Synthesis and antitumor activity of novel pyridino[2,3-d]pyrimidine urea derivatives

Article abstract of DOI:10.1002/jhet.4287

A series of novel N-(3-((6-bromopyrido[2,3-d]pyrimidin-4-yl)oxy)phenyl)pyrrolidine-1-carboxamide and 1-(3-((6-bromopyrido[2,3-d]pyrimidin-4-yl)oxy)phenyl)-3-propylurea derivatives were synthesized. Their antitumor activities against human breast carcinoma cells (MCF-7) and human colon cancer cells (HCT-116) in vitro were evaluated, using sorafenib as a positive control drug. Anticancer bioassays indicated that several compounds exhibited appreciable anticancer activity against MCF-7 and HCT-116 cells. Particularly, compounds 9g and 8b demonstrated the most significant inhibitory effect against HCT-116 and MCF-7 cells, with inhibition ratios of 25.56% and 26.46%, respectively. Additionally, the synthesized pyridine[2,3-d]pyrimidine derivatives containing a urea group moieties exhibited antitumor activities against MCF-7 and HCT-116 cells in vitro.

Full text of DOI:10.1002/jhet.4287

Received: 3 February 2021
DOI: 10.1002/jhet.4287
Revised: 20 April 2021
Accepted: 30 April 2021
A R T I C L E
Synthesis and antitumor activity of novel pyridino[2,3-d]
pyrimidine urea derivatives
Dongmei Chen1,2 | Yumei Chen1,2 | Di Yang1,2 | Zhaopeng Zheng3
Zhixu Zhou1,2,4
|
1School of Pharmaceutical Sciences,
Abstract
Guizhou University, Guiyang, PR China
2Guizhou Engineering Laboratory for
Synthetic Drugs, Guiyang, PR China
A
series of novel N-(3-((6-bromopyrido[2,3-d]pyrimidin-4-yl)oxy)phenyl)
pyrrolidine-1-carboxamide and 1-(3-((6-bromopyrido[2,3-d]pyrimidin-4-yl)oxy)
phenyl)-3-propylurea derivatives were synthesized. Their antitumor activities
against human breast carcinoma cells (MCF-7) and human colon cancer cells
(HCT-116) in vitro were evaluated, using sorafenib as a positive control drug.
Anticancer bioassays indicated that several compounds exhibited appreciable
anticancer activity against MCF-7 and HCT-116 cells. Particularly, compounds
9g and 8b demonstrated the most significant inhibitory effect against HCT-116
and MCF-7 cells, with inhibition ratios of 25.56% and 26.46%, respectively.
Additionally, the synthesized pyridine[2,3-d]pyrimidine derivatives containing
a urea group moieties exhibited antitumor activities against MCF-7 and HCT-
116 cells in vitro.
3Department of Oncology, Guizhou
Provincial People's Hospital, Guiyang, PR
China
4Department of Dermatology, Affiliated
Hospital of Guizhou Medical University,
Guiyang, PR China
Correspondence
Zhixu Zhou, School of Pharmaceutical
Sciences, Guizhou University, Guiyang
550025, PR China.
Email: zhixuzhou@126.com
Funding information
Science and Technology Fund of Guizhou
Provincial Health Department, Grant/
Award Number: gzwjkj2020-1-238;
Science and Technology Program Platform
for Talents of Guizhou Province, Grant/
Award Numbers: [2018]5781, [2016]5634;
Guizhou Provincial Natural Science
Foundation, Grant/Award Number:
[2020]1Y393
1 | INTRODUCTION
numerous pharmacologically active agents, including
drugs with anticancer properties [8–10]. For example,
As a common malignant tumor, hepatocellular carci-
noma and melanoma are devastating diseases and lead to
the occurrence of cancer-related deaths [1–3]. Although
sorafenib is currently the standard chemotherapy drug
available for the treatment of advanced hepatocellular
carcinoma (HCC), improvement of its efficacy is neces-
sary [4]. It is essential to develop therapeutic strategies
that combine sorafenib with other anticancer agents and
to design a series of novel sorafenib derivatives. Nitrogen-
containing heterocycles are pharmacologically important
scaffold and are present in numerous clinically approved
drugs [5–7]. The indazole structural unit is utilized in
indazole-based pazopanib and axitinib have been devel-
oped for application as multityrosine kinases [11–19].
Halogen bonds play a vital role in drug design, crystal
engineering, and other fields because of the noncovalent
interaction.
Urea is an organic compound that contains carbonyl
groups flanked by two nitrogen atoms with unique
hydrogen bonding capabilities, and urea derivatives can
be used as modulators of biological targets [20], prote-
ases, and epigenetic enzymes. Additionally, the
keratolytic properties of urea are also considered for its
applicability in cosmetology and dermatology, such as
J Heterocyclic Chem. 2021;1–9.
wileyonlinelibrary.com/journal/jhet
© 2021 Wiley Periodicals LLC.
1

2
CHEN ET AL.
utilization as a component of cosmetic formulas for alter-
ing skin texture to reduce roughness and discolorations
[21,22]. Halogen bonds are essential in medicinal chemis-
try as the halogenation of drugs, generally, improves both
selectivity and efficacy toward active protein sites [23].
For example, the formation of halogen bonds in ligand–
target complexes is now recognized as a kind of inter-
molecular interaction that favorably contributes to the
stability of protein–ligand complexes [24]. Therefore, we
designed a bromine substituent in the pyridino[2,3-d]
pyrimidine ring of target compounds to improve the anti-
tumor activity of the target compound and the binding
activity with the receptor protein through the interaction
of halogen bonds. Furthermore, a series of pyrido[3,4-d]
pyrimidine inhibitors demonstrated excellent potency
and kinase selectivity [25]. B-Raf and c-Abl are vital in
cancer treatment, and protein kinases c-Abl, B-Raf, and
p38α are recognized as major targets for therapeutic
intervention [26]. Owing to the importance of indazoles,
sorafenib, and halogen bond in drug development, this
study proposed that conjugation of the halogenated
pyridine[2,3-d]pyrimidine template with phenyl urea
would aid the design of novel drug candidates with
malignant tumor inhibitors.
2 | RESULTS AND DISCUSSION
2.1 | Chemistry
The detailed synthetic routes for the preparation of N-
(3-((6-bromopyrido[2,3-d]pyrimidin-4-yl)oxy)phenyl)pyrr
olidine-1-carboxamide and 1-(3-((6-bromopyrido[2,3-d]
pyrimidin-4-yl)oxy)phenyl)-3-propylurea derivatives are
SCHEME 1 Synthesis routes of 8a–8i and 9a–9m

CHEN ET AL.
3
summarized in Scheme 1. The synthesis route of the tar-
get compound was obtained by splicing the parent
nucleus (5) with the side chain of the phenolic hydroxyl
urea structure. First, the parent nucleus (5) can be pre-
pared by esterification, bromination, cyclization and
chlorination using 2-aminoniacin as the starting material.
