Discovery of biphenyl-based VEGFR-2 inhibitors. Part 3: Design, synthesis and 3D-QSAR studies

Article abstract of DOI:10.1016/j.bmc.2015.01.006

VEGFR-2 plays an essential role in angiogenesis and is a central target for anticancer drug discovery. In order to develop novel VEGFR-2 inhibitors, we designed and synthesized 33 biphenyl amides based on our previously reported lead compound. The biological results indicated that four compounds (18b, 20e, 20h and 20j) are potent VEGFR-2 inhibitors which are comparable to positive control. Compound 18b displayed the most potent VEGFR-2 inhibition with IC₅₀ value of 2.02 nM. Moreover, it exhibited promising antiproliferative activity against MCF-7 and SMMC-7721 cells with IC₅₀ values of 1.47 μM and 5.98 μM, respectively. Molecular docking and 3D-QSAR studies were also carried out. The results indicated that these biphenyl amides could serve as promising leads for further optimization as novel VEGFR-2 inhibitors.

Full text of DOI:10.1016/j.bmc.2015.01.006

Accepted Manuscript
Discovery of biphenyl-based VEGFR-2 inhibitors. Part III: Design, synthesis
and 3D-QSAR studies
Wen Lu, Pengfei Li, Yuanyuan Shan, Ping Su, Jinfeng Wang, Yaling Shi, Jie
Zhang
PII:
S0968-0896(15)00009-7
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.01.006
BMC 12009
Reference:
Bioorganic & Medicinal Chemistry
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Accepted Date:
21 December 2014
4 January 2015
5 January 2015
Please cite this article as: Lu, W., Li, P., Shan, Y., Su, P., Wang, J., Shi, Y., Zhang, J., Discovery of biphenyl-based
VEGFR-2 inhibitors. Part III: Design, synthesis and 3D-QSAR studies, Bioorganic & Medicinal Chemistry (2015),
doi: http://dx.doi.org/10.1016/j.bmc.2015.01.006
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Bioorganic & Medicinal Chemistry
jour nal homepage: www. elsevier. com
Discovery of biphenyl-based VEGFR-2 inhibitors. Part III: Design, synthesis and 3D-QSAR studies
Wen Lua,†, Pengfei Lia,b,†, Yuanyuan Shanb, Ping Sua, Jinfeng Wanga, Yaling, Shia, Jie Zhanga,∗
a School of Pharmacy, Xi'an Jiaotong University, No. 76, Yanta West Road, Xi’an, Shaanxi Province, 710061, P.R. China
b Department of Pharmacy, The First Affiliated Hospital of Medical College, Xi'an Jiaotong University, Xi'an, Shaanxi
Province, 710061, P.R. China
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
VEGFR-2 plays an essential role in angiogenesis and is a central target for
anticancer drug discovery. In order to develop novel VEGFR-2 inhibitors, we
designed and synthesized thirty-three biphenyl amides based on our previously
reported lead compound. The biological results indicated that four compounds
(18b, 20e, 20h and 20j) are potent VEGFR-2 inhibitors which are comparable
to positive control. Compound 18b displayed the most potent VEGFR-2
inhibition with IC50 value of 2.02 nM. Moreover, it exhibited promising
antiproliferative activity against MCF-7 and SMMC-7721 cells with IC50
values of 1.47 µM and 5.98 µM, respectively. Molecular docking and 3D-
Available online
Keywords:
VEGFR-2 inhibitors
Biphenyl
Amide
Anticancer
3D-QSAR
QSAR studies were also carried out. The results indicated that these biphenyl
amides could serve as promising leads for further optimization as novel
VEGFR-2 inhibitors.
2009 Elsevier Ltd. All rights reserved.
———
∗
Corresponding author. Tel/Fax: +86-29-82655451; E-mail: zhj8623@mail.xjtu.edu.cn (Jie Zhang).
† Both authors contributed equally to this work.
1

Aiming to identify novel VEGFR-2 inhibitors with
different structural features, we explored structural
modifications of our previously reported lead compound
(Figure 2). Principal changes focused on hinge binding
moiety, the oxime moiety was replaced by phenolic
hydroxyl as hydrogen bond donor and acceptor with hinge
region. Subsequently, the urea was replaced with amide
considering amide group could also form two hydrogen
bonds with DFG residues of VEGFR-2 [14]. Moreover, the
methoxyls on biphenyl scaffold were removed one or there
to reduce the steric hindrance and enhance the interaction
with VEGFR-2 [15]. Based on above rationale assessment,
we assumed that the structural modification will provide
structural diversity in the search for suitable
pharmacophoric points of potent inhibitors interaction with
VEGFR-2 [16].
1. Introduction
Angiogenesis plays a pivotal role in tumorigenesis and also
contributes to the pathogenesis and migration of
malignancies [1]. Among all angiogenic factors, vascular
endothelial growth factor receptor-2 (VEGFR-2) is the
most potent inducer acting both on angiogenesis and vessel
maintenance [2]. Anti-angiogenic therapies focus on
targeted inhibition of over-expressed VEGFR-2 with the
aim of suppressing tumor growth [3]. Inhibition of
VEGFR-2 to block angiogenesis has been an important
area of novel anticancer drug discovery for its role in
promoting cancer growth and metastasis [4].
The development of small molecule VEGFR-2 inhibitors
has attracted great interest for novel anticancer drugs
discovery. A number of potent VEGFR-2 inhibitors have
been developed and achieved clinical success in cancer
patients [5]. Considering the difference of structures, these
agents are mainly divided into two classes (defined as type
I and type II inhibitors) [6]. Type I inhibitors mimic the
ATP adenine normally target the hinge region of VEGFR-2
including sunitinib (SU11248, Pfizer), cediranib
(AZD2171, AstraZeneca), vandetanib (ZD 6474,
AstraZeneca) and so on (Figure 1) [7]. The second
generation of VEGFR-2 inhibitors (type II) binds to the
same site with type I but also extends to the additional
hydrophobic site available in DFG-out form [8]. Sorafenib
(Bay 43-9006, Bayer), tivozanib (KRN-951, Kirin), and
Vatalanib (PTK 787, Novartis) are classical type II
VEGFR-2 inhibitors (Figure 1).
<Insert Figure 2 here>
Based on previous results, we disclosed herein design and
synthesis of thirty-three biphenyl amides as novel VEGFR-
2 inhibitors. All the title compounds were evaluated for
their
tyrosine
kinase
inhibitory potency and
antiproliferative activity against two human cancer cell
lines (MCF-7 and SMMC-7721). Several molecules
exhibited comparable VEGFR-2 inhibitory activity and
antiproliferative potency with sorafenib. Furthermore,
molecular docking and 3D-QSAR with CoMFA studies
have been carried out. The possible binding modes with
VEGFR-2 and preliminary structure-activity relationships
were also discussed.
<Insert Figure 1 here>
2. Results and discussion
In our previous research, we explored structural
optimization of natural alkaloid taspine with the goal to
obtain potent VEGFR-2 inhibitors [9-10]. Finally, it was
demonstrated that taspine biphenyl derivatives could be
served as promising lead compounds [11]. In our continued
efforts to search for novel VEGFR-2 inhibitors, we have
recently described a series of biphenyl ureas as effective
anticancer agents at enzymatic and cellular levels [12].
Several promising lead compounds bearing biphenyl
scaffold have been identified for further modified [13].
2.1. Chemistry
The general synthetic routes for the preparation of title
compounds were illustrated in Scheme 1 and Scheme 2.
Up to thirty-three title compounds were prepared using
Grignard reaction and palladium-catalyzed Suzuki cross
coupling reaction as key steps [17]. The synthesis of the
first series title compounds (17a-17g, 18a-18g, 19a-19g)
employed 3'-(benzyloxy)-[1,1'-biphenyl]-3-carboxylic acid
6 and substituted anilines 8a-8c as key intermediates
(Scheme 1). An efficient synthesis of intermediate 3 was
2

