PET Imaging of the Neuropeptide Y System: A Systematic Review

Neuropeptide Y (NPY) is a vastly studied biological peptide with numerous physiological functions that activate the NPY receptor family (Y1, Y2, Y4 and Y5). Moreover, these receptors are correlated with the pathophysiology of several diseases such as feeding disorders, anxiety, metabolic diseases, neurodegenerative diseases, some types of cancers and others. In order to deepen the knowledge of NPY receptors’ functions and molecular mechanisms, neuroimaging techniques such as positron emission tomography (PET) have been used. The development of new radiotracers for the different NPY receptors and their subsequent PET studies have led to significant insights into molecular mechanisms involving NPY receptors. This article provides a systematic review of the imaging biomarkers that have been developed as PET tracers in order to study the NPY receptor family.


Introduction
Molecular imaging has been of immense value to the field of brain research, as the inaccessibility of the brain represents one of the greatest obstacles to central nervous system (CNS) studies [1]. Non-invasive imaging technology has not only played a major role in the discovery and development of CNS drugs but it has also become a prominent approach to the study and clarification of diverse neurological pathways. The uniqueness of the CNS field lies in the existence of its many potential drug targets; however, only a few are being exploited by current CNS therapeutics [2]. CNS diseases are highly heterogenous, which complicates the elucidation and research of their physiology and can lead to failure in the development of a new potential drug.
The potential of using imaging to optimize CNS drugs' discovery and development relies on various imaging techniques such as computerized tomography (CT), magnetic resonance imaging (MRI), single photon emission computed tomography (SPECT), positron emission tomography (PET) and others [3][4][5]. The main characteristics of these imaging techniques are summarized in Table 1.

Development of PET Tracers for NPY Receptors
As PET imaging is a powerful tool for drug development, especially when the target is in the central nervous system, the ability to develop successful radiotracers becomes significant as well. Radiotracers can have different roles in the drug development process: they can be used to demonstrate target delivery, to observe the change in the biochemistry of a system and to show the occupancy of a targeted site. In order to obtain an excellent radiotracer, some requirements must be fulfilled. Firstly, the radiotracer needs to have a high level of affinity towards the target, which requires the prior identification of the target of interest and then the development of ligands with high affinity to the target. Ideally, it is stipulated that the ratio B max /K d > 10 (as B max represents the target site concentration and K d the equilibrium dissociation constant of the radiotracer for that site) is met in order to have a high probability of the detection of the specific binding in vivo (even though there are some exceptions). Lipophilicity, expressed as Log P, is of relevance as it can influence the extent of the nonspecific binding activity and the ability to cross lipid membranes. Log P should be between 1 and 3.5 (though it is preferable that it is less than 3) for appropriate brain penetration and a good specific-non-specific binding ratio. In addition, the radiotracer must not be a P-glycoprotein (P-gp) substrate, as this is an efflux transporter that is substantially expressed in blood-brain barrier (BBB) penetration, blocking brain penetration. Ultimately, the radioligand should have an easy radiolabeling precursor, which means that the ligand should have chemical structural features that are suitable for radiolabeling with the appropriate radionuclide. The main goal in this step is to utilize a radiotracer with high molar activity, high radiochemical purity and high radiochemical yield. For further information about the main aspects of in vivo site-directed radiotracers, see Patel et al. [122].

NPY Y 1 Receptor
Over the past years, Y 1 receptor antagonists have been developed in order to study the diverse physiological roles of their receptors. In order to further understand the physiological role of Y 1 receptors in vivo, the development of novel PET tracers that are suitable for Y 1 receptors would allow the non-invasive imaging of the same and the determination of their occupancy. Bearing this in mind, Kameda et al. synthesized a series of 2,4-diaminopyridines and evaluated their suitability to become a PET tracer for the Y 1 receptor [123]. This investigation started with the identification of lead compound 1 ( Figure 1) as a promising PET tracer during structure-activity relationship studies. Compound 1 showed appropriate lipophilicity (Log P = 3.0) and favorable characteristics to be radiolabeled with 18 F; however, it was shown to have only a moderate level of Y 1 receptor binding affinity (Y 1 IC 50 = 21 nM). Thus, the focus of the study was on improving the affinity of the lead compound 1 towards the Y 1 receptor and its lipophilicity.
of a system and to show the occupancy of a targeted site. In order to obtain an excellent radiotracer, some requirements must be fulfilled. Firstly, the radiotracer needs to have a high level of affinity towards the target, which requires the prior identification of the target of interest and then the development of ligands with high affinity to the target. Ideally, it is stipulated that the ratio Bmax/Kd > 10 (as Bmax represents the target site concentration and Kd the equilibrium dissociation constant of the radiotracer for that site) is met in order to have a high probability of the detection of the specific binding in vivo (even though there are some exceptions). Lipophilicity, expressed as Log P, is of relevance as it can influence the extent of the nonspecific binding activity and the ability to cross lipid membranes. Log P should be between 1 and 3.5 (though it is preferable that it is less than 3) for appropriate brain penetration and a good specific-non-specific binding ratio. In addition, the radiotracer must not be a P-glycoprotein (P-gp) substrate, as this is an efflux transporter that is substantially expressed in blood-brain barrier (BBB) penetration, blocking brain penetration. Ultimately, the radioligand should have an easy radiolabeling precursor, which means that the ligand should have chemical structural features that are suitable for radiolabeling with the appropriate radionuclide. The main goal in this step is to utilize a radiotracer with high molar activity, high radiochemical purity and high radiochemical yield. For further information about the main aspects of in vivo sitedirected radiotracers, see Patel et al. [122].

NPY Y1 Receptor
Over the past years, Y1 receptor antagonists have been developed in order to study the diverse physiological roles of their receptors. In order to further understand the physiological role of Y1 receptors in vivo, the development of novel PET tracers that are suitable for Y1 receptors would allow the non-invasive imaging of the same and the determination of their occupancy. Bearing this in mind, Kameda et al. synthesized a series of 2,4-diaminopyridines and evaluated their suitability to become a PET tracer for the Y1 receptor [123]. This investigation started with the identification of lead compound 1 ( Figure 1) as a promising PET tracer during structure-activity relationship studies. Compound 1 showed appropriate lipophilicity (Log P = 3.0) and favorable characteristics to be radiolabeled with 18 F; however, it was shown to have only a moderate level of Y1 receptor binding affinity (Y1 IC50 = 21 nM). Thus, the focus of the study was on improving the affinity of the lead compound 1 towards the Y1 receptor and its lipophilicity. The SAR studies led to the identification of the potent and selective compounds 1d and 1e (Tables 3 and 4) as promising candidates for Y1 PET tracers. Firstly, the variation of the antagonist 1 right hand 2-amino group was examined and the 2-fluoro-6- The SAR studies led to the identification of the potent and selective compounds 1d and 1e (Tables 3 and 4) as promising candidates for Y 1 PET tracers. Firstly, the variation of the antagonist 1 right hand 2-amino group was examined and the 2-fluoro-6-methylenepyridine derivative showed the most potent Y 1 affinity (Y 1 IC 50 = 1.5 nM, compound 1a, Table 3). Bearing this result, this derivative was substituted with other proper functional groups in order to favor its use in radiolabeling. The replacement of the fluorine with a methyl group resulted in a powerful increase in the level of binding affinity (Y 1 IC 50 = 0.69 nM, compound 1b, Table 3) and a subtle reduction of the lipophilicity. Then, the left hand heterocycle portion of the previous compound was optimized. The 4,5-dimethyloxazole ring was replaced with a 4,5-dimethylthiazole ring, an atom of fluorine was attached to the 5-methyl group (compound 1c, Table 3) and, lastly, the fluorine was moved from the 5methyl to a 4-methyl group of the thiazole ring so as to produce compound 1d with its high affinity towards the Y 1 receptor and reduced lipophilicity (Table 3). Notably, this fluorine substitution is an additional labelling option for the incorporation of 18 F via nucleophilic substitution. Moreover, the 4-fluoromethyl-5-ethyl-thiazole derivative (compound 1e) showed an even further improvement in Y 1 receptor binding and reasonable lipophilicity (Table 3). Both compounds 1d and 1e also exhibited great selectivity towards the Y 1 receptor (Table 4). the left hand heterocycle portion of the previous compound was optimized. The 4,5dimethyloxazole ring was replaced with a 4,5-dimethylthiazole ring, an atom of fluorine was attached to the 5-methyl group (compound 1c, Table 3) and, lastly, the fluorine