CN111606969B - 一种parp1蛋白降解剂及其在抗肿瘤中的应用 - Google Patents
一种parp1蛋白降解剂及其在抗肿瘤中的应用 Download PDFInfo
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Abstract
本发明公开了一种PARP1蛋白降解剂及其在抗肿瘤中的应用。该降解剂包括结构式为
Description
技术领域
本发明属于药物化学技术领域,具体涉及一种PARP1蛋白降解剂及其在抗肿瘤中的应用。
背景技术
在整个生命活动过程中,生物体的基因组DNA经常会受到外源性和内源性因素的影响从而导致DNA损伤,这种损伤使基因组DNA变得不稳定,影响基因的正常复制和表达,进而影响生命体正常的生理活动,甚至会引起机体癌变。这种损伤主要分为内源性损伤和外源性损伤两种,其中内源性损伤包括错误复制、碱基突变、活性氧(RSO)产生和细胞自身代谢物等;外源性损伤包括化学毒物(如烷化剂等)、射线辐射等。研究表明,每个细胞平均每天发生1×104次损伤,细胞为了维持正常的生理功能存在多种DNA损伤发现和修复的机制,受损伤的DNA能够精确及时的得到修复而不影响正常生命活动。
根据DNA损伤的原因和类型大致可以分为5种:包括碱基突变、DNA交联、DNA单链断裂(single-strand breaks,SSBs)、DNA双链断裂(double-strand breaks,DSBs)和碱基错配。生命体在长期的进化过程中形成了针对不同类型DNA损伤的修复机制,具体包括直接修复、碱基切除修复(base excision repair,BER)、核苷酸切除修复(nucleotide excisionrepair,NER)、单链退火修复(single strand annealing,SSA)、错配修复(mis-matchrepair,MMR)、同源重组(Homologous recombination,HR)、和非同源末端连接(Non-homologous end joining,NHEJ)。
SSBs是DNA双螺旋链中一条链发生断裂形成的,是细胞中DNA损伤发生频率最高的一种DNA损伤,内源性因素是SSBs发生的主要原因。DNA单链断裂修复(DNA single-strandbreaks repair,SSBR)主要包括碱基切除修复(BER)、错配修复(MMR)和核苷酸切除修复(NER)。SSBs的DNA如不能及时修复通常会导致致命的DSBs。DSBs产生的主要因素是外源性因素,包括射线辐射和细胞毒性药物的刺激。DNA双链断裂修复(DNA double-strandbreaks repair,DSBR)机制主要包括同源重组(HR)和非同源末端连接(NHEJ)。DNA损伤修复机制对于维持基因组的稳定性具有十分重要的作用。细胞基因水平的改变一方面促成了细胞癌变,但另一方面又为肿瘤治疗提供了良好契机。DNA损伤修复机制对于维持基因组的稳定性至关重要,针对DNA损伤修复的不同调控机制,科学家发展了很多损伤DNA修复的来治疗肿瘤的方法,其中放疗和很多抗肿瘤药物都是通过损伤DNA来达到杀灭肿瘤的目的。因此阻断肿瘤细胞DNA修复通路是抗肿瘤药物研发的一种新策略。
1963年,Chambon发现了一种具有DNA修复能力的聚腺苷二磷酸核糖聚合酶[poly(ADP-ribose)polymerase,PARP]。经过近50年的研究,研究者们也对PARP酶家族的组成和功能有了更清晰的认知。PARP主要参与DNA单链损伤修复,也参与一些重要的细胞过程,包括染色体重塑,调控细胞凋亡、周期和免疫应答等生命过程。目前研究发现,PARP家族至少有18个成员,成员间具有一定的同源性。根据这些酶的结构域的不同分为4类(如图2.1):1)NDA损伤依赖的,包括PARP1-3,PARPs,它们通过与DNA结合域与受损的DNA结合;2)包含锚蛋白重复结构域的Tankyrases,包括Tankyrase-1,Tankyrase-2;3)CCCH型PARPs,包括PARP-7,PARP-12,PARP-13,它们包含与RNA结合的锌指结构域和有PAR结合活性的WWE(Trp-Trp-Glu)域;4)宏观PARPs,仅是单腺苷二磷酸核糖转移酶(mono-ADP-ribosyltransferase)。PRAP1和PARP2是PARP家族中主要的两类酶,其中PARP1发挥着90%以上的功能,PARP2具有与PARP1类似的功能,但两者的底物选择性不同。
PARP抑制剂是目前治疗癌症的一种有效手段,是抗肿瘤药物研发的热点和重点。PARP抑制剂与细胞毒性药物联合应用所引起的合成致死是PARP抑制剂应用的理论基础。PARP抑制剂与化疗药联用在提高化疗药效果的同时也减轻了化疗药物的不良反应。目前已经有4个PARP抑制剂被批准用于临床,其单用和联合化疗都表现出了良好的抗肿瘤的效果。还有一些分子正在进行临床试验,相信不久的将来会有很多效果更好的抑制剂用于临床。
随着PARP抑制剂研究的不断深入,在临床上也发现了一些不利于PARP抑制剂应用的问题有待进一步探讨和厘清。首先是药物长期使用的安全性问题。目前上市的4种PAPR抑制剂中除了Talazoparib外,其余3种每天都需要摄入药物300-600mg。PARP抑制剂半衰期较短,需频繁给药。在治疗过程中长期大量的摄入PARP抑制剂不仅对肿瘤细胞有杀伤作用,对于正常细胞也具有一定的杀伤作用。虽然PARP抑制剂表现出了一定的肿瘤组织选择性,但长期大量药物作用这种选择性还能不能保持有待进一步观察。在联合应用时,保证治疗效果的同时减少不良反应也是一个很重要的方面,因此,必须给PARP抑制剂确定一个合适的给药范围。其次是药物作用机制不完全清楚的问题。第三,PARP长期使用易导致肿瘤耐药性的产生,从而降低了疗效。如何出现耐药以及克服、改善耐药问题有待解决。PARP抑制剂的耐药性产生是一个复杂的过程,其中广泛接受的是BRCA1/2基因二次突变,使其蛋白功能恢复进而对PARP抑制剂耐药。另外,53BP1表达缺失和多药耐药等也使得HR功能部分恢复。癌细胞可能还存在其他耐药机制有待进一步研究,针对不同患者应设置更加精细化的治疗方案延缓和避免耐药性的产生。从PARP抑制剂在临床上的疗效也让我们更加有信心进一步开发PARP抑制剂和新的治疗肿瘤的方案。
通过降解靶点致病蛋白来治疗疾病是目前药物研发领域的一种新模式,其中蛋白降解靶向嵌合体(Proteolysis Targeting Chimera,PROTAC)技术是目前用于肿瘤靶向治疗的一项代表性的技术。PRTOAC是一个双功能的分子,由3部分组成,包括与靶蛋白结合的配体,与E3泛素蛋白连接酶结合的配体,以及连接它们的Linker。通过招募靶蛋白和E3泛素蛋白连接酶是靶蛋白多聚泛素化,进而被细胞内的泛素蛋白酶系统降解。相比于传统的小分子抑制剂,PROTAC化合物有其独特的优势。利用PROTAC技术,我们已经开发了很多在体内外高效降解治病蛋白的PROTAC化合物。目前也已经有PROTAC化合物进入临床研究。本发明利用PROTAC技术设计PARP降解剂,以期在克服小分子PARP抑制剂缺陷的同时,为PARP依赖的肿瘤靶向治疗提供一种新的方法。
发明内容
针对现有技术中的上述不足,本发明提供一种PARP1蛋白降解剂及其在抗肿瘤中的应用,提供了一种不仅能够有效抑制肿瘤细胞增殖活性,还能降解PARP1蛋白的化合物。
为实现上述目的,本发明解决其技术问题所采用的技术方案是:
其中,L为疏水连接单元,包括C4~C14的直链烷烃二酸和直链氨基酸中的至少一种;或由C4~C14的直链烷烃二酸与短链二胺形成的化合物;
B为VHL或CRBN E3泛素连接酶配体,包括VHL或其衍生物、泊马度胺或其衍生物、来那度胺或其衍生物、沙利度胺或其衍生物中的一种;
该E3泛素连接酶配体的结构通式为:
其中,Z为-CH2和-C(=O)-中的至少一种。
进一步地,疏水连接单元由直链烷烃二酸和直链氨基酸组成;E3泛素连接酶配体为VHL配体;该化合物的结构式为:
进一步地,VHL配体通过氨基或芳环与疏水连接单元连接。
进一步地,疏水连接单元为直链氨基酸;E3泛素连接酶配体为CRBN配体;该化合物的结构式为:
进一步地,直链氨基酸为丁氨酸和己氨酸中的至少一种。
进一步地,短链二胺为脂肪链二胺或聚乙二醇二胺。
进一步地,短链二胺为丙二胺、戊二胺、乙二胺、丁二胺、含氧原子的二胺或1,6己二胺。
一种抑制肿瘤细胞增殖的药物,包括上述化合物物或其药学上可接受的盐、溶剂化物、对映体。
一种PARP1蛋白降解剂,包括上述化合物或其药学上可接受的盐、溶剂化物、对映体。
进一步地,降解剂中的化合物浓度为大于1μM。
一种抗肿瘤药物,包括上述化合物或其药学上可接受的盐、溶剂化物、对映体,及其在药学中可接受的辅助成分。
进一步地,肿瘤为输卵管癌、结直肠癌、前列腺癌或食管癌。
一种联合抗肿瘤药物,包括上述化合物,以及与其联用的化疗药物。
进一步地,化疗药物为替莫唑胺或顺铂。
本发明的有益效果为:
本发明制备得到的PARP1蛋白降解剂能够有效的降解PARP1蛋白,抑制细胞增殖,并诱导肿瘤细胞凋亡。同时,其与化疗药物联用时具有增强化疗药效的作用,且几乎无生理毒性,该化合物有望为改善PARP1过度激活引起的各种疾病的治疗提供一种理想的途径。
附图说明
图1为化合物PV6~PV20对MDA-MB-436细胞内PARP1/2降解性能检测结果;
图2为PV11、PV19和PV20在不同浓度下对PARP1的降解活性检测结果;
图3为化合物PC4~PC13对MDA-MB-436细胞内PARP1/2降解性能检测结果;
图4为化合物PC14~22、PC24对MDA-MB-436细胞内PARP1/2降解性能检测结果;
图5为化合物PC18,PC22-33对MDA-MB-436(a),Capan-1(b),SW620(c)细胞内PARP1/2的降解性能检测结果;
图6为化合物SK-575对7种肿瘤细胞的细胞增殖抑制检测结果;
图7为化合物SK-575对肿瘤细胞内PARP1蛋白降解活性的检测结果;
图8为化合物SK-575对肿瘤细胞内的PARP1蛋白降解速率的检测结果;
图9为化合物SK-575在降解PARP1蛋白时稳定性的检测结果;
图10为化合物SK-575对肿瘤细胞内的PARP1蛋白降解持续性的检测结果;
图11为化合物SK-575对SW620细胞内PARP1蛋白降解性能检测结果;
图12为化合物SK-575作用后Capan-1模型的肿瘤体积变化检测结果;
图13为化合物SK-575作用后Capan-1模型的肿瘤体积检测结果;
图14为化合物SK-575作用后Capan-1模型小鼠体重变化检测结果;
图15为化合物SK-575与顺铂联合作用后Capan-1模型肿瘤体积变化检测结果;
图16为化合物SK-575与TMZ联合作用后SW620模型肿瘤体积变化检测结果;
图17为化合物SK-575与TMZ联合作用后SW620模型肿瘤体积检测结果;
图18为化合物SK-575与TMZ联合作用后SW620模型小鼠体重变化检测结果。
具体实施方式
下面对本发明的具体实施方式进行描述,以便于本技术领域的技术人员理解本发明,但应该清楚,本发明不限于具体实施方式的范围,对本技术领域的普通技术人员来讲,只要各种变化在所附的权利要求限定和确定的本发明的精神和范围内,这些变化是显而易见的,一切利用本发明构思的发明创造均在保护之列。
实施例1
1、制备PARP1配体中间体
在圆底烧瓶中加入L(4.45g,15mmol),N-Boc-哌嗪(3.35g,18mmol),加入100mLDMF后将反应液降温至0℃。搅拌下加入TEA(4.2mL,30mmol)和HATU(6.80g,18mmol)后保温反应5h。TLC监测原料反应完全后,向反应液中加入500mL水,0℃下搅拌1h。抽滤得到得大量白色固体,滤饼经少量冰水和冰EA洗涤后得到中间体粗品(6.43g,92%收率)。
在圆底烧瓶中加入上述反应粗品(6.43g,13.8mmol),加入50mL无水乙醇,快速搅拌下滴加6N HCl(6.9mL),滴加完毕后室温反应3h。TLC监测反应完全后浓缩溶剂,加入50mL水,用4N氨水调节pH>10。再用DCM萃取水层,经干燥,过柱纯化的白色固体L1(4.45g,88%收率)。
其核磁数据为:1H NMR(400MHz,d6-DMSO)δ:12.58(s,1H),8.26(d,J=7.7Hz,1H),7.96(d,J=7.9Hz,1H),7.88(t,J=7.2Hz,1H),7.82(t,J=7.4Hz,1H),7.44–7.37(m,1H),7.31(dd,J=6.4,1.7Hz,1H),7.20(t,J=9.0Hz,1H),5.75(s,1H),4.32(s,2H),3.53(s,2H),3.06(s,2H),2.70(t,J=4.8Hz,2H),2.56(t,J=4.7Hz,2H).HRMS(DART-TOF)calculated for C20H20FN4O2 +[M+H]+m/z 367.1570,found 367.1565.
2、制备VHL配体中间体
其合成路线为:
具体过程为:
在100mL圆底烧瓶中加入4-溴苯甲腈6(4.52g,25mmol),4-甲基噻唑2(4.6mL,50mmol),KOAc(4.9g,50mmol),Pd(OAc)2(56mg,0.25mmol),氮气置换3次后加入100mL DMA,150℃反应15h。反应完全后将反应液冷却到室温,加入100mL半饱和食盐水稀释后用乙酸乙酯(3×150mL)萃取,合并有机层后用饱和食盐水洗3次,合并有机层加入无水硫酸钠干燥,过滤并旋干溶剂的粗品。将粗品用PE/EA=1:1的洗脱剂过柱纯化得到淡黄色固体8(4.6g,92%收率)。1H NMR(400MHz,CDCl3)δ:8.76(s,1H),7.72(d,J=8.5Hz,2H),7.56(d,J=8.5Hz,2H),2.57(s,3H).HRMS(DART-TOF)calculated for C11H9N2S+[M+H]+m/z 201.0486,found 201.0484.
