Design and synthesis of 3-(4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridin-2- yl)-1H-quinolin-2-ones as VEGFR-2 kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.02.073

A series of 3-(4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridin-2-yl)-1H- quinolin-2-ones have been identified as a new class of VEGFR-2 kinase inhibitors. A variety of (4,5,6,7-tetrahydro-imidazo[5,4-c]pyridin-2-yl)-acetic acid ethyl esters were synthesized, and their VEGFR-2 inhibitory activity was evaluated. Described herein are the preparation of the series and the effects of the compounds on VEGFR-2 kinase activity.

Full text of DOI:10.1016/j.bmcl.2012.02.073

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2837–2842
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Bioorganic & Medicinal Chemistry Letters
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Design and synthesis of 3-(4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridin-2-yl)-
1H-quinolin-2-ones as VEGFR-2 kinase inhibitors
Sun-Young Han a,b, Jie Won Choi a,c, Jeon Yang a,d, Chong Hack Chae a, Jongkook Lee a, Heejung Jung a,
a
a,
Kwangho Lee a, Jae Du Ha a, , Hyoung Rae Kim , Sung Yun Cho
⇑
⇑
a Bio-Organic Science Division, Korea Research Institute of Chemical Technology, PO Box 107, Daejeon 305-600, Republic of Korea
b College of Pharmacy and Research Institute of Pharmaceutical Sciences, Gyeongsang National University, 816 Gil 15, Jinju-daero, Jinju, Gyeongnam 660-751, Republic of Korea
c Department of Chemistry, Sogang University, 1 Sinsoo-Dong, Mapo-Koo, Seoul 121-742, Republic of Korea
d Department of Chemistry, Chungnam National University, Yuseong, Daejeon 305-764, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 3-(4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridin-2-yl)-1H-quinolin-2-ones have been identi-
fied as a new class of VEGFR-2 kinase inhibitors. A variety of (4,5,6,7-tetrahydro-imidazo[5,4-c]pyri-
din-2-yl)-acetic acid ethyl esters were synthesized, and their VEGFR-2 inhibitory activity was
evaluated. Described herein are the preparation of the series and the effects of the compounds on VEG-
FR-2 kinase activity.
Received 2 December 2011
Revised 14 February 2012
Accepted 23 February 2012
Available online 7 March 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Cancer
VEGFR-2
KDR kinase
Imidazo[4,5-c]pyridine
Quinolin-2-one
Vascular endothelial growth factor receptor (VEGFR-2, KDR) is a
receptor tyrosine kinase that plays important roles in regulating
vascular permeability, endothelial cell proliferation and migration,
and angiogenesis under physiological conditions mediated by
vascular endothelial growth factor (VEGF).1 Upon binding of the
VEGF, VEGFR-2 dimerizes, autophosphorylates, and activates the
VEGFR-2 pathway leading to angiogenesis, the formation of new
capillaries from preexisting blood vessels, enhanced tumor
survival, and tumor migration.2 It has been postulated that the
inhibition of angiogenesis by blocking the VEGFR-2 signaling
pathway results in reduced tumor angiogenesis and tumor growth
suppression.3 Indeed, a number of inhibitors of VEGF-induced
angiogenesis have been shown to be efficacious in tumor xenograft
models.4 Developing small-molecule VEGFR-2 inhibitors for anti-
cancer therapy has been the goal of intensive drug discovery efforts
at many pharmaceutical companies.5 Sorafenib (Bayer/OSI)6 and
sunitinib (Pfizer),7 are VEGFR-2 inhibitors that are used clinically
against several types of cancer. Pazopanib (GlaxoSmithKline) and
vandetanib (AstraZeneca) recently received FDA approval for renal
cell carcinoma and medullary thyroid cancer, respectively (Fig. 1).
A variety of small-molecule inhibitors targeting VEGFR-2 have
been reported and some of them have reached late-stage clinical
development.8 Described herein is a series of tetrahydro-3H-imi-
dazo[4,5-c]pyridine-based inhibitors of VEGFR-2 that were substi-
tuted at the phenyl and tetrahydropyridine moieties. In our efforts
to discover novel small-molecule VEGFR-2 kinase inhibitors, a class
of compounds based on 3-(4,5,6,7-tetrahydro-3H-imidazo[4,5-
c]pyridin-2-yl)-1H-quinolin-2-one (1) (Fig. 2) was identified.
