Synthesis, evaluation, and mechanism study of new tepotinib derivatives as antiproliferative agents

Article abstract of DOI:10.3390/molecules24061173

Inspired by the potent inhibition activity of the c-Met (mesenchymal?epithelial transition factor) inhibitor Tepotinib, a series of new Tepotinib derivatives were synthesized and evaluated for their ability to act as antiproliferative agents to find the l

Full text of DOI:10.3390/molecules24061173

molecules
Article
Synthesis, Evaluation, and Mechanism Study of New
Tepotinib Derivatives as Antiproliferative Agents
Niu-niu Zhang , Bai-jiao An, Yan Zhou, Xing-shu Li * and Ming Yan *
Institute of Drug Synthesis and Pharmaceutical Process, School of Pharmaceutical Sciences, Sun Yat-sen
University, Guangzhou 510006, China; zhangniuniuu@yeah.net (N.-n.Z.); 15521085401@163.com (B.-j.A.);
zhouy598@mail2.sysu.edu.cn (Y.Z.)
* Correspondence: lixsh@mail.sysu.edu.cn (X.-s.L.); yanming@mail.sysu.edu.cn (M.Y.);
Tel.: +86-137-2676-2798 (X.-s.L.); +86-136-4087-9357 (M.Y.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Jean Jacques Vanden Eynde
Received: 16 February 2019; Accepted: 20 March 2019; Published: 25 March 2019
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Inspired by the potent inhibition activity of the c-Met (mesenchymal epithelial transition
−
factor) inhibitor Tepotinib, a series of new Tepotinib derivatives were synthesized and evaluated for
their ability to act as antiproliferative agents to find the leading compounds with good activity and
limited side effects. Among them, compound 31e exhibited potent antiproliferative activity (IC50
(50% inhibitory concentration) = 0.026 µM) against hepatic carcinoma 97H (human liver cancer cell)
cells and, importantly, had very low inhibitory activity against normal cells. A mechanism study
demonstrated that 31e induced G1 phase (First growth phase or G indicating gap) arrest, inhibited
the phosphorylation of c-Met and its downstream signaling component, Akt (Protein Kinase B), and
also inhibited the migration of hepatic carcinoma 97H cells.
Keywords: antiproliferative agents; c-Met inhibitor; 97H cells; low toxicity
1. Introduction
Cancer is a major disease that seriously threatens human health. According to statistics from the
World Health Organization (WHO), an estimated 9.6 million patients died from cancer in 2018 [
Therefore, the development of effective anti-tumor drugs is an important task for medicinal chemists.
Mesenchymal epithelial transition factor (c-Met), the high affinity receptor for hepatocyte growth
factor (HGF), is a unique subfamily of receptor tyrosine kinases (RTKs) [ ]. Physiologically, HGF/c-Met
signaling plays important roles in cell growth, survival, motility, and morphogenesis [ ]. However,
1].
−
2
3
aberrant c-Met activation has been observed in a wide variety of human cancers, including liver and
lung cancer, as a consequence of gene amplification or rearrangement, transcriptional regulation, as
well as autocrine or paracrine ligand stimulation [4–9]. Due to its status as a proto-oncogene and
the correlation of its dysregulation with a poor prognosis, c-Met is becoming a promising target for
cancer therapy [10]. The present c-Met kinase inhibitors are divided into three groups according to
their binding modes and structural features. Type I c-Met kinase inhibitors, such as Crizotinib A,
are adenosine triphosphate (ATP)-competitive inhibitors with a U-shaped binding mode at the ATP
binding site (Figure 1) [11,12]. Type II c-Met kinase inhibitors are usually multi-targeted agents (for
example, Cabozantinib B) (Figure 1) [13]. Type I inhibitors are generally described as specific for c-Met
kinase and more selective than class II inhibitors. However, they have limited activity against the
Tyr1230His mutation that is present in certain human tumors [4]. Type III inhibitors include other
atypical c-Met kinase inhibitors, such as Tivantinib C (Figure 1) [14].
Molecules 2019, 24, 1173; doi:10.3390/molecules24061173
www.mdpi.com/journal/molecules

Molecules 2019, 24, 1173
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H
H
N
N
F
Cl
O
O
O
N
F
N
O
O
N
HN
O
Cl
N
NH2
Crizotinib A
Cabozantinib B
O
H
N
NC
N
N
N
N
N
H
O
H
N
O
N
O
H
Tepotinib D
Tivantinib C
Figure 1. The representative c-Met kinase inhibitors of different structural types.
In 2015, Dorsch et al. reported optimized pyridazinones as being potent and selective c-Met
kinase inhibitors [15]. We found that the optimal compound, Tepotinib D (Figure 1), displays excellent
in vitro potency and in vivo anti-tumor efficacy at low doses. According to the co-crystal structure
of Tepotinib D with c-Met, it belongs to the type I c-Met kinase inhibitors. The review by Manjunath
D. Ghate et al. described the recent advances of small molecule c-Met kinase inhibitors, including
several pyridazinone derivatives, but the similar structures of our compounds have not been reported
before [16]. Inspired by the interesting scaffold of Tepotinib, we decided to synthesize and evaluate
new Tepotinib derivatives to find leading compounds with good activity and limited side effects.
Our design is shown in Figure 2 and includes the following steps: (a) Changing the position of
nitrogen on the pyridazinone ring to provide pyrazin-2(1H)-one and pyrimidin-2(1H)-one derivatives
for the study of the structure–activity relationships; (b) evaluating the anti-tumor activity of the
derivatives with or without alkyl substitution on the methylene group; (c) examining the effect of
fluorine substitution on pyridin-2(1H)-one on the anti-tumor activity; and (d) investigating the effect
of side chains on the activity. Herein, we reported the synthesis, evaluation, and optimization of new
Tepotinib derivatives as anti-tumor agents.
Changing the pyridazinone
ring structure
Modification of
O
the side chain
NC
N
N
N
N
O
Modification of
methylene group
N
Figure 2. Design of new Tepotinib derivatives.
2. Results
2.1. Chemistry
The synthesis of target compounds 11a and 11b is summarized in Scheme 1. The conventional
Suzuki coupling reaction of (3-(hydroxymethyl)phenyl)boronic acid and 2-chloro-5-fluoropyrimidine
catalyzed with PdCl2(PPh3)2 gave (3-(5-fluoropyrimidin-2-yl)phenyl)methanol
5
with

