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A series of catechol ring-fluorinated derivatives of caffeic acid phenethyl amide (CAPA) were synthesized and screened for cytoprotective activity against H2O2 induced oxidative stress in human umbilical vein endothelial cells (HUVEC). CAPA and three fluorinated analogs were found to be significantly cytoprotective when compared to control, with no significant difference in cytoprotection between caffeic acid phenethyl ester (CAPE) and CAPA., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

To determine the relationship between catechol ring modifications and the activity of caffeic acid phenethyl ester (CAPE) as a cytoprotective agent, six catechol ring-fluorinated CAPE derivatives were evaluated for their cytoprotective abilities, as well as for their antioxidant and heme oxygenase-1 (HO-1) inducing capacity in a human umbilical vein endothelial cell (HUVEC) model of oxidant stress. To ascertain the involvement of HO-1 induction in the cytoprotective effects of CAPE analogues, their ability to induce HO-1 at 20microM was determined by reverse transcriptase polymerase chain reaction, western blotting and the use of HO-1 inhibitor tin protoporphyrin IX. There was significant induction of HO-1 by CAPE derivatives. Inhibition of HO-1 enzymatic activity resulted in reduced cytoprotection. Modification of the catechol ring of CAPE by introduction of fluorine at various positions resulted in dramatic changes in cytoprotective activity. The maintenance of at least one hydroxyl group on the CAPE catechol ring and the phenethyl ester portion was required for HO-1 induction. CAPE and its derivatives were screened for their ability to scavenge intracellular reactive oxygen species generated in HUVECs by measuring 5-(and-6)-chlormethyl-2', 7'-dichlorodihydrofluorescein diacetate oxidation. The maintenance of 3, 4-dihydroxyl groups on the catechol ring was required for antioxidant activity, but antioxidant activity did not guarantee cytoprotection. Methylation or replacement of one hydroxyl group on the catechol ring of CAPE, however, provided both pro-oxidant and cytoprotective activities. These results indicate that the induction of HO-1 plays a more important role in the cytoprotective activity of CAPE derivatives than their direct antioxidant activity.

The pharmacokinetic profiles of caffeic acid phenethyl ester (CAPE) and its catechol-ring fluorinated derivative (FCAPE) were determined in rats after intravenous administration of 5, 10 or 20 mg/kg for CAPE and 20 mg/kg for FCAPE, respectively. The plasma concentrations of CAPE and FCAPE were measured using a validated liquid chromatography tandem mass spectrometric method. The pharmacokinetic parameters were estimated using non compartmental analysis (NCA) and biexponential fit. The results showed that the area under the plasma concentration-time curve for CAPE treatment increased in a proportion greater than the increase in dose from 5 to 20 mg/kg of CAPE. Total body clearance values for CAPE ranged from 42.1 to 172 ml/min/kg (NCA) and decreased with the increasing dose of CAPE. Similarly, the volume of distribution values for CAPE ranged from 1555 to 5209 ml/kg, decreasing with increasing dose. The elimination half-life for CAPE ranged from 21.2 to 26.7 min and was independent of dose. That FCAPE was distributed extensively into rat tissues and eliminated rapidly was indicated by a high value of volume of distribution and similar short elimination half-life as that of CAPE., (Copyright 2009 John Wiley & Sons, Ltd.)

A concise and efficient route to (+/-)-N'-nitrosonornicotine 5'-acetate (NNN-5'-OAc), a stable precursor of the active metabolite 5'-hydroxy(+/-)-N'-nitrosonornicotine NNN-5'-OH is reported. The synthesis utilizes sulfinimine chemistry to give (+/-)-NNN-5'-OAc in 26% yield over 4 steps.

