CN110251680B - 一种哌嗪类二乙烯基磺酰胺类链接子及其制备方法与应用 - Google Patents
一种哌嗪类二乙烯基磺酰胺类链接子及其制备方法与应用 Download PDFInfo
- Publication number
- CN110251680B CN110251680B CN201910592414.0A CN201910592414A CN110251680B CN 110251680 B CN110251680 B CN 110251680B CN 201910592414 A CN201910592414 A CN 201910592414A CN 110251680 B CN110251680 B CN 110251680B
- Authority
- CN
- China
- Prior art keywords
- compound
- piperazine
- antibody
- room temperature
- synthesis
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
- A61K47/545—Heterocyclic compounds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
- A61K47/6873—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting an immunoglobulin; the antibody being an anti-idiotypic antibody
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
- A61K49/0052—Small organic molecules
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
- C07D295/22—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with hetero atoms directly attached to ring nitrogen atoms
- C07D295/26—Sulfur atoms
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Epidemiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- General Chemical & Material Sciences (AREA)
- Nitrogen And Oxygen Or Sulfur-Condensed Heterocyclic Ring Systems (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
Description
技术领域
本发明属于生物制药及生物技术领域,涉及一种用于抗体药物偶联物的连接子及其制备方法和用途。
背景技术
抗体-药物偶联物(ADC)是靶向治疗药物的热门领域。随着用于治疗霍奇金淋巴瘤以及系统性间变性大细胞淋巴瘤(ALCL)的ADCs药物Adcetris和用于治疗乳腺癌的ADCs药物Kadcyla分别于2011和2013年上市,ADCs进入高速发展期。2017年,主要用于治疗急性髓细胞性白血病(AML)的ADCs药物 Mylotarg,经历了2000年作为首例ADCs药物上市,2010年由辉瑞公司主动撤出市场后,又重获新生。同时,辉瑞公司推出另一个用于治疗急性淋巴细胞性白血病(ALL)的ADCs药物Besponsa。2018年,用于毛细胞白血病的ADCs 药物Lumoxiti上市。另外还有超过60个ADCs正在进行临床前研究。链接子的开发在ADCs的研发中非常重要,它与ADCs的稳定性、代谢、安全性等密切相关。相较于早期的非定位偶联,定位偶联有明确的连接位点,所得ADC均一性高,有显著优势。近期发展的硫-硫键桥连技术可针对抗体链间4对硫-硫键定位修饰。
发明内容
本发明的目的是提供一种新的哌嗪类二乙烯基磺酰胺类链接子及其制备方法与应用。
为了达到上述目的,本发明提供了一种哌嗪类二乙烯基磺酰胺类链接子,其特征在于,其结构式为:
或其药学上可接受的盐;其中:L为连接基团,D为活性药物或荧光物质。
优选地,所述连接基团为羧基、胺基、炔基或叠氮中的一种。
优选地,所述活性药物为可用于抗体药物偶联物的药物。
优选地,所述活性药物为美登素类(Maytansinoids)、耳抑素肽类(Auristatins)、药物为卡其霉素类(Calicheamicins)、阿霉素类(Doxorubicins)、吡咯并苯二氮卓二聚体类(PBDs)、雷公藤甲素类(Triptolide)、秋水仙碱类(Colchicine)、康普瑞汀类(Combretastatin)、高三尖杉酯碱类(Homoharringtonine)、喜树碱类 (Camptothecin)、紫杉醇类(Paclitaxel)或雷公藤甲素类(Triptolide)中的一种。
优选地,所述活性药物的结构式为:
本发明还提供了上述哌嗪类二乙烯基磺酰胺类链接子的制备方法,其特征在于,包括以下步骤:
步骤a:化合物Ⅰ的合成:
冰浴条件下,将哌嗪-2-羧酸二盐酸盐溶于氢氧化钠水溶液中,缓慢加入二碳酸叔丁酯[(Boc)2O]的二氧六环溶液;然后室温反应12h,随后向反应体系中加入盐酸将反应液pH值调制4左右;用乙酸乙酯萃取,合并有机相,无水硫酸钠干燥,减压旋转蒸除溶剂后,得到化合物Ⅰ;
步骤b:化合物Ⅱ的合成:
将化合物Ⅰ、炔丙胺、HATU、N’N-二异丙基乙胺溶于N’N-二甲基甲酰胺中,在室温下搅拌反应12h,用水稀释反应液后,乙酸乙酯萃取,合并有机相,无水硫酸钠干燥,减压旋转蒸除溶剂后得粗品,加入乙酸乙酯和60~100目硅胶,拌匀旋干,粗品经硅胶柱层析,得到化合物Ⅱ;
步骤c:化合物Ⅲ的合成:
将化合物Ⅱ溶于二氯甲烷中,冰浴下加入三氟乙酸,逐渐升至室温,共反应 6h,减压旋转蒸除溶液后,得到化合物Ⅲ;
步骤d:化合物Ⅳ的合成:
将化合物Ⅲ和Et3N溶于DCM中,冰浴下冷却至0℃后缓慢加入2-氯乙烷磺酰氯,室温下反应20min,加入水,产物用DCM萃取,合并后的有机相用饱和NaCl洗后用无水Na2SO4干燥,减压旋蒸除去溶剂,加入CH2Cl2和60~100 目硅胶,拌匀旋干,采用乙酸乙酯/石油醚=1/1作洗脱剂,快速柱层析,得到化合物Ⅳ;
