Biomedical subjects
Publications and source records attributed to B Ebert.
[structure: see text] The first seleninic acid analogues of gamma-aminobutyric acid (GABA) and of the specific GABA(A) agonist piperidine-4-carboxylic acid (isonipecotic acid), 1 and 2, respectively, have been synthesized and shown to be potent agonists at the GABA receptors.
The excitotoxic effects of the glutamate receptor agonists kainic acid (KA) and 2-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and the corresponding neuroprotective effects of the AMPA/KA receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX) were examined in corticostriatal slice cultures. The purpose was to examine the feasibility of these cultures for excitotoxic studies, and to demonstrate possible differential excitotoxic effects of KA and AMPA on striatal and cortical neurons. Slices of dorsolateral striatum with overlying neocortex were obtained from neonatal rats and grown on semiporous membranes in serum-free medium for 3-4 weeks before exposure to KA or AMPA for 48 h. The uptake by injured cells of the fluorescent dye propidium iodide (PI) added to the culture medium was used as a quantifiable measure for neuronal degeneration and compared with efflux of the cytosolic enzyme lactate dehydrogenase (LDH) into the culture medium and loss of glutamic acid decarboxylase (GAD) activity in the tissue. Histological sections were also stained by the fluorescent dye Fluoro-Jade (FJ), for degenerating neurons and by immunocytochemical staining for gamma-aminobutyric acid (GABA). Digitized images showed a dose (0-24 microM KA, 0-6 microM AMPA) and time (0-48 h) dependent increase in PI uptake in both striatum and cortex. In other cultures exposed to KA (24 microM) or AMPA (6 microM) together with NBQX (0.1-9 microM), NBQX was found to exert a differential neuroprotective effect on striatum and cortex at low doses. NBQX was thus more protective against KA in the cortex than in the striatum, while the opposite was seen in relation to AMPA. Regarding neurodegenerative markers, PI uptake was significantly correlated with (1) LDH release into the culture medium, (2) optical density of Fluoro-Jade staining, (3) loss of GAD-activity in tissue homogenates, and (4) loss of GABA-immunostained neurons. We conclude that both differences between compounds (AMPA vs. KA) and brain areas (striatum vs. cortex) can be demonstrated in corticostriatal slice cultures, which in conjunction with an established set of markers for neuronal cell damage appears to be a feasible model for studies of the neurotoxic and neuroprotective effects of glutamate receptor agonists and antagonists.
(RS)-2-Amino-3-(5-tert-butyl-3-hydroxy-4-isoxazolyl)propionic acid (ATPA), an analogue of (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA). has previously been shown to be a relatively weak AMPA receptor agonist and a very potent agonist at the GluR5 subtype of kainic acid-preferring (S)-glutamic acid ((S)-Glu) receptors. We report here the separation of (+)- and (-)-ATPA, obtained at high enantiomeric purity (enantiomeric excess values of 99.8% and > 99.8%, respectively) using chiral chromatography, and the unequivocal assignment of the stereochemistry of (S)-(+)-ATPA and (R)-(-)-ATPA. (S)- and (R)-ATPA were characterized in receptor binding studies using rat brain membranes, and electrophysiologically using the rat cortical wedge preparation and cloned AMPA-preferring (GluR1, GluR3, and GluR4) and kainic acid-preferring (GluR5, GluR6, and GluR6 + KA2) receptors expressed in Xenopus oocytes. In the cortical wedge, (S)-ATPA showed AMPA receptor agonist effects (EC50 = 23 microM) approximately twice as potent as those of ATPA. (R)-ATPA antagonized depolarizations induced by AMPA (Ki = 253 microM) and by (S)-ATPA (Ki = 376 microM), and (R)-ATPA antagonized the biphasic depolarizing effects induced by kainic acid (Ki = 301 microM and 1115 microM). At cloned AMPA receptors, (S)-ATPA showed agonist effects at GluR3 and GluR4 with EC50 values of approximately 8 microM and at GluR1 (EC50 = 22 microM), producing maximal steady state currents only 5.4-33% of those evoked by kainic acid. (R)-ATPA antagonized currents evoked by kainic acid at cloned AMPA receptor subtypes with Ki values of 33-75 microM. (S)-ATPA produced potent agonist effects at GluR5 (EC50 = 0.48 microM). Due to desensitization of GluR5 receptors, which could not be fully prevented by treatment with concanavalin A, (S)-ATPA-induced agonist effects were normalized to those of kainic acid. Under these circumstances, maximal currents produced by (S)-ATPA and kainic acid were not significantly different. (R)-ATPA did not attenuate currents produced by kainic acid at GluR5, and neither (S)- nor (R)-ATPA showed significant effects at GluR6. (S)-ATPA as well as AMPA showed weak agonist effects at heteromeric GluR6 + KA2 receptors, whereas (R)-ATPA was inactive. Thus, (S)- and (R)-ATPA may be useful tools for mechanistic studies of ionotropic non-NMDA (S)-Glu receptors, and lead structures for the design of new subtype-selective ligands for such receptors.
