Recent Developments in PET and SPECT Radiotracers as Radiopharmaceuticals for Hypoxia Tumors

Hypoxia, a deficiency in the levels of oxygen, is a common feature of most solid tumors and induces many characteristics of cancer. Hypoxia is associated with metastases and strong resistance to radio- and chemotherapy, and can decrease the accuracy of cancer prognosis. Non-invasive imaging methods such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) using hypoxia-targeting radiopharmaceuticals have been used for the detection and therapy of tumor hypoxia. Nitroimidazoles are bioreducible moieties that can be selectively reduced under hypoxic conditions covalently bind to intracellular macromolecules, and are trapped within hypoxic cells and tissues. Recently, there has been a strong motivation to develop PET and SPECT radiotracers as radiopharmaceuticals containing nitroimidazole moieties for the visualization and treatment of hypoxic tumors. In this review, we summarize the development of some novel PET and SPECT radiotracers as radiopharmaceuticals containing nitroimidazoles, as well as their physicochemical properties, in vitro cellular uptake values, in vivo biodistribution, and PET/SPECT imaging results.


Introduction
Hypoxia is defined as a state in which supplies of oxygen (O 2 ) to tissue are insufficient for biological functions [1,2]. The unceasing growth of cancer cells leads to abnormalities in the structure and function of tumor vessels which decrease supplies to the tumor, especially the tumor interior. The proliferation of cancer cells also increases the glucose metabolism and oxygen consumption of the tumor. The net effect of inadequate supplies and the high consumption of O 2 causes hypoxia in the tumor [2,3].
Hypoxia is a common feature of most solid tumors. Oncological hypoxia often has oxygen levels below 1000 ppm [4]. Many studies have demonstrated that hypoxia promotes the malignant progression of uteri cervix cancer, prostate cancer, glioblastoma, gastric cancer, and so on [5][6][7][8][9]. Hypoxia-inducible factors (HIFs) are transcription factors that mediate the cellular response to hypoxia via the transcription of various hypoxia-inducible genes. The overexpression of HIFs is usually found in solid tumors. Aside from HIF3, which has an unclear role, HIF1 and HIF2 regulate many tumor survival and growth factors [10]. Via the mechanism involving HIFs, many hallmarks of cancer can be induced by hypoxia; therefore, tumors can adapt, overcome the lack of O 2 , invade, and metastasize [11][12][13][14][15]. Several metastases are associated with hypoxia, for instance, soft tissue sarcoma, breast cancer, gynecological cancer, and pancreatic cancer [16][17][18][19][20][21]. For many years, hypoxia has been directly or indirectly related to therapeutic resistance of radio and chemotherapy [6,[22][23][24]; thus, it can adversely affect the prognosis of cancer [9,25]. Many studies have also reported correlations between hypoxia and poor outcomes and patient mortality [5,26,27]. Owing to (0.635 MeV). Importantly, the 18 F radionuclide has a half-life of 110 min which enables more complicated radiosynthesis, longer in vivo studies, and the distribution to "satellite" PET centers lacking adequate radiochemistry facilities [36]. For instance, [ 18 F]FDG, a radiopharmaceutical analog of glucose first used for PET imaging in 1979, has become the most common radiopharmaceutical for clinical studies of cancer [109][110][111]. SPECT radiopharmaceuticals have been radiolabeled with gamma ray emitters such as 99m Tc (half-life = 6 h), 123 I (half-life = 13.3 h), and 201 Tl (half-life = 73 h) [56]. SPECT radionuclides emit lower-energy photons thus causing lower radiation exposure compared to positron-emitting nuclides; in addition, photons are more scattered and easily absorbed, resulting in a lower photon count, lower sensitivity, and higher image noise in comparison to PET [112].
Nitroimidazole has been an important pharmacophore for the development of radiopharmaceuticals for the detection of hypoxia [113,114]. Nitroimidazole is a class of compounds that includes 2-nitroimidazole, 4-nitroimidazole, 5-nitroimidazole, and metronidazole (or 2-methyl-5-nitroimidazole) ( Figure 1). These moieties are considered bioreducible and their mechanism of retention depends on the O2 levels within tissues. When entering a viable cell via diffusion, nitroimidazoles are selectively reduced by reductases into potentially reactive nitro radical anions in a process called the activation reaction. In normal oxygen levels (or under normoxic conditions), this activation reaction is reversible and the nitro radical anions can be immediately reoxidized into the parent nonradical compounds. However, the reaction does not occur in hypoxic conditions. The rate of a reoxidizing reaction depends on the oxygen concentration [114]. Therefore, with the low intracellular oxygen concentration of hypoxic conditions, the reduction of O2 and the reoxidization of nitro radical anions cannot compete with the generation of nitro radical anions ( Figure 2). Under hypoxia, further reductions of nitro radical anions take place to eventually form reactive species which covalently bind to cellular proteins or DNAs and are trapped within hypoxic cells ( Figure 2) [35,[113][114][115]. Therefore, hypoxic tissues can be differentiated from normoxic tissues. In normal oxygen levels (or under normoxic conditions), this activation reaction is reversible and the nitro radical anions can be immediately reoxidized into the parent nonradical compounds. However, the reaction does not occur in hypoxic conditions. The rate of a reoxidizing reaction depends on the oxygen concentration [114]. Therefore, with the low intracellular oxygen concentration of hypoxic conditions, the reduction of O 2 and the reoxidization of nitro radical anions cannot compete with the generation of nitro radical anions ( Figure 2). Under hypoxia, further reductions of nitro radical anions take place to eventually form reactive species which covalently bind to cellular proteins or DNAs and are trapped within hypoxic cells ( Figure 2) [35,[113][114][115]. Therefore, hypoxic tissues can be differentiated from normoxic tissues. (0.635 MeV). Importantly, the 18 F radionuclide has a half-life of 110 min which enables more complicated radiosynthesis, longer in vivo studies, and the distribution to "satellite" PET centers lacking adequate radiochemistry facilities [36]. For instance, [ 18 F]FDG, a radiopharmaceutical analog of glucose first used for PET imaging in 1979, has become the most common radiopharmaceutical for clinical studies of cancer [109][110][111]. SPECT radiopharmaceuticals have been radiolabeled with gamma ray emitters such as 99m Tc (half-life = 6 h), 123 I (half-life = 13.3 h), and 201 Tl (half-life = 73 h) [56]. SPECT radionuclides emit lower-energy photons thus causing lower radiation exposure compared to positron-emitting nuclides; in addition, photons are more scattered and easily absorbed, resulting in a lower photon count, lower sensitivity, and higher image noise in comparison to PET [112]. Nitroimidazole has been an important pharmacophore for the development of radiopharmaceuticals for the detection of hypoxia [113,114]. Nitroimidazole is a class of compounds that includes 2-nitroimidazole, 4-nitroimidazole, 5-nitroimidazole, and metronidazole (or 2-methyl-5-nitroimidazole) ( Figure 1). These moieties are considered bioreducible and their mechanism of retention depends on the O2 levels within tissues. When entering a viable cell via diffusion, nitroimidazoles are selectively reduced by reductases into potentially reactive nitro radical anions in a process called the activation reaction. In normal oxygen levels (or under normoxic conditions), this activation reaction is reversible and the nitro radical anions can be immediately reoxidized into the parent nonradical compounds. However, the reaction does not occur in hypoxic conditions. The rate of a reoxidizing reaction depends on the oxygen concentration [114]. Therefore, with the low