Biphenyl derivatives incorporating urea unit as novel VEGFR-2 inhibitors: Design, synthesis and biological evaluation

Article abstract of DOI:10.1016/j.bmc.2013.11.027

A series of novel biphenyl urea derivates were synthesized and investigated for their potential to inhibit vascular endothelial growth factor receptor-2 (VEGFR-2). In particular, A7, B3 and B4 displayed significant enzymatic inhibitory activities, with IC₅₀ values of 4.06, 4.55 and 5.26 nM. Compound A7 exhibited potent antiproliferative activity on several cell lines. SAR study suggested that the introduction of methyl at ortho-position of the biphenyl urea and tertiary amine moiety could improve VEGFR-2 inhibitory activity and antitumor effects. Molecular docking indicated that the urea moiety formed four hydrogen bonds with DFG residue. These biphenyl ureas could serve as promising lead compounds for further optimization.

Full text of DOI:10.1016/j.bmc.2013.11.027

Bioorganic & Medicinal Chemistry 22 (2014) 277–284
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Biphenyl derivatives incorporating urea unit as novel VEGFR-2
inhibitors: Design, synthesis and biological evaluation
⁄
Chen Wang, Hongping Gao, Jinyun Dong, Yanmin Zhang, Ping Su, Yaling Shi, Jie Zhang
School of Medicine, Xi’an Jiaotong University, No. 76, Yanta West Road, Xi’an, Shaanxi Province 710061, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel biphenyl urea derivates were synthesized and investigated for their potential to inhibit
vascular endothelial growth factor receptor-2 (VEGFR-2). In particular, A7, B3 and B4 displayed signifi-
cant enzymatic inhibitory activities, with IC50 values of 4.06, 4.55 and 5.26 nM. Compound A7 exhibited
potent antiproliferative activity on several cell lines. SAR study suggested that the introduction of methyl
at ortho-position of the biphenyl urea and tertiary amine moiety could improve VEGFR-2 inhibitory activ-
ity and antitumor effects. Molecular docking indicated that the urea moiety formed four hydrogen bonds
with DFG residue. These biphenyl ureas could serve as promising lead compounds for further
optimization.
Received 5 November 2013
Revised 12 November 2013
Accepted 13 November 2013
Available online 21 November 2013
Keywords:
Angiogenesis
VEGFR-2 inhibitor
Biphenyl scaffold
Urea moiety
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
H
N
H
N
O
NH
N
O
Angiogenesis plays an important role in tumor progression and
metastasis of the great majority of solid tumors. Antiangiogenesis
suppresses the growth of many types of tumor.1 It has been studied
intensively for a few decades as an effective treatment of many
types of cancer.2 Angiogenesis is regulated by many growth fac-
tors. The vascular endothelial growth factors (VEGFs) and receptor
(VEGFR-2) play essential role in angiogenesis. VEGFR-2 is the crit-
ical receptor for tumor angiogenesis. Overexpression of VEGFR-2
has been closely implicated in the angiogenesis of solid tumors.3
Therefore, VEGFR-2 inhibitors are capable of treating angiogene-
sis-related cancers. At present, numbers of small molecules had
been identified as VEGFR-2 inhibitors. There are various structure
elements of VEGFR-2 inhibitors (Fig. 1) such as urea (sorafenib),4
ABT-869;5 3-substituted indolinones (sunitinib);6 4-anilinoquinaz-
olines (ZD4190).7 Type I inhibitors constitute the majority of ATP-
competitive inhibitors and recognize mainly the active conforma-
tion of VEGFR-2. Meanwhile, type II inhibitors recognize the inac-
tive DFG (Asp-Phe-Gly)-out conformation of VEGFR-2.
Considerable effort has been made to identify potent VEGFR-2
inhibitors as therapeutic agents against cancers.
O
N
Cl
CF3
N
F
H
O
O
N
H
NH
Sorafenib
Sunitinib
F
Br
H
N
H
N
HN
N
O
O
O
F
N
NH2
N
HN
N
N
N
ZD4190
ABT-869
Figure 1. VEGFR-2 inhibitors currently approved or in clinical trials.
exploration demonstrated that biphenyl played a significant role
in the maintenance of biological activity (Fig. 2).10 Moreover,
NMR-based screening also identified biphenyl as a promising scaf-
fold to develop potential lead compound.11
Further structural optimization was performed to search for no-
vel VEGFR-2 inhibitors. It was known that urea moiety was an
important pharmacophore in type II VEGFR-2 inhibitors.12 It could
form critical hydrogen bonds with the DFG domain of VEGFR-2.
Therefore, urea unit was incorporated to biphenyl scaffold to im-
prove biological activity (Fig. 2). According to previous results,8
the biphenyls possessed poor water solubility. Therefore, tertiary
Natural alkaloid taspine could inhibit angiogenesis by inhibition
of VEGFR-2. Structural optimization of taspine afforded novel
biphenyl derivatives with potent antiangiogenic activity.8 Further
study disclosed that these biphenyls could significantly inhibit
VEGFR-2 and subsequently reduce the angiogenesis.9 SAR
⁄ Corresponding author. Tel./fax: +86 29 82655451.
E-mail address: zhj8623@mail.xjtu.edu.cn (J. Zhang).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.11.027

278
C. Wang et al. / Bioorg. Med. Chem. 22 (2014) 277–284
O
O
N
O
O
R
Skeleton
O
O
O
O
R''
O
Simplification
O
O
R3
O
O
Incorporation
N
H
N
H
Taspine
biphenyl scaffold
R2
R1
biphenyl urea
H
N
H
N
N
H
N
O
O
Cl
O
CF3
Urea moiety is known pharmacophore in VEGFR-2 inhibitors
Figure 2. Discovery of biphenyl scaffold and structures of novel biphenyl ureas.
amine was introduced to terminal aryl to improve hydrophilicity.
In present study, a series of novel biphenyl ureas were designed,
synthesized and evaluated as VEGFR-2 inhibitors. These biphenyl
derivatives bearing urea unit exhibited potent antitumor activity
and could be considered as promising lead compounds.
3. Results and discussion
The compounds were initially evaluated for their inhibitory
activity against VEGFR-2 with sorafenib (free base) as positive con-
trol. The results were summarized in Table 1. Most compounds dis-
played moderate to high inhibitory activity with IC50 values
ranging from 4.06 to 179 nM. Among them, compound A7, B3, B4
potently inhibited VEGFR-2 at nanomolar IC50 values.
2. Chemistry
Compound (A7) was the most potent in a series of para substi-
tuted phenylureas with an IC50 value of 4.06 nM, while (A3) and
(A4) showed moderate inhibitory activities with IC50 values of
17.5 and 12.8 nM, respectively. The presence of 3-morpholino-
propoxy at C-ring may be critical for the activity of these biphenyls.
B series compounds were afforded by replacement of hydrogen
with methyl. Five compounds exhibited more potent inhibitory
activity compared than their counterparts. Compounds (B3) and
(B4) displayed significant inhibitory activities with IC50 values of
4.55 and 5.26 nM, respectively. Compounds of C series were
yielded by incorporation of the R3: alkoxyamino group at 5-posi-
tion of terminal aniline. Only two compounds exhibited slightly
improvement of activity compared to corresponding analogues.
Compound (C2) was the most potent with IC50 value of 16.7 nM.
