Design and Preclinical Evaluation of Novel 18F-Labeled Phenoxyalkylguanidines Radiotracers for Norepinephrine Transporter PET Imaging

Design and Preclinical Evaluation of Novel 18F-Labeled Phenoxyalkylguanidines Radiotracers for Norepinephrine Transporter PET Imaging | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Design and Preclinical Evaluation of Novel 18 F-Labeled Phenoxyalkylguanidines Radiotracers for Norepinephrine Transporter PET Imaging Yueqi Wang, Mingxing Hu, Lili Pan, Yang Xie, Zheng Cheng, Kai Lu, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8300571/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The norepinephrine transporter (NET) is a pivotal target for imaging sympathetic nerves and neuroendocrine tumors, and positron emission tomography (PET) tracers have demonstrated substantial utility for precise clinical diagnosis among other imaging modalities. In this work, we constructed a series of aryl ¹⁸F-labeled PET tracers from the innovative phenoxyalkylguanidine scaffold, featuring longer, optimized alkoxy chains to enhance hydrophobic interactions. The tracers were efficiently prepared via the photocatalyzed 18 F-fluorination, among which the [ 18 F] o FPhOEG demonstrated high radiochemical conversion (82.8%), favorable lipophilicity (logD = -0.39), and superior NET affinity (IC 50 = 0.097 µM). In vivo evaluations revealed high and sustained target uptake (myocardial SUVmax = 8.95 in rats; tumor SUVmax = 23.35 in mice). Notably, the newly developed phenoxyalkyl core structure enabled rapid hepatic clearance, leading to significantly improved heart-to-liver and tumor-to-liver ratios (3-fold higher than [ 18 F] m FBG), which are crucial for enhancing diagnostic contrast. Additionally, the tracer was successfully produced using a commercial automated synthesis module in high RCY from a readily available precursor, guaranteeing the clinical translation of [ 18 F] o FPhOEG for cardiac and oncologic diagnosis. Nuclear Medicine & Medical Imaging Norepinephrine transporter cardiac and oncologic diagnosis phenoxyalkylguanidine 18F-fluorination PET imaging Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction The norepinephrine transporter (NET), a member of the solute carrier 6 (SLC6) family, plays a pivotal role in regulating synaptic norepinephrine (NE) levels by mediating its reuptake from the synaptic cleft into presynaptic neurons in both the central and peripheral nervous systems 1 . This mechanism is essential for maintaining noradrenergic signaling homeostasis, which governs a range of physiological functions, including mood regulation, attention, pain perception, and stress responses. Norepinephrine reuptake occurs primarily through an active, saturable, ATP-dependent process known as uptake 1 and, to a lesser extent, via a passive, non-saturable, energy-independent diffusion mechanism known as uptake 2. In addition to its physiological expression in sympathetic neurons, NET is also overexpressed in neural crest-derived tumors (NCTs), including neuroblastoma, pheochromocytoma, and paraganglioma 2,3 . As a result, NET serves as a critical target for radiopharmaceuticals, particularly for the diagnosis and treatment of neuroendocrine tumors and for imaging myocardial sympathetic innervation 4,5 . Over the past decades, major efforts have focused on developing NET-targeted radiopharmaceuticals based on norepinephrine analogs and phenylalkylguanidines, and the [ 123 I]mIBG remains the clinical gold standard for imaging myocardial sympathetic innervation and neuroendocrine tumors 3,6-10 . However, its limited resolution, challenging quantification, poor sensitivity for small metastases, high background signal, and the need for 24-hour uptake reduce its clinical effectiveness 3,11 . In contrast, the structurally similar PET tracer [ 18 F]mFBG offers markedly improved spatial resolution, sensitivity, specificity, and quantitative capability, supported by the favorable physical properties and broad availability of 18 F-fluorine. Consequently, several 18 F-labeled PET tracers, including 18 F-LMI1195 8,12 , [ 18 F]4F-mHPG 9 , [ 18 F]3F-pHPG 13 , and [ 18 F]AF78 10,14 , were developed for non-invasive assessment of sympathetic nervous system tumors. Despite this progress, most PET tracers remain structurally limited to phenylalkylguanidine variants, and further clinical translation and development are hindered by background uptake, inefficient 18 F-labeling strategies, and low radiochemical yields. Recent breakthroughs in determining the structure of NET have revealed the molecular basis of its interactions with transport substrates (NE and mIBG) and inhibitors 15-18 . These findings provide crucial insights into the design of NET-targeting drugs. NET adopts an inward-open conformation when binding mIBG, with the binding site enclosed by transmembrane helices TM1, TM3, TM6, and TM8, comprising three subsites (A, B, and C). The guanidine of mIBG forms hydrogen bonds with Phe317, Ser318, and Asp75 in subsite A. The benzyl group occupies hydrophobic pocket B formed by Val148, Tyr152, Phe317, Phe323, and Ser419, while the iodine atom engages in hydrophobic interactions with Gly149, Ser419, and Gly423 in subsite C (Figure 1B). NE binds to NET with a binding mode highly analogous to that of MIBG. Despite mIBG's longer aminoalkyl side chain, it achieves optimal interactions through conformational flexibility. Notably, substantial unoccupied space persists adjacent to hydrophobic subsites B and C, potentially accommodating bulkier aryl substituents. Reported alkoxy-substituted phenylalkylguanidine derivatives (e.g., 18 F-LMI1195 and 18 F-AF78) likely bind NET via this mechanism. Leveraging these structural insights, we initiated the development of novel NET-targeted PET tracers with enhanced imaging performance and facile radiolabeling characteristics. Our group recently established a photocatalyzed azeotropic drying-free 18 F-fluorination ( PhAzF-18 ) labeling method, enabling direct 18 F-deoxyfluorination of challenging electron-rich aryl substrates from readily accessible precursors under mild conditions 19-22 . This 18 F-labeling strategy has demonstrated outstanding potential for radiopharmaceutical discovery and clinical tracer production 23-25 . In this work, leveraging the spatial tolerance afforded by the expansive hydrophobic pocket (subsites B/C) and our efficient 18 F-labeling strategy, we designed a series of novel phenoxyalkylguanidine derivatives rather than the frequently used phenylalkylguanidine skeleton, featuring an electron-rich aromatic ring and extended linkers. Through comprehensive screening, we identified a series of novel NET-targeted phenoxyalkylguanidine PET tracers with superior diagnostic efficiency. The preferred tracer [ 18 F] o FPhOEG ([ 18 F]8- o -a) outperformed the clinically used [ 18 F] m FBG, showing significantly higher target uptake and improved target-to-background ratios in myocardial/neuroendocrine tumor models. Furthermore, its automated production was also achieved efficiently, positioning this novel NET-targeted tracer as a promising candidate for clinical translation. 2.1. Design and synthesis of phenoxyalkylguanidine PET tracers An ideal NET-targeted tracer requires high binding affinity, rapid NET transport kinetics, and prolonged retention to achieve superior target-to-background ratios. Utilizing insights derived from the NET structural framework, we have developed a series of phenoxyalkylguanidine derivatives featuring extended linkers. These structural modifications were aimed at enhancing binding affinity by optimizing complementarity with the hydrophobic cavity of NET, and increasing the electron density of the aromatic ring to improve the efficiency of 18 F-fluorine incorporation. Through streamlined Chan-Lam coupling and nucleophilic substitution reactions, we efficiently synthesized eleven phenoxyalkylguanidine-based labeling precursors. The corresponding 19 F-standards were obtained via a similar synthetic route (Scheme 1). Notably, ortho , meta -, and para -[ 18 F]fluorobenzylguanidine were also prepared using our PhAzF-18 labeling strategy for side-by-side comparison (Scheme S1). 2.2 18 F-labeling and preparation of phenoxyalkylguanidine PET tracers All precursors were successfully 18 F-radiolabeled using our reported PhAzF-18 method 22 . All precursors afforded RCCs≥30%, except for [ 18 F] 7-p-a and [ 18 F] 7-p-c , which showed relatively low radiochemical conversions (17.4% and 5%, respectively), likely due to intramolecular interactions. Notably, [ 18 F] 8- o -a , [ 18 F] 8- o -b , [ 18 F] 8- p -b , and [ 18 F] p FBG achieved excellent RCCs (>80%)(Table 1 ). After the HCl-mediated deprotection, the final products were isolated using a C18 cartridge, and all tracers were obtained as clear, colorless solutions with >95% radiochemical purity. The radiolabeled compounds were identified by co-injection with their corresponding non-radioactive standards on HPLC. Radio-HPLC chromatograms for RCC calculation and tracer analysis are provided in the SI. For all radiolabeled tracers, LogD values were determined by a shake-flask method and are shown in Table 1. We found a clear trend of increasing lipophilicity with increasing linker carbon chain length in these tracers. Notably, [ 18 F] 8- o -e , which contains an eight-carbon alkyl chain, exhibited the highest lipophilicity (2.13 ± 0.12) among these tracers. The log D of [ 18 F] o FBG was comparable to the previously reported values for [ 18 F] m FBG and [ 18 F] p FBG 26 . Table 1. The radiochemical conversion (RCC) and Log D -values of novel NET-targeted tracers. Comp. RCC a Log D Comp. RCC a Log D [ 18 F] 8- o -a 82.8% -0.39 ± 0.01 [ 18 F] 8- m -c 29.6% 0.53 ± 0.01 [ 18 F] 8- m -a 66.7% -0.21 ± 0.01 [ 18 F] 8- p -c 5.1% 0.42 ± 0.02 [ 18 F] 8- p -a 17.4% -0.18 ± 0.02 [ 18 F] 8- o -d 35.5% 0.50 ± 0.02 [ 18 F] 8- o -b 85.4% 0.01 ± 0.01 [ 18 F] 8- o -e N/A b 2.13 ± 0.12 [ 18 F] 8- m -b 66.9% 0.16 ± 0.01 [ 18 F] o FBG 51.6% -0.59 ± 0.02 [ 18 F] 8- p -b 84.4% 0.17 ± 0.01 [ 18 F] m FBG 34.8% -0.52 ± 0.01 26 [ 18 F] 8- o -c 75.2% 0.34 ± 0.01 [ 18 F] p FBG 88.9% -0.48 ± 0.01 26 a The radiochemical conversions (RCC) were calculated from the labeling of the precursors. b Extreme lipophilicity of [ 18 F] 8- o -e resulted in excessively prolonged HPLC retention time, precluding the determination of the accurate RCC. Figure 2. Static cardiac PET/CT imaging and quantitative analysis of radiotracers in healthy SD Rats at 60 min post-injection. (A) Myocardial PET images of 18 F-labeled NET-targeted tracers at 60 min post-injection in normal SD rats. (B) ROI analysis of myocardium at 60 min post-injection. Values are expressed as SUVmax. 