performed in three steps [18]. After protection of hydroxyl
group with benzyl, compound 2 was converted to Grignard
reagent, followed by esterification with trimethylborate.
Subsequent acidic hydrolysis provided 3 in high yield,
followed by palladium-catalyzed Suzuki reactions with m-
bromobenzoic acid to afford key intermediate 6 [19].
Meanwhile, intermediates (9a-9c) were obtained from
nitrophenols (7a-7c) by etherification in anhydrous DMF
in the presence of Cs2CO3, followed by palladium-
catalyzed hydrogenation [20]. Finally, the desired biphenyl
amides (17a-17g, 18a-18g, 19a-19g) were prepared via
condensation of intermediates 6 with 9a-9c to afford 10,
followed by deprotection of benzyl with H2 over Pd/C.
<Insert Scheme 1 here>
As observed in Table 1, compounds 18b, 18c and 18d
exhibited more potent VEGFR-2 inhibitory activity than
the others with IC50 values below 10 nM. However,
compounds 19b, 19c, 19d, and 19e displayed higher
antiproliferative potency with IC50 values below 10 µM.
The results demonstrated that fluorine substitution was
benefit for VEGFR-2 inhibition while chlorine substitution
contributed to antiproliferative potency against cancer
cells. In general, compounds bearing halogen showed
improved potency compared with that without substituent.
Among them, compound 18b displayed the most potent
VEGFR-2 inhibitory activity with IC50 value of 2.02 nM.
Meanwhile, it also exhibited the highest antiproliferative
potency with IC50 value of 1.47 µM against MCF-7 while
5.98 µM against SMMC-7721.
As for the title compounds of series 2 （ 20a-20l), a
procedure similar to that described above was performed.
The synthesis of key intermediate 15 was prepared from
commercially available isovanillin and m-bromophenol
(Scheme 2). Isovanillin 11 was converted to 2-
bromoisovanillin 12 by reaction with bromine [21]. After
protection of 12 with benzyl chloride, the aldehyde group
in 13 was oxidized to carboxyl affording 14. Then
brombenzene 14 and benzeneboronic acid 3 underwent
Suzuki coupling using Pd/C as catalyst. Finally, the desired
biphenyl amides (20a-20l) were prepared via condensation
of intermediates 15 with 9a-9c with EDCI as condensing
agent followed by deprotection of benzyl.
<Insert Table 2 here>
As shown in Table 2, most of title compounds exhibited
moderate to high antiproliferative activities against both
two cancer cell lines. Several of them displayed more or
similar enzymatic and cell growth inhibition activities in
comparison with sorafenib. For VEGFR-2 inhibitory
activity, 20e and 20h exhibited higher potency with IC50
values of 4.38 and 9.41 nM, respectively. Compounds 20k
and 20l showed the most potent antiproliferative activity
against MCF-7 and SMMC-7721, respectively. The results
also indicated that chlorine substitution contributed to
inhibition of cancer cell growth.
In summary, replaced the oxime with phenolic hydroxyl,
most of the title compounds displayed moderate to high
VEGFR-2 inhibitory potency. Interestingly, introduction of
halogen such as fluorine and chlorine on terminal aniline
could increase the biological activities against VEGFR-2
and two cancer cell lines. Moreover, the antiproliferative
potency of halogen-contained compounds indicated that
chlorine increased antiproliferative activity in comparison
with fluorine. Furthermore, a moderate increase in
VEGFR-2 inhibition was observed by remove of methoxyl
on biphenyl scaffold. Having demonstrated the importance
of terminal phenyl ring, the effects of ortho- and meta-
substitution of amide on potency were also investigated. In
<Insert Scheme 2 here>
2.2. Biological evaluation
The VEGFR-2 inhibitory activity and antiproliferative
potency of all title compounds were well evaluated [22].
The tyrosine kinase inhibitory activity was assayed using
well-established ADP-Glo assays as previously described.
Thirty-three title compounds were screened for their in
vitro antiproliferative activity against two cancer cell lines
by MTT method with sorafenib as positive control. The in
vitro enzymatic inhibitory activity and antiproliferative
potency associated with the structures of title compounds
were compiled in Table 1 and Table 2.
<Insert Table 1 here>
3

general, meta-substitution was somewhat favorable for
anticancer potency.
via phenolic hydroxyl and amide moieties contributed
significantly to the affinity with VEGFR-2 [23]. Molecular
docking simulation supported the initial design strategy
and suggested a common mode of interaction with ATP
pocket [24]. The two binding models demonstrated that
amide at meta-position might be favorable for affinity with
VEGFR-2 [25]. Molecular insights based on docking
suggested the favorable binding modes of these biphenyl
amides with receptor. This binding hypothesis could
provide valuable information for the discovery of novel
VEGFR-2 inhibitors. Both biological date and docking
results demonstrated that compound 18b could be a
potential and promising VEGFR-2 inhibitor.
2.3. Molecular docking
Computational molecular docking studies were carried out
to investigate the potential binding modes of title
compounds with VEGFR-2. A deeper binding mode
analysis was performed on the most potent inhibitors 20e
and 18b. Compounds 20e and 18b were docked into ATP
pocket of VEGFR-2 (PDB ID: 4ASD) by SYBYL-X 2.0.
Molecular insights based on docking indicated favorable
binding modes of 20e and 18b with ATP pocket of
VEGFR-2. The results indicated that the representative
compound 20e could tightly bind to ATP-binding hinge
region (Figure 3). In the minimized complex of 20e with
ATP site, the hydroxyl on biphenyl formed two hydrogen
bonds with the carboxyl of Val 899, with bond distances of
1.89 Å and 2.43 Å, respectively. The amide moiety formed
another two hydrogen bonds with Glu 885 and Asp 1046 in
the DFG region with distances of 2.27 Å and 1.81 Å,
respectively.
2.4. 3D-QSAR studies with CoMFA
All structures of title compounds were built and aligned
onto the common scaffold with the most potent 18b as the
template [26]. To investigate the SARs of these biphenyl
amide VEGFR-2 inhibitors, the in vitro enzymatic
inhibitory activity data were used to build 3D-QSAR
models. Twenty-eight compounds were employed and the
IC50 values were converted into pIC50 according to the
formula: pIC50 = -lgIC50. To view the field effect on the
target property, CoMFA contour maps were generated. The
contour maps could identify the important regions where
any changes in the steric and electrostatic might affect the
biological activity. 3D-QSAR contour maps of CoMFA
models were depicted in Figure 5 and Figure 6. The most
potent compound 28b was chosen to superimpose into the
contour maps to illustrate the SAR information. For
CoMFA steric contours (Figure 5), detrimental and
beneficial steric interactions are displayed in yellow and
green contours, respectively [27].
<Insert Figure 3 here>
The plausible binding mode of of 18b in the active site of
VEGFR-2 was shown in Figure 4. As we expected, the
hydroxyl on biphenyl formed a hydrogen bond with Glu
917 as a donor with distance of 2.14 Å. Meanwhile, it
formed another hydrogen bond with Cys 919 as an
acceptor with bond length of 1.94 Å. Moreover, amide
moiety formed two hydrogen bonds with Glu 885 and Asp
1046 in DFG region with distances of 2.25 Å and 1.94 Å,
respectively. It is noting that these four hydrogen bonds
were consistent with previously known VEGFR-2
inhibitors such as sorafenib. Furthermore, fluorine formed
two hydrogen bonds with amine of Glu 885 with bond
length of 2.08 Å and 2.63 Å, respectively. These two
hydrogen bonds might contribute to the more potent
biological activities of 18b.
<Insert Figure 5 here>
The contour map of the electrostatic field of CoMFA
model was depicted in Figure 6. The red contours
represented regions which led to the enhancement of
activity with electron rich groups while blue regions
represented desirability of electropositive charge groups.
<Insert Figure 6 here>
<Insert Figure 4 here>
The phenol moiety of 18b fit well into the hinge region of
ATP pocket which formed two hydrogen bonds with Glu
917 and Cys 919. The capability to form hydrogen bonds
The non cross-validated r2 for the model established was
4

0.985. The value of the variance ratio F (n1 = 5, n2 = 22)
was 290.734 while standard error of the estimate (SEE)
was 0.076. We also found in the CoMFA model that the
contributions of steric and electrostatic fields are 0.401 and
0.599, respectively.
UV light. Melting points were determined on an
electrothermic melting point apparatus which was
1
uncorrected. H-NMR spectra were measured on Bruker
Advance (400 MHz) in CDCl3 or DMSO-d6. Coupling
constants (J) are expressed in hertz (Hz) and chemical
shifts (δ) of NMR are described in parts per million (ppm)
units relative to internal control (TMS). Mass spectra were
obtained on Shimadzu HPLC-MS-QP2010 instrument [28].
4.1.1. Preparation of [3-(benzyloxy)phenyl]boronic acid
(3) [29]
3. Conclusion
As an alternative to the previously described biphenyl urea
VEGFR-2 inhibitors, we described herein the design,
synthesis and evaluation of thirty-three biphenyl amides
possessing hydroxyl group as anticancer agents. Modeling
and structure-activity relationship studies indicated that
these compounds fit well with ATP pocket of VEGFR-2.
The biological results demonstrated the potential of
biphenyl amides bearing hydroxyl as novel VEGFR-2
inhibitors. Moreover, highlighting compound 18b
exhibited potency comparable with sorafenib and might be
a promising and novel candidate.
To a suspension of m-bromophenol (1) (15.57 g, 90 mmol)
in dehydrated alcohol (300 mL) was added anhydrous
potassium carbonate (37.32 g, 270 mmol) and benzyl
chloride (11.50 mL, 99 mmol). The mixture was refluxed
for 4 h. Filtration and evaporation of alcohol was done in a
vacuum. The residue was extracted with EtOAc. The
combined organic layers were washed with water, NaOH
(2 M), brine, and HCl (2 M), dried over Na2SO4, and
concentrated to give (2) (22.02 g, 93%) as a yellow solid.
A 250 mL, two-necked, round-bottomed flask containing
dry magnesium (2.18 g, 90 mmol) and iodine (trace) was
equipped with two rubber septum stirred under the
protection of nitrogen. The solution of (2) (15.79 g, 60
mmol) in 40 mL of anhydrous THF was added slowly into
the flask through a syringe at a rate to remain reflux. After
addition finishing, the resulting mixture was kept in
refluxing for 5 h with stirring. The mixture in cooled to -
30°C, then the trimethylborate (9.36 g, 90 mmol) in 60 mL
of anhydrous THF was added slowly through a syringe.
After addition finishing, the resulting mixture was allowed
to stir at room temperature for overnight and then treated
with saturated NH4Cl solution, stirred for 1 h at room
temperature. Then THF was removed by rotary
evaporation. The aqueous layer was extracted with EtOAc
and dried over Na2SO4, filtered and concentrated to afford
the yellow crude product. The crude product was purified
by recrystallization from water affording (3) as a white
solid (7.53 g, 55%). m.p=140~142°C; EI-MS (m/z): 228.0
The binding mode of 18b with VEGFR-2 indicated that
phenolic hydroxyl group of biphenyl might be an
alternative pharmacophore binding with hinge region of
VEGFR-2. The amide could form two hydrogen bonds
with DFG residues indicated that urea moiety of lead
compound could be replaced by the amide. The results
identified two pharmacophoric groups for interacting with
VEGFR-2 and demonstrated that hydroxyl could bind to
hinge region and amide could interact with conserved Glu
885 and Asp 1046 of DFG moiety. In addition, halogen
substitution at terminal aniline led to enhanced potency in
enzymatic and cellular levels. These biphenyl amides have
strong potential to be further developed as novel VEGFR-2
inhibitors and could be a promising starting point for
further medicinal chemistry efforts.
4. Experimental
4.1. Chemistry: General procedure
Solvents and reagents were obtained from commercial
suppliers and purified according to standard procedure.
The reactions except those in aqueous media were
performed over dried glassware under nitrogen
atmosphere. All reactions were monitored by TLC on 0.25-
mm silica gel plates (fluorescence F254) and visualized with
1
(M+); H-NMR (DMSO-d6, 400 MHz):δ 5.09 (2H, s), 7.03
(1H, s), 7.25 (1H, dd, J= 7.5 Hz, 7.5 Hz), 7.31-7.45 (7H,
5

