CN112604004A - 一类抗人egfr抗体药物偶联物及其制备方法与应用 - Google Patents
一类抗人egfr抗体药物偶联物及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一类抗人EGFR抗体药物偶联物及其制备方法与应用。所述的抗人EGFR抗体药物偶联物具有式I所示结构。本发明通过利用SCT‑200全人源化单克隆抗体，通过还原抗体链间二硫键的化学方法与Linker‑MMAE偶联，构建了一类靶向于EGFR以实体瘤治疗为主的抗人EGFR抗体药物偶联物。本发明提出的新型抗体药物偶联物SCT‑200‑Linker‑MMAE，相比与SCT‑200本身，并未影响抗体的亲和力，内吞活性和靶向性，较好的保留其生物学功能，相比于SCT‑200，活性得到了明显的提高，能够显著抑制肿瘤凋亡过程中相关蛋白的表达，抑瘤效果大大增强。
Description
技术领域
本发明涉及抗人表皮生长因子受体(EGFR)抗体药物偶联物(ADC)，包括使用所述ADC的物质、制备方法及其在实体瘤治疗领域的应用。本发明属于生物技术药物领域。
背景技术
EGFR(epidermal growth factor receptor)是肿瘤治疗中的一个很重要的靶点，其在多种上皮来源的实体瘤中都呈过表达，因此，研发针对EGFR家族受体的靶向药物，成为近年来抗肿瘤治疗的主要热点。抗EGFR抗体为主要的靶向疗法，其作用机制主要是与内源性配体竞争性结合EGFR，通过抑制酪氨酸激酶的激活，促进EGFR内化等作用产生抗肿瘤效果。虽然抗体药物研究有自身独特的优势和不错市场前景，但现有的EGFR药物疗效效果都有限，靶向EGFR绝大多数情况下，长时间使用导致耐药。耐药的主要原因有EGFR蛋白胞外段突变或者EGFR下游的信号通路突变等。抗体-药物偶联物(Antibody-drug conjugate,ADC)结合了抗体的靶向性与细胞毒药物的高效细胞毒作用等优点，成为目前靶向治疗癌症的有效策略之一，同时ADC可以弥补抗体药物的不足。以EGFR为靶点的ADC药物的研究，可克服EGFR抗体产生的耐药性，提高EGFR抗体的疗效。尤其是针对实体瘤的研究，临床及市场前景广阔。然而，目前尚未有以EGFR为靶点的ADC上市药物，临床在研的靶向于EGFR的ADC有AVID-100和ABT-414和ABBV-221。然而，EGFR-ADC的研发也有一定挑战性，其中以EGFR为靶点的两个ADC IMGN-289和AMG-595也因毒性(如皮肤毒性、胃肠道毒性等)或疗效不佳等问题在临床研究中被迫终止，所以EGFR为靶点的ADC的研究机遇与挑战并存。
ADC一般由抗体(antibody)、弹头分子(warhead)和连接子(linker)3个部分组成。SCT-200抗体是一种新型靶向治疗转移性结直肠癌的全人源抗EGFR单克隆抗体(专利公布号：CN101058609A)，是通过人噬菌体库筛选获得的高亲合性100％人源抗体，具有靶向EGFR原创性序列，目前治疗转移性结直肠癌和头颈鳞癌的研究在临床Ⅱ期，临床应用可能会比嵌合抗体和人源化抗体具有更好的治疗效果和安全性。弹头分子的细胞毒性必须极高，一般要求EC50小于1nmol/L。目前，以MMAE作为弹头分子的ADC占有主导地位。MMAE是以截尾海兔中提取的天然海洋产物海兔毒素中的有效成分海兔毒素10(dolastatins 10)为先导物，是一类线性五肽细胞毒分子，在多种人癌症细胞系中，IC50为10-9～10-11mol/L，为长春碱活性的200倍。MMAE作为ADC弹头分子的优势主要有三点：(1)高细胞毒性；(2)N端单甲基取代，可与连接子相连接；(3)旁观者效应：尤其是对实体瘤治疗有益。与MMAE连接的连接子一般选用缬氨酸-瓜氨酸(Val-Cit)二肽型，与MMAE组合全称为MC-VC-PABC-MMAE，该组合为ADC临床试验中使用最广泛的Linker-Drug，上市ADC Brentuximab Vedotin(SGN-35，Adcetris)就选择该类Linker-Drug组合，使其在组织蛋白酶B中裂解，从而完全释放出MMAE。然而，VC连接子结构类型较为单一，且从结构角度出发仍存在一些问题需要改进：
(1)间隔区部分：MC会慢慢水解，失去与巯基反应的特异性；MC与抗体连接后水解产物的稳定性有待进一步改善。
(2)酶水解部分：某些血浆酶(如羧酸酯酶1C)，可以部分地消化ADC产品中的Val-Cit结构，导致药物提前脱靶。
(3)自分解部分：氨基苄氧羰基(p-aminobenzyloxycarbonyl)间隔基团类似于酯键，在血浆中的稳定性有待进一步改善。
为了克服上述问题，本发明充分采用SCT200抗体的优势，设计不同类型连接子与MMAE连接，最终制备得到新型SCT200-Linker-MMAE抗体药物偶联物。制备所得ADCs在亲和力方面和内吞方面与SCT200相当，能够上调肿瘤凋亡相关的蛋白的表达。在体内外多种实体瘤模型中，本发明的ADCs均体现了较SCT200更强的抗肿瘤活性。
发明内容
本发明的目的在于提供一类抗人EGFR抗体药物偶联物及其制备方法和抗肿瘤应用。本发明的一类抗人EGFR抗体药物偶联物对EGFR阳性的肿瘤组织具有高效靶向性、高内吞活性、高亲和活性以及高抗肿瘤活性。
为了达到上述目的，本发明采用了以下技术手段：
本发明一类抗人EGFR抗体药物偶联物，其具有式I所示结构：
其中，
所述的抗体为抗人EGFR抗体，z＝2～8；
M为能与抗体半胱氨酸巯基反应的间隔区部分，其结构为以下所示结构中的一种:
其中，n＝0,1,2,3,4或5，m＝1～10,12或24；
C为能够在酶的作用下发生水解的部分，选自二肽序列Val-Cit,Val-Ala,Val-Lys或Val-Arg；或三肽序列Ala-Val-Ala,Gly-Phe-Gly,Gly-Phe-Lys或Ala-Phe-Lys；或四肽序列Gly-Gly-Phe-Gly或Gly-Phe-Leu-Gly中的一种；
R为自分解部分，其结构为以下所示结构中的一种:
其中，优选的，所述的抗体为SCT-200单克隆抗体。
进一步的，本发明还提出了一种制备所述的抗人EGFR抗体偶联药物的方法，包括：将具有式II结构的Linker-MMAE与抗人EGFR抗体的链间二硫键发生加成反应制得所述的抗人EGFR抗体药物偶联物；
其中，优选的，所述的方法包括以下步骤：
(1)将Linker-MMAE溶于二甲基亚砜，得到连接子-细胞毒药物储备液；
(2)将还原剂溶于缓冲液中，配置还原剂储备液；
(3)将L-Cys溶于缓冲液中，制备L-Cys储备液；
(4)将抗人EGFR抗体加入步骤(2)所述的还原剂储备液中，孵育1-2h；
(5)向步骤(4)得到的反应液中加入步骤(1)的连接子-细胞毒药物储备液，孵育1-2h，制备抗人EGFR抗体药物偶联物；
(6)向步骤(5)得到的反应液中加入步骤(3)的L-Cys储备液，停止反应。
其中，优选的，所述步骤(2)中还原剂为TCEP，混合时以抗人EGFR抗体的摩尔量为基准，还原剂的摩尔量为抗体摩尔量的2-4倍。
其中，优选的，所述步骤(5)中混合时以抗人EGFR抗体的摩尔量为基准，所加Linker-MMAE的摩尔量为抗体摩尔量的4-8倍。
其中，优选的，在所述步骤(4)和步骤(5)中的反应在氮气保护下进行，步骤(4)中的反应温度为35～40℃，优选为37℃。
其中，优选的，还包括将得到的抗体药物偶联物进一步纯化的步骤，优选采用AKTApurifier蛋白质纯化系统，收集所需抗体偶联药物组分峰；收集完毕后，使用30kDa超滤管进行超滤离心，浓缩，经无菌滤膜过滤，低温储存。
更进一步的，本发明还提出了所述的抗体药物偶联物在制备用于肿瘤靶向治疗的药物中的用途，其中，所述的肿瘤为EGFR阳性实体肿瘤，包括鳞状上皮细胞癌，食管癌，鼻咽癌，肺癌，乳腺癌，胰腺癌，前列腺癌，头颈癌，结肠癌等。
以所述的抗体药物偶联物为活性成分的用于肿瘤靶向治疗的药物组合物也在本发明的保护范围之内，所述的药物组合物含有药学上有效量的本发明所述的抗体药物偶联物及药学上允许的佐剂。
相较于现有技术，本发明的有益效果是：
1、本发明制备了一系列Linker，体现了较好的血浆稳定性和酶切速率，与MMAE进行一步反应制得Linker-MMAE。
2、本发明通过利用SCT-200全人源化单克隆抗体，通过还原抗体链间二硫键的化学方法与Linker-MMAE偶联，构建了一类靶向于EGFR以实体瘤治疗为主的抗人EGFR抗体药物偶联物。优选TCEP作为还原剂，在对其反应条件进行一定优化之后，制备出平均药物抗体偶联比率(DAR)在4.0±0.5的范围内，且质量稳定可控，重复性好，收率可观。
3、本发明提出的新型抗体药物偶联物SCT-200-Linker-MMAE，相比与SCT-200本身，并未影响抗体的亲和力，内吞活性和靶向性，较好的保留其生物学功能；在体外活性评价中，相比于SCT-200，活性得到了明显的提高，IC50均在nM级别；Western Blot实验表明，相比于SCT-200，SCT-200-Linker-MMAE ADCs能够显著抑制肿瘤凋亡过程中相关蛋白的表达；在体内活性评价中，相比于SCT-200，SCT-200-Linker-MMAE ADCs的抑瘤效果大大增强。
附图说明
图1为Fmoc-二肽序列合成路线图；
图2为Fmoc-AVA合成路线图；
图3为Fmoc-GFG合成路线图；
图4为Fmoc-GGFG合成路线图；
图5为Fmoc-GFLG合成路线图；
图6为间隔区和水解区类型的Linker-MMAE合成路线图；
图7为自分解区类型的Linker-MMAE合成路线图；
其中，图7A为自分解区类型的Linker合成路线图；图7B为自分解区类型的Linker和MMAE缩合反应合成路线图；
图8为间隔区型SCT-200-Linker-MMAE的HIC-HPLC图谱；
图9为水解区型SCT-200-Linker-MMAE的HIC-HPLC图谱；
图10为自分解区型SCT-200-Linker-MMAE的HIC-HPLC图谱；
图11为SCT-200-Linker-MMAE的ESI-MS图谱；
图12为ELISA测定SCT-200-Linker-MMAE ADCs亲和力图；
图13为流式细胞法测定SCT-200-Linker-MMAE ADCs内吞速率图；
图14为Western Blot测定不同细胞EGFR表达水平图；
图15为SCT200-Linker-MMAE ADCs对凋亡相关蛋白表达的影响图；
图16为SCT200-Linker-MMAE ADCs对A431裸鼠移植瘤模型的抑瘤曲线(A)以及体重变化曲线(B)。
具体实施方式
下面结合具体实例来进一步描述本发明，本发明的优点和特点将会随着描述而更为清楚。但这些实例仅是范例性的，并不对本发明的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本发明的保护范围内。
实施例1 Linker-MMAE的合成
1.1 Fmoc-AA的合成
1.1.1 Fmoc-二肽的合成
Fmoc-L-Val-OSu的合成
根据图1中Scheme S1步骤所示，将反应产物Fmoc-L-Val(10g,29.3mmol)和HoSu(3.7g,32.3mmol)溶于100mL THF中，冰浴条件下加入DCC(6.6g,32.3mmol)，加入完毕后，撤去冰浴，室温下搅拌反应过夜。反应完毕后(石油醚：乙酸乙酯＝1：1检测)，将反应液移至冰浴，待固体完全析出后抽滤，将滤液旋蒸至泡沫状，残余物即为产物。反应产物无需进一步纯化，直接用于下步反应。
1a的合成
根据图1中Scheme S1所示，将Fmoc-L-Val-OSu(16g,36.6mmol)先溶于90mL DME中，L-Cit(6.7g,38.5mmol)溶于NaHCO3(3.2g,38.5mmol)的90mL水中成盐，再加入到反应体系中，50mL THF助溶，反应室温搅拌过夜至澄清。反应完毕后(二氯甲烷：甲醇＝10：1检测)，冰浴下加入与THF等体积的15％的柠檬酸水溶液，待反应产物白色固体完全析出，布氏漏斗抽滤至滤液澄清，滤饼抽干，真空干燥箱干燥。干燥完毕后,将白色固体研磨，用乙醚洗涤数次，抽滤，得1a，白色固体15.1g，收率为83％。分子式为C26H32N4O6,MS(ESI)m/z:497[M+H]+；1H NMR(400MHz,DMSO-d6)δ(ppm):12.48(s,1H),8.17(d,J＝7.4Hz,1H),7.90(d,J＝7.5Hz,2H),7.76(t,J＝7.1Hz,2H),7.41(q,J＝7.7Hz,3H),7.33(m,2H),6.00(s,1H),5.63(s,2H),4.30–4.21(m,3H),4.16(m,1H),3.93(m,1H),2.98–2.89(m,2H),1.99(m,1H),1.77–1.66(m,1H),1.57(m,1H),1.41(m,2H),0.88(m,6H).
