CN111217804A - 一种靶向降解ido1的protac化合物及其制备方法和应用 - Google Patents
一种靶向降解ido1的protac化合物及其制备方法和应用 Download PDFInfo
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Abstract
Description
技术领域
本发明涉及生物医药领域,具体地,本发明涉及可用于靶向降解吲哚胺2,3-加双氧酶1蛋白的化合物及其应用。
背景技术
吲哚胺2,3-双加氧酶1(IDO1)是色氨酸通过犬尿氨酸途径代谢的关键限速酶,它催化色氨酸氧化为N-甲酰基犬尿氨酸。IDO1在人体的多个组织(胎盘、胸腺、脑、肾)和细胞(巨噬细胞、树突状细胞)中广泛地表达。细胞因子如IFN-γ,TNF-α,IL-1β,IL-6可诱导IDO1表达上调。IDO1通过氧化裂解色氨酸降低机体微环境中的色氨酸浓度,同时产生犬尿氨酸、喹啉酸、3-羟基犬尿氨酸、2-氨基-3-羟基苯甲酸等代谢产物来参与免疫耐受的调节。
最初,IDO1被认为是胚胎免疫耐受中的关键免疫调节因子,可保护胎儿免受母体免疫系统的侵害。最近,大量报告证实IDO1在多种肿瘤中过表达,并且在肿瘤免疫逃逸中起重要作用。IDO1介导的癌症免疫逃逸是通过多种作用机制全面调控多种免疫细胞的结果,其中最重要的是对T细胞的调控。一方面,过表达的IDO1氧化裂解色氨酸形成色氨酸耗竭,进而激活一般性调控阻遏蛋白激酶2(GCN2)以抑制T细胞的增殖和功能。另一方面,色氨酸代谢产物也可以结合并激多种细胞的活芳烃受体(AHR),从而导致一系列生物学效应,包括调节性T细胞的增殖,树突状细胞和巨噬细胞向免疫抑制表型的转化。另外,诸如犬尿氨酸,犬尿酸和3-羟基犬尿氨酸等代谢产物也可以直接抑制T细胞的功能。IDO1介导的免疫抑制可引起多种免疫相关疾病,包括肿瘤、病毒感染、神经变性疾病、白内障、器官移植排斥、抑郁症和自身免疫性疾病等。因此,IDO1已成为癌症免疫逃逸等免疫相关疾病的重要靶点。本发明所述的化合物可靶向降解IDO1,下调IDO1蛋白水平,实现IDO1生物学功能的完全抑制,可以应用于治疗IDO1介道的免疫抑制所引起的相关疾病。
发明内容
本发明旨在克服现有技术的不足,提供一种可实现IDO1蛋白水平下调的PROTAC化合物;同时,本发明还旨在提供合成该PROTAC化合物的可行方法。
本发明上述目的通过如下技术方案实现:
一种靶向降解IDO1的PROTAC化合物,化学结构式如下:
式I中,X代表–CH2或–C=O;Y代表–CH2或–C=O;n为2~9的自然数:
优选的,
X代表–C=O;
Y代表–CH2;
n代表9;
优选的,所述化合物为:
上述靶向降解IDO1化合物在制备治疗抗癌药物中的应用。
有益效果:
本发明提供的PROTAC化合物可靶向降解Hela细胞中IDO1蛋白,因而可以用于制备治疗抗肿瘤免疫逃逸药物,其中,X代表–C=O,Y代表–CH2,n代表9的靶向降解IDO1化合物具有优异的IDO1降解活性,可以用于制备靶向降解IDO1蛋白的试剂或药物;本发明还提供了一种合成该系列IDO1靶向降解PROTAC化合物的方法。
附图说明
图1:PROTAC化合物降解活性
A:PROTAC化合物降解活性;B:不同浓度化合物2c的降解活性;C:IDO1降解率–化合物2c浓度曲线
图2:化合物2c通过泛素蛋白酶体系统降解IDO1
A:对照化合物不能降解IDO1;B:泛素蛋白酶体通路抑制剂可以影响2c降解IDO1
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IDO1化合物的合成与结构确认
合成路线:
化合物4的合成:
将氯代聚乙二醇(0.1mol)溶于四氢呋喃(250mL),溴乙酸叔丁酯(38.87g,0.2mol),加入四丁基溴化铵(32.24g,0.1mol)和叔丁醇钾(11.22g,0.1mol),并将混合物在室温搅拌过夜。将该混合物减压浓缩以除去THF,并重新溶于乙酸乙酯(200mL)中,用水洗涤(100mL×3),无水硫酸钠干燥,过滤,浓缩有机相,并通过柱层析纯化(DCM:MeOH=50:1),得到产物。
4a:Yield,15.31g,64%.1H NMR(400MHz,DMSO-d6),δ4.19(s,2H),3.99–3.85(m,4H),3.68–3.55(m,4H),1.44(s,9H).MS(ESI),Calcd.for C10H19ClO4[M+H]+:239.1,Found:239.2.
