CXCR4-targeted metal complexes for molecular imaging

CHAPTER FOURTEEN

CXCR4-targeted metal complexes for molecular imaging Isaline Renard, Stephen J. Archibald∗ Positron Emission Tomography Research Centre, Faculty of Health Sciences, University of Hull, Hull, United Kingdom *Corresponding author: e-mail address: [email protected]

Contents 1. Introduction 2. The chemokine receptor CXCR4 2.1 Chemokines and their receptors 2.2 CXCR4 and its ligand CXCL12 2.3 CXCR4: Role in cancer 3. CXCR4 binding moieties: Azamacrocycles and peptides 3.1 Tetraazamacrocyclic CXCR4 antagonists 3.2 Peptide based CXCR4 binding molecules 4. CXCR4 targeted positron emission tomography imaging probes 4.1 Antibody targeting of CXCR4: ImmunoPET 4.2 Radiolabeled peptides for CXCR4 binding 4.3 Small molecules for CXCR4 imaging 5. Conclusions and future perspectives Acknowledgments References

448 449 449 450 452 452 452 458 459 459 460 463 470 472 472

Abstract Metal ions have been used in multiple roles for the noninvasive molecular imaging of protein expression levels on the surface of cells. They can be employed to enhance binding of the complex to the protein through the formation of coordinate bonds with donor atoms on amino acid side chains. Metal ions can also, with the selection of appropriate isotopes, emit gamma rays or positrons (which annihilate to give gamma rays) that can be detected outside the body using the relevant tomographic scanners to form an image of molecular localization. Positron emission tomography is the preferred imaging modality. The chemokine receptor CXCR4 has been implicated in multiple disease states and immune system disorders but most relevant, for molecular imaging, is the correlation of high expression levels with poor prognosis in a number of cancers. CXCR4 has therefore been a target for small molecule drugs and for imaging agents. There has been considerable development work carried out over the past 20 years with particular success in the development of metal complexes that bind to CXCR4 with high affinity. Advances in Inorganic Chemistry, Volume 75 ISSN 0898-8838 https://doi.org/10.1016/bs.adioch.2019.11.002

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2020 Elsevier Inc. All rights reserved.

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The design of CXCR4 binding probes includes both peptidic and azamacrocyclic metal complexes, with the use of transition metals for binding a particularly successful avenue of research. The main radioisotopes that have been used are copper-64 and gallium-68 with some compounds translated into preclinical and clinical imaging over the past few years.

1. Introduction The CXCR4 chemokine receptor is a target in cancer imaging, with the over-expression observed in several types of cancer and directly linked to poor prognosis.1–3 The role of the chemokine receptors is to trigger cell mobilization on activation and a straightforward link can be drawn to the metastatic spread of the disease. There are also links to proliferative and radioprotective effects that can influence the continued growth of the primary tumor and its response to therapies. New combination therapies are being developed that combine chemotherapies and radiotherapy with other drugs to increase sensitivity to the treatment.4 Molecular imaging of biomarkers such as CXCR4 can also be used to select patients for treatment by determining expression levels and correlating this with the likelihood of response to therapy. Subsequently the response can then be monitored using the imaging drug. There is also an exciting possibility to switch the positron or gamma-emitting radioisotope used in imaging for an alpha or beta emitting isotope to give a targeted radionuclide treatment, with the localization in the tumor already determined using the imaging drug prior to administration. This generic approach has been tested in multiple small scale clinical trials for many years but has undergone a recent rapid expansion with Novartis purchasing imaging/therapy drug pairs for $6 billion in two company acquisitions, validating the potential of this market. CXCR4 targeted therapy combinations are in early stage clinical trials and require further development.5,6 For coordination and inorganic chemists, this area has much potential for research and development, as radio-metals offer the best flexibility for combining nuclear imaging and therapeutic agents.7 Novel metal containing molecules or chelators for attachment to biomolecules are required. In particular, macrocyclic chelators can form components of CXCR4 binding molecules. In this case, they are also involved in the binding to the protein target with coordination interactions formed between the metal center and the amino acid residue side chains (aspartates) on the accessible

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protein surface.8 Optimizing coordination chemistry interactions and the selection of the metal center can contribute to the key activity parameters for the imaging or therapeutic drug molecule: receptor residence time, binding interaction and protein internalization/recycling.9 The main research interest and clinical testing of radio-metal labeled CXCR4 antagonists has been in nuclear imaging although there is also the potential for the development of alpha or beta emitting therapeutic congeners.10,11 Positron emission tomography (PET) is the most sensitive nuclear imaging technique, which can track biochemical, physiological and pharmacological processes with a very high sensitivity (10
11–10
12 mol L
1). PET is also noninvasive and allows for quantitative measurements in vivo. With these characteristics, PET has been investigated in multiple disease types, especially cancers.12 PET relies on the use of positron-emitting radionuclides. Dependent on its energy, the emitted positron will travel a short distance (a few millimeters) within the surrounding tissues, losing energy in the process and decelerating. This positron will eventually collide with an electron resulting in annihilation and simultaneous emission of two 511 keV γ-ray photons, which travel at ca. 180 degrees from each other. The emitted photons are then simultaneously detected by the rings of detectors present in the scanner. Radio-metals that emit positrons and are utilized in molecular imaging include copper-64, gallium-68 and zirconium-89.

2. The chemokine receptor CXCR4 2.1 Chemokines and their receptors Chemokines are a family of small proteins (8–12 kDa) that belong to the superfamily of cytokines. They are responsible for directing cells throughout the body (chemotaxis) and are involved in immune and inflammatory responses.13 Structurally, chemokines contain conserved cysteine residues that form disulfide bonds which are used to classify them into four families: CC, CXC, CX3C and C, where X represents the variable amino acids between the two cysteine residues in the N-terminus of the chemokine.14 Chemokine receptors are seven-helix transmembrane G-protein coupled receptors (GPCRs). Their nomenclature follows that of their corresponding ligand.2 When chemokines bind to the extracellular loop of their receptor, the associated G-proteins are activated and trigger the corresponding signaling pathways. In normal physiology, chemokines work in a pair with their receptors to direct cells within tissues to specific locations, following a concentration gradient of the chemokine. To date, around 50 natural chemokines and

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more than 20 associated receptors have been reported.2 This redundancy indicates that some chemokines bind to multiple receptors, a process that is thought to help in the fine-tuning of this tightly regulated signaling system.15 Based on their biological function, chemokines can be divided into two main subgroups: homeostatic and inflammatory chemokines. Homeostatic chemokines regulate the trafficking and homing of different cell types to promote the development of certain tissues. As their name indicates, inflammatory chemokines regulate inflammatory processes by recruiting leukocytes and directing them to sites of inflammation.2,14 However, this classification is not mutually exclusive and some inflammatory chemokines can show homeostatic functions and vice-versa.14 Overall, chemokines and their receptors play a key role in inflammation by directing the movement of immune cells to the site of inflammation. They are also involved in hematopoiesis, angiogenesis and have been shown to play an important role in embryonic development during organogenesis.1,16 In addition to their normal physiological roles, chemokines and their receptors have been shown to be heavily involved in a variety of pathologies such as multiple sclerosis, rheumatoid arthritis, asthma, HIV infection and cancer.17 In cancer, malignant cells can overexpress some chemokine receptors, essentially “hijacking” the host’s chemokine network and using it to activate its own development with roles in tumor cell growth, proliferation and survival.18 Most importantly, chemokines and their receptors have a fundamental role in angiogenesis and progression to metastasis. The expression of the chemokine receptors on the surface of cancer cells, allows them to migrate to specific sites and to metastasize.3 Among all of the different chemokine receptors, CXCR4 has been reported to play a significant role in multiple types of cancer.15

2.2 CXCR4 and its ligand CXCL12 Investigation of CXCR4 has been a developing and expanding area of research since it was found to be a co-receptor in HIV infection in the late 1990s.19 As with the other chemokine receptors, CXCR4 is a seven-helix transmembrane GPCR (Fig. 1). It is composed of 352 amino acids and is rich in aspartic acid residues, which makes it negatively charged on the external surface at physiological pH.21,22 CXCR4 is highly expressed in various cell types such as hematopoietic cells and associated stem cells in the blood and bone marrow, as well as embryonic stem cells.23–25 So far, CXCR4 has been shown to have only one natural ligand: CXCL12 (also referred to as SDF-1; stromal cell-derived factor-1).16 The chemokine ligand CXCL12 is a

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I Y

S I G E M CHO

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NH2

T S D N Y T E E

M G E K M S D Y D G S

182 K Q G C I D O A E Y F G R I E F W A Y N E 193 Y P E N N F V N I L F R D F A N D L N L N L C W T V 262 K V A V Extracellular F I H K C A S F I V V K D L V D A A F W F P 171 V I I Q I T V H I I S F I F W Q F I S D G P T Y E L I H I S T Y Y I I P T A Y T T V V M G N V F I L A L F L P Y L T L L L L W F L F I S H C A L G A U P L I P V S V I F C C F W I G V A N L I L N G L A L G L S V G V P A L I V I H I L L F V L Y L R S I C S A Y V V L I V I T K Y Y F L V K C L T I K R D A E D G M A G L Y Intracellular Y I I K S L K A T F L L K Q L K K R S M A Q K K T H S I K L R H S S A Q H R V K G P H V R A S E S E T S T N S Q S S F H S S COOH P C

F R E E N

Fig. 1 Representation of the chemokine receptor CXCR4, showing the seven transmembrane helices, using single letter amino acid codes with key aspartate residues highlighted. Reproduced from De Clercq, E. Nat. Rev. Drug Discov. 2003, 2 (7), 581–587.

