Advances in macrocyclic chelators for positron emission tomography imaging

In recent years, radiometals have been successfully applied to medicine because of their breadth of decay properties and increased production and availability. Bifunctional chelators play a key role in radiometal‐based radiopharmaceuticals, affecting the labeling, targeting, and pharmacokinetics of bioconjugations and ensuring the stable complexation of the metal in vivo. The capacity of macrocycles to form complexes with extremely high thermodynamics, kinetics, and stability compared to acyclic chelators continues to pique the curiosity of pharmacists and biochemists among all prospective chelators utilized for the radiometal chelation. As new imaging modalities and therapeutic targets develop, the discovery of novel ligand structures with suitable chemical and biological features encourages the modification of chelators with charge and chemical properties. Herein, we present a comprehensive review of developments of macrocyclic chelators for PET radiopharmaceuticals, including innovative chelating agents with new structures and known chelating agent modifications, and their physical‐chemical properties, as well as their biological characteristics and applications in vivo.


INTRODUCTION
5][6] Its combination with anatomical imaging modalities, such as computed tomography (CT) or magnetic resonance imaging (MRI), has significantly aided in the diagnosis of disease over the past few decades. 7The coordinated efforts of biomarker identification, radiopharmaceutical development, and equipment innovation are essential to the success of PET imaging.Along with 18 F-FDG, the PET radiotracer used for routine clinical evaluation, thousands of radiopharmaceuticals have been developed for imaging biological processes and biomarkers. 8,9adiopharmaceuticals are divided into two broad categories: organically derived and metal-based. 10The former incorporate non-metal radionuclides (e.g., carbon-11, nitrogen-13, oxygen-15, fluorine-18, and iodine-123) by covalent bond formation.Most "organic" radionuclides have restricted applicability due to their short half-life and limited decay properties. 7In contrast to traditional PET radionuclides, radiometals (e.g., gallium-68, copper-64/copper-67, yttrium-86/yttrium-90, and zirconium-89) possess a wide range of half-life, breadth of decay characteristics, and relatively mild labeling conditions (rely on coordination chemistry) to better match the relative longer biological process of interest (Table 1). 11Beyond this, the ability to switch a diagnostic radiometal for a therapeutic radiometal and turn an imaging probe into a therapeutic agent is another benefit of radiometals in the creation of radiopharmaceuticals. 12 Because of increased production and sufficient purity, radiolabeling of biomolecules with metallic radionuclides have gained considerable interest for imaging with PET and radiotherapy of diseases. 8For example, gallium-68 ( 68 Ga) has become the clinical standard for the radio-labeling of somatostatin analogs (e.g., DOTATOC/TATE) and prostate-specific membrane antigen (PSMA) ligands (e.g., HBED-cc-PSMA) for the detection of neuroendocrine and prostate tumors, respectively. 13,146][17] The lutetium-177 ( 177 Lu) radiolabeled drug 177 Lu-DOTA-TATE (Lutathera), for the therapy of neuroendocrine tumors, has already been approved by the U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA) a few years ago. 18Recently, the FDA approved 177 Lu-PSMA-617 (Pluvicto) for the treatment of prostate cancer patients. 19Over the past decade, theranostic imaging has been a potent clinical tool in oncology for selecting patients who are likely to react to targeted therapies and for monitoring patients' response to treatment. 20n light of this, the use of "companion radionuclides" or "sister radioisotopes," such as yttrium-86/yttrium-90 ( 86 Y/ 90 Y), scandium-44/scandium-47 ( 44 Sc/ 47 Sc), and copper-64/copper-67 ( 64 Cu/ 67 Cu) for quantitative imaging and therapy is appealing.Other radiometals that are increasing in popularity include an alpha-emitting radionuclide actinium-225 ( 225 Ac) with applications in radionuclide therapy. 21Paramagnetic manganese-52 ( 52 Mn) with favorable electronic properties (S = 5/2, slow electron spin relaxation with T 1e values ideally on the nanosecond scale) and suitable decay characteristics are gaining interest for PET/MRI imaging. 22ost radiometal-based design radiopharmaceutical is often divided into four parts: radiometal, bifunctional chelator (BFC), linker, and bioconjugate (Figure 1). 10 While the radiometal is a label and the bioconjugate helps in accumulation at target cells, the linker and chelator integrate the two separate parts.By changing individual components to adjust drug properties, this structure offers tremendous promise for "plug-and-play" drug development.][25] Among the prospective radiometal chelators, macrocycles keep capturing the curiosity of scientists due TA B L E 1 Summary of the decay parameters, common production routes, chelators, and applications of radionuclides discussed in this review.

Average energy of decay particle (keV)
Production route  to their propensity to build complexes with very high thermodynamics, kinetics, and stability.More precise combinations of a macrocyclic platform and coordinating pendant arms have enabled for exquisite control of the chelator profiles.Recent studies found that different BFCs labeled with the same radiometal can exhibit varied pharmacokinetic characteristics. 26,27This review will focus on latest developments of macrocyclic chelators for use in PET radiopharmaceuticals (mostly in recent 5 years).It includes innovations in chelating agents with new structures and known chelating agent modifications, and their physical-chemical properties, as well as their application and biological characteristics in vivo.
