Synthesis, anticancer evaluation and docking study of vadimezan derivatives with carboxyl substitution

Article abstract of DOI:10.1039/c3md00372h

A series of xanthone analogues modified from vadimezan 6 with carboxyl substitution were synthesized as esters, amides, arylidene hydrazides, diacylhydrazides and acyl thiosemicarbazides, and their structures were confirmed by IR, ¹H NMR, MS, HRMS or elemental analysis. The in vitro anticancer activities were evaluated by the MTT method. It was found that compounds 8f, 8g and 10e were effective against A549 with an IC₅₀ at 10.8 μM, 9.4 μM and 11.5 μM respectively, and that 8e was effective against HL-60 with an IC₅₀ at 4.6 μM. Compounds 8f-h showed a significant inhibitory effect on HUVEC growth and migration in vitro, among which 8h inhibited HUVEC growth with an IC₅₀ at 6.4 μM and HUVEC migration by 67.6% and 89.7% at 2.5 μg mL^-1 and 10 μg mL ^-1 respectively. More spectacularly, docking study indicated that compound 8h might target the ATP binding site of VEGFR2. In addition, compounds 8a, 8f-h exhibited moderate in vivo antitumor efficacy against the S180 xenograft in ICR mice by 22.4-29.6% tumor weight inhibition.

Full text of DOI:10.1039/c3md00372h

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CONCISE ARTICLE
Synthesis, anticancer evaluation and docking study
of vadimezan derivatives with carboxyl
substitution†
Cite this: Med. Chem. Commun., 2014,
5, 512
Shi-Jie Zhang,a Zhi-Shan Ding,b Fu-Sheng Jiang,b Qiu-Fu Ge,c Dian-Wu Guo,c
d
e
*
Hai-Bo Li and Wei-Xiao Hu
A series of xanthone analogues modified from vadimezan 6 with carboxyl substitution were synthesized as
esters, amides, arylidene hydrazides, diacylhydrazides and acyl thiosemicarbazides, and their structures
were confirmed by IR, 1H NMR, MS, HRMS or elemental analysis. The in vitro anticancer activities were
evaluated by the MTT method. It was found that compounds 8f, 8g and 10e were effective against A549
with an IC50 at 10.8 mM, 9.4 mM and 11.5 mM respectively, and that 8e was effective against HL-60 with
an IC50 at 4.6 mM. Compounds 8f–h showed a significant inhibitory effect on HUVEC growth and
migration in vitro, among which 8h inhibited HUVEC growth with an IC50 at 6.4 mM and HUVEC
migration by 67.6% and 89.7% at 2.5 mg mLꢀ1 and 10 mg mLꢀ1 respectively. More spectacularly, docking
study indicated that compound 8h might target the ATP binding site of VEGFR2. In addition, compounds
8a, 8f–h exhibited moderate in vivo antitumor efficacy against the S180 xenograft in ICR mice by 22.4–
29.6% tumor weight inhibition.
Received 7th December 2013
Accepted 29th January 2014
DOI: 10.1039/c3md00372h
www.rsc.org/medchemcomm
synthesized as a topoisomerase II inhibitor with signicant
antiproliferative activity.12,13
Introduction
Vadimezan 6 was developed from xanthenone-4-acetic acid
(XAA),14,15 and has been recognized as a xanthone anticancer
candidate due to its excellent experimental antitumor activity as
a vascular disrupting agent that attacks the blood supply of a
cancerous tumor to cause tumor regression.16–18 Despite a phase
III failure for vadimezan 6 treating advanced non-small cell lung
cancer (NSCLC) with carboplatin or paclitaxel,19,20 study on
vadimezan 6 is continuing. Ching's group compared the activ-
ities of vadimezan analogues in murine or human cellular
models to explain why clinical responses to vadimezan 6 were
disappointing while the preclinical data were so encouraging,
and tried to search for analogues with greater clinical potential
for human use.21
Xanthones with various substituents on the three-membered
heterocyclic ring skeleton exhibit diverse biological activities,
such as antihypertensive, antioxidative, antithrombotic and
anticancer activities.1–4
Naturally existing xanthone analogues, including gentia-
kochianin 1 (Fig. 1),5 a-mangostin 2 and psorospermin 3, have
been identied to possess noticeable anticancer activities.6–8
Inspired by these natural xanthones with an excellent anti-
proliferative effect, many synthetic xanthones have been
derived from modication of xanthone scaffolds.9,10 Bisfur-
anoxanthone 4, modied from the naturally isolated psor-
ospermin 3, exhibited comparable anticancer activities to the
natural compound 3.11 Besides, in consideration of epoxy-teth-
ered natural xanthones, 1,3-bisepoxyxanthone
5
was
aGraduate School, Zhejiang Chinese Medical University, Hangzhou 310053, PR China.
E-mail: medchem@zcmu.edu.cn; Fax: +86-571-86613539; Tel: +86-571-86613663
bInstitute of Biotechnology, College of Life Science, Zhejiang Chinese Medical
University, Hangzhou 310053, PR China
cR&D Center, Hangzhou Minsheng Pharmaceutical Group Co., Ltd., Hangzhou 311100,
PR China
dLaboratory of Microbiology, Nantong Center for Disease Control and Prevention,
Nantong 226007, PR China
eCollege of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou
310032, PR China. E-mail: huyang@mail.hz.zj.cn; Fax: +86-571-88320557; Tel: +86-
571-88320557
† Electronic supplementary information (ESI) available: Characterization data of
all compounds. See DOI: 10.1039/c3md00372h
Fig. 1 Representative xanthones with anticancer activity.
