Improved automated synthesis of [18F]fluoroethylcholine as a radiotracer for cancer imaging

[(18)F]Fluoroethylcholine has been recently introduced as a promising (18)F-labelled analogue of [(11)C]choline which had been previously described as a tracer for metabolic cancer imaging with positron emission tomography (PET). Due to the practical advantages of using the longer-lived radioisotope (18)F (t(1/2)=110 min), offering the opportunity of a more widespread clinical application, we established a reliable, fully automated synthesis for its production using a modified, commercially available module. [(18)F]Fluoroethylcholine was prepared from N,N-dimethylaminoethanol by iodide catalyzed alkylation with 1-[(18)F]fluoro-2-tosylethane as alkylating agent, resulting in a total radiochemical yield of 30+/-6% after a synthesis time of 50 min. The specific activity of [(18)F]fluoroethylcholine was >55 GBq/micromol and the radiochemical purity 95-99%.     

Total synthesis and evaluation of [18F]MHMZ

Radiochemical labeling of MDL 105725 using the secondary labeling precursor 2-[(18)F]fluoroethyltosylate ([(18)F]FETos) was carried out in yields of approximately 90% synthesizing [(18)F]MHMZ in a specific activity of approximately 50MBq/nmol with a starting activity of approximately 3GBq. Overall radiochemical yield including [(18)F]FETos synthon synthesis, [(18)F]fluoroalkylation and preparing the injectable [(18)F]MHMZ solution was 42% within a synthesis time of approximately 100 min. The novel compound showed excellent specific binding to the 5-HT(2A) receptor (K(i)=9.0 nM) in vitro and promising in vivo characteristics.     

Synthesis and application of [18F]FDG-maleimidehexyloxime ([18F]FDG-MHO): a [18F]FDG-based prosthetic group for the chemoselective 18F-labeling of peptides and proteins

2-[(18)F]Fluoro-2-deoxy-D-glucose ([(18)F]FDG) as the most important PET radiotracer is available in almost every PET center. However, there are only very few examples using [(18)F]FDG as a building block for the synthesis of (18)F-labeled compounds. The present study describes the use of [(18)F]FDG as a building block for the synthesis of (18)F-labeled peptides and proteins. [(18)F]FDG was converted into [(18)F]FDG-maleimidehexyloxime ([(18)F]FDG-MHO), a novel [(18)F]FDG-based prosthetic group for the mild and thiol group-specific (18)F labeling of peptides and proteins. The reaction was performed at 100 degrees C for 15 min in a sealed vial containing [(18)F]FDG and N-(6-aminoxy-hexyl)maleimide in 80% ethanol. [(18)F]FDG-MHO was obtained in 45-69% radiochemical yield (based upon [(18)F]FDG) after HPLC purification in a total synthesis time of 45 min. Chemoselecetive conjugation of [(18)F]FDG-MHO to thiol groups was investigated by the reaction with the tripeptide glutathione (GSH) and the single cysteine containing protein annexin A5 (anxA5). Radiolabeled annexin A5 ([(18)F]FDG-MHO-anxA5) was obtained in 43-58% radiochemical yield (based upon [(18)F]FDG-MHO, n = 6), and [(18)F]FDG-MHO-anxA5 was used for a pilot small animal PET study to assess in vivo biodistribution and kinetics in a HT-29 murine xenograft model.     

Evaluation of [11C]hemicholinium-15 and [18F]hemicholinium-15 as new potential PET tracers for the high-affinity choline uptake system in the heart

