A series of N-hydroxy-N′-aminoguanidine derivatives was studied for their effects on L1210 cell growth and ribonucleotide reductase activity. With the twelve compounds studied, there was a good correlation between the inhibition of L1210 cell growth and the inhibition of ribonucleotide reductase activity. The most potent compound required concentrations of only 1.4 and 2 μM for 50% inhibition of L1210 cell growth and ribonucleotide reductase activity respectively. These guanidine analogs specifically inhibited the conversion of [14C]cytidine and deoxycytidine nucleotides in the nucleotide pool and the incorporation of [14C]cytidine into DNA without altering the incorporation of [14C]cytidine into RNA. Ribonucleotide reductase activity in drug-treated cells was reduced markedly. Iron-chelating agents did not either increase or decrease the inhibition caused by the N-hydroxy-N′-aminoguanidine derivatives. No evidence was obtained that these derivatives selectively inactivated one of the subunits of ribonucleotide reductase. These compounds appear to inhibit ribonucleotide reductase by a mechanism different from hydroxyurea or the thiosemicarbazone derivatives.

It was found that nucleoside 5'-diphosphates could serve as effectors of ribonucleotide reductase. ADP was an activator of CDP reduction; ADP reduction was activated by dGDP; GDP reduction was activated by dTDP. Conversely, dADP inhibited the reduction of CDP, UDP, GDP, and ADP; dGDP inhibited UDP and GDP reductions; and dTDP inhibited UDP reduction. The inhibition of UDP reduction by dADP, dTDP, and dGDP was at least equal to that observed for dATP, dTTP, and dGTP, respectively. In these experiments with the nucleoside diphosphates as effectors, high-pressure liquid chromatography analysis of the reaction mixtures showed that no nucleoside 5'-triphosphates were found during the reaction period which could account for the effects seen with the nucleoside diphosphates as effectors. Further experiments were carried out in which adenyl-5'-yl imidodiphosphate was used as the positive effector of CDP and UDP reductions in place of ATP. Under these conditions, CDP and UDP reductions were inhibited by dADP, dTDP, and dGDP to the same extent observed in the presence of ATP. ADP served not only as a substrate for ribonucleotide reductase but also as an activator of CDP and UDP reductions. The direct products (dNDPs) also served as positive and negative effectors. Dixon plots indicated that the dNDPs were acting as noncompetitive inhibitors with respect to the substrate. ADP increased the sedimentation velocity of the ribonucleotide reductase in a manner similar to ATP. These data are consistent with the allosteric effects seen with the nucleoside 5'-triphosphates. Additionally, from the thorough study of the role of effectors on UDP reduction, it is clear that UDP reduction was most sensitive to the negative effectors dATP, dADP, dTTP, dTDP, dGTP, and dGDP.

Ribonucleotide reductase activity is strongly regulated by nucleoside 5'-triphosphates acting as positive and negative effectors. With the use of dGTP analogs, araGTP and dITP, it was found that the structural requirements of dGTP to serve as a positive effector of ADP reductase were not the same as the requirements for dGTP to serve as a negative effector of CDP and ADP reductase activities. The dTTP analogs methylenedTTP and dideoxyTTP also gave different responses in terms of activating GDP reductase activity and inhibiting CDP and ADP reductase activities. Etheno-ATP and etheno-dATP were inactive as positive and negative effectors, respectively, of CDP reductase activity. DideoxyATP was less active than dATP as a negative effector. Formycin ATP was a very poor substitute for ATP as a positive effector of CDP reductase. These studies indicate that the effector sites are very specific in terms of binding nucleoside triphosphates as positive or negative modulators of ribonucleotide reductase activity.

Ribonucleotide reductase is a key enzyme in DNA replication and, as such, has been a target for antitumor agents. This enzyme is composed of two non-identical protein subunits which can be specifically and independently inhibited.

Experiments were carried out to test for the presence of "channeling" in L1210 cells. L1210 cells were incubated in culture in the presence of labeled cytidine and "cold" deoxycytidine and conversely, in the presence of labeled deoxycytidine and "cold" cytidine. Cytidine did not inhibit the incorporation of [14C]deoxycytidine into DNA while deoxycytidine decreased the incorporation of [14C]cytidine into DNA. Further, in L1210 cells there was not a coordinate inhibition of thymidylate synthetase when either DNA polymerase was inhibited (aphidicolin) or ribonucleotide reductase was inhibited (hydroxyurea). These data indicate that leukemia L1210 cells do not selectively channel ribonucleotides to DNA through a tightly coupled enzyme complex.

Ribonucleotide reductase from tumor cells consists of two non-identical components which can be specifically and independently inhibited. Combinations of agents directed at the individual components gave synergistic inhibition of L1210 cell growth in culture. Utilizing hydroxyurea and deoxyadenosine or IMPY and deoxyadenosine as the parent combinations, modulators were used to potentiate the effects of each of these drugs. EHNA was used to prevent the deamination of deoxyadenosine while Desferal was utilized to increase the effects of hydroxyurea and IMPY. Combinations consisting of deoxyadenosine/EHNA plus IMPY/Desferal and deoxyadenosine/EHNA plus hydroxyurea/Desferal gave synergistic inhibition of L1210 cell growth. Utilizing these combination chemotherapies, the concentrations of each of the agents could be kept to minimal, essentially non-inhibitory levels and yet still achieve complete inhibition of L1210 cell growth with the specifically generated four-drug combinations.

Combinations of inhibitors directed at the individual components of ribonucleotide reductase were studied for their effects on L1210 cell growth in culture. The combinations included pyrozoloimidazole (IMPY) plus deoxyadenosine and hydroxyurea plus deoxyadenosine. Modulators were utilized to potentiate the effects of hydroxyurea, IMPY, or deoxyadenosine. Desferal was used to modulate the activity of hydroxyurea and IMPY while erythoro-9-(2-hydroxy-3-nonyl)adenine (EHNA) was used as the modulator of deoxyadenosine metabolism. While the combinations of deoxyadenosine-EHNA, hydroxyurea-Desferal, or IMPY-Desferal caused increased growth inhibition of L1210 cells at high drug concentrations, combinations which consisted of deoxyadenosine-EHNA-IMPY-Desferal or deoxyadenosine-EHNA-hydroxyurea-Desferal gave strong synergistic inhibition of L1210 cell growth in culture at concentrations of each of the drugs which alone had minimal inhibitory effects on tumor cell growth. The four-drug combination was clearly more effective than any three-drug combination in terms of inhibition of tumor cell growth. It was also observed that the concentrations of the modulators (Desferal or EHNA) were as critical as the concentrations of hydroxyurea, IMPY, or deoxyadenosine in establishing an effective drug combination.