Design, Synthesis and Biological Evaluation of a New Series of 1-Aryl-3-{4-[(pyridin-2-ylmethyl)thio]phenyl}urea Derivatives as Antiproliferative Agents

Article abstract of DOI:10.3390/molecules24112108

To discover new antiproliferative agents with high efficacy and selectivity, a new series of 1-aryl-3-{4-[(pyridin-2-ylmethyl)thio]phenyl}urea derivatives (7a–7t) were designed, synthesized and evaluated for their antiproliferative activity against A549, HCT-116 and PC-3 cancer cell lines in vitro. Most of the target compounds demonstrated significant antiproliferative effects on all the selective cancer cell lines. Among them, the target compound, 1-[4-chloro-3-(trifluoromethyl)phenyl]-3-{4-{{[3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl]methyl}thio}phenyl}urea (7i) was identified to be the most active one against three cell lines, which was more potent than the positive control with an IC₅₀ value of 1.53 ± 0.46, 1.11 ± 0.34 and 1.98 ± 1.27 μM, respectively. Further cellular mechanism studies confirmed that compound 7i could induce the apoptosis of A549 cells in a concentration-dependent manner and elucidated compound 7i arrests cell cycle at G1 phase by flow cytometry analysis. Herein, the studies suggested that the 1-aryl-3-{4-[(pyridin-2-ylmethyl)thio]phenyl}urea skeleton might be regarded as new chemotypes for designing effective antiproliferative agents.

Full text of DOI:10.3390/molecules24112108

molecules
Article
Design, Synthesis and Biological Evaluation of a New
Series of 1-Aryl-3-{4-[(pyridin-2-
ylmethyl)thio]phenyl}urea Derivatives as
Antiproliferative Agents
Chuanming Zhang 1, Xiaoyu Tan 1, Jian Feng 1, Ning Ding 1, Yongpeng Li 1, Zhe Jin 1,
Qingguo Meng 2, Xiaoping Liu 1,* and Chun Hu 1,
*
1
Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education,
Shenyang Pharmaceutical University, Shenyang 110016, China; chuanming_zhang@yeah.net (C.Z.);
tanxiaoyuaikaoyan@163.com (X.T.); mr.fengjian@foxmail.com (J.F.); dingning2216@163.com (N.D.);
yongpengli1993@163.com (Y.L.); jinzheln@163.com (Z.J.)
Department of Pharmacy, Yantai University, Yantai 264005, China; qinggmeng@163.com
Correspondence: lxp19730107@163.com (X.L.); chunhu@syphu.edu.cn (C.H.);
Tel.: +86-24-43520246 (X.L. & C.H.)
2
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Academic Editor: Qiao-Hong Chen
Received: 30 April 2019; Accepted: 30 May 2019; Published: 4 June 2019
Abstract: To discover new antiproliferative agents with high efficacy and selectivity, a new series of
1-aryl-3-{4-[(pyridin-2-ylmethyl)thio]phenyl}urea derivatives (7a 7t) were designed, synthesized and
evaluated for their antiproliferative activity against A549, HCT-116 and PC-3 cancer cell lines in vitro
–
.
Most of the target compounds demonstrated significant antiproliferative effects on all the selective
cancer cell lines. Among them, the target compound, 1-[4-chloro-3-(trifluoromethyl)phenyl]-3-
{4-{{[3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl]methyl}thio}phenyl}urea (7i) was identified to be
the most active one against three cell lines, which was more potent than the positive control with an IC50
value of 1.53
±
0.46, 1.11
±
0.34 and 1.98
±
1.27 µM, respectively. Further cellular mechanism studies
confirmed that compound 7i could induce the apoptosis of A549 cells in a concentration-dependent
manner and elucidated compound 7i arrests cell cycle at G1 phase by flow cytometry analysis. Herein,
the studies suggested that the 1-aryl-3-{4-[(pyridin-2-ylmethyl)thio]phenyl}urea skeleton might be
regarded as new chemotypes for designing effective antiproliferative agents.
Keywords: antiproliferative agent; urea; synthesis; antiproliferative activity; apoptosis
1. Introduction
Cancer is a major public health problem in developed countries and will become the most serious
life-threatening disease worldwide in the near future [1]. Some advances in cancer treatment by
molecule-targeted drugs, such as imatinib, gefitinib, and trastuzumab, were expected to improve cancer
cure rates and also to reduce severe adverse reactions because of the high specificity of the targeted
molecules, which are expressed and have critical roles in cancer cells, but not in normal cells. However,
the clinical effect was found to be limited and did not last for a long time period because of the acquired
resistance of the tumor cells. Furthermore, these molecules often cause on-target and/or off-target
severe toxicity [2]. Therefore, the development of more target-specific therapy, with minimum toxicity,
is warranted to extend disease-free survival and improve the quality of life of cancer patients.
In recent years, proton pump inhibitors (PPIs) as potential anticancer agents were intensively
studied in cancer treatment. Lugini et al. compared the anti-tumor efficacy of different PPIs, including
omeprazole, esomeprazole, lansoprazole, rabeprazole and pantoprazole in vitro and in vivo. The
Molecules 2019, 24, 2108; doi:10.3390/molecules24112108
www.mdpi.com/journal/molecules

Molecules 2019, 24, 2108
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result indicated that all the PPIs have shown different degrees of antitumor efficacy and lansoprazole
showed a higher anti-tumor effect when compared to the other PPIs. [ ]. Recently, the research by
Zeng and Zheng et al. indicated that T-cell originated protein kinase (TOPK) activities were inhibited
by pantoprazole and ilaprazole with high affinity and selectivity [ ]. TOPK (also known as PBK or
PDZ-binding kinase) was first reported by Abe et al. in 2000 [ ], and it is a Ser/Thr protein kinase
overexpressed in hematologic tumors, breast cancer, melanoma, colorectal cancer, prostate cancer,
cervical cancer, bladder cancer and lung cancer [ 14]. The results of their studies demonstrated
3
4,5
6
7
–
that pantoprazole can suppress the growth of colorectal cancer cells as a TOPK inhibitor both in vitro
and in vivo, and also showed that the TOPK activities were inhibited by ilaprazole in HCT-116, ES-2,
A549, SW1990 cancer cells in vitro [4,5]. As shown in Figure 1, all of the PPIs molecules contain
thiomethylpyridine fragments. It can be predicted that these fragments should play an important role
in the antiproliferative activity of proton pump inhibitors.
Figure 1. Chemical structures of proton pump inhibitors (PPIs).
As known, the diaryl urea is a fragment of great importance in medicinal chemistry and can be used
for the synthesis of numerous heterocyclic compounds with diversified biological activities, including
antithrombotic [15], antimalarial [16], antibacterial [17
,18] and anti-inflammatory [19] properties, and
it is characterized by its ability to form hydrogen bond interactions with drug targets [20
–
22]. The
carbonyl oxygen atom acts as a proton acceptor while the two amide nitrogen atoms are proton donors
(Figure 2). This unique type of structure endows urea derivatives with the ability to bind a variety of
enzymes and receptors in the biological systems. Remarkably, the diaryl urea moiety is widely used in
the design of anticancer drugs, such as sorafenib, regorafenib, linifanib and tivozanib (Figure 3).
Figure 2. H-bond acceptor and donors within the diaryl urea scaffold.

Molecules 2019, 24, 2108
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Figure 3. Some anticancer drugs of the diaryl urea moiety.
Molecular hybridization strategy is a useful concept in drug design and development based on the
combination of pharmacophoric moieties of different bioactive substances to produce a new structure,
the affinity and efficacy would be improved, when compared to the parent drugs [23]. These above
interesting findings and our continuous quest to identify more potent antiproliferative agents led to
the molecular hybridization of diaryl urea and thiomethylpyridine to integrate them in one molecular
platform to generate a new hybrid, as shown in Figure 4, and expected that taking this way could get
the antiproliferative agents with highly inhibitory activity.
Figure 4. Rational design of the target compounds based on molecular hybridization strategy.
2. Results and Discussion
2.1. Chemistry
The general synthetic route is illustrated in Scheme 1. The reaction of the commercially
available 4-nitrobenzenethiol (
temperature) obtained compounds 3a
catalytic hydrogenation reduction [25]. The aryl isocyanates 6a
aromatic amines and bis(trichloromethyl)carbonate (BTC) [26]. Finally, treatment of 4a
isocyanates 6a 6e in methylene dichloride yielded 1-aryl-3-{4-[(pyridin-2-ylmethyl)thio]phenyl}urea
derivatives (7a 7t) as the target compounds [26]. The structures of the target compounds were
1
) with 2-(chloromethyl)pyridine derivatives (
3d [24], which converted to key intermediates 4a
6e were prepared by reaction between
4d with aryl
2
) in ethanol at r.t. (room
–
–4d via Pd-C
–
–
–
–
characterized by infraredspectra (IR), proton nuclear magnetic resonance spectra (1H-NMR), carbon
nuclear magnetic resonance spectra (13C-NMR), electrospray ionization mass spectra (ESI-MS) and
high-resolution mass spectra (HRMS).

