Structure-guided identification of novel VEGFR-2 kinase inhibitors via solution phase parallel synthesis

Article abstract of DOI:10.1016/j.bmcl.2006.01.063

Structural analysis of the essential binding elements of the oxindole-based kinase inhibitor (1) led to the identification of a novel class of heterocyclic-substituted pyrazolones. Knoevenagel condensation of a variety of activated methylene nucleophiles with indole or pyrrole carboxaldehydes provided a focused library of molecules, each containing elements of kinase pharmacophore probe. Initial screening for VEGFR-2 kinase inhibition eliminated several of the probes. Identification of an active pyrazolone motif and further optimization resulted in several highly potent VEGFR-2 inhibitors with cellular efficacy, anti-angiogenic activity ex vivo in rat aortic ring explant cultures, and oral anti-tumor efficacy in nude mice.

Full text of DOI:10.1016/j.bmcl.2006.01.063

Bioorganic & Medicinal Chemistry Letters 16 (2006) 2158–2162
Structure-guided identification of novel VEGFR-2 kinase
inhibitors via solution phase parallel synthesis
*
Rabindranath Tripathy, Alyssa Reiboldt, Patricia A. Messina, Mohamed Iqbal,
Jasbir Singh, Edward R. Bacon, Thelma S. Angeles, Shi X. Yang, Mark S. Albom,
Candy Robinson, Hong Chang, Bruce A. Ruggeri and John P. Mallamo
Cephalon, Inc., Discovery Research, 145 Brandywine Parkway, West Chester, PA 19380, USA
Received 18 November 2005; revised 12 January 2006; accepted 13 January 2006
Available online 3 February 2006
Abstract—Structural analysis of the essential binding elements of the oxindole-based kinase inhibitor (1) led to the identification of a
novel class of heterocyclic-substituted pyrazolones. Knoevenagel condensation of a variety of activated methylene nucleophiles with
indole or pyrrole carboxaldehydes provided a focused library of molecules, each containing elements of kinase pharmacophore
probe. Initial screening for VEGFR-2 kinase inhibition eliminated several of the probes. Identification of an active pyrazolone motif
and further optimization resulted in several highly potent VEGFR-2 inhibitors with cellular efficacy, anti-angiogenic activity ex vivo
in rat aortic ring explant cultures, and oral anti-tumor efficacy in nude mice.
ꢀ
2006 Elsevier Ltd. All rights reserved.
Despite tremendous advancement in the field of combi-
natorial chemistry relating to drug discovery, skepticism
has been expressed at the success rate of generating lead
structures against molecular targets from such large
H C
3
H C
3
N
CH3
O
H
O
+
CH3
O
N
N
H
1
N
H
1
H
H
chemical libraries. This drawback has resulted in an ini-
tiation of efforts to explore more focused libraries with
drug-like physical properties and reduced molecular
2
3
Oxindole
SU 5416
Step 1
Our Strategy
2
weight. Herein we report, a structure-guided chemical
library building process, where step-by-step structural
modifications have generated a novel class of vascular
endothelial growth factor receptor-2 (VEGFR-2) kinase
inhibitors with oral anti-tumor efficacy in nude mice.
Examination of a known class of oxindole-based inhib-
itors (1, Scheme 1) identified the key binding elements.
Retrosynthetic analysis fragmented these inhibitors into
active methylene nucleophiles and aryl aldehydes.
H
3
C
Library generation via
Knoevenagel condensation
H C
3
O
N
O
CH3
+
CH3
H
N
H
N
O
H
H
N
H
Oxindole replacement Pyrrole aldehyde
New Lead via
VEGFR-2 Kinase Assay
Step 2
3
Exploiting the classic Knoevenagel condensation intro-
duced structural diversity in a rapid fashion.
R
Replacement of pyrrole
carboxaldehyde
O
O
O
+
N
Major organic reactions (such as Aldol, Diels–Alder,
etc.,) generate typical structural patterns in products
H
N
R
H
H
Lead Optimization via
Structural Diversity
Scheme 1. Retrosynthesis of SU 5416 and kinase re-design strategy.
Keywords: VEGFR-2; Kinase; Inhibitors; Pyrazolones; Oxindole;
Anti-tumor; Angiogenesis; Knoevenagel condensation; Solution phase
parallel synthesis.
they form and these patterns become diagnostic features
in retrosynthetic analysis during synthesis of complex
substances such as natural products. Additionally, if
*
Corresponding author. Tel.: +1 610 738 6137; fax: +1 610 738
558; e-mail: rtripath@cephalon.com
4
6
0
960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.01.063