Metal complexes of 2-methyl-1H-benzimidazole-5-carboxylic acid hydrazide (4a; L(1)) and its Schiff base 2-methyl-N-(propan-2-ylidene)-1H-benzimidazole-5-carbohydrazide (5a; L(2)) with transition metal ions e.g., copper, silver, nickel, iron and manganese were prepared. The complexes formed were 1:1 or 1:2 M:L complexes and have the structural formulae [Cu(L(1))Cl(H(2)O)]Cl x 3 H(2)O (6), [Ag(L(1))NO(3)(H(2)O)] (7), [Ni(L(1))Cl(2)(H(2)O)(2)] x H(2)O (8), [Fe(L(1))Cl(3)(H(2)O)] x 3 H(2)O (9) and [Mn(L(1))(2)Cl(H(2)O)]Cl x 3 H(2)O (10) for ligand L(1), and [Cu(L(2))Cl(2)(H(2)O)(2)] x H(2)O (11), [Ag(L(2))(2)]NO(3) x H(2)O (12), [Ni(L(2))(2)Cl(2)] x 5 H(2)O (13), [Fe(L(2))(2)Cl(2)]Cl x 2 H(2)O (14) and [Mn(L(2))Cl(2)(H(2)O)(2)] x H(2)O (15) for ligand L(2). The antitumor activity of the synthesized compounds has been studied. The silver complex 7 was found to display cytotoxicity (IC(50)=2 microM) against both human lung cancer cell line A549 and human breast cancer cell line MCF-7.

The simian virus 40 (SV40) genome is a model system frequently employed for investigating eukaryotic replication. Large T-antigen (T-ag) is a viral protein responsible for unwinding the SV40 genome and recruiting necessary host factors prior to replication. In addition to duplex unwinding T-ag possesses G-quadruplex DNA helicase activity, the physiological consequence of which is unclear. However, formation of G-quadruplex DNA structures may be involved in genome maintenance and function, and helicase activity to resolve these structures may be necessary for efficient replication. We report the first real-time investigation of SV40 T-ag helicase activity using surface plasmon resonance (SPR). In the presence of ATP, T-ag was observed to bind to immobilized single-stranded DNA, forked duplex DNA, and the human telomeric foldover quadruplex DNA sequence. Inhibition of T-ag duplex helicase activity was observable in real-time and the intramolecular quadruplex was unwound.

Caffeic acid phenethyl ester (CAPE), derived from various plant sources, has been shown to ameliorate ischemia/reperfusion injury in vivo, and this has been attributed to its ability to reduce oxidative stress. Here we investigated the cytoprotection of CAPE against menadione-induced oxidative stress in human umbilical vein endothelial cells (HUVEC) to evaluate potential gene expression involvement. CAPE exhibited dose-dependent cytoprotection of HUVEC. A gene screen with microarrays was performed to identify the potential cytoprotective gene(s) induced by CAPE. Heme oxygenase-1 (HO-1) was highly upregulated by CAPE and this was confirmed with reverse transcriptase polymerase chain reaction (RT-PCR) and western blotting. Inhibition of HO-1 activity using the HO-1 inhibitor tin protoporphyrin IX (SnPPIX), resulted in loss of cytoprotection. Carbon monoxide, one of HO-1 catabolic products appeared to play a small role in CAPE protection. Caffeic acid, a potential metabolite of CAPE with similar free radical scavenging ability, however, didn't show any cytoprotective effect nor induce HO-1. These findings suggest an important role of HO-1 induction in CAPE cytoprotection against oxidant stress, which may not relate to CAPE structural antioxidant activity nor to its traditional enzymatic activity in decomposing heme but to a yet to be determined activity.

