Polyamines and the NMDA receptor: modifying intrinsic activities with aromatic substituents

Thirty-four spermidine (SPD) and spermine (SPM) derivatives with aromatic substituents were synthesized and tested as inhibitors of specific binding of the NMDA channel blocker [3H]MK-801 to membranes prepared from rat hippocampus and cerebral cortex. SPD and SPM derivatives with aromatic substituents at the primary amino groups were the most potent inhibitors (IC50 3.9-4.7 microM). These compounds most likely act directly at the NMDA ion channel, since 30 microM SPM had no pronounced influence on their inhibiting activities. SPD derivatives with aromatic substituents at the secondary amino group were either inactive or highly SPM-sensitive inhibitors (IC50 10-82 microM), depending on the size of the substituent. Our results support the hypothesis that an aromatic interaction site near the center of polyamine derivatives contributes to polyamine inverse agonism.     

1-Substituted pyrazolo[1,5-c]quinazolines as novel Gly/NMDA receptor antagonists: synthesis, biological evaluation, and molecular modeling study

A new set of 5,6-dihydro-pyrazolo[1,5-c]quinazoline-2-carboxylates (2-18), bearing different substituents (COOEt, Cl, Br, CH(3), and COOH) at position-1, were synthesized in order to investigate the influence of various groups at this specific position on Gly/NMDA receptor affinity and/or selectivity. All the herein reported compounds were evaluated for their binding at the Gly/NMDA, AMPA, and KA receptors. Some selected compounds were also tested for their functional antagonistic activity at both the AMPA and NMDA receptor-ion channels. The results obtained in this study have highlighted that a C-1 lipophilic substituent on the pyrazolo[1,5-c]quinazoline-2-carboxylate core shifts selectivity toward the Gly/NMDA receptor, while a C-1 anionic carboxylate residue is able to increase affinity toward this receptor subtype. In particular, the 2-carboxylic acids 15 and 16, bearing a chlorine atom at position-1, are not only potent (K(i)=0.18 and 0.16muM, respectively), but also highly Gly/NMDA versus AMPA selective (selectivity ratio>500). Furthermore, the 1,2-dicarboxylic acids 13 and 14 are endowed with the highest Gly/NMDA receptor binding activity (K(i)=0.09 and 0.059muM, respectively), among the pyrazoloquinazoline series of derivatives. A molecular modeling study has been carried out to better understand receptor affinity and selectivity of these new pyrazoloquinazoline derivatives.     

Mechanisms of blockade of glutamate receptor ionic channels: Paradox of 9-aminoacridine

9-Aminoacridine and tacrine differ from other channel blockers of NMDA receptors in that their binding prevents the closing of blocked channels and subsequent dissociation of the agonist. Structural determinants of aminoacridine derivatives underlying the blocking mechanism are still unknown. The aim of this study was to elucidate the effects of a dicationic 9-aminoacridine derivative and some other tricyclic compounds on NMDA receptors of rat hippocampal pyramidal neurons. All the compounds under study are voltage-dependent blockers of NMDA channels; their IC₅₀ values recorded at −80 mV vary from 1 to 50 μM. The dicationic derivatives demonstrate the same voltage dependence of the block as the monocationic derivatives. The monoand dicationic tricyclic compounds under study are weak blockers of AMPA receptor channels and differ from adamantane, phenylcyclohexyl and other dicationic derivatives that exhibit greater voltage dependence of the NMDA channel block and are able to induce effective suppression of AMPA channels. We conclude that the mechanisms of action of the tricyclic and dicationic 9-aminoacridine derivatives are different from that of 9-aminoacridine, since these compounds do not prevent closing of the blocked channels. This suggests that the binding site for 9-aminoacridine has specific properties and high selectivity with respect to ligand structure. Original Russian Text ? K.H. Kim, V.E. Gmiro, D.B. Tikhonov, L.G. Magazanik, 2007, published in Biologicheskie Membrany, 2007, Vol. 24, No. 1, pp. 96–104.     

