THE BIS(ADENOSIN-N6-YL)ALKANES, A FAMILY OF POTENTIAL DINUCLEOSIDE-POLYPHOSPHATE ANALOG PRECURSORS - CYTOTOXICITY, ADENOSINE-RECEPTOR BINDING AND METABOLISM

A series of bis(adenosin-N6-yl)alkanes, in which two adenosine residue s are linked via their N6 positions by alkyl bridges comprising betwee n 2 and 14 methylene units, were synthesized as potential precursors t o dinucleoside-polyphosphate analogues. These compounds were moderatel y cytotoxic to mammalian cells, the toxicity increasing with the lengt h of the alkyl chain. For example, the dose of bis(adenosin-N6-yl)dode cane, A[CH2]12A, leading to 50% inhibition of cell growth (ID50) for B HK fibroblasts, Walker 256 carcinoma cells and S-49 T-lymphoma cells w ere 90 +/- 8, 100 +/- 5 and 23 +/- 4 muM respectively. A significant a mount of A[CH2]12A bound to serum albumin in the growth media; thus th e ID50 for S-49 cells grown in serum-free medium was 9 +/- 2 muM. The corresponding bis-cytidine analogues were much less toxic; however the presence of a second adenosine moeity/molecule had little significant effect on cell growth when compared to N6-alkyladenosines. Toxicity t o S-49 cells was unaffected by the nucleoside-transporter inhibitor ni trobenzylthioinosine and was even higher (ID50 = 5 +/- 0.5 muM) toward s nucleoside-transport-deficient AE- 1 cells, showing that the analogu es could pass freely through the plasma membrane. Interaction with A1 adenosine receptors was shown by displacement of [H-3]N6-R-phenylisopr opyladenosine (K(d) = 6 nM) from rat adipocyte membranes, with K(i) va lues of 45, 65, 85 and 390 nM for the compounds containing 12, 8, 6 an d 4 methylene units, respectively. Affinity for human platelet membran e A2 adenosine receptors was about 100-fold less, however the compound s were weak A2 agonists, producing up to a threefold increase in intra cellular cyclic AMP in WI-38/VA-13 cells. Thus, these compounds behave , not surprisingly, as adenosine analogues. In addition, A[CH2]12A was metabolized in vitro and intracellularly by adenosine kinase (K(i) = 70 nM) and adenylate kinase to yield a number of phosphorylated deriva tives with the potential to act as diadenosine polyphosphate analogues . One of these, the bismonophosphate, was recognized by and inhibited adenylate kinase more effectively than adenosine(5')tetraphospho(5')ad enosine (Ap4A, K(i) = 3 muM).