BRN-103, a novel nicotinamide derivative, inhibits VEGF-induced angiogenesis and proliferation in human umbilical vein endothelial cells

Article abstract of DOI:10.1016/j.bmcl.2011.09.022

Anti-angiogenesis is regarded as an effective strategy for cancer treatment, and vascular endothelial growth factor (VEGF) plays a key role in the regulations of angiogenesis and vasculogenesis. In the present study, the authors synthesized five novel nicotinamide derivatives which structurally mimic the receptor tyrosine kinase inhibitor sunitinib and evaluated their anti-angiogenic effects. Transwell migration assays revealed that 2-(1-benzylpiperidin-4-yl) amino-N-(3-chlorophenyl) nicotinamide (BRN-103), among the five derivatives most potently inhibited VEGF-induced human umbilical vein endothelial cells (HUVECs). In addition, BRN-103 dose-dependently inhibited VEGF-induced migration, proliferation, and capillary-like tube formation of HUVECs and vessel sprouting from mouse aortic rings. To understand the molecular mechanisms responsible for these activities, the authors examined the effect of BRN-103 on VEGF signaling pathways in HUVECs. BRN-103 was found to suppress the VEGF-induced phosphorylation of VEGF receptor 2 (VEGR2) and the activations of AKT and eNOS. Taken together, these results suggest that BRN-103 inhibits VEGF-mediated angiogenesis signaling in human endothelial cells.

Full text of DOI:10.1016/j.bmcl.2011.09.022

Bioorganic & Medicinal Chemistry Letters 21 (2011) 6236–6241
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
BRN-103, a novel nicotinamide derivative, inhibits VEGF-induced
angiogenesis and proliferation in human umbilical vein endothelial cells
Hye-Eun Choi a,b, Min-Sang Yoo a,d, Jung-Hye Choi b,c, Jae Yeol Lee e, Je Hak Kim f, Ji Han Kim f,
Joon Kwang Lee f, Gyu Il Kim f, Yong Park f, Yong Ha Chi f, Soo Heui Paik f, Joo Han Lee f,
Kyung-Tae Lee a,b,d,
⇑
a Department of Pharmaceutical Biochemistry, College of Pharmacy, Kyung Hee University, 1 Hoegi-dong, Dongdaemun-gu, Seoul 130-701, Republic of Korea
b Department of Life and Nanopharmaceutical Science, College of Pharmacy, Kyung Hee University, 1 Hoegi-dong, Dongdaemun-gu, Seoul 130-701, Republic of Korea
c Department of Oriental Pharmaceutical Science, College of Pharmacy, Kyung Hee University, 1 Hoegi-dong, Dongdaemun-gu, Seoul 130-701, Republic of Korea
d Department of Biommedical Science, College of Medical Science, Kyung Hee University, 1 Hoegi-dong, Dongdaemun-gu, Seoul 130-701, Republic of Korea
e Research Institute for Basic Sciences and Department of Chemistry, College of Sciences, Kyung Hee University, Seoul 130-701, Republic of Korea
f Central Research Institute, Boryung Pharm. Co. Ltd, 1122-3, Shingil-Dong, Danwon-Gu, Ansan-Si, Kyungki-Do 425-839, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Anti-angiogenesis is regarded as an effective strategy for cancer treatment, and vascular endothelial
growth factor (VEGF) plays a key role in the regulations of angiogenesis and vasculogenesis. In the pres-
ent study, the authors synthesized five novel nicotinamide derivatives which structurally mimic the
receptor tyrosine kinase inhibitor sunitinib and evaluated their anti-angiogenic effects. Transwell migra-
tion assays revealed that 2-(1-benzylpiperidin-4-yl) amino-N-(3-chlorophenyl) nicotinamide (BRN-103),
among the five derivatives most potently inhibited VEGF-induced human umbilical vein endothelial cells
(HUVECs). In addition, BRN-103 dose-dependently inhibited VEGF-induced migration, proliferation, and
capillary-like tube formation of HUVECs and vessel sprouting from mouse aortic rings. To understand the
molecular mechanisms responsible for these activities, the authors examined the effect of BRN-103 on
VEGF signaling pathways in HUVECs. BRN-103 was found to suppress the VEGF-induced phosphorylation
of VEGF receptor 2 (VEGR2) and the activations of AKT and eNOS. Taken together, these results suggest
that BRN-103 inhibits VEGF-mediated angiogenesis signaling in human endothelial cells.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 26 April 2011
Revised 26 August 2011
Accepted 7 September 2011
Available online 13 September 2011
Keywords:
Anti-angiogenesis
Nicotinamide
VEGF
HUVECs
VEGR2
Angiogenesis is a crucial regulator of tumor growth and metas-
tases,1 and one of the major clinical advances in cancer treatment
made recently involves the use of anti-angiogenic drugs like suni-
tinib. Sunitinib is a multi-targeting inhibitor of receptor tyrosine
kinases (RTKs) and has been shown to be effective clinical for the
treatment of various human cancers.2,3 Recently, the US Food and
Drug Administration (FDA) approved sunitinib for the treatments
of clear cell renal cell carcinoma (ccRCC) and gastrointestinal stro-
mal tumors,4,5 and it is also being investigated intensely for the
treatment for several other cancers, including breast cancer, colo-
rectal cancer, and nonsmall cell lung cancer.6,7 Therefore, the sup-
pression of angiogenesis offers a promising approach to the
treatment of various types of solid tumors. During our continued
efforts to screen natural and synthetic compounds for anti-tumor
effects, we examined the anti-angiogenic effects of five novel syn-
thetic nicotinamide derivatives (BRN-103,8 218, 228, 229, and 279;
Fig. 1A), which mimicked sunitinib and were prepared using
synthetic methods as shown in Scheme 1 for the discovery of novel
and potent anti-angiogenic compounds.
Tumor angiogenesis is regulated by the productions of angio-
genic stimulators, such as, vascular endothelial growth factor
(VEGF), which is a key regulatory factor in the prognostic indicator
in several cancers. For this reason, the inhibition of the VEGF sig-
naling pathway has been studied as a potential therapeutic strat-
egy. VEGF receptors (VEGFRs) initiated pathways control various
steps in physiological angiogenesis,9,10 and it has been demon-
strated that VEGFR2 is the primary receptor that mediates the
angiogenic activity of VEGF via distinct signal transduction path-
ways that regulate endothelial cell proliferation, migration, differ-
entiation, and tube formation.2 Binding of VEGF to its major
receptor VEGFR2 activates receptor tyrosine kinase (RTK), which
is responsible for playing critical roles in angiogenesis. Further-
more, Tyr-1175 is the major autophosphorylation site on VEGFR22,
and phosphorylation at this site is required for the activation of
AKT, which is critically required for the subsequent stimulation
of endothelial cell migration and proliferation.11
Angiogenesis is the process of forming blood vessels from pre-
⇑
Corresponding author. Tel.: +82 2 9610860; fax: +82 2 9620860.
E-mail address: ktlee@khu.ac.kr (K.-T. Lee).
existing vessels.12 In response to VEGF (an angiogenic factor),
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.09.022

H.-E. Choi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6236–6241
6237
Figure 1. BRN compounds and the inhibition of VEGF-induced migration of HUVECs. (A) The chemical structures of the five BRN compounds examined; (B) Migration and the
Transwell assay: HUVECs were cultured in a Boyden chamber with VEGF (10 ng/ml) in the lower chamber and various BRN compounds at concentrations less than their IC90
concentrations in the upper chamber. After 24 h, cellular migrations were determined by counting cells that had migrated through the pores. The bar graphs show the
quantitative results of the average numbers of HUVECs that migrating through the membrane. Data are presented as means SD of the results of three independent
experiments. #p <0.05 versus nontreated control group, ⁄p <0.05, ⁄⁄p <0.01, ⁄⁄⁄p <0.001 versus VEGF-stimulated group; statistical significances were determined using ANOVA
and Dunnett’s post-hoc test.
Scheme 1. Reagents and conditions: (a) (i). SOCl2, DMC, reflux, 2 h; (ii). Et3N, DMC, 3-chloroaniline, reflux, 4 h, 81% (two-step yield); (b) 1-Bocpiperazine, K2CO3, xylene,
130 °C, 24 h, 89%; (c) CF3COOH, EtOH, reflux, 3 h, 95%; (d) R2-benzoyl chloride, Et3N, DMC, rt, 4–6 h, 75–83%; (e) R1-NH2, K2CO3, xylene, 130 °C, 24 h, 67–92%.
endothelial cells migrate into the surrounding extracellular matrix
where they form blood capillaries, and thus, cell migration is a
crucial step in angiogenesis and invasion. In this study, five novel
nicotinamide derivatives were preliminary screened using a Trans-
well migration assay, the most popular in vitro test of angiogene-
sis13 Cells were seeded onto the upper surface of an 8
lm pore

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H.-E. Choi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6236–6241
Figure 2. BRN-103 inhibited VEGF-induced migration and proliferation of HUVECs. (A) Representative photomicrographs of scratch wound healing assays. Using near
confluent HUVECs in a 60 mm culture dish, scratched, and incubated in 0.1% EBM medium with 10 ng/mL VEGF for 24 h in the presence or absence of various concentrations
(0.1, 0.5, 1 lM) of BRN-103 for 1 h. Representative photomicrographs of cells treated with VEGF and cells treated with VEGF together with BRN-103. Dotted lines, area
occupied by the initial scraping. Columns show mean values of three different experiments and bars represent SD. #p <0.05 versus nontreated control group, ⁄⁄⁄p <0.001
versus VEGF-stimulated group; statistical significances were compared using ANOVA and Dunnett’s post-hoc test; (B) Migration in the Transwell assay: Different
concentrations (0.1, 0.5, 1 lM) of BRN-103 were treated and tested as described in Figure 1B. Columns represent the means of three different experiments, and bars represent
SD. #p <0.05 versus nontreated control group, ⁄⁄⁄p <0.001 versus VEGF-stimulated group; statistical significances were compared using ANOVA and Dunnett’s post-hoc test;
(C) VEGF-induced cell proliferation was quantified using BrdU incorporation assays to quantify DNA synthesis in 0.1% serum-starved HUVECs in the presence or absence of
BRN-103. Columns represent the means of three different experiments, and bars represent SD. #p <0.05 versus nontreated control group, ⁄⁄⁄p <0.001 versus VEGF-stimulated
group; statistical significances were compared using ANOVA and Dunnett’s post-hoc test.
size membrane separating upper and lower chambers. The upper
chamber contained BRN-103, BRN-218, BRN-228, BRN-229, BRN-
279, or sunitinib (as a positive control) in 0.1% endothelial basal
medium (EBM), and cellular migration through the membrane
was induced when VEGF was present in the lower chamber. As
shown in Figure 1B, treatment of HUVECs with BRN compounds
at concentrations less than their IC90 values (Supplementary
Table 1) prevented cell migration. BRN-103 was found to have

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6239
Figure 3. BRN-103 inhibited VEGF-induced capillary structure formation and microvessel sprouting ex vivo. (A) After being incubated with BRN-103, HUVECs were fixed, and
tubular structures were photographed (magnification, Â100). Tube-like structures were quantified by manual counting in low power fields. Columns represent the means of
three different experiments, and bars represent SD; (B) Aortic segments isolated fromC57BL/6 mice were placed in Matrigel-covered wells and treated with VEGF in the
presence or absence of BRN-103. The data shown are representative of three independent experiments, and the photographs of microvessel sprouts from the margins of aortic
rings are representative of three separate experiments.
the greatest inhibitory effect on this migration. To further assess
the anti-migration activity of BRN-103, wound-healing and Trans-
well migration assays13,14 were performed at BRN-103 concentra-
tions of 0.1, 0.5, and 1 lM. As shown in Figure 2A and B, BRN-
103 significantly and dose-dependently inhibited VEGF-induced
HUVECs migration in both assays. Since angiogenesis requires the

