Discovery of a potent and selective ROMK inhibitor with pharmacokinetic properties suitable for preclinical evaluation

Article abstract of DOI:10.1021/ml500440u

A new subseries of ROMK inhibitors exemplified by 28 has been developed from the initial screening hit 1. The excellent selectivity for ROMK inhibition over related ion channels and pharmacokinetic properties across preclinical species support further pre

Full text of DOI:10.1021/ml500440u

Letter
pubs.acs.org/acsmedchemlett
Discovery of a Potent and Selective ROMK Inhibitor with
Pharmacokinetic Properties Suitable for Preclinical Evaluation
,
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†
†
Shawn P. Walsh,* Aurash Shahripour, Haifeng Tang, Nardos Teumelsan, Jessica Frie, Yuping Zhu,
‡
‡
‡
‡
‡
‡
Birgit T. Priest, Andrew M. Swensen, Jessica Liu, Michael Margulis, Richard Visconti,
‡
‡
‡
‡
Adam Weinglass, John P. Felix, Richard M. Brochu, Timothy Bailey, Brande Thomas-Fowlkes,
Magdalena Alonso-Galicia, Xiaoyan Zhou, Lee-Yuh Pai, Aaron Corona, Caryn Hampton,
Melba Hernandez, Ross Bentley, Jing Chen, Kashmira Shah, Joseph Metzger, Michael Forrest,
§
§
§
§
§
§
§
§
§
§
§
∥
∥
⊥
§
‡
†
Karen Owens, Vincent Tong, Sookhee Ha, Sophie Roy, Gregory J. Kaczorowski, Lihu Yang,
†
‡
§
†
Emma Parmee, Maria L. Garcia, Kathleen Sullivan, and Alexander Pasternak
†
‡
§
∥
Discovery Chemistry, Department of Pharmacology, Department of Cardiometabolic Diseases, Pharmacokinetic,
⊥
Pharmacodynamics and Drug Metabolism, Department of Chemistry Modeling and Informatics, Merck Research Laboratories,
Kenilworth, New Jersey 07033, United States
*
S Supporting Information
ABSTRACT: A new subseries of ROMK inhibitors exemplified by 28 has been developed from the initial screening hit 1. The
excellent selectivity for ROMK inhibition over related ion channels and pharmacokinetic properties across preclinical species
support further preclinical evaluation of 28 as a new mechanism diuretic. Robust pharmacodynamic effects in both SD rats and
dogs have been demonstrated.
KEYWORDS: ROMK, hypertension, heart failure, diuresis, natriuresis
iuretics represent an important class of therapeutics for the
treatment of human diseases. Thiazide diuretics, such as
hydrochlorothiazide (HCTZ), represent first line therapy for
therapeutics, no new diuretics have been disclosed to be in
development.
D
Recent efforts in our laboratory aimed at the discovery of new
mechanism diuretics have focused on the development of
selective inhibitors for the Renal Outer Medullary Potassium
uncomplicated hypertension; either alone or in combination
1
with other agents. Loop diuretics, such as furosemide, are
4,5
Channel (ROMK, Kir1.1, encoded by KCNJ1). This inward
rectifying potassium channel is expressed at the apical membrane
of epithelial cells lining two segments of the nephron: the thick
indicated for the treatment of acute pulmonary edema and
2
chronic heart failure. Approved diuretics are not without their
limitations, however. The thiazide diuretics, for example, are
associated with hypokalemia (serum potassium <3.5 mEq/L)
and elevation in fasting glucose, side effects that can be dose
limiting. Common dose-dependent adverse reactions associated
with loop diuretics include hyponatremia, hypokalemia,
hypomagnesaemia, and dehydration, thus requiring careful
ascending loop of Henle (TALH) and the cortical collecting duct
6
(
CCD). At the TALH, ROMK facilitates potassium recycling
across the luminal membrane, which is necessary for the proper
+
+
−
function of the furosemide-sensitive Na /K /2Cl cotransport-
er, the rate-determining step for sodium reuptake in this part of
the nephron. At the CCD, ROMK provides a route for potassium
secretion that is tightly coupled to sodium uptake through the
2
electrolyte monitoring in patients. Another drawback of loop
7
,8
diuretics is their short human half-lives (i.e., furosemide human
half-life ∼2 h), which are associated with robust initial diuretic
responses (impacting tolerability) followed by rebound effects;
short half-lives may also be responsible for the inferior blood
pressure efficacy of loop diuretics compared to longer half-life
amiloride-sensitive epithelial sodium channel.
