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Solution-phase synthesis of various acylguanidine derivatives and the evaluation of a small library of compounds as potential sodium channel blockers are described.
The identification and characterization of a potentially ischemia-selective and orally-active sulfoxide based NMDA ion-channel blocker showing good neuroprotective activity, (R)-(+)-N-(2-chloro-5-methylthiophenyl)-N'-(3-methylsulfinylphenyl)-N'-methylguanidine (CNS 5788), is described.
In the present investigation, the rationale for the design, synthesis, and biological evaluation of potent inhibitors of neuronal Na+ channels is described. N,N'-diaryl- and N-aryl-N-aralkylguanidine templates were locked in conformations mimicking the permissible conformations of the flexible diarylguanidinium ion (AS+, AA+, SS+). The resulting set of constrained guanidines termed "lockamers" (cyclophane, quinazoline, aminopyrimidazolines, aminoimidazolines, azocino- and tetrahydroquinolinocarboximidamides) was examined for neuronal Na+ channel blockade properties. Inhibition of [14C]guanidinium ion influx in CHO cells expressing type IIA Na+ channels showed that the aminopyrimidazoline 9b and aminoimidazoline 9d, compounds proposed to lock the N,N'-diarylguanidinium in an SS+ conformation, were the most potent Na+ channel blockers with IC50's of 0.06 microM, a value 17 times lower than that of the parent flexible compound 18d. The rest of the restricted analogues with 4-p-alkyl substituents retained potency with IC50 values ranging between 0.46 and 2.9 microM. Evaluation in a synaptosomal 45Ca2+ influx assay showed that 9b did not exhibit high selectivity for neuronal Na+ vs Ca2+ channels. The retention of significant neuronal Na+ blockade in all types of semirigid conformers gives evidence for a multiple mode of binding in this class of compounds and can possibly be attributed to a poor structural specificity of the site(s) of action. Compound 9b was also found to be the most active compound in vivo based on the high level of inhibition of seizures exhibited in the DBA/2 mouse model. The pKa value of 9b indicates that 9b binds to the channel in its protonated form, and log D vs pH measurements suggest that ion-pair partitioning contributes to membrane transport. This compound stands out as an interesting lead for further development of neurotherapeutic agents.
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Diarylguanidines, acting as NMDA receptor ion channel site ligands, represent a new class of potential neuroprotective drugs. Several diarylguanidines structurally related to N,N'-di-o-tolylguanidine (DTG), a known selective sigma receptor ligand, were synthesized and evaluated in in vitro radioligand displacement assays, with rat or guinea pig brain membrane homogenates, using the NMDA receptor ion channel site specific radioligand [3H]-(+)-5(S)-methyl-10(R),11-dihydro-5H-dibenzo[a,d]cyclohepten-5 ,10- imine (MK-801, 3), and the sigma receptor-specific radioligand [3H]-di-o-tolylguanidine (DTG, 5). This paper presents the structure-activity relationships leading to novel tri- and tetrasubstituted guanidines, which exhibit high selectivity for NMDA receptor ion channel sites and weak or negligible affinity for sigma receptors. The in vitro binding results from symmetrically substituted diphenylguanidines indicated that compounds having ortho or meta substituents (with respect to the position of the guanidine nitrogen) on the phenyl rings showed greater affinity for the NMDA receptor ion channel site compared with para-substituted derivatives. Among the group of ring substituents studied for symmetrical diarylguanidines, an isopropyl group was preferred at the ortho position and an ethyl group was preferred at the meta position. Several unsymmetrical guanidines containing a naphthalene ring on one nitrogen atom and an ortho- or a meta-substituted phenyl ring on the second nitrogen atom, e.g., N-1-naphthyl-N'-(3-ethylphenyl)guanidine (36), showed a 3-5-fold increase in affinity for the NMDA receptor ion channel site and no change in sigma receptor affinity compared to the respective symmetrical counterparts. Additional small substituents on the guanidine nitrogen atoms bearing the aryl rings resulted in tri- and tetrasubstituted guanidine derivatives which retained affinity for NMDA receptor ion channel sites but exhibited a significant reduction in their affinities for sigma receptors. For example, N-1-naphthyl-N'-(3-ethylphenyl)-N'-methylguanidine (40) showed high affinity for the NMDA receptor ion channel site (IC50 = 36 nM vs [3H]-3) and low affinity for sigma receptors (IC50 = 2540 nM vs [3H]-5). Selectivity for the NMDA receptor ion channel sites over sigma receptors appears to be dependent upon the structure of the additional substituents on the guanidine nitrogen atoms bearing the aryl groups. Methyl and ethyl substituents are most preferred in the tri- and tetrasubstituted diarylguanidines. The trisubstituted guanidine, N-1-naphthyl-N'-(3-ethylphenyl)-N'-methylguanidine (40) and its close analogues showed good in vivo neuroprotection and are potential neuroprotective drug candidates for the treatment of stroke and other neurodegenerative disorders.
