Improved radiosynthesis and preliminary in vivo evaluation of the 11C-labeled tetrazine [11C]AE-1 for pretargeted PET imaging

Pretargeted nuclear imaging based on the ligation between tetrazines and nano-sized targeting agents functionalized with trans-cyclooctene (TCO) has recently been shown to improve both imaging contrast and dosimetry in nuclear imaging of nanomedicines. Herein, we describe the improved radiosynthesis of a ¹¹C-labeled tetrazine ([¹¹C]AE-1) and its preliminary evaluation in both mice and pigs. Pretargeted imaging in mice was carried out using both a new TCO-functionalized polyglutamic acid and a previously reported TCO-functionalized bisphosphonate system as targeting agents. Unfortunately, pretargeted imaging was not successful using these targeting agents in pair with [¹¹C]AE-1. However, brain imaging in pig indicated that the tracer crossed the blood-brain-barrier. Hence, we suggest that this tetrazine scaffold could be used as a starting point for the development of pretargeted brain imaging, which has so far been a challenging task.     

Synthesis and preclinical evaluation of [18F]FSL25.1188, a reversible PET radioligand for monoamine oxidase-B

Carbon-11 labeled SL25.1188 is a promising reversible monoamine oxidase-B (MAO-B) radioligand that was recently translated for human positron emission tomography (PET) imaging. Herein, we report the development of a novel fluorinated derivative, namely, [¹⁸F](S)-3-(6-(3-fluoropropoxy)benzo[d]isoxazol-3-yl)-5-(methoxymethyl)oxazolidin-2-one ([¹⁸F]FSL25.1188; [¹⁸F]6), as a candidate ¹⁸F-labeled MAO-B radioligand, and, its subsequent preclinical evaluation in non-human primates (NHP). [¹⁸F]6 was produced and isolated (>6 GBq) with high radiochemical purity (>99%), and molar activity (>100 GBq/µmol at time of injection). Autoradiography studies conducted in post-mortem human brain sections revealed [¹⁸F]6 binding in MAO-B rich regions. PET imaging study of [¹⁸F]6 in NHP showed high brain uptake (SUV > 2.5) as well as a regional brain radioactivity distribution in accordance with MAO-B expression. [¹⁸F]6 displayed favorable in vivo kinetics, with an early peak in the time-activity curve followed by progressive wash-out from the NHP brain. Specificity of [¹⁸F]6 was investigated in a pre-treatment study with l-deprenyl (1.0 mg/kg) wherein reduced radioligand uptake was observed in all MAO-B rich regions. Results from the current preclinical investigation suggests [¹⁸F]6 is a promising MAO-B PET radioligand. Further evaluation of [¹⁸F]6 and structurally related ¹⁸F-analogs are underway to identify an optimized candidate for clinical research studies.     

Synthesis and evaluation of the novel 2-[18F]fluoro-3-propoxy-triazole-pyridine-substituted losartan for imaging AT1 receptors

The 2-[¹⁸F]fluoro-3-pent-4-yn-1-yloxypyridine ([¹⁸F]FPyKYNE) analog of the potent non-peptide angiotensin II type 1 receptor (AT₁R) blocker losartan was produced via click chemistry linking [¹⁸F]FPyKYNE to azide-modified tetrazole-protected losartan followed by TFA deprotection. Preliminary small animal imaging with positron emission tomography (PET) in rats displayed high uptake in the kidneys with good contrast to surrounding tissue. Rat metabolism displayed the presence of 23% unchanged tracer in plasma at 30 min. Upon co-administration with AT₁R blocker candesartan (2.5, 5 and 10 mg/kg), a dose-dependent reduction (47–65%) in tracer uptake was observed in the kidney, while no difference was observed following AT₂R blocker PD123,319 (5 mg/kg), indicating binding selectivity for AT₁R over AT₂R and potential for imaging AT₁R using PET.     