UK-1 is a bis(benzoxazole) natural product displaying activity against a wide range of human cancer cell lines. A simplified analog of UK-1, 4-carbomethoxy-2-(2'-hydroxyphenyl)benzoxazole, was previously found to be almost as active as UK-1 against cancer cell lines, and similar to the natural product, formed complexes with a variety of metal ions such as Mg2+ and Zn2+. A series of 4-substituted-2-(2'-hydroxyphenyl)benzoxazole analogs of this 'minimal pharmacophore' of UK-1 were prepared. The anti-cancer activity of these analogs was examined in breast and lung cancer cell lines. Spectrophotometric titrations in methanol were carried out in order to assess the ability of UK-1 and these analogs to coordinate with Mg2+ and Cu2+ ions. Although none of the new analogs were more cytotoxic than 4-carbomethoxy-2-(2'-hydroxyphenyl)benzoxazole, some analogs were identified that display similar cytotoxicity to this simplified UK-1 analog with improved water solubility. UK-1 and all of these new analogs bind Cu2+ ions better than Mg2+ ions, and the nature of the 4-substituent is important for the Mg2+ ion binding ability of these 2-(2'-hydroxyphenyl)benzoxazoles. Previous studies of a limited number of UK-1 analogs demonstrated a correlation between Mg2+ ion binding ability and cytotoxicity; however, within this series of 4-substituted-2-(2'-hydroxyphenyl)benzoxazoles the variations in cytotoxicity do not correlate with either Mg2+ or Cu2+ ion binding ability. These results, together with recent ESI-MS studies of Cu2+-mediated DNA binding by UK-1 and analogs, indicate that UK-1 and analogs may exert their cytotoxic effects by interaction with Cu2+ or other transition metal ions, rather than Mg2+, and that metal ion-mediated DNA binding, rather than metal ion binding affinity, is important for the cytotoxic effect of these compounds. The potential role of Cu2+ ions in the cytotoxic action of UK-1 is further supported by the observation that UK-1 in the presence of Cu2+ displays enhanced cytotoxicity to MCF-7 and A549 cells when compared to UK-1 alone.

The binding of a series of benzoxazole analogs with different amide- and ester-linked side chains to duplex DNA in the absence and presence of divalent metal cations is examined. All ligands were found to form complexes with Ni2+, Cu2+, and Zn2+, with 2:1 ligand/metal cation binding stoichiometries dominating for ligands containing shorter side chains (2, 6, 7, and 8), while 1:1 complexes were the most abundant for ligands with long side chains (9, 10, and 11). Ligand binding with duplex DNA in the absence of metal cations was assessed, and the long side-chain ligands were found to form low abundance complexes with 1:1 ligand/DNA binding stoichiometries. The ligands with the shorter side chains only formed DNA complexes in the presence of metal cations, most notably for 7 and 8 binding to DNA in the presence of Cu2+. The binding of long side-chain ligands was enhanced by Cu2+ and to a lesser degree by Ni2+ and Zn2+. The cytotoxicities of all of the ligands against the A549 lung cancer and MCF7 breast cancer cell lines were also examined. The ligands exhibiting the most dramatic metal-enhanced DNA binding also demonstrated the greatest cytotoxic activity. Both 7 and 8 were found to be the most cytotoxic against the A549 lung cancer cell line and 8 demonstrated moderate cytotoxicity against MCF7 breast cancer cells. Metal ions also enhanced the DNA binding of the ligands with the long side chains, especially for 9, which also exhibited the highest level of cytotoxicity of the long side-chain compounds.

The interactions between a novel enediyne [1-methyl-2-(phenylethynyl)-3-(3-phenylprop-2-ynyl)-3H-benzimidazolium] (1) and various cytosine-containing oligonucleotides were studied using electrospray ionization mass spectrometry (ESI-MS) in a flow injection analysis mode useful for small volumes. This enediyne ligand, developed as a potential alternative to the highly cytotoxic natural enediynes, some of which have been successfully used as anti-tumor agents, has previously been shown to interact with DNA through frank strand scission as well as via the formation of adducts that lead to 2'-deoxycytidine-specific cleavage. Through ESI-MS, the structures of these adducts were examined and a sequence dependence of the 2'-deoxycytidine-specific cleavage was noted. Collisionally activated dissociation of the observed adducts confirmed the strength of the interactions between the enediyne and DNA and supports a direct linkage between the enediyne and the cytosine nucleobase, likely the result of a nucleophilic attack of the phenylethynyl group by the cytosine amine.

Caffeic acid phenethyl ester (CAPE), a natural polyphenolic compound with many biological activities, has been shown to be protective against ischemia-reperfusion injury. We have synthesized six new catechol ring-fluorinated CAPE derivatives and evaluated their cytotoxic and cytoprotective effects against menadione-induced cytotoxicity in human umbilical vein endothelial cells. These results provide some insights into the structural basis of CAPE cytoprotection in this assay, which does not appear to be based solely on direct antioxidant properties.

