CN112209990A - 康普瑞汀类衍生物及其抗体药物偶联物 - Google Patents
康普瑞汀类衍生物及其抗体药物偶联物 Download PDFInfo
- Publication number
- CN112209990A CN112209990A CN202011245317.3A CN202011245317A CN112209990A CN 112209990 A CN112209990 A CN 112209990A CN 202011245317 A CN202011245317 A CN 202011245317A CN 112209990 A CN112209990 A CN 112209990A
- Authority
- CN
- China
- Prior art keywords
- antibody
- combretastatin
- compound
- cex
- derivative
- 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.)
- Granted
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06008—Dipeptides with the first amino acid being neutral
- C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
- C07K5/06034—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms
- C07K5/06052—Val-amino acid
-
- 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
- A61K31/075—Ethers or acetals
- A61K31/085—Ethers or acetals having an ether linkage to aromatic ring nuclear carbon
- A61K31/09—Ethers or acetals having an ether linkage to aromatic ring nuclear carbon having two or more such linkages
-
- 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
- A61K47/6845—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 a cytokine, e.g. growth factors, VEGF, TNF, a lymphokine or an interferon
-
- 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
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/10—Tetrapeptides
- C07K5/1002—Tetrapeptides with the first amino acid being neutral
- C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
- C07K5/1008—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Epidemiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Nitrogen And Oxygen Or Sulfur-Condensed Heterocyclic Ring Systems (AREA)
Abstract
Description
技术领域
本发明属于生物制药及生物技术领域,具体的涉及一类康普瑞汀(CA4)的衍生物,用于抗体药物偶联物的制备及其在靶向癌症治疗中的应用。
背景技术
抗体-药物偶联物(ADCs)是通过一个化学链接将活性药物分子连接到抗体上,抗体作为载体靶向运输药物分子到目标细胞。ADCs的三部分,即药物分子、链接子和抗体部分都非常重要。早期研发ADCs中所使用的药物分子倾向于毒性极高的分子,但这类ADCs会产生较大的毒副作用。近期Enhertu和Trodelvy分别于2019和2020年上市,它们均采用毒性适中的喜树碱类衍生物作为药物分子。新一代ADCs的研发中开始关注并重视毒性适中的药物分子。
康普瑞汀(CA4)是一种来自于天然产物的微管抑制剂,同时具有抑制血管生成的作用。CA4具有很强的抑制肿瘤作用,但水溶性差,难以直接成药。
发明内容
本发明所要解决的技术问题是:CA4难以直接成药的技术问题,制备一种抗体-药物偶联物及将其用于在癌症靶向治疗中。
为了解决上述技术问题,本发明采用的技术方案:
一种康普瑞汀类衍生物及其抗体药物偶联物,其特征在于,包含式Ⅰ所示的化合物或其药学上可接受的盐或溶剂合物;
式Ⅰ中,Ab表示靶向配体;L表示为链接子,用于将抗体和CA4或其衍生物连接起来;n表示药物与靶向配体的比例;X为O、NH或NR,其中R为烷烃链或PEG链。
优选地,所述式Ⅰ中,Ab为抗体。
优选地,所述式Ⅰ中,L包含可断裂和不可断裂链接子。
优选地,所述式Ⅰ中,n为1-10。
本发明还提供了上述康普瑞汀类衍生物及其抗体药物偶联物的制备方法,其特征在于,包括以下步骤:
步骤1):将康普瑞汀或其衍生物进行修饰并与化学连接子进行连接,得到康普瑞汀-链接子分子;
步骤2):将靶向配体与步骤1)获得的康普瑞汀-链接子分子进行偶联反应,合成靶向配体-药物偶联物。
本发明还提供了上述康普瑞汀类衍生物及其抗体药物偶联物在癌症靶向治疗或治疗增殖性疾病中的应用。
优选地,所述的增殖性疾病为上皮生长因子受体(Epidermal Growth FactorReceptor,EGFR)表达的病灶。
本发明提供了以CA4为药物分子,其衍生物用于抗体药物偶联物的制备及其在治疗高增殖性疾病或病灶的用途。这些疾病的特征为细胞生成一种抗原或靶点,该抗原能与抗体特异性结合。
附图说明
图1为本发明中涉及的ADCs Cex-50-5,Cex-59,Cex-70以及Cetuximab和IgG在NCI-H1975细胞中的亲和力;
图2为本发明中涉及的Cetuximab在NCI-H1975细胞中的内吞效应;
图3为本发明中涉及的ADC Cex-50-5在NCI-H1975细胞中的内吞效应;
图4为本发明中涉及的ADC Cex-70在NCI-H1975细胞中的内吞效应;
图5为本发明中涉及的ADC Cex-59在NCI-H1975细胞中的内吞效应;
图6为本发明中涉及的ADCs Cex-50-5,Cex-59,Cex-70以及Cetuximab和CA4对NCI-H1975细胞的体外增殖抑制作用;
图7为本发明中涉及的ADCs Cex-50-5,Cex-59,Cex-70以及Cetuximab和CA4对NCI-H1975-GFP细胞的体外增殖抑制作用;
图8为本发明中涉及的ADCs Cex-50-5,Cex-59,Cex-70以及Cetuximab和CA4对HCC827细胞的体外增殖抑制作用;
图9为本发明中涉及的ADCs Cex-50-5,Cex-59,Cex-70以及Cetuximab和CA4对A549细胞的体外增殖抑制作用;
图10为本发明中涉及的ADCs Cex-50-5,Cex-59,Cex-70以及Cetuximab和CA4对MDA-MB231细胞的体外增殖抑制作用;
图11为本发明中涉及的ADCs Cex-50-5,Cex-59,Cex-70以及Cetuximab和CA4对NCI-H2228细胞的体外增殖抑制作用;
图12为本发明中涉及的ADCs Cex-50-5,Cex-59,Cex-70以及Cetuximab和CA4对非小细胞肺癌NCI-H1975-GFP异种移植瘤的抑制作用;
图13为本发明中涉及的ADCs Cex-50-5,Cex-59,Cex-70以及Cetuximab和CA4对非小细胞肺癌NCI-H1975-GFP异种移植瘤的检测荧光信号;
图14为本发明中涉及的ADCs Cex-50-5,Cex-59,Cex-70以及Cetuximab和CA4对非小细胞肺癌NCI-H1975-GFP异种移植瘤小鼠体重的影响。
具体实施方式
为使本发明更明显易懂,兹以优选实施例,并配合附图作详细说明如下。
实施例1:抗体-药物偶联物的制备
(1)化合物50-5的合成
化合物50-1的合成
将康普瑞汀(316.35mg,1mmol)溶于DCM(10mL)中,随后加入对硝基苯基氯甲酸酯(403.12mg,2mmol)和吡啶(237.3mg,3mmol)。在室温下反应4h。通过TLC薄层色谱法检测反应完全。直接减压旋转蒸除溶剂后得粗品,加入5mL CH2Cl2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=3:1)得白色固体50-1(480mg,0.997mmol,99%)。ESI-HRMS计算结果:C25H24NO9[M+H]+:482.1451,实际结果:482.1410.
