CN110028475B - 基于苯并吡喃结构的新型cdk9抑制剂、其制备方法及应用 - Google Patents

基于苯并吡喃结构的新型cdk9抑制剂、其制备方法及应用 Download PDF

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CN110028475B
CN110028475B CN201910392404.2A CN201910392404A CN110028475B CN 110028475 B CN110028475 B CN 110028475B CN 201910392404 A CN201910392404 A CN 201910392404A CN 110028475 B CN110028475 B CN 110028475B
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卞金磊
李志裕
赵腾腾
王举波
吴体智
田于成
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Abstract

本发明涉及到生物医药领域，具体包括苯并呋喃结构的新型CDK9抑制剂一系列衍生物和其用途；本发明针对汉黄芩素抗肿瘤活性低，成药性差的缺点，对其母核进行改造合成了一系列化合物，特别是针对3’或4’位引入含N环状取代基，该类化合物是全新化合物，未见文献报道，同时发明人针对合成的化合物进行了一系列生物活性评价，具体来说本发明大部分化合物对CDK9具有良好的选择性，并对癌细胞具有较好的抑制活性。

Description

基于苯并吡喃结构的新型CDK9抑制剂、其制备方法及应用
技术领域
本发明涉及到生物医药领域，具体包括苯并吡喃结构的新型CDK9抑制剂一系列衍生物和其用途，具有涉及到作为CDK9抑制剂的化合物、上述化合物在药学上可接受的盐、酯、溶剂化物或前药，及上述化合物的药物组合物的用途。
技术背景
细胞周期蛋白依赖性激酶(Cyclin-dependent kinases，CDKs)是丝氨酸/苏氨酸激酶家族的一员，与调节亚基细胞周期蛋白形成异二聚体来发挥其催化功能。CDKs家族成员根据其功能的差异可以分为周期性CDKs和转录性CDKs，前者主要包括CDK1/2/4/6，控制细胞的周期进程；后者主要包括CDK7/8/9,调节mRNA的转录和加工(Malumbres M.etal.Genome Biol.,2014,15:122)。转录性CDKs的过表达或者功能增强会造成其下游特定基因的表达明显增加，特别是抗凋亡蛋白Mcl-1，从而引起肿瘤的发生(Morales F.etal.Cell Cycle,2016,15:519-27)。近年来发现非选择性的CDK抑制剂可以通过抑制CDK9的功能来达到抗肿瘤效应，因此对CDK抑制剂的研究也得到了人们的关注(Krystof V.etal.Target,Curr.Pharm.Des.,2012,18:2883–2890)。研究发现CDK9的过表达与多种肿瘤的发生、炎症的发生及病毒的复制有关，例如急性髓性白血病、乳腺癌、结肠癌和前列腺癌等及人类免疫缺陷病毒和腺病毒等(Franco L.C.et al.J.Cell Biochem.,2017,119:1273-1284)。这些发现表明了CDK9是治疗癌症的有效靶标。现在已经进入临床的CDK9抑制剂包括Flavopiridol、Dinaciclib、SNS-032和CYC065等，但都缺乏对CDK9的选择性。还有目前处于临床前研究的CDK9选择性抑制剂如LY2857785、LDC000067、BAY-1143572等。
目前研究较多的含苯并吡喃骨架的化合物主要有汉黄芩素和Flavopiridol。汉黄芩素是从汉黄芩中分离出来的，体内外药理学实验都表明汉黄芩素具有抗炎、抗氧化及抗肿瘤活性且毒性较低，在对其抗肿瘤活性的机制进行研究时发现其主要靶标是CDK9，并于2014年获得CFDA的临床批件，但汉黄芩素的抗肿瘤活性较弱(CDK9:IC50＝198μM)并且其成药性较差。Flavopiridol是非选择性的CDK抑制剂，对CDK9有较强的抑制活性(IC50＝20nM)。Flavopiridol对慢性淋巴细胞白血病、乳腺癌、肝癌及前列腺癌等多种癌症都具有一定的治疗作用，现在被FDA批准为治疗慢性淋巴细胞白血病的孤儿药，但Flavopiridol容易引起腹泻等副作用。为了进一步提高苯并吡喃类化合物对CDK9的选择性，改善其成药性，我们设计并合成了一系列含苯并吡喃类结构的CDK9小分子抑制剂。
发明内容
本发明的主要目的是寻找一类含苯并吡喃结构的，高效低毒且具有优良药物代谢性质的CDK9小分子抑制剂，用于疾病的预防和/或治疗。
本发明提供了通式(I)所示的化合物、其药学上可接受的盐、酯、溶剂化物或前药。
Figure GDA0003631929380000021
其中，所述R1为氢、
Figure GDA0003631929380000022
所述R2为氢、甲基、乙基；
所述R3为甲基、乙基、异丙基、
Figure GDA0003631929380000023
Figure GDA0003631929380000024
所述Y为N，Y的位置为所在苯环的3’位或者4’位；
所述m为1、2或者3；
所述X为O、CRa或者NRb；
所述Ra为氢、羟基或者
Figure GDA0003631929380000031
所述Rb为氢、甲基、乙基、异丙基、
Figure GDA0003631929380000032
Figure GDA0003631929380000033
本发明所述的通式(I)化合物或其药学上可接受的盐、酯、溶剂化物或前药，其特征在于，药学上可接受的盐是指通式I的化合物和药学上可接受的酸形成的盐包括无机酸盐，所述的无机酸包括：碳酸、碳酸氢根、硫酸、硫酸氢根、磷酸、磷酸一氢根、磷酸二氢根、硝酸、氯化氢、溴化氢、氢碘酸等；以及有机酸盐，所述的有机酸包括乙酸、草酸、乳酸、琥珀酸、酒石酸、柠檬酸、甲磺酸、丙酮酸、苯磺酸、对甲苯磺酸、阿魏酸、马来酸、异丁酸、丙二酸、辛二酸、扁桃酸、反丁烯二酸等类似的酸；还包括与氨基酸形成的盐等。
本发明中所优选的部分化合物结构如下：
Figure GDA0003631929380000034
Figure GDA0003631929380000041
Figure GDA0003631929380000051
Figure GDA0003631929380000061
Figure GDA0003631929380000071
Figure GDA0003631929380000081
本发明所述的通式(I)化合物或其药学上可接受的盐、酯、溶剂化物或前药用于开发以CDK9为靶点的小分子抑制剂。
一种药物组合物，其特征在于，本发明所述的通式(I)化合物或其药学上可接受的盐、酯、溶剂化物或前药，和药学上可接受的载体。
所述的一种药物组合物，其特征在于，本发明所述的通式(I)化合物或其药学上可接受的盐、酯、溶剂化物或前药和药学上可接受的辅料。
本发明所述的通式(I)化合物或其药学上可接受的盐、酯、溶剂化物或前药，或一种药物组合物，在制备预防和治疗抗病毒药物和抗肿瘤药物中的用途。
本发明所述的通式(I)化合物或其药学上可接受的盐、酯、溶剂化物或前药在制备预防和治疗抗病毒药物和抗肿瘤药物中的用途，其特征在于，所述的病毒包括人类免疫缺陷病毒、巨细胞病毒、EB病毒、腺病毒、疱疹、人T细胞淋巴细胞病毒、甲型流感病毒和登革热病毒。
本发明所述的通式(I)化合物或其药学上可接受的盐、酯、溶剂化物或前药在制备预防和治疗抗病毒药物和抗肿瘤药物中的用途，其特征在于，所述的肿瘤包括神经系统癌症、血液癌症、肝癌、肌肉癌、乳腺癌、生殖系统癌症、肺癌、胃肠癌、皮肤癌、泌尿系统癌症和食管癌。
有益效果
本发明针对汉黄芩素抗肿瘤活性低，成药性差的缺点，本发明对其母核进行改造合成了一系列的化合物，特别是针对3’或4’位引入含N环状取代基，含氮环状取代基的引入既提高了化合物对CDK9的选择性，同时改善了化合物的理化性质。该类化合物是全新化合物，未见文献报道，同时发明人针对合成的化合物做了一系列活性，具体来说本发明大部分化合物对癌细胞均有较好的抑制活性，相对于汉黄芩素有很大的提高，活性较为突出的化合物主要有L26、L27、L32和L38。
本发明部分化合物相对汉黄芩素对CDK9具有较好的抑制活性，与Flavopiridol相当，但对CDK9的选择性得到了明显的提高。特别是化合物L26对CDK9的抑制活性与Flavopiridol基本相当，其IC50值达到了3.0nM，化合物L32对CDK9/CDK2的选择性是汉黄芩53倍，是Flavopiridol的45倍。
