Visualizing reactive astrocytes: Positron emission tomography imaging ligands for imidazoline‐2 binding sites

Imidazoline‐2 binding sites (I2BS) exhibit specific expression in reactive astrocytes of the brain and are implicated in various pathophysiological processes, including analgesia, inflammation, Alzheimer's disease, Parkinson's disease, and glial tumors. However, the lack of available protein structural data poses a challenge in the exploration of I2BS functions and pharmacological characterizations, indicating the need for the discovery of selective ligands. As a non‐invasive and translational molecular imaging tool, positron emission tomography (PET) has found wide application in clinical diagnosis and drug discovery. Consequently, several PET ligands have been developed to “visualize” I2BS in the living brain, thereby elucidating the biological implications of I2BS and facilitating I2BS‐directed drug development. This review offers a comprehensive update on I2BS PET ligands, with a focus on their chemotypes and PET imaging outcomes. Furthermore, the review provides a summary of recent I2BS drug discoveries, which could serve as a catalyst for the development of more potent PET ligands.


INTRODUCTION
Imidazoline receptors or imidazoline binding sites originally refer to a family of non-adrenergic binding sites that are recognized by adrenergic compounds with an imidazoline moiety, such as clonidine, p-aminoclonidine, and idazoxan, although this has been demonstrated to be an oversimplification. [1]1b] Fortunately, recent efforts on drug discovery (mainly focusing on I 1 and I 2 receptors) demonstrate the great potential of these compounds to elicit neuroprotection and to treat various disorders such as metabolic syndrome, hypertension, chronic pain, and Alzheimer's disease (AD). [1,2]Obviously, the development of selective ligands targeting imidazoline-2 binding sites (I 2 BS) largely dominated the picture, [1] and in a recently completed phase II clinical trial, a promising I 2 BS agonist, compound CR4056 (11, Figure 7), exhibited clear analgesic activity as a first-in-class nonopioid analgesic. [3]Thus, an increasing number of studies like in vitro functional assays and in vivo pharmacological evaluations have been conducted by using these I 2 BS ligands, which in turn has boosted the identification of several promising I 2 BS agonists.Furthermore, these studies also provide novel insights into understanding the nature of I 2 BS and related pharmacological studies. [1]In parallel, the development of positron emission tomography (PET) radioligands of I 2 BS is a research priority, as it will identify the roles of this binding site in neurological diseases and facilitate I 2 BS-directed drug development.Herein, this review provides a comprehensive update on I 2 BS PET ligands by focusing on their chemotypes and PET imaging outcomes, and importantly, also offers a summary of recent I 2 BS drug discovery, which would stimulate and give new hints to the development of more potent PET ligands.

Imidazoline receptors
Initially, the imidazoline receptors were classified by using the terminology "I 1 and I 2 imidazoline sites" in 1993. [4]1b] In addition, these binding sites were located predominantly on non-mitochondrial membranes at the subcellular level. [6]In parallel with the subtype-selective drug discovery, I 1 receptors were proved to have cardiovascular and metabolic effects, [1b] such as the effects on blood pressure and heart rate, [7] anti-arrhythmia, [8] congestive heart failure, [9] as well as metabolic syndrome. [10]1b,11] As a result, it is important to note that I 2 BS refers to several different and potentially biologically diverse protein molecules, and however, their molecular identities remain elusive.With extensive research on ligand binding, I 2 BS is found in many organs including but not limited to the brain, kidney, and liver, and cell types like astrocytes, platelets, and pancreatic cells.Not all tissues express I 2 BS, and for example, skeletal muscle, heart, lung, and spleen are absent of this binding site. [11]In addition, this binding site is primarily located at the outer membrane of mitochondria at the subcellular level (Figure 1). [12]Mitochondria are one of the main sources of reactive oxygen species and reactive nitrogen species.According to existing data, I 3 receptors represent a putative binding site that is likely located on the Kir6.2subunit of ATP-sensitive potassium channels in pancreatic β-cells and may play a key role in insulin secretion. [13]1b] 1.
