CN115594766A - A conjugate and antibody-coupled drug prepared therefrom - Google Patents
A conjugate and antibody-coupled drug prepared therefromDownload PDFInfo
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Abstract
Translated fromChinese
Description
Translated fromChinese技术领域technical field
本发明涉及医药领域，具体涉及药物偶联物领域，特别涉及一种缀合物及用其制备的抗体药物偶联物。The present invention relates to the field of medicine, in particular to the field of drug conjugates, in particular to a conjugate and an antibody-drug conjugate prepared therefrom.
背景技术Background technique
抗体药物偶联物(Antibody drug conjugate，ADC)是一类新型的靶向治疗药物，由靶向特异性抗原的单克隆抗体和小分子细胞毒药物通过连接子偶联而成，其联合了抗体及细胞毒药物两者的优势，与传统的小分子抗肿瘤药物相比，具有靶向性强、细胞毒性高、降解半衰期长、毒副作用低等特点。随着
ADC一般由抗体(antibody)、小分子细胞毒素(cytotoxin)和连接子(linker)三个部分组成。其中连接子的设计对ADC药物具有重要的意义，其本身必须使得药物在血液循环系统中保持稳定，以及到达靶组织之后可以快速有效地释放活性毒素。开发ADC连接子，存在着多种重要的考量因素，包括抗体的偶联位点、每分子抗体平均偶联细胞毒素的数量(drug to antibody ratio，DAR)、连接子的可裂解性和亲水性等。连接子可分为可裂解连接子和不可裂解连接子两大类，可裂解连接子根据裂解机制的不同分为pH敏感型和酶敏感型。当前，酶敏感型连接子已经成为ADC的主流选择。研究比较成熟的酶敏感型连接子为依赖组织蛋白酶B裂解的二肽连接子，如缬氨酸-瓜氨酸(Valine-Citriline)。目前大多数ADC具有共同的结构特征，如通过马来酰亚胺接头连接。ADC药物已公开的连接子存在设计比较单一，水溶性不好等问题。ADC generally consists of three parts: antibody, small molecule cytotoxin and linker. Among them, the design of the linker is of great significance to the ADC drug, which must keep the drug stable in the blood circulation system and release the active toxin quickly and effectively after reaching the target tissue. In the development of ADC linkers, there are many important considerations, including the conjugation site of the antibody, the average number of cytotoxins per molecule of antibody (drug to antibody ratio, DAR), the cleavability and hydrophilicity of the linker sex etc. Linkers can be divided into two categories: cleavable linkers and non-cleavable linkers, and cleavable linkers are divided into pH-sensitive and enzyme-sensitive types according to the different cleavage mechanisms. Currently, enzyme-sensitive linkers have become the mainstream choice for ADCs. The more mature enzyme-sensitive linker is a dipeptide linker dependent on cathepsin B cleavage, such as Valine-Citriline. Most current ADCs share common structural features, such as linkage via maleimide linkers. The published linkers of ADC drugs have problems such as relatively simple design and poor water solubility.
关于毒素与抗体的偶联方式，目前已上市ADC药物多采用赖氨酸或半胱氨酸随机偶联，由于偶联数目和位点的不确定性，导致生成的抗体药物偶联物的不均一性，进而导致产品各组分间药物动力学性质、效价以及毒性的不均一。为了解决ADC均一性问题，近年来定点偶联技术得到了更多的青睐，如基于天然糖基化位点的GlycoConnect技术，抗体的Fc区域N297糖基化位点在酶催化下，对糖链进行切割并连接上叠氮基团，然后叠氮基团与连接子上的环辛炔通过SPAAC(strain-promoted copper-free click)反应，形成均一的DAR值为2的偶联物。Regarding the coupling method of toxins and antibodies, currently marketed ADC drugs are mostly randomly coupled with lysine or cysteine. Due to the uncertainty of the number and sites of coupling, the generated antibody-drug conjugates are inconsistent. Uniformity, which in turn leads to inhomogeneity in pharmacokinetic properties, potency and toxicity among the components of the product. In order to solve the problem of ADC uniformity, site-specific coupling technology has gained more favor in recent years, such as GlycoConnect technology based on natural glycosylation sites. After cleavage and connection of the azide group, the azide group reacts with the cyclooctyne on the linker by SPAAC (strain-promoted copper-free click) to form a uniform conjugate with a DAR value of 2.
专利文件CN108743968A公开了一种半胱氨酸改造的抗体-毒素偶联物，抗体为半胱氨酸定点插入抗体，半胱氨酸插入位点包含kappa/λ轻链恒定区轻链和IgG抗体重链恒定区重链两类。Patent document CN108743968A discloses a cysteine modified antibody-toxin conjugate, the antibody is a cysteine site-directed insertion antibody, and the cysteine insertion site contains kappa/λ light chain constant region light chain and IgG anti- There are two types of heavy chains in the constant region of the heavy chain.
但通过基因工程改造的抗体-毒素偶联物引进了药物的不确定性，需要使用还原剂还原抗体，解除抗体上改造的半胱氨酸残基上的屏蔽，并通过阳离子交换层析或超滤换液等方式去除DTT与屏蔽物，然后使用氧化剂氧化抗体，使抗体的链间二硫键重新连接，过程繁琐复杂限制了该发明的使用。However, the antibody-toxin conjugates introduced by genetic engineering introduce uncertainty in the drug, and it is necessary to use a reducing agent to reduce the antibody, release the shield on the modified cysteine residue on the antibody, and pass through cation exchange chromatography or ultrasonography. Removing DTT and shielding materials by means of filtration and replacement, and then using an oxidizing agent to oxidize the antibody to reconnect the interchain disulfide bonds of the antibody, the cumbersome and complicated process limits the application of this invention.
专利文件CN103083680B公开了一种具有聚乙二醇-氨基酸寡肽-依诺替康结构通式的药物结合物。在所述结合物中，每一个聚乙二醇端基通过氨基酸寡肽可以与多个依诺替康相连，药物的负载率大大提高。通过亲水性聚合物的改性可对依诺替康提供保护，改善药物吸收，延长作用时间，增强疗效，降低给药剂量及避免毒副作用。Patent document CN103083680B discloses a drug conjugate having a general structural formula of polyethylene glycol-amino acid oligopeptide-irnotecan. In the conjugate, each end group of polyethylene glycol can be connected with multiple irinotecans through amino acid oligopeptides, and the drug loading rate is greatly improved. The modification of the hydrophilic polymer can protect inotecan, improve drug absorption, prolong action time, enhance curative effect, reduce dosage and avoid toxic and side effects.
但此抗体药物偶联物的均一性不高，进而导致产品各组分间药物动力学性质、效价以及毒性的不均一、产品批次可重复性较差、治疗指数较低。However, the homogeneity of the antibody-drug conjugate is not high, which leads to inhomogeneity in pharmacokinetic properties, potency and toxicity among the components of the product, poor reproducibility of product batches, and low therapeutic index.
针对上述现有技术中存在的抗体偶联药物均一性较差、连接子设计较单一、水溶性不好等缺陷，迫切需要开发新的连接子技术，在增加连接子亲水性的同时达到定点偶联的目的，提高抗体药物偶联物的均一性。In view of the defects in the above-mentioned prior art, such as poor uniformity of antibody-drug conjugates, single linker design, and poor water solubility, it is urgent to develop new linker technology, which can increase the hydrophilicity of the linker while achieving fixed-point The purpose of conjugation is to improve the uniformity of antibody drug conjugates.
发明内容Contents of the invention
本发明第一方面是提供一种缀合物1，其具有式(I)所示结构：The first aspect of the present invention is to provide a
Q-X-L1-L2-D (I)QXL1 -L2 -D (I)
其中，所述Q选自：
R5和R6各自独立地具有—X1—Q1的结构，Q1选自-H、-F、-Cl、-Br、-I、-SO2、-NO2、C1-12链烷基、C3-12环烷基、C6-12芳烷基，，X1选自由单键、-O-、-S-、C1-12链烷基、C3-12环烷基、C6-12芳烷基、
X是连接基团，选自由-O-、-S-、C1-12直链/支链烷基、C3-12环烷基、C6-12芳烷基、
L1选自：直链、Y型和多分支的聚乙二醇残基中的一种；L is selected from:one of straight chain, Y-type and multi-branched polyethylene glycol residues;
当L1为Y型和多分支的聚乙二醇残基时，其可以有一个或多个分支与
L2是连接基团，为
Y3、Y4各自独立地选自：-H、C1-10直链/支链烷基、C3-10环烷基、
优选地，B为
在本发明一具体实施方式中，所述的B为
D为药物分子。D is a drug molecule.
优选的，Q为
优选的，所述的缀合物1具有式(II)所示结构：Preferably, the
其中，式(I)中的DBCO(二苯并环辛炔)接头用于与修饰抗体的叠氮基团的定点偶联；L1选自：直链、Y型和多分支的聚乙二醇残基中的一种，L1包括但不限于直链双端PEG、Y型PEG、4臂支链PEG、6臂支链PEG或8臂支链PEG，优选地，L1为直链聚乙二醇残基，具有通式(Ⅲ)所示的结构：Wherein, the DBCO (dibenzocyclooctyne) linker in the formula (I) is used for site-specific coupling with the azide group of the modified antibody; L is selected from: straight chain, Y- type and multi-branched polyethylene glycol One of the alcohol residues, L1 includes but not limited to straight-chain double-ended PEG, Y-type PEG, 4-arm branched-chain PEG, 6-arm branched-chain PEG or 8-arm branched-chain PEG, preferably, L1 is straight-chain Polyethylene glycol residue has a structure shown in general formula (Ⅲ):
其中，n1独立地选自1-30的整数，优选为1-25的整数，更优选为4-24的整数(如4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24)。Wherein,n is independently selected from an integer of 1-30, preferably an integer of 1-25, more preferably an integer of 4-24 (such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24).
在本发明的一个实施方式中，所述n1为8。In one embodiment of the present invention, saidn1 is 8.
在本发明的一个实施方式中，所述n1为24。In one embodiment of the present invention, said n1 is 24.
所述的肽类连接子包括一个或多个相同或不同的多肽残基，所述的多肽残基包括两个或两个以上相同或不同的氨基酸残基或氨基酸残基衍生物，所述的氨基酸选自：丙氨酸、精氨酸、天冬酰胺、天冬氨酸、半胱氨酸、谷氨酰胺、谷氨酸、甘氨酸、组氨酸、异亮氨酸、亮氨酸、赖氨酸、甲硫氨酸、苯丙氨酸、脯氨酸、丝氨酸、苏氨酸、色氨酸、酪氨酸、缬氨酸、瓜氨酸、鸟氨酸和胱氨酸中的一种或两种以上的组合，优选地，所述的氨基酸选自：缬氨酸、瓜氨酸、甘氨酸、丙氨酸、苯丙氨酸、酪氨酸、异亮氨酸、亮氨酸和精氨酸中的一种或两种以上的组合，所述的多肽残基包括缬氨酸-瓜氨酸、缬氨酸-丙氨酸和甘氨酸-甘氨酸-苯丙氨酸-甘氨酸中的一种。The peptide linker includes one or more identical or different polypeptide residues, and the polypeptide residues include two or more identical or different amino acid residues or amino acid residue derivatives, and the Amino acids selected from: Alanine, Arginine, Asparagine, Aspartic Acid, Cysteine, Glutamine, Glutamic Acid, Glycine, Histidine, Isoleucine, Leucine, Lys One of amino acid, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, citrulline, ornithine, and cystine Or a combination of two or more, preferably, the amino acid is selected from: valine, citrulline, glycine, alanine, phenylalanine, tyrosine, isoleucine, leucine and arginine One or a combination of two or more amino acids, the polypeptide residues include one of valine-citrulline, valine-alanine and glycine-glycine-phenylalanine-glycine .
进一步地，所述的A选自：
进一步地，所述的R1选自：-H、甲基、乙基、异丙基、叔丁基、异丁基和异戊基中的一种，所述的R2选自：-H、甲基、乙基、异丙基、叔丁基、异丁基、异戊基和
在本发明的一具体实施方式中，所述的A选自：
进一步地，所述L2选自：
优选地，所述的R2选自：-H、-C1-6直链/支链烷基、
优选地，所述的R3为(C1-6亚烷基)-芳环基，更优选地，所述的R3为(C1亚烷基)-苯基、(C2亚烷基)-苯基、(C3亚烷基)-苯基、(C4亚烷基)-苯基、(C5亚烷基)-苯基和(C6亚烷基)-苯基中的一种，特别优选地，所述的R3为(C1亚烷基)-苯基。Preferably, said R3 is (C1-6 alkylene)-aryl ring group, more preferably, said R3 is (C1 alkylene)-phenyl, (C2 alkylene )-phenyl, (C3 alkylene)-phenyl, (C4 alkylene)-phenyl, (C5 alkylene)-phenyl and (C6 alkylene)-phenyl One, particularly preferably, said R3 is (C1 alkylene)-phenyl.
优选地，所述的R4为-H、-C1-6直链/支链烷基、
在本发明一具体实施方式中，所述L2选自：
进一步地，所述的药物分子选自：鹅膏菌素、奥瑞他汀、加利车霉素、喜树碱、喜树碱衍生物和代谢物(SN-38)，隐藻霉素、道诺霉素、多拉司他丁、多柔比星、多卡霉素、埃坡霉素、埃斯帕霉素、格尔德霉素、美登素、甲氨蝶呤、单甲基奥瑞他汀E(“MMAE”)，单甲基auristatin F(“MMAF”)、吡咯并苯二氮卓、根瘤菌素、SG2285、微管溶素、长春地辛、类毒素或上述任何一种的衍生物；Further, the drug molecule is selected from: amanitin, auristatin, calicheamicin, camptothecin, camptothecin derivatives and metabolites (SN-38), cryptomycin, Dao Normycin, Dolastatin, Doxorubicin, Duocamycin, Epothilone, Espamycin, Geldanamycin, Maytansine, Methotrexate, Monomethylol Restatin E (“MMAE”), monomethyl auristatin F (“MMAF”), pyrrolobenzodiazepine, rhizobin, SG2285, tubulysin, vindesine, toxoid, or any of the above derivative;
优选地，所述药物分子选自：SN38及其衍生物、Dxd、依喜替康、MMAE、MMAF、MMAD、PBD及其衍生物、卡奇霉素和TPL中的一种。Preferably, the drug molecule is selected from one of: SN38 and its derivatives, Dxd, exitecan, MMAE, MMAF, MMAD, PBD and its derivatives, calicheamicin and TPL.
在本发明一具体实施方式中，所述的药物分子选自：
所述的DBCO连接子-药物缀合物选自：The DBCO linker-drug conjugate is selected from:
中的一种。One of.
本发明的第二方面是提供一种如通式(II)所述的缀合物1的制备方法，所述制备方法包括如下步骤：The second aspect of the present invention is to provide a preparation method of
(1)在试剂4-二甲氨基吡啶的作用下，将DBCO-NHS与
(2)将步骤(1)所得化合物1与N-羟基琥珀酰亚胺和1-(3-二甲基氨基丙基)-3-乙基碳二亚胺反应合成
(3)在试剂4-二甲氨基吡啶的作用下，将步骤(2)所得化合物2与H2N-L3-OH反应合成
(4)在试剂4-二甲氨基吡啶和对硝基氯甲酸苯酯作用下，将步骤(3)所得化合物3与
R7选自：-H、或C1-10直链/支链烷基中的一种，优选地，所述的R7为C1-5直链/支链烷基，更优选地，所述的R7为-CH3；R7 is selected from one of: -H, or C1-10 straight chain/branched chain alkyl, preferably, said R7 is C1-5 straight chain/branched chain alkyl, more preferably, The R7 is -CH3 ;
所述的R8为
Y4选自：-H、C1-10直链/支链烷基、C3-10环烷基、
本发明所述的DBCO连接子-药物缀合物可用于抗体药物偶联物的制备。The DBCO linker-drug conjugate of the present invention can be used in the preparation of antibody drug conjugates.
