Inhibition of Fc epsilon RI-mediated activation of mast cells by 2,3,4-trihydropyrimidino[2,1-a]isoquinolines

Assays based on reporter gene technology represent today an important tool in the pharmaceutical industry for discovering novel compound classes interfering with the activation and signaling of target cells after stimulation. Here we describe a reporter gene assay targeting mast cell activation of IgE plus antigen, established in an attempt to identify substances preventing type I allergy (allergic rhinitis, allergic conjunctivitis, allergic asthma, and acute and chronic urticaria). The assay is based on a murine mast cell line designated CPII, stimulation by IgE plus antigen, and a reporter gene construct with the TNF alpha promoter linked to luciferase as a read-out system. Via screening about 50,000 substances, compound 2 was found to inhibit the reporter gene induction in the submicromolar range in this assay. Analogues of compound 2 of the 2,3,4-trihydropyrimidino[2,1-a]isoquinoline type were synthesized starting from 2-alkyl-substituted benzonitriles via aminolysis with 1,3-diaminopropane, dimetalation of 2-substituted 2-phenyl-1,4,5,6-tetrahydropyrimidines with n- and sec-butylithium, reaction with carboxylic acid methyl esters, and finally acidic dehydration. From about 50 derivatives, compound 41 was selected as a lead structure with an IC50 of 0.2 microM and a TC50 of 2.7 microM. In a first profiling in secondary assays, it effectively interfered with the production of mediators such as TNF alpha, IL-4, IL-6, IL-13, and leukotriene synthesis as measured by the corresponding ELISAs. In addition, a passive cutaneous anaphylaxis in mice (a typical type I reaction) is inhibited to more than 90% by compound 41, when administered intradermally 90 min before challenge.