67-amino acid residue polypeptide expressed and secreted by multiple organs such as bone marrow, liver, kidneys, lungs and brain where it is involved in the trafficking of CXCR4-expressing cells.26 With 21% of its amino acid residues being arginine, histidine or lysine, CXCL12 is a highly basic protein and it has positive surface charges along its first and second β-strands, whereas the surface of its α-helices is predominantly negatively charged.26 These properties of the ligand are of particular interest when designing CXCR4-specific binding agents. In normal physiology, the CXCR4/CXCL12 axis has a key role in the trafficking of stem cells during embryonic development. Knockout models in mice, where either the CXCR4 or the CXCL12 gene has been deleted, have proven lethal during the embryonic stage, demonstrating that the CXCR4/CXCL12 axis is essential during organogenesis, hematopoiesis and angiogenesis at this stage.27–29 In the fully developed organism, CXCR4 plays a key role in the development and survival of hematopoietic stem cells in the bone marrow and is responsible for the trafficking of B and T cells.25,29

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2.3 CXCR4: Role in cancer Due to its role in the migration of multiple types of cells in normal physiology, it is not surprising that CXCR4 would be involved in cancer progression. So far, the over-expression of CXCR4 has been reported in more than 23 different types of cancer, including multiple myeloma, breast, ovarian and prostate cancers.27 Moreover, the CXCR4/CXCL12 pair has been shown to have an important role in tumor biology, especially tumor growth and angiogenesis with a significant influence on metastatic progression3 and it is usually associated with poor prognosis for the patients.1 Given its impact on cancer physiology and its association with particularly aggressive types of cancers, CXCR4 represents a target of high interest for the development of new cancer imaging and therapeutic agents. The successful targeting and imaging of this receptor could lead to earlier diagnosis, inform treatment selection, result in more personalized and appropriate treatment courses for the patient and ultimately better outcomes.

3. CXCR4 binding moieties: Azamacrocycles and peptides Over the last decades, CXCR4 has been the focus of intensive research aimed at the down-regulation of its activity through the development of various binding agents. The following sections detail selected examples of CXCR4-specific antagonists and the key structural components responsible for binding to the receptor. Several nonazamacrocyclic small molecules, such as IT1t30 or KRH-163631 have also been reported, but will not be discussed herein. This section focuses on azamacrocyclic and peptidic binding units.

3.1 Tetraazamacrocyclic CXCR4 antagonists 3.1.1 AMD3100 and related compounds AMD3100 was one of the early generation of tetraazamacrocyclic compounds reported to have an anti-HIV effect that was later determined to be due to binding to CXCR4.32 The mono-macrocyclic compound cyclam (1,4,8,11-tetraazacyclotetradecane) was tested for anti-HIV activity by Prof Erik De Clerq’s team at KU Leuven. One batch of the cyclam had much higher activity and this was eventually traced to a bis-cyclam impurity. The structure of this impurity was later identified showing a structure where two macrocycles were directly linked through a CdC bond.

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This compound was known but is challenging to synthesize in large amounts with high purity. Derivatives were produced with either aliphatic or aromatic spacers between the cyclam rings through N-alkylation.28 After evaluation of the anti-HIV properties of the different derivatives, it appeared the presence of the aromatic spacer led to a considerable increase in activity. The structure with the highest affinity (EC50 ¼ 3 and 9 nM against HIV-1 and HIV-2, respectively), two cyclam rings linked by a xylyl bridge, was later renamed AMD3100 due to its place in the development pipeline of Anormed, a company formed in conjunction with Johnson Matthey which had supported the initial studies (Fig. 2).20 This compound had previously been synthesized by Fabbrizzi and co-workers as part of an unrelated program looking at novel metal complexes.33 With no observed cross-interaction with other receptors, AMD3100 is a specific binder to CXCR4.34,35 This affinity can in part be explained by the electrostatic interactions between the negatively charged carboxylates of the aspartic acid residues present on the receptor at positions 171, 182, 193 and 262 and the positively charged protonated amines of the cyclam rings.20 Mutation models (produced by site directed mutagenesis) where Asp171 and Asp262 were replaced by arginine residues proved these two amino acids are especially essential for the interaction between AMD3100 and CXCR4.36 In addition to its anti-HIV properties, an unexpected side effect of AMD3100 was later observed during phase I clinical trial in healthy volunteers, showing AMD3100 to be a rapid and efficient stem cell mobilizing agent.37 By preventing CXCL12 from binding to CXCR4, AMD3100 interferes with the retention of CD34+ hematopoietic stem cells in the bone marrow, resulting in their rapid transfer to peripheral blood. These cells can be harvested and then returned to patients with hematological diseases, such as multiple myeloma, after ablation treatment.38 This property was further validated in multiple studies.39–41 AMD3100 has been approved by the US Food and Drug Administration (FDA) since 2008 and is now commercialized as Plerixafor (trade name Mozobil™) to be used in patients with

Fig. 2 Chemical structures of AMD3100 and AMD3465.

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non-Hodgkin lymphoma or multiple myeloma who would undergo autologous hematopoietic stem cell transplantation.42 With the aim to prepare a more orally bioavailable drug than AMD3100 that would be better suited for long-term anti-HIV therapy, a monocyclam derivative, AMD3465 (Fig. 2), was synthesized, replacing one of the cyclam rings of AMD3100 by an N-pyridinylmethylene group.43 This led to an eightfold increase in CXCR4 affinity in comparison to AMD3100 (as measured by a competition assay with [125I]12G5 mAb) and showed different interactions in the binding pocket of the receptor.44 SAR studies still indicated overlapping sites of interaction between AMD3100 and AMD3465, more specifically the aspartate residues Asp171 and Asp262. However, further studies using AMD3465 and other analogs showed that new interactions with histidine residues His281 and His113 are essential for CXCR4 affinity of the mono-cyclam compounds.44 AMD3465 did not achieve the desired oral bioavailability but represented an advance from AMD3100, and indicated that derivatized mono-cyclam derivatives with additional heterocycles can be used as potent anti-HIV agents by binding to CXCR4. A further opportunity for investigation in any compound containing a cyclam macrocycle, which provides an ideal tetradentate cavity for complex formation with transition metal ions, is to form the metal complexes and investigate their biological activity. 3.1.2 Metal complexes of AMD3100 Azamacrocycles, such as cyclams, are effective chelators (forming five- and six-membered chelate rings) and can form thermodynamically stable complexes with a broad range of metal ions, particularly first row transition metal ions.45,46 A series of metal complexes of AMD3100 were prepared and evaluated for their antiviral properties and their affinity for CXCR4.8,47 Metal complexes with copper(II), zinc(II) and nickel(II) are reported to show an increase in CXCR4 affinity of 7-, 36- and 50-fold, respectively.8 Other studies have shown that while zinc(II) and nickel(II) improve affinity in most cases, binding enhancement with copper(II) is more geometrically dependent and not all assays indicate enhancement of AMD3100 binding through copper(II) complex formation.48 Site directed mutagenesis indicated that the enhanced affinity of the metal complexes is particularly linked to the coordination interaction(s) of one of the metal centers with the carboxylate group of Asp262.8 For cyclam based compounds, the metal ion could form a monodentate coordinate bond with

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Fig. 3 The six possible configurations for cyclam metal complexes (with chirality of N atoms listed).

one of the carboxylate O donors and the other could form a hydrogen bond with one of the secondary amines from the macrocycle. Upon coordination with a metal center, all four nitrogen atoms of the cyclam become chiral and give rise to up to six possible configurations in solution (Fig. 3).49 In most cases, the trans-III configuration is the most thermodynamically favorable configuration, with minimum steric hindrance. However, the preferred configuration will be highly dependent on coordination number, as well as the presence of any additional ligands, and multiple configurations have been shown to co-exist in solution by NMR by Sadler and co-workers.50,51 An example of this is shown by the Zn(II) cyclam complexes, where the counter-ions appear to have an impact on configurational shift. For example, the zinc(II) complex in presence of chloride will mostly adopt the trans-III configuration, whereas the presence of acetate gives a cisV configuration. The potential to form the cis-V configuration also appears to be consistent with enhanced binding and hence increased affinity for CXCR4.51–53 3.1.3 Configurationally restricted azamacrocycles and their metal complexes To address the issue of multiple configurations in solution (binding will vary with configuration), and optimize binding affinity to the receptor, configurationally restricted azamacrocycles could be utilized to form bismacrocyclic compounds.54 The configuration of a macrocycle can be locked by the introduction of an ethylene chain between two adjacent nitrogen

