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 cla sses interfering with the activation and signaling of target cells aft er stimulation. Here we describe a reporter gene assay targeting mast cell activation by IgE plus antigen, established in an attempt to iden tify substances preventing type I allergy (allergic rhinitis, allergic conjunctivitis, allergic asthma, and acute and chronic urticaria). Th e assay is based on a murine mast cell line designated CPII, stimulati on by IgE plus antigen, and a reporter gene construct with the TNF alp ha promoter linked to luciferase as a read-out system. Via screening a bout 50 000 substances, compound 2 was found to inhibit the reporter g ene induction in the submicromolar range in this assay. Analogues of c ompound 2 of the 2,3,4-trihydropyrimidino[2,1-a]isoquinol type were sy nthesized starting from 2-alkyl-substituted benzonitriles via aminolys is with 1,3-diaminopropane, dimetalation of 2-substituted 2-phenyl-1,4 ,5,6-tetrahydropyrimidines with n- and sec-butyllithium, reaction with carboxylic acid methyl esters, and finally acidic dehydration. From a bout 50 derivatives, compound 41 was selected as a lead structure with an IC50 Of 0.2 mu M and a TC50 Of 2.7 mu M. In a first profiling in s econdary assays, it effectively interfered with the production of medi ators such as TNF alpha, IL-4, IL-6, IL-13, and leukotriene synthesis as measured by the corresponding ELISAs. In addition, a passive cutane ous anaphylaxis in mice (a typical type I reaction) is inhibited to mo re than 90% by compound 41, when administered intradermally 90 min bef ore challenge.