相对于CDK家族其他CDK来说，本发明部分化合物对CDK9具有较好的抑制活性且对CDK9的选择性也优于Flavopiridol。
本发明优选化合物L27的溶解度较好，特别是在酸性条件下(pH＝4.5)的溶解度相对于其他化合物得到了大幅度的提高在相同条件下其溶解度是汉黄芩素的500倍，Log D值也是处于合理的范围内。
选择活性较好化合物L26能通过抑制CDK9的活性，使得2位Ser磷酸化的RNA聚合酶II的表达量明显减少，并能使其下游的短促凋亡蛋白Mcl-1的表达量呈浓度依赖性的减少，使Cleaver caspase 3的表达量呈浓度依赖性的增加。这表明了化合物L26是通过抑制CDK9的活性，使得下游的促凋亡蛋白的表达增加，进而引起细胞发生凋亡的。
附图说明
图1本发明中化合物L38诱导MV4-11细胞凋亡实验结果；
图2本发明中化合物L38对CDK9及其下游蛋白及凋亡蛋白表达量的影响。
具体实施方式
下面通过具体的实施例对本发明做进一步的详细说明，但并不将本发明限制在所举的实施例范围之中。
实施例1
Figure GDA0003631929380000091
第一步：中间体I-2的制备
将间苯三酚I-1(63g，0.5mol)加入到300mL无水乙醚中，室温搅拌溶解，再依次加入无水ZnCl2(13.4g，0.1mol)和无水乙腈(50g，1.25mol)，0℃下通入干燥的HCl气体反应40h，TLC检测(V石油醚：V乙酸乙酯＝1:2)反应完全，静止待反应液温度升至室温，抽滤，滤饼烘干至恒重后溶于200mL水中，加热回流6h，析出大量固体，冷却至室温，抽滤，烘干，得白色固体60g，收率71％。m.p.119～121℃。
第二步：中间体I-3的制备
将中间体I-2(33.6g，0.2mol)加入到350mL无水丙酮，室温搅拌溶解，再加入63.48g K2CO3(0.46mol)，室温条件下滴加硫酸二甲酯(39.78mL，0.42mol)，1h左右滴加完毕，45℃继续反应4h，TLC检测(V石油醚：V乙酸乙酯＝3:1)反应完全，静止冷却至室温，抽滤，滤液倒入5倍体积的水中，用1M HCl调pH＝3-4，析出大量固体。抽滤，干燥得白色固体32.5g，收率83％。m.p.80～82℃。
第三步：中间体I-4的制备
将中间体I-3(30g，15mmol)加入到450mL乙醇，室温搅拌溶解，再加入11.39g间溴苯甲醛(92mmol)，冰浴条件下滴加7％KOH水溶液280mL(0.21mol)，滴加过程中保持温度小于5℃。1h左右滴加完毕，40℃继续反应10h，TLC检测(V石油醚：V乙酸乙酯＝6:1)反应完全，静止冷却至室温，反应液倒入10倍体积的水中，用6M HCl调pH＝3-4，析出大量固体。抽滤，烘干，得黄色固体50.1g，收率90％。m.p.190～193℃。
第四步：中间体I-5的制备
中间体I-4(16.7g，46mmol)加入到43mL无水DMSO，升温至60℃，搅拌溶解，再加入催化量的I2(0.36g，1.4mmol)，升温至130℃继续反应5h，TLC检测(V石油醚：V乙酸乙酯＝3:1)反应完全，反应液趁热倒入10倍体积的饱和硫代硫酸钠水溶液中，析出大量固体。抽滤，烘干，得淡黄色固体16.3g，收率97％。m.p.278～280℃。
第五步：中间体I-6的制备
将中间体I-5(20g，55mmol)加入到48％的氢溴酸300mL中，回流反应60h，TLC检测(V石油醚：V乙酸乙酯＝2:1)反应完全，静止冷却至室温，反应液倒入10倍体积的冰水中，析出大量固体。抽滤，滤饼用水多次洗涤至pH呈中性，干燥得粗品20.9g，聚酰胺柱层析(V石油醚：V乙酸乙酯＝2:1)得淡黄色固体15g，收率83％。m.p.192～196℃。
第六步：中间体I-7的制备
将中间体I-6(21g，64mmol)和氢氧化四甲铵(69g，38mmol)加入到450mL水中，室温搅拌溶解，再每隔20min分8次加入过硫酸钾(43g，169mmol)，加入过程中保持温度低于30℃，室温继续反应3h，TLC检测(V石油醚：V乙酸乙酯＝2:1)反应完全，抽滤，滤液用磷酸二氢钾(约30g)调pH＝6-7，析出固体，再每隔5min分3次加入45g氯化钠，室温搅拌过夜。抽滤，滤饼用20mL甲醇洗涤，烘干，得土黄色固体19g，收率60％。
第七步：中间体I-8的制备
将中间体I-7(7.8g，17.7mmol)加入到55mL DMF，室温搅拌溶解，再加入4.9g K2CO3(35.5mmol)，室温条件下滴加溴化苄(2.8mL，23mmol)，5min左右滴加完毕，60℃继续反应5h，TLC检测(V二氯甲烷：V甲醇＝10:1)反应完全，静止冷却至室温，抽滤，滤饼用20mL甲醇洗涤，滤液用6M HCl调pH＝1-2，室温搅拌过夜，析出固体。抽滤，滤饼用10mL甲醇洗涤，烘干，得橙黄色固体2.6g，收率38.8％。m.p.290～292℃。1H NMR(300MHz,DMSO-d6):δ＝12.69(s,1H,5-OH),9.13(s,1H,8-OH),7.92(d,2H,J＝9.00Hz,Ar-H),7.45(m,5H,Ar-H),7.12(d,2H,J＝9.00Hz,Ar-H),6.93(s,1H,CHCO),6.79(s,1H,Ar-H),5.34(s,2H,OCH2-Ar)ppm.
第八步：中间体I-9的制备
将中间体I-8(720mg，1.9mmol)加入到60mL乙腈，室温搅拌溶解，再加入660mgK2CO3(4.8mmol)和240mg硫酸二甲酯(2.1mmol)，回流反应4.5h，TLC检测(V石油醚：V乙酸乙酯＝2:1)反应完全，静止冷却至室温，抽滤，滤饼用20mL二氯甲烷洗涤，滤液减压蒸除溶剂，粗品用正己烷和二氯甲烷混合溶剂重结晶，得黄色固体685mg，收率92％。m.p.210～212℃。1H NMR(300MHz,DMSO-d6):δ＝12.70(s,1H,5-OH),7.93(d,2H,J＝9.00Hz,Ar-H),7.46(m,5H,Ar-H),7.10(d,2H,J＝9.00Hz,Ar-H),6.94(s,1H,CHCO),6.80(s,1H,Ar-H),5.33(s,2H,OCH2-Ar),3.85(s,3H,ArOCH3)ppm.
第九步：中间体I-10的制备
将中间体I-9(185mg,0.47mmol)加入到10mL无水DMSO，升温至60℃，搅拌溶解，再在N2保护下依次加入410.7mg吗啉(4.8mmol)和121.9mg DIEA(0.94mmol)，90℃反应10h，TLC检测(V石油醚：V乙酸乙酯＝4:1)反应完全，反应液趁热倒入10mL饱和食盐水中，静止3h，抽滤，滤饼用10mL水洗涤，最终得到黄色固体178mg，产率74.9％。m.p.220～225℃.1H NMR(300MHz,DMSO-d6):δ＝12.79(s,1H,5-OH),7.86(d,2H,J＝9.00Hz,Ar-H),7.44(m,5H,Ar-H),6.97(d,2H,J＝9.00Hz,Ar-H),6.55(s,1H,CHCO),6.46(s,1H,Ar-H),5.23(s,2H,OCH2-Ar),3.92(s,3H,ArOCH3),3.30(s,4H,N(CH 2CH2)2NH),2.95(s,4H,N(CH2CH 2)2NH)ppm.
第十步：化合物L1的制备
将中间体I-10(185mg,0.27mml)和Pd/C(60mg)加入到10mL四氢吡喃和10mL甲醇，密闭体系通入氢气，35℃反应6h，TLC检测(V乙酸乙酯：V甲醇＝10:1)反应完全，静止冷却至室温，抽滤，滤液减压蒸除溶剂，硅胶柱层析(V乙酸乙酯：V甲醇＝15:1)最终得到黄色固体78mg，产率77％。m.p.204～206℃.HRMS(ESI),[M+H]+calculated for C20H19NO6369.1212,found369.1271.1H NMR(300MHz,DMSO-d6)：＝12.53(s,1H,5-OH),10.81(s,1H,7-OH),7.48(m,3H,Ar-H),7.20(d,1H,J＝7.50Hz,Ar-H),7.05(s,1H,CHCO),6.30(s,1H,Ar-H),3.85(s,3H,ArOCH3),3.77(s,4H,N(CH2CH2)2O),3.23(s,4H,N(CH2CH2)2O)ppm.13C NMR(75MHz,DMSO-d6)：＝182.1，163.4，157.3，156.2，151.5，149.6，l31.6，129.8,127.6,118.7,116.7,112.2,105.0,103.7,99.0,66.0,61.0(2×C),48.0(2×C)ppm.IR(KBr)ν3245,2829,1657,1586,1507,1444,1356,1251,1163,1023,1010,863,837,699,671cm-1.