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atoms (side-bridge or SB, via steric effects) or two non-adjacent nitrogen atoms (cross-bridge, CB, via mechanical restriction).55 The introduction of a side-bridge in the cyclam cavity was originally reported by Wainwright.56 It was later identified that the added steric bulk results in a single configuration for the macrocycle: trans-II.57–60 This configuration was originally observed by Wainwright in the solid state,56 then later evidenced in other X-ray crystallography studies.61 The Archibald group reported the first X-ray structure of a zinc(II) complex of a SB-bicyclam showing the trans-II configuration in the solid state and it was confirmed that this was also adopted using high-field NMR.58 One aim of this approach was to increase the kinetic inertness of the metal complexes, although Boiocchi et al. reported that the non-functionalized SB-cyclam does not result in a reinforced kinetic macrocyclic effect under the conditions investigated.62 Several SB-cyclam derivatives with added pendant arm and their corresponding metal complexes have been reported with investigations ongoing to determine the impact on in vivo stability.46,58–60,63,64 The preparation of a cross-bridged cyclam (CB-cyclam) was first described by Weisman.65 Structural studies showed that the CB-cyclam can only adopt one configuration: cis-V,66–69 due to the mechanical restriction of shape, which results in an enhanced kinetic inertness of the corresponding metal complexes, where the metal center is coordinated in the cavity by four convergent nitrogen lone pairs.70 CB-cyclam was developed for applications in catalysis by Hubin and Busch in collaboration with Procter & Gamble Company.71 The synthesis of these molecules is challenging and more advanced methodologies have been developed to access a wider range of derivatives and increase efficiency.72–74 Following these observations, a cross-bridged bis macrocyclic compound with the same para-xylyl bridge as AMD3100 (CB-bicyclam) and its Cu(II) metal complexes were prepared by Archibald and co-workers and assessed for their CXCR4 affinity in vitro (Fig. 4).9 As expected, due to the absence of hydrogens on the amines, the free ligand has low affinity for the receptor (Table 1). However, once the metal centers were introduced into the rigidified cyclam cavities, the complexes showed high affinity for CXCR4, with IC50 values of 8 and 3 nM for CuCB-bicyclam and Cu2CB-bicyclam, respectively (Table 1).48 Testing was also carried out in metastatic assays to demonstrate the efficacy of this compound.75 The enhanced affinity of the Cu(II) complexes can be attributed to the rigidity of the chelator, since the cross-bridged macrocycles are locked in a cis-V configuration, the copper(II) centers will form shorter and stronger

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Fig. 4 Chemical structures of configurationally restricted macrocyclic CXCR4 antagonists.

Table 1 Affinity data for selected CXCR4 antagonists. Compound

IC50 (nM)

AMD3100

203

CuAMD3100

114

Cu2AMD3100

94

Zn2AMD3100

5

CB-Bicyclam

>5000

CuCB-Bicyclam

8

Cu2CB-Bicyclam

3

Pentixafor

94

nat

[ Ga]Ga-Ni2AMD3100-PEG-NOTA

1485a

[natGa]Ga-Ni2AMD3100-PEG-DO3A

516a

[natGa]Ga-Ni2AMD3100-PEG-p-NCS-Bz-NOTA

121a

a

Values were determined by competition binding with [125I]CXCL12. Where available, values are reported for affinity determined by the intracellular Ca2 + release assay in response to CXCL12 stimulation.

equatorial bonds with the aspartate residues present on the receptor, relative to the longer and weaker axial bonds from the non-restricted CuAMD3100 structure due to Jahn Teller distortion.9,76 Nickel(II) and cobalt(II) complexes have also been investigated as cross-bridged compounds interacting with CXCR4.77,78 Based on the results from this published work, Nimmagadda and co-workers produced cross-bridged analogs of AMD3465 which were

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complexed with copper(II) to give the RAD1 derivatives (Fig. 4). In the same way the structural constraint in Cu2CB-Bicyclam led to an increased affinity for CXCR4, the RAD1 derivatives showed an enhanced affinity for the receptor when tested in in vitro assays.79

3.2 Peptide based CXCR4 binding molecules Several CXCR4-specific peptidic agents have been developed based on the sequence and structural data on the CXCL12 chemokine, and also the screening of natural compounds isolated from other species. Peptides offer an opportunity for facile derivatization and can easily be functionalized to allow the introduction of a chelator amenable to labelling with a radio-metal, an important feature to consider when designing a radiotracer. One of the initial peptidic CXCR4 antagonists developed is T22, an 18-mer peptide (Fig. 5), derived from antimicrobial peptides isolated from horseshoe crabs.80 This peptide showed potent anti-HIV properties as well as a relatively low toxicity. Structure–activity relationships were developed and showed that the presence of the two Tyr-Arg-Lys motifs, as well as the anti-parallel β-sheet induced by the two disulfide bridges, are essential to the anti-HIV activity of the peptide. The replacement of the Trp3 residue by a 3-(2-naphthyl)-alanine (NaI) led to an increase in potency for the molecule. Later on, following the discovery of the involvement of CXCR4 in HIV infection, it was confirmed that the anti-HIV properties of T22 derived from its binding to CXCR4. In an attempt to reduce the size of the molecule, a second generation of peptides was prepared. Among the different derivatives synthesized, T140, a 14-mer peptide (Fig. 5), showed antagonistic properties against CXCR4.81 SAR studies confirmed the significant contribution of the NaI3 residue to the bioactivity of the molecule. T140 was more effective than T22 at

Fig. 5 Amino acid sequences (and intramolecular disulfide bridge sites) of CXCR4 binding peptides.

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inhibiting binding of the known CXCR4-antibody, 12G5. Alanine replacement screening allowed the identification of Arg2, NaI3, Tyr5 and Arg14 as key residues for CXCR4 binding.81 Still pursuing the goal of reducing the peptide size and increasing in vivo stability while identifying key structural features responsible for CXCR4 affinity, a third generation of peptides was developed. The approach taken was to use a cyclic pentapeptide as a template to arrange the four key amino acids identified earlier in the required proximity for optimal binding. This led to the synthesis of two isomers of interest: cyclo[NaI-GlyD-Tyr-Arg-Arg] (FC131, IC50 ¼ 8 nM) and cyclo[NaI-Gly-D-Tyr-DArg-Arg] (FC131(D-Arg), IC50 ¼ 4 nM) (Fig. 5).82 Alanine replacement studies were performed on both peptides and showed a clear reduction of CXCR4 affinity upon substitution of all of the amino acids, underlining the importance of all of the side-chain functional groups, as well as the role of the Gly5 residue in maintaining the spatial orientation of the CXCR4 peptide antagonist.83 Further SAR studies, through the N-methylation of the different amino acids, proved that the Arg3 and NaI4 residues are indispensable to CXCR4 binding affinity. The N-methylation of D-Arg2 led to a twofold increase in CXCR4 affinity (compared to its parent peptide), suggesting the orientation of the amide bond between D-Tyr1 and 2 83 D-Me-Arg is of importance for receptor interactions. The cyclisation of the peptide is beneficial as, along with the presence of D-amino acid(s) and N-Me amide bonds, it gives higher in vivo metabolic stability of the peptide, making it less susceptible to peptidase degradation.

4. CXCR4 targeted positron emission tomography imaging probes 4.1 Antibody targeting of CXCR4: ImmunoPET Antibodies have very high specificity for their target and so, despite their non-ideal high molecular weight and slow blood clearance that can result in poor tumor-to-background ratio, they are still of interest in PET probe development. They are relatively easy to engineer and can be modified to have a single conjugation site for attachment of a metal ion chelator.84 Since antibodies have a relatively long biological half-life, they need to be matched with an equally long-lived radioisotope.85 Currently, zirconium-89 (t1/2 ¼ 78.5 h, 23% β+) is the preferred choice to radiolabel antibodies, taking over from iodine-124, as it offers stable labelling and there is good worldwide availability of the isotope. The most commonly used

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chelator for zirconium(IV) is desferrioxamine B (DFO). DFO is a hexadentate acyclic ligand that offers hard oxygen donor atoms that are match with zirconium(IV), binding through its three hydroxamate groups. There is some debate over the optimal coordination number around the Zr(IV) cation which can be seven or eight coordinate but the complex formed is sufficiently stable to give acceptable results on in vivo use.86 Coordination chemistry and options for chelators of zirconium have been reviewed by Wadas et al.87 and Orvig et al.85 One example of CXCR4 PET imaging using an 89Zr-labeled antibody has been reported.88 Azad et al. conjugated the known anti-hCXCR4 antibody MDX-1338 with DFO and radiolabeled it with zirconium-89. Radiolabeling was achieved after 1 h reaction at room temperature. The probe was then tested in vivo in a mouse model using high (H1155) and low (A549) expression xenografts, showing optimal uptake 72 h postinjection, with a preference for accumulation in the high-expressing tumors. To prove CXCR4-specific accumulation in the tumor, a control antibody was also radiolabeled with zirconium-89 and showed no uptake in either tumor model. The specificity of the radiotracer was further confirmed by a blocking experiment using the unmodified CXCR4-mAb, which showed a reduction in tumor uptake. The [89Zr]CXCR4-mAb tracer was also used as part of a series of experiments to test the use of this antibody for immunotherapy. This ability to monitor response to treatment by PET imaging is of key importance to improve treatment regimens and ultimately improve therapeutic outcomes.88

4.2 Radiolabeled peptides for CXCR4 binding Peptides are easily functionalized to allow the introduction of spacers and chelators amenable to radiolabeling or the conjugation of a radiolabeled prosthetic group. In general, they cannot match antibodies in term of specificity and selectivity. However, they usually have a low toxicity and their low molecular weight and fast clearance can lead to rapid tumor accumulation to give a high quality image. One downside of using peptides in vivo can be their lack of metabolic stability and susceptibility to enzymatic degradation. This issue can however be addressed by multiple synthetic strategies such as use of nonnatural amino acids, capping, N-methylation and cyclization.89 Due to their relatively short clearance and accumulation times, peptides can be radiolabeled with short-lived radio-metals such as gallium-68 or copper-64.