化合物50-2的合成
将化合物50-1(0.480g,1mmol),N-Boc-N,N'-二甲基乙二胺(0.282g,1.5mmol)和三乙胺(0.191mL,1.5mmol)溶于二氯甲烷(10mL)中,在室温下搅拌反应过夜。用30mL水稀释反应液后,二氯甲烷萃取(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10mL CH2Cl2和2g 60-100目硅胶,拌匀旋干。粗品经硅胶柱层析(石油醚:乙酸乙酯=3:1)得白色固体50-2(0.456g,0.86mmol,86%)。ESI-HRMS计算结果:C28H39N2O8[M+H]+:531.2706,实际结果:531.3009.1H NMR(500MHz,Chloroform-d)δ7.13–7.04(m,2H),6.83(d,J=8.4Hz,1H),6.53(d,J=5.6Hz,2H),6.49–6.38(m,2H),4.10(q,J=7.1Hz,1H),3.81(d,J=17.0Hz,6H),3.70(s,6H),3.54(dt,J=19.2,4.5Hz,1H),3.44(dd,J=15.5,9.0Hz,3H),3.10(d,J=4.1Hz,1H),3.00(s,2H),2.92–2.85(m,3H),2.02(s,1H),1.44(d,J=13.2Hz,8H),1.24(t,J=7.1Hz,1H).13C NMR(126MHz,CDCl3)δ170.94,155.62,155.39,154.24,154.07,152.88,150.78,140.03,137.09,137.06,132.48,129.82,129.26,129.15,128.66,127.12,111.92,111.74,105.87,79.46,77.56,77.30,77.04,60.74,60.25,55.86,55.85,55.77,47.52,47.39,47.14,46.99,46.72,46.37,45.85,35.40,35.31,34.86,34.66,28.35,28.32,20.91,14.13.
化合物50-3的合成
将上一步所得化合物50-2(0.456g,0.86mmol)溶于二氯甲烷(10mL)中,冰浴下加入三氟乙酸(3mL,40mmol),冰浴下反应4h,直接减压旋转蒸除溶剂后得粗品,加入5mLCH2Cl2和1g 60-100目硅胶,拌匀旋干。粗品经硅胶柱层析(二氯甲烷:甲醇=10:1)得白色固体50-3(370mg,0.86mmol,99%)。ESI-HRMS计算结果:C23H31N2O9[M+H]+:431.2182,实际结果:431.2143.1H NMR(500MHz,Methanol-d4)δ7.12(d,J=8.2Hz,1H),7.05(s,1H),6.96(d,J=8.4Hz,1H),6.57–6.44(m,4H),3.80(d,J=13.4Hz,4H),3.75(s,3H),3.67(s,7H),3.37(s,3H),3.35–3.26(m,1H),3.23(t,J=5.9Hz,1H),3.11(s,1H),3.01(s,1H),2.74(d,J=12.0Hz,3H).13C NMR(126MHz,MeOD)δ155.56,154.32,152.81,150.70,150.51,139.81,139.51,136.81,132.87,130.12,129.42,128.31,127.33,123.42,123.30,111.97,111.74,105.95,59.87,55.15,55.09,48.59,48.25,48.08,47.91,47.74,47.57,47.40,47.23,46.90,46.78,45.59,45.45,34.19,32.69,32.47.
化合物50-4的合成
将上一步所得化合物50-3(88.5mg,0.206mmol),N3-PEG3-乙酸(57.652mg,0.2472mmol),HOBt(56.077mg,0.412mmol),EDCl(78.98mg,0.412mmol),N’N-二异丙基乙胺(0.102mL,0.618mmol)溶于DMF(10mL)中,在室温下搅拌反应过夜。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50-4(43.6mg,0.0675mmol),产率33%。ESI-HRMS计算结果:C31H44N5O10[M+H]+:646.3088,实际结果:646.3402。1H NMR(500MHz,Methanol-d4)δ7.35(ddt,J=8.0,5.2,2.4Hz,1H),7.27–7.16(m,2H),6.83–6.65(m,4H),4.49(dd,J=24.4,4.2Hz,1H),4.35(d,J=2.1Hz,1H),4.08–3.65(m,27H),3.56(dt,J=10.8,5.0Hz,3H),3.42–3.26(m,4H),3.26–3.13(m,5H),2.69–2.54(m,1H),2.49(s,3H),1.89(p,J=7.0Hz,1H),1.53(dd,J=16.2,6.3Hz,1H),1.31(t,J=7.2Hz,1H).13C NMR(126MHz,MeOD)δ154.36,154.30,131.43,130.64,129.69,128.51,113.40,107.32,107.24,71.79,71.52,71.10,70.30,61.13,58.01,56.47,51.76,49.51,49.34,49.17,49.00,48.83,48.66,48.49,47.62,45.29,39.08,35.80,35.34,34.97,28.95,15.78.
化合物50-5的合成
将上一步所得化合物50-4(21.8mg,0.034mmol)、2-丙炔甲酰胺-1,4-乙烯基磺酰胺哌嗪1(11.812mg,0.034mmol)、L-抗坏血酸钠(13.472mg,0.068mmol)和硫酸铜(10.853mg,0.068mmol)溶于6mL tBuOH/H2O/DMF(1/1/1)中。室温下反应1h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50-5(8.8mg,0.00886mmol),产率26%。ESI-HRMS计算结果:C43H61N8O15S2[M+H]+:993.3698,实际结果:993.3722。1H NMR(500MHz,Methanol-d4)δ8.37(d,J=13.2Hz,1H),7.20–6.94(m,8H),6.82–6.43(m,15H),6.25–5.97(m,10H),4.74–4.01(m,20H),4.00–3.41(m,119H),3.36–3.29(m,18H),3.07–2.86(m,10H),2.76(d,J=12.6Hz,2H),1.31(q,J=10.2,5.4Hz,3H).13CNMR(126MHz,MeOD)δ156.25,156.03,154.36,154.31,152.31,141.16,138.36,136.91,134.17,133.86,133.68,131.38,130.78,130.65,130.45,130.22,129.64,128.87,128.75,128.58,128.31,124.49,124.22,113.56,113.43,107.31,107.24,104.92,72.96,71.95,71.58,71.55,71.50,71.39,71.34,70.66,70.05,69.34,61.17,59.11,56.61,56.53,56.51,53.63,50.12,49.85,49.63,49.51,49.46,49.34,49.28,49.17,49.11,49.00,49.00,48.83,48.77,48.66,48.49,48.08,47.04,46.12,45.83,43.60,43.49,35.43,34.47,32.45,30.30,28.08.
(2)化合物50-6的合成
将化合物50-4(26mg,0.04mmol)、化合物对-丙炔氧基-N,N’-二乙烯基磺酰胺B1(13.182mg,0.04mmol)、L-抗坏血酸钠(15.849mg,0.08mmol)和硫酸铜(12.768mg,0.08mmol)溶于6mL tBuOH/H2O/DMF(1/1/1)中。室温下反应2h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50-6(9.4mg,0.00966mmol),产率24%。ESI-HRMS计算结果:C44H57N6O15S2[M+H]+:973.3323,实际结果:973.3289。1H NMR(500MHz,Methanol-d4)δ8.22–8.10(m,1H),7.20(d,J=8.6Hz,2H),7.16–6.91(m,7H),6.56(t,J=3.8Hz,2H),6.48(d,J=5.9Hz,2H),6.28–6.15(m,4H),5.17(d,J=5.6Hz,2H),4.57(q,J=6.6,5.9Hz,2H),4.21(d,J=25.3Hz,1H),4.06(d,J=9.8Hz,1H),3.92–3.70(m,9H),3.70–3.40(m,18H),3.37–3.33(m,3H),3.10–2.79(m,6H),1.39–1.23(m,3H).13C NMR(126MHz,MeOD)δ161.13,155.98,154.38,154.31,152.34,138.33,137.63,133.59,131.43,131.32,130.60,129.70,128.89,128.53,128.17,124.60,124.31,116.47,113.44,107.33,107.24,71.81,71.75,71.43,71.33,70.38,70.28,70.20,62.65,61.16,56.49,56.45,51.51,49.85,49.51,49.34,49.17,49.00,48.83,48.66,48.49,47.62,46.79,46.17,35.41,35.32,34.96,30.83,30.45,30.33,28.11,26.92.
