Synthesis and anticancer activity of 5'-chloromethylphosphonates of 3'-azido-3'-deoxythymidine (AZT)

A series of novel N-alkyl 5'-chloromethylphosphonates of 3'-azido-3'-deoxythymidine (6-15) was synthesized by means of phosphonylation of 3'-azido-3'-deoxythymidine (4) with P-chloromethylphosphonic ditriazolide (3) followed by a reaction with the appropriate amine. The synthesized phosphonamidates 6-15 were evaluated for their cytotoxic activity in two human cancer cell lines: oral (KB) and breast (MCF-7) using the sulforhodamine B (SRB) assay. The highest activity in KB human cancer cells was displayed by phosphonamidate 8 (IC(50)=5.8 μg/mL), however, this compound was less potent than the parent AZT (IC(50)=3.1 μg/mL). Phosphonamidate 10 showed only moderate activity (IC(50)=12.1 μg/mL) whereas the other phosphonamidates proved inactive. Similarly, the highest activity in MCF-7 human cancer cells was displayed by phosphonamidate 8 (IC(50)=3.7 μg/mL) but it proved somewhat less active than AZT (IC(50)=2.6 μg/mL). Some activity was also displayed by phosphonamidate 10 (IC(50)=12.8 μg/mL) but the other phosphonamidates were found inactive. Hydrolysis studies indicate that the synthesized phosphonamidates are likely to act as prodrugs of the parent nucleoside (AZT). Transport measurements showed that the most active phosphonamidates (8 and 10) were able to permeate across the intestinal epithelium in vitro. The apparent permeability coefficients determined in Caco-2 cell monolayers indicated that these compounds could be moderately absorbed in humans.     

In vivo toxicity of 3'-azido-3'-deoxythymidine (AZT) on CBA/Ca mice

CBA/Ca male mice were given 3'-azido-3'-deoxythymidine (AZT) in drinking water (1 mg/ml) for up to 7 weeks. Water consumption and body weight decreased significantly. Neutropenia and lymphopenia were observed during and after exposure. Significant macrocytic anemia developed and disappeared as a function of red cell life span after stopping AZT intake. A microthrombocytosis was seen. Bone marrow cellularity and spleen colony-forming unit (CFU-s) content fell, but recovered completely and quickly after terminating AZT intake. Hemopoietic stem cell function measured by 2 different methods of rescuing fatally irradiated mice was normal 4 weeks after AZT exposure, suggesting that AZT treatment does not induce a long-lasting effect in genetic control of mitotic potential of stem cells. This is in marked contrast to exposure of CBA/Ca mice to benzene and ionizing radiation.     

3'-azido-3'-deoxythymidine (AZT) transplacental perfusion kinetics and DNA incorporation in normal human placentas perfused with AZT.

Vertical transmission of the human immunodeficiency virus 1 (HIV-1) is reduced from approximately 25% to approximately 7% as a result of 3'-azido-3'-deoxythymidine (AZT) therapy given during pregnancy; however, the consequences of transplacental AZT exposure to the fetus remain unknown. To address the extent and kinetics of AZT transfer across the human placenta, perfusion studies have been performed with fresh uninfected human placentas perfused with 0.5, 1. 0 and 5.0 mg AZT/ml for 2 h using a dual recirculating single cotyledon perfusion apparatus [T.I. Ala-Kokko, P. Pienimaki, R. Herva, A.I. Hollmen, O. Pelkonen, K. V?h?kangas, Transfer of lidocaine and bupivacaine across the isolated perfused human placenta, Pharmacol. Toxicol. 77 (1995) 142-148]. For two placentas, samples of perfusion effluent were taken every 15 min from the maternal and fetal sides of the apparatus and AZT levels were determined by AZT radioimmunoassay (RIA). At the end of the perfusion, AZT-DNA incorporation into placental DNA was determined by AZT-RIA. The concentration of AZT in the fetal perfusate increased with time, along with a concomitant slow decrease in the concentration of AZT in the maternal perfusates. For three different placentas, at 2 h after the start of perfusion, AZT-DNA incorporation values (molecules of AZT/10(6) nucleotides) were 11.8 for the 0.5 mg AZT/ml perfusate, 13.7 for the 1.0 mg AZT/ml perfusion, and 42.0 for the 5 mg AZT/ml perfusion. An additional placenta perfused with 1 mg AZT/ml did not have detectable values of AZT incorporated into DNA (data not shown). The data show that AZT crosses the human placenta and becomes rapidly incorporated into DNA of placental tissue in a dose-dependent fashion, suggesting that even short exposures to this drug might induce fetal genotoxicity and might also inhibit maternal-fetal viral transmission.     

Analysis of mutations in the Tk gene of Tk(+/-) mice treated as neonates with 3'-azido-3'-deoxythymidine (AZT)

Mother-to-child transmission of the human immunodeficiency virus (HIV) is reduced by perinatal treatment with the antiretroviral nucleoside analogue 3'-azido-3'-deoxythymidine (AZT, zidovudine). AZT, however, is genotoxic and carcinogenic in mice when administered either transplacentally or neonatally, suggesting a possible cancer risk for children later in life. In a previous study we found that treating B6C3F1/Tk(+/-) mice on postnatal days 1 through 8 with intraperitoneal injections of 200 mg AZT per kg body weight per day significantly increased spleen lymphocyte mutant frequencies in the autosomal Tk gene. Allele-specific PCR of Tk mutants from treated mice indicated that 61% had lost the Tk(+) allele (loss of heterozygosity; LOH), compared with 35% of Tk mutants from control mice, a difference that was significant. In the present study, Tk mutant lymphocyte clones were analyzed further using polymorphic microsatellite markers that flank the Tk gene along the length of mouse chromosome 11. The analysis indicated that allele-loss mutations in control mice were due to either total chromosome loss, mitotic recombination, or both. The pattern of marker loss in mutants from AZT-treated mice differed significantly from the control mice and was consistent with chromosome loss, mitotic recombination, interstitial deletion, gene conversion, and an unusual discontinuous LOH. The results indicate that AZT induced a unique pattern of mutations in the Tk gene of mice and that the major mechanisms of mutation by AZT involved deletion and recombination.     

Synthesis, characterization, and in vitro assay of folic acid conjugates of 3'-azido-3'-deoxythymidine (AZT): toward targeted AZT based anticancer therapeutics

Conjugates of three components namely folic acid, poly(ethyleneglycol) and 3 '-azido-3 '-deoxythymidine (AZT) are presented. Folate-PEG units were coupled to AZT to facilitate delivery of the nucleoside into the cell. A convenient separation of the polydisperse PEGylated-folic acid regioisomers produced upon conjugation is described. This is to select for the active gamma-regioisomer over the inactive alpha-regioisomer. In vitro cytotoxicity assays were conducted against an ovarian cell line (A2780/AD) that overexpresses the folate receptor (FR) and compared to a FR free control cell line. Compared to AZT a approximately 20-fold greater potency against the resistant ovarian line was observed for the conjugates.