1b的合成
根据图1中Scheme S1所示，合成方法同1a，得白色固体纯品，两步收率为37％。分子式为C23H26N2O5，MS(ESI)m/z:411[M+H]+；1H NMR(400MHz,DMSO-d6)δ(ppm):12.48(s,1H),8.23(d,J＝7.0Hz,1H),7.90(d,J＝7.5Hz,2H),7.76(m,2H),7.42(m,3H),7.33(m,2H),4.34–4.16(m,4H),3.90(dd,J＝9.2Hz,7.1Hz,1H),1.98(m,1H),1.28(d,J＝7.2Hz,3H),0.89(m,6H).13C NMR(101MHz,DMSO-d6)δ(ppm):174.45,171.44,156.52,144.26,141.15,128.09,127.51,125.87,120.55,66.14,60.22,47.92,47.13,30.97,19.61,18.70,17.56.
1.1.2 Fmoc-三肽的合成
1c的合成
根据图2中Scheme S2所示，以2-CTC为树脂，经过固相合成，得白色固体，收率为89％。产物用乙醚洗涤、产物收率和纯度较高，无需进一步纯化。分子式为C26H31N3O6，MS(ESI)m/z:482[M+H]+；1H NMR(400MHz,DMSO-d6)δ(ppm):12.41(s,1H),8.26(d,J＝6.9Hz,1H),7.90(d,J＝7.5Hz,2H),7.72(m,2H),7.66(d,J＝9.0Hz,1H),7.57(d,J＝7.9Hz,1H),7.42(m,2H),7.34(m,2H),4.28(d,J＝6.9Hz,2H),4.22(dd,J＝8.3,6.1Hz,2H),4.18(d,J＝7.0Hz,2H),1.97(m,1H),1.27(d,J＝7.3Hz,3H),1.22(d,J＝7.2Hz,3H),0.88(d,J＝6.8Hz,3H),0.84(d,J＝6.8Hz,3H)；13C NMR(101MHz,DMSO-d6)δ(ppm):174.40,172.74,171.03,156.07,144.34,141.16,128.08,127.55,125.74,125.70,120.56,66.05,57.36,50.52,47.91,47.10,31.53,19.55,18.68,18.37,17.47.
1d的合成
根据图3中Scheme S3所示，得白色固体，收率为65％。产物用乙醚洗涤、产物收率和纯度较高，无需进一步纯化。分子式为C28H27N3O6，MS(ESI)m/z:502[M+H]+；1H NMR(400MHz,DMSO-d6)δ(ppm):12.60(s,1H),8.42(t,J＝5.9Hz,1H),8.09(d,J＝8.5Hz,1H),7.90(d,J＝7.5Hz,2H),7.71(d,J＝7.5Hz,2H),7.48(m,1H),7.42(m,2H),7.33(m,2H),7.24(d,J＝4.4Hz,4H),7.18(m,1H),4.57(m,1H),4.31–4.17(m,3H),3.79(d,J＝5.9Hz,2H),3.67(m,1H),3.52(m,1H),3.04(m,1H),2.78(m,1H)；13C NMR(101MHz,DMSO-d6)δ(ppm):171.87,171.52,144.29,141.16,138.21,129.65,128.49,128.09,127.54,126.71,125.73,120.56,66.20,54.13,47.06,43.71,41.10,38.25.
1.1.3 Fmoc-四肽的合成
1e的合成
根据图4中Scheme S4所示，得白色固体，收率为49％。产物用乙醚洗涤、产物收率和纯度较高，无需进一步纯化。分子式为C30H30N4O7，MS(ESI)m/z:559[M+H]+；1H NMR(400MHz,DMSO-d6)δ(ppm):12.55(s,1H),8.38(m,1H),8.13(d,J＝8.6Hz,1H),8.00(m,1H),7.90(d,J＝7.5Hz,2H),7.72(d,J＝7.5Hz,2H),7.60(m,1H),7.42(m,2H),7.34(d,J＝7.4Hz,2H),7.25(d,J＝4.3Hz,4H),7.18(m,1H),4.55(m,1H),4.35–4.18(m,3H),3.77(m,3H),3.63(d,J＝6.3Hz,3H),3.06(m,1H),2.77(m,1H)；13C NMR(101MHz,DMSO-d6)δ(ppm):171.84,171.51,169.79,168.92,144.31,141.17,129.62,128.52,128.09,127.55,126.72,125.71,120.58,66.22,54.28,47.08,43.92,42.23,41.12,38.12.
1f的合成
根据图5中Scheme S5所示，得白色固体，收率为75％。产物用乙醚洗涤、产物收率和纯度较高，无需进一步纯化。分子式为C34H38N4O7，MS(ESI)m/z:615[M+H]+；1H NMR(400MHz,DMSO-d6)δ(ppm):12.54(s,1H),8.18–8.07(m,2H),8.01(d,J＝8.1,1H),7.90(d,J＝7.5Hz,2H),7.71(d,J＝7.4Hz,2H),7.53(t,J＝6.1Hz,1H),7.42(t,J＝7.4Hz,2H),7.33(t,J＝7.4Hz,2H),7.23(d,J＝4.4Hz,4H),7.19–7.15(m,1H),4.57(dt,J＝9.2Hz,4.6Hz,1H),4.35(q,J＝7.8Hz,1H),4.31–4.19(m,3H),3.83–3.47(m,4H),3.03(m,1H),2.79(m,1H),1.50(q,J＝8.2Hz,7.3Hz,3H),0.86(m,6H)；13C NMR(101MHz,DMSO-d6)δ(ppm):172.62,171.52,171.16,169.36,144.29,141.16,129.72,128.45,128.09,127.54,126.66,125.71,120.57,66.23,54.14,51.30,47.06,43.76,41.46,41.07,38.01,24.51,23.48,22.11.
1.2 Linker-MMAE的合成
2a的合成
根据图6步骤所示，将1a反应产物(7.6g,15.3mmol)和PABOH(3.76g,30.6mmol)溶于140mL CH2Cl2和70mL的CH3OH混合溶剂中，避光加入EEDQ(7.5g,30.6mmol)。室温下搅拌反应，反应完毕后(二氯甲烷：甲醇＝20：1检测)旋干反应液，残余物用乙醚洗涤，抽滤，得黄色固体8.4g，收率90％。分子式为C33H39N5O6,MS(ESI)m/z:602[M+H]+；1H NMR(400MHz,DMSO-d6)δ(ppm):9.99(s,1H),8.11(d,J＝7.5Hz,1H),7.90(d,J＝7.6Hz,2H),7.75(m,2H),7.55(d,J＝8.2Hz,2H),7.43(m,3H),7.33(m,2H),7.24(d,J＝8.1Hz,2H),5.99(m,1H),5.41(s,2H),5.10(m,1H),4.43(d,J＝5.5Hz,3H),4.31(s,1H),4.25(d,J＝6.3Hz,2H),3.94(m,1H),3.03–2.88(m,2H),2.00(d,J＝9.0Hz,1H),1.76–1.65(m,1H),1.60(m,1H),1.48–1.36(m,2H),0.92–0.85(m,6H).
2b的合成
根据图6步骤所示，1b(1.87g,0.0045mmol)和PABOH(1.12g,0.009mmol)溶于无水40mL四氢呋喃中，加入HATU(2.05g,0.0054mmol)和DIEA 1.5mL，室温搅拌反应。反应完毕后，减压蒸馏，硅胶柱层析纯化，二氯甲烷:甲醇＝15:1，得淡黄色固体1.3g，收率59％。分子式为C30H33N3O5，MS(ESI)m/z:516[M+H]+；1H NMR(600MHz,DMSO-d6)δ(ppm):9.92(s,1H),8.17(d,J＝7.1Hz,1H),7.89(d,J＝7.5Hz,2H),7.75(dd,J＝10.6Hz,7.5Hz,2H),7.54(d,J＝8.1Hz,2H),7.47–7.39(m,3H),7.33(m,2H),7.24(d,J＝8.2Hz,2H),5.10(s,1H),4.43(s,3H),4.33–4.28(m,1H),4.26–4.20(m,2H),3.92(dd,J＝8.9Hz,7.1Hz,1H),2.01–1.98(m,1H),1.31(d,J＝7.1Hz,3H),0.88(m,6H).
2c的合成
根据图6步骤所示，以1c和PABOH为原料，方法同2b，得淡黄色固体，收率83％。分子式为C33H38N4O6，MS(ESI)m/z:587[M+H]+；1H NMR(400MHz,DMSO-d6)δ(ppm):9.96(s,1H),7.89(d,J＝7.5Hz,2H),7.85(d,J＝7.4Hz,1H),7.72(t,J＝7.4Hz,1H),7.58(m,2H),7.42(t,J＝7.3Hz,2H),7.35(t,J＝7.3Hz,2H),7.24(dd,J＝8.5Hz,4.0Hz,2H),6.29(s,1H),4.43(s,2H),4.29–4.10(m,2H),2.04–1.96(m,2H),1.46(m,1H),1.31(m,4H),1.25(m,5H),1.18–1.14(m,2H),0.91–0.83(m,6H).
2d的合成
根据图6步骤所示，以1d和PABOH为原料，合成方法同2b，得淡黄色固体，收率74％。分子式为C35H34N4O6，MS(ESI)m/z:502[M+H]+；1H NMR(400MHz,DMSO-d6)δ(ppm):9.78(s,1H),8.55–8.46(m,1H),8.20(d,J＝7.8Hz,1H),7.90(d,J＝7.6Hz,2H),7.70(d,J＝7.6Hz,2H),7.59(d,J＝8.1Hz,2H),7.54(d,J＝5.9Hz,1H),7.43(d,J＝7.5Hz,2H),7.32(t,J＝7.5Hz,2H),7.26(d,J＝3.4Hz,4H),6.56–6.50(m,1H),5.11(s,1H),4.54(m,1H),4.43(s,2H),4.26(m,1H),4.22(m,1H),4.02(d,J＝7.5Hz,1H),3.92(m,2H),3.73–3.67(m,1H),3.57(m,1H),3.41–3.36(m,1H),3.07(m,1H),2.90–2.82(m,1H),1.11(m,1H).
