Muitas naftoquinonas como o lapachol, podem ser encontradas em plantas da família Bignoniaceae e são conhecidas por desempenharem diversas atividades biológicas, acompanhadas, entretanto, por efeitos indesejáveis. A atividade citotóxica apresentada pelas naftoquinonas está relacionada ao aparecimento de espécies reativas de oxigênio in vivo que causam severo estresse oxidativo no interior das células. O isolapachol e a atovaquona são análogos estruturais do lapachol, sendo que a atovaquona é comercializada como fármaco para o tratamento de malária e certos tipos de pneumonia. Devido ao grande potencial biológico apresentado pelas naftoquinonas, várias tentativas no sentido de obtenção de derivados desprovidos de efeitos colaterais vêm sendo realizadas. Além disso, a determinação da segurança e eficácia dos fármacos está intimamente ligada ao estudo da formação de derivados in vivo por ocasião do metabolismo. A utilização de fungos filamentosos na predição do metabolismo que os fármacos sofreriam após administração oral, bem como de bactérias do trato gastrointestinal, pode contribuir substancialmente para a elucidação da rota metabólica de fármacos fornecendo informações sobre a geração de substâncias farmacologicamente ativas, inativas ou tóxicas e ainda sobre a produção de substâncias capazes de inibir a biotransformação de outros fármacos. Estudos de biotransformação também podem contribuir para a obtenção de novos esqueletos químicos. Dessa forma, o presente trabalho relata estudos do metabolismo microbiano do lapachol e do seu sal de potássio por bactérias do trato gastrointestinal e fungos filamentosos, além da correlação desses com as reações que ocorrem quando o isolapachol e a atovaquona são utilizados como substratos para os mesmos micro-organismos. Os experimentos de biotransformação utilizando lapachol e seu sal de potássio foram conduzidos por até dez dias, em diferentes meios de cultura, empregando-se quatro linhagens de bactérias presentes no trato gastrointestinal, além de 11 linhagens de fungos filamentosos. Foram obtidos sete metabólitos, sendo dois inéditos e dois anteriormente detectados em estudos sobre o metabolismo do lapachol em mamíferos. Durante a realização dos experimentos com o fungo filamentoso Aspergillus brasiliensis verificou-se a capacidade desse fungo em mimetizar uma reação muito importante em química orgânica, conhecida como oxidação de Hooker. As condições mais promissoras para a biotransformação do lapachol foram utilizadas nos estudos com a atovaquona e o isolapachol. A biotransformação da atovaquona possibilitou, pela primeira vez, a caracterização estrutural de um metabólito desse fármaco. Já os estudos realizados com o isolapachol permitiram inferências sobre a especificidade enzimática apresentada pelos micro-organismos avaliados. Todos os metabólitos obtidos foram submetidos aos ensaios de citotoxicidade frente a linhagens celulares normais e tumorais, o que possibilitou obter conclusões sobre a relação estrutura-atividade e sobre a citotoxicidade seletiva apresentada pelos metabólitos. Destaca-se o resultado obtido com um dos metabólitos do lapachol, ?-xiloidona, o qual se mostrou mais tóxico para a linhagem tumoral que o lapachol e não apresentou toxicidade frente à linhagem normal. O metabólito obtido a partir da biotransformação da atovaquona apresentou maior toxicidade não seletiva que a substância de partida. / Several naphthoquinones, as lapachol, can be found in the Bignoniaceae family and they present several biological activities with some unwanted effects. The cytotoxic activity displayed by naphthoquinones is correlated to the presence of reactive oxygen species, which are formed in vivo and cause severe oxidative stress within cells. Isolapachol and atovaquone are structural analogs of lapachol, and atovaquone is in the market as a drug for the treatment of malaria and some types of pneumonia. Because of the great biological potential presented by naphthoquinones, several studies have been carried out to obtain derivatives without side effects. Furthermore, the drug safety and efficacy are closely related to the study of the formation of in vivo derivatives during metabolism. The filamentous fungi and the bacteria from the gastrointestinal tract can be used in the prediction of drug metabolism after oral administration, which is an interesting tool to elucidation of the metabolic pathway of drugs, providing information on the generation of pharmacologically active, inactive or toxic substances and