Radiosynthesis and biodistribution of cyclic RGD peptides conjugated with novel [18F]fluorinated aldehyde-containing prosthetic groups

Achieving high-yielding, robust, and reproducible chemistry is a prerequisite for the (18)F-labeling of peptides for quantitative receptor imaging using positron emission tomography (PET). In this study, we extend the toolbox of oxime chemistry to include the novel prosthetic groups [(18)F]-(2-{2-[2-(2-fluoroethoxy)ethoxy]ethoxy}ethoxy)acetaldehyde, [(18)F]5, and [(18)F]-4-(3-fluoropropoxy)benzaldehyde, [(18)F]9, in addition to the widely used 4-[(18)F]fluorobenzaldehyde, [(18)F]12. The three (18)F-aldehydes were conjugated to the same aminooxy-bearing RGD peptide and the effect of the prosthetic group on biodistribution and tumor uptake studied in mice. The peptide conjugate [(18)F]7 was found to possess superior in vivo pharmacokinetics with higher tumor to blood, tumor to liver, tumor to muscle, and tumor to lung ratios than either [(18)F]10 or [(18)F]13. The radioactivity from the [(18)F]7 conjugate excreted more extensively through the kidney route with 79%id passing through the urine and bladder at the 2 h time point compared to around 55%id for the more hydrophobic conjugates [(18)F]10 and [(18)F]13. The chemical nature of a prosthetic group can be employed to tailor the overall biodistribution profile of the radiotracer. In this example, the hydrophilic nature of the ethylene glycol containing prosthetic group [(18)F]5 clearly influences the overall excretion pattern for the RGD peptide conjugate.     

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.     

New F-18 prosthetic group via oxime coupling

A novel fluorine-18 prosthetic ligand, 5-(1,3-dioxolan-2-yl)-2-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)pyridine [(18)F]2, has been synthesized. The prosthetic ligand is formed in high radiochemical yield (rcy = 71 ± 2%, n = 3) with excellent radiochemical purity (rcp = 99 ± 1%, n = 3) in a short reaction time (10 min). [(18)F]2 is a small, neutral, organic complex, easily synthesized in four steps from a readily available starting material. It can be anchored onto a target molecule containing an aminooxy functional group under acidic conditions by way of an oxime bond. We report herein two examples [(18)F]23 and [(18)F]24, potential imaging agents for β-amyloid plaques, which were labeled with this prosthetic group. This approach could be used for labeling proteins and peptides containing an aminooxy group. Biodistribution in male ICR mice for both oxime labeled complexes [(18)F]23 and [(18)F]24 were compared to that of the known β-amyloid plaque indicator, [(18)F]-AV-45, florbetapir 1. Oximes [(18)F]23 and [(18)F]24 are larger in size and therefore should reduce the blood-brain barrier (BBB) penetration. The brain uptake for oxime [(18)F]23 appeared to be reduced, but still retained some capability to cross the BBB. Oxime [(18)F]24 showed promising results after 2 min post injection (0.48% dose/gram); however, the uptake increased after 30 min post injection (0.92% dose/gram) suggesting an in vivo decomposition/metabolism of compound [(18)F]24. We have demonstrated a general protocol for the fluoride-18 labeling with a new prosthetic ligand [(18)F]2 that is tolerant toward several functional groups and is formed via chemoselective oxime coupling.     

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.     

