CN117624167A - 吡咯并苯并二氮杂卓-蒽酰亚胺杂交分子及其制备方法和用途 - Google Patents
吡咯并苯并二氮杂卓-蒽酰亚胺杂交分子及其制备方法和用途 Download PDFInfo
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- CN117624167A CN117624167A CN202210991701.0A CN202210991701A CN117624167A CN 117624167 A CN117624167 A CN 117624167A CN 202210991701 A CN202210991701 A CN 202210991701A CN 117624167 A CN117624167 A CN 117624167A
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- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 163
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical group OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 112
- 238000006243 chemical reaction Methods 0.000 claims description 91
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 84
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 71
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- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 26
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 25
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- -1 amino-substituted phenyl Chemical group 0.000 claims description 13
- 229910000160 potassium phosphate Inorganic materials 0.000 claims description 13
- 235000011009 potassium phosphates Nutrition 0.000 claims description 13
- AOSZTAHDEDLTLQ-AZKQZHLXSA-N (1S,2S,4R,8S,9S,11S,12R,13S,19S)-6-[(3-chlorophenyl)methyl]-12,19-difluoro-11-hydroxy-8-(2-hydroxyacetyl)-9,13-dimethyl-6-azapentacyclo[10.8.0.02,9.04,8.013,18]icosa-14,17-dien-16-one Chemical compound C([C@@H]1C[C@H]2[C@H]3[C@]([C@]4(C=CC(=O)C=C4[C@@H](F)C3)C)(F)[C@@H](O)C[C@@]2([C@@]1(C1)C(=O)CO)C)N1CC1=CC=CC(Cl)=C1 AOSZTAHDEDLTLQ-AZKQZHLXSA-N 0.000 claims description 12
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- FPGGTKZVZWFYPV-UHFFFAOYSA-M tetrabutylammonium fluoride Chemical compound [F-].CCCC[N+](CCCC)(CCCC)CCCC FPGGTKZVZWFYPV-UHFFFAOYSA-M 0.000 claims description 6
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Abstract
本发明公开了一种吡咯并苯并二氮杂卓‑蒽酰亚胺杂交分子及其制备方法和用途,属于化学医药技术领域。本发明为了解决现有抗体药物偶联物的弹头种类偏少,作用机制单一,长期使用容易产生耐药性的问题,提供了式I所示吡咯并苯并二氮杂卓‑蒽酰亚胺杂交分子及其制备方法和用途。实验结果表明,本发明化合物在卵巢癌细胞、胃癌细胞和乳腺癌细胞等多种肿瘤细胞中均展现出很强的抗增殖活性,并且其抗体药物偶联物具有良好的体内外抗肿瘤活性,安全性良好。
Description
技术领域
本发明属于化学医药技术领域,具体涉及一种作为抗肿瘤药用的吡咯并苯并二氮杂卓-蒽酰亚胺杂交分子及其抗体药物偶联物。
背景技术
抗体药物偶联物(Antibody-Drug Conjugates,ADCs)是一类新型肿瘤靶向治疗药物,由单克隆抗体(Antibody)、连接子/接头(Linker)和弹头(Warhead/Payload)三部分组成,其结合了抗体的高选择性和细胞毒药物高活性的优点,已成为肿瘤靶向治疗的研究热点。经过数十年的发展,ADCs的研发已取得了诸多成果,截止到2021年12月,已有12个ADCs药物被批准上市,150多个ADCs进入临床试验(参见Esnault,C.,Schrama,D.,Houben,R.,Guyétant,S.,Desgranges,A.,Martin,C.,...&Samimi,M.(2022).Antibody–DrugConjugates as an Emerging Therapy in Oncodermatology.Cancers,14(3),778.)。
弹头作为ADCs的关键组分,直接影响ADCs的治疗效果,已经成功上市和进入临床试验的ADCs,其弹头大多是微管蛋白抑制剂和DNA损伤剂,如卡奇霉素(Calicheamicins)、美登素(Maytansinoids)、澳瑞他汀(Auristatins)、吡咯并苯并二氮杂卓二聚体(PBDdimers)和喜树碱类(Camptothecins)等。虽然将这些弹头应用于ADCs获得了不错的效果,但这些弹头作用机制单一,长期使用容易产生耐药性,并且ADC的弹头种类偏少,大部分弹头结构复杂,合成难度大,费时费力。因此,开发新的ADC弹头具有重要意义。而使用“分子杂交技术”开发符合弹头要求的杂交分子,是一个很好的拓宽弹头种类的策略。杂交分子是由两种或者两种以上活性分子组合形成的单分子新化学实体,相比传统弹头,其母体结构相对简单,合成难度较低,也更容易设计出多靶点药物(参见Decker,M.(Ed.).(2017).Designof hybrid molecules for drug development.Elsevier.)。
吡咯并苯并二氮杂卓(Pyrrolobenzodiazepines,PBDs)是一类强效的DNA小沟结合剂(参见Mantaj,J.,Jackson,P.J.,Rahman,K.M.,&Thurston,D.E.(2017).Fromanthramycin to pyrrolobenzodiazepine(PBD)-containing antibody–drug conjugates(ADCs).Angewandte Chemie International Edition,56(2),462-488.)。PBD二聚体如SGD-1882、SG3199和Indolinobenzodiazepine等因其高细胞毒性已被用作ADCs的弹头,其中以SG3199为弹头的ADC药物ADCT-402已上市,其适应症为复发性或难治性弥漫性大B细胞淋巴瘤(参见Mullard,A(2021).FDA approves ADC Therapeutics'loncastuximabtesirine,ushering in a new cytotoxic payload.Nature reviews Drug discovery,20(6):414.)。除此之外,PBD单体和其他抗癌小分子“杂交”得到的杂交分子如PBD-CBI和PBD-BIA也开始被用作ADCs的弹头,这说明将PBD杂交分子用作ADCs的弹头具有良好的前景(参见Giddens,A.C.,Lee,H.H.,Lu,G.L.,Miller,C.K.,Guo,J.,Phillips,G.D.L.,...&Tercel,M.(2016).Analogues of DNA minor groove cross-linking agentsincorporating aminoCBI,an amino derivative of the duocarmycins:Synthesis,cytotoxicity,and potential as payloads for antibody–drugconjugates.Bioorganic&Medicinal Chemistry,24(22),6075-6081.和Reid,E.E.,Archer,K.E.,Shizuka,M.,McShea,M.A.,Maloney,E.K.,Ab,O.,...&Miller,M.L.(2019).Design,synthesis and evaluation of novel,potent DNA alkylating agents andtheir antibody-drug conjugates(ADCs).Bioorganic&Medicinal Chemistry Letters,29(17),2455-2458.)。
萘酰亚胺(Naphthalimides)是一类多环酰胺,其是DNA嵌入剂,可通过非共价作用嵌入DNA的碱基,从而杀伤肿瘤细胞(参见Brana,M.F.,&Ramos,A.(2001).Naphthalimidesas anticancer agents:synthesis and biological activity.Current MedicinalChemistry-Anti-Cancer Agents,1(3),237-255.)。已有Mitonafide、Amonafide、Elinafide和Bisnafide曾进入临床阶段(I/II期),但该类化合物存在骨髓抑制毒性,导致临床应用受限(参见Kamal,A.,Bolla,N.R.,Srikanth,P.S.,&Srivastava,A.K.(2013).Naphthalimide derivatives with therapeutic characteristics:A patentreview.Expert opinion on therapeutic patents,23(3),299-317.)。经结构优化后,又开发出活性更强的衍生物蒽酰亚胺(Anthracenecarbonamide)——Azonafide,VelosBio公司以其为弹头,开发了靶向类受体酪氨酸激酶孤儿受体1(ROR1)的ADC药物,目前处于临床前研究阶段(参见Sami,S.M.,Dorr,R.T.,Alberts,D.S.,&Remers,W.A.(1993).2-Substituted 1,2-dihydro-3H-dibenz[de,h]isoquinoline-1,3-diones.A new class ofantitumor agent.Journal of medicinal chemistry,36(6),765-770.和US20180369406)。
发明内容
本发明的目的在于提供吡咯并苯并二氮杂卓-蒽酰亚胺杂交分子的制备方法及其用途,以解决现有的ADC弹头种类偏少,作用机制单一,长期使用容易产生耐药性的问题。
本发明提供了式I所示化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体或其药学上可接受的盐,式I结构如下:
其中,Z选自l、m、n独立地选自1~8的整数;/>中l端与N相连,m端与O相连;
R1选自C1~C8烷基、取代或未取代的6~10元芳基;其中,所述取代的6~10元芳基的取代基选自C1~C6烷基、C1~C6烷氧基或氨基;
R2选自-H、C1~C8烷氧基或C1~C8烷巯基;
R3选自C1~C6烷基。
其中,上述化合物中,l、m、n独立地选自1~6的整数。优选的,上述化合物中,l选自1~3的整数,m选自2~6的整数,n选自2~6的整数。最优选的,上述化合物中,l为2,m选自3~5的整数,n选自3~5的整数。
其中,上述化合物中,R1选自C1~C6烷基、取代或未取代的6~10元芳基;其中,所述取代的6~10元芳基的取代基选自C1~C4烷基、C1~C4烷氧基或氨基。
优选的,上述化合物中,R1选自C1~C4烷基、取代或未取代的6元芳基;其中,所述取代的6元芳基的取代基选自C1~C4烷基、C1~C4烷氧基或氨基。
最优选的,上述化合物中,R1选自甲基、对氨基苯基或对甲氧基苯基。
其中,上述化合物中,R2选自-H、C1~C6烷氧基或C1~C6烷巯基。优选的,上述化合物中,R2选自-H、C1~C4烷氧基或C1~C4烷巯基。更优选的,上述化合物中,R2选自-H、甲氧基或甲巯基。最优选的,上述化合物中,R2为-H。
其中,上述化合物中,R3选自C1~C4烷基。优选的,上述化合物中,R3为甲基。
本发明还提供了式I所述的一些具体化合物,如下:
在式I化合物的基础上,本发明还提供了一类式II所示药物-连接子化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体或其药学上可接受的盐,其结构如下:
其中,L选自o、q独立地选自1~10的整数,p选自1~15的整数;R2如上述式I中所述;R3如上述式I中所述;Z如上述式I中所述。
其中,上述化合物中,o、q独立地选自1~8的整数,p选自2~10的整数。优选的,上述化合物中,o、q独立地选自1~7的整数,p选自6~8的整数。最优选的,上述化合物中,o为5,q为2,p为7。
其中,上述化合物中,与苯对位相连。
其中,上述化合物中,Z为l、m如上述式I中所述。
本发明还提供了式II所述的一些具体化合物,如下:
本发明还提供了上述式I或式II所述化合物的制备方法,其合成路线包括如下步骤:
a、化合物1经Suzuki偶联反应,得到化合物2:
b、化合物2经Zn粉/AcOH体系还原,得到化合物3:
c、化合物3在碱性条件下与Alloc-Cl(氯甲酸烯丙酯)反应,得化合物4:
其中,当R1不含氨基时,化合物4的R、为R1,当R1含氨基时,化合物4的R、为单N-Alloc取代的R1;
d、化合物4在酸性条件下脱去TBS(叔丁基二甲基硅基)保护基,得到化合物5:
e、化合物5经Swern氧化后关环得到化合物6:
f、化合物6在碱性条件下与TBS-OTf反应得到化合物7:
g、化合物7与LiOAc反应脱去TIPS(三异丙基硅基),得到化合物8:
h、化合物8与二碘代烷在碱性条件下反应得到化合物9:
i、化合物10与溴代烷在碱性条件下反应得到化合物11:
j、化合物11与化合物12在碱性条件下反应得到化合物13:
k、化合物13在TFA作用下脱去Boc得到化合物14:
l、化合物9和化合物12在碱性条件下反应得到化合物15:
m、化合物15在TBAF/AcOH体系下脱去TBS得到化合物16:
n、化合物16在Pd催化剂作用下脱去Alloc保护基得到产物17:
o、化合物9和化合物14在碱性条件下反应得到化合物18:
p、化合物18在TBAF/AcOH体系下脱去TBS得到化合物19:
q、化合物19在Pd催化剂作用下脱去Alloc保护基得到产物20:
r、当式I中R1为氨基取代的苯基时,化合物17或20与连接子L-H经酰胺缩合得到式II:
上述方法,步骤a中化合物1、R1-硼酸、碱和Pd催化剂在保护气氛下反应。
上述制备方法,步骤a中,化合物1与R1-硼酸的摩尔比为1:(1.0~5.0)。优选地,步骤a中,化合物1与R1-硼酸的摩尔比为1:4.0。
上述制备方法,步骤a中,化合物1与碱的摩尔比为1:(1.0~8)。优选地,步骤a中,化合物1与碱的摩尔比为1:6.0。
上述制备方法,步骤a中,化合物1与钯催化剂的摩尔比为1:0.02~0.05。优选地,步骤a中,化合物1与钯催化剂的摩尔比为1:0.05。
上述制备方法,步骤a中,Pd催化剂选自Pd(dppf)Cl2·CH2Cl2、PdCl2(PPh3)2、Pd[P(Ph)3]4、Pd(OAc)2中至少一种。上述制备方法,步骤a中,反应溶剂选自丙酮、乙腈、四氢呋喃、甲苯、DMF、1,2-二甲氧基乙烷、水中至少一种。上述制备方法,步骤a中,碱选自磷酸钾、碳酸钾、碳酸钠、氢化钠、氢氧化钡、碳酸铯中的至少一种。上述制备方法,步骤a中,反应温度为25℃。上述制备方法,步骤a中,反应在氮气保护下进行。
上述制备方法,步骤b中,反应溶剂选自甲醇或乙醇。
上述制备方法,步骤c中,碱为吡啶。
上述制备方法,步骤d中,酸为醋酸水溶液,反应溶剂选自甲醇、四氢呋喃、甲醇:四氢呋喃体积比1:1的混合物。
上述方法,步骤e中,化合物5、DMSO、草酰氯和碱,在保护气氛下反应。
上述制备方法,步骤e中,化合物5与草酰氯的摩尔比为1:(1.0~4.0)。优选地,步骤e中,化合物5与草酰氯的摩尔比为1:1.3。
上述制备方法,步骤e中,化合物5与DMSO的摩尔比为1:(1.0~8.0)。优选地,步骤e中,化合物5与DMSO的摩尔比为1:2.6。
上述制备方法,步骤e中,化合物5与碱的摩尔比为1:(1.0~8.0)。优选地,步骤e中,化合物5与碱的摩尔比为1:5.0。
上述制备方法,步骤e中,碱选自二异丙基乙胺、三乙胺中一种或两种。上述制备方法,步骤e中,反应溶剂选自二氯甲烷、氯仿、四氢呋喃中的至少一种。上述制备方法,步骤e中,反应温度为-78℃。上述制备方法,步骤e中,反应在氮气保护下进行。
上述制备方法,步骤f中,碱为2,6-二甲基吡啶。
上述制备方法,步骤g中,反应溶剂为N,N-二甲基甲酰胺和水的混合溶液。
上述制备方法,步骤h中,化合物8、二碘代烷和碱,在保护气氛下反应。
上述制备方法,步骤h中,化合物8与二碘代烷的摩尔比为1:(1.0~6.0)。优选地,步骤h中,化合物8与二碘代烷的摩尔比为1:5.0。
上述制备方法,步骤h中,化合物8与碱的摩尔比为1:(1.0~4.0)。优选地,步骤h中,化合物8与碱的摩尔比为1:1.3。
上述制备方法,步骤h中,碱选自二异丙基乙胺、三乙胺、磷酸钾、碳酸钾、碳酸钠、碳酸铯中至少一种。上述制备方法,步骤h中,反应溶剂选自N,N-二甲基甲酰胺、N,N-二甲基乙酰胺、2-丁酮、甲苯中至少一种。上述制备方法,步骤h中,反应温度为75℃。上述制备方法,步骤h中,反应在氮气保护下进行。
上述制备方法,步骤i中,化合物10、二溴代烷和碱,在保护气氛下反应。
上述制备方法,步骤i中,化合物10与二溴代烷的摩尔比为1:(1.0~6.0)。优选地,步骤i中,化合物10与二溴代烷的摩尔比为1:4.3。
上述制备方法,步骤i中,化合物10与碱的摩尔比为1:(1.0~4.0)。优选地,步骤i中,化合物10与碱的摩尔比为1:1.1。
上述制备方法,步骤i中,碱选自二异丙基乙胺、三乙胺、磷酸钾、碳酸钾、碳酸钠、碳酸铯中的至少一种。上述制备方法,步骤i中,反应溶剂选自N,N-二甲基甲酰胺、N,N-二甲基乙酰胺、2-丁酮、二氯甲烷、甲苯中的至少一种。上述制备方法,步骤i中,反应温度为45℃。上述制备方法,步骤i中,反应在氮气保护下进行。
上述制备方法,步骤j中,化合物11、化合物12和碱,在保护气氛下反应。
上述制备方法,步骤j中,化合物11与化合物12的摩尔比为1:(1.0~3.0)。优选地,步骤j中,化合物11与化合物12的摩尔比为1:1.0。
上述制备方法,步骤j中,化合物11与碱的摩尔比为1:(1.0~4.0)。优选地,步骤j中,化合物11与碱的摩尔比为1:1.5。
上述制备方法,步骤j中,碱选自二异丙基乙胺、三乙胺、磷酸钾、碳酸钾、碳酸钠、碳酸铯中的至少一种。上述制备方法,步骤j中,反应溶剂选自N,N-二甲基甲酰胺、N,N-二甲基乙酰胺、2-丁酮、甲苯中的至少一种。上述制备方法,步骤j中,反应温度为85℃。上述制备方法,步骤j中,反应在氮气保护下进行。
上述制备方法,步骤k中,反应溶剂选自二氯甲烷、氯仿、四氢呋喃、N,N-二甲基甲酰胺、N,N-二甲基乙酰胺中的至少一种。
上述制备方法,步骤l中,化合物12、化合物9和碱,在保护气氛下反应。
上述制备方法,步骤l中,化合物12与化合物9的摩尔比为1:(1.0~3.0)。优选地,步骤l中,化合物12与化合物9的摩尔比为1:1.1。
上述制备方法,步骤l中,化合物12与碱的摩尔比为1:(1.0~4.0)。优选地,步骤l中,化合物12与碱的摩尔比为1:1.5。
上述制备方法,步骤l中,碱选自二异丙基乙胺、三乙胺、磷酸钾、碳酸钾、碳酸钠、碳酸铯中的至少一种。上述制备方法,步骤l中,反应溶剂选自N,N-二甲基甲酰胺、N,N-二甲基乙酰胺、2-丁酮、甲苯中的至少一种。上述制备方法,步骤l中,反应温度为85℃。上述制备方法,步骤l中,反应在氮气保护下进行。
上述制备方法,步骤m中,反应溶剂选自二氯甲烷、氯仿、四氢呋喃、N,N-二甲基甲酰胺、N,N-二甲基乙酰胺中的至少一种。
上述方法,步骤n中化合物16、四氢吡咯和Pd催化剂,在保护气氛下反应。
上述制备方法,步骤n中,化合物16与四氢吡咯的摩尔比为1:(1.0~4.0)。优选地,步骤n中,化合物16与四氢吡咯的摩尔比为1:3.5。
上述制备方法,步骤n中,化合物16与Pd催化剂的摩尔比为1:0.02~0.10。优选地,步骤n中,化合物16与Pd催化剂的摩尔比为1:0.06。
上述制备方法,步骤n中,Pd催化剂选自Pd(dppf)Cl2·CH2Cl2、PdCl2(PPh3)2、Pd[P(Ph)3]4、Pd(OAc)2中的至少一种。上述制备方法,步骤n中,反应溶剂选自二氯甲烷、氯仿、四氢呋喃、N,N-二甲基甲酰胺中至少一种。上述制备方法,步骤n中,反应温度为25℃。上述制备方法,步骤n中,反应在氮气保护下进行。
上述制备方法,步骤o中,化合物14、化合物9和碱,在保护气氛下反应。
上述制备方法,步骤o中,化合物14与化合物9的摩尔比为1:(1.0~3.0)。优选地,步骤o中,化合物14与化合物9的摩尔比为1:1.1。
上述制备方法,步骤o中,化合物14与碱的摩尔比为1:(1.0~4.0)。优选地,步骤o中,化合物14与碱的摩尔比为1:1.5。
上述制备方法,步骤n中,碱选自二异丙基乙胺、三乙胺、磷酸钾、碳酸钾、碳酸钠、碳酸铯中至少一种。上述制备方法,步骤o中,反应溶剂选自N,N-二甲基甲酰胺、N,N-二甲基乙酰胺、2-丁酮、甲苯中至少一种。上述制备方法,步骤o中,反应温度为85℃。上述制备方法,步骤o中,反应在氮气保护下进行。
上述制备方法,步骤p中,反应溶剂选自二氯甲烷、氯仿、四氢呋喃、N,N-二甲基甲酰胺、N,N-二甲基乙酰胺中的至少一种。
上述方法,步骤q中化合物19、四氢吡咯和Pd催化剂,在保护气氛下反应。
上述制备方法,步骤q中,化合物19与四氢吡咯的摩尔比为1:(1.0~4.0)。优选地,步骤q中,化合物19与四氢吡咯的摩尔比为1:3.5。
上述制备方法,步骤q中,化合物19与Pd催化剂的摩尔比为1:0.02~0.10。优选地,步骤q中,化合物19与Pd催化剂的摩尔比为1:0.06。
上述制备方法,步骤q中,Pd催化剂选自Pd(dppf)Cl2·CH2Cl2、PdCl2(PPh3)2、Pd[P(Ph)3]4、Pd(OAc)2中至少一种。上述制备方法,步骤q中,反应溶剂选自二氯甲烷、氯仿、四氢呋喃、N,N-二甲基甲酰胺中的至少一种。上述制备方法,步骤q中,反应温度为25℃。上述制备方法,步骤q中,反应在氮气保护下进行。
上述制备方法,步骤r中,化合物17或20、连接子L-H和缩合剂,在保护气氛下反应。
上述制备方法,步骤r中,化合物17或20与连接子L-H的摩尔比为1:(1.0~5.0)。优选地,步骤r中,化合物17或20与连接子L-H的摩尔比为1:3.0。
上述制备方法,步骤r中,化合物17或20与缩合剂的摩尔比为1:(1.0~5.0)。优选地,步骤r中,化合物17或20与缩合剂的摩尔比为1:3.0。
上述制备方法,步骤r中,反应溶剂选自二氯甲烷、氯仿、甲醇、四氢呋喃、N,N-二甲基甲酰胺、二氯甲烷:甲醇体积比20:1的混合物或氯仿:甲醇体积比20:1的混合物。上述制备方法,步骤r中,反应温度为25℃。上述制备方法,步骤r中,反应在氮气保护下进行。
