Synthesis, structure-activity relationships and biological evaluation of barbigerone analogues as anti-proliferative and anti-angiogenesis agents

Article abstract of DOI:10.1016/j.bmcl.2014.04.121

A series of barbigerone analogues (7a-7w, 13a-13x) were designed, synthesized and biologically evaluated for their anti-proliferative and anti-angiogenic activities. Among these compounds, compound 13a exhibited the most potent inhibitory effect on the proliferation of HUVECs, HepG2, A375, U251, B16, and HCT116 cells (IC₅₀ = 3.80, 0.28, 1.58, 3.50, 1.09 and 0.68 μM, respectively). Compound 13a inhibited the angiogenesis in zebrafish embryo assay in a concentration-dependent manner. Furthermore, 13a also effectively inhibited the migration and capillary like tube formation of human umbilical vein endothelial cell in vitro. These results support the further investigation of this class of compounds as potential anti-proliferative and anti-angiogenesis agents.

Full text of DOI:10.1016/j.bmcl.2014.04.121

Accepted Manuscript
Synthesis, structure-activity relationships and biological evaluation of barbi-
gerone analogues as anti-proliferative and anti-angiogenesis agents
Guangcheng Wang, Fang Wang, Dong Cao, Yibin Liu, Ronghong Zhang,
Haoyu Ye, Xiuxia Li, Lin He, Zhuang Yang, Liang Ma, Aihua Peng, Mingli
Xiang, Yuquan Wei, Lijuan Chen
PII:
S0960-894X(14)00508-3
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.04.121
BMCL 21634
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
27 December 2013
15 April 2014
29 April 2014
Please cite this article as: Wang, G., Wang, F., Cao, D., Liu, Y., Zhang, R., Ye, H., Li, X., He, L., Yang, Z., Ma, L.,
Peng, A., Xiang, M., Wei, Y., Chen, L., Synthesis, structure-activity relationships and biological evaluation of
barbigerone analogues as anti-proliferative and anti-angiogenesis agents, Bioorganic & Medicinal Chemistry
Letters (2014), doi: http://dx.doi.org/10.1016/j.bmcl.2014.04.121
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Synthesis, structure-activity relationships
and biological evaluation of barbigerone
analogues as anti-proliferative and
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anti-angiogenesis agents
Guangcheng Wanga,b,1, Fang Wanga,1, Dong Caoa, Yibin Liua, Ronghong Zhanga,
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Haoyu Yea, Xiuxia Lia, Lin Hea, Zhuang Yanga,c, Liang Maa, Aihua Penga, Mingli
Xianga, Yuquan Weia, and Lijuan Chena,*
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State Key Laboratory of Biotherapy, West China Hospital, West China Medical
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School, Sichuan University, Keyuan Road 4, Gaopeng Street, Chengdu 610041, China
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b College of Chemistry and Chemical Engineering, Jishou University, Jishou 416000,
China
c College of Chemistry of Sichuan University, Chengdu, Sichuan 610064, China
1These authors contributed equally to this work.
*Corresponding Author
Lijuan Chen
Tel.: +86 28 85164063.
Fax: +86 28 85164060.
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E-mail address: chenlijuan125@163.com (L-J. Chen).
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Abstract
A series of barbigerone analogues (7a-7w, 13a-13x) were designed, synthesized and
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biologically evaluated for their anti-proliferative and anti-angiogenic activities.
Among these compounds, compound 13a exhibited the most potent inhibitory effect
on the proliferation of HUVECs, HepG2, A375, U251, B16, and HCT116 cells (IC50
= 3.80, 0.28, 1.58, 3.50, 1.09 and 0.68 µM, respectively). Compound 13a inhibited the
angiogenesis in zebrafish embryo assay in a concentration-dependent manner.
Furthermore, 13a also effectively inhibited the migration and capillary like tube
formation of human umbilical vein endothelial cell in vitro. These results support the
further investigation of this class of compounds as potential anti-proliferative and
anti-angiogenesis agents.
