CN1896064A - 海鞘素743的代谢物 - Google Patents
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Abstract
完成了由人细胞色素CYP3A4代谢的几种Et743代谢产物的纯化和结构说明。本专利中这些化合物缩写为ETM，紧接着标注数字，数字表示该化合物的近似分子量。迄今为止三个化合物已经鉴定，分别命名为ETM305，ETM775和ETM204。这些代谢物的结构如ETM305，ETM204和ETM775所示。
Description
本申请是申请日为1999年5月11日，申请号为200510054418.1发明名称为“海鞘素743的代谢物”的发明专利申请的分案申请。
本发明的背景
海鞘素(ecteinascidin本发明缩写为Et或Et′s)是从海洋被囊动物Ecteinascidia turbinata分离出来的具有很强抗肿瘤作用的药物。特别是，Et′s729，743和722已证明具有值得开发的体内抗肿瘤作用，包括对P388鼠白血病，B16黑素瘤，Lewis肺癌以及几种人肿瘤外移植物的小鼠模型的抗肿瘤作用。
美国专利号5,089,273中讲述天然的Et743的分离和特性，在此引入作为参考。美国专利号5,721,362中讲述合成的Et743的制备，在此引入作为参考。
海鞘素化合物，特别是Et′s729的抗肿瘤作用，在科学文献得到充分证明。例如，见Goldwasser等，美国肿瘤研究协会会报(Proceeding of American for Cancer Research)39：598(1998)；Kuffel等，美国肿瘤研究协会会报(Proceeding of American forCancer Research)38：596(1997)；Moore等，美国肿瘤研究协会会报(Proceeding of American for Cancer Research)38：314(1997)；Mirsalis等，美国肿瘤研究协会会报(Proceeding of American forCancer Research)38：309(1997)；Reid等，肿瘤化疗和药理学(Cancer Chemotherapy and pharmacology)38：329-334(1996)；Faircloth等，欧洲肿瘤杂志(European Journal ofCancer)，32A，Supp.1，pp.S5(1996)；Eckhardt等，美国肿瘤研究协会会报(Proceeding of American for Cancer Research)37：409(1996)；Henddriks等，美国肿瘤研究协会会报(Proceedingof American for Cancer Research)37：389(1996)；以上文献报道的公开内容在此引入作为参考。
海鞘素743(Et743)具有下面的结构：
鉴于这类化合物的令人注目的抗肿瘤作用，寻找可能具有相等或更高抗肿瘤作用的相关结构的化合物的研究一直在继续。致力于Et743的天然代谢物分离和特性研究的本发明就是这些连续研究的结果。
本发明概述
完成了由人细胞色素CYP3A4代谢的几种Et743代谢产物的纯化和结构说明。本专利中这些化合物缩写为ETM，紧接着标注数字，数字表示该化合物的近似分子量。
例如，ETM305和ETM775是由西班牙PhamaMar公司分析化学部所进行的生化研究中所获得的代谢混合物中分离出来的。由MayoClinic公司进行的类似代谢研究促成了ETM204的鉴定。这些海鞘素代谢物的结构如下：
图的简要说明
通过参考后面的图以及这个说明，可以更好理解本发明，其中：
图1是在CDCI3中ETM-SiOH-1(非极性不纯物)的1H NMR波谱图(500MHz)；
图2是ETM-SiOH-4(ETM775)的HPLC色谱图；
图3是ETM-SiOH-3(ETM305)的HPLC色谱图；
图4是ETM-SiOH-2(痕量代谢物)的HPLC色谱图；
图5是在M.B.(magic bullet[4])中ETM305的LRFAB质谱图；
图6是ETM305的ESI质谱图；
图7是在CD3OD中ETM305的1H NMR波谱图(750MHz)；
图8是ETM305的FAB/MS/MS谱图；
图9是ETM305的紫外光谱图；
图10是ETM的紫外光谱图；
图11是在M.B.(magic bullet[4])中ETM775的LRFAB质谱图；
图12是ETM775的ESI质谱图(正性模型)；
图13是ETM775的ESI质谱图(负性模型)；
图14是ETM775的FAB/MS/MS谱图(M/Z1 38-302)；
图15是ETM775的FAB/MS/MS谱图(M/Z 440-620)；
