Improved synthesis of 2′-deoxy-2′-[18F]-fluoro-1-β-d-arabinofuranosyl-5-iodouracil ([18F]-FIAU)

An improved synthesis of 2′-[¹⁸F]-fluoro-2′-deoxy-1-β-d-arabinofuranosyl-5-iodouracil ([¹⁸F]-FIAU) has been developed. The method utilizes trimethylsilyl trifluoromethanesulfonate (TMSOTf) catalyzed coupling of 2-deoxy-2-[¹⁸F]-fluoro-1,3,5-tri-O-benzoyl-d-arabinofuranose with 2,4-bis(trimethylsilyloxy)-5-iodouracil to yield the protected dibenzoyl-[¹⁸F]-FIAU. Dibenzoyl-[¹⁸F]-FIAU was deprotected with sodium methoxide to yield a mixture of α- and β-anomers in a ratio of 1:1, which were purified by HPLC. The procedure described in this article eliminates the need for HBr activation of the sugar prior to coupling with silylated iodouracil and is suitable for automation. The total reaction time was about 110 min, starting from [¹⁸F]-fluoride. The average isolated yield of the required β-anomer was 10±6% (decay corrected) with average specific activity of 125 mCi/μmol.     

In vivo evaluation of 2′-deoxy-2′-[18F]fluoro-5-iodo-1-β-D-arabinofuranosyluracil ([18F]FIAU) and 2′-deoxy-2′-[18F]fluoro-5-ethyl-1-β-D-arabinofuranosyluracil ([18F]FEAU) as markers for suicide gene expression

PURPOSE: FIAU and FEAU were evaluated in vitro and in vivo as markers for HSV1-tk gene expression. METHODS: In vitro and biodistribution studies were performed in wild type and transduced HT-29 cells using [14C]FIAU and [3H]FEAU. PET imaging was performed using [18F]FIAU and [18F]FEAU. RESULTS: In vitro uptake of [14C]FIAU in tk-positive cells was 39-fold, 49-fold, and 43-fold higher (p<0.001) than in wild type cells at 30, 60, and 120 min, respectively. Uptake of [3H]FEAU in transduced cells was 46-fold, 62-fold, and 121-fold higher (p<0.001) than in wild type cells at the same time points. In vivo uptake of [14C]FIAU at 2 h in HSV1-tk positive tumors was 15.48+/-3.94, 6.7-fold higher (p<0.001) than in wild type tumors. Uptake of [3H]FEAU in transduced tumors was 9.98+/-1.99, 5.0-fold higher (p<0.001) than in wild type tumors. Micro-PET images using [18F]FIAU and [18F]FEAU also showed very high uptake in HSV-tk tumors. CONCLUSION: [18F]FIAU and [18F]FEAU appear to be potential PET imaging agents for gene expression.     

The synthesis of radiolabelled 1-(2′-Fluoro-2′-deoxy-β-D-ribofuranosyl)-uracil and 1-(2′-chloro-2′-deoxy-β-D-ribofuranosyl)uracil

Radiolabelled derivatives of 1-(2′fluoro-2′-deoxy-β-D-ribofuranosyl)uracil 3 (2′-FUdR) and 1-(2′-chloro-2′-deoxy-β-D-ribofuranosyl)uracil 4 (2′-ClUdR) were synthesized for evaluation as diagnostic radiopharmaceuticals. 6-[³H]-2′-FUdR (21% radiochemical yield from 6-[³H]-uridine; 4.26 GBq mmol⁻¹) and 2-[¹⁴C]-2′-FUdR (25% radiochemical yield; 1.85 GBq mmol⁻¹ were prepared by reaction of anhydrous hydrogen fluoride (AHF) with 6-[³H]- or 2-[¹⁴C]-labelled 2,2′-anhydro-1-β-D-arabinofuranosyluracil 2 (2,2′-AUR). 6-[³H]-2′-ClUdR (53% radiochemical yield; 11.1 GBq mmol^-t-1) was prepared by reaction of CaCl₂ with 6-[³H]-2,2′-AUR. Reaction of [¹⁸F]-AHF with 2,2′-AUR afforded 2′-[¹⁸F]-2′-FUdR in low radiochemical yield while reaction of 2,2′-AUR with [³⁶Cl]-NaCl and trifluoroacetic acid gave 2′-[³⁶Cl]-2′-ClUdR (30% radiochemical yield; 5.46 MBq mmol⁻¹). 2′-[^34mCl]-2′-ClUdR was similarly prepared using [^34mCl]-MgCl₂ or [^34mCl]-Dowex 21-K resin in 2.7% (16.8 MBq mmol⁻¹) and 36% (0.304 GBq mmol⁻¹) radiochemical yield respectively.     