Molecules 2019, 24, 2108
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Scheme 1. Synthetic route of the target compounds 7a–7t. Reagents and conditions: (a) NaOH (aq.
2M), EtOH, r.t.; (b) H2, 1 atm, 10% Pd-C, MeOH, r.t.; (c) BTC, Et3N, CH2Cl2, r.t.; (d) intermediate 4, aryl
isocyanates 6a–6e, CH2Cl2, r.t.
2.2. Biological Evaluation
2.2.1. Antiproliferative Activity
Using sorafenib as a positive control, all of the target compounds were evaluated for the
antiproliferative activity in vitro against cancer cell lines, including A549 (lung cancer), HCT-116
(colorectal cancer), and PC-3 (prostate cancer) cell lines by MTT assay. The antiproliferative assay
results evaluated as IC50 value (Table 1) and demonstrated that several target compounds have shown
moderate to excellent potency against A549, HCT-116, and PC-3 cancer cell lines. Among the target
compounds7i showed the more potent inhibitory effect against three cancer cell lines than positive
control with IC50 values of 1.53 ± 0.46, 1.11 ± 0.34 and 1.98 ± 1.27 µM, respectively.
All the target compounds could be divided into four classes according to different substituents
on the pyridine ring (Figure 5). The analyses of the structure-activity relationships (SARs) were
summarized as follows: (1) The results of cytostatic activity assay showed that the substitutions of
the 4 (R4) and 5 (R5) positions of the C ring had a weak effect on the inhibitory activity. However, if
the 3-position (R3) hydrogen atom of the C ring was substituted by a methoxy group, the inhibitory
activity was significantly decreased. At the same time, it could be seen that the inhibitory activity was
better than other classes when the 4-position (R4) of the C ring was occupied by the trifluoroethoxy
group. (2) The substituents on the A ring had a significant effect on the inhibitory activity of each class.
When the substituents on C ring were the same, if there was no substituent on A ring, the inhibitory
activity was worst in each class, such as compound 7a, 7f, 7k and 7p. Moreover, when the substituents
in A ring were electron-withdrawing groups, such as 4-Cl or 3-CF3, the inhibitory activity was better
than that substitution of the electron-donating groups, such as 4-OCH3 in each class. Furthermore,
when the two electron-withdrawing groups coexist on the A ring, the target compounds displayed the
strongest inhibitory activity, such as compound 7d, 7i, 7n and 7s.

Molecules 2019, 24, 2108
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Figure 5. SARs summary for the target compounds.
Table 1. The chemical structures and inhibitory activities of the target compounds.
IC50 (µM) a
Compound
R3
R4
R5
R
A549
HCT-116
PC-3
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
7q
7r
H
4-chloro
4-methoxy
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-OCH3
-OCH3
-OCH3
-OCH3
-OCH3
-O(CH2)3OCH3
-O(CH2)3OCH3
-O(CH2)3OCH3
-O(CH2)3OCH3
-O(CH2)3OCH3
-OCH2CF3
-OCH2CF3
-OCH2CF3
-OCH2CF3
-OCH2CF3
-OCH3
H
H
H
H
H
H
H
H
H
12.31 ± 1.90
3.03 ± 2.79
6.35 ± 0.51
2.02 ± 2.20
2.90 ± 0.36
14.29 ± 1.77
2.23 ± 1.27
4.71 ± 0.11
1.53 ± 0.46
4.22 ± 0.99
13.37 ± 0.81
2.23 ± 1.38
5.27 ± 1.01
2.63 ± 1.25
4.88 ± 1.02
26.98 ± 4.12
14.52 ± 2.67
16.47 ± 4.51
6.23 ± 0.60
14.13 ± 1.82
2.12 ± 0.18
29.26 ± 4.53
4.80 ± 1.57
5.29 ± 0.22
1.94 ± 3.45
3.78 ± 1.41
19.31 ± 3.64
3.04 ± 0.37
12.09 ± 1.46
1.11 ± 0.34
10.69 ± 0.87
>100
3.20 ± 2.75
3.49 ± 0.78
2.51 ± 0.15
3.50 ± 0.13
45.93 ± 6.65
5.15 ± 1.01
33.10 ± 2.90
5.61 ± 0.66
17.37 ± 1.69
2.25 ± 0.71
27.22 ± 3.36
6.00 ± 0.22
6.95 ± 1.24
3.97 ± 1.02
11.48 ± 0.98
20.40 ± 2.89
6.78 ± 0.23
15.65 ± 0.69
1.98 ± 1.27
14.33 ± 3.24
>100
5.97 ± 0.55
6.95 ± 0.35
6.32 ± 1.68
7.18 ± 1.58
30.95 ± 5.41
18.57 ± 2.34
22.51 ± 3.07
10.05 ± 2.23
19.77 ± 0.74
3.60 ± 1.08
4-chloro-3-trifluoromethyl
3-trifluoromethyl
H
4-chloro
4-methoxy
4-chloro-3-trifluoromethyl
3-trifluoromethyl
H
4-chloro
4-methoxy
4-chloro-3-trifluoromethyl
3-trifluoromethyl
H
4-chloro
4-methoxy
4-chloro-3-trifluoromethyl
3-trifluoromethyl
H
-CH3
-CH3
-CH3
-CH3
-CH3
H
H
H
H
H
-OCH3
-OCH3
-OCH3
-OCH3
-OCH3
-OCH3
-OCH3
-OCH3
7s
7t
-OCH3
sorafenib
a
Inhibitory activity was assayed by exposure for 72 h to substance and expressed as the concentration
required to inhibit tumor cell proliferation by 50% (IC50). Data are presented as the means
independent experiments.
±
SEMs of three
2.2.2. Cell Apoptosis Assay
The acceptable antiproliferative activity of compound 7i promoted us to investigate its effect on
cell apoptosis. To explore the effect of compound 7i on cell apoptosis, the apoptotic analysis was
performed with Annexin V-FITC/PI double staining and analyzed with flow-cytometry calculation.
Treatment of A549 cells with compound 7i resulted in a concentration-dependent apoptosis increase,
as shown in Figure 6. Specifically, the percentage of early/primary apoptotic cells was about 4.33%
for the low concentration (1 µM) of compound 7i. When treated with high concentration (10 µM) of
compound 7i, around 29.47% of early/primary apoptosis rate was observed. While the late apoptosis
rate of A549 cells was not changed significantly with increasing concentrations.
2.2.3. Cell Cycle Analysis
The effect of compound 7i on the cell cycle was also evaluated. After treatment of A549 cells with
compound 7i for 24 h at indicated concentrations (1, 5, 10 µM), the percentage of cells in G1 phase were

Molecules 2019, 24, 2108
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60.77%, 78.05% and 90.65%, respectively (Figure 7), suggesting that compound 7i caused an obvious
G1 arrest in a concentration-dependent manner with a concomitant decrease in terms of the number of
cells in other phases of the cell cycle.
Figure 6. Compound 7i induced apoptosis of A549 cells. (A) Apoptosis effect on A549 cell line induced
by compound 7i for 24 h using Annexin V-FITC/PI double staining and flow-cytometry calculation.
The lower left quadrant represents live cells, the lower right is for early/primary apoptotic cells, upper
right is for late/secondary apoptotic cells, and the upper left represents cells damaged during the
procedure; (B) Quantitative analysis of apoptotic cells. The experiments were performed three times,
and a representative experiment is shown.
Figure 7. Effects of compound 7i on A549 cell cycle progress for 24 h. (
compound 7i at different concentrations (1 µM, 5 µM, 10 µM) for 24 h. (B
A
) Treatment of A549 cells with
) Quantitative analysis of cell
cycle. The experiments were performed three times, and a representative experiment is shown.