m), 8.04 (2H, s).
washed by Na2CO3, water, saturated sodium chloride. The
organic layer was dried over Na2SO4 to afford (8). N,N-
dimethyl-3-(4-nitrophenoxy) propan-1-amine (8) (3.92
mmol) was dissolved in 30 mL anhydrous methanol, then
0.10 g Pd/C (5%) was added to this solution. After that, the
mixture was reacted at room temperature under H2 for 4 h.
The mixture was filtered, and Pd/C was washed with
methanol for 3-4 times. The organic phases were combined
and methanol was removed by reduced pressure distillation
to yield (9a) for next step without purification.
4.1.2. 3-bromobenzoic acid (5) [30]
To a solution of m-bromobenzaldehyde (4) (5.55 g, 30
mmol) in THF (100 mL) was added distilled H2O (40 mL)
and NaH2PO4 (2.16 g, 18 mmol). The mixture was stirred
at room temperature for 10 min. NaClO2 (8.96 g, 99 mmol)
and 30% H2O2 (6.80 mL, 66 mmol) in distilled H2O (40
mL) were added into the above mixture. The mixture was
stirred at room temperature for 3 h. THF was evaporated
under vacuum and the residue was extracted with EtOAc.
The combined organic layers were washed with water and
the residue was extracted with NaOH (2 M). The aqueous
phase was acidified with concentrated HCl and the solid
was collected by filtration and dried to afford (5) (5.73 g,
95%) as a white solid. m.p=155~157°C; EI-MS (m/z):
201.1 (M+).
Compounds (9b-9u) were prepared by using the general
procedure described above.
4.1.5.
N-{4-[3-(dimethylamino)propoxy]phenyl}-3'-
hydroxybiphenyl-3-carboxamide (17a) [33]
Compound (9a) (0.89 g, 4.6 mmol), DMAP (0.24 g), and
EDCI (1.46 g, 7.6 mmol) were added in sequence to a
solution of (6) (1.16 g, 3.8 mmol) in anhydrous THF (100
mL). An excess of triethylamine (5 mL) was added
dropwise and the mixture was stirred at room temperature
for 8 h. Then the reaction mixture was quenched with
water and extracted with EtOAc. The combined organic
layers were washed with HCl (1 M), brine, saturated
NaHCO3 solution and dried over Na2SO4. After filtration
and concentration of the organic phase, crude (10) (1.19 g,
65%) was obtained. To a solution of (10) (1.19 g, 2.47
mmol) in methanol (60 mL) was added Pd/C (0.20 g),
stirred for 24 h at room temperature under the atmosphere
of hydrogen. The mixture was filtered to remove Pd/C, and
the residue was purified by column chromatography
4.1.3. 3'-(benzyloxy)biphenyl-3-carboxylic acid (6) [31]
The appropriate (3) (3.28 g, 10 mmol) and boronic acid (5)
(2.01 g, 10 mmol) was suspended in water/isopropanol
(v:v=1:2, 36 mL).Then Pd/C (0.20 g) and K2CO3 (5.02 g,
33 mmol) were added. The mixture was stirred at room
temperature for 15 min and refluxed for 15 h under
nitrogen. After the mixture was cooled to room
temperature, it was carefully acidified by addition of HCl
and extracted three times with EtOAc. The combined
organic layers were dried over Na2SO4, filtered and
concentrated under reduced pressure. The residue was
purified by column chromatography yielding (6) (1.37 g,
45%) as a white solid. m.p=142~144°C; EI-MS (m/z):
304.4 (M+).
yielding (17a) (0.92 g, 95%) as
a white solid.
1
4.1.4. N, N-dimethyl-3-(4-nitrophenoxy) propan-1-amine
(9a) [32]
m.p=150~152°C; EI-MS (m/z): 390.3 (M+). H-NMR (400
MHz, DMSO) δ 8.16 (s, 1H), 7.92 (d, J = 8.0 Hz, 1H), 7.81
(d, J = 8.0 Hz, 1H), 7.68 (d, J = 8.0 Hz, 2H), 7.60 (t, J =
4.0 Hz, 1H), 7.31 (t, J = 4.0 Hz, 1H), 7.18 (d, J = 8.0 Hz,
1H), 7.14 (s, 1H), 6.93 (d, J = 8.0 Hz, 2H), 6.82 (dd, J =
8.0Hz, 1H), 3.99 (t, J = 4.0 Hz, 2H), 2.38 (t, J = 4.0 Hz,
2H), 2.17 (s, 6H), 1.87 - 1.83 (m, 2H).
In 100 mL flask, 4-nitrophenol (1.39 g, 10 mmol) was
dissolved in 20 mL anhydrous DMF. Then 3-chloro-N, N-
dimethylpropan-1-amine hydrochloride (1.57 g, 10 mmol)
and Cs2CO3 (5.29 g, 15 mmol) were added to the solution.
Under nitrogen, the mixture was heated to 100°C, and
reacted for 2.5 h. The mixture was filtered, and the filtrate
was poured to ice water (200 mL). The aqueous phase was
extracted by EtOAc. The combined organic phase was
4.1.6. 3-(benzyloxy)-2-bromo-4-methoxybenzoic acid (22)
[10]
The title compound was prepared as we described
6

