Synthesis and evaluation of nicotinamide derivative as anti-angiogenic agents

Article abstract of DOI:10.1016/j.bmcl.2013.01.125

Previously, we have found that BRN-103, a nicotinamide derivative, inhibits vascular endothelial growth factor (VEGF)-mediated angiogenesis signaling in human endothelial cells. During our continuous efforts to identify more potent anti-angiogenic agents, we synthesized various nicotinamide derivatives and evaluated their anti-angiogenic effects. We found that 2-{1-[1-(6-chloro-5- fluoropyrimidin-4-yl)ethyl]piperidin-4-ylamino}-N-(3-chlorophenyl) pyridine-3-carboxamide (BRN-250) significantly inhibited human umbilical vascular endothelial cells (HUVECs) proliferation, migration, tube formation, and microvessel growth in a concentration range of 10-100 nM. Furthermore, BRN-250 inhibited the VEGF-induced phosphorylation and intracellular tyrosine kinase activity of VEGF receptor 2 (VEGFR2) and the activation of its downstream AKT pathway. Taken together, these findings suggest that BRN-250 be considered a potential lead compound for cancer therapy.

Full text of DOI:10.1016/j.bmcl.2013.01.125

Accepted Manuscript
Synthesis and evaluation of nicotinamide derivative as anti-angiogenic agents
Hye-Eun Choi, Jung-Hye Choi, Jae Yeol Lee, Je Hak Kim, Ji Han Kim, Joon
Kwang Lee, Gyu Il Kim, Yong Park, Yong Ha Chi, Soo Heui Paik, Joo Han
Lee, Kyung-Tae Lee
PII:
S0960-894X(13)00158-3
DOI:
http://dx.doi.org/10.1016/j.bmcl.2013.01.125
BMCL 20113
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
1 November 2012
14 January 2013
30 January 2013
Please cite this article as: Choi, H-E., Choi, J-H., Lee, J.Y., Kim, J.H., Kim, J.H., Lee, J.K., Kim, G.I., Park, Y.,
Chi, Y.H., Paik, S.H., Lee, J.H., Lee, K-T., Synthesis and evaluation of nicotinamide derivative as anti-angiogenic
agents, Bioorganic & Medicinal Chemistry Letters (2013), doi: http://dx.doi.org/10.1016/j.bmcl.2013.01.125
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[Bioorganic & Medicinal Chemistry Letters]
Synthesis and evaluation of nicotinamide derivative as anti-angiogenic
agents
Hye-Eun Choia,b, Jung-Hye Choib,c, Jae Yeol Leed, Je Hak Kime, Ji Han Kime, Joon Kwang
Lee f, Gyu Il Kime, Yong Parke, Yong Ha Chie, Soo Heui Paike, Joo Han Leee and Kyung-Tae
Lee a,b*
aDepartment of Pharmaceutical Biochemistry, bDepartment of Life and Nanopharmaceutical
Science, and cDepartment of Oriental Pharmaceutical Science,College of Pharmacy, Kyung
Hee University, 1 Hoegi-dong, Dongdaemun-gu, Seoul 130-701, Republic of Korea
dResearch Institute for Basic Sciences and Department of Chemistry, College of Sciences,
Kyung Hee University, Seoul 130-701, Republic of Korea
eCentral Research Institute, Boryung Pharm. Co. Ltd., 1122-3, Shingil-Dong, Danwon-Gu,
Ansan-Si, Kyungki-Do, 425-839, Republic of Korea
* Corresponding author.
Tel; +82-2-9610860; fax: +82-2-9620860
E-mail address: ktlee@khu.ac.kr(K-T Lee).
1

ABSTRACT
Previously, we have found that BRN-103, a nicotinamide derivative, inhibits vascular
endothelial growth factor (VEGF)-mediated angiogenesis signaling in human endothelial
cells. During our continuous efforts to identify more potent anti-angiogenic agents, we
synthesized various nicotinamide derivatives and evaluated their anti-angiogenic effects.
We found that 2-{1-[1-(6-chloro-5-fluoropyrimidin-4-yl)ethyl]piperidin-4-ylamino}-N-(3-
chlorophenyl) pyridine-3-carboxamide (BRN-250) significantly inhibited human umbilical
vascular endothelial cells (HUVECs) proliferation, migration, tube formation, and
microvessel growth in a concentration range of 10-100 nM. Furthermore, BRN-250
inhibited the VEGF-induced phosphorylation and intracellular tyrosine kinase activity of
VEGF receptor 2 (VEGFR2) and the activation of its downstream AKT pathway. Taken
together, these findings suggest that BRN-250 be considered a potential lead compound for
cancer therapy.
Keywords: Anti-angiogenesis, Nicotinamide, VEGFR, HUVECs, Receptor tyrosine kinase
2

