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A series of chemical in vitro assays are described to provide a rapid initial assessment of the in vivo stability and biological behaviour of potential new copper(II) based radiopharmaceutical agents. Chemical challenges using an excess of cysteine, glutathione (GSH) and histidine, which are models of S- and N-donor molecules found in vivo, are use...
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... simplest macrocycle in the series bears no substituents, and the structure of the Cu(II) derivative is shown in Figure 19. New macrocycles have also been constructed from amino acid components including lysine and glutamic acid ( Figure 20). These peptide based ligands are expected to have good bio-compatibility and the amino acid side residues provide pendant amino and carboxylate functional groups for subsequent conju- gation to targeting agents. ...
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... 32 Furthermore, UV−vis spectrophotometry was used as tool to assess the kinetic stability of the gallium 3.8 and indium 3.9 complexes prior to in vitro and in vivo radiolabeling studies using established assays. 33 The corresponding experimental details are reported in the SI. The stability was modeled on the metalloporphyrins in phosphate buffered solution (PBS) (pH = 7.0) and in serum with respect to the loss of metal ions. ...
Over the past decade, porphyrin derivatives have emerged as invaluable synthetic building blocks and theranostic kits for the delivery of cellular fluorescence imaging and photodynamic therapy. Tetraphenylporphyrin (TPP), its metal complexes, and related derivatives have been investigated for their use as dyes in histology and as components of multimodal imaging probes. The photophysical properties of porphyrin–metal complexes featuring radiometals have been a focus of our attention for the realization of fluorescence imaging probes coupled with radioimaging capabilities and therapeutic potential having “true” theranostic promise. We report hereby on the synthesis, radiochemistry, structural investigations, and preliminary in vitro and in vivo uptake studies on a range of functionalized porphyrin-based derivatives. In pursuit of developing new porphyrin-based probes for multimodality imaging applications, we report new functionalized neutral, polycationic, and polyanionic porphyrins incorporating nitroimidazole and sulfonamide moieties, which were used as targeting groups to improve the notoriously poor pharmacokinetics of porphyrin tags. The resulting functional metalloporphyrin species were stable under serum challenges and the nitroimidazole and sulfonamide derivatives remained fluorescent, allowing in vitro confocal studies and visualization of the lysosomal uptake in a gallium(III) sulfonamide derivative. The molecular structures of selected porphyrin derivatives were determined by single crystal X-ray diffraction using synchrotron radiation. We also investigated the nature of the emission/excitation behavior of model functional porphyrins using in silico approaches such as TD DFT in simple solvation models. The conjugation of porphyrins with the [7-13] and [7-14] fragments of bombesin was also achieved, to provide targeting of the gastrin releasing peptide receptor (GRPR). Depending on the metal, probe conjugates of relevance for single photon emission computed tomography (SPECT) or positron emission tomography (PET) probes have been designed and tested hereby, using TPP and related functional free base porphyrins as the bifunctional chelator synthetic scaffold and ¹¹¹In[In] or ⁶⁸Ga[Ga], respectively, as the central metal ions. Interestingly, for simple porphyrin conjugates good radiochemical incorporation was obtained for both radiometals, but the presence of peptides significantly diminished the radio-incorporation yields. Although the gallium-68 radiochemistry of the bombesin conjugates did not show radiochemical incorporation suitable for in vivo studies, likely because the presence of the peptide changed the behavior of the TPP-NH2 synthon taken alone, the optical imaging assays indicated that the conjugated peptide tags do mediate uptake of the porphyrin units into cells.
... The kinetic stability of 7a was studied under conditions relevant to biomedical applications. 16 The copper(II) complexes of cyclam 1, and the non-macrocyclic ligands 1,4-bis(3-aminopropyl) piperazine 4, and 1,2-bis(3-aminopropyl) ethylenediamine 3, were also prepared to compare their stability with that of 7a. The aqueous stability of 7a was rst compared with [Cu1]Cl 2 , by monitoring the UV/vis spectra of the complexes over time. ...
Single-step synthetic protocols are reported for novel macrocyclic copper(II) complexes incorporating a piperazine unit in the ligand backbone. Synchrotron X-ray structure determination confirmed the copper(II) coordination geometry in the solid state as distorted square pyramidal with chloride occupying the axial position above the N4 ligand plane. DFT calculations, electrochemical studies and UV/vis investigations in biologicalmedia all confirmed that compounds in this class have high stability, and comparewell in both thermodynamic and kinetic terms with the ubiquitous Cu(II) cyclam compounds, and are therefore of relevance for future use as synthetic scaffolds for the binding, transport and tracing of copper ions in vitro.
... blue copper proteins) requires an appreciation of how metal binding sites in proteins respond to redox processes (Rorabacher 2004). Chemical reduction followed by oxidation would seem to be crucial to the behaviour of radiolabelled copper complexes and their ability to sense hypoxia in tumours (Barnard et al. 2008), and there is also interest in the potential of copper macrocyles to act of superoxide dismutase mimetics (Fernandes et al. 2007). ...
