Nanomolar-Potency 1,2,4-Triazoloquinoxaline Inhibitors of the Kidney Urea Transporter UT-A1

Article abstract of DOI:10.1021/acs.jmedchem.8b00343

Urea transporter A (UT-A) isoforms encoded by the Slc14a2 gene are expressed in kidney tubule epithelial cells, where they facilitate urinary concentration. UT-A1 inhibition is predicted to produce a unique salt-sparing diuretic action in edema and hyponatremia. Here we report the discovery of 1,2,4-triazoloquinoxalines and the analysis of 37 synthesized analogues. The most potent compound, 8ay, containing 1,2,4-triazolo[4,3-a]quinoxaline-substituted benzenesulfonamide linked by an aryl ether, rapidly and reversibly inhibited UT-A1 urea transport by a noncompetitive mechanism with IC₅₀ ≈ 150 nM; the IC₅₀ was ～2 μM for the related urea transporter UT-B encoded by the Slc14a1 gene. Molecular modeling suggested a putative binding site on the UT-A1 cytoplasmic domain. In vitro metabolism showing quinoxaline ring oxidation prompted the synthesis of metabolically stable 7,8-difluoroquinoxaline analogue 8bl, which when administered to rats produced marked diuresis and reduced urinary osmolality. 8bl has substantially improved UT-A1 inhibition potency and metabolic stability compared with prior compounds.

Full text of DOI:10.1021/acs.jmedchem.8b00343

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Nanomolar-potency 1,2,4-triazoloquinoxaline
inhibitors of the kidney urea transporter A1 (UT-A1)
Sujin Lee, Onur Cil, Elena Diez-Cecilia, Marc O. Anderson, and Alan S Verkman
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00343 • Publication Date (Web): 28 Mar 2018
Downloaded from http://pubs.acs.org on March 28, 2018
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Nanomolarꢀpotency 1,2,4ꢀtriazoloquinoxaline
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inhibitors of the kidney urea transporter A1 (UTꢀA1)
Sujin Lee1, Onur Cil,1,2, Elena Diez-Cecilia3, Marc O. Anderson1,3, Alan S. Verkman1
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1Departments of Medicine and Physiology, Department of Pediatrics, Division of Nephrology,
3
University of California, San Francisco CA, 94143ꢀ0521, U.S.A. and Department of Chemistry
and Biochemistry, San Francisco State University, San Francisco CA, 94132ꢀ1722, U.S.A.
KEYWORDS urea transport, UTꢀA, diuretic, urinary concentrating mechanism, 1,2,4ꢀ
triazoloquinoxaline
ABSTRACT: Urea transporterꢀA (UTꢀA) isoforms encoded by the Slc14a2 gene are expressed in
kidney tubule epithelial cells where they facilitate urinary concentration. UTꢀA1 inhibition is
predicted to produce a unique, saltꢀsparing diuretic action in edema and hyponatremia. Here, we
report the discovery of 1,2,4ꢀtriazoloquinoxalines and analysis of 37 synthesized analogs. The
most potent compound, 8ay, containing 1,2,4ꢀtriazolo[4,3ꢀa]quinoxaline substituted
benzenesulfonamide linked by an aryl ether, rapidly and reversibly inhibited UTꢀA1 urea
transport by a nonꢀcompetitive mechanism with IC50 ~150 nM; IC50 was ~2 µM for the related
urea transporter UTꢀB encoded by the Slc14a1 gene. Molecular modeling suggested a putative
binding site on the UTꢀA1 cytoplasmic domain. In vitro metabolism showing quinoxaline ring
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oxidation prompted synthesis of metabolically stable 7,8ꢀdifluoroquinoxaline analog 8bl, which
when administered to rats produced marked diuresis and reduced urinary osmolality. 8bl has
substantially improved UTꢀA1 inhibition potency and metabolic stability compared to prior
compounds.
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INTRODUCTION
Urea transporters (UTs) in mammalian kidney are targets for the development of a new class
of diuretics for the treatment of edema and hyponatremia in congestive heart failure, cirrhosis,
nephrotic syndrome and other disorders associated with fluid retention.1ꢀ6 Unlike available
diuretics, UT inhibition disrupts the renal countercurrent mechanisms, which are required for the
generation of a concentrated urine, producing a diuretic response with relative saltꢀsparing.
