Synthesis and biological evaluation of ¹⁸F-labelled deuterated tropane derivatives as dopamine transporter probes

2.1 General information

Unless otherwise stated, all reagents were purchased from Energy Chemical (Shanghai, China) or J&K Scientific (Shanghai, China). Semi-preparative high-performance liquid chromatography (HPLC) consists of Waters 1525 HPLC pump, UV-2489 Detector (Waters, USA), and flow-count radioactivity detector (BioScan, USA). An analytical HPLC system was used, consisting of Waters 1525 pump, UV-2998 detector (Waters, USA), radioactive GABI Nova detector (ELYSIA RAYTEST, Germany) and an analytical C18 column (5 μm, 4.6 mm × 150 mm) was purchased from Phenomenex. Kryptfix®222 (K_2.2.2, 98.0%) was purchased from ABX (Germany). Solid-phase extraction (SPE) cartridges SepPak QMA light and SepPak C18 Plus were bought from Waters (Milford, USA). The C18 cartridge was activated using ethyl alcohol (EtOH, 20 mL), followed by sterile water (20 mL). Radioactivity was counted with a γ-counter (2480 Wizard², PerkinElmer, USA).

Compounds were purified on flash chromatography performed on silica gel (200–300 or 300–400 mesh) and detected by analytical thin-layer chromatography (TLC) using an ultraviolet (UV) lamp at 254 nm. Chemical compounds were characterized by proton and carbon nuclear magnetic resonance (¹H NMR, ¹³C NMR and ¹⁹F NMR) and mass spectrometry (MS) or gas chromatograph-mass spectrometry (GC-MS) data. NMR spectra were recorded on a Bruker spectrometer operating at 400 or 600 MHz for ¹H, 100 or 150 MHz for ¹³C, and 376 or 565 MHz for ¹⁹F acquisitions. Samples were dissolved in chloroform-d (CDCl₃) with tetramethylsilane as an internal standard and chemical shifts were expressed as ppm. J was expressed as Hz, and its splitting patterns were reported as s = singlet, d = doublet, t = triplet, m = multiple, or dd = doublet of doublets. The purities of all new compounds determined by analytical HPLC were confirmed to be more than 95% pure.

Animal studies were carried out in accordance with the guidelines put forth by the Animal Care and Ethics Committee of Jiangsu Institute of Nuclear Medicine. Male Sprague-Dawley (SD, 220–260 g) rats were provided by Cavens Laboratory Animal Technology Co. Ltd. (Jiangsu, China). Unilateral-lesioned PD model rats were induced by stereotaxic injection of 16 µg 6-hydroxydopamine (6-OHDA) (Truong et al., 2006).

2.2 Chemical synthesis

General Procedure for the Preparation of 1 and 3a-e. Compound 1 was synthesized according to the reported method (Zhao et al., 2019). Compound 3a-e was prepared according to previously reported methods (Zheng and Mulholland 1996; Gu et al., 2001). All NMR spectra and data are presented in the supporting materials.

