A number of analogs of the natural product himbacine were synthesized employing variations at the heterocyclic unit and the tether that links the heterocyclic unit to the tricyclic motif. Several of these analogs had M(2) affinity and M(1)/M(2) selectivity comparable to those of himbacine. The structural and stereochemical requirements of the heterocyclic unit for muscarinic binding are discussed in the light of these data.

Low molecular weight amide derivatives were synthesized and evaluated as M(2) receptor antagonists for the treatment of Alzheimer's disease. Isopropyl amides 19 and 31 are highly potent, selective and low molecular weight M(2) receptor antagonists with structural features different from our clinical candidate 1.

We have discovered highly potent, selective sulfide M(2) receptor antagonists with low molecular weight and different structural features compared with our phase I clinical candidate Sch 211803. Analogue 30 showed superior M(2) receptor selectivity profile over Sch 211803. More importantly, this study provided new leads for the discovery of M(2) receptor antagonists as potential drug candidates.

We previously reported the initial discovery of a novel class of stabilized benzylidene ketal M(2) receptor antagonists. This paper discusses new analogues consisting of benzamide modifications which not only improved M(2) receptor affinity and selectivity, but also enhanced the pharmacokinetic properties of the series. These changes led to the discovery of a highly potent and selective M(2) antagonist, which demonstrated in vivo efficacy and had good bioavailability in multiple species.

In search of a backup M(2) muscarinic receptor antagonist to the previously reported compound 1, we discovered compound (+)-14, which showed superior oral efficacy in animal models. The improvement of oral efficacy was achieved by modulating both the molecular weight and lipophilicity of the lead compounds.

A novel series of 2-(R)-methyl-substituted piperazines (e.g., 2) is described. They are potent M(2) selective ligands that have >100-fold selectivity versus the M(1) receptor. In the rat microdialysis assay, compound 14 showed significantly enchanced levels of acetylcholine after oral administration.

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.

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.

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.

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.

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.

Novel, selective M2 muscarinic antagonists, which replace the metabolically labile styrenyl moiety of the prototypical M2 antagonist 1 with an ether linkage, were synthesized. A detailed SAR study in this class of compounds has yielded highly active compounds that showed M2 Ki values of < 1.0 nM and >100-fold selectivity against M1, M3, and M5 receptors.

A parallel synthesis of racemic himbacine analogs was carried out by N-alkylation of various commercially available cyclic amine derivatives with the alkylating agent 4 which bears the tricyclic unit of himbacine. Several of these analogs have potency comparable to that of himbacine, albeit lacking the desired selectivity. Structure-activity relationship studies support the existence of a hydrophobic pocket in the receptor where the piperidine ring of dihydrohimbacine binds.

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 M-2 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-piperazineacetonitrile. This compound, SCH 57790, binds to cloned human M-2 receptors expressed in CHO cells with an affinity of 2.78 nM; the affinity at M-1 receptors is 40-fold lower. SCH 57790 is an antagonist at M-2 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 M-2 receptor blockade for enhancement of cognition.

Plasma and brain concentrations of the dopamine D1 receptor antagonist, SCH 39166, were measured and compared to behavioral activity in the conditioned avoidance response paradigm (CAR). SCH 39166 was administered at two behaviorally active doses (1 mg/kg, SC and 10 mg/kg, PO) and the time course for CAR activity was compared with the plasma and brain concentrations of unconjugated SCH 39166. Conjugation andN-demethylation of SCH 39166 after oral administration were also determined and first pass metabolism examined. Results from these studies demonstrated a similar time-dependent disappearance of unconjugated SCH 39166 from both the plasma and brain, independent of route of administration. Brain concentrations of SCH 39166 were approximately 5-fold higher than corresponding plasma concentrations, regardless of route. However, plasma and brain concentrations of unconjugated SCH 39166 were higher after SC administration of 1.0 mg/kg, than after PO administration of 10 mg/kg, suggesting a substantial first pass metabolism of SCH 39166. In addition, total (conjugated and unconjugated) plasma concentrations of SCH 39166 were at least 10-fold higher than unconjugated concentrations of SCH 39166 after PO administration of 10 mg/kg, demonstrating that a high proportion of drug was conjugated. Metabolism to theN-desmethyl analog, SCH 40853, was observed after PO administration of 10 mg/kg SCH 39166 and a high proportion of conjugation of the desmethyl analog was also seen. Finally, plasma concentrations of unconjugated SCH 39166 exhibited a high positive correlation (r=0.934,P<0.001) with brain concentrations of unconjugated SCH 39166. Behavioral activity in the CAR was also positively correlated with plasma concentrations (r=0.95,P<0.001) and brain concentrations (r=0.88,P<0.001) of unconjugated SCH 39166.

