CI-1017, a functionally M1-selective muscarinic agonist: design, synthesis, and preclinical pharmacology.

The five muscarinic receptor subtypes (M1-M5) are characterized by seven helices that define a transmembrane cavity which serves as the binding pocket for agonists and antagonists. The five cavities appear to be topographically different enough to permit subtype selectivity among antagonists but not among classical agonists which tend to be smaller in size than antagonists. It was reasoned that synthesis of muscarinic agonists longer/larger than their classical counterparts might result in subtype selectivity. M1 subtype selectivity was found in a class of 1-azabicyclo[2.2.1]heptan-3-one, O-(3-aryl-2-propynyl) oximes. One of these, CI-1017, improved spatial memory of hippocampally deficient mice and nbM-lesioned rats at doses of 1.0-3.2 and 0.1-0.3 mg/kg, respectively, while producing parasympathetic side effects only at very high doses (100-178 mg/kg). Additionally, CI-1017 inhibited production of amyloidogenic A beta and increased secretion of soluble APP. Thus, CI-1017, besides treating AD symptomatically, may also retard its progression. CI-1017 has recently completed phase I clinical trials.

Milameline (CI-979/RU35926): a muscarinic receptor agonist with cognition-activating properties: biochemical and in vivo characterization.

Milameline (E-1,2,5,6-tetrahydro-1-methyl-3-pyridinecarboxaldehyde, O-methyloxime monohydrochloride, CI-979, PD129409, RU35926) was characterized in vitro and evaluated for effects on central and peripheral cholinergic activity in rats and rhesus monkeys. In muscarinic binding studies, milameline displayed nanomolar affinity with an agonist ligand and micromolar affinity with antagonist ligands, with approximately equal affinities determined at the five subtypes of human muscarinic receptors (hM(1)-hM(5)) with whole cells or membranes from stably transfected Chinese hamster ovary (CHO) cells. On binding, milameline stimulated phosphatidylinositol hydrolysis in hM(1) and hM(3) CHO cells and inhibited forskolin-activated cAMP accumulation in hM(2) and hM(4) CHO cells. Additionally, it decreased K(+)-stimulated release of [(3)H]acetylcholine from rat cortical slices. Responses were not caused by the inhibition of acetylcholinesterase, and there was no significant binding to approximately 30 other neurotransmitter binding sites. In rats, milameline decreased spontaneous and scopolamine-induced swimming activity, improved water-maze performance of animals impaired by basal forebrain lesions, increased cortical blood flow, decreased core body temperature, and increased gastrointestinal motility. Electroencephalogram activity in both rats and monkeys was characterized by a predominance of low-voltage desynchronized activity consistent with an increase in arousal. Milameline also reversed a scopolamine-induced impairment of attention on a continuous-performance task in monkeys. Thus, milameline possesses a pharmacological profile consistent with that of a partial muscarinic agonist, with central cholinergic actions being produced in rats and monkeys at doses slightly lower than those stimulating peripheral cholinergic receptors.

Blockade of the MAP kinase pathway suppresses growth of colon tumors in vivo.

The mitogen-activated protein kinase pathway is thought to be essential in cellular growth and differentiation. Here we report the discovery of a highly potent and selective inhibitor of the upstream kinase MEK that is orally active. Tumor growth was inhibited as much as 80% in mice with colon carcinomas of both mouse and human origin after treatment with this inhibitor. Efficacy was achieved with a wide range of doses with no signs of toxicity, and correlated with a reduction in the levels of activated mitogen-activated protein kinase in excised tumors. These data indicate that MEK inhibitors represent a promising, noncytotoxic approach to the clinical management of colon cancer.

Design and synthesis of m1-selective muscarinic agonists: (R)-(-)-(Z)-1-Azabicyclo[2.2.1]heptan-3-one, O-(3-(3'-methoxyphenyl)-2-propynyl)oxime maleate (CI-1017), a functionally m1-selective muscarinic agonist.

The synthesis and SAR of a series of (Z)-(+/-)-1-azabicyclo[2.2. 1]heptan-3-one, O-(3-aryl-2-propynyl)oximes are described. The biochemistry and pharmacology of 24Z (PD 142505) and its enantiomers are highlighted. 24Z is functionally an m1-selective muscarinic agonist. Efficacy and m1 selectivity reside in the R enantiomer, (R)-24Z (CI-1017).