[(11)C]Hemicholinium-15 ([(11)C]HC-15) and [(18)F]hemicholinium-15 ([(18)F]HC-15) have been synthesized as new potential PET tracers for the heart high-affinity choline uptake (HACU) system. [(11)C]HC-15 was prepared by N-[(11)C]methylation of the appropriate precursor, 4-methyl-2-phenyl-morpholin-2-ol, using [(11)C]CH(3)OTf in 55-70% radiochemical yield decay corrected to end of bombardment (EOB) and 2-3Ci/mumol specific activity at end of synthesis (EOS). [(18)F]HC-15 was prepared by N-[(18)F]fluoromethylation of the precursor using [(18)F]FCH(2)OTf in 20-30% radiochemical yield decay corrected to EOB and >1.0Ci/mumol specific activity at EOS. The biodistribution of both compounds was determined in rats at 20min post-intravenous injection, and the results show the heart region uptakes 1.32+/-0.75%ID/g in R-ventricle for [(11)C]HC-15 and 1.28+/-0.81%ID/g in L-ventricle for [(18)F]HC-15, respectively. The dynamic PET imaging studies of [(11)C]HC-15 in rats were acquired 60min post-intravenous injection of the tracer using the IndyPET-II scanner. For the blocking experiments, the rats were intravenously pretreated with 3.0mg/kg of unlabeled HC-15 prior to [(11)C]HC-15 injection. [(11)C]HC-15 rat heart PET studies show rapid heart uptake to give clear heart images. The rat heart PET blocking studies found no significant blocking effect. The dynamic PET studies in normal and ablated dogs were performed using Siemens PET scanner with [(13)N]NH(3), [(11)C]HC-15, and [(18)F]HC-15. PET studies in dogs of both [(11)C]HC-15 and [(18)F]HC-15 also show significant heart uptake and give images of the heart. However, there is no significant change in [(11)C]HC-15 L-ventricle uptake following radiofrequency ablation in the dog. These results suggest that the localization of HC-15 tracers in the heart is mediated by non-specific processes, and the visualization of HC-15 tracers on the heart is related to non-specific binding of HACU.     

Synthesis and in vitro evaluation of [18F](R)-FEPAQ: a potential PET ligand for VEGFR2

Synthesis and in vitro evaluation of [(18)F](R)-N-(4-bromo-2-fluorophenyl)-7-((1-(2-fluoroethyl)piperidin-3-yl)methoxy)-6-methoxyquinazolin-4-amine ((R)-[(18)F]FEPAQ or [(18)F]1), a potential imaging agent for the VEGFR2, using phosphor image autoradiography are described. Synthesis of 2, the desfluoroethyl precursor for (R)-FEPAQ was achieved from t-butyl 3-(hydroxymethyl)piperidine-1-carboxylate (3) in five steps and in 50% yield. [(18)F]1 was synthesized by reaction of sodium salt of compound 2 with [(18)F]fluoroethyl tosylate in DMSO. The yield of [(18)F]1 was 20% (EOS based on [(18)F]F(-)) with >99% radiochemical purity and specific activity of 1-2 Ci/μmol (n=10). The total synthesis time was 75 min. The radiotracer selectively labeled VEGFR2 in slide-mounted sections of human brain and higher binding was found in surgically removed human glioblastoma sections as demonstrated by in vitro phosphor imager studies. These findings suggest [(18)F]1 may be a promising radiotracer for imaging VEGFR2 in brain using PET.     

Aromatic radiofluorination and biological evaluation of 2-aryl-6-[18F]fluorobenzothiazoles as a potential positron emission tomography imaging probe for β-amyloid plaques

To develop agents for radionuclide imaging Aβ plaques in vivo, we prepared three fluorine-substituted analogs of arylbenzothiazole class; compound 2 has a high affinity for Aβ (K(i)=5.5nM) and the specific binding to Aβ in fluorescent staining. In preparation for the synthesis of these arylbenzothiazole analogs in radiolabeled form as an Aβ plaques-specific positron emission tomography (PET) imaging probe, we investigated synthetic route suitable for its labeling with the short-lived PET radionuclide fluorine-18 (t(1/2)=110min) and diaryliodonium tosylate precursors (12, 13a-e and 14). 2-Aryl-6-[(18)F]fluorobenzothiazoles ([(18)F]1-3) were synthesized in efficiently short reaction times (40-60min) with high radiochemical yields (19-40%), purities (>95%) and specific activities (85-118GBq/μmol). Tissue distribution studies showed that high radioactivity of [(18)F]2 accumulated in the brain with rapid clearance in healthy mice. Radioactive metabolites were analyzed in brain samples of mice and corresponded to 81% of parent remained by 30min after a tail-vein injection. These results suggest that [(18)F]2 is a promising probe for evaluation of Aβ plaques imaging in brain using PET.     

18F]azadibenzocyclooctyne ([18F]ADIBO): a biocompatible radioactive labeling synthon for peptides using catalyst free [3+2] cycloaddition

N-Terminally azido-modified peptides were labeled with the novel prosthetic labeling synthon [(18)F]azadibenzocyclooctyne ([(18)F]ADIBO) using copper-free azide-alkyne [3+2]-dipolar cycloaddition in high radiochemical yields (RCYs). (18)F-Labeled [(18)F]ADIBO was prepared by nucleophilic substitution of the corresponding tosylate in 21% overall RCY (EOB) in a fully automated synthesis unit within 55 min. [(18)F]ADIBO was incubated with azide-containing peptides at room temperature in the absence of toxic metal catalysts and the formation of the triazole conjugate was confirmed. Finally, the azide-alkyne [3+2]-dipolar cycloaddition was shown to proceed with 95% radiochemical yield in ethanol within 30 min, allowing for a development of a kit-like peptide labeling approach with [(18)F]ADIBO.     