(3)化合物51-4的合成
化合物51-1的合成
将化合物康普瑞汀(0.158g,0.5mmol),溴乙酸乙酯(83.174μL,0.75mmol),碳酸钾(0.207g,1.5mmol)溶于丙酮(10mL)中,60℃加热搅拌反应回流过夜。过滤除去固体,将滤液直接减压旋转蒸除溶剂后得粗品,加入10mL CH2Cl2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=3:1)得白色固体51-1(0.159g,0.395mmol,79%)。ESI-HRMS计算结果:C22H27O7[M+H]+:403.1757,实际结果:403.1758。1H NMR(500MHz,Methanol-d4)δ6.86(d,J=1.3Hz,2H),6.75(s,1H),6.50(d,J=1.1Hz,2H),6.46–6.40(m,2H),4.46(d,J=1.2Hz,2H),4.10(qd,J=7.1,1.1Hz,2H),3.76(dd,J=24.8,1.2Hz,6H),3.63(d,J=1.3Hz,6H),1.18(td,J=7.1,1.1Hz,3H).13C NMR(126MHz,MeOD)δ170.47,154.22,150.25,148.39,138.25,134.38,131.45,130.42,130.27,124.47,116.18,113.39,107.28,67.13,62.17,61.11,56.49,56.37,49.51,49.34,49.17,49.00,48.83,48.66,48.49,29.52,14.40.
化合物51-2的合成
将上一步所得化合物51-2(0.158g,0.395mmol),LiOH(19.89mg,0.474mmol)溶于甲醇(10mL)和水(1mL)中,室温搅拌反应。通过TLC薄层色谱法检测反应完全。用稀盐酸将反应液pH值调至4,用30mL水稀释反应液后,二氯甲烷萃取(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51-2(81.3mg,0.217mmol,55%),直接用于下一步反应。ESI-HRMS计算结果:C20H23O7[M+H]+:375.1444,实际结果:375.1675.
化合物51-3的合成
将上一步所得化合物51-2(81.3mg,0.217mmol),N3-PEG3-乙酸(56.87mg,0.261mmol),HOBt(59.07mg,0.434mmol),EDCl(83.2mg,0.434mmol),N’N-二异丙基乙胺(0.113mL,0.651mmol)溶于DCM(10mL)中,在室温下搅拌反应过夜。将滤液直接减压旋转蒸除溶剂后得粗品,加入10mL CH2Cl2和1g 60-100目硅胶,拌匀旋干。粗品经硅胶柱层析(二氯甲烷:甲醇=10:1)得白色固体51-3(0.106g,0.185mmol,85%)。ESI-HRMS计算结果:C28H39N4O9[M+H]+:575.2717,实际结果:575.3059。1H NMR(500MHz,Methanol-d4)δ6.97–6.90(m,2H),6.88(t,J=1.5Hz,1H),6.54–6.44(m,4H),4.33(s,2H),3.85(d,J=1.2Hz,3H),3.74(d,J=1.2Hz,3H),3.67–3.51(m,18H),3.43(t,J=5.4Hz,2H),3.36–3.28(m,2H).13CNMR(126MHz,MeOD)δ171.04,154.28,150.57,148.38,138.40,134.39,131.80,130.55,130.28,125.16,118.02,113.26,107.40,71.62,71.51,71.34,71.09,70.49,70.42,61.19,56.53,56.44,51.73,49.51,49.34,49.17,49.00,48.83,48.66,48.49,39.96.
化合物51-4的合成
将上一步所得化合物51-3(53.2mg,0.0926mmol)、化合物2-丙炔甲酰胺-1,4-乙烯基磺酰胺哌嗪1(32.163mg,0.0926mmol)、L-抗坏血酸钠(36.69mg,0.1852mmol)和硫酸铜(29.56mg,0.1852mmol)溶于6mL tBuOH/H2O/DMF(1/1/1)中。室温下反应3h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51-4(51mg,0.0553mmol),产率60%。ESI-HRMS计算结果:C40H56N7O14S2[M+H]+:922.3327,实际结果:922.3847。1H NMR(500MHz,Methanol-d4)δ7.90(s,1H),6.93(t,J=6.7Hz,2H),6.88(d,J=1.8Hz,1H),6.71(dd,J=16.4,9.9Hz,1H),6.62–6.43(m,5H),6.22–6.06(m,3H),6.01(d,J=9.9Hz,1H),4.62–4.30(m,7H),4.11(d,J=12.6Hz,1H),3.91–3.70(m,8H),3.71–3.47(m,20H),3.42(t,J=5.4Hz,2H),3.35(s,4H),2.97(dd,J=12.6,4.2Hz,1H),2.76(td,J=11.7,11.3,3.7Hz,1H),1.50–1.19(m,1H).13C NMR(126MHz,MeOD)δ171.06,170.29,154.28,150.56,148.38,138.36,136.81,134.42,133.76,131.79,130.57,130.35,130.32,128.31,125.16,118.01,113.31,107.41,71.54,71.46,71.44,71.33,70.51,70.38,70.23,61.23,56.62,56.59,56.48,51.39,49.85,49.51,49.34,49.17,49.00,48.83,48.66,48.59,48.49,46.08,43.44,39.97,36.01.
(4)化合物52-5的合成
化合物52-1的合成
将化合物N-Boc-N-甲基乙二胺(522.72mg,3mmol),1-溴-3,6,9,12-四氧杂十三烷(271.15mg,1mmol),N’N-二异丙基乙胺(0.827mL,5mmol),碘化钠(299.78mg,2mmol)溶于DMF(20mL)中,80℃加热搅拌反应过夜。过滤除去固体,用50mL水稀释反应液后,二氯甲烷萃取(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10mL CH2Cl2和2g 60-100目硅胶,拌匀旋干。粗品经硅胶柱层析(二氯甲烷:甲醇=10:1)得白色固体52-1(0.313g,0.86mmol,86%)。ESI-HRMS计算结果:C17H37N2O6[M+H]+:365.2652,实际结果:365.2648.
化合物52-2的合成
将化合物50-1(0.481g,1mmol),上一步所得化合物52-1(0.364g,1mmol)和N’N-二异丙基乙胺(0.495mL,3mmol)溶于二氯甲烷(10mL)中,45℃加热搅拌反应回流过夜。用30mL水稀释反应液后,二氯甲烷萃取(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10mL CH2Cl2和2g 60-100目硅胶,拌匀旋干。粗品经硅胶柱层析(石油醚:乙酸乙酯=2:1)得白色固体52-2(90.8mg,0.128mmol,13%)。ESI-HRMS计算结果:C36H55N2O12[M+H]+:707.3755,实际结果:707.3817.1H NMR(500MHz,Methanol-d4)δ8.11(d,J=8.7Hz,2H),7.20–6.94(m,2H),6.88(d,J=8.7Hz,2H),6.57(d,J=4.6Hz,1H),6.49(q,J=5.3,3.8Hz,1H),3.90–3.40(m,24H),2.96–2.77(m,2H),1.56–1.20(m,8H).13C NMR(126MHz,MeOD)δ165.41,157.35,155.94,154.21,152.21,141.53,141.20,138.29,134.06,131.24,130.59,130.53,129.57,127.04,124.55,116.56,113.25,107.28,72.90,71.51,71.31,70.70,61.10,59.07,56.45,56.43,56.37,49.51,49.34,49.17,49.00,48.83,48.66,48.49,47.83,28.75,28.71,9.25.
