Discovery of a Series of 2'-α-Fluoro,2'-ß-bromo-ribonucleosides and Their Phosphoramidate Prodrugs as Potent Pan-Genotypic Inhibitors of Hepatitis C Virus.

Hepatitis C virus (HCV) nucleoside inhibitors display pan-genotypic activity, a high barrier to the selection of resistant virus, and are some of the most potent direct-acting agents with durable sustained virologic response in humans. Herein, we report, the discovery of ß-d-2'-Br,2'-F-uridine phosphoramidate diastereomers 27 and 28, as nontoxic pan-genotypic anti-HCV agents. Extensive profiling of these two phosphorous diastereomers was performed to select one for in-depth preclinical profiling. The 5'-triphosphate formed from these phosphoramidates selectively inhibited HCV NS5B polymerase with no inhibition of human polymerases and cellular mitochondrial RNA polymerase up to 100 µM. Both are nontoxic by a variety of measures and display good stability in human blood and favorable metabolism in human intestinal microsomes and liver microsomes. Ultimately, a preliminary oral pharmacokinetics study in male beagles showed that 28 is superior to 27 and is an attractive candidate for further studies to establish its potential value as a new clinical anti-HCV agent.

Exploring the intracellular pharmacokinetics of the 5-fluorouracil nucleotides during capecitabine treatment.

AIM: Three intracellularly formed metabolites are responsible for the antineoplastic effect of capecitabine: 5-fluorouridine 5'-triphosphate (FUTP), 5-fluoro-2'-deoxyuridine 5'-triphosphate (FdUTP), and 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP). The objective of this study was to explore the pharmacokinetics of these intracellular metabolites during capecitabine treatment. METHODS: Serial plasma and peripheral blood mononuclear cell (PBMC) samples were collected from 13 patients treated with capecitabine 1000 mg QD (group A) and eight patients receiving capecitabine 850 mg m(-2) BID for fourteen days, every three weeks (group B). Samples were collected on day 1 and, for four patients of group B, also on day 14. The capecitabine and 5-fluorouracil (5-FU) plasma concentrations and intracellular metabolite concentrations were determined using LC-MS/MS. Pharmacokinetic parameters were estimated using non-compartmental analysis. RESULTS: Only FUTP could be measured in the PBMC samples. The FdUTP and FdUMP concentrations were below the detection limits (LOD). No significant correlation was found between the plasma 5-FU and intracellular FUTP exposure. The FUTP concentration-time profiles demonstrated considerable inter-individual variation and accumulation of the metabolite in PBMCs. FUTP levels ranged between

Preclinical evaluation of investigational radiopharmaceutical RISAD-P intended for use as a diagnostic and molecular radiotherapy agent for prostate cancer.

BACKGROUND: The androgen receptor (AR) plays a dominant role in the pathogenesis of prostate cancer. 5-Radioiodo-3'-O-(17ß-succinyl-5α-androstan-3-one)-2'-deoxyuridin-5'-yl phosphate (RISAD-P) is an AR-targeting reagent developed for noninvasive assessment of AR and proliferative status of the AR-expressing tumors, and for molecular radiotherapy with Auger electron-emitting radionuclides. In this study, the preclinical toxicity and targeting potential of RISAD-P was evaluated. METHODS: Effects of nonradioactive ISAD-P and RISAD-P labeled with (123) I, (124) I, and (125) I were evaluated in male mice. Expanded-acute single dose toxicity studies, hematologic toxicity, liver and kidney function, pharmacokinetics, biodistribution, and imaging studies were conducted. Imaging and pilot therapy studies were conducted in transgenic mice. RESULTS: RISAD-P is not toxic at doses projected for clinical use. Its tissue distribution compares favorably with the distribution reported for (18) F-dihydrotestosterone derivatives. RISAD-P has excellent prostate cancer targeting properties. One hour after (125) IRISAD-P administration, nearly 10% of the injected dose is associated with prostate tumor. The tumor clearance is biphasic and plateaus between 24 and 48 hr post-injection. The estimated radiation doses calculated for 1 g tumor using the MIRD convention are well within the therapeutic range with values of 170, 250, 1,240 Gy × MBq(-1) × g(-1) for (125) I-, (123) I-, and (124) I-labeled RISAD-P, respectively. The transient uptake of radioactivity is observed in the genitourinary tract and stomach. Without the potassium iodide blockade, thyroid uptake is also observed. CONCLUSIONS: Biodistribution, toxicity, and radiation dosimetry studies suggest that RISAD-P holds characteristics of a promising candidate for imaging of AR expression and tumor proliferation, as well as molecular radiotherapy for metastatic or locally, regionally advanced prostate cancer.

Radiolabeled 5-iodo-3'-O-(17beta-succinyl-5alpha-androstan-3-one)-2'-deoxyuridine and its 5'-monophosphate for imaging and therapy of androgen receptor-positive cancers: synthesis and biological evaluation.

