CN112390791B - 一类dna甲基转移酶1荧光探针及其用途 - Google Patents
一类dna甲基转移酶1荧光探针及其用途 Download PDFInfo
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- C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
- C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
- C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
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Abstract
本发明属医药技术领域，涉及DNA甲基转移酶1荧光探针，具体涉及式1结构的由邻苯二甲酰亚胺类DNA甲基转移酶1(DNMT1)抑制剂与荧光素通过连接链(L)连接而成的荧光探针及其用途。本发明式1结构的荧光探针，或其药学上可以接受的盐、其立体异构体或互变异构体、或前药，表现出良好的透膜入核能力和与DNMT1的结合能力，可用于制备细胞内及细胞核内的DNMT1的定量检测制剂，为细胞病变尤其是癌变程度提供预测或诊断。
Description
技术领域
本发明属医药技术领域，涉及DNA甲基转移酶1荧光探针，具体涉及一类由邻苯二甲酰亚胺类DNA甲基转移酶1(DNMT1)抑制剂与荧光素通过连接链(L)连接而成的荧光探针，该类荧光探针表现出良好的透膜入核能力和与DNMT1的结合能力，可用于制备细胞内及细胞核内的DNMT1的定量检测制剂，为细胞病变尤其是癌变程度提供预测。
背景技术
研究显示，肿瘤的形成不仅源于基因遗传，同样也受到表观遗传修饰的影响；表观遗传学包括DNA甲基化、组蛋白共价修饰以及mRNA编辑等，其中，DNA甲基化是表观遗传中最主要的一种类型，抑癌基因的甲基化将直接破坏正常细胞的动态平衡，从而诱发癌变。
研究表明，DNA甲基化主要是指在DNA甲基转移酶(DNMTs)的催化作用下，以S-腺苷甲硫氨酸为甲基供体，将DNA的CpG岛上的胞嘧啶转变成5-甲基胞嘧啶，从而导致该基因沉默，丧失功能。DNMTs家族主要包括DNMT1，DNMT2，DNMT3A，DNMT3B，DNMT3L等，其中，DNMT1是目前研究最多的甲基转移酶，研究表明DNMT1的表达量多少与基因甲基化程度之间存在直接相关性，而抑癌基因的甲基化过度程度又与细胞的癌变程度直接相关，因此DNMT1的表达量的多少与细胞的癌变程度之间存在着相关性，即DNMT1表达量越多，细胞癌变程度越大。
目前医疗实践中，对于癌前病变的检测手段较少，且大多耗费人力物力，同时存在着敏感性与特异性较低等问题，如宫颈癌筛查方法中宫颈薄层细胞学检查(thinprepcytologic test，TCT)、高危人乳头瘤病毒(high risk human papillomavirus，HR-HPV)和第二代杂交捕获实验(hybrid capture2，HC2)检测均存在敏感性和特异性较差的问题，常导致不必要的阴道镜转诊等。因此，研制新型的癌前病变检测手段有着重要的实践意义。DNMT1是生物细胞核内一种特异性的甲基转移酶，其表达量的多少一定程度上预示着细胞的癌变程度，因此，实现对DNMT1表达量的检测将可以成为细胞或生物体健康状况的量度，甚至是癌症的预测和警告。
截至目前，关于DNMT1荧光探针的市面上及文献中均未见有报道。基于现有技术的现状，本申请的发明人将DNMT1抑制剂或其配体通过柔性连接链与适当的荧光剂连接，提供一类可通过共聚焦显微镜技术定量检测细胞核中DNMT1含量的荧光探针，本发明可用于与DNMT1高表达的细胞病变，尤其是细胞癌变的预测和诊断。
发明内容
本发明的目的在于基于现有技术的现状，提供一类可通过共聚焦显微镜技术定量检测细胞核中DNMT1含量的荧光探针，本发明中将DNMT1抑制剂或其配体通过柔性连接链与适当的荧光剂连接制成DNA甲基转移酶1荧光探针，本发明可用于与DNMT1高表达的细胞病变，尤其是细胞癌变的预测和诊断。
具体的，本发明的荧光探针具有下述通式1化合物结构，或其药学上可以接受的盐、其立体异构体或互变异构体、或前药：
其中，