previously. mp 159-161°C; EI-MS(m/z): 336.9 ([M+H]+),
1H-NMR (400MHz, CDCl3) δ ppm 3.94 (s, 3H), 5.03 (s,
2H), 6.93 (d, J=9.2 Hz, 1H), 7.37-7.56 (m, 5H), 7.87 (d,
J=9.1 Hz, 1H).
2H), 2.44 (s, 4H), 1.51 (d, J = 8.0 Hz, 4H), 1.39 (d, J = 8.0
Hz, 2H).
3'-hydroxy-N-[4-(2-morpholin-4-ylethoxy)phenyl]biphenyl-
3-carboxamide (17f)
1
The other title compounds (17b-20l) were prepared by
using the general procedure described above.
N-{4-[2-(dimethylamino)ethoxy]phenyl}-3'-hydroxy-
biphenyl-3-carboxamide (17b)
m.p.: 165-168°C. EI-MS (m/z): 418.4 [M]+. H-NMR (400
MHz, DMSO) δ 8.16 (s, 1H), 7.92 (d, J = 8.0 Hz, 1H), 7.81
(d, J = 8.0 Hz, 1H), 7.68 (d, J = 8.0 Hz, 2H), 7.58 (dd, J =
8.0 Hz, 1H), 7.31 (t, J = 4.0 Hz, 1H), 7.18 (d, J = 8.0 Hz,
1H), 7.14 (s, 1H), 6.96 (d, J = 8.0 Hz, 2H), 6.82 (dd, J =
8.0, 1H), 4.08 (t, J = 4.0 Hz, 2H), 3.61 - 3.56 (m, 4H), 2.69
(t, J = 4.0 Hz, 2H), 2.48 (s, 4H).
1
m.p.: 106-108°C. EI-MS (m/z): 376.1 [M]+. H-NMR (400
MHz, DMSO) δ 8.16 (s, 1H), 7.92 (d, J = 8.0 Hz, 1H), 7.81
(d, J = 8.0 Hz, 1H), 7.69 (d, J = 8.0 Hz, 2H), 7.60 (t, J =
4.0 Hz, 1H), 7.31 (t, J = 4.0 Hz, 1H), 7.18 (d, J = 8.0 Hz,
1H), 7.14 (s, 1H), 6.95 (d, J = 8.0 Hz, 2H), 6.83 (d, J = 8.0
Hz, 1H), 4.05 (s, 2H), 2.64 (s, 2H), 2.24 (s, 6H).
3'-hydroxy-N-[4-(3-morpholin-4-
ylpropoxy)phenyl]biphenyl-3-carboxamide (17g)
1
m.p.: 111-113°C. EI-MS (m/z): 432.3 [M]+. H-NMR (400
N-{4-[2-(diethylamino)ethoxy]phenyl}-3'-hydroxybiphenyl-
MHz, DMSO) δ 8.18 (s, 1H), 7.92 (d, J = 8.0 Hz, 1H), 7.81
(d, J = 8.0 Hz, 1H), 7.69 (d, J = 8.0 Hz, 2H), 7.59 (t, J =
4.0 Hz, 1H), 7.30 (t, J = 4.0 Hz, 1H), 7.17 (d, J = 8.0 Hz,
2H), 6.94 (d, J = 8.0 Hz, 2H), 6.83 (d, J = 8.0 Hz, 1H),
4.00 (t, J = 4.0 Hz, 2H), 3.57 (d, J = 8.0 Hz, 4H), 2.42 (t, J
= 4.0 Hz, 2H), 2.37 (s, 4H), 1.91 - 1.84 (m, 2H).
3-carboxamide (17c)
m.p.: 127~130°C. EI-MS (m/z): 404.2 [M]+. 1H-NMR (400
MHz, DMSO) δ 8.16 (s, 1H), 7.92 (d, J = 8.0 Hz, 1H), 7.81
(d, J = 8.0 Hz, 1H), 7.69 (d, J = 8.0 Hz, 2H), 7.60 (t, J =
4.0 Hz, 1H), 7.31 (t, J = 4.0 Hz, 1H), 7.18 (d, J = 8.0 Hz,
1H), 7.14 (d, J = 8.0 Hz, 1H), 6.95 (d, J = 8.0 Hz, 2H),
6.82 (dd, J = 8.0, 1H), 4.05 (t, J = 4.0 Hz, 2H), 2.64 (t, J =
4.0 Hz, 4H), 1.92 (s, 2H), 1.01 (t, J = 4.0 Hz, 6H).
3'-hydroxy-N-[4-(2-pyrrolidin-1-ylethoxy)phenyl]biphenyl-
3-carboxamide (17d)
N-{4-[3-(dimethylamino)propoxy]-3-fluorophenyl}-3'-
hydroxybiphenyl-3-carboxamide (18a)
1
m.p.: 155-158°C. EI-MS (m/z): 434.0 [M]+. H-NMR (400
MHz, DMSO) δ 8.16 (s, 1H), 7.92 (d, J = 8.0 Hz, 1H), 7.79
(dd, J = 8.0 Hz, 2H), 7.61 (dd, J = 8.0 Hz, 1H), 7.50 (d, J =
8.0 Hz, 1H), 7.31 (dd, J = 8.0 Hz, 1H), 7.24 – 7.16 (m,
2H), 7.13 (s, 1H), 6.83 (d, J = 8.0 Hz, 1H), 4.13 (s, 2H),
2.67 (s, 2H), 2.48 (d, J = 8.0 Hz, 6H), 1.39 (s, 2H).
N-{4-[2-(dimethylamino)ethoxy]-3-fluorophenyl}-3'-
hydroxybiphenyl-3-carboxamide (18b)
1
m.p.: 105-108°C. EI-MS (m/z): 402.2 [M]+. H-NMR (400
MHz, DMSO) δ 8.18 (s, 1H), 7.94 (d, J = 8.0 Hz, 1H), 7.80
(d, J = 8.0 Hz, 1H), 7.72 (d, J = 8.0 Hz, 2H), 7.59 (t, J =
4.0 Hz, 1H), 7.30 (t, J = 4.0 Hz, 1H), 7.22 - 7.13 (m, 2H),
6.96 (d, J = 8.0 Hz, 2H), 6.85 (dd, J = 8.0, 1H), 6.69 - 6.47
(m, 1H), 4.12 (t, J = 4.0 Hz, 2H), 2.96 - 2.89 (m, 2H), 2.68
(s, 4H), 1.74 (s, 4H).
m.p.: 108~110°C. EI-MS (m/z): 413.2 [M]+. 1H-NMR (400
MHz, DMSO) δ 8.21 (s, 1H), 7.91 (dd, J = 8.0 Hz, 2H),
7.78 (dd, J = 8.0 Hz, 1H), 7.62 (t, J = 4.0 Hz, 1H), 7.51 (d,
J = 8.0 Hz, 3H), 7.46 - 7.39 (m, 4H), 7.35 (d, J = 8.0 Hz,
2H), 7.21 (t, J = 4.0 Hz, 1H), 7.08 (dd, J = 8.0 Hz, 1H),
4.13 (t, J = 4.0 Hz, 2H), 2.69 (t, J = 4.0 Hz, 2H), 2.26 (s,
6H).
3'-hydroxy-N-[4-(2-piperidin-1-ylethoxy)phenyl]biphenyl-
3-carboxamide (17e)
1
m.p.: 166-169°C. EI-MS (m/z): 416.3 [M]+. H-NMR (400
MHz, DMSO) δ 8.16 (s, 1H), 7.93 (t, J = 4.0 Hz, 1H), 7.81
(d, J = 8.0 Hz, 1H), 7.68 (d, J = 8.0 Hz, 2H), 7.60 (t, J =
4.0 Hz, 1H), 7.31 (t, J = 4.0 Hz, 1H), 7.18 (d, J = 8.0 Hz,
1H), 7.14 (s, 1H), 6.95 (d, J = 8.0 Hz, 2H), 6.82 (dd, J =
8.0 Hz, 1H), 4.06 (t, J = 4.0 Hz, 2H), 2.65 (t, J = 4.0 Hz,
N-{4-[2-(diethylamino)ethoxy]-3-fluorophenyl}-3'-
hydroxybiphenyl-3-carboxamide (18c)
7

