Human uric acid transporter 1 (hURAT1): An inhibitor structure-activity relationship (SAR) study

Article abstract of DOI:10.1080/15257770.2011.594031

The current study describes the chemical synthesis of a series of (2-ethylbenzofuran-3-yl)(substituted-phenyl)methanone compounds and their subsequent in vitro testing via oocytes expressing hURAT1. The experimental data support the notion that a potent hURAT1 inhibitor requires an anion (i.e., a formal negative charge) to interact with the positively charged hURAT1 binding pocket. An anion appears to be a primary requirement in order to be a hURAT1 substrate (i.e., urate) or inhibitor. We discuss the inhibitor structure-activity relationship and how electronically donating or withdrawing groups attached to the B-ring can decrease or increase inhibitory potency, respectively. Copyright Taylor and Francis Group, LLC.

Full text of DOI:10.1080/15257770.2011.594031

This article was downloaded by: [Marquette University]
On: 19 August 2014, At: 07:09
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered
office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Nucleosides, Nucleotides and Nucleic
Acids
Publication details, including instructions for authors and
subscription information:
http://www.tandfonline.com/loi/lncn20
Human Uric Acid Transporter 1
(hURAT1): An Inhibitor Structure–Activity
Relationship (SAR) Study
M. F. Wempe a , B. Quade a , P. Jutabha b , T. Iwen a , M. Frick a , P.
J. Rice a , S. Wakui b & H. Endou b c
a School of Pharmacy , University of Colorado Denver , Anschutz
Medical Campus, Aurora, CO, USA
b Department of Pharmacology and Toxicology , Kyorin University
School of Medicine , Mitaka, Tokyo, Japan
c J-Pharma Co., Ltd. , Shinjuku, Shinjuku-ku, Tokyo, Japan
Published online: 01 Dec 2011.
To cite this article: M. F. Wempe , B. Quade , P. Jutabha , T. Iwen , M. Frick , P. J. Rice , S.
Wakui & H. Endou (2011) Human Uric Acid Transporter 1 (hURAT1): An Inhibitor Structure–Activity
Relationship (SAR) Study, Nucleosides, Nucleotides and Nucleic Acids, 30:12, 1312-1323, DOI:
10.1080/15257770.2011.594031
To link to this article: http://dx.doi.org/10.1080/15257770.2011.594031
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the
“Content”) contained in the publications on our platform. However, Taylor & Francis,
our agents, and our licensors make no representations or warranties whatsoever as to
the accuracy, completeness, or suitability for any purpose of the Content. Any opinions
and views expressed in this publication are the opinions and views of the authors,
and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content
should not be relied upon and should be independently verified with primary sources
of information. Taylor and Francis shall not be liable for any losses, actions, claims,
proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or
howsoever caused arising directly or indirectly in connection with, in relation to or arising
out of the use of the Content.
This article may be used for research, teaching, and private study purposes. Any
substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,
systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &

Conditions of access and use can be found at http://www.tandfonline.com/page/terms-
and-conditions

Nucleosides, Nucleotides and Nucleic Acids, 30:1312–1323, 2011
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2011.594031
HUMAN URIC ACID TRANSPORTER 1 (hURAT1): AN INHIBITOR
STRUCTURE–ACTIVITY RELATIONSHIP (SAR) STUDY
M. F. Wempe,1 B. Quade,1 P. Jutabha,2 T. Iwen, 1 M. Frick,1 P. J. Rice,1
S. Wakui,2 and H. Endou2,3
1School of Pharmacy, University of Colorado Denver, Anschutz Medical Campus, Aurora,
CO, USA
2Department of Pharmacology and Toxicology, Kyorin University School of Medicine,
Mitaka, Tokyo, Japan
3J-Pharma Co., Ltd., Shinjuku, Shinjuku-ku, Tokyo, Japan
The current study describes the chemical synthesis of a series of (2-ethylbenzofuran-3-
2
yl)(substituted-phenyl)methanone compounds and their subsequent in vitro testing via oocytes ex-
pressing hURAT1. The experimental data support the notion that a potent hURAT1 inhibitor re-
quires an anion (i.e., a formal negative charge) to interact with the positively charged hURAT1
binding pocket. An anion appears to be a primary requirement in order to be a hURAT1 substrate
(i.e., urate) or inhibitor. We discuss the inhibitor structure–activity relationship and how electron-
ically donating or withdrawing groups attached to the B-ring can decrease or increase inhibitory
potency, respectively.
Keywords Human uric acid transporter 1 (hURAT1; SLC22A12); inhibitor study;
structure–activity relationship (SAR)
INTRODUCTION
At the 14th International Symposium of the Purine and Pyrimidine So-
ciety (PP11) held in Japan, we presented on a series of compounds and
their ability to inhibit urate transport mediated via human uric acid trans-
porter 1 (hURAT1; SLC22A12). Humans metabolically degrade purine nu-
cleotides, nucleosides, and bases to produce urate (uric acid 1, Figure 1)
Received 17 April 2011; accepted 31 May 2011.
The research was funded by J-Pharma Co., Ltd. (Tokyo, Japan.) and also utilized services of the
Medicinal Chemistry Core facility (MCC; MFW) housed within the Department of Pharmaceutical Sci-
ences (DOPS). In part, the MCC has been funded via Colorado Clinical and Translational Sciences
Institute grant 5UL1RR025780 from the National Center for Research Resources at the National Insti-
tutes of Health (NCRR/NIH).
Address correspondence to M. F. Wempe, Department of Pharmaceutical Sciences, School of
Pharmacy, University of Colorado Denver, Anschutz Medical Campus, C238 Room V20-2105, 12850
E. Montview Blvd., Aurora, CO 80045, USA. E-mail: Michael.Wempe@ucdenver.edu
1312