Analogs of a superacidic NMDA receptor agonist, N-phthalamoyl-L-glutamic acid (PhGA): Activity and mode of interaction with the receptor recognition site

Studies of the effects of analogs of N-phthalmoyl-L-glutamic acid (PhGA, experiments on freshly isolated hippocampal neurons of rats and on mice using intracerebroventricular injection) were compared with the molecular modeling studies. Transmembrane currents evoked by applications of NMDA receptor agonists were recorded using a patch-clamp technique. Interaction of phGA derivatives with the NMDA receptors was examined by comparing their agonistic activity with that of L-aspartate. It was found that the derivatives (iso- andtere-PhGA), which contain three carboxylic groups, display a strong agonistic activity. The absence of carboxylic groups in the molecule of an agonist and lengthening of the methylene chain in the aliphatic part of the molecule result in a drop in agonistic activity.     

Reactive derivatives for affinity labeling in the ifenprodil site of NMDA receptors

To prepare thiol-reactive ifenprodil derivatives designed as potential probes for cysteine-substituted NR2B containing NMDA receptors, electrophilic centers were introduced in different areas of the ifenprodil structure. Intermediates and final compounds were evaluated by binding studies and by electrophysiology to determine the structural requirements for their selectivity. The reactive compounds were further tested for their stability and for their reactivity in model reactions; some were found suitable as structural probes to investigate the binding site and the docking mode of ifenprodil in the NR2B subunit.     

Neuroactive steroids with perfluorobenzoyl group

During an initial study in searching for the alternative derivatives suitable for photolabeling of neuroactive steroids, perfluorobenzoates and perfluorobenzamides in position 17 of 5β-androstan-3α-ol were synthesized from the corresponding 17-hydroxy and 17-amino derivatives. After transformation into glutamates or sulfates, 17α-epimers had comparable inhibitory activity at NMDA receptors to the natural neurosteroid (20-oxo-5β-pregnan-3β-yl sulfate), however, were more potent (2- to 36-fold) than their 17β-substituted analogs. In one case, fluorine in position 4' of perfluorobenzoate group was substituted with azide and activity of the final glutamate was retained comparing with the corresponding perfluorobenzoate. The series was expanded with perfluorobenzoyl derivatives of pregnanolone: Perfluorobenzamide of glutamate and perfluorobenzoate of 11α-hydroxy pregnanolone were prepared and tested. From nine tested compounds, four of them exhibit very good inhibition activity and can serve as promising leads for photolabeling experiments.     

Protection of neuronal cells against reactive oxygen species by carnosine and related compounds

Carnosine and related compounds were compared in terms of their abilities to decrease the levels of reactive oxygen species (ROS) in suspensions of isolated neurons activated by N-methyl-D-aspartic acid (NMDA) using both stationary fluorescence measurements and flow cytometry. Carnosine was found to suppress the fluorescent signal induced by ROS production and decreased the proportion of highly fluorescent neurons, while histidine showed opposite effects. N-Acetylated derivatives of both carnosine and histidine demonstrated weak (statistically indistinguishable) suppressive effects on the ROS signal. N-Methylated derivatives of carnosine suppressed intracellular ROS generation to the same extent as carnosine. This rank of effectiveness is distinct from that previously obtained for the anti-radical ability of CRCs (anserine>carnosine>ophidine). These differences suggest that the similar ability of carnosine and its N-methylated derivatives to protect neuronal cells against the excitotoxic effect of NMDA is not solely related to the antioxidant properties of these compounds.     

Comparison of the anticonvulsant activity of organic mono- and di-cations and their potential to inhibit NMDA and AMPA glutamate receptors

Effects of mono- and dicationic derivatives of adamantine and phenylcyclohexyl were studied on: (i) open channels of NMDA and AMPA glutamate receptors in the experiments on the isolated rat brain neurones, and (ii) convulsions induced by intraventricular injections of NMDA or kainate in mice. Monocations inhibited the NMDA receptors in vitro and prevented convulsions induced by NMDA in vivo, but failed to affect both the AMPA receptors and kainite-induced convulsions. Dications (IEM-1754 and IEM-1925) revealed both anti-NMDA and anti-AMPA potency in vitro, were highly effective against kainite-induced convulsions and excelled monocations in preventing the NMDA-induced ones. Evidently some steps connected with the AMPA receptor activity are involved in the genesis of the NMDA-induced convulsions. Anticonvulsant potency of IEM-1754 and IEM-1925 is comparable with those of known NMDA receptor inhibitors: memantine and MK-801. The IEM-1754 and IEM-1925 show no side effects. An incomplete correspondence between the activity in vitro and in vivo found studying some derivatives, may be due to peculiarities of their pharmacokinetics.     

A new class of N-methyl-D-aspartic acid receptor agonists and antagonists--derivatives of imidazole-4,5- and pyrazole-3,4-dicarboxylic acids]

New agonists and antagonists of the N-methyl-D-aspartic acid (NMDA) receptors were found among the derivatives of 1- or 2-alkyl-substituted imidazole-4,5- and pyrazole-3,4-dicarboxylic acids. Lipophilic surrounding of the nitrogen atom in these compounds was found to determine their ability to be either agonists or antagonists, while the distance between the terminal acidic functions was the same. An increase in the lipophilicity can also cause loss of selective action upon the NMDA receptors and occurrence of non- NMDA antagonistic activity.     

NMDA-NR2B subtype selectivity of stereoisomeric 2-(1,2,3,4-tetrahydro-1-isoquinolyl)ethanol derivatives

Enantiopure 2-(1,2,3,4-tetrahydro-1-isoquinolyl)ethanol derivatives were tested for their affinity to the ifenprodil binding site of the NMDA receptor, their potency to inhibit [3H]MK801 binding and their NMDA-NR2B subtype selectivity. The (1S,1'S)-configurated series displayed the highest affinity to the ifenprodil binding site. A reasonable potency and NMDA-NR2B subtype selectivity was found for (1S,1'S)-4c (R1=Me, R2=OMe). A high affinity to HERG K+ channels, however, suggests that (1S,1'S)-4c may involve an increased risk of cardiovascular side effects.     

Molecular electrostatic potentials in aromatic substituted 4-hydroxyquino-2-lones: Glycine/NMDA receptor antagonists

Hydroxyquinolone derivatives have proven to be useful for inhibition at the glycine binding site of N-methyl-D-aspartate (NMDA) receptor. In this work the electronic structure, molecular electrostatic potential (MESP) and vibrational characteristics of a set of C₃ substituted 4-hydroxyquino-2-lone (HQ) derivatives, which act as Glycine/NMDA receptor antagonists, have been investigated using the density functional calculations. In the optimized structures a substituent at the C₃ site of HQ tends to adopt a helical structure. MESP investigations reveal that the ligands showing better inhibition activity should possess electron-rich regions extending over the substituent and carbonyl group of HQ. A correlation of inhibitory activity to the molecular electrostatic potential topography at the carbonyl oxygen as well as to the molecular electron density topography turns out to be a significant output of the investigation. Figure Quantam chemical approach has been employed to understand the reactivity of a set of hydroxyquinolone derivatives known for their inhibition activity towards Glycine/NMDA receptor. Molecular electrostatic potential topography has been used as a tool to understand the reactivity pattern     

Synthesis and pharmacological evaluation of (2-oxaadamant-1-yl)amines

The synthesis of several (2-oxaadamant-1-yl)amines is reported. They were evaluated as NMDA receptor antagonists and several of them were more active than amantadine, but none was more potent than memantine. None of the tested compounds displayed antiviral activity. Two of the derivatives showed a significant level of trypanocidal activity.     

A Glycine Site Associated with N-Methyl-d-Aspartic Acid Receptors: Characterization and Identification of a New Class of Antagonists

Membranes from rat telencephalon contain a single class of strychnine-insensitive glycine sites. That these sites are associated with N-methyl-D-aspartic acid (NMDA) receptors is indicated by the observations that [3H]glycine binding is selectively modulated by NMDA receptor ligands and, conversely, that several amino acids interacting with the glycine sites increase [3H]N-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP) binding to the phencyclidine site of the NMDA receptor. The endogenous compound kynurenate and several related quinoline and quinoxaline derivatives inhibit glycine binding with affinities that are much higher than their affinities for glutamate binding sites. In contrast to glycine, kynurenate-type compounds inhibit [3H]TCP binding and thus are suggested to form a novel class of antagonists of the NMDA receptor acting through the glycine site. These results suggest the existence of a dual and opposite modulation of NMDA receptors by endogenous ligands.     

Synthesis and in vitro evaluation of N,N'-diphenyl and N-naphthyl-N'-phenylguanidines as N-methyl-D-aspartate receptor ion-channel ligands

A series of N,N'-diphenyl and N-naphthyl-N'-phenyl guanidine derivatives was synthesized as potential N-methyl-D-aspartate (NMDA) receptor positron emission tomography (PET) ligands. The affinity of the different compounds was determined using in vitro receptor binding assays, and their log P values were estimated using HPLC analysis. The effect of N'-3 and N'-3,5 substitution on affinity and lipophilicity was examined. The K(i) values ranged from 1.87 to 839nM, while log P values between 1.22 and 2.88 were observed.     

Two blocking sites of amino-adamantane derivatives in open N-methyl-D-aspartate channels.

Using whole-cell patch-clamp techniques, we studied the blockade of open N-methyl-D-aspartate (NMDA) channels by amino-adamantane derivatives (AADs) in rat hippocampal neurons acutely isolated by the vibrodissociation method. The rapid concentration-jump technique was used to replace superfusion solutions. A kinetic analysis of the interaction of AAD with open NMDA channels revealed fast and slow components of their blockade and recovery. Mathematical modeling showed that these kinetic components are evidence for two distinct blocking sites of AADs in open NMDA channels. A comparative analysis of different simplest models led us to conclude that these AAD blocking sites can be simultaneously occupied by two blocker molecules. The voltage dependence of the AAD block suggested that both sites were located deep in the channel pore.     

Determination of D-aspartate N-methyltransferase activity in the starfish by direct analysis of N-methyl-D-aspartate with high-performance liquid chromatography

We describe a method for the detection and quantification of D-aspartate N-methyltransferase activity. The enzyme catalyzes the S-adenosyl-L-methionine-dependent N-methylation of D-aspartate to form N-methyl-D-aspartate (NMDA). NMDA is detected directly by high-performance liquid chromatography (HPLC) of their (+)- and/or (-)-1-(9-fluorenyl)ethyl chloroformate fluorescent derivatives. The NMDA production in the assay mixture is linearly proportional to the incubation time and the amount of tissue homogenate. Using a 10 min incubation time, the method allows detection of the enzyme activity below 10 fmol/min. It can be used to analyze kinetic behavior and to quantify the enzyme from a wide variety of organisms.     

Inducible expression and pharmacological characterization of recombinant rat NR1a/NR2A NMDA receptors

In this study, we have established a non-neuronal cell line stably and inducibly expressing recombinant NMDA receptors (NRs) composed of rat NR1a/NR2A subunits. EcR-293 cells were transfected with rat NR1a and NR2A cDNAs using the inducible mammalian expression vector pIND. Cell colonies resistant for the selecting agents were picked and tested for NR2A mRNA as well as protein expression using quantitative RT-PCR and flow cytometry based immunocytochemistry. Clonal cells expressing functional NMDA receptors were identified by measuring NMDA-evoked ion currents, and NMDA-induced increase in cytosolic free calcium concentration in whole-cell patch-clamp and fluorimetric calcium measurements, respectively. One clone named D5/H3, which exhibited the highest response to NMDA, was chosen to examine inducibility of the expression and for pharmacological profiling of recombinant NR1a/NR2A NMDA receptors. To check inducibility, NR2A subunit expression in D5/H3 cells treated with the inducing agent muristerone A (MuA) was compared with that in non-induced cells. Both NR2A mRNA and protein expression was several folds higher in cells treated with the inducing agent. As part of the pharmacological characterization, we examined the activation of the expressed NR1a/NR2A receptors as a function of increasing concentration of NMDA. NMDA-evoked concentration-dependent increases in cytosolic [Ca2+] with an EC50 value of 41 +/- 1 microM. In addition, whereas the NMDA response was concentration-dependently inhibited by the channel blocker MK-801 (IC50 = 58 +/- 6 nM), NR2B subunit selective NMDA receptor antagonists were ineffective. Thus, this cell line, which stably and inducibly expresses recombinant NR1a/NR2A NMDA receptors, can be a useful tool for testing NMDA receptor antagonists and studying their subunit selectivity.     