Bartter’s
syndrome type II patients (KCNJ1 homozygotes) lack functional
ROMK expression and have a phenotype characterized by renal
Received: December 30, 2014
Accepted: May 7, 2015
3
thiazide diuretics. Despite the established importance of these
therapies and the opportunity for improvement over existing
©
XXXX American Chemical Society
A
DOI: 10.1021/ml500440u
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ACS Medicinal Chemistry Letters
Letter
9
potency was determined by measuring displacement of 35S
salt wasting, hypotension, and mild hypokalemia. A similar
phenotype has been reported from studies with the ROMK
knockout mice.
21,22
MK499 from HEK-293 cells stably expressing hERG.
9
,10
17
In addition, KCNJ1 heterozygote carriers
from the Framingham Heart Study exhibited reduced blood
pressure and a reduced risk of hypertension at age 60 when
compared with matched controls. Taken together, the evidence
suggests that selective inhibitors of ROMK should represent
novel diuretic/natriuretic agents with reduced kaliuresis,
opportunity for improved pharmacokinetic (PK) and pharma-
codynamic (PD) properties, and potential utility in the treatment
of hypertension and heart failure.
Small molecule inhibitors of ROMK were first described by
and subsequently by our
The initial lead series in our laboratories
remarkably resulted from isolation of a potent impurity 1 (Figure
) present in an otherwise inactive high throughput screening hit.
As previously reported, our initial follow-up to 1 focused on
replacement of the two nitro groups with an eye toward
improved selectivity over hERG compared to ROMK. Differ-
entiation of the ROMK potassium ion channel activity from the
hERG potassium ion channel activity was viewed as crucial due to
the association between hERG channel blockade in vitro with
QTc prolongation and risk of cardiac arrhythmias seen with a
11
23−26
broad range of drugs.
To this end, phthalide and cyano
1
2
13,14
groups (2 and 3) were identified as effective single nitro group
17
replacements, able to maintain similar ROMK inhibition to 1.
1
5,16
Denton at Vanderbilt University
own group.
As previously described, combination of these groups led to
identification of 4, which represents a >10-fold improvement in
the hERG/ROMK ratio over the initial hit; further exploration of
the structure−activity relationships (SARs) of substitutions on
the cyano-phenyl ring resulted in 5, a potent ROMK inhibitor,
which for the first time in our series provided a useful level of
selectivity over hERG (18-fold).
1
7,18
1
Following the SAR successfully applied in our acyl piperazine
18
subseries, incorporation of the optimal 4-methyl substitution
on the phthalide ring of 5 generated compound 7 (Figure 1).
This compound displayed potent ROMK inhibition (0.035 μM)
with reduced binding potency for the hERG channel (2.1 μM),
resulting in a 60-fold selectivity window. Unfortunately, when 7
was evaluated in a hERG electrophysiology (EP) assay the
potency was found to be less than 1.0 μM. Additionally, PK
evaluation in Sprague−Dawley (SD) rats revealed the compound
to have high clearance, low oral exposure, and modest half-life.
Nevertheless, 7 represented a potent ROMK inhibitor and an
interesting starting point for further optimization.
We reasoned that structural modifications capable of
attenuating the basicity of the piperazine moiety in 7 might
lead to beneficial impact on both hERG selectivity and PK
parameters. In addition, substitutions that could reduce lip-
ophilicity might also provide benefit. To this end, we next
explored substitution at the α and β carbons flanking the
nitrogens of the central piperazine ring (Table 1). On the carbon
adjacent to the phthalide phenyl ring, methyl and fluoro
substitution (8 and 9) led to modest loss of ROMK potency,
Table 1. Benzylic Substitution Structure−Activity
a
Relationship
Figure 1. Replacement of the two nitrophenyl groups in 1.