The pharmacology of the novel and selective sigma ligand, PD 144418.

The pharmacology of PD 144418 (1-propyl-5-(3-p-tolyl-isoxazol-5-yl)-1,2,3,6-tetrahydropyridine) was characterized using neurochemical, biochemical and behavioral techniques. For sigma (sigma 1 and sigma 2 respectively) sites, PD 144418 affinities were determined using whole guinea pig brain membranes with [3H](+)-pentazocine and neuroblastoma x glioma cell membranes using [3H]1,3,di-O-tolylguanidine (DTG) in the presence of 200 nM (+)-pentazocine. PD 144418 exhibited an affinity for sigma 1 of 0.08 nM (Ki) versus a K1 of 1377 nM for sigma 2 site. Additional receptor binding studies indicated that PD 144418 lacked affinity for dopaminergic, adrenergic, muscarinic and a variety of other receptors. In vitro studies indicated that PD 144418 reversed the N-methyl-D-aspartate (NMDA)-induced increase in cyclic GMP (cGMP) in rat cerebellar slices without affecting the basal levels, suggesting that sigma 1 sites may be important in the regulation of glutamine-induced actions. PD 144418 potentiated the decrease in 5-hydroxytryptophan caused by haloperidol in the mesolimbic region, but by itself had no effect in 5-hydroxytrypamine (5-HT) and dopamine (DA) synthesis. Behaviorally, similar to other sigma ligands, PD 144418 antagonized mescaline-induced scratching at doses that did not alter spontaneous motor activity. This action is suggestive of potential antipsychotic property. It exhibited no anxiolytic and antidepressant properties in the models used. These results show that PD 144418 is a very selective sigma 1 agent, devoid of any significant affinity for other receptors and that sigma 1 site may modulate actions in the CNS.

Mutations of aspartate 103 in the Hm2 receptor and alterations in receptor binding properties of muscarinic agonists.

Aspartate 103 (D103) in the third transmembrane domain of the Hm2 receptor was mutated to glutamate (D103E), asparagine (D103N), or alanine (D103A). As measured by [3H]-NMS, no significant binding was observed in D103A, while a 2-fold decrease in ligand affinity was seen in D103E and a 32-fold decrease in affinity was found in the D103N mutant. Examination of reference agonists showed greater loss of affinity in D103N than in D103E with the rank order of change being: L-607,207>carbachol>arecoline>pilocarpine>oxotremorine>McN-A-343. Of the novel 1-azabicyclo[2.2.1]-heptan-3-one oxime agonists examined, arylacetylene oximes showed little alteration in binding in either the D103E or D103N mutants, while the geometric isomers of several bicyclic aryl-ene-yne oximes showed significant changes in affinity, especially in the D103N mutant. Thus, overall size of the agonist and/or spatial orientation of the molecule within the binding pocket contribute to changes measured in binding.

Characterization of muscarinic agonists in recombinant cell lines.

Using recombinant CHO cells that express Hm1-Hm5 receptors, reference muscarinic agonists have been characterized with respect to their activity in receptor binding and second messenger assays. In whole cell [3H]-N-methyl scopolamine binding, no agonist was found to be truly subtype selective, although some showed marked differences between several of the subtypes (e.g. m1 vs. m2). As a functional index of receptor activation, phosphatidyl-inositol (PI) turnover was measured for m1, m3, and m5 receptors while inhibition of forskolin-stimulated cAMP accumulation was measured for m2 and m4 receptors. Both full and partial agonists were delineated in PI turnover, but all agonists showed similar responses on cAMP. Alkylation studies with propylbenzylcholine mustard showed that both efficacy and potency were markedly affected in the functional assays by the number of free receptors. Thus, receptor reserve appears to play a major role in the determination of subtype selectivity for agonists using functional measures. Even with these limitations, however, the use of transformed cell lines is playing a pivotal role in the discovery of selective agonists.

Synthesis and SAR of bulky 1-azabicyclo[2.2.1]-3-one oximes as muscarinic receptor subtype selective agonists.