intracellular oxygen concentration of hypoxic conditions, the reduction of O2 and the reoxidization of nitro radical anions cannot compete with the generation of nitro radical anions ( Figure 2). Under hypoxia, further reductions of nitro radical anions take place to eventually form reactive species which covalently bind to cellular proteins or DNAs and are trapped within hypoxic cells ( Figure 2) [35,[113][114][115]. Therefore, hypoxic tissues can be differentiated from normoxic tissues. Compounds bearing a nitroimidazole moiety have been widely used to detect hypoxia events in bodies. In particular, radiolabeled nitroimidazole compounds have been used for PET or SPECT imaging studies for hypoxia because the accumulation of these radiolabeled compounds in specific locations can be visualized by PET or SPECT. Thus, considerable efforts have been made to develop radiolabeled compounds (radiopharmaceuticals) containing nitroimidazole moieties for preclinical and clinical studies. Several imaging agents such as [ 18 F]FMISO [116][117][118], [ 18 F]FAZA [119], [ 18 F]EF5 [120,121], and [ 125 I]IAZA [122] (Figure 3) have been widely developed to study hypoxia; however, these radiotracers still have several drawbacks. An ideal hypoxia radiotracer should exhibit several physicochemical and biological properties. For example, radiotracers for hypoxia should have a high selectivity toward hypoxia with a low retention in normal tissues and a high retention in tumor sites. They should be non-toxic, easy to prepare, convenient [35], and have probable lipophilicity. Aside from hypoxic tissues, the degradation of radiotracers in normal tissues should only generate non-specific metabolites which cannot be trapped in these tissues [35]. Moreover, the trade-off between the absolute tumor uptake signal and the relative tumor/background ratio is also a concern [29]. Thus, various novel radiotracers have been developed to improve the effectiveness of existing radiopharmaceuticals, particularly for pharmacokinetics and biodistribution. Compounds bearing a nitroimidazole moiety have been widely used to detect hypoxia events in bodies. In particular, radiolabeled nitroimidazole compounds have been used for PET or SPECT imaging studies for hypoxia because the accumulation of these radiolabeled compounds in specific locations can be visualized by PET or SPECT. Thus, considerable efforts have been made to develop radiolabeled compounds (radiopharmaceuticals) containing nitroimidazole moieties for preclinical and clinical studies. Several imaging agents such as [ 18 F]FMISO [116][117][118], [ 18 F]FAZA [119], [ 18 F]EF5 [120,121], and [ 125 I]IAZA [122] (Figure 3) have been widely developed to study hypoxia; however, these radiotracers still have several drawbacks. An ideal hypoxia radiotracer should exhibit several physicochemical and biological properties. For example, radiotracers for hypoxia should have a high selectivity toward hypoxia with a low retention in normal tissues and a high retention in tumor sites. They should be non-toxic, easy to prepare, convenient [35], and have probable lipophilicity. Aside from hypoxic tissues, the degradation of radiotracers in normal tissues should only generate non-specific metabolites which cannot be trapped in these tissues [35]. Moreover, the trade-off between the absolute tumor uptake signal and the relative tumor/background ratio is also a concern [29]. Thus, various novel radiotracers have been developed to improve the effectiveness of existing radiopharmaceuticals, particularly for pharmacokinetics and biodistribution. In this review, the development of radiopharmaceuticals radiolabeled with various radioisotopes for PET/SPECT studies of tumor hypoxia between 2014 and the beginning of 2023 have been summarized. In particular, we describe novel radiopharmaceuticals, their physicochemical properties, in vitro biological results, in vivo biodistribution, and PET/SPECT imaging results. 18 F is a positron-emitting radioisotope with a half-life of 110 min. Up to now, 18 F is still the most widely used radioisotope for the preparation of hypoxia-targeting radiopharmaceuticals due to its proper half-life allowing for the extension of PET scans and distribution to distant facilities, as well as low positron energy (0.635 MeV), high electron intensity and high resolution [123,124]. Moreover, 18 F is small in size and chemically inert, allowing it to easily incorporate into the structures of radiotracers without greatly affecting the physicochemical and biological properties [125,126]. However, the production of 18 F requires a cyclotron which is high-cost and takes up a large space [127]. Since the development of [ 18 F]FMISO, the first radiotracer for imaging of hypoxia, various 18 F-labeled analogues of 2-nitroimidazole for hypoxia have been extensively studied both preclinically and clinically [128,129]. In this review, the development of radiopharmaceuticals radiolabeled with various radioisotopes for PET/SPECT studies of tumor hypoxia between 2014 and the beginning of 2023 have been summarized. In particular, we describe novel radiopharmaceuticals, their physicochemical properties, in vitro biological results, in vivo biodistribution, and PET/SPECT imaging results. 18 F is a positron-emitting radioisotope with a half-life of 110 min. Up to now, 18 F is still the most widely used radioisotope for the preparation of hypoxia-targeting radiopharmaceuticals due to its proper half-life allowing for the extension of PET scans and distribution to distant facilities, as well as low positron energy (0.635 MeV), high electron intensity and high resolution [123,124]. Moreover, 18 F is small in size and chemically inert, allowing it to easily incorporate into the structures of radiotracers without greatly affecting the physicochemical and biological properties [125,126]. However, the production of 18 F requires a cyclotron which is high-cost and takes up a large space [127]. Since the development of [ 18 F]FMISO, the first radiotracer for imaging of hypoxia, various 18 F-labeled analogues of 2-nitroimidazole for hypoxia have been extensively studied both preclinically and clinically [128,129] ing click reactions [130]. Ten PEG-modified compounds showed good stability in saline and human serum. The ten compounds were hydrophilic and had logp values (ranging from −1.25 ± 0.01 to −0.16 ± 0.01) lower than zero and lower than that of [ 18 Figure 4) by using click reactions [130]. Ten PEG-modified compounds showed good stability in saline and human serum. The ten compounds were hydrophilic and had logp values (ranging from −1.25 ± 0.01 to −0.16 ± 0.01) lower than zero and lower than that of [ 18 F]FMISO (0.38 ± 0.08). In vivo biodistribution studies of ten PEG-modified compounds in BALB/c mice bearing EMT-6 tumors suggested that [ 18   Lin and co-workers developed 18 F-labeled zwitterion-based ammoniomethyl-trifluoroborate bearing 2-nitroimidazole ( 18 F-AmBF3-Bu-2NI, [ 18 F]4) for imaging tumor hypoxia ( Figure 5) [131]. [ 18 F]4 had a logp value of −1.52 ± 0.02 and remained intact in mouse plasma for 1 h. At 1 h p.i., the in vivo biodistribution of [ 18 F]4 in mice with HT-29 tumors demonstrated minimal uptake in the tumor (0.54 ± 0.13%ID/g), leading to low ratios of tumor/muscle and tumor/blood ratios (0.51 ± 0.25 and 0.99 ± 0.32, respectively). At 3 h p.i., the tumor uptake was reduced to 0.19 ± 0.04%ID/g, whereas tumor/muscle and tumor/blood ratios increased (0.92 ± 0.08 and 2.62 ± 1.02, respectively). However, [ 18    Lin and co-workers developed 18 F-labeled zwitterion-based ammoniomethyl-trifluoroborate bearing 2-nitroimidazole ( 18 F-AmBF 3 -Bu-2NI, [ 18 F]4) for imaging tumor hypoxia ( Figure 5) [131]. [ 18 F]4 had a logp value of −1.52 ± 0.02 and remained intact in mouse plasma for 1 h. At 1 h p.i., the in vivo biodistribution of [ 18 F]4 in mice with HT-29 tumors demonstrated minimal uptake in the tumor (0.54 ± 0.13%ID/g), leading to low ratios of tumor/muscle and tumor/blood ratios (0.51 ± 0.25 and 0.99 ± 0.32, respectively). At 3 h p.i., the tumor uptake was reduced to 0.19 ± 0.04%ID/g, whereas tumor/muscle and tumor/blood ratios increased (0.92 ± 0.08 and 2.62 ± 1.02, respectively). However, [ 18   mice with demonstrated minimal uptake in the tumor (0.54 ± 0.13%ID/g), leading t tumor/muscle and tumor/blood ratios (0.51 ± 0.25 and 0.99 ± 0.32, respectiv the tumor uptake was reduced to 0.19 ± 0.04%ID/g, whereas tumor/m mor/blood ratios increased (0.92 ± 0.08 and 2.62 ± 1.02, respectively). How hibited lower tumor uptake and tumor/muscle and tumor/blood ratios [ 18 F]FMISO (tumor uptake = 1.84 ± 0.52%ID/g, T/M = 4.52 ± 1.36, T/B = 5.05 study of [ 18 F]4 in mice bearing HT-29 tumors also showed observable tu The fast clearance of [ 18 F]4 in normal tissues and organs was consistent wi bution results. An in vitro cellular uptake study using HT-29 cells sugge was not capable of binding or diffusing across the cell membrane; thus, it c nitroreductase inside hypoxic cancer cells.  