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CN112574216B - 一种化合物及其制备方法以及其在制备治疗抗癌药物中的应用 - Google Patents

一种化合物及其制备方法以及其在制备治疗抗癌药物中的应用 Download PDF

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CN112574216B
CN112574216B CN202011483614.1A CN202011483614A CN112574216B CN 112574216 B CN112574216 B CN 112574216B CN 202011483614 A CN202011483614 A CN 202011483614A CN 112574216 B CN112574216 B CN 112574216B
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杨诚
杨光
周红刚
张亮
李建
伦东超
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Tianjin Jikun Pharmaceutical Technology Co ltd
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Abstract

本发明创造提供了一种化合物及其制备方法以及其在制备治疗抗肿瘤药物中的应用。所述化合物的结构式为：
Figure DDA0002838769210000011
其对对FGFR(1‑3)具有强烈抑制作用，在体内外所述化合物对NCI‑H1581和SNU‑16癌细胞系均表现出显著的抗增殖作用。

Description

一种化合物及其制备方法以及其在制备治疗抗癌药物中的 应用
技术领域
本发明创造属于制药技术领域，尤其是涉及2H-吡唑并[3,4-d]嘧啶衍生物及其制备方法以及其在制备治疗抗癌药物中的应用。
背景技术
成纤维细胞生长因子受体(FGFR)是受体酪氨酸激酶(RTK)家族成员，属于受体型蛋白酪氨酸激酶，其包含四个高度保守的跨膜酪氨酸激酶：FGFR1， FGFR2，FGFR3和FGFR4。FGFR的信号传导途径为配体与受体的结合诱导FGFR 二聚化，从而引起下游信号通路(如Ras-MAPK，PI3K-Akt，STAT和PLCγ)的级联激活。FGFR在多种细胞功能中发挥重要作用，例如细胞增殖和分化以及包括发育，血管新生，体内稳态和伤口修复在内的生物学过程。1-3据研究调查，占比7.1％的癌症患者，FGFR都异常。例如，鳞状NSCLC患者中FGFR1扩增的比例最高，为20％，乳腺癌为10％，卵巢癌5％，膀胱癌3％。目前，已在胃癌(10％)，子宫内膜癌(12％)，鳞状NSCLC(5％)和三阴性乳腺癌(4％) 中均发现了FGFR2的突变，而FGFR3的突变在膀胱癌(50-60％)和骨髓瘤(15-20％)中是众所周知的。异常的FGF19/FGFR4与肝细胞癌(HCC)密切相关。4-6开发新型FGFR选择性抑制剂来阻断FGF/FGFR信号通路为FGFR相关的癌症提出了有希望的治疗策略。
研究者们致力于研发FGFR抑制剂作为治疗FGFR相关的癌症的新型抗癌剂。7首先，最初采用了几种多靶点酪氨酸激酶抑制剂(TKIs)来治疗具有FGFR 异常的相关癌症，例如dovitinib(TKI-258)，8lucitanib(E-3810)，9nintedanib (BIBF-1120)，和10ponatinib(AP-24534)。11这些早期研发的FGFR抑制剂是非选择性的。后来，已经开发了几种带有各种支架的选择性FGFR酪氨酸激酶抑制剂。一些酪氨酸激酶抑制剂目前处于不同阶段的临床研究中，例如AZD-4547 (III期)12，BGJ-398(II期)13，LY-2874455(II期)14和JNJ-42756493(II期) 15，这些选择性FGFR抑制剂是与FGFR活性形式结合的ATP竞争性抑制剂。
发明内容
有鉴于此，本发明创造旨在克服现有技术中的缺陷，提出2H-吡唑并[3,4-d] 嘧啶衍生物及其制备方法以及其在制备治疗抗癌药物中的应用。
为达到上述目的，本发明创造的技术方案是这样实现的：
一种如式(I)所示的化合物或其在药学上可接受的盐：
Figure BDA0002838769190000011
式(I)中，Ar取自取代芳环基团或取代芳杂环基团中的任意一种；
R取自H、烷烃、芳基、CF3、烷基叔胺结构中的任意一种；
Linker取自烷基、烷氧基、杂原子取代基、取代N杂环中的任意一种。
优选的，所述化合物选自以下化合物1-40中的任一种：
Figure BDA0002838769190000021
Figure BDA0002838769190000031
Figure BDA0002838769190000041
本发明还提供了所述化合物或其药学上可接受的盐的制备方法，其合成路线为：
Figure BDA0002838769190000042
。
本发明还提供了一种含有上述化合物、其药学上可接受的盐的药物组合物，其中，包含一种或多种药学上可接受的赋形剂，该药物组合物的剂型为药学上可接受的任一剂型。
本发明还提供了含有上述化合物、其药学上可接受的盐、药物组合物在用于制备治疗和/或预防癌症的药物中的用途。
优选的，所述的癌症为具有FGFR异常的相关癌症。
本发明还提供了所述的化合物、其药学上可接受的盐、权利要求4所述的药物组合物在用于制备作为不可逆的泛成纤维细胞生长因子受体(FGFR)抑制剂的用途。
在本发明中，术语“药学上可接受的辅料”包括药学上可接受的载体、赋形剂、稀释剂等，它们与药物活性成分相容。运用药学上可接受的辅料制备药物制剂对本领域普通技术人员来说是公知的。