(RS)-2-Amino-3-(3-carboxy-5-methyl-4-isoxazolyl)propionic acid (ACPA), which is a potent and selective agonist at (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptors, has previously been shown to desensitize AMPA receptors to a much lower degree than AMPA itself. We now report the synthesis of [3H]ACPA (32.5 Ci/mmol), the neurochemical and pharmacological characterization of [3H]ACPA binding, and a comparison of the distribution of [3H]ACPA, [3H]AMPA, and [3H](S)-5-fluorowillardiine binding sites in rat brain. Under equilibrium conditions, [3H]ACPA was shown to bind to a single population of receptor sites on rat brain membranes. [3H]ACPA was shown to bind with single and similar affinities (15-45 nM) to cloned AMPA receptor subunits (GluR1-4), expressed in insect cells, whereas a K(D) value of 330 nM was determined for the binding of [3H]ACPA to cloned kainic acid preferring GluR5 subunits. Whereas Bmax and K(D) values for [3H]ACPA binding, determined using filtration techniques, were different from such obtained in centrifugation assays, Bmax and K(D) values as well as association and dissociation constants were not significantly affected by the addition of the chaotropic agent KSCN. K(D) values, determined under equilibrium conditions, were, however, markedly different from K(D) values derived from kinetic data. Furthermore, the results of analyses of these kinetic data were consistent with the existence of two different populations of [3H]ACPA binding sites. The pharmacology of [3H]ACPA binding sites was characterized using a series of AMPA receptor agonists and antagonists. Whereas addition of KSCN had little effect on the affinities of AMPA receptor agonists for [3H]ACPA binding, this chaotropic agent reduced the affinities of AMPA receptor antagonists structurally related to AMPA. Based on these and previously reported data, the AMPA receptor agonists, ACPA, AMPA and (S)-5-fluorowillardiine, seem to bind to and activate AMPA receptors in a nonidentical fashion, and these three agonists together may be useful tools for studies of AMPA receptor mechanisms.