31.2 ± 1.2 MBq/nmol. 56The formation/interconversion of two different five-coordinate 68 Ga-complexes was confirmed in vitro depending on the pH conditions.Subsequently, they expanded the scope of the BFC NODIA-Me to develop the 68 Ga-labeled integrin α v ß 3 -targeting probe. 57 68 a-labeled NODIA-Me-c(RGDfK) were prepared in good yields and exhibited low liver uptake, rapid blood clearance, and renal excretion in vivo.Compared to previously described compounds, 68 Ga-NODIA-Me-c(RGDfK) showed lower affinity to tumor tissue, further causing slightly lower tumor uptake but comparable tumor-tobackground ratios.Functionalizing the imidazoles with pendant carboxylic acid arms and optimizing the spacer moiety may be one promising approach to enhance further the binding affinity and tumor targeting capabilities of the NODIA-Me conjugated probes. 58ai and coworkers contributed to the development of TACN-based chelators for 68 Ga and 64 Cu. [59][60][61][62] Bifunctional chelator NO2A tBu -N 3 , which possesses carboxylic acid and azido functional groups, enables construction of a library of multimodal and multivalent imaging probes via either standard Fmoc chemistry or strain-promoted azide-alkyne cycloaddition (SPAAC). 60Building on this molecular scaffold, a heterodimeric tracer for 68 Ga PET imaging of breast cancer and pancreatic cancer was created.It consists of arginine-glycine-aspartic (RGD) and asparagine-glycinearginine (NGR) peptides that target integrin α v β 3 and CD13, respectively. 63,64Attributed to its avidity effect, the heterodimer 68 Ga-NGR-RGD demonstrated superior binding affinity, higher tumor uptake, and longer tumor retention time compared with monomeric 68 Ga-NGR and 68 Ga-RGD.Further, Long et al. evaluated the detection efficiency of 68 Ga-NGR-RGD for ovarian tumor in contrast to 18 F-FDG. 65The results showed that in SKOV3 and ES-2 tumors, 68 Ga-NGR-RGD showed more excellent contrast than 18 F-FDG.Moreover, with abdominal metastasis models, 68 Ga-NGR-RGD PET demonstrated clear delineation in both peritoneal and liver metastases (Figure 4A).
Recent research has demonstrated the greater practicality and utility of another heterodimeric tracer, 68 Ga-yG5-RGD, targeting both CXCR4 and integrin α v β 3 , in pancreatic cancer PET imaging (Figure 4B). 66The effectiveness of monospecific tracers for tumor detection is currently constrained by tumor heterogeneity and intricate tumor-stromal interactions.These days, two brandnew PSMA/FAP heterodimers have been developed and showed great specificity and affinity to PSMA and FAP, both in vitro and in vivo. 67,68Dimeric radiotracers can target more receptors, have higher local ligand concentrations, and have better pharmacokinetics than monomeric 68 Ga-NGR-RGD and 18 F-FDG in SKOV3 and ES-2 abdominal ovarian metastasis models at 1 h p.i.In 68 Ga-NGR-RGD PET/CT imaging, several metastatic lesions with strong uptake were found in the peritoneal space (white circles).Whereas, in 18 F-FDG PET/CT imaging, there were several stripe high uptake foci.(A, bottom panel) Surgical exploration was done in the same animal after PET/CT imaging.Diffuse reddish-white nodules with a slightly firm texture were seen in the peritoneal space.Ex vivo PET imaging of excised tissues was performed.6 Reproduced under the terms of the CC-BY license. 65opyright 2022, Yu Long. 66Copyright 2022, Yaqun Jiang.
radiotracers.Therefore, bifunctional chelator NO2A tBu -N 3 may serve as a promising bridge for heterodimeric tracer to be further translated from the bench side to clinical application for early diagnosis, staging, and follow-up of tumors.
Hybrid chelators combined the benefits of both the macrocyclic and acyclic framework for the thermodynamic stability and advantageous chelating kinetics.Gai et al. developed two BFC with one or two phosphonic acid functional groups, denoted p-SCN-PhPr-NE2A1P and p-SCN-PhPr-NE2P1A. 62Both chelators were successfully conjugated with very late antigen-4 (VLA-4) targeting ligand LLP2A-PEG4 via thiourea formation.The peptidomimetic conjugates of p-SCN-Bn-NE2P1A, namely, NE2P1A-PEG4-LLP2A, showed quantitative radiolabeling with 68 Ga at mild temperatures (37 • C) and neutral pH (pH 6.8).The resultant 68 Ga-NE2P1A-PEG4-LLP2A exhibited good in vivo stability, strong tumor detection capabilities, and high retention in B16F10 cells.In this study, the chelating groups acquired a better-preorganized conformation by swapping the carboxylate group for the hard phosphonic acid group, improving the thermodynamic and kinetic stability of the complex between the NE3TA-like chela-tor and Ga 3+ .Therefore, p-SCN-Bn-NE2P1A is a highly promising BFC for developing 68 Ga-based agents, particularly for labeling compounds that cannot withstand acidic conditions and high temperatures.
Increasing the chelator stability by adding the phosphonic acid group on the pendant arm was also observed in the study by Wang et al. 69 The newly developed diphosphonate pendant armed chelators, NODP-Thia exhibited significantly enhanced complexation kinetics with Ga 3+ in comparison with dicarboxylate pendant armed counterpart NODA-Thia or NOTA.Interest in the development of chelating agents for 68 Ga 3+ has been ongoing.The most attractive 68 Ga radiotracers are those that can be quickly and easily radiolabeled in high yields at room temperature utilizing strong, dependable, and affordable radiopharmaceutical kits.
