CN1295231C溴二氢青蒿素---特别适用于治疗各种癌症的溴代二氢青蒿素|CN1295231C Artemisinin bromide---Artemisinin bromide is particularly suitable for treating various cancers
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CN1295231C溴二氢青蒿素---特别适用于治疗各种癌症的溴代二氢青蒿素
CN1295231C Artemisinin bromide---Artemisinin bromide is particularly suitable for treating various cancers
溴代二氢青蒿素
本发明公开了一种溴代二氢青蒿素，其特别适用于治疗各种癌症；申请人首创将二氢青蒿素上的3-C位甲基溴化，在二氢青蒿素的母核上直接引进杂原子；主要是以二氢青蒿素为母核，制成溴代二氢青蒿素；利用卤族元素有更强的极性，使二氢青蒿素的氧桥更容易断裂成自由基；大大增强药效，本发明溴化工艺简单，易于工业化生产，溴化有很好的专一性，使产品的合成工艺有很好的特异性，易于量化控制，对正常细胞无毒副作用，用溴代二氢青蒿素对人肝癌细胞株-Hepg2细胞毒性研究表明，溴代二氢青蒿素对体外Hepg2细胞的细胞毒IC50＜8nM。
溴代二氢青蒿素
技术领域
本发明涉及化学合成物技术领域，具体的说，是涉及二氢青蒿素的一种衍生物一溴代二氢青蒿素。
背景技术
现有从植物中提取的天然抗癌药物，由于其疗效有限和毒副作用强限制了其临床应用，始终不能成为抗癌的主力药物。
例如：紫杉醇被当今世界上公认为广谱、活性最强的抗癌药物，尤其是对子宫癌、卵巢癌、乳腺癌具有特殊的疗效，它的问世被誉为90年代国际上抗癌药三大成就之一；目前治疗妇女乳腺癌的常见用药有欧洲紫杉醇和太平洋紫杉醇；可以延长恶化时间两个月和增加存活时间3个月。
但两种紫杉醇都有副作用，病人会出现手脚麻痹的现象，太平洋紫杉醇毒性最常见且致命的副作用就是有15-20％的患者发生急性过敏性休克；副作用：抑制造血细胞，过敏，胃肠不适，以及轻度的肝脏损伤；欧洲紫杉醇常见的副作用是骨髓抑制、血球低下、疲倦等。
又如喜树碱：喜树碱是从我国喜树中提取的一种生物碱，应用于临床如消化系统肿瘤，黑色素瘤等之治疗并有部分成效；其作用机理经动力学的研究证明，喜树碱是属于细胞周期特异性药物，能使癌细胞停留于S期(DNA合成期)，阻止其进一步分裂。动物实验证明对Erlich腹水瘤细胞DNA、RNA的合成均有较明显抑制作用，上海地区用喜树碱治疗胃癌435例，有效率61％；但有严重副作用如骨髓抑制，出血性膀胱炎及消化系统之症状。
青蒿素是我国药学工作者1971年从菊科植物黄花蒿叶中提取分离到的一种具有过氧桥的倍半萜内酯类化合物；青蒿素及其衍生物是含过氧桥的倍半萜内酯类新型抗疟药，具有高效、快速、低毒、安全等特点；研究表明，青蒿素对疟原虫配子体有杀灭作用，其强度和剂量与配子体成熟度相关。
早期的研究表明，青蒿素选择性杀灭红内期疟原虫的机理主要是作用于疟原虫的膜系结构，使核膜、质膜破坏，线粒体肿胀皱缩，内外膜剥离，对核内染色物质也有一定影响，青蒿素及其衍生物通过影响线粒体的功能，阻断疟原虫营养的供应，从而达到抗疟目的；青蒿素类药应用十多年来尚未见有关抗药性的报道，在对多重抗药性恶性疟疾的治疗上，青蒿琥酯和蒿甲醚也有良好的疗效。
青蒿素及其衍生物还有以下作用：
抗卡氏肺孢子虫肺炎作用：动物实验证实，青蒿素对大鼠卡氏肺孢子虫肺炎有效；用双氢青蒿素60毫克/千克治疗大鼠卡氏肺孢子虫肺炎，大鼠存活数、存活率均高于感染组；治疗后大鼠平均肺重、平均肺重/体重比和包囊数均低于感染组，肺组织炎症反应明显减轻；进一步的研究表明，双氢青蒿素主要破坏卡氏肺孢子虫膜系结构，引起孢子虫滋养体胞浆及包囊内出现空泡，线粒体肿胀，核膜破裂，内质网肿胀，囊内小体溶解破坏等超微结构的改变。
抗孕作用：青蒿琥酯和双氢青蒿素对小鼠、金黄地鼠、大鼠及兔均有抗孕作用，金黄地鼠和豚鼠表现为流产，小鼠、大鼠和兔表现为胚胎吸收。青蒿琥酯(40毫克/千克)×5天可使妊娠大鼠血清孕酮含量下降并损伤胎膜和胎盘使胚胎坏死而终止妊娠；二氢青蒿素对体外培养的人胎膜细胞亦有直接杀伤作用；二氢青蒿素类药对胚胎有较高的选择性毒性，较低剂量即可使胚胎死亡而导致流产，但对母体子宫、卵巢和一般健康状况无明显影响，此类药有可能被开发为人工流产药物。
对肿瘤的作用：二氢青蒿素及其衍生物对鼠艾氏腹水瘤细胞和人HeLa细胞有细胞毒活性；用青蒿琥酯处理的HeLa细胞可见梯状DNA和凋亡小体。Beekman等对从9种不同组织中培养的60株肿瘤细胞进行检测发现，双氢青蒿素对白血病、黑色素瘤、结肠癌、前列腺癌和乳腺癌细胞株高度敏感，而对非小细胞肺癌、中枢神经系统肿瘤、卵巢癌和肾癌细胞株的活性较低；其抗肿瘤作用可能同二氢青蒿素与Fe2+反应产生大量自由基以及烷基化作用有关。
抗血吸虫作用：青蒿素及其多种衍生物均有抗血吸虫作用，在整个服药阶段对幼虫期的血吸虫都有杀灭作用，因此具有良好的预防效果，二氢青蒿素还能杀灭进入宿主体内的幼虫，对疫水接触者具有保护作用，用于感染日本血吸虫尾蚴后的早期治疗，可降低血吸虫感染率和感染程度，并可预防血吸虫病发生。其抗血吸虫活性基团是过氧桥，作用机理是影响虫体的糖代谢。在大规模应用青蒿琥酯预防性治疗接触疫水的人群时发现，17031人在服药后无急性血吸虫病发生，因此认为二氢青蒿素预防血吸虫病具有高效、安全、方便等特点，是目前比较理想的预防药。
治疗弓形虫感染作用：体外实验证实，青蒿素(蒿甲醚)能抑制弓形虫侵入细胞。青蒿素主要作用于虫体细胞膜、线粒体及细胞核，继而广泛损伤其膜系结构，造成核膜断裂、线粒体肿胀、空泡样变性、内质网扩张甚至出现核碎裂、核溶解现象。
对心血管的作用：青蒿素有减慢心率，抗心律失常，抑制心肌收缩力等作用。二氢青蒿素能明显对抗结扎冠脉引起的心律失常，可使氯化钙、氯仿引起的心律失常发作时间明显推迟，室颤明显减少，其作用与其抑制内向整流钾电流和浦肯野纤维瞬间外向钾电流有关。
对免疫系统的作用：双氢青蒿素、青蒿琥酯均对超适剂量免疫法诱导的供体鼠T细胞的产生有显著抑制作用，且两者都能增强受体鼠反应阶段细胞的活性。
其他：双氢青蒿素对杜氏利什曼原虫有显著抑制作用并呈剂量相关性。其机理系影响杜氏利什曼原虫前鞭毛体DNA合成，使虫体变形，核、动基体不完整，胞质内出现多个空泡，鞭毛脱落。青蒿提取物还可杀灭阴道毛滴虫和溶组织阿米巴滋养体。青蒿琥酯能松弛豚鼠气管平滑肌并非竞争性拮抗乙酰胆碱、组织胺的收缩气管作用，机理与激活腺苷酸环化酶关，且不被β肾上腺素受体噻吗洛尔所阻断。
近年来，各国的药物学家对青蒿素及其衍生物的抗癌作用进行了更广泛的研究，深层次药理、药效、毒副作用和药物动力学研究表明：
药理：用青蒿素及其衍生物对付血癌和乳腺癌细胞，发现青蒿素的选择性是其他化学疗法的100倍，而且，青蒿素可以杀死癌细胞，但不伤害周围健康细胞。“癌症细胞分裂时需要大量铁质才能复制DNA，因此癌细胞的铁质含量比正常细胞高出许多。”这些携带青蒿素衍生物的蛋白进入癌细胞后，青蒿素中的3-C位上的氧原子和12-C位上的氧原子之间形成氧桥，铁离子就被释放并与青蒿素衍生物反应，从而破坏癌细胞，氧桥断裂释放出自由基(氧原子)，自由基攻击癌细胞膜，使膜破裂而癌细胞死亡；这是把青蒿素转为抗癌药物的关键。由于癌细胞对铁的贪婪，使得药物具有很高的选择性；实验表明青蒿素标记的铁传蛋白选择和杀死癌症细胞的效率是杀死正常细胞的效率34000倍(资料出处)。
药效比较：IC50是体外癌细胞半数致死量，IC50能直观地反应药效强弱。
羟基喜树碱IC50为206u mol～305u molng；
紫杉醇IC50为8.6u mol；
二氢青蒿素IC50为24n mol(0.024 u mol)。
毒副作用：对神经系统、呼吸系统、心血管系统均无明显药理作用，仅在剂量大至40mg/kg时显示一定的镇痛、镇静作用。该药安全性较大，LD50为834.5mg/kg，化疗指数为834.0，大鼠20-180(MKD)连续30天给药，对生理、生化指标及主要脏器病理学检查均未见明显变化。特殊毒性方面，致突变实验阴性，生殖毒方面，在小鼠妊娠敏感期给药，增加吸收胎的发生，未见致畸作用。
药代动力学：小鼠灌服3H-二氢青蒿素后，血液内放射性迅速上升，半小时至1小时达到高峰，随后迅速下降，4小时降到峰值的一半，以后缓慢消失。胃肠道残留量的测量表明，半小时残留58％，1小时残留35.3％，半量消失时间约为1.2小时。经口给药后分布广泛，1小时开始达到高峰，同位素法表明各组织中胆、肝、肾最多，心、肺、脾等次之。显色法表明肌肉注射1～8小时达到高峰，肝、脑、骨、血液含量较高。口服后24小时内，80％放射性经粪、尿排出，显色法结果类似。上述结果表明二氢青蒿素进入体内后吸收快、分布广，排泄快。
1997年美国华盛顿大学生物工程系的赖亨利教授和纳伦德拉星开始设想同样的机理一定也能作用于癌症：癌症细胞分裂时需要大量铁质才能复制DNA，因此癌细胞的铁质含量比正常细胞高出许多；经研究发现，癌细胞比正常细胞含铁高5-15倍，高的达50倍，最高的白血病癌细胞居然达1000倍。赖教授称：“青蒿素不但有效，而且选择性非常强；对癌细胞有很高的毒性，但对正常细胞的影响很小。”它有可能成为无毒的高效抗癌药；哈医大的黑龙江省生物医药工程重点实验室杨宝峰、周晋教授研究发现，二氢青蒿素能有效抑制实体肿瘤细胞的增殖。他们发现，白血病细胞膜是二氢青蒿素攻击的一个主要靶点，起抗瘤机制有“凋亡”和“胀亡”两种。白血病细胞膜遭到破坏后，大量的钙离子就会进入细胞内，一方面引起细胞程序化死亡，即“凋亡”，另一方面导致细胞内的渗透压发生改变，吸收大量水分，使细胞膨胀直至死亡，即“胀亡”；这种推测在临床实验中得到了广泛支持：将若干组乳腺癌细胞和正常乳腺细胞与转铁蛋白接触，8小时以后，只剩下25％癌细胞。16小时过去以后，几乎所有癌细胞都死亡了，而正常细胞不受影响。例如一只患有严重骨癌的狗已经不能行走，在接受二氢青蒿素辅之以铁的治疗下，5天就完全恢复，赖教授的理论得到了验证。
