Discovery and in Vitro Optimization of 3-Sulfamoylbenzamides as ROMK Inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.7b00481

Inhibitors of the renal outer medullary potassium channel (ROMK) show promise as novel mechanism diuretics, with potentially lower risk of diuretic-induced hypokalemia relative to current thiazide and loop diuretics. Here, we report the identification of a novel series of 3-sulfamoylbenzamide ROMK inhibitors. Starting from HTS hit 4, this series was optimized to provide ROMK inhibitors with good in vitro potencies and well-balanced ADME profiles. In contrast to previously reported small-molecule ROMK inhibitors, members of this series were demonstrated to be highly selective for inhibition of human over rat ROMK and to be insensitive to the N171D pore mutation that abolishes inhibitory activity of previously reported ROMK inhibitors.

Full text of DOI:10.1021/acsmedchemlett.7b00481

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Letter
Discovery and In Vitro Optimization of 3-
Sulfamoylbenzamides as ROMK Inhibitors
Matthew F. Sammons, Sujay V. Kharade, Kevin J. Filipski, Markus Boehm, Aaron C. Smith,
Andre Shavnya, Dilinie P. Fernando, Matthew S. Dowling, Philip A Carpino, Neil A. Castle,
Shannon G. Zellmer, Brett M. Antonio, James R. Gosset, Anthony Carlo, and Jerod S. Denton
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.7b00481 • Publication Date (Web): 19 Jan 2018
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Discovery and In Vitro Optimization of 3-Sulfamoylbenzamides as
ROMK Inhibitors
Matthew F. Sammons,*,† Sujay V. Kharade,*,# Kevin J. Filipski,† Markus Boehm,† Aaron C. Smith,§
Andre Shavnya,§ Dilinie P. Fernando,§,‡ Matthew S. Dowling,§ Philip A. Carpino,† Neil A. Castle,○ ,‡
Shannon G. Zellmer,○,‡ Brett M. Antonio,○,‡ James R. Gosset, † Anthony Carlo,§ and Jerod S. Denton#
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† Pfizer Worldwide Research & Development, 610 Main Street, Cambridge, MA 02139, Unites States
# Department of Anesthesiology, Vanderbilt University Medical Center, Nashville, TN 37232, United States
§ Pfizer Worldwide Research & Development, Eastern Point Road, Groton, CT 06340, United States
○ Neusentis, Pfizer Worldwide Research & Development, Durham, NC 27703, United States
KEYWORDS: ROMK, Kir1.1, potassium channel inhibitors, diuretics, hypertension
ABSTRACT: Inhibitors of the renal outer medullary potassium channel
(ROMK) show promise as novel mechanism diuretics, with potentially lower
risk of diureticꢀinduced hypokalemia relative to current thiazide and loop diuꢀ
retics. Here we report the identification of
a novel series of 3ꢀ
sulfamoylbenzamide ROMK inhibitors. Starting from HTS hit 4, this series
was optimized to provide ROMK inhibitors with good in vitro potencies and
wellꢀbalanced ADME profiles. In contrast to previously reported smallꢀ
molecule ROMK inhibitors, members of this series were demonstrated to be
highly selective for inhibition of human over rat ROMK and to be insensitive
to the N171D pore mutation that abolishes inhibitory activity of previously
reported ROMK inhibitors.
Diuretics are a cornerstone of current strategies for pharmaꢀ
cological treatment of hypertension and for reduction of edeꢀ
ma and fluid retention associated with heart failure. Approxiꢀ
mately 88% of patients hospitalized with acute decompensated
heart failure receive intravenous loop diuretics.1 Thiazide diuꢀ
retics are recommended as firstꢀline agents for the treatment of
hypertension and are among the most prevalent antihypertenꢀ
sive agents used in the United States.2, 3 However, these agents
are not without limitations. Both loop and thiazide diuretics
are associated with metabolic imbalances, particularly low
levels of blood potassium (hypokalemia).4 Loop diuretics lose
efficacy upon chronic administration and high doses may be
required to achieve sufficient exposure at the site of action in
patients with impaired renal function.5 It is estimated that
among adults in the U.S. approximately 6.5 million have heart
failure, approximately 85 million are hypertensive, and the
prevalence of both conditions is projected to increase in the
coming decades.6 There is thus an urgent need to develop
novel mechanism of action pharmaceutical agents for the
treatment of these conditions that lack the limitations of curꢀ
rent diuretics.