m.p.: 135~137°C. EI-MS (m/z): 413.3 [M]+. 1H-NMR (400
MHz, DMSO) δ 8.17 (d, J = 8.0 Hz, 1H), 7.91 (dd, J = 8.0
Hz, 1H), 7.80 (d, J = 8.0 Hz, 2H), 7.60 (dd, J = 8.0 Hz,
1H), 7.52 (s, 1H), 7.38 - 7.25 (m, 1H), 7.17 (s, 3H), 6.84
(d, J = 8.0 Hz, 1H), 4.08 (d, J = 8.0 Hz, 2H), 2.79 (d, J =
8.0 Hz, 2H), 1.88 (d, J = 8.0 Hz, 4H), 0.98 (dd, J = 8.0 Hz,
6H).
1H), 7.19 (t, J = 4.0 Hz, 2H), 7.14 (s, 1H), 6.83 (dd, J = 8.0
Hz, 1H), 4.08 (t, J = 4.0 Hz, 2H), 3.59 (s, 4H), 2.41 (s,
6H), 1.92 (s, 2H).
N-{3-chloro-4-[3-(dimethylamino)propoxy]phenyl}-3'-
hydroxybiphenyl-3-carboxamide (19a)
1
m.p.: 152-155°C. EI-MS (m/z): 422.9 [M]+. H-NMR (400
MHz, DMSO) δ 8.17 (s, 1H), 7.93 (d, J = 8.0 Hz, 1H), 7.80
(d, J = 8.0 Hz, 1H), 7.69 (d, J = 8.0 Hz, 2H), 7.59 (t, J =
4.0 Hz, 1H), 7.30 (t, J = 4.0 Hz, 1H), 7.22 - 7.11 (m, 2H),
6.95 (d, J = 8.0 Hz, 2H), 6.84 (d, J = 8.0 Hz, 1H), 4.05 (t, J
= 4.0 Hz, 2H), 2.65 (t, J = 4.0 Hz, 2H), 2.24 (s, 6H), 1.89
(s, 2H).
N-[3-fluoro-4-(2-pyrrolidin-1-ylethoxy)phenyl]-3'-
hydroxybiphenyl-3-carboxamide (18d)
1
m.p.: 110-113°C. EI-MS (m/z): 420.2 [M]+. H-NMR (400
MHz, DMSO) δ 8.17 (s, 1H), 7.94 (d, J = 8.0 Hz, 1H), 7.84
- 7.76 (m, 2H), 7.60 (t, J = 4.0 Hz, 1H), 7.53 (d, J = 8.0
Hz, 1H), 7.30 (t, J = 4.0 Hz, 1H), 7.25 - 7.16 (m, 2H), 7.15
(s, 1H), 6.86 (dd, J = 8.0 Hz, 1H), 6.40 (dd, J = 8.0 Hz,
1H), 4.21 (t, J = 4.0 Hz, 2H), 3.00 (s, 2H), 2.73 (s, 4H),
1.75 (s, 4H).
N-{3-chloro-4-[2-(dimethylamino)ethoxy]phenyl}-3'-
hydroxybiphenyl-3-carboxamide (19b)
1
m.p.: 166-168°C. EI-MS (m/z): 375.9 [M]+. H-NMR (400
MHz, DMSO) δ 8.17 (s, 1H), 7.93 (d, J = 8.0 Hz, 1H), 7.80
(d, J = 8.0 Hz, 1H), 7.69 (d, J = 8.0 Hz, 2H), 7.59 (t, J =
4.0 Hz, 1H), 7.30 (t, J = 4.0 Hz, 1H), 7.21 - 7.12 (m, 2H),
6.95 (d, J = 8.0 Hz, 2H), 6.84 (d, J = 8.0 Hz, 1H), 4.05 (t, J
= 4.0 Hz, 2H), 2.65 (t, J = 4.0 Hz, 2H), 2.24 (s, 6H).
N-{3-chloro-4-[2-(diethylamino)ethoxy]phenyl}-3'-
hydroxybiphenyl-3-carboxamide (19c)
N-[3-fluoro-4-(2-piperidin-1-ylethoxy)phenyl]-3'-
hydroxybiphenyl-3-carboxamide (18e)
1
m.p.: 169-172°C. EI-MS (m/z): 434.1 [M]+. H-NMR (400
MHz, DMSO) δ 7.92 (d, J = 8.0 Hz, 1H), 7.82 (d, J = 8.0
Hz, 1H), 7.76 (dd, J = 8.0 Hz, 1H), 7.61 (t, J = 4.0 Hz,
1H), 7.50 (d, J = 8.0 Hz, 1H), 7.31 (t, J = 4.0 Hz, 1H), 7.21
- 7.16 (m, 2H), 7.14 (d, J = 8.0 Hz, 1H), 6.83 (dd, J = 8.0
Hz, 1H), 4.07 (t, J = 4.0 Hz, 2H), 3.40 (s, 2H), 2.42 - 2.32
(m, 2H), 2.16 (s, 6H), 1.87 (dd, J = 8.0 Hz, 2H).
m.p.: 208~210°C. EI-MS (m/z): 438.2 [M]+. 1H-NMR (400
MHz, DMSO) δ 8.18 (s, 1H), 7.94 (d, J = 8.0 Hz, 1H), 7.80
(d, J = 8.0 Hz, 1H), 7.74 (d, J = 8.0 Hz, 2H), 7.64 - 7.56
(m, 1H), 7.30 (t, J = 4.0 Hz, 1H), 7.21 - 7.14 (m, 2H), 6.99
(d, J = 8.0 Hz, 2H), 6.84 (dd, J = 8.0 Hz, 1H), 4.27 (s, 2H),
3.25 (s, 2H), 2.99 (s, 4H), 1.17 (t, J = 4.0 Hz, 6H).
N-[3-chloro-4-(2-pyrrolidin-1-ylethoxy)phenyl]-3'-
hydroxybiphenyl-3-carboxamide (19d)
N-[3-fluoro-4-(2-morpholin-4-ylethoxy)phenyl]-3'-
hydroxybiphenyl-3-carboxamide (18f)
1
m.p.: 158-160°C. EI-MS (m/z): 436.2 [M]+. H-NMR (400
MHz, DMSO) δ 8.15 (s, 1H), 7.92 (d, J = 8.0 Hz, 1H), 7.82
(d, J = 8.0 Hz, 1H), 7.76 (dd, J = 8.0 Hz, 1H), 7.61 (t, J =
4.0 Hz, 1H), 7.51 (t, J = 4.0 Hz, 1H), 7.31 (t, J = 4.0 Hz,
1H), 7.20 (dd, J = 8.0 Hz, 2H), 7.13 (s, 1H), 6.83 (dd, J =
8.0 Hz, 1H), 4.16 (t, J = 4.0 Hz, 2H), 3.61 - 3.55 (m, 4H),
3.36 (s, 2H), 2.71 (t, J = 4.0 Hz, 2H), 2.49 (s, 2H).
N-[3-fluoro-4-(3-morpholin-4-ylpropoxy)phenyl]-3'-
hydroxybiphenyl-3-carboxamide (18g)
1
m.p.: 145-147°C. EI-MS (m/z): 420.2 [M]+. H-NMR (400
MHz, DMSO) δ 8.17 (s, 1H), 7.93 (d, J = 8.0 Hz, 1H), 7.81
(d, J = 8.0 Hz, 1H), 7.72 (d, J = 8.0 Hz, 2H), 7.60 (t, J =
4.0 Hz, 1H), 7.30 (t, J = 4.0 Hz, 1H), 7.22 - 7.11 (m, 2H),
6.99 (d, J = 8.0 Hz, 2H), 6.83 (d, J = 8.0 Hz, 1H), 4.20 (s,
2H), 3.18 (s, 2H), 2.94 (s, 4H), 1.83 (s, 4H).
1
m.p.: 162-164°C. EI-MS (m/z): 450.0 [M]+. H-NMR (400
N-[3-chloro-4-(2-piperidin-1-ylethoxy)phenyl]-3'-
MHz, DMSO) δ 8.16 (s, 1H), 7.92 (d, J = 8.0 Hz, 1H), 7.82
(d, J = 8.0 Hz, 1H), 7.77 (dd, J = 8.0 Hz, 1H), 7.61 (t, J =
4.0 Hz, 1H), 7.51 (d, J = 8.0 Hz, 1H), 7.31 (t, J = 4.0 Hz,
hydroxybiphenyl-3-carboxamide (19e)
m.p.: 152-154°C. EI-MS (m/z): 450.3 [M]+. H-NMR (400
1
MHz, DMSO) δ 8.18 (s, 1H), 7.94 (d, J = 8.0 Hz, 1H), 7.81
8

