SCH 57790, a selective muscarinic M(2) receptor antagonist, releases acetylcholine and produces cognitive enhancement in laboratory animals.

The present studies were designed to assess whether the novel muscarinic M(2) receptor antagonist 4-cyclohexyl-alpha-[4[[4-methoxyphenyl]sulphinyl]-phenyl]-1-piperazineacetonitrile (SCH 57790) could increase acetylcholine release in the central nervous system (CNS) and enhance cognitive performance in rodents and nonhuman primates. In vivo microdialysis studies show that SCH 57790 (0.1-10 mg/kg, p.o.) produced dose-related increases in acetylcholine release from rat hippocampus, cortex, and striatum. SCH 57790 (0.003-1.0 mg/kg) increased retention times in young rat passive avoidance responding when given either before or after training. Also, SCH 57790 reversed scopolamine-induced deficits in mice in a passive avoidance task. In a working memory operant task in squirrel monkeys, administration of SCH 57790 (0.01-0.03 mg/kg) improved performance under a schedule of fixed-ratio discrimination with titrating delay. The effects observed with SCH 57790 in behavioral studies were qualitatively similar to the effects produced by the clinically used cholinesterase inhibitor donepezil, suggesting that blockade of muscarinic M(2) receptors is a viable approach to enhancing cognitive performance.

Metabolic stabilization of benzylidene ketal M(2) muscarinic receptor antagonists via halonaphthoic acid substitution.

The potential toxicological liabilities of the M(2) muscarinic antagonist 1 were addressed by replacing the methylenedioxyphenyl moiety with a p-methoxyphenyl group, resulting in M(2) selective compounds such as 3. Several halogenated naphthamide derivatives of 3 were studied in order to improve the pharmacokinetic profile via blockage of oxidative metabolism. Compound 4 demonstrated excellent M(2) affinity and selectivity, human microsomal stability, and oral bioavailability in rodents and primates.

Muscarinic agonists and antagonists in the treatment of Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by cognitive impairment and personality changes. The development of drugs for the treatment of the cognitive deficits of AD has focused on agents which counteract loss in cholinergic activity. Although symptoms of AD have been successfully treated with acetylcholinesterase inhibitors (tacrine, donepezil. rivastigmine, galanthamine), limited success has been achieved with direct M1 agonists, probably due to their lack of selectivity versus other muscarinic receptor subtypes. Muscarinic M2 antagonists have been reported to increase synaptic levels of acetylcholine after oral administration to rats (e.g. BIBN-99, SCH-57790), but their selectivity versus other muscarinic receptor subtypes is modest. Exploration of a series of piperidinylpiperidines has yielded the potent and selective M2 antagonist SCH-217443. This antagonist has excellent bioavailability in rats and dogs and shows activity in a rat model of cognition.

Facilitation of acetylcholine release and improvement in cognition by a selective M2 muscarinic antagonist, SCH 72788.

Current treatment of Alzheimer's Disease (AD) requires acetylcholinesterase inhibition to increase acetylcholine (ACh) concentrations in the synaptic cleft. Another mechanism by which ACh levels can be increased is blockade of presynaptic M2 muscarinic autoreceptors that regulate ACh release. An antagonist designed for this purpose must be highly selective for M2 receptors to avoid blocking postsynaptic M1 receptors, which mediate the cognitive effects of ACh. Structure-activity studies of substituted methylpiperadines led to the synthesis of 4-[4-[1(S)-[4-[(1,3-benzodioxol-5-yl)sulfonyl]phenyl]ethyl]-3(R)-methyl-1-piperazinyl]-4-methyl-1-(propylsulfonyl)piperidine. This compound, SCH 72788, binds to cloned human M2 receptors expressed in CHO cells with an affinity of 0.5 nM, and its affinity at M1 receptors is 84-fold lower. SCH 72788 is a functional M2 antagonist that competitively inhibits the ability of the agonist oxotremorine-M to inhibit adenylyl cyclase activity. In an in vivo microdialysis paradigm, SCH 72788 increases ACh release from the striatum of conscious rats. The compound is also active in a rodent model of cognition, the young rat passive avoidance response paradigm. The effects of SCH 72788 suggest that M2 receptor antagonists may be useful for treating the cognitive decline observed in AD and other dementias.

