Positron emission tomography imaging probes targeting chemokine receptors

Positron emission tomography (PET) is a highly sensitive nuclear medicine imaging technique. PET is used to accurately diagnose cancer and can detect early stage tumours. Molecular probes containing a positron emitting metal radioisotope (such as ⁶⁴Cu or ⁶⁸Ga) need to give a stable complex in vivo as well as targeting biomarkers or metabolic processes within the tumour. The roles of chemokine receptors in multiple disease stages have been demonstrated. The CXCR4 and CCR5 chemokine receptors have been implicated in cancer, as well as other disease states including HIV infection and chronic inflammatory diseases, including asthma and rheumatoid arthritis. Incorporation of a positron emitting radioisotope into a CXCR4 or CCR5 specific antagonist compound could allow visualisation of physiological locations with high expression levels of these receptors to characterise the disease.The small molecule CXCR4 antagonist AMD3100 (Plerixafor) has been approved for clinical use as a haematopoietic stem cell mobilising agent and also exhibits anti-HIV, anti-inflammatory and anti-tumour activity. Configurationally restricted analogues of AMD3100 complexed to metal ions have improved binding characteristics compared to AMD3100 and its metal complexes. A synthetic pathway to obtain series of configurationally restricted macrocyclic compounds (analogues of AMD3100) fixed in the trans IV configuration, has been developed and the copper(II), zinc(II) and nickel(II) complexes characterised. Their biological properties (anti-HIV, cytotoxicity and Ca²⁺ signalling inhibition) were evaluated to allow selection of compounds to be radiolabelled with ⁶⁴Cu²⁺ for evaluation as a PET imaging agent targeting CXCR4. The most active trans IV complexes, bis(zinc(II))- 1,4-xylyl bis(methyl side-bridged cyclam and bis(zinc(II))-1,4-xylyl bis(benzyl sidebridged cyclam) have EC₅₀ values of 516 and 247 nM respectively in the anti-HIV assay with cyotoxicity (CC₅₀) values of 42800 and 39600 nM respectively. However, the novel mixed metal trans II complex (copper(II)zinc(II))-1,4-xylyl bis(sidebridged cyclam) has a higher binding affinity with an EC₅₀ value of 3 nM (four times more potent than AMD3100) and cytotoxicity CC₅₀ value greater than 10 µM. Bis(zinc(II))-1,4-xylyl bis(side-bridged cyclam) was successfully radiolabelled with ⁶⁴Cu²⁺ via transmetallation to form (⁶⁴Cu (zinc(II))-1,4-xylyl bis(side-bridged cyclam) with a crude radiochemical yield of 92%.A derivative of known CCR5 antagonist TAK-779 containing a carboxylic acid functional group 2-(p-tolyl)-6,7-dihydro-5H-benzo[7]annulene-8-carboxylic acid 18 was prepared. A DO3A compound with a spacer terminating in a primary amine (tritert-butyl 2,2',2''-(10-(2-((2-aminoethyl)amino)-2-oxoethyl)-1,4,7,10- tetraazacyclododecane-1,4,7-triyl)triacetate) was successfully conjugated to compound 18 forming a potential CCR5 targeting compound that could be radiolabelled with gallium-68 for PET imaging applications. Preliminary in vitro affinity assays indicated that the modification of the structure had disrupted the CCR5 binding and some structural modification redesign may be required.Radiolabelling of the conjugate compound 2-(p-tolyl)-6,7-dihydro-5Hbenzo[7]annulene-8-amidoethyl-DOTA with gallium-68 was carried out. A crude radiochemical yield of ca. 100% was achieved to give ⁶⁸Ga-(2-(p-tolyl)-6,7-dihydro-5H-benzo[7]annulene-8-amidoethyl-DOTA) which is stable in buffer and against transferring challenge for over four hours

Acetate as a model for aspartate-based CXCR4 chemokine receptor binding of cobalt and nickel complexes of cross-bridged tetraazamacrocycles