现有技术中，上海药物研究所以二氢青蒿素酯、醚衍生物的方式来引进卤素，虞佩琳等二氢青蒿素类似物的研究、药学学报1985；20(5)：357~365，在抗癌实验(A549)中，对比物IC50是1227nM，引进溴元素的IC50是47nM，药效增强约26倍(Ying Li et al.Novel Antitumor Artemisinin DerivativesTargeting G1 Phase of the Cell Cycle.Bioorg.& Med.Chem.Lett.11(2001)5-8)。
专利权人是中国科学院上海药物研究所的中国专利02128494、名称为叔丁氧羰基二氢青蒿素、其制备方法及药物组合物，其发明提供结构式所示的叔丁氧羰基二氢青蒿素：系以二氢青蒿素为起始原料，与双叔丁基二碳酸酯在有机溶剂中进行酰化反应而制得；发明的抗寄生虫病药物组合物包含治疗有效量的叔丁氧羰基二氢青蒿素和药学上可接受的载体，与以前合成的二氢青蒿素衍生物相比，叔丁氧羰基二氢青蒿素有最大的治疗指数(＞1700)，是高效、低毒的抗寄生虫药物，在防治血吸虫病、疟疾等寄生虫病过程中可减少毒副作用的产生，尤其是在恶性疟疾的治疗中，对降低儿童死亡率有重大的意义。
申请人是浙江大学、申请号是03116762.4、发明名称为青蒿琥酯和二氢青蒿素抗血管生成作用的药物制剂及用途的中国专利申请公开了青蒿琥酯和二氢青蒿素在抗血管生成作用方面的药物制剂及其用途；该药物制剂主要含有青蒿琥酯或二氢青蒿素，其制剂形式主要为微球注射液；该发明提供的药物制剂在抗肿瘤血管生成方面有活性，可用于肿瘤血管生成及其他与血管生成有关的疾病治疗，还可用于肿瘤化疗和/或辅助化疗方面的治疗。该制剂在给药部位缓慢释放药物与吸收，延长药物作用时间；该发明以血管生成理论为背景，研究并阐明中药有效单体成分作用和机制，是发展中医药理论新的重要方向，为发现新的理论和药物作用新的靶点提供重要依据，为二氢青蒿素类药物的新用途开发提供依据。
综上所述，现有技术有以下方面的不足：
首先是青蒿素及其衍生物的医药用途虽然得到验证和肯定，但对癌症的适应症范围不够全面；
其次是现有的二氢青蒿素及其衍生物治疗癌症的药效还有待提高。
技术内容
因此，人们期待着具有更为广谱、特效、安全性高、毒副作用小和给药方式更简单的治疗癌症的药物，本发明的目的就是旨在克服上述现有技术缺限，提供一类基于二氢青蒿素的新化合物，它具有极为广谱的治疗癌症的医学效果和特别的疗效，且无任何毒副作用。
为了达到上述发明目的，本发明人研究了很多二氢青蒿素及其衍生物，并结合现有技术的教导，考虑到卤族元素取代基对药物效用有积极的影响，如溴化物具有加强药效的功能，成为许多感冒糖浆中的最佳药剂，通过无数次选择、试验，我们自主合成了溴代二氢青蒿素。
实际上，在二氢青蒿素的母核上引进杂原子是非常困难的，上海药物研究所是以二氢青蒿素酯、醚衍生物的方式来引进卤素。
溴代二氢青蒿素的化学名称为(3R，5aS，6R，8aS，9R，12S，12aR)-八氢-3-溴代亚甲基-6，9-二甲基-3，12-桥氧-12H-吡喃并[4，3-j]-1，2-苯并二塞平-10(3H)醇。
其结构式为：
本发明的主要目的是将二氢青蒿素上的3-C位甲基卤化。
合成溴代二氢青蒿素的步骤为：将二氢青蒿素溶于酯溶性溶剂，在溶液中导入溴源，进行合成反应，并去除残留溴源，后萃取提纯，并脱水结晶，得到标的化合物。
由于二氢青蒿素具有过氧桥的倍半萜内酯类结构，其应选择酯溶性溶剂对其溶解分散，较为优选的酯溶性溶剂是：乙腈、二甲基甲酰胺、乙酸、氯仿、四氯化碳。
上述的合成工艺中，所述的溴源可是单一成份的气体或液体，也可是溴化物。
液溴、溴氢酸、溴化氢气体等都可作为标的化合物的溴源导入。
在合成工艺中，当导入溴源进行溴化反应时，可加入催化剂对溴化反应进行催化。
所述的催化剂是二氧化锰、硅铝酸盐、卤化亚铜；
或是用卤钨灯直接进行照射。
本发明涉及的溴代二氢青蒿素在制备治疗癌症的药物组合物中可得到极积的应用；
本发明涉及的溴代二氢青蒿素可制成各种符合药剂学要求的各种口服剂型；
本发明涉及的溴代二氢青蒿素的可制成各种符合药剂学要求的各种外用剂型；
本发明涉及的溴代二氢青蒿素可制成各种符合药剂学要求的各种注射剂型；
所述的口服剂型包括：滴丸、速释滴丸、胶囊、颗粒、喷雾剂、口服液、片剂、骨架型缓释制剂包括：(1)不溶性(如乙基纤维素EC、聚乙烯、聚丙烯、聚硅氧烷、乙烯-醋酸乙烯共聚物、聚甲基丙烯酸甲酯等)；(2)蜡质骨架(如脂肪、蜡类物质、硬脂酸、硬脂醇、单硬脂酸甘油酯、巴西棕榈蜡、十八烷醇等)；(3)亲水凝胶(如CMC、CMC-Na、MC、PVA、HEC、SCMC、海藻酸钠、果胶、藻酸盐、琼脂、羟丙基甲基纤维素HPMC、脱乙酰壳多糖、壳多糖、半乳糖甘露聚糖等)；(4)胃内滞留片等；缓释包衣制剂包括：(1)膜控释小片、(2)微孔膜包衣片、(3)小丸剂包括：膜控小丸、骨架型小丸；
所述的注射剂型包括：注射液(普通)、冻干粉针、粉针(普通)、大输液、高浓度注射液、注射用片剂；
所述外用剂型包括：膜剂、栓剂、气雾剂、透皮制剂释药
上述剂型中：
滴丸是在中药丸剂基础上发展起来的，具有传统丸剂所没有的多种特点，故发展非常迅速。由于片剂服用量大，崩解度差，对肠胃道有刺激作用，而滴丸用舌下含服，从而大大减少了对肠胃道刺激。与其它剂型(软胶囊、冲剂、颗粒剂、胶囊剂、口服液)比较，具有比表面积大，溶出速度快的特点，这是因为滴丸可提高难溶性药物的生物利用度。由于滴丸是在骤冷条件下形成的固体分散体，药物以极小的晶粒存在，故舌下含服经舌粘膜吸收，直接进入血循环，起效快；临床应用适合于对口腔癌、喉癌的专用药。
缓释控释制剂
近年来缓释、控释制剂为开发研究热点。缓释制剂是指可以减少服药次数，提供比较平稳的血药浓度的制剂，以达到减少副作用，维持持久药效的目的。控释制剂是指通过制剂手段提供释放药物的程序，在预定的时间内药物自动按某一速度从剂型中释放与作用器官或特定的靶部位，使血药浓度长时间恒定维持在有效浓度范围内，释药均匀平衡，控释体系释药速度与时间无关，避免了传统常规制剂给药频繁所出现的“峰谷”现象，提高了临床用药安全性与有效性，可代替静脉滴注，也可根据机体需要自动控制给药速度。其目的在于寻求提供理想血药浓度的途径，提高药物的安全性和有效性。口服缓释控释制剂的研究发展很快，其种类不断增加，设计方法逐渐趋于半定量或定量化；临床应用适合于对全身各种癌症的用药。
膜剂
膜剂是近年来国内外研究和应用进展很快的剂型，临床很受欢迎，可用于口服、口眼耳鼻喉、皮肤及妇科癌症等。随着TTS(即透皮治疗系统)的不断发展，一些膜剂尤其是鼻腔、皮肤用药膜亦可起到全身作用，故在临床应用上有取代部分片剂、软膏剂和栓剂等的趋势。且具有疗程短、副作用小、药膜释放速度快的优点，是治疗各种阴道癌、宫颈癌的首选药物。由于膜剂本身体积小，重量轻，随身携带极为方便；如将生物粘附技术引入食道癌治疗的靶向制剂，所制成的磁性微球可很好地将药物粘附在癌细胞上；临床应用适合于口、咽、鼻、喉、皮肤、妇女阴道、宫颈癌的专用药。
微囊
微囊是利用天然或合成高分子材料或共聚物(囊膜材料)将药物包裹而成的一种新的剂型。药物微型包裹后利于携带，便于服用。它的优点在于可延长或控制药物的释放，制成长效制剂。囊膜有隔离外界与药物接触作用，可防止药物氧化、水解和挥发，掩盖不良气味，减少复方制剂中的配伍禁忌。也可制备特殊性能微囊(磁性微囊、PH敏感微囊)起到靶向释药作用，采用明胶冻凝法将其包成微囊，其包囊率及温室贮存稳定性较好。
微囊系利用天然的或合成的高分子材料将固体或液体药物包裹而成的直径1-500um的微小胶囊，其外型取决于囊心物质的性质和囊材凝聚的方式，微囊外面呈球状实体或呈平滑的球状膜壳形，葡萄患形及表面平滑或折叠的不规则结构等各种形状，它常用于增加药物的稳定性，掩盖药物的不良气味，改良和延缓药物的释放；临床应用适合于全身癌症的用药。
栓剂
栓剂不仅可起到局部治疗作用，而且还可通过直肠吸收到全身治疗作用，直肠给药后，药物不直接通过肝脏，可防止或减少药物在肝脏的代谢，减轻药物对肝脏的毒副作用。栓剂具有吸收快，起效迅速，生物利用度高，能维持较长时间血药浓度的优点。腔肠给药用药的有发展潜力的主渠道之一，这一领域的研究必定会推动栓剂的迅速发展；临床应用适合于肠癌、前列腺癌等。
气雾剂
气雾剂具有剂量小，分布均匀，奏效快，使用方便等特点。吸入时可减少胃肠道副作用，外用则避免对创面的刺激性，并可用定量阀门控制剂量，具有速效和定位作用。临床上主要用于幼儿不能口服或不愿口服者；本剂型临床应用适合于上皮肤癌、上呼吸道癌、肺癌等。
靶向制剂
靶向给药也是近年来国内外一个极为重要的开发热点，尤其在抗癌药物方面已取得重大发展，其原理为抗癌物与铁磁性材料包封与高分子骨架材料中，制成的超微球控释制剂在体外磁场导向下聚集滞留在靶区的癌组织上，缓慢释放药物，对癌细胞，进行有效的攻击，既可避免伤害正常细胞，又可减少用药量和降低毒性，提高疗效。磁性靶位释药系统在靶位给药方面提供了一个新的开发途径，而脂质体仍为靶向制剂中研究的热点；本剂型临床应用适合于全身各种癌症的用药。
微丸
是指直径小于2.5mm的各类丸剂，可根据不同需要将其制成速释或缓释微丸。速释微丸可使药物迅速释放。本研究的重点是缓释微丸，缓释微丸是由药物与阻滞剂混合制成或先制成普通丸芯而后再包控释膜而成。微丸压制成片，或将速释微丸与缓释微丸装于胶囊壳中制成控释胶囊剂。
微丸之有许多其他口服制剂无法相比的优点：①可通过控释微丸包衣制成缓控释制剂；②在胃肠道分布面积大，生物利用度高，刺激性小；③由于粒径小，受消化道输送食物节律影响小(如幽门关闭等)；④控释微丸可使血药浓度迅速达到疗效浓度，并维持平稳、长时间的有效浓度，血药波动小；⑤微丸的流动性好，大小均匀，易于处理(如包衣、分剂量)；⑥改善药物稳定性，掩盖不良味道；⑦适合复方制剂的配伍；本剂型临床应用适合于全身各种癌症的用药。