化合物52-3的合成
将上一步所得化合物52-2(91mg,0.129mmol)溶于二氯甲烷(10mL)中,冰浴下加入三氟乙酸(3mL,40mmol),冰浴下反应4h,直接减压旋转蒸除溶剂后得粗品体52-3(78.27mg,0.129mmol,99%),直接用于下一步反应。ESI-HRMS计算结果:C31H47N2O10[M+H]+:607.3231,实际结果:607.2978.
化合物52-4的合成
将上一步所得化合物52-3(39mg,0.0643mmol),N3-PEG3-乙酸(18mg,0.0771mmol),HOBt(37mg,0.193mmol),EDCl(27mg,0.193mmol),N’N-二异丙基乙胺(0.720mL,0.643mmol)溶于DCM(10mL)中,在室温下搅拌反应过夜。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52-4(26.7mg,0.0289mmol),产率45%。ESI-HRMS计算结果:C39H60N5O14[M+H]+:822.4137,实际结果:822.4159。1H NMR(500MHz,Methanol-d4)δ7.13(d,J=7.9Hz,1H),7.10–6.96(m,2H),6.61–6.43(m,4H),4.29(d,J=31.0Hz,1H),4.16(s,1H),3.91–3.43(m,45H),3.35(d,J=11.0Hz,5H),3.03–2.92(m,3H).13C NMR(126MHz,MeOD)δ171.99,156.20,155.95,155.77,154.36,154.31,154.29,152.30,141.17,141.06,140.99,138.36,134.25,134.17,134.13,131.44,131.36,130.73,130.65,130.62,130.58,129.64,129.62,128.86,128.66,124.53,124.50,124.43,124.27,113.45,113.35,113.32,107.31,107.28,107.22,72.96,71.74,71.67,71.61,71.57,71.53,71.48,71.44,71.35,71.08,70.87,70.65,70.41,70.32,70.10,61.14,59.09,56.67,56.49,56.46,51.81,51.75,50.04,49.51,49.34,49.17,49.00,48.83,48.66,48.49,48.16,47.85,47.60,46.97,46.91,35.56,35.29,34.32.
化合物52-5的合成
将上一步所得化合物52-4(26.7mg,0.0325mmol)、化合物2-丙炔甲酰胺-1,4-乙烯基磺酰胺哌嗪1(11.291mg,0.0325mmol)、L-抗坏血酸钠(12.877mg,0.065mmol)和硫酸铜(10.374mg,0.065mmol)溶于3mL tBuOH/H2O/DMF(1/1/1)中。室温下反应3h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52-5(22.4mg,0.0192mmol),产率51%。ESI-HRMS计算结果:C51H77N8O19S2[M+H]+:1169.4746,实际结果:1169.4477。
(5)化合物50-6的合成
化合物52-6-1的合成
将化合物6-(马来酰亚胺基)己酸琥珀酰亚胺酯(184.974mg,0.6mmol),炔丙胺(32μL,0.5mmol),三乙胺(0.083mL,0.6mmol)溶于DCM(5mL)中,在室温下搅拌反应6h。加入0.2g60-100目硅胶,拌匀旋干。粗品经硅胶柱层析(二氯甲烷:甲醇=10:1)得白色固体52-6-1(108.57mg,0.437mmol,87%)。ESI-HRMS计算结果:C13H17N2O3[M+H]+:249.1239,实际结果:249.1348。
化合物52-6的合成
将化合物52-4(20mg,0.024mmol)、化合物52-6-1(6.7mg,0.027mmol)、L-抗坏血酸钠(9.65mg,0.049mmol)和硫酸铜(7.8mg,0.049mmol)溶于3mL tBuOH/H2O/DMF(1/1/1)中。室温下反应2h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52-6(10.4mg,0.010mmol),产率40%。ESI-HRMS计算结果:C52H76N7O17[M+H]+:1170.5298,实际结果:1170.5245。1H NMR(500MHz,CD3OD)δ7.12(d,J=7.0Hz,1H),7.00(d,J=8.2Hz,2H),6.78(d,J=1.2Hz,2H),6.57(d,J=2.5Hz,2H),6.47(d,J=16.1Hz,2H),4.63(s,2H),3.93(s,2H),3.80(d,J=13.4Hz,4H),3.78–3.49(m,39H),3.37–3.27(m,6H),3.09–2.91(m,3H),1.60(d,J=41.9Hz,4H),1.31(d,J=23.0Hz,4H).13CNMR(126MHz,CD3OD)δ172.58,154.36,154.30,152.30,141.15,138.36,135.32,134.17,131.36,130.63,129.63,128.91,128.84,128.70,124.50,124.42,124.24,113.38,107.30,107.23,72.94,71.55,71.38,71.33,61.16,59.10,56.50,38.38,29.27,27.40,26.30.
(6)化合物52-7的合成
化合物52-7的合成
将化合物52-4(20mg,0.024mmol)、间-丙炔氧基-N,N’-二乙烯基磺酰胺2(8.9mg,0.027mmol)、L-抗坏血酸钠(9.65mg,0.049mmol)和硫酸铜(7.8mg,0.049mmol)溶于3mLtBuOH/H2O/DMF(1/1/1)中。室温下反应2h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52-7(19.4mg,0.017mmol),产率70%。ESI-HRMS计算结果:C52H73N6O19S2[M+H]+:1149.4372,实际结果:1149.4354。1H NMR(500MHz,CD3OD)δ8.17(d,J=16.1Hz,1H),7.34(td,J=8.1,2.3Hz,1H),7.19–7.07(m,4H),7.06–6.88(m,4H),6.52(dt,J=27.9,9.0Hz,4H),6.29–6.15(m,4H),5.18(d,J=5.3Hz,2H),4.57(dd,J=10.2,6.3Hz,2H),3.89–3.83(m,2H),3.81–3.76(m,3H),3.75–3.43(m,38H),3.32(dd,J=3.7,2.0Hz,4H),2.96–2.87(m,3H).13C NMR(126MHz,CD3OD)δ171.89,160.10,156.16,155.94,154.35,154.30,152.29,143.97,141.17,138.35,137.55,136.35,134.22,134.15,131.33,131.22,130.99,130.76,130.63,129.65,128.87,128.68,126.80,125.07,124.53,124.27,118.95,118.07,113.49,113.38,107.30,107.22,72.93,71.76,71.54,71.52,71.50,71.46,71.36,71.32,70.83,70.59,70.14,62.51,61.17,59.10,56.51,51.73,51.68,49.99,46.95,35.49,35.26,34.33.
