On the synthesis of quinone-based BODIPY hybrids: New insights on antitumor activity and mechanism of action in cancer cells.

Fluorescent quinone-based BODIPY hybrids were synthesised and characterised by NMR analysis and mass spectrometry. We measured their cytotoxic activity against cancer and normal cell lines, performed mechanistic studies by lipid peroxidation and determination of reduced (GSH) and oxidized (GSSG) glutathione, and imaged their subcellular localisation by confocal microscopy. Cell imaging experiments indicated that nor-ß-lapachone-based BODIPY derivatives might preferentially localise in the lysosomes of cancer cells. These results assert the potential of hybrid quinone-BODIPY derivatives as promising prototypes in the search of new potent lapachone antitumor drugs.

Semisynthesis of new aphidicolin derivatives with high activity against Trypanosoma cruzi.

Chagas disease continues to be a difficult disease to eradicate, largely because of the widespread populations it affects as well as the highly toxic effects of current therapies. Thus, the exploration of innovative scaffolds, ideally with distinct mechanisms of action, is urgently needed. The natural product aphidicolin and its effects on cell cycle division have been widely studied; it is a potent inhibitor of parasitic cells. In the present study, we report for the first time the semisynthesis of a series of aphidicolin derivatives, their unique structural features, and demonstration of their activity against Trypanosoma cruzi cells. Two demonstrated high potency and selectivity against parasitic amastigote cells, and thus show promise as new leads for Chagas disease treatment.

Electronic structure and gas-phase chemistry of protonated α- and ß-quinonoid compounds: a mass spectrometry and computational study.

RATIONALE: The use of quinonoid compounds against tropical diseases and as antitumor agents has prompted the search for new naturally occurring and synthetic derivatives. Among these quinonoid compounds, lapachol and its isomers (α- and ß-lapachone) serve as models for the synthesis of new compounds with biological activity, and the use of electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis as a tool to elucidate and characterize these products has furnished important information about these compounds. METHODS: ESI-MS/MS analysis under collision-induced dissociation conditions was used to describe the fragmentation mechanisms for protonated 1,4-naphthoquinone, 1,2-naphthoquinone, α-lapachone, and ß-lapachone. The B3LYP/6-31+G(d,p) model was used to obtain proton affinities, gas-phase basicities, and molecular electrostatic potential maps, thus indicating the probable protonation sites. Fragmentation pathways were suggested on the basis of the relative enthalpies of the product ions. RESULTS: The ESI-MS signals of the cationized molecules of ortho quinonoid compounds were more intense than those of the protonated molecule. Formation of the major product ions with m/z 187 from protonated α- and ß-lapachone has been attributed to a retro-Diels-Alder (RDA) reaction. CONCLUSIONS: MS/MS studies on lapachol isomers (α- and ß-lapachone) will facilitate the interpretation of the liquid chromatography (LC)-MS/MS analysis of new metabolites. MS/MS data on the 1,4-naphthoquinone, 1,2-naphthoquinone, α-lapachone and ß-lapachone core will help characterize new derivatives from in vitro/in vivo metabolism studies in complex matrices. The product ions revealed the major fragmentation mechanisms and these ions will serve as diagnostic ions to identify each studied compound.

Development of Novel Isoindolone-Based Compounds against Trypanosoma brucei rhodesiense.

This study identified the isoindolone ring as a scaffold for novel agents against Trypanosoma brucei rhodesiense and explored the structure-activity relationships of various aromatic ring substitutions. The compounds were evaluated in an integrated in vitro screen. Eight compounds exhibited selective activity against T. b. rhodesiense (IC50 <2.2 µm) with no detectable side activity against T. cruzi and Leishmania infantum. Compound 20 showed low nanomolar potency against T. b. rhodesiense (IC50 =40 nm) and no toxicity against MRC-5 and PMM cell lines and may be regarded as a new lead template for agents against T. b. rhodesiense. The isoindolone-based compounds have the potential to progress into lead optimization in view of their highly selective in vitro potency, absence of cytotoxicity and acceptable metabolic stability. However, the solubility of the compounds represents a limiting factor that should be addressed to improve the physicochemical properties that are required to proceed further in the development of in vivo-active derivatives.