was moved from the 5-methyl to a 4-methyl group of the thiazole ring so as to produce compound 1d with its high affinity towards the Y1 receptor and reduced lipophilicity (Table 3). Notably, this fluorine substitution is an additional labelling option for the incorporation of 18 F via nucleophilic substitution. Moreover, the 4-fluoromethyl-5-ethylthiazole derivative (compound 1e) showed an even further improvement in Y1 receptor binding and reasonable lipophilicity (Table 3). Both compounds 1d and 1e also exhibited great selectivity towards the Y1 receptor (Table 4). (Y1 IC50 = 0.69 nM, compound 1b, Table 3) and a subtle reduction of the lipophilicity. Then, the left hand heterocycle portion of the previous compound was optimized. The 4,5dimethyloxazole ring was replaced with a 4,5-dimethylthiazole ring, an atom of fluorine was attached to the 5-methyl group (compound 1c, Table 3) and, lastly, the fluorine was moved from the 5-methyl to a 4-methyl group of the thiazole ring so as to produce compound 1d with its high affinity towards the Y1 receptor and reduced lipophilicity (Table 3). Notably, this fluorine substitution is an additional labelling option for the incorporation of 18 F via nucleophilic substitution. Moreover, the 4-fluoromethyl-5-ethylthiazole derivative (compound 1e) showed an even further improvement in Y1 receptor binding and reasonable lipophilicity (Table 3). Both compounds 1d and 1e also exhibited great selectivity towards the Y1 receptor (Table 4). fluorine with a methyl group resulted in a powerful increase in the level of binding affinity (Y1 IC50 = 0.69 nM, compound 1b, Table 3) and a subtle reduction of the lipophilicity. Then, the left hand heterocycle portion of the previous compound was optimized. The 4,5dimethyloxazole ring was replaced with a 4,5-dimethylthiazole ring, an atom of fluorine was attached to the 5-methyl group (compound 1c, Table 3) and, lastly, the fluorine was moved from the 5-methyl to a 4-methyl group of the thiazole ring so as to produce compound 1d with its high affinity towards the Y1 receptor and reduced lipophilicity (Table 3). Notably, this fluorine substitution is an additional labelling option for the incorporation of 18 F via nucleophilic substitution. Moreover, the 4-fluoromethyl-5-ethylthiazole derivative (compound 1e) showed an even further improvement in Y1 receptor binding and reasonable lipophilicity (Table 3). Both compounds 1d and 1e also exhibited great selectivity towards the Y1 receptor (Table 4). compound 1a, Table 3). Bearing this result, this derivative was substituted with other proper functional groups in order to favor its use in radiolabeling. The replacement of the fluorine with a methyl group resulted in a powerful increase in the level of binding affinity (Y1 IC50 = 0.69 nM, compound 1b, Table 3) and a subtle reduction of the lipophilicity. Then, the left hand heterocycle portion of the previous compound was optimized. The 4,5dimethyloxazole ring was replaced with a 4,5-dimethylthiazole ring, an atom of fluorine was attached to the 5-methyl group (compound 1c, Table 3) and, lastly, the fluorine was moved from the 5-methyl to a 4-methyl group of the thiazole ring so as to produce compound 1d with its high affinity towards the Y1 receptor and reduced lipophilicity (Table 3). Notably, this fluorine substitution is an additional labelling option for the incorporation of 18 F via nucleophilic substitution. Moreover, the 4-fluoromethyl-5-ethylthiazole derivative (compound 1e) showed an even further improvement in Y1 receptor binding and reasonable lipophilicity (Table 3). Both compounds 1d and 1e also exhibited great selectivity towards the Y1 receptor (Table 4). methylenepyridine derivative showed the most potent Y1 affinity (Y1 IC50 = 1.5 nM, compound 1a, Table 3). Bearing this result, this derivative was substituted with other proper functional groups in order to favor its use in radiolabeling. The replacement of the fluorine with a methyl group resulted in a powerful increase in the level of binding affinity (Y1 IC50 = 0.69 nM, compound 1b, Table 3) and a subtle reduction of the lipophilicity. Then, the left hand heterocycle portion of the previous compound was optimized. The 4,5dimethyloxazole ring was replaced with a 4,5-dimethylthiazole ring, an atom of fluorine was attached to the 5-methyl group (compound 1c, Table 3) and, lastly, the fluorine was moved from the 5-methyl to a 4-methyl group of the thiazole ring so as to produce compound 1d with its high affinity towards the Y1 receptor and reduced lipophilicity (Table 3). Notably, this fluorine substitution is an additional labelling option for the incorporation of 18 F via nucleophilic substitution. Moreover, the 4-fluoromethyl-5-ethylthiazole derivative (compound 1e) showed an even further improvement in Y1 receptor binding and reasonable lipophilicity (Table 3). Both compounds 1d and 1e also exhibited great selectivity towards the Y1 receptor (Table 4).  methylenepyridine derivative showed the most potent Y1 affinity (Y1 IC50 = 1.5 nM, compound 1a, Table 3). Bearing this result, this derivative was substituted with other proper functional groups in order to favor its use in radiolabeling. The replacement of the fluorine with a methyl group resulted in a powerful increase in the level of binding affinity (Y1 IC50 = 0.69 nM, compound 1b, Table 3) and a subtle reduction of the lipophilicity. Then, the left hand heterocycle portion of the previous compound was optimized. The 4,5dimethyloxazole ring was replaced with a 4,5-dimethylthiazole ring, an atom of fluorine was attached to the 5-methyl group (compound 1c, Table 3) and, lastly, the fluorine was moved from the 5-methyl to a 4-methyl group of the thiazole ring so as to produce compound 1d with its high affinity towards the Y1 receptor and reduced lipophilicity (Table 3). Notably, this fluorine substitution is an additional labelling option for the incorporation of 18 F via nucleophilic substitution. Moreover, the 4-fluoromethyl-5-ethylthiazole derivative (compound 1e) showed an even further improvement in Y1 receptor binding and reasonable lipophilicity ( Table 3). Both compounds 1d and 1e also exhibited great selectivity towards the Y1 receptor (Table 4).  compound 1a, Table 3). Bearing this result, this derivative was substituted with other proper functional groups in order to favor its use in radiolabeling. The replacement of the fluorine with a methyl group resulted in a powerful increase in the level of binding affinity (Y1 IC50 = 0.69 nM, compound 1b, Table 3) and a subtle reduction of the lipophilicity. Then, the left hand heterocycle portion of the previous compound was optimized. The 4,5dimethyloxazole ring was replaced with a 4,5-dimethylthiazole ring, an atom of fluorine was attached to the 5-methyl group (compound 1c, Table 3) and, lastly, the fluorine was moved from the 5-methyl to a 4-methyl group of the thiazole ring so as to produce compound 1d with its high affinity towards the Y1 receptor and reduced lipophilicity (Table 3). Notably, this fluorine substitution is an additional labelling option for the incorporation of 18 F via nucleophilic substitution. Moreover, the 4-fluoromethyl-5-ethylthiazole derivative (compound 1e) showed an even further improvement in Y1 receptor binding and reasonable lipophilicity (Table 3). Both compounds 1d and 1e also exhibited great selectivity towards the Y1 receptor (Table 4). Afterwards, Hostetler et al. identified Y 1 -973 (compound 1e) as a promising PET tracer candidate to be radiolabeled as it fulfilled the abovementioned requirements: potency < 0.5 nM, Log P < 3.5, P-gp ratio < 3 and it was suitable for radiolabeling with 18 F. Thus, the authors were able to report the synthesis and pre-clinical evaluation of the NPY Y 1 receptor PET tracer [ 18 F]Y 1 -973 [124]. [ 18 F]Y 1 -973 was produced through the reaction of [ 18 F]KF/K 222 with the t-butyloxycarbonyl (Boc)-protected chloromethyl thiazole 2 precursor in DMSO at 130 • C using microwave heating, followed by deprotection with 1 M HCl ( Figure 2). [ 18 F]Y 1 -973 was obtained in satisfactory radiochemical yields (18 ± 13%, n = 9) with >98% radiochemical purity and high molar activity (1548 ± 859 Ci/mmol), in a total synthesis time, including purification by semi-preparative HPLC, of about 45 min [124]. Afterwards, Hostetler et al. identified Y1-973 (compound 1e) as a promising PET tracer candidate to be radiolabeled as it fulfilled the abovementioned requirements: potency < 0.5 nM, Log P < 3.5, P-gp ratio < 3 and it was suitable for radiolabeling with 18 F. Thus, the authors were able to report the synthesis and pre-clinical evaluation of the NPY Y1 receptor PET tracer [ 18 F]Y1-973 [124]. [ 18 F]Y1-973 was produced through the reaction of [ 18 F]KF/K222 with the t-butyloxycarbonyl (Boc)-protected chloromethyl thiazole 2 precursor in DMSO at 130 °C using microwave heating, followed by deprotection with 1 M HCl ( Figure 2). [ 18 F]Y1-973 was obtained in satisfactory radiochemical yields (18 ± 13%, n = 9) with >98% radiochemical purity and high molar activity (1548 ± 859 Ci/mmol), in a total synthesis time, including purification by semi-preparative HPLC, of about 45 min [124]. In vivo PET imaging studies of [ 18 F]Y1-973 in rhesus monkeys have demonstrated that this PET tracer promptly penetrates the blood-brain barrier. Baseline PET scans of this radiotracer in rhesus monkeys have shown heterogeneous distribution throughout the brain, with a pattern of uptake that is consistent with the known high NPY Y1 brain density (the highest level of uptake was in the striatum and cortical regions, there was moderate uptake in the thalamus and low uptake in the cerebellum). [ 18 F]Y1-973 also exhibited rapid kinetics in rhesus monkeys' brains, with the uptake peaking in the striatum at approximately 30 min. Additionally, it has a large binding potential that is suitable for receptor occupancy PET studies with an NPY Y1 antagonist [124].
