CN115650975A - 一种靶向降解人表皮生长因子受体2的蛋白降解靶向嵌合体化合物及其应用 - Google Patents
一种靶向降解人表皮生长因子受体2的蛋白降解靶向嵌合体化合物及其应用 Download PDFInfo
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Abstract
本发明提供了一种靶向降解人表皮生长因子受体2蛋白的蛋白降解靶向嵌合体化合物及其制备方法和应用，该化合物的结构式为：
Description
技术领域
本发明属于生物医药技术领域，具体涉及一种靶向降解人表皮生长因子受体2蛋白的蛋白降解靶向嵌合体化合物及其应用。
背景技术
受体酪氨酸激酶(RTK)是细胞外环境到细胞内信号传导的主要中介，决定了细胞的生长、分化、迁移或死亡。人表皮生长因子受体2(HER2)是HER家族的受体酪氨酸激酶之一，该家族受体激酶还包括表皮生长因子受体(EGFR)、人表皮生长因子受体3(HER3)和人表皮生长因子受体4(HER4)。HER2是由胞内激酶域、跨膜结构域以及胞外配体结合域组成的跨膜酶联受体。由于缺少与之高亲和力结合的配体，HER2通常需要与能够和配体结合的EGFR、HER3或HER4异源二聚化而被激活。但当HER2过表达时会导致细胞膜上受体浓度极高，这种情况下HER2可以不依赖配体而发生同源二聚化并诱导HER2下游信号通路的激活。多种实体瘤都存在HER2过表达或过度激活现象。HER2通过同源或异源二聚，激活固有的酪氨酸激酶活性，触发胞质结构域内特定的酪氨酸残基的自磷酸化，进而激活RAS/MAPK和PI3K/AKT等一系列细胞增殖、存活、分化、迁移相关的信号通路，导致细胞增殖异常和肿瘤发生。目前针对HER2开发了多个靶向药物，其中单克隆抗体及抗体-药物偶联物的分子太大影响了它们的细胞渗透性，限制了其在临床上的使用。小分子抑制剂虽然具有可接受的药代动力学特性、良好的口服生物利用度和较低的制造成本，但是小分子抑制剂只能抑制HER2蛋白增殖，但是无法实现HER2蛋白的降解。
蛋白降解靶向嵌合体(Proteolysis targeting chimeras，PROTAC)是一种靶向降解目标蛋白的技术，其通过E3泛素连接酶配体特异性招募E3泛素连接酶，诱导目标蛋白和E3泛素连接酶结合形成稳定的三元复合物，从而导致目标蛋白泛素化，进而被泛素蛋白酶体系统降解。PROTAC分子能有效减少目标蛋白在表达和突变过程中所获得的耐药性，在抗肿瘤药物领域中具有广阔的前景。
发明内容
针对现有技术中存在的上述问题，本发明提供一种靶向降解人表皮生长因子受体2蛋白的蛋白降解靶向嵌合体化合物及其应用，该化合物可有效解决现有的小分子抑制剂无法降解人表皮生长因子受体2蛋白的问题，且该化合物抑制人表皮生长因子受体2阳性肿瘤细胞株BT-474增殖活性优于上市药物图卡替尼。
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：
一种靶向降解人表皮生长因子受体2蛋白的蛋白降解靶向嵌合体化合物，该化合物的结构式为：
其中，HER2 Ligand为HER2蛋白的小分子配体；Linker为连接子；E3 LigaseLigand为E3泛素连接酶人小脑蛋白或希佩尔-林道蛋白的小分子配体。
进一步地，HER2蛋白的小分子配体的结构式为：
Linker的结构式为
其中n＝3–12，m＝1–5，x＝3-11，y＝1-5；
E3LigaseLigand的结构式为
进一步地，蛋白降解靶向嵌合体化合物的结构式如下：
进一步地，蛋白降解靶向嵌合体化合物的结构式如下：
上述的靶向降解人表皮生长因子受体2蛋白的蛋白降解靶向嵌合体化合物在制备治疗HER2阳性肿瘤药物中的应用。
本发明所产生的有益效果为：
1、本申请中的蛋白降解靶向嵌合体化合物可通过泛素蛋白酶体系统靶向降解肿瘤细胞中HER2蛋白，下调HER2蛋白水平，实现HER2生物学功能的完全抑制，因而可以用于治疗HER2阳性肿瘤。
具体实施方式
下面通过具体实施例进一步说明本发明，但是，应当理解为，这些实施例仅仅是用于更详细的具体说明用，而不应理解为用于以任何形式限制本发明。
本发明对试验中所使用到的材料以及实验方法进行一般性和具体性的描述。虽然为实现本发明的目的所使用的许多材料和操作方法是本领域公知的，但是本发明仍然在此作尽可能详细的描述。本领域技术人员清楚，在下文中，如未特别说明，所使用的材料和操作方法是本领域公知的。
实施例1
靶向降解HER2蛋白的PROTAC化合物的合成与结构确认
化合物3(4-{[3-甲基-4-([1,2,4]三氮杂环戊熳并[1,5-a]吡啶-7-基氧基)苯基]氨基}喹唑啉-6-酚)和化合物6(6-甲氧基-4-{[3-甲基-4-([1,2,4]三氮杂环戊熳并[1,5-a]吡啶-7-基氧基)苯基]氨基}喹唑啉-7-酚)的合成：将化合物2或5与等量的4-([1,2,4]三唑并[1,5-a]吡啶-7-基氧基)苯胺在异丙醇中回流3小时，析出沉淀，冷却后过滤，用异丙醇洗涤，干燥得到所需中间体，为黄色固体，然后，在甲醇与氨水(25％)混合溶液(体积比为1:1)中脱保护，将反应搅拌过夜，然后浓缩，得到灰白色固体。
4-{[3-甲基-4-([1,2,4]三氮杂环戊熳并[1,5-a]吡啶-7-基氧基)苯基]氨基}喹唑啉-6-酚(compound 3):Yellow solid；yield 81％；1H NMR(400MHz,DMSO-d6)δ10.12(s,1H),9.59(s,1H),8.93(d,J＝7.4Hz,1H),8.48(s,1H),8.38(s,1H),7.92(d,J＝2.5Hz,1H),7.87(dd,J＝8.8,2.6Hz,1H),7.82(d,J＝2.6Hz,1H),7.69(d,J＝8.9Hz,1H),7.45(dd,J＝9.0,2.5Hz,1H),7.20(d,J＝8.6Hz,1H),7.03(dd,J＝7.5,2.6Hz,1H),6.80(d,J＝2.6Hz,1H),2.19(s,3H).
6-甲氧基-4-{[3-甲基-4-([1,2,4]三氮杂环戊熳并[1,5-a]吡啶-7-基氧基)苯基]氨基}喹唑啉-7-酚(compound 6):Yellow solid；yield 76％；1H NMR(400MHz,DMSO-d6)δ9.56(s,1H),8.93(d,J＝7.5Hz,1H),8.41(d,J＝9.8Hz,2H),7.83(d,J＝12.6Hz,3H),7.31-6.97(m,4H),6.79(d,J＝2.6Hz,1H),3.99(s,3H),2.20(s,3H).
HER2配体A和B的合成：将化合物3或6(1eq)、1,3-二溴丙烷(5eq)、碳酸钾(2eq)添加到无水N,N-二甲基甲酰胺中，将混合物在60℃搅拌3小时，反应液用乙酸乙酯稀释，依次用水、饱和氯化钠洗涤，硫酸钠干燥，浓缩得到油状粗品，用乙醚和二氯甲烷结晶得到中间体为白色固体；然后，加入boc-哌嗪(1eq)、三乙胺(2eq)的无水N,N-二甲基甲酰胺溶液，混合物在60℃搅拌3小时，反应液用乙酸乙酯稀释，依次用水、饱和氯化钠洗涤，硫酸钠干燥，通过快速色谱法纯化。然后，用三氟乙酸的二氯甲烷溶液(体积比为1:5)脱保护，浓缩得产物。4-{[3-甲基-4-([1,2,4]三氮杂环戊熳并[1,5-a]吡啶-7-基氧基)苯基]氨基}-6-{[3-(哌嗪-1-基)丙基]氧基}喹唑啉(HER2 Ligand A):White solid；yield 52％；1H NMR(400MHz,DMSO-d6)δ9.68(s,1H),8.94(d,J＝7.5Hz,1H),8.52(s,1H),8.39(s,1H),8.02–7.91(m,1H),7.85(d,J＝5.0Hz,2H),7.74(d,J＝9.1Hz,1H),7.51(dd,J＝9.1,2.5Hz,1H),7.22(d,J＝9.4Hz,1H),7.04(dd,J＝7.4,2.7Hz,1H),6.80(d,J＝2.7Hz,1H),4.20(t,J＝6.3Hz,2H),2.79(q,J＝5.7,4.9Hz,4H),2.47(d,J＝7.1Hz,2H),2.39(s,4H),2.21(s,3H),1.99(q,J＝6.7Hz,2H).