Metabolic stability of 6,7-dialkoxy-4-(2-, 3- and 4-[18F]fluoroanilino)quinazolines, potential EGFR imaging probes

Epidermal growth factor receptors (EGFR), upregulated in many tumor types, have been a target for therapeutic development and molecular imaging. The objective of this study was to evaluate the distribution and metabolic characteristics of fluorine-18 labeled anilinoquinazolines as potential imaging agents for EGFR tyrosine kinase expression. Fluorine-18 labeled fluoronitrobenzenes were prepared by reaction of potassium cryptand [(18)F]fluoride with 1,2- and 1,4-dinitrobenzenes, and 3-nitro-N,N,N-trimethylanilinium triflate in 5min. Decay-corrected radiochemical yields of [(18)F]fluoride incorporation into the nitro-aromatic compounds were 81±2%, 44±4% and 77±5% (n=3-5) for the 2-, 3- and 4-fluoro isomers, respectively. Sodium borohydride reduction to the corresponding [(18)F]fluoroanilines was achieved with greater than 80% conversion in 5min. Coupling of [(18)F]fluoroaniline-hydrochlorides to 6,7-dimethoxy-4-chloro-quinazoline gave the corresponding 6,7-dimethoxy-4-(2-, 3- and 4-[(18)F]fluoroanilino)quinazolines in 31±5%, 17±2% and 55±2% radiochemical yield, respectively, while coupling to the 6,7-diethoxy-4-chloro-quinazoline produced 6,7-diethoxy-4-(2-, 3- and 4-[(18)F]fluoroanilino)quinazolines in 19±6%, 9±3% and 36±6% radiochemical yield, respectively, in 90min to end of synthesis from [(18)F]fluoride. Biodistribution of 2- and 4-[(18)F]fluoroanilinoquinazolines was conducted in tumor-bearing mice (MDA-MB-435 and MDA-MB-468 xenografts). Low tumor uptake (<1% injected dose per gram (ID/g) of tissue up to 3h postinjection of the radiotracers) was observed. High bone uptake (5-15% ID/g) was noted with the 4-[(18)F]fluoroanilinoquinazolines. The metabolic stabilities of radiolabeled quinazolines were further evaluated by incubation with human female cryopreserved isolated hepatocytes. Rapid degeneration of the 4-fluoro-substituted compounds to baseline polar metabolites was observed by radio-TLC, whereas, the 2- and 3-[(18)F]fluoroaniline derivatives were significantly more stable, up to 2h, corroborating the in vivo biodistribution studies. para-Substituted [(18)F]fluoroanilines, a common structural motif in radiopharmaceuticals, are highly susceptible to metabolic degradation.     

Synthesis and radiopharmacological characterization of 2beta-carbo-2'-[18F]fluoroethoxy-3beta-(4-bromo-phenyl)tropane ([18F]MCL-322) as a PET radiotracer for imaging the dopamine transporter (DAT)

The fluoroalkyl-containing tropane derivative 2beta-carbo-2'-fluoroethoxy-3beta-(4-bromo-phenyl)tropane (MCL-322) is a highly potent and moderately selective ligand for the dopamine transporter (DAT). The compound was labeled with the short-lived positron emitter (18)F in a single step by nucleophilic displacement of the corresponding tosylate precursor MCL-323 with no-carrier-added [(18)F]fluoride. The positron emission tomography (PET) radiotracer 2beta-carbo-2'-[(18)F]fluoroethoxy-3beta-(4-bromo-phenyl)tropane [(18)F]MCL-322 was obtained in decay-corrected radiochemical yields of 30-40% at a specific radioactivity of 1.6-2.4Ci/mumol (60-90GBq/mumol) at the end-of-synthesis (EOS). Small animal PET, ex vivo and in vivo biodistribution experiments in rats demonstrated a high uptake in the striatum (3.2% ID/g) 5min after injection, which increased to 4.2% ID/g after 60min. The uptake in the cerebellum was 1.8% ID/g and 0.6% ID/g after 5min and 60min post-injection, respectively. Specific binding to DAT of [(18)F]MCL-322 was confirmed by blocking experiments using the high affinity DAT ligand GBR 12909. The radiopharmacological characterization was completed with metabolite and autoradiographic studies confirming the selective uptake of [(18)F]MCL-322 in the striatum. It is concluded that the simple single-step radiosynthesis of [(18)F]MCL-322 and the promising radiopharmacological data make [(18)F]MCL-322 an attractive candidate for the further development of a PET radiotracer potentially suitable for clinical DAT imaging in the human brain.     