(RS)-5-Amino-4-(4-chlorophenyl)pentanoic acid (10) and the R-form (11) and S-form (12) of (RS)-5-amino-3-(4-chlorophenyl)pentanoic acid, which are homologues of the 4-aminobutanoic acidB (GABAB) receptor agonist (RS)-4-amino-3-(4-chlorophenyl)butanoic acid (baclofen), were synthesized. Compound 10 was synthesized by homologation at the carboxyl end of baclofen using a seven-step reaction sequence. N-Boc-protected (4R, 5R)-4-(4-chlorophenyl)-5-hydroxy-2-piperidone (18) was deoxygenated via a modified Barton-McCombie reaction to give N-Boc-protected (R)-4-(4-chlorophenyl)-2-piperidone (20), which was ring opened and deprotected to give 11.HCl. The corresponding S-enantiomer, 12.HCl, was synthesized analogously from the 4S,5S-enantiomer of 18, compound 21. The enantiomeric purities of 11.HCl (ee = 99.8%) and 12. HCl (ee = 99.3%) were determined by chiral HPLC. Compound 10 did not show detectable affinity for GABAA or GABAB receptor sites and was inactive as an agonist or an antagonist at GABAB receptors in the guinea pig ileum. Like the enantiomers of baclofen, neither 11 nor 12 showed detectable affinity for GABAA receptor sites, and in agreement with the findings for (S)-baclofen, 12 did not interact significantly with GABAB receptor sites. Compound 11 (IC50 = 7.4 +/- 0.6 microM), a homologue of (R)-baclofen (2), was shown to be some 50 times weaker than 2 (IC50 = 0.14 +/- 0.01 microM) as an inhibitor of GABAB binding. Accordingly, 11 (EC50 = 150 +/- 23 microM) was shown to be weaker than 2 (EC50 = 11 +/- 1 microM) as an inhibitor of electrically induced contractions of the guinea pig ileum. However, whereas this effect of 2 was sensitive to the GABAB antagonist, CGP35348 (4), the inhibition by 11 was not significantly affected. Furthermore, 12 (EC50 = 310 +/- 16 microM) was shown to be one-half as potent as 11 in this test system, and this effect of 12 also was insensitive to 4. The dissimilarities of the pharmacological effects of 2 and compounds 11 and 12 were emphasized by the observation that whereas 2 only inhibits the ileum contraction by 59 +/- 5%, 11 as well as 12 were shown to inhibit this response by approximately 94%. Neither 11 nor 12 appeared to affect significantly cholinergic mechanisms in the ileum, and their mechanism(s) of action remain enigmatic.
The equilibrium binding characteristics of the tritiated GABAA agonist, 5-aminomethyl-3-isothiazolol (thiomuscimol) are described. Using the filtration technique to separate bound- from free-ligand, [3H]thiomuscimol was shown to bind to the GABA(A) receptor site(s) in a saturable manner with a Kd value of 28+/-6.0 nM and a Bmax value of 50+/-4.0 fmol/mg original tissue. In parallel binding experiments, the Kd and Bmax values for [3H]muscimol were determined to be 5.4+/-2.8 nM and 82+/-11 fmol/mg original tissue, respectively. In binding assays using the centrifugation technique, Kd and Bmax values for [3H]thiomuscimol were found to be 116+/-22 nM and 154 13 fmol/mg original tissue, respectively, whereas a Kd value of 16+/-1.8 nM and a Bmax value of 155+/-8.0 fmol/mg original tissue were determined for [3H]muscimol. In comparative inhibition studies using the GABA(A) antagonist SR 95531 and a series of specific GABAA agonists, the binding sites for [3H]thiomuscimol and [3H]muscimol were shown to exhibit similar pharmacological profiles. Autoradiographic studies disclosed similar regional distribution of [3H]thiomuscimol and [3H]muscimol binding sites in rat brain. Highest densities of binding sites were detected in cortex, hippocampus, and cerebellum, whereas low densities were measured in the midbrain structures of rat cortex. In conclusion, the equilibrium GABA(A) receptor binding characteristics of [3H]thiomuscimol are very similar to those of [3H]muscimol.
Much evidence points to the involvement of N-methyl-D-aspartate (NMDA) receptors in the development and maintainance of neuropathic pain. In neuropathic pain, there is generally involved a presumed opioid-insensitive component, which apparently can be blocked by NMDA receptor antagonists. However, in order to obtain complete analgesia, a combination of an NMDA receptor antagonist and an opioid receptor agonist is needed. Recent in vitro data have demonstrated that methadone, ketobemidone, and dextropropoxyphene, in addition to being opioid receptor agonists, also are weak noncompetitive NMDA receptor antagonists. Clinical anecdotes suggest that the NMDA receptor antagonism of these opioids may play a significant role in the pharmacological action of these compounds; however, no clinical studies have been conducted to support this issue. In the present commentary, we discuss evidence for the NMDA receptor antagonism of these compounds and its relevance for clinical pain treatment; an overview of structure-activity relationships for the relevant opioids as noncompetitive NMDA receptor antagonists also is given. It is concluded that although the finding that some opioids are weak noncompetitive NMDA receptor antagonists in vitro has created much attention among clinicians, no clinical studies have been conducted to evaluate the applicability of these compounds in the treatment of neuropathic pain conditions.