Structural basis for the function and inhibition of dihydroorotate dehydrogenase from Schistosoma mansoni.

Schistosomiasis is a serious public health problem, prevalent in tropical and subtropical areas, especially in poor communities without access to safe drinking water and adequate sanitation. Transmission has been reported in 78 countries, and its control depends on a single drug, praziquantel, which has been used over the past 30 years. Our work is focused on exploiting target-based drug discovery strategies to develop new therapeutics to treat schistosomiasis. In particular, we are interested in evaluating the enzyme dihydroorotate dehydrogenase (DHODH) as a drug target. DHODH is a flavoenzyme that catalyzes the stereospecific oxidation of (S)-dihydroorotate (DHO) to orotate during the fourth and only redox step of the de novo pyrimidine nucleotide biosynthetic pathway. Previously, we identified atovaquone, used in the treatment of malaria, and its analogues, as potent and selective inhibitors against Schistosoma mansoni DHODH (SmDHODH). In the present article, we report the first crystal structure of SmDHODH in complex with the atovaquone analogue inhibitor 2-((4-fluorophenyl)amino)-3-hydroxynaphthalene-1,4-dione (QLA). We discuss three major findings: (a) the open conformation of the active site loop and the unveiling of a novel transient druggable pocket for class 2 DHODHs; (b) the presence of a protuberant domain, only present in Schistosoma spp DHODHs, that was found to control and modulate the dynamics of the inhibitor binding site; (c) a detailed description of an unexpected binding mode for the atovaquone analogue to SmDHODH. Our findings contribute to the understanding of the catalytic mechanism performed by class 2 DHODHs and provide the molecular basis for structure-guided design of SmDHODH inhibitors. DATABASE: The structural data are available in Protein Data Bank (PDB) database under the accession code number 6UY4.

Synthesis and Structure-Activity Relationships of Imidazopyridine/Pyrimidine- and Furopyridine-Based Anti-infective Agents against Trypanosomiases.

Neglected tropical diseases remain among the most critical public health concerns in Africa and South America. The drug treatments for these diseases are limited, which invariably leads to fatal cases. Hence, there is an urgent need for new antitrypanosomal drugs. To address this issue, a large number of diverse heterocyclic compounds were prepared. Straightforward synthetic approaches tolerated pre-functionalized structures, giving rise to a structurally diverse set of analogs. We report on a set of 57 heterocyclic compounds with selective activity potential against kinetoplastid parasites. In general, 29 and 19 compounds of the total set could be defined as active against Trypanosoma cruzi and T. brucei brucei, respectively (antitrypanosomal activities <10 µM). The present work discusses the structure-activity relationships of new fused-ring scaffolds based on imidazopyridine/pyrimidine and furopyridine cores. This library of compounds shows significant potential for anti-trypanosomiases drug discovery.

Fragmentation studies and electrospray ionization mass spectrometry of lapachol: protonated, deprotonated and cationized species.

Electrospray ionization mass spectrometric analysis of lapachol (2-hydroxy-3-(3-methyl-2-butenyl)-1,4-naphthoquinone) was accomplished in order to elucidate the gas-phase dissociation reactions of this important biologically active natural product. The occurrence of protonated and cationized species in the positive mode and of deprotonated species in the negative mode was explored by means of collision-induced dissociation (CID) experiments. For the protonated molecule, the H(2)O and C(4)H(8) losses occur by two competitive channels. For the deprotonated molecule, the even-electron rule is not conserved, and the radicalar species are eliminated by formation of distonic anions. The fragmentation mechanism for each ion was suggested on the basis of computational thermochemistry. Atomic charges, relative energies, and frontier orbitals were employed aiming at a better understanding of the gas-phase reactivity of lapachol. Potential energy surfaces for fragmentation reactions were obtained by the B3LYP/6-31+G(d,p) model.

Ligand-based design, synthesis and biochemical evaluation of potent and selective inhibitors of Schistosoma mansoni dihydroorotate dehydrogenase.