High levels of androgen receptor (AR) are often indicative of recurrent, advanced, or metastatic cancers. These conditions are also characterized by a high proliferative fraction. 5-Radioiodo-3'-O-(17beta-succinyl-5alpha-androstan-3-one)-2'-deoxyuridine 8 and 5-radioiodo-3'-O-(17beta-succinyl-5alpha-androstan-3-one)-2'-deoxyuridin-5'-yl monophosphate 13 target AR. They are also degraded intracellularly to 5-radioiodo-2'-deoxyuridine 1 and its monophosphate 20, respectively, which can participate in the DNA synthesis. Both drugs were prepared at the no-carrier-added level. Precursors and methods are readily adaptable to radiolabeling with various radiohalides suitable for SPECT and PET imaging, as well as endoradiotherapy. In vitro and in vivo studies confirm the AR-dependent interactions. Both drugs bind to sex hormone binding globulin. This binding significantly improves their stability in serum. Biodistribution and imaging studies show preferential uptake and retention of 8 and 13 in ip xenografts of human ovarian adenocarcinoma cells NIH:OVCAR-3, which overexpress AR. When these drugs are administered at therapeutic dose levels, a significant tumor growth arrest is observed.

Mechanism-based inactivation of thymidylate synthase by 5-(3-fluoropropyn-1-yl)-2'-deoxyuridine 5'-phosphate.

5-Fluoropropynyl-2'-deoxyuridine 5'-phosphate (3) was designed as a mechanism-based inactivator of thymidylate synthase (TS). The inhibitor was synthesized from 5-iodo-2'-deoxyuridine and propargyl alcohol by palladium-catalyzed coupling, followed by fluorination and selective phosphorylation. Incubation of TS with 3, in the presence or absence of the CH2H4folate cofactor, caused rapid, irreversible inactivation of the enzyme.

Metabolism of new anticancer oxysterol derivatives in rats.

New water soluble derivatives of oxysterols--the phosphodiesters of oxysterols and of nucleosides--have been synthesized. In vitro, these compounds share the biological properties of their parent oxysterols. Furthermore, they display anticancer activity when injected i.p. in mice bearing experimental tumors. The pharmacokinetic study described here proved that the water-soluble derivatives of oxysterols act as prodrugs releasing free oxysterol in the blood, the liver and the kidney after i.p. or i.v. injection in rats. The hydro-solubility of such compounds as well as their slow metabolism into the active principle could account for their biological activity and make them suitable as new therapeutic agents.

Methotrexate 5-aminoallyl-2'-deoxyuridine 5'-monophosphate: a potential bifunctional inhibitor of thymidylate synthase.

Mercuration of 2'-deoxyuridine 5'-phosphate (dUMP) followed by alkylation with allylamine in the presence of K2PdCl4 afforded the 5-aminoallyl deoxynucleotide, which was isolated by sequential Dowex 50 H+ and DEAE-Sephadex chromatography. Further reaction of the product with the N-hydroxysuccinimide ester of methotrexate (MTX) in dry dimethyl sulfoxide gave an MTX-aminoallyl-dUMP covalent complex separable by DEAE-Sephadex chromatography. Reprecipitation with acid from basic solution offered further purification and the structure was confirmed by elemental analysis, NMR and absorbance spectra. The product was an inhibitor of rat liver dihydrofolate reductase (I50 approximately 250 nM, cf. MTX I50 approximately 60 nM) and Lactobacillus casei thymidylate synthase. With the latter enzyme, inhibition was competitive with both nucleotide and folate substrates (Ki = 2.6 and 3.5 microM, respectively) and partial enzyme-inhibitor binary complex could be detected by gel electrophoresis. Large fluorescence changes were observed on titration of the synthase with MTX-aminoallyl-dUMP and alterations in the UV difference spectra similar to those seen on titration of the enzyme with MTX were also noted. The compound was a poor growth inhibitor for cultured murine L1210 and human CCRF-CEM cell lines, which probably reflects low cellular uptake.

Targeted drug delivery: a two-compartment growth inhibition assay demonstrates that fluorodeoxyuridine and fluorodeoxyuridine monophosphate are liposome-independent drugs.

The targeted delivery to cells by liposomes and leakage under delivery conditions of fluorodeoxyuridine (FdUR) and fluorodeoxyuridine monophosphate (FdUMP) have been evaluated using a two-compartment growth inhibition assay. Under cell culture conditions, FdUR leaks 100% from all liposomes regardless of charge or phase transition temperature. Under the same conditions, FdUMP leaks 100% from egg yolk phosphatidylglycerol liposomes, 47% from distearoylphosphatidylglycerol liposomes, 44% from egg yolk phosphatidylcholine liposomes, and 10% from distearoylphosphatidylcholine liposomes. All liposomes were prepared from a 67:33 mixture of phospholipid and cholesterol. The two-compartment assay demonstrates directly that neither of these drugs is delivered selectively to the target cells by the liposomes, suggesting that they are liposome independent drugs.