Chiral synthesis and pharmacological evaluation of NPS 1407: a potent, stereoselective NMDA receptor antagonist.

The stereoselective synthesis and biological activity of NPS 1407 (4a), (S)-(-)-3-amino-1,1-bis(3-fluorophenyl)butane, a potent, stereoselective antagonist of the NMDA receptor, are described. The racemate (4) was found to be active at the NMDA receptor in an in vitro assay, prompting the synthesis of the individual stereoisomers. The S isomer (4a) was found to be 12 times more potent than the R isomer (4b). Compound 4a demonstrated in vivo pharmacological activity in neuroprotection and anticonvulsant assays.

NPS 1506, a moderate affinity uncompetitive NMDA receptor antagonist: preclinical summary and clinical experience.

NPS Pharmaceuticals, Inc. (NPS) has synthesized a series of open-channel blockers with varying potencies at the NMDA receptor. NPS 1506 (Fig. 1) is a moderate affinity antagonist that inhibits NMDA/glycine-induced increases in cytosolic calcium in cultured rat cerebellar granule cells (IC50 = 476nM) and displaces the binding of [3H]MK-801 to rat cortical membranes (IC50 = 664nM).

Synthesis, biological activity, and absolute stereochemical assignment of NPS 1392: a potent and stereoselective NMDA receptor antagonist.

The synthesis, biological activity, and single crystal X-ray structure of NPS 1392, (R)-(-)-3,3-bis(3-fluorophenyl)-2-methylpropan-1-amine (3a), a potent, stereoselective antagonist of the NMDA receptor, are described. The NMDA receptor selectively bound the levo isomer (3a) over its enantiomer (3b), which prompted a rigorous absolute configuration assignment. NPS 1392 has the R configuration based on the single-crystal X-ray diffraction analysis of the hydroiodide salt of NPS 1392. This compound is a potential neuroprotective agent for use in the treatment of ischemic stroke.

NPS 1506, a novel NMDA receptor antagonist and neuroprotectant. Review of preclinical and clinical studies.

NPS 1506 is a moderate affinity, uncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist. NPS 1506 is neuroprotective in rodent models of ischemic stroke, hemorrhagic stroke, and head trauma, with a 2-hr window of opportunity. Neuroprotectant doses of NPS 1506 ranged from approximately 0.1-1.0 mg/kg, with peak plasma concentrations ranging from 8-80 ng/mL. Even at doses producing behavioral toxicity, NPS 1506 did not elicit MK-801-like behaviors, did not generalize to phencyclidine (PCP), and did not elicit neuronal vacuolization. In a Phase I study, intravenous (i.v.) doses of NPS 1506 from 5-100 mg were well tolerated and provided plasma concentrations in excess of those required for neuroprotection in rodents. Adverse events at the 100-mg dose included mild dizziness and lightheadedness, and mild to moderate ataxia. Neither PCP-like psychotomimetic effects nor cardiovascular effects were noted. The long plasma half-life of NPS 1506 (approximately 60 hr) suggests that a single i.v. dose will provide prolonged neuroprotection in humans.

Design, synthesis, and biological evaluation of spider toxin (argiotoxin-636) analogs as NMDA receptor antagonists.

PURPOSE: Twelve synthetic spider toxin analogs were prepared in an effort to better understand the structure-activity relationships of the polyamine portion of argiotoxin-636 (Arg-636), a noncompetitive NMDA receptor (NMDAR) antagonist. METHODS: The 1,13-diamino-4,8-diazatridecane portion of the side chain of Arg-636 was systematically modified in an effort to further our knowledge of the structural requirements for the alkyl linker spacing between the amine nitrogens. Systematic isosteric replacement of each of the amine nitrogens in the polyamine moiety with either oxygen or carbon provided a series of compounds which were evaluated in vitro for NMDAR antagonist activity. RESULTS: One-half of the heteroatoms found in Arg-636 were removed to provide analogs which maintained in vitro potency below 1 microM. However, these simplified analogs produced similar or more pronounced effects on the cardiovascular system than Arg-636 in vivo. CONCLUSIONS: In this set of analogs, a minimum of three basic nitrogens in the side chain was required for maximum potency as NMDAR antagonists. Isosteric nitrogen substitutions in the polyamine chain reduced the in vitro potency of these analogs. An analog binding-conformation model was proposed to rationalize the inactivity of these isosterically substituted analogs.

Arylamine toxins from funnel-web spider (Agelenopsis aperta) venom antagonize N-methyl-D-aspartate receptor function in mammalian brain.

