CN101333205A - 周期素依赖蛋白激酶抑制剂黄芩素哌嗪衍生物及其制法 - Google Patents
周期素依赖蛋白激酶抑制剂黄芩素哌嗪衍生物及其制法 Download PDFInfo
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- CN101333205A CN101333205A CNA2008100122840A CN200810012284A CN101333205A CN 101333205 A CN101333205 A CN 101333205A CN A2008100122840 A CNA2008100122840 A CN A2008100122840A CN 200810012284 A CN200810012284 A CN 200810012284A CN 101333205 A CN101333205 A CN 101333205A
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Abstract
一种周期素依赖蛋白激酶抑制剂黄芩素哌嗪衍生物及其制备方法，属于化学领域中药物及其合成方法与用途。这种周期素依赖蛋白激酶抑制剂黄芩素哌嗪衍生物及其制备方法，将黄芩素、甲醛溶液和哌嗪类化合物按摩尔比混合，加入为黄芩素重量倍数的甲醇，在50－70℃下搅拌缩合，将析出的沉淀滤出，用少量甲醇洗涤，干燥，得含量不少于98％(重量)的黄芩素哌嗪衍生物。该黄芩素哌嗪衍生物抑制细胞周期素依赖蛋白激酶活性强度与Flavopiridol类似，比黄芩苷提高约50多倍，并具有较强的抗肿瘤活性。本品原料来源丰富，工艺简捷，纯度高，成本低，代谢机理明确，高效低毒，与酸成盐可制成口服制剂或注射剂，有望成为高效低毒的周期素依赖蛋白激酶抑制剂类抗癌和爱滋病药物。
Description
技术领域
本发明涉及一种细胞周期素依赖蛋白激酶抑制剂黄芩素哌嗪衍生物及其制备方法，属于化学领域中药物及其合成方法与用途。
背景技术
细胞周期调控是一个非常复杂和精细的调节过程，它与细胞的分化、生长、增殖和死亡有着紧密的关系。在过去的十几年里，调控细胞周期进程的信号传导途径的发现是一个重要的里程碑。人恶性肿瘤的分子特征是细胞周期调节的失控，引起细胞分化的缺乏和细胞生长的失常，导致细胞无限增殖。其中癌症，糖尿病，病毒感染等许多疾病都与一种细胞信号传导物质一蛋白激酶的混乱有关。人类基因编码的600多种蛋白激酶已被发现，其中细胞周期素依赖蛋白激酶(CyclinDependent Kinases，Cdks)是控制细胞循环与增值过程重要调节者。Cdks是一种丝氨酸/苏氨酸的蛋白磷酸激酶，与所对应的周期蛋白(Cyclin)形成的蛋白复合物通过控制细胞循环的关键部分来实现细胞周期高效有序的运转。Cdks可以激发三磷酸腺苷(ATP)磷酸化宿主细胞蛋白的丝氨酸或苏氨酸残基而发挥作用，而磷酸化蛋白具有调节各种细胞蛋白的活性。有丝分裂是由Cdk1/Cyclin B激活驱动的理论已被广泛认可。已发现90％的肿瘤形成是由于Cdks被高度激活导致肿瘤抑制蛋白Rb被磷酸化，使Rb程路径失活。Cdks抑制剂可有效地诱导癌细胞凋亡，提高肿瘤和爱滋病化疗药物的敏感性，对Cdks的抑制成为抗肿瘤和抗艾滋病药物研究的新靶点。[Science，2004，303，1800；The AAPS Journal，2006，8，E204；CancerBiology Therapy，2003，4，S84]