实施例2-实施例10所述化合物的制备，可参考实施例1中所描述的方法和路线制备得到。
实施例2：化合物L2
Figure GDA0003631929380000131
m.p.150～153℃.HRMS(ESI),[M+H]+calculated for C23H26N2O5 410.1842,found410.1907.1H NMR(300MHz,DMSO-d6):δ＝12.62(s,1H,5-OH),7.32(m,3H,Ar-H),6.95(d,1H,J＝6.0Hz,Ar-H),6.91(s,1H,CHCO),6.26(s,1H,Ar-H),4.45(m,1H,CH(CH3)2),3.60(s,4H,Ar-N(CH 2)2),2.95(s,2H,NHCH 2),2.73(s,2H,NHCH 2),1.89(m,2H,NCH2CH 2CH2NH),1.28(d,6H,J＝6.00Hz,CH(CH 3)2)ppm.13C NMR(75MHz,DMSO-d6):δ＝181.8,163.5,159.8,156.0,150.0,148.6,131.9,130.0,125.2,114.9,113.2,108.1,104.5,102.8,99.5,73.9,50.7,48.6,47.2,46.8,28.0,22.3(2×C)ppm.IR(KBr)ν3445,2970,1652,1593,1497,1370,1322,1192,1109,1031,842,699,576,548cm-1.
实施例3：化合物L3
Figure GDA0003631929380000132
m.p.240～242℃.HRMS(ESI),[M+H]+calculated for C25H28N2O5 436.1998,found436.2065.1H NMR(300MHz,DMSO-d6):δ＝12.58(s,1H,5-OH),10.73(s,1H,7-OH),7.44(m,3H,Ar-H),7.18(d,1H,J＝7.2Hz,Ar-H),7.03(s,1H,CHCO),6.33(s,1H,Ar-H),4.83(m,1H,CH(CH2)4),3.25(s,4H,Ar-N(CH 2CH2)2N),2.50(s,4H,Ar-N(CH2CH 2)2N),2.26(s,3H,NCH3),1.68(m,8H,CH(CH 2)4)ppm.13C NMR(75MHz,DMSO-d6):δ＝182.1,163.4,157.8,155.8,151.4,150.0,131.5,129.8,125.4,118.8,116.6,112.4,104.9,103.7,99.1,84.0,54.4(2×C),47.7(2×C),45.6,32.3(2×C),23.2(2×C)ppm.IR(KBr)ν3441,2952,1651,1597,1496,1432,1375,1328,1260,1006,930,862,788,700,571cm-1.
实施例4：化合物L4
Figure GDA0003631929380000141
m.p.190～195℃.HRMS(ESI),[M+H]+calculated for C20H20N2O5 368.1372,found368.1439.1H NMR(300MHz,DMSO-d6):δ＝12.56(s,1H,5-OH),7.89(d,2H,J＝9.00Hz,Ar-H),7.14(d,2H,J＝9.00Hz,Ar-H),6.82(s,1H,CHCO),6.35(s,1H,Ar-H),3.98(s,3H,ArOCH3),3.20(s,4H,N(CH 2CH2)2NH),2.90(s,4H,N(CH2CH 2)2NH)ppm.13C NMR(75MHz,DMSO-d6):δ＝182.1,163.3,158.5,156.2,151.8,149.6,127.8(2×C),118.8,116.3,113.3(2×C),104.8,103.1,99.3,60.8,48.6(2×C),45.2(2×C)ppm.IR(KBr)ν3537,2953,1643,1605,1575,1482,1443,1386,1215,1186,1124,992,837,670,553cm-1.
实施例5：化合物L5
Figure GDA0003631929380000142
m.p.190～195℃.HRMS(ESI),[M+H]+calculated for C21H22N2O5 382.1529,found382.1599.1H NMR(300MHz,DMSO-d6):δ＝12.70(s,1H,5-OH),10.91(s,1H,7-OH),7.82(d,2H,J＝9.00Hz,Ar-H),6.99(d,2H,J＝9.00Hz,Ar-H),6.57(s,1H,CHCO),6.43(s,1H,Ar-H),4.05(s,3H,ArOCH3),3.44(s,4H,Ar-N(CH 2CH2)2N),3.01(s,3H,NCH3),2.43(s,4H,Ar-N(CH2CH 2)2N)ppm.13C NMR(75MHz,DMSO-d6):δ＝182.3,167.8,157.5,156.1,150.9,149.6,127.3(2×C),118.5,116.4,113.0(2×C),105.1,103.7,99.1,61.0,52.1(2×C),45.5(2×C),42.2ppm.IR(KBr)ν3430,2854,2648,1653,1454,1367,1228,1210,1026,921,837,780,692,583cm-1.
实施例6：化合物L6
Figure GDA0003631929380000143
m.p.207～210℃.HRMS(ESI),[M+H]+calculated for C22H24N2O6 412.1634,found412.1701.1H NMR(300MHz,DMSO-d6):δ＝12.76(s,1H,5-OH),7.92(d,2H,J＝8.90Hz,Ar-H),7.10(d,2H,J＝8.90Hz,Ar-H),6.80(s,1H,CHCO),6.31(s,1H,Ar-H),3.88(s,3H,ArOCH3),3.59(t,2H,J＝6.00Hz,NCH2CH 2OH),3.37(s,4H,Ar-N(CH 2CH2)2N),2.60(s,4H,Ar-N(CH2CH 2)2N),2.49(t,2H,J＝6.00Hz,NCH 2CH2OH)ppm.13C NMR(75MHz,DMSO-d6):δ＝181.7,163.6,157.0,156.2,153.1,149.3,127.7,127.5(2×C),118.9,114.0,113.9,103.4,101.8,98.9,60.9,60.1,58.5,52.8(2×C),46.6(2×C)ppm.IR(KBr)ν3380,2825,1656,1608,1580,1505,1392,1235,1207,1163,1102,1015,946,819,663cm-1.
实施例7：化合物L7
Figure GDA0003631929380000151
m.p.210～213℃.HRMS(ESI),[M+H]+calculated for C22H24N2O5 396.1685,found396.1710.1H NMR(300MHz,DMSO-d6):δ＝12.75(s,1H,5-OH),7.90(d,2H,J＝9.00Hz,Ar-H),7.08(d,2H,J＝9.00Hz,Ar-H),6.76(s,1H,CHCO),6.27(s,1H,Ar-H),4.38(m,1H,CH(CH3)2),3.28(s,4H,N(CH 2CH2)2NH),2.84(s,4H,N(CH2CH 2)2NH),1.30(d,6H,J＝6.09Hz,CH(CH 3)2)ppm.13C NMR(75MHz,DMSO-d6):δ＝182.0,164.4,156.3,152.1,151.1,149.8,127.6(2×C),125.1,119.0,113.1(2×C),103.4,101.7,98.8,73.8,50.7(2×C),46.6(2×C),22.3(2×C)ppm.IR(KBr)ν3379,2813,1616,1525,1506,1368,1262,1237,1119,1026,821,663,595cm-1.
实施例8：化合物L8
Figure GDA0003631929380000152
m.p.240～242℃.HRMS(ESI),[M+H]+calculated for C23H26N2O5 410.1842,found410.1890.1H NMR(300MHz,DMSO-d6):δ＝12.76(s,1H,5-OH),10.59(s,1H,7-OH),7.92(d,2H,J＝9.00Hz,Ar-H),7.11(d,2H,J＝9.00Hz,Ar-H),6.79(s,1H,CHCO),6.28(s,1H,Ar-H),4.37(m,1H,CH(CH3)2),3.34(s,4H,Ar-N(CH 2CH2)2N),3.34(s,3H,NCH3),2.45(s,4H,Ar-N(CH2CH 2)2N),1.30(d,6H,J＝6.09Hz,CH(CH 3)2)ppm.13C NMR(75MHz,DMSO-d6):δ＝181.0,167.8,157.5,156.1,150.9,149.6,127.3(2×C),118.5,116.4,113.0(2×C),105.1,103.7,99.1,74.0,52.1(2×C),45.5(2×C),42.2,22.1(2×C)ppm.IR(KBr)ν3430,2854,2648,1653,1454,1367,1228,1210,1026,921,837,780,692,583cm-1.
实施例9：化合物L9
Figure GDA0003631929380000161
m.p.265～268℃.HRMS(ESI),[M+H]+calculated for C23H24N2O6 424.1634,found424.1707.1H NMR(300MHz,DMSO-d6):δ＝12.73(s,1H,5-OH),10.56(s,1H,7-OH),8.11(s,1H,NCHO),7.94(d,2H,J＝9.00Hz,Ar-H),7.15(d,2H,J＝9.00Hz,Ar-H),6.81(s,1H,CHCO),6.29(s,1H,Ar-H),4.38(m,1H,CH(CH3)2),3.52(s,4H,Ar-N(CH 2CH2)2N),3.40(m,4H,Ar-N(CH2CH 2)2N),1.30(d,6H,J＝6.15Hz,CH(CH 3)2)ppm.13C NMR(75MHz,DMSO-d6):δ＝181.9,164.1,163.5,161.0,157.6,155.9,152.8,127.8,127.6(2×C),119.6,114.6(2×C),103.4,102.0,98.9,74.8,47.7,46.4,44.1(2×C),22.3(2×C)ppm.IR(KBr)ν3080,2972,1656,1574,1516,1433,1368,1277,1230,1202,1098,1003,968,847,825,658,559cm-1.
