Development of WNK signaling inhibitors as a new class of antihypertensive drugs

Article abstract of DOI:10.1016/j.bmc.2017.05.034

Pseudohypoaldosteronism type II (PHAII) is characterized by hyperkalemia and hypertension despite a normal glomerular filtration rate. Abnormal activation of the signal cascade of with-no-lysine kinase (WNK) with OSR1 (oxidative stress-responsive kinase 1)/SPAK (STE20/SPS1-related proline/alanine-rich kinase) and NCC (NaCl cotransporter) results in characteristic salt-sensitive hypertension. Thus, inhibitors of the WNK-OSR1/SPAK-NCC cascade are candidates for a new class of antihypertensive drugs. In this study, we developed novel inhibitors of this signal cascade from the 9-aminoacridine lead compound 1, one of the hit compounds obtained by screening our chemical library for WNK-SPAK binding inhibitors. Among the synthesized acridine derivatives, several acridine-3-amide and 3-urea derivatives, such as 10 (IC₅₀: 6.9?μM), 13 (IC₅₀: 2.6?μM), and 20 (IC₅₀: 4.8?μM), showed more potent inhibitory activity than the lead compound 1 (IC₅₀: 15.4?μM). Compounds 10 and 20 were confirmed to inhibit phosphorylation of NCC in vivo.

Full text of DOI:10.1016/j.bmc.2017.05.034

Accepted Manuscript
Development of WNK Signaling Inhibitors as a New Class of Antihypertensive
Drugs
Mari Ishigami-Yuasa, Yuko Watanabe, Takayasu Mori, Hiroyuki Masuno,
Shinya Fujii, Eriko Kikuchi, Shinichi Uchida, Hiroyuki Kagechika
PII:
S0968-0896(17)30767-8
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.05.034
BMC 13752
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
7 April 2017
15 May 2017
16 May 2017
Please cite this article as: Ishigami-Yuasa, M., Watanabe, Y., Mori, T., Masuno, H., Fujii, S., Kikuchi, E., Uchida,
S., Kagechika, H., Development of WNK Signaling Inhibitors as a New Class of Antihypertensive Drugs,
Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.05.034
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Development of WNK Signaling Inhibitors as a New Class of Antihypertensive Drugs
Mari Ishigami-Yuasa,a Yuko Watanabe,a Takayasu Mori,b Hiroyuki Masuno,a Shinya Fujii,a Eriko
Kikuchi,b Shinichi Uchida,b Hiroyuki Kagechikaa,*
aInstitute of Biomaterials and Bioengineering, Tokyo Medical and Dental University (TMDU),
b
2-3-10 Kanda-Surugadai, Chiyodaku, Tokyo 101-0062, Japan, and Department of Nephrology,
Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU),
1-5-45 Yushima, Bunkyo, Tokyo 113-8519, Japan
*Corresponding author:
Hiroyuki Kagechika
E-mail: kage.chem@tmd.ac.jp; TEL/FAX: +81-3-5280-8032/8127
Abstract
Pseudohypoaldosteronism type II (PHAII) is characterized by hyperkalemia and hypertension despite
a normal glomerular filtration rate. Abnormal activation of the signal cascade of with-no-lysine
kinase (WNK) with OSR1 (oxidative stress-responsive kinase 1)/SPAK (STE20/SPS1-related
proline/alanine-rich kinase) and NCC (NaCl cotransporter) results in characteristic salt-sensitive
hypertension. Thus, inhibitors of the WNK-OSR1/SPAK-NCC cascade are candidates for a new class
of antihypertensive drugs. In this study, we developed novel inhibitors of this signal cascade from the
9-aminoacridine lead compound 1, one of the hit compounds obtained by screening our chemical
library for WNK-SPAK binding inhibitors. Among the synthesized acridine derivatives, several
acridine-3-amide and 3-urea derivatives, such as 10 (IC50: 6.9 µM), 13 (IC50: 2.6 µM), and 20 (IC50:
4.8 µM), showed more potent inhibitory activity than the lead compound 1 (IC50: 15.4 µM).
Compounds 10 and 20 were confirmed to inhibit phosphorylation of NCC in vivo.
Keywords: antihypertensive drug, with-no-lysine kinase (WNK), acridine
1

1. Introduction
Hypertension is one of the most common diseases, affecting more than one billion people
worldwide. Most are categorized as essential hypertension, which means that the origin of the
disease is unknown. Increased blood pressure induces a remarkable increase in the risk of fatal
illnesses such as heart attack, aneurysm, and stroke owing to the increased pressure on the arterial
wall, which accelerates atherosclerosis. On the other hand, the renin-angiotensin-aldosterone system
(RAAS) is a hormone system that regulates blood pressure and fluid balance. Under normal
conditions, aldosterone production is regulated by the renin-angiotensin system and potassium
balance. Renin is released from the renal juxtaglomerular apparatus (JGA) and its production is
regulated by three major mechanisms: intrarenal baroreceptors of the afferent arterioles, solute
delivery to the macula densa cells, and the effect of sympathetic nerves on the arterioles of the JGA.1
Pseudohypoaldosteronism type II (PHAII; also known as Gordon’s disease or familial
hyperkalemic hypertension) was first reported in 1964.2 Patients with PHAII exhibit hypertension,
severe hyperkalemia, and mild metabolic acidosis. This phenotype is Cl−-dependent and is corrected
with low doses of thiazide-type diuretics.3,4 Subsequent studies showed that PHAII is transmitted in
an autosomal-dominant manner.5 The understanding of PHAII was greatly advanced with the
identification of causal mutations in the WNK1 and WNK4 genes in some patients with PHAII.6 With
no lysine (WNK) kinases are so named because of the lack of lysine in the ATP-binding cassette of
the catalytic region. Specifically, patients with WNK1 mutations have large intronic deletions that
increase the abundance of WNK1, and patients with WNK4 mutations have missense mutations that
localize to a short region near putative coiled-coil domains in a highly conserved sequence of WNK.6
The role of WNK proteins in the regulation of transporters and channels has been studied. The
WNK-oxidative stress-responsive 1 (OSR1) and STE20/SPS1-related proline-alanine-rich protein
kinase (SPAK)-NaCl cotransporter (NCC) signal cascade is constitutively activated in PHAII.7,8
Increased phosphorylation and activation of NCC cause excessive sodium reabsorption in the distal
convoluted tubules in the kidneys, resulting in salt-sensitive hypertension. Previously, the Na/K/Cl
cotransporter 1 (NKCC1) phosphorylation cascade in vascular smooth muscle cells was also found to
be an important regulator of vascular tonus.9,10 Furthermore, WNK signaling is positively controlled
by aldosterone, angiotensin II, and insulin, all of which may contribute to hypertension in patients
with hyperaldosteronism and hyperinsulinemia.11-15 Therefore, drugs that inhibit this signal cascade
are expected to help treat hypertension through dual actions (i.e., NaCl diuresis and vasodilation) and
may be particularly effective in patients with hyperaldosteronism or hyperinsulinemia.
Both OSR1 and SPAK possess a CCT domain in their C-terminus, which forms a protein fold
interacting with the Arg-Phe-Xaa-Val/Ile (RFxV/I) motif through a surface-exposed groove. WNK
kinases and SLC12A transporters (NCC/NKCCs) harbor the RFxV/I motif and directly interact with
OSR1/SPAK via the CCT domain. WNK signaling activity is known to be dependent on the
binding,16 suggesting that inhibitors of WNK binding to OSR1/SPAK would act as WNK signaling
inhibitors. Based on this principle, we previously developed a new high-throughput screening system
2

