CN111592487B - 一类含羟肟酸基团的二芳基乙烯类LSD1/HDACs双靶点抑制剂、其制备方法及应用 - Google Patents

一类含羟肟酸基团的二芳基乙烯类LSD1/HDACs双靶点抑制剂、其制备方法及应用 Download PDF

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CN111592487B
CN111592487B CN202010516850.2A CN202010516850A CN111592487B CN 111592487 B CN111592487 B CN 111592487B CN 202010516850 A CN202010516850 A CN 202010516850A CN 111592487 B CN111592487 B CN 111592487B
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段迎超
靳林峰
关圆圆
袁航
文郁康
陈书慧
秦文平
张少杰
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Xinxiang Medical University
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Abstract

本发明涉及一类含有羟肟酸基团的二芳基乙烯类LSD1/HDACs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 111976DEST_PATH_IMAGE001
其中
Figure 69478DEST_PATH_IMAGE002
优选3‑位或4‑位单取代；R2优选OH、OCH3、F、H；R3优选H、CH2OH；R4优选为H、CH3、F、OCH3；R5优选H、OH、CH2OH、CH3、F、OCH3；X为N或者CH；Y为N或者CH。本发明所述化合物对LSD1和HDAC1/6均具有较强的抑制活性，且对LSD1具有很好的选择性，多个化合物对人结肠癌HCT‑116细胞株和人胃癌MGC‑803细胞株的体外抗肿瘤活性优于上市药物SAHA。为LSD1/HDACs双靶点抑制剂类药物的研发提供了基础，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治疗药物。

Description

一类含羟肟酸基团的二芳基乙烯类LSD1/HDACs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含有羟肟酸基团的二芳基乙烯类LSD1/HDACs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)由2004年哈佛大学Shi Yang 教授首次报道，它是一个黄素腺嘌呤二核苷酸(Flavin adenine dinulcleotide,FAD) 依赖性氨基氧化酶，可以特异性的去除组蛋白H3K4的单、双甲基。LSD1的发现确认了组蛋白的甲基化与去甲基化存在动态平衡。此外，LSD1还能去除非组蛋白如p53、DNMT1、STAT3、E2F1、MYPT1、ERa和HIF-1的甲基，进一步调节其下游基因的稳定性和活性。大量的研究表明，LSD1在淋巴瘤、急髓性白血病、前列腺癌、肺癌、胃癌、雌激素受体阴性乳腺癌、口腔癌、结肠癌、滑膜肉瘤和神经母细胞瘤等多种肿瘤中的表达量显著升高，并且和肺癌、急性髓系白血病、结肠癌和乳腺癌等多种恶性肿瘤的不良预后密切相关。通过RNA干扰技术下调LSD1的表达量或用小分子抑制剂抑制LSD1的活性，均可以抑制癌细胞的增殖、转移和侵袭，是目前抗肿瘤药物研发的热点靶蛋白之一，目前已有多个LSD1小分子抑制剂处于I期、II期临床试验，用于治疗急性髓性白血病和非小细胞肺癌。
组蛋白去乙酰化酶(HDACs)是一类负责从组蛋白尾部去除乙酰基的表观遗传调控蛋白。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，HDACs家族成员的表达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HDACs的高表达呈正相关。目前已经有5种HDACs抑制剂被FDA和CFDA批准上市治疗恶性淋巴瘤、骨髓瘤等多种肿瘤，还有多个HDACs抑制剂候选药物处于临床试验。
LSD1和HDACs抑制剂均可以使肿瘤细胞内某些被外源沉默了的基因得以重新表达，诱发细胞凋亡，LSD1和HDACs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多种共抑制复合体中，参与调控多种基因的转录。LSD1活性受HDACs的调节，抑制HDACs活性后， LSD1活性也被抑制。在人乳腺癌细胞中，HDAC5可以通过上调USP28的表达促进LSD1的稳定性与去甲基化活性。在膀胱癌、乳腺癌、肺癌等多种癌症中，降低LSD1的表达量或者抑制LSD1的活性，可以显著增强癌细胞对HDACs抑制剂的敏感性。用小分子抑制剂同时抑制LSD1和HDACs的活性具有协同的抗肿瘤效应。因此，发现新型、高活性的LSD1/HDACs双靶点抑制剂，通过同时抑制LSD1、HDACs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、HDACs的生物学功能、研发新型抗肿瘤药物具有十分重要的意义。为了发现新型的LSD1/HDACs双靶点抑制剂，探索合成一类含有羟肟酸基团的二芳基乙烯类化合物，验证其LSD1、HDACs双重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/HDACs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含有羟肟酸基团的二芳基乙烯类化合物，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含羟肟酸基团的二芳基乙烯类化合物的制备方法。
本发明的再一个目的在于提供所述化合物以LSD1/HDACs为靶点在制备抗肿瘤药物中的应用。
为实现上述目的，本发明所涉及的含羟肟酸基团的二芳基乙烯类化合物的结构通式为：
Figure BDA0002530439860000021
通式I-5中，取代基
Figure BDA0002530439860000022
为单取代；R2为OH、OCH3、F、H中的任意一个；R3为H、CH2OH中的任意一个；R4为H、CH3、F、OCH3中的任意一个；R5为H、OH、CH2OH、CH3、F、OCH3中的任意一个；X为N或者CH；Y为 N或者CH。
优选：
Figure BDA0002530439860000031
3-位或4-位单取代。
优选地：通式I-5中，
Figure BDA0002530439860000032
取代基位置、R2、R3、R4、R5、X和Y所代表的取代基如下表所示：
Figure BDA0002530439860000033
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式所示：
通式I化合物的合成路线：
Figure BDA0002530439860000041
化合物I-3的制备方法:化合物I-1和取代苄基膦酸二乙酯(化合物I-2)在 N，N-二甲基甲酰胺(DMF)中，强碱性化合物存在下，室温搅拌反应，反应结束后，将反应体系倾入冰水中，抽滤，洗涤，收集固体，干燥，得化合物I-3。其中，所述强碱性化合物选自叔丁醇钾、叔丁醇钠、甲醇钠、氢化钠中的一种。
化合物I-4的制备方法:化合物I-3和各种取代苯硼酸或者吡啶硼酸，在甲苯中，碱性化合物和钯催化剂存在下，回流搅拌反应，得化合物I-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I-5的制备方法:在二氯甲烷溶液中，化合物I-4和NH2OK/NH2OH 的甲醇溶液反应，反应结束后，反应体系真空浓缩，浓缩物加入乙酸乙酯和水萃取，洗涤，有机相经柱层析分离，得化合物I-5。
本发明优点：本发明合成的含有羟肟酸基团的二芳基乙烯类化合物均具有较强的LSD1/HDACs双重抑制活性和体外抗肿瘤活性。本发明所报道的化合物对LSD1和HDAC1/6均具有较强的抑制活性，且对LSD1具有很好的选择性。多个化合物的LSD1、HDAC1的抑制IC50小于1μM，对HDAC6的抑制活性小于50nM。该类LSD1/HDACs双靶点制剂对人结肠癌HCT-116细胞株和人胃癌 MGC-803细胞株显示出较好的体外抗肿瘤活性，多个化合物的体外抗肿瘤活性优于阳性对照上市药物HDACs抑制剂SAHA。本发明化合物代表着一类具有全新结构的LSD1/HDACs双靶点抑制剂，为LSD1/HDACs双靶点抑制剂类药物的研发提供了基础，为LSD1、HDACs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
下面举实施例对本发明技术方案作详细说明。
实施例1(E)-4-(2-(2-溴-吡啶-4-基)烯基)苯甲酸甲酯(I-3a)的合成
Figure BDA0002530439860000051
在50毫升两口烧瓶中加入化合物I-1a(376mg,2.0mmol)，I-2a(601.1mg,2.1mmo)，用无水DMF10毫升溶解，冰浴下加入t-BuOK(673.3mg,6.0mmol)，加毕室温下搅拌反应0.5小时，然后将反应体系缓慢加入冰水(50mL)中，析出固体。抽滤，收集固体，硅胶柱柱层析分离纯化(石油醚：乙酸乙酯＝5:1)得化合物I-3a 396.4mg，白色固体，产率：62.3％，Mp:106-107℃。1H NMR(400 MHz,CDCl3)δ8.36(d,1H,J＝5.2Hz),8.08(d,2H,J＝8.4Hz),7.60-7.57(m, 3H),7.34-7.29(m,2H),7.04(d,1H,J＝16.4Hz),3.95(s,3H).13CNMR(101MHz, CDCl3)δ166.57,150.44,146.96,143.04,139.95,133.46,130.39,130.20,127.05, 126.87,125.16,120.09,52.27.HRMS(ESI)calcd for C15H13BrNO2[M+ H]+:318.0124,Found:318.0126.