本发明将药物组合物和药学上可接受的辅剂(如本领域普通技术人员所熟知的载体、赋形剂、稀释剂等)组合在一起，配制成各种制剂，优选为固体制剂和液体制剂，如片剂、丸剂、胶囊、粉剂、混悬剂、颗粒剂、糖浆剂、乳液剂、悬浮液等剂型以及各种缓释剂型，优选以口服给药形式。
附图说明
图1A为化合物10浓度梯度依赖性地抑制NCI-H1581细胞中FGFR1以及下游信号通路的激活；
图1B为化合物10浓度梯度依赖性地抑制SNU-16细胞中FGFR2以及下游信号通路的激活；
图2A为化合物36浓度梯度依赖性地抑制NCI-H1581细胞中FGFR1以及下游信号通路的激活；
图2B为化合物36浓度梯度依赖性地抑制SUN-16细胞中FGFR2以及下游信号通路的激活；
图3A为在NCI-H1581肿瘤细胞皮下移植瘤模型中，化合物10抑制肿瘤增殖的肿瘤体积曲线图；
图3B为在NCI-H1581肿瘤细胞皮下移植瘤模型中，化合物10处理26天后小鼠体内的肿瘤瘤重统计图；
图3C为在NCI-H1581肿瘤细胞皮下移植瘤模型中，化合物10处理过程中小鼠体重变化曲线图；
图3D为在NCI-H1581肿瘤细胞皮下移植瘤模型中，化合物10处理26天后小鼠体内肿瘤的照片图；
图4A为在NCI-H1581肿瘤细胞皮下移植瘤模型中，化合物36抑制肿瘤增殖的肿瘤体积变化曲线图；
图4B为在NCI-H1581肿瘤细胞皮下移植瘤模型中，化合物36处理26天后小鼠体内的肿瘤瘤重统计图；
图4C为在NCI-H1581肿瘤细胞皮下移植瘤模型中，化合物36处理过程中小鼠体重变化曲线图；
图4D为在NCI-H1581肿瘤细胞皮下移植瘤模型中，化合物36处理26天后小鼠体内肿瘤的照片图；
图4E为在NCI-H1581肿瘤细胞皮下移植瘤模型中，免疫组化检测不同处理组织中FGFR1的磷酸化水平。
具体实施方式
除有定义外，以下实施例中所用的技术术语具有与本发明创造所属领域技术人员普遍理解的相同含义。以下实施例中所用的试验试剂，如无特殊说明，均为常规生化试剂；所述实验方法，如无特殊说明，均为常规方法。
下面结合实施例来详细说明本发明创造。
实施例1：化合物1的制备
Figure BDA0002838769190000051
具体制备方法为：
1)化合物(1-1)的制备：
Figure BDA0002838769190000052
将3-溴-1H-吡唑并[3,4-D]嘧啶-4-胺(500mg，2.34mmol)溶解在干燥的THF(23mL)中，加入三苯基膦(2.08g，7.94mmol)，1-Boc-3-氮杂环丁烷甲醇。然后在0℃，氩气保护下，滴加偶氮二甲酸二异丙酯(1.60g，7.94mmol)。滴加完毕后，缓慢升至室温继续反应4h。反应液旋干，柱层析纯化(石油醚：乙酸乙酯＝5:1至2:1)得化合物1-1(黄色油状液体，685mg，69％)。
化合物1-1的核磁测试结果为：
1H NMR(400MHz,CDCl3)δ8.06(s,1H),7.96–7.87(m,6H),7.64–7.45(m, 9H),4.48(d,J＝7.3Hz,2H),3.97(t,J＝8.5Hz,2H),3.88–3.74(m,2H),3.19– 2.98(m,1H),1.41(s,9H).13C NMR(100MHz,CDCl3)δ163.1,163.0,156.2,155.9, 154.6,154.5,133.4,133.4,133.3,133.3,132.4,132.3,132.2,128.8,128.7,128.7, 128.6,128.6,128.5,128.4,127.8,127.4,120.9,107.1,79.3,52.9,51.5,49.7,28.8, 28.4,21.7,14.2.HRMS(ESI)calculated for C32H32BrN6NaO2P+:665.1400,found 665.1402.
2)化合物(1-2)的制备
Figure BDA0002838769190000061
将化合物1-1(5.01mg，6.74mmol)，3,5-二甲氧基苯硼酸(1.59g，8.76mmol) 溶解在1,4-二氧六环：水(9mL，v:v＝3:1)的混合溶液中，然后加入碳酸钾(1.87 g，13.5mmol)，Pd(PPh3)4(778mg，0.674mmol)。体系氩气置换3次，油浴下升温至80℃，反应过夜，反应完成后用饱和氯化钠洗(270mL)，乙酸乙酯萃取 (90mL)，有机相干燥浓缩，柱层析纯化(二氯甲烷：甲醇＝70:1至30:1)得化合物1-2(黄色油状液体，4.3g，60％)。
化合物1-2的核磁测试结果为：
1H NMR(400MHz,CDCl3)δ8.06(d,J＝3.2Hz,1H),7.89–7.71(m,6H), 7.56–7.40(m,11H),6.70–6.41(m,1H),4.58(dd,J＝7.3,3.2Hz,2H),4.11–3.96 (m,2H),3.92–3.84(m,2H),3.76(d,J＝3.1Hz,6H),3.26–3.07(m,1H),1.41(d,J ＝3.1Hz,9H).13C NMR(100MHz,CDCl3)δ163.9,163.9,163.5,163.4,160.3, 156.3,156.3,155.2,154.7,146.1,135.6,134.2,133.5,133.4,133.4,133.3,132.3, 132.2,132.0,132.0,132.0,1292,128.6,128.6,128.5,128.5,128.4,128.4,128.2, 128.2,127.8,107.9,100.6,79.3,77.4,55.3,49.5,28.9,28.4,24.5.HRMS(ESI) calculated for C40H41N6NaO4P+:723.2819,found723.2822.
