【作者】 陶云海；

【导师】 杨祥良；

【作者基本信息】 华中科技大学 ， 生物医学工程， 2006， 博士

【摘要】 γ-氨基丁酸(GABA)是哺乳动物中枢神经系统内的主要抑制性递质。GABA的降解主要由GABA转氨酶(GABA-T)催化,脱氨基生成琥珀酸半醛,再经琥珀酸半醛脱氢酶(SSADH)催化生成琥珀酸,进入三羧酸循环。增加脑内GABA浓度可以提高GABA能系统的神经抑制作用,进而可用于治疗多种神经系统疾病如癫痫、帕金森病、亨廷顿舞蹈症、Alzheimer病等,最近发现增加脑内GABA浓度还有阻断药物依赖的作用。一种增加GABA浓度的方法是设计可通过血脑屏障并选择性抑制GABA-T的化合物。因此GABA-T已经被确认为中枢神经系统药物的靶点。在本论文中,确定了对羟基苯甲醛可以作为设计GABA-T和SSADH抑制剂的先导化合物,合成并评价了两个对羟基苯甲醛类似物对GABA-T的不可逆抑制作用,研究了黄酮类化合物对这两个酶的抑制作用。另外还研究了豆腐果苷磷脂复合物的制备、理化性质及药代动力学。主要完成了以下研究工作:(1)对羟基苯甲醛表现出对GABA-T强烈的竞争性抑制(IC50=16.5μmol/L),这种抑制作用与α-酮戊二酸呈竞争性,与GABA呈非竞争性。对羟基苯甲醛也以竞争性的方式强烈地抑制SSADH(IC50=24.7μmol/L)。这种抑制作用是由于对羟基苯甲醛与α-酮戊二酸及琥珀酸半醛的结构相似性和苯环的共轭作用导致的。初步的量子化学计算表明对羟基苯甲醛与α-酮戊二酸及琥珀酸半醛的结构相似性本质上是它们前线轨道的能量和形状的相似。这些结果表明这两个酶的活性位点可以接受苯环的存在,提示对羟基苯甲醛可以作为先导化合物设计结构新颖的GABA-T和SSADH抑制剂。(2)考察了6类共17个对羟基苯甲醛类似物对GABA-T和SSADH的抑制作用,其中有8个对羟基苯甲醛类似物由本实验室合成。包括对羟基苯甲醛总计18个化合物对GABA-T和SSADH的IC50值显示出明显的构效关系。对于GABA-T的抑制,酚羟基及其对位的羰基或氨甲基是关键的。对于SSADH的抑制,酚羟基及其对位的羰基是非常重要的。4-丙烯酰苯酚对GABA-T和SSADH的IC50分别为5.48μmol/L和0.35μmol/L,4-β-氯丙酰苯酚对GABA-T和SSADH的IC50分别为3.99μmol/L和1.09μmol/L,均表现出极好的抑制活性。对羟基苯甲胺是一个强有力的GABA-T抑制剂(IC50= 15.4μmol/L),与GABA呈竞争性关系,而与α-酮戊二酸呈非竞争性关系。推测了一个统一的机制解释对羟基苯甲醛和对羟基苯甲胺对GABA-T的抑制。(3) 4-丙烯酰苯酚以时间依赖的方式强烈的不可逆抑制GABA-T,其动力学参数为KI= 470μmol/L,kinact= 0.061 min-1,kinact/KI= 0.129 (mmol/L)-1min-1。加入α-酮戊二酸可以保护这种不可逆抑制作用,表明这种抑制作用是发生在GABA-T的活性部位。巯基乙醇也可以保护这种抑制作用。提出了一种Michael加成机制解释这种不可逆抑制作用。初步的量子化学计算结果也证实了Michael加成机制的合理性。这些结果为设计更有效的GABA-T抑制剂提供了新思路。(4) 4-β-氯丙酰苯酚以时间依赖的方式强烈地不可逆抑制GABA-T,其动力学参数为KI= 24.3μmol/L,kinact= 0.0467 min-1,kinact/KI= 1.88 (mmol/L)-1min-1。加入α-酮戊二酸可以保护这种不可逆抑制作用,表明这种抑制作用是发生在GABA-T的活性部位。巯基乙醇和谷胱甘肽可以保护这种不可逆抑制作用。公认的GABA-T不可逆抑制机制不能解释这个化合物的不可逆抑制作用,因此提出了一种亲核取代机制解释这种不可逆抑制作用。虽然这个不可逆抑制机制需要进一步研究确证,但这些结果表明除了公认的两种机制还可以通过其他机制不可逆抑制GABA-T,这为设计新颖的GABA-T抑制剂提供了更广阔的空间。(5)黄酮类化合物可以显著地以剂量依赖的方式抑制GABA-T和SSADH。黄酮类化合物对GABA-T和SSADH的抑制表现出不同的构效关系。在黄芩素、黄芩苷、野黄芩苷元、野黄芩苷、槲皮素、山奈酚和芦丁等7个黄酮类化合物中,黄芩素是GABA-T最有效的抑制剂(IC50=12.8μmol/L),野黄芩苷元是SSADH最有效的抑制剂(IC50=7.2μmol/L)。黄酮类化合物对GABA-T和SSADH的抑制是非竞争性的。这些结果提示黄酮类化合物对GABA-T和SSDAH的抑制作用可能是黄酮类化合物神经药理作用的部分原因。(6)制备了豆腐果苷磷脂复合物,并通过正交设计考察了反应物初始浓度、投料比例和反应时间等因素对复合率的影响,表明最佳制备工艺是室温下以四氢呋喃为反应溶剂,豆腐果苷浓度为5 mg/mL,投料质量比例为1: 5,反应时间为2 h。X-射线衍射和差热扫描分析表明复合物呈现无定型特征,并改变了原药的相变特征;1H-NMR和IR显示了豆腐果苷的糖羟基和醛基参与复合物的结合。磷脂复合物可以明显改善豆腐果苷在水及正辛醇中的溶解性能。磷脂复合物可以显著地提高豆腐果苷在大鼠体内的生物利用度。磷脂复合物对药物吸收的改善作用与磷脂复合物不同于原药的理化性质及溶解性能有关。本论文的研究结果对于设计结构新颖的GABA-T抑制剂具有重要意义,对于阐明黄酮类化合物的药理作用具有参考价值。豆腐果苷磷脂复合物的研究为其它天然药物的新制剂研究提供了新的思路。更多还原

【Abstract】 γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian central nervous system. GABA is metabolized by the successive action of GABA transaminase (GABA-T) and succinic semialdehyde dehydrogenase (SSADH), to succinic acid, which is a substrate for the tricarboxylic acid cycle. An increase in the brain GABA concentration can enhance the inhibitory function of GABAergic system and have therapeutic applications in neurological disorders including epilepsy, Parkinson’s disease, Huntington’s chorea, and Alzheimer’s disease. Recently, it has been found that an increase in GABA also blocks the effects of drug addiction. An approach for increasing the brainGABA concentration would be to