【作者】 叶平；

【导师】 崔恒敏；

【作者基本信息】 四川农业大学 ， 基础兽医学， 2016， 博士

【摘要】 烷胺类药物主要指含有烷基胺结构的药物,其药理作用研究主要集中在机体免疫增强、促生长、抗疲劳、抗应激反应等方面的单独研究,未见免疫增强和抗疲劳作用同时进行的研究报道,其作用机制也不完全清楚,被认为是药物自身的独特结构具有的特定药理效应,也未揭示烷胺类结构所起的关键性作用。具有免疫增强或抗疲劳作用的中药含有烷胺类化学成分,这类化学成分很少被单独分离出来进行药理作用研究,缺乏相关报告,而这类化学成分具有潜在开发成新型烷胺类药物的实际应用价值。在此基础上,我们查阅了大量有关烷胺类药物和含有烷胺类化学成分中药的相关药理、临床应用研究文献,并进行了整理、分析和归纳,我们认为烷胺类化学成分同时具有抗疲劳及免疫增强双重作用,为验证这一推断,通过化学方法成功合成了二种新型烷胺类药物,即丁基异丙基胺、苯甲胺基异丙基膦酸。玛咖酰胺作为烷胺类化学成分,近几年其药理作用研究取得一定进展,故选择玛咖酰胺进行了化学合成,并作为本研究的阳性对照药物。以小鼠为实验动物,研究证实二种新型烷胺类药物属低毒性药物,并同时具有抗疲劳和免疫增强等药理作用。研究结果如下:试验一二种新型烷胺类药物的化学合成一锅法化学合成了二种新型烷胺类药物:丁基异丙基胺、苯甲胺基异丙基膦酸,并对合成工艺进行了研究。溴丁烷与异丙基胺在氢氧化钠条件下反应,合成丁基异丙基胺,收率84.6%。苯甲胺、亚磷酸、丙酮采用曼尼期反应原理,合成苯甲胺基异丙基膦酸,反应温度40℃,收率78.5%。此外,十六酸与苯甲胺反应,在碳化二亚胺及1-羟基苯并三唑催化作用下,合成了阳性对照药物玛咖酰胺,收率71.2%。核磁共振法及红外光谱仪测定了二种新型烷胺类药物丁基异丙基胺、苯甲胺基异丙基膦酸和阳性对照药物玛咖酰胺的氢谱及碳谱和红外吸收图谱,进行结构分析和鉴定,确定获得了上述烷胺类药物。毛细管法、液体密度仪和阿贝折射仪进行了三种烷胺类药物的常规理化性质测定:丁基异丙基胺,淡黄色液体、沸点122-123℃、密度0.7409g.cm-3、不溶于水:苯甲胺基异丙基膦酸,淡黄色固体、熔点178℃、溶解度24.5g/100mL;阳性对照药物玛咖酰胺,白色或灰色固体,熔点85-87℃、不溶于水。试验二二种新型烷胺类药物的毒性实验急性毒性LD0测定,结果表明丁基异丙基胺LD50为2661mg/kg,苯甲胺基异丙基膦酸LD50为3495mg/kg,阳性对照药物玛咖酰胺LD50为2851mg/kg。亚急性毒性实验,二种新型烷胺类药物丁基异丙基胺、苯甲胺基异丙基膦酸及阳性对照药物玛咖酰胺以500mg/kg体重灌胃给药,1日1次,连续15d,并设空白对照组,进行临诊、大体和组织学观察。与空白对照组比较,丁基异丙基胺组和苯甲胺基异丙基膦酸组临诊和剖检观察未见异常;脾脏、肾脏和肝脏的组织学观察,也未见异常。结果表明二种新型烷胺类药物丁基异丙基胺、苯甲胺基异丙基膦酸在给药期间是安全的,属低毒性药物。试验三二种新型烷胺类药物的抗疲劳作用研究以小鼠为研究对象,丁基异丙基胺、苯甲胺基异丙基膦酸分高低剂量组,分别为100mg/kg、200mg/kg;玛咖酰胺设为阳性对照组,剂量为50mmg/kg,生理盐水组设为空白对照。灌胃给药,1日1次,连续7d。以蒽酮法、ELISA、应激实验、交配能力测试和减肥实验等方法检测了抗疲劳相关指标。与阳性对照和空白对照比较,丁基异丙基胺、苯甲胺基异丙基膦酸可延长游泳及负重游泳时间;提高血糖、肝糖、肌肉和肝脏ATP贮备水平,降低力竭运动后血清尿素氮、乳酸含量及乳酸脱氢酶活性,其中糖原和ATP贮备水平的提高,是二种新型烷胺类药物抗疲劳及体能增强药理作用的生化基础;增强在极端冷热应激、缺氧应激条件下的存活能力和抗应激反应的能力;缩短爬背潜伏期和交配潜伏期,增加交配开始1h内的交配次数,雄性小鼠交配能力提高,性功能增强;降低肥胖模型小鼠Lee’s指数和提高血清瘦素含量,具有减肥作用。试验四二种新型烷胺类药物的免疫增强作用研究以小鼠为研究对象,丁基异丙基胺、苯甲胺基异丙基膦酸分高低剂量组,分别为100mg/kg、200mg/kg;玛咖酰胺设为阳性对照组,剂量为50mg/kg,生理盐水组设为空白对照。灌胃给药,1日1次,连续7d。以碳粒廓清实验、腹腔巨噬细胞吞噬实验、淋巴细胞玫瑰花环实验、脾脏淋巴细胞转化实验、流式细胞术、ELISA和PCR等方法观测了细胞免疫和体液免疫相关指标。与阳性对照和空白对照比较,丁基异丙基胺、苯甲胺基异丙基膦酸可提高脾脏和胸腺脏器指数,促进免疫器官发育;增强巨噬细胞吞噬功能,提高吞噬指数和吞噬率,增高T淋巴细胞活性以及脾脏淋巴细胞转化率,细胞免疫力增强;提高血清及脾脏IgA、IgG、IgM含量增高,体液免疫力增强;增高脾脏和血清 IL-2、IL-6、IL-10、IL-12、IFN-γ和 TNF-α 含量,上调脾脏 IL-2、IL-6、IL-10、IL-12、IFN-γ和TNF-α的mRNA表达。脾脏细胞因子的mRNA表达上调,是细胞因子含量增高的分子基础。结论和创新:1.三种烷胺类药物的化学合成成功,工艺可行,其中丁基异丙基胺、苯甲胺基异丙基膦酸为新合成烷胺类药物;2.二种新型烷胺类药物属低毒性烷胺类药物;3.二种新型烷胺类药物同时具有抗疲劳及免疫增强的双重作用。更多还原

【Abstract】 Alkylamine drug is a kind of drug-contained alkylamine groups including chemical drugs and traditional Chinese medicine.At present,there are no reports to show that alkylamine drugs play roles simultaneously in both anti-fatigue and immune enhancement.Based on the analysis of references about pharmacological action,clinic application of alkylamine drugs and traditional Chinese medicine-contained alkylamine groups,it can be concluded that alkylamines have roles simultaneously in