Benzofuranyl-pyran-2-ones, -pyridazines,and -pyridones from naturally occurring furochromones (visnagin and khellin)

The novel and versatile enaminones 2a,b were synthesized by treatment of visnaginone methyl ether 1a or khellinone methyl ether 1b with N,N-dimethylformamide dimethylacetal. They were reacted with hippuric acid or N-acetylglycine to yield benzofuran-5-yl-2H-pyran-2-ones 3a–d . The reaction of 2a,b with cyanoacetamide and malononitrile dimer in sodium ethoxide gave benzofuran-5-yl-pyridones 4a,b and [benzofuran-5-yl-1H-pyridine-2-ylidene] malononitrile 5a , respectively. Refluxing 2a,b with hydrazine hydrate or with hydroxyla- mine afforded benzofuran-5-yl-1H-pyrazoles 6a,b and benzofuran-5-yl-isoxazoles 7a,b , respectively. Moreover, 2a,b coupled with aryl diazonium salt in the presence of sodium hydroxide to yield 3-(benzofuran-5-yl)-2-aryl-hydrazono-3-oxo-propanals 8a,b which were excellent precursors for the synthesis of pyridazines 9–12 . © 2003 Wiley Periodicals, Inc. 15:85–91, 2004; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/hc.10219     

Photosensitized cleavage and cross-linking of pBR322 DNA with khellin and visnagin

The naturally occuring furanochromones khellin and visnagin have received considerable attention, largely because of their vasodilatory properties and of their ability (particularly that of khellin) to induce skin pigmentation upon ultraviolet light treatment of patients suffering from vitiligo. There are conflicting statements in the literature on whether or not they are capable of cross-linking DNA photochemically. Supercoiled and linear pBR322 DNA was used to probe this reaction. The results showed that both khellin and, to a greater extent, visnagin photosensitized DNA cross-linking. In addition, both photosensitizers induced extensive DNA cleavage.     

Determination of khellin and visnagin in Ammi visnaga fruits by capillary electrophoresis

A sensitive method for the simultaneous determination of phenolic xenoestrogens such as bisphenol A, 2,4-dichlorophenol, 4-tert,-butylphenol, 4-n2-pentylphenol, 4-n-hexylphenol, 4-n-heptylphenol, 4-octylphenol, 4-nonylphenol was developed using reversed-phase LC and coulometric-array detection. Stepwise gradient elution with phosphoric acid in water-acetonitrile was used. The calibration curves were linear in the range of 5.0 (or 10.0)-1000 ng ml(-1) with correlation coefficients of 0.9978-0.9999, the limits of detection were 0.01-0.02 ng ml(-1). Sample clean-up was performed by solid-phase extraction (SPE) using 3M Empore extraction disks. Three commercial sorbents, C18, SDB-XD (styrene-divinylbenzene polymer) and SDB-RPS (sulfonated styrene-divinylbenzene polymer) were compared. The highest recoveries were obtained with SDB-RPS. They were above 70% with a relative standard deviation of less than 15%. The proposed method was applied to the determination of phenolic xenoestrogens in various water samples.     

Nanosecond time-resolved infrared studies of visnagin and khellin triplets and radical ions

Time-resolved infrared spectroscopy (TRIR) and density functional theory (DFT) calculations were used to directly observe and assign the vibrational spectra of the triplet states of visnagin and khellin, and to investigate their electron-transfer chemistry. The TRIR spectra of triplet visnagin and triplet khellin, and of their radical cations and anions, were obtained upon 266 nm laser flash photolysis in acetonitrile and in deuterated acetonitrile. The radical cations were observed in the presence of chloranil, and the radical anions were formed in the presence of NaI and KSCN. The TRIR spectra are in good agreement with the calculated vibrational spectra. We did not observe the related neutral radicals by TRIR spectroscopy upon laser flash photolysis (LFP) of khellin in the presence of hydroquinone, but we found evidence for the formation of semiquinone and neutral visnagin radicals upon LFP of visnagin and hydroquinone.     