Synthesis and evaluation of novel N-fluoropyridyl derivatives of tropane as potential PET imaging agents for the dopamine transporter

A series of novel N-fluoropyridyl-containing tropane derivatives were synthesized and their binding affinities for the dopamine transporter (DAT), serotonin transporter (SERT) and norepinephrine (NET) were determined via competitive radioligand binding assays. Among these derivatives, compound 6d showed the highest binding affinity to DAT (K_i = 4.1 nM), and selectivity for DAT over SERT (5-fold) and NET (16-fold). Compound 6d was radiolabeled with Fluorine-18 in two steps. Regional brain distribution and ex vivo autoradiography studies of [¹⁸F]6d demonstrated that the ligand was selectively localized in the striatum region, where DAT binding sites are highly expressed. [¹⁸F]6d may be useful as a potential radioligand for imaging DATs with PET.     

Synthesis and evaluation of 1-[2-(4-[11C]methoxyphenyl)phenyl]piperazine for imaging of the serotonin 5-HT7 receptor in the rat brain

1-[2-(4-Methoxyphenyl)phenyl]piperazine (4) is a potent serotonin 5-HT₇ receptor antagonist (K_i = 2.6 nM) with a low binding affinity for the 5-HT_1A receptor (K_i = 476 nM). As a potential positron emission tomography (PET) radiotracer for the 5-HT₇ receptor, [¹¹C]4 was synthesized at high radiochemical yield and specific activity, by O-[¹¹C]methylation of 2′-(piperazin-1-yl)-[1,1′-biphenyl]-4-ol (6) with [¹¹C]methyl iodide. Autoradiography revealed that [¹¹C]4 showed in vitro specific binding with 5-HT₇ in the rat brain regions, such as the thalamus which is a region with high 5-HT₇ expression. Metabolite analysis indicated that intact [¹¹C]4 in the brain exceeded 90% of the radioactive components at 15 min after the radiotracer injection, although two radiolabeled metabolites were found in the rat plasma. The PET study of rats showed moderated uptake of [¹¹C]4 in the brain (1.2 SUV), but no significant regional difference in radioactivity in the brain. Pretreatment with 5-HT₇-selective antagonist SB269970 (3) did not decrease the uptake of [¹¹C]4 in the rat brain. Further studies are warranted that focus on the development of PET ligand candidates with higher binding affinity for 5-HT₇ and higher in vivo stability in brain than 4.     

Synthesis and biological evaluation of positron emission tomography radiotracers targeting serotonin 4 receptors in brain: [18F]MNI-698 and [18F]MNI-699

Two new benzodioxane derivatives were synthesized as candidates to image the serotonin 4 receptors by positron emission tomography (PET) and radiolabeled with fluorine-18 via a two-step procedure. Competition binding assays demonstrated that MNI-698 and MNI-699 had sub-nanomolar binding affinities against rat striatal 5-HT₄ receptors (K_i of 0.20 and 0.07 nM, respectively). PET imaging in rhesus monkey showed that the regional brain distribution of [¹⁸F]MNI-698 and [¹⁸F]MNI-699 were consistent with the known densities of 5-HT₄ in brain. [¹⁸F]MNI-698 and [¹⁸F]MNI-699 are among the first fluorine-18 radiotracers developed for imaging the 5-HT₄ receptors in vivo and are currently under preclinical investigation in primates for future human use.     

Improved radiosynthesis of the apoptosis marker 18F-ICMT11 including biological evaluation

We improved the specific radioactivity of the apoptosis imaging isatin derivative (18)F-ICMT11. We then evaluated (18)F-ICMT11 in EL4 tumor-bearing mice 24h after treatment with etoposide/cyclophosphamide combination therapy. Dynamic PET imaging demonstrated increased uptake in the drug-treated (0.115±0.011 SUV) compared to the vehicle-treated EL4 tumors (0.083±0.008 SUV). This effect correlated to the observed increases in apoptotic index.     

Synthesis and preliminary evaluation of a novel glutamine derivative: (2S,4S)4-[18F]FEBGln

We report the preparation of a novel glutamine derivative, (2S,4S)-2,5-diamino-4-(4-(2-fluoroethoxy)benzyl)-5-oxopentanoic acid, (2S, 4S)4-[¹⁸F]FEBGln ([¹⁸F]4), through efficient organic and radiosyntheses. In vitro assays of [¹⁸F]4 using MCF-7 cells showed that it entered cells via multiple amino acid transporter systems including system L and ASC2 transporters but not through the system A transporter. [¹⁸F]4 showed promising properties for tumor imaging and may serve as a lead compound for further optimizing and targeting the system L transporter associated with enhanced glutamine metabolism in cancer cells.     