3)化合物(1-3)的制备
Figure BDA0002838769190000071
0℃下，将化合物1-2(450mg，0.587mmol)溶解于二氯甲烷中(5mL)，然后缓慢滴加三氟乙酸(5mL)。30min后反应完全。反应液饱和NaHCO3调碱，有机相浓缩干得棕黄色油状中间体。0℃下，将该中间体溶解于5mL二氯甲烷中，然后加入TEA(98.2μL，0.704mmol)，滴加丙烯酰氯(52.2μL，0.646mmol)。室温下继续反应1h，TLC监测反应完全，反应液用饱和NaCl洗，乙酸乙酯萃取，有机相干燥浓缩，柱层析分离纯化(二氯甲烷：甲醇＝30:1至10:1)得化合物1-3(黄色油状液体，78mg，72％)。
4)化合物1的制备：将化合物1-3(100mg，0.189mmol)中加入于醋酸：四氢呋喃：水(3mL，3:1:2)的混合溶液。室温下反应18h，TLC监测反应完全。反应液用饱和碳酸氢钠溶液调碱至PH 7～8(15mL)，乙酸乙酯萃取(10mL×2)，有机相干燥浓缩，柱层析纯化(二氯甲烷：甲醇＝15:1至7:1)得化合物5a(黄色固体，57.8mg，70％)。
化合物1的核磁测试结果为：
1H NMR(400MHz,CDCl3)δ8.38(s,1H),6.80(d,J＝2.3Hz,2H),6.57(t,J＝ 2.3Hz,1H),6.33(dd,J＝17.0,1.9Hz,1H),6.17(dd,J＝17.0,10.3Hz,1H),5.76– 5.57(m,3H),4.76–4.61(m,2H),4.38–4.14(m,3H),4.02(dd,J＝10.5,5.5Hz, 1H),3.87(s,6H),3.49–3.25(m,1H).13C NMR(100MHz,CDCl3)δ165.7,161.6, 157.8,156.2,154.7,144.7,134.8,127.4,127.2,125.7,106.3,101.2,98.4,55.6,53.5, 50.8,49.4,32.7,29.1,27.9.HRMS(ESI)calculated for C20H22N6NaO3 +:417.1646, found 417.1648.
实施例2：化合物2的制备
Figure BDA0002838769190000072
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为1-Boc-3-羟甲基吡咯烷，等摩尔代替化合物(1-1)的制备方法中的1-Boc-3- 氮杂环丁烷甲醇，化合物2的核磁测试结果为：
1H NMR(400MHz,CDCl3)δ8.38(s,1H),6.81(d,J＝2.2Hz,2H),6.62–6.54 (m,2H),6.25(dd,J＝16.9,2.0Hz,1H),5.76(s,2H),5.66(dd,J＝10.6,2.0Hz,1H), 4.65(d,J＝13.3Hz,1H),4.49(t,J＝7.2Hz,2H),3.99(d,J＝13.7Hz,1H),3.86(s, 6H),3.00(t,J＝12.6Hz,1H),2.64–2.52(m,1H),1.97–1.94(m,1H),1.91(s,2H). 13C NMR(100MHz,CDCl3)δ164.5,161.5,158.0,156.0,154.6,144.6,134.9,128.5, 127.5,106.4,101.3,98.3,55.6,50.0,49.1,45.7,45.2,39.9,37.74 29.6,27.9.HRMS (ESI)calculated for C21H24N6NaO3 +:431.1802,found 431.1804.
实施例3：化合物3的制备
Figure BDA0002838769190000081
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为N-Boc-4-羟基哌啶，等摩尔代替化合物(1-1)的制备方法中的1-Boc-3-氮杂环丁烷甲醇，化合物3的核磁测试结果为：
1H NMR(400MHz,CDCl3)δ8.38(s,1H),6.80(d,J＝2.2Hz,2H),6.67–6.51 (m,2H),6.31(dd,J＝16.8,1.8Hz,1H),5.84–5.63(m,3H),5.05(tt,J＝11.3,4.2 Hz,1H),4.85(d,J＝13.4Hz,1H),4.20(d,J＝13.9Hz,1H),3.86(s,6H),3.33(t,J＝ 12.7Hz,1H),3.02–2.85(m,1H),2.34(t,J＝11.8Hz,2H),2.11(d,J＝13.0Hz,2H). 13C NMR(101MHz,CDCl3)δ161.5,157.8,155.8,154.7,154.3,144.1,135.1,132.1, 131.6,106.3,101.1,100.0,98.3,79.6,55.7,55.6,52.9,45.5,29.7,28.4,26.8.HRMS (ESI)calculated for C21H24N6NaO3 +:431.1802,found 431.1805.
实施例4：化合物4的制备
Figure BDA0002838769190000082
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为N-Boc-4-哌啶甲醇，等摩尔代替化合物(1-1)的制备方法中的1-Boc-3-氮杂环丁烷甲醇，化合物4的核磁测试结果为：
1H NMR(400MHz,CDCl3)δ8.34(s,1H),6.80(d,J＝2.3Hz,2H),6.57–6.49 (m,2H),6.23(d,J＝16.8Hz,1H),5.89(s,2H),5.64(d,J＝10.6Hz,1H),4.65(d,J ＝13.3Hz,1H),4.33(d,J＝7.1Hz,2H),3.97(d,J＝13.8Hz,1H),3.85(s,6H),3.00 (t,J＝12.9Hz,1H),2.62(t,J＝12.9Hz,1H),2.33(d,J＝11.0Hz,1H),1.66(d,J＝ 16.3Hz,3H),1.33(td,J＝14.3,7.5Hz,2H).13C NMR(100MHz,CDCl3)δ165.4, 161.5,161.5,157.9,156.0,154.7,144.3,134.9,127.9,127.9,127.4,106.4,101.1, 99.9,98.2,55.6,51.9,45.6,41.8,36.8,30.5,29.4.HRMS(ESI)calculated for C22H26N6NaO3 +:445.1959,found 445.1961.
