CN116589405A - 高效抑制自噬、逆转肿瘤多药耐药的异喹啉生物碱衍生物及制备方法和应用 - Google Patents
高效抑制自噬、逆转肿瘤多药耐药的异喹啉生物碱衍生物及制备方法和应用 Download PDFInfo
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Abstract
本发明涉及一种通过抑制自噬而逆转肿瘤多药耐药的异喹啉生物碱衍生物及制备方法和应用。所述衍生物能高效逆转胃癌、肺癌、食管癌等多种实体肿瘤细胞的多药耐药,该衍生物能抑制肿瘤细胞的自噬流,其与长春新碱、米托蒽醌、秋水仙碱、多西他赛等多种化疗药物联用,在体内外均能高效逆转肿瘤多药耐药,这类衍生物具有优异的口服生物利用度及较低的毒副作用,为自噬抑制剂作为抗肿瘤化疗增敏剂,提供了一种替代策略,可作为先导化合物用于新型耐药肿瘤逆转剂的研发。
Description
技术领域
本发明涉及医药化学领域,特别涉及一种高效抑制自噬、逆转肿瘤多药耐药的异喹啉生物碱衍生物及制备方法和应用。
背景技术
多药耐药(Multidrug resistance,MDR)是恶性肿瘤在接触一种抗癌药物后,继而对多种结构不同、作用机制各异的其他抗癌药产生耐药性。MDR在长期化疗中常常出现,极大地降低了化疗的疗效,也是大多数转移性肿瘤化疗失败的主要原因。肿瘤细胞的自噬增加是MDR的重要成因。一旦肿瘤形成,自噬能调节细胞环境的稳态,为癌细胞提供必要的生存环境,抵抗化疗后的营养压力,削弱化疗药物对细胞的杀伤作用,起到促进肿瘤发生、保护肿瘤细胞的作用,进而发展出化疗耐药性,导致难治性肿瘤和癌症复发。这种情况下,抑制自噬可使耐药癌细胞重新敏感,增强化疗药物的功效。因此,发现高效的自噬抑制剂并与传统化疗药物共同给药,长期以来被认为是有效的逆转临床多药耐药的策略。
当前只有氯喹(CQ)及其衍生物羟氯喹(HCQ)作为自噬抑制剂用于患者,但CQ或HCQ的副作用也极大地限制其在临床上的应用,因此研发一种治疗癌症的新型自噬抑制剂具有重要的临床意义。天然异喹啉生物碱具有广泛的生物活性,包括抗炎、抗肿瘤、抗氧化应激等,也被报道为MDR逆转药的主要支架。然而,异喹啉类生物碱与其他天然产物一样,有药效有限、溶解度差、代谢不稳定、毒理学性质不佳等缺点。
发明内容
本发明的目的是提供一种特异性抑制自噬逆转肿瘤多药耐药的粉异喹啉生物碱衍生物及制备方法和应用。所述衍生物能高效逆转胃癌、肺癌、食管癌等多种实体肿瘤细胞的多药耐药,该衍生物具有较低的细胞毒性,能特异性地抑制肿瘤细胞的自噬流,所述衍生物与长春新碱等多种化疗药物联用,在体内外均能高效逆转肿瘤多药耐药,这类特异性自噬抑制剂作为抗肿瘤化疗增敏剂,提供了一种替代策略,可作为先导化合物用于新型耐药肿瘤逆转剂的研发。
本发明的技术方案:
高效逆转肿瘤多药耐药的异喹啉生物碱衍生物,其具有如下通式结构:
其中:n=0或1,R为H、烷基、烯基、环烷基、环烷基烷基、芳基、芳烷基、杂芳基、杂芳基烷基、杂环基、杂环基烷基、酰基或者结构为的取代苯基的任意一种。
所述中的R1、R2、R3、R4或R5各自独立地为H、F、Cl、Br、I、三氟甲基、酯基、氰基、砜基、硝基、羟基中的任意一种;
所述化合物包括其立体异构体。
本发明采用在有机溶剂中,取代苯乙胺与取代苯乙酸在硼酸催化下,回流缩合成酰胺中间体I;而后在三氯氧磷作用下,回流脱水环合成亚胺中间体II;再经硼氢化钠还原为仲胺中间体III;仲胺中间体III经还原胺化得到中间体IV;中间体IV经钯催化的Buchwald–Hartwig交叉偶联反应得到中间体V;中间体V经三氟乙酸脱除保护剂得到中间体VI;中间体VI再经钯催化的Buchwald–Hartwig交叉偶联或者取代反应得到最终产物。
上述高效逆转肿瘤多药耐药的异喹啉生物碱衍生物的制备方法有以下步骤:
1)将3,4-二甲氧基苯乙胺,对溴苯乙酸加入到甲苯溶液中,在硼酸催化下110~150℃搅拌反应12~20小时,浓缩除去溶剂,得到反应产物,向所述反应产物中加入乙酸乙酯洗涤2~3次,过滤,得到中间体产物Ⅰ;
2)将中间体Ⅰ溶于二氯甲烷,随后加入三氯氧磷,得到反应液,加热到70~78℃,搅拌5~8小时,缓慢加入饱和碳酸氢钠水溶液对其进行淬灭,至反应溶液没有气泡生成,萃取,得到有机溶剂层,无水硫酸钠干燥,得到中间体II;
3)将中间体II溶于甲醇中,冰浴冷却至室温,缓慢加入硼氢化钠,室温下搅拌5小时,旋掉多余的无水甲醇溶液,得到中间体Ⅲ;
4)中间体III经还原胺化在NH-上引入甲基,经柱层析纯化得到中间体IV;
5)氩气环境中,将N-Boc-哌嗪或者N-Boc-高哌嗪加入到中间体IV,Pd2(dba)3,BINAP和K2CO3的甲苯(0.1M)溶液中,110℃回流反应24小时,反应溶液冷却至室温后,用硅藻土过滤,浓缩后,柱层析纯化,得到中间体V;
6)将中间体V溶于0.1M DMC中,缓慢加入三氟乙酸,室温搅拌过夜,减压蒸发除去溶剂后,柱层析纯化,得到中间体VI;
7)中间体VI经Buchwald–Hartwig交叉偶联反应或取代反应,NH上引入不同的基团,得到最终产物。
步骤1)所述3,4-二甲氧基苯乙胺:对溴苯乙酸:硼酸的摩尔比为11:11:0.88;
步骤2)所述的中间体Ⅰ:三氯氧磷的摩尔比为10~11:33;
步骤3)所述的中间体II:硼氢化钠的摩尔比为10.36:51.8;
步骤2)、步骤3)所述萃取采用二氯甲烷。
步骤4)所述还原胺化的具体方法是中间体III与37%甲醛溶液预搅半小时后,加入硼氢化钠还原;
优选地,中间体III:甲醛溶液:硼氢化钠的摩尔比为10.00~11.00:30.00~31.00:40;
步骤5)所述N-Boc-哌嗪或者N-Boc-高哌嗪:中间体IV:Pd2(dba)3:BINAP:K2CO3的摩尔比为9.0~11.0:9.0~10.0:0.99:1.98:19.8;
