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Objectives: The monocarboxylate transporters 1 and 4 (MCT1/4) are integral plasma membrane proteins that bi-directionally transport lactate as well as small monocarboxylated molecules. They are highly expressed in several tumors. BAY-8002 belongs to a class of compounds that have been identified as novel and specific MCT1 inhibitors based on functional high-throughput screening assays using a panel of cell lines highly sensitive towards MCT1 inhibition [1]. IC50 values of 1 to 12 nM and ca. 500–fold selectivity towards MCT4 have already been reported for BAY-8002 [1]. Here we designed an 18F-labeled analog of BAY-8002 ([18F]F-BAY-8002) aiming to image mainly MCT1 upregulation considering the fact that the absence of MCT4 expression in many types of cancer may not be necessarily sufficient as a single marker to predict treatment response [2]. Methods: BAY-8002 and its novel fluorinated analog (F-BAY-8002) were synthesized based on reported procedures [1]. As BAY-8002 already contains a chloro substituent which could serve as leaving group, the compound was considered as precursor for radiofluorination via a halogen-fluorine exchange approach (Figure 1A). [18F]F-BAY-8002 was radiolabeled via a one-step aromatic nucleophilic substitution reaction (SNAr) using 2-5 mg of precursor in the presence of the [18F]KF/K222/K2CO3 complex in dimethyl sulfoxide (DMSO) at 150 °C within 5 min (Figure 1B). Figure 1. (A) Synthesis of BAY-8002 and its novel fluorinated analog; (B) Radiosynthesis of [18F]F-BAY-8002. Separation of [18F]F-BAY-8002 from the chlorinated precursor was performed by semi-preparative HPLC. The tracer was finally purified via solid-phase extraction (Sep-Pak® C18 light cartridge) and formulated in 10% EtOH/saline solution to be ready for biological evaluations. Results: Despite using identical conditions [1], the novel fluorinated analog F-BAY-8002 was obtained in only 4% overall yield due to formation of by-products which have not been observed during the synthesis of BAY-8002 (35% overall yield). Due to the lack of commercially available radioligands, the MCT1 affinity (Ki) of F-BAY-8002 could not yet be determined and we therefore intend to measure the KD value of our new radiotracer by in-house established methods in near future. The novel radiotracer [18F]F-BAY-8002 was synthesized in 30 ± 9% radiochemical yields (n = 4, non-isolated, estimated by radio-HPLC) within 5 min reaction time. After purification and formulation, the final product was obtained with a radiochemical purity of > 99% (n = 1). Further radiochemical characterization of the radiotracer and the transfer of the radiosynthesis to an automated module are in progress. Conclusions: A novel 18F-labeled radioligand for potential specific MCT1-targeted imaging was developed via a straightforward fast approach in good radiochemical yields and high radiochemical purity. Notably, the labeling was successful even without protection of the carboxylic acid group resulting in a beneficial one-step instead of a two-step radiosynthesis procedure. In vitro and in vivo biological evaluation of the newly synthesized MCT1 radioligand are currently ongoing. References: [1] Quanz, M. et al. Mol Cancer Ther. 2018 17:2285-2296; [2] Le Floch, R. et al. Proc Natl Acad Sci USA. 2011 108:16663-8.

