Novel Human Urate Transporter 1 Inhibitors as Hypouricemic Drug Candidates with Favorable Druggability

Article abstract of DOI:10.1021/acs.jmedchem.0c00223

Lesinurad, a human urate transporter 1 (URAT1) inhibitor approved as a medication for the treatment of hyperuricemia associated with gout in 2015, can cause liver and renal toxicity. Here, we modified all three structural components of lesinurad by applying scaffold hopping, bioisosterism, and substituent-decorating strategies. In a mouse model of acute hyperuricemia, 21 of the synthesized compounds showed increased serum uric acid (SUA)-reducing activity; SUA was about 4-fold lower in animals treated with 44, 54, and 83 compared with lesinurad or benzbromarone. In the URAT1 inhibition assay, 44 was over 8-fold more potent than lesinurad (IC50: 1.57 μM vs 13.21 μM). Notably, 83 also displayed potent inhibitory activity (IC50 = 31.73 μM) against GLUT9. Furthermore, we also preliminarily explored the effect of chirality on the potency of the promising derivatives 44 and 54. Compounds 44, 54, and 83 showed favorable drug-like pharmacokinetics and appear to be promising candidates for the treatment of hyperuricemia and gout.

Full text of DOI:10.1021/acs.jmedchem.0c00223

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Article
Novel Human Urate Transporter 1 Inhibitors as
Hypouricemic Drug Candidates with Favorable Druggability
Tong Zhao, Qing Meng, Zhuosen Sun, Yanyu Chen, Wei Ai, Zean Zhao, Dongwei
Kang, Yue Dong, Ruipeng Liang, Ting Wu, Jianxin Pang, Xinyong Liu, and Peng Zhan
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00223 • Publication Date (Web): 08 Sep 2020
Downloaded from pubs.acs.org on September 8, 2020
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Novel Human Urate Transporter 1 Inhibitors as Hypouricemic Drug
Candidates with Favorable Druggability
Tong Zhao,†, § Qing Meng,†, § Zhuosen Sun, Yanyu Chen, Wei Ai, Zean Zhao, Dongwei Kang,
†
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Yue Dong, Ruipeng Liang, Ting Wu, Jianxin Pang, Xinyong Liu,*, † and Peng Zhan*, †
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†
‡
*, ‡
†Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of
Education), School of Pharmaceutical Sciences, Cheeloo College of Medicine, Shandong
University, 44 West Culture Road, 250012 Jinan, Shandong, PR China
‡School of Pharmaceutical Sciences, Southern Medical University, 1838 North Guangzhou Ave, 5
1
0515 Guangzhou, PR China
§These authors contributed equally to this work and should be considered as co-first authors
Abstract
Lesinurad, a URAT1 inhibitor approved as a medication for the treatment of hyperuricemia
associated with gout in 2015, can cause liver and renal toxicity. Here, we modified all three
structural components of lesinurad by applying scaffold hopping, bioisosterism and substituent
decorating strategies. In a mouse model of acute hyperuricemia, 21 of the synthesized compounds
showed increased SUA-reducing activity; SUA was about 4-fold lower in animals treated with 44,
5
4 and 83, compared with lesinurad or benzbromarone. In URAT1 inhibition assay, 44 was over
8-fold more potent than lesinurad (IC : 1.57 μM vs. 13.21 μM). Notably, 83 also displayed potent
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inhibitory activity (IC = 31.73 μM) against GLUT9. Furthermore, we also preliminarily explored
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the effect of chirality on the potency of the promising derivatives 44 and 54. Compounds 44, 54
and 83 showed favorable drug-like pharmacokinetics, and appear to be promising candidates for
the treatment of hyperuricemia and gout.
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Introduction
Uric acid, the final product of purine catabolism, is physiologically excreted in the urine.
Endogenously produced uric acid accounts for approximately 80% of the total serum uric acid
(SUA), while the remainder is derived from dietary purines. Most bacteria and mammals
decompose uric acid via uricase-mediated metabolism to afford water-soluble allantoin. However,
humans lack uricase , and uric acid is predominantly eliminated via renal excretion.1–6 Thus,
overproduction of uric acid or insufficient renal elimination causes hyperuricemia, which is
generally defined as a serum level of uric acid > 6.8 mg/dL. Hyperuricemia can be asymptomatic,
but when the serum uric acid concentration exceeds 6.8 mg/dL, monosodium urate crystallizes and
is deposited in joints or surrounding tissues -- this is the cause of gout, a severe disease that affects
millions of people worldwide, especially adult men.7,8 Hyperuricemia is also associated with a
range of chronic diseases, including hypertension, diabetes mellitus, metabolic syndrome, and
renal and cardiovascular diseases.9–11
For a long time, the preferred drugs for clinical treatment of hyperuricemia were xanthine
oxidase (XO) inhibitors, such as allopurinol (1), oxypurinol (2) and febuxostat (3) (Figure 1),12
which reduce the rate of uric acid synthesis.13,14 However, an appreciable proportion of patients do
not respond well to XO inhibitors due to serious side effects and the development of resistance.
