Nanomolar potency and metabolically stable inhibitors of kidney urea transporter UT-B

Article abstract of DOI:10.1021/jm300491y

Urea transporters, which include UT-B in kidney microvessels, are potential targets for development of drugs with a novel diuretic ('urearetic') mechanism. We recently identified, by high-throughput screening, a triazolothienopyrimidine UT-B inhibitor, 1, that selectively and reversibly inhibited urea transport with IC₅₀ = 25.1 nM and reduced urinary concentration in mice (Yao et al.J. Am. Soc. Nephrol., in press). Here, we analyzed 273 commercially available analogues of 1 to establish a structure-activity series and synthesized a targeted library of 11 analogues to identify potent, metabolically stable UT-B inhibitors. The best compound, {3-[4-(1,1-difluoroethyl)benzenesulfonyl]thieno[2,3-e][1,2,3]triazolo[1,5-a] pyrimidin-5-yl}thiophen-2-ylmethylamine, 3k, had IC₅₀ of 23 and 15 nM for inhibition of urea transport by mouse and human UT-B, respectively, and ～40-fold improved in vitro metabolic stability compared to 1. In mice, 3k accumulated in kidney and urine and reduced maximum urinary concentration. Triazolothienopyrimidines may be useful for therapy of diuretic-refractory edema in heart and liver failure.

Full text of DOI:10.1021/jm300491y

Article
pubs.acs.org/jmc
Nanomolar Potency and Metabolically Stable Inhibitors of Kidney
Urea Transporter UT-B
Marc O. Anderson,*,† Jicheng Zhang,† Yan Liu,† Chenjuan Yao,‡ Puay-Wah Phuan,‡ and A. S. Verkman‡
†Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, California 94132-4136, United States
‡Department of Medicine and Physiology, University of California, San Francisco, California 94143-0521, United States
S
* Supporting Information
ABSTRACT: Urea transporters, which include UT-B in kidney
microvessels, are potential targets for development of drugs with a
novel diuretic (‘urearetic’) mechanism. We recently identified, by
high-throughput screening, a triazolothienopyrimidine UT-B
inhibitor, 1, that selectively and reversibly inhibited urea transport
with IC50 = 25.1 nM and reduced urinary concentration in mice
(Yao et al. J. Am. Soc. Nephrol., in press). Here, we analyzed 273 commercially available analogues of 1 to establish a structure−
activity series and synthesized a targeted library of 11 analogues to identify potent, metabolically stable UT-B inhibitors. The best
compound, {3-[4-(1,1-difluoroethyl)benzenesulfonyl]thieno[2,3-e][1,2,3]triazolo[1,5-a]pyrimidin-5-yl}thiophen-2-ylmethyl-
amine, 3k, had IC50 of 23 and 15 nM for inhibition of urea transport by mouse and human UT-B, respectively, and ∼40-
fold improved in vitro metabolic stability compared to 1. In mice, 3k accumulated in kidney and urine and reduced maximum
urinary concentration. Triazolothienopyrimidines may be useful for therapy of diuretic-refractory edema in heart and liver failure.
metabolic stability. Our strategy was to deduce initial SAR from
functional testing of commercially available triazolothienopyr-
imidines, determine the site(s) of metabolism of 1, and
synthesize a library of targeted analogues. One compound with
excellent UT-B inhibition potency and in vitro metabolic
stability was further characterized and tested in mice.
INTRODUCTION
■
Urea transport across kidney tubular epithelial cells and
microvascular endothelia is required for generation of a
concentrated urine by countercurrent multiplication and
exchange mechanisms.1,2 Kidney tubules express urea trans-
porter A (UT-A) isoforms, encoded by the SLc14A2 gene, and
kidney microvessels express UT-B, encoded by the SLc14A1
gene.3−5 Knockout mice lacking UT-B6,7 or various UT-A
isoforms8−11 have reduced urinary concentrating ability,
providing a rationale for the development of UT inhibitors as
“urearetics” with a novel diuretic mechanism of action.
Conventional diuretics that target salt transporters can be of
limited efficacy in some fluid-retaining states such as advanced
congestive heart failure and cirrhosis.
RESULTS AND DISCUSSION
■
Structure−Activity Relationships of Triazolothieno-
pyrimidine UT-B Inhibitors. Initial SAR was deduced from
analysis of 273 commercially available triazolothienopyrimidine
analogues of 1. UT-B inhibition was measured by an
erythrocyte lysis assay. Of the compounds tested, 103
compounds inhibited UT-B urea permeability by >60% at 25
μM. Twelve compounds had IC50 < 500 nM, with compound 1
having the lowest IC50. Figure 1A shows concentration−
inhibition data for selected inhibitors. The data fitted well to a
single-site inhibition model with near 100% inhibition at high
concentration. Table S1 (Supporting Information) provides
IC50 for the 75 compounds (2aa−2cv) with greatest inhibition
potency, and metabolic stability data for a selected subset.
SAR analysis indicated greatest potency for thiophene-2-
methylamine at R2. Replacing the heteroaryl sulfur atom by
oxygen (thiophene → furan) increased IC50 substantially
(compare 1 and 2bo, Table S1). Bulky R2 groups containing
cyclic rings such as morpholine (2bp, IC50 = 5.6 μM) and
piperidine (IC50 > 25 μM, not shown) greatly reduced activity.
The reduced activity with inclusion of a bulky group is
consistent with our previous computational docking of 1 to a
Until recently, available UT inhibitors included urea
analogues, which have millimolar IC50, and the nonselective
membrane intercalating agent phloretin.12 Utilizing an
erythrocyte lysis-based high-throughput screen, we identified
phenylsulfoxyoxozole inhibitors of human UT-B with IC50
≈
100 nM.13 However, the phenylsulfoxyoxozoles poorly
inhibited rodent UT-B, precluding their testing in rodents.
Recently, a screen of 100 000 synthetic small molecules yielded
triazolothienopyrimidine UT-B inhibitors.14 The most potent
compound 1 reversibly inhibited mouse UT-B urea transport
with IC50 = 25.1 nM by a competitive mechanism and was
highly selective for UT-B over UT-A isoforms. Though 1 is
nontoxic, its metabolic stability was poor, requiring admin-
istration of large quantities in mice to obtain therapeutic levels
in kidney and reduce urinary concentration.
Here, we established structure−activity relationships (SARs)
of triazolothienopyrimidine UT-B inhibitors, with the goal of
identifying analogues of 1 with high potency and improved
Received: April 7, 2012
Published: June 13, 2012
© 2012 American Chemical Society
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Figure 1. UT-B inhibition potency and metabolic stability of selected triazolothienopyrimidine analogues. See Table S1 (Supporting Information)
for summary of potency and metabolic stability data for all compounds: (A) concentration−inhibition data from erythrocyte lysis assay for indicated
compounds (SE, n = 3); (B) in vitro metabolic stability data shown as kinetics of disappearance of indicated parent compounds following incubation
with hepatic microsomes and NADPH; (C) LC/MS traces showing disappearance of 1 and appearance of metabolites at m/z = 472 and 488; (D)
structure of 1 showing putative sites of metabolism.
