m, 1H),
7.00 (d, J = 8.1 Hz, 1H), 6.87 (s, 1H), 6.82 (t, J = 7.4 Hz, 1H), 6.44 (dd,
J = 17.0, 10.1 Hz, 1H), 6.25 (dd, J = 17.0, 2.0 Hz, 1H), 5.75 (dd, J =
10.1, 1.9 Hz, 1H), 4.34 (s, 2H), 3.82 (s, 3H). 13C NMR (101 MHz,
DMSO‑d6) δ 172.06, 163.18, 160.24, 157.20, 151.88, 139.49, 138.66,
131.76, 130.16, 129.14, 128.79, 126.97, 124.93, 122.01, 120.07,
119.28, 115.99, 114.91, 111.77, 110.85, 55.43, 29.44. Calcd for
amino)phenyl)acrylamide (16p) white solid, yield 64%, mp
163.4–164.1℃. 1H NMR (400 MHz, DMSO‑d6) δ 10.25 (s, 1H), 10.21 (s,
1H), 8.12 (s, 1H), 7.45 (d, J = 8.4 Hz, 2H), 7.40 (d, J = 6.5 Hz, 1H), 7.31
(dd, J = 15.1, 8.1 Hz, 4H), 6.88 (s, 1H), 6.45 (dd, J = 17.0, 10.1 Hz, 1H),
6.27 (dd, J = 17.0, 2.0 Hz, 1H), 5.76 (dd, J = 10.1, 2.0 Hz, 1H), 4.37 (s,
2H). 13C NMR (101 MHz, DMSO‑d6) δ 171.33, 170.27, 163.19, 160.31,
152.28, 139.49, 138.56, 137.29, 131.76, 131.09, 129.15, 126.97,
121.95, 120.07, 119.23, 116.12, 115.04, 111.95, 33.46. Calcd for
C
22H20F3N4O2S [M + H]+: 461.1259, found: 461.1258.
N-(3-((2-((4-fluorobenzyl)thio)-6-(trifluoromethyl)pyrimidin-4-yl)
amino)phenyl)acrylamide (16j) light yellow solid, yield 58%, mp
178.5–179.1℃. 1H NMR (400 MHz, DMSO‑d6) δ 10.26 (s, 1H), 10.21 (s,
1H), 8.13 (s, 1H), 7.44–7.38 (m, 3H), 7.34–7.26 (m, 2H), 7.11–7.06 (m,
2H), 6.88 (s, 1H), 6.45 (dd, J = 17.0, 10.1 Hz, 1H), 6.26 (dd, J = 17.0,
2.0 Hz, 1H), 5.76 (dd, J = 10.1, 2.0 Hz, 1H), 4.39 (s, 2H). 13C NMR (101
MHz, DMSO‑d6) δ 171.48, 163.18, 162.40, 160.33, 159.99, 152.24,
139.48, 138.59, 133.87, 131.75, 130.88, 129.15, 126.98, 121.97,
119.24, 115.12, 111.87, 64.98, 33.37. Calcd for C21H17F4N4OS[M +
H]+: 449.1059, found: 449.1058.
C
21H17BrF3N4OS[M + H]+: 509.0259, found: 509.0231.
N-(3-((2-((3-bromobenzyl)thio)-6-(trifluoromethyl)pyrimidin-4-yl)
amino)phenyl)acrylamide (16q) white solid, yield 54%, mp
168.1–169.3℃. 1H NMR (400 MHz, DMSO‑d6) δ 10.25 (s, 1H), 10.21 (s,
1H), 8.14 (s, 1H), 7.59 (s, 1H), 7.40 (dd, J = 10.6, 8.6 Hz, 3H), 7.31 (q, J
= 8.2 Hz, 2H), 7.23 (t, J = 7.8 Hz, 1H), 6.88 (s, 1H), 6.45 (dd, J = 17.0,
10.1 Hz, 1H), 6.27 (dd, J = 17.0, 2.0 Hz, 1H), 5.76 (dd, J = 10.1, 2.0 Hz,
1H), 4.39 (s, 2H). 13C NMR (101 MHz, DMSO‑d6) δ 171.25, 163.19,
160.27, 152.21, 140.80, 139.50, 138.53, 131.76, 131.71, 130.35,
129.82, 129.14, 127.97, 126.97, 121.96, 121.33, 119.23, 116.03,
114.99, 111.84, 33.43. Calcd for C21H17BrF3N4OS[M + H]+: 509.0259,
found: 509.0247.
N-(3-((2-((3-fluorobenzyl)thio)-6-(trifluoromethyl)pyrimidin-4-yl)
amino)phenyl)acrylamide (16 k) white solid, yield 48%, mp
145.8–146.3℃. 1H NMR (400 MHz, DMSO‑d6) δ 10.26 (s, 1H), 10.22 (s,
1H), 8.15 (s, 1H), 7.41 (d, J = 6.0 Hz, 1H), 7.34–7.27 (m, 3H), 7.21 (t, J
= 8.4 Hz, 2H), 7.08–7.01 (m, 1H), 6.89 (s, 1H), 6.46 (dd, J = 17.0, 10.1
Hz, 1H), 6.30–6.24 (m, 1H), 5.78–5.74 (m, 1H), 4.42 (s, 2H). 13C NMR
(101 MHz, DMSO‑d6) δ 171.31, 163.18, 163.10, 160.68, 160.29, 152.24,
140.76, 139.50, 138.56, 131.76, 130.08, 129.13, 126.95, 124.97,
121.96, 119.23, 115.57, 114.98, 113.68, 111.83, 33.60. Calcd for
N-(3-((2-((2-bromobenzyl)thio)-6-(trifluoromethyl)pyrimidin-4-yl)
amino)phenyl)acrylamide (16r) white solid, yield 51%, mp
180.8–181.7℃. 1H NMR (400 MHz, DMSO‑d6) δ 10.27 (s, 1H), 10.21 (s,
1H), 8.09 (s, 1H), 7.64 (d, J = 7.8 Hz, 1H), 7.52 (d, J = 7.3 Hz, 1H), 7.42
(s, 1H), 7.34–7.24 (m, 3H), 7.21 (t, J = 7.6 Hz, 1H), 6.89 (s, 1H), 6.44
(dd, J = 16.9, 10.1 Hz, 1H), 6.25 (d, J = 16.9 Hz, 1H), 5.75 (d, J = 10.2
Hz, 1H), 4.48 (s, 2H). 13C NMR (101 MHz, DMSO‑d6) δ 171.19, 163.19,
160.34, 152.24, 139.51, 138.56, 136.48, 132.66, 131.75, 131.26,
129.39, 129.19, 127.70, 126.96, 124.11, 121.97, 119.24, 116.03,
115.04, 111.88, 34.98. Calcd for C21H17BrF3N4OS[M + H]+: 509.0259,
found: 509.0266.