Validated HPLC method for simultaneous estimation of khellol glucoside,khellin and visnagin in Ammi visnaga L. fruits and pharmaceutical preparations

Tea bags including fruits of Ammi visnaga L. are used in Egypt as remedy for the treatment of kidney stones. Our study focuses on developing simple and rapid method utilising HPLC for quantitative estimation of khellol glucoside (KG), khellin (KH) and visnagin (VS) simultaneously. Their concentrations were determined in A. visnaga L. fruits at different developmental stages and in pharmaceutical formulations together with following up them during shelf life. Separation was accomplished using HPLC. Perfect resolution between KG, KH and VS was possible through using a mobile phase consisting of water:methanol:tetrahydrofuran (50:45:5, v/v/v). Peaks were detected at 245 nm. The suggested method for the determination of KG, KH and VS was successful in determining the analytes of interest without any interference of other compounds and matrix. All validation parameters were satisfactory and the procedure was relatively easy and fast as extracts are evaluated without previous steps of purification.     

Photosensitization by iodinated DNA minor groove binding ligands: Evaluation of DNA double-strand break induction and repair

Iodinated DNA minor groove binding bibenzimidazoles represent a unique class of UVA photosensitizer and their extreme photopotency has been previously characterized. Earlier studies have included a comparison of three isomers, referred to as ortho-, meta- and para-iodoHoechst, which differ only in the location of the iodine substituent in the phenyl ring of the bibenzimidazole. DNA breakage and clonogenic survival studies in human erythroleukemic K562 cells have highlighted the higher photo-efficiency of the ortho-isomer (subsequently designated UV(A)Sens) compared to the meta- and para-isomers. In this study, the aim was to compare the induction and repair of DNA double-strand breaks induced by the three isomers in K562 cells. Further, we examined the effects of the prototypical broad-spectrum histone deacetylase inhibitor, Trichostatin A, on ortho-iodoHoechst/UVA-induced double-strand breaks in K562 cells. Using γH2AX as a molecular marker of the DNA lesions, our findings indicate a disparity in the induction and particularly, in the repair kinetics of double-strand breaks for the three isomers. The accumulation of γH2AX foci induced by the meta- and para-isomers returned to background levels within 24 and 48 h, respectively; the number of γH2AX foci induced by ortho-iodoHoechst remained elevated even after incubation for 96 h post-irradiation. These findings provide further evidence that the extreme photopotency of ortho-iodoHoechst is due to not only to the high quantum yield of dehalogenation, but also to the severity of the DNA lesions which are not readily repaired. Finally, our findings which indicate that Trichostatin A has a remarkable potentiating effect on ortho-iodoHoechst/UVA-induced DNA lesions are encouraging, particularly in the context of cutaneous T-cell lymphoma, for which a histone deacetylase inhibitor is already approved for therapy. This finding prompts further evaluation of the potential of combination therapies.     

Molecular Mechanism of Photosensitization XI. Membrane Damage and DNA Cleavage Photoinduced by Enoxacin

The photosensitizing activity of enoxacin, 1-ethyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-1,8-naphthyridine-3-carboxilic acid (ENX), toward membranes and DNA has been studied, taking into account human erythrocyte photohemolysis, unilamellar liposome alterations and plasmid pBR322 DNA photocleavage. Hydroxyl radicals and an aromatic carbene generated from ENX photode-fluorination seem to be the active intermediates involved in the photosensitization process. The steady-state photolysis products do not participate in the process. The mechanism of photosensitization responsible for the membrane damage depends on the oxygen concentration and follows a different path with respect to that operative for DNA cleavage. Between oxygenated radicals, the hydroxyl seems the species mainly responsible for membrane damage, whereas DNA cleavage is mainly produced by the carbene intermediate. A molecular mechanism of the photosensitization induced by ENX is proposed.     