In 2021, Sun and co-workers prepared nitroimidazole derivative 2-[4-(carboxymethyl)-7-[2-(2-(2-nitro-1H-imidazol-1-yl)acetamido)ethyl]-1,4,7-triazanonan-1-yl]acetic acid (NOTA-NI) as a precursor and radiolabeled it with Al 18 F to produce radiotracer Al 18 F-NOTA-NI In 2021, Sun and co-workers prepared nitroimidazole derivative 2-[4-(carboxymethyl)-7-[2-(2-(2-nitro-1H-imidazol-1-yl)acetamido)ethyl]-1,4,7-triazanonan-1-yl]acetic acid (NOTA-NI) as a precursor and radiolabeled it with Al 18 F to produce radiotracer Al 18 F-NOTA-NI ([ 18 F]6) ( Figure 6) [132]. In an in vitro stability test, [   In 2021, Wu and co-workers reported 18   In 2021, Wu and co-workers reported 18     In 2022, Bernardes and co-workers developed an 18 (Figure 9) [135]. β-[ 18 F]16 was prepared in good radiochemical purity (>98%) via the radiofluorination of a precursor which was synthesized from 1,2:5,6-di-O-isopropylidene-α-D-allofuranose and had a calculated ClogP value of −1.472. The binding of β-16 to the nucleoside transporters SLC28A1, SLC28A3, and SLC29A1 was verified with half-maximum inhibitory concentration (IC50) values, which were found to be 630 ± 343, 770 ± 74, and 840 ± 22 µM, respectively. The uptake of β-[ 18 F]16 in the EMT6 and NCI-H1975 cell lines, cultured in vitro, showed a noticeable increase in hypoxic conditions compared to normoxic conditions, suggesting that β-[ 18 F]16 exhibits selectivity for hypoxic environments. At 5 h, total radioactivity uptake of β-[ 18 F]16 in EMT6 (4.98 ± 0.83) was higher than that in NCI-H1975 cells (3.73 ± 0.41) with increasing hypoxic/normoxic ratios during the study period. By using nucleoside transporter inhibitors, Kuntner and co-workers found that the cellular uptake of β-[ 18 F]16 in hypoxic tumor cells was determined by the activity of the SLC9A1 transporter. In a PET imaging study of β-[ 18 F]16 using mice bearing EMT6 tumor, at 120 min post injection (p.i.), tumor/muscle and tumor/blood ratios obtained using the isoflurane/air breathing protocol (2.13 ± 0.22 and 2.79 ± 0.33, respectively) were significantly higher than ratios obtained using the isoflurane/oxygen breathing protocol (1.22 ± 0.13  (Figure 9) [135]. β-[ 18 F]16 was prepared in good radiochemical purity (>98%) via the radiofluorination of a precursor which was synthesized from 1,2:5,6-di-O-isopropylidene-α-D-allofuranose and had a calculated ClogP value of −1.472. The binding of β-16 to the nucleoside transporters SLC28A1, SLC28A3, and SLC29A1 was verified with half-maximum inhibitory concentration (IC 50 ) values, which were found to be 630 ± 343, 770 ± 74, and 840 ± 22 µM, respectively. The uptake of β-[ 18 F]16 in the EMT6 and NCI-H1975 cell lines, cultured in vitro, showed a noticeable increase in hypoxic conditions compared to normoxic conditions, suggesting that β-[ 18 F]16 exhibits selectivity for hypoxic environments. At 5 h, total radioactivity uptake of β-[ 18 F]16 in EMT6 (4.98 ± 0.83) was higher than that in NCI-H1975 cells (3.73 ± 0.41) with increasing hypoxic/normoxic ratios during the study period. By using nucleoside transporter inhibitors, Kuntner and co-workers found that the cellular uptake of β-[ 18 F]16 in hypoxic tumor cells was determined by the activity of the SLC9A1 transporter. In a PET imaging study of β-[ 18 F]16 using mice bearing EMT6 tumor, at 120 min post injection (p.i.), tumor/muscle and tumor/blood ratios obtained using the isoflurane/air breathing protocol (2.13 ± 0.22 and 2.79 ± 0.33, respectively) were significantly higher than ratios obtained using the isoflurane/oxygen breathing protocol (1.22 ± 0. 13    cellular uptake through the nucleoside transporter ( Figure 10) [136]. The β-[ 18 17 in mice bearing CT26 colon carcinoma only showed a high uptake in the intestine (indicating rapid clearance) yet a low uptake in other organs and tissues such as blood, liver, kidneys, muscle, and CT26 colon carcinomas. Reischl and co-workers developed [ 18 F]fluoro-azomycin-2′-deoxy-β-D-ribofuranoside ([ 18 F]FAZDR, β-[ 18 F]17) as radiotracers to mimic nucleoside structure and to improve cellular uptake through the nucleoside transporter ( Figure 10) [136]. The β-[ 18 17 in mice bearing CT26 colon carcinoma only showed a high uptake in the intestine (indicating rapid clearance) yet a low uptake in other organs and tissues such as blood, liver, kidneys, muscle, and CT26 colon carcinomas. In 2019, Reischl and co-workers described the synthesis of β-2-nitroimidazole-arabinose (β-FAZA) and α-2-nitroimidazole-deoxyribose (α-FAZDR, α-[ 18 F]22) ( Figure 11) [137]. Four compounds β-FAZA, FAZA, α-FAZDR and β-FAZDR [131] (Figure 11) were radiofluorinated with 18 F to study the effect of configuration of 2-nitroimidazole pharmacophore and sugar moieties on the detection of hypoxia in tumors. In vitro cellular uptakes of the four compounds showed a good interaction of β-FAZDR with nucleoside transporters SLC28A3 and SLC28A1, and good interaction of FAZA with nucleoside transporter SLC28A1 whereas α-FAZDR was unable to interact with any transporter and β-FAZA could only inhibit transporters at high concentrations. In vivo PET imaging studies of α-[ 18   In 2019, Reischl and co-workers described the synthesis of β-2-nitroimidazole-arabinose (β-FAZA) and α-2-nitroimidazole-deoxyribose (α-FAZDR, α-[ 18 F]22) ( Figure 11) [137]. Four compounds β-FAZA, FAZA, α-FAZDR and β-FAZDR [131] (Figure 11) were radiofluorinated with 18 F to study the effect of configuration of 2-nitroimidazole pharmacophore and sugar moieties on the detection of hypoxia in tumors. In vitro cellular uptakes of the four compounds showed a good interaction of β-FAZDR with nucleoside transporters SLC28A3 and SLC28A1, and good interaction of FAZA with nucleoside transporter SLC28A1 whereas α-FAZDR was unable to interact with any transporter and β-FAZA could only inhibit transporters at high concentrations. In vivo PET imaging studies of α-[ 18     F]26 due to higher hydrophilicity. In addition, the obtained PET images were much clearer than those of [ 18 F]FDG. The biodistribution results of the two radiotracers were consistent with the PET imaging results with high tumor/muscle and tumor/blood ratios. Moreover, co-injection of the two radiotracers with 5% glucose did not significantly change their tumor uptake values, suggesting that they did not target tumors via the glucose metabolism pathway as [ 18 F]FDG. Additionally, hypoxia regions in OS732 and S180 tumors were confirmed by HIF-1α and HE staining. Two aminooxy derivatives of 2-nitroimidazole were synthesized and radiofluorinated by Chu and co-workers in 2019 to afford radiotracers [ 18  F]26 due to higher hydrophilicity. In addition, the obtained PET images were much clearer than those of [ 18 F]FDG. The biodistribution results of the two radiotracers were consistent with the PET imaging results with high tumor/muscle and tumor/blood ratios. Moreover, co-injection of the two radiotracers with 5% glucose did not significantly change their tumor uptake values, suggesting that they did not target tumors via the glucose metabolism pathway as [ 18 F]FDG. Additionally, hypoxia regions in OS732 and S180 tumors were confirmed by HIF-1α and HE staining.