图16是在CD3OD中ETM775的1H NMR波谱图(750MHz)；
图17是ETM775的紫外光谱图；
图18是ETM305的HPLC色谱图；
图19是ETM305的紫外光谱图；
图20是ETM305的ESI质谱图；
图21是ETM204的ESI质谱图；
图22是在CD3OD中ETM204的1H NMR波谱图(500MHz)；以及
图23是ETM204的ESI/MS/MS谱图。
本发明的详细说明
I.Et743的代谢研究
A.代谢混合物ETM的制备：
将Et743(50μM)与配制在含NADPH生成系统(0.4mMNADP+，25mM6-磷酸葡萄糖，0.5U/ml6-磷酸葡萄糖脱氢酶和3.3mM氯化镁)的0.1Mtris-HCI缓冲液中的浓度为0.4mg/ml的人成淋巴细胞表达的CYP3A4同种型一起孵育。在37℃孵育4小时后，加入冰冷乙腈中止反应，离心(12000g，4min)弃去固状物，上清液作HPLC分析。
B.ETM305和ETM775的纯化
2.6mg ETM(按前面A中方法制备)溶于少量CHCI3中，加到硅胶柱(8×100mm玻璃柱，填充硅胶/CHCI3浆)。硅胶柱先用CHCI3洗脱，接着用CHCI3/MeOH混合液(98，96，94，92和90％)洗脱。共收集10个试管(每管3ml)，并根据簿板色谱(TLC)结果作合并处理，产生四个级分(表1)。如1H NMR谱图(图1)所示，极性较小和非细胞毒级分(ETM-SiOH-1，2mg)由结构上与Et743不相关的脂组成。
剩下的细胞毒级分用
HPLC(PhenomenexUtracarb，ODS，10um，10×150mm，3∶1MeOH/H2O0.02M NaCI，1ml/min，Da检测：210，220，254和280nm)作进一步纯化。极性最大的级分(ETM-SiOH-4，0.2mg)产生0.1mg ETM775(图2)。ETM-SiOH-3产生0.3mg ETM305(图3)。ETM-SiOH-2由几种微量代谢物组成(图4)。
表1.ETM-SiOH级分：Rf，重量和细胞毒性
| ID# | 试管号 | Rf a | 重量 | L1210 | ||
| 500ng/mg浓度下的生长抑止率(％) | ||||||
| ETM-SiOH-1ETM-SiOH-2ETM-SiOH-3ETM-SiOH-4 | 124-56 | 0.90.5，0.70.50.3 | 2.0mg0.3mg0.4mg0.2mg | 080b303 | ||
a硅胶TLC采用CHCI3/MeOH作为流动相。b在250ng/mg浓度下的生长抑止率为30％。
C.ETM305的结构
用HRFAB/MS分析显示ETM305(对L1210细胞株的IC50为0.2ug/ml)一分子离子峰在306.0977处(图5)。该数据与其分子式C15H16NO6(Δ0.1mmu)一致。ESI/MS分析证实了ETM305的该分子量(图6)；在m/z306处观察到分子离子与其钠加合物在一起。ETM305的1HNMR波谱图(图7)对于确定其结构是非常重要的。在δ2.04，2.28和6.09处的共振现象分别与Et743中Me-6(δ 2.03)，-OCOCH3(δ2.29)和二氧-甲叉质子δ6.11，δ6.01)几乎相同。
此外，还观察到与一个-CH＝CH-NHCHO单位(δ7.09，d，1H，J＝15Hz；δ6.19，d，1H，J＝15Hz；δ8.04，s，1H)和一个附加甲基(δ2.52，s，3H)相对应的共振现象。该甲基的化学位移和乙酰苯3(δ2.55)的位移十分匹配。我们饶有兴趣地注意到ETM305的1H NMR波谱由于构象异构体围绕-NH-CHO键旋转，由二部分共振组成(4∶1比例)。正如示意图1所示，1H NMR数据连同MS数据提示，ETM305具有与一个乙烯基-甲酰胺单位和一个甲基酮相连接的Et743B-单位芳香环系统，FAB/MS/MS分析(m/z 306)支持该所提出的结构(图8)。
示意图1
D.ETM775的结构
用HRFAB/MS分析显示ETM775(对L1210细胞株的IC50为0.2ug/ml)一分子离子峰在776.2489处(图5)。该数据与其分子式C39H42N3O12S(Δ0.1mmu)一致(图11)，表明ETM775是Et743的氧化产物。正模和负模ESI/MS谱图都证实了ETM775的这一分子量(图12和图13)。因为ETM775的量有限，主要根据其质谱资料作其结构测定。对ETM775的M+H(m/z 776)的FAB/MS/MS分析，在确定该额外氧——正如由m/z 276和260(276-氧)处的峰所显示的，该额外氧以N-氧化物的形式位于N-2上——的位置方面，是重要的。在Et743中末观察到的m/z 232处的级分离子提示该甲醇胺氧被氧化为酰胺(示意图2)。正如在m/z 204(A-单位)以及m/z 224和250(C-单位)处的峰所显示的，ETM775中的A-单位和C-单位保持完整。[1]ETM775的750750Mhz 1H NMR谱图(图16)和紫外光谱图(图17)都与Et743的相似。