本发明第三方面是提供一种缀合物2，其具有式(Ⅵ)所示结构：The third aspect of the present invention is to provide a
优选地，所述缀合物2具有式(Ⅶ)所示结构：Preferably, the
优选地，所述缀合物2选自式(Ⅶ-1)、(Ⅶ-2)所示结构：Preferably, the
优选地，所述缀合物2选自：Preferably, the
其中，n选自1-30的整数(如1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30)，优选地，n选自2-15的整数。Wherein, n is selected from an integer of 1-30 (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30), preferably, n is an integer selected from 2-15.
在本发明的一个实施方式中，n为4。In one embodiment of the invention, n is 4.
在本发明的一个实施方式中，n为12。In one embodiment of the invention, n is 12.
n1独立地选自1-30的整数，优选为1-25的整数，更优选为4-24的整数(如4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24)。n is independently selected from an integer of 1-30, preferably an integer of 1-25, more preferably an integer of 4-24 (such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24).
在本发明的一个实施方式中，所述n1为8。In one embodiment of the present invention, saidn1 is 8.
在本发明的一个实施方式中，所述n1为24。In one embodiment of the present invention, said n1 is 24.
d为选自1-10的整数(如1、2、3、4、5、6、7、8、9、10)，优选地，d为选自1-5的整数；d is an integer selected from 1-10 (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10), preferably, d is an integer selected from 1-5;
在本发明的一个实施方式中，d为1。In one embodiment of the invention, d is 1.
在本发明的一个实施方式中，d为2。In one embodiment of the invention, d is 2.
L1选自：直链、Y型和多分支的聚乙二醇残基中的一种，L1包括但不限于直链双端PEG、Y型PEG、4臂支链PEG、6臂支链PEG或8臂支链PEG，当L1为Y型和多分支的聚乙二醇残基时，其可以有一个或多个分支与
优选地，L1为直链聚乙二醇残基，具有通式(Ⅲ)所示的结构：Preferably, L isa straight-chain polyethylene glycol residue with a structure shown in general formula (III):
其中，n1独立地选自1-30的整数，优选为1-25的整数，更优选为4-24的整数(如4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24)。Wherein,n is independently selected from an integer of 1-30, preferably an integer of 1-25, more preferably an integer of 4-24 (such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24).
在本发明的一个实施方式中，所述n1为8。In one embodiment of the present invention, saidn1 is 8.
在本发明的一个实施方式中，所述n1为24。In one embodiment of the present invention, said n1 is 24.
L2是连接基团，为
Y3、Y4各自独立地选自：-H、C1-10直链/支链烷基、C3-10环烷基、
优选地，B为
在本发明一具体实施方式中，所述的B为
D为药物分子。D is a drug molecule.
进一步地，所述的肽类连接子包括一个或多个相同或不同的多肽残基，所述的多肽残基包括两个或两个以上相同或不同的氨基酸残基或氨基酸残基衍生物，所述的氨基酸选自：丙氨酸、精氨酸、天冬酰胺、天冬氨酸、半胱氨酸、谷氨酰胺、谷氨酸、甘氨酸、组氨酸、异亮氨酸、亮氨酸、赖氨酸、甲硫氨酸、苯丙氨酸、脯氨酸、丝氨酸、苏氨酸、色氨酸、酪氨酸、缬氨酸、瓜氨酸、鸟氨酸和胱氨酸中的一种或两种以上的组合，优选地，所述的氨基酸选自：缬氨酸、瓜氨酸、甘氨酸、丙氨酸、苯丙氨酸、酪氨酸、异亮氨酸、亮氨酸和精氨酸中的一种或两种以上的组合，所述的多肽残基包括缬氨酸-瓜氨酸、缬氨酸-丙氨酸和甘氨酸-甘氨酸-苯丙氨酸-甘氨酸中的一种。Further, the peptide linker includes one or more identical or different polypeptide residues, and the polypeptide residues include two or more identical or different amino acid residues or amino acid residue derivatives, Described amino acid is selected from: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine acid, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, citrulline, ornithine, and cystine Preferably, the amino acid is selected from: valine, citrulline, glycine, alanine, phenylalanine, tyrosine, isoleucine, leucine acid and arginine or a combination of two or more, the polypeptide residues include valine-citrulline, valine-alanine and glycine-glycine-phenylalanine-glycine kind of.
进一步地，所述的A选自：
进一步地，所述的R1选自：-H、甲基、乙基、异丙基、叔丁基、异丁基和异戊基中的一种，所述的R2选自：-H、甲基、乙基、异丙基、叔丁基、异丁基、异戊基和
在本发明的一具体实施方式中，所述的A选自：
进一步地，所述L2选自：
优选地，所述的R1选自：-H、-C1-6直链/支链烷基、
优选地，所述的R2选自：-H、-C1-6直链/支链烷基、
优选地，所述的R3为(C1-6亚烷基)-芳环基，更优选地，所述的R3为(C1亚烷基)-苯基、(C2亚烷基)-苯基、(C3亚烷基)-苯基、(C4亚烷基)-苯基、(C5亚烷基)-苯基和(C6亚烷基)-苯基中的一种，特别优选地，所述的R3为(C1亚烷基)-苯基。Preferably, said R3 is (C1-6 alkylene)-aryl ring group, more preferably, said R3 is (C1 alkylene)-phenyl, (C2 alkylene )-phenyl, (C3 alkylene)-phenyl, (C4 alkylene)-phenyl, (C5 alkylene)-phenyl and (C6 alkylene)-phenyl One, particularly preferably, said R3 is (C1 alkylene)-phenyl.
优选地，所述的R4为-H、-C1-6直链/支链烷基、
在本发明一具体实施方式中，所述L2选自：
进一步地，所述的D为药物分子，所述的药物分子选自：鹅膏菌素、奥瑞他汀、加利车霉素、喜树碱、喜树碱衍生物和代谢物(SN-38)，隐藻霉素、道诺霉素、多拉司他丁、多柔比星、多卡霉素、埃坡霉素、埃斯帕霉素、格尔德霉素、美登素、甲氨蝶呤、单甲基奥瑞他汀E(MMAE)，单甲基auristatin F(MMAF)、吡咯并苯二氮卓、根瘤菌素、SG2285、微管溶素、长春地辛、类毒素或上述任何一种的衍生物；Further, the D is a drug molecule, and the drug molecule is selected from: amanitin, auristatin, calicheamicin, camptothecin, camptothecin derivatives and metabolites (SN-38 ), cryptomycin, daunomycin, dolastatin, doxorubicin, docarmycin, epothilone, espamycin, geldanamycin, maytansine, formazan Amhotrexate, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), pyrrolobenzodiazepine, rhizobin, SG2285, tubulysin, vindesine, toxoid, or the above Derivatives of any kind;
优选地，所述药物分子选自：SN38及其衍生物、Dxd、依喜替康、MMAE、MMAF、MMAD、PBD及其衍生物、卡奇霉素和TPL中的一种。Preferably, the drug molecule is selected from one of: SN38 and its derivatives, Dxd, exitecan, MMAE, MMAF, MMAD, PBD and its derivatives, calicheamicin and TPL.
在本发明一具体实施方式中，所述的药物分子选自：
本发明的第四方面是提供一种通式(Ⅳ)的抗体药物偶联物。The fourth aspect of the present invention is to provide an antibody-drug conjugate of general formula (IV).
其中，Ab为抗体，所述抗体包括单克隆抗体、多克隆抗体，优选为单克隆抗体，更优选为内化单克隆抗体；Wherein, Ab is an antibody, and said antibody includes monoclonal antibody and polyclonal antibody, preferably monoclonal antibody, more preferably internalized monoclonal antibody;
L1选自：直链、Y型和多分支的聚乙二醇残基中的一种，L1包括但不限于直链双端PEG、Y型PEG、4臂支链PEG、6臂支链PEG或8臂支链PEG，优选地，L1为直链聚乙二醇残基，具有通式(Ⅲ)所示的结构：L1 is selected from:one of straight chain, Y-type and multi- branched polyethylene glycol residues, L1 includes but not limited to straight-chain double-ended PEG, Y-type PEG, 4-arm branched chain PEG, 6-arm branched Chain PEG or 8-arm branched chain PEG, preferably, L isa straight-chain polyethylene glycol residue, with a structure shown in general formula (III):
其中，n1独立地选自1-30的整数，优选为1-25的整数，更优选为4-24的整数(如4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24)。Wherein,n is independently selected from an integer of 1-30, preferably an integer of 1-25, more preferably an integer of 4-24 (such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24).
在本发明的一个实施方式中，所述n1为8。In one embodiment of the present invention, saidn1 is 8.
在本发明的一个实施方式中，所述n1为24。In one embodiment of the present invention, said n1 is 24.
当L1为Y型和多分支的聚乙二醇残基时，其可以有一个或多个分支与
L2是连接基团，为
Y3、Y4各自独立地选自：-H、C1-10直链/支链烷基、C3-10环烷基、
优选地，B为
在本发明一具体实施方式中，所述的B为
D为药物分子。D is a drug molecule.
进一步地，所述的肽类连接子包括一个或多个相同或不同的多肽残基，所述的多肽残基包括两个或两个以上相同或不同的氨基酸残基或氨基酸残基衍生物，所述的氨基酸选自：丙氨酸、精氨酸、天冬酰胺、天冬氨酸、半胱氨酸、谷氨酰胺、谷氨酸、甘氨酸、组氨酸、异亮氨酸、亮氨酸、赖氨酸、甲硫氨酸、苯丙氨酸、脯氨酸、丝氨酸、苏氨酸、色氨酸、酪氨酸、缬氨酸、瓜氨酸、鸟氨酸和胱氨酸中的一种或两种以上的组合，优选地，所述的氨基酸选自：缬氨酸、瓜氨酸、甘氨酸、丙氨酸、苯丙氨酸、酪氨酸、异亮氨酸、亮氨酸和精氨酸中的一种或两种以上的组合，所述的多肽残基包括缬氨酸-瓜氨酸、缬氨酸-丙氨酸和甘氨酸-甘氨酸-苯丙氨酸-甘氨酸中的一种。Further, the peptide linker includes one or more identical or different polypeptide residues, and the polypeptide residues include two or more identical or different amino acid residues or amino acid residue derivatives, Described amino acid is selected from: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine acid, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, citrulline, ornithine, and cystine Preferably, the amino acid is selected from: valine, citrulline, glycine, alanine, phenylalanine, tyrosine, isoleucine, leucine acid and arginine or a combination of two or more, the polypeptide residues include valine-citrulline, valine-alanine and glycine-glycine-phenylalanine-glycine kind of.
进一步地，所述的A选自：
进一步地，所述的R1选自：-H、甲基、乙基、异丙基、叔丁基、异丁基和异戊基中的一种，所述的R2选自：-H、甲基、乙基、异丙基、叔丁基、异丁基、异戊基和
在本发明的一具体实施方式中，所述的A选自：、
进一步地，所述L2选自：
优选地，所述的R1选自：-H、-C1-6直链/支链烷基、
优选地，所述的R2选自：-H、-C1-6直链/支链烷基、
优选地，所述的R3为(C1-6亚烷基)-芳环基，更优选地，所述的R3为(C1亚烷基)-苯基、(C2亚烷基)-苯基、(C3亚烷基)-苯基、(C4亚烷基)-苯基、(C5亚烷基)-苯基和(C6亚烷基)-苯基中的一种，特别优选地，所述的R3为(C1亚烷基)-苯基。Preferably, said R3 is (C1-6 alkylene)-aryl ring group, more preferably, said R3 is (C1 alkylene)-phenyl, (C2 alkylene )-phenyl, (C3 alkylene)-phenyl, (C4 alkylene)-phenyl, (C5 alkylene)-phenyl and (C6 alkylene)-phenyl One, particularly preferably, said R3 is (C1 alkylene)-phenyl.
优选地，所述的R4为-H、-C1-6直链/支链烷基、
在本发明一具体实施方式中，所述L2选自：
进一步地，所述的药物分子选自：鹅膏菌素、奥瑞他汀、加利车霉素、喜树碱、喜树碱衍生物和代谢物(SN-38)，隐藻霉素、道诺霉素、多拉司他丁、多柔比星、多卡霉素、埃坡霉素、埃斯帕霉素、格尔德霉素、美登素、甲氨蝶呤、单甲基奥瑞他汀E(MMAE)，单甲基auristatin F(MMAF)、吡咯并苯二氮卓、根瘤菌素、SG2285、微管溶素、长春地辛、类毒素或上述任何一种的衍生物；Further, the drug molecule is selected from: amanitin, auristatin, calicheamicin, camptothecin, camptothecin derivatives and metabolites (SN-38), cryptomycin, Dao Normycin, Dolastatin, Doxorubicin, Duocamycin, Epothilone, Espamycin, Geldanamycin, Maytansine, Methotrexate, Monomethylol Restatin E (MMAE), monomethyl auristatin F (MMAF), pyrrolobenzodiazepine, rhizobin, SG2285, tubulysin, vindesine, toxoid or any derivative of the above;
优选地，所述药物分子选自：SN38及其衍生物、Dxd、依喜替康、MMAE、MMAF、MMAD、PBD及其衍生物、卡奇霉素和TPL中的一种。Preferably, the drug molecule is selected from one of: SN38 and its derivatives, Dxd, exitecan, MMAE, MMAF, MMAD, PBD and its derivatives, calicheamicin and TPL.
在本发明一具体实施方式中，所述的药物分子选自：
进一步地，所述的Q’选自
X是连接基团，选自由-O-、-S-、C1-12直链/支链烷基、C3-12环烷基、C6-12芳烷基、
L3选自直链或支链的C1-12亚烷基、C6-12亚芳基、C3-12环亚烷基、-S-、
L4选自直链、Y型和多分支的聚乙二醇残基中的一种；L4 is selected from one of linear, Y-type and multi-branched polyethylene glycol residues;
l选自1-50的整数。l is an integer selected from 1-50.
优选的，Q’选自
在本发明的一个实施方式中，n为4。In one embodiment of the invention, n is 4.
在本发明的一个实施方式中，n为12。In one embodiment of the invention, n is 12.
优选的，所述的抗体药物偶联物具有式(V)所示结构：Preferably, the antibody drug conjugate has a structure shown in formula (V):
其中，Ab为抗体，所述抗体包括单克隆抗体、多克隆抗体，优选为单克隆抗体，更优选为内化单克隆抗体。Wherein, Ab is an antibody, and said antibody includes monoclonal antibody and polyclonal antibody, preferably monoclonal antibody, more preferably internalized monoclonal antibody.
本发明中，抗体其形式可例如为：嵌合抗体、人源化抗体、人抗体、可与抗原结合的抗体片段(Fab、Fab’、F(ab)2、F(ab)2)、亚片段(单链构建体)或者抗体Fc融合蛋白等，优选的，所述单克隆抗体对癌症、恶性细胞、感染性生物或自身免疫性疾病相关的抗原或其表位是反应性的。In the present invention, the form of the antibody can be, for example: chimeric antibody, humanized antibody, human antibody, antibody fragment (Fab, Fab', F(ab)2, F(ab)2) that can bind to antigen, subunit Fragments (single-chain constructs) or antibody Fc fusion proteins, etc., preferably, the monoclonal antibody is reactive to antigens or epitopes associated with cancer, malignant cells, infectious organisms or autoimmune diseases.
本发明一具体实施方式中，优选的，所述单克隆抗体选自：抗HER2抗体、抗EGFR抗体、抗PMSA抗体、抗VEGFR抗体、抗CD20抗体、抗CD30抗体、抗FRα抗体、抗CD22抗体、抗CD56抗体、抗CD29抗体、抗GPNMB抗体、抗CD138抗体、抗CD74抗体、抗ENPP3抗体、抗Nectin-4抗体、抗EGFR VIII抗体、抗SLC44A4抗体、抗mesothelin抗体(抗间皮素抗体)、抗ET8R抗体、抗CD37抗体、抗CEACAM5抗体、抗CD70抗体、抗MUC16抗体、抗CD79b抗体、抗MUC16抗体、抗Muc1抗体。In a specific embodiment of the present invention, preferably, the monoclonal antibody is selected from: anti-HER2 antibody, anti-EGFR antibody, anti-PMSA antibody, anti-VEGFR antibody, anti-CD20 antibody, anti-CD30 antibody, anti-FRα antibody, anti-CD22 antibody , anti-CD56 antibody, anti-CD29 antibody, anti-GPNMB antibody, anti-CD138 antibody, anti-CD74 antibody, anti-ENPP3 antibody, anti-Nectin-4 antibody, anti-EGFR VIII antibody, anti-SLC44A4 antibody, anti-mesothelin antibody (anti-mesothelin antibody) , anti-ET8R antibody, anti-CD37 antibody, anti-CEACAM5 antibody, anti-CD70 antibody, anti-MUC16 antibody, anti-CD79b antibody, anti-MUC16 antibody, anti-Muc1 antibody.