Human Uric Acid Transporter 1 (hURAT1)
1313
FIGURE 1 Uric acid formation and the transporter-mediated distribution in humans.
and eliminate in the urine. Various renal transporter proteins are critical in
regulating uric acid serum levels.[1–3] Important transporter proteins in the
kidney are located at both the urine and blood interfaces. The urine inter-
face (apical side) includes hURAT1, the human organic anion transporter
10 (hOAT10; SLC22A13), the natrium-dependent phosphate transporter 4
(NPT4, hOATv1; SLC17A3),[4–7] and the breast cancer resistance protein
(BCRP/ABCG2).[8,9] The basolateral transporters (blood interface) include
the facilitative glucose transporter 9 (GLUT9, URATv1; SLC2A9) and human
organic anion transporter proteins 1 (hOAT1; SLC22A6) and 3 (hOAT3;
SLC22A8).[7,10–13]
Elevated uric acid (hyperuricemia) may lead to gout and produce
episodes of acute inflammatory arthritis.[4] Diet, lifestyle, and age (renal
function declines as people age) are known contributors to the production
of elevated serum urate.[14] Elevated uric acid levels throughout the body
are believed to also play a pivotal role in other diseases such as hypertension,
diabetes, chronic renal disease, and cardiovascular disease.[14–16] Therefore,
we believe that the development of uric acid transporter inhibitors will lead
to novel and therapeutically important drugs for combating various diseases.
Hence, this requires that we obtain a reliable inhibitor structure–activity re-
lationship. We recently published a chemical investigation where we probed
a variety of different (3,5-dibromo-4-hydroxyphenyl)(2-ethylbenzofuran-(#)-
yl)methanone regioisomers (# = 3, 4, or 7 position) and their ability to in-
hibit 14C-urate uptake in oocytes expressing hURAT1.[17] The current work
describes the preparation and in vitro testing of additional compounds and
further illustrates important chemical and structural requirements for po-
tent hURAT1 inhibitor activity.

1314
M. F. Wempe et al.
MATERIALS AND METHODS
Benzoyl chloride, 4-fluorobenzoyl chloride, 4-cyanobenzoyl chloride, 3-
chloro-4-methoxybenzoic acid, 3-bromo-4-methoxy-benzoic acid, and 1,2-
dimethyl-1H-indole were purchased from Sigma-Aldrich Chemical Company
(St. Louis, MO, USA). The 3-fluoro-p-anisic acid was purchased from TCI
America (Chicago, IL, USA). All other reagents were procured as previously
described.[17]
Chemical Synthesis
Compounds 1-(benzofuran-2-yl)ethanone 3, 2-ethylbenzofuran 4, (2-
ethylbenzofuran-3-yl)(4-methoxyphenyl)methanone
6,
(2-ethylbenzofuran-3-yl)
(4-hydroxyphenyl)methanone 7, (3,5-dibromo-4-hydroxyphenyl)(2-ethylbenzofuran-
3-yl)methanone 16, (3,5-dibromo-4-hydroxyphenyl)(2-ethyl-6-methoxybenzofuran-3-
yl)methanone 17, and (3,5-dibromo-4-hydroxyphenyl)(2-ethyl-6-hydroxy-benzofuran-
3-yl)methanone 18 were prepared as previously described.[17]
(2-ethylbenzofuran-3-yl)(phenyl)methanone 5: Benzofuran (4; 0.200 g,
1.37 mmol) was diluted with CS2 (4.0 mL), stirred, and cooled in an ice
bath (30 minutes). Next, benzoyl chloride (0.21 mL, 1.78 mmol) was added
drop-wise followed by tin (IV) chloride (0.21 mL, 1.78 mmol). The contents
were stirred (3.0 hours) and warmed to room temperature (RT) (21 hours).
The reaction mixture was diluted with water and extracted with EtOAc (4 ×
20 mL). The combined organic layers were washed with HCl (0.5 N, 20 mL),
water (20 mL), NaHCO3 (20 mL), and brine (20 mL). The organic phase
was dried (Na2SO4), filtered, concentrated under reduced pressure, and pu-
rified via SiO2 chromatography (100% Hex to 5% EtOAc in Hex) to give 5
(220 mg, 0.879 mmol, 64% yield). 1H-NMR (400 MHz) CDCl3: 7.84–7.82 (d,
2H), 7.63–7.59 (t, 1H), 7.51–7.47 (m, 3H), 7.39–7.37 (d, 1H), 7.31–7.26 (t,
1H), 7.21–7.17 (t, 1H), 2.94–2.88 (q, 2H), 1.36–1.32 (t, 3H); 13C-NMR (100
MHz) CDCl3: 192.0, 166.5, 153.7, 139.4, 132.6, 129.1, 128.4, 127.0, 124.3,
123.5, 121.4, 116.1, 110.9, 21.8, 12.3. LC/MS-MS: 251.1 → 105.0 m/z; GS1
and GS2 at 25, DP = 56, CE = 29, CXP = 6, tR = 4.99 minutes.
(2-ethylbenzofuran-3-yl)(4-fluorophenyl)methanone 8 was prepared with anal-
ogous methods used with compound 5, except that it was stirred at RT for
1
3 days. Compound 8 (160 mg, 0.60 mmol, 43%) was an orange oil. H-
NMR (400 MHz) CDCl3: 7.88–7.84 (t, 2H), 7.49–7.47 (d, 1H), 7.33–7.25 (m,
2H), 7.21–7.13 (m, 3H), 2.94–2.89 (q, 2H), 1.35–1.32 (t, 3H); 13C-NMR (100
MHz) CDCl3: 190.3, 166.8, 166.4, 164.3, 153.7, 135.6, 131.8 (d), 126.8, 124.4,
123.6, 121.2, 115.8 (t), 111.1, 21.8, 12.3. LC/MS-MS: 269.0 → 123.1 m/z; GS1
and GS2 at 25, DP = 61, CE = 29, CXP = 6, tR = 4.85 minutes.
4-(2-ethylbenzofuran-3-carbonyl)benzonitrile 9 was prepared using analogous
methods as compound 8. Compound 9 (16.6 mg, 0.060 mmol, 5% yield)
1
was a yellow oil. H-NMR (400 MHz) CDCl3: 7.92–7.89 (d, 2H), 7.81–7.79

