Nucleophilic substitution of nitro groups by [F]fluoride in methoxy-substituted ortho-nitrobenzaldehydes—A systematic study

As model reactions for the introduction of [¹⁸F]fluorine into aromatic amino acids, the replacement of NO₂ by [¹⁸F]fluoride ion in mono- to tetra-methoxy-substituted ortho-nitrobenzaldehydes was systematically investigated. Unexpectedly, the highly methoxylated precursors 2,3,4-trimethoxy-6-nitrobenzaldehyde and 2,3,4,5-tetramethoxy-6-nitrobenzaldehyde showed high maximum radiochemical yields (82% and 48% respectively). When the electrophilicity of the leaving group substituted carbon atom is expressed by its ¹³C NMR chemical shift a good correlation with the reaction rate at the beginning of the reaction (first min) was found (R² = 0.89), whereas the maximum radiochemical yields correlated much poorer with this electrophilicity parameter. This may be caused by side reactions becoming influencial in the further reaction course. As possible side reactions the demethylation of methoxy groups and intramolecular redox reactions could be detected by HPLC/MS.     

[F]Fluoromethyl iodide ([F]FCH2I): preparation and reactions with phenol, thiophenol, amide and amine functional groups

In this study, we report the synthesis and reactivity of [¹⁸F]fluoromethyl iodide ([¹⁸F]FCH₂I) with various nucleophilic substrates and the stabilities of [¹⁸F]fluoromethylated compounds. [¹⁸F]FCH₂I was prepared by reacting diiodomethane (CH₂I₂) with [¹⁸F]KF, and purified by distillation in radiochemical yields of 14-31% (n = 25). [¹⁸F]FCH₂I was stable in organic solvents commonly used for labeling and aqueous solution with pH 1-7, but was unstable in basic solutions. [¹⁸F]FCH₂I displayed a high reactivity with various nucleophilic substrates such as phenol, thiophenol, amide and amine. The [¹⁸F]fluoromethylated compounds synthesized by the reactions of phenol, thiophenol and tertiary amine with [¹⁸F]FCH₂I were stable for purification, formulation and storage. In contrast, the [¹⁸F]fluoromethylated compounds synthesized by the reactions of primary or secondary amines, and amide with [¹⁸F]FCH₂I were too unstable to be detected or purified from the reaction mixtures. Defluorination of these [¹⁸F]fluoromethyl compounds was a main decomposition route.     

Efficient alkali iodide promoted F-fluoroethylations with 2-[F]fluoroethyl tosylate and 1-bromo-2-[F]fluoroethane

Radiochemical ¹⁸F-fluorination yields of several compounds using the secondary labelling precursors 2-[¹⁸F]fluoroethyl tosylate ([¹⁸F]FETos) and 1-bromo-2-[¹⁸F]fluoroethane ([¹⁸F]BFE) could be considerably enhanced by the addition of an alkali iodide. The radiochemical yield of [¹⁸F]fluoroethyl choline for example could be doubled with [¹⁸F]BFE and increased from 13% to ≈80% with [¹⁸F]FETos. By addition of alkali iodide to the precursor, the ¹⁸F-fluoroethylation yields of established radiopharmaceuticals, especially in the case of automated syntheses, could be significantly increased without major changes of the reaction conditions.     

Alpha selective epoxide opening with F: synthesis of 4-(3-[F]fluoro-2-hydroxypropoxy)benzaldehyde ([F]FPB) for peptide labeling

Strained tricyclic ring systems such as epoxides are rarely used as precursors for the introduction of anionic fluorine-18 into organic compounds intended for positron emission tomography (PET). Here we report the alpha selective ring opening of epoxides for the introduction of fluorine-18 into small as well as larger biomolecules via 1- and 2-step protocols. [¹⁸F]fluoromisonidazole ([¹⁸F]MISO), a tracer for hypoxia imaging, and the tumor targeting peptide Tyr³-octreotate (TATE) were radiolabeled using epoxide opening reactions. In the latter case, the new prosthetic labeling synthon 4-(3-[¹⁸F]fluoro-2-hydroxypropoxy)benzaldehyde ([¹⁸F]FPB) has been used for ¹⁸F-introduction.     