Evidence for this mechanism comes from studies in transgenic mice lacking various UTs,7ꢀ13
from mathematical modeling of urinary concentration,14 and from rodent studies with
administration of UT inhibitors.15ꢀ17
Mammalian UTs are encoded by Slc14a2 (‘UTꢀA’ isoforms) and Slc14a1 (‘UTꢀB’ isoform)
genes. UTꢀAs are expressed in epithelial cells in kidney tubules, whereas UTꢀB is expressed in
kidney vasa recta endothelia as well as in tissues outside of the kidney including erythrocytes,
testis, urinary bladder, heart and brain.18 Of the various UT isoforms, the vasopressinꢀregulated
UTꢀA1 in inner medullary collecting duct is the principal target for UTꢀtargeted diuretic
development.19
The originally described UT inhibitors include millimolarꢀpotency urea analogs20ꢀ22 and the
nonꢀselective membrane intercalating agent phloretin.23 Using an erythrocyte lysis assay, we
originally identified highly selective UTꢀB inhibitors with IC50 down to 15 nM, which produced
a mild diuresis in mice.24, 25 Subsequently, we developed a highꢀthroughput screen to identify
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UTꢀA1 inhibitors using triply transfected MDCK cells expressing UTꢀA1, water channel
aquaporin AQP1, and a yellow fluorescence protein volume (chloride) sensor.26 Screening
produced UTꢀA1 selective inhibitors with low micromolar potency and low to modest metabolic
stability, which when delivered systemically in high doses to rats produced a diuretic response.27
A recent study reported that UTꢀA and UTꢀB double knockꢀout mice showed increased urine
output compared to the single knockꢀout mice,28 suggesting the potential utility of nonꢀselective
UT inhibitors.
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Here, we report compounds with substantially improved UTꢀA1 inhibition potency and
metabolic stability compared to prior compounds. Following highꢀthroughput screening, the
1,2,4ꢀtriazoloquinoxaline scaffold was selected for focused medicinal chemistry to optimize UTꢀ
A1 inhibition potency and pharmacological properties.
RESULTS AND DISCUSSION
Screening and scaffold selection
Collections totaling ~150,000 drugꢀlike synthetic small molecules were screened to identify
inhibitors of rat UTꢀA1 using a cellꢀbased fluorescence plate reader assay. Fig. 1 and Fig. S1
show structures of confirmed active compounds of at least 12 distinct chemical classes that
produced >80% UTꢀA1 inhibition at 25 µM. In order to select a scaffold for focused medicinal
chemistry, we assayed 80 to 150 commercially available analogs of each class (1ꢀ4, S1ꢀS7, and
8aa), with the primary goal of high potency UTꢀA1 inhibition, and a secondary goal of some
UTꢀB inhibition. A common characteristic of the UTꢀA1 inhibitors was a linear multiꢀ
heterocyclic structure such as 1 and 2. However, these linear multiꢀheterocyclic structures
showed little UTꢀB inhibition, which is also the case for 2ꢀphenylquinoline 3. Compound 4 has a
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similar thienoquinoline structure to previously reported PUꢀ48,29 and has low potency for UTꢀA1
inhibition. Another common structural motif of compounds with greatest UTꢀA1 inhibition
potency was substituted benzenesulfonamide linked to an aromatic ring, such as 5,26 6, 7 and
8aa. Of the benzenesulfonamide analogs, the 1,2,4ꢀtriazolo[4,3ꢀa]quinoxaline 8aa had greatest
UTꢀA1 inhibition potency with IC50 ~0.8 µM, while retaining modest activity against UTꢀB.
Based on considerations of potencies against UTꢀA1 and UTꢀB, drugꢀlike properties, modularity
of construction, and presence of hydrogen bond donating and accepting functionality, focused
medicinal chemistry was done here on the 4ꢀoxyphenylꢀ1,2,4ꢀtriazolo[4,3ꢀa]quinoxaline scaffold
(8). Compounds having the general 1,2,4ꢀtriazolo[4,3ꢀa]quinoxaline scaffold, though quite
distinct from those studied here, have been reported as adenosine receptor antagonists,30 and
inhibitors of folate cycle,31 BET,32 and PDE2.33
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Figure 1. (A) Structures and IC50 values of active UTꢀA1 inhibitors identified by highꢀ
throughput screening. (B) Strategy to explore structureꢀactivity relationships of 8aa analogs.
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Preliminary structureꢀactivity studies of 156 commercial analogs of 8aa indicated that a
substituted benzenesulfonamide on the 4ꢀoxyaniline group was important for UTꢀA1 inhibition
(Fig. S1). Analogs of 8aa in which the sulfonamide was replaced with NH2, OH, or H were
inactive. Compounds linked through an amide instead of sulfonamide were inactive, as were 2,4ꢀ
disubstituted benzenesulfonamides or 2ꢀthiophene sulfonamide. In general, the most active
compounds contained a 4ꢀoxyaniline moiety linked via a sulfonamide to a 2,5ꢀdisubstituted
benzene.
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Our exploration of the structureꢀactivity of this scaffold focused on four distinct components
(Fig. 1B), including: (a) modification of the X group (ether and amine linkages); (b)
benzenesulfonamide substitutions; (c) alkyl incorporation on the 1,2,4ꢀtriazole moiety; and (d)
disubstitution on the triazoloquinoxaline backbone.
Chemistry
Scheme 1 shows the synthetic route developed for 5ꢀalkylꢀ1,2,4ꢀtriazoloquinoxaline analogs in
4 steps from commercially available 2,3ꢀdichloroquinoxaline 9. Monoꢀsubstitution with
hydrazine on 2,3ꢀdichloroquinoxaline afforded hydrazinylquinoxaline 10, which was condensed
with triethyl orthoformate to give triazoloquinoxaline 11a. Nucleophilic aromatic substitution
with 3ꢀ or 4ꢀaminophenol in the presence of cesium carbonate in DMSO yielded 12a and 12b.