Angiogenesis is the process of the formation of new blood vessels from preexisting
blood vessels, and includes the destabilization of established vessels, endothelial cell
proliferation and migration, and the degradation of extra-cellular matrix, and the formation
and sprouting of new vessels.1,2,3 On the other hand, tumor angiogenesis is the process
whereby the network of blood vessels expands and penetrates cancerous growths to supply
nutrients and oxygen and remove metabolic waste from tumors. The inhibition of
angiogenesis is referred to as the fourth modality of anticancer therapy.4
Vascular endothelial growth factor (VEGF) is a crucial regulator of angiogenesis,5,6
and acts on endothelial cells as a chemotactic and mitogenic factor via endothelial cell-
specific receptors, that is vascular endothelial growth factor receptor1 (VEGFR1) (Flt-1),
VEGFR2 (Flk-1/KDR) and VEGFR3 (Flt-4), of which VEGFR2 is the major mediator of the
pro-angiogenic effects induced by VEGF.7 In addition, tumor cells release several pro-
angiogenic factors that enhance metastasis, and therefore, shorten patient survival.8 Although
antibodies against VEGF (avastin) or multi-targeting inhibitors of receptor tyrosine kinase
(RTK) (sorafenib and sunitinib) have already been approved by the US Food and Drug
Administration (FDA) for advanced renal cancer, safer and more efficient inhibitors are still
needed.9,10 The anti-angiogenic and direct anti-tumor effects of sunitinib may be important
aspects of its anti-tumor activity, at least for certain tumor types.11
In the present study, we have constructed the library of nicotinamide derivatives
using the general reaction pathway outlined in Scheme 1-2 based on the previously biological
activity of BRN-103 (a nicotinamide derivative) against angiogenesis12 and examined the
abilities of these compounds to inhibit VEGF-induced HUVEC proliferation at
concentrations less than their IC90 values (data not shown) using 5-bromo-2-deoxyuridine
(BrdU) incorporation method.13
3

O
O
O
N
Cl
N
Cl
N
Cl
H
H
c
d
H
N
NH
N
NH
N
NH
N
N
R
N
H
Boc
3
4
5a (BRN-244): R = N,N-diethylaminoethyl
5b (BRN-250): R = (6-chloro-5-fluoropyrimidino)ethyl
b
O
O
N
Cl
N
Cl
O
O
H
H
a
e
f
N
NH
N
NH
OH
N
H
Cl
N
Cl
N
Cl
N
N
H
OH
1
2
6 (BRN-201)
7 (BRN-242)
g
h
O
O
O
O
N
H
Cl
N
H
Cl
N
H
Cl
N
H
Cl
i
j
N
NH
N
NH
N
NH
N
NH
O
N
NH
N
HN
Boc
Cl
8 (BRN-137)
9
10
11 (BRN-267)
Scheme 1. Reagents and conditions: (a) i) SOCl2, DCM, reflux, 1.5 h; ii) 3-chloroaniline,
Et3N, 12 h, 82%; (b) 4-amino-1-Boc-piperidine, K2CO3, xylene, 130 oC, 24 h, 42%; (c) conc.
HCl, EtOH, reflux, 4 h, 90%; (d) N,N-diethylaminoethyl chloride or 4-(1-bromoethyl)-6-
chloro-5-fluoropyrimidine, K2CO3, ACN, reflux, 24 h, 45% for 5a and 48% for 5b; (e) 4-
amino-2,2,6,6-tetramethylpiperidine, K2CO3, xylene, 130 oC, 24 h, 82%; (f) 2-bromoethanol,
K2CO3, ACN, reflux, 18 h, 37%; (g) N-(3-chlorophenyl)-4-aminobutanamide, K2CO3, xylene,
130 oC, 24 h, 83%; (h) (R)-3-amino-1-Boc-piperidine, K2CO3, xylene, 130 oC, 24 h, 44%; (i)
conc. HCl, EtOH, reflux, 4 h, 64%; (j) benzyl chloride, K2CO3, ACN, reflux, 18 h, 68%.
The synthetic methods used to construct the nicotinamide library began with the 2-
chloronicotinic acid (1) and 2-chloro-3-cyanopyridine (12), respectively (Scheme 1 and 2). 2-
Chloronicotinic acid could be carried forward directly for the 2-chloronicotinamide derivative
(2), which was substituted with various amines to afford the corresponding 2-
aminonicotinamide derivatives [3, 6 (BRN-201), 8 (BRN-137) and 9]. In the case of
compound 3 and 9, the de-protection of Boc group and subsequent N-alkylation with N,N-
diethylaminoethyl chloride, 4-(1-bromoethyl)-6-chloro-5-fluoropyrimidine 14 and benzyl
4