The specific synthesis route was as follows: the key inter-
mediate ethyl 2-aminonicotinate (2) was prepared by
esterification using 2-aminonicotinic acid as the starting
material. The conditions of esterification are absolute
ethyl alcohol and concentrated sulfuric acid. Ethyl
2-aminonicotinate (2) was subjected to bromination with
N-bromosuccinimide (NBS) to obtain ethyl 2-amino-
5-bromonicotinate (3). 6-Bromopyrido[2,3-d]pyrimidin-4
(3H)-one (4) was obtained from ethyl 2-amino-
5-bromonicotinate (3) and formamide by cyclization.
6-Bromo-4-chloropyrido[2,3-d]pyrimidine (5) was synthe-
sized from 6-bromopyrido[2,3-d]pyrimidin-4(3H)-one (4)
via chlorinated reaction in the presence of phosphorus
oxychloride. Second, the formation of the side chain of
the phenolic hydroxyl urea structure was prepared by
their activities using MTT assay in vitro. According to the
existing literature, urea derivatives exhibit cytotoxic activ-
ities against most cancer cell lines, such as HCT-116 and
MCF-7 [27–29]. Additionally, a few compounds were
designed and showed higher activities against the HCT-
116 and MCF-7 cell lines based on the study of sorafenib.
Therefore, MCF-7 and HCT-116 cells were used as the
test cell lines in this work. The results of all compounds
are listed in Table 1.
The results showed that a few compounds exhibited
appreciable inhibitory effects against MCF-7 and HCT-
116 cells in vitro, compounds 9g, 9i, and 9l exhibited bet-
ter anticancer effects on most tested HCT-116 cells, and
compounds 9g demonstrated the best efficacy which was
better than that demonstrated by sorafenib. We totally
synthesized 22 pyridine[2,3-d]pyrimidine derivatives, and
the antiproliferative activity of the target compounds was
influence by the type of the urea substituent in the para-
position and meta-position of phenyl. Besides, Table 1
showed that the growth of MCF-7 cells was considerably
inhabited by 8b, with an inhibition ratio of 26.46%. The
compounds-associated inhibition ratio of MCF-7 cells
was in the order 8b > 8d > 9f > sorafenib. The presence
of electron-donating and high-steric-hindrance-exhibiting
urea groups (3-isopropyl and 3-tert-butyl) at the meta-
position of phenyl such as compounds 8b and 8d showed
an increase in the inhibitory potency against MCF-7 cells.
However, compounds in which the urea group were
substituted at the meta-position of the benzene ring did
not exert a remarkable effect on HCT-116 cells. Further-
more, compound 9i showed appreciable inhibitory effects
on MCF-7 and HCT-116 cells. In summary, the synthe-
sized compounds exhibited inhibitory activity against
human breast cancer cells and colon cancer cells.
3-aminophenylboronic
acid
pinacol
ester
(10)/4-aminophenylboronic acid pinacol ester (11) and
triphosgene in dichloromethane. Thereafter, 2-(3-iso
cyanatophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane
was reacted with various aromatic amine to obtain differ-
ent urea derivatives. The key intermediate phenolic
hydroxyl urea compounds were further synthesized from
aryl urea and hydrogen dioxide in methanol, as shown in
Scheme 2. The structures of the synthesized compounds
were confirmed by performing 1H NMR and MS. In addi-
tion, a few target compounds were characterized via 13C
NMR spectroscopy.
2.2 | Antitumor activity of the
synthesized compounds
3 | CONCLUSIONS
To evaluate the antitumor properties of N-(3-((6-bro-
mopyrido[2,3-d]pyrimidin-4-yl)oxy)phenyl)pyrrolidine-1-
carboxamide and 1-(3-((6-bromopyrido[2,3-d]pyrimi
A series of novel N-(3-((6-bromopyrido[2,3-d]pyrimidin-
4-yl)oxy)phenyl)pyrrolidine-1-carboxamide and 1-(3-((6-
bromopyrido[2,3-d]pyrimidin-4-yl)oxy)phenyl)-3-propylu
rea derivatives were synthesized. Their antitumor activi-
ties against human breast carcinoma cells (MCF-7) and
din-4-yl)oxy)phenyl)-3-propylurea
derivatives,
we
selected sorafenib as a positive control drug to determine
CONCLUSIO
SCHEME 2 Synthesis routes of 6a–
6i and 7a–7m
NS

4
CHEN ET AL.
TABLE 1 In vitro inhibitory activity of target compounds
the type of urea substituent present at the para-position
and meta-position of phenyl and the properties of the
group at the terminal urea are responsible for their activ-
ity against MCF-7 cells and HCT-116 cells. In summary,
the synthesized compounds exhibited antitumor activity
against human breast cancer cells and colon cancer cells.
against HCT-116 and MCF-7 cells
Inhibition rate of
Compounds HCT-116 (%)
Inhibition rate of
MCF-7 (%)
8a
0.69
5.09
5.98
5.09
—
5.38
26.46
4.24
18.47
—
8b
8c
8d
8e
4 | EXPERIMENTAL
All common reagents and solvents were used as obtained
from commercial supplies without further purifications,
unless otherwise noted. The compounds' melting points
were determined on an X-4 binocular microscope
(Beijing Tech Instrument Co., China) and were not
corrected. MS were determined on an Agilent 1100 LC–
MS in ESI mode (Agilent Technologies, Palo Alto, CA,
USA). 1H NMR and 13C NMR spectra were recorded
using a 400 MHz spectrometer, a Bruker ACF-400
(Bruker Bioscience, Billerica, MA, USA) with TMS as an
internal standard. All anhydrous solvents were dried and
purified according to standard techniques before using.
8f
—
8.14
4.24
7.46
7.63
—
8g
—
8h
8i
3.08
5.47
—
9a
9b
9c
8.53
—
11.85
7.85
5.76
3.73
15.25
1.19
11.19
10.34
12.88
1.36
9.54
4.46
14.77
9d
9e
—
0.94
—
9f
9g
25.56
7.14
15.81
9.57
—
9h
9i
4.1 | Synthesis of ethyl
2-aminonicotinate (2)
9j
9k
9l
2-Aminonicotinic acid (100 g, 723.95 mmol) was added to
a 500 mL flask, followed by ethanol (300 mL) and con-
centrated sulfuric acid (95 mL). The temperature of reac-
tion was then increased from room temperature to 90ꢀC,
stirring for 12 h. TLC was used to monitor the reaction
progress. When the reaction was complete, the
reaction liquid was poured into the ice water, and the pH
was adjusted to 8–9 using ammonia water to afford solid.