[F]Fluorophenyl organometallics as intermediates of no-carrier-added F-fluoroarylation reactions

Based on the recent availability of no-carrier-added (n.c.a.) 1-bromo-4-[¹⁸F]fluorobenzene with high radiochemical yield, the 4-[¹⁸F]fluorophenyl compounds of lithium, sodium and magnesium can now also effectively be prepared. Thus, [¹⁸F]fluoroarene reagents with a nucleophilic reaction centre are available and suitable among others for the formation of [¹⁸F]fluorophenyl compounds with electron donating substituents in the radiosynthesis of ¹⁸F-labelled complex organic structures. For these arylation reactions, however, the presence of macroscopic amounts of a haloarene as co-reactant is necessary with all n.c.a. [¹⁸F]fluorophenyl metallics. The ¹⁸F-fluoroarylation was verified for examples of aryl-carbon, -silicon, -sulphur, and -nitrogen bond formation with radiochemical yields of 20-25% related to the starting radioactivity of [¹⁸F]fluoride.     

Enantioselective syntheses of no-carrier-added (n.c.a.) (s)-4-chloro-2-[f] fluorophenylalanine and (s)-(α-methyl) -4-chloro-2-[f]fluorophenylalanine

(S)-4-Chloro-2-fluorophenylalanine and (S)-(α-methy)-4-chloro-2-fluorophenylalanine were synthesized and labeled with no carrier added (n.c.a.) fluorine-18 through a radiochemical synthesis relying on the highly enantioselective reaction between 4-chloro-2-[¹⁸F]fluorobenzyl iodide and the lithium enolate of (2S)-1-(tert-butyloxycarbonyl)-2-(tert-butyl)-3-methyl-1,3-imidazolidine-4-one for (S)-4-chloro-2-[¹⁸F]fluorophenylalanine and (2S,5S)-1-(tert-butyloxycarbonyl)-2-(tert-butyl)-3,5-dimethyl-1,3-imidazolidine-4-one for (S)-(α-methyl) -4-chloro-2-[¹⁸F] fluorophenylalanine. Quantities of about 20–25 mCi were obtained at the end of sy nthesi s, ready for injection after hydrolysis and high performance liquid chromatography (HPLC) purification, with a radiochemical yield of 17%–20% corrected to the end of bombardment after a total synthesis time of 90–105 min from [¹⁸F] fluoride. The enantiomeric excesses were shown to be 97% or more for both molecules without chiral separation and the radiochemical and chemical purities were 98% or better.     

The [F]2-Fluoro-1,3-thiazolyl Moiety - an Easily-Accessible Structural Motif for Prospective Molecular Imaging Radiotracers

2-Fluoro-1,3-thiazoles were rapidly and efficiently labeled with no-carrier-added fluorine-18 (t_1/2 = 109.7 min) by treatment of readily prepared 2-halo precursors with cyclotron-produced [¹⁸F]fluoride ion. The [¹⁸F]2-fluoro-1,3-thiazolyl moiety constitutes a new and easily-labeled structural motif for prospective molecular imaging radiotracers.     

Preparation of the novel fluorine-18-labeled VIP analog for PET imaging studies using two different synthesis methods

Vasoactive intestinal peptide (VIP) receptors are expressed on various tumor cells in much higher density than somatostatin receptors, which provides the basis for radiolabeling VIP as tumor diagnostic agent. However, fast proteolytic degradation of VIP in vivo limits its clinical application. With the aim to develop and evaluate new ligands for depicting the VIP receptors with positron emission tomography (PET), the structure modified [R^8,15,21, L¹⁷]-VIP analog was radiolabeled with ¹⁸F using two different methods. With the first method, N-4-[¹⁸F]fluorobenzoyl-[R^8,15,21, L¹⁷]-VIP ([¹⁸F]FB-[R^8,15,21, L¹⁷]-VIP 7) was produced in a decay-corrected radiochemical yield (RCY) of 33.6 ± 3%, a specific radioactivity of 255 GBq/μmol (n = 5) within 100 min in four steps. Similarly, N-4-[¹⁸F](fluoromethyl)-benzoyl-[R^8,15,21, L¹⁷]-VIP ([¹⁸F]FMB-[R^8,15,21, L¹⁷]-VIP 8) was synthesized in a RCY of 34.85 ± 5%, a specific radioactivity of 180 GBq/μmol (n = 5) within 60 min in only one step. The two products 7 and 8 were both shown good stability in HSA. Moreover, the low bone uptakes of 7 and 8 in vivo of mice showed good defluorination stability.     

