SCH 57790: a novel M2 receptor selective antagonist.

As a decrease in cholinergic neurons has been observed in Alzheimer's Disease (AD), therapeutic approaches to AD include inhibition of acetylcholinesterase to increase acetylcholine levels. Evidence suggests that acetylcholine release in the CNS is modulated by negative feedback via presynaptic M2 receptors, blockade of which should provide another means of increasing acetylcholine release. Structure-activity studies of [4-(phenylsulfonyl)phenyl]methylpiperazines led to the synthesis of 4-cyclohexyl-alpha-[4-[[4-methoxyphenyl]sulfinyl]-phenyl]-1-piperazin eacetonitrile. This compound, SCH 57790, binds to cloned human M2 receptors expressed in CHO cells with an affinity of 2.78 nM; the affinity at M1 receptors is 40-fold lower. SCH 57790 is an antagonist at M2 receptors expressed in CHO cells, as the compound blocks the inhibition of adenylyl cyclase activity mediated by the muscarinic agonist oxotremorine. This compound should be useful in assessing the potential of M2 receptor blockade for enhancement of cognition.

Application of an almost traceless linker in the synthesis of 2-alkylthiobenzimidazole combinatorial libraries.

Resin-bound triphenylphosphine was coupled to 4-fluoro-3-nitrobenzyl bromide, and 2-alkylthiobenzimidazoles were synthesized on resin in 4 steps using standard chemistries. Cleavage of the compounds from the resin was achieved with 10% NaOH in MeOH to leave a methyl group at the attachment point. A total of 47 amines and 40 electrophiles were evaluated, defining the scope of the reactions, culminating in the synthesis of an 80-member test library of high purity as determined by HPLC.

Substituted (1,2-diarylethyl)amide acyl-CoA:cholesterol acyltransferase inhibitors: effect of polar groups on in vitro and in vivo activity.

Substituted (1,2-diarylethyl)amides have been prepared and evaluated for their ability to inhibit microsomal acyl-CoA:cholesterol acyltransferase activity in vitro and to lower hepatic cholesteryl ester content in vivo in a cholesterol-fed hamster. Simple unsubstituted (diarylethyl)amides were potent inhibitors in vitro but showed poor activity in vivo. Introduction of polar groups at specific locations on the diarylethylamine moiety decreased in vitro activity but increased in vivo activity. Both effects were highly structure dependent, suggesting specific interactions which were mediating activity in each model. Optimization of these opposing effects led to compounds which were potent in both models.

Synthesis and receptor affinities of some conformationally restricted analogues of the dopamine D1 selective ligand (5R)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl- 1H-3-benzazepin-7-ol.

The synthesis of a structurally novel series of 6,6a,7,8,9,13b-hexahydro-5H-benzo[d]naphtho[2,1-b]azepines (2), conformationally restricted analogues of the dopamine D1 antagonist (5R)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepin -7-ol (SCH 23390, 1c), is described. Affinity for D1 receptors was determined by competition for rat striatal binding sites labeled by [3H]SCH 23390; affinity for D2 receptors was similarly determined by competition experiments using [3H]spiperone. Compounds in this series having the B/C-trans ring junction (2b and related analogues), where the D ring is unequivocally fixed in an equatorial orientation, possess considerably more D1 receptor affinity and selectivity vs the D2 receptor than the conformationally mobile cis stereoisomers (2a), thus leading to the conclusion that axial substituents at the 4- or 5-positions of the benzazepine nucleus are detrimental to D1 receptor affinity. Resolution and X-ray analysis demonstrated that D1 receptor affinity was preferentially associated with the (-)-6aS,13bR enantiomer of 2b.

Pharmacological profile of SCH39166: a dopamine D1 selective benzonaphthazepine with potential antipsychotic activity.