Schistosomiasis ranks second only to malaria as the most common parasitic disease worldwide. 700 million people are at risk and 240 million are already infected. Praziquantel is the anthelmintic of choice but decreasing efficacy has already been documented. In this work, we exploited the inhibition of Schistosoma mansoni dihydroorotate dehydrogenase (SmDHODH) as a strategy to develop new therapeutics to fight schistosomiasis. A series of quinones (atovaquone derivatives and precursors) was evaluated regarding potency and selectivity against both SmDHODH and human DHODH. The best compound identified is 17 (2-hydroxy-3-isopentylnaphthalene-1,4-dione) with IC50 = 23 ±â€¯4 nM and selectivity index of 30.83. Some of the new compounds are useful pharmacological tools and represent new lead structures for further optimization.

ß-amino alcohols and their respective 2-phenyl-N-alkyl aziridines as potential DNA minor groove binders.

It is known that aziridines and nitrogen mustards exert their biological activities, especially in chemotherapy, via DNA alkylation. The studied scaffold, 2-phenyl-1-aziridine, provides a distinct conformation compared to commonly used aziridines, and therefore, leads to a change in high-strained ring reactivity towards biological nucleophiles, such as DNA. The above series of compounds was tested in three breast cell lines: MCF-10, a healthy cell; MCF-7, a hormone responsive cancer cell; and MDA-MB-231, a triple negative breast cancer cell. Both aziridines and their precursors, ß-amino alcohols, showed activity towards these cells, and some of the compounds showed higher selectivity index than cisplatin, the drug used as control. When the type of cell death was investigated, the synthesized compounds demonstrated higher apoptosis and lower necrosis rates than cisplatin, and when the mechanism of action was studied, the compounds were shown to interact with DNA via its minor groove instead of alkylation or intercalation.

The 3-phenylcoumarin derivative 6,7-dihydroxy-3-[3',4'-methylenedioxyphenyl]-coumarin downmodulates the FcγR- and CR-mediated oxidative metabolism and elastase release in human neutrophils: Possible mechanisms underlying inhibition of the formation and release of neutrophil extracellular traps.

In this study, we report the ability of a set of eight 3-phenylcoumarin derivatives bearing 6,7- or 5,7-dihydroxyl groups, free or acetylated, bound to the benzopyrone moiety, to modulate the effector functions of human neutrophils. In general, (i) 6,7-disubstituted compounds (5, 6, 19, 20) downmodulated the Fcγ receptor-mediated neutrophil oxidative metabolism more strongly than 5,7-disubstituted compounds (21, 22, 23, 24), and (ii) hydroxylated compounds (5, 19, 21, 23) downmodulated this neutrophil function more effectively than their acetylated counterparts (6, 20, 22, 24, respectively). Compounds 5 (6,7-dihydroxy-3-[3',4'-methylenedioxyphenyl]-coumarin) and 19 (6,7-dihydroxy-3-[3',4'-dihydroxyphenyl]-coumarin) effectively downmodulated the neutrophil oxidative metabolism elicited via Fcγ and/or complement receptors. Compound 5 also downmodulated the immune complex-stimulated phagocytosis, degranulation of elastase, and production and release of neutrophil extracellular traps, as well as the human neutrophil chemotaxis towards n-formyl-methionyl-leucyl-phenylalanine, without altering the expression level of formyl peptide receptor type 1. Both compounds 5 and 19 did not impair the neutrophil capacity to recognize and kill Candida albicans. Docking calculations revealed that compounds 5 and 19 directly interacted with three catalytic residues - Gln-91, His-95, and Arg-239 - inside the myeloperoxidase active site. Together, these findings indicate that (i) inhibition of reactive oxygen species generation and degranulation of elastase are closely associated with downmodulation of release of neutrophil extracellular traps; and (ii) compound 5 can be a prototype for the development of novel immunomodulating drugs to treat immune complex-mediated inflammatory diseases.

Hydrophilic polymeric nanoparticles prepared from Delonix galactomannan with low cytotoxicity for ocular drug delivery.

