CN113444038B - 一类2-芳基异烟酸酰胺类lsd1/hdac双靶点抑制剂、其制备方法及应用 - Google Patents

一类2-芳基异烟酸酰胺类lsd1/hdac双靶点抑制剂、其制备方法及应用 Download PDF

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CN113444038B
CN113444038B CN202110766466.2A CN202110766466A CN113444038B CN 113444038 B CN113444038 B CN 113444038B CN 202110766466 A CN202110766466 A CN 202110766466A CN 113444038 B CN113444038 B CN 113444038B
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段迎超
张少杰
关圆圆
于童
靳林峰
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Xinxiang Medical University
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    • C—CHEMISTRY; METALLURGY
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    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
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    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
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Abstract

本发明涉及一类2‑芳基异烟酸酰胺类LSD1/HDAC双靶点抑制剂、其制备方法及在制备抗肿瘤药物中的应用，属于药物化学技术领域。所述的化合物具有如下通式：
Figure DDA0003151843380000011
其中，R1优选OH、H；R2优选OH、OCH3、F、H、CH3；R3优选H、OH、Cl、OCH3、NH2、
Figure DDA0003151843380000012
R4优选H、OH、CF3、OCH3、CH3、
Figure DDA0003151843380000013

Description

一类2-芳基异烟酸酰胺类LSD1/HDAC双靶点抑制剂、其制备方 法及应用
技术领域
本发明具体涉及一类2-芳基异烟酸酰胺类LSD1/HDAC双靶点抑制剂、其制备方法及在制备抗肿瘤药物中的应用，属于药物化学技术领域。
背景技术
组蛋白赖氨酸特异性去甲基化酶1(LSD1)是一个黄素腺嘌呤二核苷酸依赖性的氨基氧化酶，其主要功能是特异性的去除组蛋白H3K4上的单、双甲基化修饰，抑制基因的转录。通过和雌激素受体或者雄激素受体相互作用，LSD1还可以去除H3K9上的单、双甲基化修饰，从而激活下游基因的转录。此外，LSD1还能去除非组蛋白如p53、DNMT1、STAT3、 E2F1、MYPT1、ERa和HIF-1的甲基化修饰，进一步调节其下游基因的稳定性和活性。LSD1 在急性髓性白血病、前列腺癌、肺癌、胃癌、雌激素受体阴性乳腺癌、结肠癌、滑膜肉瘤和神经母细胞瘤等多种肿瘤中的表达水平显著升高，并且和肺癌、白血病、结肠癌和乳腺癌等多种恶性肿瘤的不良预后密切相关。采用RNA干扰技术敲除LSD1的表达或者用小分子化合物抑制LSD1的活性，均可以抑制肿瘤细胞的增殖、转移和侵袭，是目前抗肿瘤药物研发的热点靶标之一，目前已有IMG-7289，GSK2879552，TAK-418，ORY-2001， INCB059872，CC-90011和SP-2577等七个LSD1小分子抑制剂进入I期、II期临床试验，用于治疗急性髓系白血病和非小细胞肺癌。
组蛋白去乙酰化酶(HDAC)是一类重要的组蛋白去乙酰化酶，主要负责从组蛋白尾部赖氨酸残基上去除乙酰化修饰，沉默基因转录。在白血病、淋巴癌、宫颈癌、结直肠癌，乳腺癌等多种恶性肿瘤中，HDAC家族成员的表达水平与活性都有明显上调，并且白血病、淋巴癌、宫颈癌、结直肠癌等多种恶性肿瘤的不良预后与HDAC的表达水平呈正相关。目前已经有Vorinostat，Romidepsin，Belinostat，Panobinostat和Chidamide等5个HDAC 小分子抑制剂被FDA和CFDA批准上市，用于治疗恶性淋巴瘤、骨髓瘤等多种肿瘤。此外，还有多个HDAC抑制剂候选药物正在进行临床试验。
作为两个重要的组蛋白表观遗传调控蛋白，LSD1和HDAC都与癌症的发生、发展密切相关，并且二者之间存在着密切的串话关联关系。LSD1和HDAC1/2共同存在于NuRD、CoREST、Sin3A多种共抑制复合蛋白中，参与调控多种基因的转录。LSD1通过CoREST复合物与HDAC1以相互依赖的方式去乙酰化H4K16，从而调控胚胎干细胞和癌细胞的多能性。 LSD1与Sin3A/HDAC复合物可以协同抑制一系列促凋亡基因，维持乳腺癌对化疗的敏感性。HDAC1可以去除LSD1底物结合区K374的乙酰化修饰，进而促进LSD1和组蛋白H3的有效结合，增强LSD1的去甲基化酶活性，抑制下游靶基因的表达。在乳腺癌中，LSD1和HDAC5 的表达量均明显上调，HDAC5通过上调LSD1的去泛素酶USP28的表达，进而提高LSD1的稳定性和去甲基化活性，从而促进乳腺癌的发生和发展。相反，抑制HDAC5的活性或者敲除HDAC5则促进LSD1的泛素化降解，抑制乳腺癌细胞的增殖和侵袭。联合应用LSD1和 HDAC抑制剂对多种恶性肿瘤包括横纹肌肉瘤、胶质母细胞瘤、乳腺癌、尤文氏肉瘤和急性髓系白血病都显示出良好的协同抗肿瘤作用。因此，发现新型、高活性的LSD1/HDAC双靶点抑制剂，通过同时抑制LSD1、HDAC以及互相串话的信号通路转导，发挥“1+1>2”的协同抗肿瘤作用，有望发现新型高效的抗肿瘤先导化合物，对于研究LSD1和HDAC的生理学功能、研发新型高效的抗肿瘤药物具有十分重要的意义。为了发现新型的LSD1/HDAC双靶点抑制剂，探索合成一类2-芳基异烟酸酰胺类化合物，验证其LSD1、HDAC双靶点抑制活性和体外抗肿瘤活性为本申请的出发点，目前尚未见有该类化合物的合成、LSD1/HDAC抑制活性及抗肿瘤活性的报道。
发明内容
由上述可知，本发明的一个目的在于提供一类2-芳基异烟酸酰胺类化合物，为新药物筛选提供可能。
本发明的另一个目的在于提供此类2-芳基异烟酸酰胺类化合物的制备方法。
本发明的再一个目的在于提供所述2-芳基异烟酸酰胺类化合物以LSD1/HDAC为靶点在制备抗肿瘤药物中的应用。
为实现上述目的，本发明所涉及的2-芳基异烟酸酰胺类化合物的结构通式为：
Figure BDA0003151843370000021
通式I中，R1为OH、H中的任意一个；R2为OH、OCH3、F、H、CH3中的任意一个；R3为 H、OH、Cl、OCH3、NH2、
Figure BDA0003151843370000022
中的任意一个；R4为H、OH、CF3、OCH3、CH3、
Figure BDA0003151843370000023
Figure BDA0003151843370000024
中的任意一个；X为N或CH。
优选：通式I中，R1、R2、R3、R4和X所代表的取代基或原子如下表所示：
Figure BDA0003151843370000025
Figure BDA0003151843370000031
为实现上述第二个目的，本发明化合物的合成反应流程如下所示：
Figure BDA0003151843370000032
通式I化合物的合成路线：
化合物3的制备方法:化合物1和4-氨甲基苯甲酸甲酯盐酸盐(化合物2)在N,N- 二甲基甲酰胺(DMF)中，O-苯并三氮唑-四甲基脲六氟磷酸盐(HBTU)和碱性化合物存在下，室温搅拌反应，反应结束后，反应体系加入水和乙酸乙酯萃取，合并有机相，用水、饱和NaCl水溶液洗涤，无水硫酸钠干燥，过滤，滤液真空浓缩，浓缩物经柱层析分离，得化合物3。其中，所述强碱性化合物选自N,N-二异丙基乙胺、三乙胺中的一种。
化合物4的制备方法:化合物3和各种取代苯硼酸或取代苯硼酸频那醇酯或者取代吡啶硼酸，在甲苯中，碱性化合物和钯催化剂存在下，加热搅拌反应，反应结束后，反应体系加入水和乙酸乙酯萃取，合并有机相，用水、饱和NaCl水溶液洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，浓缩物经柱层析分离，得化合物4。其中，所述碱性化合物选自碳酸钾、碳酸钠、碳酸氢钠、碳酸铯、磷酸钾、氢化钠中的一种，所述的钯催化剂选自四(三苯基膦)钯、醋酸钯、双(二亚苄基丙酮)钯、二氯化钯中的一种。
化合物5的制备方法:在化合物4中，当R4为羟基时，化合物4和4-(溴甲基)哌啶-1-甲酸叔丁酯或者4-(2-溴乙基)吗啡啉氢溴酸盐，在DMF中，碱性化合物存在下，加热搅拌反应，反应结束后，反应体系加入水和乙酸乙酯萃取，合并有机相，用水、饱和NaCl水溶液洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，浓缩物经柱层析分离，得化合物5。其中，所述碱性化合物选自碳酸钾、碳酸钠、碳酸铯、氢氧化钠、氢氧化钾中的一种。
化合物I的制备方法:在二氯甲烷溶液中，化合物4或化合物5和NH2OK/NH2OH的甲醇溶液反应，反应结束后，反应体系真空浓缩，浓缩物加水溶解，用稀盐酸调pH为5-6，抽滤，洗涤，收集固体，用甲醇重结晶得化合物I。
本发明优点：本发明合成的2-芳基异烟酸酰胺类化合物均具有较强的LSD1/HDAC双重抑制活性和体外抗肿瘤活性，且对LSD1具有很好的选择性。本发明所报道的多个化合物对 HDAC1的IC50值小于2nM，是阳性药物SAHA的7倍。该类LSD1/HDAC双靶点抑制剂对人白血病THP-1、MOLT-4和MV4:11细胞株显示出很好的体外抗肿瘤活性，多个化合物的体外抗肿瘤活性明显优于阳性药物SAHA，特别是化合物I-5和I-8对THP-1的活性分别是SAHA 的12倍和18倍。本发明化合物代表着一类具有全新结构的高活性的LSD1/HDAC双靶点抑制剂，为LSD1/HDAC双靶点抑制剂类药物的研发提供了基础，为LSD1和HDAC的生物学功能研究提供了有效工具，可作为进一步开发的候选或者先导化合物用于开发抗肿瘤、抗病毒、抗艾滋病等疾病治疗药物，且合成方法简单，有利于推广应用。
具体实施方式
下面结合反应路线举实施例对本发明技术方案作详细说明。
实施例1 4-((2-溴异烟酸酰胺)甲基)苯甲酸甲酯(3a)的合成
Figure BDA0003151843370000041
在50mL两口烧瓶中加入化合物1a(1212.1mg,6.0mmol),化合物2(1451.9mg,7.2mmol),HBTU(2502.9mg,6.6mmol),用无水DMF(8mL)溶解,氮气保护,然后加入N,N-二异丙基乙胺(1705.9mg,13.2mmol),加毕,室温搅拌反应1.5小时,然后向反应体系中加入水和乙酸乙酯萃取,合并乙酸乙酯层,分别用水、饱和食盐水洗涤,无水硫酸钠干燥,过滤,滤液减压浓缩,浓缩物经硅胶柱柱层析分离纯化(石油醚：丙酮＝5:1) 得化合物3a(850.4mg),白色固体,产率:40.6％,Mp:114-115℃.1H NMR(400MHz, DMSO-d6)δ9.51(t,1H,J＝6.0Hz),8.57(d,1H,J＝5.2Hz),8.06(s,1H), 7.94(d,2H,J＝8.4Hz),7.85(dd,1H,J1＝1.2Hz,J2＝4.8Hz),7.48(d,2H, J＝8.4Hz),4.57(d,2H,J＝6.0Hz),3.85(s,3H).13C NMR(101MHz,DMSO-d6) δ166.53,163.77,151.75,144.86,144.65,142.37,129.78,128.79,127.99,126.11,121.72,52.56,43.10.HRMS(ESI)calcd for C15H13BrN2NaO3[M+Na]+: 371.0002,Found:371.0004.