步骤6)所述中间体V:三氟乙酸的摩尔比为6.00~8.00:20.00~50.00。
步骤7)所述取代反应,中间产物VI和氟化钾溶于乙腈(75mL,
0.1M),随后加入碘化物,室温下反应12小时,过滤,浓缩后柱层析纯化;优选地,中间产物VI:氟化钾:碘化物的摩尔比为7.52:75.2:9.02;
Buchwald–Hartwig交叉偶联反应为,氩气环境中将芳基碘化物加入到中间体VI,Pd2(dba)3,BINAP和K2CO3的甲苯溶液中,110℃回流反应24小时,反应溶液冷却至室温后,硅藻土过滤,浓缩后用柱层析纯化;优选地,所述芳基碘化物:中间体VI:Pd2(dba)3:BINAP:K2CO3的摩尔比为6.86~8.27:6.24~7.25:0.62~0.75:1.24~1.5:12.4~15.04;
步骤7)所述不同的基团为烷基、烯基、环烷基、环烷基烷基、芳基、芳烷基、杂芳基、杂芳基烷基、杂环基、杂环基烷基、酰基或者结构为的取代苯基的任意一种。
上述异喹啉生物碱衍生物在制备治疗癌症药物中的应用,
优选地,所述癌症为胃癌或肺癌或乳腺癌或胰腺癌或前列腺癌或白血病或食管癌;
优选地,所述药物为特异性自噬抑制剂;更优选地,所述抑制剂用于抗肿瘤化疗增敏剂。
上述异喹啉生物碱衍生物与长春新碱联合在制备治疗癌症药物中的应用,
优选地所述癌症为胃癌或肺癌或乳腺癌或胰腺癌或前列腺癌或白血病或食管癌。
所述药物为特异性自噬抑制剂;优选地,所述抑制剂用于抗肿瘤化疗增敏剂。
化学方法合成系列异喹啉生物碱衍生物,并将其应用于高效自噬抑制剂筛选以逆转肿瘤MDR,对于肿瘤多药耐药的联合用药治疗具有重要的意义。
本发明所述方法实现了天然异喹啉生物碱的结构精简优化,所制备的新型异喹啉生物碱衍生物,具有操作简单,安全性好,反应所需时间较短,平均只需要两天至三天即可获得最终产物,反应底物适用范围广,产物结构多样,原子经济性好,具有环境友好性且收率高等优点。采用本发明所述方法制得的异喹啉类生物碱衍生物,极大地提升了天然异喹啉生物碱(莲心碱、粉防己碱等)有限的MDR逆转活性、改善了药代动力学性质且在体内实验中未见明显毒性。
本发明所述方法制得的异喹啉生物碱衍生物具有高效的自噬抑制活性、较高的口服生物利用度以及优异的逆转肿瘤多药耐药活性的优点,是一类良好的先导物,具备进一步成药开发潜力。
申请人的实验验证:所述化合物经蛋白免疫印迹实验证明能够抑制自噬流,经肿瘤细胞毒性实验和动物荷瘤实验证明在体内外均具有优异的逆转肿瘤多药耐药活性。
附图说明
图1为OY-102逆转耐药的IC50值测定。
图2为平板克隆形成实验图。
图3为OY-102的动物实验效果。
图4为激光共聚焦显微镜观察OY-102处理细胞导致自噬体增多。
图5为透射电子显微镜观察5μMOY-102处理24小时后的超微结构图,其中,N表示细胞核;M表示线粒体;比例尺为1μm或0.5μm。
图6为蛋白质免疫印迹检测不同浓度OY-102对细胞自噬相关蛋白的影响。
图7为蛋白质免疫印迹检测不同作用时间的OY-102对细胞自噬相关蛋白的影响。
图8为蛋白质免疫印迹检测OY-102对其他耐药细胞自噬相关蛋白的影响。
图9为蛋白质免疫印迹检测细胞自噬相关蛋白(OY-102与典型的自噬诱导剂RAPA和自噬抑制剂Baf A1交叉比对效果)。
图10为蛋白质免疫印迹检测不同立体构型的OY-102对自噬相关蛋白的影响。
图11为消旋体OY-102的HPLC图。
图12为异构体(R)-OY-102的HPLC图。
图13为异构体(S)-OY-102的HPLC图。
具体实施方式
以下参照具体的实施例来说明本发明。本领域技术人员能够理解,这些实施例仅用于说明本发明的目的,其不以任何方式限制本发明的范围。
试剂:
苯乙胺、乙酸衍生物、硼酸、三氯氧磷、硼氢化钠、BINAP、Pd2(dba)3甲醛(北京伊诺凯生物科技有限公司)
其他的为市售分析纯。
本发明中所述水为蒸馏水,所述有机溶剂均为市售分析纯的极性溶剂或非极性溶剂,如:苯、甲苯、二氯甲烷、氯仿、乙腈、甲醇、四氢呋喃、石油醚、乙酸乙酯等。
实施例1
取一个250ml的圆底烧瓶,将3,4-二甲氧基苯乙胺11mmol、对溴苯乙酸11mmol和硼酸0.88mmol依次加入,之后将其溶于120ml的甲苯之中,在150℃回流条件下搅拌反应12小时。之后浓缩除去溶剂,加入30ml的乙酸乙酯对其洗涤2-3次,之后过滤掉洗涤液,得到中间产物Ⅰ(10.91mmol粗品)。
将10.91mmol的中间体Ⅰ和33mmol的三氯氧磷加入到120ml的二氯甲烷中得到反应液,将反应液加热到70℃搅拌5小时后冷却,将反应液倒入250ml烧杯中,缓慢加入饱和碳酸氢钠水溶液对其进行淬灭,直至烧杯中气泡不在大量产生,加入30ml二氯甲烷对淬灭后的反应液进行萃取,得到有机相;对有机相用无水硫酸钠进行干燥,旋去二氯甲烷即得到中间体II(10.36mmol粗品)。
取一个250ml圆底烧瓶,将10.36mmol的中间体II溶于100ml无水甲醇中,然后在冰浴条件下,缓慢加入51.8mmol的硼氢化钠,之后将圆底烧瓶密封,并在封口处扎一个气球,之后再常温条件下搅拌5小时,旋掉多余的无水甲醇溶液,得到中间体Ⅲ(10.2mmol粗品)。
将10.2mmol中间体Ⅲ和浓度为37%的30.6mmol的甲醛水溶液加入到100ml无水甲醇中,然后在冰浴条件下,缓慢加入40mmol的硼氢化钠,之后将圆底烧瓶密封,并在封口处扎一个气球,之后再常温条件下搅拌5小时,旋掉多余的无水甲醇溶液,用硅胶柱纯化(洗脱剂二氯甲烷:甲醇=80:1)即得到9.9mmol中间体IV。