Radioligand binding techniques were used to determine the affinity of a series of substituted guanidine derivatives for 1) the binding site within the ion channel of the N-methyl-D-aspartate (NMDA) receptor, as defined by displacement of MK-801 ([3H]dizocilpine) and 2) sigma sites as defined by displacement of [3H]N,N'-di-(o-tolyl)guanidine. The goal was to find ligands with high affinity and selectivity for the NMDA receptor ion-channel site. The neuroprotective activity of these compounds was assessed by their ability to protect cortical neurons from injury caused by a 5-min exposure to 500 microM glutamate in vitro. Release of lactate dehydrogenase into the culture medium by damaged neurons was used as an index of neuronal injury. The 14 compounds tested had IC50 values ranging from 37.3 nM to 12.7 microM for the NMDA receptor ion-channel site and from 8.3 nM to 7.25 microM for sigma sites. Affinity for the ion-channel site was improved by unsymmetrical substitutions on the guanidine moiety. All compounds in the series protected cortical neurons against glutamate toxicity, with EC50 values (concentration affording 50% protection) ranging from 0.38 to 28.25 microM. The neuroprotective effect of each compound was positively correlated with its ion-channel site affinity (r = 0.94); no correlation between neuroprotective efficacy and sigma site binding affinity was found (r V -0.13) establishing clearly that neuroprotection in this assay was linked to NMDA antagonist properties.
IDDC (3, 10,5-(iminomethano)-10,11-dihydro-5H-dibenzo[a,d]cycloheptene++ +) and a series of substituted derivatives were synthesized and evaluated in vitro for their ability to displace tritiated MK-801 ([3H]-2) from its specific binding site in guinea pig brain homogenate. Substitution at the 3-position of 3 with bromine, chlorine, and fluorine led to increased binding affinity. In contrast, substitution of donor groups at the 3-position gave decreased binding affinities, as did all substitutions at the 7-position and on nitrogen. Where racemic mixtures were resolved, the (+)-optical antipodes were more active than their enantiomers or racemates. The most active ligand found in this study was (+)-13e (IC50 = 15.5 +/- 4.5 nM). The affinity of (+)-13e for the PCP receptor makes it among the most potent ligands known. In vitro neuroprotection was demonstrated by 3, (+)-3, and (+)-6 (N-Me-IDDC) against glutamate-induced cell death in rat hippocampal cells.
We employed diffusion-weighted MRI (DWI) to identify regions of focal brain ischemia during the first 3 hours after permanent occlusion of the middle cerebral artery in rats. Using DWI as early as 30 minutes after the onset of ischemia, it was possible to identify the areas of brain destined to progress to infarction over the next 24 hours in untreated animals, as demonstrated by postmortem evaluation. DWI studies revealed the cerebroprotective effects of a noncompetitive N-methyl-D-aspartate receptor antagonist, CNS 1102, administered 15 minutes postocclusion, both on the cortical and caudoputaminal regions during the initial 3 hours of ischemia. Although the treatment effect lessened over the next 21 hours in a few animals with lower plasma drug levels at 3 hours, postmortem studies demonstrated a 66% reduction in the total volume of infarcted tissue with the treatment and confirmed the DWI results. T2-weighted MRI obtained at similar times revealed little or no abnormality. These results suggest that DWI provides a sensitive in vivo measure of focal cerebral ischemic injury and can assess the beneficial effects of cytoprotective therapy. DWI may be useful in the early evaluation of human stroke patients and in monitoring the effects of cerebroprotective therapies in the clinical setting.