Introduction (−)-[ 18 F]Flubatine is a PET tracer with high affinity and selectivity for the nicotinic acetylcholine α 4 β 2 receptor subtype. A clinical trial assessing the availability of this subtype of nAChRs was performed. From a total participant number of 21 Alzheimer's disease (AD) patients and 20 healthy controls (HCs), the following parameters were determined: plasma protein binding, metabolism and activity distribution between plasma and whole blood. Methods Plasma protein binding and fraction of unchanged parent compound were assessed by ultracentrifugation and HPLC, respectively. The distribution of radioactivity (parent compound+metabolites) between plasma and whole blood was determined ex vivo at different time-points after injection by gamma counting after separation of whole blood by centrifugation into the cellular and non-cellular components. In additional experiments in vitro , tracer distribution between these blood components was assessed for up to 90min. Results A fraction of 15%±2% of (−)-[ 18 F]Flubatine was found to be bound to plasma proteins. Metabolic degradation of (−)-[ 18 F]Flubatine was very low, resulting in almost 90% unchanged parent compound at 90min p.i. with no significant difference between AD and HC. The radioactivity distribution between plasma and whole blood changed in vivo only slightly over time from 0.82±0.03 at 3min p.i. to 0.87±0.03 at 270min p.i. indicating the contribution of only a small amount of metabolites. In vitro studies revealed that (−)-[ 18 F]Flubatine was instantaneously distributed between cellular and non-cellular blood parts. Discussion (−)-[ 18 F]Flubatine exhibits very favourable characteristics for a PET radiotracer such as slow metabolic degradation and moderate plasma protein binding. Equilibrium of radioactivity distribution between plasma and whole blood is reached instantaneously and remains almost constant over time allowing both convenient sample handling and facilitated fractional blood volume contribution assessment.

With the aim of producing selective radiotracers for in vivo imaging of the vesicular acetylcholine transporter (VAChT) using positron mission tomography (PET), here, we report synthesis and analysis of a new class of conformationally constrained vesamicol analogues with moderate lipophilicity. The sequential ring opening on trans -1,4-cyclohexadiene dioxide enabled an approach to synthesize 6-arylpiperidino-octahydrobenzo[1,4]oxazine-7-ols [morpholino vesamicols]. The radiosynthesis of the [ 18 F]fluoroacetyl-substituted derivative ([ 18 F]FAMV) was achieved starting from a corresponding bromo precursor [2-Bromo-1-[7-hydroxy-6-(4-phenyl-piperidin-1-yl)-octahydro-benzo[1,4]oxazin-4-yl]-ethanone] and using a modified commercial computer-controlled module system with a radiochemical yield of 27±4%, a high radiochemical purity (99%) and a specific activity of 35 GBq/μmol. In competitive binding assays using a PC12 cell line overexpressing VAChT and [ 3 H]-(−) vesamicol, 2-fluoro-1-[7-hydroxy-6-(4-phenyl-piperidin-1-yl)-octahydro-benzo[1,4]oxazin-4-yl]-ethanone (FAMV) demonstrated a high selectivity for binding to VAChT ( K i : 39.9±5.9 nM) when compared to its binding to sigma 1/2 receptors ( K i >1500 nM). The compound showed a moderate lipophilicity (logD (pH 7) =1.9) and a plasma protein binding of 49%. The brain uptake of [ 18 F]FAMV was about 0.1% injected dose per gram at 5 min after injection and decreased continuously with time. Notably, an increasing accumulation of radioactivity in the lateral brain ventricles was observed. After 1 h, the accumulation of [ 18 F]FAMV, expressed as ratio to the cerebellum, was 4.5 for the striatum, 2.0 for the cortical and 1.5 for the hippocampal regions, measured on brain slices using ex vivo autoradiography. At the present time, 75% of [ 18 F]FAMV in the plasma was shown to be metabolized to various hydrophilic compounds, as detected by high-performance liquid chromatography. The degradation of [ 18 F]FAMV was also detected in brain extracts as early as 15 min post injection (p.i.) and increased to 50% at 1 h postinjection. In conclusion, although the chemical properties of [ 18 F]FAMV and the selectivity of binding to VAChT appear to be promising indicators of a useful PET tracer for imaging VAChT, a low brain extraction, in combination with only moderate specific accumulation in cholinergic brain regions and an insufficient in vivo stability prevents the application of this compound for neuroimaging in humans.

