1,2,3-Thiadiazole substituted pyrazolones as potent KDR/VEGFR-2 kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2006.12.054

KDR kinase inhibition is considered to play an important role in regulating angiogenesis, which is vital for the survival and proliferation of tumor cells. Recently we disclosed a structure-based kinase inhibitor design strategy which led to the identification of a new class of VEGFR-2/KDR kinase inhibitors bearing heterocyclic substituted pyrazolones as the core template. Instability in a rat S9 preparation and poor iv PK profiles for most of these inhibitors necessitated exploration of new pyrazolones to identify new analogs with improved metabolic stability. Optimization of the heterocyclic moiety led to the identification of the thiadiazole series of pyrazolones (D) as potent VEGFR-2/KDR kinase inhibitors. SAR modifications, kinase selectivity profiling, and structural elements for improved PK properties were explored. Oral bioavailability up to 29% was achieved in the rat. Modeling results based on the Glide XP docking approach supported our postulation regarding the interaction of the lactam segment of the pyrazolones with the hinge region of the KDR kinase.

Full text of DOI:10.1016/j.bmcl.2006.12.054

Bioorganic & Medicinal Chemistry Letters 17 (2007) 1793–1798
1,2,3-Thiadiazole substituted pyrazolones as potent
KDR/VEGFR-2 kinase inhibitors
Rabindranath Tripathy,* Arup Ghose, Jasbir Singh, Edward R. Bacon, Thelma S. Angeles,
Shi X. Yang, Mark S. Albom, Lisa D. Aimone, Joseph L. Herman and John P. Mallamo
Cephalon Inc., 145 Brandywine Parkway, West Chester, PA 19380, USA
Received 24 October 2006; revised 12 December 2006; accepted 13 December 2006
Available online 21 December 2006
Abstract—KDR kinase inhibition is considered to play an important role in regulating angiogenesis, which is vital for the survival
and proliferation of tumor cells. Recently we disclosed a structure-based kinase inhibitor design strategy which led to the identifi-
cation of a new class of VEGFR-2/KDR kinase inhibitors bearing heterocyclic substituted pyrazolones as the core template. Insta-
bility in a rat S9 preparation and poor iv PK profiles for most of these inhibitors necessitated exploration of new pyrazolones to
identify new analogs with improved metabolic stability. Optimization of the heterocyclic moiety led to the identification of the thia-
diazole series of pyrazolones (D) as potent VEGFR-2/KDR kinase inhibitors. SAR modifications, kinase selectivity profiling, and
structural elements for improved PK properties were explored. Oral bioavailability up to 29% was achieved in the rat. Modeling
results based on the Glide XP docking approach supported our postulation regarding the interaction of the lactam segment of
the pyrazolones with the hinge region of the KDR kinase.
Ó 2007 Elsevier Ltd. All rights reserved.
Identification of novel receptor tyrosine kinase (RTK)
inhibitors continues to be an intense area of investiga-
tion in anti-tumor research,1 due to the fact that overex-
pression of some of their corresponding receptor
proteins contributes to constitutive RTK signaling,
resulting in dysregulated cell growth and cancer.2 Such
uncontrolled RTK signaling leading to tumor growth
has provided the impetus for the design of RTK inhibi-
tors as potential anti-tumor agents. The approval of
Sorafenib,3a Sunitinib,3b and Iressa4 has validated such
mechanistic approaches. One of the most extensively
studied pathways in this area is vascular endothelial
growth factor (VEGF)5 and its cell surface receptor in
human KDR (kinase domain containing receptor or
VEGFR-2)6 due to their important roles in angiogene-
sis7 which is vital for survival and proliferation of tumor
cells. KDR receptors, shown to be expressed primarily
in endothelial cells,8 upon binding to VEGF get activat-
ed and their intracellular kinase domains undergo auto-
phosphorylation, which in turn triggers signaling
pathways leading to sprouting of blood vessels toward
the tumor cells. Therefore, inhibition of KDR kinase
and subsequent blockage of angiogenesis could provide
an alternate approach to cancer therapy.9
Recently, we disclosed the identification of a novel class
of KDR kinase inhibitors (Fig. 1, A and B) comprising
heterocyclic substituted pyrazolones as the core struc-
ture.10 A structure-guided stepwise construction and
exploitation of the Knoevenagel condensation11 led us
to the identification of this series of molecules. By
appropriately modifying the heterocyclic moiety (with
1–2 heteroatoms) in C, coupled with versatility of the
aldehydes, it was possible to generate an array of very
potent and specific KDR kinase inhibitors. While the
interaction of these inhibitors with the KDR protein
at the molecular level is not fully understood, we pro-
posed, on the basis of some structure–activity relation-
ship (SAR) evidence, the structural elements essential
for kinase binding, summarized in Figure 1 (C). A major
liability of the pyrazolone series, however, was the met-
abolic instability of these compounds in rat liver S9 frac-
tion in the presence of 2 mM NADPH.12 Most of the
compounds were not detected after 30 min of incuba-
tion. Pharmacokinetic (PK) profiles in the rat also
showed similar trends and intravenous (iv) administra-
Keywords: KDR kinase; VEGFR-2 kinase; Kinase inhibitors; 1,2,3-
Thiadiazole-pyrazolones.