在500mL圆底烧瓶中加入8(3.4g,17mmol)氮气置换3次后加入无水四氢呋喃300mL降温到-10℃。缓慢滴加四氢铝锂(13.8mL,33.4mmol,2.5M,溶解于THF),滴加完毕后将反应液加热到70℃反应3h。TLC监测反应完全后,再将反应液降温到0℃,缓慢滴加2mL水淬灭反应,再加入20mLNaOH(10%)溶液搅拌30min以上,用硅藻土抽滤除去沉淀,旋干溶剂后残渣用DCM:MeOH=10:1过柱纯化得浅黄色油状物9。其核磁数据为:1H NMR(400MHz,CDCl3)δ:8.65(s,1H),7.38(q,J=8.3Hz,4H),3.90(s,2H),2.52(s,3H),1.63(s,2H).13C NMR(101MHz,CDCl3)δ150.24,148.51,143.16,131.83,130.52,129.54,127.54,46.21,16.18.HRMS(DART-TOF)calculated for C11H13N2S+[M+H]+m/z 205.0799,found 205.0798.
在100mL圆底烧瓶中加入9(1.88g,9.2mmol),10(2.13g,9.2mmol)后加入50mL干燥的DMF降温至0℃。再依次加入DIPEA(4.56mL,27.6mmol),HATU(3.84g,10.1mmol),反应5min后升至室温反应2h。反应完全后往反应液中加入100mL半饱和食盐水,用乙酸乙酯(3×100mL)萃取,合并有机层用饱和食盐水洗一次,再加入无水硫酸钠干燥,过滤并旋干溶剂的粗品。粗品用DCM:MeOH=10:1体系过柱纯化得白色固体11(3.5g,90%收率)。其核磁数据为:1H NMR(400MHz,MeOD)δ:8.91(s,1H),7.46(m,4H),4.60–4.28(m,4H),3.68–3.45(m,2H),2.50(s,3H),2.36–2.21(m,1H),2.06(m,1H),1.56–1.30(m,9H).HRMS(DART-TOF)calculated for C21H28N3O4S+[M+H]+m/z 418.1801,found 418.1803.
在100mL圆底烧瓶中加入11(3.2g,7.7mmol)后再加入20mL DCM和5mL MeOH使其溶解,加入4M HCl二氧六环溶液7mL室温反应3h。TLC监测反应完全后减压蒸去溶剂后再加入20mL DCM旋干。往旋干溶剂后的反应瓶中加入12(1.78g,7.7mmol)后加入30mL干燥的DMF降温至0℃。再依次加入DIPEA(3.8mL,23.1mmol),HATU(3.22g,8.47mmol),反应5min后升至室温反应2h。反应完全后往反应液中加入100mL半饱和食盐水,用乙酸乙酯(3×100mL)萃取,合并有机层用饱和食盐水洗一次,再加入无水硫酸钠干燥,过滤并旋干溶剂的粗品。粗品用DCM:MeOH=10:1体系过柱纯化得白色固体V1(3.35g,82%收率)。其核磁数据为:1H NMR(400MHz,CD3OD)δ:8.90(s,1H),7.47(q,J=8.2Hz,4H),4.63(t,J=8.3Hz,1H),4.59–4.26(m,4H),3.97–3.77(m,2H),2.51(s,3H),2.26(m,1H),2.13(m,1H),1.57–1.42(m,9H),1.04(m,9H).13C NMR(101MHz,CD 3OD)δ:173.01,171.50,156.39,151.41,147.64,138.84,132.01,130.11,128.98,127.55,79.28,69.70,59.41,58.96,56.62,42.32,37.49,35.41,27.33,25.55,14.43.HRMS(DART-TOF)calculated for C27H39N4O5S+[M+H]+m/z531.2641,found 531.2645.
在100mL圆底烧瓶中加入V1(1.06g,2.0mmol)后再加入10mL DCM和3mL MeOH使其溶解,加入4M HCl二氧六环溶液3mL室温反应3h。TLC监测反应完全后减压蒸去溶剂后再加入20mL DCM旋干。往旋干溶剂后的反应瓶中加入N-叔丁氧碳基-6-氨基己酸(0.46g,2.0mmol)后加入20mL干燥的DMF降温至0℃。再依次加入DIPEA(1.65mL,10mmol),HATU(0.77g,2.0mmol),反应5min后升至室温反应2h。反应完全后往反应液中加入50mL饱和食盐水,用乙酸乙酯(3×60mL)萃取,合并有机层用饱和食盐水洗一次,再加入无水硫酸钠干燥,过滤并旋干溶剂的粗品。粗品用DCM:MeOH=10:1体系过柱纯化得白色固体V2(1.1g,88%收率)。其核磁数据为:1H NMR(400MHz,CD3OD)δ:8.92(s,1H),7.56–7.38(m,4H),4.76–4.50(m,4H),4.43(d,J=15.5Hz,1H),3.99(d,J=11.0Hz,1H),3.87(dd,J=10.9,3.8Hz,1H),3.16–3.02(m,2H),2.54(s,3H),2.42–2.24(m,3H),2.22–2.10(m,1H),1.78–1.61(m,2H),1.59–1.31(m,15H),1.09(s,9H).13C NMR(101MHz,CD3OD)δ:174.42,172.97,170.95,157.03,151.42,147.63,138.87,132.03,130.11,129.00,127.64,78.42,69.71,59.44,57.59,56.69,54.51,42.41,39.92,37.57,35.23,29.31,27.64,26.16,25.86,25.34,14.76.HRMS(DART-TOF)calculated for C33H50N5O6S+[M+H]+m/z 644.3482,found 644.3484.
在100mL圆底烧瓶中加入V1(1.06g,2.0mmol)后再加入10mL DCM和3mL MeOH使其溶解,加入4M HCl二氧六环溶液3mL室温反应3h。TLC监测反应完全后减压蒸去溶剂后再加入20mL DCM旋干。往旋干溶剂后的反应瓶中加入N-叔丁氧碳基-4-氨基丁酸(0.41g,2.0mmol)后加入20mL干燥的DMF降温至0℃。再依次加入DIPEA(1.65mL,10mmol),HATU(0.77g,2.0mmol),反应5min后升至室温反应2h。反应完全后往反应液中加入50mL饱和食盐水,用乙酸乙酯(3×60mL)萃取,合并有机层用饱和食盐水洗一次,再加入无水硫酸钠干燥,过滤并旋干溶剂的粗品。粗品用DCM:MeOH=10:1体系过柱纯化得白色固体V3(0.92g,75%收率)。其核磁数据为:1H NMR(400MHz,CDCl3)δ:8.64(s,1H),7.65(s,1H),7.37–7.23(m,4H),7.19(s,1H),5.09(s,1H),4.66(s,1H),4.38(m,5H),3.99(m,1H),3.62(d,J=9.3Hz,1H),3.16–2.73(m,3H),2.46(m,3H),2.31(m,1H),2.14(m,3H),1.68(m,2H),1.36(s,9H),0.94(s,9H).13C NMR(101MHz,CDCl3)δ:173.49,171.74,171.36,156.58,150.36,148.39,138.42,131.69,130.77,129.42,128.03,79.40,70.11,58.87,58.11,56.94,43.11,39.69,36.65,35.15,33.21,28.47,26.52,16.06.HRMS(DART-TOF)calculated for C31H46N5O6S+[M+H]+m/z 616.3169,found 616.3167.
在100mL圆底烧瓶中加入13(5.0g,25mmol),7(4.6mL,50mmol),KOAc(4.9g,50mmol),Pd(OAc)2(56mg,0.25mmol),氮气置换3次后加入100mL DMA,150℃反应15h。反应完全后将反应液冷却到室温,加入100mL半饱和食盐水稀释后用乙酸乙酯(3×150mL)萃取,合并有机层后用饱和食盐水洗3次,合并有机层加入无水硫酸钠干燥,过滤并旋干溶剂的粗品。将粗品用PE/EA=1:1~EA的洗脱体系过柱纯化得到白色固体14(4.6g,83%收率)。其核磁数据为:1H NMR(400MHz,DMSO)δ:9.04(s,1H),7.67(d,J=8.1Hz,1H),7.13(s,1H),7.01(dd,J=13.9,4.9Hz,1H),2.48(s,3H).
在500mL圆底烧瓶中加入14(1.4g,6.5mmol),氮气置换3次后加入无水四氢呋喃300mL降温到-10℃。缓慢滴加四氢铝锂(,7.8mL,19.6mmol,2.5M,溶解于THF),滴加完毕后将反应液加热到50℃反应1.5h。TLC监测反应完全后,再将反应液降温到0℃,缓慢滴加2mL水淬灭反应,再加入4mLNaOH(10%)溶液搅拌30min以上,用硅藻土抽滤除去沉淀,旋干溶剂后残渣用DCM:MeOH=5:1过柱纯化得浅黄色油状物15(0.56g,40%收率)。其核磁数据为:1HNMR(400MHz,CDCl3)δ:8.65(s,1H),7.01(d,J=7.7Hz,1H),6.95(d,J=1.1Hz,1H),6.86(dd,J=7.7,1.3Hz,1H),4.17(s,2H),3.49(s,2H),2.54(s,3H).13C NMR(101MHz,CDCl3)δ:158.72,150.23,148.51,132.53,131.95,128.34,123.79,120.14,117.69,45.34,16.36.
在100mL圆底烧瓶中加入15(1.82g,8.3mmol),10(1.91g,8.3mmol)后加入40mL干燥的DMF降温至0℃。再依次加入DIPEA(4.1mL,24.9mmol),HATU(3.77g,10.0mmol),反应5min后升至室温反应2h。反应完全后往反应液中加入100mL饱和食盐水,用乙酸乙酯(3×100mL)萃取,合并有机层用饱和食盐水洗一次,再加入无水硫酸钠干燥,过滤并旋干溶剂的粗品。粗品用DCM:MeOH=10:1体系过柱纯化得白色固体16(2.4g,67%收率)。其核磁数据为:1H NMR(400MHz,CD3OD)δ:8.88(s,1H),7.34(t,J=8.0Hz,1H),6.94(dd,J=7.9,2.7Hz,2H),4.53–4.28(m,4H),3.66–3.45(m,2H),2.51(s,3H),2.34–2.22(m,1H),2.07(m,1H),1.56–1.19(m,9H).13C NMR(101MHz,CD3OD)δ:174.74,155.70,154.76,151.36,147.52,132.04,130.29,124.80,120.06,116.09,80.27,68.69,59.29,54.61,39.39,38.33,27.04,14.50.
在100mL圆底烧瓶中加入16(1.69g,3.9mmol)后再加入20mL DCM和5mL MeOH使其溶解,加入4M HCl二氧六环溶液4mL室温反应3h。TLC监测反应完全后减压蒸去溶剂后再加入20mL DCM旋干。往旋干溶剂后的反应瓶中加入12(0.90g,3.9mmol)后加入30mL干燥的DMF降温至0℃。再依次加入DIPEA(2.65mL,16mmol),HATU(1.52g,4.0mmol),反应5min后升至室温反应2h。反应完全后往反应液中加入100mL饱和食盐水,用乙酸乙酯(3×100mL)萃取,合并有机层用饱和食盐水洗一次,再加入无水硫酸钠干燥,过滤并旋干溶剂的粗品。粗品用DCM:MeOH=10:1体系过柱纯化得白色固体17(1.66g,78%收率)。其核磁数据为:1H NMR(400MHz,CD3OD)δ:8.86(s,1H),7.39(d,J=7.7Hz,1H),6.94–6.87(m,2H),4.64(t,J=8.0Hz,1H),4.59–4.26(m,4H),3.95–3.77(m,2H),2.51(s,3H),2.28–2.09(m,2H),1.48(s,9H),1.03(s,9H).13C NMR(101MHz,CD3OD)δ:173.18,171.53,156.38,155.25,151.23,147.41,132.17,131.41,129.25,124.79,120.03,115.46,79.30,69.68,59.27,58.94,56.57,38.19,37.35,35.42,27.37,25.58,14.62.
在100mL圆底烧瓶中加入17(503mg,1.37mmol)后再加入10mL DCM和2mL MeOH使其溶解,加入4M HCl二氧六环溶液2mL室温反应3h。TLC监测反应完全后减压蒸去溶剂后再加入20mL DCM旋干。往旋干溶剂后的反应瓶中加入18(152mg,1.37mmol)后加入10mL干燥的DMF降温至0℃。再依次加入DIPEA(1.1mL,6.8mmol),HATU(573mg,1.51mmol),反应5min后升至室温反应1h。反应完全后往反应液中加入50mL饱和食盐水,用乙酸乙酯(3×50mL)萃取,合并有机层用饱和食盐水洗一次,再加入无水硫酸钠干燥,过滤并旋干溶剂的粗品。粗品用DCM:MeOH=10:1体系过柱纯化得白色固体19(271mg,37%收率)。其核磁数据为:1H NMR(400MHz,CDCl3)δ:9.30(s,1H),8.64(s,1H),8.07(t,J=6.3Hz,1H),7.10(d,J=8.5Hz,1H),7.00(d,J=8.4Hz,1H),6.92(m,1H),6.84(dd,J=5.4,2.7Hz,1H),4.75–4.63(m,1H),4.50(s,1H),4.43(d,J=8.4Hz,1H),4.36(dd,J=14.6,7.1Hz,1H),4.19(dd,J=14.6,5.6Hz,1H),3.86(m,2H),3.62(dd,J=12.6,6.3Hz,1H),2.48(s,3H),2.41–2.31(m,1H),2.07(m,1H),1.66–1.57(m,1H),1.56–1.35(m,4H),0.91(s,9H).13C NMR(101MHz,CDCl3)δ:172.85,170.80,165.88,155.82,150.58,148.34,133.30,131.73,131.23,124.05,120.89,119.68,118.13,70.18,58.69,58.54,56.81,40.00,36.41,35.85,26.31,17.96,17.85,16.18,13.87.