2e的合成
根据图6步骤所示，以1e和PABOH为原料，方法同2b，得淡黄色固体，收率78％。分子式为C37H37N5O7，MS(ESI)m/z:664[M+H]+；1H NMR(400MHz,DMSO-d6)δ(ppm):9.83(m,1H),8.47(t,J＝5.9Hz,1H),8.24(d,J＝8.0Hz,1H),8.10(q,J＝5.3Hz,1H),7.89(d,J＝7.5Hz,2H),7.71(d,J＝7.5Hz,1H),7.58(d,J＝8.2Hz,2H),7.42(m,2H),7.37–7.31(m,2H),7.27–7.23(m,6H),7.19(m,1H),6.29(s,1H),5.12(s,1H),4.54(m,1H),4.44(s,2H),4.30(d,J＝6.0Hz,1H),4.23(s,1H),3.89(m,1H),3.84–3.77(m,1H),3.65(d,J＝5.8Hz,2H),3.12–3.06(m,1H),2.89(s,2H),2.74(s,1H),1.24(d,J＝3.1Hz,2H).
2f的合成
根据图6步骤所示，以1f和PABOH为原料，方法同2b，得淡黄色固体，收率66％。分子式为C41H45N5O7，MS(ESI)m/z:720[M+H]+；1H NMR(400MHz,DMSO-d6)δ(ppm):9.80(s,1H),8.19(m,2H),8.06(d,J＝8.1Hz,1H),7.90(d,J＝7.5Hz,2H),7.71(d,J＝7.5Hz,2H),7.56(m,3H),7.44–7.39(m,2H),7.35–7.30(m,2H),7.26(s,1H),7.22(s,5H),7.16(m,1H),5.09(s,1H),4.58(m,1H),4.44(s,2H),4.36–4.19(m,4H),3.88(dd,J＝5.9Hz,1.8Hz,2H),3.65(m,1H),3.54(m,1H),3.05(m,1H),2.80(m,1H),1.57(m,3H),0.88(m,6H).
4a的合成
根据图6步骤所示，3a(0.5g,1.3mmol)溶于18mL DMF中，加入MC-OSu(0.45g,1.4mmol)，室温搅拌反应过夜。反应完毕后(二氯甲烷：甲醇＝15：1检测)，旋干反应溶剂，残余物乙醚洗三遍后，抽滤得黄色固体0.69g，收率为92％。分子式为C28H40N6O7,MS(ESI)m/z:573[M+H]+；1H NMR(400MHz,DMSO-d6)δ(ppm):9.91(s,1H),8.07(d,J＝7.6Hz,1H),7.82(d,J＝8.6Hz,1H),7.55(d,J＝8.1Hz,2H),7.23(d,J＝8.1Hz,2H),7.01(s,2H),5.99(m,1H),5.42(s,2H),5.10(m,1H),4.43(d,J＝4.9Hz,2H),4.20(m,1H),3.00(m,2H),2.17(m,2H),1.97(m,1H),1.71(m,1H),1.64–1.57(m,1H),1.54–1.43(m,6H),1.20(m,2H),1.10(m,1H),0.84(m,6H).
4b的合成
根据图6步骤所示，以SMCC和3a为起始原料，合成方法同4a，得黄色固体，收率89％。分子式为C30H42N6O7,MS(ESI)m/z:599[M+H]+；1H NMR(400MHz,DMSO-d6)δ(ppm):9.91(s,1H),8.02(d,J＝7.5Hz,1H),7.72(d,J＝8.7Hz,1H),7.55(d,J＝8.1Hz,2H),7.24(d,J＝8.2Hz,2H),7.02(s,3H),5.98(m,1H),5.41(s,2H),5.10(m,1H),4.43(d,J＝5.6Hz,2H),4.16(m,1H),3.25(d,J＝7.1Hz,2H),2.98(m,2H),2.60(s,2H),2.23(m,1H),1.99(m,1H),1.73–1.60(m,6H),1.36–1.25(m,3H),0.84(m,8H).
4c的合成
根据图6步骤所示，以MC-Ph-OSu和3a为起始原料，合成方法同4a，得黄色固体，收率62％。此步产物溶解性不好，直接用乙醚和二氯甲烷混合溶剂洗涤、抽滤得到，直接进行下一步反应。
4d的合成
根据图6步骤所示，以3b和SMCC为起始原料，合成方法同4a，得黄色固体，收率67％。分子式为C27H36N4O6，MS(ESI)m/z:513[M+H]+；1H NMR(400MHz,DMSO-d6)δ(ppm):9.86(s,1H),8.10(d,J＝6.8Hz,1H),7.71(d,J＝8.6Hz,1H),7.54(d,J＝7.9Hz,2H),7.25(s,2H),7.02(s,2H),4.43(s,3H),4.14(t,J＝7.7Hz,1H),3.24(d,J＝6.9Hz,2H),2.23(m,1H),1.98(s,1H),1.75–1.50(m,6H),1.36–1.24(m,6H),0.91–0.82(m,7H).
4e的合成
根据图6步骤所示，以3c和SMCC为起始原料，合成方法同4a，得黄色固体，收率63％。分子式为C30H41N5O7，MS(ESI)m/z:584[M+H]+；1H NMR(400MHz,DMSO-d6)δ(ppm):9.96(m,1H),8.06(m,1H),7.59(d,J＝7.9Hz,1H),7.55(d,J＝8.2Hz,1H),7.23(d,J＝8.2Hz,2H),7.01(s,1H),5.12(s,1H),4.43(s,3H),4.34–4.26(m,1H),4.16(m,1H),3.24(m,1H),2.44(s,1H),2.12(m,1H),2.03–1.96(m,2H),1.71(m,2H),1.62(m,2H),1.30(d,J＝6.9Hz,3H),1.24(s,5H),1.19(m,3H),0.87–0.81(m,8H).
4f的合成
根据图6步骤所示，以3d和SMCC为起始原料，合成方法同4a，得黄色固体，收率71％。分子式为C32H37N5O7.MS(ESI)m/z:604[M+H]+；1H NMR(400MHz,DMSO-d6)δ(ppm):9.85(s,1H),8.69(s,1H),8.29(d,J＝7.3Hz,1H),8.14–8.08(m,1H),7.59(d,J＝8.2Hz,2H),7.25(d,J＝3.9Hz,6H),5.14(s,1H),4.44(s,3H),3.87(m,2H),3.78–3.72(m,1H),3.61–3.53(m,1H),3.20(d,J＝7.2Hz,2H),3.03(m,2H),2.90–2.82(m,1H),2.63–2.57(m,1H),2.42(s,1H),2.09–1.99(m,2H),1.67(m,2H),1.60(m,1H),1.51(m,1H),0.88(m,3H).
4g的合成
根据图6步骤所示，以3e和SMCC为起始原料，合成方法同4a，得黄色固体，收率72％。C34H40N6O8，MS(ESI)m/z:661[M+H]+；1H NMR(400MHz,DMSO-d6)δ(ppm):9.77(s,1H),8.38(t,J＝5.8Hz,1H),8.18(d,J＝7.9Hz,1H),8.04–7.98(m,2H),7.57(d,J＝8.2Hz,2H),7.26(d,J＝3.9Hz,4H),7.19(q,J＝4.3Hz,1H),7.01(s,2H),5.11(t,J＝5.7Hz,1H),4.54–4.47(m,2H),4.44(d,J＝5.6Hz,2H),3.88(m,2H),3.83–3.74(m,2H),3.66(d,J＝5.8Hz,2H),3.23(s,2H),3.11–3.06(m,1H),2.87–2.81(m,1H),2.11(m,1H),1.78–1.73(m,2H),1.65–1.60(m,2H),1.26(m,3H),0.90(m,3H).
4h的合成
根据图6步骤所示，以3f和SMCC为起始原料，合成方法同4a，得黄色固体，收率68％。分子式为C38H48N6O8，MS(ESI)m/z:717[M+H]+；1H NMR(400MHz,DMSO-d6)δ(ppm):9.77(s,1H),8.22–8.13(m,2H),8.00–7.94(m,2H),7.57(d,J＝8.3Hz,2H),7.26–7.19(m,6H),7.01(m,2H),5.11(t,J＝5.6Hz,1H),4.54(m,1H),4.44(m,2H),4.31(m,1H),3.87(d,J＝6.0Hz,2H),3.69(m,1H),3.54(m,1H),3.24(d,J＝7.1Hz,2H),3.04(m,1H),2.80(m,1H),2.08(m,2H),1.70(m,2H),1.64–1.58(m,3H),1.54(m,2H),1.26–1.16(m,3H),0.88(m,8H).
4i的合成
根据图6步骤所示，以3b和MC-OSu为起始原料，合成方法同4a，得黄色固体，收率70％。分子式C25H34N4O6，MS(ESI)m/z:487[M+H]+；1H NMR(400MHz,DMSO-d6)δ(ppm):9.84(s,1H),8.13(d,J＝7.0Hz,1H),7.81(d,J＝8.6Hz,1H),7.53(d,J＝8.1Hz,2H),7.23(d,J＝8.1Hz,2H),7.00(s,2H),5.08(s,1H),4.40(m,3H),4.17(t,J＝7.8Hz,1H),2.14(m,2H),1.96(m,1H),1.49(m,4H),1.35–1.14(m,7H),0.84(m,6H).
4j的合成
根据图6步骤所示，以3c和MC-OSu为起始原料，合成方法同4a，得黄色固体，收率71％。分子式为C28H39N5O7，MS(ESI)m/z:558[M+H]+；1H NMR(400MHz,DMSO-d6)δ(ppm):9.89(m,1H),8.05(t,J＝6.8Hz,1H),7.59(d,J＝8.2Hz,1H),7.54(d,J＝8.2Hz,1H),7.24(d,J＝8.0Hz,2H),7.00(s,1H),5.11(s,1H),4.43(s,2H),4.42–4.37(m,1H),4.33(t,J＝7.1Hz,1H),4.16(m,1H),2.46(s,1H),2.09(m,2H),2.00(m,2H),1.46(m,2H),1.43–1.39(m,2H),1.30(m,4H),1.24(s,4H),1.18(m,3H),0.88–0.82(m,6H).
4k的合成
根据图6步骤所示，以3d和MC-OSu为起始原料，合成方法同4a，得黄色固体，收率52％。分子式为C30H35N5O7，MS(ESI)m/z:578[M+H]+；1H NMR(400MHz,DMSO-d6)δ(ppm):9.87(s,1H),8.51(s,1H),8.26(d,J＝8.0Hz,1H),8.14(s,1H),7.59(m,2H),7.29–7.18(m,6H),5.13(s,1H),4.53–4.43(m,2H),3.95–3.86(m,2H),3.83–3.74(m,1H),3.68(m,2H),3.65–3.58(m,1H),3.43(s,2H),3.20(m,1H),3.12–3.05(m,1H),2.98(m,1H),2.91–2.82(m,1H),2.12(m,2H),1.54–1.42(m,3H),1.32–1.20(m,3H).
4l的合成
根据图6步骤所示，以3e和MC-OSu为起始原料，合成方法同4a，得黄色固体，收率51％。分子式为C32H38N6O8，MS(ESI)m/z:635[M+H]+；1H NMR(400MHz,DMSO-d6)δ(ppm):9.87(s,1H),8.53(s,1H),8.28(t,J＝8.5Hz,1H),8.17–8.11(m,1H),7.59(m,2H),7.26(t,J＝6.0Hz,5H),7.18(m,1H),5.14(s,1H),4.54–4.29(m,3H),3.97–3.75(m,3H),3.66(m,3H),3.43(s,2H),3.20(d,J＝4.0Hz,1H),3.13–2.94(m,2H),2.85(m,1H),2.17–1.98(m,2H),1.45(m,4H),1.24(m,4H).