实施例2(E)-4-(2-(2-溴-吡啶-4-基)烯基)苯甲酸甲酯(I-3b)的合成
Figure BDA0002530439860000052
按照实施例1的方法，用化合物I-2b(601.1mg,2.1mmol)替换I-2a，得目标化合物I-3b288.3 mg，白色固体，产率:45.4％，Mp:73-74℃。1H NMR(400MHz, CDCl3)δ8.33(d,1H,J＝5.2Hz),8.21(t,1H,J＝2.0Hz),8.00(dt,1H,J1＝1.2Hz, J2＝8.0Hz),7.70(dt,1H,J1＝1.6Hz,J2＝8.0Hz),7.57(s,1H),7.48(t,1H,J＝8.0 Hz),7.35-7.30(m,2H),7.03(d,1H,J＝16.4Hz),3.96(s,3H).HRMS(ESI)calcd for C15H12BrNNaO2[M+Na]+:339.9944,Found:339.9943.
实施例3(E)-3-(3-溴苯乙烯基)甲酸甲酯(I-3c)的合成
Figure BDA0002530439860000061
按照实施例1的方法，用化合物I-1b(370mg,2.0mmol)替换I-1a，得目标化合物I-3c 480mg，产率:75.6％,Mp:177-178℃.1H NMR(400MHz,DMSO-d6) δ7.97(d,2H,J＝8.4Hz),7.89(t,1H,J＝2.0Hz),7.74(d,2H,J＝8.4Hz),7.64(dt, 1H,J1＝1.2Hz,J2＝8.0Hz),7.51-7.48(m,1H),7.43(d,2H,J＝4.0Hz),7.37(t,1H,J ＝8.0Hz),3.86(s,3H).13C NMR(101MHz,DMSO-d6)δ166.41,141.90,139.66, 131.31,131.20,130.09,129.65,129.38,129.04,127.28,126.38,122.76,52.57. HRMS(ESI)calcd for C16H13BrNaO2[M+Na]+:338.9991,Found:338.9996.
实施例4(E)-3-(2-(2-(5-氟-2-羟基苯基)吡啶-4-基)烯基)苯甲酸甲酯(I-4a)的合成
Figure BDA0002530439860000062
在50毫升两口烧瓶中，加入化合物I-3b(332.2mg,1.0mmol)，甲苯(7mL),乙醇(1.5mL)，H2O(7mL)，K3PO3(1.59g,7.5mmol)，Pd(PPh3)4(58mg,0.050 mmol)和(5-氟-2-羟基)苯硼酸(202.7mg,1.3mmol)，氮气保护，95℃摄氏度搅拌反应3小时,冷却至室温，向反应体系中加入饱和NH4Cl溶液(10mL),用乙酸乙酯萃取(3X 15mL)，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，干燥完毕，抽滤，滤液真空浓缩，浓缩物用硅胶柱柱层析纯化(石油醚:乙酸乙酯＝4:1)，得化合物I-4a 290.7mg，产率:80.3％，黄白色固体， Mp:119-120℃.1H NMR(400MHz,CDCl3)δ14.06(s,1H),8.49(d,1H,J＝5.6Hz), 8.26(s,1H),8.03(d,1H,J＝7.6Hz),7.87(s,1H),7.75(d,1H,J＝7.6Hz),7.55(dd, 1H,J1＝2.8Hz,J2＝10.0Hz),7.50(t,1H,J＝7.6Hz),7.44-7.38(m,2H),7.18(d, 1H,J＝16.4Hz),7.07-7.03(m,1H),7.00-6.96(m,1H),4.43(q,2H,J＝7.2Hz),1.44 (t,3H,J＝7.2Hz).13C NMR(101MHz,CDCl3)δ166.23,157.30(d,JC,F＝2.0Hz), 156.91(d,JC,F＝236.0Hz),156.18(d,JC,F＝2.0Hz),146.34,146.28,136.12,133.33, 131.29,129.97,129.03,128.14,126.79,119.55(d,JC,F＝8.1Hz),118.99,118.91(d, JC,F＝7.1Hz),118.43(d,JC,F＝23.2Hz),116.74,111.76(d,JC,F＝24.2Hz), 61.30,14.38.HRMS(ESI)calcd for C22H19FNO3[M+H]+:364.1343,Found: 364.1345.
