Functionalized congener approach to muscarinic antagonists: analogues of pirenzepine

Abstract

The M1-selective muscarinic receptor antagonist pirenzepine 6H-pyrido[2,3-b][1,4]benzodiazepin-6-one) was derivatized to explore points of attachment of functionalized side chains for the synthesis of receptor probes and ligands for affinity chromatography. The analogues prepared were evaluated in competitive binding assays versus [3H]-N-methylscopolamine at four muscarinic receptor subtypes (m1AChR-m4AChR) in membranes from rat heart tissue and transfected A9L cells. 9-(Hydroxymethyl)pirenzepine, 8-(methylthio)pirenzepine, and a series of 8-aminosulfonyl derivatives were synthesized. Several 5-substituted analogues of pirenzepine also were prepared. An alternate series of analogues substituted on the 4-position of the piperazine ring was prepared by reaction of 4-desmethylpirenzepine with various electrophiles. An N-chloroethyl analogue of pirenzepine was shown to form a reactive aziridine species in aqueous buffer yet failed to affinity label muscarinic receptors. Within a series of aminoalkyl analogues, the affinity increased as the length of the alkyl chain increased. Shorter chain analogues were generally much less potent than pirenzepine, and longer analogues (7-10 carbons) were roughly as potent as pirenzepine at m1 receptors, but were nonselective. Depending on the methylene chain length, acylation or alkyl substitution of the terminal amine also influenced the affinity at muscarinic receptors.

Figure 1

Superimposed 500-MHz ¹H NMR spectra of 26 at various times following dissolution in aqueous medium. The time after addition of pD 7.4 potassium phosphate buffer (0.1 M) is indicated on the left side. Major resonance peaks have been assigned as (δ in ppm from tetramethylsilane): 3.22 and 3.63 (aziridinium ion); 3.42, 3.62, and 4.16 (O-phosphate ester); 3.89 (26, CH₂ α to Cl); 4.01 (25, CH₂ α to OH).

Figure 2

Kinetics of chemical transformation of the chloroethyl derivative (26) to form the aziridinium species, which subsequent leads to the hydroxyethyl (25) and O-phosphate species. Values were obtained through a nonlinear curve analysis using a computer program and the data obtained from the NMR study (Figure 1).

Scheme I^a

^aBoc = CO₂C(CH₃)₃.

Scheme II

Scheme III

Scheme IV

Scheme V

Scheme VI