Synthesis and Monkey-PET Study of (R)- and (S)-18F-Labeled 2

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Synthesis and Monkey-PET Study of (R)- and (S)-18FLabeled 2-Arylbenzoheterocyclic Derivatives as Amyloid Probes with Distinctive in vivo Kinetics Yanping Yang, Xuedan Wang, Hui Yang, Hualong Fu, Jinming Zhang, Xiaojun Zhang, Jiapei Dai, Zhiyong Zhang, Chunping Lin, Yuzhi Guo, and Mengchao Cui Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00643 • Publication Date (Web): 15 Oct 2016 Downloaded from http://pubs.acs.org on October 16, 2016

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Molecular Pharmaceutics

Synthesis and Monkey-PET Study of (R)- and (S)-18F-Labeled 2-Arylbenzoheterocyclic Derivatives as Amyloid Probes with Distinctive in vivo Kinetics† Yanping Yang,‡ Xuedan Wang,‡ Hui Yang,║ Hualong Fu,‡ Jinming Zhang,║ Xiaojun Zhang,║ Jiapei Dai,§ Zhiyong Zhang,# Chunping Lin,# Yuzhi Guo,# and Mengchao Cui*,‡

†

Dedicated to Professor Boli Liu on the occasion of his 85th birthday

‡

Key Laboratory of Radiopharmaceuticals, Ministry of Education, College of Chemistry, Beijing

Normal University, Beijing 100875, P. R. China ║

Department of Nuclear Medicine, Chinese PLA General Hospital, Beijing 100853, P. R. China

§

Wuhan Institute for Neuroscience and Neuroengineering, South-Central University for Nationalities,

Wuhan 430074, P. R. China #

Beijing ZHIBO Bio-Medical Technology Co. Ltd., Beijing 102502, P. R. China

Corresponding Author: *To whom correspondence should be addressed: Phone: 086(10)58808891, Fax: 086(10)58808891. E-mail: [email protected]

1

ACS Paragon Plus Environment


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ABSTRACT This study describes an effective strategy to improve pharmacokinetics of Aβ imaging agents, offering a novel class of (R)- and (S)-18F-labeled 2-arylbenzoheterocyclic derivatives which bear an additional chiral hydroxyl group on the side chain. These ligands displayed binding abilities toward Aβ aggregates with Ki values ranging from 3.2 to 195.6 nM. Chirality-related discrepancy was observed in biodistribution and (S)-2-phenylbenzoxazole enantiomers exhibited vastly improved brain clearance with washout ratios higher than 20. Notably, (S)-[18F]28 possessed high binding potency (Ki = 7.6 nM) and exceptional brain kinetics (9.46% ID/g at 2 min, brain2 min/brain60 min = 27.8) that is superior to well-established [18F]AV45. The excellent pharmacokinetics and low nonspecific binding of (S)-[18F]28 were testified by dynamic PET/CT scans in monkey brains. In addition, (S)-[18F]28 clearly labeled Aβ plaques both in vitro and ex vivo. These results might qualify (S)-[18F]28 to detect Aβ plaques with high signal-to-noise ratio. KEYWORDS: Alzheimer’s disease; positron emission tomography; β-amyloid plaque; molecular imaging

2


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INTRODUCTION Alzheimer’s disease (AD) is an irreversible, progressive brain disorder that causes a steady decline in memory and mental function and, eventually, the ability to implement the simplest daily tasks. AD is estimated to rank as the third leading cause of death for older people, just behind heart disease and cancer.1 The clumps of extracellular β-amyloid (Aβ) plaques and threads of intracellular neurofibrillary tangles (NFTs) may damage and destroy brain cells and are considered hallmarks of AD pathology.2 Thus, brain imaging with Aβ probes using noninvasive techniques including positron emission tomography (PET) and single photon emission computed tomography (SPECT) can enable doctors to detect the brain level of Aβ plaques and facilitate identification of early-stage AD. Early diagnosis of AD attracts mounting attention in the field of biomedical research, and a big variety of amyloid imaging agents have been tested in vivo or ex vivo to detect amyloid load in AD brains. [11C]PIB, the benchmark Aβ radiotracer, has been used in various human research protocols all around the world and is able to assess the Aβ plaques status in subjects years before Alzheimer’s symptoms appear.3,

4

Unfortunately, the short half-life of

11

C (t1/2 = 20 min) has limited its

widespread clinical application and commercialization. Thereafter a myriad of 18F-labeled Aβ probes have moved through the drug development process and the longer half-life of

18

F (t1/2 = 110 min)

permits effective broad distribution. Among them, [18F]GE067 (Flutemetamol),5 [18F]AV45 (Florbetapir)6 and [18F]BAY94-9172 (Florbetaben)7 demonstrated utility in differentiating significant amyloid burdens in AD brains as opposed to the relative absence of amyloid in subjects without AD (Figure 1). All of the three tracers have been approved by U.S. FDA and readily commercialized. These tracers constituted a step forward, but they exhibited a high level of nonspecific white matter binding which could lead to lower signal-to-noise ratio and less accurate interpretation of PET 3



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scans.8, 9 Some findings explained this issue by high lipophilicity and slow tracer clearance.10 In order to identify lower levels of amyloid and achieve earlier diagnosis, a critical need to modulate lipophilicity and fine-tune washout rates from normal brain regions existed. In addition to introducing a fluoro-pegylation (FPEG) group at one terminal of stilbene scaffold, Kung et al made another attempt to develop 2-fluoromethyl-1,3-propylenediol