6-甲氧基-4-{[3-甲基-4-([1,2,4]三氮杂环戊熳并[1,5-a]吡啶-7-基氧基)苯基]氨基}-7-{[3-(哌嗪-1-基)丙基]氧基}喹唑啉(HER2 Ligand B):White solid；yield59％；1H NMR(400MHz,DMSO-d6)δ9.54(s,1H),8.93(d,J＝7.5Hz,1H),8.48(s,1H),8.38(s,1H),7.87(s,1H),7.82(d,J＝8.1Hz,2H),7.21(d,J＝11.2Hz,2H),7.04(dd,J＝7.5,2.7Hz,1H),6.79(d,J＝2.6Hz,1H),4.19(t,J＝6.4Hz,2H),3.98(s,3H),2.99(q,J＝7.3Hz,4H),2.47(d,J＝7.0Hz,2H),2.35(t,J＝5.1Hz,4H),2.20(s,3H),1.97(q,J＝6.6Hz,2H).
HER2双官能PROTAC化合物的合成：HER2双官能PROTAC化合物通过碘代E3泛素连接酶人小脑蛋白(CRBN)/希佩尔-林道蛋白(VHL)配体与HER2配体A或B亲核取代获得，所有接头碘代的E3泛素连接酶人小脑蛋白(CRBN)/希佩尔-林道蛋白(VHL)配体均来自商业PROTAC的E3连接酶配体库；通常，1当量Tucatinib类似物A或B用1.5至2当量碘代E3泛素连接酶人小脑蛋白(CRBN)/希佩尔-林道蛋白(VHL)配体和2当量无水N,N-二异丙基乙胺在无水N,N-二甲基甲酰胺中于60℃处理3至12小时，将混合物浓缩并通过快速色谱法纯化得产物。
3-(4-{[3-(4-{3-[(4-{[3-甲基-4-([1,2,4]三氮杂环戊熳并[1,5-a]吡啶-7-基氧基)苯基]氨基}喹唑啉-6-基)氧基]丙基}哌嗪-1-基)丙基]氨基}-1-氧亚基-2,3-二氢-1H-异吲哚-2-基)六氢吡啶-2,6-二酮(H3C):White solid；yield:46％；1H NMR(400MHz,DMSO-d6)δ11.04(s,1H),9.74(s,1H),8.93(d,J＝7.5Hz,1H),8.52(s,1H),8.38(s,1H),7.99(d,J＝2.7Hz,1H),7.86(dd,J＝6.7,2.6Hz,2H),7.74(d,J＝9.1Hz,1H),7.51(dd,J＝9.0,2.5Hz,1H),7.30(t,J＝7.7Hz,1H),7.25–7.19(m,1H),7.04(dd,J＝7.5,2.7Hz,1H),6.95(d,J＝7.4Hz,1H),6.86–6.72(m,2H),5.76–5.72(m,1H),5.13(dd,J＝13.3,5.1Hz,1H),4.28–4.12(m,4H),3.23–3.13(m,3H),3.07(q,J＝7.3Hz,1H),2.94(ddd,J＝18.1,13.5,5.4Hz,2H),2.65(s,8H),2.30(qd,J＝13.2,4.5Hz,2H),2.20(s,3H),2.03(s,4H),1.87–1.74(m,2H).MS:calcd C44H47N11O5,809.4,found,[M+2H]+,405.7.
3-(4-{[5-(4-{3-[(4-{[3-甲基-4-([1,2,4]三氮杂环戊熳并[1,5-a]吡啶-7-基氧基)苯基]氨基}喹唑啉-6-基)氧基]丙基}哌嗪-1-基)戊基]氨基}-1-氧亚基-2,3-二氢-1H-异吲哚-2-基)六氢吡啶-2,6-二酮(H5C):White solid；yield:55％；1H NMR(400MHz,Chloroform-d)δ8.70(s,1H),8.52–8.41(m,2H),8.23(s,1H),7.85(d,J＝9.1Hz,1H),7.68(d,J＝2.6Hz,1H),7.57(dt,J＝6.2,2.7Hz,2H),7.47–7.40(m,1H),7.33(t,J＝7.7Hz,1H),7.24(d,J＝7.5Hz,1H),7.03(d,J＝8.6Hz,1H),6.90(dd,J＝7.5,2.6Hz,1H),6.82–6.73(m,2H),5.15(dd,J＝13.3,5.2Hz,1H),4.32–4.01(m,4H),3.22(q,J＝6.7Hz,2H),2.87–2.68(m,2H),2.50–2.27(m,11H),2.16(s,5H),1.97(t,J＝7.0Hz,3H),1.64(dt,J＝13.2,6.6Hz,2H),1.53–1.36(m,4H).MS:calcd C46H51N11O5,837.4,found,[M+2H]+,419.8.
3-(4-{[6-(4-{3-[(4-{[3-甲基-4-([1,2,4]三氮杂环戊熳并[1,5-a]吡啶-7-基氧基)苯基]氨基}喹唑啉-6-基)氧基]丙基}哌嗪-1-基)己基]氨基}-1-氧亚基-2,3-二氢-1H-异吲哚-2-基)六氢吡啶-2,6-二酮(H6C):White solid；yield:52％；1H NMR(400MHz,DMSO-d6)δ11.02(s,1H),9.69(s,1H),8.94(d,J＝7.5Hz,1H),8.53(s,1H),8.39(s,1H),7.96(d,J＝2.7Hz,1H),7.86(dq,J＝4.9,2.7Hz,2H),7.74(d,J＝9.1Hz,1H),7.51(dd,J＝9.1,2.5Hz,1H),7.31–7.18(m,2H),7.04(dd,J＝7.5,2.7Hz,1H),6.93(d,J＝7.3Hz,1H),6.80(d,J＝2.6Hz,1H),6.74(d,J＝8.1Hz,1H),5.55(q,J＝8.8,7.1Hz,1H),5.12(dd,J＝13.2,5.1Hz,1H),4.30–4.06(m,4H),3.11(d,J＝6.4Hz,1H),3.00–2.89(m,1H),2.72–2.50(m,12H),2.37–2.24(m,2H),2.21(s,3H),2.09–1.95(m,3H),1.63–1.30(m,8H).MS:calcdC47H53N11O5,851.4,found,[M+H]+,852.2.
3-(4-{[7-(4-{3-[(4-{[3-甲基-4-([1,2,4]三氮杂环戊熳并[1,5-a]吡啶-7-基氧基)苯基]氨基}喹唑啉-6-基)氧基]丙基}哌嗪-1-基)庚基]氨基}-1-氧亚基-2,3-二氢-1H-异吲哚-2-基)六氢吡啶-2,6-二酮(H7C):White solid；yield:41％；1H NMR(400MHz,DMSO-d6)δ11.02(s,1H),9.68(s,1H),8.94(d,J＝7.5Hz,1H),8.52(s,1H),8.38(s,1H),7.94(d,J＝2.7Hz,1H),7.85(dd,J＝5.9,2.8Hz,2H),7.74(d,J＝9.1Hz,1H),7.51(dd,J＝9.1,2.5Hz,1H),7.32–7.16(m,2H),7.04(dd,J＝7.5,2.7Hz,1H),6.92(d,J＝7.5Hz,1H),6.80(d,J＝2.7Hz,1H),6.73(d,J＝8.1Hz,1H),5.55(s,1H),5.11(dd,J＝13.2,5.1Hz,1H),4.31–4.00(m,4H),3.11(d,J＝6.5Hz,2H),2.93(td,J＝13.1,6.8Hz,1H),2.67–2.58(m,1H),2.50–2.26(m,10H),2.21(s,5H),2.05–1.94(m,3H),1.63–1.52(m,2H),1.41–1.24(m,8H).MS:calcd C48H55N11O5,865.4,found,[M+H]+,866.2.
3-(4-{[8-(4-{3-[(4-{[3-甲基-4-([1,2,4]三氮杂环戊熳并[1,5-a]吡啶-7-基氧基)苯基]氨基}喹唑啉-6-基)氧基]丙基}哌嗪-1-基)辛基]氨基}-1-氧亚基-2,3-二氢-1H-异吲哚-2-基)六氢吡啶-2,6-二酮(H8C):White solid；yield:38％；1H NMR(400MHz,DMSO-d6)δ11.02(s,1H),9.76(s,1H),8.95(d,J＝7.5Hz,1H),8.55(s,1H),8.39(s,1H),7.98(d,J＝2.7Hz,1H),7.84(d,J＝6.9Hz,2H),7.76(d,J＝9.1Hz,1H),7.53(dd,J＝9.1,2.5Hz,1H),7.30–7.21(m,2H),7.04(dd,J＝7.5,2.7Hz,1H),6.93(dd,J＝7.4,2.8Hz,1H),6.79(d,J＝2.6Hz,1H),6.74(d,J＝8.0Hz,1H),5.55(q,J＝11.0,8.2Hz,1H),5.12(dd,J＝13.3,5.0Hz,1H),4.28–4.09(m,5H),3.16–2.89(m,11H),2.69–2.55(m,3H),2.32(td,J＝13.1,4.5Hz,2H),2.21(s,3H),2.09–1.99(m,3H),1.54–1.60(m,4H),1.36–1.25(m,8H).MS:calcd C49H57N11O5,879.5,found,[M+H]+,880.3.