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Gallium-68 (t1/2 ¼ 68 min, 90% β+) is mostly generator produced, hence it is currently more widely available and cheaper than copper-64 (t1/2 ¼ 12.7 h, 19% β+). The main chelators of interest for gallium-68 complex formation are NOTA and DOTA derivatives. NOTA is a triazamacrocycle, derived from TACN (1,4,7-triazacyclononane), where the three amines are functionalized with acetyl groups. The hexadentate NOTA is considered as a “gold standard” for gallium complex formation resulting in efficient radiolabeling at room temperature and high in vivo stability.85 DOTA is derived from cyclen (1,4,7,10-tetraazacyclododecane) where the four amines are substituted with acetyl pendant arms. As it is a good match for multiple radio-metals, such as 68Ga, 111In or 177Lu, the octadentate DOTA is probably the most commonly used chelator in radiotracer development. It is now generally accepted that gallium(III) metal complexes with DOTA are less stable than complexes with NOTA and it exhibits slower radiolabeling kinetics, hence reactions need to be carried out at elevated temperature for efficient labeling.85 The chelator selection is dependent on the metal ion, however, it can also influence the biodistribution and in vivo properties of the radiotracer, showing in some cases DOTA can offer advantages over NOTA from this perspective.90,91 In the case of copper-64, NOTA shows good properties but specific chelators designed for copper-64 include CB-TE2A, which can form highly stable complexes under physiological conditions.69,92 These chelators are based on the structure of cyclam where two non-adjacent secondary amines in the macrocyclic ring have been functionalized with acetyl pendant arms. The cross bridged cyclam CB-TE2A is produced by configurational restriction with the addition of an ethylene bridge across the cyclam cavity.93 Although stable copper(II) complexes were prepared using the hexadentate TE2A, enhanced kinetic inertness was observed when using the more rigid cross-bridged counterpart.85,94 Gallium(III) and copper(II) radiopharmaceutical development has previously been reviewed, along with discussion of chelator selection.85,87,95 4.2.1 T140 peptide-based probes The CXCR4-specific peptide described in Section 3.2, T140, has been conjugated with the DOTA-NHS ester to give the derivative T140-2D, where a DOTA chelator was added to each of the two Lys residues.96 The resulting conjugate was radiolabeled with copper-64 at 40 °C in 20 min, showing incorporation of the radio-metal greater than 95%. The radiotracer was then assessed in vivo in mice with CHO-CXCR4 and wild type CHO xenografts.

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Unfortunately, similar to the prior observations with the fluorine-18 radiolabeled T140 tracer,97,98 [64Cu]Cu-T140-2D appeared to bind to red blood cells, leading to poor tumor-to-background ratio and high uptake in blood and abdominal region; the tumor could not be detected, with high liver and kidney uptake observed.96 Following this study, Jacobson et al. prepared two new derivatives of T140: DOTA-NFB and NOTA-NFB, where a DOTA-NHS ester or a p-SCN-Bn-NOTA was conjugated with the C-terminal Arg of NFBT140, ultimately replacing the 4-F-benzoyl group present in the original T140 sequence.99 Both compounds were radiolabeled with copper-64 and showed radiolabeling yields of greater than 97% after reaction for 20 min at 40 °C. The two radiotracers were assessed in the same tumor model as [64Cu]Cu-T140-2D. This time no retention of the tracers in the blood was observed and the CXCR4-expressing tumor was clearly visible with both tracers, with an uptake 8–10-fold higher in the CXCR4 expressing tumor than in the control tumor. Once again, high liver and kidney uptake was observed for both tracers, with a slightly higher liver uptake in the case of the NOTA derivative. Co-injection of a blocking dose of unlabeled peptide showed some reduction of tumor and liver uptake, combined with the expected increase in kidney uptake.99 4.2.2 [68Ga]Pentixafor and related derivatives In parallel to the development of the T140 derived radiotracers, Demmer et al. optimized and derivatized the structure of peptide antagonist FC131 to add a chelator amenable to gallium-68 radiolabeling.100 They started by improving both the stability and the affinity of the peptide by replacing the L-Arg2 by a D-Arg2 and N-methylating the amide bond between D-Tyr1 and D-Arg2 to give an affinity of 2 nM for CXCR4. In order to attach the DOTA, the D-Arg2 was substituted by a D-Orn2. Multiple spacers were tested to link the peptide and the DOTA and the derivative with the highest affinity had an aminomethylbenzoyl spacer (IC50 ¼ 22 nM). Finally, the DOTA moiety was conjugated to yield the final construct, later renamed CPCR4-2.100,101 The new peptide was successfully radiolabeled with gallium-68 (5 min reaction time at 95 °C) and assessed in vivo in mouse models with OH-1 human small-cell lung cancer xenografts. The radiotracer showed favorable uptake in the CXCR4-expressing cell line, resulting in high tumor-to-background ratio. A blocking experiment with a co-injection of cyclo[D-Tyr-Arg-Arg-Nal-Gly] resulted in a significant decrease in tumor uptake, confirming the CXCR4-specificity of the tracer.100

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When interpreting this data it is important to note that the [68Ga] CPCR4-2 tracer showed no binding to murine CXCR4, hence there was no background signal detected due to natural CXCR4 expression in the animal (only the human CXCR4 expressed in the tumor).102 This CXCR4-specific tracer, [68Ga]CPCR4-2, was later renamed 68 [ Ga]Pentixafor and quickly advanced toward clinical trials in cancer patients with hematological diseases, due to the well-established expression of CXCR4 in such malignancies, as well as the clinical need to assess and quantify CXCR4 expression in humans in a range of disease states (see Fig. 6).102 In one patient, originally diagnosed with lymphoma, then diagnosed with non-small cell lung cancer (NSCLC) (after lymphoma relapse), [68Ga]Pentixafor was compared to the current clinical standard for positron emission tomography imaging, [18F]FDG. These scans showed some overlap of [68Ga]Pentixafor and [18F]FDG uptake in the lymphoma region, however very low uptake of [68Ga]Pentixafor was observed in the NSCLC region, indicating the specificity of [68Ga]Pentixafor for CXCR4 expressing tumors. This study represented the first clinical evaluation of CXCR4 expression using non-invasive molecular imaging.102 Given the success of the first-inhuman trial, additional studies were performed in patients with various types of malignancies such as multiple myeloma,103 small cell lung cancer,104 breast carcinoma105 and even chronic bone infection.106 Although it is not the ideal chelator for gallium, the use of the DOTA does offer flexibility and allows for the introduction of therapeutic radioisotopes such as lutetium-177, which also bind to the chelator with sufficient stability. A 177Lu-radiolabeled analog of Pentixafor, [177Lu]Pentixather, was prepared and showed good pharmacokinetic properties, leading to favorable overall dosimetry.107 Other Pentixafor analogs were prepared, such as [68Ga] NOTA-Pentixafor108 or 18F-radiolabeled analogs in attempts to optimize properties or increase tracer availability by the use of more commonly available isotopes, but none showed sufficiently promise for clinical studies.109

4.3 Small molecules for CXCR4 imaging Small molecules can be harder to design and optimize, in terms of the structural properties, than peptides or antibodies, but they generally exhibit high metabolic stability. Their low molecular weight results in fast clearance and distribution, which can give enhanced tumor-to-background ratio. They can however sometimes present an increased risk of toxicity in vivo with off target effects. The most commonly used PET isotopes for radiolabeling

Fig. 6 [68Ga]Pentixafor-PET in patients with lymphoproliferative malignancies, and chemical structure of [68Ga]Pentixafor. A, C and E (with linked transaxial images or relevant organs B, D and F in each patient) show the [68Ga]Pentixafor-PET/CT images. (G and H) show the corresponding [18F]FDG PET of the patient depicted in (E and F). Reproduced from Wester, H.J.; Keller, U.; Schottelius, M.; Beer, A.; PhilippAbbrederis, K.; Hoffmann, F.; Simecek, J.; Gerngross, C.; Lassmann, M.; Herrmann, K.; Pellegata, N.; Rudelius, M.; Kessler, H.; Schwaiger, M. Theranostics 2015, 5 (6), 618–630.

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of small molecules are fluorine-18 and carbon-11 as they can be easily substituted into organic molecules. However, small molecules can also contain metal ions (which can be substituted for radio-metals) or they can be functionalized with a chelator to allow radio-metal labeling. Similar to peptides, the biological half-life of radiolabeled small molecules will be best matched with relatively short-lived radio-metals such as gallium-68 and copper-64. The concept of “direct radiolabeling,” in the sense that the radioisotope is directly included in the targeting moiety, is one that is rarely exploited as the metal complex needs to bind to the biological target (rather than the conjugate), and it must also be formed and purified on an appropriate timescale. 4.3.1 Mono- and bis-cyclam CXCR4 PET probes As mentioned above, AMD3100 and AMD3465 can form stable complexes with transition metals, which increases affinity for CXCR4 in most cases. It was an appropriate progression that both compounds were radiolabeled with copper-64, although analysis of the literature indicates that cyclam complexes with copper(II) are unlikely to be stable under in vivo conditions. AMD3100 was initially radiolabeled with copper-64 by Jacobson et al.110 They reported the radiochemistry and initial evaluation of the tracer in C57BL/6 immunocompetent mice. Tracer uptake was assessed both by PET scanning and ex vivo bio-distribution, showing high accumulation in the liver as well as in immune-related organs such as the spleen, bone marrow and lymph nodes.110 They investigated the specificity of the tracer through co-injection of unlabeled CuAMD3100, observing reduction in the uptake in immune-related organs as well as in the liver, which could be explained by the natural expression of murine CXCR4 in the liver.48 This is likely to be the case, however, released copper(II) ions would also be taken up into the liver, and so both processes may be occurring. The kidney uptake increased slightly following the administration of the blocking dose, which is consistent with the tracer being unable to bind to CXCR4 and being excreted. Jacobson et al. published a second study, this time evaluating [64Cu]AMD3100 in different tumor models (CHO-XR4, 3LL-XR4 and their wild type counterparts) and showed favorable uptake in CXCR4 expressing cell lines, with the tumor uptake blocked but significant liver uptake remaining, after administration of cold CuAMD3100 or noncomplexed AMD3100.111 In parallel, Nimmagadda et al. reported the in vivo evaluation of [64Cu] AMD3100 in U87-stb-CXCR4 and U87 (a high-expressing transfected