In general, the enzymatic inhibitory activities of B series com-
pounds were better than A series. The results suggested that the
methyl substitution at 2-position could slightly enhance the
The synthetic route was outlined in Scheme 1. Commercial
available vanillin was converted into bromoaldehyde (2) by reac-
tion with bromine.8 Compound (3) was obtained by reaction of
(2) with sodium formate and hydroxylamine sulfate in formic
acid.13 Aniline (6) was synthesized from (3) by methylation with
dimethyl sulfate,14 hydrolysis with NaOH15 and Hofmann rear-
rangement. Compound (9) was obtained by acylation of 3-bromo-
phenol with acetic anhydride and Fries rearrangement.16
Methylation of compound (9) with dimethyl sulfate in the pres-
ence of K2CO3 afforded (10). The Pd(dppf)Cl2-catalyzed cross-cou-
pling reaction of the pinacol ester of diboronic acid [(Me4C2O2)B–
B(O2C2Me4)] with (10) in the presence of KOAc gave (11).17 Critical
intermediate biphenyl (12) was prepared from (11) and (6) by clas-
sical Suzuki coupling reaction.18 Various substituted anilines were
treated with triphosgene (BTC) to produce various isocyanates. Fi-
nally, these isocyanates were reacted with different anilines afford-
ing various biphenyl ureas.19
Br
Br
Br
Br
Br
O
O
a
HO
O
HO
O
HO
O
e
c
O
O
O
O
b
d
NH2
CHO
CHO
CN
OH
CN
O
CONH2
1
2
4
6
3
5
O
O
O
AcO
Br
HO
Br
i
g
h
f
O
B
O
O
Br
Br
7
8
9
10
11
O
O
O
R2
O
k
O
A1~A7
B1~B7
C1~C7
j
O
O
O
H2N
N
N
R3
H
H
O
12
Scheme 1. Synthetic route of biphenyl ureas. Reagents and conditions: (a) Br2, AcOH, AcONa, Fe, rt; (b) HCOOH, HCOONa, (NH2OH)2ꢀH2SO4, 90 °C; (c) (CH3O)2SO2, K2CO3,
Me2CO, 50 °C; (d) NaOH, H2O2, EtOH, 60 °C; (e) Br2, NaOH; (f) Py, Ac2O, rt; (g) AlCl3,160 °C; (h) (CH3O)2SO2, K2CO3, acetone, 50 °C; (i) bis(pinacolato)diboron, dioxane,
Pd(pddf)Cl2, KOAc, 100 °C; (j) Pd(pddf)Cl2, Na2CO3, H2O, dioxane,100 °C; (k) triphosgene (BTC), anilines, Et3N, DCM, rt.

C. Wang et al. / Bioorg. Med. Chem. 22 (2014) 277–284
279
Table 1
Structure and VEGFR-2 inhibitory activity of biphenyl ureas
O
O
O
R3
B
C
O
N
H
N
H
A
R2
R1
R1
R2
H
R3
4-
4-
IC50 (nM)
>300
ClogP
R1
R2
R3
5-
5-
IC50 (nM)
71.4
ClogP
N
N
A1
COCH3
2.99
C1
COCH3
CH3
O
O
O
O
3.48
A2
A3
A4
COCH3
COCH3
COCH3
H
H
H
>300
17.5
12.8
3.67
3.10
3.73
C2
C3
C4
COCH3
COCH3
COCH3
CH3
CH3
CH3
16.7
17.6
>300
N
N
4.16
3.59
4.21
4-O
5-O
N
N
N
N
N
O
O
4-
5-
N
N
A5
COCH3
H
179
3.31
C5
COCH3
CH3
69.9
O
4-
O
3.80
5-
O
O
N
A6
A7
B1
COCH3
COCH3
COCH3
H
>300
4.06
2.59
2.70
3.48
C6
C7
D1
COCH3
CH3
CH3
CH3
23.9
33.3
ND⁄
O
O
4-
5-
3.08
3.19
3.87
O
O
H
COCH3
O
N
4-O
N
5-
N
N
CH3
>300
CNOHCH3
O
O
4-
4-
B2
B3
B4
COCH3
COCH3
COCH3
CH3
CH3
CH3
128
4.55
5.26
4.16
3.59
4.21
D2
D3
D4
CNOHCH3
CNOHCH3
CNOHCH3
CH3
CH3
CH3
>300
14.3
ND
N
N
4.55
3.98
4.60
O
O
4-
4-
4-O
4-O
N
N
N
N
N
O
O
4-
4-
N
N
N
B5
B6
COCH3
COCH3
COCH3
CH3
CH3
CH3
12.8
ND
3.80
3.08
D5
D6
D7
CNOHCH3
CNOHCH3
CNOHCH3
CH3
CH3
CH3
40.9
129
81.2
O
O
4.18
3.47
3.58
4-
4-
O
O
O
N
N
O
O
4-
4-
O
B7
23.8
1.06
4-O
O
4-
Sorafenib
⁄
ND = not Determined.
Table 2
Antiproliferative activity of (A7) against cancer cells
O
O
O
R3
R3
O
O
O
Compound
Cell lines (IC50
,
l
M)
O
O
O
N
EtOH
NH2OH
N
H
N
H
N
H
N
H
SGC7901a
K562b
SH-SY5Yc
MDA-MB-231d
LOVOe
6.56
11.1
Sunitinib
A7
101
80.9
0.86
2.23
5.73
10.4
35.0
ND
HO
D1~D7
B1~B7
ND: not determined.
Scheme 2. Synthetic route of D1–D7.
a
Human gastric cancer cells.
Human immortalised myelogenous leukemia line.
Human neuroblastoma cells.
Human breast cancer cells.
Human colon adenocarcinoma cell line.
b
c
d
e
activity. However, analogues belong to C series with the same
methyl substitution exhibited weaker activity. The results demon-
strated that 4-position of the urea may be privileged for the alkoxy
group substitution. Various N,N-disubstituted propoxy amine had
significant effects on the biological potency of biphenyl derivates.
3-N,N-dimethylpropoxy, 3-morpholinopropoxy, 2-piperidin-1-
ylethoxy group were preferable substitutions and might be essen-
tial for the potency of compounds.
In order to improve the affinity with VEGFR-2, the acetyl group
on the biphenyl core was converted to oximido.20 Another seven
derivatives were synthesized from (B1–B7) with the route as
shown in Scheme 2. Newly prepared compounds were also
evaluated for their inhibitory activities against VEGFR-2. The re-
sults were also summarized in Table 1. Unfortunately, the enzy-
matic inhibition activities of the newly synthesized compounds
were not enhanced. However, the biological results confirmed
our previous conclusion that the three N,N-disubstituted amines
were beneficial for activity.
In vitro antiproliferative activity of compound (A7) against
various cancer cell lines was measured by 3-(4,5-dimethylthia-

280
C. Wang et al. / Bioorg. Med. Chem. 22 (2014) 277–284
Figure 3. Docking model of A7 bound to VEGFR-2 (PDB ID: 4ASD) active site.
zol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method with
sunitinib (free base) as positive control. As shown in Table 2,
(A7) exhibited potent antiproliferative activity against four cancer
cell lines comparable to that of sunitinib. In particular, it displayed
more potent antiproliferation against SGC-7901 with IC50 value of
80.0 nM than that of sunitinib.
The most potent inhibitor (A7) was docked into the active site
of VEGFR-2 (PDB ID: 4ASD) to understand its binding mode. The
reason we chose 4ASD as receptor is that 4ASD complex contains
sorafenib as a urea containing ligand. Molecular insights based
on molecular docking indicated favorable binding interactions of
compound (A7) with the active site of VEGFR-2 (Fig. 3). 1N–H of
the urea unit formed two hydrogen bonds with Glu885 with dis-
tance of 2.55 and 1.11 Å, respectively. 3N–H could also bind to
the the side chain carboxylate of Glu885 with distance of 1.90 Å.