实施例2 4-((2-溴-5-氟异烟酸酰胺)甲基)苯甲酸甲酯(3b)的合成
Figure BDA0003151843370000051
按照实施例1的方法,用化合物1b(660.0mg,3.0mmol)替换1a,得目标化合物3b(900.1mg),白色固体,产率:81.7％,Mp:131-132℃.1H NMR(400MHz,DMSO-d6)δ 9.39(t,1H,J＝5.6Hz),8.61(s,1H),7.96(d,2H,J＝8.0Hz),7.90(d,1H, J＝5.2Hz),7.49(d,2H,J＝8.0Hz),4.56(d,2H,J＝5.6Hz),3.85(s,3H). 13C NMR(101MHz,DMSO-d6)δ166.53,161.38,155.84(d,JC-F＝259.6Hz),144.51, 140.32(d,JC-F＝27.1Hz),136.12(d,JC-F＝2.9Hz),134.71(d,JC-F＝15.2Hz), 129.80,128.83,127.91,127.87,52.58,42.94.HRMS(ESI)calcdfor C15H12BrFN2NaO3[M+Na]+:388.9908,Found:388.9904.
实施例3 4-((2-(3-羟基苯基)异烟酸酰胺)甲基)苯甲酸甲酯(4a)的合成
Figure BDA0003151843370000052
在50mL两口圆底烧瓶中,加入化合物3a(500.0mg,1.4mmol),甲苯(5mL),乙醇(5mL),H2O(1.3mL),K2CO3(359.3mg,2.6mmol),Pd(PPh3)4(162.0mg,0.14mmol) 和3-羟基苯硼酸(235.0mg,1.7mmol),氮气保护下,92℃搅拌反应4小时,反应结束后,将反应体系冷却至室温,用水和乙酸乙酯萃取,合并乙酸乙酯层,再分别用水、饱和食盐水洗涤,无水硫酸钠干燥,干燥完毕,抽滤,滤液减压浓缩,浓缩物用硅胶柱柱层析分离纯化(石油醚:乙酸乙酯＝2:1),得化合物4a(312.7mg),产率:61.5％,白色固体,Mp:185-186℃.1H NMR(400MHz,DMSO-d6)δ9.62(s,1H),9.53(t,1H,J＝ 6.0Hz),8.80(d,1H,J＝4.8Hz),8.31(d,1H,J＝1.6Hz),7.95(d,2H,J＝ 8.4Hz),7.35(dd,1H,J1＝1.6Hz,J2＝5.2Hz),7.57-7.55(m,2H),7.50(d,2H, J＝8.0Hz),7.32(t,1H,J＝8.0),6.87(dd,1H,J1＝1.6Hz,J2＝7.2Hz), 4.62(d,2H,J＝5.6Hz),3.84(s,3H).13C NMR(101MHz,DMSO-d6)δ166.56, 165.25,158.32,157.30,150.73,145.22,142.48,140.01,130.32,129.81,128.73, 127.95,120.68,117.89,116.93,113.95,52.56,43.02.HRMS(ESI)calcd for C21H19N2O4[M+H]+:363.1339,Found:363.1341.
实施例4 4-((2-(4-羟基苯基)异烟酸酰胺)甲基)苯甲酸甲酯(4b)的合成
Figure BDA0003151843370000061
按照实施例3的方法,用4-羟基苯硼酸(235.0mg,1.7mmol)替换3-羟基苯硼酸, 得化合物4b(417.4mg),白色固体,产率:81.3％,Mp:169-170℃.1H NMR(400MHz, DMSO-d6)δ9.85(s,1H),9.50(t,1H,J＝6.0Hz),8.74(d,1H,J＝5.2Hz), 8.27(s,1H),8.02(d,2H,J＝8.8Hz),7.96(d,2H,J＝8.0Hz),7.67(dd,1H, J1＝1.2Hz,J2＝4.8Hz),7.51(d,2H,J＝8.0Hz),6.91(d,2H,J＝8.4Hz), 4.63(d,2H,J＝6.0Hz),3.85(s,3H).13C NMR(101MHz,DMSO-d6)δ166.56, 165.45,159.36,157.42,150.53,145.26,142.40,129.81,129.58,128.73,128.62, 127.94,119.48,116.79,116.04,52.55,43.00.HRMS(ESI)calcd forC21H18N2NaO4 [M+Na]+:385.1159,Found:385.1158.
实施例5 4-((2-(2-羟基苯基)异烟酸酰胺)甲基)苯甲酸甲酯(4c)的合成
Figure BDA0003151843370000062
按照实施例3的方法,用2-羟基苯硼酸(235.0mg,1.7mmol)替换3-羟基苯硼酸, 得化合物4c(313mg),淡黄色固体,产率:61.7％,Mp:180-181℃.1H NMR(400MHz, DMSO-d6)δ13.69(s,1H),9.62(t,1H,J＝6.0Hz),8.79(dd,1H,J1＝0.8Hz, J2＝5.2Hz),8.61(t,1H,J＝5.2Hz),8.10(dd,1H,J1＝1.6Hz,J2＝8.4Hz), 7.96(d,2H,J＝8.4Hz),7.84(dd,1H,J1＝1.2Hz,J2＝5.2Hz),7.52(d,2H, J＝8.4Hz),7.38-7.33(m,1H),7.00-6.96(m,2H),4.65(d,2H,J＝6.0Hz), 3.85(s,3H).13C NMR(101MHz,DMSO-d6)δ166.55,164.90,159.38,157.97,147.83,145.07,143.36,132.22,129.82,128.77,128.00,127.83,120.38,119.48,119.43,118.41,117.94,52.57,43.11.HRMS(ESI)calcd for C21H17N2O4[M-H]-: 361.1194,Found:361.1190.
实施例6 4-((2-(2-甲氧基苯基)异烟酸酰胺)甲基)苯甲酸甲酯(4d)的合成
Figure BDA0003151843370000071
按照实施例3的方法,用2-甲氧基苯硼酸(258.3mg,1.7mmol)替换3-羟基苯硼酸,得化合物4d(379.9mg),无色油状物,产率:72.1％.1H NMR(400MHz,DMSO-d6)δ 9.46(t,1H,J＝6.0Hz),8.82(d,1H,J＝5.2Hz),8.26(t,1H,J＝1.2Hz), 7.96(d,2H,J＝8.0Hz),7.76-7.71(m,2H),7.49(d,2H,J＝8.0Hz),7.47- 7.42(m,1H),7.19(d,1H,J＝8.0Hz),7.09(t,1H,J＝7.6Hz),4.60(d,2H, J＝6.0Hz),3.85(s,6H).13C NMR(101MHz,DMSO-d6)δ166.56,165.55,157.19, 156.65,150.41,145.29,141.48,131.20,130.93,129.79,128.70,128.38,127.90, 122.79,121.12,119.81,112.37,56.11,52.55,43.01.HRMS(ESI)calcdfor C22H20N2NaO4[M+Na]+:399.1315,Found:399.1312.
实施例7 4-((2-(4-甲氧基苯基)异烟酸酰胺)甲基)苯甲酸甲酯(4e)的合成
Figure BDA0003151843370000072
按照实施例3的方法,用化合物4-甲氧基苯硼酸(258.3mg,1.7mmol)替换3-羟基苯硼酸,得目标化合物4e(320.5mg),白色固体,产率:60.8％,Mp:142-143℃.1H NMR(400MHz,CDCl3)δ8.66(d,1H,J＝5.2Hz),8.02(s,1H),7.95(d,2H,J ＝8.4Hz),7.92(d,2H,J＝8.8Hz),7.44(dd,1H,J1＝1.2Hz,J2＝4.8Hz), 7.35(d,2H,J＝8.0Hz),7.23(t,1H,J＝5.2Hz),6.94(d,2H,J＝8.8Hz), 4.65(d,2H,J＝6.0Hz),3.88(s,3H),3.83(s,3H).13C NMR(101MHz,CDCl3)δ 166.81,166.10,160.87,158.20,150.21,142.86,142.01,130.99,130.08,129.49, 128.32,127.66,118.25,117.29,114.22,55.38,52.22,43.79.HRMS(ESI)calcd for C22H21N2O4[M+H]+:377.1496,Found:377.1496.