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cell line and a nonexpressing cell line), as well as in MDA-MB-231 (natural low CXCR4 expression) and DU4475 (natural high CXCR4 expression) xenografts.112 In all cases, they demonstrated preferable uptake in highly expressing tumors. Each time the tumor uptake was reduced by administration of a blocking dose of AMD3100, leading to a clear lower observed signal in the tumors. Finally, and as reported by Jacobson et al., [64Cu] AMD3100 high liver uptake was observed, as well as uptake in immunerelated organs. This uptake in CXCR4-expressing organs was partly reduced upon administration of the blocking dose, however significant liver uptake could still be observed, potentially indicating an issue with the stability of the radiotracer and its ability to retain the copper ion in vivo. Archibald and co-workers recently confirmed this hypothesis through stability assays, examining the tracer across a pH range and also demonstrating the majority of the activity from [64Cu]CuAMD3100 injected in vivo was in the form of free copper(II) on excretion or extraction from the liver.48 With a similar rational as for AMD3100, AMD3465 was radiolabeled with copper-64 and assessed in vivo by De Silva et al.113 They first evaluated [64Cu] AMD3465 in transfected U87-stb-CXCR4 and nonexpressing wild type U87 xenografts, showing specific and blockable uptake in the cell line with high CXCR4 expression (with no uptake observed in U87). To further validate the tracer, they investigated its uptake in naturally expressing colorectal cancer HT29 cells, showing once that uptake in the tumor was specific and blockable. Unfortunately, as observed previously for [64Cu]AMD3100, nonblockable uptake in the liver was observed indicating low complex stability and loss of the radiolabel. Other AMD3465 derivatives amenable to 18F radiolabeling were recently described and evaluated in vivo, however, further development to give ideal properties is required.114,115 4.3.2 AMD3100 conjugates for PET imaging As an alternative to radiolabeling with copper-64, a new family of AMD3100 conjugates was developed by Poty et al. In this study, the xylyl spacer of AMD3100 was functionalized in order to introduce a linker and chelator to render the structure amenable to gallium-68 radiolabeling.116 This reduces the CXCR4 affinity but significant binding was still observed. A first generation of compounds was produced where the CXCR4targeting moiety, a Ni2AMD3100 complex, was separated from the chelator (either a DO3A or a NOTA), by a short ethylenediamine spacer (Fig. 7).116 The gallium(III) complex cold standards were prepared and evaluated for their affinity for CXCR4, confirming that the phenyl ring linking the

NH

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Fig. 7 Chemical structures of AMD3100 chelator conjugates suitable for gallium-68 radiolabeling.

117

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two cyclams in AMD3100 can be functionalized with a carboxyl group allowing further conjugation with fluorescent pharmacophores or radio-metal chelating macrocycles while retaining at least moderate affinity for the receptor. Unfortunately, these derivatives proved challenging to radiolabel, possibly due to the close proximity between imaging and targeting moieties.116 This first study paved the way to the design of a second generation of conjugates, this time increasing the length of the spacer between binding and imaging units by introducing a PEG chain. Three compounds were prepared [natGa]Ga-Ni2AMD3100-PEG-NOTA (IC50 ¼ 1485 nM), [natGa]Ga-Ni2AMD3100-PEG-DO3A (IC50 ¼ 516 nM) and [natGa] Ni2AMD3100-PEG-p-NCS-benzyl-NOTA (IC50 ¼ 121 nM) (Fig. 7).117 This new generation of derivatives proved easier to radiolabel and the tracer with the highest affinity, [68Ga]Ga-Ni2AMD3100-PEG-p-NCSbenzyl-NOTA was assessed in vivo using CXCR4-expressing H69 xenografts in mouse models, where low but blockable tumor uptake was observed. These studies offered a proof of concept that functionalizing the xylyl component of AMD3100’s core for gallium-68 radiolabeling is possible and the resulting conjugates may still retain sufficient affinity for the receptor. Further development of this approach and assessment of new radiotracer candidates in vivo is required.117 4.3.3 Configurationally restricted cyclam PET probes The inadequate stability of the copper-64 labeled complexes with AMD3100 and AMD3465 (that gave non-blockable liver uptake due to copper(II) ion release and uptake by enzymes in the liver) stimulated investigation of the higher stability cross-bridged cyclam complexes. At the same time as copper-64 work was ongoing in the Archibald group, RAD1 compounds, based on cross-bridged macrocycles included in the AMD3465 structure, were radiolabeled with copper-64 and assessed in vivo in U87stb-CXCR4 and U87 xenografts. One derivative showed specific uptake in the CXCR4-expressing tumors. This tumor uptake was blockable with a preinjection of AMD3465 (>90%). Interestingly, the liver uptake was only blockable by ca. 50% with the administration of AMD3465, indicating that despite the increased stability of the cross-bridged complex the retention of the radio-metal in vivo was not sufficient.118 This is unexpected and may be due to “out of ring” chelation where the copper(II) ion is not bound within the macrocyclic cavity and so is more easily released. The CB-bicyclam compound that had already been developed by the Archibald group was radiolabeled with copper-64 and assessed in vivo in

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U87-stb-CXCR4 and U87 xenografts in mice (Fig. 8).48 Preclinical PET/CT scanning results showed selective uptake in the CXCR4 overexpressing tumors. As with the other tracers, due to expression of murine CXCR4 in the liver, moderate liver uptake was also observed. For the first time, murine CXCR4 binding was investigated and confirmed for this tracer in a murine CXCR4 transfected cell line. To determine CXCR4specificity of the radiotracer, a blocking experiment using the preadministration of the high affinity antagonist Cu2CB-bicyclam at 5 mg/kg (a lower concentration than was used in the blocking experiments by the other research groups), which blocked both tumor and liver uptake. For this tracer, both the liver and tumor uptake were entirely blockable, demonstrating the high stability of the radiotracer and the retention of the copper ion in the macrocyclic cavity.48

Fig. 8 PET/CT scans of [64Cu]CuCB-bicyclam in mice implanted with low CXCR4 expressing tumors (U87) (left), in mice implanted with high CXCR4-expressing tumors (U87-CXCR4) (middle) and following the administration of a blocking dose of the high affinity antagonist Cu2CB-bicyclam (right). The upper arrow indicates the liver and the lower arrow indicates the tumor location.48

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In addition to its high affinity and stability, [64Cu]CuCB-bicyclam binds to both human and murine CXCR4, opening up possibilities for wider preclinical development against non-humanized disease state models and allowing cross-evaluation between multiple species as it expected to bind to CXCR4 in all species due to conservation of the binding residues. The capability to track the radioactive molecule will also potentially drive the development of therapeutic applications allowing patients to be selected that would respond to either CXCR4 antagonist chemotherapy or the use of a targeted radioisotope therapy.

5. Conclusions and future perspectives The development of CXCR4 antagonists is an excellent example of how the incorporation of metal ions into a molecular structure can be applied to enhance protein recognition and binding properties. This is based on the replacement of hydrogen bonding interactions with coordination bonds to metal ions. In order to do this effectively, the metal ion must be stably bound into the molecule but still have additional available or exchangeable coordination sites to form bonds with amino acid residue side chains, such as carboxylates or imidazole nitrogen atoms. Specifically, in this work the use of cross bridged tetraazamacrocycles provides an excellent combination of characteristics; fixing the coordination environment with a rigid chelators and enhancing the metal complex stability. The outcomes of this approach can be observed in the measurement of the biological parameters in a range of assays. Particularly important for metallodrug design are improvements in the binding energy of the interaction with the receptor that will provide more effective antagonism of the signaling process. An increase in residence time of the molecule at the biological target is also observed which correlates with improved efficacy, more infrequent dosing and an improved biological response. This is a key parameter in drug design. The metal complexes investigated in the work to develop antagonists for the CXCR4 receptor allow for comparison of the use of one or two (spatially distinct) coordination interactions, i.e., one or two metal ions. It should be noted that part of the favorable interaction with the CXCR4 receptor is due to electrostatic interactions. The comparison of one copper(II) center vs two copper(II) centers shows that the major gain comes with the addition of the first copper(II) center, although there is some

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further improvement with the addition of the second metal center the mono-metal derivative is still an effective antagonist. There are two approaches that can be taken for targeting metal radionuclides in imaging or therapeutic applications with the CXCR4 receptor. The first is the replacement of one of the metal centers already in the molecular structure of the protein binding molecule. This can be achieved by substituting the stable copper-63/65 isotopes with the positron emitting copper-64 isotope. The advantage of this approach is that it produces an identical structure to the characterized antagonist and does not introduce any additional sites for conjugation that could be metabolized or negatively affect pharmacokinetics. However, if the use of a more commonly available radio-metal isotope such as gallium-68 is required then the molecular structure can be conjugated to a further chelator (e.g., DOTA or NOTA) which can subsequently be labeled with the radio-metal. This creates a challenge as the conjugation must not significantly reduce the affinity of the molecule for the receptor and metabolic stability must be maintained. Progress has been in the determination of the best combination of antagonist molecules, selection of conjugations sites spacers and chelators but further work is needed to optimize this approach. There is an exciting future ahead for the targeting of CXCR4 in medical imaging particularly positron emission tomography as the applications are tested clinically and new molecules allowing different imaging time points (with a wider range of half-lives) are introduced. The understanding of the prognostic information is developing, along with the capability to link the imaging data to treatment selection. The ultimate treatment link is to replace the positron emitting isotope with a beta or alpha emitting isotope that would target the tumor and deliver a localized dose of ionizing radiation. The imaging drug could then be used to select patients for treatment, calculate dosimetry and monitor therapy response. None of these applications would be possible without the understanding and optimization of the coordination chemistry. This approach, where there are coordination interactions on protein binding which can be optimized has now been established, but could be further incorporated into early stage molecular design and lead selection to extend the approach beyond the targeting of CXCR4. Other chemokine receptors (there are ca. 20 with roles in multiple diseases) are likely to offer an opportunity for early success in wider adoption of this approach as their surfaces are rich in aspartate and glutamate residues that are ideal for formation of coordination interactions.