(s, 1H), 7.72 (d, J = 8.0 Hz, 1H), 7.61 (s, 1H), 7.29 (d, J =
8.0 Hz, 1H), 7.21 - 7.12 (m, 1H), 6.98 (d, J = 8.0 Hz, 1H),
6.84 (d, J = 8.0 Hz, 1H), 4.27 (d, J = 8.0 Hz, 2H), 3.05 (s,
2H), 2.84 (s, 4H), 1.65 (s, 4H), 1.46 (s, 2H).
m.p.: 145~147°C. EI-MS (m/z): 450.0 [M]+. 1H-NMR (400
MHz, DMSO) δ 7.31 (d, J = 8.0 Hz, 1H), 7.05 (t, J = 4.0
Hz, 1H), 7.01 (s, 1H), 6.97 (d, J = 8.0 Hz, 1H), 6.79 (d, J =
8.0 Hz, 1H), 6.75 (s, 1H), 6.69 (d, J = 8.0 Hz, 1H), 6.64
(dd, J = 8.0 Hz, 1H), 3.99 (t, J = 4.0 Hz, 2H), 2.81 (t, J =
4.0 Hz, 2H), 2.61 (t, J = 4.0 Hz, 4H), 1.84 (s, 6H).
3',6-dihydroxy-5-methoxy-N-[4-(2-pyrrolidin-1-
N-[3-chloro-4-(2-morpholin-4-ylethoxy)phenyl]-3'-
hydroxybiphenyl-3-carboxamide (19f)
1
m.p.: 160-162°C. EI-MS (m/z): 483.9 [M]+. H-NMR (400
MHz, DMSO) δ 8.17 (s, 1H), 7.95 (t, J = 4.0 Hz, 1H), 7.80
(d, J = 8.0 Hz, 1H), 7.71 (d, J = 8.0 Hz, 2H), 7.59 (t, J =
4.0 Hz, 1H), 7.30 (t, J = 4.0 Hz, 1H), 7.17 (d, J = 8.0 Hz,
2H), 6.96 (d, J = 8.0 Hz, 2H), 6.85 (d, J = 8.0 Hz, 1H),
4.14 (s, 2H), 3.64 (s, 4H), 2.82 (s, 2H), 2.61 (s, 4H).
N-[3-chloro-4-(3-morpholin-4-ylpropoxy)phenyl]-3'-
hydroxybiphenyl-3-carboxamide (19g)
ylethoxy)phenyl]biphenyl-2-carboxamide (20d)
m.p.: 121~124°C. EI-MS (m/z): 448.1 [M]+. 1H-NMR (400
MHz, DMSO) δ 7.32 (d, J = 8.0 Hz, 1H), 7.08 (d, J = 8.0
Hz, 1H), 7.02 (s, 1H), 6.98 (d, J = 8.0 Hz, 1H), 6.81 (d, J =
8.0 Hz, 1H), 6.75 (s, 1H), 6.70 (d, J = 8.0 Hz, 1H), 6.62 (d,
J = 8.0 Hz, 1H), 4.02 (t, J = 4.0 Hz, 2H), 2.86 (s, 2H), 2.62
(s, 4H), 1.71 (s, 4H).
1
m.p.: 115-117°C. EI-MS (m/z): 479.3 [M]+. H-NMR (400
3',6-dihydroxy-5-methoxy-N-[4-(2-piperidin-1-
MHz, DMSO) δ 8.18 (s, 1H), 7.94 (t, J = 4.0 Hz, 1H), 7.81
(d, J = 8.0 Hz, 1H), 7.69 (d, J = 8.0 Hz, 2H), 7.60 (t, J =
4.0 Hz, 1H), 7.31 (t, J = 4.0 Hz, 1H), 7.21 - 7.12 (m, 2H),
6.94 (d, J = 8.0 Hz, 2H), 6.89 - 6.80 (m, 1H), 4.00 (t, J =
4.0 Hz, 2H), 3.58 (dd, J = 8.0 Hz, 4H), 2.51 (s, 2H), 2.42
(s, 4H), 1.89 (t, J = 4.0 Hz, 2H).
ylethoxy)phenyl]biphenyl-2-carboxamide (20e)
m.p.: 180~183°C. EI-MS (m/z): 462.1 [M]+. 1H-NMR (400
MHz, DMSO) δ 7.67 (d, J = 8.0 Hz, 1H), 7.46 (d, J = 8.0
Hz, 1H), 7.41 (s, 1H), 7.03 (d, J = 8.0 Hz, 1H), 6.95 (d, J =
8.0 Hz, 1H), 6.77 (t, J = 4.0 Hz, 1H), 4.19 (s, 2H), 3.07 (s,
2H), 2.89 (d, J = 8.0 Hz, 4H), 1.64 (s, 6H).
N-{4-[3-(dimethylamino)propoxy]phenyl}-3',6-dihydroxy-
3',6-dihydroxy-5-methoxy-N-[4-(3-morpholin-4-
5-methoxybiphenyl-2-carboxamide (20a)
ylpropoxy)phenyl]biphenyl-2-carboxamide (20f)
m.p.: 188~191°C. EI-MS (m/z): 436.1 [M]+. 1H-NMR (400
MHz, DMSO) δ 7.65 (d, J = 8.0 Hz, 1H), 7.32 (d, J = 8.0
Hz, 2H), 7.01 (s, 1H), 6.91 (d, J = 8.0 Hz, 1H), 6.80 (d, J =
8.0 Hz, 2H), 6.74 (s, 1H), 6.70 (d, J = 8.0 Hz, 1H), 6.62 (d,
J = 8.0 Hz, 1H), 3.94 (t, J = 4.0 Hz, 2H), 2.75 - 2.65 (m,
2H), 2.41 (s, 6H), 1.91 (s, 2H).
m.p.: 145~148°C. EI-MS (m/z): 478.2 [M]+. 1H-NMR (400
MHz, DMSO) δ 7.65 (d, J = 8.0 Hz, 1H), 7.43 (d, J = 8.0
Hz, 2H), 7.32 (d, J = 8.0 Hz, 1H), 7.07 - 6.97 (m, 2H), 6.91
(d, J = 8.0 Hz, 1H), 6.79 (d, J = 8.0 Hz, 1H), 6.74 (s, 1H),
6.65 (d, J = 8.0 Hz, 1H), 3.97 (d, J = 8.0 Hz, 2H), 3.64 (s,
4H), 2.60 (s, 6H), 1.91 (s, 2H).
N-{4-[2-(dimethylamino)ethoxy]phenyl}-3',6-dihydroxy-5-
N-{4-[2-(dimethylamino)ethoxy]-3-fluorophenyl}-3',6-
dihydroxy-5-methoxybiphenyl-2-carboxamide (20g)
m.p.: 183~185°C. EI-MS (m/z): 440.0 [M]+. 1H-NMR (400
MHz, DMSO) δ 7.94 (s, 1H), 7.25 (d, J = 8.0 Hz, 1H), 7.25
(d, J = 8.0 Hz, 1H), 7.21 (d, J = 8.0 Hz, 1H), 7.10 (d, J =
8.0 Hz, 1H), 6.99 (d, J = 8.0 Hz, 1H), 6.67 (dd, J = 8.0, 1.6
Hz, 1H), 6.61 (d, J = 8.0 Hz, 1H), 6.58 (d, J = 8.0 Hz, 1H),
3.88 (s, 3H), 3.43 (s, 1H).
methoxybiphenyl-2-carboxamide (20b)
m.p.: 141~143°C. EI-MS (m/z): 422.1 [M]+. 1H-NMR (400
MHz, DMSO) δ 7.32 (d, J = 8.0 Hz, 1H), 7.07 (t, J = 4.0
Hz, 1H), 7.02 (s, 1H), 6.98 (d, J = 8.0 Hz, 1H), 6.81 (d, J =
8.0 Hz, 1H), 6.74 (s, 1H), 6.70 (d, J = 8.0 Hz, 1H), 6.62 (d,
J = 8.0 Hz, 1H), 4.00 (t, J = 4.0 Hz, 2H), 2.69 (t, J = 4.0
Hz, 2H), 2.28 (s, 6H).
N-{4-[2-(diethylamino)ethoxy]phenyl}-3',6-dihydroxy-5-
methoxybiphenyl-2-carboxamide (20c)
N-[3-fluoro-4-(2-piperidin-1-ylethoxy)phenyl]-3',6-
dihydroxy-5-methoxybiphenyl-2-carboxamide (20h)
9

m.p.: 165~167°C. EI-MS (m/z): 480.1 [M]+. 1H-NMR (400
MHz, DMSO) δ 7.39 (d, J = 8.0 Hz, 1H), 7.10 (s, 1H), 7.07
(s, 1H), 7.05 (s, 1H), 7.02 (s, 1H), 7.00 (s, 1H), 6.74 (s,
1H), 6.69 (d, J = 4.0 Hz, 1H), 6.63 (d, J = 8.0 Hz, 1H),
4.06 (t, J = 4.0 Hz, 2H), 2.64 (t, J = 4.0 Hz, 2H), 2.42 (s,
4H), 1.49 (s, 6H).
The VEGFR-2 inhibitory activity assay was performed
using ADP-GloTM Kinase assay kit (Promega) and
VEGFR-2 Kinase Enzyme System (Promega) according to
the manufacturer's instructions. Firstly, the VEGFR-2
reaction utilizes ATP and generates ADP. Then the ADP-
GloTM reagent is added to simultaneously terminate the
kinase reaction and deplete the remaining ATP. Finally, the
Kinase Detection Reagent is added to convert ADP to ATP
and the newly synthesized ATP is converted to light using
the luciferase reaction.
N-{3-chloro-4-[3-(dimethylamino)propoxy]phenyl}-3',6-
dihydroxy-5-methoxybiphenyl-2-carboxamide (20i)
m.p.: 191~193°C. EI-MS (m/z): 485.0 [M]+. 1H-NMR (400
MHz, DMSO) δ 7.65 (d, J = 8.0 Hz, 2H), 7.43 (s, 1H), 7.43
(s, 1H), 7.31 (d, J = 8.0 Hz, 3H), 7.01 (s, 1H), 6.89 (d, J =
8.0 Hz, 2H), 6.79 (d, J = 8.0 Hz, 3H), 6.75 (s, 1H), 6.71 (d,
J = 8.0 Hz, 2H), 6.63 (d, J = 8.0 Hz, 1H), 3.92 (t, J = 4.0
Hz, 2H), 2.46 (d, J = 8.0 Hz, 2H), 2.25 (s, 2H), 2.24 (s,
6H).
VEGFR-2 was incubated with substrates, inhibitors and
ATP in a final buffer of 25 mM HEPES (pH 7.4), 10 mM
MgCl2, 0.01% Triton X-100, 100 µg/mL BSA, 2.5 mM
DTT in 384-well plate with the total volume of 10 µL.
Then the ADP-GloTM Kinase Assay was performed in two
steps once the kinase reaction is complete. Firstly, add 5µL
ADP-Glo Reagent to stop the kinase reaction and deplete
the unconsumed ATP, leaving only ADP and a very low
background of ATP. Then incubated at room temperature
for 40 minutes and add 10µL of Kinase Detection Reagent
to convert ADP to ATP and introduced luciferase and
luciferin to detect ATP. At last, incubated at room
temperature for 30-60 minutes and measured the
luminescence with a plate-reading luminometer. The signal
was correlated with the amount of ATP present in the
reaction and was inversely correlated with the kinase
activity
N-{3-chloro-4-[2-(dimethylamino)ethoxy]phenyl}-3',6-
dihydroxy-5-methoxybiphenyl-2-carboxamide (20j)
m.p.: 138~140°C. EI-MS (m/z): 440.0 [M]+. 1H-NMR (400
MHz, DMSO) δ 7.42 (dd, J = 8.0 Hz, 1H), 7.13 (d, J = 8.0
Hz, 1H), 7.10 (s, 1H), 7.08 (s, 1H), 7.01 (d, J = 8.0 Hz,
2H), 6.76 (s, 1H), 6.70 (d, J = 8.0 Hz, 1H), 6.65 (d, J = 8.0
Hz, 1H), 4.12 (t, J = 4.0 Hz, 2H), 2.80 (s, 2H), 2.34 (s,
6H).
N-[3-chloro-4-(2-pyrrolidin-1-ylethoxy)phenyl]-3',6-
dihydroxy-5-methoxybiphenyl-2-carboxamide (20k)
m.p.: 127~129°C. EI-MS (m/z): 484.9 [M]+. 1H-NMR (400
MHz, DMSO) δ 7.64 (s, 1H), 7.56 (d, J = 8.0 Hz, 1H), 7.49
(d, J = 8.0 Hz, 1H), 7.42 (d, J = 8.0 Hz, 1H), 7.30 (d, J =
8.0 Hz, 1H), 7.15 (dd, J = 8.0 Hz, 1H), 6.93 (dd, J = 8.0
Hz, 1H), 6.75 - 6.45 (m, 1H), 4.10 - 4.01 (m, 2H), 2.81 -
2.76 (m, 2H), 2.58 - 2.42 (m, 4H), 1.69 (s, 4H).
4.3. Antiproliferative activity assay
All the title compounds were evaluated for their
antiproliferative potency against MCF-7 and SMMC-7721
using MTT method. Exponentially growing cells were
harvested and plated in 96-well plates at a concentration of
1×104 cells/well, and then incubated for 24 h at 37°C. The
cells in the wells were treated with the tested compounds
respectively at various concentrations for 48 h. Then, 20
mL MTT (5 mg/mL) was added to each well and incubated
for 4 h at 37°C. Supernatant was discarded, and 150 mL
DMSO was added to each well. Absorbance values were
determined by a microplate reader (Bio-Rad Instruments)
at 570 nm. The IC50 values were calculated according to
N-[3-chloro-4-(2-piperidin-1-ylethoxy)phenyl]-3',6-
dihydroxy-5-methoxybiphenyl-2-carboxamide (20l)
m.p.: 199~201°C. EI-MS (m/z): 496.1 [M]+. 1H-NMR (400
MHz, DMSO) δ 7.70 - 7.60 (m, 1H), 7.34 (d, J = 8.0 Hz,
1H), 7.08 (d, J = 8.0 Hz, 1H), 7.05 - 7.01 (m, 2H), 7.00 (s,
1H), 6.84 (d, J = 8.0 Hz, 1H), 6.75 (s, 1H), 6.72 - 6.67 (m,
1H), 6.63 (d, J = 8.0 Hz, 1H), 4.21 (d, J = 8.0 Hz, 2H),
3.06 (s, 2H), 2.86 (s, 4H), 1.65 (s, 6H).
4.2. VEGFR-2 inhibitory activity assays
10