实施例5：化合物5的制备
Figure BDA0002838769190000091
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为N-Boc-4-哌啶乙醇，等摩尔代替化合物(1-1)的制备方法中的1-Boc-3-氮杂环丁烷甲醇，化合物5的核磁测试结果为：
1H NMR(400MHz,CDCl3)δ8.38(d,J＝1.1Hz,1H),6.81(d,J＝2.3Hz,2H), 6.57(d,J＝2.4Hz,1H),6.44–6.36(m,2H),5.68(dd,J＝9.6,2.7Hz,1H),4.50(dd, J＝12.8,6.9Hz,2H),3.87(s,7H),3.80–3.72(m,2H),3.72–3.65(m,1H),3.57– 3.47(m,2H),3.01(d,J＝7.3Hz,1H),1.65(m,4H).13C NMR(100MHz,CDCl3)δ 166.3,162.5,158.7,156.8,155.2,145.2,136.0,128.9,128.4,107.4,102.1,99.3,56.6, 47.3,45.6,43.2,36.9,34.6,33.5,32.9,32.6,30.7,30.4.HRMS(ESI)calculated for C23H28N6NaO3 +:459.2115,found459.2115.
实施例6：化合物6的制备
Figure BDA0002838769190000092
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为1-Boc-3-羟甲基哌啶，等摩尔代替化合物(1-1)的制备方法中的1-Boc-3-氮杂环丁烷甲醇，化合物6的核磁测试结果为：
1H NMR(400MHz,CDCl3)δ8.39–8.33(m,1H),6.93–6.75(m,2H),6.63– 6.46(m,2H),6.21(dd,J＝16.9,8.7Hz,1H),5.99(s,2H),5.61(dd,J＝32.0,10.5Hz, 1H),4.53–4.27(m,3H),3.86(s,6H),3.07(dt,J＝23.7,12.4Hz,1H),2.81(dt,J＝ 44.0,12.0Hz,1H),2.35(s,1H),1.93(s,1H),1.58–1.29(m,2H),1.26(m,2H).13C NMR(100MHz,CDCl3)δ165.5,161.6,157.9,155.9,154.7,127.8,127.4,106.3, 101.3,98.2,55.6,49.8,49.7,49.5,46.5,45.8,42.7,37.4,36.4,28.7,25.2,24.1. HRMS(ESI)calculated for C22H26N6NaO3 +:445.1959,found 445.1961.
实施例7：化合物7的制备
Figure BDA0002838769190000101
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为叔丁基-4-(2-羟乙基)哌嗪-1-羧酸酯，等摩尔代替化合物(1-1)的制备方法中的1-Boc-3-氮杂环丁烷甲醇，化合物7的核磁测试结果为：
1H NMR(400MHz,CDCl3)δ8.36(s,1H),6.80(d,J＝2.3Hz,2H),6.60–6.47 (m,2H),6.26(dd,J＝16.8,1.9Hz,1H),5.67(dd,J＝10.5,1.9Hz,1H),4.57(t,J＝ 6.7Hz,2H),3.85(s,6H),3.62(s,2H),3.49(s,2H),2.96(t,J＝6.7Hz,2H),2.56(t,J ＝5.0Hz,4H).13C NMR(100MHz,CDCl3)δ165.3,161.5,157.4,155.1,154.5, 144.6,134.8,127.9,127.4,106.3,101.2,98.2,95.6,56.7,55.6,52.6,50.9,45.6,44.4, 41.8,31.9,29.1.HRMS(ESI)calculated for C22H27N7NaO3 +:460.2068,found 460.2072.
实施例8：化合物8的制备
Figure BDA0002838769190000111
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为1-BOC-4-(3-羟基丙烷)哌嗪，等摩尔代替化合物(1-1)的制备方法中的1-Boc-3- 氮杂环丁烷甲醇，化合物8的核磁测试结果为：
1H NMR(400MHz,CDCl3)δ8.36(s,1H),6.81(d,J＝2.3Hz,2H),6.58–6.50 (m,2H),5.88(s,2H),5.69(dd,J＝10.5,1.9Hz,1H),4.52(t,J＝6.9Hz,2H),3.86(s, 6H),3.69(d,J＝6.9Hz,2H),3.57(s,2H),2.51(dt,J＝10.8,6.0Hz,6H),2.21(p,J＝ 7.0Hz,2H).13C NMR(100MHz,CDCl3)δ165.3,160.3,154.4,133.4,133.3,132.2, 132.1,132.0,129.0,128.6,128.6,128.4,128.1,127.3,127.3,107.9,100.5,55.4,55.3, 53.1,52.5,45.2,26.4.HRMS(ESI)calculated for C23H29N7NaO3 +:474.2224,found 474.2226.
