6-Aminoquinolones: photostability, cellular distribution and phototoxicity.

Three selected aminoquinolones endowed with a potent antibacterial (compounds 1 and 2) and antiviral activity (compound 3) have been evaluated for their phototoxic properties in vitro. Photostability studies of these compounds indicate that compound 3 is photostable whereas compound 1 and in particular, compound 2 are rapidly photodegraded upon UVA irradiation, yielding a toxic photoproduct. Intracellular localization of these compounds has been evaluated by means of fluorescence microscopy using tetramethylrhodamine methyl ester and acridine orange, which are specific fluorescent probes for mitochondria and lysosomes, respectively. No co-staining was observed with lysosomal stain for all the test compounds. On the contrary compound 3 was found to be specifically incorporated in mitochondria. The compounds exhibited remarkable phototoxicity in two cell culture lines: human promyelocytic leukaemia (HL-60) and human fibrosarcoma (HT-1080). The quinolone-induced photodamage was also evaluated measuring the photosensitizing cross-linking in erythrocyte ghost membranes, the strand breaks activity and oxidative damage on plasmid DNA. The results show that these derivatives are able to photoinduce crosslink of erythrocytes spectrin, whereas do not significantly photocleavage DNA directly, but single strand breaks were observed after treatment of photosensitized DNA with two base excision repair enzymes, Fpg and Endo III respectively.

In vitro phototoxic properties of new 6-desfluoro and 6-fluoro-8-methylquinolones.

A representative set of potent antibacterial 6-desfluoro-8-methylquinolones, in which the C-6 fluorine atom is replaced by -NH(2) or -H, and their 6-fluoro counterparts, were investigated to evaluate their phototoxic potential and to explore the mechanism behind their phototoxicity. The capacity to photosensitize biological substrates (lipids, proteins, DNA) has been analyzed, as well as their photocytotoxicity on red blood cells and 3T3 murine fibroblasts. The results obtained show that the quinolones studied are able to photosensitize red blood cell lysis in an oxygen-dependent way and induce a high decrease in cell viability after UVA irradiation. A major correlation with phototoxicity lies in the structure of the individual antibacterials and their hydrophobicity; in particular, 6-amino derivatives are less phototoxic than corresponding unsubstituted and fluorinated compounds. Cellular phototoxicity was inhibited by the addition of free radical and hydroxyl radical scavengers (BHA, GSH and DMTU), suggesting the involvement of a radical mechanism in their cytotoxicity. A good correlation was observed between lipid peroxidation and phototoxicity, indicating that the test compounds exert their toxic effects mainly in the cellular membrane. Preliminary experiments on pBR322 DNA show that these derivatives do not photocleave DNA, differently from the two photogenotoxic fluoroquinolones, ciprofloxacin and lomefloxacin, used as reference compounds.

Acridizinium salts as a novel class of DNA-binding and site-selective DNA-photodamaging chromophores.

It was demonstrated that the interaction of the aminoacridizinium salts 2a-2d with DNA depends on the substitution pattern of the chromophore. Spectrophotometric and fluorometric titrations of the acridizinium salts 2a-2d with natural and synthetic polynucleotides reveal that the degree of interaction of the acridizinium salts 2a-2d with the nucleic acid differs significantly. The binding mode of the dyes with DNA was evaluated by circular dichroism and linear dichroism spectroscopy and compared with the parent system 2c. Whereas the 9-aminoacridizinium (2a) mainly intercalates into DNA, the salts 2b-c show a higher degree of association to the DNA backbone. The intercalated aminoacridizinium 2a caused few strand breaks upon UVA exposure, whereas the salts 2b-2d exhibit relatively efficient DNA-damaging properties. All acridizinium salts showed a sequence-selective strand cleavage for guanine-rich DNA regions.

In vitro studies of the phototoxic potential of the antidepressant drugs amitriptyline and imipramine.

Amitriptyline and imipramine, two tricyclic antidepressant drugs, have been studied to evaluate their phototoxic potential using various models. Reactive oxygen species production was investigated. A negligible production of singlet oxygen was observed for both compounds whereas a significant production of superoxide anion was noted for amitriptyline in particular. Moderate red blood cell lysis under UVA light (365 nm) was induced in the presence of the two drugs at a concentration of 50 microM. Cellular phototoxicity was investigated on a murine fibroblast cell line (3T3). The two drugs were phototoxic causing cell death at a concentration of 100 microM and a UVA dose in the range of 3.3-6.6 J/cm2. Furthermore, the two drugs photosensitized the peroxidation of linoleic acid, as monitored by the formation of dienic hydroperoxides. The presence of BHA and GSH, two free radical scavengers, significantly reduced the lipid oxidation photoinduced by the drugs, suggesting a predominant involvement of radical species. Finally, the involvement of nucleic acids in the phototoxicity mechanism was also investigated using a pBR322 plasmid DNA as a model.