N-[18F]fluoroethyl-4-piperidyl acetate ([18F]FEtP4A): A PET tracer for imaging brain acetylcholinesterase in vivo

N-[(18)F]Fluoroethyl-4-piperidyl acetate ([(18)F]FEtP4A) was synthesized and evaluated as a PET tracer for imaging brain acetylcholinesterase (AchE) in vivo. [(18)F]FEtP4A was previously prepared by reacting 4-piperidyl acetate (P4A) with 2-[(18)F]fluoroethyl bromide ([(18)F]FEtBr) at 130 degrees C for 30 min in 37% radiochemical yield using an automated synthetic system. In this work, [(18)F]FEtP4A was synthesized by reacting P4A with 2-[(18)F]fluoroethyl iodide ([(18)F]FEtI) or 2-[(18)F]fluoroethyl triflate ([(18)F]FEtOTf in improved radiochemical yields, compared with [(18)F]FEtBr under the corresponding condition. Ex vivo autoradiogram of rat brain and PET summation image of monkey brain after iv injection of [(18)F]FEtP4A displayed a high radioactivity in the striatum, a region with the highest AchE activity in the brain. Moreover, the distribution pattern of (18)F radioactivity was consistent with that of AchE in the brain: striatum>frontal cortex>cerebellum. In the rat and monkey plasma, two radioactive metabolites were detected. However, their presence might not preclude the imaging studies for AchE in the brain, because they were too hydrophilic to pass the blood-brain barrier and to enter the brain. In the rat brain, only [(18)F]fluoroethyl-4-piperidinol ([(18)F]FEtP4OH) was detected at 30 min postinjection. The hydrolytic [(18)F]FEtP4OH displayed a slow washout and a long retention in the monkey brain until the PET experiment (120 min). Although [(18)F]FEtP4A is a potential PET tracer for imaging AchE in vivo, its lower hydrolytic rate and lower specificity for AchE than those of [(11)C]MP4A may limit its usefulness for the quantitative measurement for AchE in the primate brain.     

Synthesis of 11C-labelled (R)-OHDMI and CFMME and their evaluation as candidate radioligands for imaging central norepinephrine transporters with PET

(R)-1-(10,11-Dihydro-dibenzo[b,f]azepin-5-yl)-3-methylamino-propan-2-ol ((R)-OHDMI) and (S,S)-1-cyclopentyl-2-(5-fluoro-2-methoxy-phenyl)-1-morpholin-2-yl-ethanol (CFMME) were synthesized and found to be potent inhibitors of norepinephrine reuptake. Each was labelled efficiently in its methyl group with carbon-11 (t(1/2)=20.4 min) as a prospective radioligand for imaging brain norepinephrine transporters (NET) with positron emission tomography (PET). The uptake and distribution of radioactivity in brain following intravenous injection of each radioligand into cynomolgus monkey was examined in vivo with PET. After injection of (R)-[(11)C]OHDMI, the maximal whole brain uptake of radioactivity was very low (1.1% of injected dose; I.D.). For occipital cortex, thalamus, lower brainstem, mesencephalon and cerebellum, radioactivity ratios to striatum at 93 min after radioligand injection were 1.35, 1.35, 1.2, 1.2 and 1.0, respectively. After injection of [(11)C]CFMME, radioactivity readily entered brain (3.5% I.D.). Ratios of radioactivity to cerebellum at 93 min for thalamus, occipital cortex, region of locus coeruleus, mesencephalon and striatum were 1.35, 1.3, 1.3, 1.2 and 1.2, respectively. Radioactive metabolites in plasma were measured by radio-HPLC. (R)-[(11)C]OHDMI represented 75% of plasma radioactivity at 4 min after injection and 6% at 30 min. After injection of [(11)C]CFMME, 84% of the radioactivity in plasma represented parent at 4 min and 20% at 30 min. Since the two new hydroxylated radioligands provide only modest regional differentiation in brain uptake and form potentially troublesome lipophilic radioactive metabolites, they are concluded to be inferior to existing radioligands, such as (S,S)-[(11)C]MeNER, (S,S)-[(18)F]FMeNER-D(2) and (S,S)-[(18)F]FRB-D(4), for the study of brain NETs with PET in vivo.     