4m的合成
根据图6步骤所示，以3f和MC-OSu为起始原料，合成方法同4a，得黄色固体，收率62％。分子式为C36H46N6O8，MS(ESI)m/z:691[M+H]+；1H NMR(400MHz,DMSO-d6)δ(ppm):9.77(s,1H),8.16(t,J＝6.5Hz,2H),8.02(m,2H),7.60–7.55(m,2H),7.27–7.22(m,6H),7.18(dd,J＝6.1Hz,2.6Hz,1H),7.01–6.99(m,1H),5.11(s,1H),4.54(m,1H),4.44(s,2H),4.30(m,1H),3.90–3.85(m,2H),3.71(m,1H),3.56(m,1H),3.05(m,1H),2.79(m,1H),2.08(d,J＝5.9Hz,2H),1.62(s,1H),1.55–1.44(m,7H),1.22–1.15(m,3H),0.88(m,7H).
5a的合成
根据图6步骤所示，将4a(0.69g,1.2mmol)溶于12mL DMF中，溶解后加入(PNP)2CO(1.1g,3.6mmol)和DIEA 0.5mL，室温搅拌下过夜。反应完毕后，旋干溶剂，硅胶柱分离纯化，分离条件为二氯甲烷：甲醇＝20：1，得产物为微黄色固体0.42g，收率为47％，mp:156–158℃。分子式为C35H43N7O11,HRMS[M+H]+(ESI)：found 738.3088,calcd 738.3093.1H NMR(500MHz,DMSO-d6)δ(ppm):10.09(s,1H),8.32(d,J＝9.1Hz,2H),8.14(d,J＝7.1Hz,1H),7.83(d,J＝8.6Hz,1H),7.66(d,J＝8.4Hz,2H),7.57(d,J＝9.1Hz,2H),7.41(d,J＝8.6Hz,2H),7.01(s,2H),6.00(m,1H),5.44(s,2H),5.24(s,2H),4.39(m,1H),4.20(m,1H),3.37(m,2H),2.99(m,2H),2.22–2.09(m,2H),1.96(m,1H),1.72(m,1H),1.65–1.56(m,1H),1.52–1.43(m,6H),1.22–1.15(m,2H),0.87–0.82(m,6H).
5b的合成
根据图6步骤所示，以4b和(PNP)2CO为起始原料，合成方法同5a，得黄色固体，收率66％，mp:171–173℃。分子式为C37H45N7O11,HRMS(ESI):[M+H]+found 764.3243,calcd764.3249.1H NMR(400MHz,DMSO-d6)δ(ppm):10.12(s,1H),8.38(d,J＝8.5Hz,1H),8.33–8.31(m,2H),8.29–8.25(m,1H),8.13–8.08(m,1H),7.99(d,J＝8.3Hz,2H),7.65(s,2H),7.59–7.56(m,2H),7.47(d,J＝8.3Hz,2H),7.42(d,J＝8.4Hz,2H),7.22(s,2H),6.88(d,J＝8.7Hz,1H),6.00(m,2H),5.43(s,2H),5.25(s,2H),4.44–4.36(m,2H),3.07–3.02(m,2H),2.97(m,2H),2.81(s,1H),2.16(m,1H),1.72(d,J＝7.2Hz,1H),1.64(m,1H),1.51–1.44(m,2H),1.41(m,2H),0.96(m,6H).
5c的合成
根据图6步骤所示，以4c和(PNP)2CO为起始原料，合成方法同5a，得黄色固体，收率59％,mp:148–152℃。分子式为C36H37N7O11,HRMS(ESI):[M+H]+found 744.2620,calcd744.9420.1H NMR(400MHz,DMSO-d6)δ(ppm):10.09(d,J＝10.6Hz,1H),8.32(d,J＝8.6Hz,1H),8.12–8.06(m,1H),7.72(d,J＝8.9Hz,1H),7.66(d,J＝8.1Hz,2H),7.58(d,J＝8.7Hz,2H),7.42(d,J＝8.1Hz,2H),7.02(s,2H),5.99(d,J＝6.1Hz,1H),5.42(s,2H),5.25(s,1H),4.39(q,J＝7.1Hz,1H),4.17(t,J＝7.7Hz,1H),3.25(d,J＝7.0Hz,2H),3.05–2.94(m,2H),2.23(m,1H),1.99(m,1H),1.72–1.60(m,5H),1.28(m,2H),0.84(m,6H).
5d的合成
根据图6步骤所示，以4d和(PNP)2CO为起始原料，合成方法同5a，得浅黄色固体，收率65％，mp:159–161℃。分子式为C34H39N5O10,HRMS(ESI):[M+H]+found 678.2753,calcd678.2769.1H NMR(500MHz,DMSO-d6)δ(ppm):10.04(s,1H),8.34–8.31(m,2H),8.17(d,J＝6.8Hz,1H),7.73(d,J＝8.6Hz,1H),7.66–7.63(m,2H),7.59–7.56(m,2H),7.43–7.40(m,2H),7.02(s,2H),5.24(s,2H),4.38(t,J＝7.0Hz,1H),4.15(m,1H),3.24(d,J＝7.1Hz,2H),2.23(m,1H),1.97(m,1H),1.75–1.67(m,2H),1.62(m,2H),1.31(m,4H),1.26–1.23(m,3H),0.85(m,7H).
5e的合成
根据图6步骤所示，以4e和(PNP)2CO为起始原料，合成方法同5a，得浅黄色固体，收率54％，mp:155–156℃。分子式为C37H44N6O11,HRMS(ESI):[M+H]+found 749.3128,calcd749.3141.1H NMR(400MHz,DMSO-d6)δ(ppm):10.03(s,1H),8.32(d,J＝8.8Hz,1H),8.21(d,J＝7.0Hz,1H),7.99(d,J＝7.4Hz,1H),7.64(d,J＝8.2Hz,1H),7.58(m,2H),7.42(d,J＝8.1Hz,1H),7.01(s,1H),5.25(s,1H),4.41(m,2H),4.28(m,1H),4.23–4.16(m,1H),3.24(d,J＝7.2Hz,1H),2.60(s,1H),2.21–2.08(m,1H),1.98(m,2H),1.77–1.56(m,4H),1.48(s,1H),1.37–1.22(m,8H),1.19(m,3H),1.13–1.03(m,2H),0.86(m,7H).
5f的合成
根据图6步骤所示，以4f和(PNP)2CO为起始原料，合成方法同5a，得浅黄色固体，收率51％，mp:131–133℃。分子式为C39H40N6O11,HRMS(ESI):[M+H]+found 769.2835,calcd769.2828.1H NMR(600MHz,DMSO-d6)δ(ppm):9.84(d,J＝8.7Hz,1H),8.46(m,1H),8.32(d,J＝8.6Hz,1H),8.15(d,J＝7.7Hz,1H),7.94(t,J＝6.0Hz,1H),7.67(m,2H),7.57(d,J＝8.6Hz,2H),7.42(d,J＝8.5Hz,2H),7.25(d,J＝5.0Hz,4H),7.19(s,1H),7.00(s,1H),5.25(s,1H),4.48(s,1H),3.92(m,1H),3.88–3.83(m,1H),3.73(m,1H),3.56(m,1H),3.22(t,J＝6.7Hz,2H),3.06(m,1H),2.86–2.81(m,1H),2.06(m,1H),1.63(m,4H),1.49(s,1H),1.26–1.18(m,3H),1.09(m,1H),0.87(m,3H).
5g的合成
根据图6步骤所示，以4g和(PNP)2CO为起始原料，合成方法同5a，得浅黄色固体，收率59％，mp:162–164℃。分子式为C41H43N7O12,HRMS(ESI):[M+H]+found 826.3052,calcd826.3043.1H NMR(500MHz,DMSO-d6)δ(ppm):9.95(s,1H),8.43(t,J＝5.9Hz,1H),8.34–8.31(m,2H),8.22(d,J＝8.0Hz,1H),8.03(m,2H),7.69–7.67(m,2H),7.60–7.56(m,2H),7.46–7.42(m,2H),7.27(d,J＝4.4Hz,4H),7.19(m,1H),7.01(s,2H),5.26(s,2H),4.51(m,1H),3.90(m,2H),3.77(m,1H),3.68–3.58(m,3H),3.23(d,J＝7.1Hz,2H),3.08(m,1H),2.84(m,1H),2.11(m,1H),1.78–1.71(m,2H),1.65–1.58(m,2H),1.51(m,1H),1.31–1.21(m,2H),0.93–0.85(m,2H).
5h的合成
根据图6步骤所示，以4h和(PNP)2CO为起始原料，合成方法同5a，得浅黄色固体，收率59％，mp:126–127℃。分子式为C45H51N7O12,HRMS(ESI):[M+H]+found 882.3698,calcd882.3629.1H NMR(400MHz,DMSO-d6)δ(ppm):9.93(s,1H),8.32(d,J＝8.8Hz,2H),8.19(d,J＝7.1Hz,2H),8.00–7.92(m,2H),7.67(d,J＝8.2Hz,2H),7.57(d,J＝8.8Hz,2H),7.43(d,J＝8.2Hz,2H),7.25–7.17(m,5H),7.01(s,2H),5.26(s,2H),4.54(m,1H),4.30(q,J＝7.9,7.5Hz,1H),3.89(d,J＝6.0Hz,2H),3.69(m,1H),3.54(m,1H),3.23(d,J＝7.0Hz,2H),3.04(m,1H),2.80(m,1H),2.08(m,1H),1.70(m,2H),1.60(m,3H),1.53(m,2H),1.23(m,2H),1.10(t,J＝7.0Hz,1H),0.91(m,4H),0.86(m,4H).
5i的合成
根据图6步骤所示，以4i和(PNP)2CO为起始原料，合成方法同5a，得浅黄色固体，收率63％，mp:155–157℃。分子式为C32H37N5O10,HRMS(ESI):[M+H]+found 652.2598,calcd652.2613.1H NMR(400MHz,DMSO-d6)δ(ppm):10.02(s,1H),8.32(d,J＝8.8Hz,2H),8.19(d,J＝6.9Hz,1H),7.82(d,J＝8.6Hz,1H),7.65(d,J＝8.2Hz,2H),7.58(d,J＝9.0Hz,2H),7.42(d,J＝8.2Hz,2H),7.01(s,2H),5.25(s,2H),4.40(m,1H),4.18(t,J＝7.7Hz,1H),2.15(m,2H),1.96(m,1H),1.49(m,4H),1.32(m,4H),1.19(m,2H),0.86(m,7H).
5j的合成
根据图6步骤所示，以4j和(PNP)2CO为起始原料，合成方法同5a，得浅黄色固体，收率60％，mp:134–135℃。分子式为C35H42N6O11,HRMS(ESI):[M+H]+found 723.2980,calcd723.29843.1H NMR(600MHz,DMSO-d6)δ(ppm):10.00(s,1H),8.30–8.27(m,1H),8.15(d,J＝6.8Hz,1H),7.99(d,J＝7.4Hz,1H),7.67–7.60(m,2H),7.54(d,J＝8.7Hz,1H),7.39(m,1H),6.96(m,1H),5.22(m,1H),4.33(m,2H),4.18–4.08(m,1H),3.59(m,1H),3.11(m,1H),2.10–2.03(m,2H),1.96(m,1H),1.44(q,J＝7.6Hz,3H),1.28(d,J＝6.5Hz,2H),1.26–1.20(m,11H),1.15(d,J＝7.5Hz,3H),0.86–0.79(m,5H).
5k的合成
根据图6步骤所示，以4k和(PNP)2CO为起始原料，得浅黄色固体，收率63.2％，mp:138–141℃。分子式为C37H38N6O11,HRMS(ESI):[M+H]+found743.2655,calcd 743.2671.1HNMR(600MHz,DMSO-d6)δ(ppm):9.84(s,1H),8.43(t,J＝5.8Hz,1H),8.30–8.26(m,2H),8.20–8.16(m,1H),8.00–7.96(m,1H),7.66(m,2H),7.54(d,J＝9.0Hz,2H),7.39(d,J＝8.3Hz,2H),7.23(d,J＝4.6Hz,4H),7.16(m,1H),6.95(m,1H),5.22(s,2H),4.47(m,1H),3.90(m,1H),3.87–3.81(m,1H),3.72(m,1H),3.56(m,1H),3.31(d,J＝8.0Hz,2H),3.05(m,1H),2.81(m,1H),2.03(m,2H),1.45–1.37(m,4H),1.13(m,3H).
5l的合成
根据图6步骤所示，以4l和(PNP)2CO为起始原料，得浅黄色固体，收率60％，mp:144–147℃。分子式为C39H41N7O12,HRMS(ESI):[M+H]+found800.2856,calcd 800.2886.1HNMR(500MHz,DMSO-d6)δ(ppm):9.95(s,1H),8.48–8.42(m,1H),8.35–8.30(m,2H),8.22(m,1H),8.13–8.07(m,2H),7.68(d,J＝8.3Hz,2H),7.60–7.56(m,2H),7.44(d,J＝8.6Hz,2H),7.27(d,J＝4.5Hz,4H),7.22–7.17(m,1H),7.01–6.99(m,2H),5.25(s,2H),4.52(m,1H),3.97–3.85(m,2H),3.78(m,1H),3.66(m,3H),3.08(m,1H),2.90–2.82(m,2H),2.11(m,2H),1.47(m,5H),1.19(m,2H).
5m的合成
根据图6步骤所示，以4m和(PNP)2CO为起始原料，得浅黄色固体，收率54％，mp:134–136℃。分子式为C43H49N7O12,HRMS(ESI):[M+H]+found856.3679,calcd 856.3512.1HNMR(400MHz,DMSO-d6)δ(ppm):9.92(s,1H),8.31(d,J＝8.4Hz,2H),8.24–8.12(m,2H),8.02(m,2H),7.67(d,J＝8.0Hz,2H),7.57(d,J＝8.6Hz,2H),7.43(d,J＝8.2Hz,2H),7.23(s,5H),6.99(s,2H),5.25(s,2H),4.54(s,1H),4.29(m,1H),3.98–3.82(m,2H),3.76–3.65(m,1H),3.63–3.50(m,1H),3.05(m,1H),2.79(m,1H),2.07(m,2H),1.68–1.35(m,8H),1.14(m,3H),0.88(m,6H).
M-1的合成
根据图6步骤所示，5a(93mg,0.125mmol)和MMAE(60mg,0.083mmol)溶于4mL DMF中，依次加入HoBt(10mg,0.074mmol)和0.5mL吡啶，室温下搅拌反应过夜。反应产物使用制备液相进行分离纯化。色谱条件如下：A相为H2O+0.08％TFA，B相为乙腈+0.08％TFA，0-35min B相65％等梯度洗脱，收集含有产物纯品的流份，冻干得产物80mg，收率为47％，mp:134–136℃。分子式为C68H105N11O15,HRMS(ESI):[M+H]+found 1316.7885,calcd 1316.7864。HPLC:tR＝5.838min,purity 99.2％(归一化法).1H NMR(500MHz,DMSO-d6)δ(ppm):10.05(s,1H),7.88(d,J＝7.6Hz,2H),7.73(m,2H),7.56(m,2H),7.41(dd,J＝7.6,4.1Hz,2H),7.37–7.21(m,7H),7.16(m,2H),5.16–4.95(m,4H),4.77–4.69(m,1H),4.69–4.59(m,1H),4.55–4.49(m,2H),4.46–4.38(m,2H),4.37–4.17(m,5H),4.09–3.94(m,3H),3.93–3.89(m,1H),3.80–3.75(m,1H),3.34–3.29(m,1H),3.28–3.21(m,4H),3.19(m,3H),3.11(s,2H),3.03(m,2H),2.98–2.95(m,1H),2.96–2.91(m,1H),2.86(m,3H),2.40(m,2H),2.32–2.24(m,2H),2.16–2.04(m,3H),2.04–1.91(m,3H),1.86–1.65(m,5H),1.63–1.43(m,5H),1.41–1.27(m,3H),1.09–0.94(m,6H),0.94–0.69(m,21H).
M-2的合成
根据图6步骤所示，以5b和MMAE为起始原料，合成方法同M-1，得白色固体纯品，收率41％，mp:149–151℃。分子式为C70H107N11O15,HRMS(ESI):[M+H]+found 1342.8050,calcd1342.8021.HPLC:tR＝5.964min,purity 97.2％(归一化法)。1H NMR(600MHz,DMSO-d6)δ(ppm):9.94(d,J＝12.8Hz,1H),8.28–8.24(m,1H),8.00(d,J＝7.5Hz,1H),7.86(d,J＝8.7Hz,1H),7.67(dd,J＝8.7,3.0Hz,1H),7.61–7.59(m,1H),7.54(m,2H),7.33–7.26(m,3H),7.23(m,2H),7.14(m,1H),6.97(d,J＝4.4Hz,2H),6.15–5.95(m,2H),5.08–4.93(m,3H),4.74–4.68(m,1H),4.63–4.57(m,1H),4.46(d,J＝5.9Hz,1H),4.40(m,1H),4.35(m,2H),4.23(m,2H),4.12(m,2H),3.95(m,4H),3.76–3.74(m,1H),3.59–3.49(m,2H),3.4–3.41(m,1H),3.28(d,J＝10.1Hz,1H),3.21(d,J＝8.4Hz,4H),3.16(m,3H),3.09(s,2H),3.04–2.90(m,4H),2.88–2.79(m,3H),2.38(m,1H),2.22(m,2H),2.09(m,2H),1.95(m,2H),1.77(m,2H),1.72–1.62(m,4H),1.61–1.55(m,2H),1.54–1.45(m,3H),1.41–1.39(m,1H),1.34–1.31(m,1H),1.29–1.20(m,3H),1.01(d,J＝6.6Hz,2H),0.98(d,J＝6.6Hz,2H),0.95(d,J＝6.6Hz,1H),0.90(m,2H),0.88–0.66(m,21H).
M-3的合成
根据图6步骤所示，以5c和MMAE为起始原料，合成方法同M-1，得白色固体纯品，收率35％，mp:170–173℃。分子式为C69H99N11O15,HRMS(ESI):[M+H]+found 1322.7418,calcd1322.7395.HPLC:tR＝5.853min,purity 98.7％(归一化法)。1H NMR(600MHz,DMSO-d6)δ(ppm):10.01(d,J＝11.6Hz,1H),8.35(d,J＝8.4Hz,1H),8.23(d,J＝7.5Hz,1H),7.95(d,J＝8.2Hz,2H),7.56(m,2H),7.43(d,J＝8.1Hz,2H),7.33–7.22(m,5H),7.19(s,1H),5.96(s,1H),5.39(s,2H),5.10–4.92(m,3H),4.49–4.33(m,4H),4.27–4.18(m,2H),3.96(m,3H),3.55(m,3H),3.18(m,7H),3.09(s,2H),3.04–2.90(m,4H),2.83(m,3H),2.38(m,1H),2.23(m,1H),2.10(m,3H),1.95(m,2H),1.83–1.65(m,5H),1.59(m,2H),1.55–1.41(m,3H),1.35(m,2H),1.24(m,3H),1.04–0.90(m,12H),0.87–0.71(m,16H).
C-1的合成
根据图6步骤所示，以5d和MMAE为起始原料，合成方法同M-1，得白色固体纯品，收率40％，mp:166–168℃。分子式为C67H101N9O14,HRMS(ESI):[M+H]+found 1256.7564,calcd1256.7541.HPLC:tR＝9.658min,purity 99.9％(归一化法).1H NMR(600MHz,DMSO-d6)δ(ppm):9.89(d,J＝12.3Hz,1H),8.26–8.24(m,1H),8.08(d,J＝6.9Hz,1H),8.01–7.99(m,1H),7.88–7.86(m,1H),7.67(d,J＝8.3Hz,1H),7.61–7.59(m,1H),7.54(d,J＝7.4Hz,2H),7.29(m,3H),7.23(m,2H),7.14(q,J＝7.8Hz,1H),6.97(d,J＝3.5Hz,2H),5.00(m,3H),4.71(m,1H),4.48–4.44(m,1H),4.40(d,J＝6.7Hz,1H),4.35(m,1H),4.23(m,2H),4.11(t,J＝7.7Hz,1H),3.96(m,4H),3.76–3.74(m,1H),3.59–3.49(m,6H),3.45–3.43(m,1H),3.31–3.28(m,1H),3.21(d,J＝8.4Hz,4H),3.16(m,3H),3.09(s,1H),3.01(m,1H),2.94(s,1H),2.85(d,J＝6.4Hz,1H),2.81(d,J＝6.2Hz,1H),2.38(m,1H),2.22(m,2H),2.11–2.05(m,2H),1.93(m,2H),1.82–1.73(m,2H),1.67(m,3H),1.61–1.55(m,2H),1.52–1.45(m,2H),1.26(d,J＝7.4Hz,4H),1.22(m,2H),0.98(m,6H),0.86–0.72(m,21H).
C-2的合成
根据图6步骤所示，以5e和MMAE为起始原料，合成方法同M-1，得白色固体纯品，收率54％，mp:137–139℃。分子式为C70H106N10O15,HRMS(ESI):[M+H]+found 1327.79309,calcd1327.79119.HPLC:tR＝8.952min,purity 99.4％(归一化法).1H NMR(600MHz,DMSO-d6)δ(ppm):9.94(d,J＝11.8Hz,1H),8.29(t,J＝9.5Hz,1H),8.18(d,J＝6.9Hz,1H),8.05(t,J＝7.7Hz,1H),7.97(d,J＝7.4Hz,1H),7.89(d,J＝8.7Hz,1H),7.62(d,J＝8.6Hz,1H),7.59–7.52(m,2H),7.36–7.24(m,5H),7.19–7.14(m,1H),7.01(s,1H),5.14–4.92(m,3H),4.74(s,1H),4.63(s,1H),4.50(m,1H),4.46–4.35(m,2H),4.30–4.23(m,2H),4.17(t,J＝7.5Hz,1H),3.98(m,3H),3.91–3.62(m,9H),3.57(m,2H),3.49–3.45(m,1H),3.38(q,J＝7.1Hz,1H),3.31(m,1H),3.26–3.22(m,4H),3.19(m,3H),3.12(s,1H),3.04(m,1H),2.97(s,1H),2.88(d,J＝7.5Hz,1H),2.84(d,J＝7.2Hz,1H),2.41(m,1H),2.26(m,1H),2.11(m,4H),1.96(m,2H),1.84–1.67(m,5H),1.62(m,2H),1.58–1.45(m,3H),1.27(m,5H),1.18(d,J＝7.1Hz,2H),1.08(s,1H),1.04(d,J＝6.7Hz,2H),1.01(d,J＝6.6Hz,2H),0.98(d,J＝6.6Hz,1H),0.92–0.74(m,19H).
C-3的合成
根据图6步骤所示，以5f和MMAE为起始原料，合成方法同M-1，得白色固体纯品，收率52％，mp:147–148℃。分子式为C72H102N10O15,HRMS(ESI):[M+H]+found 1347.7619,calcd1347.7599.HPLC:tR＝8.957min,purity99.8％(归一化法).1H NMR(600MHz,DMSO-d6)δ(ppm):9.79(d,J＝5.8Hz,1H),8.45(d,J＝5.9Hz,1H),8.31(d,J＝9.0Hz,1H),8.13(d,J＝8.1Hz,1H),8.05(d,J＝9.2Hz,1H),7.93(d,J＝6.1Hz,1H),7.89(d,J＝8.6Hz,1H),7.64–7.57(m,2H),7.31(d,J＝7.8Hz,3H),7.28–7.22(m,5H),7.17(m,2H),7.00(s,1H),5.40(s,1H),5.05(m,3H),4.74(s,1H),4.63(s,1H),4.48(t,J＝7.1Hz,2H),4.42(m,1H),4.27(m,1H),3.99(m,2H),3.87(m,2H),3.72(m,2H),3.56(m,2H),3.51–3.34(m,5H),3.31(m,1H),3.24(s,2H),3.22(d,J＝6.8Hz,3H),3.20(s,1H),3.17(s,1H),3.12(s,1H),3.06(m,2H),2.97(s,1H),2.90–2.79(m,4H),2.41(m,1H),2.26(m,1H),2.15–2.03(m,3H),1.98(m,2H),1.83–1.71(m,3H),1.71–1.65(m,2H),1.61–1.55(m,2H),1.54–1.47(m,2H),1.30(m,1H),1.27–1.17(m,2H),1.09(t,J＝7.0Hz,1H),1.04(d,J＝6.6Hz,1H),0.99(m,4H),0.88(d,J＝6.1Hz,2H),0.81(m,16H).
