Discovery of Dotinurad (FYU-981), a New Phenol Derivative with Highly Potent Uric Acid Lowering Activity

Article abstract of DOI:10.1021/acsmedchemlett.0c00176

To derive new uricosuric agents, novel phenol derivatives were synthesized to overcome the disadvantages of benzbromarone (BBR), attributed by its structural features. Herein, we report the discovery of new phenol derivatives with a 1,1-dioxo-1,2-dihydro-

Full text of DOI:10.1021/acsmedchemlett.0c00176

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Letter
Discovery of Dotinurad (FYU-981), a New Phenol
Derivative with Highly Potent Uric Acid Lowering Activity
Junichiro Uda, Seiichi Kobashi, Sachiho Miyata, Naoki Ashizawa, Koji Matsumoto, and Takashi Iwanaga
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.0c00176 • Publication Date (Web): 15 Jul 2020
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Discovery of Dotinurad (FYU-981), a New Phenol Derivative with
Highly Potent Uric Acid Lowering Activity
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Junichiro Uda,*, † Seiichi Kobashi, † Sachiho Miyata, † Naoki Ashizawa, ‡ Koji Matsumoto, ‡ and Takashi Iwanaga‡
† Research Laboratories 1, Medical R&D Division, FUJI YAKUHIN CO., LTD., 1-32-3, Nishiomiya, Nishi-ku, Saitama-shi,
Saitama, 331-0078, Japan
‡ Research Laboratories 2, Medical R&D Division, FUJI YAKUHIN CO., LTD., 636-1, Iidashinden, Nishi-ku, Saitama-shi,
Saitama, 331-0068, Japan
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ABSTRACT: To derive new uricosuric agents, novel phenol derivatives were synthesized to overcome the disadvantages of
benzbromarone (BBR), attributed by its structural features. Herein, we report the discovery of new phenol derivatives with
a 1,1-dioxo-1,2-dihydro-3H-1,3-benzothiazole scaffold. The selected compound 11 (dotinurad, FYU-981) demonstrated
remarkable inhibitory activity on uric acid uptake by primary human renal proximal tubule epithelial cells (RPTECs) and
URAT1-mediated uric acid transport, with weak inhibitory activity against mitochondrial respiration. Dotinurad also
displayed favorable pharmacokinetic profiles and higher potency in decreasing uric acid than BBR did in Cebus monkeys.
Dotinurad has been registered as a new uricosuric medicine in Japan. Our strategy, which focuses on the structural features
resulting in unfavorable effects, could be applied to the future developments of other drugs with disadvantages, particularly
those having a bis-aryl ketone structure.
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KEYWORDS: uricosurics, mitochondrial toxicity, benzbromarone, primary human renal proximal tubule epithelial cells,
dotinurad
a highly potent hypouricemic effect in humans, it is used as
one of the standard medicines in some countries such as
Japan, Europe, and Brazil, especially when the effect of XOI
is insufficient. However, following reports that fulminant
Hyperuricemia, a causative factor of gout, is one of the
most studied diseases globally.1 Hyperuricemia is involved
in diseases such as metabolic disorders, cardiovascular
disease, and renal dysfunctions.2, 3 There are two
significant causes of hyperuricemia, which are
hepatitis,
a rare but life-threatening disorder, was
associated with BBR administration, restriction of its use via
warnings and withdrawal occurred in Japan and Europe,
respectively.
Furthermore, BBR is an inhibitor of cytochrome P450
2C9 (CYP2C9), known as a polymorphic enzyme, and is
metabolized by the enzyme. Metabolism by CYP2C9 may
lead to drug-drug interaction and account for differences in
metabolism among individuals.8, 9 Lesinurad is only used in
combination with XOIs and an inhibitor of CYP2C9 similar
to BBR.10-12
Many studies have been carried out to investigate the
mechanisms of BBR-induced hepatic toxicity 13-19 including
the involvement of metabolism, metabolites, and
mitochondrial toxicity. Among these potential mechanisms,
mitochondrial inhibitory activity (MIA) is one of the most
promising theories for fulminant hepatitis.15, 16 Based on
these findings, to obtain compounds that possess potent
pharmacological effects without mitochondrial toxicity, we
modified BBR to overcome its disadvantages, which may be
related to a structural flaw.
overproduction of uric acid and a reduction in its renal
elimination or both. Hence, medicines used against
hyperuricemia are classified into two types based on their
mechanisms of action: xanthine oxidoreductase inhibitors
(XOIs) and uricosuric agents. Allopurinol, a representative
XOI that decreases the blood level of uric acid by inhibiting
uric acid synthesis from xanthine, has been used
worldwide as the standard medicine for a long time.