still on the production of compounds able to inhibit the biotransformation of other drugs. Biotransformation studies can also contribute to the obtention of new chemical skeletons (hits). Thus, the present work reports the study about the microbial metabolism of lapachol and its potassium salt by filamentous fungi and bacteria from the gastrointestinal tract, beyond the correlation of the reactions that occur when the isolapachol and atovaquone are used as substrates for the same microorganisms. The biotransformations of lapachol and its potassium salt were evaluated for up to ten days, in different culture media, catalyzed by four bacteria from the gastrointestinal tract and 11 filamentous fungi strains. Seven metabolites were obtained, from which two are new and two were previously detected in the mammals metabolism of lapachol. The filamentous fungus Aspergillus brasiliensis showed to be capable of mimicking the Hooker oxidation, an important organic chemistry reaction. The best conditions for the lapachol biotransformation have been used in the studies with isolapachol and atovaquone. The atovaquone biotransformation provided, for the first time, the structural characterization of a metabolite from this drug. The studies with isolapachol allowed inferences about the enzyme specificity shown by the evaluated microorganisms. All obtained metabolites were submitted to cytotoxicity assays against human cancer and tumoral cell lines. Several conclusions about the structure activity relationship and about the selective cytotoxicity showed by the metabolites were taken. It should be highlighted the obtained result with a lapachol metabolite, ?-xyloidone, which showed to be more toxic than lapachol against tumoral cell line and did not show cytotoxicity to normal cell line. The atovaquone metabolite displayed higher toxicity than pattern structure, and this activity was not selective.

This work deals with the preparation of amine derivatives of 3-chloro-5(8?)-nitro-1,4-naphthoquinone which are to be tested for anti-tubercular activity by Parke, Davis and Company.

Nucleophilic substitution reactions of alkylamines, cyclic alkylamines, and hydroxyalkylamines with 5-substituted-1,4-naphthoquinones have been studied. It has been found that the nature of the solvent employed in the reaction influences the position of mono-substitution at either the 2- or 3-position. Although both regioisomers were produced in all the reactions, protic polar solvents favoured the formation of the 3-regioisomer, whereas non-protic solvents favoured the formation of the 2-regioisomer. It has also been found that formation of 2,3-diaminoalkyl derivatives is normally unlikely. A series of hydroxyalkylamino-1,4-naphthoquinones were also synthesised. The collision-induced dissociation mass spectra of protonated hydroxyalkylamino-1,4- naphthoquinones showed fragmentation patterns which were dependent on the nature and length of the side chain and the presence and nature of the adjacent group on the 3-position on the 1,4-naphthoquinone ring. A total of 27 novel compounds were synthesised during the course of this research, the structures of which were confirmed via 1D and 2D NMR spectroscopy, mass spectrometry (ESI), IR spectroscopy and high resolution mass spectrometry (HRESIMS and HREIMS).

Nucleophilic substitution reactions of alkylamines, cyclic alkylamines, and hydroxyalkylamines with 5-substituted-1,4-naphthoquinones have been studied. It has been found that the nature of the solvent employed in the reaction influences the position of mono-substitution at either the 2- or 3-position. Although both regioisomers were produced in all the reactions, protic polar solvents favoured the formation of the 3-regioisomer, whereas non-protic solvents favoured the formation of the 2-regioisomer. It has also been found that formation of 2,3-diaminoalkyl derivatives is normally unlikely. A series of hydroxyalkylamino-1,4-naphthoquinones were also synthesised. The collision-induced dissociation mass spectra of protonated hydroxyalkylamino-1,4- naphthoquinones showed fragmentation patterns which were dependent on the nature and length of the side chain and the presence and nature of the adjacent group on the 3-position on the 1,4-naphthoquinone ring. A total of 27 novel compounds were synthesised during the course of this research, the structures of which were confirmed via 1D and 2D NMR spectroscopy, mass spectrometry (ESI), IR spectroscopy and high resolution mass spectrometry (HRESIMS and HREIMS).