The synthesis of a series of potent and efficacious 1-azabicyclo[2.2.1]heptan-3-one oxime muscarinic agonists is described. The oximes have extended appendages designed to span the cavity defined by the seven transmembrane helices of the muscarinic receptor. Some members of the series are selective for receptors of the m1 subtype. One such oxime, 31, shows affinity and functional selectivity for m1 over m2, m3, and m4 muscarinic receptor types.

Cholinergic agents: effect of methyl substitution in a series of arecoline derivatives on binding to muscarinic acetylcholine receptors.

Arecoline, arecaidine, and a series of derivatives, differing by the presence or absence of methyl groups at positions on the periphery of the molecule, were prepared, and their binding to muscarinic acetylcholine receptors was tested. On the basis of this study, muscarinic agonism for arecoline series is governed by strict structure-activity relationships, as previously observed for other agonist series. Only minor changes in nitrogen substitution were tolerated in the present series of arecoline derivatives.

4-(1,2,5,6-Tetrahydro-1-alkyl-3-pyridinyl)-2-thiazolamines: a novel class of compounds with central dopamine agonist properties.

The design, synthesis, and pharmacological properties of a novel type of 4-(1,2,5,6-tetrahydro-1-alkyl-3-pyridinyl)-2-thiazolamine with dopaminergic properties are described. In particular, 4-(1,2,5,6-tetrahydro-1-propyl-3-pyridinyl)-2-thiazolamine (4c, PD 118440) and its allyl analogue (4i, PD 120697) have been identified as orally active dopamine (DA) agonists with pronounced central nervous system effects in tests that include [3H]-haloperidol and [3H]-N-propylnorapomorphine binding, inhibition of striatal DA synthesis, inhibition of DA neuronal firing, inhibition of spontaneous locomotor activity, and reversal of reserpine-induced depression in rats. The DA autoreceptor selectivity of these heterocyclic analogues of 3-(1-propyl-3-piperidinyl)phenol (3-PPP) was also evaluated. In this series, DA agonist activity was found to be highly dependent on the size of the N-alkyl substituent, the saturation level of the six-membered ring, and the mode of attachment of the 2-aminothiazole ring.

Examination of a series of 8-[3-[bis(4-fluorophenyl)amino]propyl]-1-aryl-1,3,8- triazaspiro[4.5]decan-4-ones as potential antipsychotic agents.

8-[3-[Bis(4-fluorophenyl)amino]propyl]-1-phenyl-1,3,8- triazaspiro[4.5]decan-4-one (3) and related compounds have been shown to have antipsychotic profiles in biochemical and behavioral pharmacological test models. The dose of 3 necessary to produce catalepsy in rats is much greater than that required for activity in behavioral tests predictive of antipsychotic efficacy, for example the suppression of high base line medial forebrain bundle self-stimulation in rats. This suggests that 3 would have a reduced propensity for neurological side effects. The effects of substitution on the 1-phenyl moiety and on the N-3 nitrogen atom of the triazaspirodecanone portion of 3 were examined. Results from this study suggest that behavioral activity is sensitive to substituents on the 1-phenyl moiety while substituents on the N-3 nitrogen are more generally tolerated. In both rats and squirrel monkeys compound 3 was found to have a similar separation between doses inhibiting Sidman avoidance activity and those causing catalepsy. However, in an extrapyramidal side effect (EPS) test model using haloperidol-sensitized cebus monkeys, 3 elicited signs of EPS at doses approximating those previously determined to be efficacious.

Potential histamine H2-receptor blockers. 3- and 2-indole derivatives as immobile analogues of tautomeric forms of cimetidine.

At physiological pH, cimetidine (1a) and its analogues burimamide (1b) and metiamide (1c) exist mainly as an equilibrium mixture of tautomers. A high concentration of tautomer 2 is associated with increased H2-receptor interaction. 3-Indole derivatives (5c-f) and 2-indole derivatives (6c-f) were synthesized and tested as immobile analogues of tautomers 2 and 3, respectively. Weak competitive H2 antagonism was found in N'-cyano-N-[2-[(1H-indol-3-ylmethyl)thio]ethyl]carbamidothioic acid methyl ester (5e) and N-[2-[(1H-indol-2-ylmethyl)thio]-ethyl]-N'-methylthiourea (6c).