C
21H17F4N4OS[M + H]+: 449.1059, found: 449.1060.
N-(3-((2-((2-fluorobenzyl)thio)-6-(trifluoromethyl)pyrimidin-4-yl)
amino)phenyl)acrylamide (16 l) white solid, yield 69%, mp
165.1–166.0℃. 1H NMR (400 MHz, DMSO‑d6) δ 10.27 (s, 1H), 10.21 (s,
1H), 8.11 (s, 1H), 7.49–7.39 (m, 2H), 7.34–7.27 (m, 3H), 7.22–7.16 (m,
1H), 7.09 (td, J = 7.5, 1.0 Hz, 1H), 6.89 (s, 1H), 6.44 (dd, J = 17.0, 10.1
Hz, 1H), 6.25 (dd, J = 17.0, 2.0 Hz, 1H), 5.75 (dd, J = 10.1, 2.0 Hz, 1H),
4.43 (s, 2H). 13C NMR (101 MHz, DMSO‑d6) δ 171.19, 163.18, 161.72,
160.30, 159.27, 152.26, 139.50, 138.58, 131.74, 131.17, 129.42,
129.15, 126.95, 124.48, 124.25, 121.95, 119.22, 115.98, 114.98,
111.82, 27.87. Calcd for C21H17F4N4OS[M + H]+: 449.1059, found:
449.1054.
N-(3-((2-((2,4-dichlorobenzyl)thio)-6-(trifluoromethyl)pyrimidin-4-yl)
amino)phenyl)acrylamide (16 s) white solid, yield 75%, mp
166.2–167.1℃. 1H NMR (400 MHz, DMSO‑d6) δ 10.28 (s, 1H), 10.22 (s,
1H), 8.10 (s, 1H), 7.63 (d, J = 2.1 Hz, 1H), 7.51 (d, J = 8.3 Hz, 1H), 7.40
(d, J = 6.9 Hz, 1H), 7.34–7.27 (m, 3H), 6.89 (s, 1H), 6.45 (dd, J = 17.0,
10.1 Hz, 1H), 6.25 (dd, J = 17.0, 2.0 Hz, 1H), 5.76 (dd, J = 10.1, 2.0 Hz,
1H), 4.46 (s, 2H). 13C NMR (101 MHz, DMSO‑d6) δ 170.97, 163.18,
160.35, 152.20, 139.52, 138.52, 134.30, 134.17, 132.72, 132.32,
131.74, 129.17, 128.83, 127.19, 126.96, 121.94, 119.21, 116.04,
115.09, 111.92, 31.80. Calcd for C21H16Cl2F3N4OS[M + H]+: 499.0374,
found: 499.0514.
N-(3-((2-((4-chlorobenzyl)thio)-6-(trifluoromethyl)pyrimidin-4-yl)
amino)phenyl)acrylamide (16 m) white solid, yield 68%, mp
164.5–165.1℃. 1H NMR (400 MHz, DMSO‑d6) δ 10.25 (s, 1H), 10.22 (s,
1H), 8.13 (s, 1H), 7.39 (d, J = 8.4 Hz, 3H), 7.33 (t, J = 3.5 Hz, 2H),
7.31–7.27 (m, 2H), 6.88 (s, 1H), 6.45 (dd, J = 17.0, 10.1 Hz, 1H), 6.27
(dd, J = 17.0, 1.9 Hz, 1H), 5.78–5.72 (m, 1H), 4.39 (s, 2H). 13C NMR
(101 MHz, DMSO‑d6) δ 171.35, 163.19, 160.30, 152.29, 139.49, 138.56,
136.85, 131.75, 131.59, 130.73, 129.15, 128.17, 126.99, 121.95,
119.23, 116.09, 115.03, 111.91, 33.40. Calcd for C21H17ClF3N4OS[M +
H]+: 465.0764, found: 465.0763.
N-(3-((2-((2,6-dichlorobenzyl)thio)-6-(trifluoromethyl)pyrimidin-4-yl)
amino)phenyl)acrylamide (16 t) white solid, yield 76%, mp
179.1–179.9℃. 1H NMR (400 MHz, DMSO‑d6) δ 10.33 (s, 1H), 10.19 (s,
1H), 8.22 (s, 1H), 7.52 (d, J = 8.1 Hz, 2H), 7.47 (d, J = 7.2 Hz, 1H),
7.40–7.31 (m, 2H), 7.25 (d, J = 8.0 Hz, 1H), 6.94 (s, 1H), 6.42 (dd, J =
17.0, 10.1 Hz, 1H), 6.18 (dd, J = 17.0, 1.9 Hz, 1H), 5.71 (dd, J = 10.1,
1.9 Hz, 1H), 4.76 (s, 2H). 13C NMR (101 MHz, DMSO‑d6) δ 171.18,
N-(3-((2-((3-chlorobenzyl)thio)-6-(trifluoromethyl)pyrimidin-4-yl)
amino)phenyl)acrylamide (16n) white solid, yield 73%, mp
168.1–169.2℃. 1H NMR (400 MHz, DMSO‑d6) δ 10.26 (s, 1H), 10.22 (s,
1H), 8.15 (s, 1H), 7.44 (s, 1H), 7.39 (d, J = 6.1 Hz, 1H), 7.36–7.26 (m,
8

L. Liu et al.
Bioorganic & Medicinal Chemistry Letters 51 (2021) 128268
163.09, 160.23, 152.27, 139.46, 138.67, 135.08, 132.12, 131.71,
130.24, 129.14, 128.68, 126.82, 124.72, 121.93, 119.20, 115.78,
114.97, 111.69, 30.95. Calcd for C21H16Cl2F3N4OS[M + H]+: 499.0374,
found: 499.0373.
126.92, 124.69, 121.97, 119.24, 115.79, 114.89, 111.72, 99.36, 35.66,
10.44, 5.40. Calcd for C18H18F3N4OS[M + H]+: 395.1153, found:
395.1151.
