Ellipticine (EPT), a natural plant polyphenolic compound, has long been known for its significant anticancer and anti-HIV activities. Recent study on its photophysical properties has revealed that ellipticine has three molecular states: protonated, neutral and crystalline. Further in vitro cytotoxicity tests indicated that protonated ellipticine exhibited much higher anticancer activity than the other two states. To maximize drug therapeutic effect, a small library of ariginine-rich ionic complementary peptides derived from EAK, including EAR8-II, EAR8-a, ELR8-a, and EAR16-II, were investigated as a potential carrier to deliver prescribed protonated ellipticine for treatment of cancer. Fluorescence study demonstrated that all four peptides were able to solubilize and stabilize protonated ellipticine in aqueous solution at 5:1 mass ratio of peptide-to-ellipticine (0.5: 0.1 mg/mL) even upon 4000 times dilution. Physicochemical characteristics of peptides self-assemblies and peptide-ellipticine complexes such as particle size, surface charge, secondary structure and morphology were determined by dynamic light scattering (DLS), zeta potential, circular dichroism (CD) , atomic force microscopy (AFM) and transmission electron microscopy (TEM), respectively. Then the ellipticine maximum suspension was determined by ellipticine UV-absorption. With the help of the peptides and mechanical stirring overtime, up to 100% ellipticine could be uptaken and stabilized in the solution as protonated ellipticine. In vitro cytotoxicity tests indicated that the peptides were demonstrating significant biocompatibility without affecting the survival of two cancer cell lines, human lung carcinoma cell line A549 and breast cancer cell line MCF-7, whereas the complexes with protonated ellipticine were found to show great anticancer activity to the two cancer cell lines. The IC50 values were obtained for each of four different peptide-ellipticine complexes ranged from 0.36±0.12 to 18.90±0.46 μM. It is worth noting that the IC50 value of EAR16-ellipticine complex to MCF-7 was over 50 times higher than that one to A549, which presented that EAR16-ellipticine complex has a selective targeting activity to A549, with the lowest IC50 value of 0.36±0.12 μM among all four complexes. Such a result indicated that this library of novel arginine-rich ionic complementary peptides had a great potential to encapsulate prescribed protonated ellipticine and exhibited an excellent anticancer activity upon serial dilution in aqueous solution. Overall, the charge distribution and increased hydrophobicity of the short (8 amino acids length) peptides seemed not to affect the complex formation and its therapeutic efficacy in vitro; however, the increase in length of the peptides significantly altered the nanostructure of peptides and its complexation with ellipticine, increased the therapeutic efficacy of EAR16-EPT to A549. This work provides essential information for peptide sequence design in the development of self-assembling peptide-based delivery of hydrophobic anticancer drugs.

Tikslas: Įvertinti chinoksalino darinių struktūros įtaką jų antimikrobiniam ir priešvėžiniam aktyvumui ir atrinkti perspektyviausius junginius tolimesniems tyrimams. Darbo uždaviniai: 1. Ištirti chinoksalino darinių antimikrobinį (priešgrybelinį ir antibakterinį) aktyvumą in vitro. 2. Ištirti chinoksalino darinių priešvėžinį aktyvumą in vitro. 3. Nustatyti chinoksalino darinių antimikrobinio ir priešvėžinio aktyvumo bei cheminės struktūros ryšio dėsningumus. 4. Įvertinti chinoksalino darinių toksiškumą ir farmakokinetines savybes in silico. 5. Remiantis gautais rezultatais, atrinkti perspektyviausius chinoksalino darinius. Tyrimo metodai. Antimikrobiniam aktyvumui nustatyti naudoti in vitro metodai: skiedimo standžioje ir skystoje terpėse bei difuzijos į agarą metodas. Priešvėžinis aktyvumas įvertintas tiriant ląstelių gyvybingumo slopinimą MTT metodu. Struktūros-aktyvumo ryšys įvertintas analizuojant eksperimentinius duomenis. Junginių toksiškumas ir farmakokinetinės savybės įvertintos in silico, remiantis kompiuterinės programos ACD/I-Lab duomenimis. Tyrimo rezultatai. Aktyviausi chinoksalino dariniai Bacillus ir Staphylococcus genties bakterijų augimą slopino 16 μM koncentracija, grybelio Candida albicans – 22 μM koncentracija. Aktyviausi chinoksalino dariniai žmogaus plaučių karcinomos A549 ir glioblastomos U87 ląsteles veikė 0,5-10 μM koncentracijomis. Nustatyti antimikrobinio poveikio struktūros ryšio dėsningumai: aktyviausi junginiai chinoksalino žiedo antroje padėtyje... [toliau žr. visą tekstą] / The Aim of the Research: To evaluate the structure influence on antimicrobial and anticancer activity of quinoxaline derivatives and identify the most promising compounds for the further development. Objectives: 1. To test antimicrobial (antibacterial and antifungal) activity of quinoxaline derivatives in vitro. 