Use of cesium carbonate ensured deprotonation of the phenol to enhance nucleophilicity of the
oxygen, preventing attack by nitrogen. Nucleophilic substitution with p-, or m-phenylenediamine
in the presence of potassium carbonate yielded Nꢀsubstituted intermediates 12c and 12d.
Subsequently, 12aꢀ12d were reacted with various substituted benzenesulfonyl chlorides to give
8aaꢀ8am (Table 1). To vary substitution at the R2 position, hydrazinylquinoxaline was reacted
with various trialkyl orthoalkanoates to generate 11bꢀ11g. These intermediates were similarly
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reacted with p-aminophenol to yield aminobenzenes 12eꢀ12j. Lastly, these intermediates were
conjugated with a focused collection of substituted benzenesulfonyl chlorides to generate 8anꢀ
8bd (Table 2).
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Initial microsomal stability experiments on 8aa as described below suggested the electron rich
1,2,4ꢀtriazoloquinoxaline heteroaryl ring to be susceptible to oxidation. As such, and to explore
the importance of Part D, we devised a synthesis allowing variation of this region (Scheme 2).
Scheme 1. General synthesis of 1,2,4ꢀtriazoloquinoxalines.a
o
aReagents and conditions: (a) NH2NH2, EtOH; (b) HC(OEt)3, 100 C; (c) p-aminophenol,
o
Cs2CO3, DMSO, 50 C; (d) pꢀphenylenediamine, K2CO3, DMSO; (e) R1SO2Cl, DIPEA,
acetonitrile, 50 oC; (f) R2C(OMe)3 or R2C(OEt)3.
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Scheme 2. Synthesis of 7,8ꢀdisubstituted 1,2,4ꢀtriazoloquinoxalines.a
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aReagents and conditions: (a) NH2NH2, EtOH; (b) R2C(OEt)3 or R2C(OMe)3, 100 oC; (c) p-
aminophenol, Cs2CO3, DMSO, 50 oC; (d) R1SO2Cl, DIPEA, acetonitrile, 50 oC.
Several 6,7ꢀdisubstituted 2,3ꢀdichloroquinoxalines (13aꢀ13c) were transformed to the
hydrazine derivatives (14aꢀ14c), cyclized to the 1,2,4ꢀtriazoloquinoxalines (15aꢀ15g) using
appropriate trialkyl orthoformate or orthoalkane species, and then coupled with pꢀaminophenol
to the aminobenzene derivatives (16aꢀ16g). Lastly, a focused selection of substituted
benzenesulfonyl chlorides were coupled to generate 8beꢀ8bl (Table 3). We found some of the
1,2,4ꢀtriazoloquinoxalines (15) and 4ꢀaminophenol conjugated intermediates (16) difficult to
purify. Fortunately the sequence 15 ꢀ 16 ꢀ 8 could often be “tunneled” without purification of
each synthetic intermediate, but with the final products isolated in high purity. The selection of
the pꢀaminophenol linkage, as well as a small set of R1 and R2 groups in this series, was based on
SAR that emerged from the previous set of compounds in Tables 1 and 2.
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Table 1. UTꢀA1 inhibition of 1,2,4ꢀtriazoloquinoxalines with modifications in Parts A and
B.
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UTꢀA
IC50
Isolated
Yield
(%)
Compound
X
m-, or p-
R1
(µM)
0.8
>20
>20
>20
1.1
1.5
>20
5
O
O
p-
m-
p-
m-
p-
p-
p-
p-
p-
p-
p-
m-
p-
A
A
A
D
B
C
D
E
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35
36
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99
82
70
41
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87
44
48
8aa
8ab
8ac
8ad
8ae
8af
NH
NH
O
O
O
8ag
8ah
8ai
O
O
F
8
O
G
H
D
D
20
8aj
O
>20
>20
>20
8ak
8al
O
NH
8am
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Structureꢀactivity analysis
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UTꢀA1 inhibition data for all compounds (8aaꢀ8bl) is summarized in Tables 1ꢀ3. We first
examined the substitution pattern of the aryl linker (Part A, Table 1). The pꢀaminophenol linker
in 8aa resulted in greater UTꢀA1 inhibition potency when compared to similar compounds linked
with mꢀaminophenol (8ab), pꢀdiaminobenzene (8ac or 8am) or mꢀdiaminobenzene (8ad).
Variation of R1 (the benzenesulfonamide, Part B) showed that 2,5ꢀdisubstituted benzenes were
favored, with 5ꢀchloroꢀ2ꢀmethoxy (8aa) having greatest potency, while compounds containing 5ꢀ
methylꢀ2ꢀmethoxy (8ae) and 2,5ꢀdimethyl (8af) were less potent, and 2ꢀchloroꢀ5ꢀmethoxy (8ag)
was inactive. Compounds with a single o- or m- substituent on the benzenesulfonamide (8ahꢀ
8ak) were substantially less potent and not further explored. The R2 substituent was next varied
(Table 2), maintaining the most promising 2,5ꢀdisubstituted benzenesulfonamides at R1.