步骤e:化合物Ⅳ的合成:
将化合物Ⅳ、化合物6(参考文献R.Huang,Z.Li,Y.Sheng,J.Yu,Y.Wu,Y. Zhan,H.Chen,B.Jiang,Org.Lett.,2018,20,6526-6529)、抗坏血酸钠和硫酸铜溶于tBuOH/H2O/DMF(1/1/1)中,室温下反应3h,用制备HPLC分离得到化合物Ⅴ,即哌嗪类二乙烯基磺酰胺类链接子。
优选地,所述步骤a中哌嗪-2-羧酸二盐酸盐包括哌嗪-2-羧酸二盐酸盐、(R)- 哌嗪-2-羧酸二盐酸盐和(S)-哌嗪-2-羧酸二盐酸盐中的任意一种。
本发明提供上述哌嗪类二乙烯基磺酰胺类链接子在制备抗体药物偶联物中的应用。
本发明还提供了一种包含上述哌嗪类二乙烯基磺酰胺类链接子的靶向分子和活性药物分子偶联物,其特征在于,其结构式为:
或其药学上可接受的盐;其中:A为靶向分子,L为连接基团,D为活性药物或荧光物质。
优选地,所述靶向分子为抗体,抗体片段、替代体或变体,多肽或小分子配体中的任意一种。
更优选地,所述靶向分子为曲妥珠单抗(Trastuzumab)。
本发明中的靶向抗原包括但不限于Her2。
优选地,所述活性药物的结构式为:
与现有技术,本发明的有益效果在于:
1.本发明提供了一种新型哌嗪类二乙烯基磺酰胺类链接子的制备及其在抗体药物偶联物中的应用;
2.本发明提供的方法制备所得抗体-药物偶联物均一性高;
3.本发明提供的方法制备所得抗体-药物偶联物保持和原有抗体相近的亲和力和内吞作用;
4.本发明提供的方法制备所得抗体-药物偶联物本7、13、19与上市药物 T-DM1具有相似的肿瘤细胞杀伤作用,但是它们在Her-2低表达细胞中的细胞毒性显著小于T-DM1。
5.本发明中涉及的抗体-药物偶联物7、13、19具有更好的安全性。
附图说明
图1显示了本发明中涉及的抗体-药物偶联物的SDS-PAGE跑胶图;
其中,MW:molecular weight marker,Lane 1:Trastuzumab,Lane 2:ReducedTrastuzumab,Lane 3:抗体-药物偶联物7,Lane 4:抗体-药物偶联物13,Lane 5:抗体-药物偶联物19;
图2显示了本发明中涉及的抗体-药物偶联物的HR-ESI-MS谱图;其中,A:抗体-药物偶联物7,B:抗体-药物偶联物13,C:抗体-药物偶联物19;
图3显示了本发明中涉及的抗体-药物偶联物的SEC-HPLC谱图;
图4显示了本发明中涉及100nM的抗体-药物偶联物及曲妥珠单抗在 SK-BR-3细胞的亲和力;
图5显示了本发明中涉及的不同浓度梯度的抗体-药物偶联物及曲妥珠单抗在SK-BR-3细胞的亲和力;
图6显示了本发明中涉及100nM的抗体-药物偶联物及曲妥珠单抗在 MCF-7细胞的亲和力;
图7显示了本发明中涉及的抗体-药物偶联物及曲妥珠单抗在SK-BR-3细胞的内吞效应;
图8显示了本发明中涉及的抗体-药物偶联物、曲妥珠单抗、T-DM1、MMAE 对在SK-BR-3细胞的体外增殖抑制作用;
图9显示了本发明中涉及的抗体-药物偶联物、曲妥珠单抗、T-DM1、MMAE 对在NCI-N87细胞的体外增殖抑制作用;
图10显示了本发明中涉及的抗体-药物偶联物、曲妥珠单抗、T-DM1、MMAE 对MCF-7细胞的体外增殖抑制作用;
图11显示了本发明中涉及的抗体-药物偶联物、曲妥珠单抗、T-DM1、MMAE 对MDA-MB-231细胞的体外增殖抑制作用;
图12显示了本发明中涉及的抗体-药物偶联物、曲妥珠单抗、T-DM1、MMAE 对MDA-MB-468细胞的体外增殖抑制作用;
图13显示了本发明实施例2中抗体药物偶联物的制备流程。
具体实施方式
下面结合具体实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解,在阅读了本发明讲授的内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。
实施例1-1化合物7的制备
反应条件为:
a)氢氧化钠,1,4-二氧六环,室温,12h;
b)2-(7-氧化苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸盐(HATU),N’N-二异丙基
乙胺,N’N-二甲基甲酰胺(DMF)中,室温,12h;
c)三氟乙酸,二氯甲烷,0℃-室温,6h;
d)三乙胺(Et3N),二氯甲烷,0℃-室温,30min;
e)抗坏血酸钠,硫酸铜,tBuOH/H2O/DMF(1/1/1),室温,3h。
其具体制备步骤如下:
步骤a:化合物2的合成:
冰浴下,将2-哌嗪羧酸盐酸盐(780mg,6mmol)溶于氢氧化钠(960mg,40 mmol)水溶液中,缓慢加入二碳酸叔丁酯[(Boc)2O,2.88g,13.2mmol]的二氧六环(25mL)溶液,然后室温反应12小时;随后向反应体系中加入1M盐酸将反应液pH值调制4左右;用乙酸乙酯萃取(3*20mL),合并有机相,无水硫酸钠干燥,减压旋转蒸除溶剂后得白色固体的化合物2(880mg,2.67mmol,45%),直接用于下一部反应;ESI-HRMS Calculated for C15H27N2O6[M+H]+:331.1869, Found:331.1872.
步骤b:化合物3的合成:
将化合物2(400mg,1.21mmol),炔丙胺(100mg,1.8mmol),HATU(684 mg,1.8mmol),N’N-二异丙基乙胺(707mg,5.4mmol)溶于N’N-二甲基甲酰胺 (5mL)中,在室温下搅拌反应12小时;用100mL水稀释反应液后,乙酸乙酯萃取(3*20mL),合并有机相,无水硫酸钠干燥,减压旋转蒸除溶剂后得粗品,加入15mL乙酸乙酯和1g 60-100目硅胶,拌匀旋干,粗品经硅胶柱层析 (石油醚:乙酸乙酯=3:10)得无色油状物的化合物3(380mg,1.03mmol,85%);1HNMR(500MHz,Chloroform-d)δ6.40(d,J=119.8Hz,1H),4.51(d,J=64.1Hz, 2H),4.17–3.97(m,2H),3.87(s,2H),3.22–3.05(m,2H),2.97(s,1H),2.21(t,J= 2.6Hz,1H),1.47(d,J=11.0Hz,18H).13C NMR(126MHz,CDCl3)δ168.99, 154.67,80.50,77.29,77.04,76.78,29.36,28.29.ESI-HRMS Calculated for C18H30N3O5[M+H]+:368.2185,Found:368.2172.