We have previously shown that (S)-2-amino-3-(3-hydroxy-5-phenyl-4-isoxazolyl)propionic acid [(S)-APPA, 2] is a weak agonist at (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptors, specifically activated by (S)-AMPA (1), whereas (S)-2-amino-3-[3-hydroxy-5-(2-pyridyl)-4-isoxazolyl]propionic acid [(S)-2-Py-AMPA, 5] and (RS)-2-amino-3-[3-hydroxy-5-(2-thiazolyl)-4-isoxazolyl]propionic acid (4) are potent AMPA agonists. On the other hand, (R)-APPA (3) and (R)-2-Py-AMPA (6) have been shown to be weak AMPA antagonists. We now report the synthesis of 2-Py-AMPA (7a) and the isomeric compounds 3-Py-AMPA (7b) and 4-Py-AMPA (7c) as well as the 7a analogues, (RS)-2-amino-3-[3-hydroxy-5-(6-methyl-2-pyridyl)-4-isoxazolyl]p ropion ic acid (7d) and (RS)-2-amino-3-[3-hydroxy-5-(2-quinolinyl)-4-isoxazolyl]propionic acid (7e). Furthermore, (RS)-2-amino-3-[3-hydroxy-5-(2-furyl)-4-isoxazolyl]propionic acid (2-Fu-AMPA, 7f) and its 5-bromo-2-furyl derivative (7g) were synthesized, and (S)-2-Fu-AMPA (8) and (R)-2-Fu-AMPA (9) were prepared by semipreparative chiral HPLC resolution of 7f. HPLC analyses and circular dichroism spectroscopy indicated the absolute stereochemistry of 8 and 9 to be S and R, respectively. This was confirmed by an X-ray crystallographic analysis of 9.HCl. In receptor binding (IC50 values) and rat cortical wedge electrophysiological (EC50 values) studies, 7c (IC50 = 5.5 +/- 0.6 microM; EC50 = 96 +/- 5 microM) was shown to be markedly weaker than 7a (IC50 = 0.57 +/- 0.16 microM; EC50 = 7.4 +/- 0.2 microM) as an AMPA agonist, whereas 7b,d,e were inactive. The very potent AMPA agonist effect of 7f (IC50 = 0.15 +/- 0.03 microM; EC50 = 1.7 +/- 0. 2 microM) was shown to reside exclusively in 8 (IC50 = 0.11 +/- 0.01 microM; EC50 = 0.71 +/- 0.11 microM), whereas 9 did not interact significantly with AMPA receptors, either as an agonist or as an antagonist. 8 was shown to be photochemically active and is a potential photoaffinity label for the recognition site of the AMPA receptors. Compound 7g turned out to be a very weak AMPA receptor agonist (IC50 = 12 +/- 0.7 microM; EC50 = 160 +/- 15 microM). None of these new compounds showed detectable effects at N-methyl-d-aspartic acid (NMDA) or kainic acid receptors in vitro. The present studies have emphasized that the presence of a heteroatom in the 2-position of the heteroaryl 5-substituent greatly facilitates AMPA receptor agonist activity.