mediated salt absorption in the thick ascending limb of the
Loop of Henle (TAL) and potassium excretion in the TAL and
cortical collecting duct (CCD).9 The first reported selective,
smallꢀmolecule ROMK inhibitor VU591 (1, Figure 1) was
demonstrated to inhibit potassium transport in isolated perꢀ
fused rat collecting duct tubules with no effect on sodium
transport.10 Since this initial report, several disclosures by
investigators at Merck11ꢀ19 have established the in vivo efficaꢀ
cy of smallꢀmolecule ROMK inhibitors (Figure 1), including
Compound A (2) and clinical candidate MKꢀ7145 (3), as powꢀ
erful diuretic and natriuretic agents that, in contrast to thiaꢀ
zides and loop diuretics, have minimal impact on kaliuresis.20ꢀ
22
A number of smallꢀmolecule ROMK inhibitors have also
been disclosed in the patent literature.23
Herein, we report the discovery and in vitro optimization of
a
novel series of ROMK inhibitors based on a 3ꢀ
sulfamoylbenzamide scaffold that shows remarkable speciesꢀ
selective inhibition and an insensitivity to mutations of the
ROMK channel that ablate the inhibitory activity of other
smallꢀmolecule ROMK inhibitors.
The Pfizer compound collection was screened for inhibitors
of Tl+ uptake, a wellꢀestablished surrogate for K+ conductance
in ion channels,24 into HEKꢀ293 cells expressing the short
isoform of human ROMK (Kir1.1b or ROMK2). This isoform
was selected for screening as ROMK2 expression extends
The renal outer medullary potassium channel (ROMK,
Kir1.1) plays a major role in renal salt handling and reabsorpꢀ
tion,7 and inhibitors of ROMK show promise as novel mechaꢀ
nism diuretic agents.8 Potassium conductance via ROMK is
required for both Na+/K+/2Clꢀ coꢀtransporter (NKCC2)ꢀ
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Page 2 of 12
along the nephron in both the TAL and CCD. In contrast,
acids 5 as the acyl chlorides. Sulfonamide formation under
basic conditions then provided 3ꢀsulfamoylbenzamides.
ROMK1 is not expressed in the TAL.7
1
2
3
4
5
6
7
8
9
Key results from SAR expansion of this scaffold are preꢀ
sented in Table 1. A scan of the R1 amide region provided 3ꢀ
methylꢀ1,2,4ꢀoxadiazole 8, which was 15ꢀfold more potent
against ROMK (Tl+ flux IC50 = 440 nM) with improved metꢀ
abolic stability (HLM Clint,app,s = 30 mL/min/kg) relative to 4.
Compound 8 also showed excellent selectivity over hERG
binding (Ki > 40 µM) and functional inhibition of the hERG
channel in a patchꢀclamp assay using HEKꢀ293 cells expressꢀ
ing the hERG channel (IC50 > 100 µM). While other heterocyꢀ
cles were tolerated in this region (see SI Table 1 for more exꢀ
amples) 8 provided the largest increase in ROMK potency
relative to 4 and superior lipophilic efficiency30 (LipE) comꢀ
pared to other analogues.
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While some tolerance for variation of the R3 core benzamide
4ꢀsubstituent was observed, the presence of this substituent
was crucial for good ROMK potency. The 4ꢀunsubstituted
benzamide 9 (R3 = H) was more than 30ꢀfold less potent (Tl+
flux IC50 = 17 µM; n = 1) relative to 8. The 4ꢀmethoxy anaꢀ
logue 10 was less potent relative to 8, but with comparable
LipE (3.5 vs. 3.7) and improved metabolic stability (HLM
Clint,app,s = 10 mL/min/kg).
Figure 1. Structures of ROMK inhibitors VU591, Compound
A, and MKꢀ7145.
