Discovery of a potent and selective ROMK inhibitor with improved pharmacokinetic properties based on an octahydropyrazino[2,1-c][1,4]oxazine scaffold

Article abstract of DOI:10.1016/j.bmcl.2016.10.064

Following the discovery of small molecule acyl piperazine ROMK inhibitors, the acyl octahydropyrazino[2,1-c][1,4]oxazine series was identified. This series displays improved ROMK/hERG selectivity, and as a consequence, the resulting ROMK inhibitors do not evoke QTc prolongation in an in vivo cardiovascular dog model. Further efforts in this series led to the discovery of analogs with improved pharmacokinetic profiles. This new series also retained comparable ROMK potency compared to earlier leads.

Full text of DOI:10.1016/j.bmcl.2016.10.064

Accepted Manuscript
Discovery of a Potent and Selective ROMK Inhibitor with Improved Pharma-
cokinetic Properties Based on an Octahydropyrazino[2,1-c][1,4]oxazine Scaf-
fold
Yuping Zhu, Reynalda K. de Jesus, Haifeng Tang, Shawn P. Walsh, Jinlong
Jiang, Xin Gu, Nardos Teumelsan, Aurash Shahripour, Barbara Pio, Fa-Xiang
Ding, Sookhee Ha, Birgit T. Priest, Andrew M. Swensen, Magdalena Alonso-
Galicia, John P. Felix, Richard M. Brochu, Timothy Bailey, Brande Thomas-
Fowlkes, Xiaoyan Zhou, Lee-Yuh Pai, Caryn Hampton, Melba Hernandez,
Karen Owens, Juliann Ehrhart, Sophie Roy, Gregory J. Kaczorowski, Lihu
Yang, Emma R. Parmee, Kathleen Sullivan, Maria L. Garcia, Alexander
Pasternak
PII:
S0960-894X(16)31103-9
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.10.064
BMCL 24367
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
19 August 2016
19 October 2016
21 October 2016
Please cite this article as: 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., Alonso-Galicia, M., Felix, J.P., Brochu, R.M., Bailey,
T., Thomas-Fowlkes, B., Zhou, X., Pai, L-Y., Hampton, C., Hernandez, M., Owens, K., Ehrhart, J., Roy, S.,
Kaczorowski, 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 Octahydropyrazino[2,1-c]
[1,4]oxazine Scaffold, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.
2016.10.064
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Graphical Abstract
Discovery of a Potent and Selective ROMK
Inhibitor Based on a Octahydropyrazino[2,1-
c][1,4]oxazine Scaffold with Improved
Pharmacokinetic Properties
Leave this area blank for abstract info.
Yuping Zhu, Reynalda K. de Jesus, Haifeng Tang, Shawn P. Walsh, Jinlong Jiang, Xin Gu, Nardos Teumelsan, Aurash Shahripour, Pio Barbara, Fa-Xiang
Ding, Jessica Frie, Sookhee Ha, Birgit T. Priest, Andrew M. Swensen, Magdalena Alonso-Galicia, John P. Felix, Richard M. Brochu, Timothy Bailey,
Brande Thomas-Fowlkes, Xiaoyan Zhou, Lee-Yuh Pai, Caryn Hampton, Melba Hernandez, Karen Owens, Juliann Ehrhart, Sophie Roy, Gregory J.
Kaczorowski, Lihu Yang, Emma R. Parmee, Kathleen Sullivan, Maria L. Garcia, Alexander Pasternak