design a compound capable of permeating the blood-brain barrier that subsequently selectively inhibited GABA-T. Thus GABA-T has been validated as a target for neuroactive drugs.In this dissertation, it was established that 4-hydroxybenzaldehyde may serve as a lead structure for GABA-T and SSADH inhibitors. Based on the results, both 4-hydroxybenzaldehyde analogues were synthesized and evaluated for irreversible inhibition of GABA-T. The inhibitory effect of flavonoids on GABA-T and SSADH was also investigated. Moreover, hilicid-phospholipid complex was prepared, and its physic-chemical properties and oral pharmacokinetics in rats were investigated. The main results are as follows:(1) 4-Hydroxybenzaldehyde was shown to inhibit significantly GABA-T (IC50= 16.5μmol/L) in a competitive manner with respect toα-ketoglutarate but in a noncompetitive manner with respect to GABA. 4-Hydroxybenzaldehyde also exhibited a competitive inhibition of SSADH (IC50=24.7μmol/L). The inhibitory effects of 4-hydroxybenzaldehyde on both enzymes could result from the structural similarity between the molecule and the two enzymes’substrates, as well as the conjugative effect of benzene ring. Preliminary quantum chemical calculations suggested that the structural similarity of between 4-hydroxybenzaldehyde and the two substrates is due to the similarity of the energies and shapes of their molecular orbitals in nature. The results indicated that the presence of the benzene ring may be accepted by the active site of both enzymes, and 4-hydroxybenzaldehyde may be considered as a lead compound to design novel GABA-T and SSADH inhibitors.(2) Six groups of 4-hydroxybenzaldehyde analogues were examined as inhibitors for GABA-T and SSADH. Among them, syntheses of eight compounds were achieved in this laboratory. Investigation of 18 compounds including 4-hydroxybenzaldehyde revealed the significant structure-activity relations with regard to both enzymes inhibition. A carbonyl group or an aminomethyl group, as well as a hydroxy group at the para position of the benzene ring are important for GABA-T inhibition. A carbonyl group and a hydroxy group at the para position of the benzene ring are important for SSADH inhibition. IC50 values of 4-Acryloylphenol (6) for GABA-T and SSADH are 5.48 and 0.35μmol/L, respectively, and IC50 values of 4-β-chloropropionylphenol (7) for GABA-T and SSADH are 3.99 and 1.09μmol/L, exhibiting more potent activity than that of 4-hydroxybenzaldehyde. 4-Hydroxybenzylamine potently inhibited GABA-T (IC50= 15.4μmol/L) in a competitive manner with respect to GABA but in a noncompetitive manner with respect toα-ketoglutarate. A possible mechanism was speculated to rationalize the inhibition of GABA-T by 4-hydroxybenzaldehyde and 4-hydroxybenzylamine.(3) 4-Acryloylphenol was shown to inhibit irreversibly and potently the enzyme in a time-dependent manner with KI= 470μmol/L, kinact= 0.061 min-1 and kinact/KI= 0.129 (mmol/L)-1min-1. The inhibition was protected byα-ketoglutarate, indicating that it occurs at the active site of the enzyme.