ABSTRACT
A spirocyclic class of ROMK inhibitors was developed containing a structurally diverse heterocyclic sulfone moiety and spirocyclic core starting from lead 1. These compounds not only displayed exquisite ROMK potency but significantly improved selectivity over hERG. The lead compounds were found to have favorable pharmacokinetic properties and displayed robust diuretic, natriuretic and blood pressure lowering effects in spontaneously hypertensive rats.
Subject(s)
Diuretics/pharmacology , Drug Design , Enzyme Inhibitors/pharmacology , Heterocyclic Compounds/chemistry , Heterocyclic Compounds/pharmacology , Potassium Channels, Inwardly Rectifying/antagonists & inhibitors , Sulfones/pharmacology , Animals , Heterocyclic Compounds/chemical synthesis , Rats , Rats, Inbred SHRABSTRACT
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.
Subject(s)
Oxazines/chemistry , Oxazines/pharmacology , Potassium Channels, Inwardly Rectifying/antagonists & inhibitors , Animals , Diuresis/drug effects , Dogs , Heart Failure/drug therapy , Humans , Hypertension/drug therapy , Macaca mulatta , Oxazines/pharmacokinetics , Potassium Channels, Inwardly Rectifying/metabolism , Rats, Sprague-Dawley , Transcriptional Regulator ERG/antagonists & inhibitors , Transcriptional Regulator ERG/metabolismABSTRACT
The renal outer medullary potassium (ROMK) channel, located at the apical surface of epithelial cells in the thick ascending loop of Henle and cortical collecting duct, contributes to salt reabsorption and potassium secretion, and represents a target for the development of new mechanism of action diuretics. This idea is supported by the phenotype of antenatal Bartter's syndrome type II associated with loss-of-function mutations in the human ROMK channel, as well as, by cardiovascular studies of heterozygous carriers of channel mutations associated with type II Bartter's syndrome. Although the pharmacology of ROMK channels is still being developed, channel inhibitors have been identified and shown to cause natriuresis and diuresis, in the absence of any significant kaliuresis, on acute oral dosing to rats or dogs. Improvements in potency and selectivity have led to the discovery of MK-7145 [5,5'-((1R,1'R)-piperazine-1,4-diylbis(1-hydroxyethane-2,1-diyl))bis(4-methylisobenzofuran-1(3H)-one)], a potential clinical development candidate. In spontaneously hypertensive rats, oral dosing of MK-7145 causes dose-dependent lowering of blood pressure that is maintained during the entire treatment period, and that displays additive/synergistic effects when administered in combination with hydrochlorothiazide or candesartan, respectively. Acute or chronic oral administration of MK-7145 to normotensive dogs led to dose-dependent diuresis and natriuresis, without any significant urinary potassium losses or changes in plasma electrolyte levels. Elevations in bicarbonate and aldosterone were found after 6 days of dosing. These data indicate that pharmacological inhibition of ROMK has potential as a new mechanism for the treatment of hypertension and/or congestive heart failure. In addition, Bartter's syndrome type II features are manifested on exposure to ROMK inhibitors.
Subject(s)
Bartter Syndrome/physiopathology , Benzofurans/pharmacology , Blood Pressure/drug effects , Phenotype , Piperazines/pharmacology , Potassium Channel Blockers/pharmacology , Potassium Channels, Inwardly Rectifying/antagonists & inhibitors , Animals , Bartter Syndrome/drug therapy , Benzimidazoles/pharmacology , Benzofurans/therapeutic use , Biphenyl Compounds , Dogs , Dose-Response Relationship, Drug , Drug Synergism , Female , HEK293 Cells , Humans , Hydrochlorothiazide/pharmacology , Male , Piperazines/therapeutic use , Potassium Channel Blockers/therapeutic use , Rats , Tetrazoles/pharmacologyABSTRACT
ROMK, the renal outer medullary potassium channel, is involved in potassium recycling at the thick ascending loop of Henle and potassium secretion at the cortical collecting duct in the kidney nephron. Because of this dual site of action, selective inhibitors of ROMK are expected to represent a new class of diuretics/natriuretics with superior efficacy and reduced urinary loss of potassium compared to standard-of-care loop and thiazide diuretics. Following our earlier work, this communication will detail subsequent medicinal chemistry endeavors to further improve lead selectivity against the hERG channel and preclinical pharmacokinetic properties. Pharmacological assessment of highlighted inhibitors will be described, including pharmacodynamic studies in both an acute rat diuresis/natriuresis model and a subchronic blood pressure model in spontaneous hypertensive rats. These proof-of-biology studies established for the first time that the human and rodent genetics accurately predict the in vivo pharmacology of ROMK inhibitors and supported identification of the first small molecule ROMK inhibitor clinical candidate, MK-7145.