Molecules 2019, 24, 1173
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a yield of 68%.
Chlorination of
5
in the presence of thionyl chloride provided
2-(3-(chloromethyl)phenyl)-5-fluoropyrimidine
1b followed by cyclization provided 2a and 2b
7b. Compound 10 was synthesized by the reaction of
6
[
. On the other hand, the oxidation of ketones 1a or
17], which reacted with compound to give 7a and
and . Finally, compounds 11a and 11b were
6
8
9
prepared by the reaction of 7a, 7b, and 10 under alkaline conditions.
F
F
OH
B
N
N
Cl
N
a
b
OH
OH
Cl
N
OH
N
+
N
F
3
4
5
6
O
O
O
N
N
R1
R1
NH
e
R1
N
N
c, d
6
N
F
R2
R2
g
R2
: R1 = CN; R2 = H
1a
: R1 = CN; R2 = H
: R1 = CN; R2 = H
7b:
2a
7a
1
: R1 = R2 = F
: R = R2 = F
R
1 = CN; R2 = H
1b
2b
S
f
NH2
S
N
.HCl
Cl
N
.HCl
+
NH2 NH2
NH
10
8
9
O
N
R1
N
N
N
: R1 = CN; R2 = H
: R1 = R2 = F
11a
11b
S
N
R2
◦
Scheme 1. Reagents and conditions: a) PdCl2(PPh3)2, Na2CO3, ethanol/toluene/water, 90 C, 8 h; b)
◦
◦
SOCl2, CHCl3, reflux; c) SeO2, dioxane:H2O = 10:1, reflux, 12 h; d) NaOH,
−
30 C; e) NaH, DMF/THF,
◦
0 C—room temperature, 12 h; f) dry ethanol, reflux; g) NaOH, DMF/H2O, 60 C, 8 h, N2.
The synthesis of compounds 18, 22, and 25 is shown in Scheme 2. CH3OCH2-protected
5-bromopyrimidin-2-ol 14 reacted with (3-cyanophenyl)boronic acid to provide 15, which was
converted to 16 in the presence of 10% hydrogen chloride. Compound 16 reacted with intermediate
6
to provide 3-(1-(3-(5-fluoropyrimidin-2-yl)benzyl)-2-oxo-1. 2-dihydropyrimidin-5-yl)benzonitrile
17, which reacted with 10 to give target compound 18. Compound 22 was synthesized from 19
via the Mitsunobu reaction and Suzuki coupling reaction. The reductive amination of 23 with
formaldehyde and sodium borohydride gave compound 24, which reacted with compound 16 under
alkaline conditions to provide compound 25.

Molecules 2019, 24, 1173
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N
O
O
Br
N
Br
CN
N
N
c
a
b
O
Cl
+
N
OH
N
O
O
12
13
14
15
N
O
N
O
.
e
d
HCl
CN
N
N
CN
NH
6
N
F
16
17
N
O
N
N
CN
N
S
N
18
Z
N
OH
Z
21a:
b
N
Z =
N
N
O
O
N
f
OH
N
3
Cl
N
21b:
Cl
N
Z =
Boc
19
20a-b
21a-21b
N
O
f
16
N
N
CN
21a
N
22
O
N
NH
N
O
O
N
N
h
g
21b
Cl
N
Cl
N
23
24
N
O
d
CN
N
N
16
N
O
25
N
Scheme 2. Reagents and conditions: a) N,N-diisopropylethylamine, dry CH2Cl2, 0 ◦C—room
temperature, 6 h; b) PdCl2(PPh3)2, Na2CO3, ethanol/toluene/water, 90 ◦C, 8 h; c) 10% aq. HCl,
◦
◦
MeOH, reflux; d) K2CO3, dry DMF, 80 C, 12 h; e) NaOH, DMF/H2O, 60 C, 8 h, N2; f) PPh3, DIAD,
◦
THF, 0 C—room temperature; g) SOCl2, CHCl3, reflux; h) 35% formaldehyde, NaBH4, MeOH.
The synthesis of compounds 31a–g is summarized in Scheme 3. Intermediate 27 was prepared by
the Suzuki coupling reaction of (3-acetylphenyl)boronic acid and 2-chloro-5-fluoropyrimidine. The
reduction of compound 27 with sodium borohydride and then chlorination with thionyl chloride
produced 29. In the presence of potassium carbonate, 29 reacted with compound 16 to give 30a, which
reacted with the corresponding amino alcohols or amino thiols to produce target compounds 31a–f
(the R and S configurations of 31e were prepared and are shown in the supporting information).
The fluorine-containing target compound 31g was prepared with a similar procedure, starting from
compound 14 and intermediate 30b.

Molecules 2019, 24, 1173
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F
F
O
OH
O
N
OH
N
B
Cl
N
c
a
b
OH
N
N
+
F
N
F
26
4
27
28
N
O
Cl
N
N
N
e or f
CN
d
N
N
F
29
30a
N
N
31a :Z=
31b :Z=
31c :Z=
31d :Z=
N
O
S
S
31e: Z=
O
O
N
N
CN
N
N
N
O
O
N
N
31f: Z=
Z
O
N
O
Br
N
N
N
F
N
N
O
O
F
14
30b
F
N
O
F
N
N
N
O
F
N
31g
Scheme 3. Reagents and conditions: a) PdCl2(PPh3)2, Na2CO3, ethanol/toluene/water, 90 ◦C, 8 h; b)
◦
◦
NaBH4, MeOH, 0 C—room temperature, 1 h; c) SOCl2, CHCl3, reflux; d) K2CO3, dry DMF, 80 C, 12 h;
◦
◦
e) 31a–31b: NaOH, DMF/H2O, 60 C, 8 h, N2; f) 31c–31g: NaH, dry DMF, 0 C—room temperature, 5 h.
The synthesis of target compounds 36a
the reaction of and commercially available 5-bromo-3-fluoropyridin-2(1H)-one 32. Then, the reaction
with aryl boric acids 34a
–c is listed in Scheme 4. Compound 33 was obtained by
6
–c
afforded the key intermediates, 35a
–
c, which reacted with 10 in the presence
of NaOH to provide target compounds 36a–c.
F
F
N
Br
F
O
+
a
b
34a-c
Cl
N
NH
32
O
N
N
Br
N
6
33
F
F
F
O
O
c
N
N
N
N
R1
R1
N
N
N
S
F
35a-c
F
CN
1
36a: R1=
36b:
R =
N
N
36c: R1
=
N
Scheme 4. Reagents and conditions: a) K2CO3, dry DMF, 80 ◦C, 12 h; b) PdCl2(PPh3)2, Na2CO3,
◦
◦
DME/DMF/water, 89 C, 12 h; c) NaOH, DMF/H2O, 60 C, 8 h, N2
.

Molecules 2019, 24, 1173
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The synthesis of target compound 39 is outlined in Scheme 5. Intermediate 29 reacted with
32 to produce compound 37. Compound 38 was prepared by the Suzuki coupling reaction of 37
and (3-cyanophenyl)boronic acid. Finally, the reaction of compound 38 with 10 afforded target
compound 39.
F
F
Cl
N
b
a
O
Br
F
N
+
N
N
Br
N
H
O
N
O
F
29
32
37
F
F
O
CN
N
N
c
CN
N
N
N
N
F
S
N
38
39
Scheme 5. Reagents and conditions: a) K2CO3, dry DMF, 80 ◦C, 12 h; b) PdCl2(PPh3)2, Na2CO3,
◦
◦
DME/DMF/water, 89 C, 12 h; c) NaOH, DMF/H2O, 60 C, 8 h, N2.
2.2. Biological Evaluation
2.2.1. In Vitro Growth Inhibition of Human Cancer Cell Lines.
Hepatocellular carcinoma (HCC), a primary liver cancer with a high mortality rate, accounts for
90% of all liver cancers. It is the most common malignant tumor worldwide, especially in Asia, Africa,
and southern Europe. China is the “hardest hit” by liver cancer, and the number of annual deaths from
this condition in China accounts for about 50% of the total number of liver cancer deaths worldwide.
C-Met overexpression, mutational activation, and amplification have been found in some types of
cancer, including liver cancer [4,5]. Therefore, hepatic carcinoma 97H cells were selected for the
evaluation of the anti-tumor activity of the synthesized compounds, and the results are summarized in
Table 1. Most compounds displayed good antiproliferative activity. Among them, compound 31e
5-(3-cyanophenyl)-1-(1-(3-(5-((1-methylpiperidin-4-yl)methoxy)pyrimidin-2-yl)phenyl)ethyl)pyrimidin
-2(1H)-one, exhibited the best result with 26 nM of the IC50 value. Further study of the structure–activity
relationship indicated the pyridazinone ring or pyrimidin-2(1H)-one ring plays an important role
in antiproliferative activity. Compounds 11a and 11b, pyrazin-2(1H)-one derivatives, that were
obtained by shifting the nitrogen from the C-1 to C-5 position of pyridazin-3(2H)-one, displayed
,
poor activity (IC50 values: 9.8
which were obtained by shifting the nitrogen from the C-1 to the C-4 position, resulted in significant
improvement in anti-proliferative potency (IC50: 0.36–0.55 M). Compounds 31a , which have a
methyl substitution on the methylene carbon between pyrimidin-2(1H)-one and the benzene ring,
provided 0.3606 M to 0.026 M of the IC50 value. This indicates that the methyl substitution at
this position is favorable for antiproliferative activity. Compounds Rac-31e and (R) 31e with cyclic
piperidinylmethyloxy substitution showed excellent activity (IC50 = 0.026 M, 0.018 M). However,
the (S)-31e almost lost activity (IC50 = 1.834 M). The replacement of the pyridazinone moiety with
3-fluoropyridin-2(1H)-one moiety afforded compounds 36a . Compound 36a showed the best result
µM for 11a and 9.1 µM for 11b). Interestingly, compounds 18 and 25,
µ
–g
µ
µ
-
µ
µ
µ
–
c
of the 3-fluoropyridin-2(1H)-one derivatives, indicating that the benzonitrile group is more favorable
than the other two substitutions.