The insufficient inertness of these complexes, particularly when exposed to reducing environments (such as the hypoxic environment of tumors), results in transchelation and the unintended uptake of the radiometal in the liver. 73,74To solve this problem, cross-bridged cyclam and cyclen derivatives, such as CB-DO2A, CB-TE2A, and related systems were developed. 757][78][79][80][81][82][83] Azamacrocyclic derivatives in combination with various substituents supplying diverse donor groups have been explored in an effort to generate chelators better suited for the stable complexation of 64 Cu as well as quick complexation kinetics. 84,85ima et al. systematically investigated the behavior of azamacrocycles ligands containing picolinate groups for 64 Cu labeling and developed cyclam picolinate-based ligands, TE1PA, and its ethylene cross-bridged analog CB-TE1PA. 86,879][90] The obtained ligand p-SCNBn-TE1PA was eight times higher of the conjugation rate (5 ± 1 chelates/mAb) with 9E7.4 antibody than TE1PA (only 0.6 chelates/mAb).C-functionalization of TE1PA also led to an increase in 64 Cu radiolabeling yields and immunoreactivity of radiopharmaceutical with parent TE1PA and p-SCN-Bn-TE2A.Interestingly, the results also outperformed with commercial BFCs, p-SCN-Bn-NOTA, and p-SCN-Bn-DOTA.The persisting high liver uptake with 64 Cu-labeled 9E7.4-p-SCN-Bn-TE1PA might result from the net positive charge of the radiopharmaceuticals and the presentation of syndecan-1 in liver tissues (Figure 6A). 90In a further comparison study, the ex vivo immunoreactivity study showed that 64 Cu-9E7.4-NHS-DOTAperformed similarly with 64 Cu-9E7.4-p-SCN-Bn-TE1PA,both better than 64 Cu-9E7.4-p-SCN-Bn-NOTA. 91urther metabolic study highlighted that 64 Cu-9E7.4-p-SCN-Bn-TE1PAhas superior tumor targeting and superior resistance to transchelation by cuproproteins in the liver compared to 64 Cu-9E7.4-NHS-DOTA,especially at later times.According to these studies, the high stability of the 64 Cu-p-SCN-Bn-TE1PA complex and intestinal removal suggest that it was a lipophilic stable copper complex.
][94] Based on these findings, cyclam and cross-bridged cyclam macrocycles mono-functionalized with a 2phosphorylpyridyl side arm, TE1PyP and CB-TE1PyP were designed. 95Quantitative radiolabeling was easily achieved under incubation times of 30 min at 40 • C for TE1PyP and 85 • C for CB-TE1PyP.The biodistribution of phosphonate-appended complexes was much improved compared to their carboxylated congeners, with quick renal clearance and, most crucially, extremely little hepatic fixation (Figure 6B).This beneficial impact can be linked to the charge and lipophilicity differences brought about by phosphonic side arms.Pineau et al. continued to evolve TE1PA and CB-TE1PA into more hydrophilic and neutral bifunctional versions to avoid liver uptake. 96Two modified derivatives, TE1PA-trz-prA and CB-TE1PA-trz-prA bearing an additional carboxyl group added on the picolinate unit via a propionic acid, were developed for this aim.Such a bifunctionalization allows CB-TE1PAtrz-prA with excellent 64 Cu labeling efficiency.Dynamic PET images revealed that the complex has a short half-life distribution, and is rapidly excreted from the circulation solely by the kidneys, without liver uptake.
Researchers have studied the copper (II) coordination chemistry of cyclam and TACN derivatives bearing five-membered azaheterocycles, such as imidazole and thiazole, offering aromatic N-donors. 52,97,98Martin et al. prepared TACN-based chelator NOTI-TVA with three additional carboxylic acid groups on imidazole residue. 99On the foundation of NOTI-TVA scaffold, trimeric c(RGDfK) conjugate was then prepared by peptide bond formation.In less than 5 min of incubation time, trimer NOTI-TVA-c(RGDfK) 3 was quantitatively labeled with 64 Cu.According to the findings of PET imaging investigation, the 64 Cu-labeled trimer showed a two-fold greater and sustained tumor uptake than its equivalent monomeric portion, clearly delineating the α v ß 3 -positive U87MG tumors (Figure 6C).However, the trimer showed more tracer accumulation in the liver than the monomer, 2.75 ± 0.05%IA/g and 5.63 ± 1.61%IA/g, respectively.Considering their distribution characteristics comprehensively, higher tumor F I G U R E 6 (A) 64 Cu-9E7.4-p-SCN-Bn-TE1PAPET/CT of subcutaneous MM tumor-bearing (left) and healthy (right) mice at 24 and 48 h p.i. 90 Yellow arrows indicate tumors.(B) PET/CT imaging of BALB/c mice injected with 64 Cu-TE1PyP (left) and 64 Cu-CB-TE1PyP(right) at 2 h p.i. 95 (C) Representative transverse PET images of 64 Cu-labeled mono/trimeric c(RGDfK) of U-87MG xenograft bearing BALB/c mice at 1 and 24 h p.i., including blockade by co-injection of c(RGDfK). 99White circles indicate tumors.Reproduced with permission. 90Copyright 2018, Royal Society of Chemistry. 95Copyright 2021, ACS Publications. 99Copyright 2021, SpringerLink.absorption of the trimer may result in higher detection sensitivity.