For example, allopurinol (1) can cause severe allergic rash, stomach bleeding, fever, abdominal
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pain, diarrhea and thrombocytopenia, while febuxostat can cause severe liver damage and
cardiovascular side effects. Furthermore, in 2017, the U.S. Food and Drug Adminstration (FDA)
warned that febuxostat might increase the risk of sudden cardiac death.15–17 Uricosuric drugs are
another option for patients who are intolerant of XO inhibitors. These agents reduce reabsorption
and thus increase the excretion of uric acid. Multiple transporters are involved in reabsorption. For
example, human urate transporter 1 (URAT1, encoded by SLC22A12) and glucose transporter 9
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(GLUT9, encoded by SLC2A9) are both involved in reabsorption of urate, and high levels of
URAT1 and GLUT9 expression may cause hyperuricemia or gout.18–20 URAT1 mainly regulates
tubular reabsorption of urate through an ion-exchange mechanism with organic anions at the
apical membrane of renal proximal tubule cells.21 Inhibition of URAT1 or GLUT9 blocks
reabsorption of urate anion, thereby enhancing renal uric acid excretion, and mutations in
hURAT1 have been shown to reduce the urate-transporting activity, leading to hypouricemia.7,22,23
So far, four major uricosuric drugs have come onto the market, namely probenecid (4),
sulfinpyrazone (5), benzbromarone (6) and lesinurad (7). Lesinurad, approved in 2015 by the
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FDA, increases the excretion of uric acid by inhibiting URAT1 (Figure 2). Thus, URAT1 is an
attractive target for the development of novel uricosuric agents. Nevertheless, uricosuric drugs can
also cause side effects and may exhibit poor efficacy. For example, benzbromarone had to be
withdrawn from the European market due to its severe liver and kidney toxicity and potent
inhibition of cytochrome P450 (CYP) metabolic enzymes.24 Also, the FDA has advised that
lesinurad should be used with caution because of its severe renal toxicity and liver toxicity. This is
problematic, because the SUA-lowering activity of lesinurad is not potent. Furthermore, lesinurad
can only used in combination with the XO inhibitor febuxostat with the approval of the FDA,25-27
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because the cardiovascular toxicity of febuxostat increases the risk of co-administration.28,29
Therefore, there is still an urgent need to develop safe, potent and specific URAT1 inhibitors as
novel hypouricemic agents.
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Benzbromarone (6)
Lesinurad (7)
Figure 1. The structures of representative drugs used in the treatment of gout.
Organic
anions
Organic
anions
URAT1
OAT4
Benzbromarone
Organic anions
Diuretics
Lesinurad
urate
urate
urate
Blood
Urine
Figure 2. Mechanism of action of lesinurad.
Thus, although lesinurad is the newly approved URAT1 inhibitor and has a clear mechanism
of action, it has many shortcomings. In this context, we decided to conside it as a lead compound
and we set out to optimize its structure for improved safety and activity. Lesinurad contains three
structural components, namely the triazole ring (zone A), cyclopropyl naphthalene (zone B) and
the sulfhydryl side chain containing a carboxyl group (zone C). As regards zone A, recent work on
selective uric acid reabsorption inhibitors has suggested that the triazole moiety might mainly
serve as a scaffold to orient the pharmacophores in the proper geometry for binding to the target
enzyme, and we suspected that the brominated triazole moiety in the parent structure might
contribute to the toxicity and serious side effects. It has been reported that differences in the
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electronic and conformational features of the heterocyclic core influence the binding of inhibitors
to URAT1, as well as the toxicity.30,31 Nevertheless, there has been relatively little recent work on
the structure-activity relationship (SAR) and structural modifications of zone A.32 A classical
strategy in contemporary medicinal chemistry for ‘follow-on’-based drug discovery to obtain
novel hits, as well as to improve drug-like and safety properties, is scaffold refining via the
replacement of the central core in existing bioactive lead compounds, combined with the
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introduction of privileged substituents.33,34 However, in work reported so far, the original triazole
ring has only been replaced with different five- or six-membered heterocyclic scaffolds.35 Thus,
there is still a large chemical space to be explored in zone A; in particular, the triazole moiety can
be readily modified in terms of its ability to form hydrogen bonds, and scaffold hopping and
bioisosterism strategies are expected be applicable.32 As for zone B, cyclopropyl naphthalene is
considered to be a functional pharmacophore, having a hydrophobic or van der Waals’ interaction
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with the binding pocket of URAT1. Therefore, exploration of the SAR of this part by means of
structural diversification should be a promising approach to obtain better activity and safety.37,38
As for zone C, the carboxylic acid moiety may be a critical feature, since URAT1 is an anion
transporter. However, this group contributes to the strong acidity and high polarity of lesinurad,
which may be related to the safety. Therefore, we decided to adopt a strategy of bioisosterism to
replace the carboxylic acid in order to reduce the molecular polarity and toxicity.39, 41-42 During the
implementation of our project, some medicinal chemists have reported exploratory modifications
of zones B and C, and several promising lead compounds were obtained, as exemplified by 7a-c
(
Figure 3).36-40 Nevertheless, we envision that a more detailed and systematic evaluation of the
SAR and structure-toxicity relationships is likely to be fruitful. Therefore, in the present work, we
examined the effects of structural modification in all three zones, especially zone C, aiming to find
novel URAT1 inhibitors with lower toxicity, greater potency, and possibly new mechanisms of
action.
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Figure 3. Reported modifications of zones B and C.
