Discovery of indenopyrazoles as EGFR and VEGFR-2 tyrosine kinase inhibitors by in silico high-throughput screening

Article abstract of DOI:10.1016/j.bmcl.2007.10.084

A series of indenopyrazoles 8 and 9 were designed and synthesized as EGFR tyrosine kinase inhibitors by in silico high-throughput screening. Compounds 8b and 8d showed significant inhibition of A431 cell growth (GI₅₀ = 0.062 and 0.057 μM, respectively). Compounds 8b and 9a showed inhibitory activity toward both EGFR and VEGFR-2 (KDR) tyrosine kinases, whereas 8d inhibited VEGFR-2 tyrosine kinase, exclusively.

Full text of DOI:10.1016/j.bmcl.2007.10.084

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 285–288
Discovery of indenopyrazoles as EGFR and VEGFR-2
tyrosine kinase inhibitors by in silico high-throughput screening
Taikou Usui,a Hyun Seung Ban,a Junpei Kawada,a
Takatsugu Hirokawab and Hiroyuki Nakamuraa,*
aDepartment of Chemistry, Faculty of Science, Gakushuin University, Tokyo 171-8588, Japan
bComputational Biology Research Center, Institute of Advanced Industrial Science and Technology (AIST), Tokyo 135-0064, Japan
Received 7 July 2007; revised 28 September 2007; accepted 25 October 2007
Available online 30 October 2007
Abstract—A series of indenopyrazoles 8 and 9 were designed and synthesized as EGFR tyrosine kinase inhibitors by in silico high-
throughput screening. Compounds 8b and 8d showed significant inhibition of A431 cell growth (GI50 = 0.062 and 0.057 lM, respec-
tively). Compounds 8b and 9a showed inhibitory activity toward both EGFR and VEGFR-2 (KDR) tyrosine kinases, whereas 8d
inhibited VEGFR-2 tyrosine kinase, exclusively.
Ó 2007 Elsevier Ltd. All rights reserved.
Discovery of a pharmacologically active compound is an
essential process for development of new drugs in medic-
inal chemistry. Despite great advances in high-through-
put screening (HTS) technology, much attention has
been paid for in silico screening using virtual compound
libraries.1–3 Recently, various X-ray co-crystal struc-
tures of a target protein–ligand complex have been
reported and computational calculation technology of
a docking simulation between proteins and ligands has
also been developed based on those co-crystal struc-
tures, therefore, it may become possible to find lead
compounds toward certain target proteins without using
a large number of real chemical libraries.
(NSCLC) as EGFR tyrosine kinase inhibitors. Although
therapeutic responses to both inhibitors can persist for
as long as 2–3 years, the mean duration of response in
most cases of NSCLC is only 6–8 months.14–16 How-
ever, the mechanisms underlying acquired drug resis-
tance are not well understood.17 Therefore, the finding
of new lead compounds is still a significant requirement
in this area.
In this paper, we carried out the in silico library screen-
ing based on X-ray crystal structure of the EGFR tyro-
sine kinase domain in complex with erlotinib (PDB code
1M17)18 using Glide, a docking program,19 against a
400,000 compound library of tyrosine kinase inhibitors
from ChemBioBase.20,21 We focused on an indenopyraz-
ole framework, which was reported as cyclin dependent
kinase (CDK) inhibitor22,23 and found to be one of the
common structures among the 100 top scoring com-
pounds. The indenopyrazoles were designed as inhibi-
tors targeting to EGFR tyrosine kinase, based upon
the further detailed structure-based drug design (SBDD)
using Ligand Fit algorithm, as shown in Figure 1.
According to the docking simulation,24 the hydrogen
bonding interaction would be expected between the
nitrogen atom (N1) of the indenopyrazoles and the
MET769 amide nitrogen, and the hydroxyl group of a
benzene ring substituted at C3 position of the indenopy-
razoles and the ASP831 carboxylic acid. Therefore, we
synthesized a series of indenopyrazoles conjugated with
a variety of fragments at C3 and C5 positions.
EGFR (also known as erb-B1 or HER-1) tyrosine
kinase is one of the important kinases that play a funda-
mental role in signal transduction pathways. EGFR and
its ligands (EGF and TGF-a) have been implicated in
numerous tumors of epithelial origin4,5 and proliferative
disorders of the epidermis such as psoriasis.6 Therefore,
the design of inhibitors toward EGFR-PTK is an attrac-
tive approach for the development of new therapeutic
agents,7–10 and gefitinib (ZD-1839, Iressa)11,12 and erl-
otinib (OSI-774, Tarceva)13 have both been approved
for the treatment of non-small-cell lung cancer
Keywords: In silico screening; EGFR; VEGFR; Tyrosine kinase inhi-
bitor; Indenopyrazoles.
*
Corresponding author. Tel.: +81 339860221; fax: +81 359921029;
e-mail: hiroyuki.nakamura@gakushuin.ac.jp
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.10.084