步骤c:化合物4的合成:
将化合物3(380mg,1.03mmol)溶于二氯甲烷(4mL)中,冰浴下加入三氟乙酸(1.5mL),逐渐升至室温,共反应6h;减压旋转蒸除溶液后得黄色油状物的化合物4(282mg,1.05mmol,98%),直接用于下一步;ESI-HRMS calcd for C8H14N3O[(M+H)+]:168.1137,found:168.1135.
步骤d:化合物5的合成:
化合物4(282mg,1.05mmol)和Et3N(808mg,8.0mmol)溶于DCM(12 mL)中,冰浴下冷却至0℃后缓慢加入2-氯乙烷磺酰氯(652mg,4.0mmol),室温下反应20min,加入水(30mL),产物用DCM(3x 15mL)萃取,合并后的有机相用饱和NaCl(10mL)洗后用无水Na2SO4干燥,减压旋蒸除去溶剂,加入10mL CH2Cl2和0.4g 60-100目硅胶,拌匀旋干,采用乙酸乙酯/石油醚=1/1 作洗脱剂,快速柱层析得到产物淡黄色固体的化合物5(222.34mg,0.64mmol),产率64%;1H NMR(500MHz,DMSO-d6)δ8.54(t,J=5.5Hz,1H),6.76(ddd,J= 19.0,16.5,9.9Hz,2H),6.25–6.02(m,4H),4.39(dd,J=4.6,1.9Hz,1H),4.00– 3.82(m,3H),3.60–3.53(m,1H),3.52–3.41(m,2H),3.15(t,J=2.5Hz,1H),2.92 (dd,J=12.6,4.3Hz,1H),2.68(td,J=11.9,11.4,3.8Hz,1H).13C NMR(126MHz, DMSO)δ168.22,136.09,133.21,130.17,128.10,81.16,73.76,54.79,47.50,44.85, 42.26,40.50,40.33,40.17,40.00,39.83,39.67,39.50,28.76.ESI-HRMS calcd for C12H18N3O5S2[(M+H)+]:348.0688,found:348.0683.
步骤e:化合物7的合成:
化合物5(12mg,0.035mmol)、化合物6(参考文献R.Huang,Z.Li,Y.Sheng, J.Yu,Y.Wu,Y.Zhan,H.Chen,B.Jiang,Org.Lett.,2018,20,6526-6529)(30mg, 0.032mmol)、抗坏血酸钠(12.6mg,0.064mmol)和硫酸铜(10mg,0.064mmol) 溶于3mL tBuOH/H2O/DMF(1/1/1)中,室温下反应3h,然后用制备HPLC(固定相为C-18硅胶柱,流动相为CH3CN/H2O=10~100%,30分钟)分离得到产物白色固体的化合物7(24mg,0.01874mmol),产率53%;1H NMR(500MHz, Methanol-d4)δ8.18(dd,J=28.1,8.3Hz,1H),7.43–7.17(m,4H),6.72(dd,J=16.5,9.8Hz,1H),6.61(dt,J=11.1,7.1Hz,1H),6.28–6.09(m,2H),6.07–5.99(m, 1H),4.79–4.41(m,7H),4.41–4.03(m,4H),4.00–3.82(m,2H),3.82–3.49(m, 10H),3.51–3.23(m,10H),3.23–3.09(m,2H),3.09–2.92(m,3H),2.91–2.66(m, 1H),2.58–2.36(m,2H),2.36–2.18(m,1H),2.17–1.67(m,4H),1.66–1.50(m, 1H),1.49–1.32(m,1H),1.32–0.70(m,21H).13C NMR(126MHz,MeOD)δ 174.03,142.71,135.51,132.38,128.99,128.13,127.86,127.23,127.10,126.90, 126.72,126.58,82.05,75.91,70.42,70.05,69.95,69.15,68.33,60.61,60.23,59.54, 59.20,56.94,55.28,54.71,51.36,49.93,48.13,47.96,47.90,47.79,47.73,47.62, 47.53,47.45,47.36,47.28,47.11,46.68,44.74,44.43,44.13,42.10,33.77,31.70, 30.50,29.02,26.27,25.62,25.20,24.48,24.21,23.07,18.37,17.79,17.36,14.69, 14.44,13.79,9.53.ESI-HRMS Calculated for C59H98N11O16S2[M+H]+:1280.6634, Found:1280.6644。
实施例1-2化合物13的制备
反应条件为:
a)氢氧化钠,1,4-二氧六环,室温,12h;
b)2-(7-氧化苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸盐(HATU),N’N-二异丙基乙胺,N’N-二甲基甲酰胺(DMF)中,室温,12h;
c)三氟乙酸,二氯甲烷,0℃-室温,6h;
d)三乙胺(Et3N),二氯甲烷,0℃-室温,30min;
e)抗坏血酸钠,硫酸铜,tBuOH/H2O/DMF(1/1/1),室温,3h。
其具体制备步骤如下:
步骤a:化合物9的合成:
冰浴下,将(R)-哌嗪-2-羧酸盐酸盐8(2g,10mmol)溶于氢氧化钠(1.6g,40 mmol)水溶液中,随后加入二碳酸叔丁酯[(Boc)2O,4.4g,20.2mmol]的二氧六环(20mL)溶液,室温反应12小时;随后向反应体系中加入1M盐酸将反应液pH值调制4左右,用乙酸乙酯萃取(3*20mL),合并有机相,无水硫酸钠干燥,减压旋转蒸除溶剂后得白色固体的化合物9(2.5g,7.5mmol,77%),直接用于下一步反应;ESI-HRMS Calculated for C15H27N2O6[M+H]+:331.1869, Found:331.1872.
