2-((1H-Azol-1-yl)methyl)-N-arylbenzamides: Novel dual inhibitors of VEGFR-1/2 kinases

Article abstract of DOI:10.1016/j.bmcl.2005.11.105

Novel potent derivatives of (azol-1-yl)methyl-N-arylbenzamides with improved solubility (>3 mM) are described as ATP-competitive inhibitors of vascular endothelial growth factor receptor 2 (VEGFR-2). Many compounds display VEGFR-2 inhibitory activity reac

Full text of DOI:10.1016/j.bmcl.2005.11.105

Bioorganic & Medicinal Chemistry Letters 16 (2006) 1726–1730
2-((1H-Azol-1-yl)methyl)-N-arylbenzamides: Novel dual
inhibitors of VEGFR-1/2 kinases
Alexander S. Kiselyov,a,* Marina Semenova,b Victor V. Semenovc and Evgueni Piatnitskid
aSmall Molecule Drug Discovery, Chemical Diversity, Inc., 11558 Sorrento Valley Road, San Diego, CA 92121, USA
bInstitute of Developmental Biology, Russian Academy of Sciences, 26 Vavilov Str., 119334 Moscow, Russia
cZelinsky Institute of Organic Chemistry, Russian Academy of Sciences, 47 Leninsky Prospect, 117913 Moscow, Russia
dSmall Molecule Drug Discovery, ImClone Systems, 180 Varick Street, New York, NY 10014, USA
Received 10 November 2005; revised 29 November 2005; accepted 30 November 2005
Available online 20 December 2005
Abstract—Novel potent derivatives of (azol-1-yl)methyl-N-arylbenzamides with improved solubility (>3 mM) are described as ATP-
competitive inhibitors of vascular endothelial growth factor receptor 2 (VEGFR-2). Many compounds display VEGFR-2 inhibitory
activity reaching IC50 < 100 nM in the enzymatic assay. The compounds also inhibit the related tyrosine kinase, VEGFR-1, with
similar potencies. Several compounds containing bulky lipophilic substituents at the benzamide pharmacophore yielded 10- to
17-fold selectivity for the VEGFR-2 versus VEGFR-1 kinase.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Vascular endothelial growth factors (VEGFs) and their
respective family of receptor tyrosine kinases (VEGFRs)
are key proteins modulating angiogenesis, the formation
of new vasculature from an existing vascular network.1
Potent, specific, and non-toxic inhibitors of angiogenesis
are powerful clinical tools in oncology and ophthalmol-
ogy.2,3 Several groups in industry have developed meth-
ods for sequestering VEGF. This leads to a signal
blockade via VEGF receptors including both VEGFR-1
(Flt1) and VEGFR-2 (flk1, kinase insert domain recep-
tor, KDR)) and, subsequently to an inhibition of malig-
nant angiogenesis.
VEGFR phosphorylation and, ultimately to apoptotic
death of the aberrant endothelial cells. Drug candidates
that exhibit this mechanism of action include PTK 787
(VatalanibTM A) and ZD 6474 (VandetanibTM B). These
are Phase III and II clinical candidates, respectively,
against various cancers.4,5 The six-membered ring of a
phthalazine template in PTK 787 has been successfully
replaced with the isosteric anthranyl amide derivatives
C and D. Intramolecular hydrogen bonding was sug-
gested to be responsible for the optimal spatial orienta-
tion of pharmacophores, similar to the parent PTK
787.6
There are reports describing small-molecule inhibitors
that affect VEGF/VEGFR signaling by directly compet-
ing with the ATP-binding site of the respective intracel-
lular kinase domain. This event leads to the inhibition of
It has been suggested that the essential pharmacophores
for the VEGFR-2 activity of phthalazines and their ana-
logues include: (i) [6,6]fused (or related) aromatic sys-
tem; (ii) para- or 3,4-di-substituted aniline function in
Cl
N
Br
HN
HN
HN
HN
CF3
O
O
F
1
4
N
N
O
H
N
O
H
N
N
N
N
A
D
B
C
N
N
N
Keywords: Vascular endothelial growth factor receptor 2; Receptor tyrosine kinase; Dual kinase inhibitor; Angiogenesis; 2-((1H-Azol-1-yl)methyl)-N-
arylbenzyl amides.