透皮制剂释药
本透皮制剂主要为凝胶制剂，可持续维持本药的血药浓度24小时，避免口服和静脉给药途径的血药浓度波动；本剂型临床应用适合于全身各种癌症的用药，尤其是皮肤癌。
二氢青蒿素可直接购买，也可直接从植物中提取青蒿素，后还原成二氢青蒿素。
与现有的二氢青蒿素相比，溴代二氢青蒿素有以下几方面突出的优点：
申请人首创在在二氢青蒿素的母核上直接引进杂原子，将二氢青蒿素上的3-C位甲基溴化。
药效更强：卤族元素的极性更强，因此比二氢青蒿素的生理活性更强；按二氢青蒿素抗癌原理，其作用方式是其特有的氧桥断裂产生自由基，引起癌细胞膜破裂而杀死癌细胞；a-甲基的溴化有利于吸引3，12碳位上的氧桥断裂产生自由基，可以大大增强药效，按四川大学华西医学院用溴代二氢青蒿素对人肝癌细胞株-Hepg2细胞毒性研究表明，溴代二氢青蒿素对体外Hepg2细胞的细胞毒IC50＜8nM。
溴化工艺简单、并易于工业控制，有很好的专一性，使产品的合成工艺有很好的特异性，并且易于量化控制。
溴代二氢青蒿素无毒副作用。
附图说明
图1是溴代二氢青蒿素体用于体外癌细胞株癌细胞生存率示意图(％)。
图2是合成溴代二氢青蒿素的工艺流程图。
具体实施方式
实施例1：
本实施例描述用黄花青蒿制备青蒿素、将青蒿素还原成二氢青蒿素、用二氢青蒿素导入溴源合成溴代二氢青蒿素的过程。
黄花青蒿5000Kg用8倍乙醇24h回流浸提，浸提液注入层析硅胶柱，直到流完；用石油醚洗脱，开始为黄绿液体；洗到液体基本为无色透明或用硅胶G板薄层监测有浅蓝色荧光斑点时，换洗脱液为10％乙酸乙酯+90％石油醚洗脱，并收集其洗脱液，用上述薄层监测至无蓝色荧光斑点； 收集的洗脱液浓缩，静置过夜，收集析出的粗结晶；用30倍的50％的乙醇液重结晶，得青蒿素针状结晶；
将青蒿素10Kg溶入300L甲醇中，搅拌下加季铵盐EtNCl 4Kg，充分搅拌混均后加入硼氢化钾4Kg，加磷酸二氢钠1.65Kg，室温下反应至白色结晶析出，质液明显分离；将全部反应物倒入0℃的冰水中，静置析出白色固体；收集白色固体，用蒸馏水反复洗涤3次、干燥，得二氢青蒿素，为白色粉末状结晶。
将反应釜用N2干燥，加入100mol二氢青蒿素和1000L氯仿，溶解；加入14KgMnO2，搅匀。并于40～60℃温度下以大约16L/分钟的速度导入液溴100mol，同时进行搅拌；60分钟后，过滤反应物；向过滤物中加入1000～5000L10％的硫代硫酸钠水溶液洗涤，收集有机层；再加入5000～15000L10％的碳酸氢钠溶液洗涤，收集有机层；有机层用无水硫酸钠脱水后减压浓缩成白色结晶，晶体用正己烷洗涤，滤出结晶，干燥，得到溴代二氢青蒿素，为白色粉末状结晶。
实施例2：
本实施例描述用青蒿素还原成二氢青蒿素、用二氢青蒿素导入溴源合成溴代二氢青蒿素的过程。
市售青蒿素粗粉(与生药比为1∶4)备用加5倍50％乙醇溶解，溶解液注入层析硅胶柱，直到流完；用石油醚洗脱，开始为黄绿液体；洗到液体基本为无色透明或用硅胶G板薄层监测有浅蓝色荧光斑点时，换洗脱液为40％乙酸乙酯+60％石油醚洗脱，并收集其洗脱液，用上述薄层监测至无蓝色荧光斑点；收集的洗脱液浓缩，干燥；用乙酸乙酯精制，得青蒿素针状结晶。
将提纯后的青蒿素10Kg溶入300L50％乙醇中，搅拌下加季铵盐EtNCl 4Kg，充分搅拌混均后加入硼氢化钾4Kg，加磷酸二氢钠1.65Kg，室温下反应至白色结晶析出，质液明显分离；将全部反应物倒入0℃的冰水中，静置析出白色固体；收集白色固体，用蒸馏水反复洗涤3次、干燥，得二氢青蒿素，为白色粉末状结晶。
反应釜用N2干燥，加入100mol二氢青蒿素和1000L四氯化碳，溶解；加入14KgMnO2，搅匀。并于40℃温度下以大约16L/分钟的速度导入液溴，同时进行搅拌；90分钟后，过滤反应物；过滤物用1000～2000L的水搅拌洗涤，收集有机层；有机层用无水硫酸钠脱水后减压浓缩成白色结晶，晶体用正己烷洗涤，滤出结晶，干燥，得到溴代二氢青蒿素，为白色粉末状结晶。
实施例3溴代二氢青蒿素的制备
反应釜用N2干燥，加入100mol二氢青蒿素和1000L氯仿，溶解；加入14KgMnO2，搅匀。并于40～60℃温度下以大约16L/分钟的速度导入液溴100mol，同时进行搅拌；60分钟后，过滤反应物；向过滤物中加入1000～5000L10％的硫代硫酸钠水溶液洗涤，收集有机层；再加入5000～15000L10％的碳酸氢钠溶液洗涤，收集有机层；有机层用无水硫酸钠脱水后减压浓缩成白色结晶，晶体用正己烷洗涤，滤出结晶，干燥，得到溴代二氢青蒿素，为白色粉末状结晶。
实施例4溴代二氢青蒿素的制备
反应釜用N2干燥，加入100mol二氢青蒿素和1000L四氯化碳，溶解；加入14KgMnO2，搅匀。并于40℃温度下以大约16L/分钟的速度导入液溴，同时进行搅拌；90分钟后，过滤反应物；过滤物用1000～2000L的水搅拌洗涤，收集有机层；有机层用无水硫酸钠脱水后减压浓缩成白色结晶，晶体用正己烷洗涤，滤出结晶，干燥，得到溴代二氢青蒿素，为白色粉末状结晶。
实施例5溴代二氢青蒿素的制备
反应釜用N2干燥，加入100mol二氢青蒿素和1000L乙腈，溶解；于室温下以大约10L/分钟的速度导入液溴，同时进行搅拌；在1000W卤素灯光催化下，反应6小时；向反应物中加入1000～5000L10％的硫代硫酸钠水溶液洗涤，收集有机层；再加入5000～15000L10％的碳酸氢钠溶液洗涤，收集有机层；有机层用无水硫酸钠脱水后减压浓缩成白色结晶，晶体用正己烷洗涤，滤出结晶，干燥，得到溴代二氢青蒿素，为类白色粉末状结晶。
实施例6溴代二氢青蒿素的制备
1.5Kg二氢青蒿素和5L氯仿混合，溶解；加入14KgMnO2，搅匀。并于40℃温度下以大约16L/分钟的速度导入液溴，同时进行搅拌；90分钟后，过滤反应物；过滤物再用氯仿精制一次；用无水硫酸钠脱水后减压浓缩成白色结晶，晶体用正己烷洗涤，滤出结晶，干燥，得到溴代二氢青蒿素，为白色粉末状结晶。
实施例7
Br-DHA对人肝癌细胞株-Hepg2细胞毒性的研究报告(四川大学华西医学院2004.12.22)
1.材料：
1.1细胞株：人肝癌细胞株Hepg2购于美国ATCC，10％FBS/DMEM常规培养。
1.2受试品Br-DHA：白色粉末，平均分子量320g/mol，批号20041205。硫酸亚铁：白色粉末，批号：20041205。转铁蛋白，Sigma公司。
2.方法：
2.1细胞毒实验方法：
取对数生长期的Hepg2细胞，常规方法消化，以8×103的密度接种于24孔细胞培养板。接种24h后，接表1分别加入转铁蛋白、硫酸亚铁，培养8小时后，加入Br-DHA。药物作用72小时后，常规方法细胞计数结果见图1。
3.结果：(见下表)
4.小结
在上述实验条件下，发现Br-DHA对体外Hepg2细胞的细胞毒IC50＜8nM。
表1：Br-DHA给药剂量表
| 组别 | 转铁蛋白(nM) | 硫酸亚铁(mg) | Br-DHA(nM) |
| 空白 | ------------- | ------------- | ------------ |
| 转铁蛋白 | 880nM | 0.25 | |
| Br-DHA | 880nM880nM880nM | 0.250.010.002 | 1000408 |
注：硫酸亚铁：Br-DHA＝250ng：1nM；每剂量样本数N＝4
实施例8
Br-DHA体外抗肿瘤活性的研究(四川大学华西药学院)。
1.材料
1.1细胞株：
人肝癌细胞株Hepg2购于美国ATCC，
人肺癌细胞株A549，购于中国科学院上海细胞所。
1.2培养基：
Dulbecco’s Modified Eagle Medium(DMEM)：GIBCOBRL，Cat.No：12100-038，RPMI1640：GIBCOBRL，Cat.No：430-1800EB，
Fetal Bovine Serum：Cat.No：CH30160.03
胰酶：GIBCOBRL，Cat.No：27250-018。
1.3受试品：
Br-DHA：白色粉末，平均分子量320，大陆蓉东药业，批号：20041205，
硫酸亚铁：白色粉末，大陆蓉东药业，批号：20041205，
转铁蛋白：白色晶状粉末，平均分子量79000，Sigma。
2.方法
2.1细胞培养：
Hepg2与A549细胞分别常规培养于10％FBS/DMEM与10％FBS/RPMI1640中，2-3天换液。
2.2药物配制：
用十万分之一电子天平精密称取转铁蛋白、硫酸亚铁、BrDHA，其中转铁蛋白、硫酸亚铁直接溶于细胞培养基后过滤除菌，转铁蛋白Co＝17.6nM，硫酸业铁Co＝5mg/L；而BrDHA先溶于DMSO后，再用细胞培养基稀释到所需浓度，过滤除菌Co＝6.4mg/L。受试品的不同浓度由低比稀释配制。
转铁蛋白、硫酸亚铁、BrDHA的溶液均于临用前新鲜配制，每孔加50uL。
2.3细胞毒试验方法：
取对数生长期的Hepg2、A549细胞，常规方法消化，以8×103的密度接种于2/4孔细胞培养板。接种24h后，接表1、2分别加入转铁蛋白、相应剂量的硫酸亚铁。孵育8h后，加入各剂量的Br-DHA。药物作用72小时后，常规方法细胞计数结果见表2、3。
3.结果：见表2、表3。
4.结论：本实验表明Br-DHA对人肝癌细胞株Hepg2细胞和人肺癌细胞株A549细胞均有非常明显的体外抗肿瘤作用，对Hepg2和A549细胞的IC50分别＜8nM和31.6nM。(表2、表3见本页)
表2、Br-DHA对人肝癌细胞株--Hepg2体外抗肿瘤活性
| 组别 | 样本数 | 药物剂量 | 细胞生存率(％) | ||
| 转铁蛋白nM | 硫酸亚铁mg/L | BrDHAnM | |||
| 空白对照 | 4 | ----- | ----- | ----- | 100.0±9.1 |