Photosensitization of neoplastic cells by anthrapyrazoles

Abstract— Three anthrapyrazoles were tested for their ability to photosensitize murine leukemia cells in culture. Photosensitization was correlated with singlet oxygen yield. A cell line which exhibits anthrapyrazole resistance was also photosensitized, but the resulting photodamage was substantially less than in the drug-sensitive cell line. This result has implications with regard to repair mechanisms in drug sensitive vs resistant cells.     

Photosensitization by phenylalanine of carboxylic acids, amides, and peptides in frozen aqueous solution

Abstract— In order to elucidate the mechanism of the photosensitization of proteins and peptides by aromatic amino acids, the behaviour of aliphatic carboxylic acids and amides upon irradiation in frozen aqueous solution in the presence of phenylalanine (l-Phe) has been studied by EPR spectroscopy. EPR signals are much stronger when acids or amides are irradiated in the presence of l-Phe, showing that they are photosensitized by the aromatic amino acid. The species observed are either alkyl radicals, or radicals formed by abstraction of a hydrogen atom from a molecule of acid or arnide. A mechanism is proposed which involves the capture by carboxyl or amide carbonyl groups of hydrated electrons released by the photo-excited l-Phe, followed by the splitting of the resulting anion free radical with the formation of alkyl radicals, and transfer reactions leading to more stable free radicals. In peptides, which are also photosensitized by l-Phe, electrons are captured preferably by the carboxylic carbonyl groups.     

Photosensitization of vinyl polymerization by uranyl ions

Monomer acrylamide was used as quencher of uranyl ion fluorescence and Stern-Volmer plots were constructed at three monochromatic wavelengths of exciting radiation. The results indicated that the reaction between excited uranyl ion and monomer is one of energy transfer. The rate parameters k_p/k_p^½ and k_d′/k_i were calculated from the polymerization kinetics at high monomer concentrations. A general mechanism for the photopolymerization of vinyl monomers sensitized by uranyl ions is proposed and discussed.     

Formation of Cyclobutane Thymine Dimers from UVA Photosensitization of Pyridopsoralen Monoadducted DNA

The present report provides evidence that thymine dimerization can be UVA photosensitized at a tetranucleotide, 5′-TATT-3′, by a 7-methyl-pyrido(3, 4-c)psoralen monoadduct in DNA. The efficiency of the photoprocess depends on the tetranucleotide flanking sequences. These results demonstrate that one DNA lesion can originate the contiguous formation of a second type of lesion and emphasize the sequence-specific response to interaction of drugs with DNA. Results are related to the sensitivity of DNA to 1, 10-phenanthroline-cuprous ion complex nucleolytic activity and discussed in terms of the major role of local deformability of DNA in interaction with ligands.     

Photosensitization of Chinese hamster cells by water-soluble phthalocyanines

Abstract— Phthalocyanines are efficient photosensitizers of cultured mammalian cells and are considered for use in photodynamic therapy. The photobiological properties of chloroaluminum phthalocyanine sulfonate (AIPCS) were compared to those of the unsubstituted, water-insoluble derivative (AIPC). The development of photosensitization after addition of the dye into growth medium is ca. 8 times more rapid for AlPC than for AIPCS. Conversely, the loss of photosensitization when cells are incubated in a dye-free growth medium following a period of dye uptake, is also faster for AIPC. The dye uptake followed a kinetic behavior similar to the development of photosensitivity, but the loss of dye was too slow for both AlPC and AIPCS to explain loss of photosensitivity. When cells are incubated prior to illumination with AIPCS in phosphate buffered saline instead of growth medium, shorter time and smaller amount of dye are required to achieve the same level of photosensitization. The dependence of photosensitivity on dye concentration is linear for both AIPC and AIPCS. As already found for AIPC, photosensitization by the water-soluble derivative is also not enhanced in D₂O, suggesting that singlet oxygen is not involved in the cytotoxic response. Sodium salicylate, which was found to enhance the effect of AlPC was also effective with AIPCS. This effect is quite specific since the meta and para isomers had no effect. The metal atom complexed with the phthalocyanine ring is significant for the photobiological activity. Among the compounds tested, those containing Al or Zn are most active.     