Molecules 2019, 24, 2108
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3. Materials and Methods
3.1. Synthesis
All reagents were obtained from commercial suppliers and used without further purification.
Reaction progress was monitored by thin layer chromatography (TLC) on silica gel plates. The spots
were visualized by ultraviolet (UV) light (254 nm). The column chromatography was performed
using 200 300 mesh silica gel (Qingdao PUKE, Qingdao, China). Melting points were obtained by
−
X-5 micro-melting point apparatus (Beijing Zhongyi Boteng Technology Co., Ltd., Beijing, China) and
1
were uncorrected. H-NMR and 13C-NMR spectra were recorded on Bruker NMR spectrophotometers
(Karlsruhe, Germany) using DMSO-d6 as the solvent and TMS as the internal standard. Mass spectra
were measured with an electrospray (ESI-MS) on a Waters spectrometer (Waters Corporation, Milford,
MA, USA). High resolution mass spectrometry (HRMS) analyses were performed on an Agilent
Technologies 6530 Accurate-Mass Q-TOF Mass Spectrometer (Santa Clara, CA, USA). The purities
were determined by high-performance liquid chromatography (HPLC) using an Agilent 1100 series
HPLC (Santa Clara, CA, USA).
The original figures of 1H-NMR, 13C-NMR, MS and HRMS of all the target compounds as the
Supplementary Materials are available online.
3.1.1. General Procedure for the Preparation of 2-{[(4-nitrophenyl)thio]methyl}pyridine
Derivatives (3a–3d)
4-Nitrobenzenethiol 1 (1.55 g, 0.01 mol), and 2-(chloromethyl)pyridine hydrochloride derivatives 2
(0.01 mol) were dissolved in EtOH (100 mL), then aqueous NaOH (2M) was added dropwise. After the
addition completed, the solution was stirred for 8 h at room temperature. Upon completion, the excess
ethanol was evaporated to give the residue. A large number of white solids have been precipitated
when 200 mL of water was added. The Precipitate was filtered off and washed with water to obtain the
intermediates (3a–3d), which was used for next step without further purification.
4-(3-Methoxypropoxy)-3-methyl-2-{[(4-nitrophenyl)thio]methyl}pyridine (3a) by using compound
1 (1.55 g,
0.01 mol) and 2-(chloromethyl)-4-(3-methoxypropoxy)-3-methylpyridine hydrochloride (2.66 g,
0.01 mol), obtained a yellow solid (3.12 g) in 89.7% yield. ESI-MS (m/z): 349.3 ([M + H]+).
3-Methyl-2-{[(4-nitrophenyl)thio]methyl}-4-(2,2,2-trifluoroethoxy)pyridine (3b) by using compound 1 (1.55 g,
0.01 mol) and 2-(chloromethyl)-3-methyl-4-(2,2,2-trifluoroethoxy)pyridine hydrochloride (2.76 g,
0.01 mol), obtained a yellow solid (3.26 g) in 91.2% yield. ESI-MS (m/z): 359.1 ([M + H]+).
4-Methoxy-3,5-dimethyl-2-{[(4-nitrophenyl)thio]methyl}pyridine (3c) by using compound 1 (1.55 g, 0.01 mol)
and 2-(chloromethyl)-4-methoxy-3,5-dimethylpyridine hydrochloride (2.22 g, 0.01 mol), obtained a
yellow solid (2.69 g) in 88.5% yield. ESI-MS (m/z): 305.0 ([M + H]+).
3,4-Dimethoxy-2-{[(4-nitrophenyl)thio]methyl}pyridine (3d) by using compound 1 (1.55 g, 0.01 mol) and
2-(chloromethyl)-3,4-dimethoxypyridine hydrochloride (2.24 g, 0.01 mol), obtained a yellow solid
(2.86 g) in 93.5% yield. ESI-MS (m/z): 307.4 ([M + H]+).
3.1.2. General Procedure for the Preparation of 4-{[(pyridin-2-yl)methyl]thio}aniline
Derivatives (4a–4d)
A mixture of 3a–3d (5 mmol) and 0.1 g of preequilibrated 10% palladium/carbon in MeOH (50 mL)
was hydrogenated at room temperature and atmospheric pressure. The reaction was completely
monitored by TLC. When the reaction has completed, the mixture was filtered, and the filtrate was
evaporated to yield intermediate (4a–4d) as yellowish oil, which was used for the next step without
further purification.

Molecules 2019, 24, 2108
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4-{{[4-(3-Methoxypropoxy)-3-methylpyridin-2-yl]methyl}thio}aniline (4a) by using compound 3a (1.74 g,
5 mmol), H2 and 10% Pd-C, obtained a yellowish oil (1.52 g) in 95.4% yield. ESI-MS (m/z): 319.3
([M + H]+).
4-{{[3-Methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl]methyl}thio}aniline (4b) by using compound 3b (1.79 g,
5 mmol), H2 and 10% Pd-C, obtained a yellowish oil (1.58 g) in 96.1% yield. ESI-MS (m/z):
329.6 ([M + H]+).
4-{[(4-Methoxy-3,5-dimethylpyridin-2-yl)methyl]thio}aniline (4c) by using compound 3c (1.52 g, 5 mmol),
H2 and 10% Pd-C, obtained a yellowish oil (1.30 g) in 94.8% yield. ESI-MS (m/z): 275.2 ([M + H]+).
4-{[(3,4-Dimethoxypyridin-2-yl)methyl]thio}aniline (4d) by using compound 3d (1.53 g, 5 mmol), H2 and
10% Pd-C, obtained a yellowish oil (1.30 g) in 94.2% yield. ESI-MS (m/z): 276.1 ([M + H]+).
3.1.3. General Procedure for the Preparation of the Target Compounds (7a–7t)
To a solution of BTC (1 mmol) in CH2Cl2 (20 mL) was added dropwise to primary aromatic
amine
in CH2Cl2 (10 mL). The solvent was evaporated. The resulting residue was dissolved in CH2Cl2
(20 mL), and intermediates (4a 4d) (1 mmol) in CH2Cl2 (10 mL) was added dropwise. The mixture
5 (1 mmol) in CH2Cl2 (20 mL) followed by the dropwise addition of triethylamine (1 mL)
–
was stirred for about 3 h, monitored by TLC. After the reaction completed, the solvent was washed
with water and brine, then dried over anhydrous magnesium sulfate. The mixture was filtered, the
filtrate was evaporated and purified by silica gel chromatography (CH2Cl2/MeOH = 60/1, v/v) to obtain
target compounds.
1-{4-{{[4-(3-Methoxypropoxy)-3-methylpyridin-2-yl]methyl}thio}phenyl}-3-phenylurea (7a)
N
S
O
O
O
N
N
H
H
Compound 7a was prepared according to the general procedure by using compound 4a (0.32 g, 1 mmol)
and aniline (0.10 g, 1 mmol), obtained a white solid (0.20 g) in 45.1% yield. m.p. 105.2–106.8 ◦C. IR
(KBr, cm−1):
υ
3421.1, 2922.7, 2852.4, 1596.1, 1545.1, 1492.4, 1460.8, 1440.3, 1398.2, 1385.2, 1309.3, 1231.3,
1
1174.2, 1092.1, 1006.7, 894.6, 832.0, 799.2, 751.9, 693.4, 617.3, 507.5. H-NMR (400 MHz, DMSO-d6)
δ
8.74 (s, 1H), 8.69 (s, 1H), 8.17 (d, J = 5.6 Hz, 1H), 7.46 (s, 1H), 7.44 (s, 1H), 7.41 (s, 1H), 7.39 (s, 1H), 7.32
(s, 1H), 7.31-7.29 (m, 1H), 7.28 (s, 1H), 7.26 (s, 1H), 6.97 (t, J = 7.3 Hz, 1H), 6.90 (d, J = 5.7 Hz, 1H),
4.22 (s, 2H), 4.09 (t, J = 6.2 Hz, 2H), 3.48 (t, J = 6.2 Hz, 2H), 3.25 (s, 3H), 2.12 (s, 3H), 2.02–1.94 (m, 2H).
13C-NMR (101 MHz, DMSO-d6)
δ 163.17, 156.31, 152.87, 147.91, 140.07, 131.77, 129.23, 128.07, 122.34,
120.30, 119.09, 118.69, 106.49, 68.79, 65.48, 58.44, 31.14, 29.16, 10.88. ESI-MS (m/z): 438.4 ([M + H]+),
460.2 ([M + Na]+). HRMS (ESI) (m/z): Calcd. for C24H27N3O3S, 438.1846 ([M + H]+), found: 438.1856
([M + H]+). Purity (HPLC): 99.27%.
1-(4-Chlorophenyl)-3-{4-{{[4-(3-methoxypropoxy)-3-methylpyridin-2-yl]methyl}thio}phenyl}urea (7b)
Compound 7b was prepared according to the general procedure by using compound 4a (0.32 g, 1 mmol)
and 4-chloroaniline (0.13 g, 1 mmol), obtained a white solid (0.36 g) in 75.3% yield. m.p. 191.7–192.5 ◦C
.