2. To test anticancer activity of quinoxaline derivatives in vitro. 3. To establish relationship between structure and antimicrobial, also anticancer activity. 4. To evaluate toxicity and pharmacokinetic properties of quinoxaline derivatives in silico. 5. To identify the most promising quinoxaline derivatives with reference to experimental results. Methods. Antimicrobial activity was tested in vitro using three different methods: serial dilution in agar, in liquid broth, and diffusion into agar. Anticancer activity was investigated by evaluating cell viability using MTT assay. Structure-activity relationship was evaluated from analysis of experimental data. Toxicity and pharmacokinetic properties were predicted by using ACD/I-Lab program. Results. The most promising quinoxaline derivatives inhibited growth of Bacillus and Staphylococcus genus bacteria with 16 μM concentration, fungus Candida albicans with 22 μM concentration. The most active quinoxaline derivatives were active against human lung carcinoma A549 and glioblastoma U87 cell lines with concentrations of 0.5-10 μM. Some principles of structure – antimicrobial activity relationship were established: the most... [to full text]

Compounds from the marine environment exhibit a wide variety of biological activities, and thus hold much promise as potential drugs. The halichondrins, isolated from the Kaikoura sponge Lissodendoryx sp. are no exception to this, demonstrating potent anticancer activity. Novel cytotoxic compounds have also been isolated from the Chatham Rise sponge Lamellomorpha strongylata. Knowledge of the cellular origins of such compounds is desirable, in order to establish if the sponge or associated micro-organisms are producing the compounds of interest. Siderophores are also important molecules, which are produced on demand by bacteria in order to obtain sufficient iron necessary for their growth. Knowledge of the biosynthesis of these compounds has potential for the control of undesirable bacteria, such as the anthrax-causing pathogen Bacillus anthracis. Cell separation studies have been carried out on Lamellomorpha strongylata, locating a swinholide in sponge-associated filamentous bacteria and theonellapeptolides in sponge-associated unicellular bacteria. A microscopic analysis of dissociated cells from Lissodendoryx sp. was also undertaken. The structures of four new halichondrins (3.13 - 3.16), isolated from Lissodendoryx sp., have been determined from spectral data. All of these compounds are very similar to known B series halichondrins, with differences occurring only beyond carbon 44. As biological activity has been shown to be derived from the portion of the molecule between carbons 1 and 35, they all retain good activity in the P388 assay as expected. A new siderophore, petrobactin sulfonate (4.2), was characterised, along with three cyclic imide siderophore derivatives (4.3 - 4.5). Petrobactin sulfonate is the first marine siderophore containing a sulfonated 3,4-dihydroxy aromatic ring. The structures were elucidated from spectral data, resulting in a revision of the NMR assignments of petrobactin.

This thesis reports a study of the chemical stability and coordination chemistry of several antitumour metallocenes Cp2MCl2 (Cp = h5-C5H5; M = Ti 1, V 2, Nb 3, Mo 4), as well as derivatives of Cp2TiCl2 1, with nucleic acids, nucleic acid constituents and proteins. These studies were carried out in order to identify the biologically active species and more fully understand the molecular level mechanism of action of the antitumour metallocenes, in particular Cp2TiCl2 1, which is currently undergoing phase II clinical trials. The interactions of Cp2MoCl2 4 with four oligonucleotides were studied by 1H and 31P NMR spectroscopy. In 50 mM salt solutions of Cp2MoCl2 4, hydrolysis of the halide ligands occurred to give a solution with pD -2, containing a species in which both Cp rings remain metal bound for 24 h. At pD -7, partial hydrolysis of the Cp rings (-30percent) occurred after 24 h. Addition of an aqueous solution of Cp2MoCl2 4 in 50 mM salt to the self-complementary sequence d(CGCATATGCG)2, maintaining the pD at 6.0-7.0, showed no evidence for the formation of a metallocene-oligonucleotide complex and only peaks arising from hydrolysis of Cp2MoCl2 4 were detected. A similar result was obtained in titration experiments with the single stranded sequence d(ATGGTA) at pD 6.5-7.0. However, at pD 3.0, new signals assigned to a molybdocene-oligonucleotide complex(es), which was stable for hours at pD 3.0, were detected; while at pD -7 the complex is destabilised and only peaks arising from hydrolysis of Cp2MoCl2 4 were detected. Titration