Berlinck, R.G.S., Britton, R., Piers, E., Lim, L., Roberge, M., Rocha, R.M. and Andersen, R.J., “Granulatimide and Isogranulatimide, Aromatic Alkaloids with G2 Checkpoint Inhibition Activity Isolated from the Brazilian Ascidian Didemnum granulatum: Structure Elucidation and Synthesis, Journal of Organic Chemistry, 63, 9850-9856 (1998). Baixe o arquivo aqui.

Abstract: Crude methanol extracts of the ascidian Didemnum granulatum collected in Brazil showed activity in a new screen for G2 cell cycle checkpoint inhibitors. Bioassay-guided fractionation of the extract yielded the known alkaloids didemnimides A (1) and D (2), the new alkaloid didemnimide E (3), and a new G2 checkpoint inhibitor. Two candidate structures for the inhibitor, named granulatimide (4) and isogranulatimide (5), have been prepared via a short and efficient biomimetic synthesis involving the photolysis of didemnimide A (1). The synthesis revealed that the correct structure for the naturally occurring G2 checkpoint inhibitor is isogranulatimide (5). Granulatimide (4), the other candidate structure, was also found to be a G2 checkpoint inhibitor and it was subsequently detected in chromatographic fractions associated with purification of D. granulatum alkaloids. Granulatimide (4) and isogranulatimide (5) represent the first examples of a new class of G2 specific cell cycle checkpoint inhibitors and the first ones identified through a rational screening program.

Figura V.1 (a) Inibição do ponto de checagem G2 do ciclo celular exibida pela granulatimida, e (b) indução de morte celular em células sãs (p53+) e em células mamárias tumorais MCF-7 desprovidas do ponto de checagem G1 (p53-) quando da aplicação simultânea de diferentes intensidades de radiação g (em Gy) (Roberge et al., 1998).

Roberge, M., Berlinck, R.G.S., Xu, L., Anderson, H., Lim, L.Y., Curman, D., Stringer, C.M., Friend, S.H., Davies, P., Vincent, I., Haggarty, S.J., Kelly, M.T., Britton, R., Piers, E. and Andersen, R.J., “High Throughtput Assay for G2 Checkpoint Inhibitors and Identification of the Structurally Novel Compound Isogranulatimide”, Cancer Research, 58, 5701-5706 (1998). Baixe o artigo aqui.

Abstract: Treatment of cancer cells lacking p53 function with G2 checkpoint inhibitors sensitizes them to the toxic effects of DNA damage and has been proposed as a strategy for cancer therapy. However, few inhibitors are known, and they have been found serendipitiously. We report the development of a G2 checkpoint inhibition assay that is suitable for high-throughtput screening and its application to a screen of 1300 natural extracts. We present the isolation of a new checkpoint inhibitor, the structurally novel compound isogranulatimide. In combination with g-irradiation, isogranulatimide selectively kills MCF-7 cells lacking p53 function.

Camargo, A.J., de Oliveira, J.H.H.L., Trsic, M. and Berlinck, R.G.S., “Molecular Orbital Calculations, Experimental and Theoretical UV Spectra of Granulatimides and Didemnimides, Biologically Active Polycyclic Heteroaromatic Alkaloids from the Ascidian Didemnum granulatum”, Journal of Molecular Structure, 559, 67-77 (2001). Baixe o artigo aqui.

Abstract: A detailed computational study was performed for compounds granulatimide, isogranulatimide, didemnimides A, D, and E, using the semiempirical Austin model 1 (AM1) quantum chemical method. The electronic features and structural parameters were confronted with the inhibition of G2 cell cycle checkpoint of mammalian cancer cells. All compounds were submitted to a rigorous conformational analysis using the Tripos 5.2 force field implemented in the Spartan 5.01 program. Electronic density in specific regions of the molecules appears to play a pivotal role towards activity. The molecular planarity creates a broad negative electrostatic potential on the two sides of the active compounds (granulatimide and isogralulatimide) and a positive potential in their central core, while the non-planar compounds (didemnimides A, D, and E, which are inactive) present an asymmetric potential scattered over the molecules. These electrostatic potential features are likely to be the modulator of hydrophobicity or lipophilicity of the compounds, which appear correlated with activity. The hydrogen attached to the N atom of the pyrrole moiety of indole is more positive for active compounds than for the inactive molecules. The theoretical electronic spectra were obtained for all compounds using the configuration interaction method, with the AM1 routine. All transitions present p ® p * nature. The theoretical results are in good agreement with experimental values.

Superfícies de energia de potencial eletrostático molecular sobrepostos à densidade eletrônica total (0.002 e/au3) para os alcalóides de D. granulatum. As cores indicam -60 kcal/mol (vermelho intenso) to +46 kcal/mol (azul intenso).

Britton, R., de Oliveira, J.H.H.L., Andersen, R.J. and Berlinck, R.G.S., “Granulatimide and 6-Bromogranulatimide, Minor Alkaloids of the Brazilian Ascidian Didemnum granulatum”, Journal of Natural Products, 64, 254-255 (2001). Baixe o artigo aqui.

Abstract: Re-investigation of the extract of the ascidian Didemnum granulatum collected on the Brazilian coastline led to the isolation of two minor compounds, granulatimide (2) and 6-bromogranulatimide (3), which have been identified by analysis of their spectroscopic data. The isolation of 2 and 3 from D. granulatum corroborates previous assumptions about the occurrence of granulatimide as a natural product.