步骤b:化合物10的合成:
将化合物9(1.3g,3.9mmol),炔丙胺(440mg,7.8mmol),HATU(2.28 g,6.0mmol),N’N-二异丙基乙胺(2.555g,19.5mmol)溶于N’N-二甲基甲酰胺(10mL)中,在室温下搅拌反应12小时;用100mL水稀释反应液后,乙酸乙酯萃取(3*20mL),合并有机相,无水硫酸钠干燥,减压旋转蒸除溶剂后得粗品,加入15mL乙酸乙酯和1g 60-100目硅胶,拌匀旋干,粗品经硅胶柱层析(石油醚:乙酸乙酯=3:10)得无色油状物的化合物10(1.4g,3.8mmol,95%)。1HNMR(500MHz,DMSO-d6)δ8.46(t,J=5.5Hz,1H),4.32(d,J=69.3Hz,1H), 4.18–3.98(m,1H),3.97–3.50(m,4H),3.31(d,J=12.9Hz,1H),3.27–3.04(m, 2H),2.75(d,J=68.5Hz,1H),1.45–1.28(m,19H).13C NMR(126MHz,DMSO)δ 170.40,155.22,79.80,79.43,73.84,40.47,40.30,40.13,39.97,39.80,39.63,39.47, 28.52,28.40,28.34.ESI-HRMSCalculated for C18H30N3O5[M+H]+:368.2185, Found:368.2172.
步骤c:化合物11的合成:
将化合物10(1.4g,3.8mmol)溶于二氯甲烷(5mL)中,冰浴下加入三氟乙酸(1.5mL),逐渐升至室温,共反应6h;减压旋转蒸除溶液后得黄色油状物的化合物11(600mg,3.57mmol,94%),直接用于下一步反应;ESI-HRMS calcd for C8H14N3O[(M+H)+]:168.1137,found:168.1135.
步骤d:化合物12的合成:
化合物11(600mg,3.57mmol)和Et3N(3.03g,30mmol)溶于DCM(12mL) 中,冰浴下冷却至0℃后缓慢加入2-氯乙烷磺酰氯(1.745g,10.71mmol),室温下反应20min,加入水(30mL),产物用DCM(3x 15mL)萃取,合并后的有机相用饱和NaCl(10mL)洗后用无水Na2SO4干燥,减压旋蒸除去溶剂,加入 10mL CH2Cl2和0.8g 60-100目硅胶,拌匀旋干,采用乙酸乙酯/石油醚=1/1作洗脱剂,快速柱层析得到产物淡黄色固体的化合物12(916mg,2.64mmol),产率 74%;1H NMR(500MHz,DMSO-d6)δ8.54(t,J=5.5Hz,1H),6.76(ddd,J=19.1, 16.5,9.9Hz,2H),6.22–6.03(m,4H),4.43–4.35(m,1H),3.98–3.82(m,3H),3.59 –3.52(m,1H),3.52–3.42(m,2H),3.32(s,1H),3.15(t,J=2.5Hz,1H),2.92(dd,J =12.6,4.3Hz,1H),2.68(td,J=11.9,11.4,3.7Hz,1H).13C NMR(126MHz, DMSO)δ167.74,135.61,132.73,129.69,127.62,80.68,73.28,54.31,47.02,44.37, 41.78,40.02,39.85,39.69,39.52,39.35,39.19,39.02,28.28.ESI-HRMS calcd for C12H18N3O5S2[(M+H)+]:348.0688,found:348.0683.
步骤e:化合物13的合成:
化合物12(17mg,0.05mmol)、化合物6(46mg,0.05mmol)、抗坏血酸钠 (19mg,0.1mmol)和硫酸铜(16mg,0.1mmol)溶于3mL tBuOH/H2O/DMF (1/1/1)中,室温下反应3h,然后用制备HPLC(固定相为C-18硅胶柱,流动相为CH3CN/H2O=10~100%,30分钟)分离得到产物白色固体的化合物13(20mg, 0.016mmol),产率32%;1H NMR(500MHz,Methanol-d4)δ8.00(d,J=11.8Hz, 1H),7.45–7.16(m,6H),6.81–6.52(m,2H),6.29–5.91(m,4H),4.74–4.11(m, 14H),4.08(d,J=14.5Hz,1H),3.99–3.81(m,3H),3.80–3.51(m,14H),3.41(ddd, J=12.2,9.4,5.8Hz,1H),3.35(dd,J=4.1,1.7Hz,5H),3.29(d,J=3.4Hz,2H), 3.13(d,J=2.0Hz,1H),3.08–2.89(m,5H),2.85–2.71(m,1H),2.51(d,J=27.5 Hz,2H),2.40–2.18(m,2H),2.17–1.66(m,6H),1.66–1.52(m,1H),1.51–1.21 (m,3H),1.21–0.76(m,29H).13C NMR(126MHz,MeOD)δ174.03,170.49, 169.11,142.70,135.48,132.41,128.94,128.15,127.86,127.24,127.06,126.85, 126.71,126.55,85.27,82.05,77.48,75.91,70.35,70.07,70.00,69.21,68.75,60.59, 60.18,59.45,59.19,57.21,56.93,56.08,55.26,55.04,54.74,50.27,49.91,48.12, 48.06,47.95,47.89,47.81,47.78,47.74,47.72,47.66,47.60,47.55,47.43,47.31, 47.26,47.22,47.09,46.68,44.74,44.45,44.12,42.07,34.47,30.46,29.00,26.26, 25.59,25.19,24.46,24.21,23.06,18.36,17.76,17.36,15.59,14.66,14.55,14.42, 13.71,9.51.ESI-HRMS Calculated for C59H98N11O16S2[M+H]+:1280.6634, Found:1280.6644。
实施例1-3化合物19的制备
反应条件为:
a)氢氧化钠,1,4-二氧六环,室温,12h;
b)2-(7-氧化苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸盐(HATU),N’N-二异丙基乙胺,N’N-二甲基甲酰胺(DMF)中,室温,12h;
c)三氟乙酸,二氯甲烷,0℃-室温,6h;
d)三乙胺(Et3N),二氯甲烷,0℃-室温,30min;
e)抗坏血酸钠,硫酸铜,tBuOH/H2O/DMF(1/1/1),室温,3h。
其具体制备步骤如下:
步骤a:化合物15的合成:
冰浴下,将(S)-哌嗪-2-羧酸盐酸盐14(1g,5mmol)溶于氢氧化钠(0.7g,20 mmol)水溶液中,随后加入二碳酸叔丁酯[(Boc)2O,2.2g,10mmol]的二氧六环(10mL)溶液,室温反应12小时;随后向反应体系中加入1M盐酸将反应液pH值调制4左右,用乙酸乙酯萃取(3*20mL),合并有机相,无水硫酸钠干燥,减压旋转蒸除溶剂后得白色固体的化合物15(1.15g,3.5mmol,70%),直接用一下一部反应;ESI-HRMS Calculated for C15H27N2O6[M+H]+:331.1869, Found:331.1872.