Phenylpiperidinyl-octahydro-benzo[1,4]oxazines represent a new class of conformationally restrained vesamicol analogues. Derived from this morpholine-fused vesamicol structure, a new fluorine-18-labeled 4-fluorobenzoyl derivative ([ 18 F]FBMV) was synthesized with an average specific activity of 75 GBq/μmol and a radiochemical purity of 99%. The radiolabeling method included an exchange reaction of a 4-nitro group of the precursor by fluorine-18, a reduction procedure to eliminate excess of the nitro compound, followed by a high-performance liquid chromatography purification. [ 18 F]FBMV demonstrates (i) a moderate lipophilic character with a logD pH7.0 1.8±0.10; (ii) a considerable binding affinity to the vesicular acetylcholine transporter (VAChT) ( K i =27.5 nM), as determined using PC12 cells transfected with a VAChT cDNA, and a low affinity to σ 1,2 receptors ( K i >3000 nM); (iii) a good uptake into the rat and pig brains; (iv) a typical accumulation in the VAChT-containing brain regions; and (v) an approximately 20% reduction in cortical tracer binding after a specific cholinergic lesion using 192IgG-saporin. [ 18 F]FBMV exhibits another PET marker within the group of vesamicol derivatives that demonstrates potentials in imaging brain cholinergic deficits, while its usefulness in clinical practice must await further investigation.

Introduction Gamma amino butyric acid type A (GABA A ) receptors are involved in a variety of neurological and psychiatric diseases, which have promoted the development and use of radiotracers for positron emission tomography imaging. Radiolabeled benzodiazepine antagonists such as flumazenil have most extensively been used for this purpose so far. Recently, the non-benzodiazepine pyrazolopyrimidine derivative indiplon with higher specificity for the α 1 subtype of the GABA A receptor has been introduced for treatment of insomnia. The aim of this study was the development and biological evaluation of an 18 F-labeled derivative of indiplon. Methods Both [ 18 F]fluoro-indiplon and its labeling precursor were synthesized by two-step procedures starting from indiplon. The radiosynthesis of [ 18 F]fluoro-indiplon was performed using the bromoacetyl precursor followed by multiple-stage purification using semipreparative HPLC and solid phase extraction. Stability, partition coefficients, binding affinities and regional brain binding were determined in vitro. Biodistribution and radiotracer metabolism were studied in vivo. Results [ 18 F]Fluoro-indiplon was readily accessible in good yields (38–43%), with high purity and high specific radioactivity (>150 GBq/μmol). It displays high in vitro stability and moderate lipophilicity. [ 18 F]Fluoro-indiplon has an affinity to GABA A receptors comparable to indiplon ( K i =8.0 nM vs. 3.4 nM). In vitro autoradiography indicates high [ 18 F]fluoro-indiplon binding in regions with high densities of GABA A receptors. However, ex vivo autoradiography and organ distribution studies show no evidence of specific binding of [ 18 F]fluoro-indiplon. Furthermore, the radiotracer is rapidly metabolized with high accumulation of labeled metabolites in the brain. Conclusions Although [ 18 F]fluoro-indiplon shows good in vitro features, it is not suitable for in vivo imaging studies because of its metabolism. Structural modifications are needed to develop derivatives with higher in vivo stability.

2-[(18)F]fluoro-A-85380 (2-[(18)F]FA) is a new radioligand for noninvasive imaging of alpha4beta2* nicotinic acetylcholine receptors (nAChRs) by positron emission tomography (PET) in human brain. In most cases, quantification of 2-[(18)F]FA receptor binding involves measurement of free nonmetabolized radioligand concentration in blood. This requires an efficient and reliable method to separate radioactive metabolites from the parent compound. In the present study, three analytical methods, thin layer chromatography (TLC), high-performance liquid chromatography (HPLC) and solid phase extraction (SPE) have been tested. Reversed-phase TLC of deproteinized aqueous samples of plasma provides good estimates of 2-[(18)F]FA and its metabolites. However, because of the decreased radioactivity in plasma samples, this method can be used in humans over the first 2 h after radioligand injection only. Reliable quantification of the parent radioligand and its main metabolites was obtained using reversed-phase HPLC, followed by counting of eluted fractions in a well gamma counter. Three main and five minor metabolites of 2-[(18)F]FA were detected in human blood using this method. On average, the unchanged 2-[(18)F]FA fraction in plasma of healthy volunteers measured at 14, 60, 120, 240 and 420 min after radioligand injection was 87.3+/-2.2%, 74.4+/-3%, 68.8+/-5%, 62.3+/-8% and 61.0+/-8%, respectively. In patients with neurodegenerative disorders, the values corresponding to the three last time points were significantly lower. The fraction of nonmetabolized 2-[(18)F]FA in plasma determined using SPE did not differ significantly from that obtained by HPLC (+gamma counting) (n=73, r=.95). Since SPE is less time-consuming than HPLC and provides comparable results, we conclude that SPE appears to be the most suitable method for measurement of 2-[(18)F]FA parent fraction during PET investigations.

