Synthesis and preliminary biological evaluation of novel taspine derivatives as anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2010.03.001

Antiangiogenic therapy might represent a new promising anticancer therapeutic strategy. Taspine can significantly inhibit cell proliferation of human umbilical vein endothelial cells (HUVECs) induced by vascular endothelial growth factor-165, which is crucial for angiogenesis. In this study, a series of novel taspine derivatives were synthesized and screened for in vitro anticancer and antiangiogenesis activities. The majority of the derivatives demonstrated a moderate degree of cytotoxicity against human cancer cell lines. One of them (14) exhibited much better antiproliferative activity against CACO-2 (IC ₅₀ = 52.5 μM) and ECV304 (IC₅₀ = 2.67 μM) cells than taspine did. Some of them were also effective in antiproliferative assays against HUVECs. The in silico estimate of solubility of title compounds were higher than that of taspine.

Full text of DOI:10.1016/j.ejmech.2010.03.001

European Journal of Medicinal Chemistry 45 (2010) 2798e2805
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and preliminary biological evaluation of novel taspine derivatives
as anticancer agents
,
Jie Zhang a, Yanmin Zhang a, Yuanyuan Shan b, Na Li a, Wei Ma a, Langchong He a
*
a School of Medicine, Xi'an Jiaotong University, Xi'an, 710061, P.R. China
b The First Affiliated Hospital, Xi'an Jiaotong University, Xi'an, 710061, P.R. China
a r t i c l e i n f o
a b s t r a c t
Article history:
Antiangiogenic therapy might represent a new promising anticancer therapeutic strategy. Taspine can
significantly inhibit cell proliferation of human umbilical vein endothelial cells (HUVECs) induced by
vascular endothelial growth factor-165, which is crucial for angiogenesis. In this study, a series of novel
taspine derivatives were synthesized and screened for in vitro anticancer and antiangiogenesis activities.
The majority of the derivatives demonstrated a moderate degree of cytotoxicity against human cancer
cell lines. One of them (14) exhibited much better antiproliferative activity against CACO-2
Received 24 December 2009
Received in revised form
27 February 2010
Accepted 1 March 2010
Available online 7 March 2010
(IC50 ¼ 52.5
m
M) and ECV304 (IC50 ¼ 2.67
mM) cells than taspine did. Some of them were also effective in
Keywords:
antiproliferative assays against HUVECs. The in silico estimate of solubility of title compounds were
higher than that of taspine.
Taspine derivatives
Antiangiogenesis
Antiproliferative activity
Anticancer activity
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
healing [4]. Its anticancer and antiangiogenic properties have been
demonstrated and it could be an ideal candidate for a chemother-
Angiogenesis is a normal process in growth and development of
blood vessels as well as wound healing [1]. Tumors follow a similar
procedure to improve their own blood supply to ensure their
aberrant growth [2]. Angiogenesis is a key event of tumor
progression and metastasis and hence a target for cancer chemo-
therapy. Angiogenesis is primarily a receptor-mediated process that
is mediated by growth factors that cause signal transduction via
receptor tyrosine kinase (RTK). Vascular endothelial growth factor
(VEGF) and basic fibroblast growth factor (bFGF) have both been
implicated in tumor angiogenesis. They are potent angiogenesis
inducers in vivo and in vitro. Inhibition of the VEGF and bFGF RTKs
has provided a new paradigm in the treatment of tumors. A great
advantage of antiangiogenic agents is that they are likely to be
much less toxic than the existing chemotherapy agents. Design and
synthesis of novel small-molecule antiangiogenic agents are
underway in modern medicinal chemistry.
apeutic agent [5]. Taspine can inhibit expression of VEGF in human
umbilical vein endothelial cells (HUVECs), as well as proliferation of
HUVECs induced by VEGF-165. In a further study, He et al.
demonstrated that taspine could inhibit tumor angiogenesis, and
one of its mechanisms may be that it inhibits VEGF receptor-2
(VEGFR-2) [6]. However, some studies have demonstrated that the
use of taspine is limited by its low potency and poor solubility [7].
Poor solubility is a major problem in the biological testing of
compounds, which has made formulating the compounds a chal-
lenge for in vivo studies of efficacy and pharmacokinetics [8]. This
notwithstanding, taspine remains a good lead compound for the
design of derivatives with increased activity and solubility. Taspine
is a rigid tetracyclic molecule with a dilactonic, tertiary amine
structure (Fig. 1) [9]. To research the individual contribution of the
lactone ring and biphenyl scaffold of taspine to its biological
activity, we designed and synthesized a series of novel ring-opened
target compounds.
Alignment of multiple ligands based on shared pharmacophoric
and pharmacosteric features is a long-recognized challenge in drug
discovery and development. In this study, we applied pharmaco-
phore modeling using GALAHAD (a genetic algorithm with linear
assignment for hypermolecular alignment of datasets) to obtain
more reasonable alignment results [10]. First, we selected 15
molecules with higher anticancer activities as a training set to
Taspine was isolated from Radix et Rhizoma Leonticis (Hong Mao
Qi in Chinese) by Li and He [3]. It has many pharmacological
activities such as bacteriostatic, antibiotic, antiviral, anti-inflam-
matory, antiulcer, and cytotoxic activity, and can be used in wound
* Corresponding author. Tel./fax: þ86 29 82655451.
E-mail address: helc@mail.xjtu.edu.cn (L. He).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.03.001

J. Zhang et al. / European Journal of Medicinal Chemistry 45 (2010) 2798e2805
2799
Fig. 1. The three-dimensional (3D) structure and antiproliferative activity of Taspine.
generate a pharmacophore model for molecular alignment. Then,
a four-point pharmacophore model was generated from the 15
selected compounds. The pharmacophore features and their space
locations are shown in Fig. 2. The hypothesis contained seven
pharmacophoric features: four hydrophobic centers (cyan spheres),
two hydrogen-bond donors (green), and one hydrogen-bond
acceptor (purple). However, the title compounds did not fit the
hypothesis perfectly with a maximum fit value of 4.
the molecules has increased the solubility of the compounds while
retaining their biological activity. The second amine also mimics
the N,N-dimethylaminoethyl side chain of taspine. In this paper, we
described the synthesis and biological assay of taspine derivatives
to develop novel and active anticancer agents.