chloride to give the compound 5a (BNR-244), 5b (BRN-250)15 and 11 (BRN-267),
respectively. Compound 6 (BRN-201) containing 2,2,6,6-tetramethylpiperidine ring was
directly N-alkylated with 2-bromoethanol to provide compound 7 (BRN-242). In the case of
compound 18 (BRN-442) containing 4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylate,
this fused heterocycle (17) was easily prepared from the reaction of 4-piperidone (15) with
methyl cyanoacetate (16) in the presence of sulfur16 and could be acylated with 2-
aminonicotinic acid (14) using thionyl chloride resulted in the compounds 18 as shown in
Scheme 2.
O
CN
OH
N
NH
N
N
NH
N
CN
Cl
a
b
N
NH
O
S
N
H
NH
O
N
O
12
13
14
d
N
O
O
O
O
18 (BRN-442)
NC
c
H2N
O
HCl
NH
N
H
S
HCl
15
16
17
Scheme 2. Reagents and conditions: (a) 4-amino-1-benzylpiperidine, K2CO3, xylene, 140
oC, 40 h, 72% ; (b) KOH, IPA, reflux, 20 h, 88%; (c) sulfur, Et2NH, MeOH, 5 h, 43% (d) i)
SOCl2, DCM, reflux, 1 h; ii) compound 17, Et3N, reflux, 4 h, 83%.
It was found that some of these compounds displayed potent inhibitory effects, and
that 2-{1-[1-(6-chloro-5-fluoropyrimidin-4-yl)ethyl]piperidin-4-ylamino}-N-(3-chlorophenyl)
pyridine-3-carboxamide (BRN-250) most effectively inhibited VEGF-induced HUVEC
proliferation (Figure 1A). In addition, BRN-250 was found to inhibit VEGF-induced
HUVEC proliferation concentration-dependently and to be more potent in this respect than
5

BRN-103 (Figure 1B).
To further assess the anti-angiogenic property of BRN-250 in vitro, we examined its
inhibitory effects on the migration of endothelial cells using the wound-healing and the
Trans-well assays. As shown in Figure 2A, BRN-250 (10, 50, or 100 nM) significantly
inhibited VEGF-induced HUVEC migration in the wound-healing assay. In the Trans-well
assay – the most popular in vitro test of angiogenesis,17cells were seeded onto the upper
surface of an 8 μm pore size membrane separating upper and lower Boyden’s chambers. The
upper chamber contained BRN-250 in 0.1% endothelial basal medium (EBM), and cellular
migration through the membrane was induced when VEGF was introduced in the lower
chamber. Using this assay, we found that BRN-250 dramatically reduced cell migration
(Figure 2B). BRN-250 was found to inhibit endothelial migration dose-dependently by both
assays and to have a significant inhibitory effect at 10 nM.
Although several types of cells participate in angiogenesis, tube formation by
endothelial cells is a key step.18 Therefore, we investigated whether BRN-250 regulates
capillary tube formation by HUVECs.19 When HUVECs were seeded on growth factor
reduced two-dimensional Matrigel, robust tubular structures were formed in the presence of
VEGF (10 ng/ml). However, preincubation with BRN-250 (10, 50, or 100 nM) markedly and
dose-dependently abolished this tube formation (Figure 3A). To determine whether BRN-250
influences VEGF-induced angiogenesis ex vivo, the sprouting of vessels from rat aortic
rings20 were examined in the presence or absence of BRN-250 (10, 50, or 100 nM). As
shown in Figure 3B, VEGF significantly stimulated microvessel sprouting, which led to the
formation of a network of vessels around rat aortic rings. On the other hand, BRN-250 dose-
dependently antagonized VEGF-induced sprouting.
VEGF family members are best known as a family of potent angiogenesis factors,
6