Ischemic insults to the brain in stroke or traumatic brain injury produce excessive release of glutamate from depolarized nerve terminals. This excessive glutamate release in turn stimulates massive calcium entry into nerve cells, activating a biochemical cascade that results in cell death. A major pathway of calcium entry into depolarized nerve cells is through voltage-sensitive, high threshold calcium channels. A large fraction of this calcium entry is mediated through "R-type" calcium channels, channels resistant to blockage by dihydropyridine calcium antagonists such as nimodipine. A newly discovered compound derived from spider venom, CNS 2103, antagonizes both R-type channels and dihydropyridine-sensitive ("L-type") calcium channels. This broad spectrum of action, coupled with selectivity for calcium channels over other classes of voltage-sensitive and ligand-gated ion channels, makes CNS 2103 an interesting lead for development of drugs to treat ischemic brain injury. Activation of presynaptic ("N-type") calcium channels in nerve terminals is a primary cause of excessive neurotransmitter release in brain ischemia. Prevention of glutamate release by blockade of N-type channels in glutamatergic nerve terminals may, at an early stage in the pathophysiological cascade, abort the process leading to nerve cell death. Cambridge NeuroScience has developed a novel rapid kinetic approach for monitoring glutamate release from brain nerve terminals in vitro, and this has led to CNS 1145, a substituted guanidine that selectively blocks a kinetic component of calcium-dependent glutamate release mediated by persistent depolarization. Additional evidence suggests that CNS 1145 antagonizes presynaptic N-type calcium channels, and this may account at least in part for its ability to block glutamate release.
The conductance properties of single Cl- channels activated by glycine and gamma-aminobutyric acid (GABA) were examined in rat spinal cord neurones grown in cell culture. The majority (85%) of spinal neurones were sensitive to both glycine and GABA as were most (83%) outside-out patches tested. Glycine and GABA activated multiple conductance state Cl- channels with linear current-voltage properties when the chloride activities of the solutions bathing both sides of the membrane were similar. Glycine activated six distinct conductance states with conductances of 14, 20, 30, 43, 64 and 93 pS, whereas GABA activated five states with conductances of 13, 20, 29, 39 and 71 pS. The 30 and 43 pS states and the 20 and 29 pS states were observed most frequently with glycine and GABA, respectively. As the values of the glycine- and GABA-activated conductance states form a geometric progression when arranged in ascending order, we concluded that the channels do not consist of a cluster of identical pores. Additional conductance states (50 and 100 pS) were activated by glycine occasionally. The similarity between the conductances of the states activated by the two transmitters is consistent with the proposal that they both activate the same type of Cl- channel.
Cell-attached and whole-cell recordings were made from adult rat melanotrophs maintained in vitro by standard cell culture techniques. In cell-attached recordings the cells showed small biphasic currents which reflected spontaneous cell firing. Single channel currents often had distinct relaxations and depolarizing currents through single channels could trigger the discharge of an action potential in the cell; both observations are consistent with the high input resistance (1-10 G omega) measured in the whole-cell configuration. The discharge of action potentials occurring either spontaneously or by current injection was eliminated by tetrodotoxin or by removing Na from the external medium. A Na-dependent plateau depolarization which activated near the spike threshold was also seen. In cells exposed to tetrodotoxin and K-channel blocking agents it was possible to evoke a long-lasting (up to 20 s) action potential which was enhanced and reduced, respectively, by Ba and Cd and thus appeared to reflect currents through voltage-activated Ca channels. Small amplitude Ca-dependent depolarizations could also be evoked at membrane potentials as low as -40 mV. In cell-attached and whole-cell recordings 10 mM Ba caused the discharge of tetrodotoxin-insensitive action potentials prior to a maintained depolarization of the membrane. The low threshold for Ca-dependent depolarizations suggest that Ca influx might occur in these cells even at the resting potential. Additionally, both a Ca current and the current underlying the Na-dependent plateau depolarization may influence the rate of cell firing and in doing so further increase Ca influx through voltage-activated channels.(ABSTRACT TRUNCATED AT 250 WORDS)
Four diarylguanidine derivatives were synthesized. These compounds were found to displace, at submicromolar concentrations, 3H-labeled 1-[1-(2-thienyl)cyclohexyl]piperidine and (+)-[3H]MK-801 from phencyclidine receptors in brain membrane preparations. In electrophysiological experiments the diarylguanidines blocked N-methyl-D-aspartate (NMDA)-activated ion channels. These diarylguanidines also protected rat hippocampal neurons in vitro from glutamate-induced cell death. Our results show that some diarylguanidines are noncompetitive antagonists of NMDA receptor-mediated responses and have the neuroprotective property that is commonly associated with blockers of the NMDA receptor-gated cation channel. Diarylguanidines are structurally unrelated to known blockers of NMDA channels and, therefore, represent a new compound series for the development of neuroprotective agents with therapeutic value in patients suffering from stroke, from brain or spinal cord trauma, from hypoglycemia, and possibly from brain ischemia due to heart attack.