| 转铁蛋白 | 4 | 880 | 0.25 | ----- | 44.7±5.6 |
| BrDHA | 4 | 880 | 0.25 | 1000 | 23.3±5.3 |
| 4 | 880 | 0.05 | 200 | 32.7±14.1 | |
| 4 | 880 | 0.01 | 40 | 31.1±10.6 | |
| 4 | 880 | 0.002 | 8 | 41.6±3.6 | |
注.硫酸亚铁：BrDHA＝250ng：1nM
表3、Br-DHA对人肺癌细胞株--A549体外抗肿瘤活性
| 组别 | 样本数 | 药物剂量 | 细胞生存率(％) | ||
| 转铁蛋白nM | 硫酸亚铁mg/L | BrDHAnM | |||
| 空白对照 | 4 | ----- | ----- | ----- | 100.0±14.9 |
| 转铁蛋白 | 4 | 880 | 0.25 | ----- | 59.0±9.8 |
| BrDHA | 4 | 880 | 0.25 | 1000 | 17.4±7.5 |
| 4 | 880 | 0.05 | 100 | 39.0±5.2 | |
| 4 | 880 | 0.0025 | 10 | 58.2±5.2 | |
| 4 | 880 | 0.00025 | 1 | 59.0±1 3.0 | |
注.硫酸亚铁∶BrDHA＝250ng∶1nM。
实施例9
中国科学院成都分院分析测试中心
分析、测试结果报告单
溴化率的计算：
1、二氢青蒿素(DHA)分子量为284.34，溴原子(Br)分子量为79.90，氢原子(H)分子量为1；
2、二氢青蒿素的一个氢原子被溴原子取代后，
溴代二氢青蒿素的分子量为：(284.34-1)+79.90＝363.24
3、溴化率的计算：
79.90÷363.24＝21.996％≈22.0％。
检验报告上22.90％属正常值。...
Bromo-dihydroartemisine
The present invention discloses bromo-dihydroarteannuin which is especially suitable for treating various kinds of cancer. In the present invention, firstly, 3-C methyl of dihydroarteannuin is bromized, and hetero atoms are directly introduced into the mother nucleus of the dihydroarteannuin; the dihydroarteannuin is mainly used as the mother nucleus to prepare bromo-dihydroarteannuin; by using the high polarity of halogen elements, the oxygen bridge of the dihydroarteannuin is easily ruptured to form a free radical, and the effect of medicine is largely increased. The present invention has the advantages of simple bromination technology, easy industrialized preparation and good bromination specificity. Thus, the synthesis technology of the product has good specificity, the product is convenient for quantization control, and the product has no toxic or side effect on normal cells. Researches on the cytotoxicity of the liver cancer cell strain-Hepg2 of a human body by using bromo-dihydroarteannuin indicate that the IC50 of the bromo-dihydroarteannuin for the cytotoxin of Hepg2 cells in vitro is less than 8nM.
Br-DHA
Technical field
The present invention relates to the synthetics technical field, specifically, relate to a kind of derivative one bromo Dihydroartemisinin of Dihydroartemisinin.
Background technology
The existing natural anti-cancer drugs that extracts from plant because its curative effect finite sum toxic side effect has limited its clinical application by force, can not become anticancer main force's medicine all the time.
For example: taxol is known as wide spectrum, the strongest active cancer therapy drug in the world today, especially uterus carcinoma, ovarian cancer, mammary cancer are had special curative effect, and its appearance is described as the nineties of one of anticarcinogen three big achievements in the world; The common medication of treatment women breast cancer at present has the pure and mild Paclitaxel of European yew; Can prolong two months deterioration time and increase by 3 months survival time.
But two kinds of taxols all have side effect, and the phenomenon of hand and foot numbness can appear in patient, and the common and the most fatal side effect of Paclitaxel toxicity is exactly that acute anaphylactic shock takes place for the patient of 15-20%; Side effect: suppress hematopoietic cell, allergy, gastrointestinal upset, and slight hepar damnification; The common side effect of European yew alcohol is that bone marrow depression, blood cell are low, tired etc.
Camptothecine and for example: camptothecine is a kind of alkaloid that extracts from China camplotheca acuminata, is applied to clinically in digestive system tumor, and the treatment of melanoma etc. also has the part effect; Its mechanism of action studies have shown that through dynamic (dynamical) camptothecine is to belong to cell cycle specific agents, can make cancer cells stay in the S phase (DNA synthesis phase), stops its further division.The experimentation on animals proof all has than obvious restraining effect the synthetic of Erlich ascitic tumor cell DNA, RNA, and cancer of the stomach 435 examples, efficient 61% are treated with camptothecine in the area, Shanghai; But serious side effects such as bone marrow depression are arranged, the symptom of hemorrhagic cystitis and Digestive tract.