Production of 6-l-[18F]Fluoro-m-tyrosine in an Automated Synthesis Module for 11C-Labeling

6-l-[¹⁸F]Fluoro-m-tyrosine (6-l-[¹⁸F]FMT) represents a valuable alternative to 6-l-[¹⁸F]FDOPA which is conventionally used for the diagnosis and staging of Parkinson’s disease. However, clinical applications of 6-l-[¹⁸F]FMT have been limited by the paucity of practical production methods for its automated production. Herein we describe the practical preparation of 6-l-[¹⁸F]FMT using alcohol-enhanced Cu-mediated radiofluorination of Bpin-substituted chiral Ni(II) complex in the presence of non-basic Bu₄ONTf using a volatile iPrOH/MeCN mixture as reaction solvent. A simple and fast radiolabeling procedure afforded the tracer in 20.0 ± 3.0% activity yield within 70 min. The developed method was directly implemented onto a modified TracerLab FX C Pro platform originally designed for ¹¹C-labeling. This method enables an uncomplicated switch between ¹¹C- and ¹⁸F-labeling. The simplicity of the developed procedure enables its easy adaptation to other commercially available remote-controlled synthesis units and paves the way for a widespread application of 6-l-[¹⁸F]FMT in the clinic.     

Comparison of [18F]F-CNBI and [18F]F-CNPIFE as Positron Emission Tomography Probes for Noninvasive Imaging of Glycogen Synthase Kinase-3 in Normal Mice

Glycogen synthase kinase-3 α/β is involved in dysregulation of neuronal tau protein in Alzheimer's disease (AD). There is an unmet clinical need for a blood-brain barrier (BBB) permeable positron emission tomography (PET) probe for imaging of GSK-3α/β in the brain to understand the pathogenesis of AD. Herein, we synthesized two PET probes, [¹⁸F]F-CNBI and [¹⁸F]F-CNPIFE, and evaluated their BBB permeability and affinity towards GSK-3α/β. [¹⁹F]F-CNPIFE showed higher in-vitro binding towards GSK-3α/β (IC₅₀=19.4±2.5 nM; n=3, for GSK-3α, IC₅₀=19.4±3.8 nM; n=3, for GSK-3β) compared to [¹⁹F]F-CNBI (IC₅₀=107.6±26.0 nM; n=4, for GSK-3α, IC₅₀=105.3±18.2 nM; n=3, for GSK-3β). [¹⁸F]F-CNPIFE showed 9.5-fold higher brain uptake than [¹⁸F]F-CNBI, in normal FVB/NJ mice, which was increased by additional 1.5-fold on co-administration of [¹⁹F]F-CNPIFE with respect to [¹⁸F]F-CNBI. Overall, [¹⁸F]F-CNPIFE is a promising PET probe for GSK-3α/β imaging and warrants further evaluation in an AD mouse model.     

Bifunctional fluorescent thiacalix[4]arene based chemosensor for Cu and F ions

A new thiacalix[4]arene based fluorescent sensor bearing two dansyl groups has been synthesized in cone conformation. In CH₃CN:CH₂Cl₂ (1:1), the presence of Cu (II) induces the formation of a 1:1 metal:ligand complex, which exhibits increasing emission at 433 nm at the expense of the fluorescent emission of 1 centered at 504 nm. The detection limit of the sensor for Cu²⁺ is 2×10⁻⁷ mol L⁻¹. For anion sensing, 1 shows a high selectivity for fluoride ions over other anions tested.     