The venom of the North American funnel-web spider Agelenopsis aperta contains a variety of arylamine toxins (the alpha-agatoxins) that paralyze insects by blocking glutamatergic neuromuscular transmission. We have tested six synthetic alpha-agatoxins for their ability to antagonize glutamate receptor function in mammalian brain. These compounds produce, at submicromolar concentrations, noncompetitive inhibition of N-methyl-D-aspartate (NMDA) receptor-mediated elevations in the concentration of cytosolic free calcium in cultured rat cerebellar granule neurons. In contrast, the alpha-agatoxins are relatively weak antagonists of elevations in the cytosolic free calcium concentration induced by non-NMDA receptor agonists. The alpha-agatoxins also produce reversible suppression of the NMDA receptor-mediated excitatory postsynaptic potential in rat hippocampal slices at concentrations that have little effect on the non-NMDA receptor-mediated population spike. We conclude that the alpha-agatoxins are selective and reversible noncompetitive antagonists at NMDA receptors in mammalian brain.

Modulation of N-methyl-D-aspartate receptor-mediated increases in cytosolic calcium in cultured rat cerebellar granule cells.

The concentration of intracellular free Ca2+ ([Ca2+]i) was measured in rat cerebellar granule cells using the fluorescent indicator fura-2. Culturing the cells as monolayers on plastic squares which could be placed into cuvettes allowed measurements of [Ca2+]i to be performed on large and homogeneous populations of CNS neurons. Granule cells so cultured maintained low levels of [Ca2+]i (around 90 nM) which increased promptly upon the addition of various excitatory amino acids including N-methyl-D-aspartate (NMDA). Increases in [Ca2+]i elicited by NMDA were inhibited by Mg2+ (1 mM) and often potentiated by glycine (1 microM). The addition of TTX or strychnine (5 microM each) did not alter responses to NMDA or NMDA plus glycine. Cytosolic Ca2+ responses to NMDA/glycine were dependent on the presence of extracellular Ca2+ and were unaffected by concentrations of nifedipine or verapamil that blocked increases in [Ca2+]i elicited by K+ depolarization. Responses elicited by NMDA/glycine were inhibited competitively by 2-amino-5-phosphonovalerate or 3-((+-)-2-carboxypiperazin-4-yl)-propyl-1- phosphonic acid and non-competitively by MK-801 or Mg2+. HA-966 and 7-chlorokynurenate inhibited responses to NMDA alone and blocked competitively the potentiating effects of glycine. The results demonstrate NMDA-mediated increases in [Ca2+]i in cerebellar granule cells that arise solely from influx of extracellular Ca2+ through dihydropyridine-insensitive channels. The strict dependence of the NMDA-evoked response on extracellular Ca2+ provides little evidence for a coupling of NMDA receptors to inositol phosphate metabolism and mobilization of intracellular Ca2+. The effect of various agents on NMDA/glycine-induced increases in [Ca2+]i parallels their effects on ligand binding to or current flow through the NMDA receptor-channel complex. The measurement of cytosolic Ca2+ in this preparation of neuronal cells thus appears especially well suited for assessing, on a functional level, the regulation of NMDA receptors in the CNS.

A-69024: a non-benzazepine antagonist with selectivity for the dopamine D-1 receptor.

A-69024 HBr, 1-(2-bromo-4,5-dimethoxybenzyl)-7-hydroxy-6-methoxy-2-methyl-1,2,3,4- tetrahydroisoquinoline hydrobromide, is a selective antagonist of the dopamine D-1 receptor. A-69024 HBr shows an apparent affinity toward the D-1 receptor (identified using [125I]SCH 23390) of 12.6 (4.15-38.3) nM (mean (90% CL), n = 3); the apparent affinity toward the D-2 receptor (identified using [3H]spiroperidol is 1 290 (1,200-1,380) nM (n = 3); using [125I]lysergic acid diethylamine to identify the 5-HT1C receptor gives apparent affinity of 17,800 (9,700-32,600) nM (n = 3). In assays of adenylate cyclase activity, A-69024 HBr antagonizes the D-1 receptor with a calculated affinity of 43.9 (17.5-110) nM (n = 5), while the molecule antagonizes the D-2 receptor with a calculated affinity greater than 400 nM. Behavioral studies demonstrate that A-69024 HBr (5 mg/kg s.c.) is able to block both amphetamine-induced locomotor activity and apomorphine-induced stereotypy. Furthermore, A-69024 HBr blocks SF&F 38393-, but not quinpirole-, induced rotation in rats having unilateral 6-hydroxydopamine lesions of the substantia nigra. When administered at behaviorally effective doses. A-69024 HBr neither increases the concentration of serum prolactin nor potentiates dihydroxyphenylalanine (DOPA) accumulation in the caudate-putamen of rats pretreated with the DOPA decarboxylase inhibitor NSD 1015. Because A-69024 is a dopamine receptor antagonist discriminating between the D-1 and D-2 receptors, it may be a useful research tool.