Molecules 2019, 24, 2108
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IR (KBr, cm−1):
υ
3428.1, 2923.1, 2852.9, 1631.8, 1490.8, 1398.9, 1384.8, 1298.9, 1273.5, 1237.0, 1174.2,
1
1121.4, 1086.2, 1008.0, 881.2, 832.1, 702.8, 619.7, 506.0. H-NMR (400 MHz, DMSO-d6)
δ 8.83 (s, 1H),
8.77 (s, 1H), 8.17 (d, J = 5.6 Hz, 1H), 7.49 (d, J = 2.2 Hz, 1H), 7.47 (d, J = 2.1 Hz, 1H), 7.40 (d, J = 2.0 Hz
,
1H), 7.39 (d, J = 2.1 Hz, 1H), 7.34 (s, 1H), 7.33 (s, 1H), 7.32 (d, J = 2.1 Hz, 1H), 7.31 (s, 1H), 6.90 (d,
J = 5.7 Hz, 1H), 4.22 (s, 2H), 4.08 (t, J = 6.2 Hz, 2H), 3.48 (t, J = 6.2 Hz, 2H), 3.25 (s, 3H), 2.12 (s, 3H),
2.00–1.94 (m, 2H). 13C-NMR (101 MHz, DMSO-d6)
δ 163.09, 156.35, 152.77, 147.99, 139.09, 138.88, 131.62,
129.07, 128.41, 125.86, 120.22, 119.24, 106.47, 68.80, 65.45, 58.43, 31.14, 29.16, 10.89. ESI-MS (m/z): 473.3
([M + H]+), 495.2 ([M + Na]+). HRMS (ESI) (m/z): Calcd. for C24H26ClN3O3S, 472.1456 ([M + H]+)
found: 472.1467 ([M + H]+). Purity (HPLC): 98.66%.
,
1-(4-Methoxyphenyl)-3-{4-{{[4-(3-methoxypropoxy)-3-methylpyridin-2-yl]methyl}thio}phenyl}urea (7c)
Compound 7c was prepared according to the general procedure by using compound 4a (0.32 g,
1 mmol) and 4-methoxyaniline (0.12 g, 1 mmol), obtained a white solid (0.22 g) in 46.3% yield. m.p.
144.8–146.6 ◦C. IR (KBr, cm−1):
υ
3428.6, 2984.7, 2923.0, 2852.7, 1635.5, 1599.5, 1562.6, 1510.7, 1492.7,
1461.8, 1441.3, 1398.1, 1289.8, 1245.2, 1173.5, 1120.3, 1093.5, 1035.2, 1005.6, 800.0, 617.1, 548.4, 522.7.
1H-NMR (400 MHz, DMSO-d6)
8.64 (s, 1H), 8.47 (s, 1H), 8.16 (d, J = 5.7 Hz, 1H), 7.39 (d, J = 1.9 Hz,
δ
1H), 7.37 (d, J = 2.2 Hz, 1H), 7.35 (d, J = 2.2 Hz, 1H), 7.33 (d, J = 2.3 Hz, 1H), 7.30 (d, J = 2.1 Hz, 1H),
7.29 (d, J = 2.0 Hz, 1H), 6.89 (d, J = 5.6 Hz, 1H), 6.87 (d, J = 2.2 Hz, 1H), 6.86 (d, J = 2.2 Hz, 1H), 4.21 (s,
2H), 4.08 (t, J = 6.2 Hz, 2H), 3.71 (s, 3H), 3.48 (t, J = 6.2 Hz, 2H), 3.25 (s, 3H), 2.12 (s, 3H), 2.00–1.94
(m, 2H). 13C-NMR (101 MHz, DMSO-d6)
δ 163.06, 156.41, 154.97, 153.07, 148.01, 139.37, 133.09, 131.78,
127.82, 120.52, 120.22, 118.98, 114.44, 106.44, 68.79, 65.42, 58.43, 55.63, 31.14, 29.16, 10.88. ESI-MS (m/z):
468.4 ([M + H]+), 490.2 ([M + Na]+). HRMS (ESI) (m/z): Calcd. for C25H29N3O4S, 468.1952 ([M + H]+),
found: 468.1959 ([M + H]+). Purity (HPLC): 98.91%.
1-[4-Chloro-3-(trifluoromethyl)phenyl]-3-{4-{{[4-(3-methoxypropoxy)-3-methylpyridin-2-yl]methyl}thio}phe
nyl}urea (7d)
Compound 7d was prepared according to the general procedure by using compound 4a (0.32 g, 1 mmol)
and 3-chloro-4-(trifluoromethyl)aniline (0.20 g, 1 mmol), obtained a white solid (0.24 g) in 43.8% yield.
m.p. 136.0–137.2 ◦C. IR (KBr, cm−1):
1306.6, 1175.4, 1117.7, 1034.4, 820.6. H-NMR (400 MHz, DMSO-d6) 9.16 (s, 1H), 8.89 (s, 1H), 8.16
υ
3425.2, 2922.0, 2852.7, 1590.1, 1546.1, 1482.2, 1463.1, 1384.5,
1
δ
(d, J = 5.6 Hz, 1H), 8.10 (d, J = 2.2 Hz, 1H), 7.63 (d, J = 2.2 Hz, 1H), 7.62 (s, 1H), 7.42 (d, J = 2.0 Hz,
1H), 7.40 (d, J = 2.2 Hz, 1H), 7.33 (d, J = 2.1 Hz, 1H), 7.32 (d, J = 2.0 Hz, 1H), 6.89 (d, J = 5.7 Hz, 1H),
4.23 (s, 2H), 4.09 (t, J = 6.2 Hz, 2H), 3.48 (t, J = 6.2 Hz, 2H), 3.25 (s, 3H), 2.13 (s, 3H), 2.00–1.94 (m, 2H).
13C-NMR (101 MHz, DMSO-d6)
δ 163.08, 156.34, 152.76, 148.01, 139.76, 138.48, 132.45, 131.46, 128.91,
123.55, 122.80, 120.24, 119.57, 117.23, 106.48, 68.80, 65.44, 58.43, 31.14, 29.16, 10.89. ESI-MS (m/z): 540.2
([M + H]+), 562.0 ([M + Na]+). HRMS (ESI) (m/z): Calcd. for C25H25ClF3N3O3S, 540.1330 ([M + H]+),
found: 540.1320 ([M + H]+). Purity (HPLC): 97.33%.
1-{4-{{[4-(3-Methoxypropoxy)-3-methylpyridin-2-yl]methyl}thio}phenyl}-3-[3-(trifluoromethyl)phenyl]urea (7e
)

