Synthesis and evaluation of heteroaryl-ketone derivatives as a novel class of VEGFR-2 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.06.083

We have discovered novel inhibitors of VEGFR-2 kinase with low nanomolar potency in both enzymatic and cell-based assays. Active series are heteroaryl-ketone compounds containing a central aromatic ring with either an indazolyl or indolyl keto group in th

Full text of DOI:10.1016/j.bmcl.2008.06.083

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4344–4347
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of heteroaryl-ketone derivatives as a novel class
of VEGFR-2 inhibitors
a
a
a
Eugene L. Piatnitski Chekler a, , Reeti Katoch-Rouse , Alexander S. Kiselyov , Dan Sherman ,
*
Xiaohu Ouyang a, Ki Kim a, Ying Wang b, Yaron R. Hadari b, Jacqueline F. Doody b
a Department of Chemistry, ImClone Systems Incorporated, New York, NY 10014, USA
b Department of Protein Science, ImClone Systems Incorporated, New York, NY 10014, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We have discovered novel inhibitors of VEGFR-2 kinase with low nanomolar potency in both enzymatic
and cell-based assays. Active series are heteroaryl-ketone compounds containing a central aromatic ring
with either an indazolyl or indolyl keto group in the ortho orientation to the benzylic amine group (Fig. 1).
The best compounds were demonstrated to be inactive against a small select panel of tyrosine and serine/
threonine kinases with the exception of VEGFR-1 kinase, a close family member. In addition, the lead can-
didate 8 displayed acceptable exposure levels when administered orally to mice.
Received 8 May 2008
Revised 20 June 2008
Accepted 24 June 2008
Available online 28 June 2008
Keywords:
Angiogenesis
Ó 2008 Elsevier Ltd. All rights reserved.
Kinase inhibitor
Vascular endothelial growth factor
Angiogenesis, or formation of new blood vessels, is a highly
complex process that involves proliferation, migration, and tissue
infiltration by capillary endothelial cells from pre-existing blood
vessels. It is an important physiological process involved in embry-
onic development,1,2 follicular growth, and wound healing as well
as in pathological conditions such as tumor growth.3–6 Vascular
endothelial growth factor (VEGF),7,8 an endothelial cell-specific
mitogen, is the primary regulator of angiogenesis in vivo and it
mediates its biological effect through high-affinity VEGF tyrosine
kinase receptors, which are expressed on the surface of endothelial
cells.9 VEGF binds with high affinity to VEGFR-2 (also known as Ki-
nase Domain Region (KDR)7,10,11 to induce activation of the angio-
genic signaling pathway. Murine gene knockouts of VEGFR-2 pr
VEGF have led to embryonic lethality, a result of disorganized vas-
cular endothelial cells, indicating the pivotal role of VEGF in angi-
ogenesis.12–14 Additionally, several antagonists and inhibitors
currently in preclinical and clinical development have shown tu-
mor regression concomitantly with inhibited angiogenesis.15,16
Therefore, a direct inhibition of the kinase activity of VEGFR-2
should result in the reduction of angiogenesis and the subsequent
suppression of tumor growth.
based inhibitors recapitulated the potency and binding mode of
PTK787.17 Promising biological data for compound 4 prompted
us to proceed with the structure–activity analysis of the series,
the results of which are presented in this letter. The heteroaryl-ke-
tone series is a novel entry in this class of inhibitors which seek to
exploit a similar mode of binding to the kinase domain of VEGFR-2.
One of the key structural features of the heteroaryl-ketone series
was the ortho substitution of the central aromatic ring with a ke-
tone moiety and an anilinyl NH group (Fig. 1). We speculated that
the resulting intramolecular hydrogen bond would consequently
hold the shape of the molecule into a pseudo-bicyclic ring system.
Tight binding
R2
pocket
Hydrophobic
region
Region I
N
N
X
R3
Key hydrogen
bond interaction
to hold the
shape of the
molecule
Region II
Aromatic Ring
(Rigid)
O
H
During screening of the ImClone compound collection, we iden-
tified a heteroaryl-ketone compound 4 that demonstrated promis-
ing inhibition of VEGFR-2. We were encouraged to pursue the hit
by the precedent set by Manley et al. in which anthranilamide-
R1
Region III
X = N or CH
Variable
* Corresponding author. Tel.: +1 718 473 5557; fax: +1 484 865 9399.