Radiosynthesis and evaluation of [11C]EMPA as a potential PET tracer for orexin 2 receptors

EMPA is a selective antagonist of orexin 2 (OX₂) receptors. Previous literature with [³H]-EMPA suggest that it may be used as an imaging agent for OX₂ receptors; however, brain penetration is known to be modest. To evaluate the potential of EMPA as a PET radiotracer in non-human primate (as a step to imaging in man), we radiolabeled EMPA with carbon-11. Radiosynthesis of [¹¹C]N-ethyl-2-(N-(6-methoxypyridin-3-yl)-2-methylphenylsulfonamido)-N-(pyridin-3-ylmethyl)acetamide ([¹¹C]EMPA), and evaluation as a potential PET tracer for OX₂ receptors is described. Synthesis of an appropriate non-radioactive O-desmethyl precursor was achieved from EMPA with sodium iodide and chlorotrimethylsilane. Selective O-methylation using [¹¹C]CH₃I in the presence of cesium carbonate in DMSO at room temp afforded [¹¹C]EMPA in 1.5–2.5% yield (non-decay corrected relative to trapped [¹¹C]CH₃I at EOS) with ?95% chemical and radiochemical purities. The total synthesis time was 34–36 min from EOB. Studies in rodent suggested that uptake in tissue was dominated by nonspecific binding. However, [¹¹C]EMPA also showed poor uptake in both rats and baboon as measured with PET imaging.     

Radiosynthesis of [thiocarbonyl-11C]disulfiram and its first PET study in mice

[Thiocarbonyl-¹¹C]disulfiram ([¹¹C]DSF) was synthesized via iodine oxidation of [¹¹C]diethylcarbamodithioic acid ([¹¹C]DETC), which was prepared from [¹¹C]carbon disulfide and diethylamine. The decay-corrected isolated radiochemical yield (RCY) of [¹¹C]DSF was greatly affected by the addition of unlabeled carbon disulfide. In the presence of carbon disulfide, the RCY was increased up to 22% with low molar activity (A_m, 0.27 GBq/μmol). On the other hand, [¹¹C]DSF was obtained in 0.4% RCY with a high A_m value (95 GBq/μmol) in the absence of carbon disulfide. The radiochemical purity of [¹¹C]DSF was always >98%. The first PET study on [¹¹C]DSF was performed in mice. A high uptake of radioactivity was observed in the liver, kidneys, and gallbladder. The uptake level and distribution pattern in mice were not significantly affected by the A_m value of the [¹¹C]DSF sample used. In vivo metabolite analysis showed the rapid decomposition of [¹¹C]DSF in mouse plasma.     

Towards tropomyosin-related kinase B (TrkB) receptor ligands for brain imaging with PET: Radiosynthesis and evaluation of 2-(4-[18F]fluorophenyl)-7,8-dihydroxy-4H-chromen-4-one and 2-(4-([N-methyl-11C]-dimethylamino)phenyl)-7,8-dihydroxy-4H-chromen-4-one

The interaction of tropomyosin-related kinase B (TrkB) with the cognate ligand brain-derived neurotrophic factor (BDNF) mediates fundamental pathways in the development of the nervous system. TrkB signaling alterations are linked to numerous neurodegenerative diseases and conditions. Herein we report the synthesis, biological evaluation and radiosynthesis of the first TrkB radioligands based on the recently identified 7,8-dihydroxyflavone chemotype. 2-(4-[¹⁸F]fluorophenyl)-7,8-dihydroxy-4H-chromen-4-one ([¹⁸F]10b) was synthesized in high radiochemical yields via an efficient S_NAr radiofluorination involving a para-Michael acceptor substituted aryl followed by BBr₃-promoted double demethylation. Selective N-[¹¹C]methylation afforded 2-(4-([N-methyl-¹¹C]-dimethylamino)phenyl)-7,8-dihydroxy-4H-chromen-4-one ([¹¹C]10c) from the fully deprotected catechol-bearing normethyl precursor 13 with [¹¹C]MeOTf. In vitro autoradiography of [¹⁸F]10b with transverse rat brain sections revealed high specific binding in the cortex, striatum, hippocampus and thalamus in accordance with expected TrkB distribution. Blockade experiments with both 7,8-dihydroxyflavone (1a) and TrkB cognate ligand, BDNF, led to decreases of 80% and 85% of radioligand binding strongly supporting the hypothesis that 7,8-dihydroxyflavones exert their effect on TrkB phosphorylation via direct TrkB extracellular domain (ECD) binding. Positron emission tomography (PET) studies revealed that [¹⁸F]10b and [¹¹C]10c brain uptake is minimal and that they are rapidly eliminated from the plasma (effective plasma half-life 5–10 min) via hepatic secretion. Nevertheless, the high specific binding and TrkB specificity derived from in vitro experiments suggests that the 7,8-disubstituted flavone chemotype represents a promising scaffold for the development of TrkB radiotracers for PET.     