SCH39166 [(-)-trans-6,7,7a,8,9,13b-hexahydro-3-chloro-2-hydroxy-N-methyl- 5H-benzo[d]naptho-(2,1-b)azepine] is a benzonaphthazepine that has been evaluated as a selective D1 dopamine receptor antagonist. In vitro, SCH39166 (Ki = 3.6 nM) inhibited the binding of [3H]SCH23390 (a D1 specific compound) and blocked dopamine-stimulated adenylate cyclase (Ki = 9.1 nM); in contrast the Ki for SCH39166 to displace [3H]spiperone (D2) was greater than 1 microM and its Ki vs. [3H]-ketanserin (5-hydroxytryptamine2) binding was greater than 300 nM. In vivo, SCH39166 inhibited both rat and squirrel monkey conditioned avoidance responding (minimal effective dose = 10 and 1.78 mg/kg p.o., respectively) and had a duration of at least 6 hr in both species. In addition, SCH39166 antagonized apomorphine-induced stereotypy in rats (minimal effective dose = 10 mg/kg p.o.). These in vivo actions of SCH39166 are similar to the activity of typical dopamine antagonists. However, in contrast to D2-selective antagonists, SCH39166 failed to increase plasma prolactin levels, did not block apomorphine-induced emesis in the dog and had minimal effects on the striatal levels of homovanillic acid or dihydroxyphenylacetic acid. Furthermore, although immobility was seen after p.o. administration of SCH39166 using the inclined screen test, the drug did not cause catalepsy at doses up to 10 times its minimal effective dose in the rat conditioned avoidance response test. Additionally, SCH39166 inhibited apomorphine-induced climbing at lower doses than it inhibited apomorphine-induced sniffing in mice. The results from these latter two tests suggest that SCH39166 may have a reduced liability to produce extrapyramidal side effects. Therefore, based on this profile of activity, SCH39166 is a selective D1 dopamine receptor antagonist both in vitro and in vivo. Additionally, because this compound is longer acting in the primate than previously available D1 antagonists, it has potential utility as a clinically useful drug.

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.

Affinity chromatography of the D1 dopamine receptor from rat corpus striatum.

The D1 dopamine receptor from rat corpus striatum has been purified 200-250-fold by using a newly developed biospecific affinity chromatography matrix based on a derivative of the D1 selective antagonist SCH 23390. This compound, (RS)-5-(4-aminophenyl)-8-chloro-2,3,4,5-tetrahydro-3-methyl-1H-3-benz azepin-7-o l (SCH 39111), possesses high affinity for the D1 receptor and, when immobilized on Sepharose 6B through an extended spacer arm, was able to adsorb digitonin-solubilized D1 receptors. The interaction between the solubilized receptor and the affinity matrix was biospecific. Adsorption of receptor activity could be blocked in a stereoselective fashion [SCH 23390 greater than SCH 23388; (+)-butaclamol greater than (-)-butaclamol]. The elution of [3H]SCH 23390 activity from the gel demonstrated similar stereoselectivity for antagonist ligands. Agonists eluted receptor activity with a rank order of potency consistent with that of a D1 receptor [apomorphine greater than dopamine greater than (-)-epinephrine much greater than LY 171555 greater than serotonin]. SCH 39111-Sepharose absorbed 75-85% of the soluble receptor activity, and after the gel was washed extensively, 35-55% of the absorbed receptor activity could be eluted with 100 microM (+)-butaclamol with specific activities ranging from 250 to 450 pmol/mg of protein. The affinity-purified receptor retains the ligand binding characteristics of a D1 dopamine receptor. This affinity chromatography procedure should prove valuable in the isolation and molecular characterization of the D1 dopamine receptor.

Biochemical properties of D1 and D2 dopamine receptors.

The physiological action of dopamine are mediated by two distinct subtypes of receptors, D1 and D2 dopamine receptors. D1-receptors are linked to stimulation of adenylate cyclase whereas D2-receptors inhibit the enzyme and may also couple to other signal transduction systems such as ion channels. In order to characterize these receptors at the biochemical level we have developed specific probes for the identification and purification of these proteins. The ligand binding sites of the two receptors have been identified by photoaffinity labeling and reside on distinct polypeptides. In rat striatum, the D1 receptor binding site can be identified as a peptide of Mr = 72,000. In contrast, the D2 receptors appears to reside on an Mr = 94,000 peptide in most tissues. A larger peptide of Mr = 120,000 identified in the intermediate lobe of pituitary may represent the unproteolyzed form of this receptor. An affinity chromatography purification procedure has been developed for the D2 dopamine receptor. This procedure affords a substantial purification (greater than 1000 fold) of the receptor solubilized from bovine anterior pituitary glands with complete retention of its binding properties. These biochemical tools should eventually lead to the complete characterization of these two receptor subtypes.