Recently, new XOIs, febuxostat and topiroxostat, were
launched to provide treatment options for gout and
hyperuricemia.4
Among uricosurics that increase urinary excretion of uric
acid, probenecid and benzbromarone (BBR) have been used
for a long time in some countries, and recently, a new
uricosuric agent, lesinurad, was approved in the US and EU.
These agents act by inhibiting urate reabsorption in the
6
renal tubules.5, However, these medicines also possess
disadvantages. Probenecid, which has also been used to
maintain the blood concentration of other drugs such as
penicillin, might cause drug-drug interactions due to non-
selective inhibition of anion transporters.7 Because BBR has
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resistance protein 4 (MRP4), ATP-binding cassette sub-
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1
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family G member 2 (ABCG2), and glucose transporter 9
(GLUT9), which are involved in the transport of urate.23-25
3
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8
9
In our experimental condition, BBR, known as a selective
URAT1 inhibitor inhibited uric acid uptake into RPTECs
26
with an IC50 of single-digit micromolar and in
a
concentration-dependent manner. Hence, we believed that
the urate uptake into RPTECs is mainly due to URAT1. This
method will be reported in detail elsewhere. The
mitochondrial respiratory control ratio (RCR) was used as
an index of MIA. Both UUI (inhibitory activity of urate
uptake into RPTECs, IC50) and MIA (RCR, IC50 (M), or
inhibition (%) at 100 M) assays were performed for these
compounds (Table 1).
Figure 1. Ranking of benzbromarone (BBR) and its derivatives
based on mitochondrial toxicity and structures of tolcapone and
fenofibrate
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The association between the structure and
mitochondrial toxicity of BBR and its analogs including
amiodarone has been previously reported (Figure 1).17-19
Table 1. UUI and MIA of BBR, 1, and 2a
These studies reported that each of the structural parts
(substituted benzoic acid and benzofuran) had no or
minimal inhibitory activity on mitochondrial respiration. In
contrast, substituted benzoyl-benzofuran analogs such as
amiodarone, benzarone and BBR (Figure 1), which are
combined with these two units, had distinct inhibitory
activities and the bromo substitutions on the phenol ring
enhances it. Because these derivatives have a bis-aryl
ketone moiety known to be a radical stabilizing structure
that is associated with some adverse effects,20 we assume
the moiety affects the mitochondrial electron transfer
system. Some medicines with the bis-aryl ketone such as
tolcapone and fenofibrate (Figure 1) inhibit the
Compound
BBR
UUI a
IC50 (M)
6.8
MIA b
IC50 (M) or %
3.1 M
1
10.8
2.5
3.6 M
mitochondrial function 21, 22 and are associated with the
occurrence of hepatic toxicity. Therefore, this structure
appears to have a risk of exerting mitochondrial toxicity.
Based on the knowledge and the hypothesis stated above,
we derived two modification strategies to avoid
mitochondrial toxicity. The first is a conversion from bis-
aryl ketone structures to aryl-benzamide structures, where
the introduction of a nitrogen atom leads to a non-ketone
structure. At the same time, the other is the transformation
of a bromine atom into other electron-withdrawing
substituents. We also considered the structural features of
BBR required to show urate uptake inhibition (UUI).
2a
43%
a UUI: Inhibitory activity of urate uptake into RPTECs. b MIA:
Mitochondrial respiratory control ratio (RCR), IC50 (M), or
inhibition (%) at 100 M.
The data indicated that the UUI potency levels of both indole
1 and indoline derivative 2a are the same as that of BBR.
Although structural conversion to indole maintained high
MIA, conversion to indoline lowered it. Hence, the aromatic
indole ring might confer the same character as that of BBR
with
a bis-aryl ketone structure, while the indoline
derivative alters the character owing to the bis-aryl ketone
structure. These results suggested that modifying the bis-
aryl ketone moiety is critical to avoiding mitochondrial
toxicity. Because 2a was considered a seed compound, we
prepared 2a-derivatives by adding another substituent on
the phenol ring to investigate the compounds’ properties.
Because BBR has a plane structure and moderate acidity
with a pKa value of 5.17, similar to that of uric acid (5.75),
we assumed that BBR mimics uric acid. Therefore, electron-
withdrawing groups such as bromide on phenol might play
important roles in maintaining acidity, thereby allowing
compounds to exert urate uptake inhibitory potency.