Subsequent studies detailed the development of this initial hit
into a distinct subseries of potent and selective ROMK inhibitors
ROMK IC50
hERG IC50
#
R1
Me
R2
R3
R4
X
(μM)
(μM)
(
6). This effort resulted in the first pharmacological proof-of-
8
H
H
H
H
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
Me
H
H
C
C
C
C
C
C
C
C
C
N
0.170
0.060
0.12
2.8
2.4
9.0
9.0
3.5
1.3
1.1
1.2
2.2
6.4
biology, confirming for the first time in vivo that small molecule
9
1
1
1
1
1
1
1
F
H
ROMK inhibitors represent a new class of novel mechanism
0
1
2
3
4
5
6
OH
OMe
OEt
H
H
19
diuretics with reduced kaliuresis. In this Letter, we will describe
the parallel development of a piperazinyl diol series resulting in
ROMK inhibitors with superior potency, selectivity, and
preclinical PK properties suitable for further evaluation.
H
0.11
H
0.22
H
0.19
H
Me
Me
OH
OH
0.15
For all compounds listed, unless otherwise specified, ROMK
potency was determined as previously described using a thallium
H
0.30
H
0.070
0.049
20
flux functional assay in HEK-293 cells stably expressing ROMK
and binding potency. For the outward delayed rectifier potassium
channel (Kv11.1, human ether-a-go-go-related gene, hERG),
17
H
a
All compounds are racemic.
B
DOI: 10.1021/ml500440u
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ACS Medicinal Chemistry Letters
Letter
a
resulting in an overall erosion of the hERG selectivity. Hydroxy
or methoxy substitution (10 and 11) also resulted in a modest
loss of ROMK potency, but the corresponding loss of hERG
potency resulted in an overall improvement in the selectivity
margin (>150-fold in the case of 10). Further increase in the size
of the alkoxy substituent to ethyl (12), however, resulted in a loss
in ROMK potency and hERG margin. On the 4-CN phenyl side
of the molecule, methyl substitution adjacent to the piperazine
and mono- or dimethyl substitution at the benzylic position (13,
Scheme 1. Synthesis of Stereochemically Pure Diols 24−26
1
4, and 15) resulted in modest loss of ROMK potency and hERG
selectivity. However, hydroxyl substitution at the benzylic
position (16) largely maintained the potency and selectivity
present in 7. Guided by hERG selectivity improvements
observed upon heteroatom incorporation in our related acyl
piperazine series, we also prepared the pyridyl analogue 17. In
this case, the pyridyl leads to retention of ROMK potency with an
increased hERG/ROMK ratio (130-fold).
2
7
Based on these results, 10, 16 and 17 were selected for PK
evaluation in SD rats (0.5 mpk iv and 2 mpk po; Table 2). All
a
Table 2. SD Rat PK Properties of 7, 10, 16, and 17
7
10
33
16
40
17
40
Cl (L/kg)
72
AUCNpo (μM h kg/mg)
t1/2 (h)
0.12
1.2
21
0.46
0.83
39
0.35
1.3
37
0.74
1.5
78
Fpo (%)
a
All compounds are racemic.
a
Reagents and conditions: (a) potassium vinyltrifluoroborate,
(dppf)−CH Cl , triethylamine (TEA), ethanol (EtOH), 140
C, MW; (b) mCPBA, CH Cl , 0 °C (40−70% yield, 2 steps); (c)
three compounds showed modest improvement in clearance and
oral exposure with little effect on half-life. Oral bioavailability was
improved in all three cases; incorporation of the heterocyclic ring
in 17 lead to the most significant effect.