Improved Synthesis of 2'-deoxy-2'-[18F]fluoro-5-Methyl-1-β-DArabinofuranosyluracil ([18F]FMAU)

[18F]FMAU is an established PET probe used to monitor cellular proliferation. For clinical applications, a fully automated cGMP-compliant radiosynthesis would be preferred. However, the current synthesis of [18F]FMAU requires HBr activation of the sugar prior to the coupling with silylated uracil. This multiple step procedure makes the development of an automated protocol difficult and complicated. In this study, we report the use of Friedel-Crafts catalysts for an improved synthesis of [18F]FMAU, which also includes a significantly simplified one-pot reaction condition.     

Evaluation of 2′-deoxy-2′-[18F]fluoro-5-methyl-1-β-l-arabinofuranosyluracil ([18F]-l-FMAU) as a PET imaging agent for cellular proliferation: comparison with [18F]-d-FMAU and [18F]FLT

PURPOSE: Clevudine (L: -FMAU) an un-natural analogue of thymidine, is in clinical trials for the treatment of hepatitis B virus (HBV). L: -FMAU is phosphorylated by cellular kinases such as thymidine kinase 1 and deoxycytidine kinase, and its triphosphate form inhibits HBV deoxyribonucleic acid synthesis. Thus, L: -FMAU, radiolabeled with an appropriate isotope, may be useful for positron emission tomography (PET) imaging of tumor proliferation. We evaluated [18F]-L-FMAU as a PET imaging agent in tumor-bearing mice and compared the results with those of two other radiotracers, [18F]-d-FMAU and [18F]-FLT. METHODS: Subcutaneous xenografts of the human lung cancer cell lines H441 and H3255 were established in mice. A micro-PET scanner was used to obtain images of the tumor-bearing animals with [18F]-L-FMAU, [18F]-D-FMAU, and [18F]-FLT. RESULTS: At 2 h postinjection, the tumor uptake (% ID/g) of 18F]-L: -FMAU, 18F]-D: -FMAU, and [18F]-FLT in the faster-growing H441 cells was 3.13 +/- 1.11, 7.74 +/- 1.39, and 5.10 +/- 1.45, respectively. The corresponding values for the slower-growing H3255 cells were 1.38 +/- 0.81, 4.49 +/- 1.08, and 0.57 +/- 0.33. Tumor/muscle ratios of accumulation for [18F]-L: -FMAU, [18F]-D: -FMAU, and [18F]-FLT in H441 cells were 4.15 +/- 1.82, 3.37 +/- 1.19, and 12.94 +/- 4.38, respectively, and the corresponding values in H3255 cells were 1.62 +/- 0.50, 1.96 +/- 0.74, and 1.50 +/- 0.90. CONCLUSIONS: [18F]-L: -FMAU may be a useful agent for imaging tumor proliferation in fast-growing human lung cancers by PET.     

Herpes simplex virus thymidine kinase imaging in mice with (1-(2′-deoxy-2′-[<Superscript>18</Superscript>F]fluoro-1-β-D-arabinofuranosyl)-5-iodouracil) and metabolite (1-(2′-deoxy-2′-[<Superscript>18</Superscript>F]fluoro-1-β-D-arabinofuranosyl)-5-uracil)

Purpose  

FIAU, (1-(2′-deoxy-2′-fluoro-1-β-D-arabinofuranosyl)-5-iodouracil) has been used as a substrate for herpes simplex virus thymidine kinases (HSV-TK and HSV-tk, for protein and gene expression, respectively) and other bacterial and viral thymidine kinases for noninvasive imaging applications. Previous studies have reported the formation of a de-iodinated metabolite of ¹⁸F-FIAU. This study reports the dynamic tumor uptake, biodistribution, and metabolite contribution to the activity of ¹⁸F-FIAU seen in HSV-tk gene expressing tumors and compares the distribution properties with its de-iodinated metabolite ¹⁸F-FAU.     