*
Corresponding author. Tel.: +1 858 794 4860; fax: +1 858 794 4931; e-mail: ask@chemdiv.com
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.11.105

A. S. Kiselyov et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1726–1730
1727
Ar
O
Ar
OH
HN
HN
ii)
i)
O
O
Cl
R
Cl
1
2
4-38
MeO
MeO
MeO
Cl
N
N
N
N
N
N
N
N
R (3a-j) =
a
b
g
c
d
e
N
N
N
N
N
N
N
N
N
N
N
N
f
h
i
j
Scheme 1. Reagents and conditions: (i) DIC, ArNH2, CH2Cl2, rt, 2 h; (ii) 3a–j, K2CO3, DMF, rt, 12 h.
Table 1. Activity of N-arylbenzamides 4–38 against VEGFR-2 and VEGFR-1 kinases
Compound
Ar
R
Observed (lit.)6
a,b
VEGFR-2, IC50 (lM)
a,b
VEGFR-1, IC50 (lM)
A, PTK787
B, ZD6474
C
0.054 0.006 (0.042 0.003)
0.022 0.003 (0.017 0.003)
0.032 0.005 (0.023 0.006)
0.015 0.004 (0.009 0.001)
>10
0.14 0.02 (0.11 0.03)
0.10 0.01 (0.09 0.01)
0.17 0.05 (0.130 0.081)
0.16 0.05 (0.13 0.03)
>10
D
4
4-Cl(C6H4)
4-Cl(C6H4)
a
b
c
d
e
f
5
>10
>10
>10
>10
6
4-Cl(C6H4)
4-Cl(C6H4)
7
>10
3.55 0.50
>10
>10
>10
8
4-Cl(C6H4)
4-Cl(C6H4)
9
>10
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
4-Cl(C6H4)
4-Cl(C6H4)
g
h
i
0.092 0.008
0.047 0.005
4.28 0.66
>10
0.22 0.07
0.14 0.02
>10
4-Cl(C6H4)
4-Cl(C6H4)
j
>10
4-t-Bu(C6H4)
4-t-Bu(C6H4)
4-i-Pr(C6H4)
g
h
g
h
g
h
g
h
g
h
g
h
g
h
g
h
g
h
g
h
g
h
h
h
h
0.052 0.006
0.061 0.006
0.087 0.008
0.11 0.01
0.073 0.02
0.095 0.02
0.47 0.07
0.35 0.06
>10
0.76 0.08
0.61 0.07
0.54 0.07
0.44 0.06
1.25 0.12
1.16 0.11
0.74 0.10
0.57 0.08
>10
4-i-Pr(C6H4)
4-ClF2CO(C6H4)
4-ClF2CO(C6H4)
3-Me(C6H4)
3-Me(C6H4)
2-Me(C6H4)
2-Me(C6H4)
>10
>10
4-N-Morpholino-(C6H4)
4-N-Morpholino-(C6H4)
3,4-Cl(C6H4)
3,4-Cl(C6H4)
4-Cl-3-CF3(C6H3)
4-Cl-3-CF3(C6H3)
2-F-4-Me(C6H3)
2-F-4-Me(C6H3)
3,4-Methylenedioxy
3,4-Methylenedioxy
4-Br(C6H4)
0.28 0.04
0.21 0.04
0.066 0.008
0.056 0.008
0.31 0.03
0.44 0.05
0.29 0.03
0.21 0.04
0.13 0.02
0.23 0.04
0.44 0.08
0.38 0.06
0.69 0.09
1.55 0.22
2.12 0.35
0.34 0.04
0.30 0.05
0.22 0.04
0.26 0.05
0.41 0.07
0.69 0.09
0.34 0.05
0.49 0.06
0.18 0.03
0.36 0.05
0.95 0.11
0.77 0.09
1.14 0.15
2.53 0.31
>10
4-Br(C6H4)
4-Ph(C6H4)
4-PhO(C6H4)
4-Bn(C6H4)
a IC50 values were determined from the logarithmic concentration–inhibition curves (10 points). The values are given as means of at least two
duplicate experiments.