ABSTRACT
Following the discovery of small molecule acyl piperazine ROMK inhibitors and their initial preclinical validation as a novel diuretic agent, our group set out to discover new ROMK inhibitors with reduced risk for QT effects, suitable for further pharmacological experiments in additional species. Several strategies for decreasing hERG affinity while maintaining ROMK inhibition were investigated and are described herein. The most promising candidate, derived from the newly discovered 4-N-heteroaryl acetyl series, improved functional hERG/ROMK ratio by >10× over the previous lead. In vivo evaluation demonstrated comparable diuretic effects in rat with no detectable QT effects at the doses evaluated in an in vivo dog model.
Subject(s)
ERG1 Potassium Channel/physiology , Heterocyclic Compounds/pharmacology , Piperazines/pharmacology , Heterocyclic Compounds/chemistry , Piperazines/chemistry , Structure-Activity RelationshipABSTRACT
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.
ABSTRACT
The renal outer medullary potassium (ROMK) channel, which is located at the apical membrane of epithelial cells lining the thick ascending loop of Henle and cortical collecting duct, plays an important role in kidney physiology by regulating salt reabsorption. Loss-of-function mutations in the human ROMK channel are associated with antenatal type II Bartter's syndrome, an autosomal recessive life-threatening salt-wasting disorder with mild hypokalemia. Similar observations have been reported from studies with ROMK knockout mice and rats. It is noteworthy that heterozygous carriers of Kir1.1 mutations associated with antenatal Bartter's syndrome have reduced blood pressure and a decreased risk of developing hypertension by age 60. Although selective ROMK inhibitors would be expected to represent a new class of diuretics, this hypothesis has not been pharmacologically tested. Compound A [5-(2-(4-(2-(4-(1H-tetrazol-1-yl)phenyl)acetyl)piperazin-1-yl)ethyl)isobenzofuran-1(3H)-one)], a potent ROMK inhibitor with appropriate selectivity and characteristics for in vivo testing, has been identified. Compound A accesses the channel through the cytoplasmic side and binds to residues lining the pore within the transmembrane region below the selectivity filter. In normotensive rats and dogs, short-term oral administration of compound A caused concentration-dependent diuresis and natriuresis that were comparable to hydrochlorothiazide. Unlike hydrochlorothiazide, however, compound A did not cause any significant urinary potassium losses or changes in plasma electrolyte levels. These data indicate that pharmacologic inhibition of ROMK has the potential for affording diuretic/natriuretic efficacy similar to that of clinically used diuretics but without the dose-limiting hypokalemia associated with the use of loop and thiazide-like diuretics.
Subject(s)
Diuresis/drug effects , Diuresis/physiology , Natriuresis/drug effects , Potassium Channel Blockers/pharmacology , Potassium Channels, Inwardly Rectifying/antagonists & inhibitors , Potassium Channels, Inwardly Rectifying/physiology , Animals , CHO Cells , Cricetinae , Cricetulus , Dogs , Dose-Response Relationship, Drug , Female , HEK293 Cells , Humans , Madin Darby Canine Kidney Cells , Male , Natriuresis/physiology , Rats , Rats, Sprague-DawleyABSTRACT
A sub-class of distinct small molecule ROMK inhibitors were developed from the original lead 1. Medicinal chemistry endeavors led to novel ROMK inhibitors with good ROMK functional potency and improved hERG selectivity. Two of the described ROMK inhibitors were characterized for the first in vivo proof-of-concept biology studies, and results from an acute rat diuresis model confirmed the hypothesis that ROMK inhibitors represent new mechanism diuretic and natriuretic agents.
Subject(s)
Benzofurans/chemistry , Benzofurans/pharmacology , Potassium Channels, Inwardly Rectifying/antagonists & inhibitors , Animals , Benzofurans/pharmacokinetics , Diuresis/drug effects , Drug Discovery , Ether-A-Go-Go Potassium Channels/antagonists & inhibitors , Ether-A-Go-Go Potassium Channels/metabolism , Humans , Potassium Channels, Inwardly Rectifying/metabolism , Rats , Rats, Sprague-Dawley , Tetrazoles/chemistry , Tetrazoles/pharmacokinetics , Tetrazoles/pharmacologyABSTRACT
A series of benzazepinones were synthesized and evaluated for block of Nav1.7 sodium channels. Compound 30 from this series displayed potent channel block, good selectivity versus other targets, and dose-dependent oral efficacy in a rat model of neuropathic pain.