11C]enzastaurin, the first design and radiosynthesis of a new potential PET agent for imaging of protein kinase C

Enzastaurin (LY317615) is a potent and selective protein kinase C (PKC) inhibitor with an IC₅₀ value of ∼6 nM. [¹¹C]Enzastaurin (3-(1-[¹¹C]methyl-1H-indol-3-yl)-4-[1-[1-(2-pyridinylmethyl)-4-piperidinyl]-1H-indol-3-yl]-1H-pyrrole-2,5-dione), a new potential PET agent for imaging of PKC, was first designed and synthesized in 20-25% decay corrected radiochemical yield and 370-555 GBq/μmol specific activity at end of bombardment (EOB). The synthetic strategy was to prepare a carbon-11-labeled maleic anhydride intermediate followed by the conversion to maleimide.     

Synthesis and preliminary evaluation of 4-hydroxy-6-(3-[11C]methoxyphenethyl)pyridazin-3(2H)-one,a 11C-labeled d-amino acid oxidase (DAAO) inhibitor for PET imaging

Selective DAAO inhibitors have demonstrated promising therapeutic effects in clinical studies, including clinically alleviating symptoms of schizophrenic patients and ameliorating cognitive function in Alzheimer’s patients with early phase. Herein we report the synthesis and preliminary evaluation of a ¹¹C-labeled positron emission tomography ligand based on a DAAO inhibitor, DAO-1903 (8). ¹¹C-Isotopologue of 8 was prepared in high radiochemical yield with high radiochemical purity (>99%) and high molar activity (>37 GBq/µmol). In vitro autoradiography studies indicated that the ligand possessed high in vitro specific binding to DAAO, while in vivo dynamic PET studies demonstrated that [¹¹C]8 failed to cross the blood–brain barrier possibly due to moderate brain efflux mechanism. Further chemical scaffold optimization is necessary to overcome limited brain permeability and improve specific binding.     

Synthesis and biological evaluation of both enantiomers of [18F]flubatine,promising radiotracers with fast kinetics for the imaging of α4β2-nicotinic acetylcholine receptors

Both enantiomers of the epibatidine analogue flubatine display high affinity towards the α4β2 nicotinic acetylcholine receptor (nAChR) in vitro, accompanied by negligible interactions with diverse off-target proteins. Extended single dose toxicity studies in rodent indicated a NOEL (No Observed Effect Level) of 6.2 μg/kg for (−)-flubatine and 1.55 μg/kg for (+)-flubatine. We developed syntheses for both flubatine enantiomers and their corresponding precursors for radiolabeling. The newly synthesized trimethylammonium precursors allowed for highly efficient ¹⁸F-radiolabelling in radiochemical yields >60% and specific activities >750 GBq/μmol, thus making the radioligands practical for clinical investigation.     

