CN113185581A - 一种环十肽化合物及其在治疗新生儿缺氧缺血性脑病中的应用 - Google Patents
一种环十肽化合物及其在治疗新生儿缺氧缺血性脑病中的应用 Download PDFInfo
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Abstract
本发明涉及一种环十肽化合物及其在治疗新生儿缺氧缺血性脑病中的应用。本发明环十肽化合物能一定程度降低OGD诱导的大鼠原代星形胶质细胞氧化应激损伤和细胞凋亡;还能显著增强细胞中SIRT1的脱乙酰酶活性,是一种潜在的SIRT1激活剂,在治疗缺氧缺血损伤相关疾病方面极具潜力,有望被开发用于制备NHIE治疗药物。
Description
技术领域
本发明属于天然产物领域,具体涉及一种环十肽化合物及其在治疗新生儿缺氧缺血性脑病中的应用。
背景技术
新生儿缺氧缺血性脑病(neonatal hypoxie-ischemic encephalopathy,NHIE)是指在围产期窒息而导致脑的缺氧缺血性损伤。NHIE不仅严重威胁着新生儿的生命,更是新生儿期后病残儿中最常见的病因之一。据统计,住院的新生儿中有18.1%是NHIE患儿。我国每年出生后窒息导致的伤残婴儿高达13.6%,数量高达约30万的儿童因此变成残疾儿童。目前,NHIE的治疗选择非常有限,除用低体温疗法治疗轻度新生儿窒息所致的脑病外,临床上使用的潜在的神经保护药物,如氨基酸拮抗剂、自由基抑制剂和清除剂、生长因子、抗炎症反应药物以及抗凋亡药物等由于疗效不足和治疗诱导的副作用,并没有得到公认。
NHIE会导致人类新生儿死亡和慢性神经功能障碍,其发病机制十分复杂,机理尚未阐明。目前研究表明,NHIE与氧化应激、炎症反应、细胞凋亡和代谢障碍等诸多方面有关,其中氧化应激和细胞凋亡起着不可或缺的作用。星形胶质细胞构成大脑中大部分神经胶质细胞,是大脑发育、维持突触和维持神经修复、功能和可塑性的关键介质,星形胶质细胞还参与构成血脑屏障。因此,基于氧化应激和细胞凋亡,寻找疗效可靠的神经保护药物、保护受损星形胶质细胞成为治疗NHIE的有效策略。SIRT1是一种组蛋白去乙酰化酶,其能使蛋白质去乙酰化以调节转录、细胞凋亡、葡萄糖代谢、炎症和氧化应激。研究已发现,SIRT1过表达或激活对中枢神经系统疾病具有保护作用。Hernandez等人证明Sir1-/-小鼠在缺血后显示出比野生型小鼠更大的梗塞体积。SIRT1通过脱乙酰化的表观修饰手段负向调控氧化应激和细胞凋亡相关转录因子,有望为NHIE的临床治疗提供新的靶点。
氧糖剥夺(Oxygen-Glucose Deprivation,OGD)细胞模型是目前模拟人体缺氧缺血状态的经典离体实验模型,也是研究NHIE常用的细胞模型。建立大鼠原代星形胶质细胞OGD模型,体外模拟NHIE进行天然产物神经保护活性研究,能够为研制开发NHIE治疗药物奠定良好基础。
发明内容
先前发明人自黄花蒿内生真菌Myrothecium roridum IFB-E091分离获得一种式I结构的环肽化合物,并发现式I化合物对SGC-7901、AGS或MGC-803等肿瘤细胞表现出极强的抑制活性,同时还可抑制SGC-7901细胞迁移。为进一步研究式I化合物的药理活性,本发明提供式I化合物在预防和/或治疗新生儿缺氧缺血性脑病中的应用。
本发明提供一种式I结构的环肽化合物或其药学上可接受的盐,其特征在于式I结构如下:
本发明的另一实施方案提供一种制备上述式I化合物的方法,其特征在于包括如下步骤:
(1)将真菌Myrothecium roridum IFB-E091接入PD培养基中,于28℃、140rpm摇床培养5-7天,得种子液;
(2)将步骤(1)培养的真菌Myrothecium roridum IFB-E091进行发酵培养;将步骤(1)得到的种子液接种于固体发酵培养基中,于28-30℃静置培养28-30天,得固体发酵产物;
