Design, synthesis and anticancer activity of a new series of n-aryl-n′-[4-(Pyridin-2-ylmethoxy)benzyl]urea derivatives

Article abstract of DOI:10.3390/molecules26123496

The development of cancer treatments requires continuous exploration and improvement, in which the discovery of new drugs for the treatment of cancer is still an important pathway. In this study, based on the molecular hybridization strategy, a new structural framework with an N-aryl-N’-arylmethylurea scaffold was designed, and 16 new target compounds were synthesized and evaluated for their antiproliferative activities against four different cancer cell lines A549, MCF7, HCT116, PC3, and human liver normal cell line HL7702. The results have shown seven compounds with 1-methylpiperidin-4-yl groups having excellent activities against all four cancer cell lines, and they exhibited scarcely any activities against HL7702. Among them, compound 9b and 9d showed greatly excellent activity against the four kinds of cells, and the IC₅₀ for MCF7 and PC3 cell lines were even less than 3μM.

Full text of DOI:10.3390/molecules26123496

molecules
Article
Design, Synthesis and Anticancer Activity of a New Series of
N-aryl-N0-[4-(pyridin-2-ylmethoxy)benzyl]urea Derivatives
Shicheng Hou 1, Shishao Liang 1, Chao Zhang 1, Yingmei Han 1, Jianhui Liang 1, Hongyu Hu 1, Xingeng Zhang 1,
Chun Hu 1,
*
, Xiaoping Liu 1,* and Hong Zhang 2,
*
1
Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education,
Shenyang Pharmaceutical University, Shenyang 110016, China; houshicheng2333@163.com (S.H.);
liangshishao2@163.com (S.L.); zhangchaoylh@126.com (C.Z.); hanyingmei0613@163.com (Y.H.);
liangjianhui1502@163.com (J.L.); huhongyu64911@163.com (H.H.); zhangxg96@163.com (X.Z.)
School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang 110016, China
Correspondence: chunhu@syphu.edu.cn (C.H.); lxp19730107@163.com (X.L.); song0688@sina.com (H.Z.);
Tel.: +86-24-43520246 (C.H.)
2
*
Abstract: The development of cancer treatments requires continuous exploration and improvement,
in which the discovery of new drugs for the treatment of cancer is still an important pathway. In
this study, based on the molecular hybridization strategy, a new structural framework with an
N-aryl-N’-arylmethylurea scaffold was designed, and 16 new target compounds were synthesized
and evaluated for their antiproliferative activities against four different cancer cell lines A549, MCF7,
HCT116, PC3, and human liver normal cell line HL7702. The results have shown seven compounds
with 1-methylpiperidin-4-yl groups having excellent activities against all four cancer cell lines, and
they exhibited scarcely any activities against HL7702. Among them, compound 9b and 9d showed
greatly excellent activity against the four kinds of cells, and the IC50 for MCF7 and PC3 cell lines
were even less than 3 µM.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Hou, S.; Liang, S.; Zhang,
C.; Han, Y.; Liang, J.; Hu, H.; Zhang,
X.; Hu, C.; Liu, X.; Zhang, H. Design,
Synthesis and Anticancer Activity of
a New Series of N-aryl-N0-[4-(pyridin-
2-ylmethoxy)benzyl]urea Derivatives.
Molecules 2021, 26, 3496. https://
doi.org/10.3390/molecules26123496
Keywords: anticancer agent; urea derivative; synthesis; molecular hybridization; N-aryl-N’-arylmethy
-lurea; antiproliferative activity; cell cycle analysis
1. Introduction
Academic Editor: Qiao-Hong Chen
Nowadays, cancer has become a major challenge in human health, and a leading
cause of death [1,2]. Cancer is caused by the uncontrolled proliferation of cells, a kind of
Received: 23 April 2021
Accepted: 29 May 2021
Published: 8 June 2021
behavior unusual for cells, mostly related to some abnormal signal transduction regulation
mechanisms. In the diagnosis and treatment of cancer, many great developments have
been made, such as in the field of surgery, drug therapy from toxic drugs to targeted drugs,
etc. [3,4]. However, traditional chemotherapy drugs often have serious side effects and
adverse reactions. The emergence of small-molecule targeted drugs has eased the severe
side effects of chemotherapy drugs to a certain extent, but targeted drugs are also prone to
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
drug resistance problems with the prolonged administration time [5–7]. Therefore, it is a
great challenge and opportunity to continuously develop new candidate drug molecules to
bring new drugs to cancer treatment.
Compared with earlier targeted drugs, multi-target inhibitors can act on a variety
of different targets and inhibit different signal pathways at the same time [
multi-target inhibitors have a broader anti-tumor spectra and better prospects in clinical
practice [ ]. Urea and urea isosteres are structures that possesses both a hydrogen bond
acceptor and a hydrogen bond donor, which makes it easy to form better interactions with
drug target proteins [10]. These kinds of structures are an excellent pharmacodynamic
structure in drug molecules [11]. In many small-molecule targeted kinase inhibitors, urea
and urea isosteres, including aminopyrimidine, have been used in drug structures, some of
which showed really favorable anti-cancer activity (Figure 1) [12,13].
6,8,9]. The
Copyright:
© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
7
Molecules 2021, 26, 3496. https://doi.org/10.3390/molecules26123496
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 3496
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Figure 1. The application of urea and its isosteres in anticancer drugs.
Among the currently clinically used multi-target kinase inhibitors, sorafenib and its
derivative regorafenib with a diaryl urea structure were the prime representatives, because
they had excellent inhibitory effects on a variety of solid tumors [14
rigidity of the diaryl urea structure causes the molecular rotation to not happen freely, thus
the poor solubility of sorafenib results in low bioavailability [16 17]. This study is based on
,15]. In sorafenib, the
,
the structure of sorafenib and the retention of the urea scaffold, in which, in order to enhance
the molecular flexibility, a carbon atom was inserted into the diaryl urea structure, and the
basic urea scaffold was changed to an N-aryl-N’-benzylurea scaffold. Moreover, the diaryl
ether fragment with a pyridyl group was also modified to a 4-(pyridylmethoxy)phenyl
fragment. The nitrogen atom in the pyridine ring is believed to continue to play a key role,
for example, as a hydrogen bond acceptor with some proteins. Meanwhile, in order to keep
its position relative to the core fragment urea unchanged, the position of the nitrogen atom
in the pyridine ring linked to the core urea fragment was replaced from the 4-position to
the 2-position. Validity has been verified by a simulation using the Discovery Studio 3.0
software [18], and the distances between the nitrogen atom in the pyridine ring and the
urea moiety in sorafenib and a target compound are 10.386Å and 10.604Å, respectively
(Figure 2).
Figure 2. Spatial distance of the two nitrogen atoms in pyridine ring and urea moiety of sorafenib (left) and a target
compound (right).
Furthermore, in previous reports, some proton pump inhibitors showed anticancer
activity; lansoprazole was one of the drugs that performed well [19,20]. Based on the
molecular hybridization and structural optimization strategies, the 3-methyl-4-(2,2,2-
trifluoroethoxy)pyridin-2-ylmethyl moiety in lansoprazole and the N-aryl-N’-benzylurea