*
Corresponding author. Tel.: +1 610 738 6137; fax: +1 610 738
6558; e-mail: rtripath@cephalon.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.12.054

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R. Tripathy et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1793–1798
O
H
OH
H
O
O
N
N
N
N
H
O
O
b
N
N
a
S
N
H
N
H
N
H
N
N CH3
N CH3
4
5
6
H
N
R
R
N
O
O
A
B
O
N
S
H
Possible
d
c
CH
N
N
hydrogen bonding
sites with the enzyme
H
CH
N
N
N
O
H
O
CH
S
N
N
N
O
7
8
N
N CH3
Het
R'
Scheme 2. Reagents and conditions: (a) Cs2CO3/EtI/acetone/reflux; (b)
POCl3/DMF, 0–60 °C, 69% in two steps; (c) NaH/MeI/DMF, 0 °C-rt,
75%; (d) 3, EtOH/piperidine/heat, 82%.
N CH3
Double bond is
also critical
R
N-Methyl
substitution is
R
preferred
C
D
ylation of the indole nitrogen provided the N-methylat-
ed aldehyde 7 which was ready for Knoevenagel
condensation. Other indole aldehydes were prepared in
a similar fashion by using commercially available or lit-
erature-reported indoles. The condensation in each case
was carried out by heating equimolar quantities of pyr-
azolone and aldehyde in ethanol in the presence of a cat-
alytic amount of piperidine. Usually a precipitate is
formed within a few minutes of heating, which upon fil-
tration provides a single product14 (8k is shown in
Scheme 2) with high purity (>98%) in most of the cases.
Such a facile work-up and simple purification technique
allowed us to rapidly generate an array of compounds
utilizing a variety of indole carboxaldehydes. On the
other hand, Scheme 3 shows the synthesis of the unsub-
stituted pyrazolone 13, for which the starting 1,2,3-thia-
diazole 5-carboxylic acid ethyl ester (12) was prepared
via semicarbazone 1115 followed by rearrangement to
1,2,3-thiadiazole in presence of thionyl chloride.16 Sub-
sequent b-ketoester formation followed by cyclization
with hydrazine resulted in the pyrazolone 13. Similar
condensation with indole carboxaldehydes resulted in
product 14 (14l is shown, Scheme 3).
Figure 1. General structures of pyrazolone-based KDR kinase inhib-
itors (A and B); important structural features for activity (C) and
general structure of the proposed pyrazolones bearing 1,2,3-thiadiazole
(D).
tion of many of these compounds exhibited rapid clear-
ance.12 While the pyrazinyl derivatives (B, Fig. 1) were
among the most potent KDR kinase inhibitors against
the isolated enzyme and in cells,10 members of this class
were particularly vulnerable to metabolic instability in
the S9 assay. However, one observation from the S9
incubation studies was that some of the indole deriva-
tives which were inactive against KDR kinase were also
resistant to in vitro metabolism. Encouraged by this
observation, we further explored heterocyclic variations
which ultimately led to the identification of 1,2,3-thia-
diazole substituted class of pyrazolones (Fig. 1, D).
Herein we disclose the synthesis, SAR, improved PK
profile, and modeling results for this class of molecules.