实施例10：化合物L10
Figure GDA0003631929380000162
m.p.160～168℃.HRMS(ESI),[M+H]+calculated for C26H30N2O5 450.2188,found450.2243.1H NMR(300MHz,DMSO-d6):δ＝12.90(s,1H,5-OH),7.88(d,2H,J＝9.00Hz,Ar-H),6.90(d,2H,J＝9.00Hz,Ar-H),6.72(s,1H,CHCO),6.30(s,1H,Ar-H),4.10(m,1H,CH(CH2)5),3.65(s,4H,Ar-N(CH 2)2),3.00(s,2H,NHCH 2),2.83(s,2H,NHCH 2),1.90(m,2H,NCH2CH 2CH2NH),1.77(m,4H,CH(CH 2)5),1.52(m,2H,CH(CH 2)5),1.23(m,4H,CH(CH 2)5)ppm.13CNMR(75MHz,DMSO-d6):δ＝181.5,163.6,158.6,155.3,153.6,149.5,127.8(2×C),125.1,118.8,113.3,111.4,109.0,103.6,98.9,81.0,60.1,50.9(2×C),47.2,46.9,33.9,27.8,26.3(2×C),23.9ppm.IR(KBr)ν3217,1654,1516,1400,1105,859,546cm-1.
实施例11-实施例15所述化合物的前七步中间体的制备，可参考实施例1中所描述的方法和路线制备得到。
实施例11：化合物L11
Figure GDA0003631929380000171
第八步：中间体II-9的制备
将中间体I-7(6g，13.6mmol)加入到30mL DMF，室温搅拌溶解，再加入5.7g K2CO3(41mmol)，室温条件下滴加硫酸二甲酯(1.9g，15mmol)，5min左右滴加完毕，80℃继续反应10h，TLC检测(V二氯甲烷：V甲醇＝10:1)反应完全，静止冷却至室温，抽滤，滤饼用20mL甲醇洗涤，滤液用6M HCl调pH＝1-2，室温搅拌过夜，用300mL冰水稀释，析出大量固体。抽滤，滤饼烘干，粗品用甲醇和二氯甲烷的混合溶剂重结晶得黄色固1.0g，收率28％。m.p.155～158℃.1H NMR(300MHz,DMSO-d6):δ＝12.56(s,1H,5-OH),9.02(s,1H,8-OH),8.26(d,2H,J＝9.00Hz,Ar-H),7.46(d,2H,J＝9.00Hz,Ar-H),7.01(s,1H,CHCO),6.60(s,1H,Ar-H),3.96(s,3H,OCH3)ppm.
第九步：中间体II-10的制备
将中间体II-9(3g，10mmol)加入到20mL DMF，室温搅拌溶解，再加入3.45g K2CO3(25mmol)和1.85g溴代异丙烷(15mmol)，80℃反应8h，TLC检测(V石油醚：V乙酸乙酯＝1:1)反应完全，反应液趁热倒入10倍体积的冰水中，析出固体，静止2h，抽滤，烘干，粗品用乙醚和丙酮的混合溶剂重结晶，得黄色固体1.8g，收率52.3％。m.p.158～160℃.1H NMR(300MHz,DMSO-d6):δ＝12.59(s,1H,5-OH),8.15(d,2H,J＝9.00Hz,Ar-H),7.50(d,2H,J＝9.00Hz,Ar-H),7.02(s,1H,CHCO),6.62(s,1H,Ar-H),4.30(m,1H,CH(CH3)2),3.89(s,3H,OCH3),1.36(d,6H,J＝6.00Hz,CH(CH 3)2)ppm.
第十步：化合物L11的制备
将中间体II-10(100mg,0.29mmol)加入到10mL无水DMSO，升温至60℃，搅拌溶解，再在N2保护下依次加入250mg哌嗪(2.90mmol)和375mg DIEA(2.90mmol)，80℃反应8h，TLC检测(V石油醚：V乙酸乙酯＝2:1)反应完全，反应液趁热倒入10mL饱和食盐水中，静止3h，抽滤，滤饼用10mL水洗涤，烘干得粗品90mg，粗品用甲醇和丙酮的混合溶剂重结晶，得黄色固体64mg，收率54％。m.p.190～192℃.HRMS(ESI),[M+H]+calculated for C23H26N2O5411.1914,found411.1917.1H NMR(300MHz,DMSO-d6):δ＝12.89(s,1H,5-OH),7.91(d,2H,J＝9.00Hz,Ar-H),7.07(d,2H,J＝9.00Hz,Ar-H),6.79(s,1H,CHCO),6.55(s,1H,Ar-H),4.32(m,1H,CH(CH3)2),3.90(s,3H,OCH3),3.28(s,4H,N(CH 2CH2)2NH),2.82(s,4H,N(CH2CH 2)2NH),1.30(d,6H,J＝6.00Hz,CH(CH 3)2)ppm.13C NMR(75MHz,DMSO-d6):δ＝181.9,164.0,158.6,156.4,153.6,149.1,127.7(2×C),126.0,118.5,113.8(2×C),103.8,101.5,95.8,75.2,56.4,47.7(2×C),45.3(2×C),22.4(2×C)ppm.IR(KBr)ν3350,2938,1650,1606,1580,1513,1380,1265,1204,1105,1069,1001,828,667,590cm-1.
实施例12-实施例13所述化合物的的制备，可参考实施例11中所描述的方法和路线制备得到。
实施例12：化合物L12
Figure GDA0003631929380000181
m.p.160～168℃.HRMS(ESI),[M+H]+calculated for C24H28N2O5 425.2071,found425.2074.1H NMR(300MHz,DMSO-d6):δ＝12.88(s,1H,5-OH),7.92(d,2H,J＝9.00Hz,Ar-H),7.10(d,2H,J＝9.00Hz,Ar-H),6.80(s,1H,CHCO),6.55(s,1H,Ar-H),4.32(m,1H,CH(CH3)2),3.89(s,3H,OCH3),3.63(s,4H,N(CH 2CH2)2NH),3.35(s,4H,N(CH2CH 2)2NH),2.27(s,3H,NCH3),1.29(d,6H,J＝6.00Hz,CH(CH 3)2)ppm.13C NMR(75MHz,DMSO-d6):δ＝181.9,164.0,158.6,156.4,153.2,149.1,127.7(2×C),126.0,118.8,114.0(2×C),103.8,101.2,95.8,74.6,56.4,54.4(2×C),47.8(2×C),45.6,22.2(2×C)ppm.IR(KBr)ν3455,2971,2851,1654,1581,1519,1365,1323,1276,1253,1103,1006,944,850,806,591cm-1.
实施例13：化合物L13
Figure GDA0003631929380000191
m.p.200～202℃.HRMS(ESI),[M+H]+calculated for C24H28N2O5 425.2071,found425.2074.1H NMR(300MHz,DMSO-d6):δ＝13.04(s,1H,5-OH),7.87(d,2H,J＝9.00Hz,Ar-H),6.87(d,2H,J＝9.00Hz,Ar-H),6.72(s,1H,CHCO),6.53(s,1H,Ar-H),4.32(m,1H,CH(CH3)2),3.89(s,3H,OCH3),3.63(s,4H,Ar-N(CH 2)2),3.56(s,2H,NHCH 2),2.86(s,2H,NHCH 2),2.63(m,2H,NCH2CH 2CH2NH),1.29(d,6H,J＝6.00Hz,CH(CH 3)2)ppm.13C NMR(75MHz,DMSO-d6):δ＝181.8,163.5,159.8,156.0,151.0,149.6,128.7(2×C),126.0,118.8,114.0(2×C),103.8,101.2,95.8,73.9,56.4,50.7,48.6,47.2,46.8,28.0,22.3(2×C)ppm.IR(KBr)ν3217,1654,1516,1400,1105,859,546cm-1.
实施例14所述化合物的前九步中间体的制备，可参考实施例1和实施例11中所描述的方法和路线制备得到。
实施例14：化合物L14
Figure GDA0003631929380000192
第十步：中间体II-11的制备
中间体19(500mg，1.35mmol)和5mL DMF加入10mL耐压瓶中,室温搅拌溶解,微波反应(功率：300W)，升温至240℃反应30min。TLC检测(V石油醚：V乙酸乙酯＝2:1)反应完全，反应液倒入10mL冰水中，静止3h，析出固体，抽滤，滤饼烘干的黄色固体400mg，收率80％.1H NMR(300MHz,DMSO-d6):δ＝12.90(s,1H,5-OH),10.59(s,1H,7-OH),7.89(d,2H,J＝9.00Hz,Ar-H),7.21(d,2H,J＝9.00Hz,Ar-H),6.94(s,1H,CHCO),5.90(m,1H,-CH＝CH2),5.43(m,2H,-CH＝CH 2),4.45(m,1H,CH(CH3)2),3.31(d,J＝5.70Hz,2H,-CH 2CH＝CH2),1.29(d,6H,J＝6.00Hz,CH(CH 3)2)ppm.