3、制备CRBN配体中间体
在100mL圆底烧瓶中,依次加入3-羟基邻苯二甲酸酐(1.64g,10.0mmol)、3-氨基哌啶-2,6-二酮盐酸盐(1.64g,10.0mmol)、60mL甲苯和三乙胺(1.30mL,10.0mmol)。用油浴将所得反应混合物加热回流12小时。冷却至环境温度后,在反应体系中加入粗硅胶拌样,用DCM:EA体系柱层析纯化,得白色固体S1(2.41g,88%收率)。其核磁数据为:1H NMR(400MHz,DMSO-d6)δ(ppm)11.16(s,1H),11.08(s,1H),7.65(t,J=7.6Hz,1H),7.32(d,J=7.2Hz,1H),7.25(d,J=8.4Hz,1H),5.07(dd,J=12.8Hz,J=5.2Hz,1H),2.93-2.84(m,1H),2.61-2.46(m,3H),2.05-2.01(m,1H)。13C NMR(101MHz,DMSO-d6)δ172.75,169.96,166.98,165.78,155.43,136.35,133.12,123.52,114.34,114.26,48.62,30.93,22.01。其合成路线为:
在100mL圆底烧瓶中,将3-氟邻苯二甲酸酐(3.32g,20mmol)、3-氨基哌啶-2,6-二酮盐酸盐(3.29g,20mmol)和乙酸钠(1.97g,24mmol)在100mL乙酸中混合。将所得反应混合物在120℃加热回流12h。冷却至室温后,蒸发大部分乙酸,在残余物中加入200水,用乙酸乙酯萃取(3×200mL),合并有机层加入无水硫酸钠干燥,过滤并旋干溶剂的到灰白色固体,用少量的DCM打浆洗涤后得到灰白色固体S2(4,52g,82%收率)。其核磁数据为:1H NMR(400MHz,DMSO-d6)δ11.15(s,1H),7.98-7.93(m,1H),7.80-7.72(m,2H),5.17(dd,J=13.2Hz,J=5.2Hz,1H),2.95-2.86(m,1H),2.64-2.47(m,2H),2.10-2.06(m,1H).其合成路线为:
中间体S3的合方法参考S2的合成方法合成。其合成路线为:
在圆底烧瓶中,将S1(1.5g,5.5mmol)溶解于10mL DMF中。向搅拌的溶液中加入KI(91mg,0.55mmol)和KHCO3(826mg,8.25mmol)。然后滴加溴乙酸叔丁酯(0.98mL,6.6mmol),将所得混合物在室温下搅拌12h。在反应液中加入EtOAc(200mL),用饱和盐水洗2次后,将合并的有机层用Na2SO4干燥。过滤并减压旋干溶剂,将残余物通过快速柱色谱法用DCM:EA纯化,得到白色固体M-S7(1.7g,80%收率)。1H NMR(400MHz,CDCl3)δ(ppm)8.19(s,1H),7.66(dd,J=8.4,7.4Hz,1H),7.50(d,J=7.3Hz,1H),7.10(d,J=8.4Hz,1H),5.01-4.92(m,1H),4.78(s,2H),2.93-2.67(m,3H),2.16-2.07(m,1H),1.47(s,9H);13C NMR(101MHz,CDCl3)δ171.12,168.09,167.00,166.96,165.57,155.65,136.40,134.04,119.94,117.71,117.03,83.24,66.69,49.31,31.51,28.16,22.70.
在圆底烧瓶中,将M-S7(1.7g,4.4mmol)溶解于20mL TFA/DCM中在室温下搅拌2h。浓缩溶剂后,将残余物S7不经进一步纯化用于以下步骤。1H NMR(400MHz,DMSO-d6)δ(ppm)13.22(s,1H),11.11(s,1H),7.80(dd,J=8.6,7.3Hz,1H),7.48(d,J=7.2Hz,1H),7.40(d,J=8.5Hz,1H),5.11(dd,J=12.9,5.4Hz,1H),4.99(s,2H),2.90(m,1H),2.69-2.53(m,2H),2.05(m,1H);13C NMR(101MHz,DMSO)δ173.24,170.37,169.96,167.20,165.63,155.60,137.22,133.72,120.35,116.79,116.22,65.48,49.26,31.42,22.44。HRMS(DART-TOF)calculated for C15H12N2NaO7 +[M+Na]+m/z 355.0542,found 355.0541.
其合成路线为:
在10mL反应管中加入3-氟-N-(2,6-二氧代-3-哌啶基)邻苯二甲酰亚胺S2(552mg,2mmol),甘氨酸叔丁酯(324mg,2.4mmol),氮气置换3次后加入NMP 4mL和DIPEA(165μL),将反应置于90℃的油浴锅中反应12h。TLC监测反应完成后,在反应液中加入50mLEA稀释,依次用10%柠檬酸水溶液,饱和NaHCO3溶液洗涤,在用饱和氯化钠水溶液洗涤2次,合并有机层加入无水Na2SO4干燥,减压蒸去溶剂的得到黄绿色油状物。最后用PE/EA体系进行柱层析分离得到亮黄色固体M-S8(503mg,65%收率)。其核磁数据为:1H NMR(400MHz,CDCl3)δ:7.57(dd,J=8.5,7.1Hz,1H),7.12(d,J=7.1Hz,1H),6.94(d,J=8.5Hz,1H),5.51(s,2H),5.09(m,1H),2.94-2.66(m,3H),2.14(m,1H),1.51(s,9H)。HRMS(DART-TOF)calculated forC19H21N3NaO6+[M+Na]+m/z 410.1323,found 410.1326.
在圆底烧瓶中,将M-S7(1.7g,4.4mmol)溶解于20mL TFA/DCM中在室温下搅拌2h。浓缩溶剂后,将残余物S7不经进一步纯化用于以下步骤。1H NMR(400MHz,DMSO-d6)δ(ppm)13.22(s,1H),11.11(s,1H),7.80(dd,J=8.6,7.3Hz,1H),7.48(d,J=7.2Hz,1H),7.40(d,J=8.5Hz,1H),5.11(dd,J=12.9,5.4Hz,1H),4.99(s,2H),2.90(m,1H),2.69-2.53(m,2H),2.05(m,1H);13C NMR(101MHz,DMSO)δ173.24,170.37,169.96,167.20,165.63,155.60,137.22,133.72,120.35,116.79,116.22,65.48,49.26,31.42,22.44。HRMS(DART-TOF)calculated for C15H12N2NaO7 +[M+Na]+m/z 355.0542,found 355.0541.其合成路线为:
通用步骤C:合成中间体S9-S14
在反应瓶中加入3-氟-N-(2,6-二氧代-3-哌啶基)邻苯二甲酰亚胺S2(1eq),胺(1.2eq),氮气置换三次后加入NMP和DIPEA(2eq),将反应置于90℃的油浴锅中反应10h。TLC监测反应完成后,向反应液中加入EA稀释,依次用10%柠檬酸水溶液,饱和NaHCO 3溶液洗涤,再用饱和氯化钠水溶液洗涤2次,合并有机层加入无水Na 2SO 4干燥,减压蒸去溶剂的得到黄绿色油状物。最后用PE/EA体系进行柱层析分离得到亮黄色固体S9-S14。
S9的合成参考通用步骤C,以S2和N-Boc-1,2-乙二胺为反应原料即可得到产物S9。黄色固体,产率66%。其核磁数据为:1H NMR(400MHz,CDCl3)δ:8.04(s,1H),7.50(dd,J=8.5,7.1Hz,1H),7.12(d,J=7.1Hz,1H),6.98(d,J=8.6Hz,1H),6.39(t,J=6.0Hz,1H),4.92(dd,J=12.1,5.3Hz,1H),4.82(s,1H),3.45(q,J=5.9Hz,2H),3.36(q,J=5.7Hz,2H),2.92-2.68(m,3H),2.13(m,1H),1.45(s,9H)。HRMS(DART-TOF)calculated for C20H24N4NaO6+[M+Na]+m/z 439.1588,found 439.1592.
S10的合成参考通用步骤C,以S2和N-Boc-1,2-丁二胺为反应原料即可得到产物S10。黄色固体,产率66%。其核磁数据为:1H NMR(400MHz,CDCl3)δ:8.36(s,1H),7.47(dd,J=8.5,7.1Hz,1H),7.10-7.03(m,1H),6.87(d,J=8.6Hz,1H),6.22(t,J=5.7Hz,1H),4.94-4.84(m,1H),4.61(s,1H),3.28(q,J=6.5Hz,2H),3.20-3.10(m,2H),2.92-2.71(m,3H),2.15-2.06(m,1H),1.72-1.63(m,2H),1.63-1.53(m,2H),1.42(s,9H).HRMS(DART-TOF)calculated for C22H28N4NaO6+[M+Na]+m/z 467.1907,found 467.1901.
S11的合成参考通用步骤C,以S2和N-Boc-1,2-己二胺为反应原料即可得到产物S11。黄色固体,产率54%。1H NMR(400MHz,CDCl3)δ:8.37(s,1H),7.47(dd,J=8.5,7.1Hz,1H),7.07(d,J=7.1Hz,1H),6.86(d,J=8.5Hz,1H),6.22(t,J=5.7Hz,1H),4.90(dd,J=11.9,5.4Hz,1H),4.55(s,1H),3.25(q,J=6.6Hz,2H),3.10(q,J=6.8Hz,2H),2.92-2.71(m,3H),2.11(m,1H),1.65(m,2H),1.54-1.29(m,15H);13C NMR(101MHz,CDCl3)δ:177.48,171.29,169.63,168.58,167.75,147.10,136.23,132.62,116.75,111.51,110.01,49.00,42.68,40.57,31.53,30.13,29.28,28.55,28.33,26.74,26.59,22.93.HRMS(DART-TOF)calculated for C24H32N4NaO6+[M+Na]+m/z 495.2220,found 495.2231.
S12的合成参考通用步骤C,以S2和tert-butyl(2-(2-aminoethoxy)ethyl)carbamate为反应原料即可得到产物S12。黄色固体,产率46%。1H NMR(400MHz,CDCl3)δ:8.68(s,1H),7.49(dd,J=8.5,7.1Hz,1H),7.09(d,J=7.1Hz,1H),6.92(d,J=8.5Hz,1H),6.50(t,J=5.7Hz,1H),5.05(t,J=5.9Hz,1H),4.94(dd,J=12.1,5.3Hz,1H),3.69(t,J=5.3Hz,2H),3.55(t,J=5.2Hz,2H),3.45(q,J=5.5Hz,2H),3.38-3.25(m,2H),2.92-2.65(m,3H),2.16-2.05(m,1H),1.42(s,9H).HRMS(DART-TOF)calculated for C22H28N4NaO7+[M+Na]+m/z 483.1865,found 483.1871.
S13的合成参考通用步骤C,以S2和tert-butyl(2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate为反应原料即可得到产物S13。黄色固体,产率46%。1H NMR(400MHz,CDCl3)δ:8.73(s,1H),7.46(dd,J=8.5,7.2Hz,1H),7.07(d,J=7.1Hz,1H),6.89(d,J=8.7Hz,1H),6.50(d,J=6.9Hz,1H),5.06(s,1H),4.91(t,J=7.8Hz,1H),3.69(t,J=5.5Hz,2H),3.65-3.57(m,4H),3.53(t,J=5.2Hz,2H),3.45(q,J=5.4Hz,2H),3.29(q,J=5.5Hz,2H),2.89-2.61(m,3H),2.09(m,1H),1.40(s,9H).HRMS(DART-TOF)calculated forC24H32N4NaO8+[M+Na]+m/z 527.2118,found 527.2113.
S14的合成参考通用步骤C,以S2和tert-butyl(3-(2-(2-(3-aminopropoxy)ethoxy)ethoxy)propyl)carbamate为反应原料即可得到产物S14。黄色固体,产率57%。1HNMR(400MHz,CDCl3)δ:8.46(s,1H),7.49(dd,J=8.5,7.1Hz,1H),7.08(d,J=7.1Hz,1H),6.93(d,J=8.5Hz,1H),6.44(t,J=5.7Hz,1H),4.98(s,1H),4.91(dd,J=11.8,5.3Hz,1H),3.73-3.56(m,10H),3.53(t,J=6.0Hz,2H),3.41(q,J=6.4Hz,2H),3.21(t,J=6.5Hz,2H),2.92-2.67(m,3H),2.18-2.06(m,1H),1.93(p,J=6.3Hz,2H),1.75(p,J=6.3Hz,2H),1.43(s,9H).HRMS(DART-TOF)calculated for C28H40N4NaO9+[M+Na]+m/z 599.2693,found599.2688.
S15的合成参考通用步骤C,以S3和N-Boc-1,2-乙二胺为反应原料即可得到产物S15。黄色固体,产率71%。1H NMR(400MHz,CDCl3)δ8.65(s,1H),7.52(d,J=8.1Hz,1H),6.89(d,J=1.5Hz,1H),6.69(dd,J=8.3,1.8Hz,1H),5.56(s,1H),5.14(s,1H),4.92(dd,J=12.0,5.3Hz,1H),3.37(q,J=5.3Hz,2H),3.28(q,J=5.2Hz,2H),2.89-2.67(m,3H),2.14-2.06(m,1H),1.43(s,9H).HRMS(DART-TOF)calculated for C20H24N4NaO6+[M+Na]+m/z439.1594,found 439.1587.
在反应瓶中加入来那度胺(520mg,2mmol),N-Boc-乙醛(640mg,4mmol)和NaBH3CN(181mg,6mmol),氮气置换3次后加入10mL甲醇,将反应置于50℃的油浴锅中反应过夜。TLC监测反应完成后,在反应液中加入50mL水稀释,用EA萃取3次,合并有机层加入无水Na2SO4干燥,减压蒸去溶剂的得到白色油状物。最后用PE/EA体系进行柱层析分离得到亮白色固体S16(603mg,75%收率)。1H NMR(400MHz,CDCl3)δ8.79(s,1H),7.49(t,J=7.4Hz,1H),7.10(d,J=7.4Hz,1H),6.97(d,J=8.6Hz,1H),6.37(m,2H),4.93(m,1H),3.48(s,2H),3.45(s,4H),2.91–2.66(m,3H),2.11(m,1H),1.30–1.16(m,9H).HRMS(DART-TOF)calculated forC20H26N4NaO5+[M+Na]+m/z 425.1801,found 425.1796.其合成路线为:
在反应瓶中加入S2(552mg,2mmol)碳酸钾(552mg,4mmol)和10mL DMF,搅拌下加入碘甲烷(0.1mL),室温反应12h后加入100mL水稀释,再用EA萃取3次,合并有机层加入无水Na2SO4干燥,减压蒸去溶剂的得到黄色固体M-S17,该中间体可不经纯化进行下一步反应。
S17的合成参考通用步骤C,以M-S17和N-Boc-1,2-乙二胺为反应原料即可得到产物S17。黄色固体,产率51%(上述两步)。1H NMR(400MHz,CDCl3)δ7.54–7.42(m,1H),7.09(d,J=9.4Hz,1H),6.93(d,J=32.3,7.6Hz,1H),6.38(t,J=5.8Hz,1H),4.96–4.75(m,2H),3.49–3.30(m,4H),3.20(s,3H),3.02–2.88(m,1H),2.76(m,2H),2.14–2.05(m,1H),1.44(s,9H).13C NMR(101MHz,CDCl3)δ171.38,169.69,169.13,167.81,146.94,136.30,132.70,116.71,111.98,110.58,49.80,42.69,40.27,32.06,28.50,28.35,27.38,22.25.HRMS(DART-TOF)calculated for C21H26N4NaO6+[M+Na]+m/z 453.1750,found 453.1752.其合成路线为:
通用步骤D:合成VHL类中间体L1-L14
在25mL反应瓶中加入二酸(3mmol),再加入10mL无水DMF溶解后置于冰浴中,加入DIPEA(6mmol),搅拌下分批加入HBTU(456mg,1.2mmol,1.2eq),室温下搅拌5min,最后加入L1(366mg,1mmol),将反应置于室温下反应16h。TLC监测反应完成后,在反应液中加入50mL半饱和食盐水稀释,再用乙酸乙酯萃取(3×60mL),合并有机层用饱和氯化钠水溶液洗一次,加入无水Na2SO4干燥,过滤减压蒸去溶剂得油状粗品。最后用DCM:MeOH=10:1~5:1体系进行柱层析分离得到白色固体L2-L14。
L7的合成参考通用步骤D,以L1和1,4-丁二酸为反应原料即可得到产物L7,白色固体,产率87%。1H NMR(400MHz,CDCl3)δ:11.85(d,J=70.8Hz,1H),8.49-8.37(m,1H),7.77(m,3H),7.31(m,2H),7.03(t,J=8.8Hz,1H),4.28(s,2H),3.98-3.16(m,8H),2.75-2.47(m,4H).HRMS(DART-TOF)calculated for C24H23FN4NaO5 +[M+Na]+m/z 489.1550,found489.1548.