Molecules 2019, 24, 1173
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Table 1. Structures and antiproliferative activity of target compounds.
O
N
O
N
R1
N
N
R1
N
N
R3
N
N
R4
S
N
R2
R2
11a-11b
18-31g
F
O
N
N
R1
R3
N
S
N
36a-39
Comp.
R1
R2
R3
R4
IC50 of Against 97H cells (µM)
11a
11b
CN
F
H
F
-
-
-
-
9.810 ± 0.790
9.10 ± 0.670
18
22
CN
CN
H
H
H
H
0.443 ± 0.004
0.559 ± 0.018
25
CN
CN
H
H
H
0.361 ± 0.012
0.186 ± 0.007
31a
CH3
31b
31c
31d
CN
CN
CN
H
H
H
CH3
CH3
CH3
0.149 ± 0.012
0.216 ± 0.087
0.196 ± 0.056
(Rac)-31e
(R)-31e
(S)-31e
CN
CN
CN
H
H
H
CH3
CH3
CH3
0.026 ± 0.003
0.018 ± 0.006
1.834 ± 0.026
31f
31g
36a
36b
CN
F
H
F
-
CH3
CH3
H
0.248 ± 0.034
0.332 ± 0.044
0.149 ± 0.035
3.807 ± 0.045
-
-
-
H
36c
-
H
-
0.671 ± 0.056
39
-
-
CH3
-
-
-
0.206 ± 0.013
0.013 ± 0.008
Tepotinib
-
2.2.2. Effect of Compound 31e on Cell Cycle Progression
To evaluate the ability of compound 31e to disrupt the regulated cell cycle distribution, we
performed flow cytometry analysis to determine the arrest effect of 31e on the G1 transition, a rigorously
regulated process in the cell cycle. The results in Figure 3 show that 31e can arrest cells in the G1
phase in a dose-dependent manner. When hepatic carcinoma 97H cells were treated with 10 nM of 31e

Molecules 2019, 24, 1173
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for 24 h, the population of cells in the G1 phase (83.84%) increased compared to that of the vehicle
(DMSO, 70.42%), along with concomitant losses in the G2/M phase. This phenomenon was more
obvious at a concentration of 100 nM. A population of up to 90.88% cells was found in the G1 phase.
Correspondingly, a population of only 5.78% cells was found in the G2/M phase.
Figure 3. Effect of 31e on cell cycle progression. (A) 97H cells (3
×
105 cells/sample) were treated with
increasing concentrations of 31e for 24 h, cells were harvested, fixed with 70% ethanol, and stained
with propidium iodide (PI). The cellular DNA content was then determined by flow cytometry analysis.
(B) Quantitative analysis of the percentage of cells in each cell cycle phase was analyzed by EXPO32
ADC analysis software. The data were presented as the mean ± SEM *P < 0.05, significantly different
compared with the control by t test.
2.2.3. Inhibition of the Migration of Hepatic Carcinoma 97H Cells with Compound 31e
Tumor cell migration is one of the major causes of death in cancer patients. To evaluate the
inhibitory ability of 31e against tumor cell migration, hepatic carcinoma 97H cells (5
×
104 cells per
well) were seeded in six-well plates with or without the presence of 31e and cultured as confluent
monolayers. The results shown in Figure 4 demonstrate that 31e can inhibit tumor cells’ migration. At
a concentration of 10 nM, 31e displayed moderate inhibition of migration. Migration was significantly
suppressed when 31e was used at a dosage of 50 nM. Tumor cells’ migration was completely
suppressed with 100 nM of 31e.
Figure 4. Effect of 31e on the migration of hepatic carcinoma 97H cells. Hepatic carcinoma 97H cells
(50
×
104 cells) suspended in free serum Dulbecco’s Modified Eagle Medium (DMEM) containing 31e
for 36 h were photographed under a phase contrast microscopy (magnification: 4
results were expressed as the mean ± SD of at least three independent experiments.
×
objective). All

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2.2.4. Inhibition of Phosphorylation of c-Met and its Downstream Signaling Component, Akt, with
Compound 31e
To further study the antiproliferative mechanism of compound 31e, we analyzed its effects on
the phosphorylation of c-Met and its downstream signaling component, Akt, in hepatic carcinoma
97H cells, which overexpress c-Met. As shown in Figure 5A, when hepatic carcinoma 97H cells were
treated with compound 31e at a concentration of 10 or 50 nM for 1 h, the phosphorylation level of Akt
and c-Met was suppressed in a dose-dependent manner. When 100 nM of compound 31e was used,
the phosphorylation of both c-Met and Akt was completely inhibited. When 97H cells were treated at
the same concentration of compound 31e for 2 h, the inhibition of c-Met and Akt phosphorylation was
more obvious (Figure 5B).
Figure 5. Compound 31e inhibits c-Met phosphorylation and downstream signaling in cancer cell lines.
Cells treated with compound 31e for 1 h or 2 h were then lysed and subjected to Western blot analysis.
2.2.5. Selectivity of 31e towards Other Cancer Cells and Normal Human Cells
To evaluate the selectivity of 31e towards other cancer cells and normal human cells, the
antiproliferative activities against Pc9 (human non-small cell lung cancer cells), Hela (human epithelial
cervical cancer cells), SJSA1 (human osteosarcoma cells), LO2 (human normal cells), and HLF (human
embryonic lung fibroblast cells) were examined. As shown in Table 2, compound 31e barely inhibited
the cancer cells (Pc9, Hela, SJSA1) and normal cells (LO2, HLF), demonstrating that it exclusively
inhibits 97H cancer cells. The reason for this remains to be further investigated.
Table 2. The inhibitory activity of compound 31e against cancer cells (97H, Pc9, Hela, SJSA1), human
normal cells (LO2 and HLF), and c-Met enzyme.
IC50 (µM)
Comp.
Pc9
Hela
SJSA1
LO2
HLF
C-Met
31e
Tepotinib
42.9 ± 0.13
>50
11.22 ± 0.32
>50
12.17 ± 0.35
>50
10.33 ± 0.46
>50
9.44 ± 0.15
0.032 ± 0.06
8.105
±
0.012
0.004
±
0.001
3. Experimental Section
3.1. Chemistry
1H-NMR and 13C-NMR spectra were recorded on a Bruker AVANCE 400 or 500 spectrometer
(Karlsruhe, Germany). Chemical shifts of protons are reported in parts per million downfield from
tetramethylsilane. Peaks are labeled as single (s), broad singlet (br), doublet (d), triplet (t), double
doublet (dd), doublet of triplets (dt), or multiplet (m). The high-resolution mass spectra were analyzed
on a SHIMADZU LCMS-IT-TOF mass spectrometer. The purity of the synthesized compounds was
determined by high-performance liquid chromatography (HPLC) (Agilent, Palo Alto, CA, USA) with

