Synthesis and biological evaluation of 5-arylamino-6-chloro-1H-indazole-4,7-diones as inhibitors of protein kinase B/Akt

Article abstract of DOI:10.1016/j.bmcl.2006.08.120

A series of 5-arylamino-6-chloro-1H-indazole-4,7-diones were synthesized and evaluated for their inhibitory activity on protein kinase B/Akt. The compounds exhibited a potent Akt1 inhibitory activity. Further mechanistic study revealed that they might hav

Full text of DOI:10.1016/j.bmcl.2006.08.120

Bioorganic & Medicinal Chemistry Letters 16 (2006) 6001–6005
Synthesis and biological evaluation of 5-arylamino-6-chloro-
1H-indazole-4,7-diones as inhibitors of protein kinase B/Akt
Jong Hee Ko,b Seung Woo Yeon,b Jung Su Ryu,b Tae-Yong Kim,b
Eun-Ha Song,a Hea-Jung You,a Rae-Eun Parka and Chung-Kyu Ryua,*
aCollege of Pharmacy, Ewha Womans University, Seodaemun-ku, Seoul 120-750, Republic of Korea
bILDONG Research Laboratories, ILDONG Pharmaceutical Co. Ltd., 260-5, Eonnam-Dong,
Kuseong-Gu, Yongin, Kyongki-Do 449-910, Republic of Korea
Received 7 July 2006; revised 14 August 2006; accepted 30 August 2006
Available online 20 September 2006
Abstract—A series of 5-arylamino-6-chloro-1H-indazole-4,7-diones were synthesized and evaluated for their inhibitory activity on
protein kinase B/Akt. The compounds exhibited a potent Akt1 inhibitory activity. Further mechanistic study revealed that they
might have dual inhibitory effects on both activity and phosphorylation of Akt1 in PC-3 tumor cell line.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Akt (protein kinase B, PKB) is a serine/threonine kinase
that promotes cellular proliferation, growth, survival,
and tumor formation.1 Akt is comprised of three mam-
malian isoforms, namely Akt1, Akt2, and Akt3. Akt as
a downstream target of PI-3 kinase can induce a variety
of biological responses. Many growth factors such as
IGF-1 and PDGF bind to their receptors and lead to
activation of PI-3 kinase. PI-3 kinase phosphorylates
the Ptdlns to generate Ptdlns-3-phosphates, Ptdlns(3)P,
Ptdlns(3, 4)P2, and Ptndlns(3,4,5)P3. The Ptdlns-3-phos-
phates cause the translocation of Akt from the cyto-
plasm to the plasma membrane.1,2 Then, Akt is
activated when residues Thr308 and Ser473 are phos-
phorylated by PDK1 and PDK2. Active Akt inhibits
apoptosis and stimulates cell cycle progression by phos-
phorylating numerous targets in various cell types,
including cancer cells. Persistent activation of Akt also
occurs as a result of a deletion in tumor suppressor gene
PTEN, a negative regulator of Akt.3 Overexpression of
Akt as a result of inactivation of PTEN was found in
a variety of human tumors.4 At the genomic level, Akts
were amplified in many cancer types.5 Akt1 was ampli-
fied or overexpressed in gastric adenocarcinomas, breast
cancer, hepatocarcinoma, and prostate carcinoma and
its activation correlates to cancer progression.6,7 Conse-
quently, inhibitors of the Akt1 activity could be power-
ful anticancer agents.2
We describe herein our preliminary results on synthesis
of 1H-indazole-4,7-dione series (Fig. 1) and their Akt1
inhibitory activity. In the course of screening of 25,000
compounds offered from Korea Chemical Bank, we
identified that 1H-indazole-4,7-diones inhibited Akt1
activity in vitro. We further synthesized structurally
related derivatives to evaluate their Akt inhibitory activ-
ity and cytotoxic potential on cancer cell lines. Addition-
al mechanistic study of Akt1 inhibitory activity by those
1H-indazole-4,7-diones was also performed.