Figure 1. Pharmacophore hypothesis for the mode of binding of heteroaryl-ketones
E-mail address: piatnits@gmail.com (E.L. Piatnitski Chekler).
within the ATP-binding pocket of VEGFR-2.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.06.083

E. L. Piatnitski Chekler et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4344–4347
4345
O
H
O
N
H3C
Si CHN2
CHN2
R3
H
a
Cl
a, b
H3C
N
R3
CH3
NO2
NO2
O
2
1
16
NO2
c
17
COOH
b
R2
NH2
R2
N
N
H
N
N
R3
R3
HN
N
d
O
O
O
c, d
O
NH
NH
R1
NH2
23
24
NO2
3
R1
18
e
19-41
4-12
3
3
3
Scheme 2. Reagents and conditions: (a) 2-nitrobenzoyl chloride, EtMgBr, THF, À78
to 0 °C; (b) LiOH, MeOH, THF, rt; (c) H2, 10%Pd/C, EtOAc or EtOH; (d) R1CHO, PhH,
100 °C then Na(OAc)3BH; (e) (CH3)2SO4, NaH, THF, 0 °C.
f, c, d
e, c, d
f, c, d
13
14
15
Scheme 1. Reagents and conditions: (a) Et3N, Et2O, 0 °C; (b) Isoamyl nitrite, CH2Cl2/
Acetone; (c) H2, Pd/C, MeOH or EtOAc; (d) R1CHO, PhH, 100 °C then Na(OAc)3BH; (e)
2-Methoxyethyl bromide, K2CO3, DMF; (f) CH3I, K2CO3, DMF.
X
HN
Y
H
a
X
N
The general preparation of the heteroaryl-ketone analogs is de-
picted in Scheme 1.18 The initial strategy was to keep the top por-
tion of the molecule as an indazole moiety and vary only the
bottom portion in order to optimize the interactions in Region III.
Treatment of 2-nitro benzoylchloride 1 with trimethylsilyl diazo-
methane gave the diazo intermediate 2 that was further reacted
with anthranilic acid in the presence of isoamyl nitrite to give 2-ni-
tro ketoindazole 3. The amine moiety that was obtained via reduc-
tion of the nitro group was subjected to reductive amination with a
variety of aldehydes to provide the final compounds 4–15 (Table
1). For the indole derivatives, the synthetic route was modified
as shown in Schemes 2 and 3. A variety of Lewis acid based meth-
ods were pursued to introduce the 2-nitro acyl group in position 3
of indole ring system (Scheme 2). None of these methods produced
the desired product in appreciable yields and often gave complex
reaction mixtures. Reaction of the substituted indole 16 with the
acid chloride in the presence of the Grignard reagent gave a 1,3-
Y
O
NO2
43: X = CH, Y = N
44: X = N, Y = CH
45: X = CH, Y = N
46: X = N, Y = CH
X
b,c
HN
Y
47: X = CH, Y = N
48: X = N, Y = CH
O
NH
N
Scheme 3. Reagents and conditions: (a) 1, AlCl3, CH2Cl2; (b) H2, Pd/C, THF, EtOH; (c)
Quinoline-6-carboxaldehyde, PhH, AcOH then Na(AcO)3BH.
diacylated product which was subjected to mild hydrolysis condi-
tions to achieve a quantitative yield of the desired mono-acylated
product 17. The nitro group was then reduced and the resulting
amine moiety was subsequently subjected to reductive amination
conditions to produce a series of final target compounds 19–41
(Table 2).
Table 1
Modification of Region III in Indazole series
Compound R1
R2
KDR-HTRF Cell-based
(IC50 phosphorylation
M)a
(IC50
M)a
l
l
Furthermore, two azaindole analogs were also synthesized to
explore heteroaromatic substitution patterns in Region I (Scheme
3). A modified Lewis-acid-catalyzed acylation worked well in this
case to produce intermediates 45 and 46.19 The nitro group in
the mono-acylated products was then reduced and subjected to
reductive amination resulting in the desired compounds 47 and 48.
