Discovery of DS28120313 as a potent orally active hepcidin production inhibitor: Design and optimization of novel 4,6-disubstituted indazole derivatives

Article abstract of DOI:10.1016/j.bmcl.2017.10.031

Hepcidin has emerged as the central regulatory molecule in systemic iron homeostasis, and its inhibition could be a favorable strategy for treating anemia of chronic disease (ACD). Here, we report the design, synthesis and structure–activity relationships (SAR) of a series of 4,6-disubstituted indazole compounds as hepcidin production inhibitors. The optimization study of multi-kinase inhibitor 1 led to the design of a potent and bioavailable hepcidin production inhibitor, 32 (DS28120313), which showed serum hepcidin-lowering effects in an interleukin-6-induced acute inflammatory mouse model.

Full text of DOI:10.1016/j.bmcl.2017.10.031

Accepted Manuscript
Discovery of DS28120313 as a potent orally active hepcidin production inhib-
itor: Design and optimization of novel 4,6-disubstituted indazole derivatives
Takeshi Fukuda, Riki Goto, Toshihiro Kiho, Kenjiro Ueda, Sumie Muramatsu,
Masami Hashimoto, Anri Aki, Kengo Watanabe, Naoki Tanaka
PII:
S0960-894X(17)31016-8
https://doi.org/10.1016/j.bmcl.2017.10.031
BMCL 25360
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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Revised Date:
Accepted Date:
3 August 2017
13 October 2017
15 October 2017
Please cite this article as: Fukuda, T., Goto, R., Kiho, T., Ueda, K., Muramatsu, S., Hashimoto, M., Aki, A.,
Watanabe, K., Tanaka, N., Discovery of DS28120313 as a potent orally active hepcidin production inhibitor: Design
and optimization of novel 4,6-disubstituted indazole derivatives, Bioorganic & Medicinal Chemistry Letters (2017),
doi: https://doi.org/10.1016/j.bmcl.2017.10.031
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Discovery of DS28120313 as a potent orally active hepcidin production inhibitor:
Design and optimization of novel 4,6-disubstituted indazole derivatives
∗
Takeshi Fukuda,a, Riki Goto,b Toshihiro Kiho,c Kenjiro Ueda,d Sumie Muramatsu,d Masami Hashimoto,d
Anri Aki,b Kengo Watanabe,e and Naoki Tanaka a
a Rare Disease & LCM Laboratories, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
b Biologics & Immuno-Oncology Laboratories, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
cModality Research Laboratories, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
dPain & Neuroscience Laboratories, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
e Drug Metabolism & Pharmacokinetics Research Laboratories, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
ARTICLE INFO
ABSTRACT
Article history:
Received
Hepcidin has emerged as the central regulatory molecule in systemic iron
homeostasis, and its inhibition could be a favorable strategy for treating
anemia of chronic disease (ACD). Here, we report the design, synthesis and
structure-activity relationships (SAR) of a series of 4,6-disubstituted
indazole compounds as hepcidin production inhibitors. The optimization
study of multi-kinase inhibitor 1 led to the design of a potent and
bioavailable hepcidin production inhibitor, 32 (DS28120313), which
showed serum hepcidin-lowering effects in an interleukin-6-induced acute
inflammatory mouse model.
Revised
Accepted
Available online
Keywords:
Hepcidin
Anemia of chronic disease
Indazole
Pyrazole
2017 Elsevier Ltd. All rights reserved.
Kinase
The maintenance of serum iron levels is important
since high and low iron concentrations induce
oxidative organ damage and iron deficiency anemia, iron absorption, iron recycling by macrophages,
6,4 in addition to iron signaling. This peptide
hormone is the homeostatic regulator of intestinal
respectively.1
and iron mobilization from hepatic stores.5
Anemia of chronic disease (ACD), is the second
most prevalent form after that caused by iron
deficiency and occurs in patients with acute or
chronic immune activation. ACD, which includes
inflammation-associated anemia, is a heterogenic
anemic condition caused by chronic inflammation
from a basic disease such as rheumatoid arthritis.2
Some patients with ACD are known to present with
iron deficiency despite abundant body iron stores
(termed functional iron deficiency).
Recently, high hepcidin induction based on
inflammatory status was recognized as the cause of
functional iron deficiency.6 Hepcidin expression
deficiency is a common phenotype of hereditary
hemochromatosis. Therefore, controlling hepcidin
levels would be a promising therapeutic strategy for
treating hepcidin-induced functional iron deficiency.
Indeed, a few biologics (e.g., NOX-H94,
LY2928057, and LY2787106) are proceeding to
clinical trials for the treatment of anemia. Here, we
describe the design and optimization process aimed
at lowering the multi-kinase inhibitory activity of 1
to discover methyl [6-(3-cyclopropyl-5-methyl-1H-
Hepcidin was originally discovered as an
antibacterial peptide,3 and this hormone is inducible
by inflammatory cytokines such as interleukin (IL)-
———
∗ Corresponding author. Tel.: +81 3 3492 3131; fax: +81 3 5436 8563.
E-mail address: fukuda.takeshi.zv@daiichisankyo.co.jp (T. Fukuda)