Hofmann et al. reported a suitable 1 8 F-labeled high-molecular weight glycopeptide for the imaging of peripheral NPY Y1 receptor-positive tumors, namely breast cancer. Notably, the human Y1 receptor subtype has been discovered to be overly expressed in 85% of primary breast cancer and in 100% of lymph node metastases [125]. Despite [ 18 F]Y1-973 having remarkable properties for brain imaging of the Y1 receptor, its high lipophilicity and significant liver accumulation makes it less suitable for peripheral application for breast cancer imaging. Therefore, the authors reported the development of a full length NPY analogue bearing a glycosylation site in position 4 of the amino acid sequence and the 18 F-labeling by 18   In vivo PET imaging studies of [ 18 F]Y 1 -973 in rhesus monkeys have demonstrated that this PET tracer promptly penetrates the blood-brain barrier. Baseline PET scans of this radiotracer in rhesus monkeys have shown heterogeneous distribution throughout the brain, with a pattern of uptake that is consistent with the known high NPY Y 1 brain density (the highest level of uptake was in the striatum and cortical regions, there was moderate uptake in the thalamus and low uptake in the cerebellum). [ 18 F]Y 1 -973 also exhibited rapid kinetics in rhesus monkeys' brains, with the uptake peaking in the striatum at approximately 30 min. Additionally, it has a large binding potential that is suitable for receptor occupancy PET studies with an NPY Y 1 antagonist [124].
Hofmann et al. reported a suitable 1 8 F-labeled high-molecular weight glycopeptide for the imaging of peripheral NPY Y 1 receptor-positive tumors, namely breast cancer. Notably, the human Y 1 receptor subtype has been discovered to be overly expressed in 85% of primary breast cancer and in 100% of lymph node metastases [125]. Despite [ 18 F]Y 1 -973 having remarkable properties for brain imaging of the Y 1 receptor, its high lipophilicity and significant liver accumulation makes it less suitable for peripheral application for breast cancer imaging. Therefore, the authors reported the development of a full length NPY analogue bearing a glycosylation site in position 4 of the amino acid sequence and the 18 F-labeling by 18 F-fluoroglycosylation using click chemistry ( Figure 3) [126].
The authors selected [F 7 , P 34 ] NPY analogue as it had scientific evidence supporting its selective binding to Y 1 receptor and they then developed two peptide precursors bearing an alkyne-bearing functionality (peptides 5a and 6a, Figure 3), allowing regiospecific 18 Flabeling by 18   The authors selected [F 7 , P 34 ] NPY analogue as it had scientific evidence supporting its selective binding to Y1 receptor and they then developed two peptide precursors bearing an alkyne-bearing functionality (peptides 5a and 6a, Figure 3), allowing regiospecific 18 F-labeling by 18 F-fluoroglycosylation using click chemistry. The alkyne functionalization was obtained by the introduction of propargylglycine (Pra) at position 4 ( Figure 3). 18  Then, the remaining solution (60 mM NaOH) was adjusted to pH 8 (1M HCl) and a solution of the peptides 5a or 6a, sodium ascorbate, CuSO4 and THPTA (tris(hydroxypropyltriazolylmethyl)amine) were added. After isolation by semipreparative HPLC and subsequent solid phase extraction, [ 18 F]5b (n = 7) or [ 18 F]6b (n = 2) were obtained. The molar activities were about 40-70 GBq/μmol in overall radiochemical yields of 20-25% with a total synthesis time of 75 min. Further in vitro characterization was undertaken and the glycopeptide [ 18 F]5b showed great selectivity for Y1 receptor over the Y2 receptor and strong Y1 receptor internalization. In this study, the preclinical animal model that was used was an MCF-7 breast cancer tumor model. PET imaging studies in these animals demonstrated a specific binding uptake of the glycopeptide [ 18 F]5b from Y1 receptors present in MCF-7 tumors and that [ 18 F]5b exhibited considerably increased renal clearance properties, compared to the NPY DOTA-derivatives, showing favorable kinetics in vivo [126].
As mentioned earlier, 85% of mammary carcinomas are Y1 receptor positive. To address this issue, Keller et al. reported the exploration of 18 F-labeled compounds that were derived from the argininamide BIBP3326, which is a Y1 receptor antagonist ( Figure 4) [127].  showed great selectivity for Y 1 receptor over the Y 2 receptor and strong Y 1 receptor internalization. In this study, the preclinical animal model that was used was an MCF-7 breast cancer tumor model. PET imaging studies in these animals demonstrated a specific binding uptake of the glycopeptide [ 18 F]5b from Y 1 receptors present in MCF-7 tumors and that [ 18 F]5b exhibited considerably increased renal clearance properties, compared to the NPY DOTA-derivatives, showing favorable kinetics in vivo [126].
As mentioned earlier, 85% of mammary carcinomas are Y 1 receptor positive. To address this issue, Keller et al. reported the exploration of 18 F-labeled compounds that were derived from the argininamide BIBP3326, which is a Y 1 receptor antagonist ( Figure 4) [127]. The authors selected [F 7 , P 34 ] NPY analogue as it had scientific evidence supporting its selective binding to Y1 receptor and they then developed two peptide precursors bearing an alkyne-bearing functionality (peptides 5a and 6a, Figure 3), allowing regiospecific 18 F-labeling by 18  Then, the remaining solution (60 mM NaOH) was adjusted to pH 8 (1M HCl) and a solution of the peptides 5a or 6a, sodium ascorbate, CuSO4 and THPTA (tris(hydroxypropyltriazolylmethyl)amine) were added. After isolation by semipreparative HPLC and subsequent solid phase extraction, [ 18 F]5b (n = 7) or [ 18 F]6b (n = 2) were obtained. The molar activities were about 40-70 GBq/μmol in overall radiochemical yields of 20-25% with a total synthesis time of 75 min. Further in vitro characterization was undertaken and the glycopeptide [ 18 F]5b showed great selectivity for Y1 receptor over the Y2 receptor and strong Y1 receptor internalization. In this study, the preclinical animal model that was used was an MCF-7 breast cancer tumor model. PET imaging studies in these animals demonstrated a specific binding uptake of the glycopeptide [ 18 F]5b from Y1 receptors present in MCF-7 tumors and that [ 18 F]5b exhibited considerably increased renal clearance properties, compared to the NPY DOTA-derivatives, showing favorable kinetics in vivo [126].
