WO2019062665A1 - Linker compound, polyethylene glycol-linker conjugate and derivative thereof, and polyethylene glycol-linker-drug conjugate - Google Patents

Linker compound, polyethylene glycol-linker conjugate and derivative thereof, and polyethylene glycol-linker-drug conjugate Download PDF

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WO2019062665A1
WO2019062665A1 PCT/CN2018/106972 CN2018106972W WO2019062665A1 WO 2019062665 A1 WO2019062665 A1 WO 2019062665A1 CN 2018106972 W CN2018106972 W CN 2018106972W WO 2019062665 A1 WO2019062665 A1 WO 2019062665A1
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polyethylene glycol
linker
integer
peg
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PCT/CN2018/106972
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Chinese (zh)
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冯泽旺
王庆彬
汪进良
宋艳萍
熊艳丽
王雷敏
杜景辉
赵宣
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天津键凯科技有限公司
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Priority claimed from CN201810186781.6A external-priority patent/CN108727208B/en
Priority claimed from CN201811043884.3A external-priority patent/CN109589415B/en
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Publication of WO2019062665A1 publication Critical patent/WO2019062665A1/en
Priority to US16/833,988 priority Critical patent/US11857635B2/en

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Abstract

The present invention discloses a linker compound, a polyethylene glycol-linker conjugate and a derivative thereof, and a polyethylene glycol-linker drug conjugate. The linker compound and the polyethylene glycol conjugate and a derivative thereof can be used for drug modification, and the modification reaction is simple and easy to carry out and has a high reaction yield, and the modified drugs have a wide application range. The modified drugs gradually degrade from a conjugate chain in the body, and can stay in a lesion (such as a cancer site) for a longer period of time, achieving the purpose of sustained and controlled release, reducing frequency of administration, and greatly improving drug bioavailability and drug compliance of patients.

Description

一种连接子化合物、聚乙二醇-连接子结合物及其衍生物和聚乙二醇-连接子-药物结合物A linker compound, a polyethylene glycol-linker conjugate and a derivative thereof, and a polyethylene glycol-linker-drug conjugate 技术领域Technical field
本发明涉及医药技术领域，具体涉及一种连接子化合物、聚乙二醇-连接子结合物及其衍生物、聚乙二醇-连接子-药物结合物及其药物组合物和应用。The present invention relates to the field of medical technology, and in particular to a linker compound, a polyethylene glycol-linker conjugate and a derivative thereof, a polyethylene glycol-linker-drug conjugate, and pharmaceutical compositions and uses thereof.
背景技术Background technique
目前，在临床上，很多药物由于不适宜口服等原因(如多肽、蛋白类药物口服给药时，进入消化道后，会受各种蛋白酶、肽酶等水解环境破坏，药效降低甚至丧失，如某些药物对胃部有刺激性或不耐酸，易被胃酸破坏)，其主要给药途径为注射给药，直接注入人体组织或血管，不经过消化系统和肝脏，不会受到消化液的破坏和食物的影响，药物吸收快，血药浓度升高迅速，给药剂量精确，但在临床应用中，由于注射给药后药物常常会迅速分布全身，对病灶部位如肿瘤组织的靶向性较差，生物利用度不高，药效相对不高，且不良反应出现迅速，应对处理相对困难，另外，常常需要多次给药，且给药时需严格遵守无菌操作原则，需专业人士如医生和护士来操作，不利于病人的依从性，因此药物的临床应用常常遭遇瓶颈。At present, in clinical practice, many drugs are not suitable for oral administration. (For example, when peptides or protein drugs are administered orally, they will be destroyed by various proteases, peptidases and other hydrolyzed environments after entering the digestive tract, and the efficacy is reduced or even lost. For example, some drugs are irritating or acid-resistant to the stomach and are easily destroyed by gastric acid. The main route of administration is injection, which is directly injected into human tissues or blood vessels, without passing through the digestive system and liver, and is not affected by digestive juice. Destruction and food effects, rapid drug absorption, rapid increase in blood drug concentration, accurate dose, but in clinical applications, due to the rapid distribution of drugs throughout the body after injection, targeting of lesions such as tumor tissue Poor, low bioavailability, relatively low efficacy, rapid onset of adverse reactions, relatively difficult to handle, in addition, often requires multiple doses, and strict adherence to aseptic procedures, requiring professionals If doctors and nurses operate, it is not conducive to patient compliance, so the clinical application of drugs often encounters bottlenecks.
现有技术中科研人员常利用水溶性聚合物如聚乙二醇修饰连接药物，来延长药物的生理半衰期，降低药物的免疫原性和毒性，但药物在体内的释放和药效有时并不理想。实验中发现通过连接子(linker)将水溶性聚合物和药物连接形成聚合物-药物结合物，药物从结合物链上的降解，可以达到缓释和控释的目的，药物在病灶(例如患癌)部位停留更长时间，可降低给药频率，减少患者用药不便。如专利文献CN200680029849.5公开了一种轭合物，其中包括含有可电离氢原子的芳香族部分如芴、间隔部分和水溶性聚合物。In the prior art, researchers often use water-soluble polymers such as polyethylene glycol to modify the drug to prolong the physiological half-life of the drug and reduce the immunogenicity and toxicity of the drug, but the release and efficacy of the drug in the body is sometimes not ideal. . In the experiment, it was found that the water-soluble polymer and the drug are linked by a linker to form a polymer-drug conjugate, and the degradation of the drug from the conjugate chain can achieve the purpose of sustained release and controlled release, and the drug is in the lesion (for example, suffering from The cancer area stays longer, which can reduce the frequency of administration and reduce the inconvenience of patients. For example, the patent document CN200680029849.5 discloses a conjugate comprising an aromatic moiety containing an ionizable hydrogen atom such as a hydrazine, a spacer and a water-soluble polymer.
本申请的发明人经过大量的试验和研究，得到一种连接子化合物及其与聚乙二醇的结合物及其衍生物，其用于修饰药物时，修饰反应简单，易进行，反应产率较高，修饰药物的适用范围较宽，且修饰后的药物在体内释放速度和药效理想，可降低给药频率，大大提高药物的生物利用度和病人的依从性。The inventors of the present application have conducted a large number of experiments and studies to obtain a linker compound and a combination thereof with polyethylene glycol and a derivative thereof, which are simple to modify and easy to carry out when the drug is modified, and the reaction yield is obtained. Higher, the scope of application of the modified drug is wider, and the modified drug has an ideal release rate and efficacy in vivo, which can reduce the frequency of administration, and greatly improve the bioavailability of the drug and patient compliance.
发明内容Summary of the invention
本发明的一个目的是提供一种连接子化合物。It is an object of the invention to provide a linker compound.
本发明的另一个目的是提供一种聚乙二醇与上述连接子的结合物及其衍生物。Another object of the present invention is to provide a combination of polyethylene glycol with the above linker and derivatives thereof.
本发明的另一个目的是提供一种聚乙二醇-连接子-药物的结合物。Another object of the invention is to provide a polyethylene glycol-linker-drug combination.
本发明的另一个目的是提供包含上述结合物及其与药学上可接受的载体或添加剂的药物组合物。Another object of the present invention is to provide a pharmaceutical composition comprising the above combination and its pharmaceutically acceptable carrier or additive.
本发明的还一个目的是提供一种上述结合物、药物组合物在制备用于预防和/或治疗疾病的药物中的应用。It is still another object of the present invention to provide an application of the above-described combination or pharmaceutical composition for the preparation of a medicament for preventing and/or treating a disease.
具体地，本发明第一方面提供一种化合物，其具有如下结构：Specifically, a first aspect of the invention provides a compound having the structure:
Figure PCTCN2018106972-appb-000001
Figure PCTCN2018106972-appb-000001
其中，l为1-5的整数，Where l is an integer from 1 to 5,
Z选自：-H和羟基保护基团，Z is selected from the group consisting of: -H and a hydroxy protecting group,
A选自：氨基酸残基、多肽残基、
Figure PCTCN2018106972-appb-000002
-(CH 2) i-、-NHCO(CH 2) i-、-CONH(CH 2) i-、-(CH 2) iNH-和-CO(CR 15R 16) iNH-中的一种或多种的组合，i为0-6的整数， A is selected from the group consisting of: amino acid residues, polypeptide residues,
Figure PCTCN2018106972-appb-000002
One of -(CH 2 ) i -, -NHCO(CH 2 ) i -, -CONH(CH 2 ) i -, -(CH 2 ) i NH- and -CO(CR 15 R 16 ) i NH- Or a combination of multiples, i is an integer from 0-6,
所述氨基酸选自：甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、丝氨酸、苏氨酸、脯氨酸、天冬氨酸、天冬酰胺、谷氨酸、谷氨酰胺、赖氨酸、精氨酸、半胱氨酸、蛋氨酸、组氨酸、色氨酸、苯丙氨酸和酪氨酸，The amino acid is selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, serine, threonine, proline, aspartic acid, asparagine, glutamic acid, and glutamine. Amide, lysine, arginine, cysteine, methionine, histidine, tryptophan, phenylalanine and tyrosine,
R 1-7、R 9-11独立地选自：-H、-F、-Cl、-Br、-I、C1-6的烷基、C1-6的烷氧基、C3-6的环烷基、C1-6的烯基、C6-12的芳基、C6-12芳烷基、C3-12芳族或非芳族的杂环基、C3-12的杂环烷基和-(CH 2) l-O-Z， R 1-7 and R 9-11 are independently selected from the group consisting of: -H, -F, -Cl, -Br, -I, C1-6 alkyl, C1-6 alkoxy, C3-6 naphthenic a base, a C1-6 alkenyl group, a C6-12 aryl group, a C6-12 aralkyl group, a C3-12 aromatic or non-aromatic heterocyclic group, a C3-12 heterocycloalkyl group, and -(CH 2 ) l -OZ,
R 8和R 12独立地选自C1-6的烷基， R 8 and R 12 are independently selected from a C1-6 alkyl group,
R 13-16独立地选自：-H和C1-6的烷基， R 13-16 is independently selected from the group consisting of: -H and C1-6 alkyl groups,
B为连接基团-B 1-B 2-， B is a linking group -B 1 -B 2 -,
其中，B 1选自：-(CH 2) j-、-NHCO(CH 2) j-和-CONH(CH 2) j-，j为0-6的整数， Wherein B 1 is selected from the group consisting of: -(CH 2 ) j -, -NHCO(CH 2 ) j - and -CONH(CH 2 ) j -, and j is an integer from 0 to 6,
B 2选自：-C＝O、-C＝S、-O-、-S-、-C(O)O-、-C(O)S-、-C(S)O-和-S-S-。 B 2 is selected from the group consisting of: -C=O, -C=S, -O-, -S-, -C(O)O-, -C(O)S-, -C(S)O-, and -SS- .
所述的连接子化合物中，所述的l为1-5的整数，如1、2、3、4、5，优选为1、2或3；更优选为1。In the above linker compound, the l is an integer of from 1 to 5, such as 1, 2, 3, 4, 5, preferably 1, 2 or 3; more preferably 1.
所述的连接子化合物中，本领域技术人员可以根据实际需要选择合适的羟基保护基团，如：-CH 3、-C(CH 3) 3、-CH 2OCH 3、-COCH 3、-COC(CH 3) 3、-CH 2CH＝CH 2、-Si(CH 3) 3、
Figure PCTCN2018106972-appb-000003
Figure PCTCN2018106972-appb-000004
等。 Among the linker compounds, one skilled in the art can select a suitable hydroxy protecting group according to actual needs, such as: -CH 3 , -C(CH 3 ) 3 , -CH 2 OCH 3 , -COCH 3 , -COC (CH 3 ) 3 , -CH 2 CH=CH 2 , -Si(CH 3 ) 3 ,
Figure PCTCN2018106972-appb-000003
Figure PCTCN2018106972-appb-000004
Wait.
在本发明的一个实施例中，所述的Z为-H。In one embodiment of the invention, said Z is -H.
本发明的一个实施例中，所述的连接子化合物中，所述A为氨基酸残基，所述氨基酸选自：甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、丝氨酸、苏氨酸、脯氨酸、天冬氨酸、天冬酰胺、谷氨酸、谷氨酰胺、赖氨酸、精氨酸、半胱氨酸、蛋氨酸、组氨酸、色氨酸、苯丙氨酸和酪氨酸，优选自：甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、天冬氨酸、天冬酰胺、谷氨酸、谷氨酰胺和赖氨酸，更优选自：甘氨酸、丙氨酸和缬氨酸。In one embodiment of the present invention, in the linker compound, the A is an amino acid residue selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, and serine. , threonine, valine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, arginine, cysteine, methionine, histidine, tryptophan, benzene Alanine and tyrosine, preferably from: glycine, alanine, valine, leucine, isoleucine, aspartic acid, asparagine, glutamic acid, glutamine and lysine More preferably, it is derived from glycine, alanine and valine.
本发明的另一个实施例中，所述的连接子化合物中，所述A为
Figure PCTCN2018106972-appb-000005
优选为
Figure PCTCN2018106972-appb-000006
本发明的一个具体实施例中，所述的A为
Figure PCTCN2018106972-appb-000007
In another embodiment of the present invention, in the linker compound, the A is
Figure PCTCN2018106972-appb-000005
Preferred
Figure PCTCN2018106972-appb-000006
In a specific embodiment of the invention, the A is
Figure PCTCN2018106972-appb-000007
在本发明的另一个实施例中，所述的连接子化合物中，所述的A为-(CH 2) i-、-(CH 2) iNH-和-CO(CR 15R 16) iNH-中的一种或多种的组合。 In another embodiment of the present invention, in the linker compound, the A is -(CH 2 ) i -, -(CH 2 ) i NH- and -CO(CR 15 R 16 ) i NH - a combination of one or more of them.
优选地，所述的连接子化合物中，所述的R 1-7、R 9-11独立地选自：-H、-F、-Cl、-Br、-I、C1-6的烷基、C1-6的烷氧基和-(CH 2) l-O-Z；更优选自：-H、-F、-Cl、-CH 3、-CH 2CH 3、-CH 2CH 2CH 3、-CH(CH 3) 2、-C(CH 3) 3、-OCH 3和-(CH 2) l-O-Z；进一步优选自：-H、-F、-Cl、-CH 3、-OCH 3和-(CH 2) l-O-Z。 Preferably, in the linker compound, the R 1-7 and R 9-11 are independently selected from the group consisting of: -H, -F, -Cl, -Br, -I, C1-6 alkyl, Alkoxy group of C1-6 and -(CH 2 ) l -OZ; more preferably from: -H, -F, -Cl, -CH 3 , -CH 2 CH 3 , -CH 2 CH 2 CH 3 , -CH (CH 3 ) 2 , -C(CH 3 ) 3 , -OCH 3 and -(CH 2 ) l -OZ; further preferably from: -H, -F, -Cl, -CH 3 , -OCH 3 and -( CH 2 ) l -OZ.
在本发明的一个实施例中，所述的R 1-4均为-H。 In one embodiment of the invention, said R 1-4 are both -H.
在本发明的一个实施例中，所述的R 5-7均为-H。 In one embodiment of the invention, said R 5-7 is -H.
在本发明的一个实施例中，所述的R 9-11均为-H。 In one embodiment of the invention, said R 9-11 is -H.
优选地，所述的连接子化合物中，所述R 8和R 12独立地选自C1-4的烷基，如甲基、乙基、正丙基、异丙基、正丁基、仲丁基、异丁基或叔丁基；更优选地，所述R 8和/或R 12为甲基。 Preferably, in the linker compound, the R 8 and R 12 are independently selected from a C1-4 alkyl group, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl More preferably, the R 8 and/or R 12 is a methyl group.
优选地，所述的连接子化合物中，R 13-16独立地选自：-H和C1-3的烷基(如甲基、乙基、正丙基或异丙基)。 Preferably, in the linker compound, R 13-16 is independently selected from the group consisting of: -H and C1-3 alkyl groups (such as methyl, ethyl, n-propyl or isopropyl).
优选地，所述的连接子化合物中，R 15为-H，R 16选自：-H、甲基、乙基、正丙基和异丙基。 Preferably, in the linker compound, R 15 is -H, and R 16 is selected from the group consisting of: -H, methyl, ethyl, n-propyl and isopropyl.
在本发明的一个实施例中，所述的R 13和/或R 14为-H。 In one embodiment of the invention, said R 13 and/or R 14 is -H.
优选地，所述的连接子化合物中，i为0-3的整数，如0、1、2或3。Preferably, in the linker compound, i is an integer of 0-3, such as 0, 1, 2 or 3.
优选地，所述的连接子化合物中，j为0-3的整数，如0、1、2或3。Preferably, in the linker compound, j is an integer from 0 to 3, such as 0, 1, 2 or 3.
优选地，在本发明的一个实施例中，式Ⅰ-1中，所述A为-COCH 2NH-、-COCH(CH 3)NH-、-COCH(CH(CH 3) 2)NH-。 Preferably, in one embodiment of the invention, in Formula I-1, said A is -COCH 2 NH-, -COCH(CH 3 )NH-, -COCH(CH(CH 3 ) 2 )NH-.
优选地，所述B 2选自：-C＝O、-O-、-S-、-C(O)O-、-C(O)S-和-S-S-。 Preferably, the B 2 is selected from the group consisting of: -C=O, -O-, -S-, -C(O)O-, -C(O)S-, and -SS-.
在本发明的一个实施例中，所述的B 2为-C(O)O-或-O-。 In one embodiment of the invention, the B 2 is -C(O)O- or -O-.
在本发明另一个实施例中，所述的B为-(CH 2) jO-，j为0-3的整数，如0、1、2或3。 In another embodiment of the invention, said B is -(CH 2 ) j O-, and j is an integer from 0 to 3, such as 0, 1, 2 or 3.
在本发明的一个实施例中，式Ⅰ-2、Ⅰ-3中，所述-B-A-为-OCH 2CH 2NH-。 In one embodiment of the invention, in Formulas I-2, I-3, the -BA- is -OCH 2 CH 2 NH-.
在本发明的一个实施例中，所述连接子化合物选自如下结构：In one embodiment of the invention, the linker compound is selected from the group consisting of:
Figure PCTCN2018106972-appb-000008
Figure PCTCN2018106972-appb-000008
Figure PCTCN2018106972-appb-000009
Figure PCTCN2018106972-appb-000009
本发明另一方面还提供一种聚乙二醇-连接子结合物，其具有如下结构：Another aspect of the present invention also provides a polyethylene glycol-linker conjugate having the following structure:
PEG-X-LPEG-X-L
(Ⅱ)(II)
其中，L为本发明上述连接子，Where L is the above linker of the invention,
PEG为聚乙二醇残基，PEG is a polyethylene glycol residue,
X为连接基团，选自：-(CH 2) a-、-(CH 2) aCO-、-(CH 2) aOCO-、-(CH 2) aNHCO-、-NH(CH 2) aCO-、-(CH 2) aSO 2-、-O(CH 2) a-、-O(CH 2) aCO-、-O(CH 2) aOCO-、-O(CH 2) aNHCO-和-O(CH 2) aSO 2-中的一种或多种的组合，a为0-10的整数。 X is a linking group selected from the group consisting of: -(CH 2 ) a -, -(CH 2 ) a CO-, -(CH 2 ) a OCO-, -(CH 2 ) a NHCO-, -NH(CH 2 ) a CO-, -(CH 2 ) a SO 2 -, -O(CH 2 ) a -, -O(CH 2 ) a CO-, -O(CH 2 ) a OCO-, -O(CH 2 ) a A combination of one or more of NHCO- and -O(CH 2 ) a SO 2 -, a being an integer from 0-10.
在本发明一个实施例中，所述聚乙二醇-连接子结合物具有如下结构：In one embodiment of the invention, the polyethylene glycol-linker conjugate has the structure:
Figure PCTCN2018106972-appb-000010
Figure PCTCN2018106972-appb-000010
上式Ⅱ-1～Ⅱ-3中，所述R 1-12、l、A、B、Z等具有本发明上述定义。 In the above formulae II-1 to II-3, R 1-12 , 1, A, B, Z and the like have the above definition of the present invention.
在本发明的一个实施方式中，所述聚乙二醇-连接子结合物中，所述X选自：-(CH 2) a-、-(CH 2) aCO-、-(CH 2) aNHCO-、-NH(CH 2) aCO-、-O(CH 2) a-、-O(CH 2) aCO-和-O(CH 2) aNHCO-中的一种或多种的组合，优选为-(CH 2) a-、-(CH 2) aCO-或-(CH 2) aNHCO-。 In one embodiment of the present invention, in the polyethylene glycol-linker conjugate, the X is selected from the group consisting of: -(CH 2 ) a -, -(CH 2 ) a CO-, -(CH 2 ) a one or more of NHCO-, -NH(CH 2 ) a CO-, -O(CH 2 ) a -, -O(CH 2 ) a CO- and -O(CH 2 ) a NHCO- Combinations are preferably -(CH 2 ) a -, -(CH 2 ) a CO- or -(CH 2 ) a NHCO-.
优选地，所述聚乙二醇-连接子结合物中，a为0-5的整数，如0、1、2、3、4或5。Preferably, in the polyethylene glycol-linker conjugate, a is an integer from 0 to 5, such as 0, 1, 2, 3, 4 or 5.
在本发明的一个实施例中，所述聚乙二醇-连接子结合物中，所述X为单键、-CH 2-、-CH 2CH 2-、-CO-、-CH 2CO-或-NHCO-。 In one embodiment of the invention, in the polyethylene glycol-linker conjugate, the X is a single bond, -CH 2 -, -CH 2 CH 2 -, -CO-, -CH 2 CO- Or -NHCO-.
优选地，所述聚乙二醇-连接子结合物中，所述PEG为直链、Y型、多分支的聚乙二醇残基，例如包括单甲氧基聚乙二醇(mPEG)、直链双端PEG、Y型PEG、4臂支链PEG、6臂支链PEG或8臂支链PEG等。Preferably, in the polyethylene glycol-linker conjugate, the PEG is a linear, Y-type, multi-branched polyethylene glycol residue, for example, including monomethoxypolyethylene glycol (mPEG), Linear double-ended PEG, Y-type PEG, 4-arm branched PEG, 6-arm branched PEG or 8-arm branched PEG.