pyrazol-4-yl)-1H-indazol-4-yl]carbamate
(DS79182026, 32), a potent orally available
hepcidin production inhibitor.
As previously reported,7,8 we identified the
indazole derivative 1 as a potently active lead
compound with a half-maximal inhibitory
concentration (IC50) of 0.23 µM.9
a
b
2
3
4
5
c
d
e
6
7
8
Although compound 1 showed a hepcidin-
lowering effect following oral administration, it
inhibited numerous kinases at double the
concentration of the IC50 value (Figure 1).
f
g
9
10
Scheme 1. Synthesis of 3-heteroaryl indazole derivatives. Reagents and conditions: (a) Boc2O,
Et3N, DMAP, CH3CN, 63%; (b) Bis(pinacolato)diboron, Pd(dppf)Cl2-CH2Cl2, KOAc, 1,4-
dioxane, 97%; (c) Pd(dppf)Cl2-CH2Cl2, K3PO4-nH2O, 1,2-dimethoxyethane/H2O, 91%; (d)
CuBr, NaNO2, HBr aq., AcOH/H2O, 29%; (e) Boc2O, Et3N, DMAP, CH3CN, 89%; (f)
Heteroarylboronic acid, Pd(dppf)Cl2-CH2Cl2, K3PO4-nH2O, 1,2-dimethoxyethane/H2O, 46-83%;
(g) 4N-HCl/1,4-dioxane, 71-97%. (The Boc protected compounds were isolated as a single
regioisomer, but the regio of Boc groups were not determined.)
The transformation of amide substituents to
simple heteroaromatic rings deteriorated the
inhibitory activity (compounds 12-14). However,
the activity was effectively enhanced by
introducing a cyclic amine moiety at the pyridine
ring. Compounds 15 and 16 showed a significantly
enhanced in vitro activity.
1
IC50 = 0.23 M
µ
Figure 1.
1
IC50 values and kinase inhibitory profiles of compound
Multi-kinase inhibitors are not considered suitable
as medicines for chronic diseases and, therefore, we
decided to carry out a derivatization to reduce the
kinase inhibitory activity of our lead compound.
Hydrogen bonds are among the most important
specific interactions in biological recognition
processes. In particular, for kinases, hydrogen
bonds with hinge backbone residues are considered
the anchors of ligand binding, a generally
Table 1
. SAR of 3-heteroaryl-substituted indazole derivatives
indispensable interaction for potent enzyme
inhibition.10 We considered that the kinase
IC50
IC50
compound
R
compound
R
(µM)
(µM)
inhibitory potency of 1 was probably due to its 3-
aminoindazole moiety acting as a hinge binding
site.11 Therefore, we designed a scaffold, which
transformed the amide moiety into a heteroaromatic
ring. We thought that we could reduce the kinase
inhibitory potency by transforming the NH group at
3-position of the indazole, which is thought to play
the role of a hydrogen bond donor.
3-Heteroaryl substituted indazole derivatives were
synthesized using the process illustrated in Scheme
1. The results of the analysis of the indazole
derivatives with various functional groups on the 3-
position are summarized in Table 1.
11
0.41
14
3.1
12
2.6
2.3
15
16
0.17
13
0.12
The 4-methylpiperazin-1-yl-pyridine derivative
(16) showed good hepcidin inhibitory activity.
Although it improved slightly, compound 16
inhibited numerous kinases at a concentration that
was 2-fold the IC50 value (Figure 2).