Artemisinin be China pharmacy worker 1971 from feverfew chrysanthemum mugwort extraction separation to a kind of sesquiterpene lactones compounds with peroxide bridge; Artemisinin and derivative thereof are the novel antimalarial drugs of sesquiterpene lactones class that contains peroxide bridge, have efficient, fast, characteristics such as low toxicity, safety; Studies show that Artemisinin has killing action to gametocyte, its intensity is relevant with the gametophyte ripening degree with dosage.
Early stage studies show that, the plasmodial mechanism of Artemisinin selectively killing erythrocytic stage mainly is to act on plasmodial film structure, nuclear membrane, plasma membrane are destroyed, the mitochondrial swelling shrinkage, interior adventitia is peeled off, and coloring matter in examining is also had certain influence, and Artemisinin and derivative thereof are by the mitochondrial function of influence, the supply of blocking-up plasmodium nutrition, thus reach the antimalarial purpose; Arteannuin medicine is used and is not seen relevant drug-fast report during the last ten years as yet, and in the treatment to the multiple drug resistance pernicious malaria, Artesunate and Artemether also have good curative effect.
Artemisinin and derivative thereof also have following effect:
The effect of anti-Pneumocystis carinii pneumonia: experimentation on animals confirms that Artemisinin is effective to rat Pneumocystis carinii pneumonia; With dihydroarteannuin 60 mg/kg treatment rat Pneumocystis carinii pneumonia, survival of rats number, survival rate all are higher than infected group; Heavy, average lung weight/weight ratio of the average lung of treatment back rat and packing number average are lower than infected group, and the lung tissue inflammatory reaction obviously alleviates; Studies show that further dihydroarteannuin mainly destroys the Pneumocystis carinii film structure, cause in sporozoite trophont endochylema and the packing cavity to occur, mitochondrial swelling, nuclear membrane breaks, endoplasmic reticulum swelling, Ultrastructural changes such as the interior corpusculum dissolved destruction of capsule.
Anti-pregnant effect: Artesunate and dihydroarteannuin all have anti-pregnant effect to mouse, Golden Hamster, rat and rabbit, and Golden Hamster and cavy show as miscarriage, and mouse, rat and rabbit show as the embryo and absorb.Artesunate (40 mg/kg) * pregnant rat serum progesterone content was descended in 5 days and damage fetal membrane and placenta to make embryo necrosis and termination of pregnancy; Dihydroartemisinin also has direct killing effect to people's fetal membrane cell of vitro culture; Dihydroartemisinin class medicine has higher selective toxicity to the embryo, can make embryonic death and cause miscarriage than low dosage, but parent uterus, ovary and general health situation are not had obvious influence, and this type of medicine might be developed to the artificial abortion medicine.
Effect to tumour: Dihydroartemisinin and derivative thereof have cytotoxic activity to mouse ehrlich ascites tumor cell and human Hela cell; Visible scalariform DNA of HeLa cell and apoptotic body with the Artesunate processing.Beekman etc. detect discovery to the 60 strain tumour cells of cultivating from 9 kinds of different tissues, dihydroarteannuin is extremely sensitive to leukemia, melanoma, colorectal carcinoma, prostate cancer and breast carcinoma cell strain, and lower to the activity of nonsmall-cell lung cancer, central nerve neuroma, ovarian cancer and renal cancer cell line; Its antitumor action may be relevant with Fe2+ reaction a large amount of free radicals of generation and alkylating with Dihydroartemisinin.
Anti-schistosome function: Artemisinin and multiple derivative thereof all have anti-schistosome function; in the whole stage of taking medicine the schistosomicide of larval stage all there is killing action; therefore has the good preventing effect; Dihydroartemisinin can also be killed and enter the intravital larva of host; the contactee has provide protection to epidemic disease water; be used to infect the early treatment behind the schistosoma japonicum cercariae, can reduce schistosomicide rate and gradient of infection, and can prevent schistosomicide to take place.Its schistosomicide active group is a peroxide bridge, and the mechanism of action is the carbohydrate metabolism that influences polypide.When the crowd of large-scale application Artesunate prophylactic treatment contact epidemic disease water, find, 17031 people are taken place at the no acute schistosomiasis in back of taking medicine, therefore thinking that Dihydroartemisinin prevents characteristics such as schistosomicide has efficiently, safety, convenience, is present more satisfactory prophylactic agent.
The effect of treatment arch insect infection: experiment in vitro confirms that Artemisinin (Artemether) can suppress toxoplasma gondii and invade cell.Artemisinin mainly acts on polypide cytolemma, plastosome and nucleus, and its film structure of extensive injuries causes nuclear membrane fracture, mitochondrial swelling, the sex change of cavity sample, reticulum dilatation even nuclear fragmentation, karyolysis phenomenon occur then.
To cardiovascular effect: sweet wormwood have reducing heart rate, and anti-arrhythmia suppresses effects such as myocardial contraction.Dihydroartemisinin can obviously resist the irregular pulse that the ligation coronary artery causes, the arrhythmia episode time that calcium chloride, chloroform are caused obviously postpones, quiver and obviously reduce in the chamber, its effect is relevant with Purkinje fiber export-oriented potassium current of moment with its inhibition inward rectification potassium current.
To immune effect: dihydroarteannuin, Artesunate all have remarkable restraining effect to the generation of super suitable dosage immunization inductive donor mouse T cell, and both can both strengthen acceptor mouse step of reaction cell activity.
Other: dihydroarteannuin has remarkable restraining effect and is dosage correlation Leishmania donovani.It is synthetic that its mechanism system influence Leishmania donovani promastigote DNA, makes the polypide distortion, and nuclear, kinetoplast are imperfect, a plurality of cavitys of appearance in the kytoplasm, and flagellum comes off.Artemisinin also can be killed Trichomonas vaginalis and amoeba histolytica's trophont.Can the relax contraction tracheae effect of guinea pig tracheal smooth muscle and noncompetitive antagonism vagusstoff, histamine of Artesunate, mechanism and activated adenyl cyclase close, and are not blocked by the beta-2 adrenoceptor timolol.
In recent years, the medicine scholar of various countries has carried out research widely to the antitumous effect of Artemisinin and derivative thereof, and profound pharmacology, drug effect, toxic side effect and pharmacokinetic study show:
Pharmacology: tackle leukemia and breast cancer cell with Artemisinin and derivative thereof, find that the selectivity of Artemisinin is other chemotherapeutic 100 times, and Artemisinin can kill cancer cell, but does not injure healthy cell on every side." need a large amount of ironys ability repetition DNAs during the cancer cell division, so the irony content of cancer cells is more high than normal cell." after these albumen that carry artemisinin derivative enter cancer cells; form oxo bridge between Sauerstoffatom on the 3-C position in the Artemisinin and the Sauerstoffatom on the 12-C position; iron ion just is released and reacts with artemisinin derivative; thus destruction of cancer cells; and the oxo bridge fracture discharges free radical (Sauerstoffatom); free radical is attacked cancer cell membrane, makes film rupture and cancer cell death; This is the key that Artemisinin is transferred to cancer therapy drug.Because cancer cells makes medicine have very high selectivity to the greediness of iron; Experiment shows that the iron biography albumen of Artemisinin mark is selected and the efficient of kill cancer cell is to kill 34000 times of Normocellular efficient (data source).
Drug effect compares: IC50 is the cancer cell in vitro medium lethal dose, and IC50 can react a little less than the strong drug action intuitively.
Hydroxycamptothecine IC50 is 206u mol～305u molng;
Taxol IC50 is 8.6u mol;
Dihydroartemisinin IC50 is 24n mol (0.024 u mol).
Toxic side effect: neural system, respiratory system, cardiovascular systems are not all had obvious pharmacological action, only show certain analgesia, sedative effect during greatly to 40mg/kg at dosage.This medicine security is bigger, LD 50Be 834.5mg/kg, chemotherapeutic index is 834.0, and rat 20-180 (MKD) administration in continuous 30 days there is no considerable change to physiology, biochemical indicator and main organs pathological examination.The specific toxicity aspect, the mutagenic test feminine gender, reproduction poison aspect in mouse pregnancy critical porion administration, increases the generation that absorbs tire, does not see teratogenesis.
Pharmacokinetics: behind the mouse gavaging 3H-Dihydroartemisinin, radioactivity rises rapidly in the blood, hour peaks half an hour to 1, descends rapidly subsequently, drops to half of peak value, and slowly disappears later in 4 hours.The measurement of gi tract residual quantity shows, residual 58%, 1 hour of half an hour is residual 35.3%, partly measures extinction time and is about 1.2 hours.Widely distributed behind the oral administration, 1 hour begins to peak, and isotope method shows that courage, liver, kidney are maximum in each tissue, and the heart, lung, spleen etc. take second place.Development process shows that intramuscular injection peaked in 1～8 hour, and liver, brain, bone, blood content are higher.In oral back 24 hours, 80% radioactivity is discharged through excrement, urine, and the development process result is similar.The above results show Dihydroartemisinin enter in the body post-absorption fast, distribute extensively, drain fast.