L选自取代或未取代的C1-10亚烃基、取代或未取代的含杂原子的C1-10杂亚烃基，当L为取代或未取代的含杂原子的C1-10杂亚烃基时，所述的杂原子选自氧、氮和硫中的一个或多个；当杂原子为多个时，杂原子相同或不同；
环A选自苯环，含1-3个杂原子的五元芳香杂环，含1-3杂原子的六元芳香杂环，含6-14个原子的稠合芳香环或者芳香杂环，含3-8个碳原子的脂肪环、或者插入杂原子的含3-8个碳原子的脂肪环；当环中含有杂原子时，杂原子选自氧、氮和硫中的一个或多个；当杂原子为多个时，杂原子相同或不同；
X选自碳原子，氮原子，氧原子或者硫原子；
R1选自独立的氢、卤素、羟基、氨基，硝基，氰基、取代或未取代的含1-6个碳原子的烷基、取代或未取代的含1-6个碳原子的烷氧基，取代或未取代的含1-6个碳原子的烷氨基，取代或未取代的含1-6个碳原子的酰氨基，或者以上基团的组合；
R2选自氢，取代或未取代的含1-10个碳原子的烷基，取代或未取代的含3-6个碳原子的环烷基，取代或未取代的含2-10个碳原子的烯基；
R3选自独立的氢、卤素、羟基、氨基，硝基，氰基、取代或未取代的含1-6个碳原子的烷基、取代或未取代的含1-6个碳原子的烷氧基，取代或未取代的含1-6个碳原子的烷氨基，取代或未取代的含1-6个碳原子的酰氨基，或者以上基团的组合；
R4选自氧，连有一个取代基的亚胺基或连有两个取代基的季铵；
R5选自羟基，连有一个取代基的氨基或连有两个取代基的氨基；
所述的取代的C1-10亚烃基，所述的取代的含杂原子的C1-10的杂亚烃基，所述的取代的C1-6烃基、取代的含1-6个碳原子的烷基、取代的含1-6个碳原子的烷氧基，取代的含1-6个碳原子的烷氨基，取代的含1-6个碳原子的酰氨基、取代的含1-10个碳原子的烷基，取代的含3-6个碳原子的环烷基，取代的含2-10个碳原子的烯基中的取代是指被卤素、苯基和C1-10烷基中的一个或多个所取代，当存在多个取代基时，所述的取代基相同或不同。
本发明中，术语亚烃基是指含碳、氢两种原子，由相应的烃失去任意两个氢原子后剩下的基团https://baike.baidu.com/item/％E8％87％AA％E7％94％B1％E5％9F％BA/ 970597。术语亚烃基优选为亚烷基、亚烯基、亚炔基、亚环烷基、亚环烯基、亚环炔基或亚芳基。
本发明中，术语亚烯基是指链状亚烯基，其中碳碳双键的个数为一个或多个，碳碳双键的可位于链状亚烯基的任意位置。
本发明中，术语亚炔基是指链状亚炔基，其中碳碳三键的个数为一个或多个，碳碳三键的可位于链状亚炔基的任意位置。
本发明中，术语亚环烯基是指环状亚烯基，其中碳碳双键的个数为一个或多个，碳碳双键的可位于环状亚烯基的任意位置。
本发明中，术语亚环炔基是指环状亚炔基，其中碳碳三键的个数为一个或多个，碳碳三键的可位于环状亚炔基的任意位置。
本发明中，术语杂亚烃基是指本发明中的亚烃基的任意位置插入一个或多个杂原子，其中，亚烃基的定义同前所述。
在本发明一优选实施方案中，L选自取代或未取代的C1-10亚烃基。
在本发明一优选实施方案中，环A选自苯环，吲哚环，异吲哚环，嘧啶环，嘌呤环，呋喃环，噻酚环，吡咯环，吡啶环，恶唑环，异恶唑环，噻唑环，吡唑环，咪唑环，萘环，6-13个原子的稠合芳香杂环；当环中含有杂原子时，杂原子选自氧、氮和硫中的一个或多个；当杂原子为多个时，杂原子相同或不同。
在本发明一优选实施方案中，X选自碳原子，氮原子，氧原子。
在本发明一优选实施方案中，R1选自独立的氢、卤素、羟基、氨基，硝基，氰基、取代或未取代的含1-6个碳原子的烷基、取代或未取代的含1-6个碳原子的烷氧基，取代或未取代的含1-6个碳原子的烷氨基，取代或未取代的含1-6个碳原子的酰氨基，或者以上基团的组合。
在本发明一优选实施方案中，R2选自氢，甲基，取代或未取代的含1-10个碳原子的烷基。
在本发明一优选实施方案中，R3选自独立的氢、卤素、羟基、氨基，硝基，氰基、取代或未取代的含1-6个碳原子的烷基、取代或未取代的含1-6个碳原子的烷氧基，取代或未取代的含1-6个碳原子的烷氨基，取代或未取代的含1-6个碳原子的酰氨基，或者以上基团的组合。
在本发明一优选实施方案中，R4选自氧，连有一个取代基的亚胺基或连有两个取代基的季铵。
在本发明一优选实施方案中，R5选自羟基，连有一个取代基的氨基或连有两个取代基的氨基。