Based on these ideas, we focused on scaffold hopping and bioisosterism strategies to explore
the SARs of unexploited scaffolds bearing privileged peripheral substituents (Figure 4). Firstly,
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H-imidazole[4,5-b]pyridine, 1H-imidazole[4,5-c]pyridine and 1H-imidazole[4,5-b]pyridine, to
replace zone A in lesinurad. Then, we investigated the effect of changing the aromatic substituent
in zone B to mesitylene, naphthalene or cyanonaphthalene. Lastly, we modified zone C to obtain
diverse substituted acylsulfonamides via a bioisosterism approach. We also made various
structural modifications in zone C (increasing the length of the side chain, substituting the carbon
atom at the ortho-position of the sulfur atom with a methyl, dimethyl or cyclobutyl group, and
esterifying the terminal carboxyl group) that were expected to improve the pharmacokinetic
properties. All the newly prepared compounds were evaluated in a well developed mouse model
of acute hyperuricemia induced with hypoxanthine and potassium cyanate. A pharmacokinetics
study of the most promising compounds 44, 54 and 83 in rats indicated that they have favorable,
drug-like pharmacokinetics.
A
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or
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Target Compounds
Target Compounds
Figure 4. Design of target compounds via scaffold hopping and bioisosterism strategies.
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Based on the bioisosterism strategy, we also designed and synthesized a series of
N-acylbenzene sulfonamide analogues derived from the carboxylic acid moiety (zone C) (Figure
4
). Some of the prepared compounds showed more potent activity than lesinurad, and a
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pharmacokinetics study of the most potent compound 83 was carried out. As expected, 83 showed
favorable, drug-like pharmacokinetics. This compound also appears to be a promising candidate
for the treatment of hyperuricemia and gout.
Chemistry
The synthetic protocols for the newly designed derivatives are outlined in Schemes 1−6. As
shown in Scheme 1, 3H-imidazole[4,5-b]pyridine derivatives were synthesized by
well-established methods from commercially available 2-chloro-3-nitropyridine (8a). Treatment
of 8a with naphthalen-1-amine (9a) afforded the intermediate 10a. The nitro group of 10a was
reduced via Pd/C catalytic hydrogenation to form 11a, followed by cyclization with potassium
ethylxanthate and sodium bicarbonate to afford 12a. Nucleophilic substitution reactions of 12a
afforded 13-16, and hydrolysis with lithium hydroxide gave 17-20 (Scheme 1). The preparation
method of compound 21-28 and 29-36 were similar to those of 13-20 except that the amines (9b
and 9c) used as starting material were different. 43,44
The synthetic route to 37-46 is shown in Scheme 2. 3-Chloro-2-nitropyridine (8b) was
treated with 4-cyclopropyl-1-naphthylamine (9c) to afford intermediate 10d via
Buchwald-Hartwig coupling reaction. Then, hydrogenation reduction in the presence of Pd/C gave
11d, which was cyclized with 1,1'-thiocarbonyldiimidazole to give the key intermediate 12d,
followed by nucleophilic substitution and hydrolysis to provide 37-46.
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As shown in Scheme 3, nucleophilic substitution of 4-chloro-3-nitropyridine (8c) with
-cyclopropylnaphthalen-1-amine (9c) gave the intermediate 10e, which afforded 47-56 via
4
similar procedures to those shown in Scheme 1.45
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The synthetic route to 57-80 is shown in Scheme 4. 4-Bromo-1-naphthonitrile (9d) was
treated with 2-nitropyridin-3-amine (8d) or 3-nitropyridin-4-amine (8e) to afford intermediate 10f
and 10g via coupling reaction. Then, hydrogenation reduction in the presence of Pd/C gave 11f
and 11g, which were cyclized with 1,1'-thiocarbonyldiimidazole to give the key intermediates 12f
and 12g, followed by nucleophilic substitution and hydrolysis to provide 57-80.46
The synthetic routes to 81-96 are depicted in Scheme 5. The starting material,
4
-bromonaphthalen-1-amine (9e), was converted to 4-cyclopropylnaphthalen-1-amine (9c) via a
Suzuki coupling reaction in
a
toluene/water mixture, and 9c was reacted with
di(1H-imidazol-1-yl)methanethione
to
obtain
(10h).
afforded
the
Treatment
the
key
of
intermediate
1
-cyclopropyl-4-isothiocyanatonaphthalene
10h
with
hydrazinecarboximidamide
intermediate
5
-amino-4-(4-cyclopropylnaphthalen-1-yl)-4H-1,2,4-triazole-3-thiol (11h), which was subjected
to substitution and bromination reaction to give the intermediate methyl
-((5-amino-4-(4-cyclopropylnaphthalen-1-yl)-4H-1,2,4-triazol-3-yl)thio)acetate (12h) and
2
brominated product 13h. 13h was hydrolyzed to provide lesinurad (7). Finally, compounds 81-96
were obtained via condensation reaction with lesinurad (7).44
All novel derivatives were fully characterized by high-resolution mass spectrometry
1
(HR-MS), proton nuclear magnetic resonance ( H-NMR) spectroscopy, and carbon nuclear
1
3
magnetic resonance ( C-NMR) spectroscopy.
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11b
11c
12a
12b
12c
13-16
21-24
29-32
17-20
25-28
33-36
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Ar =
Scheme 1. Synthetic route to 3H-imidazole[4,5-b]pyridine derivatives
(
(
(
i) KF, naphthalen-1-amine or 2,4,6-trimethylaniline or 4-cyclopropylnaphthalen-1-amine, 120 ℃;
ii) Pd/C, H , EtOH, r.t.; (iii) EtOCS K, NaHCO , H O, EtOH, 70 ℃; (iv) ester, K CO , DMF, r.t.;
2
2
3
2
2
3
v) LiOH, THF, EtOH, r.t..
