Studies On Preparation Of Quinolinone Derivatives And Its Effect Of Anti-Helicobacter Pylori

Helicobacter pylori are one of the pathogenic factors of chronic gastritis,peptic ulcer,and gastric cancer.There are About 4 billion people who have been detected with Helicobacter pylori infection around the world,While the infection rate of Helicobacter pylori is 54.76%in China.Existing anti-HP treatments that using high-dose multi-antibiotics combined with bismuth and PPI for 7-14 days,which often cause intestinal flora imbalance,multiple drug resistance,constipation,and other adverse reactions,resulting in poor patient compliance and low clearance rate.Therefore,screening safe and effective anti-HP drugs is still an urgent problem to be solved.In this study,the nucleus structure of 4-quinoline was taken as the research object to synthesize a new derivative,KLT-4,by acylation,condensation,and cyclization,etc.Besides,its structure confirmation,content determination method,and anti-Hp effect were studied in vitro and in vivo.The innovative results were as follows:1.Screening out the best route and conditions to synthesize KLT-3,the key intermediate of KLT-4,by a one-step method.The feeding ratio was 1:5（5-Hydroxyanthranilic Acid:Ethyl Hexyl Ketone）,the preferred solvent was DMF,microwave power was P-80W for 5min,and then P-100W for 10min.The yield was 68.35%.2.Finished the preparation of a new derivative（KLT-4）.the optimal conditions for the preparation of KLT-4 were summarized Through the experiments as follows:the feeding molar ratio was 1:1.1（KLT-3:sodium hydroxide）,the solvent was anhydrous methanol,the reaction was refluxed at room temperature for 1 hours,and the concentration was reduced under pressure.The milky white solid KLT-4 was prepared,and the yield was 98.58%.3.The structures of KLT-4 and KLT-2 were confirmed.1)Spectral data of KLT-4:ES-MS:[M+H]⁺=282,[M-Na]^-=258;¹H NMR（600MHz,DMSO）δ7.23–7.22（d,J=9.0 Hz,2H）,6.84–6.82（m,1H）,2.59–2.57（m,2H）,1.99（s,3H）,1.58–1.55（m,2H）,1.33–1.30（m,2H）,1.28–1.26（dd,J=9.0,5.1 Hz,4H）,0.87–0.84（t,J=7.0 Hz,3H）;¹³C NMR（151MHz,DMSO）δ173.41,158.13,152.65,126.65,123.27,122.35,110.13,107.32,99.99,34.69,31.71,29.43,29.40,22.58,14.44,11.80.The confirmed structure is:sodium2-hexyl-3-methyl-4-oxo-1,4-dihydroquinoline-6-olate,referred to as KLT-4.2)Spectral data of KLT-2:ES-MS:[M+H]⁺=260,[M-H]^-=258;¹H NMR（600MHz,DMSO）δ11.14（s,1H）,9.46（s,1H）,7.49–7.16（m,2H）,7.08（dd,J=8.9,2.7Hz,1H）,2.65（q,J=7.6 Hz,2H）,2.49–2.42（m,2H）,1.41（s,2H）,1.33–1.30（m,3H）,1.24（t,J=7.5 Hz,4H）,0.87（t,J=6.8 Hz,3H）.¹³C NMR（151 MHz,DMSO）δ175.88,153.27,150.08,133.53,125.05,121.71,119.43,117.26,107.86,32.12,29.58,25.16,24.92,22.59,14.53,14.45.The confirmed structure is3-hexyl-6-hydroxy-2-methylquinolin-4（1H）-one.4.A method for the determination of KLT-4 and related substances was established.The chromatographic conditions were optimized as follows:Welchrom（?）Ulultimate XP-Phenyl（4.6×250mm,5μm）was used as a column with methanol-water as mobile phase.The flow rate was 1 ml/min.The detection wavelength was set at245nm,sample size was 10μl,and column temperature was 25℃.The linear range of KLT-4 was 0.20-2.00μg/ml.The linear regression equation was y=3754.8x+8.8803（R²=1）.The content of KLT-4 in three batches of samples was more than 97.9%.The linear range of KLT-2 was 0.20-2.00μg/ml.The linear regression equation was y=4153.2x+131.8（R²=0.9991）.The average content of the three batches of samples was 2.165%.The above method has advantages of specificity,precision,accuracy,and simple operation,and is suitable for the determination of KLT-4 and KLT-2.5.Anti-Helicobacter pylori effect of KLT-4 in vitro and in vivo.The minimum inhibitory concentration and the minimum bactericidal concentration of KLT-4 were determined to be 0.12μg/ml and 0.24μg/ml by AGAR dilution method and microdilution method.It is preliminary confirmed that KLT-4 has a selective anti-Helicobacter pylori effect.The results of animal experiments showed that the clearance rate of HP in mice was dose-dependent,and the effect of equal dose of amoxicillin was the same as that of KLT-4.