Background: 1-α-D-(5-Deoxy-5-[18F]fluoroarabinofuranosyl)-2-nitroimidazole ([18F]FAZA) is manufactured by nucleophilic radiofluorination of 1-α-D-(2’,3’-di-O-acetyl-5’-O-toluenesulfonylarabinofuranosyl)- 2-nitroimidazole (DiAcTosAZA) and alkaline deprotection to afford [18F]FAZA. High yields (>60%) under optimized conditions frequently revert to low yields (<20%) in large scale, automated syntheses. Competing side reactions and concomitant complex reaction mixtures contribute to substantial loss of product during HPLC clean-up. Objective: To develop alternative precursors for facile routine clinical manufacture of [18F]FAZA that are compatible with current equipment and automated procedures. Methods: Two new precursors, 1-α-D-(2’,3’-di-O-acetyl-5’-O-(4-nitrobenzene)sulfonyl-arabinofuranosyl)-2- nitroimidazole (DiAcNosAZA) and 1-α-D-(2’,3’-di-O-acetyl-5’-iodo-arabinofuranosyl)-2-nitroimidazole (DiAcIAZA), were synthesized from commercially-available 1-α-D-arabinofuranosyl-2-nitroimidazole (AZA). A commercial automated synthesis unit (ASU) was used to condition F-18 for anhydrous radiofluorination, and to radiofluorinate DiAcNosAZA and DiAcIAZA using the local standardized protocol to manufacture [18F]FAZA from AcTosAZA. Results: DiAcNosAZA was synthesized via two pathways, in recovered yields of 29% and 40%, respectively. The nosylation of 1-α-D-(2’,3’-di-O-acetyl-arabinofuranosyl)-2-nitroimidazole (DiAcAZA) featured a strong competing reaction that afforded 1-α-D-(2’,3’-di-O-acetyl-5’-chloro-arabinofuranosyl)-2- nitroimidazole (DiAcClAZA) in 55% yield. Radiofluorination yields were better from DiAcNosAZA and DiAcIAZA than from DiAcTosAZA, and the presence of fewer side products afforded higher purity [18F]FAZA preparations. Several radioactive and non-radioactive by products of radiofluorination were assigned tentative chemical structures based on co-chromatography with authentic reference compounds. Conclusion: DiAcClAZA, a major side-product in the preparation of DiAcNosAZA, and its deprotected analogue (ClAZA), are unproven hypoxic tissue radiosensitizers. DiAcNosAZA and DiAcIAZA provided good radiofluorination yields in comparison to AcTosAZA and could become preferred [18F]FAZA precursors if the cleaner reactions can be exploited to bypass HPLC purification.

2'-Deoxy-2'-fluorothymidine (FT) is a bioisostere of both thymidine (TdR), in which F replaces H at C-2' in the ribosyl configuration, and methyluridine, in which F replaces OH at C-2' in the ribosyl configuration. Fluorine is bioisosteric with H with respect to atomic radius and is bioisosteric with OH with respect to polarity and H-bonding as an H acceptor. The consequences of this C-2' F for H substitution on cytotoxicity, nucleoside transporter affinity, phosphorylation by thymidine kinases (TK1, TK2), cell uptake and biodistribution of FT in a murine tumor model are now reported. FT toxicity against a bank of murine and human cells was seen only at very high (˜1 mM) concentrations, although the cellular uptake of [3H]FT in these cells was comparable to that of [3H]TdR over a 24 h period. Human equilibrative nucleoside transporters (hENT1, hENT2) displayed weaker affinity for FT than for TdR, but the concentrative transporters (hCNT1, hCNT2, hCNT3) had much higher affinities for FT. FT was phosphorylated by both mitochondrial thymidine kinase (TK2) (58 % of TdR) and cytosolic thymidine kinase (TK1) (39 % of TdR). Preliminary in vivo imaging with [18F]FT in mice bearing implanted KBALB and contralateral KBALB-STK tumors showed highly selective uptake, with a tumor:blood ratio of 33 in a small herpes simplex type 1 (HSV-1 TK) expressing tumor. In conclusion, [18F]FT appears to be a strong candidate for PET imaging of viral TK transgene imaging, based on its TK1:TK2 phosphorylation differential, its selective uptake by an HSV-TK expressing murine tumor model, its interaction with nucleoside transporters and its low toxicity.