4b:Yield,19.26g,68%.1H NMR(400MHz,DMSO-d6),δ3.99(s,2H),3.91–3.65(m,4H),3.65–3.47(m,8H),1.43(s,9H).MS(ESI),Calcd.for C12H23ClO5[M+H]+:283.1,Found:283.3.
4c:Yield,19.63g,60%.1H NMR(400MHz,DMSO-d6),δ3.97(s,2H),3.93–3.75(m,4H),3.69–3.53(m,12H),1.43(s,9H).MS(ESI),Calcd.for C14H27ClO6[M+H]+:327.2,Found:327.1.
化合物5的合成:
将化合物4(0.05mmol)溶解在三氟乙酸/二氯甲烷溶液=1∶3的混合溶液(100mL)中,并在室温搅拌5h。将混合物在减压下浓缩,将残余物溶于二氯亚砜中(30mL),回流2小时,并直接浓缩以得到化合物5。
化合物7的合成:
将三甘醇(15g,0.1mol),对甲苯磺酰氯(82.4g,0.4mol)和DIPEA(64.62g,0.5mol)溶解在二氯甲烷(300mL)中,并在室温下搅拌过夜。将混合物用饱和碳酸氢钠和饱和盐水洗涤,然后用二氯甲烷萃取。浓缩有机相,用大量乙醚充分洗涤以除去杂质,得到化合物7。Yield,36.7g,73%.1H NMR(400MHz,DMSO-d6),δ7.78(d,J=8.3,4H),7.47(d,J=8.1,4H),4.18–4.08(m,4H),3.62–3.53(m,4H),3.42(d,J=6.6,8H),2.41(s,6H).MS(ESI),Calcd.for C22H30O9S2[M+H]+:503.1,Found:503.2.
化合物8的合成:
将单苄基二醇(40mmol)溶于无水THF(250mL),在冰浴下分批加入氢化钠(60%,4g,100mmol)。搅拌1小时后,滴加化合物7(4.58g,10mmol)的THF溶液。将混合物在室温搅拌过夜。用冰水淬灭反应,浓缩除去THF。将残余物溶于乙酸乙酯(200mL),并用水彻底洗涤。浓缩有机相,并通过柱层析纯化(DCM/MeOH=50:1),得到中间体二苄基二醇。然后,将二苄基乙二醇溶解在适量MeOH中,并在H2下进行脱苄基反应,得到化合物8。收率63-76%。
化合物9的合成:
将化合物8(10mmol)溶于二氯甲烷(50mL),加如DIPEA(2.59g,20mmol),并逐滴添加对甲苯磺酰氯(1.65g,8mmol)的二氯甲烷溶液,混合物在室温下搅拌过夜。将所得混合物用饱和碳酸氢钠和饱和盐水洗涤,然后用二氯甲烷萃取(60mL×3)。浓缩有机相,并通过柱层析纯化(DCM/MeOH=50:1),得到化合物9。
9a:Yield,1.99g,57%.1H NMR(400MHz,DMSO-d6),δ7.81(d,J=8.1,2H),7.52(d,J=8.0,2H),4.59(t,J=5.1,1H),4.15(dd,J=5.5,4.3,2H),3.81–3.71(m,2H),3.69–3.31(m,12H),2.45(s,3H).MS(ESI),Calcd.for C15H24O7S[M+H]+:349.2,Found:349.3.
9b:Yield,2.67g,61%.1H NMR(400MHz,DMSO-d6),δ7.79(d,J=8.3,2H),7.49(d,J=8.0,2H),4.57(t,J=5.4,1H),4.12(dd,J=5.1,3.7,2H),3.76–3.64(m,2H),3.63–3.38(m,20H),2.43(s,3H).MS(ESI),Calcd.for C19H32O9S[M+H]+:437.2,Found:437.3.
9c:Yield,2.84g,54%.1H NMR(400MHz,DMSO-d6),δ7.83(d,J=7.1,2H),7.59(d,J=6.5,2H),4.779(t,J=5.9,1H),4.12(dd,J=6.1,4.5,2H),3.87–3.71(m,2H),3.53–3.18(m,28H),2.37(s,3H).MS(ESI),Calcd.for C23H40O11S[M+H]+:525.2,Found:525.3.
9d:Yield,3.13g,51%.1H NMR(400MHz,DMSO-d6),δ7.89(d,J=6.6,2H),7.69(d,J=7.4,2H),4.45(t,J=4.9,1H),4.21(dd,J=4.1,3.2,2H),3.81–3.66(m,2H),3.69–3.29(m,28H),2.46(s,3H).MS(ESI),Calcd.for C27H48O13S[M+H]+:613.3,Found:613.4.