2. Chemistry
Our intention was to explore ring-opened derivatives, to see if
they possess increased activity and solubility. We designed and
synthesized four novel ring-opened target compounds (13e16)
(Fig. 3). We envisaged breaking the diphenyl CeC bond and lactone
CeO bond to yield four novel taspine derivatives. The preliminary
biological test demonstrated that the ester bond of the B-ring was
more important than that of D-ring in maintaining the biological
activity of these compounds.
Biphenyl compounds are known for their varied biological
activities, such as antibacterial, anti-inflammatory and anticancer
activities. Our group has investigated biphenyl compounds and RTK
inhibitors for several years. As a follow-up, a number of biphenyl
derivatives were synthesized and evaluated as anticancer agents
(24e29) (Fig. 3). The presence of chlorine and fluorine in a molecule
enhances drug persistence and lipid solubility. Recently, we have
synthesized compounds with a water-soluble secondary amine
(dimethylamino, morpholine, and piperizine) and 3-chloro-4-
flouroaniline linked to the positions 6- and 60- of the biphenyl
scaffold. The incorporation of second amines into the side chains of
Four ring-opened title compounds were synthesized by the
route outlined in Scheme 1. We used commercially available iso-
vanillin as the starting material. At first, isovanillin was oxidized to
give isovanillic acid with fused sodium hydroxide and potassium
hydroxide. The methyl ester was prepared to avoid side-reactions
of the carboxylate group. Refluxing of phenolic hydroxyl group with
prenyl bromide in the presence of K2CO3 in anhydrous acetone
yielded compound (2) [11]. A solution of (2) in N,N-dimethylaniline
was heated to reflux for 8 h to give compound (3) [12]. The prenyl
group was moved into the para-position of hydroxyl in this Claisen
rearrangement process. The next step was the coupling of (3) to the
carboxyl group of (4) by an ester bond with DCC and DMAP [13].
The oxidation of (5) with OsO4/NaIO4 produced aldehyde (6) [14],
which reacted with dimethylamine, followed by reduction with
NaBH(OAc)3 to give (7) [15]. At last, benzyl deprotection of (7) with
palladium/carbon in methanol gave the target compound (13) [16].
The other target compounds (14), (15) and (16) were synthesized in
the same way from isovanillin.
Six biphenyl derivatives were prepared by the general routes
described in Scheme 2. We also used isovanillin (17) as the starting
material. Isovanillin (17) was converted into 2-bromoisovanillin
(18) by reaction with bromine [17]. After protection of (18) as
benzyl ether [18], the aldehyde group in (19) was oxidized to the
carboxyl group to give (20) [19]. Amidation of (20) with methyl-
amine yielded the monocyclic precursor (22) for the Ullmann
reaction [20]. Biphenyl dimethane amide (23) was prepared by
dimerization of (22) by employing a classical symmetrical Ullmann
reaction [21]. Compound (24) could be readily obtained by depro-
tection of benzamide (23) under catalysis with palladium/carbon
[22]. One or two of the hydroxyl groups in (24) was etherified with
various alkyl halides in anhydrous acetone in the presence of K2CO3
to give (25) or (26) in different yields [23]. Finally, etherification of
compound (25) yielded the target compound (27), (28) or (29).
3. Results and discussion
It is not always practical to perform experimental measure-
ments, therefore, it is useful to predict molecular physicochemical
properties rapidly such as Log P, Log D, pKa, PSA, and solubility
values [24]. Before biological evaluation, we evaluated a broad
range of molecular physicochemical properties which had an
impact on drug-like ADME parameters based on chemical struc-
tures (Table 1). From Table 1, we can see that solubility values of
Fig. 2. Pharmacophore-based molecular alignment by using GALAHAD.

2800
J. Zhang et al. / European Journal of Medicinal Chemistry 45 (2010) 2798e2805
O
O
R
13 R = NMe2
COOMe 14 R = Morpholine
15 R = 3-Cl-4-F-aniline
OH
O
D
B
O
E
O
O
N
O
N
H
N
A
C
O
O
O
W =
X =
HO
MeOOC
B
D
O
O
O
O
F
Cl
O
N
Taspine
1
16
B,D,E
O
24
25
26
27
28
29
R1 = H; R2 = H
N
Y =
Z =
R1 = W; R2 = H
R1 = H; R2 = Y
R1 = W; R2 = Y
R1 = W; R2 = X
R1 = W; R2 = Z
O
CONHCH3
OR2
R1O
H3CHNOC
N
HN
O
Fig. 3. Design of ring-opened taspine derivatives.
target compounds were higher than those of taspine. Although it
was known that in silico estimates of pKa and solubility were not
exact, the calculated values suggested that there might be an
improvement in physicochemical properties.
In vitro evaluation of cytotoxicity and antiproliferative activity of
the synthesized compounds was carried out against various cancer
and normal cell lines using the 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay with taspine or gefitinib
as a control (Table 2). The antiangiogenesis screening focused on
the ability of the compounds to inhibit HUVEC growth in vitro. To
search for novel small angiogenesis inhibitors, we also tested their
antiproliferative activity against ECV304 human transformed
endothelial cells. As shown in Table 2, most of the title compounds
exhibit good growth inhibition of ECV304 proliferation in a dose-
dependent manner, with IC50 values between 2.67
1717.70 M.
mM and
m
O
O
COOMe
COOH
c
b
a
COOMe
OBn
COOMe
MeO
+
MeO
O
O
OBn
MeO
O
OH
3
2
4
5
O
O
O
O
O
N
COOMe
OBn
d
e
COOMe
OBn
13(14,15)
O
O
O
O
6
7
O
O
O
COOMe
MeO
BnO
O
O
+
c
b
BnO
MeOOC
BnO
MeOOC
MeO
O
O
COOH
O
O
OH
8
9
11
10
O
N
d
e
O
16
BnO
MeOOC
O
O
12
Scheme 1. Preparation of target compounds (13e16). Reagents and Conditions: (a) N,N-dimethylaniline, N2, reflux, 71%; (b) anhydrous THF, DCC, DMAP, (5, 71%; 10, 78%); (c) OsO4,
NaIO4, acetone/H2O/t-BuOH, (6, 32%; 11, 34%); (d) dimethylamine/Morpholine/3-Cl-4-F-aniline, THF, CH2Cl2, NaBH(OAc)3, 25e40%; (e) H2, Pd/C, 92e97%.