Figure 2. General strategy for optimization of triazolothienopyrimidines. Shown are structural determinants of triazolothienopyrimidine inhibition
activity identified from commercially available analogues (“SAR-by-catalog”) followed by synthesized of a focused library.
homology model of UT-B showing a sterically tight fit near the
amidine linkage.14 Analogues containing a flexible chain with a
terminal ether group (2ca, IC50 = 212 nM) showed moderate
activity. At R1, only compounds with alkyl- and halide-
substituted aryl sulfonyl ring were commercially available. 4-
Methylphenyl and 2,5-dimethylphenyl reduced inhibition
activity, whereas 3,4-dimethyl, 4-isopropyl, and 4-ethyl
increased activity (compare 2bo and 2cr). Halide substitution
also reduced activity (2bi). Having a two- or three-carbon
hydrophobic group at the meta- and para-position of the aryl
ring might increase activity because of tighter binding in the
hydrophobic binding pocket of UT-B. Figure 2 summarizes the
major findings of SAR analysis of commercially available
analogues of 1.
In vitro analysis of metabolic stability was done using LC/
ESI-MS following compound incubation with hepatic micro-
somes in the presence of NADPH. Figure 1B shows the kinetics
of microsomal degradation of selected compounds. Compound
1 showed rapid degradation with t1/2 ≈ 2.8 min, with
appearance of metabolic products at 16 AMU (+1 oxygen)
and 32 (+2 oxygens) (Figure 1C). As observed by the relative
growth of the peaks at m/z = 472 versus 488, the first oxidation
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a
event appears to be more rapid than the second. We
hypothesize that 1 undergoes rapid hydroxylation at either
the benzylic15 or thiophene-2-methylamine linking carbons,
positions that are thought to stabilize radical intermediates
(Figure 1D). As reported in Table S1, analogues with R1
substituted with p-chlorophenyl showed greater in vitro
metabolic stability (2bf−2bj), likely because of absence of the
benzylic hydroxylation site and also because of occupation of
the para-position, which is a known site of P450 metabolism.16
Analogues with R2 bearing the electron-rich benzylamine (2ad,
2aj, 2bc, 2bs) also showed improved metabolic stability;
however, these analogues had poor UT-B inhibition potency
compared to 1.
Table 1. UT-B Inhibition Activity and Microsomal
b
Stability of Synthesized Compounds
Synthesis of Triazolothienopyrimidine Analogues.
SARs of commercial analogues of 1 indicated thiophene-2-
methylamine as the best R2 substituent for inhibition potency.
The optimal R1 substituent was not identified. We thus
designed a library of focused analogues to fill in key elements of
the SAR that were missing from the commercially available
inhibitor set (Table 1), with the aim of maintaining or
improving UT-B inhibition potency and improving metabolic
stability. Most of the synthesized compounds were designed to
maintain the R2 substituent as thiophene-2-methylamine while
investigating a small number of alkyl (3a, 3c), alkoxy (3d, 3f),
halo (3h, 3i), and heteroaryl (3g) substituents at R1. A small
number of inhibitors with variations of R2 (substitutions for
thiophene-2-methylamine) were also synthesized (3b, 3e, and
3j) to confirm the necessity of this heterocycle. One novel
compound (3k), the 1,1-difluoroethyl analogue of 1, was
synthesized to be bioisosteric with ethyl but lacking the
benzylic hydrogens that are likely involved in the rapid
metabolism of 1.
Our general synthetic approach toward the triazolothieno-
pyrimidine scaffold is similar to that reported recently for
synthesis of 5-HT6 receptor antagonists.17 The arylsulfonyla-
cetonitrile building blocks were first synthesized (Scheme 1).
Commercially available substituted arylthiols (4a−4g) were
alkylated with bromoacetonitrile to generate the corresponding
sulfides (5a−5g), which were then oxidized with mCPBA to
give the desired arylsulfonylacetonitriles 6a−6g. An additional
variation of this building block (4-difluoroethylphenyl) was
prepared by a multistep approach (Scheme 2) because the
precursor benzenethiol was not commercially available. As such,
1-bromo-4-(1,1-difluoroethyl)benzene (7) was transformed
under Pd-catalyzed conditions with the xanthphos ligand,
analogous to the Buchwald−Hartwig reaction, to generate
sulfide ester 8. This was oxidized to sulfone 9, converted to
primary amide 10, and dehydrated using phosphorus pentoxide
to the desired 4-difluoroethylarylsulfoneacetonitrile (6h).
The second key synthetic precursor is a 3-azidothiophene
ester (Scheme 3). 3-Bromothiophene-2-carboxaldehyde (11)
was azidated by nucleophilic aromatic substitution to generate
3-azidothiophenecarboxaldehyde 12. As reported previously,14
aldehyde 12 was oxidized with the Lindgren reaction18 to
generate carboxylic acid 13, which was alkylated with isobutyl
bromide to 2-azidothiophene-1-isobutyl ester (14).
a
Determined by mouse erythrocyte lysis assay (n = 3). IC50
b
determined by fit to a single-site saturation model. Percentage of
parent compound remaining after 30 min of incubation with rat liver
microsomes.
tion step, a side reaction that was observed in early
optimization studies. Isobutyl ester was thought to afford
stability during the base-promoted cycloaddition while being
sufficiently reactive during the lactamation. The conversion of
heterocycles 15a−15h to the final library of amidine-containing
compounds (3a−3k) was afforded with PyBOP during
microwave irradiation. The use of PyBOP under microwave
irradiation conditions was based on literature using the related
The attachment of the building blocks is shown in Scheme 4.
The arylsulfonylacetonitriles 6a−6h were reacted with base to
form the nitrile enolates and coupled with the azidothiophene
ester 14 followed by in situ lactamation to generate the core
heterocycles (15a−15h). The use of isobutyl ester in 13 was to
enhance stability compared to a methyl ester in order to
prevent ester saponification during the cycloaddition−lactama-
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scaffold 15,17 gave low yields in our hands, possibly because of
the relatively small reaction scale.
