Search

An ever-increasing incidence of skin neoplastic diseases is registered. Therefore, it is important to protect the skin from the UV radiation that reaches the epidermis and dermis but also to block ROS generated by them. Our attention was attracted in developing new compounds provided with both UV filtering and antioxidant capacities. To this end, 2-phenyl-1H-benzimidazole-5-sulfonic acid (PBSA), a known UV filter, was selected as lead compound for its lack of antioxidant activity, high water solubility and good safety profile. PBSA was sequentially modified introducing hydroxyls on the phenyl ring and also substituting the functional group in position 5 of the benzimidazole ring. At the end of the synthetic study, a new, very potent class of antioxidants has been obtained. Surprisingly some of the developed molecules, while devoid of significant UV-filtering activity was endowed with potent UV-filtering booster capability if associated with known commercial UVB and UVA filters.

The phototoxicity of the new anticancer drug vandetanib was evaluated using human keratinocyte cell line, NCTC-2544. This study was started since many clinical cases of vandetanib photosensitizing reactions were recently reported in literature. Vandetanib induces a clear drop in human keratinocytes viability after cell irradiation in concentration and UV-A dose dependent mode. Since vandetanib can photolyze with the formation of two main photoproducts after UV-A exposure, the contribution of these new species was also evaluated. These two photoproducts did not have a main role in the phototoxicity of their parent drug. In our opinion, the main hypothesis for the vandetanib phototoxic potential is the formation of a very reactive specie, such as an aryl radical, which can react promptly with different targets inside the cells. In fact, a massive DNA photodamage was detected both in the in vitro DNA photocleavage experiments, and in cells. Moreover, vandetanib was able to photoinduce lipid peroxidation and protein oxidations. Vandetanib photoinduced cell death by apoptosis with the involvement of mitochondria and lysosomes.

Tubulin binding agents (TBAs) are drugs commonly used in cancer therapy as antimitotics. In the last years it has been described that TBAs, like combretastatin A-4 (CA-4), present also vascular disrupting activity and among its derivatives we identified three analogues endowed with potent microtubule depolymerizing activity, higher than that of the lead compound. In this paper we have investigated the anti-vascular activity of these derivatives. We tested the anti-angiogenic effects in human umbilical endothelial cells (HUVEC) and in vivo in chick chorioallantoic membrane assay (CAM), and in a syngeneic tumor mouse model. The three molecules, compound 1 : 1-(3,4,5-trimethoxyphenyl)-5-(4-ethoxyphenyl)-1H-1,2,4-triazole; compound 2 : (1-(3,4,5-trimethoxyphenyl)-5-(4-ethoxyphenyl)-1H-tetrazole, compound- 3 (4-amino-2-p-tolylaminothiazol-5-yl)-(3,4,5-trimethoxyphenyl)-methanone) showed a moderate effect on the growth of HUVEC cells at concentrations below 200 nM. At lower concentrations (5–20 nM), in particular compound 2 , they induced inhibition of capillary tube formation, inhibition of endothelial cell migration and affected endothelial cell morphology as demonstrated by the alteration of the microfilaments network. Moreover, they also increased permeability of HUVEC cells in a time dependent manner. In addition, compounds 1 and 3 , as well as the reference compound CA-4, inhibited VEGF-induced phosphorylation of VE-cadherin and in addition compound 3 prevented the VEGF-induced phosphorylation of FAK. In CAM assay, both compounds 2 and 3 efficiently counteracted the strong angiogenic response induced by bFGF, even at the lowest concentration used (1 pmol/egg). Moreover in a syngenic mouse model, compounds 1 – 3 after a single i.p. injection (30 mg/kg), showed a stronger reduction of microvascular density. Altogether our results identified these derivatives as potential new vascular disrupting agents candidates.

This study reports the results of an investigation of the phototoxicity mechanism induced by pitavastatin and its photoproducts, namely 6-cyclopropyl-10-fluoro-7,8-dihydrobenzo[k]phenanthridine (PP3) and 6-cyclopropyl-10-fluorobenzo[k]phenanthridine (PP4). The phototoxicity was tested in human keratinocytes cell lines NCTC-2544, and the results proved that under the same conditions, all three compounds exhibited phototoxic effects in the model tested. The reduction in cell viability was found to be both concentration- and UVA dose-dependent. A point of note is that both the photoproducts produced a dramatic decrease in cell viability with GI(50) values one order of magnitude lower compared to the parent compound. In particular, the fully aromatic derivative (PP4) showed the highest antiproliferative activity. Flow cytometric analysis indicated that pitavastatin and the photoproduct PP4 principally induced necrosis, as revealed by the large appearance of propidium iodide-positive cells and also confirmed by the rapid drop in cellular ATP levels. Further studies committed to better understanding of photoinduced cell death mechanism(s) revealed that neither pitavastatin nor PP4 induced mitochondrial depolarization or lysosomal damage, but, interestingly, extensive cell lipid membrane peroxidation along with a significant oxidation of model proteins occurred, suggesting that pitavastatin and PP4 exert their phototoxic effect mainly in the cellular membranes. The present results suggest that the phototoxicity of pitavastatin may be mediated by the formation of benzophenanthridine-like photoproducts that appear to have high potential as photosensitizers.