L8的合成参考通用步骤D,以L1和1,6-己二酸为反应原料即可得到产物L8,白色固体,产率81%。1H NMR(400MHz,DMSO-d6)δ:12.57(s,1H),8.27(d,J=7.5Hz,1H),7.94(d,J=7.7Hz,1H),7.86(t,J=7.4Hz,1H),7.80(td,J=7.5,1.3Hz,1H),7.43(t,J=6.7Hz,1H),7.35(d,J=6.4Hz,1H),7.19(t,J=9.0Hz,1H),4.32(s,2H),3.72–3.10(m,8H),2.32(d,J=25.6Hz,2H),2.19(d,J=6.0Hz,2H),1.61–1.42(m,4H).HRMS(DART-TOF)calculated forC26H27FN4NaO5 +[M+Na]+m/z 517.1863,found 517.1869.
L9的合成参考通用步骤D,以L1和1,8-辛二酸为反应原料即可得到产物L9,白色固体,产率83%。1H NMR(400MHz,DMSO-d6)δ:12.59(s,1H),12.12(br,1H),8.27(d,J=7.6Hz,1H),7.97(dd,J=8.0,1.2Hz,1H),7.90(t,J=7.6Hz,1H),7.83(td,J=7.5,1.3Hz,1H),7.44(m,1H),7.37(m,1H),7.24(t,J=9.0Hz,1H),4.34(s,2H),3.71–3.07(m,8H),2.30(dt,J=26.6,7.4Hz,2H),2.19(td,J=7.4,2.0Hz,2H),1.55–1.39(m,4H),1.33–1.25(m,4H).HRMS(DART-TOF)calculated for C28H31FN4NaO5 +[M+Na]+m/z 545.2176,found 545.2175.
L10的合成参考通用步骤D,以L1和1,10-癸二酸为反应原料即可得到产物L10,白色固体,产率78%。1H NMR(400MHz,CDCl3)δ:11.79(d,J=81.7Hz,1H),8.46(d,J=7.1Hz,1H),7.78(m,3H),7.32(m,2H),7.04(t,J=8.8Hz,1H),4.30(s,2H),3.97–3.10(m,8H),2.34(m,4H),1.69–1.53(m,4H),1.31(m,8H).HRMS(DART-TOF)calculated for C30H35FN4NaO5 +[M+Na]+m/z 573.2489,found 573.2485.
L11的合成参考通用步骤D,以L1和十一二酸为反应原料即可得到产物L11,白色固体,产率75%。1H NMR(400MHz,CDCl3)δ:11.80(d,J=75.8Hz,1H),8.39(d,J=5.0Hz,1H),7.70(m,3H),7.25(d,J=5.5Hz,2H),6.98(t,J=8.8Hz,1H),4.24(s,2H),3.90–3.07(m,8H),2.25(m,4H),1.54(m,4H),1.32–1.10(m,10H).8H).HRMS(DART-TOF)calculated forC31H37FN4NaO5 +[M+Na]+m/z 587.2646,found 587.2653.
L12的合成参考通用步骤D,以L1和十二二酸为反应原料即可得到产物L12,白色固体,产率87%。1H NMR(400MHz,CDCl3)δ:11.83(d,J=50.4Hz,1H),8.44(d,J=6.5Hz,1H),7.74(m,3H),7.31(m,2H),7.02(t,J=8.8Hz,1H),4.28(s,2H),3.91–3.14(m,8H),2.33(m,4H),1.69–1.52(m,4H),1.37–1.18(m,12H).HRMS(DART-TOF)calculated for C32H39FN4NaO5 +[M+Na]+m/z 601.2802,found 601.2802.
L13的合成参考通用步骤D,以L1和十三二酸为反应原料即可得到产物L13,白色固体,产率81%。1H NMR(400MHz,CDCl3)δ:11.80(d,J=71.3Hz,1H),8.45(d,J=5.3Hz,1H),7.87–7.69(m,3H),7.32(d,J=4.6Hz,2H),7.04(t,J=8.8Hz,1H),4.29(s,2H),3.93–3.08(m,8H),2.44–2.20(m,4H),1.61(s,4H),1.27(m,14H).HRMS(DART-TOF)calculated forC33H41FN4NaO5 +[M+Na]+m/z 615.2959,found 615.2959.
L14的合成参考通用步骤D,以L1和十四二酸为反应原料即可得到产物L14,白色固体,产率84%。1H NMR(400MHz,CDCl3)δ:11.91(d,J=30.1Hz,1H),8.43(d,J=7.5Hz,1H),7.73(d,J=12.9Hz,1H),7.39–7.22(m,1H),7.08–6.92(m,1H),4.25(d,J=17.8Hz,1H),3.92–3.13(m,1H),2.41–2.20(m,1H),1.68–1.47(m,1H),1.25(m,1H).HRMS(DART-TOF)calculated for C34H43FN4NaO5 +[M+Na]+m/z 629.3115,found 629.3110.
实施例2 PARP1 PROTAC分子的合成
通用步骤E:在25mL反应瓶中加入V1/V2/V3(0.11mmol),加入10mL DCM/MeOH(5:1)溶解后,滴加1mL 4M HCl-二氧六环溶液,室温反应2h,减压浓缩溶剂后的白色固体,向反应瓶中加入L2-L14(0.1mmol),再加入10mL无水DMF溶解后置于冰水浴中,加入DIPEA(0.2mmol),搅拌下分批加入HATU(380mg,0.1mmol),将反应置于冰水浴中反应1-2h。TLC监测反应完成后,在反应液中加入50mL半饱和食盐水稀释,再用乙酸乙酯萃取(3×60mL),合并有机层用饱和氯化钠水溶液洗一次,加入无水Na2SO4干燥,过滤减压蒸去溶剂得油状粗品。最后用DCM:MeOH=10:1~5:1体系进行柱层析分离得到白色固体。
实施例3
PV6的合成参考通用步骤E,以L7和V1为反应原料即可得到产物PV6,白色固体,产率74%。1H NMR(400MHz,DMSO)δ12.59(s,1H),8.98(s,1H),8.63–8.51(m,1H),8.27(d,J=7.7Hz,1H),7.97(d,J=7.9Hz,1H),7.90(t,J=9.9Hz,2H),7.83(t,J=7.4Hz,1H),7.49–7.32(m,6H),7.23(t,J=9.0Hz,1H),5.13(s,1H),4.53(d,J=9.2Hz,1H),4.48–4.38(m,2H),4.39–4.29(m,3H),4.28–4.17(m,1H),3.71–3.46(m,6H),3.42–3.36(m,2H),3.34(s,6H),3.17(d,J=25.0Hz,2H),2.61–2.53(m,2H),2.47–2.34(m,4H),2.09–1.98(m,1H),1.94–1.86(m,1H),0.94(s,9H).HRMS(DART-TOF)calculated for C46H51FN8NaO7S+[M+Na]+m/z 901.3483,found 901.3491.其化学结构式为:
实施例4
PV7的合成参考通用步骤E,以L8和V1为反应原料即可得到产物PV7,白色固体,产率82%。1H NMR(400MHz,DMSO)δ12.60(s,1H),8.98(s,1H),8.61–8.49(m,1H),8.27(d,J=7.7Hz,1H),7.96(d,J=7.9Hz,1H),7.93–7.78(m,3H),7.48–7.32(m,6H),7.23(t,J=9.0Hz,1H),5.14(s,1H),4.55(d,J=6.6Hz,1H),4.44(dd,J=14.7,6.1Hz,2H),4.39–4.29(m,3H),4.23(dd,J=15.9,5.3Hz,1H),3.74–3.44(m,6H),3.34(s,2H),3.17(d,J=16.1Hz,2H),2.45(s,3H),2.40–2.22(m,3H),2.22–2.10(m,1H),2.09–1.99(m,1H),1.97–1.87(m,1H),1.58–1.39(m,4H),0.94(s,9H).HRMS(DART-TOF)calculated for C48H55FN8NaO7S+[M+Na]+m/z 929.3796,found 929.3792.其化学结构式为:
实施例5
PV8的合成参考通用步骤E,以L9和V1为反应原料即可得到产物PV8,白色固体,产率79%。1H NMR(400MHz,CDCl3)δ11.42(d,J=10.6Hz,1H),8.66(s,1H),8.49–8.36(m,1H),7.82–7.64(m,3H),7.53(t,J=13.3,7.4Hz,1H),7.36–7.27(m,5H),7.00(t,J=8.9Hz,1H),6.59(d,J=8.9Hz,1H),4.72(t,J=8.0Hz,1H),4.64–4.48(m,3H),4.32(dd,J=15.1,5.3Hz,1H),4.25(s,2H),4.10(d,J=11.3Hz,1H),3.94(d,J=11.0Hz,1H),3.83–3.13(m,9H),2.54–2.38(m,4H),2.37–2.08(m,4H),1.69–1.44(m,4H),1.36–1.16(m,8H),0.95(s,9H).HRMS(DART-TOF)calculated for C50H59FN8NaO7S+[M+Na]+m/z 957.4109,found957.4105.其化学结构式为:
实施例6
PV9的合成参考通用步骤E,以L10和V1为反应原料即可得到产物PV9,白色固体,产率86%。1H NMR(400MHz,CDCl3)δ11.42(d,J=10.6Hz,1H),8.66(s,1H),8.49–8.36(m,1H),7.82–7.64(m,3H),7.53(t,J=13.3,7.4Hz,1H),7.36–7.27(m,5H),7.00(t,J=8.9Hz,1H),6.59(d,J=8.9Hz,1H),4.72(t,J=8.0Hz,1H),4.64–4.48(m,3H),4.32(dd,J=15.1,5.3Hz,1H),4.25(s,2H),4.10(d,J=11.3Hz,1H),3.94(d,J=11.0Hz,1H),3.83–3.13(m,9H),2.54–2.38(m,4H),2.37–2.08(m,4H),1.69–1.44(m,4H),1.36–1.16(m,8H),0.95(s,9H).HRMS(DART-TOF)calculated for C52H63FN8NaO7S+[M+Na]+m/z 985.4422,found985.4420.
实施例7
PV10的合成参考通用步骤E,以L11和V1为反应原料即可得到产物PV10,白色固体,产率77%。1H NMR(400MHz,CDCl3)δ11.50(d,J=22.9Hz,1H),8.65(s,1H),8.45–8.38(m,1H),7.79–7.64(m,3H),7.54(s,1H),7.37–7.25(m,6H),6.99(t,J=8.5Hz,1H),6.75–6.49(m,1H),4.71(t,J=7.9Hz,1H),4.66–4.46(m,3H),4.37–4.28(m,1H),4.24(s,2H),4.15–3.97(m,2H),3.88–3.05(m,10H),2.53–2.37(m,5H),2.36–2.09(m,6H),1.66–1.45(m,4H),1.35–1.14(m,10H),0.94(s,9H).HRMS(DART-TOF)calculated for C53H65FN8NaO7S+[M+Na]+m/z 999.4579,found 999.4578.
实施例8
PV11的合成参考通用步骤E,以L12和V1为反应原料即可得到产物PV11,白色固体,产率76%。1H NMR(400MHz,CDCl3)δ11.68(s,1H),8.64(s,1H),8.46–8.35(m,1H),7.78–7.63(m,3H),7.57(s,1H),7.30(q,J=8.3Hz,6H),6.98(t,J=8.7Hz,1H),6.68(s,1H),4.71(t,J=7.9Hz,1H),4.64–4.47(m,3H),4.30(dd,J=15.1,5.2Hz,1H),4.26–4.12(m,3H),4.07(d,J=11.2Hz,1H),3.87–3.08(m,9H),2.67–2.36(m,5H),2.35–2.04(m,5H),1.65–1.45(m,4H),1.31–1.11(m,12H),0.94(s,9H).HRMS(DART-TOF)calculated for C54H67FN8NaO7S+[M+Na]+m/z 1013.4735,found 1013.4732.
实施例9
PV12的合成参考通用步骤E,以L13和V1为反应原料即可得到产物PV12,白色固体,产率73%。1H NMR(400MHz,CDCl3)δ11.57(d,J=23.2Hz,1H),8.65(s,1H),8.47–8.34(m,1H),7.80–7.64(m,3H),7.54(s,1H),7.39–7.25(m,6H),7.03–6.93(m,1H),6.72–6.56(m,1H),4.71(t,J=7.8Hz,1H),4.64–4.48(m,3H),4.31(dd,J=15.1,5.0Hz,1H),4.24(s,2H),4.08(d,J=10.9Hz,1H),3.90–3.09(m,10H),2.51–2.37(m,4H),2.28(dt,J=26.8,6.4Hz,2H),2.22–2.07(m,3H),1.68–1.46(m,4H),1.35–1.12(m,14H),0.94(s,9H).HRMS(DART-TOF)calculated for C55H69FN8NaO7S+[M+Na]+m/z 1027.4892,found 1027.4897
实施例10
PV13的合成参考通用步骤E,以L14和V1为反应原料即可得到产物PV13,白色固体,产率61%。1H NMR(400MHz,CDCl3)δ11.28(d,J=30.0Hz,1H),8.66(s,1H),8.48–8.36(m,1H),7.80–7.64(m,3H),7.57–7.46(m,1H),7.38–7.26(m,6H),7.05–6.94(m,1H),6.54(d,J=8.8Hz,1H),4.72(t,J=8.0Hz,1H),4.63–4.47(m,3H),4.32(dd,J=15.0,5.2Hz,1H),4.25(s,2H),4.12(d,J=11.5Hz,1H),3.86–3.15(m,10H),2.52–2.41(m,4H),2.31(dt,J=26.7,7.5Hz,2H),2.23–2.10(m,4H),1.67–1.48(m,4H),1.35–1.13(m,16H),0.94(s,9H).HRMS(DART-TOF)calculated for C56H71FN8NaO7S+[M+Na]+m/z 1041.5048,found1041.5043.
实施例11
PV14的合成参考通用步骤E,以L7和V2为反应原料即可得到产物PV14,白色固体,产率80%。1H NMR(400MHz,DMSO)δ12.59(s,1H),8.98(s,1H),8.56(t,J=5.9Hz,1H),8.27(d,J=7.7Hz,1H),7.97(d,J=7.8Hz,1H),7.93–7.87(m,1H),7.86–7.80(m,2H),7.78(t,J=5.2Hz,1H),7.40(dd,J=16.8,8.3Hz,6H),7.23(t,J=8.3Hz,1H),5.14(s,1H),4.55(d,J=9.3Hz,1H),4.48–4.40(m,2H),4.40–4.29(m,3H),4.22(dd,J=15.8,5.4Hz,1H),3.76–3.35(m,9H),3.17(d,J=26.4Hz,2H),3.00(dd,J=12.3,6.2Hz,2H),2.60–2.53(m,1H),2.44(s,3H),2.31(t,J=6.8Hz,2H),2.24(dd,J=14.9,7.7Hz,1H),2.13(dd,J=14.2,6.9Hz,1H),2.08–1.98(m,1H),1.96–1.85(m,1H),1.58–1.42(m,2H),1.42–1.32(m,2H),1.29–1.19(m,2H),0.94(s,9H).HRMS(DART-TOF)calculated for C52H62FN9NaO8S+[M+Na]+m/z 1014.4324,found 1014.4319.