实施例5(E)-3-(2-(2-2-羟基苯基)吡啶-4-基)烯基)苯甲酸甲酯(I-4b)的合成
Figure BDA0002530439860000071
按照实施例4的方法，用化合物2-羟基苯硼酸(179.3mg,1.3mmol)替换5-氟-2-羟基苯硼酸，得目标化合物I-4b190.2mg，黄白色固体，产率:57.4％，Mp:142- 143℃。1H NMR(400MHz,DMSO-d6)δ14.31(s,1H),8.60(d,1H,J＝5.2Hz),8.46(s, 1H),8.28(t,1H,J＝1.6Hz),8.16(dd,1H,J1＝1.2Hz,J2＝8.0Hz),7.99(dt,1H, J1＝1.2Hz,J2＝8.0Hz),7.95(dt,1H,J1＝1.2Hz,J2＝7.6Hz),7.91(d,1H,J＝16.4Hz), 7.66(dd,1H,J1＝1.2Hz,J2＝5.2Hz),7.62(t,1H,J＝8.0Hz),7.48(d,1H,J＝16.4Hz), 7.36-7.31(m,1H),6.99-6.93(m,2H),3.91(s,3H).13C NMR(101MHz,DMSO-d6)δ 166.48,159.87,157.84,146.97,146.85,137.18,133.69,131.92,131.88,130.80, 129.93,129.76,128.36,127.51,127.41,119.94,119.22,118.43,117.31,52.77.HRMS (ESI)calcd for C21H18NO4[M+H]+:332.1281,Found:332.1288.
实施例6(E)-4-(2-(2-(5-氟-2-羟基苯基)吡啶-4-基)烯基)苯甲酸甲酯(I-4c)的合成
Figure BDA0002530439860000072
按照实施例4的方法，用I-3a(332.2mg,1.0mmol)替换I-3b，得目标化合物I- 4c241.8mg，黄白色固体，产率:69.2％，Mp:164-165℃。1H NMR(400MHz, DMSO-d6)δ14.05(s,1H),8.62(d,1H,J＝5.2Hz),8.47(s,1H),8.04-8.01(m, 3H),7.88(d,1H,J＝16.4Hz),7.81(d,2H,J＝8.4Hz),7.66(d,1H,J＝5.6Hz),7.48 (d,1H,J＝16.4Hz),7.19(td,1H,J1＝3.2Hz,J2＝8.4Hz),6.94(dd,1H,J1＝4.8Hz, J2＝8.8Hz),3.87(s,3H).13C NMR(101MHz,DMSO-d6)δ166.32,156.68(d,JC,F＝ 3.0Hz),156.02,155.60(d,JC,F＝234.3Hz),147.23,146.76,141.17,133.75,130.23, 129.83,128.81,127.79,120.80,119.68(d,JC,F＝7.1Hz),119.51(d,JC,F＝8.1Hz), 118.69(d,JC,F＝23.2Hz),117.67,113.18(d,JC,F＝25.3Hz),52.66.HRMS(ESI) calcd for C21H15FNO3[M-H]-:348.1041,Found:348.1042.
实施例7(E)-4-(2-(2-(2-羟基苯基)吡啶-4-基)烯基)苯甲酸甲酯(I-4d)的合成
Figure BDA0002530439860000081
按照实施例4的方法，用I-3a(332.2mg,1.0mmol)替换I-3b，用化合物2-羟基苯硼酸(179.3mg,1.3mmol)替换5-氟-2-羟基苯硼酸，得化合物I-4d235.6mg，产率:71.1％，黄白色固体，Mp:155-156℃。1H NMR(400MHz,DMSO-d6)δ14.25 (s,1H),8.62(d,1H,J＝5.2Hz),8.44(s,1H),8.15(dd,1H,J1＝1.6Hz,J2＝8.0Hz), 8.03(d,2H,J＝8.0Hz),7.88-7.82(m,3H),7.66(dd,1H,J1＝1.2Hz,J2＝5.2 Hz),7.53(d,1H,J＝16.4Hz),7.36-7.31(m,1H),6.99-6.93(m,2H),3.88(s,3H).13C NMR(101MHz,DMSO-d6)δ166.33,159.81,157.87,147.05,146.60,141.22, 133.52,131.93,130.22,129.81,129.02,127.81,127.44,120.00,119.25,118.42, 117.50,52.66.HRMS(ESI)calcd for C21H16NO4[M-H]-:330.1136,Found: 330.1138.
实施例8(E)-4-(2-(2-(2-甲氧基苯基)吡啶-4-基)烯基)苯甲酸甲酯(I-4e)的合成
Figure BDA0002530439860000082
按照实施例4的方法，用I-3a(332.2mg,1.0mmol)替换I-3b，用化合物2-甲氧基苯硼酸(197.5mg,1.3mmol)替换5-氟-2-羟基苯硼酸，得化合物I-4e256.3mg，白色固体，产率:74.2％，Mp:132-133℃。1H NMR(400MHz,CDCl3)δ8.68(d, 1H,J＝5.2Hz),8.05(d,2H,J＝8.4Hz),7.88(s,1H),7.77(dd,1H,J1＝2.0Hz,J2＝ 7.6Hz),7.60(d,2H,J＝8.4Hz),7.41-7.36(m,1H),7.34-7.30(m,2H),7.17(d,1H,J ＝16.4Hz),7.09(dt,1H,J1＝0.8Hz,J2＝7.6Hz),7.03(d,1H,J＝8.0Hz),3.93(s, 3H),3.88(s,3H).13C NMR(101MHz,CDCl3)δ166.72,156.95,156.90,149.85, 143.73,140.78,131.58,131.18,130.12,130.07,129.82,129.19,129.06,126.85, 122.83,121.09,118.78,111.44,55.74,52.19.HRMS(ESI)calcd for C22H20NO3[M+ H]+:346.1438,Found:346.1437.
实施例9(E)-4-(2-(2-(3-羟基苯基)吡啶-4-基)烯基)苯甲酸甲酯(I-4f)的合成
Figure BDA0002530439860000091
按照实施例4的方法，用I-3a(332.2mg,1.0mmol)替换I-3b，用化合物3-羟基苯硼酸(179.3mg,1.3mmol)替换5-氟-2-羟基苯硼酸，得化合物I-4f217.4 mg，白色固体，产率:65.6％，Mp:198-199℃。1H NMR(400MHz,DMSO-d6)δ9.60(s, 1H,J＝5.2H),8.64(d,1H,J＝5.2Hz),8.13(s,1H),8.02(d,2H,J＝8.0Hz),7.82(d, 2H,J＝8.0Hz),7.78(d,1H,J＝16.4Hz),7.60-7.54(m,3H,J＝8.4Hz),7.50(d,1H, J＝16.4Hz),7.32(t,1H,J＝8.0Hz),6.87(dd,1H,J1＝2.4Hz,J2＝8.0Hz),3.87(s, 3H).13C NMR(101MHz,DMSO-d6)δ166.36,158.23,157.15,150.38,145.27, 141.45,140.49,132.30,130.17,129.56,129.45,127.67,120.30,118.01,117.82, 116.60,113.94,52.64.HRMS(ESI)calcd for C21H16NO3[M-H]-:330.1139,Found: 330.1136.