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a TC-C18 column (250 mm
×
4.6 mm, 5
µ
m) using methanol (1/1000 diethylamine)/water mobile
phase (0.50 mL/min). Melting points were determined in open capillary tubes on a MPA100 Optimelt
automated melting point system (Stanford Research Systems, San Francisco, CA, USA,). All chemicals
were purchased from Sigma-Aldrich and Alfa Aesar chemical companies (Shanghai, China) and were
used without further purification.
Synthesis of 5-(3-cyanophenyl)-1-(3-(5-((3-(dimethylamino)propyl)thio)pyrimidin-2-yl)benzyl)
pyrazin-2(1H)-one (11a). In a 200 mL two-necked round-bottomed flask provided with a magnetic
stirrer and condenser, 50 mmol of thiourea
9 were dissolved in 60 mL of absolute ethanol. Compound
8
(3.161 mg, 20 mmol) was added in one portion in the thiourea solution. After heating at reflux
for 12 h, the reaction mixture was concentrated by rotary evaporation under reduced pressure. The
desired compound 10 was obtained as a white solid and was further used without purification [18].
NaOH (135 mg, 3.375 mmol) in 0.5 mL water was added to a mixture of 7a (287.25 mg,
0.75 mmol) and 10 (444 mg) in DMF (5 mL) under nitrogen, and the mixture was stirred at room
◦
temperature for 15 min. Then, the mixture was stirred at 60 C under nitrogen for 8 h. The
reaction mixture was allowed to cool to room temperature. The aqueous phase was extracted with
dichloromethane (30 mL
×
3). The combined organic layer was washed with H2O (15 mL) and
brine (10 mL), and then dried over anhydrous Na2SO4, filtered, and evaporated in vacuo. The
residue was purified by flash chromatography over silica gel (DCM/MeOH = 40:1–10:1) to give
5-(3-cyanophenyl)-1-(3-(5-((3-(dimethylamino)propyl)thio)pyrimidin-2-yl)benzyl)pyrazin-2(1H)-one
(11a) (224 mg, 62%) as a white solid. m.p. 106.3–107.9 ◦C. 1H-NMR (400 MHz, CDCl3)
δ 8.74 (s, 1H),
8.46–8.39 (m, 2H), 8.32 (s, 1H), 8.01 (s, 1H), 7.93 (d, J = 8.0 Hz, 1H), 7.64 (s, 1H), 7.58 (d, J = 7.7 Hz, 1H),
7.55–7.47 (m, 3H), 5.27 (s, 2H), 3.03 (t, J = 7.2 Hz, 2H), 2.43 (t, J = 7.0 Hz, 2H), 2.24 (s, 6H), 1.91–1.76
(m, 2H); 13C-NMR (101 MHz, CDCl3)
δ 161.18, 157.41, 155.35, 149.17, 138.08, 136.67, 134.95, 131.70,
131.28, 130.82, 129.79, 129.70, 129.08, 128.52, 128.04, 125.10, 118.62, 113.06, 57.87, 52.29, 45.34, 31.60,
27.04. HRMS (ESI) calculated for C27H26N6OS [M + H]+: 483.1962, found: 483.1947. Purity: 98.3%
(by HPLC).
5-(3,5-difluorophenyl)-1-(3-(5-((3-(dimethylamino)propyl)thio)pyrimidin-2-yl)benzyl)pyrazin-2(1H)
-one (11b)
.
Compound 11b was prepared via a similar procedure of 11a
.
White solid. m.p.
1
143.7–145.2 ◦C. Yield: 65%. H-NMR (500 MHz, CDCl3)
δ
8.74(s, 2H), 8.46–8.39 (m, 2H), 8.30 (s, 1H),
7.56 (s, 1H), 7.54–7.46 (m, 2H), 7.25–7.18 (m, 2H), 6.82–6.65 (m, 1H), 5.25 (s, 2H), 3.03 (t, J = 7.2 Hz, 2H),
2.42 (t, J = 7.0 Hz, 2H), 2.24 (s, 6H), 1.92–1.76 (m, 2H). 13C-NMR (126 MHz, CDCl3)
164.52 (JCF
247.5 Hz), 163.42 (JCF = 246.25 Hz), 161.21, 157.45, 155.37, 148.97, 138.74 (JCF = 10.0 Hz), 138.10, 134.92,
131.64 (J = 3.0 Hz), 130.81, 130.76, 129.76, 128.49, 128.00, 125.03, 107.79 (JCF = 26.25 Hz), 107.78
δ
=
CF
(JCF = 13.75 Hz), 103.21 (JCF = 26.25 Hz), 57.89, 52.24, 45.38, 31.65, 27.11. HRMS (ESI) calculated for
C26H25N5OF2S [M + H]+: 494.1821, found: 494.1823. Purity: 99.7% (by HPLC).
5-(3-cyanophenyl)-1-(3-(5-((3-(dimethylamino)propyl)thio)pyrimidin-2-yl)benzyl)pyrimidin-2(1H)
-one (18). Compound 18 was prepared via a similar procedure of 11a. White oil. Yield: 77.1%.
1H-NMR (500 MHz, CDCl3)
δ 8.82 (d, J = 3.2 Hz, 1H), 8.69 (s, 2H), 8.42 (s, 1H), 8.38 (d, J = 7.6 Hz, 1H),
7.99 (d, J = 3.2 Hz, 1H), 7.65 (s, 1H), 7.60 (t, J = 7.1 Hz, 2H), 7.55–7.46 (m, 3H), 5.28 (s, 2H), 3.01 (t,
J = 7.2 Hz, 2H), 2.40 (t, J = 7.0 Hz, 2H), 2.21 (s, 6H), 1.86–1.76 (m, 2H). 13C-NMR (126 MHz, CDCl3)
δ
164.70, 161.23, 157.43, 155.73, 145.11, 138.15, 135.10, 134.64, 131.48, 131.11, 130.93, 130.32, 130.14,
129.83, 129.37, 128.58, 128.18, 118.25, 116.63, 57.95, 54.44, 45.43, 31.67, 27.17. HRMS (ESI) calculated for
C27H26N6OS [M + H]+: 483.1962, found: 483.1942. Purity: 98.3% (by HPLC).
Synthesis of 5-(3-cyanophenyl)-1-(3-(5-(3-(dimethylamino)propoxy)pyrimidin-2-yl)benzyl)pyrimidin
-2(1H)-one (22). Compound 21a (157.85 mg, 0.55 mmol) and triphenylphosphine (328 mg, 1.25 mmol)
were added successively to a suspension of 16 (116.75 mg, 0.5 mmol) in THF (5 mL) under nitrogen.
A solution of diisopropyl azodicarboxylate (104.49 mg, 0.6 mmol) in THF (1 mL) was then slowly
added dropwise with ice cooling. The resultant solution was stirred at room temperature for 12 h. The