The Lai Hengli professor of Washington, DC university bioengineering dept in 1997 and Na Lundela star begin to imagine same mechanism and necessarily also can act on cancer: need a large amount of ironys ability repetition DNAs during the cancer cell division, so the irony content of cancer cells is more high than normal cell; Find that after deliberation cancer cells is than the high 5-15 of normal cell iron content times, high reaches 50 times, and the highest leukaemia cancer cell reaches 1000 times unexpectedly.Professor Lai claims: " Artemisinin is not only effective, and selectivity is very strong; Cancer cells there is very high toxicity, but very little to Normocellular influence." it might become nontoxic efficient anticarcinogen; The Yang Baofeng of Heilongjiang Province biological medicine engineering key lab, professor Zhou Jin that breathe out medical university discover that Dihydroartemisinin can effectively suppress the propagation of solid tumor cell.They find that leukemia cell's film is the main target spot that Dihydroartemisinin is attacked, and playing anti-knurl mechanism has " apoptosis " and " expand and die " two kinds.After leukemia cell's film was destroyed, a large amount of calcium ions will enter in the cell, caused programmed cell death on the one hand, i.e. " apoptosis " causes intracellular osmotic pressure to change on the other hand, absorbs large quantity of moisture, make cell expansion until death, promptly " expand and die "; This supposition has obtained extensive support in clinical experiment: some groups of breast cancer cells contacted with Transferrins,iron complexes with the normal breast cell, and after 8 hours, only remaining 25% cancer cells.After past 16 hours, nearly all cancer cells is all dead, and normal cell is unaffected.For example a dog that suffers from serious osteocarcinoma can not walk, and is accepting under the treatment that Dihydroartemisinin is aided with iron, just recovers fully in 5 days, relies professor's theory to obtain checking.
In the prior art, so the mode of Shanghai drug research Dihydroartemisinin ester, ether derivant is introduced halogen, the research of Dihydroartemisinin analogues such as Yu Peilin, Acta Pharmaceutica Sinica 1985; 20 (5): 357 ~ 365, in anticancer experiment (A549), comparison IC50 is 1227nM, the IC50 that introduces bromo element is 47nM, and drug effect strengthens about 26 times (Ying Li et al.Novel Antitumor Artemisinin DerivativesTargeting G1 Phase of the Cell Cycle.Bioorg.﹠amp; Med.Chem.Lett.11 (2001) 5-8).
The patentee is that Chinese patent 02128494, the name of Shanghai Pharmaceutical Inst., Chinese Academy of Sciences is called tert-butoxy carbonyl dihydro artemisinin, its preparation method and pharmaceutical composition, its invention provides the tert-butoxy carbonyl dihydro artemisinin shown in the structural formula: be to be starting raw material with the Dihydroartemisinin, carry out acylation reaction with dual-tert-butyl two carbonic ethers in organic solvent and make; The medicine for parasitic disease composition of invention comprises the tert-butoxy carbonyl dihydro artemisinin and the pharmaceutically acceptable carrier for the treatment of significant quantity, compared with former synthetic dihydroqinghaosu, tert-butoxy carbonyl dihydro artemisinin has maximum therapeutic index (＞1700), it is anti-parasite medicine efficient, low toxicity, in parasitosis processes such as prevention and cure of schistosomiasis, malaria, can reduce the generation of toxic side effect, especially in the treatment of pernicious malaria, significant meaning is arranged to reducing child mortality.
The applicant is that Zhejiang University, application number are 03116762.4, denomination of invention is that the pharmaceutical preparation of Artesunate and Dihydroartemisinin blood vessel formation against function and the Chinese patent application of purposes disclose Artesunate and Dihydroartemisinin pharmaceutical preparation aspect blood vessel formation against function and uses thereof; This pharmaceutical preparation mainly contains Artesunate or Dihydroartemisinin, and its dosage form is mainly microsphere injection liquid; The pharmaceutical preparation that this invention provides is having activity aspect the antineoplastic vascular generation, can be used for the disease treatment of tumor-blood-vessel growth and other and associated angiogenesis, also can be used for the treatment of chemotherapy of tumors and/or adjuvant chemotherapy aspect.Said preparation slowly discharges medicine and absorption at medicine-feeding part, prolong drug action time; This invention is a background with the vasculogenesis theory, research is also illustrated effect of Chinese medicine effective monomer component and mechanism, be the new important directions of developing Chinese medicine pharmacology opinion, provide important evidence, for the new purposes exploitation of Dihydroartemisinin class medicine provides foundation for finding the new target spot of new theory and drug effect.
In sum, prior art has the deficiency of following aspect:
Though the medicinal use that at first is Artemisinin and derivative thereof is verified and affirms, and is comprehensive inadequately to the indication scope of cancer;
Next is that the drug effect of existing Dihydroartemisinin and derivatives for treating cancer thereof is still waiting to improve.
Technology contents
Therefore, people wait in expectation have more wide spectrum, special efficacy, safe, toxic side effect is little and the medicine of the simpler treatment cancer of administering mode, purpose of the present invention just is intended to overcome above-mentioned prior art and lacks limit, the new compound of one class based on Dihydroartemisinin is provided, it has very the medical science effect and the special curative effect of the treatment cancer of wide spectrum, and without any side effects.
In order to reach the foregoing invention purpose, the inventor has studied a lot of Dihydroartemisinins and derivative thereof, and in conjunction with the instruction of prior art, consider that the haloid element substituting group has active influence to medicinal effectiveness, has the function of strengthening drug effect as bromide, become the best medicament in many flu syrup, by selecting many times, test, we have independently synthesized Br-DHA.
In fact, it is very difficult introducing heteroatoms on the parent nucleus of Dihydroartemisinin, and the institute of materia medica, Shanghai introduces halogen in the mode of Dihydroartemisinin ester, ether derivant.
The chemical name of Br-DHA be (3R, 5aS, 6R, 8aS, 9R, 12S, 12aR)-octahydro-3-bromo methylene radical-6,9-dimethyl-3,12-bridging oxygen-12H-pyrans is [4,3-j]-1 also, 2-benzo two Sai Ping-10 (3H) alcohol.
Its structural formula is:
Main purpose of the present invention is with the 3-C position methyl halogenation on the Dihydroartemisinin.
The step of synthetic Br-DHA is: Dihydroartemisinin is dissolved in the ester soluble solvent, imports the bromine source in solution, carry out building-up reactions, and remove residual bromine source, back purification by liquid extraction, and dehydration crystallization obtain the target compound.
Because Dihydroartemisinin has the sesquiterpene lactones class formation of peroxide bridge, it should select the ester soluble solvent that its dissolving is disperseed, and comparatively preferred ester soluble solvent is: acetonitrile, dimethyl formamide, acetate, chloroform, tetracol phenixin.
In the above-mentioned synthesis technique, described bromine source is the gas or the liquid of single composition, also bromide.
The bromine source that liquid bromine, bromine hydracid, bromize hydrogen gas etc. all can be used as the target compound imports.
In synthesis technique, when bromination reaction is carried out in importing bromine source, can add catalyzer bromination reaction is carried out catalysis.
Described catalyzer is Manganse Dioxide, silico-aluminate, cuprous halide;
Or directly shine with halogen tungsten lamp.
The Br-DHA that the present invention relates to can obtain extremely long-pending application in the pharmaceutical composition of preparation treatment cancer;
The Br-DHA that the present invention relates to can be made into the various various oral dosage forms that pharmaceutics requires that meet;
The Br-DHA that the present invention relates to can be made into the various various exterior-applied formulations that pharmaceutics requires that meet;
The Br-DHA that the present invention relates to can be made into the various various injection types that pharmaceutics requires that meet;
Described oral dosage form comprises: dripping pill, quick-release dripping pill, capsule, particle, sprays, oral liquid, tablet, skeleton type sustained release preparation comprise: (1) insoluble (as ethyl cellulose EC, polyethylene, polypropylene, polysiloxane, ethylene-vinyl acetate copolymer, polymethylmethacrylate etc.); (2) wax skeleton (as fat, Wax, stearic acid, stearyl alcohol, glyceryl monostearate, carnauba wax, Stearyl alcohol etc.); (3) hydrophilic gel (as CMC, CMC-Na, MC, PVA, HEC, SCMC, sodium alginate, pectin, alginate, agar, Vltra tears HPMC, chitosan, chitin, galactomannan etc.); (4) Entogastric lingering sheet etc.; The sustained release coating preparation comprises: (1) film controlled release small pieces, (2) microporous membrane coating tablet, (3) pilule comprise: film controlled piller, matrix type piller;
Described injection type comprises: injection liquid (common), freeze-dried powder, powder pin (common), infusion solutions, high density injection liquid, injection tablet;
Described exterior-applied formulation comprises: film, suppository, aerosol, preparation capable of permeating skin release
In the above-mentioned formulation:
Dripping pill grows up on the medicine pill basis, has the unexistent multiple characteristics of traditional pill, so development is very fast.Because the tablets amount is big, disintegration is poor, and intestines and stomach is had hormesis, and the dripping pill sublingual administration stimulates intestines and stomach thereby significantly reduced.Compare with other formulation (soft capsule, electuary, granule, capsule, oral liquid), it is big to have specific surface area, the characteristics that dissolution rate is fast, and this is because dripping pill can improve the bioavailability of insoluble drug.Because dripping pill is the solid dispersion that forms under quenching conditions, medicine exists with minimum crystal grain, so sublingual administration absorbs through periglottis, directly enters circulation of blood, and is rapid-action; Clinical application is suitable for the special-purpose medicine to oral carcinoma, laryngocarcinoma.
The sustained-release and controlled release preparation
Slowly-releasing, controlled release preparation are the development research focus in recent years.Sustained release preparation is meant and can reduces medicining times, and the preparation of comparison stable blood concentration is provided, and to reach the minimizing side effect, keeps the purpose of lasting drug effect.Controlled release preparation is meant by the preparation means provides the program that discharges medicine, medicine is automatically by a certain speed release and effect organ or specific target site from formulation within the predetermined time, make long-time constant the maintaining in the effective concentration scope of Plasma Concentration, the release uniform balance, control delivery drug release rate and time are irrelevant, avoided frequent " peak valley " phenomenon that occurs of traditional conventional formulation administration, clinical drug safety and validity have been improved, can replace intravenous drip, also can control injection speed automatically according to the body needs.Its purpose is to seek to provide the approach of desirable Plasma Concentration, improves the security and the validity of medicine.The researchdevelopment of oral sustained release controlled release preparation is very fast, and its kind constantly increases, and method of design is tending towards sxemiquantitative or quantification gradually; Clinical application is suitable for the medication to the various cancers of whole body.