A method for synthesis of 5-arylamino-6-chloro-1H-in-
dazole-4,7-diones 5a–h (Table 1) from commercially
available 7-nitro-1H-indazole (1) is shown in Scheme
1. 7-Nitro-1H-indazol-4-amine (2) was synthesized by
the amination of the indazole 1 with HONH2 and
R1
O
R2
R3
X
Cl
N
N
H
O
R4
R1, R2, R3, R4= H, F..,
Keywords: Akt; Inhibitor; 1H-indazole-4,7-dione; Antitumor.
*
X = NH or S
Corresponding author. Tel.: +82 2 3277 3027; fax: +82 2 3277
3051; e-mail: ckryu@ewha.ac.kr
Figure 1. 1H-Indazole-4,7-dione derivatives.
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.08.120

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J. H. Ko et al. / Bioorg. Med. Chem. Lett. 16 (2006) 6001–6005
Table 1. Structures and biological activities of 1H-indazole-4,7-diones
R1
O
R2
R3
X
N
N
H
O
Cl
R4
Compound
R1
R2
R3
R4
X
AKT1 activitya IC50 (lM)
Cytotoxicityb IC50 (lM)
KATOIIIc
HepG2
PC-3
5a
5b
5c
5d
5e
5f
F
H
H
H
NH
NH
NH
NH
NH
NH
NH
NH
S
15.8
11.8
>50
>50
16
20.2
3.9
15.8
8.9
ntd
6.3
nt
H
H
H
F
F
H
H
H
F
H
61.4
75.8
9.9
37.2
37.6
2.0
F
H
F
nt
H
F
OCH2CH3
H
nt
H
H
H
H
H
H
H
4.9
9.7
11.2
9.2
nt
5g
5h
6a
6b
3
H
CF3
H
H
12.4
13.9
>50
12.3
>50
25.0
13.8
8.9
13.7
nt
Br
CH3
Cl
H
6.1
H
Cl
CH3
H
>100
>100
>100
21.0
>100
88.7
>100
nt
nt
S
nt
nt
SWe
nt
a AKT1 inhibitory activity: active Akt1 proteins treated with test compounds were incubated with GSK3 fusion protein and subjected to Western
blot analysis, using anti-phospho GSK3a/b antibody.
b Cytotoxicity evaluation: MTT colorimetric assay.11
c Tumor cell lines tested: KATOIII (gastric adenocarcinoma), HepG2 (hepatocarcinoma), and PC-3 (prostate carcinoma).
d nt, not tested.
e SWU2009: 2-thioxo-[1,3]dithiolo[4,5-b][1,4] dithiine-5,6-dicarboxylic acid dimethyl ester.9
chloro-1H-indazole-4,7-diones 5a–h were synthesized
by nucleophilic substitution of compound 4 with appro-
priate arylamines. Most of these substitutions went as
expected and had overall high yields of 64–91%.
NH2
NO2
NH2
NH2
a
b
N
N
N
N
N
N
H
H
H
NO2
1
2
In similar manner, 5-arylthio-6-chloro-1H-indazole-4,7-
diones 6a–b were synthesized by reaction of the com-
pound 4 with appropriate arylthiols.
3
O
O
O
R1
H
N
Cl
Cl
R2
R3
d
c
N
N
Synthesized 1H-indazole-4,7-diones 5a–h and 6a–b were
evaluated in vitro for their Akt1 inhibitory activity using
a substrate phosphorylation assay. Experimental details
for this procedure are cited in Ref. 8. The IC50 values
were determined and compared to those of the positive
control SWU2009 (2-thioxo-[1,3]dithiolo[4,5-b][1,4]di-
thiine-5,6-dicarboxylic acid dimethyl ester).9 As shown
in Table 1, many of them tested exhibited a good
inhibitory activity with IC50 values in the low-micromo-
lar range. In particular, compounds 5a, 5b, 5e, 5f, 5h,
and 6b revealed potent inhibitory activity and were
superior or comparable to that of SWU2009. In
contrast, compounds 5c, 5d, and 6a did not show any
inhibitory activity up to 50 lM.