本发明一具体实施方式中，优选的，所述抗原选自：HER-2/neu、EGFR、FRα、Nectin-4、碳酸酐酶IX、B7、CCCL19、CCCL21、CSAp、BrE3、CD1、CD1a、CD2、CD3、CD4、CD5、CD8、CD11A、CD14、CD15、CD16、CD18、CD19、CD20、CD21、CD22、CD23、CD25、CD29、CD30、CD32b、CD33、CD37、CD38、CD40、CD40L、CD44、CD45、CD46、CD52、CD54、CD55、CD59、CD64、CD67、CD70、CD74、CD79a、CD80、CD83、CD95、CD126、CD133、CD138、CD147、CD154、CEACAM5、CEACAM-6、甲胎蛋白(AFP)、VEGF、ED-B纤连蛋白、EGP-1、EGP-2、EGF受体(ErbB1)、ErbB2、ErbB3、因子H、FHL-1、Flt-3、叶酸受体、Ga733、GROB、HMGB-1、缺氧诱导因子(HIF)、HM1.24、胰岛素样生长因子(ILGF)、IFN-γ、IFN-α、IFN-β、IL-2R、IL-4R、IL-6R、IL-13R、IL-15R、IL-17R、IL-18R、IL-2、IL-6、IL-8、IL-12、IL-15、IL-17、IL-18、IL-25、IP-10、IGF-1R、Ia、HM1.24、神经节糖苷、HCG、HLA-DR、CD66a-d、MAGE、mCRP、MCP-1、MIP-1A、MIP-1B、巨噬细胞移动抑制因子(MIF)、MUC1、MUC2、MUC3、MUC4、MUC5、胎盘生长因子(PIGF)、PSA、PSMA、PSMA二聚物、PAM4抗原、NCA-95、NCA-90、A3、A33、Ep-CAM、KS-1、Le(y)、间皮素、S100、腱生蛋白、TAC、Tn抗原、Thomas-Friedenreich抗原、肿瘤坏死抗原、肿瘤血管生成抗原、TNF-α、TRAIL受体(R1和R2)、VEGFR、RANTES、T101、癌干细胞抗原、补体因子C3、C3a、C3b、C5a、C5和致癌基因产物等。In a specific embodiment of the present invention, preferably, the antigen is selected from: HER-2/neu, EGFR, FRα, Nectin-4, carbonic anhydrase IX, B7, CCCL19, CCCL21, CSAp, BrE3, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD67, CD70, CD74, CD79a, CD80, CD83, CD95, CD126, CD133, CD138, CD147, CD154, CEACAM5, CEACAM-6, alpha-fetoprotein (AFP) , VEGF, ED-B fibronectin, EGP-1, EGP-2, EGF receptor (ErbB1), ErbB2, ErbB3, factor H, FHL-1, Flt-3, folate receptor, Ga733, GROB, HMGB- 1. Hypoxia-inducible factor (HIF), HM1.24, insulin-like growth factor (ILGF), IFN-γ, IFN-α, IFN-β, IL-2R, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-2, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-25, IP-10, IGF- 1R, Ia, HM1.24, ganglioside, HCG, HLA-DR, CD66a-d, MAGE, mCRP, MCP-1, MIP-1A, MIP-1B, macrophage migration inhibitory factor (MIF), MUC1, MUC2, MUC3, MUC4, MUC5, placental growth factor (PIGF), PSA, PSMA, PSMA dimer, PAM4 antigen, NCA-95, NCA-90, A3, A33, Ep-CAM, KS-1, Le(y ), mesothelin, S100, tenascin, TAC, Tn antigen, Thomas-Friedenreich antigen, tumor necrosis antigen, tumor angiogenesis antigen, TNF-α, TRAIL receptors (R1 and R2), VEGFR, RANTES, T101, Cancer stem cell antigens, complement factors C3, C3a, C3b, C5a, C5 and oncogene products, etc.
本发明的第五方面是提供一种通式(V)的抗体药物偶联物的制备方法。The fifth aspect of the present invention is to provide a preparation method of the antibody-drug conjugate of general formula (V).
所述制备方法的合成路线示意如下：The synthetic route of described preparation method is shown as follows:
进一步地，Ab为抗体，所述抗体包括单克隆抗体、多克隆抗体，优选为单克隆抗体，更优选为内化单克隆抗体；Further, Ab is an antibody, and said antibody includes monoclonal antibody and polyclonal antibody, preferably monoclonal antibody, more preferably internalized monoclonal antibody;
L1选自：直链、Y型和多分支的聚乙二醇残基中的一种，L1包括但不限于直链双端PEG、Y型PEG、4臂支链PEG、6臂支链PEG或8臂支链PEG，当L1为Y型和多分支的聚乙二醇残基时，其可以有一个或多个分支与
优选地，L1为直链聚乙二醇残基，具有通式(Ⅲ)所示的结构：Preferably, L isa straight-chain polyethylene glycol residue with a structure shown in general formula (III):
其中，n1独立地选自1-30的整数，优选为1-25的整数，更优选为4-24的整数(如4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24)。Wherein,n is independently selected from an integer of 1-30, preferably an integer of 1-25, more preferably an integer of 4-24 (such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24).
在本发明的一个实施方式中，所述n1为8。In one embodiment of the present invention, saidn1 is 8.
在本发明的一个实施方式中，所述n1为24。In one embodiment of the present invention, said n1 is 24.
L2是连接基团，为
Y3、Y4各自独立地选自：-H、C1-10直链/支链烷基、C3-10环烷基、
优选地，B为
在本发明一具体实施方式中，所述的B为
D为药物分子。D is a drug molecule.
进一步地，所述的肽类连接子包括一个或多个相同或不同的多肽残基，所述的多肽残基包括两个或两个以上相同或不同的氨基酸残基或氨基酸残基衍生物，所述的氨基酸选自：丙氨酸、精氨酸、天冬酰胺、天冬氨酸、半胱氨酸、谷氨酰胺、谷氨酸、甘氨酸、组氨酸、异亮氨酸、亮氨酸、赖氨酸、甲硫氨酸、苯丙氨酸、脯氨酸、丝氨酸、苏氨酸、色氨酸、酪氨酸、缬氨酸、瓜氨酸、鸟氨酸和胱氨酸中的一种或两种以上的组合，优选地，所述的氨基酸选自：缬氨酸、瓜氨酸、甘氨酸、丙氨酸、苯丙氨酸、酪氨酸、异亮氨酸、亮氨酸和精氨酸中的一种或两种以上的组合，所述的多肽残基包括缬氨酸-瓜氨酸、缬氨酸-丙氨酸和甘氨酸-甘氨酸-苯丙氨酸-甘氨酸中的一种。Further, the peptide linker includes one or more identical or different polypeptide residues, and the polypeptide residues include two or more identical or different amino acid residues or amino acid residue derivatives, Described amino acid is selected from: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine acid, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, citrulline, ornithine, and cystine Preferably, the amino acid is selected from: valine, citrulline, glycine, alanine, phenylalanine, tyrosine, isoleucine, leucine acid and arginine or a combination of two or more, the polypeptide residues include valine-citrulline, valine-alanine and glycine-glycine-phenylalanine-glycine kind of.
进一步地，所述的A选自：
进一步地，所述的R1选自：-H、甲基、乙基、异丙基、叔丁基、异丁基和异戊基中的一种，所述的R2选自：-H、甲基、乙基、异丙基、叔丁基、异丁基、异戊基和
在本发明的一具体实施方式中，所述的A选自：
进一步地，所述L2选自：
优选地，所述的R1选自：-H、-C1-6直链/支链烷基、
优选地，所述的R2选自：-H、-C1-6直链/支链烷基、
优选地，所述的R3为(C1-6亚烷基)-芳环基，更优选地，所述的R3为(C1亚烷基)-苯基、(C2亚烷基)-苯基、(C3亚烷基)-苯基、(C4亚烷基)-苯基、(C5亚烷基)-苯基和(C6亚烷基)-苯基中的一种，特别优选地，所述的R3为(C1亚烷基)-苯基。Preferably, said R3 is (C1-6 alkylene)-aryl ring group, more preferably, said R3 is (C1 alkylene)-phenyl, (C2 alkylene )-phenyl, (C3 alkylene)-phenyl, (C4 alkylene)-phenyl, (C5 alkylene)-phenyl and (C6 alkylene)-phenyl One, particularly preferably, said R3 is (C1 alkylene)-phenyl.
优选地，所述的R4为-H、-C1-6直链/支链烷基、
在本发明一具体实施方式中，所述L2选自：
进一步地，所述的D为药物分子，所述的药物分子选自：鹅膏菌素、奥瑞他汀、加利车霉素、喜树碱、喜树碱衍生物和代谢物(SN-38)，隐藻霉素、道诺霉素、多拉司他丁、多柔比星、多卡霉素、埃坡霉素、埃斯帕霉素、格尔德霉素、美登素、甲氨蝶呤、单甲基奥瑞他汀E(MMAE)，单甲基auristatin F(MMAF)、吡咯并苯二氮卓、根瘤菌素、SG2285、微管溶素、长春地辛、类毒素或上述任何一种的衍生物；Further, the D is a drug molecule, and the drug molecule is selected from: amanitin, auristatin, calicheamicin, camptothecin, camptothecin derivatives and metabolites (SN-38 ), cryptomycin, daunomycin, dolastatin, doxorubicin, docarmycin, epothilone, espamycin, geldanamycin, maytansine, formazan Amhotrexate, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), pyrrolobenzodiazepine, rhizobin, SG2285, tubulysin, vindesine, toxoid, or the above Derivatives of any kind;
优选地，所述药物分子选自：SN38及其衍生物、Dxd、依喜替康、MMAE、MMAF、MMAD、PBD及其衍生物、卡奇霉素和TPL中的一种。Preferably, the drug molecule is selected from one of: SN38 and its derivatives, Dxd, exitecan, MMAE, MMAF, MMAD, PBD and its derivatives, calicheamicin and TPL.
在本发明一具体实施方式中，所述的药物分子选自：
本发明第六方面是提供一种通式为(Ⅳ)的抗体药物偶联物或通式为(II)的缀合物1或通式为(Ⅵ)的缀合物2在疾病预防和/或治疗药物中的应用。The sixth aspect of the present invention is to provide an antibody drug conjugate of general formula (IV) or
进一步地，所述疾病为癌症、病原性生物感染或自身免疫性疾病。Further, the disease is cancer, pathogenic organism infection or autoimmune disease.
进一步地，所述癌症是造血肿瘤、癌、肉瘤、黑素瘤或神经胶质肿瘤。Further, the cancer is hematopoietic tumor, carcinoma, sarcoma, melanoma or glial tumor.
进一步地，所述病原性生物选自由以下组成的组：人免疫缺陷病毒(HIV)、结核分枝杆菌、无乳链球菌、耐甲氧西林金黄色葡萄球菌、嗜肺性军团病菌、酿脓链球菌、大肠杆菌、淋病柰瑟氏菌、脑膜炎奈瑟氏菌、肺炎球菌属、B型流感嗜血杆菌、苍白密螺旋体、莱姆病螺旋体、西尼罗病毒、绿脓假单胞菌、麻风分枝杆菌、流产杆菌、狂犬病毒、流感病毒、巨细胞病毒、I型单纯疱疹病毒、II型单纯疱疹病毒、人血清细小样病毒、呼吸道合胞病毒、水痘-带状疱疹病毒、乙型肝炎病毒、麻疹病毒、腺病毒、人T细胞白血病病毒、埃-巴二氏病毒、鼠白血病病毒、腮腺炎病毒、水泡性口膜炎病毒、辛德比斯病毒、淋巴细胞脉络丛脑膜炎病毒、疣病毒、蓝舌病病毒、仙台病毒、猫白血病病毒、呼长孤病毒、脊髓灰质炎病毒、猿猴病毒40、鼠乳房肿瘤病毒、登革热病毒、风疹病毒、恶性疟原虫、间日疟原虫、鼠弓形体、让氏锥虫。Further, the pathogenic organism is selected from the group consisting of: human immunodeficiency virus (HIV), Mycobacterium tuberculosis, Streptococcus agalactiae, methicillin-resistant Staphylococcus aureus, Legionella pneumophila, pyogenes Streptococcus, Escherichia coli, Neisseria gonorrhoeae, Neisseria meningitidis, Pneumococcus spp, Haemophilus influenzae type B, Treponema pallidum, Lyme disease, West Nile virus, Pseudomonas aeruginosa , Mycobacterium leprae, Bacillus abortus, rabies virus, influenza virus, cytomegalovirus, herpes simplex virus type I, herpes simplex virus type II, human serum parvovirus, respiratory syncytial virus, varicella-zoster virus, B Hepatitis virus, measles virus, adenovirus, human T-cell leukemia virus, Epstein-Barr virus, murine leukemia virus, mumps virus, vesicular stomatitis virus, Sindbis virus, lymphocytic choriomeningitis virus , wart virus, bluetongue virus, Sendai virus, feline leukemia virus, holovirus, poliovirus, simian virus 40, mouse mammary tumor virus, dengue virus, rubella virus, Plasmodium falciparum, Plasmodium vivax, Toxoplasma mouse, Trypanosoma jenny.
进一步地，所述自身免疫性疾病选自由以下组成的组：免疫介导的血小板减少症、皮肌炎、舍格伦氏综合症、多发性硬化、西登哈姆氏舞蹈症、重症肌无力、系统性红斑狼疮、狼疮性肾炎、风湿热、类风湿性关节炎、多腺体综合征、大疱性类天疱疮、糖尿病、亨-舍二氏紫癜、链球菌感染后肾炎、结节性红斑、高安氏动脉炎、阿狄森氏病、结节病、溃疡性结肠炎、多形性红斑、IgA肾病、结节性多动脉炎、强制性脊柱炎、古德帕斯丘综合征、闭塞性血栓性脉管炎、原发性胆汁性感应变、桥本甲状腺炎、甲状腺毒症、硬皮病、慢性活动性肝炎、多肌炎/皮肌炎、多软骨炎、寻常天疱疮、韦格纳氏肉芽肿病、膜性肾病、肌萎缩侧索硬化、脊髓痨、巨细胞动脉炎/多肌痛、恶性贫血、急性肾小球肾炎、纤维化肺泡炎和青少年糖尿病。Further, the autoimmune disease is selected from the group consisting of immune-mediated thrombocytopenia, dermatomyositis, Sjögren's syndrome, multiple sclerosis, Sidenham's chorea, myasthenia gravis , systemic lupus erythematosus, lupus nephritis, rheumatic fever, rheumatoid arthritis, polyglandular syndrome, bullous pemphigoid, diabetes mellitus, Henschler's purpura, poststreptococcal nephritis, nodules Erythema dermatitis, Takayasu arteritis, Addison's disease, sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy, polyarteritis nodosa, ankylosing spondylitis, Goodpasture syndrome , thromboangiitis obliterans, primary biliary strain, Hashimoto's thyroiditis, thyrotoxicosis, scleroderma, chronic active hepatitis, polymyositis/dermatomyositis, polychondritis, pemphigus vulgaris , Wegener's granulomatosis, membranous nephropathy, amyotrophic lateral sclerosis, tabes dorsalis, giant cell arteritis/polymyalgia, pernicious anemia, acute glomerulonephritis, fibrosing alveolitis, and juvenile diabetes.
本发明第七方面是提供一种疾病治疗的方法，所述方法包括向受治疗者施用本发明所述抗体药物偶联物。The seventh aspect of the present invention provides a method for disease treatment, the method comprising administering the antibody drug conjugate of the present invention to a subject.
