WO2012139487A1 - 喜树碱类化合物的聚乙二醇化衍生物 - Google Patents

喜树碱类化合物的聚乙二醇化衍生物 Download PDF

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WO2012139487A1
WO2012139487A1 PCT/CN2012/073702 CN2012073702W WO2012139487A1 WO 2012139487 A1 WO2012139487 A1 WO 2012139487A1 CN 2012073702 W CN2012073702 W CN 2012073702W WO 2012139487 A1 WO2012139487 A1 WO 2012139487A1
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compound
group
substituted
unsubstituted
camptothecin
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French (fr)
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刘克良
李思成
余晓军
冯思良
梁远军
贾启燕
韩寒
孟庆斌
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中国人民解放军军事医学科学院毒物药物研究所
成都一平医药科技发展有限公司
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Publication of WO2012139487A1 publication Critical patent/WO2012139487A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C08G65/333—Polymers modified by chemical after-treatment with organic compounds containing nitrogen
    • C08G65/33303—Polymers modified by chemical after-treatment with organic compounds containing nitrogen containing amino group
    • C08G65/33317—Polymers modified by chemical after-treatment with organic compounds containing nitrogen containing amino group heterocyclic
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C08G65/333—Polymers modified by chemical after-treatment with organic compounds containing nitrogen
    • C08G65/33396—Polymers modified by chemical after-treatment with organic compounds containing nitrogen having oxygen in addition to nitrogen
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L2203/00—Applications
    • C08L2203/02—Applications for biomedical use

Definitions

  • the present invention relates to a camptothecin-based compound, particularly a camptothecin, a 10-hydroxycamptothecin and a 10-methoxycamptothecin polyethylene glycol compound, and the use thereof in the preparation of a medicament for antitumor and the like .
  • Background technique a camptothecin-based compound, particularly a camptothecin, a 10-hydroxycamptothecin and a 10-methoxycamptothecin polyethylene glycol compound, and the use thereof in the preparation of a medicament for antitumor and the like .
  • Camptothecin (CPT, Figure 1) is a natural product isolated from Camptotheca acuminata. Since Wall et al. reported this compound in 1966, it was introduced into the clinic in the early 1970s due to its excellent anticancer activity. Considering that camptothecin is substantially insoluble in water, its carboxylate form (Fig. 1) is used as a clinically active component. In clinical trials, serious drug side effects such as diarrhea and hemorrhagic cystitis have occurred, so that clinical trials have to be terminated.
  • Topo I is the only binding target of CPT molecules, and Topo I plays a decisive role in DNA replication.
  • Topo I forms a transient single-strand gap during DNA replication, allowing semi-reserved replication of the double helix to proceed smoothly.
  • a Topo I-DNA binary complex is formed.
  • CPT and its derivatives can specifically bind to the Topo I-DNA complex in the S phase of DNA replication, eventually forming an irreversible Topo I-CPT-DNA ternary complex that terminates DNA replication and ultimately leads to cell death. Die. This mechanism has been experimentally confirmed, and the single crystal structure of the ternary composite has also been obtained.
  • camptothecin Due to the unique mechanism of action of tumor cell topoisomerase I, camptothecin has become the focus of anti-tumor drug development, especially the well-soluble and metabolically stable camptothecin derivatives have attracted wide attention.
  • the hexa- lactone ring is a key pharmacophore for the antitumor activity of camptothecin derivatives.
  • the lactone ring has a balance with the carboxylic acid form in human plasma (see Figure 1).
  • the camptothecin derivative of the carboxylic acid form has an decreased antitumor activity and an increased adverse reaction.
  • the carboxylic acid form has a binding ability to human plasma albumin 150 times that of the lactone form, the equilibrium shifts toward the carboxylic acid form, resulting in a concentration of the lactone form in the blood being much lower than that of the carboxylic acid form. Therefore, increasing the stability of metabolism in the body and reducing adverse reactions has become the focus of research on such compounds.
  • camptothecins have very poor solubility in water and are extremely unfavorable in drug delivery and metabolism. Therefore, it is also very important to find a target structure that is easily soluble in water. Summary of the invention
  • camptothecin and its derivatives especially 10-hydroxycamptothecin and 10-methoxycamptothecin
  • camptothecin and its derivatives are covalently modified with polyethylene glycol, and the activity remains unchanged.