Based on our earlier work,10 different 1,2,3-thiadiazole-
substituted pyrazolones and N-methyl-substituted in-
dole 3-carboxaldehydes were easily synthesized as
shown in Schemes 1–3. Methylthiadiazolepyrazolone
(3) was synthesized in two steps starting from the com-
mercially available 4-methyl[1,2,3]thiadiazole-5-carbox-
ylic acid ethyl ester (1) in 50% overall yield via the b-
ketoester intermediate 2 (Scheme 1).13 Scheme 2 shows
a representative example of the synthesis of N-methyl-
indole-3-carboxaldehydes starting from 4-hydroxyin-
dole (4). Selective ethyl protection of the hydroxyl group
followed by Vilsmeier formylation at the 3-position of
the indole gave compound 6 in 69% overall yield. Meth-
Using our proposed mode of binding of pyrazolones
(Fig. 1, C) with KDR kinase as a guide, we directed
structural diversity in the thiadiazole series to various in-
dole positions17 and 4-substitution of the thiadiazole
ring, respectively. The lactam unit, the exocyclic double
bond remained intact. Similarly, the indole N-methyl
group was thought to be optimal, and further variation
of such group was not pursued.
Compounds generated were tested for their ability to
inhibit the kinase activity of baculovirus-expressed hu-
man VEGFR-2 (KDR) cytoplasmic domain using
time-resolved fluorescence (TRF) assay.18 Table 1 high-
lights the SAR of methyl thiadiazoles 8 bearing 3-substi-
tuted indole moieties. These results corroborated our
earlier observation that N-methylated indoles showed
better activities than the corresponding N-unsubstituted
indoles (Table 1, 8 and 8a). While a variety of indole
substituents at different positions were tolerated, most
did not result in improved potency. The only exception
was the indole-4 position, where substituents such as Cl,
H
O
O
O
N
N
S
S
OEt
O
S
O
N
a
N
b
N
N
N
CH3
N
CH3
CH3
1
2
3
Scheme 1. Reagents and conditions: (a) NaH/methylacetate/reflux; (b)
hydrazine hydrate/ EtOH/reflux, 50% in two steps.

R. Tripathy et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1793–1798
1795
OEt
O
O
O
O
O
a
b
OEt
EtO
H2N
OEt
O
OEt
N
N
H
H
9
10
11
H
N
N
H
S
O
N
N
N
N
S
N
CH3
S
N
O
OEt
e
N
O
d
c
N
N
12
13
14
Scheme 3. Reagents and conditions: (a) dichloromethane: water/TFA; RT; (b) semicarbazide/NaOAc/water: EtOH, 58%; (c) SOCl2, 0–60 °C, 67%;
(d) i—NaH/Methylacetate/reflux, ii—Hydrazine hydrate/methanol/reflux, 22% in two steps; (e) 7, EtOH/piperidine/heat, 48%.
Table 2. Representative SAR of 1,2,3-thiadiazole substituted pyrazol-
ones-based KDR kinase Inhibitors (14a-q)
Br, alkoxy, and methyl improved potency against KDR
(Table 1, 8h–8l). Inhibitors bearing bis substituents on
the indole ring involving a 4-position also showed
potency improvements (Table 1, 8m and 8p). Com-
pounds showing <50 nM in vitro potency were tested
in the human umbilical vein endothelial cell (HU-
VEC)-based phosphorylation assays and were found to
be potent.
H
N
N
O
S
N
N
N CH3
H
R
14
The KDR kinase inhibition results and HUVEC cell
data for thiadiazoles lacking a 4-methyl group are listed
in Table 2. These compounds were found to be generally
more potent than the methylthiadiazoles. Substituents at
Compounda
R
Cell scorec
(HUVEC)
b
IC50 (nM)
14a
14b
14c
14d
14e
14f
14g
14h
14i
5-F
5-Cl
5-CN
48
34
174
73
28
110
35
16
16
8
1
2
—
—
3
5-COOCH3
5-OCH3
5-Br
Table 1. Representative examples of KDR inhibitory activity of 4-
methyl-1,2,3-thiadiazole-substituted pyrazolones (8–8p)
—
4
5-CH3
4-F
H
N
N
4
O
S
4-Cl
4-Br
2
N
N
14j
1
N R'
CH3
14k
14l
4-OCH3
4-OC2H5
4-COOCH3
13
25
22
6
4
4
14m
14n
14o
14p
14q
3
R
8
4-Br, 5-OCH3
6-Cl
4
73
12
10
—
0
Compounda
R
R0
IC50 (nM) Cell scorec
(HUVEC)
b
4-Br, 6-CH3
4,7-bis-OCH3
4
a A single geometrical isomer was isolated in each case. See Ref. 14.