and are believed to act as angiogenesis switches to trigger the expansion of quiescent tumor
tissues.21,22 Furthermore, the VEGF-related pathway has become an attractive target because
some VEGF inhibitors have already been shown to possess potent anti-tumor effects in vivo
and in vitro.23 VEGFRs and their signaling pathways represent rate-limiting steps in
physiologic angiogenesis.24,25 In particular, VEGFR2 is the primary mediator of the
angiogenic activity of VEGF via distinct signal transduction pathways that regulate
endothelial cell proliferation, migration, differentiation, and tube formation. The major
autophosphorylation site on VEGFR2 has been reported to be located at Tyr1175, and its
phosphorylation provides a docking site with the p85 subunit of PI3K and with
phosphoinositidephospholipase Cγ (PLCγ).26,27,28 Furthermore, this phosphorylation is critical
for subsequent VEGF-stimulated proliferation, chemotaxis and sprouting, and for the survival
of cultured endothelial cells in vitro and in vivo.26
Since growth factor-induced endothelial cell migration and subsequent tube
formation are known to be PI3K-AKT-dependent,29,30 activation of the AKT pathway has
been established to play a crucial role in malignant transformation, chemoresistance, and
invasiveness by inducing cell survival, growth, migration, and angiogenesis.31 Therefore, to
elucidate the molecular mechanism that underlies the anti-angiogenic effect of BRN-250, we
examined its effect on the activation of VEGFR2 and on AKT downstream of VEGFR2. As
shown in Figure 4A, BRN-250 significantly and dose-dependently suppressed the VEGF-
induced phosphorylations of VEGFR2 (Tyr1175) and AKT (Ser473) in HUVECs. Furthermore,
BRN-250 pretreatment dose-dependently suppressed the VEGF-induced intracellular tyrosine
kinase activity of VEGFR2 (Tyr1175) (Figure 4B). Sunitinib is a multi-targeting inhibitor of
receptor tyrosine kinases (RTKs) and has been shown to be effective treatment for several
human cancers.26 We found that BRN-250 was more potently inhibited the VEGF-induced
7

tyrosine kinase activity of VEGFR (Tyr1175) than sunitinib or BRN-103. Accordingly, these
results suggest that BRN-250 inhibits angiogenesis by blocking the VEGFR and AKT
signaling pathways.
In order to gain more insight of the binding modes of BRN series with a receptor of
known X-ray structure, docking studies were carried out using Molegro Virtual Docker
(MVD) 2010.4.2 for Windows.32 The reported crystal structure of VEGFR2 with
benzimidazole-urea derivative from Protein Data Bank (PDB ID 2OH4) with resolution 2.05
Å was downloaded for the present docking study.33 The active site of the receptor was
defined to include residues within a 10.0 Å radius of benzimidazole-urea atoms. The docking
wizard of MVD 2010.4.2 was used to dock the selected BRN-137, -242, -250, or
benzimidazole-urea (for docking comparison) on the active sites of VEGFR2. The most
stable docking pose was selected by Mol Dock Score predicted by the MVD scoring function
for each compound (Table 1). The most active compound BRN-250 was found to dock into
the active site of VEGFR2 with a higher Mol Dock Score and interaction energy than BRN-
137 or BRN-242, which was consistent with real experimental results (Table 1).
On finishing point of docking process, the resulting conformation poses of BRN-250
in the binding sites of VEGFR2 were considered. The detailed binding pattern of BRN-250 is
shown in Figures 5A and 5B. As shown in Figure 5A, the most binding interacted pose of
BRN-250 is shown to be docked into the active site of VEGFR2 and also its binding mode is
very similar to that of benzimidazole-urea (the control ligand), which has the extra-binding
interaction with the receptor using the 2-carbamate group of benzimidazole ring resulting in
the higher MolDock Score and interaction energy. On the other hand, BRN-250 in Figure 5B
showed four hydrogen bond interactions with Glu883 (amide NH of nicotinamide ring),
Cys917(N atom of pyrimidine ring), and Asp1044 (amide NH of nicotinamide ring & 2-amino
8

group of pyridine ring), which were similar to the three hydrogen bond interactions of
benzimidazole-urea at Cys917 and Asp1044 in x-ray crystal structure (PDB ID 2OH4 ).
In summary, nicotinamide derivatives were synthesized and evaluated with respect to
their inhibitory activities on VEGF-induced HUVEC cell proliferation. BRN-250 was found
to have greatest inhibitory activity. In addition, BRN-250 inhibited VEGF-induced HUVEC
migration, tube formation, and microvessel sprouting by interfering with the activation of
VEGFR2 and its AKT signaling. These results suggest that BRN-250 should be considered a
potential lead compound for the development of novel anti-angiogenic drugs.
9