Keywords: barbigerone; isoflavone; anti-proliferative; anti-angiogenesis
Angiogenesis, which is the growth of new blood vessels from the pre-existing
vasculature of a host, is a critical process for the growth and metastasis of most
cancerous tumors,1 and its inhibition is now a well-established therapeutic strategy for
cancer patients.2-4 Angiogenesis is a very complex process and involves a number of
distinct steps, such as endothelial cell activation, migration, proliferation, formation of
capillary tubes of endothelial cells, their invasion, and metastasis.5,6 In tumors,
angiogenesis causes the growth of new blood vessels that supply necessary oxygen
and nutrient for the growth of tumor tissue.7 It has been shown that, without
angiogenesis, a solid tumor cannot deteriorate beyond a critical size and metastasize
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to other organs.8 Therefore, inhibition of angiogenesis is a promising approach to the
development of anticancer therapy.
Barbigerone (Figure 1) is one of the naturally occurring pyranoisoflavones, which
was first isolated from the seeds of a leguminous plant Tephrosia barbigera.9 It has
been reported to have a wide range of biological activities such as antioxidant,10
antiplasmodial11 and anti-cancer activity.12,13 Our previous study has shown that
barbigerone exhibited antitumor activity by inducing apoptosis and inhibiting
angiogenesis in murine or human cancer cells.12,13 Recently, Yang and coworkers14
have reported the total synthesis of barbigerone and its four analogues (7a, 7b, 7t and
7u in this study), but they didn’t study the biological activity of these compounds. To
the best of our knowledge, there has been no study focusing on structural modification
of barbigerone associated with potential anti-cancer and anti-angiogenic activities.
During our continued efforts to screen natural and synthetic compounds for
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anti-tumor effects,
a novel series of barbigerone analogues have been designed,
synthesized and evaluated for their anti-proliferative and anti-angiogenic activities.
Please insert Figure 1 here.
In this paper, a series of barbigerone analogues (7a-7w, 13a-13x) were designed,
synthesized and biologically evaluated for their anti-proliferative and anti-angiogenic
activities. Among these compounds, compound 13a exhibited the most potent
inhibitory effect on the proliferation of HUVECs, HepG2, A375, U251, B16, and
HCT116 cells (IC50 = 3.80, 0.28, 1.58, 3.50, 1.09 and 0.68 µM, respectively).
Compound 13a inhibited the angiogenesis in zebrafish embryo assay in a
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concentration-dependent manner. Furthermore, 13a also effectively inhibited the
migration and capillary like tube formation of human umbilical vein endothelial cell
in vitro.
Barbigerone analogues 7a – 7w were prepared by the synthesis route outlined in
Scheme 1. The 4-hydroxyl of compound 1 was protected with 3,4-dihydro-2H-Pyran
in dichloromethane in the presence of pyridinium p-toluenesulfonate (PPTS) to give
the THP ether 2, which condensation with N,N-dimethylformamide dimethylacetal
(DMF-DMA) provided the enamine 3, The crude 3 was directly treated with I2 in the
presence of pyridine at room temperature for 24 h, followed by neutralizing with
saturated Na2S2O3 and extract with CHCl3, compound 4 was obtained.19,20 The
obtained compound 4 was refluxed in CH3OH-THF (1:1) in the presence of
p-toluenesulfonic acid for 1 h to give compound 5. Following a literature method,21-23
a condensation reaction between 5 and 1,1-diethoxy-3-methyl-2-butene in p-xylene in
the presence of picoline as base brought about the desired ring closure in position 8 of
5 to give the key intermediate 6, which coupling with commercial available
substituted phenylboronic acids in a Suzuki-Miyaura reaction19,20,24 led to the target
products 7a – 7w.
Please insert Scheme 1 here.
Compounds 13a – 13x were synthesized by the method shown in Scheme 2.
Having completed the synthesis of compound 4 in Scheme 1, we focus on the
synthesis of (2,4,5-trimethoxyphenyl)boronic acid 10. Trimethoxybenzene 8 was
brominated in 92.3 % yield to the bromide 9, which reacted with n-BuLi and trimethyl
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borate to provide the intermediate (2,4,5-trimethoxyphenyl)boronic acid 10.
Compound 11 was obtained by a Suzuki-Miyaura coupling reaction between of
O-THP-protected iodochromanone 4 and (2,4,5-trimethoxyphenyl)boronic acid 10.
Deprotection of the THP group was performed by treatment with p-toluenesulfonic
acid, leading to the key intermediate 12, followed by an esterification or etherification
of the liberated phenol with alkyl halide, substituted benzyl halide and substituted aryl
acid to afford the final desired products 13a – 13x.
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Please insert Scheme 2 here.
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These barbigerone analogues (7a – 7w and 13a – 13x) were evaluated for their
anti-proliferative activity in human umbilical vein endothelial cells (HUVECs),
HepG2 (hepatocellular carcinoma), A375 (melanoma), U251 (glioma), B16
(melanoma), and HCT116 (colorectal carcinoma) using MTT method. HUVECs were
used to evaluate their in vitro inhibitory effects on the proliferation of endothelial
cells that are closely related to angiogenesis. The compounds that exhibited IC50 >
10.0 µM were considered to be inactive on the respective cancer cell lines. The results
were summarized in Table 1. Among these compounds, compound 13a displayed the
most potent anti-proliferative activity than barbigerone against HUVECs, HepG2,
A375, U251, B16, and HCT116 cells.
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Please insert Table 1 here.
To study the structure – activity relationships (SAR) of barbigerone, the substitutes
of the B-ring were discussed firstly. Based on the anti-proliferative activity of 7a – 7w,
we found that the substituents of the B-ring greatly affected on anti-proliferative
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activity of the compounds in this series. The introduction of electro–withdraw groups
(CN, CF3, Cl, F, CHO, COCH3) to the B-ring has proven to be detrimental to
antitumor activity. It’s interesting to point out that 7o and 7p containing CF3 group at
para-position of the B-ring slightly decrease the anti-proliferative activity. The
replacement of the 2,4,5-trimethoxy group with various methoxyl, ethoxyl and
hydroxyl groups (3-methoxy in 7a, 3,4-dimethoxy in 7b, 4-methoxy in 7c,
2,4-dimethoxy in 7d, 2,3,4-trimethoxy in 7f, 2-F-3-methoxy in 7g, 4-hydroxyl in 7i,
3,5-dimethoxy in 7r, 2-ethoxy in 7s, 2,5-dimethoxy in 7t, 3,4,5-trimethoxy in 7u,
2-methoxy in 7v, 4-ethoxy in 7w) resulted in a remarkable decrease the
anti-proliferative activity. These results indicated the pattern of substitution in the
B-ring is closely related to the biological activity of this class of compounds. Thus,
the 2,4,5-trimethoxy group seems to be the optimal substituent on the B-ring.
Since 2,4,5-trimethoxy group is proved the most potent group in the B-ring, it was
retained during the structure-activity relationship studies focused at the C-5 position
of the A-ring. Introduction ester groups (13f and 13g) at the C-5 position, results in a
significant decrease the anti-proliferative activity. The replacement of benzopyran
ring with alkoxyl groups at the C-5 position (13a，13b，13c，13e) resulting in
increased the anti-proliferative activity, whereas 2-morpholinoethoxy group (13n)
decrease the anti-proliferative activity. The replacement of benzopyran ring with
various substituted benzoyloxyl groups (13h-13x) resulted in a maintained or
improved the anti-proliferative activity, whereas the substitution of benzyloxyl group
(13d) remarkable decreased the anti-proliferative activity. In summary, the
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information of SAR provided us a guideline to improve the inhibitory activity in the
future structural modification.
The anti-angiogenic activity of the most potent compound 13a was tested using
zebrafish embryos which represent an excellent animal model for the study of
angiogenesis.25 Zebrafish embryos were treated with barbigerone (1.25 or 2.5 µM),
13a (1.25 or 2.5 µM) or vehicle for 24 h. As shown in Figure 2, the control group had
normal vessel development, in which the subintestinal vessel (SIV) formed as a
smooth basket-like structure. In the group treated with 13a and barbigerone, the
formation of SIV was considerably inhibited compared with that of the vehicle control
group, indicating a dose-dependent inhibition pattern. Therefore, the anti-angiogenic
activity of 13a was further studied.
Please insert Figure 2 here.
Endothelial cell migration is an essential step in angiogenesis. Inhibition on this
process will block the formation of new blood vessels.26, 27 Therefore, compound 13a
was tested for possible inhibition of endothelial cell migration in a wound-healing
migration assay. As illustrated in Figure 3, the HUVECs actively migrated into the
wound area (between the two white lines) under the compound-free condition
(vehicle). At a concentration of 0.5 µM, the HUVECs migratory rates of 13a and
barbigerone were 35.36±4.13 % and 74.26±4.72 %, respectively. While the tested
compounds’ concentration reached 1.0 µM, the HUVECs migratory rates of 13a and
barbigerone were 8.45±2.91 % and 1.65±8.69 %, respectively. The results showed
that compound 13a and barbigerone exerted potent inhibitory effect on the migration
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of HUVECs in a dose-dependent manner. Compound 13a statistically exerted the
higher potent inhibitory effect on the migration of HUVECs, reaching a 4.9-fold
improvement over barbigerone at a concentration of 1.0 µM.
Please insert Figure 3 here.
In the later stages of angiogenesis, tube formation of endothelial cell is also an
important process.26 Inhibition on the formation of capillary-like tube networks will
terminate the development of new blood vessels. To further characterize the
anti-angiogenesis activity of 13a, we investigated the inhibitory effect of tube
formation by plating HUVECs on matrigel substratum. As shown in Figure 4A, in the
vehicle group, HUVECs showed high mobility on matrigel and formation of tube-like
structures was observed in 8 h. In comparison with the vehicle group, treatment of
HUVECs with 13a or barbigerone at the concentration of 1.0 µM could induce 78.21
±4.86 % and 23.74±7.6 % inhibition of tube-like structure formation respectively.
Moreover, the inhibitory rates of tube formation treated with 13a and barbigerone at a
concentration of 5.0µM were 88.33±3.5 % and 65.37±1.78 % respectively (Figure
4B). The results demonstrated that compound 13a and barbigerone could effectively
inhibit tube formation of HUVECs in a dose-dependent manner. Our observation
indicated that 13a approximately achieved a 3.3-fold improvement in the inhibition of
tube formation compared to that of barbigerone at the same concentration of 1.0 µM.
Please insert Figure 4 here.
Angiogenesis is a highly regulated process that involves a complex cascade of
events, and its inhibition is now a well-established therapeutic strategy for cancer
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patients. Herein, a series of barbigerone analogues were synthesized and their
anti-angiogenesis and anti-proliferative activities were tested. Among these
compounds, compound 13a exhibited the most potent inhibitory effect on the
proliferation of HUVECs, HepG2, A375, U251, B16, and HCT116 cells (IC50 = 3.80,
0.28, 1.58, 3.50, 1.09 and 0.68 µM, respectively). Compound 13a inhibited the
angiogenesis in zebrafish embryo assay in a concentration-dependent manner.
Furthermore, 13a also effectively inhibited the migration and capillary like tube
formation of human umbilical vein endothelial cell in vitro. In conclusion, the
preliminary in vitro anti-angiogenic activities of these compounds possess potential
for design of better future molecules targeting tumor angiogenesis. In the future
research we will be exploring for a clear structure-activity relationship of this type of
compound and studying on their mechanism of anti-angiogenesis activity.
Acknowledgements
The authors greatly appreciate the financial support from National Key Programs of
China during the 12th Five-Year Plan Period (2012ZX09103101-009) and National
Natural Science Foundation of China (81373283).
Supplementary data
Supplementary data associated with this article can be found, in the online version,
at doi: .
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Table Captions
Table 1. The anti-proliferative activities of tested compounds against HUVECs and
five cancer cell lines.
Scheme Captions
Scheme 1. Reagents and conditions: (a) DHP, PPTS, CH2Cl2, r.t., 4 h; (b) DMF-DMA,
95 °C, 3 h; (c) I2, pyridine, CHCl3, r.t., 12 h (91.7% for three steps); (d) pTsOH,
CH3OH, THF, 60 °C, 1 h (94.9%); (e) 1,1-diethoxy-3-methyl-2-butene, 3-picoline,
xylene, reflux, 24 h (48.4%); (f) ArB(OH)2, 10 % Pd/C, Na2CO3, H2O, DME, 45 °C, 1
h (49.6%–89.7%).