进一步地，所述抗体药物偶联物与选自以下一种或多种治疗方法联合施用：未共轭抗体、放射性标记抗体、药物-共轭抗体、毒素-共轭抗体、基因疗法、化疗、治疗肽、寡核苷酸、局部放疗、手术和干扰RNA疗法。Further, the antibody drug conjugate is administered in combination with one or more treatment methods selected from the following: unconjugated antibody, radiolabeled antibody, drug-conjugated antibody, toxin-conjugated antibody, gene therapy, chemotherapy, Therapeutic peptides, oligonucleotides, local radiation, surgery and interfering RNA therapy.
进一步地，所述疾病是癌症、病原性生物感染或自身免疫性疾病。Further, the disease is cancer, pathogenic organism infection or autoimmune disease.
进一步地，所述癌症是造血肿瘤、癌、肉瘤、黑素瘤或神经胶质肿瘤。Further, the cancer is hematopoietic tumor, carcinoma, sarcoma, melanoma or glial tumor.
进一步地，所述病原性生物选自由以下组成的组：人免疫缺陷病毒(HIV)、结核分枝杆菌、无乳链球菌、耐甲氧西林金黄色葡萄球菌、嗜肺性军团病菌、酿脓链球菌、大肠杆菌、淋病柰瑟氏菌、脑膜炎奈瑟氏菌、肺炎球菌属、B型流感嗜血杆菌、苍白密螺旋体、莱姆病螺旋体、西尼罗病毒、绿脓假单胞菌、麻风分枝杆菌、流产杆菌、狂犬病毒、流感病毒、巨细胞病毒、I型单纯疱疹病毒、II型单纯疱疹病毒、人血清细小样病毒、呼吸道合胞病毒、水痘-带状疱疹病毒、乙型肝炎病毒、麻疹病毒、腺病毒、人T细胞白血病病毒、埃-巴二氏病毒、鼠白血病病毒、腮腺炎病毒、水泡性口膜炎病毒、辛德比斯病毒、淋巴细胞脉络丛脑膜炎病毒、疣病毒、蓝舌病病毒、仙台病毒、猫白血病病毒、呼长孤病毒、脊髓灰质炎病毒、猿猴病毒40、鼠乳房肿瘤病毒、登革热病毒、风疹病毒、恶性疟原虫、间日疟原虫、鼠弓形体、让氏锥虫Further, the pathogenic organism is selected from the group consisting of: human immunodeficiency virus (HIV), Mycobacterium tuberculosis, Streptococcus agalactiae, methicillin-resistant Staphylococcus aureus, Legionella pneumophila, pyogenes Streptococcus, Escherichia coli, Neisseria gonorrhoeae, Neisseria meningitidis, Pneumococcus spp, Haemophilus influenzae type B, Treponema pallidum, Lyme disease, West Nile virus, Pseudomonas aeruginosa , Mycobacterium leprae, Bacillus abortus, rabies virus, influenza virus, cytomegalovirus, herpes simplex virus type I, herpes simplex virus type II, human serum parvovirus, respiratory syncytial virus, varicella-zoster virus, B Hepatitis virus, measles virus, adenovirus, human T-cell leukemia virus, Epstein-Barr virus, murine leukemia virus, mumps virus, vesicular stomatitis virus, Sindbis virus, lymphocytic choriomeningitis virus , wart virus, bluetongue virus, Sendai virus, feline leukemia virus, holovirus, poliovirus, simian virus 40, mouse mammary tumor virus, dengue virus, rubella virus, Plasmodium falciparum, Plasmodium vivax, Toxoplasma mouse, Trypanosoma junzi
进一步地，所述自身免疫性疾病选自由以下组成的组：免疫介导的血小板减少症、皮肌炎、舍格伦氏综合症、多发性硬化、西登哈姆氏舞蹈症、重症肌无力、系统性红斑狼疮、狼疮性肾炎、风湿热、类风湿性关节炎、多腺体综合征、大疱性类天疱疮、糖尿病、亨-舍二氏紫癜、链球菌感染后肾炎、结节性红斑、高安氏动脉炎、阿狄森氏病、结节病、溃疡性结肠炎、多形性红斑、IgA肾病、结节性多动脉炎、强制性脊柱炎、古德帕斯丘综合征、闭塞性血栓性脉管炎、原发性胆汁性感应变、桥本甲状腺炎、甲状腺毒症、硬皮病、慢性活动性肝炎、多肌炎/皮肌炎、多软骨炎、寻常天疱疮、韦格纳氏肉芽肿病、膜性肾病、肌萎缩侧索硬化、脊髓痨、巨细胞动脉炎/多肌痛、恶性贫血、急性肾小球肾炎、纤维化肺泡炎和青少年糖尿病。Further, the autoimmune disease is selected from the group consisting of immune-mediated thrombocytopenia, dermatomyositis, Sjögren's syndrome, multiple sclerosis, Sidenham's chorea, myasthenia gravis , systemic lupus erythematosus, lupus nephritis, rheumatic fever, rheumatoid arthritis, polyglandular syndrome, bullous pemphigoid, diabetes mellitus, Henschler's purpura, poststreptococcal nephritis, nodules Erythema dermatitis, Takayasu arteritis, Addison's disease, sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy, polyarteritis nodosa, ankylosing spondylitis, Goodpasture syndrome , thromboangiitis obliterans, primary biliary strain, Hashimoto's thyroiditis, thyrotoxicosis, scleroderma, chronic active hepatitis, polymyositis/dermatomyositis, polychondritis, pemphigus vulgaris , Wegener's granulomatosis, membranous nephropathy, amyotrophic lateral sclerosis, tabes dorsalis, giant cell arteritis/polymyalgia, pernicious anemia, acute glomerulonephritis, fibrosing alveolitis, and juvenile diabetes.
本发明提供的新型连接子-药物缀合物，可通过简单的化学方法与抗体偶联，与传统抗体药物偶联物相比，应用这种连接子得到的偶联物DAR值分布非常窄，因此生成的产品均一性高，获得的交联物单一分布的组份(DAR为4)占比80％以上；同时，本发明提供的抗体药物偶联物，裸抗和低交联度的ADC占比几乎为零(质谱检测不出DAR为0和1的组份)；此抗体药物偶联物既能够靶向EGFR抗原，又有强烈的杀伤肿瘤细胞的活性，相较于Necitumumab本身，并未影响抗体的亲和力，内吞活性和靶向性，较好的保留其生物学功能；在体外活性评价中，相较于SN38，抑瘤活性得到了明显的提高，IC50均在nM级别；并且，发明人通过大量的实验证明，本发明所述抗体药物偶联物，在治疗肿瘤方面具有一定安全性和有效性，偶联后乙二醇所赋予的亲水性可以用来调节生物分子特性；偶联物的体外肿瘤细胞增殖抑制活性较传统mc-VC-PAB偶联生物学活性、药物代谢稳定性、安全性等成药性质方面有所提高或保持；本发明在抗体偶联药物连接子中引入了PEG链，增加了细胞毒性药物的溶解性，使ADC偶联反应更充分，偶联效率更高；本发明提供的偶联方法，适用于大部分抗体，因此具有广泛的应用前景；本发明提供的偶联方法与现有偶联方法相比，本发明的基于DBCO接头的连接子-药物缀合物的优点包括但不限于：具有较快的交联速度，交联反应时间通常在2-4小时以内便可反应完毕。The novel linker-drug conjugates provided by the present invention can be coupled with antibodies through simple chemical methods. Compared with traditional antibody-drug conjugates, the DAR value distribution of the conjugates obtained by using this linker is very narrow. Therefore, the uniformity of the product generated is high, and the obtained cross-linked product has a single distribution component (DAR is 4) accounting for more than 80%; meanwhile, the antibody-drug conjugate provided by the present invention, naked antibody and ADC with low cross-linking degree The proportion is almost zero (mass spectrometry can not detect components with DAR of 0 and 1); this antibody drug conjugate can not only target the EGFR antigen, but also has a strong activity of killing tumor cells. Compared with Necitumumab itself, and The affinity, endocytic activity and targeting of the antibody are not affected, and its biological function is better preserved; in the in vitro activity evaluation, compared with SN38, the tumor inhibitory activity has been significantly improved, and the IC50 is at the nM level; and , the inventor has proved through a large number of experiments that the antibody-drug conjugate of the present invention has certain safety and effectiveness in treating tumors, and the hydrophilicity endowed by ethylene glycol after coupling can be used to adjust the characteristics of biomolecules ; The in vitro tumor cell proliferation inhibitory activity of the conjugate is improved or maintained compared with the traditional mc-VC-PAB coupling biological activity, drug metabolism stability, safety and other pharmaceutical properties; The PEG chain is introduced into the method, which increases the solubility of cytotoxic drugs, makes the ADC coupling reaction more complete, and the coupling efficiency is higher; the coupling method provided by the invention is suitable for most antibodies, so it has a wide application prospect; Compared with the existing coupling methods, the coupling method provided by the present invention, the advantages of the linker-drug conjugate based on the DBCO linker of the present invention include but are not limited to: faster cross-linking speed, and the cross-linking reaction time is generally The reaction can be completed within 2-4 hours.
本发明中所述的术语C1-10直链/支链烷基，包括甲基、乙基、C3直链/支链烷基、C4直链/支链烷基、C5直链/支链烷基、C6直链/支链烷基、C7直链/支链烷基、C8直链/支链烷基、C9直链/支链烷基、C10直链/支链烷基。The term C1-10 straight chain/branched chain alkyl described in the present invention includes methyl, ethyl, C3 straight chain/branched chain alkyl, C4 straight chain/branched chain alkyl, C5 straight chain /branched chain alkyl, C6 straight chain/branched chain alkyl, C7 straight chain/branched chain alkyl, C8 straight chain/branched chain alkyl, C9 straight chain/branched chain alkyl, C10 straight chain / branched chain alkyl.
本发明中所述的术语C1-12直链/支链烷基，包括甲基、乙基、C3直链/支链烷基、C4直链/支链烷基、C5直链/支链烷基、C6直链/支链烷基、C7直链/支链烷基、C8直链/支链烷基、C9直链/支链烷基、C10直链/支链烷基、C11直链/支链烷基、C12直链/支链烷基。The term C1-12 straight chain/branched chain alkyl described in the present invention includes methyl, ethyl, C3 straight chain/branched chain alkyl, C4 straight chain/branched chain alkyl, C5 straight chain /branched chain alkyl, C6 straight chain/branched chain alkyl, C7 straight chain/branched chain alkyl, C8 straight chain/branched chain alkyl, C9 straight chain/branched chain alkyl, C10 straight chain /branched chain alkyl, C11 straight chain/branched chain alkyl, C12 straight chain/branched chain alkyl.
本发明中所述的术语C1-5直链/支链烷基，包括甲基、乙基、C3直链/支链烷基、C4直链/支链烷基、C5直链/支链烷基。本发明中所述的术语C3-12环烷基，包括C3环烷基、C4环烷基、C5环烷基、C6环烷基、C7环烷基、C8环烷基、C9环烷基、C10环烷基、C11环烷基、C12环烷基。The term C1-5 straight chain/branched chain alkyl described in the present invention includes methyl, ethyl, C3 straight chain/branched chain alkyl, C4 straight chain/branched chain alkyl, C5 straight chain / branched chain alkyl. The term C3-12 cycloalkyl described in the present invention includes C3 cycloalkyl, C4 cycloalkyl, C5 cycloalkyl, C6 cycloalkyl, C7 cycloalkyl, C8 cycloalkane Group, C9 cycloalkyl, C10 cycloalkyl, C11 cycloalkyl, C12 cycloalkyl.
本发明中所述的术语C6-12芳环基，包括C6芳环基(苯基)、C7芳环基、C8芳环基、C9芳环基、C10芳环基、C11芳环基、C12芳环基。The termC6-12 aromatic ring group described in the present invention includesC6 aromatic ring group (phenyl),C7 aromatic ring group,C8 aromatic ring group,C9 aromatic ring group,C10 aromatic ring group, C11 aromatic ring group, C12 aromatic ring group.
本发明中所述的术语SN38是指7-乙基-10-羟基喜树碱。The term SN38 described in the present invention refers to 7-ethyl-10-hydroxycamptothecin.
本发明中所述的术语MMAE是指一甲基澳瑞他汀E。The term MMAE described in the present invention refers to monomethyl auristatin E.
本发明中所述的术语MMAF是指一甲基澳瑞他汀F。The term MMAF described in the present invention refers to monomethylauristatin F.
本发明中所述的术语MMAD是指单甲基澳瑞他汀D。The term MMAD described in the present invention refers to monomethyl auristatin D.
本发明中所述的术语PBD是指吡咯并苯并二氮杂卓。The term PBD described in the present invention refers to pyrrolobenzodiazepines.
本发明中所述的术语TPL是指雷公藤甲素。The term TPL mentioned in the present invention refers to triptolide.
本发明中所述的术语Dxd是指德鲁替康。The term Dxd described in the present invention refers to derutecan.
附图说明Description of drawings
图1为化合物1质谱图。Figure 1 is the mass spectrum of
图2为化合物1核磁图。Figure 2 is the NMR image of
图3为化合物6质谱图。Figure 3 is the mass spectrum of
图4为化合物6核磁图。Figure 4 is the NMR image of
图5为化合物7质谱图。Figure 5 is the mass spectrum of
图6为化合物7核磁图。Figure 6 is the NMR image of
图7为化合物8质谱图。Figure 7 is the mass spectrum of
图8为化合物8核磁图。Figure 8 is the NMR image of
图9为化合物9质谱图。Figure 9 is the mass spectrum of
图10为化合物9核磁图。Figure 10 is the NMR image of
图11为化合物10质谱图。Figure 11 is the mass spectrum of
图12为化合物10核磁图。Figure 12 is the NMR image of
图13为化合物11质谱图。Figure 13 is the mass spectrum of
图14为化合物11核磁图。Figure 14 is the NMR image of
具体实施方式detailed description
为了能够更清楚地理解本发明的技术内容，特举以下实施例详细说明，对本发明的技术方案进行清楚、完整地描述，其目的仅在于更好理解本发明的内容而非限制本发明的保护范围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。In order to understand the technical content of the present invention more clearly, the following examples are given in detail to describe the technical solution of the present invention clearly and completely, the purpose of which is only to better understand the content of the present invention rather than limit the protection of the present invention Scope All other embodiments obtained by persons of ordinary skill in the field without creative efforts fall within the protection scope of the present invention.
实施例1化合物1的合成The synthesis of
1.1化合物1a的合成1.1 Synthesis of Compound 1a
将PEG8(100.0g，0.27mol)溶于1000mL无水四氢呋喃中，冰水浴降温至0℃，加入固体氢氧化钠(2.2g，0.05mol)，然后向其中慢慢滴加溴乙酸叔丁酯(57.9g，0.30mol)，反应在室温下搅拌3h，液相检测大部分产物生成时停止反应。将混合物在30℃下旋转蒸发除去溶剂，用1000mL纯水溶解，用甲苯洗涤2次，收集水相，再用1000mL乙酸乙酯萃取三次。合并有机相，经无水硫酸钠干燥，过滤并浓缩，得到产品1a(95.3g，黄色油状物)，产率73％。PEG8 (100.0 g, 0.27 mol) was dissolved in 1000 mL of anhydrous tetrahydrofuran, cooled to 0° C. in an ice-water bath, solid sodium hydroxide (2.2 g, 0.05 mol) was added, and tert-butyl bromoacetate ( 57.9g, 0.30mol), the reaction was stirred at room temperature for 3h, and the reaction was stopped when most of the products were detected by liquid phase detection. The mixture was rotary evaporated at 30°C to remove the solvent, dissolved in 1000 mL of pure water, washed twice with toluene, the aqueous phase was collected, and extracted three times with 1000 mL of ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated to give product 1a (95.3 g, yellow oil) in 73% yield.
1.2化合物1b的合成1.2 Synthesis of compound 1b
将化合物1a(95.0g，0.20mol)溶于950ml二氯甲烷中，加入三乙胺(49.5g，0.49mol)，然后慢慢滴加甲基磺酰氯(34.5g，0.30mol)，滴加完成后，混合物在室温下搅拌过夜，TLC显示原料消耗完全。用500mL饱和食盐水洗涤两次，有机相用无水硫酸钠干燥，过滤并浓缩，得到产品1b(102.0g，黄色油状物)，产率91％。Compound 1a (95.0g, 0.20mol) was dissolved in 950ml of dichloromethane, triethylamine (49.5g, 0.49mol) was added, then methanesulfonyl chloride (34.5g, 0.30mol) was slowly added dropwise, and the addition was completed Afterwards, the mixture was stirred overnight at room temperature, and TLC showed complete consumption of starting material. Washed twice with 500 mL of saturated brine, the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to obtain product 1b (102.0 g, yellow oil) with a yield of 91%.