  • the toxic side effects are reduced, and its water solubility and stability are significantly increased, its half-life in the body is prolonged.
  • the present invention relates to a polyethylene glycol compound of a camptothecin compound represented by the formula (I):
  • A -(NH-CR 3 R 4 -CO)n 3 - (NH-CR 5 R 6 -CO)n 4 - (NH-CR 7 R8-CO)n5 - (NH-CR 9 Ri 0 -CO) n 6 - Z -; R 3 to.
  • B - NH-(CH 2 )n 8 -S - S-(CH 2 )n 9 -C(0) - Z-; n 8 is 2-5, n 9 is 1-4; Z is O or NH ;
  • Y - NH-(CH 2 )n 10 -SM- , n 10 is 2 - 5;
  • camptothecin compound of the formula (I) of the present invention is selected from the group consisting of camptothecin, 10-hydroxycamptothecin and 10-methoxycamptothecin.
  • the polyethylene glycol compound of the camptothecin compound represented by the formula (I) of the present invention is preferably 20-polyethylene glycol 5000 monomethyl ether succinate monoester-10-methoxycamptothecin (MC5001).
  • a further aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising at least one compound of formula (I) and a pharmaceutically acceptable carrier or excipient.
  • the invention further relates to the use of at least one compound of formula (I) for the preparation of a medicament for the prevention of tumors and the like.
  • MALDI-TOF-MS matrix-assisted laser desorption time-of-flight mass spectrometry
  • mPEG-OH according to the present invention can be purchased as a commercial reagent from Fluka, Germany, and mPEG-NH 2 can be purchased or obtained by the following reaction:
  • the present invention uses a CH 3 O PEG-OH (mPEG) having an average molecular weight of 1000, 2000, 5000 and 10000 as a raw material to synthesize a series of mPEG-modified and two-site mPEG-modified camptothecin derivatives. Each compound was analyzed by RP-HPLC and TLC as a single peak. The mass spectrometry and hydrogen spectroscopy and carbon nuclear magnetic analysis confirmed the correct structure.
  • mPEG CH 3 O PEG-OH
  • the pharmaceutical composition of the present invention is formulated into various dosage forms suitable for use in mammals, including but not limited to injections, solutions, emulsions and the like.
  • an injection prepared using mannitol as an excipient for example, an injection prepared using mannitol as an excipient.
  • the pharmaceutical compositions of the present invention can be administered in a standard manner, for example, parenteral administration including, but not limited to, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, or continuous infusion. detailed description
  • CH 3 0-PEG-OH having an average molecular weight of 1000, 2000, 5000 and 10000 used in the examples is a product of the German company Fluka, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide.
  • Hydrochloride (ED HC1 ) and dimethylaminopyridine (DMAP) are domestic analytical reagents.
  • the reaction mixture was poured into EtOAc (EtOAc)EtOAc.
  • EtOAc EtOAc
  • the chloroform was recrystallized and a small amount of crystals was obtained.
  • An appropriate amount of petroleum ether was added, and a large amount of a yellow powder solid was precipitated, and the mixture was filtered and dried under reduced pressure. Yellow powder solid.
  • the yield is about 50% (100 mg).
  • Polyethylene glycol 5000 monomethyl ether succinate monoester mPEG5000-Suc., l.Og, 0.2 mmol
  • 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride ED HCl, 120 mg, 0.4 mol
  • V,iV-dimethyl-4-aminopyridinium DMAP, 48.8 mg, 0.4 mol
  • Human gastric cancer cell line BGC-823, human breast cancer cell MCF-7, human hepatoma cell HepG2 and human colorectal cancer cell line HT29 were selected and seeded in RPMI1640 or DMEM cell culture medium containing 10% fetal bovine serum (supplemented Cyan and streptomycin were each 100 u/ml), placed in a cell culture incubator containing 5% CO 2 at 37 ° C, once every 2-3 days, 0.25% trypsin digestion, passage and collection of cells.
  • the logarithmic growth phase cells were prepared into a cell suspension of 2.5 ⁇ 10 4 /ml with RPMI1640 cell culture medium containing 10% fetal bovine serum, and added to 96-well cell culture plates at 3000 cells per well ( ⁇ ).
