Discovery of DS79182026: A potent orally active hepcidin production inhibitor

Article abstract of DOI:10.1016/j.bmcl.2017.07.004

Hepcidin has emerged as the central regulatory molecule of systemic iron homeostasis. Inhibition of hepcidin could be a strategy favorable to treating anemia of chronic disease (ACD). We report herein the synthesis and structure-activity relationships (SARs) of a series of benzisoxazole compounds as orally active hepcidin production inhibitors. The optimization study of multi kinase inhibitor 1 led to a potent and bioavailable hepcidin production inhibitor 38 (DS79182026), which showed serum hepcidin lowering effects in a mouse IL-6 induced acute inflammatory model.

Full text of DOI:10.1016/j.bmcl.2017.07.004

Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of DS79182026: A potent orally active hepcidin production
inhibitor
b
c
d
d
d
Takeshi Fukuda a, , Riki Goto , Toshihiro Kiho , Kenjiro Ueda , Sumie Muramatsu , Masami Hashimoto ,
⇑
Anri Aki b, Kengo Watanabe e, 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
c Modality Research Laboratories, Daiichi Sankyo Co, Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
d Pain & 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
a r t i c l e i n f o
a b s t r a c t
Article history:
Hepcidin has emerged as the central regulatory molecule of systemic iron homeostasis. Inhibition of hep-
cidin could be a strategy favorable to treating anemia of chronic disease (ACD). We report herein the syn-
thesis and structure-activity relationships (SARs) of a series of benzisoxazole compounds as orally active
hepcidin production inhibitors. The optimization study of multi kinase inhibitor 1 led to a potent and
bioavailable hepcidin production inhibitor 38 (DS79182026), which showed serum hepcidin lowering
effects in a mouse IL-6 induced acute inflammatory model.
Received 19 April 2017
Revised 27 June 2017
Accepted 1 July 2017
Available online xxxx
Keywords:
Hepcidin
Ó 2017 Elsevier Ltd. All rights reserved.
Anemia of chronic disease
Benzisoxazole
Pyrazole
Kinase
Hepcidin is a peptide hormone, and is known as the master reg-
ulator for systemic iron mobilization.1 The maintenance of serum
iron level is important since a high iron concentration induces
oxidative organ damage, and a low iron concentration results in
iron deficiency anemia.2 As hepcidin was originally discovered as
an antibacterial peptide,3 this hormone is inducible by inflamma-
tory cytokines such as IL-6,4 in addition to iron signaling.
at the enhancement of bioavailability and lowering of the multi
kinase inhibitory activity of 1 to discover methyl {6-[5-methyl-3-
(pyridin-2-yl)-1H-pyrazol-4-yl]-1,2-benzisoxazol-3-yl}carbamate
(DS79182026, 38), a potent orally available hepcidin production
inhibitor.
As previously reported,6 we identified the indazole derivative 1
as a potent active lead compound (IC50 = 0.13 l
M).7 Although com-
Anemia of chronic disease (ACD), which includes anemia of
inflammation, is a heterogenic anemic condition due to chronic
inflammation from a basic disease, such as rheumatoid arthritis.5
Some ACD patients are known to present iron deficiency despite
abundant body iron store (termed functional iron deficiency).
Recently, high hepcidin induction based on inflammatory status
was recognized as the cause of functional iron deficiency. Hepcidin
pound 1 showed hepcidin lowering effect in mice by the intraperi-
toneal administration, pharmacokinetic (PK) profiles supported a
lack of exposure to blood in the oral administration (Table 1).
The indispensable para-hydroxyphenyl group also might be
responsible for this low exposure because of the O-glucuronidation
via UDP-glucuronosyltransferase (UGT). As an entirely fresh start,
we started to explore the alternatives of the para-hydroxyphenyl
group.
expression deficiency is
a common phenotype of hereditary
hemochromatosis. The controlling of hepcidin level would be a
promising therapeutic strategy for treating hepcidin caused func-
tional iron deficiency. Indeed, a few such biologics (e.g. NOX-
H94, LY2928057 and LY2787106) are entering clinical trials for
treatment of anemia. Herein we describe the derivatization aimed
First, we examined the bicyclic phenolic bioisosteres. The bicyc-
lic mimetics were designed to address the phenolic H-bond donor
based on the localization afforded by the complementary fused
heterocyclic rings.8 However, the benzimidazole 3, pyrolopyridine
4 and oxindole 5 were found to be surprisingly poor mimetics.
Then, we investigated the monocyclic heteroaromatics. The
transformations to pyrrole
group deteriorated hepcidin inhibitory activity. However,
the dimethylisoxazole 8 showed moderate inhibitory activity.