such as major depression, opioid addiction, gliomas, and AD. [14]On the basis of this, I 2 BS corresponding studies are predominantly focused on the central nervous system (CNS), and as a result, several in vivo effects have been well characterized including analgesic, discriminative stimulus, neuroprotective, and hypothermic effects. [1]he expression and localization of I 2 BS in brain sections of various species including rodents, non-human primates (NHPs), and humans were carried out efficiently via autoradiography (ARG) experiments with tritiated ligands of [ 3 H]idazoxan and [ 3 H]2-BFI (Figure 2).In saturation binding assays with rat whole brain homogenates, [ 3 H]2-BFI and [ 3 H]idazoxan (in the presence of 5 μM rauwolscine to block the α 2 -adrenoceptors binding) bound to I 2 BS (the apparent single sites in a larger population >70%) with a saturated manner, and exhibited the K d values of 1.74 and 10.4 nM, respectively. [15]It should be noted that [ 3 H]idazoxan had poor selectivity toward I 2 BS and displayed equal potent binding affinities to α 2 -adrenoceptors (K d = 36 nM) as well as non-adrenergic idazoxan binding sites (K d = 19 nM), [16] the later deemed to include I 2 BS. [17]Therefore, [  Low densities were seen in the cerebellum (6.77 fmol/mg tissue), hippocampal regions (<13 fmol/mg tissue), and cortical regions (<8 fmol/mg tissue) by [ 3 H]2-BFI. [15]In addition, the ARG results of [ 3 H]2-BFI in mouse brains revealed that the specific binding was found the highest in the dorsal raphe (76.04 fmol/mg tissue), paraventricular thalamus (>63 fmol/mg tissue), accumbens nucleus (59.20 fmol/mg tissue), and lateral ventricle (54.14 fmol/mg tissue), but much lower in the cortical areas (<17 fmol/mg tissue), hippocampus (<17 fmol/mg tissue), and cerebellum (<22 fmol/mg tissue), [18] which was consistent with the binding patterns in rat brain. [15]Further explorations in NHP brain sections were accomplished with a highly potent and selective 11 C-labeled PET ligand [ 11 C]FTIMD ([ 11 C]2, Figure 4, the details were shown in Section 2 "I 2 BS PET Ligands"), and the results indicated a different expression pattern of I 2 BS from that of rodents: the specific signals (>97%) of tracer binding were found high in brain regions of the caudate, hippocampus and globus pallidus, but lowest in the cerebellum, which was confirmed by co-incubation with FTIMD and BU224 (9, Figure 7, a potent I 2 BS ligand with high selectivity and affinity). [19]urthermore, in human brain sections, the I 2 BS expression levels were roughly evaluated by [ 3 H]idazoxan, and the results revealed that high density of signals was found in brain regions of the hippocampus (up to 127 fmol/mg tissue in the gyrus dentatus), medulla oblongata of brainstem (up to 139 fmol/mg tissue in the floor IV ventricle), and nucleus caudatus of cerebral cortex (119 fmol/mg tissue), [16,17c] which was in accordance with the distribution pattern measured with [ 11 C]FTIMD in NHP brain sections. [19]he fundamental cells of the human brain are neurons and glial cells (Figure 1).Neurons are excitable cells that communicate with each other through synapses, and their normal functions are reliably supported by specialized glial cells, which generally include astrocytes, microglial cells, and oligodendrocytes. [20]Among these, astrocytes are the most abundant glial cells and have diverse functions in CNS including regulation of synaptic neurotransmitter levels and maintenance of ionic homeostasis around the neurons. [21]Astrocyte dysfunction, also known as "reactive astrogliosis," is a process through which astrocytes respond to all kinds of CNS traumas and it has emerged as a pathological marker of CNS structural disorders. [22]eactive astrocytes that undergo responses featured by functional, morphological, and molecular alterations have been implicated in many CNS pathogenic processes. [23]pecifically, reactive astrogliosis is a widely known characteristic of AD, yet its functions in AD are still unclear.Amy-loid plaques or diffuse deposits of amyloid are closely connected with reactive astrocytes, which surround them with dense layers of processes that may wall them off and serve as neuroprotective barriers. [24]Indicators of CNS trauma also include reactive astrogliosis and the emergence of glial scars, which are currently considered to be crucial in determining the long-term clinical prognosis. [25]Additionally, an increasing number of research was conducted to elucidate the role of reactive astrocytes during the response to CNS infection.For instance, reactive and scar-forming astrocytes are found in abundance around microbial abscesses, and research suggests that reactive astrocytes are important for preventing invasive microbes like Toxoplasma gondii from spreading into brain parenchyma. [26]As a result, there is renewed interest in developing reliable approaches to better characterize reactive astrocytes in vivo.Monoamine oxidases-B (MAO-B) has high expression in the outer mitochondrial membrane of astrocytes in the human brain, and many related radioligands such as [ 11 C]L-deprenyl-D 2 have been developed for the imaging of MAO-B in reactive astrocytes. [27]However, these radioligands lack astrocyte specificity, since MAO-B is also expressed in histaminergic and serotonergic neurons. [28]I 2 BS appears to be a reliable biomarker to accurately measure reactive astrocytes in the human brain in both physiological and pathological conditions.28b,29]