Subject(s)
Benzazepines/pharmacology , Neuralgia/drug therapy , Sodium Channel Blockers/pharmacology , Animals , Disease Models, Animal , RatsABSTRACT
The renal outer medullary potassium (ROMK) channel is a member of the inwardly rectifying family of potassium (Kir) channels. ROMK (Kir1.1) is predominantly expressed in kidney where it plays a major role in the salt reabsorption process. Loss-of-function mutations in the human Kir1.1 channel are associated with antenatal Bartter's syndrome type II, a life-threatening salt and water balance disorder. Heterozygous carriers of Kir1.1 mutations associated with antenatal Bartter's syndrome have reduced blood pressure and a decreased risk of developing hypertension by age 60. These data suggest that Kir1.1 inhibitors could represent novel diuretics for the treatment of hypertension. Because little is known about the molecular pharmacology of Kir1.1 channels, assays that provide a robust, reliable readout of channel activity-while operating in high-capacity mode-are needed. In the present study, we describe high-capacity, 384- and 1,536-well plate, functional thallium flux, and IonWorks electrophysiology assays for the Kir1.1 channel that fulfill these criteria. In addition, 96-well (86)Rb(+) flux assays were established that can operate in the presence of 100% serum, and can provide an indication of the effect of a serum shift on compound potencies. The ability to grow Madin-Darby canine kidney cells expressing Kir1.1 in Transwell supports provides a polarized cell system that can be used to study the mechanism of Kir1.1 inhibition by different agents. All these functional Kir1.1 assays together can play an important role in supporting different aspects of drug development efforts during lead identification and/or optimization.
Subject(s)
Drug Discovery/methods , Potassium Channel Blockers/metabolism , Potassium Channel Blockers/pharmacology , Potassium Channels, Inwardly Rectifying/antagonists & inhibitors , Potassium Channels, Inwardly Rectifying/metabolism , Animals , CHO Cells , Cricetinae , Cricetulus , Dogs , Humans , Madin Darby Canine Kidney Cells , Potassium Channel Blockers/blood , Potassium Channel Blockers/chemistry , Potassium Channels, Inwardly Rectifying/blood , Rats , Thallium/metabolismABSTRACT
The renal outer medullary potassium channel (ROMK or Kir1.1) is a putative drug target for a novel class of diuretics that could be used for the treatment of hypertension and edematous states such as heart failure. An internal high-throughput screening campaign identified 1,4-bis(4-nitrophenethyl)piperazine (5) as a potent ROMK inhibitor. It is worth noting that this compound was identified as a minor impurity in a screening hit that was responsible for all of the initially observed ROMK activity. Structure-activity studies resulted in analogues with improved rat pharmacokinetic properties and selectivity over the hERG channel, providing tool compounds that can be used for in vivo pharmacological assessment. The featured ROMK inhibitors were also selective against other members of the inward rectifier family of potassium channels.
ABSTRACT
The voltage-gated potassium channel, human Ether-à-go-go related gene (hERG), represents the molecular component of IKr, one of the potassium currents involved in cardiac action potential repolarization. Inhibition of IKr increases the duration of the ventricular action potential, reflected as a prolongation of the QT interval in the electrocardiogram, and increases the risk for potentially fatal ventricular arrhythmias. Because hERG is an appropriate surrogate for IKr, hERG assays that can identify potential safety liabilities of compounds during lead identification and optimization have been implemented. Although the gold standard for hERG evaluation is electrophysiology, this technique, even with the medium capacity, automated instruments that are currently available, does not meet the throughput demands for supporting typical medicinal chemistry efforts in the pharmaceutical environment. Assays that could provide reliable molecular pharmacology data, while operating in high capacity mode, are therefore desirable. In the present study, we describe a high-capacity, 384- and 1,536-well plate, functional thallium flux assay for the hERG channel that fulfills these criteria. This assay was optimized and validated using different structural classes of hERG inhibitors. An excellent correlation was found between the potency of these agents in the thallium flux assay and in electrophysiological recordings of channel activity using the QPatch automated patch platform. Extension of this study to include 991 medicinal chemistry compounds from different internal drug development programs indicated that the thallium flux assay was a good predictor of in vitro hERG activity. These data suggest that the hERG thallium flux assay can play an important role in supporting drug development efforts.
