Discovery of small molecule renal outer medullary potassium (ROMK

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Perspective

Discovery of small molecule renal outer medullary potassium (ROMK) channel inhibitors: A brief history of medicinal chemistry approaches to develop novel diuretic therapeutics Christopher D Aretz, Anish K Vadukoot, and Corey R. Hopkins J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 29 Apr 2019 Downloaded from http://pubs.acs.org on April 29, 2019

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Journal of Medicinal Chemistry

Discovery of small molecule renal outer medullary potassium (ROMK) channel inhibitors: A brief history of medicinal chemistry approaches to develop novel diuretic therapeutics Christopher D. Aretz, Anish K. Vadukoot, Corey R. Hopkins* Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, Omaha, NE 68198

Keywords: Inwardly-rectifying potassium channel, Kir, KCNJ, diuretics, hypertension ABSTRACT: The renal outer medullary potassium (ROMK) channel is a member of the inwardly-rectifying family of potassium (Kir, Kir1.1) channels. It is primarily expressed in two regions of the kidney, the cortical collecting duct (CCD) and the thick ascending loop of Henle (TALH).

At the CCD it tightly regulates potassium secretion while controlling potassium

recycling in TALH. As loss-of-function mutations lead to salt wasting and low blood pressure, it has been surmised that inhibitors of ROMK would represent a target for new and improved diuretics for the treatment of hypertension and heart failure. In this Perspective, we discuss and provide an overview of the medicinal chemistry approaches towards the development of small molecule ROMK inhibitors over the last decade.

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1. INTRODUCTION 1.1

Kir Channels. Inward rectifier potassium (Kir) channels belong to a large ‘superfamily’

of K+ ion channels comprised of the voltage-gated, two-pore, calcium-gated, and cyclic nucleotide-gated channels.1,2 Kir channels are widely expressed on excitable and non-excitable cell membranes and serve important roles in cell excitability and K+ homeostasis. There are seven structurally different sub-families of the Kir family identified in mammals,3 which are further divided into four functional groups: “classical” Kir channels (Kir2.x) that are constitutively active, G protein-gated Kir channels (Kir3.x) which are regulated by G protein-coupled receptors, ATPsensitive K+ channels (Kir6.x) which are tightly linked to cellular metabolism, and K+ transport channels (Kir1.x, Kir4.x, Kir5.x, and Kir7.x).4 The basic topology of the Kir family is conserved and contains four sub-units which are either homo- or heterotetramers.5 The tetramers form a canonical pore forming transmembrane domain that is selective for the movement of K+ ions.3 Each subunit is composed of two transmembrane helices (M1 and M2, also called slide-helices), the pore forming region containing the pore-helix (P), and a large cytoplasmic domain formed by the amino (N) and carboxy (C) termini (Figure 1).6 The slide-helices are believed to interact at the bilayer interface, while the large cytoplasmic domain extends the ion conduction pathway, providing docking sites for ions, proteins, and ligands.7 Interactions between the cytoplasmic domain and slide-helices are believed to be essential for the mechanical transduction of ligand binding to channel gating.8 1.2

ROMK channels. The renal outer medullary potassium (ROMK) channel is a member of

the inwardly rectifying family of potassium (Kir, Kir1.1) channels. ROMK is encoded by the KCNJ1 gene and is primarily expressed in two regions of the kidney, namely the cortical collecting duct (CCD) and the thick ascending loop of Henle (TALH).4,9 At the CCD, ROMK plays a role in 2 ACS Paragon Plus Environment

