Synthesis and cytotoxic evaluation of novel symmetrical taspine derivatives as anticancer agents

Article abstract of DOI:10.2174/157340611796150914

It has been demonstrated that taspine derivatives act as anticancer agents, thus we designed and synthesized a novel class of symmetrical biphenyl derivatives. We evaluated the cytotoxicity and antitumor activity of biphenyls against five human tumor and normal cell lines. The results indicated that the majority of the compounds exhibited anticancer activity equivalent to or greater than the positive control. Compounds (11) and (12) demonstrated the most potent cytotoxic activity with IC₅₀ values between 19.41 μM and 29.27 μM. The potent antiproliferative capabilities of these compounds against ECV304 human transformed endothelial cells indicated that these biphenyls could potentially serve as antiangiogenic agents. We also reviewed the relationship between structure and activity based on the experimental results. Our findings provide a good starting point for further development of symmetrical biphenyl derivatives as potential novel anticancer agents.

Full text of DOI:10.2174/157340611796150914

286
Medicinal Chemistry, 2011, 7, 286-294
Synthesis and Cytotoxic Evaluation of Novel Symmetrical Taspine
Derivatives as Anticancer Agents
Jie Zhang, Yanmin Zhang, Xiaoyan Pan, Sicen Wang and Langchong He*
College of Medicine, Xi’an Jiaotong University, No. 76, Yanta West Road, Xi’an, Shaanxi Province, 710061, P.R. China
Abstract: It has been demonstrated that taspine derivatives act as anticancer agents, thus we designed and synthesized a
novel class of symmetrical biphenyl derivatives. We evaluated the cytotoxicity and antitumor activity of biphenyls against
five human tumor and normal cell lines. The results indicated that the majority of the compounds exhibited anticancer ac-
tivity equivalent to or greater than the positive control. Compounds (11) and (12) demonstrated the most potent cytotoxic
activity with IC50 values between 19.41 μM and 29.27 μM. The potent antiproliferative capabilities of these compounds
against ECV304 human transformed endothelial cells indicated that these biphenyls could potentially serve as antiangio-
genic agents. We also reviewed the relationship between structure and activity based on the experimental results. Our
findings provide a good starting point for further development of symmetrical biphenyl derivatives as potential novel anti-
cancer agents.
Keywords: Symmetrical taspine derivatives, antiproliferative activity, cytotoxicity, anticancer agents, antiangiogenic agents;
VEGFR-2.
1. INTRODUCTION
We are currently engaged in a project aimed at identify-
ing novel biologically active small molecules that could
serve as powerful anticancer therapeutic candidates [9].
Biphenyl compounds are known for various biological ac-
tivities, including antibacterial, anti-inflammatory and anti-
cancer activities. We recently synthesized a series of un-
symmetrical biphenyls and evaluated their antiproliferative
activity. These unsymmetrical biphenyls exhibited potent
cytotoxicity against several human cancer cell lines. These
findings indicated that biphenyl derivatives are suitable
compounds for further optimization. Recently, we reported
the synthesis of unsymmetrical biphenyl (2), a novel ring-
opened biphenyl derivative of taspine [10]. During the
synthesis of compound (2), a novel symmetrical biphenyl
(11) was isolated as a byproduct and was also evaluated for
biological activity (Fig. 1). It was determined that compound
(11) exhibited potent anticancer activity, which attracted
considerable attention. To further investigate this finding, we
aimed to enhance the structural complexity and diversity of
compound (11) by generating novel biphenyls [11].
Cancer is a class of diseases in which a group of cells
displays uncontrolled growth, causing a cell invasion that
intrudes upon and destroys adjacent tissues, sometimes lead-
ing to metastasis and a spread to other locations in the body
via lymph or blood. It is one of the leading causes of death,
claiming more than one million victims every year [1]. Cur-
rently, cancer can be treated by chemotherapy, radiation
therapy, surgery, immunotherapy, monoclonal antibody ther-
apy, or other methods. Chemotherapy is a treatment of can-
cer through the use of anticancer cytotoxic drugs [2]. Al-
though chemotherapy may carry significant side effects,
many types of chemotherapy drugs have been developed.
Currently, development of novel chemotherapeutic agents
for cancer chemotherapy is a high priority for medicinal
chemists and is an active area of research [3].
Natural products continue to play a highly significant
role in the discovery and development of novel lead com-
pounds and new chemical entities, and this is particularly
evident in anticancer agent development [4]. Taspine (1, Fig.
1) has been identified as an important bioactive constituent
isolated from Radix et Rhizoma Leonticis (Hong Mao Qi in
Chinese) [5]. It exhibits multiple pharmacological activities;
for example, bacteriostatic, antibiotic, antiviral, anti-
inflammatory, antiulcer, cytotoxic activity, and so on [6].
The anticancer and antiangiogenic properties of taspine have
been previously demonstrated, and it has the potential to be
an ideal candidate as a chemotherapeutic agent [7]. In a re-
cent study, He et al. demonstrated that taspine was able to
inhibit tumor angiogenesis, and suggested that one of its
mechanisms might be to inhibit VEGFR-2 [8].
In this study, to further elucidate the relationship between
structure and activity for biphenyl derivatives, we describe
the synthesis of eighteen symmetrical biphenyl derivatives
(11-28) and their cytotoxic activity against five human can-
cer and normal cell lines. Among them, two compounds (11
and 12) exhibited promising growth inhibitory effects
against the tested cells with an IC50 range of 2.93-28.92 ꢀM.
Aniline that contains one or more halogen substituents
(fluoro, chloro, brome and iodo) is useful for novel antican-
cer drug design, and in addition the presence of halogen in a
molecule enhances drug persistence and lipid solubility [12].
