1,4-Dihydroindeno[1,2-c]pyrazoles as novel multitargeted receptor tyrosine kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2006.05.066

A series of 1,4-dihydroindeno[1,2-c]pyrazoles with a 3-thiophene substituent carrying a urea-type side chain were identified as potent multitargeted (VEGFR and PDGFR families) receptor tyrosine kinase inhibitors. A KDR homology model suggested that the ur

Full text of DOI:10.1016/j.bmcl.2006.05.066

Bioorganic & Medicinal Chemistry Letters 16 (2006) 4266–4271
1,4-Dihydroindeno[1,2-c]pyrazoles as novel multitargeted
receptor tyrosine kinase inhibitors
Jurgen Dinges,a,* Kimba L. Ashworth,a Irini Akritopolou-Zanze,a Lee D. Arnold,c
¨
Steven A. Baumeister,a Peter F. Bousquet,b George A. Cunha,b Steven K. Davidsen,a
Stevan W. Djuric,a Vijaya J. Gracias,a Michael R. Michaelides,a Paul Rafferty,b
Thomas J. Sowin,a Kent D. Stewart,a Zhiren Xiaa and Henry Q. Zhanga
aGlobal Pharmaceutical Research and Development, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, IL 60064-6217, USA
bAbbott Bioresearch Center, 100 Research Drive, Worcester, MA 01605-5314, USA
cOSI Pharmaceuticals, Inc., 1 Bioscience Park Drive, Farmingdale, NY 11735, USA
Received 4 May 2006; revised 17 May 2006; accepted 18 May 2006
Available online 12 June 2006
Abstract—A series of 1,4-dihydroindeno[1,2-c]pyrazoles with a 3-thiophene substituent carrying a urea-type side chain were
identified as potent multitargeted (VEGFR and PDGFR families) receptor tyrosine kinase inhibitors. A KDR homology model
suggested that the urea moiety is able to interact with a recognition motif in the hydrophobic specificity pocket of the enzyme.
Ó 2006 Elsevier Ltd. All rights reserved.
Receptor tyrosine kinases (RTKs) play a crucial role in
signal transduction as well as in cellular proliferation,
differentiation, and various regulatory mechanisms.
They consist of an extracellular ligand binding domain,
a transmembrane spanning region, and a cytoplasmic
kinase domain.1 After binding to their specific extracel-
lular growth factors, RTKs undergo dimerization and
autophosphorylation, initiating a cascade of down-
stream signaling events, which trigger a variety of cell
responses. Genetic alterations and/or stimulation
through autocrine/paracrine growth factor loops can re-
sult in RTKs that are constitutively active. These types
of disturbances in the tightly regulated signal transduc-
tion pathways have been implicated in the development
and progression of a variety of hyperproliferative diseas-
es, in particular cancer.2
factor receptors PDGFR a and b, the colony-stimulat-
ing factor-1 receptor (CSF-1R), and the stem cell factor
receptor (cKit). KDR kinase is specifically expressed in
vascular endothelial cells. It is the primary receptor for
vascular endothelial growth factor (VEGF) and plays
an essential role in tumor angiogenesis.3 Inhibition of
KDR blocks the neovascularization of tumors and has
been shown to inhibit the growth of a variety of solid tu-
mors in tumor xenograft models. Mutated cKit kinase
on the other hand is a driving factor in mastocytosis
and gastrointestinal stromal tumors.4 Wild-type cKit is
also considered to play a role in the progression of small
cell lung cancer. Here it was proposed that cKit is in-
volved in the formation of an autocrine/paracrine loop
due to coexpression of ligand and receptor within the
same tumor cell type.5 Inhibition of both, KDR and
cKit in the appropriate tumor types, has the potential
to produce antitumor effects through two distinct mech-
anisms. Inhibition of cKit should result in direct effects
on the tumor cell phenotype, while inhibition of KDR
should produce indirect effects via disruption of endo-
thelial cell function.6
Members of the split kinase domain (class III RTK)
subfamily include the vascular endothelial growth factor
receptors KDR (VEGFR2, kinase insert domain-con-
taining receptor tyrosine kinase) and FLT1 (VEGFR1,
Fms-like tyrosine kinase 1), the platelet-derived growth
In a previous report we have described our hit-to-lead
efforts in establishing 1,4-dihydroindeno[1,2-c]pyrazoles
as a novel class of KDR kinase inhibitors.7 Two key
compounds, 1 and 2 (Fig. 1), were identified with
KDR IC50 < 200 nM. We now describe our
Keywords: Cancer; Receptor tyrosine kinase; KDR kinase; Inhibitor;
1,4-Dihydroindeno[1,2-c]pyrazole.
*
Corresponding author. Tel.: +1 847 935 1448; e-mail: jurgen.
dinges@abbott.com
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.05.066

J. Dinges et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4266–4271
4267
conformation12 to create a homology model of 2 bound
into the ATP binding site of KDR kinase. Our studies
revealed that either a 20,40- or a 20,50-linked thiophene
would provide an acceptable trajectory for the urea side
chain to access the pocket. A methylene spacer between
the thiophene and the urea functionality was required to
allow the urea to form two hydrogen bonds of 3.0 and
Figure 1. Structure and KDR inhibitory potency of early lead
compounds 1 and 2.
˚
3.2 A distance to Glu 885. In addition, we proposed that
a small hydrophobic substituent in the 3-position off a
terminal phenyl ring would be beneficial because it
would project into a small hydrophobic groove formed
by Ile 892, Ile 888, Leu 889, Val 898, and Leu 1019.
Figure 2 shows compound 3 as the result of our design
efforts and its predicted binding mode.
investigations toward extending those molecules into the
hydrophobic specificity pocket of KDR kinase.
The imidazole-containing compound 1 showed excessive
inhibition of human liver microsome (HLM)-derived
CYP3A48 (IC50 = 254 nM), and so we focused on its
1-methylpiperazine-containing analogue 2 as our key
structural unit (CYP3A4 (HLM) IC50 > 10 lM). Based
on our earlier established structure–activity relation-
ships (SARs), we continued to consider compounds with
a basic side chain attached in either the 6- or 7-position.
Previous studies, focused on accessing the hydrophobic
specificity pocket of KDR kinase, identified a recogni-
tion motif for ureas in this site.9–11 So in order to plan
for the attachment of a urea functionality to our
1,4-dihydroindeno[1,2-c]pyrazoles, we utilized the
published crystal structure of cKit kinase in its inactive
The synthesis of 1,4-dihydroindeno[1,2-c]pyrazoles with
urea-type side chains is illustrated with the synthesis of 3
as a representative example (Scheme 1). Regioselective
deprotonation of the thiophene portion of 4,7 followed
by formylation with DMF, yielded aldehyde 5. For the
preparation of compounds containing 20,40-disubstitut-
ed thienyl groups, regioselectivity was achieved through
halogen–lithium exchange of the corresponding bro-
mides. After protection of the pyrazole moiety, the alde-
hyde functionality in 6 was reduced with sodium
borohydride to give the hydroxymethyl compound 7.
Direct conversion of 7 to the azide 8 using diphenyl
phosphorazidate (DPPA)13 and subsequent Staudinger
reduction afforded amine 9. Reaction of this key inter-
mediate with m-tolyl isocyanate followed by deprotec-
tion of the pyrazole led to the urea 3.
The activity of the target compounds to inhibit the phos-
phorylation of
a
peptide substrate (biotin-Ahx-
AEEEYFFLFA-amide) by KDR kinase was assessed
in an HTRFÒ assay at 1.0 mM concentration of adeno-
sine-50-triphosphate (ATP).9 Table 1 shows that the urea
linker indeed improved the potency for KDR inhibition.
However, in contrast to our previous findings, attach-
ment of the thienyl group to the 1,4-dihydroinde-
no[1,2-c]pyrazole core through its 50-position
(corresponding to the 20-position in Fig. 1) (10 and 3)
was now better tolerated than attachment through its
40-position (corresponding to the 30-position) (11 and
12). On the other hand, placing the basic side chain
in the 7-position (3 and 12), again, was only slightly
Figure 2. Model of compound 3 (green) bound to the active site of
KDR (inactive conformation, homology model based on cKit PDB
entry 1T46). Hydrogen bonds are shown in black dotted lines. H-bond
distances from the urea to Glu 885 are shown in angstroms.
Scheme 1. Reagents and conditions: (a) i—n-BuLi, THF, ꢀ78 °C; ii—DMF, THF, ꢀ78 °C to rt, 70%; (b) 4,40-dimethoxybenzhydryl chloride, Et3N,
THF, 50 °C, 80%; (c) NaBH4, MeOH/THF (2:1), 0 °C to rt, 65%; (d) DPPA, DBU, THF, 0 °C to rt, 95%; (e) i—PPh3, THF, 40 °C; ii—H2O, 40 °C,
96%; (f) m-tolyl isocyanate, CH2Cl2, rt, 62%; (g) HCl, EtOH/EtOAc (1:1), rt, 83%.

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J. Dinges et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4266–4271
Table 1. KDR inhibitory activity of a selected set of 1,4-dihydroindeno[1,2-c]pyrazoles with various substituents in 3-position
a
KDR IC50 (nM)
Compound
R1
H
R2
R3
5'
S
2'
2'
O
N
HN
10
61
180
N
N
HN
4'
O
N
S
S
HN
HN
HN
11
3
H
HN
O
N
N
H
48
N
HN
O
S
S
12
13b
H
102
N
HN
O
O
N
HN
HN
H
12285
N
S
S
N
N
14b
15b
H
H
2777
212
N
N
O
HN
O
O
O
N
N
16b
H
H
22795
S
HN S
N
N
O
17b
>50000
S
S
HN S
O
N
N
N
18b
19b
20b
H
H
H
24158
42
HN
O
N
N
N
O
S
O
HN
S
S
S
N
>50000
HN
HN
N
H
N CN
N
N
21b
22b
H
H
>50000
459
N
N
HN
O
N
HN
a Values are means of two experiments.
b Ref. 14.

J. Dinges et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4266–4271
4269
Table 2. Inhibitory activities of 1,4-dihydroindeno[1,2-c]pyrazoles with the basic side chain in 6-position and a selected set of substituents in the urea
moiety
Compound
R1
R2
R3
IC50 (nM)
FLT1
a
KDR
cKit
1387
23
10
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
H
H
2-CH3
3-CH3
4-CH3
2-CF3
444
61
24996
2897
11483
>50000
863
H
H
86
24
H
H
308
2882
34
H
H
23829
17
13
H
H
3-CF3
4-CF3
2-Cl
H
H
281
199
63
4201
>50000
1668
2275
4718
14581
27894
4953
3842
33
H
H
921
27
H
H
3,5-di-CH3
3,5-di-Cl
3-CH3
3-CH3
3-CH3
2-CH3
2-Cl
H
H
79
38
14735
139
CH3
H
H
159
4807
7458
122
180
20
CH3
CH3
H
CH3
CH3
CH3
CH3
CH3
Et
26845
5710
>50000
25
H
H
3-F, 5-CF3
3-OCF3
3-CH3
3-CH3
3-CH3
3-CH3
3-CH3
3-CH3
3-CH3
3-CH3
H
27
45
19
131
56
H
2190
75
n-Pr
i-Pr
H
35
59
H
123
21
2824
38
9746
136
c-Pr
H
i-Bu
i-Amyl
CH3O
CH3OCH2CH2
H
164
262
259
171
1590
3615
1351
2263
17239
4843
237
H
H
H
>50000
a Values are means of two experiments.
Table 3. Inhibitory activities of 1,4-dihydroindeno[1,2-c]pyrazoles with the basic side chain in 7-position and a selected set of substituents in the urea
moiety
R1
R2
R3
IC50 (nM)
FLT1
a
Compound
KDR
cKit
46
47
3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
184
159
48
3785
2056
592
613
44
101
674
69
27
96
18
38
59
19
28
17
36
H
2-CH3
3-CH3
4-CH3
3-CH3
3-CF3
3-Cl
H
48
49
50
51
52
53
54
55
56
H
104
51
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
11
12
14
20
4-F
16
17
83
25
4-OCF3
3,4-di-Cl
4-Br
18
19
36
38
4-Cl
21
64
a Values are means of two experiments.

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favored over attachment to the 6-position (10 and 11).
All efforts to identify a functionality, which could be
superior to the urea moiety, resulted in compounds with
reduced KDR potency. For instance, the acid amide 13
and the sulfonamide 16, as well as the urea mimetics 20
and 21, showed significantly diminished activities. The
acid amides 14 and 15, the sulfonamide 17, and the car-
bamates 18 and 19 represent bioisosteric replacements
for the urea functionality in the parent compounds 10
and 11. While those modifications generally were detri-
mental, it should be noted that replacement of the exter-
nal NH group of the urea (14 and 18) resulted in a larger
drop in potency than replacement of the internal NH
group (15 and 19). The activity of the carbamate 19 is
particularly noteworthy because it is equipotent to its
parent 3; however, in the KDR whole cell assay9 it only
showed an IC50 of 2 lM and therefore was not pursued
further. In comparison to 10, an 8-fold loss in activity
was observed for 22, which contained a cyclized version
of the urea.
(IC50 = 233 and 218 nM, respectively). The modifica-
tion of the N-alkyl substituent (38–45) revealed the
n- and cyclo-propyl groups as the most promising res-
idues, however, both variations again led to a slight
loss in KDR whole cell activity (IC50 = 198 nM for
39 and 171 nM for 41).
Turning our attention to 1,4-dihydroindeno[1,2-c]pyra-
zoles with the basic side chain in 7-position (Table 3),
we found a few differences in reflection to previously
established SAR trends. For compounds with a free
urea moiety, the 3-substitution on the phenyl ring,
again, is favored for KDR enzymatic activity, with the
m-tolyl urea 3 being approximately 4-fold more potent
than its unsubstituted parent compound 46. Methyla-
tion of the internal NH-group of the urea now basically
has no effect on the KDR inhibitory potency (49 vs 3),
compared to a slight loss earlier. Surprisingly, this meth-
ylation has no effect on the cKit activity although it
should prevent the formation of the more important
hydrogen bond (49 vs 31). We speculate that the basic
side chain in 7-position slightly re-orients the inhibitor
in the cKit active site, such that the external hydrogen
bond plays the more dominant role. Re-optimization
of the phenyl substitution pattern then demonstrated
that 4-substituents now become more important (52,
53, 55, and 56), which then also shifted the preference
for disubstitutions (54). Compounds 49 to 56 illustrate
that at this point, our established SAR has enabled us
to fairly routinely produce potent multitargeted RTK
inhibitors. The trifluoromethyl analogue 50 stands out
in this set, because in addition to its potency against
KDR, FLT1, and cKit, it is the most active compound
against Tie2 (IC50 = 259 nM), a more distant member
of the RTK family, which also is a target for anti-angio-
genic drug discovery.15 The KDR whole cell IC50 of 50
was determined to be 195 nM.
An investigation of the substitution pattern in the
urea moiety was initially conducted on 1,4-dihydroin-
deno[1,2-c]pyrazoles with the basic side chain in 6-po-
sition. The obtained results are summarized in Table 2.
For comparison, the table also lists the activities
for inhibition of FLT1,9 as a closely related VEGFR
family member, and cKit,9 as a representative of the
PDGFR subfamily, in addition to the KDR inhibitory
potencies. As already predicted by our computer mod-
el, compounds 10 and 23–27 demonstrate that in
mono-substituted phenyl ureas a small substituent in
3-position is preferred for KDR inhibition. This
SAR carries over for FLT1, but for cKit the 4-substi-
tution becomes more important. Substituents in the
2-position were the least favored, but gave rise to
the most selective compounds, as demonstrated by
28 (>250-fold selectivity for KDR inhibition over inhi-
bition of FLT1), 34 (about 45-fold selectivity for
KDR over both, FLT1 and cKit), and 35 (>278-fold
selectivity for KDR over cKit). In the case of disubsti-
tuted phenyl ureas, the 3- and 5-positions turned out
to be optimal (29 and 30). Replacing the phenyl
groups with aliphatic residues and aliphatic or aro-
matic heterocycles led to a complete loss of activity.
Methylation of the internal urea nitrogen diminished
the KDR potency only slightly (31: 2.6-fold), while
alkylation of the external NH-group had a more det-
rimental effect (32: 79-fold and 33: 122-fold loss). This
corresponds with our earlier observation on bioisoster-
ic replacements and confirms our computer model pre-
dictions, since the larger decrease in potency is
observed when the more optimal urea hydrogen bond
is blocked. The situation is reversed for the inhibition
of cKit, where the internal hydrogen bond appears to
play the more important role. The KDR whole cell
IC50 of 31 and its parent compound 10 were found
to be identical (146 nM), which prompted us to fur-
ther investigate N-alkylated ureas. Re-optimization
of the substitution pattern on the phenyl ring identi-
fied the ureas 36 and 37, which for the first time dis-
played comparable potencies for all three kinases, but
lost some potency in the KDR whole cell assay
In summary, 1,4-dihydroindeno[1,2-c]pyrazoles con-
taining various substituents off a thiophene ring in 3-
position were investigated to access the hydrophobic
specificity pocket in KDR kinase. Phenylurea-type side
chains were identified to be optimal. A homology
model predicted that binding of those compounds into
the ATP binding site of the inactive form of KDR
would allow the urea moiety to interact with a specific
recognition motif in the enzyme. Further optimization
of the urea-type side chain and the position of a basic
substituent on the core led to a series of multitargeted
RTK inhibitors, which displayed potent inhibition
against additional VEGFR family members (FLT1
and Tie2), as well as a member of the structurally
related PDGFR family (cKit). Future work will focus
on the evaluation of these compounds in various
in vivo efficacy models.
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