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potassium secretion, which is closely attached to the sodium uptake through the amiloridesensitive epithelial sodium channel.10 At the TALH, ROMK enables potassium recycling across the luminal membrane, a requirement for effective functioning of the furosemide-sensitive Na+/K+/2Cl- channel.11 Potassium efflux through ROMK augments the role of transporters and sodium channels, which are known targets of diuretics.12 ROMK is a homo-tetrameric membrane protein consisting of a modest extracellular domain and large cytoplasmic domain. ROMK in the plasma membrane is tightly regulated by protein tyrosine kinase (PTK), with-no-lysine-kinase 4, and serum and glucorticoid-induced kinase (SGK) respectively.13 The effect of low potassium (K+) intake on ROMK channel activity is mediated through PTK. Low dietary K+ intake induces an increase in superoxide anions, which stimulate PTK expression and tyrosine phosphorylation of ROMK channels. In addition, a recent study has shown regulation of the surface expression of ROMK channels through monoubiquitination.14 Cytosolic pH plays a critical role in the regulation of ROMK, with low pH inducing channel closure, while channel opening is favored at higher pH.15 Site specific mutations in amino and carboxy terminal domains has indicated a pH dependent modulation of the channel.16 Genetic studies on human and rodent species have provided evidence supporting ROMK as a target for novel diuretics for the treatment of hypertension and heart failure.17 Human Kir1.1 channel with loss-of-function mutations is associated with severe salt wasting, metabolic alkalosis, and hypokalemia, which are typical of antenatal Bartter's syndrome type II. Carriers of the heterozygous Kir1.1 mutations associated with antenatal Bartter's syndrome have been shown to have reduced blood pressure and a decreased risk of developing hypertension.18 Thus, it is hypothesized that pharmacological inhibition of ROMK would have the benefit of



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diuretic/natriuretic efficacy without the side-effects associated with other clinically used diuretics (e.g., dose-limiting hypokalemia).19

Figure 1: Schematic representation of Kir channel subunit: Two transmembrane helices (M1 and M2), pore-helix (P) and large cytoplasmic domain formed by the amino (N) and carboxy (C) termini.6

2. ROMK inhibitors: 2.1

Tertiapin.

Tertiapin, the first reported inhibitor, of ROMK was disclosed by Jin and

Lu in 199820 and is a small protein isolated from the venom of the honeybee. Tertiapin contains 21 amino acid residues (Figure 2) and has a highly compact structure with a high density of positively charged residues. The native tertiapin was isolated from honeybee venom and, in addition, was synthesized and both variants had identical chromatographic behaviors and were screened against ROMK and other K+ channels with comparable activities.

Tertiapin was

determined to be a very potent ROMK inhibitor with a dissociation constant of 2.0 nM; but, it was also found to be active against G-protein-gated inward rectifier K+ channel (GIRK) 1/4 with a Ki = 8.2 nM. Tertiapin is a potent, non-selective inhibitor of both ROMK and GIRK 1/4 channel.


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Following these initial studies, Felix and coworkers were able to develop tertiapin into a ROMK molecular probe.21 The key mutations were methionine-13 to glutamine and histidine-12 to lysine, which this latter substitution removed the pH dependence. Since the N-terminal region projects into the extracellular media when interacting with ROMK, it provided a physical location to attach a probe. Tertiapin was further mutated with a tyrosine-1 mutation (Figure 2), which allowed the peptide to be radiolabeled with

125I.

This mutated peptide was determined to be

biologically similar to the native peptide and the mutated peptide was further utilized as a probe for ROMK assays. In addition, tertiapin-Q, where methionine-13 is mutated to glutamine, was developed with improved oxidative stability, a known liability of tertiapin.22

Native Protein: Ala-Leu-Cys-Asn-Cys-Asn-Arg-Ile-Ile-Ile-Pro-His-Met-Cys-Trp-Lys-Lys-Cys-GlyLys-Lys Mutated Protein: Tyr-Leu-Cys-Asn-Cys-Asn-Arg-Ile-Ile-Ile-Pro-Lys-Gln-Cys-Trp-Lys-Lys-CysGly-Lys-Lys Figure 2: Amino acid sequence for tertiapin and the mutated teriapin.

2.2

Nitrophenyl inhibitors.

Although the peptide tertiapin-Q had been discovered, small

molecule discovery was progressing with groups at Vanderbilt University and Merck Research Laboratories leading the major drug discovery efforts. In 2009, Denton and coworkers developed a ROMK Tl+ flux assay,23 which they used to perform a high throughput screen (HTS) of roughly 126,000 small organic compounds and identified VU590, 1, which had an IC50 = 294 nM (Figure 3).23 The group attempted a structure activity relationship (SAR) campaign on the compound 1



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scaffold with several different aryl substitutions attempted to replace the aryl nitro groups; however, only a few were active against ROMK and none of these analogs had a sub-micromolar potency against ROMK.23 At a concentration of 10 µM, 1 had no significant effect on either Kir2.1 or Kir4.1, however, 1 had modest activity against Kir7.1 (69% inhibition). Despite the limited SAR, 1 was the first small molecule inhibitor of both ROMK and Kir7.1. O2N O N

N O

O

NO2

VU590, 1 IC50 = 294 nM

Figure 3. First small molecule inhibitor of ROMK, VU590, 1.

