(6,7-Dimethoxy-2,4-dihydroindeno[1,2-c]pyrazol-3-yl)phenylamines: Platelet-derived growth factor receptor tyrosine kinase inhibitors with broad antiproliferative activity against tumor cells

Article abstract of DOI:10.1021/jm050680m

A series of (6,7-dimethoxy-2,4-dihydroindeno[1,2-c]pyrazol-3-yl) phenylamines has been optimized to preserve both potent kinase inhibition activity against the angiogenesis target, the receptor tyrosine kinase of Platelet-Derived Growth Factor-BB (PDGF-BB

Full text of DOI:10.1021/jm050680m

J. Med. Chem. 2005, 48, 8163-8173
8163
(6,7-Dimethoxy-2,4-dihydroindeno[1,2-c]pyrazol-3-yl)phenylamines:
Platelet-Derived Growth Factor Receptor Tyrosine Kinase Inhibitors with
Broad Antiproliferative Activity against Tumor Cells
Chih Y. Ho,‡ Donald W. Ludovici,‡ Umar S. M. Maharoof,‡ Jay Mei,‡ Jan L. Sechler,‡ Robert W. Tuman,‡
Eric D. Strobel,‡ Laura Andraka,‡ Hwa-Kwo Yen,§ Gregory Leo,| Jian Li,† Harold Almond,† Hong Lu,‡
Ann DeVine,‡ Rose M. Tominovich,‡ Judith Baker,‡ Stuart Emanuel, Robert H. Gruninger,
Steven A. Middleton, Dana L. Johnson,‡ and Robert A. Galemmo, Jr.*,‡
Oncology Research Team, High Throughput Chemistry Team, Molecular Design and Informatics Team, Analytical Research
Team, Johnson & Johnson Pharmaceutical Research and Development, L.L.C., Spring House Research and Early Development
Site, Welsh and McKean Roads, Spring House, Pennsylvania 19477, and Cancer Therapeutics Team, Johnson & Johnson
Pharmaceutical Research and Development, L.L.C., Raritan Research and Early Development Site, Route 202,
Raritan, New Jersey 08869
Received July 18, 2005
A series of (6,7-dimethoxy-2,4-dihydroindeno[1,2-c]pyrazol-3-yl)phenylamines has been opti-
mized to preserve both potent kinase inhibition activity against the angiogenesis target, the
receptor tyrosine kinase of Platelet-Derived Growth Factor-BB (PDGF-BB), and to improve
the broad tumor cell antiproliferative activity of these compounds. This series culminates in
the discovery of 17 (JNJ-10198409), a compound with anti-PDGFR-â kinase activity (IC50
0.0042 µM) and potent antiproliferative activity in six of eight human tumor cell lines (IC50
0.033 µM).
)
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Introduction
Overexpression of PDGF and its receptors has been
demonstrated in human cancers including lung,10
breast,11 colorectal,12 glioma,13 and esophageal.14 PDGF
and PDGF receptors are coexpressed on tumor vascu-
lature and are up-regulated during tumor progression.13
Elevated circulating levels of PDGF are associated with
metastatic disease15 and higher microvessel counts in
human breast cancer.16 Finally, PDGF receptor is
expressed in both vascular endothelial cells and smooth
muscle cells in the tumor stroma.17
Angiogenesis, the formation of new blood vessels from
preexisting vasculature, is fundamentally important for
the physiology of neonatal development, reproduction,
and wound healing; however, the pathophysiological
contribution of this process to cancer has received much
attention.1 Folkman’s seminal observation,2 that the
sustained growth of solid tumors beyond a few mil-
limeters in diameter is dependent on the ability of tumor
cells to recruit new blood vessels from the existing
vascular network, has led to considerable efforts to
develop agents capable of inhibiting tumor-induced
angiogenesis as a therapeutic approach in oncology.3
Among the many important endogenous stimulators of
angiogenesis, PDGF-BB and its corresponding tyrosine
kinase receptor PDGFR-â play an important role in
the angiogenic process.4-6 As a proangiogenic protein,
PDGF-BB exerts both mitogenic and chemotactic effects
on microvascular endothelial cells through the PDGF
receptor tyrosine kinase-signaling pathway.5,6 In addi-
tion, PDGF-BB can induce endothelial cells to express
high levels of VEGF, another potent stimulator of
angiogenesis.7,8 PDGF-BB is an important mitogenic
factor for smooth muscle cells and pericytes, important
cell types that participate in the later stages of angio-
genesis by surrounding endothelial cells to stabilize the
newly formed microvessels.8,9
The effects of PDGF-BB are mediated through ligand
binding to its cell surface receptor tyrosine kinase,
PDGFR-â, followed by receptor dimerization and auto-
phosphorylation of the transmembrane tyrosine kinase
domain.18,19 This event activates several downstream
signaling molecules and their pathways, including phos-
pholipase-C-γ (PLC-γ), Grb2/Sos1, MAP kinase, GAP,
Src, and PI3.18-21 At the cellular level this activity
evokes a diverse set of responses, including proliferation
and chemotaxis in smooth muscle and endothelial cells
as well as changes in endothelial cell morphology. A
variety of experimental approaches have provided vali-
dation of the importance of the PDGF pathway and the
potential as a therapeutic strategy for inhibiting tumor-
induced angiogenesis and growth. These experimental
approaches include the use of PDGF receptor antibod-
ies,22 peptidomimetics of PDGF23,24 and small molecule
inhibitors of PDGF receptor tyrosine kinase activity.25-28
One of the promising new concepts in antiangiogenic
therapy is that antiangiogenic agents enhance the
remodeling or ‘normalization’ of tumor vasculature.29 It
has been proposed that a regimen that includes an
antiangiogenic agent with conventional chemotherapy
will have enhanced efficacy because the ‘normalized’
tumor vascular bed more efficiently distributes chemo-
therapeutic agents to the tumor mass.29 On the basis
Circumstantial evidence also implicates PDGF as a
driver of tumor cell proliferation in some cancers.
* Corresponding author. Telephone: 610-458-6056. Fax: 610-458-
8249. E-mail: rgalemmo@prdus.jnj.com.
‡ Oncology Research Team.
§ High Throughput Chemistry Team.
† Molecular Design and Informatics Team.
| Analytical Research Team.
Cancer Therapeutics Team.
10.1021/jm050680m CCC: $30.25 © 2005 American Chemical Society
Published on Web 11/22/2005

8164 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 26
Scheme 1. Preparation of Compounds 2-21a
Ho et al.
a (i) LHMDS, THF, RT, 12 h; (ii) NH2NH2, AcOH, reflux, 24 h, 40 to 70% for both steps; (iii) from 19 (R4 ) 3-CO2CH3): LiOH, THF,
H2O, 48 h, RT, 39%.
of this reasoning, we developed a strategy to discover
agents that would have the potential to target both
the tumor cell directly by an antiproliferative effect
and serve as an antiangiogenic agent to promote
the ‘normalization’ of the supporting tumor vascula-
ture. In this paper we report a series of compounds that
have the requisite antiproliferative activity and are
putative antiangiogenic agents due to their inhibition
of PDGFR-â tyrosine kinase.
Chemistry. The compounds used in these studies
were prepared by a one-pot, two-step procedure starting
from commercially available indan-1-ones and phenyl
thioisocyanates (Scheme 1). A solution of the indanone
and phenyl isothiocyanate in THF was added dropwise
to lithium hexamethyldisilane in THF at room temper-
ature. After 12 h, hydrazine and a small amount of
acetic acid were added to the reaction and the mixture
was heated at reflux for 24 h. Typical purified yields
for the two-step procedure were in the range of 40% to
70%. The 3-carboxyphenyl analogue 21 was prepared
in 39% yield from the corresponding methyl ester 19
by base hydrolysis.