Human Uric Acid Transporter 1 (hURAT1)
1315
(d, 2H), 7.52–7.50 (d, 1H), 7.33–7.29 (t, 1H), 7.25–7.20 (m, 2H), 2.96–2.90
(q, 2H), 1.37–1.33 (t, 3H); 13C-NMR (100 MHz) CDCl3: 190.2, 167.8, 153.7,
142.9, 132.4, 129.4, 126.1, 124.8, 123.9, 121.0, 118.0, 115.9, 115.4, 111.3, 22.0,
12.2. LC/MS-MS: 276.0 → 123.0 m/z; GS1 and GS2 at 25, DP = 71, CE = 27,
CXP = 8, tR = 4.57 minutes.
(2-ethylbenzofuran-3-yl)(3-fluoro-4-methoxyphenyl)methanone 10 was prepared
with analogous methods used with compound 8 except stirred at RT for 16
hours. Compound 10 (218 mg, 0.731 mmol, 54% yield) was a yellow oil.
1H-NMR (400 MHz) CDCl3: 7.65–7.63 (d, 2H), 7.49–7.47 (d, 1H), 7.40–7.37
(d, 1H), 7.31–7.26 (t, 1H), 7.22–7.19 (t, 1H), 7.03–6.99 (t, 1H), 3.98 (s, 3H),
2.95–2.89 (q, 2H), 1.37–1.33 (t, 3H); 13C-NMR (100 MHz) CDCl3: 189.5,
165.8, 153.6, 153.2, 151.8, 150.7, 132.1, 126.7 (t), 124.4, 123.5, 121.1, 116.8
(d), 115.8, 112.3, 111.0, 56.3, 21.8, 12.3. LC/MS-MS: 298.7 → 153.1 m/z; GS1
and GS2 at 25, DP = 41, CE = 29, CXP = 10, tR = 4.77 minutes.
(3-chloro-4-methoxyphenyl)(2-ethylbenzofuran-3-yl)methanone 11 was prepared
with analogous methods used with compound 10. Compound 11 (0.140
1
g, 0.445 mmol, 40% yield) was a light yellow oil. H-NMR (400 MHz)
CDCl3: 7.92 (s, 1H), 7.78–7.75 (d, 1H), 7.50–7.48 (d, 1H), 7.41–7.39 (d,
1H), 7.31–7.27 (t, 1H), 7.23–7.19 (t, 1H), 7.00–6.98 (d, 1H), 3.99 (s, 3H),
2.94–2.88 (q, 2H), 1.37–1.33 (t, 3H); 13C-NMR (100 MHz) CDCl3: 189.3,
165.9, 158.6, 153.6, 132.5, 131.5, 129.8, 126.9, 124.4, 123.5, 122.8, 121.1,
115.8, 111.2, 111.0, 56.4, 21.8, 12.3. LC/MS-MS: 315.0 → 169.1 m/z; GS1
and GS2 at 25, DP = 56, CE = 31, CXP = 10, tR = 4.99 minutes.
(3-bromo-4-methoxyphenyl)(2-ethylbenzofuran-3-yl)methanone 12 was prepared
with analogous methods used with compound 10. Compound 12 (0.195 g,
1
0.542 mmol, 40%) was a yellow oil. H-NMR (400 MHz) CDCl3: 8.10 (s,
1H), 7.83–7.80 (d, 1H), 7.50–7.48 (d, 1H), 7.42–7.40 (d, 1H), 7.29–7.27 (t,
1H), 7.23–7.21 (t, 1H), 6.97–6.94 (d, 1H), 3.99 (s, 3H), 2.94–2.88 (q, 2H),
1.37–1.33 (t, 3H); 13C-NMR (100 MHz) CDCl3: 189.2, 165.9, 159.5, 153.7,
134.7, 132.9, 130.6, 126.9, 124.4, 123.6, 121.2, 115.8, 111.8, 111.1, 111.0,
56.5, 21.9, 12.3. LC/MS-MS: 361.0 → 214.8 m/z; GS1 and GS2 at 25, DP =
71, CE = 31, CXP = 14, tR = 5.20 minutes.
(2-ethylbenzofuran-3-yl)(3-fluoro-4-hydroxyphenyl)methanone 13: Compound
10 (90.0 mg, 0.302 mmol) was diluted with DMF (5.0 mL). NaSEt (0.127
g, 1.51 mmol) was added and warmed (105–110◦C; 16 hours). The mixture
was then quenched with 2 volumes of NH4Cl aq and extracted with EtOAc
(4 × 20 mL). The organic phase was washed with brine, water, and then
dried (Na2SO4), filtered, and concentrated under reduced pressure, and
purified via SiO2 chromatography (4:1; Hex:EtOAc) to give 13 as a brown
oil (46 mg, 0.162 mmol, 54% yield). 1H-NMR (400 MHz) CDCl3: 7.68–7.64
(d, 1H0, 7.60–7.57 (d, 1H), 7.50–7.48 (d, 1H), 7.40–7.38 (d, 1H), 7.31–7.26
(t, 1H), 7.23–7.19 (t, 1H), 7.10–7.06 (t, 1H), 6.45 (bs, 1H), 2.95–2.90 (q,
2H), 1.37–1.33 (t, 3H); 13C-NMR (100 MHz) CDCl3: 189.9, 166.1, 153.6,
152.0, 149.6, 148.4 (d), 132.1, 127.3, 126.8, 124.4, 123.6, 121.1, 117.1 (t),