示意图2
II.Et743-Mayo代谢研究
A.M1代谢物(ETM305)
ETM样品通过C18 sep-pack柱过滤，其洗脱液(3∶1 MeOH/H2O)在氮气流下加以浓缩。所得样品经HPLC纯化(与前述的条件相同)证明有一种与ETM305相同保留时间的化合物存在(图18)。M1的紫外光谱图(图19)和ESI/MS谱图(图20)都与ETM305的相同。由此得出结论，M1代谢物具有像ETM305相同的化学结构。
B.M2代谢物(ETM204)
所提供的样品通过C18 sep-pack柱过滤，其洗脱液(3∶1 MeOH/H2O)在氮气流下加以浓缩。所得样品用FAB/MS，ESI/MS和1H NMR作分析。
C.ETM204(M2)的结构
用HRFAB/MS分析表明ETM204的分子离子峰为204.1024。该数据与其分子式C12H14NO2(Δ0.0mmu)一致。ESI/MS分析证实其分子量为204(图21)。该分子式与Et743中的A-单位的分子式相似。据此推论，ETM204的化学结构是示意图3中所示的芳香胺盐衍生物。
示意图3
ETM204的1H NMR谱图(图22)显示支持所提出的结构的共振现象：4种芳香族信号(δ9.2，s；δ7.8，d，J＝5Hz，以及δ6.8，s)和3个甲基单峰(δ4.2，δ3.9和δ2.4)。ETM204的ESI/MS/MS分析(图23)证明，与N-甲基(204-CH3)明显丢失相对应，其主峰离子为189。ETM305和ETM775的生物学研究
采用标准的实验方案，试验了化合物ETM305和ETM775对下列肿瘤细胞株的抗肿瘤作用：P388(鼠白血病)；A-549(人肺癌)；HT-29(人结肠腺癌)以及MEL-28(人恶性黑素瘤)。例如，见Bergeron等。生物化学和生物理研究通讯(Biochem Biophys resComm)1984，121(3)848-854和Schroeder等。医用化学杂志(J MedChem)，1981，24 1078-1083。这些结果在下面的表2中列出：
表2.肿瘤细胞株和抗肿瘤活性
IC50(ug/ml)
| 化合物：ETM305ETM775 | P3880.50.01 | A-5490.50.01 | HT-290.50.01 | MEL-280.250.01 |
治疗方法
本发明包括具有生物活性的化合物，因此，本发明的实施方案是以利用这样的化合物的治疗方法为目标的。如上所述，本发明的化合物体内外对肿瘤细胞株具有细胞毒作用。可以预料，体内外的这些抗肿瘤活性扩大到体内应用，同样是有效的。
这些化合物已经以基本上纯化的形式，即是在足以呈现其物理学和生物学特性的纯化水平上，被分离出来。业已发现，这些化合物具有特异的抗肿瘤活性，由于这种作用，它们作为哺乳动物尤其是人的医药剂，可能是有用的。这样，本发明的另一个方面涉及含有本发明所鉴定的这些活性化合物的药物组合物以及施用这些药物组合物的治疗方法。
如上所述，本发明的这些活性化合物具有抗肿瘤活性。因此，本发明也为患对这些化合物敏感的恶性肿瘤的任何哺乳动物提供了一种治疗方法。这种治疗方法包括给患病个体施用有治疗作用剂量的单一活性化合物，或各活性化合物的混合物，或其药物组合物。本发明也与药剂制备有关，后者包括一种或多种本发明的活性化合物作为活性成份，以及其制备方法。
药物组合物的举例包括含有适当成份的，作口服、局部施用或非肠道施用的任何固体(片剂，丸剂，胶囊剂，颗粒等)或液体(溶液，乳剂混悬液)，这些药物组合物可以含有单纯的本发明的化合物，或与其他药理学活性化合物载体联合应用。这些药物组合物非肠道施用时，可能需要是无菌的。
作为与本发明有关的术语″单位剂量″，系指适合作为动物单次剂量的物理学上独立的单位。每一单位含有经过计算能产生所需要的抗肿瘤活性而确定的活性化合物剂量，它与所需要的稀释剂即载体或赋形剂联用。本发明新颖的单位剂量的技术规格由下列条件确定，或直接取决于下列条件：(a)该活性化合物的独特特性和所要达到的特定抗肿瘤活性，(b)调配供动物抗肿瘤应用的这样的活性化合物，在技术上存在的内在限制。
单位剂量形式一般通过把冻干的或干燥的活性化合物(或其盐)分散在生理上可接受的稀释剂或赋形剂(如水、生理盐水或磷酸盐缓冲的生理盐水)中，形成水性组合物而制成。这样的稀释剂为本专业技术人员所熟知，例如，在Remington氏药剂学(Remington′sPhaarmaceutical sciences，16版，Mack Publishing公司，Easton，PA，1980，p1465-1467)一书中对此作了讨论。
单位剂量形式也可以包含一种赋形剂作为稀释剂的一部分。像弗氏(Freund′s)完全佐剂、弗氏(Freund′s)不完全佐剂和明矾都是本专业技术人员所熟知的材料，可以从几种商业途径购得。
待施用的活性化合物的量尤其取决于待处理的动物种类、实验动物的大小、肿瘤的大小(如果知道的话)、存在的肿瘤类型(例如实体)以及受试者利用该活性化合物的能力。需要施用的活性化合物的准确剂量取决于专业人员的判断，而且给每个个体确定一个准确的施用剂量，尤其在人作为被治疗的动物的场合。但是，可以根据治疗有效的血药浓度确定剂量范围，剂量范围可从约0.01μM至约100μM，优选的剂量范围从约0.1μM至约10μM。
初次给药和增强注射的适当给药方式也是可变化的。但是，有代表性的给药方式是：首次给药后，以1小时或1小时以上的间隔，通过连续的注射或其他给药方式，重复给予剂量。作为替代，能足以维持治疗有效的血药浓度的连续静脉灌注方式也是值得考虑的。
参考文献：
提供下面的背景参考文献，以协助读者了介本发明。其内容依所需程度，在此引入作为参考。
1.A)Rinehard等。有机化学杂志(J Org Chem)1990，55，4512.B)Rinehard等。美国化学杂志(J Am Chem)1996，118，9017。
2.Herbert等。J Chem SOC Perkin Trans.I，，1987，1593.
3.Pretsch等。有机化合物结构测定用波谱数据表(Tables ofSpectral Data for Structure determination of OrgannicCompounds)；Springer-Verla出版社，柏林，1989，p.H125)。
4.Rinehard等。生物化学研究通讯(Biochem Res Cmmun)，1984，124，350。
已详细阐述了本发明，包括其优化的实施方案。但是，会得到这的评价：本专业技术人员基于对本发明公开内容的理解，可以对本发明作修改和/或改进，而这仍然在本发明的范围和精神内。
Claims (1)
1.基本上纯化的，具有如下结构的ETM204：
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| MY164077A (en) * | 1999-05-13 | 2017-11-30 | Pharma Mar Sa | Compositions and uses of et743 for treating cancer |
| MY130271A (en) | 1999-05-14 | 2007-06-29 | Pharma Mar Sa | Hemisynthetic method and new compounds |
| US7919493B2 (en) * | 2000-04-12 | 2011-04-05 | Pharma Mar, S.A. | Anititumoral ecteinascidin derivatives |
| BRPI0110024B8 (pt) * | 2000-04-12 | 2021-05-25 | Pharma Mar Sa | derivados de ecteinascidina antitumorais, composição farmacêutica que os compreende e uso dos mesmos |
| US7420051B2 (en) | 2000-05-15 | 2008-09-02 | Pharma Mar, S.A. | Synthetic process for the manufacture of an ecteinaschidin compound |
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| US6809099B2 (en) | 2000-11-03 | 2004-10-26 | President And Fellows Of Harvard College | Saframycins, analogues and uses thereof |
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| US5256663A (en) * | 1986-06-09 | 1993-10-26 | The Board Of Trustees Of The University Of Illinois | Compositions comprising ecteinascidins and a method of treating herpes simplex virus infections therewith |
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