Synthesis of a fluorine-18-labelled derivative of 6-nitroquipazine,as a radioligand for the in vivo serotonin transporter imaging with PET

Considerable efforts have been engaged in the design, synthesis and pharmacological characterization of radioligands for imaging the serotonin transporter, based on its implication in several neuropsychiatric diseases, such as depression, anxiety and schizophrenia. In the 5-halo-6-nitroquipazine series, the fluoro derivative has been designed for positron emission tomography (PET). The corresponding 5-iodo-, 5-bromo- and 5-chloro N-Boc-protected quipazines as labelling precursors, as well as 5-fluoro-6-nitroquipazine as a reference compound have been synthesized. 5-[(18)F]Fluoro-6-nitroquipazine has been radiolabelled with fluorine-18 (positron-emitting isotope, 109.8 min half-life) by nucleophilic aromatic substitution from the corresponding N-Boc protected 5-bromo- and 5-chloro-precursors using K[(18)F]F-K(222) complex in DMSO by conventional heating (145 degrees C, 2 min) or microwave activation (50 W, 30-45 s), followed by removal of the protective group with TFA. Typically, 15-25 mCi (5.5-9.2 GBq) of 5-[(18)F]fluoro-6-nitroquipazine (1-2 Ci/micromol or 37-72 GBq/micromol) could be obtained in 70-80 min starting from a 550-650 mCi (20.3-24.0 GBq) aliquot of a cyclotron [(18)F]F(-) production batch (2.7-3.8% non decay-corrected yield based on the starting [(18)F]fluoride). Ex vivo studies (biodistribution in rat), as well as PET imaging (in monkey) demonstrated that 5-[(18)F]fluoro-6-nitroquipazine ([(18)F]-1d) readily crossed the blood brain barrier and accumulated in the regions rich in 5-HT transporter (frontal- and posterial cortex, striata). However, the low accumulation of the tracer in the thalamus (rat and monkey) as well as the comparable displacement of the tracer observed with both citalopram, a -HT re-uptake inhibitor and maprotiline, a norepinephrine re-uptake inhibitor (rat), indicate that 5-[(18)F]fluoro-6-nitroquipazine ([(18)F]-1d) does not have the suggested potential for PET imaging of the serotin transporter (SERT).     

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.     

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.     

Synthesis and in vivo imaging properties of [11C]befloxatone: a novel highly potent positron emission tomography ligand for mono-amine oxidase-A

Befloxatone (1, (5R)-5-(methoxymethyl)-3-[4-[(3R)-4,4,4-trifluoro-3-hydroxybutoxy]phenyl]-2-oxazolidinone) is an oxazolidinone derivative belonging to a new generation of reversible and selective mono-amine oxidase-A (MAO-A) inhibitors. In vitro and ex vivo studies have demonstrated that befloxatone is a potent, reversible and competitive MAO-A inhibitor with potential antidepressant properties. Befloxatone (1) was labelled with carbon-11 (t(12): 20.4 min) using [(11)C]phosgene as reagent. Typically, starting from a 1.2 Ci (44.4 GBq) cyclotron-produced [(11)C]CH(4) batch, 150-300 mCi (5.55-11.10 GBq) of [(11)C]befloxatone ([(11)C]-1) with a radiochemical- and chemical purity of more than 99% were routinely obtained within 20 min of radiosynthesis (including HPLC purification) with specific radioactivities of 500-2000 mCi/micromol (18.5-74.0 GBq/micromol). The results obtained in vivo with carbon-11-labelled befloxatone not only confirm the biochemical and pharmacological profile of befloxatone found in rodent and in human tissues but also point out [(11)C]befloxatone as an excellent tool for the assessment of MAO-A binding sites using positron emission tomography, a high-resolution, sensitive, non-invasive and quantitative imaging technique.     

Synthesis of a [2-pyridinyl-18F]-labelled fluoro derivative of (-)-cytisine as a candidate radioligand for brain nicotinic alpha4beta2 receptor imaging with PET

In recent years, there has been considerable effort to design and synthesize radiotracers suitable for use in Positron Emission Tomography (PET) imaging of the alpha4beta2 neuronal nicotinic acetylcholine receptor (nAChR) subtype. A new fluoropyridinyl derivative of (-)-cytisine (1), namely (-)-9-(2-fluoropyridinyl)cytisine (3, K(i) values of 24 and 3462 nM for the alpha4beta2 and alpha7 nAChRs subtypes, respectively) has been synthesized in four chemical steps from (-)-cytisine and labelled with fluorine-18 (T(1/2): 119.8 min) using an efficient two-step radiochemical process [(a). nucleophilic heteroaromatic ortho-radiofluorination using the corresponding N-Boc-protected nitro-derivative, (b). TFA removal of the Boc protective group]. Typically, 20-45 mCi (0.74-1.67 GBq) of (-)-9-(2-[18F]fluoropyridinyl)cytisine ([18F]-3, 2-3 Ci/micromol or 74-111 GBq/micromol) were easily obtained in 70-75 min starting from a 100 mCi (3.7 GBq) aliquot of a cyclotron-produced [18F]fluoride production batch (20-45% non decay-corrected yield based on the starting [18F]fluoride). The in vivo pharmacological profile of (-)-9-(2-[18F]fluoropyridinyl)cytisine ([18F]-3) was evaluated in rats with biodistribution studies and brain radioactivity monitoring using intracerebral radiosensitive beta-microprobes. The observed in vivo distribution of the radiotracer in brain was rather uniform, and did not match with the known regional densities of nAChRs. It was also significantly different from that of the parent compound (-)-[3H]cytisine. Moreover, competition studies with (-)-nicotine (5 mg/kg, 5 min before the radiotracer injection) did not reduce brain uptake of the radiotracer. These experiments clearly indicate that (-)-9-(2-[18F]fluoropyridinyl)cytisine ([18F]-3) does not have the required properties for imaging nAChRs using PET.     