2. CHEMISTRY
*Address correspondence to this author at the 54 mailbox, College of Medi-
cine, Xi’an Jiaotong University, No. 76, Yanta West Road, Xi’an, Shaanxi
Province, 710061, P.R. China; Tel/Fax: +86-29-82655451;
E-mail: helc@mail.xjtu.edu.cn
Eighteen symmetrical biphenyls were prepared by the
general procedure described in Scheme 1. We used commer-
cially available isovanillin as the starting material. Initially,
1573-4064/11 $58.00+.00
© 2011 Bentham Science Publishers Ltd.

Synthesis and Cytotoxic Evaluation of Novel Symmetrical Taspine
Medicinal Chemistry, 2011, Vol. 7, No. 4 287
O
O
O
O
N
H
H
Cl
H
N
H
N
N
N
O
O
O
F
O
O
O
O
O
O
O
O
O
F
O
O
F
N
N
O
N
H
N
H
H
Cl
Cl
O
O
O
1, Taspine
2
By-product
11
O
O
H
N
H
N
CHO
R2
O
O
R1
O
OH
CONHMe
MeHNOC
HO
O
R1
O
O
R2
MeO
N
H
N
H
OH
O
O
3
10
11-28
Fig. (1). The surprising discovery and structures of title biphenyl compounds (11-28).
CHO
CHO
CHO
Br
COOH
Br
a
b
c
MeO
MeO
Br
MeO
MeO
OH
OH
OBn
OBn
4
6
3
5
O
O
C
H
N
COCl
Br
OBn
O
N
H
d
e
f
O
BnO
MeO
Br
N
MeO
H
OBn
OBn
O
7
8
9
O
O
H
N
H
H
N
N
R2
OH
O
O
O
g
h
O
O
O
O
O
HO
R2
N
N
N
H
H
H
O
O
TM
10
Scheme 1. Preparation of target compounds (11-28)
Reagents and Conditions: (a) Fe, NaOAc, AcOH, Br2, 83%; (b) BnCl, K2CO3, 96%; (c) NaH2PO4, NaClO2, 30% H2O2, 95%; (d) SOCl2,
DMF(cat), CH2Cl2, 100%; (e) CH2Cl2, 30% CH3NH2 or diethylamine or isopropylamine, 80% ~ 87%; (f) Cu, DMF, 75%; (g) H2, Pd/C;
98%; (h) K2CO3, anhydrous ethanol, (64%~85%).
isovanillin (3) was converted into 2-bromoisovanillin (4) by
a reaction with bromine [13]. After protection of (4) as ether
(5) [14], the aldehyde group in (5) was oxidized to the car-
boxyl group yielding (6) [15]. Amidation of (6) with methy-
lamine yielded the monocyclic precursor (8) for the Ullmann
reaction [16]. Biphenyl dimethane amide (9) was prepared
by dimerization of (8) through a standard symmetrical Ull-
mann reaction [17]. Compound (10) was readily obtained by
deprotection of benzamide (9) under catalysis with palla-
dium/carbon [18]. Both of the hydroxyl groups in (10) were
etherified with various alkyl halides in anhydrous ethanol in
the presence of K2CO3 to give the title compounds in various
yields [19]. The other target compounds were prepared in an
identical manner, except for compound (6), which was ami-
dated with a different fatty amine.
3. RESULTS AND DISCUSSION
It is well understood that potential activity does not nec-
essarily guarantee acceptable bioavailability. The molecular
properties regarding adsorption, distribution, metabolism,
and excretion (ADME) are crucial for drug design, however,

288 Medicinal Chemistry, 2011, Vol. 7, No. 4
Zhang et al.
Table 1. Volsurf Descriptors of Symmetrical Biphenyl Derivatives
Lipinski’s Rule of 5
Compound
ADME Predicted (Volsurf)
MW
NRBa
NHAb
NHDc
logPd
PSAe
PPB%f
Taspine
11
369.37
731.52
762.65
764.43
686.71
784.45
662.64
662.64
626.65
558.62
664.61
787.63
818.76
820.54
742.81
840.55
720.72
815.68
846.81
5
7
0
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
2
2
0.68
5.47
5.19
6.62
3.56
5.25
3.09
4.11
3.28
1.04
2.32
6.95
6.67
8.10
5.04
6.73
3.80
5.81
5.53
74.30
153.32
153.32
153.32
171.78
153.32
153.32
153.32
153.32
153.32
179.10
153.32
153.32
153.32
171.78
153.32
179.10
135.74
135.74
29.22%
99.88%
99.85%
99.97%
98.66%
99.87%
97.46%
99.23%
97.67%
69.87%
98.61%
99.98%
99.98%
100.00%
99.81%
99.98%
99.63%
99.94%
99.93%
13
15
13
15
13
13
13
13
13
13
15
17
15
17
15
15
17
19
12
12
12
14
12
12
12
12
12
14
12
12
12
14
12
14
12
12
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
a NRB = number of rotatable bonds; b NHA = number of H-bond acceptors; c NHD = number of H-bond donors; d logP = log octanol-water partition coefficient; e PSA = polar surface
areas (Å2); f PPB% = Drug Binding to Plasma Proteins.
it is not always practical to perform the respective experi-
mental measurements. Therefore, it is useful to rapidly pre-
dict certain molecular physicochemical properties such as
LogP, polar surface areas (PSA), and plasma protein binding
rate that may indicate bioavailability [20]. Prior to biological
evaluation, we evaluated a broad range of molecular phys-
icochemical properties that potentially impact drug-like
ADME parameters based on chemical structure. All com-
pounds were filtered using Lipinski’s rule of 5 [21], and sub-
sequently tested according to the Volsurf module (SYBYL-
X 1.1) in order to calculate predicted ADME properties [22].
The ADME properties of compounds are summarized in
Table 1, including MW, logP, number of H-bond acceptors,
donors and rotatable bonds, PSA and plasma protein binding
rate. It is evident from Table 1 that the majority of the
biphenyls were neutral compounds that primarily bonded to
lipoproteins, and to a lesser extent to albumin. Compounds
(20) and (26) were weak bases (base PKa < 8.5) and pre-
dominantly bonded to alpha1-acid glycoprotein and albumin.
