In vitro and in vivo evaluation of novel chromeno[2,3-d]pyrimidinones as therapeutic agents for triple negative breast cancer.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, and the limited therapeutic options show poor efficacy in patients, associated to severe side effects and development of resistance. Considering that chromene-based scaffolds proved to be attractive candidates for cancer therapy, herein we prepared new chromeno[2,3-d]pyrimidinone derivatives by a simple two step procedure, starting from the reaction of cyanoacetamide and a salicylaldehyde. A cell viability screening in several breast cancer cell lines allowed to identify two promising compounds with IC50 values in the low micromolar range for TNBC cells. These chromenes inhibited cell proliferation, induced cell cycle arrest and triggered cell death through apoptosis. In vivo studies revealed a safe profile in invertebrate and vertebrate animal models and confirmed their capacity to inhibit tumor growth in the CAM model, inducing significant tumor regression after 4 days of treatment. The two compounds identified in this study are promising drug candidates for TNBC treatment and valuable hits for future optimization, using the versatile synthetic platform that was developed.

Imidazolylpyrrolone-Based Small Molecules as Anticancer Agents for Renal Cell Carcinoma.

An in silico study focused on known cancer-related target proteins, identified a selection of imidazo[4,5-b]pyrrolo[3,4-d]pyridines as potentially active. These compounds were prepared by a novel synthetic approach, designed and developed in-house, based on the reaction of 5-amino-4-cyanoformimidoyl imidazoles with N-substituted cyanoacetamides. The substituted imidazolylpyrrolones obtained, were cyclized intramolecularly to generate the intended imidazo[4,5-b]pyrrolo[3,4-d]pyridines in a process catalyzed by DBU. Treating the imidazolylpyrrolones with an excess of triethyl orthoformate and heating at 80 °C in the presence of acid catalysis led to imidazopyrrolodiazepines. These compounds were screened for their anticancer potential, using the renal cell carcinoma cell line model (A498 and 786-O cell lines). Two compounds exhibited IC50 values in the low micromolar range with a good selectivity index, when compared to non-neoplastic kidney cell line HK2 and the reference compounds rapamycin, cediranib and sunitinib.

Selective Cytotoxicity of Portuguese Propolis Ethyl Acetate Fraction towards Renal Cancer Cells.

Renal cell carcinoma is the most lethal cancer of the urological system due to late diagnosis and treatment resistance. Propolis, a beehive product, is a valuable natural source of compounds with bioactivities and may be a beneficial addition to current anticancer treatments. A Portuguese propolis sample, its fractions (n-hexane, ethyl acetate, n-butanol and water) and three subfractions (P1-P3), were tested for their toxicity on A498, 786-O and Caki-2 renal cell carcinoma cell lines and the non-neoplastic HK2 kidney cells. The ethyl acetate fraction showed the strongest toxicity against A498 (IC50 = 0.162 µg mL-1) and 786-O (IC50 = 0.271 µg mL-1) cells. With similar toxicity against 786-O, P1 (IC50 = 3.8 µg mL-1) and P3 (IC50 = 3.1 µg mL-1) exhibited greater effect when combined (IC50 = 2.5 µg mL-1). Results support the potential of propolis and its constituents as promising coadjuvants in renal cell carcinoma treatment.

Unravelling the anticancer potential of functionalized chromeno[2,3-b]pyridines for breast cancer treatment.

A selection of new chromeno[2,3-b]pyridines was prepared from chromenylacrylonitriles and N-substituted piperazines, using a novel and efficient synthetic procedure. The compounds were tested for their anticancer activity using breast cancer cell lines MCF-7, Hs578t and MDA-MB-231 and the non-neoplastic cell line MCF-10A for toxicity evaluation. In general, compounds showed higher activity towards the luminal breast cancer subtype (MCF-7), competitive with the reference compound Doxorubicin. The in vivo toxicity assay using C. elegans demonstrated a safe profile for the most active compounds. Chromene 3f revealed a promising drug profile, inhibiting cell growth and proliferation, inducing cell cycle arrest in G2/M phase, apoptosis and microtubule destabilization. The new compounds presented exciting bioactive features and may be used as lead compounds in cancer related drug discovery.