Radiosynthesis of a ¹⁸F-labeled 2,3-diarylsubstituted indole via McMurry coupling for functional characterization of cyclooxygenase-2 (COX-2) in vitro and in vivo

The radiosynthesis of 3-(4-[(18)F]fluorophenyl)-2-(4-methylsulfonylphenyl)-1H-indole [(18)F]-3 as potential PET radiotracer for functional characterization of cyclooxygenase-2 (COX-2) in vitro and in vivo is described. [(18)F]-3 was prepared by McMurry cyclization of a (18)F-labeled intermediate with low valent titanium and zinc via a two-step procedure in a remote controlled synthesizer unit including HPLC purification and solid phase extraction. In this way [(18)F]-3 was synthesized in 80 min synthesis time in 10% total decay corrected yield from [(18)F]fluoride in radiochemical purity >98% and a specific activity of 74-91 GBq/μmol (EOS). [(18)F]-3 was evaluated in vitro using pro-inflammatory stimulated THP-1 and COX-2 expressing tumor cell lines (FaDu, A2058, HT-29), where the radiotracer uptake was shown to be consistent with up regulated COX-2 expression. The stability of [(18)F]-3 was determined by incubation in rat whole blood and plasma in vitro and by metabolite analysis of arterial blood samples in vivo, showing with 75% of original compound after 60 min an acceptable high metabolic stability. However, no substantial tumor accumulation of [(18)F]-3 could be observed by dynamic small animal PET studies on HT-29 tumor-bearing mice in vivo. This may be due to the only moderate COX-1/COX-2 selectivity of 3 as demonstrated by both cellular and enzymatic cyclooxygenase inhibition assay in vitro. Nevertheless, the new approach first using McMurry cyclization in (18)F-chemistry gives access to (18)F-labeled diarylsubstituted heterocycles that hold promise as radiolabeled COX-2 inhibitors.     

Synthesis and in vivo evaluation of [18F]N-(2-benzofuranylmethyl)-N'-[4-(2-fluoroethoxy)benzyl]piperazine, a novel σ1 receptor PET imaging agent

N-(2-Benzofuranylmethyl)-N'-[4-(2-fluoroethoxy)benzyl]piperazine (6, σ(1)K(i)=2.6 nM) was radiolabeled with fluorine-18 to provide a potential σ(1) receptor radioligand for use in positron emission tomography (PET). Radiofluorination of the appropriate tosylate precursor furnished [(18)F]6 with a specific activity of 45 GBq/μmol, in an average radiochemical yield of 18% and greater than 98% radiochemical purity. MicroPET imaging in Papio hamadryas baboon brain revealed [(18)F]6 uptake consistent with σ receptor distribution, and specificity for σ receptors was demonstrated in a haloperidol pre-treated animal. [(18)F]6 possesses suitable properties for PET imaging of σ(1) receptors, and further investigation of this σ(1) receptor tracer is warranted.     

Radiolabeling of RGD peptide and preliminary biological evaluation in mice bearing U87MG tumors

2-[(18)F]Fluoroethyl azide ([(18)F]FEA) and terminal alkynyl modified propioloyl RGDfK were selected in this study. [(18)F]FEA was prepared by nucleophilic radiofluorination of 2-azidoethyl 4-toluenesulfonate with radiochemical yield of 71 ± 4% (n = 5, decay-corrected). We assessed the various conditions of the CuAAC reaction between [(18)F]FEA and propioloyl RGDfK, which included peptide concentration, reaction time, temperature and catalyst dosage. The (18)F-labeled-RGD peptide ([(18)F]F-RGDfK) could be obtained in 60 min by a two-step radiochemical synthesis route, with total radiochemical yield of 60 ± 2% (n = 3, decay-corrected) through click chemistry. [(18)F]F-RGDfK showed high stability in phosphate buffered saline and new-born calf serum. Micro-PET imaging at 1 h post injection of [(18)F]F-RGDfK showed medium concentration of radioactivity in tumors while much decreased concentration in tumors in the blocking group. These results showed that [(18)F]F-RGDfK obtained by click chemistry maintained the affinity and specificity of the RGDfK peptide to integrin α(v)β(3). This study provided useful information for peptide radiofluorination by using click chemistry.     