N
N
H
Cl
H
O
R4
4
5a-h
e
O
O
S
R1
R2
N
N
Cl
H
R3
6a-b
Scheme 1. Synthesis of 1H-indazole-4,7-diones. Reagents and
conditions: (a) HONH2/ EtOH/KOH/5 h/60 ꢁC; (b) H2/PtO2/EtOH/
RT/30 psi/4 h; (c) NaClO3/HCl/1 h/65 ꢁC/64–91%; (d) arylamine
(1 equiv)/EtOH/CeCl3/reflux/5 h/62–91%; (e) arylthiol (1 equiv)/EtOH/
CeCl3/reflux/5 h/55–81%.
Akt1 is activated by the phosphorylation on residues
Thr308 and Ser473. Active Akt1 then regulates down-
stream signal through the phosphorylation of cellular
substrates such as GSK3a/b, Bad, p27, p21, and Par-
4.2,10 Active Akt1 proteins treated with representive
compound 5b or 5g were incubated with a cellular sub-
strate GSK310 fusion protein and subjected to Western
blot analysis, using anti-phospho (Ser21/9) GSK3a/b
antibody. As a result, a remarkable decrease of GSK3
phosphorylation was detected with the treatment of
compound 5b or 5g (Fig. 2). Compounds 5b and 5g
KOH in EtOH in 83% yield. Compound 2 was reduced
to 1H-indazole-4,7-diamine (3) by catalytic hydrogena-
tion. The 5,6-dichloro-1H-indazole-4,7-dione (4) was
synthesized by oxidizing compound 3 with the Na-
ClO3/HCl variation in 62% yield. The 5-arylamino-6-

J. H. Ko et al. / Bioorg. Med. Chem. Lett. 16 (2006) 6001–6005
6003
bition of phosphorylation of GSK3a/b (Fig. 3). The
compounds 5b and 5g inhibited also the phosphoryla-
tion of Akt1 at Ser473, which is required for full activa-
tion of Akt1. There were no changes of total expression
levels of GSK3a/b and Akt1. These results indicate that
compounds 5b and 5g have dual inhibitory effects on
both the activity of Akt1 and the phosphorylation of
Akt1. Since Akt1 is phosphorylated on residues
Thr308 and Ser473 by PDKs in PI3 kinase pathways2,
activities of PDKs and related kinases may also be reg-
ulated by compounds 5b and 5g. Further study of 1H-
indazole-4,7-diones on other related kinases will be de-
signed and clarify the relationship.
Phospho-GSK3
Figure 2. Inhibitory effects of compounds 5b and 5g on Akt1 activity
in vitro. Active Akt1 proteins treated with compound 5b or 5g were
incubated with GSK3 fusion protein and subjected to Western blot
analysis, using anti-phospho (Ser21/9) GSK3a/b antibody.
inhibited in vitro Akt1 activity in a dose-dependent
manner.
The cytotoxicity against tumor cell lines was also inves-
tigated because Akt1 is an anti-apoptotic protein kinase
and the blockade of its activity leads to cell death. The
cytotoxic potential of 1H-indazole-4,7-diones 5a–h and
6a–b against human cancer cell lines was determined
according to NCI protocols.11 The following cell lines
were used: KATOIII (gastric adenocarcinoma), HepG2
(hepatocarcinoma), and PC-3 (prostate carcinoma), in
which Akt1 was amplified or overexpressed.6,7 Among
them tested, compounds 5b, 5e, 5f, 5g, and 5h exhibited
a good cytotoxicity. They were identified as both Akt1
inhibitor and cytotoxic agents (Table 1). However, com-
pound 6b inhibited Akt1 activity but did not exhibit
cytotoxicity.