Scheme 2. Reagents and conditions: (a) Br2, CH2Cl2, 0 °C (92.3%); (b) n-BuLi,
trimethyl borate, THF, -78 °C (37.7%); (c) 10 % Pd/C, Na2CO3, H2O, DME, 45 °C, 1
h (61.4%); (d) pTsOH, CH3OH, THF, 60 °C, 1 h (87.1%); (e) RX, K2CO3, Acetone,
r.t., overnight, or RCOOH, DCC, DMAP, CH2Cl2, r.t., overnight (18.7%-99.4%).
Figure Captions
Figure 1. Chemical structure of barbigerone.
Figure 2. Effects of 13a and barbigerone on the formation of subintestinal vessel
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(SIV) in zebrafish embryos assay. Zebrafish embryos were incubated with 13a or
barbigerone at 1.25 or 2.5 µM for 24 h.
Figure 3. Effects on the HUVECs migration. (A) HUVECs were wounded with
pipette and treated with vehicle, indicated concentrations of compound 13a or
barbigerone. At 0 h or 24 h, photographs were taken by an OLYMPUS digital camera
(magnification 50×). (B) Rates of migration impacted by compound 13a and
barbigerone on the HUVECs. Data represented the mean±standard deviation (SD)
from three independent experiments. **P <0.01; ***P <0.005.
Figure 4. Effects on the HUVECs tube formation. (A) HUVECs (1 × 104 cells)
suspended in EBM-2 containing vehicle, compound 13a (1.0 or 5.0 µM) or
barbigerone (1.0 or 5.0 µM) were added to the Matrigel. After incubation for 8 h at 37
oC, capillary networks were photographed and quantified (magnification: 100×). (B)
Rates of tube formation impacted by compound 13a or barbigerone on the HUVECs.
The number of intact tubes was counted in five randomly chosen regions and
expressed as the percentage of that of the vehicle group. The results were expressed as
mean±SD. **P <0.01; ***P <0.005.
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Table 1. The antiproliferative activities of test compounds against HUVECs and five
cancer cell lines.
IC50 (µM)
Compds
HepG2
1.77
A375
1.85
U251
4.10
B16
HCT116 HUVEC
1.23
2.36
7.45
barbigerone
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
8.90
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
2.50
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
3.55
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
3.80
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
7q
7.25
5.85
1.91
7.00
>10.0
>10.0
20
>10.0
>10.0

>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
0.28
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
1.58
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
3.50
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
1.09
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
0.68
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
3.80
7r
7s
7t
7u
7v
7w
13a
13b
13c
13d
13e
13f
13g
13h
13i
13j
13k
13l
13m
13n
13o
13p
3.45
4.75
5.90
5.10
6.60
>10.0
>10.0
9.75
4.95
3.68
6.45
4.50
8.05
1.32
1.64
6.75
2.33
2.28
2.04
2.64
9.40
5.20
9.17
>10.0
>10.0
>10.0
4.30
>10.0
>10.0
1.28
>10.0
>10.0
2.10
>10.0
>10.0
2.89
>10.0
>10.0
1.61
>10.0
>10.0
2.90
1.00
1.72
3.20
1.12
0.95
>10.0
>10.0
4.83
1.05
1.00
>10.0
2.00
1.18
1.98
1.86
1.36
2.15
2.90
2.50
>10.0
>10.0
>10.0
1.55
9.40
>10.0
8.55
>10.0
4.76
>10.0
>10.0
>10.0
10.0
6.55
>10.0
>10.0
6.18
>10.0
4.23
>10.0
7.00
>10.0
4.22
>10.0
0.99
0.98
2.14
>10.0
>10.0
21

1.23
0.94
0.75
1.69
0.87
2.18
0.89
0.61
1.30
1.02
0.98
2.02
1.12
2.85
2.28
1.14
3.51
0.47
2.44
2.40
2.28
4.18
4.40
8.90
5.40
>10.0
4.92
4.45
1.33
2.45
1.20
4.55
2.44
3.40
5.30
4.40
13q
13r
13s
13t
>10.0
>10.0
>10.0
>10.0
3.75
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
13u
13v
13w
13x
>10.0
294
295
22

296
23