实施例10(E)-4-(2-(2-(3-羟甲基苯基)吡啶-4-基)烯基)苯甲酸甲酯(I-4g)的合成
Figure BDA0002530439860000092
按照实施例4的方法，用I-3a(332.2mg,1.0mmol)替换I-3b，用化合物3-羟甲基苯硼酸(197.5mg,1.3mmol)替换5-氟-2-羟基苯硼酸，得化合物I-4g273.2mg，白色固体，产率:79.1％，Mp:184-185℃。1H NMR(400MHz,DMSO-d6)δ8.67 (d,1H,J＝4.8Hz),8.18(s,1H),8.13(s,1H),8.03-8.00(m,3H),7.83-7.75(m,3H), 7.58-7.46(m,3H),7.42(d,1H,J＝7.6Hz),5.30(t,1H,J＝5.6Hz),4.62(d,2H,J＝ 5.6Hz),3.87(s,3H).13C NMR(101MHz,DMSO-d6)δ166.38,157.26,150.46, 145.37,143.56,141.46,138.90,132.34,130.19,129.60,129.50,128.96,127.73, 127.69,125.44,125.21,120.27,118.05,63.42,52.63.HRMS(ESI)calcd for C22H20BrN2O3[M+H]+:346.1436,Found:346.1436.
实施例11(E)-4-(2-(2-(2-氟-4-甲基苯基)吡啶-4-基)烯基)苯甲酸甲酯(I-4h)的合成
Figure BDA0002530439860000101
按照实施例4的方法，用I-3a(332.2mg,1.0mmol)替换I-3b，用化合物2-氟-4- 甲基苯硼酸(197.5mg,1.3mmol)替换5-氟-2-羟基苯硼酸，得化合物I-4g 274.4mg，白色固体，产率:79.0％，Mp:139-140℃。1H NMR(400MHz,CDCl3)δ8.68(d, 1H,J＝5.2Hz),8.06(d,2H,J＝8.4Hz),7.89(t,1H,J＝8.4Hz),7.85(t,1H,J＝2.0 Hz),7.61(d,2H,J＝8.4Hz),7.37-7.32(m,2H),7.17(d,1H,J＝16.4Hz),7.10-7.07 (m,1H),7.02-6.98(m,1H),3.93(s,3H),2.41(s,3H).13C NMR(101MHz,CDCl3)δ 166.69,160.35(d,JC-F＝250.2Hz),154.18(d,JC-F＝2.5Hz),150.07,144.42,141.37 (d,JC-F＝8.5Hz),140.59,131.92,130.67(d,JC-F＝3.4Hz),130.12,129.90,128.75, 126.89,125.40(d,JC-F＝3.0Hz),124.33(d,JC-F＝11.5Hz),121.90(d,JC-F＝9.3Hz), 119.27,116.70(d,JC-F＝22.9Hz),52.22,21.20(d,JC-F＝1.7Hz).HRMS(ESI)calcd for C22H19FNO2[M+H]+:348.1394,Found:348.1396.
实施例12(E)-4-(2-([2,3’-双吡啶]-4-基)烯基)苯甲酸甲酯(I-4i)的合成
Figure BDA0002530439860000102
按照实施例4的方法，用I-3a(332.2mg,1.0mmol)替换I-3b，用化合物吡啶-3- 硼酸(159.8mg,1.3mmol)替换5-氟-2-羟基苯硼酸，得化合物I-4i 248.3mg，白色固体，产率:78.5％，Mp:152-153℃。1H NMR(400MHz,DMSO-d6)δ9.35(d,1H, J＝2.0Hz),8.71(d,1H,J＝5.2Hz),8.67(dd,1H,J1＝1.2Hz,J2＝4.8Hz),8.51(dt,1H,J1＝1.6Hz,J2＝8.0Hz),8.32(s,1H),8.02(d,2H,J＝8.0Hz),7.85-7.80(m,3H),7.61(d, 1H,J＝4.8Hz),7.56(dd,1H,J1＝4.8Hz,J2＝8.0Hz),7.49(d,1H,J＝16.4Hz),3.88(s, 3H).HRMS(ESI)calcd for C20H17N2O2[M+H]+:317.1285,Found:317.1283.
实施例13(E)-4-(2-(2’-羟基-[1,1’-双苯基]-3-基)烯基)苯甲酸甲酯(I-4j)的合成
Figure BDA0002530439860000103
按照实施例4的方法，用3c(317.18mg,1.0mmol)替换I-3b，用2-羟基苯硼酸(179.3mg,1.3mmol)替换5-氟-2-羟基苯硼酸，得化合物I-4j 159.2mg，白色固体，产率:48.2％，Mp:158-159℃。1H NMR(400MHz,DMSO-d6)δ9.57(s,1H),7.96(d, 2H,J＝8.4Hz),7.79-7.75(m,3H),7.59(dt,1H,J1＝1.2Hz,J2＝7.6Hz),7.51-7.40(m, 3H),7.37(d,1H,J＝16.4Hz),7.30(dd,1H,J1＝1.6Hz,J2＝7.6Hz),7.19(td,1H,J1＝1.6Hz,J2＝8.0Hz),6.96(dd,1H,J1＝1.2Hz,J2＝8.4Hz),6.90(td,1H,J1＝0.8Hz, J2＝7.2Hz),3.86(s,3H).13C NMR(101MHz,DMSO-d6)δ166.49,154.80,142.40, 139.62,136.73,131.97,130.86,130.08,129.67,129.14,128.84,128.65,128.16, 127.97,127.64,127.10,125.46,119.92,116.50,52.55.HRMS(ESI)calcd for C22H17O3[M-H]-:329.1183,Found:329.1187.
实施例14(E)-4-(2-(3’-羟基-[1,1’-双苯基]-3-基)烯基)苯甲酸甲酯(I-4k)的合成
Figure BDA0002530439860000111
按照实施例4的方法，用I-3c(317.18mg,1.0mmol)替换I-3b，用3-羟基苯硼酸(179.3mg,1.3mmol)替换5-氟-2-羟基苯硼酸，得化合物I-4k 234.9mg，白色固体，产率:71.1％，Mp:147-148℃。1H NMR(400MHz,DMSO-d6)δ9.57(s,1H),7.98(d, 2H,J＝8.4Hz),7.89(t,1H,J＝1.6Hz),7.77(d,2H,J＝8.4Hz),7.64(dt,1H,J1＝1.6 Hz,J2＝7.6Hz),7.55-7.53(m,1H),7.50-7.44(m,3H),7.29(t,1H,J＝8.0Hz),7.16- 7.13(m,1H),7.10(t,1H,J＝2.0Hz),6.82(dd,1H,J1＝1.6Hz,J2＝8.0Hz),3.86(s, 3H).13C NMR(101MHz,DMSO-d6)δ166.47,158.31,142.35,141.87,141.38, 137.63,131.70,130.40,130.10,129.79,128.75,128.11,127.13,127.01,126.32, 125.64,118.05,115.07,114.12,52.54.HRMS(ESI)calcd for C21H17O3[M-H]-: 329.1183,Found:329.1181.