第十步：化合物L14的制备
将中间体II-11(100mg,0.27mmol)加入到8mL无水DMSO中，升温至60℃，搅拌溶解，再在N2保护下依次加入116mg 4-甲基哌嗪(3.5mmol)和70mg DIEA(0.54mmol)，80℃反应24h，TLC检测(V石油醚：V乙酸乙酯＝2:1)反应完全，反应液趁热倒入10mL饱和食盐水中，静止3h，抽滤，滤饼用10mL水洗涤，烘干得粗品120mg，粗品用甲醇和二氯甲烷的混合溶剂重结晶，得黄色固体66mg，收率56％。m.p.203～208℃.HRMS(ESI),[M+H]+calculated for C26H30N2O5451.2188,found 451.2263.1H NMR(300MHz,DMSO-d6):δ＝13.1(s,1H,5-OH),10.59(s,1H,7-OH),7.90(d,2H,J＝9.00Hz,Ar-H),6.89(d,2H,J＝9.00Hz,Ar-H),6.80(s,1H,CHCO),5.91(m,1H,-CH＝CH2),4.96(m,2H,-CH＝CH 2),4.40(m,1H,CH(CH3)2),3.35(s,4H,N(CH 2CH2)2NH),3.31(d,J＝5.70Hz,2H,-CH 2CH＝CH2),2.48(s,4H,N(CH2CH 2)2NH),2.25(s,3H,NCH3),1.32(d,6H,J＝6.00Hz,CH(CH 3)2)ppm.13C NMR(75MHz,DMSO-d6):δ＝181.6,163.2,158.3,155.5,153.7,151.1,147.6,135.8,127.5,124.4,118.9,116.4,114.5,113.9,111.4,109.2,101.8,75.2,51.8,51.3,49.3,48.7,46.6,27.7,22.1,21.8ppm.IR(KBr)ν3128,1647,1605,1582,1514,1480,1400,1224,1108,1007,823,546cm-1.
实施例15所述化合物的前十步中间体的制备，可参考实施例1和实施例11中所描述的方法和路线制备得到。
Figure GDA0003631929380000201
实施例15：化合物L15
m.p.165～170℃.HRMS(ESI),[M+H]+calculated for C26H33N3O5 468.2454,found468.2496.1H NMR(300MHz,DMSO-d6):δ＝13.5(s,1H,5-OH),7.86(d,2H,J＝9.00Hz,Ar-H),6.86(d,2H,J＝9.00Hz,Ar-H),6.60(s,1H,CHCO),4.46(m,1H,CH(CH3)2),3.87(s,2H,CH 2N(CH3)2),3.60(s,4H,Ar-N(CH 2)2),2.82(s,2H,NHCH 2),2.77(s,2H,NHCH 2),2.56(s,6H,CH2N(CH 3)2),1.91(m,2H,NCH2CH 2CH2NH),1.27(d,6H,J＝6.00Hz,CH(CH 3)2)ppm.13C NMR(75MHz,DMSO-d6):δ＝181.5,163.1,153.8,153.1,149.2,133.3,130.5,127.6,127.3,127.1,119.5,114.0,102.9,101.1,99.8,73.5,53.6,52.3,51.8,50.6,48.2,47.0,45.6,28.9,22.5,22.4ppm.IR(KBr)ν3126,1654,1577,1517,1400,1112,859,545cm-1.
实施例16-实施例25所述化合物的制备，可参考实施例1中所描述的方法和路线制备得到。
实施例16：化合物L16
Figure GDA0003631929380000211
m.p.200～205℃.1H NMR(300MHz,DMSO-d6):δ＝12.75(s,1H,5-OH),7.92(d,2H,J＝9.00Hz,Ar-H),7.07(d,2H,J＝9.00Hz,Ar-H),6.76(s,1H,CHCO),6.30(s,1H,Ar-H),4.87(m,1H,OCH(CH2)2),3.42(m,4H,CHCH 2N&CHCH2CH 2N),3.20(s,4H,N(CH 2CH2)2NH),3.07(s,4H,N(CH2CH 2)2NH),2.10(m,2H,CHCH 2CH2N).13C NMR(75MHz,DMSO-d6):182.0,163.1,159.0,156.0,151.9,150.0,131.6,129.8,125.2,118.8,116.4,112.2,104.7,103.2,99.3,74.3,48.8(2×C),45.2(2×C),22.3(2×C)ppm.IR(KBr)ν3132,2970,1654,1603,1512,1073,547cm-1.
实施例17：化合物L17
Figure GDA0003631929380000212
m.p.250～252℃.HRMS(ESI),[M+H]+calculated for C26H32N3O5 466.2338,found466.2335.1H NMR(300MHz,DMSO-d6):δ＝12.70(s,1H,5-OH),7.77(d,2H,J＝9.00Hz,Ar-H),3.96(d,2H,J＝9.00Hz,Ar-H),6.52(s,1H,CHCO),6.28(s,1H,Ar-H),4.29(m,1H,Ar-OCH),3.38(s,4H,Ar-N(CH 2CH2)2N),2.97(s,4H,Ar-N(CH2CH 2)2N),2.59(t,2H,J＝6.00Hz,CH 2NCH3),2.37(s,3H,NCH3),2.09(s,3H,CH2NCH 3),2.60(t,2H,J＝6.00Hz,CH 2NCH3),1.76(m,4H,OCH(CH 2)2)ppm.13C NMR(75MHz,DMSO-d6):δ＝182.28,163.82,157.90,157.49,153.27,149.77,127.58,124.75,120.52,114.46,104.48,103.04,99.46,77.24,54.69,52.63,47.30,46.11,45.45,30.75ppm.IR(KBr)ν3441,2952,1597,1496,1432,1375,1328,1260,1006,930,862,788,699cm-1.
实施例18：化合物L18
m.p.190～195℃.HRMS(ESI),[M+H]+calculated for C25H24N2O6 449.1707,found449.1702.1H NMR(300MHz,DMSO-d6):δ＝12.78(s,1H,5-OH),7.81(d,2H,J＝9.00Hz,Ar-H),7.34(s,1H,Ar-H),6.72(d,2H,J＝9.00Hz,Ar-H),6.64(s,1H,CHCO),6.38(s,1H,Ar-H),6.26(m,2H,Ar-H),4.98(s,2H,OCH2-Ar),3.66(s,4H,Ar-N(CH 2)2),3.07(s,2H,NHCH 2),2.86(s,2H,NHCH 2),1.95(m,2H,NCH2CH 2CH2NH)ppm.13C NMR(75MHz,DMSO-d6):δ＝181.98,163.60,157.56,156.24,153.42,151.76,148.70,143.74,127.74,124.60,119.72,112.76,110.66,107.52,104.36,101.39,99.30,66.82,58.02,49.24,48.44,47.12,28.96ppm.IR(KBr)ν3440,2927,1652,1598,1499,1368,1026,838,772,690,591cm-1.
实施例19：化合物L19
m.p.195～198℃.HRMS(ESI),[M+H]+calculated for C24H24N4O5 449.1780,found449.1792.1H NMR(300MHz,DMSO-d6):δ＝12.80(s,1H,5-OH),11.88(s,1H,-NH-N＝),10.84(s,1H,7-OH),7.63(d,2H,J＝9.00Hz,Ar-H),7.00(d,2H,J＝9.00Hz,Ar-H),6.78(s,1H,CHCO),6.31(s,1H,Ar-H),3.41(s,4H,N(CH 2CH2)2NH),2.51(s,4H,N(CH2CH 2)2NH),2.00(s,6H,Ar-CH3)ppm.13C NMR(75MHz,DMSO-d6):δ＝181.6,163.6,156.6,156.5,153.1,148.5,138.2,127.6(2×C),125.0,118.8,113.9(2×C),103.0,102.1,98.9,54.2(2×C),46.4(2×C),9.8ppm.IR(KBr)ν3407,1653,1587,1567,1514,1375,1222,1203,1107,1000,823,566,450cm-1.
实施例20：化合物L20
m.p.177～180℃.HRMS(ESI),[M+H]+calculated for C25H26N4O5 463.1937,found463.1972.1H NMR(300MHz,DMSO-d6):δ＝12.80(s,1H,5-OH),11.85(s,1H,-NH-N＝),7.63(d,2H,J＝9.00Hz,Ar-H),7.01(d,2H,J＝9.00Hz,Ar-H),6.78(s,1H,CHCO),6.33(s,1H,Ar-H),3.40(s,4H,Ar-N(CH 2CH2)2N),2.44(s,4H,Ar-N(CH2CH 2)2N),2.23(s,3H,NCH3),1.98(s,6H,Ar-CH3)ppm.13C NMR(75MHz,DMSO-d6):δ＝181.6,163.6,156.6,156.5,153.1,148.5,138.2,127.6(2×C),125.0,118.8,113.9(2×C),103.0,102.1,98.9,54.2(2×C),46.4(2×C),9.8(2×C)ppm.IR(KBr)ν3419,1654,1587,1567,1383,1236,1221,1106,1001,882,566,453cm-1.
实施例21：化合物L21
m.p.200～202℃.HRMS(ESI),[M+H]+calculated for C23H22N4O6 451.1573,found451.1596.1H NMR(300MHz,DMSO-d6):δ＝12.70(s,1H,5-OH),10.97(s,1H,7-OH),7.88(d,2H,J＝9.00Hz,Ar-H),7.07(d,2H,J＝9.00Hz,Ar-H),6.64(s,1H,CHCO),6.51(s,1H,Ar-H),5.94(s,1H,-NH-CH＝C),4.52(s,1H,-NH-CH＝C),3.33(s,4H,Ar-N(CH 2CH2)2N),2.44(s,4H,Ar-N(CH2CH 2)2N),2.22(s,3H,NCH3)ppm.13C NMR(75MHz,DMSO-d6):182.1,165.9,163.6,159.0,150.3,147.7,131.0,128.8,127.3(2×C),121.7,111.7(2×C),104.5,103.7,99.0,57.2(2×C),52.0(2×C),46.6ppm.IR(KBr)ν3415,2972,1652,1587,1514,1379,1235,1203,1051,1004,827,664,559cm-1.