99m Tc Radiotracers for Hypoxia
99m Tc is a radionuclide-emitting gamma radiation widely used for SPECT imaging. 99m Tc possesses a favorable half-life of 6 h and low photon energy of 140 keV. In terms of convenience, compared to 18 F, 99m Tc can be obtained on-site as a pertechnetate ( 99m TcO 4 − ) by using commercial 99 Mo/ 99m Tc generators which are smaller and more affordable than cyclotrons [139]. The preparation of 99m Tc-labeled complexes through coordination reactions is often conducted smoothly with high yields. However, the obtained radiolabeled products might exhibit physical and biological properties distinctly different from their precursors due to chelates. Moreover, degradation and transchelation of the 99m Tc-labeled complexes should be noticed because these factors might affect the stability and radiopharmaceutical applications of the complexes [140].

99m Tc Radiotracers with Mono-Nitroimidazole for Hypoxia
In 2014, Rey and co-workers synthesized ligand L (2-amine-3-[2-(2-methyl-5-nitro-1Himidazol-1-yl)ethylthio] propanoic acid) bearing a metronidazole moiety and radiolabeled it with 99m Tc(CO) 3 for detecting hypoxia in tumors ( Figure 13) [141]. In vitro stability studies showed that [ 99m Tc(CO) 3 (L)] ([ 99m Tc]28) remained stable in the labelling milieu and in human plasma for 4 h. Introducing a cysteine unit to the 99m Tc complex increased its hydrophilicity (logp = −0.75 ± 0.08) and decreased protein binding, which made the pharmacokinetics of the complex more appropriate for an imaging tracer. The 99m Tc complex with an L ligand was selectively uptaken by human colon adenocarcinoma cells HCT-15 in hypoxic conditions rather than in normoxic conditions (hypoxic/normoxic ratio = 1.6 ± 0.4), indicating selectivity toward hypoxia. The in vivo biodistribution in C57BL/6 mice bearing 3LL lung carcinoma cells demonstrated good tumor uptake of [ 99m Tc]28 at 0.5 h p.i. (1.3 ± 0.4%ID/g); however, at 1 h p.i., the cellular radioactivity of [ 99m Tc]28 was decreased by half (0.5 ± 0.1%ID/g). In general, there was a low uptake and insufficient retention of [ 99m Tc]28 in all tissues and organs; nonetheless, the 99m Tc-labeled complex was rapidly eliminated from muscle, which resulted in a favorable tumor/muscle ratio of 2.0 ± 0.1%ID/g at 4 h p.i.
precursor produced the corresponding 99m Tc(CO)3 in >94% RCY, >94% radiochemical purity, and >104.8 µCi/µmol specific activity. Among nine 99m Tc complexes, three IDA-99m Tc(CO)3 complexes (logp ranging from 0.39 to 0.48) were more lipophilic than DETA-99m Tc(CO)3 (logP ranging from 0.15 to 0.28) and AEG-99m Tc(CO)3 complexes (logp ranging from −0.53 to 0.06). In vivo biodistributions of nine complexes and [ 18 F]FMISO in Swiss mice bearing HSDM1C1 murine fibrosarcoma showed that tumor uptake and tumor/blood ratio of [ 18 F]FMISO was still higher than in the nine 99m Tc complexes bearing nitroimidazoles. Noticeably, the lipophilicity of the ligands significantly affected the blood activity of the complexes. In particular, nitroimidazole-IDA-99m Tc(CO)3 complexes exhibited slow clearance from blood; thus, the tumor/blood ratios were low (from 0.18 to 0.61 at 3 h p.i.). In contrast, tumor/blood ratios of nitroimidazole-DETA-99m Tc(CO)3 complexes (from 0.84 to 1.51 at 3 h p.i.) and nitroimidazole-AEG-99m Tc(CO)3 complexes (from 1.06 to 1.78 at 3 h p.i.) were improved due to rapid blood clearance. Compared to [ 18 F]FMISO (T/B = 3.85 ± 0.23 at 3 h p.i.), the nine 99m Tc(CO)3-labeled complexes exhibited lower tumor/blood ratios.  In 2015, Chu and Sun reported the in vivo strain-promoted cyclooctyne-azide cycloaddition (SPAAC, click reaction) between 2-nitroimidazole-azide (2NIM-Az) as the hypoxia targeting agent and 99m Tc-azadibenzocyclooctyne-MAMA ( 99m Tc-AM, [ 99m Tc]37) for radiolabeling 2NIM-Az in vivo ( Figure 16) [144]. In control experiments, [ 99m Tc]37 was used as a blank control and 99m Tc-triazole-2NIM ([ 99m Tc]38) was used as a conventional control for hypoxia imaging. Biodistribution studies of a pretargeting method in male Kunming mice bearing S180 tumors with 2, 5, and 12 h intervals between injections of 2NIM-Az and [ 99m Tc]37 ( Figure 17) showed that pretargeting with a 5 h injection interval exhibited the highest tumor/muscle ratio (8.55 ± 0.57 at 8 h p.i.) as well as improved tumor uptake (0.70 ± 0.09%ID/g at 8 h p.i.) and tumor/blood ratio (1.44 ± 0.06 at 8 h p.i.). This indicated that a longer circulation time resulted in higher tumor uptake and tumor retention. Pretargeting methods gave similar tumor uptakes and tumor/blood ratio as [ 99m Tc]38. Most importantly, hypoxia pretargeting exhibited a much higher tumor/muscle ratio in comparison to [ 99m Tc]38 (0.72 ± 0.10 at 8 h p.i.), indicating that the in vivo reaction of pretargeting azide groups and 99m Tc-labeled complex could detect hypoxic tumors more effectively than a conventional imaging agent. Lower tumor uptake of [ 99m Tc]38 was due to the deactivation of 2-nitroimidazole to target hypoxia by the radiolabeled complex.      (logp = 0.38) was more lipophilic. The biodistribution of the two complexes in Swiss mice bearing fibrosarcoma tumors suggested that tumor uptake of [ 99m Tc]39 (0.70 ± 0.16%ID/g at 3 h p.i.) was significantly higher than that of the 2-NI-DETA-99m Tc(CO) 3 complex (0.24 ± 0.06%ID/g at 3 h p.i.). After 1 h p.i., the [ 99m Tc]39 complex showed slow clearance from the tumor and rapid clearance from muscles, indicating selective retention in tumors due to the reduction of the complex and non-specific binding. As a consequence of higher lipophilicity, the [ 99m Tc]39 complex exhibited slower clearance from normal tissues, tumors, and blood compared to the 2-NI-DETA-99m Tc(CO) 3   In 2016, Banerjee and co-workers prepared 99m Tc(CO)3-labeled triazole derivatives of 2-, 4-, and 5-nitroimidazole ([ 99m Tc]40a-c) for the detection of tumor hypoxia ( Figure 19) [146]. The biodistribution of three 99m Tc(CO)3 complexes and [ 18 F]FMISO in Swiss mice bearing fibrosarcoma tumors showed that tumor uptake values of the 99m Tc complexes depended on single electron reduction potential (SERP) values (indicating the ability to reduce nitroimidazole in hypoxic regions) and their lipophilicities. Among the complexes, the [ 99m Tc]40b exhibited the lowest tumor uptakes at the time points studied (30 min, 1 h, and 3 h), which was consistent with the lowest SERP value (−527 mV relative to standard hydrogen electrode (SHE)) of 4-nitroimidazole and the most rapid blood clearance because of the lowest lipophilicity (logp = −0. 52  Tc]40c (2.03 ± 0.32%ID/g). However, the results indicated that, aside from 2-nitroimidazole, 5-nitroimidazole was also a potential moiety in radiotracers for hypoxia imaging. In 2016, Banerjee and co-workers prepared 99m Tc(CO) 3 -labeled triazole derivatives of 2-, 4-, and 5-nitroimidazole ([ 99m Tc]40a-c) for the detection of tumor hypoxia ( Figure 19) [146]. The biodistribution of three 99m Tc(CO) 3 complexes and [ 18 F]FMISO in Swiss mice bearing fibrosarcoma tumors showed that tumor uptake values of the 99m Tc complexes depended on single electron reduction potential (SERP) values (indicating the ability to reduce nitroimidazole in hypoxic regions) and their lipophilicities. Among the complexes, the [ 99m Tc]40b exhibited the lowest tumor uptakes at the time points studied (30 min, 1 h, and 3 h), which was consistent with the lowest SERP value (−527 mV relative to standard hydrogen electrode (SHE)) of 4-nitroimidazole and the most rapid blood clearance because of the lowest lipophilicity (logp = −0. 52  The unexpectedly high tumor uptake of [ 99m Tc]40c was explained by the highest lipophilicity among the three complexes (logp = −0.03 ± 0.01), which led to the slowest blood clearance and longest retention in tumor cells. Due to the slow blood clearance, [ 18 F]FMISO still had a higher tumor uptake value at 30 min p.i. (4.65 ± 0.86%ID/g) than [ 99m Tc]40c (2.03 ± 0.32%ID/g). However, the results indicated that, aside from 2-nitroimidazole, 5-nitroimidazole was also a potential moiety in radiotracers for hypoxia imaging.