化合物10的合成:
将化合物9(5mmol)溶解在乙腈中(100mL),加入叠氮化钠(1.63g,25mmol)并回流24小时。浓缩除去乙腈,将残余物重新溶解在乙酸乙酯中,并用饱和盐水洗涤(50mL×3)。浓缩有机相,得到化合物10。
化合物11的合成:
将化合物10(5mmol)和10%Pd/C(0.1eq)的甲醇混合物在H 2气氛下搅拌过夜。用硅藻土过滤反应混合物,并浓缩滤液,得到化合物11。
11a:Yield,0.747g,77%.1H NMR(400MHz,DMSO-d6),δ3.71–.32(m,17H),2.75–2.62(m,2H).MS(ESI),Calcd.for C8H19NO4[M+H]+:194.1,Found:194.3.
11b:Yield,1.01g,71%.1H NMR(400MHz,DMSO-d6),δ3.86–3.37(m,25H),2.81–2.67(m,2H).MS(ESI),Calcd.for C12H27NO6[M+H]+:282.2,Found:282.4.
11c:Yield,1.45g,78%.1H NMR(400MHz,DMSO-d6),δ3.93–3.33(m,33H),2.75–2.62(m,2H).MS(ESI),Calcd.for C16H35NO8[M+H]+:370.3,Found:370.4.
11d:Yield,1.54g,67%.1H NMR(400MHz,DMSO-d6),δ3.89–3.45(m,41H),2.82–2.65(m,2H).MS(ESI),Calcd.for C20H43NO10[M+H]+:458.3,Found:458.4.
Epacadostat及其衍生物的合成:
Epacadostat及其衍生物的合成方法参考已有报道(ACS Med Chem Lett 2017;8:486–91)。
Epacadostat:1H NMR(400MHz,DMSO-d6),δ11.49(s,1H),8.88(s,1H),7.24–7.04(m,2H),6.78(ddd,J=8.9,4.2,2.7,1H),6.69(t,J=6.0,1H),6.58(s,2H),6.23(t,J=6.0,1H),3.38(t,J=6.2,2H),3.12(q,J=6.2,2H).13C NMR(100MHz,DMSO-d6),δ155.26,154.49,152.11,137.65,124.35,121.04,120.97,115.59,106.68,47.87,46.96.HR-MS,Calcd.for C11H13O4N7BrFS[M+H]+:459.9851,found:459.9858.
化合物14:HPLC>95%.1H NMR(400MHz,CDCl3),δ8.91(s,1H),7.24(t,J=8.8,1H),7.17(dd,J=6.1,2.7,1H),6.84(ddd,J=8.9,4.2,2.7,1H),6.30(t,J=5.7,1H),3.25(q,J=6.0,2H),2.81(t,J=6.3,2H).MS(ESI),Calcd.for C11H12O2N6BrF[M+H]+:359.1,Found:359.1.
Epa-8PEG:将化合物14(71.6mg,0.2mmol)溶解在无水DMF(2mL)中,加入化合物9c(209.67mg,0.4mmol)和三乙胺(40.4mg,0.4mmol),将混合物在60℃搅拌过夜。浓缩除去DMF,柱层析纯化(DCM/MeOH/NH 3·H 2O=100:5:0.5),得到化合物Epa-8PEG。Yield,79.55mg,56%,m.p.96.2–99.5℃,HPLC>95%.1H NMR(400MHz,DMSO-d6),δ11.49(s,1H),8.85(s,1H),7.26–7.00(m,2H),6.78(ddd,J=8.9,4.2,2.7,1H),6.19(t,J=5.6,1H),4.55(s,1H),3.56–3.46(m,33H),2.77(t,J=5.6,2H),2.70(t,J=5.6,2H).13C NMR(100MHz,DMSO-d6),δ170.78,156.67,156.16,155.39,153.01,140.37,139.83,138.55,138.52,125.25,121.94,121.87,116.49,116.25,107.58,107.36,72.82,70.44,70.30,70.26,70.10,60.70,60.21,48.77,47.86,44.17,21.20,14.53.HR-MS Calcd.for C27H44BrFN6O10[M+H]+:711.2286,Found:711.2360.