(7)化合物52-8的合成
将化合物52-4(20mg,0.024mmol)、对-丙炔氧基-N,N’-二乙烯基磺酰胺(8.9mg,0.027mmol)、L-抗坏血酸钠(9.65mg,0.049mmol)和硫酸铜(7.8mg,0.049mmol)溶于3mLtBuOH/H2O/DMF(1/1/1)中。室温下反应2h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52-8(15mg,0.013mmol),产率54%。ESI-HRMS计算结果:C52H73N6O19S2[M+H]+:1149.4372,实际结果:1149.4338。1H NMR(500MHz,CD3OD)δ8.17–8.09(m,1H),7.23–7.18(m,2H),7.14–6.94(m,7H),6.59–6.53(m,2H),6.47(d,J=3.8Hz,2H),6.22(dd,J=8.3,7.8Hz,4H),5.16(d,J=6.0Hz,2H),4.56(dd,J=10.6,5.5Hz,2H),4.29–4.03(m,2H),3.88–3.83(m,2H),3.80–3.76(m,3H),3.73(d,J=2.0Hz,3H),3.68–3.47(m,34H),3.32(d,J=3.1Hz,3H),2.95–2.87(m,3H).13C NMR(126MHz,CD3OD)δ171.81,161.07,156.16,155.92,154.33,154.28,152.27,144.22,141.15,138.32,137.51,134.22,134.15,133.58,131.31,130.79,130.61,129.65,128.83,128.68,128.10,126.49,124.51,124.25,118.21,116.49,113.49,113.39,107.29,107.20,72.90,71.72,71.51,71.50,71.44,71.35,71.29,70.24,70.16,70.02,62.63,61.18,59.10,56.51,51.43,49.85,35.47,35.24,34.31,0.82.
(8)化合物70的合成
化合物66的合成
将化合物27(200mg,0.31mmol),50-3(147mg,0.34mmol),1-羟基苯并三唑(62.84mg,0.465mmol),吡啶(0.179mL,2.232mmol)和N’N-二异丙基乙胺(0.189mL,1.085mmol)溶于DMF(5mL)中,在室温下搅拌反应过夜。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66(147mg,0.157mmol),产率51%。ESI-HRMS计算结果:C47H66N7O13[M+H]+:936.4719,实际结果:936.4728.1H NMR(500MHz,CD3OD)δ7.54(m,2H),7.36–7.21(m,2H),7.11(d,J=8.4Hz,1H),6.95(dd,J=11.2,5.2Hz,2H),6.59–6.40(m,4H),5.04(d,J=19.9Hz,1H),4.96(d,J=7.9Hz,1H),4.53(d,J=4.0Hz,1H),3.93–3.87(m,1H),3.83–3.70(m,7H),3.69–3.58(m,7H),3.51(dt,J=15.3,7.4Hz,3H),3.22(d,J=6.5Hz,2H),2.97–2.81(m,5H),2.04(dd,J=11.3,4.9Hz,1H),1.91(s,1H),1.77(d,J=9.1Hz,1H),1.71–1.54(m,2H),1.44(m,9H),0.95(dd,J=14.3,6.7Hz,6H).13C NMR(126MHz,CD3OD)δ174.71,172.02,169.58,162.05,158.18,157.94,156.10,154.22,152.30,141.12,139.40,138.33,134.17,133.92,131.34,130.65,130.56,130.00,129.76,129.67,128.71,128.60,126.07,124.65,124.48,121.87,121.29,121.19,116.76,113.28,113.17,112.55,107.47,107.33,80.69,68.16,67.95,61.82,61.15,61.12,59.57,56.44,56.34,54.80,35.57,35.48,35.34,35.21,35.01,31.83,31.61,31.12,30.44,28.72,27.21,19.80,18.95,18.65,17.90.
化合物67的合成
将化合物66(147mg,0.157mmol)溶于二氯甲烷(15mL)中,冰浴下加入三氟乙酸(1mL,13mmol),冰浴下反应4h后,直接减压旋转蒸除溶剂后得粗品67(41mg,0.049mmol,31%),直接用于下一步反应。ESI-HRMS计算结果:C42H58N7O11[M+H]+:836.4194,实际结果:836.4128.
化合物68的合成
将化合物67(41mg,0.049mmol),N3-PEG8-COOH(35mg,0.075mmol),HATU(28.5mg,0.075mmol)和N’N-二异丙基乙胺(0.052mL,0.3mmol)溶于DCM(5mL)中,在室温下搅拌反应过夜。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68(44mg,0.034mmol),产率70%。ESI-HRMS计算结果:C61H93N10O20[M+H]+:1285.6568,实际结果:1285.6583.1H NMR(500MHz,CD3OD)δ7.58(m,2H),7.40–7.20(m,2H),7.15–7.08(m,1H),7.06–6.93(m,2H),6.50(dt,J=20.6,10.0Hz,4H),5.04(d,J=20.4Hz,1H),4.97(d,J=8.3Hz,1H),4.53(s,1H),4.19(s,1H),3.73(dd,J=14.6,9.3Hz,8H),3.65(d,J=19.8Hz,42H),3.36(m,2H),3.24(m,2H),2.97–2.89(m,3H),2.85(d,J=10.1Hz,2H),2.59(s,2H),2.12(s,1H),1.95(s,1H),1.86–1.53(m,4H),1.31(d,J=21.5Hz,1H),0.99(m,7H).13C NMR(126MHz,CD3OD)δ174.64,173.81,171.93,161.85,157.88,156.04,154.24,154.18,152.27,141.10,139.41,138.30,134.17,131.30,130.64,130.36,129.98,129.75,129.66,128.57,124.45,121.22,113.32,107.32,104.87,71.54,71.53,71.47,71.44,71.39,71.32,71.26,71.08,68.34,68.13,61.15,60.81,56.49,54.84,51.77,47.84,46.66,37.19,35.27,31.59,30.26,19.80,18.89.
化合物70的合成
将化合物68(44mg,0.034mmol)、化合物2-丙炔甲酰胺-1,4-乙烯基磺酰胺哌嗪1(13mg,0.038mmol)、L-抗坏血酸钠(14mg,0.068mmol)和硫酸铜(12mg,0.068mmol)溶于3mLtBuOH/H2O/DMF(1/1/1)中。室温下反应2h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70(27.8mg,0.017mmol),产率50%。ESI-HRMS计算结果:C73H110N13O25S2[M+H]+:1632.7177,实际结果:1632.7153.1H NMR(500MHz,CD3OD)δ8.33(s,1H),7.65–7.50(m,2H),7.30(ddd,J=24.1,17.2,8.6Hz,2H),7.16–7.08(m,1H),7.05–6.91(m,2H),6.73(dd,J=16.3,9.8Hz,1H),6.66–6.40(m,5H),6.18(ddd,J=22.1,12.6,6.5Hz,3H),6.03(d,J=9.8Hz,1H),5.04(d,J=20.5Hz,1H),4.97(d,J=8.8Hz,1H),4.68(dd,J=9.7,5.3Hz,3H),4.59–4.47(m,3H),4.22–4.15(m,1H),4.11(d,J=12.5Hz,1H),3.94(s,2H),3.78–3.70(m,8H),3.70–3.52(m,41H),3.26(s,2H),2.97–2.89(m,3H),2.85(d,J=10.6Hz,3H),2.57(s,2H),2.17–2.07(m,1H),1.95(s,1H),1.84–1.57(m,3H),1.32(d,J=19.7Hz,2H),1.03–0.95(m,6H).13C NMR(126MHz,CD3OD)δ174.57,173.90,171.90,170.85,161.87,157.93,156.09,154.27,152.31,143.99,141.14,141.10,139.60,139.43,138.33,138.26,136.89,134.22,133.71,131.33,130.66,130.45,130.39,129.99,129.68,128.60,128.34,127.81,124.49,121.25,113.33,107.35,107.30,71.46,71.42,71.40,71.35,71.25,69.49,68.25,61.14,60.84,56.64,56.48,54.83,53.46,46.65,46.12,43.49,37.26,35.59,34.59,31.63,30.57,30.21,28.08,26.91,23.71,19.82,18.92,14.44.