The molecular polar surface areas of biphenyl derivatives
were much greater compared to taspine; in fact, the majority
were above 140, indicating that these compounds exhibit
poor cell membrane permeability. The maximum plasma
protein binding rates of target compounds were over 90%;
much higher in comparison to taspine. The results indicated
that these compounds were easily absorbed in plasma. In
general, when the bonded concentration is less than 90% of
the total plasma concentration the plasma protein binding has
little clinical importance. Plasma protein binding becomes
important when it is greater than 90%.
The most common research screening method for anti-
cancer agents employs a cytotoxicity test against a panel of
cancer cell lines [23]. All target compounds were evaluated
for cytotoxicity against several cancer cell lines in-vitro us-
ing the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay with taspine and gefitinib serving as
the positive controls. The structure and biological activity of
all symmetrical biphenyls (11-28) are summarized in Table
2.
The results (shown in Table 2) indicated that most com-
pounds exhibited potent cytotoxic activity comparable to or
stronger than that of gefitinib. Two compounds (11 and 12)
demonstrated a promising growth inhibitory effect against
tumor and normal cells with an IC50 range of 2.93-28.92 ꢀM.
Compounds (11 and 21, 3-chloro-4-fluoroaniline substitu-
tion) exhibited a greater potency than the others, including
gefitinib, against 7721 cells with IC50 values of 19.41 and

Synthesis and Cytotoxic Evaluation of Novel Symmetrical Taspine
Medicinal Chemistry, 2011, Vol. 7, No. 4 289
Table 2. Cytotoxic Activities of Title Compounds on Human Cancer and Normal Cell Lines
O
H
N
H
N
R2
O
O
R1
O
O
O
O
R1
R2
N
N
H
H
O
Cell Lines (IC50, ꢀM)
Compound
R1
R2
7721a
MCF-7 b
LOVOc
A549d
ECV304e
Taspine
2
/
/
/
/
57.58
ND
ND
ND
4.18
22.30
72.17
194.09
60.38
240.51
Cl
11
12
13
CH3
CH3
CH3
19.41
28.92
39.46
28.29
29.27
28.72
ND
ND
ND
26.70
28.69
29.16
2.93
4.08
F
CF3
Cl
Cl
18.94
14
15
CH3
CH3
186.75
62.95
141.63
46.39
ND
ND
142.84
42.46
72.11
68.07
OMe
Br
16
17
CH3
CH3
156.45
97.41
79.79
67.44
ND
ND
110.41
94.39
66.64
67.13
F
F
18
19
CH3
CH3
456.39
996.07
184.28
128.66
194.07
222.69
263.25
439.39
ND
ND
20
CH3
439.39
273.57
164.25
864.45
ND
N
F
Cl
21
22
23
isopropyl
isopropyl
isopropyl
25.41
36.18
24.14
76.55
99.83
93.15
34.86
40.22
55.51
30.53
76.98
32.15
ND
ND
ND
F
CF3
Cl
Cl
24
25
isopropyl
isopropyl
567.26
59.68
91.56
429.24
66.16
327.20
239.31
ND
ND
OMe
345.96
Br
26
isopropyl
94.81
452.38
263.18
190.41
ND
N
F

290 Medicinal Chemistry, 2011, Vol. 7, No. 4
Zhang et al.
Table 2. contd…
Cell Lines (IC50, ꢀM)
Compound
R1
R2
7721a
MCF-7 b
LOVOc
A549d
ECV304e
Cl
27
28
diethyl
54.92
134.30
59.37
70.40
ND
F
CF3
diethyl
63.79
30.21
117.52
23.75
140.99
46.30
350.96
26.60
ND
Gefitinib
/
/
27.49
ND is not determined.
a 7721, human hepatocellular carcinoma cell line; b MCF-7, human breast cancer cell line; cLOVO, human colon cancer cell line; dA549, human lung adenocarcinoma epithelial cell
line; eECV304, human umbilical vein endothelial cell line.
25.41 ꢀM, respectively. Compound (11) also exhibited po-
tent antiproliferative activity against MCF-7 cells and A549
cells with IC50 values of 28.29 and 26.70 ꢀM, respectively.
Additionally, compound (21, 3-chloro-4-fluoroaniline substi-
tution) exhibited the greatest growth inhibitory activity
against LOVO cells with an IC50 value of 34.86, which was
greater than gefitinib (IC50 = 46.30 ꢀM).
Angiogenesis is a key event of tumor progression and
metastasis and hence a target for cancer chemotherapy [25].
Angiogenesis is primarily a receptor-mediated process in
which growth factors cause signal transduction via receptor
tyrosine kinase (RTK). Vascular endothelial growth factor
(VEGF) has been implicated in tumor angiogenesis and it is
a potent angiogenesis inducer in vivo and in vitro [26]. Inhi-
bition of the VEGF RTK has provided a new paradigm in the
treatment of tumors. It is well established that antiangiogenic
agents also manifest significant inhibitory activity against
VEGFR-2. To investigate the mechanism of antiangiogene-
sis and the possible binding modes of biphenyl derivatives, a
molecular docking study of target compounds with VEGFR-
2 was performed using SYBYL-X 1.1 [27]. Compounds (11)
and (12) were docked into the kinase domain of VEGFR-2
(PDB code: 3CJF) (Fig. 2) [28]. According to our docking
simulation, shown in Fig. (2), methylamine hydrogen of (11)
formed a hydrogen bond interaction with carbonyl oxygen of
ARG 1030 with a distance of 1.96 Å. Regarding the binding
mode of compound (12), the oxygen of carboxyl formed a
H-bond to ASP 1044 with a distance of 2.20 Å, while the
fluorine atom formed a hydrogen bond with ASN 921 with a
bond length of 2.19 Å. The binding hypothesis potentially
provides valuable information for the structure-based design
of novel biphenyl derivatives.