experiments at low pD with Cp2MoCl2 4 and the dinucleotide dCG were consistent with formation of a complex arising due to coordination of molybdenum to guanine N7 and/or cytosine N3. The results obtained showed that stable oligonucleotide adducts were not formed in 50 mM salt at pD -7 and hence it is highly unlikely that formation of molybdocene-DNA adducts in vivo is the primary action that is responsible for the antitumour properties of Cp2MoCl2 4. The rate of hydrolysis of the aromatic rings of Cp2TiX2 (X equals Cl 1, OCOCH2NH3Cl 27) and the dimethylsubstituted derivatives (MeCp)2TiX2 (X equals Cl 34, OCOCH2NH3Cl 41), in aqueous solutions at pD 2-8 was studied by 1H NMR spectroscopy. Rapid hydrolysis of both the halide/glycine and Cp ligands in Cp2TiX2 (X equals Cl 1, OCOCH2NH3Cl 27) occurred and predominantly gave a precipitate at pD -7. In contrast, under the same experimental conditions, the predominant species present in aqueous solutions of (MeCp)2TiX2 (X equals Cl 34, OCOCH2NH3Cl 41) at pH 2-8 contained both MeCp rings metal bound. At pD < 5, Cp2TiX2 (X equals Cl 1, OCOCH2NH3Cl 27) and (MeCp)2TiX2 (X equals Cl 34, OCOCH2NH3Cl 41) formed similar complex(es) with purine nucleotides. However, at pD >5, stable adducts between nucleotides and Cp2TiX2 (X equals Cl 1, OCOCH2NH3Cl 27) were not formed. In contrast, (MeCp)2TiX2 (X equals Cl 34, OCOCH2NH3Cl 41) formed complex(es) with 5'-dAMP or 5'-dGMP, which were stable for 24 h. These results suggest that formation of stable chelates between (MeCp)2TiX2 (X equals Cl 34, OCOCH2NH3Cl 41) and nucleic acid constituents in vivo is possible. However, the methyl substituted derivatives 34 and 41 did not show any antitumour activity against EAT in mice when administered in either 10percentDMSO/90percentsaline or in water at pH 6.2-6.4, which suggests that the labile Cp-Ti bond present in Cp2TiCl2 1 is required for antitumour activity. The synthesis of a range of Cp substituted titanocene derivatives was investigated in an attempt to prepare derivatives with modified Cp stability in comparison to the methyl substituted derivatives. The synthesis of derivatives (CpCH2Y)2TiCl2 where Y equals ?CHO 43, ?CONMe2 44, ?NO2 45, (RCp)2TiCl2 where R equals ?COMe 46, ?COOMe 47 or ?CONMe2 48, (CpNMe2)2TiCl2 62 and (Cp(CH2)2NMe2)2TiCl2 63 was unsuccessful, due to the presence of coordinating substituents on the Cp rings and poor stability in polar, protic solvents. Hence, these derivatives were excluded from further studies. The rate of hydrolysis of the Cp rings of Cp2TiX2 (X equals Cl 1, OCOCCl3 22 and OCOCH2NH3Cl 27) in aqueous solutions, 10percentDMSO/90percentD2O and 100percent DMSO was monitored by 1H NMR spectroscopy. Rapid hydrolysis of both the carboxylate and Cp ligands of Cp2TiX2 (OCOCCl3 22 and OCOCH2NH3Cl 27) occurred in DMSO to give biologically inactive species. The rate of these reactions were concentration dependent as dilution of these samples with saline or water to give the therapeutic conditions of 10percentDMSO/90percentD2O slowed the hydrolysis chemistry. In contrast, samples of Cp2TiX2 (X equals Cl 1 and OCOCH2NH3Cl 27) dissolved in water, gave solutions containing the presumed antitumour active species in which the halide or glycine ligands have been hydrolysed but the Cp rings remain metal bound. Thus, charged X ligands may be incorporated into Cp2TiX2 and will give comparable activity to Cp2TiCl2 1 provided the samples are administered in water. The antitumour metallocenes Cp2MCl2 (M equals Ti 1, V 2, Nb 3, Mo 4) and the inactive derivative (MeCp)2TiCl2 34 were found to inhibit the relaxation of supercoiled plasmid DNA pBR322 by human topoisomerase II in vitro. These results implicated the inhibition of topoisomerase II in the mechanism of antitumour activity although there was no direct correlation between the in vitro results with biological activity against EAT in vivo. UV spectroscopy confirmed that the metallocenes Cp2MCl2 (M equals Ti 1, Mo 4) became associated with and were stabilised to hydrolysis by calf thymus DNA but not with human serum albumin. ICP-AES was used to measure the amount of metal associated with either DNA or human serum albumin after incubation with Cp2MCl2 (M equals Ti 1, Nb 3, Mo 4) and dialysis of these solution. The results confirmed that DNA stabilises or becomes associated with the metallocenes. However, errors associated with the ICP-AES measurements did not allow these results to be quantified. 1H NMR spectroscopy was used to show that the antitumour metallocene Cp2MoCl2 4 formed an adduct with glutathione 72 in the pH range 3-7 through the sulfur donor group. In comparison, the antitumour metallocenes Cp2MCl2 (M equals Ti 1, Nb 3) showed limited adduct formation with glutathione 72 at pH -3 and no adducts were detected at pH > 5.5.

Doctor of Philosophy(PhD) / The present study deals with the synthesis and characterization of six tri-palladium complexes code named MH3, MH4, MH5, MH6, MH7 and MH8 that contained two planaramine ligands bound to the central or each of the terminal metal ions. The activity of the compounds against human cancer cell lines: A2780, A2780cisR and A2780ZD0473R, cell uptake, levels of DNA-binding and nature of interaction with salmon sperm and pBR322 plasmid DNA have also been determined. Whereas cisplatin binds with DNA forming mainly intrastrand GG adduct that causes local bending of a DNA strand, the tri-palladium complexes are expected to bind with DNA forming a number of long-range interstrand GG adducts that would cause a global change in DNA conformation. Among the designed complexes, MH6 that has two 2-hydroxypyridine ligands bound to each of the two terminal palladium ions is found to be most active. The compound also has the highest cell uptake and Pd-DNA binding levels. In contrast, MH8 which has two 4-hydroxypyridine ligands bound to each of the two terminal palladium ions is found to be least active. The results indicate that, as applied to the terminal metal centres, 2-hydroxypyridine would be more activating than 4-hydroxypyridine perhaps because of greater protection provided to the terminal centres from coming in contact with the solvent molecules. In contrast, when bound to the central metal centre, 4-hydroxypyridine appears to play a slightly greater activating role than 2-hydroxypyridine or 3-hydroxypyridine, suggesting that non-covalent interactions such as hydrogen bonding associated with the ligand rather than its steric effect may be a more important determinant of antitumour property. The results illustrate structure-activity relationships and suggest that the tri-palladium complex containing two 2-hydroxypyridine ligands bound to each of the three metal centres or the compound that contains two 2-hydroxypyridine ligands bound to each of the two terminal metal centres and two 4-hydroxypyridine ligands bound to the central metal centre, may be much more active than any of the designed complexes.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / As cordiaquinonas sÃo naftoquinonas meroterpenÃides isolados de plantas pertencentes ao gÃnero Cordia com vÃrias atividades biolÃgicas descritas, incluindo atividades antifÃngica, larvicida e citotÃxica. O objetivo deste trabalho foi avaliar o potencial anticÃncer de uma cordiaquinona isolada das raÃzes da planta Cordia leucocephala. O presente estudo avaliou o potencial citotÃxico da cordiaquinona J em vÃrias linhagens de cÃlulas tumorais e normais pelo teste do MTT e seu possÃvel mecanismo de aÃÃo. A cordiaquinona J mostrou valores de CI50 variando de 4,6 a 6,8 &#956;M em cÃlulas leucÃmicas e 33,6 a 37 &#956;M em cÃlulas normais, apÃs 24 horas de incubaÃÃo. Nas cÃlulas HL-60 foi observado induÃÃo de apoptose preferencialmente pela via extrÃnseca. A induÃÃo do dano ao DNA observado pelo tratamento com a cordiaquinona J atravÃs do ensaio do cometa foi associado com a ativaÃÃo de proteÃnas quinases da via ATM/ATR. O dano ao DNA, assim como a ativaÃÃo das proteÃnas quinases da via ATM/ATR foi visualizado em cÃlulas HL-60, mas nÃo em cÃlulas normais. Estes efeitos em HL-60 podem estar relacionados com a depleÃÃo da expressÃo proteica de glutationa e de c-myc observados. O potencial anticÃncer foi confirmado in vivo atravÃs da inibiÃÃo do tumor sarcoma-180 em 72,5% apÃs o tratamento com 50 mg/kg de cordiaquinona J. O prÃ-tratamento tanto das cÃlulas quanto dos animais com N-acetil-L-cisteina inibiu todos os efeitos observados in vitro e in vivo reforÃando o papel da geraÃÃo das espÃcies reativas de oxigÃnio na atividade antitumoral da cordiaquinona J. / Cordiaquinones are meroterpenoid naphtoquinones from plants belonging to the genus Cordia with several described biological activities, including antifungal, larvicidal and cytotoxic effects. The aim of this study was to evaluate the anticancer potential of a cordiaquinone isolated from the roots of Cordia leucocephala plant. The present study evaluated the cytotoxic potential of cordiaquinone J in several tumor and normal cell lines by MTT assay and its possible mechanism of action. The study of the mechanism of action of cordiaquinones L and M, in human leukemia cells (HL-60) showed induction of cell death by apoptosis, and these effects were related to