Novel dihydrothieno[2,3-e]indazole derivatives as IκB kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.01.069

Synthesis, and structure-activity relationship (SAR) studies of the novel IKK-β inhibitors 2 and 3 characterized by a dihydrothieno[2,3-e]indazole core are presented. Compound 2t was efficacious in a mouse model of LPS-stimulated TNF-α production.

Full text of DOI:10.1016/j.bmcl.2011.01.069

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1758–1762
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel dihydrothieno[2,3-e]indazole derivatives as IjB kinase inhibitors
Hiroyasu Takahashi a, , Mariko Shinoyama a, Takashi Komine a, Muneki Nagao b, Masashi Suzuki b,
⇑
Hisatoshi Tsuchida a, Koichi Katsuyama a
a Discovery Research Laboratories, Kyorin Pharmaceutical Co., Ltd, 2399-1, Nogi, Nogi-machi, Shimotsuga-gun, Tochigi 329-0114, Japan
b Development Research Laboratories, Kyorin Pharmaceutical Co., Ltd, 1848, Nogi, Nogi-machi, Shimotsuga-gun, Tochigi 329-0114, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis, and structure–activity relationship (SAR) studies of the novel IKK-b inhibitors 2 and 3 charac-
terized by a dihydrothieno[2,3-e]indazole core are presented. Compound 2t was efficacious in a mouse
model of LPS-stimulated TNF-a production.
Received 28 October 2010
Revised 14 January 2011
Accepted 18 January 2011
Available online 22 January 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
IKK inhibitor
IKK-b
Dihydrothieno[2,3-e]indazole
NF-jB
Nuclear Factor-
jB
(NF-
jB) regulates the transcription of
We wish to report herein the synthetic details and the results of
numerous genes implicated in the induction of inflammatory and
the structure–activity relationships (SAR) studies of the novel
IKK-b inhibitors characterized by dihydrothieno[2,3-e]indazole
moiety.
immune responses and in the prevention of apoptosis.1–3 In
unstimulated cells, NF-
tive form complexed with an inhibitory protein, IkB-
sion of NF- B into the active nuclear form, composed of p50 and
p65 (Rel-A) subunits, is induced by LPS (lipopolysaccharide) or
cytokines. These stimulants activate NF- B by inducing the phos-
phorylation and degradation of IkB- , thereby allowing the rapid
translocation of NF- B from the cytoplasm to the nucleus. The en-
zyme responsible for the phosphorylation of IkB- is I B kinase
(IKK), a multisubunit complex that contains two catalytic units
(IKK- and b) and a regulatory unit (IKK-
or NEMO).4 Various
studies have indicated that IKK-b plays a dominant role in the pro-
inflammatory signal-induced phosphorylation of IkB-a 5
This re-
jB is retained in the cytoplasm as an inac-
a. The conver-
Synthesis of the dihydrothieno[2,3-e]indazole scaffold is out-
lined in Scheme 1. Formulation of 1,4-cyclohexanedione mono-eth-
ylene ketal (4) gave hydroxymethylidene ketal 5. Compound 5 was
reacted with N-alkylhydrazine derivatives to give tetrahydroindaz-
ole isomers 6 and 7 as a regioisomeric mixture.14 In addition, 6 and
7 were prepared by the alkylation of tetrahydroindazole 8 with
appropriate alkyl halides. N1- or N2-substituted regioisomers were
separated in either this or the following step by using chromatog-
raphy or recrystallization. Deprotection of ketal 6 and/or 7 under
acidic conditions gave tetrahydroindazolone 9 and/or 10. The ami-
nothiophene moiety of 11, 12 was constructed from 9 and/or 10 via
the Gewald reaction.15 One-pot condensation–cyclization se-
quences of 9, 10 with malononitrile and sulfur in the presence of
morpholine proceeded regioselectively at the C5 position to afford
dihydrothieno[2,3-e]indazole core 11 and/or 12 in a concise fash-
ion. Initial attempts to construct the tricyclic core using cyanoacet-
amide (NCCH2CONH2) or ethyl cyanoacetate (NCCH2CO2Et) instead
of malononitrile resulted in a complicated mixture. This result
means that selection of the reagent, activated nitrile, is the domi-
nant factor in this reaction. The cyano group of 11 and/or 12 was
hydrolyzed with concentrated sulfuric acid to provide carboxam-
ide 13 and/or 14, which were then converted to urea 2 and/or 3
by using sodium cyanate in the acidic condition.
j
j
a
j
a
j
a
c
.
sult indicates that selective IKK-b inhibition can provide an
effective treatment for inflammatory and autoimmune diseases.