实施例15(E)-4-(2-(5’-F-2’-羟基-[1,1’-双苯基]-3-基)烯基)苯甲酸甲酯(I-4l)的合成
Figure BDA0002530439860000112
按照实施例4的方法，用I-3c(317.18mg,1.0mmol)替换I-3b，得化合物I-4l252.9mg，白色固体，产率:72.6％，Mp:148-149℃。1H NMR(400MHz,DMSO- d6)δ9.63(s,1H),7.97(d,2H,J＝8.4Hz),7.83(t,1H,J＝2.0Hz),7.76(d,2H,J＝8.4 Hz),7.62(dt,1H,J1＝1.6Hz,J2＝8.0Hz),7.55-7.42(m,3H),7.40(d,1H,J＝16.8 Hz),7.17(dd,1H,J1＝3.2Hz,J2＝9.6Hz),7.04(td,1H,J1＝3.2Hz,J2＝8.8Hz), 6.97(dd,1H,J1＝4.8Hz,J2＝8.8Hz),3.86(s,3H).13C NMR(101MHz,DMSO-d6)δ 166.48,156.13(d,JC,F＝235.3Hz),151.14(d,JC,F＝2.0Hz),142.35,138.48,136.88, 131.79,130.09,129.60,128.94(d,JC,F＝8.1Hz),128.94,128.70,128.06,127.84, 127.09,125.98,117.37(d,JC,F＝8.1Hz),116.75(d,JC,F＝23.2Hz),115.23(d,JC,F＝ 22.2Hz),52.53.HRMS(ESI)calcd for C22H16FNO3[M-H]-:347.1089,Found: 347.1090.
实施例16(E)-4-(2-(2-(4-甲基苯基)吡啶-4-基)烯基)苯甲酸甲酯(I-4m)的合成
Figure BDA0002530439860000121
按照实施例4的方法，用I-3a(332.2mg,1.0mmol)替换I-3b，用化合物4-甲基苯硼酸(176.7mg,1.3mmol)替换5-氟-2-羟基苯硼酸，得化合物I-4m217.4 mg，白色固体，产率:81.2％，Mp:140-141℃。1H NMR(400MHz,CDCl3)δ8.66(dd, 1H,J1＝0.4Hz,J2＝5.2Hz),8.06(d,2H,J＝8.4Hz),7.93(d,2H,J＝8.4Hz),7.77- 7.76(m,1H),7.61(d,2H,J＝8.4Hz),7.36(d,2H,J＝16.4Hz),7.34-7.28(m,3H), 7.18(d,2H,J＝16.4Hz),3.93(s,3H),2.42(s,3H).13C NMR(101MHz,CDCl3)δ 166.70,158.18,150.05,144.72,140.62,139.14,136.48,131.75,130.15,129.90, 129.53,128.88,126.87,126.83,118.97,117.94,52.23,21.33.HRMS(ESI)calcd for C22H20NO2[M+H]+:330.1489,Found:330.1487.
实施例17(E)-4-(2-(2-(4-甲氧基苯基)吡啶-4-基)烯基)苯甲酸甲酯(I-4n)的合成
Figure BDA0002530439860000122
按照实施例4的方法，用I-3a(332.2mg,1.0mmol)替换I-3b，用化合物4-甲氧基苯硼酸(197.5mg,1.3mmol)替换5-氟-2-羟基苯硼酸得化合物I-4n 258.36mg，白色固体，产率:74.8％，Mp:118-119℃.1H NMR(400MHz,CDCl3)δ8.63(d, 1H,J＝5.2Hz),8.06(d,2H,J＝8.4Hz),7.99(d,2H,J＝8.8Hz),7.73(t,1H,J＝0.8 Hz),7.61(d,2H,J＝8.4Hz),7.35(d,1H,J＝16.4Hz),7.28(dd,1H,J1＝1.6Hz,J2＝5.2Hz),7.17(d,1H,J＝16.4Hz),7.02(d,2H,J＝8.8Hz),3.93(s,3H),3.87(s, 3H).13C NMR(101MHz,CDCl3)δ166.70,160.56,157.82,149.98,144.69,140.63, 131.87,131.70,130.14,129.89,128.92,128.24,126.86,118.57,117.54,114.15, 55.39,52.23.HRMS(ESI)calcd for C22H20NO3[M+H]+:346.1438,Found:346.1436.
实施例18(E)-3-(2-(2-(5-氟-2-羟基苯基)吡啶-4-基)烯基)-N-羟基苯甲酰胺(I-5a)的合成
Figure BDA0002530439860000131
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溶液，密封保存备用。在50毫升两口圆底烧瓶中，加入化合物I-4a (174.7mg,0.5mmol)，用无水二氯甲烷(3mL)溶解，氮气保护，冰浴搅拌下，慢慢滴加上步制备的NH2OK-NH2OH溶液(8.0mL)，加毕室温搅拌1.0小时.将反应体系真空浓缩，浓缩物用蒸馏水(20mL)溶解，用1N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-5a137mg，产率: 39.3％，Mp:207-208℃。1H NMR(400MHz,DMSO-d6)δ14.11(s,1H),11.33(s, 1H),9.15(s,1H),8.62(d,1H,J＝3.6Hz),8.49(s,1H),8.13(s,1H),8.06(dd,1H,J1＝2.8Hz,J2＝10.8Hz),7.91(d,1H,J＝16.4Hz),7.83(d,1H,J＝7.2Hz),7.74(d,1H,J ＝7.2Hz),7.66(d,1H,J＝2.4Hz),7.55(t,1H,J＝7.2Hz),7.43(d,1H,J＝16.4 Hz),7.20(t,1H,J＝7.2Hz),6.95(dd,1H,J1＝4.8Hz,J2＝8.4Hz).13C NMR(101MHz, DMSO-d6)δ164.43,156.71(d,JC,F＝3.0Hz),156.08,155.69(d,JC,F＝233.3Hz), 147.19,147.11,136.80,134.41,133.97,130.21,129.51,127.49,126.98,126.25, 120.68,119.71(d,JC,F＝8.1Hz),119.52(d,JC,F＝8.1Hz),118.67(d,JC,F＝23.2Hz), 117.48,113.19(d,JC,F＝24.2Hz).HRMS(ESI)calcd for C20H16FN2O3[M+H]+: 351.1139,Found:351.1138.