8
H
H
>1000
95
99
—
0
b Ref. 18a.
c Ref. 18b.
8a
8b
8c
8d
8e
8f
H
5-F
CH3
CH3
CH3
H
—
—
—
—
—
—
2
5-Cl
5-Cl
152
>300
90
5-OCH3
5-CN
4-F
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
different positions on the indole-benzene ring were also
tolerated. However, compounds bearing substituents
such as 5-CN (14c), 5-COOCH3 (14d), 5-Br (14f), and
6-Cl (14o) displayed IC50 values >50 nM. One interest-
ing finding came from the 4-bromo-substituted pyrazol-
ones (Table 2 14j, 14n, 14p). Although these compounds
were potent against the isolated enzyme, this potency
did not translate into cellular potency in at least two
cases. In contrast, similar substitution translated to high
potency in the pyrazine-pyrazolones (B), both in cells
and against the isolated enzyme.10 However, it was also
found that irrespective of the nature of the heterocyclic
group on the pyrazolones, 4-alkoxy substitution on the
>300
137
45
8g
8h
8i
4-Cl
4-Br
34
19
3
8j
4-OCH3
4-OCH2H5
4-CH3
4
8k
8l
38
58
4
—
4
8m
8n
8p
4-Br,5-OCH3
7-OCH3
23
109
20
—
3
4,7-bis-OCH3 CH3
a A single geometrical isomer was isolated in each case. See Ref. 14.
b Ref. 18a.
c Ref. 18b.

1796
R. Tripathy et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1793–1798
Table 3. IV and oral PK profile of selected pyrazolones in the rat
indole segment (Table 1, 8J, 8K; Table 2, 14k, 14l; also
Ref. 10) resulted in a good correlation between in vitro
potency and cell permeability. Moreover, a promising
PK profile also provided compelling reason to focus
on 4-alkoxy indole substituted pyrazolones. Replace-
ment of the 4-methyl group by phenyl19a in 1,2,3-thia-
diazole resulted in approximately threefold loss in
potency (Fig. 2, 15 and Table 1, 8i). Similarly, switching
to pyrroles (as in structure 16 and 17, Fig. 2) in place of
indoles, which are known and potent segments of pyraz-
olone-based kinase inhibitors,10 also did not show dra-
matic improvement in potency against KDR kinase.19b
Compound
Intrinsic (iv) Pharmacokinetics in rata
AUC
(ng*h/mL)
T1/2
(h)
Vd
(L/kg)
CL
(mL/min/kg)
8j
8k
192
1141
727
0.3
0.9
0.5
1.2
0.6
1.4
2.2
1.1
1.1
—
87
15
25
—
4.6
—
8b
8b (Oral)b
A-1 (R = F)
A-1 (Oral)c
1042
3599
507
0.23
—
a iv dosing was at 1 mg/kg (using 3% DMSO, 30% Solutol, and 67%
phosphate-buffered saline (PBS) as the vehicle, Pharmacokinetic
parameters were estimated for each rat by non-compartmental
analysis of the plasma concentration versus time data using Win-
Nonlin software (Professional Version 2.1, Pharsight Corporation,
Palo Alto, CA, 1997).
Selected compounds from both series (Tables 1 and 2)
including compound 17 were tested for their intravenous
(iv) PK profile in the rat. Unfortunately, like pyrazol-
ones A and B (Fig. 1) most of the compounds demon-
strated very low plasma levels, high clearance rates
(CL), and resulting short half-lives (t1/2). Some of the
methylthiadiazoles showed improved PK profiles com-
pared to their unsubstituted counterparts. More impor-
tantly, structural changes within the methylthiadiazole
series played an important role in improving the PK
profile. For example, simple exchange of the 4-methoxy
(8j) to 4-ethoxy on the indole ring (8k) significantly im-
proved the iv PK profile in the rat (Table 3, Fig. 4).