在氩气环境的手套箱中,将N-Boc-高哌嗪(10.89mmol,1.1eq.)加入到中间体IV(9.9mmol,1.0eq.),Pd2(dba)3(0.99mmol,0.1eq.),BINAP(1.98mmol,0.2eq.)和K2CO3(19.8mmol,2.0eq.)的甲苯(99mL,0.1M)溶液中,然后110℃回流反应24小时;反应溶液冷却至室温后,用硅藻土过滤。浓缩后用柱层析纯化,得到中间产物V(7.92mmol)。
将上述中间产物V(7.92mmol)溶于DMC(79mL,0.1M),缓慢加入三氟乙酸(39.6mL,5.0eq.),室温搅拌过夜,减压蒸发除去溶剂后用柱层析纯化,得到中间产物VI(7.52mmol)。
在氩气环境的手套箱中,将对溴碘苯(8.27mmol,1.1eq.)加入到中间体VI(7.52mmol,1.0eq.),Pd2(dba)3(0.75mmol,0.1eq.),BINAP(1.5mmol,0.2eq.)和K2CO3(15.04mmol,2.0eq.)的甲苯(75mL,0.1M)溶液中,然后110℃回流反应24小时;反应溶液冷却至室温后,用硅藻土过滤。浓缩后用柱层析纯化,得到最终产物(3.85mmol),棕色固体,总收率为35%。结构式如下所示:
1H NMR(600MHz,CDCl3)δ7.31–7.27(m,2H),6.95(d,J=8.0Hz,2H),6.67–6.61(m,2H),6.61–6.58(m,2H),6.57(s,1H),6.01(s,1H),3.84(d,J=1.7Hz,3H),3.70(dd,J=8.3,4.6Hz,1H),3.61(s,4H),3.52(d,J=1.7Hz,3H),3.44–3.34(m,4H),3.28–3.16(m,2H),2.84(dtd,J=17.9,10.6,10.1,4.0Hz,2H),2.73–2.62(m,2H),2.57(s,3H),2.08(p,J=6.4Hz,2H).13C NMR(150MHz,CDCl3)δ147.18,146.12,146.06,145.36,132.12,130.93,128.70,126.93,125.18,113.14,111.40,111.09,110.92,107.65,64.97,55.67,55.33,48.70,48.37,47.38,47.28,46.54,42.34,40.22,25.05,23.45.HRMS(ESI)m/z:calculated forC30H36 79BrN3O2[M+H]+:550.2064,found550.2055,calculated for C30H36 81BrN3O2[M+H]+:552.2043,found 552.2033.
上述化合物处理SGC7901/VCR细胞48小时后的IC50值为7.05±0.40μM,VCR处理SGC7901/VCR细胞的48小时IC50为22960.00±3500.00nM,VCR联用2.0μM上述化合物对SGC7901/VCR细胞的48小时IC50为40.26±12.51nM,相应逆转倍数为570.29。
VCR处理Eca109/VCR细胞的48小时IC50为6830.00±407.30nM,VCR联用2.0μM上述化合物对Eca109/VCR细胞的48小时IC50为49.64±4.34nM,相应逆转倍数为137.59。
实施例2
取一个250ml的圆底烧瓶,将3,4-二甲氧基苯乙胺11mmol、对溴苯乙酸11mmol和硼酸0.88mmol依次加入,之后将其溶于120ml的甲苯之中,在150℃回流条件下搅拌反应12小时。之后浓缩除去溶剂,加入30ml的乙酸乙酯对其洗涤2-3次,之后过滤掉洗涤液,得到中间产物Ⅰ(10.91mmol粗品)
将10.91mmol的中间体Ⅰ和33mmol的三氯氧磷加入到120ml的二氯甲烷中得到反应液,将反应液加热到70℃搅拌5小时后冷却,将反应液倒入250ml烧杯中,缓慢加入饱和碳酸氢钠水溶液对其进行淬灭,直至烧杯中气泡不在大量产生,加入30ml二氯甲烷对淬灭后的反应液进行萃取,得到有机相;对有机相用无水硫酸钠进行干燥,旋去二氯甲烷即得到中间体II(10.36mmol粗品)。
取一个250ml圆底烧瓶,将10.36mmol的中间体II溶于100ml无水甲醇中,然后在冰浴条件下,缓慢加入51.8mmol的硼氢化钠,之后将圆底烧瓶密封,并在封口处扎一个气球,之后再常温条件下搅拌5小时,旋掉多余的无水甲醇溶液,得到中间体Ⅲ(10.2mmol粗品)。
将10.2mmol中间体Ⅲ和浓度为37%的30.6mmol的甲醛水溶液加入到100ml无水甲醇中,然后在冰浴条件下,缓慢加入40mmol的硼氢化钠,之后将圆底烧瓶密封,并在封口处扎一个气球,之后再常温条件下搅拌5小时,旋掉多余的无水甲醇溶液,用硅胶柱纯化(洗脱剂二氯甲烷:甲醇=80:1)即得到9.9mmol中间体IV。
在氩气环境的手套箱中,将N-Boc-高哌嗪(10.89mmol,1.1eq.)加入到中间体IV(9.9mmol,1.0eq.),Pd2(dba)3(0.99mmol,0.1eq.),BINAP(1.98mmol,0.2eq.)和K2CO3(19.8mmol,2.0eq.)的甲苯(99mL,0.1M)溶液中,然后110℃回流反应24小时;反应溶液冷却至室温后,用硅藻土过滤。浓缩后用柱层析纯化,得到中间产物V(7.92mmol)。
将上述中间产物V(7.92mmol)溶于DMC(79mL,0.1M),缓慢加入三氟乙酸(39.6mL,5.0eq.),室温搅拌过夜,减压蒸发除去溶剂后用柱层析纯化,得到中间产物VI(7.52mmol)。
将上述中间产物VI(7.52mmol)和氟化钾(75.2mmol,10eq.)溶于乙腈(75mL,0.1M),随后加入6-碘-1-己炔(9.02mmol,1.2eq.)室温下反应12小时。过滤,浓缩后用柱层析纯化,得到最终产物(5.64mmol),无色油状,总收率为51%。结构式如下所示:
1H NMR(600MHz,CDCl3)δ6.92(d,J=8.6Hz,2H),6.59(d,J=8.7Hz,2H),6.56(s,1H),6.00(s,1H),3.83(s,3H),3.67(dd,J=8.2,4.8Hz,1H),3.53(s,5H),3.45(t,J=6.3Hz,2H),3.26–3.19(m,1H),3.16(dd,J=13.6,4.6Hz,1H),2.86(s,1H),2.79(s,1H),2.75(s,2H),2.71–2.57(m,4H),2.55(s,3H),2.53–2.46(m,2H),2.21(t,J=8.2Hz,2H),2.00–1.93(m,3H),1.60(p,J=6.9Hz,2H),1.53(p,J=6.8Hz,2H).13C NMR(150Hz,CDCl3)δ147.50,147.19,146.10,130.63,111.45,111.25,110.98,84.30,68.46,65.05,57.21,55.71,55.42,54.49,47.99,46.59,40.18,27.58,26.28,18.28.HRMS(ESI)m/z:calculatedfor C30H41N3O2[M+H]+:476.3272,found 476.3261.实施例3
取一个250ml的圆底烧瓶,将3,4-二甲氧基苯乙胺11mmol、对溴苯乙酸11mmol和硼酸0.88mmol依次加入,之后将其溶于120ml的甲苯之中,在150℃回流条件下搅拌反应12小时。之后浓缩除去溶剂,加入30ml的乙酸乙酯对其洗涤2-3次,之后过滤掉洗涤液,得到中间产物Ⅰ(10.91mmol粗品)
将10.91mmol的中间体Ⅰ和33mmol的三氯氧磷加入到120ml的二氯甲烷中得到反应液,将反应液加热到70℃搅拌5小时后冷却,将反应液倒入250ml烧杯中,缓慢加入饱和碳酸氢钠水溶液对其进行淬灭,直至烧杯中气泡不在大量产生,加入30ml二氯甲烷对淬灭后的反应液进行萃取,得到有机相;对有机相用无水硫酸钠进行干燥,旋去二氯甲烷即得到中间体II(10.36mmol粗品)。
取一个250ml圆底烧瓶,将10.36mmol的中间体II溶于100ml无水甲醇中,然后在冰浴条件下,缓慢加入51.8mmol的硼氢化钠,之后将圆底烧瓶密封,并在封口处扎一个气球,之后再常温条件下搅拌5小时,旋掉多余的无水甲醇溶液,得到中间体Ⅲ(10.2mmol粗品)。
将10.2mmol中间体Ⅲ和浓度为37%的30.6mmol的甲醛水溶液加入到100ml无水甲醇中,然后在冰浴条件下,缓慢加入40mmol的硼氢化钠,之后将圆底烧瓶密封,并在封口处扎一个气球,之后再常温条件下搅拌5小时,旋掉多余的无水甲醇溶液,用硅胶柱纯化(洗脱剂二氯甲烷:甲醇=80:1)即得到9.9mmol中间体IV。
在氩气环境的手套箱中,将N-Boc-哌嗪(10.89mmol,1.1eq.)加入到中间体IV(9.9mmol,1.0eq.),Pd2(dba)3(0.99mmol,0.1eq.),BINAP(1.98mmol,0.2eq.)和K2CO3(19.8mmol,2.0eq.)的甲苯(99mL,0.1M)溶液中,然后110℃回流反应24小时;反应溶液冷却至室温后,用硅藻土过滤。浓缩后用柱层析纯化,得到中间产物V(6.93mmol)。
将上述中间产物V(6.93mmol)溶于DMC(69mL,0.1M),缓慢加入三氟乙酸(34.7mL,5.0eq.),室温搅拌过夜,减压蒸发除去溶剂后用柱层析纯化,得到中间产物VI(6.24mmol)。
在氩气环境的手套箱中,将3,5-二氯碘苯(6.86mmol,1.1eq.)加入到中间体VI(6.24mmol,1.0eq.),Pd2(dba)3(0.62mmol,0.1eq.),BINAP(1.24mmol,0.2eq.)和K2CO3(12.4mmol,2.0eq.)的甲苯(68mL,0.1M)溶液中,然后110℃回流反应24小时;反应溶液冷却至室温后,用硅藻土过滤。浓缩后用柱层析纯化,得到最终产物(3.85mmol),棕色固体,总收率为35%。结构式如下所示:
1H NMR(600MHz,CDCl3)δ7.29(d,J=8.9Hz,1H),7.05–6.97(m,3H),6.87(d,J=8.2Hz,2H),6.78(dd,J=9.0,2.8Hz,1H),6.56(s,1H),6.00(s,1H),3.83(s,3H),3.70(dd,J=8.0,5.0Hz,1H),3.54(s,3H),3.33–3.27(m,4H),3.25(dd,J=7.3,3.9Hz,4H),3.23–3.14(m,2H),2.85(ddd,J=15.3,9.0,5.8Hz,1H),2.77(ddt,J=21.8,13.6,6.2Hz,2H),2.61(dt,J=15.9,4.5Hz,1H),2.55(s,3H).13C NMR(150MHz,CDCl3)δ150.49,149.25,147.12,146.06,132.71,131.62,130.48,130.39,128.84,125.54,122.35,117.35,116.39,115.47,110.96,110.95,64.76,55.63,55.39,49.48,48.76,46.58,42.46,40.18,25.19.HRMS(ESI)m/z:calculated for C29H33 35Cl2N3O2[M+H]+:526.2023,found 526.2015,calculated forC29H33 37Cl2N3O2[M+H]+:528.1993,found 528.1984.
上述化合物处理SGC7901/VCR细胞48小时后的IC50值为7.99±0.56μM,VCR处理SGC7901/VCR细胞的48小时IC50为22960.00±3500.00nM,VCR联用2.0μM上述化合物对SGC7901/VCR细胞的48小时IC50为22.70±9.49nM,相应逆转倍数为1011.45。
VCR处理Eca109/VCR细胞的48小时IC50为6830.00±407.30nM,VCR联用2.0μM上述化合物对Eca109/VCR细胞的48小时IC50为213.70±11.08nM,相应逆转倍数为31.96。
实施例4
取一个250ml的圆底烧瓶,将3,4-二甲氧基苯乙胺11mmol、对溴苯乙酸11mmol和硼酸0.88mmol依次加入,之后将其溶于120ml的甲苯之中,在150℃回流条件下搅拌反应12小时。之后浓缩除去溶剂,加入30ml的乙酸乙酯对其洗涤2-3次,之后过滤掉洗涤液,得到中间产物Ⅰ(10.91mmol粗品)
将10.91mmol的中间体Ⅰ和33mmol的三氯氧磷加入到120ml的二氯甲烷中得到反应液,将反应液加热到70℃搅拌5小时后冷却,将反应液倒入250ml烧杯中,缓慢加入饱和碳酸氢钠水溶液对其进行淬灭,直至烧杯中气泡不在大量产生,加入30ml二氯甲烷对淬灭后的反应液进行萃取,得到有机相;对有机相用无水硫酸钠进行干燥,旋去二氯甲烷即得到中间体II(10.36mmol粗品)。
取一个250ml圆底烧瓶,将10.36mmol的中间体II溶于100ml无水甲醇中,然后在冰浴条件下,缓慢加入51.8mmol的硼氢化钠,之后将圆底烧瓶密封,并在封口处扎一个气球,之后再常温条件下搅拌5小时,旋掉多余的无水甲醇溶液,得到中间体Ⅲ(10.2mmol粗品)。
将10.2mmol中间体Ⅲ和浓度为37%的30.6mmol的甲醛水溶液加入到100ml无水甲醇中,然后在冰浴条件下,缓慢加入40mmol的硼氢化钠,之后将圆底烧瓶密封,并在封口处扎一个气球,之后再常温条件下搅拌5小时,旋掉多余的无水甲醇溶液,用硅胶柱纯化(洗脱剂二氯甲烷:甲醇=80:1)即得到9.9mmol中间体IV。
在氩气环境的手套箱中,将N-Boc-哌嗪(10.89mmol,1.1eq.)加入到中间体IV(9.9mmol,1.0eq.),Pd2(dba)3(0.99mmol,0.1eq.),BINAP(1.98mmol,0.2eq.)和K2CO3(19.8mmol,2.0eq.)的甲苯(99mL,0.1M)溶液中,然后110℃回流反应24小时;反应溶液冷却至室温后,用硅藻土过滤。浓缩后用柱层析纯化,得到中间产物V(6.93mmol)。
将上述中间产物V(6.93mmol)溶于DMC(69mL,0.1M),缓慢加入三氟乙酸(34.7mL,5.0eq.),室温搅拌过夜,减压蒸发除去溶剂后用柱层析纯化,得到中间产物VI(6.24mmol)。
在氩气环境的手套箱中,将对溴碘苯(6.86mmol,1.1eq.)加入到中间体VI(6.24mmol,1.0eq.),Pd2(dba)3(0.62mmol,0.1eq.),BINAP(1.24mmol,0.2eq.)和K2CO3(12.4mmol,2.0eq.)的甲苯(68mL,0.1M)溶液中,然后110℃回流反应24小时;反应溶液冷却至室温后,用硅藻土过滤。浓缩后用柱层析纯化,得到最终产物(4.73mmol),棕色固体,总收率为43%。结构式如下所示:
1H NMR(600MHz,CDCl3)δ7.36(d,J=8.5Hz,2H),7.02(d,J=8.1Hz,2H),6.88(d,J=8.1Hz,2H),6.84(d,J=8.6Hz,2H),6.56(s,1H),5.99(s,1H),3.84(s,3H),3.71(dd,J=8.1,5.0Hz,1H),3.54(s,3H),3.27(hept,J=3.9Hz,8H),3.21(ddd,J=22.9,13.2,6.7Hz,2H),2.90–2.78(m,2H),2.75(dd,J=13.6,8.1Hz,1H),2.63(dt,J=15.9,4.5Hz,1H),2.56(s,3H).13C NMR(150MHz,CDCl3)δ150.16,149.41,147.17,146.09,131.85,131.43,130.49,128.76,125.46,117.82,116.36,112.09,110.98,64.81,55.66,55.42,49.58,49.14,42.45,40.21,25.17.HRMS(ESI)m/z:calculated for C29H34 79BrN3O2[M+H]+:536.1907,found 536.1899,calculated for C29H34 81BrN3O2[M+H]+:538.1887,found 538.1878.
上述化合物处理SGC7901/VCR细胞48小时后的IC50值为17.29±2.16μM,VCR处理SGC7901/VCR细胞的48小时IC50为22960.00±3500.00nM,VCR联用2.0μM上述化合物对SGC7901/VCR细胞的48小时IC50为373.10±104.28nM,相应逆转倍数为61.54。
VCR处理Eca109/VCR细胞的48小时IC50为6830.00±407.30nM,VCR联用2.0μM上述化合物对Eca109/VCR细胞的48小时IC50为658.30±21.45nM,相应逆转倍数为10.38。
实施例5
该实例是基于在本发明的研究方法下所得到药用价值较大的一种自噬抑制剂,又被命名为OY-102,可高效逆转肿瘤的多药耐药,具有合成简单、口服生物利用度高、逆转耐药活性高等优点。通过逆转活性测定、平板克隆形成测定和药物协同分析得到证实其与长春新碱(VCR)对耐药细胞SGC7901/VCR的优异协同抗癌作用(联用2μMOY-102,IC50=15.77nM,RF=1455.93)。在裸鼠异种移植瘤模型也表现优异的逆转耐药活性,同时具有较低的体内毒性。在机制探索中,发现OY-102能够抑制多种耐药肿瘤细胞自噬流。以下是OY-102的合成步骤:
取一个250ml的圆底烧瓶,将3,4-二甲氧基苯乙胺11mmol、对溴苯乙酸11mmol和硼酸0.88mmol依次加入,之后将其溶于120ml的甲苯之中,在150℃回流条件下搅拌反应12小时。之后浓缩除去溶剂,加入30ml的乙酸乙酯对其洗涤2-3次,之后过滤掉洗涤液,得到中间产物Ⅰ(10.91mmol粗品)。
将10.91mmol的中间体Ⅰ和33mmol的三氯氧磷加入到120ml的二氯甲烷中得到反应液,将反应液加热到70℃搅拌5小时后冷却,将反应液倒入250ml烧杯中,缓慢加入饱和碳酸氢钠水溶液对其进行淬灭,直至烧杯中气泡不在大量产生,加入30ml二氯甲烷对淬灭后的反应液进行萃取,得到有机相;对有机相用无水硫酸钠进行干燥,旋去二氯甲烷即得到中间体II(10.36mmol粗品)。
取一个250ml圆底烧瓶,将10.36mmol的中间体II溶于100ml无水甲醇中,然后在冰浴条件下,缓慢加入51.8mmol的硼氢化钠,之后将圆底烧瓶密封,并在封口处扎一个气球,之后再常温条件下搅拌5小时,旋掉多余的无水甲醇溶液,得到中间体Ⅲ(10.2mmol粗品)。
将10.2mmol中间体Ⅲ和浓度为37%的30.6mmol的甲醛水溶液加入到100ml无水甲醇中,然后在冰浴条件下,缓慢加入40mmol的硼氢化钠,之后将圆底烧瓶密封,并在封口处扎一个气球,之后再常温条件下搅拌5小时,旋掉多余的无水甲醇溶液,用硅胶柱纯化(洗脱剂二氯甲烷:甲醇=80:1)即得到9.9mmol中间体IV。
在氩气环境的手套箱中,将N-Boc-高哌嗪(10.89mmol,1.1eq.)加入到中间体IV(9.9mmol,1.0eq.),Pd2(dba)3(0.99mmol,0.1eq.),BINAP(1.98mmol,0.2eq.)和K2CO3(19.8mmol,2.0eq.)的甲苯(99mL,0.1M)溶液中,然后110℃回流反应24小时;反应溶液冷却至室温后,用硅藻土过滤。浓缩后用柱层析纯化,得到中间产物V(7.92mmol)。
将上述中间产物V(7.92mmol)溶于DMC(79mL,0.1M),缓慢加入三氟乙酸(39.6mL,5.0eq.),室温搅拌过夜,减压蒸发除去溶剂后用柱层析纯化,得到中间产物VI(7.52mmol)。
在氩气环境的手套箱中,将3,5-二氯碘苯(8.27mmol,1.1eq.)加入到中间体VI(7.52mmol,1.0eq.),Pd2(dba)3(0.75mmol,0.1eq.),BINAP(1.5mmol,0.2eq.)和K2CO3(15.04mmol,2.0eq.)的甲苯(75mL,0.1M)溶液中,然后110℃回流反应24小时;反应溶液冷却至室温后,用硅藻土过滤。浓缩后用柱层析纯化,得到最终产物(3.3mmol),棕色固体,总收率为30%。结构式如下所示:
1H NMR(600MHz,CDCl3)δ7.22(d,J=9.0Hz,1H),6.96(d,J=8.3Hz,2H),6.75(d,J=2.6Hz,1H),6.63(d,J=8.4Hz,2H),6.58–6.52(m,2H),6.05(s,1H),3.84(s,3H),3.69–3.64(m,1H),3.61(s,4H),3.54(s,3H),3.46–3.35(m,4H),3.20(ddd,J=13.2,9.2,5.1Hz,1H),3.11(dd,J=13.7,4.7Hz,1H),2.84(dt,J=15.3,7.3Hz,1H),2.77(dt,J=10.0,4.6Hz,1H),2.69(dd,J=13.7,8.1Hz,1H),2.63–2.58(m,1H),2.54(s,3H),2.08(p,J=6.0Hz,2H).13C NMR(150MHz,CDCl3)δ147.06,146.77,146.03,145.20,133.14,130.94,130.79,129.43,127.60,125.71,118.51,112.84,111.47,111.13,111.11,110.99,64.95,55.70,55.39,48.87,48.46,47.54,47.38,46.75,42.62,40.28,25.39,23.44.HRMS(ESI)m/z:calculated for C30H35 35Cl2N3O2[M+H]+:540.2179,found 540.2173,calculated forC30H35 37Cl2N3O2[M+H]+:542.2150,found 542.2142.
采用本发明所述方法可以得到立体异构体的结构式为:
其立体异构体HPLC色谱图参见图11-13。
上述化合物处理SGC7901/VCR细胞48小时后的IC50值为4.64±0.39μM,VCR处理SGC7901/VCR细胞的48小时IC50为22960.00±3500.00nM,VCR联用2.0μM上述化合物对SGC7901/VCR细胞的48小时IC50为15.77±4.22nM,相应逆转倍数为1455.93。
VCR处理Eca109/VCR细胞的48小时IC50为6830.00±407.30nM,VCR联用2.0μM上述化合物对Eca109/VCR细胞的48小时IC50为24.39±2.28nM,相应逆转倍数为280.03。
实施例6OY-102的生物活性测试部分:
1.OY-102逆转耐药活性测试
如图1A,分别在人胃癌细胞SGC7901及其耐药株细胞SGC7901/VCR中,通过CCK8法测定了VCR对两株细胞的48小时IC50值,以及在SGC7901/VCR细胞中VCR分别联用1μMOY-102、2μMOY-102、2μM莲心碱(LIEN)和2μM粉防己碱(TET)后,VCR对SGC7901/VCR细胞的48小时IC50值。此外,应用相同的实验条件,分别在人乳腺癌细胞MCF7及其耐药株细胞MCF/ADR中测定了阿霉素(ADR)对两株细胞的48小时IC50值(图1B);在人食管癌细胞Eca109及其耐药株细胞Eca109/VCR中测定了VCR对两株细胞的48小时IC50值(图1C);在人肺癌细胞A549及其耐药株细胞A549/TAX中测定了紫杉醇(TAX)对两株细胞的48小时IC50值(图1D);以及在上述耐药株细胞中分别联用1μMOY-102、2μMOY-102、2μMLIEN和2μMTET后相应抗性药物的48小时IC50值变化。由图1A-图1D可见,耐药株细胞相较对应的亲本细胞均具有较高的耐药性,尤其是A549/TAX,其次是SGC7901/VCR和MCF/ADR;而化合物OY-102呈剂量依赖的方式逆转了耐药株细胞的耐药性,尤其是SGC7901/VCR,逆转倍数最高达1455.9其次是Eca109/VCR和MCF/ADR,OY-102相较于相同浓度的天然异喹啉生物碱LIEN和TET具有更强的逆转耐药效果。