实施例19(E)-3-(2-(2-(2-羟基苯基)吡啶-4-基)烯基)-N-羟基苯甲酰胺(I-5b)的合成
Figure BDA0002530439860000141
按照实施例18的方法，用I-4b(165.7mg,0.5mmol)替换I-4a，得化合物I- 5b78mg。白色固体，产率:47.1％，Mp:187-188℃。1H NMR(400MHz,DMSO- d6)δ14.31(s,1H),11.30(s,1H),9.20(s,1H),8.60(d,1H,J＝4.0Hz),8.45(s,1H), 8.17-8.14(m,2H),7.88-7.81(m,2H),7.74(d,1H,J＝7.2Hz),7.65(d,1H,J＝3.2Hz), 7.53(t,1H,J＝7.2Hz),7.44(d,1H,J＝16.0Hz),7.34(t,1H,J＝7.2Hz),6.98-6.93(m, 2H).13C NMR(101MHz,DMSO-d6)δ164.41,164.27,159.87,157.85,146.99, 146.93,136.78,134.20,131.92,130.12,129.46,127.44,127.12,126.18,119.87, 119.24,118.43,117.29.HRMS(ESI)calcd for C21H18NO3[M+H]+:333.1234,Found: 333.1234.
实施例20(E)-4-(2-(2-(5-氟-2-羟基苯基)吡啶-4-基)烯基)-N-羟基苯甲酰胺(I-5c)的合成
Figure BDA0002530439860000142
按照实施例18的方法，用I-4c(165.7mg,0.5mmol)替换I-4a，得化合物I-5c67.6mg，白色固体，产率：38.6％，Mp:210-211℃。1H NMR(400MHz,DMSO- d6)δ14.06(s,1H),11.29(s,1H),9.10(s,1H),8.63(d,1H,J＝4.8Hz),8.47(s,1H), 8.04(dd,1H,J1＝3.2Hz,J2＝10.4Hz),7.89-7.75(m,5H),7.65(d,1H,J＝5.2Hz), 7.46(d,1H,J＝16.4Hz),7.20(td,1H,J1＝2.8Hz,J2＝8.8Hz),6.95(dd,1H,J1＝ 4.8Hz,J2＝8.8Hz).13C NMR(101MHz,DMSO-d6)δ164.19,156.69(d,JC,F＝2.0 Hz),155.69(d,JC,F＝233.3Hz),154.53,147.24,147.01,139.22,134.11,133.20, 127.99,127.79,127.54,120.74,119.74(d,JC,F＝8.1Hz),119.52(d,JC,F＝7.1Hz), 118.68(d,JC,F＝23.2Hz),117.57,113.21(d,JC,F＝25.3Hz).HRMS(ESI)calcd for C20H14FN2O3[M-H]-:349.0994,Found:349.0997.
实施例21(E)-4-(2-(2-(2-羟基苯基)吡啶-4-基)烯基)-N-羟基苯甲酰胺(I-5d)的合成
Figure BDA0002530439860000151
按照实施例18的方法，用I-4d(165.7mg,0.5mmol)替换I-4a，得化合物I-5d115mg，白色固体，产率:69.2％，Mp:206-207℃。1H NMR(400MHz, DMSO-d6)δ14.28(s,1H),11.30(s,1H),9.11(s,1H),8.61(d,1H,J＝5.2Hz),8.43 (s,1H),8.15(dd,1H,J1＝1.6Hz,J2＝8.0Hz),7.86-7.81(m,3H),7.77(d,2H,J＝ 8.0Hz),7.65(dd,1H,J1＝1.6Hz,J2＝8.0Hz),7.48(d,1H,J＝16.4Hz),7.36-7.31 (m,1H),6.98-6.93(m,2H).13C NMR(101MHz,DMSO-d6)δ164.17,159.82, 157.83,147.02,146.81,139.23,133.84,133.13,131.93,127.95,127.54,127.42, 119.92,119.24,118.42,117.34.HRMS(ESI)calcd for C20H15N2O3[M-H]-:331.1088,Found:331.1088.
实施例22(E)-4-(2-(2-(2-甲氧基苯基)吡啶-4-基)烯基)-N-羟基苯甲酰胺(I-5e)的合成
Figure BDA0002530439860000152
按照实施例18的方法，用I-4e(166.1mg,0.5mmol)替换I-4a，得化合物I-5e151.2mg，黄白色固体，产率:87.3％，Mp:176-177℃。1H NMR(400MHz, DMSO-d6)δ11.30(s,1H),9.10(s,1H),8.64(d,1H,J＝5.2Hz),7.97(s,1H),7.83- 7.55(m,7H),7.45-7.41(m,2H),7.18(d,1H,J＝8.8Hz),7.08(t,1H,J＝7.6Hz), 3.87(s,3H).13C NMR(101MHz,DMSO-d6)δ164.22,157.22,156.48,150.08, 144.32,139.41,132.84,132.37,131.17,130.61,128.88,128.57,127.85,127.47, 122.93,121.04,119.24,112.36,56.12.HRMS(ESI)calcdfor C21H19N2O3[M+H]+: 347.1390,Found:347.1394.
实施例23(E)-4-(2-(2-(3-羟基苯基)吡啶-4-基)烯基)-N-羟基苯甲酰胺(I-5f)的合成
Figure BDA0002530439860000161
按照实施例18的方法，用I-4f(165.7mg,0.5mmol)替换I-4a，得化合物I-5f82.1mg，白色固体，产率:49.4％，Mp:203-204℃。1H NMR(400MHz,DMSO- d6)δ11.29(s,1H),9.58(s,1H),9.10(s,1H),8.64(d,1H,J＝5.2Hz),8.10(s, 1H),7.83(d,2H,J＝8.0Hz),7.77-7.71(m,3H),7.59-7.53(m,3H),7.45(d,1H,J＝16.8 Hz),7.32(t,1H,J＝8.0Hz),6.87(dd,1H,J1＝2.4Hz,J2＝8.0Hz).13C NMR(101 MHz,DMSO-d6)δ164.24,158.24,157.15,150.36,145.48,140.55,139.47,132.89, 132.60,130.16,128.38,127.91,127.41,120.20,117.93,117.83,116.60, 113.96.HRMS(ESI)calcd for C20H17N2O3[M+H]+:333.1234,Found:333.1237.
实施例24(E)-4-(2-(2-(3-羟甲基苯基)吡啶-4-基)烯基)-N-羟基苯甲酰胺(I-5g)的合成
Figure BDA0002530439860000162
按照实施例18的方法，用I-4g(172.7mg,0.5mmol)替换I-4a，得化合物I-5g130.4mg，白色固体，产率:75.3％，Mp:181-182℃。1H NMR(400MHz,DMSO- d6)δ11.28(s,1H),9.09(s,1H),8.66(d,1H,J＝4.8Hz),8.16(s,1H),8.13(s,1H),8.02(d, 1H,J＝7.2Hz),7.82(d,2H,J＝8.4Hz),7.77-7.71(m,3H),7.56(d,1H,J＝4.8Hz),7.50- 7.40(m,3H),5.30(t,1H,J＝5.6Hz),4.61(d,2H,J＝5.2Hz).13C NMR(101MHz, DMSO-d6)δ164.17,157.23,150.44,145.57,143.55,139.43,138.94,132.91,132.63, 128.96,128.39,127.90,127.71,127.42,125.44,125.20,120.17,117.95,63.42.HRMS (ESI)calcd for C21H19N2O3[M+H]+:347.1390,Found:347.1389.