实施例9：化合物9的制备
Figure BDA0002838769190000112
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为1-BOC-4-(4-羟基丁烷)哌嗪，等摩尔代替化合物(1-1)的制备方法中的1-Boc-3- 氮杂环丁烷甲醇，化合物9的核磁测试结果为：
1H NMR(400MHz,CDCl3)δ8.40–8.36(m,1H),6.81(dd,J＝3.2,2.0Hz, 2H),6.54(ddd,J＝17.4,8.7,4.1Hz,2H),6.28(dt,J＝16.9,2.5Hz,1H),5.72–5.66 (m,1H),5.61(s,2H),4.46(s,2H),3.89–3.82(m,6H),3.67(s,2H),3.54(s,2H), 2.41(s,6H),2.01(s,2H),1.56(s,2H).13C NMR(100MHz,MeOD)δ169.4,166.0, 152.2,151.70,147.6,145.9,132.7,128.5,126.6,106.2,106.1,101.5,101.4,96.5, 56.6,54.7,53.5,51.1,40.5,39.1,38.5,35.2,28.3,22.7.HRMS(ESI)calculated for C24H31N7NaO3 +:488.2381,found 488.2383.
实施例10：化合物10的制备
Figure BDA0002838769190000121
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为1-BOC-4-(5-羟基戊烷)哌嗪，等摩尔代替化合物(1-1)的制备方法中的1-Boc-3- 氮杂环丁烷甲醇，化合物10的核磁测试结果为：
1H NMR(400MHz,CDCl3)δ8.38(s,1H),6.82–6.80(m,2H),6.58–6.55(m, 1H),6.53(d,J＝10.4Hz,1H),6.27(d,J＝16.7Hz,1H),5.68(d,J＝10.6Hz,1H), 5.61(s,2H),4.43(s,2H),3.86(s,6H),3.67(s,2H),3.53(s,2H),2.42–2.38(m,4H), 2.34–2.30(m,2H),2.02–1.96(m,2H),1.54(d,J＝7.4Hz,2H),1.39(d,J＝6.9Hz, 2H).13C NMR(100MHz,CDCl3)δ165.3,161.6,157.7,155.9,154.3,144.0,135.1, 127.7,127.5,106.4,101.1,100.0,98.3,58.2,55.6,53.4,52.7,47.1,45.7,41.8,40.5, 29.6,29.3,26.2,24.5.HRMS(ESI)calculated for C25H33N7NaO3 +:502.2537,found 502.2539.
实施例11：化合物11的制备
Figure BDA0002838769190000131
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为1-BOC-4-(6-羟基己烷)哌嗪，等摩尔代替化合物(1-1)的制备方法中的1-Boc-3- 氮杂环丁烷甲醇，化合物11的核磁测试结果为：
1H NMR(400MHz,CDCl3)δ8.34(d,J＝1.3Hz,1H),6.81(dd,J＝2.3,1.3Hz, 2H),6.61–6.52(m,2H),6.29(dt,J＝16.9,1.6Hz,1H),6.00(s,2H),5.69(dt,J＝ 10.5,1.6Hz,1H),4.43(t,J＝7.3Hz,2H),3.86(d,J＝1.3Hz,6H),3.69(t,J＝5.0 Hz,2H),3.56(t,J＝4.9Hz,2H),2.42(t,J＝5.0Hz,4H),2.33(t,J＝7.6Hz,2H), 1.97(t,J＝7.2Hz,2H),1.54–1.45(m,2H),1.38(t,J＝4.3Hz,4H).13C NMR(100 MHz,CDCl3)δ165.3,161.5,157.8,155.3,154.1,144.2,135.0,135.0,127.9,127.5, 127.5,106.4,106.4,101.1,98.2,58.3,55.6,53.4,52.7,47.2,45.7,41.8,29.7,27.0, 26.6,26.5.HRMS(ESI)calculated forC26H35N7NaO3 +:516.2694,found 516.2696.
实施例12：化合物12的制备
Figure BDA0002838769190000132
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为1-BOC-4-(7-羟基庚烷)哌嗪，等摩尔代替化合物(1-1)的制备方法中的1-Boc-3- 氮杂环丁烷甲醇，化合物12的核磁测试结果为：
1H NMR(400MHz,CDCl3)δ8.38(d,J＝2.8Hz,1H),6.82(t,J＝2.5Hz,2H), 6.62–6.50(m,2H),6.33–6.23(m,1H),5.77–5.56(m,3H),4.42(s,2H),3.86(d,J ＝2.8Hz,6H),3.70(s,2H),3.57(s,2H),2.50–2.37(m,4H),2.33(s,2H),1.96(s, 2H),1.47(s,2H),1.33(d,J＝27.0Hz,6H).13C NMR(101MHz,CDCl3)δ165.6, 161.8,158.1,156.1,154.5,144.3135.5,135.5,128.1,127.8,106.7,101.4,101.4,98.6, 58.7,55.9,53.7,53.1,47.5,46.0,42.2,30.0,29.9,29.3,27.6,26.9,26.8.HRMS (ESI)calculated for C27H37N7NaO3 +:530.2850,found 530.2852.
实施例13：化合物13的制备
Figure BDA0002838769190000141
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为3，4-亚甲基苯硼酸，等摩尔代替化合物(1-2)的制备方法中的3，5-二甲氧基苯硼酸，化合物13的核磁测试结果为：
1H NMR(400MHz,CDCl3)δ8.36(s,1H),7.16(d,J＝2.7Hz,1H),6.97(d,J＝ 7.9Hz,1H),6.62–6.49(m,1H),6.28(dd,J＝16.9,2.0Hz,1H),6.06(s,2H),5.86– 5.65(m,3H),4.42(t,J＝7.2Hz,2H),3.70(d,J＝7.5Hz,2H),3.55(t,J＝5.1Hz, 2H),2.43(t,J＝4.9Hz,4H),2.35(t,J＝7.6Hz,2H),1.99(td,J＝16.9,15.0,9.4Hz, 2H),1.79–1.14(m,6H).13CNMR(100MHz,CDCl3)δ165.2,157.8,155.7,154.2, 148.6,148.5,143.8,127.4,127.1,122.1,109.0,108.9,101.6,101.5,98.2,58.1,53.3, 52.7,46.9,45.6,41.7,29.5,26.1,24.5.HRMS(ESI)calculated for C24H29N7NaO3 +: 4860.2224,found 4860.2226.
