Discovery of (S,E)-3-(2-fluorophenyl)-N-(1-(3-(pyridin-3-yloxy)phenyl)ethyl)-acrylamide as a potent and efficacious KCNQ2 (Kv7.2) opener for the treatment of neuropathic pain.

Acrylamide (S)-6, a potent and efficacious KCNQ2 (Kv7.2) opener, demonstrated significant activity in two models of neuropathic pain and in the formalin test, suggesting that KCNQ2 openers may be useful in the treatment of neuropathic pain including diabetic neuropathy.

Atropisomeric 3-(beta-hydroxyethyl)-4-arylquinolin-2-ones as Maxi-K potassium channel openers.

The synthesis of a series of 3-beta-hydroxyethyl-4-arylquinolin-2-ones is described. These compounds contain hydrophilic and hydrophobic substituents ortho to the phenolic OH in the C ring of the quinolinone. Electrophysiological evaluation of the panel of compounds revealed that 11 and 16 with an unbranched ortho substituent retain activity as maxi-K ion channel openers. Members of this series of compounds can exist as stable atropisomers. Calculated estimates of the energy barrier for rotation around the aryl-aryl single bond in 3 is 31 kcal/mol. The atropisomers of (+/-)-3, (+/-)-4, and (+/-)-11 were separated by chiral HPLC and tested for their effect on maxi-K mediated outward current in hSlo injected X. laevis oocytes. The (-) isomer in each case was found to be more active than the corresponding (+) isomer, suggesting that the ion channel exhibits stereoselective activation. X-ray crystallographic structures of (+)-3 and (+)-11 were determined. Evaluation of the stability of (-)-3 at 80 degrees C in n-butanol indicated a 19.6% conversion to (+)-3 over 72 h. In human serum at 37 degrees C (-)-3 did not racemize over the course of the 30 h study.

3-[(5-Chloro-2-hydroxyphenyl)methyl]-5-[4-(trifluoromethyl)phenyl ]-1,3,4-oxadiazol-2(3H)-one, BMS-191011: opener of large-conductance Ca(2+)-activated potassium (maxi-K) channels, identification, solubility, and SAR.

Compound 8a (BMS-191011), an opener of the cloned large-conductance, Ca2+-activated potassium (maxi-K) channel, demonstrated efficacy in in vivo stroke models, which led to its nomination as a candidate for clinical evaluation. Its maxi-K channel opening properties were consistent with its structural topology, being derived by combining elements from other known maxi-K openers. However, 8a suffered from poor aqueous solubility, which complicated elucidation of SAR during in vitro evaluation. The activity of 8a in in vivo stroke models and studies directed toward improving its solubility are reported herein. Enhanced solubility was achieved by appending heterocycles to the 8a scaffold, and a notable observation was made that inclusion of a simple amino group (anilines 8k and 8l) yielded excellent in vitro maxi-K ion channel opening activity and enhanced brain-to-plasma partitioning compared to the appended heterocycles.

Pharmacological activation and inhibition of Slack (Slo2.2) channels.

The Slack (Sequence like a calcium-activated K channel) (Slo2.2) gene is abundantly expressed in the mammalian brain and encodes a sodium-activated K+ (KNa) channel. Although the specific roles of Slack channel subunits in neurons remain to be identified, they may play a role in the adaptation of firing rate and in protection against ischemic injury. In the present study, we have generated a stable cell line expressing the Slack channel, and have analyzed the pharmacological properties of these channels in these cells and in Xenopus oocytes. Two known blockers of KNa channels, bepridil and quinidine, inhibited Slack currents in a concentration-dependent manner and decreased channel activity in excised membrane patches. The inhibition by bepridil was potent, with an IC50 of 1.0 microM for inhibition of Slack currents in HEK cells. In contrast, bithionol was found to be a robust activator of Slack currents. When applied to the extracellular face of excised patches, bithionol rapidly induced a reversible increase in channel opening, suggesting that it acts on Slack channels relatively directly. These data establish an important early characterization of agents that modulate Slack channels, a process essential for the experimental manipulation of Slack currents in neurons.

Analogs of a potent maxi-K potassium channel opener with an improved inhibitory profile toward cytochrome P450 isozymes.

Quinolinone 1 is a potent maxi-K potassium channel opener. In an effort to design analogs of 1 with a better inhibitory profile toward the CYP2C9 isozyme, the two acidic sites were chemically modified independently to generate a number of analogs. These analogs were evaluated as maxi-K channel openers in vitro using Xenopus laevis oocytes expressing cloned hSlo maxi-K channels. Compounds 15, 17, and 19 showed potent activity as maxi-K channel openers and were further evaluated for inhibition of the activity of the CYP2C9 isozyme. Compounds 17 and 19 showed diminished inhibitory potency against 2C9 and also against a panel of other more common CYP isozymes.

4-Aryl-3-(mercapto)quinolin-2-ones: novel maxi-K channel opening relaxants of corporal smooth muscle.

A novel series of 4-aryl-3-(mercapto)quinolin-2-one derivatives was prepared and evaluated as openers of the cloned maxi-K channel hSlo expressed in Xenopus laevis oocytes by utilizing electrophysiological methods. The effect of these maxi-K openers on corporal smooth muscle was studied in vitro using isolated rabbit corpus cavernosum. In vivo efficacy has been demonstrated with a selective maxi-K opening relaxant in a rat model of erectile function.

Synthesis and KCNQ2 opener activity of N-(1-benzo[1,3]dioxol-5-yl-ethyl, N-[1-(2,3-dihydro-benzofuran-5-yl)-ethyl, and N-[1-(2,3-dihydro-1H-indol-5-yl)-ethyl acrylamides.

Bioisosteric replacement studies led to the identification of N-(1-benzo[1,3]dioxol-5-yl-ethyl)-3-(2-chloro-phenyl)-acrylamide ((S)-3) as a highly potent KCNQ2 opener, and 3-(2,6-difluoro-phenyl)-N-[1-(2,3-dihydro-benzofuran-5-yl)-ethyl]-acrylamide ((S)-4), and N-[1-(2,3-dihydro-1H-indol-5-yl)-ethyl]-3-(2-fluoro-phenyl)-acrylamide ((S)-5) as highly efficacious KCNQ2 openers. In contrast, their respective R enantiomers showed significantly less or no appreciable KCNQ2 opener activity even at the highest concentration tested (10 microM). Because of its high potency and moderate efficacy as well as its convenient synthesis, (+/-)-3 was selected as a reference compound for analyzing efficacies of KCNQ openers in electrophysiology studies. Compounds (S)-4 and (S)-5 demonstrated significant activity in reducing neuronal hyperexcitability in rat hippocampal slices. The synthesis and the KCNQ2 opener activity of these acrylamides are described.

3-Thio-quinolinone maxi-K openers for the treatment of erectile dysfunction.

A series of Maxi-K openers for the treatment of erectile dysfunction based on the 3-thio-quinolinone core is described. Significant levels of channel opening (up to 550% of control) are seen in transfected oocytes. Functional activity in rabbit corpus cavernosum tissue strips confirms the potential to effect therapy for ED, the effect being maximal for the 3-amino-2-hydroxy thiol side chain.

Synthesis and structure-activity relationship of acrylamides as KCNQ2 potassium channel openers.

A new class of acrylamides was synthesized, and the effects of these analogues on outward potassium current were evaluated by using two electrode voltage clamp recordings from Xenopus laevis oocytes expressing cloned mKCNQ2 channels. SAR studies indicated that the pharmacophore of the acrylamide series includes the (S) absolute configuration at the (1-phenyl)ethyl moiety and the alpha,beta-unsaturated acrylamide functionality with a free NH. This study identified (S)-N-[1-(3-morpholin-4-yl-phenyl)-ethyl]-3-phenyl-acrylamide ((S)-1) and (S)-N-[1-(4-fluoro-3-morpholin-4-yl-phenyl)-ethyl]-3-(4-fluoro-phenyl)-acrylamide ((S)-2) as KCNQ2 openers for further electrophysiological evaluations. These two acrylamides demonstrated significant activity in the cortical spreading depression model of migraine as we reported previously.

(S)-N-[1-(4-cyclopropylmethyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-ethyl]-3-(2-fluoro-phenyl)-acrylamide is a potent and efficacious KCNQ2 opener which inhibits induced hyperexcitability of rat hippocampal neurons.

(S)-N-[1-(4-Cyclopropylmethyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-ethyl]-3-(2-fluoro-phenyl)-acrylamide ((S)-2) was identified as a potent and efficacious KCNQ2 opener. This compound demonstrated significant activity in reducing neuronal hyperexcitability in rat hippocampal slices, and the inhibition mediated by (S)-2 was reversed by the KCNQ blocker linopirdine.

The synthesis and structure-activity relationships of 3-amino-4-benzylquinolin-2-ones; discovery of novel KCNQ2 channel openers.

3-amino-4-benzylquinolin-2-ones have been identified as a novel class of KCNQ2 channel openers. Synthesis and SAR is described along with their electrophysiological evaluation as activators of the cloned mKCNQ2 channel expressed in Xenopus laevis oocytes. The preliminary SAR data suggest the importance of both the trifluoromethylsulfonamido group and electron-withdrawing substituents on the quinolone nucleus for expression of KCNQ2 channel opening properties.

(S)-N-[1-(3-morpholin-4-ylphenyl)ethyl]- 3-phenylacrylamide: an orally bioavailable KCNQ2 opener with significant activity in a cortical spreading depression model of migraine.

(S)-N-[1-(3-Morpholin-4-ylphenyl)ethyl]-3-phenylacrylamide (2) was synthesized as an orally bioavailable KCNQ2 potassium channel opener. In a rat model of migraine, 2 demonstrated significant oral activity in reducing the total number of cortical spreading depressions induced by potassium chloride.

4-Aryl-3-(hydroxyalkyl)quinolin-2-ones: novel maxi-K channel opening relaxants of corporal smooth muscle targeted for erectile dysfunction.

Novel 4-aryl-3-(hydroxyalkyl)quinoline-2-one derivatives were prepared and evaluated as openers of the cloned maxi-K channel hSlo expressed in Xenopus laevis oocytes by utilizing electrophysiological methods. The effect of these maxi-K openers on corporal smooth muscle was studied in vitro using isolated rabbit corpus cavernosum. From this study, a potent maxi-K opener was identified as an effective relaxant of rabbit corporal smooth muscle and shown to be active in an in vivo animal model of male erectile function.

Novel openers of Ca2+-dependent large-conductance potassium channels: symmetrical pharmacophore and electrophysiological evaluation of bisphenols.

Electrophysiological evaluation of symmetrical analogues of the known maxi-K opener NS-004 (1) led to the discovery of bisphenols 2a, 3a and 4a as openers of cloned maxi-K channels expressed in oocytes.

The synthesis and structure-activity relationships of 4-aryl-3-aminoquinolin-2-ones: a new class of calcium-dependent, large conductance, potassium (maxi-K) channel openers targeted for post-stroke neuroprotection.

A series of 4-aryl-3-aminoquinoline-2-one derivatives was synthesized and evaluated as activators of the cloned maxi-K channel mSlo (hSlo) expressed in Xenopus laevis oocytes using electrophysiological methods. A brain penetrable activator of maxi-K channels was identified and shown to be significantly active in the MCAO model of stroke.

4,5-diphenyltriazol-3-ones: openers of large-conductance Ca(2+)-activated potassium (maxi-K) channels.

A series of diphenyl-substituted heterocycles were synthesized and evaluated by electrophysiological techniques as openers of the cloned mammalian large-conductance, Ca(2+)-activated potassium (maxi-K) channel. The series was designed from deannulation of known benzimidazolone maxi-K opener NS-004 (2) thereby providing an effective template for obtaining structure-activity-related information. The triazolone ring system was the most studied wherein 4,5-diphenyltriazol-3-one 6d (maxi-K = 158%) was identified as the optimal maxi-K channel opener.

The synthesis and characterization of BMS-204352 (MaxiPost) and related 3-fluorooxindoles as openers of maxi-K potassium channels.

3-Aryl-3-fluorooxindoles can be efficiently synthesized in two steps by the addition of an aryl Grignard to an isatin, followed by treatment with DAST. Oxindole 1 (BMS-204352; MaxiPost) can be isolated using chiral HPLC or prepared by employing chiral resolution. Cloned maxi-K channels are opened by 1, which demonstrates a brain/plasma ratio >9 in rats.

The synthesis and structure-activity relationships of 1,3-diaryl 1,2,4-(4H)-triazol-5-ones: a new class of calcium-dependent, large conductance, potassium (maxi-K) channel opener targeted for urge urinary incontinence.

A series of 1,3-diaryl 1,2,4-(4H)-triazol-5-ones was prepared and shown by electrophysiological analysis to activate a cloned maxi-K channel mSlo (or hSlo) expressed in Xenopus laevis oocytes. The effects of these structurally novel maxi-K channel openers on bladder contractile function were studied in vitro using isolated rat bladder strips pre-contracted with carbachol. Several 1,3-diaryl 1,2,4-(4H)-triazol-5-one derivatives were found to be potent smooth muscle relaxants but this activity did not completely correlate with maxi-K channel opening.

Synthesis and structure-activity relationships of 3-aryloxindoles: a new class of calcium-dependent, large conductance potassium (maxi-K) channel openers with neuroprotective properties.

A series of 3-aryloxindole derivatives were synthesized and evaluated as activators of the cloned maxi-K channel mSlo expressed in Xenopus laevis oocytes using electrophysiological methods. The most promising maxi-K openers to emerge from this study were (+/-)-3-(5-chloro-2-hydroxyphenyl)-1,3-dihydro-3-hydroxy-6-(trifluoromethyl)-2H-indol-2-one ((+/-)-8c) and its 3-des-hydroxy analogue (+/-)-11b. The individual enantiomers of (+/-)-8c were synthesized, and the maxi-K channel-opening properties were shown to depend on the absolute configuration of the single stereogenic center with the efficacy of (-)-8c superior to that of both (+)-8c and the racemic mixture when evaluated at a concentration of 20 microM. Racemic 11b exhibited greater efficacy than either the racemic 8c or the more active enantiomer in the electrophysiological evaluation. In vitro metabolic stability studies conducted with (+/-)-8c and (+/-)-11b in rat liver S9 microsomal fractions revealed significant oxidative degradation with two hydroxylated metabolites observed by liquid chromatography/mass spectrometry for each compound in addition to the production of 8c from 11b. The pharmacokinetic properties of (+/-)-8c and (+/-)-11b were determined in rats as a prelude to evaluation in a rat model of stroke that involved permanent occlusion of the middle cerebral artery (MCAO model). In the MCAO model, conducted in the spontaneously hypertensive rat, the more polar 3-hydroxy derivative (+/-)-8c did not demonstrate a significant reduction in cortical infarct volume when administered intravenously at doses ranging from 0.1 to 10 mg/kg as a single bolus 2 h after middle cerebral artery occlusion when compared to vehicle-treated controls. In contrast, intravenous administration of (+/-)-11b at a dose of 0.03 mg/kg was found to reduce the measured cortical infarct volume by approximately 18% when compared to vehicle-treated control animals. Intraperitoneal administration of (+/-)-11b at a dose of 10 mg/kg 2 h following artery occlusion was shown to reduce infarct volume by 26% when compared to vehicle-treated controls. To further probe the effects of compounds (+/-)-8c and (+/-)-11b on neurotransmitter release in vitro, both compounds were examined for their ability to reduce electrically stimulated [3H]-glutamate release from rat hippocampal slices that had been preloaded with [3H]-glutamate. Only (+/-)-11b was able to demonstrate a significant inhibition [3H]-glutamate release in this assay at a concentration of 20 microM, providing concordance with the profile of these compounds in the MCAO model. Although (+/-)-11b showed some promise as a potential developmental candidate for the treatment of the sequelae of stroke based on its efficacy in the rat MCAO model, the pharmacokinetic profile of this compound was considered to be less than optimal and was not pursued in favor of derivatives with enhanced metabolic stability.