Compound 1 itself was identified as a hit with IC50 of 1 lM for
the inhibition of VEGFR-2 kinase activity through high-throughput
screening (HTS).
Owing to the structural similarities of 1 to known benzimidaz-
olyl quinolinone 2 (IC50 of 1.7 l
M)9 and indolyl quinoxalinone 3
(IC50 of 8 nM)10, both of which possessed submicromolar potency
in the VEGFR-2 kinase assay, we sought to investigate the SAR in
ring system A and at the nitrogen of the tetrahydropyridine with
a focus on increasing intrinsic potency. In this communication,
we report the development of tetrahydro-3H-imidazo[4,5-c]pyri-
dine quinolinones as a new class of VEGFR-2 inhibitors.
According to the retrosynthetic analysis as shown in Scheme 1,
the tetrahydro-3H-imidazo[4,5-c]pyridine quinolinones 1 could be
readily prepared by condensing imidazopyridine acetates 5 with
variously substituted 2-aminobenzaldehydes 4. The corresponding
imidazopiperidine acetates 5 were envisaged to come from the
⇑
Corresponding authors. Tel.: +82 42 860 7077.
E-mail address: sycho@krict.re.kr (S.Y. Cho).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.02.073

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S.-Y. Han et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2837–2842
H
N
H
N
acid ethyl ester derivatives were synthesized by following a known
method (Scheme 4).13
CF3
Cl
O
NEt2
Me
O
Compounds were tested for VEGFR-2 inhibitory activity14 and
the results are summarized in Table 1. In the initial assessment,
inhibitory activity in the micromolar range was observed with no
substitution (1, 11, 12, 13) on the phenyl group (ring A, R2 = H)
and simple alkyl or aryl substitutions at the imidazopiperidine
nitrogen (R1 = Me, Bn, methylpiperidine) on parent compound 1
Table 2.
N
H
O
N
Me
N
H
F
NHMe
O
N
H
O
Sorafenib
Sunitinib
Of the alkyl substituents, Cbz-protected piperidine (12) slightly
improved inhibitory activity. N-substitution of the imidazopiperi-
dine by either meta- (14, 15) or para-nicotinamide (17, 18) deriva-
tives resulted in increased inhibitory activity. Introduction of
fluorine at the 6-position of the phenyl ring significantly increased
the inhibitory activity (16). 5-Fluorophenyl substitution did not
significantly improve the activity of the para- and meta-nicotin-
amide derivatives (15 and 18, respectively). However, compound
19 containing a 6-F and para-nicotinamide exhibited picomolar
inhibitory activity. To understand the SAR of N-substitution at
the imidazopiperidine, we investigated substitutions with other
amines, such as morpholine (23–27), piperazine (28–32), piperi-
dine (33–37), and pyrrolidine (38). Introduction of a chlorine (26)
at the C(6) position of the phenyl ring instead of fluorine (25) de-
creased potency. Interestingly, loss of activity was also observed
with hydrogen at C(5) (23), fluorine at C(5) (24), and methoxy at
the C(6) position (27) of the phenyl ring. Piperazine had a similar
propensity to attenuate the potency of the compounds. Introduc-
tion of a chlorine (31) or methoxy group (32) in place of the fluo-
rine (30) at the C(6) position of the phenyl ring decreased the
inhibitory activities of the piperazine derivatives. Similar results
were observed in the piperidine derivatives (35, 36). Next, we tried
to introduce a 3- or 4-pyridyl acetate in place of nicotinamide in
the derivatives. Among these pyridyl acetates, 2-pyridyl acetate
exhibited a high overall increase in potency with H or F at the
Br
F
Me
H2N
NH
S
NH
Me
N Me
MeO
O
N
O
O
N
N
N
N
N
N
Me
Me
Pazopanib
Vandetanib
Figure 1. VEGFR-2 inhibitors approved.
condensation of imidate 6 with
a
-aminoketal derivatives 7, which
could be themselves prepared through a Neber rearrangement.11
As shown in Scheme 2, classical conditions for the synthesis of
a
-aminoketal derivatives 7 involved treatment of oxime tosylates
9 with KOEt. The tosylates were derived from various ketones 8
that were treated with hydroxylamine followed by tosylation of
the corresponding oximes.