Delonix is a galactomannan polysaccharide extracted from the endosperm of Delonix regia plant. This study aims at the development of Delonix nanoparticle and assesses its potential for ocular delivery by evaluating its in-vitro stability, toxicity and cellular uptake. Fluorescent nanoparticles (BODIPY-loaded nanoparticles) were prepared by a Quality-by-Design modified nanoprecipitation technique. Optimized nanoparticles had mean sizes <240nm, PdI<0.2 and zeta potential of <-30mV. Mixture of surfactants with different hydrophilic-lipophilic balance controlled nanoparticle swelling. Nanoparticles, which were stable in the presence of simulated lachrymal fluid and lysozyme also sustained the release of BODIPY. In-vitro studies suggest no toxicity of the nanoparticles in concentration range of 100-1483.3µg/mL on retinal and corneal epithelial cells. Flow cytometry and confocal microscopy techniques showed that retinal cells but not corneal cells, uptake 18% of the nanoparticles. Therefore, Delonix nanoparticles could be a safe and promising tool for ocular drug delivery.

Lapachol, a compound targeting pyrimidine metabolism, ameliorates experimental autoimmune arthritis.

BACKGROUND: The inhibition of pyrimidine biosynthesis by blocking the dihydroorotate dehydrogenase (DHODH) activity, the prime target of leflunomide (LEF), has been proven to be an effective strategy for rheumatoid arthritis (RA) treatment. However, a considerable proportion of RA patients are refractory to LEF. Here, we investigated lapachol (LAP), a natural naphthoquinone, as a potential DHODH inhibitor and addressed its immunosuppressive properties. METHODS: Molecular flexible docking studies and bioactivity assays were performed to determine the ability of LAP to interact and inhibit DHODH. In vitro studies were conducted to assess the antiproliferative effect of LAP using isolated lymphocytes. Finally, collagen-induced arthritis (CIA) and antigen-induced arthritis (AIA) models were employed to address the anti-arthritic effects of LAP. RESULTS: We found that LAP is a potent DHODH inhibitor which had a remarkable ability to inhibit both human and murine lymphocyte proliferation in vitro. Importantly, uridine supplementation abrogated the antiproliferative effect of LAP, supporting that the pyrimidine metabolic pathway is the target of LAP. In vivo, LAP treatment markedly reduced CIA and AIA progression as evidenced by the reduction in clinical score, articular tissue damage, and inflammation. CONCLUSIONS: Our findings propose a binding model of interaction and support the ability of LAP to inhibit DHODH, decreasing lymphocyte proliferation and attenuating the severity of experimental autoimmune arthritis. Therefore, LAP could be considered as a potential immunosuppressive lead candidate with potential therapeutic implications for RA.

Oral Administration of Peptide-Based Drugs: Beyond Lipinski's Rule.

The use of peptides in therapy presents several limitations, from physicochemical characteristics to inadequate pharmacokinetic profiles for oral absorption. As peptides are gaining importance in the therapeutic arsenal, there is an increasing need to rationalize the main characteristics of this compound class in the market. Therefore, we performed an extensive analysis of all known peptide drugs and clinical candidates based on their peptide features, physicochemical and structural properties, and correlated these with their administration route and therapeutic classes. Peptide drugs are widely distributed across drug and pharmacological space, covering several therapeutic areas with structural diversity and complexity, distributed between groups of cyclic and linear compounds. Although structural and physicochemical properties are clear within these groups, we counter the consensus that cyclic peptides have better oral availability than linear peptides, as most of the orally administrated peptides have linear structures. This study and review furnishes information that could support peptide drug design, with a new cutoff of known descriptors that go beyond the Rule of Five.

Novel fluorescent lapachone-based BODIPY: synthesis, computational and electrochemical aspects, and subcellular localisation of a potent antitumour hybrid quinone.

For the first time, a fluorescent lapachone-based BODIPY was synthesised and characterised by NMR and mass spectrometry. Computational and electrochemical aspects, as well as cytotoxic activity and subcellular localisation, were studied. Confocal microscopy experiments indicated that the probe was a specific mitochondria-staining agent. These in-detail analyses were useful in understanding the cytotoxic effects and mechanism of action of this novel hybrid compound. This molecule constitutes a promising prototype owing to its potential biological activities and the new strategies aimed at mechanistic investigations in cells and in vivo, and opens up an interesting avenue of research.

Hologram quantitative structure-activity relationship and comparative molecular interaction field analysis of aminothiazole and thiazolesulfonamide as reversible LSD1 inhibitors.

BACKGROUND: LSD-1 is an enzyme that removes methyl groups from lysine residues of histone proteins. LSD-1 inhibition decreases cellular proliferation and therefore represents a therapeutic target for cancer treatment. MAO and LSD-1 are both flavin adenine dinucleotide-dependent MAOs, and the MAO inhibitor, tranylcypromine, is currently undergoing clinical trials for cancer treatment because it acts as an irreversible LSD-1 inhibitor. MATERIALS & METHODS: The present study investigated new reversible LSD-1 inhibitors, in order to develop novel selective anticancer agents. We constructed 2 and 3D quantitative structure-activity relationship models by using a series of 54 aminothiazole and thiazolesulfonamide derivatives. RESULTS: The models were validated internally and externally (q(2) , 0.691 and 0.701; r(2) , 0.894 and 0.937; r(2) test , 0.785 and 0.644, for 2 and 3D models, respectively). Fragment contribution maps, as well as steric and electrostatic contour maps were generated in order to obtain chemical information related to LSD-1 inhibition. CONCLUSION: The thiazolesulfonamide group was fundamental to the inhibition of LSD-1 by these compounds and that bulky and aromatic substituents at the thiazole ring were important for their steric and electrostatic interactions with the active site of LSD-1.

Topical Skin Cancer Therapy Using Doxorubicin-Loaded Cationic Lipid Nanoparticles and lontophoresis.

The topical administration of chemotherapeutics is a promising approach for the treatment of skin cancer; however, different pharmaceutical strategies are required to allow large amounts of drug to penetrate tumors. This work examined the potential of the anodic iontophoresis of doxorubicin-loaded cationic solid lipid nanoparticles (DOX-SLN) to increase the distribution and tumor penetration of DOX. A double-labeled cationic DOX-SLN composed of the lipids stearic acid and monoolein and a new BODIPY dye was prepared and characterized. The skin distribution and penetration of DOX were evaluated in vitro using confocal microscopy and vertical diffusion cells, respectively. The antitumor potential was evaluated in vivo through the anodic iontophoresis of DOX-SLN in squamous cell carcinoma induced in nude BALB/c mice. The encapsulation of DOX drastically altered the DOX partition coefficient and increased the distribution of DOX in the lipid matrix of the stratum corneum (SC). The association with iontophoresis created high-concentration drug reservoir zones in the follicles of the skin. Although the iontophoresis of a DOX solution increased the penetration of DOX in the viable epidermis by approximately 4-fold, the iontophoresis of cationic DOX-SLN increased the DOX penetration by approximately 50-fold. In vivo, the DOX-SLN iontophoretic treatment was effective in inhibiting tumor cell survival and tumor growth and was accompanied by an increase in keratinization and consequent cell death. These results indicate a strong and synergic effect of iontophoresis with DOX-SLN and provide a potential strategy for the treatment of skin cancer.

Transcorneal iontophoresis of dendrimers: PAMAM corneal penetration and dexamethasone delivery.

Iontophoresis of nanocarriers in the eye has been proposed to sustain drug delivery and maintain therapeutic concentrations. Fourth generation polyamidoamine (PAMAM) dendrimers are semi-rigid nanoparticles with surface groups that are easily modified. These dendrimers are known to modulate tight junctions, increase paracellular transport of small molecules and be translocated across epithelial barriers, exhibiting high uptake by different cell lines. The first aim of this study was to investigate the effect of iontophoresis on PAMAM penetration and distribution into the cornea. The second aim was to evaluate, ex vivo and in vivo, the effect of these dendrimers in dexamethasone (Dex) transcorneal iontophoresis. Anionic (PAMAM G3.5) and cationic (PAMAM G4) dendrimers were labeled with fluorescein isothiocyanate (FITC), and their distribution in the cornea was investigated using confocal microscopy after ex vivo anodal and cathodal iontophoresis for various application times. The particle size distribution and zeta potential of the dendrimers in an isosmotic solution were determined using dynamic light scattering and Nanoparticle Tracking Analysis (NTA), where the movement of small particles and the formation of large aggregates, from 5 to 100 nm, could be observed. Transcorneal iontophoresis increased the intensity and depth of PAMAM-FITC fluorescence in the cornea, suggesting improved transport of the dendrimers across the epithelium toward the stroma. PAMAM complexes with Dex were characterized by (13)C-NMR, (1)H-NMR and DOSY. PAMAM G3.5 and PAMAM G4 increased the aqueous solubility of Dex by 10.3 and 3.9-fold, respectively; however, the particle size distribution and zeta potential remained unchanged. PAMAM G3.5 decreased the Dex diffusion coefficient 48-fold compared with PAMAM G4. The ex vivo studies showed that iontophoresis increased the amount of Dex that penetrated into the cornea by 2.9, 5.6 and 3.0-fold for Dex, Dex-PAMAM G4 and Dex-PAMAM G3.5, respectively. In vivo experiments, however, revealed that iontophoresis of Dex-PAMAM-G3.5 increased Dex concentration in the aqueous humor by 6.6-fold, while iontophoresis of Dex-PAMAM G4 and Dex increased it 2.5 and 2-fold, respectively. Therefore, iontophoresis targeted PAMAM to the cornea but it is the sustained delivery of the Dex from PAMAM that prevents its rapid elimination from the aqueous humor. In conclusion, iontophoresis of PAMAM complexes represents a promising strategy for targeted and sustained topical drug delivery to the eye.

Studies on the trypanocidal activity of semi-synthetic pyran[b-4,3]naphtho[1,2-d]imidazoles from beta-lapachone.

We synthesized new naphthoimidazoles from beta-lapachone with an aromatic moiety linked to the imidazole ring, using phenylic and heterocyclic aldehydes. The most active compound against Trypanosoma cruzi had a p-methyl group linked to the phenyl ring, presenting an EC(50) value of 15.5 +/- 2.9 microM. No reliable correlation could be established with the biological activity and the structure of in the phenylic series. For the heterocyclic series, activity was associated with a three bond-distance from nitrogen to the imidazole ring, in accordance with our previous work.

Synthesis of quinoidal molecules: strategies towards bioactive compounds with an emphasis on lapachones.

Naphthoquinoidal compounds are of great interest in medicinal chemistry. In recent years, several synthetic routes have been developed to obtain bioactive molecules derived from lapachones. In this mini-review, we focus on the synthetic aspects and strategies used to design these compounds and on the biological activities of these substances for the development of drugs against the neglected diseases leishmaniasis and Chagas disease as well as malaria, tuberculosis and cancer. Three strategies used to develop bioactive naphthoquinoidal compounds are discussed: (i) C-ring modification, (ii) redox centre modification and (iii) A-ring modification. Among these strategies, reactions such as copper-catalysed azide-alkyne cycloaddition (click chemistry), palladium-catalysed cross couplings, and heterocyclisations will be discussed for the development of naphthoquinoidal compounds against Trypanosoma cruzi, Leishmania and cancer. The aim of derivatisation is the generation of novel molecules that inhibit cellular organelles/processes, generate reactive oxygen species (ROS) and increase lipophilicity to enhance penetration through the plasma membrane. Modified lapachones have emerged as promising prototypes for the development of drugs against neglected diseases and cancer.

Synthesis, cytotoxicity and in vitro antileishmanial activity of naphthothiazoles.

The leishmaniasis is a spectral disease caused by the protozoan Leishmania spp., which threatens millions of people worldwide. Current treatments exhibit high toxicity, and there is no vaccine available. The need for new lead compounds with leishmanicidal activity is urgent. Considering that many lead leishmanicidal compounds contain a quinoidal scaffold and the thiazole heterocyclic ring is found in a number of antimicrobial drugs, we proposed a hybridization approach to generate a diverse set of semi-synthetic heterocycles with antileishmanial activity. We found that almost all synthesized compounds demonstrated potent activity against promastigotes of Leishmania (Viannia) braziliensis and reduced the survival index of Leishmania amastigotes in mammalian macrophages. Furthermore, the compounds were not cytotoxic to macrophages at fivefold higher concentrations than the EC50 for promastigotes. All molecules fulfilled Lipinski's Rule of Five, which predicts efficient orally absorption and permeation through biological membranes, the in silico pharmacokinetic profile confirmed these characteristics. The potent and selective activity of semi-synthetic naphthothiazoles against promastigotes and amastigotes reveals that the 2-amino-naphthothiazole ring may represent a scaffold for the design of compounds with leishmanicidal properties and encourage the development of drug formulation and new compounds for further studies in vivo.