  • a medium containing different concentrations of the test substance was added to each well, and each concentration was set to 3 to 4 parallel wells. After 72 hours of incubation, the supernatant was discarded. Freshly prepared 0.5 mg/ml tetrazolium blue (MTT) serum-free medium was added to each well, and cultured at 37 ° C for 4 hours, and the supernatant was discarded. After dissolving in 100 ⁇ L DMSO, the light was shaken for 15 minutes, and the absorbance (OD value) was measured with a microplate reader at a detection wavelength of 570 nm and a reference wavelength of 450 nm. The concentration of the test drug was set to 10.0, 5.0, 2.5, 1.25, 0.625, 0.3125, 0.15625 g/ml, and a blank control was set. Dissolve the drug in a cell culture solution before use.
  • MTT tetrazolium blue
  • Inhibition rate (OD value of control group - OD value of administration group) / OD value of control group xl00%; drug effect index was expressed by half concentration (IC 50 ), juxtaposed The measured maximum inhibition rate (Imax) is obtained.
  • Tumor cell growth curves were fitted using MicroCal Origin software and a four-parameter Logistic program in the software to determine the half-inhibitory concentration (IC 5 Q, g /ml). The specific results are shown in Table 1.
  • HT-29 0.056 93.3 As shown in Tables 1 and 2, MC-5001 and 10-hydroxycamptothecin have comparable inhibitory activities against four tumor cells.

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Abstract

本发明涉及喜树碱类化合物的聚乙二醇化衍生物，其制备方法，含有它们的药物组合物，及其在抗肿瘤等方面的用途。

Description

喜树碱类化合物的聚乙二醇化衍生物 发明领域
本发明涉及喜树碱类化合物， 特别是喜树碱、 10-羟基喜树碱 和 10-甲氧基喜树碱的聚乙二醇化合物， 及其在制备抗肿瘤等相 关作用药物中的用途。 背景技术
喜树碱（ Camptothecin, CPT, 图 1 )是从珙桐科植物喜树 ( Camptotheca acuminata ) 中分离得到的一种天然产物。 自从 Wall等在 1966年报道了这个化合物之后， 70年代初即由于其优 异的抗癌活性被引入临床。 考虑到喜树碱在水中基本不溶， 使用 其羧酸酯形式 （图 1 )作为临床活性组分。 在临床实验中， 出现 了诸如腹泻、 出血性膀胱炎等严重药物副反应， 以致于不得不终 止临床实验。