Design and synthesis of a new [18F]fluoropyridine-based haloacetamide reagent for the labeling of oligonucleotides: 2-bromo-N-[3-(2-[18F]fluoropyridin-3-yloxy)propyl]acetamide

Based on the recently highlighted potential of nucleophilic heteroaromatic ortho-radiofluorinations in the preparation of fluorine-18-labeled radiotracers and radiopharmaceuticals for PET, a [(18)F]fluoropyridine-based bromoacetamide reagent has been prepared and used in prosthetic group introduction for the labeling of oligonucleotides. [(18)F]FPyBrA (2-bromo-N-[3-(2-[(18)F]fluoropyridin-3-yloxy)propyl]acetamide) was designed as a radiochemically feasible reagent, its pyridinyl moiety both carrying the radioactive halogen (fluorine-18) and allowing its efficient incorporation via a nucleophilic heteroaromatic substitution, and its 2-bromoacetamide function, ensuring the efficient alkylation of a phosphorothioate monoester group born at the 3'- or 5'-end of single-stranded oligonucleotides. [(18)F]FPyBrA (HPLC-purified) was efficiently prepared in 18-20% non-decay-corrected yield (based on starting [(18)F]fluoride) using a three-step radiochemical pathway in 80-85 min. The developed procedure involves (1) a high-yield nucleophilic heteroaromatic ortho-radiofluorination as the fluorine-18 incorporation-step (70-85% radiochemical yield) and uses [3-(3-tert-butoxycarbonylaminopropoxy)pyridin-2-yl]trimethylammonium trifluoromethanesulfonate as precursor for labeling, followed by (2) rapid and quantitative TFA-removal of the N-Boc-protective group and (3) condensation with 2-bromoacetyl bromide (45-65% radiochemical yield). Typically, 3.3-3.7 GBq (90-100 mCi) of HPLC-purified [(18)F]FPyBrA could be obtained in 80-85 min, starting from 18.5 GBq (500 mCi) of a cyclotron production batch of [(18)F]fluoride. [(18)F]FPyBrA was regioselectively conjugated with 9-mer and 18-mer single-stranded oligonucleotides, provided with a phosphorothioate monoester group at their 3'-end. Both natural phosphodiester DNAs and in vivo-stable 2'-methoxy and -fluoro-modified RNAs were used. Conjugation uses optimized, short-time reaction conditions (MeOH/0.1 M PBS pH 7.4, 15 min, 120 degrees C), both compatible with the chemical stability of the oligonucleotides (ONs) and the half-life of fluorine-18. Conjugated [(18)F]ONs were finally purified by RP-HPLC and desalted using a Sephadex NAP-10 column. The whole radiosynthetic procedure, including the preparation of the fluorine-18-labeled reagent, the conjugation with the oligonucleotide, and the HPLC purification and formulation lasted 140-160 min. [(18)F]FPyBrA represents a valuable alternative to the already reported N-(4-[(18)F]fluorobenzyl)-2-bromoacetamide for the design and development of oligonucleotide-based radiopharmaceuticals for PET.     

N-Succinimidyl 4-[(18)F]-fluoromethylbenzoate-labeled dimeric RGD peptide for imaging tumor integrin expression

RGD peptides, radiolabeled with (18)F, have been used in the clinic for PET imaging of tumor angiogenesis in cancer patients. RGD peptides are typically labeled using a prosthetic group such as N-succinimidyl 4-[(18)F]-fluorobenzoate ([(18)F]SFB) or 4-nitrophenyl 2-[(18)F]-fluoropropionate ([(18)F]NPFP). However, the complex radiosynthetic procedures have impeded their broad application in clinical studies. We previously radiolabeled proteins and peptides with the prosthetic group, N-succinimidyl 4-[(18)F]-fluoromethylbenzoate ([(18)F]SFMB), which was prepared in a simple one-step procedure. In this study, we labeled a PEGylated cyclic RGD peptide dimer, PEG(3)-E[c(RGDyK)](2) (PRGD2), using [(18)F]SFMB and evaluated for imaging tumor αvβ3 integrin expression with positron emission tomography (PET). [(18)F]SFMB was prepared in one step using [(18)F]fluoride displacement of a nitrobenzenesulfonate leaving group under mild reaction conditions followed by HPLC purification. The (18)F-labeled peptide, [(18)F]FMBPRGD2 was prepared by coupling PRGD2 with [(18)F]SFMB in pH 8.6 borate buffer and purified with HPLC. The direct labeling on BMBPRGD2 was also attempted. A Siemens Inveon PET was used to image the uptake of the [(18)F]FMBPRGD2 into a U87MG xenograft mouse model. [(18)F]FMBPRGD2, was prepared with a 15% overall radiochemical yield (uncorrected) in a total synthesis time of 90?min, which was considerably shorter than the preparation of [(18)F]SFB- and [(18)F]NPFP-labeled RGD peptides. The direct labeling, however, was not successful. High quality microPET images using [(18)F]FMBPRGD2 clearly visualized tumors by 15?min with good target to background ratio. Early tracer accumulation in the bladder suggests fast renal clearance. No obvious bone uptake can be detected even at 4-h time point indicating that fluorine attachment is stable in mice. In conclusion, N-succinimidyl 4-[(18)F]-fluoromethylbenzoate ([(18)F]SFMB) prosthetic group can be a good alternative for labeling RGD peptides to image αvβ3 integrin expression and for labeling other peptides.     

