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The use of synthetic model systems in elucidating pathway dependent mechanisms responsible for long-distance electron transfer is discussed. The syntheses of models composed of quinone-substituted, phenyl-linked porphyrin dimers and trimers, porphyrins with meso-substituted charge transfer complexes of Ru(bpy)3, (bpy: 2,2′-bipyridyl) and porphyrin aggregates based on multi-point hydrogen bonding are presented along with their steady-state and dynamic photophysical properties.

The synthesis of a novel cytosine-guanine ribose- and phosphate-free “artificial dinucleotide” 1 and its cytosine-cytosine analogue 2 are described. The syntheses of derivatives of these core compounds bearing porphyrin and polymeric side chains are also presented.

The synthesis of four new analogues of motexafin gadolinium (MGd), a gadolinium(III) texaphyrin complex in clinical trials for its anticancer properties, is described. These new derivatives contain either 1,2-diaminobenzene or 2,3-diaminonaphthalene subunits as the source of the imine nitrogens and bear multiple 2-[2-(2-methoxyethoxy)ethoxy]ethoxy (PEG) groups, on either meso aryl or beta-pyrrolic substituents, to increase their water solubility. All four analogues were found to be more active in vitro than the parent system MGd as judged from cell proliferation assays using the PC3 and A549 cell lines.

Motexafin gadolinium (MGd, Xcytrin®) is an anti-cancer agent that is being studied in clinical trials in various types of cancer including hematologic malignancies. MGd has been shown to generate reactive oxygen species and induce apoptosis in myeloma and lymphoma cells, and to cause an increase in intracellular levels of free zinc. We now report the effect of MGd and exogenous zinc on intracellular levels of free zinc, oxidative stress, proliferation, and cell death in human B-cell lymphoma and other hematologic cell lines. To better understand the molecular basis of these cellular responses, gene expression profiling analyses were conducted on Ramos cell cultures treated with MGd and/or zinc acetate. In cultures treated with MGd and zinc, 21% of Ramos cells were apoptotic within 8 hours of treatment as demonstrated by Annexin V staining. The apoptotic fraction increased to 30% within 12 hours and 68% by 24 hours. Analogous results were obtained using JC-1, with 38% of Ramos cells exhibiting non-aggregated (green) JC-1 fluorescence characteristic of mitochondrial dysfunction within 8 hours of combined treatment with MGd and zinc. This fraction increased to 52% by 12 hours and 74% by 24 hours. MGd and zinc similarly increased cell death in other B-cell lines (Raji, DB, DHL-4 and HF-1) tested. We also found that increased levels of oxidative stress and intracellular free zinc preceded and correlated with cell cycle arrest and apoptosis. Cultures treated with MGd or zinc acetate alone elicited transcriptional responses characterized by induction of metal response element-binding transcription factor-1 (MTF-1) and hypoxia inducible transcription factor 1 (HIF-1) regulated genes. Cultures co-treated with MGd and zinc acetate displayed further increases in the levels of MTF-1 and HIF-1 regulated transcripts and also additional transcripts regulated by NF-E2-related transcription factor 2 (NRF-2). HIF-1 activation can have negative consequences for cell growth and survival by induction of targets linked to apoptosis, whereas NRF-2 activation is characteristic of cells responding to disruption of redox balance. These data demonstrate that MGd increases intracellular free zinc and oxidative stress levels in lymphoma cells that activate adaptive survival responses but eventually lead to cell death.