Many protein kinase inhibitors act as a competitive
inhibitor of ATP binding in catalytic domain. To further
determine whether compounds 5b and 5g inhibit also
Akt1 activity in ATP-dependent manner, the inhibition
type was investigated. The assay was carried out by
using the Western blot assay with anti-phospho-
GSK3a/b antibody and GSK3 fusion protein as a sub-
strate (Figs. 4A and B).8 The inhibition of compounds
5b and 5g with respect to varying ATP concentrations
yielded double reciprocal plots converging on the y-axis
(Fig. 4). Because inhibitors 5b and 5g affected the Km
rather than the Vmax of the reaction, they can be consid-
ered ATP-competitive inhibitors of Akt1.
Further evaluation of in vivo efficacy in human tumor
xenograft was performed using one representive com-
pound 5g.13 Akt1 is constitutively active in PC-3 pros-
tate cancer cells due to the homozygous deletion of
PTEN, which is a tumor suppressor gene.14 Therefore,
a human tumor xenograft model, PC-3 prostate cancer,
was used to test the effect of Akt1 inhibitor 5g on tumor
growth in vivo, although the cytotoxicity against PC-3
cell line was similar to those against KATOIII and
HepG2 cell lines. Mice were injected with 5 mg/kg/day
Further mechanistic study on the Akt1 inhibitory activ-
ity was performed using potent compounds 5b, 5e, 5f,
5g, and 5h in cultured PC-3 cell line.12 We examined
whether these five compounds inhibit the phosphoryla-
tion of a representative cellular substrate GSK3a/b
and Akt1 itself. PC-3 cell line was treated with 10 lM
each test compound up to 5 h. These lysates were sub-
jected to Western blot analysis, using anti-phospho-
GSK3a/b (Ser21/9) antibody. Among them tested, only
two compounds 5b and 5g induced time-dependent inhi-
A
5g
5b
0
1
2
3
4
5
0
1
2
3
4
5 (hr)
Phospho-GSK3α
Phospho-GSK3β
GSK3β
1
0.84
0.83
0.58
0.38
0.36
1
0.79
0.34
0.16
0.07
0.00
B
5g
5b
0
1
2
3
4
5 0
1
2
3
4
5 (hr)
Phospho-Akt(Ser473)
Akt
1
1.08
0.76
0.47
0.39
0.16
1
0.80
0.26
0.11
0.07
0.09
Figure 3. Effects on the phosphorylation of Akt and GSK3a/b in PC-3 cell line. PC-3 cell line was treated with 10 lM compound 5b or 5g up to 5 h.
These lysates were subjected to Western blot analysis, using phospho-GSK3a/b (Ser21/9). (A) and phospho-Akt (Ser473). (B) Total GSK3b and Akt
are shown on the bottom part of A and B, respectively. The values under each blot represent the quantification of phosphorylated-GSK3 or Akt
relative to treatment with DMSO.

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J. H. Ko et al. / Bioorg. Med. Chem. Lett. 16 (2006) 6001–6005
growth compared to vehicle treatment at day 16
5.0
4.0
3.0
2.0
1.0
0.0
A
0 μM
μM
(Fig. 5). There was no difference in body weights be-
tween the treated and vehicle group throughout the
treatment period. We identified that compound 5g
exhibited the Akt1 inhibitory activity and the antitumor
effect.
5
10μM
In conclusion, 5-arylamino-6-chloro-1H-indazole-4,7-
diones 5b and 5g are considered to be new class inhibi-
tors of Akt1, and they might have dual inhibitory effects
on both activity and phosphorylation of Akt1 in the PC-
3 tumor cell line. Further pharmacological investiga-
tions of these 1H-indazole-4,7-diones and the structural
optimization are in progress in our group.
