3-(Acylamino)-5-phenyl-2H-1,4-benzodiazepines, antagonists of the peptide hormone cholecystokinin (CCK), are described. Developed by reasoned modification of the known anxiolytic benzodiazepines, these compounds provide highly potent, orally effective ligands selective for peripheral (CCK-A) receptors, with binding affinities approaching or equaling that of the natural ligand CCK-8. The distinction between CCK-A receptors on the one hand and CNS (CCK-B), gastrin, and central benzodiazepine receptors on the other is demonstrated by using the structure-activity profiles of the new compounds. Details of the binding of these agents to CCK-A receptors are examined, and the method of development of these compounds is discussed in terms of its relevance to the general problem of drug discovery.

3S(-)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-IH-1,4-benzodiazepine-3-yl)-lH-indole-2-carboxamide (L-364,718) interacted in a competitive manner with rat pancreatic cholecystokinin (CCK) receptors as determined by Scatchard analysis of the specific binding of '21I-labeled CCK. The affinity of L-364,718 for both pancreatic (IC50, 81 pM) and gallbladder (IC50, 45 pM) CCK receptors in radioligand binding assays greatly exceeded that of other reported nonpeptide CCK antagonists and was similar to that of CCK itself. In vitro functional studies utilizing CCK-induced contractions of the isolated guinea pig ileum and colon further demonstrated that L-364,718 acts as a competitive CCK antagonist, which lacks agonist activity and has a similar high affinity in these tissues (pA2, 9.9). L-364,718 exhibited a very high selectivity for peripheral CCK receptors relative to brain CCK, gastrin, and various other peptide and nonpeptide receptors in both in vitro radioligand and isolated tissue assays.In vivo, low intravenous doses of L-364,718 (0.1 mg/kg) markedly antagonized the contractions of the guinea pig gallbladder produced by intravenous administration of CCK for at least 2 hr. Administered orally, L-364,718 (ED50, 0.04 mg/kg) was highly effective as an antagonist of CCK-induced inhibition of gastric emptying in mice. The biochemical and pharmacological properties of L-364,718-namely, very high affinity and selectivity for peripheral CCK receptors, longlasting in vivo efficacy, and oral bioavailability-makes this compound a powerful tool for investigating the physiological and pharmacological actions of CCK, and possibly its role in gastrointestinal disorders.

We have identified a family of highly selective allosteric modulators of the group I metabotropic glutamate receptor subtype 5 (mGluR5). This family of closely related analogs exerts a spectrum of effects, ranging from positive to negative allosteric modulation, and includes compounds that do not themselves modulate mGluR5 agonist activity but rather prevent other family members from exerting their modulatory effects. 3,3Ј-Difluorobenzaldazine (DFB) has no agonist activity, but it acts as a selective positive allosteric modulator of human and rat mGluR5. DFB potentiates threshold responses to glutamate, quisqualate, and 3,5-dihydroxyphenylglycine in fluorometric Ca 2ϩ assays 3-to 6-fold, with EC 50 values in the 2 to 5 M range, and at 10 to 100 M, it shifts mGluR5 agonist concentration-response curves approximately 2-fold to the left. The analog 3,3Ј-dimethoxybenzaldazine (DMeOB) acts as a negative modulator of mGluR5 agonist activity, with an IC 50 of 3 M in fluorometric Ca 2ϩ assays, whereas the analog 3,3Ј-dichlorobenzaldazine (DCB) does not exert any apparent modulatory effect on mGluR5 activity. However, DCB seems to act as an allosteric ligand with neutral cooperativity, preventing the positive allosteric modulation of mGluRs by DFB as well as the negative modulatory effect of DMeOB. None of these analogs affects binding of [ 3 H]quisqualate to the orthosteric (glutamate) site, but they do inhibit [ 3 H]3-methoxy-5-(2-pyridinylethynyl)pyridine binding to the site for 2-methyl-6-(phenylethynyl)-pyridine, a previously identified negative allosteric modulator. With the use of these compounds, we provide evidence that allosteric sites on GPCRs can respond to closely related ligands with a range of pharmacological activities from positive to negative modulation as well as to neutral competition of this modulation.Metabotropic glutamate receptors (mGluRs) are G proteincoupled receptors that bind glutamate to exert a modulatory influence on neuronal excitability and synaptic transmission in the central nervous system. The eight known members of the mGluR subfamily have been divided into three groups on the basis of their sequence identity, pharmacology, and preferred signal transduction mechanism. Group I mGluRs (mGluRs 1 and 5) are primarily localized postsynaptically where they modulate ion channel activity and neuronal excitability. The group I mGluRs are coupled to G ␣q and its associated effectors, such as phospholipase C. Groups II (mGluRs 2 and 3) and III (mGluRs 4, 6, 7, and 8) are primarily located presynaptically and regulate the release of neurotransmitters, including glutamate. Group II and III mGluRs are coupled to G ␣i and its associated effectors, such as adenylate cyclase. These latter two groups are distinguished from each other by their pharmacology; selective agonists and antagonists have been identified for each group (Conn and Pin, 1997). All mGluR subtypes possess a large (ϳ560 amino acids) extracellular amino-terminal domain that contains the glutamate agonist binding site. Thus, in...

No abstract