Diradical-generating cyclizations such as the enediyne Bergman cyclization and the enyne allene Myers-Saito cyclization have been exploited by nature in the mechanism of DNA cleavage by a series of potent antitumor antibiotics. Alternative diradical-generating cyclizations have been proposed in the design of selective antitumor agents; however, little information is available concerning the utility of these alternative cyclizations in radical-based DNA cleavage chemistry. One such alternative diradical-generating cyclization, the aza-Myers-Saito cyclization of aza-enyne allenes that are derived from base-promoted isomerization of skipped aza-enediynes, has been recently reported. Here, we report the synthesis and DNA cleavage chemistry of a series of pyridinium skipped aza-enediynes (2-alkynyl-N-propargyl pyridinium salts). Efficient DNA cleavage requires the presence of the skipped aza-enediyne functionality, and optimal DNA cleavage occurs at basic pH. Within this series of compounds, the analogue bearing a p-methoxyphenyl group on the pyridinium 2-alkyne substituents was found to be the most effective DNA cleavage agent, displaying significant supercoiled DNA-nicking activity at concentrations as low as 1 microM. Detailed studies of this analogue show that DNA cleavage occurs through 4'-hydrogen-atom abstraction from the DNA backbone and oxidation of guanine bases. This is the first report of enediyne-like radical-based DNA cleavage by an agent designed to undergo an alternative diradical-generating cyclization.

Electrospray ionization mass spectrometry (ESI-MS) and spectroscopic studies in solution were used to evaluate the self-association, G-quadruplex DNA binding, and selectivity of a series of perylene diimides (PDIs) (PIPER, Tel01, Tel11, Tel12, and Tel18) or benzannulated perylene diimide ligands (Tel34 and Tel32). Fluorescence and resonance light scattering spectra of Tel01, Tel12, Tel32, and Tel34 reveal that these analogs undergo self-association in solution. UV-Vis and fluorescence titrations with G-quadruplex, duplex, or single-stranded DNA demonstrate that all the analogs, with the exception of Tel32, bind to G-quadruplex DNA, with those PDIs that are self-associated in solution showing the highest degree of selectivity for binding G-quadruplex DNA. Parallel ESI-MS analysis of the stoichiometries demonstrates the ability of the ligands, with the exception of Tel32, to bind to G-quadruplex DNA. While most ligands show major 1:1 and 2:1 binding stoichiometries as expected in the case of end-stacking, interestingly, three of the most quadruplex-selective ligands show a different behavior. Tel01 forms 3:1 complexes, while Tel12 and Tel32 only form 1:1 complexes. Collisional activation dissociation patterns are compatible with ligand binding to G-quadruplex DNA via stacking on the ends of the terminal G-tetrads. Experiments with duplex and single strand DNA were performed to assess the binding selectivities of the ligands. PIPER, Tel11, and Tel18 demonstrated extensive complexation with duplex DNA, while Tel11 and Tel18 bound to single strand DNA, confirming the lack of selectivity of these two ligands. Our results indicate that Tel01, Tel12, and Tel34 are the most selective for G-quadruplex DNA.

Electrospray ionization mass spectrometry is used to compare the metal ion binding and metal-mediated DNA binding of benzoxazole (1, 2, 3, 4) and benzimidazole (5) compounds and to elucidate the putative binding modes and stoichiometries. The observed metal versus non-metal-mediated DNA binding, as well as the specificity of DNA binding, is correlated with the biological activities of the analogs. The ESI-MS spectra for the antibacterial benzoxazole and benzimidazole analogs 4 and 5 demonstrated non-specific and non-metal-mediated binding to DNA, with the appearance of DNA complexes containing multiple ligands. The anticancer analog 2 demonstrates a clear preference for metal-mediated DNA interactions, with an apparent selectivity for Ni2+ -mediated binding over the more physiologically relevant Mg2+ or Zn2+ cations. Complexation between DNA and the biologically inactive analog 1 was not observed, either in the absence or presence of metal cations.