... In biological systems such as plasma and the cytoplasmic environment, metals may also be sequestered from complexes by interactions with a variety of ligands, especially proteins (Barnard et al. 2008). This is especially true for copper, which is known to participate in ligand exchange reactions on a rapid time scale. ...
... Research on redox active copper macrocycles to date has focused on ligands designed around thiosemicarbazones and tetra-aza-macrocycles (Barnard et al. 2008;Barbaro et al. 2000), but typically, these do not support both copper(I) and copper(II) forms. Oxidative stress and redox imbalance occur in inflammatory loci, and complexes that are able to undergo oxidation and reduction cycles would be able to report on the redox status, so long as they are relatively resistant to demetallation by proteins and other biological metal chelators. ...
A series of simple copper N(2)S(2) macrocycles were examined for their potential as biological redox sensors, following previous characterization of their redox potentials and crystal structures. The divalent species were reduced by glutathione or ascorbate at a biologically relevant pH in aqueous buffer. A less efficient reduction was also achieved by vitamin E in DMSO. Oxidation of the corresponding univalent copper species by sodium hypochlorite resulted in only partial (~65 %) recovery of the divalent form. This was concluded to be due to competition between metal oxidation and ligand oxidation, which is believed to contribute to macrocycle demetallation. Electrospray mass spectrometry confirmed that ligand oxidation had occurred. Moreover, the macrocyclic complexes could be demetallated by incubation with EDTA and bovine serum albumin, demonstrating that they would be inappropriate for use in biological systems. The susceptibility to oxidation and demetallation was hypothesized to be due to oxidation of the secondary amines. Consequently these were modified to incorporate additional oxygen donor atoms. This modification led to greater resistance to demetallation and ligand oxidation, providing a better platform for further development of copper macrocycles as redox sensors for use in biological systems.
... [19] example, approximately 1.188 (standard deviation error 0.043) and 1.209 (standard deviation 0.076), respectively, which is indicative of highly lipophilic species. These values are comparable with the reported values for the known hypoxia tracer [CuA C H T U N G T R E N N U N G (ATSM)], with a log P of approximately 1.480.202122 Compounds 1 and 2 showed similar serum-protein binding characteristics to [CuA C H T U N G T R E N N U N G (ATSM)], as demonstrated by standard radioassays; 17.3 % (standard deviation error 0.4 %) of 1 was shown to bind to serum protein after 5 min and 25.2 % (standard deviation error 2.6 %) after 1 h, whereas under the same experimental conditions, binding of 2 was 21.6 % (standard deviation error 0.5 %) after 5 min and 24.5 % (standard deviation error 1.7 %) after 1 h. ...
... Compounds 1 and 2 showed similar serum-protein binding characteristics to [CuA C H T U N G T R E N N U N G (ATSM)], as demonstrated by standard radioassays; 17.3 % (standard deviation error 0.4 %) of 1 was shown to bind to serum protein after 5 min and 25.2 % (standard deviation error 2.6 %) after 1 h, whereas under the same experimental conditions, binding of 2 was 21.6 % (standard deviation error 0.5 %) after 5 min and 24.5 % (standard deviation error 1.7 %) after 1 h. Again, these values compare well with that reported for [CuA C H T U N G T R E N N U N G (ATSM)], which had approximately 20 % serum-protein binding at 1 h.202122 Structural Investigations by ...
Copper bis(4-ethyl-3-thiosemicarbazonato) acenaphthenequinone (1) and copper bis(4-methyl-3-thiosemicarbazonato) acenaphthenequinone (2) are synthesized and characterized in solution, in the solid state, and radiolabeled. Serum-protein binding radioassays show good stability in solution and about 25 % binding to protein over 1 h, which is comparable with the hypoxia selective tracer [(64)Cu(ATSM)]. Cyclic voltammetry shows fast and reversible reduction at redox potentials similar to the values known for hypoxia-selective copper compounds. However, despite this, complex 1 does not show any hypoxic-selective uptake in HeLa cells over 1-h standard assays. Possible reasons for this are studied by using the intrinsic fluorescence of the Cu(II) complexes to determine the cellular distributions and uptake mechanism by confocal microscopy. The complexes are found to bind to the external cell membrane and disperse evenly in the cytoplasm only after a very slow cell internalization (>1 h). No significant changes in distribution are observed by fluorescence imaging under hypoxic conditions. The rate of localization in the cytoplasm contrasts with their Zn(II) analogues, which are known to have fast cell uptake (up to 20 min) and a clear localization in lysosomes and mitochondria. The cytotoxicity mechanism of 1 over 24 h against a number of adherent cell lines is seen to be by membrane disruption and is of a comparable magnitude to that of [Cu(ATSM)], as demonstrated by methyl tetrazolium (MTT) and lactate dehydrogenase (LDH) assays.
