CN116284054B - 一种海鞘素类化合物、其抗体药物偶联物及其应用 - Google Patents
一种海鞘素类化合物、其抗体药物偶联物及其应用 Download PDFInfo
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Abstract
本发明公开了一种海鞘素类化合物、其抗体药物偶联物及其应用。本发明提供了一种海鞘素类化合物或其药学上可接受的盐。本发明的抗体药物偶联物,可以实现肿瘤富集,消除或降低海鞘素作用于非疾病组织而引起的毒副作用,提高治疗效果。
Description
技术领域
本发明涉及一种海鞘素类化合物、其抗体药物偶联物及其应用。
背景技术
TMALIN™(Tumor Microenviroment Activable LINKER,肿瘤微环境可激活连接子)技术是由苏州宜联生物开发的全新linker,兼具高体内循环稳定性、可以利用肿瘤微环境和传统溶酶体进行胞外胞内双重裂解、高水溶性、能够主动进行肿瘤组织富集等多个新功能,以之形成的ADC分子会产生意想不到的效果。
海鞘素(ecteinascidins)是一类天然产物。1990年,伊利诺伊大学的KL Rinehart实验室从加勒比海鞘中分离出了六种海鞘素,而其中一种海鞘素为曲贝替定。 2015年,曲贝替定获得了FDA批准上市,用于接受过蒽环类药物治疗的患有不可切除或转移脂肪肉瘤或平滑肌肉瘤患者的治疗。除了软组织肉瘤,曲贝替定也在欧洲,加拿大等地区获得了监管机构批准用于治疗铂类药物敏感的复发性卵巢癌。后续的研究发现曲贝替定作用机制比较独特。与紫杉醇以及艾日布林抑制微管的作用机制不同,曲贝替定能够通过结合DNA来阻止细胞分裂:两个稠合的四氢异喹啉环能够与DNA小沟的特定序列产生相互作用并与之产生共价结合,使DNA的双螺旋产生异常,并干扰TC-NER系统中XPG蛋白的正常功能,从而诱发DNA双链断裂并导致细胞死亡。卢比替定是PharmaMar研发的一个海鞘素衍生物,为抗小细胞肺癌的创新药。该药物的临床数据,研究共分析了105例既往一线化疗进展的广泛期SCLC患者,用卢比替定3.2mg/m2,1小时静脉给药,3周1次。结果显示,二线治疗的客观有效率达到了35.2%,疾病控制率为68.6%。中位反应持续时间为5.3个月,患者中位生存期为9.3个月,1年的生存率为34.2%。最常见的1/2级不良反应包括疲劳(51.4%),恶心(32.4%),食欲下降(21.0%),呕吐(18.1%),腹泻(12.4%),便秘(9.5%),和中性粒细胞减少症(5.7%)。3/4级严重不良反应包括中性粒细胞减少症,贫血,疲劳,血小板减少症,发热性中性粒细胞减少症,肺炎,丙氨酸转氨酶水平升高,皮肤溃疡和腹泻。
因此,有必要寻找更好的海鞘素类药物。
发明内容
本发明所要解决的技术问题是现有的海鞘素类化合物种类较少,为此本发明提供了一种海鞘素类化合物、其抗体药物偶联物及其应用。TMALINTM技术进一步提升了抗体药物偶联物的体内循环稳定性,可在肿瘤细胞胞外胞内同时释放毒素,还能够主动进行肿瘤组织富集,且水溶性好,因此,利用该技术组合海鞘素和肿瘤靶向抗体形成的抗体药物偶联物,可以实现肿瘤富集,消除或降低海鞘素作用于非疾病组织而引起的毒副作用,提高治疗效果。
本发明提供了一种海鞘素类化合物或其药学上可接受的盐;所述的海鞘素类化合物为如下任一结构:
。
本发明还提供了一种海鞘素类化合物或其药学上可接受的盐;所述的海鞘素类化合物为如下任一结构:
。
本发明还提供了一种如式II所示的药物连接子偶联物;
其中,L’为连接子前体;
D为上述的海鞘素类化合物失去氢原子形成的基团;所述的氢原子为片段中羟基上的氢原子。
所述的连接子前体由连接子和离去基团组成,其与抗体连接后形成连接子。
所述的连接子为抗体药物偶联物领域常规的连接子,如可裂解连接子或不可裂解连接子。
在一些实施方案中,所述的连接子为如下任一结构:
其中,1位与所述的离去基团相连,2位与所述的D相连。
在一些实施方案中,所述的离去基团为甲砜基。
本发明还提供了一种药物连接子偶联物,其为如下任一结构:
。
本发明还提供了一种如式III所示的抗体药物偶联物;
其中,Tb为抗体或其抗原结合片段;
q为0-20的整数或小数,但不为0;
L为连接子;
D为上述的海鞘素类化合物失去氢原子形成的基团;所述的氢原子为片段中羟基上的氢原子。
在一些实施方案中,所述的抗体或其抗原结合片段为Fab、Fab'、F(ab')2、Fd、Fv、dAb、互补决定区片段、单链抗体(例如,scFv)、非人抗体、人源化抗体、嵌合抗体、全人抗体、前抗(Probody)、双特异性抗体或多特异性抗体。
在一些实施方案中,所述的Tb为具有内吞活性的抗体或其抗原结合片段。
在一些实施方案中,所述的Tb为具有结合肿瘤细胞表面抗原活性的抗体或其抗原结合片段。
在一些实施方案中,所述的Tb为抗B7H3抗体或其抗原结合片段、抗Trop-2抗体或者其抗原结合片段或抗Her 2抗体或其抗原结合片段。
在一些实施方案中,所述的Tb为抗B7H3抗体或其抗原结合片段,例如1D1-01抗体、2E3-02抗体、enoblituzumab、mirzotamab、omburtamab或其抗原结合片段;
所述1D1-01抗体如WO2022170971A1第194页记载;
所述2E3-02抗体如WO2022170971A1第194页记载。
在一些实施方案中,所述的Tb为抗B7H3的2E3-02抗体,所述2E3-02抗体如WO2022170971A1第194页记载。
在一些实施方案中,q选自1-12之间的任意整数,较佳地,q选自1-8之间的任意整数。
在一些实施方案中,q选自2-8之间的任意整数或小数。
在一些实施方案中,q选自2、4、6和8。
在一些实施方案中,q选自6、7和8。
所述的连接子为抗体药物偶联物领域常规的连接子,如可裂解连接子或不可裂解连接子。
在一些实施方案中,所述的连接子为如下任一结构:
其中,1位通过所述的Tb中的S原子与所述的Tb相连,2位与所述的D相连。
在一些实施方案中,如式III所示的抗体药物偶联物为如下任一结构:
其中,所述的B7H3 mAb为抗B7H3的2E3-02抗体,所述2E3-02抗体如WO2022170971A1第194页记载;q为7.9~8。
本发明还提供了一种药物组合物,其由物质X和药用辅料组成;
所述的物质X为物质X1或物质X2;所述的物质X1为上述的海鞘素类化合物或其药学上可接受的盐;所述的物质X2为上述的如式III所示的抗体药物偶联物。
本发明还提供了一种物质X在制备药物中的应用;
所述的物质X为物质X1或物质X2;所述的物质X1为上述的海鞘素类化合物或其药学上可接受的盐;所述的物质X2为上述的如式III所示的抗体药物偶联物;
所述的药物为用于治疗和/或预防与细胞活动异常相关的疾病的药物。
在一些实施方案中,所述的与细胞活动异常相关的疾病为癌症。
在一些实施方案中,所述的癌症为实体肿瘤或血液肿瘤。
在一些实施方案中,所述的癌症为与Her2、Trop2或B7H3相关的癌症。
在一些实施方案中,所述的癌症为与B7H3相关的癌症。
在一些实施方案中,所述的癌症为食管癌(例如食管腺癌和食管鳞状细胞癌)、脑瘤、肺癌(例如小细胞性肺癌和非小细胞性肺癌)、鳞状上皮细胞癌、膀胱癌、胃癌、卵巢癌、腹膜癌、胰腺癌、乳腺癌、头颈癌、子宫颈癌、子宫内膜癌、结直肠癌、肝癌、肾癌、尿路上皮癌、实体瘤、非霍奇金淋巴瘤、中枢神经系统肿瘤(例如神经胶质瘤、多形性胶质母细胞瘤、胶质瘤或肉瘤)、前列腺癌、甲状腺癌、结肠癌或黑色素瘤。
在一些实施方案中,所述的癌症为结肠癌或黑色素瘤。
本发明还提供了一种物质X在制备药物中的应用;
所述的物质X为物质X1或物质X2;所述的物质X1为上述的海鞘素类化合物或其药学上可接受的盐;所述的物质X2为上述的如式III所示的抗体药物偶联物;
所述的药物为用于治疗和/或预防癌症的药物。
在一些实施方案中,所述的癌症为实体肿瘤或血液肿瘤。
在一些实施方案中,所述的癌症为与Her2、Trop2或B7H3相关的癌症。
在一些实施方案中,所述的癌症为与B7H3相关的癌症。
在一些实施方案中,所述的癌症为食管癌(例如食管腺癌和食管鳞状细胞癌)、脑瘤、肺癌(例如小细胞性肺癌和非小细胞性肺癌)、鳞状上皮细胞癌、膀胱癌、胃癌、卵巢癌、腹膜癌、胰腺癌、乳腺癌、头颈癌、子宫颈癌、子宫内膜癌、结直肠癌、肝癌、肾癌、尿路上皮癌、实体瘤、非霍奇金淋巴瘤、中枢神经系统肿瘤(例如神经胶质瘤、多形性胶质母细胞瘤、胶质瘤或肉瘤)、前列腺癌、甲状腺癌、结肠癌或黑色素瘤。
在一些实施方案中,所述的癌症为结肠癌或黑色素瘤。
本发明还提供了一种治疗和/或预防与细胞活动异常相关的疾病的方法,其包括向患者施用治疗有效量的物质X;
所述的物质X为物质X1或物质X2;所述的物质X1为上述的海鞘素类化合物或其药学上可接受的盐;所述的物质X2为上述的如式III所示的抗体药物偶联物。
在一些实施方案中,所述的与细胞活动异常相关的疾病为癌症。
在一些实施方案中,所述的癌症为实体肿瘤或血液肿瘤。
在一些实施方案中,所述的癌症为与Her2、Trop2或B7H3相关的癌症。
在一些实施方案中,所述的癌症为与B7H3相关的癌症。
在一些实施方案中,所述的癌症为食管癌(例如食管腺癌和食管鳞状细胞癌)、脑瘤、肺癌(例如小细胞性肺癌和非小细胞性肺癌)、鳞状上皮细胞癌、膀胱癌、胃癌、卵巢癌、腹膜癌、胰腺癌、乳腺癌、头颈癌、子宫颈癌、子宫内膜癌、结直肠癌、肝癌、肾癌、尿路上皮癌、实体瘤、非霍奇金淋巴瘤、中枢神经系统肿瘤(例如神经胶质瘤、多形性胶质母细胞瘤、胶质瘤或肉瘤)、前列腺癌、甲状腺癌、结肠癌或黑色素瘤。
在一些实施方案中,所述的癌症为结肠癌或黑色素瘤。