化合物16的合成:
将来那度胺(500mg,1.93mmol),化合物5(4mmol)和DMAP(488mg,4mmol)溶于DMF(10mL),并在60℃下搅拌5h。浓缩除去DMF,柱层析纯化(DCM/MeOH=100:1),得到中间体Cl-PEGs-来那度胺。将碘化钠(628mg,9.65mmol)加入到Cl-PEG-来那度胺的乙腈溶液中,并回流24小时。浓缩除去乙腈,将残余物重新溶解在乙酸乙酯(100mL)中,并用饱和盐水(50mL×3)洗涤。浓缩有机相,得到化合物15。
16a:Yield 536.73mg,54%.1H NMR(400MHz,DMSO-d6),δ11.00(s,1H),9.67(s,1H),7.75(dd,J=7.7,1.3,1H),7.66–7.44(m,2H),5.14(dd,J=13.3,5.1,1H),4.48–4.24(m,2H),4.15(s,2H),3.78–3.61(m,8H),3.00–2.84(m,1H),2.60(dt,J=17.2,2.8,1H),2.37(qd,J=13.2,4.5,1H),2.02(dtd,J=12.5,5.2,2.2,1H).MS(ESI),Calcd.forC19H22N3O6I[M+H]+:516.1,Found:516.2.
16b:Yield 616.06mg,57%.1H NMR(400MHz,DMSO-d6),δ11.00(s,1H),9.66(s,1H),7.75(dd,J=7.7,1.3,1H),7.60–7.47(m,2H),5.14(dd,J=13.3,5.1,1H),4.53–4.26(m,2H),4.14(s,2H),3.76–3.48(m,10H),3.27(t,J=6.5,2H),2.92(ddd,J=17.2,13.6,5.4,1H),2.65–2.56(m,1H),2.37(qd,J=13.2,4.4,1H),2.05–1.97(m,1H).MS(ESI),Calcd.for C21H26 N3O7I[M+H]+:560.1,Found:560.1.
16c:Yield 606.17mg,52%.1H NMR(400MHz,DMSO-d6),δ11.02(s,1H),9.69(s,1H),7.74(dd,J=7.7,1.3,1H),7.62–7.42(m,2H),5.15(dd,J=13.3,5.1,1H),4.57–4.26(m,2H),4.14(s,2H),3.75–3.59(m,8H),3.58–3.41(m,8H),2.92(ddd,J=17.2,13.6,5.5,1H),2.67–2.56(m,1H),2.37(qd,J=13.3,4.5,1H),2.05–1.95(m,1H).MS(ESI),Calcd.forC23H30N3O8I[M+H]+:604.1,Found:604.3.
化合物1的合成:
将化合物16(0.2mmol)溶解在无水DMF(3mL)中,加入化合物14(71.6mg,0.2mmol)和三乙胺(20.2mg,0.2mmol)。将混合物在60℃下搅拌过夜。浓缩除去DMF,柱层析纯化(DCM/MeOH/NH 3·H 2O=100:5:0.5),得到化合物1。
1a:Yield 67.12mg,45%,m.p.156.3–160.1℃,HPLC>95%.1H NMR(400MHz,CD3OD),δ7.75–7.62(m,2H),7.52(t,J=7.7,1H),7.10(dd,J=6.0,2.7,1H),7.02(t,J=8.7,1H),6.82(ddd,J=8.9,4.1,2.7,1H),5.15(dd,J=13.3,5.2,1H),4.50(s,2H),4.21(d,J=7.6,2H),3.88–3.60(m,7H),3.40(t,J=6.2,2H),2.98–2.81(m,4H),2.76(ddd,J=17.6,4.7,2.4,1H),2.46(qd,J=13.2,4.7,1H),2.16(dtd,J=12.8,5.3,2.4,1H).13C NMR(100MHz,DMSO-d6),δ172.24,169.38,167.72,156.10,155.41,153.03,146.92,146.82,140.33,139.71,138.41,138.38,136.69,132.55,125.24,121.92,121.85,117.89,116.48,111.15,109.69,107.59,70.46,70.31,70.25,70.09,69.35,49.61,47.79,44.08,42.19,42.09,31.57,27.04,21.85,21.76,14.53.HR-MS Calcd.for C30H33BrFN9O8[M+H]+:746.1619,Found:746.1719.
1b:Yield 66.36mg,42%,m.p.148.6–151.9℃,HPLC>95%.1H NMR(400MHz,CD3OD),δ7.70(ddd,J=10.1,7.7,1.0,2H),7.54(t,J=7.7,1H),7.19–6.97(m,2H),6.84(ddd,J=8.8,4.1,2.7,1H),5.17(dd,J=13.3,5.2,1H),4.51(s,2H),4.24(s,2H),3.83–3.47(m,12H),3.15(t,J=6.0,2H),3.03(dd,J=6.0,4.3,2H),2.94–2.83(m,1H),2.77(ddd,J=17.6,4.7,2.4,1H),2.52–2.39(m,1H),2.24–2.12(m,1H).13C NMR(100MHz,CD3OD),δ173.27,172.28,170.14,169.77,169.28,168.43,167.90,156.41,155.74,154.01,146.82,146.76,140.57,139.48,137.67,135.83,135.70,132.59,132.49,126.37,122.50,122.43,116.90,116.71,115.52,115.29,110.66,110.55,110.13,109.91,107.50,107.28,70.27,70.23,70.20,70.16,70.14,70.12,70.10,69.89,69.24,69.09,43.15,41.90,30.82,30.34,29.34,23.74,23.56,22.34.HR-MS Calcd.for C32H37BrFN9O9[M+H]+:790.1822,Found:790.1791.