在本发明一具体实施方式中，所述聚乙二醇-连接子结合物中，所述PEG为直链聚乙二醇残基，具有通式Ⅲ或Ⅳ所示的结构：In a specific embodiment of the present invention, in the polyethylene glycol-linker conjugate, the PEG is a linear polyethylene glycol residue having a structure represented by the formula III or IV:
Figure PCTCN2018106972-appb-000011
Figure PCTCN2018106972-appb-000011
其中，p和q独立地选自1-960的整数，优选为1-480的整数。Wherein p and q are independently selected from an integer of from 1 to 960, preferably from 1 to 480.
在本发明一具体实施方式中，所述聚乙二醇-连接子结合物中，所述PEG为Y型聚乙二醇残基，具有通式Ⅴ或Ⅵ所示的结构：In a specific embodiment of the present invention, in the polyethylene glycol-linker conjugate, the PEG is a Y-type polyethylene glycol residue having a structure represented by the formula V or VI:
Figure PCTCN2018106972-appb-000012
Figure PCTCN2018106972-appb-000012
其中，i和h独立地选自1-480的整数，优选为1-240的整数。Wherein i and h are independently selected from an integer from 1 to 480, preferably from 1 to 420.
在本发明一具体实施方式中，所述聚乙二醇-连接子结合物中，所述PEG为多分支聚乙二醇残基，具有通式Ⅶ所示的结构：In a specific embodiment of the present invention, in the polyethylene glycol-linker conjugate, the PEG is a multi-branched polyethylene glycol residue having a structure represented by the formula VII:
Figure PCTCN2018106972-appb-000013
Figure PCTCN2018106972-appb-000013
其中，k是1-320的整数，优选为1-240的整数，Wherein k is an integer from 1 to 320, preferably an integer from 1 to 240,
j是3-8的整数，j is an integer of 3-8,
R是多分支聚乙二醇的核心分子，R选自：季戊四醇、寡聚季戊四醇、甲基葡萄糖苷、蔗糖、二甘醇、丙二醇、甘油和聚甘油的残基；优选地，R选自：甘油、六聚甘油、季戊四醇、二聚季戊四醇和三聚季戊四醇残基。R is a core molecule of a multi-branched polyethylene glycol, and R is selected from the group consisting of: residues of pentaerythritol, oligo-pentaerythritol, methyl glucoside, sucrose, diethylene glycol, propylene glycol, glycerol, and polyglycerol; preferably, R is selected from: Glycerin, hexaglycerol, pentaerythritol, dipentaerythritol and tripolypentaerythritol residues.
优选地，所述多分支聚乙二醇残基仅含有一个可连接位点，其具有如下结构：Preferably, the multi-branched polyethylene glycol residue contains only one connectable site having the following structure:
Figure PCTCN2018106972-appb-000014
Figure PCTCN2018106972-appb-000014
在本发明一个实施例中，所述多分支聚乙二醇残基具有如下结构：In one embodiment of the invention, the multi-branched polyethylene glycol residue has the structure:
Figure PCTCN2018106972-appb-000015
Figure PCTCN2018106972-appb-000015
其中，k是1-320的整数，优选为1-240的整数，Wherein k is an integer from 1 to 320, preferably an integer from 1 to 240,
x为1-10的整数(具体如1、2、3、4、5、6、7、8、9或10)，优选为1-6的整数。x is an integer of 1-10 (specifically, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), preferably an integer of 1-6.
在本发明的另一个实施例中，所述多分支聚乙二醇残基具有如下结构：In another embodiment of the invention, the multi-branched polyethylene glycol residue has the structure:
Figure PCTCN2018106972-appb-000016
Figure PCTCN2018106972-appb-000016
其中，k是1-320的整数，优选为1-240的整数，Wherein k is an integer from 1 to 320, preferably an integer from 1 to 240,
x为1-10的整数(具体如1、2、3、4、5、6、7、8、9或10)，优选为1-6的整数。x is an integer of 1-10 (specifically, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), preferably an integer of 1-6.
在本发明另一个实施例中，所述多分支聚乙二醇残基具有如下结构：In another embodiment of the invention, the multi-branched polyethylene glycol residue has the structure:
Figure PCTCN2018106972-appb-000017
Figure PCTCN2018106972-appb-000017
其中，k是1-320的整数，优选为1-240的整数，更优选为1-120的整数，Wherein k is an integer from 1 to 320, preferably an integer from 1 to 240, more preferably an integer from 1 to 120,
y为1-10的整数(具体如1、2、3、4、5、6、7、8、9或10)，优选为1-5的整数，更优选为1-3的整数。Y is an integer of 1-10 (specifically, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), preferably an integer of 1-5, and more preferably an integer of 1-3.
在本发明的另一个实施例中，所述多分支聚乙二醇残基具有如下结构：In another embodiment of the invention, the multi-branched polyethylene glycol residue has the structure:
Figure PCTCN2018106972-appb-000018
Figure PCTCN2018106972-appb-000018
其中，k是1-320的整数，优选为1-240的整数，更优选为1-120的整数，Wherein k is an integer from 1 to 320, preferably an integer from 1 to 240, more preferably an integer from 1 to 120,
y为1-10的整数(具体如1、2、3、4、5、6、7、8、9或10)，优选为1-5的整数，更优选为1-3的整数。Y is an integer of 1-10 (specifically, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), preferably an integer of 1-5, and more preferably an integer of 1-3.
本发明中，所述的PEG的分子量可为1-100KDa，例如1-10KDa(具体可为1、2、3、4、5、6、7、8、9、10KDa)、10-50KDa(具体可为10、15、20、25、30、35、40、45、50KDa)、50-100KDa(具体可为50、55、60、65、70、75、80、85、90、95、100KDa)等；进一步优选为10-50KDa。In the present invention, the PEG may have a molecular weight of 1-100 KDa, such as 1-10 KDa (specifically 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 KDa), 10-50 KDa (specific Can be 10, 15, 20, 25, 30, 35, 40, 45, 50 KDa), 50-100 KDa (specifically 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 KDa) And so on; further preferably from 10 to 50 KDa.
在本发明的一个实施方式中，所述聚乙二醇-连接子结合物选自如下结构：In one embodiment of the invention, the polyethylene glycol-linker conjugate is selected from the group consisting of:
Figure PCTCN2018106972-appb-000019
Figure PCTCN2018106972-appb-000019
上式Ⅱ-a～Ⅱ-e中，所述PEG和X具有本发明上述相应定义。In the above formulae II-a to II-e, the PEG and X have the above corresponding definitions of the present invention.
在本发明的一个实施例中，上式Ⅱ-a～Ⅱ-e中，所述PEG具有本发明上述通式Ⅲ、Ⅴ、Ⅵ、Ⅶ-3或Ⅶ-5的结构；In one embodiment of the present invention, in the above formulae II-a to II-e, the PEG has the structure of the above formula III, V, VI, VII-3 or VII-5 of the present invention;
在本发明的一个实施例中，上式Ⅱ-a～Ⅱ-e中，所述PEG的分子量为10-50KDa(具体可为10、15、20、25、30、35、40、45或50KDa)。In one embodiment of the present invention, in the above formulas II-a to II-e, the molecular weight of the PEG is 10-50 KDa (specifically, 10, 15, 20, 25, 30, 35, 40, 45 or 50 KDa ).
在本发明的一个实施例中，上式Ⅱ-a～Ⅱ-e中，所述X为-CH 2CO-、-CO-、-CH 2-或-CH 2CH 2-。 In one embodiment of the present invention, in the above formulae II-a to II-e, the X is -CH 2 CO-, -CO-, -CH 2 - or -CH 2 CH 2 -.
此处所述PEG可以为通式Ⅲ、Ⅴ或Ⅵ所示的具有一个可连接位点的PEG结构，其连接一个连接子，如PEG具有通式Ⅲ所示结构，X为-CO-时，Ⅱ-d的结构为
Figure PCTCN2018106972-appb-000020
所述PEG也可为通式Ⅳ或Ⅶ所示的具有两个以上可连接位点的PEG，其可通过一个或多个连接位点实现连接子的连接。在本发明的一个实施方式中，所述PEG为通式Ⅳ或Ⅶ所示的具有两个以上可连接位点的PEG时，其可通过一个连接位点与连接子连接，其他可连接位点可连接封端基(如甲氧基)，也可通过多个连接位点与连接子连接，如通式Ⅶ-1所示结构，具体如PEG为八臂聚乙二醇，X为-CH 2CO-时，Ⅱ-d的结构可为：
Figure PCTCN2018106972-appb-000021
也可为
Figure PCTCN2018106972-appb-000022
等。 The PEG described herein may be a PEG structure having a linkable site represented by Formula III, V or VI, which is linked to a linker such as PEG having the structure shown in Formula III, and when X is -CO-, The structure of II-d is
Figure PCTCN2018106972-appb-000020
The PEG can also be a PEG having two or more connectable sites as shown in Formula IV or VII, which can effect ligation of the linker through one or more ligation sites. In one embodiment of the present invention, when the PEG is a PEG having two or more connectable sites represented by Formula IV or VII, it may be linked to a linker through a linking site, and other connectable sites may be It can be linked to a blocking group (such as methoxy), or can be linked to a linker through a plurality of linking sites, such as the structure shown in Formula VII-1, such as PEG is an eight-arm polyethylene glycol, and X is -CH. 2 CO-, the structure of II-d can be:
Figure PCTCN2018106972-appb-000021
Also available for
Figure PCTCN2018106972-appb-000022
Wait.
优选地，上式Ⅱ-a～Ⅱ-e中，所述PEG具有本发明上述Ⅲ、Ⅳ、Ⅴ、Ⅵ、Ⅶ-2、Ⅶ-3、Ⅶ-4或Ⅶ-5的结构，其中，x优选为1-6的整数，y优选为1-3的整数。Preferably, in the above formulas II-a to II-e, the PEG has the structure of the above III, IV, V, VI, VII-2, VII-3, VII-4 or VII-5 of the present invention, wherein x It is preferably an integer of 1-6, and y is preferably an integer of 1-3.
本发明另一方面还提供一种聚乙二醇-连接子结合物衍生物，其具有如下结构：Another aspect of the present invention also provides a polyethylene glycol-linker conjugate which has the following structure:
PEG-X-L-P-QPEG-X-L-P-Q
(Ⅷ)(VIII)
其中，L为本发明上述连接子，Where L is the above linker of the invention,
PEG为聚乙二醇残基，PEG is a polyethylene glycol residue,
X为PEG与L的连接基团，选自：-(CH 2) a-、-(CH 2) aCO-、-(CH 2) aOCO-、-(CH 2) aNHCO-、-NH(CH 2) aCO-、-(CH 2) aSO 2-、-O(CH 2) a-、-O(CH 2) aCO-、-O(CH 2) aOCO-、-O(CH 2) aNHCO-和-O(CH 2) aSO 2-中的一种或多种的组合，a为0-10的整数， X is a linking group of PEG and L, and is selected from the group consisting of: -(CH 2 ) a -, -(CH 2 ) a CO-, -(CH 2 ) a OCO-, -(CH 2 ) a NHCO-, -NH (CH 2 ) a CO-, -(CH 2 ) a SO 2 -, -O(CH 2 ) a -, -O(CH 2 ) a CO-, -O(CH 2 ) a OCO-, -O( CH 2 ) a combination of one or more of NHCO- and -O(CH 2 ) a SO 2 -, a being an integer from 0 to 10,
P为L与封端基Q的连接基团，选自：-(CH 2) r-、
Figure PCTCN2018106972-appb-000023
-(CH 2) rO-、-(CH 2) rCO-、-(CH 2) rNH-、-(CH 2) rCONH-、-(CH 2) rNHCO-、-(CH 2) rSH-、
Figure PCTCN2018106972-appb-000024
Figure PCTCN2018106972-appb-000025
中的一种或多种的组合，r为0-10的整数， P is a linking group of L and a terminal group Q, and is selected from the group consisting of: -(CH 2 ) r -,
Figure PCTCN2018106972-appb-000023
-(CH 2 ) r O-, -(CH 2 ) r CO-, -(CH 2 ) r NH-, -(CH 2 ) r CONH-, -(CH 2 ) r NHCO-, -(CH 2 ) r SH-,
Figure PCTCN2018106972-appb-000024
Figure PCTCN2018106972-appb-000025
a combination of one or more of the following, r is an integer from 0 to 10,
Q为封端基，选自：C1-C6的烷氧基、羟基、氨基、羧基、巯基、酯基、酮基、醛基、邻二硫吡啶基、叠氮基、酰肼基、炔基、硅烷基、马来酰亚胺基和琥珀酰亚胺基，Q is a terminal group selected from the group consisting of: a C1-C6 alkoxy group, a hydroxyl group, an amino group, a carboxyl group, a thiol group, an ester group, a ketone group, an aldehyde group, an o-dithiopyridyl group, an azide group, a hydrazide group, an alkynyl group. Silyl, maleimide and succinimide groups,
R 17和R 18独立地选自：-H、C1-6的烷基、C1-6的烷氧基、C3-6环烷基和C4-10亚烷基环烷基。 R 17 and R 18 are independently selected from the group consisting of: -H, a C1-6 alkyl group, a C1-6 alkoxy group, a C3-6 cycloalkyl group, and a C4-10 alkylene cycloalkyl group.
在本发明一个实施方式中，所述衍生物具有如下结构：In one embodiment of the invention, the derivative has the structure:
Figure PCTCN2018106972-appb-000026
Figure PCTCN2018106972-appb-000026
上式Ⅷ-1～Ⅷ-3中，所述R 1-12、l、A、B、X、PEG等具有本发明上述相应定义。 In the above formulae VIII-1 to VIII-3, the above R 1-12 , 1, A, B, X, PEG and the like have the above corresponding definitions of the present invention.
在本发明的一个实施例中，所述X为单键、-CH 2-、-CH 2CH 2-、-CO-、-CH 2CO-或-NHCO-。 In one embodiment of the invention, X is a single bond, -CH 2 -, -CH 2 CH 2 -, -CO-, -CH 2 CO- or -NHCO-.
优选地，R 17和R 18独立地选自：-H、-CH 3、-CH 2CH 3、-CH 2CH 2CH 3、-OCH 3、-OCH 2CH 3和-OCH 2CH 2CH 3，更优选自：-H、-CH 3、-OCH 3和-OCH 2CH 3，在本发明一个实施例中，R 17为H，R 18为-CH 3、-OCH 3或-OCH 2CH 3，在本发明一个优选实施例中，R 17为H，R 18为-CH 3。 Preferably, R 17 and R 18 are independently selected from the group consisting of: -H, -CH 3 , -CH 2 CH 3 , -CH 2 CH 2 CH 3 , -OCH 3 , -OCH 2 CH 3 and -OCH 2 CH 2 CH 3 , more preferably from: -H, -CH 3 , -OCH 3 and -OCH 2 CH 3 , in one embodiment of the invention, R 17 is H, and R 18 is -CH 3 , -OCH 3 or -OCH 2 CH 3 , In a preferred embodiment of the invention, R 17 is H and R 18 is -CH 3 .
在本发明的一个实施例中，所述P选自：-(CH 2) r-、-(CH 2) rCH(CH 3)-、-(CH 2) rO-、-(CH 2) rCO-、-(CH 2) rNH-、-(CH 2) rCONH-、-(CH 2) rNHCO-、-(CH 2) rSH-、
Figure PCTCN2018106972-appb-000027
Figure PCTCN2018106972-appb-000028
中的一种或多种的组合。 In one embodiment of the invention, the P is selected from the group consisting of: -(CH 2 ) r -, -(CH 2 ) r CH(CH 3 )-, -(CH 2 ) r O-, -(CH 2 ) r CO-, -(CH 2 ) r NH-, -(CH 2 ) r CONH-, -(CH 2 ) r NHCO-, -(CH 2 ) r SH-,
Figure PCTCN2018106972-appb-000027
Figure PCTCN2018106972-appb-000028
a combination of one or more of them.
优选地，r为0-5的整数，如0、1、2、3、4或5。Preferably, r is an integer from 0 to 5, such as 0, 1, 2, 3, 4 or 5.
在本发明的一个具体实施例中，所述P选自：单键、-CH 2-、-CH 2CH 2-、-CH 2CH 2CH 2-、-CH 2CH 2CH 2CH 2-、-CH 2CH 2CH 2CH 2CH 2-、-CH(CH 3)-、-CH 2CH(CH 3)-、-CH 2CH 2CH(CH 3)-、-CH 2CH 2CH 2CH(CH 3)-、-CH 2CH 2CH 2CH 2CH(CH 3)-、-CH 2CH 2CH 2CH 2CH 2CH(CH 3)-、-(CH 2) rO-、-(CH 2) rCO-、-(CH 2) rNH-、-(CH 2) rCONH-、-(CH 2) rNHCO-、-(CH 2) rSH-、
Figure PCTCN2018106972-appb-000029
In a specific embodiment of the invention, the P is selected from the group consisting of: a single bond, -CH 2 -, -CH 2 CH 2 -, -CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 - , -CH 2 CH 2 CH 2 CH 2 CH 2 -, -CH(CH 3 )-, -CH 2 CH(CH 3 )-, -CH 2 CH 2 CH(CH 3 )-, -CH 2 CH 2 CH 2 CH(CH 3 )-, -CH 2 CH 2 CH 2 CH 2 CH(CH 3 )-, -CH 2 CH 2 CH 2 CH 2 CH 2 CH(CH 3 )-, -(CH 2 ) r O- , -(CH 2 ) r CO-, -(CH 2 ) r NH-, -(CH 2 ) r CONH-, -(CH 2 ) r NHCO-, -(CH 2 ) r SH-,
Figure PCTCN2018106972-appb-000029
Figure PCTCN2018106972-appb-000030
Figure PCTCN2018106972-appb-000030
Figure PCTCN2018106972-appb-000031
中的一种或多种的组合。
Figure PCTCN2018106972-appb-000031
a combination of one or more of them.
在本发明中，所述P具有两个以上连接位点时，可连接一个以上PEG-X-L-和一个以上Q，如所述P为
Figure PCTCN2018106972-appb-000032
时，上述衍生物可为
Figure PCTCN2018106972-appb-000033
等。 In the present invention, when the P has more than two linking sites, more than one PEG-XL- and more than one Q may be connected, as the P is
Figure PCTCN2018106972-appb-000032
When the above derivative is
Figure PCTCN2018106972-appb-000033
Wait.
在本发明的一个实施例中，所述Q为酯基或酮基。In one embodiment of the invention, the Q is an ester or ketone group.
在本发明的一个具体实施例中，所述的Q中，所述酯基选自：
Figure PCTCN2018106972-appb-000034
Figure PCTCN2018106972-appb-000035
和-SO 2CH 2CF 3。 In a specific embodiment of the present invention, in the Q, the ester group is selected from the group consisting of:
Figure PCTCN2018106972-appb-000034
Figure PCTCN2018106972-appb-000035
And -SO 2 CH 2 CF 3 .
在本发明的另一个具体实施例中，所述的Q中，所述酮基选自：
Figure PCTCN2018106972-appb-000036
-COCH 3和-COCH 2CH 3。 In another embodiment of the present invention, in the Q, the ketone group is selected from the group consisting of:
Figure PCTCN2018106972-appb-000036
-COCH 3 and -COCH 2 CH 3 .
在本发明的一个优选实施例中，所述Q为
Figure PCTCN2018106972-appb-000037
In a preferred embodiment of the invention, the Q is
Figure PCTCN2018106972-appb-000037
在本发明一个实施方式中中，所述衍生物具有如下结构：In one embodiment of the invention, the derivative has the structure:
Figure PCTCN2018106972-appb-000038
Figure PCTCN2018106972-appb-000038
上式Ⅷ-1～Ⅷ-3中，所述R 1-12、l、A、B、X、PEG等具有本发明上述相应定义。 In the above formulae VIII-1 to VIII-3, the above R 1-12 , 1, A, B, X, PEG and the like have the above corresponding definitions of the present invention.
在本发明的一个实施方式中，所述衍生物选自如下结构：In one embodiment of the invention, the derivative is selected from the following structures:
Figure PCTCN2018106972-appb-000039
Figure PCTCN2018106972-appb-000039
Figure PCTCN2018106972-appb-000040
Figure PCTCN2018106972-appb-000040
上式Ⅷ-a～Ⅷ-e中，所述PEG和X具有本发明上述相应定义。In the above formulae VIII-a to VIII-e, the PEG and X have the above corresponding definitions of the present invention.
在本发明的一个实施例中，上式Ⅷ-a～Ⅷ-e中，所述PEG具有本发明上述通式Ⅲ、Ⅳ、Ⅴ、Ⅵ、Ⅶ-2、Ⅶ-3、Ⅶ-4或Ⅶ-5的结构。In one embodiment of the invention, in the above formulae VIII-a to VIII-e, the PEG has the above formula III, IV, V, VI, VII-2, VII-3, VII-4 or VII of the present invention. -5 structure.
在本发明的一个实施例中，上式Ⅷ-a～Ⅷ-e中，所述PEG的分子量为10-50KDa(具体可为10、15、20、25、30、35、40、45或50KDa)。In one embodiment of the present invention, in the above formulas VIII-a to VIII-e, the PEG has a molecular weight of 10 to 50 KDa (specifically, 10, 15, 20, 25, 30, 35, 40, 45 or 50 KDa) ).
在本发明的一个实施例中，上式Ⅷ-a～Ⅷ-e中，所述X为-CH 2CO-、-CO-、-CH 2-或-CH 2CH 2-。 In one embodiment of the invention, in the above formulae VIII-a to VIII-e, the X is -CH 2 CO-, -CO-, -CH 2 - or -CH 2 CH 2 -.
优选地，上式Ⅷ-a～Ⅷ-e中，所述PEG具有本发明上述Ⅲ、Ⅳ、Ⅴ、Ⅵ、Ⅶ-2、Ⅶ-3、Ⅶ-4或Ⅶ-5的结构，其中，x为1-6的整数，y为1-3的整数。Preferably, in the above formulae VIII-a to VIII-e, the PEG has the structure of the above III, IV, V, VI, VII-2, VII-3, VII-4 or VII-5 of the present invention, wherein x An integer of 1-6, y is an integer from 1-3.