J. Zhang et al. / European Journal of Medicinal Chemistry 45 (2010) 2798e2805
2801
CHO
CHO
Br
CHO
Br
COOH
COCl
Br
a
b
c
d
MeO
MeO
MeO
MeO
Br
MeO
OH
17
OH
18
OBn
19
OBn
20
OBn
21
O
O
CONHMe
Br
e
f
g
h
OH
CONHMe
OBn
CONHMe
MeHNOC
BnO
MeHNOC
HO
MeO
OBn
O
O
22
24
23
O
O
H
N
H
N
H
H
N
N
O
O
O
O
i
O
O
O
O
O
HO
O
F
F
N
H
N
O
N
H
Cl
Cl
O
O
27
25
or
or
or
O
O
O
H
N
H
N
H
N
H
N
H
N
N
O
O
O
O
O
O
O
O
O
O
O
O
O
F
HO
F
N
N
H
N
N
N
H
Cl
Cl
H
HN
O
O
O
29
26
28
Scheme 2. Preparation of target compounds (24e29). Reagents and Conditions: (a) Fe, NaOAc, AcOH, Br2, 81%; (b) BnCl, K2CO3, 95%; (c) NaH2PO4, NaClO2, 30% H2O2, 93%; (d) SOCl2,
DMF(cat), CH2Cl2, 96%; (e) CH2Cl2, 30% CH3NH2, 85%; (f) Cu, DMF, 72%; (g) H2, Pd/C; 97%; (h) K2CO3, DMF, (26, 64%; 25, 76%); (i) K2CO3, EtOH, (27, 72%; 28, 59%; 29, 54%).
The activity of (14) was much better than that of taspine against
CACO-2 (threefold) and ECV304 (about 10-fold) cells, with IC50
values of 52.5 mM and 2.67 mM, respectively. Compound (14)
appeared to inhibit proliferation more potently in ECV304 cells, and
this may be an indication that the compound was acting through
inhibition of VEGFR-2. If the antiangiogenic properties of (14) were
confirmed, the compound could represent a novel hit for lead
optimization efforts. The biphenyl derivative (25) exhibited potent
MTT assay. The antiproliferative activity of biphenyl derivatives was
tested against six different human cancer cell lines (Table 3). The
tested compounds showed a broad spectrum of growth inhibition
activity. Preliminary results showed that six biphenyl derivatives
displayed potent antiproliferative activity, with IC50 values from
25.06 to 414.59
against A375 (IC50 ¼ 25.06
(IC50 ¼ 44.71
m
M. Compound (27) exhibited the best activity
M), MCF-7 (IC50 ¼ 60.38 M), HT-29
M) cells. The pres-
m
m
m
M) and PANC-1 (IC50 ¼ 110.43
m
inhibitory activities against CACO-2 (IC50 ¼ 20.33
m
M), A549
ence of morpholine might be important for this activity. Replace-
ment of morpholine with dimethylamine or piperizine led to
a slight decrease in activity. Compound (26) was the most active
(IC50 ¼ 73.51
m
M) and ECV304 (IC50 ¼ 72.17
m
M) cells. Among the
biphenyl derivatives, compound (28) exhibited the best anti-
proliferative activity against ECV304 cells, with an IC50 value of
57.20 mM. The results indicated that the taspine derivatives could
be served as antiangiogenic agents in cancer therapy.
We also evaluated the antiproliferative activity of biphenyl
derivatives against several other human cancer cell lines using the
compound against 7721, with an IC50 value of 78.16
compound (25) showed the best antiproliferative activity against
M) cells.
Among the two series of compounds, (14) and (28) exhibited the
highest antiproliferative activity against ECV304 cells, which might
mM, whereas
HeLa (IC50 ¼ 53.37
m
Table 1
Prediction of physicochemical properties of compounds.
CLog P
Log D (PH ¼ 7.4)
pKa
Solubility (g/L) (PH ¼ 7.4)
PSAa
Taspine
13
14
15
16
24
25
26
27
1.93
3.18
3.33
6.06
3.18
1.48
0.50
1.96
0.53
0.03
0.59
2.92
1.72
2.65
6.56
1.97
1.54
0.47
2.08
ꢀ0.71
ꢀ0.061
ꢀ1.28
8.21
0.021
0.14
0.27
0.061
0.13
36.57
0.61
22.56
2.21
0.21
74.3
94.53
9.73; 8.75
9.02; 7.28
9.01; 3.65
9.55; 8.57
9.24; 8.31
8.52
8.57; 6.76
8.62
6.76
103.76
103.32
94.53
117.12
135.22
118.59
127.46
136.69
139.49
28
29
9.29; 3.18
6.95
a
2
ꢀ
PSA ¼ Polar Surface Area (A ).

2802
J. Zhang et al. / European Journal of Medicinal Chemistry 45 (2010) 2798e2805
Table 2
Antiproliferative activities of target compounds on human cancer and normal cells.
Compound
Cell lines (IC50
CACO-2a
, mM)
A549b
ECV304c
EGFR celld
A431e
Taspine
13
14
153
110.03
52.5
4.18
453
158
180
22.3
ND
2.67
61
15.95
ND
ND
11.20
ND
ND
15
ND
ND
ND
16
3100
111
ND
ND
ND
24
25
26
27
104.18
20.33
75.85
3314.11
167.18
64.65
73.51
259.49
194.39
240.51
112.57
72.17
1364.84
ND
88.04
ND
116.02
ND
104.18
ND
75.85
ND
28
57.20
ND
ND
ND, not determined.
a
CACO-2, human epithelial colorectal adenocarcinoma cells.