Figure1. BRN-250 inhibited the VEGF-induced proliferation of HUVECs. HUVECs were
pre-treated with (A) six BRN compounds (BRN-137, -201, -242, -250, -267, or -442) at a
concentration of 100 nM and (B) various concentrations (10, 50, or 100 nM) of BRN-250 1h
prior to 24 h of VEGF treatment (10 ng/ml). VEGF-induced cell proliferation was quantified
using BrdU incorporation assays. Columns represent the means of three different experiments,
and bars represent SD. #p<0.05 vs. non-treated control group, **p<0.01, ***p<0.001 vs. VEGF-
stimulated group; statistical significances were compared using ANOVA and Dunnett’s post-
hoc test.
Figure 2. BRN-250 inhibited the VEGF-induced migration of HUVECs. (A) Representative
photomicrographs of wound healing assays. Using near confluent HUVECs in 60mm culture
dish and a scraper, wound was produced. HUVECs were pretreated with BRN-250 (10, 50 or
10 nM) for 1 h before the induction of cellular migration with VEGF (10 ng/ml). After 24 h
of incubation, picture was taken. Dotted lines show the area occupied by the initial scraping.
(B) HUVECs were cultured in a Boyden chamber, with VEGF (10 ng/ml) in the lower
chamber and various concentrations of BRN-250 in the upper chamber. After 24 h, cellular
migration was determined by counting cells migrated through the pores. The bar graphs show
average numbers of HUVECs that migrated through the membrane. Columns represent the
means of three different experiments, and bars represent SD. #p <0.05 vs.non-treated control
group, **p<0.01, ***p<0.001 vs. VEGF-stimulated group; statistical significances were
compared using ANOVA and Dunnett’s post-hoc test.
Figure 3. BRN-250 inhibited VEGF-induced capillary structure formation and microvessel
sprouting ex vivo. (A) After being incubated with BRN-250, HUVECs were fixed, and
10

tubular structures were photographed (magnification, ×100). Tube-like structures were
quantified by manual counting in low power fields. Columns represent the means of three
different experiments, and bars represent SD. #p<0.05 vs. non-treated control
group,***p<0.001 vs. VEGF-stimulated group; statistical significances were compared using
ANOVA and Dunnett’s post-hoc test. (B) Aortic segments isolated from SD Rat were placed
in Matrigel-covered wells and treated with VEGF in the presence or absence of BRN-250 (10,
50, or 100 nM). The results shown are representative of three independent experiments, and
the photographs of microvessel sprouting from the margins of aortic rings are also
representative of three separate experiments.
Figure 4. BRN-250 inhibited the VEGF-induced activations of VEGFR2 and AKT signaling
in HUVECs. (A) HUVECs were stimulated with VEGF (10 ng/ml) for the indicated time
periods with or without 1 h of BRN-250 (10, 50 or 100 nM) pretreatment. Phosphorylation of
VEGFR (5min) and Akt (30 min) were examined using Western blot analysis. (B) HUVECs
were pretreated with different concentrations of BRN-250 (10, 50 or 100 nM) 1 h prior to
being treated for 5 min with VEGF (10 ng/ml). Phosphorylation of VEGFR2 at Tyr1175 was
detected by PathScan ELISA kit. The results represent the means of three different
experiments, and bars represent SD. #p<0.05 vs. non-treated control group, ***p<0.001 vs.
VEGF-stimulated group; statistical significances were compared using ANOVA and
Dunnett’s post-hoc test.
Figure 5. The pose adopted by BRN-250 (green carbon) and the control ligand (red carbon)
in the active site of VEGF receptor 2 (PDB ID 2OH4). The figure was prepared using Ligand
Scout. (A) Three-dimensional model of the interaction of BRN-250 with the VEGFR2
11

binding site. Interacting amino acids and BRN-250 are depicted by green and red sticks,
respectively. (B) Hydrogen bond interactions are represented by white-dotted lines. The
figure was prepared using Molegro Virtual Docker (PDB ID 2OH4).
Acknowledgments
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).
12

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10.58 (1H, s ), 8.18 (1H, m, -CO-NH-CH2-), 8.64 (1H, d, J = 7.3 Hz), 8.51 (1H, s),
13

8.16 (1H, dd, J = 6.4 and 1.6 Hz), 7.87 (1H, m), 7.58 (1H, m, J = 8.4 Hz), 7.38 (1H, d,
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119.07, 116.24, 111.50, 48.32, 45.32, 45.24, 30.61, 21.46; HRMS (EI+) Calcd for
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