PET imaging of the brain serotonin transporters (SERT) with N,N-dimethyl-2-(2-amino-4-[18F]fluorophenylthio)benzylamine (4-[18F]-ADAM) in humans: a preliminary study

Purpose

The aim of this study was to assess the feasibility of using 4-[¹⁸F]-ADAM as a brain SERT imaging agent in humans.

Methods

Enrolled in the study were 19 healthy Taiwanese subjects (11 men, 8 women; age 33?±?9?years). The PET data were semiquantitatively analyzed and expressed as specific uptake ratios (SUR) and distribution volume ratios (DVR) using the software package PMOD. The SUR and DVR of 4-[¹⁸F]-ADAM in the raphe nucleus (RN), midbrain (MB), thalamus (TH), striatum (STR) and prefrontal cortex (PFC) were determined using the cerebellum (CB) as the reference region.

Results

4-[¹⁸F]-ADAM bound to known SERT-rich regions in human brain. The order of the regional brain uptake was MB (RN) > TH > STR > PFC > CB. The DVR (n?=?4, t*?=?60?min) in the RN, TH, STR and PFC were 3.00?±?0.50, 2.25?±?0.45, 2.05?±?0.31 and 1.40?±?0.13, respectively. The optimal time for imaging brain SERT with 4-[¹⁸F]-ADAM was 120?C140?min after injection. At the optimal imaging time, the SURs (n?=?15) in the MB, TH, STR, and PFC were 2.25?±?0.20, 2.28?±?0.20, 2.12?±?0.18 and 1.47?±?0.14, respectively. There were no significant differences in SERT availability between men and women (p?<?0.05).

Conclusion

The results of this study showed that 4-[¹⁸F]-ADAM was safe for human studies and its distribution in human brain appeared to correlate well with the known distribution of SERT in the human brain. In addition, it had high specific binding and a reasonable optimal time for imaging brain SERT in humans. Thus, 4-[¹⁸F]-ADAM may be feasible for assessing the status of brain SERT in humans.     

Synthesis of N,N-dimethyl-2-(2-amino-4-[18F]fluorophenylthio)benzylamine as a serotonin transporter imaging agent.

Two 403U76 analogs, N,N-dimethyl-2-(2-nitro-4-bromophenylthio)benzylamine (4) and N,N-dimethyl-2-(2,4-dinitrophenylthio)benzylamine (8) were prepared in multi-steps synthesis as precursors for the synthesis of a new serotonin transporter imaging agent, N,N-dimethyl-2-(2-amino-4-[18F]fluorophenylthio)benzylamine (12a). Reaction of 2,5-dibromonitrobenzene (1) with 2-thio-N,N-dimethylbenzamide gave N N-dimethyl-2-(2-nitro-4-bromophenylthio)benzamide (3). N,N-Dimethyl-2-(2,4-dinitrophenylthio)benzamide (6) was synthesized similarly from the reaction of 2-bromo-1,5-dinitrobenzene (2) with 2-thio-N,N-dimethylbenzamide. Reduction of 3 and 6 with BH(3)/THF gave benzylamines 4 and 8 along with their amine boranes 5 and 7. Nucleophilic substitution of 4 or 8 with K[18F]/Kryptofix 2.2.2 in DMSO at 120 degrees C followed by reduction with NaBH(4)- Cu(OAc)(2) in EtOH at 78 degrees C and purification with HPLC gave compound 12a in approximately 5-10% yield in a synthesis time of 150min from EOB. A preliminary biodistribution study in rats showed that the uptake of compound 12a in rat brain was high (approximately 1%/g) and the ratio of the uptake of compound 12a in hypothalamus (a serotonin transporter-rich area) and cerebellum (a serotonin-transporter-devoid area) was 6/1 at 1h post-injection. These results suggest that compound 12a may be a potential new serotonin transporter PET imaging agent.     