Molecules 2021, 26, 3496
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scaffold were retained into the target compounds (Figure 3) [21]. Considering the extra
space in some intracellular protein serine/threonine kinases such as BRAF kinase bind-
ing with sorafenib, a new series of N-aryl-N’-benzylurea derivatives modified with a
1-methylpiperidin-4-yl group on the 3-position of the urea scaffold were designed, which
are expected to block intracellular signal transduction and enhance their antiproliferative
activity [22,23].
Figure 3. Design of target compounds with molecular hybridization strategy.
2. Results and discussion
2.1. Chemistry
As shown in Scheme 1, the target molecules were synthesized starting with 2-(chlorome
-thyl)-3-methyl-4-(2,2,2-trifluoromethoxy)pyridine hydrochloride (
a Williamson reaction and obtained the ether compound 3, which reacted with hydroxy-
lamine to convert the oxime . The oxime was reduced by Ni-Al to obtain benzylamine
. The reaction between benzylamine and 1-methylpiperidin-4-one through reductive
amination yielded the key intermediate . Finally, the different intermediate isocyanate
, prepared by a reaction between substituting aniline or benzylamine, triphosgene, and
1) and vanillin (2) via
4
4
5
5
6
7
TEA, were mixed with compound
5 or compound 6 to yield the targeted compounds 8a–8i
and 9a–9g.
2.2. Biological Activity Evaluation
The target compounds were evaluated for their antiproliferative activity against
different human cancer cell lines, including A549 (non-small cell lung cancer cell line),
MCF-7 (breast cancer cell line), HCT116 (colon cancer cell line), PC-3 (prostate cancer cell
line) and HL7702 (human liver normal cell line) by using the MTT assay with sorafenib as
the control drug. The evaluated results as IC50 values are shown in Table 1.

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Scheme 1. Synthetic route of the target compounds. Reagents and conditions: a. K2CO3, DMF, 80 ◦C, yield 97%;
b. NH2OH HCl, NaHCO3, EtOH, H2O, yield 98%; c. Ni-Al, NaOH, EtOH, H2O, yield 95%; d. 1-methylpiperidin-4-one,
·
NaBH3CN, AcOH, MeOH yield 77%; e. amine, triphosgene, TEA, DCM; f. isocyanate 7, DCM.
Table 1. The chemical structures and inhibitory activities of the target compounds.
IC50(µM)
No.
Structure
A549
MCF7
HCT116
PC3
HL7702
8a
8b
8c
5.30 ± 1.45
>50
7.25 ± 0.87
>50
>50
17.65 ± 5.65
4.88 ± 1.94
10.98 ± 1.68
9.33 ± 1.38
29.13 ± 5.81
>50
>50
>50
11.38 ± 3.28
>50
8d
8e
26.47 ± 5.66
29.80 ± 5.09
>50
>50
9.44 ± 1.22
>50
>50
>50
>50
28.81 ± 3.11
8f
12.40 ± 0.60
13.26 ± 2.27
13.35 ± 2.78
7.84 ± 1.40
15.87 ± 0.73
>50
>50
8g
>50
>50
>50

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Table 1. Cont.
IC50(µM)
HCT116
No.
8h
Structure
A549
MCF7
PC3
HL7702
10.93 ± 2.02
9.85 ± 4.40
>50
23.31 ± 3.12
10.88 ± 2.85
>50
>50
>50
N
O
N
F3
C
O
H
H
8i
>50
>50
N
N
O
O
9a
17.53 ± 2.95
4.93 ± 0.46
2.59 ± 0.29
4.41 ± 0.14
2.90 ± 0.16
4.10 ± 0.19
>50
>50
9b
2.56 ± 0.07
3.36 ± 0.17
9c
15.76 ± 1.51
3.17 ± 0.22
4.65 ± 0.73
2.63 ± 0.08
12.90 ± 1.59
2.56 ± 0.26
12.35 ± 1.75
3.62 ± 0.27
>50
>50
9d
9e
9f
5.48 ± 4.36
6.09 ± 0.29
2.56 ± 0.16
3.18 ± 0.30
5.82 ± 0.21
5.18 ± 0.33
4.53 ± 0.33
7.39 ± 0.63
>50
>50
N
N
O
O
F3C
O
9g
6.04 ± 0.41
6.16 ± 0.46
4.23 ± 0.47
3.54 ± 0.19
2.93 ± 0.26
3.88 ± 0.36
6.25 ± 0.43
5.26 ± 0.46
>50
>50
H
N
N
O
O
sorafenib
As shown in Table 1, all the target compounds exhibited weak cytotoxic activities
against HL7702, and most of the target compounds exhibit excellent antiproliferative activ-
ity against the A549 cell line and HCT116 cell line. The IC50 values of target compounds
8c
compounds 9b
liferative activities against the MCF7 cell line and the PC3 cell line of target compounds
9a 9g with the 1-methylpiperidin-4-yl group were significantly higher than that of com-
pounds 8a 8i without the 1-methylpiperidin-4-yl group. The IC50 values of compounds
9a 9b 9d, and 9e against the MCF7 cell line and the PC3 cell line were less than 3 M and
M, respectively. Among them, target compounds 9b and 9d have shown a more potent
,
9b, and 9d against the A549 cells line were less than 5
µM, and the IC50 values of
,
9d, and 9g against the HCT116 cell line were less than 3
µM. The antipro-
–
–
,
,
µ
5
µ
antiproliferative activity against the four cancer cell lines with excellent IC50 values (under
5 µM) compared to the control drug sorafenib.
The analyses of the structure-activity relationships of the target compounds with
the 1-methylpiperidin-4-yl group were summarized as follows: (1) The introduction of
fluorine atoms on the R1 substituent of the benzene ring was mostly beneficial to the
antiproliferative activity. For example, Compounds 9a, 9b, 9d, and 9e with the fluorine
atoms in substituent on the phenyl show a better antiproliferative activity against the
MCF7 cell line and the PC3 cell line than the control drug sorafenib, especially in the MCF7

Molecules 2021, 26, 3496
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cell line. The inhibitory activities against several cell lines of compound 9f with the nitro
group were relatively weaker than that of several compounds with fluorine atoms on the
R1 substituent of the benzene ring. (2) The introduction of electron-withdrawing group
substituent R1 on the benzene ring results in an increase in antiproliferative activity. The
antiproliferative activities against all four cancer cell lines of the target compounds 9b
and 9d with a trifluoromethyl group were significantly higher than compound 9a with a
methoxy group and 9c with a trifluoromethoxy group.
The antiproliferative activities of the target compounds without the 1-methylpiperidin-
4-yl group have shown similar structure-activity relationships. For example, the antiprolif-
erative activity against the HCT116 cell line of compounds 8a
, 8b, 8c, and 8d with fluorine
atoms in substituent on the phenyl were also better than that of the other compounds
without fluorine atoms, and the IC50 value of compound 8c was less than 5 µM against the
A549 cell line.
2.3. Cell Cycle Analysis
The effect of compound 9b on the cell cycle was assayed. After treatment of MCF-
7 cells with compound 9b for 72 h at different concentrations (2.5, 5, 10, 20 M), the
µ
percentages of cells in the G2/M phase were 16.7%, 23.5%, 27.7%, and 39.3%, respec-
tively (Figure 4), indicating that compound 9b could cause an obvious G2/M arrest in a
concentration-dependent manner with a concomitant decrease in terms of the number of
cells in other phases of the cell cycle.
5 μM
0 μM
2.5 μM
20 μM
10 μM
(a)
Figure 4. Cont.