Rapid Detection of the Residual Kryptofix 2.2.2 Levels in [18F]-Labeled Radiopharmaceuticals by Ultra-Performance Liquid Chromatography Tandem Mass Spectrometry

A fast and sensitive ultra-performance liquid chromatography-tandem mass spectrometric (UPLC/MS/MS) method was developed and validated for determination of the residual levels of Kryptofix 2.2.2 (K222) in [¹⁸F]-labeled radiopharmaceuticals. The analytical time was only 3 min, and the injection volume was 5 μL. An electrospray ionization source was used in the positive mode (ESI⁺) for UPLC/MS/MS. The analytical measurements were performed in the multiple reaction monitoring (MRM) mode. The calibration curve at the spiked concentrations of 2–500 ng/mL for K222 showed good linearity. The intra- and inter-day precisions were not more than 5%. The accuracy satisfied the requirement of quality control analysis, the recoveries were found to be 80–120%. This method was successfully applied to detect the residue of K222 in [¹⁸F]-fluorodeoxyglucose [(¹⁸F)FDG], [¹⁸F]-fluoromisonizole[(¹⁸F)FMISO], 3′-deoxy-3′-[¹⁸F]-fluorothymidine [(¹⁸F)FLT], and two new [¹⁸F]-labeled radiopharmaceuticals 4-[-(2-[¹⁸F]fluoroethoxy) methyl]-1-[2-(2-methyl-5-nitro-1H- imidazol-1-yl) ethyl]-1H-1,2,3-triazole (named as ¹⁸F-BNU-1) and 4-[-(2-[¹⁸F] fluoroethoxy) methyl]-1-[2-(2-nitro-1H-imidazol-1-yl) ethyl]-1H-1,2,3-triazole (named as ¹⁸F-BNU-2) produced in our lab.     

An Improved Synthesis of N-(4-[18F]Fluorobenzoyl)-Interleukin-2 for the Preclinical PET Imaging of Tumour-Infiltrating T-cells in CT26 and MC38 Colon Cancer Models

Positron emission tomography (PET) imaging of activated T-cells with N-(4-[¹⁸F]fluorobenzoyl)-interleukin-2 ([¹⁸F]FB-IL-2) may be a promising tool for patient management to aid in the assessment of clinical responses to immune therapeutics. Unfortunately, existing radiosynthetic methods are very low yielding due to complex and time-consuming chemical processes. Herein, we report an improved method for the synthesis of [¹⁸F]FB-IL-2, which reduces synthesis time and improves radiochemical yield. With this optimized approach, [¹⁸F]FB-IL-2 was prepared with a non-decay-corrected radiochemical yield of 3.8 ± 0.7% from [¹⁸F]fluoride, 3.8 times higher than previously reported methods. In vitro experiments showed that the radiotracer was stable with good radiochemical purity (>95%), confirmed its identity and showed preferential binding to activated mouse peripheral blood mononuclear cells. Dynamic PET imaging and ex vivo biodistribution studies in naïve Balb/c mice showed organ distribution and kinetics comparable to earlier published data on [¹⁸F]FB-IL-2. Significant improvements in the radiochemical manufacture of [¹⁸F]FB-IL-2 facilitates access to this promising PET imaging radiopharmaceutical, which may, in turn, provide useful insights into different tumour phenotypes and a greater understanding of the cellular nature and differential immune microenvironments that are critical to understand and develop new treatments for cancers.     

Synthesis of <Emphasis Type="Italic">O</Emphasis>-(2-[<Superscript>18</Superscript>F]fluoroethyl)-<Emphasis Type="Italic">L</Emphasis>-tyrosine and its biological evaluation in B16 melanoma-bearing mice as PET tracer for tumor imaging

O-(2-[¹⁸F]fluoroethyl)-L-tyrosine ([¹⁸F]FET), a fluorine-18 labeled analogue of tyrosine, has been synthesized and biologically evaluated in tumor-bearing mice. The whole synthesis procedure is completed within 50 min. The radiochemical yield is about 40% (no decay corrected) and radiochemical purity more than 97% after simplified solid phase extraction. [¹⁸F]FET shows rapid, high uptake and long retention in the tumor as well as low uptake in the brain. The ratios of tumor-to-muscle (T/M) and tumor-to-blood (T/B) of [¹⁸F]FET are similar to those of [¹⁸F]FDG, but the ratios of tumor-to-brain (T/Br) are 2–3 times higher than that of [¹⁸F]FDG. Autoradiography of [¹⁸F]FET demonstrates a remarkable accumulation in melanoma with high contrast. It appears to be a probable competitive candidate for melanoma imaging with PET. Supported by the Knowledge Innovation Project of Chinese Academy of Sciences (No. KJCX1-SW-08) and the National Natural Science Foundation of China (Grant No. 30371634)     

