本文選題：疲勞 + AMPA受體調(diào)節(jié)劑�。� 參考：《西北工業(yè)大學(xué)》2014年博士論文

【摘要】：目的為尋找新型、有效、安全的抗疲勞候選化合物，本課題以AMPA受體為作用靶點(diǎn)，設(shè)計(jì)、合成一系列新型苯甲酰胺類AMPA受體調(diào)節(jié)劑。對(duì)目標(biāo)化合物進(jìn)行理化性質(zhì)研究及分子對(duì)接研究。通過(guò)藥理活性篩選，優(yōu)選出具有顯著抗疲勞活性的候選化合物，并對(duì)候選化合物進(jìn)行初步藥代動(dòng)力學(xué)和急性毒性評(píng)價(jià)，為開發(fā)具有自主知識(shí)產(chǎn)權(quán)的抗疲勞新藥提供實(shí)驗(yàn)依據(jù)。 方法設(shè)計(jì)的化合物以苯甲酰胺為結(jié)構(gòu)母核，由芳環(huán)與含氮雜環(huán)通過(guò)酰胺鍵連接而成。芳環(huán)和含氮雜環(huán)上分別引入不同取代基，重點(diǎn)考察芳環(huán)3,4-位連亞甲二氧基，芳環(huán)上不同位置、數(shù)目的甲氧基，以及不同含氮雜環(huán)對(duì)活性的影響。 采用的合成路線為活化酯法，以三氟乙酸N-琥珀酰亞胺酯為活化試劑與不同取代芳酸反應(yīng)，制備含不同取代基的苯甲酸琥珀酰亞胺酯中間體，再與相應(yīng)含氮雜環(huán)縮合制備目標(biāo)化合物；采用乙醇擴(kuò)散法，培養(yǎng)化合物單晶體；采用AutoDock4.0程序?qū)Φ玫絾尉а苌鋽?shù)據(jù)的化合物A2和D2進(jìn)行分子對(duì)接研究，考察化合物與AMPA受體的結(jié)合能力和結(jié)合模式；采用HPLC法測(cè)定各目標(biāo)化合物的純度及表觀脂水分配系數(shù)（Log P）值。 采用小鼠負(fù)重游泳和睡眠剝奪實(shí)驗(yàn)?zāi)Ｐ�，以莫達(dá)非尼和咖啡因?yàn)殛?yáng)性對(duì)照藥，，對(duì)目標(biāo)化合物的抗疲勞活性進(jìn)行篩選。灌胃給予各化合物，各化合物給藥劑量為0.2mmol/kg，連續(xù)給藥7天后，進(jìn)行負(fù)重游泳實(shí)驗(yàn)和睡眠剝奪實(shí)驗(yàn)，分別記錄小鼠負(fù)重游泳時(shí)間和睡眠剝奪時(shí)間。采用全自動(dòng)生化分析儀檢測(cè)游泳后小鼠組織糖原含量及血清中乳酸、尿氮素、超氧化物歧化酶、谷胱甘肽過(guò)氧化物酶、過(guò)氧化氫酶、乳酸脫氫酶、肌酸激酶等疲勞相關(guān)血生化指標(biāo)；采用放射性配體受體結(jié)合實(shí)驗(yàn)，測(cè)定目標(biāo)化合物與AMPA受體的親和力。 對(duì)優(yōu)選出具有顯著抗疲勞活性的化合物A2，進(jìn)行初步藥代動(dòng)力學(xué)評(píng)價(jià)，考察半衰期及生物利用度等藥代動(dòng)力學(xué)參數(shù)；采用急性毒性試驗(yàn)初步觀察化合物的毒性反應(yīng)，計(jì)算半數(shù)致死量（LD50）值。 結(jié)果本論文合成了四個(gè)系列的苯甲酰胺類19個(gè)目標(biāo)化合物，4個(gè)苯甲酸琥珀酰亞胺酯中間體及陽(yáng)性對(duì)照藥莫達(dá)非尼，共計(jì)24個(gè)化合物。所有化合物結(jié)構(gòu)經(jīng)核磁氫譜（1HNMR）、質(zhì)譜（MS）、紅外光譜（IR）及元素分析確證無(wú)誤。經(jīng)化學(xué)文摘（CA）網(wǎng)絡(luò)版SciFinder數(shù)據(jù)庫(kù)檢索，反應(yīng)路線、中間體及目標(biāo)化合物除莫達(dá)非尼和A1外均未見(jiàn)相關(guān)報(bào)道；另得到化合物A、A1、A2和D2的單晶體，經(jīng)X-ray單晶衍射分析，解析出晶體結(jié)構(gòu)；分子對(duì)接結(jié)果顯示A2和D2與AMPA受體具有較強(qiáng)的結(jié)合能力。 