N
N
N
N
NO2
NH
N
NH2
NH
N
N
NO2
Cl
N
N
O
OH
R
R
iii
iv
v
i
ii
SH
S
S
N
N
N
O
O
8b
10d
11d
12d
37-41
42-46
Scheme 2. Synthetic route to 1H-imidazole [4,5-b]pyridine derivatives
i) 4-cyclopropylnaphthalen-1-amine, Pd(OAc) , XantPhos, Cs CO , N , 1,4-dioxane, 90 ℃; (ii)
(
2
2
3
2
Pd/C, H , EtOH, r.t.; (iii) TCDI, Et N, THF, 60 ℃; (iv) ester, K CO , DMF, r.t.; (v) LiOH, THF,
2
3
2
3
EtOH, r.t..
NH2
NH
N
N
N
NO2
NH
N
NO2
N
N
N
N
N
O
OH
R
R
v
i
ii
iii
SH
iv
S
S
N
N
N
O
O
Cl
8
c
10e
12e
11e
47-51
52-56
Scheme 3. Synthetic route to 1H-imidazole[4,5-c]pyridine derivatives
(i) 4-cyclopropylnaphthalen-1-amine, NaHCO , EtOH, 60 ℃; (ii) Pd/C, H , EtOH, r.t.; (iii)
3
2
EtOCS K, NaHCO , H O, EtOH, 70 ℃; (iv) ester, K CO , DMF, r.t.; (v) LiOH, THF, EtOH, r.t..
2
3
2
2
3
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1
1
1
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1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
N
NO2
NH
N
N
NH2
NH
N
N
N
OH
N
N
O
R1
R1
R2
S
iii
iv
SH
v
vi
N
O
S
i
N
N
O
Br
CN
CN
CN
CN
CN
10f
11f
1
2f
57-62
63-68
NO2
NH
N
CN
N
NH2
NH
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
N
N
N
S
OH
ii
N
N
S
O
R1
R2
9d
v
R1
iii
iv
SH
vi
N
O
N
N
O
CN
CN
CN
75-80
CN
CN
9-74
10g
11g
12g
6
Scheme 4. Synthetic route to 4-(2-mercapto-1H-imidazo[4,5-b]pyridin-1-yl)-1-naphthonitrile
and 4-(2-mercapto-1H-imidazo[4,5-c]pyridin-1-yl)-1-naphthonitrile derivatives
(
i) 2-nitropyridin-3-amine, NaOH, CaO, DMAC, 110 ℃; (ii) 3-nitropyridin-4-amine, NaOH, CaO,
DMAC, 110 ℃;(iii) Pd/C, H , EtOH, r.t.; (iv) TCDI, Et N, THF, 60 ℃; (v) ester, K CO , DMF,
2
3
2
3
r.t.; (vi) LiOH, THF, EtOH, r.t..
N
N
N N
O
S
C
H N
SH
H2N
iv
S
N
2
N
N
NH2
NH2
O
i
ii
iii
Br
12h
9e
9c
10h
11h
v
H
N N
N
N
R
N
N
O
N
OH
S
Br
S
Br
S
O
Br
S
N
N
O
N
O
O
O
vi
vii
81-96
7
13h
Scheme 5. Synthetic route to N-acylsulfonamide derivatives
(i) cyclopropylboronic acid, Ca (PO ) , Pd(PPh ) , toluene/water (25:1), N , 100 ℃; (ii)
3
4 2
3 4
2
di(1H-imidazol-1-yl)methanethione, CH Cl , r.t.; (iii) hydrazinecarboximidamide hydrochloride,
2
2
DIEA, DMF, 50 ℃; (iv) methyl chloroacetate, K CO , DMF, r.t.; (v) NaNO , TEBA, CHBr ,
2
3
2
3
dichloroacetic acid, r.t.; (vi) LiOH, THF, EtOH, r.t.; (vii) sulfonamides, EDC·HCl, DMAP, DCM,
0 °C to r.t..