As mentioned earlier, 85% of mammary carcinomas are Y1 receptor positive. To address this issue, Keller et al. reported the exploration of 18 F-labeled compounds that were derived from the argininamide BIBP3326, which is a Y1 receptor antagonist ( Figure 4) [127].  The authors acknowledged that the bioisosteric substitution of the guanidine group in the argininamide moiety of a BIBP3226 compound (e.g., UR-MK114, 7b and UR-MK136, 7c, Figure 4) was shown to lead to the formation of highly selective and potent fluorescent ligands, bivalent ligands and tritiated radioligands for the Y1 receptor. Similarly, Keller et al. used this strategy to design new and selective [ 18 F] Y1 receptor PET ligands. After the synthesis and in vitro characterization of a series of fluorinated argininamides, they concluded that derivative 10 ( Figure 5) was the most promising candidate as it displayed the lowest lipophilicity (Log P of 3.4). Derivative 10 was synthesized using the building block 8. The radiosynthesis of [ 18 F]10 was proceeded by the treatment of excess of amine 9 with  The tracer [ 18 F]10 was then evaluated in vitro and in vivo using tumor-bearing nude mice with Y1R positive MCF-7 tumors. Its Log P was experimentally determined resulting in a value of 2.34 ± 0.03. [ 18 F]10 had fast blood clearance time at 30 and 90 min, low uptake in the liver and high uptake in the gallbladder and intestines. The tracer also showed high tumor retention and a suitable signal-to-noise ratio for PET imaging. In comparison with [ 18 F]5b, [ 18 F]10 had lower tumor uptake because of its uptake in the gallbladder. Nonetheless, [ 18 F]10 showed diminished kidney uptake when compared with the peptide tracer, which is the major advantage of the nonpeptide radioligand over the peptide tracer for PET imaging. In vivo PET imaging studies with the antagonist [ 18 F]10 in the MCF-7 nude mice revealed the visualization of Y1R-positive MCF-7 tumors [127].
Regarding these results and in order to reduce the lipophilicity of a candidate ligand and to achieve more suitable biodistribution with minimal biliary excretion, hence improving the tumor visibility in PET images, Maschauer et al. recently reported the synthesis and radiosynthesis of three BIBP3226 derivatives that were conjugated with 18 F-fluoroethoxy linkers and 18 F-fluoroglucosyl moiety and further in vitro and in vivo The tracer [ 18 F]10 was then evaluated in vitro and in vivo using tumor-bearing nude mice with Y 1 R positive MCF-7 tumors. Its Log P was experimentally determined resulting in a value of 2.34 ± 0.03. [ 18 F]10 had fast blood clearance time at 30 and 90 min, low uptake in the liver and high uptake in the gallbladder and intestines. The tracer also showed high tumor retention and a suitable signal-to-noise ratio for PET imaging. In comparison with [ 18 F]5b, [ 18 F]10 had lower tumor uptake because of its uptake in the gallbladder. Nonetheless, [ 18 F]10 showed diminished kidney uptake when compared with the peptide tracer, which is the major advantage of the nonpeptide radioligand over the peptide tracer for PET imaging. In vivo PET imaging studies with the antagonist [ 18 F]10 in the MCF-7 nude mice revealed the visualization of Y 1 R-positive MCF-7 tumors [127].
Regarding these results and in order to reduce the lipophilicity of a candidate ligand and to achieve more suitable biodistribution with minimal biliary excretion, hence improving the tumor visibility in PET images, Maschauer et al. recently reported the synthesis and radiosynthesis of three BIBP3226 derivatives that were conjugated with 18 F-fluoroethoxy linkers and 18 F-fluoroglucosyl moiety and further in vitro and in vivo characterization [128]. [ 18 F]15 was obtained through a copper-catalyzed azide-alkyne cycloaddition (CuAAc-Based) 18 F-fluoroglycosylation method between the BIBP3226 derived alkyne 11 and 6-deoxy-6[ 18 F]fluoroglucosyl azide [ 18 F]12 ( Figure 6). [ 18 F]12 was previously prepared and isolated by semi-preparative HPLC and deacetylated with NaOH, following a priorly described method [129].  17 were isolated with radioactivity yields of about 5% and 10% (in reference to [ 18 F]fluoride), molar activities between 5-6 GBq/µmol and radiochemical purity >99% in a 60 min total synthesis time ( Figure 6). characterization [128]. [ 18 F]15 was obtained through a copper-catalyzed azide-alkyne cycloaddition (CuAAc-Based) 18 F-fluoroglycosylation method between the BIBP3226 derived alkyne 11 and 6-deoxy-6[ 18 F]fluoroglucosyl azide [ 18 F]12 ( Figure 6). [ 18 F]12 was previously prepared and isolated by semi-preparative HPLC and deacetylated with NaOH, following a priorly described method [129].  17 were isolated with radioactivity yields of about 5% and 10% (in reference to [ 18 F]fluoride), molar activities between 5-6 GBq/μmol and radiochemical purity >99% in a 60 min total synthesis time ( Figure 6). The in vitro characterization of the three compounds showed that these were selective towards the Y1 receptor and revealed their weak lipophilicity, with [ 18 F] 15 17 showed specific tumor accumulation in vivo, even though there were relatively high background values in the PET images, due to the formation of radiometabolites in the blood.
Recently, the radiolabeling of the Y 1 antagonist BMS-193885 with [ 11 C] has also been reported [130]. BMS-193885 (1,4- amino]carbonyl]amino]phenyl]-2,6-dimethyl-3,5-pyridinedicarboxylic acid, dimethyl ester, 18, Figure 7) is a potent and selective NPY Y 1 receptor antagonist with good brain penetration and systemic bioavailability but poor oral bioavailability [55,131]. It was previously disclosed that it reduces food intake and body weight in animal models of obesity, being pharmacologically efficacious in the treatment of obesity in animal models [131]. Therefore gallbladder. Thus, the aim of achieving a reduced level of uptake in the gallbladder by using more hydrophilic compounds was not reached, although it was possible to reduce the lipophilicity of the analogues. Blood sample analysis by HPLC revealed that the radiotracers  17 showed specific tumor accumulation in vivo, even though there were relatively high background values in the PET images, due to the formation of radiometabolites in the blood.
Recently, the radiolabeling of the Y1 antagonist BMS-193885 with [ 11 C] has also been reported [130]. BMS-193885 (1,4- propyl]amino]carbonyl]amino]phenyl]-2,6-dimethyl-3,5-pyridinedicarboxylic acid, dimethyl ester, 18, Figure 7) is a potent and selective NPY Y1 receptor antagonist with good brain penetration and systemic bioavailability but poor oral bioavailability [55,131]. It was previously disclosed that it reduces food intake and body weight in animal models of obesity, being pharmacologically efficacious in the treatment of obesity in animal models [131]. Therefore, Kawamura    In order to obtain the tracer [ 11 C]18, an automated synthesis system that was developed in-house was used. A solution of 19 and sodium hydroxide solution in N,N-dimethylformamide (DMF) was added to a dry septum-equipped vial prior to the reaction. [ 11 C]Methyl iodide was produced by the reduction of cyclotron-produced [ 11 C]carbon di- In order to obtain the tracer [ 11 C]18, an automated synthesis system that was developed in-house was used. A solution of 19 and sodium hydroxide solution in N,Ndimethylformamide (DMF) was added to a dry septum-equipped vial prior to the reaction. [ 11 C]Methyl iodide was produced by the reduction of cyclotron-produced [ 11 C]carbon dioxide with lithium aluminum hydride, followed by iodination with hydroiodic acid. The [ 11 C]methyl iodide that was produced was then trapped in a reaction vial containing 19 in DMF. The reaction mixture was heated and held at 80 • C for 5 min. Afterwards, the solution was purified by preparative HPLC. [ 11 C]18 was synthesized approximately 30 min after the end of irradiation (EOI), with a radiochemical yield of 23 ± 3.2% (n = 6), molar activity of 87 ± 28 GBq/µmol (n = 6) and radiochemical purity >99% ( Figure 8A). The radiosynthesis of [ 11 C]22 ( Figure 8B) started with the initial production of [ 11 C]carbon dioxide using a cyclotron, followed by the subsequent reduction of [ 11 C]carbon dioxide with H 2 in the presence catalyst, generating [ 11 C]methane, the chlorination of [ 11 C]methane and the conversion of [ 11 C]carbon tetrachloride to [ 11 C]phosgene. Afterwards, aniline derivative 20 in a tetrahydrofuran (THF) solution was added to a dry septum-equipped vial just prior to radiosynthesis. [ 11 C]phosgene was trapped in the solution containing 20 at −15 • C for 1 min. The reaction mixture was heated at 30 • C for 3 min. When the solution cooled, it was added a solution containing amine derivative 21 in THF. Then, the reaction mixture was heated at 80 • C for 3 min in order to remove the THF. The crude product was then purified through the use of preparative HPLC. Finally, [ 11 C]22 was obtained at about 33 min after EOI with a radiochemical yield of 24 ± 1.5% (n = 4), molar activity of 52 ± 10 GBq/µmol (n = 4) and radiochemical purity >99% ( Figure 8B) [130].