β-Mercaptoethanol also prevented the enzyme from irreversible inhibition. The possible mechanism involving a Michael addition was speculated to elucidate the inhibition. The rationality of the mechanism was supported by the preliminary quantum chemical calculations. The results suggested future directions for the design of more potent GABA-T inhibitors.(4) 4-β-Chloropropionylphenol was shown to inhibit irreversibly and potently the enzyme in a time-dependent manner with KI= 24.3μmol/L, kinact= 0.0467 min-1, kinact/KI= 1.88 (mmol/L)-1min-1. The inhibition was protected byα-ketoglutarate, indicating that it is active site-directed.β-Mercaptoethanol and glutathione also prevented the enzyme from irreversible inhibition. Since the published mechanism can’t rationalize inactivation, a novel possible mechanism involving a nucleophilic substitution was speculated to rationalize the inactivation. Although the confirmation of the presumed mechanism requires further investigation, the results suggested that GABA-T could be inhibited irreversibly via a novel mechanism other than the two published mechanism, which give a new clue to design of more potent GABA-T inhibitors.(5) A group of flavonoids including baicalein, baicalin, scutellarein, scutellarin, quercetin, kaempferol and rutin, significantly and dose-dependently inhibited GABA-T and SSADH in a noncompetitive manner. The different structure-activity relations were observed with respect to inhibition of GABA-T and SSADH. Among the seven flavonoids, baicalein was the most potent inhibitor for GABA-T (IC50= 12.8μmol/L), and scutellarein exhibited the best inhibitory effect on SSADH (IC50= 7.2μmol/L). The results suggested that the inhibition of both enzymes by flavonoids may contribute to the beneficial effect of flavonoids on the central nervous system.(6) Helicid-phospholipid complex was prepare and the effect of such factors as the reactant concentrations, the ratio of reactants and the reactive time on the preparation was investigated via a orthogoral design, indicating the best conditions as follows: 5 mg/mL, 1: 5 and 2 h, respectively. X-ray diffraction spectra and DSC indicated that the complex exhibits an amorphous characteristic and change the transformation temperature of hilicid; 1H-NMR spectra and IR spectra suggested that hydroxy groups and aldehyde group of hilicid could interact with phospholipid. There is a great improvement in the solubility of hilicid-phospholipid complex both in water and in n-octanol. The complex significantly improved the relative bioavailability of helicid in rats. The improvement resulted from the particular physic-chemical and solubility properties of the hilicid-phospholipid complex.The results of this dissertation could be significantly useful to design novel GABA-T inhibitors, and will facilitate to elucidate the neuropharmacological actions of flavonoids. The research of helicid-phospholipid complex led to a valuable direction in development of novel formulations of natural drugs.更多还原