Biology. Our testing cascade began with an assay of
the PDGF receptor kinase activity by measuring the
ability of the kinase to phosphorylate a biotinylated
PLC1γ peptide containing the tyrosine substrate residue
for PDGF RTK bound to a Streptavidin Flashplate in
the presence of 5 µM ATP. The extent of phosphoryla-
tion was estimated from the amount of [33P] retained
after exposure to [33P]-γ-ATP. The ability of Human
Coronary Artery Smooth Muscle Cells (HCASMC) to
incorporate [14C]-thymidine upon proliferative stimula-
tion by rh-PDGF-BB was used as a measure of the
PDGF RTK inhibition activity of this series in intact
cells. The inhibition of Low Serum Growth Supplement
(LSGS)-induced proliferation of Human Umbilical Vein
Endothelial Cells (HUVEC), cells not particularly re-
sponsive to stimulation by PDGF, was used as a
measure of a compound’s non-PDGF driven antiprolif-
erative activity against nontransformed cells. A panel
of human tumor cell lines, including AsPC-1 (pancre-
atic), PC3 (prostate), H460 (lung), LoVo (colon), A375
(melanoma), LnCAP (prostate), U87MG (glioma), and
T47D (breast), was used to estimate the tumor cell
growth inhibition activity of this series. Inhibition of
tumor cell proliferation was determined by [14C]-thy-
midine incorporation following 48 h exposure to com-
pound.
Results and Discussion
Our interest was to develop antiangiogenic com-
pounds with an additional antiproliferative activity
capable of inhibiting tumor progression by controlling
both the vascularization and proliferation of the tumor
mass. Tumors may be regarded as a two-compartment
system consisting of the vasculature supporting tumor
growth composed of ‘normal’ homogeneous vascular
endothelial cells, smooth muscle cells, and pericytes that
are surrounded by colonies of neoplastic cancer cells.
To find molecules that would affect both the vascular
and transformed compartments, we identified several
compounds with the potential to inhibit the PDGFR-â
kinase-mediated angiogenic effect and then assayed
them for collateral antiproliferative activity against a
panel of human tumor cell lines.
Screening of the Johnson & Johnson compound li-
brary with an assay of PDGFR-â kinase inhibition led
to the identification of 1 as a potent inhibitor of
PDGFR-â kinase (Table 1, IC50 ) 0.017 µM). Compound
1 also had modest tumor cell antiproliferative activity
(IC50 < 10 µM) for six of eight tumor cell lines.
Analogues of 1 have been reported previously,30 never-
theless, because of the anti-PDGF and antiproliferative
activity, 1 was useful as a starting point for the design
of a novel compound series. We began by docking 1 in a
homology model of the PDGF receptor kinase ATP
binding site (Figures 1 and 2). This initial effort
indicated that the PDGF kinase ATP binding site would
readily accommodate the transmutation of compound
1 to a (2,4-dihydroindeno[1,2-c]pyrazole-3-yl)phenyl-
amine such as 2-6 (Table 1). It was apparent that an
additional H-bond donor interaction could be gained
with the hinge region of the ATP binding site and
the 3-aminophenyl substituent would be accommo-
dated in a lipophillic region of the binding pocket.
However, two issues hampered our efforts to develop a
definitive binding model for this series. First, the
pyrazole ring can form two N-H tautomers, making it
difficult to assign the H-bonding network for this
system. Second, docking the flat, rigid tricyclic ‘body’ of

PDGF Receptor Tyrosine Kinase Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 26 8165
Table 1. IC50 (µM) Values for PDGF Kinase Inhibitiona and the Antiproliferative Activity against PDGF-BB-Stimulated HCASMC,b
LSGS-Stimulated HUVEC,c and Human Tumor Cell Linesd for 1-6
R
PDGF RTK HCASMC HUVEC H460
LoVo
LnCAP
PC3
31.2
T47D
4.41
0.42
A375
ASPC1 U87MG
1
2
3
4
5
6
0.017
0.009
0.317
0.018
0.217
0.054
0.124
0.023
1.39
8.4
2.7
0.124
-
10.53
-
0.798
1.2
0.60
-
14.37
-
1.7
8.85
0.062
-
6.82
2.16
> 50
21 (2)
-
6,7-di-OCH3
H
7-OCH3
6-OCH3
5-OCH3
0.031
0.353
0.108
-
-
-
-
> 10
-
-
-
0.009
0.015
9.95
> 10
> 50
> 10
-
> 100
-
-
-
-
-
4.7
-
1.19
0.487
0.283
2.8 (2)
7.34
> 10
> 100
a The IC50 values were determined using GraphPad Prism software and 95% confidence intervals for all data were in ranges of less
than 2-fold of the reported values. The data were obtained in triplicate determinations at eight concentrations of compound in the presence
of 5 µM ATP. b HCASMC proliferation was induced by stimulation with rh-PDGF BB and quantitated by measuring [14C]-thymidine
incorporation. The IC50 values were determined using GraphPad Prism software, and 95% confidence intervals for all data were in ranges
of less than 2-fold of the reported values. The data were obtained in triplicate determinations at eight concentrations of compound. c HUVEC
proliferation was induced by stimulation with LSGS and quantitated by measuring BrdU incorporation. The IC50 values were determined
using GraphPad Prism software, and 95% confidence intervals for all data were in ranges of less than 2-fold of the reported values. The
data were obtained using six serial dilutions of each compound with eight replications for each concentration. d The effects of compounds
on tumor cells in log phase growth were measured by [14C]-thymidine incorporation. The IC50 values were determined using GraphPad
Prism software, and 95% confidence intervals for all data were in ranges of less than 2-fold of the reported values. The data were obtained
in duplicate measurements at eight serial dilutions of each compound.
Figure 1. (a and b) Two possible binding orientations for 1.
Figure 2. (a and b) Two possible binding orientations for 2.
2 in the ATP binding site gives two energetically
degenerate but opposite orientations. Our efforts to
further clarify these issues and arrive at a definitive
binding model are summarized at a later point in this
report (vide infra).
As expected, the exchange of the 3-phenyl in 1 for
the 3-aminophenyl group in 2 does preserve the
PDGFR kinase inhibition activity. When 2 is shorn
of the 6,7-dimethoxy groups, the resulting compound
3 is considerably less active as a kinase inhibitor (Table
1, PDGFR-â IC50 ) 0.317 µM). To recover this activity,
a systematic survey of the importance of methoxy
substitution on kinase inhibition was undertaken. A
series of three monomethoxy derivatives were pre-
pared: 7-methoxy (4, PDGFR-â IC50 ) 0.018 µM),
6-methoxy (5, PDGFR-â IC50 ) 0.217 µM), and 5-meth-
oxy (6, PDGFR-â IC50 ) 0.054 µM). However, best
results were obtained with the 6,7-dimethoxy substitu-
tion pattern of compound 2 (PDGFR-â IC50 ) 0.009 µM).
Furthermore, compounds 2, 4, and 6 have activity in
an assay of PDGF-BB stimulated HCASMC prolifera-
tion that parallels the activity in the isolated enzyme
assay, suggesting that cell penetration is not an issue
for this series. In a companion assay of LSGS-stimulated

8166 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 26
Ho et al.
Table 2. Effect of Substitution on 3′-Aminophenyl Group on
the IC50 (µM) Values of PDGF Kinase Inhibitiona and
PDGF-BB-Stimulated HCASMC Proliferationb
methyl (20) analogues (PDGFR-â IC50 > 0.010 µM <
0.10 µM). Poor potency was obtained with 3′-cyano
substituent (18, PDGFR-â IC50 ) 0.137 µM) and car-
boxylic acid (21, PDGFR-â IC50 > 0.8 µM).