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Table 2. UTꢀA1 inhibition of 1,2,4ꢀtriazoloquinoxalines with modifications in Part C.
UTꢀA IC50
Isolated
Compound
R1
R2
Yield (%)
(µM)
>20
>20
>20
0.5
A
B
C
A
CH3
CH3
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36
48
72
8an
8ao
8ap
8aq
CH3
CH2CH3
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B
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A
B
C
CH2CH3
CH2CH3
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0.7
0.75
0.3
0.5
0.6
1.2
1.5
0.15
0.4
0.5
0.2
0.4
0.8
8ar
8as
8at
CH(CH3)2
CH(CH3)2
CH(CH3)2
OCH2CH3
OCH2CH3
CH2CH2CH3
CH2CH2CH3
CH2CH2CH3
(CH2)3CH3
(CH2)3CH3
(CH2)3CH3
8au
8av
8aw
8ax
8ay
8az
8ba
8bb
8bc
8bd
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Short, linear and branched alkyl groups at R2 were investigated due to ease of installation,
although ethoxy was also possible using our synthetic scheme. Varying the length of alkyl group
at R2 showed that propyl (8ay) was best, while butyl (8bb) and isopropyl (8at) were comparable,
ethyl (8aq) and ethoxy (8aw) were slightly worse, and methyl (8an) was inactive. Across this
series (and compounds listed in Table 1), the 5ꢀchloroꢀ2ꢀmethoxy group at R1 appeared better
than 5ꢀmethylꢀ2ꢀmethoxy and 2,5ꢀdimethyl.
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Table 3. UTꢀA1 inhibition of 1,2,4ꢀtriazoloquinoxalines with different R3.
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Isolate
d Yield
(%)
UTꢀA
IC50 (µM)
Compound
R1
R2
R3
A
A
A
A
A
A
B
A
H
CH2CH3
CH(CH3)2
CH2CH2CH3
H
CH3
CH3
CH3
CH3
Cl
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73
99
80
70
67
40
48
2.7
1.4
1.2
0.4
>20
1.2
1.5
0.3
8be
8bf
8bg
8bh
8bi
H
F
8bj
8bk
8bl
H
F
CH2CH2CH3
F
Lastly, variation of the R3 substituent was explored (Table 3). Methyl substitution (8beꢀ8bh)
produced less potent inhibitors than the analogous unꢀsubstituted derivatives (8aa, 8aq, 8at,
8ay), although propyl at R2 (8bh) achieved the best potency in the subset. Also, we observed that
the relation between alkyl chain length and branching to inhibition potency was similar to the
analogous unꢀsubstituted analogs. A dichloro substituted compound (8bi) was inactive, while
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difluoro at R3 was tolerated in three analogs (8bjꢀ8bl). Among the R3 substituted compounds,
8bl was the most potent, preserving the propyl substituent at R2 (IC50 = 0.3 µM), although it was
less potent than the analogous unsubstituted compound 8ay (IC50 = 0.15 µM).
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Functional studies
Fig. 2A shows original fluorescence plate reader data for inhibition of UTꢀA1 urea transport in
MDCK cells. Curves are shown for zero inhibition (blue, DMSO vehicle control) and 100%
inhibition (red, phloretin), and different concentrations of 8ay. Deduced concentrationꢀinhibition
curves for 8ay, as well as for 8aa and 8bl, are shown in Fig. 2B, and Fig. 2C shows
concentrationꢀinhibition curves for rat UTꢀB, with IC50 values for 8ay, 8aa and 8bl of 2, 4 and
0.8 µM, respectively. For comparison, the previously reported thienoquinolin PUꢀ4829 showed
weak and partial UTꢀA1 inhibition, though better inhibition potency for UTꢀB.
The most potent analog 8ay was further characterized for reversibility, inhibition mechanism
and kinetics. Reversibility was studied by incubation of cells with 0.5 ꢁM 8ay for 15 min, then
washing, followed by assay of UTꢀA1 inhibition. Inhibition was fully reversed (Fig. 2D). The
IC50 for 8ay inhibition of UTꢀA1 urea transport measured using different urea concentration
gradients (200, 400 mM and 800 mM) were similar (Fig. 2E), suggesting a nonꢀcompetitive
inhibition mechanism. Measurement of the kinetics of UTꢀA1 inhibition following 8ay addition
showed little immediate inhibition and a halfꢀtime for inhibition of ~2 min (Fig. 2F), suggesting
an intracellular site of action. Cytotoxicity was not observed at a high concentration (10 µM) of
several compounds tested (Fig. 2G).