Molecules 2019, 24, 1173
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aqueous phase was extracted with dichloromethane (30 mL
×
3). The combined organic layer was
washed with H2O (20 mL) and brine (10 mL), and then dried over anhydrous Na2SO4, filtered, and
evaporated in vacuo. The residue was purified by flash chromatography over silica gel (DCM/MeOH
◦
1
= 40:1–10:1) to give 22
8.84 (d, J = 3.0 Hz, 1H), 8.45 (s, 2H), 8.40–8.33 (m, 2H), 7.91 (d, J = 3.0 Hz, 1H), 7.66 –7.57 (m, 3H),
7.56–7.47 (m, 3H), 5.29 (s, 2H), 4.18 (t, J = 6.2 Hz, 2H), 2.53 (t, J = 6.2 Hz, 2H), 2.31 (s, 6H), 2.07–2.00 (m,
2H). 13C-NMR (126 MHz, CDCl3)
164.56, 156.67, 155.65, 151.81, 144.80, 143.87, 138.53, 134.73, 134.60,
[14]. Yellow solid. m.p. 122.3–123.6 C. Yield: 45%. H-NMR (500 MHz, CDCl3)
δ
δ
131.43, 130.25, 130.20, 130.09, 129.76, 129.26, 128.14, 127.78, 118.14, 116.54, 113.67, 67.04, 55.84, 54.31,
45.37, 27.17. HRMS (ESI) calculated for C27H26N6O2 [M + H]+: 467.2190, found: 467.2185. Purity:
96.784% (by HPLC).
Synthesis of 5-(3-cyanophenyl)-1-(3-(5-((1-methylpiperidin-4-yl)methoxy)pyrimidin-2-yl)benzyl)
pyrimidin-2(1H)-one (25). 2-(3-(chloromethyl)phenyl)-5-((1-methylpiperidin-4-yl)methoxy)pyrimidine
24 (99 mg, 0.3 mmol, 1 eq.) and potassium carbonate (124 mg, 0.9 mmol, 3 eq.) were added to a
◦
suspension of 16 (77 mg, 0.33 mmol) in dry DMF (2 mL), and the mixture was stirred at 80 C for 12 h.
The reaction mixture was allowed to cool to room temperature. The aqueous phase was extracted with
dichloromethane (10 mL
×
3). The combined organic layer was washed with H2O (5 mL) and brine
(10 mL), and then dried over anhydrous Na2SO4, filtered, and evaporated in vacuo. The residue was
purified by flash chromatography over silica gel (DCM/MeOH = 30:1–10:1) to give compound 25
[
15].
1
White solid. m.p. 116.7–118.2 ◦C. Yield: 48%. H-NMR (500 MHz, CDCl3)
δ
8.84 (d, J = 2.9 Hz, 1H),
8.44 (s, 2H), 8.39–8.33 (m, 2H), 7.89 (d, J = 2.6 Hz, 1H), 7.65–7.60 (m, 2H), 7.60–7.56 (m, 1H), 7.56–7.47
(m, 3H), 5.29 (s, 2H), 3.96 (d, J = 5.6 Hz, 2H), 2.98 (d, J = 11.1 Hz, 2H), 2.34 (s, 3H), 2.04 (t, J = 11.8 Hz,
2H), 1.89–1.81 (m, 3H), 1.54–1.46 (m, 2H). 13C-NMR (126 MHz, CDCl3)
δ 164.56, 156.70, 155.63, 151.85,
144.72, 143.82, 138.52, 134.73, 134.61, 131.43, 130.25, 130.22, 130.08, 129.77, 129.26, 128.15, 127.77, 118.13,
116.53, 113.68, 73.28, 55.16, 54.30, 46.21, 35.17, 28.69. HRMS (ESI) calculated for C29H28N6O2 [M + H]+:
493.2347, found: 493.2351. Purity: 97.8% (by HPLC).
5-(3-cyanophenyl)-1-(1-(3-(5-((2-(dimethylamino)ethyl)thio)pyrimidin-2-yl)phenyl)ethyl)pyrimidin
-2(1H)-one (31a). Compound 31a was prepared via a similar procedure of 11a. Yellow oil. Yield: 66%.
1H NMR (400 MHz, CDCl3)
δ
8.78 (d, J = 3.3 Hz, 1H), 8.74 (s, 2H), 8.48 (s, 1H), 8.42 (d, J = 7.5 Hz, 1H),
7.71 (d, J = 3.3 Hz, 1H), 7.63–7.53 (m, 3H), 7.52–7.45 (m, 3H), 6.35 (q, J = 6.9 Hz, 1H), 3.08 (t, J = 7.1 Hz,
2H), 2.60 (t, J = 7.1 Hz, 2H), 2.28 (s, 6H), 1.90 (d, J = 7.0 Hz, 3H). 13C-NMR (126 MHz, CDCl3)
164.01,
δ
161.33, 157.54, 155.44, 142.40, 138.86, 138.26, 134.89, 131.45, 130.97, 130.31, 130.15, 129.84, 129.32, 128.64,
126.92, 118.22, 116.76, 113.72, 58.36, 56.32, 45.37, 32.02, 19.19. HRMS (ESI) calculated for C27H26N6OS
[M + H]+: 483.1962, found: 483.1949. Purity: 98% (by HPLC).
5-(3-cyanophenyl)-1-(1-(3-(5-((3-(dimethylamino)propyl)thio)pyrimidin-2-yl)phenyl)ethyl)pyrimidin
-2(1H)-one (31b). Compound 31b was prepared via a similar procedure of 11a. Yellow oil. Yield: 75%.
1H NMR (500 MHz, CDCl3)
δ 8.78 (d, J = 3.2 Hz, 1H), 8.73 (s, 2H), 8.49 (s, 1H), 8.42 (d, J = 7.6 Hz, 1H),
7.70 (d, J = 3.2 Hz, 1H), 7.63–7.58 (m, 1H), 7.57–7.46 (m, 5H), 6.36 (q, J = 6.9 Hz, 1H), 3.03 (t, J = 7.2 Hz,
2H), 2.42 (t, J = 7.0 Hz, 3H), 2.23 (s, 6H), 1.91 (d, J = 7.0 Hz, 3H), 1.88–1.77 (m, 2H). 13C-NMR (126
MHz, CDCl3)
δ 164.04, 161.27, 157.50, 155.49, 142.43, 138.82, 138.32, 134.88, 131.49, 131.02, 130.33,
130.31, 130.18, 129.86, 129.34, 128.66, 126.92, 118.24, 116.83, 113.74, 57.97, 56.37, 45.46, 31.70, 27.18,
19.21. HRMS (ESI) calculated for C28H28N6OS [M + H]+: 497.2118, found: 497.2108. Purity: 97.1%
(by HPLC).
Synthesis of 5-(3-cyanophenyl)-1-(1-(3-(5-(3-(dimethylamino)propoxy)pyrimidin-2-yl)phenyl)ethyl)
pyrimidin-2(1H)-one (31c). A solution of 3-(dimethylamino)propan-1-ol (113.3 mg, 1.1 mmol, 2 eq.)
in dry DMF( 5 mL) was added to a suspension of NaH (43.56 mg, 3.3 eq.) in dry DMF (5 mL) at 0 ◦C.
After 30 mins, 5-(3-cyanophenyl)-1-(1-(3-(5-fluoropyrimidin-2-yl)phenyl)ethyl)pyrimidin-2(1H)-one
30a (210 mg, 0.55 mmol, 1 eq.) was added. After the reaction mixture was stirred for 5 h, the reaction
was quenched with water (15 mL) and the mixture was extracted with dichloromethane (20 mL
×