实施例25(E)-4-(2-(2-(2-氟-4-甲基苯基)吡啶-4-基)烯基)-N-羟基苯甲酰胺(I-5h)的合成
Figure BDA0002530439860000171
按照实施例18的方法，用I-4h(173.7mg,0.5mmol)替换I-4a，得化合物I-5h115.3mg,白色固体，产率:66.2％，Mp:177-178℃。1H NMR(400MHz, DMSO-d6)δ11.31(brs,1H),9.13(brs,1H),8.68(d,1H,J＝5.2Hz),7.92(d,1H,J＝ 2.0Hz),7.87-7.81(m,3H),7.78(d,2H,J＝8.4Hz),7.65(d,1H,J＝16.4Hz),7.61 (dd,1H,J1＝1.6Hz,J2＝5.2Hz),7.46(d,1H,J＝16.4Hz),7.22-7.15(m,2H),2.39 (s,3H).13C NMR(101MHz,DMSO-d6)δ164.18,160.15(d,JC,F＝248.5Hz), 153.81(d,JC,F＝2.0Hz),150.54,145.16,141.79(d,JC,F＝4.0Hz),139.30,132.91, 132.72,131.08(d,JC,F＝4.0Hz),128.19,127.84,127.51,125.92(d,JC,F＝3.0Hz), 124.60(d,JC,F＝11.1Hz),121.90(d,JC,F＝8.1Hz),119.89,117.03(d,JC,F＝22.2 Hz),21.09.HRMS(ESI)calcd for C21H16FN2O2[M-H]-:347.1201,Found:347.1200.
实施例26(E)-4-(2-([2,3’-双吡啶]-4-基)烯基)-N-羟基苯甲酰胺(I-5i)的合成
Figure BDA0002530439860000172
按照实施例18的方法，用I-4i(158.2mg,0.5mmol)替换I-4a，得化合物I- 5i126.1mg，白色固体，产率:79.5％，Mp:152-153℃。1H NMR(400 MHz,DMSO-d6)δ11.31(s,1H),9.35(d,1H,J＝2.0Hz),9.12(s,1H),8.71(d,1H,J ＝5.2Hz),8.67(dd,1H,J1＝1.2Hz,J2＝4.4Hz),8.51(dt,1H,J1＝2.0Hz,J2＝8.0Hz), 8.31(s,1H),7.85(d,2H,J＝8.4Hz),7.82-7.75(m,3H),7.60(dd,1H,J1＝1.6Hz, J2＝5.2Hz),7.56(dd,1H,J1＝4.8Hz,J2＝8.0Hz),7.45(d,1H,J＝16.4Hz).13C NMR (101MHz,DMSO-d6)δ164.18,154.84,150.83,150.43,148.34,145.84,139.34, 134.54,134.48,133.12,133.01,128.04,127.94,127.44,124.25,121.14, 118.10.HRMS(ESI)calcd for C19H16N3O2[M+H]+:318.1237,Found:318.1236.
实施例27(E)-4-(2-(2’-羟基-[1,1’-双苯基]-3-基)烯基)-N-羟基苯甲酰胺(I-5j)的合成
Figure BDA0002530439860000181
按照实施例18的方法，用I-4j(165.2mg,0.5mmol)替换I-4a，得化合物I-5j81.7mg,白色固体，产率:49.3％，Mp:212-213℃。1H NMR(400MHz,DMSO-d6) δ11.24(s,1H),9.56(s,1H),9.05(s,1H),7.79-7.76(m,3H),7.70(d,2H,J＝8.4Hz), 7.57(dt,1H,J1＝1.6Hz,J2＝7.6Hz),7.47(dt,1H,J1＝1.6Hz,J2＝8.0Hz),7.45-7.40 (m,2H),7.35-7.29(m,2H),7.19(td,1H,J1＝1.6Hz,J2＝8.4Hz),6.97(dd,1H,J1＝1.2 Hz,J2＝8.4Hz),6.90(td,1H,J1＝1.2Hz,J2＝7.2Hz).13C NMR(101MHz,DMSO-d6) δ164.38,154.80,140.35,139.60,136.90,131.92,130.86,130.82,129.42,129.12, 128.82,128.02,127.89,127.79,126.81,125.31,119.91,116.50.HRMS(ESI)calcd for C21H16NO3[M-H]-:330.1136,Found:330.1136.
实施例28(E)-4-(2-(3’-羟基-[1,1’-双苯基]-3-基)烯基)-N-羟基苯甲酰胺(I-5k)的合成
Figure BDA0002530439860000182
按照实施例18的方法，用I-4k(165.2mg,0.5mmol)替换I-4a，得化合物I- 5k67.4mg,白色固体，产率:40.7％，Mp:187-188℃。1H NMR(400 MHz,DMSO-d6)δ11.22(s,1H),9.57(s,1H),9.06(s,1H),7.86(t,1H,J＝1.6Hz), 7.79(d,2H,J＝8.4Hz),7.71(d,2H,J＝8.4Hz),7.62(td,1H,J1＝1.6Hz,J2＝7.2 Hz),7.52(td,1H,J1＝1.6Hz,J2＝8.0Hz),7.49-7.43(m,3H),7.29(t,1H,J＝8.0Hz), 7.14(dd,1H,J1＝1.2Hz,J2＝8.0Hz),7.09(t,1H,J＝2.4Hz),6.81(dd,1H,J1＝1.6Hz, J2＝8.0Hz).13C NMR(101MHz,DMSO-d6)δ164.35,158.30,141.91,141.35,140.30, 137.80,132.01,130.56,130.40,129.78,128.37,127.80,126.85,126.78,126.17, 125.48,118.05,115.06,114.10.HRMS(ESI)calcd for C21H16NO3[M-H]-:330.1136, Found:330.1134.
实施例29(E)-4-(2-(5’-氟-2’-羟基-[1,1’-双苯基]-3-基)烯基)-N-羟基苯甲酰胺(I- 5l)的合成
Figure BDA0002530439860000191
按照实施例18的方法，用I-4l(174.2mg,0.5mmol)替换I-4a，得化合物I-5l63.9mg,白色固体，产率:36.6％，Mp:209-210℃。1H NMR(400MHz,DMSO-d6) δ11.25(s,1H),9.60(s,1H),9.05(s,1H),7.81-7.77(m,3H),7.70(d,2H,J＝8.4Hz), 7.60(dt,1H,J1＝1.6Hz,J2＝7.6Hz),7.51(dt,1H,J1＝1.6Hz,J2＝8.0Hz),7.46-7.41 (m,2H),7.36(d,1H,J＝16.8Hz),7.17(dd,1H,J1＝3.2Hz,J2＝9.6Hz),7.03(td,1H, J1＝3.2Hz,J2＝8.4Hz),6.96(dd,1H,J1＝5.2Hz,J2＝8.8Hz).13C NMR(101MHz, DMSO-d6)δ164.38,156.12(d,JC,F＝235.3Hz),151.14,140.32,138.44,137.06, 131.96,130.65,129.36,128.99(d,JC,F＝7.1Hz),128.91,128.09,127.91,127.80, 126.82,125.84,117.37(d,JC,F＝8.1Hz),116.76(d,JC,F＝23.2Hz),115.22(d,JC,F＝ 22.2Hz).HRMS(ESI)calcd for C21H15FNO3[M-H]-:348.1041,Found:348.1041.