Alternate HPLC method for the analysis of tetrabutylammonium hydroxide in [18F]fluorodeoxythymidine (FLT)

Tetrabutylammonium hydroxide is a common reagent used in the synthesis of [¹⁸F]Fluorodeoxythymidine (FLT) for positron emission tomography imaging. The British Pharmacopeia monograph for the analysis of [¹⁸F]FLT was released in 2015 incorporating a HPLC method for the analysis of tetrabutylammonium hydroxide. We describe alternate HPLC conditions that mitigate the challenges in fulfilling the system suitability requirements such as signal to noise and symmetry factor that are specified in the monograph. Our method was validated by analyzing tetrabutylammonium hydroxide at a range of concentrations to determine the linearity (R²?=?0.994), accuracy (≤9.2%) and precision (≤3.2%). Our method does not affect the analysis of [¹⁸F]FLT and it complies with all requirements in the BP monograph.     

Determination of Total Radiochemical Purity of [18F]FDG and [18F]FET by High-Performance Liquid Chromatography Avoiding TLC Method

The goal of this work was to present two high-performance liquid chromatography (HPLC) method that could be applied for the determination of the total radioactive purity of 2-deoxy-2-[¹⁸F]fluoro-D-glucose ([¹⁸F]FDG) and O-(2-[¹⁸F]fluoroethyl)-L-tyrosine ([¹⁸F]FET). The separation of [¹⁸F]fluoride ions, [¹⁸F]FET and [¹⁸F]FET intermediate was accomplished on LiChrosper RP-18, 250?×?4 mm, 5 µm (Merck) analytical column. For mobile phase 10 mM potassium dihydrogen phosphate buffer at pH7 (A) and acetonitrile (B) was used: 0–2 min: 15% B; 2–12 min: 85% B; 12–15 min: 15% B, respectively. Analysis of [¹⁸F]FDG was performed using LiChrosper 100 NH₂, 250?×?4.5 mm, 5 µm (Merck) analytical column. The initial mobile phase composition was 10 mM KH₂PO₄ buffer (pH7) and acetonitrile (15:85, v/v) and the acetonitrile ratio was decreased to 15% at 2 min after the sample injection and held for 5 min. Complete elution of [¹⁸F]fluoride ions from stationary phases could be achieved by adding 10 mg/mL K[¹⁹F]F to radioactive samples in a ratio 1:1 during the sample preparation. Recovery of [¹⁸F]fluoride ions ranged from 99.5 to 100.6%. The validation of the developed methods showed good results for linearity (r²?=?0.9981–0.9996), specificity (R_S?=?3.7–10.2), repeatability (%Area RSD_%?=?1.2–4.3%) and limit of quantitation (LOQ?=?1.6–4.5 kBq). During the cross-validation similar radiochemical purity values were obtained by the novel HPLC methods and thin layer chromatography performed according to the recommendations of the Ph. Eur. monographs.

     

Synthesis of [carbonyl-C]acetophenone via the Stille cross-coupling reaction of [1-C]acetyl chloride with tributylphenylstannane mediated by Pd2(dba)3/P(MeNCH2CH2)3N·HCl

The Stille cross-coupling reaction of [1-¹¹C]acetyl chloride with tributylphenylstannane leading to [carbonyl-¹¹C]acetophenone was studied with the goal of developing a new ¹¹C-labeling method for positron emission tomography tracer synthesis. The coupled product [carbonyl-¹¹C]acetophenone was synthesized using the Pd₂(dba)₃/P(MeNCH₂CH₂)₃N·HCl system with a 60-61% radiochemical conversion from [1-¹¹C]acetyl chloride (decay-corrected, n = 3).     

Fluoride-Promoted Ligand Exchange in Diaryliodonium Salts

Diaryliodonium salts are shown to undergo rapid, fluoride-promoted aryl exchange reactions at room temperature in acetonitrile. Aryl exchange is shown to be exquisitely sensitive to the concentration of fluoride ion in solution; fast exchange is observed as the fluoride concentration approaches a stoichiometric amount at 50 mM substrate concentration. The reaction is slowed, but not halted if benzene is the solvent, indicating that free fluoride ion or a four-coordinate anionic I(III) species may be responsible for the exchange. The fluoride-promoted aryl exchange reaction is general and allows direct measurement of the relative stabilities of diaryliodonium salts featuring different aryl substituents. The aryl exchange reaction may be of practical use for the preparation of hitherto inaccessible diaryliodonium salts, thus it also has implications for labeling radiotracers for molecular imaging with ¹⁸F-fluoride (t_1/2 = 109.7 min).     

