1 The aim of this study was the pharmacological characterization of tachykinin NKI and NK2 receptors mediating contraction in the circular muscle of the guinea-pig ileum and proximal colon. The action of substance P (SP), neurokinin A (NKA) and of the synthetic agonists [ Furthermore, an intraspecies heterogeneity of the NK2 receptor in the circular muscle of the guinea-pig ileum and colon is suggested.

The rapid evolution of combinatorial chemistry in recent years has led to a dramatic improvement in synthetic capabilities. The goal is to accelerate the discovery of molecules showing affinity against a target, such as an enzyme or a receptor, through the simultaneous synthesis of a great number of structurally diverse compounds. This is done by generating combinatorial libraries containing as many as hundreds or thousands of compounds. The need to test all these compounds led to the development of high-throughput screening (HTS) techniques, and also high-throughput analytical techniques capable of assessing the occurrence, structure and purity of the products. In order to be applied effectively to the characterization of combinatorial libraries, an analytical technique must be adequately sensitive (to analyse samples which are typically produced in nanomole amounts or less), fast, affordable and easy to automate (to minimize analysis time and operator intervention). Although no method alone can meet all the analytical challenges underlying this task, the recent progress in mass spectrometric (MS) instrumentation renders this technique an essential tool for scientists working in this area. We describe here relevant aspects of the use of MS in combinatorial technologies, such as current methods of characterization, purification and screening of libraries. Some examples from our laboratory deal with the analysis of pooled oligomeric libraries containing n x 324(n = 1, 2) compounds, using both on-line high-performance liquid chromatography/MS with an ion trap mass spectrometer, and direct infusion into a triple quadrupole instrument. In the first approach, MS and product ion MS/MS with automatic selection of the precursor were performed in one run, allowing library confirmation and structural elucidation of unexpected by-products. The second approach used MS scans to characterize the entire library and also precursor ion and neutral loss scans to detect selectively components with given structural characteristics.

In the course of a program aimed at synthesizing novel, potent NK-1 tachykinin receptor antagonists, we developed upon a bioactive model by comparing the low energy structures of a series of peptide and nonpeptide Substance P antagonists. The comparison was based on the superimposition of the aromatic rings, assuming that the rest of the molecule behaves predominantly as a template to arrange the key aromatic groups in the right spatial position. A series of 2-aminocyclohexane carboxylic acid analogues were then selected as the best templates for reproducing the postulated bioactive structure, leading to several pseudo-peptides with interesting biological activity. According to the molecular modeling, these compounds exhibit a neat parallel facing of the indolyl and naphthyl groups at about 3 A distance. Ultraviolet absorption and steady state fluorescence measurements support this conclusion, showing a linear correlation between the spectral properties and the binding affinity of these analogues. Stacking of the indole ring with naphthalene gives rise to a complex characterized by a well-defined molar extinction coefficient. Consistently, steady state and lifetime fluorescence measurements suggest that the quenching process is ascribable to ground-state interactions between the chromophores. Implications of the pi stacking propensity of aromatic groups in the biological activity of the compounds examined are briefly discussed.