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3). The combined organic layer was dried over anhydrous Na2SO4 and filtered. After the solvent
was removed in vacuo, the crude product was purified by◦column chromatography (DCM/MeOH =
1
50:1–10:1) to afford 31c as a white solid [19]. m.p. 68.3–69.9 C. Yield: 62%. H-NMR (400 MHz, CDCl3)
δ
8.78 (d, J = 3.4 Hz, 1H), 8.46 (s, 2H), 8.42 (s, 1H), 8.35 (d, J = 7.7 Hz, 1H), 7.73 (d, J = 3.4 Hz, 1H),
7.64–7.58 (m, 1H), 7.56–7.42 (m, 5H), 6.35 (q, J = 7.0 Hz, 1H), 4.21 (t, J = 6.1 Hz, 2H), 2.75 (t, J = 7.3 Hz,
2H), 2.47 (s, 6H), 2.24–2.09 (p, 2H), 1.90 (d, J = 7.0 Hz, 3H). 13C-NMR (101 MHz, CDCl3)
163.98,
δ
156.99, 155.48, 151.75, 144.00, 142.53, 138.71, 138.62, 134.92, 131.44, 130.32, 130.19, 129.77, 129.44, 129.35,
128.22, 126.55, 118.28, 116.76, 113.69, 66.75, 56.44, 55.79, 44.79, 26.49, 19.24. HRMS (ESI) calculated for
C28H28N6O2 [M + HCOO]−: 525.2256, found: 525.2238. Purity: 98.3% (by HPLC).
5-(3-cyanophenyl)-1-(1-(3-(5-(2-(dimethylamino)ethoxy)pyrimidin-2-yl)phenyl)ethyl)pyrimidin-2
(1H)-one (31d). Compound 31d was prepared via a similar procedure of 31c. White solid. m.p.
1
53.3-55.0 ◦C. Yield: 74%. H-NMR (400 MHz, CDCl3)
δ 8.80 (d, J = 3.3 Hz, 1H), 8.51 (s, 2H), 8.45 (s,
1H), 8.38 (d, J = 7.7 Hz, 1H), 7.72 (d, J = 3.4 Hz, 1H), 7.64–7.58 (m, 1H), 7.57–7.42 (m, 5H), 6.38 (q,
J = 7.0 Hz, 1H), 4.24 (t, J = 5.4 Hz, 2H), 2.83 (t, J = 5.4 Hz, 2H), 2.40 (s, 6H), 1.91 (d, J = 7.0 Hz, 3H).
13C-NMR (101 MHz, CDCl3)
δ 163.94, 156.94, 155.45, 151.78, 144.05, 142.52, 138.67, 138.59, 134.89,
131.40, 130.27, 130.15, 129.73, 129.44, 129.32, 128.19, 126.53, 118.2−4, 116.72, 113.66, 66.88, 58.10, 56.40,
45.90, 19.18. HRMS (ESI) calculated for C27H26N6O2 [M + HCOO] : 511.2099, found: 511.2074. Purity:
98% (by HPLC).
5-(3-cyanophenyl)-1-(1-(3-(5-((1-methylpiperidin-4-yl)methoxy)pyrimidin-2-yl)phenyl)ethyl)pyrimidin
-2(1H)-one (31e). Compound 31e was prepared via a similar procedure of 31c. Yellow solid. m.p.
1
98.2–99.9 ◦C. Yield: 76%. H-NMR (500 MHz, DMSO-d6)
δ
9.04 (d, J = 3.2 Hz, 1H), 8.72 (d, J = 3.2 Hz,
1H), 8.64 (s, 2H), 8.33 (s, 1H), 8.27–8.18 (m, 2H), 8.03 (d, J = 8.0 Hz, 1H), 7.81 (d, J = 7.7 Hz, 1H), 7.65 (t,
J = 7.8 Hz, 1H), 7.59–7.45 (m, 2H), 6.02 (q, J = 7.0 Hz, 1H), 4.04 (d, J = 5.8 Hz, 2H), 2.80 (d, J = 11.1 Hz,
2H), 2.16 (s, 3H), 1.92 (d, J = 7.2 Hz, 3H), 1.87 (d, J = 11.3 Hz, 2H), 1.78–1.67 (m, 3H), 1.36–1.27 (m,
2H). 13C-NMR (101 MHz, CDCl3)
δ 163.97, 156.82, 155.49, 151.98, 143.94, 142.52, 138.66, 134.92, 131.44,
130.30, 130.17, 129.77, 129.44, 129.34, 128.21, 126.52, 118.26, 116.75, 113.71, 73.39, 56.42, 55.30, 46.35,
35.26, 28.81, 19.21. HRMS (ESI) calculated for C30H30N6O2 [M + HCOO]−: 551.2412, found: 551.2402.
Purity: 98.9% (by HPLC).
5-(3-cyanophenyl)-1-(1-(3-(5-(2-morpholinoethoxy)pyrimidin-2-yl)phenyl)ethyl)pyrimidin-2(1H)-
◦
one (31f). Compound 31f was prepared via a similar procedure of 31c. White solid. m.p. 82.3–83.9 C.
Yield: 68%. H-NMR (400 MHz, CDCl3)
1
δ 8.78 (d, J = 3.4 Hz, 1H), 8.47 (s, 2H), 8.43 (s, 1H), 8.35 (d,
J = 7.7 Hz, 1H), 7.72 (d, J = 3.4 Hz, 1H), 7.62–7.56 (m, 1H), 7.56–7.42 (m, 5H), 6.35 (q, J = 7.0 Hz, 1H),
4.25 (t, J = 5.5 Hz, 2H), 3.79–3.66 (m, 4H), 2.85 (t, J = 5.5 Hz, 2H), 2.68–2.50 (m, 4H), 1.89 (d, J = 7.0 Hz,
3H). 13C-NMR (101 MHz, CDCl3)
δ 163.95, 157.05, 155.45, 151.72, 144.13, 142.50, 138.70, 138.56, 134.91,
131.42, 130.28, 130.15, 129.75, 129.46, 129.33, 128.21, 126.56, 118.25, 116.72, 113.68, 66.87, 66.77, 57.53,
56.39, 54.17, 19.21. HRMS (ESI) calculated for C29H28N6O3 [M + HCOO]−: 553.2205, found: 553.2181.
Purity: 99.6% (by HPLC).
5-(3,5-difluorophenyl)-1-(1-(3-(5-((1-methylpiperidin-4-yl)methoxy)pyrimidin-2-yl)phenyl)ethyl)
pyrimidin-2(1H)-one (31g). Compound 31g was prepared via a similar procedure of 31c. White solid.
1
m.p. 86.9–88.6 ◦C. Yield: 55%. H-NMR (500 MHz, CDCl3)
δ
8.76 (d, J = 3.2 Hz, 1H), 8.43 (s, 2H), 8.42
(s, 2H), 8.35 (d, J = 7.7 Hz, 1H), 7.67 (d, J = 3.3 Hz, 1H), 7.51 (t, J = 7.7 Hz, 1H), 7.47–7.39 (m, 1H),
6.82–6.69 (m, 3H), 6.35 (q, J = 6.9 Hz, 1H), 3.95 (d, J = 5.7 Hz, 2H), 3.00 (d, J = 11.2 Hz, 2H), 2.35 (s,
3H), 2.08 (t, J = 11.5 Hz, 2H), 1.93–1.77 (m, 6H), 1.60–1.49 (m, 2H). 13C-NMR (126 MHz, CDCl3)
δ
163.92, 163.665(JCF = 248.75 Hz), 163.565(JCF = 248.75 Hz), 156.88, 155.54, 151.94, 143.92, 142.42, 138.69,
138.65, 136.67 (JCF = 10.0 Hz), 129.74, 129.36, 128.18, 126.59, 116.74 (JCF = 2.2 Hz),108.83 (JCF = 12.5 Hz),
108.825(JCF = 26.25 Hz), 103.41 (JCF = 25.2 Hz), 73.28, 56.39, 55.20, 46.15, 35.17, 28.61, 19.18. HRMS (ESI)
calculated for C29H29N5O2F2 [M + HCOO]−: 562.2271, found: 562.2248. Purity: 99.4% (by HPLC).