Then filtered, and the filter cake was washed by water,
the solvent was dried to obtain ethyl 2-aminonicotinate
(2). White solid; yield 98.08%; 1H NMR (400 MHz,
CDCl3-d) δ: 8.26 (dd, J = 4.8, 1.9 Hz, 1H), 8.19 (dd,
J = 7.8, 1.9 Hz, 1H), 6.67 (dd, J = 7.8, 4.8 Hz, 1H), 4.40
(q, J = 7.1 Hz, 2H), 1.44 (t, J = 7.1 Hz, 3H).
17.28
—
9m
Sorafenib
17.28
human colon cancer cells (HCT-116) were evaluated in
vitro. Anticancer bio-assay highlighted the potency of the
synthesized compounds 8b, 8d, 9b, 9f, 9g, 9h, 9i, 9j, and
9l as anticancer agents against MCF-7 and HCT-116 cells.
Moreover, compound 8b with 3-tert-butyl urea exhibited
the best antiproliferative activity against MCF-7 cancer
cell lines, indicating that the use of compounds harboring
an electron-donating alkyl urea moiety at the meta-
position of the phenyl ring can result in an increase in
antiproliferative activity. However, compounds 9a–9m
with phenyl and heteroaromatic ring moieties at the ter-
minal urea were found to exhibit certain antitumor prop-
erties and showed better activity against the two tested
cell lines. Compound 9g, containing a pyridinyl moiety at
the terminal urea, showed better antiproliferative activity
against HCT-116 cells than that exhibited by sorafenib.
Furthermore, compound 9i with an electron-
withdrawing (2-trifluoromethyl)phenyl) moiety at the ter-
minal urea exhibited influence on HCT-116 and MCF-7
cells. The results of the anticancer bioassay revealed that
4.2 | Synthesis of ethyl
2-aminonicotinate (3)
Ethyl 2-aminonicotinate (2) (100 g, 601.76 mmol) was
added to a 1000 mL flask, followed by acetonitrile
(300 mL) and NBS (139 g, 10.00 mmol) that were added
slowly under an ice water bath. The mixture was then
allowed to undergo reaction at room temperature for 1 h,
and TLC was used to monitor the reaction progress. After
the reaction was complete, it was filtered, and the filter

CHEN ET AL.
5
cake was stirred well with ammonia water. The filter
cake was washed with water and then dried to obtain
ethyl 2-amino-5-bromonicotinate (3). Light yellow solid;
yield 94.93%; H NMR (400 MHz, CDCl3-d) δ: 8.29 (d,
J = 2.5 Hz, 1H), 8.27 (d, J = 2.4 Hz, 1H), 4.40 (q,
J = 7.1 Hz, 2H), 1.44 (t, J = 7.1 Hz, 3H).
3-Aminophenylboronic acid pinacol ester was added, and
the mixture was stirred continuously for 1 h until the
reaction liquid attained transparency. After the reaction
achieved completion, it was concentrated under reduced
pressure to remove the volatile components. Dic-
hloromethane (100 mL) was added to the concentrated
solution, and a mixture of triethylamine (12.7 mL) and n-
propylamine (7.53 mL) was slowly added to the flask
under an ice water bath. Then, the mixture was heated to
48ꢀC and stirred for 2 h. After the reaction was complete
via TLC, the solvent was removed by evaporation and
1 mol/L dilute hydrochloric acid (100 mL) was added.
Then, the precipitate white solid was filtered, and the fil-
ter cake was washed twice with saturated sodium bicar-
bonate, and the crude product was dried. Finally, the
crude product was recrystallized with ethanol (100 mL),
and the mixture was pumped, filtered, and dried to
obtain compound 10/11. (b) Compound 10/11 (5 g
15.14 mmol) and methanol (30 mL) were added to a
100 mL single-mouth flask, and 30% H2O2 (4.5 mL) was
slowly added. The mixture was reacted for 2 h at room
temperature. Thereafter, the reaction mixture was poured
into ice water when TLC showed it was complete. Next,
the precipitated solid was filtered, and the filter cake was
washed with water, 1-(3-hydroxyphenyl)-3-propylurea
(6a–6i, 7a–7m) were obtained after the solvent was dried.
1-(3-Hydroxyphenyl)-3-propylurea (6a). White solid;
1
4.3 | Synthesis of 6-bromopyrido[2,3-d]
pyrimidin-4(3H)-one (4)
Ethyl 2-amino-5-bromo nicotinate (3) (30 g, 122.41
mmol) and formamide (100 mL) were added to a round-
bottom flask. Thereafter, the temperature was increased
from 25ꢀC to 155ꢀC and stirring for 20 h. The reaction
mixture was poured into ice water when it was complete
by TLC. Subsequently, the precipitated solid was filtered.
The filter cake was washed with ethyl acetate and dried
to afford 6-bromopyrido[2,3-d]pyrimidin-4(3H)-one (4).
1
Yellowish-brown solid, yield 83.15%; H NMR (400 MHz,
DMSO-d6) δ: 12.75 (s, 1H), 9.05 (d, J = 2.6 Hz, 1H), 8.62
(d, J = 2.6 Hz, 1H), 8.37 (s, 1H).
4.4 | Synthesis of 6-bromo-4-chloropyrido
[2,3-d]pyrimidine (5)
1
To
a
three-necked round-bottom flask containing
(4) (20 g,
yield 81.50%; H NMR (400 MHz, DMSO-d6) δ: 9.19 (s,
6-bromopyrido[2,3-d]pyrimidin-4(3H)-one
1H), 8.26 (s, 1H), 7.01–6.93 (m, 2H), 6.70 (d, J = 8.0 Hz,
1H), 6.28 (d, J = 7.4 Hz, 1H), 6.05 (t, J = 5.6 Hz, 1H),
3.02 (q, J = 6.8 Hz, 2H), 1.43 (h, J = 7.3 Hz, 2H), 0.87 (t,
J = 7.4 Hz, 3H).
1-(tert-Butyl)-3-(3-hydroxyphenyl) urea (6b). White
solid; yield 82.56%; 1H NMR (400 MHz, DMSO-d6) δ: 9.13
(s, 1H), 8.08 (s, 1H), 6.99–6.90 (m, 2H), 6.65 (d,
J = 7.3 Hz, 1H), 6.27 (d, J = 8.1 Hz, 1H), 5.90 (s, 1H),
1.27 (s, 9H).
1-Cyclopentyl-3-(3-hydroxyphenyl) urea (6c). White
solid; yield 89.82%; 1H NMR (400 MHz, DMSO-d6) δ: 9.19
(s, 1H), 8.12 (s, 1H), 7.00–6.92 (m, 2H), 6.69 (d,
J = 7.3 Hz, 1H), 6.28 (d, J = 8.0 Hz, 1H), 6.07 (d,
J = 7.2 Hz, 1H), 3.91 (q, J = 6.6 Hz, 1H), 1.83 (dq,
J = 12.2, 6.3 Hz, 2H), 1.68–1.47 (m, 4H), 1.33 (dt,
J = 12.7, 6.2 Hz, 2H).