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Acknowledgments I.R. wishes to thank the University of Hull for a studentship. S.J.A. would like to thank the Daisy Appeal charity (grant no. DAhul2011) and for funding, and Dr. Assem Allam and his family for the generous donation to help found the PET Research Center at the University of Hull and for their continued support.

References 1. 2. 3. 4. 5. 6.

7. 8. 9. 10. 11.

12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

Zlotnik, A.; Burkhardt, A. M.; Homey, B. Nat. Rev. Immunol. 2011, 11(9), 597–606. Balkwill, F. R. J. Pathol. 2012, 226(2), 148–157. Chow, M. T.; Luster, A. D. Cancer Immunol. Res. 2014, 2(12), 1125–1131. Citrin, D. E. N. Engl. J. Med. 2017, 377(11), 1065–1075. Kircher, M.; Herhaus, P.; Schottelius, M.; Buck, A. K.; Werner, R. A.; Wester, H. J.; Keller, U.; Lapa, C. Ann. Nucl. Med. 2018, 32(8), 503–511. Habringer, S.; Lapa, C.; Herhaus, P.; Schottelius, M.; Istvanffy, R.; Steiger, K.; SlottaHuspenina, J.; Schirbel, A.; Hanscheid, H.; Kircher, S.; Buck, A. K.; Gotze, K.; Vick, B.; Jeremias, I.; Schwaiger, M.; Peschel, C.; Oostendorp, R.; Wester, H. J.; Grigoleit, G. U.; Keller, U. Theranostics 2018, 8(2), 369–383. Blower, P. J. Dalton Trans. 2015, 44(11), 4819–4844. Gerlach, L. O.; Jakobsen, J. S.; Jensen, K. P.; Rosenkilde, M. R.; Skerlj, R. T.; Ryde, U.; Bridger, G. J.; Schwartz, T. W. Biochemistry 2003, 42(3), 710–717. Khan, A.; Nicholson, G.; Greenman, J.; Madden, L.; McRobbie, G.; Pannecouque, C.; De Clercq, E.; Ullom, R.; Maples, D. L.; Maples, R. D.; Silversides, J. D.; Hubin, T. J.; Archibald, S. J. J. Am. Chem. Soc. 2009, 131(10), 3416. Maurer, S.; Herhaus, P.; Lippenmeyer, R.; Hanscheid, H.; Kircher, M.; Schirbel, A.; Maurer, H. C.; Buck, A. K.; Wester, H. J.; Einsele, H.; Grigoleit, G. U.; Keller, U.; Lapa, C. J. Nucl. Med. 2019, 60(10), 1399–1405. Herrmann, K.; Schottelius, M.; Lapa, C.; Osl, T.; Poschenrieder, A.; Hanscheid, H.; Luckerath, K.; Schreder, M.; Bluemel, C.; Knott, M.; Keller, U.; Schirbel, A.; Samnick, S.; Lassmann, M.; Kropf, S.; Buck, A. K.; Einsele, H.; Wester, H. J.; Knop, S. J. Nucl. Med. 2016, 57(2), 248–251. Mankoff, D. A.; Farwell, M. D.; Clark, A. S.; Pryma, D. A. JAMA Oncol. 2017, 3(5), 695–701. Balkwill, F. Nat. Rev. Cancer 2004, 4(7), 540–550. Zlotnik, A.; Yoshie, O. Immunity 2012, 36(5), 705–716. Chatterjee, S.; Azad, B. B.; Nimmagadda, S. In Emerging Applications of Molecular Imaging to Oncology; Pomper, M. G., Fisher, P. B., Eds.; Elsevier Academic Press Inc: San Diego, 2014; Vol. 124, pp 31–82. Horuk, R. Cytokine Growth Factor Rev. 2001, 12(4), 313–335. Viola, A.; Luster, A. D. Annu. Rev. Pharmacol. Toxicol. 2008, 48, 171–197. Nagarsheth, N.; Wicha, M. S.; Zou, W. P. Nat. Rev. Immunol. 2017, 17(9), 559–572. Feng, Y.; Broder, C. C.; Kennedy, P. E.; Berger, E. A. Science 1996, 272(5263), 872–877. De Clercq, E. Nat. Rev. Drug Discov. 2003, 2(7), 581–587. Zhou, N. M.; Luo, Z. W.; Luo, J. S.; Liut, D. X.; Hall, J. W.; Pomerantz, R. J.; Huang, Z. W. J. Biol. Chem. 2001, 276(46), 42826–42833. Wu, B. L.; Chien, E. Y. T.; Mol, C. D.; Fenalti, G.; Liu, W.; Katritch, V.; Abagyan, R.; Brooun, A.; Wells, P.; Bi, F. C.; Hamel, D. J.; Kuhn, P.; Handel, T. M.; Cherezov, V.; Stevens, R. C. Science 2010, 330(6007), 1066–1071.

CXCR4-targeted metal complexes for molecular imaging

473

23. Bleul, C. C.; Farzan, M.; Choe, H.; Parolin, C.; ClarkLewis, I.; Sodroski, J.; Springer, T. A. Nature 1996, 382(6594), 829–833. 24. Kucia, M.; Jankowski, K.; Reca, R.; Wysoczynski, M.; Bandura, L.; Allendorf, D. J.; Zhang, J.; Ratajczak, J.; Ratajczak, M. Z. J. Mol. Histol. 2004, 35(3), 233–245. 25. Pozzobon, T.; Goldoni, G.; Viola, A.; Molon, B. Immunol. Lett. 2016, 177, 6–15. 26. Crump, M. P.; Gong, J. H.; Loetscher, P.; Rajarathnam, K.; Amara, A.; ArenzanaSeisdedos, F.; Virelizier, J. L.; Baggiolini, M.; Sykes, B. D.; Clark-Lewis, I. EMBO J. 1997, 16(23), 6996–7007. 27. Balkwill, F. Semin. Cancer Biol. 2004, 14(3), 171–179. 28. Joao, H. C.; Devreese, K.; Pauwels, R.; Declercq, E.; Henson, G. W.; Bridger, G. J. J. Med. Chem. 1995, 38(19), 3865–3873. 29. Teixido, J.; Martinez-Moreno, M.; Diaz-Martinez, M.; Sevilla-Movilla, S. Int. J. Biochem. Cell Biol. 2018, 95, 121–131. 30. Thoma, G.; Streiff, M. B.; Kovarik, J.; Glickman, F.; Wagner, T.; Beerli, C.; Zerwes, H. G. J. Med. Chem. 2008, 51(24), 7915–7920. 31. Ichiyama, K.; Yokoyama-Kumakura, S.; Tanaka, Y.; Tanaka, R.; Hirose, K.; Bannai, K.; Edamatsu, T.; Yanaka, M.; Niitani, Y.; Miyano-Kurosaki, N.; Takaku, H.; Koyanagi, Y.; Yamamoto, N. Proc. Natl. Acad. Sci. U. S. A. 2003, 100(7), 4185–4190. 32. Bridger, G. J.; Skerlj, R. T.; Thornton, D.; Padmanabhan, S.; Martellucci, S. A.; Henson, G. W.; Abrams, M. J.; Yamamoto, N.; Devreese, K.; Pauwels, R.; Declercq, E. J. Med. Chem. 1995, 38(2), 366–378. 33. Ciampolini, M.; Fabbrizzi, L.; Perotti, A.; Poggi, A.; Seghi, B.; Zanobini, F. Inorg. Chem. 1987, 26(21), 3527–3533. 34. Hatse, S.; Princen, K.; Bridger, G.; De Clercq, E.; Schols, D. FEBS Lett. 2002, 527(1–3), 255–262. 35. Este, J. A.; Cabrera, C.; De Clercq, E.; Struyf, S.; Van Damme, J.; Bridger, G.; Skerlj, R. T.; Abrams, M. J.; Henson, G.; Gutierrez, A.; Clotet, B.; Schols, D. Mol. Pharmacol. 1999, 55(1), 67–73. 36. Hatse, S.; Princen, K.; Gerlach, L. O.; Bridger, G.; Henson, G.; De Clercq, E.; Schwartz, T. W.; Schols, D. Mol. Pharmacol. 2001, 60(1), 164–173. 37. Liles, W. C.; Broxmeyer, H. E.; Rodger, E.; Wood, B.; Hubel, K.; Cooper, S.; Hangoc, G.; Bridger, G. J.; Henson, G. W.; Calandra, G.; Dale, D. C. Blood 2003, 102(8), 2728–2730. 38. De Clercq, E. Biochem. Pharmacol. 2009, 77(11), 1655–1664. 39. Burroughs, L.; Mielcarek, M.; Little, M. T.; Bridger, G.; MacFarland, R.; Fricker, S.; Labrecque, J.; Sandmaier, B. M.; Storb, R. Blood 2005, 106(12), 4002–4008. 40. Martin, C.; Bridger, G. J.; Rankin, S. M. Br. J. Haematol. 2006, 134(3), 326–329. 41. Devine, S. M.; Vij, R.; Rettig, M.; Todt, L.; McGlauchlen, K.; Fisher, N.; Devine, H.; Link, D. C.; Calandra, G.; Bridger, G.; Westervelt, P.; DiPersio, J. F. Blood 2008, 112(4), 990–998. 42. De Clercq, E. Pharmacol. Ther. 2010, 128(3), 509–518. 43. Hatse, S.; Princen, K.; De Clercq, E.; Rosenkilde, M. M.; Schwartz, T. W.; Hernandez-Abad, P. E.; Skerlj, R. T.; Bridger, G. J.; Schols, D. Biochem. Pharmacol. 2005, 70(5), 752–761. 44. Rosenkilde, M. M.; Gerlach, L. O.; Hatse, S.; Skerlj, R. T.; Schols, D.; Bridger, G. J.; Schwartz, T. W. J. Biol. Chem. 2007, 282(37), 27354–27365. 45. Mewis, R. E.; Archibald, S. J. Coord. Chem. Rev. 2010, 254(15–16), 1686–1712. 46. Silversides, J. D.; Allan, C. C.; Archibald, S. J. Dalton Trans. 2007, 9, 971–978. 47. Bridger, G. J.; Skerlj, R. T. In Advances in Antiviral Drug Design; De Clercq, E. Ed.; Elsevier, 1999; Vol 3, pp 161–229.