As shown in Table 2, biphenyls without halogen substitu-
tion (14, 18, 19, and 24) were much less potent than those
containing halogen. This finding indicated that the halogen
substitution played a critical role in the activity of biphenyls.
Halogen substitution could potentially improve the antican-
cer activity of biphenyl. The cytotoxicity of two pyridine-
containing compounds (20 and 26) was too weak for further
development.
We also evaluated the antiproliferative activity of com-
pound (11) against several other human cancer cell lines
using the MTT assay. Compound (11) exhibited a broad
spectrum of growth inhibition activity against A431 (human
epithelial carcinoma cell line, IC50 = 37.90 ꢀM), A375 (hu-
man malignant melanoma cells, IC50 = 26.31 ꢀM), HT-29
(human colon carcinoma cells, IC50 = 31.03 ꢀM), Hela (hu-
man epithelial cervical cancer cells, IC50 = 33.07 ꢀM),
CACO-2 (human colonic carcinoma cell line, IC50 = 14.44
ꢀM), and PANC-1 (human pancreatic carcinoma cell line,
IC50 = 34.94 ꢀM) cells.
In the present study, eighteen novel biphenyls were de-
signed and synthesized. All of the compounds were evalu-
ated and the majority demonstrated moderate to good anti-
cancer activity. Of these compounds, (11) and (12) exhibited
the highest antiproliferation activity against several cancer
cell lines and ECV304.
The antiangiogenic activity of taspine has been previ-
ously tested using the chicken chorioallantoic membrane
neovascularisation model in vivo and the human umbilical
vein endothelial cell (HUVEC) proliferation and migration
models in vitro [24]. In this study, we used antiangiogenesis
screening to focus on the antiproliferative activity of the
compounds against HUVEC in vitro. In order to investigate
the mechanism of action of the biphenyls we also tested their
antiproliferative activity against ECV304 human trans-
formed endothelial cells. As shown in Table 2, the majority
of the title compounds exhibited potent growth inhibition of
ECV304 proliferation in a dose-dependent manner, with an
IC50 range of 2.93 - 72.17 ꢀM.
4. CONCLUSION
In summary, a novel class of symmetrical biphenyls was
investigated in this study. Preliminary bioassays indicated
that the majority of these biphenyls demonstrated potent
anticancer and antiangiogenic activity. The docking results
and the antiproliferative activity against HUVECs suggested
that these compounds could potentially serve as antiangio-
genic agents, a promising indication of the importance of
further medicinal chemistry efforts. A thorough biological
evaluation and mechanistic study of these compounds are
currently in progress and will be reported in due course.
Compounds (11) and (12) exhibited the greatest antipro-
liferative activity against ECV304 cells with IC50 values of
2.93 and 4.08 ꢀM, respectively. The results indicated that
these biphenyls could potentially serve as antiangiogenic
agents.

Synthesis and Cytotoxic Evaluation of Novel Symmetrical Taspine
Medicinal Chemistry, 2011, Vol. 7, No. 4 291
Fig. (2). Compound (11, A) and (12, B) was built and docked into the active site of VEGFR-2 (PDB code: 3CJF).
5. EXPERIMENTAL
5.2.2. 3-(benzyloxy)-2-bromo-4-methoxybenzaldehyde (5).
5.1. Antiproliferative Activity of Taspine Derivatives
To a suspension of (4) (3.47 g, 15 mmol) in dehydrated
alcohol (70 mL) was added anhydrous potassium carbonate
(6.22 g, 45 mmol) and benzyl chloride (2.30 mL, 20 mmol).
The mixture was refluxed for 4 h. Filtration and evaporation
of alcohol was done in a vacuum. The residue was extracted
with EtOAc (3ꢀ40 mL). The combined organic layers were
washed with H2O (3ꢀ30 mL), 2 M NaOH (3ꢀ30 mL), 2 M
HCl (3ꢀ30 mL) and brine (2ꢀ30 mL), dried over Na2SO4,
and concentrated to give (5) (4.62 g, 96%) as a colorless
Growth inhibitory activity was evaluated on the follow-
ing cell lines: 7721, MCF-7, LOVO, A549, ECV304, A431,
A375, CACO-2, Hela, HT-29, and PANC-1. The effects of
the compounds on cell viability were evaluated using the
MTT assay [29]. Exponentially growing cells were harvested
and plated in 96-well plates at a concentration of 1 ꢀ 104
cells/well, and incubated for 24 h at 37°C. The respective
cells in the wells were treated with target compounds at vari-
ous concentrations for 48 h. Subsequently, 20 μL MTT (5
mg/mL) was added to each well and the cells were incubated
for 4 h at 37°C. After the supernatant was discarded, 150 μL
DMSO was added to each well, and the absorbance values
were determined by a microplate reader (Bio-Rad Instru-
ments) at 490 nm.
1
solid. mp 79-81 ꢀ; EI-MS(m/z): 320.9 ([M+H]+), H NMR
(300MHz, CDCl3) ꢁ ppm 3.96 (s, 3H), 5.03 (s, 2H), 6.98 (d,
J=9.2 Hz, 1H), 7.35-7.54 (m, 5H), 7.76 (d, J=8.4 Hz, 1H),
10.27 (s, 1H).
5.2.3. 3-(benzyloxy)-2-bromo-4-methoxybenzoic acid (6).
To a solution of (5) (4.82 g, 15 mmol) in THF (60 mL)
was added distilled H2O (20 mL) and NaH2PO4 (1.08 g, 9
mmol). The mixture was stirred at room temperature for 10
min. NaClO2 (4.49 g, 50 mmol) and 30% H2O2 (3.4 mL, 33
mmol) in distilled H2O (15 mL) were added into the above
mixture. The mixture was stirred at the same temperature for
3 h. THF was evaporated under vacuum and the residue was
extrated with EtOAc (3ꢀ50 mL). The combined organic lay-
ers were washed with (3ꢀ20 mL) and the product was ex-
tracted with 2 M NaOH (5ꢀ20 mL). The aqueous phase was
acidified with concentrated HCl and the solid obtained was
collected by filtration and dried to give (6) (4.80 g, 95%) as a
white solid. mp 159-161 ꢀ; EI-MS(m/z): 336.9 ([M+H]+),
1H NMR (300MHz, CDCl3) ꢁ 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).