(3)将步骤(2)得到的固体发酵产物粉碎阴干后用体积比1:1的氯仿/甲醇混合溶剂浸提2-3次,合并浸提液后减压浓缩得到发酵产物浸膏;
(4)步骤(3)得到的发酵产物浸膏经色谱分离即得式I化合物。
步骤(1)所述PD培养基的制备方法为:去皮马铃薯200g,切碎成丁,加水煮沸30min,以纱布滤去残渣,滤液加水补足至1000mL,加20g葡萄糖溶解,分装,121℃灭菌20min,备用;培养条件为:旋转式摇床,28℃,140rpm,培养5~7天。
步骤(2)所述固体发酵培养基的配方为:15mL水、7.5g小米、7.5g麸皮、0.5g酵母膏、0.1g酒石酸钠、0.1g谷氨酸钠、0.01g绿矾和0.1mL玉米油。
步骤(4)中所述色谱分离为本领域常规的色谱分离方法,优选正相硅胶柱层析、反相硅胶柱层析、凝胶柱层析、HPLC制备中的一种或几种组合。此处所述一种或几种组合,包括同一分离手段的重复使用。所述色谱分离进一步优选将发酵产物浸膏先经正相硅胶柱层析,用氯仿/甲醇梯度洗脱(v/v 100:0→0:100),得到8个极性组分Fr.1~Fr.8;其中Fr.5组分再经正相硅胶柱层析,用氯仿/甲醇梯度洗脱(v/v 100:0→100:30)得到6个组分Fr.5-1~Fr.5-6;其中Fr.5-3组分经HPLC制备即得式I化合物。其中HPLC制备条件为:HITACHIPrimaide高效液相色谱仪,Sinochrom ODS-AP液相色谱柱(4.6×250mm,5μm),波长254nm,流动相为甲醇:水(v/v)=85:15,流速为1mL/min。
本发明的另一实施方案提供上述式I化合物或其药学上可接受的盐在制备预防和/或治疗新生儿缺氧缺血性脑病药物中的应用。
本发明的另一实施方案提供上述式I化合物或其药学上可接受的盐在制备预防和/或治疗新生儿缺氧缺血性脑病药物先导化合物中的应用。
本发明的另一实施方案提供上述式I化合物或其药学上可接受的盐在制备预防和/或治疗新生儿缺氧缺血性脑病药物候选药物中的应用。
本发明的另一实施方案提供上述式I化合物或其药学上可接受的盐在制备SIRT1激活剂中的应用。
本发明的另一实施方案提供一种药物组合物,其特征在于所述药物组合物以上述式I化合物或其药学上可接受的盐作为有效成分。该药物组合物还任选包括其他预防和/或治疗缺氧缺血性脑损伤疾病的药物。该药物组合物还任选包括药学上可接受的辅料。该药物组合物的剂型可以为固体制剂、液体制剂或半固体制剂。
与现有技术相比,本发明的优点在于式I化合物在0.01~5μg/mL浓度范围内对正常星形胶质细胞无毒性,化合物预处理能不同程度提高OGD模型大鼠星形胶质细胞的活力,且细胞培养上清液中的乳酸脱氢酶(lactate dehydrogenase,LDH)水平下降,说明式I化合物能明显减少OGD引起的大鼠星形胶质细胞死亡,对OGD损伤的星形胶质细胞表现出显著的保护作用。式I化合物能一定程度降低OGD诱导的氧化应激损伤和细胞凋亡;还能显著增强SIRT1的脱乙酰酶活性,是一种潜在的SIRT1激活剂。本发明的式I化合物对OGD损伤的大鼠原代星形胶质细胞具有显著的保护活性,在治疗缺氧缺血损伤相关疾病方面极具潜力,可用于制备NHIE治疗药物。本发明为研究和开发新的NHIE治疗药物提供了新的先导化合物。
附图说明
图1是大鼠原代星形胶质细胞的鉴定图(20×);
图2是式I化合物对正常大鼠星形胶质细胞(A)和OGD模型大鼠星形胶质细胞(B)活力的影响图(与Control组(C)比较,*p<0.05,***p<0.001;与OGD组比较,##p<0.01,###p<0.001);
图3是式I化合物对OGD模型大鼠星形胶质细胞LDH释放的影响图(与Control组比较,***p<0.001;与OGD组比较,#p<0.05);
图4是式I化合物对OGD模型大鼠星形胶质细胞中SOD和ROS水平的影响图(与Control组比较,***p<0.001;与OGD组比较,#p<0.05,###p<0.001);