本发明另一方面还提供一种聚乙二醇-连接子-药物结合物，其具有如下结构：Another aspect of the invention also provides a polyethylene glycol-linker-drug conjugate having the structure:
(PEG-X-L-Y) n-D (PEG-XLY) n -D
(Ⅸ)(IX)
其中，PEG为聚乙二醇残基，Wherein PEG is a polyethylene glycol residue,
X为PEG与L的连接基团，选自：-(CH 2) a-、-(CH 2) aCO-、-(CH 2) aOCO-、-(CH 2) aNHCO-、-NH(CH 2) aCO-、-(CH 2) aSO 2-、-O(CH 2) a-、-O(CH 2) aCO-、-O(CH 2) aOCO-、-O(CH 2) aNHCO-和-O(CH 2) aSO 2-中的一种或多种的组合，a为0-10的整数， X is a linking group of PEG and L, and is selected from the group consisting of: -(CH 2 ) a -, -(CH 2 ) a CO-, -(CH 2 ) a OCO-, -(CH 2 ) a NHCO-, -NH (CH 2 ) a CO-, -(CH 2 ) a SO 2 -, -O(CH 2 ) a -, -O(CH 2 ) a CO-, -O(CH 2 ) a OCO-, -O( CH 2 ) a combination of one or more of NHCO- and -O(CH 2 ) a SO 2 -, a being an integer from 0 to 10,
Y为L与D的连接基团，选自：-(CH 2) r-、
Figure PCTCN2018106972-appb-000041
-(CH 2) rO-、-(CH 2) rCO-、-(CH 2) rNH-、-(CH 2) rCONH-、-(CH 2) rNHCO-、-(CH 2) rSH-、
Figure PCTCN2018106972-appb-000042
Figure PCTCN2018106972-appb-000043
中的一种或多种的组合，r为0-10的整数， Y is a linking group of L and D, and is selected from the group consisting of: -(CH 2 ) r -,
Figure PCTCN2018106972-appb-000041
-(CH 2 ) r O-, -(CH 2 ) r CO-, -(CH 2 ) r NH-, -(CH 2 ) r CONH-, -(CH 2 ) r NHCO-, -(CH 2 ) r SH-,
Figure PCTCN2018106972-appb-000042
Figure PCTCN2018106972-appb-000043
a combination of one or more of the following, r is an integer from 0 to 10,
R 17和R 18独立地选自：-H、C1-6的烷基、C1-6的烷氧基、C3-6环烷基和C4-10亚烷基环烷基， R 17 and R 18 are independently selected from the group consisting of: -H, a C1-6 alkyl group, a C1-6 alkoxy group, a C3-6 cycloalkyl group, and a C4-10 alkylene cycloalkyl group.
L为本发明上述连接子，L is the above linker of the present invention,
D为含m个胺基的生物学活性剂，m为1-500的整数，D is a biologically active agent containing m amine groups, and m is an integer of from 1 to 500,
n为整数，且1≤n≤m。n is an integer and 1 ≤ n ≤ m.
在本发明的一个实施方式中，所述n＝1，此时通式Ⅸ为PEG-X-L-Y-D。In one embodiment of the invention, the n = 1 and the formula IX is PEG-X-L-Y-D.
在本发明的一个实施方式中，所述的D为含胺基的小分子生物学活性剂及其药学上可接受的盐，优选包括：阿霉素、克唑替尼、戈舍瑞林、阿糖胞苷、普鲁卡因、苯佐卡因、氯普鲁卡因、二甲卡因、多巴胺、去甲肾上腺素、克仑特罗、苯乙双胍、达拉匹林、丙舒硫胺、对氨基水杨酸、磺胺嘧啶，及其衍生物。In one embodiment of the present invention, the D is an amine group-containing small molecule biologically active agent and a pharmaceutically acceptable salt thereof, and preferably includes: doxorubicin, crizotinib, goserelin, Cytarabine, procaine, benzocaine, chloroprocaine, dimethine, dopamine, norepinephrine, clenbuterol, phenformin, dalapril, propyl sulphur Amine, p-aminosalicylic acid, sulfadiazine, and derivatives thereof.
在本发明的一个具体实施方式中，所述D为阿霉素或其衍生物，其具有如下结构：In a specific embodiment of the invention, the D is doxorubicin or a derivative thereof, which has the following structure:
Figure PCTCN2018106972-appb-000044
Figure PCTCN2018106972-appb-000044
其中，W 1选自：-H、-OH、-OCH 3和-OCH 2CH 3；优选为-H或-OCH 3，更优选为-OCH 3； Wherein W 1 is selected from the group consisting of: -H, -OH, -OCH 3 and -OCH 2 CH 3 ; preferably -H or -OCH 3 , more preferably -OCH 3 ;
W 2选自：-H、-OH、-OCO(CH 2) 5COOH和-OCO(CH 2) 2NH 2；优选为-H或-OH，更优选为-OH； W 2 is selected from the group consisting of: -H, -OH, -OCO(CH 2 ) 5 COOH and -OCO(CH 2 ) 2 NH 2 ; preferably -H or -OH, more preferably -OH;
W 3选自：-OH、-OCH 3和
Figure PCTCN2018106972-appb-000045
优选为-OH。 W 3 is selected from the group consisting of: -OH, -OCH 3 and
Figure PCTCN2018106972-appb-000045
It is preferably -OH.
在本发明的一个更实施例中，所述D为阿霉素，具有如下结构：In a further embodiment of the invention, the D is doxorubicin having the following structure:
Figure PCTCN2018106972-appb-000046
Figure PCTCN2018106972-appb-000046
在本发明的另一个实施方式中，所述D为含胺基的大分子生物学活性剂，如多肽和蛋白质类药物，所述的多肽和蛋白类药物如细胞因子(如白细胞介素、集落刺激因子、干扰素、生长因子、肿瘤坏死因子、转化生长因子-β家族或趋化因子家族等)、人血红蛋白、凝血因子、血管内皮生长因子抗体拮抗剂、蛋白类激素(如胰岛素、胰高血糖素、降钙素、下丘脑激素、垂体激素或胃肠激素等)、抗体(如单克隆抗体、多克隆抗体、二聚体、多聚体、多特异性抗体或抗体片断)、酶及辅酶类药物(苯丙氨酸裂解酶、精氨酸酶、精氨酸脱酰酶、胰核糖核酸酶、超氧化物歧化酶、天冬酰胺酶、葡糖脑苷脂酶或透明质酸酶)。In another embodiment of the present invention, the D is an amine group-containing macromolecular biological active agent such as a polypeptide and a protein drug, such as a cytokine (such as an interleukin, a colony). Stimulating factors, interferons, growth factors, tumor necrosis factors, transforming growth factor-β family or chemokine family, etc., human hemoglobin, coagulation factors, vascular endothelial growth factor antibody antagonists, protein hormones (such as insulin, pancreatic high) Glucagon, calcitonin, hypothalamic hormone, pituitary hormone or gut hormone, etc.), antibodies (such as monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies or antibody fragments), enzymes and Coenzymes (phenylalanine lyase, arginase, arginine deacylase, pancreatic ribonuclease, superoxide dismutase, asparaginase, glucocerebrosidase or hyaluronidase) ).
在所述药物结合物中，药物分子通过胺基与聚乙二醇-连接子连接，所述胺基可为肽链N-末端氨基和/或肽链内氨基酸(如赖氨酸等)残基的侧链氨基；更优选地，在所述药物结合物中，聚乙二醇-连接子通过活性碳酸酯基与药物分子的伯胺基反应形成
Figure PCTCN2018106972-appb-000047
实现连接。 In the drug conjugate, the drug molecule is linked to the polyethylene glycol-linker via an amine group, which may be an N-terminal amino group of the peptide chain and/or an amino acid (such as lysine) in the peptide chain. a side chain amino group; more preferably, in the drug conjugate, a polyethylene glycol-linker is formed by reacting an active carbonate group with a primary amine group of a drug molecule
Figure PCTCN2018106972-appb-000047
Implement the connection.
本申请中，术语“抗体”以其最广泛的含义使用并且特别覆盖单克隆抗体、多克隆抗体、二聚体、多聚体、多特异性抗体(例如：双特异性抗体)和抗体片段，只要它们表现出所需的生物活性(Miller等(2003)Jour.of Immunology,170:4854-4861)。抗体可以为鼠、人、人源化、嵌合抗体或来源于其它物种。抗体可具有任何类型(例如IgG、IgE、IgM、IgD及IgA)及类别(例如IgG1、IgG2、IgG3、IgG4、IgA1和IgA2)，其中IgM含游离氨基约450个。In the present application, the term "antibody" is used in its broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (eg, bispecific antibodies) and antibody fragments, As long as they exhibit the desired biological activity (Miller et al. (2003) Jour. of Immunology, 170: 4854-4861). The antibody can be murine, human, humanized, chimeric, or derived from other species. The antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA) and classes (e.g., IgGl, IgG2, IgG3, IgG4, IgA1, and IgA2), wherein IgM contains about 450 free amino groups.
在本发明的一个实施方式中，所述的D为细胞因子；在本发明的一个实施例中，所述的细胞因子为白介素，优选为白介素2(IL-2)，更优选为重组人白介素2(rhIL-2)。In one embodiment of the invention, the D is a cytokine; in one embodiment of the invention, the cytokine is an interleukin, preferably an interleukin 2 (IL-2), more preferably a recombinant human interleukin 2 (rhIL-2).
在本发明另一个实施方式中，所述的细胞因子为集落刺激因子，优选为粒细胞集落刺激因子，更优选为重组人粒细胞集落刺激因子。In another embodiment of the present invention, the cytokine is a colony stimulating factor, preferably a granulocyte colony stimulating factor, more preferably a recombinant human granulocyte colony stimulating factor.
在本发明的另一个实施方式中，所述的D为抗体。In another embodiment of the invention, the D is an antibody.
在本发明的一个实施方式中，所述的抗体为单克隆抗体，如抗HER2单克隆抗体，优选为重组抗HER2人源化单克隆抗体。In one embodiment of the invention, the antibody is a monoclonal antibody, such as an anti-HER2 monoclonal antibody, preferably a recombinant anti-HER2 humanized monoclonal antibody.
在本发明的另一个实施方式中，所述D为人血红蛋白。In another embodiment of the invention, the D is human hemoglobin.
在本发明的另一个实施方式中，所述D为酶及辅酶类药物，优选自：胰核糖核酸酶、超氧化物歧化酶和天冬酰胺酶。In another embodiment of the invention, the D is an enzyme and a coenzyme, preferably from: pancreatic ribonuclease, superoxide dismutase, and asparaginase.
在本发明的另一个实施方式中，所述D为蛋白类激素，优选为胰岛素。In another embodiment of the invention, the D is a protein hormone, preferably insulin.
所述聚乙二醇-连接子-药物结合物中，n为1-500的整数(如1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、20、30、40、50、60、70、80、90、100、150、200、250、300、350、400、450或500)，如所述药物为白介素2时，n可为1、2、3、4、5、6、7、8、9、10、11或12；所述药物为重组抗HER2人源化单克隆抗体时，n可为1-90的整数；所述药物为IgM时，n可为1-450的整数。In the polyethylene glycol-linker-drug combination, n is an integer from 1 to 500 ( eg 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450 or 500), if the drug is interleukin 2, n can Is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; when the drug is a recombinant anti-HER2 humanized monoclonal antibody, n may be an integer from 1 to 90; When the drug is IgM, n may be an integer from 1 to 450.
在本发明的一个实施方式中，所述Y选自：-(CH 2) r-、-(CH 2) rCH(CH 3)-、-(CH 2) rO-、-(CH 2) rCO-、-(CH 2) rNH-、 -(CH 2) rCONH-、-(CH 2) rNHCO-、-(CH 2) rSH-、
Figure PCTCN2018106972-appb-000048
Figure PCTCN2018106972-appb-000049
中的一种或多种的组合。 In one embodiment of the invention, the Y is selected from the group consisting of: -(CH 2 ) r -, -(CH 2 ) r CH(CH 3 )-, -(CH 2 ) r O-, -(CH 2 ) r CO-, -(CH 2 ) r NH-, -(CH 2 ) r CONH-, -(CH 2 ) r NHCO-, -(CH 2 ) r SH-,
Figure PCTCN2018106972-appb-000048
Figure PCTCN2018106972-appb-000049
a combination of one or more of them.
优选的，r为0-5的整数，如0、1、2、3、4或5。Preferably, r is an integer from 0 to 5, such as 0, 1, 2, 3, 4 or 5.
在本发明的一个具体实施例中，所述Y选自：单键、-CH 2-、-CH 2CH 2-、-CH 2CH 2CH 2-、-CH 2CH 2CH 2CH 2-、-CH 2CH 2CH 2CH 2CH 2-、-CH(CH 3)-、-CH 2CH(CH 3)-、-CH 2CH 2CH(CH 3)-、-CH 2CH 2CH 2CH(CH 3)-、-CH 2CH 2CH 2CH 2CH(CH 3)-、-CH 2CH 2CH 2CH 2CH 2CH(CH 3)-、-(CH 2) rO-、-(CH 2) rCO-、-(CH 2) rCONH-、-(CH 2) rNH-、-(CH 2) rSH-、
Figure PCTCN2018106972-appb-000050
In a specific embodiment of the invention, the Y is selected from the group consisting of: a single bond, -CH 2 -, -CH 2 CH 2 -, -CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 - , -CH 2 CH 2 CH 2 CH 2 CH 2 -, -CH(CH 3 )-, -CH 2 CH(CH 3 )-, -CH 2 CH 2 CH(CH 3 )-, -CH 2 CH 2 CH 2 CH(CH 3 )-, -CH 2 CH 2 CH 2 CH 2 CH(CH 3 )-, -CH 2 CH 2 CH 2 CH 2 CH 2 CH(CH 3 )-, -(CH 2 ) r O- , -(CH 2 ) r CO-, -(CH 2 ) r CONH-, -(CH 2 ) r NH-, -(CH 2 ) r SH-,
Figure PCTCN2018106972-appb-000050
Figure PCTCN2018106972-appb-000051
Figure PCTCN2018106972-appb-000051
Figure PCTCN2018106972-appb-000052
中的一种或多种的组合。
Figure PCTCN2018106972-appb-000052
a combination of one or more of them.
在本发明的另一个具体的实施例中，所述Y为-(CH 2) rCO-与单键、-CH 2-、-CH 2CH 2-、-CH 2CH 2CH 2-、-CH 2CH 2CH 2CH 2-、-CH 2CH 2CH 2CH 2CH 2-、-CH(CH 3)-、-CH 2CH(CH 3)-、-CH 2CH 2CH(CH 3)-、-CH 2CH 2CH 2CH(CH 3)-、-CH 2CH 2CH 2CH 2CH(CH 3)-、-CH 2CH 2CH 2CH 2CH 2CH(CH 3)-、-(CH 2) rO-、-(CH 2) rCONH-、-(CH 2) rNHCO-、-(CH 2) rNH-、-(CH 2) rSH-、
Figure PCTCN2018106972-appb-000053
Figure PCTCN2018106972-appb-000054
中的一种或多种的组合。 In another specific embodiment of the invention, Y is -(CH 2 ) r CO- and a single bond, -CH 2 -, -CH 2 CH 2 -, -CH 2 CH 2 CH 2 -, - CH 2 CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 CH 2 -, -CH(CH 3 )-, -CH 2 CH(CH 3 )-, -CH 2 CH 2 CH (CH 3 )-, -CH 2 CH 2 CH 2 CH(CH 3 )-, -CH 2 CH 2 CH 2 CH 2 CH(CH 3 )-, -CH 2 CH 2 CH 2 CH 2 CH 2 CH(CH 3 )- , -(CH 2 ) r O-, -(CH 2 ) r CONH-, -(CH 2 ) r NHCO-, -(CH 2 ) r NH-, -(CH 2 ) r SH-,
Figure PCTCN2018106972-appb-000053
Figure PCTCN2018106972-appb-000054
a combination of one or more of them.
在本发明的一个实施例中，所述聚乙二醇-连接子-药物结合物中，所述Y为-(CH 2) rCO-。 In one embodiment of the present invention, the polyethylene glycol - linker - drug conjugate, the Y is - (CH 2) r CO-.
在本发明的一个另一个实施例中，所述聚乙二醇-连接子-药物结合物中，所述Y为-CO-。In another embodiment of the invention, in the polyethylene glycol-linker-drug combination, the Y is -CO-.
在本发明中，所述Y具有两个以上连接位点时，可连接一个以上PEG-X-L-和D，如所述Y为
Figure PCTCN2018106972-appb-000055
时，所述聚乙二醇-连接子-药物结合物可为
Figure PCTCN2018106972-appb-000056
In the present invention, when the Y has more than two linking sites, more than one PEG-XL- and D may be connected, as the Y is
Figure PCTCN2018106972-appb-000055
The polyethylene glycol-linker-drug conjugate can be
Figure PCTCN2018106972-appb-000056
在本发明一个实施方式中，所述聚乙二醇-连接子-药物结合物具有如下结构：In one embodiment of the invention, the polyethylene glycol-linker-drug conjugate has the structure:
Figure PCTCN2018106972-appb-000057
Figure PCTCN2018106972-appb-000057
Figure PCTCN2018106972-appb-000058
Figure PCTCN2018106972-appb-000058
上式Ⅸ-1～Ⅸ-3中，所述R 1-12、l、A、B、X、PEG、Y、D等具有本发明上述相应定义。 In the above formulae IX-1 to IX-3, the above-mentioned respective definitions of the present invention are as defined above for R 1-12 , 1, A, B, X, PEG, Y, D and the like.
在本发明的一个实施例中，上式Ⅸ-1中，所述的R 1-4均为-H。 In one embodiment of the present invention, in the above formula IX-1, R 1-4 is all -H.
在本发明的一个实施例中，上式Ⅸ-2中，所述的R 5-7均为-H。 In one embodiment of the present invention, in the above formula IX-2, the R 5-7 is -H.
在本发明的一个实施例中，上式Ⅸ-3中，所述的R 9-11均为-H。 In one embodiment of the present invention, in the above formula IX-3, the R 9-11 is -H.
在本发明的一个实施例中，上式Ⅸ-2、Ⅸ-3中，所述R 8和/或R 12为甲基。 In one embodiment of the invention, in the above formula IX-2, IX-3, the R 8 and/or R 12 is a methyl group.
在本发明的一个实施例中，上式Ⅸ-1～Ⅸ-3中，所述l为1。In one embodiment of the present invention, in the above formulas IX-1 to IX-3, the l is 1.
在本发明的一个实施例中，上式Ⅸ-1中，所述A为-COCH 2NH-、-COCH(CH 3)NH-或-COCH(CH(CH 3) 2)NH-。 In one embodiment of the present invention, in the above formula IX-1, the A is -COCH 2 NH-, -COCH(CH 3 )NH- or -COCH(CH(CH 3 ) 2 )NH-.
在本发明的一个实施例中，上式Ⅸ-2、Ⅸ-3中，所述-B-A-为-OCH 2CH 2NH-。 In one embodiment of the invention, in the above formula IX-2, IX-3, the -BA- is -OCH 2 CH 2 NH-.
在本发明的一个实施例中，上式Ⅸ-1～Ⅸ-3中，所述X为单键、-CH 2-、-CH 2CH 2-、-CO-、-CH 2CO-或-NHCO-。 In one embodiment of the present invention, in the above formulas IX-1 to IX-3, the X is a single bond, -CH 2 -, -CH 2 CH 2 -, -CO-, -CH 2 CO- or - NHCO-.
在本发明的一个实施例中，上式Ⅸ-1～Ⅸ-3中，所述D为多肽和蛋白质类药物，优选为细胞因子，更优选为IL-2(如rhIL-2)，所述PEG具有本发明上述Ⅲ、Ⅴ、Ⅵ、Ⅶ-3或Ⅶ-5的结构；优选地，其中，x为1-6的整数，y为1-3的整数。In one embodiment of the present invention, in the above formulas IX-1 to IX-3, the D is a polypeptide and a protein drug, preferably a cytokine, more preferably IL-2 (such as rhIL-2), PEG has the structure of the above III, V, VI, VII-3 or VII-5 of the present invention; preferably, wherein x is an integer from 1 to 6, and y is an integer from 1 to 3.
在本发明的一个实施例中，上式Ⅸ-1～Ⅸ-3中，所述D为阿霉素或其衍生物，具有本发明上述通式Ⅹ所示结构，优选为阿霉素，其具有本发明上述通式Ⅹ-1所示结构，所述PEG具有本发明上述Ⅲ、Ⅳ、Ⅴ、Ⅵ、Ⅶ-2或Ⅶ-4的结构；优选地，其中，x为1-6的整数，y为1-3的整数。In one embodiment of the present invention, in the above formulas IX-1 to IX-3, the D is doxorubicin or a derivative thereof, and has the structure represented by the above formula X of the present invention, preferably doxorubicin, Having the structure represented by the above formula X-1 of the present invention, the PEG having the structure of the above III, IV, V, VI, VII-2 or VII-4 of the present invention; preferably, wherein x is an integer of 1-6 , y is an integer from 1-3.
在本发明的一个实施例中，上式Ⅸ-1～Ⅸ-3中，所述的PEG的分子量为10-50KDa(具体可为10、15、20、25、30、35、40、45或50KDa)。In one embodiment of the present invention, in the above formulas IX-1 to IX-3, the PEG has a molecular weight of 10 to 50 KDa (specifically, 10, 15, 20, 25, 30, 35, 40, 45 or 50KDa).
在本发明的一个具体实施方式中所述聚乙二醇-连接子-药物结合物具有如下结构：In a specific embodiment of the invention the polyethylene glycol-linker-drug conjugate has the structure:
Figure PCTCN2018106972-appb-000059
Figure PCTCN2018106972-appb-000059
上式Ⅸ-a～Ⅸ-e中，所述PEG、X、n和D具有本发明上述定义。In the above formulae IX-a to IX-e, the PEG, X, n and D have the above definitions of the present invention.