Unfortunately, both ROMK and Kir7.1 are expressed in the nephron, and since 1 does not possess selectivity against Kir7.1, it is not a suitable ROMK probe molecule. Denton and coworkers, in 2011, expanded upon compound 124 by starting with a structurally related hit from the original HTS, BNBI, 2, which was screened against ROMK using the established Tl+ flux assay (Figure 4). 2 had modest activity against ROMK (IC50 = 8 M); however, hen screened against Kir7.1, 2 had no significant effect even up to concentrations of 100 µM.24 Both 1 and 2 contain two aryl nitro benzimidazole groups tethered together by a three-atom linker. The key difference in the tethers is the absence of a central oxygen atom in 2. Introduction of the central oxygen in the linker, 3, was a key addition as this analog proved to be almost 30-fold more potent than 2, with an IC50 = 240 nM.


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O2N N

NO2

HN

N H

N BNBI, 2 IC50 = 8 M

O2N N

O

N H

NO2

HN N

VU591, 3 IC50 = 240 nM

Figure 4. Second generation ROMK inhibitors.

The selectivity of 3 was measured against a variety of inwardly rectified and voltage gated K+ channels and it was shown to have no effect on Kir7.1 at 10 µM, which was an improvement compared to 1. In addition, 3 showed no inhibition against Kir2.1, Kir2.3, and Kir4.1 at either 10 or 50 µM, and 3 only inhibited Kir6.2/SUR1 currents by 17 and 28%, respectively, which is an 150-fold improvement compared to previous compounds against Kir6.2/SUR1. Compound 3 was screened against a panel of potential off-targets, including cardiac and central nervous system ion channels and receptors, and only four targets exhibited greater than 50% radioligand displacement at 10 μM. These four targets were –aminobutyric acid (GABAA) receptor, dopamine D4 receptor, dopamine transporter, and norepinephrine transporter, and further profiling of 3 revealed that it only had activity against the GABAA receptor (IC50 = 6.2 μM), which provides a 25-fold selectivity for ROMK.24 The protein binding and metabolic stability of 3 was measured in vitro and the plasma protein binding in both human and rat plasma was compared with the anticoagulant warfarin and verapamil as controls (Table 1).24 It was determined that 3 is greater than 99% protein-bound in 7 ACS Paragon Plus Environment


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human plasma, but approximately 2% unbound in rat plasma. The metabolic stability of 3 and verapamil in human and rat liver microsomes was assessed and after 15 minutes of incubation, 60% of 3 remained in rat liver microsome and 80% of 3 remained in human liver microsomes.

Table 1: Percentage of 3, Warfain (control), and Verapamil (control) bound to human and rat serum protein and % remaining in rat and human microsomes Cmpd

Human Protein (% bound)

Rat Protein (% bound)

3 Warfarin Verapamil

99.14 ± 0.006 99.62 ± 0.002 93.41 ± 0.009

98.39 ± 0.001 99.57 ± 0003 97.14 ± 0.004

Rat Liver Microsomes (% remaining) 60 ND 24

Human Liver Microsomes (% remaining) 80 ND 28

Denton and coworkers further evaluated 3 by performing in silico docking in the binding site of ROMK25 which identified potential amino acids interacting with 3. Site directed mutagenesis of ROMK and Kir2.1 was performed to gain some understanding of the binding of 3 to ROMK and the selectivity against Kir2.1. When arginine 171 (N171) in ROMK was changed to a negatively charged residue, specifically aspartic acid or glutamic acid, the mutant channel was found to be insensitive to inhibition by 3.25 Previously, Robertson and coworkers proposed that neutralization of charged residues in the Kir channel pore would influence the electrostatic potential along the pore.6 This change could affect the interaction with K+ ions as well as charged pore blockers, and therefore, it is possible that the changes could be affecting the interaction of 3 with the pore in a similar mode. Denton and coworkers proposed that the binding site of 3 is located near N171 because in silico docking studies identified an energetically favorable interaction between 3 and N171.25 When the side chain size was increased by the addition of two methyl groups at position 171, without inducing a charge, there was a 5.5-fold loss of selectivity in the ROMK-N171Q mutant 8 ACS Paragon Plus Environment