Effects of structural differences between radioiodine-labeled 1-(2′-fluoro-2′-deoxy-d-arabinofuranosyl)-5-iodouracil (FIAU) and 1-(2′-fluoro-2′-deoxy-d-ribofuranosyl)-5-iodouracil (FIRU) on HSV1-TK reporter gene imaging

The goal of this investigation was to evaluate the effects of structural differences between FIAU and FIRU on their ability to serve as a potential tracer for reporter gene imaging. To examine the characteristics of different configurations of FIAU and FIRU, a series of evaluations were done on HSV1-TK gene-expressing cells and on mice with HSV1-TK gene-expressing tumor. The results showed that, as an imaging agent for HSV1-TK-expressing cells, radiolabeled FIAU was more efficient for in vivo imaging than FIRU.     

Fluorinated benzamide neuroleptics—2. Synthesis and radiosynthesis of (S)-N-[(1-ethyl-2-pyrrolidinyl)methyl]-5-(3-[18F]fluoropropyl)-3-substituted-2-methoxybenzamides

Synthesis of a fluorinated benzamide neuroleptic, (S)-N-[(1-ethyl-2-pyrrolidinyl)methyl]-5-(3-fluoropropyl)-2,3-dimethoxybenzamide starting from 3-(3,4-dimethoxyphenyl)-1-propanol in 20–25% overall yield is reported. Radiosynthesis of (S)-N-[(1-ethyl-2-pyrrolidinyl)methyl]-5-(3[¹⁸F]fluoropropyl)-2-methoxybenzamide([¹⁸F]FPHB) and (S)-N-[(1-ethyl-2-pyrrolidinyl)methyl]-5-(3[¹⁸F]fluoropropyl)-2,3-dimethoxybenzamide([¹⁸F]FPHB) was carried out by nucleophilic substitution reaction of (S)-N-[(1-ethyl-2-pyrrolidinyl)methyl-5-(3-tosyloxypropyl)-2-methoxybenzamide and (S)-N-[(1-ethyl-2-pyrrolidinyl)methyl]-5-(3-tosyloxypropyl)-2,3-dimethoxybenzamide respectively, with no carrier added [¹⁸F]fluoride. Both, [¹⁸F]FPHB and [¹⁸F]FPHB were obtained in approx. 20–30% yields (EOS/EOB, decay corrected). Specific activities of 900–1700 Ci/mmol for [¹⁸F]FPHB and 800–1400 Ci/mmol for [¹⁸F]FPMB were obtained by reverse phase HPLC purification. Total synthesis and purification time required for either [¹⁸F]FPHB or [¹⁸F]FPMB was 120 min from EOB.     

Automated synthesis of 2'-deoxy-2'-[18F]fluoro-5-methyl-1-β-D-arabinofuranosyluracil ([18F]-FMAU) using a one reactor radiosynthesis module

2'-Deoxy-2'-[(18)F]fluoro-5-methyl-1-β-D-arabinofuranosyluracil ([(18)F]-FMAU) is an established PET probe used to monitor cellular proliferation. For clinical applications, a fully automated cGMP-compliant radiosynthesis would be preferred. However, the current synthesis of [(18)F]-FMAU requires a multistep procedure, making the development of an automated protocol difficult and complicated. Recently, we have developed a significantly simplified one-pot reaction condition for the synthesis of [(18)F]-FMAU in the presence of Friedel-Crafts catalysts. Here, we report a fully automated synthesis of [(18)F]-FMAU based on a one reactor radiosynthesis module using our newly developed synthetic method. The product was purified on a semi-preparative high-performance liquid chromatography integrated with the synthesis module using 6% EtOH in 10 mM phosphate buffer or 8% MeCN/water. [(18)F]-FMAU was obtained in 12±3% radiochemical yield (decay corrected overall yield based on [(18)F]-F(-), n=4) with 383±33 mCi/μmol specific activity at the time of injection. The α/β anomer ratio was 4:6. The overall reaction time was about 150 min from the end of bombardment and the radiochemical purity was >99%. This automated synthesis should also be suitable for the production of other 5-substituted thymidine analogues.     