图5是式I化合物对OGD模型大鼠星形胶质细胞Bcl-2和Bax表达的影响图(与Control组比较,*p<0.05,**p<0.01;与OGD组比较,#p<0.05)(A)Western blot法检测细胞中Bax和Bcl-2蛋白的表达水平;(B)Bcl-2和Bax蛋白的相对表达量;
图6是免疫荧光法观察大鼠星形胶质细胞中SIRT1的定位图(40×);
图7是式I化合物对OGD模型大鼠星形胶质细胞SIRT1、Hif-1α和Ac-p53表达的影响图(与Control组比较,*p<0.05,***p<0.001;与OGD组比较,#p<0.05,###p<0.001)(A)Westernblot法检测细胞中SIRT1、Hif-1α和Ac-p53蛋白的表达水平;(B)SIRT1蛋白的相对表达量;(C)Hif-1α蛋白的相对表达量;(D)Ac-p53蛋白的相对表达量;
图8是式I化合物的1H-NMR谱图(CDCl3,600MHz);
图9是式I化合物的13C-NMR谱图(CDCl3,150MHz);
图10是式I化合物的HSQC谱图;
图11是式I化合物的HMBC谱图;
图12是式I化合物的1H-1H COSY谱图。
具体实施方式
为了便于对本发明的进一步理解,下面提供的实施例对其做了更详细的说明。但是这些实施例仅供更好地理解发明而并非用来限定本发明的范围或实施原则,本发明的实施方式不限于以下内容。
实施例1黄花蒿内生真菌Myrothecium roridum IFB-E091的分离与鉴定
菌株IFB-E091是2006年7月采自江苏南京郊外的健康黄花蒿Artemisia annua(Asteraceae)根部分离获得的一株内生真菌,其分离纯化按照常规方法进行,例如可按照如下步骤:
将新鲜的植株样本用自来水长时间冲洗,洗净表面的灰尘污物,稍晾干后叶片切成1cm2小块;根与茎切成1cm左右的小段,两端均需有切口。在超净工作台把上述叶片和根茎段依次浸泡于75%酒精1min,1%次氯酸钠溶液(含游离氯>2.5%)10~15min,75%酒精再1min进行表面消毒,然后以无菌水浸洗3次后在无菌滤纸上吸干水分,无菌条件下用镊子放置于分离平板表面,轻压,每皿放4片(或段),按来源依次编号,将培养皿倒置于28℃培养箱中培养,逐日观察。待菌丝从植物材料切口内长出至培养基上后,及时以接种针小心地挑取菌落边缘的尖端菌丝(连同小块培养基)转至新鲜平板,顺序编号记录,继续进行分离。根据菌落形态、颜色差异和长出时间不同,用接种针挑取各平板上菌落边缘米粒大小的琼脂块,转移至新鲜培养基上培养,重复操作直至获得纯菌落。
根据菌株IFB-E091形态学特征和18S rDNA序列比较(Genbank accessionNo.GU074399),鉴定其为Myrothecium roridum(Planta Medica,2010,76(10):1004-1006)。公众可按照本实施例中记载的分离鉴定方法或相关研究论文中记载的方法获得本发明所述“黄花蒿内生真菌Myrothecium roridum IFB-E091”。
实施例2式I化合物的分离鉴定
(1)黄花蒿内生真菌Myrothecium roridum IFB-E091的培养
首先将真菌Myrothecium roridum IFB-E091接入20个各装有400mL PD培养基(去皮马铃薯200g,切碎成丁,加水煮沸30min,以纱布滤去残渣,滤液加水补足至1000mL,加20g葡萄糖溶解,分装,121℃灭菌20min,备用)的1000mL锥形瓶中,于旋转式摇床(28℃、140rpm)振荡培养7天,得种子液。
(2)黄花蒿内生真菌Myrothecium roridum IFB-E091的发酵
将步骤(1)所得种子液接种于400个已灭菌的含固体培养基(15mL水、7.5g小米、7.5g麸皮、0.5g酵母膏、0.1g酒石酸钠、0.1g谷氨酸钠、0.01g绿矾和0.1mL玉米油)的250mL广口瓶中,每瓶15mL。然后静置于28℃的温室培养30天,得固体发酵产物。
(3)化合物的提取分离鉴定
将步骤(2)得到的固体发酵产物粉碎阴干后用氯仿/甲醇(1:1,v/v)混合溶剂浸提三次,减压去除溶剂得粗浸膏38g。粗浸膏经硅胶柱分离,氯仿/甲醇梯度洗脱(v/v 100:0→0:100)得到8个组分Fr.1~Fr.8。Fr.5再经硅胶柱分离,氯仿/甲醇梯度洗脱(v/v 100:0→100:30)得到6个组分Fr.5-1~Fr.5-6;其中Fr.5-3经HPLC制备,HITACHI Primaide高效液相色谱仪,Sinochrom ODS-AP液相色谱柱(4.6×250mm,5μm),波长254nm,流动相为甲醇:水(v/v)=85:15,流速为1mL/min,得到本发明式I化合物(20mg)。经结构确证(Marfey、LC-MS、HR-ESI-MS、一维、二维NMR等数据),式I化合物结构如下:
式I化合物分子式:C54H82N10O12,白色粉末。高分辨电喷雾质谱(HR-ESI-MS)显示[M+Na]+1085.5995和[M-H]-1061.6024,确定其分子量为1062,分子式为C54H82N10O12。其1H-和13C-NMR谱数据见表1。
表1式I化合物的1H-和13C-NMR谱数据(AVANCE 600,CDCl3,δ:ppm,J:Hz)
本发明采用Marfey法确定式I化合物的立体构型,具体方法如下:
Marfey法与LC-MS分析:化合物(1mg)溶于2mL 6N盐酸,110℃加热20h,然后冷却至室温、减压去除溶剂。水解产物溶于500μL水,用200μL 2%1-fluoro-2,4-dinitrophenyl-5-L-alaninamide(FDAA)丙酮溶液(w/v)和100μL 1N碳酸氢钠处理。混合物40℃加热6h,冷却到室温,用1N盐酸(100μL)处理至中性,所得混合物加入1000μL乙腈,即得需要分析的样品。化合物水解产物FDAA衍生物和氨基酸对照品FDAA衍生物分别用Agilent TQ-MS进行LC-MS分析,Betasil C18液相色谱柱(150×4.6mm,5μm),波长340nm,梯度洗脱(流动相A:0.1%甲酸水溶液,流动相B:0.1%甲酸乙腈溶液,洗脱梯度:10%-50%流动相B,洗脱时间75min),流速0.5mL/min。
实验发现,LC-MS不能区分NMe-L-Ala和NMe-D-Ala的FDAA衍生物,因此,这两个氨基酸的衍生化采用改良marfey法,即用FDLA(1-fluoro-2,4-dinitrophenyl-5-leuciamide)替代FDAA作为衍生化试剂。D,L-氨基酸对照品FDAA(或FDLA)衍生物的保留时间和式I化合物水解产物FDAA(或FDLA)衍生物的保留时间分别列于表2和表3中,分析表2和表3可知,式I化合物水解产物中的氨基酸全部是L-型,即式I化合物结构中的氨基酸皆是L-型氨基酸(甘氨酸无手性)。
表2氨基酸对照品衍生物的保留时间
*改良marfey法
表3式I化合物水解产物衍生物的保留时间
*改良marfey法
实施例3大鼠原代星形胶质细胞的制备和鉴定
SD(Sprague-Dawley)大鼠(扬州大学比较医学中心提供)出生后24h内制备星形胶质细胞。细胞用0.25%胰酶37℃消化10min,离心,在37℃、5%CO2条件下,采用10%胎牛血清的高糖DMEM培养基(含100units/mL penicillin G和100μg/mL streptomycin)进行培养,12~14天后获得单层星形胶质细胞,继续培养4天,备用。
实验前需要先鉴定星形胶质细胞的纯度。胶质纤维酸性蛋白(glial fibrillaryacidic protein,GFAP)是星形胶质细胞合成的重要骨架蛋白,是星形胶质细胞的特征性标志。星形胶质细胞传至3~4代时,采用免疫荧光法对细胞内的GFAP进行检测,阳性细胞会发出红色荧光。如图1所示,大多数细胞GFAP阳性(>95%),表明培养的细胞就是星形胶质细胞。
实施例4本发明式I化合物对OGD模型大鼠星形胶质细胞的保护作用
采用MTT法观察化合物对正常大鼠星形胶质细胞活力的影响。大鼠星形胶质细胞以1×104个/孔接种到96孔板,37℃、5%CO2培养箱培养至细胞融合度70%~80%,加入一定浓度的化合物处理细胞24h;然后每孔加入20μL MTT,37℃继续培养4h,每孔再加入100μLDMSO溶解甲瓒;最后采用酶标仪测定各孔490nm的吸光度。结果显示,在0.01~5μg/mL浓度范围内,式I化合物对正常星形胶质细胞无毒性,而且能不同程度促进细胞增殖;与对照组相比,0.1、0.5和1μg/mL浓度时,细胞存活率分别为117.2%(p<0.05)、127.1%(p<0.001)和117.1%(p<0.05)(图2A)。
采用MTT法观察化合物预处理对OGD模型大鼠星形胶质细胞活力的影响。大鼠星形胶质细胞以1×104个/孔接种到96孔板,37℃、5%CO2培养箱培养至细胞融合度70%~80%。对照组细胞常氧、37℃,DMEM培养基培养;OGD模型组细胞不加药物预处理的情况下,OGD 6h(5%CO2、1%O2和94%N2);药物组细胞先用一定浓度化合物预处理细胞24h,然后OGD 6h。上述各组细胞每孔加入20μL MTT,37℃继续培养4h,再加入100μL DMSO溶解甲瓒;最后采用酶标仪测定各孔490nm的吸光度。结果发现,在测试浓度范围内,式I化合物预处理能不同程度提高OGD模型星形胶质细胞的活力;与模型组细胞相比,0.5和1.0μg/mL组细胞存活率分别上升至68.5%(p<0.001)和62.9%(p<0.01),说明化合物能明显减少OGD引起的大鼠星形胶质细胞死亡,对OGD损伤的星形胶质细胞具有明显的保护作用(图2B)。
采用乳酸脱氢酶(lactate dehydrogenase,LDH)试剂盒测定细胞培养上清液中LDH的释放情况。将大鼠星形胶质细胞以约1×104个/孔的密度接种到用L-多聚赖氨酸提前包被过的96孔板中。对照组细胞常氧、37℃,DMEM培养基培养;OGD模型组细胞不加药物预处理的情况下,OGD 6h(5%CO2、1%O2和94%N2);药物组细胞先用一定浓度化合物预处理细胞24h再OGD 6h。吸出上清液,用LDH试剂盒测定上清液中的LDH水平。
如图3所示,式I化合物预处理可降低LDH释放,进一步说明式I化合物确实能减弱OGD引起的星形胶质细胞的活力丧失。因此,式I化合物在缺血缺氧性脑损伤相关疾病的治疗中可能提供更为有效的治疗方案。
实施例5本发明式I化合物能减轻OGD引起的大鼠星形胶质细胞氧化应激损伤
细胞内超氧化物歧化酶(superoxide dismutase,SOD)水平采用WST-1法测定,实验分组同LDH测定。OGD 6h后,将培养的细胞用PBS洗涤两次,离心,弃上清,加入一定量的缓冲液在冰上超声处理,然后收集细胞上清液放置于冰上,根据SOD试剂盒说明测定细胞内SOD水平。
细胞内活性氧(reactive oxygen species,ROS)水平采用2,7-DCFH-DA试剂测定,实验分组同LDH测定。细胞OGD 6h后,用冰PBS洗两遍后,消化细胞,离心后加入DCFH-DA(10μM),在37℃在黑暗中孵育30min。酶标仪测定每孔细胞的DCFH荧光(最大激发波长480nm,最大发射波长525nm)。
如图4所示,式I化合物预处理能显著逆转缺氧引起的SOD活性的降低和ROS水平的增加,说明化合物能减轻OGD引起的大鼠星形胶质细胞氧化应激损伤。
实施例6本发明式I化合物对OGD模型大鼠星形胶质细胞Bcl-2和Bax蛋白表达的影响
采用western blot法测定化合物处理后OGD模型大鼠星形胶质细胞Bcl-2和Bax蛋白的表达水平,实验分组同实施例4。OGD 6h后,用预冷的PBS洗涤细胞两次。加入相应体积的RIPA蛋白裂解液后,刮下细胞,超声处理后,置于冰上裂解30min,4℃、12500rpm离心10min。收集上清液,按照BCA试剂盒说明书定量总蛋白。使用4×loading buffer调整蛋白样品的浓度,并于95℃以上将蛋白变性5min;等量蛋白上样,浓缩胶恒压80V 30min,分离胶恒压120V至溴酚蓝指示剂跑至分离胶底部。PVDF膜置于甲醇溶液中激活30s,冰上100V转膜100~120min,并用5%脱脂奶粉缓冲液封闭4h,将膜与一抗在4℃封闭过夜。将膜用TBST清洗3次,每次10min,二抗室温孵育2h或在4℃孵育4h。显影剂A与B按1:1等比例混合后,滴在相应条带蛋白分子量处显影,并用Image J软件定量。
Western blot分析结果表明,式I化合物预处理可上调抗凋亡蛋白Bcl-2的表达,并下调促凋亡蛋白Bax的表达,说明化合物能一定程度抑制OGD导致的大鼠星形胶质细胞凋亡(图5)。
实施例7本发明式I化合物对OGD模型大鼠星形胶质细胞SIRT1的激活作用
采用免疫荧光法观察大鼠星形胶质细胞SIRT1蛋白在细胞中的定位。于24孔板中放入爬片,将星形胶质细胞传代至用L-多聚赖氨酸提前包被过的爬片中,约2×1.04个/孔,隔天换液,继续培养3天后进行实验,实验分组同实施例4。OGD 6h后,盖玻片上的细胞用PBS洗涤两次,每遍10min,并在4%PFA中固定30min。用PBS洗涤两次,将细胞在0.1%Triton-100中透化10min;在室温下,将细胞在3%BSA中封闭30min后用PBS洗涤一次。加入一抗(SIRT1),在4℃下标记过夜;将细胞用PBS洗涤两次,每次10min,然后加入荧光二抗(抗兔Alexa Fluor 594或抗鼠Alexa Fluor 488)在室温下避光孵育1h,细胞核用DAPI染色5min。在荧光显微镜下拍摄图像。免疫荧光染色分析显示,大鼠星形胶质细胞中SIRT1主要在细胞核中表达(图6)。
采用Western Blot法分析化合物对SIRT1蛋白及其底物Hif-1α和Ac-p53蛋白表达的影响,实验方法和实验分组同实施例6。结果显示,式I化合物预处理能显著提高OGD模型大鼠星形胶质细胞SIRT1蛋白的表达,且能逆转OGD引起的Hif-1α和Ac-p53蛋白的表达上升(图7),说明式I化合物能增强OGD模型大鼠星形胶质细胞SIRT1的脱乙酰酶活性,是一个潜在的SIRT1激活剂。
Claims (9)
2.一种制备权利要求1所述式I化合物的方法,其特征在于包括如下步骤:
(1)将真菌Myrothecium roridum IFB-E091接入PD培养基中,于28℃、140rpm摇床培养5-7天,得种子液;
(2)将步骤(1)培养的真菌Myrothecium roridum IFB-E091进行发酵培养;将步骤(1)得到的种子液接种于固体发酵培养基中,于28-30℃静置培养28-30天,得固体发酵产物;
(3)将步骤(2)得到的固体发酵产物粉碎阴干后用体积比1:1的氯仿/甲醇混合溶剂浸提2-3次,合并浸提液后减压浓缩得到发酵产物浸膏;
(4)步骤(3)得到的发酵产物浸膏经色谱分离即得式I化合物。
3.权利要求1所述式I化合物或其药学上可接受的盐在制备预防和/或治疗新生儿缺氧缺血性脑病药物中的应用。
4.权利要求1所述式I化合物或其药学上可接受的盐在制备预防和/或治疗新生儿缺氧缺血性脑病药物先导化合物中的应用。
5.权利要求1所述式I化合物或其药学上可接受的盐在制备预防和/或治疗新生儿缺氧缺血性脑病药物候选药物中的应用。
6.一种药物组合物,其特征在于该药物组合物以权利要求1所述式I化合物或其药学上可接受的盐作为有效成分。
7.权利要求6所述的药物组合物,其特征在于该药物组合物还任选包括其他预防和/或治疗缺氧缺血性脑损伤疾病的药物。
8.权利要求6-7任一项所述的药物组合物,其特征在于该药物组合物还任选包括药学上可接受的辅料。
9.权利要求6-8任一项所述的药物组合物,其特征在于该药物组合物的剂型可以为固体制剂、液体制剂或半固体制剂。
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