1. The chloride (Cl-) currents activated by caesium ions (Cs+), glycine and gamma-aminobutyric acid (GABA) were compared following their application to rat neurones that had been grown in cell culture. Recordings were made using the whole-cell patch clamp technique under voltage clamp conditions. 2. In spinal cord neurones, bicuculline methiodide antagonized GABA-activated currents more effectively than Cs+ - or glycine-activated currents. However, strychnine was more effective at blocking the currents activated by Cs+ or glycine than those activated by GABA. 3. Of the 3 agonists, only GABA activated currents in cells from the intermediate lobe of the rat pituitary. 4. In spinal neurones the size of the currents activated by 70 mM Cs+ was correlated to the size of the currents activated by 15 microM glycine (P less than 0.005; n = 10, Spearman's rank correlation), but there, was no significant correlation between the size of the currents activated by these agents and 10 microM GABA. 5. The joint application of glycine and Cs+ activated currents that were approximately twice as big as the sums of the currents activated by separate applications of the same doses. This synergism was consistent with Cs+ acting at the same receptor as glycine (7 microM glycine being equivalent to 31 +/- 7 mM Cs+). 6. It was concluded that Cs+ activates the same Cl- channel as the inhibitory neurotransmitter glycine.
One of the goals in studying the electrical properties of neurosecretory cells is to relate their electrical activity to the process of secretion. A central question in these studies concerns the role of transmembrane calcium ion flux in the initiation of the secretory event. With regard to the secretory process in pituitary cells, several research groups have addressed this question in vitro using mixed primary anterior pituitary cell cultures or clonal cell lines derived from pituitary tumours. Other workers, including ourselves, have used homogeneous cell cultures derived from the pituitary intermediate lobes of rats to examine the characteristics of voltage-dependent conductances, the contribution of these conductances to action potentials and their role in stimulus-secretion coupling. Pars intermedia (PI) cells often fire spontaneous action potentials whose frequency can be modified by the injection of sustained currents through the recording electrode. In quiescent cells action potentials can also be evoked by the injection of depolarizing current stimuli. At around 20 degrees C these action potentials have a duration of about 5 ms. Although most of the inward current during action potentials is carried by sodium ions, a calcium ion component can be demonstrated under abnormal conditions. Voltage-clamp experiments have revealed that the membrane of these cells contains high-threshold, L-type, Ca2+ channels and low-threshold Ca2+ channels. Since hormone release from PI cells appears not to be dependent on action potential activity but does depend on external calcium ions, it is not clear what role these Ca2+ channels play in stimulus-secretion coupling in cells of the pituitary pars intermedia. One possibility is that the low-threshold Ca2+ channels are more important to the secretory process than the high-threshold channels.