Heteroanalogues of angelicin, pyrrolo[3,2-h]quinazolines, were synthesized with the aim of obtaining new potent photochemotherapeutic agents. Many derivatives caused a significant decrease in cell proliferation in several human tumor cell lines after irradiation with UVA light (GI(50) =15.2-0.2 μM). Their phototoxicity effected apoptosis in Jurkat cells with the involvement of mitochondria (as determined by the loss of mitochondrial membrane potential and production of reactive oxygen species) and lysosomes. The phototoxicity of these compounds could be explained by lipid peroxidation.

Despite improvements in the treatment and prevention of cancer, the number of new diagnoses continues to rise; this has fuelled substantial interest in the development of new and effective chemotherapeutic agents. Compounds of the triazene class, such as dacarbazine, have been used in the clinical management of many cancer types including brain, leukemia, and melanoma. A new compound class bearing a triazenoazaindole scaffold was synthesized with the aim of identifying new antiproliferative agents. Compounds 5 a-g and 6 a-c were screened against a panel of human tumor cell lines, and two of them, 5 e and 5 f, showed cytotoxicity (GI(50) range: 2.2-8.2 μM) in all cell lines. These two compounds even maintained their cytotoxicity in some multidrug-resistant cell lines. Flow cytometry analysis demonstrated their ability to induce cell death by apoptosis with involvement of lysosomes.

Pyrrolo[3,4- h ]quinolin-2-ones were synthesized as nitrogen isosters of the angular furocoumarin angelicin, with the aim of obtaining new photochemotherapeutic agents with increased antiproliferative activity and lower undesired toxic effects. A versatile synthetic pathway was approached to allow the isolation of derivatives of the new ring system with a good substitution pattern on the pyrrole moiety. Photobiological screenings of the new compounds revealed a potent phototoxic effect and a great UVA dose dependence, reaching IC 50 values at submicromolar level. The induced cellular photocytotoxicity was related to apoptosis with the involvement of mitochondria and lysosomes, alteration of cell cycle profile and membrane lipid peroxidation.

In this paper, we have investigated the mechanism of phototoxicity of fluvastatin, an 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, in human keratinocytes cell line NCTC-2544. Fluvastatin underwent rapid photodegradation upon Ultraviolet-A (UVA) irradiation in buffered aqueous solution as shown by the changes in absorption spectra. Interestingly, no isosbestic points were observed but only a fast appearance of a spectral change, indicative of the formation of a new chromophore. The isolation and characterization of the main photoproduct revealed the formation of a polycyclic compound with a benzocarbazole-like structure. This product was also evaluated for its phototoxic potential. Cell phototoxicity was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide test after 72 h from the irradiation in the presence of fluvastatin. The results showed a reduction of the cell viability in a concentration and UVA dose-dependent manner. Surprisingly, the photoproduct showed a dramatic decrease of the cell viability that occurred at concentrations of an order of magnitude lower than the parent compound. Flow cytometric analysis indicated that fluvastatin and its main photoproduct induced principally necrosis as revealed by the large appearance of propidium iodide-positive cells and confirmed also by the rapid drop in cellular adenosine triphosphate levels. Interestingly, a rapid increase of intracellular calcium followed by an extensive cell lipid membrane peroxidation and a significant oxidation of model proteins were induced by fluvastatin and its photoproduct, suggesting that these compounds exerted their toxic effect mainly in the cellular membranes. On the basis of our results, the phototoxicity of fluvastatin may be mediated by the formation of benzocarbazole-like photoproduct that acts as strong photosensitizer.

The photostability of pitavastatin, an HMG-CoA reductase inhibitor used in the treatment of hypercholesterolemia, was investigated. The sample solution was exposed to UV-A radiation and the photodegradation process was monitored by means of spectrophotometric method and HPLC-DAD. Pitavastatin was shown to be photolabile and its photodegradation reaction followed the first-order kinetics with the rate constant k=3.54 x 10(-4)+/-9.43 x 10(-6)s(-1). The chromatograms revealed the presence of four major photoproducts (PP-1-PP-4). The separated and isolated photolytic products were identified using a mass spectrometer coupled with a time of flight (TOF) analyzer. The main reaction observed during exposure to radiation of pitavastatin was photocyclisation leading to formation of four-ring photoproducts.