Exploitation of new chalcones and 4H-chromenes as agents for cancer treatment.

Chalcone and chromene derivatives were synthesized in good yield through simple and effective reactions using innocuous solvents such as water and ethanol and high yielding aldol condensations. Generally, the reactions were performed at room temperature, leading to the isolation of highly pure compounds. These compounds were tested on breast cancer cells (MCF-7 and Hs578T) and breast non-neoplastic cells (MCF-10A). After determination of IC50 value, specific assays were performed to analyze the potential of these compounds, and those bearing halogenated substituents presented enhanced activity comparing to methoxyl or methyl groups. More specifically, the bromine atom was often present in the bioactive molecules that proceeded to the final assays and showed to be promising candidates for further studies. The selected chromene acted as a cell migration inhibitory agent and triggered regulated cell death associated with G2/M cell-arrest and microtubule destabilization. For chalcones, the results suggest an anti-proliferative activity. Also, results for combination-therapy potentiated the antitumor effect of doxorubicin and reduced cytotoxicity in MCF-10A cells.

Biological importance of structurally diversified chromenes.

Compounds incorporating the chromene scaffold are largely present in natural products and display a wide variety of biological activities. Their low toxicity combined to the broad pharmacological properties have inspired medicinal chemists in the search for new therapeutic agents. This review covers the literature between 1993 and on the biological activity of 2H- and 4H-chromenes, both from natural and synthetic origin. Includes a section that identifies a selection of chromene-based natural products, followed by recent literature on bioactive natural chromenes and the corresponding source, covering plants and fruits. Synthetic chromenes are equally important and a separate section addresses the use of these derivatives as new leads for drug discovery. Different biological targets were identified, namely those associated with anticancer, antimicrobial, anti-inflammatory, antithrombotic and antipsychotic activities.

Proteomic analysis of the action of the Mycobacterium ulcerans toxin mycolactone: targeting host cells cytoskeleton and collagen.

Buruli ulcer (BU) is a neglected tropical disease caused by Mycobacterium ulcerans. The tissue damage characteristic of BU lesions is known to be driven by the secretion of the potent lipidic exotoxin mycolactone. However, the molecular action of mycolactone on host cell biology mediating cytopathogenesis is not fully understood. Here we applied two-dimensional electrophoresis (2-DE) to identify the mechanisms of mycolactone's cellular action in the L929 mouse fibroblast proteome. This revealed 20 changed spots corresponding to 18 proteins which were clustered mainly into cytoskeleton-related proteins (Dync1i2, Cfl1, Crmp2, Actg1, Stmn1) and collagen biosynthesis enzymes (Plod1, Plod3, P4ha1). In line with cytoskeleton conformational disarrangements that are observed by immunofluorescence, we found several regulators and constituents of both actin- and tubulin-cytoskeleton affected upon exposure to the toxin, providing a novel molecular basis for the effect of mycolactone. Consistent with these cytoskeleton-related alterations, accumulation of autophagosomes as well as an increased protein ubiquitination were observed in mycolactone-treated cells. In vivo analyses in a BU mouse model revealed mycolactone-dependent structural changes in collagen upon infection with M. ulcerans, associated with the reduction of dermal collagen content, which is in line with our proteomic finding of mycolactone-induced down-regulation of several collagen biosynthesis enzymes. Our results unveil the mechanisms of mycolactone-induced molecular cytopathogenesis on exposed host cells, with the toxin compromising cell structure and homeostasis by inducing cytoskeleton alterations, as well as disrupting tissue structure, by impairing the extracellular matrix biosynthesis.

Anticancer activity of novel pyrido[2,3-b]indolizine derivatives: the relevance of phenolic substituents.