1.3化合物1c的合成1.3 Synthesis of compound 1c
将化合物1b(102.0g，0.18mol)溶于300ml水中，加入2N氢氧化钠(270mL)，反应在室温下搅拌4h，液相监控原料反应完全后，加入15％氯化铵的氨水溶液(1500mL)，该混合物在室温下搅拌2天至原料消耗完全。用600mL二氯甲烷洗涤两次，收集水相，用2N盐酸调节PH至2-3，浓缩得到白色固体，用大量二氯甲烷洗涤固体，滤液浓缩得到产品1c(50.1g，无色油状物)。Compound 1b (102.0g, 0.18mol) was dissolved in 300ml of water, 2N sodium hydroxide (270mL) was added, and the reaction was stirred at room temperature for 4h. ), the mixture was stirred at room temperature for 2 days until the starting material was completely consumed. Wash twice with 600 mL of dichloromethane, collect the aqueous phase, adjust the pH to 2-3 with 2N hydrochloric acid, concentrate to obtain a white solid, wash the solid with a large amount of dichloromethane, and concentrate the filtrate to obtain product 1c (50.1 g, colorless oil) .
1.4化合物1d的合成1.4 Synthesis of compound 1d
将化合物1c(14.3g，33.60mmol),DBCO-NHS(9.0g，22.40mmol)和4-二甲氨基吡啶(2.7g，22.40mmol)一起溶于100mL二氯甲烷中，反应室温搅拌8h，液相监测原料反应完全后，用100mL饱和食盐水洗涤一次，再用60mL10％的亚硫酸氢钠溶液洗涤两次，有机相用无水硫酸钠干燥，过滤并浓缩，得到产品1d(14.0g)，产率87％。Compound 1c (14.3g, 33.60mmol), DBCO-NHS (9.0g, 22.40mmol) and 4-dimethylaminopyridine (2.7g, 22.40mmol) were dissolved together in 100mL of dichloromethane, stirred at room temperature for 8h, and the liquid Phase monitoring After the raw materials have reacted completely, wash once with 100mL saturated brine, then wash twice with 60mL10% sodium bisulfite solution, and dry the organic phase with anhydrous sodium sulfate, filter and concentrate to obtain product 1d (14.0g). Yield 87%.
MS m/z(ESI):715.3[M+1]MS m/z(ESI):715.3[M+1]
1.5化合物1e的合成1.5 Synthesis of Compound 1e
将化合物1d(5.0g，7.00mmol)和N-羟基琥珀酰亚胺(1.0g，8.40mmol)溶于70mL二氯甲烷中，加入1-(3-二甲基氨基丙基)-3-乙基碳二亚胺(1.6g，8.40mmol)，反应室温搅拌4h，得到化合物1e的反应液，直接用于下一步反应。Compound 1d (5.0 g, 7.00 mmol) and N-hydroxysuccinimide (1.0 g, 8.40 mmol) were dissolved in 70 mL of dichloromethane, and 1-(3-dimethylaminopropyl)-3-ethane Carbodiimide (1.6g, 8.40mmol) was stirred at room temperature for 4h to obtain a reaction solution of compound 1e, which was directly used in the next reaction.
MS m/z(ESI):812.3[M+1]MS m/z(ESI):812.3[M+1]
1.6化合物1f的合成1.6 Synthesis of compound 1f
将原料Fmoc-VC-PAB(9.0g，15.0mmol)溶于30mL二甲基亚砜中，加入二乙胺(2.2g，30.0mmol)，反应室温搅拌2h，液相监控反应，原料反应完全后，加入200mL二氯甲烷，有大量固体出现，搅拌30min，过滤收集固体得到产品1f(5.0g)，产率88％。Dissolve the raw material Fmoc-VC-PAB (9.0g, 15.0mmol) in 30mL dimethyl sulfoxide, add diethylamine (2.2g, 30.0mmol), stir at room temperature for 2h, monitor the reaction in liquid phase, after the raw material reacts completely , 200mL of dichloromethane was added, a large amount of solids appeared, stirred for 30min, and the solids were collected by filtration to obtain product 1f (5.0g), with a yield of 88%.
MS m/z(ESI):380.2[M+1]MS m/z(ESI):380.2[M+1]
1.7化合物1g的合成1.7 Synthesis of Compound 1g
将化合物1f(3.0g，8.0mmol)加入上述得到的化合物1e的反应液中，加入4-二甲氨基吡啶(1.0g，8.0mmol)，反应室温搅拌2h，原料消耗完全后，加入50mL饱和食盐水洗涤，有机相用无水硫酸钠干燥，旋干溶剂，柱层析分离(DCM:MeOH 30:1-20:1)得到化合物1g(6.0g，黄色油状物)。Compound 1f (3.0g, 8.0mmol) was added to the reaction solution of compound 1e obtained above, 4-dimethylaminopyridine (1.0g, 8.0mmol) was added, and the reaction was stirred at room temperature for 2h. After the raw materials were completely consumed, 50mL of saturated salt was added Washed with water, the organic phase was dried with anhydrous sodium sulfate, spin-dried to dry the solvent, and separated by column chromatography (DCM:MeOH 30:1-20:1) to obtain compound 1g (6.0g, yellow oil).
MS m/z(ESI):1076.5[M+1]MS m/z(ESI):1076.5[M+1]
1.8化合物1h的合成1.8 Synthesis of Compound 1h
将TBS-SN38(2.5g，5.0mmol)溶于30mL二氯甲烷中，加入4-二甲氨基吡啶(0.9g，7.5mmol)，再分批次加入对硝基氯甲酸苯酯(1.2g，6.0mmol)，反应室温搅拌3h，在此反应液中加入化合物1g(6.0g，5.6mmol)，混合物在室温下搅拌过夜，TLC显示反应完成后，加入20mL二氯甲烷稀释，然后用30mL饱和食盐水洗涤，有机相用无水硫酸钠干燥，过滤并浓缩，柱层析分离(DCM:MeOH＝30：1-15:1)得到化合物1h(5.1g，黄色固体)，产率78％。Dissolve TBS-SN38 (2.5g, 5.0mmol) in 30mL of dichloromethane, add 4-dimethylaminopyridine (0.9g, 7.5mmol), and then add phenyl p-nitrochloroformate (1.2g, 6.0mmol), the reaction was stirred at room temperature for 3h, compound 1g (6.0g, 5.6mmol) was added to the reaction solution, and the mixture was stirred overnight at room temperature. Washed with water, the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated, and separated by column chromatography (DCM:MeOH=30:1-15:1) to obtain compound 1h (5.1 g, yellow solid), with a yield of 78%.
MS m/z(ESI):1608.7[M+1]MS m/z(ESI):1608.7[M+1]
1.9化合物1的合成1.9 Synthesis of
将化合物1h(5.1g，3.2mmol)溶于50mL四氢呋喃中，加入四丁基氟化铵(0.52g，2.0mmol)，室温下搅拌30min，TLC监控反应进程，反应完成后旋干溶剂，用C-18flash进行纯化制备(MeOH:H2O＝50％-90％)，得到化合物1(3.0g，黄色固体)，产率63％。图1和图2分别为化合物1质谱图和核磁图。Compound 1h (5.1g, 3.2mmol) was dissolved in 50mL tetrahydrofuran, tetrabutylammonium fluoride (0.52g, 2.0mmol) was added, stirred at room temperature for 30min, TLC monitored the reaction progress, after the reaction was completed, the solvent was spin-dried, and the solvent was washed with C -18flash was purified and prepared (MeOH:H2 O=50%-90%) to obtain compound 1 (3.0 g, yellow solid), with a yield of 63%. Figure 1 and Figure 2 are the mass spectrum and nuclear magnetic spectrum of
MS m/z(ESI):748.1[M/2+1]MS m/z(ESI):748.1[M/2+1]
1H NMR(300MHz,DMSO-d6):δ10.31(s,1H),10.08(s,1H),8.33(d,1H,J＝4.8Hz),8.05(d,1H,J＝5.4Hz),7.74(t,1H,J＝6.3Hz),7.62-7.30(m,15H),6.95(s,1H),6.02(s,1H),5.51(s,2H),5.31(s,2H),5.18-5.00(m,3H),4.44-4.25(m,2H),3.95(s,2H),3.62-3.43(m,34H),3.30-3.03(m,4H),2.52-2.50(m,2H),2.31-2.12(m,4H),2.11-1.88(m,2H),1.81-1.55(m,2H),1.53-1.31(m,2H),1.31-1.20(m,3H),0.93-0.81(m,9H)1 H NMR(300MHz,DMSO-d6):δ10.31(s,1H),10.08(s,1H),8.33(d,1H,J=4.8Hz),8.05(d,1H,J=5.4Hz) ,7.74(t,1H,J=6.3Hz),7.62-7.30(m,15H),6.95(s,1H),6.02(s,1H),5.51(s,2H),5.31(s,2H), 5.18-5.00(m,3H),4.44-4.25(m,2H),3.95(s,2H),3.62-3.43(m,34H),3.30-3.03(m,4H),2.52-2.50(m,2H ),2.31-2.12(m,4H),2.11-1.88(m,2H),1.81-1.55(m,2H),1.53-1.31(m,2H),1.31-1.20(m,3H),0.93-0.81 (m,9H)
实施例2化合物2的合成The synthesis of
2.1化合物2a的合成2.1 Synthesis of Compound 2a
将PEG8(100.0g，0.27mol)溶于1000mL无水四氢呋喃中，冰水浴降温至0℃，加入固体氢氧化钠(2.2g，0.05mol)，然后向其中慢慢滴加3-溴丙酸叔丁酯(62.7g，0.30mol)，反应在室温下搅拌3h，液相检测大部分产物生成时停止反应。将混合物在30℃下旋转蒸发除去溶剂，用1000mL纯水溶解，用甲苯洗涤2次，收集水相，再用1000mL乙酸乙酯萃取三次。合并有机相，经无水硫酸钠干燥，过滤并浓缩，得到产品2a(101.4g，黄色油状物)，产率75％。Dissolve PEG8 (100.0g, 0.27mol) in 1000mL of anhydrous tetrahydrofuran, cool to 0°C in an ice-water bath, add solid sodium hydroxide (2.2g, 0.05mol), and then slowly add 3-bromopropionic acid tert- Butyl ester (62.7g, 0.30mol), the reaction was stirred at room temperature for 3h, and the reaction was stopped when most of the products were detected by liquid phase detection. The mixture was rotary evaporated at 30°C to remove the solvent, dissolved in 1000 mL of pure water, washed twice with toluene, the aqueous phase was collected, and extracted three times with 1000 mL of ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated to give product 2a (101.4 g, yellow oil) in 75% yield.
2.2化合物2b的合成2.2 Synthesis of Compound 2b
将化合物2a(99.7g，0.20mol)溶于950ml二氯甲烷中，加入三乙胺(49.5g，0.49mol)，然后慢慢滴加甲基磺酰氯(34.5g，0.30mol)，滴加完成后，混合物在室温下搅拌过夜，TLC显示原料消耗完全。用500mL饱和食盐水洗涤两次，有机相用无水硫酸钠干燥，过滤并浓缩，得到产品2b(103.0g，黄色油状物)，产率90％。Compound 2a (99.7g, 0.20mol) was dissolved in 950ml of dichloromethane, triethylamine (49.5g, 0.49mol) was added, then methanesulfonyl chloride (34.5g, 0.30mol) was slowly added dropwise, and the addition was completed Afterwards, the mixture was stirred overnight at room temperature, and TLC showed complete consumption of starting material. Washed twice with 500 mL saturated brine, the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to obtain product 2b (103.0 g, yellow oil) with a yield of 90%.
2.3化合物2c的合成2.3 Synthesis of compound 2c
将化合物2b(103.0g，0.18mol)溶于300ml水中，加入2N氢氧化钠(270mL)，反应在室温下搅拌4h，液相监控原料反应完全后，加入15％氯化铵的氨水溶液(1500mL)，该混合物在室温下搅拌2天至原料消耗完全。用600mL二氯甲烷洗涤两次，收集水相，用2N盐酸调节PH至2-3，浓缩得到白色固体，用大量二氯甲烷洗涤固体，滤液浓缩得到产品2c(52.2g，无色油状物)。Compound 2b (103.0g, 0.18mol) was dissolved in 300ml of water, 2N sodium hydroxide (270mL) was added, and the reaction was stirred at room temperature for 4h. ), the mixture was stirred at room temperature for 2 days until the starting material was completely consumed. Wash twice with 600 mL of dichloromethane, collect the aqueous phase, adjust the pH to 2-3 with 2N hydrochloric acid, concentrate to obtain a white solid, wash the solid with a large amount of dichloromethane, and concentrate the filtrate to obtain product 2c (52.2 g, colorless oil) .
2.4化合物2d的合成2.4 Synthesis of compound 2d
将化合物2c(14.8g，33.60mmol),DBCO-NHS(9.0g，22.40mmol)和4-二甲氨基吡啶(2.7g，22.40mmol)一起溶于100mL二氯甲烷中，反应室温搅拌8h，液相监测原料反应完全后，用100mL饱和食盐水洗涤一次，再用60mL10％的亚硫酸氢钠溶液洗涤两次，有机相用无水硫酸钠干燥，过滤并浓缩，得到产品2d(13.7g)，产率84％。Compound 2c (14.8g, 33.60mmol), DBCO-NHS (9.0g, 22.40mmol) and 4-dimethylaminopyridine (2.7g, 22.40mmol) were dissolved together in 100mL of dichloromethane, stirred at room temperature for 8h, and the liquid Phase monitoring After the raw materials have reacted completely, wash once with 100mL saturated brine, then wash twice with 60mL10% sodium bisulfite solution, dry the organic phase with anhydrous sodium sulfate, filter and concentrate to obtain product 2d (13.7g), Yield 84%.
MS m/z(ESI):729.3[M+1]MS m/z(ESI):729.3[M+1]
2.5化合物2e的合成2.5 Synthesis of Compound 2e
将化合物2d(5.1g，7.00mmol)和N-羟基琥珀酰亚胺(1.0g，8.40mmol)溶于70mL二氯甲烷中，加入1-(3-二甲基氨基丙基)-3-乙基碳二亚胺(1.6g，8.40mmol)，反应室温搅拌4h，得到化合物2e的反应液，直接用于下一步反应。Compound 2d (5.1 g, 7.00 mmol) and N-hydroxysuccinimide (1.0 g, 8.40 mmol) were dissolved in 70 mL of dichloromethane, and 1-(3-dimethylaminopropyl)-3-ethane Carbodiimide (1.6g, 8.40mmol) was stirred at room temperature for 4h to obtain the reaction solution of compound 2e, which was directly used in the next reaction.
MS m/z(ESI):826.3[M+1]MS m/z(ESI):826.3[M+1]
2.6化合物2f的合成2.6 Synthesis of compound 2f
将原料Fmoc-VA-PAB(7.7g，15.0mmol)溶于30mL二甲基亚砜中，加入二乙胺(2.2g，30.0mmol)，反应室温搅拌2h，液相监控反应，原料反应完全后，加入200mL二氯甲烷，有大量固体出现，搅拌30min，过滤收集固体得到产品2f(3.9g)，产率90％。The raw material Fmoc-VA-PAB (7.7g, 15.0mmol) was dissolved in 30mL of dimethyl sulfoxide, diethylamine (2.2g, 30.0mmol) was added, and the reaction was stirred at room temperature for 2h, and the reaction was monitored by the liquid phase. , 200mL of dichloromethane was added, a large amount of solids appeared, stirred for 30min, and the solids were collected by filtration to obtain product 2f (3.9g), with a yield of 90%.