This screening campaign led to the identification of 3ꢀ
sulfamoylbenzamide 4 (Figure 2) as a ROMK inhibitor with
modest potency (Tl+ flux IC50 = 6.5 µM). Importantly, comꢀ
pound 4 demonstrated minimal inhibition of the related inward
rectifying potassium channels Kir2.1 and Kir4.1 as determined
by wholeꢀcell patchꢀclamp assays using CHO cells stably exꢀ
pressing either Kir2.1 or Kir4.1. Compound 4 also possessed
minimal affinity (Ki > 40 µM) for the humanꢀetherꢀaꢀgoꢀgo
(hERG) potassium channel (product of the hERG gene;
Kv11.1) as determined by displacement of a fluorescently
labeled derivative of dofetilide from membrane homogenates
of HEKꢀ293 cells stability expressing hERG channels.25 While
4 showed high apparent clearance in human liver microsomal
Scheme 1: Synthetic Routes Used to Prepare 3-
Sulfamoylbenzamides 8-16a
(HLM) incubations,26, 27 this selectivity profile coupled with
28
reasonable molecular weight, passive permeability (Papp
)
and
LogD29 made 4 an attractive lead for further optimization.
aReagents and conditions: (a) THF, pyridine, 50ꢀ60 °C (40ꢀ85%
yield); (b) SOCl2, DMF, CH2Cl2; then R1NH2, 2,6ꢀlutidine,
CH2Cl2 (40ꢀ46% yield); (c) R1NH2, HATU, iPr2NEt, DMF (22ꢀ
71% yield)
Figure 2. Structure and profile of HTS hit 4.
Given the high microsomal turnover and modest potency
observed for 4, we began our optimization efforts with a focus
on identifying more potent inhibitors with reduced lipophiliciꢀ
ty relative to 4. The 3ꢀsulfamoylbenzamide scaffold of 4 was
amenable to rapid SAR development via both singleton and
parallel syntheses starting from 3ꢀ(chlorosulfonyl)benzoic
Although attempts to remove or replace the sulfanilide moiꢀ
ety were not successful, variation of the sulfanilide aromatic
ring R2 substituent provided a number of compounds with
improved ROMK potency and LipE (11-13). The Nꢀ4ꢀ
(isoxazolꢀ5ꢀyl)phenyl and Nꢀ4ꢀchlorophenyl sulfonamides 11
and 12 showed ~5ꢀfold improvement in Tl+ flux potencies
(IC50 = 110 nM and 85 nM, respectively) over 8 but did not
offer improved metabolic stability. The Nꢀ4ꢀ(oxazolꢀ5ꢀ
yl)phenyl analogue 13 showed good ROMK potency (Tl+ flux
IC50 = 35 nM), but also demonstrated higher turnover in miꢀ
crosomal incubations. Other sulfanilide paraꢀsubstituents were
tolerated (see SI Table 1 for more examples), but did not offer
the balanced ADME profiles observed for 11ꢀ13.
acids
5
as illustrated in Scheme 1. The 3ꢀ(Nꢀ
arylsulfamoyl)benzoic acids 6 could be accessed by treatment
of sulfonyl chlorides 5 with anilines under basic conditions.
Subsequent HATU mediated coupling of benzoic acids 6 with
amines provided 3ꢀsulfamoylbenzamides. Alternatively, 3ꢀ
(chlorosulfonyl)benzamides 7 were prepared via amine acylaꢀ
tion following activation of 3ꢀ(Nꢀchlorosulfamoyl)benzoic
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Table 1. In Vitro ROMK Potency, hERG Potency, and ADME Properties of Benzamides 8-16
1
2
3
4
5
6
7
8
9
hROMK IC50
HLM Clint,app,s
(mL/min/kg)b (10ꢀ6 cm/s)c
Papp
(µM)a
hERG Ki
(µM)
Cmpd
R1
R2
R3
LogDd
LipE
10
11
12
13
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16
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60
(pIC50±SD)
0.44
CH3
CH3
CH3
CH3
H
30
9
20
22
8
2.7
2.3
2.3
2.3
3.0
2.2
2.4
2.9
3.7
2.5
3.5
4.7
4.0
5.3
4.2
4.1
>40
>40
>40
>40
>40
>40
>40
>40
8
(6.4±0.3)
17e
9
1.3
OCH3
CH3
CH3
CH3
CH3
CH3
10
25
35
69
<8
<9
10
11
12
13
14
15
(5.8±0.2)
0.11
6
(7.0±0.3)
0.085
Cl
19
10
12
19
(7.0±0.03)
0.035
(7.5±0.3)
0.28
(6.6±0.06)
0.086
Cl
Cl
(7.0±0.09)
0.16
(–)ꢀ16
(+)ꢀ16
OCH3
OCH3
<8
<8
16
11
2.6
2.7
4.2
2.2
28
(6.8±0.08)
13
Cl
>40
(4.9±0.1)
a IC50 values are reported as geometric means of n ≥ 2 replicates unless otherwise indicated. b Scaled apparent intrinsic clearance using
scaling method described in Ref. 23. Measured using MDCKIIꢀLE cell line as described in ref. 24. Measured at pH 7.4 using ocꢀ
tanol/buffer shakeꢀflask method as described in Ref. 26. e Data reported for a single replicate (n = 1).