A549, carcinomic human alveolar basal epithelial cells.
ECV304, human umbilical endothelial cell line.
EGFR cell, Overexpressing Epidermal Growth Factor Receptor Stably cell (HEK293/EGFR).
A431, model cell line express abnormally high levels of the EGFR.
b
c
d
e
Table 3
Antiproliferative activities of biphenyl derivatives on human cancer cells.
Compound
Cell lines (IC50, mM)
A375a
7721b
Helac
MCF-7d
HT-29e
PANC-1f
Gefitinib
24
25
26
27
20.55
105.59
217.42
174.07
25.06
242.11
ND
33.12
95.69
45.59
97.61
53.37
414.59
100.26
116.68
ND
69.16
ND
16.93
69.32
217.09
191.16
165.59
110.43
159.09
ND
200.41
213.70
122.11
44.71
133.48
47.17
109.44
78.16
194.09
124.19
125.53
109.16
83.68
60.38
123.02
ND
28
29
ND, not determined.
a
A375, human amelanotic melanoma cells.
7721, Human hepatoma cell line.
Hela, an immortal cell line.
MCF-7, human breast cancer cell line.
HT-29, human colon adenocarcinoma grade II cell line.
b
c
d
e
f
PANC-1, human pancreatic carcinoma, epithelial-like cell line.
Fig. 4. (A) The compound 14 was built and docked into the active site of VEGFR-2 (PDB ID: 3C7Q). (B) The compound 28 was built and docked into the active site of bFGFR-1 (PDB ID:
3C4F). The docking result was showed by PyMOL.
have been related to RTK inhibition. It is known that antiangiogenic
agents also manifest significant inhibitory activity against the
receptors for VEGF and bFGF. To investigate the antiangiogenesis
mechanism of taspine derivatives, a molecular docking study of
target compounds with VEGFR-2 and bFGFR-1 was performed
using SYBYL 7.0 to identify their binding mode with the enzyme.
Compounds (14) and (28) were docked into the potential binding
sites of VEGFR-2 and bFGFR-1, respectively (Fig. 4). The docking
results indicated that the binding energies of the two compounds
with VEGFR-2 and bFGFR-1 were both lower than that of taspine.
The two compounds both bound to the ATP-binding site in the cleft
between the N- and C-terminal lobes of the kinase domain. The
binding hypothesis could provide valuable information for struc-
ture-based design of taspine analogs.

J. Zhang et al. / European Journal of Medicinal Chemistry 45 (2010) 2798e2805
2803
4. Conclusions
by flash chromatography on silica gel to give (3) (2.88þg, 71.0%) as
a white solid, mp 76.0e77.0 ꢂC. EI-MS(m/z): 250.0 (M ), 1H NMR
In the present study, 10 novel ring-opened taspine derivatives
were described. All the compounds were tested and most of them
showed moderate to good anticancer activity. Compound (14),
methyl 5-[(3-hydroxy-4-methoxybenzoyl) oxy]-4-methoxy-2-(2-
morpholin-4-ylethyl)benzoate, showed the highest antiprolifera-
tion activity in CACO-2 and ECV304 cell lines. Some of the
compounds showed good antiproliferative activity against colon
(HT29), breast (MCF-7), lung (A549), rectum (CACO-2), skin (A375),
hepatoma (7721), and pancreatic (PANC-1) cancers cell lines.
In summary, the novel taspine derivatives were designed and
synthesized. Preliminary bioassays indicated that the majority of
the derivatives demonstrated moderate antiangiogenic and anti-
cancer activities. We demonstrated that the lactone ring B is
important for activity, while the lactone ring D can be opened, thus
retaining and even improving the antiproliferative properties of
taspine. The docking results and the antiproliferative activity
against HUVECs suggest that these taspine derivatives are potent
RTK inhibitors. The data indicate that these compounds could also
exhibit an antiangiogenic mechanism of action, similar to that of
taspine, and could therefore be a promising starting point for
further medicinal chemistry efforts. Our attempts to optimize the
physicochemical and biological properties of taspine analogs will
be reported in due course.
(300 MHz, CDCl3)
d
ppm 1.71 (s, 6H), 3.68 (d, 2H, J ¼ 6.0 Hz), 3.90 (s,
3H), 4.07 (s, 3H), 5.27 (t,1H, J ¼ 6.6 Hz), 5.50 (s,1H), 6.69 (s,1H), 7.51
(s, 1H).
5.2.2. Methyl 5-(3-(benzyloxy)-4-methoxybenzoyloxy)-4-methoxy-
2-(3-methylbut-2- enyl)benzoate (5)
A
solution of DCC (N,N0-dicyclohexylcarbodiimide) (0.99 g,
4.8 mmol) in 10 mL of anhydrous THF was added dropwise to
a mixture of compound (4) (1.23 g, 4.8 mmol), DMAP (4-(dime-
thylamino)pyridine) (0.05 g, 0.4 mmol) and (3) (1.0 g, 4.0 mmol) in
30 mL of anhydrous THF at 0 ꢂC. The reaction mixture was stirred at
room temperature for 48 h. The precipitated DCU (N,N0-dicyclo-
hexylurea) was filtered off and THF was evaporated in cacuo. The
residues obtained after evaporation were dissolved in 100 mL ethyl
acetate. The organic layer was washed with 2 N HCl (3 ꢁ 30 mL),
saturated NaHCO3 (3 ꢁ 30 mL) and saturated NaCl (3 ꢁ 30 mL),
dried over Na2SO4. The solvent was evaporated to give a residue
which was chromatographed on a silica column to give (5) (1.53þg,
78.0%) as white solid, mp 115.0e116.0 ꢂC. EI-MS(m/z): 489.9 (M ),
1H NMR (300 MHz, CDCl3)
d ppm 1.67 (s, 6H), 3.76 (s, 3H), 3.77 (s,
3H), 3.71 (d, 2H, J ¼ 6.0 Hz), 3.89 (s, 3H), 5.13 (s, 2H), 5.23 (t,
J ¼ 6.0 Hz, 1H), 6.79 (s, 1H), 6.89 (d, J ¼ 8.5 Hz, 1H), 7.22e7.33 (m,
5H), 7.39 (s, 1H), 7.69 (s, 1H), 7.79 (d, J ¼ 8.5 Hz, 1 H).