6 drastically lost and 4-pyridyl
⇑
7
Corresponding author.
E-mail address: fukuda.takeshi.zv@daiichisankyo.co.jp (T. Fukuda).
http://dx.doi.org/10.1016/j.bmcl.2017.07.004
0960-894X/Ó 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Fukuda T., et al. Bioorg. Med. Chem. Lett. (2017), http://dx.doi.org/10.1016/j.bmcl.2017.07.004

2
T. Fukuda et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Table 1
IC50 values and physicochemical properties and PK parameters of 1.
LogD
3.4
MSa
(%)
UGTa
(%)
Cmax
g/mL)
Tmax
(h)
AUC
(h*
(
l
l
g/mL)
89
76
2.64b
0.32c
1.33b
1.50c
6.93b
0.89c
a
b
c
Remaining (%) of the tested compound after 0.5 h incubation with mouse liver microsomes (0.5 mg/mL).
Average of two values dosed at 30 mg/kg i.p. with C57BL/6J mice (0.5% Methylcellulose, suspension).
Average of two values dosed at 30 mg/kg p.o. with C57BL/6J mice (0.5% Methylcellulose, suspension).
Surprisingly, the dimethylpyrazole 9 showed inhibitory activity
comparable to compound 2 (Table 2).
Subsequently, we investigated the combinations of the alterna-
tive pyrazole and potent benzamide moieties.6
The compounds 10 and 11 retained potent in vitro activity
(Table 3).
Table 3
SAR of 6-dimethylpyrazole derivatives.
PK parameters of compound 11 are summarized in Table 4.
Compound 11 showed high stability against UGT. PK profiles of
11 were evaluated and found to be improved compared to com-
pound 1, presumably due to the interruption of the
glucuronidation.
Indeed, 11 showed higher plasma exposure than 1 and was con-
sidered to be a suitable profile as an oral agent.
Next, the hepcidin lowering effect of compound 11 was evalu-
ated by a mouse interleukin-6 (IL-6) induced acute inflammatory
model.
Compound
R
IC50
0.33
0.26
(lM)
9
10
11
0.23
Table 2
Alternatives of para-hydroxyphenyl group.
Compound
R
IC50
(l
M)
Compound
R
IC50
>30
(lM)
2
0.40
6
3
4
>3
11
7
8
7.2
1.0
5
>30
9
0.33
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T. Fukuda et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
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Table 4
Physicochemical properties and PK parameters of 11.
LogD
MSa
(%)
UGTa
(%)
Cmaxb
g/mL)
Tmaxb
(h)
AUCb
(h* g/mL)
(
l
l
2.4
80
94
0.92
3.25
2.55
a
Remaining (%) of the tested compound after 0.5 h incubation with mouse liver microsomes (0.5 mg/mL).
Average of two values dosed at 30 mg/kg p.o. with C57BL/6J mice (0.5% Methylcellulose, suspension).
b
As hepcidin is known as an acute-phase protein responding to
tization with the intention of reducing the kinase inhibitory activ-
ity. Generally, hydrogen bonds are among the most important
specific interactions in biological recognition processes. In particu-
lar for kinases, hydrogen bonds with hinge backbone residues are
considered the anchors of ligand binding, a generally indispensable
interaction for potent enzyme inhibition.9 We considered that the
kinase inhibitory potency of 11 was probably due to its 3-aminoin-
dazole moiety acting as a hinge binding site.10
chronic inflammatory conditions, we evaluated by intravenous
injection for the acute-phase induction of hepcidin in serum levels
in response to mouse IL-6. Beforehand, we conducted a time course
study in which serum hepcidin levels began increasing as early as
1 h after injection of IL-6, reached plateau at 4 h, and stayed con-
stant until 6 h after injection (data not shown). Compound was
administered orally to 9-week-old male C57BL/6J mice 30 min
before IL-6 administration. Blood was collected at 4 h after the
IL-6 injection and the hepcidin concentration was determined.
Oral administration of 30 mg/kg doses of compound 11 inhib-
ited hepcidin production triggered by IL-6 (Fig. 1).
Therefore, we designed
a benzisoxazole scaffold, which
replaced oxygen of the indazole ring with nitrogen. The benzisox-
azole derivative 13 showed moderate hepcidin inhibitory activity
(Table 5).
Although compound 11 showed a hepcidin lowering effect in
oral administration, it inhibited many kinases at double the con-
centration of IC50 value (Fig. 2).
As expected, the kinase inhibitory potency of compound 13 was
significantly reduced at double the concentration of IC50 value
(Fig. 3).