C-4的合成
根据图6步骤所示，以5g和MMAE为起始原料，合成方法同M-1，得白色固体纯品，收率43％，mp:140–141℃。分子式为C74H105N11O16,HRMS(ESI):[M+H]+found 1404.7839,calcd1404.7814.HPLC:tR＝8.618min,purity99.2％(归一化法).1H NMR(600MHz,DMSO-d6)δ(ppm):9.82(d,J＝5.4Hz,1H),8.35(d,J＝5.9Hz,1H),8.14(d,J＝7.1Hz,1H),7.97(m,2H),7.86(d,J＝8.6Hz,1H),7.58(m,2H),7.28(d,J＝7.6Hz,3H),7.22(d,J＝4.5Hz,5H),7.14(m,2H),6.97(s,2H),5.03(m,3H),4.72(s,1H),4.61(s,1H),4.52–4.35(m,4H),4.24(m,2H),4.03–3.89(m,3H),3.84(m,2H),3.77–3.70(m,2H),3.65–3.51(m,4H),3.44(q,J＝7.8Hz,1H),3.29(m,1H),3.21(d,J＝6.3Hz,5H),3.16(m,3H),3.11–3.01(m,3H),2.95(s,1H),2.86(d,J＝8.8Hz,2H),2.82(m,2H),2.38(m,1H),2.24(m,1H),2.09(m,3H),1.95(m,2H),1.75(m,5H),1.58(m,2H),1.54–1.43(m,3H),1.30–1.18(m,4H),0.98(m,6H),0.90–0.69(m,18H).
C-5的合成
根据图6步骤所示，以5h和MMAE为起始原料，合成方法同M-1，得白色固体纯品，收率40.8％，mp:156-159℃。分子式为C78H113N11O16,HRMS(ESI):[M+H]+found 1460.84619,calcd 1460.84395.HPLC:tR＝9.837min,purity 96.648％(归一化法).1H NMR(500MHz,DMSO-d6)δ(ppm):9.88(d,J＝4.2Hz,1H),8.19(t,J＝8.7Hz,3H),8.02–7.87(m,3H),7.60(d,J＝9.1Hz,3H),7.38–7.13(m,12H),5.04(m,2H),4.69(m,1H),4.51(m,3H),4.34–4.23(m,2H),3.99(m,3H),3.87(d,J＝5.8Hz,6H),3.82–3.62(m,2H),3.53(m,3H),3.31–3.17(m,9H),3.12(s,2H),3.08–2.93(m,3H),2.83(m,4H),2.41(m,1H),2.26(m,1H),2.16–1.89(m,5H),1.85–1.65(m,4H),1.63–1.44(m,8H),1.34–1.18(m,4H),1.01(m,6H),0.90–0.76(m,22H).
C-6的合成
根据图6步骤所示，以5i和MMAE为起始原料，合成方法同M-1，得白色固体纯品，收率43％，mp:130–132℃。分子式为C65H99N9O14,HRMS(ESI):[M+H]+found 1230.7406,calcd1230.7384.HPLC:tR＝9.069min,Purity 97.9％(归一化法).1H NMR(600MHz,DMSO-d6)δ(ppm):9.90(d,J＝11.4Hz,1H),8.27(d,J＝10.1Hz,1H),8.13(d,J＝6.9Hz,1H),8.02(d,J＝8.0Hz,1H),7.86(d,J＝8.7Hz,1H),7.78(d,J＝8.6Hz,1H),7.60(d,J＝8.4Hz,1H),7.54(t,J＝6.0Hz,2H),7.32–7.21(m,5H),7.14(m,1H),6.97(s,1H),5.35(m,1H),5.08–4.94(m,2H),4.74–4.58(m,1H),4.49–4.44(m,1H),4.38(m,2H),4.24(m,1H),4.14(t,J＝7.8Hz,1H),4.01–3.90(m,2H),3.55(m,1H),3.44(q,J＝7.6Hz,1H),3.34(q,J＝7.5,7.0Hz,3H),3.28(s,1H),3.21(m,3H),3.17(s,1H),3.15(s,1H),3.09(s,2H),3.01(m,1H),2.95(s,1H),2.83(m,3H),2.38(m,1H),2.24(m,1H),2.11(m,4H),1.94(m,2H),1.80–1.67(m,3H),1.48(m,6H),1.27(d,J＝7.2Hz,3H),1.21(s,1H),1.15(m,2H),1.06(t,J＝7.4Hz,1H),1.02(d,J＝6.7Hz,2H),0.98(d,J＝6.6Hz,3H),0.95(d,J＝6.7Hz,2H),0.93–0.88(m,1H),0.86(s,1H),0.85–0.70(m,21H).
C-7的合成
根据图6步骤所示，以5j和MMAE为起始原料，合成方法同M-1，得白色固体纯品，收率50％，mp:137–139℃。分子式为C68H104N10O15,HRMS(ESI):[M+H]+found 1301.7769,calcd1301.7756.HPLC:tR＝8.818min,purity 97.7％(归一化法).1H NMR(600MHz,DMSO-d6)δ(ppm):9.97–9.90(m,1H),8.30(d,J＝8.3Hz,1H),8.16(d,J＝6.8Hz,1H),8.08–7.99(m,2H),7.89(d,J＝8.7Hz,1H),7.64(m,2H),7.56(t,J＝6.4Hz,1H),7.36–7.24(m,5H),7.16(m,1H),7.00(s,1H),5.13–4.94(m,3H),4.78–4.71(m,1H),4.63(s,1H),4.53–4.47(m,1H),4.45–4.36(m,2H),4.32(d,J＝7.2Hz,1H),4.26(m,1H),4.18(t,J＝7.5Hz,1H),4.13(t,J＝7.5Hz,1H),3.98(m,2H),3.78(m,1H),3.56(s,9H),3.38(m,3H),3.31(m,1H),3.24(m,3H),3.19(m,3H),3.12(s,1H),3.04(m,1H),2.97(s,1H),2.86(m,3H),2.41(m,1H),2.26(m,1H),2.10(m,4H),1.98(m,2H),1.77(m,3H),1.49(m,5H),1.29(d,J＝6.9Hz,3H),1.18(m,4H),1.09(t,J＝7.0Hz,1H),1.04(m,2H),1.01(d,J＝6.7Hz,2H),0.98(d,J＝6.7Hz,1H),0.91–0.73(m,19H).
C-8的合成
根据图6步骤所示，以5k和MMAE为起始原料，合成方法同M-1，得白色固体纯品，收率40％，mp:126–126℃。分子式为C70H100N10O15,HRMS(ESI):[M+H]+found 1321.7466,calcd1321.7442.HPLC:tR＝8.824min,purity 98.6％(归一化法).1H NMR(600MHz,DMSO-d6)δ(ppm):9.82(s,1H),8.44(s,1H),8.31(s,1H),8.18(s,1H),8.06(s,1H),7.99(s,1H),7.89(d,J＝8.6Hz,1H),7.61(d,J＝9.0Hz,2H),7.36–7.23(m,8H),7.17(m,2H),6.99(s,1H),5.06(m,3H),4.74(m,1H),4.63(s,1H),4.49(d,J＝7.7Hz,2H),4.42(m,1H),4.27(m,1H),3.93(m,5H),3.80–3.70(m,2H),3.62–3.53(m,2H),3.47(s,1H),3.41–3.31(m,3H),3.24(m,3H),3.19(m,2H),3.12(s,2H),3.07(m,2H),2.97(s,1H),2.85(m,4H),2.41(m,1H),2.26(m,1H),2.08(m,5H),1.84–1.68(m,3H),1.59–1.39(m,6H),1.31(s,1H),1.24(s,1H),1.16(t,J＝7.9Hz,2H),1.09(t,J＝7.2Hz,2H),1.01(m,6H),0.82(m,16H).
C-9的合成
根据图6步骤所示，以5l和MMAE为起始原料，合成方法同M-1，得白色固体纯品，收率49％，mp:154–157℃。分子式为C72H103N11O16,HRMS(ESI):[M+H]+found 1378.7682,calcd1378.7657.HPLC:tR＝8.538min,purity 99.7％(归一化法).1H NMR(600MHz,DMSO-d6)δ(ppm):9.84(d,J＝5.5Hz,1H),8.37(t,J＝5.9Hz,1H),8.31(d,J＝9.3Hz,1H),8.14(t,J＝6.7Hz,1H),8.05(t,J＝5.7Hz,1H),8.02(m,1H),7.87(d,J＝8.6Hz,1H),7.63–7.55(m,2H),7.34–7.27(m,3H),7.23(dd,J＝8.0,4.0Hz,5H),7.15(dd,J＝8.8,5.4Hz,2H),6.96(s,1H),5.11–4.93(m,3H),4.74–4.67(m,1H),4.65–4.57(m,1H),4.47(m,2H),4.44–4.37(m,1H),4.24(m,1H),3.97(m,2H),3.91(m,2H),3.84(m,4H),3.74(m,2H),3.65–3.63(m,2H),3.59(d,J＝5.5Hz,1H),3.58–3.52(m,3H),3.33(t,J＝7.1Hz,2H),3.28(m,1H),3.21(m,4H),3.17(s,1H),3.14(s,1H),3.09(s,1H),3.04(m,1H),2.95(s,1H),2.86(m,1H),2.84–2.78(m,2H),2.38(m,1H),2.23(m,1H),2.08(m,3H),2.04–1.90(m,2H),1.81–1.66(m,3H),1.55–1.40(m,6H),1.31–1.24(m,1H),1.21(d,J＝5.0Hz,1H),1.16(m,2H),1.06(t,J＝7.0Hz,1H),1.01(d,J＝6.6Hz,2H),0.98(d,J＝6.6Hz,3H),0.95(d,J＝6.6Hz,1H),0.87–0.71(m,16H).
C-10的合成
根据图6步骤所示，以5m和MMAE为起始原料，合成方法同M-1，得白色固体纯品，收率37％，mp:117–119℃。分子式为C76H111N11O16,HRMS(ESI):[M+H]+found 1434.8298,calcd1434.8283.HPLC:tR＝9.649min,purity 96.4％(归一化法).1H NMR(500MHz,DMSO-d6)δ(ppm):9.88(s,1H),8.21–8.01(m,5H),7.60(d,J＝8.1Hz,3H),7.34–7.15(m,12H),7.01(s,2H),5.04(m,2H),4.79–4.59(m,1H),4.56–4.39(m,3H),4.28(m,2H),3.98(m,3H),3.87(d,J＝5.8Hz,3H),3.79(m,2H),3.70(m,2H),3.61–3.45(m,3H),3.40–3.29(m,3H),3.21(m,8H),3.12(s,2H),3.04(m,2H),2.98(s,1H),2.87(m,3H),2.78(m,1H),2.41(m,1H),2.26(m,1H),2.13–1.96(m,5H),1.83–1.70(m,3H),1.48(m,7H),1.32–1.17(m,3H),1.06–0.98(m,5H),0.84(m,22H).
15a的合成
根据图7步骤所示，以2-CTC resin为固相载体，采用Fmoc保护方法，先将最后一个氨基酸Pro的氨基与树脂进行取代反应上载，顺序连接氨基酸，最后脱除Fmoc，与MC-OSu进行一步缩合，最后用三氟乙酸(TFA)将连接子从树脂上全部裂解上来，乙醚沉淀洗涤，抽滤，收率73％，mp:85–87℃。分子式为C28H43N7O9,HRMS(ESI):[M+H]+found 622.3166,calcd622.3195.1H NMR(400MHz,DMSO-d6)δ(ppm):12.41(s,1H),8.01–7.91(m,2H),7.78(d,J＝8.9Hz,1H),7.00(s,2H),6.00(s,1H),4.30(m,1H),4.24(m,1H),4.17(m,1H),3.80(m,1H),3.50(m,2H),3.37(t,J＝7.1Hz,2H),2.94(t,J＝6.8Hz,2H),2.23–2.04(m,4H),2.01–1.81(m,4H),1.72–1.61(m,2H),1.48(m,5H),1.41–1.33(m,2H),1.18(m,2H),1.09(m,1H),0.81(m,6H).
15b的合成
根据图7步骤所示，以2-CTC resin为固相载体，采用Fmoc保护方法，先将最后一个氨基酸Pro的氨基与树脂进行取代反应上载，顺序连接氨基酸，最后脱除Fmoc，与MC-OSu进行一步缩合，最后用三氟乙酸(TFA)将连接子从树脂上全部裂解上来，乙醚沉淀洗涤，抽滤，收率67％，mp:140–143℃。分子式为C29H45N7O9,HRMS(ESI):[M+H]+found 636.3323,calcd636.3352.1H NMR(400MHz,DMSO-d6)δ(ppm):12.40(s,1H),8.00(d,J＝7.2Hz,1H),7.89(d,J＝7.8Hz,1H),7.80(d,J＝8.8Hz,1H),7.01(s,2H),6.16–5.93(m,1H),4.48(m,1H),4.31–4.11(m,3H),3.62(m,1H),3.50(m,1H),3.37(t,J＝7.2Hz,2H),2.94(s,2H),2.13(m,3H),1.89(m,4H),1.61(m,1H),1.54–1.30(m,7H),1.27–1.06(m,7H),0.81(m,6H).
R-1的合成
根据图7步骤所示，将连接子15a与MMAE进行缩合，得到白色纯品，收率35％，mp:129–131℃。分子式为C67H108N12O15,HRMS(ESI):[M+H]+found1321.8131,calcd1321.8129.HPLC:tR＝7.535min,purity 97.0％(归一化法).1H NMR(600MHz,DMSO-d6)δ(ppm):8.51(d,J＝9.9Hz,1H),7.97–7.81(m,3H),7.77(t,J＝5.9Hz,1H),7.62(d,J＝8.4Hz,1H),7.28(d,J＝7.7Hz,2H),7.24(d,J＝7.7Hz,2H),7.15(t,J＝7.8Hz,1H),6.98(d,J＝4.5Hz,1H),5.86(m,1H),5.44–5.29(m,3H),4.92(m,1H),4.76–4.68(m,1H),4.66–4.55(m,1H),4.49(m,2H),4.40(m,1H),4.26(m,1H),4.15(m,1H),4.09–3.89(m,4H),3.72(m,2H),3.52(m,3H),3.42(m,1H),3.35(d,J＝7.2Hz,3H),3.29(s,1H),3.25–3.14(m,6H),3.10(d,J＝3.6Hz,1H),3.05–2.98(m,2H),2.95(t,J＝8.9Hz,2H),2.89(m,2H),2.79(m,1H),2.42–2.35(m,1H),2.28–2.21(m,1H),2.12(m,5H),1.94(m,3H),1.74(m,4H),1.61(m,2H),1.46(m,7H),1.31(m,3H),1.15(m,2H),1.06(m,2H),0.98(m,5H),0.78(m,21H).
R-2的合成
根据图7步骤所示，将连接子15bH与MMAE进行缩合，得到白色纯品，收率30％，mp:124–125℃。分子式为C68H110N12O15,HRMS(ESI):[M+H]+found1335.8256,calcd1335.8286.HPLC:tR＝7.621min,purity 98.9％(归一化法).1H NMR(600MHz,DMSO-d6)δ(ppm):8.53(d,J＝8.4Hz,1H),7.99(q,J＝6.7Hz,1H),7.94–7.89(m,1H),7.87(d,J＝7.0Hz,1H),7.80(d,J＝8.8Hz,1H),7.65(d,J＝8.4Hz,1H),7.31(t,J＝5.0Hz,2H),7.27(d,J＝7.5Hz,2H),7.18(t,J＝7.6Hz,1H),7.01(s,1H),5.89(d,J＝6.7Hz,1H),5.76(s,1H),5.40(m,3H),4.96(m,1H),4.74(m,1H),4.64(m,1H),4.58–4.38(m,4H),4.27–4.21(m,1H),4.16(m,1H),3.99(m,2H),3.76(m,1H),3.66(m,1H),3.57(m,1H),3.49(m,2H),3.38(d,J＝6.5Hz,3H),3.31(m,1H),3.24(m,3H),3.20(m,2H),3.16(m,2H),3.12(s,1H),3.07–3.01(m,2H),2.97(m,1H),2.92(d,J＝6.5Hz,2H),2.76(m,1H),2.45–2.38(m,1H),2.30–2.23(m,1H),2.22–2.06(m,5H),1.99(m,3H),1.91–1.68(m,5H),1.63(m,2H),1.49(m,7H),1.32(m,3H),1.17(m,4H)
实施例2 Linker-MMAE的体外血浆稳定性检测
待测Linker-MMAE配制成10mM的DMSO储备液，待用。4μL DMSO储备液加入996μL空白大鼠血浆，混合均匀后置于37℃水浴孵育。分别于0、30、60、180、360min各取样50μL，终止反应，每个反应时间点两个平行样品，置于-20℃冻存，待取样完成后统一进行处理。样品进行离心沉降蛋白处理，取上清液，使用LC-MS/MS检测MMAE的浓度，检测浓度单位(nM)。绘制释放MMAE释放量随时间变化的曲线，并计算血浆中MMAE释放速率，释放速率如表1所示。
结果表明，间隔区部分M-2最好(K＝4.0nM/hr)，其次是M-3(K＝10.9nM/hr)和M-1(K＝22.6nM/hr)。马来酰亚胺环N上α位连苯基和β位连环己基与N接链状烷基相比，对Linker-MMAE整体的稳定性有了提高，且β位连接环己基的Linker-MMAE最好。二肽水解位点：M-2(K＝4.0nM/hr)稳定性略好于C-1(K＝8.6nM/hr)。三肽水解位点：C-3(K＝8.77nM/hr)稳定性略好于C-2(K＝13.2nM/hr)。四肽水解位点：C-4(K＝14.7nM/hr)的稳定性好于C-5(K＝97.9nM/hr)，且C-5的稳定性最差。氨基酸数对血浆稳定性的影响不存在一定规律性。自分解部分Linker-MMAE在血浆中的稳定性较好，都好于M-1(K＝22.6nM/hr)，且R-2(K＝1.31nM/hr)好于R-1(K＝8.8nM/hr)。
表1 Linker-MMAE血浆中MMAE释放速率常数
实施例3 Linker-MMAE的酶切释放速率实验
待测Linker-MMAE配制成10mM的DMSO储备液，待用。将10μL储备液加入2490μL100mM L-Cys的PBS缓冲液(pH＝6)，配制成浓度为0.04mM的缓冲工作液。组织蛋白酶B，购买浓度为1.52mM，用pH＝6的PBS缓冲液稀释成0.4mM的蛋白酶溶液。1000μL浓度为0.04mM的化合物缓冲工作液与10μL浓度为0.4mM的蛋白酶溶液混合(底物与蛋白酶摩尔比为1:10)，置于37℃水浴孵育。分别于0、10、60、120、240、360分钟，终止反应，每个反应时间点两个平行样品。使用LC-MS/MS检测MMAE的浓度，检测浓度单位(nM)。
结果如表2所示，M-2水解释放最快(K＝32.3nM/hr)，其次是C-4(K＝32.1nM/hr)和C-5(K＝29.8nM/hr)，C-1(K＝25.0nM/hr)，C-2(K＝12.4nM/hr)和C-3(K＝7.4nM/hr)。自分解型Linker-MMAE经过组织蛋白酶B孵育后未检测到MMAE信号，表明R-1和R-2经水解不能完全释放出MMAE。
表2 Linker-MMAE在组织蛋白酶B中的MMAE释放速率
实施例4SCT-200-Linker-MMAE ADCs的制备
1)将Linker-MMAE(即连接子-细胞毒药物)溶于二甲基亚砜，得到连接子-细胞毒药物储备液；
2)将还原剂TCEP溶于缓冲液中，配置还原剂储备液；
3)将L-Cys溶于缓冲液中，制备L-Cys储备液；
4)将SCT-200抗体加入步骤2)所述还原剂储备液，氮气保护下37℃孵育1.5h；还原剂的摩尔量为抗体摩尔量的3倍；
5)将步骤4)所述反应液中加入步骤1)的连接子-细胞毒药物储备液，氮气保护下37℃孵育1-2.5h，制备SCT-200抗体抗人EGFR抗体药物偶联物；连接子-细胞毒药物的摩尔量为抗体摩尔量的4-8倍；
6)将步骤5)得到的反应液中加入步骤3)的储备液，停止反应。
7)将得到的抗体偶联药物SCT-200-Linker-MMAE进一步纯化的步骤，优选AKTApurifier蛋白质纯化系统，收集所需抗体偶联药物组分峰；收集完毕后，使用30kDa超滤管进行超滤离心，浓缩，经无菌滤膜过滤，低温储存。
实施例5 SCT-200-Linker-MMAE ADCs的HIC-HPLC检测
疏水层析液相色谱(HIC-HPLC)检测
色谱条件如下：
色谱系统：安捷伦1200
色谱柱：TSKgel Butul-NPR column(TOSOH),2.5μm；4.6×36mm
流动相：
A:1.5mol/L硫酸铵和25mmol/L磷酸钠水溶液(pH＝6.95)
B：75％(V/V)25mmol/L(pH＝6.95)的磷酸钠水溶液和25％(V/V)的异丙醇混合溶液
流速：0.5mL/min；进样体积：8μL；分析时间：17min；柱温：25℃；检测波长：280nm和248nm；
梯度：A相100％→0％0-15min,A相100％15.01min-17.00min，加权如表3所示，平均DAR(药物抗体偶联比率)用峰面积百分比和偶联药物个数来计算，所得图谱如图8，图9和图10所示。
表3 SCT-200-Linker-MMAE ADCs各组分比例及平均DAR
实施例6 SCT-200-Linker-MMAE ADCs的ESI-MS检测
取ADC或裸抗样品100ug，加入PNGaseF 500U 1ul，37℃酶切过夜，获得待测样品。
液质联用参数设置：
液相系统采用ACQUITY UPLC H-Class(Waters),色谱柱为ACQUITY UPLCProtein,BEH SEC,
1.7μm,2.1mm×100mm，柱温25℃，流动相为100mM乙酸铵水溶液，等度洗脱，流速为0.1ml/min。质谱检测系统为：Xevo G2-XS Qtof(Waters)，检测模式为正离子，全扫描方式。
