Design, synthesis, and BK channel-opening activity of hexahydrodibenzazepinone derivatives

In order to explore new scaffolds for large-conductance Ca2+ -activated K+ channel (BK channel) openers, we carried out molecular design and synthesis on the basis of the following two concepts: (1) introduction of a heteroatom into the dehydroabietic acid (BK channel opener) skeleton would allow easier introduction of substituents. (2) Because of the fourfold symmetrical structure of BK channels, dimeric compounds in which two pharmacophores are linked through a tether are expected to have a greater binding probability to the channels, resulting in increased channel-opening activity. Herein, we explore the usefulness of the hexahydrodibenzazepinone structure as a new scaffold for BK channel openers. The synthesized monomer compounds of hexahydrodibenzazepinone derivatives, which can be derived from dehydroabietic acid, were subjected to electrophysiological patch-clamp studies, followed by Magnus contraction-relaxation assay using rabbit urinary bladder smooth muscle strips to assess overall activities. Dimeric compounds were designed by linking the monomeric hexahydrodibenzazepinone derivatives through a diacetylenebenzene tether, and their channel-opening activities were evaluated by electrophysiological methods. Finally, we concluded that the critical structure for BK channel-opening activity is the hexahydrodibenzazepinone monomer substituted with a phenyl-bearing alkynyl substituent on the lactam amide.     

Novel BK channel openers containing dehydroabietic acid skeleton: structure-activity relationship for peripheral substituents on ring C

A series of dehydroabietic acid (DHAA, 2) derivatives was synthesized and evaluated as BK channel openers in an assay system of CHO-K1 cells expressing hBKalpha channels. Systematic modifications of the peripheral functionality of ring C of DHAA showed that the introduction of a nitro or (thio)urea group in ring C greatly enhanced the BK channel-opening activity.     

Dehydroabietic acid derivatives as a novel scaffold for large-conductance calcium-activated K+ channel openers

We found that the dehydroabietic acid structure is a new scaffold for chemical modulators of large-conductance calcium-activated K+ channels (BK channels). Structure-activity relationship (SAR) studies of the dehydroabietic acid derivatives and related non-aromatic compounds such as pimaric acid revealed the importance of the carboxyl functionality and an appropriate hydrophobic moiety of the molecules for BK channel-opening ability.     

Emerging Role of Calcium-Activated Potassium Channel in the Regulation of Cell Viability Following Potassium Ions Challenge in HEK293 Cells and Pharmacological Modulation

Emerging evidences suggest that Ca²⁺activated-K⁺-(BK) channel is involved in the regulation of cell viability. The changes of the cell viability observed under hyperkalemia (15 mEq/L) or hypokalemia (0.55 mEq/L) conditions were investigated in HEK293 cells expressing the hslo subunit (hslo-HEK293) in the presence or absence of BK channel modulators. The BK channel openers(10^-11-10^-3M) were: acetazolamide(ACTZ), Dichlorphenamide(DCP), methazolamide(MTZ), bendroflumethiazide(BFT), ethoxzolamide(ETX), hydrochlorthiazide(HCT), quercetin(QUERC), resveratrol(RESV) and NS1619; and the BK channel blockers(2x10^-7M-5x10^-3M) were: tetraethylammonium(TEA), iberiotoxin(IbTx) and charybdotoxin(ChTX). Experiments on cell viability and channel currents were performed using cell counting kit-8 and patch-clamp techniques, respectively. Hslo whole-cell current was potentiated by BK channel openers with different potency and efficacy in hslo-HEK293. The efficacy ranking of the openers at -60 mV(Vm) was BFT> ACTZ >DCP ≥RESV≥ ETX> NS1619> MTZ≥ QUERC; HCT was not effective. Cell viability after 24 h of incubation under hyperkalemia was enhanced by 82+6% and 33+7% in hslo-HEK293 cells and HEK293 cells, respectively. IbTx, ChTX and TEA enhanced cell viability in hslo-HEK293. BK openers prevented the enhancement of the cell viability induced by hyperkalemia or IbTx in hslo-HEK293 showing an efficacy which was comparable with that observed as BK openers. BK channel modulators failed to affect cell currents and viability under hyperkalemia conditions in the absence of hslo subunit. In contrast, under hypokalemia cell viability was reduced by -22+4% and -23+6% in hslo-HEK293 and HEK293 cells, respectively; the BK channel modulators failed to affect this parameter in these cells. In conclusion, BK channel regulates cell viability under hyperkalemia but not hypokalemia conditions. BFT and ACTZ were the most potent drugs either in activating the BK current and in preventing the cell proliferation induced by hyperkalemia. These findings may have relevance in disorders associated with abnormal K⁺ ion homeostasis including periodic paralysis and myotonia.     

