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.     

Synthesis of a new heterobifunctional linker, N-[4-(aminooxy)butyl]maleimide, for facile access to a thiol-reactive 18F-labeling agent

A new heterobifunctional linker containing an aldehyde-reactive aminooxy group and a thiol-reactive maleimide group, namely N-[4-(aminooxy)butyl]maleimide, was synthesized as a stable HCl salt by O-alkylation of either N-hydroxyphthalimide or N-(4-monomethoxytrityl)hydroxylamine, followed by N-alkylation of maleimide, in an overall yield of 18% (seven steps) or 29% (five steps), respectively. This heterobifunctional linker allowed a simple and efficient synthesis of a maleimide-containing thiol-reactive (18)F-labeling agent. Thus, N-[4-[(4-[(18)F]fluorobenzylidene)aminooxy]butyl]maleimide (specific activity: approximately 3000 Ci/mmol at end of synthesis) was synthesized in two steps involving the preparation of 4-[(18)F]fluorobenzaldehyde, followed by its aminooxy-aldehyde coupling reaction to the heterobifunctional linker, with an overall radiochemical yield of approximately 35% (decay corrected) within approximately 60 min from end of bombardment. Initial (18)F-labeling experiments were carried out using a thiol-containing tripeptide glutathione (GSH) and a 5'-thiol-functionalized oligodeoxynucleotide (5'-S-ODN) in phosphate-buffered saline (PBS, pH 7.5). After standing at room temperature for 10 min, the (18)F-labeled GSH and 5'-S-ODN were obtained in (18)F-labeling yields of approximately 70% and approximately 5% (decay-corrected), respectively. The heterobifunctional linker is easy to synthesize and provides a facile access to the maleimide-containing thiol-reactive (18)F-labeling agent, which could be advantageously employed in the development of (18)F-labeled biomomolecules for use with positron emission tomography.     

18F]FMDAA1106 and [18F]FEDAA1106: two positron-emitter labeled ligands for peripheral benzodiazepine receptor (PBR)

We synthesized and evaluated N-(5-fluoro-2-phenoxyphenyl)-N-(2-[(18)F]fluoromethyl-5-methoxybenzyl)acetamide ([(18)F]-FMDAA1106) and N-(5-fluoro-2-phenoxyphenyl)-N-(2-[(18)F]fluoroethyl-5-methoxybenzyl)acetamide ([(18)F]FEDAA1106) as two potent radioligands for peripheral benzodiazepine receptors (PBR). [(18)F]FMDAA1106 and [(18)F]FEDAA1106 were respectively synthesized by fluoroalkylation of the desmethyl precursor DAA1123 with [(18)F]FCH(2)I and [(18)F]FCH(2)CH(2)Br. Ex vivo autoradiograms of [(18)F]FMDAA1106 and [(18)F]FEDAA1106 binding sites in the rat brains revealed that a high radioactivity was present in the olfactory bulb, the highest PBR density region in the brain.     

1-[3-(2-[18F]fluoropyridin-3-yloxy)propyl]pyrrole-2,5-dione: design, synthesis, and radiosynthesis of a new [18F]fluoropyridine-based maleimide reagent for the labeling of peptides and proteins