We prepared the new compounds, amide-linked indole
and indoline derivatives with cyano-substituent as an
electron-withdrawing group instead of bromine atoms on
the phenol moiety, as alternatives to the acyl benzofuran
structure (compound 1 and 2a in Table 1).
The synthetic procedures of 1 and 2 are shown in Scheme
1. The synthetic methods for substituted benzoic acid as
intermediates have been reported earlier,27 and we have
described some new benzoic acids in the Supporting
Information. Acylation of indoline and 2,3-dimethylindole
was accomplished with substituted or non-substituted 4-
methoxy or 4-methoxymethoxy-3-cyanobenzoic acid and 1-
(3-dimethylaminopropyl)-3-ethylcarbodiimide
To verify our strategy, these compounds were evaluated
for their urate uptake inhibitory potency and mitochondrial
toxicity. We established a novel method to evaluate the UUI
of these compounds using primary human renal proximal
tubule epithelial cells (RPTECs) (Method: Supporting
Information). RPTECs play important roles in urate
excretion and reabsorption. In fact, they can express many
hydrochloride (EDC). The subsequent de-protection of
phenol –OH yielded the desired analogs.
transporters including urate transporter
1 (URAT1),
organic anion transporter 1 (OAT1), OAT3, multidrug
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Scheme 1. Synthesis of Indole and Indoline Analogs a
1
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Figure 2. Correlation between the MIA (RCR, IC50) and compounds’
LogP in Table 2. Compound 2a, 2d, and 2i showed ca.50% MIA at
100 M; these IC50 values are assigned as 100 M, Compound 2b
showed no MIA at 100 M; the IC50 value is assigned as 200 M.
a
Reagents and conditions: (a) EDC, HOBt in DMF, CHCl3; (b) R2 =
MOM; HCl, 1,4-Dioxane, THF or TFA, CH2Cl2, R2 = Me; LiCl, DMF, 35–
91% over two steps.
Although causes for the association between
hydrophobicity and MIA is unclear, we speculate that it was
dependent on membrane permeability and affinities to
target proteins.
The assay data of UUI and MIA, and the calculated LogP of
these derivatives are shown in Table 2.
Table 2. UUI and MIA of Compounds 1 and 2 in
Scheme 1
The association between UUI at 10 M and MIA, which
should ideally diverge from each other, is shown in Figure
3.
UUI a
MIA b
IC50 (M)
or %
LogP
Compound
R1
c
IC50
(M)
%
10.8
52.1
67.5
21.7
65.1
26.3
24.2
32.4
67.8
38.2
30.0
3.6
3.89
2.70
2.57
4.05
2.73
3.14
4.4
1
-
2a
2b
2c
2d
2e
2f
H
2.5
>100
5.3
43%
OMe
-10%
53 M
47%
I
CN
83.9
>100
31.6
8.8
SMe
40 M
6.6 M
24 M
31 M
40%
tert-Butyl
Cyclopropyl
Cl
2g
2h
2i
3.43
3.26
3.62
14.7
>100
Figure 3. Association between UUI (% inhibition at 10 M) and MIA
(RCR, IC50) derived from Table 2 and Figure 2.
CF3
a UUI: Inhibitory activity of urate uptake into RPTECs, IC50 and %
inhibition at 10 M. b MIA values are defined in Table 1. c LogP was
calculated using ChemBioDraw UltraVer.14.
Although compounds with higher UUI tended to show
higher MIA, 2a, 2c, and 2g showed promising in vitro
character: high UUI and low MIA. Similar association were
observed when UUI was evaluated at higher concentrations
(30 or 100 M). Therefore, the Cmax and urinary excretion
rate (Exc.) of 2a, 2c, and 2g were then investigated as the
pharmacokinetic (PK) screening compared to BBR (Table
3).
UUI potency seemed to be affected by substituent
characters such as steric and electric properties including
hydrophobicity. Although 2a, 2c, and 2g showed a relatively
high activity, the bulky substituent (2f) had a lower potency
compared to these three compounds presumably resulting
from the disruption of the flatness of the molecules shared
by uric acid. The electron donating (2b) and withdrawing
(2d, 2i) groups affected pKa and their potencies.
Furthermore, hydrophobicity seems to influence UUI
potency, thus UUI improved with increasing LogP values,
except for one-substituted (2a) and bulky (2f) types.