The cellular potencies of these kinase inhibitors were
studied in cell-proliferation assays using the ‘normal’
HCASMC cells (Table 2). In the HCASMC proliferation
assay, driven by exogenous PDGF-BB, all compounds
8, 14-20 inhibited 14C-labeled thymidine incorporation
over a broad range (IC50s ) 0.001 µM to 0.268 µM, Table
3), indicating good cell penetration for most of these
compounds. However, the antiproliferative activity ob-
served for many of these compounds in the LSGS-
stimulated HUVEC assay and the panel of human
tumor cells suggested a more complex cell activity
profile for this series
With only a few exceptions, the potent kinase inhibi-
tors (PDGFR-â IC50 < 0.05 µM) 8, 14-17, 19, and 20
had antiproliferative IC50 < 0.05 µM (Table 3) for all
tumor cells except the pancreatic cancer (ASPC1) and
the glioblastoma (U87MG) cell lines. Comparison of the
antiproliferative activity of 8, 14-17, 19, and 20 in the
panel of tumor cell lines with their activity against
LSGS-stimulated HUVEC proliferation suggested that,
for some of these compounds, the antiproliferative
activity may be tumor cell selective. In the LSGS-
stimulated HUVEC proliferation assay, the 3′-halo
derivatives 8, 16, 17, and the 3′-trifluoromethyl 20 were
poor inhibitors of normal cell proliferation (IC50 > 1 µM);
while 2, the 3′-methoxy 14, and the 3′-methyl 15 were
potent inhibitors (IC50 < 0.10 µM).
R
PDGF RTK
HCASMC
7
2′-Cl
0.11
-
8
3′-Cl
0.003
0.268
-
9
4′-Cl
0.659
10
11
12
13
14
15
16
17
18
19
20
21
2′,5′-di-Cl
2′,4′-di-Cl
3′,4′-di-Cl
3′,5′-di-Cl
3′-OCH3
3′-CH3
6.22 (2)
5.42 (2)
0.41 (2)
0.31 (2)
0.010
-
-
-
-
0.016 (2)
0.188
0.032 (2)
0.003 (2)
-
0.012
0.005
3′-Br
3′-F
0.0042 + 0.00063 (11)
0.137
0.033
0.038
3′-CN
3′-CO2CH3
3′-CF3
0.011 (2)
0.001
3′-CO2H
> 0.8
a The IC50 values were determined using GraphPad Prism
software, and 95% confidence intervals for all data were in ranges
of less than 2-fold of the reported values. The data were obtained
in triplicate determinations at eight concentrations of compound
in the presence of 5 µM ATP. b HCASMC proliferation was induced
by stimulation with rh-PDGF BB and quantitated by measuring
[
14C]-thymidine incorporation. The IC50 values were determined
using GraphPad Prism software, and 95% confidence intervals for
all data were in ranges of less than 2-fold of the reported values.
The data were obtained in triplicate determinations at eight
concentrations of compound.
HUVEC proliferation, a cell system not responsive to
PDGF-BB, compounds 2, 4, and 6 have potent antipro-
liferative activity not dependent on PDGF-BB. We
studied this non-PDGF-BB-mediated antiproliferative
activity in the tumor cell panel (Table 1). The potent
PDGF kinase inhibitors 4 and 6 are found to be poor
inhibitors of tumor cell proliferation. However, the 6,7-
dimethoxy compound, 2, has good activity in these
assays with IC50 e 0.64 µM in six tumor cell lines. This
suggested that the tumor cell antiproliferative activity
of 2 was not the result of its PDGFR-â kinase inhibition
activity but must be due to another mechanism. To
understand the SAR of both of these activities, a series
of 6,7-dimethoxy analogues were prepared and exam-
ined for their PDGFR-â kinase inhibition and antipro-
liferative activity against tumor cells.
The effect of substitution of the 3-aminophenyl on
PDGFR-â kinase inhibition activity was studied with a
series of mono- and dichlorophenyl derivatives (Table
2, 7-13). 2′-, 3′-, and 4′-Chlorophenyl analogues 7-9
demonstrate a clear preference for substitution in the
3′ position (8, PDGFR-â IC50) 0.003 µM). Dichloro-
phenyl analogues with substitution at the 2′,5′- and
2′,4′-positions were compounds with inhibition activity
in the micromolar range while 3′,4′- and 3′,5′- dichlo-
rophenyl substitution gives inhibitors with kinase IC50
values of several hundred nanomolar.
The potent and selective antiproliferative activity
against tumor cell lines indicated significant antitumor
potential for these compounds. Therefore, the mecha-
nism of the tumor cell selective antiproliferative activity
observed for 8, 16, 17, and 20 warranted closer exami-
nation. Compound 17 (JNJ-10198409) was chosen for
further study based upon the potent PDGFR-â kinase
inhibition (IC50) 0.0042 µM), activity in PDGF-BB
stimulated HCASMC assay (IC50) 0.002 µM), poor
activity against LSGS-stimulated HUVEC (IC50) 4.87
µM), and excellent tumor cell antiproliferative activity
(IC50 < 0.033 µM) in six of eight tumor cell lines.
Compound 17 is an ATP competitive inhibitor of
PDGFR-â kinase as exhibited by the rightward shift of
the dose response curve at increasing concentrations of
ATP (data not shown).31 In a battery of growth factor
kinase inhibition assays 17 has good activity against
PDGFR-R kinase (IC50 ) 0.045 µM, Table 4), has poor
activity against the VEGFR (IC50 ) 3.1 µM) and
bFGFR-1 kinases (IC50 ) 45.8 µM), and is inactive
against EGFR (IC50 > 100 µM) and HER-2 (IC50 > 10
µM) kinases. Like STI-571,32 17 is a potent inhibitor of
the c-Abl kinase (IC50 ) 0.022 µM). Workers at Bristol
Meyers Squibb have reported a series of indenopyrazoles
CDK4/CDK2 inhibitors with antiproliferative activity
33
against human and murine tumor cell lines. Unlike
Compounds 14-21 (Table 2) illustrate the effect of a
variety of 3′-substituents on PDGFR-â kinase inhibition
activity. Halogen substituents were preferred with the
chloro (8), fluoro (17), and bromo (16), providing the
most potent analogues (PDGFR-â IC50 < 0.010 µM).
Intermediate potency was achieved with the methoxy
(14), methyl (15), carboxymethyl (19), and trifluoro-
the Bristol Myers Squibb inhibitors, the kinase profile
for 17 indicates that this compound lacks significant
CDK inhibition activity (IC50 > 10 µM for CDKs-1, -2,
-4, and -7), suggesting inhibition of this enzyme family
cannot account for the observed antiproliferative activity
in this series. Compound 17 does have modest activity
against c-Src (IC50 ) 0.185 µM), Lck (IC50 ) 0.10 µM),

PDGF Receptor Tyrosine Kinase Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 26 8167
Table 3. IC50 (µM) Values of Inhibition of LSGS-Stimulated HUVEC Proliferationa and Human Tumor Cell Proliferationb for 2, 8,
14-17, and 20
R
HUVEC
H460
0.353
0.030
0.004
0.029
0.006
0.010 (2)
0.031
LoVo
0.124
0.012
0.004
0.019
0.006
0.017 (2)
0.028
LnCAP
PC3
T47D
A375
ASPC1
U87MG
2
8
14
15
16
17
20
H
0.031
1.73
0.011
0.008
1.48
4.87
2.50
0.60
-
0.005
0.001
0.002
0.009
0.255
0.108
0.048
0.010
0.012
0.018
0.027 (2)
0.021
0.42
0.062
1.58
0.004
0.015
0.006
0.007
0.035
2.16
21 (2)
24 (2)
> 100
> 100
> 100
13 (2)
> 100
Cl
0.030
0.033
0.042
0.037
0.032
0.44
0.427
0.355
4.450
0.331
0.592
0.290
OCH3
CH3
Br
F
CF3
a HUVEC proliferation was induced by stimulation with LSGS and quantitated by measuring BrdU incorporation. The IC50 values
were determined using GraphPad Prism software, and 95% confidence intervals for all data were in ranges of less than 2-fold of the
reported values. The data were obtained using six serial dilutions of each compound with eight replications for each concentration. b The
effects of compounds on tumor cells in log phase growth were measured by [14C]-thymidine incorporation. The IC50 values were determined
using GraphPad Prism software, and 95% confidence intervals for all data were in ranges of less than 2-fold of the reported values. The
data were obtained in duplicate measurements at eight serial dilutions of each compound.
and Fyn (IC50 ) 0.378 µM). These kinases may have
some relevance, but the broad, potent tumor cell activity
observed makes them unlikely candidates as the pri-
mary mechanism of the antiproliferative activity.
