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Alkylthio-TZTPs (3-(3-alkylthio-1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-met hylpyridines) and corresponding azabicyclic analogs were tested for m1 efficacy in cloned human m1 receptors and for antinociceptive activity in the mouse grid shock assay. The m1 (%PI) SAR were distinctly different from the analgesia and the salivation SAR, suggesting that analgesia is mediated by neither m1 nor M3 muscarinic receptors.

Xanomeline [3(3-hexyloxy-1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-me thylpyridine)] was evaluated in vivo in rat brain for effects on neurotransmitter turnover and inhibition of ex vivo binding of muscarinic radioligands. Xanomeline produced dose-related increases in the metabolite of dopamine, dihydroxyphenylacetic acid (DOPAC), in striatum. The increases in striatal DOPAC levels produced by xanomeline were antagonized by the relatively selective M1 antagonist trihexyphenidyl, suggesting that xanomeline interacts with M1 heteroreceptors on dopamine nerve terminals. Xanomeline produced small increases in striatal acetylcholine levels and did not antagonize the large increases in acetylcholine produced by the nonselective muscarinic agonist oxotremorine, indicating that xanomeline did not block M2 autoreceptors. Xanomeline inhibited ex vivo binding of muscarinic radioligands to homogenates of brain and the inhibition of ex vivo binding occurred in the same dose range as increases in DOPAC levels. Xanomeline did not appreciably induce salivation or antagonize oxotremorine-induced salivation indicating that xanomeline does not interact with M3 receptors. The effects of xanomeline on ex vivo binding and DOPAC levels lasted for about 3 hr and were evident after oral administration. An analog of xanomeline with similar in vivo effects did not inhibit acetylcholinesterase or choline acetyltransferase and inhibited choline uptake only at concentrations much higher than those required to inhibit binding. These data indicate xanomeline is selective agonist for M1 over M2 and M3 receptors in vivo in rat. It is not known whether xanomeline interacts with m4 or m5 receptors in vivo.

Xanomeline [3(3-hexyloxy-1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1- methylpyridine] has been evaluated as a muscarinic receptor agonist. In vitro, xanomeline had high affinity for muscarinic receptors in brain homogenates, but had substantially less or no affinity for a number of other neurotransmitter receptors and uptake sites. In cells stably expressing genetic m1 receptors, xanomeline increased phospholipid hydrolysis in CHO, BHK and A9 L cells to 100, 72 and 55% of the nonselective agonist carbachol. In isolated tissues, xanomeline had high affinity for M1 receptors in the rabbit vas deferens (IC50 = 0.006 nM), low affinity for M2 receptors in guinea pig atria (EC50 = 3 microM), was a weak partial agonist in guinea pig ileum and was neither an agonist nor antagonist in guinea pig bladder. In vivo, xanomeline increased striatal levels of dopamine metabolites, presumably by acting at M1 heteroreceptors on dopamine neurons to increase dopamine release. In contrast, xanomeline had only a relatively small effect on acetylcholine levels in brain, indicating that it is devoid of actions at muscarinic autoreceptors. In the gastrointestinal tract, xanomeline inhibited small intestinal and colonic motility, but increased small intestinal transmural potential difference. In contrast to the nonselective muscarinic agonist oxotremorine, xanomeline did not produce salivation, tremor nor hypothermia; it did, however, increase heart rate. The present data are consistent with the interpretation that xanomeline is a novel muscarinic receptor agonist with functional selectivity for M1 muscarinic receptors both in vitro and in vivo.

In the electrically field-stimulated rabbit vas deferens, muscarinic receptor agonists increase twitch-height by actions at postjunctional M2 receptors and decrease twitch-height by actions at prejunctional M1 receptors. In the present studies, in contrast to previous reports, muscarinic receptor agonists primarily decreased twitch-height, produced minimal increases in twitch-height, and, produced identical responses in both epididymal and prostatic tissue segments, thus permitting a more detailed investigation of the M1 receptor component of action of muscarinic receptor agonists in the rabbit vas deferens. The nonselective muscarinic receptor agonist carbachol produced biphasic effects on twitch-height in the vas deferens: lower concentrations increased twitch-height to only approximately 25-30% over control, whereas higher concentrations inhibited the twitch. The selective M1 receptor antagonist pirenzepine blocked the inhibitory effects of carbachol, and unmasked carbachol-induced increases in twitch-height. Atropine, 4-DAMP (4-diphenylacetoxy-N-methylpiperidine methiodide) and AF-DX 116 (11-2[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro- 6H-pyrido[2,3-b][1,4]benzodiazepin-6-one) blocked both the inhibitory and stimulatory effects of carbachol, but atropine and 4-DAMP were more potent in blocking the inhibitory than the stimulatory effects of carbachol, whereas the reverse was true for AF-DX 116. McN-A-343 (4-hydroxy-2-butynyl)trimethylammonium chloride, m-chlorocarbanilate) and 12 other muscarinic receptor agonists from a variety of chemical classes also produced concentration-dependent decreases in twitch-height. The log IC50s of the muscarinic receptor agonists for decreasing twitch-height were highly correlated with their log Kis for inhibiting [3H]pirenzepine (r = 0.96) and [3H]oxotremorine-M (r = 0.85) binding in rat hippocampal membranes. The present results demonstrate that the muscarinic M1 receptor mediating inhibition of twitch-height in the rabbit vas deferens has pharmacologic properties similar to the muscarinic M1 receptor in rat hippocampus.