Table 3. Pharmacokinetic Profiles of 2a, 2c, 2g, and
BBR
Exc. b (%)
5.9
Cmax a (g/mL)
0.89
Compound
R1
2a
2c
H
I
5.95
0.02
0.35
0
As shown in Figure 2, a correlation was observed between
MIA and LogP among the compounds in Table 2, in which
MIA augmented with increasing LogP values. Although
almost all compounds were close to the regression line (R2
= 0.54), compound 1 (indole derivative) showed an inferior
character with stronger MIA than the other compounds. In
contrast, 2c and 2i had weaker MIA for their LogP than the
other compounds did.
2g
Cyclopropyl
-
1.36
BBR
4.42
a Maximum concentration (Cmax). b Excretion rate in urine (%, 0–4
h) after oral administration of 3 mg/kg to rats.
To produce a pharmacological effect, special attention was
given to the Exc. (%) of compounds based on the PK profiles
of BBR in humans reported by some groups.28-33 Although
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BBR is typically eliminated from plasma at 48 h after
Table 4. Properties of Compound 7
1
2
3
4
5
6
7
8
administration and is excreted little in urine, a main
29
metabolite, 6-OH-BBR,28, remains in the plasma and is
excreted (1.2% of the dose) over 72 h in humans;30-32 this
metabolites’ URAT1 inhibitory activity was only several
times lower than that of BBR (6-OH-BBR IC50: 0.20 M, BBR
32
IC50: 0.0345 M).31,
As a long lasting effect of BBR in
UUI a
IC50
(M)
MIA b
IC50
(M)
c
Cmax
Exc. d
(%)
CYP2C
9 IC50
(M)
-
humans 33 is consistent with the sustained urinary excretion
of 6-OH-BBR, and URAT1 is present on the luminal side of
epithelial cells of the renal tubule, it is suggested that 6-OH-
BBR in urine contributes to the inhibition of uric acid
reabsorption.32, 33 Moreover, unlike BBR, the compounds in
this study should not form active metabolites to avoid
metabolic conversion by enzymes such as CYP2C9.
Therefore, we considered that the excretion of unchanged
drug with activity equivalent to that of BBR in urine is not
only important to show activity, but also for the ideal
property. Hence, to obtain candidate compounds, PK
screening was performed with a target value of >1% of
unchanged drug in 0–4 h urine in rat, based on the above 6-
OH-BBR data.
Compound
(g/mL)
8.8
19.2
6.8
24
59
3.1
2g
7
1.36
3.81
4.42
0.35
0.6
0
9
57
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BBR
0.041
a UUI and b MIA are defined in Table 1. c Cmax and d Exc. are
defined in Table 3.
Compared to 2g, compound
7 showed satisfactory
properties, with adequate UUI, weak MIA, and improved
Cmax; furthermore, 7 had a higher Exc. than 2g did and
appeared to have low CYP2C9 inhibitory activity (IC50 = 57
M). Because 7 was considered a lead compound, we
prepared 1,2-dihydro-3H-1,3-benzothiazole derivatives by
converting the phenol substituents (Table 5). Although the
synthetic method for these derivatives was previously
described,27 additional information is added in Supporting
Information.
Of the three compounds, only 2c showed a high Cmax
,
while its Exc. was low (0.02%). We concluded that the
metabolic susceptibility of the core indoline structure with
benzyl-CH2 was one of the causes of the inferior PK
profiles. Therefore, we then investigated another 1,1-
dioxo-1,2-dihydro-3H-1,3-benzothiazole derivative with -
SO2- instead of benzyl-CH2- in the core that would be of
metabolic resistance. Compound 7 of the new core
structure with cyclopropyl substituent was prepared and
compared with the indoline-type analog 2g and BBR
regarding their UUI, MIA, PK, and CYP2C9 inhibitory assays
(Table 4).
Many compounds showed potent UUI with single-digit M
(IC50) activity, whereas 8 and 10, having two electron-
withdrawing substituents, had low UUI (Table 5). However,
compared to 8 (core; -SO2-), 9 (core; -S-) maintained its UUI
potency as the substituent effect may be partially due to the
hydrophobicity of compounds. Among compounds with
high potency, 11, 14, and 15 showed Exc. >1%, and 11 had
the highest Cmax
.