本发明还提供了一类药物组合物,其是以式I和/或式II所述的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体或其药学上可接受的盐为活性成分,添加药学上可接受的辅料制备而成。
本发明还提供了上述式I和/或式II所述的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体或其药学上可接受的盐、上述药物组合物在制备抗肿瘤药物中的用途。经细胞实验,本发明化合物在胃癌、乳腺癌、卵巢癌、肺癌、胰腺癌、***癌或尿路上皮癌等多种肿瘤中均展现出很强的抗增殖活性。
本发明还提供了上述式I和/或式II所述的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体或其药学上可接受的盐、上述药物组合物在制备抗体药物偶联物中的用途。
术语定义:
本发明中提供的化合物和衍生物可以根据IUPAC(国际纯粹与应用化学联合会)或CAS(化学文摘服务社,Columbus,OH)命名***命名。
术语“烷基”是直链或支链的饱和烃基的基团。C1~C6烷基的实例包括甲基(C1)、乙基(C2)、正丙基(C3)、异丙基(C3)、正丁基(C4)、叔丁基(C4)、仲丁基(C4)、异丁基(C4)、正戊基(C5)、3-戊基(C5)、戊基(C5)、新戊基(C5)、3-甲基-2-丁基(C5)、叔戊基(C5)和正己基(C6)。除非另外指明,否则烷基的每种情况独立地任选被取代,即未被取代或被一个或多个取代基取代。“取代”是指分子中的氢原子被其它不同的原子或分子所替换。在一些实施方案中,所述C1~C6烷基是被卤素(氟、氯、溴、碘)取代的C1~C6烷基。在C1~C6烷基被取代基取代的情况中,不将取代基的碳原子数计算在内。
术语“芳基”是指在芳族环系中包含或不包含杂原子的4n+2芳族环系的基团,其中,杂原子独立地选自氮、氧和硫。除非另外指明,否则芳基的每种情况独立地任选被取代,即未被取代或被一个或多个取代基取代。
术语“烷氧基”是指基团-OR,其中R是被取代或未被取代的烷基。C1~C6烷氧基的实例包括甲氧基、乙氧基、正丙氧基、异丙氧基、正丁氧基、叔丁氧基、仲丁氧基、正戊氧基、正己氧基和1,2-二甲基丁氧基。在一些实施方案中,R是被卤基(氟、氯、溴、碘)取代的烷基。所述C1~C6烷氧基在R被取代基取代的情况中,不将取代基的碳原子数计算在内。
术语“烷巯基”是指基团R-S-,其中R是上文所定义的烷基。
术语“药学上可接受的”是指某载体、运载物、稀释剂、辅料,和/或所形成的盐通常在化学上或物理上与构成某药物剂型的其它成分相兼容,并在生理上与受体相兼容。
术语“药学上可接受的盐”是指本发明化合物与无机和/或有机酸和碱形成的酸式和/或碱式盐,也包括两性离子盐(内盐),还包括季铵盐,例如烷基铵盐。这些盐可以是在化合物的最后分离和纯化中直接得到。也可以是通过将上述化合物与一定数量的酸或碱适当(例如等当量)进行混合而得到。这些盐可能在溶液中形成沉淀而以过滤方法收集,或在溶剂蒸发后回收而得到,或在水介质中反应后冷冻干燥制得。本发明中所述盐可以是化合物的盐酸盐、硫酸盐、枸橼酸盐、苯磺酸盐、氢溴酸盐、氢氟酸盐、磷酸盐、乙酸盐、丙酸盐、丁二酸盐、草酸盐、苹果酸盐、琥珀酸盐、富马酸盐、马来酸盐、酒石酸盐或三氟乙酸盐。
本发明的某些实施方式中,本发明包括了同位素标记的化合物,所述同位素标记化合物是指与本文中所列化合物相同,但是其中的一个或多个原子被另一个原子取代,该原子的原子质量或质量数不同于自然界中常见的原子质量或质量数。可以引入本发明化合物中的同位素包括氢、碳、氮、氧、硫,即2H,3H、13C、14C、15N、17O、18O、35S。含有上述同位素和/或其它原子同位素的本发明化合物及其立体异构体,以及该化合物、立体异构体的可药用的盐均应包含在本发明范围之内。
本发明化合物或药物组合物的施用方式没有特别限制,代表性的施用方式包括(但并不限于):肠胃外(静脉内、肌肉内或皮下)、和局部给药。用于肠胃外注射的组合物可包含生理上可接受的无菌含水或无水溶液、分散液、悬浮液或乳液,和用于重新溶解成无菌的可注射溶液或分散液的无菌粉末。适宜的含水和非水载体、稀释剂、溶剂或赋形剂包括水、乙醇、多元醇及其适宜的混合物。用于局部给药的本发明化合物的剂型包括软膏剂、散剂、贴剂、喷射剂和吸入剂。活性成分在无菌条件下与生理上可接受的载体及任何防腐剂、缓冲剂,或必要时可能需要的推进剂一起混合。
本发明所述药学上可接受的辅料是指除活性成分以外包含在剂型中的物质。
本发明所述药学上可接受的辅助性成分,它具有一定生理活性,但该成分的加入不会改变上述药物组合物在疾病治疗过程中的主导地位,而仅仅发挥辅助功效,这些辅助功效仅仅是对该成分已知活性的利用,是医药领域惯用的辅助治疗方式。若将上述辅助性成分与本发明药物组合物配合使用,仍然应属于本发明保护的范围。
本发明的有益效果:
本发明提供了一类结构新颖的吡咯并苯并二氮杂卓-蒽酰亚胺杂交分子,它是通过“分子杂交技术”,以DNA小沟结合剂吡咯并苯并二氮杂卓和DNA嵌入剂蒽酰亚胺为母体,经合理设计后合成得到。体外抗肿瘤细胞增殖实验表明,本发明化合物在胃癌、乳腺癌、卵巢癌、肺癌、胰腺癌、***癌或尿路上皮癌等多种肿瘤细胞中均展示出很强的体外抗增殖活性,特别是化合物20b3表现出优越的抗肿瘤活性;并经进一步实验证明,其可通过诱导DNA形成链间交联,造成DNA损伤,最终导致细胞周期阻滞和细胞凋亡。另外,将本发明化合物制成抗体药物偶联物后,仍具有良好的体内外抗肿瘤活性,并且安全性良好。本发明为临床提供了一类新型抗体药物偶联物的弹头。
附图说明
图1为实施例6中化合物20b3诱导细胞周期抑制和细胞凋亡的结果图;其中,A为细胞周期抑制结果,药物浓度为0.33nM、1nM和3nM,药物作用时间24h;B为诱导凋亡结果,药物浓度为0.33nM、1nM和3nM,药物作用时间48h。
图2为实施例7中线性化后的pBR322质粒的DNA交联实验方案示意图;其中,A表示为未经药物作用的双链DNA(dsDNA)经热变性解链成两条互补的单链DNA(ssDNA);B表示经药物作用后,部分双链DNA会发生链间交联,这些交联的DNA在加热时表现出更强的抗变性能力,作为双链DNA迁移。
图3为实施例7中化合物20b3诱导DNA链间交联的结果图;其中,A为双链(DS)和单链(SS)DNA的1%中性琼脂糖凝胶电泳图,0(DS)是未变性、未加药的线性双链DNA,0(SS)是热变性、未加药的线性单链DNA,其余样品在进行电泳前用不同浓度的化合物20b3和奥沙利铂(阳性对照)处理后再热变性;B为化合物20b3处理后的双链DNA交联率。
图4为实施例8中的化合物20b3的荧光光谱图;其中,A为化合物20b3的三维荧光等高线图,颜色越深,代表荧光强度越强;B为化合物20b3在激发波长为488nm下的发射光谱。
图5为实施例8中化合物20b3在肿瘤细胞内的分布结果图。
图6为实施例9中化合物20b3诱导肿瘤细胞DNA损伤结果图。
图7为实施例11中T-PBA的结构图。
图8为实施例11中SDS-PAGE联合紫外荧光分析T-PBA的偶联情况图;其中,上半部分为考马斯亮蓝染色,下半部分为紫外激发荧光图。
图9为实施例12中流式细胞术检测T-PBA的靶向性结果图。
图10为实施例13中流式细胞术检测T-PBA的内化效率结果图。
图11为实施例14中T-PBA在肿瘤细胞内的分布结果图。
图12为实施例16中诱导细胞周期抑制和细胞凋亡的结果图;其中,A为细胞周期抑制结果,药物浓度为1.6nM、8nM和40nM,药物作用24h;B为诱导凋亡结果,药物浓度为1.6nM、8nM和40nM,药物作用48h。
图13为实施例17中T-PBA诱导肿瘤细胞DNA损伤结果图。
图14为实施例18中T-PBA的体内抗肿瘤活性结果图;其中,A为T-PBA在SKOV3异种移植模型中的抗肿瘤作用;B为T-PBA在NCI-N87异种移植模型中的抗肿瘤作用;箭头所指为给药时间点。
图15为实施例18中T-PBA治疗后小鼠重要器官H&E染色结果图。
具体实施方式
本发明首次合成并通过药效实验评价上述式I所示的吡咯并苯并二氮杂卓-蒽酰亚胺杂交分子。体外实验结果表明,上述杂交分子均具有良好的抗肿瘤活性。再经流式细胞术、琼脂糖凝胶电泳、激光共聚焦显微镜和Western Blot研究代表化合物20b3的作用机制后,发现该类杂交分子的靶点为DNA,并且其可通过诱导DNA形成链间交联而激活DNA损伤应答***,导致Chk2磷酸化后,进而激活Caspase信号通路,促使PARP和Caspase-3发生剪切,抑制DNA损伤修复,最终导致细胞周期阻滞和细胞凋亡。随后,我们将该类杂交分子作为弹头,与相应的连接子偶联制成如式II所示的药物-连接子之后,再与相应的抗体偶联制成抗体药物偶联物,并对其生物活性进行了评价,所得抗体偶联药物对于卵巢癌和胃癌均具有良好的体内外抗肿瘤活性,安全性良好。
实施例1:化合物9a1-9b3的合成
化合物2a的合成:
在250mL三口圆底烧瓶中加入化合物1(15.0g,21.06mmol,采用文献“Tiberghien,A.C.,...&Howard,P.W.(2016).Design and synthesis of tesirine,a clinicalantibody–drug conjugate pyrrolobenzodiazepine dimer payload.ACS medicinalchemistry letters,7(11),983-987.”所公开的方法制备)和甲苯(90mL),再加入磷酸钾(26.8g,126.36mmol)和Pd(dppf)Cl2·CH2Cl2(86.0mg,1.05mmol),置换氮气三次,然后加入甲基硼酸(5.0g,84.24mmol),置换氮气,然后在65℃下反应0.5h,TLC监测反应完全(展开剂:石油醚/乙酸乙酯=4/1,紫外显色)。冷至室温,加入水(60mL)萃取,收集有机层,用无水硫酸钠干燥,抽滤,减压浓缩,粗品用柱层析纯化(流动相:PE/EA=10/1)得黄色油状产物2a(9.85g,65.7%)。1H NMR(400MHz,CDCl3)δ7.69(s,1H),6.76(s,1H),5.51(d,J=1.9Hz,1H),4.70-4.60(m,1H),3.89(s,3H),2.83-2.72(m,1H),2.54(d,J=16.4Hz,1H),1.61(d,J=1.6Hz,3H),1.32-1.24(m,3H),1.11(s,9H),1.09(s,9H),0.90(s,9H),0.10(d,J=2.4Hz,6H).13C NMR(101MHz,CDCl3)δ156.18,145.88,137.69,127.13,123.19,123.06,116.04,110.29,110.24,59.00,56.10,56.02,36.75,25.85,18.21,17.81,13.76,12.79,-5.30.HRMS(ESI,m/z)calcd for C29H51N2O6Si2[M+H]+579.3286,found:579.3282.[α]D(CHCl3)-64.36°,c=0.26g/100mL.
化合物2b的合成:
在1000mL三口圆底烧瓶中加入化合物1(32.0g,44.93mmol),加入甲苯、乙醇和水的混合溶剂(Toluene/EtOH/H2O=256/128/128mL),再依次加入4-氨基苯硼酸频那醇酯(12.8g,58.41mmol)、碳酸钠(21.9g,206.67mmol)和Pd[P(Ph)3]4(1.04g,0.90mmol),置换氮气三次,然后在25℃下反应24h,TLC监测反应完全(展开剂:石油醚/乙酸乙酯=6/1,紫外显色)。反应液减压浓缩,除去多余的甲苯和乙醇,加入EA(500mL)萃取,收集有机层,有机层再用饱和氯化钠(200mL)洗涤,收集有机层,无水硫酸钠干燥,抽滤,减压浓缩,粗品用柱层析纯化(流动相:PE/EA=9/1-7/1)得黄色固体产物2b(13.0g,44.2%)。1H NMR(400MHz,CDCl3)δ7.74(s,1H),7.00(d,J=8.4Hz,2H),6.81(s,1H),6.58(d,J=8.5Hz,2H),6.06(s,1H),4.82-4.72(m,1H),3.94-3.85(m,4H),3.20-3.08(m,1H),2.97(d,J=16.9Hz,1H),1.33-1.27(m,3H),1.13(s,9H),1.11(s,9H),0.89(s,9H),0.11(d,J=2.9Hz,6H).13C NMR(101MHz,CDCl3)δ156.28,146.06,145.80,137.82,126.90,125.90,125.42,124.21,120.45,116.27,115.02,110.34,110.29,58.93,56.17,56.09,33.20,25.91,25.88,18.27,17.84,12.82,-5.21,-5.25.HRMS(ESI,m/z)calcd for C34H53N3NaO6Si2[M+Na]+678.3371found 678.3358.[α]D(CHCl3)-108.28°,c=0.28g/100mL.
化合物3a的合成:
在250mL单口圆底烧瓶中依次加入甲醇(80mL),乙酸(5mL),水(5mL),再于冰浴下加入锌粉(41.8g,639.78mmol),搅拌0.5h,然后滴入2a(10.0g,17.29mmol)的甲醇溶液(20mL),在5℃下反应0.5h,TLC监测反应完全(展开剂:石油醚/乙酸乙酯=5/1,紫外显色)。移至室温,抽滤(垫层硅藻土),滤饼用甲醇(75mL)洗后,再用EA洗滤饼(75mL),将滤液减压浓缩,除去甲醇后,加入乙酸乙酯(200mL),再加入水(60mL)萃取,有机层再依次用饱和碳酸氢钠(200mL)和饱和氯化钠(200mL)洗涤,收集有机层,用无水硫酸钠干燥,抽滤,减压浓缩,得黄色油状产物3a(8.6g,90.6%),此中间体不能长时间储存,应尽快用于下一步合成。1HNMR(400MHz,CDCl3)δ6.72(s,1H),6.25(s,1H),6.16(s,1H),4.70-4.58(m,1H),3.98-3.87(m,1H),3.80-3.75(m,1H),3.71(s,3H),2.71(dd,J=16.4,10.3Hz,1H),2.57-2.50(m,1H),1.68(d,J=1.6Hz,3H),1.10(s,9H),1.08(s,9H),0.89(s,9H),0.06(d,J=6.3Hz,6H).13CNMR(101MHz,CDCl3)δ165.98,148.66,142.98,140.97,125.66,121.04,113.86,112.41,109.16,62.61,58.81,56.64,36.45,25.81,18.19,17.91,13.79,12.89,-5.40.HRMS(ESI,m/z)calcd for C29H53N2O4Si2[M+H]+549.3544,found:549.3545.
化合物3b的合成:
在250mL单口圆底烧瓶中依次加入甲醇(4mL),乙酸(0.5mL)和水(0.5mL),再于冰浴下加入锌粉(11g,169.23mmol),搅拌0.5h,然后滴入化合物2b(3.0g,4.57mmol)的甲醇溶液(100mL),在5℃下反应0.5h,TLC监测反应完全(展开剂:石油醚/乙酸乙酯=3/1,紫外显色)。移至室温,抽滤(垫层硅藻土),滤饼用EA洗(100mL),将滤液减压浓缩(30℃),除去甲醇后,加入乙酸乙酯(150mL),再加入水(45mL)萃取,有机层再依次用饱和碳酸氢钠(60mL)和饱和氯化钠(60mL)洗涤,收集有机层,用无水硫酸钠干燥,抽滤,减压浓缩,得到化合物3b的粗品(2.86g,100%),将其直接用于下一步。1H NMR(400MHz,CDCl3)δ7.07(d,J=8.1Hz,2H),6.78(s,1H),6.61(d,J=8.5Hz,2H),6.28(s,1H),4.74(s,1H),4.01–3.84(m,3H),3.71(s,3H),3.13–3.02(m,1H),3.00–2.91(m,1H),1.31–1.23(m,3H),1.12(s,9H),1.10(s,9H),0.86(s,9H),0.06(s,6H).13C NMR(101MHz,CDCl3)δ166.40,148.87,145.40,143.05,141.33,125.72,124.94,123.39,115.11,113.72,111.90,109.25,62.57,58.91,56.56,32.85,25.84,18.22,17.93,12.91,-5.31,-5.33.HRMS(ESI,m/z)calcd forC34H55N3NaO4Si2[M+Na]+648.3629,found:648.3621.
化合物4a的合成:
在250mL圆底烧瓶中加入化合物3a(12g,21.88mmol),溶于二氯甲烷(96mL),再加入吡啶(3.9mL,3.8g,48.14mmol),氮气保护,-10℃下用恒压滴液漏斗缓慢滴加Alloc-Cl(2.6mL,2.9g,24.07mmol)至反应体系,滴完后于-5℃下反应1h,TLC监测反应完全(展开剂:石油醚/乙酸乙酯=4/1,紫外显色)。加入10%柠檬酸水溶液(96mL)搅拌,反应液倒入分液漏斗中,静置分层,有机层再依次用饱和碳酸氢钠(48mL)和饱和氯化钠(48mL)洗涤,有机层用无水硫酸钠干燥,抽滤,减压浓缩,即得黄色油状产物4a(13.2g,95.1%)。1H NMR(400MHz,CDCl3)δ8.59(s,1H),7.75(s,1H),6.77(s,1H),6.17(s,1H),6.00-5.89(m,1H),5.33(dd,J=17.2,1.6Hz,1H),5.22(dd,J=10.5,1.4Hz,1H),4.67-4.60(m,3H),3.87-3.77(m,2H),3.75(s,3H),2.72(dd,J=16.5,10.0Hz,1H),2.56(dd,J=16.5,4.1Hz,1H),1.68(s,3H),1.32-1.27(m,3H),1.11(d,J=7.4Hz,18H),0.88(s,9H),0.05(d,J=8.0Hz,6H).13CNMR(101MHz,CDCl3)δ165.98,148.66,142.98,140.97,125.66,121.04,113.86,112.41,109.16,62.61,58.81,56.64,36.45,25.81,18.19,17.91,13.79,12.89,-5.40.HRMS(ESI,m/z)calcd for C33H57N2O6Si2[M+H]+633.3755,found:633.3751.[α]D(CHCl3)-88.49°,c=0.39g/100mL.
化合物4b的合成:
在25mL圆底烧瓶中加入化合物3(2.86g,4.57mmol),溶于二氯甲烷(30mL),再加入吡啶(1.62mL,1.59g,20.11mmol),氮气保护,冰浴下缓慢滴加Alloc-Cl(1.07mL,1.21g,10.05mmol)至反应体系,滴完后于冰浴下反应1h,TLC监测反应完全(展开剂:石油醚/乙酸乙酯=4/1,紫外显色)。加入10%柠檬酸水溶液(30mL)搅拌,反应液倒入分液漏斗中,静置分层,有机层再依次用饱和碳酸氢钠(15mL)和饱和氯化钠(15mL)洗涤,有机层用无水硫酸钠干燥,抽滤,减压浓缩,粗品用柱层析纯化(流动相:PE/EA=90/10)得黄色油状产物4b(2.49g,68.8%)。1H NMR(400MHz,CDCl3)δ8.51(s,1H),7.73(s,1H),7.29(d,J=8.3Hz,2H),7.15(d,J=8.2Hz,2H),6.80(d,J=4.6Hz,3H),5.98–5.80(m,2H),5.34–5.13(m,4H),4.79–4.68(m,1H),4.61(d,J=5.8Hz,2H),4.57(d,J=5.5Hz,2H),4.05–3.79(m,2H),3.71(s,3H),3.12–3.03(m,1H),2.96(dd,J=15.9,3.6Hz,1H),1.31–1.22(m,3H),1.08(d,J=7.4Hz,18H),0.80(s,9H),0.01(d,J=7.7Hz,6H).13C NMR(101MHz,CDCl3)δ165.90,153.36,153.24,148.49,146.01,137.02,132.51,132.42,129.20,125.27,124.65,123.84,118.75,118.15,117.88,112.50,65.81,65.70,62.44,59.43,56.26,32.63,25.78,18.16,17.90,12.85,-5.39.HRMS(ESI,m/z)calcd for C42H63N3NaO8Si2[M+Na]+816.4051found,816.4044.[α]D(CHCl3)-35.34°,c=0.26g/100mL.
化合物5a的合成:
在250mL圆底烧瓶中加入化合物4a(3g,4.744mmol),溶于乙酸、甲醇、THF和水的混合溶剂(AcOH/MeOH/THF/H2O=21/3/3/6mL),室温搅拌5h,TLC监测反应完全(展开剂:石油醚/乙酸乙酯=2/1,紫外显色)。加入乙酸乙酯(900mL)稀释,再加入纯水(300mL*2)萃取,收集有机层,再依次用饱和碳酸氢钠(300mL*2)和饱和氯化钠(300mL*2)洗涤,有机层用无水硫酸钠干燥,抽滤,减压浓缩,粗品用柱层析纯化(流动相:PE/EA=4/1)得黄色油状产物5a(2.06g,83.8%)。1H NMR(400MHz,CDCl3)δ8.35(s,1H),7.69(s,1H),6.78(s,1H),6.15(s,1H),6.00–5.90(m,1H),5.34(dd,J=17.2,1.6Hz,1H),5.25–5.21(m,1H),4.77–4.70(m,1H),4.67–4.59(m,3H),3.86–3.80(m,2H),3.77(s,3H),2.88(dd,J=16.8,10.2Hz,1H),2.24–2.15(m,1H),1.70(s,3H),1.29(d,J=7.3Hz,3H),1.11(d,J=7.5Hz,18H).13C NMR(101MHz,CDCl3)δ166.99,153.34,148.56,146.02,132.54,132.01,124.99,122.65,117.95,112.64,66.85,65.74,56.39,37.29,17.89,13.69,12.83.HRMS(ESI,m/z)calcdfor C27H43N2O6Si[M+H]+519.2890,found:519.2882.[α]D(CHCl3)-64.02°,c=0.28g/100mL.
化合物5b的合成:
在250mL圆底烧瓶中加入化合物4b(3g,4.744mmol),溶于乙酸、甲醇、THF和水的混合溶剂(AcOH/MeOH/THF/H2O=21/3/3/6mL),室温搅拌5h,TLC监测反应完全(展开剂:石油醚/乙酸乙酯=2/1,紫外显色)。加入乙酸乙酯(900mL)稀释,再加入纯水(300mL*2)萃取,收集有机层,再依次用饱和碳酸氢钠(300mL*2)和饱和氯化钠(300mL*2)洗涤,有机层用无水硫酸钠干燥,抽滤,减压浓缩,粗品用柱层析纯化(流动相:PE/EA=4/1)得黄色油状产物5b(2.06g,83.8%)。1H NMR(400MHz,CDCl3)δ8.34(s,1H),7.69(s,1H),7.34(d,J=8.3Hz,2H),7.17(d,J=8.2Hz,2H),6.83(d,J=17.2Hz,3H),6.03–5.84(m,2H),5.39–5.17(m,4H),4.91–4.80(m,1H),4.66(d,J=5.6Hz,2H),4.60(d,J=5.7Hz,2H),3.96–3.83(m,2H),3.76(s,3H),3.32–3.21(m,1H),2.67(dd,J=15.9,3.6Hz,1H),1.35-1.27(m,3H),1.12(d,J=7.5Hz,18H).13C NMR(101MHz,CDCl3)δ167.51,153.42,153.05,148.82,146.29,137.14,132.40,132.27,128.53,125.39,124.30,123.92,118.75,118.37,118.08,112.36,65.94,65.84,61.90,56.32,56.23,33.55,17.88,12.82.HRMS(ESI,m/z)calcd forC36H49N3NaO8Si[M+Na]+702.3187,found:702.3189.[α]D(CHCl3)-65.41°,c=0.25g/100mL.