1316
M. F. Wempe et al.
115.8, 111.0, 21.8, 12.3. LC/MS-MS: 285.0 → 139.0 m/z; GS1 and GS2 at 25,
DP = 61, CE = 29, CXP = 8, tR = 4.49 minutes.
(3-chloro-4-hydroxyphenyl)(2-ethylbenzofuran-3-yl)methanone 14 was prepared
with analogous methods used with compound 13. Compound 14 (21.1 mg,
0.070 mmol, 44% yield) was a brown oil. 1H-NMR (400 MHz) CDCl3: 7.90 (s,
1H), 7.71–7.69 (d, 1H), 7.50–7.48 (d, 1H), 7.40–7.38 (d, 1H), 7.31–7.26 (t,
1H), 7.21–7.19 (t, 1H), 7.11–7.09 (d, 1H), 6.11 (bs, 1H), 2.95–2.89 (q, 2H),
1.37–1.33 (t, 3H); 13C-NMR (100 MHz) CDCl3: 189.3, 166.0, 155.3, 153.7,
132.9, 130.7, 130.4, 126.8, 124.4, 123.6, 121.1, 120.3, 116.0, 115.8, 111.1, 21.8,
12.3. LC/MS-MS: 300.9 → 155.0 m/z; GS1 and GS2 at 25, DP = 66, CE = 29,
CXP = 8, tR = 4.59 minutes.
(3-bromo-4-hydroxyphenyl)(2-ethylbenzofuran-3-yl)methanone 15 was prepared
with analogous methods used with compound 13. Compound 15 (23.2 mg,
1
0.079 mmol, 57% yield) was a light yellow oil. H-NMR (400 MHz) CDCl3:
8.06–8.05 (d, 1H), 7.76–7.74 (dd, 1H), 7.50–7.48 (d, 1H), 7.41–7.39 (d,
1H), 7.31–7.29 (t, 1H), 7.23–7.19 (t, 1H), 7.10–7.08 (d, 1H), 6.21 (bs, 1H),
2.94–2.89 (q, 2H), 1.37–1.33 (t, 3H); 13C-NMR (100 MHz) CDCl3: 189.2,
166.0, 156.3, 153.7, 133.8, 133.2, 131.1, 126.8, 124.5, 123.6, 121.1, 115.9,
115.8, 111.1, 110.5, 21.8, 12.3. LC/MS-MS: 344.9 → 198.8 m/z; GS1 and GS2
at 25, DP = 61, CE = 33, CXP = 12, tR = 4.59 minutes.
(2-ethyl-5-fluorobenzofuran-3-yl)(4-methoxyphenyl)methanone 19 was prepared
using analogous methods as compound 5 to produce a white solid (1.60
g, 5.36 mmol, 44% yield). 1-(5-fluorobenzofuran-2-yl)ethanone and 2-ethyl-5-
fluorobenzofuran were prepared using analogous methods as previously de-
1
scribed.[15] Compound 19: H-NMR (400 MHz) CDCl3: 7.84–7.81 (d, 2H),
7.41–7.38 (d, 1H), 7.09–7.06 (d, 1H), 7.01–6.96 (m, 3H), 3.90 (s, 3H),
2.93–2.87 (q, 2H), 1.35–1.31 (t, 3H); 13C-NMR (100 MHz) CDCl3: 190.0,
167.1, 163.6, 160.7, 158.3, 149.8, 131.6, 128.2 (d), 116.5, 113.8, 112.0, 111.7
(t), 107.0 (d), 55.5, 21.9, 12.2. LC/MS-MS: 299.0 → 135.0 m/z; GS1 and GS2
at 30, DP = 11, CE = 31, CXP = 8, tR = 4.69 minutes.
(2-ethyl-5-fluorobenzofuran-3-yl)(4-hydroxyphenyl)methanone 20 was prepared
with analogous methods used with compound 13. Compound 20 (550 mg,
1
1.93 mmol, 58% yield) was a white solid. H-NMR (400 MHz) CDCl3:
7.79–7.77 (d, 2H), 7.42–7.38 (dd, 1H), 7.08–7.06 (d, 1H), 7.02–6.95 (t, 1H),
6.95–6.92 (d, 2H), 6.57 (bs, 1H), 2.93–2.88 (q, 2H), 1.35–1.31 (t, 3H); 13C-
NMR (100 MHz) CDCl3: 190.7, 167.4, 160.6 (d), 158.3, 149.8, 132.0, 131.4,
128.1 (d), 115.5, 112.1, 111.9, 111.6 (t), 107.1 (d), 21.9, 12.2. LC/MS-MS:
285.0 → 121.2 m/z; GS1 and GS2 at 30, DP = 4, CE = 29, CXP = 6, tR = 4.31
minutes.
(3,5-dibromo-4-hydroxyphenyl)(2-ethyl-5-fluorobenzofuran-3-yl)methanone 21:
In a RBF/SB, NBS (0.250 g, 1.41 mmol) diluted with DCM (12.0 mL)
was mixed with DMF (0.4 mL) and the mixture was cooled in an ice
bath (10 minutes). Compound 20 (0.200 g, 0.704 mmol) in DCM (1.0
mL) was added and warmed to RT (17 hours). The reaction mixture was