88.48 mmol), phosphorus oxychloride (22 mL), and tolu-
ene (100 mL) were added, after which triethylamine
(37 mL) was slowly added using a syringe. The mixture
was heated from 25ꢀC to 155ꢀC and stirring for 3 h. Then,
the reaction mixture was poured into ice water after the
reaction achieved completion. Some insoluble substances
were filtered using diatomite, and the filter cake was the
filter cake was washed with ethyl acetate (50 mL Â 3).
After performing vacuum filtration, the filtrate was
extracted with ethyl acetate (100 mL Â 3). Thereafter, the
organic layer was dried with anhydrous sodium sulfate,
and the solvent was removed to obtain 6-bromo-
4-chloropyrido[2,3-d]pyrimidine (5). Yellow solid, yield
1
74.89%. H NMR (400 MHz, DMSO-d6) δ: 9.07 (s, 1H),
8.66 (s, 1H), 8.47 (s, 1H).
1-(3-Hydroxyphenyl)-3-isopropyl urea (6d). White
solid; yield 87.77%; 1H NMR (400 MHz, DMSO-d6) δ: 9.19
(s, 1H), 8.15 (s, 1H), 6.99–6.93 (m, 2H), 6.69 (d,
J = 8.0 Hz, 1H), 6.28 (d, J = 7.7 Hz, 1H), 5.91 (d,
J = 7.5 Hz, 1H), 3.73 (dq, J = 13.4, 6.6 Hz, 1H), 1.08 (d,
J = 6.5 Hz, 6H).
4.5 | General procedure for synthesis of
1-(3-hydroxyphenyl)-3-propylurea (6a–6i
and 7a–7m)
(a) Triphosgene (4.74 g, 15.98 mmol) and dic-
hloromethane (100 mL) were added to a 250 mL flask.
Next, the mixture was stirred for 10 min.
1-(Furan-2-ylmethyl)-3-(3-hydroxyphenyl)urea
(6e).
White solid; yield 94.39%; 1H NMR (400 MHz, DMSO-d6)
δ: 9.23 (s, 1H), 8.39 (s, 1H), 7.59 (s, 1H), 7.03–6.94 (m,

6
CHEN ET AL.
2H), 6.71 (d, J = 9.8 Hz, 1H), 6.46 (t, J = 5.7 Hz, 1H),
6.40 (d, J = 5.0 Hz, 1H), 6.31 (d, J = 8.0 Hz, 1H), 6.25 (d,
J = 3.7 Hz, 1H), 4.27 (d, J = 5.7 Hz, 2H).
N-(3-Hydroxyphenyl) pyrrolidine-1-carboxylamide (6f).
White solid; yield 85.89%; 1H NMR (400 MHz, DMSO-d6)
δ: 9.17 (s, 1H), 7.94 (s, 1H), 7.07 (t, J = 2.1 Hz, 1H), 6.97
(t, J = 8.0 Hz, 1H), 6.88 (d, J = 7.1 Hz, 1H), 6.32 (d,
J = 7.9 Hz, 1H), 3.34 (d, J = 5.5 Hz, 4H), 1.92–
1.75 (m, 4H).
1-(3,4-Dimethylphenyl)-3-(4-hydroxyphenyl) urea (7f).
White solid; yield 91.46%; H NMR (400 MHz, DMSO-d6)
1
δ: 9.04 (s, 1H), 8.32 (s, 1H), 8.25 (s, 1H), 7.21 (d, J = 2.5 Hz,
2H), 7.19 (s, 1H), 7.16–7.12 (m, 1H), 7.00 (d, J = 8.2 Hz,
1H), 6.68 (d, J = 8.8 Hz, 2H), 2.18 (s, 3H), 2.15 (s, 3H).
1-(4-Hydroxyphenyl)-3-(pyridine-2-yl) urea (7g). White
solid; yield 91.74%; 1H NMR (400 MHz, DMSO-d6) δ: 8.27
(d, J = 4.4 Hz, 1H), 7.74 (t, J = 7.0 Hz, 1H), 7.42 (d,
J = 8.4 Hz, 1H), 7.30 (d, J = 8.8 Hz, 2H), 7.08–6.95 (m,
1H), 6.74 (d, J = 8.8 Hz, 2H).
N-(3-Hydroxyphenyl) morpholine-4-carboxylamide (6g).
1
White solid; yield 95.80%; H NMR (400 MHz, DMSO-d6)
1-(3-Chlorobenzyl)-3-(4-hydroxyphenyl)urea
(7h).
δ: 8.37 (s, 1H), 7.03–6.99 (m, 1H), 6.97 (d, J = 8.0 Hz, 1H),
6.84 (d, J = 9.1 Hz, 1H), 6.34 (d, J = 8.0 Hz, 1H), 3.66–
3.53 (m, 4H), 3.44–3.37 (m, 4H).
White solid; yield 92.23%. 1-(4-Hydroxyphenyl)-
3-(2-(trifluoromethyl)phenyl) urea (7i). Gray solid; yield
93.24%; H NMR (400 MHz, DMSO-d6) δ: 10.11 (s, 1H),
9.43 (s, 1H), 7.45 (t, J = 7.7 Hz, 2H), 7.29 (d, J = 6.8 Hz,
1H), 7.24 (d, J = 7.8 Hz, 2H), 7.12 (d, J = 8.1 Hz, 1H),
7.07 (d, J = 10.1 Hz, 1H), 6.93 (d, J = 8.0 Hz, 1H), 6.47
(d, J = 7.8 Hz, 1H).
1
1-(3-Hydroxyphenyl)-3-(thiazole-2-yl) urea (6h). White
solid; yield 93.84%; 1H NMR (400 MHz, DMSO-d6) δ:
10.38 (s, 1H), 9.41 (s, 1H), 8.84 (s, 1H), 7.37 (d,
J = 3.6 Hz, 1H), 7.14–7.10 (m, 1H), 7.08 (s, 1H), 7.08–7.05
(m, 1H), 6.80 (d, J = 8.5 Hz, 1H), 6.43 (d, J = 8.1 Hz, 1H).
4-Hydroxy-N-(3-hydroxyphenyl)piperidine-1-carbox-
amide (6i). White solid; yield 93.79%. 1H NMR (400 MHz,
DMSO-d6) δ: 9.13 (s, 1H), 8.31 (s, 1H), 7.01 (t, J = 2.1 Hz,
1H), 6.96 (t, J = 8.1 Hz, 1H), 6.83 (d, J = 7.2 Hz, 1H),
6.32 (d, J = 8.0 Hz, 1H), 4.68 (d, J = 4.3 Hz, 1H), 3.80 (dt,
J = 13.0, 4.0 Hz, 2H), 3.65 (dq, J = 8.6, 4.2 Hz, 1H), 3.01
(t, J = 10.2 Hz, 2H), 1.72 (dd, J = 13.5, 4.2 Hz, 2H), 1.30
(q, J = 9.1 Hz, 2H).
