AMPA受体调节剂的设计、合成及抗疲劳活性研究

发布时间：2018-04-28 09:22

本文选题：疲劳 + AMPA受体调节剂　；参考：《西北工业大学》2014年博士论文

【摘要】：目的为寻找新型、有效、安全的抗疲劳候选化合物，本课题以AMPA受体为作用靶点，设计、合成一系列新型苯甲酰胺类AMPA受体调节剂。对目标化合物进行理化性质研究及分子对接研究。通过药理活性筛选，优选出具有显著抗疲劳活性的候选化合物，并对候选化合物进行初步药代动力学和急性毒性评价，为开发具有自主知识产权的抗疲劳新药提供实验依据。方法设计的化合物以苯甲酰胺为结构母核，由芳环与含氮杂环通过酰胺键连接而成。芳环和含氮杂环上分别引入不同取代基，重点考察芳环3,4-位连亚甲二氧基，芳环上不同位置、数目的甲氧基，以及不同含氮杂环对活性的影响。采用的合成路线为活化酯法，以三氟乙酸N-琥珀酰亚胺酯为活化试剂与不同取代芳酸反应，制备含不同取代基的苯甲酸琥珀酰亚胺酯中间体，再与相应含氮杂环缩合制备目标化合物；采用乙醇扩散法，培养化合物单晶体；采用AutoDock4.0程序对得到单晶衍射数据的化合物A2和D2进行分子对接研究，考察化合物与AMPA受体的结合能力和结合模式；采用HPLC法测定各目标化合物的纯度及表观脂水分配系数（Log P）值。采用小鼠负重游泳和睡眠剥夺实验模型，以莫达非尼和咖啡因为阳性对照药，，对目标化合物的抗疲劳活性进行筛选。灌胃给予各化合物，各化合物给药剂量为0.2mmol/kg，连续给药7天后，进行负重游泳实验和睡眠剥夺实验，分别记录小鼠负重游泳时间和睡眠剥夺时间。采用全自动生化分析仪检测游泳后小鼠组织糖原含量及血清中乳酸、尿氮素、超氧化物歧化酶、谷胱甘肽过氧化物酶、过氧化氢酶、乳酸脱氢酶、肌酸激酶等疲劳相关血生化指标；采用放射性配体受体结合实验，测定目标化合物与AMPA受体的亲和力。对优选出具有显著抗疲劳活性的化合物A2，进行初步药代动力学评价，考察半衰期及生物利用度等药代动力学参数；采用急性毒性试验初步观察化合物的毒性反应，计算半数致死量（LD50）值。结果本论文合成了四个系列的苯甲酰胺类19个目标化合物，4个苯甲酸琥珀酰亚胺酯中间体及阳性对照药莫达非尼，共计24个化合物。所有化合物结构经核磁氢谱（1HNMR）、质谱（MS）、红外光谱（IR）及元素分析确证无误。经化学文摘（CA）网络版SciFinder数据库检索，反应路线、中间体及目标化合物除莫达非尼和A1外均未见相关报道；另得到化合物A、A1、A2和D2的单晶体，经X-ray单晶衍射分析，解析出晶体结构；分子对接结果显示A2和D2与AMPA受体具有较强的结合能力。测得各个目标化合物的纯度均在98%以上，符合动物实验要求，可用于药理学实验；测得目标化合物的Log P值在0.91到2.24之间，表明化合物脂水分配系数较为合适。各化合物在实验给药期间，对小鼠体重均无影响。小鼠负重游泳实验结果显示，化合物A2、C2、A1、D1可显著延长小鼠力竭游泳时间，提高运动耐力。小鼠睡眠剥夺实验结果显示，化合物A2、A1、C3、C1、D1可显著延长小鼠睡眠剥夺时间，表明以上化合物具有显著的抗疲劳活性。游泳后小鼠组织糖原及血生化指标检测结果显示，化合物A2、C2、A1对小鼠运动疲劳后糖原含量的降低具有显著的抑制作用，表明它们可通过降低糖原的消耗从而发挥抗疲劳效果。化合物A2、A1、C2对运动疲劳后小鼠血清中乳酸及尿氮素的升高具有显著的抑制作用，表明它们可通过降低疲劳代谢产物的堆积从而缓解疲劳，其中化合物A2抗疲劳效果最为显著；放射性配体受体结合实验结果显示，化合物A2与AMPA受体的解离常数（Kd）值为2.1106nmol/L，表明其与AMPA受体具有较高的亲和力。大鼠灌胃、静脉注射给药A2后，测定相应时间点血药浓度，经计算得半衰期分别为81.34min和82.57min，生物利用度为30.8%；小鼠单次灌胃及静脉注射给予A2，给药剂量范围内均未见明显毒性反应，灌胃给药的LD50值为1.3306g/kg，静脉给药的LD50值为0.6066g/kg，表明化合物A2的毒性较低。结论本课题以AMPA受体为作用靶点，设计合成了四个系列的苯甲酰胺类AMPA受体调节剂，共计24个化合物。化合物结构经1H NMR、MS、IR及元素分析表征确证无误。经SciFinder数据库检索，其中22个为结构新颖尚未见报道的化合物。测得各目标化合物的纯度均在98%以上，测得各目标化合物的Log P值在0.91到2.24之间，表明化合物纯度达到要求、表观脂水分配系数较为合适。药效筛选结果表明化合物A2、C2、A1、D1具有显著的抗疲劳活性，其中化合物A2抗疲劳效果最为显著，且与AMPA受体的亲和力较强。候选化合物A2的药代动力学和急性毒性评价结果表明，其半衰期较合适、生物利用度较理想、毒性较低。以上研究可为抗疲劳新药的研究提供实验依据。
[Abstract]:The aim is to find new, effective and safe anti fatigue candidate compounds. A series of new benzamide AMPA receptor modulators are designed and synthesized with the AMPA receptor as the target. The physicochemical properties and molecular docking of the target compounds are studied. The candidates with significant anti fatigue activity are selected through the screening of pharmacological activity. Compounds, and preliminary pharmacokinetic and acute toxicity evaluation of candidate compounds, providing experimental evidence for developing new anti fatigue drugs with independent intellectual property rights.
The compound is composed of benzamide as the structure parent nucleus, which is composed of aromatic ring and nitrogen containing heterocyclic ring through the amide bond. The aromatic ring and nitrogen containing heterocyclic ring are introduced with different substituents respectively. The two oxygen groups of aryl ring 3,4-, different positions, the number of methoxy, and the effects of different nitrogen heterocyclic rings on the activity of aromatic rings are investigated.
The synthetic route was used as an active ester method, with the reaction of three FLUOROACETIC acid N- succinimide as activation reagent and different substituent aromatic acids to prepare succinimide intermediates containing different substituents, and then to prepare target compounds with corresponding nitrogen heterocyclic condensation. The program was used to study the molecular docking of compound A2 and D2, which obtained the single crystal diffraction data. The binding ability and binding mode of the compound and AMPA receptor were investigated. The purity of each target compound and the apparent lipid water distribution coefficient (Log P) value were measured by HPLC method.
The anti fatigue activity of the target compound was screened by the model of mouse weight swimming and sleep deprivation. The anti fatigue activity of the target compound was screened with the positive control drug of modafinil and coffee. The compound was given to each compound. The dosage of each compound was 0.2mmol/kg. After 7 days of continuous administration, the negative swimming test and sleep deprivation experiment were carried out to record the negative effects of the mice. A full automatic biochemical analyzer was used to detect the content of glycogen in the tissue of mice after swimming and the biochemical indexes of the serum lactate, urine nitrogen, superoxide dismutase, glutathione peroxidase, catalase, lactate dehydrogenase, creatine kinase and so on. The affinity between the target compound and the AMPA receptor was determined.