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.els evier.com
Discovery of a Potent and Selective ROMK Inhibitor with Improved
Pharmacokinetic Properties Based on an Octahydropyrazino[2,1-c][1,4]oxazine
Scaffold
Yuping Zhua,*, Reynalda K. de Jesusa, Haifeng Tanga, Shawn P. Walsha, Jinlong Jianga, Xin Gua, Nardos
Teumelsana, Aurash Shahripoura, Barbara Pioa, Fa-Xiang Dinga, Sookhee Haa, Birgit T. Priestb, Andrew M.
Swensenb, Magdalena Alonso-Galiciab, John P. Felixb, Richard M. Brochub, Timothy Baileyb, Brande Thomas-
Fowlkesb, Xiaoyan Zhouc, Lee-Yuh Paic, Caryn Hamptonc, Melba Hernandezc, Karen Owensd, Juliann
Ehrharte,Sophie Royc, Gregory J. Kaczorowskib, Lihu Yanga, Emma R. Parmeea, Kathleen Sullivanb, Maria L.
Garciab, Alexander Pasternaka
a Discovery Chemistry, Merck Research Laboratories, Kenilworth, NJ 07033, United States
b Department of Pharmacology, Merck Research Laboratories, Kenilworth, NJ 07033, United States
c Department of Cardiometabolic Diseases, Merck Research Laboratories, Kenilworth, NJ 07033, United States
d Pharmacokinetics, Pharmacodynamics and Drug Metabolism
e Department of Safety Assessment & Laboratory Animal Resources, Merck Research Laboratories, Kenilworth, NJ 07033, United States
, Merck Research Laboratories, Kenilworth, NJ 07033, United States
ARTICLE INFO
ABSTRACT
Article history:
Received
Following the discovery of small molecule acyl piperazine ROMK inhibitors, the acyl
octahydropyrazino[2,1-c][1,4]oxazine series was identified. This series displays improved
ROMK/hERG selectivity, and as a consequence, the resulting ROMK inhibitors do not evoke
QTc prolongation in an in vivo cardiovascular dog model. Further efforts in this series led to the
discovery of analogues with improved pharmacokinetic profiles. This new series also retained
comparable ROMK potency compared to earlier leads.
Revised
Accepted
Available online
riKeywords:
hERG
2009 Elsevier Ltd. All rights reserved.
ROMK
Hypertension
Heart failure
Diuresis
Natriuresis
recycling across the luminal membrane, providing potassium
Hypertension affects up to 25% of the adult population in
industrialized countries, about one billion people world-wide, and
the incidence increases with age. Despite the large number of
medications available, blood pressure remains largely
uncontrolled so that clinicians need to use combination therapies
for achieving target blood pressure levels. Diuretics, such as
hydrochlorothiazide (HCTZ), are widely prescribed as first line
therapy to treat uncomplicated hypertension, or as add-on
therapy.1 No new diuretics are known to be in development
despite the importance of these therapies.
cations required for function of the furosemide-sensitive
Na+/K+/2Cl- co-transporter, the rate-determining step for salt
reuptake in this part of the nephron. At the CCD, ROMK plays a
critical role in potassium homeostasis and provides a pathway for
potassium secretion that is tightly coupled to sodium uptake
through the amiloride-sensitive epithelial sodium channel.4 Based
on the dual site of action of ROMK and the potential for
differential pharmacokinetic (PK) and physicochemical properties,
we hoped that novel ROMK inhibitors would provide improved
efficacy with reduced liabilities, such as hypokalemia, compared
to the current standard-of-care diuretics. Human genetics, for
example homozygote loss of function ROMK expression
(Bartter's syndrome type II) and genetic ablation of ROMK in
rodents support these expectations, and suggest that selective
ROMK inhibitors will represent a new class of diuretic and
antihypertensive agents.5
The Renal Outer Medullary Potassium Channel (ROMK,
Kir1.1) is a member of the family of inward rectifying potassium
channels2 that plays a critical role in regulating salt and water
homeostasis. ROMK is mainly expressed on the apical membrane
of epithelial cells lining two nephron segments: the thick
ascending loop of Henle (TALH) and the cortical collecting duct
(CCD).3 At the TALH, ROMK participates in potassium