In vivo studies have shown poor brain penetration from both of these radiotracers. The synthesis of [ 11 C]22 was undertaken mainly in order to address the issue of the poor brain penetration of [ 11 C]18, as it has lower lipophilicity (Log P = 3.3) than [ 11 C]18 (Log P = 3.8) and higher affinity binding ([ 11 C]22 Ki = 2.7 nM and [ 11 C]18 Ki = 3.3 nM). Nevertheless, the radioactivity in the brain of [ 11 C]22 was much lower than that of [ 11 C]18, mostly due to its faster metabolization (high levels of [ 11 C]22 were present in the small intestine and liver). The poor brain penetration of [ 11 C]18 is mainly explained by the influence of the drug efflux transporters P-gp and BCPR. Therefore, in order to visualize the NPY Y 1 receptors in the brain using this strategy, it is crucial to develop PET tracers with superior blood-brain barrier penetration and in vivo stability. Nevertheless, this study was useful in its provision of a better understanding of the in vivo properties of 18.
Diverging from the classical methods of the radiolabeling of the Y 1 antagonists, Vall-Sagarra et al. combined the use of heterobivalent peptidic ligands (HBPLs) with [ 68 Ga] radiolabeling methods and developed a distinct approach to the development of new Y 1 radiotracers [132]. As is known, radiolabeled peptides are a great tool for tumor visualizations as they target the many receptors that are overexpressed, though normally they target a specific receptor. As different tumor lesions can express different receptor types, the tumor visualization may not be as efficacious as it was previously hypothesized. Radiolabeled heterobivalent peptide ligands (HBPLs) have the capacity to specifically target more than one receptor type, rendering them advantageous when compared to monovalent peptides, enabling better tumor visualization and improved in vivo biodistribution. As was referenced earlier, human breast cancer overexpresses 85% of NPY Y 1 receptor but, as a matter of fact, it also overexpresses the gastrin-releasing peptide receptor (GRPR) in about 75% of cases. Hence, the authors developed bispecific HBPLs and radiolabeled them with 68 Ga and then proceeded to undertake further in vitro and in vivo characterizations in order to show the general feasibility of this distinct strategy. Thus, HBPLs 23-27 and monomeric reference peptides 28 and 29 ( Figure 9) were synthesized and then radiolabeled with Ga 3+ . The 68 Ga 3+ was obtained via the elution of an 68 25 were necessary to enhanced in vivo tumor uptake and that this uptake was GRPR-and NPY Y 1 -specific. Concluding, the authors were able to demonstrate that the use of GRPR-and NPY Y 1specific HBPL were indeed beneficial to in vivo tumor uptake and visualization, compared to monospecific agents [132].
Recently, Krieger et al. have reported the synthesis of 68 Ga-radiolabeled analogues of the most promising previously described truncated NPY analogues and compared them regarding their metabolic stability [133]. Of the nearly 80 truncated peptides analogues of NPY that have been developed over the years, the authors chose the following five by virtue of their metabolic stability: linear [Pro 30 ,Lys(DOTA) 31 33 could not be obtained through these reaction conditions due to the formation of a significant number of side products. Despite the authors' efforts to find out the origin of those side products, the origins remained inconclusive and so further [ 68 Ga]33 characterization and stability evaluation was omitted. The Log P values were determined and they showed an increasing variation from the hydrophilic [ 68 Ga]30, with a Log P value of −3.61 ± 0.37, to lipophilic [ 68 Ga]32, with a Log P value of −0.48 ± 0.14 and the heterodimer [ 68 Ga]34 showed a hydrophilic character as well, with a Log P value of −2.47 ± 0.18. The stability of these four radioligands was determined by the use of a human serum and human microsomal stability assay. Regarding the human serum stability assay, the radioligands showed the formation of different hydrophilic metabolites and considerable differences concerning their rate of degradation by peptidases, which caused a great difference in the half-lives of the compounds. More recently, still focusing on in vivo receptor targeting with radiolabeled peptidebased probes, Cardoso et al. described the development and characterization of two 68 Ga-labeled NPY analogues for their potential use in breast cancer diagnosis [134]. Both of the analogues shared the same amino acid sequence: Tyr-Arg-Leu-Arg-BPA-Nle-Pro-Asn-Ile (BPA: L-4-benzoylphenylalanine), one which favors preferential binding to NPY Y 1 receptors, and were derivatized with NOTA ( Figure 10) through a lysine (35a) or an acetylated lysine (35b) linker. More recently, still focusing on in vivo receptor targeting with radiolabeled peptidebased probes, Cardoso et al. described the development and characterization of two 68 Galabeled NPY analogues for their potential use in breast cancer diagnosis [134]. Both of the analogues shared the same amino acid sequence: Tyr-Arg-Leu-Arg-BPA-Nle-Pro-Asn-Ile (BPA: L-4-benzoylphenylalanine), one which favors preferential binding to NPY Y1 receptors, and were derivatized with NOTA ( Figure 10) through a lysine (35a) or an acetylated lysine (35b) linker. The authors assessed different labelling reaction conditions, such as various peptide masses (50, 100 or 150 μg), incubation temperatures (20 and 95 °C) and reaction times (5 and 10 min). The best radiolabeling conditions were found to be 100 μg of ligand, a pH of 4.5, 5 min incubation time and 95 °C incubation temperature. With these conditions, both radiopharmaceuticals [ 68 Ga]35a and [ 68 Ga]35b showed radiochemical purities that were higher than 95% and neither gallium colloids nor 68 GaCl3 were detected. Afterwards, the physicochemical properties were evaluated. Both of the complexes were hydrophilic, with Log P values of −3.2 ± 0.1 for [ 68 Ga]35a and −2.6 ± 0.1 for [ 68 Ga]35b, though the authors argue that these Log P values should not negatively affect the cellular uptake of the complexes as it depends on the expression of the NPY receptors on the cellular surface. In general, both of the complexes showed great in vitro stability, good cellular uptake, binding affinities in the nanomolar range and high cellular internalization rate. Interestingly, both of the complexes showed significant differences in their cellular uptake and externalization rates, as well as their Log P values. As the complex [ 68 Ga]35b has a modified lysine, this makes it less hydrophilic and, consequently, increases its cellular uptake and reduces its externalization rate. Regarding their biodistribution profiles, both of the radiolabeled compounds had a high rate of blood depuration and renal excretion, being consistent with their hydrophilicity. However, the tumor uptake was moderate and ratio of tumor-to-muscle and tumor-to-blood were not as high as those which have been reported in the literature as being the most promising for diagnostic imaging. Nonetheless, these studies have shed light on the influence of the labelling technique in the development of new promising radiopharmaceuticals.