Synthesis and evaluation of 1-(4-[¹⁸F]fluoroethyl)-7-(4'-methyl)curcumin with improved brain permeability for β-amyloid plaque imaging

Alzheimer's disease is characterized by the accumulation of β-amyloid (Aβ) plaques and neurofibrillary tangles (NFTs) in the brain. We previously developed [(18)F]fluoropropylcurcumin ([(18)F]FP-curcumin), which demonstrated excellent binding affinity (K(i)=0.07 nM) for Aβ(1-40) aggregates and good pharmacokinetics in normal mouse brains. However, its initial brain uptake was poor (0.52% ID/g at 2 min post-injection). Therefore, in the present study, fluorine-substituted 4,4'-bissubstituted or pegylated curcumin derivatives were synthesized and evaluated. Their binding affinities for Aβ(1-42) aggregates were measured and 1-(4-fluoroethyl)-7-(4'-methyl)curcumin (1) had the highest binding affinity (K(i)=2.12 nM). Fluorescence staining of Tg APP/PS-1 mouse brain sections demonstrated high and specific labeling of Aβ plaques by 1 in the cortex region, which was confirmed with thioflavin-S staining of the same spots in the adjacent brain sections. Radioligand [(18)F]1 was found to have an appropriate partition coefficient (logP(o/w)=2.40), and its tissue distribution in normal mice demonstrated improved brain permeability (1.44% ID/g at 2 min post-injection) compared to that of [(18)F]FP-curcumin by a factor of 2.8 and fast wash-out from mouse brains (0.45% ID/g at 30 min post-injection). These results suggest that [(18)F]1 may hold promise as a PET radioligand for Aβ plaque imaging.     

Synthesis and radiosynthesis of [(18)F]FPhEP, a novel alpha(4)beta(2)-selective, epibatidine-based antagonist for PET imaging of nicotinic acetylcholine receptors

FPhEP (1, (+/-)-2-exo-(2'-fluoro-3'-phenyl-pyridin-5'-yl)-7-azabicyclo[2.2.1]heptane) belongs to a recently described novel series of 3'-phenyl analogues of epibatidine, which not only possess subnanomolar affinity and high selectivity for brain alpha4beta2 neuronal nicotinic acetylcholine receptors (nAChRs), but also were reported as functional antagonists of low toxicity (up to 15 mg/kg in mice). FPhEP (1, K(i) of 0.24 nM against [(3)H]epibatidine) as reference as well as the corresponding N-Boc-protected chloro- and bromo derivatives (3a,b) as precursors for labelling with fluorine-18 were synthesized in eight and nine steps, respectively, from commercially available N-Boc-pyrrole (overall yields=17% for 1, 9% for 3a and 8% for 3b). FPhEP (1) was labelled with fluorine-18 using the following two-step radiochemical process: (1) no-carrier-added nucleophilic heteroaromatic ortho-radiofluorination from the corresponding N-Boc-protected chloro- or bromo derivatives (3 a,b-1mg) and the activated K[(18)F]F-Kryptofix(222) complex in DMSO using microwave activation at 250 W for 1.5 min, followed by (2) quantitative TFA-induced removal of the N-Boc-protective group. Radiochemically pure (>99%) [(18)F]FPhEP ([(18)F]-1, 2.22-3.33 GBq, 66-137 GBq/micromol) was obtained after semi-preparative HPLC (Symmetry C18, eluent aq 0.05 M NaH(2)PO(4)/CH(3)CN, 80:20 (v:v)) in 75-80 min starting from a 18.5 GBq aliquot of a cyclotron-produced [(18)F]fluoride production batch (10-20% nondecay-corrected overall yield). In vitro binding studies on rat whole-brain membranes demonstrated a subnanomolar affinity (K(D) 660 pM) of [(18)F]FPhEP ([(18)F]-1) for nAChRs. In vitro autoradiographic studies also showed a good contrast between nAChR-rich and -poor regions with a low non-specific binding. Comparison of in vivo Positron Emission Tomography (PET) kinetics of [(18)F]FPhEP ([(18)F]-1) and [(18)F]F-A-85380 in baboons demonstrated faster brain kinetics of the former compound (with a peak uptake at 20 min post injection only). Taken together, the preliminary data obtained confirm that [(18)F]FPhEP ([(18)F]-1) has potential for in vivo imaging nAChRs in the brain with PET.     