Larger cuprous ions (d 10 ) preferentially form stable tetrahedral complexes with softer donors, can also accommodate softer donors such as thiolate and carbazone. 100,101n the late 1970s, several macrocyclic compounds with sulfur donors were developed. 102Recently, Taschner and coworkers synthesized aza/thia-macrocycle NSNS-based chelator NSNS2P and NSNS2A by introducing two Nphosphonate pendant arms or N-acetate pendant arms, respectively. 103,104NSNS2A was radiolabeled with 64 Cu 2+ , and the 60-min dynamical scan demonstrated that the PET signal from the liver peaked at approximately 8 min p.i. and remained unvaried until the end of scanning.The absence of liver clearance suggests that 64 Cu(II) was transchelated or dissociated, bound to liver proteins in hepatocytes, and was retained for a long time.In vivo study revealed a much lower stability than its tetraaza analog (TE2A), consisting of in vitro results, indicating the instability of dithiadiaza macrocyclic chelators (Figure 7A).[107][108] The DO2A2S chelator had the best affinity for copper out of the group of S-rich polyazamacrocyclic chelators (DO4S, DO3S, DO3SAm, DO2A2S, TRI4S, and TE4S).This ligand coordinated Cu 2+ and Cu + through a [4N, 2O] and [4N, 1S] donor moiety, respectively.Similar to other 64 Culabeled cyclen derivatives' behavior in vivo, 64 Cu-DO2A2S also exhibited high liver and kidney uptake. 109,110It is possible to attribute this to metabolic processing of the entire complex or transchelation processes.
By adding a methylphosphonic acid group to the macrocycle pendant arms of TETA derivatives, particularly in cross-bridged cyclams derivatives, the radiolabeling performance has been enhanced. 93,111,1124][115] Further, they prepared their bifunctional derivatives to better make use of bis(phosphinate)-cyclams' (BPC) excellent radiolabeling capabilities. 116The anchor groups (-COOH, -NH 2 , -NCS, etc.) were purposefully linked to the remote location on the bis(phosphinate) fragment at the terminal phosphorus atom.The 64 CuCl 2 F I G U R E 7 (A) Coronal PET/MR images of rats with 64 Cu-NSNS2A and 64 Cu-TE2A at 30 min p.i. 104 (B) Coronal PET images of PC3-PSCA tumor-bearing mice with 64 Cu(L 5 -7F5) and 64 Cu(NODAGA-7F5) at different time points after injection (left panel).Coronal PET/MRI images of PC3-PSCA tumor-bearing mice with 64 Cu(L 5 -7F5) at Day 2 after injection.The section was positioned through the tumor and metastasis (right panel). 116(C) Dynamic PET imaging of 64 Cu-TE1PBPON in mice.The tracer shows rapid renal excretion and efficient skeletal uptake (upper panel).4 Cu-labeled YW-7, YW-10, and YW-14, in the absence and presence of a known Aβ-specific blocking agent. 128Reproduced with permission. 104Copyright 2020, Royal Society of Chemistry. 116Copyright 2018, ACS Publications. 121Copyright 2022, Royal Society of Chemistry. 128Copyright 2021, ACS Publication.radiolabeling efficiency of the bifunctional BPC chelators and of their conjugates was comparable to that of the parent ligand, TE1PPIN.A high specific activity of 64 Cu(L 5 -7F5) was obtained by conjugating a thiocyanate BPC derivative, TE1PPIN-NCS (L 5 ), to mAb 7F5.Over the course of 2 days p.i., the prostate tumor site acquired increasing amounts of 64 Cu(L 5 -7F5) (Figure 7B).On Day 2, a metastasis at a large renal vessel was also discernible.Additionally, negligible 64 Cu activity in the liver indicated in vivo stability of the complex.The continued increase of tumor uptake and tumor-to-background ratio for 64 Cu(L 5 -7F5) leads to significantly better tumor visibility in the PET images.
8][119][120] Pazderová et al. prepared 64 Cu-TE1PBPON as bone-targeting probe. 121or healthy mice, after intravenous injection of 64 Cu-TE1PBPON, PET imaging showed rapid renal excretion and bone-selective accumulation.This tendency peaked at 30-60 min and remained stable at least 24 h.Subsequently, a similar uptake pattern was observed between 64 Cu-TE1PBPON and [ 18 F]fluoride in rat femoral defect model.Both tracers showed a higher uptake in the bone defect regions compared to the healthy reference regions (Figure 7C).Although, the diagnostic value of 64 Cu-TE1PBPON was similar to that of [ 18 F]fluoride, longer observation times can be achieved in longitudinal research with 64 Cu-TE1PBPON.