Synthesis of 3β-(4-[18F]fluoromethylphenyl)- and 3β-(2-[18F] fluoromethylphenyl)tropane-2β-carboxylic acid methyl esters: new ligands for mapping brain dopamine transporter with positron emission tomography

The synthesis of two new dopamine transporter ligands, 3β-(4-fluoromethylphenyl)tropane-2β-carboxylic acid methyl ester and 3β-(2-fluoromethylphenyl)tropane-2β-carboxylic acid methyl ester, and their spectral characterization are described. The precursors for these ligands were prepared by TiCl₄ catalyzed chloromethylation of 3β-phenyltropane-2β-carboxylic acid methyl ester followed by separation of the isomeric product mixture of 2- and 4-chloromethylphenyltropane derivatives. Reaction of the chloromethyl analogs with no-carrier-added [¹⁸F]fluoride ion followed by high performance liquid chromatography purification provided the corresponding [¹⁸F]fluoromethyltropanes, in good radiochemical yields, useful for imaging the brain dopamine transporter system in vivo with positron emission tomography.     

Nicotinic α4β2 receptor imaging agents. Part III. Synthesis and biological evaluation of 3-(2-(S)-azetidinylmethoxy)-5-(3'-18F-fluoropropyl)pyridine (18F-nifzetidine)

Thalamic and extrathalamic nicotinic α4β2 receptors found in the brain have been implicated in Alzheimer's disease, Parkinson's disease, substance abuse and other disorders. We report here the development of 3-(2-(S)-azetidinylmethoxy)-5-(3′-fluoropropyl)pyridine (nifzetidine) as a new putative high-affinity antagonist for nicotinic α4β2 receptors. Nifzetidine in rat brain homogenate assays containing α4β2 sites labeled with ³H-cytisine exhibited a binding affinity: Ki=0.67 nM. The fluorine-18 analog, 3-(2-(S)-azetidinylmethoxy)-5-(3′-¹⁸F-fluoropropyl)pyridine (¹⁸F-nifzetidine), was synthesized in 20%–40% yield, and apparent specific activity was estimated to be above 2 Ci/μmol. Rat brain slices indicated selective binding of ¹⁸F-nifzetidine to thalamus, subiculum, striata, cortex and other regions consistent with α4β2 receptor distribution. This selective binding was displaced >85% by 150 μM nicotine. Positron emission tomography (PET) imaging studies of ¹⁸F-nifzetidine in anesthetized rhesus monkey showed slow uptake in the various brain regions. Retention of ¹⁸F-nifzetidine was maximal in the thalamus and lateral geniculate followed by regions of the temporal and frontal cortex. Cerebellum showed the least uptake. Thalamus to cerebellum ratio was about 2.3 at 180 min postinjection and continued to rise. ¹⁸F-Nifzetidine shows promise as a new PET imaging agent for α4β2 nAChR. However, the slow kinetics suggests a need for >3-h PET scans for quantitative studies of the α4β2 nAChRs.     

Biodistribution and breast tumor uptake of 16α-[18F]-fluoro-17β-estradiol in rat

To evaluate the usefulness of 16alpha-[18F]-fluoro-17beta-estradiol (FES) for the assessment of estrogen receptor (ER), we examined the tissue distribution and kinetics of FES in immature female Sprague-Dawley rats and then examined FES uptake in rat breast tumors induced by 7,12-dimethylbenz(a) anthracene (DMBA). The FES uptake by the uterus, an ER-rich tissue, was highly selective and it was 3.34 +/- 0.79%ID/g at 60 minutes and 1.57 +/- 0.57%ID/g at 120 minutes after injection. The FES uptakes in ER-negative tissues were 0.12 +/- 0.05%ID/g or less and 0.05 +/- 0.03%ID/g or less, respectively. Coadministration of unlabeled beta-estradiol showed marked depression of uterine FES uptake. The FES uptake by rat breast tumors was 0.14 +/- 0.06%ID/g at 60 min and 0.12 +/- 0.09%ID/g at 120 min. The FES uptake by rat breast tumors correlated with the ER concentration (r = 0.45, p < 0.05). In conclusion, these results suggest that the FES uptake by tissue is mainly ER mediated and FES is thus useful for detecting ER positive breast tumors.     