18

F-labeled Aβ-specific imaging agents using a chain.11

side

18

F-labeled

(E)-3-fluoro-2-((4-(4-(methylamino)styryl)phenoxy)methyl)propan-1-ol ([18F]FMAPO) exhibited a moderate log P value of 2.95, high initial brain uptake (9.75% ID/g at 2 min) and fast washout rate (0.72% ID/g at 60 min), which surpassed that of [18F]AV45 (7.33% ID/g at 2 min and 1.80% ID/g at 60 min as reported).11, 12 This result suggested that the introduction of a hydroxyl group into side chain did speed up the washout rates from normal brain regions, but it must be noted that a chiral center was generated in this case, and [18F]FMAPO was bioevaluated as a racemic mixture, which may complicate the in vivo metabolism. The complex protection-deprotection reactions involved in the synthesis of FMAPO and corresponding precursors, as well as the difficult separation of [18F]FMAPO enantiomers posed special challenges, thus Kung et al forsook clinical translation of [18F]FMAPO, and decided that FPEG stilbenes ([18F]AV45 and [18F]BAY94-9172) would be more appropriate candidates.13 Kudo et al has applied similar strategy to transform

18

F-labeled NFTs tracer THK523

(18F-6-(2-fluoroethoxy)-2-(4-aminophenyl)quinoline)

to

[18F]THK5105

(18F-1-((2-(4-(dimethylamino)phenyl)quinolin-6-yl)oxy)-3-fluoropropan-2-ol) and [18F]THK5117 (18F-1-fluoro-3-((2-(4-(methylamino)phenyl)quinolin-6-yl)oxy)propan-2-ol) by replacing FPEG with a 1-fluoro-3-(oxidanyl)propan-2-ol side chain.14-16 However, [18F]THK5105 and [18F]THK5117 were 4


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both investigated as racemic mixtures. It is universally known that chirality may influence the affinity and efficacy of drugs, and individual enantiomers usually differ significantly in bioactivity, target selectivity, pharmacokinetics and pharmacodynamics profiles, and toxicity.17, 18 Besides, the use of single-enantiomer tracers can potentially result in less complex and more selective pharmacological profiles, decreasing drug interactions and undesirable effects as compared to racemic mixtures. In the field of nuclear based molecular imaging agents, it is particularly important to identify more potent enantiomers with greater target affinity and selectivity, and better pharmacological profile, which could potentially reduce the amount of radioactivity patients are exposed to and improve image quality. Therefore, Kudo et al recently developed two novel pyridylquinoline

NFTs

[18F]THK5351

tracers,

((S)-18F-1-fluoro-3-((2-(6-(methylamino)pyridin-3-yl)quinolin-6-yl)oxy)propan-2-ol) [18F]THK5317 (also known as (S)-[18F]THK5117), which are single (S)-enantiomers.19,

and 20

In

first-in-human PET studies, [18F]THK5351 and [18F]THK5317 demonstrated lower retention in white matter areas, faster kinetics and higher contrast than [18F]THK5117. Our

group

previously

reported

a

class of

FPEG

modified

2-arylbenzoxazole

and

2-arylbenzothiazole derivatives which possessed potent Aβ binding ability but slightly high lipophilicity and slow clearance from normal brain regions.21, 22 In order to reduce lipophilicity and enhance signal-to-noise ratio, herein, we applied this new approach to 2-arylbenzoxazole, 2-arylbenzothiazole and 2-arylbenzofuran scaffolds. In this study, we examined the specific binding and pharmacokinetic properties of enantiopure (R)- and (S)-18F-labeled 2-arylbenzoheterocyclic derivatives, and performed a PET/CT scan in Rhesus monkeys. The variations in biological property resulting out of chirality have been discussed in detail. 5



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------------Figure 1 -------------

EXPERIMENTAL SECTION General remarks. Commercial reagents were used for chemical synthesis without more purification. 18F¯ was kindly provided by Chinese PLA General Hospital. 1H NMR (400 MHz) and 13

C NMR (100 MHz) spectra were obtained on a Bruker Avance III NMR spectrometer in DMSO-d6

or CDCl3 solutions at room temperature. Mass spectra were recorded on a SurveyorMSQ Plus (ESI) instrument (Waltham, MA, USA). Reactions were monitored by TLC analysis (TLC Silica gel 60 F254, Merck) and products were purified through column chromatography on silica gel (54–74 µm, Qingdao Haiyang Chemical Co., Ltd). Optical purity determination was conducted on HITACHI HPLC Primaide with a Chiralpak® AS-RH column (Daicel, 5 µm, 4.6 × 150 mm). Radio-HPLC analysis was conducted on a Shimadzu SCL-20 AVP instrument equipped with a SPD-20A UV detector (λ = 254 nm) and a Bioscan Flow Count 3200 NaI/PMT γ-radiation scintillation detector. Post-mortem brain sections were acquired from the Chinese Brain Bank Center. Normal ICR mice (20–22 g, male) were purchased from Beijing Vital River Laboratory Animal Technology Co, Ltd. Transgenic mice (C57BL6, APPswe/PSEN1) and wild-type mice (C57BL6) were purchased from the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences. Rhesus monkeys used for dynamic PET/CT imaging were kindly supplied by Beijing Institute of Xieerxin Biology Resource Co, Ltd. All experiments involving use of animals were performed in conformity with 6


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guidelines approved by the animal care committee of Beijing Normal University and Chinese PLA General Hospital. Chemistry. General synthetic procedure A: Synthesis of Compounds (R)/(S)-7-12 and (R)/(S)-23-26. To a mixture of 1-6 or 19-22 (1.0 mmol), K2CO3 (3.0 mmol) and 18-crown-6 (0.1 mmol) in acetone (50 mL) was added (R)-(-)/(S)-(+)-epichlorohydrin (1.5 mmol) dropwise, and stirred for 12 h at 80 °C. The reaction mixture was concentrated by vacuum and purified by silica gel column chromatography (petroleum ether/ethyl acetate = 1/1, v/v) to afford the pure products. General synthetic procedure B: Synthesis of Compounds (R)/(S)-13-18 and (R)/(S)-27-30. To a solution of (R)/(S)-7-12 or (R)/(S)-23-26 (0.2 mmol) in toluene (15 mL) was added a 1.0 M solution of TBAF in THF (1 mL, 1.0 mmol), and stirred for 12 h at 80 °C. The reaction mixture was concentrated by vacuum and purified by silica gel column chromatography (petroleum ether/ethyl acetate = 2/1, v/v) to afford the pure products. General synthetic procedure C: Synthesis of Compounds (R)/(S)-31, 34 and 37. Compound 2, 19 or 20 (1.0 mmol) and NaOH (1.5 mmol) were added in EtOH (30 mL) and stirred at 80 °C for 1 h. After