Design and synthesis of piperidinyl piperidine analogues as potent and selective M2 muscarinic receptor antagonists.

Identification of a number of highly potent M2 receptor antagonists with >100-fold selectivity against the M1 and M3 receptor subtypes is described. In the rat microdialysis assay, this series of compounds showed pronounced enhancement of brain acetylcholine release after oral administration.

Diphenyl sulfoxides as selective antagonists of the muscarinic M2 receptor.

Structure activity studies on [4-(phenylsulfonyl)phenyl]methylpiperazine led to the discovery of 4-cyclohexyl-alpha-[4-[[4-methoxyphenyl(S)-sufinyl]phenyl]-1-pi perazineacetonitrile, 1, an M2 selective muscarinic antagonist. Affinity at the cloned human M2 receptor was 2.7 nM; the M1/M2 selectivity is 40-fold.

Diphenylsulfone muscarinic antagonists: piperidine derivatives with high M2 selectivity and improved potency.

Piperidine analogues of our previously described piperazine muscarinic antagonists are described. Piperidine analogues show a distinct structure-activity relationship (SAR) that differs from comparable piperazines. Compounds with high selectivity and improved potency for the M2 receptor have been identified. The lead compound, 12b, increases acetylcholine release in vivo. Compounds of this class may be useful for the treatment of cognitive disorders such as Alzheimer's disease (AD).

Benzylidene ketal derivatives as M2 muscarinic receptor antagonists.

Benzylidene ketal derivatives were investigated as selective M2 receptor antagonists for the treatment of Alzheimer's disease. Compound 10 was discovered to have subnanomolar M2 receptor affinity and 100-fold selectivity against other muscarinic receptors. Also, 10 demonstrated in vivo efficacy in rodent models of muscarinic activity and cognition.

Identification of the primary muscarinic autoreceptor subtype in rat striatum as m2 through a correlation of in vivo microdialysis and in vitro receptor binding data.

Muscarinic autoreceptors located on cholinergic nerve terminals are involved in the inhibitory feedback regulation of acetylcholine (ACh) release. Establishing the subtype identity of such sites provides a more complete understanding of both normal receptor function and the functional significance of receptor changes associated with various neurodegenerative diseases. In this study, a novel approach was used to identify the muscarinic autoreceptor in rat striatum. It involved the correlation of data from two different sources--in vivo microdialysis and in vitro receptor binding. Four standard muscarinic antagonists with varying binding profiles (scopolamine, pirenzepine, AF-DX116 and himbacine) were infused directly through a microdialysis probe into the striatum of conscious, freely moving rats. The objectives were to find the minimal concentration of each antagonist capable of manifesting a functional autoreceptor response (i.e., increased ACh release) and to compare the relative ability of the antagonists to bring about this effect with their relative abilities to bind to each of the cloned muscarinic receptor subtypes. The conclusion is that the muscarinic receptor mediating ACh release in rat striatum exhibits a pharmacological profile clearly consistent with it being of the m2 subtype.

Pharmacology of SCH 34826, an orally active enkephalinase inhibitor analgesic.

SCH 34826 [(S)-N-[N-[1-[[(2,2-dimethyl-1,3-dioxolan-4yl) methoxy]carbonyl]-2-phenylethyl]-L-phenylalanine]-beta-alanine] was synthesized as a p.o. active prodrug enkephalinase inhibitor. In vivo, it is de-esterified to SCH 32615 (N-[L-(-1-carboxy-2-phenyl)ethyl]-L-phenylalanyl-beta-alanine), the active constituent. In vitro, the Ki for SCH 32615 to block the degradation of Met5-enkephalin by isolated enkephalinase is 19.5 +/- 0.9 nM. In contrast, SCH 32615 did not inhibit aminopeptidase or diaminopeptidase III degradation of Met5-enkephalin up to 10 microM and did not affect angiotensin converting enzyme up to 10 microM. In vivo, p.o. administered SCH 34826 potentiated the analgesic effects of D-Ala2-Met5-enkephalinamide in mice (ED50 = 5.3 mg/kg p.o.) and rats [minimal effective dose (MED) = 1 mg/kg p.o.]; SCH 32615 had no effect up to 30 mg/kg p.o., but was active parenterally (ED50 in mice = 1.4 ng/kg sc). Direct, naloxone-reversible analgesic effects of SCH 34826 were demonstrated in the mouse low temperature hot-plate test (MED = 30 mg/kg p.o.), the mouse acetic acid-induced writhing test (MED = 30 mg/kg p.o.), the rat stress-induced analgesia test (MED = 10 mg/kg p.o.) and the modified rat yeast-paw test (MED = 100 mg/kg p.o.). Using the rat D-Ala2-Met5-enkephalinamide potentiation test the duration of action of SCH 34826 was at least 4 hs. No respiratory or gastrointestinal side effects of any consequence were noted at doses up to 100 times those active in the D-Ala2-Met-5-enkephalinamide potentiation test.