实施例14：化合物14的制备
Figure BDA0002838769190000151
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为苯并呋喃-2-硼酸，等摩尔代替化合物(1-2)的制备方法中的3，5-二甲氧基苯硼酸，化合物14的核磁测试结果为：
1H NMR(400MHz,CDCl3)δ8.39(s,1H),7.68(dd,J＝7.3,1.5Hz,1H),7.59– 7.55(m,1H),7.40–7.32(m,3H),6.53(dd,J＝16.8,10.6Hz,1H),6.28(dd,J＝16.9, 1.9Hz,1H),5.68(dd,J＝10.6,2.0Hz,1H),4.46(t,J＝7.2Hz,2H),3.69(s,2H), 3.55(s,2H),2.44(s,4H),2.36(t,J＝7.6Hz,2H),2.01(p,J＝7.4Hz,2H),1.57(q,J ＝7.6Hz,2H),1.44–1.36(m,2H).13C NMR(100MHz,CDCl3)δ165.3,163.8, 157.6,156.0 154.4,149.5,134.7,128.3,127.8,127.4,125.1,124.0,121.7,111.0, 104.3,97.9,58.1,53.3,52.7,47.3,45.6,41.7,29.5,26.1,24.4.HRMS(ESI) calculated for C25H29N7NaO2 +:482.2269,found482.2271.
实施例15：化合物15的制备
Figure BDA0002838769190000152
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为对甲氧基硼酸，等摩尔代替化合物(1-2)的制备方法中的3，5-二甲氧基苯硼酸，化合物15的核磁测试结果为：
1H NMR(400MHz,MeOD)δ8.24(s,1H),7.61(d,J＝8.6Hz,2H),7.18–7.06 (m,2H),6.82–6.64(m,1H),6.19(dd,J＝16.7,2.0Hz,1H),5.74(dd,J＝10.6,1.9 Hz,1H),4.41(t,J＝6.8Hz,2H),3.88(s,3H),3.62(s,4H),2.48(s,4H),2.39(s,2H), 1.96(q,J＝7.3Hz,2H),1.60–1.52(m,2H),1.35(q,J＝7.9Hz,2H).13C NMR(100 MHz,CDCl3)δ165.3,157.7,155.9,155.9,154.4,143.6,140.4,133.9,132.5,129.6, 127.8,126.7,125.9,124.7,64.1,58.2,53.4,52.7,48.6,47.1,29.5,26.0,24.5,15.5. HRMS(ESI)calculated for C24H31N7NaO2 +:472.2426,found 472.2428.
实施例16：化合物16的制备
Figure BDA0002838769190000161
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为4-乙酰氨基苯硼酸，等摩尔代替化合物(1-2)的制备方法中的3，5-二甲氧基苯硼酸，化合物16的核磁测试结果为：
1H NMR(400MHz,CDCl3)δ8.24(d,J＝1.3Hz,1H),8.01(s,1H),7.54–7.43 (m,3H),6.72(dd,J＝16.9,10.7Hz,1H),6.18(dt,J＝16.8,1.7Hz,1H),5.73(dt,J＝ 10.6,1.7Hz,1H),4.43(t,J＝6.7Hz,2H),3.60(q,J＝6.7,4.6Hz,4H),2.46(d,J＝ 5.2Hz,4H),2.37(t,J＝7.6Hz,2H),2.16(d,J＝1.4Hz,3H),1.97(t,J＝7.4Hz,2H), 1.57(dd,J＝11.0,4.7Hz,2H),1.34(p,J＝7.6Hz,2H).13C NMR(100MHz,MeOD) δ170.6,165.9,158.4,155.4,153.8,144.4,139.14,133.2,129.6,127.3,127.3,123.7, 120.4,120.1,100.0,97.6,57.6,52.8,52.3,45.0,41.3,28.9,25.3,23.9,22.4.HRMS (ESI)calculated for C25H32N8NaO2 +:472.2426,found 472.2428.
实施例17：化合物17的制备
Figure BDA0002838769190000171
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为4-三氟甲氧基苯硼酸，等摩尔代替化合物(1-2)的制备方法中的3，5-二甲氧基苯硼酸，化合物17的核磁测试结果为：
1H NMR(400MHz,CDCl3)δ8.27(s,1H),7.70(t,J＝7.6Hz,1H),7.63(d,J＝ 10.6Hz,1H),7.49–7.42(m,1H),7.35(dd,J＝7.8,5.3Hz,1H),6.73(dd,J＝16.8, 10.7Hz,1H),6.18(dd,J＝16.8,1.9Hz,1H),5.73(dd,J＝10.7,2.0Hz,1H),4.44(t, J＝6.8Hz,2H),3.61(d,J＝5.4Hz,4H),2.50–2.37(m,4H),2.35(d,J＝7.7Hz, 2H),1.99(q,J＝7.1Hz,2H),1.59–1.55(m,2H),1.35(s,2H).13C NMR(100MHz, CDCl3)δ166.2,158.5,156.8,155.4,150.6,143.3,136.3,131.8,128.6,128.4,127.6, 122.3,121.9,120.1,99.2,59.1,54.3,53.6,48.1,46.6,42.8,30.4,27.1,25.4.HRMS (ESI)calculated for C24H28F3N7NaO2 +:526.2143,found 526.2145.