3-(4-{[9-(4-{3-[(4-{[3-甲基-4-([1,2,4]三氮杂环戊熳并[1,5-a]吡啶-7-基氧基)苯基]氨基}喹唑啉-6-基)氧基]丙基}哌嗪-1-基)壬基]氨基}-1-氧亚基-2,3-二氢-1H-异吲哚-2-基)六氢吡啶-2,6-二酮(H9C):White solid；yield:55％；1H NMR(400MHz,DMSO-d6)δ11.02(s,1H),9.69(s,1H),8.94(d,J＝7.5Hz,1H),8.52(s,1H),8.38(s,1H),7.95(d,J＝2.7Hz,1H),7.89–7.83(m,2H),7.74(d,J＝9.1Hz,1H),7.50(dd,J＝9.1,2.5Hz,1H),7.32–7.19(m,2H),7.04(dd,J＝7.5,2.7Hz,1H),6.93(d,J＝7.4Hz,1H),6.80(d,J＝2.6Hz,1H),6.73(d,J＝8.0Hz,1H),5.54(q,J＝4.8,4.0Hz,1H),5.12(dd,J＝13.2,5.1Hz,1H),4.33–4.08(m,4H),3.10(q,J＝6.7Hz,3H),2.93(ddd,J＝18.2,13.4,5.5Hz,1H),2.66–2.58(m,1H),2.30(qd,J＝13.4,5.1Hz,11H),2.20(s,3H),2.06–1.92(m,3H),1.51–1.61(m,2H),1.42–1.24(m,12H).MS:calcdC50H59N11O5,893.5,found,[M+H]+,894.3.
3-(4-{[10-(4-{3-[(4-{[3-甲基-4-([1,2,4]三氮杂环戊熳并[1,5-a]吡啶-7-基氧基)苯基]氨基}喹唑啉-6-基)氧基]丙基}哌嗪-1-基)癸基]氨基}-1-氧亚基-2,3-二氢-1H-异吲哚-2-基)六氢吡啶-2,6-二酮(H10C):White solid；yield:56％；1H NMR(400MHz,DMSO-d6)δ11.02(s,1H),9.68(s,1H),8.94(d,J＝7.5Hz,1H),8.52(s,1H),8.38(s,1H),7.95(d,J＝2.7Hz,1H),7.85(q,J＝3.3Hz,2H),7.74(d,J＝9.1Hz,1H),7.51(dd,J＝9.1,2.5Hz,1H),7.32–7.18(m,2H),7.04(dd,J＝7.5,2.7Hz,1H),6.92(d,J＝7.4Hz,1H),6.80(d,J＝2.6Hz,1H),6.73(d,J＝8.0Hz,1H),5.55(t,J＝5.6Hz,1H),5.11(dd,J＝13.2,5.1Hz,1H),4.27–4.09(m,4H),3.17–2.85(m,6H),2.50–2.24(m,10H),2.20(s,3H),2.07–1.95(m,3H),1.53–1.60(m,2H),1.41–1.21(m,14H).MS:calcd C51H61N11O5,907.5,found,[M+H]+,908.3.
3-(4-{[12-(4-{3-[(4-{[3-甲基-4-([1,2,4]三氮杂环戊熳并[1,5-a]吡啶-7-基氧基)苯基]氨基}喹唑啉-6-基)氧基]丙基}哌嗪-1-基)十二烷基]氨基}-1-氧亚基-2,3-二氢-1H-异吲哚-2-基)六氢吡啶-2,6-二酮(H12C):White solid；yield:50％；1H NMR(400MHz,DMSO-d6)δ11.02(s,1H),9.93(s,1H),8.93(d,J＝7.5Hz,1H),8.52(s,1H),8.37(s,1H),8.08(s,1H),7.93(d,J＝5.5Hz,2H),7.72(d,J＝9.1Hz,1H),7.47(dd,J＝8.9,2.3Hz,1H),7.30–7.12(m,2H),7.02(dd,J＝7.5,2.7Hz,1H),6.92(d,J＝7.4Hz,1H),6.78(d,J＝2.6Hz,1H),6.72(d,J＝8.0Hz,1H),5.56(s,1H),5.11(dd,J＝13.2,5.1Hz,1H),4.28–4.07(m,4H),3.08(dt,J＝13.0,7.2Hz,4H),2.95–2.64(m,10H),2.30(qd,J＝13.2,4.5Hz,2H),2.19(s,3H),2.08–1.98(m,3H),1.60–1.46(m,4H),1.41–1.21(m,14H).MS:calcd C53H65N11O5,935.5,found,[M+H]+,936.3.
3-{4-[(2-{[2-(4-{3-[(4-{[3-甲基-4-([1,2,4]三氮杂环戊熳并[1,5-a]吡啶-7-基氧基)苯基]氨基}喹唑啉-6-基)氧基]丙基}哌嗪-1-基)乙基]氧基}乙基)氨基]-1-氧亚基-2,3-二氢-1H-异吲哚-2-基}六氢吡啶-2,6-二酮(H2P):White solid；yield:42％；1HNMR(400MHz,Chloroform-d)δ9.03(s,1H),8.69(s,1H),8.47(d,J＝7.4Hz,1H),8.22(s,1H),7.85(d,J＝9.1Hz,1H),7.76(d,J＝2.6Hz,1H),7.71(d,J＝2.6Hz,1H),7.57(dd,J＝8.6,2.7Hz,1H),7.41(dd,J＝9.1,2.5Hz,1H),7.33(t,J＝7.7Hz,1H),7.01(d,J＝8.6Hz,1H),6.89(dd,J＝7.5,2.6Hz,1H),6.85–6.74(m,2H),5.12(dd,J＝13.2,5.0Hz,1H),4.23–4.09(m,2H),4.05–3.93(m,2H),3.72–3.55(m,4H),3.38(s,2H),2.90–2.81(m,1H),2.72(ddd,J＝15.0,10.9,5.3Hz,1H),2.53(d,J＝4.2Hz,2H),2.45–2.24(m,10H),2.14(s,4H),2.01–1.82(m,3H).MS:calcdC45H49N11O6,839.4,found,[M+H]+,840.4.
3-(4-{[8-(4-{3-[(4-{[3-甲基-4-([1,2,4]三氮杂环戊熳并[1,5-a]吡啶-7-基氧基)苯基]氨基}喹唑啉-6-基)氧基]丙基}哌嗪-1-基)-3,6-二氧杂辛-1-基]氨基}-1-氧亚基-2,3-二氢-1H-异吲哚-2-基)六氢吡啶-2,6-二酮(H3P):White solid；yield:44％；1HNMR(400MHz,DMSO-d6)δ11.02(s,1H),9.69(s,1H),8.94(d,J＝7.5Hz,1H),8.52(s,1H),8.38(s,1H),7.95(d,J＝2.7Hz,1H),7.88–7.82(m,2H),7.74(d,J＝9.1Hz,1H),7.51(dd,J＝9.1,2.5Hz,1H),7.28(t,J＝7.7Hz,1H),7.22(d,J＝9.4Hz,1H),7.04(dd,J＝7.5,2.6Hz,1H),6.95(d,J＝7.4Hz,1H),6.83–6.77(m,2H),5.58(t,J＝5.8Hz,1H),5.12(dd,J＝13.3,5.1Hz,1H),4.28–4.11(m,4H),3.62–3.48(m,10H),3.32(d,J＝5.8Hz,4H),3.05(t,J＝7.3Hz,1H),2.91(dq,J＝13.4,6.9,5.6Hz,1H),2.68–2.57(m,2H),2.21–2.33(m,7H),2.20(s,3H),2.05–1.92(m,3H).MS:calcd C47H53N11O7,883.4,found,[M+H]+,884.2.