I BS PET LIGANDS
PET imaging technique has gained its universal value as a flexible tool for both disease diagnosis and drug discovery, especially in CNS.In parallel, the discovery of target-specific PET ligands has emerged as an important part of CNS PET imaging studies.In the development of CNS PET ligands, several key factors should be considered, such as ligand chemical structure, pharmacology, pharmacokinetics, and non-specific binding.In brief, the chemical structure of a ligand should be amenable to carbon-11 ( 11 C) and fluorine-18 ( 18 F) incorporation, and the CNS PET ligands are required to be brain permeable, which demands the ligands to have relatively small molecular weights (<500 Da) and optimal lipophilicity (shake-flask log D ranged from 1.5 to 3.5), as well as low brain efflux (ratio ≤2) caused by efflux transporters like P-glycoprotein and breast cancer resistance protein. [30]Since brain targets often have a low expression level, CNS PET ligands should have a high in vitro binding potential (B max /K d ≥10, where K d is the equilibrium constant) to adequately monitor the changes of the targets. [31] I G U R E 3 Chemical structure of [ 11 C]1 and its radiosynthesis route.
Compared with drug discovery, the development of I 2 BS PET ligands has been much lagged.To date, six PET ligands (Figures 3-6), including five 11 C-labeled ligands and an 18 F-labeled ligand, have been developed based on the chemical structure of idazoxan (7, Figure 7), which contains an imidazoline ring and has been widely used for in vitro evaluation of I 2 BS in its tritiated form.The exploration of I 2 BS selective PET ligands commenced with the preparation of [ 11 C]benazoline ([ 11 C]1, Figure 3) in 2003, which was accomplished from the precursor compound 2-naphthylmagnesium bromide using a twostep procedure with an overall radiochemical yield (RCY) of 16% (decay-corrected) in 45 min. [32]This method of 11 C-labeling has great potential to be extended to related imidazoline moieties.Despite the high affinity (K i = 0.85 nM) for I 2 BS and high selectivity (Table 1), [33] [ 11 C]1 was not further investigated for in vivo experiments including PET imaging studies, which may be attributed to its complicated radiosynthesis (Figure 3).
[ 11 C]2 has high biostability in rat brains with 98.1% parent compound remaining after 30 min post-injection. [35]In a PET study with rats, the radioactivity of [ 11 C]2 was found to have nearly homogenous distribution volume (V T ) values in various brain regions (10.90-15.09mL/cm 3 ) with slightly higher signals in the ependymal cell layer (15.09 mL/cm 3 ), hippocampus (14.25 mL/cm 3 ), and cortex (13.85 mL/cm 3 ).Moreover, pretreatment experiments with BU224 showed that the brain distribution of [ 11 C]2 was reduced in all brain regions by a similar degree (17%-34%), which indicated a moderate specific binding of [ 11 C]2 to I 2 BS in vivo. [35]Furthermore, similar PET results in a monkey brain were also observed (Figure 4): the radioactivity of [ 11 C]2 accumulated homogeneously in different brain regions including the thalamus, arcuate nucleus, occipital cortex, hippocampus, striatum, and cerebellum, and these signals were all reduced to an equal degree by the pretreatment with BU224. [19]Based on these results, [ 11  ). [36]The two compounds, metrazoline and TEIMD, have high affinity to I 2 BS with K i values of 0.37 and 1.68 nM, respectively, as well as high selectivity over α 2 -adrenoceptors and I 1 receptors (Table 1). [37]The preparation of [ 11 C]3 and [ 11 C]4 was achieved by following the same radiolabeling strategy of [ 11 C]2 (Figure 4, condition 2), which were obtained in modest yields of 9.9% and 13.6%, respectively. [36]Biodistribution results in mice showed that [ 11 C]3 and [ 11 C]4 had poor brain penetration (<1% injection dose per gram, ID/g), but the radioactivity levels were found high in peripheral tissues like the liver (>9% ID/g at 30 min postinjection) and moderate in the pancreas (∼2% ID/g at 30 min postinjection), where I 2 BS was enriched.In the case of [ 11 C]3, the signals in  [19] 2011, Wiley-Liss, Inc.).