Synthesis and evaluation of N,N-dimethyl-2-(2-amino-5-[F]fluorophenylthio)benzylamine (5-[F]-ADAM) as a serotonin transporter imaging agent

The synthesis and evaluation of a new serotonin transporter (SERT) imaging agent, N,N-dimethyl-2-(2-amino-5-[¹⁸F]fluorophenylthio)benzylamine (5-[¹⁸F]-ADAM) is reported. Nucleophilic substitution of N,N-dimethyl-2-(2-nitro-5-bromophenylthio)benzylamine with K[¹⁸F]/Kryptofix 2.2.2 in DMSO at 125°C followed by reduction with NaBH₄–Cu(OAc)₂ in EtOH at 78°C and purification with HPLC produces the desired compound with an unoptimized yield of 5–10% in a synthesis time of 150 min from EOB. The biodistribution of 5-[¹⁸F]-ADAM in rats showed a high initial uptake and relatively rapid clearance in the brain (3.221±0.762, 0.440±0.059, 0.160±0.035 and 0.028±0.003% injected dose/organ at 2, 30, 60 and 120 min after I.V. injection, respectively) with the specific binding peaking at 1 h postinjection (hypothalamus/cerebellum and hippocampus/cerebellum were 2.97 and 3.59, respectively). The initial uptake in blood, lung, kidney and heart were also high, but it cleared rapidly. The radioactivity in the femur increased with time for 5-[¹⁸F]-ADAM indicating that in vivo defluorination may occur. Metabolism studies in rats showed that 5-[¹⁸F]-ADAM was not metabolized in rat brain, but was metabolized rapidly in the blood. Blocking experiments showed that there were significant decreases in the uptake of 5-[¹⁸F]-ADAM in the brain regions (hypothalamus, hippocampus and striatum) where SERT concentrations are high when rats were pretreated with (+)McN 5652 (2 mg/kg, 5 min prior to IV injection of 5-[¹⁸F]-ADAM). These results suggest that 5-[¹⁸F]-ADAM may be a potential new serotonin transporter PET imaging agent. However, due to its rapid wash-out from the brain, defluorination in vivo and lower uptake in the brain than 4-[¹⁸F]-ADAM, 5-[¹⁸F]-ADAM may not be as useful as 4-[¹⁸F]-ADAM as a SERT imaging agent.     

An improved synthesis of 4-[18F]-ADAM,a potent serotonin transporter imaging agent

An improved synthesis of N,N-dimethyl-2-(2-amino-4-[¹⁸F]fluorophenylthio)benzylamine (4-[¹⁸F]-ADAM, 2) as a potent serotonin transporter (SERT) imaging agent is described. Molecular orbital (MO) calculation predicts that N,N-dimethyl-2-(2-nitro-4-trimethylammoniumtrifluoromethanesulfonylphenylthio)benzamide (8) is probably a better precursor than N,N-dimethyl-2-(2,4-dinitrophenylthio)benzylamine (1) for preparing 2. Radioligand 2 was synthesized by the reaction of either precursor 1 or precursor 8 with K[¹⁸F]/K_2.2.2 at 120 °C followed by reduction with BH₃ at 80 °C. The radiochemical yield (EOB) of 2 synthesized from precursor 1 and 8 was 5.7±2.4% (n=6) and 14.8±4.0% (n=5), respectively, in a synthesis time of 120 min from EOB. The specific activity of 2 was 3 Ci/μmol or 111 GBq/μmol (EOB). Thus, this new synthetic method has significantly improved the radiochemical yield of 4-[¹⁸F]-ADAM and makes this radioligand more accessible to PET Centers without a cyclotron.     

N,N-dimethyl-2-(2-amino-4-(18)F-fluorophenylthio)-benzylamine (4-(18)F-ADAM): an improved PET radioligand for serotonin transporters.