that can detect the binding by means of fluorescence correlation spectroscopy (FCS), and identified
several hit compounds among a 17,000-compound library.17 Among them, compound 1 showed the
highest inhibitory activity in the FCS assay (Figure 1). On the other hand, compound 2 bearing a
9-aminoacridine skeleton did not show inhibitory activity, which suggested that an appropriate
substituent at the 3-position of the acridine ring is necessary for inhibitory activity. Compound 1
effectively inhibited phosphorylation of SPAK and NCC in mpkDCT and that of SPAK and NKCC1
in MOVAS cells, which indicates that it can inhibit the WNK signal cascade in vivo. In this study,
therefore, we synthesized several acridine derivatives based on 1 as a lead compound in order to
clarify the structure-activity relationships of WNK-SPAK-binding inhibitors and to develop specific
inhibitors with high potency in vivo.
Figure 1. Structures of WNK-SPAK-binding inhibitor hit (1) and its derivative (2).
2. Results and Discussion
2.1. Synthesis of acridine derivatives
Our previous study showed that the 3-substituent of compound 1 is significant, as the
corresponding 3-acetamide derivative 2 did not inhibit WNK-SPAK-binding. In order to clarify the
structural requirements of the 3-substituent for WNK-SPAK-binding inhibition, we designed
compounds 3 – 5 with a different aromatic group. Further, we considered that the methyleneamino
linker at 3-position would be not stable both chemically and metabolically, we designed compounds
6 – 16 with the stable amide bond as the linking group (Scheme 1). Further, several derivatives such
as compounds 17 and 18 without the 9-amino group and 3-urea derivatives 19 – 21 with different
7-substituents were synthesized (Scheme 2). Compounds 1 and 2 were also synthesized in order to
confirm their structure and activity.
Syntheses of acridine derivatives are illustrated in Schemes 1 and 2. Ullmann reaction of
4-chloro-2-nitrobenzoic acid (22) with p-alkoxyanilines afforded N-phenyl-anthranilic acid
derivatives 23.18 Cyclization of 23 was carried out using phosphorus oxychloride to give
9-chloroacridine derivatives 24,19 which were converted to 9-aminoacridine derivatives 25 by
heating to 200°C with ammonium sulfate.20 Catalytic hydrogenation of the 3-nitro group of 25 in
DMF afforded key intermediates 26. Reductive amination of 26a with several arylaldehydes afforded
the 3-arylmethylaminoasridine derivatives 1 – 4. Acylation of 26a with various aromatic acid
chlorides afforded amide derivatives 6 – 16.
3

Scheme 1. Synthesis of acridine derivertives 1-16. Reagents and conditions: (a) p-ethoxyaniline,
K2CO3, Cu2O, Cu powder, 2-ethoxyethanol, reflux; (b) POCl3, reflux; (c) (NH4) 2SO4, phenol, reflux;
(d) H2, Pd/C, DMF; (e) AlCl3, xylene, reflux; (f) ArCHO, AcOH, NaCNBH3, MeOH for 26a; (g)
RCOCl, pyridine, DMF for 26a.
Phenol was an effective solvent for catalytic hydrogenation of 25. Treatment of
9-chloro-7-ethoxy-3-nitoroacridine (24a) under the same reaction conditions afforded
9-unsubstituted compound 27 (5%) and 9-phenoxy compound 28 (85%). Compounds 27 and 28 were
reacted with p-cyanobenzoyl chloride to give 17 and 18, respectively. Derivatives 19 and 20 bearing
a urea linkage at the 3-position of the acridine ring were obtained by reaction of 26a or 26b with
p-cyanophenyl isocyanate, respectively. Finally, 7-hydroxy derivative 21 was prepared from 26c,
which was synthesized from 26a by treatment with aluminum chloride in dry xylene at 100°C.
4

Scheme 2. Synthesis of acridine derivertives 17 - 21. Reagents and conditions: (a) H2, Pd/C, phenol;
(b) p-cyanobenzoyl chloride, pyridine, DMF; (c) p-cyanophenyl isocyanate, DMSO.
2.2. Biological activity
The inhibitory activity of the synthesized compounds on WNK-OSR1/SPAK binding was
evaluated using the FCS assay that we reported previously (Table 1).17 The lead compound 1
exhibited moderate inhibitory activity with an IC50 value of 15.4 µM, while compound 2 did not
show inhibitory activity, as reported previously.17 Among 3-arylmethylamino derivatives, compound
1 was most potent inhibitor under our assay conditions. Since the arylmethylamino group on the
acridine ring is expected to be rather unstable chemically and metabolically, we examined the
activity of 3-amide derivatives. Most of the 3-amide derivatives inhibited WNK-OSR1/SPAK
binding. This result indicates that the absence of inhibitory activity in compound 2 is due to the lack
of the aromatic ring at the amide carbonyl group. Compound 6 with the same aromatic ring
(2-furanyl moiety) as lead compound 1 showed more potent activity than 1 (its IC50 was 8.2 µM).
Replacement of the furan ring with a phenyl ring, yielding compound 7, decreased the activity, while
introduction of a 4-cyano group on the phenyl ring increased the inhibitory activity. Among the
4-substituted benzamide series 8 – 12, only the cyano group was effective, though a nitro group
slightly increased the potency. On the other hand, all 3-monosubstituted benzamide derivatives 13
and 14 showed potent inhibitory activity with IC50 values of 2.6, 3.9 and 11.8 µM, respectively.
Interestingly, 3,5-disubstituted benzamide derivative 16 showed inhibitory activity only at higher
concentrations. Replacement of the 9-amino group of 10 with a hydrogen atom or phenoxy group
resulted in loss of activity, indicating that the 9-amino group is important for the activity. Since the
amide linkage in the 3-substituent was effective, we examined derivatives with a 3-ureido moiety.
Compound 19 with a 4-cyanophenyl group on the ureido bond was insoluble under the assay
conditions, so we synthesized compounds 20 and 21 bearing a 7-methoxy or 7-hydroxy group,
5

respectively. These urea derivatives also showed potent inhibitory activity, with IC50 values of 4.8
and 2.6 µM, respectively.
Table 1. Inhibitory activity of acridine derivatives on WNK-OSR1/SPAK-binding
Compound
3-substituent (X-NH-)
7-substituent
9-substituent
(Y)
IC50 (µM)
(R)
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Me
H
1
2
2-furanylmethylamino
acetamido
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
H
15.4
Inactivea
53.5
3
2-thiophenylmethylamino
benzylamino
4
35.6
5
2,4-dimethoxyphenylamino
2-furanylcarboxamido
benzamido
21.1
6
8.2
7
28.2
8
4-chlorobenzamido
49
9
4-fluorobenzamido
36.9
10
11
12
13
14
15
16
17
18
19
20
21
4-cyanobenzamido
6.9
Inactivea
4-methoxycarbonylbenzamido
4-nitrobenzamido
20.9
3-cyanobenzamido
2.6
3-nitrobenzamido
3.9
2-naphthylcarboxamido
3,5-bis(trifluoromethyl)benzamido
3-cyanobenzamido
7.6
>100
Inactivea
Inactivea
NDb
3-cyanobenzamido
PhO
NH2
NH2
NH2
N’-(3-cyanophenyl)ureido
N’-(3-cyanophenyl)ureido
N’-(3-cyanophenyl)ureido
4.8
2.6
aInactive: inactive above the concentration of 10-5 M
bND: not determined.
We next examined the cytotoxicity of the selected compounds towards the human embryonic
kidney cell line, 293T. The IC50 values of the lead compounds 1 and 10 were 4.2 µM and 25 µM,
respectively. Mitomycin C was used as a positive control, and its IC50 value was in accordance with
previously reported values. Compound 20 did not inhibit the growth of 293T even at 100 µM.
Compounds 10 and 20 showed significantly lower cytotoxicity than lead compound 1 (Figure 2),
indicating that these may be good candidates for the development of specific WNK-SPAK-binding
6

inhibitors without side effects.
Figure 2. Evaluation of cytotoxicity toward 293-T cells of selected compounds 1 (square), 10
(triangle), 20 (open circle), and the positive control, mitomycin C (diamond and dashed line). All
fitted curves were generated by non-linear regression. Bars represent the standard deviation of the
mean.
Then, we selected compounds 10 and 20, taking into account both inhibitory activity and
solubility, for examination of the inhibitory activity toward phosphorylation in the WNK signal
cascade in vivo. Test compounds were intraperitoneally injected into C57BL/6 mice. Ninety minutes
after injection of either compound, the level of phosphorylated NCC was markedly decreased in a
dose-dependent manner. In addition, phosphorylation is inhibited similarly for NKCC1 / NKCC2
belonging to the same SLC12A3 family and being regulated by the WNK-OSR1 / SPAK signaling.
This suggests that these compounds indeed show inhibitory activity in vivo as well as in vitro.
Interestingly, phosphorylation of NCC tends to be inhibited at a lower concentration than NKCC1/2.
There are differences in the binding motifs to CCT domain (OSR1 / SPAK) among the SLC12A
transporters. NKCC 1/2 possesses RFQV (Arg-Phe-Gln-Val) motif, but NCC has RFTI (Arg-Phe-Thr
-Ile) motif. It has been confirmed that the affinity of the RFTI motif to the CCT domain is about 1/10
as compared with that of the RFQV,17 and thus it is reasonable to be inhibited at a lower
concentration. Taken together, this inhibitory effects on the phosphorylation is not due to toxic or
non-specific ones, but supports the fact that phosphorylation of SLC12A transporters is actually
inhibited via the binding inhibition.
7

Figure 3. Effect of 10 and 20 on phosphorylation in the WNK signal pathway in vivo.
3. Conclusion
In this study, we carried out structural development of our hit compound 1 previously identified
from a chemical library by high-throughput screening using fluorescence correlation spectroscopy
(FCS), in order to find novel inhibitors of WNK-OSR1/SPAK binding. Several acridine-3-amide and
3-urea derivatives, such as 10, 13, and 20, showed more potent inhibitory activity than the lead
compound 1. Although many of the acridine derivatives showed low solubility in various solvents,
we found that compounds 10 and 20 markedly decreased the level of phosphorylated NCC in vivo in
a dose-dependent manner. NCC plays a critical role in renal NaCl transport and blood pressure
maintenance. Increased phosphorylation and activation of NCC causes excessive sodium
reabsorption in the distal convoluted tubules in the kidney, which contributes to salt-sensitive
hypertension. Thus, compounds 10 and 20 are promising candidates for a new class of
antihypertensive drugs. Further studies on the structural optimization, specificity as WNK signaling
inhibitor, and in vivo activities of these acridine derivatives are on-going.
8

4. Experimental
4.1. Chemistry
All reagents were purchased from Sigma–Aldrich Chemical Co., Tokyo Kasei Kogyo Co., Wako
1
Pure Chemical Industries, and Kanto Chemical Co., Inc. H and 13C NMR spectra were recorded on a
Bruker AVANCE 400 spectrometer or a Bruker AVANCE 500 spectrometer. Mass spectra were
obtained with a Daltonics microTOF-2focus. Column chromatography was done using silica gel 60
spherical or silica gel 60 N spherical (Kanto Chemical Co., Inc.).
4.2. Synthesis
4.2.1. Synthesis of 2-[(4-ethoxyphenyl)amino]-4-nitrobenzoic acid (23a)
Potassium carbonate (2.06 g, 14.9 mmol), copper (I) oxide (85.0 mg, 0.596 mmol), and copper
powder (83.0 mg, 1.31 mmol) were added to a solution of 2-chloro-4-nitrobenzoic acid (22, 3.00 g,
14.9 mmol) and 4-ethoxyaniline (2.29 ml, 15.6 mmol) in 2-ethoxyethanol under an Ar atmosphere.
The mixture was heated at reflux overnight, then cooled to room temperature, and 1 M hydrochloric
acid was added dropwise to the mixture. The resulting precipitate was collected, washed with 1 M
hydrochloric acid and water, and dried in vacuo to yield 23a (2.80 g, 62%) as an orange powder.
1H-NMR (500 MHz, CDCl3) δ 9.37 (1 H, s), 8.13 (1 H, d, J = 8.8 Hz), 7.70 (1 H, d, J = 2.2 Hz), 7.40
(1 H, dd, J = 2.2, 8.8 Hz), 7.17 (2 H, m), 6.96 (2 H, m), 4.07 (2 H, q, J = 7.0 Hz), 1.45 (3 H, t, J = 7.0
Hz).
2-[(4-Methoxyphenyl)amino]-4-nitrobenzoic acid (23b) was prepared similarly from 22 and
4-methoxyaniline (78%). 1H-NMR (500 MHz, CD3OD) δ 8.14 (1 H, d, J = 8.7 Hz), 7.66 (1 H, d, J =
2.2 Hz), 7.41 (1 H, d, J = 2.3, 8.7 Hz), 7.23-7.21 (2 H, m), 7.04-7.01 (2 H, m), 3.84 (3 H, s).
4.2.2. Synthesis of 9-chloro-2-ethoxy-6-nitroacridine (24a)
23a (2.00 g, 6.62 mmol) was added to phosphoryl chloride (15 mL) under an Ar atmosphere. The
reaction mixture was heated at reflux overnight, then cooled to room temperature, poured into a
mixture of aqueous ammonia (100 mL), crushed ice (200 g), and methylene chloride (200 mL), and
extracted with methylene chloride. The organic layer was washed with water, dried over sodium
sulfate, and concentrated in vacuo. The residue was purified by recrystallization from chloroform to
1
yield 24a (1.76 g, 88%) as yellow needles. H-NMR (500 MHz, CDCl3) δ 9.15 (1 H, d, J = 2.2 Hz),
8.53 (1 H, d. J = 9.5 Hz), 8.35 (1 H, dd, J = 2.2, 9.5 Hz), 8.17 (1 H, d, J = 9.4 Hz), 7.58 (1 H, dd, J =
2.6, 9.4 Hz), 7.53 (1 H, d, J = 2.6 Hz), 4.30 (2 H, q, J = 7.0 Hz), 1.57 (3 H, t, J = 7.0 Hz).
1
9-Chloro-2-methoxy-6-nitroacridine (24b) was prepared similarly from 23b (53%). H-NMR (400
MHz, CD3OD) δ 9.06 (1 H, d, J = 2.3 Hz), 8.66 (1 H, dd, J = 0.3, 9.3 Hz), 8.43 (1 H, dd, J = 2.3, 9.5
Hz), 8.18 (1 H, dd, J = 0.9, 9.1 Hz), 7.70-7.69 (2 H, m), 4.10 (3 H, s).
9