c
d
With these optimized sulfonamides and benzamide cores in
place, iterative scanning of the amide R1 region led to the
identification of a series of triazoles (14ꢀ16) as ROMK inhibiꢀ
tors with wellꢀbalanced in vitro metabolic stability, passive
permeability and ROMK potency profiles. The ROMK potenꢀ
cies of 1ꢀmethylꢀ1Hꢀ1,2,3ꢀtriazoles 14 (Tl+ flux IC50 = 280
nM) and 15 (Tl+ flux IC50 = 86 nM) were similar to those of
the oxadiazole 11 and 12. However, triazoles 14 and 15 were
more stable in HLM incubations (HLM Clint,app,s < 8 and < 9
mL/min/kg, respectively) than their oxadiazole comparators.
The regioisomeric 2ꢀmethylꢀ2Hꢀ1,2,3ꢀtriazole (–)ꢀ16 (PFꢀ
ity. The enantiomeric 2ꢀmethylꢀ2Hꢀ1,2,3ꢀtriazole (+)ꢀ16 was
80ꢀfold less potent relative to (–)ꢀ16. While the more active
enantiomer (–)ꢀ16 did demonstrate weak affinity for the hERG
channel (Ki = 28 µM), it maintained the good selectivity for
ROMK inhibition over hERG channel binding observed in this
series. It should be noted that, in general, introduction of an αꢀ
methyl substituent, as found in 16, led to improved aqueous
solubility with minimal impact on ROMK potency.
Based on their attractive in vitro ADME profiles, 14 and
(–)ꢀ16 were selected for further characterization in wholeꢀcell
patchꢀclamp experiments measuring inhibition of ROMK2
currents. These data (SI Figure 1) confirmed that, as indicated
in the Tl+ flux assay, these compounds are potent inhibitors of
06807656) was also a potent ROMK inhibitor (Tl+ flux IC50
160 nM) with low HLM turnover and good passive permeabilꢀ
=
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human ROMK2. However, it was discovered that they lack wanted to test if it also plays a role in the inhibition of the
1
2
3
4
5
6
7
8
inhibitory activity against the rat ROMK2 channel in an analꢀ
ogous patchꢀclamp assay, a surprising result given the simiꢀ
larity of human and rat ROMK (> 92% identity for ROMK2)
and the similar human and rat potencies of previously reported
ROMK inhibitors.11
hKir1.1 channel by the novel inhibitors reported herein. As
previously shown, the N171D mutation results in a ROMK
channel which does not express well unless a second mutation,
K80M, is introduced.32, 33 Thus, these studies were carried out
in the K80M background. While the K80M mutation restores
the functional expression of N171 mutants it does not affect
channel inhibition by (–)ꢀ16 (SI Figure 3). Interestingly mutatꢀ
ing asparagine to aspartate at position 171 does not affect the
inhibition of ROMK currents by compound (–)ꢀ16 (Figure 3).
This behavior is in contrast to that reported for inhibitors 1 and
2, both of which show markedly lower inhibitory activity
against the N171D mutant channel.