the induction of oxidative stress. Then the study was continued only with the cordiaquinone J. The cordiaquinone J showed IC50 values ranging from 4.6 to 6.8&#956;M in leukemia cells and 33.6 to 37 &#956;M in normal cells, after 24 hours of incubation. In HL-60 cells was observed apoptosis induction preferentially by extrinsic pathway. The induction of DNA damage by cordiaquinone J observed by comet assay was associated with activation of protein kinases of ATM/ATR pathway. The DNA damage, as well as activation of protein kinases via the ATM / ATR was observed in HL-60 cells but not in normal cells. These effects in HL-60 cells may be related to the depletion of protein expression of glutathione and c-myc observed. The anticancer potential was confirmed in vivo through inhibition of sarcoma-180 tumor by 72.5% after the treatment with 50 mg/kg of cordiaquinone J. The pre-treatment of cells or animals with N-acetyl-L-cysteine abolished most of the in vitro and in vivo observed effects, reinforcing the role of reactive oxygen species generation in cordiaquinone J activity.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / A desacetilnemorona (HCRH) Ã um diterpeno abietano com anel para-quinona em sua estrutura, sendo entÃo classificada como tanshinona que sÃo frequentemente descritas pelo seu amplo espectro de atividades biolÃgicas. O diterpeno HCRH foi isolado e identificado pela primeira vez em 1971 a partir das raÃzes de plantas do gÃnero Salvia, no entanto sua atividade biolÃgica ainda nÃo havia sido descrita previamente. O objetivo deste trabalho foi avaliar o potencial anticÃncer da desacetilnemorona isolada das raÃzes da planta Hyptis carvalhoi. O presente estudo avaliou o potencial citotÃxico do diterpeno HCRH em vÃrias linhagens de cÃlulas tumorais e normais pelo teste do MTT e seu possÃvel mecanismo de aÃÃo. ApÃs 72 horas de incubaÃÃo, o composto testado apresentou valores de CI50 que variaram de 3,91 a 32,01 ÂM em cÃlulas tumorais de cÃlon (HCT-116) e leucÃmicas (HL-60), respectivamente. Enquanto que para cÃlulas normais esses valores foram de 35,68 ÂM para a linhagem V-79 e maiores que 72 ÂM para linhagens 3T3-L1 e CMSP. Em cÃlulas tumorais de cÃlon (HCT-116), o diterpeno mostrou potencial antiproliferativo de maneira tempo-dependente, induzindo aumento do nÃmero de cÃlulas na fase G0/G1 do ciclo celular e uma diminuiÃÃo expressiva da sÃntese de DNA. Esses efeitos foram acompanhados por alteraÃÃes nos nÃveis de ciclinas e CDKs, alÃm do aumento nos nÃveis das proteÃnas p21waf1/cip1 e p27kip1, independente da ativaÃÃo de p53. Dentre os eventos iniciais induzidos pelo diterpeno HCRH estÃo a geraÃÃo de espÃcies reativas de oxigÃnio (EROs) e a induÃÃo de dano ao DNA, com posterior ativaÃÃo das vias de morte. Com isso podemos sugerir que a desacetilnemorona apresenta potente atividade antiproliferativa que provavelmente se inicia com a geraÃÃo de EROs levando ao dano de DNA, o que impede a progressÃo do ciclo celular e encaminha as cÃlulas aos processos de morte por apoptose e autofagia. / The desacetylnemorone (HCRH) is an abietane diterpene with para-quinone ring in its structure, and then classified as a tanshinone that are often described by their broad spectrum of biological activities. The diterpene HCRH was isolated and identified for the first time in 1971 from roots of plants of the genus Salvia, however its biological activity has not been yet fully characterized. The aim of this study was to evaluate the anticancer potential of desacetylnemorone isolated from the roots of the plant Hyptis carvalhoi. The present study evaluated the cytotoxic potential of the diterpene HCRH in several tumor and normal cell lines using the MTT assay and its possible mechanism of action. After 72 hours of incubation, the tested compound showed IC50 values ranging from 3.91 to 32.01 ÂM in colon tumor (HCT-116) and leukemic (HL-60) cells, respectively. While for normal cells IC50 values ranged from 35.68 ÂM in V-79 to higher than 72 ÂM in 3T3-L1 and PBMC cells. In colon tumor cells (HCT-116), the diterpene showed antiproliferative potential of time-dependent manner, leading to increased number of cells in the G0/G1 phase of the cell cycle and a substantial decrease in DNA synthesis. These effects were accompanied by changes in the levels of cyclins and CDKs, in addition to the increase in the levels of proteins p21waf1/cip1 and p27kip1, independent of p53 activation. Among the initial events induced by diterpene HCRH are the generation of reactive oxygen species (ROS) and the induction of DNA damage with subsequent activation of death pathways. Thus, we can suggest that desacetylnemorone has potent antiproliferative activity associated with ROS generation leading to DNA damage, which prevents cell cycle progression and drive cells to the process of death by apoptosis and autophagy.