在本公开中,除非另有说明,否则本公开中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且,本公开中所用的细胞培养、分子遗传学、核酸化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时,为了更好地理解本公开,下面提供相关术语的定义和解释。
术语“药学上可接受的盐”是指化合物与药学上可接受的酸或碱反应得到的盐。当化合物中含有相对酸性的官能团时,可以通过在合适的惰性溶剂中用足量的药学上可接受的碱与化合物接触的方式获得碱加成盐。当化合物中含有相对碱性的官能团时,可以通过在合适的惰性溶剂中用足量的药学上可接受的酸与化合物接触的方式获得酸加成盐。具体可参见Handbook of Pharmaceutical Salts: Properties, Selection, and Use (P.Heinrich Stahl, Camille G. Wermuth, 2011, 2nd Revised Edition)。
结构片段中的“”是指该结构片段通过该位点与分子其余部分相连。例如,是指环己基。
术语“药用辅料”是指除活性药物成分以外,包含在药物制剂中的所有物质,一般分为赋形剂和附加剂两大类。具体可参见《中华人民共和国药典(2020年版)》、Handbook ofPharmaceutical Excipients (Paul J Sheskey, Bruno C Hancock, Gary P Moss,David J Goldfarb, 2020, 9th Edition)。
术语“治疗”是指消除病因或缓解症状。
术语“预防”是指降低发生疾病的风险。
术语“患者”是指需要接受治疗或预防疾病的任何动物,通常是哺乳动物,例如人类。哺乳动物包括但不限于:牛、马、羊、猪、猫、狗、小鼠、大鼠、家兔、豚鼠、猴、人类等。
术语“治疗有效量”是指给予患者的、足以有效治疗疾病的量。治疗有效量将根据化合物种类、疾病种类、疾病的严重度、患者的年龄等变化,但可由本领域技术人员视情况调整。
术语“抗体药物偶联物”是指生物活性化合物片段(药物分子)与抗体或其抗原结合片段部分连接得到的物质。在本公开的部分实施方案中,生物活性化合物片段与靶向部分通过连接子相连。所述连接子在特定环境(例如胞内低pH值环境)中或特定作用(例如溶酶体蛋白酶的作用)下能够断裂,从而使生物活性化合物片段与靶向部分或抗体或其抗原结合片段分离。在本公开的部分实施方案中,所述连接子包含可裂解或不可裂解的单元,例如肽或二硫键。在本公开的部分实施方案中,生物活性化合物片段与靶向部分或抗体或其抗原结合片段直接通过共价键相连,所述共价键在特定环境或作用下能够断裂,从而使生物活性化合物片段与抗体或其抗原结合片段部分分离。
术语“连接子”是指将生物活性化合物片段(药物分子)与抗体部分连接起来的片段。
所述的“…化合物片段”是指本领域中均知的,抗体-药物偶联物(或称抗体偶联药物(antibody-drug conjugate, ADC))中,在肿瘤组织间或肿瘤细胞内连接子裂解/降解/酶切后,能够形成具有生物活性的药物(例如小分子细胞毒药物,所述药物包括其失去一个原子或原子团后的基团)。为了避免歧义,“药物”并非仅指已获得医药监管部门审批的“药品”,还包括临床中,或者研发和学术研究中任何有潜在治疗生物活性的化合物。
对于“通过S原子与Tb相连”,本领域技术人员可以理解的是,L与打开二硫键(例如,通过还原剂TCEP还原二硫键可以打开二硫键,生成巯基-SH)后的Tb(如抗体)自身所含有巯基进行连接,也就是说,L与Tb之间的-S-并非另外再外接的硫原子。例如,其中,-S-并非另外外接的硫原子,而是打开双硫键后的Tb自身所含有巯基与L例如的1位进行连接后形成的-S-。
术语“抗体”取其最广义的解释,包括完整的单克隆抗体、多克隆抗体以及由至少两个完整抗体形成的多特异性抗体(例如双特异性抗体),只要它们具有所需的生物学活性。在本公开中,“抗体”和“免疫球蛋白”可以互换使用。
术语“单克隆抗体”指抗体来自一群基本均一的抗体,即构成该集群的各抗体完全相同,除了可能存在的少量天然突变。单克隆抗体具有针对抗原的一个决定簇(表位)的高特异性,而与其相对的多克隆抗体则包含针对不同决定簇(表位)的不同抗体。除了特异性之外,单克隆抗体的优点还在于合成时可以不受其他抗体的污染。此处修饰语“单克隆”表示该抗体的特征在于来自一个基本均一的抗体群,而不应理解成需由特殊方法制得。
在本公开的部分实施方案中,单克隆抗体还特别包括嵌合抗体,即重链和/或轻链的一部分与某种、某类或某亚类抗体相同或同源,其余部分则与另一种、另一类或另一亚类抗体相同或同源,只要它们具有所需的生物学活性(参见例如US 4,816,567;和Morrison等人,1984,PNAS,81: 6851-6855)。可用于本公开的嵌合抗体包括灵长类化(primatized)抗体,其包含来自非人灵长类(例如古猴、猩猩等)的可变区抗原结合序列和人恒定区序列。
术语“抗体片段”是指抗体的一部分,优选是抗原结合区或可变区。抗体片段的实例包括Fab、Fab′、F(ab′)2、Fd、Fv、dAb和互补决定区片段,二抗体(diabody),线性抗体和单链抗体分子。
术语“双特异性抗体”亦称为“双功能抗体偶联物”,是指由第一抗体(片段)和第二抗体(片段)通过偶联臂所形成的偶联物,该偶联物保留了各自抗体的活性,故具有双功能和双特异性。
术语“多特异性抗体”包括例如三特异性抗体和四特异性抗体,前者是具有三种不同抗原结合特异性的抗体,而后者是具有四种不同抗原结合特异性的抗体。
术语“完整抗体”指包含抗原结合可变区和轻链恒定区(CL)、重链恒定区(CH1、CH2和CH3)的抗体。恒定区可以是天然序列(例如人天然恒定区序列)或其氨基酸序列变体。完整抗体优选是具有一种或多种效应功能的完整抗体。
术语“前抗(Probody)”是一种修饰的抗体,包括一种抗体或一种抗体片段,能专门与其靶点结合,能够与掩蔽基团耦合,其中掩蔽基团指对抗体或抗体片段与其靶点的结合能力的裂解常数比没有耦合掩蔽基团的抗体或抗体片段与其靶点的结合能力的裂解常数至少大100倍或1000倍、或者10000倍。
在本公开中,非人(例如鼠)抗体的“人源化”形式指包含最少量非人免疫球蛋白序列的嵌合抗体。大多数人源化抗体是人接受者免疫球蛋白的超变区残基被置换成具有所需特异性、亲和力和功能的非人(例如小鼠、大鼠、兔或非人灵长类)超变区残基(供者抗体)。在一些实施方案中,人免疫球蛋白的框架区(FR)残基也被置换成非人残基。而且,人源化抗体还可以包含受者抗体或供者抗体中没有的残基。这些修饰是为了进一步优化抗体的性能。人源化抗体一般包含至少一个,通常是两个可变区,其中所有或几乎所有超变环(hypervanable loops)与非人免疫球蛋白的相对应,而FR则完全或几乎完全是人免疫球蛋白的序列。人源化抗体还可以包含免疫球蛋白恒定区(Fc,通常是人免疫球蛋白Fc)的至少一部分。有关细节参见例如Jones等人,1986,Nature,321: 522-525;Riechmann等人,1988,Nature,332: 323-329;和Presta,1992,Curr Op Struct Bwl 2: 593-596。
完整抗体可根据重链恒定区的氨基酸序列分为不同的“类”。主要的五类是IgA、IgD、IgE、IgG和IgM,其中几类还可以分为不同的“亚类”(同种型),例如IgG1、IgG2、IgG3、IgG4、IgA1和IgA2。抗体不同类的重链恒定区分别称为α、β、ε、γ和μ。免疫球蛋白不同类的亚基结构和三维构型是本领域中公知的。
在本公开中,尽管大多数情况下抗体中的氨基酸是L-氨基酸,但也不限于此。在一些实施方案中,抗体肽链中可以包括一个或多个D-氨基酸。包含D-氨基酸的肽在口腔、肠道或血浆中比仅包含L-氨基酸的肽更加稳定而不易降解。
本公开所用的单克隆抗体可以由许多方法生产。例如,用于本公开的单克隆抗体可以通过杂交瘤方法,使用许多物种(包括小鼠、仓鼠、大鼠和人的细胞)获得(参见例如Kohler等人,1975,Nature,256: 495),或者通过重组DNA技术制得(参见例如US 4,816,567),或者从噬菌体抗体库中分离得到(参见例如Clackson等人,1991,Nature,352: 624-628;和Marks等人,1991,Journal of Molecular Biology,222: 581-597)。
在本公开的部分实施方案中,Tb为曲妥珠单抗或帕妥珠单抗(Pertuzumab)。曲妥珠单抗是抗Her 2的单克隆抗体,其氨基酸序列是本领域技术人员已知的,其示意性序列可参见,例如CN103319599。帕妥珠单抗的示例性的重链序列和轻链序列可参见US7560111。末位Lys是容易缺失的但不影响生物活性,参见Dick, L. W.等人,Biotechnol. Bioeng.,100: 1132-1143。
在本公开的部分实施方案中,抗Trop-2抗体为记载于美国专利第7,517,964号中的RS7(即本公开Sacituzumab);以及记载于US2012/0237518中的hRS7(即本公开Sacituzumab)。可用于本公开的抗Trop-2抗体还可以通过CN103476941A中公开的载体设计、构建和构建展示抗体的抗体库的方法筛选获得,也可以索伦托医疗公司(SorrentoTherapeutics, Inc.)的G-MAB®文库进行筛选获得。
在本公开中,ErbB2和Her2/neu可互换使用,二者均表示天然序列的人Her2蛋白(Genebank登录号:X03363,参见例如Semba等人,1985,PNAS,82: 6497-6501;和Yamamoto等人,1986,Nature,319: 230-234)及其功能性衍生物,例如氨基酸序列变体。ErbB2表示编码人Her2的基因,neu表示编码大鼠p185neu的基因。在部分实施方案中,本公开的化合物或偶联物能够抑制或杀伤表达ErbB2受体的细胞,例如乳腺癌细胞、卵巢癌细胞、胃癌细胞、子宫内膜癌细胞、唾液腺癌细胞、肺癌细胞、肾癌细胞、结肠癌细胞、甲状腺癌细胞、胰腺癌细胞、膀胱癌细胞或肝癌细胞。
在本公开中Trop-2或TROP2是指人滋养层细胞表面抗原-2 (human trophoblastcell-surface antigens 2),又称为TACSTD2、M1S1、GA733-1、EGP-1,其是由许多人类肿瘤(如乳腺癌、结直肠癌、肺癌、胰腺癌、卵巢癌、前列腺癌、宫颈癌)细胞表达的细胞表面受体。在部分实施方案中,本公开的化合物或偶联物能够抑制或杀伤表达TROP2受体的细胞,例如乳腺癌细胞、结直肠癌细胞、肺癌细胞、胰腺癌细胞、卵巢癌细胞、前列腺癌细胞或宫颈癌细胞。
在不违背本领域常识的基础上,上述各优选条件,可任意组合,即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于:TMALINTM技术进一步提升了抗体药物偶联物的体内循环稳定性,可在肿瘤细胞胞外胞内同时释放毒素,还能够主动进行肿瘤组织富集,且水溶性好,因此期待利用该技术组合海鞘素和肿瘤靶向抗体形成的抗体药物偶联物,可以实现肿瘤富集,消除或降低海鞘素作用于非疾病组织而引起的毒副作用,提高治疗效果。
具体实施方式
下面通过实施例的方式进一步说明本发明,但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法,按照常规方法和条件,或按照商品说明书选择。
本公开中的缩写具有以下含义:
制备方案
以下的实施例中记载的化合物的结构通过核磁共振(1H NMR)或质谱(MS)来确定。
核磁共振(1H NMR)的测定仪器使用Bruker 400 MHz核磁共振仪;测定溶剂为氘代甲醇(CD3OD)、氘代氯仿(CDCl3)或六氘代二甲基亚砜(DMSO-d6);内标物质为四甲基硅烷(TMS)。
实施例中使用的核磁共振(NMR)谱中的缩写示于以下。
s:单峰(singlet)、d:二重峰(doublet)、t:三重峰(triplet)、q:四重峰(quartet)、dd:双二重峰(double doublet)、qd:四二重峰(quartet doublet)、ddd:双双二重峰(double double doublet)、ddt:双双三重峰(double double triplet)、dddd:双双双二重峰(double double double doublet)、m:多重峰(multiplet)、br:宽峰(broad)、J:偶合常数、Hz:赫兹、DMSO-d6:氘化二甲基亚砜。 δ值用ppm值表示。
质谱(MS)的测定仪器使用Agilent (ESI)质谱仪,型号为Agilent 6120B。
实施例1 海鞘素类生物活性化合物(Payload)合成
实施例1.1 PL-1的合成
步骤一:
冰浴下,将化合物3-(2-氨基乙基)-1H-吲哚-5-醇(1.87g,8.81mmol),溶于水(70mL)中,再加入碳酸钠(1.87g,17.61mmol),然后将溶于四氢呋喃溶剂(14mL)中的氯甲酸苄酯(1.51g,8.81 mmol)缓慢滴加进反应体系中,室温搅拌2h;LCMS监测反应;反应液用乙酸乙酯萃取,有机相经无水硫酸钠干燥,过滤,滤液减压浓缩,粗品经反相Flash纯化,得目标化合物(2.25 g)。
LCMS (ESI) [M+H]+ =311.0.
1H NMR (400 MHz, DMSO-d6) δ 10.48 (s, 1H), 8.59 (s, 1H), 7.41-7.29 (m,6H), 7.12 (d, J = 8.6 Hz, 1H), 7.04 (s, 1H), 6.84 (s, 1H), 6.59-6.57 (m, 1H),5.04 (s, 2H), 3.29-3.21 (m, 2H), 2.78-2.70 (m, 2H).
步骤二:
将化合物PL-1-2(1.5g,4.8mmol),1,2-二溴乙烷(4.6g,24.7mmol),碳酸钾(3.4g,24.7mmol)溶于丁酮(20mL)中,反应液在80 ℃下反应36h;LCMS监测反应;反应液减压浓缩,粗品经柱层析(EA:PE=3:1) 纯化,得目标化合物(830 mg)。
LCMS (ESI) [M+H]+ = 417.0.
1H NMR (400 MHz, DMSO-d6) δ 10.75 (s, 1H), 7.47-7.34 (m, 6H), 7.29 (d,J = 8.7 Hz, 1H), 7.17 (s, 1H), 7.11 (s, 1H), 6.86-6.76 (m, 1H), 5.08 (s, 2H),4.35 (t, J = 5.4 Hz, 2H), 3.84 (t, J = 5.4 Hz, 2H), 3.36-3.27 (m, 2H), 2.85(t, J = 7.4 Hz, 2H).
步骤三:
将化合物PL-1-3(2.1g,5.0mmol)溶于甲胺的乙醇溶液(50mL)中,反应在100 ℃下反应过夜;LCMS监测反应;将反应液减压浓缩,得目标化合物PL-1-4(2.0 g),直接用于下一步反应。
LCMS (ESI) [M+H]+ = 368.5.
1H NMR (400 MHz, DMSO-d6) δ 10.74 (s, 1H), 7.42 – 7.31 (m, 6H), 7.28(d, J = 8.7 Hz, 1H), 7.14 (s, 1H), 7.10 (s, 1H), 6.87 – 6.77 (m, 1H), 5.03(s, 2H), 4.22 (t, J = 4.8 Hz, 2H), 3.33 – 3.30 (m, 2H), 3.29 – 3.25 (m, 2H),2.81 (t, J = 7.5 Hz, 2H), 2.65 (s, 3H), 2.55-2.54 (m, 1H).
步骤四:
将化合物PL-1-4(2.0g,5.45mmol)和乙醇酸(828mg,10.9mmol)溶于DMF(10mL)中,加入DMTMM(3.02g,10.9mmol),DIPEA(1.4g,10.9mmol),室温反应1h;LCMS监测反应;反应液直接用反相Flash纯化(乙腈:0.05%FA的水溶液=5%-50%)得目标化合物PL-1-5(1.4 g)。
LCMS (ESI) [M+H]+ = 426.4.
1H NMR (400 MHz, DMSO-d6) δ 10.67 (d, J = 5.0 Hz, 1H), 7.42-7.27 (m,6H), 7.23 (d, J = 8.7 Hz, 1H), 7.11 (s, 1H), 7.03 (s, 1H), 6.77-6.67 (m, 1H),5.03 (s, 2H), 4.24-4.06 (m, 4H), 3.70-3.60 (m, 2H), 3.29-3.24 (m, 2H), 2.96(d, J = 21.4 Hz, 3H), 2.80 (t, J = 7.3 Hz, 2H).
步骤五:
将化合物PL-1-5(500mg,1.18mmol)和Pd/C(100mg,10%)溶于甲醇(10mL)中,氢气置换三次,反应液在氢气氛围下室温反应过夜;LCMS监测反应;反应液直接过滤,滤液减压浓缩,得目标化合物PL-1-6(260 mg)。
LCMS (ESI) [M+H]+ = 292.1.
1H NMR (400 MHz, DMSO-d6) δ 10.69 (d, J = 3.6 Hz, 1H), 7.22 (d, J =8.7 Hz, 1H), 7.09 (s, 1H), 7.02 (s, 1H), 6.75 – 6.65 (m, 1H), 4.20 – 4.05 (m,4H), 3.69 – 3.59 (m, 2H), 2.96 (d, J = 21.0 Hz, 3H), 2.88 – 2.78 (m, 2H),2.77 – 2.69 (m, 2H).
步骤六:
将化合物PL-1-6(77mg,0.26mmol),PL-1-7(50mg,0.075mmol)溶于醋酸(10mL)中,反应在室温下反应过夜;LCMS监测反应;将反应液减压浓缩,粗品经制备TLC(DCM:MeOH=20:1)纯化,得目标化合物PL-1-8(60 mg)。
LCMS (ESI) [M+H]+ = 939.6.
1H NMR (400 MHz, DMSO-d6) δ 9.96 (d, J = 3.0 Hz, 1H), 7.21 (dd, J =8.7, 2.0 Hz, 1H), 6.83 (d, J = 2.3 Hz, 1H), 6.81 (s, 1H), 6.70-6.58 (m, 1H),6.23 (d, J = 9.5 Hz, 2H), 5.17 (d, J = 5.5 Hz, 1H), 5.13-5.03 (m, 2H), 4.49(d, J = 2.4 Hz, 1H), 4.45-4.41 (m, 1H), 4.23 (d, J = 3.9 Hz, 1H), 4.17 (d, J= 5.4 Hz, 1H), 4.11-3.98 (m, 5H), 3.73 (s, 3H), 3.67-3.60 (m, 1H), 3.59-3.52(m, 4H), 3.47-3.36 (m, 2H), 3.36-3.33 (m, 1H), 3.31-3.28 (m, 1H), 3.27-3.24(m, 1H), 3.15-3.07 (m, 1H), 2.92 (d, J = 19.8 Hz, 3H), 2.89-2.85 (m, 1H),2.82-2.75 (m, 1H), 2.61-2.53 (m, 2H), 2.47-2.44 (m, 1H), 2.43-2.37 (m, 1H),2.31-2.22 (m, 6H), 2.08 (s, 3H), 1.98 (s, 3H).
步骤七:
将化合物PL-1-8(25mg,0.021mmol)溶于THF(2.5mL)中,加入稀盐酸 (2.5mL,2N),反应在室温下反应过夜;LCMS监测反应;将反应液减压浓缩,粗品经制备高效液相色谱(乙腈:0.05%FA的水溶液=5%-50%)纯化,得目标化合物PL-1(12.5 mg)。
LCMS (ESI) [M+H]+ = 895.9.
1H NMR (400 MHz, DMSO-d6) δ 9.96 (s, 1H), 8.74 (s, 1H), 7.22 (d, J =8.6 Hz, 1H), 6.83 (s, 1H), 6.71 – 6.59 (m, 1H), 6.48 (s, 1H), 6.24 (s, 1H),6.21 (s, 1H), 5.07 (d, J = 10.8 Hz, 1H), 4.46 (s, 2H), 4.20 (s, 1H), 4.17 (s,1H), 4.14 – 4.04 (m, 4H), 4.04 – 4.00 (m, 1H), 3.68 – 3.61 (m, 4H), 3.59 –3.55 (m, 1H), 3.20 (d, J = 1.7 Hz, 1H), 3.16 – 3.10 (m, 1H), 2.92 (d, J =19.9 Hz, 3H), 2.88 – 2.69 (m, 4H), 2.68 – 2.58 (m, 2H), 2.48 – 2.31 (m, 4H),2.29 (s, 3H), 2.26 (s, 3H), 2.06 (s, 3H), 1.98 (s, 3H).
实施例1.2:PL-2的合成
步骤一:
氮气保护下,将化合物PL-1(15mg,0.016mmol)溶于乙腈(3mL)中,加入去离子水(2mL),再加入硝酸银 (81mg,0.48mmol) ,将反应液于室温下避光搅拌反应过夜;LCMS监测反应;将反应液减压浓缩,粗品经制备高效液相色谱(乙腈:0.05%FA的水溶液=5%-50%)纯化,得目标化合物PL-2(5.5mg)。
LCMS (ESI) [M+H]+ = 886.1.
1H NMR (400 MHz, MeOD) δ 7.20-7.14 (m, 1H), 6.93-6.88 (m, 1H), 6.82(s, 1H), 6.80-6.74 (m, 1H), 6.29 (s, 1H), 6.12 (s, 1H), 5.40-5.29 (m, 2H),4.95 (d, J = 3.9 Hz, 1H), 4.75 (s, 1H), 4.65 (d, J = 2.8 Hz, 1H), 4.40 (s,1H), 4.37-4.29 (m, 1H), 4.23 (s, 1H), 4.22-4.17 (m, 1H), 4.12 (t, J = 5.4 Hz,2H), 3.89-3.83 (m, 1H), 3.79 (s, 3H), 3.76 (d, J = 5.8 Hz, 1H), 3.64 (t, J =4.4 Hz, 1H), 3.51-3.44 (m, 2H), 3.27-3.19 (m, 1H), 3.03 (d, J = 9.2 Hz, 3H),2.97-2.90 (m, 1H), 2.87-2.74 (m, 2H), 2.70 (s, 3H), 2.45 (s, 3H), 2.40-2.31(m, 1H), 2.30 (s, 3H), 2.24-2.15 (m, 1H), 2.04 (s, 3H).
实施例1.3:PL-3的合成
步骤一:
将化合物PL-3-1(500mg,3.62mmol),(2-溴乙基)氨基甲酸叔丁酯(970mg,4.35mmol),碳酸钾(600 mg,4.35 mmol),碘化钠(163mg,1.08mmol)溶于DMF(10mL)中,反应在60 ℃下反应4 h;LCMS监测反应;反应液直接用反相Flash纯化(乙腈:0.05%FA的水溶液=5%-50%),得目标化合物PL-3-2(400mg)。
1H NMR (400 MHz, DMSO-d 6 ) δ 9.79 (s, 1H), 9.23 (s, 1H), 7.42-7.35 (m,1H), 7.26 (d, J = 2.0 Hz, 1H), 7.12 (d, J = 8.2 Hz, 2H), 4.14-4.01 (m, 2H),3.40-3.33 (m, 2H), 1.40 (s, 9H).
步骤二:
将化合物PL-3-2(9.8g,34.9mmol)溶于DMF(50mL)中, 加入碳酸钾(14.4g,104.7mmol),然后滴加苄溴(6.6g,38.4mmol),滴加完毕,反应液在室温下反应3 h;LCMS监测反应;向反应液中加入水(50mL),然后用乙酸乙酯(100 mL*3)萃取,合并有机相,经无水硫酸钠干燥,过滤,滤液减压浓缩,粗品经柱层析(EA:PE=3:1) 纯化,得目标化合物PL-3-3(11. 0 g)。
LCMS (ESI) [M+Na]+ = 394.3.
1H NMR (400 MHz, DMSO-d6) δ 9.82 (s, 1H), 7.55 (d, J = 8.2, 1.7 Hz,1H), 7.48 (d, J = 8.6 Hz, 3H), 7.39 (t, J = 7.3 Hz, 2H), 7.35-7.31 (m, 1H),7.23 (d, J = 8.3 Hz, 1H), 6.99 (t, J = 5.2 Hz, 1H), 5.19 (s, 2H), 4.13 (t, J= 5.9 Hz, 2H), 3.40-3.33 (m, 2H), 1.37 (s, 9H).
步骤三:
将化合物PL-3-3(12.1g,33.4mmol)溶于DMF (100mL)中,降温至0 ℃,加入氢化钠(3.3g,83.5mmol),搅拌30min,再加入碘甲烷(9.5 g, 66.84 mmol),继续反应1 h;LCMS监测反应;向反应液中加入水(100mL),然后用乙酸乙酯(100 mL*3)萃取,合并有机相,经无水硫酸钠干燥,过滤,滤液减压浓缩,粗品经柱层析(EA:PE=3:1) 纯化,得目标化合物PL-3-4(8.0g)。
LCMS (ESI) [M+Na]+ = 408.2.
1H NMR (400 MHz, DMSO-d6) δ 9.83 (s, 1H), 7.56 (d, J = 8.2, 1.8 Hz,1H), 7.51 (s, 1H), 7.46 (d, J = 7.2 Hz, 2H), 7.39 (t, J = 7.2 Hz, 2H), 7.34(d, J = 7.1 Hz, 1H), 7.24 (d, J = 8.3 Hz, 1H), 5.18 (s, 2H), 4.29-4.17 (m,2H), 3.57 (t, J = 5.4 Hz, 2H), 2.87 (d, J = 13.1 Hz, 3H), 1.35 (d, J = 22.2Hz, 9H).
步骤四:
将叔丁醇钾(580mg,5.2mmol)溶于THF(10mL)中,降温至-78 ℃,对甲基苯磺酰甲基异腈(510mg,2.6mmol)溶于THF溶液(5mL),然后加入到反应液中,搅拌15min,然后再加入化合物PL-3-4(500mg,1.3mmol),继续于该温度下搅拌反应1h;LCMS监测反应;向反应液中加入甲醇(10mL),然后升温至85 ℃,回流30min;LCMS监测反应;将反应液减压浓缩,粗品用反相Flash(乙腈:0.05%FA的水溶液=5%-50%)纯化,得目标化合物PL-3-5(280 mg)。
LCMS (ESI) [M+Na]+ = 419.3.
1H NMR (400 MHz, DMSO-d6) δ 7.45 (d, J = 7.2 Hz, 2H), 7.39 (t, J = 7.2Hz, 2H), 7.34 (d, J = 7.1 Hz, 1H), 7.06 (s, 1H), 7.02 (d, J = 8.3 Hz, 1H),6.89 (d, J = 8.2 Hz, 1H), 5.08 (s, 2H), 4.10 – 4.05 (m, 2H), 3.91 (s, 2H),3.51 (t, J = 5.4 Hz, 2H), 2.85 (d, J = 12.3 Hz, 3H), 1.36 (d, J = 17.3 Hz,9H).
步骤五:
将化合物PL-3-5(2.8g,7.07mmol)溶于DCM(30mL)和乙腈(30mL)中,加入TFA(20mL),在室温下反应3 h;LCMS监测反应;反应液减压浓缩,得目标化合物PL-3-6(2.4g)。
LCMS (ESI) [M+H]+ = 297.2.
步骤六:
将化合物PL-3-6(1.0g,3.38mmol)和乙醇酸(308mg,4.07mmol)溶于DMF(40mL)中,加入DMTMM(1.4g,5.07mmol),DIPEA(1.1g,8.45mmol),加毕室温反应1 h;LCMS监测反应;反应液直接用反相Flash(乙腈:0.05%FA的水溶液=5%-50%)纯化,得目标化合物PL-3-7(1.0g)。
LCMS (ESI) [M+H]+ = 355.2.
1H NMR (400 MHz, DMSO-d6) δ 7.46 (t, J = 8.0 Hz, 2H), 7.43 – 7.37 (m,2H), 7.36 – 7.30 (m, 1H), 7.08 (s, 1H), 7.04 – 6.99 (m, 1H), 6.89 (d, J =8.0, 1.6 Hz, 1H), 5.09 (s, 2H), 4.47 – 4.37 (m, 1H), 4.19 (d, J = 5.4 Hz,1H), 4.15 – 4.07 (m, 2H), 4.05 (d, J = 5.5 Hz, 1H), 3.92 (s, 2H), 3.71 – 3.60(m, 2H), 2.92 (d, J = 12.0 Hz, 3H).
步骤七:
将化合物PL-3-7(50mg,0.14mmol)和Pd/C(10 mg,10%)溶于MeOH(5 mL)中,氢气置换三次,反应液在氢气氛围下室温反应3h;LCMS监测反应;反应液直接过滤,滤液减压浓缩,得目标化合物PL-3-8(35 mg)。
LCMS (ESI) [M+H]+ = 269.0.
步骤八:
将化合物PL-3-8(70mg,0.26mmol),PL-1-7(50mg,0.075mmol),溶于醋酸(10 mL)中,反应在室温下过夜;LCMS监测反应;将反应液减压浓缩,粗品经制备TLC(DCM:MeOH=20:1)纯化,得目标化合物PL-3-9(60 mg,)。
LCMS (ESI) [M+H]+ = 916.6.
步骤九:
将化合物PL-3-9(20mg,0.021mmol)溶于THF(2.5mL)中,加入稀盐酸(2.5mL,2N),反应在室温下过夜;LCMS监测反应;将反应液减压浓缩,粗品经制备高效液相色谱(乙腈:0.05%FA的水溶液=5%-50%)纯化,得目标化合物PL-3(10.8mg)。
LCMS (ESI) [M+H]+ = 872.9.
1H NMR (400 MHz, DMSO-d6) δ 9.46 (s, 1H), 8.93 (s, 1H), 6.56 – 6.50(m, 2H), 6.29 (d, J = 37.2 Hz, 1H), 6.23 – 6.16 (m, 2H), 5.19 (d, J = 11.6Hz, 1H), 4.63 (s, 1H), 4.56 (s, 1H), 4.26 – 4.22 (m, 2H), 4.17 (d, J = 7.1Hz, 1H), 4.07 (s, 1H), 3.84 – 3.75 (m, 2H), 3.70 – 3.67 (m, 2H), 3.65 (s,3H), 3.54 (s, 3H), 3.46 – 3.44 (m, 2H), 3.34 (d, J = 4.8 Hz, 2H), 3.22 – 3.06(m, 2H), 2.91 (d, J = 32.1 Hz, 3H), 2.73 – 2.67 (m, 2H), 2.42 – 2.36 (m, 1H),2.31 (s, 3H), 2.25 (s, 3H), 2.03 (s, 3H), 2.01 – 1.98 (m, 3H).
实施例1.4:PL-4的合成
步骤一:
氮气保护下,将化合物PL-3(20mg,0.023mmol)溶于乙腈(3mL)中,加入去离子水(2mL),再加入硝酸银(117 mg,0.69 mmol) ,将反应液于室温下避光搅拌反应过夜;LCMS监测反应;将反应液减压浓缩,粗品经制备高效液相色谱(乙腈:0.05%FA的水溶液=5%-50%)纯化,得目标化合物PL-4(6.5 mg)。
LCMS (ESI) [M+H]+ = 863.2.
1H NMR (400 MHz, DMSO-d6) 9.07 – 8.92 (m, 1H), 8.57 (s, 1H), 6.41 (s,1H), 6.38 (s, 1H), 6.30 (d, J = 11.9 Hz, 1H), 6.23 (d, J = 34.3 Hz, 1H), 6.06(s, 1H), 5.00 (d, J = 11.1 Hz, 1H), 4.80 (d, J = 10.0 Hz, 1H), 4.67 (s, 1H),4.49 – 4.41 (m, 1H), 4.36 – 4.31 (m, 1H), 4.18 – 4.06 (m, 3H), 3.98 – 3.89(m, 1H), 3.81 – 3.72 (m, 1H), 3.70 – 3.65 (m, 1H), 3.63 (s, 3H), 3.53 – 3.48(m, 1H), 3.46 (d, J = 5.1 Hz, 1H), 3.30 (s, 3H), 3.12 – 3.08 (m, 1H), 3.06 –2.99 (m, 1H), 2.90 (d, J = 20.8 Hz, 3H), 2.73 – 2.65 (m, 3H), 2.45 – 2.39 (m,1H), 2.35 – 2.29 (m, 1H), 2.27 (s, 3H), 2.20 (s, 3H), 2.19 – 2.14 (m, 1H),2.09 – 2.06 (m, 1H), 2.05 (s, 3H), 2.00 (d, J = 7.8 Hz, 1H), 1.98 (dd, J =14.4, 6.0 Hz, 4H), 2.02 – 1.95 (m, 4H).
实施例2. 连接子(Linker)合成
实施例2.1:4-((S)-2-((S)-2-(2,2-二甲基-4-(4-(2-(甲磺酰基)嘧啶-5-基)-H-1,2,3-三氮唑-1-基)丁酰胺)-3-甲基丁酰胺)-6-(二甲氨基)己酰胺)苄基 (4-硝基苯基)碳酸酯(L1)的合成
步骤一:
将化合物L1-1 (500 mg, 4.39 mmol) 溶在干燥二氯甲烷 (5 mL) 中,在0℃下滴加三溴化硼 (1.15 g, 4.6 mmol),滴加完毕升至室温,搅拌反应24 h,降温至0℃向反应液中滴加甲醇(5 mL)淬灭反应,将反应液在室温下继续搅拌24 h。向反应液中加入饱和碳酸氢钠水溶液(30 mL),然后用二氯甲烷萃取(30 mL ⅹ 3),合并有机相经无水硫酸钠干燥,过滤,减压浓缩,得目标化合物L1-2 (830 mg)。
1H NMR (400 MHz, CDCl3) δ 3.69 (s, 3H), 3.40 – 3.27 (m, 2H), 2.20 –2.09 (m, 2H), 1.21 (s, 6H).
步骤二:
将化合物L1-2 (650 mg, 3.13 mmol) 溶在DMF (10 mL) 中,加入叠氮化钠(1.02 g, 15.63 mmol),升温至80℃,搅拌反应过夜,然后降至室温,向反应液中加入水(30mL),然后用甲基叔丁基醚萃取(30 mL ⅹ 3),合并有机相,经无水硫酸钠干燥,过滤,滤液减压浓缩,得目标化合物L1-3(470 mg)。
1H NMR (400 MHz, CDCl3) δ 3.69 (s, 3H), 3.31 – 3.23 (m, 2H), 1.89 –1.81 (m, 2H), 1.22 (s, 6H).
步骤三:
将化合物L1-3 (365 mg, 2.13 mmol)、L1-4(320 mg, 2.13 mmol) 溶在叔丁醇(5 mL)和水(5 mL) 中,加入维生素C钠 (84 mg, 0.43 mmol),无水硫酸铜 (34 mg, 0.21mmol)在室温下搅拌1 h。向反应液中加入水(25 mL),然后用乙酸乙酯萃取(30 mL ⅹ 3),合并有机相,经无水硫酸钠干燥,过滤,旋干,残留物经硅胶柱色谱(PE:EA=3:1)纯化,得目标化合物L1-5(600 mg)。
LCMS (ESI) [M+H]+ = 322.1
1H NMR (400 MHz, CDCl3) δ 8.95 (s, 2H), 7.85 (s, 1H), 4.53 – 4.39 (m,2H), 3.69 (s, 3H), 2.61 (s, 3H), 2.29 – 2.17 (m, 2H), 1.31 (s, 6H).
步骤四:
将化合物L1-5 (500 mg, 1.60 mmol)溶在四氢呋喃 (5 mL)和水(5 mL) 中,加入氢氧化锂 (112 mg, 4.70 mmol),在室温下搅拌5 h。向反应液中加入1 N的盐酸调pH= 3~4,析出固体,过滤,滤饼为目标化合物L11-6(450 mg)。
LCMS (ESI) [M+H]+ = 308.0
1H NMR (400 MHz, DMSO) δ 12.43 (s, 1H), 9.06 (s, 2H), 8.76 (s, 1H),4.55 – 4.31 (m, 2H), 2.56 (s, 3H), 2.15 – 2.04 (m, 2H), 1.20 (s, 6H).
步骤五:
将化合物L1-6 (1.5 g, 4.90 mmol)溶在四氢呋喃 (10 mL)和水(10 mL) 中,加入Oxane (15.0 g, 24.5 mmol),在室温下搅拌反应过夜。将反应液倒入水(200 mL)中,过滤,滤饼即为目标化合物L1-7 (1.6 g)。
LCMS (ESI) [M+H]+ = 340.1
1H NMR (400 MHz, DMSO) δ 9.48 (s, 2H), 8.98 (s, 1H), 4.54 – 4.48 (m,2H), 3.44 (s, 3H), 2.16 – 2.09 (m, 2H), 1.21 (s, 6H).
步骤六:
将化合物L1-7 (1.9 g, 5.32 mmol)溶于DMF (40 mL)中,再加入HATU(2.13 g,5.32 mmol)和DIPEA(1.7 g, 13.3 mmol),反应液于室温下搅拌30 min,将L1-8 (1.45 g,5.32 mmol)加入到反应液中,继续搅拌反应1 h。将反应液直接经反相色谱(H2O:ACN=5%-100%)纯化,得目标化合物L1-9(1.5 g)。
LCMS (ESI) [M+H]+ = 595.5
1H NMR (400 MHz, DMSO) δ 9.51 (s, 2H), 9.05 (s, 1H), 7.89 (d, J = 7.1Hz, 1H), 7.48 (d, J = 8.6 Hz, 1H), 4.45 – 4.35 (m, 2H), 4.13 (t, 1H), 4.07-4.09 (m, 1H), 3.44 (s, 3H), 2.34 (t, J = 7.3 Hz, 2H), 2.23 (s, 6H), 2.19 –2.06 (m, 4H), 1.73 – 1.57 (m, 2H), 1.45 – 1.36 (m, 2H), 1.31 – 1.27 (m, 1H),1.22 (d, J = 9.6 Hz, 6H), 0.91 – 0.86 (m, 6H).
步骤七:
将化合物L1-9 (360 mg, 0.61 mmol),L1-10 (75 mg, 0.61 mmol)溶于DMF (5mL)中,加入HATU(230 mg, 0.61 mmol)和DIPEA(195 mg, 1.52 mmol),反应液于室温下搅拌1 h。将反应液直接经反相色谱(H2O:ACN=5%-100%)纯化,得到目标化合物L1-11(255mg)。
LCMS (ESI) [M+H]+ = 700.4
1H NMR (400 MHz, DMSO) δ 9.93 (d, J = 87.4 Hz, 1H), 9.47 (s, 1H),9.39 (s, 1H), 8.89 (d, J = 57.3 Hz, 1H), 8.54 – 8.16 (m, 1H), 7.68 (d, J =7.8 Hz, 0.5H), 7.56 – 7.48 (m, 2H), 7.47 (d, J = 8.3 Hz, 0.5H), 7.19 (d, J =8.4 Hz, 1H), 7.13 (d, J = 8.5 Hz, 1H), 5.10 – 5.04 (m, 1H), 4.44 – 4.36 (m,4H), 4.21 – 4.09 (m, 1H), 3.45 (s, 3H), 2.75 – 2.65 (m, 2H), 2.23 – 2.04 (m,4H), 1.83 – 1.63 (m, 2H), 1.59 – 1.51 (m, 2H), 1.42 – 1.35 (m, 1H), 1.35 –1.16 (m, 12H), 0.94 – 0.87 (m, 6H).
步骤八:
将化合物L1-11 (250 mg, 0.36 mmol),L1-12(288 mg, 1.43 mmol) 溶在DCM(10 mL) 中,加入三乙胺 (72 mg, 0.72 mmol),在室温下搅拌3 h。将反应液浓缩,残留物经反相色谱(H2O:ACN=5%-100%)纯化,得到L1 (120 mg)。
LCMS (ESI) [M+H]+ = 865.2;
1H NMR (400 MHz, DMSO) δ 10.04 (d, J = 73.3 Hz, 1H), 9.46 (s, 1H),9.36 (s, 1H), 8.86 (d, J = 56.6 Hz, 1H), 8.31 (d, J = 9.1 Hz, 2H), 8.08 (d, J= 9.2 Hz, 1H), 7.64 (d, J = 8.0 Hz, 1H), 7.59 – 7.55 (m, 3H), 7.42 (d, J =8.3 Hz, 1H), 7.37 (d, J = 8.5 Hz, 1H), 7.27 (d, J = 8.5 Hz, 1H), 6.83 (d, J =9.1 Hz, 1H), 5.19 (d, J = 14.8 Hz, 2H), 4.45 – 4.38 (m, 2H), 4.37 – 4.28 (m,1H), 4.21 – 4.14 (m, 1H), 3.44 (s, 3H), 2.18 – 2.12 (m, 3H), 2.10 – 2.06 (m,8H), 1.81 – 1.63 (m, 2H), 1.44 – 1.32 (m, 4H), 1.23 (d, J = 7.4 Hz, 7H), 0.94– 0.88 (m, 6H).
实施例3 毒素-连接子(payload linker)的合成
实施例3.1:DL-1的合成
步骤一:
将化合物PL-1-4 (300 mg, 0.82 mmol) 溶于二氯甲烷 (10 mL)中,再加入三乙胺 (165 mg, 1.63 mmol),(Boc)2O (267 mg, 1.23 mmol),室温搅拌反应2 h;LCMS 监测反应;向反应液中加入水 (50 mL),用二氯甲烷 (40 mL*3) 萃取,有机相经无水硫酸钠干燥,过滤,滤液减压浓缩,粗品经柱层析 (PE:EA=10:1) 纯化,得目标化合物DL-1-1 (300mg)。
LCMS (ESI) [M+H]+ =468.1.
步骤二:
将化合物DL-1-1 (200 mg,0.43 mmol) 和Pd/C (100 mg,10%)溶于甲醇 (6 mL)中,氢气置换三次,在氢气氛围下室温反应过夜;LCMS监测反应;反应液直接过滤,滤液减压浓缩,得目标化合物DL-1-2 (100 mg)。
LCMS (ESI) [M+H]+ = 334.5.
1H NMR (400 MHz, DMSO-d6) δ 10.62 (s, 1H), 7.22 (d, J = 8.7 Hz, 1H),7.08 (s, 1H), 7.01 (s, 1H), 6.71 (d, J = 8.7 Hz, 1H), 4.08 – 4.04 (m, 2H),3.54 (t, J = 5.6 Hz, 2H), 2.91-2.88 (m, 3H), 2.81 – 2.76 (m, 2H), 2.75 – 2.68(m, 2H), 1.40-1.38 (m, 9H).
步骤三:
将化合物DL-1-2 (60 mg,0.18 mmol),PL-1-7 (30 mg, 0.045 mmol)溶于醋酸(10 mL) 中,反应在室温下过夜;LCMS监测反应;将反应液减压浓缩, 粗品经制备TLC (二氯甲烷:甲醇=20: 1) 纯化,得目标化合物DL-1-3 (40 mg) 。
LCMS (ESI) [M+H]+ = 981.3.
1H NMR (400 MHz, DMSO-d6) δ 9.96 (s, 1H), 7.21 (d, J = 8.7 Hz, 1H),6.82 (d, J = 5.7 Hz, 2H), 6.66 (d, J = 9.1 Hz, 1H), 6.23 (d, J = 8.3 Hz, 2H),5.16 (d, J = 5.5 Hz, 1H), 5.10 (s, 1H), 5.08 (d, J = 7.2 Hz, 1H), 4.49 (s,1H), 4.48 – 4.39 (m, 1H), 4.25 – 4.21 (m, 1H), 4.08 (d, J = 11.0 Hz, 2H),4.03 – 3.98 (m, 2H), 3.73 (s, 3H), 3.54 (s, 3H), 3.51 – 3.47 (m, 2H), 3.42(d, J = 9.4 Hz, 1H), 3.31 – 3.28 (m, 2H), 3.26 – 3.24 (m, 1H), 3.15 – 3.08(m, 1H), 2.90-2.86 (m, 2H), 2.85 – 2.82 (m, 2H), 2.81 – 2.75 (m, 1H), 2.58(d, J = 15.0 Hz, 1H), 2.48 – 2.42 (m, 2H), 2.27 (d, J = 3.4 Hz, 6H), 2.08 (s,3H), 1.98 (s, 3H), 1.37 (d, J = 10.5 Hz, 9H).
步骤四:
氮气保护下,将化合物DL-1-3(70 mg,0.071 mmol)溶于乙腈 (15 mL) 中,加入去离子水 (3 mL),再加入硝酸银(364 mg,2.14 mmol),将反应液于室温下避光搅拌反应过夜;LCMS监测反应;将反应液减压浓缩,粗品经制备高效液相色谱(乙腈:0.05%FA的水溶液=5%-50%)纯化,得目标化合物DL-1-4 (65 mg)。
LCMS (ESI) [M+H]+ =972.4.
1H NMR (400 MHz, DMSO-d6) δ 9.91 (s, 1H), 7.21 (d, J = 8.7 Hz, 1H),6.80 (d, J = 14.0 Hz, 2H), 6.65 (d, J = 8.6 Hz, 1H), 6.22 (s, 1H), 6.15 (d, J= 9.3 Hz, 1H), 5.16 (t, J = 5.8 Hz, 1H), 5.10 – 5.03 (m, 2H), 4.68 (s, 1H),4.37 – 4.31 (m, 1H), 4.15 – 4.10 (m, 1H), 4.10 – 4.05 (m, 1H), 4.05 – 3.99(m, 3H), 3.73 (s, 3H), 3.53 (s, 3H), 3.51 – 3.46 (m, 3H), 3.35 – 3.33 (m,2H), 3.30 – 3.27 (m, 2H), 3.16 – 3.10 (m, 2H), 2.89-2.86 (m, 3H), 2.80 – 2.75(m, 2H), 2.63 – 2.56 (m, 1H), 2.47 – 2.43 (m, 2H), 2.27 (s, 3H), 2.25 (s,3H), 2.05 (d, J = 18.7 Hz, 3H), 1.97 (s, 3H), 1.38-1.35 (m, 9H).
步骤五:
将化合物DL-1-4 (60 mg,0.06 mmol) 溶于四氢呋喃 (2.5 mL) 中;加入氯化氢的1,4-二氧六环溶液 (2.5 mL,2N),反应在室温下反应过夜;LCMS监测反应;将反应液减压浓缩,得目标化合物DL-1-5 (50 mg),直接用于下一步反应。
LCMS (ESI) [M+H]+ = 828.4.
步骤六:
依次将化合物DL-1-6 (32 mg,0.084 mmol),二异丙基乙胺(22 mg,0.168 mmol)和HBTU (42 mg,0.112 mmol)加入N,N-二甲基甲酰胺(2 mL)中,随后加入化合物DL-1-5(50 mg,0.056 mmol)。反应液在室温下搅拌2 h。向反应液中加入乙酸乙酯(30 mL),用饱和食盐水(30 mL * 3)洗涤,无水硫酸钠干燥,抽滤,滤液减压浓缩,粗品经柱层析(二氯甲烷:甲醇=10:1) 纯化,得目标化合物DL-1-7(50 mg)。
LCMS (ESI) [M+H]+ = 1194.4.
步骤七:
向化合物DL-1-7 (50 mg,0.042 mmol)的N,N-二甲基甲酰胺(2 mL)溶液加入二乙胺(0.1 mL),反应液在室温下搅拌20 min。反应液除去低沸点组分后得到目标产物DL-1-8(45 mg),直接用于下一步反应。
LCMS (ESI) [M+H]+ = 972.4.
步骤八:
将化合物DL-1-8 (45 mg,0.046 mmol) 和DL-1-9 (27 mg,0.046 mmol)溶于N,N-二甲基甲酰胺 (2 mL) 中,加入HATU(26 mg,0.069 mmol),N,N-二异丙基乙胺 (12 mg,0.092 mmol) ,室温搅拌反应1 h;LCMS监测反应;反应液直接经制备高效液相色谱(乙腈:0.05% FA的水溶液=5%-50%) 纯化,得目标化合物DL-1 (18 mg)。
LCMS (ESI) [M/2+H]+ = 767.8.
1H NMR (400 MHz, DMSO-d6) δ 9.93 (s, 1H), 9.10 (s, 2H), 8.60 (t, J =6.9 Hz, 1H), 8.30 (s, 1H), 8.17 (s, 1H), 8.02 (d, J = 7.1 Hz, 1H), 7.94 (d, J= 8.6 Hz, 1H), 7.19 (d, J = 6.4 Hz, 1H), 6.82 (s, 1H), 6.64 (d, J = 8.6 Hz,1H), 6.46 (s, 1H), 6.21 (d, J = 7.5 Hz, 1H), 6.12 (s, 1H), 5.01 (d, J = 10.5Hz, 1H), 4.67 (s, 1H), 4.61 – 4.52 (m, 2H), 4.43 – 4.25 (m, 2H), 4.24 – 4.12(m, 4H), 4.12 – 4.00 (m, 4H), 3.99 – 3.94 (m, 1H), 3.77 – 3.66 (m, 3H), 3.65(s, 3H), 3.63 – 3.56 (m, 4H), 3.40 (s, 3H), 3.18 – 3.08 (m, 4H), 2.91 (d, J =40.8 Hz, 3H), 2.83 – 2.68 (m, 4H), 2.67 – 2.52 (m, 4H), 2.46 – 2.33 (m, 5H),2.28 (s, 4H), 2.27 (s, 3H), 2.25 (s, 3H), 2.04 (d, J = 14.6 Hz, 3H), 1.96 (s,3H), 1.83 – 1.77 (m, 3H), 1.69 – 1.50 (m, 2H), 1.37 – 1.30 (m, 6H), 0.85 –0.78 (m, 12H).
实施例3.2 DL-2的合成
步骤一:
将化合物DL-1-3 (40 mg,0.04 mmol)溶于四氢呋喃 (2.5 mL) 中,加入氯化氢的1,4-二氧六环溶液(2.5 mL, 2N),反应室温下搅拌过夜;LCMS监测反应;将反应液减压浓缩,得目标化合物DL-2-1 (33 mg),直接用于下一步反应。
LCMS (ESI) [M+H]+ = 837.5.
步骤二:
依次将化合物DL-1-6 (22 mg,0.057 mmol),二异丙基乙胺(15 mg,0.114 mmol)和HBTU (29 mg,0.112 mmol)加入N,N-二甲基甲酰胺(2 mL)中,随后加入化合物DL-2-1(33 mg,0.038 mmol)。反应液在室温下搅拌2 h。向反应液中加入乙酸乙酯(30 mL),用饱和食盐水(30 mL * 3)洗涤,无水硫酸钠干燥,抽滤,滤液减压浓缩,粗品经柱层析(二氯甲烷:甲醇=10:1) 纯化,得目标化合物DL-2-2(35 mg)。
LCMS (ESI) [M+H]+ = 1203.4.
步骤三:
向化合物DL-2-2 (35 mg,0.029 mmol)的N,N-二甲基甲酰胺(2 mL)溶液加入二乙胺(0.1 mL),反应液在室温下搅拌20 min。反应液除去低沸点组分后得到目标产物DL-2-3(30 mg),直接用于下一步反应。
LCMS (ESI) [M+H]+ = 981.3.
步骤四:
将化合物DL-2-3 (30 mg,0.031 mmol) 和DL-1-9 (18 mg, 0.031 mmol)溶于N,N-二甲基甲酰胺 (2 mL) 中,加入HATU(18 mg,0.047 mmol),N,N-二异丙基乙胺 (8 mg,0.062 mmol) ,室温搅拌反应1 h;LCMS监测反应;反应液直接经制备高效液相色谱(乙腈:0.05% FA的水溶液=5%-50%) 纯化,得目标化合物DL-2 (15 mg)。
LCMS (ESI) [1/2M+H]+ = 772.6.
1H NMR (400 MHz, DMSO-d6) δ 9.97 (s, 1H), 9.10 (s, 2H), 8.76 (s, 1H),8.60 (t, J = 6.6 Hz, 1H), 8.29 (s, 1H), 8.17 (t, J = 6.5 Hz,1H), 8.03 (d, J =7.1 Hz, 1H), 7.94 (d, J = 8.6 Hz, 1H), 7.20 (d, J = 8.9 Hz, 1H), 6.82 (s,1H), 6.66 (d, J = 8.5 Hz, 1H), 6.48 (s, 1H), 6.22 (d, J = 12.7 Hz, 2H), 5.07(d, J = 10.8 Hz, 1H), 4.64 – 4.54 (m, 2H), 4.45 (s, 2H), 4.24 – 4.14 (m, 4H),4.12 – 4.03 (m, 4H), 4.01 – 3.97 (m, 1H), 3.77 – 3.66 (m, 3H), 3.65 (s, 3H),3.59 (d, J = 5.6 Hz, 4H), 3.40 (s, 3H), 3.20 – 3.11 (m, 4H), 2.93 (d, 3H),2.86 – 2.72 (m, 4H), 2.69 – 2.53 (m, 4H), 2.45 – 2.42 (m, 1H), 2.41 – 2.32(m, 4H), 2.29 (s, 7H), 2.25 (s, 3H), 2.05 (s, 3H), 1.98 (s, 3H), 1.84 – 1.78(m, 2H), 1.68 – 1.50 (m, 2H), 1.38 – 1.30 (m, 6H), 0.85 – 0.78 (m, 12H).
实施例3.3:DL-3的合成
步骤一:
将化合物PL-3-5 (600 mg,1.51 mmol) 和Pd/C (400 mg)溶于甲醇 (20 mL) 中,氢气置换三次,在氢气氛围下室温反应过夜;LCMS监测反应;反应液过滤,滤液减压浓缩,得目标化合物DL-3-1 (350 mg)。
LCMS (ESI) [M+H]+ = 311.0.
1H NMR (400 MHz, DMSO-d6) δ 6.86 (d, J = 8.2 Hz, 1H), 6.70 (s, 1H),6.59 (d, J = 7.6 Hz, 1H), 4.10 – 3.98 (m, 2H), 3.50 (t, J = 5.6 Hz, 2H), 2.96– 2.84 (m, 5H), 2.74 – 2.63 (m, 2H), 1.43 – 1.33 (m, 9H).
步骤二:
将化合物DL-3-1(65 mg,0.21 mmol),PL-1-7 (35 mg, 0.053 mmol)溶于醋酸(10 mL) 中,反应在室温下反应过夜;LCMS监测反应;将反应液减压浓缩,粗品经Prep-TLC(二氯甲烷:甲醇=20: 1) 纯化,得目标化合物DL-3-2 (49 mg)。
LCMS (ESI) [M+H]+ = 958.8.
1H NMR (400 MHz, CD3OD) δ 6.80 (s, 1H), 6.43 (s, 2H), 6.29 – 6.16 (m,1H), 6.05 (s, 1H), 5.24 (d, J = 5.7 Hz, 1H), 5.17 (d, J = 5.8 Hz, 1H), 5.13(d, J = 11.6 Hz, 1H), 4.82 – 4.79 (m, 1H), 4.72 – 4.57 (m, 1H), 4.45 (d, J =2.8 Hz, 1H), 4.39 (d, J = 4.5 Hz, 1H), 4.29 (s, 1H), 4.17 (d, J = 11.6 Hz,1H), 4.07 – 3.84 (m, 2H), 3.82 (s, 3H), 3.61 (s, 3H), 3.51 – 3.46 (m, 2H),3.44 (d, J = 5.2 Hz, 1H), 3.31 – 3.29 (m, 2H),3.20 – 3.11 (m, 1H), 3.01 –2.92 (m, 2H), 2.88 (s, 3H), 2.83 – 2.75 (m, 1H), 2.68 – 2.57 (m, 1H), 2.48 –2.34 (m, 2H), 2.29 (s, 3H), 2.28 (s, 3H), 2.19 (s, 3H), 2.05 (s, 3H), 2.02 –2.00 (m, 1H),1.51 – 1.32 (m, 9H).
步骤三:
将化合物DL-3-2 (45 mg,0.04 mmol)溶于四氢呋喃 (2.5 mL) 中,加入氯化氢的1,4-二氧六环溶液 (2.5 mL, 2N),反应在室温下反应过夜;LCMS监测反应;将反应液减压浓缩,得目标化合物DL-3-3 (38 mg),直接用于下一步反应。
LCMS (ESI) [M+H]+ = 814.4.
步骤四:
依次将化合物DL-1-6 (26 mg,0.068 mmol),二异丙基乙胺(17 mg,0.135 mmol)和HBTU (34 mg,0.09 mmol)加入N,N-二甲基甲酰胺(2 mL)中,随后加入化合物DL-3-3 (38mg,0.045 mmol)。反应液在室温下搅拌2 h。向反应液中加入乙酸乙酯(30 mL),用饱和食盐水(30 mL * 3)洗涤,无水硫酸钠干燥,抽滤,滤液减压浓缩,粗品经柱层析(二氯甲烷:甲醇=10:1) 纯化,得目标化合物DL-3-4(45 mg)。
LCMS (ESI) [M+H]+ = 1180.4.
步骤五:
向化合物DL-3-4 (45 mg,0.038 mmol)的N,N-二甲基甲酰胺(2 mL)溶液加入二乙胺(0.1 mL),反应液在室温下搅拌20 min。反应液除去低沸点组分后得到目标产物DL-3-5(35 mg),直接用于下一步反应。
LCMS (ESI) [M+H]+ = 958.3.
步骤六:
将化合物DL-3-5 (35 mg,0.037 mmol) 和DL-1-9(21 mg, 0.037 mmol)溶于DMF(2 mL) 中,加入HATU (28 mg,0.074 mmol),DIPEA (10 mg,0.074 mmol) 室温反应1 h;LCMS监测反应;反应液直接经制备高效液相色谱 (乙腈:0.05% FA的水溶液=5%-50%)纯化,得目标化合物DL-3 (15 mg)。
LCMS (ESI) [M+H]+ = 1519.5.
1H NMR (400 MHz, DMSO-d6) δ 9.11 (s, 2H), 8.91 (s, 1H), 8.69 (s, 1H),8.64 – 8.53 (m, 1H), 8.18 (s, 2H), 8.06 – 7.97 (m, 1H), 7.93 (d, J = 8.4 Hz,1H), 6.43 (s, 1H), 6.38 (s, 1H), 6.32 – 6.19 (m, 2H), 6.13 (s, 1H), 5.03 (d,J = 11.8 Hz, 1H), 4.64 – 4.52 (m, 2H), 4.46 (s, 2H), 4.26 – 4.06 (m, 6H),4.04 – 3.95 (m, 1H), 3.81 – 3.64 (m, 5H), 3.63 (s, 3H), 3.58 – 3.48 (m, 4H),3.41 (s, 3H), 3.14 – 2.97 (m, 4H), 2.94 – 2.82 (m, 3H), 2.80 – 2.63 (m, 4H),2.44 – 2.32 (m, 10H), 2.28 (s, 3H), 2.20 (s, 3H), 2.19 – 2.09 (m, 2H), 2.04(s, 3H), 1.99 – 1.94 (m, 4H), 1.86 – 1.77 (m, 2H), 1.71 – 1.53 (m, 2H), 1.41– 1.32 (m, 6H), 0.86 – 0.78 (m, 12H).
实施例3.4:DL-4的合成
步骤一:
将化合物L1 (30 mg, 0.034 mmol),化合物DL-2-1 (29 mg,0.034 mmol), 溶于DMF (2 mL)中,然后再加N,N-二异丙基乙胺 (9 mg, 0.068 mmol),加完后反应液室温搅拌反应1 h;LCMS监测反应;将反应液减压浓缩,粗品经制备高效液相色谱 (乙腈:水含0.05%FA=5%-50%)纯化,得目标化合物DL-4 (13 mg)。
LCMS (ESI) [M/2+H]+ = 782.2;
1H NMR (400 MHz, DMSO-d6) δ 10.07 (s, 1H), 9.97 (s, 1H), 9.45 (s, 2H),8.93 (s, 1H), 8.26 (s, 2H), 8.11 (d, J = 7.2 Hz, 1H), 7.53 (d, J = 7.6 Hz,2H), 7.44 (d, J = 8.3 Hz, 1H), 7.25 (d, J = 8.4 Hz, 2H), 7.20 (d, J = 8.8 Hz,1H), 6.82 (d, J = 6.6 Hz, 1H), 6.69 – 6.60 (m, 1H), 6.48 (s, 1H), 6.22 (d, J= 12.6 Hz, 2H), 5.07 (d, J = 10.5 Hz, 1H), 4.96 (s, 2H), 4.47 – 4.38 (m, 5H),4.21 – 4.14 (m, 2H), 4.10 – 4.00 (m, 4H), 3.65 (s, 3H), 3.58 – 3.54 (m, 4H),3.43 (s, 3H), 3.21 – 3.18 (m, 2H), 3.15 – 3.10 (m, 2H), 2.91 (d, J = 9.6 Hz,3H), 2.86 – 2.72 (m, 4H), 2.60 (d, J = 14.6 Hz, 2H), 2.46 – 2.41 (m, 1H),2.29 (s, 3H), 2.25 (s, 3H), 2.23 – 2.20 (m, 2H), 2.19 – 2.14 (m, 2H), 2.13(s, 6H), 2.05 (s, 3H), 1.98 (s, 3H), 1.85 – 1.76 (m, 1H), 1.72 – 1.60 (m,2H), 1.43 – 1.36 (m, 2H), 1.23 (s, 6H), 0.90 – 0.85 (m, 6H).
实施例4. 抗体的制备和鉴定
本公开的抗B7H3的2E3-02抗体如WO2022170971A1第194页记载,参照WO2022170971A1实施例3.4制备获得。
实施例5. 抗体药物偶联物(ADC)的合成
实施例5.1:B7H3-ADC-01的制备
取B7H3 2E3-02抗体(4.807 mL,18.3 mg/mL),用0.0481 mL 20mM PB+100mM依地酸二钠溶液(pH 7.6)稀释,以0.5 M Na2HPO4溶液调pH至7.46,加入0.1643 mL 20 mM TCEP(三(2-羧乙基)膦,3.286 µmol)溶液混匀,室温放置90 min。然后加入DL-1(10.38 mg,11倍抗体物质摩尔数量)的二甲基亚砜(0.657mL)溶液,混匀,室温静置2 h,完毕后采用离心超滤管(Merck,Amicon Ultra-15)进行换液,缓冲液置换为pH5.9的20 mM His-Hcl缓冲溶液。得到DL-1与B7H3 2E3-02抗体的偶联产物B7H3-ADC-01。质谱法测定DAR值为8.0。
B7H3-ADC-01
DAR值测定
样品处理:取ADC样品50 μg,用超纯水稀释到 0.5 mg/ml,然后加入1M DTT 1 μl,混匀后离心取上清进样。
仪器信息
液相参数
质谱参数
实验结果
测定B7H3 2E3-02抗体轻链偶联0~1个毒素分子(LC和DAR1比例分别为0%和100%)、重链偶联0~3个毒素分子(HC、DAR1、DAR2、DAR3的比例分别为0%、0%、0%、100%),由此计算B7H3-ADC-01的抗体-药物偶联比(DAR值)为8.0。由轻重链上偶联毒素分子的分布可知,q为8。
实施例5.2:B7H3-ADC-02的制备
取B7H3 2E3-02抗体(3.967 mL,18.3 mg/mL),用0.0397 mL 20mM PB+100mM依地酸二钠溶液(pH 7.6)稀释,以0.5 M Na2HPO4溶液调pH至7.48,加入0.1356 mL 20 mM TCEP(三(2-羧乙基)膦,2.712 µmol)溶液混匀,室温放置90 min。然后加入DL-2(7.833 mg,10倍抗体物质摩尔数量)的二甲基亚砜(0.493mL)溶液,混匀,室温静置2h,完毕后采用离心超滤管(Merck,Amicon Ultra-15)进行换液,缓冲液置换为pH5.9的20 mM His-HCl缓冲溶液。得到DL-2与B7H3 2E3-02抗体的偶联产物B7H3-ADC-02。质谱法测定DAR值为7.9。
B7H3-ADC-02
DAR测定:采用与实施例5.1相同的测试方法,实验结果如下:
由轻重链上偶联毒素分子的分布可知,q可为6、7、8。
实施例6. Payload及ADC的生物活性测试
实施例6.1:Payload对体外细胞增殖的抑制活性检测
按照常规方法即37℃、5%CO2培养箱及细胞指定培养基培养SW480、A375肿瘤细胞。使用胰酶通过常规方法对SW480、A375肿瘤细胞进行消化,收集细胞并计数。用对应的检测培养基(含4% FBS)重悬细胞,将20000细胞/孔加至96孔板,100μL/孔。使用4% FBS培养基稀释待测药物,药物浓度从80nM起始,3倍稀释11个浓度梯度。已稀释的Payload分子100μL加入至含100μL培养基及细胞的96孔板。37℃ 5%CO2培养2-3天,其中A375为2天,SW480为3天。孵育结束后,每孔加入CCK8试剂20μL,反应2小时后,酶标仪检测450nM处吸光值。实验结果如表1所示。
测试结果表明,本公开的payload分子在多种肿瘤细胞中具有极强的细胞杀伤作用。
实施例6.2:ADC对体外细胞增殖的抑制活性检测
按照常规方法即37℃、5%CO2培养箱及细胞指定培养基培养SW480肿瘤细胞。使用胰酶通过常规方法对SW480肿瘤细胞进行消化,收集细胞并计数。用对应的检测培养基(含4% FBS)重悬细胞,将10000细胞/孔加至96孔板,100μL /孔。使用4% FBS培养基稀释待测药物,药物浓度从720nM起始,3倍稀释12个浓度梯度。已稀释的毒素分子100μL加入至含100μL培养基及细胞的96孔板。37℃ 5%CO2培养3天。孵育结束后,每孔加入CCK8试剂20μL,反应2小时后,酶标仪检测450nM处吸光值。实验结果如表2所示。
测试结果表明,本公开的ADC分子在SW480肿瘤细胞中具有较强的细胞杀伤作用。
Claims (10)
1.一种海鞘素类化合物或其药学上可接受的盐;所述的海鞘素类化合物为如下任一结构:
2.一种如式II所示的药物连接子偶联物;
其中,L’为连接子前体;
D为如权利要求1所述的海鞘素类化合物失去氢原子形成的基团;所述的氢原子为片段
中羟基上的氢原子;
所述的连接子前体由连接子和离去基团组成,其与抗体连接后形成连接子;
所述的连接子为如下任一结构:
其中,1位与所述的离去基团相连,2位与所述的D相连。
3.如权利要求2所述的如式II所示的药物连接子偶联物,其特征在于,所述的离去基团为甲砜基。
4.一种药物连接子偶联物,其为如下任一结构:
5.一种如式III所示的抗体药物偶联物;
其中,Tb为抗B7H3抗体或其抗原结合片段,抗Trop-2抗体或其抗原结合片段或者抗Her2抗体或其抗原结合片段;
q为0-20的整数或小数,但不为0;
L为连接子;
D为如权利要求1所述的海鞘素类化合物失去氢原子形成的基团;所述的氢原子为片段
中羟基上的氢原子;
所述的连接子为如下任一结构:
其中,1位通过所述的Tb中的S原子与所述的Tb相连,2位与所述的D相连。
6.如权利要求5所述的如式III所示的抗体药物偶联物,其特征在于,q选自6、7和8。
7.如权利要求5所述的如式III所示的抗体药物偶联物,其特征在于,其为如下任一结构:
其中,所述的B7H3 mAb为抗B7H3的2E3-02抗体;q为7.9~8。
8.一种药物组合物,其由物质X和药用辅料组成;
所述的物质X为物质X1或物质X2;所述的物质X1为如权利要求1所述的海鞘素类化合物或其药学上可接受的盐;所述的物质X2为如权利要求5~7中任一项所述的如式III所示的抗体药物偶联物。
9.一种物质X在制备药物中的应用;
所述的物质X为物质X1或物质X2;所述的物质X1为如权利要求1所述的海鞘素类化合物或其药学上可接受的盐;所述的物质X2为如权利要求5~7中任一项所述的如式III所示的抗体药物偶联物;
所述的药物为用于治疗和/或预防与细胞活动异常相关的疾病的药物。
10.一种物质X在制备药物中的应用;
所述的物质X为物质X1或物质X2;所述的物质X1为如权利要求1所述的海鞘素类化合物或其药学上可接受的盐;所述的物质X2为如权利要求5~7中任一项所述的如式III所示的抗体药物偶联物;
所述的药物为用于治疗和/或预防癌症的药物。
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