5.2. Chemistry: General Procedures
All reactions except those in aqueous media were con-
ducted according to standard techniques for the exclusion of
moisture. Solvents were purified before use according to
standard procedures. All reactions were monitored by thin
layer chromatography on 0.25-mm silica gel plates (60GF-
254) and visualized with UV light. All melting points were
determined on a Beijing micro-melting point apparatus and
were uncorrected. 1H-NMR spectra were recorded on a
Bruker AVANCF 400 MHZ instrument in CDCl3 solution
with TMS as internal standard. Mass spectra were performed
on a Shimadzu GC-MS-QP2010 instrument.
5.2.1. 2-bromo-3-hydroxy-4-methoxybenzaldehyde (4).
To a mixture of (3) (10.0 g, 66.0 mmol), NaOAc (10.80
g, 0.132 mol) and iron powder (0.34 g, 6.0 mmol) was added
glacial acetic acid (60 mL). The mixture was stirred at room
temperature for 30 min. Br2 (3.5 mL, 70.0 mol) in glacial
acetic acid (15 mL) was added dropwised into above mixture
at 23-25 ꢀ. The mixture was stirred at the same temperature
for 3 h. Ice water (150 mL) was added to the mixture and
stirred for another 1 h and filtered. The solid obtained as
dried and recrystallized from EtOH to give (4) (12.65 g,
83%) as a gray solid. mp 206-207 ꢀ; EI-MS(m/z): 230.9
5.2.4. 3-(benzyloxy)-2-bromo-4-methoxy-N-
methylbenzamide (8).
To a suspension of (6) (5.06 g, 15 mmol) in CH2Cl2 (50
mL) was added thionyl chloride (4.37 mL, 60 mmol) and a
catalytic amount of DMF. The mixture was stirred at room
temperature for 2 h. The solvent was removed by evapora-
tion in vacuo to give 5.34 g (100%) of the acid chloride (7).
To a solution of 30% aqueous methylamine (10.31 mL,
90 mmol) in THF (20 mL) was added a solution of the acid
chloride (7) (5.34 g, 15 mmol) obtained above in CH2Cl2 (15
mL) dropwised with cooling by an ice-water bath. The mix-
ture was stirred at room temperature for 2 h. The reaction
1
([M+H]+), H NMR (300MHz, CDCl3) ꢁ ppm 4.01 (s, 3H),
6.07 (s, 1H), 6.93 (d, J=8.5 Hz, 1H), 7.58 (d, J=8.5 Hz, 1H),
10.26 (s, 1H).

292 Medicinal Chemistry, 2011, Vol. 7, No. 4
Zhang et al.
mixture was diluted with AcOEt, washed with aqueous satu-
rated NaHCO3, water and brine, and dried over NaSO4. Fil-
tration and concentration in vacuo and purification by silica
gel flash chromatography (CHCl3/MeOH = 30:1) gave 4.46
g (85%) of (8) as a white solid. mp 142-144ꢀ; EI-MS(m/z):
350.9 ([M+H]+), 1H NMR (300MHz, CDCl3) ꢀ ppm 3.00 (d,
J=4.3 Hz, 3H), 3.89 (s, 3H), 5.01 (s, 2H), 6.13 (br, 1H), 6.91
(d, J=7.2 Hz, 1H), 7.37-7.56 (m, 5H), 7.55 (d, J=7.0 Hz,
1H).
Hz, 2H), 7.05-7.11 (m, 3H), 7.09 (d, J=8.4 Hz, 2H), 7.19-
7.51 (m, 2H), 7.74-7.76 (m, 1H).
Compounds (12)–(28) were prepared using the same pro-
cedure described above.
5.2.8. 5,5'-dimethoxy-N,N'-dimethyl-6,6'-bis(2-oxo-2-{[3-
(trifluoromethyl)phenyl] amino}ethoxy)biphenyl-2,2'-
dicarboxamide (12).
Yield: 67%, mp 213-215ꢀ; EI-MS(m/z): 762.1 ([M]+),
1H NMR (300MHz, CDCl3) ꢀ ppm 2.73 (s, 3H), 2.75 (s, 3H),
3.83 (s, 3H), 3.84 (s, 3H), 4.22 (d, J=14.8 Hz, 2H), 4.41 (d,
J=14.4 Hz, 2H), 6.78-6.86 (m, 4H), 7.22-7.33 (m, 5H), 7.50-
7.53 (m, 3H).
Another two intermediates were prepared in the same
way except that compound (6) was amidated with isopropy-
lamine or diethylamine.
5.2.5. 6,6'-bis(benzyloxy)-5,5'-dimethoxy-N,N'-
dimethylbiphenyl-2,2'- dicarboxamide (9).
5.2.9. 6,6'-bis{2-[(3,4-dichlorophenyl)amino]-2-oxoethoxy}-
5,5'-dimethoxy-N,N'-dimethylbiphenyl-2,2'-dicarboxamide
(13).
Yield: 64%, mp 199-201ꢀ; EI-MS(m/z): 762.2 ([M]+),
1H NMR (300MHz, CDCl3) ꢀ ppm 2.74 (s, 3H), 2.75 (s, 3H),
3.80 (s, 3H), 3.82 (s, 3H), 4.21 (d, J=14.4 Hz, 2H), 4.40 (d,
J=14.8 Hz, 2H), 6.81-6.86 (m, 4H), 7.13-7.15 (m, 2H), 7.30
(d, J=8.4 Hz, 2H), 7.48-7.51 (m, 2H).