Effect of Animal Condition and Fluvoxamine on the Result of [18F]N-3-Fluoropropyl-2β-carbomethoxy-3β-(4-iodophenyl) Nortropane ([18F]FP-CIT) PET Study in Mice

Purpose

PET (positron emission tomography) is a noninvasive imaging technique, visualizing biological aspects in vivo. In animal models, the result of PET study can be affected more prominently than in humans by the animal conditions or drug pretreatment. We assessed the effects of anesthesia, body temperature, and pretreatment with selective serotonin reuptake inhibitor on the results of [¹⁸F]N-3-fluoropropyl-2β-carbomethoxy-3β-(4-iodophenyl) nortropane ([¹⁸F]FP-CIT) PET in mice.

Methods

[¹⁸F]FP-CIT PET of C57BL/6 mice was performed in three different conditions: (1) anesthesia (isoflurane) with active warming (38°C) as a reference; (2) no anesthesia or warming; (3) anesthesia without warming at room temperature. Additional groups of mice pretreated with escalating doses of fluvoxamine (5, 20, 40, 80 mg/kg) were imaged in condition (1). The time activity curve and standardized uptake value of the striatum, cerebral cortex, and bone were compared among these conditions.

Results

In all conditions, radioactivities of the striatum and cortex tended to form a plateau after rapid uptake and washout, but that of bone tended to increase gradually. When anesthetized without any warming, all the mice developed hypothermia and showed reduced bone uptake with slightly increased striatal and cortical uptakes compared to the reference condition. In conditions without anesthesia, striatal and cortical uptakes were reduced, whereas the bone uptake showed no change. Pretreatment with fluvoxamine increased the striatal uptake and striatal specific to cortical non-specific uptake ratio, whereas the bone uptake was reduced.

Conclusion

Anesthesia, body temperature, and fluvoxamine affect the result of [¹⁸F]FP-CIT PET in mice by altering striatal and bone uptakes.     

Radiosynthesis and ex vivo evaluation of (R)-(−)-2-chloro-N-[1-11C-propyl]n-propylnorapomorphine

IntroductionSeveral dopamine D₂ agonist radioligands have been used with positron emission tomography (PET), including [¹¹C-]-(?)-MNPA, [¹¹C-]-(?)-NPA and [¹¹C]-(+)-PHNO. These radioligands are considered particularly powerful for detection of endogenous dopamine release, but they either provide PET brain images with limited contrast or have affinity for both D₂ and D₃ receptors. We here present the carbon-11 radiolabeling and ex vivo evaluation of 2-Cl-(?)-NPA, a novel PET-tracer candidate with high in vitro D₂/D₃ selectivity.Methods2-Cl-[¹¹C]-(?)-NPA and [¹¹C]-(?)-NPA were synthesized by a two step N-acylation-reduction process using [¹¹C]-propionyl chloride. Awake rats were injected with either tracer, via the tail vein. The rats were decapitated at various times, the brains were removed and quickly dissected, and plasma metabolites were measured. Radioligand specificity, and P-glycoprotein involvement in brain uptake, was also assessed.Results2-Cl-[¹¹C]-(?)-NPA and [¹¹C]-(?)-NPA were produced in high specific activity and purity. 2-Cl-[¹¹C]-(?)-NPA accumulated slower in the striatum than [¹¹C]-(?)-NPA, reaching maximum concentrations after 30 min. The maximal striatal uptake of 2-Cl-[¹¹C]-(?)-NPA (standard uptake value 0.72±0.24) was approximately half that of [¹¹C]-(?)-NPA (standard uptake value 1.37±0.18). Nonspecific uptake was similar for the two compounds. 2-Cl-[¹¹C]-(?)-NPA was metabolized quickly, leaving only 17% of the parent compound in the plasma after 30 min. The specific binding of 2-Cl-[¹¹C]-(?)-NPA was completely blocked and inhibition of P-glycoprotein did not alter the brain uptake.ConclusionEx vivo experiments showed, despite a favorable D₂/D₃ selectivity, that 2-Cl-[¹¹C]-(?)-NPA is inferior to [¹¹C]-(?)-NPA as a PET tracer in rat, because of slower brain uptake and lower specific to nonspecific binding ratio.     