Figure imgf000002_0001
图 1 喜树碱的内酯和羧酸酯形式
在 80年代， 发现了拓朴异构酶 I ( Topo I )是 CPT分子的唯 一结合目标， 而 Topo I在 DNA的复制中具有决定性的作用。
Topo I在 DNA复制过程中形成一个暂时性单链缺口， 使得 双螺旋结构的半保留复制顺利进行。 这个过程中， 形成了 Topo I-DNA二元复合物。 CPT及其衍生物可以在 DNA复制的 S期与 Topo I-DNA 复合物特异性结合， 最终形成一个不可逆的 Topo I-CPT-DNA三元复合物， 终止了 DNA的复制， 最终导致细胞死 亡。 这个机理已被实验证实， 三元复合物的单晶结构也已经得到
( PNAS, 2002 , 15387 ) 。 另外， 文章也指出， CPT在这个三元 复合物中， 起着类似于一个碱基对的作用。 从而， 对于 CPT的修 饰产物在空间上的位阻将大为降低。
由于抑制肿瘤细胞拓朴异构酶 I的独特作用机制， 喜树碱已 经成为抗肿瘤药物开发的重点， 尤其是水溶性好、 代谢稳定的喜 树碱衍生物引起了广泛的关注。
构效关系研究表明， 六元内酯环是喜树碱类衍生物抗肿瘤活 性的关键药效基团， 然而， 内酯环在人血浆中存在着与羧酸形式 的平衡 (见图 1) ,羧酸形式的喜树碱衍生物抗肿瘤活性下降并且不 良反应上升。 但是由于羧酸形式与人血浆白蛋白结合能力是内酯 形式的 150倍， 平衡向羧酸形式方向位移， 导致血液中内酯形式 的浓度远远低于羧酸形式。 因此增加体内代谢的稳定性从而降低 不良反应已经成为该类化合物研究的重点。
另外， 构效关系研究结果也指出喜树碱的 20- ( S )羟基、 D 环吡啶、 六元 E环以及分子的平面结构对于好的药效至关重要， 对于 A、 B环的修饰可以提高药效， 而其他环的修饰对于药效而 言是不矛 J的 ( Expert Opin. Ther. Patents, 2009， 195， 555-574.; Chem. Med. Chem" 2007， 2， 1807-1813 ) 。
喜树碱类天然产物在水中的溶解性能非常差， 在药物传递、 代谢过程中极其不利， 因而， 寻找易溶于水的目标结构也是非常 重要的。 发明内容
本发明人经研究现已发现喜树碱及其衍生物尤其是 10-羟基 喜树碱和 10-甲氧基喜树碱经聚乙二醇共价修饰后， 活性保持相 当， 毒副作用降低， 且显著增加其水溶性及稳定性， 延长了其在 体内的半衰期。
本发明涉及式（I )所示的喜树碱类化合物的聚乙二醇化合物：
Figure imgf000004_0001
其中， RfH, OH, OMe， 或结构 O-XrmPEG R2为结构 X2-mPEG2； 其 中 ， mPEGi 与 mPEG2 分别 独立 的 为 CH2CH2(OCH2CH2)n1-OCH3 , ηχ = 0 - 1000； 1与 2分别独立的
0 0
为一 C— (CH2)n2— ϋ-χ3， η2为 0 - 4， Χ3可以为 Α、 Β或 Υ;
A = - (NH-CR3R4-CO)n3 - (NH-CR5R6-CO)n4 - (NH-CR7R8-CO)n5 - (NH-CR9Ri0-CO)n6 - Z -； R3到 。分别独 立的为 H， 取代或未取代的 d-C6直链或者支链烷基， 取代或未 取代的 C2-C6直链或者支链烯基或者炔基，取代或未取代的 d-C6 直链或者支链烷氧基， 取代或未取代的 d-C6直链或者支链烷硫 基， 取代或未取代的 C2-C6直链或者支链烯基或者炔基氧基， 取 代或未取代的 （ 2-( 6直链或者支链烯基或者炔基， 环烷基或环链 烯基， 芳基， 杂环基； -（CH2)n7 - CO - O - mPEG; - (CH2)n7 - CO - NH - mPEG; n7为 1 - 6， mPEG定义同前； 其中， 优选 地， 杂环烷基为其环结构中含 1 - 5个（优选 1 - 3个）独立地选 自 N、 O和 S等的杂原子的环状基团； 芳基为未取代的或被独立 地选自卤素， 硝基， 羧基或 d-C4烷基的取代基单取代或二取代 或三取代的 4、 5、 6、 或 7元单环或双环芳香基团， 如苯基或者 或萘基等； 杂环基可为未取代的或被独立地选自卤素， 硝基， 羧 基或 d-C4烷基的取代基单取代或二取代的、含有 1 - 5个独立地 选自 N、 O和 S等的杂原子的 4、 5、 6、 或 7元单环或双环芳香 基团， 如吡咯基， 呋喃基， 吡啶基等； n3到 n6分别独立的为 0 或 1; Z为 O或 NH;
B = - NH-(CH2)n8-S - S-(CH2)n9-C(0) - Z-； n8为 2-5， n9为 1-4; Z为 O或 NH;
Y = - NH-(CH2)n10 -S-M- , n10为 2 - 5;
更为优选的
Μ为
Figure imgf000005_0001
, ηιο = 2ο
本发明式 （I) 中的喜树碱类化合物选自喜树碱、 10-羟基喜 树碱和 10-甲氧基喜树碱。
本发明式（I)所示的喜树碱类化合物的聚乙二醇化合物优选 是 20-聚乙二醇 5000 单甲醚琥珀酸单酯 -10-甲氧基喜树碱 ( MC5001 ) 。
本发明再一方面涉及含至少一种式（ I )化合物及药用载体或 赋形剂的药物组合物。
本发明还涉及至少一种式（ I )化合物在制备抗肿瘤等相关的 药物中的用途。
根据本发明， 在本发明中使用的缩写词具有下面的含义：
Boc-HCPT -叔丁氧羰基 -羟基喜树碱
PEG-聚乙二醇
mPEG -单甲氧基聚乙二醇
Et3N-三乙胺 RP-HPLC -反相高效液相色傳
TLC -薄层色谱
EDC-HC1 - 1-乙基 -3-(3-二甲基氨基丙基)碳二亚胺盐酸盐
DCM -二氯甲烷
DMAP - iV，iV-二甲基 -4-氨基吡
Ts-Cl -对甲苯磺酰氯
MALDI-TOF-MS -基质辅助激光解析飞行时间质谱
TCID5。-半数培养感染剂量
MDCK -犬腎细胞
MTT -四氮唑蓝
DMEM - Dulbecco极限必需培养基
根据本发明， 本发明所涉及的 mPEG - OH可以作为商品化 试剂从德国 Fluka公司购买， mPEG - NH2可以购买或通过以下 反应得到：
E¾N
mPEG— OH + Ts-Cl ^ mPEG— OTs
Figure imgf000006_0001
本发明以平均分子量为 1000、 2000、 5000 和 10000 的 CH3O PEG-OH ( mPEG ) 为原料， 合成系列单位点 mPEG修饰 以及双位点 mPEG修饰的喜树碱衍生物。各化合物经 RP - HPLC 以及 TLC分析为单一峰， 经质谱和氢谱、碳傳核磁分析， 确证结 构正确„
根据本发明， 本发明的药物组合物制成适用于哺乳动物用的 各种剂型， 包括但不限于混悬剂、 溶液剂、 乳剂等注射剂形式， 例如用甘露醇作赋形剂制成的注射剂。 根据本发明， 本发明的药 物组合物可以以标准方式施用， 例如肠胃外施用包括但不限于静 脉内的、 动脉内的、 腹膜内的、 皮下的、 肌肉内的施用或连续输 注。 具体实施方式
实施例
下述实施例代表本发明的说明性实施方案， 但本发明不受这 些实施例的限制。 实施例所用平均分子量为 1000、 2000、 5000 和 10000的 CH30-PEG-OH ( mPEG ) 为德国 Fluka公司产品， 1-乙基 -3-(3-二甲胺丙基)碳二亚胺盐酸盐 （ED HC1 ) 与二甲胺 基吡啶（DMAP ) 均为国产分析纯试剂。
实施例 1 20-聚乙二醇 5000单甲醚琥珀酸单酯 -10-甲氧基喜 树碱 ( MC5001 ) 的合成
1. 聚乙二醇单甲醚琥珀酸单酯的合成
(参考： Polymer Bulletin, 1980， 3, 347-352. )
聚乙二醇单甲醚（30g, 6mmol ) 、 琥珀酸酐（3g, 30mmol ) 以及 A V-二甲基 -4-氨基吡啶（DMAP, 300mg, 2.46mmol )置于 100ml两颈瓶中， 在氮气保护下， 加入 50ml二氯甲烷。 常温搅拌 反应 5-6天， 薄层色谱监测反应进程至反应完全。 依次用 1N HC1 ( 60mlx2 ) 、 蒸馏水（60mlx2 )洗涤， 硫酸镁干燥， 过滤， 蒸去 大量的二氯甲烷溶剂， 乙醚分散。 白色固体（20.5g ) , 收率 67%。 XH NMR ( DCC13 ) 4.26 ( t, 2H ) , 3.4-4.0 ( m, PEG ) , 3.384 ( s, 3H ) , 2.6 ( m, 4H ) 。
2. 10-甲氧基喜树碱的合成（MeO-CPT )
(参考： Bioorg. & Med. Chem. Letters, 2008， 18， 6441-6443； CN200410052756.7, page 8 ) 。
两颈瓶中加入 10-羟基喜树碱 ( 182mg, 0.5mmol )和无水碳 酸钟（210mg， 1.5mmol ) , 在氮气保护下， 加入无水丙酮 40ml, 加热至回流反应 10分钟。 加入換甲烷（ 200//L, 2.5mmol ) , 回 流反应至原料基本消失， 约 6个小时左右。
反应液倒入 100ml二氯甲烷中，依次用 1N盐酸（ 100mlx2 )、 水（100mlx2 ) 洗涤， 硫酸镁干燥， 过滤， 减压蒸干。 氯仿重结 晶， 收得晶体少量。 加入石油醚适量， 大量黄色粉末固体析出， 过滤 减压抽干。 黄色粉末固体。 收率约 50% ( lOOmg ) 。
Figure imgf000008_0001
3. 20-聚乙二醇 5000 单甲醚琥珀酸单酯 -10-甲氧基喜树碱 ( MC5001 ) 的合成
聚乙二醇 5000单甲醚琥珀酸单酯 （ mPEG5000-Suc.， l.Og, 0.2mmol ) , 1-乙基 -3-(3-二甲胺丙基)碳二亚胺盐酸盐（ ED HCl, 120mg, 0.4mol )和 V，iV-二甲基 -4-氨基吡 （DMAP， 48.8mg, 0.4mol )置于两口瓶中，在氮气保护下，加入 30ml二氯甲烷溶解。 常温搅拌反应 30分钟后， 加入 10-甲氧基喜树碱 ( MeO-CPT, 103mg, 0.28mmol ) 。 常温反应 24小时。 TLC监测至几乎不再 反应。 停止反应， 抽去大量二氯甲烷， 柱层析（^25x100， 100-200 目硅胶， 先用乙酸乙酯洗去未反应的 Boc-HCPT, 然后用甲醇： 二氯甲烷 =1:100依次上升到约 5:100 )分离， 收集产物部分， 减 压蒸去溶剂， 少量乙醚固化。 异丙醇（约 60ml )加热溶解， 冷却 析出白色固体， 过滤； 重复一次。 乙醚洗涤产物， 减压抽干。 收 得白色固体产物， 收率 66% ( 700mg ) 。 经 MALDI-TOF-MS鉴 定， 分子量在 5492左右呈正态分布， 分子量正确。
实施例 2 20-聚乙二醇 5000单甲醚琥珀酸单酯 -10-甲氧基喜 树碱 ( MC5001 )抗肿瘤活性的评价
选择人胃癌细胞 BGC-823、人乳腺癌细胞 MCF-7、人肝癌细 胞 HepG2和人大肠癌细胞 HT29四种细胞株，接种细胞于含 10% 胎牛血清的 RPMI1640或 DMEM细胞培养液中 （补充青、 链霉 素各 100u/ml ) , 置于 37°C含 5%C02的细胞培养箱中， 每 2-3天 换液一次， 0.25%胰蛋白酶消化， 传代和收集细胞。 将对数生长 期细胞， 用含 10%胎牛血清的 RPMI1640 细胞培养液配制成 2.5xl04/ml浓度的细胞悬液， 按每孔 3000细胞（ΙΟΟμΙ )加入到 96孔细胞培养板中， 培养 24小时后每孔加入含有不同浓度受试 物的培养基 ΙΟΟμΙ, 每个浓度设 3 ~ 4个平行孔。 培养 72小时后 弃上清。 每孔加入 ΙΟΟμΙ新鲜配制的 0.5mg/ml四氮唑蓝（ MTT ) 的无血清培养液， 37°C培养 4小时后弃上清。 以 lOO l DMSO溶 解， 轻度振荡 15分钟后， 用酶标仪检测吸光度（OD值） ， 检测 波长为 570nm、 参比波长为 450nm。 设置受试药品浓度为 10.0 、 5.0、 2.5、 1.25、 0.625、 0.3125、 0.15625 g/ml, 另设空白对照。 药物临用前， 用细胞培养液溶解使用。
数据处理： 抑制率 = (对照组 OD值-给药组 OD值） /对照 组 OD值 xl00%; 药物效应指标用半数浓度 ( IC50 )表示， 并列 出实测最大抑制率 （Imax ) 。 用 MicroCal Origin软件作图， 及 该软件中的四参数 Logistic程序拟合肿瘤细胞生长曲线， 求出半 数抑制浓度（IC5Q， g/ml ) 。 具体结果见表 1。
表 1 10-羟基喜树碱抑制各种受试细胞生长的作用
