KCNQ2/3 openers show differential selectivity and site of action across multiple KCNQ channels.

KCNQ2/3 voltage-gated potassium channels conduct low-threshold, slowly activating and non-inactivating currents to repolarize the neuronal resting membrane potential. The channels negatively regulate neuronal excitability and KCNQ2/3 openers are efficacious in hyperexcited states such as epilepsy and pain. We developed and utilized thallium influx assays to profile novel KCNQ2/3 channel openers with respect to selectivity across KCNQ subtypes and on requirement for tryptophan 236 of KCNQ2, a critical residue for activity of the KCNQ opener retigabine. Using distinct chemical series of openers, a quinazolinone series showed relatively poor selectivity across multiple KCNQ channels and lacked activity at the KCNQ2(W236L) mutant channel. In contrast, several novel benzimidazole openers showed selectivity for KCNQ2/3 and KCNQ2 and retain activity at KCNQ2(W236L). Profiling of several hundred KCNQ2/3 openers across multiple diverse chemical series revealed that openers show differential degrees of selectivity across subtypes, with selectivity most difficult to achieve against KCNQ2. In addition, we report the significant finding that KCNQ openers can pharmacologically differentiate between homomeric and heteromeric channels containing subtypes in common. Moreover, most openers assayed were dependent on the W236 for activity, whereas only a small number appear to use a distinct mechanism. Collectively, we provide novel insights into the molecular pharmacology of KCNQ channels by demonstrating differential selectivity and site of action for KCNQ2/3 openers. The high-throughput thallium influx assays should prove useful for rapid characterization of KCNQ openers and in guiding efforts to identify selective compounds for advancement towards the clinic.

Synthesis and in vitro activity of N-benzyl-1-(2,3-dichlorophenyl)-1H-tetrazol-5-amine P2X(7) antagonists.

Synthesis and biological evaluation of a novel class of substituted N-benzyl-1-(2,3-dichlorophenyl)-1H-tetrazol-5-amine derivatives resulted in the identification of potent P2X(7) antagonists. These compounds were assayed for activity at both the human and rat P2X(7) receptors. On the benzyl moiety, a variety of functional groups were tolerated, including both electron-withdrawing and electron-donating substituents. Ortho-substitution on the benzyl group provided the greatest potency. The ortho-substituted analogs showed approximately 2.5-fold greater potency at human compared to rat P2X(7) receptors. Compounds 12 and 38 displayed hP2X(7)pIC(50)s>7.8 with less than 2-fold difference in potency at the rP2X(7).

The antihyperalgesic activity of a selective P2X7 receptor antagonist, A-839977, is lost in IL-1alphabeta knockout mice.

The pro-inflammatory cytokine interleukin-1beta (IL-1beta) has been implicated in both inflammatory processes and nociceptive neurotransmission. Activation of P2X7 receptors is the mechanism by which ATP stimulates the rapid maturation and release of IL-1beta from macrophages and microglial cells. Recently, selective P2X7 receptor antagonists have been shown to reduce inflammatory and neuropathic pain in animal models. However, the mechanisms underlying these analgesic effects are unknown. The present studies characterize the pharmacology and antinociceptive effects of a structurally novel P2X7 antagonist. A-839977 potently (IC50=20-150 nM) blocked BzATP-evoked calcium influx at recombinant human, rat and mouse P2X7 receptors. A-839977 also potently blocked agonist-evoked YO-PRO uptake and IL-1beta release from differentiated human THP-1 cells. Systemic administration of A-839977 dose-dependently reduced thermal hyperalgesia produced by intraplantar administration of complete Freund's adjuvant (CFA) (ED50=100 micromol/kg, i.p.) in rats. A-839977 also produced robust antihyperalgesia in the CFA model of inflammatory pain in wild-type mice (ED50=40 micromol/kg, i.p.), but the antihyperalgesic effects of A-839977 were completely absent in IL-1alphabeta knockout mice. These data demonstrate that selective blockade of P2X7 receptors in vivo produces significant antinociception in animal models of inflammatory pain and suggest that the antihyperalgesic effects of P2X7 receptor blockade in an inflammatory pain model in mice are mediated by blocking the release of IL-1beta.