b Lit. IC50 values, as measured at 8 lM ATP.6

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A. S. Kiselyov et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1726–1730
Figure 1. Structural overlap between inhibitor 11 (brown), PTK787 (A, gray), and C (green).
the position 1 of phthalazine; and (iii) hydrogen bond
acceptor attached to the position 4 via an appropriate
linker (aryl or fused aryl group).4,6 To further assess
structural requirements for the dual VEGFR1/VEGFR2
activity, we designed a set of molecules that have neither
fused phthalazine system (A) nor the intramolecular
hydrogen bonding (C, D).
possible to modify compound potency against the en-
zyme. Initially, we selected benzamide pharmacophore
(4-Cl-C6H4)6 and studied the inhibitory effect of a het-
erocyclic substituent (3a–3j) on the enzymatic activity
of the resultant compounds 4–13 against VEGFR-2.
Two functions (g, h, Table 1) yielded compounds similar
in potency to PTK787 (10, 11; IC50 = 92 and 47 nM,
respectively). Weak activity was seen for the molecules
8 and 12 derived from e and i. Following these initial
data, we decided to continue optimization of the mole-
cules based on g and h. Good potency of these series
was explained by the proper alignment of the lower por-
tion of the molecule, namely pyridine-type nitrogen
atom(s) of a heterocycle (Lewis base: hydrogen bond
acceptor), with the Arg1302 moiety in the ATP-binding
pocket of VEGFR-2. This interaction may be critical for
a tight binding of a phthalazine analogue to a VEGFR-2
kinase.9,10 Furthermore, MMFF94 Force Field minimi-
zation studies suggest good overlap between series de-
scribed in this paper, as exemplified by 11 and the
development candidates PTK787 (A) and (C) (Fig. 1).
The targeted 2-((1H-azol-1-yl)methyl)-N-arylbenza-
mides 4–38 (Scheme 1) were accessed by a three-step
procedure. In the optimized procedure, o-chloromethyl
benzoic acid (1) was reacted with a series of anilines in
the presence of 1.2 equiv DIC in CH2Cl2 to give the
respective amides (2) in 76–91% yields. Alternative
amine coupling procedures involving DMAP, carbonyl
diimidazole, and benzotriazole afforded lower yields of
2. The resultant chlorides were reacted with anions gen-
erated in situ from the respective NH heterocycle (3a–j)
in DMF to furnish the targeted molecules 4–38 (45–64%
isolated yields). Notably, reactions of 2 with 3H-imi-
dazo[4,5-c]pyridine in the presence of K2CO3 furnished
products of a formal SN2 of chloride 2 with pyridine-,
instead of the anticipated imidazole-nitrogen atom at-
tack (e.g., 11, Table 1). The latter derivatives were not
detected in the reaction mixtures by LC–MS. Structures
of the resultant products were further confirmed by
NOE experiments.7
In the next step, we focused on studying SAR of the
amide portion of the molecule. The molecules substitut-
ed with p-Cl-, p-t-Bu-, and p-i-Pr- groups displayed
potencies similar to those of C and PTK787 with IC50
values of 47–92 nM in the enzymatic assay (e.g., 10,
11, and 14–17, Table 1).6 The (difluorochloro)methoxy
group (ClF2CO–, compounds 18, 19, IC50 = 73 and
95 nM, respectively) was also beneficial for VEGFR-2
inhibition. Small meta-substituents on the anilinic por-
tion of the molecule were tolerated (20, 21, IC50 = 0.47
and 0.35 lM, respectively). Similar ortho-substitution
abolished enzymatic inhibition (22, 23; IC50 > 10 lM
for both). Several di-substituted aniline fragments, for
example, 3,4-di-Cl- (26, 27; IC50 = 66 and 56 nM,
respectively), 4-Cl-3-CF3- (28, 29, IC50 = 0.31 and
0.44 lM, respectively), 2-F-4-Me- (30, 31; IC50 = 0.29
and 0.21 lM, respectively), and 3,4-methylenedioxy
groups (32, 33; IC50 = 0.13 and 0.23 lM, respectively)
also yielded potent compounds. Related ortho-fluoro
aminoaryl group has been exemplified in other VEG-
FR-2 inhibitors such as ZD 6474.5 Larger 4-substituents
Thirty-five compounds (4–38, Table 1) were tested in vi-
tro against isolated VEGFR-2. Specifically, we mea-
sured their ability to inhibit phosphorylation of a
biotinylated-polypeptide substrate (p-GAT, CIS Bio
International) in a homogeneous time-resolved fluores-
cence (HTRF) assay at an ATP concentration of
2 lM. The results were reported as a 50% inhibition con-
centration value (IC50). Literature VEGFR-2 inhibitors
(A–D) were included as internal standards for quality
control.8
As seen from Table 1, a number of 2-((1H-azol-
1-yl)methyl)-N-arylbenzamides exhibited robust inhibi-
tory activity against VEGFR-2. By varying both
amide- and (azol-1-yl)methyl substituents, it was

A. S. Kiselyov et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1726–1730
1729
3. The anti-angiogenic aptamer MacugenTM (Pegaptanib
sodium) has recently been approved to treat neovascular
age-related macular degeneration; see: Fine, S. L.; Martin,
D. F.; Kirkpatrick, P. Nat. Rev. Drug. Discov. 2005, 4,
187.