Systematic comparison of two novel,thiol-reactive prosthetic groups for <Superscript>18</Superscript>F labeling of peptides and proteins with the acylation agent succinimidyl-4-[<Superscript>18</Superscript>F]fluorobenzoate ([<Superscript>18</Superscript>F]SFB)

A systematic comparison of 4-[¹⁸F]fluorobenzaldehyde-O-(2-{2-[2-(pyrrol-2,5-dione-1-yl)ethoxy]-ethoxy}-ethyl)oxime ([¹⁸F]FBOM) and 4-[¹⁸F]fluorobenzaldehyde-O-[6-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-hexyl]oxime ([¹⁸F]FBAM) as prosthetic groups for the mild and efficient ¹⁸F labeling of cysteine-containing peptides and proteins with the amine-group reactive acylation agent, succinimidyl-4-[¹⁸F]fluorobenzoate ([¹⁸F]SFB), is described. All three prosthetic groups were prepared in a remotely controlled synthesis module. Synthesis of [¹⁸F]FBOM and [¹⁸F]FBAM was accomplished via oxime formation through reaction of appropriate aminooxy-functionalized labeling precursors with 4-[¹⁸F]fluorobenzaldehyde. The obtained radiochemical yields were 19% ([¹⁸F]FBOM) and 29% ([¹⁸F]FBAM), respectively. Radiolabeling involving [¹⁸F]FBAM and [¹⁸F]FBOM was exemplified by the reaction with cysteine-containing tripeptide glutathione (GSH), a cysteine-containing dimeric neurotensin derivative, and human native low-density lipoprotein (nLDL) as model compounds. Radiolabeling with the acylation agent [¹⁸F]SFB was carried out using a dimeric neurotensin derivative and nLDL. Both thiol-group reactive prosthetic groups show significantly better labeling efficiencies for the peptides in comparison with the acylation agent [¹⁸F]SFB. The obtained results demonstrate that [¹⁸F]FBOM is especially suited for the labeling of hydrophilic cysteine-containing peptides, whereas [¹⁸F]FBAM shows superior labeling performance for higher molecular weight compounds as exemplified for nLDL apolipoprotein constituents. However, the acylation agent [¹⁸F]SFB is the preferred prosthetic group for labeling nLDL under physiological conditions.     

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.     

A novel prosthetic group for site-selective labeling of peptides for positron emission tomography

Efficient methodologies for the radiolabeling of peptides with [(18)F]fluoride are a prerequisite to enabling commercialization of peptide-containing radiotracers for positron emission tomography (PET) imaging. It was the purpose of this study to investigate a novel chemoselective ligation reaction comprising conjugation of an [(18)F]-N-methylaminooxy-containing prosthetic group to a functionalized peptide. Twelve derivatives of general formula R1-CO-NH-Lys-Gly-Phe-Gly-Lys-OH were synthesized where R1 was selected from a short list of moieties anticipated to be reactive toward the N-methylaminooxy group. Conjugation reactions were initially carried out with nonradioactive precursors to assess, in a qualitative manner, their general suitability for PET chemistry with only the most promising pairings progressing to full radiochemical assessment. Best results were obtained for the ligation of O-[2-(2-[(18)F]fluoroethoxy)ethyl]-N-methyl-N-hydroxylamine 18 to the maleimidopropionyl-Lys-Gly-Phe-Gly-Lys-OH precursor 10 in acetate buffer (pH 5) after 1 h at 70 degrees C. The non-decay-corrected isolated yield was calculated to be 8.5%. The most encouraging result was observed with the combination 18 and 4-(2-nitrovinyl)benzoyl-Lys-Gly-Phe-Gly-Lys-OH, 9, where the conjugation reaction proceeded rapidly to completion at 30 degrees C after only 5 min. The corresponding non-decay-corrected radiochemical yield for the isolated (18)F-labeled product 27 was 12%. The preliminary results from this study demonstrate the considerable potential of this novel strategy for the radiolabeling of peptides.     

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.     

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.     

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.     

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.     