N-(3-((2-(benzylthio)-6-(trifluoromethyl)pyrimidin-4-yl)ami-no)
phenyl)acrylamide (16u) white solid, yield 59%, mp 170.6–171.1℃. 1H
NMR (400 MHz, DMSO‑d6) δ 10.25 (s, 1H), 10.21 (s, 1H), 8.14 (s, 1H),
7.45 (d, J = 19.1 Hz, 1H), 7.38 (d, J = 7.0 Hz, 2H), 7.34–7.21 (m, 5H),
6.88 (s, 1H), 6.45 (dd, J = 17.0, 10.1 Hz, 1H), 6.27 (dd, J = 17.0, 2.0 Hz,
1H), 5.77–5.73 (m, 1H), 4.41 (s, 2H). 13C NMR (101 MHz, DMSO‑d6) δ
171.62, 163.18, 160.27, 152.22, 139.48, 138.62, 137.51, 131.76,
129.13, 128.92, 128.29, 127.01, 126.95, 121.98, 119.25, 115.99,
114.94, 111.98, 34.23. Calcd for C21H18F3N4OS[M + H]+: 431.1153,
found: 431.1154.
General procedure for the synthesis of compounds 17a-z
Compound 15d (536.61
μmol, 1.00 eq) and absolute K2CO3 (804.92
μ
mol, 1.50 eq) were dissolved in 5 mL DMF and then diverse hetero-
cyclics and anilines (1.20 eq) were added, next, the temperature were
heated to 80 ◦C for 6 h. When the reaction was completed, extracted
with water and ethyl acetate, and then purified by column chromatog-
raphy to get the target compounds 17a-z. All of the target compounds
have never been reported in published articles.
4-(piperazin-1-yl)-6-(trifluoromethyl)-2-((4-(trifluorome-thyl)benzyl)
thio)pyrimidine (17a) light green solid, yield 67%, mp 147.3–148.1℃.
1H NMR (400 MHz, DMSO‑d6) δ 7.69–7.61 (m, 4H), 6.95 (s, 1H), 4.43 (s,
2H), 3.61 (s, 4H), 2.71 (s, 4H), 1.23 (s, 1H). 13C NMR (101 MHz,
DMSO‑d6) δ 170.14, 160.85, 152.96, 143.45, 129.41, 127.66, 125.06,
122.98, 122.21, 119.53, 95.94, 45.18, 33.52. Calcd for C17H17F6N4S[M
+ H]+: 423.1078, found: 423.1078.
N-(3-((2-(((2-chlorothiazol-5-yl)methyl)thio)-6-(trifluoromethyl)
pyrimidin-4-yl)amino)phenyl)acrylamide (16v) white solid, yield 76%,
mp 164.5–165.1℃. 1H NMR (400 MHz, DMSO‑d6) δ 10.34 (s, 1H), 10.22
(s, 1H), 8.17 (s, 1H), 7.51 (s, 1H), 7.41–7.27 (m, 3H), 6.92 (s, 1H), 6.45
(dd, J = 17.0, 10.1 Hz, 1H), 6.27 (dd, J = 17.0, 2.0 Hz, 1H), 5.77 (dd, J
= 10.1, 2.0 Hz, 1H), 4.60 (s, 2H). 13C NMR (101 MHz, DMSO‑d6) δ
170.63, 163.21, 160.36, 152.26, 149.62, 140.11, 139.52, 139.25,
138.40, 131.75, 129.22, 127.02, 121.94, 119.22, 116.20, 115.12,
112.00, 26.24. Calcd for C18H14ClF3N5OS2[M + H]+: 472.0280, found:
472.0279.
4-(6-(trifluoromethyl)-2-((4-(trifluoromethyl)benzyl)thio)py-rimidin-4-
yl)morpholine (17b) white solid, yield 56%, mp 154.3–155.2℃. 1H NMR
(400 MHz, DMSO‑d6) δ 7.69–7.62 (m, 4H), 7.00 (s, 1H), 4.44 (s, 2H),
3.68 (s, 4H), 3.64 (s, 4H). 13C NMR (101 MHz, DMSO‑d6) δ 170.23,
161.22, 153.07, 143.38, 129.53, 127.70, 125.12, 122.87, 122.22,
119.49, 96.20, 65.60, 33.52. Calcd for C17H16F6N3OS [M + H]+:
424.0918, found: 424.0917.
N-(3-((2-((cyclohexylmethyl)thio)-6-(trifluoromethyl)pyrimidin-4-yl)
amino)phenyl)acrylamide (16w) white solid, yield 66%, mp
186.1–187.5℃. 1H NMR (400 MHz, DMSO‑d6) δ 10.19 (s, 2H), 8.05 (s,
1H), 7.49 (s, 1H), 7.36–7.25 (m, 2H), 6.84 (s, 1H), 6.46 (dd, J = 17.0,
10.1 Hz, 1H), 6.28 (dd, J = 17.0, 1.7 Hz, 1H), 5.82–5.72 (m, 1H), 3.03
(d, J = 6.8 Hz, 2H), 1.78 (d, J = 12.4 Hz, 2H), 1.65 (d, J = 11.3 Hz, 2H),
1.60–1.50 (m, 2H), 1.21–1.08 (m, 3H), 0.96 (dd, J = 22.4, 10.9 Hz, 2H).
13C NMR (101 MHz, DMSO‑d6) δ 172.38, 163.16, 160.22, 152.25,
139.45, 138.70, 131.81, 128.97, 126.90, 121.96, 119.23, 115.83,
114.91, 111.82, 37.06, 36.86, 31.91, 25.78, 25.43. Calcd for
4-(6-(trifluoromethyl)-2-((4-(trifluoromethyl)benzyl)thio)py-rimidin-4-
yl)thiomorpholine (17c) white solid, yield 67%, mp 168.1–169.2℃. 1
H
NMR (400 MHz, DMSO‑d6) δ 7.68 (d, J = 8.8 Hz, 2H), 7.65 (d, J = 9.0
Hz, 2H), 7.03 (s, 1H), 4.43 (s, 2H), 3.99 (s, 4H), 2.60 (s, 4H). 13C NMR
(101 MHz, DMSO‑d6) δ 170.31, 160.76, 153.23, 143.40, 129.36, 127.66,
125.16, 122.88, 122.23, 119.50, 96.44, 33.56, 26.02. Calcd for
C17H16F6N3S2[M + H]+: 440.0690, found: 440.0689.