Partial structures of ketoconazole as modulators of the large conductance calcium-activated potassium channel (BK(Ca))

A series of partial structures of ketoconazole has been synthesized and tested for activity on the large conductance calcium-activated potassium channel (BK) in bovine smooth muscle cells. This has provided openers and blockers of the channel. The results suggest that the phenyl and phenoxy moieties are important for interaction with BK, whereas the imidazole group is unimportant. The properties of the phenoxy moiety seem to determine whether the compounds act to open or block the channel.     

Differential efficacy of GoSlo-SR compounds on BKα and BKαγ1–4 channels

Large conductance, voltage and Ca²⁺ activated K⁺ channels (BK channels) are abundantly expressed throughout the body and are important regulators of smooth muscle tone and neuronal excitability. Their dysfunction is implicated in various diseases including overactive bladder, hypertension and erectile dysfunction. Therefore, BK channel openers bear significant therapeutic potential to treat the above diseases. GoSlo-SR compounds were designed to be potent and efficacious BK channel openers. Although their structural activity relationships, activation in both BKα and BKαβ channels and the hypothetical mode of action of these compounds has been studied in detail in recent years, their effectiveness to open the BKαγ channels still remains unexplored. In this study, we have examined the efficacy of 3 closely related GoSlo-SR openers, GoSlo-SR-5-6 (SR-5-6), GoSlo-SR-5-44 (SR-5-44) and GoSlo-SR-5-130 (SR-5-130) using inside out patches on BKα channels coexpressed with 4 different LRRC (γ_1–4) subunits in HEK293 cells. Our data suggests that the activation effects due to SR-5-6 were not significantly affected in the presence of γ_1–4 subunits. Interestingly, the effects of more efficacious BK channel opener SR-5-44 were altered by different γ subunits. In cells expressing BKα channels, the shift in V_1/2 (ΔV_1/2) induced by SR-5-44 (3 μM) was ?76 ± 3 mV, whereas it was significantly reduced by ～70 % in BKαγ₁ channels (ΔV_1/2= ?23 ± 3, p < 0.001, ANOVA). In BKαγ₂ channels the ΔV_1/2 was ?36 ± 1 mV, which was less than that observed in BKαγ₃ and BKαγ₄ channels where the ΔV_1/2 was ?47 ± 5 mV, and ?82 ± 5 mV, respectively. Additionally, the excitatory effects of a ‘β specific’ BK channel opener, SR-5-130 were only partially restored in the patches containing BKαγ_1–4 channels. Together this data highlights that subtle modifications in GoSlo-SR structures alter their effectiveness on BK channels with accessory γ subunits and this study might provide a scaffold for the development of more tissue specific BK channel openers.     

Design and synthesis of hydroxy-alkynoic acids and their methyl esters as novel activators of BK channels

Physiological and pharmacological agents that activate large conductance, voltage-, and calcium-gated potassium (BK) channels located in the smooth muscle are effective vasodilators. Thus, activators of smooth muscle BK channels may be potential therapeutic tools to treat cardiovascular disease associated with vasoconstriction and/or impaired dilation, such as cerebrovascular spasm and constriction. We previously showed that lithocholic acid (LC) and other cholane derivatives activated smooth muscle BK channels and, thus, caused endothelium-independent cerebral artery dilation. However, clinical use of these cholane derivatives could be limited by the actions of these steroids, such as elevation of intracellular calcium and induction of apoptosis. Using LC as template, we designed and synthesized a series of hydroxy-alkynoic acids and corresponding methyl esters, as putative, non-steroid BK channel activators. Indeed, the newly synthesized compounds effectively and reversibly activated rat cerebrovascular myocyte BK channel at concentrations similar to those found effective with LC. Among all the novel compounds tested, C-10 hydroxy-alkynoic acid methyl ester appears to be the most effective activator of vascular myocyte BK channels.     