A series of 3-(3-substituted-pyrazinyl)-1,2,5,6-tetrahydro-1-methylpyridines were synthesized and found to have high affinity for central muscarinic receptors. The ability of some of these compounds to inhibit the electrically stimulated twitch of the guinea pig vas deferens indicated that the compounds were M1 agonists. M1 agonist activity was related to the length of the side chain attached to the pyrazine ring, with maximal activity being obtained with the hexyloxy side chain. The (hexyloxy)pyrazine 3f lacked M2 agonist activity as it failed to affect the guinea pig atria and was also relatively devoid of M3 agonist activity as determined by its lack of tremorogenic and sialogogic effects in mice. A comparison of the M1 agonist efficacy of these pyrazines and related 1,2,5-thiadiazoles and 1,2,5-oxadiazoles suggested that M1 efficacy was related to the magnitude of electrostatic potential located over the nitrogens of the respective heterocycles. The heteroatom directly attached to the 3 position of the pyrazine or 1,2,5-thiadiazole heterocycle markedly influenced the M1 efficacy of the compounds by determining the energetically favorably conformers for rotation about the bond connecting the tetrahydropyridyl ring and the heterocycle. A three-dimensional model for the M1-activating pharmacophore was proposed based on computational studies and the model of the muscarinic pharmacophore proposed by Schulman.

A series of novel 3-(3-substituted-1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro- 1-methylpyridines (substituted-TZTP; 5a-l, 7a-h, 8, 9c-n, 11, 13j) were synthesized and tested for central muscarinic cholinergic receptor affinity by using [3H]-oxotremorine-M (Oxo-M) and [3H]-pirenzepine (Pz) as ligands. The potency and efficacy of the compounds for the pharmacological defined M1 and M2 muscarinic receptors were determined on isolated electrically stimulated rabbit vas deferens and on spontaneously beating isolated guinea pig atria, respectively. Selected compounds were also tested for M3 activity in the isolated guinea pig ileum. The C1-8 alkoxy-TZTP 5a-l analogues all displaced [3H]-Oxo-M and [3H]-Pz with low nanomolar affinity. Depicting chain length against Oxo-M binding and against Pz binding the unbranched C1-8 alkoxy-TZTP (5a-h) derivatives produced U-shaped curves with butoxy- (5d) and (pentyloxy)-TZTP (5e) as the optimum chain length, respectively. This U-shaped curve was also seen in the ability of the compounds 5a-h to inhibit the twitch height in the vas deferens preparation. The (pentyloxy)- (5e) and the (hexyloxy)-TZTP (5f) analogues produced an over 90% inhibition of the twitch height with IC50 values in the low picomolar range. In both the atria and in the ileum preparations 5f had low efficacy and potency. With the (alkylthio)-TZTP (7a-h) analogues the structure-activity relationship was similar to the one observed with the alkoxy (5a-h) analogues, but generally 7a-h had higher receptor affinity and was more potent than the corresponding 5a-h. However, the C3-8 alkyl-TZTP (9c,e,g,h) analogues had 10-100 times lower affinity for the central muscarinic receptors than the corresponding alkoxy and alkylthio derivatives, and their efficacy in the vas deferens preparation was too low to obtain IC50 values. The unsubstituted TZTP (11) compound was a potent but nonselective muscarinic agonist. The two 3-(3-butoxy/(hexyloxy)-1,2,5-oxadiazol-4-yl)-1,2,5,6-tetrahy dro-1- methylpyridines (butoxy/hexyloxy)-OZTP; 19a/b) were also synthesized and tested. Both 19a and 19b had much lower affinity for the central muscarinic receptors than 5d and 5f, and the efficacy of 19a,b was too low to give IC50 values in the vas deferens preparation. Therefore, the C5-6 (alkyloxy)/(alkylthio)-TZTP's represent a unique series of potent functional M1 selective muscarinic agonists.

The effects on working memory of the muscarinic cholinergic agonists oxotremorine, arecoline, RS86 and pilocarpine, and the cholinesterase inhibitors physostigmine and tetrahydroaminoacadine were investigated in male F344 rats. Working memory was assessed by behavior maintained under a spatial alternation schedule of food presentation in which the interval between trials was varied from 2 to 32 sec. Under control conditions the percentage of correct responses decreased as the retention interval was varied from 2 to 32 sec. Administered alone the cholinergic agonists oxotremorine (0.01-0.1 mg/kg), arecoline (3-30 mg/kg), RS86 (0.3-3 mg/kg) and pilocarpine (0.3-3.0 mg/kg), and the cholinesterase inhibitors physostigmine (0.01-0.1 mg/kg) and tetrahydroaminoacridine (0.3-3.0 mg/kg) either had no effect on or produced dose-related deficits in working memory and decreases in response rates. The muscarinic antagonist scopolamine (0.1 mg/kg) produced retention interval-dependent decreases in the percentage of correct responding and rates of responding. The cholinergic agonists and tetrahydroaminoacridine failed to reverse the effects of scopolamine. However, physostigmine produced a dose-dependent reversal of the working-memory deficits and response-rate decreasing effects of scopolamine. The present results are consistent with the interpretation that drugs which primarily enhance M2 muscarinic cholinergic transmission are ineffective in enhancing working memory or in reversing scopolamine-induced deficits in working memory.