512 | Med. Chem. Commun., 2014, 5, 512–520
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In this study we focused on the synthesis, structural char-
acterization and preliminary evaluation of the anticancer
properties of a series of new xanthone derivatives modied from
vadimezan 6 with carboxyl substitution, in an attempt to further
explore the therapeutic potential of this tricyclic framework.
Results and discussion
Chemistry
Vadimezan 6 was synthesized by coupling the salts of 2-hydroxy-
phenylacetic acid with 2-iodo-3,4-dimethylbenzoic acid, which
was iodinated from the corresponding aniline with cuprous
chloride and tris(2-(2-methoxyethoxy)ethyl)amine (TDA-1)
catalysts to give the diacid, followed by concentrated sulfuric
acid catalyzed cyclodehydration,22 with a total yield of approxi-
mately 40% and the structure was conrmed by single crystal
crystallography.23
The esters of vadimezan 6 were obtained in moderate to
excellent yields with reactions of vadimezan and the corre-
sponding alcohols catalyzed by diphenylammonium triate
(DPAT) in reuxing toluene, as shown in Scheme 1. The appli-
cation of DPAT included merits from the viewpoint of green
chemistry and avoided acylation of vadimezan. In addition, the
products could be obtained easily by ltration aer precipita-
tion with cooling, thus simplifying the workup procedure.
Amidation of the acetic acid chain of vadimezan 6 was simply
achieved by reacting with amines using N,N0-dicyclohexyl-
carbodiimide (DCC) and N-hydroxybenzotriazole (HOBt) under
mild conditions, as shown in Scheme 2. The introduction of
HOBt in the DCC coupling was benecial in increasing the yields.
Vadimezan 6 was converted into hydrazide 9 via its ethyl
ester 7b by reuxing with excess hydrazine hydrate in ethanol,
as shown in Scheme 3. Condensation of hydrazide 9 with the
appropriate arylaldehydes afforded the corresponding arylidene
hydrazides 10a–k with E and Z conformations mixed, which
Scheme 2 Reagents and conditions: (a) R2NH2, DCC, HOBt, DMF,
60 ꢁC, 17–97%.
could be identied by resonance at different frequencies from
1H NMR spectra.
Hydrazide 9 was coupled with the corresponding carboxylic
acids to produce diacylhydrazides of vadimezan in the presence
of DCC and HOBt under mild conditions, as shown in Scheme
4. The introduction of HOBt in the DCC coupling was superior
to the use of 4-dimethylaminopyridine (DMAP) in increasing the
yields of diacylhydrazides 11a–c.
1,4-Disubstituted thiosemicarbazides 12a–c were obtained
by treating hydrazide 9 with the requisite isothiocyanates, as
shown in Scheme 5.
Scheme 1 Reagents and conditions: (a) R1OH, DPAT, toluene, reflux, 27–99%.
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Scheme 3 Reagents and conditions: (a) hydrazine hydrate, EtOH, reflux, 95%; (b) R3CHO, EtOH, reflux, 52–99%.
alkyl chains of amides, but compound 8b was most efficient
against MCF-7 and HL-60 with IC50 at 43.8 mM and 44.7 mM
respectively. Amide 8g (IC50 ¼ 9.4 mM) with a para-chloro
substituted phenyl ring was better than 8e and 3,4-dichloro
substituted 8h in its anti-A549 activity, and comparable with
meta-chloro 8f (IC50 ¼ 10.8 mM), even better than com-
bretastatin A4 (CA-4) or cisplatin (DDP). However, compound
8g (IC50 ¼ 32.1 mM) had one-fold higher activity against the
Bewo cell line as compared with 8f (IC50 ¼ 66.5 mM). It was
found that compound 8e strongly inhibited HL-60 cells, with
IC50 at 4.6 mM. Besides, compounds 8k and 8l with pyridine
substituents were not efficient against cancer cell lines, and 8k
was much weaker than 8g in general. Vadimezan amides 8n–p
with ethyl esters of amino acids seemed to have moderate
activities and their solubility was improved. The calculated
partition coefficient (log P) of compounds 8n and 8o was 3.2
and 3.4 respectively. They were predicted to have reasonable
physicochemical properties of low molecular weight, log P and
PSA.24
Scheme 4 Reagents and conditions: (a) R4COOH, DCC, HOBt, DMF,
50 ꢁC, 58–98%.
Various dinitrogen compounds containing hydrazone or
hydrazide subunits in their structure have been reported as
interesting human cancer inhibitors.25 The combination of the
xanthone nucleus with a 1-hydrazinyl-2-ylidene (–NH–N]) side
chain might be an intriguing pharmacophore for anticancer
design. Our results for the in vitro anticancer activity of aryli-
dene hydrazides 10a–k against A549 are in agreement with the
prior understanding. The anticancer activity of compounds
10g–i including para-hydroxyl substituents was greater than 100
mM against A549, except compound 10b with mono para-
hydroxyl substitution with IC50 at 35.9 mM. Comparison of the
IC50 values between compounds 10j and 10d, 10j and 10g,
Scheme 5 Reagents and conditions: (a) R5NCS, EtOH, reflux, 24–42%.