Human Uric Acid Transporter 1 (hURAT1)
1317
quenched with H2O and diluted with DCM (30 mL). The organic phase was
washed four times with H2O, NaCl aq, and then dried (Na2SO4), filtered,
and concentrated under reduced pressure, and purified twice via SiO2
chromatography (Hex:EtOAc; 4:1) to give 21 (176 mg, 0.398 mmol, 57%
yield) as a yellow solid. 1H-NMR (400 MHz) CDCl3: 7.97 (s, 2H), 7.43–7.40
(dd, 1H), 7.19–7.11 (d, 1H), 7.04–7.01 (t, 1H), 6.53 (bs, 1H), 2.88–2.84 (q,
2H), 1.36–1.33 (t, 3H); 13C-NMR (100 MHz) CDCl3: 187.4, 167.9, 160.9,
158.5, 153.3, 149.9, 133.3, 127.5 (d), 115.6, 112.5, 112.3, 111.8 (t), 110.1,
107.0 (d), 22.1, 12.1. LC/MS-MS: 442.9 → 278.8 m/z; GS1 and GS2 at 30,
DP = 21, CE = 35, CXP = 18, tR = 4.59 minutes.
(1,2-dimethyl-1H-indol-3-yl)(4-methoxyphenyl)methanone 22 was prepared
with analogous methods used with compound 5 except stirred at room tem-
perature for 16 hours. Compound 22 (215 mg, 0.770 mmol, 28% yield) was
a light brown solid. 1H-NMR (400 MHz) CDCl3: 7.80–7.78 (d, 2H), 7.38–7.36
(d, 1H), 7.33–7.31 (d, 1H), 7.23–7.19 (t, 1H), 7.10–7.06 (t, 1H), 6.95–6.93
(d, 2H), 3.89 (s, 3H), 3.74 (s, 3H), 2.60 (s, 3H); 13C-NMR (100 MHz) CDCl3:
191.7, 162.5, 143.8, 136.5, 133.8, 131.6, 127.1, 121.8, 121.1, 120.9, 113.8,
113.4, 109.1, 55.4, 29.6, 12.4. LC/MS-MS: 280.0 → 135.0 m/z; GS1 and GS2
at 30, DP = 21, CE = 29, CXP = 8, tR = 4.25 minutes.
(1,2-dimethyl-1H-indol-3-yl)(4-hydroxyphenyl)methanone 23 was prepared
with analogous methods used with compound 13. Compound 23 (40 mg, 1.50
1
mmol, 84% yield) was a white solid. H-NMR (400 MHz) CDCl3: 7.75–7.73
(d, 2H), 7.37–7.32 (t, 2H), 7.23–7.19 (t, 1H), 7.10–7.06 (t, 1H), 6.89–6.87 (d,
2H), 5.57 (bs, 1H), 3.75 (s, 3H), 2.61 (s, 3H); 13C-NMR (100 MHz) CDCl3:
191.8, 158.9, 144.0, 136.5, 133.9, 131.9, 127.1, 121.9, 121.2, 120.9, 115.0,
113.7, 109.1, 29.7, 12.4. LC/MS-MS: 266.0 → 121.1 m/z; GS1 and GS2 at 30,
DP = 21, CE = 31, CXP = 6, tR = 3.96 minutes.
(3,5-dibromo-4-hydroxyphenyl)(1,2-dimethyl-1H-indol-3-yl)methanone 24 was
prepared with analogous methods used with compound 21. Compound 24
1
(17.0 mg, 0.040 mmol 54% yield) was a white solid. H-NMR (400 MHz)
DMSO-d6: 10.7 (bs, 1H), 7.78 (s, 2H), 7.53–7.55 (d, 1H), 7.31–.7.32 (d, 1H),
7.18–7.22 (t, 1H), 7.07–7.11 (t, 1H), 3.77 (s, 3H), 2.50 (s, 3H); 13C-NMR
(100 MHz) DMSO-d6: 187.9, 154.2, 145.6, 136.8, 135.2, 133.4, 126.8, 122.4,
121.8, 120.1, 112.2, 111.8, 110.7, 30.3, 12.9. LC/MS-MS: 423.9 → 278.8 m/z;
GS1 and GS2 at 30, DP = 21, CE = 37, CXP = 18, tR = 4.26 minutes.
Functional Analysis via Oocytes Expressing hURAT1
The methods used to test compounds via oocytes expressing hURAT1
were the same as those previously described.[17]
Computational Analysis
Chemical structures were drawn using CS Chem-Draw UltraꢀR (ver-
sion 6.0.1; Cambridge Soft Corporation, Cambridge, MA, USA). The struc-
tures were copied into CS Chem3D UltraꢀR and molecular geometries were