Small molecule inhibitors of ROMK were first described by from membranes prepared from HEK-293 cells stably expressing
Denton at Vanderbilt University.6 In 2013, a novel ROMK hERG.13
inhibitor 1 (Fig. 1) with good ROMK functional potency and
Table 1:
hERG selectivity was described by our group.7 This effort resulted
Sprague Dawley (SD) rat PK properties for selected ROMK
in the first pharmacological proof-of-biology, confirming in a rat
inhibitors (0.5 mpk iv and 1 mpk po)
diuresis model that small molecule ROMK inhibitors represent a
new class of novel mechanism diuretics.8 More recently, SAR
Compound
1
2
3
efforts led to the discovery of a 4-N-tetrazole heteroaryl acetyl
series with an improved ROMK/hERG ratio.9 The best compound
in this series, 2 (Fig. 1), demonstrated a comparable diuretic effect
to 1 in rats with no detectable QTc effects in an in vivo
cardiovascular dog model9. Because of the correlation between
QTc prolongation caused by hERG inhibition and torsade de
pointes that could degenerate into ventricular fibrillation and
cause sudden cardiac death, hERG liabilities need to be
considered during drug development.10 Despite the improved
selectivity over the hERG channel, short half-lives and high
clearance rates remained an issue in these series (Table 1). Since
compounds with longer half-lives would be expected to have more
sustained pharmacodynamics (PD) effects,11 the focus of our
effort was to improve preclinical PK profiles, while retaining or
improving ROMK potency and selectivity over hERG. In this
communication, we will describe SAR efforts to block metabolic
soft spots and to introduce conformational constraints which led to
improved ROMK inhibitor PK profiles and the identification of a
novel morpholine-fused piperazine scaffold.
Cl (L/Kg)
AUCNPO
(µM h Kg/mg)
40
45
29
0.34
0.84
0.45
t1/2 (h)
0.62
33
0.44
38
0.96
28
Fpo (%)
As described in our previous work,9 benzylic substitution with
methyl on the left side of the piperazine (3) resulted in a
compound with reduced clearance rate and slightly improved half-
life, although with 5-fold lower ROMK potency as compared to 2
(Figure 1, Table 1). The reduced clearance rate suggested a
possible metabolic soft spot centered on the benzylic position;
hepatocyte stability studies on compound 1 do show left-side
oxidative metabolites, although the site of oxidation was not
definitively demonstrated. Our initial follow-up to 3 was to
introduce steric hindrance at the benzylic methyl site to block
possible metabolism14 with the aim of maintaining/improving
ROMK potency. Previous work indicated that reducing piperazine
basicity, through incorporation of an amide linkage, led to
attenuation of hERG channel potency.7 We calculated the basicity
of fusedbicyclic piperazine scaffolds incorporating an -O-, -S- and
-NH- (Fig. 2) and found a predicted reduction of basicity with -O-
or -S- containing bicycles and an increased basicity for a -N-
containing bicycle.
In addition to steric and electronic
modulation, we hoped that incorporating these features together in
ring systems would make the scaffold more rigid, and potentially
less susceptible to oxidative metabolism.
Figure 1
Discovery of 4-N-tetrazole heteroaryl acetyl ROMK inhibitor 2
with ROMK and hERG electrophysiology (EP) data12
Figure 2
Calculated pKa of fused bicyclic surrogates
For all compounds herein, ROMK activity was determined, as
previously described, using one or more of three functional
assays12: 86Rb+ flux in CHO cells stably expressing ROMK,
thallium flux in HEK-293 cells stably expressing ROMK, or
electrophysiology (EP). Potency on the hERG channel was
determined by measuring displacement of 35-S MK499 binding
To test this hypothesis, several cyclized cores incorporating -
O-, -S- and -NH- were prepared. A synthesis of a morpholine-
fused bicyclic analog is illustrated in Scheme 1.