Table 5. UUI, MIA, and Pharmacokinetic Profiles of 1,2-Dihydro-3H-1,3-benzothiazole Derivatives
*Compounds 9(n = 0), 8, 10–20 (n = 2)
R2
UUI a
MIA b
Cmax
Exc. d (%)
c
Compound
R1
IC50 (M)
IC50 (M) or %
(g/mL)
CF3
CF3
Cl
>100
13.1
>100
6.8
12%
55 M
20%
-
-
8
CN
CN
CN
Cl
3.15
1.35
9.14
4.46
4.24
1.58
1.08
6.41
7.70
4.64
3.99
1.06
0.1
23
9*
10
11
12
13
14
15
16
17
18
19
20
Cl
1.1
0.1
1.7
12.4
9.4
0.1
4.3
0.2
0
27 M
12 M
37 M
13%
CN
CN
CN
CN
CF3
F
iPr
83.3
>100
3.2
Et
ethynyl
SMe
Cl
7.1
21%
2.6
18 M
63 M
59 M
47%
Cl
33.9
5.7
Cl
SMe
OMe
OMe
CF3
Cl
17.8
4.3
49%
0.1
a UUI and b MIA are defined in Table 1. c Cmax and d Exc. are defined in Table 3.
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fractional excretion of urate at 0–4 h (FEUA; 16.5% vs. 11.5%
The URAT1 assay was performed for some compounds to
confirm the mechanism of action (Table 6). These
compounds showed URAT1 inhibitory activity (0.1 M)
along with UUI in RPTECs (10 M). A positive correlation
was observed between the two assays as shown in the
analysis of the 7 compounds in Table 6 (R2 = 0.59; Figure
4), and furthermore, in a dose-dependency test of 11 (R2 =
0.87; Figure 4-1 in Supporting Information). Thus, urate
uptake into RPTECs seems to be mainly mediated by URAT1,
as mentioned above.
1
2
3
4
5
6
7
8
of 30 mg/kg of BBR). Moreover, 11 decreased the plasma
urate level more potently than did BBR in Cebus monkeys.
This potent effect has not been reported with conventional
medicines.
Because compound 11 only weakly inhibited ABCG2 and
OAT1/3, it was shown to be a selective urate reabsorption
inhibitor (SURI), which was defined as a potent URAT1
inhibitor with minimal effect on urate secretion
transporters.34
9
10
11
12
13
14
15
16
17
18
19
20
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27
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Table 6. Urate Transporter 1 Inhibition and UUI of
1,2-Dihydro-3H-1,3-benzothiazole Derivatives and
BBR
Table 7. Properties of 11 and BBR
URAT1 inhibition a
(%)
UUI b
(%)
Compound
7
9
30
71
29
78
36
44
37
42
49
25
81
32
57
51
11 (dotinurad)
a
b
Cmax or C0
ng/mL
1085.8
± 100.4
2401.3
± 65.6
AUC0-inf
ng·h/mL
5744.7
BA. c
86.8%
Rat
10
11
13
15
BBR
11
0.3 mg/kg p.o.
11
0.3 mg/kg i.v.
± 188.8
6619.5
± 2041.8
Difference of
a URAT1 inhibitory activity; % inhibition at 0.1 M. b UUI are
defined in Table 1, % inhibition at 10 M.
b
d
e
Cebus
AUC0–24h
FEUA
ΔPUA
monkeys
μg·h/mL
(%)
from control
(mg/dL)
11
108.55
± 52.94
16.5
± 4.2
0.97
5 mg/kg p.o.
BBR
95.24
11.5
0.46
30 mg/kg p.o.
± 28.12
± 7.9
In vitro
evaluation
URAT1 inhibition
IC50 (nM)
11
37.2
BBR
190
a
b
Maximum concentration (Cmax or C0). Area under the blood
Figure 4. Correlation between the RPTEC and URAT1 assays among
the compounds in Table 6.
concentration-time curve (AUC); inf: infinity. c BA.: Bioavailability. d
FEUA: Fractional excretion of urate at 0–4 h; FEUA value of control:
8.9 ± 4.0 %. e ΔPUA: Changes in plasma urate level at 0–8 h; value of
control: 1.65 ± 0.78 mg/dL.
The dichloro-substituted derivative 11 appeared to
possess highly potent UUI and URAT1 inhibitory activities
at least equivalent to that of BBR, low MIA, and high Cmax
with moderate urinary excretion rate (>1%, 0–4 h).
Although 11 showed weak CYP2C9 inhibitory activity (IC50
= 5.7 M), its activity was thought to be sufficiently
separated from uricosuric activity based on its PK profile. As
11 (dotinurad) had a favorable balance of these parameters,
it was selected for further development. The results of the
additional in vitro and in vivo assay using rats and Cebus
monkeys are shown in Table 7 and Figure 5.
The major metabolites of 11 in humans are glucuronide
and sulfate of phenols, with excretion rates in urine during
0–72 h of 44.3% and 20.0%, respectively, in a mass balance
test.35 The formation of reactive metabolites with potential
to cause hepatotoxicity, as reported for BBR,13 was not
observed. These results suggested that 11 had a safe
metabolic pathway.
A detailed URAT1 assay of 11 showed an IC50 of 37.2 nM,
indicating slightly higher potency than that of BBR (original
data, IC50 =190 nM), similar to the equipotent inhibition in
the RPTEC assay. Compound 11 had excellent PK profiles in
both animals. In Cebus monkeys, which could be an effective
model for extrapolating the uricosuric effect in humans, 5
mg/kg of 11 showed a potent uricosuric effect with the
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ACS Medicinal Chemistry Letters
We thank Koichi Omura for his contribution to the analytical
Page 6 of 7
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work.
2
3
ABBREVIATIONS
DMF, N,N-dimethylformamide; MOM, methoxymethyl; EDC,
4
5
1-(3-dimethyl
aminopropyl)-3-ethyl
carbodiimide
hydrochloride; m-CPBA, m-chloroperbenzoic acid; TFA,
trifluoroacetic acid; CYP2C9, cytochrome P450 2C9; URAT1,
urate transporter 1; ABCG2, ATP-binding cassette sub-
family G member 2; OAT1/3, organic anion transporter 1/3;
MRP4, multidrug resistance protein 4; GLUT9, glucose
transporter 9.
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11
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50
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60
Figure 5. Changes in plasma urate level compared with the pre-
dosing value in Cebus monkeys. 11 (dotiurad); 5 and 30 mg/kg p.o.
BBR; 30 mg/kg. Values are the means + S.D. of five animals. The
average pre-dosing value of control: 2.82 mg/dL. * P<0.05, **
P<0.01, significantly different from control at each time point by
Dunnett’s test.
REFERENCES
(1) Benn, C. L.; Dua, P.; Gurrell, R.; Loudon, P.; Pike, A.; Storer,
R. I.; Vangjeli, C. Physiology of Hyperuricemia and Urate-
Lowering Treatments. Front. Med. 2018, 5, 160.
(2) Kanbay, M.; Afsar, B.; Covic, A. Uric Acid as
a
Cardiometabolic Risk Factor: To Be or Not to Be. Contrib.
Nephrol. 2011, 171, 62–67.
In conclusion, this series of studies led to the discovery of
compound 11. We proceeded to clinical trials with 11 and
obtained excellent results. The registration of 11 as a new
medicine (dotinurad: FYU-981) has been approved in Japan.
(3) Lipkpwitz, M. S. Regulation of Uric Acid Excretion by the
Kidney. Curr. Rheumatol. Rep. 2012, 14, 179–188.
(4) Diaz-Torné, C.; Perez-Herrero, N.; Perez-Ruiz, F. New
Medications in Development for the Treatment of
Hyperuricemia of Gout. Curr. Opin. Rheumatol. 2015, 27,
164–169.
We obtained new medicine by focusing on the negative
aspects of existing drugs. The method used to avoid
chemical structures that may cause unfavorable effects
could be applied to other drugs with disadvantages.
(5) Diamond, H. S.; Paolino, J. S. Evidence for a Postsecretory
Reabsorptive Site for Uric Acid in Man. J. Clin. Invest. 1973,
52, 1491–1499.
ASSOCIATED CONTENT
Supporting Information
(6) Silverman, W.; Locovei, S.; Dahl, G. Probenecid, a Gout
Remedy, Inhibits Pannexin 1 Channels. Am. J. Physiol. Cell
Physiol. 2008, 295, C761–C767.
The Supporting Information is available free of charge on
the ACS Publications website.
(7) Tanizaki, R.; Nishijima, T.; Aoki, T.; Teruya, K.; Kikuchi,
Y.; Oka, S.; Gatanaga, H. High-Dose Oral Amoxicillin Plus
Probenecid Is Highly Effective for Syphilis in Patients with
HIV Infection. Clin. Infect. Dis. 2015, 2, 177–183.
Details on the preparation of compounds 1, 2a–i (including
synthetic procedures), in vitro assays, and PK assays are
available in the Supporting Information. Although the
preparation methods for compounds 7–20 were described
previously in a patent 27 we have included some additional
chemical data (PDF).