A number of disease states including WHIM syndrome, HIV infection and cancer have been linked to the chemokine receptor CXCR4. High-affinity CXCR4 antagonist transition metal complexes of configurationally restricted bis-tetraazamacrocyclic ligands have been identified in previous studies. Recently synthesised and structurally characterised Co2+/Co3+ and Ni2+ acetate complexes of mono-macrocycle cross-bridged ligands have been used to mimic their known coordination interaction with the aspartate side chains on binding to CXCR4. Here, X-ray crystal structures for three Co2+/Co3+ acetate complexes and five Ni2+ acetate complexes are presented and demonstrate flexibility in the mode of binding to the acetate ligand concomitantly with the requisite cis-V-configured cross-bridged tetraazamacrocyle. Complexes of the smaller Co3+ metal ion exclusively bind acetate by chelating both oxygens of acetate. Larger Co2+ and Ni2+ metal ions in cross-bridged tetraazamacrocycles show a clear tendency to coordinate acetate in a monodentate fashion with a coordinated water molecule completing the octahedral coordination sphere. However, in unbridged tetraazamacrocycle acetate structures reported in the literature, the coordination preference is to chelate both acetate oxygens. We conclude that the short ethylene cross-bridge restricts the equatorial bulk of the macrocycle, prompting the metal ion to fill the equator with the larger monodentate acetate plus water ligand set. In unbridged ligand examples, the flexible macrocycle expands equatorially and generally only allows chelation of the sterically smaller acetate alone. These results provide insight for generation of optimised bis-macrocyclic CXCR4 antagonists utilising cobalt and nickel ions

Synthesis and biological evaluation of CXCR4 chemokine receptor antagonists

The CXCR4 chemokine receptor is known to be overexpressed in many types of cancer as well as being involved in several stages of metastasis, which is responsible for the majority of deaths in cancer patients. Molecular imaging techniques, such as positron emission tomography (PET), are being used in the design of early stage CXCR4 expressing cancer diagnosis agents which can be followed by the administration of CXCR4 specific drugs targeting to an individual’s specific cancer treatment requirements to prevent the spread of cancer. Incorporation of positron emitting radioisotopes such as ¹⁸F and ⁶⁴Cu into a CXCR4 specific compound allows visualisation of the locations that the drug has accumulated. This can facilitate the diagnosis of a CXCR4 expressing cancer as large volumes of emission signals will be detected in tumours. Following diagnosis, high CXCR4-affinity targeted compounds can be administrated which prevent CXCR4’s binding partner, CXCL12, activating the receptor and enabling metastasis. This work focuses on the development of high affinity CXCR4 antagonists which can be used in therapeutic and diagnostic applications.A series of configurationally restricted complexes of copper(II), zinc(II) and nickel(II), where the metal ion is coordinated to tris-macrocycles, were synthesised. Tris-macrocycles have the potential to show improved affinity for the CXCR4 receptor because the third macrocyclic ring can facilitate more interactions with the CXCR4 receptor than reported bis-macrocycles, due to the extra interactions possible with the aspartate rich receptor surface. Biological studies revealed the tris-macrocyclic series has high affinity for CXCR4. [Zn₃18] ⁶⁺ showed nanomolar activity with an IC₅₀ value of 0.84 nM, 14 times more potent than the FDA approved drug AMD3100. Another high affinity tris-macrocycle, [Zn₃19] ⁶⁺ showed an IC₅₀ value of 1.86 nM also significantly lower than ADM3100. The high affinity compounds, [Zn₃18] ⁶⁺ and [Zn₃19] ⁶⁺, were radiolabelled with ⁶⁴Cu via transmetalation. Crude-radiochemical yields (crude-RCY) of 79% and 62% were achieved for ⁶⁴CuZn₂[18] and ⁶⁴CuZn₂[19] respectively. The complexes were identified as strongly hydrophilic with calculated LogP values of -3.69 and -2.20 for ⁶⁴CuZn₂[18] and ⁶⁴CuZn₂[19] respectively.A series of rigidified bis-macrocycle copper(II) and zinc(II) complexes were synthesised and explored as imaging agents. Routes to incorporate ¹⁸F and ⁶⁸Ga into the bis-macrocycles for applications in PET imaging were explored. Mono-macrocycles were synthesised and used in a series of test reactions to evaluate a range of pendant arm functionalisations including nitro, amine, azide and alkyne. Subsequently, azide functionalised rigidified bis-macrocycle copper(II) and zinc(II) complexes for use in copper-free click reactions were synthesised which provided an effective route to incorporate ¹⁸F into macrocycles. Biological studies identified that the functionalised bis-macrocycle complexes showed high affinity towards the CXCR4 receptor. The key compound was [Zn₂32] ⁴⁺ which showed IC₅₀ values of 3.22 nM, more than 3 times lower than AMD3100.A series of surface plasmon resonance (SPR) experiments were conducted to develop a method to determine the residence time of macrocycles on the CXCR4 receptor. A range of approaches, such as intact cell immobilisation, intact cell capture and receptor capture, were studied but all methods had drawbacks therefore association and dissociation rates of macrocycles and the CXCR4 receptor were not ascertained.This work highlights important steps towards the diagnosis of CXCR4 expressing cancers with the use of highly stable tris-macroycles. Furthermore, initial steps to incorporate the most readily available radioisotope, ¹⁸F, into macrocycles have been identified. Progress towards the development of high affinity, anti-metastatic therapeutic agents, in CXCR4 expressing cancers, has been made with the synthesis and in vitro evaluation of configurationally restricted tris-macrocycle metal complexes. Key compounds in this series were [Zn₃18] ⁶⁺ and [Zn₃19] ⁶⁺ which showed significantly better affinity for the CXCR4 receptor than AMD3100 and low toxicity