R. Tripathy et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2158–2162
2159
such a structural pattern present in a molecule consti-
tutes an essential recognition motif against a target pro-
tein, then retrosynthetic analysis can play a role in a
chemical library building process. By following the reac-
tions as depicted by a retrosynthesis, an essential struc-
tural motif against that protein can be generated and
grafted on different chemical structures simply by vary-
ing the reaction components. As a result, architectural
diversity of structures with preservation of essential rec-
ognition elements and depletion of undesired segments
of the original molecule can be achieved.
O
O
H
N
H
S
N
HN
NH
N
O
NH
O
HN
O
O
O
R
4
6
7
5
H
N
N
H
O
H
O
R
N
O
N
O
Ph
Z
H
O
N
N
HN
H
CH3
8
9
H C
3
1
0
Initial hit IC50 (VEGFR 2) = 2.7 μμ M
5
Vascular endothelial growth factor (VEGF) and the cell
surface receptors in human, VEGFR-2 (also known as
Figure 1.
6
KDR; kinase domain containing receptor), are consid-
7
the 3-substituent of our lead structure 10, by using dif-
ferently substituted pyrazolones.
ered to play an important role in angiogenesis, which is
8
vital for survival and proliferation of tumor cells. In
recent years, several classes of small molecule-based
VEGFR-2 kinase inhibitors have emerged as promising
anti-angiogenic agents for possible treatment against a
wide variety of cancers. One of the pioneering classes
of compounds in this area of research belongs to
A variety of pyrazolones (6) could be generated from the
4
1
reaction of b-ketoesters (11) and hydrazine hydrate.
9
These were subsequently condensed with 3,5-dimethyl
pyrrole carboxaldehyde (3) in ethanol and using piperi-
dine as the catalyst (Scheme 2). This method of parallel
library generation in solution was found favorable, as a
single geometrical isomer of the double bond precipitat-
ed out of the reaction mixture in most cases and simple
filtration provided compounds with high purity. We
were pleased to find that the 3-substituent on the pyra-
zolones plays a dramatic role in enhancing potency
3
-substituted indolin-2-ones, developed in the laborato-
ry of Sugen, Inc. This motif was shown to interact with
the kinase at the ATP binding site (X-ray study), where
the indoline core occupies the adenine segment of the
1
0
ATP. Of particular interest is the indolin-2-one, SU
9
a
5
416 (1). The five-membered ring lactam unit of 1
forms two critical hydrogen bonding interactions with
the enzyme. Structure–activity relationship (SAR) of 1
also showed that the exocyclic double bond and substi-
tuted pyrrole unit are crucial for kinase inhibitory activ-
ity. Retrosynthetic analysis of SU 5416 (1, Scheme 1)
shows that the essential part of the pharmacophore
(
Fig. 2). In general, it was found that pyrazolone with
an attached heterocyclic ring is preferred, while two het-
ero atoms on the heterocyclic ring demonstrate greater
VEGFR-2 kinase inhibition.
(
i.e., the lactam unit and the exocyclic double bond)
belongs to a structural architecture of Knoevenagel con-
densation reaction, and the compound is easily formed
(Scheme 1) by the reaction of oxindole (2) with an
appropriate pyrrole aldehyde (3).
O
NH -NH .xH O
EtOH/reflux
H
H
O
O
2
2
2
N
N
O
N
H
CH3
R
OR'
+
H C
3
R
11
6
3
1
1
In our continuing interest in kinase library generation,
EtOH/Piperidine/Heat
a strategy to generate a novel class of VEGFR-2 kinase
inhibitors was to build in chemical diversity around the
key lactam-enone moiety of 1. Our aim was: (1) to re-
place the oxindole moiety with another cyclic lactam
Pyrazolone
Scheme 2.
(
Step 1, Scheme 1) a critical part of our design, (2) to
preserve the 3,5-dimethyl pyrrole segment, a pharmaco-
phore presentation similar to 1, and (3) select an oxin-
dole replacement and expand the scope of this strategy
by substituting the 3,5- dimethyl pyrrole segment with
various aryl aldehydes as the condensation component
in a parallel synthesis fashion (Step 2, Scheme 1).
H
H
N
N
N
N
O
O
S
N
H
H
1
0
N
N
O
2
.7 μM
CH3
CH3
H C
H C
3
3
12
57 nM
13
Commercially available or known active methylene
compounds (4–9) bearing a cyclic lactam unit were
subjected to Knoevenagel condensation with 3,5-
dimethyl pyrrole-2-carboxaldehyde (3) and the resulting
products were tested against VEGFR-2 kinase to gener-
ate a possible hit (Fig. 1). After screening a set of com-
pounds, we were able to generate our first hit (10) with a
1
63 nM
H
N
N
O
N
H
N
N
CH
3
H C
3
1
2a
14
3 nM
2
densation involving 3-phenyl-substituted pyrazolone as
.7 lM inhibitory activity,
which came from a con-
4
1
3
a single geometrical isomer. We further optimized
Figure 2.

2160
R. Tripathy et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2158–2162
After successful oxindole replacements were in hand, we
turned our attention to the 2,5 dimethyl pyrrole segment
of the molecules. Commercially available aryl carboxal-
dehydes were condensed with a series of hetero-substi-
tuted pyrazolones. After determining VEGFR-2 kinase
inhibition, we found that N-methyindole-3-carboxalde-
hyde (15) was a potential replacement for our pyrrole,
as the corresponding condensation products (data
shown for 16 and 18, Fig. 3) have comparable potencies
Structure–activity relationship (SAR) for analogs of
compounds 16 and 18, substitution on the indole ring,
showed further potency enhancement against VEGFR-
2 kinase, Table 1 recording representative examples. A
variety of substituents are tolerated on the benzene ring
of the indole nucleus, particularly at the C-4 position,
resulting in several molecules with potency <10 nM. In
another set of SAR, it was found that N-methyl group
on the indole segment is favored for VEGFR-2
1
5
16
(
14 vs 16 and 13 vs 18). Reduced kinase inhibitory
activity.
activities of the compounds 17 and 19, where the lactam
NH is arylated and the exocyclic double bond has been
saturated, respectively (Fig. 3), provide evidences
regarding the importance of the lactam-enone pharma-
In general, pyrazolone-based compounds demonstrated
better inhibition of human VEGFR-2 in human umbilical
vein endothelial (HUVEC) cell-based phosphorylation
assays than against murine VEGFR-2 (FLK-1) in SVR
1
0
cophore of the pyrazolones.
H
Ph
N
N
O
N
N
N
O
CHO
N
N CH3
N
N CH3
N
N
CH3
1
6
17
15
3
7 nM
(23% at 1 μM)
H
H
N
N
N
O
N
O
N
N
S
N CH3
S
N CH3
1
8
19
(5% at 1 μM)
4
6 nM
Figure 3.
Table 1. Representative examples of SAR of pyrazolones on the benzene ring of the indole segment
H
N
N
O
H
N
N
N
S
Het.
2
X =
Y =
N CH3
R
a
b
c
Compound
Het.
R
H
IC50 (nM)
Cell score
HUVEC
SVR
1
1
1
1
1
1
1
1
1
1
1
1
1
1
6
X
X
X
X
X
X
X
X
X
Y
Y
Y
Y
Y
37
23
10
30
17
9
—
4
—
1
1
1
1
4
4
4
4
0
0
0
0
2
6a
6b
6c
6d
6e
6f
6g
6h
8
5-F
5-Cl
4
5-OMe
4-F
4-Cl
4
4
4
4-Br
4-OMe
6
4
26
17
46
49
59
45
32
4
5,6-O–CH
H
2
–O-
–O-
4
4
8a
8b
8c
8d
5-F
5-Cl
1
—
1
4-Br
5,6-O–CH
2
4
a
b
c
A single geometrical isomer was isolated in each case. See Ref. 17.
Ref. 12a.
Ref. 12b.

