Structural development of N-(4-phenoxyphenyl)benzamide derivatives as novel SPAK inhibitors blocking WNK kinase signaling

Article abstract of DOI:10.1016/j.bmcl.2020.127408

We report here structural development of N-(4-phenoxyphenyl)benzamide derivatives as novel SPAK (STE20/SPS1-related proline/alanine-rich kinase) inhibitors. Abnormal activation of the signal cascade of with-no-lysine kinase (WNK) with OSR1 (oxidative stress-responsive kinase 1)/SPAK and NCC (NaCl cotransporter) results in characteristic salt-sensitive hypertension, and therefore inhibitors of the WNK-OSR1/SPAK-NCC cascade are candidates for antihypertensive drugs. Based on the structure of lead compound 2, we examined the SAR of N-(4-phenoxyphenyl)benzamide derivatives, and developed compound 20l as a potent SPAK inhibitor. Compounds 20l is a promising candidate for a new class of antihypertensive drugs.

Full text of DOI:10.1016/j.bmcl.2020.127408

Journal Pre-proofs
Structural development of N-(4-phenoxyphenyl)benzamide derivatives as
novel SPAK inhibitors blocking WNK kinase signaling
Shinya Fujii, Eriko Kikuchi, Yuko Watanabe, Honoka Suzuyama, Mari
Ishigami-Yuasa, Takayasu Mori, Kiyoshi Isobe, Shinichi Uchida, Hiroyuki
Kagechika
PII:
S0960-894X(20)30519-9
DOI:
Reference:
https://doi.org/10.1016/j.bmcl.2020.127408
BMCL 127408
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
4 June 2020
7 July 2020
10 July 2020
Please cite this article as: Fujii, S., Kikuchi, E., Watanabe, Y., Suzuyama, H., Ishigami-Yuasa, M., Mori, T.,
Isobe, K., Uchida, S., Kagechika, H., Structural development of N-(4-phenoxyphenyl)benzamide derivatives as
novel SPAK inhibitors blocking WNK kinase signaling, Bioorganic & Medicinal Chemistry Letters (2020), doi:
https://doi.org/10.1016/j.bmcl.2020.127408
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Structural development of N-(4-phenoxyphenyl)benzamide derivatives as novel SPAK
inhibitors blocking WNK kinase signaling
Shinya Fujii,a Eriko Kikuchi,b Yuko Watanabe,a Honoka Suzuyama,a Mari Ishigami-Yuasa,a Takayasu
Mori,b Kiyoshi Isobe,b Shinichi Uchida,b Hiroyuki Kagechikaa, *
a
Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University (TMDU), 2-3-10
Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan
b 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
Abstract: We report here structural development of N-(4-phenoxyphenyl)benzamide derivatives as novel
SPAK (STE20/SPS1-related proline/alanine-rich kinase) inhibitors. Abnormal activation of the signal
cascade of with-no-lysine kinase (WNK) with OSR1 (oxidative stress-responsive kinase 1)/SPAK and
NCC (NaCl cotransporter) results in characteristic salt-sensitive hypertension, and therefore inhibitors of
the WNK-OSR1/SPAK-NCC cascade are candidates for antihypertensive drugs. Based on the structure of
lead compound 2, we examined the SAR of N-(4-phenoxyphenyl)benzamide derivatives, and developed
compound 20l as a potent SPAK inhibitor. Compounds 20l is a promising candidate for a new class of
antihypertensive drugs.
Keywords:
Antihypertensive drugs
SPAK
WNK kinase signaling
N-(4-Phenoxyphenyl)benzamide
Recent studies on monogenic hypertensive diseases such as Liddle’s syndrome and
pseudohypoaldosteronism type II (PHAII; also known as Gordon’s disease or familial hyperkalemic
hypertension)1,2 have provided new insights into the physiological mechanisms of blood pressure
regulation. Among these conditions, PHAII is an autosomal dominant disease characterized by
hypertension, severe hyperkalemia, and mild metabolic acidosis.3 The understanding of PHAII was greatly
advanced by the identification of causal mutations in the genes encoding with-no-lysine kinase (WNK) 1
and WNK4 in some PHAII patients.4

