EVALUATION OF STEREOISOMERS OF 4-FLUOROALKYL ANALOGS OF 3-QUINUCLIDINYL BENZILATE IN IN-VIVO COMPETITION STUDIES FOR THE M1, M2, AND M3-MUSCARINIC-RECEPTOR SUBTYPES IN BRAIN

To develop a subtype selective muscarinic acetylcholine receptor (mAChR) antagonist for PET, fluorine-19 labeled alkyl analogues of quinuclidinyl benzilate (QNB) were synthesized by stereoselective reactions. To investigate these analogues for tissue subtype specificity, in vivo competitive binding studies were performed in rat brain using (R)-3-quinuclidinyl (R)-4-[I-125]Iodobenzilate (IQNB). Five, fifty, or five-hundred nmol of the non-radioactive ligands were coinjected intravenously with 8 pmol of the radioligand. Cold (R,R)-IQNB blocked (R,R)-[I-125]IQNB in a dose-dependent manner, without showing regional specificity. For the (R,S)-fluoromethyl, fluoroethyl, and -fluoropropyl derivatives, a higher percent blockade was seen at 5 and 50 nmol levels in M2 predominant tissues (medulla, pens, and cerebellum) than in M1 predominant tissues (cortex, striatum and hippocampus). The blockade pattern of the radioligand also correlated qualitatively with the percentage of M2 receptors in the region. The S-quinuclidinyl analogues showed M2 selectivity but less efficient blockade of the radioligand, indicating lower affinities. Radioligand bound to the medulla was inversely correlated to the M2 relative binding affinity of the fluoroalkyl analogues. These results indicate that the nonradioactive ligand blocks the radioligand based on the affinity of the nonradioactive ligand for a particular receptor subtype compared to the affinity of the radioligand for the same receptor subtype. Of the seven compounds evaluated, (R,S)fluoromethyl-QNB appears to show the most selectivity for the M2 subtypes in competition studies in vivo.