Discovery and biological evaluation of novel cyanoguanidine P2X(7) antagonists with analgesic activity in a rat model of neuropathic pain.

We disclose the design of a novel series of cyanoguanidines that are potent (IC(50) approximately 10-100 nM) and selective (> or = 100-fold) P2X(7) receptor antagonists against the other P2 receptor subtypes such as the P2Y(2), P2X(4), and P2X(3). We also found that these P2X(7) antagonists effectively reduced nociception in a rat model of neuropathic pain (Chung model). Particularly, analogue 53 proved to be effective in the Chung model, with an ED(50) of 38 micromol/kg after intraperitoneal administration. In addition compound 53 exhibited antiallodynic effects following oral administration and maintained its efficacy following repeated administration in the Chung model. These results suggest an important role of P2X(7) receptors in neuropathic pain and therefore a potential use of P2X(7) antagonists as novel therapeutic tools for the treatment of this type of pain.

[3H]A-804598 ([3H]2-cyano-1-[(1S)-1-phenylethyl]-3-quinolin-5-ylguanidine) is a novel, potent, and selective antagonist radioligand for P2X7 receptors.

ATP-sensitive P2X7 receptors are localized on cells of immunological origin including peripheral macrophages and glial cells in the CNS. Activation of P2X7 receptors leads to rapid changes in intracellular calcium concentrations, release of the pro-inflammatory cytokine IL-1beta, and following prolonged agonist exposure, the formation of cytolytic pores in plasma membranes. Data from gene knockout studies and recently described selective antagonists indicate a role for P2X7 receptor activation in inflammation and pain. While several species selective P2X7 antagonists exist, A-804598 represents a structurally novel, competitive, and selective antagonist that has equivalent high affinity at rat (IC50 = 10 nM), mouse (IC50 = 9 nM) and human (IC50 = 11 nM) P2X7 receptors. A-804598 also potently blocked agonist stimulated release of IL-1beta and Yo-Pro uptake from differentiated THP-1 cells that natively express human P2X7 receptors. A-804598 was tritiated ([3H]A-804598; 8.1Ci/mmol) and utilized to study recombinant rat P2X7 receptors expressed in 1321N1 cells. [3H]A-804598 labeled a single class of high affinity binding sites (Kd=2.4 nM and apparent Bmax=0.56 pmol/mg). No specific binding was observed in untransfected 1321N1 cells. The pharmacological profile for P2X antagonists to inhibit [3H]A-804598 binding correlated with their ability to block functional activation of P2X7 receptors (r=0.95, P<0.05). These data demonstrate that A-804598 is one of the most potent and selective antagonists for mammalian P2X7 receptors described to date and [3H]A-804598 is a high affinity antagonist radioligand that specifically labels rat P2X7 receptors.

Synthesis and in vitro activity of 1-(2,3-dichlorophenyl)-N-(pyridin-3-ylmethyl)-1H-1,2,4-triazol-5-amine and 4-(2,3-dichlorophenyl)-N-(pyridin-3-ylmethyl)-4H-1,2,4-triazol-3-amine P2X7 antagonists.

A novel series of aminotriazole-based P2X(7) antagonists was synthesized, and their structure-activity relationships (SAR) were investigated for activity at both human and rat P2X(7) receptors. Most compounds showed greater potency at the human receptor although several analogs were discovered with potent activity (pIC(50) > or = 7.5) at both human and rat P2X(7).

Structure-activity studies of novel cyanoguanidine ATP-sensitive potassium channel openers for the treatment of overactive bladder.

A series of novel cyanoguanidine derivatives was designed and synthesized. Condensation of N-(1-benzotriazol-1-yl-2,2-dichloropropyl)-substituted benzamides with N-(substituted-pyridin-3-yl)-N'-cyanoguanidines furnished N-{2,2-dichloro-1-[N'-(substituted-pyridin-3-yl)-N''-cyanoguanidino]propyl}-substituted benzamide derivatives. These agents were glyburide-reversible potassium channel openers and hyperpolarized human bladder cells as assessed by the FLIPR membrane potential dye (KATP-FMP). These compounds were also potent full agonists in relaxing electrically stimulated pig bladder strips, an in vitro model of overactive bladder. The most active compound 9 was evaluated for in vivo efficacy and selectivity in a pig model of bladder instability. Preliminary pharmacokinetic studies in dog demonstrated excellent oral bioavailability and a t1/2 of 15 h. The synthesis, SAR studies, and biological properties of these agents are discussed.