3-(4-{[11-(4-{3-[(4-{[3-甲基-4-([1,2,4]三氮杂环戊熳并[1,5-a]吡啶-7-基氧基)苯基]氨基}喹唑啉-6-基)氧基]丙基}哌嗪-1-基)-3,6,9-三氧杂十一烷-1-基]氨基}-1-氧亚基-2,3-二氢-1H-异吲哚-2-基)六氢吡啶-2,6-二酮(H4P):White solid；yield:56％；1H NMR(400MHz,DMSO-d6)δ11.02(s,1H),9.68(s,1H),8.94(d,J＝7.5Hz,1H),8.52(s,1H),8.39(s,1H),7.94(d,J＝2.7Hz,1H),7.84(dt,J＝5.1,2.5Hz,2H),7.74(d,J＝9.1Hz,1H),7.51(dd,J＝9.1,2.6Hz,1H),7.28(t,J＝7.7Hz,1H),7.25–7.19(m,1H),7.04(dd,J＝7.5,2.7Hz,1H),6.95(d,J＝7.4Hz,1H),6.80(q,J＝3.8,2.8Hz,2H),5.57(t,J＝5.7Hz,1H),5.11(dd,J＝13.3,5.1Hz,1H),4.26–4.10(m,4H),3.59(t,J＝5.9Hz,2H),3.56–3.48(m,10H),3.08–2.86(m,6H),2.64(dd,J＝4.6,2.5Hz,1H),2.48–2.24(m,10H),2.21(s,3H),2.07–1.92(m,3H).MS:calcd C49H57N11O8,927.5,found,[M+H]+,928.2
3-(4-{[14-(4-{3-[(4-{[3-甲基-4-([1,2,4]三氮杂环戊熳并[1,5-a]吡啶-7-基氧基)苯基]氨基}喹唑啉-6-基)氧基]丙基}哌嗪-1-基)-3,6,9,12-四氧杂十四烷-1-基]氨基}-1-氧亚基-2,3-二氢-1H-异吲哚-2-基)六氢吡啶-2,6-二酮(H5P):White solid；yield:46％；1H NMR(400MHz,DMSO-d6)δ11.02(s,1H),9.70(s,1H),8.94(d,J＝7.5Hz,1H),8.53(s,1H),8.39(s,1H),7.98(d,J＝2.6Hz,1H),7.89–7.82(m,2H),7.75(d,J＝9.1Hz,1H),7.51(dd,J＝9.1,2.5Hz,1H),7.28(t,J＝7.7Hz,1H),7.24–7.19(m,1H),7.04(dd,J＝7.5,2.7Hz,1H),6.95(d,J＝7.4Hz,1H),6.80(dd,J＝5.4,2.8Hz,2H),5.59(t,J＝5.7Hz,1H),5.11(dd,J＝13.3,5.1Hz,1H),4.28–4.09(m,4H),3.62–3.50(m,16H),3.09(q,J＝7.3Hz,3H),2.92(ddd,J＝17.2,13.5,5.4Hz,2H),2.81–2.58(m,10H),2.31(qd,J＝13.2,4.4Hz,2H),2.20(s,3H),2.00–2.06(m,3H).MS:calcd C51H61N11O9,971.5,found,[M+H]+,972.2
3-(4-{[17-(4-{3-[(4-{[3-甲基-4-([1,2,4]三氮杂环戊熳并[1,5-a]吡啶-7-基氧基)苯基]氨基}喹唑啉-6-基)氧基]丙基}哌嗪-1-基)-3,6,9,12,15-五氧杂十七烷-1-基]氨基}-1-氧亚基-2,3-二氢-1H-异吲哚-2-基)六氢吡啶-2,6-二酮(H6P):White solid；yield:41％；1H NMR(400MHz,Chloroform-d)δ9.12(d,J＝10.1Hz,1H),8.68(d,J＝1.9Hz,1H),8.48(d,J＝7.3Hz,1H),8.22(d,J＝1.6Hz,1H),7.89–7.73(m,3H),7.64(dd,J＝8.6,2.6Hz,1H),7.40(dt,J＝9.2,3.0Hz,1H),7.33–7.26(m,1H),7.21(dd,J＝7.5,2.8Hz,1H),7.02(dd,J＝8.8,3.0Hz,1H),6.88(dd,J＝7.4,2.7Hz,1H),6.82(d,J＝2.6Hz,1H),6.76(dd,J＝8.0,2.5Hz,1H),5.20–5.02(m,1H),4.65(s,1H),4.37–4.21(m,2H),4.05(dq,J＝12.4,6.3Hz,2H),3.72(dd,J＝6.2,3.6Hz,2H),3.65–3.51(m,18H),3.37(s,2H),2.89–2.41(m,14H),2.34(dq,J＝13.1,8.3,6.3Hz,1H),2.20–2.07(m,4H),1.93–1.98(m,2H).MS:calcd C53H65N11O10,1015.5,found,[M+H]+,1016.4.
(4S)-1-[(2R)-3,3-二甲基-2-{[5-(4-{3-[(4-{[3-甲基-4-([1,2,4]三氮杂环戊熳并[1,5-a]吡啶-7-基氧基)苯基]氨基}喹唑啉-6-基)氧基]丙基}哌嗪-1-基)-1-氧亚基戊基]氨基}-1-氧亚基丁基]-4-羟基-N-{[4-(4-甲基-1,3-硫杂氮杂环戊熳-5-基)苯基]甲基}四氢吡咯-2-甲酰胺(VH5C):White solid；yield:39％；1H NMR(400MHz,Chloroform-d)δ8.78(s,1H),8.68(t,J＝2.3Hz,2H),8.48(d,J＝7.4Hz,1H),8.20(d,J＝1.4Hz,1H),7.85–7.77(m,2H),7.70(d,J＝8.8Hz,1H),7.63–7.52(m,2H),7.40(dd,J＝8.9,2.2Hz,1H),7.27(d,J＝1.4Hz,2H),7.03(d,J＝8.6Hz,1H),6.89(dt,J＝7.5,2.1Hz,1H),6.80(t,J＝1.9Hz,1H),6.58(d,J＝8.7Hz,1H),4.71(t,J＝8.1Hz,1H),4.56–4.46(m,3H),4.35(dd,J＝15.1,5.6Hz,1H),4.02(q,J＝6.1,5.4Hz,3H),3.66–3.59(m,2H),2.92(dd,J＝30.4,1.5Hz,1H),2.71(d,J＝26.8Hz,7H),2.60–2.43(m,8H),2.37(dt,J＝9.3,4.9Hz,1H),2.21(d,J＝26.9Hz,7H),1.97(t,J＝6.9Hz,2H),1.26(s,2H),0.95(s,9H).MS:calcdC55H66N12O6S,1022.5,found,[M+H]+,1023.5.
(2R,4S)-1-[(2R)-3,3-二甲基-2-{[6-(4-{3-[(4-{[3-甲基-4-([1,2,4]三氮杂环戊熳并[1,5-a]吡啶-7-基氧基)苯基]氨基}喹唑啉-6-基)氧基]丙基}哌嗪-1-基)-1-氧亚基己基]氨基}-1-氧亚基丁基]-4-羟基-N-{[4-(4-甲基-1,3-硫杂氮杂环戊熳-5-基)苯基]甲基}四氢吡咯-2-甲酰胺(VH6C):White solid；yield:42％；1H NMR(400MHz,Chloroform-d)δ8.68(d,J＝2.1Hz,2H),8.49(d,J＝7.1Hz,2H),8.21(s,1H),7.84(d,J＝9.1Hz,1H),7.70(d,J＝2.6Hz,1H),7.60(dd,J＝8.6,2.6Hz,1H),7.50(d,J＝2.6Hz,1H),7.46–7.39(m,2H),7.38–7.30(m,4H),7.03(d,J＝8.6Hz,1H),6.89(dd,J＝7.5,2.6Hz,1H),6.79(d,J＝2.6Hz,1H),6.37(d,J＝9.0Hz,1H),4.69(t,J＝8.0Hz,1H),4.60–4.48(m,3H),4.34(dd,J＝15.0,5.3Hz,1H),4.08–3.96(m,3H),3.61(dd,J＝11.2,3.6Hz,1H),2.48(d,J＝17.4Hz,13H),2.29–2.09(m,8H),1.98(d,J＝6.7Hz,2H),1.59(dt,J＝13.6,6.9Hz,2H),1.40–1.46(m,2H),1.26(dd,J＝10.3,4.9Hz,2H),0.93(s,9H).MS:calcd C56H68N12O6S,1036.5,found,[M+H]+,1037.5.
(2R,4S)-1-[(2R)-3,3-二甲基-2-{[7-(4-{3-[(4-{[3-甲基-4-([1,2,4]三氮杂环戊熳并[1,5-a]吡啶-7-基氧基)苯基]氨基}喹唑啉-6-基)氧基]丙基}哌嗪-1-基)-1-氧亚基庚基]氨基}-1-氧亚基丁基]-4-羟基-N-{[4-(4-甲基-1,3-硫杂氮杂环戊熳-5-基)苯基]甲基}四氢吡咯-2-甲酰胺(VH7C):White solid；yield:40％；1H NMR(400MHz,Chloroform-d)δ8.90(s,1H),8.66(dd,J＝6.0,1.8Hz,2H),8.48(dd,J＝7.5,1.8Hz,1H),8.19(d,J＝1.8Hz,1H),7.79(dd,J＝24.4,15.1Hz,3H),7.61(d,J＝5.4Hz,2H),7.37(d,J＝9.0Hz,1H),7.27(d,J＝1.8Hz,1H),7.02(d,J＝8.7Hz,1H),6.88(dt,J＝7.6,2.3Hz,1H),6.79(d,J＝2.3Hz,1H),6.58(d,J＝8.6Hz,1H),4.70(t,J＝8.0Hz,1H),4.57–4.43(m,3H),4.40–4.30(m,1H),4.02(d,J＝8.9Hz,3H),3.62(d,J＝10.6Hz,2H),3.06–2.66(m,9H),2.48(d,J＝1.8Hz,3H),2.34–2.09(m,7H),1.95(s,2H),1.53(s,2H),1.39(td,J＝7.3,1.8Hz,2H),1.24(d,J＝9.5Hz,6H),0.95(s,9H).MS:calcd C57H70N12O6S,1050.5,found,[M+H]+,1051.6.
(2R,4S)-1-[(2R)-3,3-二甲基-2-{[8-(4-{3-[(4-{[3-甲基-4-([1,2,4]三氮杂环戊熳并[1,5-a]吡啶-7-基氧基)苯基]氨基}喹唑啉-6-基)氧基]丙基}哌嗪-1-基)-1-氧亚基辛基]氨基}-1-氧亚基丁基]-4-羟基-N-{[4-(4-甲基-1,3-硫杂氮杂环戊熳-5-基)苯基]甲基}四氢吡咯-2-甲酰胺(VH8C):White solid；yield:47％；1H NMR(400MHz,Chloroform-d)δ9.01(s,1H),8.72–8.63(m,2H),8.49(dd,J＝7.4,1.7Hz,1H),8.20(d,J＝1.7Hz,1H),7.90(d,J＝2.7Hz,1H),7.84–7.72(m,3H),7.64(t,J＝6.1Hz,1H),7.38(dd,J＝9.0,2.2Hz,1H),7.29(s,1H),7.02(d,J＝8.6Hz,1H),6.88(dt,J＝7.6,2.2Hz,1H),6.81(d,J＝2.5Hz,1H),6.52(d,J＝8.3Hz,1H),4.72(t,J＝8.2Hz,1H),4.55–4.44(m,3H),4.37(dd,J＝15.3,5.6Hz,1H),4.12(t,J＝6.3Hz,2H),4.02(d,J＝11.1Hz,1H),3.65(d,J＝9.8Hz,1H),3.18–3.01(m,6H),2.90(s,2H),2.65–2.78(m,4H),2.48(d,J＝1.7Hz,3H),2.19(d,J＝16.3Hz,6H),2.00(t,J＝6.2Hz,2H),1.53(t,J＝6.9Hz,2H),1.41(td,J＝7.4,1.8Hz,6H),1.26–1.23(m,4H),0.95(s,9H).MS:calcd C58H72N12O6S,1064.5,found,[M+H]+,1065.6.