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5-(3-cyanophenyl)-1-(3-(5-((3-(dimethylamino)propyl)thio)pyrimidin-2-yl)benzyl)-3-fluoropyridin
-2(1H)-one (36a). Compound 36a was prepared via a similar procedure of 11a. Yellow solid. m.p.
1
116.3–117.9 ◦C. Yield: 72%. H-NMR (400 MHz, CDCl3)
δ
8.74 (s, 1H), 8.43 (s, 1H), 8.41–8.37 (m, 1H),
7.64 (s, 1H), 7.62–7.52 (m, 3H), 7.52–7.49 (m, 2H), 7.45–7.41 (m, 1H), 7.37 (dd, J = 9.8, 2.3 Hz, 1H), 5.36
(s, 2H), 3.03 (t, J = 7.2 Hz, 2H), 2.45 (t, J = 7.0 Hz, 2H), 2.26 (s, 6H), 1.93–1.80 (m, 2H). 13C-NMR (126
MHz, CDCl3)
δ 161.35, 157.48, 155.62 (JCF = 26.25 Hz), 152.58 (JCF = 251.25 Hz), 137.92, 137.05, 135.88,
131.10, 130.72, 130.68, 130.23, 130.16(JCF = 3.75 Hz), 130.06, 129.66, 129.42, 128.25, 127.85, 119.54 (JCF
=
17.5 Hz), 118.29, 116.57 (JCF = 5.0 Hz), 113.46, 57.86, 52.50, 45.31, 31.65, 27.03. HRMS (ESI) calculated
for C28H26N5OFS [M + H]+: 500.1915, found: 500.1898. Purity: 98.1% (by HPLC).
1-(3-(5-((3-(dimethylamino)propyl)thio)pyrimidin-2-yl)benzyl)-5,50-difluoro-[3,30-bipyridin]-6(1H)
-one (36b). ◦Compound 36b was prepared via a similar procedure of 11a
. White solid. m.p.
δ 8.74 (s, 2H), 8.47–8.36 (m, 4H), 7.51 (d,
128.3–129.9 C. Yield: 75%. 1H-NMR (400 MHz, CDCl3)
J = 5.1 Hz, 2H), 7.45–7.42 (m, 1H), 7.40–7.33 (m, 2H), 5.37 (s, 2H), 3.04 (t, J = 7.2 Hz, 2H), 2.44 (t,
J = 7.0 Hz, 2H), 2.25 (s, 6H), 1.91–1.79 (m, 2H). 13C-NMR (126 MHz, CDCl3)
δ
161.29, 159.52 (JCF
=
257.50 Hz), 157.52, 157.45, 155.59 (JCF = 26.25 Hz), 152.66 (JCF = 252.50 Hz), 142.80 (JCF = 3.75 Hz),
137.96, 137.20 (JCF = 23.75Hz), 135.75, 130.77, 130.73, 130.36 (JCF = 5.0 Hz), 129.65, 128.26, 127.88, 120.01
(JCF = 18.75 Hz), 119.33 (JCF = 18.75 Hz), 113.96 (JCF = 6.25 Hz), 57.91, 52.48, 45.40, 31.69, 27.16. HRMS
(ESI) calculated for C26H25N5OF2S [M + H]+: 494.1821, found: 494.1807. Purity: 99.7% (by HPLC).
1-(3-(5-((3-(dimethylamino)propyl)thio)pyrimidin-2-yl)benzyl)-3-fluoro-5-(pyrimidin-5-yl)pyridin
-2(1H)-one (◦36c). Compound 36c was prepared via a similar procedure of 11a
.
White solid.
128.9–129.8 C. Yield: 78%. 1H-NMR (400 MHz, CDCl3)
δ 9.16 (s, 1H), 8.75 (s, 2H), 8.74 (s, 2H),
8.45–8.37 (m, 2H), 7.51 (d, J = 5.1 Hz, 2H), 7.44 (s, 1H), 7.36 (dd, J = 9.5, 2.3 Hz, 1H), 5.37 (s, 2H), 3.04 (t,
J = 7.2 Hz, 2H), 2.45 (t, J = 7.0 Hz, 2H), 2.26 (s, 6H), 1.93–1.80 (m,2H). 13C-NMR (126 MHz, CDCl3)
δ
161.06, 157.52, 157.25, 155.40 (JCF = 25.7 Hz), 153.68, 151.64, 137.82, 135.46, 131.62 (JCF = 10.2 Hz),
130.63, 130.16 (JCF = 4.6 Hz), 129.50, 128.71 (JCF = 12.5 Hz), 128.14, 127.73, 118.68 (JCF = 18.2 Hz), 111.65
(JCF = 5.5 Hz), 57.73, 52.35, 45.21, 31.49, 26.96. HRMS (ESI) calculated for C25H25N6OFS [M + H]+:
477.1867, found: 477.1857. Purity: 98.9% (by HPLC).
5-(3-cyanophenyl)-1-(1-(3-(5-((3-(dimethylamino)propyl)thio)pyrimidin-2-yl)phenyl)ethyl)-3-
fluoropyridin-2(1H)-one (39). Compound 39 was prepared via a similar procedure of 11a. Colorless
1
oil. Yield: 82%. H-NMR (400 MHz, CDCl3)
δ
8.75 (s, 2H), 8.49 (s, 1H), 8.40 (d, J = 7.6 Hz, 1H), 7.60–7.43
(m, 6H), 7.32 (dd, J = 9.8, 2.3 Hz, 1H), 7.22 (s, 1H), 6.62 (q, J = 7.0 Hz, 1H), 3.04 (t, J = 7.2 Hz, 2H), 2.47 (t,
J = 7.0 Hz, 2H), 2.27 (s, 6H), 1.91–1.81 (m, 5H). 13C-NMR (101 MHz, CDCl3)
δ
161.36, 157.45, 155.42(JCF
= 25.0 Hz), 152.15(JCF = 251.0 Hz), 139.61, 137.97, 137.32, 130.98, 130.76, 130.21, 130.03, 130.01, 129.53,
129.40, 128.20, 127.26 (JCF = 5.0 Hz), 126.64, 119.00 (JCF = 18.0 Hz), 118.33, 116.57 (JCF = 6.0 Hz), 113.37,
57.88, 53.78, 45.34, 31.65, 27.08, 19.23. HRMS (ESI) calculated for C29H28N5OFS [M + H]+: 514.2071,
found: 514.2066. Purity: 99% (by HPLC).
3.2. Biological Assay
3.2.1. Cell lines and Culture
The cell lines, 97H (human liver cancer cell), Pc9 (human non-small cell lung cancer), HELA
(human epithelial cervical cancer cell line), SJSA1 (human osteosarcoma cells), LO2 (human normal
liver cells), and HLF (human embryonic lung fibroblast), used in this study were purchased from
the Guangzhou ginny ou Biotechnology Co. Ltd. (Guangzhou, China). Cell lines were cultivated
in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% (v/v) heat-inactivated fetal bovine
serum (FBS), 100 units/mL penicillin, and 100 µg/mL streptomycin.