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T. Usui et al. / Bioorg. Med. Chem. Lett. 18 (2008) 285–288
O
O
O
S
O
a,b
HN
Met769
N
H
O
O
O
NH
NH
O
Ph
R1
R1
hydrophobic
pocket
Lys721
O
O
1
N
NH
3
5a,b
6a,b
ASP776
5
H
N
O
H
H
N
O
H
O
O
H
O
N
H
O
c
H
O
O
H
X
O
N
NH
Fragment 2
Asp831
O
R1
Fragment 1
X = O, NH
O
R2
7a: R1 = 4-methoxy, R2 = NH2
b: R1 = 3,4-methylenedioxy, R2 = NH2
c: R1 = 4-methoxy, R2 = OH
Figure 1. Binding simulation model of the indenopyrazoles into the
active site of EGFR tyrosine kinase domain.
d: R1 = 3,4-methylenedioxy, R2 = OH
N
The triketone derivatives 5, as precursors of indenopy-
razoles, were synthesized according to the literature
procedure with modification as shown in Scheme 1.23
3-Nitrophthalic acid 1 was reacted with acetic anhy-
dride, and the 3-nitrophthalic anhydride obtained
underwent the esterification in methanol to afford di-
methyl 3-nitrophthalate 2. Direct esterification of 1 gave
the monomethyl ester, exclusively. Hydrogenation
followed by acetylation gave the corresponding
acetamide 3 in high yields. Condensation of 3 with
acetophenones, such as 1-(4-methoxyphenyl)ethanone
4a and 1-(benzo[d][1,3]-dioxol-5-yl)ethanone 4b, gave
the corresponding triketones 5a and 5b in 14% and
20% yields, respectively.
NH
d
H
R1
O
N
NH
O
R2
8a-d
N
NH
e
8a-b
H
R1
O
N
NH
O
9a: R1 = 4-hydroxy
b: R1 = 3,4-dihydroxy
H2N
Next, phenoxy and anilino groups, as the fragment 1,
were introduced into C5 position of indenopyrazoles
through a urea bond as shown in Scheme 2. Deacryla-
tion of the amides 5a, b was carried out under acidic
methanol in reflux and the resulting anilines were treated
with phenyl chloroformate to give the corresponding
phenyl carbamates 6a and 6b in 64% and 74% yields,
respectively. Heating the carbamate 6a and 6b in
dimethylsulfoxide (DMSO) with the appropriate ani-
Scheme 2. Reagents and conditions: (a) HCl, MeOH, reflux, 76–82%;
(b) PhOC(O)Cl, Na2CO3, acetone, reflux, 64–74%; (c) aniline, DMAP,
DMSO, 80 °C, 65–91%; (d) Hydradine, p-TsOH, EtOH, reflux, 35–
47%; (e) AlCl3, EtSH, 0 °C, 22–23%.
lines gave the urea derivatives 7a–d. The pyrazole ring
formation was accomplished by treating with hydrazine
and a catalytic TsOH in refluxing ethanol to give the
indenopyrazoles 8a–d in 35–47% yields. Furthermore,
deprotection of methyl and methylenedioxy groups of
indenopyrazoles 8a and 8b was carried out using AlCl3
and EtSH to give 9a and 9b in 22% and 23% yields,
respectively.11
O
O
O
c,d
a,b
OH
OH
O
O
NO2
NO2
O
We first examined the effects of the compounds on pro-
liferation of A431 human epithelial carcinoma cell line,
which overexpresses EGFR on the cell surface. The cells
were incubated with compounds at various concentra-
tions for 72 h and cell viability was determined by
MTT assay. As shown in Table 1, the indenopyrazoles
8b and 8d reduced the proliferation of A431 cells, and
their GI50 values are 0.062 and 0.056 lM, respectively.
These results indicate that indenopyrazoles inhibit cell
proliferation at lower concentration of compounds than
tarceva (GI50 = 0.47 lM). Next, we examined in vitro
inhibitory activity of the indenopyrazoles against
EGFR, HER-2, Flt-1 (VEGFR-1), and KDR (VEG-
1
2
O
O
O
O
e
O
O
NH
NH
O
R1
O
5a: R1 = 4-methoxy
O
3
b: R1 = 3,4-methylenedioxy
Scheme 1. Reagents and conditions: (a) Ac2O, 100 °C; (b) H2SO4,
MeOH, reflux, 97%; (c) H2, Pd/C, MeOH, 90%; (d) Ac2O, Py, 100 °C,
84%; (e) acetophenones (4a–b), NaH, DMF, 90 °C, 14–20%.