inhibition ratios [34].
Acknowledgment
4.4. Molecular docking
This work was supported by the National Natural Science
Foundation of China (Grant NO. 81302737 and
81302641), and the Fundamental Research Funds for the
Central Universities (xjj2014140).
In order to understand the binding mode of the title
compounds with VEGFR-2, we performed a molecular
docking using Sybyl/Surflex-dock. The inhibitors were
docked into a ligand-binding site using a protomol-based
method. The crystal structure of VEGFR-2 was obtained
from RCSB Protein Data Bank (PDB ID: 4ASD). The
ligand and water molecules were removed and hydrogen
was added and minimized using Tripos force field and
Pullman charges. The residues in a radius 5.0 Å around
ligand were selected as active site. Compounds 20e and
18b were depicted with Sybyl/Sketch module (Tripos Inc.)
and optimized applying Powell's method with Tripos force
field with convergence criterion set at 0.05 kcal/(Åmol),
and assigned with Gasteiger-Hückel method. Other
docking parameters were kept at default [35].
Reference
[1] Kavitha, K.; Kranthi Kiran Kishore, T.; Bhatnagar,
R.S.; Nagini, S. Biochimie. 2014, 102, 56.
[2] Zhang, J.; Shan, Y.; Pan, X.; He, L. Mini. Rev. Med.
Chem. 2011, 11, 920.
[3] Caballero, J.; Muñoz, C.; Alzate-Morales, J.H.;
Cunha, S.; Gano, L.; Bergmann, R.; Steinbach, J.;
Kniess, T. Eur. J. Med. Chem. 2012, 58, 272.
[4] Xia, Y.; Song, X.; Li, D.; Ye, T.; Xu, Y.; Lin, H.;
Meng, N.; Li, G.; Deng, S.; Zhang, S.; Liu, L.; Zhu,
Y.; Zeng, J.; Lei, Q.; Pan, Y.; Wei, Y.; Zhao, Y.; Yu,
L. Sci. Rep. 2014, 4, 6031.
[5] Huang L, Huang Z, Bai Z, Xie R, Sun L, Lin K.
Future Med. Chem. 2012, 4, 1839.
[6] Zuccotto F, Ardini E, Casale E, Angiolini M. J. Med.
Chem. 2010, 53, 2681.
[7] Lintnerová, L.; García-Caballero, M.; Gregáň, F.;
Melicherčík, M.; Quesada, A.R.; Dobiaš, J.; Lác, J.;
Sališová, M.; Boháč, A. Eur. J. Med. Chem. 2014, 72,
146.
[8] Wang, C.; Gao, H.; Dong, J.; Wang, F.; Li, P.; Zhang,
J. Curr. Med. Chem. 2014, 21, 1336.
[9] Zhang, J.; Zhang, Y.; Zhang, S.; Wang, S.; He, L.
Bioorg. Med. Chem. Lett. 2010, 20, 718.
[10] Zhang, J.; Zhang, Y.; Shan, Y.; Li, N.; Ma, W.; He, L.
4.5. 3D-QSAR studies with CoMFA
The CoMFA studies were carried out using Sybyl-X 2.0.
All the structures were aligned on the common
substructure with 18b as template. CoMFA calculation was
performed using default values. The steric and electrostatic
fields were calculated at each lattice intersection of a
regularly spaced grid of 2.0 Å in all three dimensions
within defined region. A sp3 carbon atom with +1.00
charge was used as probe atom. The steric and electrostatic
fields were truncated at +30.00 kcal/mol, and the
electrostatic fields were ignored at the lattice points with
maximal steric interactions.
Eur. J. Med. Chem. 2010, 45, 2798.
[11] Zhang, J.; Zhang, Y.; Pan, X.; Wang, C.; Hu, Z.;
Wang, S.; He, L. Med. Chem. 2012, 8, 145.
[12] Wang, C.; Dong, J.; Zhang, Y.; Wang, F.; Gao, H.; Li,
P.; Wang, S.; Zhang, J. MedChemComm. 2013, 4,
1434.
[13] Wang, C.; Gao, H.; Dong, J.; Zhang, Y.; Su, P.; Shi,
Y.; Zhang, J. Bioorg. Med. Chem. 2014, 22, 277.
[14] Jiao, Y.; Xin, B.T.; Zhang, Y.; Wu, J.; Lu, X.; Zheng,
Y.; Tang, W.; Zhou, X. Eur. J. Med. Chem. 2015, 90,
170.
[15] Zhang, L.; Zheng, Q.; Yang, Y.; Zhou, H.; Gong, X.;
Zhao, S.; Fan, C. Eur. J. Med. Chem. 2014, 82, 139.
[16] Barbosa, M.L.; Lima, L.M.; Tesch, R.; Sant'Anna,
C.M.; Totzke, F.; Kubbutat, M.H.; Schächtele, C.;
Laufer, S.A.; Barreiro, E.J. Eur. J. Med. Chem. 2014,
71, 1.
[17] Dong, J.; Lu, W.; Pan, X.; Su, P.; Shi, Y.; Wang, J.;
Zhang, J. Bioorg. Med. Chem. 2014, 22, 6876.
[18] Shao, L.X.; Shi, M. Org. Biomol. Chem. 2005, 3,
1828.
PLS method was used to linearly correlate CoMFA fields
to the inhibitory activity values. The cross-validation
analysis was performed using the leave one out (LOO)
method in which one compound is removed from the
dataset and its activity is predicted using the model derived
from the rest of the dataset. The cross-validated q2 that
resulted in optimum number of components (n = 5) and
lowest standard error of prediction were considered for
further analysis. Column filtering value was set at 2.0
kcal/mol to improve the signal-to-noise ratio.
[19] Schmidt, B.; Riemer, M. J. Org. Chem. 2014, 79,
4104.
[20] Cheng, C.; Wang, X.; Xing, L.; Liu, B.; Zhu, R.; Hu,
Y. Adv. Synth. Catal. 2007, 349, 1775.
11