Selective affinity of the benzodiazepines quazepam and 2-oxo-quazepam for BZ1 binding site and demonstration of 3H-2-oxo-quazepam as a BZ1 selective radioligand.

Quazepam and 2-oxo-quazepam are novel benzodiazepines containing a trifluoroethyl substituent on the ring nitrogen at position #1. Detailed competition binding experiments (25 to 30 concs.) at 4 degrees C were undertaken with these compounds versus 3H-flunitrazepam using synaptic membranes from rat cortex or cerebellum. Unlike other benzodiazepines, both quazepam and 2-oxo-quazepam distinguished two populations of 3H-flunitrazepam binding sites in rat cortex which were present in roughly equal proportions and for which the compounds displayed a greater than 20-fold difference in affinity. In cerebellum, no such discrimination of sites was noted for 2-oxo-quazepam, but quazepam did distinguish a small, low affinity (15% of total) population of sites. 3H-2-oxo-quazepam was prepared and used in competition studies to substantiate the conclusion that these compounds discriminate two populations of benzodiazepine sites in rat cortex. This new radioligand was shown to specifically label BZ binding sites with high affinity in a saturable manner. The competition experiments were then conducted using 3H-2-oxo-quazepam at a radioligand concentration sufficiently low (0.5 nM) to ensure that only the higher affinity binding sites which 2-oxo-quazepam discriminates would be occupied. Competition experiments in both cortex and cerebellum under these conditions indicated single site binding for unlabelled quazepam and 2-oxo-quazepam in every instance. This suggests that 3H-2-oxo-quazepam should be a useful new tool for selectively labeling and studying the BZ1 population of benzodiazepine binding sites.

Relative activities of SCH 23390 and its analogs in three tests for D1/DA1 dopamine receptor antagonism.

Inhibitory activities of a series of analogs of SCH 23390 ((R)-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3- benzazepine) in which the 7-chloro group was substituted by bromo, fluoro, methyl and methoxy groups, respectively, were compared in three tests for D1 and DA1 dopamine (DA) receptor antagonism: inhibition of DA-induced renal vasodilation in the anesthetized dog (DA1 receptor model), inhibition of DA-stimulated adenylate cyclase in the striatum of adult female rats (D1 receptor model) and displacement of [3H]SCH 23390 in the striatal homogenates of male rats. In addition the D2 receptor affinity of each of the compounds chosen was assessed via displacement of [3H]spiperone binding from rat striatum. S-enantiomers of the Cl and CH3 analogs were 200- to 700-fold weaker than the respective R-enantiomers in all three tests. The activity of all the R-enantiomers was in the nanomolar range and varied no more than 8-fold in all three tests. The F analog in the ligand binding test was the only exception, which was 30-fold weaker than the C1 analog. All of the R-enantiomers studied showed much weaker affinity for the D2 receptor, as assessed by displacement of [3H]spiperone binding. Similar enantiomer selectivity and parallel affinities of the R-enantiomers in the prototype models for D1 and DA1 receptors strengthen the evidence in support of identity between the D1 and DA1 dopamine receptors. These results further indicate that displacement of SCH 23390 in the ligand binding test reflects affinity of a compound for D1 and DA1 dopamine receptors.

Characterization of the binding of 3H-SCH 23390, a selective D-1 receptor antagonist ligand, in rat striatum.