5. Experimental
5.2.3. Methyl 5-(3-(benzyloxy)-4-methoxybenzoyloxy)-4-methoxy-
2-(2-oxoethyl) benzoate (6)
5.1. Antiproliferative activity of taspine derivatives
OsO4 (23.4 mg, 5% mol) was added to a stirred solution of (5)
(0.90 g, 1.84 mmol) in 3:1:1 acetone-t-BuOHeH2O (40 mL) (the
starting material was dissolved in acetone-t-BuOH, and H2O was
added last). After stirred keeping from light at room temperature
for 30 min, NaIO4 (51.18 g, 5.52 mmol) was added in one portion.
Stirring was continued for 12 h. The reaction miture was filtered
through diatomite, washed with acetone. The filtrate was concen-
trated in vacuum under 20 ꢂC, and the residues were dissolved in
150 mL ethyl acetate. The organic layer was washed with 5% aquous
NaHSO3 (3 ꢁ 20 mL) and water (3 ꢁ 30 mL), dried over Na2SO4 and
evaporated. Flash chromatography of the residue over silica gel,
using 1:1-EtOAc-petroleum ether, gave aldehyde (6) (þ0.27 g, 32%)
as white solid, 169.5e170.5 ꢂC, EI-MS(m/z): 463.9 (M ), 1H NMR
Growth inhibitory activities were evaluated on the following cell
lines: CACO-2, A549, ECV304, EGFR cell (cell of overexpressing
Epidermal Growth Factor Receptor Stably HEK293/EGFR), A431,
A375, 7721, Hela, MCF-7, HT-29, and PANC-1. The effects of the
compounds on cell viability were evaluated using the MTT assay
[23]. 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 ꢂC. The cells in the wells were respectively treated
with target compounds at various concentrations for 48 h. Then,
20
m
L MTT (5 mg/mL) was added to each well and incubated for 4 h
L DMSO was
at 37 ꢂC. After the supernatant was discarded, 150
m
added to each well, and the absorbance values were determined by
a microplate reader (Bio-Rad Instruments) at 490 nm.
(300 MHz, CDCl3) d ppm 3.85 (s, 3H), 3.88 (s, 3 H), 3.99 (s, 3H), 4.25
(s, 2H), 5.17 (s, 2H), 6.97 (d, J ¼ 6.0 Hz, 1H), 7.28e7.37 (m, 5H), 7.44
(s, 1H), 7.69 (s, 1H), 7.72(m, 1H), 7.91 (s, 1H), 9.85 (s, 1H).
5.2. Chemistry: general procedures
5.2.4. Methyl 5-(3-(benzyloxy)-4-methoxybenzoyloxy)-2-(2-
(dimethylamino)ethyl)-4- methoxybenzoate (7)
All reactions except those in aqueous media were carried out by
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 appa-
ratus and were uncorrected. 1H-NMR spectra were recorded on
a Bruker AVANCF300 MHZ instrument in CDCl3 solution with TMS
as internal standard. Mass spectra were performed on a Shimadzu
GC-MS-QP2010 instrument.
Aldehydr (6) (0.10 g, 0.22 mmol) was dissolved in CH2Cl2
(20 mL) and methol (10 mL) under a nitrogen atmosphere at room
temperature, and dimethylamine (0.44 mmol) in THF was added.
After stirring for 4 h, NaBH(OAc)3 (0.058 g, 0.26 mmol) was grad-
ually added over 10 min to the reaction mixture. The mixture was
stirred for another 12 h at room temperature. The solvent was
evaporated in vacuuo and the residue was purified by column
chromatography to give (7) (0.042 g,þ38.0%) as a white solid, mp
147.5e148.5 ꢂC, EI-MS(m/z): 493.1 (M ), 1H NMR (300 MHz, CDCl3)
d
ppm 2.55 (s, 6H), 2.68 (t, J ¼ 7.6 Hz, 2H), 3.59 (t, J ¼ 7.9 Hz, 2H),
5.2.1. Methyl 5-hydroxy-4-methoxy-2-(3-methylbut-2-enyl)
benzoate (3)
3.85 (s, 3H), 3.87 (s, 3 H), 4.01 (s, 3H), 5.20 (s, 2H), 6.96 (s, 1H), 7.26
(s, 1H), 7.35e7.47 (m, 5H), 7.73e7.79 (m, 2H), 7.85(s, 1H).