化合物6a-6b的通用合成方法:
在100mL圆底烧瓶中加入无水DCM(25mL),氮气保护,置于-78℃搅拌,然后加入草酰氯(0.52mL,0.78g,6.145mmol),再滴入DMSO(1.0mL,1.1g,14.1mmol),然后于-78℃下搅拌10min,再滴入5a-5b(5.41mmol)的DCM溶液(20mL),此时反应液变浑浊,滴完后-78℃下搅拌15min,再滴入TEA(3.77mL,2.75g,27.17mmol),滴完后升至室温搅拌2h,TLC监测反应完全(展开剂:石油醚/乙酸乙酯=2/1,紫外显色)。加入5%柠檬酸水溶液(28mL)搅拌5分钟,分液,收集有机层,再用饱和碳酸氢钠(14mL)和水(14mL)洗涤,有机层用无水硫酸钠干燥,抽滤,减压浓缩,粗品用柱层析纯化得白色泡沫状产物6a-6b。
化合物6a的合成:
化合物5a按上述方法合成6a,粗品用柱层析纯化(流动相:PE/EA=5/1)得白色泡沫产物6a(2.28g,96.9%)。1H NMR(400MHz,CDCl3)δ7.20(s,1H),6.70(s,2H),5.74(d,J=9.6Hz,2H),5.22-5.10(m,2H),4.63-4.43(m,2H),3.92-3.82(m,4H),3.82-3.77(m,1H),2.96(dd,J=17.0,10.1Hz,1H),2.59(d,J=17.2Hz,1H),1.78(s,3H),1.27-1.20(m,3H),1.08(dd,J=7.4,2.3Hz,18H).13C NMR(101MHz,CDCl3)δ163.00,150.58,147.80,131.82,127.71,125.67,123.25,121.76,121.56,118.39,111.21,86.10,67.02,59.39,55.54,38.75,17.86,17.84,13.69,12.84.HRMS(ESI,m/z)calcd for C27H41N2O6Si[M+H]+517.2734,found:517.2729.[α]D(CHCl3)23.36°,c=0.30g/100mL.
化合物6b的合成:
化合物5b按上述方法合成6b,粗品用柱层析纯化(流动相:PE/EA=5/1)得白色泡沫产物6b(2.4g,65.4%)。1H NMR(400MHz,CDCl3)δ7.43-7.33(m,3H),7.28(d,J=3.9Hz,,1H),7.22(s,1H),7.04(s,1H),6.74(s,1H),6.02-5.90(m,1H),5.83(d,J=9.7Hz,2H),5.36(dd,J=17.2,1.6Hz,1H),5.26(dd,J=10.4,1.4Hz,1H),5.21-5.09(m,2H),4.67(d,J=5.7Hz,2H),4.60(dd,J=13.2,5.7Hz,1H),4.46(dd,J=13.8,5.4Hz,1H),3.98(td,J=10.0,3.3Hz,1H),3.85(s,3H),3.33(ddd,J=16.7,10.2,2.2Hz,1H),3.02(dd,J=16.7,1.8Hz,1H),1.28-1.22(m,3H),1.08(dd,J=7.4,2.3Hz,18H).13C NMR(101MHz,CDCl3)δ163.60,156.06,153.19,150.71,148.10,137.08,132.39,131.76,128.86,127.84,125.60,125.41,123.09,122.56,121.99,118.83,118.36,111.26,85.96,67.12,65.98,59.54,55.63,55.54,35.06,17.88,12.88.HRMS(ESI,m/z)calcd for C36H47N3NaO8Si[M+Na]+700.3030,found:700.3029.[α]D(CHCl3)93.98°,c=0.16g/100mL.
化合物7a-7b的通用合成方法:
在100mL圆底烧瓶中加入化合物6a-6b(4.36mmol),溶于无水DCM(22.5mL),再加入2,6-二甲基吡啶(2.0mL,1.84g,17.17mmol),氮气保护,冰浴下滴加TBS-OTf(3.0mL,3.45g,13.08mmol)至反应体系,滴完后升至室温反应1h,TLC监测反应完全(展开剂:石油醚/乙酸乙酯=2/1,紫外显色)。反应液加入饱和碳酸氢钠(15mL),收集有机层,再用水(15mL)洗涤,收集有机层,用无水硫酸钠干燥,抽滤,减压浓缩,粗品直接用于下一步。
化合物7a的合成:
化合物6a按照上述方法合成7a(2.635g,95.9%),得到的7a粗品为黄色油状液体,直接用于下一步反应。
化合物7b的合成:
化合物6b按照上述方法合成7b(2.646g,76.7%),得到的7b粗品为黄色油状液体,直接用于下一步反应。
化合物8a-8b的通用合成方法:
在50mL圆底烧瓶中加入化合物7a-7b(4.18mmol),溶于DMF和水的混合溶剂中(25/0.5mL)中,再加入LiOAc(0.28g,4.26mmol),25℃下搅拌10h,TLC监测反应完全(展开剂:石油醚/乙酸乙酯=2/1,紫外显色)。反应液减压浓缩(隔膜泵56℃),除去多余的DMF,加入EA(50mL)萃取,有机层再用纯水(25mL)洗涤,水层再用EA(20mL)萃取,合并有机层,依次用5%柠檬酸(25mL)和饱和氯化钠(25mL)洗涤有机层,收集有机层,无水硫酸钠干燥,抽滤,减压浓缩,粗品经柱层析纯化得白色固体8a-8b。
化合物8a的合成:
化合物7a按上述方法合成8a,粗品用柱层析纯化(流动相:PE/EA=3/1-3/2)得白色泡沫产物8a(1.84g,91.35%)。1H NMR(400MHz,CDCl3)δ7.24(s,1H),6.73(s,1H),6.68(s,1H),5.85(d,J=8.9Hz,1H),5.80-5.71(m,1H),5.14-5.04(m,2H),4.60(dd,J=13.4,5.0Hz,1H),4.42(dd,J=13.8,5.3Hz,1H),3.95(s,3H),3.75(td,J=12.8Hz,3.6Hz,1H),2.91(dd,J=17.1,10.2Hz,1H),2.38(d,J=17.3Hz,1H),1.77(s,3H),0.87(s,9H),0.23(d,J=17.2Hz,6H).13C NMR(101MHz,CDCl3)δ163.43,155.17,148.01,146.37,132.11,129.24,125.51,123.61,121.22,117.35,116.38,110.38,86.89,66.41,61.47,56.26,39.05,25.68,17.89,13.84,-4.27,-5.39.HRMS(ESI,m/z)calcd for C24H35N2O6Si[M+H]+475.2264,found:475.2260.[α]D(CHCl3)47.44°,c=0.48g/100mL.
化合物8b的合成:
化合物7b按上述方法合成8b,粗品用柱层析纯化(流动相:PE/EA=3/1-2/1)得白色固体产物8b(2.54g,95.6%)。1H NMR(400MHz,CDCl3)δ7.38(d,J=8.5Hz,3H),7.29(d,J=8.5Hz,2H),6.76(s,1H),6.71(s,1H),6.03-5.91(m,2H),5.84-5.70(m,1H),5.37(dd,J=17.2,1.5Hz,1H),5.27(dd,J=10.4,1.4Hz,1H),5.17-5.01(m,2H),4.67(d,J=5.0Hz,2H),4.61(dd,J=14.0,4.6Hz,1H),4.42(dd,J=14.0,5.2Hz,1H),3.97-3.87(m,4H),3.31(ddd,J=16.4,10.3,2.2Hz,1H),2.81(dd,J=16.3,3.5Hz,1H),0.95–0.90(s,9H),0.24(d,J=17.4Hz,6H).13C NMR(101MHz,CDCl3)δ163.89,155.17,153.17,148.24,146.45,137.12,132.40,132.04,129.34,128.93,125.55,125.31,122.99,122.67,118.81,118.33,117.46,116.46,110.41,86.66,66.51,65.94,61.46,56.30,35.11,25.69,17.94,-4.24,-5.29.HRMS(ESI,m/z)calcd for C30H38N2NaO7Si[M+Na]+658.2561,found:658.2554.[α]D(CHCl3)55.40°,c=0.33g/100mL.
化合物9a1-9b3的通用合成方法:
在10mL圆底烧瓶中加入8a-8b(0.424mmol),加入2-丁酮(2.5mL)溶解,再加入碳酸钾(0.076g,0.5483mmol)和二碘代烷(2.10mmol),氮气保护,升至75℃下搅拌16h,TLC监测反应完全(展开剂:石油醚/乙酸乙酯=3/1或2/1,紫外显色)。反应液减压浓缩,除去多余的2-丁酮,加入EA(10mL)萃取,有机层再用饱和氯化钠(3mL)洗涤,收集有机层,无水硫酸钠干燥,抽滤,减压浓缩,粗品用柱层析纯化得产物9a1-9b3。
化合物9a1的合成:
化合物8a和1,3-二碘丙烷(0.24mL,0.618g,2.10mmol)按照上述方法合成9a1,粗品经柱层析(流动相:PE/EA=9/1-8/1)得到黄色油状产物9a1(160mg,59%)。1H NMR(400MHz,CDCl3)δ7.24(s,1H),6.69(s,1H),6.64(d,J=4.7Hz,1H),5.90-5.83(m,1H),5.82-5.73(m,1H),5.15-5.05(m,2H),4.65-4.60(m,1H),4.16-4.01(m,2H),3.93-3.88(m,3H),3.79-3.70(m,1H),3.38(t,J=6.6Hz,2H),2.93(dd,J=17.0,10.3Hz,1H),2.43-2.24(m,3H),1.78(s,3H),0.88(d,J=3.8Hz,9H),0.25(d,J=4.3Hz,6H).13C NMR(101MHz,CDCl3)δ161.03,152.73,147.59,146.82,129.82,126.24,124.07,121.40,118.84,115.19,112.21,108.59,84.70,66.28,64.10,59.20,53.80,36.76,30.16,23.40,23.36,15.62,11.55,-6.44,-7.56.HRMS(ESI,m/z)calcd for C27H40IN2O6Si[M+H]+643.1700,found:643.1703.[α]D(CHCl3)90.38°,c=0.50g/100mL.
化合物9a2的合成:
化合物8a和1,4-二碘丁烷(0.28mL,0.65g,2.10mmol)按照上述方法合成9a2,粗品经柱层析(流动相:PE/EA=85/15-80/20)得到黄色油状产物9a2(193.7mg,70%)。1H NMR(400MHz,CDCl3)δ7.23(s,1H),6.69(s,1H),6.60(s,1H),5.87(d,J=8.9Hz,1H),5.82-5.70(m,1H),5.09(t,J=12.8Hz,2H),4.62(dd,J=13.6,4.7Hz,1H),4.40(dd,J=13.6,5.2Hz,1H),4.07-3.94(m,2H),3.91(s,3H),3.73(td,J=9.8,3.3Hz,1H),3.26(t,J=6.7Hz,2H),2.92(dd,J=17.0,10.3Hz,1H),2.38(d,J=16.6Hz,1H),2.07-2.01(m,2H),1.99-1.93(m,2H),1.78(s,3H),0.88(s,9H),0.24(d,J=8.7Hz,6H).13C NMR(101MHz,CDCl3)δ163.37,155.04,150.05,149.07,132.15,128.53,126.13,123.65,121.08,117.32,114.18,110.82,87.01,67.84,66.37,61.50,56.21,39.05,30.08,29.82,25.62,17.90,13.84,6.14,-4.20,-5.26.HRMS(ESI,m/z)calcd for C28H42IN2O6Si[M+H]+657.1857,found:657.1855.[α]D(CHCl3)90.38°,c=0.50g/100mL.
化合物9a3的合成:
化合物8a和1,5-二碘戊烷(0.31mL,0.68g,2.10mmol)按照上述方法合成9a3,粗品经柱层析(流动相:PE/EA=85/15-80/20)得黄色油状产物9a3(262.9mg,93%)。1H NMR(400MHz,CDCl3)δ7.23(s,1H),6.69(s,1H),6.60(s,1H),5.87(d,J=8.9Hz,1H),5.83-5.69(m,1H),5.14-5.03(m,2H),4.63(dd,J=13.7,5.0Hz,1H),4.40(dd,J=13.8,5.2Hz,1H),4.05-3.95(m,2H),3.91(s,3H),3.74(td,J=9.6,3.7Hz,1H),3.21(t,J=7.0Hz,2H),2.92(dd,J=16.8,10.4Hz,1H),2.38(d,J=16.4Hz,1H),1.93-1.85(m,4H),1.78(s,3H),1.63-1.56(m,2H),0.88(s,9H),0.24(d,J=8.1Hz,6H).13C NMR(101MHz,CDCl3)δ163.40,155.04,150.15,149.05,132.13,128.55,125.98,123.69,120.99,117.28,114.16,110.81,87.01,68.69,66.33,61.50,56.16,39.05,33.13,27.82,27.02,25.62,17.88,13.79,6.40,-4.23,-5.28.HRMS(ESI,m/z)calcd for C29H44IN2O6Si[M+H]+671.2013,found:671.2006.[α]D(CHCl3)26.91°,c=0.32g/100mL.
化合物9b1的合成:
化合物8b和1,3-二碘丙烷(0.24mL,0.618g,2.10mmol)按照上述方法合成9b1,粗品经柱层析纯化(流动相:PE/EA=85/15-67/33)得黄色油状产物9b1(248.6mg,73%)。1HNMR(400MHz,CDCl3)δ7.38(d,J=8.8Hz,3H),7.29(d,J=8.4Hz,2H),6.74(s,1H),6.68(s,1H),6.03–5.90(m,2H),5.84-5.71(m,1H),5.37(dd,J=17.2,1.5Hz,1H),5.27(dd,J=10.4,1.3Hz,1H),5.18–5.04(m,2H),4.68(d,J=5.7Hz,2H),4.65–4.60(m,1H),4.41(dd,J=13.3,6.0Hz,1H),4.11–4.04(m,2H),3.95–3.88(m,4H),3.39(t,J=6.6Hz,2H),3.35–3.28(m,1H),2.81(d,J=16.3Hz,1H),2.33(p,J=6.2Hz,2H),0.91(d,J=3.7Hz,9H),0.26(d,J=7.0Hz,6H).13C NMR(101MHz,CDCl3)δ163.75,154.99,153.14,150.13,149.18,137.19,132.39,131.98,128.76,128.57,125.99,125.48,122.89,122.62,118.74,118.21,117.53,114.50,110.88,86.74,68.55,66.45,65.84,61.48,56.16,35.06,32.38,25.66,17.89,2.23,-4.17,-5.21.HRMS(ESI,m/z)calcd forC36H46IN3NaO8Si[M+Na]+826.1997,found:826.1990.[α]D(CHCl3)152.33°,c=0.21g/100mL.
化合物9b2的合成:
化合物8b和1,4-二碘丁烷(0.28mL,0.65g,2.10mmol)按照上述方法合成9b2,粗品经柱层析纯化(流动相:PE/EA=85/15-67/33)得黄色油状产物9b2(235.6mg,68%)。1H NMR(400MHz,CDCl3)δ7.38(d,J=9.0Hz,3H),7.29(d,J=8.3Hz,2H),6.71(s,1H),6.63(s,1H),6.03-5.90(m,2H),5.83-5.71(m,1H),5.37(dd,J=17.2,1.6Hz,1H),5.27(dd,J=10.5,1.4Hz,1H),5.18-5.02(m,2H),4.68(d,J=5.8Hz,2H),4.65-4.59(m,1H),4.41(d,J=11.8Hz,1H),4.07-3.97(m,2H),3.92(s,4H),3.40-3.30(m,1H),3.27(t,J=6.7Hz,2H),2.81(d,J=16.1Hz,1H),2.08-2.02(m,2H),2.01-1.93(m,2H),0.91(s,9H),0.25(d,J=11.5Hz,6H).13C NMR(101MHz,CDCl3)δ163.80,155.00,153.11,150.30,149.14,137.12,132.38,132.03,128.83,128.57,125.76,125.49,122.96,122.55,118.74,118.25,117.38,114.19,110.83,86.75,67.84,66.43,65.86,61.44,56.10,35.07,30.03,29.77,25.62,17.89,6.08,-4.21,-5.20.HRMS(ESI,m/z)calcd for C37H48IN3NaO8Si[M+Na]+840.2153,found:840.2154.[α]D(CHCl3)85.53°,c=0.35g/100mL.
化合物9b3的合成:
化合物8b和1,5-二碘戊烷(0.31mL,0.68g,2.10mmol)按照上述方法合成9b3,粗品经柱层析纯化(流动相:PE/EA=85/15-67/33)得黄色油状产物9b3(317.2mg,90%)。1H NMR(400MHz,CDCl3)δ7.38(d,J=9.3Hz,3H),7.29(d,J=8.5Hz,2H),6.71(s,1H),6.63(s,1H),6.04-5.90(m,2H),5.83-5.71(m,1H),5.37(dd,J=17.2,1.5Hz,1H),5.27(dd,J=10.4,1.3Hz,1H),5.19-5.00(m,2H),4.68(d,J=5.7Hz,2H),4.65-4.59(m,1H),4.42(d,J=12.9Hz,1H),4.07-3.95(m,2H),3.93(s,3H),3.92-3.86(m,1H),3.38-3.27(m,1H),3.22(t,J=7.0Hz,2H),2.81(d,J=15.9Hz,1H),1.97-1.82(m,4H),1.64–1.60(m,2H),0.92(s,9H),0.25(d,J=11.0Hz,6H).13C NMR(101MHz,CDCl3)δ163.84,155.01,153.13,150.38,149.10,137.16,132.39,132.03,128.80,128.58,125.59,125.48,122.94,122.54,118.74,118.21,117.34,114.09,110.78,86.76,68.68,66.41,65.84,61.48,56.12,35.07,33.08,27.78,26.99,25.63,17.89,6.45,-4.22,-5.21.HRMS(ESI,m/z)calcd for C38H50IN3NaO8Si[M+Na]+854.2310,found:854.2313.[α]D(CHCl3)91.81°,c=0.27g/100mL.
实施例2:化合物14的合成
化合物11的合成:
在1000mL圆底烧瓶中加入N-Boc-哌嗪(10,24g,128.94mmol),再加入400mL无水DCM溶解,然后加入1,2-二溴乙烷(47.0mL,102.6g,552.0mmol)和DIEA(23.5mL,18.33g,141.8mmol),于45℃下回流96h。TLC监测反应未完全(展开剂:PE/EA=3/1,碘显),进行后处理,冷至室温,旋去DCM,加入乙酸乙酯(600mL)搅拌10分钟后抽滤,收集滤液,旋干,经减压浓缩,粗品用柱层析纯化(流动相:PE/EA=9/1-17/3)得浅黄色固体产物11(10.15g,27.0%)。1H NMR(400MHz,CDCl3)δ3.66-3.54(m,2H),3.45(s,4H),2.74(t,J=7.1Hz,2H),2.47(s,4H),1.48-1.43(m,9H).13C NMR(101MHz,CDCl3)δ153.78,81.36,60.12,55.77,52.88,28.34,28.28.
化合物13的合成:
在50mL圆底烧瓶中加入11(724mg,2.479mmol)和12(612.5mg,2.479mmol,采用文献“Yao,J.H.et.(2010).Bisanthracene bis(dicarboxylic imide)s as soluble andstable nir dyes.Chemistry-A European Journal,15(37),9299-9302.”所公开的方法制备),加入20mL无水DMF溶解,然后加入K2CO3(1.38g,9.916mmol),于85℃下回流12h。TLC监测反应完全(展开剂:PE/EA=3/1,碘显),冷至室温,旋去DMF,加入DCM(60mL)溶解,再加入水(10mL)萃取,收集有机层,水层用DCM萃取2次(20mL*2),合并有机层,有机层用无水硫酸钠干燥,抽滤,减压浓缩,粗品用柱层析纯化(流动相:DCM/EA=4/1-2/1)得黄色固体产物13(1.1g,96.7%)。1H NMR(400MHz,Chloroform-d)δ9.98(t,J=8.0Hz,1H),8.83(d,J=12.1Hz,1H),8.75(t,J=7.1Hz,1H),8.36(t,J=8.7Hz,1H),8.12(t,J=7.5Hz,1H),7.88–7.80(m,1H),7.77–7.70(m,1H),7.68–7.60(m,1H),4.46(t,J=7.1Hz,2H),3.44(s,4H),2.89–2.75(m,2H),2.62(s,4H),1.45(s,9H).13C NMR(101MHz,DMSO-d6)δ164.26,162.62,153.73,136.94,135.50,133.22,132.42,131.78,131.32,130.04,128.35,127.52,126.25,125.68,125.62,121.57,114.13,78.61,55.04,52.56,37.02,27.95,13.98.
化合物14的合成:
在50mL圆底烧瓶中加入13(700mg,1.524mmol),加入15mL氯仿溶解,然后加入三氟乙酸(1.13mL,1.737g,15.24mmol),于25℃下搅拌16h。TLC监测反应完全(展开剂:DCM/MeOH=10/1,紫外显色),加入饱和碳酸氢钠溶液(20mL)调pH至8,再加入DCM/MeOH=10/1(10mL)萃取,收集有机层,水层用DCM/MeOH=10/1萃取2次(10mL*2),合并有机层,有机层用无水硫酸钠干燥,抽滤,减压浓缩,得黄色固体产物14(0.5g,91.3%),无需提纯,直接用后续反应。
实施例3:化合物17a1-17b3的合成
化合物15a1-15b3的通用合成方法:
在25mL圆底烧瓶中加入9a1-9b3(0.336mmol),加入无水DMF(5mL)溶解,再加入碳酸钾(63.3mg,0.458mmol)和12(75.4mg,0.305mmol),氮气保护,升至85℃下搅拌20h,TLC监测反应完全(展开剂:石油醚/乙酸乙酯=3/1,紫外显色)。反应液减压浓缩,除去多余的DMF,加入EA(15mL)和纯水(5mL)萃取,水层再用EA(15mL*2)萃取,合并有机层,有机层再用饱和氯化钠(10mL*2)洗涤,收集有机层,无水硫酸钠干燥,抽滤,减压浓缩,粗品用柱层析纯化得黄色泡沫状产物15a1-15b3。
化合物15a1的合成:
化合物9a1和12按照上述方法合成15a1,粗品经柱层析(流动相:PE/EA=85/15-65/35)得黄色泡沫状产物15a1(83.7mg,36%)。1H NMR(400MHz,CDCl3)δ9.97(d,J=9.2Hz,1H),8.86(s,1H),8.75(dd,J=7.1,1.3Hz,1H),8.38(d,J=8.4Hz,1H),8.13(d,J=8.4Hz,1H),7.84-7.78(m,1H),7.77-7.71(m,1H),7.64(t,J=7.5Hz,1H),7.07(s,1H),6.66(s,1H),6.63(s,1H),5.83(d,J=8.9Hz,1H),5.74-5.63(m,1H),5.05-4.92(m,2H),4.53(t,J=6.7Hz,3H),4.37(dd,J=13.5,4.9Hz,1H),4.24-4.12(m,2H),3.69(td,J=9.1,2.8Hz,1H),3.56(s,3H),2.94-2.83(m,1H),2.42(t,J=6.6Hz,2H),2.39-2.32(m,1H),1.76(s,3H),0.79(s,9H),0.22(s,3H),0.19(s,3H).13C NMR(101MHz,CDCl3)δ165.04,163.59,163.34,154.98,150.12,148.81,136.13,134.94,133.41,133.30,132.27,131.90,131.16,129.56,128.65,128.34,128.23,126.64,126.36,125.65,125.30,123.57,122.38,120.85,117.11,115.18,113.91,110.37,86.83,67.42,66.18,61.35,55.56,38.90,37.82,27.83,25.42,17.64,13.70,-4.37,-5.48.HRMS(ESI,m/z)calcd for C43H48N3O8Si[M+H]+762.3211,found:762.3228.[α]D(CHCl3)76.47°,c=0.26g/100mL.
