1 The replacement of 4-hydroxy-N-methyl piperidine (HO NMe) in 4-diphenylacetoxy-Nmethyl piperidine (4-DAMP) metho-bromide by 4-hydroxy-but-2-ynylamines (HOCH2C= CCH2NR2) reduces the affinity for muscarine-sensitive acetylcholine receptors in guinea-pig ileum and atria. It does not abolish selectivity. The tertiary amines are more active and more selective than the corresponding quaternary salts. 2 Analogous derivatives of 4-hydroxy-but-2-ynylamines which lack the ester group (i.e. substituted 4-hydroxymethyl-propynyl amines) are less active and less selective. The quaternary compounds are more active than the tertiary bases. 3 The diphenylcarbamyl ester of 4-hydroxy-N-methylpiperidine methobromide has less than onethousandth of the activity of the diphenylacetyl ester (4-DAMP methobromide) and is not selective. 4 Although 4-diphenylacetoxy-butynyl dimethylamine is only about one-hundredth as active as 4-DAMP methobromide it appears to have comparable selectivity. It is an interesting compound because it is a tertiary amine and should cross membranes.

1 4-Diphenylacetoxy-N-(2-chloroethyl)-piperidine (4-DAMP mustard), which is known to block muscarinic M3 receptors in preference to muscarinic M2 receptors, was used to estimate the apparent affinity constants of some agonists acting at muscarinic receptors in guinea-pig ileum. Estimates for carbachol and n-pentyl-trimethyl ammonium iodide were similar to published values obtained in similar conditions: those for n-hexyl-trimethyl ammonium iodide were slightly lower. 2 The results for the agonists, n-pentyl-and n-hexyl-trimethyl ammonium iodides and for the partial agonist, n-heptyl-trimethyl ammonium iodide were not as regular as was suggested by Stephenson, though there is an overall increase in apparent affinity with chain length. 3 Estimates of apparent affinity may be affected by hexamethonium, usually present in experiments on ileum. Its absence had little effect on the results with carbachol but reduced the estimates obtained with n-pentyl trimethyl ammonium, which has strong nicotinic effects compared with its muscarinic effects. On ileum treated with tetrodotoxin the values for n-pentyl trimethyl ammonium were similar to those obtained in the presence of hexamethonium (0.28mM): slightly higher estimates of affinity were obtained in the presence of indomethacin (2.8 pM). The nicotinic effects of n-pentyl ammonium may involve the release of prostaglandins. 4 The estimates of apparent affinity did not depend on the method used to calculate them as the 'null' method and the 'operational' method give similar answers. Estimates of the transducer-ratio for the partial agonist, n-heptyl-trimethyl ammonium iodide, were numerically the same as those of its efficacy. 5 This work illustrates the use of 4-DAMP mustard as a tool for measuring the apparent affinity of agonists acting at muscarinic M3 receptors.

4-Diphenylacetoxy-N-(2-chloroethyl)-piperidine hydrochloride (I) cyclizes at neutral pH to form an aziridinium salt. The formation and breakdown of the salt depend on the temperature (in the range 25 to 37 degrees C). In solution at 30 degrees C, peak levels, corresponding to 60-80% conversion, are reached after around 60 min and the half-life exceeds 100 min. In the presence of 0.9% NaCl conversion was reduced to 45-60%. I blocks muscarinic receptors in guinea-pig ileum and atria irreversibly and it is possible to produce dose-ratios on ileum with 10 nM I which are about 100 times those on atria. After about 30 min exposure to solutions of I (prepared 15-20 min previously so that formation of aziridinium ions is well-established) the graph of log (dose-ratio) against time is linear and similar plots were obtained with two different agonists, carbachol and ethoxyethyltrimethylammonium. With results for the ileum, extrapolation of the line suggests that it does not start from zero (dose-ratio = 1): this is because of an initial relatively rapid reversible block. This early phase is similar to that seen on ileum with 10 nM 4DAMP methobromide, which is a competitive antagonist, so is probably caused by competitive block by the aziridinium ion, which closely resembles 4DAMP metho-salts. The subsequent irreversible phase should be caused by alkylation of the receptors. I is easy to make and should be a valuable tool for the study of muscarinic receptors.

1 4-Diphenylacetoxy-1:1-dimethyl cyclohexane (carbo-4-DAMP) is the carbon analogue of 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) methiodide. The compounds differ only in that the quarternary nitrogen atom in 4-DAMP methiodide is replaced by a quaternary carbon atom, which is uncharged. 2 Carbo-4-DAMP appears to act competitively at functional (M3) muscarinic receptors in guinea-pig ileum. Estimates of log affinity constant are 6.0 at 30°C and 5.9 at 37'C, i.e. the compound has 0.1% of the affinity of 4-DAMP methobromide. 3 The absence of charge makes little difference to the conformation as determined by X-ray crystallography. The bond lengths and angles are very similar, though the bonds in the cyclohexane ring of carbo-4-DAMP are consistently slightly longer than those in the piperidinium ring of 4-DAMP methiodide, and the presence of the charge slightly reduces the space between molecules. 4 The difference between the affinities of 4-DAMP methobromide and carbo-4-DAMP indicates that the contribution of coulombic forces to the binding between 4-DAMP methiodide and muscarinic (M3) receptors is at least 17 kJ mol' (4.1 kcal mol-') at 37°C. How much this is an underestimate depends upon how much hydrophobic binding is greater with the uncharged compound.

1 Lengthening the chain in diphenylacetylcholine decreases affinity for muscarinic cholinoceptors in guinea-pig ileum. Diphenylacetoxypropyldimethylamine and its quaternary trimethylammonium salt are roughly equiactive: the dimethylamine and the piperidine have some selectivity for ileum compared with atria, but are not as active nor as selective as 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) methobromide (MeBr). With the weaker diphenylacetoxybutyl compounds the base is more active than the quaternary salt. 2 The diphenylacetoxybutyl-, cis-butenyl and trans-butenyl compounds have similar affinities. The quaternary salts are less active than the tertiary bases, but they are less selective than the butynyl analogues studied in earlier work. 3 1,1-Diphenyl-1-hydroxy-2,4-hexadiynyl dimethylamine and its trimethylammonium salt are inactive in concentrations below 100 uM, as are the (+-camphor-sulphonyl ester of 4-hydroxy-N-methyl piperidine and its methiodide. The (±)-phenylcyclopentylacetyl ester of 4-hydroxy-N-methylpiperidine methobromide is more active than its cyclohexyl analogue and than 4-DAMP MeBr but it is less selective than 4-DAMP MeBr.4 The high selectivity of p-fluoro-hexahydrosila-diphenidol is confirmed but this compound has relatively low affinity (for ileum log K = 7.8). 5 The results indicate steric constraints to binding at muscarinic receptors which could be used to check molecular modelling of the receptor based on its known amino acid sequence. The group binding the charged nitrogen is probably at the mouth of a cavity which can accommodate two large rings (as in 4-DAMP MeBr) but with a depth less than about 7 A so that the rod-like hexadiynes cannot fit. Differences between types of receptor may only involve small changes in geometry secondary to differences in amino acids not directly involved in binding and the production of selectivity depends upon finding substituents which interfere with binding more at one type of receptor than at another.