在本发明的一个实施例中，上式Ⅸ-a～Ⅸ-e中，所述D为多肽和蛋白质类药物，优选为细胞因子，更优选为IL-2，所述PEG具有本发明上述Ⅲ、Ⅴ、Ⅵ、Ⅶ-3或Ⅶ-5的结构；优选地，其中，x为1-6的整数，y为1-3的整数。当PEG为通式Ⅴ、Ⅵ、Ⅶ-3或Ⅶ-5的结构时，可增加空间位阻效应，调控药物释放速度。In one embodiment of the present invention, in the above formulas IX-a to IX-e, the D is a polypeptide and a protein drug, preferably a cytokine, more preferably IL-2, and the PEG has the above III of the present invention. The structure of V, VI, VII-3 or VII-5; preferably wherein x is an integer from 1 to 6, and y is an integer from 1 to 3. When PEG is a structure of the formula V, VI, VII-3 or VII-5, the steric hindrance effect can be increased to regulate the drug release rate.
在本发明的一个实施例中，上式Ⅸ-a～Ⅸ-e中，所述D为阿霉素或其衍生物，具有本发明上述通式Ⅹ所示结构；优选地，所述D为阿霉素，其具有本发明上述通式Ⅹ-1所示结构，所述n为1。In one embodiment of the present invention, in the above formulas IX-a to IX-e, the D is doxorubicin or a derivative thereof, and has the structure represented by the above formula X of the present invention; preferably, the D is Doxorubicin having the structure represented by the above formula X-1 of the present invention, wherein n is 1.
在本发明的一个实施例中，上式Ⅸ-a～Ⅸ-e中，所述PEG的分子量为10-50KDa(具体可为10、15、20、25、30、35、40、45或50KDa)。In one embodiment of the present invention, in the above formulas IX-a to IX-e, the molecular weight of the PEG is 10-50 KDa (specifically, 10, 15, 20, 25, 30, 35, 40, 45 or 50 KDa ).
在本发明的一个实施例中，上式Ⅸ-a～Ⅸ-e中，所述X为-CH 2CO-、-CO-、-CH 2-或-CH 2CH 2-。 In one embodiment of the present invention, in the above formulae IX-a to IX-e, the X is -CH 2 CO-, -CO-, -CH 2 - or -CH 2 CH 2 -.
在本发明的一个实施例中，上式Ⅸ-a～Ⅸ-e中，所述D为IL-2，优选为rhIL-2，所述n为1-12的整数，优选为1-7的整数，具体如1、2、3、4、5、6或7。In one embodiment of the present invention, in the above formulae IX-a to IX-e, the D is IL-2, preferably rhIL-2, and the n is an integer of 1-12, preferably 1-7 An integer, such as 1, 2, 3, 4, 5, 6, or 7.
在本发明的一个更具体的实施方式中，所述药物为多肽和蛋白质类药物，优选为细胞因子，更优选为IL-2(如rhIL-2)，所述聚乙二醇-连接子-IL-2结合物具有如下结构：In a more specific embodiment of the invention, the drug is a polypeptide and a proteinaceous drug, preferably a cytokine, more preferably IL-2 (such as rhIL-2), the polyethylene glycol-linker- The IL-2 conjugate has the following structure:
Figure PCTCN2018106972-appb-000060
Figure PCTCN2018106972-appb-000060
Figure PCTCN2018106972-appb-000061
Figure PCTCN2018106972-appb-000061
Figure PCTCN2018106972-appb-000062
Figure PCTCN2018106972-appb-000062
上式中，p、q、i、h、k和x分别具有本发明通式Ⅲ、Ⅴ、Ⅵ、Ⅶ-3中的定义。In the above formula, p, q, i, h, k and x each have the definitions in the formula III, V, VI, VII-3 of the present invention.
在本发明的一个实施例中，x为1-6的整数，优选地，x为6。In one embodiment of the invention, x is an integer from 1 to 6, preferably x is 6.
在本发明的一个实施例中，上式中，所述n为1-12的整数，优选为1-7的整数，具体如1、2、3、4、5、6或7。In one embodiment of the invention, in the above formula, n is an integer from 1 to 12, preferably an integer from 1 to 7, specifically such as 1, 2, 3, 4, 5, 6, or 7.
在本发明的一个实施例中，上式中，所述IL-2为rhIL-2。In one embodiment of the invention, in the above formula, the IL-2 is rhIL-2.
在本发明的一个更具体的实施方式中，所述药物为阿霉素或其衍生物，具有本发明上述通式Ⅹ所示结构；优选地，所述D为阿霉素，其具有本发明上述通式Ⅹ-1所示结构，所述聚乙二醇-连接子-阿霉素结合物具有如下结构：In a more specific embodiment of the present invention, the drug is doxorubicin or a derivative thereof, having the structure represented by the above formula X of the present invention; preferably, the D is doxorubicin, which has the present invention The structure represented by the above formula X-1, the polyethylene glycol-linker-doxorubicin conjugate has the following structure:
Figure PCTCN2018106972-appb-000063
Figure PCTCN2018106972-appb-000063
Figure PCTCN2018106972-appb-000064
Figure PCTCN2018106972-appb-000064
Figure PCTCN2018106972-appb-000065
Figure PCTCN2018106972-appb-000065
Figure PCTCN2018106972-appb-000066
Figure PCTCN2018106972-appb-000066
上式中，DN为
Figure PCTCN2018106972-appb-000067
In the above formula, DN is
Figure PCTCN2018106972-appb-000067
p、q、k和y分别具有本发明通式Ⅲ、Ⅴ、Ⅵ、Ⅶ-3中的定义。p, q, k and y each have the definitions in the general formula III, V, VI, VII-3 of the present invention.
在本发明的一个实施例中，上式中，所述y为1-5的整数，如1、2、3、4或5。In one embodiment of the invention, in the above formula, the y is an integer from 1 to 5, such as 1, 2, 3, 4 or 5.
本发明另一方面还提供一种上述聚乙二醇-连接子-药物结合物的制备方法，包括本发明上述聚乙二醇-连接子结合物衍生物与药物反应连接的步骤。Another aspect of the present invention provides a method for producing the above polyethylene glycol-linker-drug conjugate, comprising the step of reacting the above polyethylene glycol-linker conjugate of the present invention with a drug.
优选地，所述制备方法中，所述的衍生物为活性酯，更优选地，所述衍生物的反应基团为活性碳酸酯基。Preferably, in the preparation method, the derivative is an active ester, and more preferably, the reactive group of the derivative is an active carbonate group.
在本发明的一个实施例中，所述衍生物具有上述式Ⅷ-a～Ⅷ-e的结构。In one embodiment of the invention, the derivative has the structure of the above formulae VIII-a to VIII-e.
优选地，所述制备方法中，所述药物的反应基团为胺基，更优选为伯胺基。Preferably, in the preparation method, the reactive group of the drug is an amine group, and more preferably a primary amino group.
在本发明的一个实施例中，所述药物为多肽和蛋白类药物，优选为IL-2，更优选为rhIL-2。In one embodiment of the invention, the medicament is a polypeptide and a proteinaceous drug, preferably IL-2, more preferably rhIL-2.
在本发明的一个实施例中，所述药物为阿霉素或其衍生物，具有本发明上述通式Ⅹ所示结构，优选为阿霉素，其具有本发明上述通式Ⅹ-1所示结构。In one embodiment of the present invention, the drug is doxorubicin or a derivative thereof, and has the structure of the above formula X of the present invention, preferably doxorubicin, which has the above formula X-1 of the present invention. structure.
在本发明的一个优选实施例中，所述的聚乙二醇-连接子-药物结合物为聚乙二醇-连接子-IL-2结合物，其制备方法包括本发明上述聚乙二醇-连接子结合物衍生物与IL-2反应连接的步骤。In a preferred embodiment of the present invention, the polyethylene glycol-linker-drug conjugate is a polyethylene glycol-linker-IL-2 conjugate, and the preparation method thereof comprises the above polyethylene glycol of the present invention. a step of the ligation of the linker conjugate derivative with IL-2.
优选地，所述聚乙二醇-连接子-IL-2结合物的制备方法中，所述聚乙二醇-连接子结合物衍生物与IL-2的摩尔比为1-50:1(具体如1:1、5:1、10:1、15:1、20:1、25:1、30:1、35:1、40:1、45:1或50:1)，更优选为10-30:1。Preferably, in the method for preparing the polyethylene glycol-linker-IL-2 conjugate, the molar ratio of the polyethylene glycol-linker conjugate derivative to IL-2 is 1-50:1 ( Specifically, it is 1:1, 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1 or 50:1), more preferably 10-30:1.
优选地，所述聚乙二醇-连接子-IL-2结合物的制备方法中，所述反应pH值为6.0-10.0(具体如6.0、 6.5、7.0、7.5、8.0、8.5、9.0、9.5或10.0)。Preferably, in the preparation method of the polyethylene glycol-linker-IL-2 conjugate, the reaction pH is 6.0-10.0 (specifically, such as 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5). Or 10.0).
优选地，所述聚乙二醇-连接子-IL-2结合物的制备方法中，所述反应温度为20-30℃，更优选为室温。Preferably, in the method for producing the polyethylene glycol-linker-IL-2 conjugate, the reaction temperature is 20-30 ° C, more preferably room temperature.
优选地，所述聚乙二醇-连接子-IL-2结合物的制备方法中，所述反应在缓冲液中进行，本领域技术人员可以根据所采用的反应pH值选择合适的缓冲液种类，如磷酸盐缓冲液、硼酸盐缓冲液或碳酸氢钠缓冲液等，本发明对此不作具体限定。Preferably, in the preparation method of the polyethylene glycol-linker-IL-2 conjugate, the reaction is carried out in a buffer, and a person skilled in the art can select a suitable buffer type according to the reaction pH used. For example, the phosphate buffer solution, the borate buffer solution or the sodium hydrogencarbonate buffer solution is not specifically limited in the present invention.
优选地，所述聚乙二醇-连接子-IL-2结合物的制备方法还包括终止反应的步骤，更优选地，所述终止反应步骤包括加入甘氨酸溶液，所述甘氨酸溶液浓度为0.5-2M(具体如0.5、1.0、1.5或2.0M)。Preferably, the method for preparing the polyethylene glycol-linker-IL-2 conjugate further comprises the step of terminating the reaction, and more preferably, the terminating reaction step comprises adding a glycine solution having a concentration of 0.5- 2M (specifically 0.5, 1.0, 1.5 or 2.0M).
优选地，所述聚乙二醇-连接子-IL-2结合物的制备方法还包括分离纯化反应产物的步骤。所述分离纯化的方法可采用本领域中常用的方法，如离子交换柱层析、反相高效液相色谱分离和凝胶渗透色谱分离等中的一种或多种的组合，本发明对此不作具体限定。Preferably, the method for preparing the polyethylene glycol-linker-IL-2 conjugate further comprises the step of isolating and purifying the reaction product. The separation and purification method may employ a combination of one or more of methods commonly used in the art, such as ion exchange column chromatography, reversed-phase high performance liquid chromatography separation, and gel permeation chromatography separation, etc. No specific limitation.
本发明另一方面还提供一种上述聚乙二醇-连接子-药物结合物的组合物，所述组合物中包括至少两种本发明上述具有不同n值的聚乙二醇-连接子-药物结合物。In another aspect, the present invention provides a composition of the above polyethylene glycol-linker-drug conjugate comprising at least two polyethylene glycol-linkers of the present invention having different n values. Drug conjugate.
在本发明的一个实施方式中，上述组合物中包括至少三种本发明上述具有不同n值的聚乙二醇-连接子-药物结合物。In one embodiment of the invention, at least three of the above-described polyethylene glycol-linker-drug conjugates having different n values of the invention are included in the above composition.
在本发明的一个实施方式中，上述组合物中所述药物为多肽和蛋白质类药物，优选为细胞因子，更优选为IL-2(如rhIL-2)。In one embodiment of the invention, the drug in the above composition is a polypeptide and a proteinaceous drug, preferably a cytokine, more preferably IL-2 (such as rhIL-2).
优选地，所述聚乙二醇-连接子-药物结合物聚乙二醇-连接子-IL-2结合物。Preferably, the polyethylene glycol-linker-drug conjugate polyethylene glycol-linker-IL-2 conjugate.
在本发明的一个实施例中，所述组合物包括n值为1-7的聚乙二醇-连接子-IL-2结合物。In one embodiment of the invention, the composition comprises a polyethylene glycol-linker-IL-2 conjugate having an n value of from 1 to 7.
在本发明的一个实施例中，所述组合物包括n值为1-3的聚乙二醇-连接子-IL-2结合物。In one embodiment of the invention, the composition comprises a polyethylene glycol-linker-IL-2 conjugate having an n value of 1-3.
在本发明的一个实施例中，所述组合物包括n值为3-5的聚乙二醇-连接子-IL-2结合物。In one embodiment of the invention, the composition comprises a polyethylene glycol-linker-IL-2 conjugate having an n value of 3-5.
在本发明的一个实施例中，所述组合物包括n值为4-7的聚乙二醇-连接子-IL-2结合物。In one embodiment of the invention, the composition comprises a polyethylene glycol-linker-IL-2 conjugate having an n value of 4-7.
本发明另一方面还提供一种上述聚乙二醇-连接子-药物结合物的药学上可接受的盐、异构体、前药或溶剂化物。Another aspect of the invention also provides a pharmaceutically acceptable salt, isomer, prodrug or solvate of the above polyethylene glycol-linker-drug conjugate.
本发明另一方面还提供一种包含上述聚乙二醇-连接子-药物结合物及其药学上可接受的盐、异构体、前药或溶剂化物和药学上可接受的载体或添加剂的药物组合物。Another aspect of the present invention provides a polyethylene glycol-linker-drug conjugate as described above, and a pharmaceutically acceptable salt, isomer, prodrug or solvate thereof, and a pharmaceutically acceptable carrier or additive Pharmaceutical composition.
如本文所用，“药学上可接受”一词是指在对人施用后具有生理相容性并且不会引起肠胃失调、诸如头晕的过敏反应或类似反应。添加剂可为赋形剂、崩解剂、粘结剂、润滑剂、悬浮剂、稳定剂等等中的任一种。赋形剂的例子包括乳糖、甘露醇、益寿糖、微晶纤维素、硅化微晶纤维素、粉状纤维素等等。崩解剂的例子包括低取代羟丙基纤维素、交聚维酮、羧基乙酸淀粉钠、交联羧甲基纤维素钠、淀粉等等。粘结剂的例子包括羟丙基纤维素、羟丙甲纤维素、聚维酮、共聚维酮、预胶凝淀粉等等。润滑剂的例子包括硬脂酸、硬脂酸镁、富马酰硬脂酸钠等等；润湿剂的例子包括聚氧乙烯山梨糖醇酐脂肪酸酯、泊洛沙姆、聚氧乙烯蓖麻油衍生物等等。悬浮剂的例子包括羟丙甲纤维素、羟丙基纤维素、聚维酮、共聚维酮、羧甲基纤维素钠、甲基纤维素等等。稳定剂的例子包括柠檬酸、富马酸、琥珀酸等等。另外，本发明的药物组合物还可包括阻凝剂、增味剂、乳化剂、防腐剂等等中的任一种。As used herein, the term "pharmaceutically acceptable" refers to an allergic or similar reaction that is physiologically compatible after administration to a human and does not cause a gastrointestinal disorder, such as dizziness. The additive may be any one of an excipient, a disintegrant, a binder, a lubricant, a suspending agent, a stabilizer, and the like. Examples of the excipient include lactose, mannitol, isomalt, microcrystalline cellulose, silicified microcrystalline cellulose, powdered cellulose, and the like. Examples of the disintegrant include low-substituted hydroxypropylcellulose, crospovidone, sodium starch glycolate, croscarmellose sodium, starch, and the like. Examples of the binder include hydroxypropylcellulose, hypromellose, povidone, copovidone, pregelatinized starch, and the like. Examples of the lubricant include stearic acid, magnesium stearate, sodium fumarate, and the like; examples of the wetting agent include polyoxyethylene sorbitan fatty acid ester, poloxamer, polyoxyethylene hydrazine Sesame oil derivatives and so on. Examples of suspending agents include hypromellose, hydroxypropylcellulose, povidone, copovidone, sodium carboxymethylcellulose, methylcellulose, and the like. Examples of the stabilizer include citric acid, fumaric acid, succinic acid, and the like. Further, the pharmaceutical composition of the present invention may further comprise any one of an anti-coagulant, a flavor enhancer, an emulsifier, a preservative, and the like.
本发明所述的药物组合物可以为片剂(包括糖衣片剂、膜包衣片剂、舌下片剂、口腔崩解片、口腔片剂等等)、丸剂、粉剂、颗粒剂、胶囊剂(包括软胶囊、微胶囊)、锭剂、糖浆剂、液体、乳剂、混悬剂、控制释放制剂(例如，瞬时释放制剂、缓释制剂、缓释微囊)、气雾剂、膜剂(例如，口服崩解膜剂、口腔粘膜-粘附膜剂)、注射剂(例如，皮下注射、静脉注射、肌内注射、腹膜内注射)、静脉滴注剂、透皮吸收制剂、软膏剂、洗剂、粘附制剂、栓剂(例如，直肠栓剂、阴道栓剂)、小药丸、鼻制剂、肺制剂(吸入剂)、眼睛滴剂等等、口服或胃肠外制剂(例如，静脉内、肌内、皮下、器官内、鼻内、皮内、滴注、脑内、直肠内等给药形式、给药至肿瘤的附近和直接给药至病变处)。优选地，所述的药物组合物为注射剂。The pharmaceutical composition of the present invention may be a tablet (including a sugar-coated tablet, a film-coated tablet, a sublingual tablet, an orally disintegrating tablet, an oral tablet, etc.), a pill, a powder, a granule, a capsule. (including soft capsules, microcapsules), lozenges, syrups, liquids, emulsions, suspensions, controlled release preparations (for example, transient release preparations, sustained release preparations, sustained release microcapsules), aerosols, films ( For example, an oral disintegrating film, an oral mucosa-adhesive film agent, an injection (for example, subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection), an intravenous drip, a transdermal absorption preparation, an ointment, a wash Agent, adhesive preparation, suppository (for example, rectal suppository, vaginal suppository), small pill, nasal preparation, lung preparation (inhalation), eye drops, etc., oral or parenteral preparation (for example, intravenous, intramuscular) , subcutaneous, intra-organ, intranasal, intradermal, instillation, intracerebral, intrarectal, etc., administered to the vicinity of the tumor and directly administered to the lesion). Preferably, the pharmaceutical composition is an injection.
本发明所述的药学上可接受的辅料优选为药学上可接受的注射剂辅料，例如等渗的无菌盐溶液(磷酸二氢钠、磷酸氢二钠、氯化钠、氯化钾、氯化钙、氯化镁等，或上述盐的混合物)，或所述药物组合物是干燥的例如是冷冻干燥的组合物，其适当地通过加入无菌水或生理盐水形成可注射溶质。The pharmaceutically acceptable excipients of the present invention are preferably pharmaceutically acceptable injectable excipients, such as isotonic sterile saline solutions (sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, potassium chloride, chlorination). Calcium, magnesium chloride, or the like, or a mixture of the above salts, or the pharmaceutical composition is a dry, for example, freeze-dried composition, which is suitably formed into an injectable solute by the addition of sterile water or physiological saline.
本发明另一方面还提供一种上述连接子化合物、聚乙二醇-连接子结合物在制备聚乙二醇-连接子结合物衍生物中的应用。Another aspect of the present invention also provides the use of the above linker compound, polyethylene glycol-linker conjugate, in the preparation of a polyethylene glycol-linker conjugate derivative.
优选地，所述的衍生物为活性酯，更优选为活性碳酸酯。Preferably, the derivative is an active ester, more preferably an activated carbonate.
本发明另一方面还提供一种上述连接子化合物、聚乙二醇-连接子结合物及其衍生物在修饰药物中的应用。Another aspect of the present invention also provides the use of the above-described linker compound, polyethylene glycol-linker conjugate and derivatives thereof in modifying a medicament.
优选的，所述的应用为制备聚乙二醇-连接子-药物结合物；所述药物具有本发明上述定义。Preferably, the use is for the preparation of a polyethylene glycol-linker-drug conjugate; the medicament having the above definition of the invention.
本发明另一方面还提供一种上述连接子化合物、聚乙二醇-连接子结合物及其衍生物、聚乙二醇-连接子-药物结合物及其药学上可接受的盐、异构体、前药或溶剂化物、药物组合物在制备预防和/或治疗疾病的药物中的应用。In another aspect, the present invention provides a linker compound, a polyethylene glycol-linker conjugate and a derivative thereof, a polyethylene glycol-linker-drug conjugate, a pharmaceutically acceptable salt thereof, and a heterogeneous Use of a body, prodrug or solvate, pharmaceutical composition for the preparation of a medicament for the prevention and/or treatment of a disease.
优选地，所述的疾病为肿瘤、自身免疫疾病、病毒性疾病或细菌性疾病。Preferably, the disease is a tumor, an autoimmune disease, a viral disease or a bacterial disease.
优选地，所述肿瘤疾病包括肾细胞癌、黑色素瘤、恶性血管内皮细胞瘤、皮肤T细胞瘤、卵巢癌、乳腺癌、膀胱癌、肺癌、神经胶质瘤、神经母细胞瘤、肝癌、毛细胞白血病、髓样母细胞白血病、结肠癌、癌性胸腹腔积液或非霍奇金淋巴瘤等。Preferably, the tumor disease comprises renal cell carcinoma, melanoma, malignant vascular endothelial cell tumor, cutaneous T cell tumor, ovarian cancer, breast cancer, bladder cancer, lung cancer, glioma, neuroblastoma, liver cancer, hair Cell leukemia, myeloid leukemia, colon cancer, cancerous pleural effusion or non-Hodgkin's lymphoma.
优选地，所述自身免疫疾病包括类风湿关节炎、系统性红斑狼疮和干燥综合征。Preferably, the autoimmune disease comprises rheumatoid arthritis, systemic lupus erythematosus and Sjogren's syndrome.
优选地，所述病毒包括肝炎病毒、乳头状病毒、HSV、HIV、EBv、冠状病毒和流感病毒等，更优选为肝炎病毒，如HBV或HCV。Preferably, the virus comprises hepatitis virus, papillomavirus, HSV, HIV, EBv, coronavirus and influenza virus, etc., more preferably hepatitis virus such as HBV or HCV.