Synthesis of L-[β-11C]amino acids using immobilized enzymes

L-[β-¹¹C]-3,4-dihydroxyphenylalanine(L-[β-¹¹C]DOPA) and L-[β-¹¹C]-5-hydroxytryptophan(L-[β-¹¹C]-5-HTP) were synthesized in one step with immobilized thermostable enzymes (alanine racemase, D-amino acid oxidase, and β-tyrosinase or tryptophanase) on an aminopropyl-CPG carrier in a single column and by passing D,L-[3-¹¹C]alanine through the column with coenzymes and other substrates. L-[β-¹¹C]DOPA and L-[β-¹¹C]-5-HTP could be obtained at yields of 53% and 60%, respectively, by optimizing the amounts and the ratios of the enzymes used, the reaction temperature, the pH, and the flow rate. Moreover, the same immobilized enzyme column could be used repeatedly.     

Synthesis of 11C-labeled (±)-5-methyl-10, 11-dihydro-5H-dibenzo[a,d]cyclohepten-5, 10-imine [(±)-[11C]MK801]

The synthesis of C5 labeled (±)-5-[¹¹C]methyl-10, 11-dihydro-5H-dibenzo[a,d]cyclo-hepten-5, 10-imine [(±)-[¹¹C]MK801] has been accomplished via alkylation of (±)-10, 11-dihydro-5H-dibenzo[a,d]cyclohepten-5, 10-imine-N-t-butylformamidine [(±)-5-des-methyl MK801 formamidine). The ¹¹C labeling is accomplished by reaction of the anion of (±)-5-des-methyl MK801 formamidine, generated with s-butyllithium, and [¹¹C]methyl iodide.     

Preliminary evaluation of 1′-[18F]fluoroethyl-β-D-lactose ([18F]FEL) for detection of pancreatic cancer in nude mouse orthotopic xenografts

IntroductionEarly detection of pancreatic cancer could save many thousands of lives. Non-invasive diagnostic imaging, including PET with [¹⁸F]FDG, has inadequate resolution for detection of small (2–3 mm) pancreatic tumours. We demonstrated the efficacy of PET imaging with an ¹⁸F-labelled lactose derivative, [¹⁸F]FEDL, that targets HIP/PAP, a biomarker that is overexpressed in the peritumoural pancreas. We developed another analogue, 1-[¹⁸F]fluoroethyl lactose ([¹⁸F]FEL), which is simpler to synthesise, for the same application. We conducted a preliminary evaluation of the new probe and its efficacy in detecting orthotopic pancreatic carcinoma xenografts in mice.MethodsXenografts were developed in nude mice by injecting L3.6pl/GL⁺ pancreatic carcinoma cells into the pancreas of each mouse. Tumour growth was monitored by bioluminescence imaging (BLI); accuracy of BLI tumour size estimates was verified by MRI in two representative mice. When the tumour size reached approximately 2–3 mm, the animals were injected with [¹⁸F]FEL (3.7 MBq) and underwent static PET/CT scans. Blood samples were collected at 2, 5, 10, 20 and 60 min after [¹⁸F]FEL injection to track blood clearance. Following imaging, animals were sacrificed and their organs and tumours/pancreatic tissue were collected and counted on a gamma counter. Pancreas, including tumour, was frozen, sliced and used for autoradiography and immunohistochemical analysis of HIP/PAP expression.ResultsTumour growth was rapid, as observed by BLI and MRI. Blood clearance of [¹⁸F]FEL was bi-exponential, with half-lives of approximately 3.5 min and 40 min. Mean accumulation of [¹⁸F]FEL in the peritumoural pancreatic tissue was 1.29 ± 0.295 %ID/g, and that in the normal pancreas of control animals was 0.090 ± 0.101 %ID/g. [¹⁸F]FEL was cleared predominantly by the kidneys. Comparative analysis of autoradiographic images and immunostaining results demonstrated a correlation between [¹⁸F]FEL binding and HIP/PAP expression.Conclusion[¹⁸F]FEL may be useful for non-invasive imaging of early-stage pancreatic tumours by PET. The results warrant further studies.