3][124][125] However, these 11 Cand 18 F-radiolabeled agents are hampered by their short physical half-life and complex synthesis, and discovery of novel 64 Cu PET imaging agents would be useful for both diagnostic and medication development.To that end, Mirica's group developed the first-generation BFCs based on macrocyclic TACN and 2,11-diaza[3.3]-(2,6)pyridinophane(N4) ligand frameworks. 126,127These compounds exhibited specific binding to Aβ aggregates both in the in vitro and ex vivo brain sections of AD mice.To increase the lipophilicity and facilitate brain uptake, Wang et al. used the method of employing one ester derivative of the carboxylate pendant arm connected to the TACN backbone (termed, YW-7, -8, -9, -10, and -14). 128In ex vivo autoradiography study, only limited background intensity was observed in WT mouse brain sections.However, the 64 Cu-labeled YW-7 through YW-14 complexes were shown to have varying degrees of enhanced intensity in 5xFAD mouse brain slices.Due to its neutral makeup, the 64 Cu complex of the carboxylic acid BFC YW-14 displayed a lower intensity than that of YW-7 (t-butyl ester) and YW-10 (methyl ester) (Figure 7D).Further, in vivo biodistribution experiments were conducted, and the result suggested that identifiable brain uptake could be observed within 2 min after injection and, more importantly, followed by rapid washout from the brains to provide a potential contrast.
They further modify the structure by connecting two ester carboxylate pendant arms to the TACN backbone. 129lthough the Cu 2+ complexes of the dicarboxylic acid YW-13 have more specificity to the Aβ plaques than dicarboxylate esters, a negative log D value of YW-13 was not anticipated to pass through the blood-brain barrier (BBB).To increase lipophilicity, they designed a series of BFC derivatives containing a TACN macrocyclic ligand and carboxylate ester arms. 130One of them, 64 Cu-YW-15-Me, showed moderate brain uptake in wildtype mice (0.69 ± 0.08%ID/g).Huang et al. developed second-generation multifunctional compounds containing the 2-monomethylamino-pyridyl fragment (HYR-16) to more effectively reduce the metal-induced Aβ toxicity. 131t is demonstrated that the HYR-16 has the ability to redirect the toxic Cu 2+ -mediated Aβ 2 oligomerization into the creation of less toxic Aβ 2 aggregates.The neutral nature of these 64 Cu complexes makes them unable to counteract the positive charge of the Cu 2+ ion, and hence restricts the BBB permeability.It is presumably why the brain uptake of these 64 Cu complexes are slightly lower than that described before. 132ignificant researches have been explored on copper compounds and targeting vectors.4][135] The production of ligands that can stably chelate 64 Cu is still a topic of intense study, but the best option also relies on the size, polarity, and stability of the targeting vector in vivo.

ZIRCONIUM-89
7][138][139][140] The high charge density, Lewis acidity, and chemical hardness of the Zr 4+ make the preference for hard oxygen donors, including oxalate and polyhydroxamates.2][143] Although the most often used chelator for 89 Zr radiolabeling is hexadentate siderophore DFO-an acyclic, hydroxamic acid-based compound (Figure 8). 144,145Some preclinical studies indicated that due to the unsaturated coordination sphere of 89 Zr-DFO, the release of 89 Zr 4+ ion from the radiopharmaceutical and deposition in bone (typically ≈ 5%ID/g) had been reported during prolonged imaging times. 146For clinical applications, this may increase background signal, hinder the interpretation of clinical imaging data, and impede treatment planning. 147n 2017, Pandya et al. initially investigated tetraazamacrocycle-based chelator DOTA and derivatives thereof as 89 Zr chelators. 148In vitro EDTA challenge and biodistribution studies demonstrated that 89 Zr-DOTA forms more stable complexes than 89 Zr-DFO.The crystal structure revealed that nitrogen atoms are also engaged in the complex formation with the oxophilic Zr 4+ ion and saturate coordination sphere around the ion together.A drawback to DOTA is the requirement of consistent high temperature (90 • C, 45 min).In a recent paper, the same group compared a set of macrocyclic chelators with different sizes and flexibility of the polyazamacrocycle ring (TETA, TRITA, PCTA, and NOTA), and assessed how these structures affected the radiochemistry and stability. 149Only smaller and more preorganized chelator NOTA and PCTA could be radiolabeled with 89 Zr under milder conditions.Moreover, 89 Zr-NOTA and 89 Zr-PCTA demonstrated improved in vivo behavior with quick perfusion and clearance from the blood pool and liver tissue, as well as strong in vitro stability.These innovative findings demonstrated that polyazamacrocycles are efficient 89 Zr chelating agents and may serve as a novel research strategy for more potent and safer immuno-PET agents.
Sun et al. developed new chelating agents (NOTHA, NODHA, and NODHA-PY) constructed on TACN. 64At the same time point, NOTHA and NODHA were more effective in binding 89 Zr than NODHA-PY. 89Zr-DFO and 89 Zr-NODHA were stable in human serum for at least 7 days, while a significant quantity of 89 Zr was lost from 89 Zr-NOTHA (∼5% release) and 89 Zr-NODHA-PY (<12% release) over 7 days.In biodistribution studies, 89 Zr-NODHA displayed minimal radioactivity in the blood and better renal clearance at all time points.However, at the 24 h time point, 89 Zr-NODHA was still present in the bones in a non-negligible amount (1.55%ID/g).Chong's group synthesized a novel chelator DA-18C6-BHA, which could rapidly bind to 89 Zr at mild reaction conditions (10 min, >98% radiolabeling efficiency). 66 89Zr-DA-18C6-BHA displayed higher complex stability under excess EDTA solution and in vivo than 89 Zr-DFO.The in vivo evidence indicates that 89 Zr-DA-18C6-BHA displayed a great biodistribution profile with quick blood clearance and minimal absorption in normal tissues, including bone.