Carbonyl group identified to be involved in hydrogen bond with
the backbone NH of Asp1046 with distance of 1.74 Å. Length of
three hydrogen bonds was less than 2 Å and they could signifi-
cantly improve the affinity with VEGFR-2.
5. Experimental
5.1. Chemistry: general procedure
Solvents and reagents were purified according to the standard
procedures. All reactions except those in aqueous media were car-
ried out by standard techniques for the exclusion of moisture.
Anhydrous reactions were carried out under nitrogen atmosphere.
Reactions were monitored by thin layer chromatography on 0.25-
mm silica gel plates (60GF-254) and visualized with UV light. Melt-
ing points were determined on electrothermal melting point appa-
ratus and are uncorrected. 1H NMR spectra were measured at
400 MHz on a Bruker Advance AC 400 instrument. Mass spectra
were obtained on a Shimadzu HPLC–MS-QP2010 instrument.
5.1.1. 3-Bromo-4-hydroxy-5-methoxybenzaldehyde (2)
To a mixture of (1) (20.0 g, 0.13 mol), NaOAc (21.6 g, 0.26 mol)
and iron powder (0.68 g, 0.01 mol) was added glacial acetic acid
(120 mL). The mixture was stirred at room temperature for
30 min. Br2 (7 mL, 0.14 mol) in glacial acetic acid (30 mL) was
added dropwise into the above mixture at 23–258 °C. The mixture
was stirred at the same temperature overnight. Ice-water (250 mL)
was added to the mixture and stirred for another 1 h. The solid ob-
tained was dried and recrystallized from EtOH to give (2) (24.6 g,
81%) as gray solid. Mp: 159–160 °C, MS (EI) [M]+: m/z = 230, 1H
NMR (400 MHz, CDCl3) d ppm: 7.38 (s, 1H), 6.62 (s, 1H), 4.00 (s,
3H).
4. Conclusion
In conclusion, twenty-eight novel biphenyl urea derivates were
designed, synthesized and evaluated as novel VEGFR-2 inhibitors.
Several derivatives exhibited potent inhibitory activity against
VEGFR-2. Three of them (A7, B3 and B4) displayed significant enzy-
matic inhibitory activities, with IC50 values of 4.06, 4.55 and
5.26 nM. Moreover, compound (A7) exhibited good antiprolifera-
tive activities against K562, SY5Y and LOVO cell lines. SAR study
disclosed that methyl at 2-position of terminal aniline could im-
prove biological activity. Furthermore, the favorable position for
substitution of the aminoalkoxy group was the 4-position to the
urea group. 3-N,N-Dimethylpropoxy, 3-morpholinopropoxy, 2-
piperidin-1-ylethoxy groups were the most preferred substitutions
on the phenyl group joined to urea group. Docking study showed
four hydrogen bonds between urea unit and active site of VEGFR-
2 as discussed above. These results confirmed as known that urea
moiety played a vital role in the biological activity.21–23 In sum-
mary, these biphenyl ureas have strong potential to be further
developed as novel VEGFR-2 inhibitors. Further structural optimi-
zation of these promising anticancer agents will be reported in
due course.
5.1.2. 3-Bromo-4-hydroxy-5-methoxybenzonitrile (3)
A mixture of (2) (20.7 g, 90 mmol), sodium formate (26.5 g,
300 mmol), and formic acid (150 mL) was heated to 90 °C. To the
above mixture was added hydroxylamine sulfate (8.88 g, 54 mmol)
in six equal portions at 30 min intervals, and the mixture was
heated at 90 °C for 5 h. The reaction was cooled to room tempera-
ture and poured to a solution of sodium chloride (100 g) in water
(400 mL). The resultant solid was collected by filtration and dried
to give an off-white solid (3) (18.0 g, 86%). Mp: 142–144 °C, MS
(EI) [M]+: m/z = 227, 1H NMR (400 MHz, CDCl3) d ppm: 7.07 (s,
1H), 6.42 (s, 1H), 3.98 (s, 3H).
5.1.3. 3-Bromo-4,5-dimethoxybenzonitrile (4)
A mixture of (3) (13.5 g, 60 mmol), potassium carbonate (24.9 g,
180 mmol) and acetone (100 mL) was stirred at 50 °C for 30 min.

C. Wang et al. / Bioorg. Med. Chem. 22 (2014) 277–284
281
Dimethyl sulfate (6.60 mL, 66 mmol) was added dropwise into the
above mixture at 50 °C. After completion of reaction, the solid was
filtered off and the solvent was evaporated. The solid obtained was
dried to give off-white solid (4) (13.5 g, 94%). Mp: 122–123 °C. MS
(EI) [M]+: m/z = 243, 1H NMR (400 MHz,CDCl3) d ppm: 7.50 (s, 1H),
7.12 (s, 1H), 3.94 (s, 3H), 3.92 (s, 3H).
MS (EI) [M]+: m/z = 229, 1H NMR (400 MHz, CDCl3) d ppm: 7.65 (d,
J = 8.0 Hz, 1H), 7.18 (s, 1H), 7.15 (d, J = 4.0 Hz, 1H), 3.94 (s, 3H), 2.61
(s, 3H).
5.1.9. 1-(50-Amino-20,3,30-trimethoxybiphenyl-4-yl)ethanone
(12)
A flask charged with Pd(pddf)Cl2 (0.37 g, 0.5 mmol), KOAc
(1.95 g, 20 mmol), and the pinacol ester of diboron (1.40 g,
5.5 mmol) and (10) (1.15 g, 5 mmol) was flushed with nitrogen.
1,4-dioxane (20 mL) were then added. After being stirred at
100 °C for 5 h under nitrogen atmosphere, the mixture was cooled
to room temperature. Compound (6) (0.81 g, 3.5 mmol), H2O
(6 mL) and 1,4-dioxane (5 mL) were then added and the mixture
was refluxed overnight under nitrogen. The product was extracted
with AcOEt (30 mL ꢂ 3), washed with water, and dried over Na2-
SO4. After filtration and concentration in vacuo, the residues was
purified by silica gel flash chromatography (PE/AcOEt = 3:1) gave
(12) (0.98 g, 65%) as slight yellow solid. Mp: 116–117 °C,MS (EI)
[M]+: m/z = 301, 1H NMR (400 MHz, CDCl3) d ppm: 7.81 (d,
J = 8.0 Hz, 1H), 7.27 (s, 1H), 7.15 (d, J = 8.0 Hz, 1H), 6.35 (s, 1H),
6.30 (s, 1H), 3.94 (s, 3H), 3.89 (s, 3H), 3.52 (s, 3H), 2.67 (s, 3H).
5.1.4. 3-Bromo-4,5-dimethoxybenzamide (5)
To a solution of (4) (8.03 g, 33 mol) in ethanol (150 mL) was
added NaOH (1.60 g, 40 mmol) and 30% H2O2 (17 mL, 600 mmol).
The mixture was stirred at 60 °C for 1 h. After completion of reac-
tion, the mixture was acidized with concentrated HCl. Ethanol was
evaporated and residues was poured into water and filtered. The
white solid obtain was dried to give (5) (7.70 g, 89.5%). Mp: 156–
157 °C, MS (EI) [M]+: m/z = 259, 1H NMR (400 MHz, CDCl3) d
ppm: 7.53 (s, 1H), 7.45 (s, 1H), 3.95 (s, 3H), 3.93 (s, 3H).