Using the chemistry outlined in Scheme 1, or with some
variations, a series of analogues were prepared to explore the
effect of cyclized cores on ROMK potency, selectivity over
hERG, and PK profiles. While most analogues containing the -O-
cyclized cores had similar or better ROMK potency than the
uncyclized compound 3, compounds with the -S- and -N-
containing cores resulted in loss of ROMK inhibitory activity
(Table 2). Surprisingly, ROMK electrophysiology potency for the
four diastereomers 4-7 was quite similar despite the fact that
different orientations for the phthalide pharmacophore were
expected. We speculate that the cis-isomers may readily flip to a
morpholine boat conformation, resulting in a similar positioning
of the phthalide pharmacophore.
basicity (4-7) had similar or better selectivities over hERG (303-
fold to 690-fold) than the uncyclized analog 3 (345-fold).
Compounds with reduced
A
Scheme 1. Synthesis of morpholine-fused bicyclic analogs
Reagents and conditions: (a) Potassium vinyltrifloroborate,
PdCl2(dppf)-CH2Cl2, triethylamine (TEA), ethanol (EtOH), reflux,
10 hr, 100%; (b) mCPBA, CH2Cl2, 0 °C, 70% yield; (c) EtOH,
100 °C, MW, 40%; (d) Cyanomethylenetributyl phosphorane,
Benzene, 100 °C, 18 hr; HPLC separation, ChiralPac® AD
4.6x250 mm, 25 °C, 19% (R,S), 7% (S,S) (e) 4 N HCl/Dioxane, 18
hr, 25 °C, 100% (f) EDC, HOBt, DCM, 25 °C, 3 hr, 63%.
compound with increased basicity, 9, had reduced selectivity
(157-fold). The stronger hERG potency of 8 (IC50 8.9 M) led to
diminished interest in the fused thiomorpholines. Data for 6 (642-
fold) and 7 (870-fold) confirmed an improvement of ROMK
selectivity over hERG compared to the uncyclized analog 3 (345-
fold). The (R,S) isomer 5 had a similar selectivity (303-fold)
compared to 3 (345-fold).
Table 2: ROMK inhibitors with fused bicyclic core
ROMK*
hERG binding*
hERG binding /
hERG EP*
hERG EP/
Bicycle =
#
4
EP IC50 (µM)
IC50 (µM)
ROMK EP (fold)
IC50 (µM)
ROMK EP (fold)
0.105
0.065
53.4
21.1
508
325
NA
NA
303
(S,R)
(R,S)
(S,S)
(R,R)
19.7
5
6
0.088
0.086
0.100
26.8
59.4
8.9
305
690
89
56.5
74.9
NA
642
870
NA
7
8
(R,S)
(S,S)
9
0.140
22.0
157
NA
NA
*Control compound used in all assays; STD < 20%
Compounds 5-7 were further evaluated in SD rats (0.5 mpk iv
and mpk po, Table 3) in order to determine their
pharmacokinetic (PK) profiles. Compounds 5 and 7 possessed
the most favorable overall features, namely good in vitro
1

potency, >100x in vitro selectivity over hERG, excellent oral
bioavailability, with reduced clearance rates, and higher
exposures than 3.
Bicycle =
ROMK*
EP IC50
(µM)
hERG
EP*
IC50 (µM)
hERG EP/
ROMK EP
(fold)
=
5
Table 3: SD rat PK properties for selected ROMK inhibitors
(0.5 mg/kg IV and 1.0 mg/kg PO)
0.065
19.7
303
Compounds
3
5
6
7
Cl (L/Kg)
AUCNpo (µM h Kg/mg)
t1/2 (h)
29
14
54
20
0.057
0.205
0.036
75.9
NA
~10
1,332
NA
10
11
0.45 2.05
0.96 1.0
0.32
0.67
46
2.02
0.80
100
Fpo (%)
28
82
We next focused on modifying the left side of the mopholine
containing bicyclic core of 5-7, hoping to identify compounds
with an extended half-life and equivalent or improved selectivity
over hERG. We explored replacement of the phthalide with
pharmacophores which had been successfully used in our
previous work.7 The compounds were prepared in a similar
fashion as described in Scheme 1. Compounds 10 and 21 had
improved ROMK selectivity over hERG (1,332-fold to 548-fold)
compared to 5 (303-fold), (Table 4). Compounds 12, and 16
were comparable to 5 (303-fold). Further PK st0.85udies (Table
5) of compounds with similar or better ROMK/hERG selectivity
revealed that most displayed comparable or improved PK profiles
to those of 5-7. Compounds 16 and 21 had the longest half-lives
(1.46 and 1.64 h, respectively) with good oral bioavailabilities
(69% and 67%, respectively). SAR trends indicated that in the
morpholine fused bicyclic series, 6-fluoro-2-methyl-benzonitrile
(in 12 and 16) and 2-methyl-benzonitrile (in 13, 17 and 21) were
the best left side moieties with respect to improving half-life.
However, the isochromanone pharmacophore (in 10), provided
the best ROMK / hERG selectivity.
~278
12
13
14
15
16
17
18
19
0.050
0.045
0.072
0.041
0.112
0.083
0.104
NA
>10
>10
12.5
17.6
~10
NA
NA
>222
>139
305
157
~120
NA
Table 4:
SAR of left-side
pharmacophores
0.060
0.027
>10
>167
548
20
21
14.8
*Control compound used in all assays; STD < 20%