Radiosynthesis and evaluation of a fluorine-18 labeled radioligand targeting vesicular acetylcholine transporter

Vesicular acetylcholine transporter (VAChT) is a reliable biomarker for assessing the loss of cholinergic neurons in the brain that is associated with cognitive impairment of patients. 5-Hydrotetralin compound (±)-5-OH-VAT is potent (K_i?=?4.64?±?0.32?nM) and selective for VAChT (>1800-fold and 398-fold for σ₁ and σ₂ receptor, respectively) with favorable hydrophilicity (LogD?=?1.78), while (?)-5-OH-VAT originally serves as the radiolabeling precursor of (?)-[¹⁸F]VAT, a promising VAChT radiotracer with a logD value of 2.56. To evaluate (?)-5-OH-[¹⁸F]VAT as a radiotracer for VAChT, we performed in vitro binding assay to determine the potency of the minus enantiomer (?)-5-OH-VAT and plus enantiomer (+)-5-OH-VAT, indicating that (?)-5-OH-VAT is a more potent VAChT enantiomer. Radiosynthesis of (?)-5-OH-[¹⁸F]VAT was explored using three strategies. (?)-5-OH-[¹⁸F]VAT was achieved with a good yield (24?±?6%) and high molar activity (～37?GBq/µmol, at the end of synthesis) using a microwave assisted two-step one-pot procedure that started with di-MOM protected nitro-containing precursor (?)-6. MicroPET studies in the brain of nonhuman primate (NHP) suggest that (?)-5-OH-[¹⁸F]VAT readily penetrated the blood brain barrier and specifically accumulated in the VAChT-enriched striatum with improved washout kinetics from striatum compared to [¹⁸F]VAT. Nevertheless, the lower target to non-target ratio may limit its use for in vivo measurement of the VAChT level in the brain.     

Development and characterization of a promising fluorine-18 labelled radiopharmaceutical for in vivo imaging of fatty acid amide hydrolase

Fatty acid amide hydrolase (FAAH), the enzyme responsible for terminating signaling by the endocannabinoid anandamide, plays an important role in the endocannabinoid system, and FAAH inhibitors are attractive drugs for pain, addiction, and neurological disorders. The synthesis, radiosynthesis, and evaluation, in vitro and ex vivo in rat, of an ¹⁸F-radiotracer designed to image FAAH using positron emission tomography (PET) is described.Fluorine-18 labelled 3-(4,5-dihydrooxazol-2-yl)phenyl (5-fluoropentyl)carbamate, [¹⁸F]5, was synthesized at high specific activity in a one-pot three step reaction using a commercial module with a radiochemical yield of 17–22% (from [¹⁸F]fluoride). In vitro assay using rat brain homogenates showed that 5 inhibited FAAH in a time-dependent manner, with an IC₅₀ value of 0.82 nM after a preincubation of 60 min. Ex vivo biodistribution studies and ex vivo autoradiography in rat brain demonstrated that [¹⁸F]5 had high brain penetration with standard uptake values of up to 4.6 and had a regional distribution which correlated with reported regional FAAH enzyme activity. Specificity of binding to FAAH with [¹⁸F]5 was high (>90%) as demonstrated by pharmacological challenges with potent and selective FAAH inhibitors and was irreversible as demonstrated by radioactivity measurements on homogenized brain tissue extracts.We infer from these results that [¹⁸F]5 is a highly promising candidate radiotracer with which to image FAAH in human subjects using PET and clinical studies are proceeding.     

Synthesis and in vitro evaluation of small-molecule [18F] labeled gonadotropin-releasing hormone (GnRH) receptor antagonists as potential PET imaging agents for GnRH receptor expression

Two novel small molecule gonadotropin-releasing hormone (GnRH) receptor antagonists (12 and 13) of the furamide-class were synthesized and evaluated in vitro for their receptor binding affinities for the rat GnRH receptor. Radiolabeling with no carrier added fluorine-18 of the appropriate precursors was investigated in a one-step reaction. Log P (Octanol/PBS pH 7.4) and serum stability of the compounds were investigated. The antagonists showed low nM affinity for the rat GnRH receptor. ¹⁸F-radiolabled compounds were obtained in high radiochemical purity (>95%) and specific activity (>75 GBq/μmol). These findings suggest this class of compounds holds promise as potential probes for PET targeting of GnRH-receptor expression.     