实施例8 4-((2-(4-甲基苯基)异烟酸酰胺)甲基)苯甲酸甲酯(4f)的合成
Figure BDA0003151843370000073
按照实施例3的方法,用化合物4-甲基苯硼酸(231.1mg,1.7mmol)替换3-羟基苯硼酸,得目标化合物4f(321.4mg),白色固体,产率:63.7％,Mp:152-153℃.1H NMR (400MHz,CDCl3)δ9.54(d,1H,J＝6.0Hz),8.80(d,1H,J＝4.8Hz),8.36(s, 1H),8.06(d,2H,J＝8.4Hz),7.96(d,2H,J＝8.0Hz),7.75(dd,1H,J1＝1.6 Hz,J2＝5.2Hz),7.51(d,2H,J＝8.0Hz),7.34(d,2H,J＝8.0Hz),4.63(d, 2H,J＝6.0Hz),2.38(s,3H).HRMS(ESI)calcd forC22H21N2O3[M+H]+:361.1547, Found:361.1544.
实施例9 4-((2-(4-三氟甲基苯基)异烟酸酰胺)甲基)苯甲酸甲酯(4g)的合成
Figure BDA0003151843370000081
按照实施例3的方法,用4-三氟甲基苯硼酸(332.1mg,1.7mmol)替换3-羟基苯硼酸,得化合物4g(356.0mg),产率:63.3％,白色固体,Mp:154-155℃.1H NMR(400 MHz,CDCl3)δ8.81(d,1H,J＝4.8Hz),8.16(t,1H,J＝1.2Hz),8.13(d,2H, J＝8.4Hz),7.99(d,2H,J＝8.4Hz),7.72(d,2H,J＝8.4Hz),7.58(dd,1H, J1＝1.2Hz,J2＝4.8Hz),7.39(d,2H,J＝8.4Hz),6.97(t,1H,J＝6.0Hz), 4.71(d,2H,J＝6.0Hz),3.90(s,3H).13C NMR(101MHz,CDCl3)δ166.78, 165.59,157.02,150.65,142.64,142.31,141.67,131.33(q,JC-F＝32.7Hz), 130.15,129.63,127.72,127.31,125.82(q,JC-F＝3.7Hz),124.05(q,JC-F＝273.3Hz),119.71,118.48,52.26,43.91.HRMS(ESI)calcd for C22H18F3N2O3[M+H]+: 415.1264,Found:415.1265.
实施例10 4-((2-(4-吗啉基苯基)异烟酸酰胺)甲基)苯甲酸甲酯(4h)的合成
Figure BDA0003151843370000082
按照实施例3的方法,用化合物4-吗啉苯硼酸频哪醇酯(491.6mg,1.7mmol)替换3-羟基苯硼酸,得化合物4h(199.3mg),白色固体,产率:33.0％,Mp:189-190℃.1H NMR(400MHz,CDCl3)δ8.73(dd,1H,J1＝1.2Hz,J2＝5.2Hz),8.05-8.01(m, 3H),7.98(d,2H,J＝9.2Hz),7.43-7.39(m,3H),6.98(d,2H,J＝8.8Hz), 6.71(t,1H,J＝6.0Hz),4.73(d,2H,J＝6.0Hz),3.91(s,3H),3.88(t,4H, J＝5.2Hz),3.25(t,4H,J＝5.2Hz).13C NMR(101MHz,CDCl3)δ166.72, 166.07,158.38,152.16,150.27,142.78,141.92,130.18,129.71,129.49,127.96, 127.73,117.72,116.94,115.10,66.77,52.19,48.54,43.87.HRMS(ESI)calcd for C25H26N3O4[M+H]+:432.1918,Found:432.1917.
实施例11 4-((2-(3-吗啉基苯基)异烟酸酰胺)甲基)苯甲酸甲酯(4i)的合成
Figure BDA0003151843370000091
按照实施例3的方法,用化合物3-吗啉苯硼酸频哪醇酯(491.6mg,1.7mmol)替换3-羟基苯硼酸,得化合物4i(211.4mg),白色固体,产率:35.0％,Mp:155-156℃.1H NMR(400MHz,CDCl3)δ8.74(d,1H,J＝5.2Hz),8.10(s,1H),7.98(d,2H,J ＝8.4Hz),7.62(t,1H,J＝2.0Hz),7.51(dd,1H,J1＝1.6Hz,J2＝5.2Hz), 7.44(d,1H,J＝7.6Hz),7.38(d,2H,J＝8.0Hz),7.34(d,1H,J＝7.6Hz), 7.04(d,1H,J＝5.2Hz),6.98(dd,1H,J1＝2.0Hz,J2＝8.0Hz),4.69(d,2H, J＝6.0Hz),3.89(s,3H),3.86(t,4H,J＝4.8Hz),3.22(t,4H,J＝4.8Hz). 13C NMR(101MHz,CDCl3)δ166.79,165.93,158.84,151.85,150.28,142.80,142.04,139.40,130.12,129.65,129.56,127.71,118.99,118.58,118.21,116.86,114.21,66.91,52.24,49.27,43.85.HRMS(ESI)calcd for C25H26N3O4[M+H]+: 432.1918,Found:432.1918.
实施例12 4-((2-(4-(4-甲基-1-哌嗪基)苯基)异烟酸酰胺)甲基)苯甲酸甲酯(4j)的合成
Figure BDA0003151843370000092
按照实施例3的方法,用化合物4-(4-甲基-1-哌嗪基)苯硼酸频哪醇酯(531.8mg,1.7mmol)替换3-羟基苯硼酸,得化合物4j(94.6mg),白色固体,产率:15.2％,Mp: 167-168℃.1H NMR(400MHz,CDCl3)δ9.51(t,1H,J＝6.0Hz),8.73(d,1H,J ＝5.2Hz),8.27(s,1H),8.04(d,2H,J＝8.4Hz),7.96(d,2H,J＝8.0Hz), 7.64(dd,1H,J1＝1.6Hz,J2＝5.2Hz),7.50(d,2H,J＝8.0Hz),7.05(d,2H, J＝8.4Hz),4.62(d,2H,J＝6.0Hz),3.85(s,3H),3.25(t,4H,J＝5.2Hz), 2.46(t,4H,J＝5.2Hz),2.23(s,3H).13C NMR(101MHz,CDCl3)δ166.56,165.46,157.33,152.19,150.54,145.29,142.30,129.81,128.71,128.40,127.94,119.29,116.53,115.14,54.95,52.57,47.75,46.25,42.99.HRMS(ESI)calcd forC26H29N4O3[M+H]+:445.2234,Found:445.2236.
实施例13 4-((2-(4-吗啉磺酰基)苯基)异烟酸酰胺)甲基)苯甲酸甲酯(4k)的合成
Figure BDA0003151843370000101
按照实施例3的方法,用化合物4-吗啉磺酰基苯硼酸(460.9mg,1.7mmol)替换3-羟基苯硼酸,得化合物4k(189.3mg),白色固体,产率:38.2％,Mp:216-217℃.1H NMR(400MHz,DMSO-d6))δ9.59(t,1H,J＝6.0Hz),8.91(d,1H,J＝5.2Hz), 8.51(t,1H,J＝1.2Hz),8.43(d,2H,J＝8.8Hz),7.96(d,2H,J＝8.4Hz), 7.91-7.87(m,3H),7.52(d,2H,J＝8.4Hz),4.65(d,2H,J＝6.0Hz),3.85(s, 3H),3.65(t,4H,J＝4.4Hz),2.92(t,4H,J＝4.4Hz).13C NMR(101MHz,DMSO- d6)δ166.55,165.07,155.49,151.17,145.11,143.02,142.94,135.43,129.81, 128.79,128.74,128.10,127.99,121.83,119.05,65.76,52.56,46.38,43.08. HRMS(ESI)calcd for C25H24N3O6S[M-H]-:494.1391,Found:494.1392.
实施例14 4-((2-(3-氯-4-甲氧基苯基)异烟酸酰胺)甲基)苯甲酸甲酯(4l)的合成
Figure BDA0003151843370000102
按照实施例3的方法,用化合物3-氯-4-甲氧基苯硼酸(316.9mg,1.7mmol)替换3-羟基苯硼酸得化合物4l(207.1mg),白色固体,产率:36.0％,Mp:154-155℃.1H NMR(400MHz,CDCl3)δ8.75(dd,1H,J1＝1.2Hz,J2＝5.2Hz),8.10(d,1H,J＝2.0 Hz),8.04-8.01(m,3H),7.92(dd,1H,J1＝2.0Hz,J2＝8.4Hz),7.48(dd,1H,J1＝1.6Hz,J2＝5.2Hz),7.42(d,2H,J＝8.4Hz),7.01(d,1H,J＝8.8Hz), 6.75(t,1H,J＝5.6Hz),4.73(d,2H,J＝6.0Hz),3.96(s,3H),3.92(s,3H). 13C NMR(101MHz,CDCl3)δ166.80,165.87,156.84,156.07,150.34,142.76, 142.13,131.76,130.10,129.54,128.80,127.71,126.40,123.01,118.78,117.30, 112.00,56.26,52.24,43.86.HRMS(ESI)calcd for C22H20ClN2O4[M+H]+:411.1106, Found:411.1105.
实施例15 4-((2-(3,4-二甲氧基苯基)异烟酸酰胺)甲基)苯甲酸甲酯(4m)的合成
Figure BDA0003151843370000103
按照实施例3的方法,用化合物3,4-二甲氧基苯硼酸(309.4mg,1.7mmol)替换3-羟基苯硼酸得化合物4m(275.4mg),黄色固体,产率:48.4％,Mp:132-133℃.1H NMR(400MHz,CDCl3)δ8.69(d,1H,J＝5.2Hz),8.09(t,1H,J＝1.2Hz),7.95(d, 2H,J＝8.4Hz),7.65(d,1H,J＝2.0Hz),7.52(d,1H,J＝2.0Hz),7.49(dd, 1H,J1＝1.2Hz,J2＝5.2Hz),7.40-7.34(m,3H),6.88(d,1H,J＝8.4Hz),4.67 (d,2H,J＝5.2Hz),3.93(s,3H),3.89(s,3H),3.88(s,3H).13C NMR(101MHz, CDCl3)δ166.80,166.06,158.09,150.33,150.14,149.26,142.91,142.01, 131.34,130.06,129.46,127.66,119.65,118.37,117.50,111.02,109.81,55.96, 55.93,52.22,43.80.HRMS(ESI)calcd for C23H21N2O5[M-H]-:405.1456,Found: 405.1456.
实施例16 4-((2-(2,4-二甲基苯基)异烟酸酰胺)甲基)苯甲酸甲酯(4n)的合成
Figure BDA0003151843370000111
按照实施例3的方法,用化合物2,4-二甲基苯硼酸(258.5mg,1.7mmol)替换3-羟基苯硼酸,得目标化合物4n(400.4mg),白色固体,产率:80.4％,Mp:88-89℃.1H NMR(400MHz,CDCl3)δ8.76(d,1H,J＝5.2Hz),8.00(d,2H,J＝8.4Hz), 7.73(dd,1H,J1＝1.2Hz,J2＝2.0Hz),7.54(dd,1H,J1＝1.6Hz,J2＝5.2Hz), 7.38(d,2H,J＝8.4Hz),7.26-7.25(m,1H),7.09(s,2H,J＝7.6Hz),7.09(s, 1H),7.07(d,1H,J＝8.0Hz),6.84(t,1H,J＝5.6Hz),4.69(d,2H,J＝5.6 Hz),3.90(s,3H),2.36(s,3H),2.32(s,3H).13C NMR(101MHz,CDCl3)δ166.76,165.87,161.35,149.93,142.77,141.50,138.61,136.71,135.68, 131.72130.14,129.61,129.57,127.69,126.73,121.33,118.52,52.22,43.82, 21.18,20.25.HRMS(ESI)calcd for C23H23N2O3[M+H]+:375.1703,Found:375.1701.
实施例17 4-((2-(2-氟-4-甲基苯基)异烟酸酰胺)甲基)苯甲酸甲酯(4o)的合成
Figure BDA0003151843370000112
按照实施例3的方法,用2-氟-4-甲基苯硼酸(269.4mg,1.8mmol)替换3-羟基苯硼酸,得目标化合物4o(490.1mg),白色固体,产率:89.3％,Mp:126-127℃.1H NMR (400MHz,CDCl3)δ8.76(d,1H,J＝5.2Hz),8.06(dd,1H,J1＝1.2Hz,J2＝2.4 Hz),7.98(d,2H,J＝8.0Hz),7.84(t,1H,J＝8.4Hz),7.54(dd,1H,J1＝ 1.6Hz,J2＝4.8Hz),7.37(d,2H,J＝8.0Hz),7.07-7.01(m,1H),6.95(d,1H,J ＝12.8Hz),4.68(d,2H,J＝5.6Hz),3.90(s,3H),2.39(s,3H).13C NMR(101 MHz,DMSO-d6)δ162.05,161.15,155.56(d,JC-F＝250.7Hz),149.85(d,JC-F＝2.5 Hz),145.63,138.10,137.24(d,JC-F＝8.6Hz),137.05,125.84(d,JC-F＝3.1Hz), 125.33 124.75 122.88 120.75(d JC-F＝3.0Hz)118.86(d JC-F＝11.3Hz)116.52(d,JC-F＝9.7Hz),114.76,112.00(d,JC-F＝22.8Hz)47.45,39.04,16.46. HRMS(ESI)calcd for C22H20FN2O3[M+H]+:379.1451,Found:379.1451.
实施例18 4-((2-(3-氯-4-吗啉苯基)异烟酸酰胺)甲基)苯甲酸甲酯(4p)的合成
Figure BDA0003151843370000121
按照实施例3的方法,用3-氯-4-吗啉苯硼酸(410.5mg,1.7mmol)替换3-羟基苯硼酸,得目标化合物4p(355mg),白色固体,产率:76.2％,Mp:147-148℃.1H NMR(400 MHz,CDCl3)δ8.70(dd,1H,J1＝0.8Hz,J2＝5.2Hz),8.06(d,2H,J＝2.0Hz), 7.97(d,2H,J＝8.4Hz),7.85(dd,1H,J1＝2.4Hz,J2＝8.4Hz),7.51(dd,1H, J1＝1.6Hz,J2＝4.8Hz),7.37(d,2H,J＝8.4Hz),7.25(t,1H,J＝5.6Hz), 7.05(d,1H,J＝8.8Hz),4.68(d,2H,J＝6.0Hz),3.90(s,3H),3.88(t,4H, J＝4.8Hz),3.10(t,4H,J＝4.8Hz),1.82(s,1H).13C NMR(101MHz,CDCl3)δ 166.79,165.83,156.81,150.37,150.03,142.82,142.14,133.93,130.10,129.52, 129.32,128.94,127.70,126.12,120.21,118.97,117.54,67.05,52.25,51.46,43.84.HRMS(ESI)calcd for C25H23ClN3O4[M-H]-:464.1383,Found:464.1380.
实施例19 4-((2-(2-甲基-4-氨基苯基)异烟酸酰胺)甲基)苯甲酸甲酯(4q)的合成
Figure BDA0003151843370000122
按照实施例3的方法,用2-甲基-3-氨基苯硼酸(256.6mg,1.7mmol)替换3-羟基苯硼酸,得化合物4q(420.5mg),无色油状物,产率:80％.1H NMR(400MHz,CDCl3)δ 8.63(dd,1H,J1＝0.8Hz,J2＝5.2Hz),7.89(d,2H,J＝8.4Hz),7.63(dd,1H, J1＝0.8Hz,J2＝1.6Hz),7.48(dd,1H,J1＝1.6Hz,J2＝5.2Hz),7.28(d,2H, J＝8.4Hz),7.17(t,1H,J＝6.0Hz),6.98(t,1H,J＝8.0Hz),6.65(d,2H,J ＝7.6Hz),4.56(d,2H,J＝6.0Hz),3.81(s,3H),1.96(s,3H),1.16(s,2H). 13C NMR(101MHz,CDCl3)δ166.84,165.91,161.63,149.73,145.34,142.94, 141.61,140.64,130.05,129.43,127.61,126.44,121.63,120.14,120.09,118.95, 115.50,52.23,43.72,14.08.HRMS(ESI)calcd for C22H22N3O3[M+H]+:376.1656,Found:376.1656.
实施例20 4-((6'-甲氧基-[2,3'-吡啶]-4-甲酰胺基)甲基)苯甲酸甲酯(4r)的合成
Figure BDA0003151843370000131
按照实施例3的方法,用2-甲氧基-5-吡啶硼酸(236.5mg,1.6mmol)替换3-羟基苯硼酸,得化合物4r(353.2mg),黄色固体,产率:72.6％,Mp:156-157℃.1H NMR(400 MHz,DMSO-d6)δ9.50(t,1H,J＝5.2Hz),8.95(s,1H),8.81(d,1H,J＝4.8 Hz),8.43(d,1H,J＝8.4Hz),8.36(s,1H),7.96(d,2H,J＝7.6Hz),7.76(d, 1H,J＝4.8Hz),7.51(d,2H,J＝7.6Hz),6.98(d,1H,J＝8.4Hz),4.63(d, 2H,J＝5.6Hz),3.93(s,3H),3.85(s,3H).13C NMR(101MHz,DMSO-d6)δ 166.54,165.23,164.75,155.15,150.88,146.11,145.16,142.67,137.86,129.80, 128.74,128.10,127.96,120.50,117.55,111.09,53.95,52.56,43.02.HRMS(ESI) calcd for C21H18N3O4[M-H]-:376.1303,Found:376.1302.
实施例21 4-((5-氟-2-(对甲苯基)异烟酸酰胺)甲基)苯甲酸甲酯(4s)的合成
Figure BDA0003151843370000132
按照实施例3的方法,用3b(514.0mg,1.4mmol)替换3a,用4-甲基苯硼酸(244.4mg,1.7mmol)替换3-羟基苯硼酸,得化合物4s(450.29mg),白色固体,产率: 85.0％,Mp:135-136℃.1H NMR(400MHz,CDCl3)δ8.61(d,1H,J＝2.0Hz),8.36 (d,1H,J＝6.0Hz),8.04(d,2H,J＝8.4Hz),7.91(d,2H,J＝8.0Hz),7.43(d, 2H,J＝8.4Hz),7.29(d,2H,J＝8.0Hz),7.19-7.12(m,1H),4.76(d,2H,J＝ 6.4Hz),3.92(s,3H),2.41(s,3H).13C NMR(101MHz,CDCl3)δ166.73,161.73 (d,JC-F＝2.6Hz),155.45(d,JC-F＝256.5Hz),155.13(d,JC-F＝4.4Hz),142.55, 139.56,138.90,138.62,134.80,130.18,129.66,127.79(d,JC-F＝10.4Hz), 127.59,126.76,121.09,52.20,43.93,21.30.HRMS(ESI)calcd for C22H20FN2O3[M+ H]+:379.1452,Found:379.1450.
实施例22 4-((5-氟-2-(4-甲氧基苯基)异烟酸酰胺)甲基)苯甲酸甲酯(4t)的合成
Figure BDA0003151843370000133
按照实施例21的方法,用化合物4-甲氧基苯硼酸(260.0mg,1.7mmol)替换3-羟基苯硼酸,得化合物4t(387.1mg),白色固体,产率:70.1％,Mp:128-129℃.1H NMR (400MHz,CDCl3)δ8.58(d,1H,J＝2.4Hz),8.32(d,1H,J＝6.0Hz),8.04(d, 2H,J＝8.0Hz),7.96(d,2H,J＝8.8Hz),7.43(d,2H,J＝8.4Hz),7.20-7.13 (m,1H),7.00(d,2H,J＝8.8Hz),4.76(d,2H,J＝6.4Hz),3.92(s,3H),3.87 (s,3H).13C NMR(101MHz,CDCl3)δ166.74,161.78(d,JC-F＝2.7Hz),160.80, 155.22(d,JC-F＝256.1Hz),154.81(d,JC-F＝4.0Hz),142.56,138.65(d,JC-F＝ 28.0Hz),130.23,130.17,129.66,128.23,127.79(d,JC-F＝10.3Hz),127.58,120.58,114.28,55.39,52.19,43.92.HRMS(ESI)calcd for C22H20FN2O4[M+H]+: 395.1407,Found:395.1400.
实施例23 4-((2-(4-(2-吗啉基乙氧基)苯基)异烟酸酰胺)甲基)苯甲酸甲酯(5a)的合成
Figure BDA0003151843370000141
在50mL两颈圆底烧瓶中加入碳酸铯(358.4mg,1.1mmol),加入无水N,N-二甲基甲酰胺(5mL),搅拌下加入化合物4b(362.4mg,1.0mmol)和化合物4-(2-溴乙基)吗啡啉氢溴酸盐(330.0mg,1.2mmol),70℃加热搅拌反应1小时.反应结束后,反应体系加入水和乙酸乙酯萃取,合并乙酸乙酯层,用水、饱和食盐水洗涤,无水硫酸钠干燥,干燥完毕,过滤,滤液减压浓缩,浓缩物经硅胶柱柱层析分离纯化(石油醚:丙酮＝4:1)得化合物5a(385.7mg),白色固体,产率:81.1％,Mp:132-133℃.1HNMR(400MHz,CDCl3) δ8.70(dd,1H,J1＝0.4Hz,J2＝5.2Hz),8.04(dd,1H,J1＝0.8Hz,J2＝1.6 Hz),7.98(d,2H,J＝8.4Hz),7.95(d,2H,J＝8.8Hz),7.45(dd,1H,J1＝1.6 Hz,J2＝9.2Hz),7.38(d,2H,J＝8.0Hz),7.10(t,1H,J＝5.6Hz),6.97(d, 2H,J＝6.8Hz),4.68(d,2H,J＝5.6Hz),4.15(t,2H,J＝5.6Hz),3.90(s,3H),3.73(t,4H,J＝4.4Hz),2.82(t,2H,J＝5.6Hz),2.59(t,4H,J＝4.4 Hz).13C NMR(101MHz,CDCl3)δ166.79,166.06,159.99,158.13,150.23,142.87, 142.03,131.18,130.09,129.51,128.33,127.67,118.28,117.33,114.84,66.89, 65.81,57.57,54.08,52.23,43.80.HRMS(ESI)calcd for C27H30N3O5[M+H]+: 476.2180,Found:476.2175.
实施例24叔丁基4-((4-(4-((4-(甲氧基羰基)苯基)氨基甲酰基)-2-吡啶基)苯氧基)甲基)-1-哌啶羧酸酯(5b)的合成
Figure BDA0003151843370000142
按照实施例23的方法,用化合物4-(溴甲基)哌啶-1-甲酸叔丁酯(140.2mg,1.2mmol)替换4-(2-溴乙基)吗啡啉氢溴酸盐,得化合物5b(253.0mg),白色固体,产率:45.2％,Mp:105-106℃.1H NMR(400MHz,CDCl3)δ8.71(d,1H,J＝4.8Hz),8.05 (t,1H,J＝1.2Hz),8.00(d,2H,J＝8.4Hz),7.96(d,2H,J＝8.8Hz),7.46 (dd,1H,J1＝1.6Hz,J2＝5.2Hz),7.40(d,2H,J＝8.4Hz),7.07(t,1H,J＝ 6.0Hz),6.95(d,2H,J＝8.8Hz),4.70(d,2H,J＝6.0Hz),4.15(brs,2H), 3.90(s,3H),3.83(d,2H,J＝6.4Hz),2.73(brs,2H),2.03-1.91(m,1H), 1.84-1.79(m,2H),1.46(s,9H),1.31-1.20(m,2H).13C NMR(101MHz,CDCl3)δ166.75,166.03,160.33,158.22,154.90,150.25,142.88,142.05,131.08,130.11,129.60,128.34,127.69,118.19,117.26,114.74,79.48,72.38,52.18,43.82, 36.20,28.86,28.47.HRMS(ESI)calcd for C32H36N3O6[M-H]-:558.2610,Found: 558.2609.
实施例25 N-(4-(羟基氨基甲酰基)苄基)-2-(3-羟基苯基)异烟酸酰胺(I-1)的合成
Figure BDA0003151843370000151
冰浴、搅拌下,将氢氧化钾的无水甲醇溶液(7.0g,45mL甲醇)慢慢滴加入盐酸羟胺的无水甲醇溶液中(5.84g,20mL甲醇),加毕,搅拌5分钟,过滤得NH2OK-NH2OH甲醇溶液,密封保存备用。在50mL两口圆底烧瓶中,加入化合物4a(362.4mg,1.0mmol), 用无水二氯甲烷(5mL)溶解,氮气保护、冰浴搅拌下,慢慢滴加上步制备的NH2OK-NH2OH溶液(16.5mL),加毕室温搅拌1.0小时.将反应体系减压浓缩,浓缩物用蒸馏水(20mL)溶解,用稀HCl调pH 5-6,有固体析出,过滤,收集固体,固体用甲醇重结晶得化合物I- 1(171.5mg),白色固体,产率:47.2％,Mp:145-146℃.1H NMR(400MHz,DMSO-d6) δ11.22(s,1H),9.64(s,1H),9.50(t,1H,J＝6.0Hz),9.04(s,1H),8.80(d, 1H,J＝4.8Hz),8.31(t,1H,J＝1.2Hz),7.76-7.73(m,3H),7.59-7.56(m,2H), 7.43(d,2H,J＝8.4Hz),7.32(t,1H,J＝8.0Hz),6.88(dd,1H,J1＝1.6Hz, J2＝8.0Hz),4.58(d,2H,J＝5.6Hz).13C NMR(101MHz,DMSO-d6)δ165.20, 158.32,157.28,150.71,142.78,142.56,140.02,131.91,130.33,127.67,127.47, 120.68,117.89,116.93,113.94,43.01.HRMS(ESI)calcd for C20H17N3NaO4[M+ Na]+:386.1111,Found:386.1112.
实施例26 N-(4-(羟基氨基甲酰胺)苄基)-2-(4-羟基苯基)异烟酸酰胺(I-2)的合成
Figure BDA0003151843370000152
按照实施例25的方法,用4b(362.4mg,1.0mmol)替换4a,得化合物I-2(223.1 mg),白色固体,产率:61.4％,Mp:161-162℃.1H NMR(400MHz,DMSO-d6)δ11.22 (s,1H),9.85(s,1H),9.46(t,1H,J＝5.6Hz),9.05(s,1H),8.74(d,1H,J＝ 8.4Hz),8.25(s,1H),8.01(d,2H,J＝8.8Hz),7.74(d,2H,J＝8.0Hz),7.66 (dd,1H,J1＝0.8Hz,J2＝4.8Hz),7.43(d,2H,J＝8.4Hz),6.90(d,2H,J＝ 8.8Hz),4.58(d,2H,J＝5.6Hz).13C NMR(101MHz,DMSO-d6)δ165.39,164.50, 159.35,157.40,150.52,142.83,142.47,131.90,129.58,128.62,127.67,127.47, 119.49,116.78,116.04,42.99.HRMS(ESI)calcd for C20H16N3O4[M-H]-:362.1146, Found:362.1147.
实施例27 N-(4-(羟基氨基甲酰胺)苄基)-2-(2-羟基苯基)异烟酸酰胺(I-3)的合成
Figure BDA0003151843370000161
按照实施例25的方法,用4c(362.4mg,1.0mmol)替换4a,得化合物I-3(252.9 mg),产率:69.6％,黄色固体,Mp:192-193℃.1H NMR(400MHz,DMSO-d6)δ13.68 (s,1H),11.22(s,1H),9.56(t,1H,J＝6.0Hz),9.04(s,1H),8.78(d,1H,J＝ 5.2Hz),8.59(s,1H),8.09(dd,1H,J1＝1.6Hz,J2＝8.4Hz),7.82(dd,1H,J1＝1.2Hz,J2＝5.2Hz),7.75(d,2H,J＝8.4Hz),7.44(d,2H,J＝8.4Hz),7.35 (td,1H,J1＝1.6Hz,J2＝7.6Hz),6.99-6.95(m,2H),4.60(d,2H,J＝5.6Hz). 13C NMR(101MHz,DMSO-d6)δ164.85,159.37,157.97,147.82,143.47,142.62, 132.21,131.99,127.84,127.72,127.48,120.38,119.49,119.46,118.40,117.95, 43.12.HRMS(ESI)calcd for C20H16N3O4[M-H]-:362.1146,Found:362.3349.
实施例28 N-(4-(羟基氨基甲酰基)苄基)-2-(2-甲氧基苯基)异烟酸酰胺(I-4)的合成
Figure BDA0003151843370000162
按照实施例25的方法,用4d(376.4mg,1.0mmol)替换4a,得化合物I-4(232.8 mg),产率:61.7％,白色固体,Mp:189-190℃.1H NMR(400MHz,DMSO-d6)δ11.21 (s,1H),9.40(t,1H,J＝6.0Hz),9.03(d,1H,J＝2.0Hz),8.80(d,1H,J＝ 4.8Hz),8.24(t,1H,J＝1.6Hz),7.74-7.71(m,4H),7.47-7.39(m,3H),7.18 (d,1H,J＝7.6Hz),7.09(td,1H,J1＝1.2Hz,J2＝7.6Hz),4.55(d,2H,J＝ 5.6Hz),3.84(s,3H).13C NMR(101MHz,DMSO-d6)δ165.49,164.52,157.19, 156.62,150.40,142.88,141.57,131.89,131.20,130.93,128.39,127.61,127.46, 122.79,121.13,119.81,112.39,56.13,42.98.HRMS(ESI)calcd for C21H19N3NaO4[M +Na]+:400.1268,Found:400.1269.
实施例29 N-(4-(羟基氨基甲酰基)苄基)-2-(4-甲氧基苯基)异烟酸酰胺(I-5)的合成
Figure BDA0003151843370000171
按照实施例25的方法,用4e(376.4mg,1.0mmol)替换4a,得化合物I-5(201.1 mg),白色固体,产率:53.3％,Mp:203-204℃.1H NMR(400MHz,DMSO-d6)δ11.23 (s,1H),9.52(t,1H,J＝5.6Hz),9.04(s,1H),8.77(d,1H,J＝5.2Hz),8.32 (s,1H),8.13(d,2H,J＝8.8Hz),7.74(d,2H,J＝8.0Hz),7.70(d,1H,J＝ 4.4Hz),7.43(d,2H,J＝8.4Hz),7.08(d,2H,J＝8.8Hz),4.58(d,2H,J＝ 5.6Hz),3.83(s,3H).13C NMR(101MHz,DMSO-d6)δ165.32,164.53,160.91, 157.04,150.62,142.83,142.57,131.92,131.12,128.55,127.68,127.49,119.86, 117.12,114.67,55.74,43.00.HRMS(ESI)calcd for C21H20N3O4[M+H]+:378.1448,Found:378.1446.
实施例30 N-(4-(羟基氨基甲酰胺)苄基)-2-(4-甲基苯基)异烟酸酰胺(I-6)的合成
Figure BDA0003151843370000172
按照实施例25的方法,用4f(360.4mg,1.0mmol)替换4a,得化合物I-6(215.39mg),白色固体,产率:59.6％,Mp:218-219℃.1H NMR(400MHz,DMSO-d6) δ11.22(s,1H),9.49(t,1H,J＝6.0Hz),9.04(s,1H),8.80(d,1H,J＝ 5.2Hz),8.35(s,1H),8.06(d,2H,J＝8.0Hz),7.76-7.73(m,3H),7.43(d, 2H,J＝8.0Hz),7.34(d,2H,J＝8.0Hz),4.59(d,2H,J＝5.6Hz),2.38(s, 3H).HRMS(ESI)calcd for C21H20N3O3[M+H]+:362.1499,Found:362.1494.
实施例31 N-(4-(羟基氨基甲酰胺)苄基)-2-(4-三氟甲基苯基)异烟酸酰胺(I-7)的合成
Figure BDA0003151843370000173
按照实施例25的方法,用4g(414.4mg,1.0mmol)替换4a,得化合物I-7(196.1 mg),白色固体,产率:47.2％,Mp:233-234℃.1H NMR(400MHz,DMSO-d6)δ11.22 (s,1H),9.54(t,1H,J＝6.0Hz),9.05(s,1H),8.89(d,1H,J＝4.8Hz), 8.48(t,1H,J＝1.2Hz),8.38(d,2H,J＝8.4Hz),7.91(d,2H,J＝8.4Hz), 7.86(dd,1H,J1＝1.2Hz,J2＝4.8Hz),7.74(d,2H,J＝8.4Hz),7.44(d,2H,J ＝8.4Hz),4.60(d,2H,J＝6.0Hz).13C NMR(101MHz,DMSO-d6)δ165.01, 155.62,151.09,142.96,142.69,142.44,131.95,129.97(q,JC-F＝31.9Hz),127.92,127.70,127.48,126.24(q,JC-F＝3.4Hz),124.72(q,JC-F＝273.2Hz), 123.36,121.70,118.76,43.05.HRMS(ESI)calcd for C21H16F3N3NaO3[M+Na]+: 438.1036,Found:438.1037.
实施例32 N-(4-(羟基氨基甲酰胺)苄基)-2-(4-吗啉基苯基)异烟酸酰胺(I-8)的合成
Figure BDA0003151843370000181
按照实施例25的方法,用4h(431.5mg,1.0mmol)替换4a,得化合物I-8(281.9 mg),黄色固体,产率:65.2％,Mp:158-159℃.1H NMR(400MHz,DMSO-d6)δ11.26 (s,1H),9.51(t,1H,J＝6.0Hz),9.08(s,1H),8.78(d,1H,J＝4.8Hz),8.32 (s,1H),8.10(d,2H,J＝8.4Hz),7.79(d,2H,J＝8.4Hz),7.69(d,1H,J＝ 4.8Hz),7.48(d,2H,J＝8.0Hz),7.12(d,2H,J＝4.8Hz),4.63(d,2H,J＝ 5.6Hz),3.81(t,4H,J＝4.4Hz),3.27(t,4H,J＝4.4Hz).13C NMR(101MHz, DMSO-d6)δ165.42,164.55,157.30,152.30,150.52,142.85,142.47,131.94,128.91,127.97,127.66,127.48,119.37,116.65,114.99,66.48,48.17,43.01. HRMS(ESI)calcd for C24H23N4O4[M-H]-:431.1725,Found:431.1725.
实施例33 N-(4-(羟基氨基甲酰胺)苄基)-2-(3-吗啉基苯基)异烟酸酰胺(I-9)
Figure BDA0003151843370000182
按照实施例25的方法,用4i(431.5mg,1.0mmol)替换4a,得化合物I-9(346.8 mg),黄色固体,产率:80.2％,Mp:107-108℃.1H NMR(400MHz,DMSO-d6)δ11.14 (s,1H),9.56(s,1H),9.11(s,1H),8.80(d,1H,J＝4.8Hz),8.35(s,1H), 7.75-7.71(m,4H),7.58(d,1H,J＝7.2Hz),7.43-7.36(m,3H),7.07(d,1H,J ＝8.0Hz),4.91(s,2H),3.77(t,4H,J＝4.0Hz),3.19(t,4H,J＝4.0Hz).13C NMR(101MHz,Pyr-d5)δ166.04,158.29,152.12,150.40,143.27,142.71,139.83, 132.97,129.65,127.94,127.68,120.44,118.42,116.69,114.20,66.68,49.03, 43.53.HRMS(ESI)calcd for C24H23N4O4[M-H]-:431.1725,Found:431.1725.
实施例34 N-(4-(羟基氨基甲酰胺)苄基)-2-(4-(4-甲基哌嗪-1-基)苯基)异烟酸酰胺(I- 10)的合成
Figure BDA0003151843370000191
按照实施例25的方法,用4j(444.5mg,1.0mmol)替换4a,得化合物I-10(129.3mg),红色固体,产率:29.1％,Mp:197-198℃.1H NMR(400MHz,DMSO-d6)δ11.21 (s,1H),9.46(t,1H,J＝6.0Hz),9.04(s,1H),8.72(d,1H,J＝4.8Hz),8.26 (s,1H),8.03(d,2H,J＝8.8Hz),7.73(d,2H,J＝8.4Hz),7.63(dd,1H,J1＝ 1.2Hz,J2＝4.8Hz),7.42(d,2H,J＝8.0Hz),7.05(d,2H,J＝8.8Hz),4.57 (d,2H,J＝6.0Hz),3.25(t,4H,J＝4.8Hz),2.46(t,4H,J＝4.8Hz),2.23 (s,3H).13C NMR(101MHz,DMSO-d6)δ165.40,164.47,157.32,152.18,150.52, 142.85,142.39,131.90,128.42,127.94,127.66,127.46,119.29,116.53,115.14, 54.94,47.75,46.24,42.98.HRMS(ESI)calcd for C25H28N5O3[M+H]+:446.2187,Found:446.2184.
实施例35 N-(4-(羟基氨基甲酰胺)苄基)-2-(4-(4-吗啉磺酰基)苯基)异烟酸酰胺(I-11) 的合成
Figure BDA0003151843370000192
按照实施例25的方法,用4k(495.6mg,1.0mmol)替换4a,得化合物I-11(268.6mg),黄色固体,产率:54.1％,Mp:160-161℃.1H NMR(400MHz,DMSO-d6))δ11.23 (s,1H),9.55(t,1H,J＝5.2Hz),9.05(s,1H),8.90(d,1H,J＝4.8Hz),8.50 (s,1H),8.43(d,2H,J＝8.4Hz),7.91-7.86(m,3H),7.75(d,2H,J＝8.0Hz), 7.44(d,2H,J＝8.0Hz),4.60(d,2H,J＝5.2Hz),3.65(t,4H,J＝4.0Hz), 2.92(t,4H,J＝4.0Hz).13C NMR(101MHz,DMSO-d6)δ165.00,164.49,155.46, 151.17,143.03,142.99,142.70,135.32,131.95,128.75,128.11,127.71,127.49, 121.84,119.05,65.75,46.37,43.06.HRMS(ESI)calcd forC24H24N4NaO6S[M+Na]+: 519.1309,Found:519.1307.