步骤b:化合物16的合成:
将化合物15(1.15g,3.5mmol),炔丙胺(578mg,10.5mmol),HATU(1.9 g,5.0mmol),N’N-二异丙基乙胺(2.2g,17mmol)溶于N’N-二甲基甲酰胺(12 mL)中,在室温下搅拌反应12小时;用100mL水稀释反应液后,乙酸乙酯萃取(3*20mL),合并有机相,无水硫酸钠干燥,减压旋转蒸除溶剂后得粗品,加入15mL乙酸乙酯和1g 60-100目硅胶,拌匀旋干。粗品经硅胶柱层析(石油醚:乙酸乙酯=3:10)得无色油状物的化合物16(830mg,2.26mmol,65%);1HNMR(500MHz,DMSO-d6)δ8.46(s,1H),4.32(d,J=68.7Hz,1H),4.17–3.97 (m,1H),3.96–3.55(m,4H),3.23(d,J=13.2Hz,1H),3.13(s,1H),2.86(t,J=64.5 Hz,1H),1.35(d,J=6.7Hz,19H).13C NMR(126MHz,CDCl3)δ171.19,169.17, 154.66,81.45,80.45,79.32,77.41,77.16,76.90,71.77,60.42,56.44,54.19,43.43, 42.66,42.16,29.31,28.77,28.35,28.31,27.89,21.07,14.22,1.91,0.03.ESI-HRMS Calculated for C18H30N3O5[M+H]+:368.2185,Found:368.2172.
步骤c:化合物17的合成:
将化合物16(830mg,2.26mmol)溶于二氯甲烷(3mL)中,冰浴下加入三氟乙酸(1.5mL),冰浴下反应6h,减压旋转蒸除溶液后得黄色油状物的化合物17(378mg,2.26mmol,100%),直接用于下一步;ESI-HRMS calcd for C8H14N3O[(M+H)+]:168.1137,found:168.1135.
步骤d:化合物18的合成:
化合物17(378mg,2.26mmol)和Et3N(2.28g,22.6mmol)溶于DCM(10 mL)中,冰浴下冷却至0℃后缓慢加入2-氯乙烷磺酰氯(1.474g,9.04mmol),室温下反应20min,加入水(30mL),产物用DCM(3x 15mL)萃取,合并后的有机相用饱和NaCl(10mL)洗后用无水Na2SO4干燥,减压旋蒸除去溶剂,加入10mL CH2Cl2和0.4g 60-100目硅胶,拌匀旋干。采用乙酸乙酯/石油醚=1/1 作洗脱剂,快速柱层析得到产物淡黄色固体18(541mg,1.56mmol),产率69%;1H NMR(500MHz,Chloroform-d)δ6.75(t,J=5.4Hz,1H),6.64(dd,J=16.5,9.8 Hz,1H),6.46(dd,J=16.5,9.9Hz,1H),6.30(dd,J=16.5,14.5Hz,2H),6.07(dd,J =9.9,8.1Hz,2H),4.49(dt,J=3.7,1.6Hz,1H),4.22(dt,J=12.8,1.8Hz,1H),4.17 –3.93(m,3H),3.67(dddt,J=60.2,12.4,3.6,1.7Hz,2H),3.28(ddd,J=13.6,12.0, 3.5Hz,1H),2.96(dd,J=12.8,4.0Hz,1H),2.83(td,J=12.1,3.4Hz,1H),2.26(t,J =2.5Hz,1H).13C NMR(126MHz,CDCl3)δ167.22,135.14,132.90,129.73,128.77, 78.92,77.41,77.16,76.91,72.17,55.69,45.58,44.24,42.35,29.78.ESI-HRMS calcd for C12H18N3O5S2[(M+H)+]:348.0688,found:348.0683.
步骤e:化合物19的合成:
化合物18(18mg,0.052mmol)、化合物6(48mg,0.052mmol)、抗坏血酸钠(20mg,0.105mmol)和硫酸铜(18mg,0.105mmol)溶于3mL tBuOH/H2O/DMF (1/1/1)中,室温下反应3h,然后用制备HPLC(固定相为C-18硅胶柱,流动相为CH3CN/H2O=10~100%,30分钟)分离得到产物白色固体19(18mg,0.014 mmol),产率27%。1H NMR(500MHz,Methanol-d4)δ8.00(d,J=11.8Hz,1H), 7.42–7.17(m,5H),6.78–6.56(m,2H),6.24–5.98(m,4H),4.79–4.50(m,7H), 4.50–4.01(m,6H),3.99–3.80(m,3H),3.81–3.49(m,13H),3.41(ddd,J=12.2,9.4,5.8Hz,1H),3.35(dd,J=4.1,1.7Hz,5H),3.29(d,J=3.4Hz,2H),3.22–3.09 (m,2H),3.08–2.89(m,4H),2.87–2.70(m,1H),2.51(d,J=27.5Hz,2H),2.40– 1.22(m,11H),1.23–0.73(m,27H).13C NMR(126MHz,MeOD)δ174.03,170.49, 169.11,142.70,135.48,132.41,128.94,128.15,127.86,127.24,127.06,126.85, 126.71,126.55,89.72,85.27,82.05,77.48,75.91,70.35,70.07,70.00,69.21,68.75, 60.59,60.18,59.45,59.19,57.21,56.93,56.08,55.26,55.04,54.74,50.27,49.91, 48.12,48.06,47.95,47.89,47.81,47.78,47.74,47.72,47.66,47.60,47.55,47.43, 47.31,47.26,47.22,47.09,46.68,44.74,44.45,44.12,42.07,34.47,30.46,29.00, 26.26,25.59,25.19,24.46,24.21,23.06,18.36,17.76,17.36,15.59,14.66,14.55, 14.42,13.71,9.51.ESI-HRMSCalculated for C59H98N11O16S2[M+H]+:1280.6634, Found:1280.6644。
实施例2:抗体药物偶联物的制备
如图13所示,本实施例提供了抗体-药物偶联物7、抗体-药物偶联物13、抗体-药物偶联物19的制备方法。
具体反应条件为:
a)三(2-羧乙基)膦室温(TCEP),磷酸盐缓冲液(PBS),室温,2h;