The first stage of exploring the SAR of heteroaryl-ketones was to
modify Region III (Table 1). The pyridyl group in 4 gave an encour-
4
5
6
7
8
4-Pyridyl
4-Pyrazolyl
5-Indazolyl
6-Indazolyl
6-Quinolinyl
6-Isoquinolinyl
4-Acetylaminophenyl
piperonyl
5-Benzimidazolyl
6-Quinolinyl
6-Indazolyl
H
H
H
H
H
H
H
H
1.6 0.4
>10
0.80 0.1
0.73 0.19 0.074 0.004
0.23 0.03 0.13 0.3
10.0 2.0
>10
>10
3.0 0.1
8.5 1.5
0.87 0.12
—
0.12 0.3
9
—
—
10
11
12
13
14
2.6 0.3
0.31 0.05
H
CH3
b
>10
aging cell-based phosphorylation assay IC50 of 0.87 lM; however,
2-Methoxy 0.56 0.07 1.8 0.5
ethyl
CH3
the corresponding pyrazole derivative 5 turned out to be inactive.
It was assumed that the nitrogen in a smaller group such as pyra-
zole was not within reach of the key interacting amino acid.20 This
hypothesis could also be confirmed via the presence of a larger
indazole group (e.g., 6 and 7) where the nitrogens in the indazole
moieties are most likely picking up the key interaction resulting
15
6-Indazolyl
0.25 0.04 0.30 0.08
a
IC50 values were determined from the logarithmic concentration–inhibition
point (at least eight points). The important key values are given as the mean of at
least two duplicate experiments.
b
Precipitation was observed for this compound under the assay conditions.

4346
E. L. Piatnitski Chekler et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4344–4347
Table 2
Table 3
Modifications of Region I and III in Indole series
Correlation of VEGFR-1 and VEGFR-2-binding data
Compound R1
R2 R3
KDR-HTRF
Cell-based phosphorylation
(IC50 lM)
Compound
VEGFR-2 (IC50
l
M)
VEGFR-1 (IC50 lM)
a
a
(IC50
lM)
4
6
7
8
1.6
0.8
0.73
0.23
1.9
19
20
21
22
23
4-Pyridyl
H
H
H
H
H
H
H
H
H
5-
CH3
9.0 1.0
0.61b
2.1 0.7
0.46
0.27
0.30
5-Indazolyl
6-Indazolyl
6-Quinolinyl
6-Quinolinyl
0.24 0.07
0.17 0.01
1.0 0.3
0.14b
2.0 0.5
1.9 0.15
0.16 0.02
0.54b
0.18 0.03
0.73 0.15
0.70 0.01
—
(e.g., 24) resulted in a smaller change in activity as compared to the
non-methylated compound 23 indicating that a small hydrophobic
group is tolerable at that site. This notion is reiterated by the 7-
methyl analogues 26 and 36 which retain activity. The azaindole
analogs 47 and 48 were found to be inactive, suggesting that a
nitrogen atom in the ring is not tolerated in the positions facing
hydrophobic regions of the protein backbone (Fig. 1).20 Overall,
the SAR trend was similar in both the indazole and indole cases,
where the 6-indazolyl group in Region III consistently gave slightly
better activity than the corresponding quinolinyl group.
Characterization of the heteroaryl-ketones indicates that this
series is highly selective for VEGF family receptors. When
compounds 4 and 6–8 were screened against a panel of several
tyrosine and serine/threonine kinases (EGFR, ErbB2, Raf-1, c-Met,
IGF1-R, InsR, CDK2, and PKB), the only kinase besides VEGR-2 that
was targeted was VEGFR-1, a close family member (Table 3). More-
over, inhibition of VEGFR-2 correlates well with that seen with
VEGFR-1, indicating that this series would target both receptors in-
volved in angiogenesis. ATP competition experiments demon-
strated that this series acts as a direct and reversible competitor
of ATP (data not shown). Representative compounds were not toxic
to a pool of human hepatocytes or cytotoxic to NIH3T3 fibroblast
24
25
26
27
28
29
30
6-Quinolinyl CH3 5-
CH3
6-
6-Quinolinyl
6-Quinolinyl
6-Quinolinyl
6-Quinolinyl
6-Quinolinyl
6-Quinolinyl
H
H
H
H
H
H
0.94 0.06
2.0b
CH3
7-
CH3
2-
CH3
4-
OCH3
5-
>10
>10
—
1.9b
0.45 0.15
0.66 0.11
OCH3
6-
1.9 0.3
OCH3
5-F
6-F
5-Cl 3.9 0.8
5-
31
32
33
34
6-Quinolinyl
6-Quinolinyl
6-Quinolinyl
6-Indazolyl
H
H
H
H
5.9b
1.9b
1.0 0.2
0.81 0.02
0.56 0.03
0.052 0.015
0.87 0.13
0.25 0.09
0.95 0.6
>10
CH3
6-
CH3
7-
CH3
2-
35
36
37
6-Indazolyl
6-Indazolyl
6-Indazolyl
H
H
H
0.076 0.005
0.355 0.05
—
CH3
5-F
6-F
38
39
40
41
6-Indazolyl
6-Indazolyl
6-Indazolyl
6-
H
H
H
H
1.8 0.7
1.5 0.5
0.25 0.03
0.25 0.02
0.83 0.06
—
cells (GI50 > 100 lM).