PdCl
2
2
2
°
2
2
ChiralPac AD-H SFC chromatography, 10% EtOH/CO , flow rate
2
2
00 mL/min, 100 bar, 25 °C; (d) N-Boc-piperazine, EtOH, 150 °C,
MW (50−90% yield); (e) (i) trifluoroacetic acid, CH C , rt, (ii)
2
2
Next we turned to combining the hydroxy substitution and to
producing compounds as single enantiomers (Scheme 1). Suzuki
cross coupling of 18 with the potassium vinyl trifluoroborate salt
followed by m-chloroperbenzoic acid (mCPBA) epoxidation
provided the racemic epoxide 19. Chiral supercritical fluid
chromatography (SFC) separation of the enantiomers followed
by reaction of the individual epoxides with Boc-piperazine led to
Na CO (aq) (85−90% yield); (f) NBS, H O/t-butanol (2:1), 40 °C,
2
3
2
then addition of NaOH at 0 °C (44−85% yield); (g) 20 + 22, EtOH,
50 °C, MW (58−92% yield).
1
para- or meta-positions relative to the pyridine nitrogen resulted
in compounds with improved ROMK potency, with the
exception of the p-trifluoromethyl and m-chloro groups (39,
40, 47, and 48). Of the ortho-substituents explored, only the
methyl group (35 and 36) provided improved ROMK potency
than (R,S)-24. Overall, the best balance of ROMK potency and
hERG selectivity was achieved using the p-methoxy group
((R,S)-28). Deletion of the pyridyl nitrogen from (R,R)-27 and
(R,S)-28 provided compounds with similar ROMK potency but
reduced hERG selectivity (29 and 30), underscoring the
importance of the heterocycle. No additivity was observed
when combining the best para-substituent with the best ortho-
substituent (OMe and Me, 43 and 44). Finally, anticipating the
p-methoxy group might undergo metabolism in vivo, we explored
the size of the alkoxy group and incorporation of the trideutero-
methoxy group (45, 46, 49, and 50).
In addition to the Th flux and S-MK499 assays, compounds
were evaluated in ROMK and hERG EP assays (Table 4). There
was good correlation of potency in both the ROMK and hERG
EP assays, with three ROMK inhibitors identified with EP ratios
of greater than 1000-fold. To understand how this level of in vitro
selectivity translated in vivo, (R,S)-28 was evaluated in
anesthetized guinea pigs (n = 3). No significant change in QT
interval was observed following IV infusion of (R,S)-28 at 2 mpk
(average peak unbound plasma concentration = 2.2 μM).
28
the individual hydroxy enantiomers. We found that optimal
selectivity for the terminal aminolysis of this and related styrene
epoxides was achieved under thermal conditions in a microwave
reactor in the absence of coordinating acid. Hydrolysis of the t-
butyl carbamate was followed by basic workup to provide (R)-
and (S)-20 as their free amine bases. Synthesis of epoxides (R)-
and (S)-22 began with vinylation of the aryl bromide 21;
however, epoxidation of the resulting olefins with mCPBA led to
completive N-oxide formation. Alternatively, reaction with N-
bromosuccinimide (NBS) and sodium hydroxide (NaOH) led to
one-pot formation of the desired epoxides following in situ
cyclization of the intermediate hydroxy bromides. Chiral SFC
separation led to isolation of the individual epoxide enan-
2
9
tiomers. Reaction of the individual epoxides (R)- and (S)-22
with (R)- and (S)-20 provided the four individual products
shown in Scheme 1.
35
Using the chemistry outlined in Scheme 1, a series of
analogues was prepared to examine the effect of substitution on
the 4-CN pyridyl ring (Table 3). Because the initial analogues
made with (S)-20 piperazine were substantially less potent, only
(
R)-20 was used for subsequent analogues. Of the initial
analogues, (R,S)-23 provides the best combination of ROMK
potency and hERG selectivity. In general, substitution in the
C
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Table 3. Substitution around 5-CN pyridyl ring
a
Absolute configuration assigned using vibrational circular dichroism (VCD) spectroscopy. The other compounds obtained as single isomers, but
with undetermined absolute stereochemistry.