Hypoxic pulmonary vasoconstriction does not affect hydrostatic pulmonary edema formation.

We studied the effects of regional hypoxic pulmonary vasoconstriction (HPV) on lobar flow diversion in the presence of hydrostatic pulmonary edema. Ten anesthetized dogs with the left lower lobe (LLL) suspended in a net for continuous weighing were ventilated with a bronchial divider so the LLL could be ventilated with either 100% O2 or a hypoxic gas mixture (90% N2-5% CO2-5% O2). A balloon was inflated in the left atrium until hydrostatic pulmonary edema occurred, as evidenced by a continuous increase in LLL weight. Left lower lobe flow (QLLL) was measured by electromagnetic flow meter and cardiac output (QT) by thermal dilution. At a left atrial pressure of 30 +/- 5 mmHg, ventilation of the LLL with the hypoxic gas mixture caused QLLL/QT to decrease from 17 +/- 4 to 11 +/- 3% (P less than 0.05), pulmonary arterial pressure to increase from 35 +/- 5 to 37 +/- 6 mmHg (P less than 0.05), and no significant change in rate of LLL weight gain. Gravimetric confirmation of our results was provided by experiments in four animals where the LLL was ventilated with an hypoxic gas mixture for 2 h while the right lung was ventilated with 100% O2. In these animals there was no difference in bloodless lung water between the LLL and right lower lobe. We conclude that in the presence of left atrial pressures high enough to cause hydrostatic pulmonary edema, HPV causes significant flow diversion from an hypoxic lobe but the decrease in flow does not affect edema formation.

Chronic scopolamine treatment and brain cholinergic function.

Scopolamine was either continuously infused or injected once daily into C3H mice. Chronic infusion resulted in mice that were supersensitive to the hypothermia and tremor produced by the muscarinic agonist, oxotremorine. Chronic scopolamine infusion did not alter brain acetylcholinesterase (AChE) or choline acetyltransferase (ChAT) activities but it did produce an increase in brain muscarinic receptors, as measured by quinuclidinyl benzilate (QNB) binding. The maximal increase in QNB binding was seen at the 0.2 mg/kg/hr dose. Further increase in dose resulted in a return to control QNB binding in all brain regions studied except cortex. These animals were still supersensitive to oxotremorine, suggesting a dissociation between receptor number and response to agonist. Animals injected once daily for 10 days with 5 mg/kg exhibited an increase in QNB binding while no increase was seen at 20 mg/kg/day. Chronic oxotremorine infusion resulted in tolerance to the hypothermia-producing effects of oxotremorine. This was accompanied by a decrease in brain QNB binding. Coinfusion of scopolamine with oxotremorine blocked both the tolerance development and receptor changes. These experiments demonstrate that chronic scopolamine treatment can elicit an increase in brain muscarinic receptors which is accompanied by supersensitivity to agonists. However, this effect is not clearly dose related, and a strict relationship between receptor number and agonist response does not exist.

Hydralazine does not inhibit canine hypoxic pulmonary vasoconstriction.

Clinical experience with hydralazine has led to conflicting data concerning its effect on the pulmonary vasculature. We studied the effects of hydralazine on the hypoxic pulmonary vasoconstrictor response in 9 dogs challenged with inhalation of 10% oxygen in the presence and absence of hydralazine. Prior to administration of the drug, hypoxia increased cardiac output from 174 +/- 13 to 209 +/- 21 ml/kg/min (p less than 0.05) and pulmonary artery pressure from 9 +/- 1 to 19 +/- 1 mmHg (p less than 0.05). After hydralazine, cardiac output rose during normoxia to 275 +/- 30 and during hypoxia to 305 +/- 34 ml/kg/min (p less than 0.05). Pulmonary artery pressure continued to respond to hypoxia, rising from 11 +/- 1 to 21 +/- 1 mmHg (p less than 0.05) in the presence of hydralazine. Hydralazine reduced pulmonary vascular resistance during normoxia from 173 +/- 14 to 136 +/- 13 dynes X s X cm-5 (p less than 0.05) but even after the drug, pulmonary vascular resistance rose sharply during hypoxia. There was no significant difference in the response to hypoxia of pulmonary artery pressure or pulmonary vascular resistance after hydralazine when compared with that before hydralazine. In a second set of 6 dogs, we repeated these experiments but volume-depleted the dogs after the administration of hydralazine to prevent the passive pulmonary vasodilation that occurs because of the rise in cardiac output with the drug. We again found no inhibition of hypoxic pulmonary vasoconstriction by hydralazine. Finally, we administered sodium nitroprusside to 4 dogs using the same model and found a significant inhibition of hypoxic pulmonary vasoconstriction. Hydralazine, unlike nitroprusside, does not inhibit the pulmonary vascular response to hypoxia.