The in vitro activity of previously reported ROMK inhibiꢀ
tors has mostly been evaluated using the longer Kir1.1 splice
variant, Kir1.1a (ROMK1), which possesses 19 additional
amino acids at the Nꢀterminus relative to ROMK2. As shown
in Table 2, the inhibitory activity of compounds 12, 14, and (–
)ꢀ16 was maintained in testing against ROMK1. In a Tl+ꢀflux
assay, compounds 12, 14, and (–)ꢀ16 were able to inhibit
hROMK1 in a concentration dependent manner. These results
were further confirmed using the gold standard patchꢀclamp
electrophysiology technique, with all three compounds showꢀ
ing greater ROMK inhibitory potency in the patchꢀclamp asꢀ
say relative to the Tl+ꢀflux assay. In our experience, the shift in
apparent potencies between these assay formats is typical beꢀ
havior from small molecule ROMK inhibitors. The species
disconnect was maintained in testing against the longer Kir1.1
splice variant as well. In the Tl+ꢀflux assay, known Kir1.1
inhibitor 1 was able to inhibit rat ROMK1 with potency simiꢀ
lar to that previously reported for inhibition of human
ROMK1 (IC50 = 420 nM and 240 nM for rat and human
ROMK, respectively). However, compounds 12, 14, and (–)ꢀ
16 did not have any effect at concentrations up to 30 µM (for
representative concentrationꢀresponse curves see SI Figure 2).
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In summary, a series of 3ꢀ(sulfamoyl)benzamide ROMK inꢀ
hibitors has been discovered following identification of 4 as a
hit from a highꢀthroughput screening campaign of the Pfizer
compound collection. Optimization of this series has provided
a number of compounds with wellꢀbalanced in vitro ADME
properties and ROMK inhibitory potency. In contrast to previꢀ
ously reported smallꢀmolecule ROMK inhibitors, these comꢀ
pounds lack inhibitory activity at the rat ROMK channel. The
inhibitory activity of (–)-16 is insensitive to the introduction of
the N171D mutation in the ROMK conduction pore that greatꢀ
ly diminishes the activity of other smallꢀmolecule inhibitors.
Taken together, these results suggest that the series of inhibiꢀ
tors described herein interact with ROMK in a mode distinct
from previously reported inhibitors. Future work is planned to
elucidate the nature of the interaction of ROMK with these
inhibitors.
Table 2. In Vitro Rat and Human ROMK1 Potency for
Select Inhibitors
hROMK
hROMK Tl+ flux rROMK Tl+flux
patchꢀclamp
IC50 (nM)a
IC50 (µM)a
(pIC50±SD)
Cmpd
IC50 (nM)b
(95% CI)
(pIC50±SD)
744
0.420
N.D.c
1
(6.1±0.3)
(6.4±0.4)
777
62
>30
12
14
(6.1±0.06)
(61ꢀ64)
1042
104
>30
>30
(5.9±0.3)
(31ꢀ117)
391
61
(–)ꢀ16
(6.4±0.03)
(63ꢀ171)
Figure 3. Inhibition of human ROMK K80M and
K80M/N171D currents by compound (–)ꢀ16. Wholeꢀcell curꢀ
rents in HEK293 cells transiently expressing human ROMK
K80M (open bars) or human ROMK K80M/N171D (dark
bars) channels were examined under voltage clamp conditions
as described in the Supporting Information. (N.S = statistically
not significant)
a
IC50 values are reported as geometric means of n≥3 repliꢀ
cates unless otherwise indicated. Reported IC50 values are
b
derived from global fit to a five point concentrationꢀresponse
curve with n≥4 for each concentration. c Not Determined.
Based on in silico docking and functional analysis of
ROMK mutants, it has been proposed that 1 binds in the
ROMK conduction pore at a site ~6.5 Å below the channel
selectivity filter in the proximity of N171 and in contact with
two adjacent ROMK subunits.31 Consistent with this model,
the ROMK N171D mutant is insensitive to inhibition by 1. A
similar result has been reported for 2, which inhibits the
N171D mutant with ~90ꢀfold lower potency relative to its
activity against the wildꢀtype channel.22 Since N171 has been
identified as a conserved part of the small molecule binding
site for these previously discovered ROMK inhibitors, we
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website.
Details for the synthesis of key intermediates and analytical data
for compounds 8ꢀ16; Experimental details for ROMK1 Tl+ꢀflux
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and electrophysiology experiments; Representative Tl+ꢀflux conꢀ
(7) Welling, P. A.; Ho, K. A comprehensive guide to the ROMK
potassium channel: form and function in health and disease. Am. J.
Physiol.: Renal Physiol. 2009, 297, F849ꢀF863.
(8) Denton, J. S.; Pao, A. C.; Maduke, M. Novel diuretic targets.
Am. J. Physiol.: Renal Physiol. 2013, 305, F931ꢀF942.