本发明中，所述的取代的C1-10亚烃基，所述的取代的含杂原子的C1-10的杂亚烃基，所述的取代的C1-6烃基中的取代是指被卤素、苯基和C1-10烷基中的一个或多个所取代，当存在多个取代基时，所述的取代基相同或不同。
本发明中，术语亚烃基是指含碳、氢两种原子，由相应的烃失去任意两个氢原子后剩下的基团https://baike.baidu.com/item/％E8％87％AA％E7％94％B1％E5％9F％BA/ 970597。术语亚烃基优选为亚烷基、亚烯基、亚炔基、亚环烷基、亚环烯基、亚环炔基或亚芳基。
本发明中，术语亚烯基是指链状亚烯基，其中碳碳双键的个数为一个或多个，碳碳双键的可位于链状亚烯基的任意位置。
本发明中，术语亚炔基是指链状亚炔基，其中碳碳三键的个数为一个或多个，碳碳三键的可位于链状亚炔基的任意位置。
本发明中，术语亚环烯基是指环状亚烯基，其中碳碳双键的个数为一个或多个，碳碳双键的可位于环状亚烯基的任意位置。
本发明中，术语亚环炔基是指环状亚炔基，其中碳碳三键的个数为一个或多个，碳碳三键的可位于环状亚炔基的任意位置。
本发明中，术语杂亚烃基是指本发明中的亚烃基的任意位置插入一个或多个杂原子，其中，亚烃基的定义同前所述。
在本发明一优选实施方案中，式1所示荧光探针选自以下任一所示化合物：
本发明提供了荧光探针的制备方法，本发明中以实施例1和实施例4代表，按下述流程制得通式1化合物结构的荧光探针，
本发明的进一步目的是式1所示荧光探针、或其药学上可以接受的盐、其立体异构体或互变异构体、或前药在制备对各类细胞内DNA甲基转移酶1(DNMT1)定性和定量检测制剂中的用途。
本发明的进一步目的是式1所示荧光探针、或其药学上可以接受的盐、其立体异构体或互变异构体在制备用于与DNMT1高表达相关疾病的诊断或预测的药物中的应用；所述的与DNMT1高表达相关疾病优选为各种恶性肿瘤，更优选为宫颈癌，口腔粘膜癌，皮肤癌等。
附图说明
图1.小分子RG108与DNMT1蛋白的亲和力拟和结果。
图2.实施例与DNMT1蛋白的亲和力测定实时传感图。
图3：荧光探针对不同DNMT1表达量的Hela细胞的定量检测。
图4.实施例1的荧光探针在临床细胞中的应用。
具体实施例
本发明中以实施例1和实施例4代表，按下述流程制备实施例1-15所示荧光探针：
(S)-2-(4-羟基-1,3-二氧代异吲哚啉-2-基)-3-(1H-吲哚-3-基)丙酸甲酯(3)的制备
取化合物1(L-色氨酸甲酯盐酸盐))在50毫升圆底烧瓶中加入540mg(2.1mmol)，加入15毫升乙酸，150mg乙酸钾(1.53mmol)，然后加入化合物2(4-羟基邻苯甲酸酐)450mg(2.75mmol)。将溶剂加热至145℃，27小时后，反应完成。蒸发溶剂，得到黄色固体，硅胶柱色谱，得到纯产物506.6mg，收率66％.1H-NMR(DMSO-d6,400MHz)δ＝11.22(s,1H),10.80(s，1H),7.60(d，J＝4Hz，1H),7.50(d,J＝4Hz,1H),7.24(d,J＝4Hz,1H),7.18(d,J＝4Hz，1H),7.13(d,J＝4Hz,1H),6.98(s,2H),6.88(d,J＝4Hz,1H),5.19(s,1H),3.72(s,3H),3.53(s,2H)；ESI-MS m/z 365.1(M+H+).
甲基-(S)-2-(4-(2-((叔丁氧基羰基)氨基)乙氧基)-1,3-二氧代异吲哚-2-基)-3-(1H-吲哚-3-基)丙酸乙酯(4)的制备
取化合物3 500mg(1.37mmol)，加入20ml DMF，K2CO3 220mg(1.65mmol)，KI 274mg(1.65mmol)，Boc-保护的2-溴乙胺370mg(1.65mmol)，80℃下反应。24小时后，反应结束。加入饱和盐水，用EA萃取，蒸发有机相，得到固体，硅胶柱色谱，得到纯产物(4)346mg，回收原料144mg，收率71％.1H-NMR(DMSO-d6,400MHz)δ＝7.91(s,1H),7.51(dd,J＝8Hz,2H),7.28(d,J＝4Hz,1H),7.20(d,J＝4Hz,1H),7.04(d,J＝4Hz,2H),6.99(d,J＝8Hz,1H),6.96(s,1H),5.22(s,1H),5.15(t,J＝8Hz,1H),4.07(s,2H),3.71(s,3H),3.65(d,2H),3.49(s,1H),1.37(s,9H)；ESI-MS m/z 530.3(M+Na+).
(S)-2-(4-(2-((叔丁氧基羰基)氨基)乙氧基)-1,3-二氧代异吲哚啉-2-基)-3-(1H-吲哚-3-基)丙酸(5)的制备
化合物4 0.66mmol溶于3ml EA中，加入LiI 351.5mg(2.63mmol)，并在80℃回流.1天后，在瓶壁上观察到黄色固体。加入20ml EA后沉淀出大量固体，过滤并用EA洗涤并干燥，得到纯固体化合物5:97mg，收率31％.1H-NMR(DMSO-d6,400MHz)δ＝10.60(s，1H))，7.66(t，J＝8Hz，1H)，7.47(d，J＝8Hz，1H)，7.37(d，J＝8Hz，1H)，7.28(d，J＝8Hz，1H)，7.24(d，J＝8Hz，1H)，7.00(d，J＝8Hz，1H)，6.96(d，J＝8Hz，1H)，6.92(d，J＝8Hz，1H)，6.86(s，1H)，4.57(s，1H)，4.13(s，2H)，3.59(s，2H)，3.27(s，2H)，1.37(s，9H)；ESI-MS m/z 492.2(M-H+)。
(S)-2-(4-(2-氨基乙氧基)-1,3-二氧代异吲哚啉-2-基)-3-(1H-吲哚-3-基)丙酸(6)的制备
化合物5(0.3mmol)溶于2ml DCM：三氟乙酸＝20：1的溶剂中，并在室温下搅拌。2小时后，反应完成。减压除去溶剂，得到粗产物化合物6约117mg，未经纯化，直接进行下一步缩合反应，ESI-MS m/z 394.2(M+H+)。
(S)-5-(3-(2-((2-(1-羧基-2-(1H-吲哚-3-基)乙基)-1,3-二氧代异吲哚啉-4-基)氧基)乙基)硫脲基)-2-(6-羟基-3-氧代-3H-黄嘌呤-9-基)苯甲酸(实施例1)的制备
取化合物6(0.18mmol)，DMF 1ml，K2CO3 30mg(0.22mmol)，然后加入FITC 35mg(0.09mmol)并在室温下反应2小时。通过加入乙醚使固体沉淀，经反相柱色谱，得到10mg目标化合物(实施例1)，收率14.2％(化合物结构确证见实施例1)
(S)-2-(4-(2-氨基乙氧基)-1,3-二氧代异吲哚啉-2-基)-3-(1H-吲哚-3-基)丙酸甲酯(7)的制备
化合物4(1.24mmol)溶于6ml DCM/三氟乙酸盐＝20/1的溶剂中，搅拌12小时，减压除去溶剂，柱色谱法纯化，得到化合物7 120mg，产率86.4％.1H-NMR(DMSO-d6,400MHz)δ＝10.90(s，1H)，8.53(s，1H)，8.15(s，1H)，7.52(s，1H)，7.39(s，1H)，7.33(s，1H)，7.26(s，1H)，7.17(s，1H)，7.07(s，2H)，7.00(s，1H)，4.61(s，1H)，4.15(s，2H)，3.60(s，3H)，3.45(s，2H)，3.19(s，2H)；ESI-MS m/z 408.2(M-H+)。
(S)-5-(3-(2-((2-(3-(1H-吲哚-3-基)-1-甲氧基-1-氧代丙-2-基)-1,3-二氧代异吲哚啉-4-氧乙基)硫脲基)-2-(6-羟基-3-氧代-3H-黄嘌呤-9-基)苯甲酸(实施例4)的制备
取化合物7(0.36mmol)，DMF 1ml，K2CO3 94加入mg(0.67mmol)，然后加入化合物FITC 108.8mg(0.28mmol)，室温下搅拌2小时。加入乙醚使固体沉淀，并进行柱外反相柱色谱，得到实施例4，红色固体25mg，收率11.2％(化合物结构确证见实施例4)。
实施例1.
制得(S)-5-(3-(3-((2-(1-羧基-2-(1H-吲哚-3-基)乙基)-1,3-邻苯二甲亚酰-4-基)氧基)乙基)硫脲基)-2-(6-羟基-3-氧代-3H-呫吨-9-基)苯甲酸
1H-NMR(DMSO-d6,400MHz)δ＝10.75(s,2H),10.20(s,2H),10.15(s,4H),8.28(s,2H),7.85(d,J＝8Hz,2H),7.78(d,J＝8Hz,2H),7.74(d,J＝8Hz,2H),7.58(d,J＝8Hz,2H),7.49(s,4H),7.36(d,J＝8Hz,2H),7.25(s,2H),7.19(s,1H),7.10(s,1H),7.01(s,4H),6.91(s,2H),6.68(s,4H),6.60(s,4H),6.57(s,4H),5.05(s,2H),4.33(d,J＝12Hz,4H),3.89(d,J＝12Hz,4H),3.53(s,4H)(共两组氢)；ESI-MS m/z 783.2(M-H+).。
实施例2.
制得(S)-5-(3-(3-((2-(1-羧基-2-(1H-吲哚-3-基)乙基)-1,3-邻苯二甲亚酰-4-基)氧基)丙基)硫脲基)-2-(6-羟基-3-氧代-3H-呫吨-9-基)苯甲酸
1H-NMR(DMSO-d6,400MHz)δ＝10.74(s,1H),10.14(s,2H),9.93(s,1H),8.28(s,1H),7.86(s,1H),7.79(s,1H),7.73(s,1H),7.57(s,1H),7.47(s,1H),7.40(d,J＝8Hz,1H),7.34(s,1H),7.25(d,J＝8Hz,1H),7.00(s,2H),6.90(s,1H)，6.67(s,2H)，6.60(s,2H)，6.57(s,2H)，5.04(s,1H)，4.15(s,2H),3.52(s,4H),1.99(s,2H)；13C-NMR(DMSO-d6,150MHz):δ(ppm)194.26,185.10,184.43,179.96,170.28,168.11,167.44,166.73,166.16,165.33,64.67,163.26,159.26,157.03,155.50,153.50,151.63,146.46,136.81,135.84,132.74,128.83,126.76,124.92,123.10,120.78,119.80,119.54,118.22,117.74,117.09,115.78,115.01,112.44,111.25,109.63,102.04,82.03,66.38,52.10,40.29,27.40,23.82；ESI-MSm/z 797.2(M+H+).。
实施例3.
制得(S)-5-(3-(3-((2-(1-羧基-2-(1H-吲哚-3-基)乙基)-1,3-邻苯二甲亚酰-4-基)氧基)己基)硫脲基)-2-(6-羟基-3-氧代-3H-呫吨-9-基)苯甲酸
1H-NMR(DMSO-d6,400MHz)δ＝10.75(s,2H),10.16(s,4H),10.03(s,1H),9.97(s,1H),8.25(s,1H),8.19(d,J＝8Hz,1H),7.88(d,J＝8Hz,1H),7.79(d,J＝8Hz,1H),7.73(s,2H),7.59(s,1H),7.47(m,4H),7.33(d,J＝8Hz,2H),7.26(d,J＝8Hz,2H),7.18(d,J＝8Hz,1H),7.01(s,4H),6.91(s,2H),6.68(s,4H),6.60(s,4H),6.58(s,4H),5.04(s,2H),4.14(d,J＝8Hz,4H),3.16(t,J＝8Hz,8H),1.72(m,4H),1.55(m,4H),1.38(m,4H),1.25(m,4H)；ESI-MS m/z 839.3(M+H+).。
实施例4.
制得(S)-5-(3-(3-((2-(1-甲氧羰基-2-(1H-吲哚-3-基)乙基)-1,3-邻苯二甲亚酰-4-基)氧基)乙基)硫脲基)-2-(6-羟基-3-氧代-3H-呫吨-9-基)苯甲酸
1H-NMR(DMSO-d6,400MHz)δ＝10.75(s,2H),10.20(s,2H),10.15(s,4H),8.30(s,1H),8.22(s,1H),7.85(d,J＝8Hz,1H),7.79(d,J＝8Hz,1H),7.73(d,J＝8Hz,2H),7.55(d,J＝8Hz,1H),7.48(d,J＝8Hz,4H),7.36(m,2H),7.24(d,J＝8Hz,2H),7.16(d,J＝8Hz,1H),7.00(d,J＝8Hz,4H),6.88(t,J＝8Hz,2H),6.66(s,4H),6.59(s,4H),6.56(s,4H),5.16(s,2H),4.33(d,J＝12Hz,4H),3.87(d,J＝12Hz,4H),3.69(s,6H),3.65(s,4H)；ESI-MS m/z797.2(M-H+).。
实施例5.
制得(S)-5-(3-(3-((2-(1-甲氧羰基-2-(1H-吲哚-3-基)乙基)-1,3-邻苯二甲亚酰-4-基)氧基)丙基)硫脲基)-2-(6-羟基-3-氧代-3H-呫吨-9-基)苯甲酸
1H-NMR(DMSO-d6,400MHz)δ＝11.03(s,1H),10.88(s,1H),10.33(s,2H),8.95(s,1H),8.46(s,1H),7.85(d,J＝8Hz,1H),7.76(d,J＝8Hz,1H),7.49(d,J＝8Hz,2H),7.33(d,J＝8Hz,1H),7.26(d,J＝8Hz,1H),7.16(d,J＝8Hz,1H),7.02(t,J＝8Hz,2H),6.91(d,J＝8Hz,1H),6.74(s,2H),6.57(s,4H)，5.16(s,1H)，4.28(s,2H)，3.69(s,4H)，3.52(s,3H)，2.04(s,2H)；13C-NMR(DMSO-d6,150MHz):δ(ppm)180.53,169.29,168.49,166.52,165.83,165.06,162.12,159.44,157.70,155.62,152.44,151.69,146.45,141.65,136.95,135.85,132.60,128.78,128.52,126.72,126.26,123.74,123.34,120.76,119.81,118.22,117.73,115.68,115.21,115.10,112.47,111.29,110.71,109.56,109.49,109.03,102.10,66.53,52.49,51.76,31.04,29.66,27.76,23.91；ESI-MS m/z 811.2(M+H+).。
实施例6.
制得(S)-5-(3-(3-((2-(1-甲氧羰基-2-(1H-吲哚-3-基)乙基)-1,3-邻苯二甲亚酰-4-基)氧基)己基)硫脲基)-2-(6-羟基-3-氧代-3H-呫吨-9-基)苯甲酸
1H-NMR(DMSO-d6,400MHz)δ＝10.77(s,2H),10.15(s,4H),9.99(s,1H),9.92(s,1H),8.24(s,1H),8.15(s,2H),7.87(d,J＝8Hz,1H),7.79(s,2H),7.72(s,2H),7.57(s,1H),7.45(m,4H),7.32(d,J＝8Hz,2H),7.25(d,J＝8Hz,2H),7.17(d,J＝8Hz,1H),7.02(s,4H),6.89(s,2H),6.61(s,4H),6.57(s,4H),5.16(s,2H),4.13(d,J＝8Hz,4H),3.69(s,6H),3.46(d,J＝8Hz,8H),1.72(m,4H),1.59(m,4H),1.40(m,4H),1.31(m,4H)；ESI-MS m/z 853.3(M+H+).。
实施例7.
制得(S)-4-(3-(2-((2-(1-羧基-2-(1H-咪唑-3-基)乙基)-1,3-邻苯二甲亚酰-4-基)氧基)乙基)硫脲基)-2-(6-羟基-3-氧代-3H-呫吨-9-基)苯甲酸
1H-NMR(DMSO-d6,400MHz)δ＝12.94(s,1H),10.54(s,1H),10.14(s,2H),8.28(s,1H),7.86(s,1H),7.79(s,1H),7.76(s,1H),7.73(s,1H),7.71(d,J＝8Hz,1H),7.57(s,1H),7.47(s,1H),7.40(d,J＝8Hz,1H),6.78(d,J＝8Hz,1H),6.67(s,2H),6.60(s,2H),6.57(s,2H),5.04(s,1H),4.15(s,2H),3.56(s,4H)；ESI-MS m/z 735.2(M+H+).。
实施例8.
制得(S)-4-(3-(2((2-(1-羧基-2-(2,4-二氧代-3,4-二氢嘧啶-1(2H)-基)乙基)-1,3-二恶吲哚啉-4-基)氧基)乙基)硫脲基)-2-(6-羟基-3-氧代-3H-黄嘌呤-9-基)苯甲酸
1H-NMR(DMSO-d6,400MHz)δ＝11.23(s,1H),10.75(s,1H),10.14(s,2H),8.28(s,1H),7.79(s,1H),7.76(s,1H),7.73(s,1H),7.57(s,1H),7.47(s,1H),7.44(d.J＝8Hz,1H),7.40(d.J＝8Hz,1H),6.67(s,2H),6.60(s,2H),6.57(s,2H),5.77(d.J＝8Hz,1H),5.04(s,1H),4.15(s,2H),3.22(s,4H)；ESI-MS m/z 778.2(M+H+).。
实施例9.
制得(S)-4-(3-(2-((2-(2-(1H-苯并[d]咪唑-3-基)-1-羧乙基)-1,3-二恶吲哚啉-4-基)(氧)乙基)硫脲基-2-(6-羟基-3-氧代-3H-黄嘌呤-9-基)苯甲酸
1H-NMR(DMSO-d6,400MHz)δ＝10.89(s,1H),10.14(s,2H),8.28(s,1H),8.28(s,1H),7.79(s,1H),7.76(s,1H),7.73(s,1H),7.57(s,1H),7.47(s,1H),7.40(d.J＝8Hz,1H),7.25(d,J＝8Hz,1H),7.00(s,2H),6.90(s,1H),6.67(s,2H),6.60(s,2H),6.57(s,2H),5.04(s,1H),4.15(s,2H),3.12(s,4H)；ESI-MS m/z 784.3(M+H+).。
实施例10.
制得(S)-4-(3-(2-((2-(2-(1H-苯并[d]咪唑-3-基)-1-羧乙基)-1,3-二恶吲哚啉-4-基)(氧)乙基)硫脲基-2-(6-羟基-3-氧代-3H-黄嘌呤-9-基)苯甲酸
1H-NMR(DMSO-d6,400MHz)δ＝11.54(s,1H),10.14(s,2H),8.28(s,1H),7.92(s,1H),7.79(s,1H),7.76(s,1H),7.73(s,1H),7.57(s,1H),7.47(s,1H),7.40(d,J＝8Hz,1H),7.25(d,J＝8Hz,1H),7.14(d,J＝8Hz,1H),7.00(s,1H),6.67(s,2H),6.60(s,2H),6.57(s,2H),5.04(s,1H),4.15(s,2H),3.99(s,4H)；ESI-MS m/z 786.2(M+H+).。
实施例11.
制得(S)-4-(3-(2-((2-(2-(5-溴-1H-苯并[d]咪唑-1-基)-1-羧乙基)-1,3-二恶吲哚啉-4-基)(氧)乙基)硫脲基-2-(6-羟基-3-氧代-3H-黄嘌呤-9-基)苯甲酸
1H-NMR(DMSO-d6,400MHz)δ＝10.23(s,1H),10.14(s,2H),8.28(s,1H),7.79(s,1H),7.76(s,1H),7.73(s,1H),7.60(s,1H),7.57(s,1H),7.47(s,1H),7.40(d,J＝8Hz,1H),7.30(s,1H),7.22(d,J＝8Hz,1H),7.13(d,J＝8Hz,1H),6.67(s,2H),6.60(s,2H),6.57(s,2H),5.04(s,1H),4.15(s,2H),3.78(s,4H)；ESI-MS m/z 862.1(M+H+).。
实施例12.