Effect of aldehyde and methoxy substituents on nucleophilic aromatic substitution by [F]fluoride

For a series of benzaldehydes only with a leaving group or with both a leaving group and a single methoxy substituent ¹⁸F-fluorination via nucleophilic aromatic substitution (S_NAr) was studied in DMF and Me₂SO. In general, the radiochemical yields were clearly higher in DMF than in Me₂SO. In the fluorodehalogenation reaction (leaving group: halogen = Br, Cl), extremely low radiochemical yields were observed in Me₂SO (<1%). By monitoring labeling reactions using HPLC, oxidation of the aldehyde function of the precursor was detected. Especially, 2-bromobenzaldehyde was oxidized fastest in Me₂SO (within 3 min reaction time, 90% of the precursor was consumed; radiochemical yield = 1.0 ± 0.5%); however, in DMF oxidation was always kept at a low level during the entire reaction (<5% of the precursor was oxidized; radiochemical yield = 73.0 ± 0.2%). In DMF, nitrobenzaldehydes with a methoxy substituent (methoxy group in meta-position to the nitro group) were labeled with good radiochemical yields (4-methoxy-2-nitrobenzaldehyde: 87 ± 3%; 2-methoxy-4-nitrobenzaldehyde: 83 ± 3%; 2-methoxy-6-nitrobenzaldehyde: 79 ± 4%) comparable to the non-substituted nitrobenzaldehydes (2-nitrobenzaldehyde: 84 ± 3%; 4-nitrobenzaldehyde: 81 ± 5%). Moreover, for structurally similar compounds, radiochemical yields showed a good correlation with ¹³C-NMR ppm values of the aromatic carbon atom bearing the leaving group.     

Synthesis of [18F]F-γ-T-3, a Redox-Silent γ-Tocotrienol (γ-T-3) Vitamin E Analogue for Image-Based In Vivo Studies of Vitamin E Biodistribution and Dynamics

Vitamin E, a natural antioxidant, is of interest to scientists, health care pundits and faddists; its nutritional and biomedical attributes may be validated, anecdotal or fantasy. Vitamin E is a mixture of tocopherols (TPs) and tocotrienols (T-3s), each class having four substitutional isomers (α-, β-, γ-, δ-). Vitamin E analogues attain only low concentrations in most tissues, necessitating exacting invasive techniques for analytical research. Quantitative positron emission tomography (PET) with an F-18-labeled molecular probe would expedite access to Vitamin E’s biodistributions and pharmacokinetics via non-invasive temporal imaging. (R)-6-(3-[¹⁸F]Fluoropropoxy)-2,7,8-trimethyl-2-(4,8,12-trimethyltrideca-3,7,11-trien-1-yl)-chromane ([¹⁸F]F-γ-T-3) was prepared for this purpose. [¹⁸F]F-γ-T-3 was synthesized from γ-T-3 in two steps: (i) 1,3-di-O-tosylpropane was introduced at C6-O to form TsO-γ-T-3, and (ii) reaction of this tosylate with [¹⁸F]fluoride in DMF/K₂₂₂. Non-radioactive F-γ-T-3 was synthesized by reaction of γ-T-3 with 3-fluoropropyl methanesulfonate. [¹⁸F]F-γ-T-3 biodistribution in a murine tumor model was imaged using a small-animal PET scanner. F-γ-T-3 was prepared in 61% chemical yield. [¹⁸F]F-γ-T-3 was synthesized in acceptable radiochemical yield (RCY 12%) with high radiochemical purity (>99% RCP) in 45 min. Preliminary F-18 PET images in mice showed upper abdominal accumulation with evidence of renal clearance, only low concentrations in the thorax (lung/heart) and head, and rapid clearance from blood. [¹⁸F]F-γ-T-3 shows promise as an F-18 PET tracer for detailed in vivo studies of Vitamin E. The labeling procedure provides acceptable RCY, high RCP and pertinence to all eight Vitamin E analogues.