化合物15a2的合成:
化合物9a2和12按照上述方法合成15a2,粗品用柱层析纯化(流动相:PE/EA=85/15-65/35)得黄色泡沫状产物15a2(180mg,76%)。1H NMR(400MHz,CDCl3)δ10.02(d,J=9.1Hz,1H),8.85(s,1H),8.76(dd,J=6.9,1.2Hz,1H),8.37(d,J=8.1Hz,1H),8.13(d,J=8.4Hz,1H),7.86-7.81(m,1H),7.78-7.72(m,1H),7.68-7.62(m,1H),7.22(s,1H),6.71-6.62(m,2H),5.90-5.82(m,1H),5.81-5.70(m,1H),5.15-5.03(m,2H),4.66-4.58(m,1H),4.45-4.31(m,3H),4.16-4.04(m,2H),3.91-3.85(m,3H),3.79-3.70(m,1H),2.98-2.86(m,1H),2.42-2.33(m,1H),2.07-2.00(m,4H),1.77(s,3H),0.88(d,J=10.0Hz,9H),0.24(d,J=8.3Hz,6H).13C NMR(101MHz,CDCl3)δ165.01,163.59,163.49,155.11,150.37,149.10,136.31,135.09,133.44,132.32,132.11,131.28,129.72,128.68,128.58,128.20,126.66,126.44,125.88,125.38,123.73,123.66,122.36,121.03,117.40,115.10,114.25,110.80,87.02,68.87,66.36,61.51,56.18,40.07,39.06,26.76,25.65,24.84,17.89,13.84,-4.21,-5.26.HRMS(ESI,m/z)calcd for C44H49N3NaO8Si[M+Na]+798.3187,found:798.3182.[α]D(CHCl3)124.95°,c=0.28g/100mL.
化合物15a3的合成:
化合物9a3和12按照上述方法合成15a3,粗品用柱层析纯化(流动相:PE/EA=85/15-65/35)得黄色泡沫状产物15a3(77mg,32%)。1H NMR(400MHz,CDCl3)δ10.04(d,J=9.1Hz,1H),8.87(s,1H),8.78(dd,J=7.1,1.3Hz,1H),8.39(d,J=7.8Hz,1H),8.14(d,J=8.4Hz,1H),7.88-7.82(m,1H),7.76(dd,J=8.4,7.1Hz,1H),7.69-7.62(m,1H),7.21(s,1H),6.68(s,1H),6.60(s,1H),5.85(d,J=9.0Hz,1H),5.79-5.70(m,1H),5.12-5.05(m,2H),4.63-4.58(m,1H),4.44-4.38(m,1H),4.35-4.29(m,2H),4.07–3.98(m,2H),3.87(s,3H),3.76–3.71(m,1H),2.94-2.87(m,1H),2.40–2.33(m,1H),1.99-1.86(m,4H),1.77(s,3H),1.70-1.63(m,2H),0.82(s,9H),0.22-0.18(m,6H).13C NMR(101MHz,CDCl3)δ165.08,163.62,163.51,155.10,150.33,149.03,136.30,135.07,133.51,133.45,132.38,132.11,131.31,129.72,128.73,128.56,128.26,126.72,126.46,125.73,125.41,123.73,122.45,121.00,17.36,115.21,113.99,110.74,87.01,68.88,66.35,61.52,56.08,40.42,39.05,28.75,27.95,25.59,23.74,17.84,13.83,-4.24,-5.28.HRMS(ESI,m/z)calcd forC45H52N3O8Si[M+H]+790.3524,found:790.3524.[α]D(CHCl3)51.80°,c=0.26g/100mL.
化合物15b1的合成:
化合物9b1和12按照(25)中所述方法合成15b1,粗品用柱层析纯化(流动相:PE/EA=90/10-60/40)得黄色泡沫状产物15b1(126.6mg,45%)。1H NMR(400MHz,CDCl3)δ9.97(d,J=9.2Hz,1H),8.86(s,1H),8.75(dd,J=7.1,1.3Hz,1H),8.39(d,J=8.2,1H),8.13(d,J=8.5Hz,1H),7.81(ddd,J=9.2,6.6,1.4Hz,1H),7.75(dd,J=8.3,7.0Hz,1H),7.64(t,J=7.2Hz,1H),7.40-7.34(m,3H),7.29(s,1H),7.09(s,1H),6.72(s,1H),6.65(s,1H),6.02-5.88(m,2H),5.75-5.63(m,1H),5.40-5.34(m,1H),5.27(dd,J=10.4,1.3Hz,1H),5.05-4.96(m,2H),4.67(d,J=5.7Hz,2H),4.61-4.50(m,3H),4.37(dd,J=13.7,4.9Hz,1H),4.27-4.15(m,2H),3.85(td,J=9.8,3.6Hz,1H),3.56(s,3H),3.34-3.24(m,1H),2.82-2.73(m,1H),2.43(p,J=6.4Hz,2H),0.82(s,9H),0.24(s,3H),0.20(s,3H).13C NMR(101MHz,CDCl3)δ165.22,163.96,163.79,155.12,153.16,150.55,149.07,137.11,136.33,135.12,133.57,133.48,132.43,131.98,131.33,129.74,128.94,128.82,128.57,128.40,126.79,126.52,125.50,125.47,123.05,123.01,122.54,122.47,118.76,118.31,118.23,117.35,115.34,114.15,110.59,86.76,67.62,66.43,65.91,61.55,55.73,37.97,35.09,27.96,25.60,17.82,-4.20,-5.24.HRMS(ESI,m/z)calcd for C52H54N4NaO10Si[M+Na]+945.3507,found:945.3508.[α]D(CHCl3)254.38°,c=0.23g/100mL.
化合物15b2的合成:
化合物9b2和12按照(25)中所述方法合成15b2,粗品用柱层析纯化(流动相:PE/EA=90/10-60/40)得黄色泡沫状产物15b2(148.5mg,52%)。1H NMR(400MHz,CDCl3)δ10.03(d,J=9.1Hz,1H),8.87(s,1H),8.78(dd,J=7.0,1.3Hz,1H),8.39(d,J=8.4Hz,1H),8.14(d,J=8.4Hz,1H),7.90-7.80(m,1H),7.76(dd,J=8.4,7.1Hz,1H),7.70-7.63(m,1H),7.42-7.34(m,3H),7.29(d,J=8.4Hz,2H),6.69(d,J=16.0Hz,2H),6.05-5.90(m,2H),5.83-5.71(m,1H),5.37(dd,J=17.2,1.5Hz,1H),5.27(dd,J=10.4,1.3Hz,1H),5.18-5.01(m,2H),4.67(d,J=5.7Hz,2H),4.66-4.59(m,1H),4.38(t,J=7.0Hz,3H),4.10(d,J=7.2Hz,2H),3.90(s,4H),3.37-3.25(m,1H),2.88-2.75(m,1H),2.04(d,J=6.1Hz,4H),0.90(s,9H),0.24(s,6H).13C NMR(101MHz,CDCl3)δ165.04,163.90,163.62,155.06,153.11,150.60,149.17,137.11,136.35,135.10,133.47,132.38,132.34,131.98,131.28,129.71,128.83,128.70,128.61,128.24,126.64,126.42,125.52,125.44,125.38,122.97,122.47,122.37,118.75,118.20,118.15,117.43,115.14,114.26,110.78,86.76,68.86,66.41,65.82,61.50,56.07,40.03,35.04,26.69,25.62,24.79,17.87,-4.24,-5.23.HRMS(ESI)calcdfor C53H56N4O10Si[M+Na]+959.3766found 959.3660.
化合物15b3的合成:
化合物9b3和12按照(25)中所述方法合成15b3,粗品用柱层析纯化(流动相:PE/EA=90/10-60/40)得黄色泡沫状产物15b3(153.6mg,53%)。1H NMR(400MHz,CDCl3)δ10.03(d,J=9.2Hz,1H),8.86(s,1H),8.78(dd,J=7.1,1.3Hz,1H),8.38(d,J=8.4Hz,1H),8.14(d,J=8.5Hz,1H),7.89-7.80(m,1H),7.75(dd,J=8.4,7.0Hz,1H),7.65(dd,J=8.3,6.6Hz,1H),7.42-7.34(m,3H),7.30(s,1H),7.24(s,1H),6.73(s,1H),6.64(s,1H),6.03-5.89(m,2H),5.80-5.68(m,1H),5.41-5.32(m,1H),5.27(dd,J=10.4,1.3Hz,1H),5.16-4.99(m,2H),4.68(d,J=5.7Hz,2H),4.66-4.58(m,1H),4.45-4.37(m,1H),4.33(t,J=7.6Hz,2H),4.11-3.99(m,2H),3.94-3.83(m,4H),3.36-3.24(m,1H),2.88-2.74(m,1H),2.02-1.94(m,2H),1.91-1.84(m,2H),1.72-1.64(m,2H),0.86(s,9H),0.22(d,J=10.1Hz,6H).13C NMR(101MHz,CDCl3)δ165.12,163.98,163.67,155.11,153.17,150.63,149.16,137.15,136.36,135.12,133.55,133.49,132.44,132.41,132.03,131.34,129.75,128.91,128.77,128.65,128.30,126.73,126.48,125.50,125.43,123.04,122.48,118.81,118.72,118.27,118.21,117.47,115.24,114.09,110.79,86.81,68.93,66.45,65.89,61.58,56.20,40.43,35.09,28.75,27.96,25.64,23.74,17.88,-4.20,-5.18.HRMS(ESI,m/z)calcd forC54H58N4NaO10Si[M+Na]+973.3820,found:973.3823.[α]D(CHCl3)54.69°,c=0.31g/100mL.
化合物16a1-16b3的通用合成方法:
在10mL圆底烧瓶中加入15a1-15b3(0.263mmol),加入无水THF(3mL)溶解,再加入TBAF(1mol/L in THF,0.45mL,0.447mmol)和冰乙酸(39μL,41mg,0.684mmol),氮气保护,于25℃下搅拌16h,TLC监测反应完全(展开剂:DCM/MeOH=20/1,紫外显色)。反应液减压浓缩,除去多余的THF,加入DCM(10mL),再加入饱和碳酸氢钠(5mL)萃取,水层再用DCM(10mL*2)萃取,合并有机层,有机层再用饱和氯化钠(10mL*2)洗涤,收集有机层,无水硫酸钠干燥,抽滤,减压浓缩,粗品用制备TLC板纯化得黄色固体产物16a1-16b3.
化合物16a1的合成:
化合物15a1按照上述方法合成16a1,粗品用制备TLC板纯化(展开剂:DCM/MeOH=20/1)得黄色固体产物16a1(163.4mg,96%)。1H NMR(400MHz,CDCl3)δ9.96(d,J=9.2Hz,1H),8.84(s,1H),8.76-8.69(m,1H),8.37(d,J=8.3Hz,1H),8.12(d,J=8.4Hz,1H),7.87-7.78(m,1H),7.73(dd,J=8.3,7.1Hz,1H),7.64(t,J=7.6Hz,1H),7.05(s,1H),6.79(s,1H),6.73(s,1H),5.74(d,J=9.7Hz,2H),5.05(t,J=14.4Hz,2H),4.65-4.57(m,1H),4.50(t,J=6.8Hz,2H),4.46-4.36(m,1H),4.28-4.18(m,2H),3.85-3.76(m,1H),3.54-3.44(m,4H),3.01-2.90(m,1H),2.65-2.56(m,1H),2.39(m,2H),1.78(s,3H).13C NMR(101MHz,CDCl3)δ165.17,163.84,162.91,155.97,150.15,148.81,136.32,135.13,133.55,133.49,132.40,131.75,131.37,129.72,128.77,128.34,128.03,126.73,126.52,125.46,125.04,123.21,122.48,121.65,117.96,115.31,114.24,110.61,86.23,67.71,66.79,59.51,55.70,38.77,38.00,27.83,13.75.HRMS(ESI,m/z)calcd for C37H34N3O8[M+H]+648.2346,found:648.2344.[α]D(CHCl3)90.91°,c=0.14g/100mL.化合物16a2的合成:
化合物15a2按照上述方法合成16a2,粗品用制备TLC板纯化(展开剂:DCM/MeOH=20/1)得黄色固体产物16a2(139.1mg,80%)。1H NMR(400MHz,CDCl3)δ10.02(d,J=9.2Hz,1H),8.86(s,1H),8.77(d,J=7.0Hz,1H),8.39(d,J=8.4Hz,1H),8.14(d,J=8.4Hz,1H),7.89–7.83(m,1H),7.76(t,J=7.7Hz,1H),7.66(t,J=7.6Hz,1H),7.21(s,1H),6.74(s,1H),6.71(s,1H),5.24–5.08(m,2H),4.72–4.64(m,1H),4.68(dd,J=13.3,5.6Hz,1H),4.54–4.42(m,1H),4.36(t,J=6.9Hz,2H),4.18–4.06(m,2H),3.88(s,3H),3.86–3.81(m,1H),3.02–2.91(m,1H),2.65–2.56(m,1H),2.04–1.94(m,4H),1.78(s,3H).13C NMR(101MHz,CDCl3)δ165.00,163.67,162.96,155.96,150.42,148.86,136.34,135.13,133.49,133.45,132.31,131.80,131.34,129.72,128.65,128.19,126.64,126.45,125.39,125.03,123.30,123.24,122.30,121.68,118.07,115.07,114.07,110.83,86.23,68.91,66.79,59.51,56.11,40.06,38.78,26.74,24.69,13.75.HRMS(ESI,m/z)calcd for C38H35N3NaO8[M+Na]+684.2322,found:684.2316.[α]D(CHCl3)48.00°,c=0.18g/100mL.
化合物16a3的合成:
化合物15a3按照上述方法合成16a3,粗品用制备TLC板纯化(展开剂:DCM/MeOH=20/1)得黄色固体产物16a3(136.7mg,77%)。1H NMR(400MHz,CDCl3)δ10.03(d,J=9.1Hz,1H),8.87(s,1H),8.82-8.75(m,1H),8.39(d,J=8.3Hz,1H),8.14(d,J=8.4Hz,1H),7.90-7.83(m,1H),7.76(dd,J=8.4,7.0Hz,1H),7.65(t,J=7.5Hz,1H),7.22(s,1H),6.72(d,J=8.9Hz,2H),5.78(d,J=9.8Hz,2H),5.20-5.05(m,2H),4.70-4.62(m,1H),4.50–4.40(m,1H),4.36-4.27(m,2H),4.14-4.03(m,2H),3.88(s,3H),3.85-3.80(m,1H),3.05-2.91(m,1H),2.64-2.56(m,1H),2.03-1.95(m,2H),1.92-1.85(m,2H),1.79(s,3H),1.69-1.64(m,2H).13C NMR(101MHz,CDCl3)δ165.08,163.78,162.95,155.90,150.38,148.74,136.37,135.14,133.57,133.52,132.37,131.74,131.35,129.72,128.72,128.26,128.11,126.67,126.45,125.43,124.83,123.25,122.37,121.59,117.99,115.18,113.71,110.74,86.20,68.90,66.73,59.53,56.12,40.36,38.74,28.49,27.69,23.48,13.71.HRMS(ESI,m/z)calcd for C39H37N3NaO8[M+Na]+698.2478,found:698.2478.[α]D(CHCl3)54.29°,c=0.23g/100mL.
化合物16b1的合成:
化合物15b1按照上述方法合成16b1,粗品用制备TLC板纯化(展开剂:DCM/MeOH=20/1)得黄色固体产物16b1(157.3mg,74%)。H NMR(400MHz,CDCl3)δ9.95(dd,J=9.1,1.0Hz,1H),8.83(s,1H),8.72(dd,J=7.1,1.3Hz,1H),8.36(dd,J=8.5,1.4Hz,1H),8.12(d,J=8.7Hz,1H),7.82(ddd,J=9.2,6.6,1.4Hz,1H),7.73(dd,J=8.4,7.1Hz,1H),7.64(t,J=7.6Hz,1H),7.36(d,J=9.4Hz,3H),7.29(s,1H),7.07(s,1H),6.86(s,1H),6.77(s,1H),6.02–5.91(m,1H),5.82(d,J=9.6Hz,1H),5.78–5.64(m,1H),5.40–5.33(m,1H),5.29–5.24(m,1H),5.16–4.94(m,2H),4.67(d,J=5.7Hz,2H),4.65–4.58(m),4.50(t,J=6.8Hz,2H),4.47–4.38(m,1H),4.30–4.20(m,2H),4,01–3.93(m,1H),3.51(s,3H),3.38–3.28(m,1H),3.09–3.00(m,1H),2.44–2.35(m,2H).13C NMR(101MHz,CDCl3)δ165.11,163.86,163.49,155.92,153.26,150.47,148.91,137.17,136.32,135.14,133.48,132.43,132.33,131.73,131.36,129.72,128.72,128.69,128.26,126.69,126.51,125.53,125.43,124.77,123.12,123.11,122.38,118.78,118.68,118.32,118.27,117.97,115.20,114.38,110.62,86.08,67.78,66.84,65.93,59.65,55.75,38.03,35..01,27.85.HRMS(ESI,m/z)calcd forC46H40N4NaO10[M+Na]+831.2642,found:831.2638.[α]D(CHCl3)121.56°,c=0.23g/100mL.
化合物16b2的合成:
化合物15b2按照上述方法合成16b2,粗品用制备TLC板纯化(展开剂:DCM/MeOH=20/1)得黄色固体产物16b2(168.7mg,78%)。1H NMR(400MHz,CDCl3)δ9.93(d,J=9.1Hz,1H),8.74(s,1H),8.70(dd,J=7.1,1.3Hz,1H),8.30(dd,J=8.5,1.2Hz,1H),8.06(d,J=8.3Hz,1H),7.85–7.76(m,1H),7.69(dd,J=8.4,7.0Hz,1H),7.65–7.57(m,1H),7.35(d,J=11.9Hz,3H),7.24(t,J=4.3Hz,2H),7.09(s,1H),6.80(s,1H),6.03–5.72(m,3H),5.39–5.32(m,1H),5.27–5.22(m,1H),5.19–5.07(m,2H),4.66(d,J=5.4Hz,3H),4.56(s,1H),4.50–4.41(m,1H),4.35–4.27(m,2H),4.12(t,J=6.4Hz,2H),4.04–3.95(m,1H),3.88(s,3H),3.35–3.23(m,1H),3.06–2.96(m,1H),2.04–1.97(m,4H).13C NMR(101MHz,CDCl3)δ165.06,163.76,163.45,155.90,153.16,150.64,148.91,137.06,136.43,135.19,133.55,133.51,132.35,131.71,131.37,129.75,128.73,128.69,128.25,126.64,126.46,125.51,125.40,124.66,123.09,123.06,122.47,122.31,118.76,118.68,118.29,118.11,115.11,114.10,110.81,86.04,68.89,66.85,65.90,59.57,56.14,40.07,34.97,26.69,24.66.HRMS(ESI,m/z)calcd for C47H42N4NaO10[M+Na]+845.2799,found:845.2801.[α]D(CHCl3)114.63°,c=0.19g/100mL.
化合物16b3的合成:
化合物15b3按照上述方法合成16b3,粗品用制备TLC板纯化(展开剂:DCM/MeOH=20/1)得黄色固体产物16b3(138.6mg,63%)。1H NMR(400MHz,CDCl3)δ9.92(t,J=7.9Hz,1H),8.76–8.65(m,2H),8.32–8.23(m,1H),8.04(t,J=6.4Hz,1H),7.82–7.74(m,1H),7.72–7.63(m,1H),7.63–7.54(m,1H),7.35(d,J=8.4Hz,3H),7.25–7.20(m,3H),6.78(s,1H),5.99–5.83(m,2H),5.82–5.68(m,1H),5.37–5.29(m,1H),5.25–5.19(m,1H),5.11(s,1H),5.08(s,1H),4.64(d,J=5.7Hz,3H),4.47–4.37(m,1H),4.30–4.21(m,2H),4.09–3.93(m,3H),3.87(s,3H),3.33–3.22(m,1H),3.04–2.93(m,1H),1.99–1.92(m,2H),1.90–1.81(m,2H),1.68–1.59(m,2H).13C NMR(101MHz,CDCl3)δ165.06,163.82,163.49,155.82,153.17,150.66,148.84,137.10,136.39,135.16,133.59,133.48,132.36,131.70,131.35,129.72,128.67,128.21,126.64,126.44,125.50,125.41,124.52,123.02,123.01,122.48,122.30,118.71,118.66,118.26,118.00,117.95,115.10,113.83,110.75,86.03,68.93,66.76,65.86,59.63,56.14,40.38,34.96,28.48,27.67,23.49.HRMS(ESI,m/z)calcd forC48H44N4O10[M+Na]+859.2955,found:859.2955.[α]D(CHCl3)142.80°,c=0.13g/100mL.