To a solution of (8) (7.00 g, 20 mmol) in anhydrous DMF
(25 mL) was added freshly activated copper powder (12.8 g,
200 mmol) under nitrogen atmosphere, and the mixture was
refluxed for 4 h. The mixture was filtered and the residual
DMF was poured into 150 mL of ice-water. The residue was
extracted with CHCl3 (3ꢁ70 mL), and the combined organic
layers were washed with 2 M HCl (3ꢁ50 mL) and brine
(2ꢁ50 mL), dried over Na2SO4, and concentrated. The resi-
due was chromatographed on silica gel (PE/AcOEt = 1:1) to
give (9) (4.06 g, 75%) as a white solid. mp 178-180ꢀ; EI-
5.2.10. 5,5'-dimethoxy-6,6'-bis{2-[(4-
methoxyphenyl)amino]-2-oxoethoxy}-N,N'- dimethyl-
biphenyl-2,2'-dicarboxamide (14).
1
MS(m/z): 540.1 ([M]+), H NMR (300MHz, CDCl3) ꢀ ppm
Yield: 75%, mp 114-116ꢀ; EI-MS(m/z): 681.9 ([M]+),
1H NMR (300MHz, CDCl3) ꢀ ppm 2.72 (s, 3H), 2.73 (s, 3H),
3.79 (s, 3H), 3.80 (s, 3H), 4.20 (d, J=14.6 Hz, 2H), 4.39 (d,
J=14.8 Hz, 2H), 6.87(d, J=8.8 Hz, 2H), 7.27-7.36 (m, 4H),
7.45-7.50 (m, 4H), 7.78-7.79 (m, 2H).
2.65 (d, J=4.6 Hz, 6H), 3.87 (s, 6H), 4.73 (d, J=10.9 Hz,
1H), 4.82 (d, J=10.8 Hz, 1H), 6.94-7.19 (m, 12H), 7.33 (d,
J=8.4 Hz, 2H).
5.2.6. 6,6'-dihydroxy-5,5'-dimethoxy-N,N'-
dimethylbiphenyl-2,2'-dicarboxamide (10).
5.2.11. 6,6'-bis{2-[(4-bromophenyl)amino]-2-oxoethoxy}-
5,5'-dimethoxy-N,N'- dimethylbiphenyl-2,2'-dicarboxamide
(15).
Yield: 77%, mp 243-245ꢀ; EI-MS(m/z): 684.0 ([M]+),
1H NMR (300MHz, CDCl3) ꢀ ppm 2.74 (s, 3H), 2.75 (s, 3H),
3.82 (s, 3H), 3.83 (s, 3H), 4.17 (d, J=14.4 Hz, 2H), 4.40 (d,
J=14.4 Hz, 2H), 6.81-6.86 (m, 2H), 6.95-6.97 (m, 2H), 7.08-
7.14 (m, 2H), 7.40-7.43 (m, 2H), 7.52-7.55 (m, 2H).
To a solution of (9) (5.41 g, 10.0 mmol) in anhydrous
methanol (150 mL) was added 10% Pd/C (0.50 g) (10%)
under hydrogen atmosphere. The mixture was stirred at room
temperature until no starting material could be observed by
TLC. Pd/C was filtered and washed with methanol (600 mL)
and acetone (400 mL). Then the combined filtrates were
evaporated under vacuum to give (10) (3.53 g, 98%) as a
1
gray solid. mp 166-168ꢀ; EI-MS(m/z): 360.1 ([M]+), H
NMR (300MHz, CDCl3) ꢀ ppm 2.74 (d, J=4.2 Hz, 6H), 3.92
(s, 6H), 6.89 (d, J=8.4 Hz, 2H), 7.16 (d, J=8.5 Hz, 2H).
5.2.12. 6,6'-bis{2-[(4-bromophenyl)amino]-2-oxoethoxy}-
5,5'-dimethoxy-N,N'- dimethylbiphenyl-2,2'-dicarboxamide
(16).
Yield: 80%, mp 100-102ꢀ; EI-MS(m/z): 661.9 ([M]+),
1H NMR (300MHz, CDCl3) ꢀ ppm 2.74 (s, 6H), 3.83 (s, 6H),
4.21 (d, J=14.8 Hz, 2H), 4.41 (d, J=14.8 Hz, 2H), 6.78-6.86
(m, 4H), 7.21-7.34 (m, 6H), 7.41-7.52 (m, 2H).
5.2.7. 6,6'-bis{2-[(3-chloro-4-fluorophenyl)amino]-2-
oxoethoxy}-5,5'-dimethoxy- N,N'-dimethylbiphenyl-2,2'-
dicarboxamide (11).
To a suspension of (24) (3.60 g, 10.0 mmol) in dehy-
drated alcohol (100 mL) was added anhydrous potassium
carbonate (8.29 g, 60.0 mmol). The mixture was stirred at
room temperature for 30 min and then 2-chloro-N-(3-chloro-
4-fluorophenyl)acetamide (5.55 g, 25.0 mmol) was added.
The reaction mixture was refluxed for 10 h. Filtration and
evaporation of alcohol was done in a vacuum. The residue
was extracted with EtOAc (3ꢁ50 mL). The combined or-
ganic layers were washed with H2O (3ꢁ20 mL), 2 M NaOH
(3ꢁ20 mL), 2 M HCl (3 ꢁ20 mL) and brine (2ꢁ20 mL), dried
over Na2SO4 and solvent was removed. The crude product
was purified by silica gel column chromatography to give
(11) (5.27 g, 72%) as a white crystalline powder, mp 199-
201ꢀ; EI-MS(m/z): 732.2 ([M]+), 1H NMR (300MHz,
CDCl3) ꢀ ppm 2.74 (s, 3H), 2.76 (s, 3H), 3.86 (s, 6H), 4.20
(d, J=14.6 Hz, 2H), 4.40 (d, J=14.6 Hz, 2H), 6.88(d, J=8.5
5.2.13. 6,6'-bis{2-[(3-fluorophenyl)amino]-2-oxoethoxy}-
5,5'-dimethoxy-N,N'- dimethylbiphenyl-2,2'-dicarboxamide
(17).