We previously generated a PHAII model mice bearing an aspartic acid to alanine mutation (D561A) in
WNK4, which is one of the causative mutations found in PHAII patients, and we established that the
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 The increased phosphorylation and activation of NCC induce excessive NaCl
reabsorption in the kidney and causes salt-sensitive hypertension. WNK signaling is positively regulated by
aldosterone, angiotensin II, and insulin, which may contribute to hypertension in patients with
hyperaldosteronism and hyperinsulinemia.7–11 Recent studies also support the notion that
WNK-OSR1/SPAK-NKCC1 phosphorylation and activation in vascular smooth muscle cells can play a
pivotal role in the regulation of vascular tonus.12 Therefore, compounds that inhibit this signal cascade are
expected to reduce hypertension through dual actions (i.e., NaCl diuresis and vasodilation), and may be
particularly effective to treat patients with hyperaldosteronism or hyperinsulinemia.
Based on these considerations, we have focused on the development of WNK-OSR1/SPAK-NCC signal
cascade inhibitors.13 We recently developed inhibitors of WNK binding to OSR1/SPAK, such as 1, as
candidate antihypertensive agents (Figure 1).14 In the present study, we focused on SPAK kinase, because
SPAK knockout in mice was not fatal, and the knockout mice displayed hypotension with low levels of
NCC and NKCC1 phosphorylation in the kidney and aorta, respectively. In addition to hypertension, recent
studies have revealed that SPAK has therapeutic potential for treating multiple diseases including
neurological diseases,15 inflammatory colitis, and cystic fibrosis.16
NH2
O
O
N
N
H
NC
1
Cl
HO
H
N
Cl
O
O
Br
Cl
2
Figure 1. Structures of our developed WNK-OSR1/SPAK binding inhibitor (1) and the hit compound (2)
discovered during SPAK inhibitor screening.
We previously developed a screening system for SPAK kinase inhibitors using ELISA, and identified
aryloxybenzanilide derivative 2 as a hit.17 Compound 2 also lowered the phosphorylation level of NCC in

vivo, and was considered a promising lead compound for further structural development. Compound 2
consists of three parts, namely, the left aryloxy moiety, the central benzene moiety, and the right benzoyl
moiety. Recently, based on the structure of our hit compound 2, Zhang et al. reported the development of a
SPAK inhibitor ZT-1a for brain disorders .18 Here, we investigated the structure-activity relationship (SAR)
of each part of the N-(4-aryloxyphenyl)benzamide scaffold.
The synthetic routes are summarized in Schemes 1, 2 and 3. Scheme 1 shows the synthesis of
compounds bearing various aryloxy moieties. Aromatic nucleophilic substitution (SNAr) of compound 3
and various phenols afforded the corresponding diphenyl ethers 4a-4j, and hydrogenation of the nitro group
of 4a-4j gave amines 5a-5j, respectively. Amide condensation between amines 5a-5j and
3,5-dichloro-2-methoxybenzoic acid afforded compounds 6a-6j, and then removal of the methyl group
afforded the desired compounds 7a-7j, respectively. Compounds 11a and 11b bearing a bicyclic group as
the aryloxy moiety were prepared similarly (Scheme 1).
Cl
Cl
MeO
H
N
HO
NO2
R
H
3
a
c
d
R1
N
4
2
Cl
Cl
R1
R1
F
O
O
Cl
O
5
O
O
Cl
Cl
3
Cl
4a-j
5a-j
(R = NO2)
(R = NH2)
6a-j
7a-j
b
7a (R1 = H)
7f
7g
(R1 = 3-CF3)
(R1 = 2-OH)
(R1 = 3-OH)
(R1 = 4-OH)
7b (R1 = 4-Cl)
7c (R1 = 2,4-Cl2) 7h
7d (R1 = 3,5-Cl2) 7i
7e (R1 = 2-CF3)
(R1 = 3,5-(OH)2)
7j
e
Cl
Cl
MeO
HO
R
H
N
H
11a
11b
Ar=
HO
d
c
N
Cl
Cl
Ar
O
Ar
O
Ar
O
Ar=
O
O
Cl
Cl
Cl
8a,b
9a,b
(R = NO2)
(R = NH2)
b
10a,b
11a,b
Scheme 1. Reagents and conditions: (a) R1-Ph-OH, K2CO3, DMF, rt, 80%-quant.; (b) Fe powder, HCl,
EtOH- H2O, reflux, 70%-quant.; (c) 3,5-dichloro-2-methoxybenzoic acid, (COCl)2, DMF, CH2Cl2, rt, then
5a-5j, Et3N, THF, rt, 60%-quant.; (d) BBr3, CH2Cl2, rt, 37%-88%, (e) ArOH, K2CO3, DMF, rt, 98%-quant.
Compounds modified at the central benzene moiety were synthesized in the same way as 7a-7j, namely,
by construction of diaryl ethers via SNAr reaction to afford compounds 13a-13g, followed by
hydrogenation of the nitro group to afford compounds 14a-14g, amide condensation, and removal of the
methyl group to give the desired compounds 16a-16g, respectively (Scheme 2).