There has been considerable interest in the development of PET radioligands that are useful for imaging serotonin transporter (SERT) in the living human brain. For the last decade, (11)C-(+)McN5652 has been the most promising PET agent for studying SERT in humans. However, this agent has some limitations. Recently, a new promising SERT PET radioligand, 3-(11)C-amino-4-(2-dimethylaminomethylphenylsulfanyl)benzonitrile, has been reported. We recently reported the synthesis of a new (18)F-labeled SERT PET radioligand, N,N-dimethyl-2-(2-amino-4-(18)F-fluorophenylthio)benzylamine (4-(18)F-ADAM), which may have advantages over (11)C-labeled radioligands. The purpose of this study was to evaluate this newly developed (18)F-labeled PET radioligand as a promising agent for studying SERT in the living human brain. METHODS: This agent was evaluated by studying its in vitro binding to different monoamine transporters, its in vivo biodistributions in rats, its integrity and pharmacologic profiles in rat brain, and its distribution in a female baboon brain. RESULTS: In vitro binding assays showed that 4-F-ADAM displayed high affinity to SERT sites (inhibition constant = 0.081 nmol/L, using membrane preparations of LLC-PK1 cells expressing the specific transporter) and showed more than 1,000- and 28,000-fold selectivity for SERT over norepinephrine transporter and dopamine transporter, respectively. Biodistribution of 4-(18)F-ADAM in rats showed a high initial uptake and slow clearance in the brain (2.13%, 1.90%, and 0.95% injected dose per organ at 2, 30, and 60 min after intravenous injection, respectively), with the specific binding peaking at 2 h after injection (hypothalamus/cerebellum = 12.49). The uptake in blood, muscle, lung, kidney, and liver was also initially high but cleared rapidly. The radioactivity in the femur increases with time for 4-(18)F-ADAM, indicating that in vivo defluorination may occur. In vivo metabolism studies in rats showed that 4-(18)F-ADAM was not metabolized in rat brain (>96% of radioactivity was recovered as parent compound at 1 h after injection). However, it metabolized rapidly in the blood. Less than 7% of the radioactivity recovered from plasma was the parent compound, with the majority of radioactivity in the plasma not extractable by ethyl acetate. Blocking studies showed significant decreases in the uptake of 4-(18)F-ADAM in the brain regions (hypothalamus, hippocampus, and striatum) where SERT concentrations are high when rats were pretreated with (+)McN5652 (2 mg/kg 5 min before intravenous injection of 4-(18)F-ADAM). However, changes in the uptake of 4-(18)F-ADAM in these brain regions were less significant when rats were pretreated with either methylphenidate or nisoxetine. The baboon study showed that uptake of 4-(18)F-ADAM in the midbrain peaked at approximately 1 h after injection and then declined slowly. The ratios of the radioactivity in the midbrain to that in the cerebellum (where the concentration of SERT is low) at 2 and 3 h after injection were 3.2 and 4.2, respectively. CONCLUSION: 4-(18)F-ADAM is suitable as a PET radioligand for studying SERT in the living brain. Further characterization of this new radioligand in humans is warranted.     

A high yield robotic synthesis of 9-(4-[18F]-fluoro-3-hydroxymethylbutyl)guanine ([18F]FHBG) and 9-[3-[18F]fluoro-1-hydroxy-2-propoxy)methyl]guanine([18F] FHPG) for gene expression imaging.

The aim of this study was to develop an automated synthesis of 9-(4-[(18)F]-fluoro-3-hydroxymethylbutyl)guanine ([(18)F]FHBG) and 9-[(3-[(18)F]fluoro-1-hydroxy-2-propoxy)methyl]guanine ([(18)F]FHPG) using a Scanditronix Anatech RB III robotic system. [(18)F]HF was produced via (18)O(p, n)(18)F using a Scanditronix MC17F cyclotron. On average, a typical run produced [(18)F]FHBG and [(18)F]FHPG with an uncorrected radiochemical yield of 19% and 16%, respectively, at end of synthesis (EOS) from irradiation of 95% enriched [(18)O]water. The total synthesis time was 80 min. The retention time of [(18)F]FHBG and [(18)F]FHPG (the radio-peak) was 3.9 and 4.0 min, respectively, which was consistent with the [(19)F]FHBG and [(19)F]FHPG ultraviolet peak. The radiochemical purity was greater than 97%. A robotic, automated method for [(18)F]FHBG and [(18)F]FHPG radiosynthesis is therefore feasible. The radiation burden for the operator can be reduced as much as possible. Sufficient radioactivities of [(18)F]FHBG and [(18)F]FHPG could be obtained for non-invasive monitoring the expression of transfected gene in vivo with positron emission tomography (PET).     