(2R,4S)-1-[(2R)-3,3-二甲基-2-{[9-(4-{3-[(4-{[3-甲基-4-([1,2,4]三氮杂环戊熳并[1,5-a]吡啶-7-基氧基)苯基]氨基}喹唑啉-6-基)氧基]丙基}哌嗪-1-基)-1-氧亚基壬基]氨基}-1-氧亚基丁基]-4-羟基-N-{[4-(4-甲基-1,3-硫杂氮杂环戊熳-5-基)苯基]甲基}四氢吡咯-2-甲酰胺(VH9C):White solid；yield:38％；1H NMR(400MHz,Chloroform-d)δ8.80(s,1H),8.66(d,J＝9.0Hz,2H),8.49(d,J＝7.4Hz,1H),8.19(d,J＝3.8Hz,1H),7.80(d,J＝9.4Hz,2H),7.69(dd,J＝8.7,2.7Hz,1H),7.62(d,J＝2.8Hz,1H),7.56(t,J＝6.1Hz,1H),7.38(dd,J＝9.1,2.4Hz,1H),7.28(s,2H),7.02(d,J＝8.6Hz,1H),6.88(dt,J＝6.5,3.2Hz,1H),6.80(t,J＝3.1Hz,1H),6.46(d,J＝8.9Hz,1H),4.68(d,J＝8.0Hz,1H),4.60–4.46(m,3H),4.35(dd,J＝15.1,5.5Hz,1H),4.06(dt,J＝17.2,8.9Hz,3H),3.62(dd,J＝11.3,3.6Hz,1H),2.72(s,6H),2.60(t,J＝7.1Hz,2H),2.48(d,J＝3.9Hz,5H),2.37(dd,J＝10.5,4.5Hz,2H),2.16(d,J＝7.5Hz,6H),1.99(q,J＝7.6,7.2Hz,2H),1.51(dd,J＝13.6,6.4Hz,4H),1.24(d,J＝16.2Hz,10H),0.94(s,10H).MS:calcdC59H74N12O6S,1078.5,found,[M+H]+,1079.6.
(4S)-1-[(2R)-3,3-二甲基-2-{[10-(4-{3-[(4-{[3-甲基-4-([1,2,4]三氮杂环戊熳并[1,5-a]吡啶-7-基氧基)苯基]氨基}喹唑啉-6-基)氧基]丙基}哌嗪-1-基)-1-氧亚基癸基]氨基}-1-氧亚基丁基]-4-羟基-N-{[4-(4-甲基-1,3-硫杂氮杂环戊熳-5-基)苯基]甲基}四氢吡咯-2-甲酰胺(VH10C):White solid；yield:48％；1H NMR(400MHz,Chloroform-d)δ8.68(s,2H),8.50(d,J＝7.5Hz,1H),8.42(s,1H),8.21(s,1H),7.84(d,J＝9.1Hz,1H),7.68(d,J＝2.6Hz,1H),7.60(dd,J＝8.6,2.6Hz,1H),7.49(d,J＝2.6Hz,1H),7.47–7.39(m,2H),7.39–7.29(m,4H),7.03(d,J＝8.6Hz,1H),6.90(dd,J＝7.5,2.6Hz,1H),6.79(d,J＝2.6Hz,1H),6.29(d,J＝9.0Hz,1H),4.69(t,J＝7.9Hz,1H),4.59–4.49(m,3H),4.35(dd,J＝15.0,5.4Hz,1H),4.11–3.99(m,3H),3.62(dt,J＝11.1,5.1Hz,1H),2.58–2.41(m,13H),2.32–2.25(m,2H),2.20–2.10(m,6H),2.00(d,J＝6.6Hz,2H),1.49–1.40(m,4H),1.24(s,12H),0.93(s,9H).MS:calcd C60H76N12O6S,1092.5,found,[M+H]+,1093.6.
(4S)-1-[(2R)-3,3-二甲基-2-{[11-(4-{3-[(4-{[3-甲基-4-([1,2,4]三氮杂环戊熳并[1,5-a]吡啶-7-基氧基)苯基]氨基}喹唑啉-6-基)氧基]丙基}哌嗪-1-基)-1-氧亚基十一烷基]氨基}-1-氧亚基丁基]-4-羟基-N-{[4-(4-甲基-1,3-硫杂氮杂环戊熳-5-基)苯基]甲基}四氢吡咯-2-甲酰胺(VH11C):White solid；yield:57％；1H NMR(400MHz,Chloroform-d)δ8.67(d,J＝7.6Hz,3H),8.49(d,J＝7.6Hz,1H),8.20(s,1H),7.81(d,J＝9.1Hz,1H),7.76(s,1H),7.67(d,J＝8.8Hz,1H),7.59(s,1H),7.50(t,J＝6.1Hz,1H),7.42–7.30(m,5H),7.02(d,J＝8.8Hz,1H),6.89(dd,J＝7.5,2.7Hz,1H),6.80(d,J＝2.6Hz,1H),6.39(d,J＝8.9Hz,1H),4.69(t,J＝7.9Hz,1H),4.60–4.47(m,3H),4.35(dd,J＝12.1,5.4Hz,1H),4.12–3.99(m,3H),3.62(dd,J＝11.4,3.5Hz,1H),2.65(s,6H),2.57(d,J＝7.3Hz,3H),2.49(s,3H),2.41(t,J＝8.3Hz,3H),2.16(d,J＝9.1Hz,6H),2.01(d,J＝6.9Hz,2H),1.48–1.41(m,4H),1.22(d,J＝7.4Hz,14H),0.94(s,9H).MS:calcd C61H78N12O6S,1106.6,found,[M+H]+,1107.7.
(4R)-1-[(2R)-3,3-二甲基-2-{[12-(4-{3-[(4-{[3-甲基-4-([1,2,4]三氮杂环戊熳并[1,5-a]吡啶-7-基氧基)苯基]氨基}喹唑啉-6-基)氧基]丙基}哌嗪-1-基)-1-氧亚基十二烷基]氨基}-1-氧亚基丁基]-4-羟基-N-{[4-(4-甲基-1,3-硫杂氮杂环戊熳-5-基)苯基]甲基}四氢吡咯-2-甲酰胺(VH12C):White solid；yield:47％；1H NMR(400MHz,Chloroform-d)δ8.69(d,J＝4.5Hz,2H),8.50(d,J＝7.5Hz,1H),8.21(s,1H),8.10(s,1H),7.85(d,J＝9.1Hz,1H),7.70(d,J＝2.6Hz,1H),7.62(dd,J＝8.6,2.7Hz,1H),7.48–7.39(m,2H),7.39–7.29(m,5H),7.06(d,J＝8.6Hz,1H),6.90(dd,J＝7.5,2.6Hz,1H),6.82(d,J＝2.6Hz,1H),6.21(d,J＝8.9Hz,1H),4.70(t,J＝7.9Hz,1H),4.54(dt,J＝13.0,5.2Hz,3H),4.34(dd,J＝15.0,5.3Hz,1H),4.16–4.02(m,3H),3.60(dd,J＝11.3,3.6Hz,1H),2.53(d,J＝14.6Hz,10H),2.35–2.28(m,3H),2.22–2.09(m,8H),2.02(q,J＝6.8Hz,2H),1.62–1.53(m,2H),1.40–1.48(m,2H),1.29–1.20(m,16H),0.93(s,9H).MS:calcd C62H81N12O6S,1120.6,found,[M+H]+,1121.6.
(4R)-4-羟基-1-[(7R)-7-(2-甲基丙-2-基)-1-(4-{3-[(4-{[3-甲基-4-([1,2,4]三氮杂环戊熳并[1,5-a]吡啶-7-基氧基)苯基]氨基}喹唑啉-6-基)氧基]丙基}哌嗪-1-基)-5,8-二氧亚基-6-氮杂-3-氧杂辛-8-基]-N-{[4-(4-甲基-1,3-硫杂氮杂环戊熳-5-基)苯基]甲基}四氢吡咯-2-甲酰胺(VH2P):White solid；yield:37％；1H NMR(400MHz,Chloroform-d)δ8.73–8.65(m,3H),8.49(d,J＝7.5Hz,1H),8.20(s,1H),7.83(d,J＝9.2Hz,1H),7.72(d,J＝2.6Hz,1H),7.60(dd,J＝8.6,2.6Hz,1H),7.53–7.38(m,3H),7.37–7.27(m,5H),7.04(d,J＝8.7Hz,1H),6.90(dd,J＝7.5,2.6Hz,1H),6.80(d,J＝2.6Hz,1H),4.68(t,J＝8.0Hz,1H),4.60–4.48(m,3H),4.31(dd,J＝12.2,5.3Hz,1H),4.05–3.86(m,5H),3.63(tt,J＝9.9,4.6Hz,3H),2.64–2.40(m,17H),2.19(s,4H),1.95d,J＝6.8Hz,2H),0.96(s,9H).MS:calcd C54H64N12O7S,1024.5,found,[M+H]+,1025.4.