Subject(s)
Ether-A-Go-Go Potassium Channels/antagonists & inhibitors , High-Throughput Screening Assays , Potassium Channel Blockers/pharmacology , Action Potentials/drug effects , Animals , CHO Cells , Cricetinae , Cricetulus , Ether-A-Go-Go Potassium Channels/genetics , Ether-A-Go-Go Potassium Channels/physiology , HEK293 Cells , Humans , Patch-Clamp Techniques , Thallium/metabolismABSTRACT
Analogs of the previously reported voltage gated sodium channel blocker CDA54 were prepared in which one of the amide functions was replaced with aromatic and non-aromatic heterocycles. Replacement of the amide with an aromatic heterocycle resulted in significant loss of sodium channel blocking activity, while non-aromatic heterocycle replacements were well tolerated.
Subject(s)
Isoxazoles/chemistry , Isoxazoles/pharmacology , Sodium Channel Blockers/chemistry , Sodium Channel Blockers/pharmacology , Animals , Isoxazoles/therapeutic use , Models, Molecular , Molecular Structure , Pain/drug therapy , Rats , Rats, Sprague-Dawley , Sodium Channel Blockers/therapeutic use , Spinal Nerves/drug effects , Structure-Activity RelationshipABSTRACT
A series of novel isoxazole voltage gated sodium channel blockers have been synthesized and evaluated. Substitutions on the benzylic position of benzamide were investigated to determine their effect on Na(v)1.7 inhibitory potency. The spirocyclobutyl substitution had the most significant enhancement on Na(v)1.7 inhibitory activity.
Subject(s)
Isoxazoles/therapeutic use , Pain/drug therapy , Sodium Channel Blockers/therapeutic use , Sodium Channels/metabolism , Animals , Cell Line , Chronic Disease , Humans , Isoxazoles/chemistry , Isoxazoles/pharmacology , Pain/immunology , Rats , Sodium Channel Blockers/chemistry , Sodium Channel Blockers/pharmacology , Spinal Nerves/drug effects , Structure-Activity RelationshipABSTRACT
The K(ir) family of potassium-selective ion channels is characterized by their inward (anomalous) rectifying current-voltage relationship. K(ir) channels are widely expressed in mammalian cells and through their role in regulation of the cell membrane potential have been implicated in diverse physiological functions. To enable the identification of novel K(ir) channel inhibitors, a fluorescence resonance energy transfer (FRET)-based membrane potential assay was developed using a Chinese hamster ovary cell line stably expressing a human K(ir) channel. The FRET-based assay incorporates the use of two dyes {N-(6-chloro-7-hydroxycoumarin-3-carbonyl)-dimyristoylphosphatidylethanolamine (CC2-DMPE) and bis(1,3-diethylthiobarbiturate)trimethine oxonol [DiSBAC(2)(3)]} to track changes in membrane potential, thus enabling all of the advantages of ratiometric readout: reduced inaccuracies arising from well-to-well variation in cell number, dye loading, signal intensities, and plate inconsistencies. The assay was miniaturized to a 1,536-well microtiter plate format and read on a fluorometric imaging plate reader (FLIPR(Tetra), Molecular Devices, Sunnyvale, CA). The assay was automated and utilized to perform a primary high-throughput screening campaign to identify novel inhibitors of the K(ir) channel.
Subject(s)
Drug Evaluation, Preclinical/methods , Fluorescence Resonance Energy Transfer/methods , G Protein-Coupled Inwardly-Rectifying Potassium Channels/drug effects , Potassium Channel Blockers/pharmacology , Animals , Barbiturates , Bee Venoms/pharmacology , CHO Cells , Coumarins , Cricetinae , Cricetulus , Ethanolamines , Fluorescent Dyes , G Protein-Coupled Inwardly-Rectifying Potassium Channels/metabolism , Humans , Isoxazoles , Kinetics , Membrane Potentials/physiology , ThiobarbituratesABSTRACT
A series of 3-amino-1,5-benzodiazepinones were synthesized and evaluated as potential sodium channel blockers in a functional, membrane potential-based assay. One member of this series displayed subnanomolar, state-dependent sodium channel block, and was orally efficacious in a mouse model of epilepsy.