The first radiosynthesis of [11C]AZD8931 as a new potential PET agent for imaging of EGFR,HER2 and HER3 signaling

The reference standard AZD8931{2-(4-((4-((3-chloro-2-fluorophenyl)amino)-7-methoxyquinazolin-6-yl)oxy)piperidin-1-yl)-N-methylacetamide} (11a) was synthesized from methyl 4,5-dimethoxy-2-nitrobenzoate or ethyl 4,5-dimethoxy-2-nitrobenzoate and 2-chloro-N-methylacetamide in 11 steps with 2–5% overall chemical yield. The precursor N-desmethyl-AZD8931{2-(4-((4-((3-chloro-2-fluorophenyl)amino)-7-methoxyquinazolin-6-yl)oxy)piperidin-1-yl)acetamide} (11b) was synthesized from methyl 4,5-dimethoxy-2-nitrobenzoate or ethyl 4,5-dimethoxy-2-nitrobenzoate and 2-bromoacetamide in 11 steps with 2–4% overall chemical yield. The target tracer [¹¹C]AZD8931 {2-(4-((4-((3-chloro-2-fluorophenyl)amino)-7-methoxyquinazolin-6-yl)oxy)piperidin-1-yl)-N-[¹¹C]methylacetamide} ([¹¹C]11a) was prepared from N-desmethyl-AZD8931 (11b) with [¹¹C]CH₃OTf under basic condition (NaH) through N-[¹¹C]methylation and isolated by HPLC combined with solid-phase extraction (SPE) in 40–50% radiochemical yield based on [¹¹C]CO₂ and decay corrected to end of bombardment (EOB) with 370–1110 GBq/μmol specific activity at EOB.     

Synthesis and biological evaluation of 2-(3,4-dimethoxyphenyl)-6-(2-[18F]fluoroethoxy)benzothiazole ([18F]FEDBT) for PET imaging of breast cancer

Given the ever-present demand for improved PET radiotracer in oncology imaging, we have synthesized 2-(3,4-dimethoxyphenyl)-6-(2-[¹⁸F]fluoroethoxy)benzothiazole ([¹⁸F]FEDBT), a fluorine-18-containing fluoroethylated benzothiazole to explore its utility as a PET imaging tracer. [¹⁸F]FEDBT was prepared via kryptofix-mediated nucleophilic substitution of the tosyl group precursor. Fractionated ethanol-based solid-phase (SPE cartridge-based) purification afforded [¹⁸F]FEDBT in 60% radiochemical yield (EOB), with radiochemical purity in excess of 98% and the specific activity was 35 GBq/μmol. The radiotracer displayed clearly higher cellular uptake ratio in various breast cancer cell lines MCF7, MDA-MB-468 and MDA-MB-231. However, both biodistribution and microPET studies have showed an higher abdominal accumulation of [¹⁸F]FEDMBT and the tumor/muscle ratio of 1.8 was observed in the MDA-MB-231 xenograft tumors mice model. Further the lipophilic improvement is needed for the reducement of hepatobilliary accumulation and to promote the tumor uptake for PET imaging of breast cancer.     

Synthesis, radiosynthesis and in vivo preliminary evaluation of [11C]LBT-999, a selective radioligand for the visualisation of the dopamine transporter with PET

LBT-999 (8-((E)-4-fluoro-but-2-enyl)-3beta-p-tolyl-8-aza-bicyclo[3.2.1]octane-2beta-carboxylic acid methyl ester), a cocaine derivative belonging to a new generation of highly selective dopamine transporter (DAT) ligands, and its corresponding carboxylic acid derivative, the latter used as precursor for labelling both with tritium and the positron-emitter carbon-11 (half-life: 20.38 min), were synthesized from (R)-cocaine. [(3)H]LBT-999 (>99% radiochemically pure, specific radioactivity of 3.1 TBq/mmol) was prepared from [(3)H]methyl iodide, allowing its in vitro pharmacological evaluation (K(D): 9 nM for DAT and IC(50) > 1000 nM for SERT and NET). Routine production batches of 4.5-9.0 GBq of iv injectable solutions of [(11)C]LBT-999 (with specific radioactivities ranging from 30 to 45 GBq/mumol) were prepared in 25-30 min (HPLC purification and formulation included) using the efficient methylation reagent [(11)C]methyl triflate. The preliminary in vivo pharmacological evaluation of [(11)C]LBT-999, using both biodistributions in rats and brain imaging in monkeys with positron emission tomography (PET), clearly illustrates that this ligand is an excellent candidate for quantification with PET of DAT in humans.