Synthesis and biological evaluation of 5-substituted 1,4-dihydroindeno[1,2-c]pyrazoles as multitargeted receptor tyrosine kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2007.03.031

We report the synthesis and biological evaluation of 5-substituted 1,4-dihydroindeno[1,2-c]pyrazoles as multitargeted kinase inhibitors. Initial efforts focused on the development of selective KDR inhibitors, while later strategies involved the improvement of potency toward multiple kinase targets. Thus, several compounds were identified as potent KDR, Flt1, Flt3, and c-Kit inhibitors.

Full text of DOI:10.1016/j.bmcl.2007.03.031

Bioorganic & Medicinal Chemistry Letters 17 (2007) 3136–3140
Synthesis and biological evaluation of 5-substituted
1,4-dihydroindeno[1,2-c]pyrazoles as multitargeted
receptor tyrosine kinase inhibitors
Irini Akritopoulou-Zanze,* Daniel H. Albert, Peter F. Bousquet, George A. Cunha,
Christopher M. Harris, Maria Moskey,ꢀ Jurgen Dinges,
Kent D. Stewart and Thomas J. Sowin
Global Pharmaceutical Research and Development, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, IL 60044, USA
Received 7 February 2007; revised 9 March 2007; accepted 12 March 2007
Available online 15 March 2007
Abstract—We report the synthesis and biological evaluation of 5-substituted 1,4-dihydroindeno[1,2-c]pyrazoles as multitargeted
kinase inhibitors. Initial efforts focused on the development of selective KDR inhibitors, while later strategies involved the improve-
ment of potency toward multiple kinase targets. Thus, several compounds were identified as potent KDR, Flt1, Flt3, and c-Kit
inhibitors.
Ó 2007 Elsevier Ltd. All rights reserved.
Receptor tyrosine kinases (RTKs) are important regula-
tors of cell survival, migration, and proliferation as well
as angiogenesis and their over-expression or deregula-
tion leads to uncontrollable cellular signaling and can-
cer. Platelet-derived growth factor receptors PDGFR
(PDGFR-a, PDGFR-b, Flt3, CSF1R, and cKit) and
vascular endothelial growth factor receptors VEGFR
(KDR, Flt1, and Flt4) are RTK subfamilies that play
key roles in tumor angiogenesis and therefore have been
targeted for the development of anti-cancer therapies.
inhibitor of KDR, Flt1, PDGFR-a, PDGFR-b, Flt3,
CSF1R, cKit, and RET), which was approved by the
FDA for the treatment of GIST and advanced renal-cell
carcinoma (RCC), and Nexavar4 (an inhibitor of KDR,
Flt1, PDGFR-b, c-Kit, RET, and Raf isoforms) for
N
N
NH
3
NH
7
6
N
N
4
5
S
S
Initial strategies involved single target therapies and
resulted in the FDA approval of Avastin (a humanized
monoclonal antibody targeting VEGF, the growth fac-
tor that stimulates VEGFRs) for the treatment of meta-
static colorectal cancer.1 Gleevec (a Bcr-Abl inhibitor
approved for the treatment of chronic myelogenous leu-
kemia) is also a PDGFR and c-Kit inhibitor, and has
been approved for the treatment of gastrointestinal stro-
mal tumors (GIST).2 Subsequent approaches involved
the use of multitargeted inhibitors such as Sutent3 (an
1 KDR IC50 = 1.2 μM
2 KDR IC50 = 180 nM
N
NH
HN
N
HN
S
N
O
3 KDR IC50 = 48 nM
N
NH
N
N
Keywords: Tricyclic pyrazoles; 1,4-dihydroindeno[1,2-c]pyrazoles;
Multitargeted kinase inhibitors; KDR inhibitors; Flt1 inhibitors; Flt3
inhibitors; c-Kit inhibitors; PDGFR inhibitors; VEGFR inhibitors.
S
O
4 KDR IC50 = 63 nM
*
Corresponding author. Tel.: +1 8479375006; fax: +1 847 9350310;
e-mail: irini.zanze@abbott.com
Present address: 5 Margaret Lane, Lee, NH 03824, USA.
Figure 1. Structures and KDR inhibitory activity of various 1,4-
dihydroindeno[1,2-c]pyrazoles.
ꢀ
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.03.031