(2S,4R)-4-羟基-1-[(10R)-10-(2-甲基丙-2-基)-1-(4-{3-[(4-{[3-甲基-4-([1,2,4]三氮杂环戊熳并[1,5-a]吡啶-7-基氧基)苯基]氨基}喹唑啉-6-基)氧基]丙基}哌嗪-1-基)-8,11-二氧亚基-9-氮杂-3,6-二氧杂十一烷-11-基]-N-{[4-(4-甲基-1,3-硫杂氮杂环戊熳-5-基)苯基]甲基}四氢吡咯-2-甲酰胺(VH3P):White solid；yield:39％；1HNMR(400MHz,Chloroform-d)δ8.70–8.64(m,3H),8.49(d,J＝7.5Hz,1H),8.19(s,1H),7.83(d,J＝9.1Hz,1H),7.70(d,J＝2.6Hz,1H),7.64–7.50(m,3H),7.42(dd,J＝9.1,2.5Hz,1H),7.38–7.28(m,5H),7.04(d,J＝8.6Hz,1H),6.90(dd,J＝7.5,2.6Hz,1H),6.79(d,J＝2.6Hz,1H),4.71(t,J＝8.0Hz,1H),4.60–4.45(m,3H),4.36(dd,J＝11.1,5.5Hz,1H),4.08–3.92(m,5H),3.71–3.56(m,7H),2.62–2.40(m,17H),2.18(s,4H),1.97(t,J＝6.8Hz,2H),0.95(s,9H).MS:calcd C56H68N12O7S,1068.5,found,[M+H]+,1069.5.
(2S,4R)-4-羟基-1-[(10R)-10-(2-甲基丙-2-基)-1-(4-{3-[(4-{[3-甲基-4-([1,2,4]三氮杂环戊熳并[1,5-a]吡啶-7-基氧基)苯基]氨基}喹唑啉-6-基)氧基]丙基}哌嗪-1-基)-8,11-二氧亚基-9-氮杂-3,6-二氧杂十一烷-11-基]-N-{[4-(4-甲基-1,3-硫杂氮杂环戊熳-5-基)苯基]甲基}四氢吡咯-2-甲酰胺(VH4P):White solid；yield:49％；1HNMR(400MHz,Chloroform-d)δ8.67(d,J＝1.6Hz,2H),8.59(s,1H),8.49(d,J＝7.5Hz,1H),8.17(s,1H),7.83(d,J＝9.0Hz,1H),7.71(d,J＝2.6Hz,1H),7.65–7.56(m,2H),7.56–7.50(m,1H),7.43(dd,J＝9.2,2.5Hz,1H),7.40–7.29(m,5H),7.04(dd,J＝8.7,3.2Hz,1H),6.90(dd,J＝7.5,2.6Hz,1H),6.80(d,J＝2.7Hz,1H),4.70(t,J＝8.0Hz,1H),4.59–4.45(m,3H),4.37(dd,J＝11.1,5.5Hz,1H),4.11–3.92(m,5H),3.70–3.54(m,11H),2.69–2.36(m,17H),2.13(dd,J＝13.4,8.2Hz,4H),1.99(d,J＝6.6Hz,2H),0.95(s,9H).MS:calcd C58H72N12O7S,1112.5,found,[M+H]+,1113.6.
(4R)-4-羟基-2-[(16R)-16-(2-甲基丙-2-基)-1-(4-{3-[(4-{[3-甲基-4-([1,2,4]三氮杂环戊熳并[1,5-a]吡啶-7-基氧基)苯基]氨基}喹唑啉-6-基)氧基]丙基}哌嗪-1-基)-14,17-二氧亚基-15-氮杂-3,6,9,12-四氧杂十七烷-17-基]-N-{[4-(4-甲基-1,3-硫杂氮杂环戊熳-5-基)苯基]甲基}环戊烷甲酰胺(VH5P):White solid；yield:42％；1H NMR(400MHz,Chloroform-d)δ8.67(s,2H),8.53–8.46(m,2H),8.18(s,1H),7.83(d,J＝9.1Hz,1H),7.73(d,J＝2.6Hz,1H),7.65–7.55(m,2H),7.50(d,J＝2.6Hz,1H),7.43(dd,J＝9.1,2.5Hz,1H),7.39–7.28(m,5H),7.04(d,J＝8.7Hz,1H),6.90(dd,J＝7.5,2.6Hz,1H),6.81(d,J＝2.6Hz,1H),4.71(t,J＝7.9Hz,1H),4.58–4.46(m,3H),4.37(dd,J＝11.0,5.5Hz,1H),4.12–3.93(m,5H),3.71–3.54(m,15H),2.63–2.43(m,17H),2.19(s,4H),2.00(d,J＝6.5Hz,2H),0.94(s,9H).MS:calcd C60H76N12O7S,1156.6,found,[M+H]+,1157.5.
(2S,4R)-4-羟基-1-[(19R)-19-(2-甲基丙-2-基)-1-(4-{3-[(4-{[3-甲基-4-([1,2,4]三氮杂环戊熳并[1,5-a]吡啶-7-基氧基)苯基]氨基}喹唑啉-6-基)氧基]丙基}哌嗪-1-基)-17,20-二氧亚基-18-氮杂-3,6,9,12,15-五氧杂二十烷-20-基]-N-{[4-(4-甲基-1,3-硫杂氮杂环戊熳-5-基)苯基]甲基}四氢吡咯-2-甲酰胺(VH6P):White solid；yield:32％；1H NMR(400MHz,Chloroform-d)δ8.67(s,2H),8.54–8.46(m,2H),8.19(s,1H),7.83(d,J＝9.1Hz,1H),7.72(d,J＝2.6Hz,1H),7.66–7.48(m,3H),7.42(dd,J＝9.1,2.4Hz,1H),7.38–7.27(m,6H),7.04(d,J＝8.6Hz,1H),6.90(dd,J＝7.5,2.6Hz,1H),6.81(d,J＝2.5Hz,1H),4.70(t,J＝8.0Hz,1H),4.59–4.47(m,3H),4.37(dd,J＝11.1,5.5Hz,1H),4.11–3.93(m,5H),3.67–3.55(m,19H),2.69–2.36(m,17H),2.13(dd,J＝13.4,8.1Hz,4H),2.00(d,J＝6.4Hz,2H),0.95(s,9H).MS:calcd C62H80N12O7S,1200.6,found,[M+H]+,1201.6.
2-(2,6-二氧亚基六氢吡啶-3-基)-5-{[6-(4-{3-[(4-{[3-甲基-4-([1,2,4]三氮杂环戊熳并[1,5-a]吡啶-7-基氧基)苯基]氨基}喹唑啉-6-基)氧基]丙基}哌嗪-1-基)己基]氨基}异吲哚-1,3-二酮(H6C4):Cyan solid；yield:40％；1H NMR(400MHz,Chloroform-d)δ8.68(s,1H),8.50(d,J＝7.4Hz,1H),8.36(s,1H),8.23(s,1H),7.84(d,J＝9.1Hz,1H),7.70(d,J＝2.6Hz,1H),7.63(dd,J＝8.6,2.6Hz,1H),7.48(d,J＝8.3Hz,2H),7.42(dd,J＝9.1,2.4Hz,1H),7.02(d,J＝8.6Hz,1H),6.93–6.83(m,2H),6.78(d,J＝2.5Hz,1H),6.67(dd,J＝8.3,2.2Hz,1H),5.07(t,J＝5.4Hz,1H),4.90(dd,J＝12.0,5.3Hz,1H),4.13–4.07(m,2H),3.14(q,J＝6.5Hz,2H),2.85–2.69(m,4H),2.56–2.48(m,6H),2.32(t,J＝7.7Hz,2H),2.16(s,3H),2.11–1.96(m,4H),1.61(q,J＝7.2Hz,2H),1.48(t,J＝7.5Hz,2H),1.35–1.27(m,6H).MS:calcd C47H51N11O6,865.4,found,[M+H]+,866.4.
2-(2,6-二氧亚基六氢吡啶-3-基)-5-(乙基氨基)异吲哚-1,3-二酮6-{[3-(4-丁基哌嗪-1-基)丙基]氧基}-4-{[3-甲基-4-([1,2,4]三氮杂环戊熳并[1,5-a]吡啶-7-基氧基)苯基]氨基}喹唑啉(H7C4):Cyan solid；yield:38％；1H NMR(400MHz,Chloroform-d)δ8.80(s,1H),8.66(s,1H),8.50(d,J＝7.4Hz,1H),8.22(s,1H),7.81(dd,J＝5.9,3.3Hz,2H),7.78–7.68(m,2H),7.50(d,J＝8.3Hz,1H),7.40(dd,J＝9.1,2.4Hz,1H),7.02(d,J＝8.7Hz,1H),6.93–6.85(m,2H),6.81(d,J＝2.6Hz,1H),6.72(dd,J＝8.3,2.2Hz,1H),5.32(d,J＝5.0Hz,1H),4.90(dd,J＝12.0,5.4Hz,1H),4.17(t,J＝6.3Hz,2H),3.15(q,J＝6.5Hz,2H),2.96(q,J＝7.4Hz,4H),2.84–2.72(m,2H),2.55(t,J＝7.5Hz,6H),2.35(dd,J＝9.3,6.2Hz,2H),2.17(s,3H),2.13–2.05(m,1H),2.01(t,J＝7.0Hz,2H),1.61(t,J＝7.2Hz,2H),1.50(q,J＝7.4Hz,2H),1.41(t,J＝7.4Hz,8H).MS:calcd C48H53N11O6,879.4,found,[M+H]+,880.4.
2-(2,6-二氧亚基六氢吡啶-3-基)-5-{[8-(4-{3-[(4-{[3-甲基-4-([1,2,4]三氮杂环戊熳并[1,5-a]吡啶-7-基氧基)苯基]氨基}喹唑啉-6-基)氧基]丙基}哌嗪-1-基)辛基]氨基}异吲哚-1,3-二酮(H8C4):Cyan solid；yield:43％；1H NMR(400MHz,Chloroform-d)δ9.23(s,1H),8.65(s,1H),8.50(d,J＝7.4Hz,1H),8.21(s,1H),7.93(t,J＝2.4Hz,2H),7.86(dd,J＝8.8,2.7Hz,1H),7.79(d,J＝9.1Hz,1H),7.52(d,J＝8.3Hz,1H),7.38(dd,J＝9.1,2.4Hz,1H),7.02(d,J＝8.6Hz,1H),6.95(d,J＝2.1Hz,1H),6.92–6.74(m,3H),5.61(t,J＝5.1Hz,1H),4.90(dd,J＝11.7,5.5Hz,1H),4.27(t,J＝6.2Hz,2H),3.16(q,J＝6.7Hz,2H),2.88–2.65(m,11H),2.55(t,J＝8.0Hz,2H),2.18(s,3H),2.07(dt,J＝14.3,8.3Hz,3H),1.50(t,J＝7.4Hz,14H).MS:calcd C49H55N11O6,893.4,found,[M+H]+,894.5.
2-(2,6-二氧亚基六氢吡啶-3-基)-5-(甲基氨基)异吲哚-1,3-二酮6-{[3-(4-庚基哌嗪-1-基)丙基]氧基}-4-{[3-甲基-4-([1,2,4]三氮杂环戊熳并[1,5-a]吡啶-7-基氧基)苯基]氨基}喹唑啉(H9C4):Cyan solid；yield:45％；1H NMR(400MHz,Chloroform-d)δ8.67(s,2H),8.50(d,J＝7.5Hz,1H),8.22(s,1H),7.82(d,J＝9.1Hz,1H),7.77(d,J＝2.6Hz,1H),7.71(dd,J＝8.7,2.6Hz,1H),7.64(d,J＝2.7Hz,1H),7.51(d,J＝8.3Hz,1H),7.41(dd,J＝9.1,2.5Hz,1H),7.02(d,J＝8.6Hz,1H),6.93–6.86(m,2H),6.80(d,J＝2.6Hz,1H),6.71(dd,J＝8.4,2.2Hz,1H),5.15(t,J＝5.4Hz,1H),4.90(dd,J＝12.1,5.4Hz,1H),4.13(dt,J＝17.0,6.8Hz,2H),3.16(q,J＝6.5Hz,2H),2.82–2.43(m,11H),2.33(q,J＝6.3,4.7Hz,2H),2.17(s,3H),2.10–1.97(m,3H),1.61(t,J＝7.3Hz,2H),1.47(dq,J＝13.1,7.0Hz,2H),1.32(t,J＝7.3Hz,14H).MS:calcd C50H57N11O6,907.5,found,[M+H]+,908.4.
2-(2,6-二氧亚基六氢吡啶-3-基)-5-{[10-(4-{3-[(4-{[3-甲基-4-([1,2,4]三氮杂环戊熳并[1,5-a]吡啶-7-基氧基)苯基]氨基}喹唑啉-6-基)氧基]丙基}哌嗪-1-基)癸基]氨基}异吲哚-1,3-二酮(H10C4):Cyan solid；yield:46％；1H NMR(400MHz,Chloroform-d)δ8.69(s,1H),8.50(d,J＝7.5Hz,1H),8.23(s,1H),8.16(s,1H),7.88–7.81(m,1H),7.70(d,J＝2.6Hz,1H),7.63(dd,J＝8.6,2.7Hz,1H),7.52(d,J＝8.3Hz,1H),7.43(d,J＝7.5Hz,2H),7.03(d,J＝8.6Hz,1H),6.90(dd,J＝7.5,2.5Hz,2H),6.79(d,J＝2.6Hz,1H),6.69(dd,J＝8.4,2.2Hz,1H),4.94–4.82(m,2H),4.11(t,J＝6.4Hz,2H),3.17(q,J＝6.6Hz,2H),2.78–2.49(m,11H),2.37–2.31(m,2H),2.17(s,3H),2.11–1.99(m,3H),1.62(p,J＝7.1Hz,2H),1.48(t,J＝7.8Hz,2H),1.41–1.23(m,14H).MS:calcd C51H59N11O6,921.5,found,[M+H]+,922.5.
2-(2,6-二氧亚基六氢吡啶-3-基)-5-{[6-(4-{3-[(6-甲氧基-4-{[3-甲基-4-([1,2,4]三氮杂环戊熳并[1,5-a]吡啶-7-基氧基)苯基]氨基}喹唑啉-7-基)氧基]丙基}哌嗪-1-基)己基]氨基}异吲哚-1,3-二酮(CH6C4):Cyan solid；yield:46％；1H NMR(400MHz,DMSO-d6)δ8.66(s,1H),8.50(d,J＝7.4Hz,1H),8.24(s,1H),8.11(s,1H),7.64–7.49(m,3H),7.39(s,1H),7.29(s,1H),7.00(d,J＝8.6Hz,1H),6.89(q,J＝3.0Hz,2H),6.79–6.64(m,2H),5.00(t,J＝5.3Hz,1H),4.90(dd,J＝12.1,5.4Hz,1H),4.18(q,J＝8.7,7.7Hz,2H),3.94(s,3H),3.16(q,J＝6.4Hz,2H),2.90–2.53(m,11H),2.40(t,J＝7.3Hz,2H),2.14(s,3H),2.11–2.04(m,3H),1.46–1.33(m,10H).MS:calcd C48H53N11O7,895.4,found,[M+H]+,896.4.
2-(2,6-二氧亚基六氢吡啶-3-基)-5-(乙基氨基)异吲哚-1,3-二酮7-{[3-(4-丁基哌嗪-1-基)丙基]氧基}-6-甲氧基-4-{[3-甲基-4-([1,2,4]三氮杂环戊熳并[1,5-a]吡啶-7-基氧基)苯基]氨基}喹唑啉(CH7C4):Cyan solid；yield:48％；1H NMR(400MHz,Chloroform-d)δ8.66(s,1H),8.50(d,J＝7.4Hz,1H),8.23(s,1H),8.01(d,J＝9.2Hz,1H),7.61(d,J＝2.7Hz,1H),7.55(dd,J＝8.5,3.5Hz,2H),7.29(s,2H),7.02(d,J＝8.7Hz,1H),6.94–6.85(m,2H),6.76(d,J＝2.6Hz,1H),6.71(dd,J＝8.4,2.2Hz,1H),4.98–4.77(m,2H),4.20(t,J＝6.6Hz,2H),3.96(s,3H),3.18(q,J＝6.6Hz,2H),2.90–2.52(m,11H),2.37(t,J＝7.9Hz,2H),2.16(s,3H),2.12–2.04(m,3H),1.56–1.32(m,11H).MS:calcd C49H55N11O7,909.4,found,[M+H]+,910.4.
2-(2,6-二氧亚基六氢吡啶-3-基)-5-{[8-(4-{3-[(6-甲氧基-4-{[3-甲基-4-([1,2,4]三氮杂环戊熳并[1,5-a]吡啶-7-基氧基)苯基]氨基}喹唑啉-7-基)氧基]丙基}哌嗪-1-基)辛基]氨基}异吲哚-1,3-二酮(CH8C4):Cyan solid；yield:49％；1H NMR(400MHz,Chloroform-d)δ8.66(s,1H),8.50(d,J＝7.5Hz,1H),8.23(s,1H),7.94(s,1H),7.60(d,J＝2.6Hz,1H),7.54(dd,J＝8.5,3.1Hz,2H),7.31(d,J＝16.7Hz,2H),7.02(d,J＝8.7Hz,1H),6.90(dd,J＝7.2,2.5Hz,2H),6.76(d,J＝2.6Hz,1H),6.70(dd,J＝8.4,2.2Hz,1H),4.90(dd,J＝12.1,5.4Hz,1H),4.81(t,J＝5.4Hz,1H),4.20(t,J＝6.6Hz,2H),3.95(s,3H),3.18(q,J＝6.6Hz,2H),2.80–2.50(m,11H),2.36(d,J＝7.7Hz,2H),2.16(s,3H),2.09(dt,J＝13.6,5.3Hz,3H),1.56–1.30(m,14H).MS:calcd C50H57N11O7,923.4,found,[M+H]+,924.4.