Xu et al. reported a new bifunctional chelating agent, Dar, containing multiple donors such as pyridine nitrogen, aliphatic nitrogen, and phenolic oxygen. 151Dar-PSMA-617 complexes were successfully labeled with 68 Ga, 89 Zr, and 177 Lu at room temperature with high RCY and purity.In vitro and in vivo data demonstrated that radiolabeled-Dar-PSMA-617 exhibited comparable, or even better, tumor uptake and antitumor efficacy with radiolabeled-DOTA/DFO-PSMA-617 (Figure 9B).The development of Dar may provide a useful platform in precision theranostics of tumors, because there is currently no BFC that can radiolabel with diagnostic and therapeutic radionuclides under mild conditions.Despite attempts have been made to enhance chelation chemistry that can sequester the bone-seeking 89 Zr with high in vivo stability, DFO continues to be the go-to chelator for clinical application. 152,153 I G U R E 1 0 (A) Comparative PET/CT images in PSMA − and PSMA + tumor-bearing mice of 44 Sc(picaga)-DUPA in comparison to 44 Sc-DOTA-DUPA at 90 min p.i. (left panel).Biodistribution study with different probes at 120 min p.i. (right panel). 161Representative decay-corrected coronal PET/MRI images were obtained from 4 h to 10 days post injection of 52 Mn-DOTAGA-bevacizumab.White arrows: subcutaneously growing KB-3-1 cervix tumors. 178Reproduced with permission. 161Copyright 2019, Royal Society of Chemistry.Reproduced under the terms of the CC-BY license. 178Copyright 2023, Csaba Csikos.
SCANDIUM-44 44 Sc has emerged as a novel PET radioisotope with ultimate emission characteristics and half-life well suited to the pharmacokinetics of small molecules and peptides. 154,155Additionally, the emission properties of 47 Sc are comparable to 177 Lu. 156 Its preclinical in vitro and in vivo researches have been motivated by the significant benefits of the 44 Sc/ 47 Sc pair as a theranostic combination and the improved availability of 44 Sc. 157 DOTA is currently considered the gold standard Sc 3+ chelator with high stability and pM value.However, its complexation kinetics is slow, requiring at least 30 min of radiolabeling at 70 • C-95 • C. 158,159 Sc 3+ is the smallest rare earth metal acid.It is slightly bigger than Ga 3+ and has a high predilection (I A = 10.49) for chemically hard donor ion, such as carboxylates, aliphatic amines. 160Some people have investigated scandium's compatibility with the smaller macrocycle because of its small size.Vaughn et al. studied a set of mixed carboxylic acid/picolinic acid donor arms on TACN for the chela-tion of 44 Sc (Figure 10A). 161Substituting the mpatcn ligand via a functionalized glutarate pendant arm afforded a BFC that was incorporated a PSMA targeting vector, 2-[3-(1,3dicarboxypropyl)ureido]pentanedioic acid (DUPA).The resulting conjugate picaga-DUPA chelated 44 Sc at room temperature with high yield.Similar to the 18 F-based imaging probe DCFPyL, which is presently undergoing phase 3 clinical trials, 44 Sc(picaga)-DUPA demonstrated high binding affinity with PSMA. 44Sc(picaga)-DUPA clearly depicted PSMA + xenografts in PET scans.The tumor uptakes of 44 Sc(picaga)-DUPA and 44 Sc(DOTA)-DUPA at 120 min p.i. were 13.8 ± 0.6%ID/g and 2.8 ± 1.3%ID/g, respectively.Interestingly, 44 Sc(picaga)-DUPA had significantly lower kidney and splenic uptakes than MIP-1427 (a 99m Tc-based PSMA-inhibitor), providing greater feasibility for future therapeutic applications with 47 Sc.
3][164][165][166] However, it has also been discovered that AAZTA may form rather stable complexes with Sc 3+ and Ga 3+ (log K ML = 27.7 and = 21.2, respectively). 167,168Lisowski et al. provided an overview of the design, synthesis, and uses of AAZTA-derived chelators in the 68 Ga and 44 Sc PET imaging.Despite not being a new class of chelators, AAZTA's applications for 44 Sc make it a desirable candidate for developing new radiopharmaceuticals.

MANGANESE-52
52g Mn is an intriguing radiometal for immuno-PET because of its long half-life and low positron energy that offers favorable resolution up to 1.2 mm for imaging, which is crucial in small animal imaging. 169Additionally, the Mn 2+ ion's 5/2 spin quantum number, long electronic relaxation time, quick water-exchange rate, and low in vivo toxicity make it the ideal T1 agent. 170,171][174] Mn 2+ , a hard transition metal (I A = 7.09) with highspin d 5 , prefers hexa-and heptadentate coordination. 175,176OTA have been successfully employed as chelators for Mn 2+ radioisotopes and showed high in vitro and in vivo inertness. 177,178Graves et al. originally published the in vivo assessment of 52 Mn-labeled mAb in 2015. 177PET imaging in mice bearing 4T1 xenograft tumors showed peak tumor uptake of 18.7 ± 2.7%ID/g at 24 h p.i., with a delayed blood clearance over time and a relatively significant bone and spleen uptake (>10%ID/g, 120 h p.i.).More recently, Csikos et al. successfully synthesized a 52 Mn-DOTAGA-bevacizumab PET probe with high RCY and suitable stability properties for in vivo PET/MR imaging in a VEGF-A expressing KB-3-1 cervix carcinoma tumor-bearing mouse. 178The quantitative PET images showed that the KB-3-1 tumors were recognizable from 4 h after injection, and the accumulation of radiopharmaceuticals increased with time and started to plateau 2-3 days (Figure 10B).Although it gradually declined over time, tumor-to-organ ratios remained high until the end of the study, and traditional Mn 2+ target organs such as the thyroid, liver, pancreas, and kidney had little tracer uptake.