the [ 99m Tc]40b exhibited the lowest tumor uptakes at the time points studied (30 min, 1 h, and 3 h), which was consistent with the lowest SERP value (−527 mV relative to standard hydrogen electrode (SHE)) of 4-nitroimidazole and the most rapid blood clearance because of the lowest lipophilicity (logp = −0.52 ± 0.01). Even though [ 99m Tc]40a had the highest SERP value (−418 mV relative to SHE), the [ 99m Tc]40c exhibited the highest tumor uptake values at 30 min p.i. (2.03 ± 0.32%ID/g) and at 1 h p.i. (1.45 ± 0.08%ID/g). At 3 h p.i., [ 99m Tc]40c exhibited a similar tumor uptake (0.81 ± 0.06 %ID/g) as [ 99m Tc]40a (0.75 ± 0.14%ID/g). The unexpectedly high tumor uptake of [ 99m Tc]40c was explained by the highest lipophilicity among the three complexes (logp = −0.03 ± 0.01), which led to the slowest blood clearance and longest retention in tumor cells. Due to the slow blood clearance, [ 18 F]FMISO still had a higher tumor uptake value at 30 min p.i. (4.65 ± 0.86%ID/g) than [ 99m Tc]40c (2.03 ± 0.32%ID/g). However, the results indicated that, aside from 2-nitroimidazole, 5-nitroimidazole was also a potential moiety in radiotracers for hypoxia imaging.  The three complexes also had low plasma protein binding, which allowed more unbound 99m Tc complexes to diffuse into tumor cells. In vitro cellular uptake studies using A549 human lung cancer cells showed that all 99m Tc complexes exhibited hypoxia selectivity. In particular, [ 99m Tc]41 showed the highest cellular uptake in both hypoxic and normoxic conditions (12.58 ± 0.73 and 40.87 ± 4.74, respectively); however, its hypoxic/normoxic ratio (3.25 ± 0.08) was still lower than that of [ 99m Tc]43 (4.47 ± 0.10). In normoxic conditions, [ 99m Tc]41 had a higher cellular uptake than [ 99m Tc]42 and [ 99m Tc]43, indicating the effect of the asparagine unit on cellular uptake status. The in vivo biodistribution in BALB/c nude mice bearing A549 cells suggested that the three 99m Tc-labeled complexes were excreted from the intestine and kidneys due to high uptake in these organs.   and the corresponding nitroimidazole ligands using cyclic voltammetry indicated that the metal complex did not affect the ability of nitroimidazole ligands to be reduced by hypoxic cells. Both [ 99m Tc]44 and [ 99m Tc]45 were highly lipophilic with logp values of 0.94 ± 0.1 and 0.97 ± 0.07, respectively. Biodistribution in Swiss mice bearing fibrosarcoma tumors showed fast clearance of the 99m Tc-labeled complexes from blood and high uptakes in the liver and intestine due to high lipophilicity. The low retention of the 99m Tc-labeled complexes in blood resulted in low tumor uptakes (0. 34   In 2017, Banerjee and co-workers reported the synthesis of two 99m Tc-labeled complexes, [ 99m Tc(NS3)(2NimNC)] ([ 99m Tc]44) and [ 99m Tc(NS3)(MetNC)] ([ 99m Tc]45) bearing 4 + 1′ mixed-ligands ( Figure 21) [148].   In 2020, 99m Tc-2-MBI ([ 99m Tc]46) was reported by Zhang and co-workers ( Figure 22) [149].
[ 99m Tc]72 complex had good stability in vitro in saline and in mice serum for 24 h. The complex exhibited lipophilicity with a logp value of 1.512. According to an electrophoresis study, [ 99m Tc]46 was neutral, suggesting high compatibility with living tissues. Comparing cellular uptake values using S180 cells of [ 99m Tc]46 in hypoxic and aerobic conditions, the 99m Tc-labeled complex exhibited selectivity for hypoxia with significantly higher accumulation in hypoxic cells than in aerobic cells during the period studied (6 h). Biodistribution studies in Balb/c mice bearing S180 tumor showed that [ 99m Tc]46 exhibited higher accumulation in tumors compared to normal tissues at different time points from 30 min to 24 h. The 99m Tc-labeled complex exhibited the highest tumor uptake at 4 h p.i. (12.94 ± 2.09%ID/g); however, at 24 h p.i. tumor/muscle ratio (4.14 ± 0.77) and tumor/blood ratio (3.91 ± 0.63) were found to be the highest. Metabolic stability study of [ 99m Tc]72 in mice showed intact 99m Tc-labeled complex in blood and urine; however, the 99m Tc-complex decomposed in the liver, indicating metabolism via the hepatobiliary system and excretion via the urinary system, which was also consistent with the biodistribution results of [ 99m Tc]46. Scintigraphy imaging studies in mice bearing S180 tumors also showed a high accumulation of [ 99m Tc]46 in tumors with a tumor/normal tissue ratio of 3.39 ± 0.38, which were in accordance with the biodistribution studies of [ 99m Tc]46. The complex exhibited lipophilicity with a logp value of 1.512. According to an elect phoresis study, [ 99m Tc]46 was neutral, suggesting high compatibility with living tissu Comparing cellular uptake values using S180 cells of [ 99m Tc]46 in hypoxic and aerobic c ditions, the 99m Tc-labeled complex exhibited selectivity for hypoxia with significan higher accumulation in hypoxic cells than in aerobic cells during the period studied (6 Biodistribution studies in Balb/c mice bearing S180 tumor showed that [ 99m Tc]46 exhibi higher accumulation in tumors compared to normal tissues at different time points fr 30 min to 24 h. The 99m Tc-labeled complex exhibited the highest tumor uptake at 4 h (12.94 ± 2.09%ID/g); however, at 24 h p.i. tumor/muscle ratio (4.14 ± 0.77) and tumor/blo ratio (3.91 ± 0.63) were found to be the highest. Metabolic stability study of [ 99m Tc]72 mice showed intact 99m Tc-labeled complex in blood and urine; however, the 99m Tc-comp decomposed in the liver, indicating metabolism via the hepatobiliary system and exc tion via the urinary system, which was also consistent with the biodistribution results [ 99m Tc]46. Scintigraphy imaging studies in mice bearing S180 tumors also showed a h accumulation of [ 99m Tc]46 in tumors with a tumor/normal tissue ratio of 3.39 ± 0.38, wh were in accordance with the biodistribution studies of [ 99m Tc]46.   (50) were developed by Zhang and co-workers ( Figure 23) [150]. In vitro studies showed that the three 99m Tc-labeled complexes remained stable in saline and in mouse serum, and all were hydrophilic. In particular, [ 99m Tc]47 was more hydrophilic (logp = −3.02 ± 0.08) than [ 99m Tc]48 (logp = −0.76 ± 0.03) and [ 99m Tc]49 (logp = −1.73 ± 0.02), indicating the effect of the co-ligand on the hydrophilicity of the complex. In vitro cellular uptake studies of the three complexes using S180 cells suggested significantly higher cellular uptake values in hypoxic conditions than in aerobic conditions at all the studied time points (1, 2, and 4 h), indicating selectivity toward hypoxia. The biodistribution of the three 99m Tc-labeled complexes in Kunming mice bearing S180 tumors showed high uptake values in kidneys, suggesting excretion mainly via the urinary pathway. Among the three 99m Tc-labeled complexes, [ 99m Tc]47 exhibited the highest tumor uptake value (1.05 ± 0.27%ID/g at 2 h p.i.). Therefore, this complex was chosen for SPECT/CT imaging studies. In SPECT/CT imaging studies, at 2 h p.i., the uptake of [ 99m Tc]47 was