Identification of the binding subunit of the D1-dopamine receptor by photoaffinity crosslinking.

The D1-dopamine receptor from rat striatum has been successfully identified by photoaffinity crosslinking using a newly synthesized radioiodinated derivative of the selective D1-antagonist SCH-23390. This compound, (R,S)-5-(3'-aminophenyl)-8-chloro-2,3,4,5-tetrahydro-3-methyl-[1H]-3- benzazepin-7-ol(SCH-38548), has been radioiodinated by a chloramine T procedure yielding three radioiodinated products. One of these separated congeners (with Rf = 0.35 on thin layer chromatography; CH2Cl2/MeOH/triethylamine; 82.5:17.5:0.01) binds reversibly to rat striatal membranes with high affinity (KD approximately equal to 200 pM), appropriate stereoselectivity, and D1-dopaminergic specificity. [125I]SCH-38548 can be covalently incorporated into a peptide of Mr approximately equal to 72,000 using the heterobifunctional crosslinking reagent N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate. Covalent incorporation of [125I]SCH-38548 into the Mr approximately equal to 72,000 peptide can be blocked by dopaminergic agents with D1-dopaminergic specificity (for agonists: SKF-38393 greater than apomorphine greater than dopamine; for antagonists: SCH-23390 much much greater than, SCH-23388 and cis-flupentixol much much greater than trans-flupentixol). The D1-dopaminergic selectivity and specificity of the labeling were further demonstrated by the fact that other antagonists such as domperidone, ketanserin, phentolamine, and alprenolol did not compete for the covalent labeling of the Mr approximately equal to 72,000 peptide. These results indicate that the ligand-binding subunit of the D1-dopamine receptor resides on peptide distinct from that of the D2-dopamine receptor (Mr = 94,000). This new radioligand should be useful in the molecular characterization of the D1-dopaminergic receptor from various sources.

Pharmacological effects of SCH 30497--a novel analgesic substance.

SCH 30497 (2-[3-(1,2,3,6-tetrahydro-4-2(methylphenyl)-pyridin-1-yl) -propyl]-1,2,4-triazolo[4,3-a] pyridin-3-(2H)-one) was tested for analgesic effects in the rat, mouse and squirrel monkey. SCH 30497 showed dose-related analgesic effects in the rat yeast-paw test; at peak times the ED7sec (95% confidence limits) in mg/kg via the oral, subcutaneous, intramuscular and intravenous routes of administration were as follows, respectively: 4.7 (2.1-9.8), 5.7 (3.4-9.6), 6.4 (4.1-9.8) and 1.4 (0.6-3.2). SCH 30497 was also analgesic in the acetic acid-induced writhing test in mice (ED50 = 1.9 mg/kg p.o.) and the squirrel monkey shock titration test (ED50 = 14.5 mg/kg p.o.). It was inactive in the mouse (100 mg/kg p.o.) and rat (40 mg/kg p.o.) tail-flick tests. Thus, SCH 30497 was efficacious versus chemical, mechanical and electrical nociceptive stimuli. Naloxone antagonism of the analgesic effects of SCH 30497 was species specific with significant inhibition observed only in the rat and not in the mouse or monkey. SCH 30497 did not produce Straub tail or hyperactivity in mice. Twice daily dosing at 30 mg/kg p.o. to rats for 5 days failed to produce tolerance; in separate experiments, daily injections for 10 days at 20 or 100 mg/kg p.o. failed to induce signs of dependence following naloxone challenge. SCH 30497-induced analgesia was not attenuated in rats previously made tolerant to narcotics by implantation of a morphine pellet. SCH 30497 showed a weak ability to displace 3H-Met5-enkephalin from its binding sites on rat brain membranes (IC50 = 48 microM). SCH 30497 (100 microM) did not affect prostaglandin synthesis in vitro. In vivo, the drug did not have anti-inflammatory or ulcerogenic effects up to 80 mg/kg p.o. Acute behavioral, neurological and autonomic side effects were primarily depressant in rodents and occurred at doses greater than 15 times those that were analgesically relevant. Moderate doses in the monkey (2.5 times the ED50) and high doses in mice produced convulsions. It is hypothesized that SCH 30497-like drugs represent a new class of analgesics based on this unique pharmacological spectrum of activity.