2.3 Cardiac PET imaging in normal rats The cardiac PET imaging protocol was designed to assess the myocardial radiotracer distribution of 11 newly synthesized tracers and to identify promising candidates for cardiac sympathetic nerve imaging. As control and comparison, we also subjected three benzylguanidine analogs to PET imaging studies under the same experimental conditions. Figure 2-A shows representative static cardiac PET images of healthy rats using the aforementioned tracers at 60 min after tracer injection. In coronal images, all tracers visualized the myocardium, with the left ventricular wall being most prominent. This demonstrates the tolerance of the expansive hydrophobic pocket formed by subsites B/C toward tracers with extended linkers. Among them, [ 18 F] 8- o -a exhibited the highest myocardial uptake compared to all the other ligands (Figure 2-B), with a maximum standardized uptake (SUV max ) value of 8.95 ± 0.62 (n=3), followed by [ 18 F] 8- p -b and [ 18 F] 8- o -c . To investigate the mechanisms of the PET tracer uptake in the rat hearts, we found pretreatment with selective uptake-1 blocking agents desipramine (DMI, 2 mg/kg) intravenously via tail vein 10 min before the radiotracer injection decreased the radiotracer uptake in the myocardium, whereas pretreatment with phenoxybenzamine (PhB, 25 mg/kg intravenously) could significantly reduce myocardial uptake, counting approximately 50% decrease (Fig. SX). These results indicated that myocardial tracer accumulation in rats might result from the synergistic contribution of both uptake-1 and uptake-2, consistent with previous reports 5,12 . Besides, the radioactivity levels in the adjacent liver and lung were also evaluated. In the lungs, only [ 18 F] 8- o -b and [ 18 F] 8- p -b showed slight bilateral radioactivity, whereas the other tracers showed even lower uptake, resulting in favorable heart-to-lung ratios (Fig. SX). The liver, as another important background organ, showed variable uptake levels among the different compounds (Fig. SX). [ 18 F] o FBG had the highest liver uptake, with liver activity exceeding that of the heart at 60 min p.i., resulting in a heart-to-liver ratio of only 0.60. In contrast, [ 18 F] 8- o -c demonstrated significantly higher heart-to-liver uptake ratios (7.42 at 60 min p.i.). Figure 3 shows the 60-minute dynamic PET images (A) and time-activity curves (B) of [ 18 F] m FBG , [ 18 F] 8-o-a , and [ 18 F] 8-o-c in a rat following tracer injection. All tracers are rapidly distributed to the myocardium in the first frame (0-5 min) after injection. From the early images, the left ventricular wall was clearly delineable and remained visible until the last frame (50-60 min). Time-activity curves analysis of myocardium further showed that [ 18 F] 8- o -a and [ 18 F] 8- o -c sustained high and stable radioactivity throughout the scan time of 60 min with only a mild decline, whereas [ 18 F] m FBG decreased by approximately 50%, exhibiting the uptake level of myocardium comparable to liver at the late phase. In addition, different background-clearance patterns were observed among the three tracers. [ 18 F] 8- o -c exhibited rapid clearance from both liver and lungs, resulting in superior contrast. In comparison, [ 18 F] 8- o -a showed elevated pulmonary uptake peaking in the early phase (0-10 min), followed by rapid clearance from the lungs. In the same experiments, [ 18 F] m FBG showed persistent, higher hepatic retention with minimal washout over time. Notably, [ 18 F] 8- o -a and [ 18 F] 8- o -c achieved favorable heart-to-liver ratios and were 3-fold higher than that of [ 18 F] m FBG (P<0.05) at the end of the study, facilitating the evaluation of inferior wall activity. 2.4 Tumor PET imaging in mice To further evaluate the ability to localize neuroendocrine tumors arising from the sympathetic system, static PET imaging was performed 30, 60, 120, and/or 240 min after injection of 18 F-labeled tracers in rat pheochromocytoma (PC-12) tumor-bearing mice (n = 3, Figure SX-SX). Representative maximum-intensity projection (MIP) images at 60 min p.i. for newly synthesized tracers are shown in Figure 4A. The majority of the evaluated tracers showed significant tumor uptake and favorable tumor-to-background contrast, except for [ 18 F] o FBG and [ 18 F] 8- o / m / p -c . In addition, these eight tracers that exhibited significant tumor accumulation also showed uptake in adrenal glands and organs and tissues highly innervated by sympathetic neurons, including the myocardium, thyroid, salivary glands, and brown adipose tissue 27,28 . All tracers showed predominant renal clearance with evident bladder accumulation, and no detectable bone uptake, supporting their in vivo stability. The SUVmax values of PC-12 tumors and main organs, including muscles, blood, liver, lung, myocardium, and bone in mice for all 12 radioligands, are presented in Figure 4A and Table 2. Among the benzylguanidine-based tracers ([ 18 F] o/m/p FBG ), the [ 18 F] m FBG exhibited remarkable radioactivity uptake in PC-12 tumors, with the SUVmax of 22.13 ± 1.95 (n = 3) (Figure 4B). In contrast, within the newly developed phenoxyalkylguanidine-based tracers, the ortho -fluoro derivatives showed markedly higher uptake in PC-12 tumors than fluorination on the meta - and para-position, with [ 18 F] 8- o -a reaching the highest level in all tracers, yielding a maximum SUV of 23.35 ± 4.35 at 60 min p.i. (n = 3). Moreover, we observed that the uptake of [ 18 F] 8- o / m / p -c in PC-12 tumors was markedly lower than that of [ 18 F] 8- o / m / p -a/b , suggesting that extension of the linker carbon chain reduced the affinity of the tracers for NET in mice. Accordingly, [ 18 F] 8- o -d and [ 18 F] 8- o -e , with further extended linkers, were not tested in PC-12 tumor-bearing mice. Interestingly, while the [ 18 F] 8- o / m / p -c performed well in rat cardiac imaging, their capacity in mouse tumor models was significantly compromised, showing a distinctly different biodistribution. In PC-12 tumor-bearing mice, uptake was low in tumors as well as in organs with dense sympathetic innervation. This discrepancy may be attributed to the species variations for the norepinephrine uptake mechanisms, with the high existence of the nonneural uptake-2 mechanism in rats compared to humans and other species 12 . Table 2. Quantitative imaging analysis of NET-targeted tracers at 60 min p.i. in PC-12 models Comp. Tumor Muscle Blood Liver Lung Myocardium Bone [ 18 F] o FBG 4.11 ± 1.00 0.24 ± 0.06 0.30 ± 0.03 3.39 ± 0.38 0.23 ± 0.03 0.54 ± 0.09 0.36 ± 0.12 [ 18 F] m F BG 22.13 ± 1.95 0.53 ± 0.07 1.03 ± 0.08 1.70 ± 0.06 0.44 ± 0.04 2.24 ± 0.06 0.64 ± 0.07 [ 18 F] p FBG 10.31 ± 1.67 0.22 ± 0.05 0.60 ± 0.01 1.60 ± 0.18 0.36 ± 0.03 0.98 ± 0.13 0.53 ± 0.09 [ 18 F]8- o -a 23.35 ± 4.35 0.14 ± 0.02 1.26 ± 0.19 0.65 ± 0.18 0.55 ± 0.10 3.75 ± 0.86 0.44 ± 0.13 [ 18 F]8- m -a 10.14 ± 1.32 0.16 ± 0.03 1.10 ± 0.02 0.57 ± 0.06 0.40 ± 0.04 2.68 ± 0.52 0.63 ± 0.09 [ 18 F]8- p -a 10.30 ± 0.78 0.21 ± 0.02 1.25 ± 0.04 1.82 ± 0.12 0.50 ± 0.04 0.46 ± 0.15 0.44 ± 0.07 [ 18 F]8- o -b 22.66 ± 3.16 0.26 ± 0.08 1.20 ± 0.16 0.51 ± 0.16 0.80 ± 0.09 4.31 ± 1.72 0.43 ± 0.10 [ 18 F]8- m -b 8.29 ± 4.09 0.19 ± 0.06 1.11 ± 0.03 0.46 ± 0.04 0.47 ± 0.12 3.75 ± 0.36 0.46 ± 0.09 [ 18 F]8- p -b 9.09 ± 2.09 0.18 ± 0.03 1.50 ± 0.12 1.15 ± 0.07 0.56 ± 0.07 4.25± 0.32 0.41 ± 0.03 [ 18 F]8- o -c 2.41 ± 0.86 0.15 ± 0.01 0.36 ± 0.02 0.16 ± 0.02 0.21 ± 0.03 0.74 ± 0.08 0.34 ± 0.03 [ 18 F]8- m -c 0.69 ± 0.12 0.16 ± 0.02 0.39 ± 0.03 0.38 ± 0.05 0.26 ± 0.04 0.82 ± 0.07 0.47 ± 0.06 [ 18 F]8- p -c 1.06 ± 0.44 0.20 ± 0.05 0.51 ± 0.04 0.51 ± 0.07 0.40 ± 0.11 1.11 ± 0.23 0.32 ± 0.04 2.5 Characteristics and biodistribution in tumor-bearing mice. After evaluation of the potential for in vivo PET imaging in rat and mice of the 18 F-labeled radioligands, 18 F-labeled 1-(2-(2-fluorophenoxy)ethyl)guanidine ([ 18 F] 8- o -a) , namely [ 18 F] o FPhOEG, was selected to the further investigate its stability, affinity and biodistribution in tumor-bearing mice due to superior cardiac and tumor uptake, as well as for comparison with the widely used NET-targeted PET tracer [ 18 F] m FBG . As shown in Figure 5A, the [ 18 F] o FPhOEG showed excellent in vitro stability in PBS and rat serum at 37 °C for up to 4 hours, as confirmed by radio-HPLC analysis with no evidence of defluorination. NET binding affinity studies were performed with SK-N-SH cells expressing NET using [ 18 F] m FBG as the radioligand and increasing concentrations of [ 19 F] o FPhOEG and [ 19 F] m FBG to compete for uptake. The IC 50 values were 0.097 μM for o FPhOEG and 0.254 μM for m FBG , respectively. Representative competitive uptake curves are shown in Figure 5B. In the cellular uptake assay (Figure 5C), we found that [ 18 F] o FPhOEG showed higher uptake in SK-N-SH cells than [ 18 F] m FBG at all four time points (15, 30, 60, and 120 min). In addition, pretreatment with the selective uptake-1 inhibitor desipramine (DMI) further confirmed that tracer accumulation was NET-mediated. In the blocking group, DMI (10 μM) significantly inhibited the uptake of both tracers, with an inhibition rate exceeding 95%, confirming their specific binding to NET-expressing cells. Figure 5D showed the in vivo performance of [ 18 F] o FPhOEG vs [ 18 F] mFBG in PC-12 xenografts at 30, 60, 120, and 240 min p.i. The PET/CT imaging demonstrated that both tracers rapidly accumulated in tumors and maintained their retention for up to 240 min without significant washout. Quantitative analysis indicated that tumor uptake for both tracers peaked at 120 min ([ 18 F] o FPhOEG : SUVmax = 25.30 ± 3.88; [ 18 F] m FBG : 21.96 ± 2.95; Figure X-cd), with no statistically significant differences observed at any time point (P > 0.05; Figure SX). In addition, [ 18 F] o FPhOEG exhibited lower muscle and hepatic distribution compared with [ 18 F] m FBG (Figure SX). As a result, [ 18 F] o FPhOEG achieved comparable tumor uptake but provided markedly improved tumor-to-background contrast relative to [ 18 F] m FBG (Figure XX). Subsequent biodistribution results of [ 18 F] o FPhOEG were consistent with the ROI analysis, showing the highest radioactivity accumulation in PC-12 tumors, followed by brown adipose tissue, myocardium and adrenal glands (Figure 5E and Table SX). [ 18 F] o FPhOEG also exhibited rapid blood clearance following intravenous injection, with residual radioactivity 1.23 ± 0.21%ID/g at 30 min. Similarly, early pulmonary uptake followed by rapid clearance of the tracer was also observed in mice, consistent with its behavior in rats. Thus, [ 18 F] o FPhOEG demonstrated excellent stability, high specificity for NET-expressing cells, and superior diagnostic performance, enabling rapid and accurate detection of sympathetic system-derived neuroendocrine tumors with high contrast in preclinical models. 2.6 Automatic Production and PET/CT studies in Rhesus Macaque The clinical translational potential of [ 18 F] o FPhOEG was further evaluated by an established automated production process using an integrated system comprising the commercial AllinOne radiosynthesis module and an LED reactor (ProBox), as previously reported 23,29 , to confirm its reproducibility and applicability for routine production. Details of the setup configuration and automated synthesis process are provided in Table SX and Figure SX. The [ 18 F] o FPhOEG was successfully synthesized and isolated in 29.4% RCY (decay corrected) in 75 min with a molar activity (A m ) of 64.76 GBq/μmol. Given that the hearts of monkeys and humans lack the nonneural uptake-2 pathway, nonhuman primates serve as an appropriate model for preclinical evaluation of sympathetic nerve tracers 12,30 . A 60-minute dynamic PET/CT study was subsequently performed in a healthy male rhesus macaque to characterize the cardiac and adjacent background distribution pattern of [ 18 F] o FPhOEG . The sequential cardiac PET MIP images of [ 18 F] o FPhOEG in the rhesus macaque were presented in Figure 6A. The 3 slice orientations and polar map of the left ventricle (LV) at 50 min p.i. are available in Figure 6B and 6C. The TAC curve showed the dynamic quantitative SUV mean value in four major tissues and organs of the tracer (Figure 6D). As shown in Figure 6A, [ 18 F] o FPhOEG rapidly distributed to the kidney, spleen, heart and blood pool, pancreas and liver within the first 2 min after injection. Thereafter, radioactivity in the spleen and lungs declined quickly, whereas hepatic uptake gradually increased, reaching its maximum at approximately 10-20 min. After 20 min, renal activity had largely cleared, allowing the adrenal glands to become clearly discernible. Uptake of [ 18 F] o FPhOEG into myocardium was similarly rapid; the ventricular wall was already well delineable on the second frame image (1-2 min) from the blood pool owing to rapid tracer clearance from circulation, and myocardial activity remained essentially stable throughout the 60-minute scan. Furthermore, to more comprehensively characterize the spatial distribution of [ 18 F] o FPhOEG within the myocardium, we performed quantitative circumferential analysis on a series of reoriented cardiac PET images. Shown are short axis (SA), vertical long axis (VLA) and horizontal long axis (HLA) views (Figure 6B) and polar maps (Figure 6C) of the left ventricle (LV), which not only define the regional variation of [ 18 F] o FPhOEG myocardial uptake in the rhesus macaque but also facilitate precise localization and quantitative assessment of potentially affected myocardial segments. The TAC analysis in the myocardium showed consistently high and sustained tracer retention over the 60-minute scan, peaking at 17.5 minutes (SUVmean = 9.94 ± 1.30) and exhibiting minimal washout over time (~ 0.2% per minute). A gradual increase in hepatic radioactivity was observed until 12.5 min p.i., after which tracer levels declined with hepatic clearance. The peak heart-to-liver ratio of 2.80 was recorded at 50 min p.i. Moreover, radioactivity in the blood pool and lungs cleared rapidly within 2 minutes and subsequently remained at a low level. Despite the high-quality cardiac images obtained with [ 18 F] 8- o- c in SD rats, its myocardial behavior in rhesus macaques was entirely different, showing only minimal distribution in the myocardium (Figure SX). This finding further confirms that elongation of the alkyl linker substantially reduces NET affinity and the species-dependent differences in norepinephrine uptake pathways. Furthermore, the results suggested that myocardial uptake measured in rats is not necessarily a reliable predictor of left ventricular concentrations in primates or humans, demonstrating that rat-only evaluation is insufficient for assessing cardiac sympathetic nerve tracers. In contrast, [ 18 F] o FPhOEG exhibited favorable kinetics and imaging properties for quantitative PET studies of cardiac sympathetic innervation. Subsequently, whole-body static PET/CT scans at 60, 120, and 240 min (Figure 7A) after [ 18 F] o FPhOEG injection were performed in the same rhesus macaque to further evaluate the biodistribution and meta bolic profile of [ 18 F] o FPhOEG in a nonhuman primate. TAC analysis showed the uptake in the major organs of the tracer (Figure 7B and 7C). Representative transverse PET and PET/CT fused images at 240 min p.i., illustrating organs with prominent uptake, are presented in Figure 7D. As shown in Figure 7, [ 18 F] o FPhOEG mainly accumulated in the bladder, heart, adrenal glands, spleen, salivary glands, pancreas, and liver, while lower activity was detected in the brain, lungs, intestine, bone, and muscle in all PET images. Over the course of the scan, the tracer was cleared by the urinary system, leading to accumulation of activity in the bladder. Uptake in the adrenal and salivary glands remained consistently high throughout the scan period, whereas radioactivity in the heart, spleen, liver, and pancreas gradually decreased over time. Notably, radioactivity in bone remained low at all time points, indicating no in vivo radio-defluorination and confirming the meta bolic stability of the tracer in nonhuman primates. Human radiation absorbed dose estimates were derived from the biodistribution of [ 18 F] o FPhOEG in the rhesus macaques at three imaging time points using OLINDA/EXM (version 2.2.3). The absorbed dose estimates (mSv/MBq) for the reference adult male model of OLINDA/EXM are summarized in Table SX. The urinary bladder wall received the highest radiation dose (0.469 mGy/MBq), followed by the prostate, due to its proximity to the bladder with substantial tracer accumulation. The effective dose of the whole body was 0.0261 mGy/MBq. These results of in vivo distribution and radiation absorbed dose estimates in non-human primates warranted further clinical translation of [ 18 F] o FPhOEG in humans. Conclusions In this study, we successfully designed, synthesized, and systematically evaluated a series of novel 18 F-labeled phenoxyalkylguanidine NET-targeted PET tracers based on structure-based rational drug design and an efficient organic photocatalyzed 18 F-labeling strategy. All tracer precursors were synthesized via concise, robust routes and efficiently 18 F-labeled using our PhAzF-18 method. The optimized [ 18 F] o FPhOEG showed good radiochemical yield, higher NET binding affinity, and excellent pharmacokinetic properties, exhibiting remarkable cardiac and tumor uptake in PET imaging. Compared to [ 18 F] m FBG , [ 18 F] o FPhOEG exhibited markedly higher and more sustained myocardial uptake in SD rats and comparable tumor accumulation in PC12 tumor-bearing mice, and significantly increased heart-to-liver and tumor-to-liver ratios (3-fold). These findings suggest that [ 18 F] o FPhOEG shows rapid hepatic clearance across the three animal models, which is crucial for minimizing liver interference with inferior wall assessment in cardiac PET and enhancing the visualization of hepatic metastases in tumor PET. Moreover, the tracer was successfully produced using a commercial automated module, highlighting its manufacturability and translational potential. In conclusion, these preclinical results make [ 18 F] o FPhOEG a highly promising NET-targeted PET tracer for clinical applications in assessing cardiac sympathetic innervation denervation, such as in heart failure, and in evaluating neuroendocrine tumors arising from the sympathetic nervous system. 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Patterns of salivary gland uptake in I-131 MIBG scintigraphy. Revista Española de Medicina Nuclear 25 , 198-201, doi:https://doi.org/10.1157/13088418 (2006). Wang, Y. et al. Photocatalyzed (18)F-Fluorination: A Streamlined Radiolabeling Approach for Rapid Adoption and Automation. Org Lett 27 , 7224-7229, doi:10.1021/acs.orglett.5c02193 (2025). Raffel, D. M. et al. Radiolabeled Phenethylguanidines: Novel Imaging Agents for Cardiac Sympathetic Neurons and Adrenergic Tumors. Journal of Medicinal Chemistry 50 , 2078-2088, doi:10.1021/jm061398y (2007). Scheme Scheme 1 is available in the Supplementary Files section. Additional Declarations The authors declare potential competing interests as follows: The authors have filed a provisional patent on the basis of the research in this manuscript. Supplementary Files floatimage2.png Scheme 1 General synthetic route of phenoxyalkylguanidines labeling precursors and 19 F-standards. (a) Cu(OAc) 2 , Et 3 N, DCM, RT, 24 h, 20%-35%; (b) K 2 CO 3 , CH 3 CN, 80℃, 49%-78%, 10 h; (c) TFA, DCM, rt, 2-5 h; (d) N, N ′-Di-Boc-1H-pyrazole-1-carboxamidine, K 2 CO 3 , CH 3 CN, rt, 12 h, 71%-95%. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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Representative \u003csup\u003e18\u003c/sup\u003eF-labeled NET-targeted PET tracers derived from phenylalkylguanidine. B. Overlap of the binding mode of NE (green, PDB: 8HFF) and MIBG (cyan, PDB: 8XB3) with NET. C. Construction of novel \u003csup\u003e18\u003c/sup\u003eF-labeled phenoxyalkylguanidines NET-targeted PET tracers. \u003cem\u003ePhAzF-18\u003c/em\u003e, photocatalyzed azeotropic drying-free \u003csup\u003e18\u003c/sup\u003eF-fluorination.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8300571/v1/9716e4c8a09b27491b070e6d.png"},{"id":97869214,"identity":"8e531b17-205b-4b73-9cc2-eb4ca8ef6d0a","added_by":"auto","created_at":"2025-12-10 10:03:24","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":396292,"visible":true,"origin":"","legend":"\u003cp\u003eStatic cardiac PET/CT imaging and quantitative analysis of radiotracers in healthy SD Rats at 60 min post-injection. (A) Myocardial PET images of \u003csup\u003e18\u003c/sup\u003eF-labeled NET-targeted tracers at 60 min post-injection in normal SD rats. (B) ROI analysis of myocardium at 60 min post-injection. Values are expressed as SUVmax.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8300571/v1/128e2ec5987ae0633f4ce6c4.png"},{"id":97900570,"identity":"413852a8-de28-4871-8851-3540e315a5c1","added_by":"auto","created_at":"2025-12-10 15:45:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":628461,"visible":true,"origin":"","legend":"\u003cp\u003eDynamic micro-PET/CT image analysis of [\u003csup\u003e18\u003c/sup\u003eF]\u003cem\u003e\u003cstrong\u003em\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eFBG\u003c/strong\u003e, [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e8-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-a\u003c/strong\u003e and [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e8-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-c\u003c/strong\u003e in healthy SD rats (n = 3). (A) Coronal and fused PET/CT images of [\u003csup\u003e18\u003c/sup\u003eF]\u003cem\u003e\u003cstrong\u003em\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eFBG\u003c/strong\u003e [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e8-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-a\u003c/strong\u003e and [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e8-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-c\u003c/strong\u003e during the 60 min dynamic scan. (B) Time-activity curves for [\u003csup\u003e18\u003c/sup\u003eF]\u003cem\u003e\u003cstrong\u003em\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eFBG\u003c/strong\u003e, [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e8-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-a\u003c/strong\u003e and [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e8-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-c\u003c/strong\u003e. Values are expressed as SUVmax.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8300571/v1/5e3d5c1508b7863d3627ac48.png"},{"id":97899944,"identity":"82b17663-878a-45c1-9ae1-33fbdf17b768","added_by":"auto","created_at":"2025-12-10 15:45:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":744017,"visible":true,"origin":"","legend":"\u003cp\u003eMicro-PET/CT imaging and quantitative analysis of 12 tracers in PC-12 tumor-bearing mice. (A) The MIP images of \u003csup\u003e18\u003c/sup\u003eF-labeled tracers at 60 min post-injection. (B) ROI analysis of \u003csup\u003e18\u003c/sup\u003eF-labeled tracers in PC-12 tumors at 60 min p.i. Values are expressed as SUVmax (mean ± SD, n = 3). Tumors are indicated by white dashed circles.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8300571/v1/eeb0051f2f7e80141b30bd26.png"},{"id":97869216,"identity":"d8102ae1-2f90-4b98-9491-3464d191b4c4","added_by":"auto","created_at":"2025-12-10 10:03:24","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":515955,"visible":true,"origin":"","legend":"\u003cp\u003e(A) \u003cem\u003eIn vitro\u003c/em\u003e stability of [\u003csup\u003e18\u003c/sup\u003eF]\u003cem\u003e\u003cstrong\u003eo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eFPhOEG\u003c/strong\u003e in PBS (a) and fresh rat serum (b) at 37 ℃ over 1-4 h using radio-HPLC. (B) Competitive inhibition curves of [\u003csup\u003e18\u003c/sup\u003eF]\u003cem\u003e\u003cstrong\u003em\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eFBG\u003c/strong\u003e uptake in SK-N-SH cells treated with increasing concentrations of cold reference \u003cem\u003e\u003cstrong\u003eo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eFPhOEG\u003c/strong\u003e (red) and \u003cem\u003e\u003cstrong\u003em\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eFBG\u003c/strong\u003e (blue). Uptake values are expressed as a percentage of control [\u003csup\u003e18\u003c/sup\u003eF]\u003cem\u003e\u003cstrong\u003em\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eFBG\u003c/strong\u003e uptake. (C) Cell uptake of [\u003csup\u003e18\u003c/sup\u003eF]\u003cem\u003e\u003cstrong\u003eo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eFPhOEG \u003c/strong\u003e(a) and [\u003csup\u003e18\u003c/sup\u003eF]\u003cem\u003e\u003cstrong\u003em\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eFBG\u003c/strong\u003e (b) in SK-N-SH cells (SK-N-SH) and in SK-N-SH cells with pretreatment with 10 μM DMI (SN-N-SH blocked) after 15, 30, 60 and 120 min incubation at 37 °C. ****P \u0026lt; 0.0001. Data are presented as mean ± SD (n = 3). (D) Representative PET/CT images were obtained at 30, 60, 120 and 240 min p.i. of [\u003csup\u003e18\u003c/sup\u003eF]\u003cem\u003e\u003cstrong\u003eo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eFPhOEG \u003c/strong\u003e(a) and [\u003csup\u003e18\u003c/sup\u003eF]\u003cem\u003e\u003cstrong\u003em\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eFBG\u003c/strong\u003e (b) in PC-12 tumor-bearing mice (tumors are indicated by orange circles). Top row, MIP. Second row, coronal images (Cor) of tumors. Bottom row, transverse images (Tra) of tumors. Tumor, muscle, and liver ROIs were manually delineated for quantitative analysis. Shown are SUVmax values of tumor and muscle uptake at four time points after injection of [\u003csup\u003e18\u003c/sup\u003eF]\u003cem\u003e\u003cstrong\u003eo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eFPhOEG \u003c/strong\u003e(c) and [\u003csup\u003e18\u003c/sup\u003eF]\u003cem\u003e\u003cstrong\u003em\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eFBG\u003c/strong\u003e (d), and the corresponding tumor-to-liver ratios (e) in PC-12 tumor-bearing mice. ns, \u003cem\u003eP\u003c/em\u003e\u0026gt;0.05; *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.005. (E) Tissue biodistribution of [\u003csup\u003e18\u003c/sup\u003eF]\u003cem\u003e\u003cstrong\u003eo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eFPhOEG\u003c/strong\u003e in PC-12 tumor-bearing mice. (n = 5 per time point).