化合物17a1-17b3的通用合成方法:
在10mL圆底烧瓶中加入化合物16a1-16b3(0.155mmol)溶于无水DCM(3mL),再加入四氢吡咯(45μL,39mg,0.548mmol),氮气保护,再加入Pd[P(Ph)3]4(11.5mg,0.01mmol),氮气保护,室温下搅拌0.5h,TLC监测反应完全(展开剂:二氯甲烷/甲醇=20/1,紫外显色)。加入DCM(10mL)稀释,再加入饱和氯化铵(4mL)萃取,水层再用DCM(10mL*2)萃取,合并有机层,有机层再用饱和氯化钠(10mL)洗涤,收集有机层,无水硫酸钠干燥,抽滤,减压浓缩,粗品用制备TLC板纯化得黄色固体产物17a1-17b3。
化合物17a1的合成
化合物16a1按照上述方法合成17a1,粗品用制备TLC板纯化得黄色固体产物17a1(68.4mg,81%)。17a1的纯度通过Waters Alliance e2695 HPLC***(搭配Waters 2996二极管矩阵检测器),XBridge C18(4.6mm×150mm,5μm)反相柱进行检测,检测条件:流动相A为含0.1%甲酸的乙腈,流动相B为含0.1%甲酸的水,梯度洗脱(0min,流动相A为10%;0-10min,流动相A上升为100%;10-11min,流动相A保持在100%;11-15min,流动相A为10%),流速:0.6mL/min,温度:50℃,进样量:10μL,检测波长:264nm,后续合成的化合物纯度均采用此方法,测得17a1的HPLC纯度97.05%。1H NMR(400MHz,CDCl3)δ9.96(d,J=9.1Hz,1H),8.85(s,1H),8.73(dd,J=7.1,1.3Hz,1H),8.37(dd,J=8.4,1.3Hz,1H),8.12(d,J=8.4Hz,1H),7.84-7.78(m,1H),7.77-7.75(m,1H),7.63(ddd,J=7.9,6.7,1.1Hz,1H),7.28(s,1H),6.79(s,1H),6.71(d,J=1.8Hz,1H),4.53(t,J=6.7Hz,2H),4.33-4.28(m,1H),4.22-4.16(m,1H),3.44(s,3H),3.19-3.09(m,1H),2.98-2.88(m,1H),2.44(t,J=6.6Hz,2H),1.82(s,3H),0.88(t,J=6.7Hz,2H).13C NMR(101MHz,CDCl3)δ165.11,163.75,162.50,160.87,151.00,147.77,140.12,136.14,134.95,133.42,133.34,132.30,131.16,129.62,128.69,128.22,126.72,126.39,125.36,123.54,122.47,121.43,119.11,115.27,111.39,110.65,67.57,55.58,53.75,39.20,38.08,27.88,13.63.HRMS(ESI,m/z)calcd for C33H28N3O5[M+H]+546.2029,found:546.2030.[α]D(CHCl3)279.85°,c=0.13g/100mL.
化合物17a2的合成
化合物16a2按照上述方法合成17a2,粗品用制备TLC板纯化得黄色固体产物17a2(60.7mg,70%)。1H NMR(400MHz,CDCl3)δ10.01(d,J=9.1Hz,1H),8.84(s,1H),8.76(dd,J=7.1,1.4Hz,1H),8.37(d,J=8.4Hz,1H),8.13(d,J=8.4Hz,1H),7.84(dd,J=8.5,6.7Hz,1H),7.76-7.71(m,1H),7.67-7.62(m,1H),7.46(s,1H),6.82(s,1H),6.74(d,J=1.8Hz,1H),4.38(t,J=6.9Hz,2H),4.26-4.17(m,2H),4.16-4.09(m,1H),3.95(s,1H),3.89(s,3H),3.22-3.10(m,1H),3.00-2.89(m,1H),2.07–2.00(m,4H),1.83(d,J=1.7Hz,3H).13CNMR(101MHz,CDCl3)δ165.13,163.73,162.60,160.91,151.13,147.94,140.19,136.38,135.13,133.53,132.41,132.03,131.32,129.73,128.77,128.33,126.74,126.45,125.41,123.59,122.45,121.45,119.20,115.25,111.72,110.81,68.73,56.15,53.80,40.06,39.24,26.66,24.74,13.64.HRMS(ESI,m/z)calcd for C35H33N3NaO6[M+CH3OH+Na]+614.2267,found:614.2264.HPLC purity 99.34%.[α]D(CHCl3)46.90°,c=0.13g/100mL.
化合物17a3的合成
化合物16a3按照上述方法合成17a3,粗品用制备TLC板纯化得黄色固体产物17a3(48.0mg,54%)。1H NMR(400MHz,CDCl3)δ10.02(d,J=9.2Hz,1H),8.84(s,1H),8.76(d,J=6.9Hz,1H),8.36(d,J=8.4Hz,1H),8.12(d,J=8.5Hz,1H),7.84(ddd,J=8.9,6.7,1.5Hz,1H),7.79(d,J=4.0Hz,1H),7.77-7.71(m,1H),7.54(d,J=6.0Hz,1H),7.47(s,1H),6.79(s,1H),6.74(d,J=2.0Hz,1H),4.32(t,J=7.6Hz,2H),4.17-4.04(m,2H),4.16-4.06(m,2H),3.89(s,3H),3.21-2.10(m,1H),3.00-2.89(m,1H),2.03-1.96(m,2H),1.93-1.87(m,2H),1.83(s,3H),1.70-1.65(m,2H).13C NMR(101MHz,CDCl3)δ165.05,163.62,162.59,160.92,151.16,147.91,140.22,136.24,135.02,133.41,132.33,131.24,129.67,128.68,128.21,126.70,126.39,125.36,123.57,122.41,121.44,119.11,115.18,112.58,111.70,110.67,68.83,56.14,53.79,40.37,39.22,28.71,27.87,23.64,13.63.HRMS(ESI,m/z)calcd for C35H32N3O5[M+H]+574.2342,found:574.2338.HPLC purity 99.26%.[α]D(CHCl3)43.33°,c=0.15g/100mL.
化合物17b1的合成
化合物16b1按照上述方法合成17b1,粗品用制备TLC板纯化得黄色固体产物17b1(73.3mg,76%)。1H NMR(400MHz,CDCl3)δ9.98(d,J=9.1Hz,1H),8.87(s,1H),8.74(d,J=7.0Hz,1H),8.38(d,J=8.3Hz,1H),8.14(d,J=8.5Hz,1H),7.84–7.79(m,1H),7.77–7.72(m,1H),7.67–7.62(m,1H),7.30(d,J=5.7Hz,1H),7.21(d,J=8.3Hz,3H),6.81(s,1H),6.68(d,J=8.1Hz,2H),4.55(t,J=6.6Hz,3H),4.36–4.23(m,3H),3.44(s,3H),3.40–3.28(m,2H),2.49–2.41(m,2H).13C NMR(101MHz,CDCl3/CDOD3=10/1)δ165.38,164.75,164.31,164.23,152.56,151.82,145.22,142.19,138.83,136.89,136.69,135.46,133.67,132.51,131.49,129.91,128.87,128.45,126.59,126.15,126.02,125.54,124.04,123.26,122.42,121.17,119.53,114.25,112.27,107.46,67.60,58.65,55.81,38.18,35.83,27.95.HRMS(ESI,m/z)calcd for C38H31N4O5[M+H]+623.2294,found:623.2286.HPLC purity 97.38%.[α]D(CHCl3)538.42°,c=0.13g/100mL.
化合物17b2的合成
化合物16b2按照上述方法合成17b2,粗品用制备TLC板纯化得黄色固体产物17b2(85.8mg,87%)。1H NMR(400MHz,CDCl3)δ10.03(d,J=9.1Hz,1H),8.87(s,1H),8.78(d,J=6.8Hz,1H),8.38(d,J=8.5Hz,1H),8.14(d,J=8.6Hz,1H),7.88–7.83(m,1H),7.78–7.73(m,1H),7.69-7.63(m,1H),7.49(s,1H),7.33(s,1H),7.21(d,J=8.7Hz,2H),6.84(s,1H),6.68(d,J=8.3Hz,2H),4.44–4.30(m,3H),4.24–4.13(m,2H),3.91(s,3H),3.86–3.78(m,1H),3.60–3.52(m,1H),3.41–3.29(m,1H),2.05–1.94(m,4H).13C NMR(101MHz,CDCl3/CD3OD=10/1)δ164.97,164.58,163.78,163.05,152.63,151.88,145.12,142.15,138.85,136.95,136.48,135.25,133.46,132.21,131.28,129.72,128.56,128.05,126.34,125.97,125.82,125.29,123.85,123.03,122.03,121.02,119.31,114.51,112.46,107.30,68.58,58.51,56.17,40.03,35.72,26.64,24.58.HRMS(ESI,m/z)calcd for C39H33N4O5[M+H]+637.2451,found:637.2449.HPLC purity 95.77%.[α]D(CHCl3)121.34°,c=0.16g/100mL.
化合物17b3的合成
化合物16b3按照上述方法合成17b3,粗品用制备TLC板纯化得黄色固体产物17b3(76.6mg,76%)。1H NMR(400MHz,CDCl3)δ10.04(d,J=9.0Hz,1H),8.86(s,1H),8.78(d,J=6.9Hz,1H),8.38(d,J=8.3Hz,1H),8.14(d,J=8.7Hz,1H),7.88–7.83(m,1H),7.78–7.71(m,1H),7.67–7.62(m,1H),7.49(s,1H),7.33(s,1H),7.21(d,J=8.8Hz,2H),6.81(s,1H),6.68(d,J=8.1Hz,2H),4.41–4.27(m,3H),4.16-4.09(m,2H),3.99–3.81(m,4H),3.60–3.51(m,1H),3.39–3.30(m,1H),2.03–1.97(m,2H),1.93–1.85(m,2H),1.69–1.64(m,2H).13C NMR(101MHz,CDCl3/CD3OD=10/1)δ164.75,164.58,163.59,163.04,152.73,151.97,145.04,142.08,139.01,137.08,136.29,135.10,133.27,132.01,131.17,129.63,128.36,127.83,126.26,125.94,125.79,125.18,123.88,122.93,121.89,120.90,119.15,114.56,112.46,107.07,68.65,58.50,56.18,40.31,35.72,28.57,27.62,23.43.HRMS(ESI,m/z)calcd forC40H34N4O5[M+H]+651.2607,found:651.2603.HPLC purity 95.53%.[α]D(CHCl3)166.62°,c=0.17g/100mL.
实施例4:化合物20a1-20b3的合成
化合物18a1-18b3的通用合成方法:
在25mL圆底烧瓶中加入9a1-9b3(0.336mmol),加入无水DMF(5mL)溶解,再加入碳酸钾(116.1mg,0.84mmol)和14(108.0mg,0.437mmol),氮气保护,升至85℃下搅拌20h,TLC监测反应完全(展开剂:石油醚/乙酸乙酯=2/1和DCM/CH3OH=10/1,紫外显色)。反应液减压浓缩,除去多余的DMF,加入EA(15mL)和纯水(5mL)萃取,水层再用EA(15mL*2)萃取,合并有机层,有机层再用饱和氯化钠(10mL*2)洗涤,收集有机层,无水硫酸钠干燥,抽滤,减压浓缩,粗品用柱层析纯化得黄色固体产物18a1-18b3。化合物18a1的合成:
化合物9a1和14按照上述方法合成18a1,粗品经柱层析(流动相:DCM/CH3OH=60/1-45/1)得黄色泡沫状产物18a1(169.3mg,57.7%)。1H NMR(400MHz,CDCl3)δ9.92(d,J=9.1Hz,1H),8.74(s,1H),8.69(dd,J=7.0,1.3Hz,1H),8.29(d,J=8.3Hz,1H),8.06(d,J=8.4Hz,1H),7.80(ddd,J=9.3,6.6,1.5Hz,1H),7.69(dd,J=8.4,7.0Hz,1H),7.60(dd,J=8.2,6.6Hz,1H),7.29(s,1H),6.69(s,1H),6.62(s,1H),5.87(d,J=8.9Hz,1H),5.82–5.70(m,1H),5.14–5.03(m,2H),4.67–4.57(m,1H),4.46–4.37(m,3H),4.11–3.98(m,2H),3.90(s,3H),3.79–3.68(m,1H),3.00–2.84(m,2H),2.82–2.67(m,6H),2.64–2.48(m,6H),2.42–2.33(m,1H),2.12–2.04(m,2H),1.77(s,3H),0.88(s,9H),0.24(d,J=6.2Hz,6H).13C NMR(101MHz,CDCl3)δ165.04,163.63,163.40,155.04,150.23,149.05,136.27,135.06,133.50,133.40,132.37,132.09,131.26,129.68,128.74,128.56,128.29,126.71,126.43,125.97,125.39,123.67,122.44,120.99,117.31,115.25,114.26,110.80,87.00,67.48,66.32,61.50,56.11,55.68,54.95,53.12,53.04,39.03,37.61,26.19,25.63,17.87,13.78,-4.22,-5.29.HRMS(ESI,m/z)calcd for C49H60N5O8Si[M+H]+874.4211,found:874.4214.[α]D(CHCl3)72.66°,c=0.30g/100mL.
化合物18a2的合成:
化合物9a2和14按照上述方法合成18a2,粗品用柱层析纯化(流动相:DCM/CH3OH=60/1)得黄色泡沫状产物18a2(189.6mg,63.6%)。1H NMR(400MHz,CDCl3)δ10.01(d,J=9.0Hz,1H),8.87(s,1H),8.76(dd,J=7.0,1.3Hz,1H),8.39(d,J=8.4Hz,1H),8.14(d,J=8.4Hz,1H),7.85(ddd,J=9.1,6.6,1.4Hz,1H),7.75(dd,J=8.4,7.1Hz,1H),7.65(ddd,J=8.0,6.7,1.1Hz,1H),7.21(s,1H),6.68(s,1H),6.58(s,1H),5.86(d,J=8.9Hz,1H),5.80-5.70(m,1H),5.15-4.99(m,2H),4.66–4.57(m,1H),4.46(t,J=6.9Hz,3H),4.43–4.36(m,1H),4.07–3.92(m,2H),3.89–3.80(m,3H),3.77–3.68(m,1H),3.32–2.15(m,16H),1.90–1.85(m,2H),1.77(s,3H),0.87(s,9H),0.23(d,J=10.3Hz,6H).13C NMR(101MHz,CDCl3)δ165.05,163.64,163.42,155.05,150.21,149.00,136.28,135.07,133.51,133.41,132.38,132.10,131.27,129.69,128.75,128.53,128.30,126.72,126.44,125.85,125.40,123.68,122.45,120.97,117.28,115.26,114.01,110.73,87.00,68.76,66.31,61.49,57.99,56.08,55.68,53.08,52.96,39.03,37.61,26.85,25.61,23.14,17.86,13.78,-4.24,-5.30.HRMS(ESI,m/z)calcd for C50H62N5O8Si[M+H]+888.4368,found:888.4363.[α]D(CHCl3)53.56°,c=0.26g/100mL.
化合物18a3的合成:
化合物9a3和14按照上述方法合成18a3,粗品用柱层析纯化(流动相:DCM/CH3OH=60/1)得黄色泡沫状产物18a3(202.7mg,66.9%)。1H NMR(400MHz,CDCl3)δ10.02(d,J=9.1Hz,1H),8.88(s,1H),8.77(dd,J=7.1,1.3Hz,1H),8.40(d,J=8.2Hz,1H),8.15(d,J=8.5Hz,1H),7.90–7.81(m,1H),7.76(dd,J=8.4,7.1Hz,1H),7.66(t,J=7.5Hz,1H),7.22(d,J=2.7Hz,1H),6.68(s,1H),6.58(s,1H),5.86(d,J=8.8Hz,1H),5.79–5.69(m,1H),5.13-5.02(m,2H),4.66–4.58(m,1H),4.46(t,J=6.9Hz,2H),4.43–4.36(m,1H),4.03-3.92(m,2H),3.88(s,3H),3.78–3.68(m,1H),3.27-2.23(m,16H),1.91–1.82(m,2H),1.77(s,3H),1.54–1.46(m,2H),0.87(s,9H),0.23(d,J=9.7Hz,6H).13C NMR(101MHz,CDCl3)δ164.97,163.57,163.43,155.05,150.28,148.99,136.20,135.01,133.42,133.34,132.30,132.10,131.21,129.64,128.68,128.55,128.20,126.68,126.40,125.79,125.35,123.68,122.38,120.94,117.26,115.17,114.01,110.74,87.00,68.91,66.30,61.50,58.41,56.12,55.70,53.20,53.09,39.04,37.62,28.79,26.38,25.61,24.02,17.86,13.78,-4.25,-5.29.HRMS(ESI,m/z)calcd for C51H64N5O8Si[M+H]+902.4524,found:902.4518.[α]D(CHCl3)60.46°,c=0.34g/100mL.
化合物18b1的合成:
化合物9b1和14按照上述方法合成18b1,粗品用柱层析纯化(流动相:DCM/CH3OH=50/1-30/1)得黄色泡沫状产物18b1(184.5mg,52.4%)。1H NMR(400MHz,CDCl3)δ10.02(d,J=9.2Hz,1H),8.86(s,1H),8.76(d,J=7.0Hz,1H),8.38(d,J=8.4Hz,1H),8.14(d,J=8.4Hz,1H),7.88–7.81(m,1H),7.75(t,J=7.7Hz,1H),7.69–7.61(m,1H),7.37(d,J=9.5Hz,3H),7.30(s,1H),7.25(s,1H),6.73(s,1H),6.64(s,1H),6.04–5.89(m,2H),5.83–5.70(m,1H),5.42–5.32(m,1H),5.27(d,J=10.5Hz,1H),5.19–4.97(m,2H),4.71–4.57(m,3H),4.53–4.32(m,3H),4.14–3.97(m,2H),3.96–3.84(m,4H),3.38–3.25(m,1H),3.05–2.30(m,13H),2.10–1.98(m,2H),0.91(s,9H),0.25(d,J=9.5Hz,6H).13C NMR(101MHz,CDCl3)δ164.93,163.86,163.55,155.04,153.14,150.58,149.16,137.22,136.17,134.98,133.36,133.30,132.43,132.25,132.01,131.16,129.62,128.73,128.62,128.14,126.64,126.36,125.58,125.41,125.31,122.93,122.48,122.32,118.73,118.18,117.39,115.11,114.26,110.83,86.79,67.59,66.40,65.79,61.50,56.13,55.77,54.93,53.34,53.21,37.60,35.06,26.36,25.67,17.90,-4.20,-5.19.HRMS(ESI,m/z)calcd for C58H67N6O10Si[M+H]+1035.4688,found:1035.4680.[α]D(CHCl3)132.46°,c=0.23g/100mL.
化合物18b2的合成:
化合物9b2和14按照上述方法合成18b2,粗品用柱层析纯化(流动相:DCM/CH3OH=50/1-30/1)得黄色泡沫状产物18b2(189.6mg,63.6%)。1H NMR(400MHz,CDCl3)δ10.00(d,J=9.2Hz,1H),8.85(s,1H),8.75(dd,J=7.1,1.3Hz,1H),8.38(dd,J=8.5,1.3Hz,1H),8.13(d,J=8.4Hz,1H),7.85(ddd,J=9.2,6.6,1.4Hz,1H),7.75(dd,J=8.4,7.1Hz,1H),7.65(ddd,J=8.0,6.6,1.1Hz,1H),7.38(t,J=7.9Hz,3H),7.29(s,1H),7.23(s,1H),6.78(s,1H),6.61(s,1H),6.04-5.89(m,2H),5.82-5.70(m,1H),5.42-5.33(m,1H),5.27(dd,J=10.5,1.3Hz,1H),5.16-5.01(m,2H),4.71-4.59(m,3H),4.49-4.37(m,3H),4.07-3.94(m,2H),3.93–3.83(m,4H),3.37-3.24(m,1H),2.99–2.51(m,13H),1.93–1.81(m,4H),0.91(s,9H),0.25(d,J=12.6Hz,6H).13C NMR(101MHz,CDCl3)δ165.09,163.89,163.69,155.04,153.11,150.49,149.12,137.08,136.35,135.12,133.54,133.46,132.40,132.05,131.30,129.73,128.90,128.78,128.64,128.34,126.73,126.46,125.57,125.47,125.42,123.03,122.45,118.78,118.83,118.28,117.39,115.27,114.08,110.79,86.79,68.78,66.41,65.89,61.52,57.95,56.13,55.68,53.03,52.86,37.59,35.09,26.85,25.66,23.06,17.91,-4.20,-5.18.HRMS(ESI,m/z)calcd for C59H69N6O10Si[M+H]+1049.4844,found:1049.4843.[α]D(CHCl3)9.21°,c=0.25g/100mL.
化合物18b3的合成:
化合物9b3和14按照上述方法合成18b3,粗品用柱层析纯化(流动相:DCM/CH3OH=50/1-30/1)得黄色泡沫状产物18b3(223.8mg,62.7%)。1H NMR(400MHz,CDCl3)δ10.01(d,J=9.2Hz,1H),8.86(s,1H),8.76(dd,J=7.1,1.3Hz,1H),8.39(d,J=8.4Hz,1H),8.14(d,J=8.4Hz,1H),7.90-7.81(m,1H),7.75(dd,J=8.4,7.1Hz,1H),7.65(dd,J=8.3,6.7Hz,1H),7.39(d,J=5.8Hz,2H),7.31–7.29(m,1H),7.24(d,J=1.3Hz,1H),6.75-6.68(m,1H),6.62(s,1H),6.03–5.90(m,2H),5.78–5.71(m,1H),5.41–5.33(m,1H),5.29-5.25(m,1H),5.15–5.01(m,2H),4.71-4.59(m,3H),4.50-4.37(m,3H),4.05–3.94(m,2H),3.93–3.85(m,4H),3.37-3.27(m,1H),3.01–2.38(m,13H),1.92–1.83(m,2H),1.74–1.64(m,2H),1.55–1.48(m,2H),0.91(s,9H),0.25(d,J=12.3Hz,6H).13C NMR(101MHz,CDCl3)δ164.97,163.89,163.60,155.05,153.16,150.58,149.12,137.22,136.23,135.03,133.37,132.44,132.29,132.04,131.21,129.66,128.78,128.65,128.19,126.67,126.39,125.49,125.43,125.34,122.96,122.48,122.35,118.76,118.76,118.21,117.36,115.13,114.07,110.80,86.80,68.95,66.41,65.82,61.52,58.41,56.16,55.74,53.20,53.10,37.61,35.09,28.80,26.39,25.66,24.03,17.90,-4.21,-5.17.HRMS(ESI,m/z)calcd for C60H71N6O10Si[M+H]+1063.5001,found:1063.4995.[α]D(CHCl3)124.95°,c=0.23g/100mL.
化合物19a1-19b3的通用合成方法
在10mL圆底烧瓶中加入19a1-19b3(0.263mmol),加入无水THF(3mL)溶解,再加入TBAF(1mol/L in THF,0.53mL,0.526mmol)和冰乙酸(45μL,47mg,0.789mmol),氮气保护,于25℃下搅拌16h,TLC监测反应完全(展开剂:DCM/MeOH=15/1,紫外显色)。反应液减压浓缩,除去多余的THF,加入DCM(10mL),再加入饱和碳酸氢钠(5mL)萃取,水层再用DCM(10mL*2)萃取,合并有机层,有机层再用饱和氯化钠(10mL*2)洗涤,收集有机层,无水硫酸钠干燥,抽滤,减压浓缩,粗品用制备TLC板纯化得黄色固体产物19a1-19b3.
化合物19a1的合成:
化合物18a1按照上述方法合成19a1,粗品用制备TLC板纯化(展开剂:DCM/MeOH=15/1)得黄色固体产物19a1(163.6mg,81.9%)。1H NMR(400MHz,CDCl3)δ10.01(d,J=9.2Hz,1H),8.86(s,1H),8.76(dd,J=7.1,1.3Hz,1H),8.44-8.35(m,1H),8.14(d,J=8.4Hz,1H),7.85(ddd,J=9.2,6.6,1.4Hz,1H),7.75(dd,J=8.4,7.1Hz,1H),7.69-7.62(m,1H),7.21(s,1H),6.70(s,2H),5.76(d,J=9.9Hz,2H),5.17-5.01(m,2H),4.70-4.62(m,1H),4.46(t,J=7.1Hz,3H),4.09-3.99(m,2H),3.88(s,3H),3.85-3.80(m,1H),3.01-2.92(m,1H),2.86-2.43(m,13H),1.92-1.83(m,2H),1.77(s,3H).13C NMR(101MHz,CDCl3)δ165.09,163.67,162.95,155.85,150.35,148.79,136.39,135.16,133.55,133.50,132.41,131.86,131.32,129.73,128.78,128.34,128.27,126.72,126.47,125.43,125.15,123.29,122.43,121.58,117.91,115.25,114.09,110.84,86.12,67.58,66.67,59.64,56.14,55.65,54.72,53.20,53.09,38.88,37.59,26.23,13.73.HRMS(ESI,m/z)calcd for C43H46N5O8[M+H]+760.3346,found:760.3339.[α]D(CHCl3)85.11°,c=0.30g/100mL.
化合物19a2的合成:
化合物18a2按照上述方法合成19a2,粗品用制备TLC板纯化(展开剂:DCM/MeOH=15/1)得黄色固体产物19a2(174.3mg,85.7%)。1H NMR(400MHz,CDCl3)δ10.00–9.89(m,1H),8.81–8.66(m,2H),8.37–8.26(m,1H),8.13–8.02(m,1H),7.88–7.56(m,3H),7.34–7.27(m,1H),6.84–6.69(m,2H),5.91–5.71(m,2H),5.20–5.05(m,2H),4.74–4.60(m,1H),4.52–4.36(m,3H),4.15–4.05(m,2H),3.96–3.82(m,4H),3.05–2.92(m,1H),2.84–2.43(m,13H),2.08–1.99(m,2H),1.82(s,1H),1.78(s,2H),1.31–1.22(m,2H).13C NMR(101MHz,CDCl3)δ165.08,163.67,162.97,155.82,150.38,148.74,136.37,135.15,133.53,133.46,132.39,131.87,131.32,129.73,128.77,128.32,128.25,126.71,126.46,125.41,125.03,123.29,122.43,121.51,117.80,115.24,113.87,110.75,86.08,68.98,66.63,59.67,58.02,56.12,55.61,53.19,53.07,38.87,37.61,27.08,23.18,13.72.HRMS(ESI,m/z)calcd for C44H48N5O8[M+H]+774.3503,found:774.3502.[α]D(CHCl3)39.15°,c=0.39g/100mL.
化合物19a3的合成:
化合物18a3按照上述方法合成19a3,粗品用制备TLC板纯化(展开剂:DCM/MeOH=15/1)得黄色固体产物19a3(131.7mg,63.6%)。1H NMR(400MHz,CDCl3)δ9.93(d,J=9.2Hz,1H),8.75(s,1H),8.69(d,J=7.0Hz,1H),8.30(d,J=8.3Hz,1H),8.06(d,J=8.4Hz,1H),7.84-7.75(m,1H),7.69(t,J=7.7Hz,1H),7.61(t,J=7.5Hz,1H),7.23(s,1H),6.72(s,2H),5.87-5.71(m,2H),5.23-5.03(m,2H),4.71-4.61(m,1H),4.49-4.35(m,3H),3.99(d,J=6.7Hz,2H),3.90(s,3H),3.86-3.78(m,1H),3.03-2.91(m,1H),2.83-2.69(m,5H),2.66-2.44(m,6H),2.36(t,J=7.8Hz,2H),1.89-1.80(m,2H),1.78(s,3H),1.60-1.52(m,2H),1.48-1.41(m,2H).13C NMR(101MHz,CDCl3)δ164.99,163.59,163.01,155.75,150.36,148.74,136.31,135.09,133.48,133.42,132.35,131.96,131.28,129.69,128.73,128.33,128.27,126.70,126.44,125.39,125.12,123.30,122.40,121.59,117.73,115.19,113.88,110.78,86.05,68.95,66.57,59.81,58.38,56.15,55.60,53.14,53.09,38.92,37.61,28.79,26.28,23.96,13.75.HRMS(ESI,m/z)calcd for C45H50N5O8[M+H]+788.3659,found:788.3658.[α]D(CHCl3)20.07°,c=0.45g/100mL.
化合物19b1的合成:
化合物18b1按照上述方法合成19b1,粗品用制备TLC板纯化(展开剂:DCM/MeOH=15/1)得黄色固体产物19b1(115.7mg,47.8%)。1H NMR(400MHz,CDCl3)δ9.97(d,J=9.1Hz,1H),8.81(s,1H),8.72(d,J=7.0Hz,1H),8.34(d,J=8.3Hz,1H),8.09(d,J=8.4Hz,1H),7.79(dd,J=9.2,6.6Hz,1H),7.71(t,J=7.7Hz,1H),7.59(t,J=7.5Hz,1H),7.40(s,1H),7.34(d,J=8.4Hz,2H),7.28(s,1H),7.25(s,1H),6.95(s,1H),6.82(s,1H),6.02-5.87(m,2H),5.84-5.72(m,1H),5.40-5.32(m,1H),5.26(d,J=10.4Hz,1H),5.12(d,J=11.3Hz,2H),4.66(d,J=6.0Hz,3H),4.45(t,J=7.3Hz,3H),4.16-4.07(m,2H),4.04-3.97(m,1H),3.91(s,3H),3.39-3.29(m,1H),3.09-3.01(m,1H),2.84-2.66(m,6H),2.62-2.42(m,7H),2.05-1.98(m,2H).13C NMR(101MHz,CDCl3)δ165.18,163.73,163.44,155.81,153.10,150.67,148.89,137.02,136.47,135.22,133.60,133.56,132.45,132.37,131.82,131.39,129.76,128.90,128.83,128.40,126.70,126.50,125.57,125.46,124.81,122.95,122.63,122.46,118.77,118.34,117.99,115.30,114.15,110.88,85.94,67.65,66.76,65.92,59.63,56.18,55.70,54.62,53.30,53.14,37.60,35.11,26.21.HRMS(ESI,m/z)calcd forC52H53N6O10[M+H]+921.3823,found:921.3823.[α]D(CHCl3)35.68°,c=0.32g/100mL.
化合物19b2的合成:
化合物18b2按照上述方法合成19b2,粗品用制备TLC板纯化(展开剂:DCM/MeOH=15/1)得黄色固体产物19b2(222.9mg,90.7%)。1H NMR(400MHz,CDCl3)δ10.00(d,J=9.2Hz,1H),8.85(d,J=5.1Hz,1H),8.75(dt,J=7.1,1.8Hz,1H),8.41-8.34(m,1H),8.12(d,J=8.3Hz,1H),7.87-7.78(m,1H),7.73(t,J=7.8Hz,1H),7.62(t,J=7.6Hz,1H),7.39(s,1H),7.36(d,J=8.2Hz,2H),7.29(s,1H),7.24(s,1H),6.81(s,1H),6.03-5.92(m,1H),5.88-5.72(m,2H),5.42-5.33(m,1H),5.29-5.25(m,1H),5.12(d,J=11.7Hz,2H),4.71-4.61(m,3H),4.45(t,J=7.1Hz,3H),4.14-3.98(m,3H),3.89(s,3H),3.39-3.29(m,1H),2.97-2.34(m,13H),1.92-1.85(m,2H),1.80-1.72(m,2H).13C NMR(101MHz,CDCl3)δ165.20,163.76,163.46,155.79,153.10,150.68,148.83,137.00,136.49,135.23,133.61,133.54,132.46,132.37,131.82,131.40,129.78,128.90,128.83,128.41,126.71,126.49,125.55,125.45,124.68,122.86,122.63,122.47,118.74,118.34,117.90,115.30,113.90,110.80,85.92,69.09,66.73,65.92,59.65,58.01,56.16,55.63,53.28,53.12,37.65,35.09,27.11,23.17.HRMS(ESI,m/z)calcd for C53H55N6O10[M+H]+935.3980,found:935.3982.[α]D(CHCl3)176.00°,c=0.16g/100mL.
化合物19b3的合成:
化合物18b3按照上述方法合成19b3,粗品用制备TLC板纯化(展开剂:DCM/MeOH=15/1)得黄色固体产物19b3(218.5mg,87.6%)。1H NMR(400MHz,CDCl3)δ10.00(d,J=9.2Hz,1H),8.85(d,J=5.1Hz,1H),8.75(dt,J=7.1,1.8Hz,1H),8.41-8.34(m,1H),8.12(d,J=8.3Hz,1H),7.87-7.78(m,1H),7.73(t,J=7.8Hz,1H),7.62(t,J=7.6Hz,1H),7.39(s,1H),7.36(d,J=8.2Hz,2H),7.29(s,1H),7.24(s,1H),6.87–6.70(m,2H),6.03-5.92(m,1H),5.91–5.74(m,2H),5.40-5.33(m,1H),5.29-5.24(m,1H),5.12(d,J=11.7Hz,2H),4.71-4.61(m,3H),4.45(t,J=7.1Hz,3H),4.14-3.98(m,3H),4.07-3.95(m,3H),3.41-3.29(m,1H),3.12-03(m,1H),2.95-2.35(m,13H),1.91-1.82(m,2H),1.69-1.58(m,2H),1.50-1.43(m,2H).13C NMR(101MHz,CDCl3)δ165.07,163.67,163.50,155.75,153.14,150.61,148.83,137.07,136.42,135.18,133.55,133.52,132.41,131.93,131.37,129.75,128.85,128.79,128.47,128.35,126.71,126.49,125.55,125.44,124.83,123.03,122.57,122.42,118.75,118.30,117.84,115.23,114.00,110.85,85.90,68.91,66.70,65.89,59.82,58.19,56.18,55.51,52.97,52.72,37.60,35.11,28.61,25.84,23.82.HRMS(ESI,m/z)calcd forC54H57N6O10[M+H]+949.4136,found:949.4133.[α]D(CHCl3)32.59°,c=0.38g/100mL.
化合物20a1-20b3的通用合成方法:
在10mL圆底烧瓶中加入化合物19a1-19b3(0.155mmol)溶于无水DCM(3mL),再加入四氢吡咯(45μL,39mg,0.548mmol),氮气保护,再加入Pd[P(Ph)3]4(11.5mg,0.01mmol),氮气保护,室温下搅拌0.5h,TLC监测反应完全(展开剂:二氯甲烷/甲醇=15/1,紫外显色)。加入DCM(10mL)稀释,再加入饱和氯化铵(4mL)萃取,水层再用DCM(10mL*2)萃取,合并有机层,有机层再用饱和氯化钠(10mL)洗涤,收集有机层,无水硫酸钠干燥,抽滤,减压浓缩,粗品用制备TLC板纯化得黄色固体产物20a1-20b3。
化合物20a1的合成:
化合物19a1按照上述方法合成20a1,粗品用制备TLC板纯化得黄色固体产物20a1(70.6
mg,69.3%)。1H NMR(400MHz,CDCl3)δ9.95(d,J=9.2Hz,1H),8.77(s,1H),8.71(d,J=7.1
Hz,1H),8.31(d,J=8.4Hz,1H),8.08(d,J=8.5Hz,1H),7.81–7.78(m,1H),7.70(t,J=7.7Hz,1H),7.61(t,J=7.7Hz,1H),7.49(s,1H),6.82(s,1H),6.73(s,1H),4.44(t,J=7.3Hz,2H),4.27–4.20(m,1H),4.19-4.06(m,2H),3.91(d,J=10.2Hz,3H),3.86–3.78(m,1H),3.22–3.10(m,1H),3.00–2.90(m,1H),2.81–2.69(m,5H),2.57-2.47(m,7H),2.09–2.01(m,2H),1.82(s,3H).13C NMR(101MHz,CDCl3)δ164.94,164.92,163.54,162.66,160.85,151.04,147.88,140.21,136.19,135.00,133.32,132.24,131.20,129.63,128.61,128.13,126.63,126.38,125.32,123.54,122.31,121.48,119.25,115.08,111.73,110.84,67.39,56.15,55.67,54.85,53.78,53.48,53.12,39.21,37.57,26.22,13.63.HRMS(ESI,m/z)calcd for C40H44N5O6[M+CH3OH+H]+690.3292,found:690.3291.HPLC purity 95.54%.[α]D(CHCl3)123.21°,c=0.16g/100mL.化合物20a2的合成:
化合物19a2按照上述方法合成20a2,粗品用制备TLC板纯化得黄色固体产物20a2(80.5mg,77.4%)。1H NMR(400MHz,CDCl3)δ10.05-9.97(m,1H),8.86(s,1H),8.76(dd,J=7.1,1.3Hz,1H),8.38(dd,J=8.4,1.4Hz,1H),8.14(d,J=8.4Hz,1H),7.85(ddd,J=9.3,6.6,1.4Hz,1H),7.75(dd,J=8.4,7.0Hz,1H),7.65(ddd,J=8.0,6.7,1.1Hz,1H),7.49(s,1H),6.78(s,1H),6.74(q,J=1.9Hz,1H),4.46(t,J=7.1Hz,2H),4.29-4.19(m,1H),4.16-3.99(m,3H),3.91(s,3H),3.21-3.12(m,1H),2.99-2.91(m,1H),2.88-2.68(m,7H),2.66-2.48(m,5H),1.92-1.87(m,2H),1.83(s,3H),1.79-1.73(m,2H).13C NMR(101MHz,CDCl3)δ165.10,163.69,162.65,160.91,151.10,147.92,140.19,136.33,135.11,133.56,133.47,132.42,131.30,129.72,128.80,128.36,126.76,126.47,125.43,123.59,122.49,121.44,119.19,115.33,111.70,110.66,68.80,58.08,56.16,55.72,53.80,53.27,53.18,39.23,37.68,26.91,23.26,13.63.HRMS(ESI,m/z)calcd for C41H46N5O6[M+CH3OH+H]+704.3448,found:704.3448.HPLC purity 95.28%.[α]D(CHCl3)273.96°,c=0.15g/100mL.
化合物20a3的合成:
化合物19a3按照上述方法合成20a3,粗品用制备TLC板纯化得黄色固体产物20a3(80.5mg,77.4%)。1H NMR(400MHz,CDCl3)δ10.02–9.90(m,1H),8.84–8.66(m,2H),8.39–8.27(m,1H),8.14–8.02(m,1H),7.80(s,1H),7.75–7.66(m,1H),7.66–7.58(m,1H),7.49(d,J=2.2Hz,1H),6.81–6.69(m,2H),4.49–4.37(m,2H),4.27–4.19(m,1H),4.10–3.98(m,2H),3.93(s,3H),3.83(dd,J=6.9,2.9Hz,1H),3.22–3.10(m,1H),2.99–2.91(m,1H),2.81–2.68(m,5H),2.65–2.44(m,5H),2.43–2.35(m,2H),1.94–1.80(m,2H),1.83(s,3H),1.62–1.54(m,2H),1.52–1.44(m,2H).13C NMR(101MHz,CDCl3)δ164.85,163.48,162.66,160.83,151.08,147.85,140.19,136.14,134.96,133.27,133.25,132.17,131.15,129.60,128.54,128.05,126.56,126.33,125.27,123.50,122.22,121.48,119.14,114.98,111.67,110.63,68.82,58.33,56.14,55.65,53.77,53.12,52.95,39.19,37.55,28.73,26.21,23.92,13.62.HRMS(ESI,m/z)calcd for C42H48N5O6[M+CH3OH+H]+718.3605,found:718.3589.[α]D(CHCl3)49.24°,c=0.22g/100mL.化合物20b1的合成:
化合物19b1按照上述方法合成20b1,粗品用制备TLC板纯化得黄色固体产物20b1(74.4mg,65.4%)。1H NMR(400MHz,DMSO-d6)δ9.79(d,J=9.7Hz,1H),9.14–9.03(m,1H),8.62–8.46(m,2H),8.21(d,J=8.7Hz,1H),7.89–7.75(m,2H),7.66(t,J=8.0Hz,1H),7.45–7.34(m,1H),7.33–7.25(m,1H),7.22–7.12(m,1H),7.08(d,J=8.0Hz,1H),6.64–6.48(m,2H),6.45–6.32(m,1H),5.34–5.09(m,2H),4.62–4.49(m,1H),4.27–4.07(m,3H),4.00–3.85(m,2H),3.73–3.61(m,3H),3.22(s,2H),2.64–2.47(m,7H),2.44–2.27(m,5H),1.94–1.80(m,2H).13C NMR(101MHz,CDCl3/CD3OD=10/1)δ164.92,164.64,163.73,163.12,151.82,146.17,145.12,142.07,136.50,135.29,133.45,133.35,132.22,131.34,129.77,128.58,128.10,126.42,126.06,125.92,125.34,124.05,123.30,122.99,122.08,120.96,114.81,112.56,109.06,107.37,67.26,58.63,56.30,56.27,55.58,54.97,52.92,37.43,35.83,26.14.HRMS(ESI,m/z)calcd for C45H47N6O6[M+CH3OH+H]+767.3557,found:767.3553.HPLCpurity 95.53%.[α]D(CHCl3)226.58°,c=0.19g/100mL.
化合物20b2的合成:
化合物19b2按照上述方法合成20b2,粗品用制备TLC板纯化得黄色固体产物20b2(53.7mg,46.3%)。1H NMR(400MHz,CDCl3)δ10.05–9.95(m,1H),8.85(s,1H),8.76(d,J=7.2Hz,1H),8.37(d,J=8.3Hz,1H),8.13(d,J=8.4Hz,1H),7.88-7.82(m,1H),7.74(t,J=7.7Hz,1H),7.64(t,J=7.5Hz,1H),7.52(d,J=4.1Hz,1H),7.35–7.30(m,1H),7.23–7.13(m,2H),6.81(s,1H),6.65(t,J=9.2Hz,2H),4.50–4.42(m,2H),4.41-4.34(m,1H),4.30–4.18(m,1H),4.14–3.99(m,2H),3.93(s,3H),3.59–3.50(m,1H),3.33–3.24(m,2H),2.86–2.70(m,6H),2.66–2.42(m,6H),1.91–1.86(m,2H),1.76–1.69(m,2H).13C NMR(101MHz,CDCl3)δ165.00,163.62,162.66,161.11,151.20,147.96,146.23,140.23,136.27,135.06,133.43,133.38,132.31,131.24,129.68,128.69,128.23,126.63,126.39,126.08,125.98,125.35,123.82,123.42,122.37,120.66,119.05,115.08,111.71,110.72,68.79,58.03,56.23,56.16,55.69,53.78,53.15,37.62,29.31,26.88,23.16.HRMS(ESI,m/z)calcd forC46H49N6O6[M+CH3OH+H]+781.3714,found:781.3710.HPLC purity 97.75%.[α]D(CHCl3)93.79°,c=0.15g/100mL.
化合物20b3的合成:
化合物19b3按照上述方法合成20b3,粗品用制备TLC板纯化得黄色固体产物20b3(94.5mg,80%)。1H NMR(400MHz,CDCl3)δ12.30(s,1H),10.00(d,J=9.2Hz,1H),9.75(s,1H),8.89(s,1H),8.77(d,J=7.1Hz,1H),8.41(d,J=8.5Hz,1H),8.16(d,J=8.5Hz,1H),7.90–7.84(m,1H),7.77(t,J=7.7Hz,1H),7.67(t,J=7.5Hz,1H),7.49(s,1H),7.33(s,1H),7.22(d,J=8.1Hz,2H),6.77(s,1H),6.68(d,J=8.2Hz,2H),4.50–4.33(m,3H),4.18–3.99(m,3H),3.90(s,3H),3.81(s,1H),3.54(s,1H),3.37(s,1H),3.35–3.25(m,2H),3.24–3.14(m,2H),3.09–2.98(m,2H),2.94–2.64(m,6H),1.94–1.84(m,2H),1.67–1.61(m,2H),1.58–1.52(m,2H).13C NMR(101MHz,CDCl3)δ165.22,163.82,162.70,161.12,151.07,147.93,146.14,140.25,136.52,135.26,133.69,133.59,132.52,131.46,129.83,128.90,128.50,126.73,126.55,126.15,126.03,125.50,123.79,123.61,122.52,120.79,119.21,115.15,111.76,110.75,68.65,60.40,56.17,55.40,53.81,52.64,44.93,37.50,35.57,28.46,24.38,23.77.HRMS(ESI,m/z)calcd for C47H51N6O6[M+CH3OH+H]+795.3870,found:795.3878.HPLC purity 98.77%.[α]D(CHCl3)100.85°,c=0.15g/100mL.