Film
Film is research both at home and abroad in recent years and uses the formulation that makes much progress, and clinical being popular can be used for oral, mouthful diseases of eye, ear, nose and throat, skin and gynecological cancer etc.Along with the continuous development of TTS (being transdermal therapeutic system), some films especially nasal cavity, dermatologic film also can play general action, so the trend that replaces part tablet, ointment and suppository etc. is arranged in clinical application.And having short treating period, side effect is little, medicine film release rate is fast advantage, is the choice drug of the various carcinomas of vagina of treatment, cervical cancer.Because the volume of film own is little, in light weight, it is very convenient to carry; As the bioadhesion technology being introduced the targeting preparation of esophagus cancer treatment, made magnetic microsphere can stick to medicine on the cancer cells well; Clinical application is suitable for the special-purpose medicine of mouth, pharynx, nose, larynx, skin, woman vagina, cervical cancer.
Micro-capsule
Micro-capsule is an a kind of new formulation of utilizing natural or synthesized polymer material or multipolymer (capsule film material) that the medicine parcel is formed.Be beneficial to behind the miniature parcel of medicine and carry, be convenient to take.Its advantage is to prolong or to control the release of medicine, makes prolonged action preparation.Cyst membrane has isolates extraneous and medicine contact action, can prevent oxidation of drug, hydrolysis and volatilization, covers unpleasant odor, reduces the incompatibility in the compound preparation.Also can prepare property micro-capsule (magnetic micro-capsule, the responsive micro-capsule of PH) and play the targeting drug release effect, adopt the gelatin congealing method that it is bundled into micro-capsule, its packing rate and greenhouse package stability are better.
Micro-capsule system utilizes tiny capsules natural or that the synthetic macromolecular material wraps up solid or liquid medicine the diameter 1-500um that forms, its external form depends on the character of core material and the mode of capsule material cohesion, the micro-capsule outside is spherical entity or is level and smooth spherical putamina shape, grape is suffered from shape and surface smoothing or folding different shapes such as erratic composition, it is usually used in increasing stability of drug, cover the unpleasant odor of medicine, improve and delay the release of medicine; Clinical application is suitable for the medication of whole body cancer.
Suppository
Suppository not only can play local therapeutic effects, but also can absorb the whole body therapeutic effect by rectum, and behind the rectal administration, medicine directly by liver, can not prevent or reduce the metabolism of medicine at liver, alleviates the toxic side effect of medicine to liver.Suppository has and absorbs soon, and onset is rapid, and the bioavailability height can be kept the advantage of long period Plasma Concentration.Coelenteron administration medication one of main channel of development potentiality arranged, the research in this field must promote developing rapidly of suppository; Clinical application is suitable for intestinal cancer, prostate cancer etc.
Aerosol
Characteristics such as it is little that aerosol has dosage, is evenly distributed, and it is fast to prove effective, easy to use.Can reduce gastrointestinal side effect during suction, the pungency to the surface of a wound is then avoided in external application, and available quantitative valve control dosage, has quick-acting and positioning action.Being mainly used in the child clinically can not be oral or be reluctant oral person; The clinical application of this formulation is suitable for skin carcinoma, upper respiratory tract cancer, lung cancer etc.
Targeting preparation
Target administration also is a domestic and international in recent years very important exploitation focus, especially aspect cancer therapy drug, obtained significant development, its principle be anticarcinogen and ferromagnetic substance seal with the high-molecular bone frame material in, the ultra micro ball controlled release preparation of making is assembled under external magnetic field guiding and is trapped on the cancerous tissue of target area, slowly discharges medicine, to cancer cells, effectively attack, both can avoid injuring normal cell, and can reduce dosage again and reduce toxicity, improve curative effect.Magnetic target position medicine releasing system provides a new development approach aspect targeting of drugs, and liposome still is the focus of studying in the targeting preparation; The clinical application of this formulation is suitable for the medication of the various cancers of whole body.
Micropill
Be meant all kinds of pills of diameter, can be made into quick-release or sustained release pellet according to different needs less than 2.5mm.Fast release micropill can make medicine discharge rapidly.The emphasis of this research is a sustained release pellet, and sustained release pellet is to be mixed and made into or to make earlier common ball core by medicine and retarding agent then to wrap release-controlled film again and form.Micropill compacting or is loaded on fast release micropill and sustained release pellet and makes Extencap in the capsule shell in flakes.
The advantage that has many other oral preparations to compare of micropill: 1. can make sustained-release preparation by the controlled release micro pill dressing; 2. big at the gi tract distribution area, the bioavailability height, pungency is little; 3. because particle diameter is little, be subjected to digestive tube to carry the food rhythm and pace of moving things to influence little (close as pylorus etc.); 4. controlled release micro pill can make Plasma Concentration reach curative effect concentration rapidly, and keeps steady, long effective concentration, and the fluctuation of blood medicine is little; 5. the good fluidity of micropill is evenly big or small, is easy to handle (as dressing, divided dose); 6. improve medicine stability, cover undesirable taste; 7. the compatibility that is fit to compound preparation; The clinical application of this formulation is suitable for the medication of the various cancers of whole body.
The preparation capable of permeating skin release
This preparation capable of permeating skin is mainly gel preparation, and the sustainable Plasma Concentration of keeping this medicine 24 hours is avoided oral and the blood concentration fluctuation intravenously administrable approach; The clinical application of this formulation is suitable for the medication of the various cancers of whole body, especially skin carcinoma.
Dihydroartemisinin can directly be bought, and also can directly extract Artemisinin from plant, after be reduced into Dihydroartemisinin.
Compare with existing Dihydroartemisinin, Br-DHA has the outstanding advantage of following several respects:
Applicant's initiative is directly introduced heteroatoms on the parent nucleus at Dihydroartemisinin, with the 3-C position methyl bromination on the Dihydroartemisinin.
Drug effect is stronger: the polarity of haloid element is stronger, and is therefore stronger than the physiologically active of Dihydroartemisinin; Press the anticancer principle of Dihydroartemisinin, its mode of action is that its distinctive oxo bridge fracture produces free radical, causes that cancer cell membrane breaks and kill cancer cell; The bromination of a-methyl helps attracting 3, oxo bridge fracture on 12 carbon potentials produces free radical, can strengthen drug effect greatly, with Br-DHA human hepatoma cell strain-Hepg2 Study of cytotoxicity is shown that by West China medical college of Sichuan University Br-DHA is to the cell toxicant IC50＜8nM of external Hepg2 cell.
Bromination technology is simple and be easy to Industry Control, and good specificity is arranged, and makes the synthesis technique of product that excellent specificity be arranged, and is easy to quantified controlling.
Br-DHA has no side effect.
Description of drawings
Fig. 1 is that the Br-DHA body is used for cancer cell in vitro strain cancer cells survival rate synoptic diagram (%).
Fig. 2 is the process flow sheet of synthetic Br-DHA.
Embodiment
Embodiment 1:
Present embodiment is described with the chrysanthemum sweet wormwood and is prepared Artemisinin, Artemisinin is reduced into Dihydroartemisinin, imports the process that Br-DHA is synthesized in the bromine source with Dihydroartemisinin.
Chrysanthemum sweet wormwood 5000Kg injects the chromatography silicagel column with 8 times of ethanol 24h backflow lixiviates, vat liquor, up to having flowed; Use the sherwood oil wash-out, begin to be yellowish green liquid; When washing liquid and be substantially water white transparency or with the monitoring of silica gel G plate thin layer light blue fluorescence spot being arranged, changing elutriant is 10% ethyl acetate+90% sherwood oil wash-out, and collects its elutriant, monitors to there not being the blue-fluorescence spot with above-mentioned thin layer; The elutriant of collecting concentrates, and standing over night is collected the coarse crystallization of separating out; With 30 times 50% ethanol liquid recrystallization, Artemisinin needle crystal;
Artemisinin 10Kg is dissolved in the 300L methyl alcohol, add quaternary ammonium salt EtNCl 4Kg under stirring, fully stir the back that is mixed and add POTASSIUM BOROHYDRIDE 4Kg, add biphosphate sodium 1.65Kg, react under the room temperature to white crystals and separate out, matter liquid obviously separates; The total overall reaction thing poured in 0 ℃ the frozen water, leave standstill and separate out white solid; Collect white solid,, get Dihydroartemisinin, be the white powder crystallization with distilled water repetitive scrubbing 3 times, drying.
With reactor N2 drying, add 100mol Dihydroartemisinin and 1000L chloroform, dissolving; Add 14KgMnO2, stir evenly.And the speed with about 16L/ minute imports liquid bromine 100mol under 40～60 ℃ of temperature, stirs simultaneously; After 60 minutes, the filtering reaction thing; The sodium thiosulfate solution washing that in filtrate, adds 1000～5000L10%, collected organic layer; The sodium hydrogen carbonate solution washing that adds 5000～15000L10% again, collected organic layer; Organic layer with anhydrous sodium sulfate dehydration after concentrating under reduced pressure become white crystals, crystal washs with normal hexane, leaches crystallization, drying obtains Br-DHA, is the white powder crystallization.
Embodiment 2:
Present embodiment is described with Artemisinin and is reduced into Dihydroartemisinin, imports the process that Br-DHA is synthesized in the bromine source with Dihydroartemisinin.
Commercially available Artemisinin meal (with the crude drug ratio be 1: 4) standbyly add 5 times of 50% dissolve with ethanol, lysate injects the chromatography silicagel column, up to having flowed; Use the sherwood oil wash-out, begin to be yellowish green liquid; When washing liquid and be substantially water white transparency or with the monitoring of silica gel G plate thin layer light blue fluorescence spot being arranged, changing elutriant is 40% ethyl acetate+60% sherwood oil wash-out, and collects its elutriant, monitors to there not being the blue-fluorescence spot with above-mentioned thin layer; The elutriant of collecting concentrates, drying; Refining with ethyl acetate, get Artemisinin needle crystal.
Artemisinin 10Kg after purifying is dissolved in the 300L50% ethanol, add quaternary ammonium salt EtNCl 4Kg under stirring, fully stir the back that is mixed and add POTASSIUM BOROHYDRIDE 4Kg, add biphosphate sodium 1.65Kg, react under the room temperature to white crystals and separate out, matter liquid obviously separates; The total overall reaction thing poured in 0 ℃ the frozen water, leave standstill and separate out white solid; Collect white solid,, get Dihydroartemisinin, be the white powder crystallization with distilled water repetitive scrubbing 3 times, drying.