FPyME (1-[3-(2-fluoropyridin-3-yloxy)propyl]pyrrole-2,5-dione) was designed as a [(18)F]fluoropyridine-based maleimide reagent for the prosthetic labeling of peptides and proteins via selective conjugation with a thiol (sulfhydryl) function. Its pyridinyl moiety carries the radioactive halogen (fluorine-18) which can be efficiently incorporated via a nucleophilic heteroaromatic substitution, and its maleimido function ensures the efficient alkylation of a free thiol function as borne by cysteine residues. [(18)F]FPyME (HPLC-purified) was prepared in 17-20% non-decay-corrected yield, based on starting [(18)F]fluoride, in 110 min using a three-step radiochemical pathway. The developed procedure involves (1) a high-yield nucleophilic heteroaromatic ortho-radiofluorination on [3-(3-tert-butoxycarbonylaminopropoxy)pyridin-2-yl]trimethylammonium trifluoromethanesulfonate as the fluorine-18 incorporation step, followed by (2) rapid and quantitative TFA-induced removal of the N-Boc-protective group and (3) optimized maleimide formation using N-methoxycarbonylmaleimide. Typically, 4.8-6.7 GBq (130-180 mCi) of radiochemically pure [(18)F]FPyME ([(18)F]-1) could be obtained after semipreparative HPLC in 110 min starting from a cyclotron production batch of 33.3 GBq (900 mCi) of [(18)F]fluoride (overall radiochemical yields, based on starting [(18)F]fluoride: 28-37% decay-corrected). [(18)F]FPyME ([(18)F]-1) was first conjugated with a small model hexapeptide ((N-Ac)KAAAAC), confirming the excellent chemoselectivity of the coupling reaction (CH(2)SH versus CH(2)NH(2)) and then conjugated with two 8-kDa proteins of interest, currently being developed as tumor imaging agents (c-AFIM-0 and c-STxB). Conjugation was achieved in high yields (60-70%, isolated and non-decay-corrected) and used optimized, short-time reaction conditions (a 1/9 (v/v) mixture of DMSO and 0.05 M aq Tris NaCl buffer (pH 7.4) or 0.1 M aq PBS (pH 8), at room temperature for 10 min) and purification conditions (a gel filtration using a Sephadex NAP-10 cartridge or a SuperDex Peptide HR 10/30 column), both compatible with the chemical stability of the proteins and the relatively short half-life of the radioisotope concerned. The whole radiosynthetic procedure, including the preparation of the fluorine-18-labeled reagent, the conjugation with the protein and the final purification took 130-140 min. [(18)F]FPyME ([(18)F]-1) represents a new, valuable, thiol-selective, fluorine-18-labeled reagent for the prosthetic labeling with fluorine-18 of peptides and proteins. Because of its excellent chemoselectivity, [(18)F]FPyME offers an interesting alternative to the use of the nonselective carboxylate and amine-reactive [(18)F]reagents and can therefore advantageously be used for the design and development of new peptide- and protein-based radiopharmaceuticals for PET.     

Synthesis and evaluation of novel F-18 labeled 4-aminoquinazoline derivatives: potential PET imaging agents for tumor detection

Three novel (18)F-labeled 4-aminoquinazoline derivatives, N-(3-chloro-4-fluorophenyl)-6-(2-[(18)F]fluoroethoxy)-7-methoxyquinazolin-4-amine([(18)F]1), N-(3-ethynylphenyl)-6-(2-[(18)F]fluoroethoxy)-7-methoxyquinazolin-4-amine([(18)F]2), and N-(3-bromophenyl)-6-(2-[(18)F]fluoroethoxy)-7-methoxyquinazolin-4-amine([(18)F]3) were synthesized and radiolabeled by two-step reaction with overall radiochemical yield of 21-24% (without decay corrected). Then we carried out their biodistribution experiments in S180 tumor-bearing mice. Results showed that they had certain concentration accumulation in tumor and fast clearance from muscle and blood. It was encouraging that [(18)F]3 was competitive among three (18)F-labeled 4-aminoquinazoline derivatives in some aspects such as tumor/muscle uptake ratio reaching 7.70 at 60 min post-injection, tumor/blood uptake ratio reaching 6.61 at 120 min post-injection. So we compared radioactivity characteristics of [(18)F]3 with those of [(18)F]-FDG and L-[(18)F]-FET in the same animal model. The absolute radioactivity uptake of [(18)F]3 in tumor reached 3.31 at 60 min p.i., which was slightly higher than [(18)F]-FDG (2.16) and L-[(18)F]-FET (2.75) at the same time phase. For [(18)F]3, tumor/muscle uptake ratio peaked 7.70 at 60 min, which was obviously superior to those of [(18)F]-FDG and L-[(18)F]-FET at all time points. The tumor/brain uptake ratios of [(18)F]3 were 10.36, 17.42, 41.11 at 30 min, 60 min and 120 min post-injection, respectively, and are much higher than those of L-[(18)F] FET (2.54, 2.92 and 2.95) and [(18)F]-FDG (0.61, 1.02 and 1.33) at the same time points. All these results indicate that [(18)F]3 is promising to become a potential PET tumor imaging agent.     