The aim of the present study was to identify molecular analogs of angelicin (ANG) able to increase erythroid differentiation of K562 cells and expression of gamma-globin genes in human erythroid precursor cells, with low effects on apoptosis. ANG-like molecules are well-known photosensitizers largely used for their antiproliferative activity in the treatment of different skin diseases (i.e., psoriasis, vitiligo, eczema, and mycosis fungoides). To verify the activity of these derivatives, we employed three experimental cell systems: (1) the human leukemic K562 cell line, (2) K562 cell clones stably transfected with a pCCL construct carrying green-EGFP under the gamma-globin gene promoter, and (3) the two-phase liquid culture of human erythroid progenitors isolated from normal donors and beta-thalassemia patients. The results of our study suggest that trimethyl ANG is a powerful inducer of erythroid differentiation, compared with known inducers, such as ANG, cytosine arabinoside, mithramycin, and cisplatin. These data could have practical relevance, because pharmacologically mediated regulation of human gamma-globin gene expression, with the consequent induction of fetal hemoglobin, is considered a potential therapeutic approach in hematological disorders including beta-thalassemia and sickle cell anemia.

A convenient synthesis of the pyrano[2,3-e]isoindol-2-one ring system, an heteroanalogue of angelicin, is reported. Our synthetic approach consists of the annelation of the pyran ring on the isoindole moiety using 5-dialkylamino- or 5-hydroxymethylene intermediates as building blocks. The photoantiproliferative activity of the new derivatives was studied. Some of them bearing the benzyl group at the 8 position were active with IC(50) in the micromolar range. Cell cytotoxicity involves apoptosis, alteration of cell cycle profile and membrane photodamage.

A new series of pyrazolo[3,4-h]quinolines, heteroanalogues of angelicin was conveniently prepared with a broad substitution pattern. A large number of derivatives was obtained and the cellular photocytotoxicity was evaluated in vitro against 5 different human tumor cell lines with GI50 values reaching the nanomolar level (14.52e0.04 mM). Selected compounds were able to photoinduce a massive cell death with the involvement of mitochondria. Their photodamage cellular targets were proteins and lipids and they did not cause any kind of DNA photodamage. This latter event is of considerable importance in the modulation of long term side effects, generally associated with the use of classical furocoumarins.

The antiproliferative activity, upon UVA irradiation, of two tetracyclic derivatives of benzo[b]thiophenes and pyridines, a benzo[b]thienopyridopyrimidone (1) and a thienocarboline (2), has been investigated in a panel of human tumor cell lines. The two compounds present a remarkable cytotoxicity after UVA irradiation (365 nm), reaching an IC50 of 0.1 microM in the leukaemia cell lines and 0.3-0.5 microM in the solid tumour cell lines. Their effect on the cell cycle was measured by flow cytometry in Jurkat cells. The compounds induce cell cycle perturbations and trigger a massive apoptosis as revealed by the externalisation of Annexin V-targeted residues at the outer plasmatic membrane. Furthermore the drugs induce, upon UVA irradiation significant variations of the mitochondrial potential (Deltapsi(mt)) measured by flow cytometry using the fluorochrome JC-1. In addition we characterized the mitochondrial production of reactive oxygen species (ROS) using the probe dihydroethidine (HE) and the oxidations of the mitochondrial phospholipid cardiolipin using the interacting probe nonyl acridine orange (NAO). Both compounds stimulate the production of ROS, and remarkably induce oxidation of cardiolipin. We have investigated the DNA-binding properties of these two compounds by means of UV-Vis spectroscopy and fluorescence. The two compounds exhibit a low affinity toward the macromolecule. The mode of binding was also investigated by means of flow linear dichroism (LD) which has revealed that the two compounds do not efficiently intercalate into DNA. Finally, the DNA-photocleavaging properties of the test compounds were studied on pBR322 plasmid DNA as a model. Only compound 1 is able to induce a significant production of single strand breaks only after digestion with the base excision repair enzyme Endo III. Altogether these data suggest that DNA is not a preferential target of these molecules and other subcellular structures may be responsible for their high phototoxic activity.

A new class of compounds analogues of geiparvarin is described: aldolic condensation of 3(2H)-furanones and 7-(2-oxoethoxy)coumarins followed by a very efficient dehydration protocol led to the title compounds which show good antitumoral activity against several human cell lines.

An efficient synthesis (yields >70%) of linear 7H-furo[3,2-g]chromen-7-one derivatives (psoralens or furocoumarins) has been carried out starting from ring-substituted 2-(coumarin-7-yl)oxyaldehydes; moreover, the phototoxicity of these compounds has been tested on a cultured cell line of murine fibroblast. © 2002 Elsevier Science Ltd. All rights reserved.

