Structural Development of Salicylanilide-Based SPAK Inhibitors as Candidate Antihypertensive Agents

Article abstract of DOI:10.1002/cmdc.202100273

Hypertension is an important target for drug discovery. We have focused on the with-no-lysine kinase (WNK)-oxidative stress-responsive 1 (OSR1) and STE20/SPS1-related proline-alanine-rich protein kinase (SPAK)-NaCl cotransporter (NCC) signal cascade as a potential target, and we previously developed a screening system for inhibitors of WNK-OSR1/SPAK-NCC signaling. Herein we used this system to examine the structure-activity relationship (SAR) of salicylanilide derivatives as SPAK kinase inhibitors. Structural design and development based on our previous hit compound, aryloxybenzanilide derivative 2, and the veterinary anthelmintic closantel (3) led to the discovery of compound 10 a as a potent SPAK inhibitor with reduced toxicity. Compound 10 a decreased the phosphorylation level of NCC in mouse kidney in vivo, and appears to be a promising lead compound for a new class of antihypertensive drugs.

Full text of DOI:10.1002/cmdc.202100273

Accepted Article
Title: Structural development of salicylanilide-based SPAK inhibitors as
candidate antihypertensive agents
Authors: Shinya Fujii, Eriko Kikuchi, Honoka Suzuyama, Yuko
Watanabe, Mari Ishigami-Yuasa, Hiroyuki Masuno, Takayasu
Mori, Kiyoshi Isobe, Shinichi Uchida, and Hiroyuki Kagechika
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.202100273
Link to VoR: https://doi.org/10.1002/cmdc.202100273
01/2020

10.1002/cmdc.202100273
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Structural development of salicylanilide-based SPAK inhibitors
as candidate antihypertensive agents
Shinya Fujii,[a] Eriko Kikuchi,[b] Honoka Suzuyama,[a] Yuko Watanabe,[a] Mari Ishigami-Yuasa,[a] Hiroyuki
Masuno,[a] Takayasu Mori,[b] Kiyoshi Isobe,[b] Shinichi Uchida,[b] Hiroyuki Kagechika*[a]
[a]
[b]
Dr. S. Fujii, H. Suzuyama, Y. Watanabe, M. Ishigami-Yuasa, H. Masuno, Prof. H. Kagechika
Institute of Biomaterials and Bioengineering
Tokyo Medical and Dental University (TMDU)
2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan
E-mail: kage.chem@tmd.ac.jp
Dr. E. Kikuchi, Dr. T. Mori, Dr. K. Isobe, Prof. S. Uchida
Department of Nephrology, Graduate School of Medical and Dental Sciences
Tokyo Medical and Dental University (TMDU)
1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan
Supporting information for this article is given via a link at the end of the document.
Abstract: Hypertension is an important target for drug discovery.
We have focused on the with-no-lysine kinase (WNK)-oxidative
stress-responsive
1 (OSR1) and STE20/SPS1-related proline–
alanine-rich protein kinase (SPAK)-NaCl cotransporter (NCC) signal
cascade as a potential target, and we previously developed a
screening system for inhibitors of WNK-OSR1/SPAK-NCC signaling.
Herein we used this system to examine the structure-activity
relationship (SAR) of salicylanilide derivatives as SPAK kinase
inhibitors. Structural design and development based on our previous
hit compound, aryloxybenzanilide derivative 2, and the veterinary
anthelmintic closantel (3) led to the discovery of compound 10a as a
potent SPAK inhibitor with reduced toxicity. Compound 10a
decreased the phosphorylation level of NCC in mouse kidney in vivo,
and appears to be a promising lead compound for a new class of
antihypertensive drugs.