Table 2. SAR of 4-substituted indazole derivatives
4
5
IC50
(µM)
IC50
(µM)
16 IC50 = 0.12 µM
compound
R
compound
R
H
Figure 2.
16
23
4.1
29
2.7
1.2
IC50 values and kinase inhibitory profiles of compound
24
25
26
1.0
30
31
32
We estimated that the hydrogen atom of the newly
introduced pyridine ring functioned as a hydrogen
bond donor. Therefore, we designed a new scaffold,
which transferred the substituent from the 3- to 4-
position of indazole. We hypothesized that the
kinase inhibitory potency could be decreased by
removing substituents that could function as
hydrogen bond donors.
0.45
0.19
0.34
O
O
H
N
0.093
*
33
34
35
0.66
3.0
The 4-substituted indazole derivatives were
synthesized using the process illustrated in Scheme
2.
27
28
1.9
2.2
(5-)
O
H
N
>10
a
b
O
*
17
18
19
5
Furthermore, the methyl urea group showed good
activity and, surprisingly, the methyl carbamate
group showed a significant increase in the in vitro
activity (compounds 31 and 32).
c
d
e
20
21
The steric tolerance on the carbamate group was
narrow, and the conversion to an ethyl carbamate
group (33) considerably attenuated the activity.
In addition, transferring the substituent to the 5-
position of indazole completely abrogated the in
vitro activity.
22
Scheme 2.
Synthesis of 4-substituted indazole derivatives. Reagents and conditions: (a)
Boc2O, Et3N, DMAP, CH3CN, 55%; (b) Bis(pinacolato)diboron, Pd(dppf)Cl2-CH2Cl2, KOAc,
1,4-dioxane, 99%; (c) Pd(dppf)Cl2-CH2Cl2, K3PO4-nH2O, 1,2-dimethoxyethane/H2O, 72%; (d)
acyl chloride, pyridine, CH2Cl2 or alkyl chloroformate, pyridine, CH2Cl2 or isocyanate, Et3N,
THF, 27-61%; (e) 4N-HCl/1,4-dioxane, 16-90%. (The Boc protected compounds were isolated
as a single regioisomer, but the regio of Boc groups were not determined.)
The results of the analysis of the indazole
derivatives with various functional groups on the 4-
position are summarized in Table 2.
Compound 23 that lacked a substituent at the 3-
position showed a considerably weaker activity
than its parent compound, but compound 24 in
which the amide moiety migrated to the 4-position
showed moderate activity. The compounds with
sterically smaller substituents on the amide group
showed higher activity (25 and 26). The
Next, the effects of various substituents on the
pyrazole were examined. The 4-methoxycarbonyl
aminoindazole derivatives with various substituents
on the pyrazole ring were synthesized using the
process illustrated in Scheme 3.12
a
19
9
36
introduction of the 4-piperidynylaryl group, which
showed high activity at the 3-position, limited the
enhancement of activity (compound 27).
c
b
The reverse amide, primary amine, and methane
sulfonamide derivatives (28–30) exhibited weaker
activity than that of the parent compound.
37
38
Scheme 3. Synthesis of 6-substituted indazole derivatives. Reagents and
conditions: (a) Methyl chloroformate, pyridine, CH2Cl2, 92%; (b) Pd(dppf)Cl2-
CH2Cl2, K3PO4-nH2O, 1,2-dimethoxyethane/H2O, 31-90%; (c) 4N-HCl/1,4-
dioxane, 20-70%.

Table 4. Physicochemical properties and pharmacokinetic (PK) parameters
of DS28120313
The results of the analysis of the substituted
pyrazole derivatives are summarized in Table 3.
Cmaxb
(µg/mL)
AUCb
(h*µg/mL)
MSa
(%)
PB
(free %)
Tmaxb
(h)
LogD
Table 3. SAR of 6-substituted indazole derivatives
3.1
84
24
4.94
0.67
14.7
a Remaining (%) test compound after 0.5 h incubation with mouse liver
microsomes (0.5 mg/mL).
b Average of two values dosed at 30 mg/kg orally (p.o.) in C57BL/6J mice
(0.5% methylcellulose suspension).
The appropriately lipophilic compound
IC50
IC50
DS28120313 possessed good metabolic stability
and low plasma protein binding (fu,p = 24%).
Further, DS28120313 showed high plasma
exposure in mice and was considered to have a
suitable profile as an oral agent. Next, the hepcidin-
lowering effect of DS28120313 was evaluated
using an IL-6-induced acute inflammatory mouse
model.
Hepcidin is known as an acute-phase protein
produced in response to chronic inflammatory
conditions. Therefore, we evaluated the acute-phase
induction of hepcidin in serum in response to
intravenous injection of mouse IL-6. Prior to this
evaluation, we conducted a time-course study
where serum hepcidin levels began increasing as
early as 1 h after the injection of IL-6, plateaued at
4 h, and then remained constant until 6 h after the
injection (data not shown). DS28120313 was
administered orally to 9-week-old male C57BL/6J
mice 30 min before IL-6 administration. Blood
samples were collected 4 h after the IL-6 injection,
and the hepcidin concentration was determined.
compound
R
compound
R
(µM)
(µM)
32
c-Pr
0.093
0.15
0.18
0.19
3.9
43
CO2Me
0.13
39
40
41
42
Me
Et
44
45
46
47
CO2H
CONHMe
Ph
>10
2.0
i-Pr
1.1
CH2OMe
0.32
The alkyl groups showed high activity, but the
ether substituent deteriorated the inhibitory activity
(compounds 39–42). In addition, the compound
with the ester group showed high activity, but that
of compounds with a carboxylic acid or amide
group was greatly attenuated (compounds 43–45).
The introduction of a phenyl or 2-pyridyl group,
which produced compounds that showed high
activity in the derivatization of benzisoxazole
scaffold, limited the enhancement of activity
(compounds 46 and 47).8
Since we acquired compound 32 that possessed
high inhibitory activity, we performed a kinase
profiling assay of this candidate. As expected, the
kinase inhibitory potency of 32 was significantly
reduced at double the concentration of its IC50
value (Figure 3).
32 IC50 = 0.093 µM
Figure 4.
Effect of compound DS28120313. The compound was
administered to mice at dose of 30 mg/kg (p.o., 0.5% methylcellulose,
suspension, n = 4) before IL-6 treatment. #, p < 0.05 vs Saline treated
group (t-test), ***, p < 0.001 vs 0.5% MC treated group (t-test).
Figure 3. IC50 values and kinase inhibitory profiles of compound 32
Thus, the potently active hepcidin production
inhibitor 32, named DS28120313, was selected for
further investigations.
To evaluate the in vivo efficacy, we assessed the
pharmacokinetic (PK) parameters of DS28120313
(Table 4).
DS28120313 significantly reduced the blood
hepcidin level at an oral dose of 30 mg/kg (Figure
4).
In conclusion, we discovered a series of 4,6-
disubstituted indazole derivatives as potent and