The association of the naphtho[1,2-b]quinolizinium bromide (5a) and naphtho[2,1-b]quinolizinium bromide (5b) with DNA and the propensity of these cationic arenes to damage DNA after UV-A irradiation have been studied. Spectrophotometric and fluorimetric titrations show that the two isomers 5a and 5b bind to DNA (K approximately 10(5) M(-1)). The highest affinity was observed for GC base pairs. The mode of binding was investigated by CD and LD spectroscopy. Whereas quinolizinium 5a exclusively intercalates in DNA, the isomer 5b exhibits a deviation from perfect intercalation into the double helix. Moreover, efficient DNA damage was observed on UV-A irradiation in the presence of the quinolizinium salts. Primer extension analysis indicates that the photocleavage takes place preferentially at guanine-rich regions.

A new series of pyrrolo[3,4- h ]quinazolines was conveniently prepared with a broad substitution pattern. A large number of derivatives was obtained and the cellular cytotoxicity was evaluated in vitro against 5 different human tumor cell lines with GI 50 values reaching the low micromolar level (1.3–19.8 μM). These compounds were able to induce cell death mainly by apoptosis through a mitochondrial dependent pathway. Selected compounds showed antimitotic activity and a reduction of tubulin polymerization in a concentration-dependent manner. Moreover, they showed anti-angiogenic properties since reduced in vitro endothelial cell migration and disrupted HUVEC capillary-like tube network in Matrigel.

Derivatives of new ring systems 11H-pyrido[3',2':4,5]pyrrolo[3,2-c]cinnoline and pyrido[3',2':4,5]pyrrolo[1,2-c][1,2,3]benzotriazine have been prepared from the key intermediates 2-(1H-pyrrolo[2,3-b]pyridin-2-yl)anilines in excellent yields (94-99%) and screened by the National Cancer Institute (Bethesda, MD) on about 60 human tumor cell lines derived from nine cancer cell types. The tested compounds exhibited antiproliferative activity against all the human cell lines, showing comparable MG_MID (mean graph midpoint) values in the range of 0.74-1.15 μM. A particular efficacy was observed against the leukemia subpanel (GI50 = 0.73-0.0090 μM). Flow cytometric analysis of the cell cycle demonstrated an increase in the percentage of cells in the G2/M phase. The compounds caused apoptosis of the cells, mitochondrial depolarization, generation of reactive oxygen species, and activation of caspase-3, caspase-8, and caspase-9. Moreover, they acted as topoisomerase I inhibitors.

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 μM. Cellular phototoxicity was investigated on a murine fibroblast cell line (3T3). The two drugs were phototoxic causing cell death at a concentration of 100 μM and a UVA dose in the range of 3.3–6.6 J/cm 2 . 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.

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.

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.

Exposure of aqueous solutions of the antitumor drug vandetanib to UV-A light results in the photochemical degradation. Two main photodegradation products were identified by HPLC–MS analysis and their structures were elucidated, after their isolation by HPLC, on the basis of LC–MS and NMR spectra. The photoproducts derived from a simple debromination (N-(2-fluorophenyl)-6-methoxy-7-((1-methylpiperidin-4 yl)methoxy)quinazolin-4-amine, FP3) or from the loss of the bromide atom followed by the solvent addition (N-(4-hydroxy-2-fluorophenyl)-6-methoxy-7-((1-methylpiperidin-4 yl)methoxy)quinazolin-4-amine, FP2). At our knowledge this is the first report about the photodegradation of vandetanib.

Highlights ► Furocoumarins photoinduce erythroid differentiation in K562 cells with UVA. ► Furocoumarins photoinduce high increase in α-, ζ-, ε-, and γ-globin mRNA sequences. ► Furocoumarin erythrodifferentiation is connected with DNA photodamage. ► 5,5′-Dimethylpsoralen photoproducts present differentiating properties., Differentiation-therapy is an important approach in the treatment of cancer, as in the case of erythroid induction in chronic myelogenous leukemia. Moreover, an important therapeutic strategy for treating beta-thalassemia and sickle-cell anemia could be the use of drugs able to induce erythroid differentiation and fetal hemoglobin (HbF) accumulation: in fact, the increased production of this type of hemoglobin can reduce the clinical symptoms and the frequency of transfusions. An important class of erythroid differentiating compounds and HbF inducers is composed by DNA-binding chemotherapeutics: however, they are not used in most instances considering their possible devastating side effects. In this contest, we approached the study of erythrodifferentiating properties of furocoumarins. In fact, upon UV-A irradiation, they are able to covalently bind DNA. Thus, the erythrodifferentiation activity of some linear and angular furocoumarins was evaluated in the experimental K562 cellular model system. Quantitative real-time reverse transcription polymerase-chain reaction assay was employed to evaluate the accumulation of different globin mRNAs. The results demonstrated that both linear and angular furocoumarins are strong inducers of erythroid differentiation of K562 cells. From a preliminary screening, we selected the most active compounds and investigated the role of DNA photodamage in their erythroid inducing activity and mechanism of action. Moreover, some cytofluorimetric experiments were carried out to better study cell cycle modifications and the mitochondrial involvement. A further development of the work was carried out studying the erythroid differentiation of photolysis products of these molecules. 5,5′-Dimethylpsoralen photoproducts induced an important increase in γ-globin gene transcription in K562 cells.