Photosensitization of DNA damage by a new cationic pyropheophorbide derivative: sequence-specific formation of a frank scission

Pyropheophorbides are red-absorbing porphyrin-like photosensitizers that may interact with DNA either by intercalation or by external binding with self-stacking according to the value of the nucleotide to chromophore molar ratio (N/C). This article reports on the nature and sequence selectivity of the DNA damage photoinduced by a water-soluble chlorhydrate of aminopyropheophorbide. First, this pyropheophorbide is shown to induce on irradiation the cleavage of phiX174 DNA by both Type-I and -II mechanisms, suggested by scavengers and D2O effects. These conclusions are then improved by sequencing experiments performed on a 20-mer oligodeoxynucleotide (ODN) irradiated at wavelengths >345 nm in the presence of the dye, N/C varying from 2.5 to 0.5. Oxidation of all guanine residues to the same extent is observed after piperidine treatment on both single- and double-stranded ODN. Moreover, unexpectedly, a remarkable sequence-selective cleavage occurring at a 5'-CG-3' site is detected before alkali treatment. This frank break is clearly predominant for a low nucleotide to chromophore molar ratio, corresponding to a self-stacking of the dye along the DNA helix. The electrophoretic properties of the band suggest that this lesion results from a sugar oxidation, which leads via a base release to a ribonolactone residue. The proposal is supported by high-performance liquid chromatography-matrix-assisted laser desorption-ionization mass spectrometry experiments that also reveal other sequence-selective frank scissions of lower intensity at 5'-GC-3' or other 5'-CG-3' sites. This sequence selectivity is discussed with regard to the binding selectivity of cationic porphyrins.     

Photosensitization by Norfloxacin is a Function of pH

Abstract— Norfloxacin is a fluoroquinolone (FQ) antibiotic that has been reported to cause cutaneous photosensitivity in animals and occasionally in humans. We have studied the fluorescence and singlet oxygen (¹O₂)-generating properties of norfloxacin. Upon UV excitation the drug fluoresces in water, and the relative intensities of two major fluorescence bands at ca 420 and 450 nm are affected by pH. The overall quantum yield of fluorescence (φ_F) is also strongly pH dependent: φ_F is low in 0.2 N HC1 solution (0.2), increasing steeply to 0.12 at pH 4, then gradually decreasing to 0.01 at pH 10. The changes in φ_F are accompanied by changes in fluorescence lifetime from 0.6 ns at pH 1 to 1.8 ns at pH 4. Norfloxacin exhibits phosphorescence in low temperature glasses. The formation of a triplet state at room temperature is also suggested by ¹O₂ phosphorescence in aerobic D₂O. This phosphorescence is “self-quenched” by norfloxacin itself with an efficiency that is pH dependent: k_q is 7.9 ×10⁶M^?1s^?1 at pD 4, decreases to 1.9 × 10⁶M^?1 s^?1 at pD 7.5 but then increases about 20-fold in alkaline D₂O solutions. This quenching causes the observed ¹O₂ production by norfloxacin (0.1 mM) to show a maximum at around pH 8–9. However, after correction for self-quenching, the quantum yield of ¹O₂ production (φ_so), measured by using perinaphthenone as a standard, yielded the following values: φ_so is about 0.07 in 0.2 N DCl solution, 0.08 at pH 7.5 and then increases smoothly to ～ 0.2 in 0.1 M NaOD solution. The relatively high, unquenched ¹O₂ production at physiological pH 7.4 (φ_so～ 0.08) suggests that ¹O₂ reactions may play an important role in the cutaneous phototoxicity of norfloxacin and other FQ antibiotics.     