MS m/z(ESI):294.1[M+1]MS m/z(ESI):294.1[M+1]
2.7化合物2g的合成2.7 Synthesis of compound 2g
将化合物2f(2.4g，8.0mmol)加入上述得到的化合物2e的反应液中，加入4-二甲氨基吡啶(1.0g，8.0mmol)，反应室温搅拌2h，原料消耗完全后，加入50mL饱和食盐水洗涤，有机相用无水硫酸钠干燥，旋干溶剂，柱层析分离(DCM:MeOH 30:1-20:1)得到化合物2g(5.6g，黄色油状物)。Compound 2f (2.4g, 8.0mmol) was added to the reaction solution of compound 2e obtained above, 4-dimethylaminopyridine (1.0g, 8.0mmol) was added, and the reaction was stirred at room temperature for 2h. After the raw materials were completely consumed, 50mL of saturated salt was added After washing with water, the organic phase was dried with anhydrous sodium sulfate, spin-dried to dry the solvent, and separated by column chromatography (DCM:MeOH 30:1-20:1) to obtain compound 2g (5.6g, yellow oil).
MS m/z(ESI):1004.5[M+1]MS m/z(ESI):1004.5[M+1]
2.8化合物2h的合成2.8 Synthesis of compound 2h
将TBS-SN38(2.5g，5.0mmol)溶于30mL二氯甲烷中，加入4-二甲氨基吡啶(0.9g，7.5mmol)，再分批次加入对硝基氯甲酸苯酯(1.2g，6.0mmol)，反应室温搅拌3h，在此反应液中加入化合物2g(5.6g，5.6mmol)，混合物在室温下搅拌过夜，TLC显示反应完成后，加入20mL二氯甲烷稀释，然后用30mL饱和食盐水洗涤，有机相用无水硫酸钠干燥，过滤并浓缩，柱层析分离(DCM:MeOH＝30：1-15:1)得到化合物2h(5.8g，黄色固体)，产率76％。Dissolve TBS-SN38 (2.5g, 5.0mmol) in 30mL of dichloromethane, add 4-dimethylaminopyridine (0.9g, 7.5mmol), and then add phenyl p-nitrochloroformate (1.2g, 6.0mmol), the reaction was stirred at room temperature for 3h, compound 2g (5.6g, 5.6mmol) was added to the reaction solution, and the mixture was stirred overnight at room temperature. Washed with water, the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated, and separated by column chromatography (DCM:MeOH=30:1-15:1) to obtain compound 2h (5.8 g, yellow solid), with a yield of 76%.
MS m/z(ESI):1536.7[M+1]MS m/z(ESI):1536.7[M+1]
2.9化合物2的合成2.9 Synthesis of
将化合物2h(5.8g，3.8mmol)溶50mL四氢呋喃中，加入四丁基氟化铵(0.52g，2.0mmol)，室温下搅拌30min，TLC监控反应进程，反应完成后旋干溶剂，用C-18flash进行纯化制备(MeOH:H2O＝50％-90％)，得到化合物2(3.5g，黄色固体)，产率65％。Compound 2h (5.8g, 3.8mmol) was dissolved in 50mL tetrahydrofuran, tetrabutylammonium fluoride (0.52g, 2.0mmol) was added, stirred at room temperature for 30min, TLC monitored the reaction progress, after the reaction was completed, the solvent was spin-dried, and the solvent was washed with C- 18flash was purified and prepared (MeOH:H2 O=50%-90%) to obtain compound 2 (3.5 g, yellow solid) with a yield of 65%.
MS m/z(ESI):711.8[M/2+1]MS m/z(ESI):711.8[M/2+1]
1H NMR(300MHz,DMSO-d6):δ10.31(s,1H),10.08(s,1H),8.33(d,1H,J＝4.8Hz),8.05(d,1H,J＝5.4Hz),7.74(t,1H,J＝6.3Hz),7.62-7.30(m,15H),6.95(s,1H),5.01(s,2H),4.80-4.74(m,3H),4.64(s,2H),4.34(d,1H,J＝5.1Hz),4.22(s,2H),3.67-3.62(m,4H),3.52-3.49(m,30H),3.26(t,2H,J＝6.3Hz),2.76-2.71(m,1H),2.61-2.54(m,4H),2.35(t,2H,J＝6.6Hz),1.96(q,2H,J＝5.1Hz),1.49(d,3H,J＝4.8Hz),1.18(t,3H,J＝6.3Hz),0.96-0.89(m,9H)1 H NMR(300MHz,DMSO-d6):δ10.31(s,1H),10.08(s,1H),8.33(d,1H,J=4.8Hz),8.05(d,1H,J=5.4Hz) ,7.74(t,1H,J＝6.3Hz),7.62-7.30(m,15H),6.95(s,1H),5.01(s,2H),4.80-4.74(m,3H),4.64(s,2H ),4.34(d,1H,J=5.1Hz),4.22(s,2H),3.67-3.62(m,4H),3.52-3.49(m,30H),3.26(t,2H,J=6.3Hz) ,2.76-2.71(m,1H),2.61-2.54(m,4H),2.35(t,2H,J=6.6Hz),1.96(q,2H,J=5.1Hz),1.49(d,3H,J =4.8Hz),1.18(t,3H,J=6.3Hz),0.96-0.89(m,9H)
实施例3化合物3的合成The synthesis of
3.1化合物3a的合成3.1 Synthesis of compound 3a
将1g(1.08g，1.0mmol)溶于10mL二氯甲烷中，氮气保护下，冷却至5℃以下，依次滴加对硝基氯甲酸苯酯(406mg，2.0mmol)的二氯甲烷溶液和吡啶(160mg，2.0mmol)，滴加完毕后室温下搅拌过夜，TLC显示反应完成后，加入二氯甲烷稀释，然后用饱和食盐水洗涤，有机相用无水硫酸钠干燥，过滤并浓缩，得到化合物3a(800mg)的粗品，直接用于下一步反应。Dissolve 1g (1.08g, 1.0mmol) in 10mL of dichloromethane, under nitrogen protection, cool to below 5°C, add dropwise a dichloromethane solution of phenyl p-nitrochloroformate (406mg, 2.0mmol) and pyridine (160mg, 2.0mmol), stirred overnight at room temperature after the dropwise addition, after TLC showed that the reaction was complete, dichloromethane was added to dilute, then washed with saturated brine, the organic phase was dried with anhydrous sodium sulfate, filtered and concentrated to obtain compound The crude product of 3a (800 mg) was directly used in the next reaction.
MS m/z(ESI):1241.5[M+1]MS m/z(ESI):1241.5[M+1]
3.2化合物3的合成3.2 Synthesis of
将3a(745mg,0.6mmol)和SN38(182mg,0.5mol)溶于10ml干燥的N,N-二甲基甲酰胺中,冷却至0℃，加入4-二甲氨基吡啶(122mg，1.0mmol)和1-羟基苯并三氮唑(27mg，0.2mmol)，室温搅拌过夜。在反应液中加入乙酸乙酯，用饱和食盐水洗涤，无水硫酸钠干燥，过滤并浓缩，经柱层析分离纯化得到化合物3(374mg，淡黄色固体)，产率51％。Dissolve 3a (745mg, 0.6mmol) and SN38 (182mg, 0.5mol) in 10ml of dry N,N-dimethylformamide, cool to 0°C, add 4-dimethylaminopyridine (122mg, 1.0mmol) and 1-hydroxybenzotriazole (27mg, 0.2mmol), stirred overnight at room temperature. Ethyl acetate was added to the reaction solution, washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated, separated and purified by column chromatography to obtain compound 3 (374 mg, light yellow solid), yield 51%.
MS m/z(ESI):1494.6[M+1]MS m/z(ESI):1494.6[M+1]
1H NMR(300MHz,DMSO-d6):δ9.70(s,1H),8.33(d,2H,J＝4.8Hz),8.01(d,1H,J＝5.4Hz),7.65-7.24(m,14H),7.07(t,1H,J＝6.5Hz),6.74(s,1H),6.02(t,1H,J＝5.1Hz),5.45(s,2H),5.01(s,2H),4.77(s,1H),4.75(s,2H),4.64(s,2H),4.60(t,1H,J＝5.4Hz),4.36-4.32(m,1H),4.22(s,2H),3.67(t,2H,J＝4.8Hz),3.52-3.3.49(m,34H),3.28(t,2H,J＝6.6Hz),3.14(t,2H,J＝6.0Hz),2.73-2.55(m,5H),1.87(t,2H,J＝6.0Hz),1.53-1.48(m,2H),1.18(t,3H,J＝5.4Hz),0.96-0.86(m,9H)1 H NMR (300MHz, DMSO-d6): δ9.70(s, 1H), 8.33(d, 2H, J=4.8Hz), 8.01(d, 1H, J=5.4Hz), 7.65-7.24(m, 14H), 7.07(t, 1H, J=6.5Hz), 6.74(s, 1H), 6.02(t, 1H, J=5.1Hz), 5.45(s, 2H), 5.01(s, 2H), 4.77( s,1H),4.75(s,2H),4.64(s,2H),4.60(t,1H,J=5.4Hz),4.36-4.32(m,1H),4.22(s,2H),3.67(t ,2H,J=4.8Hz),3.52-3.3.49(m,34H),3.28(t,2H,J=6.6Hz),3.14(t,2H,J=6.0Hz),2.73-2.55(m, 5H), 1.87(t, 2H, J=6.0Hz), 1.53-1.48(m, 2H), 1.18(t, 3H, J=5.4Hz), 0.96-0.86(m, 9H)
实施例4化合物4的合成The synthesis of
4.1化合物4a的合成4.1 Synthesis of compound 4a
将Fmoc-Gly-Gly-Phe-Gly-OH(558mg，1mmol)溶于10mL无水N,N-二甲基甲酰胺中，加入2-乙氧基-1-乙氧碳酰基-1,2-二氢喹啉(370mg，1.5mmol)，搅拌30分钟，再加入对氨基苯甲醇(185mg，1.5mmol)，反应在室温下搅拌过夜，液相检测大部分产物生成时停止反应。加入二氯甲烷搅拌，过滤，滤饼用二氯甲烷洗涤，得到产品4a(498mg，白色固体)，产率75％。Dissolve Fmoc-Gly-Gly-Phe-Gly-OH (558 mg, 1 mmol) in 10 mL of anhydrous N,N-dimethylformamide, add 2-ethoxy-1-ethoxycarbonyl-1,2 -Dihydroquinoline (370mg, 1.5mmol), stirred for 30 minutes, then added p-aminobenzyl alcohol (185mg, 1.5mmol), the reaction was stirred overnight at room temperature, and the reaction was stopped when most of the products were detected by liquid phase detection. Add dichloromethane and stir, filter, and wash the filter cake with dichloromethane to obtain product 4a (498mg, white solid), with a yield of 75%.
4.2化合物4b的合成4.2 Synthesis of compound 4b
将化合物4a(664mg，1mmol)溶于3mL无水N,N-二甲基甲酰胺中，加入2mL二乙胺，反应室温搅拌4h，液相监控反应，原料反应完全后，浓缩反应液，经中压制备纯化得到产品4b(353mg，白色固体)，产率80％。Compound 4a (664mg, 1mmol) was dissolved in 3mL of anhydrous N,N-dimethylformamide, 2mL of diethylamine was added, the reaction was stirred at room temperature for 4h, and the reaction was monitored by the liquid phase. The product 4b (353 mg, white solid) was obtained by medium-pressure preparative purification with a yield of 80%.
MS m/z(ESI):442.2[M+1]MS m/z(ESI):442.2[M+1]
4.3化合物4c的合成4.3 Synthesis of compound 4c
将化合物4b(663mg，1.5mmol)和化合物1e(812mg,1mmol)的反应液中，加入4-二甲氨基吡啶(183mg，1.5mmol)，反应室温搅拌2h，原料消耗完全后，加入50mL饱和食盐水洗涤，有机相用无水硫酸钠干燥，旋干溶剂，柱层析分离(DCM:MeOH 30:1-20:1)得到化合物4c(925mg，黄色油状物)。Add 4-dimethylaminopyridine (183 mg, 1.5 mmol) to the reaction solution of compound 4b (663 mg, 1.5 mmol) and compound 1e (812 mg, 1 mmol), and stir at room temperature for 2 h. After the raw materials are completely consumed, add 50 mL of saturated salt Washed with water, the organic phase was dried with anhydrous sodium sulfate, spin-dried to dry the solvent, and separated by column chromatography (DCM:MeOH 30:1-20:1) to obtain compound 4c (925 mg, yellow oil).
MS m/z(ESI):1138.5[M+1]MS m/z(ESI):1138.5[M+1]
4.4化合物4d的合成4.4 Synthesis of compound 4d
将SN38(784mg，2mmol)和4-二甲氨基吡啶(366mg，3mmol)溶于8mL二氯甲烷中，氮气保护下加入三光气(268mg)，室温搅拌反应5分钟，向反应液中加入Boc-DMEA(456mg,2.5mmol)的二氯甲烷溶液(2mL)，室温搅拌反应5分钟，LCMS显示原料反应完全。反应液用水洗涤2次，得到粗品4d(黄色固体，910mg),产率75％。Dissolve SN38 (784 mg, 2 mmol) and 4-dimethylaminopyridine (366 mg, 3 mmol) in 8 mL of dichloromethane, add triphosgene (268 mg) under nitrogen protection, stir at room temperature for 5 minutes, and add Boc- A dichloromethane solution (2 mL) of DMEA (456 mg, 2.5 mmol) was stirred at room temperature for 5 minutes. LCMS showed that the starting material was completely reacted. The reaction solution was washed twice with water to obtain the crude product 4d (yellow solid, 910 mg), with a yield of 75%.
MS m/z(ESI):607.2[M+1]MS m/z(ESI):607.2[M+1]
4.5化合物4e的合成4.5 Synthesis of compound 4e
将化合物4d(910mg,1.5mmol)溶于二氯甲烷(4mL)和三氟乙酸(1mL)的混合溶剂中，氮气保护下室温搅拌反应2小时，LCMS显示原料基本反应完全。反应液加入乙腈稀释，低温浓缩，中压制备液相纯化得到产物4e(黄色固体，622mg，产率82％)。Compound 4d (910mg, 1.5mmol) was dissolved in a mixed solvent of dichloromethane (4mL) and trifluoroacetic acid (1mL), and stirred at room temperature for 2 hours under nitrogen protection. LCMS showed that the reaction of the starting material was almost complete. The reaction solution was diluted with acetonitrile, concentrated at low temperature, and purified by medium-pressure preparative liquid phase to obtain product 4e (yellow solid, 622 mg, yield 82%).
MS m/z(ESI):507.2[M+1]MS m/z(ESI):507.2[M+1]
4.6化合物4的合成4.6 Synthesis of
将化合物4c(1.38g，1mmol)溶于6mL二氯甲烷中，加入4-二甲氨基吡啶(180mg，1.5mmol)，再分批次加入对硝基氯甲酸苯酯(240mg，1.2mmol)，反应室温搅拌3h，在此反应液中加入化合物4e(557mg，1.1mmol)，混合物在室温下搅拌过夜，TLC显示反应完成后，加入二氯甲烷稀释，然后用饱和食盐水洗涤，有机相用无水硫酸钠干燥，过滤并浓缩，柱层析分离得到化合物4(1.35g，黄色固体)，产率80％。Compound 4c (1.38g, 1mmol) was dissolved in 6mL of dichloromethane, 4-dimethylaminopyridine (180mg, 1.5mmol) was added, and phenyl p-nitrochloroformate (240mg, 1.2mmol) was added in batches, The reaction was stirred at room temperature for 3 h, compound 4e (557 mg, 1.1 mmol) was added to the reaction solution, and the mixture was stirred overnight at room temperature. After TLC showed that the reaction was complete, dichloromethane was added to dilute, then washed with saturated brine, and the organic phase was washed with Dry over sodium sulfate, filter and concentrate, and separate by column chromatography to obtain compound 4 (1.35 g, yellow solid), with a yield of 80%.