实施例12
PV15的合成参考通用步骤E,以L8和V2为反应原料即可得到产物PV15,白色固体,产率77%。1H NMR(400MHz,DMSO)δ12.60(s,1H),8.98(s,1H),8.56(t,J=5.9Hz,1H),8.27(d,J=7.7Hz,1H),7.97(d,J=7.7Hz,1H),7.89(t,J=7.5Hz,1H),7.86–7.79(m,2H),7.73(s,1H),7.41(dd,J=17.3,8.4Hz,6H),7.23(t,J=8.9Hz,1H),5.14(d,J=3.0Hz,1H),4.56(d,J=9.3Hz,1H),4.44(dd,J=14.5,7.1Hz,2H),4.39–4.31(m,3H),4.23(dd,J=15.9,5.4Hz,1H),3.74–3.36(m,9H),3.17(d,J=14.3Hz,2H),3.00(s,2H),2.45(s,3H),2.34(s,1H),2.31–2.21(m,2H),2.13(dd,J=14.4,7.4Hz,1H),2.09–2.01(m,2H),1.96–1.86(m,1H),1.56–1.43(m,6H),1.41–1.32(m,2H),1.28–1.19(m,3H),0.94(s,9H).HRMS(DART-TOF)calculated for C54H66FN9NaO8S+[M+Na]+m/z 1042.4637,found 1042.4634.
实施例13
PV16的合成参考通用步骤E,以L9和V2为反应原料即可得到产物PV16,白色固体,产率75%。1H NMR(400MHz,DMSO)δ12.60(s,1H),8.98(s,1H),8.56(t,J=5.8Hz,1H),8.27(d,J=7.6Hz,1H),7.97(d,J=7.7Hz,1H),7.89(t,J=7.5Hz,1H),7.86–7.80(m,2H),7.71(s,1H),7.40(dd,J=16.9,8.3Hz,6H),7.23(t,J=9.0Hz,1H),5.14(s,1H),4.55(d,J=9.3Hz,1H),4.44(dd,J=13.5,7.3Hz,2H),4.39–4.30(m,3H),4.22(dd,J=15.9,5.3Hz,1H),3.72–3.37(m,9H),3.16(d,J=15.2Hz,2H),3.00(d,J=5.4Hz,2H),2.45(s,3H),2.37–2.21(m,3H),2.13(dd,J=13.8,6.7Hz,1H),2.06–1.98(m,2H),1.96–1.87(m,1H),1.54–1.42(m,6H),1.40–1.32(m,2H),1.29–1.17(m,6H),0.94(s,9H).HRMS(DART-TOF)calculated for C56H70FN9NaO8S+[M+Na]+m/z 1070.4950,found 1070.4947.
实施例14
PV17的合成参考通用步骤E,以L10和V2为反应原料即可得到产物PV17,白色固体,产率70%。1H NMR(400MHz,DMSO)δ8.98(s,1H),8.57(t,J=6.0Hz,1H),8.28(d,J=7.6Hz,1H),7.95(t,J=9.6Hz,1H),7.93–7.78(m,3H),7.71(t,J=5.3Hz,1H),7.41(dd,J=18.3,8.3Hz,6H),7.23(t,J=9.0Hz,1H),5.15(d,J=3.4Hz,1H),4.56(d,J=9.3Hz,1H),4.45(dd,=15.2,6.8Hz,2H),4.41–4.30(m,4H),4.23(dd,J=16.0,5.4Hz,1H),3.75–3.37(m,9H),3.17(d,J=15.7Hz,2H),3.01(dd,J=12.4,6.2Hz,2H),2.45(s,3H),2.38–2.20(m,3H),2.14(dd,J=14.4,7.1Hz,1H),2.03(t,J=7.4Hz,2H),1.97–1.88(m,1H),1.48(s,6H),1.42–1.32(m,2H),1.33–1.10(m,10H),0.95(s,9H).HRMS(DART-TOF)calculated forC58H74FN9NaO8S+[M+Na]+m/z 1098.5263,found 1098.5261.
实施例15
PV18的合成参考通用步骤E,以L12和V2为反应原料即可得到产物PV18,白色固体,产率69%。1H NMR(400MHz,CDCl3)δ11.70(s,1H),8.63(s,1H),8.40(d,J=6.9Hz,1H),7.81–7.58(m,4H),7.30(dd,J=17.7,9.2Hz,6H),6.99(d,J=8.1Hz,1H),6.86(s,1H),6.10(s,1H),4.71(t,J=7.9Hz,1H),4.66–4.48(m,3H),4.48–4.36(m,1H),4.31(dd,J=15.4,5.3Hz,1H),4.24(s,2H),4.07(d,J=10.9Hz,1H),3.83–3.17(m,10H),3.12(d,J=6.1Hz,2H),2.53(s,2H),2.44(s,3H),2.42–2.12(m,5H),2.07(t,J=7.3Hz,2H),1.65–1.45(m,6H),1.45–1.34(m,2H),1.32–1.12(m,14H),0.94(s,9H).HRMS(DART-TOF)calculated forC60H78FN9NaO8S+[M+Na]+m/z 1126.5576,found 1126.5576.
实施例16
PV19的合成参考通用步骤E,以L12和V3为反应原料即可得到产物PV19,白色固体,产率57%。1H NMR(400MHz,CDCl3)δ11.59(d,J=14.6Hz,1H),8.58(s,1H),8.40–8.24(m,1H),7.77–7.60(m,3H),7.58(t,J=5.7Hz,1H),7.24(q,J=8.4Hz,6H),6.95(d,J=8.4Hz,1H),6.42(t,J=5.2Hz,1H),4.67(t,J=8.0Hz,1H),4.53–4.40(m,3H),4.33(s,1H),4.25(dd,J=15.2,5.2Hz,1H),4.18(s,2H),4.02(d,J=11.1Hz,1H),3.78–3.01(m,12H),2.38(s,3H),2.34–2.23(m,2H),2.23–2.07(m,4H),2.02(t,J=6.0Hz,2H),1.73–1.61(m,2H),1.59–1.35(m,3H),1.31(d,J=6.6Hz,2H),1.19–1.07(m,10H),0.90(s,9H).HRMS(DART-TOF)calculated for C58H74FN9NaO8S+[M+Na]+m/z 1098.5263,found 1098.5257.
实施例17
合成L15:参考通用步骤E,以L12和甘氨酸叔丁酯为反应原料即可得到产物L15,白色固体,产率51%。1H NMR(400MHz,CDCl3)δ11.39(d,J=23.9Hz,1H),8.54–8.36(m,1H),7.71(dd,J=11.8,5.6Hz,3H),7.30(d,J=5.2Hz,2H),7.01(t,J=8.9Hz,1H),6.11(s,1H),4.27(s,2H),3.91(d,J=5.1Hz,2H),3.83–3.14(m,8H),2.29(dt,J=28.5,7.4Hz,2H),2.19(t,J=7.6Hz,2H),1.68–1.49(m,4H),1.44(s,9H),1.35–1.14(m,12H).
PV20的合成:在反应瓶中加入L15(0.1mmol)和V1(0.1mmol),加入10mL DCM/MeOH(5:1)溶解后,滴加2mL 4M HCl-二氧六环溶液,室温反应2h,减压浓缩溶剂后的白色固体混合物,向反应瓶中加入10mL无水DMF溶解后置于冰水浴中,加入DIPEA(0.5mmol),搅拌下分批加入HATU(380mg,0.1mmol),将反应置于冰水浴中反应1h。TLC监测反应完成后,在反应液中加入50mL半饱和食盐水稀释,再用乙酸乙酯萃取(3×60mL),合并有机层用饱和氯化钠水溶液洗一次,加入无水Na2SO4干燥,过滤减压蒸去溶剂得油状粗品。最后用DCM:MeOH=10:1体系进行柱层析分离得到白色固体PV20,产率43%。1H NMR(400MHz,CDCl3)δ11.60(s,1H),8.64(s,1H),8.46–8.34(m,1H),8.07(s,1H),7.79–7.60(m,4H),7.38–7.22(m,6H),7.06–6.88(m,2H),4.77(t,J=8.0Hz,1H),4.71(d,J=9.1Hz,1H),4.55(dd,J=15.5,6.6Hz,2H),4.36–4.21(m,3H),4.16(s,1H),4.04–3.93(m,3H),3.81–3.13(m,9H),2.46(s,3H),2.36–2.20(m,5H),2.21–2.09(m,2H),1.65–1.45(m,4H),1.28–1.13(m,12H),0.98(s,9H).HRMS(DART-TOF)calculated for C56H70FN9NaO8S+[M+Na]+m/z 1070.4950,found 1070.4953.
实施例18
通用步骤H:合成PC4-PC13
在25mL反应瓶中加入N-Boc-丁氨酸/己氨酸(1mmol),再加入10mL无水DMF溶解后置于冰浴中,加入DIPEA(2mmol),搅拌下分批加入HATU(380mg,1mmol,1.0eq),室温下搅拌5min,再加入L1(366mg,1mmol),将反应置于室温下反应1h。TLC监测反应完成后,在反应液中加入50mL半饱和食盐水稀释,再用乙酸乙酯萃取(3×60mL),合并有机层用饱和氯化钠水溶液洗一次,加入无水Na2SO4干燥,过滤减压蒸去溶剂得油状粗品。最后用DCM:MeOH=10:1体系进行柱层析分离得到白色固体L16-L17。
实施例19
合成L16:参考通用步骤H,以L1和N-Boc-4-氨基丁酸为反应原料即可得到产物L16,白色固体,产率82%。1H NMR(400MHz,CDCl3)δ:11.66(d,J=19.7Hz,1H),8.49–8.37(m,1H),7.71(dd,J=10.5,4.0Hz,3H),7.40–7.22(m,2H),7.00(dd,J=12.3,5.9Hz,1H),4.90(s,1H),4.27(s,2H),3.87–3.21(m,8H),3.14(m,2H),2.35(dt,J=28.7,7.0Hz,2H),1.88–1.74(m,2H),1.39(m,8H).HRMS(DART-TOF)calculated for C29H34FN5NaO5 +[M+Na]+m/z574.2442,found 574.2437.
实施例20
合成L17:参考通用步骤H,以L1和N-Boc-6-氨基己酸为反应原料即可得到产物L17,白色固体,产率86%。1H NMR(400MHz,CDCl3)δ:1H NMR(400MHz,CDCl3)δ11.53(d,J=27.4Hz,1H),8.50–8.39(m,1H),7.72(dd,J=11.4,5.3Hz,1H),7.39–7.22(m,1H),7.01(t,J=9.0Hz,1H),4.66(s,1H),4.27(s,1H),3.92–3.16(m,1H),3.08(d,J=6.0Hz,1H),2.40–2.19(m,1H),1.72–1.55(m,1H),1.55–1.26(m,1H),1.26–1.19(m,1H).HRMS(DART-TOF)calculated for C31H38FN5NaO5 +[M+Na]+m/z 602.2755,found 602.2751.
实施例21
合成L18:参考通用步骤E,以L16和N-Boc-4-氨基丁酸为反应原料即可得到产物L18,白色固体,产率74%。1H NMR(400MHz,CDCl3)δ:11.66(d,J=19.8Hz,1H),8.49–8.37(m,1H),7.71(dd,J=10.6,4.0Hz,3H),7.40–7.22(m,2H),7.00(dd,J=12.3,5.9Hz,1H),4.90(s,1H),4.27(s,2H),3.87–3.21(m,8H),3.14(dd,J=11.1,5.8Hz,2H),2.35(dt,J=28.7,7.0Hz,2H),1.88–1.74(m,2H),1.39(d,J=5.3Hz,8H).HRMS(DART-TOF)calculated forC33H41FN6NaO6 +[M+Na]+m/z 659.2969,found 659.2974.
实施例22
合成L19:参考通用步骤E,以L16和N-Boc-6-氨基己酸为反应原料即可得到产物L19,白色固体,产率77%。1H NMR(400MHz,CDCl3)δ:11.29(m,1H),8.51–8.36(m,1H),7.83–7.68(m,3H),7.42–7.28(m,2H),7.02(t,J=8.8Hz,1H),6.34(t,J=26.9Hz,1H),4.70(s,1H),4.28(s,2H),3.88–3.18(m,11H),3.17–2.98(m,2H),2.39(dt,J=27.4,6.6Hz,2H),2.30–1.92(m,4H),1.90–1.76(m,2H),1.59(d,J=6.6Hz,2H),1.51–1.35(m,14H),1.31(dd,J=14.4,7.9Hz,3H),1.25(d,J=11.7Hz,2H).HRMS(DART-TOF)calculated forC35H45FN6NaO6 +[M+Na]+m/z 687.3282,found 687.3282.
实施例23
合成L20:参考通用步骤E,以L17和N-Boc-4-氨基丁酸为反应原料即可得到产物L20,白色固体,产率70%。1H NMR(400MHz,CDCl3)δ:11.44(d,J=35.8Hz,1H),8.54–8.39(m,1H),7.73(dd,J=11.7,8.1Hz,3H),7.31(dd,J=8.3,5.3Hz,2H),7.01(t,J=8.9Hz,1H),5.99(d,J=18.7Hz,1H),4.73(s,1H),4.27(s,2H),3.86–3.14(m,10H),3.06(dd,J=12.7,6.3Hz,2H),2.40–2.16(m,4H),2.13(t,J=7.5Hz,2H),1.61(dd,J=14.7,7.4Hz,4H),1.53–1.36(m,14H),1.35–1.26(m,4H).HRMS(DART-TOF)calculated for C35H45FN6NaO6 +[M+Na]+m/z 687.3282,found 687.3283.
实施例24
合成L21:参考通用步骤E,以L17和N-Boc-6-氨基己酸为反应原料即可得到产物L21,白色固体,产率70%。1H NMR(400MHz,CDCl3)δ:11.51(m,1H),8.48–8.38(m,1H),7.81–7.60(m,3H),7.36–7.27(m,2H),7.02(dd,J=12.1,5.4Hz,1H),6.50(s,1H),5.00(s,1H),4.27(s,2H),3.87–2.99(m,13H),2.46–2.22(m,3H),2.18(t,J=6.4Hz,3H),1.85–1.69(m,2H),1.58(d,J=24.4Hz,2H),1.55–1.45(m,2H),1.45–1.28(m,12H).HRMS(DART-TOF)calculated for C37H49FN6NaO6 +[M+Na]+m/z 715.3595,found 715.3600.