(8) Lee, M. H.; Graham, G. G.; Williams, K. M.; Day, R. O. A
Benefit-Risk Assessment of Benzbromarone in the
Treatment of Gout. Was its Withdrawal from the Market in
the Best Interest of Patients? Drug Saf. 2008, 31, 643–665.
AUTHOR INFORMATION
Corresponding Authors
*E-mail: j-uda@fujiyakuhin.co.jp
ORCID
(9) Sistonen, J.; Fuselli, S.; Palo, J. U.; Chauhan, N.; Padh, H.;
Sajantila A. Pharmacogenetic Variation at CYP2C9,
CYP2C19, and CYP2D6 at Global and Microgeographic
Scales. Pharmacogenet. Genomics. 2009, 19, 170–179.
Junichiro Uda: 0000-0001-8143-5249
Author Contributions
(10) Dalbeth, N.; Jones, G.; Terkeltaub, R.; Khanna, D.;
Kopicko, J.; Bhakta, N.; Adler, S.; Fung, M.; Storgard, C.;
Baumgartner, S.; Perez-Ruiz, F. Lesinurad, a Selective Uric
Acid Reabsorption Inhibitor, in Combination with
Febuxostat in Patients with Tophaceous Gout: Findings of a
Phase III Clinical Trial. Arthritis Rheumatol. 2017, 69, 1903–
1913.
The manuscript was written through the contributions of all
authors.
Funding Sources
No external funding was received for this study.
Notes
(11) Abhishek, A. New Urate-Lowing Therapies. Curr. Opin.
Rheumatol. 2018, 30, 177–182.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
(12) Dean, L. Lesinurad Therapy and CYP2C9 Genotype,
Medical
Genetics
Summaries
[Internet].
https://www.ncbi.nlm.nih.gov/books/NBK537366/
(accessed March 13, 2020). Created: February 11, 2019.
6
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ACS Medicinal Chemistry Letters
(13) McDonald, M. G.; Rettie, A. E. Sequential Metabolism
and Bioactivation of the Hepatotoxin Benzbromarone:
Formation of Glutathione Adducts from Catechol
(26) Tan P. K.; Ostertag, T. M.; Miner J. N. Mechanism of High
Affinity Inhibition of the Human Urate Transporter URAT1.
Sci. Rep. 2016, 6, 34995.
1
2
3
4
5
6
7
8
a
Intermediate. Chem. Res. Toxicol. 2007, 20, 1833–1842.
(27) Kobashi, S.; Uda, J.; Miyata, S.; Inoue, T.; Ashizawa, N.;
Matsumoto, K.; Taniguchi, T.; Iwanaga, T.; Nagata, O. Novel
Phenol Derivative. US 8,367,843 B2, February 5, 2013.
(14) Ohe, T.; Umezawa, R.; Kitagawara, Y.; Yasuda, D.;
Takahashi, K.; Nakamura, S.; Abe, A.; Sekine, S.; Ito, K.;
Okunushi, K.; Morio, H.; Furihata, T.; Anzai, N.; Mashino, T.
Synthesis of Novel Benzbromarone Derivatives Designed to
Avoid Metabolic Activation. Bioorg. Med. Chem. Lett. 2018,
28, 3708–3711.
(28) Walter-Sack, I.; de Vries, J. X.; von Bubnoff, A.;
Pfleilschifter, V.; Raedsch, R. Biotransformation and Uric
Acid Lowering Effect of Benzbromarone in Patients with
Liver Cirrhosis - Evidence for Active Benzbromarone
Metabolites? Eur. J. Med. Res. 1995, 1, 16–20.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(15) Ai, Q.; Jing, Y.; Jiang, R.; Lin, L.; Dai, J.; Che, Q.; Zhou, D.;
Jia, M.; Wan, J.; Zhang, L. Rotenone, a Mitochondrial
(29) Walter-Sack, I.; de Vries, J. X.; Ittensohn, A.; Raedsch, R.
Biliary Excretion of Benzbromarone and its Hydroxylated
Main Metabolites in Humans. Eur. J. Med. Res. 1998, 3, 45–
49.
Respiratory
Complex
I
Inhibitor,
Ameliorates
Lipopolysaccharide/D-Galactosamine-Induced Fulminant
Hepatitis in Mice. Int. Immunopharmacol. 2014, 21, 200–
207.
(30) Oikawa, T.; Kunishima, C.; Adachi, Y.; Sato, Y.; Okamura,
Y.; Tanaka, N.; Hosoda, T. Metabolism Study of
Benzbromarone: In vivo Metabolism and Pharmacokinetics
in Healthy Volunteers. J. New Rem.  Clin. 2004, 53, 682–
691.