[reaction: see text] On the basis of density functional calculations, the isomerization of skipped azaenediynes (C-alkynyl-N-propargylimines) to azaenyne allenes and subsequent rapid aza-Myers-Saito cyclization to alpha,5-didehydro-3-picoline were predicted. We prepared the N-propargylimine of 1-phenyl-3-tri(isopropyl)silylprop-2-yn-1-one, which undergoes proto-desilylation and isomerization to an azaenyne allene when treated with tetrabutylammonium fluoride. In the presence of 1,4-cyclohexadiene, this azaenyne allene affords 6-phenyl-3-picoline and other products corresponding to the trapping of an alpha,5-didehydro-3-picoline diradical.

Human telomeres are comprised of d(TTAGGG) repeats that are capable of forming G-quadruplex DNA structures. Ligands that bind to and stabilize these G-quadruplex DNA structures are potential inhibitors of the cancer cell-associated enzyme telomerase. Other potential biological uses of G-quadruplex targeting ligands have been proposed. One particularly challenging aspect of the contemplated uses of G-quadruplex targeting ligands is their selectivity for G-quadruplex DNA versus double-stranded DNA structures. We have previously reported the observation that two structurally related 3,4,9,10-perylenetetracarboxylic acid diimide-based G-quadruplex DNA ligands, PIPER [N,N'-bis(2-(1-piperidino)ethyl)-3,4,9,10-perylenetetracarboxylic acid diimide] and Tel01 [N,N'-bis(3-(4-morpholino)propyl)-3,4,9,10-perylenetetracarboxylic acid diimide], have different levels of G-quadruplex DNA binding selectivity at pH 7 as determined by absorbance changes in the presence of different DNA structures [Kerwin, S. M., Chen, G., Kern, J. T., and Thomas, P. W. (2002) Bioorg. Med. Chem. Lett. 12, 447-450]. Here we report that the less G-quadruplex DNA selective ligand PIPER can unwind double-stranded, closed circular plasmid DNA, as determined by a topoisomerase I assay. A model for the interaction of Tel01 with the G-quadruplex DNA structure formed by d(TAGGGTTA) was determined from NMR experiments. This model is similar to the previously published model for PIPER bound to the same G-quadruplex DNA and failed to provide a structural basis for the observed increased selectivity of Tel01 interaction with G-quadruplex DNA. In contrast, investigation into the aggregation state of Tel01 and PIPER as well as other 3,4,9,10-perylenetetracarboxylic acid diimide analogues bearing basic side chains demonstrates that ligand aggregation is correlated with G-quadruplex DNA binding selectivity. For all six analogues examined, those ligands that were aggregated at pH 7 in 70 mM potassium phosphate, 100 mM KCl, 1 mM EDTA buffer also demonstrated G-quadruplex DNA binding selectivity under these buffer conditions. Ligands that were not aggregated under these conditions display much lower levels of G-quadruplex DNA selectivity. The aggregation state of these ligands is extremely sensitive to the buffer pH. Tel01, which is aggregated at pH 7, is not aggregated at pH 6.4, where it demonstrates only modest G-quadruplex DNA binding selectivity, and PIPER in pH 8.5 buffer is both aggregated and highly G-quadruplex DNA-selective. To our knowledge, these studies demonstrate the first DNA structure selectivity as achieved through pH-mediated ligand aggregation. The potential impact of these findings on the selectivity of other classes of G-quadruplex DNA ligands is discussed.

