Synthesis And Antitumor Activity Of Naphthalimide Derivatives With Selective Inhibitory Effect In Non-small Cell Lung Cancer

The molecular modification based on lead compounds is an important research direction in medicinal chemistry.Naphthalimide derivatives have attracted much attention in antitumor drug research due to its special rigid planar structure.Many naphthalimide derivatives,such as Amonafide,have evaluated under clinical trials.However,it failed to come into the market due to its toxic side effects,solubility and drug resistance.In vivo,Amonafide is easily metabolized by the enzyme N-acetyltransferase 2（NAT2）to toxic derivative N-acetyl-Amonafide,which is the main reason for its toxic and side effects.Therefore,the modification of 3-arylamines in the naphthalamide ring may be the key of molecular design that it is really suitable for clinical use.In this study,three naphthalimide derivatives were synthesized by using 2/3/4-fluorobenzoyl chloride to modify 3-position aromatic amines of naphthalimide ring.Their antitumor activities and structure-activity relationship were studied in depth.Their targets and molecular mechanisms were preliminarily defined.These provide important theoretical basis and data reference for clinical research and drug preparation of naphthalimide derivatives.The specific research content is as follows:1.Three naphthalimide compounds MX-C2,MX-C3 and MX-C4 were synthesized from 3-position aromatic amines of naphthalimide ring modified by 2/3/4-fluorobenzoyl chloride.Their structures were characterized by nuclear magnetic resonance（NMR）,high resolution mass spectrometry（HR-MS）,high performance liquid chromatography（HPLC）and Fluorescence spectroscopy.2.The antitumor activities and structure-activity relationship of three compounds were studied in vitro.1)Inhibitory effects of MX-C2,MX-C3 and MX-C4 on the proliferation of tumor cells and normal cells were detected by MTT assay.The results showed that the compounds of MX-C2,MX-C3 and MX-C4 had selective inhibitory effects on lung cancer cell lines.Especially MX-C4 had the most significant selective inhibitory effect on wild type p53 lung cancer cell lines NCI-H460 and its IC₅₀ value was 1.39±0.31μM.However,the antitumor activity of the three compounds on p53-deficient lung cancer cell lines H1299 was not obvious and their IC₅₀values were more than 20μM.The toxicity and side effects on normal cell lines（HL-7702,LX2）were less than that of Amonafide,and their IC₅₀ values were more than 20μM.2)Subcellular organelle separation and mass spectrometry were used to analysis the ability of MX-C2,MX-C3 and MX-C4 to enter cells and their enrichment in mitochondria and cytoplasm.Morphology,apoptosis,Caspase-3 and ATP levels at corresponding time point of mass spectrometry were detected by other biological means.The results showed that the ability of MX-C2,MX-C3 and MX-C4 to enter cells was different,and the location of MX-C2,MX-C3 and MX-C4 in mitochondria and cytoplasm were different as well.The ability of MX-C2 to enter cells was weakly,and it is so instable in complex cellular environments that couldn’t be detected in mitochondria and cytoplasm.MX-C3 can enter the cytoplasm in 1 hour but takes longer to enter the mitochondria.While MX-C4 enters the cell in a shorter time,and it can directly enter the mitochondria.MX-C4 has the ability to target the mitochondria.After 48 hours,MX-C4 reaches saturation in the mitochondria before it escapes into the cytoplasm.3)The effects of MX-C2,MX-C3 and MX-C4 on the cell cycle of HCC-827 were examined by flow cytometry and Western blot.And the apoptosis of HCC-827 cells were also examined by morphological staining,Annexin V-FITC/PI double staining,mitochondrial membrane potential（JC-1）and Caspase-3 activity.The ATP levels in HCC-827 cells were measured by ATP detection kit.The results showed that the three compounds have different effects on the cell cycle arrest of HCC-827 cells.While MX-C4 can arrest the cell cycle of HCC-827 cell in a dose-dependent manner in S phase.However,MX-C2 and MX-C3 can arrest the cell cycle in S,G2 phase.Moreover,MX-C2,MX-C3 and MX-C4 can induce apoptosis through activating Caspase-3 mediated mitochondrial pathway in HCC-827 cells.MX-C2 can decrease the ATP level in a dose-dependent manner,while MX-C3 and MX-C4 can decrease the ATP level at low concentration,but increase it at high concentration in HCC-827 cells,which shows structure-activity difference obviously.3.The target of compound MX-C4 was studied preliminarily.1)MTT assay and Western blot were used to detect the relationship between the anti-tumor activity of MX-C4 and p53.The results showed that the anti-tumor effect of MX-C4 was related to p53,interfering with the expression of p53 could significantly reduce the sensitivity of MX-C4 to tumor cells.2)The interaction of MX-C4 and DNA was detected by DNA relaxation and Western blot,and the results showed that MX-C4 did not activate p53 by causing DNA damage.3)The interaction between MX-C4 and p-p53was analyzed by immunoprecipitation combined with mass spectrometry（Co-IP-MS）,the results showed that MX-C4 could directly interact with p53.4)Immunofluorescence and subcellular organelle separation combined with western blot were used to determine whether MX-C4 could promote the release of AIF and cytochrome C,as well as its effect on the expression level of Bcl-2 family proteins.The results showed that MX-C4 induced apoptosis through a non-AIF-dependent signaling pathway.5)Immunofluorescence was used to detect the effect of MX-C4 on Bak conformation activation,and the distribution of p-p53 and Bak in mitochondria and cytoplasm was detected by Western blot.The results showed that MX-C4 could induce Bak conformation activation,and p-p53 or Bak accumulated in mitochondria in a dose-dependent manner.6)The interaction between Bcl-xl and Bak,and the interaction between Bcl-xl and p-p53 were detected by Co-IP.The results showed that MX-C4 could activate Bak conformation and induce apoptosis by reorganizing Bak-Bcl-xl complex.