The nature of the antiproliferative effect of 17 was
assessed by a cell counting experiment using a trypan
blue exclusion assay with the H460 human lung carci-
noma cell line. Unlike the nonselective kinase inhibitor
staurosporine and the mitosis inhibitor colchicine, after
24 h treatment 17 did not cause the cell number to drop
below the original cell colony plating density until the
concentration of 17 exceeded 1.0 µM. This demonstrates
that the low nanomolar antiproliferative activity ob-
served for 17 in tumor cell lines appears to be a
cytostatic, not a cytotoxic, phenomena up to concentra-
tions of 1.0 µM. In this experiment the cytotoxic
threshold for the staurosporine control was apparent at
concentrations of 0.01 µM and for the colchicine control
at 0.1 µM.31
Binding Model. Compounds 1 and 2 were minimized
in a homology model of the ATP binding site of the
PDGF receptor tyrosine kinase based upon the pub-
lished crystal structure of the VEGF receptor tyrosine
kinase (PDB code: 1VR2).34 The homology model of
PDGFR-â kinase was constructed using the Composer
module in Sybyl v6.7 (Tripos Inc., St. Louis, MO). The
sequences of PDGFR-â kinase and VEGF-R2 kinase are
56% identical and a sequence alignment of these two
proteins is included in Supporting Information. Com-
pounds 1 and 2 were docked in the ATP binding site of
the PDGFR-â kinase homology model using Glide v2.0
(Schro¨dinger LLC., Portland, OR). Some 5000 confor-
mations were generated for each ligand by the Monte
Carlo method, and the 30 best poses were kept for
further scoring based on an E-model scoring function.
The complexes were minimized using AMBER94 force
field with coordinates of backbone atoms fixed.
was found for the presence of the pyrazole N(2)H
tautomer. There was a 3:1 doubling of the resonances
for the pyrazole N(1)H and exocyclic -NH-, suggesting
hindered rotation about the pyrazole-N(H)-phenyl
dihedral and the presence of two rotomers of 17 on the
NMR time scale.35
We present here a model that assumes the N(1)H
tautomer is the bioactive form of the pyrazole ring for
1 and 2. While this tautomer is the major form found
in solution based upon our NMR study, this evidence
does not definitively elucidate the bioactive tautomeric
form bound to the protein. However, the N(1)H tau-
tomer does allow the modeled compounds to maintain
a complementary hydrogen bond donor-acceptor net-
work with the ‘hinge’ region of the ATP binding pocket.
In the docking orientation illustrated in Figures 1a and
2a for 1 and 2, respectively, the tricyclic pyrazole portion
of both molecules are directed along the lipophillic
channel occupied by the triphosphate moiety of ATP,
an orientation similar to that observed for the BMS
indenopyrazoles in a crystal complex with CDK 2.33
With this orientation we postulate a H-bonding network
between the Glu542 carbonyl and the pyrazole N(1)-H
for 1 and 2, a H-bond between the amide NH of Tyr543
and the pyrazole N(2) of 1 and 2, and for 2 there is an
additional H-bond between the aniline N-H and the
carbonyl of Met544. The alternative docking for 1 and
2 envisions a ‘Tarceva-like’ orientation36 of the 6,7-
dimethoxy-2,4-dihydroindeno[1,2-c]pyrazole ring system
with the 6,7-dimethoxy group directed toward the highly
variable ‘selectivity region’ at the solvent interface. For
compounds 1 and 2 the pyrazole N(1)-H is anchored
by a H-bond with the carbonyl of Met544 and the
pyrazole N(2) supplies an H-bond acceptor interaction
with the amide N-H of Tyr543. For compound 2, the
additional aniline N-H can bind with the Glu542
carbonyl.
From the start it was apparent that two equi-
energetic docking orientations were available for 1
(Figures 1a,b) and 2 (Figures 2a,b). This picture is
further complicated by the presumed ability of the
pyrazole heterocycle to exist in two tautomeric forms.
However, in 1D proton, 2D ROESY, 2D [1H]-[13C]
gradient-HMBC, and 2D [1H]-[15N] gradient HMBC
NMR experiments on the related analog 17, no evidence
Conclusion
We have shown that a series of tricyclic pyrazoles with
potent inhibition activity against the angiogenesis
target PDGFR-â kinase can be optimized to enhance a
cellular antiproliferative activity unrelated to the origi-
nal target kinase. This was accomplished by following
in parallel the structure-activity relationships for both

8168 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 26
Ho et al.
Compounds 2-16 and 18-21 were prepared by essentially
the same procedure from the appropriate starting materials.
The starting materials, method of purification, spectral data,
and analytical data are listed below:
PDGFR-â kinase inhibition and cell antiproliferative
activity with a panel of both ‘normal’ and tumor cells.
From this series, compound 17 (JNJ 10198409) emerged
based on the PDGFR-â kinase inhibition activity and
selective tumor cell antiproliferative effect. A cell count-
ing experiment demonstrates the tumor cell antiprolif-
erative activity of 17 is cytostatic, not cytotoxic, in
nature at concentrations up to 1.0 µM, well above the
low nanomolar IC50 observed for the antiproliferative
effect.31 Both kinase and nonkinase biomolecular targets
are under consideration as potential drivers of the
antiproliferative activity, but the mechanism of action
is still a subject of active investigation.
Characterization of 17 in our kinase selectivity screen
demonstrates that this compound has activity against
c-Abl kinase similar to that reported for STI-571. With
a kinase inhibition profile similar to STI-571 combined
with potent antiproliferative activity, 17 may represent
an improved agent for the treatment of the current
clinical indication for STI-571, chronic myelogenous
leukemia (CML), and a potential therapeutic approach
for patients with STI-571-resistant CML.37 In a broader
context, 17 represents a class of antitumor agents
endowed with a dual mechanism of action: an antian-
giogenic effect based upon the potent PDGFR-â kinase
inhibition and a cytostatic effect, due to the potent,
selective antiproliferative activity in human tumor cell
lines. Further work describing the in vitro and in vivo
activity of this dual mechanism agent will be reported
in due course.
(6,7-Dimethoxy-2,4-dihydroindeno[1,2-c]pyrazol-3-yl)-
phenylamine, Trifluoroacetate Salt (2). This material was
prepared by the method described for 17 starting from 5,6-
dimethoxyindan-1-one and phenyl isothiocyanate. The desired
product was isolated by reverse phase hplc using a C18 column
and eluting with a gradient of CH3CN/H2O with 0.05% TFA
to yield a solid: MS m/z 308 (M+ + H); [1H]-NMR(DMSO-d6)
δ: 3.40 (2H, s, CH2), 3.80 (3H, s, CH3), 3.85 (3H, s, CH3), 6.75
(1H, t, CH), 7.10-7.30 (6H, m, CH), 8.35 (1H, s, NH), 12.0
(1H, s, NH); HPLC purity: system 1, >99%, retention time:
4.0 min; system 2, 97.2% (214 nm) and 97.2% (254 nm),
retention time: 4.40 min; HRMS calcd for C18H17N3O2(M+
H) 308.139902, found 308.138883.
+
(2,4-Dihydroindeno[1,2-c]pyrazol-3-yl)phenylamine (3).
