Purpose. This study is concerned with cellular delivery/generation of
2'-azido-2'-deoxyuridine and -deoxycytidine diphosphate (N3UDP or N3CD
P), potent inhibitors of ribonucleotide reductase. It characterizes th
e phosphorylation steps involved in the conversion of 2'-azido-2'-deox
yuridine (N(3)Urd) and 2'-azido-2'-deoxycytidine (N(3)Cyd) to the corr
esponding diphosphates and explores a prodrug approach in cellular del
ivery of the inhibitor which circumvents the requirement of deoxynucle
oside kinases. Methods. Cell growth of CHO and 3T6 cells of known deox
ycytidine kinase level was determined in the presence of N(3)Urd and N
(3)Cyd. Activity of ribonucleotide reductase was determined in the pre
sence of the azidonucleosides as well as their mono- or di-phosphates
in a Tween 80-containing permeabilizing buffer. A prodrug of 5'-monoph
osphate of N(3)Urd was prepared and its biological activity was evalua
ted with CHO cells as well as with cells transfected with deoxycytidin
e kinase. Results. N(3)Urd failed to inhibit the growth of both cell l
ines, while N(3)Cyd was active against 3T6 cells and moderately active
against CHO cells. These results correlate with the deoxycytidine kin
ase levels found in the cells. Importance of the kinase was further es
tablished with the finding that the nucleoside analogs were inactive a
s reductase inhibitors in a permeabilized cell assay system while thei
r mono- and di-phosphates were equally active. The prodrug was active
in cell growth inhibition regardless of the deoxycytidine kinase level
. Conclusions. The azidonucleosides become potent inhibitors of the re
ductase by two sequential phosphorylation steps. The present study ind
icates that the first step to monophosphate is rate-limiting, justifyi
ng a prodrug approach with the monophosphate.