Configurationally restricted bis-tetraazamacrocyclic complexes: chemokine receptor antagonists

The chemokine receptor CXCR4 is a trans-membrane protein which has been implicated in many physiological and pathological processes including cancer, rheumatoid arthritis and most significantly HIV replication. CXCR4 plays a vital role in embryonic development but is not essential at the post-development stage; therefore, it has been identified as a potential therapeutic target.Bis-macrocyclic drugs (e.g. AMDS 100) bind to aspartate residues on the CXCR4 surface and inhibit HIV replication by blocking the interaction of gp!20/gp41 with the protein. The incorporation of transition metals (e.g. zinc(II) and copper(II)) into the macrocyclic cavity increases anti-viral potency. The addition of a bridging ethylene unit to the macrocyclic framework locks the complex into a single configuration, potentially optimising the interaction with the receptor.A series of configurationally restricted macrocyclic compounds have been prepared utilising bis-aminal chemistry. Characterisation by X-ray crystallography and X-ray absorption spectroscopy has confirmed that the complexes possessing an ethylene bridge between adjacent nitrogen atoms are fixed in the trans-II configuration and that complexes containing an ethylene bridge between non-adjacent nitrogen atoms adopt the cis-V configuration. In addition, solution EXAFS has been used as a model to probe the binding of the complexes to aspartate residues on the receptor surface.The zinc(II) trans-II and copper(II) cis-V complexes reported here are more potent against HIV replication than AMDS 100 (IC₅₀ values against IIIB ; 0.00208 µM, 0.00491 µM and 0.018 µM respectively), confirming the importance of coordination interactions for potent binding to CXCR4 and also validating the strategy of configurationally fixing the macrocyclic unit for optimising receptor binding. It is believed that both thermodynamic and kinetic properties are important for effective binding to CXCR4

Applying in silico Tools to the Discovery of Novel CXCR4 Inhibitors

International audienceThe process of HIV entry begins with the binding of the viral envelope glycoprotein gp120 to both the CD4 receptor and one of the CXCR4 or CCR5 chemokine coreceptors. There is currently considerable interest in developing novel ligands which can bind to these coreceptors and hence block virus-cell fusion. This article reviews the use of different in silico structure-based and ligand-based virtual screening (VS) tools for the discovery of potential HIV entry inhibitors for the CXCR4 receptor. More specifically, it discusses homology modelling, de novo design, docking, QSAR analyses, pharmacophore modelling, and similarity searches. Results from retrospective VS of a library of known CXCR4 inhibitors taken from the literature and from prospective VS of a combinatorial virtual library are reviewed. The structures of active compounds found by these approaches, as well as CXCR4 inhibitors currently in development are also discussed