Introduction
Hypertension affects more than one billion people worldwide,
and can lead to potentially fatal diseases such as heart attack,
stroke, and aneurysmal rupture. Therefore, there is a need for
effective antihypertensive agents. To develop such agents, it is
important to understand the mechanisms of blood pressure
regulation. For example, pseudohypoaldosteronism type II
(PHAII) is a monogenic hypertensive disease,[1,2] also known as
familial hyperkalemic hypertension or Gordon’s disease. PHAII
is an autosomal dominant disease characterized by
hypertension, severe hyperkalemia, and mild metabolic
acidosis,[3] and is associated in at least some patients with
mutations in the with-no-lysine kinases WNK1 and WNK4
genes.[4] Specifically, patients with WNK1 mutations have large
intronic deletions that increase the abundance of WNK1, and
patients with WNK4 mutations have missense mutations
localized in a short region near putative coiled-coil domains in a
highly conserved sequence of WNK. WNK kinases are so
named because of the lack of lysine in the ATP-binding cassette
of the catalytic region.
Figure 1. Structures of our developed WNK-OSR1/SPAK binding inhibitor (1)
and hit compounds 2 and closantel (3) discovered by SPAK inhibitor screening.
We previously generated PHAII model mice (Wnk4D561A/+
knock-in mice) that carry the same mutation as PHAII patients,
and we discovered that constitutive activation of the WNK-
oxidative stress-responsive 1 (OSR1) and STE20/SPS1-related
proline–alanine-rich protein kinase (SPAK)-NaCl cotransporter
(NCC) signal cascade is the major pathogenic mechanism of
PHAII.[5,6] WNK-OSR1/SPAK-NCC signaling is constitutively
activated in PHAII, and the increased phosphorylation and
activation of NCC cause excessive sodium reabsorption in the
distal convoluted tubules in the kidneys, resulting in salt-
sensitive hypertension. WNK signaling is positively controlled by
aldosterone, angiotensin II, and insulin, which may contribute to
hypertension in patients with hyperaldosteronism and
hyperinsulinemia.[7–11] In addition, WNK-OSR1/SPAK-NKCC1
phosphorylation and activation in vascular smooth muscle cells
play a central role in the regulation of vascular tonus.[12-15]
Consequently, compounds that inhibit this signal cascade are
1
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10.1002/cmdc.202100273
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expected to reduce hypertension through dual actions, namely Results and Discussion
NaCl diuresis and vasodilation, and may be effective to treat
patients with hyperaldosteronism or hyperinsulinemia. In
addition to hypertension, recent studies have revealed that
SPAK inhibitors have therapeutic potential for treating multiple
diseases, including neurological diseases,[16,17] inflammatory
colitis, and cystic fibrosis.[18] On the basis of these
considerations, we have investigated the development of WNK-
OSR1/SPAK-NCC signaling inhibitors as a novel class of
antihypertensive agents.[19,20] For example, we previously
developed inhibitors of WNK binding to OSR1/SPAK, such as
1.[21] We also developed a screening system for SPAK kinase
inhibitors using ELISA, obtaining aryloxybenzanilide derivative 2
as a hit compound,[20] and we recently reported on the structure-
Design and synthesis
Compounds 2 and 3 share a salicylanilide moiety as a common
structural motif. Our previous SAR study based on compound 2
also indicated that the 2-hydroxyl group of the benzoyl group is
important for the SPAK-inhibitory activity. In addition, closantel
(3) exhibited inhibitory activity toward NCC phosphorylation in
vivo.[20] Therefore, we considered that the 3,5-diiodosalicylanilide
structure is a promising pharmacophore for SPAK inhibitors.
Here, we designed and synthesized
a
series of 3,5-
diiodosalicylanilide derivatives as hybrid structures based on
benzanilide 2 and closantel (3), and compared them with 3,5-
dichlorosalicylanilide derivatives.
activity relationship of 2.[22] In addition, we found that
a
veterinary anthelmintic closantel (3) exhibited SPAK-inhibitory
activity.[20] Interestingly, the core structures of 2 and 3 are similar.
However, overdosing of 3 causes various adverse effects,
including neurotoxicity.[23-25] In this study, therefore, we
investigated the structural development and SAR of
salicylanilide-based SPAK inhibitors, focusing on hybrid
structures of compounds 2 and 3, with the aim of finding potent
SPAK inhibitors with reduced toxicity, or a more favorable ratio
of activity to toxicity.