Table 5
SD rat PK properties for selected ROMK inhibitors
(0.5 mg/kg IV and 1.0 mg/kg PO)
Table 7
SD rat PK properties selected ROMK inhibitors
(0.5 mg/kg IV and 1.0 mg/kg PO)
Compound
Compounds
10
17
12
22
16
14
18
26
21
13
10
17
1.4
23
15
2.25
24
49
0.18
Cl (L/Kg)
AUCNpo
(µM h Kg/mg)
t1/2 (h)
Cl (L/Kg)
AUCNpo
1.4
1.0
1.8
1.8
2.0
0.85
65
0.75
98
0.49
12
(µM*h*kg/mg)
t1/2 (h)
Fpo (%)
0.85 1.05
65 57
1.46 0.97
69 100
1.64
67
In the 6-fluoro-2-methyl-benzonitrile series, the pyridine
regioisomer of the (R,S) and (S,S) bicyclic series (analogs 25 and
28), the pyrimidine analog (27), and the pyridazine analog (30)
showed similar ROMK/hERG selectivity (25 278-fold, 28 284-
fold, 27 303-fold, 30, 392-fold, Table 8). The pyrimidine analog
29 had the best selectivity for ROMK.
Fpo (%)
Having optimized the left side of the scaffold, we next
explored the right side. Small molecule inhibition of the hERG
channel is often associated with π-stacking and hydrophobic
interactions between inhibitors and aromatic residues in the
hERG cavity.15 Accordingly, we tried to change the position of
the nitrogen within the the phenyl ring or incorporate an
additional nitrogen to see if this would modulate hERG activity.
We found that in the isochromanone series (Table 6), compound
10 had the best in vitro profile (potency and hERG selectivity).
The PK profiles of analogs 23 and 24 had no obvious advantage
over 10 (Table 7).
Table 8:SAR of isosteres
*Control compound used in all assays; STD < 20%
ROMK*
EP IC50
(µM)
hERG*
EP
hERG EP/
ROMK EP
Ar =
Bicyclic=
#
IC50 (µM) (fold)
Table 6: SAR of isosteres
ROMK*
EP IC50 EP IC50 EP/ROMK
hERG*
hERG
25
0.073
0.018
0.033
0.070
0.039
0.086
0.107
20.3
5.9
278
328
~303
284
646
392
115
Bicyclic=
Ar =
#
(R,S)
(µM)
(µM)
EP (fold)
26
27
28
29
30
10
22
0.057
75.9
1,332
(R,S)
(R,S)
(S,S)
(S,S)
(S,S)
(R,S)
(R,S)
~10
19.9
25.2
33.7
12.3
0.080
>30
>375
23
24
0.165
0.260
41.5
>90
252
(S,R)
(R,S)
>346
*Control compoud used in all assays; STD < 20%
31
(S,S)
Compounds with good ROMK potency, hERG EP (IC50 >10
Compounds with good ROMK potency, hERG EP (IC50 >10 µM)

