CN1332716C - High molecular antineoplastic cisplatin medicine and preparation method thereof - Google Patents

High molecular antineoplastic cisplatin medicine and preparation method thereof Download PDF

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CN1332716C
CN1332716C CN 200510072155 CN200510072155A CN1332716C CN 1332716 C CN1332716 C CN 1332716C CN 200510072155 CN200510072155 CN 200510072155 CN 200510072155 A CN200510072155 A CN 200510072155A CN 1332716 C CN1332716 C CN 1332716C
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rpeg2y
cisplatin
integer
polymer
structure
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CN1698903A (en )
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黄骏廉
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北京市海格里斯科技有限公司
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Abstract

本发明以双臂RPEG In the present invention, arms RPEG

Description

一种高分子抗肿瘤顺铂药物及其制备方法 A polymer anti-tumor drugs cisplatin and its preparation method

技术领域 FIELD

本发明涉及一种药品,特别涉及一种以双臂聚乙二醇为载体的高分子抗肿瘤顺铂药物及其制备方法,即以双臂RPEG2Y-(简称:双臂聚乙二醇)为载体,通过官能团化反应对小分子抗肿瘤药物顺铂进行改性。 The present invention relates to a pharmaceutical, more particularly to a double-arm polyethylene glycol as a carrier polymer cisplatin anti-tumor and preparation method thereof, that arms RPEG2Y- (abbreviation: polyethylene glycol arms) of vector, by reaction of a functional group on the small molecule anticancer drugs cisplatin modified.

背景技术 Background technique

自从1969年B.Rosenberg和V.Camp首次报道顺式-二氯二氨合铂(简称顺铂)具有广谱的抗癌活性以来,顺铂在癌症治疗中的应用和研究得到了迅速的发展,目前已经成为各国普遍临床使用的抗肿瘤药物。 Since 1969 B.Rosenberg and V.Camp first reported cis - since dichloro-diammine platinum (cisplatin for short) has a broad spectrum of antitumor activity, cisplatin and research applications in cancer treatment has been rapid development It has now become a national anticancer drugs commonly used clinically. 然而,该药物存在毒副作用大、在体内代谢速度快、易被快速清除等缺点,因而限制了顺铂治疗指数的进一步提高。 However, the presence of toxic side effects of the drug, in vivo metabolism fast speed, susceptible to rapid clearance and other shortcomings, thus limiting further improve the therapeutic index of cisplatin. 因此,降低顺铂的毒副作用,延长其在体内的代谢半衰期已经成为提高顺铂疗效的当务之急。 Therefore, reducing the side effects of cisplatin, extending its metabolism in vivo half-life has become imperative to improve the efficacy of cisplatin. 而把其载体化则无疑为此提供了一条有效的途径。 The carrier of the doubt to its aims to provide an effective way.

聚乙二醇(PEG)是一种水溶性的两亲聚合物,具有良好的生物相容性、非免疫原性、无毒等特点,因此,它是顺铂改性的理想的大分子载体。 Polyethylene glycol (PEG) is a water soluble amphiphilic polymer, having a good biocompatibility, non-immunogenic, non-toxic characteristics, therefore, it is desirable to cisplatin-modified macromolecular carrier . 与相同分子量的线型PEG相比,双臂及多臂PEG由于其有较大的流体力学体积,较小的静态黏度,预计对药物会产生更为有效的生理作用。 Compared with the same molecular weight linear PEG, multi-arm PEG arms, and because of its large hydrodynamic volume, the smaller the static viscosity, expected to generate more effective physiological effects of drugs. 目前尚未见到类似报导。 We have yet to see similar reports.

发明内容 SUMMARY

本发明的目的是获得一种以双臂聚乙二醇为载体的高分子抗肿瘤顺铂药物及其制备方法。 Object of the present invention is to obtain a double-arm polyethylene glycol as a carrier polymer tumor and preparation method thereof cisplatin antibody.

本发明以双臂聚乙二醇(RPEG2Y-)作为载体,通过其官能端基二酸的螯合作用对小分子抗肿瘤药物顺铂进行改性,化学通式为RPEG2Y-BC,其中RPEG2Y-简称为双臂聚乙二醇,B为螯合顺铂的带有两个羧酸基团的结构,C为被修饰的小分子药物顺铂,其结构式如下: In the present invention, polyethylene glycol arms (RPEG2Y-) as a carrier, is modified, the general chemical formula of a small molecule anti-tumor drugs cisplatin by chelation its functional end groups diacid RPEG2Y-BC, wherein RPEG2Y- referred to as a polyethylene glycol arms, B is a chelate structure with cisplatin two carboxylic acid groups, C is the modified small molecule drugs cisplatin, having the formula as follows: or 其中R为10个碳以下的直链烷烃、异丙基或苄基;R1为CH或苯基;R2为1~9个碳原子的烃基;a、b、c为0~9的整数;k为0~6的整数;s、t是使RPEG2-中的RO-(CH2CH2O)s-及RO-(CH2CH2O)t-部分的Mn约为2000~80000的整数; Wherein R is C 10 or less linear alkane, isopropyl or benzyl; Rl is CH or phenyl; R2 is a hydrocarbon group having 1 to 9 carbon atoms; a, b, c is an integer from 0 to 9; K is an integer of 0 to 6; s, t is the so RPEG2- RO- (CH2CH2O) s- and RO- (CH2CH2O) Mn t- integer portion is about 2,000 to 80,000;

W为O、S、 W is O, S, NH之一,同一分子中W可为不同基团;Y为 One NH, W may be the same molecule, different radicals; Y is NH、S、O、 NH, S, O, CH=、N=之一;V为 CH =, N one =; V is NH、S、O、 NH, S, O, =HC、=N之一。 = HC, = one N.

上述双臂聚乙二醇(RPEG2Y-)载体结构中,R为甲基尤为优选。 Polyethylene glycol arms above (RPEG2Y-) support structure, R is a methyl group is particularly preferable.

上述高分子抗肿瘤药物的分子量为2000-160000。 Molecular weight of the polymer of antineoplastic 2000-160000.

上述高分子抗肿瘤药物的制备方法,是将用常规方法制备的官能化双臂聚乙二醇(RPEG2Y-X)和含有丙二酸酯结构的化合物反应,然后水解得到含丙二酸结构的化合物RPEG2Y-B,最后RPEG2Y-B和氯原子被硝酸根取代的顺铂反应得到目标产物RPEG2Y-BC。 The method of preparing the above polymer antineoplastic agents, is polyethyleneglycol functionalized with arms (RPEG2Y-X) prepared by a conventional method and a compound comprising structure malonate, malonate followed by hydrolysis to give structure compound RPEG2Y-B, and finally RPEG2Y-B and chlorine atoms are substituted with cisplatin nitrate to afford the desired product RPEG2Y-BC.

该化学反应利用了现有技术中现有反应条件而得。 The prior art utilizes the chemical reaction in the reaction conditions existing obtained.

上述高分子抗肿瘤药物中的载体双臂RPEG是以线性RPEG为原料,通过合成RPEG活性酯,利用多步反应分别在水相以及油相中与例如赖氨酸的ω及α位的胺基偶合得到的。 The polymer carrier arms antineoplastic RPEG RPEG is linear as a raw material for synthesis RPEG active esters, the use of multi-step reactions, respectively, in the aqueous phase and an oil phase with an amine e.g. lysine at position α and ω the resulting coupling. 双臂树杈状PEG的合成方法在本领域中已有描述,例如:Yamsuki等,Agric.Bio.Chem.1988,52,2185-2196;Monfardidi等,BioconjugateChem.1995,6,62-69。 PEG-shaped arms branches synthesis have been described in the art, for example: Yamsuki et, Agric.Bio.Chem.1988,52,2185-2196; Monfardidi the like, BioconjugateChem.1995,6,62-69.

上述高分子抗肿瘤药物制备是将双臂RPEG上的羧酸官能团与带丙二酸酯结构的化合物如氨基丙二酸二乙酯连接,然后水解得到丙二酸结构,最后与氯原子被取代为硝酸根的顺铂偶合。 The polymer prepared antitumor agent is a carboxylic acid functional group on arms RPEG malonate compound with a structure, such as Diethyl connected, followed by hydrolysis to give malonic acid structure, eventually substituted with a chlorine atom as nitrate cisplatin coupling. 以单甲氧基聚乙二醇(mPEG)和赖氨酸(lysine)制备的双臂聚乙二醇为例,该高分子抗癌药物的合成路线如下: In monomethoxy polyethylene glycol (mPEG) and lysine (lysine) was prepared as an example polyethylene glycol arms, the polymer anticancer drugs scheme is as follows:

关于线形PEG连接顺铂的方法在本领域中已有描述,例如Yuichi等,Macromol.Biosci.2001,1,No.8,355-363。 The method of connecting a linear PEG on cisplatin been described in the art, like e.g. Yuichi, Macromol.Biosci.2001,1, No.8,355-363.

上述高分子抗肿瘤药物中小分子药物的接药率在90%-99%之间。 Small molecule dose rate then the polymer antineoplastic drugs between 90% -99%.

上述的高分子抗肿瘤药物RPEG2Y-BC及制备方法,所述的RPEG2-的结构是: The above-described polymer and antineoplastic RPEG2Y-BC preparation, RPEG2- the structure is:

其中R为10个碳以下的直链烷烃、异丙基或苄基;R1为CH或苯基;a、b、c为0~9的整数;k为0~6的整数;s、t、为10~2000的整数;W为O、S、NH Wherein R is C 10 or less linear alkane, isopropyl or benzyl; Rl is CH or a phenyl group; a, b, c is an integer of 0-9; K is an integer of 0 to 6; s, t, is an integer of 10 to 2,000; W is O, S, NH 之一,同一分子中W可为不同基团。 One of W in the same molecule may be a different group.

例如: E.g: 等。 Wait.

上述双臂聚乙二醇RPEG2Y-的结构中R是甲基尤为优选。 Polyethylene glycol arms above RPEG2Y- structure in which R is methyl are particularly preferred.

上述的高分子抗肿瘤药物及制备方法中,Y可以为 Antineoplastic above polymer and production method, Y may be NH、S、O、 NH, S, O, CH=、N=之一; CH =, N = one;

上述的高分子抗肿瘤药物及制备方法中,官能化的双臂聚乙二醇为RPEG2CO-X时,其中的X可以为下述结构之一:-H-OH-NH2 Antineoplastic above polymer and preparation method, the functionalized polyethylene glycol for the arms when RPEG2CO-X, where X can be one of the following structures: -H-OH-NH2 上述的高分子抗肿瘤药物RPEG2Y-BC及制备方法中,B的结构可以是下述之一: The above-described polymer and antineoplastic RPEG2Y-BC preparation, structure B may be one of the following: 其中R2为1~9个碳原子的烃基;V为 Wherein R2 is a hydrocarbon group having 1 to 9 carbon atoms; V is NH、S、O、 NH, S, O, =HC、=N之一。 = HC, = one N.

例如: E.g:

等。 Wait.

上述的高分子抗肿瘤药物及制备方法中,C的结构是两个氯原子被取代的顺铂。 Antineoplastic above polymer and production method, structure C is substituted with two chlorine atoms in cisplatin.

上述高分子抗肿瘤药物以双臂RPEG为载体,通过化学反应,将小分子抗肿瘤药物顺铂与载体偶合。 In the polymer arms RPEG antitumor agent as a carrier by a chemical reaction, small molecule anti-tumor drugs cisplatin and coupled to the carrier. 和相同分子量的线性RPEG比较,双臂RPEG具有更大的流体力学体积,这样就更有效的减少了药物在生物体内失活或被酶水解的速度。 Linear RPEG and the same molecular weight compared RPEG arms has a larger hydrodynamic volume, this more effectively reduces the deactivation rate of the drug or enzymatic hydrolysis in vivo. 同时通过丙二酸的螯合作用与顺铂连接生成的稳定的六元环,使药物的释放更加缓慢,增加了药物在体内的半衰期。 Simultaneously connected by chelation with cisplatin malonic acid produced a stable six-membered ring, so that the release the drug more slowly, increasing the drug's half-life in vivo.

上述具有通式RPEG2CO-BC的双臂mPEG顺铂偶合物(DImPEG-Pt)经体外细胞毒性实验表明,该药物保留了相当程度的抗肿瘤活性;同时,与相同分子量的线性mPEG顺铂偶和物相比,具有通式RPEG2CO-BC的高分子抗肿瘤药物具有更高的体外细胞毒活性,这也说明了双臂mPEG比线性mPEG更适合于对小分子药物的改性,可以更大限度的减少药物失活的程度。 MPEG arms above cisplatin conjugate (DImPEG-Pt) having the general formula RPEG2CO-BC cells via in vitro toxicity tests showed that the drug retains a considerable degree of antitumor activity; the same time, even with the same linear mPEG molecular weight Cisplatin and compared thereof, having the general formula RPEG2CO-BC polymer having higher antitumor cytotoxic activity in vitro, which also shows more than a linear mPEG mPEG arms suitable for modification of small molecule drugs, a greater extent reduction in the degree of inactivation of the drug.

附图说明 BRIEF DESCRIPTION

图1是双臂聚乙二醇改性的顺铂(DImPEG-Pt)系列的剂量反应曲线图图2是单臂聚乙二醇改性的顺铂(mPEG-Pt)系列的剂量反应曲线图具体实施方式实施例一RPEG2CO-BC的合成方法(以单甲氧基聚乙二醇(mPEG)分子量2000为例)1.将10g mPEG(Mn=2000,5mmol)溶于约150mL甲苯,在氮气保护下常压蒸除绝大部分甲苯,以除去mPEG中所含的微量水分。 FIG 1 is a dual-arm polyethylene glycol-modified cisplatin (DImPEG-Pt) series of dose-response curves of FIG. 2 is a graph showing the dose response of a single arm of cisplatin-modified polyethylene glycol (mPEG-Pt) series Examples of a synthetic method RPEG2CO-BC (in mono methoxy polyethylene glycol (mPEG) molecular weight of 2000, for example) DETAILED DESCRIPTION embodiment 1 the 10g mPEG (Mn = 2000,5mmol) was dissolved in about 150mL of toluene, under a nitrogen under atmospheric pressure under most of the toluene was distilled off to remove traces of moisture contained in the mPEG. 冷至室温后,向其中加入约200mL无水THF,加热微沸至mPEG完全溶解。 After cooling to room temperature, adding about 200mL of THF anhydrous, it was heated to a gentle boil mPEG completely dissolved. 随后向mPEG溶液中滴加0.2mol/L的萘钠溶液直至溶液呈灰绿色。 Followed by dropwise addition of 0.2mol / L sodium naphthalene solution until the solution was added to mPEG grayish green. 取5.24mL氯乙酸乙酯(6.03g,50mmol)用50mL THF稀释后加入反应溶液中,室温下搅拌反应4h。 The reaction solution was added ethyl taken 5.24mL (6.03g, 50mmol) chloride was diluted with 50mL THF, the reaction was stirred at room temperature for 4h. 反应液经浓缩后用无水乙醚沉淀,所得粗产物加到300mL 0.1mol/L NaOH溶液中,50℃搅拌12h。 After precipitation with anhydrous ethyl ether the reaction was concentrated, the resulting crude product was added 300mL 0.1mol / L NaOH solution, stirred for 12h 50 ℃. 然后用1mol/L HCl调节pH=3,用CH2Cl2萃取三次,合并萃取液浓缩后用无水乙醚沉淀。 Then adjusted pH = 3 with 1mol / L HCl, extracted three times with CH2Cl2, and precipitated with anhydrous ethyl ether extracts were combined and concentrated. 再经两次溶解-过滤-沉淀后,真空干燥,得mPEG-CH2COOH 9.5g,产率92%。 And then the two dissolution - filtration - the precipitate was dried under vacuum to afford mPEG-CH2COOH 9.5g, 92% yield. 经核磁共振测定,产物中mPEG-CH2COOH摩尔含量为95%。 As determined by NMR, mPEG-CH2COOH molar content of the product was 95%. (1HNMR(CDCl3)mPEG-CH2COOH,δ=4.15) (1HNMR (CDCl3) mPEG-CH2COOH, δ = 4.15)

2.将上述9.5g mPEG-CH2COOH(Mn=2058,4.6mmol)溶于约150mL甲苯,在氮气保护下常压蒸除绝大部分甲苯,以除去其中所含的微量水分。 2. The above 9.5g mPEG-CH2COOH (Mn = 2058,4.6mmol) was dissolved in about 150mL of toluene under nitrogen pressure most of the toluene was distilled off to remove traces of moisture contained therein. 冷至室温后,向其中加入约120mL无水二氯甲烷溶解mPEG-CH2COOH。 After cooling to room temperature, and thereto was added 120mL of dry dichloromethane was dissolved about mPEG-CH2COOH. 随后向溶液中分别加入N-羟基琥珀酰亚胺(NHS)1.06g(9.2mmol),二环己基碳二亚胺(DCC)2.86g(13.8mmol),于冰水浴下搅拌反应12h。 Followed by addition of N- hydroxysuccinimide were added to the solution (NHS) 1.06g (9.2mmol), dicyclohexyl carbodiimide (DCC) 2.86g (13.8mmol), the reaction was stirred under ice-water bath 12h. 反应液经浓缩后用无水乙醚沉淀,所得粗产物在加热下溶于约40mL干燥甲苯,过滤,除去其中的不溶物后用无水乙醚沉淀。 After precipitation with anhydrous ethyl ether the reaction was concentrated, the resulting crude product was dissolved under heating to about 40mL of dry toluene, filtered, after which the insolubles were removed by precipitation with anhydrous ether. 再经两次溶解-过滤-沉淀后,真空干燥,得mPEG-CH2COOSu(mPEG-琥珀酰亚胺碳酸酯)9.0g,产率90%。 And then the two dissolution - filtration - the precipitate was dried under vacuum to afford mPEG-CH2COOSu (mPEG- succinimidyl carbonate) 9.0 g of, 90% yield. 经紫外光谱法来测定,产物中琥珀酰亚胺碳酸酯的摩尔含量为94%。 Was determined by UV spectroscopy, molar content in the product succinimidyl carbonate was 94%.

3.称取赖氨酸(Lysine)1.46g(10mmol)溶于80mL pH=8的硼酸、硼砂缓冲溶液中。 3. Weigh Lys (Lysine) 1.46g (10mmol) was dissolved in 80mL pH = boric acid, borax buffer 8. 将第2步中制得的mPEG-CH2COOSu4.3g(2mmol)在1h内分批逐步加入,期间通过滴加0.2mol/L的NaOH溶液保持溶液的pH值为8(用pH计监控)。 Step 2 The resulting mPEG-CH2COOSu4.3g (2mmol) was gradually added portionwise over 1h, pH by dropwise addition of 0.2mol / L NaOH solution to maintain the solution was 8 (monitored with pH meter) period. 室温继续搅拌反应12h后,在冰水浴下将溶液的pH值用盐酸调至2-3,用二氯甲烷萃取产物。 The reaction After 12h, the solution under ice-water bath and stirring was continued at room temperature the pH of 2-3, and the product was extracted with dichloromethane raised hydrochloride. 反复萃取三次,将萃取液合并,浓缩,于无水乙醚中沉淀。 Repeatedly extracted three times, the extracts were combined, concentrated, precipitated into anhydrous ether. 再溶解-过滤-沉淀二次后,真空干燥,得到产物mPEG-CH2CO-Lysine,用于下一步反应。 Redissolved - filtration - After the precipitation twice and dried under vacuum to give the product mPEG-CH2CO-Lysine, for the next reaction.

4.将上步反应所得产物mPEG-CH2CO-Lysine溶于约80mL无水二氯甲烷中,滴加三乙胺调节溶液pH值至8-9(将溶液滴至湿润的精密pH试纸测得)。 Step 4. The resulting reaction product of the mPEG-CH2CO-Lysine was dissolved in about 80mL of dry methylene chloride, triethylamine was added dropwise to adjust the solution pH 8-9 (solution dropwise to wet pH test paper precision) . 在3小时内分批逐步加入第2步中制得的mPEG-CH2COOSu 4.7g(2.2mmol),并不断滴加三乙胺,保持溶液的pH值为8-9。 Portionwise over 3 hours to gradually added in step 2 prepared in mPEG-CH2COOSu 4.7g (2.2mmol), and continuously dropwise addition of triethylamine while maintaining the solution pH of 8-9. 然后将反应液升温至40℃,回流搅拌48小时后,过滤、浓缩,于无水乙醚中沉淀。 The reaction was then warmed to 40 ℃, after stirring at reflux for 48 hours, filtered, concentrated, precipitated into anhydrous ether. 所得产物再溶解-过滤-沉淀二次后,真空干燥,得产物8.0g。 The resulting product was redissolved - filtration - After the precipitation twice and dried in vacuo to give the product 8.0g. 经GPC测定,其中双臂mPEG2-CHCO-Lysine的含量约为80%。 Determined by GPC, wherein the content of arms mPEG2-CHCO-Lysine about 80%.

将上述产物配成10%的水溶液,选择合适孔径的超滤膜,在1.5个大气压下,超滤分离,即可提纯双臂mPEG,最终得产物mPEG2-CHCO-Lysine5.0g。 The above product was formulated into a 10% aqueous solution, select the appropriate membrane pore size, at 1.5 atmospheres, ultrafiltration, can be purified arms of mPEG, to give the final product mPEG2-CHCO-Lysine5.0g.

5.第4步中制得的将5.0g mPEG2-CHCO-Lysine(Mn=4226,1.2mmol)溶于约100mL甲苯溶液,在氮气保护下常压蒸除绝大部分甲苯,以除去其中所含的微量水分。 5. Step 4 will be prepared 5.0g mPEG2-CHCO-Lysine (Mn = 4226,1.2mmol) was dissolved in about 100mL toluene solution under nitrogen pressure most of the toluene was distilled off to remove contained therein trace moisture. 冷至室温后,向其中加入约120mL无水二氯甲烷溶解mPEG2-CHCO-Lysine。 After cooling to room temperature, and thereto was added 120mL of dry dichloromethane was dissolved about mPEG2-CHCO-Lysine. 随后向溶液中分别加入N-羟基琥珀酰亚胺(NHS)0.28g(2.4mmol),二环己基碳二亚胺(DCC)0.75g(3.6mmol),于冰水浴下搅拌反应12h。 Followed by addition of N- hydroxysuccinimide (NHS) 0.28g (2.4mmol) were added to the solution, dicyclohexyl carbodiimide (DCC) 0.75g (3.6mmol), under ice-water bath the reaction was stirred 12h. 反应液经浓缩后用无水乙醚沉淀,所得粗产物在加热下溶于约40mL干燥甲苯,过滤,除去其中的不溶物。 After precipitation with anhydrous ethyl ether the reaction was concentrated, the resulting crude product was dissolved under heating to about 40mL of dry toluene and filtered to remove insoluble substances. 再经两次溶解-过滤-沉淀后,真空干燥,得mPEG2-CHCO-Lysine-OSu 4.5g,产率90%。 And then the two dissolution - filtration - the precipitate was dried in vacuo to give mPEG2-CHCO-Lysine-OSu 4.5g, 90% yield. 经紫外光谱法来测定,产物中琥珀酰亚胺碳酸酯的摩尔含量为98%。 Was determined by UV spectroscopy, molar content in the product succinimidyl carbonate was 98%.

6.称取0.7g氨基丙二酸二乙酯(DEAM)(3.3mmol),溶于50mL无水二氯甲烷中,加入三乙胺1mL,冰水浴下搅拌2小时。 6. Weigh 0.7g of diethyl (DEAM) (3.3mmol), was dissolved in 50mL anhydrous dichloromethane, was added triethylamine 1mL, with stirring under ice-cooling for 2 hours. 将4.5g第5步中制得的mPEG2-CHCO-Lysine-OSu(1.1mmol)溶于约80mL无水二氯甲烷中,滴加三乙胺调节溶液pH值至8-9(将溶液滴至湿润的精密pH试纸测得)。 4.5g of Step 5 prepared in mPEG2-CHCO-Lysine-OSu (1.1mmol) was dissolved in about 80mL of dry methylene chloride, triethylamine was added dropwise to adjust the solution pH 8-9 (solution dropwise to precision wet pH test paper). 冰水浴下将前面反应的氨基丙二酸二乙酯溶液加入到mPEG2-CHCO-Lysine-OSu溶液中。 The ice-water bath amino previous reaction was added to a solution of diethyl malonate mPEG2-CHCO-Lysine-OSu solution. 0℃下反应12小时后,过滤、浓缩,于无水乙醚中沉淀。 After 12 hours of reaction at 0 deg.] C, filtered, concentrated, precipitated into anhydrous ether. 所得产物再溶解-过滤-沉淀二次后,真空干燥,得产物4.3g。 The resulting product was redissolved - filtration - After the precipitation twice and dried in vacuo to give the product 4.3g. 经核磁共振测定,其中mPEG2-CHCO-Lysine-DEAM的含量约为80%。 As determined by nuclear magnetic resonance, wherein the content of mPEG2-CHCO-Lysine-DEAM about 80%. (1H NMR(CDCl3)COOCH2CH3,δ=1.30)7.冰水浴下将第6步中制得的4.3g mPEG2-lysine-DEAM(0.98mmol)溶于NaOH甲醇溶液(甲醇92mL,1mol/L NaOH 13.5mL)中,搅拌2h。 (1H NMR (CDCl3) COOCH2CH3, δ = 1.30) 7. The ice-water bath prepared in step 6 of 4.3g mPEG2-lysine-DEAM (0.98mmol) was dissolved in methanol solution of NaOH (methanol 92mL, 1mol / L NaOH 13.5 mL), and stirred for 2h. 用1mol/L HCl调节pH=6,40℃旋转蒸发溶剂,然后加入二氯甲烷溶解,加入无水硫酸镁干燥。 Adjusted with 1mol / L HCl pH = 6,40 ℃ rotary evaporation, then dissolved in methylene chloride was added, dried over anhydrous magnesium sulfate. 过滤,将滤液浓缩,于无水乙醚中沉淀。 Filtered, and the filtrate was concentrated and precipitated into anhydrous ether. 再溶解沉淀两次后,真空干燥,得产物(mPEG2-CHCO-Lysine-DA)4.0g,产率95%。 After redissolving precipitated twice, and dried in vacuo to give the product (mPEG2-CHCO-Lysine-DA) 4.0g, 95% yield. 经核磁共振测定,其中mPEG2-CHCO-Lysine-DA含量为75%。 As determined by nuclear magnetic resonance, wherein mPEG2-CHCO-Lysine-DA content of 75%. (核磁中1.30处的峰消失)8.将顺铂1.35g(4.5mmol)溶于约200mL去离子水,加热至60℃使之完全溶解。 (1.30 in NMR peak disappeared) 8. Cisplatin 1.35g (4.5mmol) was dissolved in approximately 200mL of deionized water and heated to 60 deg.] C to complete dissolution. 再加入硝酸银1.52g(9mmol),于60℃避光搅拌反应6h后滤掉生成的氯化银沉淀。 Silver nitrate was added 1.52g (9mmol), stirred at 60 deg.] C in the dark for 6h the reaction of silver chloride precipitate generated was filtered off. 向滤液中加入4.0gmPEG2-CHCO-Lysine-DA(0.92mmol),继续于60℃避光搅拌反应24h后,用二氯甲烷萃取产物。 To the filtrate was added 4.0gmPEG2-CHCO-Lysine-DA (0.92mmol), the reaction was continued at 60 deg.] C in the dark After stirring 24h, the product was extracted with dichloromethane. 反复萃取三次,将萃取液合并,浓缩,于无水乙醚中沉淀。 Repeatedly extracted three times, the extracts were combined, concentrated, precipitated into anhydrous ether. 再溶解-过滤-沉淀二次后,真空干燥,得产物mPEG2-CHCO-Lysine-Pt产率82%。 Redissolved - filtration - the second precipitate, and dried in vacuo to give the product mPEG2-CHCO-Lysine-Pt 82% yield. 经原子吸收光谱法测定,产物中顺铂的摩尔含量为87.1%。 As determined by atomic absorption spectrometry, product cisplatin content of 87.1 mol%.

实施例二RPEGCO-BC-Pt的制备(以mPEG分子量4000为例)称取10g mPEG(Mn=4000,2.5mmol),以实施例一相同的方法制备端基为羧基的mPEG-CH2COOH,连接氨基丙二酸二乙酯,水解,最后合成mPEG-CH2CO-Lysine-Pt。 Preparation Example two RPEGCO-BC-Pt (in Example mPEG MW 4000) Weigh 10g mPEG (Mn = 4000,2.5mmol), to implement a method of preparing the same embodiment is carboxy end group mPEG-CH2COOH, amino connector malonate, hydrolysis, and finally synthesizing mPEG-CH2CO-Lysine-Pt. 产率为81%,最终产物中顺铂的摩尔含量为85.2%。 81% yield in the final product cisplatin content of 85.2% by mole.

实施例三RPEG2CO-BC的合成方法(以单甲氧基聚乙二醇(mPEG)分子量80000为例)将16g mPEG(Mn=80000,0.2mmol),以实施例一相同的方法,得mPEG-CH2CO-Lysine-Pt 4.5g,总产率28%。 Synthesis Example three RPEG2CO-BC (in monomethoxy polyethylene glycol (mPEG) Case 80,000 molecular weight) The 16g mPEG (Mn = 80000,0.2mmol), in the same manner as in Example embodiment a yield mPEG- CH2CO-Lysine-Pt 4.5g, 28% overall yield. 最终产物中顺铂的摩尔含量为80.4%。 The final product cisplatin content of 80.4 mol%.

按照以上实施例的步骤,本发明合成了三种分子量的高分子药物,用于体外细胞毒性实验。 Following the procedure of the above embodiment, the present invention is a polymer synthesized molecular weight of three kinds of drugs for in vitro cytotoxicity assays. 合成了载体分子量为4000、6000和9400的三种双臂mPEG(简称:DImPEG)顺铂偶合物:DImPEG-4000-Pt、DImPEG-6000-Pt、DImPEG-9400-Pt以及三种线性mPEG顺铂偶合物:mPEG-4000-Pt、mPEG-6000-Pt、mPEG-9400-Pt。 Synthesis of a molecular weight of 4000, 6000 and the carrier 9400 mPEG three arms (abbreviation: DImPEG) cisplatin conjugate: DImPEG-4000-Pt, DImPEG-6000-Pt, DImPEG-9400-Pt cisplatin and three kinds of linear mPEG conjugate: mPEG-4000-Pt, mPEG-6000-Pt, mPEG-9400-Pt. 以上各种药物中顺铂的摩尔含量列于下表1:表.1大分子药物中铂的摩尔含量 All of the above in the molar content of the drug cisplatin listed in Table 1: Table .1 macromolecular drugs molar content of platinum

上述药物的体外细胞毒性实验过程及结果见实施例四。 Vitro toxicity experiment results of the above drugs and cells, see Example IV.

上述药物的急性毒性实验过程及结果见实施例五。 Acute toxicity test results of the above processes and drug see Example V.

实施例四将C6人乳腺癌细胞株用含10%胎牛血清的DMEM培养液在37℃,5%CO2条件下培养,传代。 Example C6 The four human breast cancer cell lines were cultured in DMEM containing 10% fetal bovine serum at 37 ℃, 5% CO2 conditions, passaged embodiment. 将生长旺盛的C6细胞经0.25%胰蛋白酶消化后,配成终浓度为70000个细胞/mL的细胞悬液,将该悬液按100uL/孔均匀加入96孔平底培养板中。 The vigorous growth of C6 cells were digested with 0.25% trypsin, to a final concentration of 70,000 cells / mL in suspension, the suspension by 100 uL / ​​hole uniformly added to 96 well flat bottom culture plate. 每孔细胞数为7000个。 The number of cells per well into 7000. 在37℃,5%CO2,湿度100%的条件下孵育24h。 For 24h at 37 ℃, 5% CO2, 100% humidity conditions. 将上述各大分子药物溶于DMEM培养液,用0.22um微孔滤膜过滤。 The above-described major molecule drugs dissolved in DMEM, filtered through a 0.22um microporous membrane. 所得药物溶液以大分子药物中顺铂的含量为基准,稀释成不同浓度,折算成小分子顺铂分别为0.02ug/mL,0.2ug/mL,2ug/mL,20ug/mL,200ug/mL,2mg/mL。 The resultant drug solution to the content of macromolecular drug cisplatin as a reference diluted into different concentrations, converted into smaller molecules cisplatin was 0.02ug / mL, 0.2ug / mL, 2ug / mL, 20ug / mL, 200ug / mL, 2mg / mL. 向上述96孔平底培养板中加入不同浓度的药物100uL/孔,培养2天, 换液,培养一天。 To the above 96-well flat bottom plates with different concentrations of the drug 100uL / well and cultured for 2 days, medium was changed, cultured for one day. 每孔加入20uL 0.5%四氮唑(MTT)溶液,4小时后, 吸出上清液,加入150uL二甲基亚砜(DMSO),振荡10min。 After each well was added 20uL 0.5% tetrazolium (MTT) solution was 4 hours, the supernatant was aspirated, 150uL was added dimethyl sulfoxide (DMSO), shaken 10min. 用酶标仪检测各孔570nm处的吸光值(OD值),记录结果,按下列公式计算细胞存活率:细胞存活率=实验组OD值/对照组OD值×100%从附图中可求出各药物对癌细胞的半数抑制浓度(IC50),根据大分子药物不同的接药率,以顺铂的含量为基准,各药物的IC50值列于表.2。 With the absorbance (OD) of each well of ELISA plate reader at 570nm, recording the results, cell viability was calculated according to the following equation: Cell viability = OD value of experimental group / OD value of control group × 100% can be evaluated from the drawings each of the drugs to cancer cells half maximal inhibitory concentration (an IC50), depending on the dose rate of macromolecular drugs access to the content of cisplatin as a reference, an IC50 value of each drug are listed in table .2.

表.2 DImPEG-Pt与mPEG-Pt系列药品的IC50值 Table .2 IC50 value DImPEG-Pt series with mPEG-Pt drugs

从表中可以看出,在同系列药物中,载体PEG的分子量未对药物的体外细胞毒性产生规律性的影响,但DImPEG-Pt系列药物的IC50值却均低于相同分子量的mPEG-Pt系列药物。 As can be seen from the table, in the same series of drugs, the carrier is not generated molecular weight of PEG effects on the regularity of the in vitro cytotoxic drugs, but DImPEG-Pt series IC50 values ​​of drugs but were lower than the same molecular weight mPEG-Pt series drug. 这说明了相同分子量的DImPEG-Pt药物比mPEG-Pt药物体现出了更好的体外抗肿瘤活性。 This illustrates the same molecular weight DImPEG-Pt drugs reflects better antitumor activity than mPEG-Pt drugs.

图1是双臂聚乙二醇改性的顺铂(DImPEG-Pt)系列的剂量反应曲线图,图2是单臂聚乙二醇改性的顺铂(mPEG-Pt)系列的剂量反应曲线图,本发明以细胞存活分数对药物的剂量作图,得到了上述几个药物的剂量反应曲线。 FIG 1 is a polyethylene glycol-modified dual-arm dose-response curves in FIG cisplatin (DImPEG-Pt) series, FIG. 2 is a single-arm polyethylene glycol-modified cisplatin (mPEG-Pt) series of dose-response curve FIG present invention in surviving fraction plotted against the dose of the drug, the dose response curves obtained above several drugs. 同期本发明也测得了对照样品顺铂的剂量反应曲线。 The present invention is also measured over the same period had the dose response curve control sample cisplatin. 从图中可以看出,不同分子量的DImPEG-Pt和mPEG-Pt在体外对C6细胞都有较好的抗肿瘤活性。 As can be seen from the figure, the different molecular weights DImPEG-Pt and mPEG-Pt antitumor activity in vitro C6 cells are preferred.

实施例五为了检验顺铂经PEG修饰后的急性毒性变化,测定了上述高分子药物的LD50。 Fifth Embodiment In order to examine the acute toxicity of cisplatin by Changes after PEG modification, the polymer was determined LD50 medicament.

TA1小白鼠随机分成5组,每组20只,雌雄各半,分别用顺铂,接了线型和两臂PEG的顺铂给药,用ip×1治疗方案,设五个计量组,给药后观察即时反应。 TA1 mice were randomly divided into 5 groups, n = 20, male and female, were treated with cisplatin, then administration of platinum and cis-linear arms of the PEG, ip × 1 with the treatment regimen, provided five measurement group to after the drug was observed immediate reaction. 记录二周内小鼠的死亡分布。 Death distribution record two weeks the mice. 以24小时为死亡高峰。 For the death of a 24-hour peak. 死亡动物尸检未发现肉眼可见的,明显的实质性的病变。 Dead animals autopsy found no visible, significant substantive lesions. 二周后存活动物一般情况良好。 After two weeks the surviving animals are generally in good condition. 实验结果用Bliss方法计算,如表3所示。 The results calculated by the Bliss method, as shown in Table 3.

