the invention relates to novel compounds which are both inhibitors of the enzyme phosphodiesterase 4 (pde4) and antagonists of the muscarinic m3 receptor.processes for the preparation of such compounds, compositions containing them and their use in therapy.determination of binging m3 cho ki cells expressing the cloned human m3 receptor (swissprot p20309) were harvested in phosphate buffered saline and ca + + and mg + + and collectionbound by centrifugation at 1500 rpm for three minutes.the pellets were resuspended in ice cold buffer (tris hcl (ph 7.4, 15 mm, 0.3 mm 2 mm mgcl2, edta, egta and by 1 mm) and gdp (politron adjustment 5 for 15 s).the crude membrane fraction was collected by centrifugation after two stages of 40000 g for 20 min at 4 degrees c, separated by a wash step in the buffer.the pellets obtained were finally resuspended in buffer b (75 mm tris hcl, ph 7.4, 12.5 mm).0.3 mm mgcl2, edta, egta, 2 mm, 250 mm sucrose) and aliquots were stored at - 80 degrees celsius.on the day of the experiment, and the membranes were resuspended in buffer (50 mm tris hcl, ph 7.4, mgcb 2.5 mm edta (1 mm).the frequency nonselective muscarinic ligand [3h] - n-methyl scopolamine (mol). pharmacol.45: 1899 - 1907) was used to mark the sites of binding to m3.the experiments were carried out in double bond (concentration curves in ten points) in 96 well plates at a concentration of radioligand to 0.1 - 0.3 nm.nonspecific binding was determined in the presence of n-methyl scopolamine cold 10 um. the sample volume (ml) were incubated at room temperature for 90 minutes.the reaction was stopped by rapid filtration through the plates unifilter gf / b and two washes with buffer (0.75 ml), cold (using a harvester filtermate harvester.the radioactivity on the filters was measured in a scintillation counter (perkin elmer) tricarb 2500 microplate.representative compounds of the invention, when they were tested in one of the protocols mentioned above, showed an ic50 of less than 100 nm.re