优选地，所述细菌性疾病如麻风病、肺结核等。Preferably, the bacterial disease is leprosy, tuberculosis or the like.
优选地，所述应用为本发明所述的聚乙二醇-连接子-IL-2结合物在制备增强手术、放疗或化疗后的肿瘤患者的机体免疫功能的药物中的应用。Preferably, the application is the use of the polyethylene glycol-linker-IL-2 conjugate of the present invention in the preparation of a medicament for enhancing the immune function of a tumor patient after surgery, radiation therapy or chemotherapy.
本发明另一方面还提供一种向一名个体施用上述药物组合物的方法。Another aspect of the invention also provides a method of administering the above pharmaceutical composition to an individual.
本发明提供的连接子化合物及其与聚乙二醇的结合物及其衍生物，可用于修饰药物，且修饰反应简单，容易进行，反应产率较高，修饰药物的适用范围较宽。本发明的提供的多肽和蛋白类药物通过非肽连接子与聚乙二醇的结合物，特别是一种白介素(如白介素2)通过非肽连接子与聚乙二醇的结合物，药物可以从结合物结构中降解分离，可实现缓释和控释，降低给药频率，大大提高药物的生物利用度和病人的依从性。特别是，本发明的发明人对结合物的偶合度进行了更深入的研究，得到偶联度明确的结合物或其混合物，有利于后续药效的优化和药理的研究。The linker compound provided by the invention and the combination thereof with polyethylene glycol and the derivative thereof can be used for modifying a drug, and the modification reaction is simple, easy to carry out, the reaction yield is high, and the modified drug has a wide application range. The polypeptide and protein drug provided by the invention can be combined with a polyethylene glycol by a non-peptide linker, especially an interleukin (such as interleukin 2), and the drug can be combined with a polyethylene glycol. Degradation and separation from the structure of the conjugate can achieve sustained release and controlled release, reduce the frequency of administration, and greatly improve the bioavailability of the drug and patient compliance. In particular, the inventors of the present invention conducted a more in-depth study on the degree of coupling of the conjugate, and obtained a conjugate having a well-coupled degree or a mixture thereof, which is advantageous for optimization of subsequent effects and pharmacological studies.
附图说明DRAWINGS
图1所示为实施例8中各反应组制备的mPEG-L5-rhIL-2(20K)的RP-HPLC色谱图，检测波长：214nm。从上到下分别为：1-反应1组，2-反应2组，3-反应3组，4-反应4组，5-反应5组，6-反应6组。Fig. 1 is a RP-HPLC chromatogram of mPEG-L5-rhIL-2 (20K) prepared in each reaction group of Example 8, and the detection wavelength was 214 nm. From top to bottom are: 1-reaction 1 group, 2-reaction 2 group, 3-reaction 3 group, 4-reaction 4 group, 5-reaction 5 group, 6-reaction group 6 group.
图2所示为实施例8中反应3组制备的mPEG-L5-rhIL-2(20K)的SEC-MALS谱图。Figure 2 is a SEC-MALS spectrum of mPEG-L5-rhIL-2 (20K) prepared in the reaction group 3 of Example 8.
图3所示为实施例8中反应3组制备的mPEG-L5-rhIL-2(20K)经阳离子交换层析后的色谱图。Figure 3 is a chromatogram of mPEG-L5-rhIL-2 (20K) prepared by the reaction of Group 3 in Example 8 after cation exchange chromatography.
图4所示为实施例8中反应3组制备的mPEG-L5-rhIL-2(20K)纯化后穿透峰及各分段收集偶联物的RP-HPLC色谱图。从上到下分别为：1-穿透，2-A8，3-A7，4-A6，5-A5，6-A4，7-A3。Figure 4 is a RP-HPLC chromatogram of the purified peak of the mPEG-L5-rhIL-2 (20K) prepared in the reaction group 3 of Example 8 and the conjugates collected in each fraction. From top to bottom are: 1-penetration, 2-A8, 3-A7, 4-A6, 5-A5, 6-A4, 7-A3.
图5所示为实施例14得到的药效学研究实验结果。Figure 5 shows the results of the pharmacodynamic study obtained in Example 14.
图6所示为实施例23得到的药效学研究实验结果。Figure 6 shows the results of a pharmacodynamic study obtained in Example 23.
具体实施方式Detailed ways
除非另有定义，本发明中所使用的所有的技术和科学术语具有与本发明涉及领域的技术人员通常理解的相同的含义，如，“多肽和蛋白类药物”是指用于预防、治疗和诊断的多肽和蛋白质类物质，其中，多肽是α-氨基酸以肽键连接在一起形成的化合物，也可以是蛋白质水解的中间产物；N条多肽链按一定的空间结构缠绕纠结构成蛋白质。多肽和蛋白类药物按药物结构分类可分为：氨基酸及其衍生物类药物、多肽和蛋白质类药物、酶和辅酶类药物、核酸及其降解物和衍生物类药物、糖类药物、脂类药物、细胞生长因子和其他生物制品类药物。Unless defined otherwise, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art to which the invention relates, such as "polypeptide and protein drugs" means for prevention, treatment, and Diagnosed polypeptides and proteinaceous materials, wherein the polypeptide is a compound formed by linking alpha-amino acids with peptide bonds, or may be an intermediate product of protein hydrolysis; N polypeptide chains are entangled into a protein according to a certain spatial structure. Peptides and protein drugs can be classified according to their drug structure: amino acids and their derivatives, peptides and protein drugs, enzymes and coenzymes, nucleic acids and their degradants and derivatives, sugars, lipids. Drugs, cell growth factors and other biological products.
本发明中所述的IL-2可以是天然的、重组蛋白(如重组人白介素2)或同样具有天然IL-2功能的突变体(如“重组人白介素-2(IL-2)突变体克隆及在巴斯德毕赤酵母系统中的表达与纯化”，刘堰，博士学位论文，中所述的“IL-2-C125A/L18M/L19S”)，也包括通过组织培养、蛋白质合成、细胞培养(天然、重组细胞或突变体)方法得到的产品。天然、重组IL-2或突变体的提取和分离方法是本领域技术人员所熟知的。The IL-2 described in the present invention may be a natural, recombinant protein (e.g., recombinant human interleukin 2) or a mutant having the same function as a native IL-2 (e.g., "recombinant human interleukin-2 (IL-2) mutant clone. And expression and purification in the Pichia pastoris system", Liu Wei, Ph.D. thesis, "IL-2-C125A/L18M/L19S"), also includes tissue culture, protein synthesis, cells Products obtained by culturing (natural, recombinant cells or mutants) methods. Methods for the extraction and isolation of natural, recombinant IL-2 or mutants are well known to those skilled in the art.
本发明中所述的英文缩写及其代表含义如下：The English abbreviations and their representative meanings described in the present invention are as follows:
IL-2：白介素2；rhIL-2：重组人白介素2；HSV：单纯孢疹病毒；HIV：人类免疫缺陷病毒；HBV：乙型肝炎病毒；HCV：丙型肝炎病毒；EBv：人类孢疹病毒4型。IL-2: interleukin 2; rhIL-2: recombinant human interleukin 2; HSV: simple herpesvirus; HIV: human immunodeficiency virus; HBV: hepatitis B virus; HCV: hepatitis C virus; EBv: human herpesvirus Type 4.
下面将结合本发明实施例，对本发明的技术方案进行清楚、完整地描述，显然，所描述的实施例仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。The technical solutions of the present invention will be described clearly and completely in conjunction with the embodiments of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative efforts are within the scope of the present invention.
本发明所用的化合物原料可以商购，也可以根据公开的制备方法进行制备，其并不限制本发明的范围。The starting materials of the compounds used in the present invention are either commercially available or can be prepared according to the disclosed preparation methods without limiting the scope of the invention.
实施例中所用的聚乙二醇及其衍生物由北京键凯科技股份有限公司提供，除非指明，分子量均为20K。其他为市售试剂。The polyethylene glycol and its derivatives used in the examples were supplied by Beijing Key Kai Technology Co., Ltd., and unless otherwise specified, the molecular weight was 20K. Others are commercially available reagents.
实施例1：连接链(L)的合成Example 1: Synthesis of Linker Chain (L)
Figure PCTCN2018106972-appb-000068
Figure PCTCN2018106972-appb-000068
将BOC-氨基酸(92.2mmol)和N,N-二环己基碳二亚胺(DCC，23.8g，115.3mmol)加到二氯甲烷(500mL)中，冰水浴冷却，再加入对羟基苯甲醇(11.4g，92.2mmol)，加完后撤去冰浴，室温反应过夜。过滤，滤饼用乙酸乙酯洗涤，滤液蒸干得粗品，柱层析纯化后得到产物1。BOC-amino acid (92.2 mmol) and N,N-dicyclohexylcarbodiimide (DCC, 23.8 g, 115.3 mmol) were added to dichloromethane (500 mL), cooled in ice water, and then p-hydroxybenzyl alcohol ( 11.4 g, 92.2 mmol), after the addition was completed, the ice bath was removed and allowed to react at room temperature overnight. Filtration, the filter cake was washed with ethyl acetate, and the filtrate was evaporated to dryness.
1a:19.7g，收率76.0％。 1H NMR:(CDCl 3)：8.75(s,1H),7.22(d,2H),7.05(d,2H),4.87(s,2H),3.74(s,2H),1.52(s,9H)。 1a: 19.7 g, yield 76.0%. 1 H NMR: (CDCl 3 ): 8.75 (s, 1H), 7.22 (d, 2H), 7.05 (d, 2H), 4.87 (s, 2H), 3.74 (s, 2H), 1.52 (s, 9H) .
1b:20.3g，收率74.8％。 1H NMR:(CDCl 3)：8.74(s,1H),7.21(d,2H),7.05(d,2H),4.88(s,2H),3.77(m,1H),1.51(s,9H),1.27(d,3H)。 1b: 20.3 g, yield 74.8%. 1 H NMR: (CDCl 3 ): 8.74 (s, 1H), 7.21 (d, 2H), 7.05 (d, 2H), 4.88 (s, 2H), 3.77 (m, 1H), 1.51 (s, 9H) , 1.27 (d, 3H).
1c:21.6g，收率72.5％。 1H NMR:(CDCl 3)：8.75(s,1H),7.22(d,2H),7.05(d,2H),4.87(s,2H),3.61(d,1H),2,82(m,1H),1.52(s,9H),1.06(d,6H)。 1c: 21.6 g, yield 72.5%. 1 H NMR: (CDCl 3 ): 8.75 (s, 1H), 7.22 (d, 2H), 7.05 (d, 2H), 4.87 (s, 2H), 3.61 (d, 1H), 2, 82 (m, 1H), 1.52 (s, 9H), 1.06 (d, 6H).
将化合物1(39.1mmol)溶于二氯甲烷(250mL)中，加入三氟乙酸(50mL)，加完后室温搅拌过夜，浓缩，残分中加入二氯甲烷，再蒸干，反复三次，最后加乙醚沉淀，过滤，得产物L。Compound 1 (39.1 mmol) was dissolved in dichloromethane (250 mL), trifluoroacetic acid (50 mL) was added, and the mixture was stirred at room temperature overnight, concentrated, dichloromethane was added to the residue, and evaporated to dryness. Precipitate with diethyl ether and filter to give the product L.
L1:11.1g，收率96.7％。L1:11.1g, yield 96.7%.
L2:11.6g，收率97.1％。L2: 11.6 g, yield 97.1%.
L3:12.7g，收率96.3％。L3: 12.7 g, yield 96.3%.
实施例2：单甲氧基聚乙二醇乙酸与连接链的结合物(mPEG-L-40K)的合成Example 2: Synthesis of a combination of monomethoxypolyethylene glycol acetic acid and a linking chain (mPEG-L-40K)
Figure PCTCN2018106972-appb-000069
Figure PCTCN2018106972-appb-000069
将单甲氧基聚乙二醇-乙酸(mPEG-CM，40K，5g，0.125mmol)、化合物L(0.25mmol，实施例1制备)和1-羟基苯并三氮唑(HOBt，16.9mg，0.125mmol)加到反应瓶中，用二氯甲烷溶解，再加入二异丙基乙基胺(45.2mg，0.35mmol)，搅拌均匀，冰浴冷却后分批加入(EDCI，47.9mg，0.25mmol)，加完后体系自然升到室温，反应过夜。次日浓缩后残分用异丙醇结晶，抽滤，干燥后得产品mPEG-L。Monomethoxy polyethylene glycol-acetic acid (mPEG-CM, 40K, 5g, 0.125mmol), compound L (0.25mmol, prepared in Example 1) and 1-hydroxybenzotriazole (HOBt, 16.9 mg, 0.125 mmol) was added to the reaction flask, dissolved in dichloromethane, then diisopropylethylamine (45.2 mg, 0.35 mmol) was added, stirred well, cooled in ice-bath and then added in portions (EDCI, 47.9 mg, 0.25 mmol) After the addition, the system naturally rose to room temperature and reacted overnight. After concentration on the next day, the residue was crystallized from isopropanol, suction filtered, and dried to give the product mPEG-L.
mPEG-L1(40K):4.6g，收率92.4％。mPEG-L1 (40K): 4.6 g, yield 92.4%.
mPEG-L2(40K):4.5g，收率90.8％。mPEG-L2 (40K): 4.5 g, yield 90.8%.
mPEG-L3(40K):4.7g，收率93.7％。mPEG-L3 (40K): 4.7 g, yield 93.7%.
实施例3：连接链L5的制备Example 3: Preparation of Linker L5
连接链L5的合成路线如下所示：The synthetic route of the link L5 is as follows:
Figure PCTCN2018106972-appb-000070
Figure PCTCN2018106972-appb-000070
化合物(2)的合成：Synthesis of Compound (2):
3,4-二羟基苯甲醛(10g，72.5mmol)溶于乙腈(150mL)中，加入碳酸氢钠(8g，94.3mmol)，升温至60℃，加入溴苄(12.4g，72.5mmol)，然后升温至80℃搅拌过夜。浓缩除去乙腈，残分中加入10％盐酸水溶液(200mL)，用乙酸乙酯萃取(150mL*3)，合并后干燥，过滤，浓缩，残分用柱层析纯化得灰白色固体10g(收率60％)。 1HNMR:(CDCl 3)：δ9.82(s,1H),7.48-7.40(m,7H),7.05(m,1H),6.02(s,1H),5.21(s,2H)。 3,4-Dihydroxybenzaldehyde (10 g, 72.5 mmol) was dissolved in acetonitrile (150 mL), sodium bicarbonate (8 g, 94.3 mmol) was added, warmed to 60 ° C, and benzyl bromide (12.4 g, 72.5 mmol) was added, then The temperature was raised to 80 ° C and stirred overnight. The acetonitrile was concentrated to remove the residue. EtOAc (EtOAc) (EtOAc) %). 1 H NMR: (CDCl 3 ): δ 9.82 (s, 1H), 7.48-7.40 (m, 7H), 7.05 (m, 1H), 6.02 (s, 1H), 5.21. (s, 2H).
化合物(3)的合成：Synthesis of Compound (3):
化合物(2)(5g，21.9mmol)溶于DMF(80mL)中，加入碳酸钾(7.6g，54.75mmol)，碘化钾(0.73g，4.38mmol)，搅拌10分钟，加入ATN-1(9g，28.47mmol)，升至70℃搅拌过夜。后处理，往反应液中加入氯化铵饱和溶液(400mL)，用乙酸乙酯萃取(150mL*3)，合并后干燥，过滤，浓缩，残分用柱层析纯化得白色固体5g(收率61％)。 1H NMR:(CDCl 3)：δ9.85(s,1H),7.47-7.36(m,7H),7.03(m,1H),5.24(s,2H),5.07(s,1H),4.17(m,2H),3.59(m,2H),1.46(s,9H)。 Compound (2) (5 g, 21.9 mmol) was dissolved in DMF (80 mL), EtOAc (EtOAc, EtOAc, EtOAc, EtOAc Methyl), stirred to 70 ° C and stirred overnight. After work-up, a saturated solution of ammonium chloride (400 mL) was added to the reaction mixture, and the mixture was combined with ethyl acetate (150 mL*3), dried, filtered, concentrated, and the residue was purified by column chromatography 61%). 1 H NMR: (CDCl 3 ): δ 9.85 (s, 1H), 7.47-7.36 (m, 7H), 7.03 (m, 1H), 5.24 (s, 2H), 5.07 (s, 1H), 4.17 ( m, 2H), 3.59 (m, 2H), 1.46 (s, 9H).
化合物(4)的合成：Synthesis of Compound (4):
将化合物(3)(5g，13.5mmol)溶于四氢呋喃(100mL)中，室温加入硼氢化钠(0.77g，20.25mmol)，室温搅拌2小时后冷却到0℃，加入醋酸(2mL)淬灭反应，减压浓缩除去溶剂，残分用柱层析纯化，得无色油状物4g(收率80％)。 1H NMR:(DMSO-d6)：δ7.38-7.05(m,8H),6.80(m,1H),5.09(s,2H),5.06(m,1H),4.40(m,2H),3.98(m,2H),3.31(m,2H),1.38(s,9H)。 The compound (3) (5 g, 13.5 mmol) was dissolved in tetrahydrofuran (100 mL), sodium borohydride (0.77 g, 20.25 mmol) was added at room temperature, stirred at room temperature for 2 hours, then cooled to 0 ° C, and then quenched with acetic acid (2 mL) The solvent was concentrated under reduced pressure. 1 H NMR: (DMSO-d6): δ 7.38-7.05 (m, 8H), 6.80 (m, 1H), 5.09 (s, 2H), 5.06 (m, 1H), 4.40 (m, 2H), 3.98 (m, 2H), 3.31 (m, 2H), 1.38 (s, 9H).
化合物(5)的合成：Synthesis of Compound (5):
化合物(4)(1g，2.7mmol)溶于甲醇(8mL)中，加入Pd/C(10％，0.3g)，再通入氢气反应过夜。过滤，滤液浓缩，残分用柱层析纯化，得产品0.6g(收率78％). 1H NMR:(DMSO-d6)：δ8.37(s,1H),7.09(s,1H),6.71(s,1H),6.65(d,1H),6.56(d,1H),4.45(s,2H),3.89(m,2H),3.31(m,2H),1.38(s,9H)。 Compound (4) (1 g, 2.7 mmol) was dissolved in methanol (8 mL). Pd/C (10%, 0.3 g) The filtrate was concentrated and residue purified by column chromatography minutes, to give 0.6 g of product (yield 78%) 1 H NMR:. (DMSO-d6): δ8.37 (s, 1H), 7.09 (s, 1H), 6.71 (s, 1H), 6.65 (d, 1H), 6.56 (d, 1H), 4.45 (s, 2H), 3.89 (m, 2H), 3.31 (m, 2H), 1.38 (s, 9H).
化合物(6)的合成：Synthesis of Compound (6):
将化合物(5)(0.6g，2.1mmol)溶于丙酮(7mL)，加入碳酸钾(0.58g，4.2mmol)，体系冷却到0℃，加入醋酸酐(235mg，2.3mmol)，缓慢升至室温反应3小时。后处理，加入氯化铵水溶液(30mL)，乙酸乙酯萃取，合并干燥浓缩，过柱得产物0.5g(收率73％)。 1H NMR:(DMSO-d6)：δ7.12(s,1H),6.89(s,1H),6.65(d,1H),6.56(d,1H),4.45(s,2H),3.89(m,2H),3.31(m,2H),2.14(s,3H),1.38(s,9H)。 Compound (5) (0.6 g, 2.1 mmol) was dissolved in acetone (7 mL), potassium carbonate (0.58 g, 4.2 mmol) was added, the system was cooled to 0 ° C, and acetic anhydride (235 mg, 2.3 mmol) was added and slowly warmed to room temperature. Reaction for 3 hours. After work-up, an aqueous solution of ammonium chloride (30 mL) was added, and ethyl acetate was evaporated, and then concentrated, dried and concentrated to give a product (yield: 73%). 1 H NMR: (DMSO-d6): δ 7.12 (s, 1H), 6.89 (s, 1H), 6.65 (d, 1H), 6.56 (d, 1H), 4.45 (s, 2H), 3.89 (m) , 2H), 3.31 (m, 2H), 2.14 (s, 3H), 1.38 (s, 9H).
化合物(L5)的合成：Synthesis of compound (L5):
将化合物(6)(0.5g，1.5mmol)溶于二氯甲烷(7mL)中，加入三氟乙酸(4mL),室温搅拌1小时。浓缩除去溶剂，残分中加入乙醚，有固体析出，过滤，干燥得固体产物0.3g，收率86％。 1H NMR:(DMSO-d6)：δ8.30(s,1H),6.89(s,1H),6.65(d,1H),6.56(d,1H),4.45(s,2H),3.85(m,2H),3.29(m,2H),2.12(s,3H)。 Compound (6) (0.5 g, 1.5 mmol) was dissolved in dichloromethane (7 mL). The solvent was removed by concentration, diethyl ether was added to the residue, and a solid was precipitated, which was filtered and dried to give a solid product (0.3 g). 1 H NMR: (DMSO-d6): δ 8.30 (s, 1H), 6.89 (s, 1H), 6.65 (d, 1H), 6.56 (d, 1H), 4.45 (s, 2H), 3.85 (m) , 2H), 3.29 (m, 2H), 2.12 (s, 3H).