4. Bold, G.; Altmann, K.-H.; Jorg, F.; Lang, M.; Manley, P.
W.; Traxler, P.; Wietfeld, B.; Bruggen, J.; Buchdunger, E.;
Cozens, R.; Ferrari, S.; Pascal, F.; Hofmann, F.; Martiny-
Baron, G.; Mestan, J.; Rosel, J.; Sills, M.; Stover, D.;
Acemoglu, F.; Boss, E.; Emmenegger, R.; Lasser, L.;
Masso, E.; Roth, R.; Schlachter, C.; Vetterli, W.; Wyss,
D.; Wood, J. M. J. Med. Chem. 2000, 43, 2310.
5. (a) Hennequin, L. F.; Stokes, E. S. E.; Thomas, A. P.;
Johnstone, C.; Ple, P. A.; Ogilvie, D. J.; Dukes, M.;
Wedge, S. R.; Kendrew, J.; Curwen, J. O. J. Med. Chem.
2002, 45, 1300; (b) Wedge, S. R.; Kendrew, J.; Hennequin,
L. F.; Valentine, P. J.; Barry, S. T.; Brave, S. R.; Smith, N.
R.; James, N. H.; Dukes, M.; Curwen, J. O.; Chester, R.;
Jackson, J. A.; Boffey, S. J.; Kilburn, L. L.; Barnett, S.;
Richmond, G. H. P.; Wadsworth, P. F.; Walker, M.;
Bigley, A. L.; Taylor, S. T.; Cooper, L.; Beck, S.;
Juergensmeier, J. M.; Ogilvie, D. J. Cancer Res. 2005,
65, 4389.
Table 2. Compounds 11, 14, 18, 19, and 26 are ATP-competitive
inhibitors of VEGFR-2
Compound
Ki at IC50 (lM)
Ki at IC90 (lM)
11
14
18
19
26
0.09
0.13
0.16
0.22
0.17
0.11
0.15
0.21
0.19
0.21
on the arylamide portion of the molecule led to a dimin-
ished potency against the enzyme (34–38). For example,
4-Br derivatives (34, 35) lost almost 5-fold of activity
compared to the 4-Cl analogues 10, 11. Phenyl, phen-
oxy, and benzyl derivatives (36–38) furnished only mod-
erate potency against VEGFR-2. We speculated that
these functions cannot be properly accommodated in
the tight hydrophobic pocket of VEGFR-2.9 Similar
observation has been reported by the other group.4
Three selected VEGFR-2 inhibitors, namely 11, 14, 18,
19, and 26, all tested ATP-competitive in the radioas-
say11 (see Table 2).
6. (a) Manley, P. W.; Furet, P.; Bold, G.; Bruggen, J.;
¨
Mestan, J.; Meyer, T.; Schnell, C.; Wood, J. J. Med.