2.3 Radiochemistry

[¹⁸F]4a-e and [¹⁸F]6a-e were prepared by a two-step one-pot automatic radiosynthesis of the tosylate precursor according to the previously reported method (Koivula et al., 2005) on a commercially available PET-MF-2 V-IT-1 (Beijing PET Technology, China) [¹⁸F]fluoride multifunction synthesis module (Scheme 2) with slight modification. [¹⁸F]fluoride was produced by the cyclotron via the ¹⁸O (p, n)¹⁸F nuclear reaction and trapped on a SepPak QMA light cartridge. [¹⁸F]F^- trapped in the cartridge was eluted by a stock solution (15 mg of K₂₂₂ in 800 µL of acetonitrile (CH₃CN)/3.0 mg of K₂CO₃ in 100 µL of water). The [¹⁸F]F^- elution solution was evaporated at 105 °C for 3 min in a stream of nitrogen. Afterward, CH₃CN (1.0 mL) was added to the reaction tube and the reaction mixture was dried twice at 105 °C under N₂. Precursor 1 or 7 (10.0 mg), in CH₃CN (500 µL) was added, and the mixture was heated at 90 °C for 15 min. After cooling to room temperature, dry DMF solution containing 3 (12.0 mg) was added, the mixture was heated to 135 °C for 25 min, and then cooled to room temperature. Next, the reaction was quenched with 1.0 mL HPLC solvent, and the crude product was purified by HPLC. HPLC purification was performed on a Phenomenex Luna C18 column (10 mm × 250 mm, 5 µm) with a mobile phase of CH₃CN/H₂O (60/40, v/v) (0.1% triethylamine, TEA) at a flow rate of 4.0 mL/min. The retention time of [¹⁸F]4a-e or [¹⁸F]6a-e was ∼ 20.0 min, the required radioactive product was collected and diluted with 30 mL H₂O, transferred to a C18 cartridge, and then the cartridge was washed with 10 mL H₂O. The trapped [¹⁸F]4a-e or [¹⁸F]6a-e was eluted with 2.0 mL EtOH and the final product was formulated in a solution containing 10% EtOH in 0.9% saline (0.5 mL, 10% ethanol/normal saline) prior to administration. The total radiosynthesis time was in the range of 90–110 min. The product was identified by co-injection of the corresponding non-radioactive ligand and its radiochemical purity was measured by an analytical HPLC containing a Gemini NX-C18 column (3 mm × 150 mm, 5 µm) with a mobile phase of methanol/water/trifluoroacetic acid (MeOH/H₂O/TFA, 40/60/0.1, v/v/v) at 1.0 mL/min flowing rate. The molar activity was calculated by a calibration curve of the non-radioactive compound through HPLC with a UV detector.

2.4 In vitro stability

The in vitro stability assay of all ¹⁸F-labelled probes was performed by evaluating their radiochemical purity using a radio-HPLC system. The [¹⁸F]4a-e or [¹⁸F]6a-e (37 MBq) were added to fetal bovine serum (FBS, 500 µL) and phosphate-buffered saline (PBS, pH 7.4, 500 µL), respectively. At indicated time points (0, 1, 2, 4, and 6 h), the serum samples (100 µL) were mixed with MeOH (100 µL) at each time point and centrifuged at 14,000×g in an Eppendorf 5415R centrifuge (Eppendorf, Germany) for 5 min. The radiochemical purity (RCP) of ¹⁸F-labelled probes in supernatant and the PBS solution was determined by radio-HPLC.

2.5 Distribution coefficient (logD_7.4)

The values of logD_7.4 were determined by the conventional shaking-flask method with n-octanol and PBS as a fractionation system (n = 3). [¹⁸F]4a-e or [¹⁸F]6a-e (18.5 KBq) was added into a two-phase mixture of PBS (pH 7.4, 3.0 mL) and n-octanol (3.0 mL) and vortexed for 5 min. The PBS and the n-octanol phase were separated via centrifugation (5 min, 1000 r/min). Then, the PBS (1.0 mL) and n-octanol (1.0 mL) solutions were pipetted into γ-counting tubes and determined by a γ-counter, respectively. The logD values were calculated by the formula: logD = log (CPM_n-octanol/CPM_PBS). The above experiment was repeated 5 times to obtain an average logD value.

2.6 In vitro competitive binding assay

The SD rats were decapitated under ether anesthesia and striata were removed immediately and homogenized through a high throughput group grinder in 50 mM Tris-HCl buffer (pH 7.4) containing 120 mM NaCl, 2 mM KCl, 1 mM CaCl₂, and 1 mM MgCl₂. After centrifuging at 14,000 g at 4 °C for 5 min, the supernatant was discarded. The precipitate was diluted into 100 mg/mL of striatum homogenate in Tris-HCl buffer, which was divided into multiple tissue samples with a volume of 20 µL (2 mg of striatum homogenate). A competitive binding assay was performed with 20 µL striatal homogenate, 20 µL ^99mTc-TRCDAT-1 (∼18.5 KBq, 925KBq/mL), a known DAT binding ligand (Bao et al., 2000), and 20 µL of non-radioactivity competing ligands (non-radioactivity compound 4a-e and 6a-e, 10^-6 to 10^-12 M) in 440 µL Tris-HCl buffer. The non-specific binding was determined in the presence of DAT inhibitor (CFT, 20 µM) (Milius et al., 1991). After incubating at 37 °C for 60 min, these mixtures were centrifuged for 5 min (14,000×g/min), and the supernatant and precipitates were separated respectively. Tris-HCl buffer (400 µL) was added to wash the striatum precipitates and centrifugation for 5 min (14,000×g/min) again. The bound and the free radioactivity were separated by centrifuging and measured with a γ-counter separately. Data of competition experiments were analyzed using the nonlinear least-square curve fitting program to determine semi-inhibitory concentrations (IC₅₀) with GraphPad Prism.

2.7 Cell uptake and blocking assays

The human neuroblastoma (SH-SY5Y) cells (1 × 10⁵ cells/100 mL) were grown in 24-well plates and assigned to three groups: control, CFT blocking, and DTBZ pretreated group. The control group was added with [¹⁸F]4a-e (37 KBq), CFT blocking group was added with [¹⁸F]4a-e and CFT (20 μM), and DTBZ (Kilbourn et al., 1996) (VMAT2 inhibitor) pretreated group was added with [¹⁸F]4a-e and DTBZ (20 μM). The cells were preincubated with 20 µM DTBZ for 10 min. Then the cells were incubated at 37 °C for 15, 30, 60, 90 and 120 min, respectively. After reaching the expected incubation time, the cells were washed twice with 1.0 mL of ice-cold PBS. Subsequently, the cells were lysed and collected with sodium hydroxide (1.0 mol/mL). The radioactivity in the cell lysates and supernatants were measured via a γ-counter. The percentage of the cell counts was calculated by the cell counts and the total counts.

2.8 In vivo PET imaging studies

A microPET system (Inveon 5000, Germany) was used for real-time scanning of rat brain. The SD rats were fixed to a small animal PET scanner bed under 2% isoflurane anesthesia. After adjusting the location of the brain, dynamic PET scans were acquired from 0 to 120 min after tail vein injection of [¹⁸F]4a-e or [¹⁸F]6a-e (∼10 MBq, 0.5 mL, 10 % ethanol/saline) respectively. After the scanning, the obtained imaging data was segmented into a sequence of 43 frames: 10 × 0.5 min, 5 × 1 min, 10 × 2 min and 18 × 5 min. The dynamic images were obtained by reconstruction with the filtered back projection (FBP) algorithm. Image analyses were performed using ASIPro VM software (Concorde Microsystems) and time-activity curves (TACs) of the regions of interest (ROIs, striatum and cerebellum) were plotted from the dynamic images.

To verify the specific binding of [¹⁸F]4e to DAT in vivo, DAT inhibitor CFT (1.0 mg/kg) was injected into normal rats at 40 min after [¹⁸F]4e injection and dynamic imaging was performed for 120 min.

Additionally, to further verify the binding specificity of [¹⁸F]4e, microPET scans of DAT blocking and semi-PD model rats were performed. The time window of these PET scans was set at 40–70 min post tracer injection since a relatively high and stable ST/CB values were observed from the TACs of [¹⁸F]4e in normal rats. The control and unilateral-lesioned PD model rats were injected with [¹⁸F]14e (∼10 MBq). The blocking rats were co-injected with [¹⁸F]14e (∼10 MBq) and CFT (1.0 mg/kg). All rats were scanned at 40 min after injection and continued for 30 min (40–70 min). Brain imaging images were collected and analyzed using ASIPro VM software.

2.9 Ex vivo autoradiography with [¹⁸F]4e

The specific binding ability of [¹⁸F]4e to DAT was further confirmed with ex vivo autoradiography. The control male rat was injected with [¹⁸F]4e (∼185 MBq) and sacrificed 40 min later, the brains were immediately dissected and frozen at −25 °C and sliced into 20 µm coronal sections with a cryostat microtome (HM525, Thermo, Germany). The slices were exposed to the imaging plate for 1 h and scanned with a phosphine screen imager. Then images were attained using Cyclone Plus Storage Phosphor System (Perkin Elmer, USA), and ROIs (striatum and cerebellum) were drawn manually. Ex vivo autoradiography was quantified as ratios of the striatum to the cerebellum. For blocking studies, the rat was co-injected with [¹⁸F]4e and CFT (1 mg/kg), and the exposure and imaging procedures were the same as described above.

2.10 Biodistribution with [¹⁸F]6e and [¹⁸F]4e

All SD rats were injected with a solution of [¹⁸F]6e or [¹⁸F]4e (∼18.5 MBq/rat) to examine the biodistribution. These rats were sacrificed at 5, 15, 30, 60 and 120 min post injection (n = 5). The blood (200 μL) was obtained from the carotid artery. The brain tissues including the striatum, cerebellum, hippocampus and cortex were dissected and other organs of interest (heart, liver, spleen, lung, kidney, stomach, small intestines, testis, bone, muscle, pancreas) were quickly removed and weighed. Then radioactivity of these samples was measured with a γ-counter. The percentage of the injected dose per gram (%ID/g) of brain tissues and the percentage of the injected dose per organ (%ID) of the interested organs were calculated with sample counts and decay-corrected 1% predose counts (100-fold dilution of injection).

2.11 Metabolite analyses with [¹⁸F]6e and [¹⁸F]4e

Radio-metabolites were analyzed using HPLC slightly modified from previously reported methods (Jin et al., 2018). Two-months-old rats were injected with [¹⁸F]4e or [¹⁸F]6e (∼185 MBq, 0.5 mL, 10% ethanol/normal saline) intravenously and sacrificed at 5, 15, 30, 60 and 120 min (n = 3) post injection. Plasma, striatum and cerebellum samples were rapidly collected. The samples were homogenized in 0.5 mL MeOH containing 1.0 mg/ml FP-CIT (20 µM), the homogenates were centrifuged at 14,000 g at 4 °C in an Eppendorf 5415R centrifuge for 5 min and the supernatant and the precipitate were collected. The sample extraction efficiency was calculated by measuring the radioactivity counts of the supernatant and the precipitate. The supernatant of the centrifuged samples was injected into the analytic HPLC system for the analysis of in vivo stability. The mobile phase was composed of MeOH/H₂O/TFA (40/60/0.1, v/v/v), and the flow rate was 1.0 mL/min. The HPLC fractions at 0.5 min intervals were collected with an automatic collector for 20 min. Then, each fraction tube was counted separately using a well γ-counter. The percentages of the parent tracer were calculated as the ratio of the radioactivity of the parent compound fractions to the total amount of radioactivity collected.

2.12 Statistical analysis

Unless otherwise stated, all data were presented as mean ± standard deviation (sd) as indicated in the legends of the figures. To determine the statistical significance of the experiments, t-test was used and p-values of <0.05 were considered significant.

3.1 Chemical synthesis

Syntheses of the non-radioactive ligands 4a-e and 6a-e are shown in Scheme 1. Briefly, 3-fluoropropyl-1,1,2,2,3,3-d₆ 4-methylbenzenesulfonate (2) was synthesized from propane-1,3-diyl-d₆ bis (4-methylbenzenesulfonate) (1) using nucleophilic fluorination with TBAF in 50% yields. Subsequent alkylation of compound 3a-e with 2 or 5 gave compounds 4a-e and 6a-e respectively in 40–60% yields.

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Scheme 1

Design and synthesis of non-radioactive fluorinated standards.

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Scheme 2

Radiosynthesis of [¹⁸F]4a-e and [¹⁸F]6a-e.

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3.2 Radiosynthesis, lipophilicity, and in vitro stability

All ¹⁸F-labelled ligands ([¹⁸F]4a-e and [¹⁸F]6a-e) were radio-synthesized by a two-step one-pot approach with nortropane precursors reacting with [¹⁸F]fluoropropyl tosylate ([¹⁸F]5) or deuterated [¹⁸F]fluoropropyl tosylate ([¹⁸F]2) (Scheme 2). The decay-corrected radiochemical yields (RCYs) of ligands were obtained between 10.6 ± 2.8% and 35.1 ± 5.4% (Table S1), for an averaged synthesis time of 90–110 min. All the RCPs were >99% (Fig. S2) and the molar activities were >20 GBq/μmol (Table S1). As a final step, a sterile 0.9% saline solution of ¹⁸F-labelled ligands was prepared for in vitro and in vivo experiments.

The radiochemical purity of all ¹⁸F-labelled ligands remained at least 96% after being incubated in FBS and PBS for 6 h at 37 °C (Fig. S3). The LogD values of all ¹⁸F-labelled probes were measured to be 0.60–2.50 (n = 3) (Table S2).

3.3 In vitro binding assay

Upon synthesis, compounds 4a-e and 6a-e were compared for their in vitro binding activity toward DAT. For this purpose, we determined their ability to inhibit DAT activity using of IC₅₀ values against ^99mTc-TRODAT-1, a verified DAT tracer (Bao et al., 2000). As shown in Table 1, compounds 4a-e exhibited good potency with IC₅₀ values between 2 and 17 nM. The compounds FP-CIT-d₆ (4e) and FP-CIT (6e) bearing an iodophenyl on tropane demonstrated the highest potency among these compounds, with an IC₅₀ value of 2.7 nM and 3.2 nM respectively. The competition curves of the 10 compounds are shown in Fig. S1.

Table 1 In vitro binding assay results of 4a-e and 6a-e.

compound	R	X	IC50 (nM)
4a	D	H	17.56
4b	D	F	14.56
4c	D	Cl	5.89
4d	D	CH3	7.60
4e	D	I	2.67
6a	H	H	20.53
6b	H	F	15.87
6c	H	Cl	5.59
6d	H	CH3	7.26
6e	H	I	3.16

3.4 Cellular uptake and blocking assays

The cell uptake of [¹⁸F]4a-e was assessed in SH-SY5Y cells. The binding specificity of [¹⁸F]4a-e was preliminarily evaluated in cells treated with DAT inhibitor CFT and VMAT2 inhibitor DTBZ. We observed that [¹⁸F]4e showed the highest cell uptake (12%) and CFT inhibition efficacy (∼72%) among [¹⁸F]4a-e. However, the differences in cell uptake among [¹⁸F]4a-e were not significant (P > 0.05). Contrary to the CFT inhibition effect, little difference of [¹⁸F]4a-e uptake was found between the DTBZ group and the control group within 120 min (Fig. S4).

3.5 MicroPET

After the synthesis of ¹⁸F-labelled ligands, dynamic PET acquisition was carried out in SD rats (n = 3). Fig. 2a-d and Fig. S5 show TACs of PET imaging with [¹⁸F]4a-e and [¹⁸F]6a-e. Among them, [¹⁸F]4c and [¹⁸F]4e showed greater average standardized uptake values (SUV) (4.06 ± 1.45 and 3.76 ± 1.73, respectively) of striatum and target-to-background (striatum/cerebellum) standardized uptake value ratios (SUVr) (3.16 ± 1.62 and 3.20 ± 1.73) (Table 2). However, [¹⁸F]4e showed the highest maximum SUVr of 6.84. Furthermore, the TACs of [¹⁸F]4e since 20 min showed higher striatum/cerebellum (ST/CB) than those of other ligands until the end of the PET scan (Fig. S5).

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Fig. 2

Time-activity curves of PET imaging with [¹⁸F]6e or [¹⁸F]4e in rat brain. The TACs of brain SUV (a-b) and SUVr (c-d) of [¹⁸F]6e and [¹⁸F]4e in the rat. The TACs of brain SUV (e) and SUVr (f) of [¹⁸F]4e in control (non-blocking) or blocking (injected CFT at 40 min) rat. Data are expressed as mean ± sd (n = 3).

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Considering the lowest IC₅₀ and highest SUVr (ST/CB) maximum of PET imaging results of [¹⁸F]4e, we selected [¹⁸F]4e for further in vivo PET study and compared with its nondeuterated compound [¹⁸F]6e. Fig. 3a-b illustrated representative PET images of [¹⁸F]4e and [¹⁸F]6e in rat brains. Summation images between 0 and 120 min after the radioligand injection showed a high signal in the striatum, a region with a high DAT concentration. Low radioactivity was observed in the cerebellum, a region with low DAT expression. As shown in the TACs of brain regions (Fig. 2b), SUV of the striatum gradually increased after [¹⁸F]4e injection, peaked at 5 min (SUV = 6.68) and then reduced steadily. The SUVr of [¹⁸F]4e reached 6.84 at around 90 min (Fig. 2d). The average SUV and SUVr of [¹⁸F]4e were 4.06 ± 1.45, and 3.20 ± 1.73 within 120 min, respectively (Table 2).

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Fig. 3

Coronal microPET images obtained from SD rats injected with [¹⁸F]4e (a) or [¹⁸F]6e (b) at 0–120 min. (c) Representative PET images of the control group, CFT blocking group and semi-PD model group at 40–70 min post injection. The striatum and lateral skull regions were highlighted with ellipses and arrows, respectively. (d) Quantification of PET of ST/CB. Data are expressed as mean ± sd (n = 3). ****P < 0.0001.

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To further demonstrate the targeting specificity of [¹⁸F]4e in vivo, a blocking test was performed with DAT inhibitor CFT (Milius et al., 1991) (1 mg/kg) at 40 min after the probe injection, and the result showed that the striatum uptake and TAC ratio reduced dramatically after CFT injection (Fig. 2e-f). The specificity of the probe to DAT was thus proved. Representative images and quantitative analysis of the control group, CFT blocking group were shown in Fig. 3c-d.

To investigate the ability of [¹⁸F]4e to detect the DAT deficit, the semi-PD model rats (lesioned in the right ST with 6-OHDA) were used for microPET imaging. As shown in Fig. 3c, the radioactivity of the un-lesioned side of the striatum was similar with control rats (ST_unlesioned/CB = 3.21 ± 0.46), while the lesioned side showed dramatically reduced radioactivity similar to background (ST_lesioned/CB = 1.04 ± 0.09) (Fig. 3d).

3.6 Ex vivo autoradiography

Fig. 4 showed representative ex vivo autoradiograms of [¹⁸F]4e on the rat brain slices. Control rat slice images showed high uptake in the DAT-rich striatum. A low signal was seen in the DAT non-rich regions such as the cerebellum and cortex (CX). When rats were co-injected with CFT, the radioactivity of [¹⁸F]4e in the striatum of these slices declined dramatically. In the control rat, the ST/CB and ST/CX value of the control group was 11.28 ± 0.83 and 7.18 ± 0.57, respectively. Contrarily, no visual difference in radioactivity levels among the striatum, cortex and cerebellum of brain slices in the blocking rat was observed. The ST/CB and ST/CX of the blocking group significantly decrease to 1.22 ± 0.09 and 1.04 ± 0.07, respectively (****P < 0.0001). These results indicated that the binding pattern of [¹⁸F]4e in rat brain was specific to DAT.

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Fig. 4

Ex vivo autoradiography of [¹⁸F]4e in control or blocking rat brain. ST: striatum, CB: cerebellum, CX: cortex. ****P < 0.0001 (n = 4).

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3.7 Biodistribution

Biodistribution studies quantify tracer accumulation in organs and determine metabolic pathways. Fig. 5 shows the radioactivity distribution over 120 min after injection of [¹⁸F]6e and [¹⁸F]4e in rats.

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Fig. 5

Biodistribution of [¹⁸F]6e (a) and [¹⁸F]4e (b) in SD rats. The ratio of uptake in the striatum to the cerebellum for [¹⁸F]6e and [¹⁸F]4e (c). Data were expressed as % injected dose/organ and expressed as mean ± sd (n = 5).

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These studies demonstrated [¹⁸F]4e has reversible binding, as well as [¹⁸F]6e, in the peripheral main organs, with a maximum uptake between 5 and 15 min. The liver had the highest uptake with 12.85 ± 2.02 %ID at 15 min, similar to [¹⁸F]6e. Initial accumulation in the kidneys of [¹⁸F]4e was 2.05 ± 0.33 %ID, which was compatible with [¹⁸F]6e (Table S3, S4). It was observed that radioactivity in other organs decreased gradually after the initial uptake (Fig. 5a-b). Furthermore, the radioactivity of bone (<0.09 %ID) were quite low for both radiotracers within 120 min.

In brain tissues, High radioactivity was shown in the striatum since 5 min, striatum uptake was the highest after injection, while low radioactivity was observed in the cerebellum and cortex. The radioactivity of [¹⁸F]4e in the striatum and cerebellum respectively reached 0.95 ± 0.16 and 0.75 ± 0.32 % ID/g at 5 min and decreased to 0.21 ± 0.15 and 0.04 ± 0.01 % ID/g at 120 min. [¹⁸F]4e displayed a higher ST/CB ratio of 5.89 ± 1.23 at 60 min, 5.84 ± 1.36 at 120 min (Fig. 5c, Table S5, S6), while the ST/CB of [¹⁸F]6e were 4.42 ± 0.86 and 4.54 ± 0.07 at 60 min and 120 min respectively. These results showed that the ratio of ST/CB of [¹⁸F]4e was higher than that of [¹⁸F]6e between 60 min and 120 min.

3.8 Metabolite analysis

The extraction efficiency of the total radioactivity was 85 ± 5% (Table S7). Radio-metabolites of [¹⁸F]6e or [¹⁸F]4e in rat brain and plasma were analyzed at 5, 15, 30, 60 and 120 min after tracer injection through HPLC (Fig. S7). The percentage of unmetabolized parental [¹⁸F]4e in plasma decreased throughout the time course, from 85 ± 9% at 5 min to 15 ± 5% at 120 min. Meanwhile, intact [¹⁸F]6e in plasma showed lower concentration from 5 to 120 min as compared with [¹⁸F]4e, from 48 ± 7% at 5 min to 6 ± 5% at 120 min post injection. In the brain striatum, the parent compound of [¹⁸F]4e accounted for about 84 ± 4% at 120 min, whereas the percentage of [¹⁸F]6e was only 65 ± 3% at the same time point post injection. In the cerebellum, [¹⁸F]4e also possessed higher intact tracer concentration than [¹⁸F]6e, for example, 56 ± 6% vs 15 ± 5% at 120 min post injection (Fig. S7 and Table S8). The results demonstrated that metabolic rates of [¹⁸F]4e in plasma, striatum and cerebellum were slower than [¹⁸F]6e (Fig. 6).

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Fig. 6

Metabolism analysis of intact [¹⁸F]4e (red) and [¹⁸F]6e (blue) in rat plasma, striatum and cerebellum (% of total radioactivity, mean ± sd, n = 3).

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