1-(4-Hydroxyphenyl)-3-(pyrazine-2-yl) urea (7j). White
1
solid; yield 85.7%; H NMR (400 MHz, DMSO-d6) δ: 8.97
(s, 1H), 8.34–8.28 (m, 1H), 8.24 (d, J = 2.6 Hz, 1H), 7.29
(d, J = 8.7 Hz, 2H), 6.79–6.71 (m, 2H).
1-(4-Chlorobutyl)-3-(4-hydroxyphenyl)
urea
(7k).
White solid; yield 95.90%.
1-Cyclohexyl-
3-(4-hydroxyphenyl) urea (7l). White solid; yield 91.10%;
1H NMR (400 MHz, DMSO-d6) δ: 8.91 (s, 1H), 7.93 (s,
1H), 7.12 (d, J = 8.8 Hz, 2H), 6.62 (d, J = 8.8 Hz, 2H),
5.87 (d, J = 7.9 Hz, 1H), 3.52–3.38 (m, 1H), 1.78 (dd,
J = 12.2, 3.2 Hz, 2H), 1.71–1.60 (m, 2H), 1.53 (dd, J = 8.3,
3.9 Hz, 1H), 1.35–1.23 (m, 2H), 1.21–1.06 (m, 3H).
1-(tert-Butyl)-3-(4-hydroxyphenyl) urea (7m). Gray
solid; yield 92.83%; 1H NMR (400 MHz, DMSO-d6) δ: 8.91
(s, 1H), 7.89 (s, 1H), 7.22–7.03 (m, 2H), 6.63 (d,
J = 8.5 Hz, 2H), 5.82 (s, 1H), 1.29 (s, 9H).
1-(3,4-Dimethoxyphenyl)-3-(4-hydroxy-phenyl)
urea
(7a). White solid; yield 88.41%. 1H NMR (400 MHz,
DMSO-d6) δ: 9.04 (s, 1H), 8.36 (s, 1H), 8.22 (s, 1H), 7.21
(s, 1H), 7.19 (d, J = 2.1 Hz, 2H), 6.84 (d, J = 2.0 Hz, 2H),
6.68 (d, J = 8.9 Hz, 2H), 3.72 (d, J = 10.8 Hz, 6H).
1-(4-(Dimethylamino)phenyl)-3-(4-hydroxyphenyl) urea
(7b). White solid; yield 84.32%. 1H NMR (400 MHz,
DMSO-d6) δ: 9.02 (s, 1H), 8.15 (d, J = 5.6 Hz, 2H), 7.23
(d, J = 9.0 Hz, 2H), 7.19 (d, J = 8.8 Hz, 2H), 6.68 (d,
J = 6.9 Hz, 2H), 6.66 (d, J = 6.7 Hz, 2H), 2.82 (s, 6H).
1-(4-Hydroxyphenyl)-3-propylurea (7c). White solid;
4.6 | General procedure for synthesis of
8a–8i and 9a–9m
1
yield 87.70%. H NMR (400 MHz, DMSO-d6) δ: 8.88 (s,
1H), 7.99 (s, 1H), 7.12 (d, J = 8.9 Hz, 2H), 6.61 (d,
J = 8.9 Hz, 2H), 5.93 (t, J = 5.7 Hz, 1H), 3.00 (q,
J = 6.8 Hz, 2H), 1.41 (h, J = 7.4 Hz, 2H), 0.86 (t,
J = 7.4 Hz, 3H).
1-(2,4-Difluorophenyl)-3-(4-hydroxyphenyl) urea (7d).
White solid; yield 96.30%; 1H NMR (400 MHz, DMSO-d6)
δ: 9.10 (s, 1H), 8.69 (s, 1H), 8.37 (s, 1H), 8.08 (td, J = 9.3,
6.2 Hz, 1H), 7.29 (ddd, J = 11.7, 8.9, 2.9 Hz, 1H), 7.21 (d,
J = 8.8 Hz, 2H), 7.03 (t, J = 9.5 Hz, 1H), 6.69 (d,
J = 8.8 Hz, 2H).
N-(4-Hydroxyphenyl)morpholine-4-carboxylamide (7e).
White solid; yield 95.67%; 1H NMR (400 MHz, DMSO-d6)
δ: 9.02 (s, 1H), 8.24 (s, 1H), 7.18 (d, J = 8.9 Hz, 2H), 6.64
(d, J = 8.9 Hz, 2H), 3.66–3.55 (m, 4H), 3.44–3.35 (m, 4H).
(1) Potassium carbonate (0.89 g, 6.44 mmol) and N,N-
dimethylformamide (10 mL) was added to a 100 mL
flask, and then 1-(3-hydroxyphenyl)-3-propylurea (6a)
was added to the mixture with stirring. After reacting
30 min, 6-bromo-4-chloropyrido[2,3-d]pyrimidine (5) was
slowly added and stirred for 1 h at room temperature.
The reaction mixture was poured into ice water when
TLC showed it was complete and the some solid was pre-
cipitate. Then precipitated solid was filtered, and the fil-
ter cake was washed with water and dried to afford
1-(3-((6-bromopyrido [2,3-d]pyrimidin-4-yl)oxy)phenyl)-
3-propylurea (8a).
1-(3-((6-Bromopyrido[2,3-d]pyrimidin-4-yl)oxy)phenyl)
-3-propylurea (8a). Light solid; yield 67.60%; mp 195–

CHEN ET AL.
7
198ꢀC; 1H NMR (400 MHz, DMSO-d6) δ: 9.35 (d,
J = 1.6 Hz, 1H), 9.06 (d, J = 1.2 Hz, 1H), 8.95 (s, 1H),
8.64 (d, J = 9.6 Hz, 1H), 7.58 (s, 1H), 7.34 (t, J = 8.0 Hz,
1H), 7.20 (d, J = 8.3 Hz, 1H), 6.87 (d, J = 7.9 Hz, 1H),
6.22 (t, J = 4.8 Hz, 1H), 3.03 (dd, J = 12.2, 6.1 Hz, 2H),
1.44 (dd, J = 14.2, 7.2 Hz, 2H), 0.87 (t, J = 7.3 Hz, 3H).
ESI-MS, m/z: 424.04[M + H]+.
1-(3-((6-Bromopyrido[2,3-d]pyrimidin-4-yl)oxy)phenyl)
-3-(tert-butyl)urea (8b). Yellow solid; yield 72.04%; mp
215–218ꢀC; 1H NMR (400 MHz, DMSO-d6) δ: 9.33 (d,
J = 2.4 Hz, 1H), 9.03 (d, J = 2.5 Hz, 1H), 8.92 (s, 1H),
7.57 (s, 1H), 7.35 (t, J = 8.2 Hz, 1H), 7.13 (d, J = 8.0 Hz,
1H), 6.88 (d, J = 8.0 Hz, 1H), 6.06 (s, 1H), 1.28 (s, 9H).
