Synthesis Of Azeliragon Analogues And Isolation As Well As Anti-inflammatory Activity Of Polysaccharides From Nervilia Fordii

The first part of the thesis is the design,synthesis and in vitro activity evaluation of the receptor for advanced glycation end products（RAGE）inhibitor Azeliragon triazole analogues.RAGE is an immunoglobulin on the cell membrane,which is a pattern recognition receptor.It has been proved to be closely related to the occurrence and development of many chronic diseases such as Alzheimer’s disease,inflammation,and tumors.Inhibiting the combination of excessive activation of RAGE and brain Aβcan reduce neuroinflammation,thereby delaying neurodegenerative diseases and reducing damage to the nervous system.Azeliragon’s mechanism of action is to reduce the entry of Aβinto the brain and at the same time reduce the inflammation in the brain.It is currently known as the drug with the best comprehensive curative effect on RAGE,but the results of clinical trials show that it has a certain degree of toxicity and is anti-neuro-inflammation.Activity is not very good.It is necessary to optimize its structure to obtain compounds with high efficiency and low toxicity.We replaced the imidazole in the center of Azeliragon’s structure with triazole,and kept the 2 phenyl groups attached to the center of the structure,and performed corresponding group substitutions or structure for the 3 substituents on the triazole and 2 phenyl groups.A total of 35 new compounds were synthesized,and their structures were characterized by nuclear magnetic resonance（NMR）as well as high resolution mass spectrum（HRMS）,and their anti-neuro-inflammatory activities were tested.The results show that some compounds have a better protective effect on BV-2 cells under the action of Aβ_1-42,and significantly increase the cell survival rate.Among them,the cell survival rate of KC-48reaches 81.80±5.34%,which is comparable to the control group.There is a significant difference than.The effect of the compound on the production of TNF-αand IL-6 by BV-2 cells under the action of Aβ1-42.The experimental results suggest that some compounds bind to RAGE and prevent Aβ_1-42 from killing cells,TNF-αand IL-6.The release amount increased,and was closer to the blank group than the value of the Aβ_1-42 model group.In addition,RAGE is closely related to the occurrence,development,and metastasis of breast cancer.Blocking RAGE is of great significance to the prevention and treatment of breast cancer.In vitro activity experiments show that a total of 13 Azeliragon analogs have good growth inhibitory effects on the human triple-negative breast cancer cell line SUM149,with IC50 values ranging fromμM to n M,and the most active KC-37 is 0.220±0.034μM;the other compounds have lower cytotoxic activity,and the inhibition rate at the maximum test concentration（10μM）is less than 50%.The anti-breast cancer structure-activity relationship suggests that the substituent R1 is very sensitive to the length of the carbon chain and the charge distribution,and only n-butyl substitution has better activity.The diethylamine propoxy group substituted by R2 is an important group.The commonality of R3 is that most of the aromatic rings are connected with strong electron withdrawing groups.Combining the results of pharmacological experiments with the calculated data of the molecular properties of the compound,compounds with higher activity can be found has a large number of rotatable keys.It is possible that these compounds are more"softer"and can better fit the active pockets of RAGE ligands.The specific reasons need to be further explored.The second part of the thesis is the separation,purification,structure analysis and in vitro anti-inflammatory activity test of two homogeneous polysaccharides of Nervilia fordii.Nervilia fordii is a common medicine for clearing away heat and detoxification with the same folk medicine and food in Guangdong,Guangxi and Southeast Asia.It is often used clinically for the treatment of pulmonary inflammatory diseases,but the material basis of its medicinal effect has not yet been elucidated.We