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proliferation of endothelial cells, we tested whether incubation
with BRN-103 inhibits VEGF-induced HUVECs proliferation using
the BrdU incorporation assay,15 and it was found that BRN-103 sig-
nificantly and concentration-dependently inhibited this prolifera-
tion by inhibiting DNA synthesis (Fig. 2C). Sunitinib was also
found to significantly inhibit VEGF-induced cell migration and pro-
liferation, but this was less than that exhibited by BRN-103.
Although several types of cells participate in angiogenesis, tube
formation by endothelial cells is a key step.16 Therefore, we
investigated how BRN-103 regulates capillary tube formation by
HUVECs.17 When HUVECs were seeded on growth factor-reduced
two-dimensional Matrigel, robust tubular structures were formed
in the presence of VEGF. However, preincubation with BRN-103
markedly and dose-dependently abolished this tube formation
(Fig. 3A). To determine whether BRN-103 influences VEGF-induced
angiogenesis ex vivo, the sprouting of vessels from mouse aortic
rings18 were examined in the presence or absence of BRN-103. It
was found that VEGF significantly stimulated microvessel sprout-
ing, which led to the formation of a network of vessels around
the aortic rings (Fig. 3B), and that the addition of BRN-103 dose-
dependently antagonized VEGF-induced sprouting.
The VEGF signaling pathway plays important roles in migration,
proliferation, and tube formation by endothelial cells, which are all
required for angiogenesis.2,19 VEGF binds to its receptors, VEGFR1
and VEGFR2 (KDR/Flk), which has stronger tyrosine kinase activity
than VEGFR1, and also transduces the major signals for vascular
endothelial responses during angiogenesis.20,21 The PI3K/AKT and
RAF/MEK-ERK pathways have been demonstrated to regulate endo-
thelial cell migration, proliferation, growth, and survival.22,23 In
particular, AKT regulates endothelial nitric oxide synthase (eNOS)
activation,24 which stimulates vasodilation, vascular remodeling,
and angiogenesis,25 whereas ERK (extracellular signal-related
kinase) is required for the regulations of the endothelial cell cycle
and endothelial proliferation, growth, migration, and apoptosis.26
In addition, after being stimulated by extracellular growth factor,
activated ERK is also required for eNOS activation.22,27
To elucidate the mechanism that underlies the anti-angiogenic
effect of BRN-103, we examined signaling molecules and pathways
using Western blotting assays. As shown in Figure 4A, the VEGF-in-
duced phosphorylation of VEGFR2 at Tyr-1175 was suppressed
dose-dependently by BRN-103 (Fig. 4A), suggesting that the anti-
angiogenic properties of BRN-103 may be at least partially due to
VEGR2 inhibition.. In addition, BRN-103 significantly and concen-
tration-dependently suppressed the VEGF-triggered phosphoryla-
tions of AKT (Ser473) and eNOS (Ser1172), whereas it only mildly
inhibited VEGF-induced phosphorylation of ERK (Thr202/Tyr204) in
HUVECs. Therefore, we propose that BRN-103 inhibits angiogenesis
by blocking the AKT and eNOS signaling pathways. In contrast,
sunitinib inhibited the VEGF-mediated phosphorylations of
VEGFR2, ERK and eNOS, but not the VEGF-mediated phosphoryla-
tion of AKT.
In conclusion, the novel nicotinamide derivative BRN-103 was
found to inhibit endothelial cell migration, proliferation, tube for-
mation, and capillary formation by interfering with the activation
of the VEGF receptor and its downstream signaling of AKT, ERK,
and eNOS. These results suggest that BRN-103 should be consid-
ered a potential lead compound for the development of novel
anti-angiogenic drugs.
Figure 4. BRN-103 inhibited the VEGF-triggered activations of VEGFR2 and VEGFR2
signaling in HUVECs. (A) Cells were pretreated with different concentrations (0.1, 0.5,
or 1 lM) of BRN-103 for 1 h, then treated with VEGF (10 ng/ml), and incubated for
Acknowledgments
5 min. Protein extracts were then harvested and subjected to Western blot analysis
using specific anti-phosphorylated VEGFR2 antibody. VEGFR and b-actin were used
as internal controls. The immunoblot shown is representative of three separate
experiments; (B) 0.1% serum-starved HUVECs were pretreated with different
This research was supported by Basic Research Program
through the National Research Foundation of Korea (NRF) funded
by the Ministry of Education, Science and Technology
(2009-0088135).
concentrations (0.1, 0.5, or 1 lM) of BRN-103 for 1 h, then treated with VEGF
(10 ng/ml), and incubated for 5 min (for p-ERK), 30 min (for p-AKT) and 1 h (p-eNOS).
Protein extracts were then harvested and subjected to Western blot analysis using
specific p-AKT, AKT, p-ERK, ERK, p-eNOS or eNOS antibodies. The immunoblot shown
is representative of three separate experiments. Columns represent the means of
three different experiments, and bars represent SD. #p <0.05 versus nontreated
control group, ⁄⁄p <0.01, ⁄⁄⁄p <0.001 versus VEGF-stimulated group; statistical
significances were compared using ANOVA and Dunnett’s post-hoc test.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.09.022.

H.-E. Choi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6236–6241
6241
antibody. After addition of peroxidase conjugated secondary antibody,
substrate and stop solution were added. The amount of BrdU incorporated
was determined by measuring the absorbance at 450 nm.
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