I. Akritopoulou-Zanze et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3136–3140
3137
RCC. Whether the multitargeted inhibitors have the
advantage of blocking more than one of the signaling
pathways essential to tumor survival and growth,5 the
exact combination of activities against the various
kinases, and which kinase target combination is required
for efficacious therapeutics is still a fruitful area of
investigation.
We have previously described the discovery of 1,4-
dihydroindeno[1,1-c]pyrazoles as a novel class of VEG-
FR and PDGFR inhibitors. Starting from the high
throughput screening hit 1 (Fig. 1), compound 2 was
identified as a lead compound with acceptable binding
and whole cell activity.6 Attachment of a urea side chain
to access the hydrophobic specificity pocket in KDR led
to a series of potent multitargeted (KDR, Flt1, cKit,
Tie2) inhibitors such as 3.7 Introduction of an acetylenic
side chain led to the discovery of 4, which was effica-
cious in animal tumor models.8
KDR:Lys920
KDR:Phe918
KIT:Tyr672
KDR:Val 916
KIT:Thr670
KIT:Cys674
In this report, we describe our efforts to explore the 5
position of compound 1. Since in the early stages of
the project one of the goals was to develop KDR selec-
tive inhibitors, we have attempted to improve the KDR
over c-Kit selectivity by structure based drug design. A
careful examination of the ATP binding sites of KDR
and c-Kit revealed a prominent difference in the aspara-
gine 923 residue in KDR versus aspartic acid 677 in
c-Kit (Fig. 2). We reasoned that placement of acidic
functionalities in close vicinity to these residues would
KDR:Phe921
KIT:Tyr675
5
KDR:Val 898
KIT:Ile653
KDR:Thr926
KIT:Asn680
KDR:Asn923
KIT:Asp 677
Figure 2. Overlay of KDR (green) and KIT (orange) protein crystal
structures (PDB entries 1VR2 and 1T45, respectively) with a model of
Compound 1 bound in ATP-site. Hydrogen bonds between the
inhibitor and the hinge region are shown with black dotted lines.
Select positions of residue difference between KIT and KDR kinases
are indicated. The residue difference at KDR 923/KIT 677 was targeted
with the compounds of this study utilizing substituents at position 5.
Table 1. Kinase inhibitory activity of 5-amide analogs
N
NH
S
R1
Compound
R1
KDR
a
IC50
(lM)
Flt1
IC50
c-Kit
a
IC50
(lM)
a
O
O
(lM)
a
b
N
O
O
CO2H
R3
9a
7.04
3.47
>50
N
R4
O
CO2H
HO
HO
CO2H
PhO
5
8
6
7
O
O
O
N
NH
9b
10a
9c
4.84
27.89
0.58
3.08
17.57
0.37
>50
7.18
5.61
N
N
S
S
R3
N
9
c, d
O
R4
HO
N
NH
O
O
e
HO
HO
N
H
O
S
R3
N
10
R4
9d
0.41
0.44
0.24
0.27
5.15
1.67
N
H
O
Scheme 1. Reagents and conditions: (a) AlCl3, NaCl, 160 °C, 2 h, 44%;
(b) R3R4NH, PS-DCC, HOBT, DMF; (c) 1—NaH, benzene, reflux,
overnight; 2—NH2NH2ÆH2O, EtOH, reflux, 1 h, 10–37% in three steps
from compound 6; (d) 1:1 TFA/CH2Cl2 for Boc protected acids or
2:2:1 1 M LiOH/MeOH/THF for esters, 11–55%; (e) LAH, THF,
reflux, 33–50%.
HO
10d
N
H
a IC50 values are based on seven-point curves, performed in duplicate.