制得(S)-4-(3-(2-((2-(2-(6-氨基-9H-嘌呤-9-基)-1-羧乙基)-1,3-二恶吲哚啉-4-基)(氧)乙基)硫脲基-2-(6-羟基-3-氧代-3H-黄嘌呤-9-基)苯甲酸
1H-NMR(DMSO-d6,400MHz)δ＝10.27(s,1H),10.14(s,2H),9.94(s,2H),8.28(s,1H),8.18(s,1H),8.03(s,1H),7.79(s,1H),7.76(s,1H),7.73(s,1H),7.57(s,1H),7.47(s,1H),7.40(d,J＝8Hz,1H),6.67(s,2H)，6.60(s,2H),6.57(s,2H),5.04(s,1H),4.15(s,2H),2.98(s,4H)；ESI-MS m/z 801.2(M+H+).。
实施例13.
制得(S)-4-(3-(2-((2-(1-羧基-2-(1H-吲哚-3-基)乙基)-1,3-二恶吲哚啉-4-基)-氮烷基)乙基)硫脲基)-2-(6-羟基-3-氧代-3H-黄嘌呤-9-基)苯甲酸
1H-NMR(DMSO-d6,400MHz)δ＝10.56(s,1H),10.14(s,2H),9.93(s,1H),8.28(s,1H),7.86(s,1H),7.79(s,1H),7.73(s,1H),7.57(s,1H),7.47(s,1H),7.40(d,J＝8Hz,1H),7.34(s,1H),7.25(d,J＝8Hz,1H),7.00(s,2H),6.90(s,1H),6.67(s,2H),6.60(s,2H),6.57(s,2H),5.04(s,1H),4.15(s,2H),3.12(s,4H)；ESI-MS m/z 782.2(M+H+).。
实施例14.
制得(S)-5-(3-(3-(2-(1-羧基-2-(1H-吲哚-3-基)乙基)-1,3-二恶吲哚啉-4-基)丙基)硫脲基)-2-(6-羟基-3-氧代-3H-黄嘌呤-9-基)苯甲酸
1H-NMR(DMSO-d6,400MHz)δ＝10.90(s,1H),10.14(s,2H),9.93(s,1H),8.28(s,1H),7.86(s,1H),7.79(s,1H),7.73(s,1H),7.57(s,1H),7.47(s,1H),7.40(d,J＝8Hz,1H),7.34(s,1H),7.25(d,J＝8Hz,1H),7.00(s,2H),6.90(s,1H),6.67(s,2H),6.60(s,2H),6.57(s,2H),5.04(s,1H),4.15(s,2H),3.52(s,4H),1.97(s,2H)；ESI-MS m/z 781.3(M-H+).。
实施例15.
制得(S)-N-(9-(2-羧基-5-(3-(2-((2-(1-羧基-2-(1H-吲哚-3-基)乙基)-1,3-二恶吲哚啉-4-基)氧基)乙基)硫脲基)苯基)-6-(二乙基氨基)-3H-呫吨-3-亚基)-N-乙基乙烷胺
1H-NMR(DMSO-d6,400MHz)δ＝10.64(s,1H),9.93(s,1H),8.28(s,1H),7.86(s,1H),7.79(s,1H),7.73(s,1H),7.57(s,1H),7.47(s,1H),7.40(d,J＝8Hz,1H),7.34(s,1H),7.25(d,J＝8Hz,1H),7.00(s,2H),6.90(s,1H),6.67(s,2H),6.60(s,2H),6.57(s,2H),5.04(s,1H),4.15(s,2H),4.07(s,4H),3.52(s,4H),3.34(s,4H),1.41(s,6H)，1.32(s,6H)；ESI-MS m/z 894.5(M-H+).。
实施例16.
荧光探针与DNMT1的结合能力SPR实验结果：
实验操作：在pH分别为5.5、5.0、4.5和4.0的10mM醋酸钠缓冲液与终浓度为50.7μg/mL的DNMT1蛋白的结合能力进行检测，根据结果选择最佳偶联pH值，在该浓度下将DNMT1蛋白与CM5芯片进行偶联，再将DNMT1蛋白与小分子荧光探针的亲和力测定，以DNMT1抑制剂RG108为阳性对照，对亲和力测定数据进行分析；
实验结果：DNMT1蛋白与CM5芯片的最佳偶联pH值为5.0。小分子荧光探针与DNMT1之间的相互作用如表1和图1-2所示；
结果显示：所有测试小分子荧光探针与DNMT1之间的相互作用力显著高于RG108与DNMT1之间的相互作用力，高出近两个数量级。
表1：小分子荧光探针与DNMT1酶之间的结合能力
实施例17.小分子荧光探针在HeLa细胞中的显像
实验操作：以实施例1为例，小分子荧光探针与构建DNMT1高表达Hela细胞(高出3.5倍)和普通Hela细胞共孵育4h，并以DAPI染色后，共聚焦荧光显微镜拍照，以ImageJ软件进行定量分析；
实验结果：从直观的共聚焦图片以及间接的定量参数来看，该探针在DNMT1高表达的细胞中的富集能力显著高于普通细胞90％以上，如图3和表2所示，结果表明，所述小分子荧光探针可用于细胞内DNMT1含量的定性和定量检测。
表2.实施例1-6在不同细胞的细胞核中荧光强度比较
实施例18.小分子荧光探针在临床病理细胞中的显像
以实施例1中的(S)-5-(3-(3-((2-(1-羧基-2-(1H-吲哚-3-基)乙基)-1,3-邻苯二甲亚酰-4-基)氧基)乙基)硫脲基)-2-(6-羟基-3-氧代-3H-呫吨-9-基)苯甲酸为例，进行与临床二级病变的宫颈脱落细胞和正常宫颈脱落细胞共孵育4h后，并以DAPI染色后以ImageJ进行定量；实验结果显示：子宫颈病变细胞的DNMT1表达量高于正常细胞，也高于其他的癌变细胞；将该探针应用于临床上发生了二级病变的宫颈细胞，结果如图4和表3所示，探针在细胞核中的聚集程度远高于正常宫颈脱落细胞，高出了360％；结果表明本探针能实现靶向DNMT1的能力，并且具有临床应用价值，可实现在医疗中对于宫颈病变细胞病变程度的预测和诊断。
表3.实施例1的荧光探针在不同临床细胞的细胞核中荧光强度比较
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Claims (4)
1.一类下述通式1所示化合物，或其药学上可以接受的盐：
其中，
L选自未取代的C1-10亚烷基；
环A选自吲哚环；
X选自氧原子；
R1选自氢；
R2选自氢或者甲基；
R3选自氢；
R4选自氧；
R5选自羟基。
2.如权利要求1所述的通式1所示化合物，或其药学上可以接受的盐，其特征在于，
所述的式1所示化合物选自以下任一所示化合物：
3.一种如权利要求1或2所述的式1所示化合物、或其药学上可以接受的盐在制备宫颈癌细胞内DNA甲基转移酶1定性定量检测制剂中的应用。
4.一种如权利要求1或2所述的式1所示化合物、或其药学上可以接受的盐在制备用于与甲基转移酶1高表达的宫颈癌的诊断和预测的药物中的应用。
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017039318A1 (en) * | 2015-09-01 | 2017-03-09 | Kainos Medicine, Inc. | Benzimidazole derivatives for dna methylation inhibitors |
| CN106536509A (zh) * | 2014-06-16 | 2017-03-22 | 基础应用医学研究基金会 | 作为组蛋白甲基转移酶和dna甲基转移酶的双重抑制剂的新型化合物 |
| CN106977425A (zh) * | 2017-04-01 | 2017-07-25 | 清华大学深圳研究生院 | 具有dna甲基转移酶和组蛋白去乙酰化酶抑制活性的异羟肟酸衍生物及其制备方法与应用 |
-
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106536509A (zh) * | 2014-06-16 | 2017-03-22 | 基础应用医学研究基金会 | 作为组蛋白甲基转移酶和dna甲基转移酶的双重抑制剂的新型化合物 |
| WO2017039318A1 (en) * | 2015-09-01 | 2017-03-09 | Kainos Medicine, Inc. | Benzimidazole derivatives for dna methylation inhibitors |
| CN106977425A (zh) * | 2017-04-01 | 2017-07-25 | 清华大学深圳研究生院 | 具有dna甲基转移酶和组蛋白去乙酰化酶抑制活性的异羟肟酸衍生物及其制备方法与应用 |
Non-Patent Citations (4)
| Title |
|---|
| Hydrolysis of Phthalyl Amino Acid Esters of Fluoroscein in the Presence of Leucine Aminopeptidasel (36421);JOHN J. THO MAS et al.;《HYDROLYSIS OF ESTERS OF FLUORESCEIN》;19711231;第140卷;第179-182页 * |
| Imaging Nanometer-Sized a-Synuclein Aggregates by Superresolution Fluorescence Localization Microscopy;M. Julia Roberti et al.;《Biophysical Journal》;20120430;第102卷;第1598-1607页 * |
| Multicolor Far-Field Fluorescence Nanoscopy through Isolated Detection of Distinct Molecular Species;Mariano Bossi et al.;《Nano Letters》;20080722;第8卷(第8期);第2463-2468页 * |
| Rhodamine Spiroamides for Multicolor Single-Molecule Switching Fluorescent Nanoscopy;Vladimir N. Belov et al.;《Chem. Eur. J.》;20091231;第15卷;第10762-10776页 * |
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