New benzoquinolizin-5-one derivatives as furocoumarin analogs: DNA-interactions and molecular modeling studies.

Three derivatives of 1H,5H and 3H,5H-benzo[ij]quinolizin-5-one (BQZ1), previously prepared by chemical synthesis with the aim of obtaining furocoumarin analogs, have been studied. These are able to intercalate inside DNA and by subsequent irradiation with UVA light, to photoreact with DNA. Compound I (10-methoxy-7-methyl-1H,5H-benzo[ij]quinolizin-5-one) has a potentially photoreactive 2,3 double bond because of its conjugation with the pyridine ring of quinolinone, while compounds II (10-acetoxy-7-methyl-3H,5H-benzo[ij]quinolizin-5-one) and III (10-methoxy-7-methyl-3H,5H-benzo[ij]quinolizin-5-one) have a potentially photoreactive 1,2 double bond conjugated with the benzene ring of quinolinone. Compounds I and III, having a tricyclic planar structure, intercalate inside the DNA, while compound II cannot intercalate efficiently because of the steric hindrance of the acetoxy group in 10, lying outside the plane of the molecule and rotated by an angle of 77.6 degrees with respect to the tricyclic plane. The photoreaction of BQZ with DNA structure, as already known for psoralen and angelicin derivatives, consists of a [2 + 2] photocycloaddition reaction with the pyrimidine bases. The main photoadduct between the 2,3 double bond of I and the 5,6 double bond of thymine has been isolated and characterized by NMR, showing a cis-anti structure. Theoretical calculations, using AM1 Hamiltonian, have been carried out to describe the photocycloaddition reaction mechanism better. From a theoretical point of view, in the case of BQZ both the 1,2 or 2,3 double bonds and the 6,7 double bond may be involved in the [2 + 2] photocycloaddition. Spin densities and molecular orbital symmetries of compound I, in its triplet state, suggest that the 2,3 double bond interacts favorably with the 5,6 double bond of thymine moiety. On the contrary, the acetoxy substituent in position 10 of II seems to play a negative role in the DNA intercalation process.

Photochemical and photobiological studies on methylthioangelicins.

4,4',5'-Trimethyl-1'-thioangelicin (1) and 4,6,4',5'-tetramethyl-1'-thioangelicin (2), two newly synthesised isosters of furocoumarins having a sulfur atom in their five-membered ring, were studied in terms of interactions with DNA, both in the ground state and after UVA light absorption. The compounds were able to intercalate the macromolecule and to photobind efficiently, forming C4-cycloadducts with thymine. The antiproliferative effect of this binding was shown in Ehrlich and HeLa cells and by T2 phage inactivation. Tests on Salmonella typhimurium indicated low mutagenic activity. In particular, compound 1 has photobiological activity comparable with that of 4,6,4'-trimethylangelicin, but is less mutagenic.

4-tert-butylperoxymethyl-9-methoxypsoralen as intercalating photochemical alkoxyl-radical source for oxidative DNA damage.

We describe the synthesis of a novel psoralen peroxide 1 that generates on irradiation (350 nm) alkoxyl radicals, namely tert-butoxyl radicals, as confirmed by electron spin resonance studies with the spin trap 5,5-dimethyl-pyrroline-N-oxide. The radical source intercalates into the DNA, which has been demonstrated by linear-flow-dichroism measurements. Thus, the alkoxyl radicals are formed advantageously directly in the DNA matrix. In supercoiled pBR322 DNA, the generation of strand breaks by the photochemically or metal-catalyzed generated alkoxyl radicals is demonstrated. Photosensitization by the psoralen chromophore was excluded because similar substances that do not release radicals caused no DNA damage, nor were the photoproducts of the peroxide 1 active. With calf thymus DNA, 8-oxoGua and small amounts of guanidine-releasing products, e.g. oxazolone, were observed. However, in these reactions the photoproduct also displayed some DNA-oxidizing capacity.

DNA damage by tert-butoxyl radicals generated in the photolysis of a water-soluble, DNA-binding peroxyester acting as a radical source.