C
21H24F3N4OS[M + H]+: 437.1623, found: 437.1622.
4-(4-methylpiperazin-1-yl)-6-(trifluoromethyl)-2-((4-(triflu-oromethyl)
benzyl)thio)pyrimidine (17d) light green solid, yield 67%, mp
94.5–95.1℃; 1H NMR (400 MHz, DMSO‑d6) δ 7.65 (s, 2H), 7.64 (d, J =
9.8 Hz, 2H), 6.99 (s, 1H), 4.43 (s, 2H), 3.67 (s, 4H), 2.32 (s, 4H), 2.19 (s,
3H). 13C NMR (101 MHz, DMSO‑d6) δ 170.21, 160.99, 153.08, 143.40,
129.40, 127.35, 125.12, 122.87, 122.23, 119.50, 96.15, 53.94, 45.44,
33.55. Calcd for C18H19F6N4S[M + H]+: 437.1235, found: 437.1233.
4-(4-ethylpiperazin-1-yl)-6-(trifluoromethyl)-2-((4-(trifle-oromethyl)
benzyl)thio)pyrimidine (17e) white solid, yield 59%, mp 84.5–85.3℃. 1H
NMR (400 MHz, DMSO‑d6) δ 7.68–7.62 (m, 4H), 6.99 (s, 1H), 4.43 (s,
2H), 3.67 (s, 4H), 2.35 (d, J = 5.7 Hz, 4H), 2.32 (d, J = 7.1 Hz, 2H), 1.01
(t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, DMSO‑d6) δ 170.29, 160.79,
153.06, 143.40, 129.38, 127.65, 125.12, 122.87, 122.23, 96.10, 51.72,
51.33, 43.76, 33.54, 11.78. Calcd for C19H21F6N4S[M + H]+: 451.1391,
found: 451.1390.
N-(3-((2-(((tetrahydro-2H-pyran-4-yl)methyl)thio)-6-(trifluoro-
methyl)pyrimidin-4-yl)amino)phenyl)acrylamide (16x) white solid, yield
51%, mp 103.5–104.1℃. 1H NMR (400 MHz, DMSO‑d6) δ 10.21 (s, 2H),
8.06 (s, 1H), 7.48 (d, J = 6.6 Hz, 1H), 7.35–7.26 (m, 2H), 6.85 (s, 1H),
6.46 (dd, J = 17.0, 10.1 Hz, 1H), 6.28 (dd, J = 16.9, 1.9 Hz, 1H), 5.77
(dd, J = 10.1, 1.9 Hz, 1H), 3.80 (dd, J = 11.3, 2.8 Hz, 2H), 3.20 (t, J =
10.9 Hz, 2H), 3.07 (t, J = 5.8 Hz, 2H), 1.78 (ddt, J = 14.9, 7.7, 3.7 Hz,
1H), 1.65 (d, J = 12.8 Hz, 2H), 1.26–1.20 (m, 2H). 13C NMR (101 MHz,
DMSO‑d6) δ 172.14, 163.21, 160.26, 152.22, 139.45, 138.66, 131.78,
129.03, 126.98, 121.97, 119.22, 115.93, 114.96, 111.87, 66.71, 36.23,
34.50, 31.84. Calcd for C20H22F3N4O2S[M + H]+:439.1416, found:
439.1416.
N-(3-((2-(((tetrahydrofuran-2-yl)methyl)thio)-6-(trifluorome-thyl)pyr-
imidin-4-yl)amino)phenyl)acrylamide (16y) yellow solid, yield 53%, mp
94.6–95.1℃. 1H NMR (400 MHz, DMSO‑d6) δ 10.28 (s, 1H), 10.22 (s,
1H), 8.08 (s, 1H), 7.45 (d, J = 6.6 Hz, 1H), 7.33 (dt, J = 13.0, 7.7 Hz,
2H), 6.89–6.84 (m, 1H), 6.51–6.42 (m, 1H), 6.28 (dd, J = 17.0, 1.8 Hz,
1H), 5.77 (dd, J = 10.1, 1.9 Hz, 1H), 4.30 (d, J = 4.3 Hz, 1H), 3.62–3.56
(m, 2H), 2.06–1.74 (m, 4H), 1.26 (dd, J = 16.7, 12.9 Hz, 2H). 13C NMR
(101 MHz, DMSO‑d6) δ 171.00, 163.22, 160.29, 151.89, 139.52, 138.49,
131.80, 129.19, 126.99, 121.90, 119.17, 115.95, 114.97, 111.76, 61.56,
44.65, 37.65, 33.70, 28.95. Calcd for C19H19F3N4O2S [M + Na]+:
447.1079, found: 447.1087.
4-(4-phenylpiperazin-1-yl)-6-(trifluoromethyl)-2-((4-(triflu-oromethyl)
benzyl)thio)pyrimidine (17f) yellow solid, yield 82%, mp 92.3–93.2℃.
1H NMR (400 MHz, DMSO‑d6) δ 7.66 (d, J = 7.0 Hz, 4H), 7.24 (t, J = 7.6
Hz, 2H), 7.06 (s, 1H), 6.97 (d, J = 8.1 Hz, 2H), 6.82 (t, J = 7.2 Hz, 1H),
4.46 (s, 2H), 3.85 (s, 4H), 3.20 (s, 4H). 13C NMR (101 MHz, DMSO‑d6) δ
170.28, 161.04, 153.13, 150.52, 143.40, 129.43, 128.95, 127.74,
125.10, 122.88, 122.25, 119.52, 119.27, 115.67, 96.28, 47.79, 33.60.
Calcd for C23H21F6N4S[M + H]+: 499.1391, found: 499.1390.
tert-butyl 4-(6-(trifluoromethyl)-2-((4-(trifluoromethyl)benzyl) thio)
pyrimidin-4-yl)piperazine-1-carboxylate (17 g) white solid, yield 81%,
mp 117.2–118.1℃. 1H NMR (400 MHz, DMSO‑d6) δ 7.66 (s, 4H), 6.99
(s, 1H), 4.44 (s, 2H), 3.69 (s, 4H), 3.39 (s, 4H), 1.42 (s, 9H). 13C NMR
(101 MHz, DMSO‑d6) δ 170.26, 161.09, 153.75, 153.13, 143.37, 129.42,
127.70, 125.07, 122.87, 122.21, 119.48, 96.24, 79.19, 33.58, 27.96.