both anti-fatigue and immune enhancement.In order to validate the above-mentioned conclusion,two new type alkylamines including N-butyl-N-isopropylamine and benzyl amine isopropyl phosphonic acid were synthesized by chemical method.In recent years,the pharmacological action of macamides has been observed and thus,macamides,as a positive control drug,was chemically synthesized in our experiment.The mice were used as the experimental animals to confirm that two new type alkylamine drugs were low toxic and have roles simultaneously in both anti-fatigue and immune enhancement.The results were as follows:Experiment 1 Chemical synthesis of two new type alkylamine drugsTwo new type alkylamines,e.g.,N-isopropyl-N-butylamine and(2-(benzylamino)propan-2-yl)phosphonic acid,were chemically synthesized by one pot method.N-isopropyl-N-butylamine was synthesized by the reaction of bromobutane with isopropylamine in alkaline conditions,and its yield was 84.6%.(2-(benzylamino)propan-2-yl)phosphonic acid was synthesized with benzylamine,phosphorous acid and acetone by the mannes reaction principle,and its yield was 78.5%.Additionally,under the catalysis of 1-hydroxybenzotriazole and carbodiimide,macamides was synthesized with hexadecanoic acid and benzylamine,and its yield was 71.2%.Two new type alkylamine drugs were identified by mass spectrometry and IFNrared spectroscopy.Also,the conventional physical and chemical properties of two new type alkylamine drugs were determined by capillary tube method,liquid density measuring apparatus and Abbe refractometer.The results showed that N-isopropyl-N-butylamine was light yellow liquid and was insoluble in water,and its boiling point and density were 122-123 ℃ and 0.7409g/cm3.(2-(benzylamino)propan-2-yl)phosphonic acid was light yellow solid,and its melting point and solubility were 178 ℃ and 24.5g/100mL.Macamides was white or gray solid and was insoluble in water,and its melting point was 85-87 ℃.Experiment 2 Toxicity of two new type alkylamine drugsLD50 of acute toxicity was measured by intragastrical administration in mice.LD50 of N-isopropyl-N-butylamine was 2661 mg/kg,LD50 of(2-(benzylamino)propan-2-yl)phosphonic acid was 3495 mg/kg,and LD50 of macamides was 2851 mg/kg.In the subacute toxicity,500mg/kg body weight intragastric administration with continuous administration of 15 days(once a day)showed no abnormal changes of the clinical and gross observation.Histologically,no abnormal changes were also found in the liver,spleen and kidney of the two new type alkylamine groups when compared to control groups.Experiment 3 Anti-fatigue test of two new type alkylamine drugs in miceThe mice were treated with N-isopropyl-N-butylamine and(2-(benzylamino)propan-2-yl)phosphonic