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.     

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.     

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.     

Oxalic acid supported Si-18F-radiofluorination: one-step radiosynthesis of N-succinimidyl 3-(di-tert-butyl[18F]fluorosilyl)benzoate ([18F]SiFB) for protein labeling

N-Succinimidyl 3-(di-tert-butyl[(18)F]fluorosilyl)benzoate ([(18)F]SiFB), a novel synthon for one-step labeling of proteins, was synthesized via a simple (18)F-(19)F isotopic exchange. A new labeling technique that circumvents the cleavage of the highly reactive active ester moiety under regular basic (18)F-labeling conditions was established. In order to synthesize high radioactivity amounts of [(18)F]SiFB, it was crucial to partially neutralize the potassium oxalate/hydroxide that was used to elute (18)F(-) from the QMA cartridge with oxalic acid to prevent decomposition of the active ester moiety. Purification of [(18)F]SiFB was performed by simple solid-phase extraction, which avoided time-consuming HPLC and yielded high specific activities of at least 525 Ci/mmol and radiochemical yields of 40-56%. In addition to conventional azeotropic drying of (18)F(-) in the presence of [K(+)?2.2.2.]C(2)O(4), a strong anion-exchange (SAX) cartridge was used to prepare anhydrous (18)F(-) for nucleophilic radio-fluorination omitting the vacuum assisted drying of (18)F(-). Using a lyophilized mixture of [K(+)?2.2.2.]OH resolubilized in acetonitrile, the (18)F(-) was eluted from the SAX cartridge and used directly for the [(18)F]SiFB synthesis. [(18)F]SiFB was applied to the labeling of various proteins in likeness to the most commonly used labeling synthon in protein labeling, N-succinimidyl-4-[(18)F]fluorobenzoate ([(18)F]SFB). Rat serum albumin (RSA), apo-transferrin, a β-cell-specific single chain antibody, and erythropoietin were successfully labeled with [(18)F]SiFB in good radiochemical yields between 19% and 36%. [(18)F]SiFB- and [(18)F]SFB-derivatized RSA were directly compared as blood pool imaging agents in healthy rats using small animal positron emission tomography. Both compounds demonstrated identical biodistributions in healthy rats, accurately visualizing the blood pool with PET.     

Synthesis and evaluation of N-(5-fluoro-2-phenoxyphenyl)-N-(2-[(18)F]fluoromethoxy-d(2)-5-methoxybenzyl)acetamide: a deuterium-substituted radioligand for peripheral benzodiazepine receptor

N-(5-Fluoro-2-phenoxyphenyl)-N-(2-[(18)F]fluoromethoxy-d(2)-5-methoxybenzyl)acetamide ([(18)F]2) is a potent ligand (IC(50): 1.71 nM) for peripheral benzodiazepine receptor (PBR). However, in vivo evaluation on rodents and primates showed that this ligand was unstable and rapidly metabolized to [(18)F]F(-) by defluorination of the [(18)F]fluoromethyl moiety. In this study, we designed a deuterium-substituted analogue, N-(5-fluoro-2-phenoxyphenyl)-N-(2-[(18)F]fluoromethoxy-d(2)-5-methoxybenzyl)acetamide ([(18)F]5) as a radioligand for PBR to reduce the in vivo metabolic rate of the non-deuterated [(18)F]2. The design principle was based on the hypothesis that the deuterium substitution may reduce the rate of defluorination initiated by cleavage of the C-H bond without altering the binding affinity for PBR. The non-radioactive 5 was prepared by reacting diiodomethane-d(2) (CD(2)I(2), 6) with a phenol precursor 7, followed by treatment with tetrabutylammonium fluoride. The ligand [(18)F]5 was synthesized by the alkylation of 7 with [(18)F]fluoromethyl iodide-d(2) ([(18)F]FCD(2)I, [(18)F]9). Compound 5 displayed a similar in vitro affinity to PBR (IC(50): 1.90 nM) with 2. In vivo evaluation demonstrated that [(18)F]5 was metabolized by defluorination to [(18)F]F(-) as a main radioactive component, but its metabolic rate was slower than that of [(18)F]2 in the brain of mice. The deuterium substitution decreased the radioactivity level of [(18)F]5 in the bone of mouse, augmented by the percentage of specific binding to PBR in the rat brain determined by ex vivo autoradiography. However, the PET image of [(18)F]5 for monkey brain showed high radioactivity in the brain and skull, suggesting a possible species difference between rodents and primates.     

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.     

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 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.     

Syntheses of F-18 labeled fluoroalkyltyrosine derivatives and their biological evaluation in rat bearing 9L tumor

We hereby report the synthesis of four fluorine-18 labeled tyrosine derivatives, 3-(2-[(18)F]fluoroethyl)tyrosine ([(18)F]1, [(18)F]ortho-FET), 3-(3-[(18)F]fluoropropyl)tyrosine ([(18)F]2, [(18)F]ortho-FPT) O-methyl-[3-(2-[(18)F]fluoroethyl)]tyrosine ([(18)F]3, [(18)F]MFET), and O-methyl-[3-(3-[(18)F]fluoropropyl)]tyrosine ([(18)F]4, [(18)F]MFPT). The fluorine-18 labeled tyrosine derivatives were prepared by the displacement reaction of the ethyl and propyl tosylates with K[(18)F]/K2.2.2 in acetonitrile under no-carrier-added (NCA) conditions, followed by hydrolysis with 4N HCl. The biological properties of labeled compounds were evaluated in rats bearing 9L tumor after an intravenous injection and PET image was obtained. The tumor/blood and tumor/brain ratios were 2.06, 2.92 for [(18)F]1, 2.25, 4.05 for [(18)F]2, 2.88, 1.90 for [(18)F]3, and 2.00, 2.60 for [(18)F]4 at 60 min post injection, respectively. The PET image showed localized accumulation of PET tracers in 9L glioma of the rat.     

Synthesis, biological evaluation and radiochemical labeling of a dansylhydrazone derivative as a potential imaging agent for apoptosis

To develop a small molecule-based tracer for in vivo apoptosis imaging, dansylhydrazone (DFNSH) was synthesized in 93% yield in less than 30 min. The biological evaluation showed that DFNSH selectively binds to paclitaxel-induced apoptotic cancer cells. The high magnification fluorescent images demonstrate that DFNSH is localized within the cytoplasm of cells that bound Alexa 488 labeled annexin V on the plasma membrane. [(18)F]-DFNSH ([(18)F]-3) was synthesized and isolated in 50-60% radiochemical yields, based on [K/K(222)](18)F, with a synthesis time of 50 min (EOB). The straightforward preparation of fluorine-18 labeled 3 makes it a promising tracer for PET imaging of apoptosis.     

Synthesis and evaluation of [18F]Fluorobutyl ethacrynic amide: a potential PET tracer for studying glutathione transferase

[(18)F]Flurobutyl ethacrynic amide ([(18)F]FBuEA) was prepared from the precursor tosylate N-Boc-N-[4-(toluenesulfonyloxy)butyl]ethacrynic amide with a radiochemical yield of 3%, a specific activity of 48 GBq/μmol and radiochemical purity of 98%. Chemical conjugation of [(18)F]FBuEA with glutathione (GSH) via a self-coupling reaction and enzymatic conjugation under catalysis of glutathiontransferase alpha (GST-α) and π provided about 41% yields of radiochemical conjugated product [(18)F]FBuEA-GSH, 85% and 5-16%, respectively. The catalytic selectivity of this tracer toward GST-alpha was addressed. Positron emission tomography (PET) imaging of [(18)F]FBuEA in normal rats showed that a homogeneous pattern of radioactivity was distributed in the liver, suggesting a catalytic role of GST. By contrast, PET images of [(18)F]FBuEA in rats with thioacetamide-induced cholangiocarcinoma displayed a heterogeneous pattern of radioactive accumulation with cold spots in tumor lesions. PET imaging with [(18)F]FBuEA could be used for early diagnosis of hepatic tumor with a low GST activity as well as liver function.     

