Development And Properties Of Aromatic Nitrogen Mustard-photosensitizer Conjugates

Objective:To explore the novel single-molecule anti-tumor leading compounds of chemotherapy-photodynamic therapy and explore their in vivo and in vitro properties.Method:Aromatic nitrogen mustard-（benzylidene）cycloalkanone conjugates were prepared by the Cross-Aldol condensation reaction of aromatic nitrogen mustard with bis-（benzylidene）cycloalkanone.Using phthalic anhydride,3-diethylaminophenol,cyclohexanone and aromatic nitrogen mustard as raw materials to develop rhodamine conjugations by Friedel-Crafts acylation,Cross-Aldol condensation reaction,etc.Their structures were characterized by modern analytical methods,such as ¹H NMR,¹³C NMR,FT-IR,high resolution mass spectrometry and X-ray diffraction.MTT assay was used to investigate the darktoxicity and phototoxicity of the conjugates on A549（human lung adenocarcinoma）,HepG2（human liver cancer）and B16F10（mouse melanoma）cell lines.The optical properties were investigated by UV-Vis spectrophotometer and fluorescence spectrophotometer.The aromatic nitrogen mustard-（benzylidene）cyclobutanone conjugates and their contrast molecules were calculated at the B3LYP and 6-311G+level with Gaussian09 software.For aromatic nitrogen mustard-rhodamine conjugates:the produce of singlet oxygen in vitro was investigated by DPBF method,and the properties of mitochondrial targeting and near-infrared fluorescence imaging were studied by Fluorescence inverted microscope on HepG2 cells and B16F10 cells.Two methods of tail vein injection and tumor site injection were applied to investigate the synergistic antitumor activity and fluorescence imaging performance of the aromatic nitrogen mustard-rhodamine conjugations on the C57 mice with B16F10 tumor model,and the H&E staining method（heart,liver,spleen,lung,kidney）and serum biochemical indicators（liver function and renal function）were used to test the safety of conjugates in vivo.Results:It successfully developed three aromatic nitrogen mustard-（benzylidene）cyclobutanone conjugates and six their contrast molecules,and one aromatic nitrogen mustard-rhodamine conjugate and two its contrast molecules,which were confirmed by modern analytical method.In vitro performance tests showed that the conjugates showed high phototoxicity to the tested cell lines and had synergistic effect of chemotherapy-photodynamic therapy.Quantum chemical calculations indicated that the nitrogen mustard-（benzylidene）cyclobutanone conjugate and the contrast molecule had similar frontier orbitals and similar energy differences.The in vitro optical properties confirmed that the aromatic nitrogen mustard-rhodamine conjugate had a fluorescence emission wavelength in the near-infrared region and had good fluorescence quantum yield and well singlet oxygen production.Fluorescence inverted microscopy confirmed that the aromatic nitrogen mustard-rhodamine conjugate had mitochondrial targeting in HepG2 and B16F10 cells,and real-time near-infrared fluorescence imaging in B16F10 cells.Both of two injections ways showed that the aromatic nitrogen mustard-rhodamine conjugate had chemotherapy-PDT synergistic anti-tumor effect and real-time near-infrared fluorescence imaging in vivo,and had no obvious toxicity to major organelles,liver function and renal function.Conclusion:The developed aromatic nitrogen mustard-（benzylidene）cyclobutanone conjugates and aromatic nitrogen mustard-rhodamine conjugate have chemotherapy-p hototherapy synergistic anti-tumor effects.Aromatic nitrogen mustard-rhodamine conj ugate also shows mitochondria-targeted drug delivery,near-infrared fluorescence ima ging,and in vivo biosafety,which provides new design ideas and research foundatio n for developing new single-molecule antitumor drugs of chemotherapy-phototherapy synergistic dual therapy.