DOTA has the tendency to form stable complexes with Mn 2+ ; however, lack of an inner sphere water molecule in nat Mn-DOTA results in no MR contrast enhancement. 179Ndiaye et al. have investigated bispidine (3,7-diazabicyclo[3.3.1]nonane)derivatives with the goal of significantly enhancing the kinetic inertness of Mn 2+ complex while ensuring good relaxation characteristics and water solubility. 180,181The strongly preorganized structure of the 2,4-pyridyl-disubstituted bispidol ligand L6 provides its Mn 2+ complex with exceptional kinetic inertness.
In addition, L6 could be quantitatively radiolabeled with 52 Mn at neutral pH 7, and kept stable in various media over 18 h (>91%).Recently, they evaluated bispidine ligands bearing an acetate function (L7) or a phosphonate (L8) on the bicycle for the complexation of 55 / 52 Mn 2+ .L8 showed excellent radiolabeling properties with 52 Mn under pH 7 in 15 min at room temperature.The outstanding stability and kinetic inertness of complexes endorse the great potential of Mn 2+ -bispidines as MRI and PET imaging agents.
Manganese is a versatile transition metal and its isotopes are suitable for MR, PET, and PET/MR imaging. 182The radiochemistry community is faced with a relatively new challenge in the complexation of Mn isotopes employing hexa-and heptadentate chelators to prepared complexes with great in vivo stability.

OTHER RADIONUCLIDES
The PET radionuclide 18 F is by far the most widely used for small molecule imaging.This area of study has advanced quickly since McBride et al.'s initial publication on the use of metal (aluminum) fluorine complexes for radiolabeling with [ 18 F]fluoride in 2009. 183,1841][192] Because TACN ring size are suitable for Al 3+ (0.53 Å), pentadentate ligand form mononuclear octahedral complexes with aluminum and possess a free single coordination site for binding fluorine. 193Recently, Wang et al. developed pentadentate AlF-chelator p-SCN-PhPr-NODA. 194As a proof of concept, p-SCN-PhPr-NODA were easily conjugated to Glu-urea-Lys via thiourea bond formation, and successfully radiofluorinated via Al 18 F chelating process.Biodistribution study and MicroPET scan in healthy mice demonstrated excellent in vivo stability of Al 18 F-labeled construct, due to the long propyl chain in the structure reducing potential steric hindrance during the Al 18 F chelation.The β − particle-emitting radionuclides 90 Y and 177 Lu are powerful radionuclides ideal for targeted radiotherapy.Additionally, 86 Y has attracted interest as a surrogate isotope of yttrium that might be utilized as a matched pair for targeted radionuclide treatment with the β − -emitter 90 Y. 195-197 177 Lu also possesses an imageable γ-emission, allowing quantitative SPECT imaging applications. 198,199 177Lu and 86/90 Y are radionuclides with a prevalent oxidation state (+3) and a preferred coordination number of 8 or 9.The affinity of Lu 3+ for ionic bonds (I A = 10.07) is characteristic of the lanthanides, which prefer oxygen-donating groups. 160,200As previously indicated, the chemistry of Y 3+ (I A = 10.64) is quite similar to that of the lanthanides, and DOTA and DTPA analogs are the most often employed chelators for 177 Lu and 86/90 Y radiotracers.3p-C-NETA, a ligand possessing both a parent macrocyclic NODA backbone and a flexible acyclic tridentate pendant arm, has been reported by Chong and coworkers to be a promising chelator in terms of kinetics and stability for 90 Y and 177 Lu. 201,202 90 Y and 177 Lu-labeled 3p-C-NETA-trastuzumab exhibited excellent in vitro stability and pharmacokinetic properties in tumor-bearing mice.Surprisingly, Cleeren et al. found that NODA-based chelator 3p-C-NETA is a multipurpose chelator that can be employed for both targeted radionuclide therapy ( 177 Lu, 213 Bi, and 225 Ac) and diagnostic applications (Al 18 F-method and 68 Ga).As proof of concept, throughout the investigation, [ 18 F]AlF-and 177 Lu-3p-C-NETA-complex demonstrated good in vitro stability in both PBS and human serum.