clearly observed in S180 tumors. High uptakes of [ 99m Tc]47 in other tissues were consistent with the biodistribution results of this 99m Tc-labeled complex. In 2022, Su and Chu reported four cyclopentadienyl 99m Tc(CO)3 complexes containing 2-nitroimidazole moieties in which 2-nitroimidazoles and cyclopentadienyls were linked via carbon chains of different lengths ( Figure 24) [151]. Among the four 99m Tc-labeled complexes, [ 99m Tc]51d containing two 2-nitroimidazole groups was the most lipophilic (logp = 0.66 ± 0.02); thus, it could enter the cell more easily than the three complexes containing one 2-nitroimidazole group, and exhibited higher in vitro cellular uptake in hypoxic cells (40.0 ± 2.0%). Extending the carbon chains in the three complexes [ 99m Tc]51a-c resulted in higher lipophilicity and easier diffusion through the cell membrane, yet low cellular uptake values and low hypoxic selectivity. [ 99m Tc]51d showed the best selectivity toward hypoxia among the four complexes (hypoxic/aerobic uptake ratio =  In 2022, Su and Chu reported four cyclopentadienyl 99m Tc(CO) 3 complexes containing 2-nitroimidazole moieties in which 2-nitroimidazoles and cyclopentadienyls were linked via carbon chains of different lengths ( Figure 24) [151]. Among the four 99m Tc-labeled complexes, [ 99m Tc]51d containing two 2-nitroimidazole groups was the most lipophilic (logp = 0.66 ± 0.02); thus, it could enter the cell more easily than the three complexes containing one 2-nitroimidazole group, and exhibited higher in vitro cellular uptake in hypoxic cells (40.0 ± 2.0%). Extending the carbon chains in the three complexes [ 99m Tc]51a-c resulted in higher lipophilicity and easier diffusion through the cell membrane, yet low cellular uptake values and low hypoxic selectivity. [ 99m Tc]51d showed the best selectivity toward hypoxia among the four complexes (hypoxic/aerobic uptake ratio =
Pharmaceutics 2023, 15, x FOR PEER REVIEW 21 o Biodistribution studies in mice bearing S180 tumors showed no significant difference tumor uptake values at 4 h p.i. of the three 99m Tc-labeled complexes (from 0.71 ± 0.14 1.00 ± 0.26%ID/g) and 99m Tc-2P2 (0.86 ± 0.22%ID/g). However, the tumor/muscle ratios PEG-modified complexes (from 5.56 ± 1.10 to 7.20 ± 2.37) were significantly higher th those of 99m Tc-2P2 (T/M = 3.24 ± 0.65 at 4 h p.i.). Additionally, the three PEG-modifi complexes also exhibited improved tumor/blood ratios (from 1. 66     Biodistribution studies in mice bearing S180 tumors showed no significant difference in tumor uptake values at 4 h p.i. of the three 99m Tc-labeled complexes (from 0.71 ± 0.14 to 1.00 ± 0.26%ID/g) and 99m Tc-2P2 (0.86 ± 0.22%ID/g). However, the tumor/muscle ratios of PEG-modified complexes (from 5.56 ± 1.10 to 7.20 ± 2.37) were significantly higher than those of 99m Tc-2P2 (T/M = 3.24 ± 0.65 at 4 h p.i.). Additionally, the three PEG-modified complexes also exhibited improved tumor/blood ratios (from 1. 66    In 2020, Zhang and co-workers developed two 99m Tc(CO) 3 complexes bearing isocyanide derivative of 4-nitroimidazole (73) (Figure 34) [168] by using a synthetic pathway reported by Denny and co-workers in 1994 [169] In 2018, Zhang and co-workers developed four 99m Tc-labeled complexes containing 2-nitroimidazole isocyanide derivatives ( Figure 35) [170]. Four complexes [ 99m Tc]74a-d exhibited stability in both the labeling milieu and mouse serum and exhibited hydrophilicity. In particular, logP values increased when the number of CH2 groups increased from two groups in [ 99m Tc]74a (−2.65 ± 0.14) to five groups in [ 99m Tc]74d (−0.45 ± 0.05). In vitro cellular uptake studies of four 99m Tc-labeled complexes using S180 cells showed significantly higher cellular uptakes in hypoxic than aerobic conditions, suggesting selectivity for hypoxia. The biodistribution of four [ 99m Tc]74a-d complexes in Kunming mice bearing S180 tumors showed relatively high tumor uptakes and low muscle uptakes at 2 h p.i., resulting in high tumor/muscle ratios. Among the four 99m Tc-labeled complexes, [ 99m Tc]74c exhibited the highest tumor uptake (0.83 ± 0.14%ID/g at 2 h p.i.) and the highest tumor/muscle ratio (5.05 at 2 h p.i.). SPECT/CT images of [ 99m Tc]74c were in accordance with its biodistribution results with an observable accumulation of [ 99m Tc]74c in tumor regions (ROI ratio = 5.39 ± 0.67). The high accumulation of this complex was also observed in the liver and kidneys.  In 2018, Zhang and co-workers developed four 99m Tc-labeled complexes containing 2-nitroimidazole isocyanide derivatives ( Figure 35) [170]. Four complexes [ 99m Tc]74a-d exhibited stability in both the labeling milieu and mouse serum and exhibited hydrophilicity. In particular, logP values increased when the number of CH 2 groups increased from two groups in [ 99m Tc]74a (−2.65 ± 0.14) to five groups in [ 99m Tc]74d (−0.45 ± 0.05). In vitro cellular uptake studies of four 99m Tc-labeled complexes using S180 cells showed significantly higher cellular uptakes in hypoxic than aerobic conditions, suggesting selectivity for hypoxia. The biodistribution of four [ 99m Tc]74a-d complexes in Kunming mice bearing S180 tumors showed relatively high tumor uptakes and low muscle uptakes at 2 h p.i., resulting in high tumor/muscle ratios. Among the four 99m Tc-labeled complexes, [ 99m Tc]74c exhibited the highest tumor uptake (0.83 ± 0.14%ID/g at 2 h p.i.) and the highest tumor/muscle ratio (5.05 at 2 h p.i.). SPECT/CT images of [ 99m Tc]74c were in accordance with its biodistribution results with an observable accumulation of [ 99m Tc]74c in tumor regions (ROI ratio = 5.39 ± 0.67). The high accumulation of this complex was also observed in the liver and kidneys. In 2018, Zhang and co-workers developed four 99m Tc-labeled complexes containing 2-nitroimidazole isocyanide derivatives ( Figure 35) [170]. Four complexes [ 99m Tc]74a-d exhibited stability in both the labeling milieu and mouse serum and exhibited hydrophilicity. In particular, logP values increased when the number of CH2 groups increased from two groups in [ 99m Tc]74a (−2.65 ± 0.14) to five groups in [ 99m Tc]74d (−0.45 ± 0.05). In vitro cellular uptake studies of four 99m Tc-labeled complexes using S180 cells showed significantly higher cellular uptakes in hypoxic than aerobic conditions, suggesting selectivity for hypoxia. The biodistribution of four [ 99m Tc]74a-d complexes in Kunming mice bearing S180 tumors showed relatively high tumor uptakes and low muscle uptakes at 2 h p.i., resulting in high tumor/muscle ratios. Among the four 99m Tc-labeled complexes, [ 99m Tc]74c exhibited the highest tumor uptake (0.83 ± 0.14%ID/g at 2 h p.i.) and the highest tumor/muscle ratio (5.05 at 2 h p.i.). SPECT/CT images of [ 99m Tc]74c were in accordance with its biodistribution results with an observable accumulation of [ 99m Tc]74c in tumor regions (ROI ratio = 5.39 ± 0.67). The high accumulation of this complex was also observed in the liver and kidneys.