the liver and pancreas could be significantly blocked by coinjection with BU224, but not moxonidine (an agonist for α 2 -adrenoceptors and I 1 receptors) and efaroxan (an antagonist for α 2 -adrenoceptors and I 1 receptors).On the contrary, [ 11 C]4 exhibited high nonspecific binding, of which the radiotracer uptake in the liver and pancreas was not blocked by BU224.Metabolite analysis showed that [ 11 C]3 displayed moderate biostability in the liver and pancreas with 41% and 61% remaining unchanged at 30 min postinjection, respectively.PET scans in mice demonstrated that the highest radioactivity levels of [ 11 C]3 were observed in the liver and pancreas, and the signals were significantly reduced by 50.8% and 56.3%, respectively, with the coinjection of BU224, suggesting its specific binding to I 2 BS in vivo. [36]However, no further PET studies with [ 11 C]3 in higher species were reported.
(V T = 41.9-105.7 mL/cm 3 ). [38]In porcine brains, [ 11 C]5 showed heterogeneous uptake in different brain regions with the highest in the thalamus (V T = 49.7 mL/cm 3 ) and lowest in the cerebellum (V T = 26.6 mL/cm 3 ), which is in accordance with the known distribution of I 2 BS, and this radioactivity accumulation was remarkably blocked by BU224 in a dose-dependent manner. [41]Similarly, the distribution of [ 11 C]5 in NHPs was the highest in the globus pallidus (V T = 114 mL/cm 3 ) and the lowest in the cerebellum (V T = 48.1 mL/cm 3 ). [43]These results indicated that [ 11 C]5 owned much higher specificity to I 2 BS than [ 11 C]2.In further human PET imaging with healthy male volunteers, reversible kinetics and a heterogeneous radioactivity distribution were discovered for [ 11 C]5 with the highest radioactivity in the striatum (V T = 105 mL/cm 3 ) and the lowest in the cerebellum (V T = 41 mL/cm 3 ), which is consistent with the findings in porcine and NHPs.Moreover, as shown in Figure 5, the brain uptake was dose-dependently reduced throughout the brain by pretreatment with idazoxan, but no obvious uptake reduction was found with isocarboxazid (a nonselective MAO inhibitor). [38]As the first I 2 BS-specific PET tracer that has been advanced to clinical research, [ 11 C]5 was employed in clinical studies in patients with F I G U R E 6 (A) Two-step radiosynthesis of [ 18 F]6.(B) Coronal rat brain positron emission tomography (PET) images of [ 18 F]6 under the conditions of baseline and BU224-blockade.HIP, hippocampus; HTH, hypothalamus.(Reproduced with permission. [47]2014, Elsevier Inc.).
neurodegenerative diseases to assess the changes of I 2 BS expressed on activated astrocytes.Compared with healthy control, early Parkinson's disease (PD) patients showed an increased uptake of [ 11 C]5 in the frontal, temporal, parietal, and occipital cortical regions, while moderate/advanced PD patients had a reduced uptake across the whole brain.These results also indicated that the reactive astrocytes have clinical relevance with the development of cognitive impairment. [44]In addition, PET studies of [ 11 C]5 in AD patients revealed higher tracer uptake in the frontal, temporal, occipital, and medial temporal lobes of amyloid-β (Aβ)-positive cognitively impaired subjects, indicating an involvement of astrogliosis in the pathophysiology of AD. [45] A similar link was also observed (Figure 5): the Aβ-positive patients showed higher tracer uptake compared to controls except in the temporal lobe, where reduced [ 11 C]5 uptake was observed.These observations are consistent with a potential mechanistic correlation TA B L E 1 Properties and positron emission tomography (PET) imaging results of imidazoline-2 binding sites (I 2 BS) PET ligands.

Affinity
a Calculated using the online ALOGPS 2.1 program. [57 ]tween Aβ deposition and astrocyte reactivity.Additionally, these new findings support the hypothesis that early Aβ deposition in the temporal lobe may trigger sustained pro-inflammatory astrocyte reactivity, which eventually results in astrocyte dystrophy and amyloid-associated neuropathology. [39]Since there are few reports about the clinical PET studies of [ 11 C]5, further trials are required to verify its utility in exploring astrocyte reactivity. [46]ompared with 11 C, 18 F has the advantages of a longer half-life (109.8 vs. 20.4min) for multistep synthesis and off-site use and lower positron energy (650 vs. 960 keV) to provide PET images in higher spatial resolution. [48]nspired by the excellent properties of [ 11 C]5, the structurally analog BU99018 (6, or FEBU, Figure 6) that has an N-fluoroethyl moiety was developed as an 18 F-labeled I 2 BS PET ligand.As shown in Table 1, compound 6 exhibited both comparable binding affinity to I 2 BS (K i = 2.6 nM) and selectivity over α 2 -adrenoceptors with that of 5. [40] As shown in Figure 6, [ 18 F]6 was prepared by the reaction of N-demethylated precursor and [ 18 F]fluoroethyl bromide in two steps with a modest yield of 10.1% (decay-corrected). [47]Biodistribution results in mice demonstrated that [ 18 F]6 could efficiently penetrate the BBB with a high initial brain uptake of ∼8%ID/g, and the radioactivity uptake was significantly blocked by coinjection of the two I 2 BS ligands BU224 and 2-BFI.In a PET study of rats, the highest uptake of [ 18 F]6 was found in brain regions like the hypothalamus (high I 2 BS expression) and hippocampus (low I 2 BS expression), and moderate in the cerebellum (low I 2 BS expression), which was only partially in accordance with distribution of I 2 BS in rodent brain. [15,18]Pretreatment with BU224 homogeneously reduced the area under the curve [60-90 min] in the hypothalamus, hippocampus, and cerebellum by 77%, 68%, and 75% of the control, respectively, indicating limited specific binding of [ 18 F]6 to I 2 BS in vivo (Figure 6).In addition, [ 18 F]6 showed good biostability in mice brains with >90% remaining unchanged at 60 min postinjection. [47]However, the PET study in higher species with [ 18 F]6 has not yet been reported.
In conclusion, the development of I 2 BS is still in its infancy, and all of the reported ligands are derived from the chemical structure of idazoxan.[ 11 C]1 was the first developed I 2 BS PET ligand but was not further evaluated due to its complicated radiosynthesis, whilst [ 11 C]3 and [ 11 C]4 failed to penetrate BBB and could be only used for PET imaging of I 2 BS in peripheral tissues.I 2 BS PET imaging in the brain was commenced with [ 11 C]2, which showed good specific binding to I 2 BS in vivo.Of note, [ 11 C]5 is the most widely used ligand in clinics to evaluate the reactive astrocytes in patients with AD, PD, and cognitive impairment, and more clinical research is warranted.To date, [ 18 F]6 is the first and only 18 F-labeled I 2 BS PET ligand and shows moderate specific binding and good biostability in rats, and PET study in NHPs is required to verify its utility in imaging I 2 BS.These PET ligands have small molecular weights <250 Da and moderate clogP values (1.90-3.19),which are optimal for BBB penetration.However, among the tested ligands, [ 11 C]2, [ 11 C]5, and [ 18 F]6 could readily penetrate the BBB (Table 1), while  1) show weak brain entry (≤1% ID/g), which was possibly induced by their interactions with brain efflux pumps on the murine BBB.Therefore, there is clearly a need for I 2 BS PET ligands that are designed based on new chemical scaffolds, aiming to improve the I 2 BS-binding properties and brain kinetics.Furthermore, the differences in I 2 BS expression levels among species should also be taken into consideration in the development of I 2 BS PET ligands.

PERSPECTIVES FOR I 2 BS PET LIGAND DEVELOPMENT
Since no protein structural data are available, the exploration of the I 2 BS functions and pharmacological characterizations relies on the discovery of related selective ligands, which are especially devoid of I 1 receptors and α 2 -adrenoceptors' affinity. [49]Idazoxan (7, Figure 7) is an imidazoline antagonist for α 2 -adrenoceptors and its binding potential to I 2 BS was first mentioned in 1987. [11]bviously, this compound has poor selectivity over α 2adrenoceptors.From this starting point, various families of benzo-fused heterocyclic compounds including benzodioxane, benzofuran, and benzoxazine derivatives were developed. [49]Among them, the benzofuran compound 2-BFI (8, Figure 7) exhibited highly active and selective binding properties with pK i values of 8.89 and 4.57 to I 2 BS and α 2 -adrenoceptors, respectively. [50]As a result, the tritiated [ 3 H]8 has been widely used in binding and autoradiographic studies as an important I 2 BS selective radioligand. [15]By replacing the benzo fused heterocyclic ring with a quinoline nucleus, compound BU224 (9, Figure 7) was developed as a potent I 2 BS ligand with a high affinity of 8.68 (pK i ), which also showed improved selectivity over I 1 receptors (I 2 /I 1 = 832) than benazoline (1, Figure 3). [51]In addition, BU224 has been well accepted as a blocking ligand in I 2 BS studies both in vitro and in vivo.LSL60101 (garsevil, 10, Figure 7) was an analog of 2-BFI, which has an imidazole ring that is connected by position 2 and was first described as an I 2 BS-selective ligand involved in astrocyte activation and neuronal regeneration. [52]Compared with 2-BFI, compound 10 showed decreased affinity (pK i = 6.67) to I 2 BS and lowered selectivity toward α 2 -adrenoceptors (Table 2), which was further confirmed by its analogs. [52]In addition, CR4056 (11, Figure 7), which has a 1H-imidazol moiety, is a first-inclass I 2 BS ligand characterized by potent analgesic activity in both animal models and humans and is in clinical phase II studies for osteoarthritis and postoperative dental pain. [3,53]Compound 11 exhibited strong agonist efficiency but with a low affinity of 596 nM (IC 50 , [ 3 H]2-BFI assay) and poor selectivity over MAO-A (IC 50 = 358 nM). [54]As a result, more modifications are needed to develop I 2 BS PET tracers of imidazole-like structure.According to the 3D-QSAR study, the idazoxan-based ligands are characterized by a planar ligand conformation that contains the following elements including a non-substituted imidazoline cycle (p1) and a 2.5 Å wide aromatic moiety (p2) that has a dihedral angle of 142 • between them (Figure 8A).In addition, an extremity at 2.9 Å from the imidazoline C2 atom following the interplane intersection line and the other extremity following a line at 150 • with respect to the interplane intersection line were also required. [55]Changing the p1 cycle from imidazoline to imidazole (compound 10) resulted in a significantly reduced affinity to I 2 BS, which was possibly induced by the electrostatic alteration. [52]urrently, in most cases, the development of I 2 BS agents usually proceeds by synthesizing compounds based on the scaffolds of idazoxan, in which the chemical structure is connected by position 2 of the imidazoline ring without additional substitution in other positions. [49]The chemical structures of these 2-imidazoline-like ligands are relatively restricted.Fortunately, Carmen Escolano et al. have made a great breakthrough in the discovery of I 2 BS ligands with novel backbones.In 2017, they reported a family of (2-imidazolin-4-yl)phosphonate compounds (12-14, Figure 7) as potent I 2 BS ligands. [56]These ligands are highly substituted 2-imidazoline-containing compounds which feature substituents in the 1-, 4-, and 5-positions, and showed much higher affinity to I 2 BS than idazoxan (pK i >8, Table 2), as well as higher selectivity over α 2adrenoceptors.Among them, compound 12 displayed the highest selectivity over α 2 -adrenoceptors (I 2 /α 2 = 10233) as well as a high affinity (pK i = 8.19).In addition, further modification in 4-position with the incorporation of a phenyl substituent led to the analog 13, which has an outstanding affinity to I 2 BS (pK i = 9.42), but a reduced selectivity over α 2 -adrenoceptors (I 2 /α 2 = 457).These results indicate that the (2-imidazolin-4-yl)phosphonate might be considered a suitable scaffold with a great neuroprotective potential acting at I 2 BS.2c] The pharmacological profiling led to the identification of compound 14 (Figure 7), which displayed outstanding affinity to I 2 BS (pK i = 8.61, [ 3 H]2-BFI assay) and excellent selectivity index regarding I 1 receptors and α 2 -adrenoceptors (Table 2).The further 3D-QSAR study revealed that the incorporation of a phenyl ring to the N-maleimide group may positively correlate with I 2 BS binding activity.Contrarily, the introduction of bulkier substituents such as naphthol to the N-maleimide group adversely impacts the affinity for I 2 BS and may reduce the efficacy of I 2 ligands.Additionally, further introduction of two steric hot spots (var183), such as halogen atoms (3-chloro-4-fluoro) in  (Reproduced with permission. [55]2000, American Chemical Society) (B) Representation of positive interactions (in red) of a derivative of 14 in 3D-QSAR model; the steric hot spots are presented in green and hydrophobic regions in yellow.2c] 2020, American Chemical Society).
the N-phenyl ring at a distance range of 6.00-6.40Å, may significantly improve the I 2 BS binding activity and selectivity (Figure 8B).

CONCLUSIONS
PET permits the noninvasive quantification of biological and pharmacological processes, and thus, PET imaging of astrocytes is indispensable for a better understanding of their role in disease.However, PET imaging of reactive astrocytes is still in its infancy, and more sensitive and

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare no conflict of interest.

F I G U R E 1
Neurons and glial cells are fundamental cells of the human brain and the subcellular localization of imidazoline-2 binding sites (I 2 BS) in reactive astrocytes.The glial cells include astrocytes, microglial cells, and oligodendrocytes.I 2 BS is located on astrocyte mitochondrial membranes.
C]2 only exhibited moderate specific binding in the living brain and was not pursued for further human PET studies.Subsequently, two analogs ([ 11 C]metrazoline and [ 11 C]TEIMD, [ 11 C]3 and [ 11 C]4, respectively, Figure 4) of [ 11 C]2 were developed, in which the benzene ring and imidazoline ring were spaced by ethylene ([ 11 C]3) or ethyl linkage ([ 11 C]4

F
I G U R E 5 (A) Radiosynthesis [ 11 C]5.(B) Typical brain positron emission tomography (PET) images of [ 11 C]5 in healthy volunteers under baseline conditions and dose-dependent blockade by idazoxan.(Reproduced with permission.

[ 11 C
]3 and [ 11 C]4 with higher clogP values (Table K i (I 1 ) >10,000 nM K i (α 2 ) = 537.0nM [2c] F I G U R E 8 (A) Pharmacophoric elements of idazoxan-based imidazoline-2 binding sites (I 2 BS) ligands, template molecule: compound 1. [2c] specific biomarkers are required.Currently, two PET ligands [ 11 C]L-deprenyl-D 2 and [ 11 C]BU99008 ([ 11 C]5)have been used for neuroimaging of astrocytes, which target MAO-B and I 2 BS, respectively.Since MAO-B is also expressed in serotonergic neurons, I 2 BS is a more promising biomarker because of its higher specificity for astrocytes.However, studies are required to clarify the biology of I 2 BS, and extensively, its relevance in disease states.In addition, tracers with improved reproducibility of V T measurements and faster kinetics are needed in PET imaging of I 2 BS, and thus the development of ligands with new structural framework is needed.In all, this review summarized the reported I 2 BS PET ligands to date, as well as the recent I 2 BS drug discovery, which would pave the way for the development of more potent PET ligands and further stimulate drug discovery in the field of I 2 BS.A U T H O R C O N T R I B U T I O N SW.L., Y.Q., J.C., and Y.L. contributed to writing the original draft; X.D. contributed to the investigation; S.H.L. and H.F. contributed to conceptualization and investigation; W.L., S.H.L., and H.F. contributed to data accuracy; S.H.L. and H.F. contributed to the manuscript's supervision, review, and editing.A C K N O W L E D G M E N T SWe thank the Beijing Municipal Natural Science Foundation (No. 7224366) and National Natural Science Foundation of China (No. 22306014) to Hualong Fu. 3 3 H]2-BFI is preferable for in vitro ARG studies of I 2 BS, and the results in rat brain sections showed that I 2 BS had high expression levels in the anterior olfactory nucleus, hypothalamic regions, and midbrain.As measured by [ 3 H]2-BFI, the B max (the maximum concentration of target binding sites) values were 102.15, 112.09, 77.71, and 88.75 fmol/mg tissue in brain regions of the surface of the dorsal third ventricle, arcuate hypothalamic nucleus, optic chiasm/supraoptic decuss, and interpeduncular nucleus, respectively, which was confirmed by the specific binding of [ 3 H]idazoxan in the same regions, yet in the presence of rauwolscine.