A solution of (2) (4.06 g, 16.24 mmol) in N,N-dimethylaniline
(20 mL) was refluxed under nitrogen for 8 h, then cooled to room
temperature, added ethyl acetate (150 mL). The organic layer was
washed with 2 N HCl (5 ꢁ 30 mL), saturated NaHCO3 (2 ꢁ 30 mL)
and saturated NaCl (2 ꢁ 30 mL), dried over Na2SO4. The solvent was
filtered and concentrated to give pale yellow oil, which was purified
5.2.5. Methyl 2-(2-(dimethylamino)ethyl)-5-(3-hydroxy-4-
methoxybenzoyloxy)-4- methoxybenzoate (13)
Compound (7) (0.16 g, 0.32 mmol) and Pd/C (0.02 g) in meth-
anol (25 mL) were stirred at room temperature under H2 atmo-
sphere for 48 h. After completion of the reaction, the solid materials

2804
J. Zhang et al. / European Journal of Medicinal Chemistry 45 (2010) 2798e2805
were separated by filtration over Celite followed by washed with
methanol. After removal of the solvent, the crude product was
purified by column chromatography to give (13) (0.12 g, 97.0%) as
a white solid, mp 112.5e114.5 ꢂC, EI-MS(m/z): 403.1 (Mþ), 1H NMR
stirred at the same temperature for 3 h. THF was evaporated under
vacuum and the residue was extrated with EtOAc (2 ꢁ 80 mL). The
combined organic layers were washed with (3 ꢁ 30 mL) and the
product was extracted with 2 M NaOH (5 ꢁ 30 mL). The aqueous
phase was acidified with concentrated HCl and the solid obtained
was collected by filtration and dried to give (20) (9.37 g, 93%) as
a white solid. mp 159e161 ꢂC; EI-MS(m/z): 336.9 ([M þ H]þ), 1H
(300 MHz, CDCl3)
d
ppm 2.52(s, 6H), 2.71 (t, J ¼ 7.3 Hz, 2H), 3.62 (t,
J ¼ 7.0 Hz, 2H), 3.84 (s, 3H), 3.89 (s, 3 H), 3.98 (s, 3H), 6.89 (s, 1H),
7.08 (d, J ¼ 8.7 Hz, 1H), 7.54 (d, J ¼ 9.0 Hz, 1H), 7.67 (s, 1H), 7.81 (s,
1H).
NMR (300 MHz, CDCl3)
d ppm 3.94 (s, 3H), 5.03 (s, 2H), 6.93 (d,
Compounds (14), (15) and (16) were prepared by using the
general procedure described above.
J ¼ 9.2 Hz, 1H), 7.37e7.56 (m, 5H), 7.87 (d, J ¼ 9.1 Hz, 1H).
5.2.9. 3-(Benzyloxy)-2-bromo-4-methoxy-N-methylbenzamide
(22)
5.2.5.1. Methyl-5-[(3-hydroxy-4-methoxybenzoyl)oxy]-4-methoxy-
2-(2-morpholin-4-ylethyl)benzoate (14). mp 121.0e124.5 ꢂC, EI-MS
To a suspension of (20) (5.04 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 evaporation in vacuo to give 5.31 g (100%)
of the acid chloride of (20).
(m/z): 445.2 (Mþ), 1H NMR (300 MHz, CDCl3)
d ppm 2.55e2.63 (m,
4H), 2.82 (t, J ¼ 8.0 Hz, 2H), 3.42e3.49 (m, 4H), 3.71 (t, J ¼ 7.8 Hz,
2H), 3.82 (s, 3H), 3.90 (s, 3 H), 3.97 (s, 3H), 6.76 (s, 1H), 7.00 (d,
J ¼ 9.0 Hz, 1H), 7.42 (d, J ¼ 8.7 Hz, 1H), 7.73 (s, 1H), 7.87 (s, 1H).
To a solution of 30% aqueous methylamine (10.31 mL, 90 mmol)
in THF (20 mL) was added a solution of the acid chloride (21)
(5.31 g, 15 mmol) obtained above in CH2Cl2 (15 mL) dropwise with
cooling by an ice-water bath. The mixture was stirred at room
temperature for 2 h. The reaction mixture was diluted with AcOEt,
washed with aqueous saturated NaHCO3, water and brine, and
dried over NaSO4. Filtration and concentration in vacuo and puri-
fication by silica gel flash chromatography (CHCl3/MeOH ¼ 30:1)
gave 4.46 g (85%) of (22) as a white solid. mp 142e144 ꢂC; EI-MS(m/
5.2.5.2. Methyl-2-{2-[(3-chloro-4-fluorophenyl)amino]ethyl}-5-[(3-
hydroxy-4-methoxybenzoyl) oxy]-4-þmethoxybenzoate (15). mp
101.0e103.5 ꢂC, EI-MS(m/z): 503.7 (M ), 1H NMR (300 MHz, CDCl3)
d
ppm 2.49 (t, J ¼ 7.5 Hz, 2H), 3.52 (t, J ¼ 7.2 Hz, 2H), 3.81 (s, 3H),
3.87 (s, 3 H), 3.95 (s, 3H), 6.88 (d, J ¼ 7.5 Hz, 1H), 6.94 (s, 1H),
6.97e7.10 (m, 2H), 7.19e7.21 (m, 1H), 7.54 (d, J ¼ 9.0 Hz, 1H), 7.68 (s,
1H), 7.77 (s, 1H).
5.2.5.3. 2-Methoxy-5-(methoxycarbonyl)phenyl-2-[2-(dimethyla-
z): 350.9 ([M þ H]þ), 1H NMR (300 MHz, CDCl3)
d ppm 3.00 (d,
mino)ethyl]-5-hydroxy-4-methoxybenzþoate
(16). mp
J ¼ 4.3 Hz, 3H), 3.89 (s, 3H), 5.01 (s, 2H), 6.13 (br, 1H), 6.91 (d,
J ¼ 7.2 Hz, 1H), 7.37e7.56 (m, 5H), 7.55 (d, J ¼ 7.0 Hz, 1H).
107.0e109.5 ꢂC, EI-MS(m/z): 402.9 (M ), 1H NMR (300 MHz, CDCl3)
d
ppm 2.62 (s, 6H), 3.55 (t, J ¼ 6.6 Hz, 2H), 3.83 (s, 3H), 3.85 (s, 3 H),
3.98 (s, 3H), 4.01 (t, J ¼ 6.9 Hz, 2H), 6.66 (s, 1H), 7.13 (d, J ¼ 9.0 Hz,
5.2.10. 6,60-Bis(benzyloxy)-5,50-dimethoxy-N,N0-dimethylbiphenyl-
2,20- dicarboxamide(23)
1H), 7.72 (s, 1H), 7.86 (d, J ¼ 9.0 Hz, 1H), 7.93 (s, 1H).
To a solution of (22) (3.49 g, 10 mmol) in anhydrous DMF
(25 mL) was added freshly activated copper powder (6.4 g,
100 mmol) under nitrogen atmosphere, and the mixture was
refluxed for 4 h at 150e160 ꢂC. The mixture was filtered and DMF
was evaporated under vacuum. The residue was extrated with
CHCl3 (3 ꢁ 50 mL), and the combined organic layers were washed
with 2 M HCl (3 ꢁ 30 mL) and brine (2 ꢁ 30 mL), dried over Na2SO4,
and concentrated. The residue was chromatographed on silica gel
(PE/AcOEt ¼ 1:1) to give (23) (1.95 g, 72%) as a white solid. mp
178e180 ꢂC; EI-MS(m/z): 540.1 ([M]þ), 1H NMR (300 MHz, CDCl3)
5.2.6. 2-Bromo-3-hydroxy-4-methoxybenzaldehyde (18)
To
a mixture of (17) (20.0 g, 0.132 mol), NaOAc (21.59 g,
0.263 mol) and iron powder (0.68 g, 0.012 mol) was added glacial
acetic acid (120 mL). The mixture was stirred at room temperature
for 30 min. Br2 (7.0 mL, 0.14 mol) in glacial acetic acid (30 mL) was
added dropwise into the above mixture at 23e25 ꢂC. The mixture
was stirred at the same temperature for 3 h. Ice-water (250 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
(18) (24.59 g, 81%) as a gray solid. mp 206e207 ꢂC; EI-MS(m/z):
d
ppm 2.65 (d, J ¼ 4.6 Hz, 6H), 3.87 (s, 6H), 4.73 (d, J ¼ 10.9 Hz, 1H),
230.9 ([M þ H]þ), 1H NMR (300 MHz, CDCl3)
d
ppm 4.01 (s, 3H), 6.07
4.82 (d, J ¼ 10.8 Hz, 1H), 6.94e7.19 (m, 12H), 7.33 (d, J ¼ 8.4 Hz, 2H).
(s, 1H), 6.93 (d, J ¼ 8.5 Hz, 1H), 7.58 (d, J ¼ 8.5 Hz, 1H), 10.26 (s, 1H).
5.2.11. 6,60-Dihydroxy-5,50-dimethoxy-N,N0-dimethylbiphenyl-2,20-
dicarboxamide (24)
5.2.7. 3-(Benzyloxy)-2-bromo-4-methoxybenzaldehyde (19)
To a suspension of (18) (6.90 g, 30 mmol) in dehydrated alcohol
(120 mL) was added anhydrous potassium carbonate (12.44 g,
90 mmol) and benzyl chloride (5.20 mL, 45 mmol). The mixture
was refluxed for 4 h. Filtration and evaporation of alcohol was done
in a vacuum. The residue was extrated with EtOAc (2 ꢁ 70 mL). The
combined organic layers were washed with H2O (3 ꢁ 40 mL), 2 M
NaOH (3 ꢁ 40 mL), 2 M HCl (3 ꢁ 40 mL) and brine (2 ꢁ 40 mL),
dried over Na2SO4, and concentrated to give (19) (9.12 g, 95%) as
a yellow solid. mp 79e81 ꢂC; EI-MS(m/z): 320.9 ([M þ H]þ), 1H NMR
To a solution of (23) (3.11 g, 5.76 mmol) in anhydrous methanol
(100 mL) was added 10% Pd/C (0.31 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 (400 mL). Then the combined filtrates were
evaporated under vacuum to give (24) (2.0þ1 g, 97%) as a gray solid.
mp 166e168 ꢂC; EI-MS(m/z): 360.1 ([M] ), 1H NMR (300 MHz,
CDCl3)
d
ppm 2.74 (d, J ¼ 4.2 Hz, 6H), 3.92 (s, 6H), 6.89 (d, J ¼ 8.4 Hz,
2H), 7.16 (d, J ¼ 8.5 Hz, 2H).
(300 MHz, CDCl3)
d
ppm 3.96 (s, 3H), 5.03 (s, 2H), 6.98 (d, J ¼ 9.2 Hz,
1H), 7.35e7.54 (m, 5H), 7.76 (d, J ¼ 8.4 Hz, 1H), 10.27 (s, 1H).
5.2.12. 6-Hydroxy-5,50-dimethoxy-N,N0-dimethyl-60-(2-morpholin-
4 -ylethoxy)biphenyl-2,20- dicarboxamide (26)
5.2.8. 3-(Benzyloxy)-2-bromo-4-methoxybenzoic acid (20)
A solution of (24) (3.13 g, 8.7 mmol) and potassium carbonate
(2.41 g, 17.5 mmol) in DMF (50 mL) was stirred at room tempera-
ture for 30 min and then 4-(3-chloropropyl)morpholine was added.
This mixture was stirred at 80 ꢂC in a water bath for 10 h under
nitrogen atmosphere. The reaction was poured on crushed ice-
water mixture and extracted with ethyl acetate (4 ꢁ 50 mL).
To a solution of (19) (9.60 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 (6.8 mL, 66 mmol) in distilled H2O
(25 mL) were added into the above mixture. The mixture was

J. Zhang et al. / European Journal of Medicinal Chemistry 45 (2010) 2798e2805
2805
Organic layers were combined, extracted with brine (3 ꢁ 30 mL),
dried over Na2SO4 and solvent was removed. The crude material
was crystallized from AcOEt/PE to afford (26) (2.64þg, 64%) as
a white solid. mp 172e175 ꢂC; EI-MS(m/z): 473.2 ([M] ), 1H NMR
cocrystal structure. Here molecule docking was performed using
the Sybyl/FlexX (Tripos Inc.) based on the crystal structures of
VEGFR-2 and bFGFR-1 taken from the Protein Data Bank. Hydro-
gens were added and minimized using the Tripos force field and
Pullman charges. Model compounds were constructed with the
Sybyl/Sketch module (Tripos Inc.) and optimized using Powell's
method with the Tripos force field with convergence criterion set at
(300 MHz, CDCl3)
d
ppm 2.14e2.35 (m, 6H), 2.63 (d, J ¼ 4.2 Hz, 3H),
2.76 (d, J ¼ 4.3 Hz, 3H), 3.61e3.65 (m, 4H),3.86 (s, 3H), 3.92 (s, 3H),
4.11e4.15 (m, 2H), 6.84 (d, J ¼ 8.4 Hz, 1H), 6.89 (d, J ¼ 8.5 Hz, 1H),
7.14 (d, J ¼ 8.4 Hz, 1H), 7.38 (d, J ¼ 8.8 Hz, 1H).
ꢀ
}
0.05 kcal/(A mol), and assigned with the Gasteiger-Huckel method
ꢀ
Compound (8) was prepared in the same way.
[25]. The residues in a radius of 6.5 A around BIBF 1120 (the ligand
of VEGFR-2 in the crystal structure 3C7Q) in VEGFR-2, and C4F (the
ligand of bFGFR-1 in the crystal structure 3C4F) in bFGFR-1 were
selected as the active site. Other docking parameters implied in the
program were kept at default. Virtual docking of VEGFR-2 and
bFGFR-1 in complex with the inhibitors provided a basis for further
studies aimed at identifying inhibitors of VEGF- and bFGF-induced
angiogenesis, and valuable information for structure-based design
of second generation inhibitors.
5.2.12.1. 6-{2-[(3-Chloro-4-fluorophenyl)amino]-2-oxoethoxy}-60-
hydroxy-5,50-dimethoxy-N,N0- dimethylbiphenyl-2,20-dicarboxamide
(25). Yield, 76%, white crystalline powder, mp 159e162 ꢂC; EI-MS
(m/z): 545.1 ([M]þ), 1H NMR (300 MHz, CDCl3)
d ppm 2.63 (s, 3H),
2.84 (s, 3H), 3.83 (s, 3H), 3.91 (s, 3H), 4.59 (s, 1H), 4.64(s, 1H), 5.72
(br, 1H), 6.33(br, 1H), 6.72 (d, J ¼ 7.8 Hz, 1H), 6.95 (d, J ¼ 7.9 Hz, 1H),
7.08 (d, J ¼ 8.7 Hz, 1H), 7.26e7.29 (m, 3H), 8.00 (d, J ¼ 6.3 Hz, 1H),
8.92 (s, 1H).
5.2.13. 6-{2-[(3-Chloro-4-fluorophenyl)amino]-2-oxoethoxy}-5,50-
dimethoxy-N,N0-dimethyl-60- (2-morpholin-4-ylethoxy)biphenyl-
2,20-dicarboxamide (27)
Acknowledgments
This work was supported by the National Natural Science
Foundation (NNSF) of China (Grant Number 30901839 and
30730110). We also thank Prof. Wenfang Xu and Huawei Zhu for the
pharmacophore modeling and molecular docking study. We are
grateful to Prof. Zongru Guo for practical guidance during the
course of the project.
To a suspension of (25) (0.30 g, 0.55 mmol) in dehydrated
alcohol (50 mL) were added anhydrous potassium carbonate
(0.46 g, 3.3 mmol) and N-(2-chloroethyl)morpholine hydrochloride
(0.21 g, 1.1 mmol). The mixture was refluxed for 8 h. Filtration and
evaporation of alcohol was done in a vacuum. The residue was
extrated with EtOAc (2 ꢁ 30 mL). The combined organic layers were
washed with H2O (3 ꢁ10 mL), 2 M NaOH (3 ꢁ 10 mL), 2 M HCl
(3 ꢁ 10 mL) and brine (2 ꢁ 10 mL), dried over Na2SO4, and
concentrated to give (27) (0.25 g, 72%) as a white crystalline
powder, mp 165e167 ꢂC; EI-MS(m/z): 658.1 ([M]þ), 1H NMR
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d
ppm 2.18e2.33 (m, 6H), 2.62 (d, J ¼ 3.8 Hz, 3H),
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5.2.13.1. 6-{2-[(3-Chloro-4-fluorophenyl)amino]-2-oxoethoxy}-3,30-dime-
tIhoxy-N,N0-dimethyl-60- (2-morpholin-4-ylethoxy)biphenyl-2,20-dicarbox-
1
amide (28). Yield, 59%, mp 138e141 ꢂC; EI-MS(m/z): 617.1 ([M]þ), H
NMR (300 MHz, CDCl3)
d
ppm 2.38 (s, 6H), 2.67 (d, J ¼ 4.2 Hz, 3H), 2.72
(t, J ¼ 6.3 Hz, 2H), 2.88(d, J ¼ 3.9 Hz, 3H), 3.84 (s, 3H), 3.92 (s, 3H), 4.14 (t,
J ¼ 6.0 Hz, 2H), 4.72 (s, 1H), 4.75 (s, 1H), 6.84 (d, J ¼ 8.2 Hz, 1H), 6.92 (d,
J ¼ 8.1 Hz, 1H), 7.13 (d, J ¼ 8.5 Hz, 2H), 7.69e7.74 (m, 3H), 8.01 (d,
J ¼ 8.3 Hz, 2H).
5.2.13.2. 6-{2-[(3-Chloro-4-fluorophenyl)amino]-2-oxoethoxy}-5,50-
dimethoxy-N,N0-dimethyl-60-(2-piperazin-1-ylethoxy)biphenyl-2,20-
dicarboxamide (29). Yield, 54%, mp 192e195 ꢂC; EI-MS(m/z): 657.1
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d ppm 2.54e2.67 (m, 6H), 2.78 (s,
3H), 3.07(s, 3H), 3.42e3.57 (m, 4H), 3.78 (s, 3H), 3.91 (s, 3H), 4.57 (s,
1H), 4.62(s, 1H), 6.80 (d, J ¼ 8.3 Hz, 1H), 6.98 (d, J ¼ 8.0 Hz, 1H), 7.06
(d, J ¼ 8.8 Hz, 2H), 7.43 (d, J ¼ 8.3 Hz, 2H), 7.91e7.99 (m, 3H).
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5.3. Molecular modeling
Molecular docking method was often applied to predict the
binding conformation of ligand when there was no available
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