The preparation of imidazopiperidine acetates 512 is outlined in
Scheme 3. Imidazopiperidine 10 was reacted with suitable acyl ha-
lides to afford imidazopiperidine acetates 5. A variety of N-substi-
tuted
(4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridin-2-yl)acetic
N
N
O
N
N
H
N
H
N
H
A
N
H
N
H
O
N
H
O
1
2
3
Figure 2. Structures of VEGFR-2 inhibitors 1–3.
N
R1
EtO OEt
N
N
O
R2
R2
NH2Cl
OEt
NH2
R1
CHO
NH2
N
N
N
H
EtO2C
+
+
EtO2C
N
H
N
H
R1
4
5
7
6
Scheme 1. Retrosynthetic analysis of tetrahydro-3H-imidazo[4,5-c]pyridine quinolinones.
EtO OEt
NOTs
O
NH2
c
a, b
N
N
N
R1
R1
R1
7a: R1=Me
7b: R1=Bn
7c: R1=
8
9
N
Cbz
Scheme 2. Preparation of
a-aminoketal derivatives 7 via a Neber rearrangement. Reagents and conditions: (a) NH2OH, K2CO3, EtOH, reflux, 80–90%; (b) TsCl, pyridine, rt, 90%;
(c) K, EtOH, Na2SO4, 60 °C, 40–90%.

S.-Y. Han et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2837–2842
2839
5a: R1=Me (56%)
NH.HCl
OEt
N
O
O
EtO2C
a
NH2
5b: R1=Bn (46%)
EtO2C
+
N
R1
N
H
5c: R1=NCbz-piperidine (13%)
N
R1
7
b
R1=Bn
N
EtO2C
c
N
EtO2C
h
N
EtO2C
H
N
N
N
N
H
N
O
N
H
NH
R4
N
H
O
5n: R4= 3-methyl
5d: 2-pyridyl
5e: 3-pyridyl
10
5o: R4 = 4-methoxy
g
d,e
f,e
N
EtO2C
N
EtO2C
N
N
EtO2C
N
N
N
O
N
R3
N
N
H
H
N
H
N
O
O
R3
5g: R3=diethyl
5l: 2-pyridyl
5f
5m: 4-pyridyl
5h: R3=morpholinyl
5i: R3=N-Me piperazinyl
5j: R3=piperidinyl
5k: R3=pyrrolidinyl
Scheme 3. Preparation of imidazopiperidine acetates 5. Reagents and conditions: (a) 4 M-HCl/dioxane, EtOH, reflux, 72%; (b) Pd(OH)2, EtOH, 60 psi, 92%; (c)
pyridinecarboxylic acid, HOBT, EDCI, N-methylmorpholine, rt, 32–40%; (d) 4-chloromethylbenzoyl chloride, Et3N, CH2Cl2, 0 °C, 62%; (e) amine, K2CO3, KI, THF, rt, 63%; (f)
ClCH2COCl, Et3N, CH2Cl2, 0 °C, 42%; (g) pyridinylacetic acid, HOBT, EDCI, N-methylmorpholine, 40–50%; (h) isocyanates, CH2Cl2, rt, 55–62%.
N
R1
N
O
R2
6
R2
piperidine
4
5
8
CHO
3
N
EtO2C
xylene, reflux
N
H
2
+
1
N
R1
N
H
NHR
7
N
H
4 R=H, Boc
1
5
Scheme 4. Synthesis of tetrahydro-3H-imidazo[4,5-c]pyridine quinolinones.
C(6) position of the phenyl ring. When modifying the core with 4-
pyridyl acetate, chlorine (43) at the C(6) position of the phenyl ring
in place of H (41) or F (42) gave rise to inhibitory activity in the
picomolar range.
crystal structure of VEGFR-2 (pdb code: 3VHE) by using the Glide
software with standard precision (SP) protocols.20 The representa-
tive binding mode of most active compound 19 was shown in Fig-
ure 3.
Measurement of CYP (cytochrome P450) inhibitory activity and
microsomal stability were performed to obtain a preliminary eval-
uation of the toxicities of selected compounds. The data revealed
excellent microsomal stabilities and no significant CYP inhibitory
activity except from compound 42 with respect to 2C9, 3A4 and
2D6. 19 and 21 inhibited 2C9 and 3A4, respectively.