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Results and Discussion
The activity of the synthesized compounds to lower serum uric acid (SUA) was evaluated using
our established acute hyperuricemia model in mice, in which high SUA levels are maintained for
more than 6 hours.47 Benzbromarone and lesinurad were used as positive control drugs. The
results are summarized in Tables 1-4.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 1. Structures of the series I compounds and serum uric acid concentrations in acute
hyperuricemia model mice treated with these compounds
4
4
5
4
5
5
N
O
N
O
R2
6
R1
R2
N
O
6
R1
6
R2
R1
N
7
S
N
7
S
N
N
7
S
N
O
N
O
O
1
3-20
21-28
2
9-36
Compds
R
1
R
2
SUA (μM) a,b
1187.60 ± 97.49
815.40 ± 131.62
1253.17 ± 92.61
1137.33 ± 125.76
1207.50 ± 130.25
1217.00 ± 136.92
1252.50 ± 75.27
1229.00 ± 150.16
Decrease ratio (DR) % c
1
1
3
4
Et
Et
Et
Et
H
4.03
37.38
-1.84
8.54
15
1
6
17
2.25
1
1
2
8
9
0
H
1.40
H
-.178
0.32
H
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
21
Et
Et
Et
Et
H
1255.20 ± 183.98
-2.20
11.19
22.34
41.56
18.26
13.05
37.04
-4.96
41.96
23.73
40.34
2.61
2
2
2
2
2
2
2
3
4
5
6
7
1107.75 ± 195.15
983.25 ± 122.64
768.80 ± 61.17
1028.75 ± 43.87
1087.00 ± 101.37
819.25 ± 100.77
1288.00 ± 211.53
764.33 ± 49.56
967.80 ± 86.63
782.40 ± 67.21
1203.50 ± 94.83
745.00 ± 76.95
1220.67 ± 139.62
763.20 ± 248.75
1241.83 ± 36.75
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
H
H
28
H
2
9
Et
Et
Et
Et
H
30
3
3
3
3
3
3
1
2
3
4
5
6
43.69
1.07
H
H
42.06
-0.83
H
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Model a
Vehicle b
-
-
-
-
-
-
-
-
1232.60 ± 55.74
116.50 ± 9.85
717.60 ± 65.35
818.40 ± 87.15
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Benzbromarone
46.14
37.11
Lesinurad
a
Model: mice with acute hyperuricemia induced with xanthine and potassium oxonate, and not
b
treated with test compound (i.e., untreated hyperuricemic mice). Vehicle: Mice not treated with
c
xanthine and potassium oxonate, or test compound (i.e, untreated normal mice). Decrease ratio =
(
Model SUA – Compound SUA) ÷ (Model SUA – Vehicle SUA)
Table 2. Structures of the series II and III compounds and serum uric acid concentrations in
acute hyperuricemia model mice treated with these compounds
4
N
5 N 4
5
N
N
O
O
R2
6
R1
R2
6
7
R1
S
S
7
N
N
O
O
3
7-46
47-56
Compds
R
1
R
2
SUA (μM)
Decrease ratio (DR) % c
3
7
Et
Et
Et
777.20 ± 59.87
776.80 ± 45.97
759.40 ± 45.08
33.56
33.60
35.37
38
3
9
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
40
41
Et
Et
658.80 ± 78.90
45.58
47.90
760.70 ± 106.64
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
4
2
3
H
H
H
H
768.00 ± 39.05
759.20 ± 128.97
192.80 ± 49.81
994.80 ± 41.38
34.50
35.39
92.89
11.47
44
4
4
4
5
6
7
H
495.30 ± 57.42
62.18
Et
Et
Et
Et
706.00 ± 46.34
641.20 ± 94.12
620.60 ± 123.7
431.60 ± 114.6
40.79
47.37
49.46
68.65
48
4
9
50
51
52
Et
699.60 ± 48.61
41.35
H
H
H
692.40 ± 68.60
553.60 ± 126.7
225.00 ± 89.18
42.17
56.26
89.62
5
3
54
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56
H
H
734.60 ± 142.0
37.89
72.99
388.80 ± 72.69
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Model a
Vehicle b
-
-
-
-
-
-
-
-
1108.00 ± 43.93
122.80 ± 11.96
753.20 ± 32.86
841.20 ± 55.16
-
-
Benzbromarone
36.00
27.06
Lesinurad
a
Model: mice with acute hyperuricemia induced with xanthine and potassium oxonate, and not
b
treated with test compound (i.e., untreated hyperuricemic mice). Vehicle: Mice not treated with
c
xanthine and potassium oxonate, or test compound (i.e, untreated normal mice). Decrease ratio =
(
Model SUA – Compound SUA) ÷ (Model SUA – Vehicle SUA)
First, we examined the effects of mesitylene, naphthalene and 1-cyclopropylnaphthalene
substitution (zone B) at the imidazole nitrogen atom on the activity, in combination with a range
of R and R substituents in the side chain (13 - 36). As shown in Table 1, some compounds
1
2
exhibited high potency for reducing SUA. In particular, 24 (768.8 μM), 27 (768.8 μM), 29 (764.33
μM), 31 (782.40 μM), 33 (745.00 μM) and 35 (763.20 μM) were as potent as or more potent than
lesinurad (818.40 μM). Examination of the SAR indicates that the order of potency for
replacement of the aromatic ring was as follows: 1-cyclopropylnaphthalene > mesitylene >
naphthalene.
Next, we retained the preferred 1-cyclopropylnaphthalene structure of Zone B, and applied a
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
bioisosterism strategy to modify Zone A, using 1H-imidazole[4,5-c]pyridine (series II) and
1
H-imidazole (series III) as replacements for 3H-imidazole[4,5-b]pyridine. As shown in Table 2,
all the novel 1H-imidazole[4,5-c]pyridine and 1H-imidazole derivatives except 45 showed
significant SUA-lowering activity, and eight of them were more active than lesinurad and
benzbromarone. Among them, 44 (192.80 μM) and 54 (225.00 μM) were the most potent
SUA-reducers, being over three times more potent than the reference drugs. SAR analysis
indicated that the position of the N atom in the fused ring significantly influenced the activity.
Thus, when the N atom is at the 4 or 5 position of pyridimazole, the potency of the derivatives is
significantly greater than that of the counterparts with the N atom at the 7 position, and the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
-position is superior to the 4-position; for example, 48 > 38 > 30, while 50 > 40 > 32, 53 > 43 >
34 and 56 > 46 (Table 2).