Another encouraging example was methylthiadiazole
compound 8b (Figs. 3, 4). By blocking a possible meta-
bolic site on the indole 5-position (5-H replaced with 5-
F), the iv PK profile was improved significantly. More
strikingly, this compound also demonstrated 29% oral
bioavailability (BA) after a 5 mg/kg po dose in the rat
(Figs. 3, 4). Despite modest potency for 8b and poor cell
permeability, we selected this compound for PK profil-
ing as a proof-of-concept molecule based on favorable
iv PK data previously obtained with an analogous com-
pound (A-1, R = F, Figs. 3 and 4) in the thiazole series,
bearing a 5-fluoroindole group (Table 3). However, A-1
showed low oral bioavailability (ꢀ3%). Therefore, a
methylthiadiazole substituent on the pyrazolone ring
showed better bioavailability than the corresponding
thiazole-based pyrazolone, at least in a comparable sys-
tem. Figure 4 summarizes the PK results.
b Oral dosing was at 5 mg/kg, Cmax = 356 ng/mL (using 50% Tween 80,
40% propylene carbonate, and 10% propylene glycol).
c Oral dosing was at 5 mg/kg, Cmax = 139 ng/mL (using 50% Tween 80,
40% propylene carbonate, and 10% propylene glycol).
10000
Compound 8b iv
Compound 8b po
1000
100
10
Bioavailability = 29%
1
0
1
2
3
4
5
6
7
Time (h)
Figure 3. Plasma levels of compound 8b in the rat (1 mg/kg iv, 5 mg/kg
po).
Modeling results based on the Glide XP docking ap-
proach20 supported our postulation regarding interac-
tion of the lactam segment of the pyrazolones with the
hinge region of the KDR kinase. The lactam ring of
the pyrazolone has one hydrogen donor NH flanked
by two hydrogen acceptors, that is, the carbonyl O
and N. This may lead to two possible binding modes
of the pyrazolone ring in the hinge region: either the
hydrogen acceptor O or the N may bind with the
Table 4 shows the selectivity profile of a representative
inhibitor, 8k, against a panel of tyrosine kinases. 8k
demonstrates potent, low-nanomolar inhibition of
KDR, VEGFR3, TRK A, and FLT3. Other kinases
such as SRC, VEGFR1, c-Met, and Tie-2 are only
weakly inhibited.
H
N
H
N
H
N
N
O
N
N
O
O
H
N
H
N
S
S
S
N
CH3
N
N
N
N
N
Ph
N
CH3
Br
IC50 = 56 nM
IC50 = 197 nM
IC50 = 89 nM
17
16
15
Figure 2. KDR kinase binding results of pyrazolones: (a) 4-phenyl thiadiazole (15); (b) thiadiazoles bearing pyrrole segments (16, 17).

R. Tripathy et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1793–1798
1797
H
H
H
N
N
N
N
H
N
O
N
O
O
S
N
N
S
S
N
O
N
S
N
N
N
N CH3
N
CH3
N CH3
N CH3
CH3
O
N
N CH3
CH3
O
H3C
H3C
F
F
14h
(Poor i.v. PK)
8h
(Improved i.v. PK)
A-1
(Good i.v. PK
Low oral BA)
8b
(Oral Bioavailability
29%)
Figure 4. Summary of structural modifications to improve PK.
Table 4. Kinase inhibition profiles of the pyrazolone 8k
In conclusion, we have identified two series of 1,2,3-thia-
diazole substituted pyrazolones as potent and cell-per-
meable KDR kinase inhibitors. Although 4-methyl
substituted thiadiazolepyrazolones were less robust in
potency than their unsubstituted counterpart, they
showed an improved PK profile. We have identified
structural moieties (4-ethoxy, 5-fluoro on the indole
ring) responsible for improved iv PK profile in the rat.
Compound 8b showed 29% oral bioavailability in rats.
Modeling results supported the hypothesis regarding
the mode of interaction of pyrazolone-based inhibitors
with KDR kinase. Anti-tumor properties from this class
of molecules will be reported in due course.
Kinase
IC50(nM)
Kinase
IC50(nM)
KDR
38
>3000
16
Tie-2
>10000
3700
36
VEGFR1
VEGFR3
SRC
c-Met
TRK A
FLT3
463
19
Cys-917 backbone NH. There are four VEGFR-2 struc-
tures in the protein databank (1VR2, 1Y6A, 1Y6B, and
1YWN). 1VR2 is the apo-protein structure; 1YWN has
a ligand bound DFG-out structure. The other two were
intermediate structures where the Phe of DFG was nei-
ther ‘in’ nor ‘out’. To understand the binding modes of
these pyrazolones, we used Glide XP docking approach
and the 1Y6B structure.
Acknowledgments
During the ‘protein preparation’ however, the missing
part of the P-loop (Ala-842 and Phe-843) was built, fol-
lowing the conformation of 1YWN, followed by a con-
strained minimization. The Glide docking study also
showed two major binding modes as discussed above.