2-Fluoropyridine prosthetic compounds for the 18F labeling of bombesin analogues

Acetylene-bearing 2-[¹⁸F]fluoropyridines [¹⁸F]FPy5yne and PEG-[¹⁸F]FPyKYNE were prepared via efficient nucleophilic heteroaromatic [¹⁸F]fluorination of their corresponding 2-trimethylammoniumpyrdinyl precursors. The prosthetic groups were conjugated to azide- and PEG₃-modified bombesin(6–14) analogues via copper-catalyzed azide–alkyne cycloaddition couplings to yield mono- and di-mini-PEGylated ligands for PET imaging of the gastrin- releasing peptide receptor. The PEG₃- and PEG₂/PEG₃-bearing ¹⁸F peptides showed decreased lipophilicity relative to an analogous non-mini-PEGylated ¹⁸F peptide. Assessment of water-soluble peptide pharmacokinetics and tumour-targeting capabilities in a mouse model of prostate cancer is currently underway.     

Synthesis of a fluorine-18 labeled derivative of epibatidine for in vivo nicotinic acetylcholine receptor PET imaging

Epibatidine (exo-2-(2'-chloro-5'-pyridyl)-7-azabicyclo[2.2.1]heptane), a natural compound isolated from the skin of the Ecuadorian poison frog Epipedobates tricolor, is the most potent nicotinic acetylcholine receptor (nAChR) agonist reported to date. In order to visualize and quantify in vivo these receptors in human brain using Positron Emission Tomography (PET), [18F]norchlorofluoroepibatidine (exo-2-(2'-[18F]fluoro-5'-pyridyl)-7-azabicyclo[2.2.1]heptane), a fluorine-18 (t(1/2): 110 min) radiolabeled derivative of epibatidine has been designed. The corresponding 2'-bromo-, 2'-iodo- and 2'-nitro exo-2-(5'-pyridyl)-7-azabicyclo[2.2.1]heptane analogues as labeling precursors, as well as norchlorofluoroepibatidine as a reference compound have been synthesized by reductive, stereoselective, palladium-catalyzed Heck-type coupling between an N-Boc protected azanorbornene and the corresponding halopyridine. [18F]Norchlorofluoroepibatidine has been radiolabeled with fluorine-18 by nucleophilic aromatic substitution from the corresponding Boc-protected halo- and nitro precursors using [18F]FK-K222 complex in DMSO by conventional heating (at 150-180 degrees C for 10 min) or microwave activations (at 100 Watt, for 1 to 2.5 min), followed by TFA-removal of the protective group. Typically, using the microwave activation procedure, 60-80 mCi (2.22-2.96 GBq) of pure [18F]norchlorofluoroepibatidine could be obtained in less than 2 h (110-115 min) from the bromo labeling precursor, with specific radioactivities of 1.5-2.5 Ci/micromol (55.5-92.5 GBq/micromol) calculated for End of Bombardment. The preliminary PET experiments in baboon (Papio papio) with [18F]norchlorofluoroepibatidine show a high uptake and a rapid accumulation of the radiotracer into the brain within 30 min. In the thalamus, a nAChR rich area, uptake of radioactivity reached a maximum at 40 min (10% I.D./100 mL tissue). The ratio of radioactivity thalamus/cerebellum (the latter being a nAChR poor area) was 2 at 40 min and increased with time, up to 4.3 at 160 min. Its specific regiodistribution and its high ratio of specific-to-nonspecific binding confirm the ideal profile of [18F]norchlorofluoroepibatidine as a suitable radioligand for PET imaging of nAChRs in the brain.     

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.     

Radiosynthesis of the tumor hypoxia marker [18F]TFMISO via O-[18F]trifluoroethylation reveals a striking difference between trifluoroethyl tosylate and iodide in regiochemical reactivity toward oxygen nucleophiles