(9) Kharade, S. V.; Flores, D.; Lindsley, C. W.; Satlin, L. M.; Denꢀ
ton, J. S. ROMK inhibitor actions in the nephron probed with diuretꢀ
ics. Am. J. Physiol.: Renal Physiol. 2016, 310, F732ꢀF737.
(10) Bhave, G.; Chauder, B. A.; Liu, W.; Dawson, E. S.; Kadakia,
R.; Nguyen, T. T.; Lewis, L. M.; Meiler, J.; Weaver, C. D.; Satlin, L.
M.; Lindsley, C. W.; Denton, J. S. Development of a Selective Smallꢀ
Molecule Inhibitor of Kir1.1, the Renal Outer Medullary Potassium
Channel. Mol. Pharmacol. 2011, 79, 42ꢀ50.
centationꢀresponse curves for compounds 1, 12, 14, and (ꢀ)ꢀ16;
Effect of ROMK1 K80M mutation on inhibition by (ꢀ)ꢀ16. (PDF)
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AUTHOR INFORMATION
Corresponding Authors
* Eꢀmail: matthew.sammons@pfizer.com
* Eꢀmail: sujay.v.kharade@vanderbilt.edu
Present Addresses
9
‡
D.F.: 360 Binney St., Cambridge, MA, 02142, United
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chu, R.; AlonsoꢀGalicia, M.; Kaczorowski, G. J.; Roy, S.; Yang, L.;
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Priest, B. T.; Swensen, A. M.; AlonsoꢀGalicia, M.; Felix, J. P.; Broꢀ
chu, R. M.; Bailey, T.; ThomasꢀFowlkes, B.; Zhou, X.; Pai, L. Y.;
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J.; Yang, L.; Garcia, M. L.; Pasternak, A. Discovery of a novel subꢀ
class of ROMK channel inhibitors typified by 5ꢀ(2ꢀ(4ꢀ(2ꢀ(4ꢀ(1Hꢀ
Tetrazolꢀ1ꢀyl)phenyl)acetyl)piperazinꢀ1ꢀyl)ethyl)isobenzofuranꢀ
1(3H)ꢀone. Bioorg. Med. Chem. Lett. 2013, 23, 5829ꢀ5832.
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J.; Zhu, Y.; Priest, B. T.; Swensen, A. M.; Liu, J.; Margulis, M.; Visꢀ
conti, R.; Weinglass, A.; Felix, J. P.; Brochu, R. M.; Bailey, T.;
ThomasꢀFowlkes, B.; AlonsoꢀGalicia, M.; Zhou, X.; Pai, L.ꢀY.; Coꢀ
rona, A.; Hampton, C.; Hernandez, M.; Bentley, R.; Chen, J.; Shah,
K.; Metzger, J.; Forrest, M.; Owens, K.; Tong, V.; Ha, S.; Roy, S.;
Kaczorowski, G. J.; Yang, L.; Parmee, E.; Garcia, M. L.; Sullivan, K.;
Pasternak, A. Discovery of a Potent and Selective ROMK Inhibitor
with Pharmacokinetic Properties Suitable for Preclinical Evaluation.
ACS Med. Chem. Lett. 2015, 6, 747ꢀ752.
(14) Walsh, S. P.; Shahripour, A.; Tang, H.; de Jesus, R. K.; Teꢀ
umelsan, N.; Zhu, Y.; Frie, J.; Priest, B. T.; Swensen, A. M.; Alonsoꢀ
Galicia, M.; Felix, J. P.; Brochu, R. M.; Bailey, T.; ThomasꢀFowlkes,
B.; Zhou, X.; Pai, L.ꢀY.; Hampton, C.; Hernandez, M.; Owens, K.;
Ehrhart, J.; Roy, S.; Kaczorowski, G. J.; Yang, L.; Garcia, M. L.;
Pasternak, A. Differentiation of ROMK potency from hERG potency
in the phenacetyl piperazine series through heterocycle incorporation.
Bioorg. Med. Chem. Lett. 2016, 26, 2339ꢀ2343.