5.1.5. 3-Bromo-4,5-dimethoxyaniline (6)
Br2 (0.9 mL) was added dropwise into a solution of NaOH
(2.70 g, 68 mmol) in distill water (54 mL) at ꢁ5 °C and was stirred
for another 10 min. (5) (3.6 g, 14 mmol) was added into the above
solution in batches and stirring continued for 20 min. The mixture
was warmed to room temperature and kept for 30 min. Then the
suspension was heated at 40 °C for 1 h. The mixture was cool to
room temperature, poured into water and extracted with AcOEt
(50 mL ꢂ 3). The organic layer was collected and washed with
water, brine, and dried over Na2SO4. Filtration and concentration
in vacuo afforded crude product. Further purification by silica gel
flash chromatography (PE/AcOEt = 2:1) gave (6) (1.90 g, 60%) as
yellow solid. Mp: 90–91 °C, MS (EI) [M]+: m/z = 233, 1H NMR
(400 MHz, CDCl3) d ppm: 6.48 (s, 1H), 6.23 (s, 1H), 3.83 (s, 3H),
3.79 (s, 3H).
5.1.10. N-(40-Acetyl-30,5,6-trimethoxybiphenyl-3-yl)-N0-{4-[2-
(dimethylamino)ethoxy] phenyl}urea (A1)
Triphosgene (0.36 g, 1.2 mmol) was dissolved in anhydrous
CH2Cl2 (15 mL) and the mixture was stirred on the ice-bath for
15 min. A solution of the (12) (0.90 g, 3 mmol) in anhydrous CH2Cl2
(10 mL) was added dropwise to the above mixture and stirring
continued for 20 min. Et3N (0.36 mL, 2.58 mmol) diluted with CH2-
Cl2 (5 mL) was then added into the mixture. Stirring was continued
for 20 min and a solution of Et3N (0.36 mL, 2.58 mmol), 4-(2-
(dimethylamino)ethoxy)aniline (0.54 g, 3 mmol) in anhydrous
CH2Cl2 (20 mL) was added. After completion of the action, the reac-
tion was quenched with dilute Na2CO3. The organic layer was
washed with water and brine, and dried over Na2SO4. After filtra-
tion and concentration in vacuo, the residues was purified by silica
gel flash chromatography (CH2Cl2/MeOH = 30:1) yielding (A1).
yield 33%, mp: 107–110 °C, MS (ESI) [M+H]+: m/z = 508, 1H NMR
(400 MHz,CDCl3) d ppm: 7.71 (d, J = 8.0 Hz, 2H), 7.38 (s, 1H), 7.25
(s, 1H), 7.21 (s, 1H), 7.06 (d, J = 8.0 Hz, 1H), 6.90 (s, 1H), 6.63 (d,
J = 8.0 Hz, 2H), 4.14 (s, 2H), 3.88 (s, 3H), 3.84 (s, 3H), 3.51 (s, 3H),
3.27 (s, 2H), 2.75 (s, 6H), 2.64 (s, 3H).
5.1.6. 3-Bromophenyl acetate (8)
Compound (7) (10.0 g, 58 mmol) was dissolved in pyri-
dine(30 mL) under ice-bath and was stirred for 30 min. Acetic
anhydride (8.3 mL, 88 mmol) was added dropwise into above mix-
ture at the same temperature. The ice-bath was removed after
dropping and the mixture was stirred for 2 h. Concentrated HCl
and ice-water (300 mL) was added to the above reaction solution
to neutralize PH to 7. The mixture was extracted with AcOEt
(100 mL ꢂ 3). The organic layer was collected and washed with
brine and dried over Na2SO4. Filtration and concentration in vacuo
yield yellow oil (8) (10.15 g, 74%).
5.1.11. Compounds A2–A7, B1–B7 and C1–C7 were also prepared
by using the general procedure described above
5.1.7. 1-(4-Bromo-2-hydroxyphenyl)ethanone (9)
5.1.11.1. N-(40-Acetyl-30,5,6-trimethoxybiphenyl-3-yl)-N0-{4-[2-
In a 250 mL round bottom flask (8) (5.49 g, 26 mmol) and anhy-
drous AlCl3 (6.92 g, 51 mmol) was mixed thoroughly on oil bath at
160 °C for 2 h. Reaction mixture was poured into ice water and
concentrated hydrochloric acid solution was added to break com-
plex formed during reaction. The mixture was taken in AcOEt
(50 mL ꢂ 3). The organic layer was combined and washed with
water and brine, and dried over Na2SO4. After filtration and con-
centration in vacuo, the residues was purified by silica gel flash
chromatography (PE/AcOEt = 30:1) gave (9) (3.85 g, 70%) as white
solid. Mp: 36–38 °C, MS (EI) [M]+: m/z = 213, 1H NMR (400 MHz,
CDCl3) d ppm: 7.60 (d, J = 8.0 Hz, 1H), 7.20 (s, 1H), 7.07 (d,
J = 8.0 Hz, 1H), 2.63 (s, 3H).
(diethylamino)ethoxy]phenyl}urea (A2).
Yield 35%, mp:
107–109 °C. MS (ESI) [M+H]+: m/z = 536, 1H NMR (400 MHz, CDCl3)
d ppm: 7.73 (d, J = 8.0 Hz, 1H), 7.43 (s, 1H), 7.32 (d, J = 8.0 Hz, 2H),
7.23 (s, 1H), 7.09 (d, J = 8.0 Hz, 1H), 6.94 (s, 1H), 6.62 (d, J = 8.0 Hz,
2H), 4.28 (s, 2H), 3.90 (s, 3H), 3.86 (s, 3H), 3.52 (s, 3H), 3.36 (s, 2H),
3.19 (s, 4H), 2.64 (s, 3H),1.27 (s, 6H).
5.1.11.2. N-(40-Acetyl-30,5,6-trimethoxybiphenyl-3-yl)-N0-{4-[3-
(dimethylamino)propoxy]phenyl}urea (A3).
Yield 40%, mp:
132–134 °C, MS (ESI) [M+H]+: m/z = 522, 1H NMR (400 MHz, CDCl3)
d ppm:7.76 (d, J = 8.0 Hz, 1H), 7.47 (s, 1H), 7.36 (d, J = 8.0 Hz, 2H),
7.25 (s, 1H), 7.12 (d, J = 8.0 Hz, 1H), 6.92 (s, 1H), 6.63 (d,
J = 8.0 Hz, 2H), 3.92 (s, 3H), 3.90 (s, 3H), 3.71 (t, J = 8.0 Hz, 2H),
3.55 (s, 3H), 3.15 (t, J = 8.0 Hz, 2H), 2.80 (s, 6H), 2.66 (s, 3H),
2.21–2.23 (m, 2H).
5.1.8. 1-(4-Bromo-2-methoxyphenyl)ethanone (10)
A mixture of (9) (2.14 g, 10 mmol), potassium carbonate (4.14 g,
30 mmol), and acetone (100 mL) was stirred at 50 °C for 30 min.
Dimethyl sulfate (1.1 mL,12 mmol) was added dropwise into the
above mixture at 50 °C. After completion of reaction, the solid
was filtered off and the solvent was evaporated. The solid obtained
was dried to give (10) (2.12 g, 75%) as yellow solid. Mp: 60–61 °C,
5.1.11.3. N-(40-Acetyl-30,5,6-trimethoxybiphenyl-3-yl)-N0-[4-(2-
piperidin-1-ylethoxy)phenyl]urea (A4).