3138
I. Akritopoulou-Zanze et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3136–3140
result in KDR selective molecules. Modeling of com-
pound 1 in the active sites of KDR and c-Kit suggested
that 5-substituted analogs were best in accessing these
residues. Thus, we embarked in the development of
new synthetic routes to access such molecules as shown
in Scheme 1. Initial formation of indanone 6 was estab-
lished with a Friedel-Crafts reaction of the neat
reagents. Since direct formation of the pyrazole ring
from compound 6 failed, we resorted in forming amides
7 first, using select amines containing a Boc or ester pro-
tected acid. Amides 7 readily reacted with phenyl thio-
phene-3-carboxylate 8 to provide, upon deprotection,
the free carboxylic acid compounds 9. Further LAH
reduction yielded compounds 10.
group was reduced, and the resulting amino group was
protected with an acetyl group. Formation of pyrazoles
with plain phenyl thiophene-3-carboxylate 8, deprotec-
tion of the amine group, and amidation reactions pro-
vided the final products 19.
Overall, reverse amides 19 were more potent and re-
tained the same selectivity profile previously observed
with compounds 9 (Table 2). Substituents carrying
hydrogen bond acceptor groups such as the oxygen
in 19b were more potent than simple aliphatic chains
such as in 19a. However, the positioning of the oxy-
gen group was crucial as observed in compounds
19b and 19c, and also in heterocycles 19f and 19g.
Sulfonamides (19d) were also well tolerated as well
as aromatic groups (19h), although in this case we
observe an increase in c-Kit binding. The improved
potency and selectivity of analogs bearing a hydrogen
The compounds were tested against KDR, Flt1, and
c-Kit in an HTRF assay format at 1 mM ATP.9 We
have selected a variety of cyclic and acyclic carboxylic
acids in order to probe the KDR/c-Kit site. Table 1
summarizes some of our findings. The compounds were
equipotent in Flt1 but were typically 10-fold less potent
in c-Kit with the exception of compound 10a. The pres-
ence of the carboxylic acid moiety seams to somewhat
affect the selectivity, as shown in examples 9d and its
reduced version 10d. However, the overall activity of
these inhibitors was moderate at best and their challeng-
ing preparation prompted us to explore the 5-substi-
tuted reverse amides. These compounds were prepared
from nitro indane 11 following Scheme 2. Oxidation of
11 yielded indanone 12, which was protected, the nitro
Table 2. Kinase inhibitory activity of 5-reverse amide analogs
N
NH
S
NH
R
Compound
R
KDR
a
IC50
(lM)
Flt1
IC50
c-Kit
a
IC50
(lM)
a
(lM)
19a
19b
19c
19d
0.56
0.29
9.54
O
O
O
O
O
a
c
b
O
0.07
0.65
0.31
0.03
0.45
0.29
1.58
2.55
4.39
NO2
11
NO2
12
O
NO2
13
O
O
PhO
O
S
O
O
R2
C
O
8 R2 = H
S
O
O
16 R2 = PhOCH2C
d
19e
19f
19g
19h
19i
0.05
0.11
0.06
0.10
0.92
0.08
0.09
0.07
0.05
0.71
1.12
1.10
0.9
O
e
NH
O
NH2
O
14
15
N
N
NH
NH
O
f, g
O
R
R2
S
S
NH
R5
NH
O
N
O
O
O
17 R2 = H
18 R2 = PhOCH2C
19 R2 = H
C
20 R2 = PhOCH2C
C
0.18
Scheme 2. Reagents and conditions: (a) Cr2O3, AcOH, rt, 5 h, 51%; (b)
ethylene Glycol, cat p-TsOH, Benzene, Dean Stark, overnight, 96%; (c)
H2 60 psi, Raney Ni, 1:1 MeOH/EtOAc, 20 min, 96%; (d) 1—AcCl,
pyridine, 30 min; 2—p-TsOH, acetone, H2O, reflux, 1 h, 76% in two
steps; (e) 1—NaH, Benzene, reflux, overnight; 2—NH2NH2ÆH2O,
EtOH, reflux, 1 h, 23% in two steps; (f) concd HCl, MeOH, reflux, 3 h,
82%; (g) R5COCl, pyridine, 10 min, 2–58%.
O
N
4.33
N
O
a IC50 values are based on seven-point curves, performed in duplicate.

I. Akritopoulou-Zanze et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3136–3140
3139
Table 3. Kinase inhibitory activity of position 5-analogs containing acetylenic thiophene substituents
N
NH
S
O
NH
R
Compound
R
KDR
a
IC50
(lM)
CSF1R
a
IC50
(lM)
Flt1
IC50
Flt3
IC50
cKit
IC50
Tie2
Lck
IC50
Fyn
IC50
Hck
IC50
Lyn
IC50
Src
IC50
a
a
a
a
a
a
a
a
a
IC50
(lM)
(lM)
(lM)
(lM)
(lM)
(lM)
(lM)
(lM)
(lM)
O
O
20a
20b
20c
0.053
0.113
0.016
0.153
0.175
40.595
13.025
24.747
16.038
13.681
30.27
20.806
43.311
O
O
0.270
0.036
1.271
0.190
0.136
0.020
0.624
0.065
1.003
0.127
>50
>50
>50
>50
>50
>50
>50
O
O
O
21.382
22.986
30.587
O
O
20d
20e
0.263
0.010
2.975
0.045
0.153
0.006
1.108
0.022
0.619
0.049
>50
26.529
30.963
>50
>50
>50
>50
>50
>50
>50
>50
O
O
O
N
4.877
N
a IC50 values are based on seven-point curves, performed in duplicate.
bond accepting group may be rationalized by the
aminoacid difference cited above, that is, KDR has
a nearby hydrogen bond donor (Asn 923) whereas
c-Kit (Asp 677) cannot make an analogous hydrogen
bond.
In conclusion, we have discovered a series of 5-substi-
tuted 1,4-dihydroindeno[1,2-c]pyrazoles as potent mult-
itargeted kinase inhibitors.
Acknowledgments
Encouraged by these results we selected several 5-
reverse amide compounds to build more elaborate
analogs incorporating the acetylenic moiety found
in compound 4. It was expected based on modeling
analysis that the two substituents would have addi-
tive effect on activity as the 5-substituents were
extending into the ribose pocket and the acetylenic
moiety probed the hydrophobic pocket. Compounds
20 were synthesized according to Scheme 2 using
the acetylenic thiophene benzoyl ester 16,10 instead
of 8. In direct comparisons of unsubstituted and
acetylenic analogs such as 19b versus 20a, 19e versus
20c, and 19i versus 20e, the acetylenic analogs were
more potent inhibitors across the same panel of
kinases. However, the gains in potency resulting from
interactions with the hydrophobic site also led to
erosion of the c-Kit selectivity. One explanation for
the lack of selectivity is possible shifting of the
5-substituent position in analogs bearing the acetyl-
enic extension (Table 3).
The authors thank the Abbott High Throughput
Purification and Structural Chemistry groups for their
support with purification and analytical services,
respectively.
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and 62 nM, respectively).9

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