(16) Meyer, J. N.; Leuthner, T. C.; Luz, A. L. Mitochondrial
Fusion, Fission, and Mitochondrial Toxicity. Toxicology.
2017, 391, 42–53.
(17) Spaniol, M.; Bracher, R.; Ha, H. R.; Follath, F.;
Krähenbühl, S. Toxicity of Amiodarone and Amiodarone
Analogues on Isolated Rat Liver Mitochondria. J. Hepatol.
2001, 35, 628–636.
(31) Oikawa, T.; Kunishima, C.; Matsumoto, K.; Ishikawa, S.;
Endou, H. The Inhibitory Effect of Benzbromarone and 6-
Hydroxybenzbromarone on Urate Transporter (URAT1). J.
New Rem.  Clin. 2005, 54, 645–650.
(18) Kaufmann, P.; Török, M.; Hänni, A.; Roberts, P.; Gasser,
R.; Krähenbühl, S. Mechanisms of Benzarone and
Benzbromarone-Induced Hepatic Toxicity. Hepatology.
2005, 41, 925–935.
(32) Endou, H.; Oikawa, T. Medicinal Compositions
Containing 6-Hydroxybenzbromarone or Salts Thereof. US
7,521,570 B2, April 21, 2009.
(19) Waldhauser, K. M.; Török, M.; Ha, H. R.; Thomet, U.;
Konrad, D.; Brecht, K.; Follath, F.; Krähenbühl, S.
Hepatocellular, Toxicity and Pharmacological Effect of
Amiodarone and Amiodarone Derivatives. J. Pharmacol. Exp.
Ther. 2006, 319, 1413–1423.
(33) Jain, A. K.; Ryan, J. R.; McMahon, F. G.; Noveck, R. J. Effect
of Single Oral Doses of Benzbromarone on Serum and
Urinary Uric Acid. Arthritis. Rheum. 1974, 17, 149–157.
(34) Taniguchi, T.; Ashizawa, N.; Matsumoto, K.; Saito, R.;
Motoki, K.; Sakai, M.; Chikamatsu, N.; Hagihara, C.; Hashiba,
M.; Iwanaga, T. Pharmacological Evaluation of Dotinurad, a
Selective Urate Reabsorption Inhibitor. J. Pharmacol. Exp.
Ther. 2019, 371, 162–170.
(20) Barratt, M. D. Structure-Activity Relationships, and
Prediction of the Phototoxicity and Phototoxic Potential of
New Drugs. Altern. Lab. Anim. 2004, 32, 511–524.
(21) Brunmair, B.; Lest, A.; Staniek, K.; Gras, F.; Scharf, N.;
Roden, M.; Nohl, H.; Waldhäusl, W.; Fürnsinn, C. Fenofibrate
Impairs Rat Mitochondrial Function by Inhibition of
Respiratory Complex I. J. Pharmacol. Exp. Ther. 2004, 311,
109–114.
(35) Omura, K.; Miyata, K.; Kobashi, S.; Ito, A.; Fushimi, M.;
Uda, J.; Sasaki, T.; Iwanaga, T.; Ohashi, T. Ideal
Pharmacokinetic Profile of Dotinurad as a Selective Urate
Reabsorption Inhibitor. Drug Metab. Pharmacokinet. 2020,
35, 313-320.
(22) Grünig, D.; Felser, A.; Bouitbir, J.; Krähenbühl, S. The
Catechol-O-Methyltransferase Inhibitors Tolcapone and
Entacapone Uncouple and Inhibit the Mitochondrial
Respiratory Chain in HepaRG Cells. Toxicol. in Vitro 2017,
42, 337–347.
(23) Xu, L.; Shi, Y.; Zhuang, S.; Liu, N. Recent Advances on
Uric Acid Transporters. Oncotarget 2017, 8, 100852–
100862.
(24) Xiao, J.; Zhang, X.; Fu, C.; Yang, Q.; Xie, Y.; Zhang, Z.; Ye,
Z. Impaired Na+−K+-ATPase Signaling in Renal Proximal
Tubule Contributes to Hyperuricemia-induced Renal
Tubular Injury. Exp. Mol. Med. 2018, 50, e452.
(25) McDonagh, E. M.; Thorn, C. F.; Callaghan, J. T.; Altman,
R. B.; Klein, T. E. PharmGKB summary: Uric Acid-Lowering
Drugs Pathway, Pharmacodynamics. Pharmacogenet.
Genomics 2014, 24, 464–476.
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