N,N-Dimethyl-2-(2-amino-4-methylthiophenylthio)benzylamine (SMe-ADAM, 1) is a highly potent and selective inhibitor of the serotonin transporter (SERT). This compound was labeled with carbon-11 by methylation of the S-desmethyl precursor 10 with [(11)C]methyl iodide to obtain the potential positron emission tomography (PET) radioligand [(11)C]SMe-ADAM. The radiochemical yield was 27 +/- 5%, and the specific radioactivity was 26-40 GBq/micromol at the end of synthesis. Ex vivo and in vivo biodistribution experiments in rats demonstrated a rapid accumulation of the radiotracer in brain regions known to be rich in SERT, such as the thalamus/hypothalamus region (3.59 +/- 0.41%ID/g at 5 min after injection). The specific uptake reached a thalamus to cerebellum ratio of 6.74 +/- 0.95 at 60 min postinjection. The [(11)C]SMe-ADAM uptake in the thalamus was significantly decreased by pretreatment with fluoxetine to 38 +/- 11% of the control value. Furthermore, no metabolites of [(11)C]SMe-ADAM could be detected in the SERT-rich regions of the rat brain. It is concluded that [(11)C]SMe-ADAM may be a suitable PET ligand for SERT imaging in the living brain.

This paper reports the synthesis, biological evaluation, in vitro and ex vivo autoradiography of the first Tc-99m ligand with subnanomolar affinity for the 5-HT(1A) receptor and a remarkably high affinity for the alpha1-adrenergic receptor. The neutral "3+1" mixed-ligand complex combines 4-(6-mercaptohexyl)-1-(2-methoxyphenyl)piperazine as monodentate and 3-(N-methyl)azapentane-1,5-dithiol as tridentate unit with oxotechnetium(V). The analogous rhenium complex was synthesized for complete structural characterization and used in receptor binding assays. In competition experiments both complexes display subnanomolar affinity for the 5-HT(1A) receptor (IC(50)0.24 nM for Re, 0.13 nM for Tc) but also very high affinities for the alpha1-adrenergic receptor (IC(50) 0.05 nM for Re, 0.03 nM for Tc). Biodistribution studies show a brain uptake in rat of 0.22% ID five minutes post injection. In vitro autoradiographic studies in rat brain and postmortem human brain indicate accumulation of the Tc-99m complex in brain areas which are rich in 5-HT(1A) receptors or in alpha1-adrenergic receptors. This in vitro enrichment can be blocked respectively by the 5-HT(1A) receptor agonist 8-OH-DPAT or by prazosin hydrochloride, an alpha1-adrenergic receptor antagonist. Ex vivo autoradiographic studies in rats show a slight accumulation of the Tc-99m complex in 5-HT(1A) receptor-rich areas of the brain, which could not be blocked, as well as in regions rich in alpha1-adrenergic receptors, which could be blocked by prazosin hydrochloride.

The present study describes the synthesis of the [18F]fluoromethyl analogue of (+)-McN5652 ([18F]FMe-McN) as a new potential tracer for the serotonin transporter. In vitro binding studies have shown that FMe-McN displays only slightly lower affinity for the serotonin transporter (K(i) = 2.3 +/- 0.1 nM) than (+)-McN5652 (K(i) = 0.72 +/- 0.2 nM). The radiofluorinated tracer [18F]FMe-McN was prepared by reaction of normethyl (+)-McN5652 with the fluoromethylation agent [18F]bromofluoromethane in an overall radiochemical yield of 5 +/- 1% (decay-corrected, related to [18F]fluoride) and with high specific radioactivity (200-2,000 GBq/micromol at the end of synthesis).