Calcd for C22H25F6N4O2S[M + H]+: 523.1602, found:523.1603.
4-(4-(pyridin-2-yl)piperazin-1-yl)-6-(trifluoromethyl)-2-((4-(tri-
N-(3-((2-((cyclopropylmethyl)thio)-6-(trifluoromethyl)pyrimidin-4-yl)
amino)phenyl)acrylamide (16z) white solid, yield 70%, mp 90.5–91.7℃.
1H NMR (400 MHz, DMSO‑d6) δ 10.20 (s, 1H), 10.19 (s, 1H), 8.10 (s,
1H), 7.47 (d, J = 6.9 Hz, 1H), 7.35–7.25 (m, 2H), 6.85 (s, 1H), 6.47 (dd,
J = 17.0, 10.1 Hz, 1H), 6.28 (dd, J = 17.0, 2.0 Hz, 1H), 5.77 (dd, J =
10.1, 2.0 Hz, 1H), 3.08 (d, J = 7.2 Hz, 2H), 1.12 (ddd, J = 10.2, 6.0, 2.7
Hz, 1H), 0.54–0.46 (m, 2H), 0.30–0.23 (m, 2H). 13C NMR (101 MHz,
DMSO‑d6) δ 172.41, 160.16, 152.28, 139.41, 138.72, 131.80, 129.05,
fluoromethyl)benzyl)thio)pyrimidine (17 h) light yellow solid, yield 82%,
9

L. Liu et al.
Bioorganic & Medicinal Chemistry Letters 51 (2021) 128268
mp 116.2–117.0℃. 1H NMR (400 MHz, DMSO‑d6) δ 8.14 (d, J = 3.8 Hz,
1H), 7.67 (s, 4H), 7.57 (t, J = 7.8 Hz, 1H), 7.04 (s, 1H), 6.85 (d, J = 8.6
Hz, 1H), 6.67 (t, J = 5.8 Hz, 1H), 4.46 (s, 2H), 3.82 (s, 4H), 3.60 (s, 4H).
13C NMR (101 MHz, DMSO‑d6) δ 170.18, 161.09, 158.49, 153.06,
147.54, 143.42, 137.57, 129.45, 127.72, 125.10, 122.87, 122.26,
119.53, 113.22, 107.08, 96.28, 43.82, 33.58. Calcd for C22H20F6N5S[M
+ H]+: 500.1344, found: 500.1345.
trimethoxyphenyl)pyrimidin-4-amine (17p) brown solid, yield 74%, mp
146.4–147.2℃. 1H NMR (400 MHz, DMSO‑d6) δ 10.17 (s, 1H), 7.63 (d,
J = 7.9 Hz, 2H), 7.59 (d, J = 7.8 Hz, 2H), 6.99 (s, 2H), 6.82 (s, 1H), 4.48
(s, 2H), 3.75 (s, 6H), 3.64 (s, 3H). 13C NMR (101 MHz, DMSO‑d6) δ
171.11, 160.33, 152.87, 142.86, 134.18, 129.74, 128.23, 127.83,
127.51, 125.52, 125.13, 124.68, 122.82, 121.95, 119.22, 100.19, 60.07,
55.73, 33.52. Calcd for C22H20F6N3O3S[M + H]+: 520.1130, found:
520.1130.
4-(4-(pyrimidin-2-yl)piperazin-1-yl)-6-(trifluoromethyl)-2-((4-(tri-
fluoromethyl)benzyl)thio)pyrimidine (17i) white solid, yield 83%, mp
138.3–138.9℃. 1H NMR (400 MHz, DMSO‑d6) δ 8.41 (d, J = 4.2 Hz,
2H), 7.67 (s, 4H), 7.04 (s, 1H), 6.68 (t, J = 4.2 Hz, 1H), 4.46 (s, 2H), 3.82
(s, 8H). 13C NMR (101 MHz, DMSO‑d6) δ 170.26, 161.12, 161.00,
157.93, 153.08, 143.40, 129.45, 127.72, 125.11, 122.87, 122.25,
119.52, 110.45, 96.29, 42.60, 33.59. Calcd for C21H19F6N6S[M + H]+:
501.1296, found: 501.1295.
4-methyl-2-((6-(trifluoromethyl)-2-((4-(trifluoromethyl)ben-zyl)thio)
pyrimidin-4-yl)thio)thiazole (17q) white solid, yield 88%, mp
86.4–87.2℃. 1H NMR (400 MHz, DMSO‑d6) δ 7.76 (s, 1H), 7.66 (d, J =
8.5 Hz, 3H), 7.54 (d, J = 7.9 Hz, 2H), 4.38 (s, 2H), 2.40 (s, 3H). 13C NMR
(101 MHz, DMSO‑d6) δ 171.49, 153.99, 153.33, 150.17, 142.30, 142.23,
129.54, 127.98, 125.11, 122.78, 121.70, 118.62, 110.16, 33.54, 16.58.
Calcd for C17H12F6N3S3[M + H]+: 468.0098, found: 468.0097.
4-(pyrimidin-2-ylthio)-6-(trifluoromethyl)-2-((4-(trifluoro-methyl)
benzyl)thio)pyrimidine (17r) white solid, yield 85%, mp 102.5–103.4℃.
1H NMR (400 MHz, DMSO‑d6) δ 8.85 (d, J = 4.8 Hz, 2H), 8.32 (s, 1H),
7.67 (d, J = 8.1 Hz, 2H), 7.64 (d, J = 8.3 Hz, 2H), 7.52 (t, J = 4.8 Hz,
1H), 4.47 (s, 2H). 13C NMR (101 MHz, DMSO‑d6) δ 171.26, 170.55,
165.61, 158.85, 153.66, 142.31, 129.74, 127.96, 125.14, 121.50,
120.36, 118.76, 113.08, 33.81. Calcd for C17H11F6N4S2[M + H]+:
449.0329, found: 449.0330.
N-(4-fluorophenyl)-6-(trifluoromethyl)-2-((4-(trifluorometh-yl)benzyl)
thio)pyrimidin-4-amine (17j) yellow solid, yield 77%, mp
123.5–124.1℃. 1H NMR (400 MHz, DMSO‑d6) δ 10.22 (s, 1H),
7.68–7.55 (m, 6H), 7.20 (t, J = 8.6 Hz, 2H), 6.81 (s, 1H), 4.45 (s, 2H).