Neurogenic Detrusor Overactivity Is Associated with Decreased Expression and Function of the Large Conductance Voltage- and Ca2+-Activated K+ Channels

Patients suffering from a variety of neurological diseases such as spinal cord injury, Parkinson’s disease, and multiple sclerosis often develop neurogenic detrusor overactivity (NDO), which currently lacks a universally effective therapy. Here, we tested the hypothesis that NDO is associated with changes in detrusor smooth muscle (DSM) large conductance Ca²⁺-activated K⁺ (BK) channel expression and function. DSM tissue samples from 33 patients were obtained during open bladder surgeries. NDO patients were clinically characterized preoperatively with pressure-flow urodynamics demonstrating detrusor overactivity, in the setting of a clinically relevant neurological condition. Control patients did not have overactive bladder and did not have a clinically relevant neurological disease. We conducted quantitative polymerase chain reactions (qPCR), perforated patch-clamp electrophysiology on freshly-isolated DSM cells, and functional studies on DSM contractility. qPCR experiments revealed that DSM samples from NDO patients showed decreased BK channel mRNA expression in comparison to controls. Patch-clamp experiments demonstrated reduced whole cell and transient BK currents (TBKCs) in freshly-isolated DSM cells from NDO patients. Functional studies on DSM contractility showed that spontaneous phasic contractions had a decreased sensitivity to iberiotoxin, a selective BK channel inhibitor, in DSM strips isolated from NDO patients. These results reveal the novel finding that NDO is associated with decreased DSM BK channel expression and function leading to increased DSM excitability and contractility. BK channel openers or BK channel gene transfer could be an alternative strategy to control NDO. Future clinical trials are needed to evaluate the value of BK channel opening drugs or gene therapies for NDO treatment and to identify any possible adverse effects.     

The discovery of a new class of large-conductance Ca2+-activated K+ channel opener targeted for overactive bladder: synthesis and structure-activity relationships of 2-amino-4-azaindoles

2-Amino-4-azaindoles have been identified as a structurally novel class of BK(Ca) channel openers. Their synthesis from 2-chloro-3-nitropyridine is described together with their in vitro properties assessed by 86Rb(+) efflux and whole-cell patch-clamp assays using HEK293 cells stably transfected with the BK(Ca) alpha subunit. In vitro functional characterization of BK(Ca) channel opening activity was also assessed by measurement of relaxation of smooth muscle tissue strips obtained from Landrace pig bladders. The preliminary SAR data indicate the importance of steric bulk around the 2-amino substituent.     

Reactive oxygen species impair Slo1 BK channel function by altering cysteine-mediated calcium sensing

Vascular dysfunction is a hallmark of many diseases, including coronary heart disease, stroke and diabetes. The underlying mechanisms of these disorders, which are intimately associated with inflammation and oxidative stress caused by excess reactive oxygen species (ROS), have remained elusive. Here we report that ROS are powerful inhibitors of vascular smooth muscle calcium-dependent Slo1 BK or Maxi-K potassium channels, an important physiological determinant of vascular tone. By targeting a cysteine residue near the Ca(2+) bowl of the BK alpha subunit, H(2)O(2) virtually eliminates physiological activation of the channel, with an inhibitory potency comparable to a knockout of the auxiliary subunit BK beta 1. These results reveal a molecular structural basis for the vascular dysfunction involving oxidative stress and provide a solid rationale for a potential use of BK openers in the prevention and treatment of cardiovascular disorders.     