We next investigated the effect of the side chain on the activity by altering its length and
modifying the substituents (Zone C in Figure 3). We found that increasing the length of the side
chain did not greatly affect the potency. For example, compounds 18, 22, 26, 38, 43 and 48, which
contain a propyl group, show comparable SUA-reducing activity to the corresponding short-chain
analogues 17, 21, 25, 37, 42 and 47, respectively. On the other hand, the presence of a methyl
group or a dimethyl group on the carbon atom adjacent to the sulfur atom (R ) did influence the
1
activity. For example, among the acids (R = H), methyl substitution was the best choice, since 27,
2
3
5, 44 and 54 were more potent SUA reducers than the unsubstituted or dimethyl-substituted
compounds (28, 36, 45 and 54). On the other hand, among the esters (R = Et), the order of
2
*
*
potency for substitution at the side chain was as follows: -C (CH ) > C HCH > -CH - (24 > 23 >
3
2
3
2
2
1, 40 > 39 > 37, and 50 > 49 > 47). In other words, greater steric size of the substituent is
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associated with greater SUA-reducing activity. A plausible explanation is that the conformation in
the binding pocket of URAT1 might be stabilized via increased hydrophobic interaction.
Interestingly, most of the acid-containing compounds were more potent than the esters in the
mouse model of acute hyperuricemia (e.g., 49 > 44, 49 > 54, 23 > 27). This suggests that the esters
were not hydrolyzed in this animal model, at least on the time scale of this experiment.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 3. Structures of the series II and III compounds and serum uric acid concentrations in
acute hyperuricemia model mice treated with these compounds
4
N
5 N 4
5
N
N
O
O
R2
6
R1
R2
6
7
R1
S
S
7
N
N
O
O
CN
7-68
CN
69-80
5
Decrease ratio (DR) % c
Compds
R
1
R
2
SUA (μM)
5
5
5
7
8
9
Me
Me
Me
Me
Et
970.60 ± 67.77
992.80 ± 66.09
1012.20 ± 82.74
937.20 ± 82.21
940.20 ± 61.73
9.51
7.17
5.12
60
13.03
12.71
6
6
1
2
Et
732.88 ± 166.66
34.56
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
63
H
H
H
H
H
568.20 ± 93.82
51.91
20.30
71.62
95.01
3.67
6
6
6
6
4
5
6
7
868.20 ± 54.99
381.20 ± 117.06
159.20 ± 19.61
1026.00 ± 73.51
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
8
H
484.40 ± 148.80
60.74
69
Me
Me
Me
Me
Me
922.80 ± 74.55
975.80 ± 34.42
952.00 ± 92.01
891.00 ± 139.99
974.60 ± 69.33
14.54
8.96
7
7
7
7
0
1
2
3
11.47
17.90
9.09
7
4
Et
923.00 ± 70.58
14.44
7
7
7
5
6
7
H
H
H
H
494.80 ± 121.30
954.60 ± 78.59
575.60 ± 61.80
796.60 ±40.44
59.22
11.19
51.13
27.84
78
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79
80
H
H
916.20 ± 50.08
15.24
27.36
801.20 ± 88.54
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Model a
Vehicle b
-
-
-
-
-
-
-
-
1060.83 ± 113.13
111.83 ± 11.47
587.13 ± 66.86
757.33 ± 93.05
-
-
Benzbromarone
49.94
31.93
Lesinurad
a
Model: mice with acute hyperuricemia induced with xanthine and potassium oxonate, and not
b
treated with test compound (i.e., untreated hyperuricemic mice). Vehicle: Mice not treated with
c
xanthine and potassium oxonate, or test compound (i.e, untreated normal mice). Decrease ratio =
(
Model SUA – Compound SUA) ÷ (Model SUA – Vehicle SUA)
Next, we tested the activity of compounds bearing a cyano group instead of the cyclopropyl
moiety (Series IV). As shown in Table 3, some compounds exhibited high potency in reducing
SUA, but the overall activity was not significantly improved compared with that of compounds in
Series II and III. Among them, 66 (159.20 μM), with the N atom was at the 4 position of
pyridimazole, was the most potent derivative, being 2.8 times more potent than lesinurad.
Meanwhile, most of the acid-containing compounds were more potent than the esters, which is
consistent with the results in series I-III (e.g. 63 > 57, 65 > 59). Among the acids, replacement of
the hydrogen in the ortho-position of sulfydryl resulted in more potent activity. However, among
derivatives having the N atom at the 5 position of pyridimazole, most were inactive, and only a
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
few compounds, such as compounds 75 (494.80 μM), 77 (575.60 μM) and 78 (796.60 μM),
showed moderate SUA-reducing potency. Thus, the position of the N atom appears to be critical
for the interaction of these compounds with URAT1.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 4. Structures of the series V compounds and serum uric acid concentrations in acute
hyperuricemia model mice treated with these compounds
H
N
N
R
O
N
S
Br
S
O
N
O
81-96
Compds
R
SUA (μM)
Decrease ratio (DR) % c
64.65%
81
474.28 ± 40.84
366.17 ± 43.93
133.30 ± 49.72
175.02 ± 53.97
352.95 ± 41.47
453.07 ± 69.66
322.65 ± 66.18
481.57 ± 60.43
F
8
8
8
8
8
8
2
3
4
5
6
7
75.17%
Br
F
97.84%
F
93.78%
Cl
76.46%
NO2
66.71%
79.41%
S
88
63.94%
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Cl
S
8
9
589.48 ± 108.17
53.44%
9
9
9
0
1
2
300.93 ± 34.21
1025.08 ± 70.15
891.30 ± 91.42
1015.30 ± 89.90
1077.43 ± 110.11
958.25 ± 122.49
983.80 ± 294.7
81.53%
11.04%
24.06%
11.99%
5.94%
17.54%
15.1%
-
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
93
9
4
95
9
6
Model a
Vehicle b
Lesinurad
-
-
-
1138.48 ± 125.93
111.13 ± 13.89
726.77 ± 105.94
-
40.07%
a
Model: mice with acute hyperuricemia induced with xanthine and potassium oxonate, and not
b
treated with test compound (i.e., untreated hyperuricemic mice). Vehicle: Mice not treated with
c
xanthine and potassium oxonate, or test compound (i.e, untreated normal mice). Decrease ratio =
(Model SUA – Compound SUA) ÷ (Model SUA – Vehicle SUA)
Finally, we tested the SUA-lowering activity of the newly synthesized acyl sulfonamide
compounds in vivo. Ten compounds showed significant activity. Among them, compounds 83 and
8
4 were the most potent, reducing SUA to 133.30 μM and 175.02 μM, respectively (reduction
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9
1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
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3
3
3
3
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5
5
5
5
5
5
5
6
ratios of 97.84% and 93.78%, respectively).