SCH 39166 is a novel benzonaphthazepine that is a selective dopamine D1 receptor antagonist. It is currently undergoing clinical trials in humans for possible use as an antipsychotic medication. To date, no studies have been presented describing the metabolism of SCH 39166. Therefore, the present studies investigated the possible in vitro glucuronidation of SCH 39166 from both rodent and squirrel monkey liver microsomes. Comparisons were made with the classic D1 antagonist, SCH 23390, a benzazepine which undergoes extensive glucuronidation in the rat. Additionally, dose response and duration of action of SCH 39166 on the conditioned avoidance paradigm (CAR) in both rats and squirrel monkeys were compared to the dose response and duration of action of SCH 23390 in these species. Results demonstrated that H-3-SCH 39166 was glucuronidated by both rat and monkey liver microsomes. In studies with rat liver microsomes, the rate of glucuronidation of SCH 39166 and the affinity for the glucuronosyltransferase enzyme were equivalent to those of SCH 23390. In vivo behavioral studies also indicated no differences in either the dose-response curves or the time course of behavioral effects for either Dl antagonist after oral administration in the rat. In contrast, the rate of glucuronidation in squirrel monkey liver microsomes of SCH 23390 was 3-4 times faster than that of SCH 39166. Moreover, these findings were consistent with in vivo behavioral studies in squirrel monkeys, in which the duration of action of SCH 23390 was much shorter than SCH 39166.

SCH 23390 is a novel benzazepine that selectively blocks dopamine receptors of the D1 subtype. Glucuronidation of this selective D1 antagonist was studied in vitro using rat liver microsomes. Methods to separate SCH 23390 glucuronide from SCH 23390 were developed which utilized either HPLC techniques or solvent extraction of SCH 23390 with 3-heptanone. Formation of a SCH 23390 glucuronide was confirmed upon incubation of SCH 23390 and UDPGA with naive rat liver microsomes. Liver enzyme activity for SCH 23390 glucuronidation was also enhanced after addition of the detergents, Lubrol or Triton X-100, to the naive liver microsomes. Kinetic analyses indicated an apparent Vmax and Km for UDPGA as 120.9 pmol/mg protein/min and 0.63 mM, and an apparent Vmax and Km for SCH 23390 as 282.4 pmol/mg protein/min and 0.41 microM. Further characterization of the liver enzyme responsible for the glucuronidation of SCH 23390 revealed a stereoselective substrate preference similar to that seen with the D1 dopamine receptor. Substrate inhibition studies indicated that SCH 23390, haloperidol, apomorphine, and alpha-naphthol demonstrated the highest affinity for the glucuronosyltransferase enzyme. However, (-)-sulpiride, raclopride, and endogenous substrates such as dopamine, serotonin, epinephrine, and norepinephrine demonstrated low affinity for the liver enzyme. These studies describe a rat liver glucuronosyltransferase with a unique substrate specificity toward selected dopaminergic agents. Finally, induction profiles revealed that neither phenobarbital (100 mg/kg, ip, for 3 days), beta-naphthoflavone (100 mg/kg, ip, for 4 days), nor 3-methylcholanthrene (80 mg/kg, ip, for 4 days) enhanced liver glucuronosyltransferase activity for SCH 23390 glucuronidation.