AC-[3-ALKYLTHIOPROLINE(31)] AND AC-[4-ALKYLTHIOPROLINE(31)] CCK4 ANALOGS - SYNTHESIS AND IMPLICATIONS FOR THE CCK-B RECEPTOR-BOUND CONFORMATION

It has been reported that substitution of the Met(31) residue in Boc-CCK4 (Boc-Trp(30)-Met(31)-Asp(32)Phe(33)-NH2, CCK33 numbering) by trans-3-propyl-L-proline yields a highly potent and selective CCK-B agonist. To further explore the structural requirements of the Met(31) side chain in the receptor-bound conformation of CCK4, we have synthesized several Ac-CCK4 analogs containing substitution of Met(31) by 3- and 4-(alkylthio)-substituted proline derivatives. To this end we have developed novel synthetic routes to enantiomerically pure N-Boc-4-cis- and -trans-(methylthio)prolines and racemic N-Boc-3-cis and -trans-[(4-methylbenzyl)thio]prolines. The protected mercaptoprolines were incorporated into Ac-CCK4 analogs using SPPS and were alkylated using various electrophiles following cleavage from the solid support. Binding assays reveal that 3-(alkylthio)prolines analogs have higher affinities at the CCK-B receptor than the corresponding 4-(alkylthio)proline analogs, and that trans-3-(alkylthio)proline analogs had higher affinities than corresponding cis-3-(alkylthio)proline analogs. Within both the cis- and trans-3-(alkylthio)proline series, the order of potency was found to be Me < Et < n-Pr. The trans-3-(n-propylthio)-L-proline analog demonstrates a higher affinity than that reported for Boc-CCK4[trans-3-propyl-L-Pro(31)]. Comparison of the low-energy structures calculated for several high-affinity Ac-CCK4 analogs reveal a common geometry which we propose to be the CCK-B receptor-bound conformation. This model shows grouping ofthe hydrophobic side chains of Trp, Met, and Phe at one side of the molecule and the hydrophilic side chain of Asp and the C-terminal carboxamide at the other side.