2-(2'-((Dimethylamino)methyl)-4'-(3-[(18)F]fluoropropoxy)-phenylthio)benzenamine for positron emission tomography imaging of serotonin transporters

INTRODUCTION: A new (18)F ligand, 2-(2'-((dimethylamino)methyl)-4'-(3-[(18)F]fluoropropoxy)-phenylthio)benzenamine ([(18)F]1), for positron emission tomography (PET) imaging of serotonin transporters (SERT) was evaluated. METHODS: Binding affinity was determined through in vitro binding assays with LLC-PK1 cells overexpressing SERT, NET or DAT (LLC-SERT, LLC-NET and LLC-DAT) and with rat cortical homogenates. Localization and selectivity of [(18)F]1 binding in vivo were evaluated by biodistribution, autoradiography and A-PET imaging studies in rats. RESULTS: This compound displayed excellent binding affinity for SERT in vitro with K(i)=0.33 and 0.24 nM in LLC-SERT and rat cortical homogenates, respectively. Biodistribution studies with [(18)F]1 showed good brain uptake (1.61% dose/g at 2 min postinjection), high uptake into the hypothalamus (1.22% dose/g at 30 min) and a high target-to-nontarget (hypothalamus to cerebellum) ratio of 9.66 at 180 min postinjection. Pretreatment with a SERT selective inhibitor considerably inhibited [(18)F]1 binding in biodistribution studies. Ex vivo autoradiography reveals [(18)F]1 localization to brain regions with high SERT density, and this binding was blocked by pretreatment with SERT selective inhibitors. Small animal PET (A-PET) imaging in rats provided clear images of tracer localization in the thalamus, midbrain and striatum. In A-PET chasing experiments, injecting a SERT selective inhibitor 75 min post-tracer injection causes a dramatic reduction in regional radioactivity and the target-to-nontarget ratio. CONCLUSION: The results of the biological studies and the ease of radiosynthesis with moderately good radiochemical yield (RCY=10-35%) make [(18)F]1 an excellent candidate for SERT PET imaging.     

Synthesis and evaluation of 2-amino-5-(4-[18F]fluorophenyl)pent-4-ynoic acid ([18F]FPhPA): A novel 18F-labeled amino acid for oncologic PET imaging

Introduction¹⁸ F-labeled amino acids are important PET radiotracers for molecular imaging of cancer. This study describes synthesis and radiopharmacological evaluation of 2-amino-5-(4-[¹⁸ F]fluorophenyl)pent-4-ynoic acid ([¹⁸ F]FPhPA) as a novel amino acid radiotracer for oncologic imaging.Methods¹⁸ F]FPhPA was prepared using Pd-mediated Sonogashira cross-coupling reaction between 4-[¹⁸ F]fluoroiodobenzene ([¹⁸ F]FIB) and propargylglycine. The radiopharmacological profile of [¹⁸ F]FPhPA was evaluated in comparison with O-(2-[¹⁸ F]fluoroethyl)-L-tyrosine ([¹⁸ F]FET) using the murine breast cancer cell line EMT6 involving cellular uptake studies, radiotracer uptake competitive inhibition experiments and small animal PET imaging.Results¹⁸ F]FPhPA was prepared in 42 ± 10% decay-corrected radiochemical yield with high radiochemical purity >95% after semi-preparative HPLC purification. Cellular uptake of L-[¹⁸ F]FPhPA reached a maximum of 58 ± 14 % radioactivity/mg protein at 90 min. Lower uptake was observed for racemic and D-[¹⁸ F]FPhPA.Radiotracer uptake inhibition studies by synthetic and naturally occurring amino acids suggested that Na⁺-dependent system ASC, especially ASCT2, and Na⁺-independent system L are important amino acid transporters for [¹⁸ F]FPhPA uptake into EMT6 cells. Small animal PET studies demonstrated similar high tumor uptake of [¹⁸ F]FPhPA in EMT6 tumor-bearing mice compared to [¹⁸ F]FET reaching a maximum standardized uptake value (SUV) of 1.35 after 60 min p.i.. Muscle uptake of [¹⁸ F]FPhPA was higher (SUV_30min = 0.65) compared to [¹⁸ F]FET (SUV_30min = 0.40), whereas [¹⁸ F]FPhPA showed a more rapid uptake and clearance from the brain compared to [¹⁸ F]FET.ConclusionL-[¹⁸ F]FPhPA is the first ¹⁸ F-labeled amino acid prepared through Pd-mediated cross-coupling reaction.Advances in Knowledge and Implications for patient CareL-[¹⁸ F]FPhPA displayed promising properties as a novel amino acid radiotracer for molecular imaging of system ASC and system L amino acid transporters in cancer.     