Our results demonstrate that most of the compounds modified with an aromatic ring or a
thiophene ring at the end of acylsulfonamide moiety have greater activity than those modified with
a fatty acyl chain (83 > 91, 84 > 92, etc.). For example, a benzene ring containing a substituent
increased the potency: 83 > 81, 84 > 81, and 87 > 81. The presence of halogens on the benzene
ring resulted in much higher activity than the presence of a methyl group (83 > 87). Derivatives
having a thiophene ring or a chlorothiophene ring also showed considerable activity.
In general, the modification of the acylsulfonamide moiety appears to be an effective
approach to enhance the SUA-reducing activity of lead compounds.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Evaluation of serum uric acid-lowering activity in rats
Some representative derivatives were further evaluated for the ability to reduce SUA in another
animal species (rats), in order to confirm their potency. Lesinurad was selected as positive control
drug. The results are showed in Table 5.
Table 5. Serum uric acid concentrations in rats treated with compounds 44 and 54
Compds
SUA (μM)
220.16 ± 82.75
240.42 ± 77.76
467.32 ± 85.26
890.30 ± 166.18
92.14 ± 17.66
Decrease ratio (DR) %
44
83.96
81.42
52.99
-
5
4
Lesinurad
Model
Vehicle
-
Compounds 44 (220.16 μM) and 54 (240.42 μM) both appear to be promising candidates, being
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about 1.6 times more potent than the reference drug lesinurad. The results in rats were consistent
with those in mice.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Inhibition of human URAT1
We next examined the inhibition of human URAT1 by some representative compounds. As shown
in Table 6, 44 inhibited the URAT1-mediated uptake of 14C-UA with an IC50 of 1.57 μM, being
more than 8-fold more potent than lesinurad (IC = 13.21 μM). The inhibitory activities of all the
5
0
compounds tested were consistent with their relative activities in the acute hyperuricemia model in
mice, supporting the idea that URAT1 is the target molecule of these compounds for
hypouricemic activity, as expected. Furthermore, the representative acyl-sulfonamide compounds
8
3 and 84 showed good inhibitory activity against URAT1 with IC values of 4.17 μM and 7.29
50
μM respectively. Notably, 83 and 84 also exhibited significant inhibitory activity towards the
transporter GLUT9, which plays an important synergistic role in the reabsorption of uric acid,
with IC values of 31.73 μM and 32.20 μM, respectively. This may be one important reason why
5
0
compounds 83 and 84 showed good activity in vivo. Thus, we have identified acylsulfonamide
compounds as dual-target inhibitors.
Table 6. IC50 values of some representative compounds for URAT1-mediated 14C-UA
transport, and inhibitory activity towards GLUT9
Transporter
URAT1
Substrate
14C-UA
Compds
IC (μM)
50
Lesinurad
13.21 ± 1.35
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2
2
2
2
2
2
2
2
3
3
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3
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3
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4
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5
5
5
5
5
5
5
6
35
>30
5
4
6.14 ± 0.61
1.57 ± 0.38
4.17 ± 0.29
7.29 ± 0.59
31.73 ± 3.92
32.20 ± 2.26
0
1
2
3
4
5
6
7
8
9
0
1
2
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8
9
0
1
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8
9
0
1
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8
9
0
1
2
3
4
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8
9
0
44
8
8
3
4
GLUT9
-
-
83
84
CYP-Inhibitory Activity
Drug discovery efforts generally include in vitro assays early in the screening funnel for
measurement of cytochrome P450 (CYP) inhibition. The most effective compounds 44, 54 and 83
were evaluated for the ability to inhibit CYP drug-metabolizing enzymes in vitro. As summarized
in Table 7, 44 displayed no significant inhibition of CYP1A2 (IC > 50.0 μM), CYP2C9 (IC =
5
0
50
2
0.0 μM), CYP2C19 (IC50 = 48.7 μM), CYP2D6 (IC50 > 50.0 μM) or CYP3A4M (IC50 > 50.0),
and 54 also showed no significant inhibition of CYP1A2 (IC > 50.0 μM), CYP2C19 (IC > 50.0
5
0
50
μM), CYP2D6 (IC > 50.0 μM) or CYP3A4M (IC = 36.5), though it did inhibit CYP2C9 (IC
50
5
0
50
=
2.39 μM). Similarly, compound 83 does not inhibit the activity of CYP1A2 (IC50 > 50.0 μM),
CYP2C19 (IC50 = 20.6 μM) or CYP2D6 (IC50 > 50.0 μM). Importantly, more than half of all
drugs are metabolized by CYP3A4, and thus inhibition of CYP3A4 by any new therapeutic is
highly undesirable due to the potential for drug-drug interactions (DDI) in patients, the results of