实施例22：化合物L22
m.p.178～180℃.HRMS(ESI),[M+H]+calculated for C23H22N4O5S 467.1344,found 467.1362.1H NMR(300MHz,DMSO-d6):δ＝12.99(s,1H,5-OH),10.70(s,1H,7-OH),7.93(d,2H,J＝9.00Hz,Ar-H),7.12(d,2H,J＝9.00Hz,Ar-H),6.93(s,1H,CHCO),6.74(s,1H,Ar-H),6.61(m,3H,Ar-H&NH2),3.44(s,4H,Ar-N(CH 2CH2)2N),2.59(s,4H,Ar-N(CH2CH 2)2N),2.32(s,3H,NCH3)ppm.13C NMR(75MHz,DMSO-d6):182.1,168.9,163.6,161.8,160.8,152.7,137.7,130.5,126.9,127.3(2×C),111.7(2×C),108.0,104.5,104.0,99.3,57.2(2×C),52.0(2×C),46.6ppm.IR(KBr)ν3392,1655,1601,1512,1356,1209,1109,825,701,467cm-1.
实施例23：化合物L23
Figure GDA0003631929380000231
m.p.164～168℃.HRMS(ESI),[M+H]+calculated for C26H28N4O6 493.2042,found493.2083.1H NMR(300MHz,DMSO-d6):δ＝12.80(s,1H,5-OH),11.92(s,1H,-NH-N＝),10.93(s,1H,7-OH),7.63(d,2H,J＝9.00Hz,Ar-H),7.00(d,2H,J＝9.00Hz,Ar-H),6.78(s,1H,CHCO),6.30(s,1H,Ar-H),4.50(s,1H,NCH2CH2OH),3.54(m,2H,NCH2CH 2OH),3.33(s,4H,Ar-N(CH 2CH2)2N),2.55(s,4H,Ar-N(CH2CH 2)2N),2.44(t,2H,J＝6.00Hz,NCH 2CH2OH),1.97(s,6H,Ar-CH3)ppm.13C NMR(75MHz,DMSO-d6):181.6,163.7,156.6,156.5,153.1,148.5,138.0,127.6(2×C),125.0,118.7,113.7(2×C),103.0,101.9,98.9,60.1,58.5,52.8(2×C),48.6,46.5(2×C),10.1(2×C)ppm.IR(KBr)ν3412,2914,1654,1584,1513,1370,1223,1203,1106,1000,824,562cm-1.
实施例24：化合物L24
m.p.250～258℃.HRMS(ESI),[M+H]+calculated for C29H27N5O5 526.2012,found526.2083.1H NMR(300MHz,DMSO-d6):δ＝12.80(s,1H,5-OH),11.91(s,1H,-NH-N＝),10.95(s,1H,7-OH),8.15(m,1H,Ar-H),7.66(d,2H,J＝9.00Hz,Ar-H),7.57(m,1H,Ar-H),7.06(d,2H,J＝9.00Hz,Ar-H),6.89(m,1H,Ar-H),6.80(s,1H,CHCO),6.68(m,1H,Ar-H),6.32(s,1H,Ar-H),3.66(s,4H,Ph-N(CH 2CH2)2N),3.48(s,4H,Ph-N(CH2CH 2)2N),1.99(s,6H,Ar-CH3)ppm.13C NMR(75MHz,DMSO-d6):181.6,163.7,158.7,156.5(2×C),153.0,148.5,147.6,137.9,137.6,127.7(2×C),124.9,118.9,113.8,113.2,107.2,103.1,102.0,98.8,46.2(2×C),44.1(2×C),8.4(2×C)ppm.IR(KBr)ν3411,3167,2360,1652,1584,1563,1508,1434,1354,1227,1202,1103,998,953,844,818,773cm-1.
实施例25：化合物L25
m.p.200～202℃.HRMS(ESI),[M+H]+calculated for C29H26N4O7 543.1835,found543.1861.1H NMR(300MHz,DMSO-d6):δ＝12.79(s,1H,5-OH),11.92(s,1H,-NH-N＝),10.96(s,1H,7-OH),7.88(s,1H,Ar-H),7.65(d,2H,J＝9.00Hz,Ar-H),7.04(m,3H,Ar-H),6.80(s,1H,CHCO),6.66(m,,1H,Ar-H),6.31(s,1H,Ar-H),3.82(s,4H,Ar-N(CH 2CH2)2N),3.47(s,4H,Ar-N(CH2CH 2)2N),1.98(s,6H,Ar-CH3)ppm.13C NMR(75MHz,DMSO-d6):181.6,163.6,158.3,156.6,156.5,152.7,148.5,146.9,144.9,137.9,127.7(2×C),124.9,119.1,119.0,115.9,114.0,113.8,111.4,103.1,102.1,98.9,59.9(2×C),46.5(2×C),10.0(2×C)ppm.IR(KBr)ν3392,3163,2923,1581,1515,1433,1368,1279,1227,1103,1012,825,755,559cm-1.
实施例26-实施例27所述化合物的制备，可参考实施例1和实施例11中所描述的方法和路线制备得到。
实施例26：化合物L26
Figure GDA0003631929380000251
m.p.200～203℃.HRMS(ESI),[M+H]+calculated for C26H28N4O5 477.2093,found477.2124.1H NMR(300MHz,DMSO-d6):δ＝12.94(s,1H,5-OH),11.90(s,1H,-NH-N＝),7.72(d,2H,J＝8.60Hz,Ar-H),7.04(d,2H,J＝8.60Hz,Ar-H),6.86(s,1H,CHCO),6.61(s,1H,Ar-H),3.88(s,3H,ArOCH3),3.40(s,4H,Ar-N(CH 2CH2)2N),2.60(s,4H,Ar-N(CH2CH 2)2N),2.35(s,3H,NCH3),1.96(s,6H,Ar-CH3)ppm.13C NMR(75MHz,DMSO-d6):182.3,172.5,164.4,158.2,157.5,148.3,138.3,137.0,128.7,128.2,126.1,123.5,114.5,104.0,102.5,96.4,57.1,54.2(2×C),46.4(2×C),9.1(2×C)ppm.IR(KBr)ν3425,2936,1656,1600,1514,1432,1375,1339,1223,1206,1126,999,816,661,567cm-1.
实施例27：化合物L27
Figure GDA0003631929380000252
m.p.210～212℃.HRMS(ESI),[M+H]+calculated for C25H26N4O5 463.1937,found463.1973.1H NMR(300MHz,DMSO-d6):δ＝12.94(s,1H,5-OH),11.89(s,1H,-NH-N＝),7.72(d,2H,J＝8.60Hz,Ar-H),7.02(d,2H,J＝8.60Hz,Ar-H),6.85(s,1H,CHCO),6.61(s,1H,Ar-H),3.88(s,3H,ArOCH3),3.32(s,4H,Ar-N(CH 2CH2)2N),2.90(s,2H,Ar-N(CH2CH 2)2N),2.71(s,2H,Ar-N(CH2CH 2)2N),1.95(s,6H,Ar-CH3)ppm.13C NMR(75MHz,DMSO-d6):181.8,163.8,157.7,157.0,147.8,137.8,127.9,127.7(2×C),125.6,118.9,114.0,103.5,102.0,95.9,56.5,46.1,45.5,45.4,44.1,9.5(2×C)ppm.IR(KBr)ν3410,2923,1655,1598,1513,1373,1224,1205,1124,999,825,567cm-1.
实施例28-实施例40所述化合物的前四步中间体的制备，可参考实施例1中所描述的方法和路线制备得到。
实施例28：化合物L28
Figure GDA0003631929380000261
第五步：中间体VI-6的制备
将中间体I-5(15g，50mmol)加入到75mL无水DMF，冰浴至0℃，搅拌溶解，再加入NBS(8.93g，50mmol)，缓慢升温至室温继续反应2h，TLC检测(V石油醚：V乙酸乙酯＝1:2)反应完全，抽滤，滤饼用10mL甲醇洗涤。取出滤饼用20mL甲醇打浆，抽滤，滤饼干燥至恒重的白色固体15.36g，收率80％。
第六步：中间体VI-7的制备
将中间体VI-6(2g，4.26mmol)、吡咯烷(8.72mmol)、CsCO3(2.84g，8.72mmol)、(Pd)2(dba)3(120mg，0.13mmol)和rac-BINAP(244mg，0.39mmol)加入50mL甲苯中，在N2保护下回流36h。TLC检测(V乙酸乙酯：V二氯甲烷＝20:1)反应完全，缓慢冷却至室温，减压蒸馏得到深棕色粘稠固体。固体用20ml二氯甲烷分散，用甲磺酸水溶液调至pH＝4-5，静置，分液得水层。水层用NaHCO3水溶液调至pH＝8-9，用20mL二氯甲烷萃取，二氯甲烷层用无水Na2SO4干燥，抽滤，滤液减压蒸馏得到黄棕色固体，干燥至恒重得黄棕色固体218mg，收率10.73％。
第七步：中间体VI-8的制备
将中间体VI-7(218mg，0.57mmol)溶于2mL二氯甲烷中，冰浴降至0℃以下。在N2保护下加入三溴化硼(0.27mL，2.85mmol)，撤去冰浴，缓慢升至室温反应1h。TLC检测(V二氯甲烷：V甲醇＝15:1)反应完全。在冰浴条件下缓慢滴加10mL甲醇淬灭反应，减压蒸馏得棕色粗品。粗品用10mL二氯甲烷溶解，用NaHCO3水溶液调至pH＝8-9，静置分液，二氯甲烷层用10mL饱和NaCl水溶液洗涤，无水Na2SO4干燥，抽滤，滤液减压蒸馏，得淡黄色固体183mg，收率86.8％；HRMS(ESI),[M+H]+calculated for C20H20FN2O4 371.1407,found 371.1397；1HNMR(300MHz,DMSO-d6)δ(ppm):8.23(q,J＝6.00Hz,2H,Ar-H),7.48(t,J＝9.00Hz,2H,Ar-H),7.03(s,1H,Ar-H),6.55(s,1H,Ar-H),3.91(s,3H,-OCH3),3.02(m,8H,-CH2-).