Even though a number of groups have reported structurally dis-
tinct IKK-b selective inhibitors,6–8 thiophene amino carboxamide-
based inhibitors9–11 as compound 1 have attracted considerable
interest, because of their remarkable pharmacologic activity.12
In our research to find an original scaffold for IKK inhibitors, we
developed the novel dihydrothieno[2,3-e]indazole derivatives 2
and 3 (Fig. 1).13 This functionalized tricyclic template mimics the
topological disposition of the thiophene amino carboxamide-based
IKK inhibitors.
Compounds synthesized by the above methodology were eval-
⇑
Corresponding author.
E-mail address: hiroyasu.takahashi@mb.kyorin-pharm.co.jp (H. Takahashi).
uated for their ability to inhibit the IKK-
a and IKK-b catalyzed
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.01.069

H. Takahashi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1758–1762
1759
Figure 1.
Scheme 1. Reagents and conditions: (a) HCO2-Me, NaH, THF/MeOH, reflux, 2 h; (b) RNHNH2, MeOH, 0 °C, 1 h; (c) NH2NH2, H2O, MeOH, 0 °C, 1 h; (d) RX, Cs2CO3, CH3CN, 80 °C,
1–3 h; (e) 1 N HCl, acetone, 60 °C, 1 h; (f) NCCH2CN, S8, morpholine, EtOH–THF, rt, 1 h; (g) concd H2SO4, 40 °C, 3 h; (h) NaCNO, AcOH–H2O, rt, 2 h.
phosphorylation of IkB-
used to measure the ability of compounds to inhibit LPS-induced
TNF-
production in human monocyte THP-1 cells.16
a
substrate, and a cell-based assay was
tendency between in vitro activity and the substitution position
is reversed in the cell-based assay. 2-Substituted derivatives 2a,
b showed modest cellular activity at 5.2 and 2.5 lM, but their
a
The results shown in Table 1 reveal the effects of the substitu-
tion position on the indazole moiety. Substitution of the N-2 or
N-3 position with an n-propyl or p-fluorobenzyl group was toler-
ated in the enzyme assay, and two sets of compounds, 2a, b and
3a, b, showed moderate to good potency (the IC50’s range was from
potency was five- to eight-fold greater than the cellular activity
of 3-substituted 3a, b. This means the gain in the intrinsic potency
of 3-substituted derivatives 3a, b did not translate sufficiently to
enhanced cellular activity. We therefore decided to focus our fur-
ther investigations on 2-substituted derivatives.
0.2 to 2.9
l
M). Compound 3b was over ten times more potent than
According to the above investigation, our methodology to
synthesize N2-substituted isomers 2c–w was modified in a regio-
selective manner (Scheme 2). For the regioselective synthesis of
N2-substituted intermediate 6, bis-electrophilic hydroxyketone 5
was converted to enol-acetate 15, which was expected to react
the n-propyl-substituted derivative 3a and five times more potent
than the 2-substituted isomer 2b. These results suggest a tentative
SAR for the indazole substituent, with N3-substitution showing
more potent enzyme activity than that of N2-substitution. This
Table 1
IKK-b inhibitory activities and cell-based assay of the N-2 and N-3 substituted derivatives
H2N
O
H2N
O
NH
NH
3
3
S
S
O
O
R
N
N
N
H2N
H2N
1
N
2
1
2
R
2a,b
3a,b
Compound
R
In vitro IC50 (lM)
IKK-b
THP-1
2a
3a
n-Pr
2.9
2.2
5.2
42
2b
3b
0.9
0.2
2.5
12
F

1760
H. Takahashi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1758–1762
Scheme 2. Reagents and conditions: (a) HCO2Me, NaOEt, THF, 0 °C, reflux, 3 h, then Ac2O, rt, 1 h; (b) EtOH, reflux, 1 h; (c) TFA, CH2Cl2, rt, 2 h (the ratio of the desired 6 to its
regioisomer 7 was approximately 10:1); (d) 1 N HCl, acetone, 60 °C, 1 h; (e) NCCH2CN, S8, morpholine, EtOH–THF, rt, 1 h; (f) concd H2SO4, 40 °C, 3 h (g) NaCNO, AcOH–H2O, rt,
2 h.
