Membrane-permeant derivatives of cyclic AMP optimized for high potency, prolonged activity, or rapid reversibility

A novel membrane-permeant derivative of cAMP, cAMP acetoxymethyl ester (cAMP/AM), was synthesized via silylated intermediates. Its ability to induce Cl^- secretion by T84 cells, a human colon cancer cell line, was compared with that of two other membrane-permeant cAMP derivatives that were recently introduced, N⁶,O²-dibutyryl-cAMP acetoxymethyl ester (bt₂cAMP/AM) and S(p)-5,6-dichlorobenzimidazole-1-β-D-ribofuranoside 3',5'-cyclic phosphorothioate (S(p)-5,6-DCl-cBIMPS). All of these compounds are powerful activators of Cl^- secretion when applied extracellularly, with EC₅₀ values of 60 μM, 0.7 μM, and 3 μM, respectively. However, cAMP/AM was expected to be readily degraded inside cells, in contrast to the cyclophosphodiesterase- resistant S(p)-5,6-DCl-cBIMPS or the only slowly metabolizable N⁶-butyryl- cAMP derived from bt₂cAMP/AM. Reversibility of cAMP/AM action was demonstrated by wash-out experiments; Cl^- secretion induced by high doses of cAMP/AM (100 μM) could be quickly abolished by rinsing of the cells, whereas similar experiments with bt₂cAMP/AM and S(p)-5,6-DCl-cBIMPS showed much slower decreases. Even more sensitive to residual cAMP derivatives was the synergistic effect of carbachol, which was applied after the incubation with membrane-permeant derivatives and their subsequent wash-out. Although doses of cAMP derivatives that barely activated Cl^- secretion were readily capable of inducing a synergistic response with carbachol, cells incubated with high doses of cAMP/AM (100 μM) and subsequently washed showed only a nonsynergistic carbachol response, in contrast to cells incubated with bt₂cAMP/AM or S(p)-5,6-DCl-cBIMPS. We therefore characterize cAMP/AM as a membrane-permeant derivative of cAMP that is easily metabolizable inside cells and hence is most useful for applications where a transient intracellular cAMP signal is desired. In contrast, completely nonmetabolizable S(p)-5,6-DCl-cBIMPS seems to be more useful in longer incubations that require steady levels of cAMP-dependent protein kinase activation. bt₂cAMP/AM combines the advantages of intracellular trapping by ester hydrolysis and reduced cyclophosphodiesterase sensitivity of its active intracellular product, which probably lead to its particularly high potency.