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for 3 (versus ROMK-WT). When an asparagine residue was added to Kir2.1 at position 172, it increased sensitivity to 3 by three-fold. Further substitutions or modifications to smaller hydrophobic residues of the adjacent pore-lining residues resulted in loss of 3 sensitivity. Lastly, the double mutation, V168A/N171Q, resulted in a greater loss of 3 sensitivity when compared to the N171Q modification alone. Additional mutations in the pore of ROMK failed to reduce the sensitivity towards 3 which supported their computational modeling results that indicated 3 interacts with both residues concurrently.25 2.3

Piperazine-containing inhibitors. Merck Research Laboratories has also actively

pursued small molecule inhibitors for ROMK. Tang and co-workers reported a novel class of selective small molecule ROMK inhibitors starting from an HTS of their internal sample collection.11 The initial hit molecule, 4, (Figure 5) showed moderate potency towards ROMK (IC50 = 5.2 µM) in a functional 86Rb+ efflux assay but was found to be selective against cardiac Kir2.1 (IC50 > 100 µM) and kidney Kir2.3 (IC50 > 100 µM). Upon further purification by HPLC, 4 lost activity towards ROMK, but led to the isolation of a minor impurity, 5, which showed good ROMK inhibitory activity (IC50 = 0.052 µM) and selectivity over Kir2.1 and Kir2.3 channels (IC50 > 100 µM). Unfortunately, it was ~10-fold more potent on human ether-a-go-go-related gene (hERG) channel (IC50 = 0.005 µM) - a rapidly activating delayed rectifier potassium channel important for cardiac repolarization.11,26 Exploratory chemistry around 5, which included replacement of nitro groups with bioisoteres, phthalide groups, modification of linker lengths, introduction of methyl substitution around core piperazine ring was initiated. This subsequently led to the identification of multiple active moieties which underwent further chemical modifications to improve the selectivity over the hERG channel. Compound 6, identified through SAR, maintained activity against ROMK, but had approximately 3.5-fold selectivity over hERG channel and showed good 9 ACS Paragon Plus Environment


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rat bioavailability (%F = 64). Compared to 5, 6 displayed lower total clearance and a reasonable oral exposure in rat, however, with comparable half-life (T1/2) (Table 2). Hence, 6 was used as a small molecule probe for the evaluation of ROMK inhibitors in animal models of diuresis and hypertension.11

Figure 5. HTS hit-to-lead development of ROMK inhibitor.

Table 2. Rat PK properties of compounds 5 and 6. Properties CL (mL/min/kg) AUCNpo (µM∙h∙kg/mg) T1/2 (h)

5 261 0.023 1.9

6 59 0.46 1.5 10

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Fpo (%)

14

64

In a separate SAR study, the core scaffold 5 was developed into a separate series of potent and selective small molecule ROMK inhibitors by replacement of the first nitro group with a phthalide group, 7 (as in 6) and then the right-hand nitro group was replaced with 4-(1H-tetrazol1-yl)phenyl pharmacophore, 8 (Figure 6).27 Combining the phthalide and tetrazole along with an amide functionality in order to reduce HERG liabilities, by modulating the basicity of the nitrogen group, resulted in analogs 9 and 10. These two compounds displayed good potency on the ROMK channel, improved HERG selectivity, but the rat PK still remained problematic (short T1/2, high CL) (Table 3).27



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Figure 6. Discovery of phenyl tetrazole amide series as potent ROMK inhibitors. Table 3. Rat PK properties for compounds 9 and 10: Properties CL (mL/min/kg) AUCNpo (µM∙h∙kg/mg) T1/2 (h) Fpo (%)