474

Isaline Renard and Stephen J. Archibald

48. Burke, B. P.; Miranda, C. S.; Lee, R. E.; Renard, I.; Nigam, S.; Clemente, G. S.; D’huys, T.; Ruest, T.; Domarkas, J.; Thompson, J. A.; Schols, D.; Hubin, T. J.; Cawthorne, C. J.; Archibald, S. J. J. Nucl. Med. 2020, 61(1), 123–128. https://doi. org/10.2967/jnumed.118.218008, in press. 49. Bosnich, B.; Poon, C. K.; Tobe, M. L. Inorg. Chem. 1965, 4(8), 1102–1108. 50. Hunter, T. M.; Paisey, S. J.; Park, H. S.; Cleghorn, L.; Parkin, A.; Parsons, S.; Sadler, P. J. J. Inorg. Biochem. 2004, 98(5), 713–719. 51. Hunter, T. M.; McNae, I. W.; Simpson, D. P.; Smith, A. M.; Moggach, S.; White, F.; Walkinshaw, M. D.; Parsons, S.; Sadler, P. J. Chem. A Eur. J. 2007, 13(1), 40–50. 52. Liang, X. Y.; Parkinson, J. A.; Weishaupl, M.; Gould, R. O.; Paisey, S. J.; Park, H. S.; Hunter, T. M.; Blindauer, C. A.; Parsons, S.; Sadler, P. J. J. Am. Chem. Soc. 2002, 124(31), 9105–9112. 53. Liang, X. Y.; Weishaupl, M.; Parkinson, J. A.; Parsons, S.; McGregor, P. A.; Sadler, P. J. Chem.-Eur. J. 2003, 9(19), 4709–4717. 54. Timmons, J. C.; Hubin, T. J. Coord. Chem. Rev. 2010, 254(15–16), 1661–1685. 55. Hubin, T. J. Coord. Chem. Rev. 2003, 241(1–2), 27–46. 56. Wainwright, K. P. Inorg. Chem. 1980, 19(5), 1396–1398. 57. Kowallick, R.; Neuburger, M.; Zehnder, M.; Kaden, T. A. Helv. Chim. Acta 1997, 80(3), 948–959. 58. Valks, G. C.; McRobbie, G.; Lewis, E. A.; Hubin, T. J.; Hunter, T. M.; Sadler, P. J.; Pannecouque, C.; De Clercq, E.; Archibald, S. J. J. Med. Chem. 2006, 49(21), 6162–6165. 59. Khan, A.; Greenman, J.; Archibald, S. J. Curr. Med. Chem. 2007, 14(21), 2257–2277. 60. McRobbie, G.; Valks, G. C.; Empson, C. J.; Khan, A.; Silversides, J. D.; Pannecouque, C.; De Clercq, E.; Fiddy, S. G.; Bridgeman, A. J.; Young, N. A.; Archibald, S. J. Dalton Trans. 2007, (43), 5008–5018. 61. Siegfried, L.; Kowallick, R.; Kaden, T. A. Supramol. Chem. 2001, 13(2), 357–367. 62. Boiocchi, M.; Bonizzoni, M.; Fabbrizzi, L.; Foti, F.; Licchelli, M.; Poggi, A.; Taglietti, A.; Zema, M. Chem. Eur. J. 2004, 10(13), 3209–3216. 63. Plutnar, J.; Havlickova, J.; Kotek, J.; Hermann, P.; Lukes, I. New J. Chem. 2008, 32(3), 496–504. 64. Khan, A.; Silversides, J. D.; Madden, L.; Greenman, J.; Archibald, S. J. Chem. Commun. 2007, (4), 416–418. 65. Weisman, G. R.; Rogers, M. E.; Wong, E. H.; Jasinski, J. P.; Paight, E. S. J. Am. Chem. Soc. 1990, 112(23), 8604–8605. 66. Hubin, T. J.; McCormick, J. M.; Alcock, N. W.; Clase, H. J.; Busch, D. H. Inorg. Chem. 1999, 38(20), 4435–4446. 67. Niu, W. J.; Wong, E. H.; Weisman, G. R.; Lam, K. C.; Rheingold, A. L. Inorg. Chem. Commun. 1999, 2(8), 361–363. 68. Hubin, T. J.; Alcock, N. W.; Busch, D. H. Acta Crystallogr. Sect. C: Cryst. Struct. Commun. 2000, 56, 37–39. 69. Wong, E. H.; Weisman, G. R.; Hill, D. C.; Reed, D. P.; Rogers, M. E.; Condon, J. S.; Fagan, M. A.; Calabrese, J. C.; Lam, K. C.; Guzei, I. A.; Rheingold, A. L. J. Am. Chem. Soc. 2000, 122(43), 10561–10572. 70. Niu, W. J.; Wong, E. H.; Weisman, G. R.; Hill, D. C.; Tranchemontagne, D. J.; Lam, K. C.; Sommer, R. D.; Zakharov, L. N.; Rheingold, A. L. Dalton Trans. 2004, (21), 3536–3547. 71. Hubin, T. J.; McCormick, J. M.; Collinson, S. R.; Buchalova, M.; Perkins, C. M.; Alcock, N. W.; Kahol, P. K.; Raghunathan, A.; Busch, D. H. J. Am. Chem. Soc. 2000, 122(11), 2512–2522. 72. Abdulwahaab, B. H.; Burke, B. P.; Domarkas, J.; Silversides, J. D.; Prior, T. J.; Archibald, S. J. J. Org. Chem. 2016, 81(3), 890–898.