Molecules 2021, 26, 3496
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80
60
40
20
0
G1
S
G2/M
concentrations of treatment
(b)
Figure 4. Effects of compound 9b on MCF7 cell cycle progress for 24 h. (
a) Treatment of MCF7 cells with compound 9b at different
concentrations (0 µM, 2.5 µM, 5 µM, 10 µM, 20 µM) for 24 h. (b) Quantitative analysis of cell cycle.
3. Materials and Methods
3.1. Chemistry
3.1.1. General Information
All reagents were obtained from commercial suppliers and used without further
purification. The progress of the reactions was monitored by thin-layer chromatography
(TLC) on silica gel plates and the spots visualized under ultraviolet (UV) light (254 nm).
The column chromatography was performed using 200–300 mesh silica gel (Qingdao
Haiyang, Qingdao, China). Mass spectra were measured with an electrospray (ESI-MS) on
an Agilent 1100 Series LC/MSD Trap (Agilent Corporation, Santa Clara, CA, USA). 1H-
NMR and 13C-NMR spectra were recorded on Bruker NMR spectrophotometers (Karlsruhe,
Germany) using DMSO-d6 as the solvent. The IR spectra were measured using a Bruker
Fourier number infrared spectrometer (Agilent Corporation, Santa Clara, CA, USA). 1H-
NMR, 13C-NMR, ESI-MS and HRMS spectra of the target compounds are available in the
Supplementary Material (Figures S1–S80).
3.1.2. Synthesis of 3-methoxy-4-[(3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl)methoxy]
benzaldehyde (3)
A mixture of 2-(chloromethyl)-3-methyl-4-(2,2,2-trifluoroethoxy)pyridine hydrochlo-
ride
1
(27.6 g, 100 mmol), vanilli◦n
2 (15.22 g, 100 mmol), K2CO3 (69 g, 0.5 mol) and
DMF(100 mL) was stirred at 80 C overnight. The reaction was monitored by TLC
(PE:EA = 3:1). The mixture was poured into 750 mL of water and stirred for 30 min
until the K2CO3 was completely dissolved. The solids that precipitated out were filtered,
washed with 2 mol/L NaOH aqueous solution and water, then dried to obtain a white
solid 34.54 g. The yield was 97%, ESI-MS: 356.3([M + H]+).
3.1.3. Synthesis of (E)-3-methoxy-4-((3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl)
methoxy)benzaldehyde oxime (4)
A mixture of hydroxylamine hydrochloride (3.06 g, 44 mmol) and NaHCO3 (3.69 g,
44 mmol) in water (50 mL) was stirred at room temperature until no gas was released. A solu-
tion of 3-methoxy-4-((3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl)methoxy)benzaldehyde
3
(14.21 g,40 mmol) in EtOH (50 mL) was added into the mixture and continued string for 3 h.
The progress of the reaction can be confirmed by TLC. EtOH was removed under reduced

Molecules 2021, 26, 3496
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pressure, and the white solid was precipitated, filtered off with suction, and washed with
water. After drying, the white solid obtained was 14.6 g, with a yield of 98%, ESI-MS:
371.3([M + H]+).
3.1.4. Synthesis of (3-methoxy-4-((3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-
yl)methoxy)phenyl)methanamine (5)
An aqueous solution of NaOH (5 mol/L, 60 mL) was added into the solution of 3-
methoxy-4-((3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl)methoxy)benzaldehyde oxime
4
(11.10 g,30 mmol) in EtOH (50 mL) under an ice bath. Nickel-aluminum alloy (10 g) was
slowly added into the mixture in several times, during which a lot of gas was generated.
Then slowly returned to room temperature and stirred overnight. The progress of the
reaction was monitored by TLC. After removing the solid by suction filtration, EtOH was
distilled off under reduced pressure. The residual solution was extracted by EA, and the
organic phase was washed by water and brine, then dried by Na2SO4. After the solvent
was removed under reduced pressure, the white solid obtained was 10.14 g, with a yield of
95%, ESI-MS: 357.2([M + H]+).
3.1.5. Synthesis of N-(3-methoxy-4-((3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl)
methoxy)benzyl)-1-methylpiperidin-4-amine (6)
A mixture of 1-methylpiperidin-4-one (2.26 g, 20 mmol), (3-methoxy-4-((3-methyl-
4-(2,2,2-trifluoroethoxy)pyridin-2-yl)methoxy)phenyl)methanamine(7.12 g, 20 mmol) 5,
AcOH and MeOH was stirred for 1h at room temperature. NaBH3CN was added in 3 times,
during which a lot of gas was generated and stirring was continued for 3 h. The progress of
the reaction was monitored by TLC. After MeOH was distilled off under reduced pressure,
a paste mixture was obtained. An aqueous solution of NaOH (2 mol/L) was added to the
mixture and stirred until the paste dissolved. The solution was extracted by EA, and the
organic phase was washed by water and brine, then dried by Na2SO4. After the solvent
was removed under reduced pressure, a yellowish oil of 7.02 g was obtained, with a yield
of 77%, 454.2([M + H]+).
3.1.6. General Procedure for the Synthesis of the Target Urea Derivatives
Triphosgene (0.20 g, 0.67 mmol) was dissolved in 10 mL DCM, a solution of substituted
aniline or benzylamine (2 mmol) in 10 mL DCM was slowly dropped in during stirring.
There were solids that gradually precipitated out. Then a solution of TEA (0.4 g, 4 mmol) in
DCM (10 mL) was slowly dropped into the mixture, the solids gradually dissolved, and the
solution of substituted isocyanate 7 was obtained. The solution of
5 or 6 (2 mmol) in 10 mL
DCM was added. After the reaction was completed, the mixture was washed by water
and brine and dried by Na2SO4. After DCM was distilled off under reduced pressure, the
mixture was purified by silica gel chromatography (DCM:EA = 5:1, v/v) to afford 8a
and silica gel chromatography (DCM:MeOH = 20:1, v/v) to afford 9a–9g.
–8i
1-(3-methoxy-4-((3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl)methoxy)benzyl)-3-(4-(trifluorom
◦
ethoxy)phenyl)urea (8a), white powder 0.99 g, yield 89%; m.p.: 158.8–159.3 C; MS: 560.4([M
1
+ H]+); H NMR (400 MHz, DMSO-d6)
δ 8.73 (s, 1H), 8.34 (d, J = 5.7 Hz, 1H), 7.54–7.47
(m, 2H), 7.23 (d, J = 8.6 Hz, 2H), 7.14 (d, J = 5.7 Hz, 1H), 7.05 (d, J = 8.2 Hz, 1H), 6.94 (d,
J = 2.0 Hz, 1H), 6.81 (dd, J = 8.2, 2.0 Hz, 1H), 6.59 (t, J = 5.9 Hz, 1H), 5.14 (s, 2H), 4.92 (q,
J = 8.8 Hz, 2H), 4.23 (d, J = 5.7 Hz, 2H), 3.74 (s, 3H), 2.22 (s, 3H).; 13C NMR (101 MHz,
DMSO-d6)
δ 161.82, 155.94, 155.51, 149.63, 148.02, 147.18, 142.55, 140.26, 133.67, 124.25
(q, J = 277.9 Hz), 122.08, 121.99, 120.69 (q, J = 254.9 Hz), 119.69, 119.19, 114.27, 112.07,
108.02, 71.56, 65.11 (q, J = 34.7 Hz), 55.95, 43.10, 10.30; IR: 3399, 3053, 3008, 2971, 2829, 1706,
1556, 1507, 1454, 1416, 1256, 1220, 1194, 1154, 1015, 911, 847, 796, 672, 645, 544; HRMS:
558.146914([M − H]−) for C25H22F6N3O5, 560.161467([M + H]+) for C25H24F6N3O5.
1-(3-chloro-4-fluorophenyl)-3-(3-methoxy-4-((3-methyl-4-(2,2,2-trifluoroe◦thoxy) pyridin-2-yl)meth
oxy)benzyl)urea (8b), white powder 0.86 g, yield 82%; m.p.: 167.8–169.1 C; MS:528.3([M + H]+);