Subject(s)
Anticonvulsants/pharmacology , Benzodiazepinones/pharmacology , Epilepsy/drug therapy , Ether-A-Go-Go Potassium Channels/antagonists & inhibitors , Sodium Channel Blockers/pharmacology , Animals , Anticonvulsants/chemical synthesis , Anticonvulsants/pharmacokinetics , Benzodiazepinones/chemical synthesis , Benzodiazepinones/pharmacokinetics , Electrophysiology , Electroshock , Epilepsy/metabolism , Ether-A-Go-Go Potassium Channels/metabolism , Fluorescence Resonance Energy Transfer , Humans , Mice , Molecular Structure , Rats , Sodium Channel Blockers/chemical synthesis , Sodium Channel Blockers/pharmacokineticsABSTRACT
A series of imidazopyridines were evaluated as potential sodium channel blockers for the treatment of neuropathic pain. Several members were identified with good hNa(v)1.7 potency and excellent rat pharmacokinetic profiles. Compound 4 had good efficacy (52% and 41% reversal of allodynia at 2 and 4h post-dose, respectively) in the Chung rat spinal nerve ligation (SNL) model of neuropathic pain when dosed orally at 10mg/kg.
Subject(s)
Pyridines/chemistry , Pyridines/pharmacology , Sodium Channel Blockers/chemistry , Sodium Channel Blockers/pharmacology , Sodium Channels/metabolism , Analgesics/chemistry , Analgesics/pharmacology , Animals , Inflammation/drug therapy , Molecular Structure , NAV1.7 Voltage-Gated Sodium Channel , Pain/drug therapy , Rats , Sodium Channel Blockers/pharmacokinetics , Structure-Activity RelationshipABSTRACT
Voltage-gated sodium channels (Nav1) transmit pain signals from peripheral nociceptive neurons, and blockers of these channels have been shown to ameliorate a number of pain conditions. Because these drugs can have adverse effects that limit their efficacy, more potent and selective Nav1 inhibitors are being pursued. Recent human genetic data have provided strong evidence for the involvement of the peripheral nerve sodium channel subtype, Nav1.7, in the signaling of nociceptive information, highlighting the importance of identifying selective Nav1.7 blockers for the treatment of chronic pain. Using a high-throughput functional assay, novel Nav1.7 blockers, namely, the 1-benzazepin-2-one series, have recently been identified. Further characterization of these agents indicates that, in addition to high-affinity inhibition of Nav1.7 channels, selectivity against the Nav1.5 and Nav1.8 subtypes can also be achieved within this structural class. The most potent, nonselective member of this class of Nav1.7 blockers has been radiolabeled with tritium. [3H]BNZA binds with high affinity to rat brain synaptosomal membranes (Kd = 1.5 nM) and to membranes prepared from HEK293 cells stably transfected with hNav1.5 (Kd = 0.97 nM). In addition, and for the first time, high-affinity binding of a radioligand to hNav1.7 channels (Kd = 1.6 nM) was achieved with [3H]BNZA, providing an additional means for identifying selective Nav1.7 channel inhibitors. Taken together, these data suggest that members of the novel 1-benzazepin-2-one structural class of Nav1 blockers can display selectivity toward the peripheral nerve Nav1.7 channel subtype, and with appropriate pharmacokinetic and drug metabolism properties, these compounds could be developed as analgesic agents.
Subject(s)
Benzazepines/chemistry , Sodium Channel Blockers/chemistry , Sodium Channels/physiology , Animals , Benzazepines/metabolism , Benzazepines/pharmacology , Binding, Competitive , Cell Line , Cell Membrane/metabolism , Electrophysiology , Humans , Membrane Potentials/drug effects , Molecular Structure , NAV1.7 Voltage-Gated Sodium Channel , NAV1.8 Voltage-Gated Sodium Channel , Radioligand Assay , Rats , Sodium Channel Blockers/metabolism , Sodium Channel Blockers/pharmacology , Sodium Channels/drug effects , Sodium Channels/metabolism , Synaptosomes/metabolismABSTRACT
A series of benzazepinones were synthesized and evaluated as hNa(v)1.7 sodium channel blockers. Several compounds from this series displayed good oral bioavailability and exposure and were efficacious in a rat model of neuropathic pain.
Subject(s)
Benzodiazepinones/chemical synthesis , Benzodiazepinones/therapeutic use , Neuralgia/drug therapy , Sodium Channel Blockers/chemical synthesis , Sodium Channel Blockers/therapeutic use , Sodium Channels/drug effects , Animals , Benzodiazepinones/pharmacokinetics , Biological Availability , Disease Models, Animal , Dogs , Drug Evaluation, Preclinical , Molecular Structure , NAV1.7 Voltage-Gated Sodium Channel , Rats , Sodium Channel Blockers/pharmacokinetics , Sodium Channels/chemistryABSTRACT
A series of benzodiazepines and benzazepinones were synthesized and evaluated as potential sodium channel blockers in a functional, membrane potential-based assay. One member of the benzazepinone series, compound 47, displayed potent, state-dependent block of hNa(v)1.7, and was orally efficacious in a rat model of neuropathic pain.