Synthesis and in Vitro Characterization of Radioiodinatable Benzodiazepines Selective for Type 1 and Type 2 Cholecystokinin Receptors

Abstract

Radiolabeled antagonists of specific peptide receptors identify a higher number of receptor binding sites than agonists and may thus be preferable for in vivo tumor targeting. In this study, two novel radioiodinated 1,4-benzodiazepines, (9), were developed. They were characterized in vitro as high affinity selective antagonists at cholecystokinin type 1 and 2 (CCK 1 and CCK 2 ) receptors using receptor binding, calcium mobilization, and internalization studies. Their binding to human tumor tissues was assessed with in vitro receptor autoradiography and compared with an established peptidic CCK agonist radioligand. The 125 I-labeled CCK 1 receptor-selective compound 9 often revealed a substantially higher amount of CCK 1 receptor binding sites in tumors than the agonist 125 I-CCK. Conversely, the radioiodinated CCK 2 receptor-selective compound 7 showed generally weaker tumor binding than 125 I-CCK. In conclusion, compound 9 is an excellent radioiodinated non-peptidic antagonist ligand for direct and selective labeling of CCK 1 receptors in vitro. Moreover, it represents a suitable candidate to test antagonist binding to CCK 1 receptorexpressing tumors in vivo.

Molecular Basis for Binding and Subtype Selectivity of 1,4-Benzodiazepine Antagonist Ligands of the Cholecystokinin Receptor

“…Benzodiazepines-The reagents that are the focus of this work are the 1,4-benzodiazepine antagonists of CCK receptors that differ only in the stereochemistry (S or R) of their 3-position side chains while exhibiting selective binding to either the type 1 cholecystokinin receptor (CCK1R) (BDZ-1, identified previously as compounds 5 and 9 (29)) or the type 2 cholecystokinin receptor (CCK2R) (BDZ-2, identified previously as compounds 3 and 7 (29)). The 1,4-benzodiazepines with an S-orientation, (S)-1-(3-iodophenyl)-3-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e] [1,4]diazepin-3-yl)urea, represent the BDZ-1 radioligand with incorporation of radioactive iodine ( 125 I) and BDZ-1 with non-radioactive 127 I, whereas those with an R-orientation, (R)-1-(3-iodophenyl)-3-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e] [1,4]diazepin-3-yl)urea, represent the BDZ-2 radioligand with 125 I and BDZ-2 with 127 I (Fig.…”

“…The 1,4-benzodiazepines with an S-orientation, (S)-1-(3-iodophenyl)-3-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e] [1,4]diazepin-3-yl)urea, represent the BDZ-1 radioligand with incorporation of radioactive iodine ( 125 I) and BDZ-1 with non-radioactive 127 I, whereas those with an R-orientation, (R)-1-(3-iodophenyl)-3-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e] [1,4]diazepin-3-yl)urea, represent the BDZ-2 radioligand with 125 I and BDZ-2 with 127 I (Fig. 1) (29). These were prepared by oxidative iodination of precursor compounds (S) [1,4]diazepin-3-yl)-3(3(trimethylstannyl)phenyl)urea, respectively, as we described previously (29).…”

“…These were prepared by oxidative iodination of precursor compounds (S) [1,4]diazepin-3-yl)-3(3(trimethylstannyl)phenyl)urea, respectively, as we described previously (29). These compounds have been fully characterized chemically and pharmacologically (29) and have the advantage of providing radioligands for competition binding assays with direct binding to the allosteric site within the helical bundle of CCK receptors.…”

“…The current work utilized a focused chimeric receptor approach targeting these six residues and took advantage of a unique pair of subtype-selective radioiodinatable 1,4-benzodiazepine ligands that could be used as direct probes of this intramembranous interhelical ligand-binding site (29). Direct binding analysis was performed for an extensive series of chimeric CCK1R/CCK2R constructs to determine the contributions of each of these residues in small molecule ligand selectivity and to demonstrate the reversal of selectivity via distinct combinations of substitutions.…”

Cholecystokinin-induced satiety, a key gut servomechanism that is affected by the membrane microenvironment of this receptor

“…42,43 The allosteric nature of this small-molecule-binding site was further confirmed using pharmacologic analyses, including the kinetics of ligand dissociation and the impact of orthosteric and possible allosteric ligands on the functions of each other. [42][43][44] Radioiodinated small-molecule CCK1R ligands have been developed 45,46 that provide a means for direct displacement from this allosteric pocket to establish the molecular basis for the binding of small-molecule ligands. This was successfully applied to antagonist ligands to gain insights into the inactive conformation of this pocket.…”