b)化合物7或者13或者19,PBS/二甲亚砜,室温,12h。
将化合物7,13,19分别溶于DMSO配成10mM的溶液;曲妥珠单抗 (Trastuzumab)(上海环耀生物科技有限公司,货号:180288-69-1)溶于PBS (pH=7.4)缓冲溶液配成2.5mg/mL的溶液,TCEP溶于纯水配成10mM的溶液 (用NaOH/H3PO4调节PH为7.0)。
在微孔板(330uL LABTIDE 96Round Well)的四个孔中分别加入100uL的曲妥珠单抗溶液,0.83uL的TCEP溶液,然后放在微孔板振荡器上室温反应2 小时。随后加入1.67uL(10eq.)化合物7或者13或者19的DMSO溶液,然后放在微孔板振荡器上室温下反应12小时后;通过Zeba脱盐柱(ZebaTM Spin Desalting Columns,7K MWCO,0.5mL)除去过量的小分子化合物,得到抗体抗体-药物偶联物,分别命名为抗体-药物偶联物7、抗体-药物偶联物13、抗体-药物偶联物19。用UPLC-MS(型号ABsciex 4600)分析反应结果。
图1显示了本发明中涉及的抗体-药物偶联物的SDS-PAGE跑胶图;
其中,MW:molecular weight marker,Lane 1:Trastuzumab,Lane 2:ReducedTrastuzumab,Lane 3:抗体-药物偶联物7,Lane 4:抗体-药物偶联物13,Lane 5:抗体-药物偶联物19;
图2显示了本发明中涉及的抗体-药物偶联物的HR-ESI-MS谱图;其中,A:抗体-药物偶联物7,B:抗体-药物偶联物13,C:抗体-药物偶联物19;
图3显示了本发明中涉及的抗体药物偶联物的SEC-HPLC谱图;
如图1~3所示,本发明中所制备得到的抗体-药物偶联物7、13、19均一性高。
实施例3:抗体-药物偶联物的亲和力测定
在本实施例中,研究了曲妥珠单抗(Trastuzumab)、抗体-药物偶联物7、抗体-药物偶联物13、抗体-药物偶联物19对肿瘤细胞系的亲和作用。
以下实验中所用的试剂及耗材来源于:Goat Anti-Human IgG H&L 购置于Abcam公司,DAPI购置于Cell Signaling公司,face清洗液 (PBS+1%BSA),人乳腺癌细胞SK-BR-3和MCF-7细胞来自美国模式培养物集存库ATCC。本实施例中使用流式细胞术(flow cytometry,FCM)来分析抗体及 ADC对肿瘤细胞系的亲和作用。FCM是利用流式细胞仪在细胞分子水平上通过单克隆抗体对单个细胞进行多参数、快速的定量分析、分选的技术。Trastuzumab 可以特异性结合在靶细胞的Her-2抗原位点,然后用特异的荧光二抗标记Trastuzumab,利用FCM检测荧光二抗的荧光强度可以间接指示Trastuzumab与靶细胞的抗原位点的结合能力。
分别收集SK-BR-3、MCF-7细胞至15mL离心管,在4℃下1200rpm离心 3min,弃上清;用face洗液洗涤细胞1次,分装到1.5mL离心管(每个管大于1×105个细胞)中,在4℃下1200rpm离心3min,去上清。
每个样品管内分别加入200μLTrastuzumab单抗、抗体-药物偶联物7、抗体- 药物偶联物13、抗体-药物偶联物19(浓度分别设置为:0.01、0.03、0.1、0.3、 1、3、10、30、100、300nM),在冰上孵育30min。
在4℃下1200rpm离心3min,去上清,用face洗液洗涤细胞3次;每个样品管内加入200μL DAPI(0.1μg/mL),冰上孵育15min后在CytoFLEX(Beckman Coulter)流式细胞仪上进行检测分析。
如图4所示,本发明中涉及Trastuzumab单抗、抗体-药物偶联物7、抗体- 药物偶联物13、抗体-药物偶联物19在Her-2高表达细胞株SK-BR-3细胞中的亲和力,在100nM浓度下,Trastuzumab抗体药物偶联物7、13、19与SK-BR-3 细胞亲和力和曲妥珠单抗与SK-BR-3细胞亲和力相同。
图5显示了本发明中涉及的不同浓度梯度的Trastuzumab抗体药物偶联物及曲妥珠单抗在SK-BR-3细胞的亲和力,Trastuzumab抗体药物偶联物7、13、19 和曲妥珠单抗与SK-BR-3细胞亲和力拟合曲线具有高度一致性。
图6显示了本发明中涉及的Trastuzumab抗体药物偶联物及曲妥珠单抗在 Her-2低表达细胞株MCF-7细胞的亲和力,在100nM浓度下,抗体-药物偶联物 7、13、19和曲妥珠单抗与MCF-7细胞具有相同的亲和力,并且显著低于SK-BR-3 细胞。因此对曲妥珠单抗的上述改造没有影响其在靶细胞中的亲和力。
实施例4:抗体-药物偶联物的内吞作用测定
在本实施例中,研究了肿瘤细胞系对曲妥珠单抗(Trastuzumab)、抗体-药物偶联物7、抗体-药物偶联物13、抗体-药物偶联物19的内吞作用。
以下实验中所用的试剂及耗材来源于:Goat Anti-Human IgG H&L 购置于Abcam公司,DAPI购置于Cell Signaling公司,face清洗液(PBS+1% BSA),人乳腺癌细胞SK-BR-3和MCF-7细胞来自美国模式培养物集存库ATCC。本实施例中使用流式细胞术(flow cytometry,FCM)来分析肿瘤细胞系对抗体及ADC的内吞作用。
Trastuzumab抗体结合在靶细胞的Her-2抗原位点后会发生内 吞作用进入细胞内。细胞在37℃可以正常内 吞,在4℃细胞绝大多数生理活动受到抑制,视为不内 吞。Trastuzumab抗体与肿瘤细胞分别在4℃和37℃孵育一段时间,该时间段内的内 吞量=(4℃细胞表面抗体量-37℃细胞表面抗体量)/4℃细胞表面抗体量×100%。
分别收集SK-BR-3和MCF-7细胞至15mL离心管,在4℃下1200rpm离心3min,弃上清;用face洗液洗涤细胞1次,分装到1.5mL离心管(每个管大于1×105个细胞)中,在4℃下1200rpm离心3min,去上清。
每个样品管内加入200μLTrastuzumab、抗体-药物偶联物7、抗体-药物偶联物13、抗体-药物偶联物19(浓度为均10nM),在4℃孵育30min。
在4℃下1200rpm离心3min,去上清,用face洗液洗涤细胞3次;每个样品管内加入200μL DAPI(0.1μg/mL),冰上孵育15min后在CytoFLEX(Beckman Coulter)流式细胞仪上进行检测分析,结果如图8所示。
图8显示了本发明中涉及的抗体-药物偶联物7、13、19及曲妥珠单抗在Her-2 高表达细胞株SK-BR-3细胞的内吞效应。10nM的抗体-药物偶联物7、13、19 及曲妥珠单抗在SK-BR-3细胞中经过3小时的内 吞效率分别为32.77%、34.39%、 32.22%及35.64%。因此,对曲妥珠单抗的上述改造没有影响其在靶细胞中的内 吞效率。