Motexafin gadolinium (MGd, Xcytrin®) is an anti-cancer agent that selectively localizes in tumors and promotes redox stress by oxidizing intracellular reducing species. MGd, at low concentrations, induces expression of metallothioneins (MT) and zinc transporter 1 (ZnT-1) transcripts in vitro. In the present study, we describe the effects of MGd on zinc ion homeostasis, thioredoxin reductase activity and cytotoxicity in lymphoma cell lines. Human lymphoma (Ramos, DHL-4, HF-1) cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum. Zinc (0–100 μM) and 0–25 μM MGd were added for 4–6 hr. Medium was exchanged and thioredoxin reductase activity was assessed by measuring the rate of lipoate reduction (Biaglow, Anal. Biochem.281:77, 2000). In other experiments, cells were treated with MGd and zinc, and analyzed by flow cytometry using annexin-V, propidium iodide, and the ion-specific fluorescent probe, FluoZin-3-AM™. RNA from treated cultures was harvested and metallothionein and ZnT-1 induction assessed by Northern blotting. Treatment with MGd and zinc led to synergistic increases in free intracellular zinc levels, inhibition of lipoate reduction, MT and ZnT-1 induction, and cytotoxicity. In DHL-4, exposure to 10 μM MGd and 50 μM zinc for 3 hr led to a 2.5-fold increase in FluoZn-3 fluorescence, relative to treatment with zinc alone. In Ramos, this treatment led to 1.4, 2.6, 5.2, and 30-fold increase in FluoZn-3 fluorescence after 2, 4, 6, or 24 hr. There was a 2-fold increase in annexin-V positive Ramos cells after 6 hr, and an 11-fold increase in propidium iodide permeable cells by 24 hr. The rate of lipoate reduction decreased to 69%, 47%, and 58% of control in Ramos, DHL-4, and HF-1 cell lines after 5 hr under these conditions. MT and ZnT-1 transcript levels were elevated within 4 hr after treatment with MGd, zinc, or the combination, and remained elevated for at least 24 hr. For example, in HF-1, MT transcript levels were increased 20, 40, and 173-fold by MGd, zinc, or the combined treatment after 8 hr. These observations support the characterization of MGd as a redox cycling agent that increases the intracellular availability of free zinc. This activity leads to inhibition of thioredoxin reductase and, ultimately, induction of cell death. The proposed mechanism of action supports the use of this agent alone or in combination with various chemotherapy agents that induce redox stress.

Sapphyrins are pentapyrrolic metal-free expanded porphyrins that localize to tumors. We have previously demonstrated that sapphyrins induce apoptosis in a variety of hematologic tumor cell lines including lymphomas (Ramos, DHL-4, HF-1), leukemias (Jurkat, HL-60), and myelomas (8226/S, 1-310, C2E3, 1-414). Through chemical modification of the parent compound, PCI-2000, a number of derivatives were generated and tested for induction of apoptosis in Ramos cells. PCI-2000 and one of the more potent apoptosis-inducing derivatives, PCI-2050, were injected into CD-1 nude mice bearing Ramos xenografts. Animals were sacrificed 48 hrs after injection and analyzed for drug uptake in the tumor, liver and spleen using flow cytometry. For PCI-2000, the relative uptake was spleen>tumor>liver. For PCI-2050 the relative uptake was tumor>spleen>liver, suggesting that PCI-2050 preferentially localizes in tumors compared to PCI-2000. Tumor cells isolated from PCI-2050 treated animals grew less well in culture and had more apoptotic cells than those derived from PCI-2000 or control animals. Uptake of PCI-2050 into xenograft tumor cells and tumor cell killing was dose dependent. PCI-2050 (10 umol/kg x 2 days in a row) was administered to Ramos xenograft bearing animals that were then monitored for tumor growth. In both minimal tumor (animals treated before tumor was palpable) and established tumor (palpable tumor) models, PCI-2050 reduced tumor growth by 60–75%. Alternative dosing strategies revealed that split dosing (allowing 1 or more days between doses) was more efficacious in tumor control than dosing 2 days in a row. At the doses used in this study, there was no myelosuppression or lymphosuppression, hepatic or renal abnormalities as assessed by complete blood count and comprehensive serum chemistry analysis, respectively. Our work demonstrates that PCI-2050 induces apoptosis in tissue culture and inhibits tumor growth in an animal tumor model while exhibiting minimal toxicity. PCI-2050 and other sapphyrin derivatives will be further evaluated as potential anti-cancer agents.

The synthesis of the cytosine-guanine linked dimer1, and its symmetric guanine-guanine analogue2 are described. Compound1 is the first homogeneous ribose-free system to be prepared and characterized in which molecular recognition by complementary guanine-cytosine base-pairing might be possible. It is thus prototypical of a new and potentially large class of ‘artificial oligonucleotides’.

The putative antiviral agent, (S)-2-amino-7-(2,3-dihydroxy-propyl) purine (1b), and its achiral analogue, 2-amino-7-(2-hydroxyethyl) purine (2), were synthesized by a procedure involving alkylation at N7 of guanosine followed by deribosylation and deoxygenation. Evidence for the stereochemical integrity of the former preparation was obtained from the X-ray diffraction structure of the novel tricyclic compound, (S)-6H, 7H, 8H-2-amino-7-hydroxy-[1,4] oxazepino [1,2,3-d, elpurine (17, obtained by a similar synthetic sequence. Compound (1a), a regioisomer of the known antiviral agent, (S)-9-(2,3-dihydroxypropyl) adenine ((S)-DHPA), was tested and found to be inactive in tissue culture against herpes virus type-2, rotavirus, poliovirus, and parainfluenza virus.