Table 2
Table 3
SAR of 2-alkylsubstituted derivatives
SAR of 2-alkylaminosubstituted series
H2N
H2N
O
O
NH
NH
S
S
O
O
N
N
H2N
H2N
N
N
R
R
Compound
R
In vitro IC50
IKK-b
(lM)
Compound
R
In vitro IC50
IKK-b
(lM)
THP-1
THP-1
2c
2d
2e
i-Pr
n-Bu
n-Hex
2.3
2.7
8.4
4.6
4.0
NTa
2l
4.6
7.1
5.1
N
Me
2f
4.2
4.5
5.6
2g
>30
2m
2.5
N
N
N
2h
4.8
2.3
24
N
2na
2oa
3.0
2.2
1.7
0.5
2i
2j
9
N
11
NTa
MeO
Me
N
2k
8.8
4.6
NTa
5.1
N
2p
4.8
1.2
Me
N
2l
N
Me
2qa
>10
NTb
N
a
NT = not tested.
a
Racemic form.
NT = not tested
b
preferably with nucleophiles at the b-position. Actually, the con-
densation of 15 with the various N-Boc-protected alkylhydrazines
16, followed by deprotection of the N-Boc group with TFA gave the
desired condensation products 6c–y regioselectively. Conversion
from 6c–y to 2c–y was achieved as described above.
decreased the potency. A benzyl substituent could be replaced by
a 2-, 3-, or 4-pyridylmethyl substituent that showed moderate en-
zyme potency (IC50’s range was from 2.3 to 4.8 lM). Incorporation
The enzyme activity and cellular potency on various 2-alkylsub-
stituted derivatives 2c–l are shown in Table 2. Substitution at the
2-position with isopropyl, n-butyl, or benzyl groups was tolerated
(2c–d, 2f), whereas elongation of the alkyl chain to n-hexyl 2e
of hydrophilic substituents such methoxyethyl 2j and dimethyl-
aminoethyl derivatives 2k resulted in decreased enzyme potency
compared with 2d. In contrast, N-benzyl N-methylaminoethyl-
substituted derivative 2l showed moderate enzyme and cellular

H. Takahashi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1758–1762
1761
Table 4
SAR of 3-piperidinyl series
H2N
O
NH
S
O
N
N
H2N
R
N
Compound
R
In vitro IC50 (lM)
IKK-b
IKK-
a
THP-1
2o
2r
2.2
2.5
>100
>100
0.5
0.6
4.4
0.8
Figure 2. Effect of 2t on TNF-a concentrations in LPS-stimulated mice.
Cl
Cl
five-fold over that of 2r. The chloro atom of 2s could be replaced by
other polar functional groups such amino or hydroxyl groups 2v,
2w with enzyme potency. On the other hand, the potency of
p-methoxy-substituted 2x was decreased to less than half that of
2w, and the more hindered acetamide-substituted 2y also dropped
in potency. These results suggest that there are polar interaction(s)
around the benzylic p-positions. Compounds 2r, 2t–w showed a
2s
2t
0.5
1.7
41
F
>100
F
F
F
2u
3.1
NTa
0.3
good cellular potency IC50 of around 0.5
lM, and some compounds
F
F
showed moderate to good kinase selectivity for IKK-
a
(18 ꢀ over
80-fold). Among these novel dihydrothieno[2,3-e]indazole deriva-
tives, 2,4-difluoro-substituted 2t offered the preferred overall pro-
file with IKK-b potency, with more than 50-fold selectivity over
2v
2w
2x
0.5
0.9
1.9
9
0.5
0.7
3.8
H2N
HO
MeO
F
NTa
>100
IKK-
The in vivo efficacy of the selected compounds 2t was next as-
sessed in a mouse model of LPS-stimulated TNF-
production.17
Mice were dosed orally with 2t at 3 mg/kg and 10 mg/kg, 1 h prior
to LPS administration. The TNF- levels were then measured
90 min later, as shown in Figure 2, and oral dosing of 2t at
10 mg/kg was found to inhibit TNF- production in a dose-depen-
a and good cellular potency.