Molecules 2021, 26, 3496
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1H NMR (400 MHz, DMSO-d6)
δ
8.72 (s, 1H), 8.34 (d, J = 5.7 Hz, 1H), 7.77 (dd, J = 6.8,
2.4 Hz, 1H), 7.32–7.18 (m, 2H), 7.14 (d, J = 5.7 Hz, 1H), 7.04 (d, J = 8.2 Hz, 1H), 6.93 (d,
J = 2.0 Hz, 1H), 6.80 (dd, J = 8.2, 1.9 Hz, 1H), 6.63 (t, J = 5.8 Hz, 1H), 5.13 (s, 2H), 4.91 (q,
J = 8.7 Hz, 2H), 4.22 (d, J = 5.7 Hz, 2H), 3.74 (s, 3H), 2.22 (s, 3H).; 13C NMR (101 MHz,
DMSO-d6)
δ 161.81, 155.94, 155.44, 152.33 (d, J = 240.0 Hz), 149.59, 148.04, 147.15, 138.26 (d,
J = 2.8 Hz), 124.27 (q, J = 277.4 Hz), 122.05, 119.69, 119.44 (d, J = 18.9 Hz), 119.31, 118.23
(d, J = 6.5 Hz), 117.13 (d, J = 21.3 Hz), 114.26, 112.08, 108.06, 71.55, 65.08 (q, J = 34.2 Hz),
56.00, 43.09, 10.35; IR: 3313, 2944, 2883, 1641, 1564, 1500, 1477, 1420, 1390, 1308, 1258, 1209,
1164, 1131, 1008, 970, 911, 862, 800, 757, 647, 576, 445; HRMS: 526.116220([M
−
H]−) for
C24H21ClF4N3O4, 528.130773([M + H]+) for C24H23ClF4N3O4.
1-(3-methoxy-4-((3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl)methoxy)benzyl)-3-(4-(trifluorom
ethyl)phenyl)urea (8c), white powder 0.78 g, yield 72%; m.p: 169.9–171.0 ◦C; MS:544.5([M +
H]+), 566.1([M + Na]+); 1H NMR (400 MHz, DMSO-d6)
δ 8.95 (s, 1H), 8.34 (d, J = 5.7 Hz, 1H),
7.59 (q, J = 8.8 Hz, 4H), 7.14 (d, J = 5.7 Hz, 1H), 7.05 (d, J = 8.2 Hz, 1H), 6.95 (d, J = 2.0 Hz,
1H), 6.82 (dd, J = 8.2, 1.9 Hz, 1H), 6.69 (t, J = 5.8 Hz, 1H), 5.14 (s, 2H), 4.91 (q, J = 8.7 Hz, 2H),
4.24 (d, J = 5.7 Hz, 2H), 3.74 (s, 3H), 2.22 (s, 3H). 13C NMR (101 MHz, DMSO-d6)
δ 161.81,
155.93, 155.25, 149.61, 148.03, 147.19, 144.64, 133.47, 126.37, 125.09 (q, J = 270.6 Hz), 124.26
(q, J = 277.5 Hz), 122.06, 119.73, 117.72, 114.26, 112.10, 108.05, 71.55, 65.09 (q, J = 35.0 Hz),
55.98, 43.10, 10.31; IR: 3414, 3376, 2940, 2886, 1703, 1686, 1581, 1534, 1512, 1477, 1408, 1321,
1256, 1220, 1180, 1155, 1135, 1102, 1063, 1008, 979, 862, 842, 812, 595, 554;HRMS: 542.151999
([M − H]−) for C25H22F6N3O4, 544.166552([M + H]+)+ for C25H24F6N3O4.
1-(3-methoxy-4-((3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl)methoxy)benzyl)-3-(3-(trifluorom
◦
ethyl)phenyl)urea (8d), white powder 0.83 g, yield 76%; m.p.: 153.2–154.2 C; MS:544.2([M +
H]+), 542.0([M
−
H]−); 1H NMR (400 MHz, DMSO-d6)
δ
8.90 (s, 1H), 8.34 (d, J = 5.7 Hz,
1H), 7.98 (s, 1H), 7.52 (d, J = 8.3 Hz, 1H), 7.45 (t, J = 7.9 Hz, 1H), 7.23 (d, J = 7.6 Hz, 1H), 7.14
(d, J = 5.7 Hz, 1H), 7.04 (d, J = 8.2 Hz, 1H), 6.94 (d, J = 1.9 Hz, 1H), 6.81 (dd, J = 8.2, 1.9 Hz,
1H), 6.68 (t, J = 5.9 Hz, 1H), 5.13 (s, 2H), 4.91 (q, J = 8.7 Hz, 2H), 4.23 (d, J = 5.8 Hz, 2H), 3.74
(s, 3H), 2.22 (s, 3H). 13C NMR (101 MHz, DMSO-d6)
δ 161.82, 155.94, 155.46, 149.62, 148.02,
147.18, 141.80, 133.59, 130.11, 124.73 (q, J = 272.3 Hz), 124.25 (q, J = 277.4 Hz), 122.07, 121.62,
119.69, 117.68, 114.28, 114.06, 112.08, 108.02 (d, J = 4.4 Hz), 71.56, 65.10 (q, J = 34.2 Hz), 55.97
(d, J = 3.2 Hz), 43.08, 10.33; IR: 3412, 3374, 2940, 2876, 1702, 1582, 1551, 1514, 1477, 1442,
1380, 1341, 1312, 1255, 1221, 1182, 1158, 1−111, 1067, 1028, 1007, 979, 891, 864, 813, 796, 702,
664, 597, 552; HRMS: 542.151999([M
C25H24F6N3O4.
−
H] ) for C25H22F6N3O4+, 544.161552 ([M + H]+) for
1-(3-methoxy-4-((3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl)methoxy)benzyl)-3-(4-methoxyph
enyl)urea (8e), −white powder 0.59 g, yield 58%; m.p.: 179.5–180.3 ◦C; MS:566.2 ([M + H]+),
504.0([M
−
H] ); 1H NMR (400 MHz, DMSO-d6)
δ 8.28 (s, 1H), 7.29 (d, J = 8.9 Hz, 2H), 7.14
(d, J = 5.7 Hz, 1H), 6.93 (d, J = 1.9 Hz, 1H), 6.81 (dd, J = 9.0, 7.1 Hz, 3H), 6.40 (t, J = 5.9 Hz,
1H), 5.13 (s, 2H), 4.92 (q, J = 8.8 Hz, 2H), 4.20 (d, J = 5.8 Hz, 2H), 3.70 (s, 3H), 2.22 (s, 3H). 13
NMR (101 MHz, DMSO-d6) 161.80, 155.96, 155.86, 154.40, 149.57, 148.05, 147.08, 134.08,
C
δ
133.97, 124.28 (d, J = 277.9 Hz), 122.05, 119.91, 119.64, 114.33, 114.27, 112.06, 108.07, 71.58,
65.08 (q, J = 34.5 Hz), 55.99, 55.59, 43.09, 10.36; IR: 3312, 2940, 2839, 1631, 1571, 1508, 1467,
1417, 1376, 1363, 1308, 1271, 1241, 1160, 1136, 1030, 973, 862, 827, 669, 578, 524, 423; HRMS:
504.175179 ([M − H]−) for C25H25F3N3O5, 506.189732 ([M + H]+) for C25H27F3N3O5.
1-(3-methoxy-4-((3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl)methoxy)benzyl)-3-(3-nitropheny
l)urea (8f), yell−ow powder 0.31 g, yield 30%; m.p.: 168.9–170.5 ◦C; MS: 521.2 ([M + H]+),
519.0([M
−
H] ); 1H NMR (400 MHz, DMSO-d6)
δ 9.08 (s, 1H), 8.52 (t, J = 2.2 Hz, 1H), 8.33
(d, J = 5.8 Hz, 1H), 7.75 (d, J = 8.3 Hz, 1H), 7.66 (d, J = 8.4 Hz, 1H), 7.50 (dd, J = 9.1, 7.3 Hz,
1H), 7.13 (d, J = 5.9 Hz, 1H), 7.04 (d, J = 8.2 Hz, 1H), 6.96–6.92 (m, 1H), 6.85–6.71 (m, 2H),
5.13 (s, 2H), 4.91 (q, J = 8.8 Hz, 2H), 4.24 (d, J = 5.9 Hz, 2H), 3.74 (s, 3H), 2.21 (s, 3H). 13
C
NMR (101 MHz, DMSO-d6) δ 161.83, 155.93, 155.35, 149.63, 148.59, 148.02, 147.20, 142.28,
133.51, 130.31, 124.26 (q, J = 277.8 Hz), 124.16, 122.07, 119.74, 115.95, 114.32, 112.14, 112.06,
108.05, 71.56, 65.11 (q, J = 34.4 Hz), 56.02, 43.11, 10.34; IR: 3410, 3010, 2943, 2882, 2832, 1701,

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1584, 1527, 1503, 1480, 1383, 1347, 1318, 1264, 1207, 1161, 1138, 1122, 1033, 1000, 980, 868,
814, 794, 735, 671, 612, 585, 557, 445; HRMS: 519.149693 ([M
−
H]−) for C24H22F3N4O6,
521.164246([M + H]+) for C24H24F3N4O6.
1-(3-methoxy-4-((3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl)metho◦xy)benzyl)-3-(4-meth oxy-
benzyl)urea (8g), white powder 0.39 g, yield 38%; m.p.: 153.0–154.2 C; MS: 520.2([M + H]+),
1
517.9([M
−
H]−); H NMR (400 MHz, DMSO-d6)
δ
9.08 (s, 1H), 8.52 (t, J = 2.2 Hz, 1H),
8.33 (d, J = 5.8 Hz, 1H), 7.75 (d, J = 8.3 Hz, 1H), 7.66 (d, J = 8.4 Hz, 1H), 7.50 (dd, J = 9.1,
7.3 Hz, 1H), 7.13 (d, J= 5.9 Hz, 1H), 7.04 (d, J = 8.2 Hz, 1H), 6.96–6.92 (m, 1H), 6.85–6.71
(m, 2H), 5.13 (s, 2H), 4.91 (q, J = 8.8 Hz, 2H), 4.24 (d, J = 5.9 Hz, 2H), 3.74 (s, 3H), 2.21 (s,
3H); 13C NMR (101 MHz, DMSO-d6)
δ 161.82, 158.54, 158.46, 155.99, 149.57, 148.04, 146.99,
134.47, 133.30, 124.28 (q, J = 277.7 Hz), 122.06, 119.46, 114.26, 114.07, 111.86, 108.08, 71.61,
65.10 (q, J = 34.9 Hz), 55.92, 55.51, 43.25, 42.90, 10.36; IR: 3349, 3301, 2949, 2925, 2884, 2832,
1605, 1579, 1561, 1515, 1468, 1424, 1363, 1363, 1363, 1275, 1251, 1169, 1156, 1133, 1103, 1038,
975, 863, 816, 728, 637, 561; HRMS: 518.190829 ([M
−
H]−) for C26H27F3N3O5, 520.205382
([M + H]+) for C26H29F3N3O5.
1-(4-ethoxybenzyl)-3-(3-methoxy-4-((3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl)methoxy) ben-
◦
zyl)urea (8h), white powder 0.57 g, yield 53%; m.p.: 148.9–149.9 C; MS: 534.3 ([M + H]+); 1H
NMR (400 MHz, DMSO-d6) 8.34 (d, J = 5.7 Hz, 1H), 7.20–7.11 (m, 3H), 7.02 (d, J = 8.2 Hz,
δ
1H), 6.90–6.81 (m, 3H), 6.75 (dd, J = 8.3, 1.9 Hz, 1H), 6.30 (td, J = 6.1, 2.3 Hz, 2H), 5.13
(s, 2H), 4.92 (q, J = 8.8 Hz, 2H), 4.15 (d, J = 5.8 Hz, 4H), 3.99 (q, J = 7.0 Hz, 2H), 3.71 (s,
3H), 2.22 (s, 3H), 1.31 (t, J = 7.0 Hz, 3H); 13C NMR (101 MHz, DMSO-d6)
δ 161.82, 158.48,
157.77, 155.96, 149.54, 148.02, 146.96, 134.46, 133.16, 128.76, 124.28 (q, J= 277.7, 277.3 Hz),
122.06, 119.44, 114.56, 114.21, 111.81, 108.08, 71.57, 65.08 (q, J = 34.5 Hz), 63.39, 55.89, 43.24,
42.89, 15.11, 10.36; IR: 3331, 2978, 2927, 2883, 1609, 1579, 1556, 1516, 1471, 1423, 1390, 1275,
1249, 1148, 1025, 974, 861, 815, 753, 728, 639, 574, 548; HRMS: 532.206479 ([M
−
H]−) for
C27H29F3N3O5, 534.221032([M + H]+) for C27H31F3N3O5.
1-(4-(dimethylamino)benzyl)-3-(3-methoxy-4-((3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2yl)meth
oxy)benzyl)urea (8i), white powder 0.73 g, yield 69%; m.p.: 159.4–161.4 ◦C; MS:533.9 ([M
+ H]+), 555.4([M + Na]+); 1H NMR (400 MHz, DMSO-d6)
δ 8.34 (d, J = 5.6 Hz, 1H), 7.14
(d, J = 5.7 Hz, 1H), 7.11–7.06 (m, 2H), 7.02 (d, J = 8.2 Hz, 1H), 6.87 (d, J = 2.0 Hz, 1H), 6.74
(dd, J = 8.2, 1.9 Hz, 1H), 6.69–6.65 (m, 2H), 6.22 (dt, J = 21.9, 5.9 Hz, 2H), 5.12 (s, 2H), 4.92
(q, J = 8.7 Hz, 2H), 4.15 (d, J = 5.9 Hz, 2H), 4.10 (d, J = 5.8 Hz, 2H), 3.71 (s, 3H), 2.85 (s,
6H), 2.22 (s, 3H). 13C NMR (101 MHz, DMSO-d6)
δ 161.81, 158.46, 155.98, 149.97, 149.55,
148.04, 146.96, 134.50, 128.77, 128.49, 124.28 (q, J = 277.7 Hz), 122.05, 119.44, 114.22, 112.85,
111.82, 108.08, 71.59, 65.08 (q, J = 34.3 Hz), 55.92, 43.23, 43.06, 10.36; IR: 3336, 2940, 2918,
2879, 1613, 1570, 1517, 1468, 1421, 1308, 1256, 1233, 1175, 1137, 1039, 1011, 969, 922, 854, 810,
−
736, 650, 564; HRMS: 531.222464 ([M
C27H32F3N4O4.
−
H] ) for C27H30F3N4O4, 522.237017 ([M + H]+) for
1-(3-methoxy-4-((3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl)methoxy)benzyl)-1-(1-methylpip
eridin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea (9a), white powder 0.79 g, yield 60%; m.p.:
127.3–129.4 ◦C; MS:657.2([M + H]+); 1H NMR (400 MHz, DMSO-d6)
δ 8.52 (s, 1H), 8.33
(d, J = 5.6 Hz, 1H), 7.58–7.51 (m, 2H), 7.23 (d, J = 8.6 Hz, 2H), 7.14 (d, J = 5.7 Hz, 1H),
7.03 (d, J = 8.3 Hz, 1H), 6.90 (d, J = 2.0 Hz, 1H), 6.75 (dd, J = 8.2, 1.9 Hz, 1H), 5.11 (s, 2H),
4.91 (q, J = 8.7 Hz, 2H), 4.52 (s, 2H), 4.10 (tt, J = 12.0, 4.0 Hz, 1H), 3.70 (s, 3H), 2.83 (d,
J = 11.6 Hz, 2H), 2.20 (d, J = 5.4 Hz, 6H), 2.06 (d, J = 10.0 Hz, 2H), 1.70 (tt, J = 12.4, 6.7 Hz,
2H), 1.55 (d, J = 14.4 Hz, 2H). 13C NMR (101 MHz, DMSO-d6)
δ 161.81, 155.73, 149.48, 148.05,
146.93, 143.15, 140.30, 133.64, 124.26(q, J =276.1Hz), 121.50, 118.77, 120.66 (q, J = 253.5 Hz),
111.28, 108.07, 71.55, 65.11 (q, J = 34.8, 34.0 Hz), 55.95, 55.08, 53.09, 45.99, 45.70, 45.16, 39.69,
29.90, 10.36; IR: 3397, 2950, 2848, 2799, 1648, 1584, 1513, 1470, 1416, 1377, 1293, 1256, 1227,
1204, 1159, 1132, 1031, 982, 921, 847, 825, 800, 754, 536; HRMS: 655.236063 ([M
−
H]−) for
C31H33F6N4O5, 657.250616 ([M + H]+) for C31H35F6N4O5.
1-(3-methoxy-4-((3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl)methoxy)benzyl)-1-(1-methylpipe
ridin-4-yl)-3-(4-(trifluoromethyl)phenyl)urea (9b), white powder 0.73 g, yield 57%; m.p.: 138.4–

Molecules 2021, 26, 3496
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140.5 ◦C; MS: 641.1([M + H]+), 321.5 ([M + 2H]2+), 639.5([M
DMSO-d6)
−
H]−); 1H NMR (400 MHz,
8.73 (s, 1H), 8.33 (d, J = 5.6 Hz, 1H), 7.67 (d, J = 8.6 Hz, 2H), 7.58 (d, J = 8.7 Hz,
δ
2H), 7.14 (d, J = 5.7 Hz, 1H), 7.03 (d, J = 8.3 Hz, 1H), 6.89 (d, J = 2.0 Hz, 1H), 6.75 (dd, J = 8.3,
2.0 Hz, 1H), 5.10 (s, 2H), 4.91 (q, J = 8.7 Hz, 2H), 4.54 (s, 2H), 4.16–4.07 (m, 1H), 3.70 (s, 3H),
2.84 (d, J = 10.9 Hz, 2H), 2.19 (s, 6H), 2.06 (t, J = 10.0 Hz, 2H), 1.72 (q, J = 12.6, 11.8 Hz,
2H), 1.57 (d, J = 11.4 Hz, 2H). 13C NMR (101 MHz, DMSO-d6)
δ 161.81, 155.92, 155.50,
149.49, 148.05, 146.97, 144.86, 133.47, 125.91 (q, J = 3.9 Hz), 125.08 (q, J = 270.9 Hz), 124.26
(q, J = 277.6, 277.1 Hz), 122.16 (q, J = 31.8 Hz), 122.08, 118.76, 114.21, 111.26, 108.06, 71.54,
65.11 (q, J = 34.4 Hz), 55.94, 55.06, 53.24, 45.70, 45.25, 29.88, 10.34; IR: 3387, 2943, 2846, 2802,
1650, 1584, 1513, 1468, 1416, 1378, 1313, 1250, −1224, 1162, 1131, 1063, 1030, 1016, 981, 862,
843, 811, 753, 577; HRMS: 639.241148 ([M
for C31H35F6N4O4.
−
H] ) for C31H33F6N4O4, 641.255701([M + H]+)
1-(3-methoxy-4-((3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl)methoxy)benzyl)-3-(4-methoxyph
enyl)-1-(1-methylpiperidin-4-yl)urea (9c), white powder 0.71 g, yield 59%; m.p.: 179.1–180.8;
MS:603.2([M + H]+); 1H NMR (400 MHz, DMSO-d6) δ 8.33 (d, J = 5.7 Hz, 1H), 8.12 (s, 1H),
7.33–7.26 (m, 2H), 7.14 (d, J = 5.7 Hz, 1H), 7.04 (d, J = 8.3 Hz, 1H), 6.90 (d, J = 2.0 Hz, 1H),
6.84–6.74 (m, 3H), 5.11 (s, 2H), 4.91 (q, J = 8.7 Hz, 2H), 4.49 (s, 2H), 4.08 (tt, J = 11.3, 3.9 Hz,
1H), 3.71 (d, J = 1.9 Hz, 6H), 2.80 (d, J = 11.0 Hz, 2H), 2.21 (s, 3H), 2.17 (s, 3H), 2.06–1.95 (m,
2H), 1.67 (qd, J = 12.1, 3.9 Hz, 2H), 1.54 (d, J = 12.0 Hz, 2H). 13C NMR (101 MHz, DMSO-d6)
δ
161.81, 156.08, 155.96, 155.07, 149.46, 148.06, 146.87, 133.99, 133.88, 124.27 (q, J = 278.0 Hz),
122.70, 122.06, 118.80, 114.19, 113.89, 111.29, 108.08, 71.57, 65.10 (q, J = 33.8 Hz), 55.96, 55.59,
55.28, 52.98, 46.03, 45.03, 30.19, 10.38; IR: 3403, 2943, 2836, 2788, 2759, 1642, 1583, 1511,
1467, 1446, 1416, 1373, 1295, 1250, 1220, 1159, 1128, 1034, 1011, 962, 860, 824, 737, 666, 576,
542, 441; HRMS: 637.241006 ([M + Cl]−) for C31H37ClF3N4O5, 603.278881 ([M + H]+) for
C31H38F3N4O5.
1-(3-methoxy-4-((3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl)methoxy)benzyl)-1-(1-methylpip
eridin-4-yl)-3-(3-(trifluoromethyl)phenyl)urea (9d), white powder 0.75 g, yield 59%; m.p.:
160.4–162.2 ◦C; MS: 641.2([M + H]+); 1H NMR (400 MHz, DMSO-d6)
δ 8.69 (s, 1H), 8.33
(d, J = 5.7 Hz, 1H), 7.91 (d, J = 2.0 Hz, 1H), 7.74 (dd, J = 8.2, 2.0 Hz, 1H), 7.46 (t, J = 8.0
Hz, 1H), 7.27 (d, J = 7.8 Hz, 1H), 7.14 (d, J = 5.7 Hz, 1H), 7.04 (d, J = 8.3 Hz, 1H), 6.90 (d,
J = 2.0 Hz, 1H), 6.76 (dd, J = 8.3, 2.0 Hz, 1H), 5.11 (s, 2H), 4.91 (q, J = 8.7 Hz, 2H), 4.54
(s, 2H), 4.11 (tt, J = 12.1, 3.9 Hz, 1H), 3.70 (s, 3H), 2.84 (d, J = 11.2 Hz, 2H), 2.20 (s, 6H),
2.05 (d, J = 13.2 Hz, 2H), 1.72 (tt, J = 12.4, 6.8 Hz, 2H), 1.57 (d, J = 11.8 Hz, 2H). 13C NMR
(101 MHz, DMSO-d6) δ 161.81, 155.93, 155.63, 149.48, 148.06, 146.94, 141.89, 133.50, 129.78,
129.51 (q, J = 32.3, 31.7 Hz), 124.76 (d, J = 272.5 Hz), 124.26 (q, J = 278.9, 278.5 Hz), 123.84,
118.75, 118.39 (d, J = 3.9 Hz), 116.39 (d, J = 4.4 Hz), 114.22, 111.25, 108.08, 71.54, 65.10 (q,
J = 34.6 Hz), 55.94, 55.05, 53.16, 46.04, 45.68, 45.21, 29.82, 10.35; IR: 3385, 2940, 2839, 2791,
2771, 2735, 1645, 1583, 1513, 1493, 1468, 1444, 1376, 1326, 1247, 1222, 1152, 1122, 1030, 1000,
−
972, 909, 835, 788, 749, 701, 667, 540, 458; HRMS: 639.241148 ([M
−
H] ) for C31H33F6N4O4,
641.255701 ([M + H]+) for C31H35F6N4O4.
3-(3-chloro-4-fluorophenyl)-1-(3-methoxy-4-((3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl)meth
oxy)benzyl)-1-(1-methylpiperidin-4-yl)urea (9e), white powder 0.69 g, yield 55%; m.p.: 176.2–
◦
1
177.7 C; MS:625.2([M + H]+); H NMR (400 MHz, DMSO-d6)
δ 8.54 (s, 1H), 8.33 (d,
J = 5.6 Hz, 1H), 7.74 (dd, J = 6.9, 2.6 Hz, 1H), 7.41 (ddd, J = 9.2, 4.4, 2.6 Hz, 1H), 7.27 (t,
J = 9.1 Hz, 1H), 7.14 (d, J = 5.7 Hz, 1H), 7.04 (d, J = 8.3 Hz, 1H), 6.89 (d, J = 2.0 Hz, 1H),
6.75 (dd, J = 8.2, 1.9 Hz, 1H), 5.11 (s, 2H), 4.91 (q, J = 8.7 Hz, 2H), 4.51 (s, 2H), 4.08 (tt,
J = 12.1, 3.9 Hz, 1H), 3.71 (s, 3H), 2.83 (d, J = 11.2 Hz, 2H), 2.20 (d, J = 2.9 Hz, 6H), 2.05
(d, J = 11.7 Hz, 2H), 1.71 (qd, J = 12.3, 3.7 Hz, 2H), 1.59–1.51 (m, 2H). 13C NMR (101 MHz,
DMSO-d6) δ 161.81, 155.93, 155.62, 152.83 (d, J = 240.9 Hz), 149.46, 148.06, 146.93, 138.30,
133.52, 124.27 (q, J = 277.7 Hz), 122.06, 121.79, 120.59 (d, J = 6.5 Hz), 119.01 (d, J = 18.2 Hz),
118.75, 116.71 (d, J = 21.4 Hz), 114.20, 111.26, 108.08, 71.54, 65.10 (q, J = 34.6 Hz), 55.97,
55.09, 53.16, 45.77, 45.19, 29.89, 10.37; IR: 3403, 3365, 2940, 2881, 2822, 1702, 1598, 1515, 1448,

Molecules 2021, 26, 3496
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1407, 1370, 1319, 1260, 1217, 1151, 1107, 1063, 965, 906, 842, 785, 713, 593, 509, 455; HRMS:
623.205307 ([M − H]−) for C30H33ClF4N4O4, 625.219923 ([M + H]+) for C30H35ClF4N4O4.
1-(3-methoxy-4-((3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl)methoxy)benzyl)-1-(1-methylpipe
ridin-4-yl)-3-(3-nitrophenyl)urea (9f), yellow powder 0.79 g, yield 64%; m.p.: 157.5–159.1
◦C; MS:618.2([M + H]+); 1H NMR (400 MHz, DMSO-d6)
δ 8.86 (s, 1H), 8.47 (t, J = 2.3 Hz,
1H), 8.33 (d, J = 5.7 Hz, 1H), 7.92 (dd, J = 8.2, 2.0 Hz, 1H), 7.79 (dd, J = 8.1, 2.2 Hz, 1H),
7.51 (t, J = 8.2 Hz, 1H), 7.13 (d, J = 5.7 Hz, 1H), 7.04 (d, J = 8.3 Hz, 1H), 6.91 (d, J = 2.0 Hz,
1H), 6.76 (dd, J = 8.3, 2.0 Hz, 1H), 5.10 (s, 2H), 4.91 (q, J = 8.7 Hz, 2H), 4.55 (s, 2H), 4.10 (tt,
J = 11.9, 4.0 Hz, 1H), 3.71 (s, 3H), 2.80 (d, J = 11.0 Hz, 2H), 2.20 (s, 3H), 2.16 (s, 3H), 2.00
(t, J = 11.3 Hz, 2H), 1.71 (qd, J = 12.1, 3.8 Hz, 2H), 1.61–1.52 (m, 2H); 13C NMR (101 MHz,
DMSO-d6)
δ 161.80, 155.93, 155.52, 149.48, 148.28, 148.06, 146.95, 142.46, 133.45, 129.97,
126.24, 124.27 (q, J = 277.6 Hz), 122.06, 118.74, 116.58, 114.26, 114.21, 111.25, 108.07, 71.54,
65.10 (q, J = 34.4 Hz), 55.97, 55.21, 53.48, 46.01, 45.24, 30.08, 10.37; IR: 3366, 2940, 2842, 2781,
1657, 1585, 1512, 1467, 1426, 1376, 1343, 1248, 1222, 1161, 1131, 1032, 1011, 967, 861, 824, 737,
−
667, 584, 454; HRMS: 616.238842 ([M
C30H35F3N5O6.
−
H] ) for C30H33F3N5O6, 618.253395 ([M + H]+) for
3-(4-ethoxybenzyl)-1-(3-methoxy-4-((3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl)methoxy)ben
◦
zyl)-1-(1-methylpiperidin-4-yl)urea (9g), white powder 0.41 g, yield 33%; m.p.: 119.0–121.2 C;
1
MS 631.3([M + H]+); H NMR (400 MHz, DMSO-d6)
δ 8.34 (d, J = 5.7 Hz, 1H), 7.13 (dd,
J = 15.2, 7.1 Hz, 3H), 7.01 (d, J = 8.3 Hz, 1H), 6.84–6.75 (m, 4H), 6.72 (dd, J = 8.3, 2.0 Hz, 1H),
5.11 (s, 2H), 4.92 (q, J = 8.7 Hz, 2H), 4.36 (s, 2H), 4.18 (d, J = 5.6 Hz, 2H), 3.98 (q, J = 6.9 Hz,
3H), 3.63 (s, 3H), 2.79 (d, J = 11.0 Hz, 2H), 2.21 (s, 3H), 2.17 (s, 3H), 2.00 (t, J = 11.8 Hz, 2H),
1.61 (qd, J = 12.2, 3.8 Hz, 2H), 1.48 (d, J = 9.9 Hz, 2H), 1.32 (d, J = 7.0 Hz, 3H). 13C NMR
(101 MHz, DMSO-d6) δ 161.82, 157.98, 157.62, 155.99, 149.46, 148.06, 146.82, 134.24, 133.60,
128.63, 124.28 (q, J = 277.9 Hz), 122.08, 118.80, 114.39, 114.08, 111.14, 108.09, 71.61, 65.12 (q,
J = 34.4 Hz), 63.37, 55.79, 55.18, 52.47, 45.79, 44.88, 43.52, 29.99, 15.12, 10.38; IR: 3348, 2937,
2881, 2837, 2793, 1612, 1584, 1509, 1477, 1447, 1395, 1374, 1292, 1253, 1162, 1131, 1033, 1003,
971, 916, 804, 772, 577; HRMS: 665.272306 ([M + Cl]−) for C33H41ClF3N4O5, 631.310182
([M + H]+) for C33H42F3N4O5.
3.2. Biological Evaluation
3.2.1. Antiproliferative Activity Assays
The antiproliferative activities of target compounds were determined using a standard
MTT assay [24
–
27]. Exponentially growing cells A549 (1.5
103 cells/well), HCT-116 (800 cells/well), PC-3 (2.0 103 cells/well) and HL7702
103 cells/well) were seeded into 96-well plates and incubated for 24 h to allow the
×
103 cells/well), MCF-7
(2.2
(5.0
×
×
×
cells to attach. Then, a fresh medium containing various concentrations of the candidate
compounds was added to each well. The cells were then incubated for 96 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). The
optical densities (OD) at 570 nm were measured, and the IC50 of the target compounds was
calculated by using GraphPad Prism 5.0 software to perform nonlinear fitting with the cell
survival rate under different concentrations of the compounds.
3.2.2. Cell Cycle Analysis
As for the flow cytometric analysis of DNA content, 1
×
105 MCF-7 cells in exponential
growth were treated with different concentrations of compound 9b for 24 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 a flow
cytometer (Becton, Dickinson, Franklin Lakes, NJ, USA) [28]. Data were analyzed using
Flowjo software v9.0.

Molecules 2021, 26, 3496
13 of 14
4. Conclusions
In summary, based on the structure of sorafenib and lansoprazole, 16 target N-aryl-N’-
arylmethylurea derivatives were designed with molecular hybridization and synthesized,
and their antiproliferative activities were assayed. The target compounds 9b and 9d
have shown excellent antiproliferative activities against all four kinds of tumor cell lines
(non-small cell lung cancer A549, breast cancer MCF-7, colon cancer HCT116, prostate
cancer PC-3). All target compounds have demonstrated weak cytotoxic activities against
human liver normal cell line HL7702. The biological assay results showed these target
compounds with the 1-methylpiperidin-4-yl group on the 3-position of urea in the target
compounds and substituents containing fluorine atoms on the phenyl ring exhibit potently
antiproliferative activities. The cell cycle evaluation has shown that compound 9b could
cause an obvious G2/M arrest in a concentration-dependent manner.
1
Supplementary Materials: The following are available online, H-NMR, 13C-NMR, ESI-MS and
HRMS of the target compounds (Figures S1–S80).
Author Contributions: Proposal for the subject: C.H. and X.L.; synthetic work and the characteriza-
tion of all target compounds: S.H., S.L., C.Z., Y.H., J.L., H.H. and X.Z.; biological assays: S.H., X.Z.
and H.Z.; preparation of the manuscript: S.H. and C.Z.; review and correction for the manuscript:
C.H., X.L. and H.Z. All authors have read and agreed to the published version of the 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).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on request from the
corresponding authors.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of all target compounds are available from the authors.
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