The most frequently occurring binding mode had the
pyrazolone O bound to the Cys-917 NH and the thiadia-
zole ring near the salt bridge Lys-866 (Fig. 5). These
studies revealed no clear SAR to differentiate between
the two binding modes. It is also possible that the nature
of substitution may dictate one over the other.
We thank Kurt A. Josef for his help in NMR experi-
ments, Sheryl L. Meyer, and Chrysanthe M. Spais for
providing purified recombinant proteins.
References and notes
1. For reviews, see (a) Bou¨ıs, D.; Kusumanto, Y.; Meijer, C.;
Mulder, N. H.; Hosper, G. A. P. Pharmacol. Res. 2006, 53,
89; (b) Kim, D. N.; Lu, B.; Hallahan, D. E. Curr. Opin.
Investig. Drugs 2004, 5, 597; (c) Cohen, P. Curr. Opin.
Chem. Biol. 1999, 3, 459.
´
2. (a) Bennasroune, A.; Gardin, A.; Aunis, D.; Cremel, G.;
Hubert, P. Critical Rev. Oncol./Hematol. 2004, 50, 23; (b)
Christensen, J. G.; Schreck, R.; Burrows, J.; Kuruganti,
P.; Chan, E.; Le, P.; Chen, J.; Wang, X.; Ruslim, L.;
Blake, R.; Lipson, K. E.; Ramphal, J.; Do, S.; Cui, J. J.;
Cherrington, J. M.; Mendel, D. B. Cancer Res. 2003, 63,
7345.
3. (a) Wilhelm, S.; Carter, C.; Lynch, M.; Lowinger, T.;
Dumas, J.; Smith, R. A.; Schwartz, B.; Simantov, R.;
Kelley, S. Nat. Rev. Drug Discov. 2006, 5, 835; (b)
Cabebe, E.; Wakelee, H. Drugs Today (Barc) 2006, 42,
387.
4. McKillop, D.; Partridge, E. A.; Kemp, J. V.; Spence, M.
P.; Kendrew, J.; Barnett, S.; Wood, P. G.; Giles, P. B.;
Patterson, A. B.; Bichat, F.; Guilbaud, N.; Stephens, T. C.
Mol. Cancer Ther. 2005, 4, 641.
5. Jakeman, L. B.; Armanini, M.; Phillips, H. S.; Ferrara, N.
Endocrinology 1993, 133, 848.
6. (a) De Vries, C.; Escobedo, J. A.; Ueno, H.; Houck, K.;
Ferrara, N.; Williams, L. T. Science 1992, 255, 989; (b)
Terman, B. I.; Dougher-Vermazen, M.; Carrion, M. E.;
Dimitrov, D.; Armellino, D. C.; Gospodarowicz, D.; Bohlen,
P. Biochem. Biophys. Res. Commun. 1992, 187, 1579.
Figure 5. The two possible binding modes often found by Glide for the
various pyrazolones. The distance of the pyrazolone NH from the Glu-
915 (CO) was 1.86A in the more frequent binding mode.

1798
R. Tripathy et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1793–1798
7. Klagsbrun, M.; Moses, M. A. Chem. Biol. 1999, 6, R217.
Sciences #1244-105, Boston, MA). After a few minutes,
the fluorescence of the resulting solution was measured
using the Perkin–Elmer EnVision 2100 (or 2102) multila-
bel plate reader. Inhibition data were analyzed using IDBS
ActivityBase and IC50 curves were generated using XLFit
3. IC50 values were reported as the average of at least two
independent determinations.; (b) Subconfluent HUVECs
were serum-starved by replacing media with EBM-2
(endothelial cell basal medium, serum-free; Clonetics
#CC-3156; BioWhittaker, Walkersville, MD) containing
0.05% BSA for 1 h at 37 °C, during which time the test
compound (50 nM) or DMSO (control) was added to the
cells. Human VEGF (Clonetics #CC-4114) was then
added to the cells at a concentration of 10 ng/mL for
5 min. Cells were lysed in radioimmunoprecipitation assay
(RIPA) buffer containing 1 mM activated sodium vana-
date and protease inhibitors (Protease Inhibitor Cocktail
Set III; Calbiochem, San Diego, CA), sheared with a 27
gauge syringe, and then centrifuged at 12,000g for 15 min.