(9)化合物59的合成
化合物45的合成
将Boc-GGFG-COOH(444mg,1.02mmol)和PAB-OH(201mg,1.63mmol)溶于二氯甲烷(40mL)和甲醇(20mL)的混合溶液中,随后加入EEDQ(388.8mg,1.57mmol),在室温下避光搅拌反应2天。反应结束后直接减压旋转蒸除溶剂后得粗品,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45(170mg,0.31mmol),产率31%。ESI-HRMS计算结果:C27H36N5O7[M+H]+:542.2615,实际结果:542.2611.1H NMR(500MHz,DMSO-d6)δ9.78(s,1H),8.42(t,J=5.7Hz,1H),8.20(d,J=7.9Hz,1H),7.94(t,J=5.4Hz,1H),7.57(d,J=8.5Hz,2H),7.31–7.22(m,6H),7.22–7.12(m,1H),6.99(t,J=5.9Hz,1H),5.10(t,J=5.7Hz,1H),4.53(td,J=9.3,4.6Hz,1H),4.44(d,J=5.7Hz,2H),3.97–3.74(m,3H),3.63(dd,J=16.7,5.4Hz,1H),3.56(d,J=5.9Hz,2H),3.08(dd,J=13.8,4.5Hz,1H),2.82(dd,J=13.8,9.7Hz,1H),1.36(d,J=19.5Hz,9H).13C NMR(126MHz,DMSO-d6)δ171.44,169.67,168.95,167.35,155.84,137.84,137.46,137.42,129.18,128.13,127.00,126.33,118.88,78.15,62.64,54.31,48.63,43.26,42.74,41.85,37.43,28.21.
化合物49的合成
将化合物45(165mg,0.3mmol)溶于无水四氢呋喃(10mL)中,冰浴下加入对硝基苯基氯甲酸酯(140mg,0.7mmol)和吡啶(0.3mL,3.8mmol)。在室温下搅拌反应18h。直接减压旋转蒸除溶剂后得粗品,加入5mL CH2Cl2和1g 60-100目硅胶,拌匀旋干。粗品经硅胶柱层析(二氯甲烷:甲醇=10:1)得白色固体49(65mg,0.092mmol,31%)。ESI-HRMS计算结果:C34H39N6O11[M+H]+:707.2677,实际结果:707.2692.1H NMR(500MHz,DMSO-d6)δ9.93(s,1H),8.44(t,J=5.7Hz,1H),8.34–8.28(m,2H),8.21(d,J=7.9Hz,1H),7.94(t,J=5.3Hz,1H),7.67(d,J=8.5Hz,2H),7.60–7.53(m,2H),7.43(d,J=8.6Hz,2H),7.29–7.23(m,4H),7.22–7.14(m,1H),6.99(t,J=5.9Hz,1H),5.25(s,2H),4.52(td,J=9.2,4.8Hz,1H),3.90(qd,J=16.6,5.8Hz,2H),3.79(dd,J=16.7,5.7Hz,1H),3.63(dd,J=16.7,5.3Hz,1H),3.54(t,J=13.7Hz,2H),3.08(dd,J=13.8,4.5Hz,1H),2.82(dd,J=13.7,9.8Hz,1H),1.35(d,J=18.7Hz,9H).13C NMR(126MHz,DMSO-d6)δ171.48,169.65,168.96,167.68,155.82,155.31,151.97,145.18,139.26,137.82,129.53,129.17,128.12,126.32,125.42,122.62,119.05,78.13,70.27,43.23,42.76,41.81,37.40,28.19.
化合物51的合成
将化合物49(65mg,0.092mmol),化合物50-3(50mg,0.116mmol),1-羟基苯并三唑(16.6mg,0.123mmol),吡啶(0.2mL,2.5mmol)和N’N-二异丙基乙胺(0.2mL,1.2mmol)溶于DMF(5mL)中,在室温下搅拌反应过夜。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51(50mg,0.05mmol),产率54%。ESI-HRMS计算结果:C51H64N7O14[M+H]+:998.4511,实际结果:998.4505.1H NMR(500MHz,DMSO-d6)δ9.87(d,J=10.7Hz,1H),8.43(s,1H),8.20(d,J=7.8Hz,1H),7.94(t,J=5.1Hz,1H),7.57(dd,J=27.2,7.9Hz,2H),7.26(d,J=4.4Hz,6H),7.16(ddd,J=29.3,9.3,2.8Hz,2H),7.06–6.94(m,3H),6.60–6.40(m,4H),5.08–4.86(m,2H),4.52(s,1H),3.92–3.70(m,8H),3.58(ddd,J=25.2,13.7,7.4Hz,12H),3.12–2.74(m,10H),1.35(d,J=18.2Hz,9H).13C NMR(126MHz,DMSO-d6)δ171.45,169.65,168.93,167.52,155.83,153.39,153.05,152.60,150.73,139.63,138.46,137.84,136.73,132.04,129.17,128.46,128.32,128.12,126.91,126.31,123.15,119.00,105.90,103.67,78.13,60.09,60.04,59.99,55.85,55.75,55.55,54.28,46.32,43.24,42.73,41.83,37.43,34.54,34.15,28.19.
化合物55的合成
将化合物51(50mg,0.05mmol)溶于二氯甲烷(4mL)中,冰浴下加入三氟乙酸(1mL,13.2mmol),冰浴下继续反应4h后,直接减压旋转蒸除溶剂后得粗品55(45mg,0.049mmol,99%),直接用于下一步反应。ESI-HRMS计算结果:C46H56N7O12[M+H]+:898.3987,实际结果:898.3975.
化合物57的合成
将上一步所得化合物55(45mg,0.049mmol),N3-PEG4-COOH(15mg,0.055mmol),HATU(23mg,0.06mmol)和N’N-二异丙基乙胺(0.033mL,0.2mmol)溶于DCM(5mL)中,在室温下搅拌反应过夜。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57(18.2mg,0.016mmol),产率32%。ESI-HRMS计算结果:C56H73N10O17[M+H]+:1157.5155,实际结果:1157.5192.1H NMR(500MHz,CD3OD)δ7.68–7.49(m,2H),7.44–6.74(m,11H),6.50(ddd,J=34.0,14.0,9.1Hz,3H),5.17–4.94(m,2H),4.55(dt,J=13.7,6.8Hz,1H),4.10–3.98(m,3H),3.96–3.82(m,6H),3.82–3.70(m,7H),3.70–3.57(m,20H),3.56–3.42(m,3H),3.36–3.32(m,2H),3.27–3.18(m,1H),3.06–2.83(m,6H).13C NMR(126MHz,CD3OD)δ174.12,173.78,172.35,171.93,169.51,156.11,154.66,154.22,152.31,139.39,138.53,134.24,131.36,130.66,130.36,130.27,129.99,129.76,129.69,129.55,128.60,127.83,124.50,121.30,121.17,121.06,113.29,107.34,104.84,72.02,71.53,71.52,71.51,71.45,71.33,71.26,71.06,68.20,61.13,56.96,56.65,56.45,56.36,51.75,47.83,44.16,43.69,43.34,38.07,35.51.