-0.05
0.05
0.1
0
-1.0
1/[ATP]
20.0
B
0 μM
5 μM
Acknowledgment
15.0
10.0
5.0
This study was supported by a grant of the Korea Sci-
ence and Engineering Foundation (KOSEF R06-2002-
011-01004-0).
10 μM
References and notes
1. Scheid, M. P.; Woodgett, J. R. FEBS Lett. 2003, 546, 108.
2. Cheng, J. Q.; Lindsley, C. W.; Cheng, G. Z.; Yang, H.;
Nicosia, S. V. Oncogene 2005, 24, 7482.
0.0
-0.05
0
0.05
0.1
3. Nicholson, K. M.; Anderson, N. G. Cell. Signalling 2002,
14, 381.
1/[ATP]
-5.0
4. Vivanco, I.; Sawyers, C. Nat. Rev. 2002, 2, 489.
5. Staal, S. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 5034.
6. Nakatani, K.; Thompson, D. A.; Barthel, A.; Sakaue, H.;
Liu, W.; Weigel, R. J.; Roth, R. A. J. Biol. Chem. 1999,
274, 21528.
Figure 4. Double reciprocal plots of the rate of Akt1 catalytic activity
against the substrate ATP. The kinase activity of Akt1 was assayed at
various concentrations of ATP in the presence of increasing concen-
trations of compound 5g (A) or 5b (B). Akt activity was determined
and expressed as the mean intensities measured by densitometric
analysis. Data points were collected in triplicate, plotted on Linewe-
aver–Burk double reciprocal plots, and fitted to linear regression.
7. Goswami, A.; Ranganathan, P.; Rangnekar, V. M. Cancer
Res. 2006, 66, 2889.
8. Materials: Anti-phospho-GSKa/b antibody, anti-phos-
pho-Akt antibody, anti-Akt antibody, anti-GSKb anti-
body, Akt1/PKB kinase, and GSK3 fusion protein were
purchased from Cell Signaling Technology Inc. (Beverly,
MA). KATOIII, HepG2, and PC-3 cell lines were
purchased from Korean cell line bank. MTT [1-(4,5-
5g
Vehicle
2500
2000
1500
1000
500
#
dimethylthiazol-2-yl)-2,4-diphenyl-2H-tetrazolium
bro-
#
mide] and other reagents were purchased from Sigma
and Aldrich (St. Louis, MO). Cell culture: The cell lines
were maintained in Dulbecco’s modified essential medium
containing 10% heat-inactivated (30 min at 56 ꢁC) fetal
bovine serum. The cell lines were incubated at 5% CO2 and
95% humidity in a 37 ꢁC chamber. Akt kinase assay
in vitro: Akt1/PKB protein kinase (10 U/lL) was incubat-
ed at 37 ꢁC for 10 min in reaction buffer (20 mM HEPES,
14.3 mM NaCl, 1 mM EDTA, and 2 mM DTT) contain-
ing the test compounds dissolved in DMSO. This mixture
was added 2 mM ATP 3 lL and GSK3 fusion protein
(1 mg/mL) 1 lL and incubated at 37 ꢁC for 30 min. The
reaction was stopped by incubation for 5 min at 70 ꢁC.
This sample was separated by 12% SDS–PAGE and
transferred to PVDF membranes by Western blotting or
Dot blotting. After blocking by incubation in TBST
(10 mM Tris–HCl, pH 7.5, 170 mM NaCl, and 0.5%
Tween 20) containing 5% (w/v) skim milk for 10 min,
membrane was incubated overnight at 4 ꢁC with primary
antibody (1:1000). Subsequent to washing with TBST,
membrane was incubated with horseradish peroxidase-
conjugated secondary antibody (1:2000) and then washed.
*
*
0
0
2
4
6
8
10
12
14
16
18
Days
Figure 5. Antitumor efficacy of compound 5g in sc PC-3 prostate
cancer xenograft model. PC-3 cells were sc injected into nude mice.