9 40 0.34 0.62 33

10 43 0.48 0.50 52

The Merck team also published multiple patents reporting the synthesis and evaluation of a novel class of fused, bicyclic piperazine based inhibitors, 11-16 (Figure 7).28-31 These fused bicyclic piperazine scaffolds would reduce basicity, based off calculated pKa, and increase rigidity of the compounds.32 This steric and electronic modulation was theorized to result in compounds being less susceptible to oxidative metabolism. The analogs with the octahydropyrazino[2,1c][1,4]oxazine core, e.g., 13, had similar or improved ROMK potency, when compared to 9. Unfortunately, the octahydro-2H-pyrazino[1,2-a]pyrazine, 11, and octahydropyrazino[2,1c][1,4]thiazine, 12, analogs resulted in a decrease in ROMK potency. Each of the diastereomers of the octahydropyrazino[2,1-c][1,4]oxazine, 13, core had similar ROMK potency with the R,S and R,R diastereomers possessing the most favorable properties; good in vitro ROMK potency and selectivity, oral bioavailability, reduced clearance rates, and higher exposures when compared to 9 (Figure 7). Replacement of phthalide functional group with previously established pharmacophores, resulted in several different analogs. Two of these analogs, 14 and 15, had improved ROMK selectivity over hERG when compared to (R,S)-13,32 with 14 having the best ROMK selectivity of the series, 75.9-fold selectivity for ROMK over hERG. The second analog series, the 2-methylbenzonitrile, e.g., 15, was explored with the addition of a fluorine and inclusion of various heteroaromatic ring linkers between the acetamide and tetrazole groups. Of the heteroaromatic 12 ACS Paragon Plus Environment

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ring linkers explored (pyridines, pyrimidine, and pyridazine) 16, with a pyridine linker, had a favorable rat PK profile, potent ROMK activity, and good selectivity over hERG. O

O

O

O

N

N

X

N O 9

N

N O

N

N N

ROMK IC50 = 0.11 M hERG IC50 = 14 M

N

X = N, 11 X = S, 12

N

N N

O O

O

H

O

N N N

O

N

O H O

13 ROMK IC50 = 0.065-0.105 M hERG IC50 = 21.1-59.4 M

N

N

O

N

14 ROMK IC50 = 0.057 M hERG IC50 = 75.9 M

N N

N

N N

F H H

H

N

O H

N

O

N

NC

N

NC

O

15 ROMK IC50 = 0.060 M hERG IC50 = 14.8 M

N

N

N N

N O

16 ROMK IC50 = 0.073 M hERG IC50 = 20.3 M

N

N

N N

Figure 7. Discovery of morpholine-fused bicyclic ROMK inhibitors Compound 16 was evaluated in functional assays against other ion channels, and was determined to be highly selective for ROMK. Against other inward rectifying potassium channels, Kir2.1, Kir4.1, and Kir7.1, the IC50 was greater than 100 µM and it was also tested against cardiac



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channels Nav1.5 and Cav1.2 (IC50 >30 M). In a panel of 115 off-target enzyme and radioligand binding assay, only somatostatin sub-type 2 receptor (IC50 = 4.1 µM) showed any activity, which provides roughly a 60-fold in vitro selectivity for ROMK. No measurable CYP (3A4, 2D6, 2C9) inhibition by 16 was observed and liver microsome and hepatocyte stability and protein plasma binding for 16 was measured across multiple different species (Table 4). The in vivo PK profile of 16 was evaluated across preclinical species and was found to be consistent with oral QD dosing (Table 5).32

Table 4. Microsome stability, hepatocyte stability, and protein plasma binding for 16. Properties Microsome Stability (%parent 45 min.) Hepatocyte Stability (%parent @ 90 min.) Protein Plasma Binding (% free fraction)

Rat

Dog

Rhesus

Human

85

82

72

89

-

-

92

99

48

52

60

58

Table 5. PK properties of 16 in rat, dog, and rhesus Properties Dose. iv/po (mg/kg) Cl (mL/min/kg) AUCNpo (µM∙h∙kg/mg) t1/2 (h) Fpo (%)

Rat 1/2 21 0.79 1.5 46

Dog (Beagle) 1/2 2.5 16.7 17.3 100

Rhesus 1/2 4.0 6.0 5.0 67

In a parallel approach, Walsh and co-workers at Merck Research Laboratories reported the development of a piperazinyl diol series with superior potency, selectivity and PK properties as ROMK inhibitors.33 Following the SAR studies on their previously reported 7, they replaced the right-hand nitro group with a 3-methoxy-4-cyanophenyl moiety, generating 17, a similarly potent ROMK inhibitor, and, with a promising 18-fold selectivity over hERG (Figure 8). SAR studies 14 ACS Paragon Plus Environment