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Figure 2. In vitro characterization of UTꢀA inhibitors. (A) Original fluorescence data showing
inhibition of rat UTꢀA1 by different concentrations of 8ay. Positive (100% inhibition, phloretin,
red curve) and negative (0% inhibition, DMSO vehicle, blue curve) curves shown. (B)
Concentrationꢀinhibition data for UTꢀA1 inhibition by indicated compounds (mean ± S.E.M., n =
3). (C) Concentrationꢀdependence data for UTꢀB inhibition by indicated compounds (mean ±
S.E.M., n = 3). (D) Reversibility study. Cells were incubated with 8ay at 0.5 µM for 15 min,
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washed for 15 min, and then assayed for UTꢀA1 inhibition. (E) Urea competition. Experiments
done as in A but with by different urea concentrations (200, 400 and 800 mM). (F) Kinetic study.
Experiments done as in A but at different times after addition of 0.5 µM 8ay. (G) Cytotoxicity
measured by Alamar Blue assay in transfected MDCK cells incubated for 24 h with 10 µM 8aa,
8ay or 8bl (mean ± S.E.M., n = 3). Vehicle control shown.
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Molecular docking
Fig. 3 shows a docked conformation of 8bl bound to a previously described homology model
of the UTꢀA1 cytoplasmic domain.26 In the docked conformation the central triazoloquinoxaline
heterocycle is positioned near several hydrophobic residues around the entrance of the
cytoplasmic channel, including Leu652, Pro705, Leu895, Val898. The hydrophobic propyl (R2
substituent) projects deeper into the cavity, surrounded by the side chains of Gln599, Phe602,
Tyr655, and Phe832. The 4ꢀoxyaniline linker group extends outward from the channel, near
Ser696 and Val706, while the substituted benzenesulfonamide resides completely outside of the
channel, making contacts with the cytoplasmic surface of the protein (nearest to Asp644, Leu895
and Ser896). Notably, a broader series of active analogs of 8bl were also docked (data not
shown), giving a similar binding orientation of the triazoloquinoxaline with alkyl substituents at
R2 projecting into the channel. For comparison, a docked pose of inactive analog 8am is
provided in Fig. S3 (Supporting Information), in which the triazoquinoxaline and substituted
benzenesulfonamide ring systems are flipped into an opposite orientation, and this compound
lacks a hydrophobic substituent extending into the channel.
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Figure 3. Computational modeling of triazoloquinoxaline inhibitor bound to a homology
model of rat UTꢀA1. Zoomedꢀout (left) and zoomedꢀin (right) representations of 8bl interacting
with the UTꢀA1 cytoplasmic domain, showing complementary fit of the overall scaffold and
projection of the hydrophobic propyl group into the channel.
In vitro metabolic stability
In vitro metabolic stability was measured by compound incubation with rat hepatic microsomes
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at 37 C for different times in the presence of NADPH, following by LC/MS analysis. Fig. 4A
shows the time course of metabolism for relatively potent quinoxalines 8ay, 8bh, and 8bl (each
containing propyl at R2). Compounds 8ay (R3 = H) and 8bh (R3 = CH3) were rapidly degraded at
15 min with 40% and 25% remaining, respectively, which contrasted with 8bl (R3 = F), which
was substantially more stable, with 60% remaining at 30 min. Degradation of 8ay (m/z = 524)
was paralleled with an increase in signal at m/z = 540 (Fig. 4B), consistent with hydroxylation,
suggesting that the fluoro substituent in 8bl (R3 = F) prevented hydroxylation at this position
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(Fig. 4C), explaining its relative stability compared to 8ay. The rapid metabolism of 8bh was not
explored, but could be due to hydroxylation of the two benzylic methyl groups. In vivo studies in
rats were done using the relatively stable and potent 8bl.
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Figure 4. In vitro metabolic stability in rat hepatic microsomes. (A) Compounds 8ay, 8bh and
8bl at 5 ꢁM were incubated for indicated times with 1 mg/mL rat hepatic microsomes in the
presence of NADPH, and parent compound assayed by LC/MS (mean ± S.E.M., n = 3). (B)
LC/MS profile showing disappearance of 8ay over 30 min incubation (left) and appearance of
metabolite at +16 Da (right). (C) Schematic of potential sites of metabolism.
Rat pharmacokinetics and efficacy
After preliminary testing of testing different administration routes (oral, intraperitoneal,
intravenous), intravenous administration of 8bl at 4 mg/kg yielded serum concentrations greater
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than 2 µM for 3 hours (Fig. 5A left), and urine concentrations of 6ꢀ8 µM for 3 hours (Fig. 5A
right), each well above the IC50 of 0.3 µM for UTꢀA1 inhibition by 8bl determined in vitro.
Intravenous administration of 8bl at 4 mg/kg significantly increased 3ꢀhour urine volume and
reduced urine osmolality compared to preꢀtreatment values (Fig. 5B and C), whereas
administration of vehicle did not significantly change 3ꢀhour urine volume or osmolality.
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Figure 5. In vivo pharmacokinetics of 8bl and diuretic action in rats. (A) Kinetics of serum 8bl
concentration following intravenous administration of 4 mg/kg 8bl (left). 8bl concentration in
urine collected for 3 h (right). (B, C) 3ꢀhour urine volume (B) and osmolality (C) before and
after intravenous administration of 8bl or vehicle (mean ± S.E.M., n = 4). *P < 0.01, **P < 0.05
compared to ꢀ3 to 0 h.