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Compound 7e was prepared according to the general procedure by using compound 4a (0.32 g, 1 mmol)
and 3-(trifluoromethyl)aniline (0.16 g, 1 mmol), obtained a white solid (0.27 g) in 53.1% yield. m.p.
136.1–137.9 ◦C. IR (KBr, cm−1):
υ
3327.8, 2958.7, 2927.8, 2859.0, 2377.4, 2350.2, 2311.0, 1724.0, 1648.5,
1585.5, 1552.2, 1492.5, 1465.2, 1397.5, 1338.6, 1295.8, 1230.8, 1166.2, 1116.1, 1092.4, 1068.9, 1005.4, 890.0,
804.1, 732.9, 699.2, 602.3, 505.8. 1H-NMR (400 MHz, DMSO-d6)
9.05 (s, 1H), 8.84 (s, 1H), 8.17 (d,
δ
J = 5.6 Hz, 1H), 8.09 (s, 0H), 8.00 (d, J = 2.3 Hz, 1H), 7.57 (d, J = 8.4 Hz, 1H), 7.51 (t, J = 7.9 Hz, 1H),
7.42 (d, J = 2.0 Hz, 1H), 7.40 (s, 1H), 7.33 (d, J = 2.1 Hz, 1H), 7.32 (d, J = 2.1 Hz, 1H), 7.30 (s, 1H), 6.90
(d, J = 5.7 Hz, 1H), 4.23 (s, 2H), 4.09 (t, J = 6.2 Hz, 2H), 3.48 (t, J = 6.2 Hz, 2H), 3.25 (s, 3H), 2.13 (s,
3H), 2.01–1.94 (m, 3H). 13C-NMR (101 MHz, DMSO-d6)
δ 163.11, 156.32, 152.85, 147.97, 140.98, 138.68,
131.55, 130.34, 129.10, 128.64, 122.30, 120.25, 119.40, 118.53, 106.46, 68.79, 65.44, 58.42, 31.13, 29.16, 10.88.
ESI-MS (m/z): 506.3 ([M + H]+). HRMS (ESI) (m/z): Calcd. for C25H26F3N3O3S, 506.1720 ([M + H]+),
found: 506.1728 ([M + H]+). Purity (HPLC): 97.09%.
1-{4-{{[3-Methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl]methyl}thio}phenyl}-3-phenylurea (7f)
Compound 7f was prepared according to the general procedure by using compound 4b (0.33 g, 1 mmol)
◦
and aniline (0.10 g, 1 mmol), obtained a white solid (0.18 g) in 39.9% yield. m.p. 143.3–145.1 C.
IR (KBr, cm−1):
υ 3424.1, 2923.9, 2852.6, 1687.8, 1639.9, 1600.0, 1548.5, 1495.9, 1441.2, 1399.4, 1384.7,
1307.8, 1284.4, 1266.4, 1232.1, 1176.6, 1112.2, 970.6, 915.9, 854.7, 836.1, 801.5, 783.1, 751.0, 696.2, 657.1,
1
618.8, 574.4. H-NMR (400 MHz, DMSO-d6)
δ 8.73 (s, 1H), 8.67 (s, 1H), 8.24 (d, J = 5.7 Hz, 1H), 7.46
(s, 1H), 7.44 (s, 1H), 7.41 (s, 1H), 7.39 (s, 1H), 7.32 (s, 1H), 7.30 (s, 1H), 7.28 (s, 1H), 7.26 (s, 1H), 7.03
(d, J = 5.7 Hz, 1H), 6.97 (t, J = 7.3 Hz, 1H), 4.89 (q, J = 8.7 Hz, 2H), 4.25 (s, 2H), 2.16 (s, 3H). 13C-NMR
(101 MHz, DMSO-d6)
δ 161.62, 157.24, 152.87, 148.00, 140.05, 139.22, 131.90, 129.23, 127.85, 125.66,
122.90, 122.35, 120.44, 119.10, 118.71, 107.07, 64.92, 31.13, 10.74. ESI-MS (m/z): 448.4 ([M + H]+), 470.2
([M + Na]+). HRMS (ESI) (m/z): Calcd. for C22H20F3N3O2S, 448.1301 ([M + H]+), found: 448.1295
([M + H]+). Purity (HPLC): 97.04%.
1-(4-Chlorophenyl)-3-{4-{{[3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl]methyl}thio}phenyl}urea (7g)
Compound 7g was prepared according to the general procedure by using compound 4b (0.33 g,
1 mmol) and 4-chloroaniline (0.13 g, 1 mmol), obtained a white solid (0.29 g) in 61.0% yield. m.p.
203.6–205.2 ◦C. IR (KBr, cm−1):
υ
3424.1, 2984.9, 2923.1, 2851.9, 2350.0, 2311.0, 1611.4, 1548.6, 1491.9,
1440.3, 1399.6, 1384.9, 1370.1, 1311.1, 1268.3, 1172.6, 1111.7, 1051.4, 1004.4, 897.2, 798.3, 668.5, 615.4.

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1H-NMR (400 MHz, DMSO-d6)
δ
8.81 (s, 1H), 8.75 (s, 1H), 8.23 (d, J = 5.7 Hz, 1H), 7.48 (d, J = 2.1
Hz, 1H), 7.47 (d, J = 2.2 Hz, 1H), 7.40 (d, J = 2.0 Hz, 1H), 7.38 (s, 1H), 7.33 (d, J = 2.1 Hz, 1H), 7.32
(d, J = 2.2 Hz, 1H), 7.31 (s, 1H), 7.30 (d, J = 1.9 Hz, 1H), 7.03 (d, J = 5.7 Hz, 1H), 4.88 (q, J = 8.7 Hz,
2H), 4.25 (s, 2H), 2.16 (s, 3H). 13C-NMR (101 MHz, DMSO-d6)
δ 161.62, 157.22, 152.77, 148.00, 139.08,
138.98, 131.80, 129.07, 128.11, 125.87, 122.90, 120.43, 120.23, 119.23, 107.08, 65.09, 31.14, 10.74. ESI-MS
(m/z): 482.6 ([M + H]+), 504.3 ([M + Na]+). HRMS (ESI) (m/z): Calcd. for C22H19ClF3N3O2S, 482.0911
([M + H]+), found: 482.0916 ([M + H]+). Purity (HPLC): 99.19%.
1-(4-Methoxyphenyl)-3-{4-{{[3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl]methyl}thio}phenyl}urea (7h)
Compound 7h was prepared according to the general procedure by using compound 4b (0.33 g,
1 mmol) and 4-methoxyaniline (0.12 g, 1 mmol), obtained a white solid (0.22 g) in 45.9% yield. m.p.
171.3–172.1 ◦C. IR (KBr, cm−1):
υ
3383.8, 2922.1, 2851.4, 2377.6, 2349.6, 1703.0, 1656.7, 1619.2, 1591.3,
1546.4, 1511.1, 1492.7, 1465.5, 1399.3, 1312.1, 1264.5, 1231.2, 1175.1, 1112.8, 1040.3, 1006.7, 970.2, 918.1,
897.2, 831.5, 799.7, 658.3. 1H-NMR (400 MHz, )
8.64 (s, 1H), 8.47 (s, 1H), 8.16 (d, J = 5.7 Hz, 1H),
δ
7.39 (d, J = 1.9 Hz, 1H), 7.37 (d, J = 2.2 Hz, 1H), 7.35 (d, J = 2.2 Hz, 1H), 7.33 (d, J = 2.3 Hz, 1H), 7.30
(d, J = 2.1 Hz, 1H), 7.29 (d, J = 2.0 Hz, 1H), 6.89 (d, J = 5.6 Hz, 1H), 6.87 (d, J = 2.2 Hz, 1H), 6.86 (d,
J = 2.2 Hz, 1H), 4.91-4.85 (m, 2H), 4.21 (s, 2H), 3.71 (s, 3H), 2.12 (s, 3H). 13C-NMR (101 MHz, DMSO-d6)
δ
161.84, 156.03, 154.81, 153.88, 153.39, 148.09, 133.72, 133.38, 125.65, 121.84, 120.37, 120.33, 115.40,
114.43, 108.04, 65.10, 55.63, 31.13, 10.44. ESI-MS (m/z): 478.3 ([M + H]+). HRMS (ESI) (m/z): Calcd. for
C23H22F3N3O3S, 478.1407 ([M + H]+), found: 478.1408 ([M + H]+). Purity (HPLC): 99.66%.
1-[4-Chloro-3-(trifluoromethyl)phenyl]-3-{4-{{[3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl]methyl}thio}phe
nyl}urea (7i)
Compound 7i was prepared according to the general procedure by using compound 4b (0.33 g, 1 mmol)
and 3-chloro-4-(trifluoromethyl)aniline (0.20 g, 1 mmol), obtained a white solid (0.27 g) in 49.5% yield.
m.p. 142.0–143.0 ◦C. IR (KBr, cm−1):
υ
3422.3, 2922.1, 2852.6, 1587.4, 1547.6, 1480.8, 1419.0, 1309.8,
1
1263.9, 1177.2, 1111.0, 1035.5, 974.7, 819.3, 618.3. H-NMR (400 MHz, DMSO-d6) δ 9.17 (s, 1H), 8.90 (s,
1H), 8.24 (d, J = 5.7 Hz, 1H), 8.10 (d, J = 2.2 Hz, 1H), 7.64 (dd, J = 8.9, 2.2 Hz, 1H), 7.61 (d, J = 8.7 Hz,
1H), 7.42 (d, J = 2.0 Hz, 1H), 7.40 (d, J = 2.2 Hz, 1H), 7.33 (d, J = 2.2 Hz, 1H), 7.32 (d, J = 2.1 Hz, 1H),
7.04 (d, J = 5.7 Hz, 1H), 4.89 (q, J = 8.7 Hz, 2H), 2.16 (s, 3H). 13C-NMR (101 MHz, DMSO-d6)
δ 161.61,
157.18, 152.75, 148.00, 139.74, 138.60, 132.42, 131.64, 128.58, 127.33, 127.02, 125.65, 123.53, 122.80, 120.44,
119.55, 117.28, 117.22, 107.07, 64.79, 39.69, 10.72. ESI-MS (m/z): 550.1, 552.1, 553.1 ([M + H]+). HRMS
(ESI) (m/z): Calcd. for C23H18ClF6N3O2S, 550.0785 ([M + H]+), found: 550.0769 ([M + H]+). Purity
(HPLC): 99.97%.
1-{4-{{[3-Methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl]methyl}thio}phenyl}-3-[3-(trifluoromethyl)phenyl]
urea (7j)

