Molecular probes for muscarinic receptors: derivatives of the M1-antagonist telenzepine

Abstract

Functionalized congeners of the M1-selective muscarinic antagonist telenzepine (4,9-dihydro-3-methyl-4-[(4-methyl-1-piperazinyl)acetyl]-10H- thieno[3,4-b][1,5]benzodiazepin-10-one) were developed and found to bind to the receptor with affinities (Ki values) in approximately the nanomolar range. The derivatives contain a 10-aminodecyl group, which provides a nucleophilic functionality for further derivatization. The attachment of a spacer chain to the distal piperazinyl nitrogen was based on previous findings of enhanced affinity at muscarinic receptors in an analogous series of alkylamino derivatives of pirenzepine [J. Med. Chem. (1991) 34, 2133-2145]. The telenzepine derivatives contain prosthetic groups for radioiodination, protein cross-linking, photoaffinity labeling, and fluorescent labeling and biotin for avidin complexation. The affinity for muscarinic receptors in rat forebrain (mainly m1 subtype) was determined in competitive binding assays vs [3H]-N-methylscopolamine. A (p-aminophenyl)-acetyl derivative for photoaffinity labeling had a Ki value of 0.29 nM at forebrain muscarinic receptors (16-fold higher affinity than telenzepine). A biotin conjugate displayed a Ki value of 0.60 nM at m2-receptors and a 5-fold selectivity versus forebrain. The high affinity of these derivatives makes them suitable for the characterization of muscarinic receptors in pharmacological and spectroscopic studies, for peptide mapping, and for histochemical studies.

Figure 1

Inhibition curves for the displacement of [³H]-N-methylscopolamine by PAPA-TAC (6), ASA-TAC (8), biotinyl-TAC (9), and the eosine isothiocyanate conjugate of TAC (15). Specific counts are shown versus concentration of the inhibitor for rat forebrain membranes (●) and rat heart membranes (○). Inhibition of [³H]NMS binding was determined by incubating the analog and membranes with 0.5 nM [³H] NMS in phosphate-buffered saline at pH 7.2 at 37 °C for 60 min. Nonspecific binding was determined by coincubation with 1 μM atropine.

Scheme I^a. Synthesis of Functionalized Congeners of Telenzepine

^aReagents: (a) N,N -diisopropylethylamine, α-chloroethyl chloroformate; (b) 1,10-dibromodecane, N,N-diisopropylethylamine; (c) NH₃; (d) isothiocyanates or activated carboxylic esters.