1. The macroscopic currents induced in cultured rat melanotrophs by exogenous gamma-aminobutyric acid (GABA) were analysed using the patch clamp recording technique. 2. Using various concentrations of intra- and extracellular chloride it was demonstrated that the conductance activated by GABA was chloride selective. Since these currents were blocked with bicuculline and enhanced with chlordiazepoxide the involvement of GABAA receptors similar to those in the CNS is indicated. 3. When chloride was symmetrically distributed across the membrane the voltage/current relationship was linear; pronounced rectification of GABA mediated currents was evident when there was an asymmetrical distribution of chloride. 4. With concentrations of GABA greater than 10 microM a fading of the current was seen during prolonged (5-10 s) applications. This effect appeared to be due to a decline of conductance rather than a shift of the chloride equilibrium potential. 5. Values for the Hill coefficient derived from dose-response curves suggested that the binding of 2 molecules of GABA to the receptor is required for the activation of the chloride channel. 6. There was no indication of a direct, GABAB receptor-mediated change of conductance.
1. Caesium ions (Cs+), applied extracellularly, caused a decrease in the input resistance of cultured spinal cord (s.c.) neurones and depolarized the neurones when they contained 140 mM-CsCl. 2. The reversal potential for Cs+-activated currents shifted 56 mV on average for a 10-fold reduction in the intracellular chloride ion (Cl-) activity, indicating that the Cs+-activated currents were specific to Cl-. 3. The activation of Cl- currents by Cs+ was not due to the depolarization-evoked release of neurotransmitter from presynaptic terminals. We therefore suggest that Cs+ were acting directly on the extracellular surface of the s.c. neurones to activate Cl- currents. 4. Cs+-activated currents showed desensitization in the presence of 140 mM-Cs+. 5. The log-log plot of the dose-response data could be fitted with a straight line with a slope of 1.7 +/- 0.4 (n = 6), indicating that at least 2 Cs+ were needed to activate a single Cl- channel. The KD of the Cs+-induced response was greater than 69 mM. 6. In outside-out patches Cs+ activated single Cl- channels. These channels were not activated by sodium or potassium ions. 7. The Cs+-activated channels displayed a total of five distinct conductance states which had mean conductances of 20, 30, 43, 66 and 92 pS. The 30 and 43 pS states were the most frequently occurring states. 8. The conductance states of the Cs+-activated channel have the same conductances as those reported for gamma-aminobutyric acid (GABA)- and glycine-activated channels in rat s.c. neurones. We therefore conclude that Cs+ activate the same type of Cl- channel as GABA and glycine through an unidentified receptor.
This report describes the techniques we have developed to record changes in intracellular calcium concentration which take place during action potentials in the cell body of rat dorsal root ganglion neurones in vitro. The photoprotein, aequorin, was microinjected into the cell body of individual sensory neurones and light output from Ca2+-activated aequorin molecules was recorded with a photomultiplier tube attached to a modified inverted microscope. Aspects of the technology outlined here include: cell culture methods; a flow chamber for electrophysiological experiments on cell culture preparations; modifications to our inverted microscope; use of 150 mM KCl-filled microelectrodes; and an electronic device for processing the photomultiplier output. Some preliminary results are presented.
A two-microelectrode voltage-clamp technique was used to record membrane current responses induced by the application of the neutral amino acids, gamma-aminobutyric acid (GABA) and glycine, to mouse spinal neurones maintained in cell culture. Membrane current responses to GABA were depressed by the convulsant agents, bicuculline, picrotoxin and pentylenetetrazole. Membrane current responses to glycine were depressed by the convulsant compound, strychnine. Analysis of the fluctuations in membrane current about the mean response during prolonged applications of GABA revealed that neither the conductance nor the average open-time of GABA-activated ion-channels was altered in the presence of picrotoxin or bicuculline. Pentylenetetrazole caused a reduction in the open-time of GABA-activated ion-channels but this effect alone was too small to account for the depression of the current response. A similar analysis of glycine-induced responses in the presence of strychnine revealed that the depression of glycine responses by strychnine was not associated with any changes in the conductance or average open-time of glycine-activated ion-channels. We therefore conclude that the convulsant compounds act by reducing the rate of activation of ion-channels by the neutral amino acids either through a reduction in the number of free receptors or by a change in the kinetics of channel activation.