Pancreatic cancer is a complex malignancy arising from the accumulation of genetic and epigenetic defects in the affected cells. Standard chemotherapy for patients with advanced disease shows only modest effects and is associated with considerable toxicity. Overexpression or aberrant activation of members of the epidermal growth factor receptor tyrosine kinase family, which includes EGFR and HER-2, occurs frequently and is associated with multiple drug resistance and decreased patient survival. In this study, we have investigated the therapeutic potential of AS104, a novel compound of the triazene class, with potential inhibitory effects on EGFR. We found that treatment of cells with AS104 causes significant reduction of cell growth and metabolic activity in four human pancreatic cancer cell lines. Furthermore, we show that the AS104-mediated induction of apoptotic cell death is associated with stimulation of autophagy in a dose-dependent manner. Treatment of cells with AS104 results in significant down-regulation of EGFR and HER-2 expression and activity and subsequent inhibition of downstream signaling proteins. Quantitative RT-PCR analysis and assays with proteasome inhibitors revealed that AS104 regulates the expression of EGFR and HER-2 at the transcriptional level. These findings provide for the first time experimental evidence for efficacy of AS104 in the simultaneous transcriptional repression of EGFR and HER-2 genes and suggest that AS104 may have therapeutic potential in the treatment of pancreatic cancers that express high levels of the aforementioned receptor tyrosine kinases.

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.

Chronic inflammatory response in the airway tract of patients affected by cystic fibrosis is characterized by an excessive recruitment of neutrophils to the bronchial lumina, driven by the chemokine interleukin (IL)-8. We previously found that 5-methoxypsoralen reduces Pseudomonas aeruginosa -dependent IL-8 transcription in bronchial epithelial cell lines, with an IC50 of 10 μM (Nicolis E, Lampronti I, Dechecchi MC, Borgatti M, Tamanini A, Bezzerri V, Bianchi N, Mazzon M, Mancini I, Giri MG, Rizzotti P, Gambari R, Cabrini G. Int Immunopharmacol 9: 1411–1422, 2009). Here, we extended the investigation to analogs of 5-methoxypsoralen, and we found that the most potent effect is obtained with 4,6,4′-trimethylangelicin (TMA), which inhibits P. aeruginosa -dependent IL-8 transcription at nanomolar concentration in IB3–1, CuFi-1, CFBE41o−, and Calu-3 bronchial epithelial cell lines. Analysis of phosphoproteins involved in proinflammatory transmembrane signaling evidenced that TMA reduces the phosphorylation of ribosomal S6 kinase-1 and AKT2/3, which we found indeed involved in P. aeruginosa -dependent activation of IL-8 gene transcription by testing the effect of pharmacological inhibitors. In addition, we found a docking site of TMA into NF-κB by in silico analysis, whereas inhibition of the NF-κB/DNA interactions in vitro by EMSA was observed at high concentrations (10 mM TMA). To further understand whether NF-κB pathway should be considered a target of TMA, chromatin immunoprecipitation was performed, and we observed that TMA (100 nM) preincubated in whole living cells reduced the interaction of NF-κB with the promoter of IL-8 gene. These results suggest that TMA could inhibit IL-8 gene transcription mainly by intervening on driving the recruitment of activated transcription factors on IL-8 gene promoter, as demonstrated here for NF-κB. Although the complete understanding of the mechanism of action of TMA deserves further investigation, an activity of TMA on phosphorylating pathways was already demonstrated by our study. Finally, since psoralens have been shown to potentiate cystic fibrosis transmembrane conductance regulator (CFTR)-mediated chloride transport, TMA was tested and found to potentiate CFTR-dependent chloride efflux. In conclusion, TMA is a dual-acting compound reducing excessive IL-8 expression and potentiating CFTR function.

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.

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.

An efficient synthesis (yields >70%) of linear 7H-furo[3,2-g]chromen-7-one derivatives (psoralens or furocoumarins) has been carried out starting from ring-substituted 2-(coumarin-7-yl)oxyaldehydes; moreover, the phototoxicity of these compounds has been tested on a cultured cell line of murine fibroblast.

In the search for new photochemotherapeutic agents, a series of derivatives of the ring system pyrrolo[3,2-h]quinoline--bioisosters of the angular furocoumarin angelicin--were synthesized through a four-step synthetic approach, in reasonable overall yields. Eight of the synthesized derivatives showed a remarkable phototoxicity against a panel of four human tumor cell lines and a great dose UV-A dependence, reaching IC₅₀ values at submicromolar level. The mode of cellular death photoinduced by pyrrolo[3,2-h]quinolines was evaluated through a series of flow cytometric analysis and other tests were performed to clarify their mechanism of action.