4.2.3. Synthesis of 9-amino-2-ethoxy-6-nitroacridine (25a)
Ammonium sulfate (0.990 g, 7.55 mmol) was added to a solution of 24b (1.14 g, 3.77 mmol) in
phenol (38 mL) under an Ar atmosphere. The reaction mixture was heated at reflux for 4 h, then
cooled to room temperature, and methylene chloride (150 mL) and 1 M sodium hydroxide (50 mL)
were added to the mixture. The precipitate was collected by filtration, washed with 1 M sodium
1
hydroxide and water, and dried in vacuo to yield 25a (1.02 g, 95%) as a red powder. H-NMR (400
MHz, DMSO-d6) δ 8.62 (1 H, d, J = 2.3 Hz), 8.59 (1 H, d, J = 9.4 Hz), 7.96 (1 H, dd, J = 2.4, 9.5 Hz),
7.89 (2 H, s), 7.86 (1 H, d, J = 9.4 Hz), 7.72 (1 H, d, J = 2.4 Hz), 7.45 (1 H, dd, J = 2.6, 9.3 Hz), 4.22
(2 H, q, J = 7.0 Hz), 1.44 (3 H, t, J = 7.0 Hz).
1
9-Amino-2-ethoxy-6-nitroacridine (25b) was prepared similarly from 24b (quant). H-NMR (400
MHz, CD3OD) δ 8.75 (1 H, d, J = 2.3 Hz), 8.47 (1 H, d, J = 9.8 Hz), 8.03 (1 H, dd, J = 2.5, 9.5 Hz),
7.88 (1 H, d, J = 9.4 Hz), 7.60 (1 H, d, J = 2.6 Hz), 7.48 (1 H, dd, J = 2.8, 9.4 Hz), 4.00 (3 H, s).
4.2.4. Synthesis of 6,9-diamino-2-ethoxyacridine (26a)
25a (1.05 g, 3.72 mmol) was hydrogenated in DMF (18.5 mL) with 10% palladium on carbon (100
mg) under an H2 atmosphere overnight. Water was added to the mixture and the solution was filtered
through Celite. Brine was added to the filtrate. The resulting yellow precipitate was collected by
filtration, and dried in vacuo to yield 26a (726 mg, 77%) as a yellow powder, which was purified by
recrystallization from DMF. 1H-NMR (400 MHz, DMSO-d6) δ 8.15 (1 H, d, J = 9.3 Hz), 7.74 (1 H, d,
J = 2.5 Hz), 7.62 (1 H, d, J = 9.2 Hz), 7.53 (1 H, dd, J = 2.6, 9.2 Hz), 6.95 (1 H, dd, J = 2.2, 9.3 Hz),
6.66 (1 H, d, J = 2.1 Hz), 4.21 (2 H, q, J = 7.0 Hz), 1.50 (3 H, t, J = 7.0 Hz).
6,9-Diamino-2-methoxyacridine (26b) was prepared similarly from 25b (quant). 1H-NMR (400 MHz,
CD3OD) δ 8.14 (1 H, d, J = 9.3 Hz), 7.74 (1 H, d, J = 2.5 Hz), 7.60 (1 H, d, J = 9.3 Hz), 7.52 (1 H,
dd, J = 2.6, 9.2 Hz), 6.94 (1 H, dd, J = 2.2, 9.3 Hz), 6.63 (1 H, d, J = 2.2 Hz), 3.96 (1 H, s).
4.2.5. Synthesis of 6,9-diamino-2-hydroxyacridine (26c)
Aluminum chloride (341 mg, 2.56 mmol) was added to a solution of 26a (50.0 mg, 0.198 mmol) in
dry xylene (4 mL), and the mixture was heated under reflux. The yellow mixture turned red, then
black. After 1 h, the solution was diluted with cold water (2 mL). The yellow precipitate was
collected by filtration, and washed with water and ether to yield 26c (27.0 mg, 60%) as a yellow
powder. 1H-NMR (400 MHz, CD3OD) δ 8.10 (1 H, d, J = 9.3 Hz), 7.58 (1 H, d, J = 2.2 Hz), 7.44 (1
H, dd, J = 0.3, 9.0 Hz), 7.43 (1 H, dd, J = 2.6, 9.2 Hz), 6.90 (1 H, dd, J = 2.2, 9.3 Hz), 6.61 (1 H, d, J
= 2.2 Hz).
4.2.6.
Typical
procedure
for
3-arylmethylamino
derivatives:
Synthesis
of
9-amino-2-ethoxy-6-(2-furanylmethylamino)acridine (1)
10

Acetic acid (1.32 mL), 2-furaldehyde (0.260 mL, 3.19 mmol) and sodium cyanoborohydride (252 mg,
4.01 mmol) were added to a solution of 26a (200 mg, 0.790 mmol) in methanol (4 mL) under an Ar
atmosphere. The mixture was stirred at 50°C overnight, then poured into cold water, and extracted
with ethyl acetate. The organic layer was washed with water and brine, dried over sodium sulfate, and
evaporated. Water was added to the residue, and the resulting yellow precipitate was collected by
filtration, washed with water and dried in vacuo to yield 1 (202 mg, 77%) as a yellow powder, which
1
was purified by recrystallization from hexane / ethanol. Mp 210°C (dec); H-NMR (500 MHz,
CD3OD) δ 8.09 (1 H, d, J = 9.4 Hz), 7.65 (1 H, d, J = 2.5 Hz), 7.57 (1 H, d, J = 9.2 Hz), 7.48 (1 H,
dd, J = 2.6, 9.2 Hz), 7.47 (1 H, m), 6.94 (1 H, dd, J = 2.3, 9.4 Hz), 6.57 (1 H, d, J = 2.2 Hz), 6.38 (2
H, m), 4.47 (2 H, s), 4.17 (2 H, q, J =7.0 Hz), 1.47 (3 H, t, J = 7.0 Hz); 13C-NMR (125 MHz,
CD3OD) δ 156.8, 156.2, 155.2, 153.0, 143.7, 143.7, 136.0, 127.9, 126.1, 120.7, 117.7, 113.8, 111.6,
108.8, 105.1, 104.3, 93.4, 65.5, 41.0, 15.2; HRMS (ESI+) calcd for C20H20N3O2 (M+H)+ 334.1550.
found 334.1541.
Compounds 3 – 5 were prepared similarly from 26a.
1
9-Amino-2-ethoxy-6-(2-thiophenylmethylamino)acridine (3): 68%; H-NMR (400 MHz, CD3OD) δ
8.13 (1 H, d, J = 9.4 Hz), 7.71 (1 H, d, J = 2.4 Hz), 7.58 (1 H, d, J = 9.2 Hz), 7.51 (1 H, dd, J = 2.4,
9.2 Hz), 7.31 (1 H, dd, J = 1.2, 5.1 Hz), 7.13 (1 H, dd, J = 1.0, 3.4 Hz), 7.00 (2 H, m), 6.57 (1 H, d, J
= 2.4 Hz), 4.67 (2 H, s), 4.13 (2 H, q, J = 7.0 Hz), 1.46 (3 H, t, J = 7.0 Hz); 13C-NMR (125 MHz,
CD3OD) δ 156.7, 156.0, 155.0, 143.5, 143.0, 135.9, 128.1, 127.8, 126.8, 126.1, 126.0, 120.6, 117.7,
113.7, 105.1, 104.2, 93.6, 65.5, 43.1, 15.2; HRMS (ESI+) calcd for C20H20N3OS (M+H)+ 350.1322.
found 350.1317.
1
9-Amino-2-ethoxy-6-(2-phenylmethylamino)acridine (4): 44%; Mp 230°C (dec); H-NMR (400
MHz, CD3OD) δ 8.12 (1 H, d, J = 9.4 Hz), 7.70 (1 H, d, J = 2.4 Hz), 7.54 (1 H, d, J = 10.2 Hz), 7.49
(1 H, dd, J = 2.4, 10.2 Hz), 7.41 (2 H, d, J = 7.2 Hz), 7.37 (2 H, t, J = 7.2 Hz), 7.27 (1 H, t, J = 7.2
Hz), 7.00 (1 H, dd, J = 2.4, 10.2 Hz), 6.44 (1 H, d, J = 2.4 Hz), 4.45 (2 H, s), 4.07 (2 H, q, J = 7.0
Hz), 1.44 (3 H, t, J = 7.0 Hz); 13C-NMR (125 MHz, CD3OD) δ 156.8, 156.1, 155.6, 143.8, 139.5,
136.0, 129.9, 128.6, 127.8, 126.1, 120.6, 117.8, 113.9, 105.0, 104.4, 93.4, 65.6, 47.9, 15.2; HRMS
(ESI+) calcd for C22H22N3O (M+H)+ 344.1757. found 344.1748.
1
9-Amino-6-[(2,4-dimethoxyphenyl)methylamino]-2-ethoxyacridine (5): 30%; H-NMR (500 MHz,
CD3OD) δ 8.05 (1 H, d, J = 9.4 Hz), 7.63 (1 H, d, J = 2.5 Hz), 7.51 (1 H, d, J = 9.2 Hz), 7.44 (1 H,
dd, J = 2.5, 9.2 Hz), 7.18 (1 H, d, J = 8.4 Hz), 6.95 (1 H, dd, J = 2.2, 9.4 Hz), 6.58 (1 H, d, J = 2.4
Hz), 6.47 (1 H, dd, J = 2.4, 8.3 Hz), 6.39 (1 H, d, J = 2.0 Hz), 4.38 (2 H, s), 4.15 (2 H, q, J = 7.0 Hz),
3.87 (3 H, s), 3.77 (3 H, s), 1.47 (3 H, t, J = 7.0 Hz); 13C-NMR (125 MHz, CD3OD) δ 162.3, 160.0,
156.7, 155.9, 155.7, 143.9, 135.9, 130.3, 127.7, 125.9, 120.6, 118.9, 117.9, 113.8, 105.5, 104.8,
104.4, 99.6, 93.0, 65.5, 56.1, 56.0, 42.9, 15.2; HRMS (ESI+) calcd for C24H26N3O3 (M+H)+ 404.1969.
11

found 404.1959.
4.2.7.
Typical
procedure
for
3-amide
derivatives:
Synthesis
of
N-(9-amino-2-ethoxyacridin-6-yl)benzamide (7)
Benzoyl chloride (50.0 µL, 0.594 mmol) was added to a mixture of 26a (100 mg, 0.396 mmol) and
pyridine (3 drops) in DMF (4 mL) under an Ar atmosphere. The mixture was stirred at room
temperature overnight, and then poured into water (20 mL). The resulting yellow precipitate was
collected by filtration, washed with hot water and ethyl acetate, and dried in vacuo to yield 7 (62.2
mg, 42%) as a yellow powder, which was purified by recrystallization from DMSO / methylene
chloride. Mp 279°C (dec); 1H-NMR (400 MHz, DMSO-d6) δ 10.5 (1 H, s), 8.37 (1 H, s), 8.33 (1 H, d,
J = 9.3 Hz), 8.01 (2 H, m), 7.73 (1 H, d, J = 9.3 Hz), 7.68-7.55 (7 H, m), 7.35 (1 H, 9.2 Hz), 4.18 (2
H, q, J = 7.0 Hz), 1.43 (3H, t, J = 7.0 Hz); 13C-NMR (125 MHz, DMSO-d6) δ 166.0, 153.4, 148.5,
147.5, 144.8, 139.8, 135.0, 131.7, 129.4, 128.5, 127.8, 124.0, 123.4, 117.3, 115.5, 112.9, 109.6,
101.3, 63.6, 14.7; HRMS (ESI+) calcd for C22H20N3O2 (M+H)+ 358.1550. found 358.1552.
Compounds 2, 6, 8 – 16 were prepared similarly from 26a.
1
N-(9-Amino-2-ethoxyacridin-6-yl)acetamide (2): H-NMR (400 MHz, DMSO-d6) δ 10.8 (1 H, s),
9.64 (2 H, br d), 8.58 (1 H, d, J = 9.4 Hz), 8.50 (1 H, d, J = 1.8 Hz), 8.01 (1 H, d, J = 2.5 Hz), 7.80 (1
H, d, J = 9.3 Hz), 7.62 (1 H, dd, J = 2.5, 9.3 Hz), 7.48 (1 H, dd, J = 2.0, 9.3 Hz), 4.19 (2 H, q, J = 7.0
Hz), 1.48 (3 H, t, J = 7.0 Hz); 13C-NMR (125 MHz, DMSO-d6) δ 170.0, 155.4, 154.8, 144.5, 140.1,
134.8, 127.4, 125.5, 120.4, 116.9, 112.3, 107.0, 104.2, 103.8, 64.2, 24.4, 14.5; HRMS (ESI+) calcd
for C17H18N3O2 (M+H)+ 296.1394. found 296.1389.
1
N-(9-Amino-2-ethoxyacridin-6-yl)furan-2-carboxamide (6): H-NMR (500 MHz, DMSO-d6) δ10.9
(1 H, s), 9.63 (2 H, s), 8.63 (1 H, s), 8.62 (1 H, d, J = 1.7 Hz), 8.03 (1 H, dd, J = 1.6, 6.7 Hz), 8.02 (1
H, d, J = 2.0 Hz), 7.83 (1 H, d, J = 9.2 Hz), 7.81 (1 H, dd, J = 2.0, 9.4 Hz), 7.64 (1 H, dd, J = 2.3, 9.3
Hz), 7.60 (1 H, d, J = 3.5 Hz), 6.77 (1 H, dd, J = 1.7, 3.6 Hz), 4.21 (2 H, q, J = 7.0 Hz), 1.42 (3 H, t,
J = 7.0 Hz); 13C-NMR (125 MHz, DMSO-d6) δ 156.9, 155.2, 154.8, 146.8, 144.1, 140.2, 140.1,
135.2, 127.4, 125.3, 120.7, 117.7, 116.3, 112.5, 112.4, 107.4, 105.9, 103.7, 64.2, 14.5; HRMS (ESI+)
calcd for C20H18N3O3 (M+H)+ 348.1343. found 348.1347.
N-(9-Amino-2-ethoxyacridin-6-yl)-4-chlorobenzamide (8): Mp >300°C; 1H-NMR (400 MHz,
DMSO-d6) δ11.0 (1 H, s), 9.55 (2 H, s), 8.64 (1 H, s), 8.61 (1 H, d, J = 9.4 Hz), 8.08 (2 H, d, J = 8.6
Hz), 8.00 (1 H, d, J = 2.1 Hz), 7.84 (1 H, d, J = 8.1 Hz), 7.78 (1 H, d, J = 9.1 Hz), 7.66 (2 H, d, J =
8.6 z), 7.61 (1 H, dd, J = 2.2, 9.3 Hz), 4.20 (2 H, q, J = 7.0 Hz), 1.42 (3 H, t, J = 7.0 Hz); 13C-NMR
(125 MHz, DMSO-d6) δ 165.6, 155.0, 154.7, 143.8, 140.8, 137.1, 133.0, 131.4, 130.0, 128.6, 127.1,
125.1, 122.8, 117.7, 112.4, 107.7, 103.4, 69.8, 14.5; HRMS (ESI+) calcd for C22H19ClN3O2 (M+H)+
392.1160. found 392.1160.
12

N-(9-Amino-2-ethoxyacridin-6-yl)-4-fluorobenzamide (9): Mp >300°C; 1H-NMR (500 MHz,
DMSO-d6) δ 10.8 (1 H, s), 9.05 (2 H, s), 8.59 (1 H, s), 8.52 (1 H, d, J = 9.2 Hz), 8.11 (2 H, m), 7.90
(1 H, s), 7.80 (1 H, d, J = 9.2 Hz), 7.73 (1 H, dd, J = 1.6, 9.3 Hz), 7.57 (1 H, d, J = 8.7 Hz), 7.43 (2 H,
m), 4.20 (2 H, q, J = 7.0 Hz), 1.43 (3 H, t, J = 7.0 Hz); 13C-NMR (125 MHz, DMSO-d6) δ 165.5,
164.3 (d, JCF = 274 Hz), 163.4, 154.6, 130.9, 130.8 (d, JCF = 9.6 Hz), 124.8, 117.7, 115.6 (d, JCF =
21.9 Hz), 112.5, 109.3, 107.9, 103.1, 64.1, 14.5; HRMS (ESI+) calcd for C22H19FN3O2 (M+H)+
376.1456. found 376.1453.
1
N-(9-Amino-2-ethoxyacridin-6-yl)-4-cyanobenzamide (10): H-NMR (400 MHz, CD3OD) δ 8.73 (1
H , d, J = 1.9 Hz), 8.46 (1 H, d, J = 9.4 Hz), 8.15 (2 H, m), 7.95 (2 H, m), 7.81 (1 H, d, 2.5 Hz), 7.78
(1 H, d, J = 9.3 Hz), 7.70 (1 H, dd, J = 2.0, 9.3 Hz), 7.64 (1 H, dd, J = 2.6, 9.3 Hz), 4.24 (2 H, q, J =
7.0 Hz), 1.51 (3 H, t, J = 7.0 Hz) ; 13C-NMR (125 MHz, DMSO-d6) δ 165.4, 155.2, 154.8, 143.8,
138.3, 132.6, 128.8, 127.5, 125.3, 120.4, 118.2, 117.7, 114.4, 112.4, 107.7, 106.2, 103.5, 64.2, 14.5;
HRMS (ESI+) calcd for C23H19N4O2 (M+H)+ 383.1503. found 383.1496.
N-(9-Amino-2-ethoxyacridin-6-yl)-4-(methoxycarbobyl)benzamide (11): 1H-NMR (500 MHz,
DMSO-d6) δ 8.71 (1 H, d, J = 1.3 Hz), 8.60 (1 H, d, J = 9.4 Hz), 8.15 (4 H, br s), 7.99 (1 H, d, J =
1.8 Hz), 7.82 (1 H, d, J = 9.2 Hz), 7.76 (1 H, dd, J = 1.9, 9.3 Hz), 7.68 (1 H, dd, J = 2.3, 9.1 Hz),
4.22 (2 H, q, J = 7.0 Hz), 3.97 (3 H, s), 1.44 (3 H, t, J = 7.0 Hz); 13C-NMR (125 MHz, DMSO-d6) δ
166.0, 165.6, 155.4, 154.9, 144.2, 140.0, 138.3, 134.8, 132.6, 129.3, 128.4, 127.6, 125.3, 120.6,
117.8, 112.4, 107.5, 105.9, 103.6, 64.2, 52.6, 14.5; HRMS (ESI+) calcd for C24H22N3O4 (M+H)+
416.1605. found 416.1617.
1
N-(9-Amino-2-ethoxyacridin-6-yl)-4-nitorobenzamide (12): Mp 187°C (dec); H-NMR (500 MHz,
DMSO-d6) δ 10.8 (1 H, s), 8.41 (2 H, d, J = 7.2 Hz), 8.37 (1 H, d, J = 1.7 Hz), 8.34 (1 H, d, J = 9.3
Hz), 8.25 (2 H, d, J = 7.2 Hz), 7.74 (1 H, d, J = 9.3 Hz), 7.64 (2 H, m), 7.56 (2 H, br s), 7.34 (1 H, dd,
J = 2.6, 9.3 Hz), 4.18 (2 H, q, J = 7.0 Hz), 1.43 (3 H, t, J = 7.0 Hz); 13C-NMR (125 MHz, DMSO-d6)
δ 163.3, 154.4, 149.2, 148.4, 147.6, 145.3, 140.7, 139.2, 129.9, 129.4, 123.9, 123.6, 123.6, 117.2,
117.1, 116.4, 112.9, 109.9, 101.1, 63.6, 14.7; HRMS (ESI+) calcd for C22H19N4O4 (M+H)+ 403.1401.
found 403.1393.
1
N-(9-Amino-2-ethoxyacridin-6-yl)-3-cyanobenzamide (13): H-NMR (500 MHz, DMSO-d6) δ 11.1
(1 H, s), 9.58 (2 H, s), 8.64 (1 H, d, J = 1.5 Hz), 8.61 (1 H, d, J = 9.3 Hz), 8.49 (1 H, s), 8.32 (1 H, d,
J = 8.0 Hz), 8.12 (1 H, d, 7.7 Hz), 7.98 (1 H, d, J = 2.2 Hz), 7.83 (1 H, d, J = 9.4 Hz), 7.79 (1 H, d, J
= 7.8 Hz), 7.75 (1 H, dd, J = 1.8, 9.3 Hz), 7.63 (1 H, dd, J = 2.4, 9.3 Hz), 4.20 (2 H, q, J = 7.0Hz),
1.42 (3 H, t, J = 7.0 Hz); 13C-NMR (125 MHz, DMSO-d6) δ 164.9, 155.2, 154.8, 143.9, 140.1, 135.6,
13

135.3, 132.9, 131.7, 130.0, 127.4, 125.3, 120.9, 118.3, 117.7, 112.4, 111.7, 107.6, 106.4, 103.7, 103.6,
64.2, 14.5; HRMS (ESI+) calcd for C23H19N4O2 (M+H)+ 383.1503. found 383.1492.
N-(9-Amino-2-ethoxyacridin-6-yl)-3-nitrobenzamide (14): Mp >300°C; 1H-NMR (400 MHz,
DMSO-d6) δ 11.2 (1 H, s), 9.61 (2 H, s), 8.82 (1 H, t, J = 1.9, 2.0 Hz), 8.62 (2 H, m), 8.47 (2 H, m),
7.97 (1 H, d, J = 2.4 Hz), 7.88 (1 H, d, J = 8.0, 8.0 Hz), 7.83 (1 H, d, J = 9.3 Hz), 7.78 (1 H, dd, J =
1.9, 9.3 Hz), 7.61 (1 H, dd, J = 2.5, 9.2 Hz), 4.19 (2 H, q, J = 7.0 Hz), 1.41 (3 H, t, J = 7.0 Hz);
13C-NMR (125 MHz, DMSO-d6) δ 164.7, 155.4, 155.0, 147.9, 144.0, 140.1, 135.7, 135.2, 134.6,
130.5, 127.6, 126.9, 125.5, 122.9, 120.8, 117.9, 112.5, 107.7, 106.4, 103.7, 64.4, 14.6; HRMS (ESI+)
calcd for C22H19N4O4 (M+H)+ 403.1401. found 403.1398.
N-(9-Amino-2-ethoxyacridin-6-yl)naphthalene-2-carboxamide (15): Mp >300°C; 1H-NMR (500
MHz, DMSO-d6) δ 11.1 (1 H, s), 9.48 (2 H, s), 8.73 (1 H, s), 8.70 (1 H, s), 8.61 (1 H, d, J = 9.2 Hz),
8.16-8.03 (4 H, m), 7.99 (1 H, s), 7.83 (2 H, m), 7.71-7.63 (3 H, m), 4.21 (2 H, q, J = 7.0 Hz), 1.43 (3
H, t, J = 7.0 Hz); 13C-NMR (125 MHz, DMSO-d6) δ 166.8, 154.9, 154.7, 144.2, 140.8, 134.5, 132.0,
131.6, 129.3, 129.1, 128.6, 128.3, 127.8, 127.2, 127.1, 125.2, 125.2, 124.5, 121.2, 117.8, 112.4,
107.6, 106.4, 103.5, 64.2, 14.5; HRMS (ESI+) calcd for C26H22N3O2 (M+H)+ 408.1707. found
408.1696.
1
N-(9-Amino-2-ethoxyacridin-6-yl)-3,5-bis(trifluoromethyl)benzamide (16): Mp >300°C; H-NMR
(500 MHz, DMSO-d6) δ 8.67 (3 H, m), 8.63 (1 H, d, J = 9.3 Hz), 8.45 (1 H, s), 8.00 (1 H, d, J = 2.5
Hz), 7.84 (1 H, d, J = 9.3 Hz), 7.75 (1 H, dd, J = 1.8, 9.3 Hz), 7.68 (1 H, dd, J = 2.5, 8.8 Hz), 4.21 (2
H, q, J = 7.0 Hz), 1.43 (3 H, t, J = 7.0 Hz); 13C-NMR (125 MHz, DMSO-d6) δ 163.9, 155.5, 154.9,
143.8, 139.9, 136.6, 135.0, 130.6 (q, J = 32.5 Hz), 129.0, 127.7, 125.8, 125.4, 125.0 (q, J = 271 Hz),
120.6, 117.8, 112.4, 107.7, 106.3, 103.6, 64.2, 14.5; HRMS (ESI+) calcd for C24H18F6N3O2 (M+H)+
494.1298. found 494.1300.
4.2.8. Synthesis of 6-amino-2-ethoxyacridine (27) and 6-amino-2-ethoxy-9-phenoxyacridine (28)
24a (100 mg, 0.331 mmol) was hydrogenated in phenol (4 mL) with 10% palladium on carbon (10.0
mg) under an H2 atmosphere overnight. Then, 1 M sodium hydroxide was added, and the mixture
was extracted with ethyl acetate. The organic layer was washed with water, dried over sodium sulfate,
and concentrated in vacuo. The residue was purified by silica gel column chromatography (eluent;
methylene chloride: methanol 7 : 1 to methylene chloride : methanol : aqueous ammonia 175 : 25 : 1)
to give 27 (4.0 mg, 5%) as a brown powder and 28 (95 mg, 85%) as a yellow powder.
27: 1H-NMR (500 MHz, CD3OD) δ 8.78 (1 H, s), 7.88 (1 H, d, J = 9.5 Hz), 7.85 (1 H, d, J = 9.4Hz),
7.52 (1 H, d, J = 9.5 Hz), 7.39 (1 H, s), 7.18 (1 H, dd, J = 1.8, 9.1 Hz), 6.95 (1 H, s), 4.20 (2 H, q, J =
7.0 Hz), 1.49 (3 H, t, J = 7.0 Hz).
28: 1H-NMR (500 MHz, CD3OD) δ 7.86 (1 H, d, J = 9.2 Hz), 7.75 (1 H, d, J = 9.2 Hz), 7.38 (1 H, dd,
14

J = 2.8, 9.4 Hz), 7.26 (2 H, m), 7.08-6.98 (4 H, m), 6.87 (2 H, d, J = 8.8 Hz), 3.89 (2 H, q, J = 7.2
Hz), 1.31 (3 H, t, J = 7.2 Hz); 13C-NMR (125 MHz, CDCl3) δ 159.2, 155.3, 153.3, 150.7, 148.1,
147.4, 130.4, 129.8, 125.5, 123.6, 122.3, 120.2, 119.3, 115.5, 115.4, 107.0, 98.9, 63.6, 14.5; HRMS
(ESI+) calcd for C21H19N2O2 (M+H)+ 331.1441. found 331.1442.
4.2.9. 4-Cyano-N-(2-ethoxyacridin-6-yl)benzamide (17)
4-Cyanobenzoyl chloride (33.4 mg, 0.202 mmol) was added to a mixture of 27 (24.0 mg, 0.101
mmol) and pyridine (3 drops) in DMF (4 mL) under an Ar atmosphere. The mixture was stirred at
room temperature overnight, and then poured into water (10 mL). The resulting yellow precipitate
was collected by filtration, washed with hot water and ethyl acetate, and dried in vacuo to yield 17
(5.5 mg, 81%) as a brown powder, which was purified by recrystallization from methanol / ethyl
1
acetate. H-NMR (400 MHz, CD3OD) δ 8.79 (1 H, s), 8.69 (1 H, d, J = 1.9 Hz), 8.14 (2 H, d, J = 8.2
Hz), 8.05 (1 H, d, J = 9.1 Hz), 8.02 (1 H, d, J = 9.4 Hz), 7.92 (2 H, d, J = 8.2 Hz), 7.85 (1 H, dd, J =
2.1, 9.1 Hz), 7.50 (1 H, dd, J = 2.8, 9.4 Hz), 7.35 (1 H, d, J = 2.7 Hz), 4.23 (2 H, q, J = 7.0 Hz), 1.50
(3 H, t, J = 7.0 Hz); 13C-NMR (125 MHz, DMSO-d6) δ 164.8, 155.4, 147.4, 145.9, 139.2, 138.8,
133.4, 132.5, 130.3, 128.7, 128.4, 126.6, 125.5, 123.8, 121.6, 118.3, 116.3, 114.0, 104.5, 63.5, 14.6;
HRMS (ESI+) calcd for C23H18N3O2 (M+H)+ 368.1394. found 368.1392.
4-Cyano-N-(2-ethoxy-9-phenoxyacridin-6-yl)benzamide (18) was prepared similarly from 28 in 39%.
1H-NMR (500 MHz, DMSO-d6) δ 10.90 (2 H, s), 8.80, (1 H, d, J = 1.9 Hz), 8.17 (2 H, d, J = 8.5 Hz),
8.11 (1 H, d, J = 9.5 Hz), 8.07 (2 H, d, J = 8.0 Hz), 7.99 (1 H, d, J = 9.3 Hz), 7.85 (1 H, dd, J = 2.0,
9.3 Hz), 7.53 (1 H, dd, J = 2.8, 9.5 Hz), 7.33 (2 H, t, J = 8.0 Hz), 7.12 (1 H, d, J = 2.8 Hz), 7.09 (1 H,
t, J = 7.4, 7.5 Hz), 6.92-6.90 (2 H, d, J = 8.0 Hz), 4.03 (2 H, q, J = 7.0 Hz), 1.31 (3 H, t, J = 7.0 Hz);
13C-NMR (125 MHz, DMSO-d6) δ 164.9, 158.7, 155.8, 152.2, 148.8, 147.6, 139.6, 138.7, 132.6,
131.1, 130.2, 128.7, 125.7, 122.8, 122.3, 119.9, 118.3, 117.1, 116.6, 115.5, 114.1, 98.2, 63.6, 14.2;
HRMS (ESI+) calcd for C29H22N3O3 (M+H)+ 460.1656. found 460.1652.
4.2.10. Synthesis of N-(9-Amino-2-ethoxyacridin-6-yl)-N’-(4-cyanophenyl)urea (19)
4-Cyanophenyl isocyanate (60mg, 0.451 mmol) was added to a solution of 26a (52 mg, 0.218 mmol)
in DMSO (1 mL) under an Ar atmosphere. The reaction mixture was stirred at 100°C overnight, and
then poured into water (20 mL) and ethyl acetate (20 mL). The resulting yellow precipitate was
collected by filtration, washed with hot water, and dried in vacuo to yield 19 (18.0 mg, 20%) as a
1
yellow powder. Mp >300°C; H-NMR (500 MHz, DMSO-d6) δ11.20 (1 H, s), 11.02 (1 H, s), 10.23
(1H, s), 8.95 (2H, br s), 8.38 (1H, d, J = 9.0 Hz), 8.08 (1H, s), 7.72-7.61 (4 H, m), 7.56 (2 H, d, J =
8.6 Hz), 7.50 (2 H, m), 4.11 (2H, q, J = 7.0 Hz), 1.41 (3H, t, J = 7.0 Hz); 13C-NMR (125 MHz,
DMSO-d6) δ 155.2, 153.7, 152.0, 144.8, 143.7, 140.3, 133.8, 133.5, 133.4, 127.0, 125.7, 120.1, 119.3,
118.2, 118.1, 116.4, 112.9, 106.2, 106.1, 103.9, 103.9, 102.6, HRMS (ESI+) calcd for C23H20N5O2
(M+H)+ 398.1612. found 398.1610.
15

