Exploring the potential for subtype-selective muscarinic agonists in glaucoma.

Pilocarpine has been used to lower intraocular pressure (IOP) in glaucoma patients for more than 100 years. Since the identification of five muscarinic receptor subtypes, there has been an interest in separating the IOP-lowering effects from the ocular side effects of pupil constriction and lens accommodation. However, all these actions seem to be mediated by the M3 receptor. A novel muscarinic receptor agonist, AGN 199170, that has no activity on the M3 subtype was compared to pilocarpine in a monkey glaucoma model. This compound lowered IOP suggesting that muscarinic agonists targeted at muscarinic receptors other than the M3 subtype may be able to selectively lower IOP.

Structure-activity studies of conantokins as human N-methyl-D-aspartate receptor modulators.

The activities of conantokin-G (con-G), conantokin-T (con-T), and several novel analogues have been studied using polyamine enhancement of [3H]MK-801 binding to human glutamate-N-methyl-D-aspartate (NMDA) receptors, and their structures have been examined using CD and 1H NMR spectroscopy. The potencies of con-G[A7], con-G, and con-T as noncompetitive inhibitors of spermine-enhanced [3H]MK-801 binding to NMDA receptor obtained from human brain tissue are similar to those obtained using rat brain tissue. The secondary structure and activity of con-G are found to be highly sensitive to amino acid substitution and modification. NMR chemical shift data indicate that con-G, con-G[D8, D17], and con-G[A7] have similar conformations in the presence of Ca2+. This consists of a helix for residues 2-16, which is kinked in the vicinity of Gla10. This is confirmed by 3D structure calculations on con-G[A7]. Restraining this helix in a linear form (i.e., con-G[A7,E10-K13]) results in a minor reduction in potency. Incorporation of a 7-10 salt-bridge replacement (con-G[K7-E10]) prevents helix formation in aqueous solution and produces a peptide with low potency. Peptides with the Leu5-Tyr5 substitution also have low potencies (con-G[Y5,A7] and con-G[Y5,K7]) indicating that Leu5 in con-G is important for full antagonist behavior. We have also shown that the Gla-Ala7 substitution increases potency, whereas the Gla-Lys7 substitution has no effect. Con-G and con-G[K7] both exhibit selectivity between NMDA subtypes from mid-frontal and superior temporal gyri, but not between sensorimotor and mid-frontal gyri. Asn8 and/or Asn17 appear to be important for the ability of con-G to function as an inhibitor of polyamine-stimulated [3H]MK-801 binding, but not in maintaining secondary structure. The presence of Ca2+ does not increase the potencies of con-G and con-T for NMDA receptors but does stabilize the helical structures of con-G, con-G[D8,D17], and, to a lesser extent, con-G[A7]. The NMR data support the existence of at least two independent Ca2+-chelating sites in con-G, one involving Gla7 and possibly Gla3 and the other likely to involve Gla10 and/or Gla14.

Heteroaryl analogues of AMPA. 2. Synthesis, absolute stereochemistry, photochemistry, and structure-activity relationships.

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.

Aryl and cycloalkyl analogues of AMPA: synthetic, pharmacological and stereochemical aspects.

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.

Molecular pharmacology of 4-substituted glutamic acid analogues at ionotropic and metabotropic excitatory amino acid receptors.

The pharmacology of (2S,4R)-4-methylglutamic acid, (2S,4S)-4-methylglutamic acid and (S)- and (R)-4-methyleneglutamic acids (obtained in high chemical and enantiomeric purity from racemic 4-methyleneglutamic acid by chiral HPLC using a Crownpak CR(+) column), was examined in binding experiments using rat brain ionotropic glutamate receptors, and in functional assays using cloned metabotropic glutamate (mGlu) receptors. As a notable result of these studies, (2S,4R)-4-methylglutamic acid and (2S,4S)-4-methylglutamic acid were shown to be selective for kainic acid receptors and mGlu receptors (subtypes 1alpha and 2), respectively, whereas (S)-4-methyleneglutamic acid showed high but rather non-selective affinity for the (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA), kainic acid, NMDA and mGlu receptors (subtypes 1alpha and 2). Although none of the compounds were specific for any of the receptor subtypes, the results demonstrate that each of these structurally related compounds has a distinct pharmacological profile.