As multi kinase inhibitors are not considered to be suitable as
medicines for chronic diseases, we decided to carry out the deriva-
Since we were able to find that the benzisoxazole scaffold had a
lower kinase inhibitory activity, we then examined the substituent
at the 3-position of benzisoxazole.
3,6-Disubstituted benzisoxazole derivatives were synthesized
as illustrated in Scheme 1. Starting from a bromopyrazole 14,
ortho-fluorobenzonitrile 16 was obtained by Boc-protection and
Suzuki-coupling reaction. Cyclization with acetohydroxamic acid
yielded 3-amino-6-substituted benzisoxazole intermediate 17
accompanying Boc-deprotection. After reprotection of the pyra-
zole, amidation with acid chloride yielded 3-amide benzisoxazole
derivative 19.
Table 5
In vitro activity of indazole and benzisoxazole scaffolds.
Compound
X
IC50 (lM)
Fig. 1. Effect of compound 11. 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 < 0.001 vs Saline treated group (t-test), ***, p < 0.001 vs 0.5% MC treated
group (t-test).
12
13
NH
O
0.082
0.29
Fig. 2. IC50 values and kinase inhibitory profiles of compound 11.
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T. Fukuda et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Fig. 3. IC50 values and kinase inhibitory profiles of compound 13.
Scheme 2. Synthesis of 2-Aryl-5-methyl pyrazoles. Reagents and conditions:
(a) Acetone, Sodium methoxide, THF, quant.; (b) Hydrazine monohydrate, EtOH,
69–94%; (c) N-bromosuccinimide, CH2Cl2/CCl4, 55–100%; (d) 3,4-Dihydro-2H-pyran,
p-Toluenesulfonic acid monohydrate, THF, 69–94%.
Scheme 1. Synthesis of 6-substituted benzisoxazole derivatives. Reagents and
conditions: (a) Boc2O, Et3 N, DMAP, THF, 63–99%; (b) 4-Cyano-3-fluorophenyl-
boronic acid, Pd(dppf)Cl2-CH2Cl2, K3PO4-nH2O, 1,2-dimethoxyethane/H2O, 7–58%;
(c) Acetohydroxamic acid, potassium tert-butoxide, DMF, 58–100%; (d) Boc2O,
Et3 N, DMAP, THF, 24–95%; (e) acyl chloride, pyridine, CH2Cl2 or alkyl chlorofor-
mate, pyridine, CH2Cl2 or Ethyl isocyanate, Et3 N, THF, 20–95%; (f) 4N-HCl/dioxane,
71–97%. (The Boc protected compounds were single regioisomer, but the regio of
Boc groups were not determined.)
thetic routes are summarized in Scheme 2. Arylmethylpyrazole
26 was prepared from aryl ester 24 by Claisen condensation and
following cyclization with hydrazine monohydrate. The THP-pro-
tected bromopyrazole 28 was obtained from 26 by bromination
and treatment of 2,3-dihydropyran with p-Toluenesulfonic acid.11
The objective derivants were provided by the similar method illus-
trated in Scheme 1 with the use of bromopyrazole 28.
The results of methylpyrazole derivatives variously substituted
are summarized in Table 7.
As a consequence of tolerability of alkyl substituents, the bulk-
iness negatively affected the in vitro activity (compound 31, 32).
Moreover, various polar groups such as ether, alcohol, ester, and
amide deteriorated the activity (compounds 33–36).
Presuming by the results of the effective cyclopropyl group, the
sp2-carbon character of the substituent was important for
enhancement of the activity. As expected, the installation of the
phenyl group resulted in a moderate activity. So it was found that
the aromatic ring enhanced inhibitory activity, and we next
focused on various heteroaromatic ring transformations. Results
of the nitrogen-containing heteroaromatic ring, 2-pyridyl deriva-
tive 38, provided a significant leap to in vitro activity.
Using isocyanate and alkyl chloroformate in place of acyl chlo-
ride gave urea and carbamate derivatives. Finally, deprotection of
the Boc group with an acidic condition gave the objective
compounds.
The results of benzisoxazole derivatives variously functional-
ized on the 3-position are summarized in Table 6.
A primary amine derivative 21 showed a weaker activity. On the
other hand, it was found that the carbamate derivative 22 showed
a similar activity to compound 13. Moreover, urea 23 maintained a
moderate activity.
Next, various substituents on the pyrazole were examined.
Regarding the preparation of various bromopyrazole 28, the syn-
Table 6
SAR of 3-substituted benzisoxazole derivatives.
We next turned our attention to the introduction of a sub-
stituent to the versatile 2-pyridyl ring (Table 8).
