Two new types of unsymmetrical bis(thiosemicarbazone) proligands and their neutral zinc(II) and copper(II) complexes have been synthesized. These bifunctional ligands both chelate the metal ions and provide pendent amino groups that can be readily functionalized with biologically active molecules. Functionalization has been demonstrated by the synthesis of three water-soluble glucose conjugates of the new zinc(II) bis(thiosemicarbazonato) complexes, and their copper(II) analogues have been prepared in aqueous solution via transmetalation. A range of techniques including NMR, electron paramagnetic resonance, cyclic voltammetry, high-performance liquid chromatography (HPLC), UV/vis, and fluorescence emission spectroscopy have been used to characterize the complexes. Four compounds, including two zinc(II) complexes, have been characterized by X-ray crystallography. The connectivity and conformation of the glucose conjugates have been assigned by NMR spectroscopy. Time-dependent density functional theory calculations have been used to assign the electronic transitions of the copper(II) bis(thiosemicarbazonato) chromophore. Two copper-64-radiolabeled complexes, including one glucose conjugate, have been prepared and characterized using radio-HPLC, and transmetalation is shown to be a viable method for radiolabeling compounds with copper radionuclides. Preliminary cell washout studies have been performed under normoxic conditions, and the uptake and intracellular distribution have been studied using confocal fluorescence microscopy.

64 Cu-diacetyl-bis(N 4 -methylthiosemicarbazonate), 64 Cu-ATSM, continues to be investigated clinically as a PET agent both for delineation of tumor hypoxia and as an effective indicator of patient prognosis, but there are still aspects of the mechanism of action that are not fully understood. Methods: The retention of radioactivity in tumors after administration of 64 Cu-ATSM in vivo is substantially higher for tumors with a significant hypoxic fraction. This hypoxia-dependent retention is believed to involve the reduction of Cu-ATSM, followed by the loss of copper to cellular copper processing. To shed light on a possible role of copper metabolism in hypoxia targeting, we have compared 64 Cu retention in vitro and in vivo in CaNT and EMT6 cells or cancers after the administration of 64 Cu-ATSM or 64 Cu-acetate. Results: In vivo in mice bearing CaNT or EMT6 tumors, biodistributions and dynamic PET data are broadly similar for 64 Cu-ATSM and 64 Cu-acetate. Copper retention in tumors at 15 min is higher after injection of 64 Cu-acetate than 64 Cu-ATSM, but similar values result at 2 and 16 h for both. Colocalization with hypoxia as measured by EF5 immunohistochemistry is evident for both at 16 h after administration but not at 15 min or 2 h. Interestingly, at 2 h tumor retention for 64 Cuacetate and 64 Cu-ATSM, although not colocalizing with hypoxia, is reduced by similar amounts by increased tumor oxygenation due to inhalation of increased O 2 . In vitro, substantially less uptake is observed for 64 Cu-acetate, although this uptake had some hypoxia selectivity. Although 64 Cu-ATSM is stable in mouse serum alone, there is rapid disappearance of intact complex from the blood in vivo and comparable amounts of serum bound activity for both 64 Cu-ATSM and 64 Cu-acetate. Conclusion: That in vivo, in the EMT6 and CaNT tumors studied, the distribution of radiocopper from 64 Cu-ATSM in tumors essentially mirrors that of 64 Cu-acetate suggests that copper metabolism may also play a role in the mechanism of selectivity of Cu-ATSM.

New M(II) bis(thiosemicarbazonato) complexes (M = Ni(II), Cu(II) and Zn(II)) featuring allyl groups at the exocyclic nitrogens have been synthesised. The complexes were characterised in solution by spectroscopic methods and their solid state structures determined by single crystal X-ray diffraction using synchrotron radiation. The Zn(II) complex was found to be intrinsically fluorescent and soluble in biocompatible media. The uptake of this Zn(II) complex in HeLa, MCF-7 and IGROV cancer cells was monitored by fluorescence microscopies (epi- and confocal fluorescence imaging). The radiolabelling to (64)Cu(II) bis(thiosemicarbazonato) complex was performed cleanly by transmetallation from the corresponding Zn(II) species using (64)Cu(OAc)(2).

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.

Bis(thiosemicarbazones) have been of interest to chemists for over fifty years; they display antitumour, antibiotic and antiviral properties. Recently it has become apparent that they may also provide a convenient way of labelling biologically active molecules by using metallic radionuclides and/or fluorescence. Although apparently simple, the synthesis of bis(thiosemicarbazone) ligands can be problematic. This article provides a summary of the published literature, based on the synthetic strategies used and indicates some of the difficulties that may arise.