13C NMR (101 MHz, DMSO‑d6) δ 171.06, 160.29, 152.22, 142.92,
134.45, 129.52, 127.76, 127.44, 125.53, 123.00, 121.93, 119.20,
115.64, 115.41, 33.53. Calcd for C19H13F7N3S [M + H]+: 448.0718,
found: 448.0719
N-(p-tolyl)-6-(trifluoromethyl)-2-((4-(trifluoromethyl)benz-yl)thio)pyr-
imidin-4-amine (17 k) yellow solid, yield 72%, mp 112.3–113.2℃. 1H
NMR (400 MHz, DMSO‑d6) δ 10.12 (s, 1H), 7.64 (d, J = 8.1 Hz, 2H), 7.59
(d, J = 8.0 Hz, 2H), 7.44 (d, J = 5.8 Hz, 2H), 7.16 (d, J = 7.9 Hz, 2H),
6.80 (s, 1H), 4.44 (s, 2H), 2.28 (s, 3H). 13C NMR (101 MHz, DMSO‑d6) δ
171.04, 143.00, 135.56, 129.55, 129.31, 127.74, 127.42, 125.55,
125.04, 122.85, 121.97, 121.10, 120.10, 119.22, 33.50, 20.40. Calcd for
4-(pyridin-2-ylthio)-6-(trifluoromethyl)-2-((4-(trifluoromethyl) benzyl)
thio)pyrimidine (17 s) white solid, yield 80%, mp 77.3–78.1℃. 1H NMR
(400 MHz, DMSO‑d6) δ 8.67 (d, J = 3.9 Hz, 1H), 7.92 (t, J = 7.6 Hz, 1H),
7.85 (d, J = 7.7 Hz, 1H), 7.64 (d, J = 11.8 Hz, 3H), 7.51 (d, J = 6.0 Hz,
1H), 7.47 (d, J = 8.2 Hz, 2H), 4.30 (s, 2H). 13C NMR (101 MHz,
DMSO‑d6) δ 172.97, 171.23, 153.16, 150.87, 149.92, 142.31, 138.27,
130.21, 129.55, 127.91, 125.12, 124.60, 121.43, 118.70, 110.55, 33.51.
Calcd for C18H12F6N3S2[M + H]+: 448.0377, found: 448.0377.
N-(1H-tetrazol-5-yl)-6-(trifluoromethyl)-2-((4-(trifluorome-thyl)
benzyl)thio)pyrimidin-4-amine (17 t) white solid, yield 62%, mp
175.1–175.9℃. 1H NMR (400 MHz, DMSO‑d6) δ 16.14 (s, 1H), 11.95 (s,
1H), 7.66 (s, 4H), 7.38 (s, 1H), 4.52 (s, 2H). 13C NMR (101 MHz,
DMSO‑d6) δ 171.59, 159.42, 153.81, 142.98, 129.74, 127.82, 125.09,
122.83, 121.82, 119.01, 99.77, 33.60. Calcd for C14H10F6N7S [M + H]+:
422.0623, found: 422.0623.
C
20H16F6N3S [M + H]+: 444.0969, found: 444.0970
N-(4-methoxyphenyl)-6-(trifluoromethyl)-2-((4-(trifluorometh-yl)
benzyl)thio)pyrimidin-4-amine (17 l) light yellow solid, yield 69%, mp
104.5–105.1℃. 1H NMR (400 MHz, DMSO‑d6) δ 10.05 (s, 1H), 7.63 (d,
J = 7.6 Hz, 2H), 7.58 (s, 2H), 7.46 (s, 2H), 6.94 (d, J = 8.2 Hz, 2H), 6.76
(s, 1H), 4.42 (s, 2H), 3.75 (s, 3H). 13C NMR (101 MHz, DMSO‑d6) δ
170.99, 160.24, 156.09, 143.09, 129.56, 127.71, 127.40, 127.08,
125.55, 125.04, 122.89, 121.99, 119.21, 114.09, 55.18, 33.59. Calcd for
C
20H16F6N3OS[M + H]+: 460.0918, found: 460.0919.
4-((1-phenyl-1H-tetrazol-5-yl)thio)-6-(trifluoromethyl)-2-((4-(tri-
fluoromethyl)benzyl)thio)pyrimidine (17u) white solid, yield 57%, mp
106.5–107.1℃. 1H NMR (400 MHz, DMSO‑d6) δ 8.01 (s, 1H), 7.62 (d, J
= 7.7 Hz, 4H), 7.58 (d, J = 6.8 Hz, 3H), 7.51 (d, J = 7.9 Hz, 2H), 4.27 (s,
2H). 13C NMR (101 MHz, DMSO‑d6) δ 171.59, 168.72, 153.60, 146.58,
141.77, 133.05, 130.83, 129.60, 129.43, 127.91, 124.92, 122.76,
121.21, 118.44, 111.13, 33.56. Calcd for C20H13F6N6S2[M + H]+:
515.0547, found: 515.0551.
N-(3-chloro-4-fluorophenyl)-6-(trifluoromethyl)-2-((4-(triflu-oro-
methyl)benzyl)thio)pyrimidin-4-amine (17 m) yellow solid, yield 67%, mp
132.4–133.2℃. 1H NMR (400 MHz, DMSO‑d6) δ 10.36 (s, 1H), 7.96 (d,
J = 5.8 Hz, 1H), 7.63 (dd, J = 15.2, 8.5 Hz, 4H), 7.48 (d, J = 8.7 Hz, 1H),
7.40 (t, J = 9.0 Hz, 1H), 6.84 (s, 1H), 4.47 (s, 2H). 13C NMR (101 MHz,
DMSO‑d6) δ 171.14, 160.14, 152.33, 142.72, 135.46, 129.54, 127.83,
125.52, 125.11, 122.82, 122.44, 121.86, 121.23, 119.36, 117.13,
116.92, 33.58. Calcd for C19H12ClF7N3S[M + H]+: 482.0329, found:
482.0328.
2-((6-(trifluoromethyl)-2-((4-(trifluoromethyl)benzyl)thio) pyrimidin-
4-yl)thio)-1,3,4-thiadiazole (17v) white solid, yield 65%, mp
123.1–124.0℃. 1H NMR (400 MHz, DMSO‑d6) δ 9.89 (s, 1H), 8.05 (s,
1H), 7.66 (d, J = 7.8 Hz, 2H), 7.56 (d, J = 7.9 Hz, 2H), 4.41 (s, 2H). 13C
NMR (101 MHz, DMSO‑d6) δ 171.58, 168.71, 158.78, 155.81, 153.34,
141.96, 129.65, 128.03, 125.19, 122.78, 121.37, 111.24, 33.70. Calcd
for C15H9F6N4S3[M + H]+: 454.9894, found: 454.9922.