BACKGROUND/AIM: The potential of indolizine derivatives as anticancer agents has been shown through recent studies. Herein, we present our experimental results, showing that pyrido[2,3-b]indolizine derivatives are effective against colorectal cancer (CRC) cell lines. MATERIALS AND METHODS: Several pyrido[2,3-b]indolizine derivatives were synthesized and their anticancer potential was evaluated against three CRC cell lines and two normal fibroblast cultures. RESULTS: Our experiments identified 4-(3,4)-dihydroxyphenyl)-2-phenylpyrido[2,3-b]indolizine-10-carbonitrile (4f) as being active against all CRC cell lines at concentrations non-cytotoxic against fibroblast cultures. Additionally, cell-cycle analysis indicated that pyrido[2,3-b]indolizines can affect cell-cycle progression, with treated cells accumulating in the S- and G2/M-phase. CONCLUSION: The hydroxyl groups in both the 3- and 4- positions of the aromatic substituent on C4 of the indolizine nucleus are crucial for activity against CRC cell lines. Further manipulation of the number and position of hydroxyl substituents on the aromatic rings may lead to improved anticancer activity of this class of compounds.

Synthesis of novel chromene scaffolds for adenosine receptors.

A one-pot procedure was developed for the synthesis of novel 3-[amino(methoxy)methylene]-2-oxo-3,4-dihydro-2H-chromen-4-yl)-3-cyanoacetamides and chromeno[3,4-c]pyridine-1-carbonitriles from the reaction of 2-oxo-2H-chromene-3-carbonitriles and cyanoacetamides. These chromene derivatives were identified as new scaffolds for adenosine receptors and the hits 3a, 3c, 5a, and 5b were found.

IFN-gamma-dependent activation of macrophages during experimental infections by Mycobacterium ulcerans is impaired by the toxin mycolactone.

Buruli ulcer, caused by Mycobacterium ulcerans infections, is a necrotizing skin disease whose pathogenesis is associated with the exotoxin mycolactone. Despite the relevance of this emergent disease, little is known on the immune response against the pathogen. Following the recent demonstration of an intramacrophage growth phase for M. ulcerans, we investigated the biological relevance of IFN-gamma and the antimycobacterial mechanisms activated by this cytokine in M. ulcerans-infected macrophages. Three M. ulcerans strains were tested: 5114 (mutant mycolactone-negative, avirulent strain); 94-1327 (intermediate virulence); and 98-912 (high virulence). We show in this study that IFN-gamma is expressed in mouse-infected tissues and that IFN-gamma-deficient mice display increased susceptibility to infection with strains 5114 and, to a lesser extent, 94-1327, but not with the highly virulent strain. Accordingly, IFN-gamma-activated cultured macrophages controlled the proliferation of the avirulent and the intermediate virulent strains. Addition of mycolactone purified from strain 98-912 to cultures of IFN-gamma-activated macrophages infected with the mycolactone-negative strain led to a dose-dependent inhibition of the IFN-gamma-induced protective mechanisms, involving phagosome maturation/acidification and increased NO production, therefore resulting in increased bacterial burdens. Our findings suggest that the protection mediated by IFN-gamma in M. ulcerans-infected macrophages is impaired by the local buildup of mycolactone.

Novel nitrogen compounds enhance protection and repair of oxidative DNA damage in a neuronal cell model: comparison with quercetin.

Oxidative DNA damage has been described as an important type of damage that occurs in neuronal cells, with severe implications in many neurodegenerative diseases and in aging. We have previously reported the protection of four new synthetic nitrogen compounds (FMA4, FMA7, FMA762 and FMA796) against oxidative stress conditions. In this work, we studied their effects on oxidative DNA damage induced in rat pheochromocytoma (PC12) cells, using the Comet assay, and compared them with a natural antioxidant, quercetin. Among the compounds tested, FMA762 and FMA796 were the most effective in preventing tert-butylhydroperoxide (t-BHP)-induced formation of DNA strand breaks and in improving the cells' capacity to repair this kind of damage. These effects were similar to the ones of quercetin, a flavonoid with known antioxidant activity. Moreover, contrarily to quercetin, they increased the repair capacity of oxidised bases induced with the photosensitiser Ro 19-8022. This effect seems to be mediated by an increase in DNA repair enzymes activity, assessed by the in vitro BER assay, but no regulation at the expression of OGG1 and APE1 genes was detected. In addition to other properties previously found for the nitrogen compounds, they now prove their effectiveness against oxidative stress-induced DNA damage in the neuronal cell model used.

Protective role of new nitrogen compounds on ROS/RNS-mediated damage to PC12 cells.

Reactive oxygen (ROS) and nitrogen (RNS) species are known to be involved in many degenerative diseases. This study reports four new nitrogen compounds from organic synthesis, identified as FMA4, FMA7, FMA762 and FMA796, which differ mainly by the number of hydroxyl groups within their phenolic unit. Their potential role as antioxidants was evaluated in PC12 cells by assessing their protection against oxidative and nitrosative insults. The four compounds, and particularly FMA762 and FMA796, were able to protect cells against lipid peroxidation and intracellular ROS/RNS formation to a great extent. Their protective effects were likely mediated by their free radicals scavenging ability, as they appeared to be involved neither in the induction of natural antioxidant enzymes like GSH-PX and SOD, nor in the inhibition of NOS. Nevertheless, these results suggest a promising potential for these compounds as ROS/RNS scavengers in pathologies where oxidative/nitrosative stress are involved.

The condensation of salicylaldehydes and malononitrile revisited: synthesis of new dimeric chromene derivatives.

The reaction of salicylic aldehydes with malononitrile was reinvestigated, and the reaction pathway was followed by 1H NMR spectroscopy. A delicate control of the experimental conditions allowed the synthesis of 2-imino-2H-chromene-3-carbonitriles 1, (2-amino-3-cyano-4H-chromen-4-yl)malononitriles 2, 4-amino-5-imino-2,7-dimethoxy-5H-chromeno[3,4-c]pyridine-1-carbonitrile 12, and (4,5-diamino-1-cyano-1,10b-dihydro-2H-chromeno[3,4-c]pyridin-2-ylidene)malononitrile 13. Two novel 2-iminochromene dimers, with structures 8 and 9, were isolated and fully characterized. The activity of compound 8a on Aspergillus spp. growth and on ochratoxin A production was evaluated. The results of the bioassays indicate that compound 8a, applied at concentrations of 2 mM, totally inhibited the growth of the fungi tested. Ochratoxin A production by Aspergillus alliaceus was reduced by about 93% with a 200 microM solution of this compound. A moderate inhibitory effect was observed for the analogous structure 8b, and no inhibition was registered for compounds 2 and 1, used as synthetic precursors of the dimeric species 8.

Oxidative stress protection by newly synthesized nitrogen compounds with pharmacological potential.

In this study we used new nitrogen compounds obtained by organic synthesis whose structure predicted an antioxidant potential and then an eventual development as molecules of pharmacological interest in diseases involving oxidative stress. The compounds, identified as FMA4, FMA5, FMA7 and FMA8 differ in the presence of hydroxyl groups located in the C-3 and/or C-4 position of a phenolic unit, which is possibly responsible for their free radicals' buffering capacity. Data from the DPPH discoloration method confirm the high antiradical efficiency of the compounds. The results obtained with cellular models (L929 and PC12) show that they are not toxic and really protect from membrane lipid peroxidation induced by the ascorbate-iron oxidant pair. The level of protection correlates with the drug's lipophilic profile and is sometimes superior to trolox and equivalent to that observed for alpha-tocopherol. The compounds FMA4 and FMA7 present also a high protection from cell death evaluated in the presence of a staurosporine apoptotic stimulus. That protection results in a significant reduction of caspase-3 activity induced by staurosporine which by its turn seems to result from a protection observed in the membrane receptor pathway (caspase-8) together with a protection observed in the mitochondrial pathway (caspase-9). Taken together the results obtained with the new compounds, with linear chains, open up perspectives for their use as therapeutical agents, namely as antioxidants and protectors of apoptotic pathways. On the other hand the slight pro-oxidant profile obtained with the cyclic structures suggests a different therapeutic potential that is under current investigation.