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 [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.     

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.     

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.     

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.     

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 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.     

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.     

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%.     

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.     

Synthesis and preliminary evaluation of [(18)F]-fluoro-(2S)-Exaprolol for imaging cerebral beta-adrenergic receptors with PET

Cerebral beta-adrenergic receptors (beta-ARs) are of interest in several disorders including Parkinson's disease, Alzheimer's disease and in particular major depressive disorder. Development of a positron emission tomography (PET) ligand for imaging beta-ARs would allow the quantification of these receptors in the living human brain so as to better understand both the pathophysiology of depression and how to improve treatment. Currently there are no radioligands suitable for this purpose. In an attempt to achieve this goal, we prepared [(18)F]-labeled (2S)-1-(1-fluoropropan-2-ylamino)-3-(2-cyclohexylphenoxy)propan-2-ol (fluoro-Exaprolol; (2S)-1). Radiolabeling with fluorine-18 was accomplished via preparation of a precursor containing a tosyl leaving group (10), and utilizes the 2-oxazolidinone group to simultaneously protect both the amine and hydroxy groups. The oxazolidinone was readily removed with lithium aluminum hydride following a nucleophilic [(18)F]-fluoride for tosyl displacement to prepare [(18)F]-(2S)-1 in 31% radiochemical yield (uncorrected for decay), with >98% radiochemical purity in <1h. The specific activity of the formulated product was 927 mCi/micromol and the log P (pH 7.4) was 2.97. Preliminary biological evaluations in conscious rats indicated that [(18)F]-(2S)-1 had good brain uptake for imaging (0.8-1.3% injected dose/gram (% ID/g) of wet tissue, 5 min post-injection of the radiotracer) with a slow washout (>0.5% ID/g at 60 min post-injection) in all brain regions. Pharmacological challenges indicate that the binding is largely non-specific, as administration of Propranolol, authentic (2S)-1, or WAY 100635 prior to injection of [(18)F]-(2S)-1 did not block uptake of the radiotracer. These results indicate that [(18)F]-(2S)-1 is not a suitable candidate for PET imaging of cerebral beta-ARs.     

WAY-100635 antagonist-induced plasticity of 5-HT receptors: regulatory differences between a stable cell line and an in vivo native system

We present evidence that the 5-hydroxytryptamine(1A) (5-HT(1A)) receptor antagonist, N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]-ethyl}-N-(2-pyridinyl)cyclohexanecarboxamide (WAY-100635), can induce receptor internalization in a human (h)5-HT(1A) receptor Chinese hamster ovary (CHO-K1) cell system. Exposure of h5-HT(1A) CHO cells to WAY-100635 decreased the cell-surface h5-HT(1A) receptor density in a way that was both time (24-72 h) and concentration (1-100 nm) dependent.[(3)H]WAY-100635 and [(3)H]8-hydroxy-dipropylaminotetralin ([(3)H]8-OH-DPAT) saturation analyses demonstrated a significant reduction (50-60%) in total h5-HT(1A) receptor number in the WAY-100635-treated (100 nm; 72 h) compared with control cells. In WAY-100635-treated cells, the 8-OH-DPAT-mediated inhibition of forskolin (FSK)-stimulated cAMP accumulation was right-shifted and the maximal inhibitory response of 8-OH-DPAT was impaired compared with control cells. Similar results were obtained for 8-OH-DPAT-mediated Ca(2+) mobilization after WAY-100635 treatment. h5-HT(1A) receptors labeled with [(3)H]WAY-100635, as well as [(3)H]4-(2'-Methoxy)-phenyl-1-[2'-(N-2'-pyridinyl)-p-fluorobenzamido]ethyl-piperazine (MPPF), exhibited a time-dependent rate of cellular internalization that was blocked by endocytotic suppressors and was pertussis-toxin insensitive. In contrast, quantitative autoradiographic studies demonstrated that chronic treatment of rats with WAY-100635 for two weeks produced a region-specific increase in the 5-HT(1A) receptor density. In conclusion, prolonged exposure of an h5-HT(1A) cell-based system to the 5-HT(1A) antagonist, WAY-100635, induced a paradoxical internalization of cell surface receptor resulting in depressed functional activity. This suggests that an antagonist can influence 5-HT(1A) receptor recycling in vitro differently to in vivo regulatory conditions.     

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.     

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.     

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.