monkeys, (by applying allometric scaling) was estimated to be 24
hr (with some uncertainty given the significantly longer dog half-
life, despite similar metabolic profiles between species). This is a
significantly longer projected half-life than the most commonly
used loop diuretics, furosemide (human half-life = 0.5-2 hr) and
torsemide (human half-life = 3 hr).16 The enhanced half-life may
provide a PK-PD advantage with regard to the peak diuretic
effects associated with loop diuretics, by providing a reduction in
peak-to-trough exposures.
and ROMK/hERG selectivity (>200) were further examined to
determine their pK profiles in SD rats (Table 9). A general trend
towards increased half-life was observed in the (R,S) bicyclic
series when the nitrogen was positioned adjacent to the acetamide
linker (25) or when the number of nitrogens in the ring was
increased (27). Although compound 27 had a slightly better PK
profile with a half-life in SD rats of 1.4 hr and good oral
bioavailability, the potency in hERG EP (IC50 ~10 µM) made it
less favorable than compound 25 (hERG EP IC50 20.3 µM,
ROMK/hERG selectivity 278-fold). An opposite effect was
observed in the (S,S) bicyclic series (16, 28-29). Although
compound 29 had the largest selectivity ratio in the 6-fluoro-2-
methyl-benzonitrile series, the relative shorter half-life made it
less attractive than 25.
Table 10
PK properties of 25 in dog and rhesus monkey
Species
Dose
IV
Cl
AUCNpo
t1/2
Fpo
Table 9SD or Wistar rat PK properties selected ROMK
inhibitors
(L/Kg) (µM h Kg/mg)
(h) (%)
(PO)
Compounds
Dog (Beagle) 1 (2)
Rhesus 1 (2)
2.5
4.0
16.7
6.0
17.3 100
12
25
1/2
27
16
28
29
5.0
67
Dose iv/po
(mg/kg)
0.5/1
0.5/1
0.5/1
0.5/1
1/2
To assess its pharmacodynamic effects, 25 was dosed orally
QD for 3 days in spontaneously hypertensive rats (SHR) at 3, 10
and 30 mg/kg (Table 11).17 Compound 25 demonstrated better
efficacy than HCTZ for 24 hour diuresis at day 1, and a
comparable blood pressure lowering at day 3 at all three doses.
Cl (L/Kg)
AUCNpo
22
21
12.4
4.64
14
20.7
19.7
1.01
1.0
0.79
1.80
(µM h
Kg/mg)
t1/2 (h)
1.05
57
1.5
46
1.4
1.46
69
0.96
68
1.36
54
Table 11
Diuresis and blood pressure lowering of 25 in SHR
Fpo (%)
~100
Compound
25
(3)
1.4
25
(10)
1.6
25
(30)
1.8
HCTZ
(25 )
Given its favorable rat PK profile, potent ROMK
activity and good selectivity over hERG, compound
25 was selected for further evaluation in functional
assays for other related inward rectifying potassium
channels (Kir2.1, Kir4.1 and Kir7.1, all IC50 >100
µM) as well as the cardiac channels Nav1.5 and
Cav1.2 (both with IC50 > 30 µM) and was found to
have excellent selectivity for ROMK over other ion
channels. Selectivity of 25 was further evaluated in
a panel of 115 off-target enzyme and radioligand
binding assays at Ricerca Biosciences, LLC. The
sole finding with >50% inhibition at 10 µM was the
somatostatin sub-type 2 receptor, with an IC50 of
4.1 µM (~60-fold in vitro selectivity for ROMK).
Compound 25 was also evaluated for inhibition of a
panel of CYP’s (3A4, 2D6, 2C9) and found to have
no significant inhibition at the concentrations tested
(IC50’s >50 µM). Microsome stability (% parent @
45 min) for 25 was determined to be 85% in rat,
82% in dog, 72% in rhesus and 89% in human.
Hepatocyte stability (%parent @ 90 min) for 25
was determined to be 92% in rhesus and 99% in
human, indicating minimal in vitro metabolism.
Plasma protein binding (% free fraction) for 25 was
determined to be 48% in rat, 52% in dog, 60% in
rhesus, and 58% in human plasma. Compound 25
had excellent permeability in LLC-PK1 cells (Papp
28). Inhibitor 25 was selected for further PK profiling in two
additional species (dog and rhesus monkey; Table 10). The
compound had PK profiles across preclinical species consistent
with oral QD dosing. In addition, the human half-life projection
of 25 based on observed preclinical PK in rat, dog and rhesus
(Dose, mg/kg)
Diuresis fold-
increase*24 hr
1.05
δSBP (mmHg)*
(day 3)
-9
-16
-16
-12
*Compared to vehicle; n = 5 per group, on day 3 for blood pressure lowering
and day 1 for diuresis
To determine how the in vitro hERG selectivity translated in
vivo, compound 25 was further evaluated in an anesthetized
cardiovascular guinea pig (GP) model (n = 3).18 No significant
change in the QTc interval was observed following IV infusion
of 25, with
a maximal average peak unbound plasma
concentration of 83 µM.
In conclusion, following the discovery of compound 3 and the
initial preclinical validation of small molecule ROMK inhibitors
as a novel diuretic agents in rats, we set out to discover new
ROMK inhibitors with comparable ROMK potency, low risk for
QTc effects, and improved pharmacokinetic properties. The
most promising candidate to emerge from this work, 25, had
significantly improved half-life in three preclinical species, with
comparable ROMK potency and an improved ROMK/hERG
selectivity ratio over previously reported compound 3. In vivo
evaluation of 25 demonstrated a blood pressure lowering effect
comparable to HCTZ at a lower dose (10 mpk vs. 25 mpk of
HCTZ) and no detectable QTc effects when evaluated in an in
vivo cardiovascular GP model. Future development of this series
of ROMK inhibitors will be the subject of subsequent
publications.

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