实施例25
合成PC4:参考通用步骤E,以L16和S7为反应原料即可得到产物PC4,白色固体,产率71%。1H NMR(400MHz,CDCl3)δ:11.26(d,J=30.9Hz,1H),9.66(s,1H),8.41(dd,J=6.2,1.8Hz,1H),7.78–7.65(m,4H),7.65–7.55(m,1H),7.54–7.46(m,1H),7.37–7.23(m,2H),7.19(d,J=8.4Hz,1H),7.01(t,J=8.5Hz,1H),4.98(m,1H),4.64(s,2H),4.26(s,2H),3.85–3.15(m,10H),2.92–2.70(m,3H),2.49–2.31(m,2H),2.13(s,1H),1.98–1.84(m,2H).HRMS(DART-TOF)calculated for C39H36FN7NaO9 +[M+Na]+m/z 788.2456,found 788.2458.
实施例26
合成PC5:参考通用步骤E,以L17和S7为反应原料即可得到产物PC5,白色固体,产率79%。1H NMR(400MHz,CDCl3)δ:11.13(d,J=33.8Hz,1H),9.85(s,1H),8.48–8.40(m,1H),7.81–7.65(m,4H),7.62–7.48(m,2H),7.35–7.26(m,2H),7.20(d,J=8.4Hz,1H),7.02(t,J=8.7Hz,1H),5.08–4.87(m,1H),4.64(q,J=14.2Hz,2H),4.27(s,2H),3.85–3.15(m,10H),2.93–2.67(m,3H),2.43–2.25(m,2H),2.19–2.09(m,1H),1.72–1.52(m,4H),1.45–1.31(m,2H).HRMS(DART-TOF)calculated for C41H40FN7NaO9 +[M+Na]+m/z 816.2769,found816.2766.
实施例27
合成PC6:参考通用步骤E,以L18和S7为反应原料即可得到产物PC6,白色固体,产率74%。1H NMR(400MHz,DMSO)δ:12.58(s,1H),11.11(s,1H),8.26(dd,J=7.8,1.1Hz,1H),7.98(m,2H),7.93–7.85(m,1H),7.86–7.74(m,3H),7.49(d,J=7.1Hz,1H),7.46–7.33(m,3H),7.23(t,J=9.0Hz,1H),5.12(dd,J=12.9,5.4Hz,1H),4.76(d,J=8.6Hz,2H),4.33(s,2H),3.69–2.98(m,12H),2.95–2.82(m,1H),2.66–2.47(m,2H),2.31(dt,J=27.4,6.9Hz,2H),2.14–1.95(m,3H),1.72–1.54(m,4H).HRMS(DART-TOF)calculated for C43H43FN8NaO10 +[M+Na]+m/z 873.2984,found 873.2984.
实施例28
合成PC7:参考通用步骤E,以L19和S7为反应原料即可得到产物PC8,白色固体,产率82%。1H NMR(400MHz,CDCl3)δ:10.92(d,J=32.3Hz,1H),10.30(s,1H),8.48–8.39(m,1H),7.82–7.66(m,4H),7.61–7.49(m,2H),7.36–7.24(m,3H),7.20(d,J=8.4Hz,1H),7.03(t,J=8.8Hz,1H),6.44–6.33(m,1H),4.99(dd,J=11.9,5.6Hz,1H),4.72–4.56(m,2H),4.27(s,2H),3.84–3.13(m,12H),2.92–2.69(m,3H),2.39(dt,J=30.4,6.2Hz,2H),2.26–2.05(m,3H),1.67–1.50(m,4H),1.45–1.32(m,3H).HRMS(DART-TOF)calculated forC45H47FN8NaO10 +[M+Na]+m/z 901.3297,found 901.3294.
实施例29
合成PC8:参考通用步骤E,以L20和S7为反应原料即可得到产物PC8,白色固体,产率78%。1H NMR(400MHz,CDCl3)δ:11.23(d,J=36.5Hz,1H),9.75(s,1H),8.42(dd,J=6.6,2.5Hz,1H),7.80–7.62(m,5H),7.51(d,J=7.3Hz,1H),7.35–7.24(m,2H),7.21(d,J=8.4Hz,1H),7.02(t,J=8.7Hz,1H),6.61–6.42(m,1H),5.03–4.89(m,1H),4.65(s,2H),4.27(s,2H),3.82–3.11(m,12H),2.91–2.71(m,3H),2.30(dt,J=30.6,7.0Hz,2H),2.21(t,J=7.2Hz,2H),2.17–2.14(m,1H),1.93–1.78(m,2H),1.67–1.54(m,2H),1.53–1.41(m,2H),1.39–1.26(m,3H).HRMS(DART-TOF)calculated for C45H47FN8NaO10 +[M+Na]+m/z 901.3297,found 901.3296.
实施例30
合成PC9:参考通用步骤E,以L21和S7为反应原料即可得到产物PC9,白色固体,产率76%。1H NMR(400MHz,CDCl3)δ:11.15(d,J=38.7Hz,1H),10.08(s,1H),8.50–8.38(m,1H),7.81–7.66(m,4H),7.61–7.47(m,2H),7.37–7.26(m,3H),7.20(d,J=8.4Hz,1H),7.03(t,J=8.8Hz,1H),6.05(s,1H),5.00(dd,J=11.7,5.9Hz,1H),4.64(q,J=14.2Hz,2H),4.28(s,2H),3.86–3.11(m,13H),2.95–2.66(m,3H),2.32(dt,J=30.7,7.1Hz,2H),2.24–2.09(m,3H),1.70–1.53(m,6H),1.54–1.42(m,2H),1.43–1.26(m,4H).HRMS(DART-TOF)calculated for C47H51FN8NaO10 +[M+Na]+m/z 929.3610,found 929.3614.
实施例31
合成PC10:参考通用步骤E,以L16和S8为反应原料即可得到产物PC10,黄色固体,产率82%。1H NMR(400MHz,CDCl3)δ:11.00(d,J=18.7Hz,1H),9.42(d,J=12.7Hz,1H),8.42(d,J=7.5Hz,1H),7.83–7.66(m,3H),7.50–7.43(m,1H),7.36–7.24(m,3H),7.13(d,J=6.9Hz,1H),7.04(t,J=8.8Hz,1H),6.82–6.65(m,2H),5.00–4.84(m,1H),4.28(s,2H),3.93(d,J=5.7Hz,2H),3.76–3.09(m,10H),2.92–2.65(m,3H),2.28(dd,J=27.6,21.1Hz,2H),2.15–2.03(m,1H),1.88–1.77(m,2H).HRMS(DART-TOF)calculated for C39H37FN8NaO8 +[M+Na]+m/z 787.2616,found 787.2613.
实施例32
合成PC11:参考通用步骤E,以L17和S8为反应原料即可得到产物PC11,黄色固体,产率78%。1H NMR(400MHz,CDCl3)δ:11.24(d,J=36.8Hz,1H),9.52(d,J=59.2Hz,1H),8.40(d,J=7.9Hz,1H),7.83–7.63(m,3H),7.47(t,J=7.8Hz,1H),7.29(d,J=7.3Hz,3H),7.11(d,J=7.1Hz,1H),7.02(t,J=8.8Hz,1H),6.98–6.88(m,1H),6.78(d,J=8.5Hz,1H),6.73(t,J=5.9Hz,1H),4.95(dd,J=11.8,5.7Hz,1H),4.26(s,2H),3.92(d,J=5.6Hz,2H),3.83–3.08(m,10H),2.90–2.66(m,3H),2.24(dt,J=33.6,6.7Hz,2H),2.16–2.04(m,1H),1.64–1.49(m,2H),1.49–1.38(m,2H),1.32–1.20(m,2H).HRMS(DART-TOF)calculated forC41H41FN8NaO8 +[M+Na]+m/z 815.2924,found 815.2927.
实施例33
合成PC12:参考通用步骤E,以L20和S8为反应原料即可得到产物PC12,黄色固体,产率73%。1H NMR(400MHz,CDCl3)δ:10.90(d,J=70.0Hz,1H),9.31(s,1H),8.48–8.36(m,1H),7.84–7.66(m,3H),7.49(t,J=7.5Hz,1H),7.38–7.28(m,2H),7.14(d,J=6.8Hz,1H),7.05(t,J=8.7Hz,1H),6.85–6.69(m,2H),6.36–6.24(m,1H),5.02–4.86(m,1H),4.28(s,2H),3.94(d,J=5.0Hz,2H),3.80–3.11(m,12H),2.95–2.69(m,3H),2.31(dt,J=12.6,6.6Hz,2H),2.19–2.04(m,3H),1.78(dt,J=12.3,6.0Hz,2H),1.66–1.56(m,2H),1.47(dd,J=12.9,6.4Hz,2H),1.38–1.29(m,2H).HRMS(DART-TOF)calculated for C45H48FN9NaO9 +[M+Na]+m/z 900.3451,found 900.3461.
实施例34
合成PC13:参考通用步骤E,以L21和S8为反应原料即可得到产物PC13,黄色固体,产率81%。1H NMR(400MHz,CDCl3)δ:10.96(d,J=55.6Hz,1H),9.39(s,1H),8.49–8.39(m,1H),7.83–7.67(m,3H),7.50(t,J=7.8Hz,1H),7.38–7.27(m,2H),7.15(d,J=7.1Hz,1H),7.04(t,J=8.6Hz,1H),6.97–6.86(m,1H),6.80(d,J=8.5Hz,1H),6.74(t,J=5.9Hz,1H),6.04–5.91(m,1H),5.05–4.88(m,1H),4.28(s,2H),3.94(d,J=5.9Hz,2H),3.82–3.14(m,12H),2.93–2.67(m,3H),2.32(dt,J=31.2,6.7Hz,2H),2.18–2.01(m,3H),1.68–1.40(m,8H),1.37–1.17(m,4H).HRMS(DART-TOF)calculated for C47H52FN9NaO9 +[M+Na]+m/z928.3764,found 928.3767.
实施例35
合成PC14:参考通用步骤E,以L7和S10为反应原料即可得到产物PC14,黄色固体,产率62%。1H NMR(400MHz,CDCl3)δ:11.36(s,1H),9.61(s,1H),8.42(s,1H),7.78–7.62(m,3H),7.41(t,J=7.7Hz,1H),7.36–7.24(m,2H),6.99(t,J=8.9Hz,2H),6.81(d,J=8.5Hz,1H),6.65–6.42(m,1H),6.16(s,1H),4.99–4.81(m,1H),4.25(s,2H),3.94–2.99(m,12H),2.89–2.57(m,5H),2.50(s,2H),1.71–1.45(m,4H).HRMS(DART-TOF)calculated forC41H41FN8NaO8 +[M+Na]+m/z 815.2929,found 815.2927.
实施例36
合成PC15:参考通用步骤E,以L8和S10为反应原料即可得到产物PC15,黄色固体,产率65%。1H NMR(400MHz,CDCl3)δ:11.04(d,J=28.7Hz,1H),9.33(s,1H),8.44(d,J=6.5Hz,1H),7.82–7.63(m,3H),7.45(t,J=7.8Hz,1H),7.31(dd,J=12.0,7.0Hz,2H),7.03(dd,J=13.7,7.9Hz,2H),6.85(d,J=8.5Hz,1H),6.41(d,J=31.0Hz,1H),6.20(t,J=4.7Hz,1H),5.01–4.85(m,1H),4.27(s,2H),3.87–3.12(m,12H),2.91–2.67(m,3H),2.33(dt,J=33.2Hz,2H),2.21(s,2H),2.15–2.07(m,1H),1.63(d,J=7.0Hz,8H).HRMS(DART-TOF)calculated for C43H45FN8NaO8 +[M+Na]+m/z 843.3242,found 843.3237.
实施例37
合成PC16:参考通用步骤E,以L9和S10为反应原料即可得到产物PC16,黄色固体,产率54%。1H NMR(400MHz,CDCl3)δ:10.78(d,J=37.2Hz,1H),9.02(s,1H),8.45(d,J=6.3Hz,1H),7.83–7.63(m,3H),7.47(t,J=7.8Hz,1H),7.38–7.27(m,2H),7.05(dd,J=17.4,7.8Hz,2H),6.87(d,J=8.5Hz,1H),6.21(s,1H),5.83(d,J=19.8Hz,1H),4.93(dd,J=11.7,5.3Hz,1H),4.28(s,2H),3.85–3.18(m,12H),2.94–2.65(m,3H),2.30(dt,J=30.6,6.8Hz,2H),2.20–2.06(m,3H),1.72–1.51(m,8H),1.40–1.27(m,4H).HRMS(DART-TOF)calculated for C45H49FN8NaO8 +[M+Na]+m/z 871.3555,found 871.3556.
实施例38
合成PC17:参考通用步骤E,以L10和S10为反应原料即可得到产物PC17,黄色固体,产率59%。1H NMR(400MHz,CDCl3)δ:10.65(d,J=35.2Hz,1H),8.87(s,1H),8.52–8.39(m,1H),7.83–7.63(m,3H),7.54–7.42(m,1H),7.39–7.25(m,2H),7.05(dd,J=18.5,7.7Hz,2H),6.88(d,J=8.5Hz,1H),6.22(t,J=5.4Hz,1H),5.67(s,1H),4.92(dd,J=11.9,5.4Hz,1H),4.28(s,2H),3.97–3.05(m,12H),2.95–2.68(m,3H),2.30(dt,J=29.7,7.2Hz,2H),2.20–2.05(m,3H),1.76–1.51(m,8H),1.34–1.21(m,8H).HRMS(DART-TOF)calculated forC47H53FN8NaO8 +[M+Na]+m/z 899.3868,found 899.3868.
实施例39
合成PC18:参考通用步骤E,以L12和S10为反应原料即可得到产物PC18,黄色固体,产率62%。1H NMR(400MHz,CDCl3)δ:11.33(d,J=23.8Hz,1H),9.26(s,1H),8.43(d,J=5.9Hz,1H),7.78–7.64(m,3H),7.44(t,J=7.7Hz,1H),7.37–7.27(m,2H),7.03(d,J=6.8Hz,2H),6.84(d,J=8.5Hz,1H),6.20(s,1H),5.93(s,1H),4.97–4.84(m,1H),4.26(s,2H),3.87–3.11(m,12H),2.90–2.66(m,3H),2.29(d,J=28.8Hz,2H),2.15–2.04(m,3H),1.74–1.46(m,8H),1.36–1.11(m,14H).HRMS(DART-TOF)calculated for C49H57FN8NaO8 +[M+Na]+m/z 927.4181,found 927.4186.
实施例40
合成PC19:参考通用步骤E,以L7和S9为反应原料即可得到产物PC19,黄色固体,产率49%。1H NMR(400MHz,CDCl3)δ:11.07(d,J=7.7Hz,1H),9.53(s,1H),8.43(d,J=5.0Hz,1H),7.82–7.65(m,3H),7.44(t,J=7.8Hz,1H),7.37–7.28(m,2H),7.03(dd,J=15.5,7.8Hz,2H),6.98–6.85(m,2H),6.43(s,1H),4.93(dd,J=11.7,5.5Hz,1H),4.27(s,2H),3.87–3.13(m,12H),2.91–2.56(m,5H),2.52(s,2H),2.15–2.05(m,1H).HRMS(DART-TOF)calculated for C39H37FN8NaO8 +[M+Na]+m/z 787.2616,found 787.2620.