Two different types of channels are targets for potassium channel openers in Xenopus oocytes

K+ channel openers elicit K+ currents in follicle-enclosed Xenopus oocytes. The most potent activators are the pinacidil derivatives P1075 and P1060. The rank order of potency to activate K+ currents in follicle-enclosed oocytes was: P1075 (K0.5:5 microM) greater than P1060 (K0.5:12 microM) greater than BRL38227 (lemakalim) (K0.5:77 microM) greater than RP61410 (K0.5:100 microM) greater than (-)pinacidil (K0.5:300 microM). Minoxidil sulfate, nicorandil, RP49356 and diazoxide were ineffective. Activation by the K+ channel openers could be abolished by the antidiabetic sulfonylurea glibenclamide. It was not affected by the blocker of the Ca(2+)-activated K+ channels charybdotoxin. The various K+ channel openers failed to activate glibenclamide-sensitive K+ channels in defolliculated oocytes, but BRL derivatives (K0.5 for BRL38226 is 150 microM) and RP61419 inhibited a background current. The channel responsible for this background current is K+ permeable but not fully selective for K+. It is resistant to glibenclamide. It is inhibited by Ba2+, 4-aminopyridine, Co2+, Ni2+ and La3+.     

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.     

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.     

Anticancer effects of a novel class rosin-derivatives with different mechanisms

In this Letter, the anticancer activity of novel rosin-derivatives introducing indicated side chains at position C18 of dehydroabietic acid (DHAA) was reported. Gratifyingly, all of these derivatives showed significantly cytotoxicity toward diverse human carcinoma cell lines. We found the compound 4 could induce cell membrane damage at high concentration as well as cell apoptosis at low concentration. However, compound 5, attachment of quinidine to dehydroabietic acid via thiourea bond, only induced apoptotic cell death. In addition, all these active compounds induced apoptosis mainly through mitochondrial-dependent pathway. Interestingly, compound 5 exhibited the highest anticancer activity and little toxicity to normal cells compared with the other derivatives. Therefore, 5 merits further investigation as a potential agent for future anticancer treatment.     

Direct chemotactic effect of bradykinin and related peptides-significance of amino- and carboxyterminal character of oligopeptides in chemotaxis of tetrahymena pyriformis.

The chemotactic character of the nonapeptide bradykinin (BK1-9) and its derivatives was studied in the eukaryotic ciliated model Tetrahymena pyriformis. The results demonstrate that BK1-9 has a direct and ligand-specific chemoattractant effect (maximal at 10(-11) m) without any intermediate substance as is essential in some mammalian test systems. Evaluation of the chemotactic effect elicited by derivatives showed that the presence of N- and C-terminal arginines can influence chemotactic potency of the molecule via expression of pyrrolidine and aromatic ring structures of terminal amino acid residues. Removal of the N-terminal Arg (expression of Pro) results in a significant decrease in chemotaxis (BK2-9), while further truncation of the C-terminal, causing expression of the aromatic ring of Phe (BK2-8), results in a highly chemoattractant variant. A single pyrrolidine ring on the C-terminus BK1-7 also has a positive effect on the chemotactic character, however further truncation (BK1-6, BK1-5) causes the chemoattractant character to become chemorepellent. Study of chemotactic selection with BK derivatives supports our previous findings that only phylogenetically selected ligands or their close derivatives are able to induce long-term selection with chemotaxis.     

Cardiovascular and biological effects of K+ channel openers, a class of drugs with vasorelaxant and cardioprotective properties