化合物59的合成
将化合物57(18.2mg,0.016mmol)、化合物2-丙炔甲酰胺-1,4-乙烯基磺酰胺哌嗪1(6mg,0.017mmol)、L-抗坏血酸钠(6.4mg,0.032mmol)和硫酸铜(5.12mg,0.032mmol)溶于3mL tBuOH/H2O/DMF(1/1/1)中。室温下反应2h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59(3mg,0.002mmol),产率12%。ESI-HRMS计算结果:C68H90N13O22S2[M+H]+:1504.5765,实际结果:1504.5795.1H NMR(500MHz,CD3OD)δ8.07(d,J=7.3Hz,1H),7.55(dd,J=31.9,23.9Hz,2H),7.45–6.76(m,11H),6.75–6.66(m,1H),6.58(ddd,J=10.6,9.1,4.2Hz,2H),6.53–6.42(m,2H),6.17(ddd,J=16.5,5.1,3.3Hz,2H),6.11(dd,J=10.0,3.4Hz,1H),6.01(dd,J=9.9,3.4Hz,1H),5.06(t,J=14.9Hz,1H),4.66–4.51(m,5H),4.42(dd,J=15.5,6.1Hz,1H),4.17–3.97(m,4H),3.96–3.83(m,8H),3.82–3.70(m,8H),3.69–3.53(m,23H),3.24(dd,J=13.7,5.1Hz,1H),3.09–2.82(m,8H),2.79–2.69(m,1H).13C NMR(126MHz,CD3OD)δ174.14,173.78,172.33,171.94,170.60,154.29,152.31,138.53,136.81,133.68,131.37,130.43,130.29,130.00,129.69,129.57,128.77,128.61,128.35,127.86,126.13,124.51,121.21,121.09,113.33,107.35,72.03,71.43,71.35,71.29,71.23,69.90,61.15,56.66,56.47,52.25,46.09,43.46,38.12,35.41.
药物-链接子与抗体偶联合成抗体药物偶联物
在微孔板(330μL LABTIDE 96Round Well)中分别加入100μL的西妥昔单抗溶液(PBS(pH=7.4)缓冲溶液,2.5mg/mL),0.83μL的三(2-羧乙基)膦(TCEP)溶液(溶于纯水配成10mM的溶液,用NaOH/H3PO4调节PH为7.0),然后放在微孔板振荡器上室温反应2小时。随后分别加入1.67μL(10eq.10mM DMSO溶液)上述制备的50-5、50-6、52-4、52-5、52-6、52-7、52-8、59和70,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(ZebaTMSpinDesalting Columns,7K MWCO,0.5mL)除去过量的小分子化合物。分别得到相应的抗体-药物偶联物。用UPLC-MS(型号ABsciex 4600)分析反应结果。
实施例2:抗体-药物偶联物的亲和力和内吞作用测定
在本实施例中,研究了Cetuximab、Cex-50-5、Cex-59、Cex-70对肿瘤细胞系的亲和和内吞作用。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。受试物与细胞分别在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%。
以下实验中所用的试剂及耗材来源于:西妥昔单抗(Cetuximab)(上海环耀生物科技有限公司,货号:205923-56-4),Goat Anti-Human IgG H&L(FITC)购置于Abcam公司,DAPI购置于Cell Signaling公司,face清洗液(PBS+1%BSA)。仪器:CytoFLEX FlowCytometer。
本实施例中使用流式细胞术(flow cytometry,FCM)来分析抗体及ADCs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。Cetuximab可以特异性结合在靶细胞的EGFR抗原位点,然后用特异的荧光二抗标记Cetuximab,利用FCM检测荧光二抗的荧光强度可以间接指示Cetuximab与靶细胞的抗原位点的结合能力。
(1)、细胞收集:收集NCI-H1975细胞至15mL离心管,1500rpm离心3min,弃上清;
(2)、用face洗液(PBS+0.1%BSA)洗涤细胞1次,分装到1.5mL离心管(每个EP管保证不少于2×105个活细胞),1500rpm离心3min,去上清;
(3)、NCI-H1975细胞用不同浓度(100、20、4、0.8、0.16、0.032、0.0064、0.00128nM)的200μL抗体/ADCs溶液于冰上孵育细胞30min;期间轻微吹吸细胞2次防止细胞沉底;每种细胞设置PBS对照组;
(4)、NCI-H1975细胞的37℃内吞组样品用face洗液清洗3次,转移到37℃恒温箱里孵育3h后放回冰上;4℃组样品仍在冰上孵育,face洗液清洗3次;
(6)、每个样品用200μL的DAPI(1μg/mL)的PBS溶液稀释,置于冰上15min后上机检测。
实验结果如图1、图2所示,本发明中涉及的ADCs Cex-50-5,Cex-59,Cex-70与原抗Cetuximab具有同等的亲和力和内吞作用。
实施例3: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Cetuximab、ADCs Cex-50-5、Cex-59、Cex-70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、胰蛋白酶-EDTA、胎牛血清、重组人胰岛素、100×青链霉素、1×PBS(pH7.4)、CCK8显色试剂购置于Gibco公司;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:细胞系:NCI-H1975、NCI-H1975-GFP、HCC827、A549、MDA-MB-231、NCI-H2228、293T。
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NCI-H1975(5×103)、NCI-H1975-GFP(5×103)、HCC827(6×103)、A549(3×103)、MDA-MB-231(6×103)、NCI-H2228(5×103)、293T(3×103)以3-6×103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8-10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%
实验结果:图6-11显示了本发明中涉及的ADCs Cex-50-5,Cex-59,Cex-70以及Cetuximab和CA4对NCI-H1975、NCI-H1975-GFP、HCC827、A549、MDA-MB231、NCI-H2228细胞的体外增殖抑制作用。本发明中涉及的ADCs Cex-50-5,Cex-59,Cex-70能够显著抑制肿瘤细胞的生长,尤其对EGFR敏感的HCC827细胞。ADCs Cex-50-5,Cex-59,Cex-70、西妥昔单抗和MMAE在不同细胞中的IC50(nM)如表1所示。
表1
实施例4:抗体-药物偶联物的体内抗肿瘤生物活性测定
实验动物:63只SPF级别4-5周龄NCG雄性小鼠,适应环境5-7d,随机取56只小鼠,在小鼠右侧腋下皮下接种5×106个无支原体污染的NCI-H1975-GFP活细胞,生长7d即可看小颗粒实体瘤,生长到第15d肿瘤大小生长到150mm3左右开始正式实验处理,选取肿瘤大小均一的48只小鼠进行实验。
实验分组:
①空白对照组PBS,(n=8);
②小分子药物对照组(CA4,0.3mg/kg BW),(n=8);
③西妥昔抗体对照组(10mg/kg BW),(n=8);
④Cex-50-5(10mg/kg BW),(n=8);
⑤Cex-59(10mg/kg BW),(n=8);
⑥Cex-70(10mg/kg BW),(n=8)。
⑦健康组(CK),非荷瘤小鼠正常生长不给予任何药物处理(n=7)
注射方法:尾静脉注射,每只200μL体积
仪器:小鼠尾静脉注射血管显示仪
每4d给药一次,连续给药3次
每4d监测体重一次,连续监测至末期
每4d监测肿瘤大小一次(游标卡尺),连续监测至末期。
肿瘤体积计算方法:体积=长×宽×宽/2
采血:最后一次给药后第4天眼眶取血300μL,室温静置4小时后5000g离心15分钟取上层血清,-80℃冻存备用。
实验结果:图12、13显示了本发明中涉及的ADCs Cex-50-5,Cex-59,Cex-70以及Cetuximab和CA4对非小细胞肺癌NCI-H1975-GFP异种移植瘤的抑制作用。ADCs Cex-50-5,Cex-59,Cex-70处理组表现出比原抗Cetuximab及小分子药物CA4更能显著抑制人非小细胞肺癌NCI-H1975-GFP异种移植小鼠皮下肿瘤生长的作用,且效果持久。
图14显示了本发明中涉及的ADCs Cex-50-5,Cex-59,Cex-70以及Cetuximab和CA4对非小细胞肺癌NCI-H1975-GFP异种移植小鼠体重的影响。ADCs Cex-50-5,Cex-59,Cex-70对小鼠体重基本没影响,说明它们没有明显的毒副作用。
Claims (7)
2.如权利要求1所述的康普瑞汀类衍生物及其抗体药物偶联物,其特征在于,所述式Ⅰ中,Ab为抗体。