Yield 37%, mp:
114–116 °C, MS (ESI) [M+H]+: m/z = 548, 1H NMR (400 MHz, CDCl3)
d ppm: 7.81(d, J = 8.0 Hz,1H), 7.53 (s, 1H), 7.32 (s, 1H), 7.27 (s, 2H),

282
C. Wang et al. / Bioorg. Med. Chem. 22 (2014) 277–284
7.18 (s, 1H), 7.07 (d, J = 8.0 Hz, 1H), 6.88 (d, J = 8.0 Hz, 2H), 4.11 (t,
J = 6.0 Hz, 2H), 3.91 (s, 6H), 3.56 (s, 3H), 2.81 (t, J = 6.0 Hz, 2H), 2.70
(s, 3H), 2.56 (s, 4H), 1.65 (s, 4H), 1.48 (s, 2H).
5.1.11.11.
methyl-4-(2-pyrrolidin-1-ylethoxy)phenyl]urea (B5).
N-(40-Acetyl-30,5,6-trimethoxybiphenyl-3-yl)-N0-[2-
Yield
45%, mp: 161–164 °C, MS (ESI) [M+H]+: m/z = 548, 1H NMR
(400 MHz, CDCl3) d ppm: 7.72 (d, J = 8.0 Hz, 1H), 7.44 (s, 1H),
7.38 (d, J = 8.0 Hz, 1H), 7.23 (s, 1H), 7.07 (d, J = 8.0 Hz, 1H), 6.95
(s, 1H), 6.56 (s, 1H), 6.52 (d, J = 8.0 Hz, 1H), 4.23 (s, 2H), 3.90 (s,
3H), 3.84 (s, 3H), 3.71 (s, 2H), 2.52 (s, 3H), 3.39 (s, 2H), 2.99 (s,
2H), 2.64 (s, 3H), 2.13 (s, 3H), 2.06 (s, 4H).
5.1.11.4. N-(40-Acetyl-30,5,6-trimethoxybiphenyl-3-yl)-N0-[4-(2-
pyrrolidin-1-ylethoxy)phenyl]urea (A5).
Yield 41%, mp:
113–115 °C, MS (ESI) [M+H]+: m/z = 534, 1H NMR (400 MHz, CDCl3)
d ppm: 7.80 (d, J = 8.0 Hz, 1H), 7.51 (s, 1H), 7.36 (s, 1H), 7.26 (d,
J = 4.0 Hz, 2H), 7.08 (d, J = 8.0 Hz, 1H), 6.88 (d, J = 8.0 Hz, 2H),
6.63 (s, 1H), 4.12 (t, J = 6.0 Hz, 2H), 3.92 (s, 6H), 3.56 (s, 3H), 2.96
(t, J = 6.0 Hz, 2H), 2.71 (s, 4H), 2.69 (s, 3H), 1.85 (s, 4H).
5.1.11.12.
methyl-4-(2-morpholin-4-ylethoxy)phenyl]urea (B6).
N-(40-Acetyl-30,5,6-trimethoxybiphenyl-3-yl)-N0-[2-
Yield
44%, mp: 110–113 °C, MS (ESI) [M+H]+: m/z = 564, 1H NMR
(400 MHz, CDCl3) d ppm: 7.50 (s, 1H), 7.36 (d, J = 12.0 Hz, 1H),
7.24 (s, 1H), 7.05 (d, J = 8.0 Hz, 1H), 6.81 (s, 1H), 6.78 (d,
J = 8.0 Hz, 1H), 6.62 (d, J = 8.0 Hz, 1H), 6.58 (s, 1H), 4.11 (t,
J = 6.0 Hz, 2H), 3.91 (s, 3H), 3.90 (s, 3H), 3.77 (t, J = 4.0 Hz, 4H),
3.54 (s, 3H), 2.85 (t, J = 6.0 Hz, 2H), 2.67 (s, 3H), 2.62 (s, 4H), 2.28
(s, 3H).
5.1.11.5. N-(40-Acetyl-30,5,6-trimethoxybiphenyl-3-yl)-N0-[4-(2-
morpholin-4-ylethoxy)phenyl]urea (A6).
Yield 35%, mp:
123–126 °C, MS (ESI) [M+H]+: m/z = 550, 1H NMR (400 MHz, CDCl3)
d ppm: 7.81 (d, J = 8.0 Hz, 1H), 7.57 (s, 1H), 7.44 (s, 1H), 7.31 (s, 1H),
7.27 (s, 1H), 7.06 (d, J = 8.0 Hz, 1H), 6.86 (d, J = 8.0 Hz, 2H), 6.59 (s,
1H), 4.13 (t, J = 6.0 Hz, 2H), 3.91 (s, 6H), 3.79 (t, J = 4.0 Hz, 4H), 3.55
(s, 3H), 2.87 (t, J = 6.0 Hz, 2H), 2.71 (s, 3H), 2.67 (s, 4H).
5.1.11.13.
methyl-4-(3-morpholin-4-ylpropoxy)phenyl]urea
(B7).
Yield 30%, mp: 130–133 °C, MS (ESI) [M+H]+: m/z = 578,
N-(40-Acetyl-30,5,6-trimethoxybiphenyl-3-yl)-N0-[2-
5.1.11.6. N-(40-Acetyl-30,5,6-trimethoxybiphenyl-3-yl)-N0-[4-(3-
morpholin-4-ylpropoxy)phenyl]urea (A7).
Yield 39%, mp:
107–109 °C, MS (ESI) [M+H]+: m/z = 564, 1H NMR (400 MHz, CDCl3)
d ppm: 7.82 (d, J = 8.0 Hz, 1H), 7.55 (s, 1H), 7.31 (s, 1H), 7.27 (s, 1H),
7.15 (s, 1H), 7.07 (d, J = 8.0 Hz, 1H), 6.88 (d, J = 8.0 Hz, 2H), 6.59 (s,
1H), 4.00 (t, J = 6.0 Hz, 2H), 3.92 (s, 6H), 3.77 (t, J = 4.0 Hz, 4H), 3.56
(s, 3H), 2.70 (s, 3H), 2.58 (t, J = 8.0 Hz, 2H), 2.54 (s, 4H), 1.97–2.02
(m, 2H).
1H NMR (400 MHz, CDCl3) d ppm:7.42 (s, 1H), 7.29 (d, J = 8.0 Hz,
1H), 7.23 (s, 1H), 7.08(d, J = 8.0 Hz, 1H), 6.92 (s, 1H), 6.79 (s, 1H),
6.75 (d, J = 8.0 Hz, 1H), 6.65 (d, J = 4.0 Hz, 1H), 4.00(t, J = 6.0 Hz,
2H), 3.92 (s, 3H), 3.92 (s, 3H), 3.81 (t, J = 4.0 Hz, 4H), 3.53 (s, 3H),
2.70 (t, J = 8.0 Hz, 2H), 2.29 (s, 3H), 2.08 (s, 4H), 2.02–2.06 (m, 2H).
5.1.11.14. N-(40-Acetyl-30,5,6-trimethoxybiphenyl-3-yl)-N0-{5-[2-
5.1.11.7. N-(40-Acetyl-30,5,6-trimethoxybiphenyl-3-yl)-N0-{4-[2-
(dimethylamino)et-oxy]-2-methylphenyl}urea (C1).
Yield
(dimethylamino)ethoxy]-2-methylphenyl}urea (B1).