Scheme 1. General Synthesis of Arylsulfonylacetonitrile
Building Blocks
a
Potency and Metabolic Stability of Triazolothienopyr-
imidine Analogues. The IC50 and in vitro metabolic stability
of synthesized analogues were determined (Table 1). In
general, analogues lacking stabilized benzylic carbon for
hydroxylation showed improved metabolic stability (3c, 3d,
3i). Fortunately, 3k showed both excellent inhibition potency
and metabolic stability and was further characterized. UT-B
inhibition by 3k was measured by stopped-flow light scattering,
which provides a definitive measure of compound potency. The
assay measures the kinetics of cell volume following rapid
mixing of an erythrocyte suspension with a urea-containing
solution. Figure 3A shows representative light scattering data
a
Reagents and conditions: (a) bromoacetonitrile, K2CO3, DMF, 2 h,
rt; (b) mCPBA, CH2Cl2, 0 °C, 2 h.
Scheme 2. Synthesis of the
Difluoroethylarylsulfonylacetonitrile Building Block
a
a
Reagents and conditions: (a) methyl thioacetate, Pd[PPh3]4,
xanthphos ligand, DIPEA, 1,4-dioxane, 135 °C, 16 h; (b) mCPBA,
CH2Cl2, 0 °C, 2 h; (c) NH3 in MeOH, 50 °C, 16 h; (d) phosphorus
pentoxide, toluene, 75 °C, 1 h.
Scheme 3. Synthesis of the Azidothiophenyl Ester Building
a
Block
a
Reagents and conditions: (a) NaN3, DMSO, 65 °C, 48 h; (b) sodium
chlorite, sulfamic acid, H2O/acetone (1:1), 0 °C, 30 min; (c) isobutyl
bromide, Cs2CO3, DMF, 80 °C, 6 h.
Figure 3. UT-B inhibition and in vitro metabolic stability of 3k. (A)
Stopped-flow light scattering measurement of urea permeability in
mouse erythrocytes. Erythrocyte suspensions were mixed with an
equal volume of urea-containing solution to give a 100 mM inwardly
directed urea gradient. Inward urea flux is seen as decreasing scattered
light intensity. Erythrocytes were incubated with indicated concen-
trations of 3k for 10 min prior to measurements. (B) Concentration−
inhibition data for mouse (top) and human (bottom) erythrocytes
(SE, n = 3). Fitted IC50 values were 23 nM (mouse) and 15 nM
(human). (C) In vitro metabolic stability in hepatic microsomes. (left)
LC/MS showing kinetics of disappearance of 3k. (right) Kinetics of 3k
disappearance, with data for 1 shown for comparison (SE, n = 3).
Scheme 4. Generation of Inhibitors via [2 + 3]
Cycloaddition and Microwave-Assisted Dehydrative
Amidine Coupling
a
a
Reagents and conditions: (a) NaOEt, EtOH, 30 min, rt; (b) amines,
PyBOP, CH3CN, microwave, 100 °C, 30 min.
for inhibition of UT-B urea transport in mouse erythrocytes.
Each curve consists of a rapid upward phase, representing
osmotic cell shrinkage, followed by a slower downward phase,
representing urea (and water) influx. The kinetics of the
downward phase was greatly slowed by 3k in a concentration-
dependent manner. Figure 3B summarizes concentration−
inhibition data for mouse and human UT-B. Deduced IC50
values were 23 and 15 nM, respectively.
reagent BOP without heating;19,20 however, we found that the
low-temperature conditions were not successful with triazolo-
thienopyrimidines in the presence of BOP or PyBOP. With
microwave assistance, PyBOP gave conversion to the desired
amidine products, which was chosen over BOP because of its
carcinogenic byproduct hexamethylphosphoramide (HMPA).
POCl3, which has been reported to activate the lactam in
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Figure 4. In vivo pharmacology and urearetic efficacy of 3k. (A) LC/MS analysis of 3k concentration in blood. Standards are shown at the left and
inset. Also shown are original LC/MS traces and deduced 3k concentrations in blood, following intraperitoneal bolus administration of 400 μg of 3k
(SE, 3 mice per group). (B) Concentrations of 3k in kidney and urine for mice studied in (A). (C) Concentrations of 3k and 1 in blood at 6 h after
intraperitoneal administration of 200 μg of 3k and 200 μg of 1 (SE, 3 mice). (D) Urine osmolality in mice after intraperitoneal administration of 400
μg of 3k or formulation alone followed 1 h later by dDAVP (SE, 4 mice, (∗) P < 0.01).
Figure 3C showed in vitro metabolic stability in hepatic
microsomes. In parallel experiments, the t1/2 for disappearance
of 3k was ∼120 min, substantially slower than t1/2 of ∼2.8 min
for disappearance of 1 shown for comparison. Thus, compared
to 1, compound 3k is ∼40-fold more stable in vitro while
retaining comparable potency.
The urearetic activity of 3k was assayed by measurement of
urine osmolality in mice under maximal antidiuretic stimulation
by the vasopressin analogue dDAVP. dDAVP was adminis-
trated 1 h after intraperitoneal injection of formulation, without
or with 3k. Figure 4D shows that dDAVP increased urine
osmolality to >3 Osm/kg H2O in control (formulation alone)
mice, which was reduced by ∼0.5 Osm/kg H2O in mice
receiving 3k. This reduction in urine osmolality is similar to
that produced by UT-B gene knockout.6,14
In Vivo Mouse Studies. The pharmacokinetics and renal
accumulation of 3k were measured in mice following
intraperitoneal bolus administration of 400 μg of 3k in a
formulation chosen for efficient dissolution and absorption.
Blood and kidney tissues were obtained at different times and
3k was assayed by LC/MS after organic extraction, comparing
with standards. Figure 4A show the LC/MS calibration for
blood measurements (left), and the kinetics of 3k concen-
tration (right). Figure 4B shows the kinetics of 3k
concentration in kidney and urine. These data show high
concentrations (>1 μM) of 3k for many hours, well above its
IC50 of ∼25 nM for inhibition of UT-B urea transport. In a
separate comparative study, mice received 200 μg of 3k and 1
at the same time by intraperitoneal injection. At 6 h, the 3k
concentration was ∼3-fold greater than that of 1 in blood
(Figure 4C).