Synthesizing bioactive small molecules by structural modification of 1,4-naphthoquinone was the primary goal of this research. Several bioactive compounds with anticancer, antifungal, and antibacterial activities were synthesized. All the synthetic protocols were optimized in such ways that do not require cumbersome purification. First, a new protocol for the synthesis of NQM111 was developed. NQM111 is a highly potent anticancer agent developed in our laboratory, but the old protocol does not provide enough quantity for in vivo study. Therefore, a new safe and improved method was developed which provides enough quantity for in vivo study. The second project involves the synthesis of 1,4-naphthoquinone conjugated with an aromatic group. These compounds are a highly potent anticancer agent with ~8-fold selectivity towards cancer cell lines than the non-cancer cell line. A mode of action study of this compound was identified, and it was observed that these compounds generate reactive oxygen species,which triggers apoptosis. The final project involves the synthesis of 1,4-naphthoquinone based antifungal, and antibacterial compounds. These compounds are multi-cationic in nature with a hydrophobic tail. Six different analogs with varying hydrophobic tails were synthesized and tested for their antibacterial and antifungal activity. These compounds showed excellent activity against wide range of fungi including resistant strains.

Submitted by Maria Medeiros (maria.dilva1@ufcg.edu.br) on 2018-06-25T14:25:31Z No. of bitstreams: 1 JACKSON BORBA DA CRUZ - TESE (PPGCEMat) 2015.pdf: 4572910 bytes, checksum: 529c14d2c73d94769063607a9a287e65 (MD5) / Made available in DSpace on 2018-06-25T14:25:31Z (GMT). No. of bitstreams: 1 JACKSON BORBA DA CRUZ - TESE (PPGCEMat) 2015.pdf: 4572910 bytes, checksum: 529c14d2c73d94769063607a9a287e65 (MD5) Previous issue date: 2015-05-22 / As neoplasias constituem um conjunto de doenças que se caracterizam por uma massa anormal de tecido com crescimento descontrolado e excessivo em relação aos demais tecidos normais. De acordo com o comportamento biológico, elas podem ser classificadas em benignas ou malignas (câncer). Segundo o Instituto Nacional de Câncer (INCA), são esperados 576 mil casos novos de câncer para 2014 e 2015 no Brasil. O combate ao câncer é feito com medidas preventivas, diagnóstico precoce e tratamento. O sistema de liberação controlada de fármacos através da utilização de biomateriais poliméricos associados a compostos com ação antineoplásica pode ser empregado como alternativa de tratamento para esta patologia. Desta forma, este trabalho tem como objetivo a síntese e caracterização de arcabouços de quitosana utilizados como carreadores da droga antitumoral (1,4- Naftoquinona), cuja taxa de liberação poderá ser controlada pela utilização de um agente reticulante como o tripolifosfato de sódio (TPP), com a perspectiva da confecção de um sistema de liberação controlada de fármacos antitumorais. O método consiste na solubilização da quitosana em ácido acético, adição do fármaco, congelamento, liofilização e reticulação com TPP. Todas as amostras foram caracterizadas por Difração de Raios X (DRX), Microscopia Eletrônica de Varredura (MEV), Espectroscopia de Energia Dispersiva de Raios X (EDS), Espectroscopia na Região do Infravermelho com Transformada de Fourier (FTIR) e Biodegradação Enzimática, A microscopia (MEV) evidenciou a formação de arcabouços porosos de quitosana (Q), quitosana com TPP (QT), quitosana com 1,4 – Naftoquinona (QN) e quitosana com TPP e 1,4 – Naftoquinona (QNT). Já no EDS foi observada a presença de elementos químicos como sódio, fósforo, nitrogênio, carbono e oxigênio. A reticulação dos arcabouços, comprovada pelo FTIR, DRX, Termogravimetria, Grau de Intumescimento e EDS, aumentou a taxa de degradação dos mesmos demonstrada pelo ensaio de Biodegradação Enzimática. A incorporação do fármaco foi confirmada por DRX, FTIR, Grau de Intumescimento e Termogravimetria. Desta forma, pode-se concluir que houve à formação de arcabouços reticulados e não reticulados porosos, com propriedades morfológicas e físico-químicas que podem contribuir para carrear fármacos antineoplásicos, sendo possível controlar a taxa de degradação dos mesmos e consequentemente a liberação do fármaco. / The neoplasias are a group of disorders characterized by an abnormal mass of tissue which has uncontrolled and excessive growth when compared to normal tissue. According to their biological behavior, they can be classified as benign or