Conversion of N-(2-chloroethyl)-4-piperidinyl diphenylacetate (4-DAMP mustard) to an aziridinium ion and its interaction with muscarinic receptors in various tissues.
A 2-chloroethylamine derivative [N-(2-chloroethyl)-4-piperidinyl diphenylacetate (4-DAMP mustard)] of the selective muscarinic antagonist N,N-dimethyl-4-piperidinyl diphenylacetate (4-DAMP) was synthesized, and its conversion to an aziridinium ion and interaction with muscarinic receptors was investigated. When dissolved in aqueous solution at pH 7.4 and 37 degrees, 4-DAMP mustard released an equivalent amount of chloride. The release of chloride was consistent with a first-order process having a half-time of 5.7 min. The aziridinium ion reached a peak concentration at 32 min, corresponding to 75% of the initial concentration of 4-DAMP mustard. When homogenates of rat brain, heart, and submaxillary gland were incubated with 4-DAMP mustard (9 nM) for 1 hr, washed extensively, and then assayed for muscarinic receptor binding properties, a 56% decrease in the binding capacity of N-[3H]methylscopolamine in the heart and brain and a 71% decrease in the gland were observed, without a significant change in the dissociation constants. The affinity of 4-DAMP mustard and its transformation products for muscarinic receptors was determined in competitive binding experiments with N-[3H] methylscopolamine, and the results show that the aziridinium ion of 4-DAMP mustard was the most potent form, compared with the parent 2-chloroethylamine (4-DAMP mustard) and the alcoholic hydrolysis product. The rates of receptor alkylation by 4-DAMP mustard were measured in the rat heart and gland. Virtually no alkylation (less than 1%) occurred in the heart at a 4-DAMP mustard concentration of 1.6 nM, after 30 min, whereas almost 50% alkylation was observed in the gland under the same conditions. Almost complete alkylation of receptors in the gland could be achieved at a 4-DAMP mustard concentration of 200 nM, after 1 hr. Treatment of the isolated rat ileum with 4-DAMP mustard caused an irreversible blockade of contractions elicited by the muscarinic agonist oxotremorine-M, and this blockade persisted after extensive washing. The results presented here show that 4-DAMP mustard forms an aziridinium ion that binds irreversibly to muscarinic receptors and exhibits selectivity for M3, compared with M2 muscarinic receptors.
Thomas
E A
EA
Department of Pharmacology, College of Medicine, University of California, Irvine 92717.
Hsu
H H
HH
Griffin
M T
MT
Hunter
A L
AL
Luong
T
T
Ehlert
F J
FJ
D000478:Alkylation; D000818:Animals; D001388:Aziridines / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D004158:Diphenylacetic Acids / Q000737:chemistry / Q000378:metabolism*; D004347:Drug Interactions; D006863:Hydrogen-Ion Concentration; D007082:Ileum / Q000187:drug effects / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D009206:Myocardium / Q000378:metabolism; D019832:N-Methylscopolamine; D010095:Oxotremorine / Q000494:pharmacology; D010880:Piperidines / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000378:metabolism*; D012602:Scopolamine Derivatives / Q000037:antagonists & inhibitors / Q000378:metabolism; D013363:Submandibular Gland / Q000187:drug effects / Q000378:metabolism; D013696:Temperature
D001388:Aziridines; D004158:Diphenylacetic Acids; D010880:Piperidines; D011976:Receptors, Muscarinic; D012602:Scopolamine Derivatives; C074183:N-(2-chloroethyl)-4-piperidinyl diphenylacetate; C033132:aziridine; D010095:Oxotremorine; D019832:N-Methylscopolamine