ESI-MS, m/z: 416.07[M À H]À.
mp 222–224ꢀC; 1H NMR (400 MHz, DMSO-d6) δ: 9.32 (d,
J = 2.6 Hz, 1H), 9.03 (d, J = 2.6 Hz, 1H), 8.92 (s, 1H),
7.53 (s, 1H), 7.37 (s, 1H), 7.36 (d, J = 1.5 Hz, 1H), 6.96–
6.90 (m, 1H), 3.61–3.56 (m, 4H), 3.43–3.39 (m, 4H). 13C
NMR (101 MHz, DMSO-d6) δ: 167.31, 159.27, 158.27,
157.82, 155.36, 152.48, 142.42, 135.57, 129.85, 118.96,
117.39, 115.39, 113.12, 112.52, 66.44, 44.63. ESI-MS, m/z:
428.04 [M À H]À.
1-(3-((6-Bromopyrido[2,3-d]pyrimidin-4-yl)oxy)phenyl)
-3-(thiazol-2-yl)urea (8h). Yellow solid; yield 45.64%; mp
230–232ꢀC; 1H NMR (400 MHz, DMSO-d6) δ: 9.34 (d,
J = 2.5 Hz, 1H), 9.06 (d, J = 2.5 Hz, 1H), 8.94 (s, 1H),
7.66 (s, 1H), 7.45 (t, J = 8.1 Hz, 1H), 7.37 (d, J = 3.4 Hz,
1H), 7.33 (d, J = 8.8 Hz, 1H), 7.11 (s, 1H), 7.04 (d,
J = 9.5 Hz, 1H). ESI-MS, m/z: 442.98 [M À H]À.
1-(3-((6-Bromopyrido[2,3-d]pyrimidin-4-yl)oxy)phenyl)-
3-cyclopentylurea (8c). Yellow solid; yield 46.29%; mp 173–
176ꢀC; 1H NMR (400 MHz, DMSO-d6) δ: 9.34 (d,
J = 2.6 Hz, 1H), 9.04 (d, J = 2.6 Hz, 1H), 8.93 (s, 1H), 7.55
(t, J = 2.1 Hz, 1H), 7.36 (t, J = 8.1 Hz, 1H), 7.19 (d,
J = 9.2 Hz, 1H), 6.89 (dd, J = 7.8, 1.8 Hz, 1H), 3.97–3.82
(m, 1H), 1.83 (td, J = 12.2, 6.5 Hz, 2H), 1.67–1.58 (m, 2H),
1.57–1.48 (m, 2H), 1.41–1.30 (m, 2H). 13C NMR (101 MHz,
DMSO-d6) δ: 167.32, 159.24, 158.27, 157.83, 155.08, 152.76,
142.48, 135.58, 130.14, 118.93, 115.51, 114.52, 112.54,
111.21, 51.35, 33.23, 23.61. ESI-MS, m/z: 426.06[M À H]-.
1-(3-((6-Bromopyrido[2,3-d]pyrimidin-4-yl)oxy)phenyl)
-3-isopropylurea (8d). Yellow solid; yield 77.26%; mp 221–
224ꢀC; 1H NMR (400 MHz, DMSO-d6) δ: 9.04 (d,
J = 2.6 Hz, 1H), 8.93 (s, 1H), 7.55 (t, J = 2.1 Hz, 1H), 7.35
(t, J = 8.1 Hz, 1H), 7.19 (dd, J = 8.6, 2.3 Hz, 1H), 6.88
(dd, J = 8.0, 2.2 Hz, 1H), 3.73 (dd, J = 13.0, 6.5 Hz, 1H),
1.09 (d, J = 6.5 Hz, 6H). ESI-MS, m/z: 400.04[M À H]À.
1-(3-((6-Bromopyrido[2,3-d]pyrimidin-4-yl)oxy)phenyl)
-3-(furan-2-ylmethyl)urea (8e). Yellow solid; yield 82.29%;
N-(3-((6-bromopyrido[2,3-d]pyrimidin-4-yl)oxy)phenyl)
-4-hydroxypiperidine-1-carboxamide (8i). Brown solid;
yield 80.83%; mp 207–208ꢀC; 1H NMR (400 MHz, DMSO-
d6) δ:9.33 (d, J = 2.1 Hz, 1H), 9.03 (d, J = 2.2 Hz, 1H),
8.92 (s, 1H), 7.54 (s, 1H), 7.41–7.26 (m, 2H), 6.91 (d,
J = 6.6 Hz, 1H), 3.81 (d, J = 12.9 Hz, 2H), 3.66 (s, 1H),
3.04 (t, J = 11.7 Hz, 2H), 1.73 (d, J = 12.7 Hz, 2H), 1.29
(dd, J = 20.8 Hz, H), 1.19 (d, J = 23.5 Hz, 2H). ESI-MS,
m/z: 442.05 [M À H]À.
1-(4-((6-Bromopyrido[2,3-d]pyrimidin-4-yl)oxy)phenyl)
-3-(3,4-dimethoxyphenyl)urea (9a). Gray solid; yield
39.05%; mp 233–235ꢀC; 1H NMR (400 MHz, DMSO-d6) δ:
9.35 (d, J = 2.5 Hz, 1H), 9.06 (d, J = 2.5 Hz, 1H), 8.93 (d,
J = 4.3 Hz, 1H), 8.81 (s, 1H), 8.63 (s, 1H), 7.57 (d,
J = 8.9 Hz, 2H), 7.27 (d, J = 9.0 Hz, 2H), 7.23 (s, 1H),
6.88 (t, J = 6.0 Hz, 2H), 3.74 (s, 3H), 3.70 (d, J = 6.9 Hz,
3H). 13C NMR (101 MHz, DMSO-d6) δ: 171.22, 159.21,
157.85, 155.69, 153.26, 149.23, 146.61, 138.50, 135.56,
133.86, 122.46, 119.68, 118.89, 112.95, 112.54, 110.67,
1
mp 197–200ꢀC; H NMR (400 MHz, DMSO-d6) δ: 9.37 (s,
105.78,
104.42,
56.33,
55.83.
ESI-MS,
m/z:
1H), 9.08 (d, J = 1.6 Hz, 1H), 8.97 (s, 1H), 8.80 (s, 1H),
7.60 (s, 2H), 7.38 (t, J = 8.1 Hz, 1H), 7.25 (d, J = 7.9 Hz,
1H), 6.92 (d, J = 7.8 Hz, 1H), 6.66 (t, J = 4.6 Hz, 1H),
6.42 (s, 1H), 6.28 (d, J = 2.0 Hz, 1H), 4.30 (d, J = 4.9 Hz,
2H). ESI-MS, m/z: 462.02 [M À H]À.