实施例30(E)-4-(2-(2-(4-甲基苯基)吡啶-4-基)烯基)-N-羟基苯甲酰胺(I-5m)的合成
Figure BDA0002530439860000192
按照实施例18的方法，用I-4m(164.7mg,0.5mmol)替换I-4a，得化合物I- 5m106.9mg,白色粉末，产率:64.7％,Mp:230-231℃.1H NMR(400MHz,DMSO- d6)δ11.29(s,1H),9.11(s,1H),8.63(d,1H,J＝5.2Hz),8.15(s,1H),8.06(d,2H,J＝ 7.6Hz),7.82(d,2H,J＝8.0Hz),7.76-7.72(m,3H),7.52(d,1H,J＝5.2Hz),7.44(d, 1H,J＝16.8Hz),7.33(d,2H,J＝8.0Hz),2.38(s,3H).13C NMR(101MHz,DMSO- d6)δ164.12,157.05,150.39,145.48,139.42,139.14,136.36,132.90,132.58,129.81, 128.37,127.89,127.39,126.95,120.02,117.49,40.37,40.16,39.95,39.74,39.53, 21.32.HRMS(ESI)calcd forC21H17N2O2[M-H]-:329.1296,Found:329.1298.
实施例31(E)-4-(2-(2-(4-甲氧基苯基)吡啶-4-基)烯基)-N-羟基苯甲酰胺(I-5n)的合成
Figure BDA0002530439860000201
按照实施例18的方法，用I-4n(172.7mg,0.5mmol)替换I-4a，得化合物I-5n107mg，白色固体,产率:61.8％,Mp:241-242℃.1H NMR(400MHz,DMSO-d6) δ11.32(brs,1H),9.13(brs,1H),8.61(d,1H,J＝5.2Hz),8.14-8.11(m,3H),7.84(d, 1H,J＝8.0Hz),7.77-7.71(m,3H),7.49(dd,1H,J1＝1.6Hz,J2＝5.2Hz),7.46(d,1H, J＝16.4Hz),7.08(d,1H,J＝8.0Hz),3.84(s,3H).13C NMR(101MHz,DMSO-d6)δ 164.21,160.67,156.85,150.30,145.40,139.47,132.85,132.48,131.59,128.47, 128.42,127.90,127.39,119.49,117.10,114.55,55.70.HRMS(ESI)calcd for C21H17N2O3[M-H]-:345.1245,Found:345.1244.
实施例32本发明所合成羟肟酸化合物的LSD1、HDAC1抑制活性评价
(一)重组蛋白水平LSD1和HDAC1/6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1.5mL 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。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 BDA0002530439860000202
试验结果采用SPSS软件计算IC50值。
2、HDAC1/6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20mM 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15μL1X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、实验结果
表1重组蛋白水平LSD1和HDAC1/6抑制活性测定结果
Figure BDA0002530439860000211
Figure BDA0002530439860000221
a N.D.:未测定。
b15nM的抑制率为85％。
从上表实验结果可以看出，本发明化合物对LSD1和HDAC1/6均具有较好的抑制活性，IC50值在纳摩尔至微摩尔水平。其中化合物I-5d、I-5f、I-5g、I-5h、I-5i 和I-5j对HDAC1的抑制活性均小于1.0μM；化合物I-5d、I-5e、I-5f、I-5g、I-I- 5h和I-5i对HDAC6的抑制活性均小于50nM。化合物I-5d对LSD1和HDAC1/6的抑制活性均小于1.0μM，具有进一步的开发价值。本发明的化合物代表着一类结构全新的LSD1/HDACs双靶点抑制剂，为LSD1/HDACs双靶点抑制剂类药物的研发提供了基础，为LSD1和HDACs的生物学功能研究提供了有效工具。
(二)体外抗肿瘤活性测定
1、实验方法
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(10μM、2.5μM、0.625μM、0.1563μM、0.0391μM、0.0098μM、 0.0024μM、0.006μM)，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40ul CellTiter-GloReagent，振荡混匀2分钟，室温继续孵育10分钟后，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％。
2、实验结果
表2体外抗肿瘤活性评价结果
Figure BDA0002530439860000231
a人类结肠癌细胞，b人类胃癌细胞
实验结果表明:化合物对所测试的两种细胞株均表现出较好的抗肿瘤活性。其中化合物I-5d、I-5f、I-5g和I-5h对人HCT-116结肠癌细胞的抑制活性优于阳性对照上市药物SAHA；化合物I-5d和I-5h对人MGC-803胃癌细胞的IC50值分别是1.42μM和0.77μM，抑制活性优于SAHA，其中化合物I-5h的活性是SAHA的2 倍。化合物I-5d和I-5h对所测试的两种细胞株抑制活性均优于阳性对照SAHA, 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。
我们进一步在两种白血病细胞株(THP-1、K562)和三种实体瘤细胞株(人肺癌A-549细胞株、人胃癌SGC-7901细胞株、人乳腺癌MCF-7)上评价了化合物I- 5d和I-5h的体外抗肿瘤活性。
表3化合物I-5d、I-5h体外抗肿瘤活性评价结果
Figure BDA0002530439860000241
实验结果表明:除人乳腺癌MCF-7细胞株外，化合物I-5d、I-5h对另外四种细胞株均具有较好抑制活性，其中对THP-1、K562和SGC-7901三种细胞株的抑制活性强于阳性对照SAHA.
(三)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4化合物I-5d、I-5h对MAO-A/B抑制活性评价结果
Figure BDA0002530439860000242
Figure BDA0002530439860000251
aN.D.:未检测
实验结果表明:化合物I-5d对LSD1的同源蛋白MAO-A和MAO-B具有较弱的抑制活性，对LSD1的选择性大于36.7倍。化合物I-5h在100μM的浓度对MAO-A 和MAO-B没有明显抑制活性，对LSD1的选择性大于61倍。结果显示化合物对I- 5d、I-5h对LSD1很好的选择性。

Claims (3)

1.一类含有羟肟酸基团的二芳基乙烯类化合物，其特征在于，选以下化合物其中之一:
Figure FDA0003694655840000011
Figure FDA0003694655840000021
2.如权利要求1所述的一类含有羟肟酸基团的二芳基乙烯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所述的一类含有羟肟酸基团的二芳基乙烯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抗结肠癌，肺癌，胃癌药物。
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