Chem. 2002, 45, 5697; (b) Manley, P. W.; Bold, G.;
Fendrich, G.; Furet, P.; Mestan, J.; Meyer, T.; Meyhack,
B.; Strauss, A.; Wood, J. Cell Mol. Biol. Lett. 2003, 8, 532;
(c) Altmann, K.-H.; Bold, G.; Furet, P.; Manley, P. W.;
Wood, J. M.; Ferrari, S.; Hofmann, F.; Mestan, J.; Huth,
All compounds were also tested in the HTRF format
against VEGFR-1. The results in Table 1 indicate that
VEGFR-2 active N-arylbenzamides display good activity
against VEGFR-1 as well. For the most active com-
pounds, the IC50 values were in the 0.14–0.70 lM range.
This outcome could be of benefit in the clinical setting
as both receptors are reported to mediate VEGF signaling
in the angiogenesis.12 Notably, several compounds con-
taining bulky lipophilic substituents at the benzamide
pharmacophore (14–16, 18, and 19) yielded 10- to 17-fold
selectivity for the VEGFR-2 versus VEGFR-1 kinase.
This observation suggests that it is possible to develop
VEGFR-2 specific inhibitors decoupled from the VEG-
FR-1 activity. Further screening of 4–38 against a number
of other receptor (IGF1R, InR, FGFR1, Flt3, ErbB1,
ErbB2, EphB4, and c-Met) and cytosolic (PKA, GSK3b,
PKB/Akt, bcr-Abl, and Cdk2/5) kinases in an HTRF for-
mat indicated no significant cross-reactivity (PI < 40%,
triplicate measurements) at a screening concentration of
10 lM.
A.; Kruger, M.; Seidelmann, D.; Menrad, A.; Haberey,
¨
M.; Thierauch, K.-H. US Patent 6,878,720 B2, 2005.
7. Similar reactivity of 3H-imidazo[4,5-c]pyridine toward
electrophiles has been reported, see: (a) Ra, W. W. K.;
Pacherb, K. G. R. Tetrahedron 1992, 48, 10549; (b)
Penning, T. D.; Chandrakumar, N. S.; Desai, B. N.;
Djuric, S. W.; Gasiecki, A. F.; Malecha, J. W.; Miyashiro,
J. M.; Russell, M. A.; Askonas, L. J.; Gierse, J. K.;
Harding, E. I.; Highkin, M. K.; Kachur, J. F.; Kim, S. H.;
Villani-Price, D.; Pyla, E. Y.; Ghoreishi-Haack, N. S.;
Smith, W. G. Bioorg. Med. Chem. Lett. 2003, 13, 1137,
Significant NOE’s for 11;
Cl
HN
O
δ 4.95
H
H
δ 8.39
δ8.55
δ 7.33
H
H
N
H
In summary, we have described a series of 2-((1H-azol-
1-yl)methyl)-N-arylbenzamides as potent ATP-competi-
tive inhibitors of the VEGFR-2 receptor. These
compounds are also inhibitors of the VEGFR-1 receptor.
All potent compounds are stable towards hydrolysis and
display good solubility (>3 mM) in the screening buffer.
The analogues presented in this Letter are potentially
useful in the treatment of conditions such as cancer.
Further details on their biological properties, such as
cell-based and functional activity, together with murine
oral exposure data, will be presented in due course.
N
N
Tests revealed that all potent compounds were stable
toward hydrolysis with amines (morpholine, piperazine),
alkoxy- and hydroxide anions at reflux in MeOH/H2O and
upon prolonged storage in DMSO. Furthermore, they
displayed good solubility in the screening buffer (>3 mM)
as indicated by HPLC studies.
Analytical data for the selected compounds 8: N-(4-
Chlorophenyl)-2-((5,6-dimethoxy-1H-benzo[d]imidazol-
1-yl))benzamide; mp 165–166 ꢁC; 1H NMR (400 MHz,
DMSO-d6) d, ppm: 3.76 (s, 6H, OMe), 4.88 (s, 2H, CH2),
7.11 (s, 2H), 7.20 (d, J = 8.4 Hz, 1H), 7.23 (m, 1H), 7.26
(d, J = 8.4 Hz, 2H), 7.40 (m, 1H), 7.61 (d, J = 8.4 Hz, 2H),
7.75 (d, J = 8.4 Hz, 1H), 8.01 (s, 1H), 13.20 (br s, exch
D2O, 1H, NH); 13C NMR (100 MHz, DMSO-d6) d, ppm:
47.5, 56.6,100.9, 122.6, 126.0, 127.3, 127.5, 128.7, 129.1,
129.4, 131.7, 132.1, 133.3, 133.9, 135.2, 141.4, 143.5, 165.0;
ESI MS (M+1): 423, (MÀ1): 421; HRMS, exact mass
calcd for C23H20ClN3O3: 421.1193. Found: 421.1185.