cooling

down

to

room

temperature,

(S)-(+)-3-chloropropane-1,2-diol

or

(R)-(-)-3-chloropropane-1,2-diol (1.4 mmol) was added, and stirred for 3 h at 80 °C. The reaction mixture was concentrated by vacuum and purified by silica gel column chromatography (petroleum ether/ethyl acetate = 1/2, v/v) to afford the pure products. General synthetic procedure D: Synthesis of Compounds (R)/(S)-32, 35 and 41. A solution of (R)/(S)-31, 34 or 40 (0.5 mmol) in anhydrous pyridine (10 mL) was stirred in an ice bath, and tosyl chloride (0.8 mmol) was slowly added. The reaction mixture was stirred for 5 h in an ice bath and concentrated under reduced pressure. After pyridine was removed by vacuum, the mixture was 7



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extracted with CH2Cl2 (3 × 10 mL). The organic layer was dried over anhydrous MgSO4, and the filtrate was concentrated by vacuum. The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate = 1/2, v/v) to afford the pure products. General synthetic procedure E: Synthesis of Compounds (R)/(S)-33, 36 and 42. To a solution of (R)/(S)-32, 35 or 41 (0.05 mmol) in CH2Cl2 (20 mL) was added pyridinium toluene-4-sulphonate (0.3 mmol) and 3,4-dihydro-2H-pyran (0.6 mmol). The mixture was stirred for 5 h at 40 °C. After the reaction was completed, the mixture was concentrated by vacuum and extracted with CH2Cl2 (3 × 10 mL). The organic layer was dried over anhydrous MgSO4, and the filtrate was concentrated by vacuum. The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate = 2/1, v/v) to afford the pure products. The yields, optical purities, NMR and MS data are provided in the Supporting Information. Radiochemistry. [18F]Fluoride trapped on a QMA cartridge was eluted into a reaction vessel with 1 mL of Kryptofix 222/K2CO3 solution (13 mg of Kryptofix 222 and 1.1 mg of K2CO3 in MeCN/H2O, 4/1). The solvent was removed under a stream of nitrogen gas at 120 °C, and then the residue was dried azeotropically with 1 mL of anhydrous acetonitrile three times. A solution of the OH-THP-protected tosylate precursors ((R)/(S)-33, 36 or 42, 5.0 mg) in MeCN (1.5 mL) was added into the reaction vessel and heated for 8 min at 100 °C. Then 0.2 mL of aqueous HCl (1 M) was added and heated for additional 5 min at 100 °C. The reaction vessel was allowed to cool to room temperature, and then the mixture was adjusted to pH 8–9 with an aqueous solution of NaHCO3. 10.0 mL of water was added and the mixture was passed through a preconditioned Sep-Pak Plus-C18 cartridge (Bonna-Agela Technologies). The cartridge was washed with 10 mL of water, and then the trapped compound was eluted with acetonitrile (2 × 1 mL) and subjected to HPLC purification on a 8


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Venusil MP C18 column (Bonna-Agela Technologies, 5 µm, 10 × 250 mm). The HPLC fraction containing the desired radiotracer was collected and concentrated under reduced pressure. The final product was diluted with 3 mL of saline for biological assessments containing 10% ethanol. The total radiosynthesis took 60 min. After HPLC purification, radiochemical purity and specific activity were determined by HPLC.

Biological Evaluation. In vitro binding assay using Aβ aggregates, partition coefficient determination, in vivo biodistribution, in vivo metabolism, in vitro and ex vivo autoradiography were all performed according to reported procedures.22, 23 PET/CT scans in rhesus monkeys: Two male rhesus monkeys were used for PET/CT imaging of (S)-[18F]28 (9 years old, 15 kg) and [18F]AV45 (8 years old, 7.5 kg). Monkey was anesthetized by intramuscular injection of ketamine hydrochloride. Anesthesia was then maintained by the administration of a mixture of isoflurane (3%–5%), oxygen (6%), and medical air with endotracheal incubation. A venous catheter was inserted into a hind limb, and the monkey was positioned on the bed of the PET (uMI 510, United Imaging, China). After completion of the transmission scan, subjects received 90 MBq of (S)-[18F]28 or [18F]AV45 as an intravenous bolus injection. Dynamic brain PET scans (0 to 60 min) started immediately after injection, and whole-body scans were obtained from 60 to 66 min. Three-dimensional ordered subsets expectation maximization (3D OSEM) algorithm was used for image reconstruction. Time frames for brain PET imaging were 6 × 20 s, 6 × 30 s, 5 × 60 s, 5 × 120 s, 10 × 240 s. Regions of interest (ROIs) outlines were drawn manually on the whole brain, frontal cortex, parietal cortex, temporal cortex, cerebellum and white matter using the CT templates. The resulting ROI was then transferred to co-registered PET images. 9



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Standardized Uptake Values (SUVs) were calculated for all ROIs by normalization of injected dose and body weight. Time−activity curves (TACs) were calculated from this.