第八步：化合物L28的制备
将中间体VI-8(174mg，0.49mmol)，1-甲基哌嗪(490mg，4.9mmol),DIPEA(632mg，4.9mmol)溶于2mL DMSO中，升温至120-130℃反应1.5h。TLC检测(V二氯甲烷：V甲醇＝10:1)反应完全。冷却至室温，缓慢滴加10mL H2O淬灭反应，用二氯甲烷萃取，二氯甲烷层用10mL饱和食盐水洗涤，无水Na2SO4干燥，抽滤，滤液减压蒸馏，过柱纯化的淡黄色固体163mg，收率73.9％；HRMS(ESI),[M+H]+calculated for C25H30N3O4 436.2236,found 436.2243；76.5％；1HNMR(300MHz,DMSO-d6)δ(ppm):13.10(d,1H,Ar-OH),7.91(q,J＝6.00Hz,2H,Ar-H),7.08(t,J＝9.00Hz,2H,Ar-H),6.80(t,J＝12.00Hz,1H,Ar-H),6.44(d,J＝42.00Hz,1H,Ar-H),3.88(s,3H,-OCH3),3.35(m,8H,-N-CH2-),3.21(s,2H,-CH2-),2.50(d,4H,-CH2-),2.25(s,3H,-CH3),1.97(s,2H,-CH2-).
实施例29-实施例40所述化合物的制备，可参考实施例28中所描述的方法和路线制备得到。
实施例29：化合物L29
Figure GDA0003631929380000271
HRMS(ESI),[M+H]+calculated for C25H30N3O5 452.2185,found 452.2196；1H NMR(300MHz,chloroform-d)δ(ppm):13.02(s,1H,Ar-OH),7.93(d,J＝6.00Hz,2H,Ar-H),7.03(d,J＝9.00Hz,2H,Ar-H),6.58(s,1H,Ar-H),6.40(s,1H,Ar-H),3.92(s,3H,O-CH3),3.89(t,J＝3.00Hz,4H,-O-CH2-),3.50(t,J＝6.00Hz,4H,-N-CH2-),3.20(t,J＝9.00Hz,4H,-N-CH2-),2.70(t,J＝6.00Hz,4H,-N-CH2-),2.47(s,3H,-CH3).
实施例30：化合物L30
Figure GDA0003631929380000272
HRMS(ESI),[M+H]+calculated for C25H28N5O4 462.2141,found 462.2158；1HNMR(300MHz,DMSO-d6)δ(ppm):7.53(d,J＝9.00Hz,2H,Ar-H),7.35(s,1H,N-CH＝),6.90(d,J＝9.00Hz,2H,Ar-H),6.69(s,1H,Ar-H),6.54(s,1H,-C-CH＝),5.48(d,J＝3.00Hz,1H,Ar-H),3.85(s,3H,-N-CH3),3.53(s,3H,-OCH3),3.29(m,4H,-CH2-),2.51(m,4H,-CH2-),2.21(s,3H,-CH3).
实施例31：化合物L31
Figure GDA0003631929380000281
HRMS(ESI),[M+H]+calculated for C26H32N3O4 450.2393,found 450.2392；1H NMR(300MHz,MeOH-d)δ(ppm):7.85(d,J＝9.00Hz,2H,Ar-H),6.98(d,J＝9.00Hz,2H,Ar-H),6.47(s,1H,Ar-H),6.36(s,1H,Ar-H),3.85(s,3H,-OCH3),3.32(s,4H,-N-CH2-),3.25(d,J＝3.00Hz,4H,-N-CH2-),3.00(s,4H,-N-CH2-),2.54(d,J＝3.00Hz,4H,-N-CH2-),2.30(s,3H,-NCH3).
实施例32：化合物L32
Figure GDA0003631929380000282
HRMS(ESI),[M+H]+calculated for C25H31N4O4 451.2345,found 451.2330；1H NMR(300MHz,chloroform-d)δ(ppm):12.99(s,1H,Ar-OH),7.94(d,J＝9.00Hz,2H,Ar-H),7.02(d,J＝9.00Hz,2H,Ar-H),6.95(s,1H,Ar-H),6.37(s,1H,Ar-H),3.90(s,3H,-OCH3),3.45(s,4H,-N-CH2-),3.10(s,4H,-N-CH2-),2.66(s,4H,-N-CH2-),2.43(s,3H,-N-CH3),1.71(m,6H,-CH2-).
实施例34：化合物L34
Figure GDA0003631929380000283
HRMS(ESI),[M+H]+calculated for C21H21FNO5 386.1404,found 386.1418；1HNMR(300MHz,DMSO-d6)δ(ppm):12.88(s,1H,Ar-OH),8.21(q,J＝6.00Hz,2H,Ar-H),7.49(t,J＝9.00Hz,2H,Ar-H),7.02(s,1H,Ar-H),6.55(s,1H,Ar-H),4.67(s,1H,-C-OH),3.90(s,3H,-OCH3),3.71(m,1H,-CH-),2.92(m,4H,-CH2-),1.71(m,4H,-CH2-).
实施例35：化合物L35
Figure GDA0003631929380000291
HRMS(ESI),[M+H]+calculated for C22H24FN2O4 399.1720,found 399.1731；1HNMR(300MHz,chloroform-d)δ(ppm):12.80(s,1H,Ar-OH),8.02(t,J＝6.00Hz,2H,Ar-H),7.25(d,J＝9.00Hz,2H,Ar-H),6.63(s,1H,Ar-H),6.40(s,1H,Ar-H),3.91(s,3H,-OCH3),3.05(m,4H,-CH2-),2.85(m,2H,-CH-),1.12(d,J＝6.00Hz,6H,-CH3),0.89(d,J＝9.00Hz,1H,-NH-).
实施例36：化合物L36
Figure GDA0003631929380000292
HRMS(ESI),[M+H]+calculated for C23H24FN2O7S 491.1288,found 491.1293；1HNMR(300MHz,chloroform-d)δ(ppm):12.87(s,1H,Ar-OH),7.94(d,J＝6.00Hz,2H,Ar-H),7.34(d,J＝9.00Hz,2H,Ar-H),6.65(s,1H,Ar-H),6.43(s,1H,Ar-H),4.19(m,2H,-OCH 2CH3),3.96(s,3H,-OCH3),3.50(m,8H,-CH2-),1.37(t,J＝9.00Hz,3H,-CH3).
实施例37：化合物L37
Figure GDA0003631929380000293
HRMS(ESI),[M+H]+calculated for C20H20FN2O5S 419.1077,found 419.1091；1HNMR(300MHz,chloroform-d)δ(ppm):12.85(s,1H,Ar-OH),7.97(q,J＝3.00Hz,2H,Ar-H),7.29(d,J＝18.00Hz,2H,Ar-H),6.64(s,1H,Ar-H),6.43(s,1H,Ar-H),3.95(s,3H,-OCH3),3.65(t,J＝9.00Hz,4H,-CH2-),3.28(d,J＝6.00Hz,4H,-CH2-).
实施例38：化合物L38
Figure GDA0003631929380000301
HRMS(ESI),[M+H]+calculated for C24H29N4O4 437.2189,found 437.2154；1H NMR(300MHz,DMSO-d6)δ(ppm):7.96(d,J＝12.00Hz,2H,Ar-H),7.08(d,J＝9.00Hz,2H,Ar-H),6.72(s,1H,Ar-H),6.47(s,1H,Ar-H),3.88(s,3H,-OCH3),3.75(s,8H,-CH2-),3.86(d,J＝18.00Hz,8H,-CH2-).
实施例39：化合物L39
Figure GDA0003631929380000302
HRMS(ESI),[M+H]+calculated for C24H28N3O5 438.2029,found 438.2041；1H NMR(300MHz,DMSO-d6)δ(ppm):8.00(d,J＝12.00Hz,2H,Ar-H),7.09(t,J＝6.00Hz,2H,Ar-H),6.77(s,1H,Ar-H),6.48(s,1H,Ar-H),3.87(s,3H,-OCH3),3.74(m,4H,-O-CH2-),3.03(t,J＝6.00Hz,4H,-N-CH2-),2.86(t,J＝3.00Hz,4H,-N-CH2-),2.51(s,4H,-N-CH2-).
