Synthesis, theoretical calculation and evaluation of the tyrosinase activity of isobenzofuranones

Ftalidas e seus derivados são um grupo de metabólitos secundários que apresentam bioatividades variadas, tais como anti-inflamatória, antibacteriana, inseticida, anticâncer, fitotóxica e fungicida. Uma subclasse de ftalidas que possui bioatividade inclui as tetraidroisobenzofuranonas. Considerando a potencial aplicação biológica dessa classe de compostos, novos derivados de isobenzofuranonas foram sintetizadas. A rota sintética escolhida para a síntese teve como material de partida a (S)-5-(hidroximetil)furan-2(5H)-ona (6) obtida em seis etapas a partir do D-manitol. A essa lactona (6) foram feitas reações com anidrido acético e cloreto de benzoíla, produzindo o acetato de (S)-(5-oxo- 2,5-diidrofuran-2-il)metila (7) e o benzoato de (S)-(5-oxo-2,5-diidrofuran-2- il)metila (8), respectivamente. A reação de Diels Alder entre o ciclopentadieno e as -lactonas ,-insaturadas (6, 7 e 8) foi escolhida como a etapa chave para a obtenção dos derivados de isobenzofuranonas. Foram obtidos os adutos (3S,3aS,4R,7S,7aR)-3-(hidroximetil)-3a,4,7,7a-tetraidro-4,7-metanoisobenzo- furan-1-(3H)-ona (9a), (3S,3aS,4S,7R,7aR)-3-(hidroximetil)-3a,4,7,7a-tetraidro- 4,7-metanoisobenzofuran-1(3H)-ona (9b), acetato de ((1S,3aR,4S,7R,7aS)-3- oxo-1,3,3a,4,7,7a-hexahidro-4,7-metanoisobenzofuran-1-il)metila (10a), acetato de((1S,3aR,4R,7S,7aS)-3-oxo-1,3,3a,4,7,7a-hexahidro-4,7-metanoisobenzo - furan-1-il)metila (10b), benzoato de ((1S,3aR,4R,7S,7aS)-3-oxo-1,3,3a,4,7,7a- hexahidro-4,7-metanoisobenzofuran -1-il)metila (11a) e benzoato de ((1S,3aR,4R,7S,7aS)-3-oxo-1,3,3a,4,7,7a-hexahidro-4,7-metanoisobenzofuran- 1-il)metila (11b) com rendimentos acima de 82%. Nos adutos 9a, 9b, 10a e 11a foram realizadas reações de hidrogenação catalítica e epoxidação, sendo obtidos 8 adutos modificados com rendimentos que variaram de 65 a 98%. Além disso, modificações na hidroxila do composto 9a também foram realizadas, obtendo-se os compostos ((1S,3aR,4S,7R,7aS)-3-oxo-1,3,3a,4,7,7a- hexaidro-4,7-metanoisobenzofuran-1-il)metil-metanosulfonato (20), (3S,3aS,4R,7S,7aR)-3- (azidometil)-3a,4,7,7a-tetraidro-4,7-metanoisobenzofuran-1(3H)-ona (21) e (3S,3aS,4R,7S,7aR)-3-(bromometil)-3a,4,7,7a-tetraidro -4,7-metanoisobenzo- furan-1(3H)-ona (22). Os compostos sintetizados foram caracterizados por espectrometria de massas, espectroscopia no IV e de RMN de 1 H e de RMN de 13 C, além das técnicas de RMN bidimensionais: COSY, NOESY, HSQC e HMBC. Adicionalmente, cálculos computacionais (DFT) foram utilizados para a elucidação estrutural de dois dos novos epóxidos sintetizados, (1aR,2R,2aR,5S,5aS,6S,6aS)-5-(hidroximetil)hexa-hidro-2,6-metano-oxireno [2,3-f]isobenzofurano-3(1aH)-ona (16) e (1aS,2S,2aR,5S,5aS,6R,6aR)]-5- (hidroximetilo)hexa-hidro-2,6-metanooxireno[2,3-f]isobenzofurano-3(1aH)-ona (17). Além das análises dos espectros de RMN 2D, as atribuições das configurações relativas das estruturas dos epóxidos também foram inferidas a partir de informações obtidas por cálculos teóricos. Como consequência da síntese enantiosseletiva a partir de um poliol natural, as configurações absolutas dos epóxidos 16 e 17 também foram definidas. Por fim, para avaliar a atividade biológica, testes de inibição da enzima tirosinase utilizando os substratos L-tirosina e L-DOPA foram realizados com os 20 compostos sintetizados. Os compostos 8 e 11b apresentaram porcentagens de inibição superior a 50% tanto para a L-tirosina quanto para a L-DOPA e para esses compostos foram determinados os valores de IC 50 . O valor obtido para o composto 8 foi IC 50 = 4,23 μM e para o cicloaduto 11b o valor obtido foi IC 50 = 4,03 μM, enquanto que ácido kójico (controle positivo) apresentou IC 50 = 6,45 μM. Testes de viabilidade celular realizados com células de melanoma B16F10 demonstraram que os compostos 8 e 11b não possuem efeito citotóxico significativo nas concentrações avaliadas. Em seguida, foi avaliado o efeito inibitório dos compostos quanto ao teor de melanina nas células de melanoma B16F10. O composto 8 numa concentração de 50 μM diminuiu o teor de melanina em 23% enquanto que na concentração de 100 μM houve uma diminuição de 39%. Já a substância 11b apresentou inibição no teor de melanina nas concentrações de 50 μM e 100 μM, com porcentagens inibitórias de 25% e 45%, respectivamente. Com isso, os compostos avaliados 8 e 11b demonstraram ser promissores como agentes despigmentantes da pele, uma vez que apresentaram valores de IC 50 inferiores ao obtido para o ácido kójico, equilibraram