References and notes
1. (a) Klagsbrun, M.; Moses, M. A. Chem. Biol. 1999, 6,
R217; (b) Risau, W. Nature 1997, 386, 671.
2. The anti-angiogenic antibody AvastinTM (Bevacizumab)
has recently been approved to treat colorectal cancer; see:
Culy, C. Drugs Today 2005, 41, 23.

1730
A. S. Kiselyov et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1726–1730
Elemental analysis Calcd for C23H20ClN3O3: C, 65.48; H,
10. In order to establish N-arylbenzamide pharmacophores
essential for VEGFR-2 activity, we have prepared seven
analogues (39–45) of the potent inhibitors 10 and 14 (see
below).
4.78; N, 9.96. Found: C, 65.25; H, 4.97; N, 9.79. 10: 2-
((1H-Pyrrolo[3,2-c]pyridin-1-yl)methyl)-N-(4-chlorophe-
nyl)benzamide; mp 174–175 ꢁC; 1H NMR (400 MHz,
DMSO-d6) d, ppm: 5.04 (s, 2H, CH2), 6.22 (d, J = 6.8 Hz,
1H), 6.68 (d, J = 6.8 Hz, 1H), 7.15 (d, J = 8.0 Hz, 1H),
7.19 (d, J = 8.4 Hz, 2H), 7.28 (m, 1H), 7.33 (d, J = 8.8 Hz,
1H), 7.41 (m, 1H), 7.60 (d, J = 8.4 Hz, 2H), 7.82 (d,
J = 8.0 Hz, 1H), 8.33 (d, J = 8.8 Hz, 1H), 8.49 (s, 1H),
13.30 (br s, exch. D2O, 1H, NH); 13C NMR (100 MHz,
DMSO-d6) d, ppm: 51.5, 98.9, 112.4, 121.6, 122.5, 124.9,
125.2, 127.4, 129.0, 129.5, 130.3, 132.2, 133.5, 133.9, 135.8,
141.6, 148.2, 148.6, 165.6; ESI MS (M+1): 363, (MÀ1):
361; HRMS, exact mass calcd for C21H16ClN3O:
361.0982. Found: 361.0974. Elemental analysis Calcd for
C21H16ClN3O: C, 69.71; H, 4.46; N, 11.61. Found: C,
69.52; H, 4.27, N, 11.44. 11: 2-((5H-imidazo[4,5-c]pyridin-
5-yl)methyl)-N-(4-chlorophenyl)benzamide; mp 181–
183 ꢁC, 1H NMR (400 MHz, DMSO-d6), d, ppm: 4.95
(s, 2H, CH2), 7.17 (d, J = 8.0 Hz, 1H), 7.21 (m, 1H), 7.26
(d, J = 8.4 Hz, 2H), 7.33 (d, J = 8.8 Hz, 1H), 7.40 (m, 1H),
7.55 (d, J = 8.4 Hz, 2H), 7.68 (s, 1H), 7.75 (d, J = 8.4 Hz,
1H), 8.39 (s, 1H), 8.55 (d, J = 8.8 Hz, 1H), 13.30 (br s,
exch D2O, 1H, NH); 13C NMR (100 MHz, DMSO-d6) d,
ppm: 49.9, 112.6, 122.8, 126.1, 127.0, 128.5, 129.4, 130.3,
131.6, 133.4, 133.9, 135.7, 135.9, 139.8, 146.5, 147.1, 150.6,
165.2; ESI MS (M + 1): 364,(M À 1): 362; HRMS, exact
mass calcd for C20H15ClN4O: 362.0934. Found: 362.0924.