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Compound 7j was prepared according to the general procedure C by using compound 4b (0.33 g,
1 mmol) and 3-(trifluoromethyl)aniline (0.16 g, 1 mmol), obtained a white solid (0.34 g) in 66.1% yield.
m.p. 174.7–176.4 ◦C. IR (KBr, cm−1):
υ 3421.4, 2985.6, 2924.1, 2852.9, 2349.2, 2311.0, 1614.9, 1491.8,
1445.1, 1399.1, 1339.8, 1313.1, 1288.0, 1264.6, 1231.2, 1173.2, 1114.6, 1071.2, 1006.3, 976.0, 832.4, 798.2,
1
700.6, 616.4. H-NMR (400 MHz, DMSO-d6)
δ 9.07 (s, 1H), 8.87 (s, 1H), 8.24 (d, J = 5.7 Hz, 1H), 8.01 (d,
J = 2.0 Hz, 1H), 7.60–7.54 (m, 1H), 7.51 (t, J = 7.9 Hz, 1H), 7.43 (d, J = 2.0 Hz, 1H), 7.41 (d, J = 2.1 Hz,
1H), 7.33 (d, J = 2.1 Hz, 1H), 7.33–7.31 (m, 1H), 7.30 (d, J = 1.7 Hz, 1H), 7.04 (d, J = 5.7 Hz, 1H), 4.89 (q,
J = 8.7 Hz, 2H), 4.26 (s, 2H), 2.16 (s, 3H). 13C-NMR (101 MHz, DMSO-d6)
δ 161.66, 157.16, 152.86, 147.96,
140.97, 138.80, 131.76, 130.35, 130.15, 129.84, 128.31, 122.30, 120.47, 119.40, 118.54, 114.59, 107.09, 65.09,
49.05, 31.13, 10.73. ESI-MS (m/z): 516.2 ([M + H]+). HRMS (ESI) (m/z): Calcd. for C23H19F6N3O2S,
516.1175 ([M + H]+), found: 516.1174 ([M + H]+). Purity (HPLC): 97.29%.
1-{4-{[(4-Methoxy-3,5-dimethylpyridin-2-yl)methyl]thio}phenyl}-3-phenylurea (7k)
Compound 7k was prepared according to the general procedure by using compound 4c (0.27 g, 1
mmol) and aniline (0.10 g, 1 mmol), obtained a white solid (0.23 g) in 57.3% yield. m.p. 188.1–188.9 ◦C.
IR (KBr, cm−1):
υ
3422.4, 2923.8, 2852.4, 2351.0, 2321.9, 1644.0, 1597.4, 1553.7, 1494.2, 1441.5, 1398.0,
1
1312.9, 1270.7, 1237.5, 1173.3, 1127.7, 1073.5, 1002.5, 798.1, 755.7, 738.0, 694.0, 616.4. H-NMR (400 MHz,
DMSO-d6)
δ 8.72 (s, 1H), 8.67 (s, 1H), 8.12 (s, 1H), 7.45 (d, J = 1.3 Hz, 1H), 7.44-7.42 (m, 1H), 7.40
(d, J = 2.1 Hz, 1H), 7.39 (d, J = 2.1 Hz, 1H), 7.31 (d, J = 2.2 Hz, 1H), 7.29 (d, J = 2.1 Hz, 1H), 7.28 (s,
1H), 7.26 (d, J = 1.6 Hz, 1H), 6.97 (t, J = 7.4 Hz, 1H), 4.21 (s, 2H), 3.70 (s, 3H), 2.18 (s, 6H). 13C-NMR
(101 MHz, DMSO-d6) δ 163.86, 155.80, 152.86, 148.97, 140.05, 139.16, 131.86, 129.24, 127.98, 125.19, 125.03,
122.35, 119.11, 118.70, 60.17, 31.15, 13.38, 11.34. ESI-MS (m/z): 394.6 ([M + H]+), 416.3 ([M + Na]+)
.
HRMS (ESI) (m/z): Calcd. for C22H23N3O2S, 394.1584 ([M + H]+), found: 394.1586 ([M + H]+). Purity
(HPLC): 99.89%.
1-(4-Chlorophenyl)-3-{4-{[(4-methoxy-3,5-dimethylpyridin-2-yl)methyl]thio}phenyl}urea (7l)
Compound 7l was prepared according to the general procedure by using compound 4c (0.27 g, 1 mmol)
and 4-chloroaniline (0.13 g, 1 mmol), obtained a white solid (0.28 g) in 66.2% yield. m.p. 206.8–208.2 ◦C
.
IR (KBr, cm−1):
υ
3422.5, 2923.0, 2852.0, 2377.1, 2349.6, 2310.8, 1630.4, 1547.7, 1491.7, 1439.7, 1399.2,
1
1385.1, 1309.9, 1270.9, 1235.5, 1173.0, 1124.1, 1051.3, 1004.6, 832.1, 798.2, 702.1, 668.3, 617.0. H-NMR
(400 MHz, DMSO-d6)
δ 8.81 (s, 1H), 8.75 (s, 1H), 8.11 (s, 1H), 7.48 (d, J = 2.1 Hz, 1H), 7.46 (d, J = 2.2 Hz,
1H), 7.40 (d, J = 2.0 Hz, 1H), 7.38 (d, J = 2.2 Hz, 1H), 7.33 (d, J = 2.1 Hz, 1H), 7.32 (s, 1H), 7.31 (s,

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1H), 7.30 (d, J = 2.1 Hz, 1H), 4.21 (s, 2H), 3.70 (s, 3H), 2.18 (s, 3H), 2.18 (s, 3H). 13C-NMR (101 MHz,
DMSO-d6) δ 163.86, 155.79, 152.77, 148.97, 139.08, 138.94, 131.76, 129.07, 127.98, 128.25, 125.87, 125.19,
125.03, 120.23, 119.25, 60.17, 31.14, 13.37, 11.33. ESI-MS (m/z): 428.7 ([M + H]+). HRMS (ESI) (m/z):
Calcd. for C22H22ClN3O2S, 428.1194 ([M + H]+), found: 428.1199 ([M + H]+). Purity (HPLC): 99.53%.
1-{4-{[(4-Methoxy-3,5-dimethylpyridin-2-yl)methyl)]thio}phenyl}-3-(4-methoxyphenyl)urea (7m)
Compound 7m was prepared according to the general procedure by using compound 4c (0.27 g,
1 mmol) and 4-methoxyaniline (0.12 g, 1 mmol), obtained a white solid (0.21 g) in 49.2% yield. m.p.
171.4–172.6 ◦C. IR (KBr, cm−1):
1397.7, 1311.5, 1292.4, 1270.1, 1240.4, 1173.4, 1073.2, 1053.2, 1031.3, 1003.9, 828.2, 797.9, 616.3. H-NMR
υ
3422.5, 2921.1, 2850.5, 1642.5, 1593.2, 1547.5, 1493.7, 1468.2, 1439.0,
1
(400 MHz, DMSO-d6)
δ 8.68 (s, 1H), 8.50 (s, 1H), 8.15 (s, 1H), 7.39 (d, J = 1.9 Hz, 1H), 7.37 (d, J = 2.1 Hz,
1H), 7.35 (d, J = 2.0 Hz, 1H), 7.33 (d, J = 2.2 Hz, 1H), 7.29 (d, J = 2.1 Hz, 1H), 7.27 (d, J = 1.9 Hz, 1H),
6.87 (d, J = 2.3 Hz, 1H), 6.86 (d, J = 2.2 Hz, 1H), 4.21 (s, 2H), 3.73 (s, 3H), 3.71 (s, 3H), 2.20 (s, 3H), 2.17
(s, 3H). 13C-NMR (101 MHz, DMSO-d6)
δ 164.47, 155.40, 154.98, 153.08, 148.27, 139.64, 133.09, 132.28,
127.19, 125.70, 125.46, 125.41, 120.49, 118.95, 118.40, 114.46, 60.30, 55.65, 31.14, 13.45, 11.35. ESI-MS
(m/z): 424.3 ([M + H]+). HRMS (ESI) (m/z): Calcd. for C23H25N3O3S, 424.1689 ([M + H]+), found:
424.1698 ([M + H]+). Purity (HPLC): 96.88%.
1-[4-Chloro-3-(trifluoromethyl)phenyl]-3-{4-{[(4-methoxy-3,5-dimethylpyridin-2-yl)methyl]thio}phenyl}urea
(7n)
Compound 7n was prepared according to the general procedure by using compound 4c (0.27 g, 1 mmol)
and 3-chloro-4-(trifluoromethyl)aniline (0.20 g, 1 mmol), obtained a white solid (0.35 g) in 70.1% yield.
m.p. 152.3–153.1 ◦C. IR (KBr, cm−1):
1384.6, 1311.1, 1265.2, 1229.0, 1174.6, 1130.8, 1073.5, 1031.7, 823.3, 619.9. H-NMR (400 MHz, DMSO-d6)
υ
3422.2, 2921.6, 2852.2, 1719.2, 1593.8, 1546.4, 1480.4, 1419.8,
1
δ
9.18 (s, 1H), 8.91 (s, 1H), 8.12 (s, 1H), 8.10 (d, J = 2.0 Hz, 1H), 7.64 (d, J = 8.9 Hz, 1H), 7.62 (s, 1H), 7.42
(d, J = 2.1 Hz, 1H), 7.40 (d, J = 2.1 Hz, 1H), 7.33 (s, 1H), 7.31 (d, J = 1.9 Hz, 1H), 4.22 (s, 2H), 3.70 (s, 3H),
2.19 (s, 3H), 2.18 (s, 3H). 13C-NMR (101 MHz, DMSO-d6)
164.07, 154.69, 154.49, 153.03, 149.00, 140.01,
δ
132.84, 132.38, 126.45, 126.36, 124.20, 123.34, 122.49, 121.03, 115.40, 60.18, 31.13, 13.43, 11.00. ESI-MS
(m/z): 496.1; 497.1; 498.1; 499.1 ([M + H]+). HRMS (ESI) (m/z): Calcd. for C23H21ClF3N3O2S, 496.1068
([M + H]+), found: 496.1066 ([M + H]+). Purity (HPLC): 98.56%.
1-{4-{[(4-Methoxy-3,5-dimethylpyridin-2-yl)methyl]thio}phenyl}-3-[3-(trifluoromethyl)pHenyl]urea (7o)