MS m/z(ESI):1670.7[M+1]MS m/z(ESI):1670.7[M+1]
实施例5化合物5的合成The synthesis of
5.1化合物5a的合成5.1 Synthesis of Compound 5a
将Boc-SN38(984mg，2mmol)和4-二甲氨基吡啶(366mg，3mmol)溶于8mL二氯甲烷中，氮气保护下加入三光气(268mg)，室温搅拌反应5分钟，向反应液中加入Boc-DMEA(456mg,2.5mmol)的二氯甲烷溶液(2mL)，室温搅拌反应5分钟，LCMS显示原料反应完全。反应液用水洗涤2次，得到粗品5a(黄色固体，543mg),产率77％。Dissolve Boc-SN38 (984 mg, 2 mmol) and 4-dimethylaminopyridine (366 mg, 3 mmol) in 8 mL of dichloromethane, add triphosgene (268 mg) under nitrogen protection, stir at room temperature for 5 minutes, and add A dichloromethane solution (2 mL) of Boc-DMEA (456 mg, 2.5 mmol) was stirred at room temperature for 5 minutes, and LCMS showed that the starting material was completely reacted. The reaction solution was washed twice with water to obtain the crude product 5a (yellow solid, 543 mg), with a yield of 77%.
MS m/z(ESI):707.3[M+1]MS m/z(ESI):707.3[M+1]
5.2化合物5b的合成5.2 Synthesis of Compound 5b
将化合物5a(1.1g,1.5mmol)溶于二氯甲烷(4mL)和三氟乙酸(1mL)的混合溶剂中，氮气保护下室温搅拌反应2小时，LCMS显示原料基本反应完全。反应液加入乙腈稀释，低温浓缩，中压制备液相纯化得到产物5b(黄色固体，645mg，产率85％)。Compound 5a (1.1g, 1.5mmol) was dissolved in a mixed solvent of dichloromethane (4mL) and trifluoroacetic acid (1mL), and stirred at room temperature for 2 hours under nitrogen protection. LCMS showed that the reaction of the starting material was almost complete. The reaction solution was diluted with acetonitrile, concentrated at low temperature, and purified by medium-pressure preparative liquid phase to obtain product 5b (yellow solid, 645 mg, yield 85%).
MS m/z(ESI):507.2[M+1]MS m/z(ESI):507.2[M+1]
5.3化合物5的合成5.3 Synthesis of
将化合物1g(1.1g，1mmol)溶于6mL二氯甲烷中，加入4-二甲氨基吡啶(180mg，1.5mmol)，再分批次加入对硝基氯甲酸苯酯(240mg，1.2mmol)，反应室温搅拌3h，在此反应液中加入化合物5b(557mg，1.1mmol)，混合物在室温下搅拌过夜，TLC显示反应完成后，加入二氯甲烷稀释，然后用饱和食盐水洗涤，有机相用无水硫酸钠干燥，过滤并浓缩，柱层析分离得到化合物5(1.3g，黄色固体)，产率81％。Compound 1g (1.1g, 1mmol) was dissolved in 6mL of dichloromethane, 4-dimethylaminopyridine (180mg, 1.5mmol) was added, and phenyl p-nitrochloroformate (240mg, 1.2mmol) was added in batches, The reaction was stirred at room temperature for 3 h, compound 5b (557 mg, 1.1 mmol) was added to the reaction solution, and the mixture was stirred overnight at room temperature. After TLC showed that the reaction was complete, dichloromethane was added to dilute, and then washed with saturated brine, and the organic phase was washed with Dry over sodium sulfate, filter and concentrate, and separate by column chromatography to obtain compound 5 (1.3 g, yellow solid), with a yield of 81%.
MS m/z(ESI):1608.7[M+1]MS m/z(ESI):1608.7[M+1]
实施例6化合物6的合成The synthesis of
【含有N3官能团和NHS官能团的PEG衍生物】[PEG derivatives containing N3 functional groups and NHS functional groups]
6.1化合物6a的合成6.1 Synthesis of Compound 6a
将十二甘醇(546.0g,1.0mol)溶于四氢呋喃(5V)中，冰水浴降温，加入叔丁醇钾(11.2g,0.1mol)，然后滴加丙烯酸叔丁酯(38.4g,0.3mol)的四氢呋喃溶液(3V)，自然回温，将混合物搅拌过夜。体系用二氯甲烷萃取，氯化铵水溶液洗涤，经无水硫酸钠干燥，过滤并浓缩，得到产物6a(136.5g)，其不经进一步纯化直接使用。Dodecaethylene glycol (546.0g, 1.0mol) was dissolved in tetrahydrofuran (5V), cooled in an ice-water bath, potassium tert-butoxide (11.2g, 0.1mol) was added, and tert-butyl acrylate (38.4g, 0.3mol) was added dropwise ) in tetrahydrofuran (3V), warmed naturally, and the mixture was stirred overnight. The system was extracted with dichloromethane, washed with aqueous ammonium chloride, dried over anhydrous sodium sulfate, filtered and concentrated to give product 6a (136.5 g), which was used without further purification.
6.2化合物6b的合成6.2 Synthesis of Compound 6b
将化合物6a(136.5g,0.25mol)溶于二氯甲烷(7V)中，冰水浴降温，加入三乙胺(303.6g,3.0mol)，然后滴加对甲苯磺酰氯(286.0g,1.5mol)的二氯甲烷溶液(3V)，自然回温，将混合物搅拌过夜。体系用等体积1N稀盐酸洗涤，再用饱和碳酸氢钠洗涤，有机相经无水硫酸钠干燥，过滤并浓缩，得到产物6b(227.2g)，其不经进一步纯化直接使用。Compound 6a (136.5g, 0.25mol) was dissolved in dichloromethane (7V), cooled in an ice-water bath, triethylamine (303.6g, 3.0mol) was added, and p-toluenesulfonyl chloride (286.0g, 1.5mol) was added dropwise A solution of dichloromethane (3V) was allowed to warm up naturally, and the mixture was stirred overnight. The system was washed with an equal volume of 1N dilute hydrochloric acid and then with saturated sodium bicarbonate, and the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to give product 6b (227.2 g), which was used without further purification.
6.3化合物6c的合成6.3 Synthesis of compound 6c
将化合物6b(227g,0.27mol)溶于四氢呋喃(10V)中，加入叠氮基三甲基硅烷(172.8g,1.5mol)和四丁基氟化铵三水合物(559.0g,2.0mol),混合物在50℃下搅拌过夜。反应加水淬灭，用二氯甲烷萃取，有机相经无水硫酸钠干燥，过滤并浓缩，并通过硅胶柱层析纯化得到产物6c(115.0g)，产率60％。Compound 6b (227g, 0.27mol) was dissolved in tetrahydrofuran (10V), and azidotrimethylsilane (172.8g, 1.5mol) and tetrabutylammonium fluoride trihydrate (559.0g, 2.0mol) were added, The mixture was stirred overnight at 50 °C. The reaction was quenched with water, extracted with dichloromethane, the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated, and purified by silica gel column chromatography to obtain product 6c (115.0 g), yield 60%.
6.4化合物6d的合成6.4 Synthesis of Compound 6d
将化合物6c(115.0g,0.16mol)溶解于2N稀盐酸(5V)中，室温反应至液相检测结束。水相加入15％氯化钠，用等体积二氯甲烷萃取3次，合并有机相，过滤并浓缩，得到产物6d(106.2g)。Compound 6c (115.0 g, 0.16 mol) was dissolved in 2N dilute hydrochloric acid (5V), and reacted at room temperature until the liquid phase detection was completed. The aqueous phase was added with 15% NaCl, extracted three times with an equal volume of dichloromethane, and the organic phases were combined, filtered and concentrated to give the product 6d (106.2 g).
6.5化合物6的合成6.5 Synthesis of
将化合物6d(106.2g,0.16mol)溶于二氯甲烷(10V)中，冰水浴降温，加入N-羟基丁二酰亚胺(120.8g,1.1mol)，然后滴加二环己基碳二亚胺(227.0g,1.1mol)的二氯甲烷溶液(3V)，滴加完毕后室温反应，直至液相检测反应结束。将体系过滤，滤液浓缩，经硅胶柱层析纯化得到产物6(85.9g,淡黄色液体),产率70％。图3和图4分别为化合物6的质谱图和核磁图。Compound 6d (106.2g, 0.16mol) was dissolved in dichloromethane (10V), cooled in an ice-water bath, N-hydroxysuccinimide (120.8g, 1.1mol) was added, and then dicyclohexylcarbodiimide was added dropwise A methylene chloride solution (3V) of amine (227.0 g, 1.1 mol) was added dropwise and reacted at room temperature until the liquid phase detection reaction was completed. The system was filtered, the filtrate was concentrated, and purified by silica gel column chromatography to obtain product 6 (85.9 g, light yellow liquid), with a yield of 70%. Figure 3 and Figure 4 are the mass spectrum and nuclear magnetic spectrum of
MS m/z(ESI):758.6[M+18]1H NMR(300MHz,CDCl3):δ3.87(t,2H,J＝6.3Hz),3.71-3.68(m,44H),3.41(t,2H,J＝4.8Hz),2.92(t,2H,J＝6.3Hz),2.89-2.84(m,4H)MS m/z(ESI):758.6[M+18]1H NMR(300MHz, CDCl3):δ3.87(t,2H,J=6.3Hz),3.71-3.68(m,44H),3.41(t,2H ,J=4.8Hz),2.92(t,2H,J=6.3Hz),2.89-2.84(m,4H)
实施例7化合物7的合成The synthesis of
7.1化合物7a的合成7.1 Synthesis of compound 7a
将NH2-PEG24-PA(38.5g，33.60mmol),DBCO-NHS(9.0g，22.40mmol)和4-二甲氨基吡啶(2.7g，22.40mmol)一起溶于100mL二氯甲烷中，反应室温搅拌8h，液相监测原料反应完全后，用100mL饱和食盐水洗涤一次，再用60mL10％的亚硫酸氢钠溶液洗涤两次，有机相用无水硫酸钠干燥，过滤并浓缩，得到产品7a(25.8g)，产率82％。Dissolve NH2-PEG24-PA (38.5g, 33.60mmol), DBCO-NHS (9.0g, 22.40mmol) and 4-dimethylaminopyridine (2.7g, 22.40mmol) together in 100mL of dichloromethane, and stir at room temperature 8h, after the liquid phase monitors that the raw materials have reacted completely, wash once with 100mL saturated brine, then wash twice with 60mL10% sodium bisulfite solution, dry the organic phase with anhydrous sodium sulfate, filter and concentrate to obtain product 7a (25.8 g), yield 82%.
MS m/z(ESI):1433.8[M+1]MS m/z(ESI):1433.8[M+1]
7.2化合物7b的合成7.2 Synthesis of compound 7b
将化合物7a(10.0g，7.00mmol)和N-羟基琥珀酰亚胺(1.0g，8.40mmol)溶于70mL二氯甲烷中，加入1-(3-二甲基氨基丙基)-3-乙基碳二亚胺(1.6g，8.40mmol)，反应室温搅拌4h，得到化合物7b的反应液，直接用于下一步反应。Compound 7a (10.0 g, 7.00 mmol) and N-hydroxysuccinimide (1.0 g, 8.40 mmol) were dissolved in 70 mL of dichloromethane, and 1-(3-dimethylaminopropyl)-3-ethane Carbodiimide (1.6g, 8.40mmol) was stirred at room temperature for 4h to obtain the reaction solution of compound 7b, which was directly used in the next reaction.
MS m/z(ESI):1530.8[M+1]MS m/z(ESI):1530.8[M+1]
7.3化合物7c的合成7.3 Synthesis of compound 7c
将原料Fmoc-VC-PAB(9.0g，15.0mmol)溶于30mL二甲基亚砜中，加入二乙胺(2.2g，30.0mmol)，反应室温搅拌2h，液相监控反应，原料反应完全后，加入200mL二氯甲烷，有大量固体出现，搅拌30min，过滤收集固体得到产品7c(5.0g)，产率88％。Dissolve the raw material Fmoc-VC-PAB (9.0g, 15.0mmol) in 30mL dimethyl sulfoxide, add diethylamine (2.2g, 30.0mmol), stir at room temperature for 2h, monitor the reaction in liquid phase, after the raw material reacts completely , 200mL of dichloromethane was added, a large amount of solids appeared, stirred for 30min, and the solids were collected by filtration to obtain product 7c (5.0g), with a yield of 88%.
MS m/z(ESI):380.2[M+1]MS m/z(ESI):380.2[M+1]
7.4化合物7d的合成7.4 Synthesis of compound 7d
将化合物7c(3.0g，8.0mmol)加入上述得到的化合物7b的反应液中，加入4-二甲氨基吡啶(1.0g，8.0mmol)，反应室温搅拌2h，原料消耗完全后，加入50mL饱和食盐水洗涤，有机相用无水硫酸钠干燥，旋干溶剂，柱层析分离(DCM:MeOH 30:1-20:1)得到化合物7d(11.0g，黄色油状物)。Compound 7c (3.0g, 8.0mmol) was added to the reaction solution of compound 7b obtained above, 4-dimethylaminopyridine (1.0g, 8.0mmol) was added, and the reaction was stirred at room temperature for 2h. After the raw materials were completely consumed, 50mL of saturated salt was added After washing with water, the organic phase was dried with anhydrous sodium sulfate, spin-dried to dry the solvent, and separated by column chromatography (DCM:MeOH 30:1-20:1) to obtain compound 7d (11.0 g, yellow oil).
MS m/z(ESI):1795.0[M+1]MS m/z(ESI):1795.0[M+1]
7.5化合物7e的合成7.5 Synthesis of compound 7e
将TBS-SN38(2.5g，5.0mmol)溶于30mL二氯甲烷中，加入4-二甲氨基吡啶(0.9g，7.5mmol)，再分批次加入对硝基氯甲酸苯酯(1.2g，6.0mmol)，反应室温搅拌3h，在此反应液中加入化合物7d(10.0g，5.6mmol)，混合物在室温下搅拌过夜，TLC显示反应完成后，加入20mL二氯甲烷稀释，然后用30mL饱和食盐水洗涤，有机相用无水硫酸钠干燥，过滤并浓缩，柱层析分离(DCM:MeOH＝30：1-15:1)得到化合物7e(8.4g，黄色固体)，产率72％。Dissolve TBS-SN38 (2.5g, 5.0mmol) in 30mL of dichloromethane, add 4-dimethylaminopyridine (0.9g, 7.5mmol), and then add phenyl p-nitrochloroformate (1.2g, 6.0mmol), the reaction was stirred at room temperature for 3h, compound 7d (10.0g, 5.6mmol) was added to the reaction solution, the mixture was stirred overnight at room temperature, after TLC showed that the reaction was complete, 20mL of dichloromethane was added to dilute, and then diluted with 30mL of saturated salt Washed with water, the organic phase was dried with anhydrous sodium sulfate, filtered and concentrated, separated by column chromatography (DCM:MeOH=30:1-15:1) to obtain compound 7e (8.4g, yellow solid), yield 72%.