Molecular Mechanism of Drug Photosensitization. IX. Effect of Inorganic Ions on DNA Cleavage Photosensitized by Naproxen

Abstract— Photocleavage of DNA induced by naproxen and the correlated protective effect by some inorganic ions have been considered. The presence of a DNA complex is suggested and only associated naproxen seems to be responsible for the cleavage, for which the quantum yield of single strand breaks was calculated. The inorganic ions I^-, Mn²⁺, Co²⁺ and Cu²⁺ decrease naproxen-photoin-duced DNA cleavage. Iodide acts by a heavy atom mechanism, thus inhibiting naproxen photolysis and decreasing the amount of free radicals responsible for the photocleavage both in aerobic and anaerobic conditions. Metallic ions protect only within a range of concentrations, as for higher amounts damaging processes are observed. The protective efficiency of cations decreases with the increase of free drug concentration in the bulk of the solution, due to their involvement in the scavenging of naproxen radicals generated by photolysis of the free drug. In the presence of EDTA the cations show a better protective action. The most likely hypothesis is an inhibiting effect on the damaging processes via a redox cycle. The different behaviors of copper and of the two other cations can be justified by the influence of redox potentials of free and complexed metals and by the superoxide dis-mutase-like activity of copper.     

Photosensitization of 2'-deoxyadenosine-5'-monophosphate by pterin

UV-A radiation (320-400 nm) induces damages to the DNA molecule and its components through photosensitized reactions. Pterins, heterocyclic compounds widespread in biological systems, participate in relevant biological processes and are able to act as photosensitizers. We have investigated the photosensitization of 2'-deoxyadenosine-5'-monophosphate (dAMP) by pterin (PT) in aqueous solution under UV-A radiation. The effect of pH was evaluated, the participation of oxygen was investigated and the products analyzed. Kinetic studies revealed that the reactivity of dAMP towards singlet oxygen (1O2) is very low and that this reactive oxygen species does not participate in the mechanism of photosensitization, although it is produced by PT upon UV-A excitation. In contrast, analysis of irradiated solutions by means of electrospray ionization mass spectrometry strongly suggested that 8-oxo-7,8-dihydro-2'-deoxyadenosine-5'-monophosphate (8-oxo-dAMP) was produced, indicating that the photosensitized oxidation takes place via a type I mechanism (electron transfer).     

DNA Damage and Apoptosis Induced by Photosensitization of 5,10,15,20-Tetrakis (N-methyl-4-pyridyl)-21H,23H-porphyrin via Singlet Oxygen Generation

Cancer photodynamic therapy (PDT) requires photosensitizers that efficiently and selectively destroy tumor cells. We investigated 5,10,15,20-tetrakis ( N -methyl-4-pyridyl)-21 H ,23 H -porphyrin (TMPyP) as a potential cancer treatment. Confocal fluorescence microscopy showed that TMPyP was localized in the nuclei, whereas 5-aminolevulinic acid (ALA)-derived protoporphyrin IX (PPIX) was localized diffusely in the cytoplasm of human leukemia (HL-60) cells. In HL-60 cells under UVA irradiation, TMPyP effectively induced apoptosis. Moreover, 8-oxo-7,8-dihydro-2'-deoxyguanosine, an oxidative product of 2'-deoxyguanosine, was accumulated in the DNA of cells treated with photoirradiated TMPyP, whereas only small amounts were observed in ALA-treated cells in the presence of UVA light. TMPyP and UVA caused extensive damage at every guanine residue in DNA fragments obtained from the human p 53 tumor suppressor gene and the c-Ha- ras -1 proto-oncogene, whereas PPIX induced little DNA damage under these conditions. Electron spin resonance spectroscopy using a singlet oxygen (¹O₂) probe and D₂O showed that photoexcited TMPyP generated ¹O₂. These results suggest that photoexcited TMPyP reacts with oxygen to generate ¹O₂, which in turn, oxidizes guanine residues. Taken together, the results demonstrated that TMPyP was localized in the nucleus where it was photosensitized to induce DNA damage, suggesting that TMPyP may have clinical utility as a nucleus-targeted PDT.