实施例41
合成PC20:参考通用步骤E,以L8和S9为反应原料即可得到产物PC20,黄色固体,产率50%。1H NMR(400MHz,CDCl3)δ:11.08(d,J=29.3Hz,1H),9.49(s,1H),8.42(d,J=6.1Hz,1H),7.86–7.62(m,3H),7.44(t,J=7.7Hz,1H),7.37–7.26(m,2H),7.13(s,1H),7.07–6.98(m,2H),6.93(d,J=8.5Hz,1H),6.47(d,J=19.2Hz,1H),4.92(d,J=5.8Hz,1H),4.27(s,2H),3.85–3.12(m,12H),2.91–2.64(m,3H),2.45–2.02(m,5H),1.71–1.49(m,4H).HRMS(DART-TOF)calculated for C41H41FN8NaO8 +[M+Na]+m/z 815.2924,found 815.2933.
实施例42
合成PC21:参考通用步骤E,以L9和S9为反应原料即可得到产物PC 21,黄色固体,产率57%。1H NMR(400MHz,CDCl3)δ:11.07(d,J=30.2Hz,1H),9.42(d,J=15.9Hz,1H),8.51–8.33(m,1H),7.84–7.62(m,3H),7.44(t,J=7.4Hz,1H),7.37–7.26(m,2H),7.04(dd,J=12.5,7.8Hz,2H),6.94(d,J=8.6Hz,1H),6.51(d,J=31.9Hz,1H),6.40(s,1H),5.01–4.84(m,1H),4.27(s,2H),3.85–3.16(m,12H),2.91–2.67(m,3H),2.28(dt,J=32.5,6.7Hz,2H),2.18–2.04(m,3H),1.67–1.46(m,4H),1.35–1.19(m,6H).HRMS(DART-TOF)calculatedfor C43H45FN8NaO8 +[M+Na]+m/z 843.3237,found 843.3234.
实施例43
合成PC22:参考通用步骤E,以L10和S9为反应原料即可得到产物PC22,黄色固体,产率55%。1H NMR(400MHz,CDCl3)δ:11.03(d,J=32.2Hz,1H),9.27(d,J=18.2Hz,1H),8.44(d,J=6.4Hz,1H),7.83–7.62(m,3H),7.46(t,J=7.7Hz,1H),7.38–7.27(m,2H),7.04(dd,J=15.8,7.6Hz,2H),6.96(d,J=8.5Hz,1H),6.40(s,2H),5.00–4.84(m,1H),4.27(s,2H),3.88–3.09(m,12H),2.90–2.67(m,3H),2.29(d,J=31.1Hz,2H),2.18–2.04(m,3H),1.67–1.46(m,4H),1.36–1.15(m,8H).HRMS(DART-TOF)calculated for C45H49FN8NaO8 +[M+Na]+m/z871.3550,found 871.3554.
实施例44
合成PC23:参考通用步骤E,以L11和S9为反应原料即可得到产物PC23,黄色固体,产率60%。1H NMR(400MHz,CDCl3)δ:11.18(d,J=28.5Hz,1H),9.32(d,J=11.4Hz,1H),8.49–8.29(m,1H),7.84–7.61(m,3H),7.44(t,J=7.8Hz,1H),7.38–7.26(m,2H),7.03(dd,J=12.5,7.8Hz,2H),6.95(t,J=7.3Hz,1H),6.50(d,J=21.0Hz,1H),6.39(s,1H),4.93(dd,J=12.1,5.4Hz,1H),4.26(s,2H),3.89–3.10(m,12H),2.91–2.64(m,3H),2.29(dt,J=29.9,7.0Hz,2H),2.19–2.07(m,3H),1.67–1.44(m,4H),1.31–1.15(m,10H).HRMS(DART-TOF)calculated for C46H51FN8NaO8 +[M+Na]+m/z 885.3706,found 885.3706.
实施例45
合成PC24:参考通用步骤E,以L12和S9为反应原料即可得到产物PC 24,黄色固体,产率58%。1H NMR(400MHz,CDCl3)δ:11.17(d,J=25.5Hz,1H),9.29(s,1H),8.42(d,J=6.4Hz,1H),7.85–7.61(m,3H),7.44(t,J=7.7Hz,1H),7.37–7.25(m,2H),7.14–6.99(m,2H),6.96(d,J=8.5Hz,1H),6.47(s,1H),6.39(s,1H),5.01–4.82(m,1H),4.26(s,2H),3.86–3.10(m,12H),2.90–2.65(m,3H),2.39–2.20(m,2H),2.17–2.06(m,3H),1.67–1.45(m,4H),1.33–1.09(m,12H).HRMS(DART-TOF)calculated for C47H53FN8NaO8 +[M+Na]+m/z899.3863,found 899.3861.
实施例46
合成PC25:参考通用步骤E,以L13和S9为反应原料即可得到产物PC25,黄色固体,产率64%。1H NMR(400MHz,CDCl3)δ:11.05(d,J=30.3Hz,1H),9.15(d,J=20.5Hz,1H),8.48–8.34(m,1H),7.82–7.63(m,3H),7.51–7.40(m,1H),7.38–7.27(m,2H),7.04(dd,J=16.0,7.6Hz,2H),6.97(d,J=8.6Hz,1H),6.38(d,J=13.3Hz,2H),4.93(dd,J=12.1,5.4Hz,1H),4.27(s,2H),3.88–3.17(m,12H),2.92–2.67(m,3H),2.30(dt,J=29.0,7.2Hz,2H),2.20–2.04(m,3H),1.66–1.46(m,4H),1.33–1.15(m,14H).HRMS(DART-TOF)calculatedfor C48H55FN8NaO8 +[M+Na]+m/z 913.4019,found 913.4028.
实施例47
合成PC26:参考通用步骤E,以L14和S9为反应原料即可得到产物PC26,黄色固体,产率67%。1H NMR(400MHz,CDCl3)δ:10.90(d,J=31.1Hz,1H),9.03(d,J=21.6Hz,1H),8.51–8.36(m,1H),7.84–7.62(m,3H),7.50–7.41(m,1H),7.37–7.27(m,2H),7.11–6.91(m,3H),6.47–6.36(m,1H),6.28(s,1H),4.93(dd,J=12.0,5.5Hz,1H),4.27(s,2H),3.92–3.10(m,12H),2.93–2.65(m,3H),2.31(dt,J=28.4,7.3Hz,2H),2.19–2.06(m,3H),1.68–1.50(m,4H),1.31–1.13(m,16H).HRMS(DART-TOF)calculated for C49H57FN8NaO8 +[M+Na]+m/z927.4176,found 927.4189.
实施例48
合成PC27:参考通用步骤E,以L12和S11为反应原料即可得到产物PC27,黄色固体,产率65%。1H NMR(400MHz,CDCl3)δ:11.02(d,J=26.4Hz,1H),8.96(s,1H),8.53–8.36(m,1H),7.72(dd,J=11.9,5.6Hz,3H),7.46(t,J=7.8Hz,1H),7.39–7.27(m,2H),7.03(dd,J=17.1,7.7Hz,2H),6.85(d,J=8.5Hz,1H),6.21(t,J=5.4Hz,1H),5.75–5.54(m,1H),4.92(dd,J=11.7,5.3Hz,1H),4.27(s,2H),3.89–3.12(m,12H),2.91–2.66(m,3H),2.30(dt,J=28.6,7.2Hz,2H),2.19–2.04(m,3H),1.71–1.54(m,6H),1.53–1.45(m,2H),1.44–1.18(m,16H).HRMS(DART-TOF)calculated for C51H61FN8NaO8 +[M+Na]+m/z 955.4489,found955.4496.
实施例49
合成PC28:参考通用步骤E,以L12和S12为反应原料即可得到产物PC28,黄色固体,产率49%。1H NMR(400MHz,CDCl3)δ:10.77(d,J=31.3Hz,1H),8.97(d,J=29.3Hz,1H),8.52–8.41(m,1H),7.86–7.63(m,3H),7.56–7.44(m,1H),7.37–7.27(m,2H),7.10(d,J=7.1Hz,1H),7.03(t,J=8.7Hz,1H),6.91(d,J=8.5Hz,1H),6.56(t,J=5.3Hz,1H),6.17(s,1H),4.91(dd,J=12.0,5.3Hz,1H),4.28(s,2H),3.94–3.15(m,16H),2.97–2.65(m,3H),2.31(dt,J=28.1,7.3Hz,2H),2.21–2.05(m,3H),1.65–1.49(m,4H),1.32–1.12(m,12H).HRMS(DART-TOF)calculated for C49H57FN8NaO9 +[M+Na]+m/z 943.4125,found 943.4136.
实施例50
合成PC29:参考通用步骤E,以L12和S13为反应原料即可得到产物PC29,黄色固体,产率58%。1H NMR(400MHz,CDCl3)δ:10.66(d,J=34.0Hz,1H),9.05(d,J=31.7Hz,1H),8.51–8.37(m,1H),7.83–7.66(m,3H),7.55–7.42(m,1H),7.38–7.27(m,2H),7.09(d,J=7.1Hz,1H),7.04(t,J=8.6Hz,1H),6.89(d,J=8.5Hz,1H),6.51(t,J=5.4Hz,1H),6.08(s,1H),4.97–4.84(m,1H),4.28(s,2H),3.95–3.07(m,20H),2.92–2.64(m,3H),2.31(dt,J=14.4,7.4Hz,2H),2.19–2.06(m,3H),1.69–1.48(m,4H),1.33–1.16(m,12H).HRMS(DART-TOF)calculated for C51H61FN8NaO10 +[M+Na]+m/z 987.4387,found 987.4396.
实施例51
合成PC30:参考通用步骤E,以L12和S14为反应原料即可得到产物PC30,黄色固体,产率54%。1H NMR(400MHz,CDCl3)δ:10.82(d,J=29.0Hz,1H),8.95(d,J=21.5Hz,1H),8.53–8.35(m,1H),7.83–7.61(m,3H),7.47(t,J=7.8Hz,1H),7.38–7.27(m,2H),7.04(dd,J=16.7,7.5Hz,2H),6.91(d,J=8.6Hz,1H),6.44(t,J=5.5Hz,1H),6.22(s,1H),4.91(dd,J=11.8,5.4Hz,1H),4.27(s,2H),3.97–3.08(m,24H),2.91–2.65(m,3H),2.30(dt,J=27.7,7.0Hz,2H),2.19–2.04(m,3H),1.96–1.88(m,2H),1.79–1.70(m,2H),1.66–1.49(m,4H),1.35–1.17(m,12H).HRMS(DART-TOF)calculated for C55H69FN8NaO11 +[M+Na]+m/z1059.4962,found 1059.4962.
实施例52
合成PC31:参考通用步骤E,以L12和S15为反应原料即可得到产物PC31,黄色固体,产率67%。1H NMR(400MHz,CDCl3)δ:10.95(d,J=36.3Hz,1H),9.14(d,J=17.3Hz,1H),8.50–8.35(m,1H),7.85–7.64(m,3H),7.51(d,J=8.2Hz,1H),7.31(dd,J=12.4,5.5Hz,2H),7.04(t,J=8.5Hz,1H),6.86(d,J=1.8Hz,1H),6.68(dd,J=8.4,1.9Hz,1H),6.41(t,J=5.9Hz,1H),5.74(t,J=4.6Hz,1H),4.94(dd,J=11.9,5.4Hz,1H),4.28(s,2H),3.93–3.13(m,12H),2.93–2.63(m,3H),2.31(dt,J=13.3,6.9Hz,2H),2.22–2.07(m,3H),1.68–1.47(m,4H),1.32–1.15(m,12H).HRMS(DART-TOF)calculated for C47H53FN8NaO8 +[M+Na]+m/z 899.3863,found 899.3866.
实施例53
合成PC32:参考通用步骤E,以L12和S16为反应原料即可得到产物PC32,黄色固体,产率61%。1H NMR(400MHz,CD3OD)δ:8.41–8.33(m,1H),7.94(d,J=7.8Hz,1H),7.90–7.77(m,2H),7.53–7.45(m,1H),7.39(s,1H),7.32(dd,J=10.4,5.0Hz,1H),7.16(t,J=9.0Hz,1H),7.08(dd,J=7.2,4.3Hz,1H),6.86(d,J=8.0Hz,1H),5.16(dd,J=12.7,2.7Hz,1H),4.38(s,2H),4.27(s,2H),3.85–3.40(m,8H),3.39–3.25(m,4H),3.00–2.84(m,1H),2.84–2.71(m,1H),2.56–2.30(m,3H),2.19(t,J=7.3Hz,3H),1.72–1.47(m,4H),1.35–1.12(m,12H).HRMS(DART-TOF)calculated for C47H55FN8NaO7 +[M+Na]+m/z 885.4070,found885.4074.
实施例54
合成PC33:参考通用步骤E,以L12和S17为反应原料即可得到产物PC33,黄色固体,产率57%。1H NMR(400MHz,CDCl3)δ:10.73(d,J=21.8Hz,1H),8.53–8.33(m,1H),7.73(dd,J=14.1,7.0Hz,3H),7.48(t,J=7.8Hz,1H),7.38–7.27(m,2H),7.14–6.94(m,3H),6.39(s,1H),6.07(s,1H),4.92(dd,J=12.1,5.5Hz,1H),4.27(s,2H),3.88–3.22(m,12H),3.19(s,3H),3.01–2.89(m,1H),2.82–2.68(m,2H),2.30(dt,J=12.9,6.8Hz,2H),2.16(t,J=7.6Hz,2H),2.11–2.02(m,1H),1.67–1.51(m,4H),1.35–1.17(m,12H).HRMS(DART-TOF)calculated for C48H55FN8NaO8 +[M+Na]+m/z 913.4019,found 913.4026.