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CONCLUSION
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The 1,2,4ꢀtriazolo[4,3ꢀa]quinoxaline analogs identified and characterized here are substantially
more potent and metabolically stable than previously described UTꢀA1 inhibitors. Inhibition of
UTꢀA1ꢀmediated urea transport was rapid, reversible and nonꢀcompetitive, and probably
involves inhibitor binding at the cytoplasmic surface of UTꢀA1 near but distinct from the urea
binding site. Following structureꢀactivity analysis, which resulted in improved metabolic stability
by difluoro substitution on triazoloquinoxaline backbone, 8bl was found to have favorable
pharmacokinetics in rats and produced a diuretic response. These results support further
preclinical development of 1,2,4ꢀtriazolo[4,3-a]quinoxalines as firstꢀinꢀclass diuretics targeting
kidney urea transporters.
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EXPERIMENTAL SECTION
General. All solvents and chemicals were used as purchased without further purification.
Reagents are purchased from SigmaꢀAldrich (St. Louis, MO), Alfa Aesar (Haverhill, MA), TCI
America (Portland, OR), and Maybridge. Reaction progress was monitored on Merck precoated
silica gel plates (with fluorescence at 254 nm) using ethyl acetate/nꢀhexane as solvent system.
Column chromatography was done with Fluka silica gel 60 (230−400 mesh). Proton (1H) and
carbon (13C) NMR spectra were recorded on a Bruker Avance 300 (300 MHz for 1H; 75 MHz for
1
13
13C) or 500 (500.13 MHz for H; 125.76 MHz for C) using CDCl3, CD3OD, or DMSOꢀd6 as
solvents. Chemical shifts are given in parts per million (ppm). HRMS was performed using a
hybrid quadrupole Orbitrap mass analyzer, QExactive (Thermo, Bremen, Germany) with an
electrospray ionization source. The mass resolution was set as 70000 at m/z 200, and mass
accuracy was better than 3 ppm. Purity of all final compounds was 95% or higher as determined
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by high performance liquid chromatography (HPLC) using UV absorbance at 254 nm. HPLC
was done on a Xterra MS C18 column (2.1 mm × 100 mm, 3.5 ꢁm) with 0.2 mL/min
water/acetonitrile (containing 0.1% formic acid), 24 min linear gradient, 5−95% acetonitrile.
Nꢀ(4ꢀ([1,2,4]triazolo[4,3-a]quinoxalinꢀ4ꢀyloxy)phenyl)ꢀ5ꢀchloroꢀ2ꢀmethoxybenzene
sulfonamide 8aa
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To a suspension of 4ꢀoxyphenylquinoxaline amine 12a (70 mg, 0.25 mmol) in acetonitrile (7
mL) was added DIPEA (0.07 mL, 0.5 mmol) and 2ꢀmethoxyꢀ5ꢀchlorobenzenesulfonyl chloride
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(61 mg, 0.25 mmol). The mixture was stirred overnight at 60 C. After cooling to rt, water (7
mL) was added to the reaction mixture, resulting in the formation of the product as a precipitate,
which was dried in vacuo afforded product as yellow solid (70 mg, 58%). 1H NMR (300 MHz,
DMSOꢀd6) δ 10.28 (brs, 1H), 10.16 (s, 1H), 8.37 (d, 1H, J = 8.0 Hz), 7.74 (d, 1H, J = 2.7 Hz),
7.70 (d, 1H, J = 2.7 Hz), 7.68ꢀ7.56 (m, 3H), 7.31 (d, 2H, J = 9.0 Hz), 7.31ꢀ7.26 (m, 1H), 7.21 (d,
2H, J = 9.0 Hz), 3.91 (s, 3H); 13C NMR (75 MHz, DMSOꢀd6) δ 155.8, 151.4, 148.6, 138.7,
138.6, 135.6, 135.1, 134.1, 129.6, 128.5, 128.2, 128.1, 127.8, 124.5, 124.1, 123.0, 121.9, 116.9,
115.5, 57.0; HRMS m/z (ESI) calcd for C22H16ClN5O4S (M + H)+: 482.0673, found: 482.0685.
2ꢀChloroꢀ3ꢀhydrazinylquinoxaline 10
A solution of 2,3ꢀdichloroquinoxaline 9 (14.0 g, 69.6 mmol) and hydrazine monohydrate (3.5
mL, 70.0 mmol) in ethanol (250 mL) was stirred overnight at ambient temperature. The resulting
precipitate was filtered using a Büchner funnel under reduced pressure, then washed with iceꢀ
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cold ethanol (20 mL) and dried in vacuo. Yellow powder, 13.3 g, 97.8%. H NMR (300 MHz,
CDCl3) δ 7.88 (dd, 1H, J = 1.2, 8.1 Hz), 7.85 (dd, 1H, J = 1.2, 8.4 Hz), 7.65 (ddd, 1H, J = 1.2,
1.5, 8.4 Hz), 7.48 (ddd, 1H, J = 1.5, 8.1, 8.4 Hz), 4.18 (brs, 1H).