The selected compounds were examined for their inhibitory
activity on the proliferation of human umbilical vein endothelial
cells (HUVEC) induced by the VEGF. The inhibitory activity of com-
pounds on cell proliferation was measured using a Premix WST-1
cell proliferation assay kit.18 In addition, the selected compounds
were evaluated for inhibitory activity against HUVEC tube forma-
tion by Image-Pro Plus (Media Cybernetics) image analysis.19 As
shown in Table 3, selected compounds 19 and 21 showed weak
inhibitory activity for HUVEC proliferation. Compound 19 exhib-
ited marginal inhibitory activities for HUVEC tube formation at
The quinolinone compounds fit tightly into the ATP binding
cavity of the VEGFR-2 in inactive conformation with similar hydro-
gen bondings to Sutent in KIT kinase.21 Hydrogen bonding interac-
tions are observed between the carbonyl of the quinolinone ring
and N of C919 in the hinge region (3.12 Å). The hydrogen bonding
between the NH of the quinolinone and the main chain carbonyl of
E917 is somewhat blocked by the collision of ring A with V916 and
V899, and this may be the cause of low potency of this compounds.
The fluorine at the C(6) position of ring A makes additional hydro-
gen bonding with NH of K868 (3.43 Å). Without this interaction,
the potency is lowered as shown in series (Table 1, compounds
(14–19), (24–26), (28–30), (33–35), and (39, 40). The chlorine
makes better interaction with K868 than methoxy group, as shown
in series (Table 1, compounds 26, 27, 31, 32, 36, 37). The pyridi-
none ring stackes into the hydrophobic cleft comprised of A866,
L1035 and C1045, and the piperidine ring makes hydrophobic
10
lM and no activity was observed for 21 (data not shown). The
interaction with the side chain of L840 and Ca
of G922. The R1 sub-
low activities for HUVEC proliferation and tube formation of 19
and 21 was presumably attributed to the low degree of cell pene-
tration (Table 4).
stituents extend into the solvent exposed region. The docking
study indicated that the quinolinone compounds could fit into
the binding site and could be potent VEGFR-2 inhibitors.
To provide a rational for the experimental in vitro activities of
the quinolinone derivatives, compounds were docked into the
In summary, we identified
3H-imidazo[4,5-c]pyridin-2-yl)-1H-quinolin-2-one scaffold that
a novel 3-(4,5,6,7-tetrahydro-

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S.-Y. Han et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2837–2842
Table 1
SAR of the 3-(4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridin-2-yl)-1H-quinolin-2-one derivatives
N R1
N
R2
4
5
8
3
6
N
H
2
1
7
N
O
H
1
Compounds
R1
R2
H
VEGFR-2 IC50
1
(lM)
Compounds
R1
R2
H
VEGFR-2 IC50
0.3
(l
M)14
N
N
N
N
N
O
1
Me
28
N
N
N
N
N
O
O
O
O
11
12
13
Bn
H
H
H
6
29
30
31
5-F
6-F
6-Cl
1.05
N
Cbz
0.6
5
0.013
0.019
NH
O
14
15
16
H
0.2
32
33
34
6-OMe
1.1
N
O
O
O
O
N
N
5-F
6-F
0.6
H
0.016
0.74
N
N
0.001
5-F
O
O
O
O
O
O
O
O
O
O
O
N
N
N
N
17
18
19
20
H
0.2
35
36
37
38
6-F
0.008
0.022
0.28
0.4
N
5-F
6-F
H
0.8
6-Cl
6-OMe
H
N
N
0.0003
0.0013
N
N
21
22
6-F
H
0.0049
0.3
39
40
H
0.0013
0.0007
N
N
O
6-F
N
O
O
O
O
O
O
O
O
O
O
O
O
N
N
N
23
24
25
H
0.3
41
42
43
H
0.0067
0.0086
0.0005
N
N
N
N
5-F
6-F
1.1
6-F
6-Cl
0.0043
26
27
6-Cl
0.035
0.76
44
45
H
H
0.0021
0.046
N
H
OMe
O
O
O
N
6-OMe
N
H

S.-Y. Han et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2837–2842
2841
Table 2
Effects of compounds 19 and 21 on CYP inhibitory activity and microsomal stability
CYP (% remaining activity at 10
l
M)15
Microsomal stability
Permeability17 (PAMPA)
(% remaining activity at
10 l
M; 30 min incubation)16
Human
1A2
3A4
2C9
2C9
2D6
nta
16
19
21
42
86
96
89
58
83
89
16
33
99
53
94
28
92
77
100
35
67
90
100
21
96
99
92
93
nt
À6.2
À6.8
nt
a
Not tested.