2.平板克隆形成实验考察OY-102的逆转耐药活性
在SGC7901/VCR、MCF/ADR和Eca109/VCR细胞中,采用0.5μM的OY-102与0.5μM或1μM相应抗性药(VCR或DOX),处理细胞72小时后细胞聚落形成情况。如图2,0.5μM的OY-102和0.5μM抗癌药几乎对细胞没有毒性,但两者联用几乎完全杀死了耐药株细胞。
3.OY-102在SD大鼠静脉注射和口服后的药代动力学参数
申请人测定了OY-102在SD大鼠静脉注射(1mg/kg)和口服(10mg/kg)后的药代动力学参数。非房室模型计算的药代动力学参数如表1所示,静脉给药组(iv)和口服给药组(op)分别在给药0.10和4.0小时后达到血药浓度峰值(Tmax)。iv组相应的峰值浓度(Cmax)是op组的近2.6倍。两组末端消除速率常数(λz)均较低,而半衰期(t1/2)相对较长(9小时作用),以及平均停留时间(MRT0-inf)也相对较长,表明OY-102在iv和op中作用时间相对较长。op组较iv组的药浓度-时间曲线下的面积(AUC0-t和AUC0-inf)更大,以及在此基础上计算OY-102的口服生物利用度为62.29%。表明OY-102的口服生物利用度较高,由此确定了动物研究的有效剂量:OY-102单用药组(10mg/kg/2d);OY-102低剂量(5mg/kg/2d)联用VCR(0.5mg/kg/2d)。
表1 OY-102的口服和静脉注射的药代动力学参数
4.动物实验结果
利用SGC7901/VCR细胞建立了裸鼠异种移植瘤模型,检测OY-102在体内的肿瘤MDR逆转活性。分为四个治疗组溶剂对照组(10mL/kg/2d,灌胃)、VCR组(0.5mg/kg/2d,尾静脉)、OY-102组(10mg/kg/2d,灌胃)以及VCR(0.5mg/kg/2d)/OY-102(5mg/kg/2d)联合治疗组。治疗3周后,OY-102与VCR联合治疗能有效抑制体内肿瘤增殖(图3A和图3B);10mg/kg的OY-102单独治疗后也观察到抗肿瘤增殖的作用(图3A和图3B);各组裸鼠体重在治疗期间无显著变化(图3C)。肿瘤的病理和TUNEL分析显示,OY-102/VCR联用给药和OY-102单独治疗,增加了肿瘤组织中核固缩、空泡和凋亡细胞的比例,而Ki-67增殖染色显著减少。肝组织HE染色显示,OY-102与VCR联合给药没有明显的肝毒性(图3D)。这些结果表明,OY-102是一种有效的抗癌试剂和MDR逆转剂,可以逆转肿瘤的VCR耐药并抑制SGC7901/VCR异种移植瘤的生长,且没有明显的肝毒性和全身毒性。
5.OY-102增加细胞内自噬体的数量
为了确定化合物OY-102是否会影响人类细胞的自噬,申请人用荧光蛋白EGFP标记自噬标志蛋白LC3,构建了EGFP-LC3质粒,并用激光共聚焦扫描显微镜观察细胞中EGFP-LC3蛋白的表达情况来反映自噬体的积累情况。首先分别转染EGFP-LC3质粒于SGC7901/VCR和MCF/ADR细胞24小时后,加或不加5μMOY-102处理细胞24小时,随后用激光共聚焦扫描显微镜观察自噬体的积累情况。如图4,用化合物OY-102处理细胞会导致SGC7901/VCR和MCF/ADR细胞中EGFP-LC3荧光斑点的形成显著增加,表明细胞中自噬体增多。
6.透射电子观察OY-102对肿瘤细胞自噬的影响
为了进一步确定化合物OY-102对SGC7901/VCR和MCF7/ADR细胞的自噬影响,使用透射电子显微镜(TEM)直接观察细胞内的自噬体的积累情况。如图5,与对照组细胞相比,经5μM OY-102处理24小时后的癌细胞中自噬体增多,还观察到处理组肿胀的线粒体和线粒体自噬也明显增加。
7.蛋白免疫印迹观察OY-102对肿瘤细胞自噬相关蛋白的影响
为了验证细胞自噬体数量的增多是出于药物诱导的自噬体生成增多,还是自噬体降解受阻导致其积累增加。申请人通过蛋白质免疫印迹用LC3B抗体同时检测LC3B-I和LC3B-II,以及SQSTM1抗体检测p62,旨在验证OY-102导致细胞自噬体数量增多是由于诱导自噬引起还是抑制自噬引起。如图6A-图6C和图7A-图7C,申请人研究了化合物OY-102对SGC7901/VCR和MCF/ADR细胞中LC3B转化和p62表达的影响,发现OY-102处理细胞后的LC3B-II和p62均呈剂量和时间依赖性积累(图6B、图6C为图6A的三次重复后的LC3B-II/β-Actin和p62/β-Actin相对于阴性对照的比值的统计图;图7B、图7C为图7A三次重复后的LC3B-II/β-Actin和p62/β-Actin相对于阴性对照的比值的统计图(平均值±SD,n=3;ns表示P>0.05,*表示P<0.05,**表示P<0.01,***表示P<0.001))。由此推测OY-102是一种自噬抑制剂,导致LC3B-II和p62降解受阻,而不是自噬诱导剂(LC3B-II增加和p62减少)。
此外,申请人同样在耐药株细胞A549/TAX、SGC7901/DDP和Eca109/VCR中分别测定了化合物OY-102对细胞中LC3B和p62水平的影响。如图8,OY-102处理上述耐药株细胞后,均表现为LC3B-II和p62的积累,表明OY-102对不同耐药株细胞均能表现为自噬抑制的作用。
8.OY-102与经典自噬抑制剂和自噬诱导剂的对比
申请人引入了经典的自噬诱导剂雷帕霉素(Rapamycin,RAPA),RAPA通过抑制mTOR-ULK途径介导的自噬抑制作用进而诱导自噬,以及自噬抑制剂巴弗洛霉素A1(bafilomycin A1,Baf A1),通过抑制溶酶体酸化抑制自噬体降解。如图9A-图9C所示,单独OY-102处理细胞与Baf A1相似促进LC3B-II和p62均增加,与RAPA单独处理细胞不同(LC3B-II轻微增加而p62减少);同时OY-102能抑制RAPA诱导的自噬,即OY-102和RAPA联用进一步增加LC3B-II表达和反转p62表达增加,处理24小时后更为明显(图9B、C为9A的三次重复后的LC3B-II/β-Actin和p62/β-Actin相对于阴性对照的百分比值。(平均值±SD,n=3;ns表示P>0.05,*表示P<0.05,**表示P<0.01,***表示P<0.001))。由此表明化合物OY-102与BafA1相似是一种自噬抑制剂,而不同于RAPA的自噬体促进降解作用,同时OY-102能抑制RAPA诱导的自噬。
9.OY-102的S构型活性优于R构型
通过手性拆分得到了S构型((S)-OY-102)和R构型((R)-OY-102)。如图10A-图10C,5μM的(S)-OY-102、(R)-OY-102和OY-102分别对SGC7901/VCR和MCF/ADR细胞的自噬相关蛋白(p62和LC3B)表达进行检测,发现(S)-OY-102对自噬相关蛋白的影响强于(R)-OY-102(图10B、C为图10A三次重复后的LC3B-II/β-Actin和p62/β-Actin的统计图(平均值±SD,n=3))。
此外,申请人通过CCK8法测定了异构体的对SGC7901/VCR细胞的48小时IC50值,以及1μM的OY-102、(S)-OY-102和(R)-OY-102联用不同浓度的VCR对SGC7901/VCR细胞的48小时IC50值。如表2,单用的细胞毒性和联用的逆转活性均是(S)-OY-102高于(R)-OY-102。由此表明(S)-OY-102的细胞毒性、MDR逆转活性和自噬调节活性均优于异构体(R)-OY-102和消旋体OY-102,将会是后续药物进一步开发的重点关注构型。
表2不同立体构型的OY-102单用药以及VCR联用1μMOY-102的48小时IC50值和逆转倍数
结论:
本发明所述异喹啉生物碱衍生物,能够抑制自噬、高效逆转肿瘤细胞多药耐药。其中以OY-102为代表的衍生物,在体内外均表现出及其优异的逆转活性,并且,OY-102具有优异的口服生物利用度。进一步的机理研究证实OY-102是一种高效的肿瘤细胞自噬抑制剂,能够通过抑制自噬流而发挥逆转耐药的作用。本发明所述制备方法的成熟稳定的合成路线,所述衍生物强效的逆转活性以及优异的口服生物利用度,证明其具有进一步开发成耐药肿瘤增敏剂的应用潜力。
Claims (10)
1.一种高效逆转肿瘤多药耐药的异喹啉生物碱衍生物,其特征在于:该衍生物具有如下通式结构:
其中:n=0或1,R为H、烷基、烯基、环烷基、环烷基烷基、芳基、芳烷基、杂芳基、杂芳基烷基、杂环基、杂环基烷基、酰基或者结构为的取代苯基的任意一种。
2.根据权利要求1所述的化合物,其特征在于:所述中的R1、R2、R3、R4或R5各自独立地为H、F、Cl、Br、I、三氟甲基、酯基、氰基、砜基、硝基、羟基中的任意一种。
3.根据权利要求1-2任一所述的化合物,其特征在于:所述化合物包括其立体异构体。
4.权利要求1-2任一所述的化合物的制备方法,其特征在于,有以下步骤:
1)将3,4-二甲氧基苯乙胺,对溴苯乙酸加入到甲苯溶液中,在硼酸催化下110~150℃搅拌反应12~20小时,浓缩除去溶剂,得到反应产物,向所述反应产物中加入乙酸乙酯洗涤2~3次,过滤,得到中间体产物Ⅰ;
2)将中间体Ⅰ溶于二氯甲烷,然后加入三氯氧磷得到反应液,加热到70~78℃,搅拌5~8小时,缓慢加入饱和碳酸氢钠水溶液对其进行淬灭,至反应溶液没有气泡生成,萃取,得到有机溶剂层,无水硫酸钠干燥,得到中间体Ⅱ;
3)将中间体Ⅱ溶于甲醇中,冰浴冷却至室温,缓慢加入硼氢化钠,室温下搅拌5小时,旋掉多余的无水甲醇溶液,得到中间体Ⅲ;
4)中间体III经还原胺化在NH-上引入甲基,经柱层析纯化得到中间体IV;
5)氩气环境中,将N-Boc-哌嗪或者N-Boc-高哌嗪加入到中间体IV,Pd2(dba)3,BINAP和K2CO3的甲苯(0.1M)溶液中,110℃回流反应24小时,反应溶液冷却至室温后,用硅藻土过滤,浓缩后,柱层析纯化,得到中间体V;
6)将中间体V溶于0.1M DMC中,缓慢加入三氟乙酸,室温搅拌过夜,减压蒸发除去溶剂后,柱层析纯化,得到中间体VI;
7)中间体VI经Buchwald–Hartwig交叉偶联反应或取代反应,NH上引入不同的基团,得到最终产物。
5.权利要求4所述的方法,其特征在于:步骤1)所述3,4-二甲氧基苯乙胺:对溴苯乙酸:硼酸的摩尔比为11:11:0.88;
步骤2)所述的中间体Ⅰ:三氯氧磷的摩尔比为10~11:33;
步骤3)所述的中间体Ⅱ:硼氢化钠的摩尔比为10.36:51.8;
步骤2)、步骤3)所述萃取采用二氯甲烷。
6.权利要求4所述的方法,其特征在于:步骤4)所述还原胺化的具体方法是中间体III与37%甲醛溶液预搅半小时后,加入硼氢化钠还原;
优选地,中间体III:甲醛溶液:硼氢化钠的摩尔比为10.00~11.00:30.00~31.00:40;
步骤5)所述N-Boc-哌嗪或者N-Boc-高哌嗪:中间体IV:Pd2(dba)3:
BINAP:K2CO3的摩尔比为9.0~11.0:9.0~10.0:0.99:1.98:19.8;
步骤6)所述中间体V:三氟乙酸的摩尔比为6.00~8.00:20.00~50.00。
7.权利要求4所述的方法,其特征在于:步骤7)所述取代反应为,中间产物VI和氟化钾溶于乙腈(75mL,0.1M),随后加入碘化物,室温下反应12小时,过滤,浓缩后柱层析纯化;优选地,中间产物VI:氟化钾:碘化物的摩尔比为7.52:75.2:9.02;
Buchwald–Hartwig交叉偶联反应,氩气环境中将芳基碘化物加入到中间体VI,Pd2(dba)3,BINAP和K2CO3的甲苯溶液中,110℃回流反应24小时,反应溶液冷却至室温后,硅藻土过滤,浓缩后用柱层析纯化;优选地,所述芳基碘化物:中间体VI:Pd2(dba)3:BINAP:K2CO3的摩尔比为6.86~8.27:6.24~7.25:0.62~0.75:1.24~1.5:12.4~15.04;
步骤7)所述不同的基团为烷基、烯基、环烷基、环烷基烷基、芳基、芳烷基、杂芳基、杂芳基烷基、杂环基、杂环基烷基、酰基或者结构为的取代苯基的任意一种。
8.权利要求1-2任一所述的异喹啉生物碱衍生物在制备治疗癌症药物中的应用,
优选地,所述癌症为胃癌或肺癌或乳腺癌或胰腺癌或前列腺癌或白血病或食管癌;
优选地,所述药物为特异性自噬抑制剂;更优选地,所述抑制剂用于抗肿瘤化疗增敏剂。
9.权利要求1-2任一所述的异喹啉生物碱衍生物与长春新碱联合在制备治疗癌症药物中的应用,
优选地所述癌症为胃癌或肺癌或乳腺癌或胰腺癌或前列腺癌或白血病或食管癌。
10.根据权利求8所述的应用,其特征在于:所述药物为特异性自噬抑制剂;优选地,所述抑制剂用于抗肿瘤化疗增敏剂。
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