5-Cl 1.1 0.3
>10
When 8 was administered to mice (interperitoneally, 30 mg/kg
H
Isoquinolinyl
6-Quinolinyl
6-Quinolinyl
in a solution of 5% ethanol, 5% Tween-80, 5% PEG400 and 85% PBS),
plasma concentration of 2.4 lM was present at 1 h post-injection;
47
48
H
H
5-(N) >10
7-(N) 7.8 0.3
—
>10
however, at 4 h compound concentration was below detection lim-
its. Mass spectroscopy revealed that 8 was glucuronidated, pre-
sumably leading to its rapid elimination. Therefore, we explored
indole compound 24 with an N-methyl indole moiety that should
prevent the formation of glucuronidation adduct. When this com-
pound was also dosed interperitoneally, plasma levels at the 1 h
a
IC50 values were determined from the logarithmic concentration–inhibition
point (at least eight points). The important values are given as the mean of at least
two duplicate experiments.
Precipitation was observed for the compound under the enzymatic assay con-
ditions hence the data for n = 1 are depicted. However, the corresponding cellular
data are shown for n = 2.
b
time point (2.9
the plasma concentration of 24 was ꢀ1.1
with no glucuronidation products detectable. Moreover, both ana-
l
M) were similar to those seen with 8. Notably,
l
M at the 4-h time point
in the observed activity. The substitution of these heterocyclic moi-
eties with a carbonyl group (10) resulted in a negative outcome.
The 6-Quinolynyl group as R1 would position the basic nitrogen
proximal to the 5- and 6-inadzolyl nitrogen atoms. Thus, the quin-
olinyl and indazolyl moieties were found to be optimized groups in
Region III that resulted in nanomolar activity. Incorporation of a
solubilizing chain (methoxyethyl group) into Region I led to enzy-
matically similarly potent compound 14, but a significant loss of
cellular potency was observed.
In the interest of synthetic tractability, we confined our explo-
ration of Region I SAR to the indole derivatives (Table 2). The quin-
olinyl and indazolyl groups were maintained in Region III as they
conferred superior potency compared to other moieties (Table 2).
With the quinolinyl group in Region III, the 5-methylindole deriv-
ative 23 was found to be the most active in the cell-based assay
logues 8 and 24 demonstrated plasma exposure (ꢀ1.8
lM at 1 h)
upon oral administration.
We have described the synthesis and biological activity of a no-
vel series of potent VEGFR-2 inhibitors based on a heteroaryl-ke-
tone scaffold. This series demonstrated potent cell-based
inhibition of KDR autophosphorylation and selectivity against a
group of tyrosine and serine/threonine kinases. Compounds bear-
ing the 6-indazolyl group in Region III were slightly more potent
compared to analogs with the corresponding quinolinyl group.
However, the quinolinyl analog 8 showed oral exposure in mice.
The most active compounds 7, 34, and 35 were comparable or
more inhibitory to VEGFR-2 receptor than the standard clinical
candidate ZD647421 (IC50 100 nM) in our cellular phosphorylation
assay.
with an IC50 = 0.16 lM. With the indazolyl in Region III, the 5-
and 6-methyl compounds showed comparable activity in cellular
assay (34 and 35). The loss of activity observed in analogs where
the indole was substituted in the 2 position with a methyl group
(27 and 37) could be attributed either to tight spacing in that re-
gion of the kinase active site or to dihedral angle alteration be-
tween the indole group and the central phenyl ring. However,
the substitution of the free NH group in indole with a methyl group
Acknowledgments
The authors thank E. Wu, C. Balagtas, R. Rolser, K. H. Yu, S. Patel
and Dr. D. Milligan for providing in-vitro data on the compounds.
We also acknowledge helpful discussions with Dr. M. Duncton,
Dr. W. Wong, L. Smith, Dr. K. Kim and Dr. J. Kawakami. We also
thank Dr. M. Labelle for proof reading the document.

E. L. Piatnitski Chekler et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4344–4347
4347
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