SUMMARY AND CONCLUSIONS
■
SAR analysis of triazolothienopyrimidine UT-B inhibitors,
followed by rational synthesis of a focused library, identified
3k as having excellent UT-B inhibition potency, with IC50 of 23
and 15 nM for mouse and human UT-B, respectively, and ∼40-
fold improved in vivo metabolic stability compared with 1. The
urearetic activity of 3k was demonstrated in mice. In addition
to prior data from knockout mice, the potential utility of UT-B
inhibitors in humans is supported by the urinary concentrating
defect in rare humans lacking UT-B.21,22 UT inhibitors have a
fundamentally different mechanism-of-action from conven-
tional diuretics, which target kidney tubule salt transporters,
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mmol, 0.2 equiv). The mixture was heated at 135 °C under argon
for 16 h. After cooling to room temperature, the mixture was filtered
and the filtrate was concentrated under reduced pressure. The mixture
was purified by flash column chromatography (hexanes/ethyl acetate =
6:1) to afford the sulfide (8) (yield 99%). The sulfide (1084 mg, 4.40
mmol) was dissolved in CH2Cl2 (44 mL) and oxidized by treatment
with mCPBA (4341 mg, 17.61 mmol, 4.0 equiv) at 0 °C for 2 h. The
reaction mixture was taken up in CH2Cl2 (150 mL) and was then
washed with aqueous 5% Na2SO3 (3 × 50 mL). The organic layer was
washed with brine, dried over Na2SO4, and concentrated in vacuo to
and so may act in synergy. Diuretics are used widely to increase
renal salt and water excretion in fluid overload conditions such
as congestive heart failure, cirrhosis, and nephrotic syndrome
and when vasopressin levels are inappropriately high such as in
the syndrome of inappropriate secretion of antidiuretic
hormone (SIADH). By disrupting countercurrent mechanisms,
UT inhibitors alone, or in combination with loop diuretics, may
induce a diuresis in states of refractory edema where
conventional diuretics are not effective.23−25
Several caveats are noted for further preclinical development
of UT inhibitors. Demonstration of efficacy is needed in
suitable animal disease models of fluid retention associated with
heart or liver failure. Because UT-B is also expressed outside
the kidney and mild extrarenal phenotypes have been reported
in UT-B knockout mice,26,27 thorough in vivo evaluation of
inhibitor toxicity is necessary. The absence of extrarenal
phenotypes in humans lacking UT-B is reassuring in this
regard. Finally, we note that maximum urearetic action might
be achieved by nonselective UT inhibitors that target UT-A
isoforms as well.
1
yield 9 as a colorless oil. H NMR (CDCl3, 500 MHz): δ 1.97 (t, J =
15.0 Hz, 3H), 3.74 (s, 3H), 4.16 (s, 2H), 7.74 (d, J = 5.0 Hz, 2H), 8.04
(d, J = 10.0 Hz, 2H). 13C NMR (CDCl3, 125 MHz): δ 25.9 (t, J = 28.8
Hz), 53.4, 61.1, 126.0 (t, J = 6.3 Hz), 129.2, 134.6, 144.3, 162.9.
ESI(+)-HRMS [M + Na]+ calculated 301.0322, observed 301.0319 for
C11H12F2O4S.
[4-(1,1-Difluoroethyl)benzenesulfonyl]acetonitrile (6h). The
precursor methyl ester (9) (1232 mg, 4.43 mmol, 1.0 equiv) was
added to a methanol solution of NH3 (7 N) (15 mL, 132.8 mmol, 30
equiv) in the presence of catalytic NaCN (0.71 mg, 0.44 mmol, 0.1
equiv) in a sealed vial, and the mixture was heated at 50 °C for 16 h.
The reaction mixture was extracted with CH2Cl2, washed with brine,
and then concentrated in vacuo. The crude product was washed with
ether and filtered to give the primary amide 10 which was used directly
without purification or characterization. Amide 10 (500 mg, 1.90
mmol, 1.0 equiv) was dehydrated with phosphorus pentoxide (16175
mg, 56.98 mmol, 30 equiv) in toluene (95 mL) at 75 °C for 1 h. The
reaction mixture was added to ice−water (300 mL) and then extracted
with CH2Cl2 (200 mL). The aqueous layer was extracted with
additional CH2Cl2 (3 × 50 mL), and the final organic layer was dried
over Na2SO4. Purification by flash column chromatography (hexanes/
ethyl acetate = 3:1) yielded the arylsulfonylacetonitrile 6h as white
needle-like crystals (146 mg, yield 31%). 1H NMR (CDCl3, 300
MHz): δ 1.97 (t, J = 18.0 Hz, 3H), 4.08 (s, 1H), 7.80 (d, J = 9.0 Hz,
2H), 8.12 (d, J = 6.0 Hz, 2H). 13C NMR (acetone-d6, 75 MHz): δ 24.6
(t, J = 29.3 Hz), 44.7, 111.2, 126.1 (t, J = 6.0 Hz), 129.2, 139.1,
144.2(t, J = 26.3 Hz). ESI(+)-HRMS [M + Na]+ calculated 268.0220,
observed 268.0190 for C10H9F2NO2S.
METHODS
■
Chemistry. General Information. All solvents used in reactions
were anhydrous and obtained from commercial sources unless
otherwise specified. H and 13C NMR spectra were recorded on a
1
1
Bruker DRX 300 MHz or Bruker Avance 500 MHz spectrometer. H
NMR chemical shifts are relative to TMS (δ = 0.00 ppm) or CDCl3 (δ
= 7.26 ppm). 13C NMR chemical shifts are relative to CDCl3 (δ =
77.23 ppm). Products were purified by flash column chromatography
on silica gel (230−400 mesh). The eluent used for purification is
reported in parentheses. Low-resolution LC/MS was performed using
a Waters Micromass ZQ instrument or an Agilent-1100 HPLC
instrument equipped with an Agilent 1956B mass spectrometer. High-
resolution mass spectra were obtained by the University of Notre
Dame Mass Spectrometry and Proteomics Facility (Notre Dame, IN)
using ESI either by direct infusion on a Bruker micrOTOF-II or by LC
elution via an ultrahigh pressure Dionex RSLC with C18 column
coupled with a Bruker micrOTOF-Q II. Microwave-assisted organic
synthesis was performed using a Biotage Isolera instrument. Thin layer
chromatography (TLC) was done on aluminum sheet silica gel Merck
60F254. Commercial triazolothienopyrimidine analogues were pur-
chased from ChemDiv (San Diego, CA). Some intermediates (6a−6g)
are commercially available but were synthesized for the purpose of this
study. Purity of all biologically evaluated compounds was >95% by RP-
HPLC (254 nm) unless otherwise noted.
General Conditions for Conversion of Substituted Arylthiols
(4) to Substituted Arylsulfonylacetonitriles (6). Bromoacetoni-
trile was dissolved in DMF (0.4 M), stirred in an ice bath, and then
treated with substituted benzenethiol (4) (0.95 equiv) and K2CO3 (2
equiv), and the mixture was allowed to stir for 2 h at 0 °C. The
reaction mixture was taken up into excess H2O and extracted three
times with Et2O. The combined organic extracts were washed twice
with water and brine, followed by concentration in vacuo to generate
the intermediate aryl sulfide acetonitrile (5) in ∼90% yield. The sulfide
was then dissolved in CH2Cl2 (0.4 M) and treated with mCPBA (77%
peroxybenzoate, 2 equiv). The mixture was stirred at 0 °C under argon
for 2 h to give complete conversion as monitored by TLC. The
reaction mixture was quenched with excess sodium sulfite solution and
extracted twice with CH2Cl2. The organic layer was washed with brine,
dried over Na2SO4, and concentrated in vacuo to generate 6.