N-(2,4-dichlorophenyl)-6-(trifluoromethyl)-2-((4-(trifluorome-thyl)
benzyl)thio)pyrimidin-4-amine (17n) yellow solid, yield 68%, mp
135.8–136.5℃. 1H NMR (400 MHz, DMSO‑d6) δ 10.01 (s, 1H), 7.77 (s,
1H), 7.70 (d, J = 8.6 Hz, 1H), 7.58 (d, J = 7.9 Hz, 2H), 7.46 (d, J = 8.7
Hz, 1H), 7.40 (d, J = 7.7 Hz, 2H), 6.94 (s, 1H), 4.31 (s, 2H). 13C NMR
(101 MHz, DMSO‑d6) δ 170.96, 161.20, 152.56, 143.01, 133.65, 130.74,
129.62, 129.43, 129.29, 128.78, 127.76, 124.89, 122.82, 121.88,
119.15, 99.13, 33.44. Calcd for C19H12Cl2F6N3S[M + H]+: 498.0033,
found: 498.0034.
2-methyl-5-((6-(trifluoromethyl)-2-((4-(trifluoromethyl)benzyl) thio)
pyrimidin-4-yl)thio)-1,3,4-thiadiazole (17w) white solid, yield 82%, mp
143.4–144.1℃. 1H NMR (400 MHz, DMSO‑d6) δ 7.99 (s, 1H), 7.66 (d, J
= 8.0 Hz, 2H), 7.55 (d, J = 7.9 Hz, 2H), 4.41 (s, 2H), 2.75 (s, 3H). 13
C
N1-(6-(trifluoromethyl)-2-((4-(trifluoromethyl)benzyl)thio) pyrimidin-
4-yl)benzene-1,3-diamine (17o) brown liquid, yield 68%. 1H NMR
(400 MHz, DMSO‑d6) δ 9.95 (s, 1H), 7.70–7.58 (m, 4H), 7.00 (t, J = 7.9
Hz, 1H), 6.81 (s, 1H), 6.77 (s, 1H), 6.37 (d, J = 7.7 Hz, 1H), 5.38 (s, 2H),
4.45 (s, 2H). 13C NMR (101 MHz, DMSO‑d6) δ 170.98, 160.42, 149.32,
143.15, 138.62, 129.69, 129.22, 127.70, 127.38, 125.04, 122.85,
121.99, 119.26, 110.35, 108.99, 106.68, 33.51. Calcd for C19H14F6N4S
[M + H]+: 445.0922, found: 445.0923.
NMR (101 MHz, DMSO‑d6) δ 171.60, 170.68, 169.22, 157.00, 154.88,
153.37, 142.07, 129.57, 128.00, 125.18, 121.37, 111.05, 33.66, 15.36.
Calcd for C16H11F6N4S3[M + H]+: 469.0050, found: 469.0048.
2-((6-(trifluoromethyl)-2-((4-(trifluoromethyl)benzyl)thio) pyrimidin-
4-yl)thio)-4,5-dihydrothiazole (17x) yellow solid, yield 83%, mp
107.2–108.1℃. 1H NMR (400 MHz, DMSO‑d6) δ 8.85 (s, 1H), 7.70 (s,
4H), 4.78 (t, J = 7.5 Hz, 2H), 4.57 (s, 2H), 3.51 (t, J = 7.5 Hz, 2H). 13
C
NMR (101 MHz, DMSO‑d6) δ 201.67, 171.55, 159.60, 154.12, 142.45,
6-(trifluoromethyl)-2-((4-(trifluoromethyl)benzyl)thio)-N-(3,4,5-
129.66, 127.97, 125.25, 121.61, 118.87, 104.68, 57.86, 33.90, 28.35.
10

L. Liu et al.
Bioorganic & Medicinal Chemistry Letters 51 (2021) 128268
Calcd for C16H12F6N3S3[M + H]+: 456.0098, found: 456.0099.
N-(6-methoxypyridin-3-yl)-6-(trifluoromethyl)-2-((4-(triflu-oromethyl)
benzyl)thio)pyrimidin-4-amine (17y) white solid, yield 72%, mp
129.6–130.3℃. 1H NMR (400 MHz, DMSO‑d6) δ 8.78 (s, 1H), 8.65 (d, J
= 5.0 Hz, 2H), 7.96 (s, 1H), 7.90 (d, J = 5.1 Hz, 2H), 7.56 (d, J = 8.0 Hz,
2H), 7.47 (d, J = 8.0 Hz, 2H), 4.36 (s, 2H). 13C NMR (101 MHz,
DMSO‑d6) δ 171.51, 170.49, 153.34, 153.31, 152.98, 152.74, 150.38,
142.08, 142.07, 139.74, 129.42, 127.99, 125.05, 124.60, 120.10,
110.72, 33.54. Calcd for C21H13F6N4S3[M + H]+: 531.0207, found:
531.0208.
for 24 h. After that, collected cells, fixed with pre-cooled 70% ethanol at
4℃ overnight and stained with 20 mg/mL PI and 20 mg/mL RNase A.
Then cell suspension was incubated for 40 min in the dark. Finally,
samples were analyzed for DNA content with flow cytometry (Becton,
Dickinson and Company, NJ).
Scratch assay
H1975 cells in the logarithmic growth phase were seeded in 6-well
plates at a density of 3.5 × 105 cells per well. After 24 h incubation,
5-(pyridin-4-yl)-2-((6-(trifluoromethyl)-2-((4-(trifluorometh-yl)benzyl)
thio)pyrimidin-4-yl)thio)thiazole (17z) light yellow solid, yield 77%, mp
92.9–93.7℃. 1H NMR (400 MHz, DMSO‑d6) δ 10.14 (s, 1H), 8.34 (s,
1H), 7.87 (d, J = 8.3 Hz, 1H), 7.62 (d, J = 7.8 Hz, 2H), 7.57 (d, J = 7.4
Hz, 2H), 6.84 (d, J = 8.8 Hz, 1H), 6.79 (s, 1H), 5.76 (s, 1H), 4.42 (s, 2H),
3.86 (s, 3H). 13C NMR (101 MHz, DMSO‑d6) δ 171.09, 160.62, 152.21,
142.93, 139.88, 129.50, 128.91, 127.71, 127.45, 125.52, 125.00,
122.82, 121.93, 119.20, 110.25, 53.18, 33.52. Calcd for C19H16F6N4OS
[M + H]+:461.0871, found:461.0870.
the cell surface was scratched by a 200 μL pipet tip. Cells were washed
three or four times with PBS and cultured in RPMI-1640 medium con-
taining 2% FBS. Taken photos with a microscope as control groups. After
cells were treated with different concentrations (0, 2, 4 μM) of com-
pound 17v for 24 or 48 h, take photos again as experimental groups.
Photoshop was used to process the images.
DAPI staining assay
H1975 cells in the logarithmic growth phase were seeded in 6-well
Cell culture and reagents
plates at 2 × 105 cells per well. After 24 h incubation, cells were
treated with different concentrations (0, 2, 4 μM) of compound 17v for
RPMI-1640 and MTT were purchased from Solarbio. Fetal Bovine
Serum (FBS) was purchased from Tianhang Biotechnology Co. Annexin
V-FITC/PI Apoptosis Detection Kit and Cell Cycle Detection Kit were
bought from Keygen Biotech. PC-3 (human prostate cancer cell line),
MGC-803 (human gastric cancer cell line), MCF-7 (human breast cancer
cell line) and H1975 (non-small cell lung cancer cell line) were obtained
from our laboratories (originally purchased from the CCTCC). The an-
tibodies used in western blotting (Bcl-2, Bax and p53) were all pur-
chased from CST. All cancer cells were cultured in RPMI 1640 medium
with 10% FBS and all of them were cultured at 37℃ in a humidified
incubator of 95% air and 5% CO2. Compounds were dissolved in DMSO
to prepare a stock solution with an initial concentration of 10 mM.
48 h. After that, the cells were washed two or three times with PBS and
fixed with pre-cooled methanol at 4℃ for 10 min, then incubated in the
DAPI dye solution in the dark. Photos were taken with a fluorescence
microscope (Nikon Eclipse TieS, Nikon Ltd, Japan), and the images were
processed with Photoshop.
Analysis of apoptosis by flow cytometry
H1975 cells in the logarithmic growth phase were seeded in 6-well
plates at a density of 1.5 × 105 cells per well. After 24 h incubation,
cells were treated with different concentrations (0, 1, 2, 4 μM) of com-
pound 17v for 48 or 72 h. The cells were collected, washed with PBS and
measured with the Annexin V-FITC/PI Apoptosis Detection Kit (Keygen
Biotech, China), according to the protocol. Then, the cells were detected
using the FAC-SCalibur flow cytometer (BD Biosciences, San Jose, CA,
USA), and data was analyzed by the Flow Jo software.
MTT assay
Cells in the logarithmic growth phase were seeded in 96-well plates
at 3000–5000 cells per well. After the cells were cultured for 24 h at
37℃, removed the medium and added 200 μL of medicated medium at
Western blot analysis
different concentrations to each well. After 72 h incubation, 20 μL of
MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide,
Solarbio) was added to each well at a final concentration of 5 mg/mL,
and cells were cultured for 4 h in a 37℃ incubator. After that, the me-
H1975 cells were seeded in cell culture dishes (100 mm). After 24 h
incubation, the cells were treated with different concentrations (0, 1, 2,
4 μM) of compound 17v for 48 h. Then, the cells were harvested with
dium was aspirated, 150 μL DMSO was then added to each well, and the
trypsin and lysed with cell/tissue RIPA lysis buffer (Beyotime Biotech-
nology) for 30 min. The supernatant was determined by a BCA Protein
Assay kit (Beyotime Biotechnology), denatured at 100 ◦C for 10 min, and
incubated at ꢀ 20 ◦C for Western Blot. After being separated by SDS-
PAGE, proteins were transferred to nitrocellulose (NC) membranes and
blocked by 5% skim milk at room temperature for 2 h. The membranes
were then incubated overnight at 4 ◦C with primary antibodies. After
washing the membrane with the secondary antibody (1: 10000) at room
temperature for 2 h, the membranes were washed with PBST. Blots
exposure was performed with an ECL chemiluminescence solution. The
data were analyzed using ImageJ software.
plate was shaken on the shaker for 10 min until the formazan dissolved
completely. The absorbance was measures at a wavelength of 490 nm
with a microplate reader, and the cell survival rate was measured. The
date of IC50 was calculated using SPASS 16.0 software, and the results
were Mean ± SD of three independent experiments.
Colony formation assay
Cells in the logarithmic growth phase were seeded in a 6-well plate at
1000 cells per well. After approximately 24 h incubation, different
concentrations (0, 1, 2, 4 μM) of compound 17v were treated for 9 days.
After washing 2–3 times with PBS, cells were fixed with ice methanol for
30 min and stained with crystal violet for 30 min. Images were photo-
graphed after drying the plate. After that, morphological analysis was
performed through microscopy (Leica EZ4W, China).
Molecular docking
Molecular docking study was performed with Molecular Operating
Environment software (MOE 2020). The co-crystal structure of the small
molecule with the EGFR (PDB ID: 5GNK) was obtained from the RCSB
protein database (www.rcsb.org). The “QuickPrep” function was used
for structural correction and protonation of receptor proteins. After that,
all solvent molecules were deleted and the protonation states of the
ionizable residues with pKa = 7 were set up. We used the co-crystal
ligand site as the docking site. The highest score conformation of the
Cell cycle analysis
H1975 cells in the logarithmic growth phase were seeded in 6-well
plates at a concentration of 2 × 105 per well. After 24 h incubation,
treated with different concentrations (0, 1, 2, 4 μM) of compound 17v
11

L. Liu et al.
Bioorganic & Medicinal Chemistry Letters 51 (2021) 128268
11 Woyach JA. Next Generation Small Molecules in Chronic Lymphocytic Leukemia.
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Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
13 Li X, Zuo Y, Tang G, et al. Discovery of a series of 2,5-diaminopyrimidine covalent
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Acknowledgments
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Project supported by the National Natural Science Foundation of
China (No. U1904163) and this work was supported by National Key
Research Program of Proteins (No. 2018YFE0195100) and Opening
Fund from State Key Laboratory of Esophageal Cancer Prevention &
Treatment (No. K2020000X).
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12