Biodistribution, toxicity and radiation dosimetry studies of the serotonin transporter radioligand 4-[18F]-ADAM in rats and monkeys

Purpose  

4-[¹⁸F]-ADAM is a potent serotonin transport imaging agent. We studied its toxicity in rats and radiation dosimetry in monkeys before human studies are undertaken.     

4-[18F]fluoroarylalkylethers via an improved synthesis of n.c.a. 4-[18F]fluorophenol

This paper describes the improved synthesis of n.c.a. 4- 18F]fluorophenol for the preparation of 18F-labeled alkylarylethers. Nucleophilic fluorination of substituted benzophenone derivatives yielded n.c.a. 4- 18F]fluoro-4'-substituted benzophenones with 80- 90% RCY, which were converted to benzoic acid phenylesters by treatment with peracetic acid. Strong electron-withdrawing substituents like nitro, cyano and trifluoromethyl favor a fluorophenyl-to-oxygen migration resulting in the formation of corresponding benzoic acid fluorophenylesters. N.c.a. 18F]fluorophenol is almost quantitatively formed after hydrolysis and can easily be converted with alkylhalides into n.c.a. 18F]fluoroarylalkylethers.     

Fully automated synthesis of 4-[18F]fluorobenzylamine based on borohydride/NiCl2 reduction

Introduction4-[¹⁸F]Fluorobenzylamine ([¹⁸F]FBA) is an important building block for the synthesis of ¹⁸F-labeled compounds. Synthesis of [¹⁸F]FBA usually involves application of strong reducing agents like LiAlH₄ which is challenging to handle in automated synthesis units (ASUs). Therefore, alternative methods for the preparation of [¹⁸F]FBA compatible with remotely-controlled syntheses in ASUs are needed.Methods¹⁸F]FBA was prepared in a remotely-controlled synthesis unit (GE TRACERlab? FX) based on Ni(II)-mediated borohydride exchange resin (BER) reduction of 4-[¹⁸F]fluorobenzonitrile ([¹⁸F]FBN). [¹⁸F]FBA was used for the synthesis of novel thiol-reactive prosthetic group 4-[¹⁸F]fluorobenzyl)maleimide [¹⁸F]FBM and Hsp90 inhibitor 17-(4-[¹⁸F]fluorobenzylamino)-17-demethoxy-geldanamycin [¹⁸F] GA.Results[¹⁸F]FBA could be prepared in high radiochemical yield greater than 80% (decay-corrected) within 60 min. In a typical experiment, 7.4 GBq of [¹⁸F]FBA could be obtained in high radiochemical purity of greater than 95% starting from 10 GBq of cyclotron-produced n.c.a. [¹⁸F]fluoride. [¹⁸F]FBA was used for the preparation of 4-[¹⁸F]fluorobenzyl)maleimide as a novel prosthetic group for labeling of thiol groups as demonstrated with tripeptide glutathione. [¹⁸F]FBA was also used as building block for the syntheses of small molecules as exemplified by the preparation of Hsp90 inhibitor 17-(4-[¹⁸F]fluorobenzylamino)-17-demethoxy-geldanamycin.ConclusionThe described remotely-controlled synthesis of [¹⁸F]FBA will significantly improve the availability of [¹⁸F]FBA as an important and versatile building block for the development of novel ¹⁸F-labeled compounds containing a fluorobenzylamine moiety.     