Isobutyl 3-Azidothiophene-2-carboxylate (14). Compound
1314 (5.8 g, 34.3 mmol) was treated with 1-bromo-2-methylpropane
(4.47 mL, 1.2 equiv) and cesium carbonate (7.82 g, 0.7 equiv) in
anhydrous DMF (0.1 M) and heated to 80 °C for 6 h under argon.
The mixture was cooled, dissolved in H2O (150 mL), and extracted
with 1:1 diethyl ether/ethyl acetate (3 × 200 mL). The organic layer
was washed with distilled H2O (5 × 100 mL), dried over Na2SO4, and
concentrated in vacuo to afford compound 14 as an amber oil (6.77 g,
1
yield 77%). H NMR (CDCl3, 500 MHz): δ 1.00 (d, J = 10 Hz, 6H),
2.05 (m, 1H), 4.06 (d, J = 5 Hz, 2H), 6.91 (d, J = 5 Hz, 1H), 7.46 (d, J
= 5 Hz, 1H). 13C NMR (CDCl3, 125 Hz): δ 19.8, 28.6, 71.8, 118.3,
122.8, 131.6, 142.8, 161.6. ESI(+)-LRMS [M + Na]+ calculated 248.0,
observed 247.8 for C9H11N3O2S.
General Conditions for Coupling of Substituted Arylsulfo-
nylacetonitriles (6a−h) with Azidothiophene Ester (14) To
Generate Cycloadducts (15a−h). Sodium ethoxide (2 mmol scale
typically, 10 equiv based on starting material 14) was dissolved in
absolute ethanol (0.3 M), and then a substituted arylsulfonylacetoni-
trile (6) was added (1.5 equiv based on 14). The mixture was stirred
for 15 min at room temperature. Next, the 3-azidothiophene ester
starting material (14) was added (1.0 equiv), and the mixture was
stirred for 30 min at room temperature. TLC and HPLC analysis
confirmed the consumption of starting material and formation of
product. Solid NaHCO3 (25 equiv) was added to neutralize the excess
alkoxide base, and the reaction mixture was taken up into CHCl3 and
washed three times with aqueous 1 M HCl. The products were
generally purified by crystallization with chloroform, with additional
purification steps as specified.
[4-(1,1-Difluoroethyl)phenylsulfonyl]acetic Acid Methyl
Ester (9). 1-Bromo-4-(1,1-difluoroethyl)benzene (7) (1000 mg, 4.52
mmol, 1.0 equiv) was treated with mercaptoacetic acid methyl ester
(0.809 mL, 9.05 mmol, 2.0 equiv) in 1,4-dioxane (45 mL) and N-
ethyldiisopropylamine (1.495 mL, 9.05 mmol, 2.0 equiv), tetrakis-
(triphenylphosphine)palladium(0) (523 mg, 0.45 mmol, 0.1 equiv),
9,9-dimethyl-4,5-bis(diphenylphosphino)xanthene (451 mg, 0.91
3-(4-Isopropylbenzenesulfonyl)-4H-thieno[2,3-e][1,2,3]-
triazolo[1,5-a]pyrimidin-5-one (15a). The crude product was
purified by flash column chromatography using a gradient solvent
system (CH2Cl2/Et3N = 100:1 → CH2Cl2/CH3OH/Et3N = 100:2:1).
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Yellow solid, yield 15%. 1H NMR (DMSO-d6, 300 MHz): δ 1.16 (d, J
= 6 Hz, 6H), 2.93−3.07 (m, 1H), 7.25(d, J = 6 Hz, 1H), 7.42 (d, J = 9
Hz, 2H), 7.94 (d, J = 9 Hz, 2H), 8.05 (d, J = 6 Hz, 1H). ESI(+)-
HRMS [M + H]+ calculated 375.0585, observed 375.0574 for
C16H14N4O3S2. This compound is available commerically.
precipitation and trituration with cold CHCl3. Additional or alternative
purification measures are provided as indicated.
[3-(4-Isopropylbenzenesulfonyl)thieno[2,3-e][1,2,3]triazolo-
[1,5-a]pyrimidin-5-yl]thiophen-2-ylmethylamine (3a). Crude
product was purified by flash column chromatography using
CH2Cl2/acetic acid (v/v = 100:1) to elute the starting materials
first, then changing to CH2Cl2/CH3OH/acetic acid (v/v/v = 100:2:1).
Yellow solid, yield 21%. 1H NMR (DMSO-d6, 300 MHz): δ 1.17 (d, J
= 9 Hz, 6H), 2.91−2.95 (m, 1H), 4.97 (d, J = 6 Hz, 2H), 7.03−7.04
(m, 1H), 7.23−7.24 (m, 1H), 7.39 (d, J = 9 Hz, 2H), 7.45−7.46 (m,
1H) 7.95−7.98 (m, 1H), 7.99−8.01 (m, 2H), 8.42 (d, J = 6 Hz, 1H),
9.48 (t, J = 3 Hz, 1H). 13C NMR (CDCl3, 125 Hz): δ 24.6, 34.6, 117.5,
126.5, 128.0, 128.3, 128.4, 138.0, 140.5, 155.5, 156.5. ESI(+)-HRMS
[M + H]+ calculated 470.0779, observed 470.0782 for C21H19N5O2S3.
Furan-2-ylmethyl-[3-(4-isopropylbenzenesulfonyl)thieno-
[2,3-e][1,2,3]triazolo[1,5-a]pyrimidin-5-yl]amine (3b). Crude
product was purified by flash column chromatography using
CH2Cl2/acetic acid (v/v = 100:1) to elute the starting materials
first, then changing to CH2Cl2/CH3OH/acetic acid (v/v/v = 100:2:1).