1c:Yield 66.72mg,40%,m.p.142.8–143.9℃,HPLC>95%.1H NMR(400MHz,CD3OD),δ7.78–7.64(m,2H),7.54(t,J=7.7,1H),7.11(dd,J=6.0,2.7,1H),7.03(t,J=8.7,1H),6.83(ddd,J=8.8,4.1,2.7,1H),5.16(dd,J=13.3,5.1,1H),4.50(s,2H),4.19(s,2H),3.81–3.44(m,17H),2.95–2.76(m,5H),2.51–2.39(m,1H),2.17(dtd,J=12.9,5.3,2.4,1H).13C NMR(100MHz,CD3OD),δ173.20,170.19,169.80,169.29,168.44,167.90,155.73,154.04,146.78,146.73,140.60,139.47,137.62,135.83,135.70,132.46,126.44,122.56,122.49,116.88,116.70,115.51,115.28,110.69,110.59,110.12,109.89,107.51,107.29,70.22,70.16,70.09,70.06,69.85,69.23,68.46,42.71,41.87,31.34,30.81,30.34,29.38,23.66,22.33,13.03.HR-MS Calcd.for C34H41BrFN9O10[M+H]+:834.2144,Found:834.2029.
化合物19的合成:
将化合物17(2mmol)溶解在DMF(10mL)中,加入化合物11(3mmol)和三乙胺(202mg,2mmol)。将混合物在90℃下搅拌过夜。浓缩除去DMF,将残余物重新溶解在乙酸乙酯中,并用饱和盐水洗涤。浓缩有机层,柱层析纯化(DCM/MeOH=100:1),得到化合物18。然后,在DIPEA存在下,在二氯甲烷中,用过量的甲苯磺酰氯处理化合物18。在室温搅拌过夜后,混合物分别用1M HCl,饱和碳酸氢钠和饱和盐水洗涤。浓缩除去溶剂,得到化合物19。
Pom-8PEG:Yield 725.32mg,58%,m.p.113.1–116.9℃,HPLC>95%.1H NMR(400MHz,DMSO-d6),δ11.08(s,1H),7.58(dd,J=8.6,7.1,1H),7.15(d,J=8.6,1H),7.04(d,J=7.0,1H),6.60(t,J=5.8,1H),5.06(dd,J=12.9,5.4,1H),4.55(t,J=5.4,1H),3.70–3.41(m,32H),2.89(ddd,J=17.4,14.0,5.4,1H),2.68–2.49(m,2H),2.04(ddt,J=13.0,5.6,3.3,1H).13C NMR(100MHz,DMSO-d6),δ173.24,170.50,169.41,167.76,146.90,136.69,132.57,117.92,111.14,109.73,72.81,70.32,70.28,70.25,69.96,69.37,60.69,55.37,49.05,42.20,31.45,22.62.MS(ESI),HR-MS Calcd.for C29H43N3O12[M+H]+:626.2847,Found:626.2947.
19a:Yield 688.9mg,57%.1H NMR(400MHz,DMSO-d6),δ11.09(s,1H),7.84–7.71(m,2H),7.58(dd,J=8.6,7.1,1H),7.47(d,J=8.1,2H),7.21–6.99(m,2H),6.59(s,1H),5.05(dd,J=12.9,5.4,1H),4.10(dd,J=5.5,3.3,2H),3.75–3.42(m,14H),2.96–2.82(m,1H),2.64–2.52(m,2H),2.41(s,3H),2.06–1.96(m,1H).MS(ESI),Calcd.for C28H33N3O10S[M+H]+:604.2,Found:604.3.
19b:Yield 637mg,46%.1H NMR(400MHz,DMSO-d6),δ11.09(s,1H),7.87–7.72(m,2H),7.59(dd,J=8.5,7.0,1H),7.48(d,J=8.0,2H),7.15(d,J=8.6,1H),7.04(d,J=7.0,1H),6.60(t,J=5.9,1H),5.06(dd,J=12.9,5.4,1H),4.18–4.02(m,2H),3.71–3.43(m,22H),2.89(ddd,J=17.3,14.0,5.4,1H),2.66–2.52(m,2H),2.42(s,3H),2.10–1.96(m,1H).MS(ESI),Calcd.for C32H41N3O12S[M+H]+:692.3,Found:692.4.
19c:Yield 639.85mg,41%.1H NMR(400MHz,DMSO-d6),δ11.08(s,1H),7.86–7.72(m,2H),7.59(dd,J=8.6,7.1,1H),7.48(d,J=8.0,2H),7.15(d,J=8.6,1H),7.04(d,J=7.0,1H),6.60(t,J=5.8,1H),5.05(dd,J=12.9,5.4,1H),4.10(dd,J=5.6,3.1,2H),3.66–3.47(m,30H),2.94–2.83(m,1H),2.55(s,2H),2.42(s,3H),2.09–1.96(m,1H).MS(ESI),Calcd.for C36H49N3O14S[M+H]+:780.3,Found:780.3.
19d:Yield 780.66mg,45%.1H NMR(400MHz,DMSO-d6),δ11.09(s,1H),7.84–7.75(m,2H),7.59(dd,J=8.5,7.1,1H),7.48(d,J=8.0,2H),7.15(d,J=8.6,1H),7.05(d,J=7.0,1H),6.60(t,J=5.8,1H),5.06(dd,J=12.9,5.4,1H),4.12(s,2H),3.66–3.49(m,38H),2.89(ddd,J=17.5,14.1,5.4,1H),2.64–2.53(m,2H),2.42(s,3H),2.07–1.98(m,1H).MS(ESI),Calcd.for C40H57N3O16S[M+H]+:867.3,Found:867.5.
19c-CH3:Yield 762.53mg,48%.1H NMR(400MHz,DMSO-d6),δ7.88–7.71(m,2H),7.59(dd,J=8.5,7.1,1H),7.48(d,J=8.0,2H),7.15(d,J=8.6,1H),7.04(d,J=7.0,1H),6.60(t,J=5.8,1H),5.05(dd,J=12.9,5.4,1H),4.11(dd,J=5.6,3.1,2H),3.66–3.47(m,30H),3.05–2.99(s,3H),2.94–2.83(m,1H),2.55(s,2H),2.42(s,3H),2.09–1.96(m,1H).MS(ESI),Calcd.for C37H51N3O14S[M+H]+:794.3,Found:794.3.
化合物2的合成:
将化合物19(0.2mmol)溶解在无水DMF(3mL)中,加入化合物14(71.6mg,0.2mmol)和三乙胺(20.2mg,0.2mmol)。将混合物在60℃下搅拌过夜。浓缩除去DMF,柱层析纯化(DCM/MeOH/NH 3·H 2O=100:5:0.5),得到化合物2。
2a:Yield 71.12mg,45%,m.p.147.7–151.1℃,HPLC>95%.1H NMR(400MHz,DMSO-d6),δ11.48(s,1H),8.85(s,1H),7.62–7.45(m,1H),7.29–6.96(m,4H),6.77(ddd,J=8.8,4.1,2.7,1H),6.59(t,J=5.8,1H),6.19(d,J=5.1,1H),5.05(dd,J=12.9,5.4,1H),4.32(q,J=7.1,0.5H),4.03(q,J=7.1,0.5H),3.72–3.39(m,12H),3.27(d,J=5.4,2H),2.88(ddd,J=17.7,13.8,5.3,1H),2.73(d,J=27.6,4H),2.51(d,J=1.9,3H),2.03(s,1H).13CNMR(100MHz,DMSO-d6),δ173.26,170.53,169.41,167.76,156.15,146.89,140.38,139.82,138.52,136.69,132.57,129.54,129.21,125.22,121.97,117.91,116.51,111.16,109.74,70.26,70.10,69.36,49.04,48.67,47.73,31.45.HR-MS Calcd.for C32H37N9O9[M+H]+:790.1882,Found:790.2073.
2b:Yield 80.79mg,46%,m.p.138.2–141.5℃,HPLC>95%.1H NMR(400MHz,CD3OD),δ7.52(dd,J=8.6,7.1,1H),7.12(dd,J=6.0,2.7,1H),7.08–6.98(m,3H),6.85(ddd,J=8.8,4.1,2.7,1H),5.03(dd,J=12.4,5.5,0.5H),4.80–4.69(m,0.5H),3.70(t,J=5.3,2H),3.66–3.54(m,18H),3.47(t,J=5.6,4H),2.96(t,J=6.1,2H),2.91–2.63(m,5H),2.09(ddd,J=12.6,5.1,2.8,1H).13C NMR(100MHz,CD3OD),δ173.28,170.19,169.30,167.90,155.73,154.04,146.73,140.60,139.47,137.62,135.83,135.70,132.46,126.44,122.56,122.49,116.88,116.70,115.51,110.69,110.59,109.89,107.51,107.29,70.23,70.16,70.09,70.06,69.85,69,23,68.46,42.72,41.87,30.81,30.34,22.34.HR-MSCalcd.for C36H45BrFN9O11[M+H]+:878.2414,Found:878.2639.
2c:Yield 71.51mg,37%,m.p.123.1–126.5℃,HPLC>95%.1H NMR(400MHz,CD3OD),δ7.53(dd,J=8.6,7.0,1H),7.18–6.99(m,4H),6.85(ddd,J=8.8,4.1,2.7,1H),5.04(dd,J=12.5,5.5,0.5H),4.74(t,J=7.8,0.5H),3.72–3.43(m,32H),2.96–2.60(m,7H),2.10(ddd,J=12.6,5.2,2.8,1H).13C NMR(100MHz,CD3OD),δ173.27,170.16,169.28,167.91,155.73,146.82,140.58,139.47,137.66,135.82,135.68,132.49,126.39,122.51,122.44,116.89,116.70,115.51,115.27,110.65,110.55,109.91,107.50,107.28,70.26,70.23,70.21,70.16,70.13,70.10,69.89,69.25,69.12,43.17,41.89,30.81,29.34,22.34.HR-MS Calcd.for C40H53BrFN9O13[M+H]+:966.2930,,Found:966.3238.
2d:Yield 69.58mg,33%,m.p.93.1–96.4℃,HPLC>95%.1H NMR(400MHz,CD3OD),δ7.53(ddd,J=9.8,7.1,2.8,1H),7.19–6.99(m,4H),6.85(ddd,J=8.7,4.1,2.7,1H),5.04(dd,J=12.5,5.5,0.5H),4.75(d,J=7.8,0.5H),3.71(t,J=5.3,2H),3.66–3.57(m,34H),3.46(dt,J=17.3,5.7,4H),2.98–2.59(m,7H),2.10(ddd,J=12.6,5.1,2.8,1H).13C NMR(100MHz,CD3OD),δ173.26,167.90,155.73,146.82,140.57,139.48,137.67,135.83,135.69,132.49,126.37,122.50,122.43,116.90,116.70,115.52,110.66,110.55,109.91,107.51,107.28,70.27,70.23,70.21,70.17,70.14,70.13,70.11,69.89,69.24,69.09,43.15,41.90,30.82,30.34,29.35,22.34.HR-MS Calcd.for C44H61BrFN9O15[M+H]+:1054.3455,Found:1054.3734.
2c-CH3:Yield 80.38mg,41%,m.p.132.7–135.4℃,HPLC>95%.1H NMR(400MHz,DMSO-d6),δ7.59(dd,J=8.6,7.1,1H),7.21–7.10(m,3H),7.05(d,J=7.1,1H),6.79(dt,J=8.9,3.4,1H),5.13(dd,J=13.0,5.4,1H),3.63(t,J=5.4,2H),3.53–3.40(m,28H),3.27(t,J=6.1,2H),3.03(s,3H),2.99–2.89(m,1H),2.77(dt,J=12.2,3.9,3H),2.68(t,J=5.7,2H),2.57(td,J=13.3,4.6,1H),2.11–2.01(m,1H).13C NMR(100MHz,DMSO-d6),δ172.24,170.78,170.25,169.38,167.72,156,16,156.10,155.41,153.03,1146.82,140.33,139.71,138.41,138.38,136.69,132.55,125.25,121.92,121.85,117.89,116.25,111.15,109.72,109.69,107.59,107.37,70.46,70.31,70.25,70.09,69.35,60.21,55.34,49.61,48.71,47.79,44.18,44.08,42.19,42.09,31.57,27.04,21.85,21.76,21.19,14.53.HR-MS Calcd.for C41H55BrFN9O13[M+H]+:980.3087,Found:980.3167.