F
a
F
a
O
NTa
NTa
a
2y
>10
Me
N
H
F
dent manner. Other potent compounds 2v and 2w were not effec-
tive in this in vivo assay.
a
NT = not tested.
In summary, we developed a novel series of IKK-b inhibitors
possessing a dihydrothieno[2,3-e]indazole core. This series shows
potent in vitro activity in relevant biological assays, and compound
2t was efficacious in a mouse inflammation model. Further inves-
tigation of the pharmacological profiles of these novel compounds
is in progress.
potency. This result suggests that the N-benzyl group of 2l would
be a key fragment for further optimization.
Therefore, we anticipated that the orientation effect of the
N-benzyl moiety might be effective for increasing potency. As
such, various cyclic amino derivatives 2m–q were prepared in
the same manner as described in Scheme 2 (Table 3). In this ser-
ies, four- to six-membered cyclic amino derivatives showed
moderate enzyme potency, whereas the activity of seven-mem-
bered azepanyl derivative 2q dropped. In this series, 3-piperidi-
nyl analog 2o showed two-fold more potent enzyme activity
Acknowledgments
We gratefully acknowledge Hiromi Fujiya and Megumi Wada
for their support and synthetic contributions.
We are also thank Masahiro Ueno and Hitoshi Inoue for their
useful discussions.
(IC50’s of 2.2
lar activity at IC50’s of 0.5
l
M) than 2l. Compound 2o also showed good cellu-
References and notes
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a preferable combination with regard to enzyme and cellular
potency.
In addition to investigating N-benzyl derivatives, we embarked
upon further exploration of the benzyl group of 2o. The enzyme
and cell-based activities of 3-piperidinyl derivatives are shown in
Table 4. To assess the kinase selectivity, some compounds of the
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After the incubation, the plate was washed three times with PBS-0.05%
Tween20. PBS containing 3% BSA (200 L/well) was added to the plate and
incubated for 60 min at 25 °C. After incubation, the plate was washed three
times with PBS-0.05% Tween20. The assay buffer (90 L/well) containing IKK,
25 mM Tris (pH 7.4), 2 mM DTT, 0.01% Tween 20, and the test compound
solution (5 L/well) containing 1% DMSO were added to the plate and
incubated for 10 min at 25 °C. Reactions were initiated by adding a solution
(5 L/well) of 60 M ATP and 200 mM MgCl2 and incubated for 60 min at
25 °C. After the incubation, the plate was washed three times with PBS-0.05%
Tween 20. Mouse monoclonal antibody for phospho IkB- (1:7000, Cell
signaling, 100 L/well) was then added to the plate and incubated for 16 h at
l
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4 °C. After the incubation, the plate was washed three times with PBS-0.05%
Tween 20. HRP-conjugated rabbit polyclonal antibody for mouse IgG (1:7000,
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GE Healthcare, 100
25 °C. After the plate was washed, the amount of phosphorylation of IkB-
substrate was measured using TMB reagent (100 L/well).
Measurement of TNF- inhibitory activity: Human monocyte THP-1 cells
(2 Â 105 cells/well) was incubated with serum-free RPMI-1640 containing
the test compounds and LPS (0.2 g/well) for hr at 37 °C. TNF-
concentrations in the conditioned media were measured by ELISA methods.
17. Female DBA/2 mice were administered 2r by peroral gavage. After 60 min, LPS
(1 g/body) dissolved in 0.2 mL of saline was injected intraperitoneally. After
90 min, plasma TNF- levels were analyzed with a commercial TNF- ELISA kit.
lL/well) was added to the plate and incubated for 60 min at
a
l
16. Measurement of IKK-
inhibitory activity of the compounds was determined with the following assay
that measures the phosphorylation of IkB- substrate by the respective
kinases. The enzymes used in the assay were human IKK- and IKK-b. Biotin-
conjugated IkB- peptide (Bio-IkB peptide) was used as the IkB- substrate (21
amino acid residues). In brief, 0.2 M Bio-IkB peptide solution (100 L/well)
was added to a 96-well avidine-coated plate and incubated for 60 min at 25 °C.
a
and IKK-b inhibitory activity: The IKK-
a
and IKK-b
a
a
l
6
a
a
a
a
l
l
l
a
a