Tissue distribution, autoradiography, and metabolism of 4-(2'-methoxyphenyl)-1-[2' -[N-2"-pyridinyl)-p-[(18)F]fluorobenzamido]ethyl]piperazine (p-[(18)F]MPPF), a new serotonin 5-HT(1A) antagonist for positron emission tomography: An In vivo study in rats

The in vivo behavior of 4-(2'-methoxyphenyl)-1-[2'-[N-(2"-pyridinyl)-p-[(18)F]fluorobenzamido ]ethyl]-piperazine (p-[(18)F]MPPF), a new serotonin 5-HT(1A) antagonist, was studied in awake, freely moving rats. Biodistribution studies showed that the carbon-fluorine bond was stable in vivo, that this compound was able to cross the blood-brain barrier, and that a general diffusion equilibrium could account for the availability of the tracer. The great quantity of highly polar metabolites found in plasma did not contribute to the small amounts of metabolites found in hippocampus, frontal cortex, and cerebellum. Exvivo p-[(18)F]MPPF and in vitro 8-hydroxy-2-(di-n-[(3)H]propylamino)tetralin autoradiography were compared both qualitatively and quantitatively. Qualitative evaluation proved that the same brain regions were labeled and that the p-[(18)F]MPPF labeling is (a) in total agreement with the known distribution of 5-HT(1A) receptors in rats and (b) characterized by very low nonspecific binding. Quantitative comparison demonstrated that the in vivo labeling pattern obtained with p-[(18)F]MPPF cannot be explained by differences in regional blood flow, capillary density, or permeability. The 5-HT(1A) specificity of p-[(18)F]MPPF and binding reversibility were confirmed in vivo with displacement experiments. Thus, this compound can be used to evaluate parameters characterizing 5-HT(1A) binding sites in the brain.     

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.     

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.     

Quantitative analysis and comparison study of [18F]AlF-NOTA-PRGD2, [18F]FPPRGD2 and [68Ga]Ga-NOTA-PRGD2 using a reference tissue model

With favorable pharmacokinetics and binding affinity for α(v)β(3) integrin, (18)F-labeled dimeric cyclic RGD peptide ([(18)F]FPPRGD2) has been intensively used as a PET imaging probe for lesion detection and therapy response monitoring. A recently introduced kit formulation method, which uses an (18)F-fluoride-aluminum complex labeled RGD tracer ([(18)F]AlF-NOTA-PRGD2), provides a strategy for simplifying the labeling procedure to facilitate clinical translation. Meanwhile, an easy-to-prepare (68)Ga-labeled NOTA-PRGD2 has also been reported to have promising properties for imaging integrin α(v)β(3). The purpose of this study is to quantitatively compare the pharmacokinetic parameters of [(18)F]FPPRGD2, [(18)F]AlF-NOTA-PRGD2, and [(68)Ga]Ga-NOTA-PRGD2. U87MG tumor-bearing mice underwent 60-min dynamic PET scans following the injection of three tracers. Kinetic parameters were calculated using Logan graphical analysis with reference tissue. Parametric maps were generated using voxel-level modeling. All three compounds showed high binding potential (Bp(ND)?=?k(3)/k(4)) in tumor voxels. [(18)F]AlF-NOTA-PRGD2 showed comparable Bp(ND) value (3.75±0.65) with those of [(18)F]FPPRGD2 (3.39±0.84) and [(68)Ga]Ga-NOTA-PRGD2 (3.09±0.21) (p>0.05). Little difference was found in volume of distribution (V(T)) among these three RGD tracers in tumor, liver and muscle. Parametric maps showed similar kinetic parameters for all three tracers. We also demonstrated that the impact of non-specific binding could be eliminated in the kinetic analysis. Consequently, kinetic parameter estimation showed more comparable results among groups than static image analysis. In conclusion, [(18)F]AlF-NOTA-PRGD2 and [(68)Ga]Ga-NOTA-PRGD2 have comparable pharmacokinetics and quantitative parameters compared to those of [(18)F]FPPRGD2. Despite the apparent difference in tumor uptake (%ID/g determined from static images) and clearance pattern, the actual specific binding component extrapolated from kinetic modeling appears to be comparable for all three dimeric RGD tracers.