NPY Y2 Receptor
Winterdahl et al. reported the development of the first and, to date, the only PET radiotracer for the Y2 receptor [135]. The authors took advantage of the suitable characteristics of the Y2 receptor antagonist JNJ-31020028 (36, Figure 11) and radiolabeled this compound using [ 11 C]CH3I in order to visualize the Y2 receptors in the living brain. JNJ-31020028 (N-(4-{4-[2-(diethylamino)-2-oxo-1-phenylethyl]piperazin-1-yl}-3-fluorophenyl)-2-pyridin-3-ylbenzamide) is a Y2 receptor antagonist with high affinity and selectivity for the receptors, poor oral availability (6%) in rats but high bioavailability subcutaneously (100%) and a half-life of 0.83 h. It penetrates the blood-brain barrier and occupies The authors assessed different labelling reaction conditions, such as various peptide masses (50, 100 or 150 µg), incubation temperatures (20 and 95 • C) and reaction times (5 and 10 min). The best radiolabeling conditions were found to be 100 µg of ligand, a pH of 4.5, 5 min incubation time and 95 • C incubation temperature. With these conditions, both radiopharmaceuticals [ 68 Ga]35a and [ 68 Ga]35b showed radiochemical purities that were higher than 95% and neither gallium colloids nor 68 GaCl 3 were detected. Afterwards, the physicochemical properties were evaluated. Both of the complexes were hydrophilic, with Log P values of −3.2 ± 0.1 for [ 68 Ga]35a and −2.6 ± 0.1 for [ 68 Ga]35b, though the authors argue that these Log P values should not negatively affect the cellular uptake of the complexes as it depends on the expression of the NPY receptors on the cellular surface. In general, both of the complexes showed great in vitro stability, good cellular uptake, binding affinities in the nanomolar range and high cellular internalization rate. Interestingly, both of the complexes showed significant differences in their cellular uptake and externalization rates, as well as their Log P values. As the complex [ 68 Ga]35b has a modified lysine, this makes it less hydrophilic and, consequently, increases its cellular uptake and reduces its externalization rate. Regarding their biodistribution profiles, both of the radiolabeled compounds had a high rate of blood depuration and renal excretion, being consistent with their hydrophilicity. However, the tumor uptake was moderate and ratio of tumor-to-muscle and tumor-to-blood were not as high as those which have been reported in the literature as being the most promising for diagnostic imaging. Nonetheless, these studies have shed light on the influence of the labelling technique in the development of new promising radiopharmaceuticals.

NPY Y 2 Receptor
Winterdahl et al. reported the development of the first and, to date, the only PET radiotracer for the Y 2 receptor [135]. The authors took advantage of the suitable characteristics of the Y 2 receptor antagonist JNJ-31020028 (36, Figure 11) and radiolabeled this compound using [ 11 C]CH 3 I in order to visualize the Y 2 receptors in the living brain. JNJ-31020028 (N-(4-{4-[2-(diethylamino)-2-oxo-1-phenylethyl]piperazin-1-yl}-3-fluorophenyl)-2-pyridin-3-ylbenzamide) is a Y 2 receptor antagonist with high affinity and selectivity for the receptors, poor oral availability (6%) in rats but high bioavailability subcutaneously (100%) and a half-life of 0.83 h. It penetrates the blood-brain barrier and occupies 90% (at 10 mg/kg) of the Y 2 receptors [76]. The radiosynthesis of N-[ 11 C]-methyl-JNJ-31020028 ([ 11 C]37) was performed in an semiautomated system wherein [ 11 C]methyl iodide was produced from [ 11 C]CO 2 . [ 11 C]CH 3 I was trapped in a solution of 36 and NaH dissolved in DMF and THF. The reaction occurred over the course of 3 min at 50 • C (Figure 11). The crude product was then purified by preparative HPLC and the radiolabeled compound [ 11 C]37 was obtained with radiochemical purity > 98% and molar activity > 100 GBq/µmol (the radiochemical yield was not disclosed).
Molecules 2022, 27, x FOR PEER REVIEW 19 of 32 90% (at 10 mg/kg) of the Y2 receptors [76]. The radiosynthesis of N-[ 11 C]-methyl-JNJ-31020028 ([ 11 C]37) was performed in an semiautomated system wherein [ 11 C]methyl iodide was produced from [ 11 C]CO2. [ 11 C]CH3I was trapped in a solution of 36 and NaH dissolved in DMF and THF. The reaction occurred over the course of 3 min at 50 °C ( Figure  11). The crude product was then purified by preparative HPLC and the radiolabeled compound [ 11 C]37 was obtained with radiochemical purity > 98% and molar activity > 100 GBq/μmol (the radiochemical yield was not disclosed). The novel PET tracer [ 11 C]37 was then assessed by the use of autoradiography in brain sections and by the in vivo PET imaging of the pig brain. The radiographic results have demonstrated that high densities of [ 11 C]37 were found in the hippocampus and cerebellum, this is a finding which agrees with the anatomical distribution of the Y2 receptors that has been described in other animal species. The radiotracer [ 11 C]37 was also shown to be distributed rapidly into the brain and metabolized slowly in the bloodstream. The authors also concluded that PET studies using cyclosporine improved the target-to-background ratio of [ 11 C]37, enabling the estimation of the pharmacokinetic parameters. This is due to the inhibition of the efflux transporter P-gp by cyclosporine as PET studies without cyclosporine gave a value of less than 1 for the ratio between the area under the wholebrain time-activity curve and the plasma time-activity curve of the radiotracer [ 11 C]37, suggesting efflux transport at the blood-brain barrier [135].
Furthermore, Andersen et al. described the [ 11 C]carbonyl labeling of the same compound, JNJ-31020028 (36), by using palladium-aryl oxidative addition complexes in carbonylation reactions with [ 11 C]carbon monoxide [136]. [ 11 C-Carbonyl]JNJ-31020028 ([ 11 C]36) was achieved with the best radiochemical yield of 25% (Table 5) and radiochemical purity of 55%, when the reaction of the amino precursor 38 was undertaken with the XantPhos ligated aryl palladium complex 39 and guanidine base Me-TDB (6 equiv.) in dioxane, at 95 °C for 10 min. To this end, the uses of different bases and ligated aryl palladium complexes and their correspondents 11 CO trapping efficiency and [ 11 C]36 radiochemical purity and yield were evaluated. In a first attempt, when NEt3 (1 equiv.) was added to the reaction of the amino precursor 38 with PPh3 ligated aryl palladium complex, no traces of 11 CO trapping were observed ( Table 5, entry 1). The implementation of the XantPhos ligated aryl palladium complex allowed the formation of the desired product [ 11 C]36, though with low radiochemical yield, when DBU (4 equiv.) was used in the reaction (Table 5, entry 2). An increase in the reaction temperature led to a higher 11 CO trapping efficiency but lower radiochemical purity of the product (Table 5, entry 3). A series of different organobases were also tested. The use of DBN, proton-sponge and TMGN bases (4 equiv.) led to a low level of 11 CO gas trapping ( Table 5, entries 4-6); however, the use of the guanidine base Me-TDB (4 equiv.) increased the reactivity profoundly and significantly higher values for the trapping efficiency and radiochemical purity of the product were observed (Table 5, entry 7). The augmentation of the temperature of the reaction did not have a beneficial impact ( Table 5,  The novel PET tracer [ 11 C]37 was then assessed by the use of autoradiography in brain sections and by the in vivo PET imaging of the pig brain. The radiographic results have demonstrated that high densities of [ 11 C]37 were found in the hippocampus and cerebellum, this is a finding which agrees with the anatomical distribution of the Y 2 receptors that has been described in other animal species. The radiotracer [ 11 C]37 was also shown to be distributed rapidly into the brain and metabolized slowly in the bloodstream. The authors also concluded that PET studies using cyclosporine improved the target-to-background ratio of [ 11 C]37, enabling the estimation of the pharmacokinetic parameters. This is due to the inhibition of the efflux transporter P-gp by cyclosporine as PET studies without cyclosporine gave a value of less than 1 for the ratio between the area under the wholebrain time-activity curve and the plasma time-activity curve of the radiotracer [ 11 C]37, suggesting efflux transport at the blood-brain barrier [135]. Furthermore, Andersen et al. described the [ 11 C]carbonyl labeling of the same compound, JNJ-31020028 (36), by using palladium-aryl oxidative addition complexes in carbonylation reactions with [ 11 C]carbon monoxide [136]. [ 11 C-Carbonyl]JNJ-31020028 ([ 11 C]36) was achieved with the best radiochemical yield of 25% (Table 5) and radiochemical purity of 55%, when the reaction of the amino precursor 38 was undertaken with the Xant-Phos ligated aryl palladium complex 39 and guanidine base Me-TDB (6 equiv.) in dioxane, at 95 • C for 10 min. To this end, the uses of different bases and ligated aryl palladium complexes and their correspondents 11 CO trapping efficiency and [ 11 C]36 radiochemical purity and yield were evaluated. In a first attempt, when NEt 3 (1 equiv.) was added to the reaction of the amino precursor 38 with PPh 3 ligated aryl palladium complex, no traces of 11 CO trapping were observed ( Table 5, entry 1). The implementation of the XantPhos ligated aryl palladium complex allowed the formation of the desired product [ 11 C]36, though with low radiochemical yield, when DBU (4 equiv.) was used in the reaction (Table 5, entry 2). An increase in the reaction temperature led to a higher 11 CO trapping efficiency but lower radiochemical purity of the product (Table 5, entry 3). A series of different organobases were also tested. The use of DBN, proton-sponge and TMGN bases (4 equiv.) led to a low level of 11 CO gas trapping ( Table 5, entries 4-6); however, the use of the guanidine base Me-TDB (4 equiv.) increased the reactivity profoundly and significantly higher values for the trapping efficiency and radiochemical purity of the product were observed (Table 5, entry 7). The augmentation of the temperature of the reaction did not have a beneficial impact ( Table 5, entry 8). Finally, the increase in the guanidine base amount to 6 equivalents led to the formation of [ 11 C-carbonyl]JNJ-31020028 ([ 11 C]36) with a good purity profile and acceptable radiochemical yield on an average of three reproducible reactions (Table 5, entry 9). with a good purity profile and acceptable radiochemical yield on an average of three reproducible reactions (Table 5, entry 9).