实施例5:抗体-药物偶联物的体外细胞增殖生物活性测定
在本实施例中,研究了Trastuzumab、抗体-药物偶联物7、抗体-药物偶联物 13、抗体-药物偶联物19、T-DM1(Roche,100mg)、MMAE(ChemShuttle,2.5 g)对肿瘤细胞系增殖的作用。
以下实验中所用的试剂及耗材来源于:RPMI1640培养基、DMEM培养基、 MEM培养基、0.25%胰蛋白酶-EDTA、胎牛血清、重组人胰岛素、100×青链霉素、1×PBS(pH 7.4)、CCK8显色试剂购置于Gibco公司;人肿瘤细胞系SK-BR-3、 NCI-N87、MCF7、MDA-MB-231、MDA-MB-468来自美国模式培养物集存库ATCC;10cm培养皿(Corning)及96孔细胞培养板(Corning);多功能酶标仪 (SpectraMax i3)。
本实施例使用CCK8比色法来评价待测物的抗增殖作用。Cell Counting Kit-8(简称CCK-8)试剂中含有WST-8【化学名:2-(2-甲氧基-4-硝基苯基)-3-(4- 硝基苯基)-5-(2,4-二磺酸苯)-2H-四唑单钠盐】。它在电子载体1-甲氧基-5-甲基吩嗪鎓硫酸二甲酯(1-Methoxy PMS)的作用下被细胞中的脱氢酶还原为具有高度水溶性的黄色甲瓒产物(Formazan dye)。生成的甲瓒物的数量与活细胞的数量成正比,在450nm波长下产生最大吸收峰。因此可利用这一特性直接进行细胞增殖和毒性分析。
本实例选用的细胞系有:SK-BR-3、NCI-N87、MCF-7、MDA-MB-231、 MDA-MB-468。
SK-BR-3、MDA-MB-231、MDA-MB-468细胞在含有10%胎牛血清和1%青链霉素的DMEM培养基中,MCF7细胞在含有10%胎牛血清、1%青链霉素、 1%NEAA和10μg/L胰岛素的MEM培养基中,NCI-N87细胞在含有10%胎牛血清和1%青链霉素的RPMI1640培养基中,所有细胞于37℃、5%二氧化碳培养箱中培养至对数生长期,将处于对数生长期的上述细胞以2~5×103个细胞每孔的密度接种至96孔培养板,每孔100μL,培养24小时后加入不同浓度的药物处理72小时,药物原始浓度为500nM,以5倍稀释制备10个浓度,每个浓度设置3个复孔,并设相应浓度的溶媒对照及无细胞培养基孔。作用结束后,每孔加入10μL CCK8试剂,在于37℃,5%二氧化碳培养箱中继续培养一段1~4 小时。然后在450nm波长下测定吸光度(OD值)。
抑制率(%)=(OD对照-OD给药)/(OD对照-OD空白)×100%
图8~12显示了本发明中涉及的抗体-药物偶联物7、13、19,曲妥珠单抗,上市的抗体-药物偶联物的阳性对照T-DM1以及小分子毒性药物MMAE对在 SK-BR-3、NCI-N87、MCF-7、MDA-MB-231和MDA-MB-468细胞的体外增殖抑制作用。
表1本发明中涉及的曲妥珠单抗,抗体-药物偶联物7、13、19,T-DM1 及MMAE在SK-BR-3、NCI-N87、MCF-7、MDA-MB-231和MDA-MB-468细胞中的IC50(nM)
结果显示,本发明提供的方法制备所得抗体-药物偶联物本7、13、19与上市药物T-DM1具有相似的肿瘤细胞杀伤作用,但是它们在Her-2低表达细胞中的细胞毒性显著小于T-DM1。因此,本发明中涉及的抗体-药物偶联物7、13、 19具有更好的安全性。
Claims (2)
其中:A为曲妥珠单抗;
所述哌嗪类二乙烯基磺酰胺类链接子的制备方法,包括以下步骤:
步骤a:化合物Ⅰ的合成:
冰浴条件下,将哌嗪-2-羧酸二盐酸盐溶于氢氧化钠水溶液中,缓慢加入二碳酸叔丁酯[(Boc)2O]的二氧六环溶液;然后室温反应12h,随后向反应体系中加入盐酸将反应液pH值调制4左右;用乙酸乙酯萃取,合并有机相,无水硫酸钠干燥,减压旋转蒸除溶剂后,得到化合物Ⅰ;
步骤b:化合物Ⅱ的合成:
将化合物Ⅰ、炔丙胺、HATU、N’N-二异丙基乙胺溶于N’N-二甲基甲酰胺中,在室温下搅拌反应12h,用水稀释反应液后,乙酸乙酯萃取,合并有机相,无水硫酸钠干燥,减压旋转蒸除溶剂后得粗品,加入乙酸乙酯和60~100目硅胶,拌匀旋干,粗品经硅胶柱层析,得到化合物Ⅱ;
步骤c:化合物Ⅲ的合成:
将化合物Ⅱ溶于二氯甲烷中,冰浴下加入三氟乙酸,逐渐升至室温,共反应6h,减压旋转蒸除溶液后,得到化合物Ⅲ;
步骤d:化合物Ⅳ的合成:
将化合物Ⅲ和Et3N溶于DCM中,冰浴下冷却至0℃后缓慢加入2-氯乙烷磺酰氯,室温下反应20min,加入水,产物用DCM萃取,合并后的有机相用饱和NaCl洗后用无水Na2SO4干燥,减压旋蒸除去溶剂,加入CH2Cl2和60~100目硅胶,拌匀旋干,采用乙酸乙酯/石油醚=1/1作洗脱剂,快速柱层析,得到化合物Ⅳ;
步骤e:化合物Ⅳ的合成:
2.如权利要求1所述的靶向分子和活性药物分子偶联物,其特征在于,所述步骤a中哌嗪-2-羧酸二盐酸盐包括哌嗪-2-羧酸二盐酸盐、(R)-哌嗪-2-羧酸二盐酸盐和(S)-哌嗪-2-羧酸二盐酸盐中的任意一种。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910592414.0A CN110251680B (zh) | 2019-07-03 | 2019-07-03 | 一种哌嗪类二乙烯基磺酰胺类链接子及其制备方法与应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910592414.0A CN110251680B (zh) | 2019-07-03 | 2019-07-03 | 一种哌嗪类二乙烯基磺酰胺类链接子及其制备方法与应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN110251680A CN110251680A (zh) | 2019-09-20 |
CN110251680B true CN110251680B (zh) | 2022-10-18 |
Family
ID=67923908
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201910592414.0A Active CN110251680B (zh) | 2019-07-03 | 2019-07-03 | 一种哌嗪类二乙烯基磺酰胺类链接子及其制备方法与应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN110251680B (zh) |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2009009434A1 (en) * | 2007-07-06 | 2009-01-15 | University Of Chicago | Polymers and uses thereof |