Synthesis of the 3,5-diiodosalicylanilide derivatives is shown in
Schemes 1, 2 and 3. Scheme 1 shows the synthesis of
compounds 7 and 10 bearing various aryloxy moieties. Anilines
5a-e were prepared from compound 4 as described in our
previous paper (Figure S1).[22] Condensation between anilines
5a-e and 3,5-diiodo-2-methoxybenzoic acid afforded compounds
6a-e, and the removal of the O-methyl group by boron tribromide
afforded the designed compounds 7a-e, respectively.
Compounds 10a and 10b, bearing a bicyclic group as the
aryloxy moiety, were similarly prepared (Scheme 1).
Scheme 1. Reagents and conditions: (a) 3,5-diiodo-2-methoxybenzoic acid, EDC, HOBt, DMF, 40 °C, 60%-quant.; (b) BBr3, CH2Cl2, rt, 37%-88%
2
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Scheme 2. Reagents and conditions: (a) phenol, K2CO3, DMF, rt, 97%-quant.; (b) Fe powder, HCl, EtOH- H2O, reflux, 72%-94%.; (c) 3,5-diiodo-2-
methoxybenzoic acid, EDC, HOBt, DMF, 40 °C, 60%-quant.; (d) BBr3, CH2Cl2, rt, 37%-88%.
Scheme 3. Reagents and conditions: (a) 4-fluoronitrobenzene, K2CO3, DMF, rt, 97%; (b) Fe powder, HCl, EtOH-H2O, reflux, 72%; (c) method A: 3,5-dichloro-2-
methoxybenzoic acid, (COCl)2, DMF, CH2Cl2, rt, then 20, Et3N, THF, rt.; method B: 3,5-diiodo-2-methoxybenzoic acid, EDC, HOBt, DMF, 40 °C; (d) BBr3, CH2Cl2,
rt, 37%-88%.
Table 1. Biological activity of salicylanilide derivatives 7, 10, 22 and 23.
ELISA
mpkDCT
activity[b]
mpkDCT
Compound
R =
1-Br-naphth-2-yl
X
CC50/IC50
7.7
LogP[e]
7.49
IC50 (μM)
0.26[a]
CC50 (μM)
2
Cl
―
I
+++
2.0
7.9
Closantel (3)
―
0.77
2.6
+++
+
10
8.50
7.26
8.38
8.38
6.87
6.87
7.87
8.57
5.27
6.27
7a
Ph
n.t.[c]
n.t.[c]
3.3
9.8
13
n.d.[d]
n.d.[d]
3.7
7b
7c
2,4-Cl2−C6H3
3,5-Cl2−C6H3
2-OH−C6H4
4-OH−C6H4
7-OH-naphth-2-yl
tetrahydronaphth-2-yl
4-OH−C6H4
7-OH-naphth-2-yl
1.0
+
0.90
4.2
+
7d
7e
n.t.[c]
2.3
4.2
+
3.1
10a
10b
22
0.30
+++
-
8.3
3.2
7.2
7.3
28
1.6
2.0
Cl
5.6[a]
5.1[a]
-
1.3
23
+++
1.4
[a] Taken from our previous report.[22] [b] Activity toward mpkDCT cells is indicated as follows: +++, IC50 value between 1.6 μM and 3.2 μM; +, IC50 value between
6.3 μM and 13 μM; -, IC50 value larger than 25 μM or no significant activity. [c] Not tested, [d] Not determined. [e] Calculated with ChemDraw15.
Synthesis of compounds 14 bearing various substituents on
the central benzene ring is shown in Scheme 2. These
compounds were synthesized in a similar manner to that
described for the preparation of compounds 7. Namely,
condensation between 3,5-diiodo-2-methoxybenzoic acid and
anilines 12a-e prepared from 11a-e gave 13a-e, and the removal
of the O-methyl group afforded the designed compounds 14a-e,
respectively (Scheme 2). Compounds 20 and 21 are derivatives
of compound 10a without the chlorine atom, and were
synthesized similarly from 15 as a starting material (Scheme 3).