实施例18：化合物18的制备
Figure BDA0002838769190000172
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为2-萘硼酸，等摩尔代替化合物(1-2)的制备方法中的3，5-二甲氧基苯硼酸，化合物18的核磁测试结果为：
1H NMR(400MHz,CDCl3)δ8.41(s,1H),8.17(s,1H),8.03(d,J＝8.4Hz,1H), 7.93(dd,J＝9.3,3.6Hz,2H),7.82(d,J＝8.4Hz,1H),7.58(dd,J＝6.2,3.2Hz,2H), 6.51(dd,J＝16.8,10.5Hz,1H),6.27(d,J＝16.8Hz,1H),5.67(d,J＝10.7Hz,1H), 4.49(t,J＝7.2Hz,2H),3.68(s,2H),3.51(d,J＝15.2Hz,2H),2.37(dd,J＝19.5, 12.3Hz,6H),2.04(dd,J＝14.6,7.4Hz,2H),1.57(dd,J＝14.7,7.5Hz,2H),1.42 (dd,J＝14.9,7.9Hz,2H).13C NMR(100MHz,CDCl3)δ165.28,157.63,155.97, 154.54,149.88,142.42,135.37,135.35,130.91,127.75,127.50,127.24,126.68, 121.64,121.38,121.00,118.73,98.31,58.16,53.43,52.73,47.17,45.72,41.87,29.53, 26.24,24.52.HRMS(ESI)calculated forC27H31N7NaO+:492.2482,found 492.2484.
实施例19：化合物19的制备
Figure BDA0002838769190000181
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为4-甲基萘硼酸，等摩尔代替化合物(1-2)的制备方法中的3，5-二甲氧基苯硼酸，化合物19的核磁测试结果为：
1H NMR(400MHz,CDCl3)δ8.37(s,1H),8.10(d,J＝8.4Hz,1H),7.91(d,J＝ 8.3Hz,1H),7.63–7.41(m,4H),6.52(dd,J＝16.8,10.5Hz,1H),6.26(dd,J＝16.8, 1.7Hz,1H),5.67(dd,J＝10.5,1.7Hz,1H),4.50(t,J＝7.1Hz,2H),3.66(s,2H), 3.49(d,J＝16.9Hz,2H),2.78(s,3H),2.36(dd,J＝19.3,12.0Hz,6H),2.13–1.98 (m,2H),1.57(dt,J＝14.9,7.6Hz,2H),1.42(dt,J＝15.0,7.6Hz,2H).13C NMR (100MHz,CDCl3)δ165.27,159.72,157.75,155.67,154.23,144.16,134.49,130.52, 127.93,127.88,127.43,120.40,120.11,115.46,114.30,98.54,98.31,63.68,58.15, 53.31,52.69,47.03,41.74,29.71,29.54,26.11,24.50,24.04.HRMS(ESI)calculated for C28H33N7NaO+:570.1587,found570.1590.
实施例20：化合物20的制备
Figure BDA0002838769190000191
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为4-溴萘硼酸，等摩尔代替化合物(1-2)的制备方法中的3，5-二甲氧基苯硼酸，化合物20的核磁测试结果为：
1H NMR(400MHz,CDCl3)δ8.38(s,1H),8.11(d,J＝8.4Hz,1H),7.92(d,J＝ 8.3Hz,1H),7.64–7.42(m,4H),6.53(dd,J＝16.8,10.5Hz,1H),6.27(dd,J＝16.8, 1.7Hz,1H),5.68(dd,J＝10.5,1.7Hz,1H),4.51(t,J＝7.1Hz,2H),3.67(s,2H), 3.50(d,J＝17.0Hz,2H),2.37(dd,J＝19.3,12.0Hz,6H),2.13–1.99(m,2H),1.58 (dt,J＝14.9,7.6Hz,2H),1.43(dt,J＝15.0,7.6Hz,2H).13C NMR(100MHz,CDCl3) δ165.16,157.62,157.55,155.88,154.26,149.40,134.60,128.22,127.80,127.72, 127.32,125.04,123.91,121.64,121.60,110.91,104.17,99.87,97.77,58.01,53.21, 52.59,47.20,45.47,41.63,29.34,24.31.HRMS(ESI)calculated for C27H30BrN7NaO+:570.1587,found 570.1590.
实施例21：化合物21的制备
Figure BDA0002838769190000192
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为2,5-二甲氧基苯硼酸，等摩尔代替化合物(1-2)的制备方法中的3，5-二甲氧基苯硼酸，化合物21的核磁测试结果为：
1H NMR(400MHz,CDCl3)δ8.35(s,1H),7.07(s,1H),7.02(d,J＝8.6Hz,2H), 6.56(dd,J＝16.8,10.6Hz,1H),6.28(d,J＝16.8Hz,1H),5.70(d,J＝7.6Hz,1H), 4.44(t,J＝7.1Hz,2H),3.81(d,J＝11.0Hz,6H),3.69(s,2H),3.55(s,2H),2.47– 2.32(m,6H),2.03(dd,J＝14.0,7.0Hz,2H),1.61–1.52(m,2H),1.41(dd,J＝14.7, 7.6Hz,2H).13C NMR(100MHz,CDCl3)δ165.28,158.41,155.55,154.40,153.90, 150.49,140.79,127.83,127.47,123.11,117.10,116.10,113.43,100.37,58.17,56.76, 55.85,53.35,52.72,47.06,45.66,41.80,29.55,26.21.HRMS(ESI)calculated for C25H33N7NaO3 +:502.2537,found 502.2539.