18F]fluoro-deoxy-glucose folate: a novel PET radiotracer with improved in vivo properties for folate receptor targeting

The folate receptor (FR) is upregulated in various cancer types (FR-α isoform) and in activated macrophages (FR-β isoform) which are involved in inflammatory and autoimmune diseases, but its expression in healthy tissues and organs is highly restricted to only a few sites (e.g kidneys). Therefore, the FR is a promising target for imaging and therapy of cancer and inflammation using folate-based radiopharmaceuticals. Herein, we report the synthesis and evaluation of a novel folic acid conjugate with improved properties suitable for positron emission tomography (PET). [(18)F]-fluoro-deoxy-glucose folate ([(18)F]3) was synthesized based on the click chemistry approach using 2-deoxy-2-[(18)F]fluoroglucopyranosyl azide and a folate alkyne derivative. The novel radiotracer [(18)F]3 was produced in good radiochemical yields (25% d.c.) and high specific radioactivity (90 GBq/μmol). Compared to previously published (18)F-folic acid derivatives, an increase in hydrophilicity was achieved by using a glucose entity as a prosthetic group. Biodistribution and PET imaging studies in KB tumor-bearing mice showed a high and specific uptake of the radiotracer in FR-positive tumors (10.03 ± 1.12%ID/g, 60 min p.i.) and kidneys (42.94 ± 2.04%ID/g, 60 min p.i.). FR-unspecific accumulation of radioactivity was only found in the liver (9.49 ± 1.13%ID/g, 60 min p.i.) and gallbladder (17.59 ± 7.22%ID/g, 60 min p.i.). No radiometabolites were detected in blood, urine, and liver tissue up to 30 min after injection of [(18)F]3. [(18)F]-fluoro-deoxy-glucose-folate ([(18)F]3) is thus a promising PET radioligand for imaging FR-positive tumors.     

Site-specific labeling of annexin V with F-18 for apoptosis imaging

Annexin V is useful in detecting apoptotic cells by binding to phosphatidylserine (PS) that is exposed on the outer surface of the cell membrane during apoptosis. In this study, we examined the labeling of annexin V-128, a mutated form of annexin V that has a single cysteine residue at the NH 2 terminus, with the thiol-selective reagent (18)F-labeling agent N-[4-[(4-[(18)F]fluorobenzylidene)aminooxy]butyl]maleimide ([(18)F]FBABM). We also examined the cell binding affinity of the (18)F-labeled annexin V-128 ([(18)F]FAN-128). [(18)F]FBABM was synthesized in two-step, one-pot method modified from literature procedure. (Toyokuni et al., Bioconjugate Chem. 2003, 14, 1253-1259). The average yield of [(18)F]FBABM was 23 +/- 4% (n = 4, decay-corrected) and the specific activity was approximately 6000 Ci/mmol. The total synthesis time was approximately 92 min. The critical improvement of this study was identifying and then developing a purification method to remove an impurity N-[4-[(4-dimethylaminobenzylidene)aminooxy]butyl]maleimide 4, whose presence dramatically decreased the yield of protein labeling. Conjugation of [(18)F]FBABM with the thiol-containing annexin V-128 gave [(18)F]FAN-128 in 37 +/- 9% yield (n = 4, decay corrected). Erythrocyte binding assay of [(18)F]FAN-128 showed that this modification of annexin V-128 did not compromise its membrane binding affinity. Thus, an in vivo investigation of [ (18)F]FAN-128 as an apoptosis imaging agent is warranted.