Pharmacology
Inhibition on cancer cell growth in vitro. The in vitro anti- respectively, showed that meta-hydroxyl and para-methoxyl
cancer activity of vadimezan esters 7a–q was generally weak groups might be crucial to maintain anti-A549 activity. Among
with all IC50 values beyond 100 mM, except ester 7m, whose IC50 these arylidene hydrazides, compound 10e seemed to be the
against BGC-823 was 81.5 mM as shown in Table 1. It is thought best against these cancer cells, with IC50 at 68.4 mM against
that these esters acted as prodrugs of vadimezan 6, which have Ishikawa, 11.5 mM against A549, 76.39 mM against Bewo,
no in vitro anticancer activity as reported, and could be hydro- 36.2 mM against HeLa, and 20.8 mM against HL-60.
lyzed to vadimezan 6 in the body to exert the anticancer activity
in vivo.
In addition, the anticancer activity of some compounds (7m,
10a, 10e, 11a–c and 11e) against Jeko-1 and SK-BR-3 was further
Compared with amides 8a, 8b and 8c, there seemed a tested, and the preliminary results indicated that all their IC50
tendency that the anti-A549 activity decreased with longer side values were beyond 10 mg mLꢀ1
.
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Table 1 IC50 values of selected compounds against cancer cell linesa (mM)
Compound
Ishikawa
A549
Bewo
HeLa
Siha
MCF-7
HL-60
BEL-7402
NCI-460
BGC-823
7m
8a
8b
8e
8f
8g
8h
8i
8j
8k
8n
8o
8p
10a
10b
10c
10d
10e
10f
10j
10k
6
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
61.87
>100
>100
>100
>100
>100
91.16
68.41
>100
>100
>100
>100
20.10
16.03
>100
28.88
46.86
77.56
10.82
9.39
>100
>100
>100
>100
66.45
32.05
>100
>100
>100
>100
>100
26.22
45.45
67.15
73.70
84.86
>100
76.39
>100
>100
>100
>100
12.74
41.85
>100
>100
>100
>100
25.44
>100
18.72
94.23
>100
>100
>100
>100
>100
>100
>100
>100
>100
36.22
>100
>100
>100
>100
14.83
8.90
>100
91.99
>100
>100
36.09
46.65
96.13
32.35
63.24
>100
>100
98.03
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
44.55
>100
>100
43.77
>100
>100
>100
>100
>100
>100
95.94
46.38
40.61
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
25.51
18.73
>100
>100
44.72
4.59
>100
>100
nt
>100
nt
nt
nt
nt
nt
>100
>100
nt
>100
nt
nt
nt
nt
nt
81.48
>100
nt
>100
nt
nt
nt
nt
nt
91.23
67.58
42.44
76.13
93.77
80.87
>100
79.86
90.86
52.79
>100
>100
>100
20.83
>100
>100
>100
>100
<0.03
21.46
92.00
25.79
>100
>100
42.92
35.00
39.56
21.35
35.91
50.74
>100
11.52
15.66
54.94
86.78
>100
12.52
15.59
nt
nt
nt
nt
nt
nt
>100
>100
>100
nt
>100
nt
>100
nt
>100
>100
>100
nt
>100
>100
>100
nt
>100
nt
>100
nt
>100
>100
>100
nt
76.42
>100
>100
nt
>100
nt
>100
nt
>100
>100
>100
nt
CA-4
DDP
16.86
8.90
9.43
a
Values presented are means of three experiments; compounds with IC50 larger than 100 mM against all tested cell lines are not listed; nt ¼ not
tested.
An overall view of the obtained results shows that amides 8a– formation assay, the in vitro cytotoxicity of selected compounds
p and arylidene hydrazides 10a–k were most active, and that the against HUVEC was assayed by MTT. The IC50 values of selected
introduction of esters as 7a–q, diacylhydrazides as 11a–e or acyl compounds against HUVEC are listed in Table 2. The dose–
thiosemicarbazides as 12a–c did not seem to signicantly response curves of HUVEC inhibition with the tested
increase the in vitro activity. No enantioselectivity was observed compounds are demonstrated in Fig. 2, indicating a linear
from comparison of enantiomers 12b and 12c.
increase in the HUVEC inhibition rate in a concentration-
Inhibition on HUVEC growth and migration in vitro. dependent manner; compounds 8g and 8h were most active and
Although angiogenesis is a complex process, tube formation 8f seemed weaker.
and cell migration are critical for endothelial cells in the orga-
The morphological changes in HUVEC cells treated with
nization of new blood capillaries in tumors. Compounds 8e–h compounds 8f–h at 12.5 mg mLꢀ1 for 24 h were photographed as
and 10e showing the best in vitro anticancer activity were further shown in Fig. 3. Compared with the control, compounds 8g and
evaluated for their antiangiogenic effects by tube formation
assay, and vadimezan 6 was included in the assay for compar-
ison. Tube formation assay was performed at non-cytotoxic
doses of each compound against the human vein umbilical cell
line (HUVEC). To identify non-cytotoxic doses for tube
Table 2 IC50 values of selected compounds against HUVECa (mM)
Compound
HUVEC inhibition
8e
8f
8g
8h
10e
6
>20
11.10 ꢂ 2.54
6.87 ꢂ 1.24
6.40 ꢂ 2.21
>20
>20
Fig.
2 Dose–response curves of HUVEC inhibition with test
a
Values presented are means ꢂ SD.
compounds 8f–h.
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Fig. 3 Morphological changes in HUVEC cells (A) control, 24 h; (B) treated with 8f (12.5 mg mLꢀ1), 24 h; (C) treated with 8g (12.5 mg mLꢀ1), 24 h;
(D) treated with 8h (12.5 mg mLꢀ1), 24 h.