(RS)-2-Amino-3-(4-butyl-3-hydroxyisoxazol-5-yl)propionic acid (Bu-HIBO, 6) has previously been shown to be an agonist at (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) receptors and an inhibitor of CaCl2-dependent [3H]-(S)-glutamic acid binding (J. Med. Chem. 1992, 35, 3512-3519). To elucidate the pharmacological significance of this latter binding affinity, which is also shown by quisqualic acid (3) but not by AMPA, we have now resolved Bu-HIBO via diastereomeric salt formation using the diprotected Bu-HIBO derivative 11 and the enantiomers of 1-phenylethylamine (PEA). The absolute stereochemistry of (S)-Bu-HIBO (7) (ee = 99.0%) and (R)-Bu-HIBO (8) (ee > 99.6%) were established by an X-ray crystallographic analysis of compound 15, a salt of (R)-PEA, and diprotected 8. Circular dichroism spectra of 7 and 8 were recorded. Whereas 7 (IC50 = 0.64 microM) and 8 (IC50 = 0.57 microM) were equipotent as inhibitors of CaCl2-dependent [3H]-(S)-glutamic acid binding, neither enantiomer showed significant affinity for the synaptosomal (S)-glutamic acid uptake system(s). AMPA receptor affinity (IC50 = 0.48 microM) and agonism (EC50 = 17 microM) were shown to reside exclusively in the S-enantiomer, 7. Compounds 7 and 8 did not interact detectably with kainic acid or N-methyl-D-aspartic acid (NMDA) receptor sites. Neither 7 nor 8 affected the function of the metabotropic (S)-glutamic acid receptors mGlu2 and mGlu4a, expressed in CHO cells. Compound 8 was shown also to be inactive at mGlu1 alpha, whereas 7 was determined to be a moderately potent antagonist at mGlu1 alpha (Ki = 110 microM) and mGlu5a (Ki = 97 microM). Using the rat cortical wedge preparation, the AMPA receptor agonist effect of 7 was markedly potentiated by coadministration of 8 at 21 degrees C, but not at 2-4 degrees C. These observations together indicate that the potentiation of the AMPA receptor agonism of 7 by 8 is not mediated by metabotropic (S)-glutamate receptors but rather by the CaCl2-dependent (S)-glutamic acid binding system, which shows the characteristics of a transport mechanism. After intravenous administration in mice, 7 (ED50 = 44 mumol/kg) was slightly more potent than AMPA (1) (ED50 = 55 mumol/kg) and twice as potent as Bu-HIBO (6) (ED50 = 94 mumol/kg) as a convulsant, whereas 8 was inactive. After subcutaneous administration in mice, Bu-HIBO (ED50 = 110 mumol/kg) was twice as potent as AMPA (ED50 = 220 mumol/kg) as a convulsant. Since 7 and Bu-HIBO (EC50 = 37 microM) are much weaker than AMPA (EC50 = 3.5 microM) as AMPA receptor agonists in vitro, the presence of a butyl group in the molecules of Bu-HIBO and 7 seems to facilitate the penetration of these compounds through the blood-brain barrier.
In order to elucidate whether opioid analgesics available on the Scandinavian market also act as noncompetitive N-methyl-D-aspartate (NMDA) antagonists, a series of commercially available opioids were screened for their affinity in [3H](RS)-5-methyl-10, 11-dihydro-5H-dibenzo[a,d]cycloheptene-5,10-imine ([3H]MK-801) binding assays and potential inhibitory actions on responses to NMDA in the rat cortical wedge preparation. Of the screened compounds (codeine, dextropropoxyphene, etorphine, fentanyl, and morphine), only dextropropoxyphene, with an IC50 value in [3H]MK-801 binding of 5 microM, was found to be active. Further characterization of the interaction of dextropropoxyphene with the NMDA response in the rat cortical wedge preparation illustrated the noncompetitive NMDA antagonist activity of dextropropoxyphene. Analysis of the dextropropoxyphene inhibition curve of NMDA gave an IC50 value of 190 microM and a Hill slope of 0.8.
We have previously shown that (RS)-2-amino-3-(3-hydroxy-5-phenylisoxazol-4-yl)propionic acid (APPA, 2) is a functional partial agonist at the (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) subtype of excitatory amino acid receptors, reflecting that (S)-APPA is a full agonist and (R)-APPA a competitive antagonist at AMPA receptors. We have now synthesized and pharmacologically characterized (RS)-2-amino-3-[3-hydroxy-5-(2-fluorophenyl)isoxazol-4-yl]propioni c acid (2-F-APPA, 5a), 3-F-APPA (5b), 4-F-APPA (5c), (S)-4-F-APPA (6), (R)-4-F-APPA (7), and the fully and partially, respectively, saturated APPA (2) analogues, (RS)-2-amino-3-(3-hydroxy-5-cyclohexylisoxazol-4-yl)propionic acid (5d) and compound 5e containing a 1-cyclohexenyl ring. The absolute stereochemistry of 6 and 7 was established on the basis of comparative circular dichroism studies on 6, 7, and (S)- and (R)-APPA. 4-F-APPA (5c), (S)-4-F-APPA (6), 5d, and 5e were shown to selectively inhibit [3H]AMPA binding and to activate AMPA receptors. Whereas (S)-4-F-APPA (6) showed full AMPA receptor agonism, (R)-4-F-APPA (7) was an AMPA receptor antagonist. Co-administration of (S)- and (R)-4-F-APPA to the rat cortical wedge preparation produced functional partial AMPA receptor agonism. Semi empirical calculations showed that the magnitude of the torsional angle of the bond connecting the two rings in the series of nonannulated bicyclic AMPA analogues appears to be of importance for the potency and efficacy of these compounds.