实施例4：单甲氧基聚乙二醇乙酸与连接链的结合物(mPEG-L5-NHS-20K)的合成Example 4: Synthesis of a combination of monomethoxypolyethylene glycol acetic acid and a linking chain (mPEG-L5-NHS-20K)
Figure PCTCN2018106972-appb-000071
Figure PCTCN2018106972-appb-000071
mPEG-L5的合成：Synthesis of mPEG-L5:
将mPEG(20K)-CM-NHS(2g，0.1mmol)溶于无水二氯甲烷中，低温冷却至0℃，加入DIPEA(78mg，0.6mmol)，然后加入化合物L5，缓慢升至室温搅拌过夜。反应液浓缩后残分用异丙醇结晶，抽滤，干燥后得产品mPEG-L5(1.8g，90％)。 1H NMR:(DMSO-d6)：δ8.10(s,1H),6.85(s,1H),6.72(d,1H),6.65(d,1H),5.34(s,2H),4.20(m,2H),3.65(m,1800H),3.51(m,2H),3.24(s,3H),2.78(m,4H),2.12(s,3H)。 mPEG(20K)-CM-NHS (2 g, 0.1 mmol) was dissolved in anhydrous dichloromethane, chilled to 0 ° C, then DIPEA (78 mg, 0.6 mmol) was added, then compound L5 was added and slowly warmed to room temperature overnight. . After the reaction mixture was concentrated, the residue was crystallised from isopropyl alcohol, filtered, and dried to give the product m.s. 1 H NMR: (DMSO-d6): δ 8.10 (s, 1H), 6.85 (s, 1H), 6.72 (d, 1H), 6.65 (d, 1H), 5.34 (s, 2H), 4.20 (m) , 2H), 3.65 (m, 1800H), 3.51 (m, 2H), 3.24 (s, 3H), 2.78 (m, 4H), 2.12 (s, 3H).
mPEG-L5-NHS的合成：Synthesis of mPEG-L5-NHS:
将化合物mPEG-L5(1g，0.05mmol)加到反应瓶中，用二氯甲烷(6mL)溶解，N 2保护下冷却，加入琥珀酰亚胺碳酸酯(19.0mg，0.075mmol)，搅拌溶解后再加入DIPEA(12.9mg，0.1mmol)，加完后撤去冷浴，室温反应过夜。反应液浓缩，残分用异丙醇结晶，得产品mPEG-L5-NHS。 1H NMR:(DMSO-d6)：δ8.10(s,1H),6.85(s,1H),6.72(d,1H),6.65(d,1H),4.65(s,2H),4.15(m,2H),3.65(m,1800H),3.29(m,2H),3.24(s,3H), The compound mPEG-L5 (1 g, 0.05 mmol) was added to a reaction flask, dissolved in dichloromethane (6 mL), cooled under N 2 and then succinimide carbonate (19.0 mg, 0.075 mmol) Further, DIPEA (12.9 mg, 0.1 mmol) was added, and after cooling, the cold bath was removed and allowed to react at room temperature overnight. The reaction solution was concentrated, and the residue was crystallized from isopropyl alcohol to give the product mPEG-L5-NHS. 1 H NMR: (DMSO-d6): δ 8.10 (s, 1H), 6.85 (s, 1H), 6.72 (d, 1H), 6.65 (d, 1H), 4.65 (s, 2H), 4.15 (m) , 2H), 3.65 (m, 1800H), 3.29 (m, 2H), 3.24 (s, 3H),
2.12(s,3H)。2.12 (s, 3H).
实施例5：Y-PEG-L5-NHS-20K的合成Example 5: Synthesis of Y-PEG-L5-NHS-20K
Figure PCTCN2018106972-appb-000072
Figure PCTCN2018106972-appb-000072
其制备方法参考实施例4。The preparation method is referred to Example 4.
实施例6：U-PEG-L5-NHS-20K的合成Example 6: Synthesis of U-PEG-L5-NHS-20K
Figure PCTCN2018106972-appb-000073
Figure PCTCN2018106972-appb-000073
其制备方法参考实施例4。The preparation method is referred to Example 4.
实施例7：8arm-PEG-L5-NHS-20K的合成Example 7: Synthesis of 8arm-PEG-L5-NHS-20K
Figure PCTCN2018106972-appb-000074
Figure PCTCN2018106972-appb-000074
其制备方法参考实施例4。The preparation method is referred to Example 4.
实施例8：mPEG-L5-rhIL-2(20K)的合成Example 8: Synthesis of mPEG-L5-rhIL-2 (20K)
Figure PCTCN2018106972-appb-000075
Figure PCTCN2018106972-appb-000075
在氮气吹扫下，将在-20℃下储存的mPEG-L5-NHS(20K，实施例4制备)升温至室温。在DMSO中制备mPEG-L5-NHS(20K)储备溶液(200mg/mL)，加入rhIL-2溶液中(反应分组、反应条件如反应物mPEG-L5-NHS：rhIL-2摩尔比(以下简称为PEG：rhIL-2摩尔比)及具体反应pH如表1所示)。混合物中rhIL-2的终浓度是0.5mg/mL。将碳酸氢钠缓冲液(1M，pH9.0)分别添加至混合物以达到20mM的终浓度，室温下反应2h以提供缀合物。2h后，分别添加1M甘氨酸(pH6.0)至终浓度100mM来终止反应。mPEG-L5-NHS (20K, prepared in Example 4) stored at -20 ° C was warmed to room temperature under a nitrogen purge. Prepare mPEG-L5-NHS (20K) stock solution (200mg/mL) in DMSO and add it to rhIL-2 solution (reaction grouping, reaction conditions such as reactant mPEG-L5-NHS:rhIL-2 molar ratio (hereinafter referred to as PEG: rhIL-2 molar ratio) and specific reaction pH are shown in Table 1. The final concentration of rhIL-2 in the mixture was 0.5 mg/mL. Sodium bicarbonate buffer (1 M, pH 9.0) was added to the mixture separately to reach a final concentration of 20 mM, and reacted at room temperature for 2 h to provide a conjugate. After 2 h, 1 M glycine (pH 6.0) was added to a final concentration of 100 mM to terminate the reaction.
对终止后的反应体系开展RP-HPLC测定不同反应组的偶联情况，根据偶联物的迁移情况(左移代表偶联度高)分析评价偶联效率和偶联度，RP-HPLC谱图如图1所示，由图1可知，偶联度及偶联效率均为反应3组〉反应4组〉反应5组〉反应2组〉反应6组〉反应1组。进一步开展SEC+MALS分析反应3组，评价偶联度，结果如图2所示。RP-HPLC was used to determine the coupling of different reaction groups after the termination of the reaction system. The coupling efficiency and coupling degree were evaluated according to the migration of the conjugate (the left shift represents the high coupling degree). RP-HPLC spectrum As shown in Fig. 1, it can be seen from Fig. 1 that the coupling degree and the coupling efficiency are all three groups of reactions: 4 groups of reactions > 5 groups of reactions > 2 groups of reactions > 6 groups of reactions > 1 group of reactions. Further, three groups of SEC+MALS analysis reactions were carried out, and the coupling degree was evaluated. The results are shown in Fig. 2.
本实施例制备的偶联物mPEG-L5-rhIL-2(20K)的偶联度结果如表1所示。The coupling degree results of the conjugate mPEG-L5-rhIL-2 (20K) prepared in this example are shown in Table 1.
表1：实施例8制备的偶联物的SEC-MALS偶联度结果表Table 1: Results of SEC-MALS coupling degree of the conjugate prepared in Example 8
反应1组 Reaction group 1 反应2组 Reaction 2 反应3组 Reaction 3 groups 反应4组Reaction 4 groups 反应5组Reaction 5 groups 反应6组Reaction 6 groups
投料摩尔比Feeding molar ratio 10：110:1 20：120:1 30：130:1 30：130:1 30：130:1 30：130:1
反应pHReaction pH 9.89.8 9.89.8 9.89.8 8.08.0 7.27.2 6.06.0
偶联度 Coupling degree 1,2,31,2,3 1,2,31,2,3 4～74~7 3～53~5 1,2,31,2,3 1,2,31,2,3
比例proportion 〉90％〉90% 〉90％〉90% 〉90％〉90% 〉90％〉90% 〉70％〉70% 〉80％〉80%
上表所获产品可以分为三类：偶联度为4-7的mPEG-L5-rhIL-2、偶联度为3-5的mPEG-L5-rhIL-2和偶联度为1-3的mPEG-L5-rhIL-2，最优含量可控制在大于90％。The products obtained in the above table can be divided into three categories: mPEG-L5-rhIL-2 with a coupling degree of 4-7, mPEG-L5-rhIL-2 with a coupling degree of 3-5, and a coupling degree of 1-3. The optimal content of mPEG-L5-rhIL-2 can be controlled to be greater than 90%.
接着分别将终止反应的混合物用纯化水稀释以提供低于0.5mS/cm(25℃)的电导率。使用冰醋酸将pH值调节至4.0，然后进行柱层析纯化，如图3所示。结合RP-HPLC分析(图4)，PEG在穿透峰中富集，mPEG-L5-rhIL-2在0～50％B(0～0.5M NaCl梯度)中洗脱，分段收集各洗脱峰(A3～A8)，观察到偶联度由高到低分布。The reaction-terminated mixture was then separately diluted with purified water to provide a conductivity of less than 0.5 mS/cm (25 ° C). The pH was adjusted to 4.0 using glacial acetic acid and then purified by column chromatography as shown in FIG. Combined with RP-HPLC analysis (Fig. 4), PEG was enriched in the breakthrough peak, and mPEG-L5-rhIL-2 was eluted in 0-50% B (0-0.5 M NaCl gradient). Peaks (A3 to A8) were observed to have a high to low distribution.
实施例9：mPEG-L5-rhIL-2(40K)的合成Example 9: Synthesis of mPEG-L5-rhIL-2 (40K)
其制备方法参照实施例8。The preparation method is referred to in Example 8.
实施例10：Y-PEG-L5-rhIL-2(20K)的合成Example 10: Synthesis of Y-PEG-L5-rhIL-2 (20K)
Figure PCTCN2018106972-appb-000076
Figure PCTCN2018106972-appb-000076
其制备方法参照实施例8。The preparation method is referred to in Example 8.
实施例11：U-PEG-L5-rhIL-2(20K)的合成Example 11: Synthesis of U-PEG-L5-rhIL-2 (20K)
Figure PCTCN2018106972-appb-000077
Figure PCTCN2018106972-appb-000077
其制备方法参照实施例8。The preparation method is referred to in Example 8.
实施例12：8arm-PEG-L5-rhIL-2(20K)的合成Example 12: Synthesis of 8arm-PEG-L5-rhIL-2 (20K)
Figure PCTCN2018106972-appb-000078
Figure PCTCN2018106972-appb-000078
其制备方法参照实施例8。The preparation method is referred to in Example 8.
实施例13：mPEG-L1-rhIL-2(20K)的合成Example 13: Synthesis of mPEG-L1-rhIL-2 (20K)
Figure PCTCN2018106972-appb-000079
Figure PCTCN2018106972-appb-000079
在氮气吹扫下，将在-20℃下储存的mPEG-L1-NHS(20K，其制备方法参考实施例2)升温至室温。在DMSO中制备mPEG-L1-NHS(20K)储备溶液(200mg/mL)，加入rhIL-2溶液中(PEG：rIL-2摩尔比分别为1:1、3:1、10：1、30:1)。混合物中rhIL-2的终浓度是0.5mg/mL。将碳酸氢钠缓冲液(1M，pH9.0)分别添加至混合物以达到20mM的终浓度，室温下反应2h以提供缀合物。2h后，分别添加1M甘氨酸(pH6.0)至终浓度100mM来终止反应。接着分别将终止反应的混合物用纯化水稀释以提供低于0.5mS/cm(25℃)的电导率。使用冰醋酸将pH值调节至4.0，然后进行柱层析纯化。mPEG-L1-NHS (20K, prepared in the same manner as in Example 2) stored at -20 ° C was warmed to room temperature under a nitrogen purge. The mPEG-L1-NHS (20K) stock solution (200 mg/mL) was prepared in DMSO and added to the rhIL-2 solution (PEG:rIL-2 molar ratios were 1:1, 3:1, 10:1, 30: 1). The final concentration of rhIL-2 in the mixture was 0.5 mg/mL. Sodium bicarbonate buffer (1 M, pH 9.0) was added to the mixture separately to reach a final concentration of 20 mM, and reacted at room temperature for 2 h to provide a conjugate. After 2 h, 1 M glycine (pH 6.0) was added to a final concentration of 100 mM to terminate the reaction. The reaction-terminated mixture was then separately diluted with purified water to provide a conductivity of less than 0.5 mS/cm (25 ° C). The pH was adjusted to 4.0 using glacial acetic acid and then purified by column chromatography.
实施例14：经本发明修饰的IL-2的药效学研究Example 14: Pharmacodynamic study of IL-2 modified by the present invention
采用实施例8的反应3组条件进行PEG与rIL-2偶联，偶联混合物经离子交换色谱纯化，分离得到PEG与rIL-2偶联物进行药效学实验。采用C57BL/6小鼠的皮下黑色素瘤B16模型评估mPEG-L5-rhIL-2偶联物(以下简称为PEG-rhIL-2)的抑制肿瘤效果。实验过程如下：每只5-6周龄的C57BL/6小鼠在背部中部植入10 6个B16细胞。肿瘤生长至可测量尺寸后，实验动物随机分组，每组6只，以不同剂量浓度和剂量方案对小鼠施用测试化合物：rhIL-2、PEG-rhIL-2及溶剂对照。每隔一天测量体重和肿瘤体积。药效实验分组如表2所示，实验结果如表3和图5所示。 The PEG was coupled with rIL-2 using the reaction 3 conditions of Example 8. The coupled mixture was purified by ion exchange chromatography, and the conjugate of PEG and rIL-2 was isolated for pharmacodynamic experiments. The tumor suppressing effect of mPEG-L5-rhIL-2 conjugate (hereinafter abbreviated as PEG-rhIL-2) was evaluated using a subcutaneous melanoma B16 model of C57BL/6 mice. The experimental procedure was as follows: Each 5-6 week old C57BL/6 mouse was implanted with 10 6 B16 cells in the middle of the back. After tumor growth to measurable size, experimental animals were randomized into groups of 6 rats, and test compounds were administered to mice at different dose concentrations and dosage regimens: rhIL-2, PEG-rhIL-2, and solvent control. Body weight and tumor volume were measured every other day. The pharmacodynamic experiment grouping is shown in Table 2, and the experimental results are shown in Table 3 and Figure 5.
表2：药效实验分组Table 2: Group of pharmacodynamic experiments
分组Grouping 剂量浓度Dose concentration 施用途径Route of administration 频次frequency
溶剂对照组Solvent control N/AN/A IVIV 注射一次Injection once
rhIL-2组rhIL-2 group 1mg/Kg1mg/Kg IVIV 每天一次，注射5天Once a day, 5 days of injection
PEG-rhIL-2组PEG-rhIL-2 group 1mg/Kg1mg/Kg IVIV 注射一次Injection once
表3：药效实验结果Table 3: Results of pharmacodynamic experiments
Figure PCTCN2018106972-appb-000080
Figure PCTCN2018106972-appb-000080
由数据可知，给药第9天时，与溶剂对照组肿瘤体积(1260mm 3)相比，经本发明修饰的rhIL-2组肿瘤体积(310mm 3)大大减小，与未修饰的rhIL-2组肿瘤体积(650mm 3)相比，经本发明修饰后的mPEG-L5-rhIL-2组的抑瘤效果提高了27％，且其给药频次大大降低，作为抗肿瘤药物具有降低给药频率、大大提高药物的生物利用度和病人的依从性的优势。 It is seen from the data, the administration day 9, compared to the vehicle control group Tumor volume (1260mm 3), according to the present invention by a modified rhIL-2 group tumor volume (310mm 3) greatly reduced, unmodified rhIL-2 group Compared with the tumor volume (650 mm 3 ), the anti-tumor effect of the mPEG-L5-rhIL-2 group modified by the present invention was increased by 27%, and the frequency of administration was greatly reduced, and the anti-tumor drug has a reduced frequency of administration, Greatly improve the bioavailability of drugs and the advantages of patient compliance.
实施例15 单甲氧基聚乙二醇-阿霉素结合物(mPEG-L-Dox(40K))的制备Example 15 Preparation of monomethoxypolyethylene glycol-doxorubicin conjugate (mPEG-L-Dox (40K))
Figure PCTCN2018106972-appb-000081
Figure PCTCN2018106972-appb-000081
将化合物mPEG-L(0.075mmol，实施例2制备)加到反应瓶中，用二氯甲烷(30mL)溶解，N 2保护下冷却，加入琥珀酰亚胺碳酸酯(23.0mg，0.09mmol)，搅拌溶解后再加入三乙胺(10.1mg，0.1mmol)，加完后撤去冷浴，室温反应过夜。反应液浓缩，残分用异丙醇结晶，得产品mPEG-L-NHS。 Compound mPEG-L (0.075mmol, prepared in Example 2) was added to the reaction flask, was dissolved with dichloromethane (30 mL), cooled under N 2, was added succinimidyl carbonate (23.0mg, 0.09mmol), After stirring and stirring, triethylamine (10.1 mg, 0.1 mmol) was added. After the addition, the cold bath was removed and allowed to react at room temperature overnight. The reaction solution was concentrated, and the residue was crystallized from isopropyl alcohol to give the product mPEG-L-NHS.
mPEG-L1-NHS(40K):2.6g，收率88.5％。mPEG-L1-NHS (40K): 2.6 g, yield 88.5%.
mPEG-L2-NHS(40K):2.7g，收率89.2％。mPEG-L2-NHS (40K): 2.7 g, yield: 89.2%.
mPEG-L3-NHS(40K):2.6g，收率87.9％。mPEG-L3-NHS (40K): 2.6 g, yield 87.9%.
将化合物mPEG-L-NHS(0.06mmol，上步制得)溶于二氯甲烷(25mL)中，N 2保护下冷却，加入二异丙基乙基胺(12.9mg，0.1mmol)，搅拌均匀后再加入盐酸阿霉素(52.2mg，0.09mmol)，加完后室温搅拌5小时。反应液浓缩，残分用异丙醇结晶，抽滤，干燥得红色固体产品。 Compound mPEG-L-NHS (0.06mmol, prepared by the step) was dissolved in dichloromethane (25mL), the cooling under N 2, was added diisopropylethyl amine (12.9mg, 0.1mmol), stir Then, doxorubicin hydrochloride (52.2 mg, 0.09 mmol) was added, and the mixture was stirred at room temperature for 5 hours after the addition. The reaction mixture was concentrated, and the residue was crystallised from isopropyl alcohol.
mPEG-L1-Dox(40K)：2.0g，收率84.9％。 1H NMR:(DMSO-d6)：δ8.84(s,1H),8.68(S,1H),7.62(m,1H),7.53(d,1H),7.33(d,2H),7.16(d,3H),5,52(s,2H),5.12(t,1H),4.83(s,2H),4.61(s,2H),4.47(s 2H),4.32(t,1H),4.06(m,1H),3.96(m,1H),3.89(m,1H),3.65(m,3600H),3.41(m,5H),3.27(m,1H),2.28(d,2H),2.05(d,2H)。 mPEG-L1-Dox (40K): 2.0 g, yield 84.9%. 1 H NMR: (DMSO-d6): δ 8.84 (s, 1H), 8.68 (S, 1H), 7.62 (m, 1H), 7.53 (d, 1H), 7.33 (d, 2H), 7.16 (d) , 3H), 5, 52 (s, 2H), 5.12 (t, 1H), 4.83 (s, 2H), 4.61 (s, 2H), 4.47 (s 2H), 4.32 (t, 1H), 4.06 (m) , 1H), 3.96 (m, 1H), 3.89 (m, 1H), 3.65 (m, 3600H), 3.41 (m, 5H), 3.27 (m, 1H), 2.28 (d, 2H), 2.05 (d, 2H).
mPEG-L2-Dox(40K)：2.1g，收率85.7％。 1H NMR:(DMSO-d6)：δ8.82(s,1H),8.67(S,1H),7.62(m,1H),7.53(d,1H),7.33(d,2H),7.16(d,3H),5,52(s,2H),5.12(t,1H),4.83(s,2H),4.59(d,1H),4.47(s 2H),4.32(t,1H),4.06(m,1H),3.96(m,1H),3.89(m,1H),3.65(m,3600H),3.41(m,5H),3.27(m,1H),2.28(d,2H),2.05(d,2H),1.58(d,3H)。 mPEG-L2-Dox (40K): 2.1 g, yield 85.7%. 1 H NMR: (DMSO-d6): δ 8.82 (s, 1H), 8.67 (S, 1H), 7.62 (m, 1H), 7.53 (d, 1H), 7.33 (d, 2H), 7.16 (d) , 3H), 5, 52 (s, 2H), 5.12 (t, 1H), 4.83 (s, 2H), 4.59 (d, 1H), 4.47 (s 2H), 4.32 (t, 1H), 4.06 (m) , 1H), 3.96 (m, 1H), 3.89 (m, 1H), 3.65 (m, 3600H), 3.41 (m, 5H), 3.27 (m, 1H), 2.28 (d, 2H), 2.05 (d, 2H), 1.58 (d, 3H).
mPEG-L3-Dox(40K)：2.1g，收率84.6％。 1H NMR:(DMSO-d6)：δ8.83(s,1H),8.65(S,1H),7.62(m,1H),7.53(d,1H),7.33(d,2H),7.16(d,3H),5,52(s,2H),5.12(t,1H),4.83(s,2H),4.54(d,1H),4.47(s 2H),4.32(t,1H),4.06(m,1H),3.96(m,1H),3.89(m,1H),3.65(m,3600H),3.41(m,5H),3.27(m,1H),2.28(d,2H),2.05(d,2H),1.34(d,3H),1.16(d,6H)。 mPEG-L3-Dox (40K): 2.1 g, yield 84.6%. 1 H NMR: (DMSO-d6): δ 8.83 (s, 1H), 8.65 (S, 1H), 7.62 (m, 1H), 7.53 (d, 1H), 7.33 (d, 2H), 7.16 (d) , 3H), 5, 52 (s, 2H), 5.12 (t, 1H), 4.83 (s, 2H), 4.54 (d, 1H), 4.47 (s 2H), 4.32 (t, 1H), 4.06 (m) , 1H), 3.96 (m, 1H), 3.89 (m, 1H), 3.65 (m, 3600H), 3.41 (m, 5H), 3.27 (m, 1H), 2.28 (d, 2H), 2.05 (d, 2H), 1.34 (d, 3H), 1.16 (d, 6H).