RESULTS Chemistry. Scheme 1 outlined the synthesis of fluorinated 2-arylbenzoheterocyclic derivatives. The starting chemicals including 1–6 and 19–22 were synthesized according to previously reported procedures.22, 24, 25 The preparation of fluorinated 2-arylbenzothiazoles and 2-arylbenzoxazoles was first performed by reacting (R)-(-)- or (S)-(+)-epichlorohydrin with compounds 1–6 and 19–22 in the presence of K2CO3 to afford chiral epoxide derivatives (R)/(S)-7–12 and (R)/(S)-23–26 in yields of 11.2–70.7%. Subsequently, the epoxides were converted into the desired (R)/(S)-13–18 and (R)/(S)-27–30 in yields of 7.4–63.3%, by regioselective ring opening of epoxide with tetrabutylammonium fluoride (TBAF) in toluene. Enantiomeric purities of target compounds (R)/(S)-13–18 and (R)/(S)-27–30 were determined to be higher than 97% by chiral HPLC analysis. Detailed data and chiral HPLC chromatograms are provided in Table S1 and Figure S1. Scheme 2 outlines the synthesis of tetrahydropyran (THP) protected tosylate precursors for (R)/(S)-14, 28 and 27. The free hydroxyl groups in 2, 20 and 19 were coupled with (S)-(+)- or (R)-(-)-3-chloropropane-1,2-diol in EtOH to produce (S)/(R)-31, 34 and 37 in yields of 39.6–89.2%. For the N-monomethylated compounds, the free hydroxyl groups of (R)/(S)-37 were first protected using tert-butyldimethylsilyl chloride (TBDMSCl), and the methylamino groups were subsequently protected with a tert-butyloxycarbonyl (Boc) group. (R)/(S)-40 were obtained by deprotection of the TBS groups in (R)/(S)-39 with TBAF in tetrahydrofuran (THF). Regioselective monotosylation of the free hydroxyl moieties of (R)/(S)-31, 34 and 40 with p-tosyl chloride (TsCl) in pyridine, which 10


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was used as the solvent and base, gave (R)/(S)-32, 35 and 41 in yields of 7.0-62.3%. Since the outward free hydroxyl group of these diols exposed to less steric hindrance, favorable regioselectivity was achieved for the synthesis of monotosylates (R)/(S)-32, 35 and 41. Then, the residual free hydroxy group of (R)/(S)-32, 35 and 41 were readily protected with a THP group to produce the final tosylate precursors (R)/(S)-33, 36 and 42.

------------Scheme 1 Scheme 2 -------------

In vitro binding assay using Aβ aggregates. The binding affinity of these fluorinated (R)/(S)-2-arylbenzoheterocyclic derivatives to Aβ42 aggregates was quantitatively assessed by an inhibition binding assay using [125I]4-(6-iodoimidazo[1,2-a]pyridin-2-yl)-N,N-dimethylaniline ([125I]IMPY) as the competing radio-ligand. Table 1 summarizes the inhibition constants of ligands (R)/(S)-13–18 and 27–30. (R)/(S)-2-arylbenzoheterocyclic derivatives inhibited the binding of [125I]IMPY with Ki values varying from 3.2 to 195.6 nM. Most importantly, ligands (R)-14, 18, 28 and (S)-14, 15, 16, 18, 28 exhibited potent binding affinities with Ki values lower than 20 nM, which were comparable to that of IMPY (Ki = 12.5 nM) and AV45 (Ki = 8.8 nM), highlighting potential for the detection of Aβ plaques in the AD brain.

------------11



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Table 1 -------------

Radiochemistry. As illustrated in Scheme 3, each of the OH-THP-protected tosylate precursors was reacted with [18F]fluoride/K2CO3/Kryptofix 222 in anhydrous acetonitrile for 8 min at 100 °C. Then the mixture was treated with 0.2 mL of aqueous HCl (1 M) to remove the THP and Boc group, giving (R)/(S)-[18F]14, 27, 28 in radiochemical yield of 16–37% (decay corrected). The total radiosynthesis took 60 min. After HPLC purification, the radiochemical purity was determined to be greater than 98%. Specific activity was estimated to be approximately 80 GBq/µmol at the end of synthesis. Co-injection HPLC analysis verified the identities of these radiofluorinated tracers by retention time comparison with corresponding nonradioactive ligands (Table S2 and Figure S2). The preparation of [18F]AV45 and corresponding precursor was implemented according to previously reported procedures.26

------------Scheme 3 -------------

In vivo biodistribution. The lipophilicity of these 2-arylbenzoheterocyclic derivatives was moderate, with calculated cLog P values ranging between 2.33 and 3.99 (Table 1). As shown in Table 2, the Log D values of three pairs of radiofluorinated ligands ((R)/(S)-[18F]14, 27 and 28) measured by n-octanol/buffer shake flask method also lied within optimal Log D range (1.5–4.0). 12


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Lipophilicity in this range would be expected to provide good blood brain barrier (BBB) permeability with low plasma protein binding and metabolic liability. Then in vivo biodistribution of (R)/(S)-[18F]14, 27 and 28 was implemented in normal ICR mice to evaluate the pharmacokinetic properties. Line charts of brain and intestine uptake were shown in Figure 2, while detailed in vivo biodistribution data were summarized in Table S3−S6 in Supporting Information. Enantiopure (R)/(S)-[18F]14, 27 and 28 penetrated the BBB with high initial brain uptakes of 6.67–13.41% ID/g at 2 min post-injection (p.i.) and reduced to 0.29–3.43% ID/g at 60 min p.i.. Surprisingly, 2-phenylbenzoxazole enantiomers exhibited similar initial brain uptake but significant different washout rates at later time points. (R)-[18F]27 and (R)-[18F]28 displayed moderate washout rates with brain2 min/brian60 min ratio of 6.1 and 5.8, whereas (S)-[18F]27 and (S)-[18F]28 exhibited dramatically improved clearance profiles with several-folds higher brain2 min/brian60 min ratio of 23.0 and 27.8, respectively (Table 2). A similar chirality-related biodistribution pattern was observed for 2-phenylbenzothiazole derivatives, but the clearance for both (R)-[18F]14 (brain2 min/brian60 min = 3.9) and (S)-[18F]14 (brain2 min/brian60 min = 5.3) were relatively slow compared to 2-phenylbenzoxazoles. Racemic [18F]28 exhibited medial washout rate with brain2 min/brian60 min ratio of 8.9, approximating to the calculated ratio of 9.7 using Formula 1.

Brain2 min/Brain60 min Ratio of a Racemate =

R2 min+S2 min (1) R60 min+S60 min

R2 min and S2 min represent brain uptake of (R)- and (S)-enantiomer at 2 min p.i.. R60 min and S60 min represent brain uptake of (R)- and (S)-enantiomer at 60 min p.i..

Compared to corresponding (R)-enantiomers, (S)-enantiomers showed more remarkable intestinal 13



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accumulation of radioactivity over time. Additionally, sustained low degrees of bone uptake were observed during the entire 60 min investigation time, suggesting little defluorination in vivo.

------------Figure 2 Table 2 -------------

In vivo metabolism in mice. To investigate the chirality influence on pharmacokinetics, ((R)-[18F]28 and (S)-[18F]28 were subjected to in vivo metabolism study in normal ICR mice. Brain, plasma, liver, urine and feces samples were acquired at 2, 10 and 30 min p.i., and then analyzed with non-chiral reverse phase radio-HPLC. The recovery rate of radioactivity was approximately 70%. Across the evaluation time points, four corresponding radioactive metabolite species were detected for each (R)/(S)-[18F]28, all of which were more hydrophilic than the parent tracer with shorter retention times. Figure 3 illustrated relative percentage of the radioactive parents and key metabolites to the total radioactivity determined in brain, plasma and liver samples. Detailed metabolism data were provided in Table S7 and S8. (R)/(S)-[18F]28 exhibited good in vivo biostability in the brain with more than 80% of radioactivity attributable to parent tracers at 10 min p.i.. In plasma, similar results were observed for (R)/(S)-[18F]28 where intact forms constituted 68.4% and 60.3% of the total radioactivity at 2 min p.i., and steadily decreased to 3.9% and 8.6% at 30 min p.i., respectively. However, (R)/(S)-[18F]28 exhibited variable metabolism profile in liver, the main metabolic organ. The amount of radioactivity attributable to unmetabolized (S)-[18F]28 was approximately 3-folds 14


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lower than that of (R)- [18F]28 at 2 and 10 min p.i.. In addition, the vast majority of radioactivity was deemed to be metabolite [18F]Ms-1 (82.5%, 95.5% and 98.9% at 2, 10 and 30 min p.i., respectively), whereas metabolite [18F]MR-1 accounted for much smaller proportion of radioactivity (41.9%, 73.1% and 61.2% at 2, 10 and 30 min p.i., respectively). HPLC retention times suggested that metabolites [18F]Ms-1 and [18F]MR-1 (tR = 2.8 min) in liver were much more hydrophilic than the corresponding intact enantiomers. Radioactivity in urine samples was low at the early stages and then increased remarkably with the time passing. There were no parents in urine and all the radioactivity was presented as hydrophilic metabolites [18F]MS-1, [18F]MS-2 and [18F]MR-1, [18F]MR-2. This result implied that the hydrophilic metabolites were excreted predominantly through urine. In feces, the amount of radioactivity stayed low throughout the test time.

------------Figure 3 -------------

In vitro autoradiography. To further test the binding potential of (R)/(S)-[18F]28 to Aβ plaques, in vitro autoradiography was conducted on brain sections from AD (n = 3), cerebral amyloid angiopathy (CAA) patients (n = 2) and Tg mouse (n = 1). As shown in Figure 4, enantiopure (S)-[18F]28 displayed clear labeling of three forms of Aβ deposits including dense-core plaques (red arrows), diffuse plaques (purple arrows) on the cerebral cortex, and plaques accumulating within blood vessel walls (yellow arrows). The presence and location of these hot spots was found to be in conformity with fluorescent flecks costained with DANIR 3b, a near-infrared fluorescent compound 15



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

targeting Aβ deposits. In both human postmortem brain sections and mice brain sections, low non-specific binding was observed in white matter that lies beneath the cortex, indicating high signal sensitivity to track amyloid levels. In contrast, the healthy control subject and wild-type mouse displayed no binding signals. Furthermore, the counterpart (R)-[18F]28 showed specific binding to Aβ plaques in a similar manner with results illustrated in Figure S3.

------------Figure 4 -------------

PET/CT scans in rhesus monkeys. Encouraged by favorable binding ability and kinetic profiles of (S)-[18F]28 in rodent models, dynamic PET/CT scans were performed in rhesus monkey brains after an intravenous injection of (S)-[18F]28 (90 MBq) and [18F]AV45 (90 MBq), respectively. Figure 5 illustrated summed PET images integrated over 0–10, 10–20, 30–40, and 50–60 min p.i. of both tracers for comparison, as well as TACs derived from entire 60 min dynamic PET data. (S)-[18F]28 exhibited a relatively uniform regional distribution pattern throughout the brain compared with [18F]AV45 (Figure 5a). Frontal, parietal and temporal cortex TACs illustrated similar initial brain uptakes with SUVs of 3.88–4.69 peaked at 2–3 min but a significantly faster clearance for (S)-[18F]28 than that for [18F]AV45. The washout ratios of 3.2, 2.3 and 2.3 for [18F]AV45, which were consistent with the reported cortex washout profiles,12 were approximately only half of that for (S)-[18F]28 (5.7, 5.2 and 5.6, respectively). Besides the faster clearance from brain cortical regions examined, (S)-[18F]28 also exhibited a considerable lower degree of uptake and retention in 16


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cerebellum and white matter areas, with about 1.5 and 1.2 lower SUVs at the end of the study. The lower uptake into cerebellum and white matter resulted in lower overall whole brain uptake of (S)-[18F]28 (3.09 SUV) in comparison to [18F]AV45 (3.81 SUV). The whole brain washout ratio of 3.6 for (S)-[18F]28 was confirmed to be faster than that for [18F]AV45 (washout ratio = 2.1). The whole body image integrated over 60–66 min p.i. suggested that radioactivity mainly accumulated in the liver, bladder, and kidney (Figure S4). In line with the findings made in mice, (S)-[18F]28 seemed resistant toward defluorination, on the contrary, [18F]AV45 showed considerable accumulation of radioactivity in the bone, suggesting substantial defluorination in vivo (Figure S4).

------------Figure 5 -------------

Ex vivo autoradiography. As (S)-[18F]28 provided the best features with regard to binding ability and kinetics profile, ex vivo autoradiographic studies were implemented to evaluate its binding behavior to Aβ plaques in vivo. A Tg mouse and an age-matched wild-type control received 37 MBq of (S)-[18F]28 through the tail vein. The mice were sacrificed at 20 min p.i., and the brains were quickly removed, frozen and sectioned for ex vivo autoradiography. Autoradiography data verified the brain uptake of (S)-[18F]28 seen in biodistribution studies and represented a distinct binding to Aβ plaques in the cortex, cerebellum, and hippocampus regions of Tg mouse brain slices (Figure 6). The binding pattern of autoradiography channel is consistent with fluorescent staining results using DANIR 3b. Conversely, no apparent Aβ labeling was observed in brain slices from 17



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Page 18 of 36

wild-type mouse.

------------Figure 6 -------------

DISCUSSION To modulate lipophilicity and fine-tune pharmacokinetics of Aβ-specific PET imaging agents, we replaced the conventional FPEG chain with a 1-fluoro-3-(oxidanyl)propan-2-ol side chain. The structures of newly designed (R)/(S)-2-arylbenzoheterocyclic derivatives bear a free hydrophilic hydroxyl terminal, but a chiral center was induced in this case. Traditionally, the racemates were resolved into its individual enantiomers by chiral separation using supercritical fluid chromatography (SFC) on a Chiralpak AD column.27 In this work, enantiopure (R)-(-)-/(S)-(+)-epichlorohydrin and (S)-(+)-/(R)-(-)-3-chloropropane-1,2-diol were employed as starting compounds to provide enantiopure final products as reported.28-30 High binding affinity (Ki ≤ 20 nM) is an important prerequisite for an ideal Aβ imaging probe. As expected, these fluorinated (R)/(S)-2-arylbenzoheterocyclic derivatives maintained binding abilities toward Aβ aggregates. In particular, ligands (R)-14, 18, 28 and (S)-14, 15, 16, 18, 28 appeared to be qualified for Aβ labeling with Ki values lower than 20 nM. Besides, interesting structure-activity relationships were observed. The N,N-dimethylated derivatives ((R)/(S)-14, 16, 18, 28, 30) exhibited higher binding affinities than the corresponding N-monomethylated derivatives ((R)/(S)-13, 15, 17, 27, 29), in accordance with structure-activity relationships of stilbene,31, 18


32

benzothiazole,22

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benzoxazole,22 and benzofuran33 derivatives containing a para-aminophenyl or para-aminopyridyl group. Introducing an electronegative nitrogen atom into the aminophenyl group reduces the binding affinity for Aβ aggregates (Ki, 13 < 15, 14 < 16, 27 < 29, 28 < 30). In addition, the (R)/(S)-2-pyridylbenzoheterocyclic derivatives displayed enantiomer-related differences in binding ability toward Aβ aggregates, with (R)-enantiomers having more than 2-fold higher Ki values, except for (R)/(S)-29 (Ki = 195.6 and 186.8 nM, respectively). Conversely, chirality did not affect the binding affinity of (R)/(S)-2-phenylbenzoheterocyclic significantly that (R)/(S)-enantiomers exhibited comparable Ki values, except for (R)/(S)-28 (Ki = 15.5 and 7.6 nM, respectively). (R)/(S)-14 and (R)/(S)-28, which displayed phenomenal binding ability, were selected for

18

F

radiolabeling from corresponding 2-arylbenzothiazole and 2-arylbenzoxazole libraries. In order to investigate biological effect of substituent moiety, (R)/(S)-27 (R = H) with a N-Methylamino group were radiolabeled for biological comparison with ligands (R)/(S)-28 (R = Me), which bear a N, N-dimethylamino group. We firstly applied a reported nucleophilic epoxide opening reaction with 18

F¯ to prepare the desired

18

F-labeled 2-phenylbenzoheterocyclic ligands from its epoxide

precursors in selected solvents (CH3CN or t-amyl-OH) and base systems (K2CO3 or K2(COO)2).34 Unfortunately, the radiochemical yields were calculated to be less than 1%. Thus, an alternative radiolabeling procedure was employed in which the OH-THP-protected tosylates were used as the precursors. In in vivo biodistribution, enantiopure (S)-[18F]27 and (S)-[18F]28 exhibited extraordinary faster clearance from normal brain region than their antipodes, (R)-[18F]27 and (R)-[18F]28. In intestine, (S)-[18F]27 and (S)-[18F]28 showed more remarkable accumulation of radioactivity over time, indicating faster excretion of radioactivity through intestine, which may be in conformity with faster 19



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brain clearance profiles. Similar stereospecific pharmacokinetic properties were observed for other drugs.35, 36 The metabolism results of (R)/(S)-[18F]28 that there was a higher proportion of polar and hydrophilic metabolite [18F]Ms-1, which can be readily absorbed by the intestine and discharged from the body through urination, may explain the faster brain clearance of (S)-[18F]28. In comparison to previously reported FPEG 2-arylbenzoheterocyclic derivatives (brain2 min/brian60 min = 1.8 – 4.7),21, 22

adding a free hydroxyl terminal into the side chain did accelerate the brain clearance. In particular,

(S)-2-phenylbenzoxazoles ((S)-[18F]27 and (S)-[18F]28) achieved approximately 10-folds increase in the brain clearance ratio. In brief, (S)-[18F]28 stands out with a combination of robust Aβ-binding capacity (Ki = 7.6 ± 2.8 nM), high initial brain uptake (brain2 min = 9.46% ID/g), rapid brain clearance (brain2

min/brain60 min

= 27.8) and little defluorination (bone60

min

= 1.13% ID/g), significantly

overstepping that of [18F]AV45 (Ki = 8.8 ± 1.5 nM, brain2 min = 6.59% ID/g, brain2 min/brain60 min = 3.3, bone60 min = 3.32% ID/g) under the identical experimental conditions. These results demonstrate high potential of the single (S)-enantiomer, (S)-[18F]28, in achieving preferable signal-to-noise ratio and support it as a promising Aβ imaging candidate. Dynamic PET/CT scans in rhesus monkeys further confirmed the high BBB penetration ability and excellent washout kinetics for (S)-[18F]28. Although the improvement in clearance profile was less apparent than biodistribution results in normal ICR mice, the whole brain washout ratio of 3.6 for (S)-[18F]28 was confirmed to be faster than that for [18F]AV45 (washout ratio = 2.1). This discrepancy may be explained by interspecies difference between rodent animals and non-human primates. Besides the faster clearance from all examined brain cortex regions (frontal cortex, parietal cortex, temporal cortex), (S)-[18F]28 also exhibited lower retention in white matter and cerebellum, which is usually used as a reference area. This lower retention suggested lower degree of nonspecific 20


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binding, and might be an advantage over clinical 18F amyloid radiotracers whose white matter background are relatively high. Additionally, (S)-[18F]28 exhibited little defluorination in vivo, but [18F]AV45 showed remarkable bone accumulation of radioactivity. Reported PET imaging data of [18F]AV45 in humans also showed a high level of radioactivity retention in the bones,37 which can cause undesired signals, and thereby interfere with the quantification of specific signals during brain PET scan. Finally, in vitro and ex vivo autoradiography results demonstrated that (S)-[18F]28 did pass through the BBB and selectively label Aβ plaques with little nonspecific binding. In conclusion, these preliminary results support (S)-[18F]28 as a promising Aβ imaging candidate. The clinical investigations of (S)-[18F]28 in AD patients and healthy volunteers that are under way will clarify its usefulness for the early diagnosis of AD pathology. Additionally, the hydroxylation of the fluoroalkoxy side chain has proven to be effective in improving pharmacokinetics, and it may be applicable in other 18F-labeled imaging agents.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. In vitro autoradiography, in vivo biodistribution, metabolism, PET/CT data, as well as supplementary data for chemical synthesis.

AUTHOR INFORMATION Corresponding Author E-mail: [email protected]. Tel/Fax: 086(10)58808891. 21



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Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This study was supported by the National Science and Technology Major Projects for Major New Drugs Innovation and Development (Grant No. 2014ZX09507007-002), the National Natural Science Foundation of China (Grant No. 21571022, 21201019 and 30670586), and the Research Fund for the Doctoral Program of Higher Education of China (Grant No. 20130003120012).

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Table 1. Inhibition constants for the binding of [125I]IMPY to Aβ42 aggregates.

a

Ligands

Position

Chiral center

R

X

Y

cLog Pa

Ki (nM)b

(R)-13 (S)-13 (R)-14 (S)-14 (R)-15 (S)-15 (R)-16 (S)-16 (R)-17 (S)-17 (R)-18 (S)-18

6 6 6 6 6 6 6 6 6 6 6 6

R S R S R S R S R S R S

H H Me Me H H Me Me H H Me Me

S S S S S S S S O O O O

CH CH CH CH N N N N CH CH CH CH

26.0 ± 6.5 27.2 ± 2.9 3.7 ± 1.1 3.2 ± 1.1 45.4 ± 11.8 9.9 ± 2.8 27.2 ± 7.1 11.1 ± 5.4 103.2 ± 18.6 128.6 ± 40.2 11.0 ± 1.5 10.8 ± 2.6

(R)-27

5

R

H

O

CH

3.80 3.80 3.99 3.99 3.06 3.06 3.31 3.31 3.42 3.42 3.09 3.09 3.42

(S)-27 (R)-28 (S)-28 (R)-29 (S)-29 (R)-30 (S)-30 IMPY AV45

5 5 5 5 5 5 5 -

S R S R S R S -

H Me Me H H Me Me -

O O O O O O O -

CH CH CH N N N N -

3.42 3.09 3.09 2.40 2.40 2.39 2.39 4.43 3.61

85.3 ± 5.2 15.5 ± 3.2 7.6 ± 2.8 195.6 ± 49.1 186.8 ± 39.0 57.4 ± 7.6 27.2 ± 6.3 12.5 ± 1.4 8.8 ± 1.5

67.3 ± 5.5

Calculated with the online ALOGPS 2.1 program. bMeasured in triplicate with values given as the mean ± SD.

26


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Table 2. Comparison of binding affinities and kinetic properties. a

Ligands

Log D

Ki (nM)

18

4.11 ± 0.08 3.97 ± 0.18 3.57 ± 0.11 3.46 ± 0.06 3.89 ± 0.12 3.84 ± 0.07 -

3.7 ± 1.1 3.2 ± 1.1 67.3 ± 5.5 85.3 ± 5.2 15.5 ± 3.2 7.6 ± 2.8 8.8 ± 1.5

(R)-[ F]14 (S)-[18F]14 (R)-[18F]27 (S)-[18F]27 (R)-[18F]28 (S)-[18F]28 Racemic [18F]28 [18F]AV45

Brain uptake (% ID/g)b 2 min

60 min

Brain2 min/Brain60 min ratio

13.41 ± 2.00 8.95 ± 0.61 7.74 ± 1.00 6.67 ± 0.34 9.16 ± 0.34 9.46 ± 0.58 10.93 ± 0.25 6.59 ± 0.36

3.43 ± 0.58 1.69 ± 0.31 1.27 ± 0.15 0.29 ± 0.06 1.58 ± 0.10 0.34 ± 0.03 1.23 ± 0.10 2.02 ± 0.19

3.9 5.3 6.1 23.0 5.8 27.8 8.9 3.3

a

a

Measured in triplicate with values given as the mean ± SD. bEach value represents the mean ± SD for 5–6 mice at

each interval.

OH

N NH

18

S

HO

18

O

F

F

3

18

NH

X

F

[18 F]BAY94-9172: X = CH [18F]AV45: X = N

[18F]GE067

18

F

F

∗

NH [18F]FMAPO (Racemate )

OH 18

O

O

∗

OH 18

O

F

O ( S)

N

N

N R

[18 F]THK523

NH 2

N Me

[18F]THK5105: R = Me [18F]THK5117: R = H

[18F]THK5351: X = N X [18F]THK5317: X = CH

N H

Me

Mirror N 18

F

O

n

Y

X

Y N R

N R

N X

X = S, O; Y = CH, N R = H, CH3; n = 1-3

5 O 6

(S) 18

F

OH

18

(R) F

5 O OH 6

N X

Y N R

( R)/(S )-2-arylbenzoheterocyclic derivatives X = S, O; Y = CH, N; R = H, CH3

Figure 1. Chemical structures of Aβ and tau PET probes and the newly designed (R)/(S)-18F-labeled 2-arylbenzoheterocyclic derivatives.

27



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 2. Line charts of brain and intestinal uptake of (R)/(S)-[18F]14, 27, 28 and [18F]AV45 in normal ICR mice at different post-injection time points. Error bars represent standard deviations for 5–6 mice at each interval.

28


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Figure 3. Percentages of parent tracers extracted from the brain (a), plasma (b) and liver (c) of ICR mice after intravenous injection of (R)- or (S)-[18F]28. (d) Percentages of metabolites [18F]MR-1 and [18F]MS-1 extracted from liver. n = 2 for each data point and error bars represent standard deviations.

29



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 4. In vitro autoradiography of (S)-[18F]28 on brain sections from five AD/CAA patients, a healthy human (male, 84 years old, frontal lobe), a Tg mouse (C57BL6, APPswe/PSEN1, male, 11 months old, whole brain, transverse view) and a wild-type mouse (C57BL6, male, 11 months old, whole brain, transverse view). AD/CAA brain section information: AD1, male, 91 years old, temporal lobe; AD2, female, 64 years old, temporal lobe; AD3, female, 71 years old, temporal lobe; CAA1, male, 70 years old, temporal lobe; CAA2, female, 68 years old, frontal lobe. The presence and location of plaques were confirmed by fluorescence staining using DANIR 3b. Red and blue squares mark areas for magnification as presented in bottom panels. Red arrows, purple arrows and yellow arrows indicate dense-core deposits, diffuse deposits, and deposits accumulating along the vessel walls, respectively. 30


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Figure 5. Dynamic PET/CT study of (S)-[18F]28 and [18F]AV45 in normal rhesus monkeys. (a) Transverse, coronal, and sagittal CT and summed PET images of monkey head after administration of (S)-[18F]28 and [18F]AV45. Color coding depicts mean SUVs integrated over 0–10, 10–20, 30–40, and 50–60 min after injection. (b–c) TACs of (S)-[18F]28 and [18F]AV45 in frontal cortex, parietal cortex, temporal cortex, whole brain, cerebellum and white matter for the entire 60 min dynamic PET scan.

31



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 6. Ex vivo plaque labeling of (S)-[18F]28. (a) Tg mouse (C57BL6, APPswe/PSEN1, male, 15 months old). (b) Age-matched wild-type mouse (C57BL6, male, 15 months old). Fluorescence costaining using DANIR 3b presented on the right panel confirmed the presence and location of plaques.

32


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Scheme 1. Reagents and conditions: (a) K2CO3, 18-crown-6, acetone, reflux; (b) TBAF (1 M in THF), toluene, 80 °C.

33



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Page 34 of 36

Precursors of (R)/(S)-14 5

5

N

HO

N

6

a

5 c

OH

TsO

O OTHP

S

TsO

Precursors of (R)/(S)-28 HO

HO

N

OTHP O (R) 5 6

TsO

(R)/(S)-32

N S (S)-33

OH O

TsO

N

5 6

a

OH (R)-(-)/(S)-(+)

O 20

c

Cl

N S

O OH

OH HO

N

5 6

O OTHP

(R)-33

TsO

N

6

(S)

N

6

(R)/(S)-31 5

N

b

S

N

6

(R)

N

O

2

5

N

6 HO

OH (R)-(-)/(S)-(+)

S

HO

Cl

O

b

N

N

5 6

O

N O

(R)/(S)-34

TsO

N N O

OTHP O (S) 5 6

(R)-36

(R)/(S)-35

N N O (S)-36

Precursors of (R)/(S)-27 OH HO

HO

N

5 6

HO

NH

TBSO

OTBS O

f

HO

O

Boc

OTHP O (R) 5 6

TsO N

O

Boc

NH O (R)/(S)-38

b

N N O

Boc

(R)/(S)-40

N

N

OH O 5 6

(R)/(S)-39

TsO

5 6

(R)/(S)-37

N O

OTBS O

d

NH

OH N

5 6

c

TBSO

N

5 6

a

OH (R)-(-)/(S)-(+)

O 19

e

Cl

O

OTHP O (S) 5 6

(R)-42

TsO

O 5 6

N N O

Boc

(R)/(S)-41

N N O

Boc

(S)-42

Scheme 2. Reagents and conditions: (a) NaOH, EtOH, H2O, 80 °C; (b) TsCl, pyridine, 0 °C; (c) PPTS, CH2Cl2, 3,4-dihydro-2H-pyran, 40 °C; (d) TBDMSCl, CH2Cl2, imidazole, 40 °C; (e) (Boc)2O, THF, 85 °C; (f) TBAF, THF, 30 °C.

34


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Scheme 3. Reagents and conditions: (a) Kryptofix 222, K2CO3 or K2(COO)2, t-amyl-alcohol, 100 °C or 110 °C, 5/10/30 min; (b) Kryptofix 222, K2CO3, min; (c) 1 M HCl (aq), 100 °C, 5 min, NaHCO3.

35


18

18

F¯, MeCN or

F¯, MeCN, 100 °C, 8


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

30x10mm (300 x 300 DPI)


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Synthesis and Monkey-PET Study of (R)- and (S)-18F-Labeled 2

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