Table 4. EP Evaluation of Selected Inhibitors
IC s >50 μM; Kir4.1, Kir7,1 IC s >10 μM) as well as the cardiac
50 50
channels Nav 1.2 and Cav 2.1 (>30 μM) and found to have
excellent selectivity for ROMK. (R,S)-28 was also evaluated for
6
23
28
27
35
12
41
46
50
13
ROMK EP IC50
nM)
64
18
54
40
inhibition of a panel of CYPs (3A4, 2D6, 2C9; IC s >50 μM)
(
50
hERG EP IC50
μM)
4.5
70
12
33
14
14
12
30
and found to have a signal only against 2C8 (IC50 = 8.2 μM).
Plasma protein binding (% free fraction) in rat and dog for (R,S)-
(
hERG/ROMK
667
1222
1166
254
300
2307
28 was 43% and 83%, respectively, with excellent permeability
(
Papp = 23). Finally, rat and dog ROMK Th flux IC s for (R,S)-
50
With several potent and selective ROMK inhibitors identified,
we next turned to evaluating the PK profiles in SD rats (1 mpk iv
and 2 mpk po; Table 5). All of the diol inhibitors evaluated
demonstrated improvement in Cl, t , oral exposure, and oral
28 were found to be 0.014 and 0.042 μM, respectively.
(R,S)-28 was selected for further PK profiling in two
additional species (dog and rhesus monkey; Table 6). The
1
/2
bioavailability versus the previously reported 6.
Table 6. PK Properties of (R,S)-28 in Multiple Species
(
R,S)-28 was further evaluated in functional assays for several
related inward rectifying potassium channels (Kir 2.3. Kir2.1,
Table 5. SD Rat PK for Selected ROMK Inhibitors
6
23
28
31
35
50
Cl (L/kg)
40
23
25
58
dose IV/PO
(mpk)
CL
AUCNpo
t1/2
Fpo
species
(L/kg)
(μM h kg/mg)
(h)
(%)
AUCNpo (μM h kg/mg)
t1/2 (h)
0.34
0.62
33
1.6
2.0
96
0.84
1.6
65
1.3
1.4
81
1.2
1.5
91
dog
0.5/1
1/2
5.6
25
5.6
5.9
7.2
84
30
Fpo (%)
rhesus
0.51
D
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compound was found to have PK profiles across preclinical
species consistent with an oral QD therapeutic. In addition,
human half-life projection of (R,S)-28 based on observed
preclinical PK in rat, dog, and rhesus monkeys by applying
allometric scaling is estimated to be 10 h, significantly longer than
the most commonly used loop diuretics furosemide and
torsemide. This potentially provides a PK−PD advantage with
regard to the peak diuretic effects associated with loop diuretics,
by providing a reduction in peak-to-trough exposures.
Notes
The authors declare no competing financial interest.
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(
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8 across preclinical species support continued evaluation of this
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ASSOCIATED CONTENT
Supporting Information
Synthesis and characterization data for compounds 7, (R,S)-23,
R,R)-27, (R,S)-28, 35, 41, 46, and 50. Additional details
regarding assay correlation. The Supporting Information is
available free of charge on the ACS Publications website at DOI:
0.1021/ml500440u.
■
*
S
1.1
Channel. Mol. Pharmacol. 2011, 79, 42.
(17) Tang, H.; Walsh, S. P.; Yan, Y.; de Jesus, R. K.; Shahripour, A.;
(
Teumelsan, N.; Zhu, Y.; Ha, S.; Owens, K. A.; Thomas-Fowlkes, B. S.;
Felix, J. P.; Liu, J.; Kohler, M.; Priest, B. T.; Bailey, T.; Brochu, R.;
Alonso-Galicia, M.; Kaczorowski, G. J.; Roy, S.; Yang, L.; Mills, S. G.;
Garcia, M. L.; Pasternak, A. Discovery of Selective Small Molecule
ROMK Inhibitors as Potential New Mechanism Diuretics. ACS Med.
Chem. Lett. 2012, 3, 367.
1
AUTHOR INFORMATION
Corresponding Author
E-mail: shawn_walsh@merck.com.
