CN115466266A - mTOR蛋白降解靶向嵌合体及其制备方法和应用 - Google Patents
mTOR蛋白降解靶向嵌合体及其制备方法和应用 Download PDFInfo
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Abstract
本申请属于医药技术领域,具体涉及一种mTOR蛋白降解靶向嵌合体及其制备方法和应用。本发明的mTOR蛋白降解靶向嵌合体化合物表现出强大的mTOR降解能力和mTOR抑制能力并抑制MCF‑7的增殖。化合物P1可以减少mTOR下游蛋白p‑S6(Ser240/244)和p‑AKT(Ser473)的表达,进一步研究表明,我们的化合物通过诱导自噬抑制癌细胞生长,而对细胞凋亡或细胞周期没有影响。
Description
技术领域
本申请属于医药技术领域,具体涉及一种mTOR蛋白降解靶向嵌合体及其制备方法和应用。
背景技术
雷帕霉素机制靶蛋白(mechanistic target of rapamycin,mTOR)是一种丝氨酸/苏氨酸激酶,属于磷酸肌醇3-激酶相关激酶(PIKK)家族。mTOR复合物mTORC1和mTORC2作为营养、能量和氧化还原感受器,均参与调控PI3K/AKT/mTOR通路。PI3K/AKT下游mTORC1的激活,促进了蛋白质、脂质和核苷酸的产生,这有利于细胞的生存、生长和增殖;mTORC2通过调节蛋白激酶,包括AKT等,促进细胞的增殖和存活。研究发现,mTOR异常活跃在各种类型的癌症中,增加了细胞增殖和新陈代谢,从而促进肿瘤的启动和发展。
mTOR与致癌通路的密切关系使其成为肿瘤治疗的一个有效靶点。目前,mTOR抑制剂已被用于多种癌症的治疗,但仍存在潜在毒性的问题。高效mTOR选择性抑制剂MLN0128(也称为INK128,Sapaniertib,TAK-228)在各种人类肿瘤异种移植模型中显示出强大的体内外活性,已经对骨肉瘤、软组织肉瘤、乳腺癌和原发渗出性淋巴瘤等实体肿瘤进行了临床试验。然而,由于继发毒性导致剂量减少等原因,MLN0128在转移性去势抵抗性前列腺癌患者中的临床疗效有限。
蛋白质水解靶向嵌合体(PROTACs)是一种双功能分子,可通过泛素-蛋白酶体途径靶向蛋白质降解。从结构上看,PROTAC由两个功能部分组成,一端是高度特异性结合目的蛋白的“弹头”,另一端是E3连接酶识别的配体,二者通过连接子连接。目前,多种与恶性肿瘤有关的蛋白激酶作为PROTAC靶标均取得良好的效果,包括磷酸肌醇3-激酶(PI3K)、蛋白激酶B(AKT)、癌基因融合蛋白(BCR-ABL)、细胞周期蛋白依赖性激酶(CDK)、表皮生长因子受体(EGFR)、间变性淋巴瘤激酶(ALK)等。然而,该技术在靶向mTOR降解方面鲜有报道。
发明内容
本发明旨在至少在一定程度上解决相关技术中的技术问题之一。为此,本发明的目的在于提供一种mTOR蛋白降解靶向嵌合体及其制备方法和应用。
为此,本发明提出了mTOR蛋白降解靶向嵌合体,所述mTOR蛋白降解靶向嵌合体的结构式如下:
优选的,所述mTOR蛋白降解靶向嵌合体的结构式如下:
或者,优选的,所述mTOR蛋白降解靶向嵌合体的结构式如下:
或者,优选的,所述mTOR蛋白降解靶向嵌合体的结构式如下:
上述的mTOR蛋白降解靶向嵌合体的制备方法,包括如下步骤:
根据本发明的具体实施方式,本发明提出了mTOR蛋白降解靶向嵌合体在制备mTOR降解剂或者mTOR抑制剂中的应用。
根据本发明的具体实施方式,本发明还提出了mTOR蛋白降解靶向嵌合体在制备预防和/或治疗肿瘤药物中的应用。
根据本发明的具体实施方式,本发明还提出了mTOR蛋白降解靶向嵌合体在制备预防和/或治疗乳腺癌药物中的应用。
根据本发明的具体实施方式,本发明还提出了mTOR蛋白降解靶向嵌合体在制备诱导MCF-7细胞的自噬药物中的应用。优选化合物P1在制备诱导MCF-7细胞的自噬药物中的应用。
本发明所述的化合物在临床上的给药方式可以采用口服、注射等方式。
本发明的化合物临床所用剂量为0.01-1000mg/天,也可根据病情的轻重或剂型的不同偏离此范围。
另外,根据本发明上述具体实施方式提出的药物及其应用,还可以具有如下附加的技术特征:
一种药物组合物,含有权利要求1所述的mTOR蛋白降解靶向嵌合体和药学上可接受的辅料。
上述的组合物在制备mTOR抑制剂中的应用。
本发明所述药学上可接受的辅料,是指制备不同剂型时加入所需的各种常规辅料,例如稀释剂、黏合剂、崩解剂、助流剂、润滑剂、矫味剂、包合材料、吸附材料等以常规的制剂方法制备成任何一种常用的口服制剂,例如可以是颗粒剂、散剂、片剂、胶囊剂、丸剂、口服液、汤剂、滴丸剂等。
本发明的附加方面和优点将在下面的描述中部分给出,部分将从下面的描述中变得明显,或通过本发明的实践了解到。
本发明的有益效果:
药理实验结果表明:
(1)基于PROTAC的策略,合成了MLN0128与pomalidomide的偶联物并对其生物学机制进行了评价。化合物表现出强大的mTOR抑制能力并抑制MCF-7的增殖。化合物P1可以减少mTOR下游蛋白p-S6(Ser240/244)和p-AKT(Ser473)的表达,进一步研究表明,我们的化合物通过诱导自噬抑制癌细胞生长,而对细胞凋亡或细胞周期没有影响。
(2)通过新的合成路线合成了纯度95%以上目标化合物P1-P3。目标化合物的合成如路线1所示:以pyrazolo[3,4-d]pyrimidin-4-amine的卤代物为原料,通过Suzuki偶联反应和取代反应得到吡唑环的N-1位取代的MLN0128衍生物5,8,11,化合物8脱保护得到9。化合物11水解得到12。根据文献报道的方法,以pomalidomide13为原料,通过酰化、叠氮化和还原反应得到pomalidomide的叠氮衍生物15和氨基衍生物16。pomalidomide与Br-PEG3-t-butyl ester17发生取代反应得到化合物18。中间体5与叠氨化物15进行click反应,得到化合物P1。中间体12与16缩合得到化合物P2。中间体9与18缩合得到化合物P3。目标化合物P1-P3的纯度通过核磁和高压液相鉴定在95%以上。
(3)化合物对MCF-7细胞的mTOR蛋白降解实验结果表明,化合物P1能够诱导MCF-7细胞的mTOR蛋白降解,且化合物P1对mTOR蛋白的降解作用具有时间依赖性。
附图说明
图1为目标化合物P1-P3对人乳腺癌细胞MCF-7的抗细胞增殖活性图;
图2为不同浓度的P1对MCF-7细胞状态的影响图;
图3为化合物P1作用后的MCF-7细胞中mTOR下游蛋白p-S6(Ser240/244)和p-AKT(Ser473)的表达量变化图;
图4为化合物P1对MCF-7细胞周期的影响图;
图5为化合物P1对MCF-7细胞凋亡的影响图;
图6为化合物P1对MCF-7细胞自噬标志蛋白LC3A/B的影响图。
图7为化合物MLN0128,Pomalidomide,P1-P3作用后的MCF-7细胞中mTOR蛋白的表达量变化图。
图8为化合物MLN0128,P1作用后的MCF-7细胞中mTOR蛋白的表达量随时间变化图。
图9为化合物MLN0128,Pomalidomide,蛋白酶体抑制剂MG-132预处理后,又经化合物P1作用的MCF-7细胞的mTOR蛋白的表达量变化图。
具体实施方式
下面结合附图和具体实施方式来对本申请作更进一步的说明,以便本领域的技术人员更了解本申请,但这些实施例仅用于说明本发明而不用于限制本发明的范围。而且,除非另有说明,各方法步骤的编号仅为鉴别各方法步骤的便利工具,而非为限制各方法步骤的排列次序或限定本发明可实施的范围,其相对关系的改变或调整,在无实质变更技术内容的情况下,当亦视为本发明可实施的范畴。
为了更好的理解上述技术方案,下面更详细地描述本发明的示例性实施例。虽然显示了本发明的示例性实施例,然而应当理解,可以以各种形式实现本发明而不应被这里阐述的实施例所限制。相反,提供这些实施例是为了能够更透彻地理解本发明,并且能够将本发明的范围完整的传达给本领域的技术人员。
下列实施例中所述试验方法,如无特殊说明,均为常规方法;所述试剂和生物材料,如无特殊说明,均可从商业途径获得。
实施例1
4-氨基-3-溴-1H-吡唑并[3,4-d]嘧啶-1-羧酸叔丁酯(2)的制备
将化合物1(5g,23.3mmol),DMAP(0.28g,0.01mmol)溶于无水DCM(100mL)中,室温下一次性加入Boc2O(5.58g,25.63mmol),继续搅拌12小时,TLC跟踪检测(EA;PE=1:1,Rf=0.5)化合物1完全消失,反应液真空旋干,剩余物用60mL甲基叔丁基醚与乙酸乙酯混合液洗(MTBE/EA=5:1),得到白色固体化合物2(4.1g,13.06mmol产率:56.1%)。
实施例2
5-(4-氨基-1H-吡唑并[3,4-d]嘧啶-3-基)苯并[d]恶唑-2-胺(4)的制备
将化合物2(1g,3.18mmol),2-胺基苯并恶唑-5-硼酸酯(1.25g,4.78mmol),碳酸钠(0.67g,6.36mmol),Pd(pph3)4(0.3g,0.26mmol)溶于dioxane与水的混合溶液中(50ml,10:1),真空置换N2三次,加热到90℃,N2保护条件下反应12小时,TLC跟踪检测(EA,Rf=0.3)化合物2完全消失,冷却到室温,加入20mL水,EA萃取3次,每次10ml,有机相饱和食盐水洗一次,无水硫酸钠干燥,过滤,旋干得到粗品。粗品用EA(10mL)洗涤得到灰色化合物4(0.48g,1.80mmol,产率:56.6%)。1H NMR(400MHz,DMSO)δ8.18(s,1H),7.57(s,2H),7.46(d,J=8.1Hz,1H),7.41(s,1H),7.23(d,J=8.1Hz,1H).
实施例3
5-(4-氨基-1-(丙-2-yn-1-基)-1H-吡唑[3,4-d]嘧啶-3-基)苯并[d]恶唑-2-胺(5)的制备
将化合物4(0.45g,1.68mmol),碳酸钠(0.35g,3.37mmol)溶于无水DMF(10mL)中,一次性加入将化合物3-bromoprop-1-yne(0.80g,6.74mmol),室温反应12小时,LCMS检测化合物4反应完全,加入20ml水,EA萃取3次,有机相饱和食盐水反洗一次,无水硫酸钠干燥,旋干,得到粗品化合物5(0.1g,纯度约73%)。1H NMR(400MHz,DMSO)δ8.28(s,1H),7.57(s,2H),7.48(d,J=8.0Hz,1H),7.41(s,1H),7.25(d,J=8.1Hz,1H),5.19(s,2H),3.39(s,1H).
实施例4
叔丁基(4-(4-氨基-3-碘-1H-吡唑并[3,4-d]嘧啶-1-基)氨基甲酸酯(7)的制备
将化合物6(0.52g,1.9mmol),tert-butyl(4-bromobutyl)carbamate(0.50g,1.9mmol)和碳酸钾(0.36g,2mmol)置于反应瓶中,加入二甲基乙酰胺DMA(4ml),25℃反应3小时,TLC检测反应(EA,RF=0.39),加入50ml水,MTBE(10ml*3),合并有机相,食盐水反洗,干燥,旋干得到化合物0.38g粉末状固体,固体用MTBE洗,得到0.25g粗品化合物7。
实施例5
叔丁基(4-(4-氨基-3-(2-氨基苯并[d]恶唑-5-基)-1H-吡唑基[3,4-d]嘧啶-1-基)氨基甲酸酯(8)的制备
将化合物7(0.24g,0.56mmol)、2-胺基苯并恶唑-5-硼酸酯(0.22g,0.83mmol)和碳酸钠(0.15g,1.1mmol)置于装有DME6ml/水3ml的50ml单口瓶中,再加入四三苯基磷钯(0.1g,0.087mmol),氮气置换3次,加热到110℃,反应3小时,TLC检测反应(EA,Rf=0.36)显示化合物7完全消失,冷却到室温,反应液中加入20mlEA,分液,水相用EA10ml萃取2次,合并有机相,饱和食盐水反洗一次,干燥,过滤,滤液旋干得到粗品。粗品经过薄层板制备纯化得到0.12g白色固体化合物8(产率:48.9%)。1H NMR(600MHz,DMSO)δ8.23(s,1H),7.55(s,2H),7.46(d,J=8.1Hz,1H),7.40(d,J=1.3Hz,1H),7.23(dd,J=8.1,1.5Hz,1H),6.80(t,J=5.6Hz,1H),4.32(t,J=6.9Hz,2H),2.96–2.90(m,2H),1.86–1.78(m,2H),1.38–1.29(m,11H).ESI-MS m/z C21H26N8O3[M+H]+:439.10.
实施例6
5-(4-氨基-1-(4-氨基丁基)-1H-吡唑[3,4-d]嘧啶-3-基)苯并[d]恶唑-2-胺(9)的制备
将化合物8(0.1g,0.23mmol)溶于2ml无水DCM中,室温下再加入4N/L的盐酸气乙酸乙酯溶液1ml,25度反应12小时,TLC检测反应(EA,RF=0.36)显示化合物8完全消失,旋干得到白色固体化合物9(0.13g)直接投下一步。
实施例6
叔丁基3-(2-(4-氨基-3-碘-1H-吡唑并[3,4-d]嘧啶-1-基)乙氧基)丙酸酯(10)的制备
将化合物6(0.26g,1mmol),化合物tert-butyl 3-(2-(2-bromoethoxy)ethoxy)propanoate(0.25g,0.85mmol)和碳酸钾(0.36g,2mmol)置于反应瓶中,加入DMA(4ml),加热到80℃反应3小时,TLC检测反应(EA,RF=0.4),冷却到室温,加入50ml水,MTBE(10ml*3),合并有机相,食盐水反洗,干燥,旋干得到化合物0.38g粉末状固体,固体用MTBE重结晶得到类黄色固体0.25g化合物10(产率:62%)。1H NMR(600MHz,DMSO)δ8.19(s,1H),4.41(t,J=5.5Hz,2H),3.81(t,J=5.6Hz,2H),3.51–3.43(m,4H),3.40–3.36(m,2H),3.34(s,2H),2.34(t,J=6.1Hz,2H),1.36(s,9H).
实施例8
叔丁基3-(2-(4-氨基-3-(2-氨基苯并[d]恶唑-5-基)-1H-吡唑基[3,4-d]嘧啶-1-基)乙氧基)丙酸酯(11)的制备
将化合物10(1.4g,2.9mmol)、2-胺基苯并恶唑-5-硼酸酯(1.4g,5.38mmol)和碳酸钠(1.6g,15mmol)置于装有DME18ml/水9ml的单口瓶中,再加入四三苯基磷钯(0.3g,0.26mmol),氮气置换3次,加热到110℃,反应3小时,TLC检测反应(EA,Rf=0.32)显示化合物10完全消失,冷却到室温,反应液中加入20mlEA,分液,水相用EA10ml萃取2次,合并有机相,饱和食盐水反洗一次,干燥,过滤,滤液旋干得到粗品。粗品经过薄层板制备纯化得到0.5g白色固体粉末化合物11(产率:35%)。1H NMR(600MHz,DMSO)δ8.24(s,1H),7.54(s,2H),7.47(d,J=8.1Hz,1H),7.41(d,J=1.5Hz,1H),7.24(dd,J=8.1,1.7Hz,1H),4.47(t,J=5.7Hz,2H),3.88(t,J=5.7Hz,2H),3.53–3.46(m,4H),3.43–3.40(m,2H),2.32(t,J=6.2Hz,2H),1.35(s,9H).ESI-MS m/z C23H29N7O5[M+H]+:484.10,[M+Na]+:506.10.
实施例9
3-(2-(2-(4-氨基-3-(2-氨基苯并[d]恶唑-5-基)-1H-吡唑基[3,4-d]嘧啶-1-基)乙氧基)丙酸(12)的制备
将化合物11(0.2g,0.41mmol)置于反应瓶中,加入2mlTFA及催化量的Et3SiH,25度反应12小时,TLC检测反应(PE:EA=1:1,Rf=0.5)显示化合物11完全消失,旋干反应液,加入MTBE(5ml),PE(10ml),搅拌1小时,有固体析出,倒出上清液,再加入加入MTBE(5ml),PE(10ml),搅拌1小时,过滤得到黄色固体化合物粗品12(0.12g,产率:70%),直接投下步。
实施例10
6-溴-N-(2-(2,6-二氧哌啶-3-基)-1,3-二氧异喹啉-4-基)己酰胺(14)的制备
将化合物13(5g,18.3mmol)与化合物6-bromohexanoyl chloride(3.9g,18.3mmol)溶于绝对无水THF(80mL)中,N2保护条件下,加热回流2小时,TLC跟踪检测(EA,Rf=0.5)化合物13完全消失.反应液真空旋干,剩余物用EA重结晶得到白色固体化合物14(6.5g,产率:78%)。1H NMR(400MHz,CDCl3)δ9.41(s,1H),8.81(d,J=8.5Hz,1H),8.40(s,1H),7.71(t,J=7.9Hz,1H),7.54(d,J=7.3Hz,1H),5.08–4.82(m,1H),3.41(t,J=6.5Hz,2H),2.97–2.85(m,1H),2.83–2.70(m,2H),2.48(t,J=7.4Hz,2H),2.24–2.13(m,1H),1.96–1.85(m,2H),1.82–1.73(m,2H),1.59–1.47(m,2H).ESI-MS m/z C19H20BrN3O5[M-H]-:447.90,449.80,.
实施例11
6-叠氮-N-(2-(2,6-二氧哌啶-3-基)-1,3-二氧异喹啉-4-基)己酰胺(15)的制备
将化合物14(3.46g,7.69mmol)溶于无水DMF(60mL),室温下一次性加入NaN3(1g,15.3moL),N2保护条件下,加热到80℃继续反应12小时,TLC跟踪检测(EA:PE=1:1,Rf=0.5)化合物14完全消失.反应液冷却到室温,加入200ml水,EA萃取3次,每次用100mL,EA相再用饱和食盐水(100mL)反洗,无水硫酸钠干燥,过滤,真空旋干,剩余物加入MTBE重结晶得到白色固体化合物15(3.15g,产率:99.4%),经核磁确认。1H NMR(400MHz,CDCl3)δ9.41(s,1H),8.81(d,J=8.5Hz,1H),8.46(s,1H),7.70(t,J=7.9Hz,1H),7.54(d,J=7.3Hz,1H),5.01–4.88(m,1H),3.28(t,J=6.9Hz,2H),2.94–2.84(m,1H),2.83–2.74(m,2H),2.47(t,J=7.4Hz,2H),2.19–2.12(m,1H),1.82–1.73(m,2H),1.68–1.61(m,2H),1.51–1.44(m,2H).ESI-MS m/z C19H20N6O5[M-H]-:411.10.
实施例12
6-氨基-N-(2-(2,6-二氧哌啶-3-基)-1,3-二氧异喹啉-4-基)己酰胺(16)的制备
将化合物15(1.24g,3mmol)和三苯基膦(1.18g,4.5mmol)溶于15mL THF/1M(4:1)中的溶液中,在室温下搅拌4小时,旋转蒸发除去THF,加乙酸乙酯萃取,保留水层浓缩,得到粗品16(0.6g,产率:52%)。ESI-MS m/zC19H22N4O5[M+H]+:387.10.
实施例13
3-(2-(2-(2,6-二氧哌啶-3-基)-1,3-二氧异吲哚啉-4-基)氨基)乙氧基)乙氧基)丙酸(18)的制备
将化合物13(0.15g,0.55mmol)、溴代-三聚乙二醇-丙酸叔丁酯17(0.22g,0.65mmol)和K2CO3(0.09g,0.65mmol)溶于无水DMF(3ml)中,75℃下加热24小时。冷却到室温,加入50ml水,EA萃取3次,有机相饱和食盐水反洗一次,无水硫酸钠干燥,旋干得到粉末状固体。粗品溶于CH2Cl2(5mL)溶液中,加入TFA(3mL),室温下搅拌2小时,旋干后加水稀释,乙酸乙酯洗,MTBE洗,水相冻干得到化合物18(0.12g,产率:46%)。
实施例14
6-(4-((4-氨基-3-(2-氨基苯并[d]恶唑-5-基)-1H-吡唑[3,4-d]嘧啶-1-基)甲基)-1H-1,2,3-三唑-1-基)-N-(2-(2,6-二氧哌啶-3-基)-1,3-二氧异吲哚-4-基)己酰胺(P1)的制备
将化合物5(90mg,0.295mmol),化合物15(121mg,0.295mmol),CuSO4.5H2O(49mg,0.20mmol),抗坏血酸钠(5.8mg,0.03mmol)溶于THF与水的混合溶液中(5ml,10:1),室温搅拌12小时,LCMS检测反应完全,加入20ml水,EA萃取3次,有机相饱和食盐水反洗一次,无水硫酸钠干燥,旋干,经制备液相提纯(cloumn:Phenomenex Luna 80*30mm*3um;mobilephase:(water-CAN);B%:15%-45%,8min),冻干得到白色固体化合物P1(60.2mg,产率:28.4%)。1H NMR(600MHz,DMSO-d6)δ11.15(s,1H),9.70(s,1H),9.29(s,1H),8.62(s,1H),8.51(s,1H),8.44(d,J=8.4Hz,1H),8.19(s,1H),7.85–7.80(m,1H),7.61(t,J=8.0Hz,2H),7.50(s,1H),7.34(d,J=8.2Hz,1H),5.70(s,2H),5.14(dd,J=12.9,5.4Hz,2H),4.33(t,J=7.1Hz,2H),2.95–2.84(m,1H),2.61(dd,J=14.2,2.8Hz,1H),2.57–2.51(m,1H),2.44(t,J=7.4Hz,2H),2.10–2.02(m,1H),1.88–1.79(m,2H),1.67–1.59(m,2H),1.33–1.25(m,2H).13C NMR(151MHz,DMSO-d6)δ172.79,171.86,169.81,167.62,166.68,152.72,151.57,147.87,146.85,141.46,136.47,136.10,131.48,127.21,126.42,124.01,121.85,118.37,117.11,114.28,109.96,96.80,49.28,48.90,42.50,36.17,30.94,29.41,25.35,24.10,21.99,0.15,0.12.LC/MS(ESI)calcd for C34H32N13O6[M+H]+m/z718.2599,found718.2662.
实施例15
6-(3-(2-(4-氨基-3-(2-氨基苯并[d]恶唑-5-基)-1H-吡唑并[3,4-d]嘧啶-1-基)乙氧基)丙胺基)-N-(2-(2,6-二氧哌啶-3-基)-1,3-二氧异喹啉-4-基)己酰胺(P2)的制备
将化合物12(60mg,0.14mmol),HATU(79.8mg,0.21mmol),TEA(29mg,0.28mmol)和化合物16(54mg,0.14mmol)溶于3ml无水DMF中,25度反应12小时,LCMS显示反应完全,反应液直接高压液相制备(cloumn:Phenomenex Luna 80*30mm*3um;mobile phase:(water(HCl)-CAN);B%:15%-45%,8min),冻干得到白色固体化合物P2(48mg,产率:43.1%)。1HNMR(600MHz,DMSO)δ11.15(s,1H),9.69(s,1H),8.70(s,2H),8.59(s,1H),8.45(d,J=8.3Hz,1H),7.86–7.78(m,2H),7.62(dd,J=14.0,7.6Hz,2H),7.53(s,1H),7.38(d,J=8.0Hz,1H),5.17–5.12(m,1H),4.57(s,2H),3.91(t,J=4.7Hz,2H),3.53–3.46(m,4H),3.39(s,2H),3.04–2.98(m,2H),2.95–2.82(m,4H),2.61(d,J=16.2Hz,1H),2.45(t,J=7.3Hz,2H),2.22(t,J=6.2Hz,2H),2.10–2.04(m,1H),1.64–1.56(m,2H),1.42–1.36(m,2H),1.34–1.27(m,2H).13C NMR(151MHz,DMSO)δ172.80,171.97,169.82,169.77,167.69,166.68,161.94,160.86,152.46,151.94,147.61,147.23,146.61,136.53,136.12,131.46,127.40,126.30,122.08,118.32,117.01,114.09,110.15,96.60,69.42,69.36,67.88,66.85,48.92,46.94,38.28,36.47,36.12,30.95,28.86,25.93,24.50,22.00.LC/MS(ESI)calcdfor C38H41N11O9[M+H]+m/z796.3197,found 796.3235.
实施例16
N-(4-(4-氨基-3-(2-氨基苯并[d]恶唑-5-基)-1H-吡唑基[3,4-d]嘧啶-1-基)丁基)-3-(2-(2-(2-(2-二氧哌啶-3-基)-1,3-二氧异喹啉-4-基)乙氧基)丙酰胺(P3)的制备
将化合物9(0.1g,0.29mmol),HATU(168mg,0.44mmol),TEA(58.6mg,0.58mmol)和化合物18(138mg,0.29mmol)溶于3ml无水DMF中,25度反应12小时,LCMS显示反应完全,反应液直接高压液相制备(cloumn:Phenomenex Luna 80*30mm*3um;mobile phase:(water(HCl)-CAN);B%:10%-40%,8min),冻干得到白色固体化合物P3(90mg,产率:38.9%)。1HNMR(600MHz,DMSO)δ11.09(s,1H),9.77–9.43(m,4H),8.62(s,1H),8.20(s,1H),7.93(t,J=5.4Hz,1H),7.71(d,J=8.3Hz,1H),7.59–7.54(m,2H),7.45(dd,J=8.3,1.4Hz,1H),7.12(d,J=8.6Hz,1H),7.01(d,J=7.0Hz,1H),5.06–5.02(m,1H),4.41(t,J=6.4Hz,2H),3.60(t,J=5.4Hz,2H),3.55–3.52(m,4H),3.50–3.47(m,2H),3.46–3.43(m,4H),3.43–3.40(m,2H),3.07–3.02(m,2H),2.91–2.83(m,1H),2.61–2.54(m,2H),2.26(t,J=6.4Hz,2H),2.04–1.99(m,1H),1.88–1.82(m,2H),1.41–1.34(m,2H).13C NMR(151MHz,DMSO)δ172.94,170.18,170.15,169.00,167.38,160.74,152.36,151.37,146.81,146.67,146.46,146.26,136.34,135.40,132.12,128.02,123.30,117.55,113.42,110.92,110.77,109.26,96.67,69.83,69.80,69.58,68.94,66.91,48.63,46.95,41.77,37.80,36.21,31.06,26.43,26.20,22.23.LC/MS(ESI)calcd for C38H44N11O9[M+H]+m/z798.3323,found 798.3370.
实施例17
4.2.分子对接
分子对接实验使用SYBYL-X 2.0(Tripos)的Surflex-Dock模块完成。选用mTOR晶体结构4JT5作为对接受体。通过移除水分子、加氢和残基修复进行蛋白准备,选用AMBER7FF99力场对蛋白进行能量优化。根据原始配体生成对接口袋,将准备好的配体对接到口袋中。使用PyMOL软件或SYBYL得到用于描述结合模式的图像。
实验例生物实验部分
实验例1细胞增殖抑制试验
人乳腺癌细胞MCF-7使用含10%胎牛血清(FBS)的DMEM培养基,在37℃、5% CO2的细胞孵箱中培养。将细胞接种到96孔板中,每孔4000个细胞(190μL),置CO2细胞孵箱中,待细胞贴壁生长过夜后,加入不同浓度的化合物(10μL),每个浓度设定三个复孔,并设定相应浓度DMSO对照孔,化合物作用68h后,加入20μL 5mg/mL的MTT,4h后完全吸去培养液,注意不要吸掉孔板底部的甲瓒颗粒,加100μLDMSO溶解甲瓒颗粒,振荡器振荡2-5min,酶标仪490nm检测OD值。数据统计分析。
实验例2激酶活性及选择性实验
mTOR激酶抑制活性由KinaseProfilerTMService Assay测定。mTOR(h)与50mMHEPES pH 7.5,1mM EGTA,0.01% Tween 20,2mg/mL substrate,3mM MnCl2 and[γ-33P-ATP](浓度约500cpm/pmol)共同孵育。反应由MnATP的混合加入引发。室温孵育40分钟后,加入3%磷酸溶液停止反应。取10μL的反应液滴在P30过滤器垫上,用75mM的磷酸洗三次,每次5分钟,甲醇干燥,闪烁计数仪测定。
实验例3蛋白印迹(Western Blot)实验
细胞接种于六孔盘中,待细胞贴壁后不同时间给药,加药处理完毕后,加入RIPA裂解液收集细胞,提取蛋白,用BCA蛋白浓度测定试剂盒测蛋白浓度,取等浓度蛋白样品加入溴酚蓝裂解液处理,沸水浴变性15min。制备10% SDS凝胶,配制完毕后,将煮好的蛋白离心,上样,加样完毕,选择恒压75V电压电泳45min。当染料前沿进入分离胶后,调整电压为120V继续电泳直至溴酚蓝染料前沿下至凝胶末端处,即停止电泳。电泳结束后,取出凝胶,切除浓缩胶。转膜,PVDF膜用甲醇浸泡活化,按照顺序由阴极至阳极平放三张滤纸-凝胶-PVDF膜-三张滤纸,放入转膜槽中,在槽的一边放置冰盒降温,转膜槽加满transferbuffer,放入4℃冰箱内,连接好电极,接通电流,200mA转膜1h。转膜结束后,切膜、洗膜后,将膜用5%的脱脂牛奶封闭1h,将膜取出,洗膜略晾干放入杂交袋中,两边封口,分别加入p-S6(Ser240/244)一抗、p-AKT(Ser473)一抗、LC3A/B一抗和β-actin一抗,4℃摇床放置过夜。洗膜后加入二抗稀释液,室温下孵育30min,洗后显影。观察分析蛋白表达变化的关系。
实验例4流式细胞术实验
细胞接种于六孔板中,待细胞贴壁后不同浓度给药,24h后收集细胞,PBS洗,75%的乙醇固定。PBS洗掉乙醇后,每管加100μL RNase A,37℃水浴30min,加入400μL PI染色剂染色,4℃避光30min,上机检测。
将不同浓度给药处理的细胞收集,PBS洗后,加入500μL的Binding Buffer悬浮细胞,加入5μL Anneexin V-FITC混匀后,加入5μL Propidium Iodide,混匀。室温、避光反应5-15min。在一小时之内,上机检测。
实验例结果:
1.激酶活性mTOR kinase inhibition
为了评估合成的化合物与mTOR的结合力,进行了激酶活性测试。化合物P1-P3对mTOR的抑制率见表1.化合物P1-P3均对mTOR有抑制活性,证实了我们的假设,说明在MLN0128吡唑环的N-1位引入基团对于mTOR结合力的影响较小,是Linker的合适连接位置。化合物P1对mTOR的抑制活性最好,IC50为0.087μM,linker延长后的化合物P2和P3活性均有不同程度的下降,说明PROTAC分子的linker不宜过长。
表1化合物对mTOR抑制活性
2.抗肿瘤细胞增殖活性Anti-proliferative activity
采用MTT比色法评价了目标化合物P1-P3对人乳腺癌细胞MCF-7的抗细胞增殖活性,实验结果(见图1)显示:P1-P3的抗增殖活性均呈现出浓度依赖性,且在25μM下的抑制率(处理72h)达到90%。观察不同浓度的P1对MCF-7细胞状态的影响,发现与DMSO对照组相比,用10μM P1处理6h后细胞状态变化不明显,用25μM P1处理6h后细胞萎缩明显,细胞增殖减缓(见图2)。
表2
3.细胞内活性Cellular inhibition activity against mTOR
为了验证化合物在细胞内对mTOR的抑制活性,通过Western Blot检测了化合物P1作用后的MCF-7细胞中mTOR下游蛋白p-S6(Ser240/244)和p-AKT(Ser473)的表达量变化(见图3),发现随着P1处理时间的延长,p-AKT(Ser473)和p-S6(Ser240/244)蛋白的表达减少。说明,化合物P1能够抑制细胞内mTOR下游通路。
4.对细胞周期和凋亡的影响Effect on cell cycle and apoptosis
为了进一步探究该类化合物的作用机制,采用流式细胞术检测了化合物P1对MCF-7细胞周期和细胞凋亡的影响。结果(见图4和图5)发现:与MLN0128能够引起细胞周期阻滞和细胞调亡不同,MCF-7经P1处理24小时之后,与空白对照组相比,细胞周期和细胞凋亡没有明显变化。
5.化合物对自噬的影响
考虑到mTOR与细胞自噬相关,我们通过western bolt检测到自噬标志蛋白LC3A/B-I和LC3A/B-II的转化,数据初步表明P1可以诱导MCF-7细胞的自噬。在人类肿瘤中,自噬扮演着十分重要的作用,这种作用是环境依赖性的。作为一个细胞存活的通路,自噬的激活能够防止长期的组织损伤和细胞死亡,这就能降低肿瘤发生的风险。P1可以诱导MCF-7细胞的自噬,是预防乳腺癌进行深入药理研究的理想化合物。
5.化合物对MCF-7细胞的mTOR蛋白降解实验
为了验证P1能够诱导MCF-7细胞的mTOR蛋白降解,MLN0128和pomalidomide不诱导MCF-7细胞的mTOR蛋白降解。我们用western blotting分析检测了DMSO(空白对照)、MLN0128、pomalidomide和P1对MCF-7细胞中mTOR蛋白的表达水平的影响,并进行了灰度值的分析。
实验方法步骤:
蛋白印迹(Western Blot)实验:将细胞接种于六孔盘中,待细胞贴壁后不同时间给药,加药处理完毕后,加入RIPA裂解液收集细胞,提取蛋白,用BCA蛋白浓度测定试剂盒测蛋白浓度,取等浓度蛋白样品加入溴酚蓝裂解液处理,沸水浴变性15min。制备10% SDS凝胶,配制完毕后,将煮好的蛋白离心,上样,加样完毕,选择恒压75V电压电泳45min。当染料前沿进入分离胶后,调整电压为120V继续电泳直至溴酚蓝染料前沿下至凝胶末端处,即停止电泳。电泳结束后,取出凝胶,切除浓缩胶。转膜,PVDF膜用甲醇浸泡活化,按照顺序由阴极至阳极平放三张滤纸-凝胶-PVDF膜-三张滤纸,放入转膜槽中,在槽的一边放置冰盒降温,转膜槽加满transfer buffer,放入4℃冰箱内,连接好电极,接通电流,200mA转膜1h。转膜结束后,切膜、洗膜后,将膜用5%的脱脂牛奶封闭1h,将膜取出,洗膜略晾干放入杂交袋中,两边封口,分别加入mTOR一抗和β-actin一抗,4℃摇床放置过夜。洗膜后加入二抗稀释液,室温下孵育30min,洗后显影。观察分析蛋白表达变化的关系。通过Image J软件测定蛋白印迹条带的灰度值。
结果:发现与DMSO(空白对照,灰度值为24248)相比,用MLN0128和pomalidomide处理后的细胞内的mTOR水平均没有明显变化(蛋白印迹条带宽度和灰度相似,灰度值分别为30507和26872),说明MLN0128和pomalidomide没有诱导MCF-7细胞的mTOR蛋白降解。而与DMSO(空白对照,灰度值为24248)相比,用P1处理后的细胞内的mTOR水平明显下降(蛋白印迹条带变弱,灰度值降低为7994),说明P1能够诱导MCF-7细胞的mTOR蛋白降解。结果见表3和图7。
表3化合物MLN0128,Pomalidomide,P1-P3作用后的MCF-7细胞中mTOR蛋白的表达量灰度值
向MCF-7细胞中加入P1,处理不同时间(6h,12h,24h)后,用western blotting分析细胞内mTOR蛋白的表达水平。结果发现,随着P1处理时间的延长,细胞中mTOR蛋白的表达水平逐渐降低。与DMSO处理的空白对照组(灰度值为23903)相比,P1处理6h后MCF-7细胞的mTOR水平下降(灰度值降低为9060),处理12h后mTOR水平继续下降(灰度值降低为7457),处理24h后mTOR水平下降更明显(灰度值降低为3392)。这说明,化合物P1对mTOR蛋白的降解作用具有时间依赖性。结果见表4和图8。
表4化合物MLN0128,P1作用后的MCF-7细胞中mTOR蛋白的表达量随时间变化的灰度值
为了确定P1是否通过泛素-蛋白酶体系统诱导mTOR蛋白的降解,在用P1处理MCF-7细胞之前,用蛋白酶体抑制剂MG-132预处理MCF-7细胞。结果发现,与DMSO处理的空白对照组(灰度值为24853)相比,直接用P1处理MCF-7细胞,细胞内mTOR表达水平明显降低(灰度值降低为6690);经蛋白酶体抑制剂MG-132预处理后,再加入P1处理细胞,细胞内mTOR水平没有降低(灰度值为22326)。说明,抑制蛋白酶体会阻断P1对mTOR的降解作用,P1诱导的mTOR降解依赖于泛素-蛋白酶体系统。结果见表5和图9。
表5化合物MLN0128,Pomalidomide,蛋白酶体抑制剂MG-132预处理后,又经化合物P1作用的MCF-7细胞的mTOR蛋白的表达量灰度值
在本说明书的描述中,参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中,对上述术语的示意性表述不应理解为必须针对的是相同的实施例或示例。而且,描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。此外,本领域的技术人员可以将本说明书中描述的不同实施例或示例进行接合和组合。
尽管上面已经示出和描述了本发明的实施例,可以理解的是,上述实施例是示例性的,不能理解为对本发明的限制,本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。
Claims (10)
6.权利要求1-4任一权利要求书所述的mTOR蛋白降解靶向嵌合体在制备mTOR降解剂或者mTOR抑制剂中的应用。
7.权利要求1-4任一权利要求书所述的mTOR蛋白降解靶向嵌合体在制备预防和/或治疗肿瘤药物中的应用。
8.权利要求1-4任一权利要求书所述的mTOR蛋白降解靶向嵌合体在制备预防和/或治疗乳腺癌药物中的应用。
9.权利要求1-2任一权利要求书所述的mTOR蛋白降解靶向嵌合体在制备诱导MCF-7细胞的自噬药物中的应用。
10.一种药物组合物,其特征在于,含有权利要求1所述的mTOR蛋白降解靶向嵌合体和药学上可接受的辅料。
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