Synthesis and characterisation of novel azamacrocyclic chelators for biomedical applications : biological activity and radiolabelling

Azamacrocyclic derivatives that contain nitrogen donors have been used for decades in analytical, industrial and medical applications. They are good hosts for metal ions, anions, neutral molecules and organic cation guests. Azamacrocyclic complexes that are biologically active have been used in the identification of diseased tissues. Transition metal macrocyclic complexes have also received significant attention due to their pharmacological properties such as toxicity against bacterial and fungal growth. Many macrocyclic complexes have been reported to have anti-inflammatory properties.A number of azamacrocyclic ligands have been used in the design of bifunctional chelators (BFCs) that have been utilised in molecular imaging. Incorporation of a positron emitting metal radioisotope such as 68Ga requires a BFC to form a stable complex in vivo and for covalent bond formation (conjugation) with a targeting moiety. Macrocyclic complexes are essential to a number of biological systems and have shown affinity for the CXCR4 chemokine receptor. Studies confirmed that CXCR4 is an important factor in the migration, invasiveness, proliferation and metastasis of tumours and overexpression of CXCR4 has been shown in twenty three different human tumours including non-small cell lung cancer, ovarian cancer, prostate cancer, colorectal cancer and breast cancer.In this work three different classes of azamacrocycles have been synthesised. The first group is a series of configurationally restricted copper(II), zinc(II) and nickel(II) mono-ring macrocycles bearing benzimidazole derivatised pendant arms. The antifungal activity has been determined for both ligands and their transition metal complexes. Most of the compounds tested showed a recognisable activity. The zinc(II) complexes of the ligands (side-bridged cyclen benzimidazole and side-bridged cyclen 4-nitrobenzyl benzimidazole) showed the highest antifungal activity of the compounds tested.The second class of azamacrocyclic derivatives synthesised comprises five novel bifunctional chelators based on benzimidazole TACN and NO2A derivatives. Four 68Ga complexes of the following ligands: NO2A benzimidazole, TACN tris 4-nitrobenzyl benzimidazole, NO2A 4-nitrobenzyl benzimidazole and NO2A 4-aminobenzyl benzimidazole, have been synthesised in radiochemical yields of 73%, 55%, 25% and 37% respectively at RT with a 5 minute reaction time.The third type of azamacrocyclic ligands are C-functionalised bis-tetraazamacrocyclic derivatives to be used as CXCR4 antagonists. Four new C-functionalised bis-azamacrocycles that have an amino group to allow for subsequent conjugation were synthesised: 4-aminobenzyl-C-functionalised side-bridged bis-cyclam, 4-aminobenzyl-C-functionalised side-bridged bis-cyclam cyclen, 4-aminobenzyl-C-functionalised bis-cyclam and 4-aminobenzyl-C-functionalised bis-cyclam cyclen. Zinc(II), nickel(II) and copper(II) complexes of the 4-nitrobenzyl-C-functionalised side-bridged bis-cyclam were synthesised and characterised as CXCR4 antagonists. A selection of the synthesised compounds was biologically evaluated in a number of assays (displacement assays, anti-HIV assays, cytotoxicity assays and calcium(II) signalling assays) with the free ligands showing activity and the metal complexes also acxtive and significantly more potent

Towards the synthesis of multifunctional constructs : coupling PET and PDT for the targeted diagnosis and therapy of CXCR4 expressing tumours