The photolysis of the water-soluble perester 1 leads to tert-butoxyl radicals as confirmed by EPR studies with the spin trap 5, 5-dimethylpyrroline N-oxide (DMPO). In the presence of DNA, oxidative cleavage of the latter was demonstrated by the formation of strand breaks in supercoiled pBR 322 DNA and by a substantial decrease of the melting temperature of salmon testes DNA. Guanidine, released from, for example, oxazolone and oxoimidazolidine on base treatment, was observed with calf thymus DNA and 2'-deoxyguanosine. These DNA modifications were effectively inhibited by the radical scavenger di-tert-butylcresol or the hydrogen atom donor glutathione. Photosensitization by the arene chromophore was excluded since the corresponding ester 2 caused no DNA damage, nor were the photoproducts of the perester 1 active. The efficacy of the perester 1 in oxidizing DNA derives from the fact that the tert-butoxyl radicals are photolytically generated in the immediate vicinity of the DNA, due to electrostatic binding of the cationic perester to the DNA, as confirmed by fluorescence measurements. These results demonstrate that the photolysis of perester 1 provides a suitable source of tert-butoxyl radicals in aqueous media, a necessary prerequisite for biochemical investigations.

Characterization of psoralen-oleic acid cycloadducts and their possible involvement in membrane photodamage.

By UVA irradiation of an ethanol solution of psoralen and oleic acid, four main photoproducts have been isolated and characterized: two have cis,cis structure; the other two are trans,cis. The same adducts have been isolated from the photoreaction of psoralen with beta-oleoyl-gamma-stearoyl-1-alpha-phosphatidylcholine followed by enzymatic hydrolysis with phospholipase A2. The four isomers stimulate protein kinase C to almost the same extent.

Photobiological properties of 1'-thieno-4,6,4'-trimethylangelicin.

Some photobiological properties of 1'-thieno-4,6,4'-trimethylangelicin (TTMA), a new isoster of 4,6,4'-trimethylangelicin (TMA) were studied in comparison with the parent compound. The TTMA absorbs UVA light and photobinds in vitro to DNA more efficiently than TMA; however, in Ehrlich cells in vivo TTMA linked to DNA to a lesser extent than the parent compound. In general, the formation of damage into DNA is in line with this last result: In fact, TTMA and TMA form equivalent amounts of interstrand cross-links (ISC) both in vitro in linearized PM2 DNA and in vivo in HeLa cells. In this system TTMA induces DNA-protein cross-links (DPC) more efficiently than TMA; on the contrary, no significant amounts of single-strand breaks were detected with both compounds. The antiproliferative activity of TTMA is consistent with these results, being only slightly more pronounced than that of TMA. Experiments carried out using double irradiation demonstrated that these drugs are capable of inducing antiproliferative effects by biphotonic reactions, including the formation of both ISC and DPC. Thus, replacement of the oxygen atom by a sulfur increases the UV absorption of the drug and its capacity to photobind to DNA in vitro but does not yield a comparable enhancement of its photosensitizing properties in vivo; this might be due to various reasons, for instance to an increase in the lipophilic character that could modify the behavior in vivo.

Dark and photochemical interactions of dimethyltetrahydrobenzoangelicin with DNA.

A study of dark interaction and photoreaction between 4,6-dimethyltetrahydrobenzoangelicin (THBA) and DNA is described. 4,6-Dimethyltetrahydrobenzoangelicin is a furocoumarin derivative in which 4' and 5' carbons are linked by a four-methylene bridge. In spite of the bulky aliphatic ring, THBA forms a complex with DNA in the dark and, on UVA irradiation, reacts with pyrimidine bases of DNA yielding monoadducts only involving its furan side double bond. Two main photoproducts form: they derive from a C4-cycloaddition to thymine and cytosine, respectively, and account for 56% and 39% of the total photoreaction yield. Both show cis-syn configuration. Two other isomers, one with thymine and one with cytosine, formed with so much lower yield (ca 3 and 1%, respectively) that their structure could not be assigned. Furthermore, in spite of its angular structure, THBA induces a small number of crosslinks in DNA.

1-Thiopsoralen, a new photobiologically active heteropsoralen. Photophysical, photochemical and computer aided studies.

1-Thiopsoralen (7H-thieno[3,2-g]benzofuran-7-one) 1, a lead compound of a series of heteropsoralens, was investigated. The electronic transitions involved were studied. Fluorescence quantum yield is very low, while laser flash photolysis showed that the triplet state is practically the sole transient of 1. Fluorescence quantum yield (phi F) and triplet lifetime (tau F) as well as triplet quantum yield (phi T) and lifetime (tau T) were determined. The production of singlet oxygen was also evaluated by photophysical measurements. Photophysical data suggest that DNA photobinding of 1, owing to short fluorescence lifetime value and high triplet quantum yield, occurs likely through triplet mechanism. Interactions between 1 and DNA were studied both in the ground and the excited state. In the ground state 1 undergoes intercalation inside duplex DNA. This fact is also supported by molecular modeling studies. By UVA-light activation 1 photobinds covalently to DNA forming mono and diadducts. The furan side 1-thymine monoadduct, isolated from DNA photomodified by thiopsoralen, shows a cis-syn stereochemistry, in agreement with quantum mechanics studies. Compound 1 photobinds also with linolenic acid, component of lecithins, giving a C4-cycloaddition, and supporting that this compound also induces photolesions at the level of cell membrane, like psoralen. Compound 1 exhibits strong skin-phototoxicity.