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around the phthalide ring of 17 led to the identification of 18, which represented a potent ROMK inhibitor with 60-fold selectivity over the hERG channel. Unfortunately, compound 18 displayed high total clearance and low oral exposures with moderate half-life (Table 6).33 For the carbon adjacent to the phthalide phenyl ring of 18, methyl and fluorine substitution led to a modest loss of ROMK potency; however, there was a moderate improvement on hERG selectivity (Figure 8). Hydroxy or methoxy substitution resulted in a modest loss of ROMK potency, but led to a significant improvement in the hERG selectivity margin (>150-fold). Modification of 18 with the addition of a hydroxy group at the benzylic position and inclusion of a heteroatom into the righthand ring (e.g., 19), maintained the potency and selectivity comparable to compound 18. Guided by their selectivity improvement towards hERG through heteroatom introduction, the pyridine analogue, 19, maintained ROMK potency while significantly improving selectivity against hERG (130-fold). Compound 19 possessed a total clearance of 40 mL/min/kg and oral bioavailability in rat was improved (Table 6).33



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Figure 8. Development of piperazinyl diol series of ROMK inhibitors. Table 6. Rat PK properties of compounds 18 and 19: Properties CL (mL/min/kg) AUCNpo (µM∙h∙kg/mg) T1/2 (h) Fpo (%)

18 72 0.12 1.2 21

19 40 0.74 1.5 78

Compound 19 was further optimized by the addition of a second hydroxy group, yielding 20.33 As the active analogues contained the (R,S)-isomer, further analogues were designed to retain the same stereochemistry. In general, substitutions, methoxy for example, at the para- or meta16 ACS Paragon Plus Environment

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positions with respect to the pyridine nitrogen in 19 resulted in an improvement of ROMK potency, while ortho- substitutions led to a decrease in ROMK potency, with the notable exception of a methyl group. Deletion of the pyridyl nitrogen led to decrease in overall selectivity towards hERG channel although it maintained ROMK potency. Compound 20 is a potent and selective ROMK inhibitor with 531-fold selectivity over the hERG channel and was selective against several other inward rectifying potassium channels (Kir2.3, Kir2.1; IC50 > 50 µM; Kir4.1, Kir2.3 Kir7.1; IC50 > 10 µM). In addition, the in vivo PK profile of 20 was determined in rat, dog and rhesus monkey and was found to be consistent with an oral QD therapeutic across preclinical species (Table 7).33

Table 7. PK properties of Compound 20 Properties Dose IV/PO (mpk) CL (mL/min/kg) AUCNpo (µM∙h∙kg/mg) T1/2 (h) Fpo (%)

20 Rat 1/2 31 0.84 1.6 65

Dog 0.5/1 5.6 5.6 5.9 84

Rhesus 1/2 25 0.51 7.2 30

Tang and coworkers describe the development of a subseries of novel ROMK inhibitors by merging SAR data from their previously published piperazine carboxamide and piperazine diamine series (Figure 6).34 Initial SAR studies on the phthalide phenyl ring of 9 revealed substitution at the 4-position of the phthalide, for example a methyl group, is well tolerated, 21 (Figure 9). A significant improvement in hERG selectivity (>1000-fold) was observed when a fluorine is introduced at the benzylic position, 22, presumably by modulation of the nitrogen basicity. Unfortunately, 22 displayed poor rat PK properties (high CL, short T1/2) (Table 7).



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O

O

O

O

N

N

N

N O 9 ROMK IC50 = 0.11 µM hERG IC50 = 14 µM hERG/ROMK = 127

O 21 ROMK IC50 = 0.19 µM hERG IC50 = 20 µM hERG/ROMK = 100

N N N N

N N N N

O O N F

N O

22 ROMK IC50 = 0.069 µM hERG IC50 = 68 µM hERG/ROMK = 1000

N N N N

Figure 9. hERG modulation in ROMK inhibitors. Table 7. Rat PK data of 22 Rat PK: 1 mpk IV, 2 mpk PO Cl Vdss AUCN po t1/2 F% po