Yield: 78%, mp 108-110ꢀ; EI-MS(m/z): 662.2 ([M]+),
1H NMR (300MHz, CDCl3) ꢀ ppm 2.74 (s, 3H), 2.75 (s, 3H),
3.83 (s, 6H), 4.26 (d, J=14.8 Hz, 2H), 4.43 (d, J=14.4 Hz,
2H), 6.87(d, J=8.8 Hz, 2H), 7.26-7.46 (m, 8H), 7.85-7.87 (m,
2H).
5.2.14. 6,6'-bis(2-anilino-2-oxoethoxy)-5,5'-dimethoxy-
N,N'-dimethylbiphenyl- 2,2'-dicarboxamide (18)
Yield: 85%, mp 110-112ꢀ; EI-MS(m/z): 626.3 ([M]+),
1H NMR (300MHz, CDCl3) ꢀ ppm 2.70 (s, 3H), 2.71 (s, 3H),

Synthesis and Cytotoxic Evaluation of Novel Symmetrical Taspine
Medicinal Chemistry, 2011, Vol. 7, No. 4 293
3.80 (s, 3H), 3.81 (s, 3H), 4.24 (d, J=14.4 Hz, 2H), 4.42 (d,
J=14.8 Hz, 2H), 7.09-7.14 (m, 6H), 7.35-7.49 (m, 4H), 7.68-
7.75 (m, 4H).
5.2.21. 6,6'-bis{2-[(4-bromophenyl)amino]-2-oxoethoxy}-
N,N'-diisopropyl-5,5'-dimethoxybiphenyl-2,2'-
dicarboxamide (25).
Yield: 81%, mp 207-209ꢀ; EI-MS(m/z): 840.0 ([M]+),
1H NMR (300MHz, CDCl3) ꢀ ppm 1.04 (d, J=6.7 Hz, 12H),
1.32-1.35 (m, 2H), 3.80 (s, 3H), 3.81 (s, 3H), 4.26 (d, J=14.8
Hz, 2H), 4.44 (d, J=14.8 Hz, 2H), 6.78-6.83 (m, 2H), 6.95-
7.12 (m, 4H), 7.47-7.58 (m, 4H).
5.2.15. 6,6'-bis[2-(isopropylamino)-2-oxoethoxy]-5,5'-
dimethoxy-N,N'- dimethylbiphenyl-2,2'-dicarboxamide (19)
Yield: 76%, mp 168-170ꢀ; EI-MS(m/z): 557.8 ([M]+),
1H NMR (300MHz, CDCl3) ꢀ ppm 1.05 (t, J=6.7 Hz, 6H),
1.06 (t, J=6.7 Hz, 6H), 1.42 (m, 2H), 2.72 (s, 3H), 2.73 (s,
3H), 3.80 (s, 3H), 3.81 (s, 3H), 4.20 (d, J=14.6 Hz, 2H), 4.41
(d, J=14.8 Hz, 2H), 6.87(d, J=8.5 Hz, 2H), 7.12 (d, J=8.4
Hz, 2H).
5.2.22. 6,6'-bis{2-[(6-fluoropyridin-3-yl)amino]-2-
oxoethoxy}-N,N'-diisopropyl-5,5' -dimethoxybiphenyl-2,2'-
dicarboxamide (26)
Yield: 84 %, mp 145-146ꢀ; EI-MS(m/z): 719.9 ([M]+),
1H NMR (300MHz, CDCl3) ꢀ ppm 1.00 (d, J=6.7 Hz, 12H),
1.28-1.33 (m, 2H), 3.84 (s, 3H), 3.85 (s, 3H), 4.21 (d, J=14.8
Hz, 2H), 4.47 (d, J=14.8 Hz, 2H), 6.88(d, J=8.7 Hz, 2H),
7.13 (d, J=8.7 Hz, 2H), 8.50-8.58 (m, 1H), 8.69-8.75 (m,
2H).
5.2.16. 6,6'-bis{2-[(6-fluoropyridin-3-yl)amino]-2-
oxoethoxy}-5,5'-dimethoxy- N,N'-dimethylbiphenyl-2,2'-
dicarboxamide (20)
Yield: 65 %, mp 100-102ꢀ; EI-MS(m/z): 664.1 ([M]+),
1H NMR (300MHz, CDCl3) ꢀ ppm 2.73 (s, 3H), 2.74 (s, 3H),
3.82 (s, 6H), 4.22 (d, J=14.4 Hz, 2H), 4.44 (d, J=14.8 Hz,
2H), 6.87(d, J=8.8 Hz, 2H), 7.12 (d, J=8.4 Hz, 2H), 8.37-
8.50 (m, 2H), 8.60-8.65 (m, 1H).
5.2.23. 6,6'-bis{2-[(3-chloro-4-fluorophenyl)amino]-2-
oxoethoxy}-N,N,N',N'- tetraethyl-5,5'-dimethoxybiphenyl-
2,2'-dicarboxamide (27).
Yield: 85% mp 179-181ꢀ; EI-MS(m/z): 814.2 ([M]+),
1H NMR (300MHz, CDCl3) ꢀ ppm 0.97 t, J=6.7 Hz, 6H ,
1.05 (t, J=6.7 Hz, 6H), 3.00 (q, J=7.0 Hz, 2H), 3.09 (q, J=7.2
Hz, 2H), 3.90 (s, 6H), 4.29 (d, J=14.8 Hz, 2H), 4.44 (d,
J=14.8 Hz, 2H), 6.94(d, J=8.6 Hz, 2H), 7.07-7.12 (m, 3H),
7.24 (d, J=8.6 Hz, 2H), 7.37-7.57 (m, 2H), 7.77-7.85 (m,
1H).
5.2.17. 6,6'-bis{2-[(3-chloro-4-fluorophenyl)amino]-2-
oxoethoxy}-N,N'-diisopropyl- 5,5'-dimethoxybiphenyl-2,2'-
dicarboxamide (21).