Copper complexes are of medicinal and biological interest, including as anticancer drugs designed to cleave intracellular biomolecules by O2 activation. To exhibit such activity, the copper complex must be redox active and resistant to dissociation. Metallothioneins (MTs) and glutathione (GSH) are abundant in the cytosol and nucleus. Because they are thiol‐rich reducing molecules with high CuI affinity, they are potential competitors for a copper ion bound in a copper drug. Herein, we report the investigation of a panel of CuI/CuII complexes often used as drugs, with diverse coordination chemistries and redox potentials. We evaluated their catalytic activity in ascorbate oxidation based on redox cycling between CuI and CuII, as well as their resistance to dissociation or inactivation under cytosolically relevant concentrations of GSH and MT. O2‐activating CuI/CuII complexes for cytosolic/nuclear targets are generally not stable against the GSH/MT system, which creates a challenge for their future design.
The development of novel radiopharmaceuticals for imaging and therapy requires rapid and reproducible in vitro assays to estimate their in vivo stability and dissociation behaviour. In general, these assays should allow an assessment of decomplexation of the radiometal from the radiopharmaceuticals. In the past, a series of chemical challenges has been widely used to estimate complex stability under non-physiological and non-radiotracer conditions providing limited information on the potential in vivo stability. In contrast, we herein present two independent in vitro methods to measure the stability of radiocopper complexes under physiologically relevant conditions. To quantify and compare the dissociation behaviour of six well-established 64Cu chelators (TETA, DOTA, NOTA, Cyclam, diamSar and EDTA), we combine a protein challenge experiment considering the stability of the chelators in the presence of human superoxide dismutase with a serum assay measuring the stability of the radiometal complexes against human serum. Unlike HPLC- and TLC-based analytical techniques, we describe the stability assessments by standard gel electrophoretic procedures, which allow a timesaving workflow as well as simultaneous processing and comparative analysis of a variety of copper-containing chelators and conjugates thereof. [64Cu]Cu-diamSar is the most kinetically stable ligand, whereas the acyclic chelator [64Cu]Cu-EDTA underwent an almost complete complex dissociation. Furthermore, kinetic stability studies in human serum carried out for [64Cu]Cu-diamSar revealed no substantial time-dependent influence under commonly used labelling conditions. Both described assays, the protein challenge experiment as well as the serum stability assay, are not restricted to radiocopper, but may be adopted for other radiometal containing chelators.
One of the pathological hallmarks of Alzheimer's disease is the presence of amyloid-β plaques in the brain and the major constituent of these plaques is aggregated amyloid-β peptide. New thiosemicarbazone-pyridylhydrazine based ligands that incorporate functional groups designed to selectively bind amyloid-β plaques have been synthesized. The new ligands form stable four coordinate complexes with a positron-emitting radioactive isotope of copper, (64)Cu. Two of the new Cu(II) complexes include a functionalized styrylpyridine group and these complexes bind selectively to amyloid-β plaques in samples of post-mortem human brain tissue. Strategies to increase brain uptake by functional group manipulation have led to a (64)Cu complex that effectively crosses the blood-brain barrier in mice. The new complexes described in this manuscript provide insight into strategies to deliver metal complexes to amyloid-β plaques.
Metal ions have been implicated in several neurodegenerative diseases, including Alzheimer's disease, as their dyshomeostasis may lead to production of reactive oxygen species as well as increased toxicity of amyloid protein aggregates. In this work, we present design and synthesis of three novel multifunctional hydroxypyridinone ligands, , , and , bearing benzothiazole and benzoxazole functionalities. We study the ability of these compounds to bind metal ions Cu(ii), Zn(ii), and Fe(iii), as well as their antioxidant activity and cytotoxicity. Additionally, we determine the pro-ligands' (compounds prior to chelation) propensity to target amyloid protein. Through these studies we determine the effect of combining amyloid- and metal-binding functionalities within the HPO scaffold on different aspects of AD pathology.
Four 2-acetylpyridine 4N-alkyl thiosemicarbazones, and their Ga(III) and In(III) complexes have been prepared and characterised by fluorescence, UV–Vis, IR, 1H and 13C NMR spectroscopy, mass spectrometry and X-ray crystallographic analysis. Comparison of the crystal structures gave an insight into the nature of the complexes formed, demonstrating a preference for [ML2]+ type complexes with gallium and [MLX3] species with indium. Stability studies on two candidates indicated that complex [InL3Cl2MeOH] was stable to chemical degradation for prolonged periods in human serum, giving this complex potential for further biological evaluation.
![a] Eirini Fragogeorgi](https://i1.rgstatic.net/ii/profile.image/272459416993839-1441970795803_Q64/Eirini-Fragogeorgi.jpg)
The reaction of the C-functionalized cyclam chelating agent 1,4,8,11-tetraazacyclotetradecane-6-carboxylic acid (1) with CuCl 2 generated a stable and neutral complex 2, which was characterized by elemental analysis, UV/Vis and IR spec-troscopy, electrospray ionization mass spectrometry (ESI-MS), EPR spectroscopy and X-ray crystallography. The sec-ondary amine groups of 1 were protected to generate 3, which was further conjugated with the bombesin (BN) deriv-ative H 2 N-(Ornithine) 3 -BN(2–14) by a solid phase peptide synthesis method. After cleavage from the resin and depro-tection, the resulting product 5 was obtained and charac-terized with ESI-MS and NMR spectroscopy, and was sub-sequently complexed under mild conditions with CuCl 2 to