To establish accurate and early stage tumour diagnosis, quantitative molecular imaging modalities such as positron emission tomography (PET) are being employed. Molecular probes containing a positron emitting isotope (e.g. ¹⁸F, ⁶⁴Cu) are designed to target biomarkers or metabolic processes within the tumour. A therapeutic molecule could then be conjugated to this diagnostic probe to create a multifunctional construct (MFC). MFCs are a new concept in drug design offering the potential for both diagnosis and therapy. This work exploits the chelating properties of saturated tetraazamacrocycles known herein as macrocycles as well as their known affinity for the chemokine receptor CXCR4, a cell surface receptor involved in embryonic development and cell migration known to be overexpressed in at least twenty three different tumour types, along with the photodynamic activity of porphyrin molecules to create novel MFCs with diagnostic and therapeutic properties.A series of configurationally restricted copper(II), zinc(II) and nickel(II) mono-ring macrocycles bearing a methylamine pendant arm are presented. Monomacrocycles possess the potential for improved pharmacological properties. Biological studies assessing the affinity and potency of these macrocycles as CXCR4 antagonists demonstrated that zinc(II) monomacrocycles were the most potent in their class. A side bridged (SB) zinc(II) monocyclen compound; [Zn9]²⁺ synthesised in 64% yield, demonstrated the highest affinity for CXCR4 in its class showing a 2.5-fold higher affinity in comparison to the clinically licensed antagonist AMD3100 (IC₅₀ = 12 nM vs. 31 nM).In response to the high affinity observed for a meta-substituted biscyclen ligand (L48) a novel cross bridged (CB) meta-substituted biscyclen compound bearing a methylamine pendant arm 20 was synthesised along with its copper(II), nickel(II) and zinc(II) complexes in 77%, 88% and 90% yields, respectively. These metal complexes displayed high affinity for CXCR4 when competed with a CXCR4 specific antibody (% inhibition >85%) and required sub-micromolar concentrations to reduce the ‘normal’ signalling of CXCR4 by 50%. [Cu₂20]⁴⁺ has the potential to be a targeted PET probe through the use of ⁶⁴Cu and so a detailed biological evaluation into its binding mode was undertaken. These assays concluded that [Cu₂20]⁴⁺ exerts its antagonistic effect by locking the receptor on the surface of the cell, stopping the internalisation and recycling processes, which would result in formation of a new receptor. An analogous meta-substituted biscyclam ligand (28) and its copper(II), zinc(II) and nickel(II) complexes were synthesised in 74%, 58% and 46% yields, respectively. These metal complexes also demonstrated high affinity for CXCR4 (IC₅₀ <0.1 μM). In fact [Cu₂28]⁴+ showed unexpectedly high affinity for CXCR4 demonstrating almost a 3-fold higher affinity than AMD3100 (IC₅₀ = 11nM vs. 31 nM), the potential of [Cu₂28]⁴⁺ as a targeted PET probe should also be considered.Various organic spacer arms were attached to bismacrocycle 20, to produce a CXCR4 targeting probe with a functional arm for further conjugation which would also act to prevent steric hindrance. An acidic terminating chain was successfully attached to bismacrocycle 20 in quantitative yield, via ring-opening of diglycolic anhydride as well as an amine terminating variant (93% yield). A biotin tag was also attached to bimacrocycle 20 and its copper(II) complex was successfully isolated in a 94% yield. Addition of a spacer arm was found to have no significantly detrimental effect on binding affinity to CXCR4.A series of known water and organic soluble metalloporphyrins were synthesised. Subsequent conjugation to macrocyclic compounds proved challenging but analytical data for the conjugation of bismacrocycle 20 and metalloporphyrin [Zn59] indicated a successful reaction had occurred. This reaction represents important steps towards the synthesis of MFCs with diagnostic and therapeutic properties

Development and evaluation of CXCR4 receptor targeted probes for medical imaging applications