实施例5:CCK-8法检测杂交分子的体外细胞毒性
DMEM、RMPI1640、胎牛血清购自Gibco BRL公司(Invitrogen Corporation,USA)。人卵巢癌细胞系SKOV3、人胃癌细胞系NCI-N87和人乳腺癌细胞系MDA-MB-231均购于ATCC(American type culture collection),并采用文献推荐的方法进行培养,即MDA-MB-231细胞和SKOV3细胞用10%胎牛血清的DMEM培养基培养,NCI-N87细胞用含10%胎牛血清的RPMI-1640培养基培养。所用测试化合物均由实验室自行制备。CCK-8试剂盒购买自日本同仁化学研究所。
取对数生长期的人乳腺癌细胞系MDA-MB-231、人卵巢癌细胞系SKOV3和人胃癌细胞系NCI-N87,用含10%胎牛血清的DMEM培养液调整各细胞密度,SKOV3细胞密度为3000个/孔,NCI-N87细胞密度为5000个/孔,MDA-MB-231密度为4000个/孔,于96孔培养板中加入100μL单细胞悬液/孔,于37℃,5%CO2条件下培养。
过夜细胞贴壁后,加入不同浓度的各测试化合物,本发明中待检测的杂交分子有17a1-17b3和20a1-20b3,选用奥沙利铂为阳性对照。这些化合物溶解于DMSO作为母液保存于4℃冰箱待用,其中杂交分子的母液浓度为10mM,奥沙利铂的母液浓度为100mM。本发明的体外细胞毒性研究设置8个梯度浓度,杂交分子浓度设为1000nM、500nM、100nM、20nM、4nM、0.8nM、0.16nM、0.032nM,奥沙利铂的浓度设为500μM、50μM、10μM、2μM、0.4μM、80nM、16nM、3.2nM,需在生物安全柜内用相应的培养基稀释适量体积的母液并梯度稀释至特定浓度。由于本发明在本实验中不吸除96孔板中各孔的培养基,而是直接加入等体积(100μL)的化合物溶液与细胞共孵育,因而实际上需加倍配制各化合物梯度溶液,当然只需初始浓度加倍即可。
化合物与细胞共孵育72h后,往各96孔板内加入CCK-8试剂,每孔20μL,加药时注意不要在孔内产生气泡,加完后置37℃避光孵育约2h,期间注意观察96孔板颜色梯度。然后将96孔板置于酶联免疫检测仪450nm下检测各孔的OD值,计算各孔的抑制率,计算公式如下:抑制率%=(A对照孔-A化合物孔)/(A对照孔-A空白孔)×100%,其中:
A对照孔为含有细胞、CCK-8和培养基,不含化合物的孔测得的OD值;
A化合物孔为含有细胞、CCK-8、培养基和化合物的孔测得的OD值;
A空白孔为含有CCK-8和培养基,不含细胞和化合物的孔测得的OD值;
各化合物各孔的抑制率输入Graphpad prism软件即可计算各化合物对于各细胞的IC50值(半数抑制浓度,即当50%的细胞增殖被抑制时对应的化合物/药物浓度)。重复上述实验操作3次以上,综合分析获得这些杂交分子的体外毒性数据,结果见下表1:
表1杂交分子的体外细胞毒性(72h)
P<0.05,差异具有统计学意义。
实验结果表明,我们设计合成的杂交分子均具有良好的抗肿瘤活性(0.17-221.2nM),均远高于奥沙利铂(4.1μM-11.2μM),并且除17a1、17a2和17a3外,这些杂交分子的活性也高于文献之前报道的PBD-萘酰亚胺杂交分子(0.1μM-0.1mM)。在所有测试的杂交分子中,20b3表现出优越的抗肿瘤活性(0.17-0.94nM),其合成产率也是最高的,另外,20b3位于PBD的C环的对苯氨基,可引入ADC的linker,有利于后续ADC的制备。因此,20b3是一个有潜力的候选弹头。
实施例6:20b3抑制肿瘤细胞周期和诱导凋亡
收集对数生成期的SKOV3细胞,计数,铺6孔板,细胞密度为50万个/孔,体积为2mL/孔,置恒温细胞培养箱(37℃,5%CO2)中培养。候选化合物20b3(10mM in DMSO)用培养基稀释至0.33nM、1nM和3nM待用。细胞培养24h后,吸除6孔板中的培养基,将新配制的20b3溶液加至各6孔板中,体积为2mL/孔,阴性对照加入等体积培养基,各6孔板放回培养箱中继续培养24h。然后将细胞上清全部收集到15mL离心管中,剩余贴壁细胞加1mL胰酶消化后也一起收集到离心管内,离心(1000g,5min),去除上清液。接着加入1mL预冷的PBS,重悬细胞,转移至1.5mL离心管,离心(4℃,1000g,5min),吸除上清,再加入1mL预冷的70%乙醇,轻轻吹打混匀,置4℃冰箱固定过夜后再次离心(4℃,1000g,5min),吸除上清。紧接着往每管细胞样品中加入0.5mL新鲜配制的PI染色液(根据说明书配制),缓慢并充分重悬细胞,37℃避光温浴30min后转至流式上样管中,用流式细胞仪(安捷伦,Novocyte2070R)检测和分析20b3对肿瘤细胞周期的抑制情况。
细胞凋亡实验中细胞培养和细胞收集基本同细胞周期实验,不同的是凋亡实验铺板密度为30万个/孔,20b3作用时间为48h,以及在消化细胞时采用不含EDTA的胰酶。提前从-20℃冰箱中取出Annexin V-FITC细胞凋亡检测试剂盒,解冻,根据说明书配制适量染色液,冰上避光保存待用。需设置4份阴性对照组,用作流式检测参数调节的对照样品,即Annexin V-PI双无、Annexin V-PI双染、Annexin V单染和PI单染。临时配好染色液后,加210μL至各细胞样品中,室温避光孵育15min后转至流式上样管,并在1h内进行流式细胞仪检测,分析20b3对肿瘤细胞的凋亡诱导效应。
20b3抑制细胞周期和诱导凋亡的实验结果如图1所示。由结果可知,在细胞周期实验中,空白对照组的S期比例为17.96%,经0.33nM、1nM和3nM的20b3作用后,肿瘤细胞的S期比例明显上升,并且呈现浓度依赖效应,分别为19.76%、26.01%和50.45%。而在凋亡实验中,空白对照组的凋亡细胞(Annexin V+)比例为9.65%,而经0.33nM、1nM和3nM的20b3作用后,凋亡细胞的比例明显上升,且呈浓度依赖性,分别为9.71%、16.11%和58.68%。细胞周期和凋亡实验结果表明,20b3能剂量依赖性地抑制细胞有丝***于S期,诱导细胞凋亡,发挥抗肿瘤作用。
实施例7:20b3诱导的DNA链间交联
使用限制性内切酶HindIII-HF(星选酶)切割pBR322质粒,使其线性化后加入EtOH沉淀DNA,再使用基因组DNA提取试剂盒回收质粒,NanoDrop 2000测定其浓度并稀释至100ng/μL待用。配制20b3母液(1mM in DMSO),并用超纯水稀释配成50、25、2、15、10和5μM的溶液,奥沙利铂溶液同法配制成100、10和1μM,两者均在-20℃保存待用。取11个200μL PCR管,依次编号为1-11,并加入1μL线性化的pBR322质粒DNA(100ng/μL),然后在1-2号管中加入1μL含有5%DMSO的超纯水,3-8号管中分别加入1μL的5、10、15、20、25和50μM的20b3溶液,9-11号管中分别加入1、10和100μM的奥沙利铂溶液,最后所有PCR管均用TEOA缓冲液补足至10μL,涡旋振荡,瞬离,置37℃下孵育2h后,将1号管置于冰上,2-11号管置于PCR仪上92℃加热3min后,立即置于冰上。随后往各PCR管中分别加入2μL上样缓冲液(6×),混匀后取6μL加入1%琼脂糖凝胶的胶孔中,100V电泳45min后取出凝胶,在Bio-Rad凝胶成像***中观察和调整电泳图像,扫描成像,用ImageJ进行灰度分析,计算各样品组DNA的交联率,实验方案如图2所示。
实验结果如图3所示,使用ImageJ对各条带进行灰度分析,计算出0μM、0.01μM、0.1μM、0.5μM、1μM、2.5μM和5μM的20b3对应的DNA交联率为0%、1.6%、20.3%、64.4%、81.6%、95.1%和98.8%,而1μM、10μM和100μM的奥沙利铂对应的DNA交联率为2.3%、17.3%和100.0%,这说明20b3可诱导DNA的链间交联,并且随着经不同浓度的20b3处理后,DNA的链间交联率呈现剂量依赖性。另外,可以看到,20b3为5μM时的交联率和阳性对照药奥沙利铂为100μM时的交联率基本相当,表明20b3的链间交联能力约是奥沙利铂的20倍。
实施例8:20b3的细胞内分布
配制500μM的20b3的DMSO母液,取适量体积加入到1cm石英比色皿中,室温放置10min后,选择三维扫描方式,设置扫描参数,对样品进行三维扫描,然后使用OringinPro2019b软件处理数据和绘图,获得20b3的三维荧光等高线图。培养、收集对数生长期的SKOV3和MDA-MB-231细胞,铺入35mm激光共聚焦培养皿,细胞密度均为10万个/皿,体积为2mL/皿,置培养箱中孵育24h。吸除培养皿中旧的培养基,将新配制好的25nM的20b3溶液加至共聚焦培养皿中,2mL/皿,然后将培养皿放回培养箱继续培养12h。再次吸除旧的培养基,1mL 1×PBS清洗三次,Hoechst33342(PBS 1:100稀释,1mL)染色细胞核,37℃孵育0.5h。再次吸除旧的培养基,1mL 1×PBS清洗三次后加1mL PBS,于激光共聚焦显微镜选择合适的激发光通道观察。
在本发明研究中,我们使用荧光分光光度计测得20b3的三维荧光光谱数据。由检测结果可知(图4),20b3的最大激发波长为468nm,发射光谱范围为480-560nm,在激发波长为464-550nm下的发射光谱具有较强的荧光强度,而480-560nm的发射光主要呈现绿光,因此我们可以选择488nm作为共聚焦显微镜的激发波长,此时在镜下的20b3则呈现绿色荧光。另一方面,我们使用DNA荧光染料Hoechst 33342进行细胞核染色,其在405nm的激发波长下呈现蓝光,而在488nm的激发波长下不显色,因此结合20b3的荧光特性,可以在共聚焦显微镜下观察20b3进入细胞后在细胞内的分布情况。由激光共聚焦显微镜下观察结果可知(图5),20b3的绿色荧光主要出现在细胞核内,在胞浆中基本无分布,表明20b3进入肿瘤细胞后,主要分布在细胞核,也进一步证明20b3的作用靶点为细胞核内的DNA。
实施例9:20b3诱导肿瘤细胞DNA损伤
培养、收集对数生长期的SKOV3和NCI-N87细胞,铺6孔板,细胞密度分别为25万个/孔和50万个/孔,体积为2mL/皿,置培养箱中孵育24h。吸除6孔板中的培养基,将新配制的20b3溶液(浓度分别为12.5、2.5、0.5nM、0.1nM和0.02nM)加至各6孔板中,体积为2mL/孔,阴性对照加入等体积培养基,各6孔板放回培养箱继续培养48h。预冷的PBS洗涤2次,细胞刮刀刮下细胞至1.5mL离心管中,离心(4℃,3500rpm,5min),弃去上清,加入适量RIPA裂解液,充分混匀,冰上裂解2h,离心(4℃,13000g,20min),上清即为细胞总蛋白。吸取适量上清,使用BCA蛋白定量试剂盒测定总蛋白浓度,其余加入适量5×SDS-PAGE蛋白上样缓冲液,并在100℃下加热5min,分装保存于-20℃备用。
配制分离胶(10%,5ml),加入玻璃板的空隙中,立即封上饱和异丙醇与水的混合液,待分离胶凝固后,倒去上层液体,并用滤纸吸干。配制浓缩胶(5%,2mL),加至分离胶上层,***梳齿。将新配制好的SDS-PAGE凝胶放入电泳槽,加入电泳缓冲液后拔掉梳子,依次加入Marker和蛋白样品(30μg),进行电泳。装配转膜“三明治”,将转移槽置于冰水浴中,放入“三明治”,加入转膜缓冲液至完全没过“三明治”,进行转膜。转膜结束后取出杂交膜,用适量的TBST洗膜,再将膜转移至5%脱脂牛奶中,摇床上室温封闭2h。封闭结束后将膜取出,加入5%脱脂牛奶稀释后的一抗(按1:1000比例稀释),4℃摇晃孵育过夜。
然后用适量的TBST洗膜3次,5min/次,将膜取出后加入TBST稀释后的辣根过氧化物酶标记的二抗(按1:5000比例稀释),37℃摇晃孵育1h。接着再次用适量的TBST洗膜3次,5min/次。最后将ECL显色底物Solution A和Solution B按1:1的比例混合,加到膜上后使用化学发光成像仪进行检测。
本发明通过Western Blot研究20b3对DNA损伤及凋亡相关蛋白表达水平的影响。由实验结果可知(图6),经20b3作用48h后,和空白对照组相比,SKOV3和NCI-N87细胞中的γ-H2AX的表达水平显著提升,并呈现浓度依赖性,表明20b3可诱导肿瘤细胞发生DNA损伤。此外,还可发现细胞中的p-Chk2表达也增加了,说明DNA损伤发生后,激活了DNA修复相关通路,使Chk2磷酸化。进一步还可以发现,细胞中的PARP和Caspase-3的表达水平下降,而其切割产物Cleaved Caspase-3和Cleaved PARP的表达水平升高,并呈现浓度依赖性。这些结果表明,20b3可通过诱导DNA损伤,激活DNA损伤应答***,磷酸化Chk2后,进而激活Caspase信号通路,促使PARP和Caspase-3发生剪切,抑制DNA损伤修复,最终导致细胞周期阻滞和细胞凋亡。
实施例10:药物-连接子21的合成:
在25mL圆底烧瓶中依次加入2-乙氧基-1-乙氧碳酰基-1,2-二氢喹啉(EEDQ)(48.7mg,0.20mmol)和Mc-Val-Ala-OH(75mg,0.20mmol),溶于二氯甲烷和甲醇的混合溶剂中(DCM/MeOH=20/1,2mL),氮气保护,室温搅拌0.5h后,再置于冰浴下搅拌5分钟,然后加入化合物20b3(50mg,0.066mmol)的溶液(DCM/MeOH=20/1,3mL),撤去冰浴,室温下搅拌16h,TLC监测反应完全(展开剂:二氯甲烷/甲醇=15/1,紫外显色)。减压浓缩,除去溶剂,粗品用少量二氯甲烷和甲醇的混合溶剂(DCM/MeOH=10/1)溶解后,经制备TLC板纯化得黄色固体产物21(33mg,44.7%)。1H NMR(400MHz,CDCl3)δ9.92–9.82(m,1H),8.70–8.60(m,2H),8.24(d,J=9.2Hz,1H),8.01(d,J=7.8Hz,1H),7.79–7.72(m,1H),7.69–7.61(m,2H),7.60–7.54(m,2H),7.49(d,J=8.4Hz,1H),7.41(s,1H),7.30(s,1H),7.27–7.10(m,3H),6.66(s,2H),4.39(d,J=7.4Hz,3H),3.92(s,2H),3.79(s,1H),3.48–3.33(m,5H),2.77(d,J=7.6Hz,6H),2.57(s,3H),2.48–2.37(m,3H),2.32–2.19(m,2H),1.91–1.75(m,2H),1.71–1.37(m,15H),1.28–1.23(m,2H),0.99–0.90(m,6H).13C NMR(101MHz,CDCl3)δ174.07,173.39,172.19,171.78,170.85,170.83,164.94,163.58,162.73,161.36,153.55,151.40,148.59,147.94,145.34,140.32,137.57,136.25,135.05,134.04,133.37,132.26,131.23,129.65,128.64,128.17,126.63,126.38,125.34,122.31,120.10,118.71,115.10,111.68,110.73,68.87,58.33,56.18,55.64,53.13,52.97,50.45,37.56,37.48,29.66,28.76,28.27,28.17,26.37,26.24,26.19,25.27,25.00,23.93,19.23,18.90,18.52,18.19.HRMS(ESI,m/z)calcd for C65H76N9O11[M+CH3OH+H]+1158.5664,found:1158.5616.HPLC purity98.20%.[α]D(CHCl3)15.45°,c=0.13g/100mL.
实施例11:抗体偶联物T-PBA的制备和偶联情况分析
本实验前需先配制ADC偶联缓冲液和ADC保存缓冲液,4℃保存备用。将适量Trastuzumab冻干粉末溶于注射用水作为抗体原液,控制浓度在20mg/mL左右,冻存于-80℃备用。临用前采用AKTA Purifier 100蛋白纯化***将抗体原液置换到ADC偶联缓冲液(14.4mM NaH2PO4+5.6mM Na2HPO4+20mM NaCl+1mM EDTA,pH 7.40)中,然后用30KDMillipore超滤浓缩管进行浓缩,期间采用Nanaodrop 2000测定抗体浓度,控制在10mg/ml左右,-20℃保存备用。
偶联反应前,需先用TCEP(1.74mM in ADC偶联缓冲液,临用现配)部分还原抗体的链间二硫键,本发明中抗体和TCEP的摩尔比为1:2,反应在1.5mL离心管中进行,1个反应体系的体积为0.8mL,使抗体终浓度为5mg/mL,反应离心管横放置于摇床(37℃,120rpm)反应1.5h。还原反应完成后,于生物安全柜内直接往反应体系中加入新配制的药物-连接子21的DMF溶液,药物-连接子21和抗体的摩尔比为4:1,且整个反应体系中DMF的占比为10%,即需加入88.9μL浓度为1.361mg/mL的DMF溶液。偶联反应离心管横放置于摇床(25℃,120rpm)反应。偶联反应2h后,离心(4℃,4000rpm,5min),取上清至新的1.5mL离心管,然后使用脱盐柱(HiTrapTM Desalting 5mL)联合AKTA Purifier 100蛋白纯化***将偶联反应样品从ADC偶联缓冲液置换到ADC保存缓冲液(14.4mM NaH2PO4+5.6mM Na2HPO4+50mM NaCl,pH 7.40)中,使用30KD Millipore超滤浓缩管进行浓缩,使样品浓度为10mg/mL左右,分装,标记名称为T-PBA,以及浓度和时间,置-80℃保存待用。
抗体偶联物T-PBA制备完成后,本发明采用SDS-PAGE联合紫外荧光和紫外双波长法分析T-PBA的偶联情况。
在SDS-PAGE实验中,Trastuzumab和T-PBA用PBS稀释至合适浓度,各均分成两份,每份10μg,然后分别加入3μL 5×变性还原和非变性非还原蛋白上样缓冲液混匀,补加UP水至15μL,再取与5×变性还原蛋白上样缓冲液混匀的样品于100℃下煮3min,将所有制备好的样品瞬时离心,4℃保存待用。
样品准备完后,将新配制的SDS-PAGE凝胶(方法同前)放入电泳槽,加入电泳缓冲液后拔掉梳子,向每个泳道加入3μg或10μL刚制好的样品和Marker,开始电泳。注意观察电泳情况,待溴酚兰条带跑至玻璃板底部时终止电泳。电泳完成后,用拨片剥下玻璃板上的SDS-PAGE胶,用清水洗净,将其平放于Bio-Rad凝胶扫描仪的载胶台上,用紫外激发并采集经紫外激发后的SDS-PAGE胶发光图像。然后再将SDS-PAGE胶放入考马斯亮蓝染色液中浸没,置于摇床轻摇,染色过夜后取出,加入脱色液浸没,置于摇床轻摇脱色,脱色干净后,将SDS-PAGE胶置于凝胶扫描仪上扫描并采集考染结果。
本发明采用紫外双波长法分析T-PBA的偶联情况时需先测定药物-连接子21和Trastuzumab在280nm及335nm下的摩尔吸光系数(ε),其中280nm为抗体的特征吸收波长,335nm药物-连接子21的特征吸收波长。抗体在280nm下的摩尔相关系数可查询为219123,而抗体在335nm下无吸收,此时的摩尔消光系数记为0。测定药物-连接子21这两个波长下的摩尔吸光系数(ε)的方法如下:将药物-连接子21配制成0.025mg/mL的DMF/H2O溶液,然后在紫外分光光度计上测定此溶液在280nm及335nm下的吸光度值,平行测定三次取平均值。然后根据Lambert-Beer定律A=Ecl计算弹头-linker在280nm和335nm下消光系数和/>及摩尔消光系数/>和/>
待T-PBA制备完成后,即可测定其在280nm及335nm的吸光度值,然后根据UV-DAR计算公式计算T-PBA的抗体药物偶联比(即偶联率,DAR),公式如下: 其中,cD为药物-连接子21的浓度,cAb为抗体浓度,/>为抗体在280nm下摩尔吸收系数,A280为T-PBA在280nm下的吸光度值,A335为T-PBA在335nm下吸光度值,/>为抗体在335nm下摩尔吸收系数(无吸收),/>为药物-连接子21在335nm下摩尔吸收系数,/>为药物-连接子21在280nm下摩尔吸收系数。
在本发明中,我们基于将抗体链间二硫键部分还原的巯基偶联方式制备获得了抗体偶联物T-PBA(图7),然后采用SDS-PAGE联合紫外荧光和紫外双波长法分析T-PBA的偶联情况。
由于药物-连接子21的结构具有刚性平面结构和大π键,使得T-PBA除具备抗体本身的属性外,还具有强荧光特性。因此,本发明可采用SDS-PAGE联合紫外检测法分析T-PBA的偶联情况。检测结果如图8所示,上图为考马斯亮蓝染色,下图为紫外激发荧光图。在右上方的非还原性电泳中,Trastuzumab和T-PBA的主条带分子量都为150KD左右,条带清晰,纯度符合后续实验要求。在左上方的还原性电泳图中,Trastuzumab和T-PBA均被还原成两个片段,分子量分别为50KD(重链)与25KD(轻链),在T-PBA的轻链略上方的小条带是由于偶联上弹头后分子量增加所致。在下面的紫外激发荧光图中,右下方的还原性电泳显示T-PBA在150KD位置有荧光,而Trastuzumab在150KD处未观察到荧光,这说明抗体成功偶联上了药物-连接子21。而在左下方的还原性电泳图中,T-PBA分别在50KD与25KD处都显出荧光,50KD处的条带比25KD处的条带荧光更强,而Trastuzumab在这两个条带位置没有观察到荧光,说明抗体的轻链和重链上均偶联上了药物-连接子21,但主要偶联在重链上。
在本发明研究中,我们还采用紫外双波长法分析T-PBA的偶联情况。我们先测得了0.025mg/mL的药物-连接子21在280nm和335nm下的吸光度,计算平均值后,推算出消光系数和/>再算出药物-连接子21的摩尔消光系数/>和/>(εD=M×ED,E单位L/(g·cm),M40=1126),最终算得/>和/>分别为15764L/(mol·cm)和19818L/(mol·cm)。另外,我们也测得了Trastuzumab的/>为219123L/(mol·cm),而Trastuzumab在335nm下无吸收,/>为0。制备获得T-PBA后,我们测得T-PBA在280nm和335nm下的吸光度A280和A335,将各项数据代入UV-DAR计算公式:/>最终算得T-PBA的平均DAR为2.26,测算结果见表2。
表2 T-PBA的UV-DAR测定结果
实施例12:T-PBA的靶向性