A simplified one-pot synthesis of 9-[(3-[18F]fluoro-1-hydroxy-2-propoxy)methyl]guanine([18F]FHPG) and 9-(4-[18F]fluoro-3-hydroxymethylbutyl)guanine ([18F]FHBG) for gene therapy

9-[(3-[18F]Fluoro-1-hydroxy-2-propoxy)methyl]guanine ([18F]FHPG, 2) has been synthesized by nucleophilic substitution of N(2)-(p-anisyldiphenylmethyl)-9-[[1-(p-anisyldiphenylmethoxy)-3-toluenesulfonyloxy-2-propoxy]methyl]guanine (1) with potassium [18F]fluoride/Kryptofix 2.2.2 followed by deprotection with 1 N HCl and purification with different methods in variable yields. When both the nucleophilic substitution and deprotection were carried out at 90 degrees C and the product was purified by HPLC (method A), the yield of compound 2 was 5-10% and the synthesis time was 90 min from EOB. However, if both the nucleophilic substitution and deprotection were carried out at 120 degrees C and the product was purified by HPLC, the yield of compound 2 decreased to 2%. When compound 2 was synthesized at 90 degrees C and purified by Silica Sep-Pak (method B), the yield increased to 10-15% and the synthesis time was 60 min from EOB. Similarly, 9-(4-[18F]fluoro-3-hydroxymethylbutyl)guanine ([18F]FHBG, 4) was synthesized with method A and method B in 9% and 10-15% yield, respectively, in a synthesis time of 90 and 60 min, respectively, from EOB. Compound 2 was relatively unstable in acidic medium at 120 degrees C while compound 4 was stable under the same condition. Both compound 2 and compound 4 had low lipid/water partition coefficient (0.126 +/- 0.022, n=5 and 0.165 +/- 0.023, n=5, respectively). Although it contains non-radioactive ganciclovir ( approximately 5-30 microg) as a chemical by-product, compound 2 synthesized by method B has a similar uptake in 9L glioma cells as that synthesized by method A, and is a potential tracer for imaging herpes simplex virus thymidine kinase gene expression in tumors using PET. Similarly, compound 4 synthesized by method B contains approximately 10-25 microg of penciclovir as a chemical by-product. Thus, the simplified one pot synthesis (method B) is a useful method for synthesizing both compound 2 and compound 4 in good yield for routine clinical use, and the method is readily amenable for automation.     

Fully automated one-pot synthesis of [18F]fluoromisonidazole

A (18)F-labeled fluoromisonidazole (1H-1-(3-[(18)F]fluoro-2-hydroxypropyl)-2-nitroimidazole, [(18)F]FMISO) was prepared via a one-pot, two-step synthesis procedure using a modified commercial Tracerlab FX(F-N) synthesis module. Nucleophilic fluorination of the precursor molecule 1-(2'-nitro-1'-imidazolyl)-2-O-tetrahydropyranyl-3-O-toluenesulphonylpropanediol using no-carrier-added [(18)F]fluoride, followed by hydrolysis of the protecting group with 1 mol/L HCl and purification with Sep-Paks instead of HPLC, gave [(18)F]FMISO. The overall radiochemical yield with no decay correction was greater than 40%, the whole synthesis time was less than 40 min and the radiochemical purity was greater than 95%. The new automated synthesis procedure can be applied to the fully automated synthesis of [(18)F]FMISO using a commercial FDG synthesis module.     

Preparation and biological evaluation of 2-amino-6-[18F]fluoro-9-(4-hydroxy-3-hydroxy-methylbutyl) purine (6-[18F]FPCV) as a novel PET probe for imaging HSV1-tk reporter gene expression

INTRODUCTION: 2-Amino-6-[(18)F]fluoro-9-(4-hydroxy-3-hydroxy-methylbutyl) purine (6-[(18)F]FPCV) was prepared via a one-step nucleophilic substitution and evaluated as a novel probe for imaging the expression of herpes simplex virus type 1 thymidine kinase (HSV1-tk) reporter gene. METHODS: Log P of 6-[(18)F]FPCV was calculated in octanol/phosphate-buffered saline (PBS). Stability studies were performed in PBS and bovine serum albumin (BSA). Cell uptake was performed at various time points in wild-type cells and transduced cells. For in vivo studies, tumors were grown in nude mice by inoculation with C6 cells, wild type and tk positive. The radiotracer was intravenously injected to animals, and micro-PET imaging was performed. Biodistribution of 6-[(18)F]FPCV was performed on another group of animals at different time points. RESULTS: Log P of 6-[(18)F]FPCV was -0.517. 6-[(18)F]FPCV was fairly stable in PBS and BSA at 6 h. The tracer uptake in C6-tk cells was 5.5-18.8 times higher than that in wild-type cells. The plasma half-life of 6-[(18)F]FPCV was as follows: alpha t(1/2)=1.2 min and beta t(1/2)=73.7 min. The average ratio of tumor uptake between the transduced tumor and the wild-type tumor was 1.69 at 15 min. CONCLUSION: Biological evaluation showed that 6-[(18)F]FPCV is a potential probe for imaging HSV1-tk gene expression. However, its in vivo defluorination may limit its application in PET imaging of gene expression.     