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Compound 7o was prepared according to the general procedure by using compound 4c (0.27 g, 1 mmol)
and 3-(trifluoromethyl)aniline (0.16 g, 1 mmol), obtained a white solid (0.30 g) in 64.7% yield. m.p.
156.9–158.1 ◦C. IR (KBr, cm−1):
1398.4, 1369.9, 1338.2, 1311.7, 1271.2, 1229.2, 1172.1, 1124.2, 1072.1, 1002.9, 797.9, 698.2, 616.0. H-NMR
υ
3420.6, 2984.8, 2922.8, 2851.8, 2350.3, 2321.1, 1609.8, 1491.8, 1443.2,
1
(400 MHz, DMSO-d6)
1H), 7.51 (t, J = 7.8 Hz, 1H), 7.42 (d, J = 1.9 Hz, 1H), 7.40 (d, J = 2.2 Hz, 1H), 7.33 (s, 1H), 7.32 (s, 1H), 7.30
(d, J = 2.6 Hz, 1H), 4.22 (s, 2H), 3.70 (s, 3H), 2.19 (s, 3H), 2.18 (s, 3H). 13C-NMR (101 MHz, DMSO-d6)
δ 9.04 (s, 1H), 8.84 (s, 1H), 8.12 (s, 1H), 8.00 (d, J = 2.0 Hz, 1H), 7.57 (d, J = 8.8 Hz,
δ
163.88, 155.76, 152.85, 148.95, 140.97, 138.73, 131.69, 130.35, 128.51, 125.21, 125.05, 123.31, 122.32, 119.43,
118.58, 114.64, 60.16, 31.13, 13.36, 11.33. ESI-MS (m/z): 462.3 ([M + H]+). HRMS (ESI) (m/z): Calcd. for
C23H22F3N3O2S, 462.1458 ([M + H]+), found: 462.1469 ([M + H]+). Purity (HPLC): 99.79%.
1-{4-{[(3,4-Dimethoxypyridin-2-yl)methyl]thio}phenyl}-3-phenylurea (7p)
Compound 7p was prepared according to the general procedure by using compound 4d (0.28 g, 1 mmol)
and aniline (0.10 g, 1 mmol), obtained a white solid (0.18 g) in 45.8% yield. m.p. 127.7–128.5 ◦C. IR
(KBr, cm−1):
υ
3287.2, 2937.3, 1654.0, 1593.7, 1548.0, 1487.0, 1442.7, 1421.2, 1379.4, 1297.9, 1270.4, 1231.6,
1
1175.0, 1071.7, 997.3, 932.8, 829.0, 782.9, 742.9, 692.4, 618.5, 516.5. H-NMR (400 MHz, DMSO-d6)
δ 8.72
(s, 1H), 8.67 (s, 1H), 8.12 (d, J = 5.5 Hz, 1H), 7.49–7.45 (m, 1H), 7.44 (s, 1H), 7.41 (d, J = 2.0 Hz, 1H), 7.40
(d, J = 2.2 Hz, 1H), 7.33 (d, J = 2.0 Hz, 1H), 7.31 (d, J = 1.9 Hz, 1H), 7.29 (d, J = 7.7 Hz, 1H), 7.28–7.25 (m,
1H), 7.03 (d, J = 5.5 Hz, 1H), 6.97 (t, J = 7.3 Hz, 1H), 4.17 (s, 2H), 3.87 (s, 3H), 3.74 (s, 3H). 13C-NMR
(101 MHz, DMSO-d6)
δ 158.52, 152.88, 151.53, 145.72, 143.40, 138.98, 131.39, 129.24, 128.54, 122.33,
119.17, 118.69, 108.29, 60.99, 56.34, 36.37, 31.14. ESI-MS (m/z): 396.3 ([M + H]+), 418.2 ([M + Na]+)
.
HRMS (ESI) (m/z): Calcd. for C21H21N3O3S, 396.1376 ([M + H]+), found: 396.1380 ([M + H]+). Purity
(HPLC): 99.90%.
1-(4-Chlorophenyl)-3-{4-{[(3,4-dimethoxypyridin-2-yl)methyl]thio}phenyl}urea (7q)
Compound 7q was prepared according to the general procedure by using compound 4d (0.28 g,
1 mmol) and 4-chloroaniline (0.13 g, 1 mmol), obtained a white solid (0.21 g) in 49.1% yield. m.p.
141.7–142.9 ◦C. IR (KBr, cm−1):
υ
3345.3, 3096.8, 2924.2, 2852.2, 1711.6, 1631.2, 1590.8, 1535.1, 1490.0,
1449.2, 1427.9, 1399.4, 1300.6, 1284.5, 1237.1, 1195.2, 1174.0, 1087.1, 1067.2, 996.9, 828.3, 703.0, 509.1.
1H-NMR (400 MHz, DMSO-d6)
8.83 (s, 1H), 8.76 (s, 1H), 8.11 (d, J = 5.5 Hz, 1H), 7.49 (s, 1H), 7.47 (s,
1H), 7.41 (s, 1H), 7.39 (s, 1H), 7.34-7.31 (m, 2H), 7.03 (d, J = 5.5 Hz, 1H), 4.17 (s, 2H), 3.87 (s, 3H), 3.74
(s, 3H). 13C-NMR (101 MHz, DMSO-d6)
158.52, 152.78, 151.50, 145.71, 143.40, 139.10, 138.75, 131.29,
δ
δ
129.07, 128.79, 125.85, 120.21, 119.30, 108.30, 60.99, 56.34, 36.29, 31.14. ESI-MS (m/z): 430.6 ([M + H]+)
,
452.1 ([M + Na]+). HRMS (ESI) (m/z): Calcd. for C21H20ClN3O3S, 430.0987 ([M + H]+), found: 430.0993
([M + H]+). Purity (HPLC): 99.33%.
1-{4-{[(3,4-Dimethoxypyridin-2-yl)methyl]thio}phenyl}-3-(4-methoxyphenyl)urea (7r)