The preliminary pharmacokinetic evaluation of A2, a compound with significant anti fatigue activity, was conducted to investigate the pharmacokinetic parameters of half-life and bioavailability, and the toxicity of compounds was preliminarily observed by acute toxicity test, and the value of half lethal dose (LD50) was calculated.
Results four series of benzamides, 19 target compounds, 4 benzoate succinimide intermediates and a positive control drug, modafenil, were synthesized in this paper. The total structure of all compounds was confirmed by nuclear magnetic hydrogen spectrum (1HNMR), mass spectrometry (MS), infrared spectroscopy (IR) and elemental analysis. The Chemical Abstract (CA) network SciFi Nder database retrieval, reaction routes, intermediates and target compounds were not reported except for modafinil and A1, and the single crystals of compound A, A1, A2 and D2 were obtained by X-ray single crystal diffraction analysis, and the crystal structure was analyzed. The docking results showed that A2 and D2 were strongly binding to AMPA acceptors.
The purity of each target compound is above 98%, which meets the requirements of animal experiments and can be used in pharmacological experiments. The Log P value of the target compounds is between 0.91 and 2.24, indicating that the partition coefficient of the compound is more suitable.
The results showed that compounds A2, C2, A1, D1 could significantly prolong the swimming time and exercise endurance of mice. The results of sleep deprivation experiment in mice showed that compounds A2, A1, C3, C1, D1 could significantly prolong the sleep deprivation time of mice, indicating the above compounds. The results of the detection of glycogen and blood biochemical indexes after swimming showed that compound A2, C2, A1 had significant inhibitory effect on the decrease of glycogen content in mice after exercise fatigue, indicating that they could reduce the consumption of glycogen to exert the effect of anti fatigue. Compound A2, A1, C2 were used to the blood of mice after exercise fatigue. The increase of lactic acid and nitrogen in the urine has a significant inhibitory effect, indicating that they can reduce fatigue by reducing the accumulation of fatigue metabolites, and compound A2 has the most significant anti fatigue effect. The results of radioligand binding assay show that the dissociation constant (Kd) of the compound A2 and AMPA is 2.1106nmol/L. It has a high affinity with the AMPA receptor.
The blood drug concentration in the corresponding time point was measured at the corresponding time point after perfusion of A2 in rats. The half life time was 81.34min and 82.57min respectively. The bioavailability was 30.8%, and the mice were given a single gastric perfusion and intravenous injection of A2. There was no obvious toxic reaction in the dose range. The LD50 value of the gavage was 1.3306g/kg, and the LD50 value of the intravenous administration was equal to that of the intravenous administration. 0.6066g/kg shows that the toxicity of compound A2 is low.
Conclusion four series of benzamide AMPA receptor modulators were designed and synthesized with the AMPA receptor as the target target. The compound structure was confirmed by 1H NMR, MS, IR and elemental analysis, and 22 of the compounds were found in the SciFinder database. The purity of the compound is above 98%, and the Log P value of each target compound is between 0.91 and 2.24. It shows that the purity of the compound has reached the requirement and the apparent lipid water distribution coefficient is more suitable. The results of drug effect screening show that the compounds A2, C2, A1, D1 have significant anti fatigue activity, and the anti fatigue effect of the chemical compound A2 is most significant and is related to the affinity of AMPA receptor. The pharmacokinetics and acute toxicity evaluation of the candidate compound A2 showed that the half-life of the compound was more suitable, the bioavailability was ideal, and the toxicity was low. The above study could provide the experimental basis for the research of anti fatigue new drugs.

【学位授予单位】：西北工业大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R914.5;R96

【参考文献】