Yield
47%, mp: 119–121 °C, MS (ESI) [M+H]+: m/z = 522, 1H NMR
(400 MHz, CDCl3) d ppm: 7.74 (d, J = 8.0 Hz, 1H), 7.26 (s, 2H),
7.15 (d, J = 8.0 Hz, 1H), 7.12 (s, 1H), 7.07 (s, 1H), 6.87 (d,
J = 8.0 Hz, 1H), 6.37 (d, J = 8.0 Hz, 1H), 4.31 (s, 2H), 3.92 (s, 3H),
3.85 (s, 3H), 3.54 (s, 3H), 3.18 (s, 2H), 2.66 (s, 3H), 2.22 (s, 3H),
1.34 (s, 6H).
32%, mp: 141–143 °C, MS (ESI) [M+H]+: m/z = 522, 1H NMR
(400 MHz, CDCl3) d ppm: 7.78 (d, J = 8.0 Hz, 1H), 7.47 (s, 1H),
7.33 (d, J = 8.0 Hz, 1H), 7.23 (s, 1H), 7.06 (d, J = 8.0 Hz, 1H), 6.82
(s, 1H), 6.78 (d, J = 8.0 Hz, 1H), 6.60 (d, J = 4.0 Hz, 1H), 4.06 (t,
J = 6.0 Hz, 2H), 3.91 (s, 3H), 3.90 (s, 3H), 3.54 (s, 3H), 2.76 (t,
J = 6.0 Hz, 2H), 2.67 (s, 3H), 2.37 (s, 6H), 2.27 (s, 3H).
5.1.11.15. N-(40-Acetyl-30,5,6-trimethoxybiphenyl-3-yl)-N0-{5-[2-
5.1.11.8. N-(40-Acetyl-30,5,6-trimethoxybiphenyl-3-yl)-N0-{4-[2-
(diethylamino)ethoxy]-2-methylphenyl}urea(C2).
Yield
(diethylamino)ethoxy]-2-methylphenyl}urea
(B2).
Yield
42%, mp: 95–97 °C, MS (ESI) [M+H]+: m/z = 550, 1H NMR (400 MHz,
CDCl3) d ppm: 7.68 (s, 1H), 7.66 (s, 1H), 7.35 (d, J = 8.0 Hz, 1H), 7.23
(s, 1H), 7.09 (t, J = 6.0 Hz, 2H), 7.02 (s, 1H), 6.57 (d, J = 12.0 Hz, 1H),
4.27 (s, 2H), 3.93 (s, 3H), 3.84 (s, 3H), 3.55 (s, 3H), 3.37 (s, 2H), 3.15
(s, 4H), 2.57 (s, 3H), 2.09 (s, 3H), 1.22 (s, 6H).
33%, mp: 137–139 °C, MS (ESI) [M+H]+: m/z = 550, 1H NMR
(400 MHz, CDCl3) d ppm: 7.73 (d, J = 8.0 Hz, 1H), 7.49 (s, 1H),
7.41(d, J = 8.0 Hz, 1H), 7.24 (s, 1H), 7.09 (d, J = 8.0 Hz, 1H), 6.91 (s,
1H), 6.58 (s, 1H), 6.55 (d, J = 8.0 Hz, 1H), 4.29 (s, 2H), 3.90 (s, 3H),
3.86 (s, 3H), 3.53 (s, 3H), 3.31 (s, 2H), 3.14–3.19 (m, 4H), 2.65 (s,
3H), 2.16 (s, 3H), 1.33 (t, J = 6.0 Hz, 6H).
5.1.11.16. N-(40-Acetyl-30,5,6-trimethoxybiphenyl-3-yl)-N0-{5-[3-
(dimethylamino)propoxy]-2-methylphenyl}urea
(C3).
5.1.11.9. N-(40-Acetyl-30,5,6-trimethoxybiphenyl-3-yl)-N0-{4-[3-
(dimethylamino)propoxy]-2-methylphenyl}urea
Yield 40%, mp: 103–105 °C, MS (ESI) [M+H]+: m/z = 536,
1H NMR (400 MHz, DMSO-d6) d ppm: 7.67(s,1H), 7.60(s,1H),
7.34(d, J = 8.0 Hz,1H), 7.24 (s, 1H), 7.12 (d, J = 8.0 Hz, 1H), 7.04 (d,
J = 8.0 Hz, 1H), 6.99 (d, J = 8.0 Hz, 1H), 6.50 (d, J = 8.0 Hz, 1H),
3.94 (s, 5H), 3.85 (s, 3H), 3.55 (s, 3H), 2.57 (s, 3H), 2.46 (s, 2H),
2.24 (s, 6H), 2.18 (s, 3H), 1.85–1.88 (m, 2H).
(B3).
Yield 35%, mp: 137–139 °C, MS(ESI) [M+H]+: m/z = 536,
1H NMR (400 MHz, CDCl3) d ppm: 7.78 (d, J = 8.0 Hz, 1H), 7.48 (s,
1H), 7.32 (d, J = 8.0 Hz, 1H), 7.23 (s, 1H), 7.06 (d, J = 8.0 Hz, 1H),
6.79 (s, 1H), 6.76 (d, J = 8.0 Hz, 1H), 6.60 (s, 1H), 3.99 (t,
J = 4.0 Hz, 2H), 3.90 (s, 3H), 3.89 (s, 3H), 3.54 (s, 3H), 2.66 (s, 3H),
2.49 (t, J = 6.0 Hz, 2H), 2.29 (s, 6H), 2.27 (s, 3H), 1.97 (t, J = 6.0 Hz,
2H).
5.1.11.17.
methyl-5-(2-piperidin-1-ylethoxy)phenyl]urea (C4).
N-(40-Acetyl-30,5,6-trimethoxybiphenyl-3-yl)-N0-[2-
Yield
31%, mp: 111–113 °C, MS (ESI) [M+H]+: m/z = 562, 1H NMR
(400 MHz, DMSO-d6) d ppm: 7.70 (s, 1H), 7.69 (d, J = 8.0 Hz, 1H),
7.35 (d, J = 8.0 Hz, 1H), 7.23 (s, 1H), 7.09 (t, J = 4.0 Hz, 2H), 7.01
(d, J = 8.0 Hz, 1H), 6.57 (d, J = 8.0 Hz, 1H), 4.30 (s, 2H), 3.94 (s,
3H), 3.84 (s, 3H), 3.55 (s, 3H), 3.40 (s, 4H), 3.03 (s, 2H),2.57 (s,
3H), 2.22 (s, 3H), 1.76 (s, 4H), 1.53 (s, 2H).
5.1.11.10.
methyl-4-(2-piperidin-1-ylethoxy)phenyl]urea (B4).
N-(40-Acetyl-30,5,6-trimethoxybiphenyl-3-yl)-N0-[2-
Yield
36%, mp: 181–182 °C, MS (ESI) [M+H]+: m/z = 562, 1H NMR
(400 MHz, DMSO-d6) d ppm: 7.66 (d, J = 8.0 Hz, 1H), 7.51 (d,
J = 8.0 Hz, 1H), 7.34 (s, 1H), 7.24 (s, 1H), 7.12 (d, J = 8.0 Hz, 1H),
6.96 (s, 1H), 6.78 (d, J = 8.0 Hz, 1H), 6.72 (d, J = 8.0 Hz, 1H), 3.96
(s, 2H), 3.93 (s, 3H), 3.83 (s, 3H), 3.54 (s, 3H), 3.36 (s, 4H), 2.57
(s, 3H), 2.35 (t, J = 12.0 Hz, 2H), 2.20 (s, 3H), 2.15 (s, 4H), 1.81–
1.85 (m, 2H).
5.1.11.18.
methyl-5-(2-pyrrolidin-1-ylethoxy)phenyl]urea (C5).