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investigation of the inhibition of hepatic CYP drug-metabolizing enzymes suggest that that 44, 54
and 83 have low potential for hepatotoxicity and can be considered for further in vivo evaluation
in rats.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 7. Inhibitory effects of 44 and 54 on CYP1A2, CYP2C9, CYP2C19, CYP2D6 and
CYP3A4M
CYP
standard inhibitor
IC50 (μM)
Compd
IC50 (μM)
Compd
IC50 (μM)
IC50 (μM)
Compd
isozyme
1
A2
α-Naphthoflavone
Sulfaphenazole
(+)-N-3-benzylnirvanol
Quinidine
0.216
0.609
0.227
0.134
0.0375
44
44
44
44
44
> 50
20.0
48.7
> 50
> 50
54
54
54
54
54
> 50
2.39
> 50
> 50
36.5
83
83
83
83
83
> 50
0.636
20.6
2C9
2
C19
2D6
> 50
> 50
3
A4M
Ketoconazole
In Vivo Pharmacokinetic Study
The pharmacokinetic profiles of compounds 44, 54 and 83 were examined in Wistar rats (Table 8
and Figure 5). After a single 1 mg/kg iv dose of 44 and 54, the half-life (t ), mean clearance rate
1
/2
−1
(
CL) and mean residence time (MRT) values were 3.05 h, 264.0 mL h−1 kg , 2.70 h and 2.27 h,
−1
3
18.3 mL h−1 kg , 2.86 h, respectively. At the dose of 20 mg/kg orally, compound 44 was rapidly
absorbed with a time-to-maximum-concentration (Tmax) of 2 h, a t1/2 of 3.4 h, a MRT of 5.3 h, a
maximum concentration (Cmax) of 15.54 μg/mL, and an area under curve (AUC ) of 53153.7
0-t
ng/mL•h. The corresponding values for 54 were 0.5 h, 3.1 h, and 4.1 h, 21.6 μg/mL and 58249.1
ng/mL•h. Notably, 44 and 54 exhibited extremely high oral bioavailability of 76.3% and 93.6%,
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2
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4
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4
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5
5
5
5
5
5
5
5
5
5
6
respectively. After a single 1 mg/kg iv dose of 83, the t1/2 value, CL value and MRT value were
5
.05 h, 327.6 mL h−1 kg−1 and 5.05 h. Administration of 83 at 10 mg/kg resulted in Tmax of 1.83 h
with a Cmax of 3390 ng/mL. The t1/2 value of 83 was 3.43 h, and the MRT was 5.55 h. In addition,
the oral bioavailability of 83 was 63.4%, which is sufficient for a drug candidate. Overall, 44, 54
and 83 appear to have favorable drug-like properties.
0
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2
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2
2
2
2
2
2
2
3
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3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
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5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. Plasma concentration−time profiles of 44, 54 and 83 in rats following oral or
intravenous administration.
Table 8. Pharmacokinetic parameters of 44, 54 and 83a
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1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
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3
3
3
3
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6
Parameter
Compound
AUC(0-t)
AUC(0-∞)
MRT(0-∞)
t1/2
Unit
p.o.
54b
-
44b
53153.7
88846.0
5.3
83c
19425.3
19612.7
5.6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ng/mL•h
58249.1
65123.8
4.6
ng/mL•h
h
h
3.4
3.1
3.4
Tmax
h
2
0.5
1.8
Cmax
μg/mL
15.5
76.3
21.6
93.6
i.v.d
3.4
F
%
63.4
Parameter
Compounds
AUC(0-t)
AUC(0-∞)
MRT(0-∞)
t1/2
Unit
-
44
3484.9
3911.3
2.7
54
83
3029.0
3094.3
4.3
ng/mL•h
ng/mL•h
h
3111.7
3324.4
2.86
2.3
h
3.1
5.0
CL
mL/h/kg
264.0
318.3
327.6
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a
d
b
c
PK parameters (mean ± SD, n = 5). Dosed orally at 20 mg/kg. Dosed orally at 10 mg/kg.
Dosed intravenously at 1 mg/kg
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Safety Assessment
Acute Toxicity Assessment of 44, 54 and 83 in Healthy Mice
To examine in vivo safety, we performed a single-dose acute toxicity test of 44, 54 and 83 in
healthy Kunming mice. After intragastric administration of 44, 54 and 83 at the dose of 500
mg/kg, no mouse died up to one week, whereas 20% of the mice died in the group given 250
mg/kg lesinurad. These results suggest that the three candidate compounds show lower acute
toxicity than the approved drug lesinurad, at least under the conditions of our study. We also
observed no behavioral abnormality of the treated animals (lethargy, clonic convulsion, anorexia,
or ruffled fur). In addition, there was no marked difference in weight gain of male or female mice
over the 1-week period (Figure 6), compared with the vehicle control (P > 0.05), supporting the
idea that 44, 54 and 83 show low toxicity.
(A)
(B)
Figure 6. Time courses of body weight of (A) male mice and (B) female mice after administration
of test compounds.
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