[21] Zhang, J.; Zhang, Y.; Pan, X.; Wang, S.; He, L. Med.
Chem. 2011, 7, 286.
[22] Ji, Z.; Ahmed, A.A.; Albert, D.H.; Bouska, J.J.;
Bousquet, P.F.; Cunha, G.A.; Diaz, G.; Glaser, K.B.;
Guo, J.; Harris, C.M.; Li, J.; Marcotte, P.A.; Moskey,
M.D.; Oie, T.; Pease, L.; Soni, N.B.; Stewart, K.D.;
Davidsen, S.K.; Michaelides, M.R. J. Med. Chem.
2008, 51, 1231.
[23] Frey, R.R.; Curtin, M.L.; Albert, D.H.; Glaser, K.B.;
Pease, L.J.; Soni, N.B.; Bouska, J.J.; Reuter, D.;
Stewart, K.D.; Marcotte, P.; Bukofzer, G.; Li, J.;
Davidsen, S.K.; Michaelides, M.R. J. Med. Chem.
2008, 51, 3777.
[24] Shi, L.; Wu, T.T.; Wang, Z.; Xue, J.Y.; Xu, Y.G. Eur.
J. Med. Chem. 2014, 84, 698.
[25] Shi, L.; Wu, T.T.; Wang, Z.; Xue, J.Y.; Xu, Y.G.
Bioorg. Med. Chem. 2014, 22, 4735.
[26] Mohan Krishna, K.; Inturi, B.; Pujar, G.V.; Purohit,
M.N.; Vijaykumar, G.S. Eur. J. Med. Chem. 2014, 84,
516.
[27] Pan, X.; Wang, F.; Zhang, Y.; Gao, H.; Hu, Z.; Wang,
S.; Zhang, J. Bioorg. Med. Chem. 2013, 21, 2527.
[28] Gao, H.; Su, P.; Shi, Y.; Shen, X.; Zhang, Y.; Dong,
J.; Zhang, J. Eur. J. Med. Chem. 2015, 90, 232.
[29] Rosen, B.M.; Wilson, D.A.; Wilson, C.J.; Peterca, M.;
Won, B.C.; Huang, C.; Lipski, L.R.; Zeng, X.; Ungar,
G.; Heiney, P.A.; Percec, V. J. Am. Chem. Soc. 2009,
131, 17500.
[30] Wang, J.Q.; Gao, M.; Miller, K.D.; Sledge, G.W.;
Zheng, Q.H. Bioorg. Med. Chem. Lett. 2006, 16,
4102.
[31] Schmidt, B.; Riemer, M.; Karras, M. J. Org. Chem.
2013, 78, 8680.
[32] Pan, X.; Dong, J.; Gao, H.; Wang, F.; Zhang, Y.;
Wang, S.; Zhang, J. Chem. Biol. Drug Des. 2014, 83,
592.
[33] Biava, M.; Battilocchio, C.; Poce, G.; Alfonso, S.;
Consalvi, S.; Di Capua, A.; Calderone, V.; Martelli,
A.; Testai, L.; Sautebin, L.; Rossi, A.; Ghelardini, C.;
Di Cesare Mannelli, L.; Giordani, A.; Persiani, S.;
Colovic, M.; Dovizio, M.; Patrignani, P.; Anzini, M.
Bioorg. Med. Chem. 2014, 22, 772.
[34] Wang, F.; Lu, W.; Zhang, T.; Dong, J.; Gao, H.; Li,
P.; Wang, S.; Zhang, J. Bioorg. Med. Chem. 2013,
21, 6973.
[35] Zhang, J.; Shan, Y.; Pan, X.; Wang, C.; Xu, W.; He,
L. Chem. Biol. Drug. Des. 2011, 78, 709.
12

Captions for Illustrations
Figure Captions
Figure 1. Structures of Type I and Type II VEGFR-2 inhibitors.
Figure 2. Design strategy and structures of title compounds.
Figure 3. Predicted binding mode of 20e with VEGFR-2.
Figure 4. Predicted binding mode of 18b with VEGFR-2.
Figure 5. The steric contour maps of CoMFA.
Figure 6. The electrostatic contour maps of CoMFA.
Scheme Captions
Scheme 1. Preparation of compounds 17a-19g.
Reagents and Conditions: (a) BnCl, K2CO3, C2H5OH, reflux; (b) Mg/I2, THF, reflux; (c)
B(OCH3)3, 2M HCl; (d) H2O2, NaClO2, NaH2PO4; (e) Pd/C, K2CO3, i-PrOH, H2O, reflux; (f)
Cs2CO3, DMF, reflux; (g) Pd/C, H2, CH3OH; (h) EDCI, DMAP, THF; (i) Pd/C, H2, CH3OH.
Scheme 2. Preparation of compounds 20a-20l.
Reagents and Conditions: (a) BnCl, K2CO3, C2H5OH, reflux; (b) Mg/I2, THF, reflux; (c)
B(OCH3)3, 2M HCl; (d) Br2, Fe, CH3COOH, CH3COONa (e) BnCl, K2CO3, C2H5OH, reflux; (f)
H2O2, NaClO2, NaH2PO4; (g) Pd/C, K2CO3, i-PrOH, H2O, reflux (h) Cs2CO3, DMF, reflux; (i)
Pd/C, H2, CH3OH; (j) EDCI, DMAP, THF; (k) Pd/C, H2, CH3OH.
Table Captions
Table 1. Structures and biological activity of biphenyl amides (Series 1)
Table 2. Structures and biological activity of biphenyl amides (Series 2)

Figure 1. Structures of Type I and Type II VEGFR-2 inhibitors.

Figure 2. Design strategy and structures of title compounds.

Figure 3. Predicted binding mode of 20e with VEGFR-2.

Figure 4. Predicted binding mode of 18b with VEGFR-2.

Figure 5. The steric contour maps of CoMFA.

Figure 6. The electrostatic contour maps of CoMFA.

Scheme 1. Preparation of compounds 17a-19g.
Reagents and Conditions: (a) BnCl, K2CO3, C2H5OH, reflux; (b) Mg/I2, THF, reflux; (c)
B(OCH3)3, 2M HCl; (d) H2O2, NaClO2, NaH2PO4; (e) Pd/C, K2CO3, i-PrOH, H2O, reflux; (f)
Cs2CO3, DMF, reflux; (g) Pd/C, H2, CH3OH; (h) EDCI, DMAP, THF; (i) Pd/C, H2, CH3OH.

Scheme 2. Preparation of compounds 20a-20l.
Reagents and Conditions: (a) BnCl, K2CO3, C2H5OH, reflux; (b) Mg/I2, THF, reflux; (c)
B(OCH3)3, 2M HCl; (d) Br2, Fe, CH3COOH, CH3COONa (e) BnCl, K2CO3, C2H5OH, reflux; (f)
H2O2, NaClO2, NaH2PO4; (g) Pd/C, K2CO3, i-PrOH, H2O, reflux (h) Cs2CO3, DMF, reflux; (i)
Pd/C, H2, CH3OH; (j) EDCI, DMAP, THF; (k) Pd/C, H2, CH3OH.

Table 1. Structures and biological activity of biphenyl amides (Series 1)
Compound
17a
R1
H
H
H
H
H
H
H
F
R2
MCF-7(μM) SMMC-7721(μM) VEGFR-2(nM)
7.85
3.91
9.28
15.85
404.03
12.58
11.29
949.58
229.52
11.61
5.98
624.66
ND*
17b
17c
16.20
6.12
359.63
31.98
214.87
62.52
54.26
14.48
2.02
17d
17e
7.15
17f
28.05
266.11
1.94
17g
18a
18b
F
1.47
18c
F
5.16
9.62
8.74
18d
F
16.68
2.51
18.69
21.47
103.19
664.72
1008.84
6.79
4.86
18e
F
44.32
129.25
ND
18f
F
83.76
187.55
47.05
2.22
18g
F
19a
Cl
Cl
Cl
Cl
Cl
Cl
Cl
85.32
27.58
158.46
17.85
ND
19b
19c
1.41
3.81
19d
1.34
7.13
19e
1.04
9.76
19f
14.55
2.25
40.64
133.25
9.96
77.45
125.82
0.85
19g
Sorafenib
1.88
* ND = Not Determined.

Table 2. Structures and biological activity of biphenyl amides (Series 2)
Compound
20a
R1
H
H
H
H
H
H
F
R2
MCF-7(μM)SMMC-7721(μM)VEGFR-2(nM)
64.46
590.48
52.70
357.05
155.62
13.74
149.32
11.67
84.53
822.41
133.22
69.19
82.82
626.44
349.54
20.95
988.21
88.99
13.77
15.14
9.96
71.89
344.61
69.40
182.82
4.38
20b
20c
20d
20e
20f
15.04
ND
20g
20h
F
9.41
20i
Cl
Cl
Cl
Cl
1116.46
1309.59
1.22
ND*
20j
130.17
22.85
94.69
0.85
20k
20l
2.71
Sorafenib
1.88
* ND = Not Determined.

Discovery of biphenyl-based VEGFR-2 inhibitors. Part Ⅲ: Design,
synthesis and 3D-QSAR studies
Wen Lua,†, Pengfei Lia,b,†, Yuanyuan Shanb, Ping Sua, Jinfeng Wanga, Yaling, Shia, Jie Zhanga,
a
School of Pharmacy, Xi'an Jiaotong University, No. 76, Yanta West Road, Xi’an, Shaanxi
Province, 710061, P.R. China
b
Department of Pharmacy, The First Affiliated Hospital of Medical College, Xi'an Jiaotong
University, Xi'an, Shaanxi Province, 710061, P.R. China
These biphenyl amides have potential to be further developed as novel VEGFR-2 inhibitors and
could be a promising starting point for further medicinal chemistry efforts.
 Corresponding author. Tel/Fax: +86-29-82655451; E-mail: zhj8623@mail.xjtu.edu.cn (Jie Zhang).
† Both authors contributed equally to this work.