This material was prepared by the method described for 17
starting from indan-1-one and phenyl isothiocyanate. The
product was isolated by silica gel chromatography with 2%
methanol:methylene chloride. Compound 3 was subsequently
crystallized from methylcyclohexane:toluene as a white solid.
mp 148-149 °C; MS m/z 248 (M + H)+; [1H]-NMR (DMSO-d6)
δ: 3.5 (s, 2H), 6.75 (t, 1H), 7.0-7.3 (m, 5H), 7.35 (t, 1H), 7.5
(dd, 2H), 8.4 (s, 1H), and 12.3 ppm (s, 1H); HPLC purity:
system 2, 98.9% (214 nm) and 97.2% (254 nm), retention
time: 4.70 min; HRMS calcd for C16H13N3 (M+ + H) 248.118773,
found 248.118420.
(7-Methoxy-2,4-dihydroindeno[1,2-c]pyrazol-3-yl)phen-
ylamine, Trifluoroacetate Salt (4). This material was
prepared by the method described for 17 starting from 6-meth-
oxyindan-1-one and phenyl isothiocyanate. The target com-
pound was purified by reverse phase HPLC with a C18 column
and eluted with a gradient of CH3CN/H2O with 0.05% TFA to
give a TFA salt: MS m/z 278 (M+ + H); [1H]-NMR (DMSO-d6)
δ: 3.30 (s, 2H), 3.71 (s, 3H), 6.79 (m, 2H), 7.14 (m, 5H), 7.34
(d, 1H); HPLC purity: system 1, >99%, retention time: 4.5
min; system 2, 98.0% (214 nm) and 98.0% (254 nm), retention
time: 4.84 min; HRMS calcd for C17H15N3O (M+ + H)
278.12337, found 278.128760.
(6-Methoxy-2,4-dihydroindeno[1,2-c]pyrazol-3-yl)phen-
ylamine, (5). This material was prepared by the method
described for 17 starting from 5-methoxyindan-1-one and
phenyl isothiocyanate. MS m/z 278 (M+ + H); [1H]-NMR
(DMSO-d6) δ: 3.32 (br. s, 2H), 2.79 (s, 3H), 6.69-6.89 (m, 1H),
6.91-6.92 (d, 1H), 7.14-7.19 (m, 5H), 7.42-7.45 (d, 1H), 8.35
(br s, 1H); HPLC purity: system 1, >99%, retention time: 4.4
min; system 2, 97.6% (214 nm) and 97.8% (254 nm), retention
time: 4.62 min; HRMS calcd for C17H15N3O (M+ + H)
278.129337, found 278.128924.
Experimental Section
High-resolution mass spectra were obtained with a Auto
Spec Micromass; low resolution mass spectral analyses were
1
performed with an Agilent 1100 Series LC/MS. The H NMR
used in this study was a 360 MHz Bruker AM 360WB. Two
HPLC systems were used for product purity assessment;
HPLC system 1: Hewlett-Packard model 1050 HPLC with a
3.3 mm × 50 mm Supelco 3 µm AZB + C18 column, eluting
with a mobile phase gradient of 4: 96 acetonitrile: water (0.1%
TFA) to 100: 0 acetonitrile:water (0.1% TFA) over 0.5 min at
a flow rate of 1.2 mL: min for a total run time of 9.5 min;
HPLC system 2: Hewlett-Packard model 1100 HPLC with a
4.4 mm × 30 mm Luna 3 µm C18(2)100R column, eluting with
a mobile phase gradient of 4: 96 acetonitrile:water (0.1% TFA)
to 100: 0 acetonitrile:water (0.1% TFA) over 0.5 min at a flow
rate of 1.2 mL: min for a total run time of 9.5 min.
(5-Methoxy-2,4-dihydroindeno[1,2-c]pyrazol-3-yl)phen-
ylamine, Trifluoroacetate Salt (6). This material was
prepared by the method described for 17 starting from 4-meth-
oxyindan-1-one and phenyl isothiocyanate. The crude mixture
was purified to give 6 by reverse phase hplc and eluted from
a C18 column with a gradient of CH3CN/H2O with 0.05% TFA
to give a TFA salt: MS m/z 278 (M+ + H); [1H]-NMR (DMSO-
d6) δ: 3.35 (s, 2H), 3.82 (s, 3H), 6.72 (t, 1H), 6.94 (d, 1H), 7.20
(m, 5H), 7.34 (t, 1H); HPLC purity: system 1, >99%, retention
time: 4.6 min; system 2, 98.0% (214 nm) and 98.6% (254 nm),
Preparation of Study Compounds: 3-Fluoro-N-(6,7-
dimethoxy-2,4-dihydroindeno[1,2-c]pyrazol-3-yl)phenyl-
amine (17). A mixture of 5,6-dimethoxyindan-1-one (3.0 g,
0.0154 mol) and 3-fluorophenyl isothiocyanate (2.4 g, 0.0157
mol) in THF (3.0 mL) was added to lithium hexamethyldisilane
(15.4 mL, 0.0154 mol) dropwise at room temperature. The
reaction mixture was stirred for 12 h. Hydrazine (0.75 mL,
0.0154 mol) and acetic acid (0.96 mL) were added to the
reaction mixture, which was then heated at the reflux tem-
perature for 24 h. The resulting mixture was added to water
(30 mL) and then extracted with CH2Cl2 (3 × 30 mL). The
organic layers were combined and washed sequentially with
aqueous NaHCO3 solution (30 mL), water (30 mL), and brine
solution (30 mL), dried (Na2SO4), and solvent was removed in
vaccuo. The residue was dissolved in ethyl acetate and
decolorized with charcoal and recrystallized to give compound
17 (3.5 g, 0.0107 mol, 69%) as an off white solid; mp 171-
173.5 °C MS: m/z 326 (M + H)+; [1H]-NMR (DMSO-d6) δ: 3.40
(s, 2H), 3.80 (s, 3H), 3.81 (s, 3H), 6.48-6.55 (t,1H), 7.1-7.2
(m 5H), 7.3-7.4 (d, 1H), 8.8 (s, 1H); HPLC purity (retention
time): >99% (4.1 min); Calcd for C18H16FN3O2: C, 66.45; H,
4.96; N, 12.92: Found: C, 66.19; H, 4.76; N, 12.79.
retention time: 4.80 min; HRMS calcd for C17H15N3O (M+
H) 278.129337, found 278.129477.
+
2-Chloro-N-(6,7-dimethoxy-2,4-dihydroindeno[1,2-c]-
pyrazol-3-yl)phenylamine, Trifluoroacetate Salt (7). This
material was prepared by the method described for 17 starting
from 5,6-dimethoxyindan-1-one and 2-chlorophenyl isothiocy-
anate. Elution of the crude material with reverse phase hplc
using a C18 column and a gradient of CH3CN/H2O with 0.05%
TFA gave pure 7 as a solid: MS m/z 341 (M+ + H); [1H]-NMR
(DMSO-d6) δ: 3.40 (2H, s, CH2), 3.80 (3H, s, CH3), 3.85 (3H,
s, CH3), 6.90 (1H, t, CH), 7.10-7.30 (4H, m, CH), 7.40 (1H, d,
CH), 7.90 (1H, s, NH); HPLC purity: system 1, >99%,
retention time: 4.5 min; system 2, 98.8% (214 nm) and 98.9%

PDGF Receptor Tyrosine Kinase Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 26 8169
(254 nm), retention time: 4.82 min; HRMS calcd C18H16ClN3O2
ether solution was added to precipitate the compound 13,
which was collected and dried under vacuum at 60 °C. mp >
270 °C; MS m/z 376 and 378 (M+ + H); [1H]-NMR (DMSO-d6)
δ: 3.44 (s, 2H), 3.83 (s, 3H), 3.82 (s, 3H),6.93 (s, 1H),7.19 (s,
1H), 7.21 (s, 1H), 7.35 (s, 2H); HPLC purity: system 1, >99%,
retention time: 5.7 min; system 2, 96.8% (214 nm) and 99.6%
(254 nm), retention time: 5.83 min; HRMS calcd for C18H15-
Cl2N3O2 (M+ + H) 376.061957, found 376.061027.