Molecules 2019, 24, 1173
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3.2.2. Kinase Inhibition Assay.
The c-Met enzymes and the Z’-LYTE Kinase Kit were purchased from Invitrogen. The tested
compounds were used in concentrations of 0.001 to 10 mM. Briefly, 1 L of an inhibitor or 5% DMSO,
L of enzyme, and 2 L of substrate/ATP mix were added to a 384-well low volume plate L and
incubated at room temperature for 60 min. Then, 5 L of ADP-Glo Reagent was added, and the
mixture was incubated at room temperature for 40 min. Finally, 10 L of Kinase Detection Reagent
µ
2
µ
µ
µ
™
µ
was added, and the mixture was incubated at room temperature for 30 min. The plate was measured
on a multifunction microplate reader (Molecular Devices, Flex Station 3), and the luminescence was
recorded (integration time 0.5–1 s). Curve fitting and data presentations were performed using Graph
Pad Prism version 5.0 (GraphPad Inc., La Jolla, CA, USA). Every experiment was repeated at least
three times.
3.2.3. MTT (Thiazolyl Blue Tetrazolium Bromide) Assay.
For the cytotoxicity assay, the cells grown in the logarithmic phase were seeded into 96-well plates
(5
×
103 cells/well) for 24 h, and then exposed to different concentrations of the test compounds for
48 h. After the attached cells had been incubated with 5 mg/mL MTT (Sigma, St. Louis, MO, USA)
for another 4 h, the suspension was discarded and subsequently the dark blue crystals (formazan)
were solubilized in dimethyl sulfoxide (DMSO). Then, the absorbance was measured at 570 nm using
a multifunction microplate reader (Molecular Devices, Flex Station 3) (Thermo Fisher Scientific, San
Jose, CA, USA), and each experiment was performed at least in triplicate. The cytotoxic effects of each
compound were expressed as the IC50 values, which represents the drug concentration required to
cause 50% tumor cell growth inhibition. This was calculated with GraphPad Prism Software version
5.02 (GraphPad Inc., La Jolla, CA, USA).
3.2.4. Western Blot Analysis.
97H cells seeded in 60-mm dishes at a density of 5
×
105 cells/well were incubated with or
without compound 31e at the indicated concentrations for 4 h. After incubation, the cells were washed
twice with ice-cold PBS, and then lysed in RIPA lysis buffer (Radio Immunoprecipitation Assay)
containing 150 mM NaCl, 50 mM Tris (pH 7.4), 1% (w/v) sodium deoxycholate, 1% (v/v) Triton X-100,
0.1% (w/v) SDS, and 1 mM EDTA (ethylenediaminetetraacetic acid) (Beyotime, Shanghai, China).
◦
The lysates were incubated at 0 C for 30 min, and vortexed every 10 min intermittently, then the
total proteins were harvested by centrifuging at 12,500 g for 15 min. The protein concentrations were
determined by a BCA (bicinchoninic acid) Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL,
USA), and then the protein extracts were reconstituted in loading buffer containing 62 mM Tris-HCl,
2% SDS (sodium dodecyl sulfate), 10% glycerol, and 5%
China), and boiled at 100 C for 3 min. An equal amount of the proteins (40
β
-mercaptoethanol (Beyotime, Shanghai,
◦
µg) was separated
by 8%–12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and were
transferred to nitrocellulose membranes (Amersham Biosciences, Little Chalfont, Buckinghamshire,
UK). After blocking with 5% non-fat dried milk in TBS containing 1% Tween-20 for 90 min at room
temperature, the membranes were incubated overnight with specific primary antibodies (Abcam,
◦
London, England) at 4 C. After three washes in TBST, the membranes were incubated with the
appropriate HRP-conjugated (Horseradish Peroxidase-conjugated) secondary antibodies at room
temperature for 2 h. The blots were developed with enhanced chemiluminescence (Pierce, Rockford,
IL, USA) and detected by an LAS4000 imager (GE Healthcare, Waukesha, WI, USA).
3.2.5. Cell Cycle Analysis
97H cells were seeded in 6-well plates (3
absence of compound 31e at the indicated concentrations for 24 h. Then, cells were harvested by
×
105 cells/well) and incubated in the presence or
centrifugation and fixed in 70% ice-cold ethanol overnight. The ethanol was removed the next day,

Molecules 2019, 24, 1173
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and then the cells were resuspended in the ice-cold PBS and treated with RNAse A (Keygen Biotech,
◦
Nanjing, China) at 37 C for 30 min, followed by incubation with the DNA staining solution propidium
◦
iodide (PI) (Keygen Biotech, Nanjing, China) at 4 C for 30 min. About 10,000 events were detected by
flow cytometry (Epics XL, Beckman Coulter, Fullerton, CA, USA) at 488 nm. The data regarding the
number of cells in different phases of the cell cycle were analyzed by EXPO32 ADC analysis software
(Beckman Coulter, Fullerton, CA, USA).
3.2.6. Anti-Cell-Migration Study
97H cells were plated in a 6-well culture dish at 5
non-migrated cells were scraped off the upper surface of the membrane with a 10
×
104 cells/dish and grown for 24 h, and the
L pipette. The
µ
medium was then replaced with 10% serum DMEM medium and treated with compound 31e at the
indicated concentrations for another 36 h. After washing with phosphate buffer solution (PBS), the cell
images were immediately detected by a Zeiss LSM 570 laser scanning confocal microscope (Carl Zeiss,
Jena, Germany).
4. Conclusions
We synthesized a series of new Tepotinib derivatives to evaluate their ability to act as
antiproliferative agents. The optimal compound, 31e, in which pyridazinone is replaced with
the pyrimidin-2(1H)-one moiety and methyl substitution on the methylene moiety of Tepotinib is
introduced, exhibited potent antiproliferative activity (IC50: 0.026 µM) against hepatic carcinoma 97H
cells. The mechanism study indicated that 31e induces G1 phase arrest and inhibits the phosphorylation
of c-Met and its downstream signaling component, Akt, in a dose-dependent manner. Overall,
the current study demonstrates that 31e is a promising leading compound in the development of
anticancer agents. Further structural optimization and the evaluation of its in vivo anti-tumor activity
are in progress.
Supplementary Materials: The spectra of 1H-NMR and 13C-NMR for the target compounds are available online.
Author Contributions: Project administration, N.-n.Z., B.-j.A., Y.Z. and X.-s.L.; Writing–original draft, N.-n.Z. and
X.-s.L.; Writing–review & editing, X.-s.L. and M.Y.
Funding: This research was funded by the National Natural Science Foundation of China (No. 21772240), the
Guangzhou Science Technology and Innovation Commission (201707010210).
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors.
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