4ꢀchloroꢀ[1,2,4]triazolo[4,3,a]quinoxaline 11a
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A mixture of hydrozinylquinoxaline 10 (6.0 g, 30.8 mmol) and triethylorthoformate (50 mL,
300 mmol) was stirred at 100 oC for 1 h. After cooling, the reaction mixture was filtered using a
Büchner funnel under reduced pressure, then washed with cyclohexane (10 mL) and dried in
vacuo. Yellow solid, 4.0 g, 63%. 1H NMR (300 MHz, DMSOꢀd6) δ 10.2 (s, 1H), 8.46 (dd, 1H, J
= 0.9, 8.2 Hz), 8.04 (dd, 1H, J = 0.9, 8.1 Hz), 7.88ꢀ7.70 (m, 2H); 13C NMR (75 MHz, DMSOꢀd6)
δ 139.3, 138.9, 134.8, 130.7, 129.4, 128.6, 128.4, 125.3, 117.2; LRMS (ESI) m/z 205 [M+H]+.
4ꢀ([1,2,4]triazolo[4,3-a]quinoxalinꢀ4ꢀyloxy)aniline 12a
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To a solution of quinoxaline 11a (300 mg, 1.5 mmol) in DMSO (4 mL) was added 4ꢀ
aminophenol (160 mg, 1.5 mmol) and cesium carbonate (580 mg, 1.8 mmol). The mixture was
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heated to 80 C for 1.5 h. After cooling to rt, the reaction mixture was diluted with CH2Cl2 (10
mL), acidified with 1N HCl, and the resulting precipitate was filtered using a Büchner funnel
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under reduced pressure. Olive green solid (300 mg, 74%). H NMR (300 MHz, DMSOꢀd6) δ
10.20 (d, 1H, J = 5.4 Hz), 8.36 (d, 1H, J = 7.9 Hz), 7.68ꢀ7.55 (m, 3H), 7.05 (d, 2H, J = 8.7 Hz),
6.67(d, 2H, J = 8.7 Hz), 5.14 (s, 2H); 13C NMR (75 MHz, DMSOꢀd6) δ 152.1, 147.1, 142.2,
138.79, 138.70, 134.5, 128.2, 128.1, 127.5, 124.4, 122.5, 116.8, 114.7, LRMS (ESI) m/z 278
[M+H]+.
Cell culture. As described previously,34 MDCK cells stably expressing rat UTꢀA1, YFPꢀ
H148Q/V163S and human AQP1 were grown in Dulbecco’s modified Eagle medium (DMEM)
containing 10% fetal bovine serum (FBS), penicillin G (100 U/mL), streptomycin (100 ꢁg/mL),
zeocin (500 ꢁg/mL), geneticin (750 ꢁg/mL) and hygromycin (500 ꢁg/mL) at 37 °C, 5% CO2. A
cell clone with bright YFP fluorescence and relatively low AQP1 expression was used to
maximize dynamic range.
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In vitro functional studies. Reversibility of UTꢀA1 inhibition was tested by preꢀincubating
the UTꢀA1ꢀexpressing MDCK cells with 8ay (0.5 µM) for 10 min and then washing the cells
with PBS prior to assay. The kinetics of UTꢀA1 inhibition was measured by adding 8ay (0.5 µM)
at different times prior to assay. The urea concentrationꢀdependence of UTꢀA1 inhibition was
studied from inhibitor concentrationꢀresponse data using different of urea gradients.
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UTꢀB inhibition measurements. As described,34 whole rat blood was diluted to a hematocrit
of ~1.5% in PBS containing 1.25 M acetamide and 5 mM glucose. Erythrocyte suspensions (100
ꢁL) were added to 96ꢀwell roundꢀbottom microplates and test compounds (1 ꢁL) were added.
After 15 min incubation, 20 ꢁL of the erythrocyte suspension was added rapidly to each well.
Following vigorous mixing, erythrocyte lysis was quantified by absorbance (Abs) at 710 nm.
Nonꢀlysed controls (isosmolar buffer) and lysed controls (0.7 mM phloretin) were added in each
plate as negative and positive controls, respectively. Percentage erythrocyte lysis was calculated
as: % lysis = 100% (Absneg – Abstest)/(Absneg – Abspos).
Cell toxicity. Transfected MDCK cells on black 96ꢀwell Costar microplates with clear plastic
bottoms were cultured for 24 h at 37 °C and incubated with test compounds (10 µM) for 24 h.
Cell viability was assayed using AlamarBlue (Pierce, Rockford, IL).