malignant (cancer). According to the National Cancer Institute (NCI), it is expected that there will be 576,000 new cancer cases in Brazil during 2014 and 2015. The fight against cancer is done with preventive measures, early diagnosis and treatment. The controlled release system of drugs through the use of polymeric biomaterials associated with the compounds that have an antineoplastic action can be used as an alternative treatment for this disease. Thus, this work has as the objective, the synthesis and characterization of chitosan scaffolds used as carriers for antitumor drugs (1,4 Naftoquinona), whose release rate can be controlled by using a crosslinking agent such as sodium tripolyphosphate (TPP), with the prospect of making a controlled release system for antitumor drugs. The method comprises in the solubility of chitosan in acetic acid, drug addition, freezing the material, lyophilization and crosslinking with TPP. All samples were characterized by X-Ray Diffraction (XRD), Scanning Electron Microscopy (SEM), Energy Dispersive X-ray Spectroscopy (EDS), Fourier Transform Infrared Spectroscopy (FTIR), and Enzymatic Biodegradation. The Microscopy (SEM) showed the formation of porous scaffolds of chitosan (Q), chitosan with TPP (CT), chitosan with 1.4 - Naftoquinona (QN) and chitosan with TPP and 1.4 - Naftoquinona (QNT). EDS showed the presence of chemical elements such as sodium, phosphorus, nitrogen, carbon and oxygen. The crosslinking of the scaffolds, proven by FTIR, XRD, Thermogravimetry, Degree of Swelling and EDS, increased its rate of degradation thereof, as demonstrated by the Enzymatic Biodegradation test. The incorporation of the drug was confirmed by XRD, FTIR, Degree of Swelling and Thermogravimetry. Thus, it can be concluded that there was the formation of crosslinked and non-crosslinked porous scaffolds, with morphological and physicochemical properties that can contribute to the carrying of antineoplastic drugs, being possible to control their degradation rate and consequently drug release.

FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / As quinonas sÃo metabÃlitos de ampla distribuiÃÃo na natureza que possuem diversas atividades farmacolÃgicas de importÃncia clÃnica. A naftoquinona α-lapachona demonstrou potencial como protÃtipo para o desenvolvimento de substÃncias com propriedades anticÃncer, como relatado pelo nosso grupo de pesquisa, em que o seu derivado tetrahidropirano (THP) apresentou citotoxicidade e seletividade significante contra a linhagem de melanoma MDA-MB-435. Assim, o objetivo deste trabalho foi avaliar o mecanismo de aÃÃo envolvido na citotoxicidade da α-lapachona e derivado THP em cÃlulas de melanoma MDA-MB-435. Inicialmente, avaliou-se a citotoxicidade da alfa-lapachona e derivado THP em 8 linhagens tumorais de mama e melanoma, atravÃs do ensaio do MTT, mostrando CI50 de 1,37 e 8,18 ÂM, respectivamente, apÃs 72 horas de incubaÃÃo. A seletividade do derivado THP no ensaio de Alamar Blue, demonstrou que este se apresentou 2,6 vezes menos citotÃxico para cÃlulas normais quando comparado Ãs cÃlulas tumorais. Estudos do mecanismo de morte celular na linhagem tumoral MDA-MB-435 indicaram que o derivado THP causou reduÃÃo de cÃlulas viÃveis associado com o aumento de cÃlulas nÃo-viÃveis por induÃÃo da perda de integridade da membrana plasmÃtica nas concentraÃÃes de 5 e 10 ÂM apÃs 24 horas de incubaÃÃo. A atividade citotÃxica do derivado THP nÃo està relacionada a uma fase especÃfica do ciclo celular, ativaÃÃo de caspases efetoras e formaÃÃo de espÃcies reativas de oxigÃnio, sugerindo a ocorrÃncia de um processo necrÃtico a partir de 6 horas de tratamento, demonstrado pela avaliaÃÃo da integridade de membrana. Assim, os resultados exibidos sugerem que a introduÃÃo do radical tetrahidropirano na molÃcula da α-lapachona aumenta a citotoxicidade em cÃlulas de melanoma MDA-MB-435, via necrose, o que reforÃa a importÃncia de naftoquinonas, como protÃtipo para o desenvolvimento de novos compostos sintÃticos com atividade antitumoral / Quinone metabolites are widely distributed in nature showing various pharmacological activities of clinical importance. The naphthoquinone α-lapachone has been shown to be suitable as a prototype for the development of substances with anticancer properties, as reported by our group to tetrahydropyran derivative (THP), which demonstrated significant cytotoxicity and selectivity against MDA-MB-435 melanoma line. The aim of the present work was to evaluate the mechanism of action involved in the cytotoxicity of α-lapachone and its THP derivative against MDA-MB-435 melanoma cells. Initially, we evaluated the cytotoxicity of α-lapachone and its THP derivative against 8 cell lines, by the MTT assay, showing IC50 of 1.37 and 8.18 ÂM to breast and melanoma lines, respectively, after 72 hours of incubation. The selectivity of the THP derivative in Alamar Blue assay, demonstrated that THP is 2.6 times less cytotoxic to normal cells as compared to tumor cells. Studies on the mechanism of cell death in MDA-MB-435 tumor line showed that the THP derivative caused a reduction on viable cells associated with an increase of non-viable cells by inducing loss of membrane integrity in concentrations of 5 and 10 ÂM. The cytotoxic activity of THP was independent of cell cycle, activation of effector caspases and formation of reactive oxygen species, suggesting the occurrence of a necrotic process after 6 hours of treatment, demonstrated by evaluation of membrane integrity. Thus, the data suggest that a tetrahydropyran group introduction in α-lapachone molecule enhances the cytotoxicity of MDA-MB-435 in melanoma cells, via necrosis, which reinforces the importance of naphthoquinones as prototypes for the development of new synthetic compounds with antitumor activity

Submitted by Biblioteca da Faculdade de Farmácia (bff@ndc.uff.br) on 2017-04-04T18:00:06Z No. of bitstreams: 1 Cardoso, Mariana Filomena do Carmo [Dissertação, 2012].pdf: 7461706 bytes, checksum: 1ba99c29719229ef773ca5d72b10c91f (MD5) / Made available in DSpace on 2017-04-04T18:00:06Z (GMT). No. of bitstreams: 1 Cardoso, Mariana Filomena do Carmo [Dissertação, 2012].pdf: 7461706 bytes, checksum: 1ba99c29719229ef773ca5d72b10c91f (MD5) / Esse trabalho descreve uma nova metodologia sintética de novos derivados pirazólicos análogos a 2,2-dimetil-2,3-di-hidronafto[1,2-b]furan-4,5-diona (nor-β-lapachona), através da inserção do núcleo pirazólico a posição C-3 da nor-β-lapachona. Nesta dissertação foram sintetizados 16 (dezesseis) substâncias inéditas, sendo oito da família 3-pirazolil-2,2-dimetil-2,3-di-hidronafto[1,2-b]furan-4,5-diona contendo o núcleo pirazólico acoplado à naftoquinona os quais foram submetidos a testes biológicos para avaliação de suas atividades citotóxicas in vitro contra quatro linhagens de células tumorais humanas e uma linhagem de células normais humanas. Todas as amostras mostraram-se ativas para as linhagens tumorais e não apresentaram hemólise. A metodologia clássica para a substituição nucleofílica no carbono 3 da nor-β-lapachona desenvolvida pelo nosso grupo de pesquisa mostrou-se pouco eficaz, levando a baixos rendimentos com formação de vários produtos colaterais. Desta forma, realizou-se um estudo metodológico a fim de se viabilizar a síntese de uma família de 3-pirazolil-nor-β-lapachonas com rendimentos satisfatórios. Assim, após várias modificações nos parâmetros reacionais, observou-se que o melhor intermediário sintético era o 3-hidroxi-2,2-dimetil-2,3-di-hidronafto[1,2-b]furan-4,5-diona / This paper describes a new synthetic methodology to new pyrazole derivatives analogous to the 2,2-dimethyl-2,3-dihidronaphtho-[1,2-b]-furan-4 ,5-dione (nor-β-lapachone) by inserting the core pyrazolic on the C-3 position of the nor-β-lapachone. In this essay were synthesized 16 (sixteen) new compounds, being eight 3-pyrazolyl-2,2-dimethyl-2,3-dihidronaphtho [1,2-b]-furan-4 ,5-dione family containing core pyrazolic naphthoquinone attached to which were submitted to biological tests to evaluate their in vitro cytotoxic activities against four human tumor cell lines and normal human cell line. All samples were active for tumor cell lines and showed no hemolysis. The classical methodology for the nucleophilic substitution at carbon 3 of the nor-β-lapachone developed by our research group proved to be ineffective, leading to low yields with the formation of various side products. Thus, there was a methodological study in order to facilitate the synthesis of a family of 3-pyrazolyl-nor-β-lapachones with satisfactory yields. Then, after the various modifications on the reaction parameters, it was found that the better synthetic intermediate was the 3-hydroxy-2,2-dimethyl-2,3-dihidronaphtho-[1,2-b]-furan-4 ,5-dione

This thesis describes a series of naphthoquinone reactions employing pyridine carboxylic acid derivatives (nicotinic acid derivatives). The products of these reactions will be tested by Parke, Davis and Company for their activity against the tubercle bacillus and other pathogenic microorganisms.

Submitted by (lucia.rodrigues@ufrpe.br) on 2017-02-15T15:40:19Z No. of bitstreams: 1 Wilson Silva do Nascimento.pdf: 1102619 bytes, checksum: f3fd4580e9425c165601806a91092eda (MD5) / Made available in DSpace on 2017-02-15T15:40:19Z (GMT). No. of bitstreams: 1 Wilson Silva do Nascimento.pdf: 1102619 bytes, checksum: f3fd4580e9425c165601806a91092eda (MD5) Previous issue date: 2011-02-15 / In the present study was performed the synthesis of a new series of 1,2,3- triazole derivative 1,4-disubstituted naphthoquinone group containing the position of a heterocyclic ring from the reaction of 1,3-dipolar cycloaddition between the two precursor azido-1,4-naphthoquinone and 10 terminal alkynes, using a method that employs the use of CuI as the catalytic species for the regioselective formation of the triazole and acetonitrile as solvent. Other methods were also tested, including the one described by using Sharpless reducing environment, however, these methods were less effective or not promoted the formation of the triazole ring. Two of these triazoles had obtained the hydroxyl groups present in its structure acetylated methodology developed in our laboratory using acetic anhydride and montmorillonite K-10 by ultrasound, a total number of 12 new structures of [1,2,3]-triazole 1,4- disubstituted. A second route to obtain 1,4-disubstituted triazoles connected to 1,4- naphthoquinone was proposed from the reaction between 2-ethynyl-1,4- naphthoquinone and azido compound, for it was synthesized the 2-(3-hydroxy-3- metilbutinil)-1,4-naphthoquinone. However, the subsequent reaction of deprotection of this compound, as well as the synthesis of the precursor 2-trimethylsilyl-1,4- naphthoquinone did not work. All the products of unknown structures were characterized by 1H NMR and 13C NMR, elemental analysis, LC-MS and infrared. / No presente trabalho foi realizada a síntese de uma nova série de derivados 1,2,3-triazólicos 1,4-dissubstituídos contendo o grupo naftoquinona na posição 1 deste anel heterocíclico a partir da reação de cicloadição 1,3-dipolar entre o precursor 2-azido-1,4-naftoquinona e 10 alcinos terminais, utilizando um método que emprega o uso de CuI como espécie catalítica para a formação regiosseletiva do anel triazólico e acetonitrila como solvente. Outros métodos também foram testados, entre eles o descrito por Sharpless que utiliza meio redutor, no entanto, estes métodos mostraram-se menos eficiente ou não promoveram a formação do anel triazólico. Dois destes triazóis obtidos tiveram os grupos hidroxilas presente em sua estrutura acetilados por metodologia desenvolvida em nosso laboratório utilizando anidrido acético e montmorillonite K-10 em ultra som, totalizando uma série de 12 novas estruturas de [1,2,3]-triazóis 1,4-dissubstituídos. Uma segunda rota para a obtenção dos derivados triazólicos 1,4-dissubstituídos ligados à 1,4- naftoquinona foi proposta a partir da reação entre o 2-etinil-1,4-naftoquinona e azido composto, para isso, foi sintetizado o 2-(3-hidroxi-3metilbutinil)-1,4-naftoquinona. No entanto, a subseqüente reação de desproteção deste composto, assim como, a síntese do precursor 2-trimetilsilil-1,4-naftoquinona não funcionou. Todos os produtos obtidos de estruturas inéditas foram caracterizados por espectroscopia de RMN 1H e RMN 13C, análise elementar, LC-MS e infravermelho.