Whole-cell patch-clamp recordings from single cultured cortical neurones have been used to study the action of (RS)-2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl+ ++]propionic acid (ATPO), which has previously been proposed to be a potent selective antagonist of 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptors. ATPO competitively reduced peak responses evoked by semi-rapid applications of AMPA (Ki = 16 microM) but had variable effects on plateau responses, which were on average unchanged. Following blockade of AMPA receptor desensitization by cyclothiazide (CTZ, 100 microM), the plateau responses were reduced by ATPO to a similar extent as the peak responses, indicating that ATPO reduces desensitization of AMPA receptors. Semi-rapid application of kainic acid (KA) and the KA receptor-selective agonist, (2S,4R)-4-methylglutamic acid (MeGlu) evoked non-desensitizing responses which were competitively antagonized by ATPO (Ki values: 27 and 23 microM, respectively). Responses to MeGlu were unaffected by CTZ (100 microM), but potentiated 3 fold following blockade of KA receptor desensitization by concanavalin A (Con A, 300 microg ml(-1)). Responses of spinal cord neurones to MeGlu were blocked by ATPO to a similar extent before and after blockade of KA receptor desensitization by Con A. Although selectively potentiated by Con A, plateau responses to MeGlu were reduced by 69.6% by the AMPA selective antagonist, GYKI 53655 (10 microM). The remaining component was further reduced by ATPO with a Ki of 36 microM, which was not significantly different from that in the absence of GYKI 53655, but was greater than that on responses to AMPA. It is concluded that ATPO is a moderate-potency competitive inhibitor of naturally expressed non-NMDA receptors.
The opioid, ketobemidone, has previously been shown to be a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist. In Denmark, ketobemidone is available in a formulation which contains ketobemidone and a spasmolytic compound, (RS)-3-dimethylamino-1,1-diphenylbut-1-ene, hydrochloride (A29), in a one to five ratio. Using in vitro receptor binding techniques and an in vitro electrophysiological preparation consisting of rat cerebral cortex, we have characterized the interaction between A29 and the different glutamate receptor subtypes. A29 selectively inhibited binding of the non-competitive NMDA receptor antagonist 3H-MK-801 with a Ki value 16 +/- 4.5 microM, but was inactive in assays measuring affinities for other glutamate receptors. In agreement with the binding studies, A29 was found to selectively inhibit responses to NMDA in the rat cortical wedge preparation, whereas responses to kainate and AMPA were unaffected. Analysis of dose response curves showed A29 to be a NMDA receptor antagonist with an IC50 value of 100 microM versus responses to 10 microM NMDA. The inhibitory effects of ketobemidone and A29 on responses to 10 microM NMDA were additive. These data show that the combination of A29 and ketobemidone exert more potent antagonism at the NMDA receptor than does ketobemidone alone.
In general, death of a person--also in cases of a so called "sudden death"--is followed by a certification of death, normally by the physician who attended the patient in his last illness, but doctors in external e.g. extraclinical emergency care systems as well are liable to fill in death certificates concerning these unknown patients whose sudden and in general unexpected cases of death mostly happened under dubious circumstances. Thus, doctors in extraclinical care are highly susceptible to miss cases of homicide, cases with relevant forensic and statistical aspects or claims of relatives concerning insurance premiums of the underwriter. By demonstrating three different case reports of "sudden-death"-incidents, the circumstances of death are illuminated and consequences are shown.
A number of 3-isoxazolol bioisosteres, 7a-i, of (S)-glutamic acid (Glu), in which the methyl group of (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA, 1) was replaced by different 5-membered heterocyclic rings, were synthesized. Comparative in vitro pharmacological studies on this series of AMPA analogues were performed using receptor binding assays (IC50 values) and the electrophysiological rat cortical slice model (EC50 values). None of these compounds showed detectable affinity for the N-methyl-D-aspartic acid subtype of Glu receptors. Some of the compounds were weak inhibitors of [3H]kainic acid binding. The inhibitory effects on [3H]AMPA binding and agonist potencies at AMPA receptors of 7a-i were strictly dependent on the structure, electrostatic potential, and methyl substitution of the heterocyclic 5-substituent. Thus, while 7a (IC50 = 0.094 microM; EC50 = 2.3 microM) was approximately equipotent with AMPA (IC50 = 0.023 microM; EC50 = 5.4 microM), (RS)-2-amino-3-[3-hydroxy-5-(1H-imidazol-2-yl)isoxazol-4-yl]propio nic acid (7b) (IC50 = 48 microM; EC50 = 550 microM) was some 2 orders of magnitude weaker than AMPA, and (RS)-2-amino-3-[3-hydroxy-5-(1-methyl-1H-imidazol-2-yl)-isoxazol-4 -yl] propionic acid (7c) (IC50 > 100 microM; EC50 > 1000 microM) was inactive. Furthermore, (RS)-2-amino-3-[3-hydroxy-5-(2-methyl-2H-tetrazol-5-yl)isoxazol -4-yl] propionic acid (7i) (IC50 = 0.030 microM; EC50 = 0.92 microM) was more potent than AMPA, whereas its N-1 methyl isomer, (RS)-2-amino-3-[3-hydroxy-5-(1-methyl-1H-tetrazol-5-yl)isoxazol -4-yl] propionic acid (7h) (IC50 = 54 microM; EC50 > 1000 microM) was inactive as an AMPA agonist. A quantitative structure-activity relationship (QSAR) analysis revealed a positive correlation between receptor affinity, electrostatic potential near the nitrogen atom at the "ortho" position of the heterocyclic 5-substituent, and the rotational energy barrier around the bond connecting the two rings. We envisage that a hydrogen bond between the protonated amino group and an ortho-positioned heteroatom of the ring substituent at the 5-position stabilize receptor-active conformations of these AMPA analogues.
The enantiomers of the potent non-competitive NMDA receptor antagonist ketamine and its major metabolite, norketamine were evaluated as NMDA receptor antagonists using the rat cortical wedge preparation and the neonatal rat spinal cord preparation, respectively, for electrophysiological studies and [3H](RS)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-im ine ([3H]MK801) in homogenate binding experiments. In agreement with earlier studies (S)-ketamine (Ki 0.3 microM) was found to possess a 5 times higher affinity for the NMDA receptor complex than (R)-ketamine (Ki 1.4 microM). (S)-Norketamine (Ki 1.7 microM) had approximately an 8 times higher affinity than (R)-norketamine (Ki 13 microM) in the inhibition of [3H]MK-801 binding. All compounds inhibited responses to NMDA in the rat cortical wedge preparation and the hemisected neonatal rat spinal cord, being approximately four times more potent in the cortex than in the spinal cord except for (R)-norketamine being only twice as potent. In light of the clinically obtained concentrations of norketamine after oral administration of ketamine, these data strongly suggest that (S)-norketamine may contribute significantly to the clinical activity of (S)-ketamine, especially when given orally.