Cl
Cl
HO
MeO
NO2
R
H
H
N
d
a
c
N
5
Cl
Cl
F
O
O
R2
12a-g
O
R2
2
O
O
R2
16a-g
16e
R2
3
13a-g
14a-g
(R = NO2)
(R = NH2)
15a-g
b
16a (R2 = H)
(R2 = 3-Me)
(R2 = 3-CF3)
(R2 = 2-OH)
16b (R2 = 2-Cl)
16f
16c (R2 = 3,5-Cl2) 16g
16d (R2 = 3-Br)
Scheme 2. Reagents and conditions: (a) phenol, K2CO3, DMF, rt, 97%-quant.; (b) Fe powder, HCl, EtOH-
H2O, reflux, 72%-94%.; (c) 3,5-dichloro-2-methoxybenzoic acid, (COCl)2, DMF, CH2Cl2, rt, then 14a-14g,
Et3N, THF, rt, 86%-96%; (d) BBr3, CH2Cl2, rt, 37%-88%.
Scheme 3 shows the synthesis of compounds with various benzoyl moieties. SNAr reaction between
compounds 3 and 17 gave diaryl ether 18, and then hydrogenation afforded amine 19. Amide condensation
using various carboxylic acids gave the desired compounds 20a-20k and 21. Dihydroxy derivatives 20l-20o
were prepared by removal of the methyl groups of the dimethoxy derivatives 20h-k, respectively.
R3
4
5
3
2
H
R
N
a
c
6
O
O
OH
O
Br
Cl
Br
17
Br
Cl
18
19
(R = NO2)
(R = NH2)
20a-k
b
(R3 = H)
(R3 = 2-OH-4-CF3)
(R3 = 2-OH-5-NO2)
(R3 = 3-OH-4-Me)
(R3 = 2,3-(OMe)2)
(R3 = 3,4-(OMe)2)
(R3 = 3,5-(OMe)2)
(R3 = 2,6-(OMe)2)
20a
20b
20c
20d
20e
20f
20g
20h
20i
20j
20k
(R3 = 2-OH)
(R3 = 3-OH)
(R3 = 4-OH)
e
HO
N
H
(R3 = 2,3-(OH)2)
(R3 = 3,4-(OH)2)
(R3 = 3,5-(OH)2)
(R3 = 2,6-(OH)2)
20l
N
d
20m
20n
20o
20h-k
20l-o
O
O
Br
Cl
21
Scheme 3. Reagents and conditions: (a) 3, K2CO3, DMF, rt, 71%; (b) Fe powder, HCl, EtOH- H2O, reflux,
94%; (c) Method A: Ar-COOH, EDC, HOBt, DMF, 40˚C, 19-99%; Method B: Ar-COOH, (COCl)2, DMF,
CH2Cl2, rt, then 19, Et3N, THF, rt, 7-95%; Method C: Ar-COOH, PCl3, DMAP, toluene, 100˚C, 24-29%
(d) BBr3, CH2Cl2, rt, 37-88%, (e) 2-OH-Py-3-COOH, EDC, HOBt, DMF, 40˚C, 25%
The SPAK-inhibitory activity of the synthesized compounds was assessed by means of ELISA. Table 1
summarizes the SAR of the left aryloxy moiety. Non-substituted phenyl derivative 7a showed inhibitory
activity with an IC50 value of 4.5 μM, and the introduction of two chlorine atoms (7c, 7d) or one
trifluoromethyl group (7e, 7f) at the phenyl group increased the potency. Among the synthesized phenoxy
derivatives 7a-7j, dihydroxyphenyl derivative 7k exhibited the most potent activity with an IC50 value of

0.65 μM. Naphthyl derivative 11a bearing a 7-hydroxy group instead of the 1-bromo group of compound 2
exhibited lower activity than 2. Tetrahydronaphthyl derivative 11b also exhibited only moderate potency.
Although clear relationship between the substituent and activity was not observed, increase of
hydrophobicity in this moiety increased the inhibitory activity. Among the tested aryloxy groups, the
1-bromonaphth-2-yl group of compound 2 was the most promising substituent (Table 1).
Table 1. SPAK-inhibitory activity of synthesized compounds 7a-7j, 11a and 11b.
Compound Ar =
IC50 (μM)
0.26
4.5
2
1-Br-naphth-2-yl
7a
7b
7c
7d
7e
7f
Ph
4-Cl−C6H4
4.9
2,4-Cl2−C6H4
3,5-Cl2−C6H4
2-CF3−C6H4
3-CF3−C6H4
2-OH−C6H4
3-OH−C6H4
4-OH−C6H4
3,5-(OH)2−C6H3
7-OH-naphth-2-yl
1.4
2.7
1.1
1.0
7g
7h
7i
2.0
3.5
5.6
7j
0.65
5.1
11a
11b
tetrahydronaphth-2-yl 2.8
Next, the SAR of the central benzene moiety was investigated. Compound 16a without a substituent at
the central benzene ring exhibited an IC50 value of 5.7 μM. Compounds bearing chlorine (7a, 16b, and
16c), bromine (16d), a trifluoromethyl group (16f) and a hydroxyl group (16g) were more potent than 16a,
whereas compound 16e bearing a methyl group exhibited low activity. The results suggested that
electron-withdrawing character but not hydrophobicity is favorable for this moiety, and that the
introduction of a chlorine atom at the central benzene ring is also favorable (Table 2).

Table 2. SPAK-inhibitory activity of synthesized compounds 16a-16g.
Cl
HO
R
H
N
5
Cl
O
2
O
3
Compounds
2a
R
–
IC50 (μM)
0.26
4.5
7a
3-Cl
H
16a
5.7
16b
16c
2-Cl
1.5
3,5-Cl2
3-Br
1.8
16d
16e
1.5
3-Me
3-CF3
2-OH
17
16f
2.4
16g
2.1
aThe structure is shown in Figure 1.
Finally, the SAR of the right benzoyl moiety was examined (Table 3). Non-substituted benzoyl
derivative 20a exhibited no SPAK-inhibitory activity. Among the hydroxybenzoyl derivatives, 2-hydroxy
(20b) and 3-hydroxy (20c) derivatives did not exhibit activity, while 4-hydroxy derivative 20d showed an
IC50 value of 2.1 μM. Although 20b showed no activity, compounds bearing a 2-hydroxy group, such as
20e, 20f, 20l, 20o, as well as the lead compound 2 exhibited significant activity. These results indicate that
the combination of a 2-hydroxy group and another substituent is important for SPAK-inhibitory activity.
Dihydroxybenzoyl derivatives 20l and 20o exhibited significant activity, whereas the corresponding
dimethoxybenzoyl derivatives showed no activity. 2-Hydroxynicotinoyl derivative 21 was also inactive.
Among the tested compounds, 2,3-dihydroxybenzoyl derivative 20l exhibited the most potent activity with
an IC50 value of 0.050 μM (Table 3). Substituents on the benzoyl moiety affected the inhibitory activity
more powerfully than other substituents, and therefore the 2-hydroxybenzoyl substructure could be the
pharmacophore of these SPAK inhibitors. The results implied the potential hydrogen bonding between
SPAK and the compound at 2- and 3-positions. Although the binding site of the inhibitors on the protein
has not been identified, these SAR findings could provide a design rationale for development of novel and
potent SPAK inhibitors.

Table 3. SPAK-inhibitory activity of synthesized compounds 20a-20o and 21.
H
N
Ar
O
O
Br
Cl
Compounds Ar
IC50 (μM)
0,26
>50
>50
>50
2.1
2
2-OH-3,5-Cl2-C6H2
Ph
20a
20b
20c
20d
20e
20f
20g
20h
20i
20j
20k
20l
20m
20n
20o
21
2-OH−C6H4
3-OH−C6H4
4-OH−C6H4
2-OH-4-CF3−C6H3
2-OH-5-NO2−C6H3
3-OH-4-Me−C6H3
2,3-(OMe)2−C6H3
3,4-(OMe)2−C6H3
3,5-(OMe)2−C6H3
2,6-(OMe)2−C6H3
2,3-(OH)2−C6H3
3,4-(OH)2−C6H3
3,5-(OH)2−C6H3
2,6-(OH)2−C6H3
2-OH-pyridin-3-yl
2.3
0.11
4.4
>50
>50
>50
3.2
0.050
>50
>50
0.24
>50
In conclusion, based on the structure of lead compound 2, we examined the SAR of
N-(4-phenoxyphenyl)benzamide derivatives for inhibition of SPAK. The results suggested that
2-hydroxybenzoyl substructure is a key pharmacophore, and we developed compound 20l as a potent
SPAK inhibitor. The SAR information obtained in this study could provide a design rationale for the
development of potent SPAK inhibitors as candidate antihypertensive agents. Further structural
development including the combination of substituents and biological investigations such as in vivo PK/PD
studies are in progress.
Acknowledgments
This work was partly supported by JSPS Core-to-Core Program, A. Advanced Research Networks, and
Japan Agency for Medical Research and Development (AMED) under Grant Number JP20am0101098
(Platform Project for Supporting Drug Discovery and Life Science Research, BINDS).

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Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may
be considered as potential competing interests:
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Structural development of N-(4-phenoxyphenyl)-
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Shinya Fujii, Eriko Kikuchi, Yuko Watanabe, Honoka Suzuyama, Mari Ishigami-Yuasa, Takayasu Mori,
Kiyoshi Isobe, Shinichi Uchida, Hiroyuki Kagechika,

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