■
*
(18) Tang, H.; de Jesus, R. K.; Walsh, S. P.; Zhu, Y.; Yan, Y.; Priest, B.
T.; Swensen, A. M.; Alonso-Galicia, M.; Felix, J. P.; Brochu, R. M.;
E
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ACS Medicinal Chemistry Letters
Letter
Bailey, T.; Thomas-Fowlkes, B.; Zhou, X.; Pai, L.-Y.; Hampton, C.;
Hernandez, M.; Owens, K.; Roy, S.; Kaczorowski, G. J.; Yang, L.; Garcia,
M. L.; Pasternak, A. Discovery of a Novel Sub-Class of ROMK Channel
Inhibitors Typified by 5-(2-(4-(2-(1H-Tetrazole-1-yl)phenyl)acetyl)-
piperazin-1-yl)ethyl)isobenzofuran-13H-one. Bioorg. Med. Chem. Lett.
2
013, 23, 5829.
(
19) Garcia, M. L.; Priest, B. T.; Alonso-Galicia, M.; Zhou, X.; Felix, J.
P.; Brochu, R. M.; Bailey, T.; Swensen, A.; Pai, L.-Y.; Xiao, J.; Hernandez,
M.; Hoagland, K.; Owens, K.; Tang, H.; de Jesus, R.; Roy, S.;
Kaczorowski, G. J.; Pasternak, A. Pharmacologic Inhibition of the Renal
Outer Medullary Potassium Channel Causes Diuresis and Natriuresis in
the absence of Kaliuresis. J. Pharm. Exp. Ther. 2014, 348, 153.
(
20) Felix, J. P.; Priest, B. T.; Solly, S.; Bailey, T.; Brochu, R. M.; Liu, C.
J.; Kohler, M. G.; Kiss, L.; Alonso-Galicia, M.; Tang, H.; Pasternak, A.;
Kaczorowski, G. J.; Garcia, M. L. The Inward Rectifying Potassium
Channel Kir1.1; Development of Functional Assays to Identify and
Characterize Channel Inhibitors. Assay Drug Dev. Technol. 2012, 10,
417.
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21) Wang, J.; Della Penna, K.; Wang, H.; Karczewski, J.; Connolly, T.
M.; Koblan, K. S.; Bennett, P. B.; Salata, J. J.Functional and
Pharmacological Properties of Canine ERG Channels. Am. J. Phys.
2
(
003, 284, 256.
22) Further details regarding standard deviations and number of assay
runs are available in the Supporting Information.
23) Cavero, I.; Mestre, M.; Guillon, J.-M.; Crumb, W. Drugs that
(
Prolong QT Interval as an Unwanted Effect; Assessing their Likelihood
of Inducing Hazardous Cardiac Dysrhythmias. Expert Opin. Pharmac-
other. 2000, 1, 947.
(
24) Sanguinetti, M. C.; Jiang, C.; Curran, M. E.; Keating, M. T. A
Mechanistic Link Between an Inherited and Aquired Cardiac
Arrhythmia; HERG Encodes the IKr Potassium Channel. Cell 1995,
81, 299 ..
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25) Fermini, B.; Fossa, A. A. Nat. Rev. The impact of Drug Induced
QT Prolongation on Drug Discovery and Development. Nat. Rev. Drug
Discovery 2003, 2, 439.
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26) Pearlstein, R.; Vaz, R.; Rampe, D. J. Understanding the Structure-
activity Relationship of the Human Ether-a-go-go-related Gene Cardiac
K+ Channel. J. Med. Chem. 2006, 49, 4801.
(
27) Full description of this related series will be the subject of another
publication.
28) The absolute stereochemistry of each enantiomer was inferred
based on X-ray structure determination of a derivative made from (R)-
(
1
2
0 and by Mosher ester and Trost ester H NMR analysis of a derivative
made from the t-butyl carbamate of (R)-21.
29) The absolute stereochemistry of each enantiomer was determined
using vibrational chircular dichromism (VCD).
30) Description of the dog diuresis model will be included separately
as part of a full pharmacology paper.
(
(
F
DOI: 10.1021/ml500440u
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX