Membrane affinity and metabolism of N⁴-palmitoyl-1-β-D-arabinofuranosylcytosine into cultured KB cells

N⁴-Palmitoyl-1-β-D-arabinofuranosylcytosine (N⁴-palmitoyl-ara-C), a lipophilic derivative of 1-β-D-arabinofuranosylcytosine, possessed an affinity for KB cell plasma membrane. Approximately 15 to 25% of the drug incorporated into KB cells was retained in plasma membrane when the cells were treated with the drug for 1 to 32 hr at 10 μM, the concentration required for 50% inhibition of cell growth. Less than 5.3% of the drug was found in the plasma membrane when the cells were treated with 1-β-D-arabinofuranosylcytosine. N⁴-Palmitoyl-ara-C in the membrane fraction cosedimented with plasma membrane in a sucrose density gradient at 4°, indicating a close association between the drug and the membrane. The affinity of N⁴-palmitoyl-ara-C for plasma membrane probably contributes to the efficient uptake rate and the strong cytotoxic effect of N⁴-palmitoyl-ara-C reported previously. The metabolites of N⁴-palmitoyl-ara-C in KB cells, treated with the drug for 32 hr at the concentration required for 50% inhibition of cell growth (10 μM), were analyzed by diethylaminoethyl Sepharose CL-6B column chromatography and thin-layer and paper chromatography. This analysis showed that over 98% of the drug present in the KB cell was N⁴-palmitoyl-ara-C. The active metabolites, 1-β-D-arabinofuranosylcytosine, 1-β-D-arabinofuranosylcytosine 5'-monophosphate, N⁴-palmitoyl-1-β-D-arabinofuranosylcytosine 5'-monophosphate, and 1-β-D-arabinofuranosylcytosine 5'-triphosphate, were found in amounts of 0.41, 0.37, 0.17, and 0.05%, respectively, of the total drug found in the cells. Also found were the inactive metabolites 1-β-D-arabinofuranosyluracil and 1-β-D-arabinofuranosylcytosine diphosphate choline in amounts of 0.61 and 0.29%, respectively.