3-Methoxy-N-(6,7-dimethoxy-2,4-dihydroindeno[1,2-c]-
pyrazol-3-yl)phenylamine, Hydrochloride Salt (14). This
material was prepared by the method described for 17 starting
from 5,6-dimethoxyindan-1-one and 3-methoxyphenyl isothio-
cyanate. The crude was treated with saturated hydrochloric
acid in ether solution to yield an off white solid: MS m/z 338
(M+ + H); [1H]-NMR (DMSO-d6) δ: 3.46 (s, 2H), 3.74 (s, 3H),
3.81 (s, 3H), 3.82 (s, 3H), 6.50-6.52 (d, 1H), 6.75-6.77 (d, 1H),
6.77 (s, 1H), 7.17-7.27 (m, 3H); HPLC purity: system 2, 100%
(214 nm) and 100% (254 nm), retention time: 4.52 min; HRMS
calcd for C19H19N3O3 (M+ + H) 338.150467, found 338.149426.
3-Methyl-N-(6,7-dimethoxy-2,4-dihydroindeno[1,2-c]-
pyrazol-3-yl)phenylamine, (15). This material was prepared
by the method described for 17 starting from 5,6-dimethoxy-
indan-1-one and 3-methylphenyl isothiocyanate. The crude
material was recrystallized in ethyl acetate and decolorized
with charcoal to yield 15 as an off white solid: MS m/z 322
(M+ + H); [1H]-NMR (DMSO-d6) δ: 2.24 (s, 3H), 3.78 (s, 3H),
3.82 (s, 3H), 6.54-6.56 (d, 1H), 7.04-7.19 (m, 5H), 8.24 (br s,
1H); HPLC purity: system 1, >99%, retention time: 4.3 min;
system 2, 99.0% (214 nm) and 99.2% (254 nm), retention
time: 4.72 min; HRMS calcd for C19H19N3O2 (M+ + H)
322.155552, found 322.154514.
(M+ + H) 342.100930, found 342.101458.
3-Chloro-N-(6,7-dimethoxy-2,4-dihydroindeno[1,2-c]-
pyrazol-3-yl)phenylamine, Trifluoroacetate Salt (8). This
material was prepared by the method described for 17 starting
from 5,6-dimethoxyindan-1-one and 3-chlorophenyl isothiocy-
anate. The product was purified further by reverse phase
HPLC with a C18 column eluted with a gradient of CH3CN/
H2O with 0.05% TFA to yield 8 as a fluffy white solid: MS
m/z 341 (M+ + H); [1H]-NMR (DMSO-d6) δ: 3.40 (2H, s, CH2),
3.80 (3H, s, CH3), 3.85 (3H, s, CH3), 6.80 (1H, d, CH), 7.10-
7.25 (4H, m, CH), 7.40 (1H, s, CH), 8.80 (1H, s, NH); HPLC
purity: system 1, >99%, retention time: 4.7 min; system 2,
100% (214 nm) and 100% (254 nm), retention time: 4.97 min;
HRMS calcd C18H16ClN3O2 (M+ + H) 342.100930, found
342.100880.
4-Chloro-N-(6,7-dimethoxy-2,4-dihydroindeno[1,2-c]-
pyrazol-3-yl)phenylamine, Hydrochloride Salt (9). This
material was prepared by the method described for 17 starting
from 5,6-dimethoxyindan-1-one and 4-chlorophenyl isothiocy-
anate. The product was purified by reverse phase HPLC with
a C18 column and eluted with a gradient of CH3CN/H2O with
0.05% TFA. Compound 9 was a white solid: MS m/z 341 (M+
+ H)+; [1H]-NMR (DMSO-d6) δ: 3.50 (2H, s, CH2), 3.80 (6H,
s, CH3), 7.30 (2H, d, CH), 7.30 (1H, s, CH), 7.35 (1H, s, CH),
7.40 (2H, d, CH); HPLC purity: system 1, >99%, retention
time: 4.6 min; system 2, 99.0% (214 nm) and 99.7% (254 nm),
retention time: 4.93 min; HRMS calcd C18H16ClN3O2 (M+
H) 342.100930, found 342.101085.
+
2,5-Dichloro-N-(6,7-dimethoxy-2,4-dihydroindeno[1,2-
c]pyrazol-3-yl)phenylamine, Trifluoroacetate Salt (10).
This material was prepared by the method described for 17
starting from 5,6-dimethoxyindan-1-one and 2,5-dichlorophen-
yl isothiocyanate. Compound 10 was isolated by reverse phase
HPLC with a C18 column eluting with a gradient of CH3CN/
3-Bromo-N-(6,7-dimethoxy-2,4-dihydroindeno[1,2-c]-
pyrazol-3-yl)phenylamine, Trifluoroacetate Salt (16).
This material was prepared by the method described for 17
starting from 5,6-dimethoxyindan-1-one and 3-bromophenyl
isothiocyanate. Compound 16 was purified by reverse phase
HPLC with a C18 column eluted with a gradient of CH3CN/
H2O with 0.05% TFA to give a TFA salt: MS m/z 375 (M+
+
H); [1H]-NMR (DMSO-d6) δ: 3.45 (2H, s, CH2), 3.80 (3H, s,
CH3), 3.85 (3H, s, CH3), 6.85 (1H, d, CH), 7.20 (1H, s, CH),
7.25 (1H, s, CH), 7.40 (1H, d, CH), 7.45 (1H, s, NH), 8.00 (1H,
s, CH); HPLC purity: system 1, >99%, retention time: 5.4
min; system 2, 100% (214 nm) and 98.4% (254 nm), retention
time: 5.64 min; HRMS calcd for C18H15Cl2N3O2 (M+ + H)
376.061957, found 376.060153.
H2O with 0.05% TFA to give a TFA salt; MS m/z 385 (M+
+
H); [1H]-NMR (DMSO-d6) δ: 3.41 (s, 2H), 3.77 (s, 3H), 3.79 (s,
3H), 6.90 (dd, 1H), 7.14 (m, 3H), 7.22 (s, 1H), 7.49 (s, 1H), 8.81
(br s, 1H); HPLC purity: system 1, >99%, retention time: 4.8
min; system 2, 100% (214 nm) and 99.7% (254 nm), retention
time: 5.06 min; HRMS calcd for C18H16BrN3O2 (M+ + H)
386.050413, found 386.048508.
2,4-Dichloro-N-(6,7-dimethoxy-2,4-dihydroindeno[1,2-
c]pyrazol-3-yl)phenylamine, Trifluoroacetate Salt (11).
This material was prepared by the method described for 17
starting from 5,6-dimethoxyindan-1-one and 2,4-dichlorophen-
yl isothiocyanate. Crude 11 was further purified by reverse
phase HPLC by eluting from a C18 column with a gradient of
CH3CN/H2O with 0.05% TFA to give pure 11 as a TFA salt:
MS m/z 375 (M+ + H); [1H]-NMR (DMSO-d6) δ: 3.41 (s, 2H),
3.80 (s, 3H), 3.81 (s, 3H), 7.25 (s, 1H), 7.35 (s, 1H), 7.40 (d,
2H), 7.65 (d, 1H); HPLC purity: system 1, >99%, retention
time: 5.5 min; system 2, 100% (214 nm) and 99.8% (254 nm),
retention time: 5.25 min; HRMS calcd for C18H15Cl2N3O2 (M+
+ H) 376.061957, found 376.060828.
3,4-Dichloro-N-(6,7-dimethoxy-2,4-dihydroindeno[1,2-
c]pyrazol-3-yl)phenylamine, Hydrochloride Salt (12).