NPY Y5 Receptor
In the literature, the first reported research using a PET ligand for the Y5 receptor, performed by Erondu et al. [42], aimed to test if NPY Y5 antagonism would lead to weight loss in overweight and obese patients. In order to verify this hypothesis, MK-0557 [42] (40, Figure 12), a potent selective and orally available Y5 antagonist, was used. In this case, these PET studies were aimed at providing complementary data about the MK-0557 dosage by studying the Y5 receptor occupancy after the oral administration of 40. The selective Y5 PET ligand [ 11 C]MK-0233 ([ 11 C]41), which had been developed and validated in the rhesus monkey [137], was used in human volunteers. [ 11 C]41 has a similar structure to the Y5 antagonist 40 and it is also extremely selective to this receptor ( Figure 12). The longterm weight loss study with MK-0557 showed that, even though this NPY Y5 antagonist had a favorable clinical safety profile, the magnitude of the induced weight loss was not clinically meaningful as the degree of weight loss after 52 weeks of treatment with 40 was significantly less compared to other weight loss drugs, the report showed [42]. These clinical studies provided a better clarification of the role of the NPY Y5 receptor in human energy homeostasis and its utility as a target for anti-obesity drug therapy. The authors also concluded that, for the purposes of future anti-obesity drug development programs, targeting only the NPY Y5 receptor was unlikely to produce therapeutic efficacy [42].

NPY Y 5 Receptor
In the literature, the first reported research using a PET ligand for the Y 5 receptor, performed by Erondu et al. [42], aimed to test if NPY Y 5 antagonism would lead to weight loss in overweight and obese patients. In order to verify this hypothesis, MK-0557 [42] (40, Figure 12), a potent selective and orally available Y 5 antagonist, was used. In this case, these PET studies were aimed at providing complementary data about the MK-0557 dosage by studying the Y 5 receptor occupancy after the oral administration of 40. The selective Y 5 PET ligand [ 11 C]MK-0233 ([ 11 C]41), which had been developed and validated in the rhesus monkey [137], was used in human volunteers. [ 11 C]41 has a similar structure to the Y 5 antagonist 40 and it is also extremely selective to this receptor ( Figure 12). The longterm weight loss study with MK-0557 showed that, even though this NPY Y 5 antagonist had a favorable clinical safety profile, the magnitude of the induced weight loss was not clinically meaningful as the degree of weight loss after 52 weeks of treatment with 40 was significantly less compared to other weight loss drugs, the report showed [42]. These clinical studies provided a better clarification of the role of the NPY Y 5 receptor in human energy homeostasis and its utility as a target for anti-obesity drug therapy. The authors also concluded that, for the purposes of future anti-obesity drug development programs, targeting only the NPY Y 5 receptor was unlikely to produce therapeutic efficacy [42]. had a favorable clinical safety profile, the magnitude of the induced weight loss was not clinically meaningful as the degree of weight loss after 52 weeks of treatment with 40 was significantly less compared to other weight loss drugs, the report showed [42]. These clinical studies provided a better clarification of the role of the NPY Y5 receptor in human energy homeostasis and its utility as a target for anti-obesity drug therapy. The authors also concluded that, for the purposes of future anti-obesity drug development programs, targeting only the NPY Y5 receptor was unlikely to produce therapeutic efficacy [42]. The synthesis and radiolabeling of compound 41 with [ 11 C] was later published by Takahashi et al. [138]. The authors reported a series of trans-3-oxospiro[(aza)isobenzofuran-1(3H),1 -cyclohexane]-4 -carboxamide derivatives and examined their binding affinities towards the Y 5 receptor and their brain penetrability. Compound 41 showed the most promising biological profile for becoming a Y 5 PET tracer as it had a good Y 5 binding affinity of 1.5 ± 0.3, an acceptable Log P value of 2.79 and was selective to the Y 5 receptor (human Y 1 , Y 2 and Y 4 IC 50 > 10 µm). Moreover, its susceptibility to human and mouse P-glycoprotein transporters was studied and it was concluded that the precursor 41 was not a substrate of the human P-gp transporter. Thus, compound 41 was selected to be radiolabeled with [ 11 C] in order to become a novel Y 5 PET tracer. Succinctly, the synthesis of 41 starts with the reaction of 3-bromopyridine 42 with LDA followed by 1,4-cyclohexanedione monoethyleneketal and acid mediated hydrolysis so as to obtain the ketone 43 ( Figure 13). This was then stereoselectively reduced by NaBH 4 , providing the cis-alcohol 44. The trans carboxylic acid 45 was achieved by the mesylation of alcohol 44, followed by the reaction with NEt 4 CN and acid mediated hydrolysis. Compound 45 was then coupled with 2amino-5-o-fluorophenylpyrimidine in order to afford the precursor 46. The subsequent radiolabeling of precursor 46 can then be achieved by its reaction with [ 11 C]CO in the presence of Pd(PPh 3 ) 4 , providing [ 11 C]41 as a product [137,138]. No statements of the yield or molar activity of the radiolabeled product were disclosed. The synthesis and radiolabeling of compound 41 with [ 11 C] was later published by Takahashi et al. [138]. The authors reported a series of trans-3-oxospiro[(aza)isobenzofuran-1(3H),1′-cyclohexane]-4′-carboxamide derivatives and examined their binding affinities towards the Y5 receptor and their brain penetrability. Compound 41 showed the most promising biological profile for becoming a Y5 PET tracer as it had a good Y5 binding affinity of 1.5 ± 0.3, an acceptable Log P value of 2.79 and was selective to the Y5 receptor (human Y1, Y2 and Y4 IC50 > 10 μm). Moreover, its susceptibility to human and mouse Pglycoprotein transporters was studied and it was concluded that the precursor 41 was not a substrate of the human P-gp transporter. Thus, compound 41 was selected to be radiolabeled with [ 11 C] in order to become a novel Y5 PET tracer. Succinctly, the synthesis of 41 starts with the reaction of 3-bromopyridine 42 with LDA followed by 1,4-cyclohexanedione mono-ethyleneketal and acid mediated hydrolysis so as to obtain the ketone 43 (Figure 13). This was then stereoselectively reduced by NaBH4, providing the cis-alcohol 44.
