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A seriesof twenty four acyclic unsaturated 2,6-substututed purines 5a-20b were synthesized. These compounds were evaluated for cytotoxic activity against NCI-60 DTP human tumor cell line screen at 10µM concentration. N9-[(Z)-4'-chloro-2'-butenyl-1'-yl]-2,6-dichloropurine(5a), N9-[4'-chloro-2'-butynyl-1'-yl]-2,6-dichloropurine(10a), N9-[(E)-2',3'-dibromo-4'-chloro-2'-butenyl-1'-yl]-6-methoxypurine(14) and N9-[4'-chloro-2'-butynyl-1'-yl]-6-(4-methoxyphenyl)-purine(19) exhibited highly potent cytotoxic activity with GI50 values in the 1–5 µM range for most human tumor cell lines. Other compounds exhibited moderate activity.