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8300571/v1/f1777e39521d10ee59ea6c6e.png"},{"id":97869219,"identity":"5e77e3e4-4362-4f4d-9aee-bed48c1ecc82","added_by":"auto","created_at":"2025-12-10 10:03:24","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":492640,"visible":true,"origin":"","legend":"\u003cp\u003eDynamic cardiac PET imaging of [\u003csup\u003e18\u003c/sup\u003eF]\u003cem\u003e\u003cstrong\u003eo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eFPhOEG\u003c/strong\u003e in healthy rhesus macaques. (A) Representative MIP images of [\u003csup\u003e18\u003c/sup\u003eF]\u003cem\u003e\u003cstrong\u003eo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eFPhOEG\u003c/strong\u003e PET imaging at different time points. Abbreviations: H = Heart, L = Lung, Li = Liver, K = Kidney, S = Spleen, A = Adrenal gland, P = Pancreas. (B) The short axis (SA), horizontal long axis (HLA) and vertical long axis (VLA) views of the left ventricle (LV). (C) The polar map (left) and quantification of 17 segments (right) of LV using PMOD V4.004 Cardiac PET module. (D) Time-activity curves of myocardium (heart), liver, lung, and blood pool assessed by PMOD V4.004 via manual ROI delineation on dynamic PET images. Values are expressed as SUVmean.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8300571/v1/c0b2ab3446d53001a7d8d79f.png"},{"id":97900516,"identity":"57b54173-532f-481f-854e-f8082bce5302","added_by":"auto","created_at":"2025-12-10 15:45:36","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":553192,"visible":true,"origin":"","legend":"\u003cp\u003eWhole-body static PET/CT scans at 60, 120, and 240 min p.i. of [\u003csup\u003e18\u003c/sup\u003eF]\u003cem\u003e\u003cstrong\u003eo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eFPhOEG\u003c/strong\u003e in a healthy male rhesus macaque. (A) Whole-body MIP images obtained at 60, 120 and 240 min p.i. of [\u003csup\u003e18\u003c/sup\u003eF]\u003cem\u003e\u003cstrong\u003eo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eFPhOEG\u003c/strong\u003e. Abbreviations: SA = Salivary glands, H = Heart, Li = Liver, S = Spleen, A = Adrenal gland, Pe = Renal pelvis. (B-C) ROI analysis of [\u003csup\u003e18\u003c/sup\u003eF]\u003cem\u003e\u003cstrong\u003eo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eFPhOEG\u003c/strong\u003e in major organs. Values are expressed as SUVmean. (D) Representative transverse PET and PET/CT fused images of parotid gland, myocardium, right adrenal gland, right renal pelvis and left adrenal gland at 240 min p.i.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8300571/v1/ea5b1f53b42fd4244de05186.png"},{"id":98443158,"identity":"6b4e50d6-1485-4769-852f-41e697f4c034","added_by":"auto","created_at":"2025-12-17 17:12:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6615600,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8300571/v1/95065ca6-88ac-4026-bcb6-2e0a53c1b41b.pdf"},{"id":97899416,"identity":"d20368f6-91db-4337-a99d-99cd3c723eaa","added_by":"auto","created_at":"2025-12-10 15:44:27","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":293473,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1\u003c/strong\u003e General synthetic route of phenoxyalkylguanidines labeling precursors and \u003csup\u003e19\u003c/sup\u003eF-standards. (a) Cu(OAc)\u003csub\u003e2\u003c/sub\u003e, Et\u003csub\u003e3\u003c/sub\u003eN, DCM, RT, 24 h, 20%-35%; (b) K\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e, CH\u003csub\u003e3\u003c/sub\u003eCN, 80℃, 49%-78%, 10 h; (c) TFA, DCM, rt, 2-5 h; (d) \u003cem\u003eN, N\u003c/em\u003e′-Di-Boc-1H-pyrazole-1-carboxamidine, K\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e, CH\u003csub\u003e3\u003c/sub\u003eCN, rt, 12 h, 71%-95%.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8300571/v1/9b5acfd716bc56f774aed0e7.png"}],"financialInterests":"The authors declare potential competing interests as follows: The authors have filed a provisional patent on the basis of the research in this manuscript.","formattedTitle":"\u003cp\u003eDesign and Preclinical Evaluation of Novel \u003csup\u003e18\u003c/sup\u003eF-Labeled Phenoxyalkylguanidines Radiotracers for Norepinephrine Transporter PET Imaging\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe norepinephrine transporter (NET), a member of the solute carrier 6 (SLC6) family, plays a pivotal role in regulating synaptic norepinephrine (NE) levels by mediating its reuptake from the synaptic cleft into presynaptic neurons in both the central and peripheral nervous systems\u003csup\u003e1\u003c/sup\u003e. This mechanism is essential for maintaining noradrenergic signaling homeostasis, which governs a range of physiological functions, including mood regulation, attention, pain perception, and stress responses. Norepinephrine reuptake occurs primarily through an active, saturable, ATP-dependent process known as uptake 1 and, to a lesser extent, via a passive, non-saturable, energy-independent diffusion mechanism known as uptake 2. In addition to its physiological expression in sympathetic neurons, NET is also overexpressed in neural crest-derived tumors (NCTs), including neuroblastoma, pheochromocytoma, and paraganglioma\u003csup\u003e2,3\u003c/sup\u003e. As a result, NET serves as a critical target for radiopharmaceuticals, particularly for the diagnosis and treatment of neuroendocrine tumors and for imaging myocardial sympathetic innervation \u003csup\u003e4,5\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eOver the past decades, major efforts have focused on developing NET-targeted radiopharmaceuticals based on norepinephrine analogs and phenylalkylguanidines, and the [\u003csup\u003e123\u003c/sup\u003eI]mIBG remains the clinical gold standard for imaging myocardial sympathetic innervation and neuroendocrine tumors\u003csup\u003e3,6-10\u003c/sup\u003e. However, its limited resolution, challenging quantification, poor sensitivity for small metastases, high background signal, and the need for 24-hour uptake reduce its clinical effectiveness\u003csup\u003e3,11\u003c/sup\u003e. In contrast, the structurally similar PET tracer [\u003csup\u003e18\u003c/sup\u003eF]mFBG offers markedly improved spatial resolution, sensitivity, specificity, and quantitative capability, supported by the favorable physical properties and broad availability of \u003csup\u003e18\u003c/sup\u003eF-fluorine. Consequently, several \u003csup\u003e18\u003c/sup\u003eF-labeled PET tracers, including \u003csup\u003e18\u003c/sup\u003eF-LMI1195\u003csup\u003e8,12\u003c/sup\u003e, [\u003csup\u003e18\u003c/sup\u003eF]4F-mHPG\u003csup\u003e9\u003c/sup\u003e, [\u003csup\u003e18\u003c/sup\u003eF]3F-pHPG\u003csup\u003e13\u003c/sup\u003e, and [\u003csup\u003e18\u003c/sup\u003eF]AF78\u003csup\u003e10,14\u003c/sup\u003e, were developed for non-invasive assessment of sympathetic nervous system tumors. Despite this progress, most PET tracers remain structurally limited to phenylalkylguanidine variants, and further clinical translation and development are hindered by background uptake, inefficient \u003csup\u003e18\u003c/sup\u003eF-labeling strategies, and low radiochemical yields.\u003c/p\u003e\n\u003cp\u003eRecent breakthroughs in determining the structure of NET have revealed the molecular basis of its interactions with transport substrates (NE and mIBG) and inhibitors\u003csup\u003e15-18\u003c/sup\u003e. These findings provide crucial insights into the design of NET-targeting drugs. NET adopts an inward-open conformation when binding mIBG, with the binding site enclosed by transmembrane helices TM1, TM3, TM6, and TM8, comprising three subsites (A, B, and C). The guanidine of mIBG forms hydrogen bonds with Phe317, Ser318, and Asp75 in subsite A. The benzyl group occupies hydrophobic pocket B formed by Val148, Tyr152, Phe317, Phe323, and Ser419, while the iodine atom engages in hydrophobic interactions with Gly149, Ser419, and Gly423 in subsite C (Figure 1B). NE binds to NET with a binding mode highly analogous to that of MIBG. Despite mIBG\u0026apos;s longer aminoalkyl side chain, it achieves optimal interactions through conformational flexibility. Notably, substantial unoccupied space persists adjacent to hydrophobic subsites B and C, potentially accommodating bulkier aryl substituents. Reported alkoxy-substituted phenylalkylguanidine derivatives (e.g., \u003csup\u003e18\u003c/sup\u003eF-LMI1195 and \u003csup\u003e18\u003c/sup\u003eF-AF78) likely bind NET via this mechanism. Leveraging these structural insights, we initiated the development of novel NET-targeted PET tracers with enhanced imaging performance and facile radiolabeling characteristics.\u003c/p\u003e\n\u003cp\u003eOur group recently established a photocatalyzed azeotropic drying-free \u003csup\u003e18\u003c/sup\u003eF-fluorination \u0026nbsp;(\u003cem\u003ePhAzF-18\u003c/em\u003e) labeling method, enabling direct \u003csup\u003e18\u003c/sup\u003eF-deoxyfluorination of challenging electron-rich aryl substrates from readily accessible precursors under mild conditions\u003csup\u003e19-22\u003c/sup\u003e. This \u003csup\u003e18\u003c/sup\u003eF-labeling strategy has demonstrated outstanding potential for radiopharmaceutical discovery and clinical tracer production\u003csup\u003e23-25\u003c/sup\u003e. In this work, leveraging the spatial tolerance afforded by the expansive hydrophobic pocket (subsites B/C) and our efficient \u003csup\u003e18\u003c/sup\u003eF-labeling strategy, we designed a series of novel phenoxyalkylguanidine derivatives rather than the frequently used phenylalkylguanidine skeleton, featuring an electron-rich aromatic ring and extended linkers. Through comprehensive screening, we identified a series of novel NET-targeted phenoxyalkylguanidine PET tracers with superior diagnostic efficiency. The preferred tracer [\u003csup\u003e18\u003c/sup\u003eF]\u003cem\u003eo\u003c/em\u003eFPhOEG ([\u003csup\u003e18\u003c/sup\u003eF]8-\u003cem\u003eo\u003c/em\u003e-a)\u0026nbsp;outperformed the clinically used [\u003csup\u003e18\u003c/sup\u003eF]\u003cem\u003em\u003c/em\u003eFBG, showing significantly higher target uptake and improved target-to-background ratios in myocardial/neuroendocrine tumor models. Furthermore, its automated production was also achieved efficiently, positioning this novel NET-targeted tracer as a promising candidate for clinical translation.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003e2.1. Design and synthesis of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ephenoxyalkylguanidine\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;PET tracers\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAn ideal NET-targeted tracer requires high binding affinity, rapid NET transport kinetics, and prolonged retention to achieve superior target-to-background ratios. Utilizing insights derived from the NET structural framework, we have developed a series of phenoxyalkylguanidine derivatives featuring extended linkers. These structural modifications were aimed at enhancing binding affinity by optimizing complementarity with the hydrophobic cavity of NET, and increasing the electron density of the aromatic ring to improve the efficiency of \u003csup\u003e18\u003c/sup\u003eF-fluorine incorporation. Through streamlined Chan-Lam coupling and nucleophilic substitution reactions, we efficiently synthesized eleven phenoxyalkylguanidine-based labeling precursors. The corresponding \u003csup\u003e19\u003c/sup\u003eF-standards were obtained via a similar synthetic route (Scheme 1). Notably, \u003cem\u003eortho\u003c/em\u003e, \u003cem\u003emeta\u003c/em\u003e-, and \u003cem\u003epara\u003c/em\u003e-[\u003csup\u003e18\u003c/sup\u003eF]fluorobenzylguanidine were also prepared using our \u003cem\u003ePhAzF-18\u003c/em\u003e labeling\u003cem\u003e\u0026nbsp;\u003c/em\u003estrategy for side-by-side comparison (Scheme S1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 \u003csup\u003e18\u003c/sup\u003eF-labeling and preparation of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ephenoxyalkylguanidine\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;PET tracers\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll precursors were successfully \u003csup\u003e18\u003c/sup\u003eF-radiolabeled using our reported \u003cem\u003ePhAzF-18\u003c/em\u003e method\u003csup\u003e22\u003c/sup\u003e. All precursors afforded RCCs\u0026ge;30%, except for [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e7-p-a\u003c/strong\u003e and [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e7-p-c\u003c/strong\u003e, which showed relatively low radiochemical conversions (17.4% and 5%, respectively), likely due to intramolecular interactions. Notably, [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e8-\u003cem\u003eo\u003c/em\u003e-a\u003c/strong\u003e, [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e8-\u003cem\u003eo\u003c/em\u003e-b\u003c/strong\u003e, [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e8-\u003cem\u003ep\u003c/em\u003e-b\u003c/strong\u003e, and [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e\u003cem\u003ep\u003c/em\u003eFBG\u003c/strong\u003e achieved excellent RCCs (\u0026gt;80%)(Table \u003cstrong\u003e1\u003c/strong\u003e). After the HCl-mediated deprotection, the final products were isolated using a C18 cartridge, and all tracers were obtained as clear, colorless solutions with \u0026gt;95% radiochemical purity. The radiolabeled compounds were identified by co-injection with their corresponding non-radioactive standards on HPLC. Radio-HPLC chromatograms for RCC calculation and tracer analysis are provided in the SI.\u003c/p\u003e\n\u003cp\u003eFor all radiolabeled tracers, LogD values were determined by a shake-flask method and are shown in Table 1. We found a clear trend of increasing lipophilicity with increasing linker carbon chain length in these tracers. Notably, [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e8-\u003cem\u003eo\u003c/em\u003e-e\u003c/strong\u003e, which contains an eight-carbon alkyl chain, exhibited the highest lipophilicity (2.13 \u0026plusmn; 0.12) among these tracers. The log\u003cem\u003eD\u0026nbsp;\u003c/em\u003eof [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e\u003cem\u003eo\u003c/em\u003eFBG\u003c/strong\u003e was comparable to the previously reported values for [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e\u003cem\u003em\u003c/em\u003eFBG\u003c/strong\u003e and [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e\u003cem\u003ep\u003c/em\u003eFBG\u003c/strong\u003e\u003csup\u003e26\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1.\u0026nbsp;\u003c/strong\u003eThe radiochemical conversion (RCC) and Log\u003cem\u003eD\u003c/em\u003e-values of novel NET-targeted tracers.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eComp.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRCC\u003csup\u003ea\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLog\u003cem\u003eD\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eComp.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRCC\u003csup\u003ea\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLog\u003cem\u003eD\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e[\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e8-\u003cem\u003eo\u003c/em\u003e-a\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e82.8%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e-0.39 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e[\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e8-\u003cem\u003em\u003c/em\u003e-c\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e29.6%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e0.53 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e[\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e8-\u003cem\u003em\u003c/em\u003e-a\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e66.7%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e-0.21 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e[\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e8-\u003cem\u003ep\u003c/em\u003e-c\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e5.1%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e0.42 \u0026plusmn; 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e[\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e8-\u003cem\u003ep\u003c/em\u003e-a\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e17.4%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e-0.18 \u0026plusmn; 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e[\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e8-\u003cem\u003eo\u003c/em\u003e-d\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e35.5%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e0.50 \u0026plusmn; 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e[\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e8-\u003cem\u003eo\u003c/em\u003e-b\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e85.4%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e0.01 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e[\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e8-\u003cem\u003eo\u003c/em\u003e-e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003eN/A\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e2.13 \u0026plusmn; 0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e[\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e8-\u003cem\u003em\u003c/em\u003e-b\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e66.9%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e0.16 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e[\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e\u003cem\u003eo\u003c/em\u003eFBG\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e51.6%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e-0.59 \u0026plusmn; 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e[\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e8-\u003cem\u003ep\u003c/em\u003e-b\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e84.4%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e0.17 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e[\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e\u003cem\u003em\u003c/em\u003eFBG\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e34.8%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e-0.52 \u0026plusmn; 0.01\u003csup\u003e26\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e[\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e8-\u003cem\u003eo\u003c/em\u003e-c\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e75.2%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e0.34 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e[\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e\u003cem\u003ep\u003c/em\u003eFBG\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e88.9%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e-0.48 \u0026plusmn; 0.01\u003csup\u003e26\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003ea\u003c/sup\u003eThe radiochemical conversions (RCC) were calculated from the labeling of the precursors.\u003csup\u003e\u0026nbsp;b\u003c/sup\u003eExtreme lipophilicity of [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e8-\u003cem\u003eo\u003c/em\u003e-e\u003c/strong\u003e resulted in excessively prolonged HPLC retention time, precluding the determination of the accurate RCC.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 2.\u0026nbsp;\u003c/strong\u003eStatic cardiac PET/CT imaging and quantitative analysis of radiotracers in healthy SD Rats at 60 min post-injection. (A) Myocardial PET images of \u003csup\u003e18\u003c/sup\u003eF-labeled NET-targeted tracers at 60 min post-injection in normal SD rats. (B) ROI analysis of myocardium at 60 min post-injection. Values are expressed as SUVmax.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 Cardiac PET imaging in normal rats\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cardiac PET imaging protocol was designed to assess the myocardial radiotracer distribution of 11 newly synthesized tracers and to identify promising candidates for cardiac sympathetic nerve imaging. As control and comparison, we also subjected three benzylguanidine analogs to PET imaging studies under the same experimental conditions. Figure 2-A shows representative static cardiac PET images of healthy rats using the aforementioned tracers at 60 min after tracer injection. In coronal images, all tracers visualized the myocardium,\u0026nbsp;with the left ventricular wall being most prominent.\u0026nbsp;This demonstrates the tolerance of the expansive hydrophobic pocket formed by subsites B/C toward tracers with extended linkers. Among them, [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e8-\u003cem\u003eo\u003c/em\u003e-a\u003c/strong\u003e exhibited the highest myocardial uptake compared to all the other ligands (Figure 2-B), with a maximum standardized uptake (SUV\u003csub\u003emax\u003c/sub\u003e) value of 8.95 \u0026plusmn; 0.62 (n=3), followed by [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e8-\u003cem\u003ep\u003c/em\u003e-b\u003c/strong\u003e and [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e8-\u003cem\u003eo\u003c/em\u003e-c\u003c/strong\u003e. To investigate the mechanisms of the PET tracer uptake in the rat hearts, we found pretreatment with selective uptake-1 blocking agents desipramine (DMI, 2 mg/kg) intravenously via tail vein 10 min before the radiotracer injection decreased the radiotracer uptake in the myocardium,\u0026nbsp;whereas pretreatment with phenoxybenzamine (PhB, 25 mg/kg intravenously) could significantly reduce myocardial uptake, counting\u0026nbsp;approximately 50% decrease (Fig. SX). These results indicated that myocardial tracer accumulation in rats might result from the synergistic contribution of both uptake-1 and uptake-2, consistent with previous reports\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003csup\u003e5,12\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBesides, the radioactivity levels in the adjacent liver and lung were also evaluated. In the lungs, only [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e8-\u003cem\u003eo\u003c/em\u003e-b\u003c/strong\u003e and [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e8-\u003cem\u003ep\u003c/em\u003e-b\u003c/strong\u003e showed slight bilateral radioactivity, whereas the other tracers showed even lower uptake, resulting in favorable heart-to-lung ratios (Fig. SX). The liver, as another important background organ, showed variable uptake levels among the different compounds (Fig. SX). [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e\u003cem\u003eo\u003c/em\u003eFBG\u003c/strong\u003e had the highest liver uptake, with liver activity exceeding that of the heart at 60 min p.i., resulting in a heart-to-liver ratio of only 0.60. In contrast, [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e8-\u003cem\u003eo\u003c/em\u003e-c\u003c/strong\u003e demonstrated significantly higher heart-to-liver uptake ratios (7.42 at 60 min p.i.).\u003c/p\u003e\n\u003cp\u003eFigure 3 shows the 60-minute dynamic PET images (A) and time-activity curves (B) of [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e\u003cem\u003em\u003c/em\u003eFBG\u003c/strong\u003e, [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e8-o-a\u003c/strong\u003e, and [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e8-o-c\u003c/strong\u003e in a rat following tracer injection. All tracers are rapidly distributed to the myocardium in the first frame (0-5 min) after injection. From the early images, the left ventricular wall was clearly delineable and remained visible until the last frame (50-60 min). Time-activity curves analysis of myocardium further showed that [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e8-\u003cem\u003eo\u003c/em\u003e-a\u003c/strong\u003e and [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e8-\u003cem\u003eo\u003c/em\u003e-c\u003c/strong\u003e sustained high and stable radioactivity throughout the scan time of 60 min with only a mild decline, whereas [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e\u003cem\u003em\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eFBG\u003c/strong\u003e decreased by approximately 50%, exhibiting the uptake level of myocardium comparable to liver at the late phase. In addition, different background-clearance patterns were observed among the three tracers. [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e8-\u003cem\u003eo\u003c/em\u003e-c\u003c/strong\u003e exhibited rapid clearance from both liver and lungs, resulting in superior contrast. In comparison, [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e8-\u003cem\u003eo\u003c/em\u003e-a\u003c/strong\u003e showed elevated pulmonary uptake peaking in the early phase (0-10 min), followed by rapid clearance from the lungs. In the same experiments, [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e\u003cem\u003em\u003c/em\u003eFBG\u003c/strong\u003e showed persistent, higher hepatic retention with minimal washout over time. Notably, [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e8-\u003cem\u003eo\u003c/em\u003e-a\u003c/strong\u003e and [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e8-\u003cem\u003eo\u003c/em\u003e-c\u003c/strong\u003e achieved favorable heart-to-liver ratios and were 3-fold higher than that of [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e\u003cem\u003em\u003c/em\u003eFBG\u0026nbsp;\u003c/strong\u003e(P\u0026lt;0.05) at the end of the study, facilitating the evaluation of inferior wall activity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4 Tumor PET imaging in mice\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further evaluate the ability to localize neuroendocrine tumors arising from the sympathetic system, static PET imaging was performed 30, 60, 120, and/or 240 min after injection of \u003csup\u003e18\u003c/sup\u003eF-labeled tracers in rat pheochromocytoma (PC-12) tumor-bearing mice (n = 3, Figure SX-SX). Representative maximum-intensity projection (MIP) images at 60 min p.i. for newly synthesized tracers are shown in Figure 4A. The majority of the evaluated tracers showed significant tumor uptake and favorable tumor-to-background contrast, except for [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e\u003cem\u003eo\u003c/em\u003eFBG\u0026nbsp;\u003c/strong\u003eand [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e8-\u003cem\u003eo\u003c/em\u003e/\u003cem\u003em\u003c/em\u003e/\u003cem\u003ep\u003c/em\u003e-c\u003c/strong\u003e. In addition, these eight tracers that exhibited significant tumor accumulation also showed uptake in adrenal glands and organs and tissues highly innervated by sympathetic neurons, including the myocardium, thyroid, salivary glands, and brown adipose tissue \u003csup\u003e27,28\u003c/sup\u003e. All tracers showed predominant renal clearance with evident bladder accumulation, and no detectable bone uptake, supporting their \u003cem\u003ein vivo\u003c/em\u003e stability. The SUVmax values of PC-12 tumors and main organs, including muscles, blood, liver, lung, myocardium, and bone in mice for all 12 radioligands, are presented in Figure 4A and Table 2. Among the benzylguanidine-based tracers ([\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e\u003cem\u003eo/m/p\u003c/em\u003eFBG\u003c/strong\u003e), the [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e\u003cem\u003em\u003c/em\u003eFBG\u003c/strong\u003e exhibited remarkable radioactivity uptake in PC-12 tumors, with the SUVmax of 22.13 \u0026plusmn; 1.95 (n = 3) (Figure 4B). In contrast, within the newly developed phenoxyalkylguanidine-based tracers, the \u003cem\u003eortho\u003c/em\u003e-fluoro derivatives showed markedly higher uptake in PC-12 tumors than fluorination\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eon the \u003cem\u003emeta\u003c/em\u003e- and para-position, with [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e8-\u003cem\u003eo\u003c/em\u003e-a\u0026nbsp;\u003c/strong\u003ereaching the highest level in all tracers, yielding a maximum SUV of 23.35 \u0026plusmn; 4.35 at 60 min p.i. (n = 3).\u0026nbsp;Moreover, we observed that the uptake of [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e8-\u003cem\u003eo\u003c/em\u003e/\u003cem\u003em\u003c/em\u003e/\u003cem\u003ep\u003c/em\u003e-c\u003c/strong\u003e in PC-12 tumors was markedly lower than that of [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e\u0026nbsp;8-\u003cem\u003eo\u003c/em\u003e/\u003cem\u003em\u003c/em\u003e/\u003cem\u003ep\u003c/em\u003e-a/b\u003c/strong\u003e, suggesting that extension of the linker carbon chain reduced the affinity of the tracers for NET in mice. Accordingly, [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e8-\u003cem\u003eo\u003c/em\u003e-d\u003c/strong\u003e and [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e8-\u003cem\u003eo\u003c/em\u003e-e\u003c/strong\u003e,\u0026nbsp;with further extended linkers,\u0026nbsp;were not tested in PC-12 tumor-bearing mice.\u0026nbsp;Interestingly, while the [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e8-\u003cem\u003eo\u003c/em\u003e/\u003cem\u003em\u003c/em\u003e/\u003cem\u003ep\u003c/em\u003e-c\u003c/strong\u003e performed well in rat cardiac imaging, their capacity in mouse tumor models was significantly compromised, showing a distinctly different biodistribution. In PC-12 tumor-bearing mice, uptake was low in tumors as well as in organs with dense sympathetic innervation. This discrepancy may be attributed to the species variations for the norepinephrine uptake mechanisms, with the high existence of the nonneural uptake-2 mechanism in rats compared to humans and other species\u003csup\u003e12\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2. Quantitative imaging analysis of NET-targeted tracers at 60 min p.i. in PC-12 models\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv align=\"center\"\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eComp.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.5833%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTumor\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.4167%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMuscle\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBlood\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLiver\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLung\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.5%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMyocardium\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.5%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBone\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e[\u003csup\u003e18\u003c/sup\u003eF]\u003cem\u003eo\u003c/em\u003eFBG\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.5833%;\"\u003e\n \u003cp\u003e4.11 \u0026plusmn; 1.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.4167%;\"\u003e\n \u003cp\u003e0.24 \u0026plusmn; 0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e0.30 \u0026plusmn; 0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e3.39 \u0026plusmn; 0.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e0.23 \u0026plusmn; 0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e0.54 \u0026plusmn; 0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e0.36 \u0026plusmn; 0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e[\u003csup\u003e18\u003c/sup\u003eF]\u003cem\u003em\u003c/em\u003eF\u003cem\u003eBG\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.5833%;\"\u003e\n \u003cp\u003e22.13 \u0026plusmn; 1.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.4167%;\"\u003e\n \u003cp\u003e0.53 \u0026plusmn; 0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e1.03 \u0026plusmn; 0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e1.70 \u0026plusmn; 0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e0.44 \u0026plusmn; 0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e2.24 \u0026plusmn; 0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e0.64 \u0026plusmn; 0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e[\u003csup\u003e18\u003c/sup\u003eF]\u003cem\u003ep\u003c/em\u003eFBG\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.5833%;\"\u003e\n \u003cp\u003e10.31 \u0026plusmn; 1.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.4167%;\"\u003e\n \u003cp\u003e0.22 \u0026plusmn; 0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e0.60 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e1.60 \u0026plusmn; 0.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e0.36 \u0026plusmn; 0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e0.98 \u0026plusmn; 0.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e0.53 \u0026plusmn; 0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e[\u003csup\u003e18\u003c/sup\u003eF]8-\u003cem\u003eo\u003c/em\u003e-a\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.5833%;\"\u003e\n \u003cp\u003e23.35 \u0026plusmn; 4.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.4167%;\"\u003e\n \u003cp\u003e0.14 \u0026plusmn; 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e1.26 \u0026plusmn; 0.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e0.65 \u0026plusmn; 0.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e0.55 \u0026plusmn; 0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e3.75 \u0026plusmn; 0.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e0.44 \u0026plusmn; 0.13\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e[\u003csup\u003e18\u003c/sup\u003eF]8-\u003cem\u003em\u003c/em\u003e-a\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.5833%;\"\u003e\n \u003cp\u003e10.14 \u0026plusmn; 1.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.4167%;\"\u003e\n \u003cp\u003e0.16 \u0026plusmn; 0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e1.10 \u0026plusmn; 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e0.57 \u0026plusmn; 0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e0.40 \u0026plusmn; 0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e2.68 \u0026plusmn; 0.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e0.63 \u0026plusmn; 0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e[\u003csup\u003e18\u003c/sup\u003eF]8-\u003cem\u003ep\u003c/em\u003e-a\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.5833%;\"\u003e\n \u003cp\u003e10.30 \u0026plusmn; 0.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.4167%;\"\u003e\n \u003cp\u003e0.21 \u0026plusmn; 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e1.25 \u0026plusmn; 0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e1.82 \u0026plusmn; 0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e0.50 \u0026plusmn; 0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e0.46 \u0026plusmn; 0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e0.44 \u0026plusmn; 0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e[\u003csup\u003e18\u003c/sup\u003eF]8-\u003cem\u003eo\u003c/em\u003e-b\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.5833%;\"\u003e\n \u003cp\u003e22.66 \u0026plusmn; 3.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.4167%;\"\u003e\n \u003cp\u003e0.26 \u0026plusmn; 0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e1.20 \u0026plusmn; 0.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e0.51 \u0026plusmn; 0.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e0.80 \u0026plusmn; 0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e4.31 \u0026plusmn; 1.72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e0.43 \u0026plusmn; 0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e[\u003csup\u003e18\u003c/sup\u003eF]8-\u003cem\u003em\u003c/em\u003e-b\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.5833%;\"\u003e\n \u003cp\u003e8.29 \u0026plusmn; 4.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.4167%;\"\u003e\n \u003cp\u003e0.19 \u0026plusmn; 0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e1.11 \u0026plusmn; 0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e0.46 \u0026plusmn; 0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e0.47 \u0026plusmn; 0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e3.75 \u0026plusmn; 0.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e0.46 \u0026plusmn; 0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e[\u003csup\u003e18\u003c/sup\u003eF]8-\u003cem\u003ep\u003c/em\u003e-b\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.5833%;\"\u003e\n \u003cp\u003e9.09 \u0026plusmn; 2.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.4167%;\"\u003e\n \u003cp\u003e0.18 \u0026plusmn; 0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e1.50 \u0026plusmn; 0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e1.15 \u0026plusmn; 0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e0.56 \u0026plusmn; 0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e4.25\u0026plusmn; 0.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e0.41 \u0026plusmn; 0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e[\u003csup\u003e18\u003c/sup\u003eF]8-\u003cem\u003eo\u003c/em\u003e-c\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.5833%;\"\u003e\n \u003cp\u003e2.41 \u0026plusmn; 0.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.4167%;\"\u003e\n \u003cp\u003e0.15 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e0.36 \u0026plusmn; 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e0.16 \u0026plusmn; 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e0.21 \u0026plusmn; 0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e0.74 \u0026plusmn; 0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e0.34 \u0026plusmn; 0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e[\u003csup\u003e18\u003c/sup\u003eF]8-\u003cem\u003em\u003c/em\u003e-c\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.5833%;\"\u003e\n \u003cp\u003e0.69 \u0026plusmn; 0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.4167%;\"\u003e\n \u003cp\u003e0.16 \u0026plusmn; 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e0.39 \u0026plusmn; 0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e0.38 \u0026plusmn; 0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e0.26 \u0026plusmn; 0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e0.82 \u0026plusmn; 0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e0.47 \u0026plusmn; 0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e[\u003csup\u003e18\u003c/sup\u003eF]8-\u003cem\u003ep\u003c/em\u003e-c\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.5833%;\"\u003e\n \u003cp\u003e1.06 \u0026plusmn; 0.44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.4167%;\"\u003e\n \u003cp\u003e0.20 \u0026plusmn; 0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e0.51 \u0026plusmn; 0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e0.51 \u0026plusmn; 0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e0.40 \u0026plusmn; 0.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e1.11 \u0026plusmn; 0.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e0.32 \u0026plusmn; 0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003e2.5 Characteristics and biodistribution in tumor-bearing mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter evaluation of the potential for \u003cem\u003ein vivo\u003c/em\u003e PET imaging in rat and mice of the \u003csup\u003e18\u003c/sup\u003eF-labeled radioligands, \u003csup\u003e18\u003c/sup\u003eF-labeled 1-(2-(2-fluorophenoxy)ethyl)guanidine ([\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e8-\u003cem\u003eo\u003c/em\u003e-a)\u003c/strong\u003e, namely [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e\u003cem\u003eo\u003c/em\u003eFPhOEG,\u003c/strong\u003e was selected to the further investigate its stability, affinity and biodistribution in tumor-bearing mice due to superior cardiac and tumor uptake, as well as for comparison with the widely used NET-targeted PET tracer [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e\u003cem\u003em\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eFBG\u003c/strong\u003e. As shown in Figure 5A, the [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e\u003cem\u003eo\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eFPhOEG\u0026nbsp;\u003c/strong\u003eshowed excellent \u003cem\u003ein vitro\u003c/em\u003e stability in PBS and rat serum at 37 \u0026deg;C for up to 4 hours, as confirmed by radio-HPLC analysis with no evidence of defluorination. NET binding affinity studies were performed with SK-N-SH cells expressing NET using [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e\u003cem\u003em\u003c/em\u003eFBG\u003c/strong\u003e as the radioligand and increasing concentrations of [\u003csup\u003e19\u003c/sup\u003eF]\u003cstrong\u003e\u003cem\u003eo\u003c/em\u003eFPhOEG\u003c/strong\u003e and [\u003csup\u003e19\u003c/sup\u003eF]\u003cstrong\u003e\u003cem\u003em\u003c/em\u003eFBG\u003c/strong\u003e to compete for uptake. The IC\u003csub\u003e50\u003c/sub\u003e values were 0.097 \u0026mu;M for \u003cstrong\u003e\u003cem\u003eo\u003c/em\u003eFPhOEG\u003c/strong\u003e and 0.254 \u0026mu;M for \u003cstrong\u003e\u003cem\u003em\u003c/em\u003eFBG\u003c/strong\u003e, respectively. Representative competitive uptake curves are shown in Figure 5B. In the cellular uptake assay (Figure 5C), we found that [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e\u003cem\u003eo\u003c/em\u003eFPhOEG\u003c/strong\u003e showed higher uptake in SK-N-SH cells than [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e\u003cem\u003em\u003c/em\u003eFBG\u003c/strong\u003e at all four time points (15, 30, 60, and 120 min). In addition, pretreatment with the selective uptake-1 inhibitor desipramine (DMI) further confirmed that tracer accumulation was NET-mediated. In the blocking group, DMI (10 \u0026mu;M) significantly inhibited the uptake of both tracers, with an inhibition rate exceeding 95%, confirming their specific binding to NET-expressing cells. Figure 5D showed the \u003cem\u003ein vivo\u003c/em\u003e performance of [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e\u003cem\u003eo\u003c/em\u003eFPhOEG\u003c/strong\u003e vs [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e\u003cem\u003emFBG\u003c/em\u003e\u003c/strong\u003e in PC-12 xenografts at 30, 60, 120, and 240 min p.i. The PET/CT imaging demonstrated that both tracers rapidly accumulated in tumors and maintained their retention for up to 240 min without significant washout. Quantitative analysis indicated that tumor uptake for both tracers peaked at 120 min ([\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e\u003cem\u003eo\u003c/em\u003eFPhOEG\u003c/strong\u003e: SUVmax = 25.30 \u0026plusmn; 3.88; [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e\u003cem\u003em\u003c/em\u003eFBG\u003c/strong\u003e: 21.96 \u0026plusmn; 2.95; Figure X-cd), with no statistically significant differences observed at any time point (P \u0026gt; 0.05; Figure SX). In addition, [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e\u003cem\u003eo\u003c/em\u003eFPhOEG\u003c/strong\u003e exhibited lower muscle and hepatic distribution compared with [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e\u003cem\u003em\u003c/em\u003eFBG\u003c/strong\u003e (Figure SX). As a result, [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e\u003cem\u003eo\u003c/em\u003eFPhOEG\u003c/strong\u003e achieved comparable tumor uptake but provided markedly improved tumor-to-background contrast relative to [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e\u003cem\u003em\u003c/em\u003eFBG\u003c/strong\u003e (Figure XX). Subsequent biodistribution results of [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e\u003cem\u003eo\u003c/em\u003eFPhOEG\u003c/strong\u003e were consistent with the ROI analysis, showing the highest radioactivity accumulation in PC-12 tumors, followed by brown adipose tissue, myocardium and adrenal glands (Figure 5E and Table SX). [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e\u003cem\u003eo\u003c/em\u003eFPhOEG\u003c/strong\u003e also exhibited rapid blood clearance following intravenous injection, with residual radioactivity 1.23 \u0026plusmn; 0.21%ID/g at 30 min. Similarly, early pulmonary uptake followed by rapid clearance of the tracer was also observed in mice, consistent with its behavior in rats. Thus, [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e\u003cem\u003eo\u003c/em\u003eFPhOEG\u003c/strong\u003e demonstrated excellent stability, high specificity for NET-expressing cells, and superior diagnostic performance, enabling rapid and accurate detection of sympathetic system-derived neuroendocrine tumors with high contrast in preclinical models.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6 Automatic Production and PET/CT studies in Rhesus Macaque\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe clinical translational potential of [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e\u003cem\u003eo\u003c/em\u003eFPhOEG\u003c/strong\u003e was further evaluated by an established automated production process using an integrated system comprising the commercial AllinOne radiosynthesis module and an LED reactor (ProBox), as previously reported \u003csup\u003e23,29\u003c/sup\u003e, to confirm its reproducibility and applicability for routine production. Details of the setup configuration and automated synthesis process are provided in Table SX and Figure SX. The [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e\u003cem\u003eo\u003c/em\u003eFPhOEG\u003c/strong\u003e was successfully synthesized and isolated in 29.4% RCY (decay corrected) in 75 min with a molar activity (A\u003csub\u003em\u003c/sub\u003e) of 64.76 GBq/\u0026mu;mol. Given that the hearts of monkeys and humans lack the nonneural uptake-2 pathway, nonhuman primates serve as an appropriate model for preclinical evaluation of sympathetic nerve tracers\u003csup\u003e12,30\u003c/sup\u003e. A 60-minute dynamic PET/CT study was subsequently performed in a healthy male rhesus macaque to characterize the cardiac and adjacent background distribution pattern of [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e\u003cem\u003eo\u003c/em\u003eFPhOEG\u003c/strong\u003e. The sequential cardiac PET MIP images of [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e\u003cem\u003eo\u003c/em\u003eFPhOEG\u003c/strong\u003e in the rhesus macaque were presented in Figure 6A. The 3 slice orientations and polar map of the left ventricle (LV) at 50 min p.i. are available in Figure 6B and 6C. The TAC curve showed the dynamic quantitative SUV mean value in four major tissues and organs of the tracer (Figure 6D). As shown in Figure 6A, [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e\u003cem\u003eo\u003c/em\u003eFPhOEG\u003c/strong\u003e rapidly distributed to the kidney, spleen, heart and blood pool, pancreas and liver within the first 2 min after injection. Thereafter, radioactivity in the spleen and lungs declined quickly, whereas hepatic uptake gradually increased, reaching its maximum at approximately 10-20 min. After 20 min, renal activity had largely cleared, allowing the adrenal glands to become clearly discernible. Uptake of [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e\u003cem\u003eo\u003c/em\u003eFPhOEG\u003c/strong\u003e into myocardium was similarly rapid; the ventricular wall was already well delineable on the second frame image (1-2 min) from the blood pool owing to rapid tracer clearance from circulation, and myocardial activity remained essentially stable throughout the 60-minute scan. Furthermore, to more comprehensively characterize the spatial distribution of [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e\u003cem\u003eo\u003c/em\u003eFPhOEG\u003c/strong\u003e within the myocardium, we performed quantitative circumferential analysis on a series of reoriented cardiac PET images. Shown are short axis (SA), vertical long axis (VLA) and horizontal long axis (HLA) views (Figure 6B) and polar maps (Figure 6C) of the left ventricle (LV), which not only define the regional variation of [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e\u003cem\u003eo\u003c/em\u003eFPhOEG\u0026nbsp;\u003c/strong\u003emyocardial uptake in the rhesus macaque but also facilitate precise localization and quantitative assessment of potentially affected myocardial segments. The TAC analysis in the myocardium showed consistently high and sustained tracer retention over the 60-minute scan, peaking at 17.5 minutes (SUVmean = 9.94 \u0026plusmn; 1.30) and exhibiting minimal washout over time (~ 0.2% per minute). A gradual increase in hepatic radioactivity was observed until 12.5 min p.i., after which tracer levels declined with hepatic clearance. The peak heart-to-liver ratio of 2.80 was recorded at 50 min p.i. Moreover, radioactivity in the blood pool and lungs cleared rapidly within 2 minutes and subsequently remained at a low level. Despite the high-quality cardiac images obtained with [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e8-\u003cem\u003eo-\u003c/em\u003ec\u003c/strong\u003e in SD rats, its myocardial behavior in rhesus macaques was entirely different, showing only minimal distribution in the myocardium (Figure SX). This finding further confirms that elongation of the alkyl linker substantially reduces NET affinity and the species-dependent differences in norepinephrine uptake pathways. Furthermore, the results suggested that myocardial uptake measured in rats is not necessarily a reliable predictor of left ventricular concentrations in primates or humans, demonstrating that rat-only evaluation is insufficient for assessing cardiac sympathetic nerve tracers. In contrast, [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e\u003cem\u003eo\u003c/em\u003eFPhOEG\u0026nbsp;\u003c/strong\u003eexhibited favorable kinetics and imaging properties for quantitative PET studies of cardiac sympathetic innervation.\u003c/p\u003e\n\u003cp\u003eSubsequently, whole-body static PET/CT scans at 60, 120, and 240 min (Figure 7A) after [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e\u003cem\u003eo\u003c/em\u003eFPhOEG\u003c/strong\u003e injection were performed in the same rhesus macaque to further evaluate the biodistribution and \u003cem\u003emeta\u003c/em\u003ebolic profile of [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e\u003cem\u003eo\u003c/em\u003eFPhOEG\u003c/strong\u003e in a nonhuman primate. TAC analysis showed the uptake in the major organs of the tracer (Figure 7B and 7C). Representative transverse PET and PET/CT fused images at 240 min p.i., illustrating organs with prominent uptake, are presented in Figure 7D. As shown in Figure 7, [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e\u003cem\u003eo\u003c/em\u003eFPhOEG\u003c/strong\u003e mainly accumulated in the bladder, heart, adrenal glands, spleen, salivary glands, pancreas, and liver, while lower activity was detected in the brain, lungs, intestine, bone, and muscle in all PET images. Over the course of the scan, the tracer was cleared by the urinary system, leading to accumulation of activity in the bladder. Uptake in the adrenal and salivary glands remained consistently high throughout the scan period, whereas radioactivity in the heart, spleen, liver, and pancreas gradually decreased over time. Notably, radioactivity in bone remained low at all time points, indicating no \u003cem\u003ein vivo\u003c/em\u003e radio-defluorination and confirming the \u003cem\u003emeta\u003c/em\u003ebolic stability of the tracer in nonhuman primates. Human radiation absorbed dose estimates were derived from the biodistribution of [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e\u003cem\u003eo\u003c/em\u003eFPhOEG\u003c/strong\u003e in the rhesus macaques at three imaging time points using OLINDA/EXM (version 2.2.3). The absorbed dose estimates (mSv/MBq) for the reference adult male model of OLINDA/EXM are summarized in Table SX. The urinary bladder wall received the highest radiation dose (0.469 mGy/MBq), followed by the prostate, due to its proximity to the bladder with substantial tracer accumulation. The effective dose of the whole body was 0.0261 mGy/MBq. These results of \u003cem\u003ein vivo\u003c/em\u003e distribution and radiation absorbed dose estimates in non-human primates warranted further clinical translation of [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e\u003cem\u003eo\u003c/em\u003eFPhOEG\u003c/strong\u003e in humans.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn this study, we successfully designed, synthesized, and systematically evaluated a series of novel \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eF-labeled phenoxyalkylguanidine NET-targeted PET tracers based on structure-based rational drug design and an efficient organic photocatalyzed \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eF-labeling strategy. All tracer precursors were synthesized via concise, robust routes and efficiently \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eF-labeled using our \u003cem\u003ePhAzF-18\u003c/em\u003e method. The optimized [\u003csup\u003e18\u003c/sup\u003eF]\u003cb\u003eo\u003c/b\u003e\u003cb\u003eFPhOEG\u003c/b\u003e showed good radiochemical yield, higher NET binding affinity, and excellent pharmacokinetic properties, exhibiting remarkable cardiac and tumor uptake in PET imaging. Compared to [\u003csup\u003e18\u003c/sup\u003eF]\u003cb\u003em\u003c/b\u003e\u003cb\u003eFBG\u003c/b\u003e, [\u003csup\u003e18\u003c/sup\u003eF]\u003cb\u003eo\u003c/b\u003e\u003cb\u003eFPhOEG\u003c/b\u003e exhibited markedly higher and more sustained myocardial uptake in SD rats and comparable tumor accumulation in PC12 tumor-bearing mice, and significantly increased heart-to-liver and tumor-to-liver ratios (3-fold). These findings suggest that [\u003csup\u003e18\u003c/sup\u003eF]\u003cb\u003eo\u003c/b\u003e\u003cb\u003eFPhOEG\u003c/b\u003e shows rapid hepatic clearance across the three animal models, which is crucial for minimizing liver interference with inferior wall assessment in cardiac PET and enhancing the visualization of hepatic metastases in tumor PET. Moreover, the tracer was successfully produced using a commercial automated module, highlighting its manufacturability and translational potential. In conclusion, these preclinical results make [\u003csup\u003e18\u003c/sup\u003eF]\u003cb\u003eo\u003c/b\u003e\u003cb\u003eFPhOEG\u003c/b\u003e a highly promising NET-targeted PET tracer for clinical applications in assessing cardiac sympathetic innervation denervation, such as in heart failure, and in evaluating neuroendocrine tumors arising from the sympathetic nervous system.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cspan\u003eAll the animal experiments in the manuscript were performed following the protocol approved by the Animal Care and Use Committees guidelines in West China Hospital and Sichuan University (20240627002 for rodent animals and 20240701001 for rhesus monkey).\u003c/span\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eTorres, G. E., Gainetdinov, R. R. \u0026amp; Caron, M. G. 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M.\u003cem\u003e et al.\u003c/em\u003e Radiolabeled Phenethylguanidines:\u0026thinsp; Novel Imaging Agents for Cardiac Sympathetic Neurons and Adrenergic Tumors. \u003cem\u003eJournal of Medicinal Chemistry\u003c/em\u003e \u003cstrong\u003e50\u003c/strong\u003e, 2078-2088, doi:10.1021/jm061398y (2007).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Scheme ","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Department of Nuclear Medicine and Clinical Nuclear Medicine Research Lab, West China Hospital, Sichuan University, Chengdu, Sichuan, 610041, China","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Norepinephrine transporter, cardiac and oncologic diagnosis, phenoxyalkylguanidine,18F-fluorination, PET imaging","lastPublishedDoi":"10.21203/rs.3.rs-8300571/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8300571/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe norepinephrine transporter (NET) is a pivotal target for imaging sympathetic nerves and neuroendocrine tumors, and positron emission tomography (PET) tracers have demonstrated substantial utility for precise clinical diagnosis among other imaging modalities. In this work, we constructed a series of aryl \u0026sup1;⁸F-labeled PET tracers from the innovative phenoxyalkylguanidine scaffold, featuring longer, optimized alkoxy chains to enhance hydrophobic interactions. The tracers were efficiently prepared via the photocatalyzed \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eF-fluorination, among which the [\u003csup\u003e18\u003c/sup\u003eF]\u003cb\u003eo\u003c/b\u003e\u003cb\u003eFPhOEG\u003c/b\u003e demonstrated high radiochemical conversion (82.8%), favorable lipophilicity (logD = -0.39), and superior NET affinity (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.097 \u0026micro;M). In vivo evaluations revealed high and sustained target uptake (myocardial SUVmax\u0026thinsp;=\u0026thinsp;8.95 in rats; tumor SUVmax\u0026thinsp;=\u0026thinsp;23.35 in mice). Notably, the newly developed phenoxyalkyl core structure enabled rapid hepatic clearance, leading to significantly improved heart-to-liver and tumor-to-liver ratios (3-fold higher than [\u003csup\u003e18\u003c/sup\u003eF]\u003cem\u003em\u003c/em\u003eFBG), which are crucial for enhancing diagnostic contrast. Additionally, the tracer was successfully produced using a commercial automated synthesis module in high RCY from a readily available precursor, guaranteeing the clinical translation of [\u003csup\u003e18\u003c/sup\u003eF]\u003cb\u003eo\u003c/b\u003e\u003cb\u003eFPhOEG\u003c/b\u003e for cardiac and oncologic diagnosis.\u003c/p\u003e","manuscriptTitle":"Design and Preclinical Evaluation of Novel 18F-Labeled Phenoxyalkylguanidines Radiotracers for Norepinephrine Transporter PET Imaging","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-10 10:03:19","doi":"10.21203/rs.3.rs-8300571/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"be8a0813-5cd8-4cce-a297-cba6cf170caf","owner":[],"postedDate":"December 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":59403858,"name":"Nuclear Medicine \u0026 Medical Imaging"}],"tags":[],"updatedAt":"2025-12-10T10:03:19+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-10 10:03:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8300571","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8300571","identity":"rs-8300571","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}