37%, mp: 102–104 °C, MS (ESI) [M+H]+: m/z = 548, 1H NMR
N-(40-Acetyl-30,5,6-trimethoxybiphenyl-3-yl)-N0-[2-
Yield

C. Wang et al. / Bioorg. Med. Chem. 22 (2014) 277–284
283
(400 MHz,CDCl3) d ppm: 7.43 (s, 1H), 7.26 (d, J = 4.0 Hz, 1H), 7.24
(s, 1H), 7.08 (d, J = 8.0 Hz, 2H), 7.03 (d, J = 4.0 Hz, 1H), 6.99 (d,
J = 4.0 Hz, 1H), 6.34 (d, J = 4.0 Hz, 1H), 4.12 (t, J = 6.0 Hz, 2H), 3.92
(s, 3H), 3.91 (s, 3H), 3.56 (s, 3H), 2.98 (t, J = 6.0 Hz, 2H), 2.76 (s,
4H), 2.67 (s, 3H), 2.20 (s, 3H), 1.85 (s, 4H).
3H), 2.15 (s, 3H), 1.96–2.01 (m, 2H). 13C NMR (101 MHz, DMSO-
d6) d 157.18, 155.26, 154.40, 153.72, 153.25, 140.88, 140.29,
136.91, 134.87, 131.48, 130.61, 129.30, 126.59, 124.60, 121.28,
116.49, 112.60, 112.23, 111.39, 103.38, 66.29, 60.80, 56.19, 56.11,
55.98, 45.68, 27.47, 18.51, 15.74.
5.1.11.19.
methyl-5-(2-morpholin-4-ylethoxy)phenyl]urea (C6).
N-(40-Acetyl-30,5,6-trimethoxybiphenyl-3-yl)-N0-[2-
Yield
5.1.15. N-{40-[(1E)-N-Hydroxyethanimidoyl]-30,5,6-
trimethoxybiphenyl-3-yl}-N0-[2-methyl-4-(2-piperidin-1-
ylethoxy)phenyl]urea (D4)
40%, mp: 120–124 °C, MS (ESI) [M+H]+: m/z = 564, 1H NMR
(400 MHz, CDCl3) d ppm: 7.77(d, J = 8.0 Hz, 1H), 7.39 (s, 1H),7.33
(s, 1H), 7.24 (s, 1H), 7.05 (d, J = 8.0 Hz, 1H), 6.97 (d, J = 4.0 Hz,
1H), 6.75 (d, J = 4.0 Hz, 1H), 6.52 (d, J = 4.0 Hz, 1H), 4.19 (t,
J = 6.0 Hz, 2H), 3.90 (s, 3H), 3.88 (s, 3H), 3.85(t, J = 4.0 Hz, 4H),
3.55 (s, 3H), 3.01 (s, 2H), 2.86 (s, 4H), 2.67 (s, 3H), 2.19 (s, 3H).
Yield 93%, mp: 158–160 °C, MS (ESI) [M+H]+: m/z = 577, 1H
NMR(400 MHz,CDCl3) d ppm: 7.35 (d, J = 8.0 Hz, 1H), 7.20(d,
J = 8.0 Hz, 1H), 7.15 (d, J = 8.0 Hz, 1H), 7.12 (s, 1H), 6.87 (s, 1H),
6.70 (s, 3H), 3.97 (t, J = 6.0 Hz, 2H), 4.21 (t, J = 6.0 Hz, 2H), 3.83 (s,
3H), 3.78 (s, 3H), 3.54 (s, 3H), 2.84 (t, J = 6.0 Hz, 2H), 2.63 (s, 4H),
2.26 (s, 3H), 2.16 (s, 3H), 1.65–1.68 (m, 4H), 1.48 (s, 2H). 13C
NMR (101 MHz, DMSO-d6) d 157.18, 155.05, 154.40, 153.65,
153.26, 140.93, 140.26, 136.82, 134.87, 131.44, 130.67, 129.30,
126.60, 124.54, 121.28, 116.59, 112.60, 112.32, 111.41, 103.40,
66.03, 60.81, 57.81, 56.13, 55.99, 54.82, 25.93, 24.29, 18.49, 15.74.
5.1.11.20.
methyl-5-(3-morpholin-4-ylpropoxy)phenyl]urea
(C7).
N-(40-Acetyl-30,5,6-trimethoxybiphenyl-3-yl)-N0-[2-
Yield 38%, mp: 97–100 °C, MS (ESI) [M+H]+: m/z = 578,
1H NMR (400 MHz, CDCl3) d ppm: 7.58 (s, 1H), 7.42(d, J = 4.0 Hz,
1H), 7.31 (s, 1H), 7.24 (s, 1H), 7.03 (d, J = 8.0 Hz, 2H), 6.60 (d,
J = 4.0 Hz, 1H), 6.55 (d, J = 4.0 Hz, 1H), 3.99 (t, J = 6.0 Hz, 2H), 3.89
(s, 6H), 3.73 (t, J = 4.0 Hz, 4H), 3.54 (s, 3H), 2.68 (s, 3H), 2.52 (d,
J = 8.0 Hz, 2H), 2.49 (s, 4H), 2.20 (s, 3H), 1.93–1.99 (m, 2H).
5.1.16. N-{40-[(1E)-N-Hydroxyethanimidoyl]-30,5,6-
trimethoxybiphenyl-3-yl}-N0-[2-methyl-4-(2-pyrrolidin-1-
ylethoxy)phenyl]urea (D5)
Yield 92%, mp: 140–142 °C, MS (ESI) [M+H]+: m/z = 563, H-
1
5.1.12. N-{4-[2-(Dimethylamino)ethoxy]-2-methylphenyl}-N0-
{40-[(1E)-N-hydro-xyl-ethanimidoyl]-30,5,6-
trimethoxybiphenyl-3-yl}urea (D1)
NMR(400 MHz, CDCl3) d ppm:7.13–7.18 (m, 3H), 7.09 (s, 1H),
6.83 (d, J = 8.0 Hz, 1H), 6.76 (d, J = 8.0 Hz, 2H), 6.70 (s, 1H), 3.97
(t, J = 6.0 Hz, 2H), 4.22 (t, J = 6.0 Hz, 2H), 3.81 (s, 3H), 3.77 (s, 3H),
3.54 (s, 3H), 2.95 (t, J = 6.0 Hz, 2H), 2.73 (s, 4H), 2.55 (s, 3H), 2.16
(s, 3H), 1.85 (s, 4H). 13C NMR (101 MHz, DMSO-d6) d 157.18,
155.09, 154.40, 153.70, 153.25, 140.90, 140.28, 136.88, 134.87,
131.50, 130.65, 129.30, 126.59, 124.61, 121.28, 116.53, 112.60,
112.24, 111.40, 103.40, 67.30, 60.80, 56.12, 55.98, 54.86, 54.47,
23.62, 18.51, 15.74.