Table 3
Results of VEGF-induced HUVEC anti-proliferative activity18 for 19 and 21
Control
VEGF (20 ng/ml)
VEGF + compound 19
VEGF + compound 21
0.1
lM
1
l
M
10
71
lM
0.1
l
M
1
l
M
10
79
lM
100
179
120
72
92.3
82
at KRICT for providing us with technical assistance on the CYP
activity and microsomal stability experiments.
Table 4
Effects of compound 19 and 21 on HUVEC tube formation (% of control)19
Control
Compound 19
Compound 21
References and notes
1
l
M
10
62
lM
1
l
M
10
99
lM
1. (a) Olsson, A. K.; Kimberg, A.; Kreuger, J.; Claesson-Welsh, L. Nat. Rev. Mol. Cell
Biol. 2006, 7, 359; (b) Ferrara, N.; Gerber, H.; LeCouter, J. Nat. Med. 2003, 9, 669;
(c) Carmeliet, P. Nat. Med. 2003, 9, 653–660; (d) Yang, J. C.; Haworth, L.; Sherry,
R. M.; Hwu, P.; Schwartzentruber, D. J.; Topalian, S. L.; Steinberg, S. M.; Chen, H.
X.; Rosenberg, S. A. N. Eng. J. Med. 2003, 349, 427.
100
93
102
2. Ferrara, N.; Gerber, H.; LeCouter, J. Nat. Med. 2003, 6, 669.
3. (a) Cross, M. J.; Claesson-Welsh, L. Trends Pharmacol. Sci. 2001, 22, 201; (b)
McMahon, G. Oncologist 2000, 5, 3; (c) Prewett, M.; Huber, J.; Li, Y.; Santiago, A.;
O’Connor, W.; King, K.; Overholser, J.; Hooper, A.; Pytowski, B.; Witte, L.;
Bohlen, P.; Hicklin, D. J. Cancer Res. 1999, 59, 5209.
4. (a) Cohen, M. H.; Gootenberg, J.; Keegan, P.; Pazdur, R. Oncologist 2007, 12, 356;
(b) Sandler, A.; Gray, R.; Perry, M. C.; Brahmer, J.; Schiller, J. H.; Dowlati, A.;
Lilenbaum, R.; Johnson, D. H. N. Eng. J. Med. 2006, 355, 2542; (c) Cardones, A. R.;
Banez, L. L. Curr. Pharm. Des. 2006, 12, 387.
5. (a) Boyer, S. J. Curr. Top. Med. Chem. 2002, 2, 973; (b) Traxler, P. Expert Opin.
Ther. Targets 2003, 2, 215.
6. (a) Atkins, M.; Jones, C. A.; Kirkpatrick, P. Nat. Rev. Drug Disc. 2006, 5(4), 279–
280; (b) Strumberg, D. Drugs Today 2005, 41, 773.
7. Ng, R.; Chen, E. X. Curr. Clin. Pharmacol. 2006, 1, 223–228.
8. (a) Rosen, L. S.; Kurzrock, R.; Mulay, M.; Van Vugt, A.; Purdom, M.; Ng, C.;
Silverman, J.; Koutsoukos, A.; Sun, Y.-N.; Bass, M. B.; Xu, R. Y.; Polverino, A.;
Wiezorek, J. S.; Chang, D. D.; Benjamin, R.; Herbst, R. S. J. Clin. Oncol. 2007,
25(17), 2369; (b) Harmange, J.; Weiss, M.; Germain, J.; Polverino, A. J.; Borg, G.;
Bready, J.; Buckner, W.; Chen, D.; Choquette, D.; Coxon, A.; DeMelfi, T.; DiPietro,
L.; Doerr, N.; Estrada, J.; Fellows, I.; Flynn, J.; Graceffa, R. F.; Harriman, S. P.;
Kaufman, S.; La, D. S.; Long, A.; Martin, M. W.; Neervannan, S.; Patel, V. F.;
Potashman, M.; Regal, K.; Roveto, P. M.; Schrag, M. L.; Starnes, C.; Tasker, A.;
Teffera, Y.; Wang, L.; White, R. D.; Whittington, D.; Zanon, R. J. Med. Chem. 2008,
51, 1649; (c) Weiss, M. M.; Harmange, J.; Polverino, A. J.; Bauer, D.; Berry, L.;
Berry, V.; Borg, G.; Bready, J.; Bretz, A.; Chen, D.; Chi, V.; Choquette, D.; Coxon,
A.; DeMelfi, T.; Doerr, N.; Estrada, J.; Fellows, I.; Flynn, J.; Graceffa, R. F.;
Harriman, S. P.; Kaufman, S.; La, D. S.; Long, A.; Neervannan, S.; Patel, V. F.;
Potashman, M.; Regal, K.; Roveto, P. M.; Schrag, M. L.; Starnes, C.; Tasker, A.;
Teffera, Y.; Whittington, D.; Zanon, R. J. Med. Chem. 2008, 51, 1668–1680; (d)
Mendel, D.; Laird, D.; Xin, X.; Louie, S.; Christensen, J.; Li, G.; Schreck, R.;
Abrams, T.; Ngai, T.; Lee, L.; Murray, L.; Carver, J.; Chan, E.; Moss, K.; Haznedar,
J.; Sukbuntherng, J.; Blake, R.; Sun, L.; Tang, C.; Miller, T.; Shirazian, S.;
McMahon, G.; Cherrington, J. Clin. Cancer Res. 2003, 9, 327.
Figure 3. Docking model of VEGFR-2 with compound 19. The side chains of binding
site are shown as lines and labeled with their residue name. Hydrogen bondings are
shown in yellow dashed lines. Parts of the P-loop residues are omitted for clarity.
exhibited potent VEGFR-2 inhibitory activity. Compounds 19 and
21 had marginal inhibitory activities for HUVEC proliferation and
tube formation with stable microsomal stabilities and low degrees
of binding to CYPs. Introduction of F and Cl at the C(6) position of
the phenyl ring dramatically increased VEGFR-2 inhibitory activity.
9. Renhowe, P. A.; Pecchi, S.; Shafer, C. M.; Machajewski, T. D.; Jazan, E. M.; Taylor,
C.; Antonios-McCrea, W.; McBride, C. M.; Frazier, K.; Wiesmann, M.; Lapointe,
G. R.; Feucht, P. H.; Warne, R. L.; Heise, C. C.; Menezes, D.; Aardalen, K.; Ye, H.;
He, M.; Le, V.; Vora, J.; Jansen, J. M.; Wernette-Hammond, M. E.; Harris, A. L. J.
Med. Chem. 2009, 52, 278.
10. Fraley, M. E.; Arrington, K. L.; Buser, C. A.; Ciecko, P. A.; Coll, K. E.; Fernandes, C.;
Hartman, G. D.; Hoffman, W. F.; Lynch, J. J.; McFall, R. C.; Rickert, K.; Singh, R.;
Smith, S.; Thomas, K. A.; Wong, B. K. Bioorg. Med. Chem. Lett. 2004, 14, 351.
11. (a) Neber, P. W.; Burgard, A.; Their, W. Ann. Chem. 1936, 1, 277; (b) LaMattina, J.
L.; Suleske, R. T. Synthesis 1980, 1, 329; (c) LaMattina, J. L.; Sulske, R. T. Org.
Synth. 1986, 64, 19; (d) Itoh, K.; Oka, Y. Chem. Pharm. Bull. 1983, 2016, 31; (e)
Diez, A.; Voldoire, A.; Lopez, I.; Rubiralta, M.; Segarra, V.; Pages, L.; Palacios, J.
M. Tetrahedron 1995, 51, 5143.
Acknowledgments
This work was supported in part by a Grant for the anti-cancer
research program from the Korea Research Institute of Chemical
Technology (KRICT) and the Basic Science Research Program from
NRF (2011-0010374, S.-Y.H.) funded by the government of Korea
(MEST). We thank the Drug Discovery Technology Platform team
12. Ha, J. D.; Lee, S. J.; Nam, S. Y.; Kang, S. K.; Cho, S. Y.; Ahn, J. H.; Choi, J.-K.
Tetrahedron Lett. 2006, 47, 6201.

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S.-Y. Han et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2837–2842
13. For the reaction between 2-benzoimidazole-2-ylacetate and 2-
aminobenzaldehyde: Fraley, M. E.; Hambaugh, S. R.; Hungate, R. W. U.S.