A novel benzazepine, SCH 23390, has recently been described as a very potent and selective dopamine D-1 receptor antagonist based on its potent inhibition of dopamine sensitive adenylate cyclase and its selective displacement of 3H-piflutixol from rat striatal receptor sites. In the present study, the in vitro binding of 3H-SCH 23390 to specific striatal receptor sites has been characterized. Binding was saturable and stereospecific, and the results of both saturation and competition studies are consistent with the binding of 3H-SCH 23390 to a single striatal site. A KD of 0.53 nM was obtained through Scatchard analysis. Relative potencies of a variety of neuroleptics in competing with 3H-SCH 23390 and also 3H-spiperone support an interpretation that the single site to which 3H-SCH 23390 binds is the D-1 dopamine receptor. Also, the binding capacity of 3H-SCH 23390 (69 pmoles/gm wet weight) is in agreement with published values for the binding capacities of 3H-piflutixol and 3H-flupentixol. These data, coupled with the low level of non-specific binding encountered with this radioligand (4-8% of total binding at normally employed ligand concentration of 0.3 nM), its high specific activity and its negligible binding to plastic and glass surfaces make it ideally suited for studying interactions with this receptor.

Selective affinity of 1-N-trifluoroethyl benzodiazepines for cerebellar type 1 receptor sites.

In binding studies with rat brain membranes, 1,4-benzodiazepines containing a trifluoroethyl moeity at the 1-N position, including halazepam and quazepam, had significantly higher affinities for binding sites in cerebellum than in cortex. This selectivity for cerebellar sites is not a property of benzodiazepines without the trifluoroethyl moiety, but is similar to that seen with the triazolopyridazines. Since halazepam and quazepam, like the triazolopyridazines, have behavioral effects in animals at doses much lower than those that cause ataxia, it is tempting to attribute this separation of pharmacologic activities to differential activity at subpopulations of benzodiazepine receptors. Further work is necessary to clarify this possibility.

Potentiation of the hypoglycemic effect of insulin by thiorphan, an enkephalinase inhibitor.

Insulin administration to rats produced a dose-related hypoglycemia. When insulin and the enkephalinase (Enk'ase) inhibitor thiorphan (30 or 100 mg/kg s.c.) were co-administered, there was a potentiation of the hypoglycemic response to insulin; these doses of thiorphan alone had no significant effect on plasma glucose. When tested in vitro against isolated Enk'ase, both insulin and its beta-chain inhibited the catabolism of [Met5]enkephalin. Theoretically, thiorphan blocked the catabolism of insulin by inhibiting Enk'ase. Alternatively, thiorphan acted as an inhibitor of another insulin-catabolizing enzyme having similar substrate requirements as Enk'ase.

Relationship of yeast-injection induced changes in brain met-enkephalin and analgesia.

Subplantar injection of Brewer's yeast induces a hyperalgesia that is associated with an increase in the level of striatal Met-enkephalin (ME); there was no change in the hypothalamus of periaqueductal gray. To test the relationship between striatal ME and analgesia, naloxone (10, 3, 0.5 mg/kg, SC) or thiorphan (100 micrograms, ICV) were administered. Neither drug caused a potentiation or a reduction in the hypersensitivity. These data suggest that an increase in striatal does not result in altered pain sensitivity in this model.

Sulfhydryl reactivity: mechanism of action of several antiviral compounds--selenocystine, 4-(2-propinyloxy)-beta-nitrostyrene, and acetylaranotin.

The addition of 5 mM dithiothreitol to a cell-free assay system for influenza ribonucleic acid (RNA) polymerase activity reverses the inhibitory activity otherwise possessed by three established antiviral compounds: selenocystine, 4-(2-propinyloxy)-beta-nitrostyrene, and acetylaranotin. Although 50% or greater enzyme inhibitory activity is repeatedly achieved for these compounds at a concentration of approximately 50 mug/ml (0.1 to 0.25 mM) in the absence of dithiothreitol, no inhibition is seen in its presence at inhibitor concentrations as high as 200 mug/ml. Against the deoxyribonucleic acid-directed RNA polymerases of Escherichia coli and chicken embryo cells, acetylaranotin and 4-(2-propinyloxy)-beta-nitrostyrene caused very little inhibition. Only selenocystine significantly inhibited these two enzymes in the absence of reducing agent, but to an extent substantially less than that obtained against the viral enzyme. These results appear to suggest that influenza RNA polymerase is uniquely sensitive to a variety of structurally diverse antiviral compounds as a consequence of their sulfhydryl reactivity-a fact which might aid in the search for and development of more potent chemotherapeutic agents.