实施例36 N-(4-(羟基氨基甲酰胺)苄基)-2-(3-氯-4-甲氧基苯基)异烟酸酰胺(I-12)的合成
Figure BDA0003151843370000193
按照实施例25的方法,用4l(410.9mg,1.0mmol)替换4a,得化合物I-12(264.4mg),黄色固体,产率:64.2％,Mp:177-178℃.1H NMR(400MHz,DMSO-d6)11.22(s, 1H),9.54(t,1H,J＝6.0Hz),9.01(s,1H),8.79(d,1H,J＝5.2Hz),8.38(s, 1H),8.23(d,1H,J＝2.4Hz),8.15(dd,1H,J1＝2.0Hz,J2＝8.4Hz),7.75- 7.72(m,3H),7.43(d,2H,J＝8.0Hz),7.31(d,1H,J＝8.8Hz),4.58(d,2H, J＝6.0Hz),3.94(s,3H).13C NMR(101MHz,DMSO-d6)δ165.14,164.36,155.97, 155.60,150.74,142.75,142.71,131.99,128.36,127.67,127.46,127.19,122.06, 120.56,117.36,113.43,56.77,43.01.HRMS(ESI)calcd forC21H17ClN3O4[M-H]-: 410.0913,Found:410.0914.
实施例37 N-(4-(羟基氨基甲酰胺)苄基)-2-(3,4-二甲氧基苯基)异烟酸酰胺(I-13)的合成
Figure BDA0003151843370000201
按照实施例25的方法,用4m(406.4mg,1.0mmol)替换4a,得化合物I-13(306.8mg),黄色固体,产率:75.3％,Mp:210-211℃.1H NMR(400MHz,DMSO-d6)δ11.23 (s,1H),9.49(t,1H,J＝5.2Hz),9.06(s,1H),8.78(d,1H,J＝4.8Hz), 8.31(s,1H),7.76-7.69(m,5H),7.43(d,2H,J＝8.0Hz),7.10(d,1H,J＝8.4 Hz),4.59(d,2H,J＝5.2Hz),3.87(s,3H),3.83(s,3H).13C NMR(101MHz, DMSO-d6)δ165.17,164.75,164.50,155.14,150.87,146.11,142.75,137.88, 131.93,128.11,127.69,127.47,120.51,117.56,111.09,53.96,43.02.HRMS(ESI) calcd for C22H21N3NaO5[M+Na]+:430.1373,Found:430.1373.
实施例38 N-(4-(羟基氨基甲酰胺)苄基)-2-(2,4-二甲基苯基)异烟酸酰胺 (I-14)的合成
Figure BDA0003151843370000202
按照实施例25的方法,用4n(374.4mg,1.0mmol)替换4a,得化合物I-14(82.9 mg),白色固体,产率:22.0％,Mp:196-197℃.1H NMR(400MHz,DMSO-d6)δ11.20 (s,1H),9.43(t,1H,J＝6.0Hz),9.03(s,1H),8.79(d,1H,J＝5.2Hz),7.90 (s,1H),7.75(dd,1H,J1＝1.6Hz,J2＝5.2Hz),7.72(d,2H,J＝8.0Hz),7.40 (d,2H,J＝8.0Hz),7.35(d,1H,J＝8.0Hz),7.15-7.11(m,2H),4.54(d,2H, J＝6.0Hz),2.33(s,3H),2.31(s,3H).13C NMR(101MHz,DMSO-d6)δ165.22, 160.45,150.12,142.73,142.06,138.25,137.27,135.82,131.97,131.87,130.15, 127.65,127.42,127.01,121.60,119.79,43.00,21.19,20.58.HRMS(ESI)calcdfor C22H20N3O3[M-H]:374.1510,Found:374.1509.
实施例39 N-(4-(羟基氨基甲酰胺)苄基)-2-(2-氟-4-甲基苯基)异烟酸酰胺(I-15)的合成
Figure BDA0003151843370000211
按照实施例25的方法,用4o(378.4mg,1.0mmol)替换4a,得化合物I-15(281.8mg),白色固体,产率:74.3％,Mp:189-190℃.1H NMR(400MHz,DMSO-d6)δ11.21 (s,1H),9.48(t,1H,J＝6.0Hz),9.09(s,1H),8.85(d,1H,J＝5.2Hz),8.18 (s,1H),7.87(t,1H,J＝8.0Hz),7.81(dd,1H,J1＝1.6Hz,J2＝5.2Hz),7.73 (d,2H,J＝8.0Hz),7.41(d,2H,J＝8.0Hz),7.23-7.17(m,2H),4.56(d,2H, J＝5.6Hz),2.39(s,3H).13C NMR(101MHz,DMSO-d6)δ165.12,164.43,160.18 (d,JC-F＝249.4Hz),153.92(d,JC-F＝2.4Hz),150.85,142.73,142.36,142.24(d, JC-F＝8.6Hz),131.96,131.05(d,JC-F＝3.2Hz),127.66,127.44,126.05(d,JC-F＝ 2.8Hz),124.09(d,JC-F＝11.4Hz),121.79(d,JC-F＝8.7Hz),120.51,117.12(d,JC-F＝21.8Hz),43.03,21.11.HRMS(ESI)calcd for C21H17FN3O3[M-H]-:378.1259, Found:378.1257.
实施例40 N-(4-(羟基氨基甲酰胺)苄基)-2-(3-氯-4-吗啉苯基)异烟酸酰胺(I-16)的合成
Figure BDA0003151843370000212
按照实施例25的方法用,4p(465.9mg,1.0mmol)替换4a,得化合物I-16(318.4mg),黄色固体,产率:68.2％,Mp:185-186℃.1H NMR(400MHz,DMSO-d6)) δ11.20(s,1H),9.51(t,2H,J＝5.2Hz),8.80(d,1H,J＝5.2Hz),8.37(s, 1H),8.21(d,1H,J＝1.6Hz),8.11(dd,1H,J1＝1.6Hz,J2＝8.4Hz),7.76- 7.73(m,3H),7.43(d,2H,J＝8.4Hz),7.29(d,1H,J＝8.4Hz),4.59(d,2H, J＝5.2Hz),3.77(t,4H,J＝4.4Hz),3.07(t,4H,J＝4.4Hz).13CNMR(101 MHz,DMSO-d6)δ165.13,164.37,155.59,150.80,150.10,142.74,142.68,134.20,132.02,128.90,128.21,127.66,127.44,126.79,121.35,120.71, 117.55,66.73,51.58,43.03.HRMS(ESI)calcd for C24H22ClN4O4 -[M-H]:465.1335, Found:465.1335.
实施例41 N-(4-(羟基氨基甲酰胺)苄基)-2-(3-氨基-2-甲基苯基)异烟酸酰胺(I-17)
Figure BDA0003151843370000221
按照实施例25的方法,用4q(375.4mg,1.0mmol)替换4a,得化合物I-17(335.0mg),黄色固体,产率:89.0％,Mp:143-144℃.1H NMR(400MHz,DMSO-d6)δ11.22 (s,1H),9.44(t,1H,J＝6.0Hz),9.04(s,1H),8.79(d,1H,J＝4.0Hz),7.84 (s,1H),7.76-7.71(m,3H),7.40(d,2H,J＝7.2Hz),7.00(t,1H,J＝6.8Hz), 6.73(d,1H,J＝7.6Hz),6.60(d,1H,J＝7.2Hz),5.02(brs,2H),4.54(d,2H, J＝5.6Hz),1.98(s,3H).13C NMR(101MHz,DMSO-d6)δ165.23,164.53,161.60, 150.00,147.67,142.83,141.78,141.00,131.89,127.66,127.46,126.22,121.83, 119.70,119.26,118.28,114.70,42.99,14.66.HRMS(ESI)calcdfor C21H21N4O3[M +H]+:377.1608,Found:377.1608.
实施例42 N-(4-(羟基氨基甲酰胺)苄基)-6'-甲氧基-[2,3'-二吡啶]-4-甲酰胺(I-18) 的合成
Figure BDA0003151843370000222
按照实施例25的方法,用4r(377.4mg,1.0mmol)替换4a,得化合物I-21(312.9mg),白色固体,产率:82.7％,Mp:196-197℃.1H NMR(400MHz,DMSO-d6)δ11.23 (s,1H),9.47(t,1H,J＝5.6Hz),9.06(s,1H),8.95(d,1H,J＝2.0Hz),8.81 (d,1H,J＝4.8Hz),8.43(dd,1H,J1＝2.4Hz,J2＝8.8Hz),8.36(s,1H),7.76- 7.74(m,3H),7.44(d,2H,J＝8.0Hz),6.98(d,1H,J＝8.8Hz),4.59(d,2H, J＝4.6Hz),3.93(s,3H).13C NMR(101MHz,DMSO-d6)δ165.17,164.75,164.50, 155.14,150.87,146.11,142.75,137.88,131.93,128.11,127.69,127.47,120.51, 117.56,111.09,53.96,43.02.HRMS(ESI)calcd for C20H17N4O4[M-H]-:377.1255, Found:377.1257.
实施例43 5-羟基-N-(4-(羟基氨基甲酰胺)苄基)-2-(4-甲基苯基)异烟酸酰胺(I-19)的合成
Figure BDA0003151843370000223
按照实施例25的方法,用4s(378.4mg,1.0mmol)替换4a,得化合物I-19(118.0mg),黄色固体,产率:31.1％,Mp:235-236℃.1H NMR(400MHz,DMSO-d6)δ12.11 (s,1H),11.21(s,1H),9.69(t,1H,J＝6.0Hz),9.03(s,1H),8.41(s,1H), 8.29(s,1H),7.92(d,2H,J＝8.0Hz),7.74(d,2H,J＝8.0Hz),7.44(d,2H, J＝8.4Hz),7.28(d,2H,J＝8.0Hz),4.61(d,2H,J＝6.0Hz),2.35(s,3H). 13C NMR(101MHz,DMSO-d6)δ167.38,164.49,153.44,147.66,142.30,140.87, 138.08,135.90,132.03,129.78,127.70,127.53,126.11,123.45,117.35,42.79, 21.23.HRMS(ESI)calcd for C21H18N3O4[M-H]-:376.1303,Found:376.1303.
实施例44 5-羟基-N-(4-羟基氨基甲酰胺)苄基)-2-(4-甲氧基苯基)异烟酸酰胺(I-20)的合成
Figure BDA0003151843370000231
按照实施例25的方法,用4t(394.4mg,1.0mmol)替换4a,得化合物I-20(186.6mg),黄色固体,产率:47.2％,Mp:224-225℃.1H NMR(400MHz,DMSO-d6)δ12.02 (s,1H),11.23(s,1H),9.64(t,1H,J＝4.8Hz),9.05(s,1H),8.41(s,1H), 8.26(s,1H),7.97(d,2H,J＝8.8Hz),7.75(d,2H,J＝8.4Hz),7.45(d,2H, J＝8.0Hz),7.04(d,2H,J＝8.8Hz),4.62(d,2H,J＝5.6Hz),3.81(s,3H). 13C NMR(101MHz,DMSO-d6)δ167.38,164.50,159.95,153.04,147.59,142.33, 140.73,132.01,131.24,127.70,127.53,123.55,116.93,114.54,55.65,42.78. HRMS(ESI)calcd for C21H18N3O5[M-H]-:392.1252,Found:392.1251.
实施例45 N-(4-(羟基氨基甲酰胺)苄基)-2-(4-(2-吗啉基乙氧基)苯基)异烟酸酰胺(I- 21)的合成
Figure BDA0003151843370000232
按照实施例25的方法,用5a(475.5mg,1.0mmol)替换4a,得化合物I-21(401.2mg),白色固体,产率:84.2％,Mp:110-111℃.1H NMR(400MHz,DMSO-d6)δ11.22 (s,1H),9.48(t,1H,J＝6.0Hz),9.05(s,1H),8.70(d,1H,J＝4.8Hz),8.31 (s,1H),8.11(d,2H,J＝8.4Hz),7.74(d,2H,J＝7.6Hz),7.69(d,1H,J＝ 4.4Hz),7.43(d,2H,J＝8.0Hz),7.09(d,2H,J＝8.4Hz),4.58(d,2H,J＝ 4.8Hz),4.16(t,2H,J＝5.6Hz),3.59(t,4H,J＝4.8Hz),3.38(brs,4H), 2.72(t,2H,J＝5.6Hz).13C NMR(101MHz,DMSO-d6)δ165.31,164.52,160.13, 157.02,150.63,142.82,142.55,131.91,131.10,128.54,127.67,127.48,119.87, 117.08,115.21,66.65,65.89,57.47,54.11,43.00.HRMS(ESI)calcd forC26H27N4O5[M-H]-:475.1987,Found:475.1990.
实施例46叔丁基4-((4-(4-((4-(羟基氨基甲酰胺)苄基)氨基甲酰基)-2-吡啶基)苯氧基) 甲基)哌啶-1-羧酸酯(I-22)的合成
Figure BDA0003151843370000241
按照实施例25的方法,用5b(559.7mg,1.0mmol)替换4a,得化合物I-22(455.8mg),白色固体,产率:81.3％,Mp:122-123℃.1H NMR(400MHz,DMSO-d6)δ11.23 (s,1H),9.52(t,1H,J＝6.0Hz),9.06(s,1H),8.76(d,1H,J＝4.0Hz),8.32 (s,1H),8.11(d,2H,J＝8.0Hz),7.75(d,2H,J＝7.6Hz),7.70(d,1H,J＝ 4.8Hz),7.43(d,2H,J＝7.6Hz),7.07(d,2H,J＝8.0Hz),4.58(d,2H,J＝ 5.6Hz),4.00-3.97(m,2H),3.91(d,2H,J＝5.2Hz),2.81-2.68(m,2H),1.97- 1.91(m,1H),1.77(d,2H,J＝12.4Hz),1.41(s,9H),1.22-1.10(m,2H).13C NMR(101MHz,DMSO-d6)δ165.30,164.52,160.33,157.02,154.36,150.62, 142.83,142.54,131.90,131.06,128.54,127.68,127.48,119.86,117.08,115.16, 78.99,72.27,43.00,35.83,28.77,28.57.HRMS(ESI)calcd for C31H35N4O6[M-H]-: 559.2562,Found:559.2561.
本发明所合成化合物的LSD1、HDAC1和THP-1抑制活性评价 (一)LSD1、HDAC1和THP-1的抑制活性评价：
1、LSD1抑制活性评价实验方法
样品为实施例所合成的上述化合物纯化而得；样品储备液：称取3-5mg样品置于1.5 mL EP管中，然后用DMSO配制成浓度是20mM的溶液，-20℃避光保存，实验时根据所需浓度用DMSO稀释。将待测样品与LSD1蛋白于室温孵育后，加入LSD1反应底物H3K4me2并孵育反应，最后加入荧光染料Amplex和辣根过氧化酶HRP室温孵育，在酶标仪上激发光 530nm，发射光590nm检测荧光数值。
Figure BDA0003151843370000242
试验结果采用SPSS软件计算IC50值。
2、HDAC1抑制活性评价实验方法
称取3-5mg样品置于1.5mL EP管中，然后用DMSO配制成浓度是10mM的溶液，- 20℃避光保存，实验时根据所需浓度用DMSO稀释。制备1X缓冲液(改良Tris缓冲液)。 HDAC酶用1X缓冲液稀释至1.67X终浓度。将胰蛋白酶和乙酰肽底物混合制成底物溶液，用1X缓冲液稀释至2.5X终浓度。用Echo550将250nL待测化合物转移到384孔板上。然后，将15μL的酶液加入384孔板中，与待测化合物在室温下预孵育15min。用15 μL1X缓冲液作为阴性对照。然后将10μL底物溶液加入384孔中以启动反应。用 EnVision在激发波长355nm和发射波长460nm处检测荧光强度。
3、体外抗肿瘤活性测定
样品为实施例所合成的化合物；样品储备液：称取3-5mg样品置于1.5mL EP管中，然后用DMSO配制成浓度为10mmol/L溶液，-20℃避光保存放置，实验时根据所需浓度利用培养基稀释。
取对数生长期的细胞，消化计数后，用培养基调整细胞密度，以4000-5000个细胞/孔接种至96孔板中，每孔100μL，培养24h后，加入用培养基稀释好的不同浓度的药物(10 μM、2.5μM、0.625μM、0.1563μM、0.0391μM、0.0098μM、0.0024μM、0.006 μM)，每个浓度设3个复孔，另设空白对照组及阳性对照组。药物作用72h后，每孔加入40 μL CellTiter-GloReagent，振荡混匀2min，室温继续孵育10min后，酶标仪检测荧光强度，计算抑制率，计算公式如下:
抑制率(％)＝(最大荧光强度-给药组荧光强度)/(最大荧光强度-最小荧光强度)×100％。
4、实验结果
表1 LSD1、HDAC1和THP-1抑制活性评价结果
Figure BDA0003151843370000251
Figure BDA0003151843370000261
a N.D.:未测定。
b 30nM浓度时的抑制率为83％。
从上表实验结果可以看出，本发明化合物对LSD1和HDAC1均具有较好的抑制活性，IC50值在纳摩尔至微摩尔水平。大部分化合物对HDAC1的IC50小于10nM,优于阳性对照SAHA，特别是化合物I-2，I-8，I-10，I-16和I-21对HDAC1的IC50小于2nM，是阳性药物SAHA的7倍。体外抗肿瘤活性评价显示多个化合物对THP-1白血病细胞具有很好的抑制活性，其中化合物I-5,I-8和I-15对THP-1的IC50小于1μM,明显优于SAHA。特别是化合物I-5和I-8对THP-1 的活性分别是SAHA的12倍和18倍。
化合物I-5，I-8和I-15对THP-1的抑制活性显著优于阳性对照SAHA，我们进一步在两种白血病细胞株(MOLT-4、MV4:11)上评价了化合物I-5，I-8和I-15的体外抗肿瘤活性。
表2 化合物I-5和I-8体外抗肿瘤活性评价结果
Figure BDA0003151843370000262
实验结果表明化合物I-5、I-8和I-15对MOLT-4和MV4:11两种白血病细胞株均具有很好的抑制活性，IC50值均小于0.5μM。本发明化合物代表着一类结构全新的LSD1/HDAC双靶点抑制剂，多个化合物具有很好的体外抗肿瘤活性，可作为进一步开发的候选或者先导化合物，应用于制备抗癌药物，为LSD1/HDAC双靶点抑制剂类药物的研发提供了基础，为LSD1和 HDAC的生物学功能研究提供了有效工具。
(二)重组蛋白水平MAO-A/B抑制活性评价
1、实验方法：样品为实施例所合成的上述化合物纯化而得；样品储备液：称取3-5mg 样品置于1.5mL EP管中，然后用DMSO配制成浓度是20mM的溶液，-20℃避光保存，实验时根据所需浓度用DMSO稀释。Clorgyline和R-(-)-deprenyl分别作为MAO-A和MAO-B活性评价的阳性对照药物。根据制造商的使用方法，使用Promega的商品化MAO-Glo检测试剂盒测定对MAO-A和MAO-B的抑制活性。
2、实验结果：
表3 化合物I-5和I-8对MAO-A/B抑制活性评价结果
Figure BDA0003151843370000271
aN.D.:未检测
实验结果表明化合物I-5和I-8对LSD1的同源蛋白MAO-A和MAO-B没有抑制活性，对LSD1 具有很好的选择性。

Claims (3)

1.一类2-芳基异烟酸酰胺类化合物，其特征在于，选自以下化合物其中之一:
Figure FDA0003810434310000011
Figure FDA0003810434310000021
2.如权利要求1所述的一类2-芳基异烟酸酰胺类化合物在药物制备中的应用，其特征在于，将其作为活性成分用于LSD1/HDAC双靶点抑制剂类药物的制备。
3.如权利要求2所述的一类2-芳基异烟酸酰胺类化合物在药物制备中的应用，其特征在于，将其作为活性成分用于制备急性髓系白血病的治疗药物。
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