实施例40：化合物L40
Figure GDA0003631929380000303
1H NMR(300MHz,DMSO-d6)δ(ppm):8.00(d,J＝9.00Hz,2H,Ar-H),7.00(d,J＝9.00Hz,3H,Ar-H),6.79(s,1H,Ar-H),6.49(s,1H,Ar-H),3.89(s,3H,-OCH3),3.55(t,J＝6.00Hz,5H,-N-CH2-),3.00(m,4H,-N-CH2-),2.90(q,J＝6.00Hz,8H,-CH2-),2.48(q,J＝6.00Hz,4H,-CH2-),1.85(d,J＝12.00Hz,2H,-CH2-),1.45(m,2H,-CH2-).
下面是此发明部分化合物的生物药理学实验和结果：
一、本发明部分化合物对癌细胞的生长抑制活性
实验材料：
a.MTT溶液的配置
将250mg MTT，加入到50mL灭菌后的1×PBS(0.01mol/L pH＝7.4)中，避光搅拌30min，用0.22μm的微孔滤膜过滤，然后分装于EP管中，-20℃避光保存。
b.细胞培养:
细胞株：HepG2、A549、HCT116和THP-1
细胞培养材料：DMEM液体培养基，1640液体培养基,10％胎牛血清，0.25％胰酶，1×PBS
c.其他：
分析纯DMSO溶液，细胞计数板，96孔细胞培养板
实验仪器：
酶联免疫检测仪
实验步骤：
(1)铺板
接种细胞：用0.25％胰酶将细胞消化，用含10％FBS的细胞培养基配成单个细胞悬液。以每孔4×103～104个细胞接种于96孔培养板中，每孔体积100μL。培养细胞：将培养板放入细胞培养箱，37℃、5％二氧化碳及饱和湿度条件下，培养24小时。
(2)加药
显微镜下观察细胞已贴壁且呈正常生长形态，即可进行给药：吸弃培养板中的旧培养液，加入含药物的培养液继续在细胞培养箱内培养72h。
(3)呈色
培养72h后，每孔加入20μL MTT溶液(5mg/mL)，37℃继续孵育4h，去除上清液，每孔加入150μL DMSO，震荡10min，使甲瓒充分溶解。
(4)比色
492nm波长，在酶联免疫检测仪上测定各孔光吸收值，记录实验结果。
(5)实验结果分析
实验结果如下表所示。
表1本发明部分化合物对癌细胞的生长抑制活性
Figure GDA0003631929380000321
Figure GDA0003631929380000331
由表1可见，本发明大部分化合物对癌细胞均有较好的抑制活性，相对于汉黄芩素有很大的提高，活性较为突出的化合物主要有L26、L27、L32和L38。
二、本发明部分化合物对激酶CDK9和CDK2的抑制活性
实验原理和实验方法：
CDK9/cyclinT、CDK2/CyclinA复合物与底物肽8mM MOPS、0.2mM EDTA、10mM醋酸镁和10μΜγ-[33P]-ATP一起孵育。加入Mg-ATP混合物后，室温反应40分钟，加入3％磷酸溶液中止反应。将反应液等分点样到P30滤垫上，用75mM磷酸洗涤3次，每次5min；用甲醛洗涤1次。然后干燥并闪烁计数。通过在不同浓度的化合物存在的情况下，在60mM的HEPES-氢氧化钠(pH 7.5)中3mM的氯化镁，3mM的氯化锰，3μM的钠-原钒，1.2mM DTT，50μg/mL PEG20000和1μM的ATP中将CDK9-cyclin T、CDK2-Cyclin A复合物与重组底物RBERCHK tide在30℃条件下孵育2h。通过使用磷酸化特异性抗体和western blot分析检测磷酸化底物。
实验结果如下表所示。
表2本发明部分化合物对激酶CDK9和CDK2的IC50值
Figure GDA0003631929380000341
NA表示化合物对CDK2没有抑制活性。
由表2可见，本发明部分化合物相对汉黄芩素对CDK9具有较好的抑制活性，与Flavopiridol相当，但对CDK9的选择性得到了明显的提高。特别是化合物L26对CDK9的抑制活性与Flavopiridol基本相当，其IC50值达到了3.0nM，化合物L32对CDK9/CDK2的选择性是汉黄芩53倍，是Flavopiridol的45倍。
三、本发明部分化合物对不同CDKs的选择性
表3本发明部分化合物对不同CDKs的选择性
Figure GDA0003631929380000351
由表3可见，相对于CDK家族其他激酶，本发明部分化合物对CDK9具有较好的抑制活性且对CDK9的选择性也优于汉黄芩素以及临床II期药物Flavopiridol。
四、本发明部分化合物的类药性参数
表4本发明部分化合物的类药性参数
Figure GDA0003631929380000352
由表4可见，本发明优选化合物L27的溶解度较好，特别是在酸性条件下(pH＝4.5)的溶解度相对于其他化合物得到了大幅度的提高在相同条件下其溶解度是汉黄芩素的500倍，Log D值也是处于合理的范围内。
五、我们选取了活性较好的化合物L26，对其进行了诱导MV4-11细胞凋亡的实验。
实验方法：将处于对数生长期的MV4-11细胞接种于6孔板内，将L26化合物配制成不同的浓度梯度并给与细胞，24小时后收集细胞，PBS洗涤一次，然后加入细胞凋亡染色液，30分钟后用流失细胞仪进行检测。
结果如下：
图1本发明中化合物L26诱导MV4-11细胞凋亡实验结果
由图1可见，化合物L26能够以浓度依赖性的方式诱导MV4-11细胞凋亡，当化合物L26的浓度为1μM时可以使约40％的细胞发生凋亡。
六、我们用WB的方法对化合物L26的抗肿瘤作用机制进行了研究，我们考察了化合物L26对CDK9及其下游蛋白表达量的影响。
实验方法：将处于对数生长期的MV4-11细胞接种于6孔板内，并给与不同浓度梯度的化合物L26,4小时后收集细胞，提取蛋白，以DMSO作为阴性对照，β-actin作为内参。
结果如下：
由图2可见，化合物L26能通过抑制CDK9的活性，使得2位Ser磷酸化的RNA聚合酶II的表达量明显减少，并能使其下游的短促凋亡蛋白Mcl-1的表达量呈浓度依赖性的减少，使Cleaver caspase 3的表达量呈浓度依赖性的增加。这表明了化合物L26是通过抑制CDK9的活性，使得下游的促凋亡蛋白的表达增加，进而引起细胞发生凋亡的。

Claims (8)

1.一种如通式(I)所示的化合物或其药学上可接受的盐
Figure FDA0003656403020000011
其中，所述R1为氢、
Figure FDA0003656403020000012
所述R2为氢、甲基或乙基；
所述R3为甲基、乙基、异丙基、
Figure FDA0003656403020000013
Figure FDA0003656403020000014
所述Y为N，Y的位置为所在苯环的3’位或4’位；
所述m为1、2或者3；
所述X为O、CRa或NRb；
所述Ra为氢、羟基或
Figure FDA0003656403020000015
所述Rb为氢、甲基、乙基、异丙基、
Figure FDA0003656403020000021
Figure FDA0003656403020000022
2.根据权利要求1所述的化合物 或其药学上可接受的盐，其特征在于药学上可接受的盐是指通式(I)的化合物和药学上可接受的酸形成的盐，包括无机酸盐和有机酸盐；其中所述的无机酸包括：碳酸、碳酸氢根、硫酸、硫酸氢根、磷酸、磷酸一氢根、磷酸二氢根、硝酸、氯化氢、溴化氢或氢碘酸；所述的有机酸包括乙酸、草酸、乳酸、琥珀酸、酒石酸、柠檬酸、甲磺酸、丙酮酸、苯磺酸、对甲苯磺酸、阿魏酸、马来酸、异丁酸、丙二酸、辛二酸、扁桃酸、反丁烯二酸或氨基酸。
3.根据权利要求1或2所述化合物或其药学上可接受的盐，其特征在于：所述的化合物为如下结构任意一项：
Figure FDA0003656403020000023
Figure FDA0003656403020000031
Figure FDA0003656403020000041
Figure FDA0003656403020000051
Figure FDA0003656403020000061
4.一种药物组合物，其特征在于，权利要求1-3任意一种化合物或其药学上可接受的盐，和药学上可接受的辅料。
5.根据权利要求1-3任意一项所述的化合物或其药学上可接受的盐和权利要求4所述的药物组合物在制备以CDK9为靶点的小分子抑制剂药物中的应用。
6.根据权利要求1-3任意一项所述的化合物或其药学上可接受的盐和权利要求4所述的药物组合物在制备预防或治疗抗病毒药物和抗肿瘤药物中的应用。
7.根据权利要求6所述的应用，其特征在于：所述的病毒包括HIV病毒、巨细胞病毒、EB病毒、腺病毒、疱疹、人T细胞淋巴细胞病毒、甲型流感病毒和登革热病毒。
8.根据权利要求6所述的应用，其特征在于：所述的肿瘤包括神经胶质瘤、白血病、淋巴癌、肝癌、肌肉癌、乳腺癌、前列腺癌、肺癌、胃肠癌、皮肤癌、卵巢癌和食管癌。
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