天然黄酮类化合物广泛存在于天然植物中，具有多种生物活性，是天然的Cdks抑制剂。但是，由于天然黄酮类化合物的水溶性差，生物利用度低，生物活性相对较弱。通过对天然黄酮结构修饰，增强其生物活性，具有重要的实用价值。1994年美国国家卫生研究院癌症研究所(NCI)在全球大规模天然抗癌药物筛选中，从Dysoxylum binectariferum的茎和树皮中分离得到一种含氮的黄酮Rohitukine，显示具有一定的抗癌活性。以此为先导化合物，Aventis公司研制合成了第一个作为高效低毒的Cdks抑制剂Flavopiridol。Flavopiridol是一种黄酮胺基衍生物。最早被认为是酪氨酸蛋白激酶抑制剂，但后来发现其抑制Cdks活性更强，而对其它激酶作用较弱，它也是mRNA转录抑制剂。因此，Flavopiridol成为美国第一个针对细胞周期进入临床试验的可口服抗癌药物，已经进入II期临床试验。美国Iowa州立大学的研究人员同时还发现Flavopiridol可阻碍爱滋病病毒HIV-1在细胞内的增殖，能显著提高化疗药物的敏感性，显示了良好的抗癌和爱滋病应用前景。由于Flavopiridol对P-TEFb酶具有高度的抑制力，很小剂量即能对HIV-1病毒产生极强的杀伤力。[Cancer Biology Therapy，2003，4，S84，77，146；Curr.Pharm.Des.，2006，12，1949]一个有趣的发现是细胞凋亡抑制剂survivin被Cdk1/Cyclin B磷酸化，可取消其抗凋亡活性，而survivin只在恶性肿瘤中表达，不在成熟的组织中表达。Flavopiridol通过抑制Cdk1/Cyclin B而降低survivin在细胞中的浓度。NCI的Louis Staudt等用DNA芯片技术揭示了Flavopiridol诱导癌细胞凋亡及其抑制HIV增殖机理为能够关闭大多数基因转录mRNA，然而它并没有杀死各种类型的细胞，只是有选择性的针对癌细胞。这是由于使癌细胞无节制生长的RNA分子是非常短命的，完全依赖mRNA的持续合成，一旦Flavopiridol进入细胞，mRNA持续时间短的基因产生mRNA的速度没有降解的快，这种mRNA迅速被降解，而正常细胞mRNA分子寿命长的却仍旧存在。Flavopiridol为ATP选择性Cdks抑制剂与细胞毒类抗癌药物不同，并直接杀伤细胞，而是通过抑制细胞生长诱导癌细胞凋亡，这就意味着虽然作用的对象是一种所有人体细胞中普遍存在的生物过程，但由于其所需的药用剂量很小，因此对人体正常细胞几乎没有伤害。[Genome Biology，2001，2(10)，0041.1]Flavopiridol能有效提高化疗药物的敏感性，具有明显的减毒增效协同以及抑制“多药耐药性”作用，也可用于癌症晚期治疗，能明显改善患者生存质量，延缓生命。但是Flavopiridol具有手性结构，合成工艺复杂，产率较低，成本较高，价格昂贵，同时还存在着严重腹泻等不良反应。尽管如此，作为ATP选择性Cdks抑制剂Flavopiridol的发现还是给癌症和艾滋病的治疗带来了新的理念与希望。
黄芩为唇形科植物Scutellaria baicalensis Geoygi，主要成分为黄芩苷，具有抑菌，抗炎，抗变态反应，抗氧化，降血脂，抗血栓等作用。作为天然的Cdks抑制剂，其水解产物黄芩素活性更强，能诱导细胞调亡，抑制细胞增殖，抑制HIV复制与逆转录酶，在抗肿瘤和抗艾滋病等方面显示出有独特的作用，受到了广泛关注。[Eur.J.Pharm.，2001，425，165；Anticancer Res.，2005，25，959]但是黄芩素不溶于水，容易被氧化，生物利用度低，生物活性强度达不到临床需要，因此，本发明希望通过对黄芩素结构修饰来寻找新型高效低毒的ATP选择性Cdks抑制剂。