CXCR4-targeted metal complexes for molecular imaging

475

73. Lewis, E. A.; Allan, C. C.; Boyle, R. W.; Archibald, S. J. Tetrahedron Lett. 2004, 45(15), 3059–3062. 74. Silversides, J. D.; Burke, B. P.; Archibald, S. J. C. R. Chim. 2013, 16(6), 524–530. 75. Panneerselvam, J.; Jin, J.; Shanker, M.; Lauderdale, J.; Bates, J.; Wang, Q.; Zhao, Y. D.; Archibald, S. J.; Hubin, T. J.; Ramesh, R. PLoS One, 2015, 10(3), e0122439. 76. Maples, R. D.; Cain, A. N.; Burke, B. P.; Silversides, J. D.; Mewis, R. E.; D’Huys, T.; Schols, D.; Linder, D. P.; Archibald, S. J.; Hubin, T. J. Chem. A Eur. J. 2016, 22(36), 12916–12930. 77. Cain, A. N.; Freeman, T. N. C.; Roewe, K. D.; Cockriel, D. L.; Hasley, T. R.; Maples, R. D.; Allbritton, E. M. A.; D’Huys, T.; van Loy, T.; Burke, B. P.; Prior, T. J.; Schols, D.; Archibald, S. J.; Hubin, T. J. Dalton Trans. 2019, 48(8), 2785–2801. 78. Smith, R.; Huskens, D.; Daelemans, D.; Mewis, R. E.; Garcia, C. D.; Cain, A. N.; Freeman, T. N. C.; Pannecouque, C.; De Clercq, E.; Schols, D.; Hubin, T. J.; Archibald, S. J. Dalton Trans. 2012, 41(37), 11369–11377. 79. Woodard, L. E.; Nimmagadda, S. J. Nucl. Med. 2011, 52(11), 1665–1669. 80. Masuda, M.; Nakashima, H.; Ueda, T.; Naba, H.; Ikoma, R.; Otaka, A.; Terakawa, Y.; Tamamura, H.; Ibuka, T.; Murakami, T.; Koyanagi, Y.; Waki, M.; Matsumoto, A.; Yamamoto, N.; Funakoshi, S.; Fujii, N. Biochem. Biophys. Res. Commun. 1992, 189(2), 845–850. 81. Tamamura, H.; Omagari, A.; Oishi, S.; Kanamoto, T.; Yamamoto, N.; Peiper, S. C.; Nakashima, H.; Otaka, A.; Fujii, N. Bioorg. Med. Chem. Lett. 2000, 10(23), 2633–2637. 82. Fujii, N.; Oishi, S.; Hiramatsu, K.; Araki, T.; Ueda, S.; Tamamura, H.; Otaka, A.; Kusano, S.; Terakubo, S.; Nakashima, H.; Broach, J. A.; Trent, J. O.; Wang, Z. X.; Peiper, S. C. Angew. Chem. Int. Edit. 2003, 42(28), 3251–3253. 83. Ueda, S.; Oishi, S.; Wang, Z. X.; Araki, T.; Tamamura, H.; Cluzeau, J.; Ohno, H.; Kusano, S.; Nakashima, H.; Trent, J. O.; Peiper, S. C.; Fujii, N. J. Med. Chem. 2007, 50(2), 192–198. 84. Aluicio-Sarduy, E.; Ellison, P. A.; Barnhart, T. E.; Cai, W. B.; Nickles, R. J.; Engle, J. W. J. Label. Compd. Radiopharm. 2018, 61(9), 636–651. 85. Price, E. W.; Orvig, C. Chem. Soc. Rev. 2014, 43(1), 260–290. 86. Holland, J. P.; Vasdev, N. Dalton Trans. 2014, 43(26), 9872–9884. 87. Wadas, T. J.; Wong, E. H.; Weisman, G. R.; Anderson, C. J. Chem. Rev. 2010, 110(5), 2858–2902. 88. Azad, B. B.; Chatterjee, S.; Lesniak, W. G.; Lisok, A.; Pullambhatla, M.; Bhujwalla, Z. M.; Pomper, M. G.; Nimmagadda, S. Oncotarget 2016, 7(11), 12344–12358. 89. Vinogradov, A. A.; Yin, Y. Z.; Suga, H. J. Am. Chem. Soc. 2019, 141(10), 4167–4181. 90. Notni, J.; Pohle, K.; Wester, H. J. EJNMMI Res. 2012, 2, 28. 91. Lin, M.; Welch, M. J.; Lapi, S. E. Mol. Imaging Biol. 2013, 15(5), 606–613. 92. Lewis, E. A.; Boyle, R. W.; Archibald, S. J. Chem. Commun. 2004, 19, 2212–2213. 93. Silversides, J. D.; Smith, R.; Archibald, S. J. Dalton Trans. 2011, 40(23), 6289–6297. 94. Boswell, C. A.; Sun, X. K.; Niu, W. J.; Weisman, G. R.; Wong, E. H.; Rheingold, A. L.; Anderson, C. J. J. Med. Chem. 2004, 47(6), 1465–1474. 95. Burke, B. P.; Clemente, G. S.; Archibald, S. J. J. Label. Compd. Radiopharm. 2014, 57(4), 239–243. 96. Jacobson, O.; Weiss, I. D.; Szajek, L. P.; Niu, G.; Ma, Y.; Kiesewetter, D. O.; Farber, J. M.; Chen, X. Y. Theranostics 2011, 1, 251–262. 97. Jacobson, O.; Weiss, I.; Farber, J.; Chen, X. Y. J. Nucl. Med. 2010, 51, 282. 98. Jacobson, O.; Weiss, I. D.; Kiesewetter, D. O.; Farber, J. M.; Chen, X. Y. J. Nucl. Med. 2010, 51(11), 1796–1804. 99. Jacobson, O.; Weiss, I. D.; Szajek, L. P.; Niu, G.; Ma, Y.; Kiesewetter, D. O.; Peled, A.; Eden, H. S.; Farber, J. M.; Chen, X. Y. J. Control. Release 2012, 157(2), 216–223.

476

Isaline Renard and Stephen J. Archibald

100. Demmer, O.; Gourni, E.; Schumacher, U.; Kessler, H.; Wester, H. J. ChemMedChem 2011, 6(10), 1789–1791. 101. Gourni, E.; Demmer, O.; Schottelius, M.; D’Alessandria, C.; Schulz, S.; Dijkgraaf, I.; Schumacher, U.; Schwaiger, M.; Kessler, H.; Wester, H. J. J. Nucl. Med. 2011, 52(11), 1803–1810. 102. Wester, H. J.; Keller, U.; Schottelius, M.; Beer, A.; Philipp-Abbrederis, K.; Hoffmann, F.; Simecek, J.; Gerngross, C.; Lassmann, M.; Herrmann, K.; Pellegata, N.; Rudelius, M.; Kessler, H.; Schwaiger, M. Theranostics 2015, 5(6), 618–630. 103. Philipp-Abbrederis, K.; Herrmann, K.; Knop, S.; Schottelius, M.; Eiber, M.; Luckerath, K.; Pietschmann, E.; Habringer, S.; Gerngross, C.; Franke, K.; Rudelius, M.; Schirbel, A.; Lapa, C.; Schwamborn, K.; Steidle, S.; Hartmann, E.; Rosenwald, A.; Kropf, S.; Beer, A. J.; Peschel, C.; Einsele, H.; Buck, A. K.; Schwaiger, M.; Gotze, K.; Wester, H. J.; Keller, U. EMBO Mol. Med. 2015, 7(4), 477–487. 104. Lapa, C.; Luckerath, K.; Rudelius, M.; Schmid, J. S.; Schoene, A.; Schirbel, A.; Samnick, S.; Pelzer, T.; Buck, A. K.; Kropf, S.; Wester, H. J.; Herrmann, K. Oncotarget 2016, 7(8), 9288–9295. 105. Vag, T.; Steiger, K.; Rossmann, A.; Keller, U.; Noske, A.; Herhaus, P.; Ettl, J.; Niemeyer, M.; Wester, H. J.; Schwaiger, M. EJNMMI Res. 2018, 8, 90. 106. Bouter, C.; Meller, B.; Sahlmann, C. O.; Staab, W.; Wester, H. J.; Kropf, S.; Meller, J. J. Nucl. Med. 2018, 59(2), 320–326. 107. Schottelius, M.; Osl, T.; Poschenrieder, A.; Hoffmann, F.; Beykan, S.; Hanscheid, H.; Schirbel, A.; Buck, A. K.; Kropf, S.; Schwaiger, M.; Keller, U.; Lassmann, M.; Wester, H. J. Theranostics 2017, 7(9), 2350–2362. 108. Poschenrieder, A.; Schottelius, M.; Schwaiger, M.; Wester, H. J. EJNMMI Res. 2016, 6, 36. 109. Poschenrieder, A.; Osl, T.; Schottelius, M.; Hoffmann, F.; Wirtz, M.; Schwaiger, M.; Wester, H. J. Tomography 2016, 2(2), 85–93. 110. Jacobson, O.; Weiss, I. D.; Szajek, L.; Farber, J. M.; Kiesewetter, D. O. Bioorg. Med. Chem. 2009, 17(4), 1486–1493. 111. Weiss, I. D.; Jacobson, O.; Kiesewetter, D. O.; Jacobus, J. P.; Szajek, L. P.; Chen, X. Y.; Farber, J. M. Mol. Imaging Biol. 2012, 14(1), 106–114. 112. Nimmagadda, S.; Pullambhatla, M.; Stone, K.; Green, G.; Bhujwalla, Z. M.; Pomper, M. G. Cancer Res. 2010, 70(10), 3935–3944. 113. De Silva, R. A.; Peyre, K.; Pullambhatla, M.; Fox, J. J.; Pomper, M. G.; Nimmagadda, S. J. Nucl. Med. 2011, 52(6), 986–993. 114. Amor-Coarasa, A.; Kelly, J. M.; Singh, P. K.; Ponnala, S.; Nikolopoulou, A.; Williams, C.; Vedvyas, Y.; Jin, M. M.; Warren, J. D.; Babich, J. W. Molecules 2019, 24(8), 1612. 115. Brickute, D.; Braga, M.; Kaliszczak, M. A.; Barnes, C.; Lau, D.; Carroll, L.; Stevens, E.; Trousil, S.; Alam, I. S.; Nguyen, Q. D.; Aboagye, E. O. Mol. Pharm. 2019, 16(5), 2106–2117. 116. Poty, S.; Desogere, P.; Goze, C.; Boschetti, F.; D’Huys, T.; Schols, D.; Cawthorne, C.; Archibald, S. J.; Maecke, H. R.; Denat, F. Dalton Trans. 2015, 44(11), 5004–5016. 117. Poty, S.; Gourni, E.; Desogere, P.; Boschetti, F.; Goze, C.; Maecke, H. R.; Denat, F. Bioconjug. Chem. 2016, 27(3), 752–761. 118. Woodard, L. E.; De Silva, R. A.; Azad, B. B.; Lisok, A.; Pullambhatla, M.; Lesniak, W. G.; Mease, R. C.; Pomper, M. G.; Nimmagadda, S. Nucl. Med. Biol. 2014, 41(7), 552–561.