58 mL/min/kg 1.4 L/Kg 0.23 μM*h 0.35 h 36

Further SAR studies on the symmetrical compound, 5, was initiated. Replacement of both aryl nitro groups with the phthalide moieties (e.g., 23) showed improved selectivity against hERG (Figure 10).34 Incorporation of methyl groups to both of the phthalide group, 24, led to even greater ROMK selectivity, ROMK/hERG ratio of 39. Greater improvements in ROMK/hERG ratio were obtained by the addition of a hydroxy group, 25. The symmetric diol, 26, was active; however, the enantiomerically pure 27 displayed remarkable potency and selectivity towards ROMK (IC50 = 9 nM; selectivity = 2440). The three stereoisomers (R,R, S,S and R,S) showed similar potency


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and selectivity towards hERG channel and in vivo rat PK studies of these isomers displayed similar oral exposure and half-lives. Compound 27 was further subjected to PK profiling in higher species (Table 8) and it displayed significant improvement in half-life in rhesus when compared to the dog. Among the three stereoisomers, 27 (R,R-isomer) displayed remarkable diuresis and naturiuesis when compared to the S,S-stereoisomer (Table 9). 27 was also selective towards other members of the Kir family (Kir2.3, Kir2.1, Kir4.1, Kir2.3 Kir7.1; IC50 > 30 µM) and showed good selectivity towards other cardiac ion channels (Cav1.0, Nav1.5; IC50 > 30 µM). In summary 27, renamed as MK-7145, demonstrated improved hERG selectivity, dose dependent lowering of blood pressure and was selected as the first small molecule ROMK inhibitor to enter clinical development.35 MK-7145 entered into three separate clinical trials looking at effects on blood pressure in male patients with hypertension (NCT01370655), pharmacokinetics following single dose in patients with moderate renal insufficiency (NCT01832103), and safety and tolerability study in patients with renal insufficiency and heart failure with renal insufficiency (NCT01558674).36 Unfortunately, second and third trials have been withdrawn and terminated, respectively. The first trial (NCT01370655) has been completed, however, there has been no communication from Merck regarding the results or further progression of this compound.



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O O 2N

O N

N

N

N

O

5 ROMK IC50 = 0.052 µM hERG IC50 = 0.005 µM hERG/ROMK = 0.1

NO2

O

23 ROMK IC50 = 0.089 µM hERG IC50 = 1.9 µM hERG/ROMK = 21 O

O

O

O

N

N

OH N

N

O

O 24 ROMK IC50 = 0.076 µM hERG IC50 = 3.0 µM hERG/ROMK = 39

25 ROMK IC50 = 0.034 µM hERG IC50 = 5.2 µM hERG/ROMK = 153

O

O

O

O

O

O N OH

N

OH N

OH

OH N

O 26 ROMK IC50 = 0.045 µM hERG IC50 = 23 µM hERG/ROMK = 510

O

O

27 (MK-7145) ROMK IC50 = 0.009 µM hERG IC50 = 22 µM hERG/ROMK = 2440

O

Figure 10. Discovery of the clinical candidate, MK-7145, 27. Table 8. PK properties of compound 27. Properties

27 Dose: 1 mpk IV, 2 mpk PO Rat Dog 56 56 3.8 7.5 0.14 0.32 1.1 1.7 21 51

Cl (mL/min/kg) Vdss (L/kg) AUCNpo (µM∙h∙kg/mg) T1/2 (h) Fpo (%)

Rhesus 29 6.7 0.30 4.0 25

Table 9. Acute (4 h) Diuresis and Natriuresis of 27 in SD rat after oral dosing Dose

Diuresis fold increase

0.3 mg/kg

6.4

Natriuresis fold increase 6.2

Kaliuresis fold increase 2.1 20

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1 mg/kg 3 mg/kg 10 mg/kg

8.6 9.0 9.4

8.6 8.0 8.5

1.8 1.4 1.7

Bristol-Myers Squibb published a series of patents covering their entries into the ROMK field in 2017 and 2018. The compounds contained the familiar phthalide group as seen in the previous Merck compounds; however, they significantly modified the central portion of the molecule (Figure 11). The group utilized a 5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-2-yl) core scaffold, 28-29, with a pendant indole or indazole group to produce potent ROMK inhibitors (IC50 = 28 and 38 nM, respectively).37 Next, the group has reported on acyclic analogs (replacing the piperazine with a secondary amine, 30-31) with pendant five-membered heterocycles, which also produced very potent compounds ( 40 M MW = 395/LogD = 3.0/LipE = 2.2 HLM Clint,app,s =120 mL/min/kg Papp = 15x10-6 cm/s

43

Figure 14. Initial HTS hit by Pfizer.