N-(9-Amino-2-methoxyacridin-6-yl)-N’-(4-cyanophenyl)urea
(20)
and
N-(9-amino-2-hydroxyacridin-6-yl)-N’-(4-cyanophenyl)urea (21) were prepared similarly from 26b
and 26c, respectively.
20: 27%; Mp >300°C; 1H-NMR (400 MHz, DMSO-d6) δ 11.22 (1 H, s), 11.04 (1 H, s), 9.17 (1 H, s),
8.39 (1 H, d, J = 9.1 Hz), 8.08 (1 H, s), 7.71-7.47 (8 H, m), 3.87 (3 H, s); 13C-NMR (125 MHz,
DMSO-d6) δ 155.0, 154.3, 152.3, 152.1, 145.2, 144.5, 144.2, 140.5, 135.2, 133.3, 132.9, 126.5,
124.9, 120.7, 119.4, 119.3, 118.3, 118.2, 117.2, 112.0, 106.2, 103.4, 103.0, 102.6, 55.9; HRMS
(ESI+) calcd for C22H18N5O2 (M+H)+ 384.1455. found 384.1448.
1
21: 28%; Mp >300°C; H-NMR (400 MHz, DMSO-d6) δ 10.42 (1 H, s), 10.24 (1 H, s), 10.20 (1 H,
s), 9.32 (2 H, s), 8.50 (1 H, d, J = 9.4 Hz), 8.22 (1 H, d, J = 2.0 Hz), 7.80-7.67 (6 H, m), 7.58 (1 H,
dd, J = 2.4, 9.1 Hz), 7.37 (1 H, dd, J = 2.0, 9.3 Hz); 13C-NMR (125 MHz, DMSO-d6) δ 155.5, 154.1,
152.4, 145.2, 140.7, 134.2, 133.9, 133.8, 127.4, 126.1, 120.5, 119.6, 118.6, 118.5, 113.3, 106.6, 106.5,
104.3, 104.3, 103.0; HRMS (ESI+) calcd for C21H14N5O2 (M+H)+ 368.1153. found 368.1159.
4.3. Biology
4.3.1. Expression and purification of GST–SPAK-CCT in Escherichia coli.17
DNA encoding residues 452–553 of rat SPAK (CCT, almost identical to residues 442–545 of human
SPAK) was cloned into pGEX6P-1 vector (Promega). E. coli BL21 cells were transformed with
recombinant GST-fusion SPAK (GST–SPAK-CCT) protein expression vector, and seeded into 1 liter
of 2YT broth [1.6% (w/v) tryptone/1% (w/v) yeast extract/0.5% NaCl] containing 100 µg/ml
ampicillin. Culture was continued at 37°C until the absorbance at 600 nm reached 0.6. Then IPTG (1
mM) was added, and the cells were cultured for an additional 16 h at 28°C. Cells were isolated by
centrifugation and resuspended in 40 ml of ice-cold PBS, followed by sonication (Tomy Ultrasonic
Disrupter UD-201). Lysates were centrifuged at 4°C for 5 min at 10000 x g. The GST-fusion protein
was affinity-purified on 1.2 ml of glutathione–Sepharose beads and eluted in elution buffer
containing 83 mM Tris/HCl, 150 mM KOH and 30 mM glutathione.
4.3.2. Cytotoxicity assay
HEK293T cells were seeded in a 96-well plate at a density of 1 x 105 cells/ml in a volume of 200 µL
per well, then various concentrations of compounds were added and the plate was incubated for 40
hours. Cell Counting Kit-8 reagent (10 µL) (Dojindo, CK04, Japan) was added to each well and
incubation was continued for 3.5 hours at 37℃ under 5% CO2. Absorbance was measured at 450 nm
using a microplate reader (ARVO MX, PerkinElmer Inc.). Dose–response curves were fitted by
non-linear regression to a sigmoidal dose–response model, and the value of IC50 was calculated with
KaleidaGraph 4.0J.
16

4.3.3. FCS assay.17
Fluorescent TAMRA (6-carboxytetramethylrhodamine)-labeled WNK4 peptide covering the RFQV
motif [WNK4-RFQV (TAMRA-SEEGKPQLVGRFQVTSSK)] and TAMRA-labeled WNK4 peptide
in which the RFXV motif was mutated [WNK4-AFQV (TAMRA-SEEGKPQLVGAFQVTSSK)] as a
negative control were prepared (Hokkaido System Science). The TAMRA-labeled peptides were
incubated with various concentrations of GST–SPAK-CCT (1–65 µM) in reaction buffer consisting
of 1×PBS with 0.05% Tween 20 at room temperature (25°C) for 30 min, and FCS measurements of
single-molecule fluorescence were performed using a FluoroPoint-light analytical system
(Olympus).21 The confocal volume for detection of fluorescence signals in this equipment is in the
range of a single femtolitre. Therefore, TAMRA-labeled peptide was diluted so that a single
fluorophore was likely to be present in the confocal space. The final concentration of each peptide in
the FCS assays was 2.7 nM. The concentration of GST–SPAK-CCT used for screening was 0.5 µM,
since at this concentration the binding reaction was not saturated and was reproducibly detected. The
concentration of DMSO (used as a solvent of compounds) was 1% in the final assay solutions. The
assay was performed in a 384-well plate. All experiments were performed with a data acquisition
time of 15 s, and the measurements were repeated five times per sample. Kd and IC50 values were
calculated by Origin 8.1 data analysis and graphing software (OriginLab).
4.3.4. Animal Studies
C57BL/6 mice (10 weeks old) were maintained with standard laboratory chow and water. The
Animal Care and Use Committee of Tokyo Medical and Dental University approved the
experimental protocol. In an acute treatment study, compounds were administered intraperitoneally
at a dose of 5 mg/kg, 10 mg/kg or 20 mg/kg in 100 µL of DMSO; the control mice were
administered 100 µL of DMSO alone. The mice were euthanized 90 minutes after injection. The
kidneys were removed and rapidly homogenized in a 10-fold mass excess of ice-cold 1% (w/v)
Triton X-100 lysis buffer. Whole homogenates without the nuclear fraction (600 x g) were
collected and denatured at 60°C for 20 minutes as previously reported,3 then sonicated, and
centrifuged at 6000 x g for 5 minutes at 4°C. A 120 µl aliquot of the supernatant was denatured at
60°C for 20 minutes. The total protein concentration was determined by the Bradford method using
BSA as a standard, and lysates were stored at -80°C.
4.3.5. Immunoblotting
Kidneys were dissected from the mice. Quantitative immunoblotting was performed as described
previously.8 Membranes were blocked with blocking reagent and reacted with the following primary
antibodies: anti-total SPAK (Cell Signaling Technology), anti-phosphorylated SPAK22 anti-(total
NCC),23 anti-phospho-NCC (pSer71)12, anti-total NKCC1 (T4) (Hybridoma Bank, University of
Iowa, Iowa City, IA), anti-phosphorylated NKCC1 (pThr206 in mouse NKCC1),24 anti-total NKCC2
(Alpha Diagnostic, San Antonio, TX), and anti-phosphorylated NKCC2 (termed pT2 antibody;
17

kindly provided by K. Mutig).25 Alkaline phosphatase-conjugated anti-mouse IgG antibodies
(Promega) were used as secondary antibodies for colorimetric detection. The intensities of the bands
were analysed and quantified by ImageJ (NIH) software.
Acknowledgement
This work was partly supported by JSPS Core-to-Core Program, A. Advanced Research Networks,
and Platform Project for Supporting in Drug Discovery and Life Science Research (Platform for
Drug Discovery, Informatics, and Structural Life Science) from Japan Agency for Medical Research
and Development (AMED).
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Graphical Abstract
Development of WNK Signaling Inhibitors as a New Class of Antihypertensive Drugs
Mari Ishigami-Yuasa, Yuko Watanabe, Takayasu Mori, Hiroyuki Masuno, Shinya Fujii, Eriko
Kikuchi, Shinichi Uchida, Hiroyuki Kagechika
21