Clarified cell lysates were immunoprecipitated with anti-
VEGFR-2 antibody for 1 h, followed by incubation with
Protein A Sepharose for another hour at 4 °C and analysis
by SDS–PAGE. Western blotting was performed using
4G10 anti-phosphotyrosine antibody (Upstate #05-321;
Lake Placid, NY) and phosphorylated proteins were
visualized using enhanced chemiluminescence (ECL)
(Amersham #RPN2106, Piscataway, NJ). The percentage
inhibition was calculated by analyzing scanned autoradio-
graphs on a densitometer. Scores were based on decrease in
protein band density compared with VEGF-stimulated
control (no inhibitor) as follows: 0 = no decrease; 1 = 1–
25%; 2 = 26–50%; 3 = 51–75%; 4 = 76–100%.
8. Strawn, L. M.; McMahon, G.; App, H.; Schreck, R.;
Kuchler, W. R.; Longhi, M. P.; Hui, T. H.; Tang, C.;
Levitzki, A.; Gazit, A.; Chen, I.; Keri, G.; Orfi, L.; Risau,
W.; Flamme, I.; Ullrich, A.; Hirth, K. P.; Shawver, L. K.
Cancer Res. 1996, 56, 3540.
9. Paz, K.; Zhu, Z. Front. Biosci. 2005, 10, 1415.
10. Tripathy, R.; Reiboldt, A.; Messina, P. A.; Iqbal, M.;
Singh, J.; Bacon, E. R.; Angeles, T. S.; Yang, S. X.;
Albom, M. S.; Robinson, C.; Chang, H.; Ruggeri, B. A.;
Mallamo, J. P. Bioorgan. Med. Chem. Lett. 2006, 16, 2158.
11. March, J. In Advance Organic Chemistry, 3rd ed.; John
Wiley & Sons, Inc.: New York, 1985; p 835.
12. Unpublished results.
13. Battegay, M.; Wolff, A. Bull. Soc. Chim. 1923, 33, 1481.
14. The configuration of the double bond is assumed to be Z
in all cases based on H-NMR chemical shift correlations
1
and x-structure of a related pyrazolone (see Ref. 10).
15. Wisliconus, W. Justus Liebigs Ann. Chem. 1907, 356, 46.
16. Shafiee, A.; Lalezari, I.; Mirrashed, M.; Nercesian, D. J.
Heterocyclic Chem. 1977, 14, 567, and references there in.
17. Indole-2- position was not explored, as
a methyl
substituent in the pyrazine series (B-1, R = 2-methyl) was
not tolerated (only 7% inh. at 300 nM), whereas the des-
methyl compound, B-2, R = H, had an IC50 value of
37 nM against KDR10.
18. (a) The VEGFR-2/KDR assay was performed in a
microtiter plate format using time-resolved fluorescence
(TRF). Briefly, each 96-well plate (Costar # 3922; Corn-
ing, NY) was coated with 20 lg/mL of substrate solution
(recombinant human GST-PLC-c/GST) in Tris-buffered
saline (TBS). The KDR assay mixture (total vol-
ume = 100 lL/well) containing 20 mM Hepes, pH 7.2,
40 lM ATP, 10 mM MnCl2, 0.1% BSA, and variable
concentrations of test compound (diluted in DMSO; 2.5%
DMSO final in assay) was then added to the assay plate.
Enzyme (30 ng/mL KDR) was added and the reaction was
allowed to proceed at 37 °C for 15 min. The phosphory-
lated product was detected by incubating with Eu-N1-
labeled PY100 antibody (Perkin–Elmer Life Sciences
#AD0160, Boston, MA) at 37 °C for 1 h, followed by
addition of enhancement solution (Perkin–Elmer Life
19. (a) The pyrazolone precursor for 1516 was prepared in a
similar method as outlined in Scheme 3.; (b) The inhibitors
16 and 17 were prepared by the condensation of 3,5-
dimethyl-1H-pyrrole-2-carboxaldehydealdehyde
and
4,5,6,7-tetrahydroindole-2-carboxaldehyde with the pyra-
zolones 3 and 13, respectively.
20. Friesner, R. A.; Banks, J. L.; Murphy, R. B.; Halgren, T.
A.; Klicic, J. J.; Mainz, D. T.; Repasky, M. P.; Knoll, E.
H.; Shelley, M.; Perry, J. K.; Shaw, D. E.; Francis, P.;
Shenkin, P. S. J. Med. Chem. 2004, 47, 1739.