This material was prepared by the method described for 17
starting from 5,6-dimethoxyindan-1-one and 3,4-dichlorophen-
yl isothiocyanate. The crude was treated with saturated HCl
in ether solution to yield hydrochloride salt: MS m/z 375 (M+
+ H); [1H]-NMR (DMSO-d6) δ: 3.41 (s, 2H), 3.81 (s, 6H), 7.18
(m, 3H), 7.49 (d, 1H), 7.61 (s, 1H); HPLC purity: system 1,
>99%, retention time: 5.3 min; system 2, 99% (214 nm) and
99.4% (254 nm), retention time: 5.49 min; HRMS calcd for
C18H15Cl2N3O2 (M+ + H) 376.061957, found 376.060461.
3-Cyano-N-(6,7-dimethoxy-2,4-dihydroindeno[1,2-c]-
pyrazol-3-yl)phenylamine, Trifluoroacetate Salt (18).
This material was prepared by the method described for 17
starting from 5,6-dimethoxyindan-1-one and 3-cyanophenyl
isothiocyanate. Reverse phase HPLC was used to further
purify 18 by elution from a C18 column with a gradient of CH3-
CN/H2O with 0.05% TFA to give the TFA salt of the product
as a fluffy white solid: MS m/z 333 (M + H)+; [1H]-NMR
(DMSO-d6) δ: 3.45 (2H, s, CH2), 3.80 (3H, s, CH3), 3.85 (3H,
s, CH3), 7.15 (1H, s, CH), 7.20 (1H, d, CH), 7.25 (1H, s, CH),
7.40 (1H, t, CH), 7.50 (1H, d, CH), 7.80 (1H, s, CH), 9.00 (1H,
s, NH); HPLC purity: system 1, >99%, retention time: 4.2
min; system 2, 95.6% (214 nm) and 97.7% (254 nm), retention
time: 5.54 min; HRMS calcd for C19H16N4O2 (M+ + H)
333.135151, found 333.134670.
3-Carbomethoxy-N-(6,7-dimethoxy-2,4-dihydroindeno-
[1,2-c]pyrazol-3-yl)phenylamine, (19). This material was
prepared by the method described for 17 starting from 5,6-
dimethoxyindan-1-one and 3-carbomethoxyphenyl isothiocy-
anate. The crude was recrystallized in ethyl acetate to give a
light brown solid: MS m/z 366 (M + H)+; [1H]-NMR (DMSO-
d6) δ: 3.40 (s, 2H), 3.79 (s, 3H), 3.83 (s, 3H), 3.83 (s, 3H), 7.13
(s, 1H), 7.21 (s, 1H), 7.32 (br s, 1H), 7.34 (br s, 1H), 8.68 (br s,
1H); HPLC purity: system 1, >99% retention time: 4.2 min;
system 2, 99.0% (214 nm) and 98.4% (254 nm), retention
time: 4.56 min; HRMS calcd for C20H19N3O4 (M+ + H)
366.145381, found 366.144026.
3,5-Dichloro-N-(6,7-dimethoxy-2,4-dihydroindeno[1,2-
c]pyrazol-3-yl)phenylamine, Hydrochloride Salt (13).
This material was prepared by the method described for 17
starting from 5,6-dimethoxyindan-1-one and 3,5-dichlorphenyl
isothiocyanate. The crude material was recrystallized from
ethanol. The first and second crops of solid were combined and
dissolved in hot CH3CN, to which an equal volume of HCl-
3-Trifluoromethyl-N-(6,7-dimethoxy-2,4-dihydroindeno-
[1,2-c]pyrazol-3-yl)phenylamine, Trifluoroacetate Salt
(20). This material was prepared by the method described for

8170 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 26
Ho et al.
Table 4. Kinase Panel Inhibition Data for 17
17 starting from 5,6-dimethoxyindan-1-one and 3-trifluoro-
methylphenyl isothiocyanate. Compound 20 was further
purified by a reverse phase HPLC fitted a C18 column and
eluted with a gradient of CH3CN/H2O with 0.05% TFA to yield
the trifluoroacetate salt as a fluffy white solid: MS m/z 376
(M+ + H); [1H]-NMR (DMSO-d6) δ: 3.42 (s, 2H), 3.78 (s, 3H),
3.80 (s, 3H), 7.05 (d, 1H0, 7.17 (s, 1H), 7.22 (s, 1H), 7.40 (m,
2H), 7.68 (s, 1H), 8.92 (br s, 1H); HPLC purity: system 1,
>99%, retention time: 5.2 min; system 2, 98.4% (214 nm) and
99.4% (254 nm), retention time: 5.25 min; HRMS calcd for
C19H16F3N3O3 (M+ + H) 376.127287, found 376.125758.
3-Carboxy-N-(6,7-dimethoxy-2,4-dihydroindeno[1,2-c]-
pyrazol-3-yl)phenylamine, Trifluoroacetate Salt (21). To
a flask under argon was added 0.16 g (0.33 mmol) of compound
19, 4.5 mL of THF, 1 mL of H2O, and 0.043 g (1.0 mmol) of
lithium hydroxide monohydrate. The reaction mixture was
stirred at room temperature for 2 days. The solvent was
evaporated, and then water and a drop of TFA were added.
This material was purified by reverse phase HPLC with a C18
column and eluted with a gradient of CH3CN/H2O with 0.05%
TFA to give 0.06 g (39%) of 21 as a TFA salt: MS m/z 352 (M+
+ H); [1H]-NMR (DMSO-d6) δ : 3.4 (s, 2H), 3.8-3.9 (2s, 6H),
7.1 (s, 1H), 7.2 (s, 1H), 7.3-7.5 (m, 2H), 7.8 (s, 1H), 8.8 (bs,
1H); HPLC purity: system 1, >99%; system 2, 100% (214 nm)
and 86.5% (254 nm), retention time: 3.96 min; HRMS calcd
for C19H17N3O4 (M+ + H) 352.129731, found 352.128941.
kinase
IC50 (µM)
PDGFR-â
PDGFR-R
VEGFR
HER-2
EGFR
0.0042
0.045
3.1
>10
>100
45.8
0.022
>100
>10
>100
bFGFR1
c-ABL
CDK1
CDK2
CDK4
CDK7
56.7
0.185
0.100
0.378
c-SRC
LCK
FYN
PKA
>100
GSK3
13
>100
>100
>100
MAPK
IRK
FAK
Casein K1
Casein K2
Calmodulin K
7.2
>100
>10
for an individual compound was measured using the above
procedure. The IC50 for PDGF-R kinase activity refers to the
concentration of an inhibitor at which the activity of the
PDGF-R kinase is reduced by one-half as compared with
reactions containing no inhibitor.
Inhibition of PDGF-Receptor Kinase Activity. PDGF-R
kinase activity was assayed by the ability of the kinase to
phosphorylate a consensus sequence of its target proteins,
PLCγ in a cell-free system, in particular, a PLC1 peptide
comprising the tyrosine residue where the phosphorylation
occurs was used for the assay.
Kinase Selectivity. Assays and Substrates. Selectivity
versus additional kinases (Table 4) was assessed using in-
house assays as described above for the PDGFR kinase assay
but incorporating appropriate modifications to the assay buffer
and substrates for each respective kinase enzyme.38 Com-
pounds were further evaluated for kinase selectivity in the
Upstate panel of kinases (Upstate, L.L.C., Charlottesville, VA).
Cell Proliferation Assay in Normal Cells in the Pres-
ence of PDGF Stimulation. The effect of compounds on cell
proliferation in normal human primary cells, in particular,
cryopreserved human coronary artery smooth muscle cells
(HCASMC), in the presence of PDGF stimulation was tested
based on incorporation of [14C]-thymidine into DNA of cells.
The following materials were purchased from their respec-
tive sources:
Recombinant human PDGF beta homodimer, rhPDGF-BB
purchased from R&D System (Minneapolis, MN, Cat. No:
220-BB); cryopreserved human coronary artery smooth muscle
cells (HCASMC), tissue culture medium for HCASMC, and
smooth muscle growth supplement (SMGS) purchased from
Cascade Biologics (Portland, OR, HCASMC Cat. No: C-017-
5C; Medium 231 Cat. No: M-231-500; and SMGS Cat. No:
S-007-25); 96-well CytoStar tissue culture treated scintillating
microplates purchased from Amersham (Piscataway, NJ, Cat.
No: RPNQ0160); methyl 14C-thymidine at 56 mCi/mmol (250
µCi/2.5 mL) purchased from NEN (Cat. No.: NEC568); DMSO
from Sigma (St. Louis, MO, Cat. No: D-5879); sterile reagent
reservoirs from Costar (VWR International, Inc., West Chester,
PA, Cat. No: 4870); Dulbecco’s PBS from Gibco (Cat. No:
14190-136); backing tape white plate cover for bottom of
CytoStar plate from Packard (Cat. No: 6005199).
HCASMC were seeded at approximately 4000 cells/well in
a volume of 100 µL of complete Medium 231 with SMGS. Cells
were grown for 48 h until they reached approximately
80% confluence. They were rendered quiescent by incuba-
tion in SMGS-free Medium 231 for 24 h. Cell culture media
was replenished with SMGS-free Medium 231 containing
rhPDGF-BB at 50 ng/mL in a total volume of 100 µL/well,
and 1 µL of test compounds in serially diluted concentra-
tions in 100% DMSO was added to each well. For the
maximum growth control wells, only 1 µL of 100% DMSO was
added; for minimum growth (blank) wells, 1 µL of 10 mM
cycloheximide was added to each well. After incubation for 24
h, 20 µL of [14C]-thymidine mix was added to each well and
The following reagents were prepared for the assay:
10× Kinase Buffer (500 mM Tris-HCl pH ) 8, 100 mM
MgCl2, 1 mM Na3VO4); 10 mM DTT (final concentration at
1 mM in assay); 10 mM ATP (final concentration at 5 µM
in assay); [33P]-γ-ATP (Cat. No.: NEG/602H. 2000-3000
Ci/mmol) purchased from NEN; purified, soluble, recombinant
PDGF-receptor beta enzyme comprising the tyrosine kinase
domain (from amino acid 545 to 1106 of GenBank Access NO:
AAA36427) at 0.4 mg/mL; enzyme dilution buffer (50 mM Tris-
HCl pH ) 8.0, 0.1% BSA); wash/stop buffer (PBS +100 mM
EDTA); NEN Streptavidin Flashplate (cat#: SMP-103) which
binds to the biotinylated PLC1 peptide but not the PDGF-R
enzyme; PLC1 peptide (Biotin-KHKKLAEGSAYEEV-Amide)
at 1 mM in 50 mM Tris-HCL with pH of 8.0.
Reagents were first mixed according to the following regi-
men: 1100 µL of 10× Kinase Buffer, 1100 µL of 10 mM DTT,
5.5 µL of 10 mM cold ATP, 2.75 µL of 1 mM PLC1 Peptide 8.8
µL of [33P]-γ-ATP (10 mCi/ml), and 5475 µL of H2O. The above
mixture was dispensed into each well of a Flashplate at 70
µL/well. To test the effect of a compound on PDGF-R kinase
activity, the test compound either in a fixed concentration or
in serially diluted concentrations in 100% DMSO was added
to appropriate wells at 1 µL/well. Enzyme PDGF-R was diluted
in enzyme dilution buffer. The kinase reaction was initiated
by adding 30 µL of diluted PDGF-R enzyme solution to each
well on the Flashplate containing radioactive ATP and PLC1,
except wells of column 12 rows E through H, which were used
to calculate the plate background. The Flashplate was swirled
to mix and was incubated at 30 °C for 60 min. Then, the
reaction mixture was decanted and the Flashplate was washed
three times each with 200 µL of wash/stop buffer. Subse-
quently, each well on the Flashplate was filled with 200 µL of
wash/stop buffer. The amount of [33P] retained in each well
was measured using a Packard TopCount after the plate was
sealed with a transparent plate sealer. When a test compound
inhibited the PDGF-R kinase activity, the well containing such
a compound contained less [33P] as compared to the well
without the compound. The percentage of inhibition of the test
compound on PDGF-R kinase activity is defined as the amount
of [33P] retained in the well containing the compound divided
by the amount of [33P] in the well without the compound. To
test the potency of inhibition of present compounds, an IC50

PDGF Receptor Tyrosine Kinase Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 26 8171
the [14C]-thymidine mix was made according to the following
regimen: 220 µL of [14C]-thymidine, 1980 µL of SMGS-free
Medium 231. Cells were incubated for an additional 24 h in
media containing test compounds, rhPDGF-BB and [14C]-
thymidine. Then, the reaction mixture was discarded and the
plate was washed three times each with 200 µL of PBS.
Subsequently, each well on the plate was filled with 200 µL of
PBS. The top of the plate was sealed with transparent plate
sealer, and white plate backing sealers were applied to the
bottom of plates. The retained [14C] inside each well was
measured using a Packard Top Count. The amount of [14C]
retained in a well correlates to the proliferation of cells inside
the well. When a test compound inhibited rhPDGF-BB-induced
HCASMC proliferation, the well containing such a compound
retained less [14C] as compared to the maximum growth control
wells without the compound. To test the potency of inhibition,
the IC50 of an individual compound on the inhibition of
rhPDGF-BB-induced HCASMC proliferation was measured
using the above procedure. The IC50 refers to the concentration
of the test compound at which the amount of rhPDGF-BB-
induced HCASMC proliferation is reduced by one-half as
compared to the maximum growth control wells without the
compound.
Supporting Information Available: Detailed experi-
mental procedures and spectra are provided for the NMR
experiments described for 17. The sequence alignment used
to develop the homology model of the PDGF RTK ATP binding
site is also included. This material is available free of charge
via the Internet at http://pubs.acs.org.
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PDGF Receptor Tyrosine Kinase Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 26 8173
15N] gradient-HMBC experiment that gave a 3-bond correlation
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[
from the major aniline NH to the major pyrazole N2. The HMBC
experiment did not suppress the one bond NH coupling so
doublets were observed for the N1 and aniline nitrogens.
Proton-nitrogen correlations were not observed for the minor
isomer. In the 2D [1H]-[13C] gradient-HMBC data the pyrazole
NH did not show correlations to carbons outside of the pyrazole
ring and thus could not be used to further confirm the ROESY
results. ROEs were seen from the aniline NH and the fluoro-
aromatic protons to the methylene protons on the dihydroindeno-
[1,2-c]pyrazole ring, suggesting that both the “in” and “out”
orientations were accessible on the NMR time scale. Hindered
rotation about the aniline-NH-pyrazole bond is believed to be
the cause for the doubling of the resonances. The proton spectra
of 17 in CDCl3 or AcCN-d3 did not show a doubling of the
resonances as observed in DMSO-d6. In deuteriochloroform the
NH resonances were broadened almost into the baseline and in
deuterioacetonitrile the NH resonances were broad, as well, as
one of the fluorophenyl protons. For a full disclosure of the
experimental details, see the Supporting Information.
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(35) For 17 dissolved in DMSO-d6 the 1D proton spectrum showed
two resonances for each of the NH protons in a 3:1 ratio, and
this doubling was also observed for the fluorophenyl aromatic
protons and the methylene protons on the dihydroindeno[1,2-c]-
pyrazole ring system. The singlets for the methoxy protons
attached at C-6 and the methylene protons on C-4 were
broadened. The ROESY spectrum showed exchange peaks
between the two sets of resonances. There was an ROE from
the downfield NH to H8 confirming its assignment as the
pyrazole NH at the 1-position. This was confirmed by a 2D [1H]-
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J.; Allen, R.; Perry, M.; Jolliffe, L.; Middleton, S. A. A Vascular
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JM050680M