of targeted anticancer therapies. CA Cancer J Clin. 2013;63:249–279.
11. Li Xing et al. Kinase hinge binding scaffolds and their hydrogen bond
patterns. Bioorg Med Chem. 2015;23:6520–6527.
orally bioavailable inhibitors of hepcidin
production. Starting from the multi-kinase inhibitor
1, we developed a 4,6-disubstituted indazole as a
scaffold with a lower kinase inhibitory activity than
its parent compound by transforming the hinge
binding site.
12. McLaughlin M, et al. The THP-protected pyrazoles were isolated as
single regioisomer. The regiochemistry were determined by comparing
1
the chemical shift of 2-position of tetrahydropyran with reported H
NMR data. J Org Chem. 2008;73:4309–4312.
The optimization of substituents at the 4- and 6-
positions produced DS28120313, which possessed
potent in vitro activity. Furthermore, DS28120313
inhibited hepcidin production in HepG2 cells and
lowered the serum hepcidin levels in an IL-6
induced acute inflammatory mouse model.
The exact target molecule underlying the
mechanism of action is not known, but
DS28120313 was found to be a promising
compound as an oral agent with in vivo efficacy.
Both DS28120313 and DS791820268 showed
potent in vitro activity and strong ability to inhibit
hepcidin production in IL-6-induced high-hepcidin
model mouse by oral administration, we consider
both as promising compounds. We are planning to
evaluate in higher order in vivo model(e.g. anemia
amelioration models) and we will comprehensively
assess the qualities of both compounds.
And, the target identification to elucidate the
mechanism of action of these compounds is
ongoing and will be reported in due course.
Acknowledgments
We would like to thank Dr. Nakayama, Dr.
Machinaga for their technical assistance and helpful
discussions.
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(http://dx.doi.org/10.1016/j.bmcl.2017.03.056)
8.
9.
Fukuda T. et al. Discovery of DS79182026: A potent orally active
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3722. (http://dx.doi.org/10.1016/j.bmcl.2017.07.004)
Hepcidin inhibitory activity of each compound was tested in HepG2 cell
line, stimulated with 50 ng/mL of BMP6 for Hepcidin mRNA
expression induction. Data fit for IC50 was determined using GraphPad
Prism’s nonlinear regression equation.
10. Grace K. Dy et al. Understanding, recognizing, and managing toxicities

Graphical Abstract
Discovery of DS28120313 as a potent orally
active hepcidin production inhibitor: Design
and optimization of novel 4,6-disubstituted
indazole derivatives
Leave this area blank for abstract info.
Takeshi Fukuda, Riki Goto, Toshihiro Kiho, Kenjiro Ueda, Sumie Muramatsu, Masami Hashimoto,
Anri Aki, Kengo Watanabe, and Naoki Tanaka
32 (DS28120313)
Hepcidin mRNA expression
IC50 = 0.093µM