Installation of a methyl group at the 3- and 4-positions of pyr-
idine decreased activity (compounds 42, 43). In contrast, the 6-
methylpyridine derivative 44 retained potent activity, but a bulky
substituent at the 6-position weakened the activity (compound
46). These results encouraged us to further optimize compound 38.
Next, the substituent of carbamate at the 3-position was exam-
ined (Table 9).
Linear substituents such as methyl carbamate 38 and ethyl car-
bamate 47 were acceptable, but the branched isopropyl carbamate
48 was slightly less active. Meanwhile, fluoroethyl carbamate 49
and methoxyethyl carbamate 50 showed preservation of the favor-
able hepcidin inhibitory activity.
Compound
R
IC50 (lM)
13
0.29
21
22
1.4
0.21
23
0.72
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T. Fukuda et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
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Table 7
SAR of 6-substituted benzisoxazole derivatives.
Compound
R
IC50
(lM)
Compound
R
IC50
(l
M)
22
29
30
31
c-Pr
Me
Et
0.21
0.58
0.46
0.91
35
36
37
38
CO2Me
CONHMe
Ph
2.9
4.4
0.54
0.039
i-Pr
32
33
t-Bu
>10
1.1
39
40
0.35
0.21
CH2OMe
34
CH(OH)Me
1.2
41
2.9
Table 8
SAR of 2-pyridyl pyrazole derivatives.
Compound
R
IC50
(
lM)
Compound
R
IC50 (lM)
38
42
43
H
3-Me
4-Me
0.039
4.8
2.6
44
45
46
6-Me
6-OMe
6-i-Pr
0.042
0.40
2.6
Table 9
SAR of substituted carbamate derivatives.
Compound
R
IC50
(lM)
Compound
R
IC50 (lM)
38
47
48
Me
Et
i-Pr
0.039
0.040
0.16
49
50
FCH2CH2-
MeOCH2CH2-
0.047
0.069
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T. Fukuda et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Fig. 4. IC50 values and kinase inhibitory profiles of compound 38.
Table 10
Physicochemical properties and PK parameters of DS79182026.
LogD
MSa
(%)
PB
(free%)
Cmaxb
g/mL)
Tmaxb
(h)
(l
2.5
87
17
9.17
1.0
AUCb
Vdc
(L/kg)
Clc
BAb
(%)
(h*lg/mL)
(mL/min/kg)
36.2
0.91
14.5
100
a
b
c
Remaining (%) of the tested compound after 0.5 h incubation with mouse liver microsomes (0.5 mg/mL).
Average of two values dosed at 30 mg/kg p.o. with C57BL/6J mice (0.5% Methylcellulose, suspension).
Average of two values dosed at 30 mg/kg i.v. with C57BL/6J mice (92 mM SBE7-b-CyD, solution).
DS79182026 significantly reduced blood hepcidin level at a
dose of 30 mg/kg by the oral administration.
In conclusion, we discovered a series of benzisoxazole deriva-
tives as potent and orally bioavailable hepcidin production inhibi-
tors. Starting from the multi kinase inhibitor 1, we acquired the
benzisoxazole as a scaffold with a lower kinase inhibition by trans-
formation of the hinge binding site.
With the optimization of substituents at the 3- and 6-positions,
DS79182026 was found to possess potent in vitro activity.
DS79182026 inhibited hepcidin production in HepG2 cells and
lowered serum hepcidin levels in an IL-6 induced mouse acute
inflammatory model.
The target molecule of the mechanism of action is not known
exactly, but DS79182026 was found to be a promising compound
as an oral agent showing in vivo efficacy.
The target identification to understand the mechanism of action
of these compounds is ongoing and will be reported in due course.
Acknowledgments
Fig. 5. Effect of DS79182026. 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).
We would like to thank Dr. Nakayama, Dr. Machinaga for their
technical assistance and helpful discussions.
A. Supplementary data
In addition, the highly potent compound 38 retained its low
kinase inhibitory activity through the derivatization at the pyra-
zole ring (Fig. 4).
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2017.07.
004.
Thus, orally active hepcidin production inhibitor 38, named
DS79182026, was selected for further investigations.
Pharmacokinetic (PK) parameters of DS79182026 were evalu-
ated (Table 10).
Appropriate lipophilic compound DS79182026 possessed good
metabolic stability and low plasma protein binding (fu,p = 17%).
DS79182026 showed high plasma exposure in mice and was con-
sidered to have a suitable profile as an oral agent.
Next, the hepcidin lowering effect of DS79182026 was evalu-
ated by mouse acute inflammatory model (Fig. 5).
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