An SAR campaign was conducted with the focus on reducing the lipophilicity of 43 (LipE = 2.2). Various heterocycles were evaluated in the R1 amide region (Figure 15)46 and fivemembered ring systems, e.g., 3-methyl-1,2,4-oxadiazole, 44, which had a 15-fold increase in potency against ROMK (Tl+ flux IC50 = 440 nM) were the best. This compound had an improved metabolic stability relative to 43 (44, CL = 30 mL/min/kg) and maintained excellent selectivity over hERG (Ki > 40 μM and IC50 > 100 μM). The LipE was also improved when compared to 43 (LipE 3.7).47 There was some tolerance for R3 substitution of the core benzamide ring. When the methyl group was exchanged for a methoxy group, 45, the potency was decreased (IC50 = 1.3 μM) but had a comparable LipE (3.7) to 44 and had improved metabolic stability with a CL of 10 mL/min/kg. The presence of a 4-substitution, R3, is critical for ROMK potency as the 4unsubstituted (R3 = H, IC50 = 17 M) was more than 30-fold less potent than 44.



Page 28 of 41

Further variation of R2 substitutions with improved ROMK potency and LipE were identified, 46 and 47. Compound 46 showed a 5-fold increase in ROMK potency in Tl+ flux assays when compared to 44 and 47 was even more potent; however, the metabolic stability was significantly worse.46 Attempts to replace the sulfonamide moiety were unsuccessful. Further optimization of the R1 amide moiety led to the identification of several triazoles, 48 and (-)-49, with balanced in vitro metabolic stability, passive permeability, and ROMK potency profiles. Compound 48 had similar ROMK potency as 46 but was more stable in HLM incubations than the oxadiazoles (CL < 9 mL/min/kg). Lastly, (-)-49 was identified as a potent ROMK inhibitor with low HLM turnover and a good passive permeability. Interestingly, its enantiomer, (+)-49, was 80-fold less potent than (-)-49. Compound (-)-49 did have a weak affinity for hERG (Ki = 28 μM); however, the selectivity for ROMK over hERG was acceptable, and the incorporation of the α-methyl group led to improved aqueous solubility.46 Based on the overall properties, (-)-49 was selected for further analysis by patch-clamp experiment and it was shown to be potent inhibitor of human ROMK (IC50 = 160 nM); however and surprisingly, it lacked inhibitory activity against rat ROMK channel. This result was surprising because of the greater than 92% homology of human and rat ROMK and the similar potency for both human and rat ROMK in previously reported inhibitors. This species disconnect was maintained when these compounds were tested against the longer Kir1.1 splice variant. It had no effect on rat Kir1.1 at concentrations up to 30 μM. Work by the Denton laboratory has identified N171 as a conserved part of the small molecule binding site for previously discovered ROMK inhibitors. Sammons and coworkers analyzed if this residue plays a role in the inhibition of ROMK by compound (-)-49.46 When the asparagine was mutated to an aspartate at position 171, there was no loss in ROMK potency which 28 ACS Paragon Plus Environment

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is in contrast to the previously reported ROMK inhibitors. This result implies that the compounds from this series interact with ROMK in a mode distinct from previously reported inhibitors.



R1 NH

O

O R3

O N

O N

N

N

R2

O S N O O H

O S N O H

S N O H

44 hROMK IC50 = 0.44 M HLM CL = 30 mL/min/kg Papp = 20x10-6 cm/s LogD = 2.7 LipE = 3.7 hERG >40 M

O

O N

O N

N

N


NH

O

NH

NH

O

NH

O

Page 30 of 41

O O S N O H

Cl


O S N O H 47 hROMK IC50 = 0.035 M HLM CL = 69 mL/min/kg Papp = 10x10-6 cm/s LogD = 2.2 LipE = 5.3 hERG >40 M N N N

N N N O

O

NH

O S N O H

N

Cl

48 hROMK IC50 = 0.086 M HLM CL 40 M

NH

O S N O O H 49 hROMK IC50 = 0.16 M HLM CL

Discovery of small molecule renal outer medullary potassium (ROMK

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