225 Ac (t 1/2 = 240 h) and its daughter isotopes 213 Bi (t 1/2 = 45.6 min) are considered to be the most promising therapeutic radionuclides.With the biggest ionic radii within the f-block, actinium is the most basic trivalent ion known and is only chemically stable in the trivalent oxidation state (Ac 3+ ). 203 225Ac can easily be complexed with the DOTA chelator, but requires high temperatures (80 • C-95 • C) or microwave assistance.5][206][207] Macropa was found to rapidly bind kBq activities of 225 Ac at room temperature leading to a complex with high in vivo stability.Therefore, macropa might offer a valuable alternative for 225 Ac labeling of heat-sensitive bio(macro)molecules.The difficulties in maintaining intact complexation of 225 Ac and chemically different daughter radionuclides are the main problems with current 225 Ac utilization.The major limitation of α-emitting radiometal is the lack of an organic or inorganic framework to bind radionuclide stably, especially in the presence of high decay energy.Recent advance in macrocycles for α-emitting radiometal in target alpha therapy will be reviewed elsewhere.

DISCUSSION
In recent years, with the popularization of PET equipment and improved availability of medical cyclotrons, the number of radionuclide production and use sites have significantly expanded worldwide.These advancements have sparked a surge in research into positron-emitting radiometals, which were previously unavailable but had desirable properties for nuclear imaging and therapy.
A number of promising metal-based radiopharmaceuticals that have been approved by the FDA/EMA have also greatly increased confidence in the pharmaceutical industry.
Radiopharmaceuticals labeling with a radiometal generally requires BFC, as metal ions can form insoluble hydroxides at physiological pH.The existence of BFC can, on the one hand, form kinetically stable complexes with the radiometal, and on the other hand, play a role of covalent attachment of targeting vector.The characteristics of BFC, including geometry, lipophilicity, and overall charge, significantly influence the pharmacokinetics of targeted radiopharmaceuticals. 26,27,208 Usually, hydrophilic BFCs and anionic complexes tend to be excreted via the kidneys, lipophilic complexes accumulate in the liver, cationic complexes exhibit affinity to the heart, and electroneutrality is an essential requirement for a compound to cross the BBB. 207Different BFCs with various donor atoms and frameworks are needed for various radiometals.In turn, the dependence of the properties of radioactive tracers on the chemical structure makes it possible for radiochemists to continue to develop and upgrade novel BFCs.
When a new chelator is synthesized for the chelation of radiometal ions, or an old chelator is reused with new radiometal ions, initial screening tests are usually carried out through simple radiolabeling to determine many factors: what temperature is required (room temperature is preferred), how long does a reaction take (as soon as possible), and whether the chelator can effectively combine the radiometal ion and radiolabeling in high yields (quantitative is best). 30In order to obtain the optimized property and application of each radiometal-chelate complex, an arduous balancing work needs to be carried out.It is necessary to study and provide different chelates of various properties, and understand their coordination chemistry with the radioactive metal to be labeled.
When a chelator with a radiolabeling property suited for a certain radiometal ion is found during early screening, additional studies must be undertaken under conditions relevant to in vivo translation.Micro PET/SPECT imaging may be used to track the metabolism of these radiometalchelate complexes, and the radioactive distribution of tissues can be measured and calculated in the biodistribution experiments for the percentage of injected dose of radioactivity per gram.High thermodynamic stability does not always imply excellent radioactive labeling conditions and in vivo features.The major goal of assessing and choosing a chelator is to find an effective bifunctional chelating system that strikes a compromise between fast complexation kinetics and high kinetic inertness of the production. 209,210The most important consideration, even more so than the metal-chelate complex's perfect thermodynamic stability, is in vivo kinetic inertness. 30he selection of a chelator is dictated by the radiometal, which in turn is determined by the intended use (for diagnosis and radiotherapy of diseases).There are chelators that can be used widely in conjunction with various radiometals (such as DOTA for radiolabeling with 68 Ga, 111 In, or 177 Lu), but it is also necessary to develop optimum chelating systems that are specifically tailored to distinct radiometals.Whether developing new chelators or labeling new radioactive metals with old chelators, the ultimate goal is to prepare a new generation of radiopharmaceuticals to address the medical need for early cancer detection and systemic targeted therapy.

F I G U R E 2
Selected macrocyclic chelators and their respective BFCs.

F
I G U R E 3 (A, top panel) Whole-body PET images of ApoE KO mice.From left to right, 68 Ga-HDL, 68 Ga-PCTA-DSPE, and 18 F-FDG radiotracers.(A, bottom panel) Images of aorta and heart of ApoE KO mice: (a) macroscopic view; (b) 3D PET image.The heart was removed before the dissection of the aorta; its position is indicated with a red dashed circle.Atheromatous plaques are highlighted in yellow.(B) Ex vivo accumulation of (B, top panel) Static PET/CT images of BxPC3 xenograft tumor mice at 30 min p.i. of 68 Ga-yG5-RGD, 68 Ga-yG5, and 68 Ga-RGD.Arrows indicate the location of tumors.(B, bottom panel) Tumor uptakes determined by quantitative ROI analysis of PET/CT images (n = 4; *p < .05,**p < .01,***p < .001).

F I G U R E 5
Structures of macrocyclic chelators for copper-64.
Description: (bo high ) skeletal regions with the highest bone uptake; (bo low ) skeletal regions with the lowest bone uptake; (bo def ) critical bone defect in the right femur; (bo ref ) healthy bone reference region in the left femur; (ht) heart; (ki) kidney; (bl) bladder; (SUV) standardized uptake value; (RT) radiotracer; (L) left; (R) right.(D) Autoradiography images of brain sections of WT and 5xFAD mice treated with