acid with 100 mg/kg and 200 mg/kg,respectively.The positive control group was set with macamides by 50mg/kg.The normal saline group was set as the blank control group.The experiment was performed by intragastrical administration with 1 day 1 time,continuous 7d.Parameters of anti-fatigue were detected by using methods of anthrone-sulfate,ELISA,stress test,male sexual function test and weight loss test.The results showed that two new type alkylamine drugs,e.g.,N-isopropyl-N-butylamine and(2-(benzylamino)propan-2-yl)phosphonic acid made mice prolong swimming time,and improve the survival ability under extreme cold,heat stress and hypoxia stress,and improve mating ability,shorten the latent period of mating,and increase mating frequency within 1h from the beginning of mating when compared to control groups.The blood glucose and hepatic glycogen contents,and muscle ATP and hepatic ATP levels were increased,and the serum urea nitrogen contents,lactic acid contents and lactate dehydrogenase activity after exhaustive exercise were decreased in the two new type alkylamine drugs groups.The improvement ofglycogen and ATP contents was the basis of pharmacological action of the two new type alkylamine drugs in anti-fatigue.At the same time,the Lee’s index was lower and serum leptin contents were higher in two new type alkylamine drugs groups than those in control groups.Experiment 4 Immune enhancement of two new type alkylamine drugsThe methods were as same as those in anti-fatigue study.The immune parameters were detected by using methods of carbon clearance test,macrophage phagocytosis test,lymphocyte E-rosettes assay,T lymphocyte transformation test,flow cytometer,ELISA and PCR.When compared to control groups,the two new type alkylamine drugs could increase the under-mentioned immune indexes,e.g.,splenic and thymic growth indexes which showed that the two new type alkylamine drugs could improve splenic and thymic development,and macrophage phagocytosis function,T lymphocyte activity,splenic lymphocyte transformation rate,spleen and serum IgA,IgG,IgM contents,and splenic and serum IL-2,IL-6,IL-10,IL-12,IFN-y and TNF-a contents.Also,the mRNA expression of cytokines in the spleen was up-regulated,which was the molecular basis of the increased cytokine contents.Conclusions and innovationsFirstly,the two new type alkylamine drugs,e.g,N-butyl-N-isopropylamine and benzyl amine isopropyl phosphonic acid,are successfully synthesized by chemical method.Secondly,the two new type alkylamine drugs are low toxic.Last,the two new type alkylamine drugs have roles simultaneously in both anti-fatigue and immune enhancement.The above-mentioned conclusions and innovations are firstly reported at home and abroad.更多还原