494.05 [M À H]À.
1-(4-((6-Bromopyrido[2,3-d]pyrimidin-4-yl)oxy)phe-
nyl)-3-(4-(dimethylamino)phenyl)urea (9b). Brown solid;
yield 11.32%; mp 229–230ꢀC; 1H NMR (400 MHz, DMSO-
d6) δ: 9.30 (s, 1H), 9.02 (s, 1H), 8.90 (s, 1H), 8.65 (s, 1H),
8.32 (s, 1H), 7.52 (d, J = 7.9 Hz, 2H), 7.25 (d, J = 9.9 Hz,
4H), 6.70 (d, J = 8.7 Hz, 2H), 2.81 (s, 6H). ESI-MS, m/z:
477.07 [M À H]À.
N-(3-((6-Bromopyrido[2,3-d]pyrimidin-4-yl)oxy)phenyl)
pyrrolidine-1-carboxamide (8f). Yellow solid; yield 69.70%;
1
mp 215–218ꢀC; H NMR (400 MHz, DMSO-d6) δ: 9.34 (d,
J = 2.6 Hz, 1H), 9.05 (d, J = 2.6 Hz, 1H), 8.94 (s, 1H), 7.61
(t, J = 2.1 Hz, 1H), 7.44 (d, J = 8.3 Hz, 1H), 7.35 (t,
J = 8.1 Hz, 1H), 6.91 (dd, J = 7.9, 2.1 Hz, 1H), 3.36 (t,
J = 6.5 Hz, 4H), 1.85 (t, J = 6.5 Hz, 4H). 13C NMR
(101 MHz, DMSO-d6) δ: 167.33, 159.27, 158.27, 157.84,
154.12, 152.45, 142.68, 135.55, 129.74, 118.96, 117.22,
114.99, 112.88, 112.50, 46.18, 25.46. ESI-MS, m/z:
412.04 [M À H]À.
1-(4-((6-Bromopyrido[2,3-d]pyrimidin-4-yl)oxy)phenyl)
-3-propylurea (9c). Yellow solid; yield 70.05%; mp 200–
201ꢀC; 1H NMR (400 MHz, DMSO-d6) δ: 9.33 (d,
J = 2.6 Hz, 1H), 9.04 (d, J = 2.6 Hz, 1H), 8.92 (s, 1H),
8.57 (s, 1H), 7.49 (s, J = 9.0 Hz, 2H), 7.22 (d, J = 9.0 Hz,
2H), 6.21 (t, J = 5.7 Hz, 1H), 3.09–3.03 (dd, 2H), 1.46 (dd,
J = 7.2 Hz, 2H), 0.89 (t, J = 7.4 Hz, 3H). 13C NMR
(101 MHz, DMSO-d6) δ: 167.60, 159.18, 158.23, 157.86,
155.71, 146.12, 139.12, 135.55, 122.37, 118.93, 112.54,
41.36, 23.47, 11.82. ESI-MS, m/z: 424.04 [M + H]+.
N-(3-((6-Bromopyrido[2,3-d]pyrimidin-4-yl)oxy)phenyl)
morpholine-4-carboxamide (8g). Pink solid; yield 84.71%;

8
CHEN ET AL.
1-(4-((6-Bromopyrido[2,3-d]pyrimidin-4-yl)oxy)phenyl)
mp 198–199ꢀC; 1H NMR (400 MHz, DMSO-d6) δ: 9.32 (d,
J = 2.6 Hz, 1H), 9.03 (d, J = 2.6 Hz, 1H), 8.92 (s, 1H),
7.53 (d, J = 1.9 Hz, 1H), 7.35 (s, 1H), 7.33 (d, J = 8.1 Hz,
1H), 6.90 (dt, J = 6.3, 2.3 Hz, 1H), 1.62 (d, J = 12.5 Hz,
1H), 1.59 (d, J = 8.2 Hz, 1H), 1.51 (dd, J = 19.1, 7.0 Hz,
3H), 1.12 (d, J = 6.9 Hz, 3H). ESI-MS, m/z: 450.90
[M + H]+.
-3-(2,4-difluorophenyl)urea (9d). Yellow solid; yield
62.67%; mp 240–242ꢀC; 1H NMR (400 MHz, DMSO-d6) δ:
9.34 (d, J = 2.5 Hz, 1H), 9.06 (d, J = 2.5 Hz, 1H), 8.93 (s,
1H), 8.12–8.00 (m, 1H), 7.56 (dd, J = 8.8 Hz, 2H), 7.37–
7.30 (m, 2H), 7.30 (s, 1H), 7.08 (t, J = 8.1 Hz, 1H). ESI-
MS, m/z: 494.00 [M À H]À.
N-(4-((6-bromopyrido[2,3-d]pyrimidin-4-yl)oxy)phenyl)
morpholine-4-carboxamide (9e). Yellow solid; yield
70.25%; mp 224–226ꢀC; 1H NMR (400 MHz, DMSO-d6) δ:
9.34 (d, J = 2.6 Hz, 1H), 9.04 (d, J = 2.6 Hz, 1H), 8.93 (s,
1H), 7.60–7.50 (m, 2H), 7.29–7.21 (m, 2H), 3.66–3.60 (m,
4H), 3.48–3.41 (m, 4H). ESI-MS, m/z: 428.04 [M À H]À.
1-(4-((6-Bromopyrido[2,3-d]pyrimidin-4-yl)oxy)phenyl)
-3-(3,4-dimethylphenyl)urea (9f). Yellow solid; yield
61.28%; mp 249–250ꢀC; 1H NMR (400 MHz, DMSO-d6) δ:
9.34 (d, J = 2.5 Hz, 1H), 9.05 (d, J = 2.5 Hz, 1H), 8.93 (s,
1H), 8.76 (s, 1H), 8.53 (s, 1H), 7.56 (d, J = 8.9 Hz, 2H),
7.29 (d, J = 8.9 Hz, 2H), 7.24 (s, 1H), 7.18 (d, J = 7.6 Hz,
1H), 7.05 (d, J = 8.2 Hz, 1H), 2.20 (s, 3H), 2.16 (s, 3H)
ESI-MS, m/z: 462.06 [M À H]À.