The chemokine receptor CXCR4 has been shown to be overexpressed in over 23 different types of cancers, making it an attractive target for therapeutic and imaging agents. Molecular imaging techniques, such as positron emission tomography (PET) and single photon emission tomography (SPECT), are being used for the development of CXCR4 receptor targeted cancer diagnostic agents. This work exploits the properties of tetraazamacrocycles and their known affinity for the chemokine receptor CXCR4. The inclusion of copper-64, gallium-68, fluorine-18, technetium-99m and optical probes allows the high affinity macrocyclic compounds to be developed as imaging agents.Copper-64 labelled CXCR4 targeted PET tracers have been reported in the literature (AMD3100 and AMD3465), however, they suffer from transchelation and loss of the radiolabel in vivo. Novel tetraazamacrocyclic tracers [64Cu][Cu5(OAc)](OAc) and [64Cu][Cu25(OAc)2](OAc)2 were produced in a decay corrected RCY of 41.6 ± 4.6% and 75.4 ± 1.5%. Calcium signalling assays gave an IC50 value of 60 nM and 4 nM, respectively. In vitro cell binding experiments showed that [64Cu][Cu5(OAc)](OAc) had a higher affinity for the CXCR4 receptor when compared to other CXCR4 imaging tracers. In vivo experiments carried out in mice revealed the tracer [64Cu][Cu5(OAc)](OAc) to be specific to CXCR4 with an 8-fold higher uptake seen in the CXCR4 positive cell lines compared to the negative; (23.6 ± 2.7; 3.0 ± 0.5 respectively). Importantly no liver uptake was seen when a blocking dose was administered, indicating the improved stability of the CB cyclam structure.Several approaches for developing a novel gallium-68 CXCR4 targeted PET tracer were investigated. In vitro and in vivo data showed that the inclusion of DOTAGA in [Zn229(OAc)2](OAc)2 resulted in a decrease in affinity. Alternative PEG chain spacer and pretargeted approaches were investigated to overcome this issue. A THP analogue of Pentixafor (P5) was synthesised and radiolabelled in a 76.8% decay corrected RCY. In vivo studies were carried out and showed that the lipophilicity of the THP moiety is detrimental to the biodistribution of the tracer. Inorganic radiofluorination was attempted for the synthesis of fluorine-18 labelled CXCR4 targeting PET probes; with the antagonist [Zn2AlF43(OAc)2](OAc)2 showing high affinity towards the receptor (IC50 = 20 nM).Novel CXCR4 targeted SPECT tracers such as [99mTc][Cu2Tc51(OAc)2](OAc)2 (IC50 = 16 nM) were synthesised and radiolabelled at up to 46.0% RCY. Optical imaging CXCR4 probes were also developed including a novel aza-BODIPY labelled peptide (P7). The antagonist maintained high affinity (IC50 = 42 nM) towards the CXCR4 receptor with initial confocal experiments indicating that the NIR probe is worthy of further investigation

Identification of Anti-Malarial Compounds as Novel Antagonists to Chemokine Receptor CXCR4 in Pancreatic Cancer Cells

Despite recent advances in targeted therapies, patients with pancreatic adenocarcinoma continue to have poor survival highlighting the urgency to identify novel therapeutic targets. Our previous investigations have implicated chemokine receptor CXCR4 and its selective ligand CXCL12 in the pathogenesis and progression of pancreatic intraepithelial neoplasia and invasive pancreatic cancer; hence, CXCR4 is a promising target for suppression of pancreatic cancer growth. Here, we combined in silico structural modeling of CXCR4 to screen for candidate anti-CXCR4 compounds with in vitro cell line assays and identified NSC56612 from the National Cancer Institute's (NCI) Open Chemical Repository Collection as an inhibitor of activated CXCR4. Next, we identified that NSC56612 is structurally similar to the established anti-malarial drugs chloroquine and hydroxychloroquine. We evaluated these compounds in pancreatic cancer cells in vitro and observed specific antagonism of CXCR4-mediated signaling and cell proliferation. Recent in vivo therapeutic applications of chloroquine in pancreatic cancer mouse models have demonstrated decreased tumor growth and improved survival. Our results thus provide a molecular target and basis for further evaluation of chloroquine and hydroxychloroquine in pancreatic cancer. Historically safe in humans, chloroquine and hydroxychloroquine appear to be promising agents to safely and effectively target CXCR4 in patients with pancreatic cancer