Yellow solid, yield 3%. 1H NMR (CDCl3, 500 MHz): δ 1.20 (d, J = 5
Hz, 6H), 2.92−2.94 (m, 1H), 4.20−4.23 (m, 2H), 7.05 (s, 1H), 7.17
(t, J = 5 Hz, 1H), 7.35 (d, J = 5 Hz, 2H), 7.53 (m, 1H), 8.14 (d, J = 5
Hz, 2H), 8.19 (d, J = 5 Hz, 1H), 8.36 (d, J = 5 Hz, 2H). This
compound is commercially available and has also been reported.17
T h i o p h e n - 2 - y l m e t h y l - [ 3 - ( 4 - t r i fl u o r o m e t h y l -
benzenesulfonyl)thieno[2,3-e][1,2,3]triazolo[1,5-a]pyrimidin-
5-yl]amine (3c). Crude product was purified by flash column
chromatography using CH2Cl2/acetic acid (v/v = 100:1) to elute the
starting materials first, then changing to CH2Cl2/CH3OH/Et3N (v/v/
v = 100:1:1). White solid, yield 26%. 1H NMR (CDCl3, 300 MHz): δ
5.17 (d, J = 6 Hz, 2H), 7.04−7.05 (m, 1H), 7.51 (d, J = 6 Hz, 1H),
7.53 (d, J = 6 Hz, 1H), 7.68−7.71 (m, 2H), 7.93 (d, J = 6 Hz, 1H),
8.00 (d, J = 6 Hz, 1H), 8.33−8.35 (m, 2H). ESI(+)-HRMS [M + H]+
calculated 400.9990, observed 400.9982 for C14H7F3N4O3S2.
3-(4-Trifluoromethylbenzenesulfonyl)-4H-thieno[2,3-e]-
[1,2,3]triazolo[1,5-a]pyrimidin-5-one (15b). The crude product
was purified by flash column chromatography, using a gradient solvent
system (CH2Cl2/Et3N = 100:1 → CH2Cl2/CH3OH/Et3N = 100:2:1).
The combined organics were taken up into chloroform, washed twice
with aqueous 1 M HCl, dried over Na2SO4, and concentrated in vacuo
1
to yield pure product. White solid, yield 5%. H NMR (CDCl3, 500
MHz): δ 7.85 (d, J = 5 Hz, 1H), 7.86 (d, J = 10 Hz, 2H), 8.06 (d, J = 5
Hz, 1H,), 8.25 (d, J = 10 Hz, 2H). ESI(+)-HRMS [M + H]+ calculated
400.9990, observed 400.9982 for C14H7F3N4O3S2.
3-(4-Trifluoromethoxybenzenesulfonyl)-4H-thieno[2,3-e]-
[1,2,3]triazolo[1,5-a]pyrimidin-5-one (15c). The crude product
was purified by flash column chromatography (CH2Cl2/CH3OH/Et3N
= 100:2:1). The combined organics were taken up into chloroform,
washed twice with aqueous 1 M HCl, dried over Na2SO4, and
concentrated in vacuo to yield pure product. White solid, yield 12%.
1H NMR (CDCl3, 500 MHz): δ 7.37 (d, J = 5 Hz, 1H), 7.73 (d, J = 5
Hz, 2H), 8.16 (d, J = 10 Hz, 1H), 8.30 (d, J = 10 Hz, 2H).
3-(4-Methoxybenzenesulfonyl)-4H-thieno[2,3-e][1,2,3]-
triazolo[1,5-a]pyrimidin-5-one (15d). The crude product was
purified by flash column chromatography (CH2Cl2/CH3OH/Et3N =
100:2:1). The combined organics were taken up into chloroform,
washed twice with aqueous 1 M HCl, dried over Na2SO4, and
concentrated in vacuo to yield pure product. Yellow solid, yield 32%.
1H NMR (CDCl3, 300 MHz): δ 3.87 (s, 3H), 7.02 (d, J = 15 Hz, 2H),
7.84 (d, J = 10 Hz, 1H), 8.02 (d, J = 10 Hz, 1H,), 8.03 (d, J = 10 Hz,
2H). ESI(+)-HRMS [M + H]+ calculated 363.0221, observed
363.0216 for C14H10N4O4S2.
3-(Thiophene-2-sulfonyl)-4H-thieno[2,3-e][1,2,3]triazolo-
1
T h i o p h e n - 2 - y l m e t h y l - [ 3 - ( 4 -
[1,5-a]pyrimidin-5-one (15e). Brown solid, yield 11%. H NMR
trifluoromethoxybenzenesulfonyl)thieno[2,3-e][1,2,3]triazolo-
(CDCl3, 300 MHz): δ 7.15−7.18 (m, 1H), 7.74 (d, J = 6 Hz, 1H),
7.86 (d, J = 6 Hz, 1H), 7.91 (d, J = 6 Hz, 1H), 8.04 (d, J = 6 Hz, 1H).
ESI(+)-HRMS [M + H]+ calculated 338.9680, observed 338.9677 for
C11H6N4O3S3.
3-(4-Fluorobenzenesulfonyl)-4H-thieno[2,3-e][1,2,3]triazolo-
[1,5-a]pyrimidin-5-one (15f). White solid, yield 43%. 1H NMR
(CDCl3, 500 MHz): δ 7.85 (d, J = 5 Hz, 1H), 8.05 (d, J = 5 Hz, 1H),
8.13 (m, 2H). ESI(+)-HRMS [M + Na]+ calculated 372.9842,
observed 372.9832 for C13H7FN4O3S2.
[1,5-a]pyrimidin-5-yl]amine (3d). Additional purification involved
1
prep-RP-HPLC. Yield 2%. H NMR (CDCl3, 500 MHz): δ 5.17 (d, J
= 5 Hz, 2H), 5.80 (t, J = 5 Hz, 1H), 7.03−7.05 (m, 1H), 7.22 (d, J = 5
Hz, 1H), 7.25 (d, J = 10 Hz, 2H), 7.30 (d, J = 5 Hz, 1), 7.93 (d, J = 5
Hz, 1H), 8.00 (d, J = 5 Hz, 1H), 8.26 (d, J = 10 Hz, 2H). ESI(+)-
HRMS [M + H]+ calculated 512.0132, observed 512.0139 for
C19H12F3N5O3S3.
[3-(4-Isopropylbenzenesulfonyl)thieno[2,3-e][1,2,3]triazolo-
[1,5-a]pyrimidin-5-yl]-(4-methoxybenzyl)amine (3e). The crude
product was purified by flash column chromatography using CH2Cl2/
acetic acid (v/v = 100:1) to elute the starting materials first and then
changing to CH2Cl2/CH3OH/acetic acid (v/v/v = 100:2:1). White
3-(4-Bromobenzenesulfonyl)-4H-thieno[2,3-e][1,2,3]-
1
triazolo[1,5-a]pyrimidin-5-one (15g). Yellow solid, yield 53%. H
NMR (CDCl3, 500 MHz): δ 7.72 (d, J = 10 Hz, 2H), 7.85 (d, J = 5
Hz, 1H), 7.97 (d, J = 10 Hz, 2H), 8.05 (d, J = 5 Hz, 1H). ESI(+)-
HRMS [M + Na]+ calculated 432.9041, observed 432.9018 for
C13H7BrN4O3S2. This compound has been reported.17
1
solid, yield 20%. H NMR (CDCl3, 300 MHz): δ 1.21 (d, 6 H, J = 6
Hz), 2.89−2.93 (m, 1H), 4.92 (d, J = 6 Hz, 2H), 6.93 (d, J = 9 Hz,
2H), 7.17 (d, J = 3 Hz, 1H), 7.42 (d, J = 9 Hz, 2H), 7.89−7.91 (m,
2H), 7.97−7.98 (m, 1H), 8.12 (d, J = 9 Hz, 2H). LC/ESI-LRMS [M +
H]+ calculated 494.1 observed 494.0 for C24H23N5O3S2.
