Benzo [a] phenoxazinium chlorides: Synthesis, antifungal activity, in silico studies and evaluation as fluorescent probes

Four new benzo [a] phenoxazinium chlorides with combinations of chloride, ethyl ester and methyl as terminals of the amino substituents were synthesized. These compounds were characterized and their optical properties were studied in absolute dry ethanol and water. Their antiproliferative activity was tested against Saccharomyces cerevisiae in a broth microdilution assay, along with an array of 36 other benzo[a]phenoxazinium chlorides. Minimum Inhibitory Concentration (MIC) values between 1.56 and > 200 tM were observed. Fluorescence microscopy studies, used to assess the intracellular distribution of the dyes, showed that these benzo[a]phenoxazinium chlorides function as efficient and site specific probes for the detection of the vacuole membrane. The added advantage of some of the compounds, that displayed the lower MIC values, was the simultaneous staining of both the vacuole membrane and the perinuclear membrane of endoplasmic reticulum (ER). Molecular docking studies were performed on the human membrane protein oxidosqualene cyclase (OSC), using the crystal structure available on PDB (code 1W6K). The results showed that these most active compounds accommodated better in the active sites of ER enzyme OSC suggesting this enzyme as a potential target. As a whole, the results demonstrate that the benzo [a] phenoxazinium chlorides are interesting alternatives to the available commercial dyes. Changes in the substituents of these compounds can tailor both their staining specificity and antimicrobial activity.
Thanks are due to the Fundacao para a Ciencia e Tecnologia (FCT, Portugal) for financial support to the NMR portuguese network (PTNMR, Bruker Avance III 400-Univ. Minho), FCT and FEDER (European Fund for Regional Development)-COMPETE-QREN-EU for financial support to Research Centres CQ/UM [PEst-C/QUI/UI0686/2013 (FCOMP-01-0124-FEDER-037302)], CBMA (project PEst-OE/BIA/UI4050/2014, and UID/BIA/04050/2019) and CFUM [PEst-C/FIS/UI0607/2011(F-COMP-01-0124-FEDER-022711)]. Post-doctoral grant to B. R. Raju (SFRH/BPD/62881/2009) is also acknowledged to FCT, POPH-QREN, FSE. We are also thankful to J. Nosek from the Department of Biochemistry, Faculty of Natural Sciences, Comenius University in Bratislava, Slovakia, for giving the strain S. cerevisiae W303-1A-pDF01-VBA1-YEGFP. To B. Westermann from Department of Biology, Faculty of Biology, Chemistry and Earth Sciences, University of Bayreuth, Germany, for the strain S. cerevisiae W303-1B-pYX232mtGFP. By providing the strain S. cerevisiae BY4741-SEC66-GFP we are thankful to E. O'Shea from Department of Molecular and Cellular Biology, Faculty of Arts and Sciences, Harvard University, Massachusetts, U.S.A. Finally, we would like to acknowledge to P. Coutinho from the Department of Physics, School of Sciences, University of Minho, Portugal, for kindly provide the salmon sperm DNA used in this work.