Elemental analysis Calcd for C20H15ClN4O: C, 66.21; H,
4.17; N, 15.44. Found: C, 65.96; H, 4.03, N, 15.22.5.
Cl
Cl
HN
N
HN
N
N
N
HN
N
O
O
O
N
N
N
N
39
40
41
42
Cl
Cl
Cl
N
N
O
N
HN
N
O
O
H
N
N
H
N
N
45
43
44
Of these, only the benzimidazole 44 (rigid isostere of the
secondary amide bond in 10) displayed marginal activity
against VEGFR-2 (IC50 = 2.66 0.40 lM). Compounds
39 and 40 were also hydrolytically unstable upon storage
in DMSO and screening media (LC–MS analysis). Substi-
tution at the central benzene ring (naphthalene 41) was
not tolerated. Tertiary amide (42), ester (43), and 5-aryl
benzimidazole derivative (45) were inactive in the enzy-
matic assay (PI < 20% at 10 lM). Based on these observa-
tions, we concluded that both secondary amide and
flexible benzylic linker are required for the activity of
the N-arylbenzamide derivatives. In addition, we found
that N-(4-chlorophenyl)benzamides modified with 5-mem-
bered heterocycles (imidazole and tetrazole) were inactive
against both VEGFR-2 and VEGFR-1. This was presum-
ably due to lack of interaction of the respective 2-((1H-
azol-1-yl)methyl) group with the Arg1032 moiety.
8. VEGFR tyrosine kinase inhibition was determined by
measuring the phosphorylation level of poly-Glu-Ala-Tyr-
biotin (pGAT-biotin) peptide in a homogeneous time-
resolved fluorescence (HTRF) assay. Into a black 96-well
Costar plate was added 2 ll/well of 25· compound in
100% DMSO (final concentration in the 50 ll kinase
reaction was typically 1 nM to 10 lM). Next, 38 ll of
reaction buffer (25 mM Hepes, pH 7.5, 5 mM MgCl2,
5 mM MnCl2, 2 mM DTT, and 1 mg/ml BSA) containing
0.5 mmol pGAT-biotin and 3–4 ng KDR enzyme was
added to each well. After 5–10 min preincubation, the
kinase reaction was initiated by the addition of 10 ll of
10 lM ATP in reaction buffer, after which the plate was
incubated at room temperature for 45 min. The reaction
was stopped by addition of 50 ll KF buffer(50 mM Hepes,
pH 7.5, 0.5 M KF, and 1 mg/ml BSA) containing 100 mM
EDTA and 0.36 lg/ml PY20K (Eu-cryptate labeled anti-
phosphotyrosine antibody, CIS Bio International) was
added and after an additional 2 h incubation at room
temperature, the plate was read in a RUBYstar TRF reader.
9. (a) McTigue, M. A.; Wickersham, J. A.; Pinko, C.;
Showalter, R. E.; Parast, C. V.; Tempczyk-Russell, A.;
Gehring, M. R.; Mroczkowski, B.; Kan, C. C.; Villafran-
ca, J. E.; Appelt, K. Structure 1999, 7, 319; (b) Berman, H.
M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T. N.;
Weissig, H.; Shindyalov, I. N.; Bourne, P. E. The Protein
Data Bank, Nucleic Acids Res. 2000, 28, 235–242.
11. Competition assays were conducted with varying concen-
trations (0–500 lM) of ATP. Specifically, five different
concentrations of [32P]ATP were incubated with VEGFR-
2 in the absence, IC50 or IC90 concentration of the
inhibitors for 45 min at RT. A double reciprocal graph of
the degree of phosphorylation (1/cpm) against ATP
concentration (1/[ATP]) was plotted. The data were
analyzed by a non-linear least-squares program to deter-
mine kinetic parameters using GraphPad software. Deter-
mined Ki values for the five selected compounds are listed
in Table 2.
12. (a) Hanahan, D.; Folkman, J. Cell 1996, 86, 353;
(b) Folkman, J.; Klagsburn, M. Science 1987, 235,
442; (c) Zachary, I. Biochem. Soc. Trans. 2003, 31,
1171; (d) Eskens, F. Br. J. Cancer 2004, 90, 1.