3.如权利要求1所述的康普瑞汀类衍生物及其抗体药物偶联物,其特征在于,所述式Ⅰ中,L包含可断裂和不可断裂链接子。
4.如权利要求1所述的康普瑞汀类衍生物及其抗体药物偶联物,其特征在于,所述式Ⅰ中,n为1-10。
5.权利要求1-4任意一项所述的康普瑞汀类衍生物及其抗体药物偶联物的制备方法,其特征在于,包括以下步骤:
步骤1):将康普瑞汀或其衍生物进行修饰并与化学连接子进行连接,得到康普瑞汀-链接子分子;
步骤2):将靶向配体与步骤1)获得的康普瑞汀-链接子分子进行偶联反应,合成靶向配体-药物偶联物。
6.一种权利要求1-4任意一项所述的康普瑞汀类衍生物及其抗体药物偶联物在癌症靶向治疗或治疗增殖性疾病中的应用。
7.如权利要求6所述的应用,其特征在于,所述的增殖性疾病为上皮生长因子受体表达的病灶。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202011245317.3A CN112209990B (zh) | 2020-11-10 | 2020-11-10 | 康普瑞汀类衍生物及其抗体药物偶联物 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202011245317.3A CN112209990B (zh) | 2020-11-10 | 2020-11-10 | 康普瑞汀类衍生物及其抗体药物偶联物 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN112209990A true CN112209990A (zh) | 2021-01-12 |
CN112209990B CN112209990B (zh) | 2022-10-14 |
Family
ID=74056690
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202011245317.3A Active CN112209990B (zh) | 2020-11-10 | 2020-11-10 | 康普瑞汀类衍生物及其抗体药物偶联物 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN112209990B (zh) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113549106A (zh) * | 2021-07-14 | 2021-10-26 | 淮阴工学院 | 一种康普瑞汀衍生物及其制备方法和应用 |
CN114409562A (zh) * | 2021-02-08 | 2022-04-29 | 四川百利药业有限责任公司 | 一种ca4衍生物及其配体-药物偶联物 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002066631A1 (fr) * | 2001-02-21 | 2002-08-29 | Denka Seiken Co., Ltd. | Anticorps capables de reconnaitre helicobacter pylori et procede de detection de helicobacter pylori |
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 |
CN110269943A (zh) * | 2019-07-29 | 2019-09-24 | 上海科技大学 | 一种靶向于egfr的抗体-药物偶联物及其制备方法与应用 |
-
2020
- 2020-11-10 CN CN202011245317.3A patent/CN112209990B/zh active Active
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002066631A1 (fr) * | 2001-02-21 | 2002-08-29 | Denka Seiken Co., Ltd. | Anticorps capables de reconnaitre helicobacter pylori et procede de detection de helicobacter pylori |
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 |
CN110269943A (zh) * | 2019-07-29 | 2019-09-24 | 上海科技大学 | 一种靶向于egfr的抗体-药物偶联物及其制备方法与应用 |
Non-Patent Citations (2)
Title |
---|
T.DAVIDOV 等: "Carbonic Anhydrase 4 (CA4) and Crystallin Alpha-B (CRYAB) Immunoreactivity May Distinguish Benign From Malignant Thyroid Nodules in Patients With Indeterminate Thyroid Cytology", 《JOURNAL OF SURGICAL RESEARCH》 * |
马娟娟 等: "血管抑制剂康普瑞汀和康普瑞汀磷酸二钠纳米制剂研究进展", 《国际药学研究杂志》 * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114409562A (zh) * | 2021-02-08 | 2022-04-29 | 四川百利药业有限责任公司 | 一种ca4衍生物及其配体-药物偶联物 |
WO2022166719A1 (zh) * | 2021-02-08 | 2022-08-11 | 四川百利药业有限责任公司 | 一种ca4衍生物及其配体-药物偶联物 |
CN113549106A (zh) * | 2021-07-14 | 2021-10-26 | 淮阴工学院 | 一种康普瑞汀衍生物及其制备方法和应用 |
CN113549106B (zh) * | 2021-07-14 | 2023-05-26 | 淮阴工学院 | 一种康普瑞汀衍生物及其制备方法和应用 |
Also Published As
Publication number | Publication date |
---|---|
CN112209990B (zh) | 2022-10-14 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US6825166B2 (en) | Molecular conjugates for use in treatment of cancer | |
CN112209990B (zh) | 康普瑞汀类衍生物及其抗体药物偶联物 | |
JPH10513187A (ja) | 高分子量のポリマーを基剤とするプロドラッグ | |
AU2002254400A1 (en) | Molecular conjugates for use in treatment of cancer | |
CN109464654B (zh) | 鹅膏毒肽类抗体偶联物 | |
CN104861039B (zh) | 一种酞菁基化合物、制备方法及作为单、双光子荧光探针在癌症靶向及线粒体标记中的应用 | |
KR20220038601A (ko) | 캄토테신 약물 및 그의 항체 접합체 | |
CN111001012A (zh) | 一种亲水碳酸酯型抗体偶联药物 | |
CN113979912B (zh) | 两种前列腺特异性膜抗原靶向荧光探针及其制备方法与应用 | |
CN111925311A (zh) | 肿瘤造影化合物、其制备方法及在肿瘤诊断成像中的应用 | |
Chellan et al. | Bioactive half-sandwich Rh and Ir bipyridyl complexes containing artemisinin | |
CN113713117B (zh) | 一种白蛋白结合型肿瘤环境响应型抗肿瘤前体药物及其制备方法和应用 | |
CN111868021A (zh) | 分枝型单分散聚乙二醇、中间体及其制备方法 | |
CN110269943B (zh) | 一种靶向于egfr的抗体-药物偶联物及其制备方法与应用 | |
CN114573504A (zh) | 一种含N-OH键的β-榄香烯衍生物及其制备方法和应用 | |
CN111205350A (zh) | 作为组蛋白脱乙酰酶抑制剂的大环硫代酸酯前体药物 | |
CN112480212A (zh) | 靶向肝细胞生长因子的高亲和肽及其应用 | |
WO2003055887A1 (en) | Conjugated porphyrin, chlorin or bacteriochlorin chromophore | |
CN106243114A (zh) | 分子靶向氮杂芳香环轴向取代酞菁配合物及制备方法 | |
CN108250125B (zh) | 一种肿瘤靶向探针化合物及其合成和应用 | |
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 | |
CN106795169B (zh) | 光己醚化合物及其药物组合物和用途 | |
CN108676002B (zh) | 9-氮(6’-氨基)己基杂双环[3.3.1]壬烷偶联罗丹明b类化合物及用途 | |
CN114632079B (zh) | 一种基于青蒿素的铁池靶向分子影像探针制备及其应用 | |
CN116813704B (zh) | 一种肿瘤靶向荧光分子探针及其应用 |
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 |