有关黄酮衍生物US-A-5849733公开了Flavopiridol的硫代和氧代衍生物作为Cdks抑制剂用于治疗增生疾病。US-A-5116954公开了具有抗肿瘤和免疫调节活性的黄酮类化合物。CD16686131A(WO2004/004632)公开了作为Cdks抑制剂的黄酮衍生物。CD1427003A公开了具有抑制蛋白激酶C活性的黄芩素8-位取代的甲胺类衍生物及其制备方法，但并未提到黄芩素哌嗪衍生物。到目前为止，国内外专利和文献中均尚未涉及到用黄芩素与甲醛和哌嗪类化合物缩合成新结构类型的黄芩素哌嗪衍生物，以及作为Cdks抑制剂的用途。
发明内容
本发明的目的是开发一种周期素依赖蛋白激酶抑制剂黄芩素哌嗪衍生物及其制备方法，这种具有抑制Cdks作用的黄芩素哌嗪衍生物与酸成盐，并与药学上可以接受的辅料配合制成口服片剂或注射剂，用于抑制肿瘤生长并诱导肿瘤细胞凋亡，在预防和治疗癌症及爱滋病方面具有重要用途。
本发明解决其技术问题所采用的技术方案是：一种周期素依赖蛋白激酶抑制剂黄芩素哌嗪衍生物，具有下述结构通式：
其中，哌嗪上的取代基R为CH3，CH2CH3，CH2CH2OH，CH2CH2SH，CH2COOH，CH2CHOHCH2OH，CH2CHOHCH2SH或CH2CHSHCH2OH。
所述的周期素依赖蛋白激酶抑制剂黄芩素哌嗪衍生物制备方法：将黄芩素、甲醛溶液和哌嗪类化合物按摩尔比为1∶1.2∶1.2混合，加入为黄芩素重量10-40倍的甲醇，在50-70℃下搅拌缩合1-6小时，将析出的沉淀滤出，用少量甲醇洗涤，干燥，得含量不少于98％(重量)的黄芩素哌嗪衍生物。其中，所用黄芩素纯度是影响产物纯度和收率的关键因素。
所述哌嗪类化合物选自哌嗪，甲基哌嗪，乙基哌嗪，哌嗪乙醇，哌嗪巯基乙醇，哌嗪乙酸，哌嗪丙二醇，哌嗪巯基-2丙醇-3或哌嗪巯基-3丙醇-2。
所述抗癌和爱滋病药物的黄芩素哌嗪衍生物制成片剂。
片剂的配方为：黄芩素哌嗪衍生物1摩尔，甲磺酸盐1-2摩尔，淀粉为黄芩素哌嗪衍生物重量0.5-2倍，制成每片含黄芩素哌嗪衍生物200-500mg的黄色薄膜衣片。
片剂的制备方法为：称取黄芩素哌嗪衍生物，甲磺酸，加用适量乙醇润湿，加入淀粉，制粒，干燥，压片，用丙烯酸树脂和柠檬黄色素包薄膜衣。
制备原理：黄芩素哌嗪衍生物可与甲磺酸中和，形成一种弱离子对药物，可使稳定性和生物利用度得到提高。
所述抗癌和爱滋病药物的黄芩素哌嗪衍生物制成针剂。
针剂的配方为：黄芩素哌嗪衍生物1摩尔，柠檬酸为黄芩素哌嗪衍生物重量1-2倍，羟丙基-β-环糊精1-2摩尔，制成每瓶含黄芩素哌嗪衍生物10-50mg的注射用粉针剂。
制备方法：黄芩素哌嗪衍生物，柠檬酸，羟丙基-β-环糊精，加注射用水溶解，超声处理20分钟，用氢氧化钠调节pH 3.5-5.5，过滤，分装，冻干，即得。
制备原理：用羟丙基-β-环糊精与黄芩素哌嗪衍生物形成包合物，提高其稳定性和溶解度，临用前用葡萄糖注射液溶解稀释后，供静脉给药。
黄芩素哌嗪衍生物的制备原理：黄芩素仅能在C8位发生亚甲基哌嗪衍生物取代反应，几乎无副产物，反应产物单一。当反应原料黄芩素含量大于85％时，主要产物黄芩素哌嗪衍生物可直接从反应液中析出，纯度高达98％(重量比)以上，而其它黄芩黄酮苷元亚甲基哌嗪衍生物取代产物的脂溶性较大，仍然溶于反应溶液中。
黄芩素哌嗪衍生物的结构设计：在黄芩素衍生物对Cdk1/Cyclin B抑制作用筛选与构效关系研究中发现新结构类型化合物黄芩素哌嗪衍生物具有良好的应用前景。据此优化设计出系列新的黄芩素哌嗪衍生物，所述的哌嗪类化合物选自哌嗪，甲基哌嗪，乙基哌嗪，哌嗪乙醇，哌嗪巯基乙醇，哌嗪乙酸，哌嗪丙二醇，哌嗪巯基-2丙醇-3或哌嗪巯基-3丙醇-2。
黄芩素衍生物对Cdk1/Cyclin B抑制作用筛选：该筛选生化分析技术是基于酶对磷酸化和非磷酸化底物水解的灵敏性不同的一种荧光共振能量转移技术(Fluorescence resonance energy transfer FRET)。测定采用一种合成的缩氨酸底物，这种底物是由带有荧光供体香豆素和受体荧光素共同组成的FRET。在初级反应中，蛋白激酶将ATP的γ磷酸基转移到一个丝氨酸或者苏氨酸残余羟基上，然而当蛋白激酶抑制剂存在时，磷酸化受阻。在接下来的反应中，加入一种定点切断酶，淬灭了蛋白激酶反应，这种定点切断酶能定点识别和切断未被磷酸化的底物。没有磷酸化的底物FRET系统被断开，而磷酸化底物依然维持FRET。断开的和没有断开的底物在400nm波长激发，分别在445nm和520nm波长有发射波长，计算发射比，如果底物被磷酸化后发射比会比较低表明很少或者没有蛋白激酶的抑制作用，发射比高则表明底物没有被磷酸化有蛋白激酶的抑制作用。
将待测黄芩素衍生物用二甲基亚砜溶解，制成每1ml含1mg的溶液，再依次用4％二甲基亚砜和蛋白激酶缓冲液稀释成不同浓度，Cdk1/Cyclin B抑制活性按Invitrogen公司蛋白激酶分析试剂盒操作方法测定。黄芩素衍生物对Cdk1/CyclinB抑制作用(ATP为10μM)IC50筛选结果见表1。结果显示：黄芩苷IC50为14.36μM对Cdk1/Cyclin B活性显示有轻度抑制作用。当被甲酯化成黄芩苷甲酯IC50为11.64μM，抑制活性略有提高。当被水解为黄芩素IC50为6.53μM，活性比黄芩苷提高1倍多。槲皮素由于C2苯坏上为含有两个亲水性酚羟基，脂溶性降低，活性较弱。黄芩素C8单胺亚甲基衍生物IC50分别为1.05～1.28μM，活性比黄芩素提高约5-6倍。含有2个以上杂原子新结构类型化合物黄芩素哌嗪衍生物IC50分别为0.25～0.27μM，活性比黄芩素提高了约20倍，比黄芩苷提高了约50倍，作用机理和作用强度与Flavopiridol相近(IC50为0.30μM)，为选择性ATP结合位点Cdks抑制剂，而有别于非选择性Cdks毒剂星孢菌素。其中，8-N-甲基哌嗪亚甲基黄芩素对Cdk1/Cyclin B活性抑制作用测定结果曲线见图1，横坐标为8-N-甲基哌嗪亚甲基黄芩素浓度nM，纵坐标为对Cdk1/Cyclin B活性抑制率％。研究果显示黄芩素哌嗪衍生物具有较强的抑制Cdk1/Cyclin B活性作用，在预防和治疗癌症和爱滋病方面具有良好应用前景。
表1.黄芩素衍生物对Cdk1/Cyclin B抑制作用IC50筛选结果
构效关系分析推论：
(1)C8位含氮取代基的存在与位置对Cdk1/Cyclin B的抑制活性起到了全关重要的作用，且随着杂原子数目增多以及脂水分配系数降低，活性增强。这是由于N原子占据了Cdk1/Cyclin B的ATP结合口袋中氨基位。
(2)C4＝O，C5-OH，C7-OH是必需基团，酚羟基的缺失以及甲基化都会影响到抑制活性，增加C6-OH，活性增强，这是由于该组酸性基团占据了Cdk1/Cyclin B的ATP结合口袋中磷酸位。
(3)C2位为疏水取代基团是必须的，再引入卤素等疏水性基团，活性增加，而羟基等亲水基团的引入，可使活性消失。这种脂溶性基团是ATP所不具有的，它可能与Cdk1/Cyclin B的疏水性口袋融合，导致其构型发生明显的改变。
8-N-甲基哌嗪亚甲基黄芩素抑制人癌细胞生长并诱导凋亡作用：8-N-甲基哌嗪亚甲基黄芩素对人宫颈癌Hela、人乳腺癌MCF-7、人胃癌BGC823细胞株在24、48、72小时MTT法测定半数抑制浓度IC50，以及在IC50浓度下，作用于癌细胞48小时后，用AnnexinV-PI双染检测流式细胞仪分析早期凋亡细胞的比例，晚期凋亡细胞和坏死细胞比例，测定结果见表2。结果表明：8-N-甲基哌嗪亚甲基黄芩素具有较强的抑制癌细胞生长和诱导凋亡作用，其中诱导早期癌细胞调亡比例远大于晚期，Cdks抑制是诱导凋亡的关键机制，在医疗上可用于制备抗癌药物。
表2.8-N-甲基哌嗪亚甲基黄芩素对人癌细胞生长抑制与诱导凋亡
8-N-甲基哌嗪亚甲基黄芩素家兔体内主要代谢过程：高效液相-质谱-紫外光谱(LC-MS-DAD)检测结果表明：8-N-甲基哌嗪亚甲基黄芩素进入家兔体内后，迅速分布到各组织，与蛋白和组织有较高的结合率，并经历了较强的肝脏首过效应。代谢过程主要为甲基化和葡萄糖醛酸化。主要代谢产物为6-甲氧基-8-N-甲基哌嗪亚甲基黄芩苷，其次为6-甲氧基-8-N-甲基哌嗪亚甲基黄芩素。代谢物尿液中排泄体外，由胆汁分泌到肠道的6-甲氧基-8-N-甲基哌嗪亚甲基黄芩苷在肠道微生物β-葡萄糖醛酸水解酶的作用下被水成6-甲氧基-8-N-甲基哌嗪亚甲基黄芩素，部分被吸收入血，形成肝肠循环，另一部分则由粪便直接排除体外。
本发明的有益效果是：这种周期素依赖蛋白激酶抑制剂黄芩素哌嗪衍生物及其制备方法，将黄芩素、甲醛溶液和哌嗪类化合物按摩尔比混合，加入为黄芩素重量倍数的甲醇，在50-70℃下搅拌缩合，将析出的沉淀滤出，用少量甲醇洗涤，干燥，得含量不少于98％(重量)的黄芩素哌嗪衍生物。该黄芩素哌嗪衍生物抑制细胞周期素依赖蛋白激酶活性强度与Flavopiridol类似，比黄芩苷提高约50多倍，并具有较强的抗肿瘤活性。本品原料来源丰富，工艺简捷，纯度高，成本低，代谢机理明确，高效低毒，与酸成盐可制成口服制剂或注射剂，有望成为高效低毒的周期素依赖蛋白激酶抑制剂类抗癌和爱滋病药物。
附图说明
图1是8-N-甲基哌嗪亚甲基黄芩素对Cdk1/Cyclin B活性抑制测定曲线，横坐标为8-N-甲基哌嗪亚甲基黄芩素浓度nM，纵坐标为对Cdk1/Cyclin B活性抑制率％。
具体实施方式
下面结合实施例对本发明作进一步的说明。
实施例1：
8-N-甲基哌嗪亚甲基黄芩素的制备：取黄芩素100g，加入甲醇2L，甲基哌嗪45g，37％甲醛溶液36.5ml，在55℃下搅拌4小时，将析出的沉淀滤出，用少量甲醇洗涤，干燥，得到浅黄色固体113g，含量为99.9％。m.p.：280～282℃；UV：λmax(乙醇)278，330nm；MS：(API*ES)m/z 383.1[M+H]+，405.1[M+Na]+；IR：3400，3050，2925，2861，1637，1570，1509cm-1；1H NMR(DMSO-d6，400MHz)：δ2.14(s，3H，CH3)，2.62(s，8H，N-CH2)，3.44(s，7-OH)，3.96(s，2H，8-CH2)，6.87(s，1H，3-H)，7.46～7.54(m，3H，ArH)，8.01～8.02(m，2H，ArH)，8.11(s，6-OH)，12.64(s，1H，5-OH)。
实施例2
8-N-乙醇哌嗪亚甲基黄芩素的制备：取无水哌嗪90g，加入无水甲醇2L，氯乙醇70g，无水碳酸钾150g，溴化钾5g，加热回流1小时，过滤，在滤液中加入黄芩素270g，37％甲醛溶液100ml，在60℃下搅拌3小时，蒸出部分溶剂，放置，将析出的沉淀滤出，用少量甲醇洗涤，干燥，得到浅黄色固体350g，含量为99.5％。MS：(API-ES)m/z 413.2[M+H]+，435.2[M+Na]-；1H NMR(DMSO-d 6，400MHz)：δ2.70(s，8H，N-H2)，3.16(s，1H，7-OH)，3.99(s，2H，8-CH2)，4.15(d，2H，CH2-OH)，6.75(s，1H，3-H)，7.56～7.60(m，3H，Ar-H)，8.06-8.07(m，2H，Ar-H)，12.65(s，1H，5-OH)。
实施例3
8-N-甲基哌嗪亚甲基黄芩素片剂的制备：称取8-N-甲基哌嗪亚甲基黄芩素382g，加入甲磺酸96g，用适量乙醇润湿，加入适量淀粉，制粒，干燥，压成每片含200mg的素片，用丙烯酸树脂和柠檬黄包黄色薄膜衣。
实施例4
注射用8-N-乙醇哌嗪亚甲基黄芩素制备：称取8-N-乙醇哌嗪亚甲基黄芩素412g，枸橼酸824g，羟丙基-β-环糊精3000g，加注射用水5000ml溶解，超声处理20分钟，用氢氧化钠调节pH 4.5，过滤，分装，冻干，制成每瓶含黄芩素哌嗪衍生物50mg的注射用粉针剂，即得。
Claims (5)
1、一种周期素依赖蛋白激酶抑制剂黄芩素哌嗪衍生物，其特征在于：具有下述结构通式：
其中，哌嗪上的取代基R为CH3，CH2CH3，CH2CH2OH，CH2CH2SH，CH2COOH，CH2CHOHCH2OH，CH2CHOHCH2SH或CH2CHSHCH2OH。
2、据权利要求1所述的周期素依赖蛋白激酶抑制剂黄芩素哌嗪衍生物制备方法，其特征在于：将黄芩素、甲醛溶液和哌嗪类化合物按摩尔比为1∶1.2∶1.2混合，加入为黄芩素重量10-40倍的甲醇，在50-70℃下搅拌缩合1-6小时，将析出的沉淀滤出，用少量甲醇洗涤，干燥，得含量不少于98％(重量)的黄芩素哌嗪衍生物。
3、据权利要求2所述的周期素依赖蛋白激酶抑制剂黄芩素哌嗪衍生物制备方法，其特征在于：所述哌嗪类化合物选自哌嗪，甲基哌嗪，乙基哌嗪，哌嗪乙醇，哌嗪巯基乙醇，哌嗪乙酸，哌嗪丙二醇，哌嗪巯基-2丙醇-3或哌嗪巯基-3丙醇-2。
4、据权利要求1所述的周期素依赖蛋白激酶抑制剂黄芩素哌嗪衍生物，其特征在于：所述抗癌和爱滋病药物的黄芩素哌嗪衍生物制成片剂；片剂的配方为：黄芩素哌嗪衍生物1摩尔，甲磺酸盐1-2摩尔，淀粉为黄芩素哌嗪衍生物重量0.5-2倍，制成每片含黄芩素哌嗪衍生物200-500mg的黄色薄膜衣片；片剂的制备方法为：称取黄芩素哌嗪衍生物，甲磺酸，加用适量乙醇润湿，加入淀粉，制粒，干燥，压片，用丙烯酸树脂和柠檬黄色素包薄膜衣。
5、据权利要求1所述的周期素依赖蛋白激酶抑制剂黄芩素哌嗪衍生物，其特征在于：所述抗癌和爱滋病药物的黄芩素哌嗪衍生物制成针剂；针剂的配方为：黄芩素哌嗪衍生物1摩尔，柠檬酸为黄芩素哌嗪衍生物重量1-2倍，羟丙基-β-环糊精1-2摩尔，制成每瓶含黄芩素哌嗪衍生物10-50mg的注射用粉针剂；制备方法：称取黄芩素哌嗪衍生物，柠檬酸，羟丙基-β-环糊精，加注射用水溶解，超声处理20分钟，用氢氧化钠调节pH 3.5-5.5，过滤，分装，冻干。
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