have separated and purified two homogeneous polysaccharides from Nervilia fordii.One of them is protein-bound polysaccharide NFP-A4,and one is water-soluble polysaccharide NFP-1.The purity and physicochemical properties analysis,structural identification and in vitro immunomodulatory activity research were carried out.The results show that the molecular weight of NFP-A4 is about 980 KDa and the purity is 98.5%.NFP-1 has a molecular weight of 950 k Da and a purity of97.8%.NFP-A4 is composed of galacturonic acid（Gal A）,galactose（Gal）,glucose（Glc）,arabinose（Ara）,glucuronic acid（Glc A）,rhamnose（Rha）and mannose（Man）.The molar ratio is 3.9:3.7:3.6:2.8:2.1:1.3:1 and contains trace amounts of xylose（Xyl）.The types and ratios of monosaccharides mainly contained in NFP-1 are:arabinose:galactose:rhamnose:galacturonic acid=2.1:2.0:1.0:0.4.It also contains a small amount of mannose,glucose and glucuronic acid.The neutral polysaccharide content of NFP-A4 is 77.67%,the uronic acid content is 11.56%,and the protein content is 12.83%.The main connection types of the glycosyl are→3,4-Gal-1→,→3-Glc-1→,→3-Ara-1→,and→4,6-Gal-1→.The D-galactose in NFP-1 has two glycosidic bond connection methods:T-β-Galp-（1→and→4）-β-Galp-（1→,T represents the terminal of the sugar chain.At the same time,NFP-The D-arabinose in 1 has two glycosidic bond connection methods:→5）-α-Araf-（1→and→3）-α-Araf-（1→.Methylation and GC-MS analysis results indicate that,Rhamnose also has two glycosidic bond connection methods:→2）-α-Rhap-（1→and→2,4）-α-Rhap-（1→.The structural unit of NFP-1 can be determined as→4）-α-Rhap-（1→4）-β-Galp-（1→）.（2→4）-α-Galp A-（1→2）-α-Rhap-（1→2）-α-Rhap-（4→1）-β-Galp-T and two branched chains→2,4)-α-Rhap-（1→5）-α-Araf-（1→3）-α-Araf-（1→,and→2,4）-α-Rhap-（1→4）-β-Galp-(1→.In vitro anti-inflammatory activity experiments show that when the concentration of these two polysaccharides is lower than the highest tested dose,there is no cytotoxicity to macrophages.Compared with the control group,the levels of pro-inflammatory mediators such as TNF-α,IL-6,IL-1β,IL-10 and NO released by RAW264.7 macrophages activated by LPS（1μg/m L）significantly increased（P<0.01）.After that,the release of TNF-α,IL-6,IL-1βand NO from RAW264.7 macrophages activated by LPS treated with different concentrations of NFP-1 were significantly lower than that of the LPS control group.And the IL-10 release of RAW264.7 macrophages activated by LPS treated with different concentrations of NFP-1 was significantly higher than that of the LPS control group.The experimental results confirm that NFP-1 has the potential for immune regulation,especially the regulation of inflammation.In addition,compared with the control group containing only NFP-A4,the secretion of NO and the production of TNF-αand IL-6 were significantly reduced in the test group with anti-TLR4.Compared with the control group containing only NFP-A4,the secretion of NO and the production of TNF-αand IL-6 were significantly reduced in the test group using TAK-242 and NFP-A4.However,no significant changes were found in the test group using other antibodies.The results show that TLR4 is a pattern recognition receptor that NFP-A4 binds to macrophages.NFP-A4 can significantly increase the phosphorylation of JNK,ERK and p38in a concentration-dependent manner.Compared with the NFP-A4 test group,the phosphorylation of JNK,ERK and p38 was significantly down-regulated.In the test group containing NFP-A4 in combination with the JNK inhibitor SP600125,the ERK inhibitor PD98059 or the p38 inhibitor SB20350,NO,TNF-α,IL-6 is significantly reducedThe activation of macrophages by NFP-A4 is related to the MAPK signaling pathway.Here we propose a possible mechanism of action:NFP-A4 interacts with the TLR4 receptor on macrophages,and initiates the downstream signaling cascade through the MAPK signaling pathway,and ultimately regulates the secretion of NO and the production of certain cytokines.In vitro studies have shown that NFP-A4 can regulate the immune response of macrophages with or without LPS stimulation.That is to say,these two polysaccharides have dual functions of immune regulation.