MS m/z(ESI):1164.1[M/2+1]MS m/z(ESI):1164.1[M/2+1]
7.6化合物7的合成7.6 Synthesis of
将化合物7e(7.4g，3.2mmol)溶于50mL四氢呋喃中，加入四丁基氟化铵(0.52g，2.0mmol)，室温下搅拌30min，TLC监控反应进程，反应完成后旋干溶剂，用C-18flash进行纯化制备(MeOH:H2O＝50％-90％)，得到化合物7(4.4g，黄色固体)，产率60％。图5和图6分别为化合物7质谱图和核磁图。Compound 7e (7.4g, 3.2mmol) was dissolved in 50mL tetrahydrofuran, tetrabutylammonium fluoride (0.52g, 2.0mmol) was added, stirred at room temperature for 30min, TLC monitored the reaction progress, after the reaction was completed, the solvent was spin-dried, and the solvent was washed with C -18flash was purified and prepared (MeOH:H2 O=50%-90%) to obtain compound 7 (4.4 g, yellow solid) with a yield of 60%. Figure 5 and Figure 6 are the mass spectrum and nuclear magnetic spectrum of
MS m/z(ESI):1107.4[M/2+1]MS m/z(ESI):1107.4[M/2+1]
实施例8化合物8的合成The synthesis of
将化合物1(1.5g，1.0mmol)溶于10mL二氯甲烷中，加入化合物6(0.78g，1.05mmol)，室温下搅拌3h，HPLC显示原料化合物1反应完全，旋蒸除去溶剂得到粗品。用少量DCM溶解粗品，慢慢加入甲基叔丁基醚至有固体析出，搅拌30分钟后，过滤得到化合物8(1.8g，黄色固体)，产率82％。图7和图8分别为化合物8质谱图和核磁图。Compound 1 (1.5g, 1.0mmol) was dissolved in 10mL of dichloromethane, compound 6 (0.78g, 1.05mmol) was added, stirred at room temperature for 3h, HPLC showed that the reaction of the starting
MS m/z(ESI):1118.3[M/2+1]MS m/z(ESI):1118.3[M/2+1]
实施例9化合物9的合成The synthesis of
【含有N3官能团和NHS官能团的PEG衍生物】[PEG derivatives containing N3 functional groups and NHS functional groups]
9.1化合物9a的合成9.1 Synthesis of compound 9a
将四甘醇(194g,1.0mol)溶于四氢呋喃(5V)中，冰水浴降温，加入叔丁醇钾(11.2g,0.1mol)，然后滴加丙烯酸叔丁酯(38.4g,0.3mol)的四氢呋喃溶液(3V)，自然回温，将混合物搅拌过夜。体系用二氯甲烷萃取，氯化铵水溶液洗涤，经无水硫酸钠干燥，过滤并浓缩，得到产物9a(80.6g)，其不经进一步纯化直接使用。Dissolve tetraethylene glycol (194g, 1.0mol) in tetrahydrofuran (5V), cool down in an ice-water bath, add potassium tert-butoxide (11.2g, 0.1mol), and then dropwise add tert-butyl acrylate (38.4g, 0.3mol) Tetrahydrofuran solution (3V), naturally warmed up, and the mixture was stirred overnight. The system was extracted with dichloromethane, washed with aqueous ammonium chloride, dried over anhydrous sodium sulfate, filtered and concentrated to give product 9a (80.6 g), which was used without further purification.
9.2化合物9b的合成9.2 Synthesis of compound 9b
将化合物9a(80.6g,0.25mol)溶于二氯甲烷(7V)中，冰水浴降温，加入三乙胺(303.6g,3.0mol)，然后滴加对甲苯磺酰氯(286.0g,1.5mol)的二氯甲烷溶液(3V)，自然回温，将混合物搅拌过夜。体系用等体积1N稀盐酸洗涤，再用饱和碳酸氢钠洗涤，有机相经无水硫酸钠干燥，过滤并浓缩，得到产物9b(128.7g)，其不经进一步纯化直接使用。Compound 9a (80.6g, 0.25mol) was dissolved in dichloromethane (7V), cooled in an ice-water bath, triethylamine (303.6g, 3.0mol) was added, and p-toluenesulfonyl chloride (286.0g, 1.5mol) was added dropwise A solution of dichloromethane (3V) was allowed to warm up naturally, and the mixture was stirred overnight. The system was washed with an equal volume of 1N dilute hydrochloric acid and then with saturated sodium bicarbonate, and the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to give product 9b (128.7 g), which was used without further purification.
9.3化合物9c的合成9.3 Synthesis of compound 9c
将化合物9b(128g,0.27mol)溶于四氢呋喃(10V)中，加入叠氮基三甲基硅烷(172.8g,1.5mol)和四丁基氟化铵三水合物(559.0g,2.0mol),混合物在50℃下搅拌过夜。反应加水淬灭，用二氯甲烷萃取，有机相经无水硫酸钠干燥，过滤并浓缩，并通过硅胶柱层析纯化得到产物9c(55.5g)，产率60％。Compound 9b (128g, 0.27mol) was dissolved in tetrahydrofuran (10V), and azidotrimethylsilane (172.8g, 1.5mol) and tetrabutylammonium fluoride trihydrate (559.0g, 2.0mol) were added, The mixture was stirred overnight at 50 °C. The reaction was quenched with water, extracted with dichloromethane, the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated, and purified by silica gel column chromatography to obtain product 9c (55.5 g), yield 60%.
9.4化合物9d的合成9.4 Synthesis of compound 9d
将化合物9c(55.5g,0.16mol)溶解于2N稀盐酸(5V)中，室温反应至液相检测结束。水相加入15％氯化钠，用等体积二氯甲烷萃取3次，合并有机相，过滤并浓缩，得到产物9d(46.5g)。Compound 9c (55.5 g, 0.16 mol) was dissolved in 2N dilute hydrochloric acid (5V), and reacted at room temperature until the liquid phase detection was completed. The aqueous phase was added with 15% NaCl, extracted three times with an equal volume of dichloromethane, and the organic phases were combined, filtered and concentrated to give product 9d (46.5 g).
9.5化合物9的合成9.5 Synthesis of
将化合物9d(46.5g,0.16mol)溶于二氯甲烷(10V)中，冰水浴降温，加入N-羟基丁二酰亚胺(120.8g,1.1mol)，然后滴加二环己基碳二亚胺(227.0g,1.1mol)的二氯甲烷溶液(3V)，滴加完毕后室温反应，直至液相检测反应结束。将体系过滤，滤液浓缩，经硅胶柱层析纯化得到产物9(43.4g,淡黄色液体),产率70％。图9和图10分别为化合物9的质谱图和核磁图。Compound 9d (46.5g, 0.16mol) was dissolved in dichloromethane (10V), cooled in an ice-water bath, N-hydroxysuccinimide (120.8g, 1.1mol) was added, and then dicyclohexylcarbodiimide was added dropwise A methylene chloride solution (3V) of amine (227.0 g, 1.1 mol) was added dropwise and reacted at room temperature until the liquid phase detection reaction was completed. The system was filtered, the filtrate was concentrated, and purified by silica gel column chromatography to obtain product 9 (43.4 g, light yellow liquid), with a yield of 70%. Figure 9 and Figure 10 are the mass spectrum and nuclear magnetic spectrum of
MS m/z(ESI):406.3[M+18]MS m/z(ESI):406.3[M+18]
实施例10化合物10的合成The synthesis of
将化合物1(1.5g，1.0mmol)溶于10mL二氯甲烷中，加入化合物9(0.41g，1.05mmol)，室温下搅拌3h，HPLC显示原料化合物1反应完全，旋蒸除去溶剂得到粗品。用少量DCM溶解粗品，慢慢加入甲基叔丁基醚至有固体析出，搅拌30分钟后，过滤得到化合物10(1.6g，黄色固体)，产率85％。图11和图12分别为化合物10质谱图和核磁图。Compound 1 (1.5g, 1.0mmol) was dissolved in 10mL of dichloromethane, compound 9 (0.41g, 1.05mmol) was added, stirred at room temperature for 3h, HPLC showed that the reaction of the starting
MS m/z(ESI):942.4[M/2+1]MS m/z(ESI):942.4[M/2+1]
实施例11化合物11的合成The synthesis of
将化合物1(1.5g，1.0mmol)溶于10mL二氯甲烷中，加入化合物9(0.41g，1.05mmol)，室温下搅拌3h，HPLC显示原料化合物1反应完全，旋蒸除去溶剂得到粗品。用少量DCM溶解粗品，慢慢加入甲基叔丁基醚至有固体析出，搅拌30分钟后，过滤得到化合物11(2.1g，黄色固体)，产率83％。图13和图14分别为化合物11质谱图和核磁图。Compound 1 (1.5g, 1.0mmol) was dissolved in 10mL of dichloromethane, compound 9 (0.41g, 1.05mmol) was added, stirred at room temperature for 3h, HPLC showed that the reaction of the starting
MS m/z(ESI):1301.5[M/2+1]MS m/z(ESI):1301.5[M/2+1]
实施例12抗体偶联物的制备The preparation of
12.1两步法制备抗体偶联药物12.1 Two-step preparation of antibody-drug conjugates
1.mAb与化合物6反应过程1. Reaction process of mAb and
【抗体与含有N3官能团和NHS官能团的PEG衍生物反应，使抗体带有N3官能团标记。】[Antibody reacts with PEG derivatives containing N3 functional group and NHS functional group, so that the antibody is labeled with N3 functional group. 】
按照mAb与化合物6的摩尔比为1:20的投料比进行反应。先量取208μL的mAb溶液(浓度：9.6mg/mL)置于1.5mL的离心管中，则量取的mAb为2mg。使用5mM pH3.0的PBS溶液配制10mg/mL的化合物6溶液，量取20μL加入到离心管中，充分混合，震荡反应2h。反应完毕后用超滤的方式去除未反应的化合物6。The reaction was carried out according to the molar ratio of mAb to compound 6 which was 1:20. First measure 208 μL of mAb solution (concentration: 9.6 mg/mL) and place it in a 1.5 mL centrifuge tube, then the measured mAb is 2 mg. A 10 mg/
2.mAb-PEG12与linker-drug反应过程2. Reaction process between mAb-PEG12 and linker-drug
取制备好的mAb-PEG12样品，测定蛋白浓度为2.05mg/mL，量取490μL样品溶液置于1.5mL的离心管中。称量10mg linker-drug，溶解在DMSO中，制备成1mg/mL的溶液。按照mAb与linker-drug投料比为1:4计算，量取41μL化合物1溶液加入到离心管中，再向离心管中加入510μL的PBS和59μL的DMSO，保持蛋白浓度为1mg/mL，水与DMSO的比例为10:1，充分混合，震荡反应2h。反应完毕后用超滤的方式去除未反应的小分子，如下表所示Take the prepared mAb-PEG12 sample, determine the protein concentration to be 2.05 mg/mL, measure 490 μL of the sample solution and place it in a 1.5 mL centrifuge tube. Weigh 10 mg linker-drug, dissolve in DMSO, and prepare a 1 mg/mL solution. According to the calculation that the ratio of mAb to linker-drug is 1:4, measure 41 μL of
表1Linker-drug的种类以及对应的ADC分子的名称Table 1 Types of Linker-drugs and names of corresponding ADC molecules
其中，化合物1为实施例1制备，化合物4为实施例4制备，DBCO-VC-PAB-SN38，为自制，纯度90％，MS m/z(ESI):1087.4[M+1]，结构式如下所示：Among them,
12.2一步法制备抗体偶联药物12.2 One-step preparation of antibody-drug conjugates
按照mAb与化合物10的摩尔比为1:20的投料比进行反应。先量取1mL的mAb溶液(浓度：2.0mg/mL)置于1.5mL的离心管中，则量取的mAb为2mg。使用DMSO配制5.21mg/mL的化合物11溶液，量取100μL加入到离心管中，充分混合，震荡反应2h。反应完毕后用超滤的方式去除未反应的化合物11，得到ADC4。The reaction was carried out according to the molar ratio of mAb to compound 10 of 1:20.
实施例13DAR值的测量The measurement of embodiment 13DAR value
用液相的方法检测实施例12所制ADC的DAR值。The DAR value of the ADC prepared in Example 12 was detected by a liquid phase method.
液相色谱设置如下：The LC settings were as follows:
表2液相色谱条件Table 2 Liquid Chromatography Conditions
溶解实施例12制备的ADC1、ADC2、ADC3、ADC4，按照表2中的液相色谱条件对ADC1、ADC2、ADC3、ADC4进行检测，该液相色谱条件下SN38与ADC1、ADC2、ADC3、ADC4分离度大于1.5。制备不同浓度标准曲线，建立SN38峰面积与浓度之间的线性标准曲线，分别对ADC 1、ADC 2、ADC3和ADC4上的SN38进行定量。根据ADC的抗体浓度和SN38的浓度计算ADC上SN38的偶联个数。测定实施例12制备的ADC 1的DAR值为4，ADC2的DAR值为4，ADC3的DAR值为2，ADC4的DAR值为4，ADC 1、ADC2和ADC4的DAR值均高于ADC3，实验结果表明，相较于不含PEG基团的连接子，该发明获得的ADC1、ADC2和ADC4的DAR值更高。Dissolve the ADC1, ADC2, ADC3, and ADC4 prepared in Example 12, and detect ADC1, ADC2, ADC3, and ADC4 according to the liquid chromatography conditions in Table 2. Under the liquid chromatography conditions, SN38 is separated from ADC1, ADC2, ADC3, and ADC4 The degree is greater than 1.5. Prepare standard curves of different concentrations, establish a linear standard curve between the peak area of SN38 and the concentration, and quantify SN38 on
实施例14ADC与EGFR的亲和力测定The affinity determination of embodiment 14 ADC and EGFR
ADC药物的亲和力用SPR的方法进行检测，试验仪器为GE公司的产品biacore。简单操作步骤为：将EGFR抗原偶联到CM芯片上。用不同浓度的ADC药物或单抗药物测试ADC药物的抗体或游离的抗体与抗原的亲和力。结果显示实施例12制备的ADC1、ADC2和ADC3，其抗体的亲和力下降不是很明显，ADC1和ADC2的亲和力高于ADC3。The affinity of the ADC drug is detected by the method of SPR, and the test instrument is biacore, a product of GE Company. The simple operation steps are: coupling the EGFR antigen to the CM chip. Use different concentrations of ADC drugs or monoclonal antibody drugs to test the affinity of ADC drug antibodies or free antibodies to antigens. The results showed that ADC1, ADC2 and ADC3 prepared in Example 12 had less obvious decrease in the affinity of the antibodies, and the affinity of ADC1 and ADC2 was higher than that of ADC3.
表3实验结果：Table 3 Experimental results:
实施例15体外细胞毒性实验Example 15 In vitro cytotoxicity test
用BXPC-3(人胰腺癌细胞)细胞进行体外细胞毒性测试。试验操作如下：In vitro cytotoxicity tests were performed with BXPC-3 (human pancreatic cancer cell) cells. The test operation is as follows:
1、96孔板每孔加入细胞100μL(留2个空白组不加细胞，加入同体积的培养基)。细胞置于37℃的5％CO2细胞培养箱中培养24h。细胞毒性实验每孔加入100μL约含10000个细胞。1. Add 100 μL of cells to each well of the 96-well plate (keep 2 blank groups without adding cells, and add the same volume of medium). The cells were cultured in a 5% CO2 incubator at 37°C for 24 hours. For cytotoxicity experiments, add 100 μL to each well to contain about 10,000 cells.
2、每孔加入10μL不同浓度ADC或SN38的药物。2. Add 10 μL of ADC or SN38 drugs with different concentrations to each well.
3、将96孔板在37℃，含5％CO2空气及100％湿度的细胞培养箱中孵育24小时。3. Incubate the 96-well plate at 37°C for 24 hours in a cell culture incubator containing 5% CO2 air and 100% humidity.
4、每孔加入10μL的CCK-8溶液。37℃，5％CO2培养箱中孵育3h。4. Add 10 μL of CCK-8 solution to each well. Incubate for 3 hours at 37°C in a 5% CO2 incubator.
5、酶标仪测定450nm处的吸光度。5. Measure the absorbance at 450nm with a microplate reader.
6、结果分析：6. Result analysis:
A.细胞存活率：将各测试孔的OD值减去本底OD值(空白组)，各重复孔的OD值取平均数±SD。细胞存活率％＝(加药细胞OD/对照细胞OD)×100％。A. Cell survival rate: subtract the background OD value (blank group) from the OD value of each test well, and take the mean ± SD of the OD value of each repeated well. Cell viability%=(OD of drug-added cells/OD of control cells)×100%.
B.求出T/C＝50％时的药物浓度(IC50)及T/C＝10％时的药物浓度(IC90)。B. Determine the drug concentration (IC50) when T/C=50% and the drug concentration (IC90) when T/C=10%.
结果显示实施例12制备的ADC 1和ADC2的IC50值均小于ADC3的IC50值，小于SN38的IC50值。The results showed that the IC50 values of
表4细胞毒性结果：Table 4 Cytotoxicity Results:
结果表明，该发明获得的ADC1和ADC2不仅能够有效地发挥EGFR抗体部分的生物学功能，还兼有SN38对肿瘤细胞的高效杀伤活性。相较于传统的不含PEG的二肽linker制备得到的ADC3，本发明得到的含有PEGlinker的ADC1和ADC2具有更优的活性。The results show that the ADC1 and ADC2 obtained by the invention can not only effectively play the biological function of the EGFR antibody part, but also have the high-efficiency killing activity of SN38 on tumor cells. Compared with ADC3 prepared by the traditional dipeptide linker without PEG, the ADC1 and ADC2 containing PEG linker obtained by the present invention have better activity.
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