实施例3~17、25~54制备得到的化合物与其相应的结构式见表1。
表1制备得到的化合物
实施例53
1、PV6~PV20体外肿瘤细胞增殖抑制实验
选用MDA-MB-436(BRCA1突变,人乳腺癌细胞系)和Capan-1(BRCA2突变,人胰腺癌细胞系)细胞对PV6~PV20进行了细胞增殖抑制实验,结果见表1。
表1 PV6~PV20化合物体外肿瘤细胞增殖抑制实验
注:aIC50为3次实验平均值。
由表1数据可知,随着链长的增加,化合物对这两种肿瘤细胞的增殖抑制活性也会增加。其中,PV11对这两种细胞的抑制活性好,分别是44nM和71nM,比阳性化合物olaparib弱2-3倍。
同样的规律也表现在化合物PV14-20的活性结果中。但是,PV18的活性相较PV11降低了2-3倍。该结果表明,PARP PROTACs化合物需具有合适的链长和特殊种类的Linker组成时才能表现出较好的细胞增殖抑制活性。同时,进一步的制备得到了化合物PV19,PV20。细胞抑制活性结果表明,PV19和PV20的活性相较于PV11都有明显的下降。
PV5和PV11具有相同的链长,区别只在于PV5用两个氧原子替换了碳原子,根据表1数据可知,PV5的活性相较于PV11有很大的降低。综合上述结果,采用相对疏水的碳链设计合成PARP PROTACs分子有助于提高化合物和酶的结合力,从而发挥更好的生物活性。
2、MDA-MB-436细胞内PARP1降解实验
采用浓度为1μM的PV6~PV20作用24h后,Western blot检测化合物对MDA-MB-436细胞内PARP1蛋白含量的影响。其结果见图1。如图1所示,PV11,PV12,PV19和PV20在1μM浓度下表现出了弱的降解活性,其中PV19的降解活性最好,能降解约70%的PARP1。但是这一类PROTACs分子都没有表现出对PARP2的降解活性。
进一步研究了PV11、PV19和PV20在0.01、0.1、1μM浓度下对PARP1的降解活性。其结果见图2。如图2所示,PV11,PV12,PV19和PV20仅在1μM浓度下表现出了弱的降解活性,其中PV19的降解活性最好,能降解约70%的PARP1。这类化合物的细胞增殖抑制活性与降解活性并没有表现出对应的增减一致性,说明它们在细胞内主要发挥了PARP1抑制剂的作用,发挥降解蛋白的作用较弱。
为了进一步研究这些分子降解PARP1蛋白的途径,预先添加蛋白酶体以抑制剂MG132作用细胞2h,再加入PROTACs化合物作用细胞。结果显示,添加蛋白酶体抑制剂后能够有效抑制蛋白的降解,表明这类化合物是通过细胞内的蛋白酶体途径降解蛋白的。
3、PC类化合物体外活性研究
(1)细胞增殖抑制实验
选用MDA-MB-436(BRCA1突变,人乳腺癌细胞系)和Capan-1(BRCA2突变,人胰腺癌细胞系)细胞对PC4~PC33进行了细胞增殖抑制实验,结果见表2。
表2 PC4~PC33化合物体外肿瘤细胞增殖抑制实验
注:aIC50为3次实验平均值。
由表2数据可知,PC4和PC5的细胞抑制活性数据显示其IC50大于10μM(表2.6)。PC8,PC9对两种肿瘤细胞表现出了弱的抑制活性,分别是阳性药olaparib的40-50倍。并且,随着链长的增加,化合物对细胞的抑制活性也随之增加,PC18和PC24对细胞增殖有很好的抑制作用。其中PC18对MDA-MB-436和Capan-1细胞的IC50分别为34nM和117nM,约为阳性药olaparib的2-4倍。采用乙二胺合成的化合物PC24表现出了优于PC18的抑制活性,对上述两种肿瘤细胞的IC50分别为19nM和56nM,是Olaparib的1-2倍。
为了进一步提高化合物的细胞增殖抑制活性和降解PARP蛋白的活性,将PROTACs分子中连接CRBN配体的O原子替换为NH得到了PC10-PC13。细胞抑制活性结果显示短链的PROTACs分子PC10和PC11表现出了微弱的抑制活性,而PC12和PC13具有和其对应衍生物PC10和PC11相似的细胞活性。由此可见,改变连接位置原子类型并不能明显提高化合物的细胞增殖抑制活性。
(2)PARP1蛋白降解性能
在1μM的浓度下评估了PC4~PC13化合物对MDA-MB-436细胞内PARP1/2蛋白的降解能力(图3b),Western blot结果显示,短链化合物PC4和PC5对胞内PARP1/2都没有降解。随着链长的增加,PC7表现出了弱的降解活性。PC8,PC9在1μM的浓度下可以降解MDA-MB-436细胞内80%以上的PARP1蛋白,但对PARP2蛋白没有降解效果。
为了进一步评估PROTACs分子的降解PARP蛋白的效力,探究了化合物PC9和PC13在0.001-10μM浓度下作用细胞24h降解MDA-MB-436细胞胞内PARP1蛋白的活性(图3c)。Western blot结果显示,在高浓度下(1μM/10μM),PC9和PC13都能够很好地降解PARP1蛋白,最大降解达到了90%以上,而在低浓度下(0.01μM/0.001μM)无明显的蛋白降解。总体上,PC9和PC13对胞内PARP1的降解具有浓度依赖性,PC13的降活性略好于PC9。
由此说明,PC9和PC13能够很好的降解MDA-MB-436细胞胞内的PARP1蛋白,但并没有表现出较好细胞增殖抑制活性。再选用MDA-MB-436细胞做了进一步研究,用1μM浓度的PROTACs分子作用细胞1-5天后对胞内蛋白含量做免疫印迹实验分析,发现PC9在1μM下作用细胞1天能够明显降解胞内PARP1蛋白,但作用2天只有少量的蛋白降解,作用3-5天后已经不能降解蛋白(图3d)。
根据以上数据可知,PC9和PC13能够很好的降解MDA-MB-436细胞胞内的PARP1蛋白,但并没有表现出较好细胞增殖抑制活性。再选用MDA-MB-436细胞做了进一步研究,用1μM浓度的PROTACs分子作用细胞1-5天后对胞内蛋白含量做免疫印迹实验分析,发现PC9在1μM下作用细胞1天能够明显降解胞内PARP1蛋白,但作用2天只有少量的蛋白降解,作用3-5天后已经不能降解蛋白(图3d)。在同样的条件下对PC13也进行了免疫印迹研究,结果显示随着作用时间的增加,化合物降解PARP1蛋白的能力在下降。由此推断,PC9和PC13稳定性是影响其发挥细胞抑制活性和蛋白降解活性的关键,CRBN配体连接位点采用NH基团得到的PROTACs分子的稳定性由于O原子取代的分子。影响其稳定性的原因可能有:1)细胞内某些酶对化合物的降解;2)培养基及其血清中的某些化合物影响了PC9和PC13的稳定性等。
(3)研究了1μM浓度下PC14~PC22和PC24作用细胞24h后,这些化合物对MDA-MB-436细胞内PARP1/2蛋白的降解情况,结果见图4。如图4所示,Western blot结果显示,PC18表现出了弱的降解活性,PC22和PC24表现出了对PARP1蛋白明显的降解,特别是PC24能够降解细胞内超过99%以上的蛋白,其结果优于之前得到的PC13。该系列化合物并未显示出对PARP2的降解。
(4)为了筛选出降解最优的化合物,选择对MDA-MB-436细胞增殖抑制活性(IC50<100nM)较好的化合物进行了不同浓度的梯度的研究。选用了MDA-MB-436(BRCA1突变,人乳腺癌细胞系),Capan-1(BRCA2突变,人胰腺癌细胞系)和SW620(人结直肠癌细胞系)细胞进行进行了4个浓度(1,10,100,1000nM)的免疫印迹实验,结果见表3和图5。
表3化合物PC18,22-33在肿瘤细胞中降解PARP1蛋白的情况
通过3次平衡实验,发现PC24,PC25和PC26对胞内的PARP1都有很好的降解,最大降解大于99%。考虑到PROTACs化合物分子量相较抑制剂有了很大的增加,选择分子量更小的PC24作为最优化合物,命名为SK-575。
SK-575能在10-1000nM浓度下对MDA-MB-436和SW620细胞内PAPR1蛋白都能降解胞内大于99%以上的蛋白,降解Capan-1细胞内PARP1的能力相对较弱,能在100-1000nM浓度下降解95%以上的PARP1蛋白,这可能与SK-575透过Capan-1细胞膜的能力有关。然后又合成了SK-575的N-甲基化分子PC33,其在作用浓度下对PARP1蛋白并未表现出降解活性,这也证明了化合物是通过泛素-蛋白酶体系统降解蛋白的。
实施例54 SK-575体外活性研究
在对PROTAC化合物进行细胞增殖抑制试验时测试的到SK-575抑制MDA-MB-436和Capan-1细胞增殖的活性分别为19nM和44nM。为了进一步研究SK-575对其他肿瘤细胞的增殖抑制活性,选用HCC1937(BRCA1突变,人乳腺癌细胞系),22Rv1(BRCA1突变,人前列腺癌细胞系),MDA-MB-468(PTEN突变,人乳腺癌细胞系),PC-3(PTEN突变,人前列腺癌细胞系),LnCap(PTEN突变,人前列腺癌细胞系),SW620(人结直肠癌细胞系)和MDA-MB-231(人三阴性乳腺癌癌细胞系)这7种细胞对其进行了细胞活性测试,其结果见表4和图6。
表4 SK-575对肿瘤细胞的增殖抑制活性(IC50 aμM)
根据表4和图6的检测结果可知,SK-575表现出了优于阳性药olaparib的细胞增殖抑活性。
然后进一步考察了SK-575在MDA-MB-436,Capan-1和SW620细胞中降解PARP1蛋白的DC50,其结果见图7。如图7所示,SK-575在这三种细胞中都表现出了较高的降解活性,半数降解DC50分别为1.17,8.40和0.487nM,特别是在人结直肠癌SW620细胞中的DC50达到了pM。
同时研究了SK-575(30nM)在上述三种细胞中降解PARP1的速率,其结果见图8;如图8所示,SK-575作用MDA-MB-436和SW620细胞1h即可降解胞内近60%的PARP1蛋白,作用2h小时可降解细胞内超过90%的PARP1蛋白,作用4h即可最大限度降解PARP1蛋白。SK-575在Capan-1细胞内降解蛋白速率相对较慢,作用6h可达到最大降解。由此可见,SK-575能在细胞内快速降解PARP1蛋白。
还研究了SK-575降解蛋白的持续性(参见图9),如图9所示,在100nM浓度下,其在MDA-MB-436细胞中能持续作用细胞5天。由此可见,SK-575在培养基环境中能够稳定存在,且不易被细胞内的酶代谢。SK-575这一性质明显优于前期筛选的化合物PC9和PC13。
最后我们研究了SK-575作用MDA-MB-436细胞2h后PARP1蛋白的变化情况(参见图10),我们发现SK-575作用于细胞2h后,洗去培养基中的化合物仍能够维持降解较高,或者说细胞并不能在较短时间补偿的合成PARP1蛋白,这也进一步说明SK-575作用于细胞降解蛋白的持续性较高。
实施例55 SK-575的体内抗肿瘤活性研究
1、SK-575降解肿瘤组织内蛋白的研究
将SW620细胞8×105个/孔接种到BABL/c裸鼠(雄性,5周龄)右腋皮下,待肿瘤长到300-400mm3时,将荷瘤小鼠分为溶剂组(2只),给药组(1h,3h,6h,24h,每组3只)。给药组每只小鼠腹腔注射SK-575(25mg/kg)后分别在1h,3h,6h,24h处死小鼠,剥离肿瘤组织提取蛋白进行Western bolt实验分析PARP1蛋白含量,对照组两只小鼠给予溶剂3h后处死小鼠,剥离肿瘤组织进行分析,结果见图11。如图11所示,Western blot结果显示,给药后1h,SK-575即可降解肿瘤组织内70%的PARP1蛋白,这说明化合物SK-575经腹腔注射给药后能迅速吸收进入血循环在体内快速降解PARP1蛋白。化合物在6h降解蛋白达到最大值,并可以维持作用效果24h。由此可见,PROTACs化合物SK-575能够在体内快速降解PARP1蛋白,并可以持续降解蛋白发挥作用,这也弥补了SK-575在代谢快的缺陷。因此,SK-575可进行进一步体内抗肿瘤研究。
2、SK-575抗肿瘤活性研究
使用BALB/c裸鼠接种Capan-1细胞建立了异种移植肿瘤模型,待肿瘤生长到150mm3时随机分为4组:空白组(ip),阳性药组(Olaparib,100mg/kg,po)和SK-575给药组(高剂量组50mg/kg,ip,低剂量组25mg/kg,ip),每组6只。阳性药组每天给药1次,持续21天。SK-575高、低剂量组每周第1-5天给药5次,持续3周,一共给药15次。结果如图12和13所示。
如图12和13所示,SK-575能够剂量依赖性的抑制肿瘤生长,当腹腔注射剂量为25和50mg/kg时,SK-575对肿瘤的抑制率分别为68.0%和74.2%,而阳性药Olaparib给药100mg/kg时的肿瘤抑制率为68.5%。化合物SK-575在低剂量,低的给药频率下可达到和阳性药Olaparib相同的抑制活性,证明了PROTACs化合物SK-575在体内能够抑制Capan-1模型肿瘤的生长。
为了检测化合物的毒性,对小鼠给药期间的体重变化进行了分析,结果如图14。如图14所示,在给药期间并未发现小鼠死亡。小鼠体重无明显的变化,能够正常的进食进水,说明化合物SK-575对小鼠没有明显的毒性。实验结束后,解剖小鼠我们发现,SK-575高剂量给药组有两只小鼠腹膜上有未被吸收的药物(化合物SK-575显亮黄色),说明该化合物进入体内后不易被吸收,持续给药使得未被吸收化合物SK-575在小鼠腹腔内蓄积。
3、SK-575联合顺铂对肿瘤的生长抑制作用
使用BALB/c裸鼠接种了Capan-1细胞建立了异种移植肿瘤模型,179,180待肿瘤生长到150mm3时分为5组:空白组(ip),顺铂单用组(6mg/kg,ip),阳性药Olaparib和顺铂联用组(Olaparib,100mg/kg,po;顺铂,6mg/kg,ip)和SK-575和顺铂联用组(SK-575高剂量组50mg/kg,ip,低剂量组25mg/kg,ip;顺铂6mg/kg,ip),每组6只。SK-575高、低剂量组和阳性药Olaparib组在第1-5天连续给药5次,在第3天时同时给予顺铂一次,并检测给药后的效果,其结果见图15。
如图15所示,SK-575能够剂量依赖性的增强顺铂抑制肿瘤生长的活性,当腹腔注射剂量为25和50mg/kg时,SK-575表现出了明显的增加顺铂杀伤肿瘤的作用,并且高剂量组表现出了优于Olaparib的效果。
4、SK-575联合DNA烷化剂TMZ对肿瘤的生长抑制作用
使用BALB/c裸鼠接种了人结直肠癌SW620细胞建立了异种移植肿瘤模型,待肿瘤生长到150mm3时分为7组:空白组(ip),SK-575单用组(10mg/kg,ip),TMA单用组(50mg/kg,ip),阳性药Olaparib和TMA联用组(Olaparib,100mg/kg,po;TMZ,50mg/kg,ip)和SK-575和TMZ联用组(SK-575高剂量组10mg/kg,ip/po,低剂量组5mg/kg,ip;TMZ50mg/kg,po),每组6只。联合用药组连续给药5天,每次给药时先给予SK-575和Olaparib 40min后,再灌胃给药TMZ,持续观察小鼠48天,记录数据,其结果见图16和图17。
如图16和17所示,腹腔注射SK-575表现出了明显的增加TMZ杀伤肿瘤的效果,其中SK-575高剂量组的效果稍优于同等剂量的Olaparib。虽然SK-575灌胃后只有很少的化合物被吸收,但在本实验中给予灌胃给药SK-575的小鼠肿瘤生长也被抑制,优于TMZ单用组。
接着,我们考察了SK-575和TMZ联用的毒性。和Capan-1肿瘤模型中SK-575联合顺铂用药一样,SK-575联合TMZ用药也表现出了明显的动物体重的减轻,Olaparib和SK-575高剂量腹腔注射组表现出了明显的毒性,给药结束后第3天出现了最大体重减轻,达到了15%,但给药后10天以内动物体重均可恢复(参见图18)。实验过程中未发现小鼠死亡。
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