Heteroaryl analogues of AMPA. Synthesis and quantitative structure-activity relationships.

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.

Determination of the solution structures of conantokin-G and conantokin-T by CD and NMR spectroscopy.

Conantokin-G and conantokin-T are two paralytic polypeptide toxins originally isolated from the venom of the fish-hunting cone snails of the genus Conus. Conantokin-G and conantokin-T are the only naturally occurring peptidic compounds which possess N-methyl-D-aspartate receptor antagonist activity, produced by a selective non-competitive antagonism of polyamine responses. They are also structurally unusual in that they contain a disproportionately large number of acid labile post-translational gamma-carboxyglutamic acid (Gla) residues. Although no precise structural information has previously been published for these peptides, early spectroscopic measurements have indicated that both conantokin-G and conantokin-T form alpha-helical structures, although there is some debate whether the presence of calcium ions is required for these peptides to adopt this fold. We now report a detailed structural study of synthetic conantokin-G and conantokin-T in a range of solution conditions using CD and 1H NMR spectroscopy. The three-dimensional structures of conantokin-T and conantokin-G were calculated from 1H NMR-derived distance and dihedral restraints. Both conantokins were found to contain a mixture of alpha- and 310 helix, that give rise to curved and straight helical conformers. Conantokin-G requires the presence of divalent cations (Zn2+, Ca2+, Cu2+, or Mg2+) to form a stable alpha-helix, while conantokin-T adopts a stable alpha-helical structure in aqueous conditions, in the presence or absence of divalent cations (Zn2+, Ca2+, Cu2+, or Mg2+).

A new highly selective metabotropic excitatory amino acid agonist: 2-amino-4-(3-hydroxy-5-methylisoxazol-4-yl)butyric acid.

The homologous series of acidic amino acids, ranging from aspartic acid (1) to 2-aminosuberic acid (5), and the corresponding series of 3-isoxazolol bioisosteres of these amino acids, ranging from (RS)-2-amino-2-(3-hydroxy-5-methylisoxazol-4-yl)acetic acid (AMAA, 6) to (RS)-2-amino-6-(3-hydroxy-5-methylisoxazol-4-yl)hexanoic acid (10), were tested as ligands for metabotropic excitatory amino acid receptors (mGlu1 alpha, mGlu2, mGlu4a, and mGlu6). Whereas AMAA (6) and (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propinoic acid (AMPA, 7) are potent and highly selective agonists at N-methyl-D-aspartic acid (NMDA) and AMPA receptors, respectively, the higher homologue of AMPA (7), (RS)-2-amino-4-(3-hydroxy-5-methylisoxazol-4-yl)butyric acid (homo-AMPA, 8), is inactive at ionotropic excitatory amino acid receptors. Homo-AMPA (8), which is a 3-isoxazolol bioisostere of 2-aminoadipic acid (3), was, however, shown to be a specific and rather potent agonist at mGlu6, approximately 4 times weaker than the nonselective excitatory amino acid receptor agonist (S)-glutamic acid. 2-Aminoadipic acid (3), which shows a complex excitatory amino acid synaptic pharmacology, was an agonist at mGlu6 as well as mGlu2. AMPA (7) and the higher homologue of homo-AMPA (8), (RS)-2-amino-5-(3-hydroxy-5-methylisoxazol-4-yl)pentanoic acid (9), showed relatively weak agonist effects at mGlu6. It is concluded that homo-AMPA (8) is likely to be a useful tool for studies of the pharmacology and physiological role of mGlu6. We describe a new versatile synthesis of this homologue of AMPA and the synthesis of compound 10.