The trans carboxylic acid 45 was achieved by the mesylation of alcohol 44, followed by the reaction with NEt4CN and acid mediated hydrolysis. Compound 45 was then coupled with 2-amino-5-o-fluorophenylpyrimidine in order to afford the precursor 46. The subsequent radiolabeling of precursor 46 can then be achieved by its reaction with [ 11 C]CO in the presence of Pd(PPh3)4, providing [ 11 C]41 as a product [137,138]. No statements of the yield or molar activity of the radiolabeled product were disclosed.    [139]. The authors sought to enhance the reactivity of [ 11 C]CO by improving its solubility via chemical complexation to CO-binding molecules in a solution. They were able to achieve this by forming a copper [ 11 C]carbonyl complex, copper(I)tris(3,5-dimethylpyrazolyl)borate-[ 11 C]carbonyl (Cu(Tp*)[ 11 C]CO), which was then used in the [ 11 C]carbonylation reaction that led to the formation of the radiolabeled compound [ 11 C]41. Hence, the radiosynthesis of [ 11 C]41 was produced through the ringclosing [ 11 C]carbonylation reaction between pyridyl-bromide and the hydroxyl functional groups of the precursor 46 and mediated by the catalyst Pd(PPh 3 ) 4 . In this work, the authors synthesized [ 11 C]41 in two different ways: one using a microfluidic methodology and one using a low-pressure technique. Regarding the first method, the authors analyzed the temperature and flow rate's influence on the radiolabeling reaction and, so as to maximize the yields and minimize the synthesis reaction time that were required in order to obtain enough radiotracer [ 11 C]41 to perform the PET studies, the reactions were performed at 180 • C with a flow rate of 100 µL/min. This led to the production of [ 11 C]MK-0233 with a molar activity of 100 ± 30 GBq/µmol, radiochemical purity > 99% and a decay-corrected radiochemical yield of 7.2 ± 0.7% in 27 min of synthesis time. Concerning the second method, a 3 mL vial was used as the reaction vessel and, despite some problems and subsequent optimizations related to the apparatus, the radiolabeled compound was achieved with a molar activity of 100 ± 15 GBq/µmol, radiochemical purity > 99% and a decay-corrected radiochemical yield of 7.1 ± 2.2%, also in 27 min. The authors had initially predicted a greater difference between the results of these methods, but the radiochemical yields were almost identical and they were produced in the same amount of time. Nevertheless, the feasibility of using microfluidics for solutions-based carbonylations proved it to the be a suitable method for PET tracers' production, with the potential to surpass the vial method.
More recently, Kumar et al. reported the identification and radiolabeling of four potent and selective Y 5 receptor antagonists that could emerge as Y 5 receptor PET tracers. The selected four candidates (47, 48, 49 and 50, Figure 14) showed high affinities towards the receptor and were suitable for radiolabeling with [ 18 F]fluoride via nucleophilic substitution reactions [140].  41 in two different ways: one using a microfluidic methodology and one using a low-pressure technique. Regarding the first method, the authors analyzed the temperature and flow rate's influence on the radiolabeling reaction and, so as to maximize the yields and minimize the synthesis reaction time that were required in order to obtain enough radiotracer [ 11 C]41 to perform the PET studies, the reactions were performed at 180 °C with a flow rate of 100 μL/min. This led to the production of [ 11 C]MK-0233 with a molar activity of 100 ± 30 GBq/μmol, radiochemical purity > 99% and a decaycorrected radiochemical yield of 7.2 ± 0.7% in 27 min of synthesis time. Concerning the second method, a 3 mL vial was used as the reaction vessel and, despite some problems and subsequent optimizations related to the apparatus, the radiolabeled compound was achieved with a molar activity of 100 ± 15 GBq/μmol, radiochemical purity > 99% and a decay-corrected radiochemical yield of 7.1 ± 2.2%, also in 27 min. The authors had initially predicted a greater difference between the results of these methods, but the radiochemical yields were almost identical and they were produced in the same amount of time. Nevertheless, the feasibility of using microfluidics for solutions-based carbonylations proved it to the be a suitable method for PET tracers' production, with the potential to surpass the vial method. More recently, Kumar et al. reported the identification and radiolabeling of four potent and selective Y5 receptor antagonists that could emerge as Y5 receptor PET tracers. The selected four candidates (47, 48, 49 and 50, Figure 14) showed high affinities towards the receptor and were suitable for radiolabeling with [ 18 F]fluoride via nucleophilic substitution reactions [140]. The authors performed the non-radiolabeling experiments using KF/krytofix/K2CO3 in DMSO at 180 °C. Regarding the radiolabeling experiments, both conventional heating and microwave irradiation were tested. The radiolabeling of the compound 47 did not provide any radiolabeled product under conventional heating, although under microwave irradiation [ 18 F]47 was obtained in <2% isolated RCY ( Table 6). The same results   48 showed good BBB penetration and was retained in several brain regions such as the caudate, putamen and cortical regions. The distribution of this radiotracer was shown to be compliant with the acknowledged distribution of the Y 5 receptor and [ 11 C]MK0233 in monkeys and human subjects. [ 18 F]LuAE00654 also demonstrated itself to be of rapid clearance and its specificity was proven by blocking experiments using the Y 5 ligand LuA44608. Concerning the radiotracer [1 8 F]50, even though it penetrated the baboons' blood-brain barriers, it showed little retention of radioactivity in the brain, fast washout and a faster metabolization in comparison to [ 48) showed great promise as a PET radiotracer for the Y 5 receptor and could be used for the development of novel drugs aiming at the NPY Y 5 receptor functions.

Conclusions
NPY has gained a lot of interest from the scientific community over the years and has been broadly studied and biologically and pharmacologically characterized due to its involvement in the pathophysiology of several diseases, especially brain diseases. To further understand its functioning, PET imaging of the NPY receptors has been investigated. For that purpose, several radiotracers were developed for three of the NPY receptors (Y1, Y2 and Y5). In this paper, we have highlighted some approaches to the chemical and radiolabeling development of the different radiotracers for the NPY receptors and the subsequent PET studies and results, which were mainly used in tumor visualization and receptor visualization associated with obesity and other hypothalamic disorders.
Despite the promising results that have been obtained so far with the pre-clinical and clinical studies, which were crucial to the better understanding of the NPY receptors in the several pathologies, it is clear that further investigation is still needed. The advances in NPY system PET imaging have been occurring at a slower rate as there is still some difficulty in not only developing novel radiotracers that combine good radiolabeling methods with good pharmacological profiles but also in expanding the preclinical development to the clinical area. Nevertheless, PET imaging of the NPY system holds a lot of potential to further the developments in and insights into this topic and to support the <2% 25%

Conclusions
NPY has gained a lot of interest from the scientific community over the years and has been broadly studied and biologically and pharmacologically characterized due to its involvement in the pathophysiology of several diseases, especially brain diseases. To further understand its functioning, PET imaging of the NPY receptors has been investigated. For that purpose, several radiotracers were developed for three of the NPY receptors (Y1, Y2 and Y5). In this paper, we have highlighted some approaches to the chemical and radiolabeling development of the different radiotracers for the NPY receptors and the subsequent PET studies and results, which were mainly used in tumor visualization and receptor visualization associated with obesity and other hypothalamic disorders.
Despite the promising results that have been obtained so far with the pre-clinical and clinical studies, which were crucial to the better understanding of the NPY receptors in the several pathologies, it is clear that further investigation is still needed. The advances in NPY system PET imaging have been occurring at a slower rate as there is still some difficulty in not only developing novel radiotracers that combine good radiolabeling methods with good pharmacological profiles but also in expanding the preclinical development to the clinical area. Nevertheless, PET imaging of the NPY system holds a lot of potential to further the developments in and insights into this topic and to support the 25%

Conclusions
NPY has gained a lot of interest from the scientific community over the years and has been broadly studied and biologically and pharmacologically characterized due to its involvement in the pathophysiology of several diseases, especially brain diseases. To further understand its functioning, PET imaging of the NPY receptors has been investigated. For that purpose, several radiotracers were developed for three of the NPY receptors (Y 1 , Y 2 and Y 5 ). In this paper, we have highlighted some approaches to the chemical and radiolabeling development of the different radiotracers for the NPY receptors and the subsequent PET studies and results, which were mainly used in tumor visualization and receptor visualization associated with obesity and other hypothalamic disorders.
Despite the promising results that have been obtained so far with the pre-clinical and clinical studies, which were crucial to the better understanding of the NPY receptors in the several pathologies, it is clear that further investigation is still needed. The advances in NPY system PET imaging have been occurring at a slower rate as there is still some difficulty in not only developing novel radiotracers that combine good radiolabeling methods with good pharmacological profiles but also in expanding the preclinical development to the clinical area. Nevertheless, PET imaging of the NPY system holds a lot of potential to further the developments in and insights into this topic and to support the discovery and development of new promising CNS drugs that are able to target this important family of receptors in the years to come. Funding: The authors wish to thank the financial support from Portuguese National Funds via FCT-Fundação para a Ciência e a Tecnologia-UIDB/04539/2020, UIDP/04539/2020, UIDB/4950/2020, UIDP/4950/2020 and LA/P/0058/2020. Inês C. F. Fonseca also thanks FCT and ICNAS Pharma for her PhD grant PD/BDE/150334/2019. Acknowledgments: Not applicable.

Conflicts of Interest:
The authors declare no conflict of interest.