測(cè)得各個(gè)目標(biāo)化合物的純度均在98%以上，符合動(dòng)物實(shí)驗(yàn)要求，可用于藥理學(xué)實(shí)驗(yàn)；測(cè)得目標(biāo)化合物的Log P值在0.91到2.24之間，表明化合物脂水分配系數(shù)較為合適。 各化合物在實(shí)驗(yàn)給藥期間，對(duì)小鼠體重均無(wú)影響。小鼠負(fù)重游泳實(shí)驗(yàn)結(jié)果顯示，化合物A2、C2、A1、D1可顯著延長(zhǎng)小鼠力竭游泳時(shí)間，提高運(yùn)動(dòng)耐力。小鼠睡眠剝奪實(shí)驗(yàn)結(jié)果顯示，化合物A2、A1、C3、C1、D1可顯著延長(zhǎng)小鼠睡眠剝奪時(shí)間，表明以上化合物具有顯著的抗疲勞活性。游泳后小鼠組織糖原及血生化指標(biāo)檢測(cè)結(jié)果顯示，化合物A2、C2、A1對(duì)小鼠運(yùn)動(dòng)疲勞后糖原含量的降低具有顯著的抑制作用，表明它們可通過(guò)降低糖原的消耗從而發(fā)揮抗疲勞效果。化合物A2、A1、C2對(duì)運(yùn)動(dòng)疲勞后小鼠血清中乳酸及尿氮素的升高具有顯著的抑制作用，表明它們可通過(guò)降低疲勞代謝產(chǎn)物的堆積從而緩解疲勞，其中化合物A2抗疲勞效果最為顯著；放射性配體受體結(jié)合實(shí)驗(yàn)結(jié)果顯示，化合物A2與AMPA受體的解離常數(shù)（Kd）值為2.1106nmol/L，表明其與AMPA受體具有較高的親和力。 大鼠灌胃、靜脈注射給藥A2后，測(cè)定相應(yīng)時(shí)間點(diǎn)血藥濃度，經(jīng)計(jì)算得半衰期分別為81.34min和82.57min，生物利用度為30.8%；小鼠單次灌胃及靜脈注射給予A2，給藥劑量范圍內(nèi)均未見(jiàn)明顯毒性反應(yīng)，灌胃給藥的LD50值為1.3306g/kg，靜脈給藥的LD50值為0.6066g/kg，表明化合物A2的毒性較低。 結(jié)論本課題以AMPA受體為作用靶點(diǎn)，設(shè)計(jì)合成了四個(gè)系列的苯甲酰胺類AMPA受體調(diào)節(jié)劑，共計(jì)24個(gè)化合物。化合物結(jié)構(gòu)經(jīng)1H NMR、MS、IR及元素分析表征確證無(wú)誤。經(jīng)SciFinder數(shù)據(jù)庫(kù)檢索，其中22個(gè)為結(jié)構(gòu)新穎尚未見(jiàn)報(bào)道的化合物。測(cè)得各目標(biāo)化合物的純度均在98%以上，測(cè)得各目標(biāo)化合物的Log P值在0.91到2.24之間，表明化合物純度達(dá)到要求、表觀脂水分配系數(shù)較為合適。藥效篩選結(jié)果表明化合物A2、C2、A1、D1具有顯著的抗疲勞活性，其中化合物A2抗疲勞效果最為顯著，且與AMPA受體的親和力較強(qiáng)。候選化合物A2的藥代動(dòng)力學(xué)和急性毒性評(píng)價(jià)結(jié)果表明，其半衰期較合適、生物利用度較理想、毒性較低。以上研究可為抗疲勞新藥的研究提供實(shí)驗(yàn)依據(jù)。
[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.

【學(xué)位授予單位】：西北工業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R914.5;R96

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