Compound (B1) (0.40 g, 0.69 mmol) was dissolved in ethanol
20 mL and the solution was heated to 50 °C. After being stirred
for 15 min, hydroxylamine hydrochloride was added to the above
mixture and stirring continued for 1 h. The mixture was poured
into cool water and neutralized with Na2CO3. The product was ex-
tracted with CH2Cl2 (20 mL ꢂ 3), washed with water, and dried
over Na2SO4. Filtration and concentration in vacuo to afford (D1)
(0.38 g, 88%). Mp: 155–157 °C, MS(ESI) [M+H]+:m/z = 537, 1H
NMR (400 MHz, DMSO-d6) d ppm: 7.61 (d, J = 8.0 Hz, 1H), 7.33 (s,
1H), 7.26 (d, J = 8.0 Hz, 1H), 7.12 (s, 1H), 7.01 (d, J = 4.0 Hz, 1H),
6.95 (s, 1H), 6.84 (s, 1H), 6.78 (d, J = 4.0 Hz, 1H), 4.23 (s, 2H), 3.83
(s, 6H), 3.08 (s, 2H), 3.55 (s, 3H), 2.70 (s, 6H), 2.24 (s, 3H), 2.09
(s, 3H). 13C NMR (101 MHz, DMSO-d6) d 157.14, 154.41, 153.85,
153.74, 153.21, 140.78, 140.32, 136.99, 134.92, 131.63, 131.05,
129.30, 126.58, 123.99, 121.25, 116.78, 112.56, 111.24, 103.40,
63.30, 60.82, 56.11, 56.04, 55.99, 53.73, 43.51, 18.79, 15.76.
5.1.17. N-{40-[(1E)-N-Hydroxyethanimidoyl]-30,5,6-
trimethoxybiphenyl-3-yl}-N0-[2-methyl-4-(2-morpholin-4-
ylethoxy)phenyl]urea (D6)
Yield 89%, mp: 151–153 °C, MS (ESI) [M+H]+: m/z = 579, H-
1
NMR (400 MHz, CDCl3) d ppm:7.32 (s, 1H), 7.24 (d, J = 8.0 Hz,
1H), 7.19 (d, J = 8.0 Hz, 1H), 7.16 (s, 1H), 6.90 (d, J = 4.0 Hz, 1H),
6.74 (d, J = 8.0 Hz, 2H), 6.66 (d, J = 4.0 Hz, 1H), 4.18 (t, J = 6.0 Hz,
2H), 3.85 (s, 3H), 3.78 (s, 7H), 3.54 (s, 3H), 2.86 (t, J = 6.0 Hz, 2H),
2.68 (s, 4H), 2.27 (s, 3H), 2.20 (s, 3H). 13C NMR (101 MHz, DMSO-
d6) d 157.18, 155.01, 154.41, 153.64, 153.26, 140.94, 140.26,
136.81, 134.88, 131.40, 130.70, 129.31, 126.60, 124.50, 121.28,
116.61, 112.60, 112.31, 111.41, 103.41, 66.63, 65.88, 60.81, 57.54,
56.13, 55.98, 54.10, 18.49, 15.74.
5.1.13. N-{4-[2-(Diethylamino)ethoxy]-2-methylphenyl}-N0-{40-
[(1E)-N-hydroxyethanimidoyl]-30,5,6-trimethoxybiphenyl-3-
yl}urea (D2)
Yield 85%, mp: 155–157 °C, MS (ESI) [M+H]+:m/z = 565, 1H NMR
(400 MHz, CDCl3) d ppm: 7.20 (d, J = 8.0 Hz, 2H), 7.15 (s, 1H), 7.08
(s, 1H), 6.88 (d, J = 8.0 Hz, 1H), 6.73 (d, J = 8.0 Hz, 2H), 6.69 (s, 1H),
4.16 (t, J = 6.0 Hz, 2H), 3.82 (s, 3H), 3.77 (s, 3H), 3.53 (s, 3H), 2.91 (t,
J = 6.0 Hz, 2H), 2.71–2.76 (m, 4H), 2.26 (s, 3H), 2.06 (s, 3H), 1.12 (t,
J = 6.0 Hz, 6H). 13C NMR (101 MHz, DMSO-d6) d 157.17, 155.07,
154.41, 153.63, 153.26, 140.90, 140.26, 136.81, 134.88, 131.41,
130.63, 129.31, 126.58, 124.52, 121.28, 116.53, 112.58, 112.28,
111.38, 103.37, 67.21, 60.81, 56.12, 55.98, 51.82, 47.46, 18.49,
15.76, 12.23.
5.1.18. N-{40-[(1E)-N-Hydroxyethanimidoyl]-30,5,6-
trimethoxybiphenyl-3-yl}-N0-[2-methyl-4-(3-morpholin-4-
ylpropoxy)phenyl]urea (D7)
Yield 88%, mp: 154–156 °C, MS (ESI) [M+H]+: m/z = 593, 1H NMR
(400 MHz, DMSO-d6) d ppm: 7.52 (d, J = 8.0 Hz, 1H), 7.31 (s, 1H),
7.26 (d, J = 8.0 Hz, 1H), 7.13 (s, 1H), 6.04 (d, J = 8.0 Hz, 1H), 6.94 (s,
1H), 6.78 (s, 1H), 6.72 (d, J = 8.0 Hz, 1H), 3.96 (t, J = 6.0 Hz, 2H),
3.83 (s,6H), 3.57 (t, J = 4.0 Hz, 4H), 3.55 (s, 3H), 2.41 (t, J = 8.0 Hz,
2H), 2.36 (s, 4H), 2.20 (s, 3H), 2.10 (s, 3H), 1.82–1.88 (m, 2H). 13C
NMR (101 MHz, DMSO-d6) d 157.18, 155.24, 154.41, 153.65,
153.26, 140.94, 140.26, 136.81, 134.88, 131.44, 130.58, 129.31,
126.60, 124.57, 121.29, 116.55, 112.60, 112.28, 111.41, 103.42,
66.68, 66.28, 60.81, 56.13, 55.99, 55.37, 53.86, 26.42, 18.48, 15.74.
5.1.14. N-{4-[3-(Dimethylamino)propoxy]-2-methylphenyl}-N0-
{40-[(1E)-N-hydroxyethanimidoyl]-30,5,6-trimethoxybiphenyl-
3-yl}urea (D3)
Yield 89%, mp: 139–141 °C, MS (ESI) [M+H]+: m/z = 551, 1H
NMR (400 MHz, CDCl3) d ppm: 7.29 (s, 1H), 7.19 (d, J = 8.0 Hz,
1H), 7.14 (d, J = 8.0 Hz, 1H), 7.11 (s, 1H), 6.86 (d, J = 8.0 Hz, 2H),
6.71 (s, 1H), 6.61 (s, 1H), 3.97 (t, J = 6.0 Hz, 2H), 3.81 (s, 3H), 3.75
(s, 3H), 3.50 (s, 3H), 2.50 (t, J = 6.0 Hz, 2H), 2.29 (s, 6H), 2.24 (s,
5.2. Kinase assay24
The ability of compounds to inhibit the phosphorylation of a
peptide substrate by VEGFR-2 was evaluated in a microtiter plate

284
C. Wang et al. / Bioorg. Med. Chem. 22 (2014) 277–284
format using homogeneous time-resolved fluorescence (HTRF).
Firstly, kinase (Km = 0.003767 ng/ L) and substrate
(Km = 121.4 nM) were separately added to a 384-well plate, and
variable concentrations of compounds (diluted in buffer) were
then added to the assay plate. ATP (2 lL, Km = 1.332 lM) was
added and the reaction was allowed to proceed at 37 °C for
30 min. The TK-antibody labeled with Eu3+-cryptate and streptavi-
din-XL665 were then added with EDTA to detect the phosphory-
lated product at room temperature for 1 h. Then the fluorescence
was measured at 615 nm (cryptate) and 665 nm (XL665) using
the Perkin-Elmer victor 2030 multilabel plate reader. Finally, the
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Acknowledgments
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This work was supported by the National Natural Science Foun-
dation (NNSF) of China (Grant Nos. 81302641, 81302737 and
81230079), the Fundamental Research Funds for the Central Uni-
versities, and Natural Science Basic Research Plan in Shaanxi Prov-
ince of China (Program No. 2011K15-05-02).