baixa toxicidade e potente atividade inibitória do teor de melanina em células de melanoma B16F10.Phthalides and their derivatives are a group of secondary metabolites that exhibit varied bioativities, such as anti-inflammatory, antibacterial, insecticidal, anticancer, phytotoxic and fungicidal. A subclass of phthalides having bioactivity includes tetrahydroisobenzofuranones. Considering the potential biological application of this class of compounds, new isobenzofuranone derivatives have been synthesized. The synthetic route chosen for the synthesis was as a starting material (S)-5-(hydroxymethyl)furan-2(5H)-one (6) obtained in six steps from D-mannitol. Acetic anhydride and benzoyl chloride were reacted with lactone (6) to give (S)-(5-oxo-2,5-dihydrofuran-2-yl)methyl acetate (7) and (S)- (5-oxo-2,5-dihydrofuran-2-yl)methyl benzoate (8), respectively. The Diels Alder reaction between the cyclopentadiene and -unsaturated -lactones (6, 7 and 8) was chosen as the key step for the preparation of the isobenzofuranone derivatives. The adducts were obtained (3S3aS,4R,7S,7aR)-3-(hydroxymethyl)- 3a,4,7,7a-tetrahydro-4,7-methanoisobenzofuran -1(3H)-one (9a), (3S,3aS,4S,7R,7aR)-3-(hydroxymethyl)-3a,4,7,7a-tetrahydro-4,7-methanoiso- benzofuran-1(3H)-one (9b), ((1S,3aR,4S,7R,7aS)-3-oxo-1,3,3a,4,7,7a- hexahydro -4,7-methanoisobenzofuran -1-yl)methyl acetate (10a), ((1S,3aR,4R,7S,7aS)-3-oxo-1,3,3a,4,7,7a-hexahydro-4,7-methanoisobenzo - furan-1-yl)methyl acetate (10b), ((1S,3aR,4S,7R,7aS)-3-oxo-1,3,3a,4,7,7a- hexahydro-4,7-methanoisobenzofuran-1-yl)methyl benzoate (11a) and ((1S,3aR,4R,7S,7aS)-3-oxo-1,3,3a,4,7,7a-hexahydro-4,7-methanoisobenzo - furan-1-yl)methyl benzoate (11b) with yields above 82%. In the adducts 9a, 9b, 10a and 11a reactions of catalytic hydrogenation and epoxidation were carried out, and eight modified adducts were obtained in yields ranging from 65 to 98%. In addition, modifications to the hydroxyl of compound 9a were also performed to give the (1S, 3aR, 4S, 7R, 7aS) -3-oxo-1,3,3a, 4,7,7a-hexahydro-4,7- methanoisobenzofuran-1-yl)methylmethanesulfonate (20), (3S,3aS,4R,7S,7aR)- 3-(azidomethyl)-3a,4,7,7a-tetrahydro-4,7-methanoisobenzofuran-1(3H)-one (21) and the (3S,3aS,4R,7S,7aR)-3-(Bromomethyl)-3a,4,7,7a-tetrahydro-4,7- methanoisobenzofuran-1(3H)-one (22). The synthesized compounds were characterized by mass spectrometry, IR spectroscopy and 1 H NMR and 13 C NMR, as well as two-dimensional NMR techniques: COSY, NOESY, HSQC and HMBC. In addition, computational calculations (DFT) were used for the structural elucidation of two of the new synthesized epoxides (1aR,2R,2aR,5S,5aS,6S,6aS)-5-(hydroxymethyl)hexahydro-2,6-methano- oxireno[2,3-f]isobenzofuran-3(1aH)-one (16) and (1aS,2S,2aR,5S,5aS,6R,6aR)- 5-(hydroxymethyl)hexahydro-2,6-methanooxireno[2,3-f]isobenzofuran-3(1aH)- one (17). In addition to the analyzes of the 2D NMR spectra, the attributions of the relative configurations of the structures of the epoxides were also inferred from information obtained by theoretical calculations. As a consequence of the enantioselective synthesis from a natural polyol, the absolute configurations of epoxides 16 and 17 were also defined. Finally, to evaluate the biological activity, test of inhibition of the enzyme tyrosinase using the substrates L- tyrosine and L-DOPA were performed with the 20 compounds synthesized. Compounds 8 and 11b showed percentages of inhibition greater than 50% for both L-tyrosine and L-DOPA and for these compounds the IC 50 values. The value obtained for compound 8 was IC 50 = 4.23 μM and for cycloadduct 11b the value obtained was IC 50 = 4.23 μM, whereas kojic acid (positive control) showed IC 50 = 6.45 μM. Cell viability tests performed with B16F10 melanoma cells demonstrated that compounds 8 and 11b have no significant cytotoxic effect at the concentrations evaluated. Next, the inhibitory effect of the compounds on melanin content in B16F10 melanoma cells was evaluated. Compound 8 at a concentration of 50 μM decreased the melanin content by 23% while at the concentration of 100 μM there was a decrease of 39%. Substance 11b showed inhibition of melanin content at concentrations of 50 μM and 100 μM, with inhibitory percentages of 25% and 45%, respectively. Thus, the evaluated compounds 8 and 11b were shown to be promising as depigmenting agents of the skin, since they had lower IC 50 values than that obtained for kojic acid, low toxicity and potent inhibitory activity of melanin content in B16F10 melanoma cells.Coordenação de Aperfeiçoamento de Pessoal de Nível Superio