The increased resistance of hypoxic cells to all forms of cancer therapy presents a major barrier to the successful treatment of most solid tumors. Inhibition of the essential kinase Checkpoint kinase 1 (Chk1) has been described as a promising cancer therapy for tumors with high levels of hypoxia-induced replication stress. However, as inhibition of Chk1 affects normal replication and induces DNA damage, these agents also have the potential to induce genomic instability and contribute to tumorigenesis. To overcome this problem, we have developed a bioreductive prodrug, which functions as a Chk1/Aurora A inhibitor specifically in hypoxic conditions. To achieve this activity, a key functionality on the Chk1 inhibitor (CH-01) is masked by a bioreductive group, rendering the compound inactive as a Chk1/Aurora A inhibitor. Reduction of the bioreductive group nitro moiety, under hypoxic conditions, reveals an electron-donating substituent that leads to fragmentation of the molecule, affording the active inhibitor. Most importantly, we show a significant loss of viability in cancer cell lines exposed to hypoxia in the presence of CH-01. This novel approach targets the most aggressive and therapy-resistant tumor fraction while protecting normal tissue from therapy-induced genomic instability.

The detection and segmentation of adherent eukaryotic cells from brightfield microscopy images represent challenging tasks in the image analysis field. This paper presents a free and open-source image analysis package which fully automates the tasks of cell detection, cell boundary segmentation, and nucleus segmentation in brightfield images. The package also performs image registration between brightfield and fluorescence images. The algorithms were evaluated on a variety of biological cell lines and compared against manual and fluorescence-based ground truths. When tested on HT1080 and HeLa cells, the cell detection step was able to correctly identify over 80% of cells, whilst the cell boundary segmentation step was able to segment over 75% of the cell body pixels, and the nucleus segmentation step was able to correctly identify nuclei in over 75% of the cells. The algorithms for cell detection and nucleus segmentation are novel to the field, whilst the cell boundary segmentation algorithm is contrast-invariant, which makes it more robust on these low-contrast images. Together, this suite of algorithms permit brightfield microscopy image processing without the need for additional fluorescence images.Finally our sephaCe application, which is available at http:// www.sephace.com, provides a novel method for integrating these methods with any motorised microscope, thus facilitating the adoption of these techniques in biological research labs.

Combretastatins are stilbene-based, tubulin depolymerization agents with selective activity against the tumor vasculature; two variants (A-1 and A-4) are currently undergoing clinical trials. Combretastatin A-1 (CA1) has a greater antitumor effect than combretastatin A-4 (CA4). We hypothesized that this reflects the enhanced reactivity conferred by the second (ortho) phenolic moiety in CA1. Oxidation of CA1 by peroxidase, tyrosinase, or Fe(III) generates a species with mass characteristics of the corresponding ortho-quinone Q1. After administration of CA1-bis(phosphate) to mice, the hydroquinone-thioether conjugate Q1H2-SG, formed from the nucleophilic addition of GSH to Q1, was detected in liver. In competition, electrocyclic ring closure of Q1, over a few minutes at pH 7.4, leads to a second ortho-quinone product Q2, characterized by exact mass and NMR. This product was also generated by human promyelocytic leukemia (HL-60) cells in vitro, provided that superoxide dismutase was added. Q2 is highly reactive toward glutathione (GSH) and ascorbate, stimulating oxygen consumption in a catalytic manner. Free radical intermediates formed during autoxidation of CA1 were characterized by EPR, and the effects of GSH and ascorbate on the signals were studied. Pulse radiolysis was used to initiate selective one-electron oxidation or reduction and provided further evidence, from the differing absorption spectra of the radicals formed on oxidation of CA1 or reduction of Q2, that two different quinones were formed on oxidation of CA1. The results demonstrate fundamental differences between the pharmacological properties of CA1 and CA4 that provide two possible explanations for their differential activities in vivo: oxidative activation to a quinone intermediate likely to bind to protein thiols and possibly to nucleic acids and stimulation of oxidative stress by enhancing superoxide/hydrogen peroxide production. The observation of the GSH conjugate Q1H2-SG in vivo provides a new marker for oxidative metabolism of relevance to current clinical trials of CA1-bis(phosphate) (OXi4503).