9-((2-phosphonylmethoxy)ethyl)guanine and 2--deoxyadenosine-triphosphate

N6-Cyclopropyl-PMEDAP (cPr-PMEDAP) is a novel derivative of the acyclic nucleoside phosphonate 9-(2-phosphonylmethoxyethyl)-2,6-diaminopurine (PMEDAP). Its cytostatic activity was found to be 8- to 20-fold more pronounced than that of PMEDAP and equivalent to that of the guanine derivative 9-(2-phosphonylmethoxyethyl)guanine (PMEG) against a variety of tumor cell lines. Unlike PMEDAP, but like PMEG, cPr-PMEDAP was equally cytostatic to wild-type and 9-(2-phosphonylmethoxyethyl)adenine/PMEDAP-resistant variants of the human erythroleukemia K562 and the murine leukemia L1210 cell lines. Also, cPr-PMEDAP and PMEG proved to be equipotent inducers of K562 and rat choriocarcinoma RCHO cell differentiation, whereas the differentiation-inducing activity of PMEDAP was 5- to 25-fold less pronounced. Furthermore, compared to PMEDAP, cPr-PMEDAP and PMEG were 10- to 25-fold more potent in inhibiting the progression of K562 cells through the S phase of the cell cycle, resulting in a marked accumulation of the four 2'-deoxyribonucleoside 5'-triphosphate pools. The biological effects of cPr-PMEDAP, but not PMEDAP, were reversed by the adenylate deaminase inhibitor 2'-deoxycoformycin (dCF). Formation of the deaminated derivative of cPr-PMEDAP (i.e. PMEG) was demonstrated in crude extracts from K562 and L1210 cells and in metabolism studies with radiolabeled cPr-PMEDAP and PMEG. This is the very first example of an acyclic nucleoside phosphonate analogue that is susceptible to deamination. However, cPr-PMEDAP was not recognized as a substrate by purified adenosine deaminase or by adenylate deaminase. These findings might point to an as yet unidentified cellular enzyme, sensitive to dCF but different from the common adenosine and AMP deaminases. Our data demonstrate the superior antiproliferative and differentiation-inducing effects of cPr-PMEDAP on tumor cells, as compared to the parent compound PMEDAP, based on the unique metabolic properties of this novel compound.

Topics: Adenine; Adenosine Deaminase; Adenosine Triphosphate; AMP Deaminase; Animals; Antineoplastic Agents; Cell Cycle; Cell Differentiation; Cell Division; Deoxyadenine Nucleotides; Drug Interactions; Drug Screening Assays, Antitumor; Erythrocytes; Guanine; Humans; K562 Cells; Leukemia L1210; Mice; Organophosphonates; Organophosphorus Compounds; Pentostatin; Prodrugs; Rats; Tumor Cells, Cultured

Incubation of CEM cells for 24 h with the guanine, 2,6-diaminopurine, and adenine nucleotide analogs of the 9-(2-phosphonylmethoxyethyl) series, 9-(2-phosphonylmethoxyethyl)guanine (PMEG), 9-(2-phosphonylmethoxyethyl)-2,6-diaminopurine (PMEDAP), and 9-(2-phosphonylmethoxyethyl)adenine (PMEA), was found to inhibit DNA synthesis 50% at concentrations of 1, 6, and 25 microM, respectively. Possible reasons for the marked differences were investigated, including cellular transport of the analogs, different efficiencies of intracellular phosphorylation, differential effects on 2'-deoxynucleotide (dNTP) pools, and differences in the affinities of the cellular DNA polymerases for the diphosphate derivatives of the drugs. No significant differences in cellular uptake were found among the analogs; however, they did differ in the efficiency of phosphorylation, i.e., CEM cells were found to accumulate higher levels of PMEG-diphosphate (PMEGpp) than PMEDAP-diphosphate (PMEDAPpp) or PMEA-diphosphate (PMEApp). Treatment of cells with any of the nucleotide analogs resulted in increased dNTP pools, with PMEG producing the greatest increase. All three analogs had the greatest effect on the dATP pool size, whereas the dGTP pool size was not significantly affected. Comparison of the ratios of nucleotide analog diphosphates to their corresponding dNTPs under conditions where DNA synthesis is inhibited 50% suggested that cellular DNA polymerases were approximately twice as sensitive to PMEGpp than to PMEDAPpp and 5-fold more sensitive to PMEGpp than to PMEApp. Consistent with this hypothesis, examination of the efficiencies with which the replicative DNA polymerases alpha, delta, and epsilon incorporated the analogs showed that DNA polymerase delta, the most sensitive of the DNA polymerases, incorporated PMEGpp twice as efficiently as PMEDAPpp and 7-fold more efficiently than PMEApp.

Topics: Adenine; Antineoplastic Agents; Deoxyadenine Nucleotides; DNA; DNA Polymerase I; DNA Polymerase II; DNA Polymerase III; Guanine; Humans; Nucleic Acid Synthesis Inhibitors; Organophosphonates; Organophosphorus Compounds; Tumor Cells, Cultured