实施例16 聚乙二醇乙酸与连接链L3的结合物(PEG-L3(20K))的制备Example 16 Preparation of a combination of polyethylene glycol acetic acid and a linker L3 (PEG-L3 (20K))
Figure PCTCN2018106972-appb-000082
Figure PCTCN2018106972-appb-000082
将聚乙二醇-乙酸(PEG-CM，20K，5 g，0.25mmol)、化合物L3(168.5m g，0.5mmol，实施例1制备)和1-羟基苯并三氮唑(HOBt，67.6mg，0.5mmol)加到反应瓶中，用二氯甲烷溶解，再加入二异丙基乙基胺(193.6mg，1.5mmol)，搅拌均匀，冰浴冷却后分批加入(EDCI，191.7mg，1mmol)，加完后体系自然升到室温，反应过夜。次日浓缩后残分用异丙醇结晶，抽滤，干燥后得产品PEG-L3(20K)4.8g，收率96.0％。 Polyethylene glycol - acetic acid (PEG-CM, 20K, 5 g, 0.25mmol), compound L3 (168.5m g, 0.5mmol, prepared in Example 1) and 1-hydroxybenzotriazole triazole (HOBt, 67.6mg , 0.5 mmol) was added to the reaction flask, dissolved in dichloromethane, then diisopropylethylamine (193.6 mg, 1.5 mmol) was added, stirred well, cooled in an ice bath and then added in portions (EDCI, 191.7 mg, 1 mmol) After the addition, the system naturally rose to room temperature and reacted overnight. After concentration on the next day, the residue was crystallized from isopropyl alcohol, filtered, and dried to give </RTI></RTI></RTI>
实施例17 聚乙二醇乙酸与连接链L3及阿霉素的结合物(PEG-L3-D ox(20K))的制备 Example 17 Preparation of a combination of polyethylene glycol acetic acid with a linker L3 and doxorubicin (PEG-L3-D o x (20K))
Figure PCTCN2018106972-appb-000083
Figure PCTCN2018106972-appb-000083
将化合物PEG-L3(2g，0.1mmol，实施例16制备)加到反应瓶中，用二氯甲烷(40mL)溶解，N 2保护下冷却，加入琥珀酰亚胺碳酸酯(51.2mg，0.2mmol)，搅拌溶解后再加入三乙胺(30.3mg，0.3mmol)，加完后撤去冷浴，室温反应过夜。反应液浓缩，残分用异丙醇结晶，得产品PEG-L3-NHS(20K)1.8g，收率88.5％。 Compound PEG-L3 (2g, 0.1mmol, prepared in Example 16) was added to the reaction flask, was dissolved with dichloromethane (40 mL), cooled under N 2, was added succinimidyl carbonate (51.2mg, 0.2mmol After stirring and stirring, triethylamine (30.3 mg, 0.3 mmol) was added. After the addition, the cold bath was removed, and the reaction was allowed to proceed overnight at room temperature. The reaction solution was concentrated, and the residue was crystallised from isopropyl alcohol to give the product PEG-L3-NHS (20K) 1.8 g, yield: 88.5%.
将化合物PEG-L3-NHS(1.6g，0.08mmol，上步制得)溶于二氯甲烷(30mL)中，N 2保护下冷却，加入二异丙基乙基胺(19.4mg，0.15mmol)，搅拌均匀后再加入盐酸阿霉素(69.6mg，0.12mmol)，加完后室温搅拌5小时。反应液浓缩，残分用异丙醇结晶，抽滤，干燥得红色固体产品PEG-L3-Dox(20K)1.3g，收率81.2％。 1H NMR:(DMSO-d6)：δ8.81(s,2H),8.68(S,2H),7.62(m,2H),7.53(d,2H),7.33(d,8H),7.16(d,4H),5,52(s,4H),5.12(t,2H),4.83(s,4H),4.54(d,2H),4.32(t,2H),4.06(m,2H),3.96(s,6H),3.71(m,2H),3.67(m,1800H),3.41(s,4H),3.27(m,2H),2,97(m,2H),2.39(S,4H),2.28(d,4H),2.05(d,4H),1.34(d,6H),1.16(d,12H)。 The compound PEG-L3-NHS (1.6g, 0.08mmol, prepared by the step) was dissolved in dichloromethane (30mL), N 2 protection under cooling, was added diisopropylethyl amine (19.4mg, 0.15mmol) After stirring evenly, doxorubicin hydrochloride (69.6 mg, 0.12 mmol) was added, and the mixture was stirred at room temperature for 5 hours after the addition. The reaction mixture was concentrated, and the residue was crystallised from isopropyl alcohol, filtered, and dried to give a red solid product PEG-L3-Dox (20K) 1.3 g. 1 H NMR: (DMSO-d6): δ 8.81 (s, 2H), 8.68 (S, 2H), 7.62 (m, 2H), 7.53 (d, 2H), 7.33 (d, 8H), 7.16 (d) , 4H), 5, 52 (s, 4H), 5.12 (t, 2H), 4.83 (s, 4H), 4.54 (d, 2H), 4.32 (t, 2H), 4.06 (m, 2H), 3.96 ( s,6H), 3.71 (m, 2H), 3.67 (m, 1800H), 3.41 (s, 4H), 3.27 (m, 2H), 2, 97 (m, 2H), 2.39 (S, 4H), 2.28 (d, 4H), 2.05 (d, 4H), 1.34 (d, 6H), 1.16 (d, 12H).
实施例18 4arm-PEG-L3(20K)的制备Example 18 Preparation of 4arm-PEG-L3 (20K)
Figure PCTCN2018106972-appb-000084
Figure PCTCN2018106972-appb-000084
将4arm-PEG-乙酸(20K，5g，0.25mmol)、化合物L3(674mg，2.0mmol，实施例1制备)和1-羟基苯并三氮唑(HOBt，135.1mg，1mmol)加到反应瓶中，用二氯甲烷溶解，再加入二异丙基乙基胺(129.1mg，1.0mmol)，搅拌均匀，冰浴冷却后分批加入(EDCI，191.7mg，1mmol)，加完后体系自然升到室温，反应过夜。次日浓缩后残分用异丙醇结晶，抽滤，干燥后得产品4arm-PEG-L3(20K)：4.7g，收率94.0％。4arm-PEG-acetic acid (20K, 5g, 0.25mmol), compound L3 (674mg, 2.0mmol, prepared in Example 1) and 1-hydroxybenzotriazole (HOBt, 135.1 mg, 1 mmol) were added to the reaction flask. Dissolve in dichloromethane, add diisopropylethylamine (129.1 mg, 1.0 mmol), stir well, cool in ice bath, add in portions (EDCI, 191.7 mg, 1 mmol), after the addition, the system naturally rises to The reaction was allowed to proceed overnight at room temperature. After concentration on the next day, the residue was crystallized from isopropyl alcohol, filtered, and dried to give product 4arm-PEG-L3 (20K): 4.7 g, yield 94.0%.
实施例19 4arm-PEG-L3-Dox(20K)的制备Example 19 Preparation of 4arm-PEG-L3-Dox (20K)
Figure PCTCN2018106972-appb-000085
Figure PCTCN2018106972-appb-000085
将化合物4arm-PEG-L3(2g，0.1mmol，实施例18制备)加到反应瓶中，用二氯甲烷(40mL)溶解，N 2保护下冷却，加入琥珀酰亚胺碳酸酯(1.02g，0.4mmol)，搅拌溶解后再加入三乙胺(60.6mg，0.6mmol)，加完后撤去冷浴，室温反应过夜。反应液浓缩，残分用异丙醇结晶，得产品4arm-PEG-L3-NHS(20K)1.8g，收率90.0％。 The compound was added to 4arm-PEG-L3 (2g, 0.1mmol, prepared in Example 18) the reaction flask, was dissolved with dichloromethane (40 mL), cooled under N 2, was added succinimidyl carbonate (1.02 g of, 0.4 mmol), after stirring and stirring, triethylamine (60.6 mg, 0.6 mmol) was added. After the addition, the cold bath was removed and allowed to react at room temperature overnight. The reaction solution was concentrated, and the residue was crystallized from isopropyl alcohol to give the product 4arm-PEG-L3-NHS (20K) 1.8 g, yield 90.0%.
将化合物4arm-PEG-L3-NHS(1.5g，0.075mmol，上步制得)溶于二氯甲烷(30mL)中，N 2保护下冷却，加入二异丙基乙基胺(77.5mg，0.6mmol)，搅拌均匀后再加入盐酸阿霉素(261mg，0.45mmol)，加完后室温搅拌5小时。反应液浓缩，残分用异丙醇结晶，抽滤，干燥得红色固体产品4arm-PEG-L3-Dox(20K)1.3g，收率86.7％。 1H NMR:(DMSO-d6)：δ8.82(s,4H),8.67(s,4H),7.62(m,4H),7.53(d,4H),7.33(d,8H),7.16(d,12H),5,51(s,8H),5.12(t,4H),4.83(s,8H),4.55(t,4H),4.33(m,12H),4.06(m,4H),3.95(s,12H),3.67(m,1800H),3.55(t,8H),3.47(s,4H),3.28(m,4H),3.08(m,4H),2.27(d,8H),2.04(m,8H),1.68(t,8H),1.42(d,12H),1.16(d,24H)。 The compound was cooled under 4arm-PEG-L3-NHS ( 1.5g, 0.075mmol, prepared on step) was dissolved in dichloromethane (30mL), N 2 protection, was added diisopropylethyl amine (77.5mg, 0.6 Methyl), after stirring evenly, doxorubicin hydrochloride (261 mg, 0.45 mmol) was added, and the mixture was stirred at room temperature for 5 hours. The reaction mixture was concentrated, and the residue was crystallised from isopropyl alcohol, filtered, and dried to give a red solid product 4arm-PEG-L3-Dox (20K) 1.3 g, yield 86.7%. 1 H NMR: (DMSO-d6): δ 8.82 (s, 4H), 8.67 (s, 4H), 7.62 (m, 4H), 7.53 (d, 4H), 7.33 (d, 8H), 7.16 (d) , 12H), 5, 51 (s, 8H), 5.12 (t, 4H), 4.83 (s, 8H), 4.55 (t, 4H), 4.33 (m, 12H), 4.06 (m, 4H), 3.95 ( s, 12H), 3.67 (m, 1800H), 3.55 (t, 8H), 3.47 (s, 4H), 3.28 (m, 4H), 3.08 (m, 4H), 2.27 (d, 8H), 2.04 (m) , 8H), 1.68 (t, 8H), 1.42 (d, 12H), 1.16 (d, 24H).
实施例20  8arm-PEG-L4-NHS(20K)的制备Example 20 Preparation of 8arm-PEG-L4-NHS (20K)
Figure PCTCN2018106972-appb-000086
Figure PCTCN2018106972-appb-000086
8-arm-PEG-N 3(20K，2g，0.1mmol)、化合物L4(参照中国专利申请CN201510354709.6中所述方法制备，220mg，1mmol)、维生素C(440mg，2.5mmol)加到N,N-二甲基甲酰胺(20mL)中，快速搅拌使其溶解，然后加入五水合硫酸铜(250mg，1mmol)的水溶液(4.4mL，2.2mL/g PEG)，室温反应过夜，用异丙醇沉淀得到1.8g产物。 8-arm-PEG-N 3 (20K, 2g, 0.1mmol), compound L4 (prepared by the method described in Chinese patent application CN201510354709.6, 220mg, 1mmol), vitamin C (440mg, 2.5mmol), added to N, In N-dimethylformamide (20 mL), it was dissolved by rapid stirring, then an aqueous solution of copper sulfate pentahydrate (250 mg, 1 mmol) (4.4 mL, 2.2 mL/g PEG) was added and allowed to react overnight at room temperature with isopropanol. The precipitate gave 1.8 g of product.
将化合物8arm-PEG-L4(2g，0.1mmol，上步制得)加到反应瓶中，用二氯甲烷(40mL)溶解，N 2保护下冷却，加入琥珀酰亚胺碳酸酯(1.02g，0.4mmol)，搅拌溶解后再加入三乙胺(60.6mg，0.6mmol)，加完后撤去冷浴，室温反应过夜。反应液浓缩，残分用异丙醇结晶，得产品8arm-PEG-L4-NHS(20K)1.7g，收率85％。 Compound 8arm-PEG-L4 (2g, 0.1mmol, prepared by the step) was added to the reaction flask, was dissolved with dichloromethane (40 mL), cooled under N 2, was added succinimidyl carbonate (1.02 g of, 0.4 mmol), after stirring and stirring, triethylamine (60.6 mg, 0.6 mmol) was added. After the addition, the cold bath was removed and allowed to react at room temperature overnight. The reaction mixture was concentrated, and the residue was crystallised from isopropyl alcohol to give EtOAc (yield:
实施例21 8arm-PEG-L4-Dox(20K)的制备Example 21 Preparation of 8arm-PEG-L4-Dox (20K)
Figure PCTCN2018106972-appb-000087
Figure PCTCN2018106972-appb-000087
将化合物8arm-PEG-L4-NHS(20K，1.5g，0.075mmol，实施例20制备)溶于二氯甲烷(30mL)中，N 2保护下冷却，加入二异丙基乙基胺(77.5mg，0.6mmol)，搅拌均匀后再加入盐酸阿霉素(261mg，0.45mmol)，加完后室温搅拌5小时。反应液浓缩，残分用异丙醇结晶，抽滤，干燥得红色固体产品8arm-PEG-L4-Dox(20K)1.4g，收率93.3％。 The compound was cooled under 8arm-PEG-L4-NHS ( 20K, 1.5g, 0.075mmol, prepared in Example 20) was dissolved in dichloromethane (30mL) in, N 2 protection, was added diisopropylethyl amine (77.5 mg , 0.6 mmol), after stirring evenly, doxorubicin hydrochloride (261 mg, 0.45 mmol) was added, and the mixture was stirred at room temperature for 5 hours. The reaction mixture was concentrated, and the residue was crystallised from isopropyl alcohol, filtered, and dried to give a red solid product of 8 </ RTI></RTI></RTI></RTI></RTI><RTIgt;
实施例22 4arm PEG-L5-Dox(20K)的制备Example 22 Preparation of 4arm PEG-L5-Dox (20K)
Figure PCTCN2018106972-appb-000088
Figure PCTCN2018106972-appb-000088
4arm PEG-L5的合成Synthesis of 4arm PEG-L5
将4arm PEG(20K)-CM-NHS(2g，0.1mmol)溶于无水二氯甲烷中，低温冷却至0℃，加入DIPEA(78mg，0.6mmol)，然后加入化合物L5(实施例3制备)，缓慢升至室温搅拌过夜。反应液浓缩后残分用异丙醇结晶，抽滤，干燥后得产品4arm PEG-L5(1.8g，90％)。 1H NMR:(DMSO-d6)：δ8.11(s,4H),6.86(s,4H),6.74(d,4H),6.65(d,4H),5.35(s,8H),4.20(m,8H),3.66(m,1800H),3.52(m,4H),3.24(s,6H),2.78(m,8H),2.13(s,6H)。 4arm PEG(20K)-CM-NHS (2g, 0.1mmol) was dissolved in anhydrous dichloromethane, chilled to 0 ° C, DIPEA (78 mg, 0.6 mmol) was added, then compound L5 was added (prepared in Example 3) Stir slowly to room temperature and stir overnight. After the reaction mixture was concentrated, the residue was crystallized from isopropyl alcohol, filtered, and dried to give the product 4arm PEG-L5 (1.8 g, 90%). 1 H NMR: (DMSO-d6): δ 8.11 (s, 4H), 6.86 (s, 4H), 6.74 (d, 4H), 6.65 (d, 4H), 5.35 (s, 8H), 4.20 (m) , 8H), 3.66 (m, 1800H), 3.52 (m, 4H), 3.24 (s, 6H), 2.78 (m, 8H), 2.13 (s, 6H).
4arm PEG-L5-NHS的合成Synthesis of 4arm PEG-L5-NHS
将化合物4arm PEG-L5(1.5g，0.075mmol，上步制得)加到反应瓶中，用二氯甲烷(30mL)溶解，N 2保护下冷却，加入琥珀酰亚胺碳酸酯(68.0mg，0.3mmol)，搅拌溶解后再加入DIPEA(51.6mg，0.4mmol)，加完后撤去冷浴，室温反应过夜。反应液浓缩，残分用异丙醇结晶，得产品4armPEG-L5-NHS(1.4g，93％)。 1H NMR:(DMSO-d6)：δ8.12(s,4H),6.85(s,4H),6.72(d,4H),6.65(d,4H),4.65(s,8H),4.15(m,8H),3.65(m,1800H),3.29(m,8H),3.24(s,12H),2.12(s,9H)。 Compound 4arm PEG-L5 (1.5g, 0.075mmol , prepared on step) was added to the reaction flask, was dissolved with dichloromethane (30 mL), cooled under N 2, was added succinimidyl carbonate (68.0 mg, After the mixture was stirred and dissolved, DIPEA (51.6 mg, 0.4 mmol) was added. After the addition, the cold bath was removed and allowed to react at room temperature overnight. The reaction mixture was concentrated, and the residue was crystallised from isopropyl alcohol to give the product 4arm PEG-L5-NHS (1.4 g, 93%). 1 H NMR: (DMSO-d6): δ 8.12 (s, 4H), 6.85 (s, 4H), 6.72 (d, 4H), 6.65 (d, 4H), 4.65 (s, 8H), 4.15 (m) , 8H), 3.65 (m, 1800H), 3.29 (m, 8H), 3.24 (s, 12H), 2.12 (s, 9H).
4arm PEG-L5-Dox的合成Synthesis of 4arm PEG-L5-Dox
将化合物4arm PEG-NHS(1g，0.05mmol，上步制得)加到反应瓶中，用二氯甲烷(20mL)溶解， N 2保护下冷却，加入二异丙基乙基胺，搅拌均匀后再加入盐酸阿霉素(348.8mg，0.3mmol)，加完后室温反应过夜。反应液浓缩，残分用异丙醇结晶，抽滤，干燥得棕红色固体产品4arm PEG-L5-Dox(0.84g，84％)。 1H NMR:(DMSO-d6)：δ8.81(s,4H),8.67(s,4H),7.63(m,4H),7.53(d,4H),7.34(d,8H),7.17(d,12H),5,52(s,8H),5.12(t,4H),4.83(s,8H),4.55(t,4H),4.33(m,12H),4.06(m,4H),3.95(s,12H),3.67(m,1800H),3.55(t,8H),3.47(s,4H),3.28(m,4H),3.09(m,4H),2.26(d,8H),2.05(m,8H),1.66(t,8H),1.43(d,12H),1.17(d,24H)。 The compound 4arm PEG-NHS (1 g, 0.05 mmol, obtained in the above step) was added to a reaction flask, dissolved in dichloromethane (20 mL), cooled under N 2 and added to diisopropylethylamine. Further, doxorubicin hydrochloride (348.8 mg, 0.3 mmol) was added, and the reaction was allowed to proceed overnight at room temperature after the addition. The reaction mixture was concentrated, and the residue was crystallised from isopropyl alcohol, filtered, and dried to give a brown brown solid product 4arm PEG-L5-Dox (0.84 g, 84%). 1 H NMR: (DMSO-d6): δ 8.81 (s, 4H), 8.67 (s, 4H), 7.63 (m, 4H), 7.53 (d, 4H), 7.34 (d, 8H), 7.17 (d) , 12H), 5, 52 (s, 8H), 5.12 (t, 4H), 4.83 (s, 8H), 4.55 (t, 4H), 4.33 (m, 12H), 4.06 (m, 4H), 3.95 ( s, 12H), 3.67 (m, 1800H), 3.55 (t, 8H), 3.47 (s, 4H), 3.28 (m, 4H), 3.09 (m, 4H), 2.26 (d, 8H), 2.05 (m) , 8H), 1.66 (t, 8H), 1.43 (d, 12H), 1.17 (d, 24H).
实施例23 HCT116肿瘤异位移植小鼠模型中肿瘤生长抑制研究Example 23 Tumor growth inhibition study in a mouse model of HCT116 tumor ectopic transplantation
实验目的：Purpose:
检测药物对HCT116肿瘤异位移植小鼠模型的肿瘤生长抑制作用。The tumor growth inhibition effect of the drug on the HCT116 tumor ectopic transplantation mouse model was examined.
药物分组：Drug grouping:
G1：溶媒；G1: solvent;
G2：阳性药物阿霉素(市售)；G2: positive drug doxorubicin (commercially available);
G3：4armPEG-L5-Dox(实施例22制备)。G3: 4arm PEG-L5-Dox (prepared in Example 22).
实验方法：experimental method:
细胞培养：Cell culture:
收到HCT116细胞(市售)后吸除原培养液，加入10mL新鲜的培养液。当细胞达到～90％的汇合时收集细胞，进行传代。移除培养液后加入10mL EDTA/PBS溶液，室温放置5min，吸去EDTA/PBS溶液，将3mL0.25℅的胰酶(37℃)混匀铺在细胞表面，然后立即吸除并将已无介质的培养瓶放入培养箱直至细胞从培养瓶壁分离(5min)。加入20mL含10％FBS的DMEM并轻轻吹打将细胞混匀。用计数板记数并用10％FBS的DMEM稀释，将40mL细胞悬液转移至150mm的细胞培养皿，然后放于培养箱。隔天换液，当细胞达到～80％的汇合后传代，传代方法同上。After receiving HCT116 cells (commercially available), the original culture solution was aspirated, and 10 mL of fresh culture solution was added. Cells were harvested and passaged when the cells reached ~90% confluence. After removing the culture solution, add 10 mL EDTA/PBS solution, leave it at room temperature for 5 min, aspirate the EDTA/PBS solution, and mix 3 mL of 0.25 c/o trypsin (37 ° C) on the cell surface, then immediately aspirate and no The culture flask of the medium was placed in the incubator until the cells were separated from the wall of the culture flask (5 min). Add 20 mL of DMEM containing 10% FBS and gently pipette to mix the cells. The cells were counted by counting plates and diluted with 10% FBS in DMEM, and 40 mL of the cell suspension was transferred to a 150 mm cell culture dish, which was then placed in an incubator. Change the liquid every other day, when the cells reach ~80% confluence and pass the passage, the subculture method is the same as above.