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Compound 7r was prepared according to the general procedure by using compound 4d (0.28 g,
1 mmol) and 4-methoxyaniline (0.12 g, 1 mmol), obtained a white solid (0.20 g) in 47.9% yield. m.p.
179.0–180.6 ◦C. IR (KBr, cm−1):
υ
3428.5, 2985.2, 2923.2, 2851.3, 1630.7, 1587.2, 1557.0, 1510.3, 1490.9,
1442.6, 1398.6, 1299.7, 1270.7, 1232.4, 1173.6, 1072.2, 1033.0, 1000.9, 934.0, 829.1, 799.5, 617.4, 549.0, 523.3.
1H-NMR (400 MHz, DMSO-d6)
8.62 (s, 1H), 8.46 (s, 1H), 8.11 (d, J = 5.5 Hz, 1H), 7.39 (d, J = 2.0 Hz,
δ
1H), 7.38 (d, J = 2.2 Hz, 1H), 7.35 (d, J = 2.2 Hz, 1H), 7.33 (d, J = 2.2 Hz, 1H), 7.31 (d, J = 2.2 Hz, 1H),
7.30 (d, J = 2.0 Hz, 1H), 7.03 (d, J = 5.5 Hz, 1H), 6.87 (d, J = 2.2 Hz, 1H), 6.85 (d, J = 2.2 Hz, 1H), 4.15
(s, 2H), 3.87 (s, 3H), 3.74 (s, 3H), 3.71 (s, 3H). 13C-NMR (101 MHz, DMSO-d6)
δ 158.51, 154.98, 153.07,
151.55, 145.73, 143.40, 139.22, 133.08, 131.45, 128.24, 120.54, 119.06, 114.46, 108.30, 60.99, 56.35, 55.65,
36.43, 31.15. ESI-MS (m/z): 426.3 ([M + H]+). HRMS (ESI) (m/z): Calcd. for C22H23N3O4S, 426.1482
([M + H]+), found: 426.1489 ([M + H]+). Purity (HPLC): 98.84%.
1-[4-Chloro-3-(trifluoromethyl)phenyl]-3-{4-{[(3,4-dimethoxypyridin-2-yl)methyl]thio}pHenyl}urea (7s)
Compound 7s was prepared according to the general procedure by using compound 4d (0.28 g, 1 mmol)
and 3-chloro-4-(trifluoromethyl)aniline (0.20 g, 1 mmol), obtained a white solid (0.32 g) in 64.5% yield.
m.p. 188.1–189.2 ◦C. IR (KBr, cm−1):
1306.1, 1229.2, 1175.6, 1132.0, 1068.8, 1033.0, 824.9. H-NMR (400 MHz, DMSO-d6)
υ
3425.5, 2921.9, 2852.4, 1589.9, 1545.2, 1485.2, 1419.2, 1384.4,
1
δ
9.15 (s, 1H), 8.88 (s,
1H), 8.11 (d, J = 5.5 Hz, 1H), 8.10 (d, J = 2.2 Hz, 1H), 7.64 (d, J = 8.8 Hz, 1H), 7.62–7.58 (m, 1H), 7.42
(d, J = 2.0 Hz, 1H), 7.41 (d, J = 2.2 Hz, 1H), 7.34 (d, J = 2.2 Hz, 1H), 7.33 (d, J = 2.0 Hz, 1H), 7.03 (d,
J = 5.5 Hz, 1H), 4.17 (s, 2H), 3.88 (s, 3H), 3.74 (s, 3H). 13C-NMR (101 MHz, DMSO-d6)
δ 158.52, 152.76,
151.47, 145.73, 143.40, 139.76, 138.36, 132.44, 131.12, 129.28, 123.53, 122.78, 119.62, 117.24, 108.32, 61.00,
56.35, 36.17, 31.14. ESI-MS (m/z): 498.2 ([M + H]+). HRMS (ESI) (m/z): Calcd. for C22H19ClF3N3O3S,
498.0861 ([M + H]+), found: 498.0844 ([M + H]+). Purity (HPLC): 98.10%.
1-{4-{[(3,4-Dimethoxypyridin-2-yl)methyl]thio}phenyl}-3-[3-(trifluoromethyl)phenyl]urea (7t)
Compound 7t was prepared according to the general procedure by using compound 4d (0.28 g, 1 mmol)
and 3-(trifluoromethyl)aniline (0.16 g, 1 mmol), obtained a white solid (0.21 g) in 44.3% yield. m.p.
198.4–199.8 ◦C. IR (KBr, cm−1):
υ
3422.2, 2985.4, 2377.5, 2349.8, 2320.7, 2024.8, 1712.9, 1594.1, 1564.4,
1537.2, 1491.3, 1445.8, 1399.4, 1370.2, 1316.3, 1273.6, 1230.1, 1173.8, 1124.9, 1068.3, 1002.1, 932.4, 892.8,
828.2, 798.3, 743.3, 697.8, 615.6. 1H-NMR (400 MHz, DMSO-d6)
9.04 (s, 1H), 8.83 (s, 1H), 8.11 (d,
δ

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J = 5.5 Hz, 1H), 8.01 (d, J = 2.0 Hz, 1H), 7.60–7.54 (m, 1H), 7.51 (t, J = 7.9 Hz, 1H), 7.43 (d, J = 2.0 Hz,
1H), 7.41 (d, J = 2.2 Hz, 1H), 7.34 (d, J = 2.2 Hz, 1H), 7.32 (d, J = 2.5 Hz, 1H), 7.30 (s, 1H), 7.03 (d,
J = 5.6 Hz, 1H), 4.17 (s, 2H), 3.88 (s, 3H), 3.75 (s, 3H). 13C-NMR (101 MHz, DMSO-d6)
δ 158.52, 152.87,
151.50, 145.72, 143.41, 140.98, 138.55, 131.22, 130.34, 129.08, 122.31, 119.49, 118.56, 114.65, 108.31, 60.99,
56.34, 36.24, 31.12. ESI-MS (m/z): 464.2 ([M + H]+). Purity (HPLC): 98.85%.
3.2. Biological Evaluation
3.2.1. Antiproliferative Activity Assays
The antiproliferative activities of target compounds were determined using a standard MTT
assay [27
PC-3 (8
–
×
30]. Exponentially growing cells A549 (3
×
103 cells/well), HCT-116 (1
×
104 cells/well) and
103 cells/well) were seeded into 96-well plates and incubated for 24 h to allow the cells to
attach. After 24 h of incubation, the culture medium was removed and fresh medium containing
various concentrations of the candidate compounds was added to each well. The cells were then
incubated for 72 h, thereafter MTT assays were performed and cell viability was assessed at 570 nm by
a microplate reader (ThermoFisher Scientific (Shanghai) Instrument Co., Ltd., Shanghai, China).
3.2.2. Cell Apoptosis Assay
A549 cells were seeded into a 6-well plate (2
×
105/well) and incubated for 24 h. Then the cells
were treated with different concentrations of the tested compound 7i for 24 h. Thereafter, the cells
were collected and the Annexin-V-FITC/PI apoptosis kit (Biovision, Milpitas, CA, USA) was used
according to the manufacturer’s protocol. The cells were analyzed by Accuri C6 flow cytometric
(Becton Dickinson, Franklin Lakes, NJ, USA) [31].
3.2.3. Cell Cycle Analysis
For flow cytometric analysis of DNA content, 5
×
105 A549 cells in exponential growth were
treated with different concentrations of the compound 7i for 24 or 48 h. After an incubation period, the
cells were collected, centrifuged and fixed with ice cold ethanol (70%). The cells were then treated with
buffer containing RNAse A and 0.1% Triton X-100 and then stained with the propidium iodide (PI).
The samples were analyzed on Accuri C6 flow cytometer (Becton Dickinson). [32].
4. Conclusions
In summary, a new series of 1-aryl-3-{4-[(pyridin-2-ylmethyl)thio]phenyl}urea derivatives were
designed and synthesized based on molecular hybridization strategy. Majority of target compounds
showed moderate to good growth inhibition against the tested cancer cells. Particularly, compound 7i
exhibited more potent antiproliferative activity than well-known anticancer drug sorafenib against all
three cancer cell lines (A549, HCT-116 and PC-3). The preliminary mechanism investigation showed
that compound 7i could induce A549 cells to apoptosis, and halted cell cycle progression at the
G1 phase. The SARs illustrated that these target compounds in this work might serve as bioactive
fragments, and compound 7i could be used as a lead compound for the development of potent cancer
chemotherapeutic agents in the drug discovery process.
Supplementary Materials: The following are available online. 1H-NMR, 13C-NMR, ESI-MS and HRMS of the
target compounds, respectively.
Author Contributions: C.Z., X.T., J.F., Y.L., N.D. and Z.J. contributed to the synthetic work and the characterization
of all target compounds. C.Z. and Z.J. the preparation of the manuscript. C.Z., X.T. and Q.M. performed the
biological assays. X.L. and C.H. proposed the studies and contributed to their design, as well as to the writing of
the manuscript. All authors have read and approved the final manuscript.
Funding: This work was supported by the National Science Foundation of China (Grant No. 21342006), the
Program for Innovative Research Team of the Ministry of Education of China (Grant No. IRT_14R36), the Natural

Molecules 2019, 24, 2108
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Science Foundation of Liaoning Province, China (Grant No. 201602695), and the Scientific Research Foundation of
Department of Education of Liaoning Province, China (Grant No. L2015517).
Conflicts of Interest: The authors confirm that this article content has no conflict of interest.
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Sample Availability: Samples of all the target compounds are available from the authors.
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