Reactor N2 drying adds 100mol Dihydroartemisinin and 1000L tetracol phenixin, dissolving; Add 14KgMnO2, stir evenly.And the speed with about 16L/ minute imports the liquid bromine under 40 ℃ of temperature, stirs simultaneously; After 90 minutes, the filtering reaction thing; Filtrate is with the water agitator treating of 1000～2000L, collected organic layer; Organic layer with anhydrous sodium sulfate dehydration after concentrating under reduced pressure become white crystals, crystal washs with normal hexane, leaches crystallization, drying obtains Br-DHA, is the white powder crystallization.
The preparation of embodiment 3 Br-DHAs
Reactor N2 drying adds 100mol Dihydroartemisinin and 1000L chloroform, dissolving; Add 14KgMnO2, stir evenly.And the speed with about 16L/ minute imports liquid bromine 100mol under 40～60 ℃ of temperature, stirs simultaneously; After 60 minutes, the filtering reaction thing; The sodium thiosulfate solution washing that in filtrate, adds 1000～5000L10%, collected organic layer; The sodium hydrogen carbonate solution washing that adds 5000～15000L10% again, collected organic layer; Organic layer with anhydrous sodium sulfate dehydration after concentrating under reduced pressure become white crystals, crystal washs with normal hexane, leaches crystallization, drying obtains Br-DHA, is the white powder crystallization.
The preparation of embodiment 4 Br-DHAs
Reactor N2 drying adds 100mol Dihydroartemisinin and 1000L tetracol phenixin, dissolving; Add 14KgMnO2, stir evenly.And the speed with about 16L/ minute imports the liquid bromine under 40 ℃ of temperature, stirs simultaneously; After 90 minutes, the filtering reaction thing; Filtrate is with the water agitator treating of 1000～2000L, collected organic layer; Organic layer with anhydrous sodium sulfate dehydration after concentrating under reduced pressure become white crystals, crystal washs with normal hexane, leaches crystallization, drying obtains Br-DHA, is the white powder crystallization.
The preparation of embodiment 5 Br-DHAs
Reactor N2 drying adds 100mol Dihydroartemisinin and 1000L acetonitrile, dissolving; Speed with about 10L/ minute under room temperature imports the liquid bromine, stirs simultaneously; Under the photochemical catalysis of 1000W halogen lamp, reacted 6 hours; The sodium thiosulfate solution washing that in reactant, adds 1000～5000L10%, collected organic layer; The sodium hydrogen carbonate solution washing that adds 5000～15000L10% again, collected organic layer; Organic layer with anhydrous sodium sulfate dehydration after concentrating under reduced pressure become white crystals, crystal washs with normal hexane, leaches crystallization, drying obtains Br-DHA, is the crystallization of off-white powder shape.
The preparation of embodiment 6 Br-DHAs
1.5Kg Dihydroartemisinin and 5L chloroform mix, dissolving; Add 14KgMnO2, stir evenly.And the speed with about 16L/ minute imports the liquid bromine under 40 ℃ of temperature, stirs simultaneously; After 90 minutes, the filtering reaction thing; Filtrate is refining once with chloroform again; Become white crystals with concentrating under reduced pressure behind the anhydrous sodium sulfate dehydration, crystal washs with normal hexane, leaches crystallization, and drying obtains Br-DHA, is the white powder crystallization.
Embodiment 7
Br-DHA is to the Cytotoxic research report of human hepatoma cell strain-Hepg2 (2004.12.22 of West China medical college of Sichuan University)
1. material:
1.1 cell strain: human hepatoma cell strain Hepg2 purchases the ATCC in the U.S., and 10%FBS/DMEM is conventional to be cultivated.
1.2 be subjected to test product Br-DHA: white powder, molecular-weight average 320g/mol, lot number 20041205.Ferrous sulfate: white powder, lot number: 20041205.Transferrins,iron complexes, Sigma company.
2. method:
2.1 cellulotoxic experiment method:
The Hepg2 cell of taking the logarithm vegetative period, ordinary method digestion is with 8 * 10 3Density be inoculated in 24 porocyte culture plates.Behind the inoculation 24h, connect table 1 and add Transferrins,iron complexes, ferrous sulfate respectively, cultivate after 8 hours, add Br-DHA.Behind the drug effect 72 hours, the ordinary method cell counting the results are shown in Figure 1.
3. result: (seeing the following form)
4. brief summary
Under above-mentioned experiment condition, find the cell toxicant IC50＜8nM of Br-DHA to external Hepg2 cell.
Table 1:Br-DHA dosage table
| Group | Transferrins,iron complexes (nM) | Ferrous sulfate (mg) | Br-DHA(nM) |
| Blank | ------------- | ------------- | ------------ |
| Transferrins,iron complexes | 880nM | 0.25 | |
| Br-DHA | 880nM 880nM 880nM | 0.25 0.01 0.002 | 1000 40 8 |
Annotate: ferrous sulfate: Br-DHA=250ng:1nM; Every dosage sample number N=4
Embodiment 8
The research of Br-DHA anti tumor activity in vitro (HuaXi college of pharmacy, SiChuan University).
1. material
1.1 cell strain:
Human hepatoma cell strain Hepg2 purchases the ATCC in the U.S.,
Human lung carcinoma cell line A549 purchases the Shanghai cell institute in the Chinese Academy of Sciences.
1.2 substratum:
Dulbecco’s Modified Eagle Medium(DMEM)：GIBCOBRL，Cat.No：12100-038，RPMI1640：GIBCOBRL，Cat.No：430-1800EB，
Fetal Bovine Serum：Cat.No：CH30160.03
Pancreatin: GIBCOBRL, Cat.No:27250-018.
1.3 be subjected to test product:
Br-DHA: white powder, molecular-weight average 320, continent Rong east medicine company, lot number: 20041205,
Ferrous sulfate: white powder, continent Rong east medicine company, lot number: 20041205,
Transferrins,iron complexes: white crystalline powder, molecular-weight average 79000, Sigma.
2. method
2.1 cell cultures:
Hepg2 and A549 cell routine respectively are incubated among 10%FBS/DMEM and the 10%FBS/RPMI1640, change liquid in 2-3 days.
2.2 medicine preparation:
Take by weighing Transferrins,iron complexes, ferrous sulfate, BrDHA with 100,000/electronic balance precision, wherein Transferrins,iron complexes, ferrous sulfate directly are dissolved in the degerming of cell culture medium after-filtration, Transferrins,iron complexes C o=17.6nM, sulfate industry iron C o=5mg/L; And after BrDHA is dissolved in DMSO earlier, be diluted to desired concn with cell culture medium again, filtration sterilization C o=6.4mg/L.Prepared than dilution by low by the different concns of test product.
All in facing with preceding fresh preparation, every hole adds 50uL to the solution of Transferrins,iron complexes, ferrous sulfate, BrDHA.
2.3 cytotoxicity test method:
The Hepg2 that takes the logarithm vegetative period, A549 cell, ordinary method digestion is with 8 * 10 3Density be inoculated in 2/4 porocyte culture plate.Behind the inoculation 24h, connect the ferrous sulfate that table 1,2 adds Transferrins,iron complexes, corresponding dosage respectively.After hatching 8h, add the Br-DHA of each dosage.Behind the drug effect 72 hours, the ordinary method cell counting the results are shown in Table 2,3.
3. result: see Table 2, table 3.
4. conclusion: this experiment shows that Br-DHA all has very obvious in-vitro antitumor action to human hepatoma cell strain Hepg2 cell and human lung carcinoma cell line A549 cell, to the IC of Hepg2 and A549 cell 50Difference＜8nM and 31.6nM.(table 2, table 3 are seen this page or leaf)
Table 2, Br-DHA are to human hepatoma cell strain--the Hepg2 anti tumor activity in vitro
| Group | Sample number | Drug dose | Cells survival rate (%) | ||
| Transferrins,iron complexes nM | Ferrous sulfate mg/L | BrDHAnM | |||
| Blank | 4 | ----- | ----- | ----- | 100.0±9.1 |
| Transferrins,iron complexes | 4 | 880 | 0.25 | ----- | 44.7±5.6 |
| BrDHA | 4 | 880 | 0.25 | 1000 | 23.3±5.3 |
| 4 | 880 | 0.05 | 200 | 32.7±14.1 | |
| 4 | 880 | 0.01 | 40 | 31.1±10.6 | |
| 4 | 880 | 0.002 | 8 | 41.6±3.6 | |
Annotate. ferrous sulfate: BrDHA=250ng:1nM
Table 3, Br-DHA are to human lung carcinoma cell line--the A549 anti tumor activity in vitro
| Group | Sample number | Drug dose | Cells survival rate (%) | ||
| Transferrins,iron complexes nM | Ferrous sulfate mg/L | BrDHAnM | |||
| Blank | 4 | ----- | ----- | ----- | 100.0±14.9 |
| Transferrins,iron complexes | 4 | 880 | 0.25 | ----- | 59.0±9.8 |
| BrDHA | 4 | 880 | 0.25 | 1000 | 17.4±7.5 |
| 4 | 880 | 0.05 | 100 | 39.0±5.2 | |
| 4 | 880 | 0.0025 | 10 | 58.2±5.2 | |
| 4 | 880 | 0.00025 | 1 | 59.0±1 3.0 | |
Annotate. ferrous sulfate: BrDHA=250ng: 1nM.
Embodiment 9
Analytical Test Center, Chengdu Branch, Chinese Academy of Sciences
Analysis, test result report
The calculating of bromination rate:
1, Dihydroartemisinin (DHA) molecular weight is 284.34, and bromine atoms (Br) molecular weight is 79.90, and hydrogen atom (H) molecular weight is 1;
2, after Dihydroartemisinin hydrogen atom is replaced by bromine atoms,
The molecular weight of Br-DHA is: (284.34-1)+and 79.90=363.24
3, the calculating of bromination rate:
79.90÷363.24＝21.996％≈22.0％。
22.90% belongs to normal value on the survey report....
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