1318
M. F. Wempe et al.
generated in the Gaussian Z-matrix style via the CS MOPAC application. For
each compound, an Austin-Model (AM1) semiempirical calculation (closed
shell, tight convergence criteria) was conducted using Gaussian© software
packet (Gaussian, Inc., Carnegie, PA, USA).[18] Next, gas phase ab initio ge-
ometry optimizations were performed using Gaussian 03 at the Hartree–Fock
(HF) level of theory using the 6-311G basis set.[19,20]
RESULTS AND DISCUSSION
As summarized in Figure 2 and Table 1, we prepared and tested a
series of (2-ethylbenzofuran-3-yl)(3,4,5-substituted-phenyl)methanone com-
pounds not previously described.[17] These compounds and the in vitro data
illustrate very important structural requirements in order for a molecule
to become a potent hURAT1 inhibitor. We chemically modified the para-
position of the C-ring[17] and subsequently probed 14C-urate (10.0 µM)
transport inhibition (50 µM test compound). The functional group trans-
formation from hydrogen 5, to methoxy 6, to phenol 7 afforded a profound
inhibitory effect. Phenol (7) was fairly potent and subsequently tested at
a variety of concentrations to generate an in vitro IC50 value (2.80 0.18
µM; Table 1). The insightful modification of methoxy 6 to phenol 7 sug-
gested the notion that a good inhibitor required a hydrogen bonding inter-
action in this region of the molecule. However, the fabrication and testing
of fluoro 8 and nitrile 9—two molecules that may participate in hydrogen
bonding—showed that they were not appreciable hURAT1 inhibitors. From
FIGURE 2 General synthesis of (2-ethylbenzofuran-3-yl)(3,4,5-(substituted)-phenyl)methanones and
(3,5-dibromo-4-hydroxyphenyl)(1,2-dimethyl-1H-indol-3-yl)methanone.

Human Uric Acid Transporter 1 (hURAT1)
1319
TABLE 1 Compound and inhibition data summary
% Inhibition
at 50 µM
Entry
R1
R2
R3
R4
R5
IC50
5
6
7
8
9
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
H
H
H
H
H
H
H
H
H
H
H
H
O-Me
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Br
Br
Br
H
H
Br
H
H
Br
H
O-Me
OH
F
CN
O-Me
O-Me
O-Me
OH
OH
OH
OH
OH
OH
O-Me
OH
OH
O-Me
OH
H
H
H
H
H
F
Cl
Br
F
Cl
Br
Br
Br
Br
H
H
Br
H
H
Br
13.5
13.3
90.8
10.4
23.6
29.4
52.4
22.2
97.9
98.8
99.1
99.9
99.9
99.8
27.2
98.0
100
ND
ND
2.80 0.18 µM
ND
ND
ND
ND
ND
10
11
12
13
14
15
16
17
18
19
20
21
22∗
23∗
24∗
2.41 0.09 µM
874 56 nM
814 160 nM
26 3 nM
111 14 nM
138 88 nM
ND
F
F
H
H
H
1.59 0.11 µM
6
4 nM
H
H
H
74.0
95.6
100
45.21 0.39 µM
2.05 0.12 µM
401 116 nM
OH
∗See Figure 2 for the complete chemical structure.
these data, the concept of hydrogen bonding was clearly not the predominate
interaction to afford a potent inhibitor. Next, we continued to probe these
concepts by modifying the C-Ring and preparing monohalogen methoxy
analogues where X = F, Cl, and Br; compounds 10, 11, and 12, respectively.
Interesting enough, while there was a moderate inhibitory enhancement by
incorporating a monohalogen (chloro-methoxy performed the best at 50
µM), these methoxy analogues were still weak inhibitors. These data also
support the idea that a phenol may be required in the C-ring. This concept
becomes very clear after preparing and testing phenol analogues 13, 14, and
15. These compounds demonstrate a halogen/steric effect where modifi-
cation to the chloro 14 and bromo 15 analogues produced submicromolar
(<1.0 µM) hURAT1 inhibitors (874 and 814 nM, respectively). Lastly, the
incorporation of an additional bromine atom ortho to the phenol to afford
dibromo-analogue 16 (benzbromarone) generated a very potent inhibitor
(26 nM). While also potent inhibitors, 6-methoxy 17 and 6-hydroxy 18 (the
major in vivo metabolite of 16) were less potent than 16. Thus, it appears
that an electron donating group on the B-ring decreases inhibitor potency.
This notion appears to be supported by comparing 5-fluoro analogues 19,
20, and 21. As was the case with going from 6 to 7 and then to 16, going
from methoxy analogue 17 to phenol 18, and subsequently to dibromo 21,
produced a dramatic increase in inhibitory potency. The inductively with-
drawn 5-fluoro analogue 21 became superior to 16, having an IC50 of 6