实施例2式1a,1b,1c,2a,2b,2c,2d所示PROTAC化合物在蛋白免疫印迹实验水平的生物活性测试
细胞处理:取对数生长期Hela细胞于6孔板中,使用IFN-γ诱导IDO1表达上调,并同时使用实施例PROTAC化合物处理;孵育相应时间后收细胞。
细胞全蛋白抽提:
收集细胞:将处理后的细胞于培养基中刮下,充分混悬后300g离心5分钟收集,PBS洗一遍后,弃去PBS。
裂解细胞:每一样品加入100μL的2×Loading Buffer,充分震荡混匀,100℃变性5分钟,混匀后于-20℃保存或直接用于Western Blot检测。5×Loading Buffer的配方为:250mM Tris-HCl(pH6.8),10%(W/V)SDS,0.5%(W/V)溴酚蓝,50%(V/V)甘油,5%(W/V)β-巯基乙醇(2-ME)。2×Loading Buffer的制备是将1.5倍体积的dd水加入到5×LoadingBuffer中即得。
蛋白免疫印迹实验(Western Blot,WB)检测的具体步骤如下:
1)配制合适浓度的SDS-PAGE胶:分离胶的浓度为10%,浓缩胶的浓度为5%。
2)制备样品:根据实验要求制备蛋白样品,离心、混匀并上样于SDS-PAGE胶上样孔中。根据蛋白定量结果适量调整上样体积。
3)电泳:接通电源,蛋白样品在浓缩胶中电压为80伏特,待蛋白样品进入分离胶时,把电压调整为120伏特继续电泳。待溴酚蓝几乎完全跑出PAGE胶时终止电泳。
4)转膜:电泳结束后取下凝胶,按下列顺序安装转膜装置:(负极)、滤纸、凝胶、活化的PVDF膜、滤纸、(正极)。然后夹紧转移装置置于转膜缓冲液中,最后放入冰盒,置于4℃冷库100V恒压通电40分钟。
5)封闭:转膜结束后,取出PVDF膜,将膜浸没在含5%的脱脂奶粉的TBST缓冲液里,室温下摇床振荡1小时。
6)一抗孵育:封闭结束后,用TBST缓冲液荡洗3次,然后加入适度稀释比例的一抗,4℃过夜。将PVDF膜用TBST缓冲液荡洗3次,每次振荡5分钟。
7)二抗孵育:弃去TBST缓冲液,加入稀释的二抗,室温下摇床振荡1小时。弃去二抗,将PVDF膜用TBST缓冲液荡洗3次,每次振荡5分钟。
8)曝光:将ECL显色底物均匀覆盖在PVDF膜上,曝光成像。
本发明实施例的化合物对IDO1的降解活性如下:在Hela细胞系中,在蛋白免疫印迹实验(WB)结果中可以明显观察到式2c所示化合物对IDO1蛋白的降解作用,如图1所示(A:PROTAC化合物:10μmol/L,IFN-γ:5ng/mL,处理时间:24小时。B:IFN-γ:5ng/mL,处理时间24小时)。2c的半数降解浓度为2.84μmol/L,IDO1的最大降解率为93%(图1C)。
在Rescue实验中,蛋白免疫印迹实验(WB)结果中可以明显观察到5μmol/L的对照化合物Epa-8PEG,Pom-8PEG(5),Epacadostat(Epa),2c-CH3处理均不能引起IDO1蛋白水平下降(图2A:IFN-γ:5ng/mL,处理时间24小时),式2c所示化合物可显著降低IDO1蛋白水平。使用IDO1蛋白竞争性抑制剂Epa,E3泛素连接酶竞争性抑制剂泊马渡胺(Pomalidomide,Pom),蛋白酶体通路抑制剂MG132和MLN4924预处理Hela细胞2小时显著影响式2c所示化合物对IDO1蛋白的降解作用,结果如图2B所示(IFN-γ:5ng/mL,式2c浓度:5μmol/L,Pom浓度:1μmol/L,Epa浓度:1μmol/L,MG132浓度:1μmol/L,MLN4929浓度:1μmol/L,处理时间:24小时)。
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CN112430229A (zh) * | 2020-11-24 | 2021-03-02 | 福建医科大学 | 一种靶向降解parp蛋白的化合物及其制备方法与应用 |
CN115650975A (zh) * | 2022-08-23 | 2023-01-31 | 四川大学华西医院 | 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 |
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Cited By (7)
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CN112430229A (zh) * | 2020-11-24 | 2021-03-02 | 福建医科大学 | 一种靶向降解parp蛋白的化合物及其制备方法与应用 |
CN112430229B (zh) * | 2020-11-24 | 2023-03-21 | 福建医科大学 | 一种靶向降解parp蛋白的化合物及其制备方法与应用 |
CN115650975A (zh) * | 2022-08-23 | 2023-01-31 | 四川大学华西医院 | 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 |
CN115974862A (zh) * | 2023-01-30 | 2023-04-18 | 四川大学华西医院 | 一种基于protac原理的hl化合物及其制备方法和应用 |
CN115974862B (zh) * | 2023-01-30 | 2024-04-19 | 四川大学华西医院 | 一种基于protac原理的hl化合物及其制备方法和应用 |
CN116444454A (zh) * | 2023-06-16 | 2023-07-18 | 中国医学科学院医药生物技术研究所 | N-羟基脒衍生物及制备方法和应用、肿瘤免疫治疗药物 |
CN116444454B (zh) * | 2023-06-16 | 2023-09-12 | 中国医学科学院医药生物技术研究所 | N-羟基脒衍生物及制备方法和应用、肿瘤免疫治疗药物 |
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