Selectivity, binding stoichiometry, and mode of binding of Tel01, distamycin A, and diethylthiocarbocyanine iodide (DTC) to the parallel stranded G4-quadruplex [d(T2G5T)]4 were investigated by ESI-MS. The first drug/quadruplex complexes observed by ESI-MS are described. Tel01, distamycin A, and DTC all form complexes with quadruplex DNA, but only Tel01 is completely selective for quadruplex versus duplex oligonucleotide under the conditions employed. Previous solution determinations of the binding mode of Tel01 and distamycin A to quadruplex oligonucleotides indicate that Tel01 interacts through end-stacking with guanine tetrads of quadruplex DNA, while distamycin A interacts by binding to quadruplex grooves. When these two different drug/quadruplex complexes are subjected to collisionally activated dissociation in a mass spectrometer, the observed fragmentation patterns are distinct. Tel01/quadruplex complexes undergo facile loss of drug and dissociation to single-strand oligonucleotide ions, while distamycin/quadruplex complexes fragment into single-strand oligonucleotide ions in which the drug molecule is retained. Dissociation patterns for DTC/quadruplex complexes are similar to those of distamycin; therefore, it is concluded that DTC interacts with [d(T2G5T)]4 through groove-binding. These ESI-MS results are applicable to both the identification and characterization of G-quadruplex interactive agents and may also be useful in probing unusual DNA structures.

The (Z)-hex-1,5-diyne-3-ene reactive core common to the enediyne antitumor antibiotics undergoes a Bergman cyclization after proper activation to afford reactive diradical intermediates that are responsible for initiating DNA cleavage. Direct modification of the enediyne core has been proposed as a method to permit cancer cell-specific triggering of the diradical-generating cyclization. For example, 3-aza-3-ene-1,5-diynes undergo an aza-Bergman cyclization to afford the fleeting 2,5-didehydropyridine diradicals. While protonation of these aza-enediynes can afford products of diradical trapping, the hydrolytic instability of the 3-aza-3-ene-1,5-diyne moiety prevents its use in pH-triggered DNA cleaving anticancer agents. Recently, more hydrolytically stable systems incorporating the 4-aza-3-ene-1,6-diyne moiety were developed. We report here studies of the 4-aza-3-ene-1,6-diyne-containing benzimidazolium salt AZB002 [1-methyl-2-(phenylethynyl)-3-(3-phenylprop-2-ynyl)-3H-benzimidazolium tetrafluoroborate] and two structurally related heterocycles that lack the aza-enediyne functionality, AZB016 [1,3-dimethyl-2-(phenylethynyl)-3H-benzimidazolium triflate] and AZB004 [3-methyl-2-(phenylethynyl)benzothiazolium triflate]. The interaction of these compounds with supercoiled DNA, a double-stranded DNA fragment, and a short DNA duplex oligonucleotide was investigated. There are three distinct DNA interactions exhibited by AZB002: a frank strand scission leading to the relaxation of supercoiled DNA and formation of at least two different DNA adducts, one of which leads to cytosine-specific cleavage after piperidine/heat treatment. In contrast, analogues lacking the aza-enediyne functionality either fail to interact with DNA (AZB016) or cleave DNA at guanine residues, presumably through alkylation of the N-7 position (AZB004). We also investigated the cytotoxicity of AZB002 and the related heterocyclic compounds AZB004 and AZB016 and find that only the DNA interactive compounds AZB002 and AZB004 display significant cytotoxicity. In particular, AZB002 is cytotoxic against a wide range of cancer cell lines.

Ribosome inhibiting proteins, RIPs, are a widespread family of toxic enzymes. Ricin is a plant toxin used as a poison and biological warfare agent; shiga toxin is a homologue expressed by pathogenic strains of E. coli. There is interest in creating effective antidote inhibitors to this class of enzymes. RIPs act by binding and hydrolyzing a specific adenine base from rRNA. Previous virtual screens revealed that pterins could bind in the specificity pocket of ricin and inhibit the enzyme. In this paper we explore a range of compounds that could serve as better platforms for inhibitor design. This establishes the importance of key hydrogen bond donors and acceptors for active-site complementarity. 8-Methyl-9-oxoguanine is a soluble compound that has the best inhibitory properties of any platform tested. The X-ray structure of this complex revealed that the inhibitor binds in an unexpected way that provides insight for future design. Several inhibitors of ricin were also shown to be inhibitors of shiga toxin, suggesting this program has the potential to develop effective antidotes to an important form of food poisoning.