10-羟基喜树碱
细胞
半数抑制浓度 IC5。（ g/ml ) 最大抑制效应 （Imax, % )
BGC-823 >10 41.3
MCF-7 〈0.15625 72
HepG2 0.164 82
HT-29 〈0.15625 93.3% 表 2 MC-5001抑制各种受试细胞生长的作用
MC-5001
细胞
半数抑制浓度 IC5。（ g/ml ) 最大抑制效应 （Imax, % )
BGC-823 >10 43.7
MCF-7 0.148 73.1
HepG2 0.429 81.5
HT-29 0.056 93.3 由表 1和表 2显示， MC-5001与 10-羟基喜树碱对四种肿瘤 细胞的抑制活性相当。

Claims

权利要求
1. 式（I ) 所示的喜树碱类化合物的聚乙二醇化合物:
Figure imgf000011_0001
其中， RfH, OH, OMe， 或结构 O-XrmPEG R2为结构 X2-mPEG2； 其 中 ， mPEGi 与 mPEG2 分别 独立 的 为 CH2CH2(OCH2CH2)n1-OCH3 , ηχ = 0 - 1000； 1与 2分别独立的
0 0
为一 C— (CH2)n2— ϋ-χ3， η2为 0 - 4， Χ3可以为 Α、 Β或 Υ;
A = - (NH-CR3R4-CO)n3 - (NH-CR5R6-CO)n4 - (NH-CR7R8-CO)n5 - (NH-CR9Ri0-CO)n6 - Z -； R3到 。分别独 立的为 H， 取代或未取代的 d-C6直链或者支链烷基， 取代或未 取代的 C2-C6直链或者支链烯基或者炔基，取代或未取代的 d-C6 直链或者支链烷氧基， 取代或未取代的 d-C6直链或者支链烷硫 基， 取代或未取代的 C2-C6直链或者支链烯基或者炔基氧基， 取 代或未取代的 （ 2-( 6直链或者支链烯基或者炔基， 环烷基或环链 烯基， 芳基， 杂环基； -（CH2)n7 - CO - O - mPEG; - (CH2)n7 - CO - NH - mPEG; n7为 1 - 6， mPEG定义同前； 其中， 优选 地， 杂环烷基为其环结构中含 1 - 5个（优选 1 - 3个）独立地选 自 N、 O和 S等的杂原子的环状基团； 芳基为未取代的或被独立 地选自卤素， 硝基， 羧基或 d-C4烷基的取代基单取代或二取代 或三取代的 4、 5、 6、 或 7元单环或双环芳香基团， 如苯基或者 或萘基等； 杂环基可为未取代的或被独立地选自卤素， 硝基， 羧 基或 d-C4烷基的取代基单取代或二取代的、含有 1 - 5个独立地 选自 N、 O和 S等的杂原子的 4、 5、 6、 或 7元单环或双环芳香 基团， 如吡咯基， 呋喃基， 吡啶基等； n3到 n6分别独立的为 0 或 1; Z为 O或 NH;
B = - NH-(CH2)n8-S - S-(CH2)n9-C(0) - Z-； n8为 2-5， n9为 1-4; Z为 O或 NH;
Y = - NH-(CH2)n10 -S-M- , n10为 2 - 5;
更为优选的
M为
Figure imgf000012_0001
2. 权利要求 1的化合物，其中所述 A中的 n3 = n4 = ii5 = ii6 = 0 时， 则 Z为 O或 NH。
3. 权利要求 1的化合物， 其中所述 B中的 n8 = 2, n9=l时， 则 Z为 O或 NH。
4. 权利要求 1的化合物， 其中所述 Y中的 M为
Figure imgf000012_0002
则 n10 = 2。
5. 权利要求 1的化合物， 其中 =Η， OH, OMe。
6. 权利要求 1 的化合物， 其中所述式（I) 中的喜树碱类化合 物选自喜树碱、 10-羟基喜树碱和 10-甲氧基喜树碱。
7. 权利要求 6 中的化合物， 其中所述式 （I) 所示的喜树碱类 化合物的聚乙二醇化合物是 20-聚乙二醇 5000单甲醚琥珀酸单酯 -10-甲氧基喜树碱。
8. 药物组合物， 其含有权利要求 1 的至少一种式 （I ) 的化合 物及药物载体或赋形剂。
9. 权利要求 1-8中任一项的化合物或组合物在制备抗肿瘤等 相关作用的药物中的用途。
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