Synthesis of 3,5-dichlorosalicylanilide derivatives 22, 23 and
24a-d was described in our previous report.[22]
was incubated with GST-SPAK [T233E] in the presence of GST-
MO25a. NKCC2 phosphorylation was detected by anti-p-NKCC2
(pThr100/105) antibodies.[20] We further investigated the
inhibitory effect on NCC phosphorylation using a cell-based
assay with mouse renal distal tubule-derived (mpkDCT) cells,
which endogenously express NCC. We also examined
cytotoxicity with the same cell line.
First, we examined the structure-activity relationship of the
aryloxy group at the left terminal of compounds 2 (Table 1).
Compound 7a without any substituent on the phenoxy group
exhibited moderate inhibitory activity with an IC50 value of 2.6
μM in ELISA. Introduction of two chlorine atoms (7b and 7c)
enhanced the activity, but 7c exhibited higher cytotoxicity than 3
toward mpkDCT cells, resulting in a lower CC50/IC50 ratio than
that of 3. Though introduction of a hydroxyl group (7d and 7e)
reduced the cytotoxicity, the SPAK-inhibitory activity of 7d and
7e were less than the lead compounds, resulting in low
CC50/IC50 ratio. The corresponding 3,5-dichlorosalicylanilide
Biological activity
The SPAK-inhibitory activity of the synthesized compounds was
assessed by means of our previously developed ELISA. The
ELISA plate was coated with GST-NKCC2 [1–174] and the plate
3
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10.1002/cmdc.202100273
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derivative 22 bearing a 4-hydroxyphenyl group also exhibited a
low CC50/IC50 ratio. These results indicated that introduction of
the hydrophilic functionality reduces the cytotoxicity, but the
decrease of hydrophobicity is unfavorable for inhibitory activity.
On the basis of these considerations, we investigated the activity
and toxicity of hydroxynaphthyl derivative 10a, and found that it
exhibited potent SPAK-inhibitory activity in vitro (IC50; 0.30 μM),
as well as a significant inhibitory effect on NCC phosphorylation
in mpkDCT cells, and an improved CC50/IC50 ratio. The
corresponding 3,5-dichlorosalicylanilide derivative 23 also
exhibited inhibitory activity in mpkDCT cells, though its CC50/IC50
ratio was significantly smaller than that of 10a (Table 1).
we investigated the importance of the chlorine atom of the 7-
hydroxynaphthoxy derivative 10a, which exhibits potent activity
and a large CC50/IC50 value. Table 3 shows the biological activity
of the 7-hydroxynaphthoxy derivatives. In contrast to the
phenoxy derivatives shown in Table 2, compound 20 lacking the
chlorine atom was significantly less potent than 3-chloro
derivative 10a, and did not inhibit NCC phosphorylation in
mpkDCT cells. The corresponding 3,5-dichlorosalicylanilide
derivative 21 also showed no inhibitory activity in mpkDCT cells.
These results indicated that the 3-chloro substitution is essential
for the inhibitory activity of the 7-hydroxynaphthoxy derivatives in
mpkDCT cells, in contrast to the result for 14a.
Next, we investigated the structure-activity relationship of the
central benzene moiety (Table 2). Removal of the chlorine atom
of compound 7a, yielding compound 14a, increased the
inhibitory activity (IC50 1.7 μM in ELISA assay). Introduction of a
bromine (14c), trifluoromethyl (14d) or hydroxyl group (14e)
enhanced the inhibitory activity in ELISA assay, but these
compounds did not inhibit NCC phosphorylation in mpkDCT
cells. Similar results were observed with the 3,5-
dichlorosalicylanilide derivatives 24, namely, compounds
bearing a bromine (24b), trifluoromethyl (24c) or hydroxyl group
(24d) exhibited more potent activity than 3-chloro derivative 24a,
but did not inhibit NCC phosphorylation in mpkDCT cells.
We next examined NCC phosphorylation in mouse kidney
using closantel (3) as the positive control in order to establish
whether compound 10a is effective in vivo. As shown in Figure 2,
phosphorylation level of NCC were decreased in the kidneys at
30 minutes after intraperitoneal injection of 10a (20 mg/kg), and
had recovered by 100 minutes after administration. These
results suggest that 10a inhibited SPAK kinase in vivo, and the
inhibition was reversible. We previously reported that the SPAK-
inhibitory activity of closantel were short-acting and reversible.[20]
Thus, our findings indicate that 10a functions as a SPAK
inhibitor in the same manner as closantel, but has an improved
activity-toxicity profile.
Since compound 14a without a chlorine atom on the central
benzene ring showed higher potency than 3-chloro derivative 7a,
Table 2. Biological activity of 3,5-diiodosalicylanilide derivatives 14a-14e and 3,5-dichlorosalicylanilide derivatives 24a-24d.
ELISA
mpkDCT
activity[b]
mpkDCT
Compound
R =
X
CC50/IC50
LogP[e]
8.50
IC50 (μM)
CC50 (μM)
Closantel (3)
7a
―
―
0.77
+++
7.9
10
3-Cl
H
I
2.6
+
n.t.[c]
2.5
4.1
2.2
2.0
7.3
2.8
7.2
5.9
7.3
n.d.[d]
1.5
0.8
3.7
2.9
8.1
0.6
4.8
2.5
3.5
7.26
6.70
7.26
7.53
7.62
6.31
5.66
5.93
6.02
4.71
14a
1.7
+++
14b
2-Cl
3-Br
3-CF3
2-OH
3-Cl
3-Br
3-CF3
2-OH
5.4
+++
14c
0.60
0.70
0.90
4.5[a]
1.5[a]
2.4[a]
2.1[a]
-
14d
-
14e
-
24a
Cl
+++
24b
-
-
-
24c
24d
[a] Taken from our previous report.[22] [b] Activity toward mpkDCT cells is indicated as follows: +++, IC50 value between 1.6 μM and 3.2 μM; +, IC50 value between
6.3 μM and 13 μM; -, IC50 value larger than 25 μM or no significant activity. [c] Not tested, [d] Not determined. [e] Calculated with ChemDraw15.
4
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Table 3. Biological activity of 7-hydroxynaphthoxy derivatives 10a, 20, 21 and 23.
ELISA
mpkDCT
activity[b]
mpkDCT
Compound
R =
X
CC50/IC50
LogP[e]
IC50 (μM)
0.77
CC50 (μM)
Closantel (3)
―
Cl
H
―
+++
+++
-
7.9
8.3
7.6
6.3
7.3
10
28
8.50
7.87
7.31
5.71
6.27
10a
20
I
0.30
2.5
3.0
2.1
1.4
21
H
Cl
3.0
5.1[a]
-
23
Cl
+++
[a] Taken from our previous report.[22] [b] Activity toward mpkDCT cells is indicated as follows: +++, IC50 value between 1.6 μM and 3.2 μM; -, IC50 value larger
than 25 μM or no significant activity. [c] Not tested, [d] Not determined. [e] Calculated with ChemDraw15.
Keywords: antihypertensive drugs • hypertension • salicylanilide
• SPAK • WNK
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In this study, we investigated the structure-activity relationship
(SAR) and structural development of salicylanilide derivatives as
SPAK kinase inhibitors, focusing on hybrid structures designed
on the basis of lead compounds 2 and 3, with the aim of finding
a potent inhibitor of WNK-OSR1/SPAK-NCC signaling that
shows an improved toxicity profile. Specifically, we designed and
synthesized a series of 3,5-diiodosalicylanilide derivatives as
candidate SPAK inhibitors. Among them, we discovered
compound 10a as a potent SPAK inhibitor with an improved
activity-toxicity profile compared to the lead compounds.
Compound 10a reduced the phosphorylation level of NCC in
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inhibition, like closantel (3). Compound 10a appears to be a
promising candidate for a new class of antihypertensive drugs.
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10.1002/cmdc.202100273
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We developed novel salicylanilide derivative 10a as a potent SPAK inhibitor with an improved activity-toxicity profile. Compound 10a
reduced the phosphorylation level of NCC in mouse kidney in vivo. Compound 10a appears to be a promising candidate for a new
class of antihypertensive drugs.
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