Angular furoquinolinones, psoralen analogs: novel antiproliferative agents for skin diseases. Synthesis, biological activity, mechanism of action, and computer-aided studies.

With the aim of obtaining new potential photochemotherapeutic agents, having increased antiproliferative activity and decreased undesired effects, we have prepared some new furoquinolinones. Two of them have been studied in detail: 1,4,6,8-tetramethyl-2H-furo[2,3-h]-quinolin-2-one (8), and 4,6,8,9-tetramethyl-2H-furo[2,3-h]quinolin-2-one (10). These compounds form a molecular complex with DNA, undergoing intercalation inside the duplex macromolecule, as shown by linear flow dichroism. The complexed ligands, by subsequent irradiation with UV-A light, photobind with the macromolecule forming only monocycloadducts with thymine with cis-syn configuration. In order to evaluate the electronic effects induced by the nitrogen atom in position 1 of 8, semiempirical calculations have been performed on both 4,6,4'-trimethylangelicin (TMA) and 8. The results obtained do not clearly differentiate between the two molecules which, at this level of approximation, show the possibility of photoreaction with both the 3,4- and 8,9-olefinic bonds for 8 and the 3,4- and 4',5'-bonds for TMA. In the lower energy conformation of intercalated 8, the furan ring is turned toward the minor groove of the polynucleotide, in such a way that photoreaction of this ring with thymine is favored. These compounds unexpectedly inhibit DNA and RNA synthesis in Ehrlich cells, in the dark. They also show a strong photoantiproliferative activity, 2 orders of magnitude higher than 8-methoxypsoralen (8-MOP), the most used drug for photochemotherapy. Their mutagenic activity on Escherichia coli is similar to that of TMA and 8-MOP. On the basis of these results, the compounds should deserve evaluation of their activity in the treatment of hyperproliferative skin diseases.

Sulphur and selenium analogues of psoralen as novel potential photochemotherapeutic agents.

Some heteropsoralens, obtained by replacing one or both the intracyclic oxygen atoms with sulphur and/or selenium, were studied. In preliminary tests, these compounds showed strong photobiological activity, in some cases more than two orders of magnitude higher than that of psoralen. Heteropsoralens containing sulphur undergo intercalation inside duplex DNA, showing evident affinity for the macromolecule; when selenium replaces furan oxygen, the psoralen isoster also undergoes intercalation but with lower efficiency, while psoralen isosters in which pyrone oxygen is replaced by selenium practically do not intercalate. Parallel behaviour was also observed for DNA photobinding and crosslink formation. The cycloadduct between furan selenium and pyrone sulphur isoster and thymine was isolated and characterized. The capacity of the various psoralen isosters to generate singlet oxygen and superoxide radical anion was studied. For the former the yield varies markedly for the various compounds, while for the latter the yield is similar for all compounds.

Improvement of dissolution characteristics of psoralen by cyclodextrins complexation.

Psoralen was found to form an inclusion complex with beta-cyclodextrin (beta CD), heptakis(2,6-di-O-methyl)-beta-cyclodextrin (DM beta CD) and hepatakis(2,3,6-tri-O-methyl)-beta-cyclodextrin (TM beta CD). Phase solubility studies revealed the formation of a 1:1 molar complexes. The stability constant were 663 M-1 for beta CD, 603 M-1 for DM beta CD and 69.6 M-1 for TM beta CD. The formation of complexes in the solid state was confirmed by spectroscopic analyses, X-ray diffractometry and differential thermal data. The solubility and dissolution rate of the complexed forms were improved, particularly for the DM beta CD complex. The strength of binding of psoralen to DNA was not influenced by complexation with cyclodextrins.

Pyrroloquinolinone methylderivatives, furocoumarin analogues: interaction with biomolecules and computer-aided studies.