Fully automated synthesis of [18F]fluoromisonidazole using a conventional [18F]FDG module

We developed a new fully automated method for the synthesis of [¹⁸F]fluoromisonidazole ([¹⁸F]FMISO) by modifying a commercial FDG synthesizer and its disposable fluid pathway. A three-step procedure was used to prepare the tosylate precursor, 1-(2′-nitro-1′-imidazolyl)-2-O-tetrahydrofuranyl-3-O-toluenesulfonylpropanediol. Using glycerol as the starting material, the precursor was synthesized with a yield of 21%. The optimal labeling conditions for the automated synthesis of [¹⁸F]FMISO was 10 mg of precursor in acetonitrile (2 ml heated at 105°C for 360 s, followed by heating at 75°C for 280 s and hydrolysis with 1 N HCl at 105°C for 300 s. Using 3.7 GBq of [¹⁸F]F⁻ as a starting activity, [¹⁸F]FMISO was obtained with high end-of-synthesis (EOS) radiochemical yields of 58.5±3.5% for 60.0±5.2 min with high-performance liquid chromatography (HPLC) purification. When solid-phase purification steps were added, the EOS radiochemical yields were 54.5±2.8% (337±25 GBq/μmol) for 70.0±3.8 min (n=10 for each group, decay-corrected). With a high starting radioactivity of 37.0 GBq, we obtained radiochemical yields of 54.4±2.9% and 52.8±4.2%, respectively (n=3). The solid-phase purification removed unreacted [¹⁸F]fluoride and polar impurities before the HPLC procedure. Long-term tests showed a good stability of 98.2±1.5%. This new automated synthesis procedure combines high and reproducible yields with the advantage of using a disposable cassette system.     

Improved synthesis of [18F]FLETT via a fully automated vacuum distillation method for [18F]2-fluoroethyl azide purification

The synthesis of [¹⁸F]2-fluoroethyl azide and its subsequent click reaction with 5-ethynyl-2′-deoxyuridine (EDU) to form [¹⁸F]FLETT was performed using an iPhase FlexLab module. The implementation of a vacuum distillation method afforded [¹⁸F]2-fluoroethyl azide in 87±5.3% radiochemical yield. The use of Cu(CH₃CN)₄PF₆ and TBTA as catalyst enabled us to fully automate the [¹⁸F]FLETT synthesis without the need for the operator to enter the radiation field. [¹⁸F]FLETT was produced in higher overall yield (41.3±6.5%) and shorter synthesis time (67 min) than with our previously reported manual method (32.5±2.5% in 130 min).     

An improved preparation of [18F]FPBM: A potential serotonin transporter (SERT) imaging agent