Occurrence of N-methyl- -aspartate in bivalves and its distribution compared with that of N-methyl- -aspartate and , -aspartate

The presence of N-methyl- -aspartate (NMLA) was demonstrated in bivalves, Corbicula sandai and Tapes japonica. To our knowledge, this is the first report on the occurrence of NMLA in animal tissues. NMLA in bivalve tissues was identified according to the following findings; (a) its derivatives with (+)- and (−)- 1-(9-fluorenyl)ethyl chloroformate (FLEC) behaved identically with those of authentic NMLA, respectively, on high-performance liquid chromatography (HPLC), (b) its derivatives with (+)- and (−)- FLEC behaved identically with (−)- and (+)-FLEC derivatives of authentic N-methyl- -aspartate (NMDA), respectively, on HPLC and (c) its behavior on thin-layer chromatography was the same as those of authentic NMLA. We also describe the distribution of NMDA, and - and -aspartate, to which N-methylaspartate enantiomers are structurally related. NMDA was more widely dirtributed than NMLA in bivalves. These bivalves containing NMLA showed lower -aspartate contents and /( + ) ratios of aspartate, than the bivalves containing NMDA.     

Localization and interaction of N-methyl-D-aspartate and non-N-methyl-D-aspartate receptors of lamprey spinal neurons.

Small volumes of N-Methyl-D-Aspartate (NMDA) and non-NMDA excitatory amino acid receptor agonists were applied to localized regions of the dendritic trees of lamprey spinal neurons along their medial-lateral axis to obtain a spatial map of glutamate receptor distribution. Voltage clamp and frequency domain methods were used to obtain quantitative kinetic data of the voltage dependent ionic channels located both on the soma and on highly branched dendritic membranes. Pressure pulses of NMDA applied to the most peripheral regions of the dendritic tree elicited large somatic impedance increases, indicating that the most peripheral dendrites are well supplied with NMDA receptors. Experiments done with kainate did not elicit somatic responses to agonist applications on peripheral dendrites. The data obtained are consistent with the hypothesis that the activation of NMDA receptors by exogenous glutamate is significantly modified by the simultaneous activation of non-NMDA receptors, which shunts the NMDA response. The non-NMDA shunting hypothesis was tested by a combined application of kainate and NMDA to mimic the action of glutamate showing that the shunting effect of non-NMDA receptor activation virtually abolished the marked voltage dependency typical of NMDA receptor activation. These data were interpreted with a compartmental neuronal model having both NMDA and non-NMDA receptors.     

Effects of felbamate,kynurenic acid derivatives and nmda antagonists on in vitro kainate-induced epileptiform activity

The effects of the novel anticonvulsant felbamate, which binds to the 5–7 dichlorokynurenic binding sites, were tested towards the CA1 epileptiform activity induced in rat hippocampal slices by kainic acid. The effects of the kynurenic acid derivatives 7-chlorokynurenic acid and 5–7-dichlorokynurenic acid and of the NMDA antagonists COS 19755, MK-801 and ketamine were also studied for comparison. Slice perfusion with 1 μM kainic acid produced within 30 min the development of an evoked CA1 epileptiform bursting made up by an increase in amplitude of the primary population spikes followed by the appearance of secondary epileptiform population spikes. Slice perfusion with CGS 19755 (100 μM) or MK-801 (100 μM) or ketamine (100 μM) failed to affect within 30 min the CA1 epileptiform activity due to kainic acid. On the contrary, slice perfusion with felbamate (1.3–1.6 mM) or 7-chlorokynurenic acid (100 μM) or 5–7-dichlorokynurenic acid (100 μM) produced within 30 min a significative (P < 0.05) decrease of the kainate-induced epileptiform bursting duration. The results indicate that felbamate and kynurenic acid derivatives but not NMDA antagonists present an inhibitory effect against the epileptiform activity due to kainic acid.