2-(2,6-二氧亚基六氢吡啶-3-基)-5-(甲基氨基)异吲哚-1,3-二酮7-{[3-(4-庚基哌嗪-1-基)丙基]氧基}-6-甲氧基-4-{[3-甲基-4-([1,2,4]三氮杂环戊熳并[1,5-a]吡啶-7-基氧基)苯基]氨基}喹唑啉(CH9C4):Cyan solid；yield:41％；1H NMR(400MHz,Chloroform-d)δ8.66(s,1H),8.50(d,J＝7.4Hz,1H),8.23(s,1H),8.00(s,1H),7.60(d,J＝2.7Hz,1H),7.54(d,J＝8.4Hz,2H),7.28(d,J＝5.5Hz,2H),7.01(d,J＝8.7Hz,1H),6.90(dd,J＝7.7,2.5Hz,2H),6.76(d,J＝2.6Hz,1H),6.70(dd,J＝8.4,2.2Hz,1H),4.92–4.80(m,2H),4.19(t,J＝6.5Hz,2H),3.94(s,3H),3.17(q,J＝6.5Hz,2H),2.78–2.53(m,11H),2.41(d,J＝7.2Hz,2H),2.15(s,3H),2.08(t,J＝7.4Hz,3H),1.53–1.27(m,16H).MS:calcdC51H59N11O7,937.5,found,[M+H]+,938.4.
2-(2,6-二氧亚基六氢吡啶-3-基)-5-{[10-(4-{3-[(6-甲氧基-4-{[3-甲基-4-([1,2,4]三氮杂环戊熳并[1,5-a]吡啶-7-基氧基)苯基]氨基}喹唑啉-7-基)氧基]丙基}哌嗪-1-基)癸基]氨基}异吲哚-1,3-二酮(CH10C4):Cyan solid；yield:45％；1H NMR(400MHz,Chloroform-d)δ8.66(s,1H),8.50(d,J＝7.5Hz,1H),8.23(s,1H),8.03(s,1H),7.62(s,1H),7.55(t,J＝7.4Hz,2H),7.35(d,J＝7.7Hz,1H),7.29(s,1H),7.05–6.96(m,1H),6.94–6.86(m,2H),6.79–6.68(m,2H),4.88(t,J＝6.2Hz,2H),4.19(t,J＝6.5Hz,2H),3.95(s,3H),3.18(q,J＝6.5Hz,2H),2.80–2.56(m,11H),2.46(d,J＝8.3Hz,2H),2.15(s,3H),2.08(q,J＝7.0Hz,3H),1.30(d,J＝17.2Hz,18H).MS:calcd C52H61N11O7,951.5,found,[M+H]+,952.5.
分别对实施例1中制得的靶向降解HER2蛋白的PROTAC化合物的抗BT-474细胞株增殖活性、降解活性进行测试，具体测试方法如下，具体测试结果见表1-2。
1、抗BT-474细胞株增殖活性测试
收集对数生长期细胞重悬后用细胞计数仪细胞计数，按照96孔板每孔9000个细胞取适量的细胞悬液与培养基混匀后铺板，梯队浓度双官能PROTAC化合物作用72小时后，每孔加入CCK8各10μL，避光孵育2到4小时，用酶标仪在450nm的波长下检测每孔对应的吸光度值；
细胞抑制率＝100％-(加药组吸光度平均值/空白对照组吸光度平均值)×100％，分析计算得到半数抑制浓度IC50。
2、HER2降解活性测试
取对数生长期BT-74细胞于6孔板中，使用500nMHER2双官能PROTAC化合物处理24小时后，用预冷的PBS洗3次细胞，后用刮板收集细胞，加入适量的含蛋白酶抑制剂RIPA裂解液于冰上裂解细胞15分钟，经低温超声后，离心(4℃，13000rpm，15分钟)收集上清，将上清与SDS-PAGE蛋白上样缓冲液(十二烷基硫酸钠-聚丙烯酰胺凝胶)混合后置于沸水中煮沸10分钟，蛋白样品经10％的SDS-PAGE电泳分离后转移至PVDF(聚偏二氟乙烯)膜上，脱脂牛奶室温封闭一小时，以β-actin为内参蛋白，使用相应的一抗和PVDF膜4℃孵育过夜，使用相应的辣根酶标记的二抗进一步标记后，通过化学发光法使免疫印迹显色，通过相应蛋白免疫印迹灰度的比较，判断HER2双官能PROTAC化合物降解HER2的活性。
表1：靶向降解HER2蛋白的PROTAC化合物的抗增殖活性和降解活性
表2：靶向降解HER2蛋白的PROTAC化合物的抗增殖活性和降解活性
PROTAC化合物通过诱导靶蛋白、E3泛素连接酶与其形成三元复合物，将靶蛋白引入泛素蛋白酶体系统，使靶蛋白被泛素化修饰进而被降解。稳定三元复合物的形成是靶蛋白有效降解的关键，而PROTAC化合物结构中连接靶蛋白与E3泛素连接酶配体的连接子及连接位点至关重要。通过表1和表2中的检测结果得知，本发明所涉及的HER2蛋白的PROTAC化合物通过考察不同的连接子和连接方式，获得了可引起有效HER2蛋白水平下降的PROTAC化合物，并通过调控HER2蛋白水平来有效抑制HER2蛋白功能。
Claims (5)
1.一种靶向降解人表皮生长因子受体2蛋白的蛋白降解靶向嵌合体化合物，其特征在于，该化合物的结构式为：
其中，HER2 Ligand为人表皮生长因子受体2蛋白的小分子配体；Linker为连接子；E3Ligase Ligand为E3泛素连接酶人小脑蛋白或希佩尔-林道蛋白的小分子配体。
2.如权利要求1所述的靶向降解人表皮生长因子受体2蛋白的蛋白降解靶向嵌合体化合物，其特征在于，人表皮生长因子受体2蛋白的小分子配体的结构式为：
Linker的结构式为
其中n＝3–12，m＝1–5，x＝3-11，y＝1-5；
E3 Ligase Ligand的结构式为
3.如权利要求1或2所述的靶向降解人表皮生长因子受体2蛋白的蛋白降解靶向嵌合体化合物，其特征在于，所述蛋白降解靶向嵌合体化合物的结构式如下：
4.如权利要求3所述的靶向降解人表皮生长因子受体2蛋白的蛋白降解靶向嵌合体化合物，其特征在于，所述蛋白降解靶向嵌合体化合物的结构式如下：
5.如权利要求1所述的靶向降解人表皮生长因子受体2蛋白的蛋白降解靶向嵌合体化合物在制备治疗人表皮生长因子受体2阳性肿瘤药物中的应用。
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101356171A (zh) * | 2005-11-15 | 2009-01-28 | 阿雷生物药品公司 | 作为erbbi型受体酪氨酸激酶抑制剂用于治疗增殖性疾病的n4-苯基-喹唑啉-4-胺衍生物和相关化合物 |
| CN106456795A (zh) * | 2014-03-03 | 2017-02-22 | 辛塔医药品有限公司 | 靶向治疗学 |
| CN106543185A (zh) * | 2016-11-10 | 2017-03-29 | 吉林大学 | 一种靶向泛素化降解plk1和brd4蛋白的化合物及其应用 |
| CN110753693A (zh) * | 2016-12-23 | 2020-02-04 | 阿尔维纳斯运营股份有限公司 | Egfr蛋白水解靶向嵌合分子和相关使用方法 |
| CN111217804A (zh) * | 2020-02-21 | 2020-06-02 | 四川大学华西医院 | 一种靶向降解ido1的protac化合物及其制备方法和应用 |
| CN113372342A (zh) * | 2020-03-10 | 2021-09-10 | 暨南大学 | 蛋白降解靶向嵌合体及其应用 |
| CN113735828A (zh) * | 2021-09-07 | 2021-12-03 | 南方医科大学 | 一种靶向降解egfr的化合物及其制备方法和应用 |
| WO2022006386A1 (en) * | 2020-07-02 | 2022-01-06 | Enliven Therapeutics, Inc. | Alkyne quinazoline derivatives as inhibitors of erbb2 |
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101356171A (zh) * | 2005-11-15 | 2009-01-28 | 阿雷生物药品公司 | 作为erbbi型受体酪氨酸激酶抑制剂用于治疗增殖性疾病的n4-苯基-喹唑啉-4-胺衍生物和相关化合物 |
| CN106456795A (zh) * | 2014-03-03 | 2017-02-22 | 辛塔医药品有限公司 | 靶向治疗学 |
| CN106543185A (zh) * | 2016-11-10 | 2017-03-29 | 吉林大学 | 一种靶向泛素化降解plk1和brd4蛋白的化合物及其应用 |
| CN110753693A (zh) * | 2016-12-23 | 2020-02-04 | 阿尔维纳斯运营股份有限公司 | Egfr蛋白水解靶向嵌合分子和相关使用方法 |
| CN111217804A (zh) * | 2020-02-21 | 2020-06-02 | 四川大学华西医院 | 一种靶向降解ido1的protac化合物及其制备方法和应用 |
| CN113372342A (zh) * | 2020-03-10 | 2021-09-10 | 暨南大学 | 蛋白降解靶向嵌合体及其应用 |
| WO2022006386A1 (en) * | 2020-07-02 | 2022-01-06 | Enliven Therapeutics, Inc. | Alkyne quinazoline derivatives as inhibitors of erbb2 |
| CN113735828A (zh) * | 2021-09-07 | 2021-12-03 | 南方医科大学 | 一种靶向降解egfr的化合物及其制备方法和应用 |
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