T. Usui et al. / Bioorg. Med. Chem. Lett. 18 (2008) 285–288
287
Table 1. Enzyme and cellular inhibitory activity assay results for
compounds 8 and 9
According to our docking simulation as shown in Figure
3,27,28 hydrogen bond interactions were observed be-
tween the N1 nitrogen of 8b and the backbone-NH of
Asn921, and the aniline nitrogen atoms of 8b and the
carboxyl oxygen of Glu883. In the case of the binding
mode of 9a, a hydrogen bond between the phenol group
of 9a and the carboxyl oxygen of Glu848 was observed
in addition to two hydrogen bonds observed in the bind-
ing mode of 8b. In both cases, the benzene ring of the
indenopyrazole framework was located near Leu1033.
Compound GI50a (lM)
Kinase inhibitionc (%)
A431b
EGFR HER2 Flt-1 KDRd
8a
8b
1.59
0.062
>10
3
61
7
6
28
8
16
60
9
42
88 (0.38 lM)
44
8c
8d
0.056
>10
4
2
13
36
10
—
34
70 (0.70 lM)
73 (0.65 lM)
58
9a
9b
65
50
81
30
7
4.79
0.47
NDe
3
Tarceva
AAL 993
—
—
38
95 (0.039 lM)
In conclusion, the indenopyrazoles were found as
EGFR tyrosine kinase inhibitors by the in silico high-
a Concentration required to inhibit cell growth by 50%.
b EGFR overexpressing human epidermoid carcinoma cell.
c Kinase assay was performed at a 1 lM concentration of compounds.
d IC50 values are indicated in the parenthesis.
e Not determined.
throughput screening using
a 400,000 compound
library, and a series of indenopyrazoles 8 and 9 were de-
signed and synthesized. Although 8b, 9a, and 9b showed
moderate inhibitory activity toward EGFR tyrosine
kinase, no inhibitory activity was observed in com-
pounds 8a, 8c, and 8d. Unexpectedly, significant inhibi-
tion of VEGFR-2 tyrosine kinase was observed in
compounds 8b, 8d, and 9a at 1 lM concentration. The
significant growth inhibition toward A431 cells was ob-
served in indenopyrazoles 8b and 8d, and their GI50 val-
ues were much lower than that of tarceva. According to
the western blotting analysis, compounds 8b and 8d pos-
sessed inhibitory activities toward CDKs in A431 cells,
therefore the significant cell growth inhibition property
FR-2) tyrosine kinases by measuring the levels of phos-
phorylation of the tyrosine kinase-specific ligand pep-
tides at 1 lM concentration of compounds.25
Compounds 8b, 9a, and 9b showed moderate inhibition
of EGFR tyrosine kinase (ꢀ50%), whereas compounds
8a, 8c, and 8d did not show inhibitory activity toward
EGFR tyrosine kinase. Interestingly, indenopyrazoles
synthesized here possessed relatively selective inhibitory
activity toward KDR tyrosine kinase, and compounds
8b and 8d, especially, showed high inhibitory activity
(88% and 73%, respectively) at 1 lM.
Since a significant growth inhibition of compounds 8b
and 8d toward A431 cells was observed in Table 1, we
next examined CDK inhibitory activity of compounds
8a–d and 9a–b by Western blotting analysis of retino-
blastoma tumor suppressor gene (Rb), which undergoes
phosphorylation by activated CDKs. The results are
shown in Figure 2. Compounds 8b and 8d, which exhib-
ited a significant growth inhibition toward A431 cells,
showed a potent inhibition of the phosphorylation of
Rb at 1 lM concentration of compounds in A431 cells.
Tarceva did not show a potent inhibition of the phos-
phorylation of Rb, therefore, the significant growth
inhibition of compounds 8b and 8d toward A431 cells
may be caused by inhibitions of CDKs.
In order to better understand the structure–activity rela-
tionship between the VEGFR2 tyrosine kinase inhibi-
tion and the possible binding modes of the
indenopyrazoles 8b and 9a, we performed molecular
docking experiments of 8b and 9a with the ligand-bind-
ing site of VEGFR2 kinase (PDB code 1Y6A).26
Figure 3. An overlay of indenopyrazoles 8b (gray) and 9a (green)
docked into the active site of VEGFR2 kinase domain. Docking model
was calculated by the DS modeling 1.7 based on the X-ray analysis
data of VEGFR2 tyrosine kinase in complex with the ligand8 (PDB
code: 1Y6A).
N
NH
pRb
H
O
N
NH
O
N
α-Tubulin
O
-
8a
8b
8c
8d 9a
9b
10 Tar
Compound 10
Figure 2. Western blotting analysis of phosphorylation of Rb in A431 cells. A431 cells were incubated for 12 h in the presence of indenopyrazoles or
tarceva (Tar) at 1 lM concentration and the levels of each protein were detected with the specific antibody. Compound 10, which was reported as a
CDK inhibitor,22 was used as a positive control.

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