Patent 6479512 B1, 2002.
concentration of 21 was 2 lM. After pre-incubation of reaction mixture at
37 °C for 5 min, the reaction was initiated by addition NADPH regeneration
solution (BD Biosciences) and the reaction was terminated by adding three
times volume of ice-cold acetonitrile with imipramine (80 ng/ml) as internal
standard at single-time-point 30 min. After pretreatment of biological samples
with vortex and centrifuging, the samples were analyzed by LC/MS/MS system
(AB Sciex 2000 Qtrap).
14. VEGFR-2 kinase assay: Inhibition of kinase activity against recombinant VEGFR-
2
protein was measured using homogeneous time-resolved fluorescence
(HTRF) assays. Recombinant proteins containing VEGFR-2 kinase domain
were purchased from Millipore. Optimal enzyme, ATP, and substrate
concentrations were established using HTRF KinEASE kit (Cisbio) according
to the manufacturer’s instructions. Assays are composed of the VEGFR-2
enzyme mixed with serially diluted compounds and peptide substrates in a
kinase reaction buffer (250 mM HEPES (pH 7.0), 0.5 mM orthovanadate, 0.05%
BSA, 0.1% NaN3, 5 mM MgCl2, 1 mM MnCl2, 1 mM DTT, supplement enzymatic
buffer). Following the addition of reagents for detection, the Time Resolved-
Fluorescence Resonance Energy Transfer (TR-FRET) signal was measured using
an EnVision multi-label reader (Perkin Elmer). Dose–response curves were
generated to determine IC50 using Prism version 5.01 (GraphPad).
17. Ottaviani, G.; Martel, S.; Carrupt, P.-A. J. Med. Chem. 2006, 49, 3948.
18. In vitro VEGF-induced HUVEC proliferation assay: Pooled HUVECs (Lonza) were
grown in a 1:1 mixture of EBM-2 medium with EGM-2 supplements and used
at passage eight or lower. HUVECs were placed at a density of 10,000 cells/well
on
a collagen type I precoated 96-well plate. After 24 h, the cells were
incubated for 24 h in the presence of rhVEGF (20 ng/mL) in the presence of the
compound under examination. Cells were assayed for proliferation by the
Premix WST-1 proliferation assay system (Takara).
19. In vitro angiogenesis assay (tube formation): 24-well plates were coated with
200 mL of a 1:1 mixture of Matrigel (10 mg/mL) and EBM-2 medium with
EGM-2 supplements and incubated at 37 °C for 1 h. HUVECs (Lonza) were
placed at a density of 40,000/well and incubated for 16–18 h. After incubation,
the plates were fixed and images were captured using a Nikon camera (50Â)
and analyzed using Image-Pro Plus software (Media Cybernetics).
20. Friesner, R. A.; Banks, J. L.; Murphy, R. B.; Halgren, T. A.; Klicic, J. J.; Mainz, D. T.
J. Med. Chem. 2004, 47, 1739.
21. Gajiwala, K. S.; Wu, J. C.; Christensen, J.; Deshmukh, G. D.; Diehl, W.; DiNitto, J.
P.; English, J. M.; Greig, M. J.; He, Y. A.; Jacques, S. L.; Lunney, E. A.; McTigue, M.;
Molina, D.; Quenzer, T.; Wells, P. A.; Yu, X.; Zhang, Y.; Zou, A.; Emmett, M. R.;
Marshall, A. G.; Zhang, H. M.; Demetri, G. D. Proc. Natl. Acad. Sci. U.S.A. 2009,
106, 1542.
15. CYP450 inhibition assay: The potential of 21 to inhibit major human
cytochrome P450 (CYP450) enzymes was evaluated using
a recombinant
CYP450 inhibition kit (Invitrogen Vivid). The IC50 values of 21 for each CYP
isoform was determined from the inhibition of major five CYP450 isoform such
as CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 by fluorescence detection
in 96-well format. The studies were conducted in duplicate over a 20 min
incubation period with 20 min pre-incubation at room temperature. The IC50
values were estimated using Prism Software.
16. Microsomal stability test: NADPH dependent oxidative metabolism of 21 in
human and rat liver microsomes was determined on single-time-point 30 min.
The reaction mixtures consisted of human or rat liver microsomes (0.5 mg/ml;
BD Gentest) in 100 mM potassium phosphate buffer (pH 7.4) and final