R. Tripathy et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2158–2162
2161
cell-based phosphorylation assays. The data for HUVEC
100
75
*
**
versus SVR (SVEN 1 ras, murine endothelial pancreatic
islet cells) are comparable for the inhibitors bearing 4-
methoxy and halogen substituents on the indole ring of
the pyrazine-based inhibitors (16e–g, Table 1). The cell
activities for methylenedioxy group at 4 and 5 positions
were also found to be good (16h and 18d, Table 1).
*
*
*
*
5
0
5
0
2
A number of these compounds have shown significant
anti-angiogenic activity ex vivo in rat aortic ring explant
4
nM
40nM
100nM
400nM
1
8
cultures, inhibition of HUVEC capillary tube forma-
1
Figure 4. Dose-related effects of the pyrazolone VEGFR-2 kinase
inhibitor 18d on angiogenesis (microvessel sprouting) in serum-free
collagen gel cultures of rat aortic ring explants maintained in serum-
free MCDB 131 medium in the absence of exogenous VEGF. Values
are the percent inhibition of microvessel growth during the peak phase
of growth (day 8) relative to untreated controls; n = 8 replicates/
concentration. Inter-assay variability <10%. **p < 0.01 relative to
untreated controls by the Student Newman–Keuls test.
9
tion in vitro (data not shown), and significant anti-tu-
mor efficacy following oral administration to nude mice
2
0
bearing SVR murine angiosarcoma xenografts. Data
have been shown for a representative compound 18d,
which was easily synthesized from the reaction of the
pyrazolone 22 with the indole aldehyde 24 by heating
in ethanol in the presence of piperidine (Scheme 3).
Selectivity profile against a representative panel of ki-
nases for 18d is shown in Table 2. While poor enzymatic
inhibitions for 18d were observed against PKC, Trk A,
and CDK1/Cyclin B1, significant activities were ob-
served against other VEGFR isozymes.
700
Vehicle (Tween:PG, 1:1) p.o. BID,n=10
6
5
4
3
00
00
00
00
18d, 30 mg/kg/dose p.o.BID, n=10
The inhibitor 18d showed dose-related inhibition in
microvessel growth relative to untreated control during
peak phase of vessel growth, with 55%, 66% (p < 0.01),
*
200
***
**
1
00
0
**
***
7
5% (p < 0.01), and 90% (p < 0.001) inhibition observed
**
4
at 4, 40, 100, and 400 nM, respectively (Fig. 4), clearly
indicating anti-angiogenic properties associated with
pyrazolone-based VEGFR-2 kinase inhibitors.
0
2
6
8
10
12
Days of Treatment
Figure 5. Effects of VEGF-R2 kinase inhibitor, 18d on the growth of
SVR murine angiosarcoma xenografts in nude mice.
More importantly, compound 18d was also tested for
anti-tumor efficacy on the growth of SVR tumors
(
Fig. 5) in nude mice. The compound 18d was dosed
day four of dosing and extending throughout the study
2
relative to vehicle treated controls (Fig. 5).
1
orally at 30 mg/kg twice a day for 10 days. Significant
inhibition in tumor growth was noticed beginning at
In conclusion, we have demonstrated a structure-guided
library building process in solution phase, where we
have grafted key structural elements of a known kinase
inhibitor on different structural classes which resulted in
the identification and optimization of a novel class of
VEGFR-2 kinase inhibitors. SAR on pyrazolone class
of molecules resulted in inhibitors which were extremely
potent against the isolated enzyme as well as in cells.
Some of these molecules demonstrated anti-angiogenic
activity ex vivo in rat aortic ring explant cultures and
oral anti-tumor efficacy in nude mice (shown for com-
pound 18d).
NaH/Toluene
Diethyl
Carbonate
H
O
N
O
O
N
O
NH -NH .xH O
2
2
2
S
CH3
S
O
Ethanol/reflux
N
reflux
N
S
2
1
N
20
22
(
51% in two steps)
H
2
2
N
N
O
O
o
Piperidine
Ethanol
1
2
. POCl /DMF/ 60 C
3
O
O
H
S
. NaH/DMF/MeI
O
O
N
N CH3
N
0oC
9
H
N
CH3
23
2
4
O
O
(
49% in two steps)
1
8d
(
78%)
Scheme 3. Synthesis of 18d, a compound selected for anti-tumor and
anti-angiogenic evaluation.
Acknowledgments
We thank Dr. Jeffry L. Vaught for his support and
encouragement. We also thank Mr. Kurt A. Josef for
his help in some NMR experiments.
Table 2. Kinase selectivity profile for 18d
Kinase assay
% Inhibition (IC50 in nM)
PKC
Trk A
13% at 1 lM
44% at 1 lM
(>10,0000)
References and notes
Cdk 1/Cyclin B1
VEGFR-1
VEGFR-3
25% at 300 nM
63% at 300 nM
1
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(
stimulated VEGFR2 phosphorylation in HUVEC or
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VEGF-stimulated control (no inhibitor) as measured by a
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3 = 51–75%; 4 = 76–100%.
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6
21. Murine SVR endothelial cells (1 · 10 ) were injected into
1
1
1. Tripathy, R.; Learn, K. S.; Reddy, D. R.; Iqbal, M.;
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2. Compounds were tested for their ability to inhibit the
kinase activity of baculovirus-expressed human VEGFR2
the right flank of the female athymic nude mice. At day
5 post-implantation when palpable tumors were con-
firmed, mice were randomized into treatment groups
(n = 8–10) and administered 18d at the specified con-
centration, orally, twice a day. Statistical analyses were
done using the Mann–Whitney Sum test with *p 6 0.05,
(
KDR) cytoplasmic domain using a modification of the
ELISA-based assay described for trkA. (a) Angeles, T. S
et al. Anal. Biochem. 1996, 236, 49, Detection was
performed utilizing time-resolved fluorescence (TRF).
IC50 values are reported as the average of at least two
p 6 0.005, and
*
p 6 0.001 relative to vehicle con-
***
*
trols. A denotes
50 mg/kg.
for 18d at 30 mg/kg and
for at
***
*
*