(15) Zhu, Y.; de Jesus, R. K.; Tang, H.; Walsh, S. P.; Jiang, J.; Gu,
X.; Teumelsan, N.; Shahripour, A.; Pio, B.; Ding, F.ꢀX.; Ha, S.;
Priest, B. T.; Swensen, A. M.; AlonsoꢀGalicia, M.; Felix, J. P.; Broꢀ
chu, R. M.; Bailey, T.; ThomasꢀFowlkes, B.; Zhou, X.; Pai, L.ꢀY.;
Hampton, C.; Hernandez, M.; Owens, K.; Ehrhart, J.; Roy, S.; Kaꢀ
czorowski, G. J.; Yang, L.; Parmee, E. R.; Sullivan, K.; Garcia, M. L.;
Pasternak, A. Discovery of a potent and selective ROMK inhibitor
with improved pharmacokinetic properties based on an octahydropyꢀ
razino[2,1ꢀc][1,4]oxazine scaffold. Bioorg. Med. Chem. Lett. 2016,
26, 5695ꢀ5702.
(16) Chobanian, H. R.; Guo, Y.; Pio, B.; Tang, H.; Teumelsan, N.;
Clements, M.; Frie, J.; Ferguson, R.; Guo, Z.; ThomasꢀFowlkes, B. S.;
Felix, J. P.; Liu, J.; Kohler, M.; Priest, B.; Hampton, C.; Pai, L.ꢀY.;
Corona, A.; Metzger, J.; Tong, V.; Joshi, E. M.; Xu, L.; Owens, K.;
Maloney, K.; Sullivan, K.; Pasternak, A. The design and synthesis of
novel spirocyclic heterocyclic sulfone ROMK inhibitors as diuretics.
Bioorg. Med. Chem. Lett. 2017, 27, 1109ꢀ1114.
(17) Hampton, C.; Zhou, X.; Priest, B. T.; Pai, L.ꢀY.; Felix, J. P.;
ThomasꢀFowlkes, B.; Liu, J.; Kohler, M.; Xiao, J.; Corona, A.; Price,
O.; Gill, C.; Shah, K.; Rasa, C.; Tong, V.; Owens, K.; Ormes, J.;
Tang, H.; Roy, S.; Sullivan, K. A.; Metzger, J. M.; AlonsoꢀGalicia,
M.; Kaczorowski, G. J.; Pasternak, A.; Garcia, M. L. The Renal Outer
Medullary Potassium Channel Inhibitor, MKꢀ7145, Lowers Blood
Pressure, and Manifests Features of Bartter’s Syndrome Type II Pheꢀ
notype. J. Pharmacol. Exp. Ther. 2016, 359, 194ꢀ206.
‡
N.A.C., S.G.Z, and B.M.A.: 4222 Emperor Blvd., Suite
350, Durham, NC, 27703, United States.
Author Contributions
The manuscript was written through contributions of all authors.
All authors have given approval to the final version of the manuꢀ
script.
ACKNOWLEDGMENT
The authors would like to thank James Bradow for SFC resolution
and analysis support. We would also like to thank Beth Vetelino
and Steve Coffey for synthetic support.
ABBREVIATIONS
ROMK, renal outer medullary potassium channel; TAL, thick
ascending limb of the Loop of Henle; CCD, cortical collecting
duct; hERG, human etherꢀaꢀgoꢀgo gene; HLM, human liver miꢀ
crosomes; Papp, passive permeability; HEKꢀ293, human embryonic
kidney 293 cells; CHO, Chinese hamster ovary; DMAP, 4ꢀ
dimethylaminopyridine;
HATU,
1ꢀ
[bis(dimethylamoni)methylene]ꢀ1H-1,2,3ꢀtriazolo[4,5ꢀb]ꢀ
pyridiniumꢀ3ꢀoxide hexafluorophosphate; LipE, lipophilic effiꢀ
ciency; ADME, absorption, distribution, metabolism, and
exctretion; SAR, structureꢀactivity relationship; THF, tetrahydroꢀ
furan; DMF, N,Nꢀdimethylformamide.
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Figure 1. Structures of ROMK inhibitors VU591, Compound A, and MK-7145.
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Figure 2. Structure and profile of HTS hit 4.
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Scheme 1: Synthetic Routes Used to Prepare 3-Sulfamoylbenzamides 8-16a
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Table 1. In Vitro ROMK Potency, hERG Potency, and ADME Properties of Benzamides 8-17
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Figure 3. Inhibition of human ROMK K80M and K80M/N171D currents by compound (–)-16. Whole-cell
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Supporting Information. (N.S = statistically not significant)
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