1320
M. F. Wempe et al.
FIGURE 3 Resonance-stabilized anions of urate and compounds 15 and 16.
4 nM (Table 1). In addition, we investigated the influence of replacing the
oxygen atom within the benzofuran functionality with a nitrogen atom to
afford (1,2-dimethyl-1H-indol-3-yl)methanone compounds 22, 23, and 24.
Interestingly enough, compounds 22 and 23 were more potent inhibitors
than 6 and 7, respectively. However, compound 24 was not as potent as 16.
These examples also illustrate the importance of the A-ring heteroatom and
the influence of electronics and substituted groups.
As depicted in Figure 3, urate, a weak acid in blood and urine, has a pKa
of approximately 5.75 and 5.35, respectively, and therefore predominates
in the anionic form. As an anion, 25 functions as a substrate of various or-
ganic anion transporters such as hOAT1 and hOAT3, as well as NPT4 and
hURAT1. Furthermore, the data supports the notion that hURAT1 has a
binding pocket possessing a positive charge, and that Coulombic interac-
tions are required for substrate and/or inhibitor recognition. Comparing
the increase in inhibitory potential going from monobromo 15 to dibromo
16, the change in molecular acidity (more acidic, lower pKa) produces
a shift in equilibrium (i.e., Le Chatelier’s principle) to afford resonance-
stabilized anions 26 and 27, respectively. The more acidic 16 produces a
higher concentration of anion 27, and thus is a better inhibitor. Further-
more, phenols are well known to undergo phenol-keto tautomerism and

Human Uric Acid Transporter 1 (hURAT1)
1321
FIGURE 4 Computed HF/6-311G natural population analysis (NPA) charge versus pKa and hURAT1
inhibitory potential; ꢀ = phenol; ꢁ = chlorophenol; ꢂ = bromophenol; • = dichlorophenol; ꢃ =
dibromophenol; ꢄ = urate; ◦ = test compounds.
thus, their equilibrium may be influenced by the chemical environment.[21]
We compared the gas phase energies of 15 and 16 and their correspond-
ing keto tautomers 28 and 29, respectively. The keto tautomers were com-
puted (HF/6-311G) to be 23.4 and 24.6 kcal/mol higher in energy than
their corresponding phenols, respectively. To further illustrate the impor-
tance of the anion phenomena, we conducted additional computational
modeling using various phenols (phenol, o-chloro-phenol, o-bromo-phenol,
2,6-dichlorophenol, and 2,6-dibromophenol) with well-known pKa values
(9.95, 8.48, 8.42, 6.79, and 6.67, respectively). The anions were computed
and the charges (Natural Population Analysis; obtained via Pop=NPA and
SCF=Tight) of the phenolic oxygen at the HF/6-311G basis set were plotted
(Figure 4). A linear relationship between the charge and experimental pKa
exists. The charge corresponding to the urate nitrogen anion 25 was also
included. The theoretical charges for compounds 7, 13–16, and 21 were
then plotted. These data (Figure 4) further illustrate the importance of the
phenol functionality and its intrinsic acidity to afford a higher concentration
of anions, and thus enhance inhibitor potency.
In conclusion, the experimental data support the notion that a potent
hURAT1 inhibitor requires an anion (i.e., a formal negative charge) to in-
teract with the positively charged binding pocket. An anion appears to be
a primary requirement in order to be a hURAT1 substrate (i.e., urate) or
inhibitor (i.e., 16). The difference between the molecules being a substrate
as opposed to an inhibitor relates to molecular size. For example, 1 has
3
˚
a computed Connolly Solvent-Excluded Volume (CSEV) of 92.3 A, while
3
˚
compound 16 has a CSEV of 253.1 A . In our hands, compound 16 was
not readily transported by hURAT1 and inhibitor interactions occurred at