接种细胞悬液制备：Inoculation of cell suspension preparation:
细胞接种当天检查细胞达到～80％的汇合，提前换液。3小时后用吸量管移去培养液后，每个皿加入20mL EDTA/PBS溶液，室温放置5min，吸去EDTA/PBS溶液，将3mL 0.25℅的胰酶(37℃)混匀铺在细胞表面，然后立即吸除并将已无介质的培养瓶放入培养箱直至细胞从培养瓶壁分离(5min)。加入20mL含10％FBS的DMEM并轻轻吹打将细胞混匀。将细胞悬液转移将离心管室温离心5min后，移去上清，再加入无血清的DMEM轻轻混匀，,再次离心，移去上清，加入少量冰冷PBS轻轻混匀。用计数板记数并用PBS稀释至2×10 7细胞/Ml，放置于冰上直至接种。 On the day of cell inoculation, the cells were examined to reach ~80% confluence and the cells were changed in advance. After 3 hours, remove the culture solution with a pipette, add 20 mL of EDTA/PBS solution to each dish, leave it at room temperature for 5 min, aspirate EDTA/PBS solution, and mix 3 mL of 0.25 c/o trypsin (37 ° C) on the cells. The surface was then immediately aspirated and the medium-free flask was placed in the incubator until the cells were separated from the flask wall (5 min). Add 20 mL of DMEM containing 10% FBS and gently pipette to mix the cells. Transfer the cell suspension. Centrifuge the tube at room temperature for 5 min. Remove the supernatant, add the serum-free DMEM, mix gently, centrifuge again, remove the supernatant, and mix gently by adding a small amount of ice-cold PBS. The cells were counted with a counting plate and diluted to 2 × 10 7 cells/ml with PBS, and placed on ice until inoculation.
肿瘤接种：Tumor inoculation:
小鼠右前腋皮下接种HCT116细胞，每只动物接种200μLPBS细胞悬液，总共接种30只动物，接种后每周测量2次肿瘤体积，当平均肿瘤体积达100-200mm 3时挑选18只肿瘤大小接近的动物，随机分为3组，每组6只动物。分组后24内小时开始给药，每次给药前称量动物体重以调整给药量。最后一次给药以后继续观察测量动物，每周测量2次体重及肿瘤大小，肿瘤体积(mm 3)计算按公式：V＝0.5(a×b 2)，其中a代表长径，b代表宽径。某组动物肿瘤平均体积大于2000mm 3即处死改组动物。组间肿瘤大小的差异将使用t检验比较，P<0.05将视为统计学意义上的差异。 HCT116 cells were inoculated subcutaneously into the right anterior humerus, and each animal was inoculated with 200 μL of PBS cell suspension. A total of 30 animals were inoculated. The tumor volume was measured twice a week after inoculation. When the average tumor volume reached 100-200 mm 3 , 18 tumors were selected to be close in size. Animals were randomly divided into 3 groups of 6 animals each. Dosing was started within 24 hours after grouping, and the body weight of the animals was weighed before each administration to adjust the dose. The animals were continuously observed after the last administration, and the body weight and tumor size were measured twice a week. The tumor volume (mm 3 ) was calculated according to the formula: V = 0.5 (a × b 2 ), where a represents the long diameter and b represents the broad diameter. . The average volume of tumors in a certain group of animals was greater than 2000 mm 3 , that is, the animals were sacrificed. Differences in tumor size between groups will be compared using the t test, and P < 0.05 will be considered as a statistically significant difference.
实验结果：Experimental results:
实验结果如图6所示，阳性药物阿霉素和化合物4armPEG-L5-Dox均有明显的抗肿瘤效果，并且化合物4arm PEG-L5-Dox的效果比阿霉素更佳，且随着给药时间延长，效果差距逐渐增大，这很可能与化合物4arm PEG-L5-Dox中阿霉素以聚乙二醇为载体，可使药物在肿瘤部位停留更长时间并可达到缓释控释效果有关。给药过程中动物体重下降可以接受，该化合物有进一步研究的价值。The experimental results are shown in Figure 6. The positive drug doxorubicin and the compound 4armPEG-L5-Dox have significant anti-tumor effects, and the compound 4arm PEG-L5-Dox is better than doxorubicin, and with the administration The time is prolonged, and the effect gap is gradually increased. This is likely to be related to the compound 4arm PEG-L5-Dox, which uses polyethylene glycol as a carrier to allow the drug to stay in the tumor for a longer period of time and achieve sustained release and controlled release. related. The weight loss of the animal during administration is acceptable and the compound has further research value.
本发明实施例中所述的“偶联度”是指每个IL-2分子上偶联的PEG分子数目(即本发明中通式Ⅸ中所述的n值)，如，偶联度为4的mPEG-L5-rhIL-2，是指每个rhIL-2分子上偶联4个mPEG-L5链段；偶联度为4-7的mPEG-L5-rhIL-2，是指含有偶联度为4的mPEG-L5-rhIL-2、偶联度为5的mPEG-L5-rhIL-2、偶联度为6的mPEG-L5-rhIL-2和偶联度为7的mPEG-L5-rhIL-2的混合物。The "degree of coupling" as used in the examples of the present invention means the number of PEG molecules coupled on each IL-2 molecule (i.e., the value of n described in the formula IX in the present invention), for example, the degree of coupling is 4 mPEG-L5-rhIL-2 refers to the coupling of 4 mPEG-L5 segments on each rhIL-2 molecule; mPEG-L5-rhIL-2 with a coupling degree of 4-7, which means coupling mPEG-L5-rhIL-2 with degree 4, mPEG-L5-rhIL-2 with coupling degree of 5, mPEG-L5-rhIL-2 with coupling degree of 6, and mPEG-L5- with coupling degree of 7 a mixture of rhIL-2.
本发明实施例17、19、21和22的反应式中所述DN的结构为
Figure PCTCN2018106972-appb-000089
The structures of the DNs in the reaction formulas of Examples 17, 19, 21 and 22 of the present invention are
Figure PCTCN2018106972-appb-000089
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换等，均应包含在本发明的保护范围之内。The above is only the preferred embodiment of the present invention, and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc., which are within the spirit and principles of the present invention, should be included in the scope of the present invention. within.

Claims (13)

  1. 一种聚乙二醇-连接子-药物结合物，其具有如下结构：A polyethylene glycol-linker-drug conjugate having the following structure:
    (PEG-X-L-Y) n-D (PEG-XLY) n -D
    (Ⅸ)(IX)
    其中，PEG为聚乙二醇残基，Wherein PEG is a polyethylene glycol residue,
    X为PEG与L的连接基团，选自：-(CH 2) a-、-(CH 2) aCO-、-(CH 2) aOCO-、-(CH 2) aNHCO-、-NH(CH 2) aCO-、-(CH 2) aSO 2-、-O(CH 2) a-、-O(CH 2) aCO-、-O(CH 2) aOCO-、-O(CH 2) aNHCO-和-O(CH 2) aSO 2-中的一种或多种的组合，a为0-10的整数， X is a linking group of PEG and L, and is selected from the group consisting of: -(CH 2 ) a -, -(CH 2 ) a CO-, -(CH 2 ) a OCO-, -(CH 2 ) a NHCO-, -NH (CH 2 ) a CO-, -(CH 2 ) a SO 2 -, -O(CH 2 ) a -, -O(CH 2 ) a CO-, -O(CH 2 ) a OCO-, -O( CH 2 ) a combination of one or more of NHCO- and -O(CH 2 ) a SO 2 -, a being an integer from 0 to 10,
    Y为L与D的连接基团，选自：-(CH 2) r-、
    Figure PCTCN2018106972-appb-100001
    -(CH 2) rO-、-(CH 2) rCO-、-(CH 2) rNH-、-(CH 2) rCONH-、-(CH 2) rNHCO-、-(CH 2) rSH-、
    Figure PCTCN2018106972-appb-100002
    Figure PCTCN2018106972-appb-100003
    中的一种或多种的组合，r为0-10的整数，     Y is a linking group of L and D, and is selected from the group consisting of: -(CH 2 ) r -,
    Figure PCTCN2018106972-appb-100001
    -(CH 2 ) r O-, -(CH 2 ) r CO-, -(CH 2 ) r NH-, -(CH 2 ) r CONH-, -(CH 2 ) r NHCO-, -(CH 2 ) r SH-,
    Figure PCTCN2018106972-appb-100002
    Figure PCTCN2018106972-appb-100003
    a combination of one or more of the following, r is an integer from 0 to 10,
    R 17和R 18独立地选自：-H、C1-6的烷基、C1-6的烷氧基、C3-6环烷基和C4-10亚烷基环烷基， R 17 and R 18 are independently selected from the group consisting of: -H, a C1-6 alkyl group, a C1-6 alkoxy group, a C3-6 cycloalkyl group, and a C4-10 alkylene cycloalkyl group.
    D为含m个胺基的生物学活性剂，m为1-500的整数，D is a biologically active agent containing m amine groups, and m is an integer of from 1 to 500,
    n为整数，且1≤n≤m，n is an integer and 1≤n≤m,
    L为连接子，其具有如下结构：L is a linker having the following structure:
    Figure PCTCN2018106972-appb-100004
    Figure PCTCN2018106972-appb-100004
    其中，l为1-5的整数，Where l is an integer from 1 to 5,
    A选自：氨基酸残基、多肽残基、
    Figure PCTCN2018106972-appb-100005
    -(CH 2) i-、-NHCO(CH 2) i-、-CONH(CH 2) i-、-(CH 2) iNH-和-CO(CR 15R 16) iNH-中的一种或多种的组合，i为0-6的整数，     A is selected from the group consisting of: amino acid residues, polypeptide residues,
    Figure PCTCN2018106972-appb-100005
    One of -(CH 2 ) i -, -NHCO(CH 2 ) i -, -CONH(CH 2 ) i -, -(CH 2 ) i NH- and -CO(CR 15 R 16 ) i NH- Or a combination of multiples, i is an integer from 0-6,
    R 1-7、R 9-11独立地选自：-H、-F、-Cl、-Br、-I、C1-6的烷基、C1-6的烷氧基、C3-6的环烷基、C1-6的烯基、C6-12的芳基、C6-12芳烷基、C3-12芳族或非芳族的杂环基、C3-12的杂环烷基和-(CH 2) l-O-Z， R 1-7 and R 9-11 are independently selected from the group consisting of: -H, -F, -Cl, -Br, -I, C1-6 alkyl, C1-6 alkoxy, C3-6 naphthenic a base, a C1-6 alkenyl group, a C6-12 aryl group, a C6-12 aralkyl group, a C3-12 aromatic or non-aromatic heterocyclic group, a C3-12 heterocycloalkyl group, and -(CH 2 ) l -OZ,
    R 8和R 12独立地选自C1-6的烷基， R 8 and R 12 are independently selected from a C1-6 alkyl group,
    R 13-16独立地选自：-H、C1-6的烷基， R 13-16 is independently selected from the group consisting of: -H, C1-6 alkyl,
    B为连接基团-B 1-B 2-， B is a linking group -B 1 -B 2 -,
    其中，B 1选自：-(CH 2) j-、-NHCO(CH 2) j-和-CONH(CH 2) j-，j为0-6的整数， Wherein B 1 is selected from the group consisting of: -(CH 2 ) j -, -NHCO(CH 2 ) j - and -CONH(CH 2 ) j -, and j is an integer from 0 to 6,
    B 2选自：-C＝O、-C＝S、-O-、-S-、-C(O)O-、-C(O)S-、-C(S)O-和-S-S-。 B 2 is selected from the group consisting of: -C=O, -C=S, -O-, -S-, -C(O)O-, -C(O)S-, -C(S)O-, and -SS- .
  2. 如权利要求1所述的结合物，其特征在于，所述D为多肽和蛋白类药物，所述多肽和蛋白类药物为细胞因子、人血红蛋白、凝血因子、血管内皮生长因子抗体拮抗剂、蛋白类激素、抗体或酶及辅酶类药物；优选地，所述D为细胞因子，更优选为白介素；进一步优选地，所述D为IL-2，优选为rhIL-2。The conjugate according to claim 1, wherein said D is a polypeptide and a protein drug, and said polypeptide and protein drug are cytokines, human hemoglobin, blood coagulation factor, vascular endothelial growth factor antibody antagonist, protein A hormone, an antibody or an enzyme and a coenzyme; preferably, said D is a cytokine, more preferably an interleukin; further preferably, said D is IL-2, preferably rhIL-2.
  3. 如权利要求1所述的结合物，其特征在于，所述D为含胺基的小分子生物学活性剂及其药学上可接受的盐，优选包括：阿霉素、克唑替尼、戈舍瑞林、阿糖胞苷、普鲁卡因、苯佐卡因、氯普鲁卡因、二甲卡因、多巴胺、去甲肾上腺素、克仑特罗、苯乙双胍、达拉匹林、丙舒硫胺、对氨基水杨酸、磺胺嘧啶，及其衍生物；The conjugate according to claim 1, wherein said D is an amine group-containing small molecule biologically active agent and a pharmaceutically acceptable salt thereof, preferably comprising: doxorubicin, crizotinib, and ge Serelin, cytarabine, procaine, benzocaine, chloroprocaine, dimethine, dopamine, norepinephrine, clenbuterol, phenformin, dalapril , propyl sulphide, p-aminosalicylic acid, sulfadiazine, and derivatives thereof;
    优选地，所述D为阿霉素或其衍生物，其具有如下结构：Preferably, the D is doxorubicin or a derivative thereof having the following structure:
    Figure PCTCN2018106972-appb-100006
    Figure PCTCN2018106972-appb-100006
    其中，W 1选自：-H、-OH、-OCH 3和-OCH 2CH 3；优选为-H或-OCH 3，更优选为-OCH 3； Wherein W 1 is selected from the group consisting of: -H, -OH, -OCH 3 and -OCH 2 CH 3 ; preferably -H or -OCH 3 , more preferably -OCH 3 ;
    W 2选自：-H、-OH、-OCO(CH 2) 5COOH和-OCO(CH 2) 2NH 2；优选为-H或-OH，更优选为-OH； W 2 is selected from the group consisting of: -H, -OH, -OCO(CH 2 ) 5 COOH and -OCO(CH 2 ) 2 NH 2 ; preferably -H or -OH, more preferably -OH;
    W 3选自：-OH、-OCH 3和
    Figure PCTCN2018106972-appb-100007
    优选为-OH；     W 3 is selected from the group consisting of: -OH, -OCH 3 and
    Figure PCTCN2018106972-appb-100007
    Preferably it is -OH;
    更优选地，所述D为阿霉素，其具有如下结构：More preferably, said D is doxorubicin having the following structure:
    Figure PCTCN2018106972-appb-100008
    Figure PCTCN2018106972-appb-100008
  4. 如权利要求2所述的结合物，其特征在于，n为1-12的整数，优选为1-7的整数。The combination according to claim 2, wherein n is an integer of from 1 to 12, preferably an integer of from 1 to 7.
  5. 如权利要求1-4任一项所述的结合物，其特征在于，连接子中，所述R 5-7均为-H； The conjugate according to any one of claims 1 to 4, wherein, in the linker, the R 5-7 is -H;
    所述的R 9-11均为-H； The R 9-11 is -H;
    所述R 8和/或R 12为甲基； The R 8 and/or R 12 is a methyl group;
    所述l为1；The l is 1;
    连接子L-a中，所述A为-COCH 2NH-、-COCH(CH 3)NH-或-COCH(CH(CH 3) 2)NH-； In the linker La, the A is -COCH 2 NH-, -COCH(CH 3 )NH- or -COCH(CH(CH 3 ) 2 )NH-;
    连接子L-b或L-c中，所述-B-A-为-OCH 2CH 2NH-。 In the linker Lb or Lc, the -BA- is -OCH 2 CH 2 NH-.
  6. 如权利要求1-5任一项所述的结合物，其特征在于，所述X选自：-(CH 2) a-、-(CH 2) aCO-、-(CH 2) aNHCO-、-NH(CH 2) aCO-、-O(CH 2) a-、-O(CH 2) aCO-、-O(CH 2) aNHCO-中的一种或多种的组合；和/或， The conjugate according to any one of claims 1 to 5, wherein the X is selected from the group consisting of: -(CH 2 ) a -, -(CH 2 ) a CO-, -(CH 2 ) a NHCO- a combination of one or more of -NH(CH 2 ) a CO-, -O(CH 2 ) a -, -O(CH 2 ) a CO-, -O(CH 2 ) a NHCO-; /or,
    所述Y为-(CH 2) rCO-与单键、-CH 2-、-CH 2CH 2-、-CH 2CH 2CH 2-、-CH 2CH 2CH 2CH 2-、-CH 2CH 2CH 2CH 2CH 2-、-CH(CH 3)-、-CH 2CH(CH 3)-、-CH 2CH 2CH(CH 3)-、-CH 2CH 2CH 2CH(CH 3)-、-CH 2CH 2CH 2CH 2CH(CH 3)-、-CH 2CH 2CH 2CH 2CH 2CH(CH 3)-、-(CH 2) rO-、-(CH 2) rCONH-、-(CH 2) rNHCO-、-(CH 2) rNH-、-(CH 2) rSH-、
    Figure PCTCN2018106972-appb-100009
    Figure PCTCN2018106972-appb-100010
    中的一种或多种的组合。     The Y is -(CH 2 ) r CO- and a single bond, -CH 2 -, -CH 2 CH 2 -, -CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 CH 2 -, -CH(CH 3 )-, -CH 2 CH(CH 3 )-, -CH 2 CH 2 CH(CH 3 )-, -CH 2 CH 2 CH 2 CH ( CH 3 )-, -CH 2 CH 2 CH 2 CH 2 CH(CH 3 )-, -CH 2 CH 2 CH 2 CH 2 CH 2 CH(CH 3 )-, -(CH 2 ) r O-, -( CH 2 ) r CONH-, -(CH 2 ) r NHCO-, -(CH 2 ) r NH-, -(CH 2 ) r SH-,
    Figure PCTCN2018106972-appb-100009
    Figure PCTCN2018106972-appb-100010
    a combination of one or more of them.
  7. 如权利要求1-6任一项所述的结合物，其特征在于，所述X为单键、-CH 2-、-CH 2CH 2-、-CO-、-CH 2CO-或-NHCO-；和/或，所述的Y为-CO-。 The conjugate according to any one of claims 1 to 6, wherein X is a single bond, -CH 2 -, -CH 2 CH 2 -, -CO-, -CH 2 CO- or -NHCO -; and / or, said Y is -CO-.
  8. 如权利要求1-7任一项所述的结合物，其特征在于，所述结合物具有如下结构：The conjugate according to any one of claims 1 to 7, wherein the conjugate has the following structure:
    Figure PCTCN2018106972-appb-100011
    Figure PCTCN2018106972-appb-100011
    Figure PCTCN2018106972-appb-100012
    Figure PCTCN2018106972-appb-100012
  9. 如权利要求1-8任一项所述的结合物，其特征在于，所述PEG为直链聚乙二醇残基，其具有通式Ⅲ或Ⅳ所示的结构：The conjugate according to any one of claims 1 to 8, wherein the PEG is a linear polyethylene glycol residue having a structure represented by the formula III or IV:
    Figure PCTCN2018106972-appb-100013
    Figure PCTCN2018106972-appb-100013
    其中，p和q独立地选自1-960的整数；Wherein p and q are independently selected from an integer from 1 to 960;
    或，or,
    所述PEG为Y型聚乙二醇残基，其具有通式Ⅴ或Ⅵ所示的结构：The PEG is a Y-type polyethylene glycol residue having a structure represented by the formula V or VI:
    Figure PCTCN2018106972-appb-100014
    Figure PCTCN2018106972-appb-100014
    其中，i和h独立地选自1-480的整数；Wherein i and h are independently selected from an integer from 1 to 80;
    或，or,
    所述PEG为多分支聚乙二醇残基，其具有通式Ⅶ所示的结构：The PEG is a multi-branched polyethylene glycol residue having the structure shown in Formula VII:
    Figure PCTCN2018106972-appb-100015
    Figure PCTCN2018106972-appb-100015
    其中，k是1-320的整数，Where k is an integer from 1 to 320,
    j是3-8的整数，j is an integer of 3-8,
    R是多分支聚乙二醇的核心分子，R选自：季戊四醇、寡聚季戊四醇、甲基葡萄糖苷、蔗糖、二甘醇、丙二醇、甘油和聚甘油的残基。R is a core molecule of a multi-branched polyethylene glycol, and R is selected from the group consisting of: pentaerythritol, oligo-pentaerythritol, methyl glucoside, sucrose, diethylene glycol, propylene glycol, glycerin, and polyglycerol residues.
  10. 如权利要求9所述的结合物，其特征在于，所述多分支聚乙二醇残基具有如下结构：The conjugate according to claim 9, wherein said multi-branched polyethylene glycol residue has the following structure:
    Figure PCTCN2018106972-appb-100016
    Figure PCTCN2018106972-appb-100016
    优选地，所述多分支聚乙二醇残基具有如下结构：Preferably, the multi-branched polyethylene glycol residue has the following structure:
    Figure PCTCN2018106972-appb-100017
    Figure PCTCN2018106972-appb-100017
    Figure PCTCN2018106972-appb-100018
    Figure PCTCN2018106972-appb-100018
    其中，x和y独立地选自1-10的整数。Wherein x and y are independently selected from an integer from 1 to 10.
  11. 如权利要求1-10任一项所述的结合物，其特征在于，所述的PEG的分子量为1-100KDa，优选为10-50KDa。The conjugate according to any one of claims 1 to 10, wherein the PEG has a molecular weight of from 1 to 100 KDa, preferably from 10 to 50 KDa.
  12. 一种药物组合物，其包含权利要求1-11任一项所述的聚乙二醇-连接子-药物结合物。A pharmaceutical composition comprising the polyethylene glycol-linker-drug conjugate of any of claims 1-11.
  13. 一种权利要求1-11任一项所述的聚乙二醇-连接子-药物结合物或权利要求12所述的药物组合物在制备预防和/或治疗疾病的药物中的应用；Use of the polyethylene glycol-linker-drug conjugate according to any one of claims 1 to 11 or the pharmaceutical composition according to claim 12 for the preparation of a medicament for preventing and/or treating diseases;
    优选地，所述的疾病为肿瘤、自身免疫疾病、病毒性疾病或细菌性疾病。Preferably, the disease is a tumor, an autoimmune disease, a viral disease or a bacterial disease.
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