3-(4-Difluoroethylbenzenesulfonyl)-4H-thieno[2,3-e][1,2,3]-
1
triazolo[1,5-a]pyrimidin-5-one (15h). White solid, yield 74%. H
NMR (CDCl3, 300 MHz): δ 1.91 (t, J = 18.0 Hz, 3H), 7.70 (d, J = 9.0
Hz, 2H), 7.84 (d, J = 6.0 Hz, 1H), 8.04 (d, J = 3.0 Hz, 1H), 8.17 (d, J
= 9.0 Hz, 2H). ESI(+)-HRMS [M + H]+ calculated 397.0240,
observed 397.0251 for C15H10F2N4O3S2.
[3-(4-Methoxybenzenesulfonyl)thieno[2,3-e][1,2,3]triazolo-
[1,5-a]pyrimidin-5-yl]thiophen-2-ylmethylamine (3f). The crude
product was purified by flash column chromatography (CH2Cl2/
1
CH3OH/acetic acid = 120:1:1). Brown solid, yield 36%. H NMR
General Conditions for Coupling of Heterocyclic Lactams
(15a−h) with Primary Amines To Generate Triazolothienopyr-
imidines (3a−k). Heterocyclic lactam starting material (15a−h) (1
equiv) was dissolved in anhydrous CH3CN (0.3 M) and treated with
primary amine (3 equiv) and DBU (3 equiv) and PyBOP (3 equiv),
and the mixture was irradiated by microwave at 100 °C for 30 min.
The reaction was monitored by HPLC, showing consumption of
starting material and formation of tentative product. The crude
reaction mixture was treated with aqueous 1 M HCl (10 equiv) and
stirred for 30 min at room temperature. Aqueous 1 M sodium
carbonate solution was carefully added to adjust the pH to 9. The
crude aqueous reaction mixture was extracted with ethyl acetate. The
organic layer was washed three times with aqueous 1 M HCl, dried
over Na2SO4, and concentrated in vacuo. The product was purified by
(CDCl3, 500 MHz): δ 3.82 (s, 3H), 5.17 (d, J = 10 Hz, 2H), 6.90−
6.93 (m, 2H), 7.04 (d, J = 5 Hz, 1H), 7.23 (d, J = 5 Hz, 1H), 7.30 (dd,
J = 10 Hz, 5 Hz, 1H), 7.91 (d, J = 10 Hz, 1H), 8.00 (d, J = 10 Hz, 1H),
8.16 (d, J = 15 Hz, 2H). 13C NMR (CDCl3, 125 MHz): δ 41.0, 50.3,
114.7, 114.8, 118.1, 126.4, 127.9, 128.0, 134.0, 134.2, 139.2, 139.5,
140.2, 153.2, 164.1. ESI(+)-HRMS [M + H]+ calculated 458.0415,
observed 458.0421 for C19H15N5O3S3.
[3-(Thiophene-2-sulfonyl)thieno[2,3-e][1,2,3]triazolo[1,5-a]-
pyrimidin-5-yl]thiophen-2-ylmethylamine (3g). White solid,
1
yield 10%. H NMR (CD3OD, 500 MHz): δ 5.09 (s, 2H), 6.98 (dd,
J = 5 Hz, 1H), 7.08 (dd, J = 5 Hz, 1H), 7.23 (d, 1H), 7.29 (d, J = 5 Hz,
1H), 7.80 (dd, J = 10 Hz, 5 Hz, 1H), 7.82 (dd, J = 5 Hz, 1H), 7.96 (d,
J = 5 Hz, 1H), 8.25 (d, J = 5 Hz, 1H).
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[3-(4-Fluorobenzenesulfonyl)thieno[2,3-e][1,2,3]triazolo-
[1,5-a]pyrimidin-5-yl]thiophen-2-ylmethylamine (3h). Yellow
solid, yield 43%. H NMR (CDCl3, 500 MHz): δ 5.16 (d, J = 5 Hz,
2H), 5.74 (s, 1H), 7.04 (s, 1H), 7.11 (t, J = 5 Hz, 2H), 7.22(s, 1H),
7.31 (d, J = 5 Hz, 1H), 7.92 (d, J = 5 Hz, 1H), 8.00 (d, J = 5 Hz, 1H),
8.23(t, J = 5 Hz, 2H).
buffer (100 mM) containing 1 mM NADPH. The mixture was then
chilled on ice, and 0.5 mL of ice-cold ethyl acetate was added. Samples
were centrifuged for 15 min at 3000 rpm. The supernatant was
evaporated to dryness, and the residue was dissolved in 150 μL of
mobile phase (acetonitrile:/water, 3:1, containing 0.1% formic acid)
for LC/MS. Reverse-phase HPLC separations were carried out using a
Waters C18 column (2.1 mm × 100 mm, 3.5 mm particle size)
equipped with a solvent delivery system (Waters model 2690, Milford,
MA). The solvent system consisted of a linear gradient from 5% to
95% acetonitrile run over 16 min (0.2 mL/min flow rate).
Mouse Studies. 3k was formulated in 5% DMSO, 2.5% Tween-80,
and 2.5% PEG400 in H2O, as described.14 Mice (age 8−10 weeks, 25−
35 g) were administered 300 μL of formulation (without or with 3k)
by intraperitoneal injection. Urine, blood, and kidney samples were
collected and processed as described.14 dDAVP ([1-deamino-8-D-
arginine]vasopressin, 1 μg/kg) was given 1 h after formulation. Urine
osmolality was measured in water-diluted urine samples by freezing-
point osmometry. Kidneys were homogenized in acetic acid (100 μL
per 1 g of tissue) and ethyl acetate (10 mL per 1 g of tissue). The
homogenate was centrifuged at 3000 rpm for 15 min. Calibration
standards were prepared in kidney homogenates from untreated mice
to which was added known amounts of 3k. The ethyl acetate
containing supernatant was dried under nitrogen and the residue
dissolved in acetonitrile/H2O (3:1) containing 0.1% formic acid.
HPLC was done on a Xterra MS C18 column (2.1 mm × 100 mm, 3.5
μm particle size; Waters, Milford, MA) connected to a solvent delivery
system (model 2690, Waters). The solvent system consisted of a linear
gradient from 5% to 95% acetonitrile containing 0.1% formic acid over
16 min (0.2 mL/min flow). 3k was detected by absorbance at 262 nm.