Quantitation of tolerance development after chronic oxotremorine treatment.

A new procedure was developed to quantitate the tolerance which develops as mice are chronically infused with the muscarinic agonist, oxotremorine. Cumulative dose-response curves were constructed for the effects of oxotremorine on body temperature and rotarod performance by administering sequential injections to individual animals. These dose-response curves compare favorably to those constructed by injecting individual animals with one of several doses. The sequential injection technique was used to assess the magnitude of tolerance development to oxotremorine. A linear relationship between oxotremorine infusion rate (dose) and magnitude of change of the ED50 value for impairment of rotarod performance was observed, with animals receiving an infusion rate of 1.0 mg/kg/hr showing a 24-fold increase in ED50. Dose-response curves for tolerant animals were parallel to those constructed for naive animals. The oxotremorine dose required to decrease body temperature to 35 degrees C (ED35 degrees) was 80-fold greater than control in the group treated with 1.0 mg/kg/hr. The dose-response curves for tolerant animals were not parallel to those seen in naive animals. Time courses of recovery from a challenge dose of oxotremorine suggest little change in metabolism occurred during chronic infusion. Chronic oxotremorine infusion resulted in a decrease in the total number of QNB binding sites. Both high- and low-affinity sites were reduced in number. Since no change in K1 for the muscarinic agonist, carbamylcholine, was observed, it seems unlikely that a change occurs in the affinity of the muscarinic receptor for agonists. Significant change in receptor number was detected only in animals that received higher doses of oxotremorine. Chronic oxotremorine treatment had no effect on choline uptake by synaptosomes prepared from any of five brain regions.

Genetic influences on cholinergic drug response.

Three mouse strains were tested for oxotremorine effects on open-field activity and body temperature. Open-field activity was depressed less severely in C3H mice than in C57BL, which were less affected than DBA. While no differences in the hypothermic effects of oxotremorine were observed 15 min after injection, the time courses of the drug effect on body temperature indicated that C3H were less affected than C57BL or DBA. No differences in the activities of acetylcholinesterase of choline acetyltransferase were found among the three strains in cortex, cerebellum, hindbrain (pons-medulla), or total midbrain. While no differences in muscarinic receptor levels were found in the four large brain areas, finer dissection of the midbrain revealed small differences in total receptor number in striatum, hippocampus, and remaining midbrain areas. C3H mice exhibited greater QNB binding than C57BL and DBA mice in striatum; DBA mice exhibited greater QNB binding in hippocampus than C57BL (C3H mice were not different from either strain); and C57BL had less QNB binding than the other two strains in midbrain. All of these differences were small (20% or less). No differences in KD were observed. The inhibition of receptor binding by either oxotremorine or nicotine was the same in all strains, but the IC50 for oxotremorine varied from region to region. While behavioral differences in the effects of oxotremorine are clear, there is no obvious biochemical explanation for these differences.

Cholinergic adaptations to chronic oxotremorine infusion.

The development of tolerance to cholinergic agonists such as oxotremorine is a well established phenomenon. The hypothesis that such tolerance may be explained by a decrease in the number of affinity of muscarinic receptors was tested by chronically treating C3H mice with oxotremorine. Chronic treatment was achieved by continuously infusing oxotremorine via an indwelling i.v. catheter. Doses ranged from 0.03 to 1.0 mg/kg/hr. Clear tolerance was observed in that symptoms such as salivation, lacrimation and muscle tremor decreased or disappeared during the infusion period. Similarly, chronically treated animals exhibited minimal hypothermia or impairment of rotarod performance when challenged with an oxotremorine dose which significantly depressed both of these measures in naive animals. The activities of the enzymes, acetylcholinesterase and choline acetyltransferase, as well as the binding of [3H]-3-quinuclidinyl benzilate in seven brain regions, were assessed. Chronic oxotremorine treatment failed to alter acetyltransferase activity in any of the brain regions. Choline acetyltransferase activity was only marginally decreased in several brain regions. A significant decrease in maximal [3H]-3-quinudidinyl binding was observed in six of the regions examined. No alteration in [3H]-3-quinuclidinyl affinity was detected. Tolerance to oxotremorine was detected at doses which failed to alter choline acetyltransferase activity or receptor number. These data support the observations of others who noted that chronic muscarinic stimulation results in a decrease in muscarinic receptors, but suggest the importance of mechanisms other than decreased receptor number in early stages of tolerance development.