When the tumors reached an average size of about 200 mm2, mice were
treated with either vehicle (control) or 5 mg/kg/day compound 5g.
Each measurement represents an average of 6 tumors. Compound 5g
inhibits PC-3 tumor growth by 27.1% for 16 days. *p < 0.05; #p < 0.01.
compound 5g until day 16. The treatment with
compound 5g (5 mg/kg/day) led to significant antitumor
effect, showing 27.1% (p < 0.01) inhibition of tumor

J. H. Ko et al. / Bioorg. Med. Chem. Lett. 16 (2006) 6001–6005
6005
Membrane was incubated with ECL (Amersham Bio.,
UK) substrate reagent and exposed to X-ray film. Phos-
phorylated GSK3 fusion protein was quantified by a
densitometric analysis using ImageMaster 2D platinum
v5.0 (Amersham Bio., UK). For the investigation of
inhibitor type, the initial velocity v was obtained as the
intensities of phosphorylated GSK3 substrates (arbitrary
unit/30 min). Data points were collected in triplicate,
plotted on Lineweaver–Burk double reciprocal plots, and
fitted to linear regression.
loaded on a 12% acrylamide/bisacrylamide gel, separated
by electrophoresis, and transferred to PVDF membrane,
preincubated in 5% skim milk TBST for 10 min, and
incubated with following antibodies against: phospho-
GSK3a/b antibody, anti-phospho-Akt antibody, anti-Akt
antibody, and anti-GSK3b antibody. Immunoreactive
bands were detected and quantified by the same method
described for the quantification of phosphorylated GSK3
fusion protein at Akt kinase assay in vitro.
13. Evaluation of in vivo antitumor effect with human tumor
xenograft: PC-3 prostate cells were inoculated (106 cells/
site) in the flank of male nude mice, 4 weeks of age, and
mice were maintained in a pathogen-free environment.
Tumors were measured twice weekly using a digital
caliber, and tumor volumes were calculated using the
formula length · (width)2/2. Once tumors reached
200 mm3, mice were randomly assigned to groups
(n = 6). Compound 5g was dissolved in cremopore solu-
tion (ethanol 7%, cremopore 7%, and 84% distilled water)
and injected iv at the dose of 5 mg/kg per mouse per
0.2 mL daily for 16 days. Statistical analysis: The results
are shown as means SD of the number (n) of experi-
ments. Statistical analysis of difference was determined by
a one-way analysis of variation (ANOVA). Differences
were accepted as statistically significant at a p value of
*p < 0.05 and #p < 0.01.
9. Ko, J. H.; Yeon, S. W.; Lee, H. S.; Kim, T. Y.; Noh, D.
Y.; Shin, K. S.; Hong, S. K.; Kang, S. S. Yakhak Hoeji
2004, 48, 105.
10. Ali, A.; Hoeflich, K. P.; Woodgett, J. R. Chem. Rev. 2001,
101, 2527.
11. Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.;
McMahon, J.; Vistica, D.; Warren, T. W.; Bokesch, H.;
Kenney, S.; Boyd, M. R. J. Natl. Cancer Inst. 1990, 82,
1107.
12. Western blot analysis for inhibition of Akt1 and GSK3a/b
phosphorylation: PC-3 cells (2 · 106) were incubated in 35-
mm culture dishes in DEME with 10% FBS in a 5% CO2
atmosphere at 37 ꢁC for one day. The cells were incubated
with compounds at 10 lM in DMEM up to 5 h. The
culture media were aspirated, and the cells were lysed in
50 mM Tris–HCl, pH 7.6, 180 mM NaCl, 2 mM EDTA,
1% Triton X-100, protease inhibitor cocktail, and
Na3VO4. Seventy micrograms of total cell lysates was
14. McMenamin, M. E.; Soung, P.; Perera, S.; Kaplan, I.;
Loda, M.; Sellers, W. R. Cancer Res. 1999, 59, 4291.