Novel and potent 3-(2,3-dichlorophenyl)-4-(benzyl)-4H-1,2,4-triazole P2X7 antagonists.

Structure-activity relationship (SAR) studies were conducted around early tetrazole-based leads 3 and 4. Replacements for the tetrazole core were investigated and the pendant benzyl substitution was reoptimized with a triazole isostere. Triazole-based P2X(7) antagonists were identified with similar potency to the lead compound 4 but with improved physiochemical properties. Compound 12 was active in a rat model of neuropathic pain.

Structure-activity relationship studies on a series of novel, substituted 1-benzyl-5-phenyltetrazole P2X7 antagonists.

1-Benzyl-5-aryltetrazoles were discovered to be novel antagonists for the P2X(7) receptor. Structure-activity relationship (SAR) studies were conducted around both the benzyl and phenyl moieties. In addition, the importance of the regiochemical substitution on the tetrazole was examined. Compounds were evaluated for activity to inhibit calcium flux in both human and rat recombinant P2X(7) cell lines using fluorometric imaging plate reader technology. Analogues were also assayed for their ability to inhibit IL-1beta release and to inhibit P2X(7)-mediated pore formation in human THP-1 cells. Compound 15d was advanced to efficacy studies in a model of neuropathic pain where significant reversal of mechanical allodynia was observed at doses that did not affect motor coordination.

Functional characterization of large conductance calcium-activated K+ channel openers in bladder and vascular smooth muscle.

Calcium activated K(+) channels (K(Ca) channels) are found in a variety of smooth muscle tissues, the most characterized of which are the large conductance K(Ca) channels (BK(Ca) or maxi-K(+) channels). Recent medicinal chemistry efforts have identified novel BK(Ca) openers including 2-amino-5-(2-fluoro-phenyl)-4-methyl-1H-pyrrole-3-carbonitrile (NS-8), BMS-204352 and its analog 3-(5-chloro-2-hydroxy-phenyl)-3-hydroxy-6-trifluoromethyl-1,3-dihydro-indol-2-one (compound 1), and 5,7-dichloro-4-(5-chloro-2-hydroxy-phenyl)-3-hydroxy-1H-quinolin-2-one (compound 2). Although these compounds are effective BK(Ca) openers as shown by electrophysiological methods, little is known about their effects on smooth muscle contractility. In this study, the responsiveness of structurally diverse BK(Ca) openers-NS-8, compounds 1 and 2 and the well characterized nonselective NS-1619-was assessed using segments of endothelium denuded rat aorta, rat and guinea pig detrusor precontracted with extracellular K(+), and Landrace pig detrusor stimulated by electrical field. In all preparations, the compounds tested inhibited or completely abolished contractions with similar potencies (-logIC(50) values: 3.8 to 5.1). In rat aorta, in the presence of 80 mM K(+), the compounds significantly shifted the concentration-response curve to the right compared with those obtained in 30 mM K(+). These data are consistent with K(+) channel (BK(Ca) channel) activation as the underlying mechanism of relaxation by compounds that share the electrophysiological property of BK(Ca) current activation. The similar potencies at detrusor and vascular smooth muscle suggest that the achievement of smooth muscle selectivity in vitro with the representative compounds examined in this study may prove to be a challenge when targeting BK(Ca) channels for smooth muscle indications such as overactive bladder.

Design and synthesis of novel cyanoguanidine ATP-sensitive potassium channel openers for the treatment of overactive bladder.

Thiourea derivatives were identified as glyburide-reversible potassium channel openers through high-throughput screening. Based on these findings, a number of novel cyanoguanidines were designed and synthesized, which hyperpolarized human bladder K(ATP) channels. These agents are potent full agonists in relaxing electrically-stimulated pig bladder strips. The synthesis, SAR and biological properties of these agents are discussed.