The MRI hypoxia marker trifluoromisonidazole (TFMISO) [1-(2-nitro-1H-imidazol-1-yl)-3-(2,2,2-trifluoroethoxy)propan-2-ol] was successfully labeled with (18)F to expand its role into a bimodal PET/MRI probe. (18)F-Labeling was achieved via a three-step procedure in which 2,2,2-[(18)F]trifluoroethyl p-toluenesulfonate prepared by (18)F-(19)F exchange served as the [(18)F]trifluoroethylating agent. The O-[(18)F]trifluoroethylation reaction proceeded efficiently to give the intermediate 1,2-epoxy-3-(2,2,2-[(18)F]trifluoroethoxy)propane, with approximately 60% of (18)F incorporated from the tosylate precursor, which was condensed with 2-nitroimidazole to yield [(18)F]TFMISO. Approximately 40% of the [(18)F]trifluoroethyl tosylate precursor was converted into the final product. In stark contrast, 2,2,2-[(18)F]trifluoroethyl iodide failed to produce [(18)F]TFMISO, giving instead 1,1-[(18)F]difluoro-2-iodoethoxy and 1-[(18)F]fluoro-2-iodovinyloxy analogs of [(18)F]TFMISO. Thus, this investigation has identified 2,2,2-[(18)F]trifluoroethyl tosylate as an excellent [(18)F]trifluoroethylating agent, which can convert efficiently an alcohol into the corresponding [(18)F]trifluoroethyl ether.     

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.     

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.     

Novel radiosynthesis of PET HSV-tk gene reporter probes [18F]FHPG and [18F]FHBG employing dual Sep-Pak SPE techniques

Positron emission tomography (PET) herpes simplex virus thymidine kinase (HSV-tk) gene reporter probes 9-[(3-[(18)F]fluoro-1-hydroxy-2-propoxy)methyl]guanine ([(18)F]FHPG) and 9-(4-[(18)F]fluoro-3-hydroxymethylbutyl)guanine ([(18)F]FHBG) were prepared by nucleophilic substitution of the appropriate tosylated precursors with [(18)F]KF/Kryptofix 2.2.2 followed by a quick deprotection reaction and purification with a simplified dual Silica Sep-Pak solid-phase extraction (SPE) method in 15-30% radiochemical yield.     

3,4,6-Tri-O-acetyl-2-deoxy-2-[18F]fluoroglucopyranosyl phenylthiosulfonate: a thiol-reactive agent for the chemoselective 18F-glycosylation of peptides

3,4,5-Tri-O-acetyl-2-[18F]fluoro-2-deoxy-d-glucopyranosyl 1-phenylthiosulfonate (Ac3-[18F]FGlc-PTS) was developed as a thiol-reactive labeling reagent for the site-specific 18F-glycosylation of peptides. Taking advantage of highly accessible 1,3,4,6-tetra-O-acetyl-2-deoxy-2-[18F]fluoroglucopyranose, a three-step radiochemical pathway was investigated and optimized, providing Ac3-[18F]FGlc-PTS in a radiochemical yield of about 33% in 90 min (decay-corrected and based on starting [18F]fluoride). Ac3-[18F]FGlc-PTS was reacted with the model pentapeptide CAKAY, confirming chemoselectivity and excellent conjugation yields of >90% under mild reaction conditions. The optimized method was adopted to the 18F-glycosylation of the alphavbeta3-affine peptide c(RGDfC), achieving high conjugation yields (95%, decay-corrected). The alphavbeta3 binding affinity of the glycosylated c(RGDfC) remained uninfluenced as determined by competition binding studies versus 125I-echistatin using both isolated alphavbeta3 and human umbilical vein endothelial cells (Ki = 68 +/- 10 nM (alphavbeta3) versus Ki = 77 +/- 4 nM (HUVEC)). The whole radiosynthetic procedure, including the preparation of the 18F-glycosylating reagent Ac3-[18F]FGlc-PTS, peptide ligation, and final HPLC purification, provided a decay-uncorrected radiochemical yield of 13% after a total synthesis time of 130 min. Ac3-[18F]FGlc-PTS represents a novel 18F-labeling reagent for the mild chemoselective 18F-glycosylation of peptides indicating its potential for the design and development of 18F-labeled bioactive S-glycopeptides suitable to study their pharmacokinetics in vivo by positron emission tomography (PET).