Fig. 4 Effect of 8f–h on HUVEC cell migration (A) control, 0 h; (B) control, 24 h; (C) treated with 8f (2.5 mg mLꢀ1), 24 h; (D) treated with 8f (10 mg
mLꢀ1), 24 h; (E) treated with 8g (2.5 mg mLꢀ1), 24 h; (F) treated with 8g (10 mg mLꢀ1), 24 h; (G) treated with 8h (2.5 mg mLꢀ1), 24 h; (H) treated with
8h (10 mg mLꢀ1), 24 h.
Table 3 Inhibition of HUVEC cell migration treated with 8f–ha
8h signicantly changed the conguration of HUVEC cells,
while vadimezan 6 at less than 500 mM (i.e. 141 mg mLꢀ1) did not
Compound
Concentration (mg mLꢀ1
)
Migration inhibition (%)
induce any morphological change in HUVEC cells as Kim's
group reported,26 suggesting that these derivatives of vadimezan
might have a different mechanism of action at the molecular
level.
8f
2.5
10
2.5
10
2.5
10
32.58 ꢂ 8.68
58.25 ꢂ 6.47
45.62 ꢂ 9.66
65.85 ꢂ 10.21
67.58 ꢂ 6.58
89.68 ꢂ 11.25
8g
8h
The effect of compounds 8f–h on HUVEC migration was
measured by the wound healing migration assay. As shown in
Fig. 4, there was a signicant reduction in the ability of 8h
(10 mg mLꢀ1) treated cells to migrate into the empty space. The
migration rate (%) is listed in Table 3, showing that the inhib-
a
Values presented are means ꢂ SD.
itory effect of 8h on HUVEC migration was 89.7% at 10 mg mLꢀ1
.
Even at 2.5 mg mLꢀ1, the reduction in migration of 8h treated the VEGFR2 kinase domain revealed a possible hydrogen bond
HUVEC cells was comparable to that of 8g treated HUVEC cells between the central carbonyl group of vadimezan 6 and the
at 10 mg mLꢀ1. These observations indicated that these backbone amide of CYS919 in the inter-lobe linker region of the
compounds, especially 8h, could block in vitro angiogenesis by kinase domain. Knowing that compound 8h exhibited anti-
inhibiting endothelial cell migration.
angiogenic activity, docking study of 8h into the ATP binding
In silico molecular docking. Buchanan et al. found that site of VEGFR2 and EGFR was performed using AutoDock 4.0 to
vadimezan 6 was a multi-kinase inhibitor targeting vascular stimulate a binding model, based on a Lamarckian genetic
endothelial growth factor receptor 2 (VEGFR2) in particular with algorithm (LGA) method.28,29 The VEGFR2 model was derived
an IC50 value at 11 mM, which may contribute to the effect of from the crystal structure of a pyridyl-pyrimidine benzimidazole
vadimezan 6 on the vasculature.27 Further docking studies into bound VEGFR2 obtained from RCSB (PDB : 3EWH).30 The
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Fig. 5 (A) Binding model of 8h with VEGFR2 displaying the interacted amino acid residue; (B) docking model of 8h in the kinase domain of
VEGFR2.
Table 4 In vivo antitumor efficacy of compounds against the S180 xenograft in micea
Group
Dose (mg kgꢀ1
)
ARb
No. of animalsc
BWd (g)
BWLe (%)
TWf (g)
TWIg (%)
Control
6
/
30
Ig
Ig
Ig
Ig
Ig
Ig
Ig
Ig
11
7
7
7
7
7
7
7
31.77 ꢂ 2.02
21.84 ꢂ 4.97**
29.07 ꢂ 3.57
28.49 ꢂ 2.91*
28.53 ꢂ 2.32**
28.37 ꢂ 2.64*
27.84 ꢂ 2.17*
27.68 ꢂ 3.21*
/
2.23 ꢂ 0.41
0.53 ꢂ 0.22**
2.14 ꢂ 0.36
1.66 ꢂ 0.42*
1.82 ꢂ 0.57
1.73 ꢂ 0.37*
1.61 ꢂ 0.48*
1.57 ꢂ 0.53*
/
31.25
8.50
76.23
4.04
7b
8a
8e
8f
8g
8h
200
200
200
200
200
200
10.32
10.20
10.70
12.37
12.87
25.56
18.39
22.42
27.80
29.60
a
Values presented are means ꢂ SD. Statistical analysis: Student's t-test.* p < 0.05 vs. control, ** p < 0.01 vs. control. b Administration route. c Number
d
e
of mice tested in the group. Average body weight aer drug treatment. Percentage of mice body weight loss vs. control group, BWL% ¼ 100 ꢀ
f
g
(mean BWtreated/mean BWcontrol ꢃ 100). Average tumor-weight aer drug treatment. Percentage of tumor-weight inhibition vs. the control
group, TWI% ¼ 100 ꢀ (mean TWtreated/mean TWcontrol ꢃ 100).
docking results as shown in Fig. 5 indicated that compound 8h which demonstrated 76.1% suppression of tumor growth at
penetrated through the Asp–Glu channel and extended to the 30 mg kgꢀ1
backpocket of VEGFR2. In addition to hydrophobic interaction,
compound 8h was also involved in hydrogen bond interaction
.
with GLU885 and the distance of the hydrogen bond was 3.03 A.
˚
Conclusion
Compound 8h displayed a total binding energy value of ꢀ10.4
kcal molꢀ1. However, under this minimum energy conforma- In order to nd vadimezan derivatives with potential anti-
tion docking pose, hydrogen bonding between 8h and CYS919 cancer activity, a series of xanthone analogues modied from
was not observed, and the distance between the central carbonyl vadimezan 6 with carboxyl substitution were synthesized as
group of 8h and the backbone amide of the CYS919 residue was esters, amides, arylidene hydrazides, diacylhydrazides and acyl
˚
calculated to be 7.2 A.
thiosemicarbazides, and their in vitro anticancer activities
The EGFR model derived from the crystal structure of erlo- were screened across a broad range of cancer cell lines.
tinib bound EGFR obtained from RCSB (PDB : 1M17)31 was also Compounds 8f, 8g and 10e were most effective against A549
applied to dock with 8h, which partially occupied the ATP with IC50 at 10.8 mM, 9.4 mM and 11.5 mM respectively, and 8e
binding pocket of EGFR. Further in vitro tyrosine kinase inhi- with IC50 at 4.6 mM against HL-60. Compounds 8f–h showed a
bition assay was undertaken.
signicant inhibitory effect on in vitro HUVEC growth and
In vivo xenogra inhibition. The in vivo antitumor efficacy of migration, among which 8h inhibited HUVEC growth with IC50
xanthones was also evaluated against the S180 xenogra model at 6.4 mM and HUVEC migration by 67.6% and 89.7% at 2.5 mg
in comparison with vadimezan 6 and 0.9% saline solution used mLꢀ1 and 10 mg mLꢀ1, respectively. More interestingly, dock-
as a blank control. The results presented in Table 4 showed that ing study indicated that compound 8h might target the ATP
compounds 8a, 8f, 8g and 8h with an intragastric (ig) admin- binding site of VEGFR2. In addition, compounds 8a, 8f–h
istration route of 200 mg kgꢀ1 of body weight once daily for exhibited moderate in vivo antitumor efficacy against the S180
consecutive six days exhibited moderate experimental thera- xenogra in ICR mice by 22.4–29.6% tumor weight inhibition.
peutic efficacy of 25.6%, 22.4%, 27.8% and 29.6%, respectively, The results extend the previous understanding of this class of
and compound 7b showed no signicant suppression of S180 compounds, conrm the possibility for modication on the
tumor growth, unlike the prodrug rationale. None of the carboxylic acid group, and may help further design xanthone
compounds showed superior potency to vadimezan 6, derivatives.
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Ar-H2), 7.31 (d, J ¼ 8.0 Hz, 1H, Ar-H7), 4.27 (s, 2H, NHNH2), 3.82
(s, 2H, Ar-CH2), 2.450 (s, 3H, Ar-CH3), 2.446 (s, 3H, Ar-CH3); EI-
MS m/z (%): 296 (M+, 53), 265 (72), 237 (100), 209 (10), 194 (6),
178 (6), 165 (17), 152 (4); anal. calcd for C17H16N2O3 : C, 68.91;
H, 5.44; N, 9.45%; found: C, 68.83; H, 5.38; N, 9.61%.
Experimental section
Chemistry
Full details about the instruments and reagents used are given
in the electronic supplementary information (ESI†) along with
characterization data for all compounds. Given below are
protocols for the synthesis of representative compounds.
Methyl 2-(5,6-dimethyl-9-oxo-9H-xanthen-4-yl)acetate (7a).
Vadimezan 6 (141 mg, 0.5 mmol), methanol (32 mg, 1.0 mmol)
and DPAT (32 mg, 0.1 mmol) in toluene (15 mL) were heated to
reux for 2 h. Evaporation of the solvent under reduced pres-
sure gave a crude product, which was puried by recrystalliza-
tion from ethanol to give 7a as a white solid, 146 mg (yield:
N0-(2-Hydroxybenzylidene)-2-(5,6-dimethyl-9-oxo-9H-xanthen-
4-yl)acetohydrazide (10a). A mixture of 2-(5,6-dimethyl-9-oxo-
9H-xanthen-4-yl)acetohydrazide 9 (44 mg, 0.15 mmol) and
2-hydroxybenzaldehyde (20 mg, 0.165 mmol) in ethanol (20 mL)
was reuxed for 6 h. The reaction mixture was cooled and the
precipitated solid was ltered, dried and recrystallized from
ethanol to give compound 10a as a white solid, 59 mg (yield:
99.2%); Mp: 247–249 C; IR nmax (KBr)/cmꢀ1: 3247, 3031, 1688,
ꢁ
1639, 1620, 1490, 1414, 1273, 1226, 752; 1H NMR (DMSO-d6, 400
MHz) d: 12.03 and 11.54 (both s, total 1H, NH), 11.10 and 10.12
(both s, total 1H, OH), 8.47 and 8.37 (both s, total 1H, N]CH),
8.11 and 8.09 (both d, J ¼ 8.8 Hz, total 1H, Ar-H1), 7.93 (d, J ¼ 8.0
Hz, 1H, Ar-H8), 7.84 and 7.83 (both d, J ¼ 6.5 Hz, total 1H, Ar-
H3), 7.69 and 7.54 (both d, J ¼ 7.6 Hz, total 1H, Ar'), 7.45 and
7.43 (both t, J ¼ 7.6 Hz, total 1H, Ar-H2), 7.30–7.23 (m*, 1H + 1H,
Ar-H7 + Ar'), 6.91 and 6.77 (both t, J ¼ 7.5 Hz, total 2H, Ar'), 4.40
and 4.03 (both s, total 2H, Ar-CH2), 2.44, 2.40, 2.39 and 2.33 (all
s, total 6H, 2 ꢃ Ar-CH3) *overlap; EI-MS m/z (%): 400 (M+, 28),
296 (11), 281 (9), 265 (30), 238 (100), 209 (15), 194 (9), 165 (28);
anal. calcd for C24H20N2O4 : C, 71.99; H, 5.03; N, 7.00%; found:
C, 71.86; H, 4.97; N, 7.11%.
98.6%); Mp: 188–189 C; IR nmax (KBr)/cmꢀ1: 2952, 1736, 1651,
ꢁ
1601, 1414, 1333, 1226, 1173, 771; 1H NMR (CDCl3, 500 MHz) d:
8.29 (dd, J1 ¼ 1.5 Hz, J2 ¼ 8.0 Hz, 1H, Ar-H1), 8.09 (d, J ¼ 8.0 Hz,
1H, Ar-H8), 7.64 (dd, J1 ¼ 1.3 Hz, J2 ¼ 7.3 Hz, 1H, Ar-H3), 7.35 (t,
J ¼ 7.5 Hz, 1H, Ar-H2), 7.21 (d, J ¼ 8.0 Hz, 1H, Ar-H7), 4.00 (s,
2H, Ar-CH2), 3.74 (s, 3H, OCH3), 2.46 (s, 3H, Ar-CH3), 2.45 (s, 3H,
Ar-CH3); EI-MS m/z (%): 296 (M+, 61), 238 (18), 237 (100), 236
(45), 209 (26), 165 (20).
Compounds 7b–7q were prepared by using the same proce-
dure for 7a, with the corresponding alcohols.
2-(5,6-Dimethyl-9-oxo-9H-xanthen-4-yl)-N-propylacetamide
(8a). Vadimezan 6 (141 mg, 0.5 mmol), DCC (124 mg, 0.6 mmol),
HOBt (81 mg, 0.6 mmol) and propan-1-amine (36 mg, 0.6 mmol)
were heated to 60 ꢁC in DMF (20 mL) for 24 h, and the mixture
was cooled to room temperature overnight to precipitate DCU.
Aer ltration, the ltrate was poured into ice-water to precip-
itate a white solid. The crude product was collected by ltration
and recrystallized from ethanol to afford compound 8a as a
white solid, 105 mg (yield: 64.9%); Mp: 187–188 ꢁC; PHPLC
96.9%, tR ¼ 3.94 min, (CH3CN : H2O ¼ 8 : 2, Tf ¼ 1.0, l ¼ 240
nm); IR nmax (KBr)/cmꢀ1: 3292, 2962, 1653, 1602, 1413, 1332,
1212, 763; 1H NMR (CDCl3, 500 MHz) d: 8.31 (d, J ¼ 8.0 Hz, 1H,
Ar-H1), 8.10 (d, J ¼ 8.0 Hz, 1H, Ar-H8), 7.69 (d, J ¼ 6.0 Hz, 1H, Ar-
H3), 7.38 (t, J ¼ 7.0 Hz, 1H, Ar-H2), 7.23 (d, J ¼ 8.0 Hz, 1H, Ar-
H7), 5.53 (br, 1H, NH), 3.95 (s, 2H, Ar-CH2), 3.23–3.19 (m, 2H,
NHCH2), 2.47 (s, 6H, 2 ꢃ Ar-CH3), 1.47–1.43 (m, 2H, CH2CH3),
0.80 (t, J ¼ 7.3 Hz, 3H, CH2CH3); EI-MS m/z (%): 323 (M+, 29),
239 (18), 238 (100), 237 (11), 223 (18), 209 (11), 195 (11), 165 (9);
anal. calcd for C20H21NO3 : C, 74.28; H, 6.55; N, 4.33%; found:
C, 74.39; H, 6.61; N, 4.29%.
Compounds 10b–10k were prepared by using the same
procedure for 10a, with the corresponding aldehydes.
(E)-N0-Pent-3-enoyl-N-2-(5,6-dimethyl-9-oxo-9H-xanthen-4-yl)-
acetohydrazide (11a). Hydrazide 9 (44.4 mg, 0.15 mmol), DCC
(37.1 mg, 0.18 mmol), HOBt (24.3 mg, 0.18 mmol) and (E)-pent-3-
ꢁ
enoic acid (15.0 mg, 0.15 mmol) were heated to 50 C in DMF
(15 mL) for 12 h, and the mixture was cooled overnight to
precipitate DCU. Aer ltration, the ltrate was poured into ice-
water to precipitate a white solid. The crude product was
collected by ltration and recrystallized from ethanol to afford
compound 11a as a pale white solid, 55.8 mg (yield: 98.4%); Mp:
187–190 C; IR nmax (KBr)/cmꢀ1: 3327, 2928, 2851, 1627, 1602,
ꢁ
1576, 763; 1H NMR (DMSO-d6, 400 MHz) d: 10.17 (d, J ¼ 1.6 Hz,
1H, NH), 9.86 (d, J ¼ 1.6 Hz, 1H, NH), 8.08 (dd, J1 ¼ 1.2 Hz, J2 ¼
8.0 Hz, 1H, Ar-H1), 7.92 (d, J ¼ 8.4 Hz, 1H, Ar-H8), 7.83 (d, J ¼ 7.0
Hz, 1H, Ar-H3), 7.42 (t, J ¼ 7.6 Hz, 1H, Ar-H2), 7.30 (d, J ¼ 8.4 Hz,
1H, Ar-H7), 5.58–5.43 (m, 2H, CH]CH), 3.92 (s, 2H, Ar-CH2),
2.83 (d, J ¼ 6.0 Hz, 2H, COCH2), 2.46 (s, 3H, Ar-CH3), 2.44 (s, 3H,
Ar-CH3), 1.60 (d, J ¼ 6.0 Hz, 3H, CH3); EI-MS m/z(%): 378 (M+, 65),
323 (11), 296 (49), 265 (100), 237 (100), 209 (20), 194 (11), 165 (31);
EI-HRMS : M+ 378.1587 for C22H22N2O4 (calcd 378.1580).