数据采集采用MassLynx 4.1(Waters)软件，并经过MaxEnt I去卷积处理。模型参数设置分辨率为2-3.5Da。最小强度比率设置60％，输出分辨率设置为1Da。算法迭代参数设置为20，结果如图11所示。
实施例7 SCT-200-Linker-MMAE ADCs的亲和活性检测
包被抗原：用PBS稀释抗原至2μg/mL，每孔加入50μL包被，保鲜膜包好4℃过夜。洗板：板内液体甩干，加入PBST 200μL/孔，洗3次，每次摇3-5min。封闭：每孔加入1％的BSA，200μL/孔，4℃过夜。洗板：板内液体甩干，加入PBST 200μL/孔，洗3次，每次摇3-5min。加入待测样品，PBST做稀释液，每孔加入50μL，每个浓度平行3次，37℃孵育1h。洗板：板内液体甩干，加入PBST 200μL/孔，洗3次，每次摇3-5min，拍干ELISA板子。孵二抗：每孔加50μL二抗(1：5000稀释)，37℃孵育1h。洗板：板内液体甩干，加入PBST 200μL/孔，洗3次，每次摇3-5min，拍干ELISA板子。每孔加TMB显色液(避光反应)100μL，室温避光显色15-20min。终止反应：每孔加入2M的H2SO4 100μL，由蓝色变为黄色，终止反应。酶标仪在405nM波长测定OD值，加终止液后立即进行检测。
结果显示，结果如图12所示，裸抗体SCT-200和ADCs与抗原亲和力呈现浓度依赖，说明基于半胱氨酸偶联的方法制备的SCT200-Linker-MMAE ADCs的亲和力与抗体SCT-200的亲和力相近，基本保持原有抗原抗体的亲和力。抗原抗体之间的亲和力来于ADC抗体本身，连接子的改变对其基本不产生影响。
实施例8 SCT-200-Linker-MMAE ADCs的内吞活性检测
收集细胞：KYSE520细胞用胰酶消化，4℃离心5min沉淀细胞，细胞重悬浓度为1×107/mL。将细胞50μL/管分装，每组平行3个样品。一抗结合：使用FACS染色缓冲液稀释一抗样品，使一抗终浓度为20μg/mL。冰浴孵育1h后，用FACS染色缓冲液洗涤细胞，4℃离心5min去上清。抗体内化：每个离心管加入200μL FACS染色缓冲液，在37℃孵育2h，同时设置4℃的样品管作为阴性对照。加入2mL冰浴FACS染色缓冲液终止内化。标记二抗：使用Alexa Fluor488标记的羊抗人IgG(H+L)二抗，4℃避光孵育30min。加入FACS染色缓冲液洗涤细胞，4℃离心5min沉淀细胞去上清，重复洗涤两次。将处理好的细胞重悬于500μL冰浴PBS，流式细胞仪检测荧光强度。
流式细胞法检测SCT200-Linker-MMAE ADCs的内化速率，测定了KYSE520细胞中各个ADCs的内化速率。分别将ADCs在4℃孵育30min和37℃孵育2h，细胞表面结合抗体的内化水平通过计算37℃孵育样品相对于4℃孵育对照品的平均荧光强度(MFI)降低水平得出。
t时间点的％MFI＝37℃孵育样品的MFI×100/4℃孵育样品的MFI；
t时间点的内化百分比＝100-t时间点％MFI
结果如图13和表4所示，连接子的改变对于内吞速率没有明显的影响。
表4 SCT200-Linker-MMAE ADCs的内化速率
实施例9 Western Blot实验检测肿瘤细胞EGFR表达水平
(1)样品制备和细胞蛋白抽提，细胞在直径60mm细胞培养皿中生长至密度80％-90％，PBS清洗细胞两次后，加入150μL含有蛋白酶抑制剂和磷酸酶抑制剂的蛋白裂解液，刮下细胞，4℃、12000rpm离心20min，离心完成后取上清。
(2)BCA法蛋白定量后用蛋白裂解液调整蛋白浓度，加入5×上样缓冲液，99℃水浴锅煮10min后，-20℃保存。
(3)Western Blot实验
上样：待检测蛋白样品上样，蛋白上样量为30μg/孔。两侧分别加入6μL和3μL蛋白Marker；电泳：通过预染蛋白marker和溴酚蓝的位置来确定电泳停止时间，约70min；湿转法转膜：转膜条件为390mA恒流70min，使用0.45μm孔径的PVDF膜；封闭：将转好蛋白的膜完全浸在5％脱脂牛奶-TBST中室温轻摇1h；一抗孵育：用5％脱脂牛奶-TBST稀释一抗，EGFR和β-actin抗体都按照1：1000稀释，在4℃孵育过夜；洗膜：回收一抗，-20℃保存。在TBST中洗膜5次，每次5min；二抗孵育：用5％脱脂奶粉-TBST稀释二抗，山羊抗兔IgG(H+L)和山羊抗小鼠IgG(H+L)抗体按照1:10000稀释，室温轻摇2h。洗膜：TBST洗膜5次，每次5min。曝光：将ECL发光液加至膜上后，避光反应30s-1min后曝光条带。
实验结果如图14所示，A431,FaDu,AsPc-1,HCC827,KYSE520,KYSR450为EGFR表达细胞株，Raji和Daudi为EGFR不表达细胞株。
实施例10 SCT-200-Linker-MMAE ADCs的体外抗肿瘤活性测定
取对数生长期的细胞离心重悬计数，并以1×104至3×104/孔接种于96孔中，37℃培养2h。然后，加入不同浓度的LR004-VC-MMAE(LR004和MMAE作为对照品)，每个药物浓度设置3个平行孔。37℃孵育72h后，每孔加入20μL CCK8试剂继续培养1-2h。观察颜色反应，并用酶标仪测定450nm处的吸光值。实验过程中分别设置空白组(不含细胞)及阴性对照组(无药物处理)，按下列公式计算细胞的存活率：细胞存活率＝(加药组A450值-空白组A450值)/(对照组A450值-空白组A450值)×100％。IC50值计算使用SPSS软件。
结果表明，如表5和表6所示，在细胞实验中，SCT-200-Linker-MMAE ADCs体现了较强的抗肿瘤活性，且对EGFR不表达细胞株没有抗肿瘤作用，展现了其靶向选择性。
表5 SCT200-Linker-MMAE ADC四株肿瘤细胞抗肿瘤活性(IC50,nM)
表6 SCT200-Linker-MMAE ADC另外四株肿瘤细胞抗肿瘤活性(IC50,nM)
实施例11 SCT-200-Linker-MMAE ADCs对肿瘤凋亡过程中相关蛋白表达的影响
检测SCT200-Linker-MMAE ADCs对肿瘤凋亡相关蛋白的表达的影响，根据上述实验结果选择了SCT200-M-2,SCT200-C-2和SCT200-C-4进行凋亡蛋白相关性研究。将A431细胞分别经0.01,0.1和1μg/mL的上述ADCs、1μg/mL SCT200和1nM MMAE处理24h，收集样品经Western Blot检测。
在细胞周期中，p53的调节功能主要体现在G1和G2/M期，与转录激活作用密切相关，结果如图15所示，在所有被检测样品中，MMAE与ADCs对p-p53均有上调作用；当DNA受到损伤时，可以使caspase-3活化(cleaved-caspase-3)和PARP失活，MMAE(1nM)可活化caspase-3，SCT200(1μg/mL)活化效果不明显，但是ADCs可以明显上调cleaved-caspase-3，并且呈浓度依赖；PARP为DNA损伤修复酶，当细胞凋亡时导致PARP失活，被cleaved-caspase-3剪切为cleaved-PARP。cleaved-PARP表达水平有所不同，control和SCT200条带不明显，MMAE的条带较浅，而ADCs 1μg/mL可以明显上调cleaved-PARP表达水平，并且呈浓度依赖。结果表明，ADCs可以通过P-P53,caspase-3和cleaved-PARP依赖的凋亡通路诱导细胞凋亡。
实施例12 SCT-200-Linker-MMAE ADCs对A431裸鼠移植瘤模型的药效学评价
6-8周BALB/c裸鼠右侧腋下皮下注射5×106/100μL的A431细胞。当肿瘤长至第七天时，裸鼠肿瘤体积约为110mm3，随机分为5组，分别为对照组，SCT-200(12mg/kg)，SCT-200-M-1(4mg/kg)，SCT-200-M-2(4mg/kg),SCT-200-C-2(4mg/kg)和SCT-200-C-4(4mg/kg)每组6只裸鼠，每隔3天尾静脉给药一次，共给药4次，肿瘤抑制曲线如图16所示。结果显示，四组ADC组均以4mg/kg的剂量给药显示出了较强的抑瘤效果，与对照组和SCT-200相比具有显著性差异(P<0.01)。另外，SCT200-M-2(4mg/kg),SCT200-C-2(4mg/kg)和SCT200-C-1(4mg/kg)三组与SCT200-M-1(4mg/kg)组抗肿瘤效果较强，且抗肿瘤效果相当，没有显著性差异。
Claims (10)
1.一类抗人EGFR抗体药物偶联物，其特征在于，所述的抗人EGFR抗体药物偶联物具有式I所示结构：
其中，
所述的抗体为抗人EGFR抗体，z＝2～8；
M为能与抗体半胱氨酸巯基反应的间隔区部分，其结构为以下所示结构中的一种:
其中，n＝0,1,2,3,4或5，m＝1～10,12或24；
C为能够在酶的作用下发生水解的部分，选自二肽序列Val-Cit,Val-Ala,Val-Lys或Val-Arg；或三肽序列Ala-Val-Ala,Gly-Phe-Gly,Gly-Phe-Lys或Ala-Phe-Lys；或四肽序列Gly-Gly-Phe-Gly或Gly-Phe-Leu-Gly中的一种；
R为自分解部分，其结构为以下所示结构中的一种:
2.如权利要求1所述的一类抗人EGFR抗体药物偶联物，其特征在于，所述的抗体为SCT-200单克隆抗体。
3.一种制备权利要求1所述的抗人EGFR抗体偶联药物的方法，其特征在于，包括：将具有式II结构的Linker-MMAE与抗人EGFR抗体的链间二硫键发生加成反应制得所述的抗人EGFR抗体药物偶联物；
4.如权利要求3所述的方法，其特征在于，包括以下步骤：
(1)将Linker-MMAE溶于二甲基亚砜，得到连接子-细胞毒药物储备液；
(2)将还原剂溶于缓冲液中，配置还原剂储备液；
(3)将L-Cys溶于缓冲液中，制备L-Cys储备液；
(4)将抗人EGFR抗体加入步骤(2)所述的还原剂储备液中，孵育1-2h；
(5)向步骤(4)得到的反应液中加入步骤(1)的连接子-细胞毒药物储备液，孵育1-2h，制备抗人EGFR抗体药物偶联物；
(6)向步骤(5)得到的反应液中加入步骤(3)的L-Cys储备液，停止反应。
5.根据权利要求4所述的方法，其特征在于，所述步骤(2)中还原剂为TCEP，混合时以抗人EGFR抗体的摩尔量为基准，还原剂的摩尔量为抗体摩尔量的2-4倍。
6.根据权利要求34所述的方法，其特征在于，所述步骤(5)中混合时以抗人EGFRSCT-200抗体的摩尔量为基准，所加Linker-MMAE连接子-细胞毒药物的摩尔量为抗体摩尔量的4-8倍。
7.根据权利要求4所述的方法，其特征在于，在所述步骤(4)和步骤(5)中的反应在氮气保护下进行，步骤(4)中的反应温度为35～40℃，优选为37℃。
8.根据权利要求4所述的方法，其特征在于，还包括将得到的抗体药物偶联物进一步纯化的步骤，优选采用AKTA purifier蛋白质纯化系统，收集所需抗体偶联药物组分峰；收集完毕后，使用30kDa超滤管进行超滤离心，浓缩，经无菌滤膜过滤，低温储存。
9.权利要求1或2所述的抗体药物偶联物在制备用于肿瘤靶向治疗的药物中的用途，其中，所述的肿瘤为EGFR阳性实体肿瘤，包括鳞状上皮细胞癌，食管癌，鼻咽癌，肺癌，乳腺癌，胰腺癌，前列腺癌，头颈癌，结肠癌等。
10.以权利要求1或2所述的抗体药物偶联物为活性成分的用于肿瘤靶向治疗的药物组合物，其特征在于，含有药学上有效量的权利要求1或2所述的抗体药物偶联物及药学上允许的佐剂。
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