131 I-/ 125 I Radiotracers for Hypoxia
Similar to 18 F, radioactive iodine can also be directly incorporated into various molecules while retaining its biological properties. Radionuclide 124 I has a long half-life of 4 days which enables distant distribution as well as long-term PET imaging studies [172,173]. 131 I emits beta irradiation and is used for the preparation of SPECT imaging agents. 125 I, a SPECT radionuclide-emitting photon irradiation, has a much longer half-life than 124 I (59.5 days) [174]. This long half-life of 125 I might not be favorable for in vivo SPECT imaging due to the long exposure of the body to radiation. As a radioactive halogen like 18 F, the yields of radioiodination reactions might not always be high, limiting the application of radioactive iodine. Additionally, iodine radionuclides are produced using a cyclotron, which is another drawback.
In 2019, Chu and co-workers developed 131

131 I-/ 125 I Radiotracers for Hypoxia
Similar to 18 F, radioactive iodine can also be directly incorporated into various molecules while retaining its biological properties. Radionuclide 124 I has a long half-life of 4 days which enables distant distribution as well as long-term PET imaging studies [172,173]. 131 I emits beta irradiation and is used for the preparation of SPECT imaging agents. 125 I, a SPECT radionuclide-emitting photon irradiation, has a much longer half-life than 124 I (59.5 days) [174]. This long half-life of 125 I might not be favorable for in vivo SPECT imaging due to the long exposure of the body to radiation. As a radioactive halogen like 18 F, the yields of radioiodination reactions might not always be high, limiting the application of radioactive iodine. Additionally, iodine radionuclides are produced using a cyclotron, which is another drawback.
In 2019, Chu and co-workers developed 131  In 2020, to study how introducing a second 2-nitroimidazole group in the structure of radiotracers affected the detection of hypoxia, Chu and co-workers synthesized eight 131   In 2020, to study how introducing a second 2-nitroimidazole group in the structure of radiotracers affected the detection of hypoxia, Chu and co-workers synthesized eight 131  In vitro cellular uptakes of eight 131 I-labeled radiotracers using S180 tumor cells showed that [ 131 64 Cu is one of the copper radioisotopes used in molecular imaging and radiotherapy. With a half-life of 12.7 h, many 64 Cu-labeled radiotracers were developed for the PET imaging of many types of cancers [178]. The production of 64 Cu-labeled radiotracers is mainly carried out via the coordination of a variety of chelators to 64 Cu, which can be obtained from a cyclotron or reactor. Similar to 99m Tc, the coordination of 64 Cu might be unstable, which will affect the stability of the radiotracer in vivo. In addition, the inconvenience of using a cyclotron to generate 64 Cu is also another drawback of 64 Culabeled radiotracers.  64 Cu is one of the copper radioisotopes used in molecular imaging and radiotherapy. With a half-life of 12.7 h, many 64 Cu-labeled radiotracers were developed for the PET imaging of many types of cancers [178]. The production of 64 Cu-labeled radiotracers is mainly carried out via the coordination of a variety of chelators to 64 Cu, which can be obtained from a cyclotron or reactor. Similar to 99m Tc, the coordination of 64 Cu might be unstable, which will affect the stability of the radiotracer in vivo. In addition, the inconvenience of using a cyclotron to generate 64 Cu is also another drawback of 64 Cu-labeled radiotracers.

64 Cu Radiotracers for Hypoxia
In 2016, Yang and co-workers reported a 64

Conclusions and Perspectives
Hypoxia, a low level of oxygen, is a common feature in solid tumors. Tumor hypoxia has been considered a negative factor in the treatment of cancer due to the resistance to    64 Cu is one of the copper radioisotopes used in molecular imaging and radiothera With a half-life of 12.7 h, many 64 Cu-labeled radiotracers were developed for the PET i aging of many types of cancers [178]. The production of 64 Cu-labeled radiotracers mainly carried out via the coordination of a variety of chelators to 64 Cu, which can obtained from a cyclotron or reactor. Similar to 99m Tc, the coordination of 64 Cu might unstable, which will affect the stability of the radiotracer in vivo. In addition, the inc venience of using a cyclotron to generate 64 Cu is also another drawback of 64 Cu-labe radiotracers.

64 Cu Radiotracers for Hypoxia
In 2016, Yang and co-workers reported a 64 Cu-labeled complex containing bis(2troimidazole) ( 64 Cu-BMS2P2, [ 64 Cu]90) ( Figure 39) [179]. In vitro stability studies show that [ 64 Cu]90 remained stable (>90% radiochemical purity) in saline for 60 h. An in vi cellular uptake study of [ 64 Cu]90 using HUH-7 cancer cells showed that, at different ti points from 1 h to 4 h, and [ 64 Cu]90 exhibited significantly higher cellular uptakes in poxic conditions than in normoxic conditions. The hypoxic/normoxic ratio of [ 64 Cu]90 w highest at 3 h (2.59). In addition, the cellular uptake of [ 64 Cu]90 in hypoxic conditions w higher than that of 64 Cu-BMS181321 containing one nitroimidazole group [180], indicat that adding a second nitroimidazole group increased cellular uptake in hypoxic con tions. An in vivo PET imaging study of [ 64 Cu]90 using mice bearing A549 tumors show a clearly observed uptake of [ 64 Cu]90 in tumors, intestines, and liver. CA9 immunohis chemistry demonstrated a high expression of CA9 in tumors and the expression of C was also consistent with the results of PET imaging, indicating the hypoxic specificity [ 64 Cu]90.

Conclusions and Perspectives
Hypoxia, a low level of oxygen, is a common feature in solid tumors. Tumor hypo has been considered a negative factor in the treatment of cancer due to the resistance

Conclusions and Perspectives
Hypoxia, a low level of oxygen, is a common feature in solid tumors. Tumor hypoxia has been considered a negative factor in the treatment of cancer due to the resistance to radiotherapy and chemotherapy it causes. Therefore, an accurate assessment of the hypoxia status of tumors before aggressive cancer treatments is crucial to reduce poor outcomes and mortality.
Molecular imaging methods for the detection of tumor hypoxia have received growing attention due to their non-invasive nature, repeatability, uniformity, and ability to detect biological processes in vivo [181]. In recent years, many radiotracers for targeting hypoxia were studied in vitro on hypoxic cancer cell lines and in vivo in animals bearing hypoxic tumors.
[ 18 F]FMISO, a hypoxia marker for PET imaging, has been used to evaluate hypoxia in many clinical studies [182] and was also involved as a control for hypoxia in many studies. However, it still has several drawbacks including slow tumor uptake, low tumor/normal tissues ratios, and non-specific metabolisms producing undesired metabolites [35]. Hence, the development of novel radiotracers with better physicochemical and biological properties, and the improvement of hypoxia imaging effectiveness are necessary for successful clinical applications in the future.
In the development of novel hypoxia radiotracers, several important criteria should be met to achieve high-quality PET or SPECT images, namely, high accumulation and retention at tumor sites yet low uptakes in blood and normal tissues. Many efforts have been made to obtain the probable pharmacokinetics of hypoxia radiotracers. Recently developed 18 F-radiotracers for hypoxia can be categorized into two main groups: radiotracers with linkers and radiotracers with carbohydrate structures. For example, [ 18 F]FMISO, and [ 18 F]EF5, two commonly used radiotracers, are radiotracers with linkers. These types of tracers have a simple structure and a good hydrophilic property as well as a high uptake in the hypoxia region. Second, radiotracers with carbohydrate structures are also used in the PET study. [ 18 F]FAZA, which is another commonly used radiotracer, is a radiotracer with a carbohydrate structure. These types of tracers showed a high hydrophilic property due to hydroxy groups and a high uptake in the hypoxia region. On the other hand, 99m Tc-radiotracers can be classified based on the number of nitroimidazole moieties present in their structures, specifically, mono-nitroimidazole, di-nitroimidazole, and multi-nitroimidazole. The main structural difference of recently developed 99m Tclabeled radiotracers is the number of nitroimidazole and related linkers or chelates which affected the properties of the radiotracers. For example, the addition of more nitroimidazole can increase the accumulation of nitroimidazole-bearing radiotracers in the hypoxia region.
In recent studies, there has been a noticeable trend towards adjusting the hydrophilicity of radiotracers in order to achieve higher tumor uptake and tumor/background contrast compared to the common radiotracer [ 18 F]FMISO. Notably, in many developed radiotracers, the superior radiotracer with the highest tumor uptake or the highest tumor/normal tissue ratios is often more properly hydrophilic than the other radiotracers in the group.
In several studies, the effect of linkers on the hydrophilicity and biological properties of radiotracers has been investigated. The length of the linkers has revealed a great impact on the lipophilicity of radiotracers, resulting in notable changes in their uptake and retention in tumor and normal tissues. In this approach, PEG chains are commonly used to connect the nitroimidazole moieties to the rest of the tracer containing the radioisotope, thereby leading to the proper hydrophilicity of the radiotracers compared to those without a PEG chain. Similarly, extending the CH 2 chains also makes the radiotracers more lipophilic. In addition, adding benzene moieties can also increase the lipophilicity of the radiotracer while adding more nitroimidazole moieties might affect the hydrophilicity of the radiotracers, depending on their overall structure. The advantage of highly hydrophilic radiotracers is that they have a fast clearance from blood; thus, their tumor/blood ratios were significantly increased, PET/SPECT image contrasts were greatly improved and the radiotracers were excreted via renal routes rapidly. However, increasing hydrophilicity is not always correlated with the proper pharmacokinetics and the best tumor/background contrast, as in the cases of [ 99m Tc]30, [ 99m Tc]37, and [ 131 I]85. This might be explained that these radiotracers were cleared from blood too quickly that they did not have enough time to absorb into cancer cells, as well as not being lipophilic enough to enter cancer cells by diffusion through phospholipid bilayers. In contrast, highly lipophilic radiotracers can be retained in cancer and normal tissues. Additionally, their long retention in normal tissues significantly reduced the tumor/normal tissues ratios and PET/SPECT image contrast. Therefore, in the development of novel radiotracers for hypoxia, it is important to adjust the hydrophilicity of the radiotracers in order to find the radiotracer with optimal hydrophilicity and pharmacokinetics.
We believe that criteria other than hydrophilicity can also affect the pharmacokinetics of the radiotracers and should be considered when developing novel radiotracers for hypoxia. Adding multiple nitroimidazole moieties is also a commonly employed approach in the design of novel radiotracers to enhance better uptake in the hypoxia regions of tumors. Notably, multiple nitroimidazole units can be added into a radiolabeled complex bearing bifunctional chelators to capture radioactive transition metals like 64 18 F has been the most used radioisotope in the development of hypoxia radiotracers for many decades due to its small size and inert characteristics [183]. Most of the 18 F radiotracers were prepared via nucleophilic substitution reactions. However, in order to prepare 18 F radiotracers, big and expensive cyclotrons are required. Thus, a generator, which is a more simple and easy-to-handle piece of equipment to produce radionuclides such as 99m Tc, is also popular nowadays. It is clear that besides the most common radioisotope 18 F, coordination of the 99m Tc core to a bifunctional chelate has received growing attention recently due to many reasons. First, the convenience of generators over cyclotrons has made the preparation of 99m Tc-labeled radiotracers easier for both research and clinical purposes. Secondly, 99m Tc-labeled radiotracers are highly versatile owing to the use of bifunctional chelates, which are diverse and extensively studied [184][185][186][187][188]. Thus, future research in developing novel 99m Tc-labeled radiotracers for hypoxia should consider employing a variety of bifunctional chelates and nitroimidazole moieties. Thirdly, hydrophilic 99m Tc-labeled radiotracers can be synthesized from the corresponding precursors containing several hydrophilic groups, whereas the radiofluorination of precursors bearing several hydrophilic groups (mostly via nucleophilic substitution) is more difficult. Moreover, 99m Tc cores are varied in oxidation states, for example, 99m Tc(I) ([ 99m Tc(CO) 3 ] + core), 99m Tc(III) ( 99m Tc 3+ core), 99m Tc(V) ([ 99m TcN] 2+ core, [ 99m TcO] 3+ core), which showed different biodistributions. However, tumor uptake values and tumor/background ratios seem to depend on many factors rather than only the oxidation state of the 99m Tc core. core). Therefore, side-by-side studies are still needed to study the effect of 99m Tc cores on the biological properties of 99m Tc-radiotracers for hypoxia. The main limitation of 99m Tc-labeled radiotracers is that SPECT imaging offers a lower sensitivity and accuracy compared to PET imaging [189,190]. Nonetheless, 99m Tc-labeled radiotracers have less stable coordination than the covalent bonds of radioactive fluorine and iodine.
Despite the advantages and favorable physicochemical and biological properties of the summarized 99m Tc-labeled radiotracers, there is still a lack of clinical trials conducted for these radiotracers. However, some 18 18 F-labeled radiotracers with proper linkers, carbohydrates, and nucleic acid could be used for clinical study. Validating PET/SPECT imaging tracers for hypoxia poses several challenges. In addition to common requirements such as non-toxicity, high uptake, and rapid clearance, the radiotracers must exhibit a suitable biodistribution specific to the different tumor types. As a result, many radiotracers lack universality across various types of cancers [191,192]. However, after solving these issues, radiolabeled radiotracers can be successfully used for clinical study.
In recent years, the synthesis and evaluation of 131/124 I-and 64 Cu-labeled radiotracers are still limited, which might be due to their long half-life and production using cyclotrons. Thus, further research into the imaging techniques and equipment is needed to develop improved radiotracers with these radioactive isotopes in the imaging of tumor hypoxia.
Each radiotracer mentioned in this review has its advantages and disadvantages in lipophilicity, hypoxic selectivity, tumor uptake, tumor/blood contrast, tumor/muscle contrast, etc. Thus, the discovery of better novel hypoxia-targeting agents as imaging agents for wide applications in clinical hypoxia imaging is still needed.
Moreover, in order to visualize hypoxia through molecular imaging, several studies must be achieved for better molecular imaging in the future. First, proper radiotracers with more specificity and selectivity to hypoxia should be developed for a variety of applications and should be applied to clinical study. Thus, future studies should focus on the production of novel promising structures. Second, the preparation process for radiotracers should be achieved via more simple and efficient steps. Particularly, a short, low-cost, and environmentally synthetic process is useful. Third, radiolabeling protocol should also be easier and more effective; thus, desired radiolabeled compounds should be obtained with high radiochemical yields. Fourth, improved imaging techniques and equipment should also be developed to visualize hypoxia more clearly. It is expected that many scientists will endeavor to synthesize and evaluate novel hypoxia radiotracers with better properties for clinical application in the future. We believe that this review provides an overall picture of recent developments in new radiotracers for hypoxia.