Compounds 11b–11e were prepared by using the same
procedure for 11a, with the corresponding acids.
Compounds 8b–8p were prepared by using the same proce-
dure for 8a, with the corresponding amines.
2-(5,6-Dimethyl-9-oxo-9H-xanthen-4-yl)acetohydrazide (9). A
mixture of ethyl 2-(5,6-dimethyl-9-oxo-9H-xanthen-4-yl)acetate
7b (155 mg, 0.5 mmol), ethanol (10 mL) and 85% hydrazine
hydrate (2.3 mL) was reuxed for 24 h, and more hydrazine
hydrate was added if necessary to reach completion. The
mixture was cooled and ltered to give compound 9 as a white
solid, 140 mg (yield: 94.6%); Mp: 239–241 ꢁC; IR nmax (KBr)/
cmꢀ1: 3290, 3063, 2915, 1654, 1639, 1618, 1602, 1413, 1332,
1228, 758; 1H NMR (DMSO-d6, 500 MHz) d: 9.34 (s, 1H, NHNH2),
8.08 (dd, J1 ¼ 1.5 Hz, J2 ¼ 8.0 Hz, 1H, Ar-H1), 7.93 (d, J ¼ 8.0 Hz,
1H, Ar-H8), 7.77 (d, J ¼ 6.5 Hz, 1H, Ar-H3), 7.41 (t, J ¼ 7.5 Hz, 1H,
4-Allyl-1-(2-(5,6-dimethyl-9-oxo-9H-xanthen-4-yl)acetyl)thio-
semicarbazide (12a). A mixture of 2-(5,6-dimethyl-9-oxo-9H-
xanthen-4-yl)acetohydrazide 9 (59 mg, 0.2 mmol) and 3-iso-
thiocyanatoprop-1-ene (20 mg, 0.2 mmol) in ethanol (10 mL)
was reuxed for 12 h. The mixture was cooled and the precipi-
tated solid was ltered, dried and recrystallized from ethanol to
give compound 12a as a white solid, 19 mg (yield: 24.1%); Mp:
518 | Med. Chem. Commun., 2014, 5, 512–520
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MedChemComm
167–170 C; IR nmax (KBr)/cmꢀ1: 3250, 3068, 2973, 1698, 1644,
ꢁ
In vitro HUVEC growth inhibition assay. HUVEC cells were
seeded on 96-well plates at 2 ꢃ 104 cells per well and incubated
in a 5% CO2 incubator at 37 ꢁC for 24 h, followed by treatment
with selected compounds 6, 8e–h, and 10e at concentrations of
5 mL, 10 mL, 15 mL, 20 mL, and 25 mL in DMSO solution and
incubation under 5% CO2 at 37 ꢁC for 48 h. Mitochondrial
metabolism was measured as a marker for cell growth by adding
20 mL per well MTT (5 mg mLꢀ1 in medium). Crystals formed
were dissolved in 150 mL of DMSO. The absorbance was deter-
mined using a microplate reader at 570 nm.
In vitro HUVEC migration inhibition assay. HUVEC cells
were seeded on 24-well plates at 2 ꢃ 104 cells per well and
incubated in a 5% CO2 incubator at 37 ꢁC until 80% of the well
bottom was spread by cells. The cell layers were wounded with a
10 mL plastic pipette tip and incubated in the presence of 8f–h.
Cell migration into the wounded area was photographed at
0 and 24 h. The migration inhibitory rate was calculated by
Image-Pro Plus 6.0 soware.
1601, 1542, 1494, 1414, 1336, 1215, 763; 1H NMR (DMSO-d6, 500
MHz) d: 10.11 (brs, 1H, C(O)NHNH), 9.39 (s, 1H, C(O)NHNH),
8.13 (brs, 1H, C(S)NH), 8.10 (dd, J1 ¼ 2.0 Hz, J2 ¼ 8.0 Hz, 1H, Ar-
H1), 7.94 (d, J ¼ 8.0 Hz, 1H, Ar-H8), 7.81 (d, J ¼ 7.0 Hz, 1H, Ar-
H3), 7.43 (t, J ¼ 7.5 Hz, 1H, Ar-H2), 7.32 (d, J ¼ 8.5 Hz, 1H,
Ar-H7), 5.87–5.79 (m, 1H, CH2CH]CH2), 5.14 (dd, J1 ¼ 1.5 Hz, J2
¼ 17.5 Hz, 1H, ¼CHH, cis), 5.06 (dd, J1 ¼ 1.0 Hz, J2 ¼ 10.0 Hz,
1H, ¼CHH, trans), 4.13–4.09 (brs, 2H, CH2CH]CH2), 3.97 (s,
1H, Ar-CHH), 3.96 (s, 1H, Ar-CHH), 2.48 (s, 3H, Ar-CH3), 2.46 (s,
3H, Ar-CH3); EI-MS m/z (%): 384 ([M ꢀ C + H]+, 3), 368 (3), 353
(2), 339 (1), 296 (14), 265 (21), 256 (15), 237 (36); ESI-HRMS: [M ꢀ
H]ꢀ 394.1229 for C21H21N3O3S–H (calcd 394.1225).
Compounds 12b and 12c were prepared by using the same
procedure for 12a, with the corresponding isothiocyanates.
Pharmacology
Cancer cell lines endometrial Ishikawa, lung A549 and NCI-
460, chorion Bewo, cervical HeLa and Siha, breast MCF-7 and
SK-BR-3, leukemia HL-60, liver BEL-7402, stomach BGC-823
and lymphomas Jeko-1 were obtained from Shanghai Insti-
tutes for Biological Science of the Chinese Academy of
Sciences. S180 cells were provided by the Zhejiang Academy of
Medical Science. ICR mice were provided by the Zhejiang
Center of Laboratory Animals, male, 18–22 g, and acclima-
In silico molecular docking. Molecule 8h was taken for
prediction of the 3D structure, and the energy was minimized
for exible docking. Gasteiger charges were added and
nonpolar hydrogens were merged to carbon atoms by Auto-
DockTools (ADT 1.4.5) and converted into the pdbqt format for
the docking run. The 3D structures of VEGFR2 (3EWH) and
EGFR (1M17) were obtained from the Protein Data Bank at the
website: http://www.pdb.org/. Ligands and nonreceptor atoms
such as water and ions were removed, and Kollman charges
were assigned and saved in the pdbqt format as the receptor le.
The grid maps dening the search region in the docking
process were calculated with AutoGrid and had dimensions of
ꢁ
tized to the laboratory environment at 25 C prior to experi-
mentation with food and water ad libitum. All animal
experiments were performed in compliance with the relevant
laws and institutional guidelines, and approved by the
committee on animal research at the Hangzhou Minsheng
Pharmaceutical Group.
Cell cultures. DMEM and RPMI1640 were purchased from
Gibco, and MTT from Sigma. Each human cancer cell line was
maintained in the standard medium and cultivated as a
monolayer in DMEM supplemented with 800 (U vꢀ1) penicillin,
0.1% (w/v) streptomycin and 10% (v/v) fetal bovine serum (FBS).
Cultures were maintained at 37 ꢁC, 5% CO2 in a humidied
atmosphere for 3–5 d. HUVEC was grown on RPMI1640 medium
containing 10% (v/v) FBS.
In vitro cancer cell growth inhibition assay. Cancer cells,
treated with trypsin–EDTA solution, were seeded into 96-well
plates at 106 cells per well, incubated in a 5% CO2 incubator at
37 ꢁC for 24 h, and treated with compounds synthesized at
different concentrations in DMSO solution. Mitochondrial
metabolism was measured as a marker for cell growth by adding
10 mL per well MTT (5 mg mLꢀ1 in medium) with 48 h incu-
bation at 37 ꢁC. Crystals formed were dissolved in 150 mL DMSO.
The absorbance was determined using a microplate reader at
570 nm. The absorbance data were converted into a cell inhi-
bition percentage, and compared with DMSO treated cells
˚
40 ꢃ 140 ꢃ 140 A centered by the predened active site of the
protein. The LGA parameters were set as default values with 50
GA runs. The best affinity modes were screened by binding
energy scores and analyzed by PyMol program.
In vivo xenogra inhibition assay. To initiate tumors, S180
cells (2 ꢃ 106 cells per animal) were injected subcutaneously into
the right anks of ICR male mice. Sixty mice with near average-
sized tumors were randomly assigned to seven experimental
groups treated with vadimezan 6 (30 mg kgꢀ1 of body weight), 7b
(200 mg kgꢀ1), 8a (200 mg kgꢀ1), 8e (200 mg kgꢀ1), 8f (200 mg
kgꢀ1), 8g (200 mg kgꢀ1) and 8h (200 mg kgꢀ1), and a control
group with eleven mice treated with a vehicle of 0.9% saline
solution (0.1 mL/10 g of body weight) once daily through the ig
administration route from the second day aer establishing the
mouse model for consecutive six days. The tumor bearing
animals were observed everyday and euthanized aer experiment
and the solid tumors were weighed for calculating the tumor
weight inhibition of compounds in the S180 xenogra model.
Acknowledgements
(control culture). The percentage of inhibition was calculated by The authors thank the Center of Analysis & Measurement of
the following equation: percentage inhibition ¼ (1 ꢀ At/Ac) ꢃ Zhejiang University and College of Chemistry and Life Sciences,
100, where At and Ac represent the absorbance in treated and Zhejiang Normal University for characterization of the
control cultures, respectively. The drug concentration causing compounds and partial nancial support. S.-J. Zhang is grateful
50% cell inhibition (IC50) was determined by interpolation from to the research fund from the Zhejiang Chinese Medical
dose–response curves.
University for nancial support (2011ZY26).
This journal is © The Royal Society of Chemistry 2014
Med. Chem. Commun., 2014, 5, 512–520 | 519

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Concise Article
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520 | Med. Chem. Commun., 2014, 5, 512–520
This journal is © The Royal Society of Chemistry 2014