培养SKOV3(HER2 3+)、NCI-N87(HER2 3+)和MDA-MB-231(HER2 0-1+)细胞,当细胞处于对数生长期时,每皿加入2mL胰酶,消化细胞约4min后,加入2mL含血清的培养基终止消化,收集至15mL离心管,离心(800rpm,3min),弃上清。然后加入10mLPBS重悬细胞,再次离心(800rpm,3min),弃上清,再加适量PBS重悬细胞,计数,将细胞悬液分装至1.5mL离心管中,106个活细胞/管,每种细胞3管。分管后,再次离心(4℃,3500rpm,3min),吸除上清,置冰上,往各管加入100μL一抗(即为Trastuzumab和T-PBA,临用前用预冷的PBS稀释至10μg/mL,PBS作为阴性对照),混悬细胞,冰上孵育40min。然后往各管加入500μL预冷的PBS,将细胞轻轻吹打混匀,离心(4℃,3500rpm,3min),吸除上清,重复2-3次后各加入100μL二抗(即为山羊抗人IgG/FITC,临用前用预冷的PBS按照1:200稀释成适量体积),混悬细胞,冰上孵育40min后用预冷的PBS洗涤2-3次。最后向各管加入350μL预冷的PBS,轻轻混匀细胞,转至流式进样管,进行流式细胞仪(Novocyte2070R)检测和分析。
经过上述实验操作和数据分析,获得研究结果如图9所示(红色为PBS对照组,绿色为Trastuzumab,蓝色为T-PBA)。由图可知,T-PBA和Trastuzumab对于高表达HER2的SKOV3和NCI-N87细胞均展现出很强的结合能力,而对于低表达HER2的MDA-MB-231细胞的结合能力都非常弱,表明偶联上小分子化合物(药物-连接子21)后,抗体对于HER2的靶向性没有明显改变。
实施例13:T-PBA的内化效率
培养、收集对数生长期的SKOV3(HER2 3+)和NCI-N87(HER2 3+)细胞,各分装至9个1.5mL离心管中,共18管。然后往没管中加入100μL一抗(即为Trastuzumab和T-PBA,临用前用预冷的PBS稀释至10μg/mL,PBS作为阴性对照),混悬细胞,冰上孵育40min后用预冷的PBS洗涤2-3次,每次500μL。随后向各管中加入100μL预冷的PBS,混悬细胞,置37℃孵箱孵育0h、1.5h和4h,使抗体内化。孵育到指定时间点后,停止孵育,离心(4℃,3500rpm,3min),吸除上清,再往各管中加入二抗(即为山羊抗人IgG/FITC,临用前用预冷的PBS按照1:200稀释成适量体积),混悬细胞,冰上孵育40min后同法用预冷的PBS洗涤2-3次,每次500μL。最后往各管中加入350μL预冷的PBS,混匀细胞,转至流式进样管中,进行流式细胞仪(Novocyte 2070R)检测和分析。
经过上述实验操作和数据分析,获得研究结果如图10所示(最左边的黄色线代表PBS组,红色线代表内化0h,绿色线代表内化1.5h,蓝色线代表内化4h)。由图可知,随着孵育时间增加,Trastuzumab和T-PBA的内化程度也逐渐增加。通过计算可得,在SKOV3细胞中,Trastuzumab和T-PBA 1.5h的内化效率分别为13.74%和14.65%,4h的内化效率分别为21.16%和28.15%;在NCI-N87细胞中,Trastuzumab和T-PBA 1.5h的内化效率为17.69%和15.52%,4h的内化效率分别为25.79%和21.05%。Trastuzumab和T-PBA在两种细胞中的内化效率均相近,表明偶联上小分子化合物(药物-连接子21)后,抗体的内化水平没有明显改变。
实施例14:T-PBA的细胞内分布
培养、收集对数生长期的SKOV3和MDA-MB-231细胞铺入35mm激光共聚焦培养皿,细胞密度均为10万个/皿,2mL/皿。置孵箱(37℃,5%CO2)培养24h后,吸除培养基,加入临用前用培养基稀释至1μM的T-PBA溶液,2mL/皿,放回孵箱继续孵育12h后,吸除培养基,用1×PBS清洗三次,每次1mL。然后加入1mL用无血清无抗生素的DMEM培养基稀释20000倍的Lyso-Tracker(Red)标记溶酶体,置37℃孵育0.5h后,吸除培养基,1×PBS清洗三次,每次1mL。清洗完后加入1mL用PBS稀释100倍的Hoechst33342进行细胞核染色,置37℃孵育0.5h。孵育结束后,吸除培养基,再次同法用1×PBS清洗三次。最后加入1mL PBS,于激光共聚焦显微镜下观察。
本发明已经证明T-PBA的弹头20b3在488nm激发波长下呈现绿光,因此在本实验中,我们选择在488nm激发波长下观察T-PBA内化后的细胞内分布情况,并用在405nm的激发波长下可呈现蓝光的Hoechst 33342染料进行细胞核定位,在561nm的激发波长下会呈现红光的Lyso-Tracker Red染料进行溶酶体定位。由实验结果可知(图11),T-PBA与细胞共孵育12h后,其在高表达HER2的SKOV3细胞内的分布显著高于低表达HER2的MDA-MB-231细胞,且主要分布于溶酶体。上述结果表明,T-PBA经HER2介导的内化作用进入肿瘤细胞,并被转运至溶酶体降解,释放弹头20b3后发挥杀伤效应。
实施例15:T-PBA的体外细胞毒性
方法基本同杂交分子体外细胞毒性实验(实施例5),但T-PBA的梯度浓度设置为1000nM、200nM、40nM、8nM、1.6nM、0.32nM、0.064nM、0.0128nM。此外,还同时检测了弹头20b3和药物-连接子21的体外细胞毒性,浓度设置同T-PBA。
实验结果表明,T-PBA对HER2高表达的SKOV3和NCI-N87细胞均有强效的杀伤活性,其IC50为纳摩尔水平(IC50=5.4-56.1nM),而对于HER2低表达的MDA-MB-231细胞,T-PBA的活性则明显降低,其IC50为344.6nM。在梯度浓度范围内,Trastuzumab没有发挥抗肿瘤作用,其IC50大于1000nM(表3)。除此之外,我们还检测了弹头(20b3)和弹头-linker(40)的体外抗肿瘤活性以进行对比。显然,弹头20b3对测试细胞具有强大的杀伤活性,其IC50为亚纳摩尔水平(IC50=0.17-0.94nM),而药物-连接子21的活性相比弹头明显降低,其IC50为纳摩尔水平(IC50=237.8-341.7),其活性降低了2-3个数量级,这有可能是因为弹头连上linker后,封闭了其药效团氨基,导致活性下降。这些实验结果表明,弹头20b3和药物-连接子21对于不同肿瘤细胞的杀伤效果没有明显的特异性,但制成ADC后,ADC的杀伤效果具有细胞特异性,其活性与肿瘤细胞的HER2表达量相关。
表3 T-PBA、弹头的体外抗肿瘤活性
实施例16:T-PBA抑制肿瘤细胞周期和诱导凋亡
方法基本同20b3抑制肿瘤细胞周期实验和诱导凋亡(实施例6),但T-PBA设置的浓度为1.6nM、8nM和40nM。
由肿瘤细胞周期抑制实验结果可知(图12),空白对照组的S期比例为24.56%,而经不同浓度(1.6nM、8nM和40nM)的T-PBA作用后,细胞的S期比例明显上升,且呈浓度依赖性,分别为34.46%、43.09%和54.82%,而细胞的G0/G1期和G2/M期比例有所降低。由肿瘤细胞凋亡实验结果可知(图x),空白对照组的凋亡细胞(Annexin V+)比例为0.14%,而经不同浓度的T-PBA(1.6nM、8nM和40nM)作用后,凋亡细胞的比例明显上升,且呈剂量依赖性,分别为15.42%、26.86%和36.81%。细胞周期和凋亡实验结果表明,T-PBA和20b3一样,浓度依赖性地抑制细胞有丝***于S期,诱导细胞凋亡,发挥抗肿瘤作用。
实施例17:T-PBA诱导DNA损伤
方法基本同20b3诱导DNA损伤实验(实施例7),但T-PBA设置的浓度为125nM、25nM、5nM、1nM和0.2nM。
由实验结果可知(图13),经T-PBA作用48h后,和空白对照组相比,SKOV3和NCI-N87细胞中的γ-H2AX的表达水平显著增加并呈现浓度依赖性,表明T-PBA可导致SKOV3和NCI-N87细胞发生DNA损伤。另外,细胞中的p-Chk2表达也增加了,说明DNA损伤发生后,激活了DNA损伤应答***,活化Chk2。除此之外,细胞中的PARP和Caspase-3的表达水平下降,而其切割产物Cleaved Caspase-3和Cleaved PARP的表达水平升高,并呈现浓度依赖性。这些结果表明,T-PBA经内化进入细胞后,可经溶酶体途径释放弹头小分子20b3,进而诱导DNA损伤,激活DNA损伤应答***,磷酸化Chk2后,进而激活Caspase信号通路,促使PARP和Caspase-3发生剪切,抑制DNA损伤修复,最终导致细胞周期阻滞和细胞凋亡。
实施例18:T-PBA的体内抗肿瘤活性
本发明构建人卵巢癌SKOV3细胞和人胃癌NCI-N87细胞的Balb/c裸鼠皮下荷瘤模型来评价T-PBA的体内抗肿瘤活性,具体实验方法如下:
提前购买6-8周龄的Balb/c无胸腺雌性裸鼠(体重18-20g)在动物房适应性饲养1周,期间培养SKOV3细胞和NCI-N87细胞至实验所需用量。然后收集对数生长期细胞至50mL离心管内,离心(800rpm,3min),去除上清液,用无血清无抗生素的培养基(双无培养基)适量清洗细胞3次。接着加入适量双无培养基重悬细胞,计数,将SKOV3细胞密度调为108个活细胞/mL,NCI-N87细胞密度调为2.5×107个活细胞/mL。细胞收好后,立即在裸鼠右上肢背部皮下进行接种,100μL/只。接种后,密切观测肿瘤体积,待肿瘤平均体积生长至约200mm3时,每个模型随机分成5组,即对照组(生理盐水)、裸抗组(10mg/kg)、T-PBA(10mg/kg)、T-PBA(5mg/kg)和T-PBA(1mg/kg),每组6只裸鼠。
分组完成后即开始尾静脉注射给药(药液提前于生物安全柜内配制好),100μL/只,每3天给药一次,连续给药4次。从第1次给药开始,每3-4天用电动游标卡尺测量肿瘤长径、短径(单位:mm),根据公式计算肿瘤体积(mm3),即肿瘤体积=1/2×长径×短径2,同时用电子天平称量裸鼠体重(单位:g),观察裸鼠的生理和生活状况。当肿瘤大面积溃烂或体积达到2500mm3时,结束治疗,处死小鼠,当出现显著治疗效果时,可以延长观察时间。根据肿瘤体积和裸鼠体重变化,利用Graphpad Prism软件绘制药物抑瘤曲线和裸鼠体重变化曲线,计算观察时间终点时的抑瘤率(对照组和实验组平均肿瘤体积的差值与照组平均肿瘤体积的百分比),分析评价药物治疗效果。
由动物实验结果可知(图14),在SKOV3模型中,与对照组相比,高、中和低剂量组的抑瘤率分别为91.5%、70.1%和57.5%,呈现剂量依赖性;在NCI-N87模型中,与对照组相比,高、中和低剂量组的抑瘤率分别为97.1%、88.1%和50.2%,也呈现剂量依赖性。这些结果表明,T-PBA在10mg/kg和5mg/kg剂量下均能显著延缓肿瘤生长,具有良好的抗肿瘤效果。另外,整个治疗期间,移植瘤裸鼠状态良好,体重没有明显降低,说明T-PBA在给药剂量下没有明显的毒副作用。
上述实验至终点时,取出对照组、抗体组(10mg/kg)和T-PBA(10mg/kg)裸鼠的心、肝、脾、肺和肾,固定于4%多聚甲醛中,送成都里来生物科技有限公司进行H&E染色。结果如图15所示,与对照组相比,抗体组和T-PBA给药组的各器官均未发现明显的组织病变,表明T-PBA在治疗剂量下无明显毒副作用,具有良好的安全性。
Claims (14)
1.式I所示化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体或其药学上可接受的盐,其特征在于:结构如下:
其中,Z选自l、m、n独立地选自1~8的整数;/>中l端与N相连,m端与O相连;
R1选自C1~C8烷基、取代或未取代的6~10元芳基;其中,所述取代的6~10元芳基的取代基选自C1~C6烷基、C1~C6烷氧基或氨基;
R2选自-H、C1~C8烷氧基或C1~C8烷巯基;
R3选自C1~C6烷基。
2.根据权利要求1所述的化合物,其特征在于:l、m、n独立地选自1~6的整数;优选的,l选自1~3的整数,m选自2~6的整数,n选自2~6的整数;最优选的,l为2,m选自3~5的整数,n选自3~5的整数。
3.据权利要求1所述的化合物,其特征在于:R1选自C1~C6烷基、取代或未取代的6~10元芳基;其中,所述取代的6~10元芳基的取代基选自C1~C4烷基、C1~C4烷氧基或氨基;
优选的,R1选自C1~C4烷基、取代或未取代的6元芳基;其中,所述取代的6元芳基的取代基选自C1~C4烷基、C1~C4烷氧基或氨基;
最优选的,R1选自甲基、对氨基苯基或对甲氧基苯基。
4.根据权利要求1所述的化合物,其特征在于:R2选自-H、C1~C6烷氧基或C1~C6烷巯基;
优选的,R2选自-H、C1~C4烷氧基或C1~C4烷巯基;
更优选的,R2选自-H、甲氧基或甲巯基;
最优选的,R2为-H。
5.根据权利要求1所述的化合物,其特征在于:R3选自C1~C4烷基;优选的,R3为甲基。
6.根据权利要求1~5任一项所述的化合物,其特征在于:所述式I化合物选自:
7.式II所示化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体或其药学上可接受的盐,其特征在于:结构如下:
其中,L选自o、q独立地选自1~10的整数,p选自1~15的整数;
R2如权利要求1或4所述;R3如权利要求1或5所述;Z如权利要求1或2所述。
8.根据权利要求7所述的化合物,其特征在于:至少满足下列的一项:
o、q独立地选自1~8的整数,p选自2~10的整数;优选的,o、q独立地选自1~7的整数,p选自6~8的整数;最优选的,o为5,q为2,p为7;
与苯对位相连;
Z为l、m如权利要求1或2所述。
9.根据权利要求7或8所述的化合物,其特征在于:所述式II化合物选自:
10.权利要求1~9任一项所述化合物的制备方法,其特征在于:包括如下步骤:
a、化合物1经Suzuki偶联反应,得到化合物2:
b、化合物2经Zn粉/AcOH体系还原,得到化合物3:
c、化合物3在碱性条件下与Alloc-Cl反应,得化合物4:
其中,当R1不含氨基时,化合物4的R、为R1,当R1含氨基时,化合物4的R、为单N-Alloc取代的R1;
d、化合物4在酸性条件下脱去TBS保护基,得到化合物5:
e、化合物5经Swern氧化后关环得到化合物6:
f、化合物6在碱性条件下与TBS-OTf反应得到化合物7:
g、化合物7与LiOAc反应脱去TIPS,得到化合物8:
h、化合物8与二碘代烷在碱性条件下反应得到化合物9:
i、化合物10与溴代烷在碱性条件下反应得到化合物11:
j、化合物11与化合物12在碱性条件下反应得到化合物13:
k、化合物13在TFA作用下脱去Boc得到化合物14:
l、化合物9和化合物12在碱性条件下反应得到化合物15:
m、化合物15在TBAF/AcOH体系下脱去TBS得到化合物16:
n、化合物16在Pd催化剂作用下脱去Alloc保护基得到产物17:
o、化合物9和化合物14在碱性条件下反应得到化合物18:
p、化合物18在TBAF/AcOH体系下脱去TBS得到化合物19:
q、化合物19在Pd催化剂作用下脱去Alloc保护基得到产物20:
r、当式I中R1为氨基取代的苯基时,化合物17或20与连接子L-H经酰胺缩合得到式II:
11.根据权利要求10所述的制备方法,其特征是:满足以下至少一项:
步骤a,化合物1、碱和Pd催化剂,在保护气氛下反应;
步骤a,化合物1与的摩尔比为1:(1.0~5.0);优选地,步骤a,化合物1与/>的摩尔比为1:4.0;
步骤a,化合物1与碱的摩尔比为1:(1.0~8);优选地,步骤a,化合物1与碱的摩尔比为1:6.0;
步骤a,Pd催化剂选自Pd(dppf)Cl2·CH2Cl2、PdCl2(PPh3)2、Pd[P(Ph)3]4、Pd(OAc)2中一种或两种以上;
步骤a,化合物1与钯催化剂的摩尔比为1:0.02~0.05;优选地,步骤a,化合物1与钯催化剂的摩尔比为1:0.05;
步骤a,反应溶剂选自丙酮、乙腈、四氢呋喃、甲苯、DMF、1,2-二甲氧基乙烷、水中一种或两种以上;
步骤a,碱选自磷酸钾、碳酸钾、碳酸钠、氢化钠、氢氧化钡、碳酸铯中的至少一种;
步骤a,反应温度为25℃;
步骤a,反应在氮气保护下进行;
步骤b,反应溶剂选自甲醇或乙醇;
步骤c,碱为吡啶;
步骤d,酸为醋酸水溶液,反应溶剂选自甲醇、四氢呋喃、甲醇:四氢呋喃体积比1:1的混合物;
步骤e,化合物5、DMSO、草酰氯和碱,在保护气氛下反应;
步骤e,化合物5与草酰氯的摩尔比为1:(1.0~4.0);优选地,步骤e,化合物5与草酰氯的摩尔比为1:1.3;
步骤e,化合物5与DMSO的摩尔比为1:(1.0~8.0);优选地,步骤e,化合物5与DMSO的摩尔比为1:2.6;
步骤e,化合物5与碱的摩尔比为1:(1.0~8.0);优选地,步骤e化合物5与碱的摩尔比为1:5.0;
步骤e,碱选自二异丙基乙胺、三乙胺中一种或两种;
步骤e,反应溶剂选自二氯甲烷、氯仿、四氢呋喃中的至少一种;
步骤e,反应温度为-78℃;
步骤e,反应在氮气保护下进行;
步骤f,碱为2,6-二甲基吡啶;
步骤g,反应溶剂为N,N-二甲基甲酰胺和H2O的混合溶液;
步骤h,化合物8、二碘代烷和碱,在保护气氛下反应;
步骤h,化合物8与二碘代烷的摩尔比为1:(1.0~6.0);优选地,步骤h,化合物8与二碘代烷的摩尔比为1:5.0;
步骤h,化合物8与碱的摩尔比为1:(1.0~4.0);优选地,步骤h,化合物8与碱的摩尔比为1:1.3;
步骤h,碱选自二异丙基乙胺、三乙胺、磷酸钾、碳酸钾、碳酸钠、碳酸铯中的至少一种;
步骤h,反应溶剂选自N,N-二甲基甲酰胺、N,N-二甲基乙酰胺、2-丁酮、甲苯中的至少一种;
步骤h,反应温度为75℃;
步骤h,反应在氮气保护下进行;
步骤i,化合物10、二溴代烷和碱,在保护气氛下反应;
步骤i,化合物10与二溴代烷的摩尔比为1:(1.0~6.0);优选地,步骤i,化合物10与二溴代烷的摩尔比为1:4.3;
步骤i,化合物10与碱的摩尔比为1:(1.0~4.0);优选地,步骤i,化合物10与碱的摩尔比为1:1.1;
步骤i,碱选自二异丙基乙胺、三乙胺、磷酸钾、碳酸钾、碳酸钠、碳酸铯中的至少一种;
步骤i,反应溶剂选自N,N-二甲基甲酰胺、N,N-二甲基乙酰胺、2-丁酮、二氯甲烷、甲苯中的至少一种;
步骤i,反应温度为45℃;
步骤i,反应在氮气保护下进行;
步骤j,化合物11、化合物12和碱,在保护气氛下反应;
步骤j,化合物11与化合物12的摩尔比为1:(1.0~3.0);优选地,步骤j,化合物11与化合物12的摩尔比为1:1.0;
步骤j,化合物11与碱的摩尔比为1:(1.0~4.0);优选地,步骤j,化合物11与碱的摩尔比为1:1.5;
步骤j,碱选自二异丙基乙胺、三乙胺、磷酸钾、碳酸钾、碳酸钠、碳酸铯中的至少一种;
步骤j,反应溶剂选自N,N-二甲基甲酰胺、N,N-二甲基乙酰胺、2-丁酮、甲苯中的至少一种;
步骤j,反应温度为85℃;
步骤j,反应在氮气保护下进行;
步骤k,反应溶剂选自二氯甲烷、氯仿、四氢呋喃、N,N-二甲基甲酰胺、N,N-二甲基乙酰胺中的至少一种;
步骤l,化合物12、化合物9和碱,在保护气氛下反应;
步骤l,化合物12与化合物9的摩尔比为1:(1.0~3.0);优选地,步骤l,化合物12与化合物9的摩尔比为1:1.1;
步骤l,化合物12与碱的摩尔比为1:(1.0~4.0);优选地,步骤l,化合物12与碱的摩尔比为1:1.5;
步骤l,碱选自二异丙基乙胺、三乙胺、磷酸钾、碳酸钾、碳酸钠、碳酸铯中的至少一种;
步骤l,反应溶剂选自N,N-二甲基甲酰胺、N,N-二甲基乙酰胺、2-丁酮、甲苯中的至少一种;
步骤l,反应温度为85℃;
步骤l,反应在氮气保护下进行;
步骤m,反应溶剂选自二氯甲烷、氯仿、四氢呋喃、N,N-二甲基甲酰胺、N,N-二甲基乙酰胺中的至少一种;
步骤n,化合物16、四氢吡咯和Pd催化剂,在保护气氛下反应;
步骤n,化合物16与四氢吡咯的摩尔比为1:(1.0~4.0);优选地,步骤n,化合物16与四氢吡咯的摩尔比为1:3.5;
步骤n,Pd催化剂选自Pd(dppf)Cl2·CH2Cl2、PdCl2(PPh3)2、Pd[P(Ph)3]4、Pd(OAc)2中的至少一种;
步骤n,化合物16与Pd催化剂的摩尔比为1:0.02~0.10;优选地,步骤n,化合物16与Pd催化剂的摩尔比为1:0.06;
步骤n,反应溶剂选自二氯甲烷、氯仿、四氢呋喃、N,N-二甲基甲酰胺中的至少一种;
步骤n,反应温度为25℃;
步骤n,反应在氮气保护下进行;
步骤o,化合物14、化合物9和碱,在保护气氛下反应;
步骤o,化合物14与化合物9的摩尔比为1:(1.0~3.0);优选地,步骤o,化合物14与化合物9的摩尔比为1:1.1;
步骤o,化合物14与碱的摩尔比为1:(1.0~4.0);优选地,步骤o,化合物14与碱的摩尔比为1:1.5;
步骤o,碱选自二异丙基乙胺、三乙胺、磷酸钾、碳酸钾、碳酸钠、碳酸铯中的至少一种;
步骤o,反应溶剂选自N,N-二甲基甲酰胺、N,N-二甲基乙酰胺、2-丁酮、甲苯中的至少一种;
步骤o,反应温度为85℃;
步骤o,反应在氮气保护下进行;
步骤p,反应溶剂选自二氯甲烷、氯仿、四氢呋喃、N,N-二甲基甲酰胺、N,N-二甲基乙酰胺中的至少一种;
步骤q,化合物19、四氢吡咯和Pd催化剂,在保护气氛下反应;
步骤q,化合物19与四氢吡咯的摩尔比为1:(1.0~4.0);优选地,步骤q,化合物19与四氢吡咯的摩尔比为1:3.5;
步骤q,Pd催化剂选自Pd(dppf)Cl2·CH2Cl2、PdCl2(PPh3)2、Pd[P(Ph)3]4、Pd(OAc)2中的至少一种;
步骤q,化合物19与Pd催化剂的摩尔比为1:0.02~0.10;优选地,步骤q,化合物19与Pd催化剂的摩尔比为1:0.06;
步骤q,反应溶剂选自二氯甲烷、氯仿、四氢呋喃、N,N-二甲基甲酰胺中的至少一种;
步骤q,反应温度为25℃;
步骤q,反应在氮气保护下进行;
步骤r,化合物17或20、连接子L-H和缩合剂,在保护气氛下反应;
步骤r,化合物17或20与连接子L-H的摩尔比为1:(1.0~5.0);优选地,步骤r,化合物17或20与连接子L-H的摩尔比为1:3.0;
步骤r,化合物17或20与缩合剂的摩尔比为1:(1.0~5.0);优选地,步骤r,化合物17或20与缩合剂的摩尔比为1:3.0;
步骤r,反应溶剂选自二氯甲烷、氯仿、甲醇、四氢呋喃、N,N-二甲基甲酰胺、二氯甲烷:甲醇体积比20:1的混合物或氯仿:甲醇体积比20:1的混合物;
步骤r,反应温度为25℃;
步骤r,反应在氮气保护下进行。
12.药物组合物,其特征在于:以权利要求1~9任一项所述的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体或其药学上可接受的盐为活性成分,添加药学上可接受的辅料制备而成。
13.权利要求1~9任一项所述的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体或其药学上可接受的盐、权利要求12所述的药物组合物在制备抗肿瘤药物中的用途;优选的,所述肿瘤为胃癌、乳腺癌、卵巢癌、肺癌、胰腺癌、***癌或尿路上皮癌。
14.权利要求1~9任一项所述的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体或其药学上可接受的盐、权利要求12所述的药物组合物在制备抗体药物偶联物中的用途。
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