Several new chemical entities (RP 52891, cromakalim and its derivatives) are potent and specific openers of vascular K+ channels. This mechanism is also shared, at least partially, by drugs such as minoxidil, diazoxide, pinacidil and nicorandil. The opening of plasmalemma K+ channels produces loss of cytosolic K+. This effect results in cellular hyperpolarization and functional vasorelaxation. In normotensive or hypertensive rats, K+ channel activators decrease aortic blood pressure (by producing a directly mediated fall in systemic vascular resistance) and reflexly increase heart rate. The former effect is not modified by specific blockers of classical vascular receptors but it is completely antagonized by the hypoglycemic sulphonylurea, glibenclamide, an established blocker of ATP-regulated K+ channels. K+ channel openers produce selective coronary vasodilatation and afford functional and biochemical protection to the ischemic myocardium. This salutary effect is mediated via cardiac K+ channel modulation and may result from an improved myocardial oxygen balance in the ischemic region. K+ channel openers increase plasma renin activity in animals as well as in man. However, only diazoxide, but not cromakalim or RP 52891, lowers plasma insulin concentration. The dose of glibenclamide entirely blocking the latter effect is over 50-fold smaller than that antagonizing the hypotensive and hyper-reninemic responses to diazoxide. In conclusion, K+ channel activators are potent vasorelaxant and cardioprotective agents possessing an original mechanism of action which is the opening of plasmalemma ATP-regulated K+ channels. Their clinical use as antihypertensive agents may be accompanied by undesirable effects (characteristic of peripheral vasodilators) which are likely to be attenuated or avoided by controlled release formulations. However, inasmuch as low doses of K+ channel openers may be sufficient to produce selective coronary artery dilatation and cardioprotection, these compounds could be of particular value in treating patients with coronary artery disease efficaciously and possibly without adverse cardiovascular effects.     

Novel oxime and oxime ether derivatives of 12,14-dichlorodehydroabietic acid: design, synthesis, and BK channel-opening activity

Oxime ether derivatives of the benzylic ketone of 12,14-dichlorodehydroabietic acid (diCl-DHAA, 4b) were synthesised, and their BK channel-opening activity was evaluated in an assay system of CHO-K1 cells expressing hBKalpha channels. Oxime ether structure on the B ring of diCl-DHAA significantly increased the BK channel-opening activity.     

Synthesis and vasorelaxant activity of new 1,4-benzoxazine derivatives potassium channel openers

As part of a search for new potassium channel openers, the synthesis and vasorelaxant activity of new 1,4-benzoxazine derivatives derived from transformation of the benzopyran skeleton of cromakalim were described. Several new 1,4-benzoxazine derivatives were provided with significant vasorelaxant activity with an overall pharmacological behavior similar to CRK (1f, 1i, 2d, 2e, 2f and 2i).     

Synthesis and pharmacological evaluation of 4H-1,4-benzothiazine-2-carbonitrile 1,1-dioxide and N-(2-cyanomethylsulfonylphenyl)acylamide derivatives as potential activators of ATP sensitive potassium channels

1,2,4-Thiadiazine derivatives, like 3-methyl-7-chlorobenzo-4H-1,2,4-thiadiazine 1,1-dioxide, diazoxide and 7-chloro-3-isopropylamino-4H-benzo-1,2,4-thiadiazine 1,1-dioxide, BPDZ 73, are potent openers of Kir6.2/SUR1 K(ATP) channels. To explore the structure-activity relationship of this series of K(ATP) openers, 4H-1,4-benzothiazine-2-carbonitrile 1,1-dioxide and N-(2-cyanomethylsulfonylphenyl)acylamide derivatives were synthesized from 2-acetylamino-5-chloro-benzenesulfonic acid pyridinium salt or 2-aminobenzenethiols. The 4H-1,4-benzothiazine-2-carbonitrile 1,1-dioxide derivatives (e.g., 7-chloro-3-isopropylamino-4H-1,4-benzothiazine-2-carbonitrile 1,1-dioxide, 3f) were found to activate K(ATP) channels as indicated by their ability to hyperpolarize beta cell membrane potential, to inhibit glucose-stimulated insulin release in vitro and to increase ion currents through Kir6.2/SUR1 channel as measured by patch clamp. The potency and efficacy of, for example, 3f is however significantly reduced compared to the corresponding 4H-1,2,4-benzothiadiazine 1,1-dioxide derivatives. Opening of the 4H-1,2,4-thiadiazine ring to get (e.g., 2-cyanomethylsulfonyl-4-fluorophenyl) carbamic acid isopropyl ester (4c) gives rise to compounds, which are able to open K(ATP) channels but with considerable reduced potency compared to, for example, diazoxide. Compound 3a, 7-chloro-3-methyl-4H-1,4-benzothiazine-2-carbonitrile 1,1-dioxide, which inhibits insulin release in vitro from beta cells and rat islets, reduces plasma insulin levels and blood pressure in anaesthetized rats upon intravenous administration.