Pyrroloquinolinones, furocoumarin analogues, contain a divinilbenzene moiety, suggesting possible photoreactivity. Quantum mechanics calculations indicate that the pyrrole-side double bond exhibits strong photoreactivity, while the pyridone-side double bond is only poorly photoreactive. Intercalation models obtained by molecular mechanics calculations suggest that, in the cis-syn intercalation arrangement, the pyridone-side double bond is well aligned with the nearby thymine, supporting possible C4-cycloaddition with the 5,6 double bond of thymine, while the pyrrole-side double bond assumes an unfavourable position for photobinding. These data suggest that photoreaction between the pyridone-side and thymine double bonds may takes place, although with very low yield. Experimental evidence concerning DNA-photobinding exhibited by 2,6-dimethyl-9-methoxy-4H-pyrroloquinolinone (Compound I) confirms theoretical predictions. The formation of C4-cycloadducts between the pyridone side double bond and thymine also takes place with very low yield. Compound I shows marked BSA photobinding, suggesting that pyrroloquinolinones may photoreact with proteins. The three pyrroloquinolinones examined show high yields of singlet oxygen generation, suggesting that photobiological effects may be obtained through this photodynamic pathway, rather than through DNA photobinding.

Psoralen-fatty acid cycloadducts activate protein kinase C (PKC) in human platelets.

The C4-photocycloadduct psoralen-linolenic acid activates protein kinase C (PKC), in human platelets, which in turn phosphorylates 47 kDa protein, representing its major substrate in these cells. This behaviour is similar to that shown by diacylglycerol, a main second messenger responsible of various biological effects, e.g. of melanogenesis.

8-Azapsoralen derivatives: isolation and characterization of the furan-side cycloadducts with DNA.

The formation of C4-cycloadducts by photoreaction of eight methyl derivatives of 8-azapsoralen with DNA was studied. The main reaction involves the furan ring of the compounds and the 5,6 double bond of thymine giving cis-syn adducts, although a minor amount of a cycloadduct with cytosine was also isolated for 4,4'-dimethylazapsoralen. The role of the nitrogen atom appears to depend on the electronic effect, leading to a decrease in the reactivity of the pyrone ring. As with psoralens, the methyl groups increase both the lipophilicity and, with the exception of the 5,4',5'-trimethyl derivative, the photoreactivity. However, methyl groups do not appear to influence the chemistry of the photoaddition.

Azapsoralens-DNA interactions: crystal structure characterization of furan-side monoadduct and computer-aided studies.

In this paper a theoretical study, concerning molecular mechanics optimised structures, obtained by quantum mechanics as well as molecular mechanics calculations was carried out with the aim of correlating the theoretical model of the interactions between azapsoralens and DNA with the data experimentally obtained. The theoretical model suggests that both furan-side and pyrone-side double bonds may be involved in the cycloaddition with pyrimidines (although the cycloaddition at the level of furan is preferred), and is in line with the capacity of these compounds to form inter-strand cross-links. Moreover, concerning the theoretical intercalation model calculations on 3,4,4',5'-tetramethylazapsoralen intercalated inside a polynucleotide, they suggest a cis-syn arrangement between furan-side of the intercalated ligand and the above situated thymine, with which, under light activation, a cycloadduct may take place, having a cis-syn steric arrangement. Also this datum is in agreement with the cis-syn regio and stereochemistry of the isolated 4,4',5'-trimethylazapsoralen-thymine cycloadduct. Finally, from theoretical data, the role of nitrogen seems not important: in fact only small differences were found with the corresponding methylpsoralens so that the small differences observed may be mainly attributed to steric rather than to electronic effects. In general a good correlation between the theoretical model and the experimental data was observed.

New mono- and bis-intercalating compounds between DNA base pairs.

With the aim to obtain new bis-intercalating agents in DNA a series of compounds was prepared linking two identical tricyclic moieties (two 4,5'-8-trimethylpsoralen (TMP)) or different (one TMP and one pyrido[3,2-b]quinoline) through a hydrocarburic aminated flexible chai. Bis-psoralen-amines as well as psoralen-pyrydoquinolin-amines were obtained. For comparison the corresponding mono-psoralen-amines were also prepared condensing a TMP moiety with various hydrocarburic aminated chains. Melting profiles of the complexes between bis-psoralen-amines or psoralen-pyridoquinolin-amines and DNA evidence two different thermal transitions, which can be correlated with bis-intercalation which takes place, at least in part, inside duplex DNA. On the other hand mono-psoralen-amines evidenced only one thermal transition, in line with mono-intercalation. Bis-intercalating agents complexed with DNA, under UVA irradiation photoconjugate covalently to the macromolecule, even if to a lower extent in comparison with mono-intercalating agents. Moreover these bis-intercalating agents in the photoreaction with DNA form inter-strand cross-links; also in this case bis-intercalating agents are less active than mono-intercalating agents.