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Increased glycolysis and overexpression of glucose transporters (GLUTs) are physiological characteristics of human malignancies. Based on the so-called Warburg effect, 18flurodeoxyglucose-positron emission tomography (FDG-PET) has successfully developed as clinical modality for the diagnosis and staging of many cancers. To leverage this glucose transporter mediated metabolic disparity between normal and malignant cells, in the current report, we focus on the fluorine substituted series of glucose, mannose and galactose-conjugated (trans-R,R-cyclohexane-1,2-diamine)-2-flouromalonato-platinum(II) complexes for a comprehensive evaluation on their selective tumor targeting. Besides highly improved water solubility, these sugar-conjugates presented improved cytotoxicity than oxaliplatin in glucose tranporters (GLUTs) overexpressing cancer cell lines and exhibited no cross-resistance to cisplatin. For the highly water soluble glucose-conjugated complex (5a), two novel in vivo assessments were conducted and the results revealed that 5a was more efficacious at a lower equitoxic dose (70% MTD) than oxaliplatin (100% MTD) in HT29 xenograft model, and it was significantly more potent than oxaliplatin in leukemia-bearing DBA/2 mice as well even at equimolar dose levels (18% vs 90% MTD). GLUT inhibitor mediated cell viability analysis, GLUT1 knockdown cell line-based cytotoxicity evaluation, and platinum accumulation study demonstrated that the cellular uptake of the sugar-conjugates was regulated by GLUT1. The higher intrinsic DNA reactivity of the sugar-conjugates was confirmed by kinetic study of platinum(II)-guanosine adduct formation. The mechanistic origin of the antitumor effect of the fluorine complexes was found to be forming the bifunctional Pt-guanine-guanine (Pt-GG) intrastrand cross-links with DNA. The results provide a rationale for Warburg effect targeted anticancer drug design.

Two novel fluorescent bioprobes, namely, 6N-Gly-Cy3 and 6N-Gly-Cy5, were designed and synthesized for real-time glucose transport imaging as well as potentially useful tracer for galactokinase metabolism. The structure of the bioprobes was fully characterized by 1H NMR, 13C NMR, IR, and HRMS. The fluorescence properties, glucose transporter (GLUT) specificity, and the quenching and safety profiles were studied. The cellular uptake of both bioprobes was competitively diminished by d-glucose, 2-deoxy-d-glucose and GLUT specific inhibitor in a dose-dependent manner in human colon cancer cells (HT29). Comparison study results revealed that the 6N-derived bioprobes are more useful for real-time imaging of cell-based glucose uptake than the structurally similar fluorescent tracer 6-NBDG which was not applicable under physiological conditions. The up to 96 h long-lasting quenching property of 6N-Gly-Cy5 in HT29 suggested the potential applcability of the probe for cell labeling in xenograft transplantation as well as in vivo animal imaging studies.

Two novel cyanine-based 1-amino-1-deoxy-β-glucose conjugates (Glu-1N-Cy3 and Glu-1N-Cy5) were designed, synthesized and their fluorescence characteristics were studied. Both Glu-1N-Cy3 and Glu-1N-Cy5 accumulate in living HT29 human colon cancer cells, which overexpress glucose transporters (GLUTs). The cellular uptake of the bioprobes was inhibited by natural GLUT substrate d-glucose and 2-deoxy-d-glucose. The GLUT specificity of the probes was validated with quercetin, which is both a permeant substrate via GLUTs and a high-affinity inhibitor of GLUT-mediated glucose transport. Competitive fluorometric assay for GLUT substrate cell uptake revealed that Glu-1N-Cy3 and Glu-1N-Cy5 are 5 and 10 times more sensitive than 2-NBDG, a leading fluorescent glucose bioprobe. This study provides fundamental data supporting the potential of these two conjugates as new powerful tools for GLUT-mediated theranostics, in vitro and in vivo molecular bioimaging and drug R&D.

Malignant neoplasms exhibit a higher rate of glycolysis than normal cells; this is known as the Warburg effect. To target it, a galactose-conjugated (trans-R,R-cyclohexane-1,2-diamine)-2-chloromalonato-platinum(II) complex (Gal-Pt) was designed, synthesized, and evaluated in five human cancer cell lines and against two different xenograft tumour models. Gal-Pt exhibits much higher aqueous solubility (over 25 times) and improved cytotoxicity than oxaliplatin, especially in human colon (HT29) and lung (H460) cancer cell lines. The safety profile of Gal-Pt was investigated in vivo by exploring the maximum tolerated dose (MTD) and animal mortality rate. The ratios of the animal lethal dosage values to the cytotoxicity in HT29 (LD50/IC50) showed that Gal-Pt was associated with an increased therapeutic index by over 30-fold compared to cisplatin and oxaliplatin. We evaluated in vivo antitumor activity by single agent intravenous treatment comparison studies of Gal-Pt (50 mg/kg as 65% MTD) and cisplatin (3 mg/kg, as 80% MTD) in a H460 lung cancer xenograft model, and with oxaliplatin (7 mg/kg, as 90% MTD) in a HT29 colon cancer xenograft model. The results show that Gal-Pt was more efficacious against H460 than cisplatin, and had superior potency in HT29 cells compared to oxaliplatin under nontoxic dosage conditions. The dependency between cytotoxicity of Gal-Pt and glucose transporters (GLUTs) was investigated by using quercetin as an inhibitor of GLUTs in HT29 cells. The cytotoxic potency of Gal-Pt was highly reduced by the inhibitor, suggesting that the uptake of Gal-Pt was regulated by glucose transporters. The GLUT mediated transportability and cellular uptake of Gal-Pt was also demonstrated using a fluorescent glucose bioprobe in HT29 competition assay.

Novel cis-2-methylmalonato(trans-R,R-cyclohexane-1,2-diamine)platinum(II) glycoconjugates derived from different sugar motifs, namely, glucose (Glu-Me-Pt), mannose (Man-Me-Pt) and galactose (Gal-Me-Pt) were designed and synthesized based on the third generation clinical drug oxaliplatin for potential glucose transporters (GLUTs) mediated tumor targeting. All platinum(II) glycoconjugates were characterized by 1H NMR, 13C NMR, IR, HRMS as well as 195Pt-NMR analysis. Despite their substantial improvement in water solubility, the conjugates exhibited comparable or better in vitro cytotoxicities than oxaliplatin determined in six different human cancer cell lines. Glu-Me-Pt has been shown to be more effective than cisplatin and oxaliplatin with improved therapeutic index in leukemia-bearing DBA/2 mice model. The potential GLUT transportability of the complexes was investigated using cell-based fluorescent competition assay and GLUT inhibitor mediated cell viability analysis in GLUT over-expressing HT29 cell line. Each sugar motif was found to be useful to enable the platinum(II) complexes as substrate for GLUT mediated cell uptake. In vitro DNA adduct formation analysis has been investigated for the first time for this class of compounds to reveal the intrinsic differences in antitumor activity between the malonatoplatinum(II) glycoconjugates and oxaliplatin. The intrinsic DNA reactivity of the platinum(II)-sugar conjugates was found as Gal-Me-Pt > Glu-Me-Pt > Man-Me-Pt ≈ oxaliplatin by kinetic study on the formation of platinum(II) adducts with guanosine-5′-monophosphate (5′-GMP). The results from this study demonstrate the usefulness of glucose, mannose and galactose as alternative sugar motif on glycoconjugation for GLUT mediated drug design and pharmaceutical R&D, and the obtained fundamental results also support the potential of the GLUT targeted platinum(II)-sugar conjugates as lead compounds for further pre-clinical evaluation.