Homology modeling and docking computations. A homology model of rat UTꢀA1 was
previously generated, as described.26 The homology models were prepared for docking using the
FREDꢀRECEPTOR utility (Version 2.2.5, OpenEye Scientific, Santa Fe, NM,
http://www.eyesopen.com), with cytoplasmic and extracellular domains defined with 10 cubic Å
boxes. Structures of the relevant inhibitor molecules were converted to SMILES strings,
transformed to threeꢀdimensional conformations, and minimized using PIPELINE PILOT
(Accelrys, San Diego, CA). Single conformations were passed through MOLCHARGE (Version
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1.5.0, OpenEye Scientific) to apply MMFF charges, and through OMEGA (Version 2.4.6,
OpenEye Scientific) to generate multiꢀconformational libraries, which were docked using FRED
(Version 2.2.5, OpenEye Scientific), using consensus scoring functions ChemGauss3,
ChemScore, OEChemScore, ScreenScore, ShapeGauss, PLP, and ZapBind. Bound complexes
were visualized using PYMOL (Schrödinger, San Diego, CA).
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In vitro metabolic stability. Compounds (5 ꢁM) were preꢀincubated for 5 min at 37 °C with
rat liver microsomes (SpragueꢀDawley, male; 1 mg protein/mL; SigmaꢀAldrich, St. Louis, MO)
in PBS (0.1 M, pH 7.4), then 1 mM NADPH was added and incubated for specified times at 37
°C. The mixture was then chilled on ice, and 0.5 mL of iceꢀcold EtOAc was added. Samples
were centrifuged for 15 min at 3,000 rpm, the supernatant evaporated to dryness, and the residue
was dissolved in 100 ꢁl mobile phase (acetonitrile: water [3:1], containing 0.1% formic acid) for
LC/MS. Reverseꢀphase HPLC separations were carried out using a Xterra MS C18 column (2.1
mm x 100 mm, 3.5 ꢁm) equipped with a solvent delivery system (Waters model 2695, Milford,
MA) using a linear gradient from 5 to 95% acetonitrile containing 0.1% formic acid, run over 24
min at 0.2 mL/min flow rate. Peak area/ internal area ratio was plotted over incubation time.
MassLynx 4.1 was used for analyzing data.
Rat pharmacokinetics. Procedures herein were approved by the Committee on Animal
Research at the University of California, San Francisco. Rats (Wistar males, 250ꢀ300 g) were
purchased from Charles River Laboratories (Wilmington, MA). Rats were acclimated by placing
them individually in metabolic cages for 3 consecutive days (6 hours per day) before the
experiments. Compound 8bl was administered intravenously using a 24ꢀgauge catheter at 4
mg/kg in saline containing 20% dimethylacetamide and 1.28 mg/mL NaOH in saline (total
administered volume ~1 mL per rat). Blood samples (250 µL) were collected via tail vein
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puncture at 15, 30, 60, 120 and 180 minutes in plain tubes with no anticoagulant, and serum was
separated by centrifugation at 5,000 rpm for 15 min. Urine was collected in metabolic cages for 3
hours after injection at room temperature. Serum and urine samples were kept frozen at ꢀ20 °C
until analysis. Calibration standards were prepared in urine and plasma from control (nonꢀ
treated) rats to which known amounts of pure 8bl were added. The mixtures were centrifuged for
20 min at 13,200 rpm and 90 µL of supernatants were taken for LC/MS. LC/MS analysis was
performed with Waters 2695 separation module coupled to a Waters Micromass ZQ mass
spectrometer, using ESIꢀpositive ionization, and masses ranging from 100 to 1500 Da (40V cone
voltage).
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Diuretic studies in rats. Compound 8bl was administered to rats as described above for
pharmacokinetics studies. Rats were placed in metabolic cages individually with water and food
available ad libitum before and during experiments. Spontaneously voided urine was collected
for 3 hours and then compound or vehicle treatment was given intravenously. Urine was
collected for 3 hours after treatments. Urine volume was quantified and urine osmolality was
measured using freezing point depression osmometry (Precision Systems).
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website.
Structures of active UTꢀA1 inhibitors identified by highꢀthroughput screening, experimental
details of in vitro microsomal stability assay, and synthesis details and spectroscopic
characterization of synthesized conmpounds (PDF).
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Molecular strings and data for all final inhibitor candidates (CSV).
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AUTHOR INFORMATION
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Corresponding Author
*Alan S. Verkman, M.D., Ph.D., 1246 Health Sciences East Tower, University of California, San
Francisco, CA 94143ꢀ0521, U.S.A.; Phone 415 476ꢀ8530; Fax 415 665ꢀ3847; Eꢀmail
Alan.Verkman@ucsf.edu; www.ucsf.edu/verklab
Notes
The authors declare no competing financial interest.
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval
to the final version of the manuscript.
ACKNOWLEDGMENT
This
work
was
supported
by
National
Institutes
of
Health
grants
DK101373, DK72517, DK099803, and EY13574. The authors thank to Dr. Robert Yen of the
SFSU Mass Spectrometry Facility for HRMS measurements, funded by National Science
foundation grant CHEꢀ1228656.
ABBREVIATIONS
UT, Urea Transporter; DIPEA, N,N-diisopropyl ethylamine; DMSO, dimethyl sulfoxide; SAR,
structureꢀactivity relationship; MDCK, MadinꢀDarby canine kidney; AQP1, Aquaporinꢀ1; PBS,
phosphate buffered saline.
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