Cyclo-(3-methylsaligenyl)-5-O-[1-(2,4-difluoro-5-[(125) I] iodophenyl)-2-deoxy-beta-D-ribofuranosyl] phosphate (cycloSal- dRF[I-125] IB) was radioiodinated with sodium [I-125] iodide via copper-catalyzed isotope exchange in 48% radiochemical yield. cycloSal-dRF[I-125] IB was found to be incorporated into the cytoplasmic nucleic acid and mitochondrial fractions of murine KBALB and K-STK (engineered to express HSV-1 thymidine kinase) cells in cell culture. Uptake was greater than that for either the corresponding nucleoside dRF[I-125] IB or [I-125] IUdR. These in vitro studies support a mechanism of metabolic activation to the free nucleotide, thereby effecting TK-bypass. Pharmacokinetic studies in rats reflect a complex interplay of tissue depot effects, hepatobiliary recycling, and metabolism. Biodistribution studies in tumor-bearing mice provide further evidence for lipophilic depot effects and hepatobiliary recirculation, with no evidence for active (metabolic) accumulation in any tissue.

1-(2-Deoxy-β-D-ribofuranosyl)-2,4-difluoro-5-iodobenzene (dRFIB) is a putative bioisostere of iododeoxyuridine (IUdR). The advantages of dRFIB over IUdR for in vivo studies include resistance to both phosphorolytic cleavage of the nucleoside bond and de-iodination. dRFIB was radioiodinated (dRF123/125IB) by copper-catalyzed exchange using commercial sodium [123/125I]iodide. The in vivo biodistribution of dRF[125I]IB in BALBc mice and imaging of dRF[123I]IB in Sprague-Dawley rats are reported. In vivo data for rats show rapid clearance of radioactivity from blood (>95%ID in 15 minutes), extensive excretion in urine (56%ID/24 hours), concentration in the hepatobiliary-small intestine system and very little fecal excretion (∼3%ID/24 hours). Pharmacokinetic data for dRF[125I]IB (i.v. 48.7 ug/kg) in rats (t1/2[h] = 0.51 ± 0.14, AUCinf[μg.min/mL] = 3.7 ± 0.4, Cl[L/kg/h] = 0.75 ± 0.12, Vss[L/kg] = 0.96 ± 0.18) confirm previously reported dose-dependent pharmacokinetics. Scintigraphic images of rats dosed with dRF[123I]I were compatible with rapid soft-tissue clearance and extensive accumulation of radioactivity in bladder/urine and liver/small intestine. In tumor-bearing mice, thyroid and stomach radioactivity was indicative of moderate deiodination. An unidentified polar radioactive metabolite was detected in serum.

Radiolabelled nucleosides have potential applications as cell proliferation markers and as substrates for the Herpes simplex thymidine kinase (HSV-TK) reporter gene used in gene therapy. 2'-Deoxy-2'-[F-18]fluorothymidine ([F-18]FT) is highly-selective for HSV-TK relative to mammalian TK and is therefore of interest as a gene therapy reporter probe. [F-18]FT was prepared via the SN2 displacement of nosyl on 3-tert-butoxycarbonyl-1-(3',5'-di-O-benzoyl-2'-O-p-nitrophenylsulfonyl-beta-D-arabinofuranosyl)thymine by [F-18]fluoride anion, followed by deprotection. This radiofluorination precursor was synthesized from commercially-available 5-methyluridine in 11.5 % chemical yield (6 steps; Scheme 2). Fluorination of the nosylate using cyclotron-produced [F-18]fluoride as the no-carrier-added activated K[F-18]F-Kryptofix(222) complex in acetonitrile at 95 C-omicron, with a reaction time of 10 min, afforded crude [F-18]FT in radiochemical yields (RCY) ranging from 4.2 to 15.6 percent (9.2 +/- 3.8 %; n = 20). Radio TLC of the crude radiofluorinated product showed 36 to 78 percent (59.1 +/- 17.2 %; n = 20) [F-18]FT, recovered with radiochemical purity of 88 to 98 percent (93.8 +/- 3.2 %; n = 20). Using a GE TracerLab FX synthesis unit and a manually-loaded HPLC, synthesis and purification times of 60 +/- 5 min afforded overall recovered radiochemical yields averaging 5.5 +/- 1.9 % (n = 20). The identity of [F-18]FT was confirmed by TLC co-development, and HPLC co-elution with authentic FT.An authentic sample of reference 2'-deoxy-2'-fluorothymidine (FT) was prepared by direct fluorination of the 2'-arabino compound 1-(3',5'-di-O-trityl-beta-D-arabinofuranosyl)thymine with DAST, followed by removal of the trityl protecting groups with trifluoroacetic acid, in 4 steps from 2,2'-anhydro-1-(-D-arabinofuranosyl)thymine, in 43 % overall chemical yield.