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Oxotremorine (Ia) and its succinimide analogue (IIa) have been substituted in the pyrrolidine ring with a methyl group in the 2- or 3-positions. The compounds are oxotremorine antagonists. The 2-methyl-substituted enantiomers show stereoselectivity, the S-isomers being the most active.

A series of structural analogues of the potent oxotremorine-like agent N-(4-pyrrolidino-2-butynyl)-N-methyl-acetamide (1) was investigated for muscarinic activity in the isolated guinea pig ileum. Substitution of larger alkyl groups for the acetyl methyl group of 1 results in an attenuation of muscarinic potency. The observation that the agonist N-(4-dimethylamino-2-butynyl)-N-methylpropionamide (6) has a dissociation constant (KA = 5.1 X 10(-5) M), estimated after elimination of spare receptors with dibenamine, similar to that of the antagonist N-(4-dimethylamino-2-butynyl)-N-methyl-2,2-dimethylpropionamide (11) suggests that the decrease in muscarinic agonist activity with increasing substitution is due mainly to a loss of efficacy. The N-methyl group of 1 is essential for muscarinic activity since its replacement by a hydrogen atom or an ethyl group yields antagonists.

Two mustard analogues of oxotremorine, N-[4-(2-chloroethylmethylamino)-2-butynyl]-2-pyrrolidone (BM 123) and N-[4-(2-chloromethylpyrrolidino)-2-butynyl]-2-pyrrolidone (BM 130), were synthesized. BM 123 and BM 130 cyclize in neutral aqueous solution to aziridinium ions. These aziridinium ions are potent stimulants of the guinea pig ileum. This agonist activity is unaffected by hexamethonium but is inhibited by methylatropine and by pretreatment of solutions of BM 123 and BM 130 with thiosulfate. The parent 2-chloroalkylamines and the alcohols formed by hydrolysis of the aziridinium ions have very weak pharmacological effects. Incubation of the isolated guinea pig ileum for 30 min with BM 123 at 20 microM and 2 microM caused 94% and 48% receptor alkylation, respectively, as calculated from the shift in the agonist dose-response curve. Similar incubations with BM 130 at 20 microM and 5 microM alkylated 85% and 63% of the receptors. No recovery from this apparent blockade was observed over a 4-hr time period. These calculated receptor occupancies by BM 123 and BM 130 agreed with those estimated from reduction of [3H] (-)-3-quinuclidinyl benzilate binding to homogenates of the ileum following exposure to BM 123 and BM 130. Methylatropine (20 nM) protected against the irreversible actions of BM 123 and BM 130. In homogenates of the guinea pig ileum, BM 123 and BM 130 also caused a selective reduction in the binding capacity of [3H]N-methylscopolamine without significantly affecting the apparent affinity. This inhibitory effect persisted after extensive washing. BM 130 was a weak agonist at nicotinic receptors of the frog rectus abdominis muscle, whereas BM 123 was almost inactive. In conclusion, BM 123 and BM 130 are potent and specific muscarinic agonists that bind irreversibly to muscarinic receptors.

A number of secondary and tertiary amines as well as quaternary ammonium compounds, obtained by structural modification of the amino group of oxotremorine (1) and its acetamide analogue (14), were investigated for muscarinic and antimuscarinic activity in vivo and in vitro. For the quaternary ammonium analogues, decrease of in vitro muscarinic potency is well correlated with increase of the size of the quaternary ammonium group, as estimated from increments in apparent molal volumes. A similar decrease of muscarinic potency with increasing substitution at the nitrogen atom is generally observed for the secondary and tertiary amines. For the latter the reduction in muscarinic activity appears to be due to loss of efficacy, since the higher homologues are partial agonists or antagonists. There is a highly significant correlation between muscarinic activity in vitro and central tremorogenic activity of the tertiary amines.

1 The enantiomers of some analogues of the central muscarinic agent, oxotremorine, were prepared and investigated for tremorogenic and tremorolytic activity in intact mice and for muscarinic and antimuscarinic activity on the isolated ileum of the guinea-pig. 2 The R-isomers were more potent than the S-isomers both in vivo and in vitro regardless of whether the compounds are agonists, partial agonists or competitive antagonists. 3 It is suggested that in the oxotremorine series, agonists and antagonists interact with a common receptor site, in contrast to classical muscarinic antagonists which are believed to bind also to accessory receptor areas, located close to the agonist binding site.

Some of the central and peripheral effects of oxotremorine (OT) and its azetidine analogue, N-[4-azetidinyl)-2-butynyl]-2-pyrrolidone (BM 120), were compared in mice and rats. BM 120 was found to be about twice as potent as OT and to have a somewhat longer duration of action. All its effects were antagonized by pretreatment with atropine sulphate. BM 120 acted as powerful muscarinic agonist on the isolated guinea pig ileum.

The stereoisomers of some analogues of oxotremorine containing two chiral centres, one in the 1-position of the butynyl chas are oxotremorine antagonists. They show a marked stereoselectivity, which depends mainly on the configuration of the chiral centre in the butynyl chain and to a lesser extent on the configuration of that in the pyrrolidine ring.