Yield: 79% mp 201-203ꢀ; EI-MS(m/z): 786.1 ([M]+),
1H NMR (300MHz, CDCl3) ꢀ ppm 1.07 (d, J=6.8 Hz, 12H),
1.32-1.35 (m, 2H), 3.88 (s, 6H), 4.27 (d, J=14.8 Hz, 2H),
4.46 (d, J=14.4 Hz, 2H), 6.92(d, J=8.5 Hz, 2H), 7.09-7.14
(m, 3H), 7.22 (d, J=8.4 Hz, 2H), 7.35-7.62 (m, 2H), 7.79-
7.84 (m, 1H).
5.2.24. 5,5'-dimethoxy-N,N'-dimethyl-6,6'-bis(2-oxo-2-{[3-
(trifluoromethyl)phenyl] amino}ethoxy)biphenyl-2,2'-
dicarboxamide (22).
Compounds (12)–(28) were prepared using the same pro-
cedure described above.
1
Yield: 81%, mp 76-78ꢀ; EI-MS(m/z): 846.8 ([M]+), H
5.2.18. 5,5'-dimethoxy-N,N'-dimethyl-6,6'-bis(2-oxo-2-{[3-
(trifluoromethyl)phenyl] amino}ethoxy)biphenyl-2,2'-
dicarboxamide (22).
NMR (300MHz, CDCl3) ꢀ ppm 0.99 t, J=6.9 Hz, 6H ,
1.09 (t, J=6.9 Hz, 6H), 3.09 (q, J=7.2 Hz, 2H), 3.27 (q, J=7.4
Hz, 2H), 3.87 (s, 6H), 4.27 (d, J=14.8 Hz, 2H), 4.45 (d,
J=14.4 Hz, 2H), 6.83-6.92 (m, 4H), 7.23-7.35 (m, 5H), 7.57-
7.65 (m, 3H).
1
Yield: 82%, mp 94-96ꢀ; EI-MS(m/z): 817.8 ([M]+), H
NMR (300MHz, CDCl3) ꢀ ppm 1.06 (d, J=6.9 Hz, 12H),
1.30-1.33 (m, 2H), 3.84 (s, 3H), 3.85 (s, 3H), 4.26 (d, J=14.6
Hz, 2H), 4.44 (d, J=14.8 Hz, 2H), 6.82-6.89 (m, 4H), 7.21-
7.30 (m, 5H), 7.52-7.55 (m, 3H).
5.3. Molecular Modeling
The molecular docking method was frequently applied to
predict the binding conformation of the ligands if no cocrys-
tal structure was available. Molecule docking was performed
using the Sybyl/Surflex (Tripos Inc.) based on the crystal
structures of VEGFR-2 taken from the Protein Data Bank
(PDB code 3CJF). The 3D-structures of docking compounds
were constructed with the Sybyl/Sketch module and opti-
mized using Powell’s method. Energy minimization was
performed using the Tripos force field with a convergence
criterion of 0.005 kcal/(Å·mol) and a maximum of 1000 it-
erations and Gasteiger-Hückel charges. A non-bonded cutoff
distance of 8 Å was used to determine the intramolecular
interaction [30]. The VEGFR-2 was prepared using the Pro-
tein Preparation tool in the Sybyl-X Biopolymer module.
The ligand within the active site and all of the water mole-
cules were removed while the inhibitor (SAV) was extracted.
The residues within a radius of 6.5 Å around the SAV (the
ligand of VEGFR-2 in the crystal structure 3CJF) in
VEGFR-2 were selected as the active sites. Other docking
parameters of the program were maintained at a default set-
5.2.19. 6,6'-bis{2-[(3,4-dichlorophenyl)amino]-2-
oxoethoxy}-5,5'-dimethoxy-N,N'-dimethylbiphenyl-2,2'-
dicarboxamide (23).
Yield: 69%, mp 108-110ꢀ; EI-MS(m/z): 820.2 ([M]+),
1H NMR (300MHz, CDCl3) ꢀ ppm 1.03 (d, J=6.7 Hz, 12H),
1.32-1.35 (m, 2H), 3.81 (s, 3H), 3.82 (s, 3H), 4.24 (d, J=14.4
Hz, 2H), 4.41 (d, J=14.8 Hz, 2H), 6.87-6.92 (m, 4H), 7.15-
7.19 (m, 2H), 7.29 (d, J=8.4 Hz, 2H), 7.42-7.45 (m, 2H).
5.2.20. N,N'-diisopropyl-5,5'-dimethoxy-6,6'-bis{2-[(4-
methoxyphenyl)amino]-2 -oxoethoxy}biphenyl-2,2'-
dicarboxamide (24).
Yield: 75%, mp 214-216ꢀ; EI-MS(m/z): 741.9 ([M]+),
1H NMR (300MHz, CDCl3) ꢀ ppm 1.02 (d, J=6.8 Hz, 12H),
1.29-1.32 (m, 2H), 3.80 (s, 6H), 4.22 (d, J=14.8 Hz, 2H),
4.38 (d, J=14.8 Hz, 2H), 6.85(d, J=8.7 Hz, 2H), 7.25-7.34
(m, 4H), 7.46-7.51 (m, 4H), 7.77-7.80 (m, 2H).

294 Medicinal Chemistry, 2011, Vol. 7, No. 4
Zhang et al.
Med. Chem., 2002, 45, 5058-5068.
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the inhibitors has provided a basis for further studies aimed
at identifying inhibitors of VEGF-induced angiogenesis, and
has provided valuable information regarding the structure-
based design of novel biphenyl VEGFR-2 TK inhibitors.
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ACKNOWLEDGEMENTS
This work was supported by the National Natural Science
Foundation (NNSF) of China (Grant Number 30901839 and
30730110).
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Received: November 25, 2010
Revised: February 17, 2011
Accepted: April 06, 2011