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Muitos compostos isolados de plantas apresentam diversos tipos de atividades biolÃgicas, como tem sido o caso de proteÃnas conhecidas como lectinas. Essas proteÃnas fazem parte de um grupo de molÃculas que reconhecem e se ligam a carboidratos contidos na superfÃcie das cÃlulas de forma especÃfica e reversÃvel. Nas Ãltimas dÃcadas as lectinas vÃm se tornando ferramentas promissoras com aÃÃo antitumoral e antiviral. Esse trabalho objetivou investigar a possÃvel aÃÃo anticancerÃgena e antiviral das lectinas isoladas das sementes de Canavalia brasiliensis (ConBr), Canavalia marÃtima (ConM), Dioclea lasiocarpa (DLasiL) e Dioclea sclerocarpa (DSclerL), o possÃvel mecanismo inicial de aÃÃo anticancerÃgena da lectina DLasiL, alÃm de estudar a estrutura da DLasiL atravÃs das tÃcnicas de fluorescÃncia e dicroÃsmo circular e seu mecanismo para atividade anticÃncer. A lectina DLasiL foi particularmente caracterizada, apresentando 237 resÃduos de aminoÃcidos com alta similaridade com as lectinas da subtribo Diocleinae. RessonÃncia paramagnÃtica de elÃtrons mostrou sinal caracterÃstico da presenÃa do Ãon manganÃs (Mn2+), enquanto medidas de ICP-MS confirmaram a quantidade deste Ãon, alÃm de cÃlcio (Ca2+), cuja quantidade foi inferior ao medido nas outras lectinas. Estudos de fluorescÃncia intrÃnseca indicaram melhor acessibilidade aos triptofanos por molÃculas neutras, provando que a vizinhanÃa possuui carÃter nÃo carregado, enquanto que fluorescÃncia extrÃnseca usando Bis-ANS ilustrou a alteraÃÃo conformacional provocada pela interaÃÃo a aÃÃcares, concluindo que à possÃvel utilizar esse tipo de medida para a constataÃÃo desse fenÃmeno. Medidas de DicroÃsmo Circular confirmam a significativa estabilidade tÃrmica da DLasiL no qual perdeu 50% de sua atividade a 72 oC. Para investigar a aÃÃo antitumoral das lectinas em estudo, cÃlulas de carcinoma de ovÃrio humano (A2780), carcinoma caucasiano de pulmÃo humano (A549), carcinoma de mama humano (MCF7), carcinoma de prÃstata humano (PC3) foram cultivadas com DLasiL. AlÃm disso, foi determinada a viabilidade celular. Os resultados mostraram que as lectinas foram efetivas em inibir o crescimento celular das linhangens testadas utilizando concentraÃÃo nanomolar. Dentre as lectinas testadas, a mais efetiva em inibir o crescimento celular foi a DLasiL, demonstrando um maior Ãndice de citotoxicidade contra cÃlulas da linhagem A2780 com IC50 de 52 nM. O mecanismo de aÃÃo da DLasiL foi investigado atravÃs de ensaios especÃficos de apoptose. Dados de ciclo celular mostraram que a DLasiL apresenta mudanÃas significativas nos nÃveis S e G2/M. Adicionalmente, ensaios com uma sÃrie de conjuntos de vÃrus apresentaram resultados bastante promissores. / Several compounds isolated from plants, including proteins called lectins, have exhibited many types of biological activities. These proteins are specific recognizing and binding to carbohydrates found onto the cell surface. During the last decades, lectins have become promising sources for antitumor and antiviral studies. This study aimed to investigate legumineous lectins, ConBr (Canavalia brasiliensis), ConM (Canavalia maritima), DLasiL (Dioclea lasiocarpa) and DSclerL (Dioclea sclerocarpa) as potential anti-cancer and anti-viral agents, as well as studying structurally DLasiL using fluorescence, circular dichroism and its initial mechanism of anticancer activity. DLasiL lectin was characterized showing 237 amino acid residues with high similarity to lectin from the Diocleinae subtribe. Electron paramagnetic resonance of DLasiL showed a typical signal for manganese (Mn+2), while ICP-MS provided its actual amount along with calcium (Ca2+), whose values were below those found for the other lectins. Intrinsic fluorescence studies using three quenchers showed a better accessibility of neutral species to tryptophans of DLasiL, while extrinsic fluorescence using bis-ANS exhibited a dose-dependent conformational changes promoted by sugars. Circular dichroism showed very expressive thermal stability of DLasiL with mid-point for thermal denaturation at 72 ÂC. These lectins were used to treat human ovarian carcinoma (A2780), Human Lung Carcinoma (A549), Human Breast Carcinoma (MCF7), Human Prostate Carcinoma (PC3) and cell viability was determined. These proteins showed potent activity on inhibiting cell growth at nanomolar concentrations. Among these lectins, DLasiL was the most effective showing potent cytotoxicity against A2780 cell line with IC50 of 52 nM. The mechanism of cytotoxicity action of DLasiL was investigated by specific apoptosis assay. Cell cycle studies showed DLasiL causes significant changes in the levels of S and G2/M. A virus screening investigation was carried out showing promissing antiviral activity.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-06-15T14:22:16Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertaçao final-Colaço.pdf: 884713 bytes, checksum: fca04fb02a3890a222aaff75e2759910 (MD5) / Made available in DSpace on 2016-06-15T14:22:16Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertaçao final-Colaço.pdf: 884713 bytes, checksum: fca04fb02a3890a222aaff75e2759910 (MD5) Previous issue date: 2012-10-29 / CNPq / O câncer é uma das principais causas de morte no mundo e alberga intensos e crescentes investimentos em pesquisa oriundos dos setores público e privado. Considerando os alarmantes indicadores epidemiológicos registrados e a visível carência de medicamentos mais eficazes e menos tóxicos para esta patologia, o Laboratório de Planejamento e Síntese de Fármacos vem, desde o final da década de 1980, desenvolvendo moléculas com potenciais propriedades anticâncer. A meta é minimizar o impacto dessa doença no Brasil e no mundo. Para tanto, conhecendo-se as propriedades anticâncer de derivados da acridina, este trabalho teve como objetivo preparar, determinar as características físico-químicas, caracterizar estruturalmente e avaliar a atividade anticâncer de oito moléculas originais derivadas da acridina. Nos ensaios in vitro foram utilizadas linhagens celulares T47D, NG97, HEPG2, Jurkat e Raji. Para a realização das sínteses, foram realizadas reações de N-alquilação, condensação, ciclização e adição de Michael. As moléculas obtidas foram comprovadas através de métodos espectroscópicos no infravermelho, ressonância magnética nuclear de hidrogênio e espectrometria de massas. Os resultados revelam que o composto 3-(acridin-9-ilmetil)-5-(5-bromo-1H-indol-3-ilmetileno)-tiazolidana-2,4-diona (LPSF/AA-29) se destacou por apresentar menores valores de viabilidade celular para as linhagens T47D, HEPG2 e NG97 de células testadas, chamando atenção para a dose de 50 μM que foi a melhor. Também foi possível observar que o substituinte triazol presente no composto LPSF/AA-48 possibilitou uma maior toxicidade frente as linhagens Raji e Jurkat, principalmente na dose de 100 μM. / Cancer is a leading cause of death in the world and is home to intense and increasing investments in research coming from the public and private sectors. Whereas the alarming epidemiological indicators recorded and visible lack of more effective and less toxic drugs for this condition, the Laboratory Planning and Synthesis of Pharmaceuticals has, since the late 1980s, developing molecules with potential anticancer properties. The goal is to minimize the impact of this disease in Brazil and worldwide. To do so, knowing the anticancer properties of acridines derivatives, this study aimed to prepare, determine the physico-chemical characteristics, structurally characterize and evaluate the anticancer activity of eight original molecules derived from acridines. In vitro cell lines T47D, NG97, HEPG2, Jurkat and Raji were used. To perform the synthesis, reactions of N-alkylation, condensation, Michael addition and cyclization were performed. The molecules obtained were confirmed by spectroscopic methods in the infrared, nuclear magnetic resonance and mass spectrometry. The results show that compound 3 - (acridin-9-ylmethyl)-5-(5-bromo-1H-indol-3-ylmethylene)-tiazolidana-2,4-dione (LPSF/AA-29) stood out with lower values for T47D cell viability, and HEPG2 NG97 tested cell lines, calling attention to the dose of 50 mM which was the best. Was also observed that the triazole substituent present in the compound LPSF/AA-48 allowed greater toxicity across the Raji and Jurkat cell lines, especially at a dose of 100 mM.

Orientador: Anita Jocelyne Marsaioli / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Quimica / Made available in DSpace on 2018-08-04T15:47:17Z (GMT). No. of bitstreams: 1 Lapis_MireleSanchesFernandes_M.pdf: 6468600 bytes, checksum: 6af92e23ef4b2fdd6be4740a8ba9f443 (MD5) Previous issue date: 2005 / Resumo: Este trabalho teve como objetivo elucidar algumas interações ecológicas de espécies de Orchidaceae e Guttiferae. No primeiro capítulo, é descrita a determinação da composição química das fragrâncias florais de cinco espécies de orquídeas pertencentes ao complexo Maxillaria madida (M. juergensii, M. madida, M. vernicosa, M. ferdinandiana e M. pachyphylla), através da técnica de headspace dinâmico e análise por CG/EM. Monoterpenóides e sesquiterpenóides não oxigenados são os principais compostos voláteis emitidos e provavelmente responsáveis pela atração dos insetos polinizadores. No segundo capítulo, demos continuidade ao estudo da composição química do látex dos frutos de C. grandiflora iniciado por Cláudio A. G. da Câmara. Este estudo resultou no isolamento de 3 compostos, sendo 2 inéditos e derivados do ácido cis-d-tocotrienólico. Estes compostos quando submetidos ao teste em CCD com b-caroteno, apresentaram propriedades antioxidantes. No último capítulo deste trabalho, é apresentada a análise da composição química das resinas florais do híbrido interespecífico de C. weddelliana X paralicola, comparada com a das resinas florais de seus parentais. Observou-se que todos os compostos isolados também estão presentes nas espécies genitoras, variando apenas suas abundâncias. Finalmente, foi avaliada a atividade antineoplástica das resinas florais de C. grandiflora, C. hilariana, C. renggerioides, C. fluminensis, C. spiritu-sanctensis, C. lanceolata, C. nemorosa hermafrodita e C. paralicola, em 8 diferentes linhagens de células. Os resultados revelaram que os metabólitos secundários (benzofenonas poliisopreniladas) presentes nestas espécies são eficientes agentes antitumorais e o mecanismo de ação destes compostos envolve a inibição de topoisomerases. / Abstract: The aim of this research was the elucidation of ecological interactions in Orchidaceae and Guttiferae species. The first chapter describes the floral fragrance chemical compositions of five orchid species belonging to the Maxillaria madida group (M. juergensii, M. madida, M. vernicosa, M. ferdinandiana and M. pachyphylla) obtained by applying dynamic headspace sampling technique and GC/MS analysis. Monoterpenoids and sesquiterpenoids without oxygen in its structure are the main emitted volatile compounds and probably responsible for the pollinator attractions. In the second chapter the investigation of Clusia grandiflora fruit latex chemical composition initiated some years ago by Dr Cláudio A. G. da Câmara was concluded with the isolation of novel compounds derived from tocotrienolic acid. Two of which (methyl-(2Z, 6E, 10E)-13-(6-hydroxy-2,7,8-trimethyl-3, 4-dihydro-2H-2chromenyl)-2,6,10-trimethyl-2,6,10-tridecatrienoate and methyl-(2Z, 6E,10E)-13-(6-formyl-5-hydroxy-2,8-dimethyl -3, 4-dihydro-2H-2-chromenyl)-2,6,10-trimethyl-2,6,10-tridecatrienoate) are novel secondary metabolites which have antioxidant properties revealed by b-carotene TLC test. Evaluation of the interespecific hybrid of Clusia weddelliana x paralicola floral resin and revealed that the chemical constituents in the parents and hybrid species were identical with different relative abundances. Finally, we have evaluated the anticancer activity of the floral resins of C. grandiflora, C. hilariana, C. renggerioides, C. fluminensis, C. spiritu-sanctensis, C. lanceolata, C. nemorosa hermafrodita and C. paralicola in eigth cell lines. The results showed that the secondary metabolites (polyisoprenylated benzophenones) were efficient antitumor agents by topoisomerase inhibition. / Mestrado / Quimica Organica / Mestre em Química