表.3 DImPEG-Pt与mPEG-Pt系列药品的LD50值 Table .3 LD50 value DImPEG-Pt series with mPEG-Pt drugs

从表中可以看出,顺铂通过PEG修饰后,其急性毒性明显降低。 As it can be seen from the table, cisplatin modified by PEG, which significantly reduced the acute toxicity.

Claims (5)

  1. 1.一种高分子抗肿瘤药物,其特征在于,化学通式为RPEG2Y-BC:其中:RPEG2-的结构式如下: 1. A polymer antineoplastic drug, wherein the chemical formula of RPEG2Y-BC: wherein: RPEG2- the following structural formula: 其中,R为10个碳以下的直链烷烃、异丙基或苄基;R1为CH或苯基;a、b、c为0~9的整数;k为0~6的整数;s、t是使RPEG2-中的RO-(CH2CH2O)s-及RO-(CH2CH2O)t-部分的Mn约为2000~80000的整数;W为O、S、 Wherein, R 10 is a straight-chain alkane carbons or less, isopropyl or benzyl; Rl is CH or a phenyl group; a, b, c is an integer of 0-9; K is an integer of 0 to 6; s, t It is to make the RPEG2- RO- (CH2CH2O) s- and Mn t- portion RO- (CH2CH2O) an integer of from about 2,000 to 80,000; W is O, S, NH、之一,同一分子中W可为不同基团;其中,B的结构是下述之一: NH, one of the same molecule may be different W groups; wherein B is one of the following structures: R2为1~9个碳原子的烃基; R2 is a hydrocarbon group having 1 to 9 carbon atoms; 所述C的结构是两个氯原子被取代的顺铂,结构式如下: The structure C is substituted with two chlorine atoms of cisplatin, the following structural formula:
  2. 2.如权利要求1所述的高分子抗肿瘤药物,其特征在于R为甲基。 2. The polymer composition of the antitumor agent according to claim 1, characterized in that R is methyl.
  3. 3.如权利要求1至2任一所述的高分子抗肿瘤药物,其特征在于,是按照以下步骤制备的:官能化双臂聚乙二醇RPEG2Y-X和含有丙二酸酯结构的化合物反应,然后水解得到含丙二酸结构的化合物RPEG2Y-B,最后RPEG2Y-B和氯原子被硝酸根取代的顺铂反应得到目标产物RPEG2Y-BC,其中,X为下述结构之一: 3. The antitumor agent to a polymer according to any of claims 1 and 2, wherein, prepared according to the following steps: a functionalized polyethylene glycol arms RPEG2Y-X and a compound structure containing malonate the reaction, followed by hydrolysis to give compound RPEG2Y-B malonate structure, and finally RPEG2Y-B and chlorine atoms are substituted with cisplatin nitrate to afford the desired product RPEG2Y-BC, wherein, X is one of the following structures:
  4. 4.如权利要求1至3任一所述的高分子抗肿瘤药物的制备方法,其特征在于,包括以下步骤:官能化双臂聚乙二醇RPEG2Y-X和含有丙二酸酯结构的化合物反应,然后水解得到含丙二酸结构的化合物RPEG2Y-B,最后RPEG2Y-B和氯原子被硝酸根取代的顺铂反应得到目标产物RPEG2Y-BC,其中,X为下述结构之一: 4. The production method according to any one of a polymer 31 antitumor agent as claimed in claim, characterized in that it comprises the steps of: functionalized polyethylene glycol arms RPEG2Y-X and a compound structure containing malonate the reaction, followed by hydrolysis to give compound RPEG2Y-B malonate structure, and finally RPEG2Y-B and chlorine atoms are substituted with cisplatin nitrate to afford the desired product RPEG2Y-BC, wherein, X is one of the following structures: 其中,所述的RPEG2-的结构式如下: Wherein said RPEG2- formula as follows: 其中R为10个碳以下的直链烷烃、异丙基或苄基;R1为CH或苯基;a、b、c为0~9的整数;k为0~6的整数;s、t是使RPEG2-中的RO-(CH2CH2O)s-及RO-(CH2CH2O)t-部分的Mn约为2000~80000的整数;W为O、S、NH、 Wherein R is C 10 or less linear alkane, isopropyl or benzyl; Rl is CH or a phenyl group; a, b, c is an integer of 0-9; K is an integer of 0 to 6; s, t is the so RPEG2- RO- (CH2CH2O) s- and Mn t- portion RO- (CH2CH2O) is an integer of from about 2000 to 80,000; W is O, S, NH, 之一,同一分子中W可为不同基团; One in the same molecule may be different W groups; 所述的B的结构是下述之一: The structure B is one of the following: 其中R2为1~9个碳原子的烃基; Wherein R2 is a hydrocarbon group having 1 to 9 carbon atoms; C的结构是两个氯原子被取代的顺铂,结构式如下: Structure C is substituted with two chlorine atoms of cisplatin, the following structural formula:
  5. 5.如权利要求4所述的高分子抗肿瘤药物的制备方法,其特征在于,R为甲基。 5. The polymer preparation method 4 antitumor agent as claimed in claim wherein, R is methyl.
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