1-(4-((6-Bromopyrido[2,3-d]pyrimidin-4-yl)oxy)phenyl)
-3-cyclohexylurea (9l). Yellow solid; yield 31.78%; mp
194–195ꢀC; 1H NMR (400 MHz, DMSO-d6) δ: 9.34 (d,
J = 2.6 Hz, 1H), 9.04 (d, J = 2.6 Hz, 1H), 8.93 (s, 1H),
8.43 (s, 1H), 7.47 (d, J = 9.0 Hz, 2H), 7.21 (d, J = 9.0 Hz,
2H), 6.11 (d, J = 7.8 Hz, 1H), 3.58–3.38 (m, 1H), 1.92–
1.75 (m, 2H), 1.75–1.60 (m, 2H), 1.30 (dd, J = 17.0,
7.1 Hz, 2H), 1.17 (dd, J = 9.3, 4.7 Hz, 4H). ESI-MS, m/z:
440.07 [M À H]+.
1-(4-((6-Bromopyrido[2,3-d]pyrimidin-4-yl)oxy)phenyl)
-3-(tert-butyl)urea (9m). Yellow solid; yield 76.04%; mp
213–215ꢀC; 1H NMR (400 MHz, DMSO-d6) δ: 9.30 (d,
J = 2.6 Hz, 1H), 9.01 (d, J = 2.6 Hz, 1H), 8.88 (s, 1H),
8.35 (s, 1H), 7.43 (d, J = 8.9 Hz, 2H), 7.19 (d, J = 8.9 Hz,
2H), 6.01 (s, 1H), 1.28 (s, 9H). ESI-MS, m/z:
416.07 [M À H]À.
1-(4-((6-Bromopyrido[2,3-d]pyrimidin-4-yl)oxy)phenyl)
-3-(pyridin-2-yl)urea (9g). Yellow solid; yield 78.64%; mp
235–238ꢀC; 1H NMR (400 MHz, DMSO-d6) δ: 9.34 (d,
J = 2.6 Hz, 1H), 9.05 (d, J = 2.6 Hz, 1H), 8.93 (s, 1H),
8.30 (d, J = 5.7 Hz, 1H), 7.82–7.71 (m, 1H), 7.71–7.57 (m,
2H), 7.49 (dd, J = 8.2, 5.8 Hz, 1H), 7.32 (d, J = 8.9 Hz,
2H), 7.03 (dd, J = 7.1, 5.8 Hz, 1H). ESI-MS, m/z:
435.02 [M À H]À.
4.7 | Antitumor activity
The antiproliferative activity of the target compounds
was evaluated against human breast carcinoma cells
(MCF-7) and human colon cancer cell (HCT-116) using
the standard MTT assay in vitro. The title compounds
were dissolved in proper DMSO to obtain different
diluted solution as blank control and commercial reagent
sorafenib was used as positive control to evaluate the
antiproliferative activity of the target compounds.
The cancer cell line was cultured in Dulbecco's modified
Eagle's medium (DMEM) supplemented with 10% fetal
bovine serum (FBS). Approximately, 4 Â 103 cells,
suspended in DMEM medium, were plated onto each
well of a 96-well plate and incubated in 5% CO2 at 37ꢀC
for 12 h. The tested compound at the final concentration
of 100 μmol/L was added to the culture medium, and the
cell cultures were continued for 24 h. Fresh MTT (20 μL)
was added to each well at a terminal concentration of
5 mg/mL and incubated with cells at 37ꢀC for 4 h but
needed keeping in the dark. The formazan crystals were
dissolved in 150 mL DMSO for each well, and the absor-
bancy at 492 nm (for absorbance of MTT formazan) and
630 nm (for the reference wavelength) were measured
with an ELISA reader. The compound was tested three
times. The OD value of each well at 490 nm was detected
by using a microplate reader. The cellular viability of
every well (%) = OD dosed/OD normalÂ100.
1-(4-((6-Bromopyrido[2,3-d]pyrimidin-4-yl)oxy)phenyl)
-3-(3-chlorobenzyl)urea (9h). Light yellow solid; yield
76.50%; mp 249–251ꢀC; 1H NMR (400 MHz, DMSO-d6) δ:
9.33 (s, 1H), 9.04 (s, 1H), 8.92 (s, 1H), 7.71 (s, 1H), 7.56
(d, J = 7.9 Hz, 2H), 7.34–7.19 (m, 4H), 7.03 (d,
J
=
7.6 Hz, 1H), 1.15 (s, 2H). ESI-MS, m/z:
483.21 [M À H]À.
1-(4-((6-Bromopyrido[2,3-d]pyrimidin-4-yl)oxy)phenyl)
-3-(2-(trifluoromethyl)phenyl)urea (9i). Yellow solid; yield
94.00%; mp 202–205ꢀC; 1H NMR (400 MHz, DMSO-d6) δ:
9.33 (t, J = 2.5 Hz, 1H), 9.04 (dd, J = 4.3, 2.6 Hz, 1H),
8.92 (d, J = 4.1 Hz, 1H), 7.59 (d, J = 1.9 Hz, 1H), 7.57 (d,
J = 2.0 Hz, 1H), 7.54–7.49 (m, 1H), 7.34–7.26 (m, 2H),
7.25–7.17 (m, 2H), 6.73–6.66 (m, 1H). ESI-MS, m/z:
504.37 [M + H]+.
1-(4-((6-Bromopyrido[2,3-d]pyrimidin-4-yl)oxy)phenyl)
-3-(pyrazin-2-yl)urea (9j). Yellow solid; yield 80.09%; mp
232–234ꢀC; 1H NMR (400 MHz, DMSO-d6) δ: 9.34 (d,
J = 2.6 Hz, 1H), 9.05 (d, J = 2.7 Hz, 1H), 9.05 (d,
J = 1.4 Hz, 1H), 8.93 (s, 1H), 8.35–8.31 (m, 1H), 8.27 (d,
J = 2.6 Hz, 1H), 7.63 (d, J = 8.9 Hz, 2H), 7.34 (d,
J = 8.9 Hz, 2H). ESI-MS, m/z: 440.03 [M + H]+.
1-(4-((6-Bromopyrido[2,3-d]pyrimidin-4-yl)oxy)phenyl)
-3-(3-chloropropyl)urea (9k). Yellow solid; yield 84.00%;

CHEN ET AL.
9
[14] E. E. W. Cohen, L. S. Rosen, E. E. Vokes, M. S. Kies, A. A.
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ACKNOWLEDGMENTS
This work was supported by Guizhou Provincial Natural
Science Foundation ([2020]1Y393) and the Science and
Technology Program Platform for Talents of Guizhou
Province ([2016]5634 and [2018]5781). The Science and
Technology Fund of Guizhou Provincial Health Depart-
ment (gzwjkj2020-1-238).
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DATA AVAILABILITY STATEMENT
The data that support the findings of this study are avail-
able from the corresponding author upon reasonable
request. The data that supports the findings of this study
are available in the supporting information of this article.
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ORCID
Zhixu Zhou https://orcid.org/0000-0002-9633-9521
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How to cite this article: D. Chen, Y. Chen,
D. Yang, Z. Zheng, Z. Zhou, J Heterocyclic Chem
2021, 1. https://doi.org/10.1002/jhet.4287