IntroductionIn vivo positron emission tomography (PET) imaging of the serotonin transporter (SERT) is a valuable tool in drug development and in monitoring brain diseases with altered serotonergic function. We have developed a two-step labeling reaction for the preparation of the high serotonin affinity ligand [¹⁸F]FPBM ([¹⁸F]2-(2′-((dimethylamino)methyl)-4′-(3-fluoropropoxy)phenylthio)benzenamine, 1).MethodTo improve and automate the radiolabeling of [¹⁸F]FPBM, 1, an intermediate, [¹⁸F]3-fluoropropyltosylate, [¹⁸F]4, was prepared first, and then it was reacted with the phenol precursor (4-(2-aminophenylthio)-3-((dimethylamino)methyl)phenol, 3) to afford [¹⁸F]FPBM, 1. To optimize the labeling, this O-alkylation reaction was evaluated under different temperatures, using different bases and varying amounts of precursor 3. The desired product was obtained after a solid phase extraction (SPE) purification.ResultsThis two-step radiolabeling reaction successfully produced the desired [¹⁸F]FPBM, 1, with an excellent radiochemical purity (> 95%, n = 8). Radiochemical yields were between 31% and 39% (decay corrected, total time of labeling: 70 min, n = 8). The SPE purification cannot completely remove pseudo-carriers in the final dose of [¹⁸F]FPBM, 1. The concentrations of major pseudo-carriers were measured by UV-HPLC (476–676, 68–95 and 50–71 μg for precursor 3, O-hydroxypropyl and O-allyloxy derivatives, 5 and 6, respectively). To investigate the potential inhibition of SERT binding of these pseudo-carriers, we performed in vitro competition experiments evaluated by autoradiography. Known amounts of ‘standard’ FPBM, 1, of the pseudo-carriers, 5 and 6, were added to the HPLC-purified [¹⁸F]1 dose. The inhibition of ‘standard’ FPBM, 1, binding to the SERT binding sites, using monkey brain sections, were measured (EC₅₀ = 13, 46, 7.1 and 8.3 nM, respectively for 1, precursor 3, O-hydroxypropyl and O-allyloxy derivative of 3).ConclusionAn improved radiolabeling method by a SPE purification for preparation of [¹⁸F]FPBM, 1, was developed. The results suggest that it is feasible to use this labeling method to prepare [¹⁸F]FPBM, 1, without affecting in vivo SERT binding.     

Enantioselective synthesis of no-carrier-added (NCA) 6-[18F]fluoro-L-DOPA.

The application of a chiral phase-transfer-catalyst (PTC) in the synthesis of N.C.A. 6-[18F]fluoro-L-DOPA has been recently developed. The 6-trimethylammoniumveratraldehyde triflate precursor and PTC (O-allyl-N-(9)-anthracenylcinchonidinium bromide) were synthesized and successful synthesis route was developed for the preparation of 6-[18F]fluoro-L-DOPA with high radiochemical yields (4-9%, decay uncorrected) and short synthesis time (80min). The radiochemical purity was over 99% and no D-isomer was detected by HPLC analysis using a chiral mobile phase.     

Alkyl-fluorinated thymidine derivatives for imaging cell proliferation II. Synthesis and evaluation of N3-(2-[18F]fluoroethyl)-thymidine

We prepared N(3)-(2-[(18)F]fluoroethyl)-thymidine ([(18)F]NFT202) and examined its potential as a positron emission tomography (PET) ligand for imaging cellular proliferation. [(18)F]NFT202 was synthesized from 3',5'-di-O-toluoyl-N(3)-(2-p-toluenesulfoxyethyl)-thymidine in a two-step reaction. N(3)-(2-fluoroethyl)-[2-(14)C]thymidine ([(14)C]NFT202) was also synthesized from [2-(14)C]thymidine in a one-step reaction. Whereas [(18)F]NFT202 did not accumulate in mouse Lewis lung carcinoma tumors, 3'-[(18)F]3'-fluoro-3'-deoxythymidine ([(18)F]FLT) showed significantly high uptake. To clarify this unexpected result, we evaluated the cell uptake of [(14)C]NFT202 in vitro. The uptake was approximately eight times higher in thymidine kinase 1 (TK1)(+) clones (L-M cells) than in TK1-deficient mutant L-M(TK(-)) cells (P<.01, Student's t test). In addition, we observed a positive correlation between tracer uptake and the S-phase fraction. However, the net in vitro tumor cell uptake of [(14)C]NFT202 was lower than that of [2-(14)C]3'-fluoro-3'-deoxythymidine. [(14)C]NFT202 was not effectively incorporated into the DNA fraction and was indeed washed out from tumor cells. These results clearly showed that [(18)F]NFT202 did not surpass the performance of [(18)F]FLT. We therefore conclude that [(18)F]NFT202 is not a suitable PET ligand for imaging tumor cell proliferation.