1322
M. F. Wempe et al.
the apical (urine) side.[17] Lastly, electronically withdrawing group(s) on
the B-ring appear to enhance inhibitor potency and are likely helpful for
avoiding and/or reduceing the P450 metabolism associated with the B-ring.
For example, hydroxy 18 is the major metabolite of compound 16 in vivo.
Our current efforts are focused on developing B-ring-modified analogs and
blocking unwanted the CYP-catalyzed metabolism believed to be responsible
for unwanted drug–drug interactions with respect to this chemical template.
Furthermore, we are studying other transporter proteins known to be in-
volved in urate re-absorption and secretion (e.g., URATv1, hOAT’s).
REFERENCES
1. Becker, M.A.; Jolly, M. Hyperuricemia and associated diseases. Rheum. Dis. Clin. N. Am. 2006, 32,
275–293.
2. Anzai, N.; Kanai, Y.; Endou, H. New insights into renal transport of urate. Curr. Opin. Rheumatol.
2007, 19, 151–157.
3. Kutzing, M.K.; Firestein, B.L. Altered uric acid levels and disease states. J. Pharmacol. Exp. Ther. 2008,
324, 1–7.
4. Shin, H.J.; Takeda, M.; Enomoto, A.; Fujimura, M.; Miyazaki, H.; Anazi, N.; Endou, H. Interactions
of urate transporter URAT1 in human kidney with uricosuric drugs. Nephrology 2011, 16, 156–162.
5. Enomoto, A.; Kimura, H.; Chairoungdua, A.; Shigeta, Y.; Jutabha, P.; Cha, S.H.; Hosoyamada,
M.; Takeda, M.; Sekine, T.; Igarashi, T.; Matsuo, H.; Kikuchi, Y.; Oda, T.; Ichida, K.; Hosoya, T.;
Shimokata, K.; Niwa, T.; Kanai, Y.; Endou, H. Molecular identification of a renal urate anion ex-
changer that regulates blood urate levels. Nature 2002, 417, 447–452.
6. Roch-Ramel, F.; Guisan, B. Renal transport of urate in humans. News Physiol. Sci. 1999, 14, 80–84.
7. Mount, D.B.; Kwon, C.Y.; Zandi-Nejad, K. Renal urate transport. Rheum. Dis. Clin. N. Am. 2006, 32,
313–331.
8. Huls, M.; Brown, C.D.A.; Windass, A.S.; Sayer, R.; van den Heuvel, J.J.M.W.; Heemskerk, S.; Russel,
F.G.M.; Masereeuw, R. The breast cancer resistance protein transporter ABCG2 is expressed in the
human kidney proximal tubule apical membrane. Kidney Int. 2008, 73, 220–225.
9. Woodward, O.M.; Ko¨ttgen, A.; Coresh, J.; Boerwinkle, E.; Guggino, W.B.; Ko¨ttgen, M. Identification
of a urate transporter, ABCG2, with a common functional polymorphism causing gout. PNAS 2009,
106, 10338–10342.
10. Jutabha, P.; Anzai, N.; Kitamura, K.; Taniguchi, A.; Kaneko, S.; Yan, K.; Yamada, H.; Shimada,
H.; Kimura, T.; Katada, T.; Fukutomi, T.; Tomita, K.; Urano, W.; Yamanaka, H.; Seki, G.; Fujita,
T.; Moriyama, Y.; Yamada, A.; Uchida, S.; Wempe, M. F.; Endou, H.; Sakurai, H. Human sodium-
phosphate transporter 4 (hNPT4/SLC17A3) as a common renal secretory pathway for drugs and
urate. J. Biol. Chem. 2010, 285, 35123–35132.
11. Anzai, N.; Ichida, K.; Jutabha, P.; Kimura, T.; Babu, E.; Jin, C.J.; Srivastava, S.; Kitamura, K.; Hisatome,
I.; Endou, H.; Sakurai, H. Plasma urate level is directly regulated by a voltage-driven urate efflux
transporter URATv1 (SLC2A9) in humans. J. Biol. Chem. 2008, 283, 26834–26838.
12. Caulfield, M.J.; Munroe, P.B.; O’Neill, D.; Witkowska, K.; Charchar, F.J.; Doblado, M.; Evans, S.;
Eyheramendy, S.; Onipinla, A.; Howard, P.; Shaw-Hawkins, S.; Dobson, R.J.; Wallace, C.; Newhouse,
S.J.; Brown, M.; Connell, J.M.; Dominiczak, A.; Farrall, M.; Lathrop, G.M.; Samani, N.J.; Kumari,
M.; Marmot, M.; Brunner, E.; Chambers, J.; Elliott, P.; Kooner, J.; Laan, M.; Org, E.; Veldre, G.;
Viigimaa, M.; Cappuccio, F.P.; Ji, C.; Lacone, R.; Strazzullo, P.; Moley, K.H.; Cheeseman, C. SLC2A9
is a high-capacity urate transporter in humans. PLoS Med. 2008, 5, e197.
13. Sekine, T.; Watanabe, N.; Hosoyamada, M.; Kanai, Y.; Endou, H. Expression cloning and characteri-
zation of a novel multispecific organic anion transporter. J. Biol. Chem. 1997, 272, 18526–18529.
14. Saag, K.G.; Choi, H. Epidemiology, risk factors, and lifestyle modifications for gout. Arthr. Res. Ther.
2006, 8(Suppl. 1:S2).
15. Feig, D.I.; Kang, D.H.; Johnson, R.J. Uric acid and cardiovascular risk. N. Engl. J. Med. 2008, 359,
1811–1821.

Human Uric Acid Transporter 1 (hURAT1)
1323
16. Anzai, N.; Jutabha, P.; Endou, H. Renal solute transporters and their relevance to serum urate
disorder. Curr. Hypertens. Rev. 2010, 6, 148–154.
17. Wempe, M.F.; Jutabha, P.; Quade, B.; Iwen, T.J.; Frick, M.M.; Rice, P.J.; Anzai, N.; Endou, H. Devel-
oping potent human uric acid transporter 1 (hURAT1) inhibitor. J. Med. Chem. 2011, 54, 2701–2713.
18. Frisch, M.J.; Trucks, G.W.; Schlegel, H.B.; Scuseria, G.E.; Robb, M.A.; Cheeseman, J.R.; Montgomery,
J.A.; Vreven, T.; Kudin, K.N.; Burant, J.C. Gaussian 03 Revision C.02, Gaussian, Inc., Wallingford, CT,
2003.
19. Hehre, W.J.; Stewart, R.F.; Pople, J.A. Self-consistent molecular-orbital methods. I. Use of Gaussian
expansions of Slater-type atomic orbitals. J. Chem. Phys. 1969, 51, 2657–2665.
20. Collins, J.B.; Schleyer, PvR.; Binkley, J.S.; Pople, J.A. Self-consistent molecular orbital methods. XVII.
Geometries and binding energies of second-row molecules. A comparison of three basis sets. J. Chem.
Phys. 1976, 64, 5142–5151.
21. Smith, M.B.; March, J. March’s Advanced Organic Chemistry Reactions, Mechanisms, and Structure, 6th ed.,
John Wiley & Sons, Inc., Hoboken, NJ, 2007, pp. 107–109.