Induction of terminal erythroid differentiation can be an efficient strategy to inhibit proliferation of chronic myelogenous leukemia cells. Psoralens, well-known photo-chemotherapeutic agents, were found to be efficient at inducing erythroid differentiation of K562 cells, an in vitro cell line isolated from the pleural effusion of a patient with chronic myelogenous leukemia in blast crisis. The effects of crude pre-irradiated solutions of 5-methoxypsoralen on erythroid differentiation of human leukemic K-562 cells were evaluated. The major photoproduct was characterized and analyzed, and it was found to induce erythroid differentiation of K562 cells and inhibit NF-kappaB/DNA interactions.

4,6-Dimethylbenzoangelicin, obtained by fusing a benzene ring at the furan side of 4,6-dimethylangelicin, was studied in terms of crystal structure and interactions with DNA in both ground and excited states. 4,6-Dimethylbenzoangelicin has a planar structure and forms a molecular complex with DNA, undergoing intercalation inside the double helix. Under UVA irradiation, it photoconjugates covalently with the macromolecule, showing a DNA photobinding rate slightly lower than that of 8-methoxypsoralen, involving however only its 3,4 double bond, i.e. behaving as a pure monofunctional agent. The parameters of dark binding and photobinding were determined, and two C4 cycloadducts with thymine were isolated and characterized.

In this review we summarize the structure and biological effects of linear and angular psoralens. These compounds exhibit interesting biological effects on the cell cycle, apoptosis and differentiation. These molecules should be considered promising drugs in the therapy of several diseases, including psoriasis, mycosis fungoides and cancer. Also, preclinical data demonstrate a possible use of these molecules for the treatment of beta-thalassemia and other hematological disorders.

— Photochemical and photobiological properties of a new isoster of psoralen, 4, 4′, 5′-trimethyl-8-azapsoralen (4, 4′, 5′-TMAP), have been studied. This compound shows a high DNA-photobinding rate, higher than that of 8-methoxypsoralen (8-MOP), forming both monoadducts and inter-strand cross-links. The yield of cross-links, however, is markedly lower than that of 8-MOP. Antiproliferative activity of 4, 4′, 5′-TMAP, in terms of DNA synthesis inhibition in Ehrlich ascites tumor cells, is higher than that of 8-MOP. Mutagenic activity on E. coli WP2 R46+ cells appeared similar to or even lower than that of 8-MOP. This new compound applied on depilated guinea pig skin and irradiated with UVA did not show any skin-phototoxicity. On the basis of these properties 4, 4′, 5′-TMAP appears to be a potential photochemotherapeutic agent.

Isoindoloquinoxalines 4 and 5 were obtained by refluxing 2-(2'-aminoaryl)-1-cyanoisoindoles 3a- e in acetic or formic acid. All derivatives were screened by the National Cancer Institute (Bethesda, MD) for the in vitro one dose primary anticancer assay against a 3-cell line panel. Compounds 4a- e, screened against a panel of about 60 human tumor cell lines, showed remarkable antineoplastic activity; they had GI 50 values in the low micromolar or submicromolar range and reached, in the case of 4c, nanomolar concentrations on 88% of the 59 tested cell lines. Flow cytometric analysis of cell cycle after treatment with 4c demonstrated an arrest of the cell cycle in G2/M phase. This effect was accompanied with apoptosis of the cells, mitochondrial depolarization, generation of reactive oxygen species, and activation of caspase-3 and caspase-9. Moreover, 4c induced a clear increase in the mitotic index, inhibited microtubule assembly in vitro, and interestingly also acted as a topoisomerase I inhibitor.

A new class of compounds, the thiopyrano[2,3-e]indol-2-ones, bioisosters of the angular furocoumarin angelicin, was synthesized with the aim of obtaining new photochemotherapeutic agents. In particular 7,8-dimethyl-thiopyranoindolone 6c s showed a remarkable phototoxicity and a great dose UVA dependence reaching IC(50) values at submicromolar level. This latter photoinduced a massive apoptosis and a remarkable photodamage to lipids and proteins. Although it did not intercalate DNA, it was able to cause photooxidation of DNA bases.

In this review we summarize the structure and biological effects of linear and angular psoralens. These compounds exhibit very interesting biological effects on cell cycle, apoptosis and differentiation. These molecules should be considered as promising drugs in the therapy of several diseases, including psoriasis, mycosis fungoides, cancer. In addition, pre-clinical data demonstrate a possible employment of these molecules for the treatment of β-thalassemia.

Psoralens, also known as furocoumarins, are a well-known class of photosensitizers largely used in the therapy of various skin disease. In this study we have evaluated the effects of crude pre-irradiated solutions of furocoumarins derivatives on (a) erythroid differentiation and apoptosis of human leukemic K562 cells and (b) hemoglobin synthesis in cultures of human erythroid progenitors derived from the peripheral blood. To prove the activity of a mixture of photoproducts generated by UVA irradiation of the three psoralen derivatives 5-methoxypsoralen (5-MOP) 8-methoxypsoralen (8-MOP), and angelicin (ANG), we employed the human leukemic K562 cell line and the two-phase liquid culture procedure for growing erythroid progenitors. The results obtained demonstrate that pre-irradiated solutions of psoralen derivatives significantly induce erythroid differentiation of K562 cells irrespective of the type of derivative used, suggesting that the active photoproduct(s) share a common structure. Interestingly, solutions of psoralens irradiated in anaerobic conditions do not exhibits erythroid inducing ability, indicating that the effect is mostly due to photooxidized psoralen products. In erythroid precursor cells, psoralens photolysis products stimulates at low concentrations an increase of hemoglobin A and hemoglobin F. Altogether, these data suggest that photoproducts of psoralen warrant further evaluation as potential therapeutic drugs in beta-thalassaemia and sickle cell anaemia.

The interactions and the photosensitizing activity of three antimalarial drugs quinine (Q), mefloquine (MQ) and quinacrine (QC) toward DNA was studied. Evidences obtained by absorption and emission spectroscopy and by linear dichroism measurements indicate that these derivatives bind the macromolecule with a high affinity (binding constants Ka approximately 10(5) M(-1)). The absorption characteristics of the drugs changed markedly by addition of DNA and their fluorescence was quenched with rate constants higher than that of diffusion. The geometry of binding involves predominantly the intercalation into the double helix. The DNA photocleavage properties of antimalarials was investigated using plasmid DNA as a model, at different [drug]/ [DNA] ratios. The results indicate that mainly MQ and Q are able to induce significant photodamage to DNA. In particular the marked effect of the former drug is evidenced after treatment of photosensitized DNA by two base excision repair enzymes, formamydo-pyrimidine glycosilase (Fpg) and Endonuclease III (Endo III). From a mechanistic point of view, experiments carried out in different experimental conditions indicate that these drugs photoinduce DNA damage through singlet oxygen and/or radical cation production. These findings are further supported by the determination of two photoproducts of 2'-deoxyguanosine, which are diagnostic for Type I and Type II pathways, namely 2,2-diamino(2-deoxy-beta-D-erythro-pentofuranosyl)-4-amino-5(2H)-oxazolone and (R,S)4-hydroxy-8-oxo-4,8-dihydro-2'-deoxyguanosine (4-OH-8-oxo-dGuo). Laser flash photolysis experiments carried out in the presence of DNA indicates that the excitation produces mainly the triplet state for Q and the triplet and radical cation for QC. Moreover the singlet and triplet states and radical cations of the drugs are quenched by 2'-deoxyguanosine monophosphate. The absorbances of these transients decrease with increasing DNA concentration.

This article describes the results of a coupled photophysical and photobiological study aimed at understanding the phototoxicity mechanism of the antimalarial drugs amodiaquine (AQ), primaquine (PQ) and chloroquine (CQ). Photophysical experiments were carried out in aqueous solutions by steady-state and time-resolved spectrometric techniques to obtain information on the different decay pathways of the excited states of the drugs and on the transient species formed upon laser irradiation. The results showed that all three drugs possess very low fluorescence quantum yields (10(-2)-10(-4)). Laser flash photolysis experiments proved the occurrence of photoionization processes leading to the formation of a radical cation in all three systems. In the case of AQ the lowest triplet state was also detected. Together with the photophysical properties the photobiological properties of the antimalarial drugs were investigated under UV irradiation, on various biological targets through a series of in vitro assays. Phototoxicity on mouse 3T3 fibroblast and human keratinocyte cell lines NCTC-2544 was detected for PQ and CQ but not for AQ. In particular, PQ- and CQ-induced apoptosis was revealed by the externalization of phosphatidylserine. Furthermore, upon UV irradiation, the drugs caused significant variations of the mitochondrial potential (Deltapsi(mt)) measured by flow cytometry. The photodamages produced by the drugs were also evaluated on proteins, lipids and DNA. The combined approaches were useful in understanding the mechanism of phototoxicity induced by these antimalarial drugs.

Psoralens, also known as furocoumarins, are a class of photosensitizers largely used in the therapy of various skin diseases. In this study we have evaluated the combined effects of UVA irradiation and furocoumarins derivatives on (a) erythroid differentiation and apoptosis of human leukemia K562 cells and (b) globin gene expression in cultures of human erythroid progenitors derived from the peripheral blood. To prove the activity of a series of linear and angular furocoumarins derivatives, we employed the human leukemia K562 cell line and the two-phase liquid culture procedure for growing erythroid progenitors. Quantitative real-time reverse transcription polymerase-chain assay (Q-RT-PCR) was employed for quantification of the accumulation of globin mRNAs. The results obtained demonstrate that both linear and angular furocoumarins are strong inducers of erythroid differentiation of K562 cells. From a preliminary screening, we have selected two derivatives, 5-methoxypsoralen (5-MOP) and trimethylangelicin (TMA), for which we have investigated their mechanism of action. The cell cycle analysis showed that these derivatives induce, after irradiation, a cell cycle arrest in the G2/M phase, followed by apoptosis. Mitochondrial depolarisation and caspases activation seem to be involved in the mechanism of cell death. In erythroid precursor cells, psoralens in combination with UVA irradiation, stimulate at very low concentrations a preferential increase of γ-globin mRNA. Altogether, these data suggest that psoralen derivatives warrant further evaluation as potential therapeutic drugs in β-thalassemia and sickle cell anemia.

Two antibacterial fluoroquinolones, levofloxacin and moxifloxacin, were investigated to evaluate their photophysical properties and to explore the mechanism of their phototoxicity. Photophysical experiments were carried out in aqueous solution by stationary and time-resolved fluorimetry, and by laser flash photolysis, to obtain information on the various decay pathways of the excited states of the drugs and on transient species formed upon irradiation. The results obtained show that levofloxacin is able to photosensitize red blood cell lysis in an oxygen-independent way and induce a high decrease in cell viability after UVA irradiation, although to a lesser degree than the racemic mixture ofloxacin. Moxifloxacin, which is an 8-MeO-substituted fluoroquinolone, is less phototoxic than the other compounds. Cellular phototoxicity was inhibited by the addition of superoxide dismutase, catalase, and free radical and hydroxyl radical scavengers (BHA, GSH, mannitol, and DMTU), indicating the involvement of superoxide anion and/or a radical mechanism in their cytotoxicity. A good correlation was observed between lipid peroxidation, protein photodamage, and cellular phototoxicity, indicating that test compounds exert their toxic effects mainly in the cellular membrane. Experiments carried out on pBR322 DNA show that these derivatives do not significantly photocleave DNA directly, but single-strand breaks were evidenced after treatment of photosensitized DNA by two base-excision-repair enzymes, and Endo III.

A new class of compounds, the pyrrolo[2,3-h]quinolin-2-ones, nitrogen isosters of the angular furocoumarin Angelicin, was synthesized with the aim of obtaining new photochemotherapeutic agents with increased antiproliferative activity and lower undesired toxic effects than the lead compound. Two synthetic pathways were approached to allow the isolation both of the dihydroderivatives 10-17 and of the aromatic ring system 23. Compounds 10-17 showed a remarkable phototoxicity and a great UVA dose dependence reaching IC(50) values at submicromolar level. 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. A weak co-staining was observed with mitochondrial stain, whereas a specific localization in lysosomes was observed. Studies directed to elucidate the mode of action of this series of compounds revealed that they do not intercalate with DNA and do not induce photodamage to the macromolecule. On the contrary, they induce significative photodamage to lipids and proteins.

A series of geiparvarin analogues modified on the unsaturated alkenyloxy bridge, where a H-atom replaced the 3'-Me group, were synthesized and evaluated against a panel of human tumor cell lines in vitro. These compounds demonstrated a stronger increase in growth inhibitory activity when compared to the parent compound geiparvarin (8). In particular, the activity increased even further in the series of demethylated compounds when a Me substituent in the coumarin moiety is introduced. On the contrary, the same modifications exerted on the parent compound led to an activity reduction. Interestingly, the new derivatives proved to be fully inhibitory to drug-resistant cell lines, thus suggesting that they are not subject to the pump-mediating efflux of antitumor drugs. On the basis of their cytotoxic profiles, the most-active compounds were selected for further biological evaluation. The extracellular acidification rate by the new geiparvarin analogues was measured with the Cytosensor microphysiometer. The new derivatives significantly increased the acidification rate during the 24-48 h of incubation in a concentration-dependent manner. Cell-cycle analysis, evaluated by flow cytometry, revealed a strong apoptotic induction by these compounds confirmed by DNA laddering and observation by electron microscopy. Interestingly, the apoptotic pathway did not appear to be mediated by the activation of caspase-3.

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.

Treatment of cells with UVA radiation in combination with linear psoralens induces a cell-cycle block and subsequent apoptosis, whereas angular derivatives produce apopotosis without affecting the cellular cycle.

The title compound exhibits a selective enhancement of its fluorescence intensity in the presence of AT-rich DNA.