A number of 3-isothiazolol bioisosteres of glutamic acid (1) and analogs of the AMPA receptor agonist, (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA, 2a), including (RS)-2-amino-3-(3-hydroxy-5-methylisothiazol-4-yl)propionic acid (thio-AMPA, 2b), were synthesized. Comparative in vitro pharmacological studies on this series of 3-isothiazolol and the corresponding 3-isoxazolol amino acids were performed using a series of receptor binding assays (IC50 values) and the electrophysiological rat cortical slice model (EC50 values). Whereas 2a (IC50 = 0.04 +/- 0.005 microM, EC50 = 3.5 +/- 0.2 microM) is markedly more potent than the tert-butyl analog ATPA (3a) (IC50 = 2.1 +/- 0.16 microM, EC50 = 34 +/- 2.4 microM) in [3H]AMPA binding and electrophysiological studies, 2b (IC50 = 1.8 +/- 0.13 microM, EC50 = 15.0 +/- 2.4 microM) was approximately equipotent with thio-ATPA (3b) (IC50 = 0.63 +/- 0.07 microM, EC50 = 14 +/- 1.3 microM). (RS)-2-Amino-3-(3-hydroxyisoxazol-5-yl)propionic acid (HIBO, 4a) was approximately equipotent with its thio analog 4b, whereas 4-Br-HIBO (5a) (IC50 = 0.65 +/- 0.12 microM, EC50 = 22 +/- 0.6 microM) turned out to be much more potent than the corresponding 3-isothiazolol 5b (IC50 = 17 +/- 2.2 microM, EC50 = 500 +/- 23 microM). 2b (ED50 = 130 mumol/kg) was more potent than 2a (220 mumol/kg) as a convulsant after subcutaneous administration in mice. The protolytic properties of 2a,b-4a,b were determined using 13C NMR spectroscopy. For each pair of compounds, the alpha-amino acid groups showed similar protolytic properties, whereas the 3-isoxazolol moieties typically showed pKa values 2 units lower than those of the 3-isothiazolols. Accordingly, calculations of ionic species distributions revealed pronounced differences between 3-isoxazolol and 3-isothiazolol amino acids. No simple correlation between activity as AMPA agonists in vitro and pKa values of these compounds was apparent. On the other hand, the relative potencies of AMPA (2a) and thio-AMPA (2b) in vitro and in vivo may reflect that these compounds predominantly penetrate the blood-brain barrier as net uncharged diprotonated ionic species.
We have previously shown that whereas (RS)-2-amino-3-(3-hydroxy-5-phenylisoxazol-4-yl)propionic acid (APPA) shows the characteristics of a partial agonist at (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) receptors, (S)-APPA is a full AMPA receptor agonist and (R)-APPA a weak competitive AMPA receptor antagonist. This observation led us to introduce the new pharmacological concept, functional partial agonism. Recently we have shown that the 2-pyridyl analogue of APPA, (RS)-2-amino-3-[3-hydroxy-5-(2-pyridyl)isoxazol-4-yl]propionic acid (2-Py-AMPA), is a potent and apparently full AMPA receptor agonist, and this compound has now been resolved into (+)- and (-)-2-Py-AMPA (ee > or = 99.0%) by chiral HPLC using a Chirobiotic T column. The absolute stereochemistry of the enantiomers of APPA has previously been established by X-ray analysis, and on the basis of comparative studies of the circular dichroism spectra of the enantiomers of APPA and 2-Py-AMPA, (+)- and (-)-2-Py-AMPA were assigned the (S)- and (R)-configuration, respectively. In a series of receptor binding studies, neither enantiomer of 2-Py-AMPA showed detectable affinity for kainic acid receptor sites or different sites at the N-methyl-D-aspartic acid (NMDA) receptor complex. (+)-(S)-2-Py-AMPA was an effective inhibitor of [3H]AMPA binding (IC50 = 0.19 +/- 0.06 microM) and a potent AMPA receptor agonist in the rat cortical wedge preparation (EC50 = 4.5 +/- 0.3 microM) comparable with AMPA (IC50 = 0.040 +/- 0.01 microM; EC50 = 3.5 +/- 0.2 microM), but much more potent than (+)-(S)-APPA (IC50 = 5.5 +/- 2.2 microM; EC50 = 230 +/- 12 microM). Like (-)-(R)-APPA (IC50 > 100 microM), (-)-(R)-2-Py-AMPA (IC50 > 100 microM) did not significantly affect [3H]AMPA binding, and both compounds were weak AMPA receptor antagonists (Ki = 270 +/- 50 and 290 +/- 20 microM, respectively).