Mass spectra were acquired on a mass spectrometer (Alliance HT
2790 + ZQ, Waters) using positive ion detection. For analysis of blood
and urine, fluids were diluted with equal volumes of water and
extracted with ethyl acetate.
1
[3-(4-Bromobenzenesulfonyl)thieno[2,3-e][1,2,3]triazolo-
[1,5-a]pyrimidin-5-yl]thiophen-2-ylmethylamine (3i). White
1
solid, yield 35%. H NMR (CDCl3, 500 MHz): δ 5.16 (d, J = 5 Hz,
2H), 5.78 (t, J = 5 Hz, 1H), 7.04−7.06 (m, 1H), 7.32−7.33 (m, 1H),
7.33 (m, 1H), 7.58 (d, J = 10 Hz, 2H), 7.93 (d, J = 5 Hz, 1H), 8.00 (d,
J = 5 Hz, 1H), 8.05 (d, J = 10 Hz, 2H). This compound is
commercially available and has been reported.17
[3-(4-Isopropylbenzenesulfonyl)thieno[2,3-e][1,2,3]triazolo-
[1,5-a]pyrimidin-5-yl]naphthalen-1-ylmethylamine (3j). Crude
material was purified by flash column chromatography using CH2Cl2/
acetic acid (v/v = 100:1) to elute the starting materials, then changed
to CH2Cl2/CH3OH/acetic acid (v/v/v = 100:2:1). White solid, yield
32%. 1H NMR (CDCl3, 500 MHz): δ 1.19 (d, J = 5 Hz, 6H,), 2.88 (m,
1H), 5.46 (d, J = 5 Hz, 2H), 5.61 (t, J = 5 Hz, 1H), 7.47−7.51 (m,
1H), 7.53 (d, J = 5 Hz, 2H), 7.55 (s, 1H), 7.56 (s, 1H), 7.62 (d, J = 5
Hz, 1H), 7.91 (d, J = 5 Hz, 1H), 7.95 (d, J = 5 Hz, 1H), 7.98 (d, J = 5
Hz, 1H), 8.10 (d, J = 5 Hz, 1H), 8.13 (d, J = 10 Hz, 2H). LC/ESI-
LRMS [M + H]+ calculated 514.1, observed 514.0 for C27H23N5O2S2.
[3-[4-(1,1-Difluoroethyl)benzenesulfonyl]thieno[2,3-e]-
[1,2,3]triazolo[1,5-a]pyrimidin-5-yl]thiophen-2-ylmethylamine
(3k). After microwave irradiation, the reaction mixture was treated
with aqueous 1 M HCl (10 equiv) and stirred for 10 min and then
extracted three times with CH2Cl2. Combined organics were washed
three times with aqueous 1 M HCl, dried over Na2SO4, and
concentrated in vacuo. The product was precipitated by the addition of
CH3OH and isolated by vacuum filtration. The product 3k was further
purified by trituration with a small amount of CHCl3 to give a white
solid. Yield 15%. 1H NMR (acetone-d6, 300 MHz): δ 1.94 (s, J = 18.0
Hz, 3H), 5.15 (s, 2H), 7.01 (dd, J = 9.0, 3.0 Hz, 1H), 7.30−7.31 (m,
1H), 7.36−7.39 (m, 1H). 7.72 (d, J = 6.0 Hz, 2H), 7.97 (d, J = 6.0 Hz,
1H), 8.26 (d, J = 9.0 Hz, 2H), 8.34 (d, J = 6.0 Hz, 1H). 13C NMR
(acetone-d6, 125 MHz): δ 25.0 (t, J = 28.8 Hz), 39.7, 114.5, 116.7,
125.3, 125.8 (t, J = 5.0 Hz), 127.0, 127.1, 127.0, 131.4, 135.6, 139.1,
141.0, 141.9, 142.6 (t, J = 27.5 Hz), 144.6, 153.7. ESI(+)-HRMS [M +
H]+ calculated 492.0356, observed 492.0423 for C15H10F2N4O3S2.
Blood Collection. Whole blood was collected from 8- to 12-week-
old (25−35 g) wild-type mice in a CD1 genetic background by orbital
puncture following subcutaneous injection of sodium heparin.
Procedures were approved by the UCSF Committee on Animal
Research. Human venous blood was collected into heparinized tubes,
stored at 4 °C, and used within 48 h.
ASSOCIATED CONTENT
* Supporting Information
■
S
Table S1 summarizing potency and metabolic stability data for
commercially available analogues of 1. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: 415-338-6495. Fax: 415-405-0377. E-mail: marc@sfsu.
■
edu.
Notes
Erythrocyte Lysis Assay of UT-B Inhibition. Whole blood was
diluted to a hematocrit of ∼1% in PBS containing 1.25 M acetamide
and 5 mM glucose. Then 100 μL of the erythrocyte suspension was
added to each well of a 96-well round-bottom microplate to which
inhibitor was added from DMSO stock solution. After 10 min of
incubation, 20 μL of the erythrocyte suspension was added rapidly to
one well of a 96-well plate (Costar, Corning, NY) containing 180 μL
of isosmolar buffer (PBS containing 1% DMSO). Erythrocyte lysis was
quantified by absorbance at 710 nm, as described.13 Percentage
erythrocyte lysis was calculated using control values from the same
plate as % lysis = 100 × (Aneg − Atest)/(Aneg − Apos), where Atest is the
absorbance from a test well.
Stopped-Flow Measurement of Erythrocyte Urea Perme-
ability. Measurements were done using a Hi-Tech Sf-51 instrument
(Wiltshire, U.K.). Whole blood (mouse or human) was diluted in PBS
(hematocrit of ∼0.5%), incubated with inhibitor for 5 min, and then
subjected to a 100 mM inwardly directed urea gradient. The kinetics of
increasing cell volume caused by urea influx was measured as the time-
course of 90° scattered light intensity at 530 nm. Urea permeability
and percentage inhibition were computed as described.13
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by Grants DK35124, DK72517,
DK86125, EB00415, HL73856, and EY13574 from the
National Institutes of Health.
ABBREVIATIONS USED
■
UT, urea transporter; mCPBA, m-chloroperoxybenzoic acid;
PyBOP, benzotriazol-1-yloxytripyrrolidinophosphonium hexa-
fluorophosphate; BOP, benzotriazole-1-yloxy-tris-
(dimethylamino)phosphonium hexafluorophosphate; HMPA,
hexamethylphosphoramide; xanthphos, 4,5-bis-
(diphenylphosphino)-9,9-dimethylxanthene; DMF, N,N-dime-
thylformamide; DMSO, dimethylsulfoxide
REFERENCES
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