CN107400072A (zh) * | 2017-06-30 | 2017-11-28 | 上海科技大学 | 一种双乙烯磺酰胺连接子及其制备和应用 |
CN108530512A (zh) * | 2018-03-28 | 2018-09-14 | 上海科技大学 | 乙烯磺酰胺链接子及其应用 |
-
2019
- 2019-07-03 CN CN201910592414.0A patent/CN110251680B/zh active Active
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2009009434A1 (en) * | 2007-07-06 | 2009-01-15 | University Of Chicago | Polymers and uses thereof |
CN107400072A (zh) * | 2017-06-30 | 2017-11-28 | 上海科技大学 | 一种双乙烯磺酰胺连接子及其制备和应用 |
CN108530512A (zh) * | 2018-03-28 | 2018-09-14 | 上海科技大学 | 乙烯磺酰胺链接子及其应用 |
Non-Patent Citations (1)
Title |
---|
An expedient strategy for the diversity-oriented synthesis of macrocyclic compounds with natural product-like characteristics;Joe J. Ciardiello等;《Tetrahedron》;20161231;第72卷;第3567-3578页 * |
Also Published As
Publication number | Publication date |
---|---|
CN110251680A (zh) | 2019-09-20 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN107250104B (zh) | 新型成像组合物及其用途 | |
KR20200064059A (ko) | 프로그램가능한 수지상 약물 | |
CN103732595B (zh) | 叶酸的白蛋白‑结合实体缀合物 | |
Chatzisideri et al. | Synthesis and biological evaluation of a Platinum (II)-c (RGDyK) conjugate for integrin-targeted photodynamic therapy | |
KR20200067132A (ko) | 프로그램가능한 중합체성 약물 | |
US10000494B2 (en) | Small-molecule Hsp90 inhibitors | |
CN109200291A (zh) | 一种靶向于egfr的抗体偶联药物及其制备方法和其用途 | |
CN108290864A (zh) | 一种蛋白激酶抑制剂及其制备方法和医药用途 | |
CN110143961A (zh) | 一种基于vhl配体诱导bet降解的吡咯并吡啶酮类双功能分子化合物 | |
Karges et al. | Synthesis and Characterization of an Epidermal Growth Factor Receptor‐Selective RuII Polypyridyl–Nanobody Conjugate as a Photosensitizer for Photodynamic Therapy | |
US10011587B2 (en) | Multivalent ligands targeting VEGFR | |
CN110312517A (zh) | 促黄体激素释放激素受体(lhrh-r)缀合物及其用途 | |
CN114933633B (zh) | 一种特异性识别fgfr4的天然肽探针及其应用 | |
CN110256313A (zh) | 一种光敏剂前药化合物及其制备方法和应用 | |
CN112209990B (zh) | 康普瑞汀类衍生物及其抗体药物偶联物 | |
WO2020187913A1 (en) | Small molecule photosensitizers for photodynamic therapy | |
Xiao et al. | Transporter-targeted bile acid-camptothecin conjugate for improved oral absorption | |
CN111643676B (zh) | 一种双特异性二聚体、双特异性二聚体-药物偶联物及其应用 | |
JP2021508737A (ja) | 細胞標的複合体の調製のための遷移金属ベースの官能性部分 | |
CN110251680B (zh) | 一种哌嗪类二乙烯基磺酰胺类链接子及其制备方法与应用 | |
Martínez‐Alonso et al. | A Novel Near‐IR Absorbing Ruthenium (II) Complex as Photosensitizer for Photodynamic Therapy and its Cetuximab Bioconjugates | |
Wei et al. | Site-specific construction of triptolide-based antibody-drug conjugates | |
Li et al. | Discovery of novel antibody-drug conjugates bearing tissue protease specific linker with both anti-angiogenic and strong cytotoxic effects | |
Lu et al. | Log P analyzation-based discovery of GSH activated biotin-tagged fluorescence probe for selective colorectal cancer imaging | |
WO2023245857A1 (zh) | 一种辣椒素衍生化光敏剂及其制备方法与应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |