Design, synthesis and biological evaluation of novel hydroxy-phenyl-1H-benzimidazoles as radical scavengers and UV-protective agents.

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.

Pitavastatin, a new HMG-CoA reductase inhibitor, induces phototoxicity in human keratinocytes NCTC-2544 through the formation of benzophenanthridine-like photoproducts.

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.

Trimethylangelicin reduces IL-8 transcription and potentiates CFTR function.

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 IC(50) 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.

Photoreactivity of 5-fluorouracil under UVB light: photolysis and cytotoxicity studies.

The photodegradation of the chemotherapeutic agent 5-fluorouracil (5-FU) under UVB light was studied both in aqueous and methanol solutions and in systemic and topical formulations. As monitored by HPLC, photodegradation in solution takes place in a concentration dependent manner; thus, the solution for parenteral administration (10(-1) M) showed negligible loss of the active principle. On the contrary, the commercial cream containing 5% of 5-FU showed low stability under UVB exposure. When dissolved either in water or methanol, 5-FU yields two photoproducts which have been characterized as two isomers coming from the addition of the solvent to the 5,6 double bond of the drug. As a consequence, photomodified 5-FU loses its antiproliferative activity on HCT-15 and HeLa cells. MS analysis showed that photoaddition occurred with nucleophilic amino acids, such as cysteine and serine, while susceptible amino acids (cysteine and methionine) were oxidized. In fact, high production of the superoxide anion under UVB light as well as photooxidation of BSA suggests protein photodamage as a mechanism of photosensitization. Indeed, some phototoxicity was shown in experiments on NCTC keratinocytes and MCF-7 resistant cells irradiated with UVB light. The interactions with these biological targets may contribute to skin phototoxicity and photoallergy induced by 5-FU in vivo.

Pyrrolo[3,4-h]quinolinones a new class of photochemotherapeutic agents.

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.

Synthesis of pyrrolo[3,2-h]quinolinones with good photochemotherapeutic activity and no DNA damage.

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.

Pyrano[2,3-e]isoindol-2-ones, new angelicin heteroanalogues.

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.

Isoindolo[2,1-a]quinoxaline derivatives, novel potent antitumor agents with dual inhibition of tubulin polymerization and topoisomerase I.

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.

Thiopyrano[2,3-e]indol-2-ones: angelicin heteroanalogues with potent photoantiproliferative activity.

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.

Photophysical properties and photobiological behavior of amodiaquine, primaquine and chloroquine.

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.

Pyrrolo[3',2':6,7]cyclohepta[1,2-b]pyridines with potent photo-antiproliferative activity.

Pyrrolo[3',2':6,7]cyclohepta[1,2-b]pyridines were synthesized as a new class of tricyclic system in which the pyridine ring is annelated to a cycloheptapyrrole scaffold, with the aim of obtaining new photosensitizing agents with improved antiproliferative activity and lower undesired toxic effects. A versatile synthetic pathway was approached, which allowed the isolation of derivatives of the title ring system with a good substitution pattern on the pyrrole moiety. Photobiological studies revealed that the majority of the new compounds showed a potent cytotoxic effect upon photoactivation with light of the proper wavelength, especially when decorated with a 2-ethoxycabonyl group and a N-benzyl substituted moiety, with EC50 values reaching the submicromolar level. The mechanism of action was evaluated.

Induction of apoptosis by photoexcited tetracyclic compounds derivatives of benzo[b]thiophenes and pyridines.

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.

Vascular disrupting activity of combretastatin analogues.

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 200nM. At lower concentrations (5-20nM), 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 (1pmol/egg). Moreover in a syngenic mouse model, compounds 1-3 after a single i.p. injection (30mg/kg), showed a stronger reduction of microvascular density. Altogether our results identified these derivatives as potential new vascular disrupting agents candidates.

Pyrazolo[3,4-h]quinolines promising photosensitizing agents in the treatment of cancer.

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 (14.52-0.04 µM). 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.

Vandetanib-induced phototoxicity in human keratinocytes NCTC-2544.

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.

Photochemical and photobiological studies on furoquinazolines as new psoralen analogs.

Linear (L) and angular (A) 4',5'-dimethylfuroquinazolines (FQZs) were synthesized and studied as furocoumarin analogs. These molecules proved to be photounstable with a photodegradation extent correlated to UVA light doses. Both compounds did intercalate inside the DNA double helix, but were not able to photobind DNA bases under UVA irradiation. This behavior was further rationalized through docking studies. The photosensitizing effects of these compounds were evaluated on Jurkat tumor cells and NCTC-2544 human keratinocytes, with and without antioxidants, to demonstrate the involvement of a photodynamic mechanism. Indeed, significant amounts of singlet oxygen and superoxide anion were generated in the presence of both compounds, that account for the oxidative damage induced to some isolated biological substrates (DNA, amino acids, proteins and lipids). Photophysical studies by use of a flash photolysis set up showed detectable triplet population and production of singlet reactive oxygen species for linear furoquinazoline, which can be responsible for the oxidation of biological substrates, and therefore can affect the cell proliferation.

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: two new ring systems with antitumor activity.

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.

Synthesis of a new class of pyrrolo[3,4-h]quinazolines with antimitotic activity.

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 GI50 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.

Isolation and structure elucidation of the main UV-A photoproducts of vandetanib.

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.

Induction of erythroid differentiation and increased globin mRNA production with furocoumarins and their photoproducts.

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.