实施例22：化合物22的制备
Figure BDA0002838769190000201
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为3,4-二甲氧基苯硼酸，等摩尔代替化合物(1-2)的制备方法中的3，5-二甲氧基苯硼酸，化合物22的核磁测试结果为：
1H NMR(400MHz,CDCl3)δ8.39(s,1H),7.27(s,1H),6.89(s,2H),6.64– 6.50(m,1H),6.29(d,J＝16.6Hz,1H),5.70(d,J＝10.2Hz,3H),4.43(d,J＝6.9Hz, 2H),3.94(s,3H),3.91(s,3H),3.69(s,2H),3.56(s,2H),2.38(dd,J＝22.3,15.1Hz, 6H),2.03–1.98(m,2H),1.57(d,J＝6.9Hz,2H),1.40(d,J＝7.0Hz,2H).13C NMR (100MHz,CDCl3)δ165.34,165.29,157.82,155.94,155.91,155.87,154.22,154.05, 153.99,144.18,138.75,128.59,127.92,127.42,105.53,98.33,61.03,58.18,56.33, 53.39,52.70,47.06,29.61,26.19,24.53.HRMS(ESI)calculated for C25H33N7NaO3 +: 502.2537,found 502.2539.
实施例23：化合物23的制备
Figure BDA0002838769190000211
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为3,4,5-三甲氧基苯硼酸，等摩尔代替化合物(1-2)的制备方法中的3，5-二甲氧基苯硼酸，化合物23的核磁测试结果为：
1H NMR(400MHz,CDCl3)δ8.30(s,1H),6.82(d,J＝1.3Hz,2H),6.47(ddd,J ＝16.8,10.5,1.3Hz,1H),6.19(dt,J＝16.8,1.6Hz,1H),5.87(s,2H),5.60(dt,J＝ 10.5,1.6Hz,1H),4.36(t,J＝7.3Hz,2H),3.86(d,J＝1.3Hz,6H),3.83(d,J＝1.3 Hz,3H),3.60(t,J＝5.0Hz,2H),3.47(t,J＝5.0Hz,2H),2.33(t,J＝5.1Hz,4H), 2.27(d,J＝7.5Hz,2H),1.93(q,J＝7.6Hz,2H),1.49(t,J＝7.6Hz,2H),1.33(d,J＝ 7.6Hz,2H).13C NMR(100MHz,CDCl3)δ165.2,158.0,155.8,154.2,153.9,144.1, 138.8,128.6,128.6,128.4,127.7,127.5,105.6,98.3,61.0,58.2,56.3,53.4,52.7,47.0, 45.7,41.9,29.7,29.6,26.3,24.5.HRMS(ESI)calculated for C26H35N7NaO4 +: 532.2643,found 536.2645.
实施例24：化合物24的制备
Figure BDA0002838769190000212
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为3-三氟甲基苯硼酸，等摩尔代替化合物(1-2)的制备方法中的3，5-二甲氧基苯硼酸，化合物24的核磁测试结果为：
1H NMR(400MHz,CDCl3)δ8.37(s,1H),7.68–7.54(m,3H),7.33(d,J＝6.5 Hz,1H),6.59–6.47(m,1H),6.25(dd,J＝16.8,4.6Hz,1H),5.95–5.59(m,3H), 4.43(d,J＝6.6Hz,2H),3.64(d,J＝9.2Hz,2H),3.52(s,2H),2.39(d,J＝5.7Hz, 4H),2.32(d,J＝7.3Hz,2H),1.98(q,J＝7.1Hz,2H),1.56(s,2H),1.42–1.32(m, 2H).13C NMR(100MHz,CDCl3)δ165.3,157.7,155.8,154.5,149.8,142.5,135.3, 130.9,127.7,127.5,126.7,121.4,121.0,98.3,58.1,53.4,52.7,47.2,45.7,41.9,29.7, 29.5,26.2,24.5.HRMS(ESI)calculatedfor C24H28F3N7NaO+:510.2205,found 510.2207.
实施例25：化合物25的制备
Figure BDA0002838769190000221
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为3-异丙氧基苯硼酸，等摩尔代替化合物(1-2)的制备方法中的3，5-二甲氧基苯硼酸，化合物25的核磁测试结果为：
1H NMR(400MHz,CDCl3)δ8.15(d,J＝1.2Hz,1H),7.36(t,J＝7.9Hz,1H), 7.11(dd,J＝8.2,5.0Hz,2H),7.00–6.95(m,1H),6.70–6.60(m,1H),6.09(dt,J＝ 16.8,1.6Hz,1H),5.64(dt,J＝10.6,1.6Hz,1H),4.60(q,J＝6.0Hz,1H),4.32(t,J＝ 6.8Hz,2H),3.53(t,J＝6.3Hz,4H),2.38(s,4H),2.30(t,J＝7.6Hz,2H),1.87(d,J ＝7.1Hz,2H),1.48(t,J＝7.8Hz,2H),1.25(dd,J＝6.0,1.3Hz,6H),1.22(d,J＝7.4 Hz,2H).13C NMR(100MHz,MeOD)δ166.0,158.6,158.5,155.4,153.7,144.7, 134.0,130.2,127.3,127.3,120.1,116.4,115.5,100.0,97.6,69.9,57.6,52.8,52.3, 46.4,45.0,41.3,28.9,25.2,23.9,21.0.HRMS(ESI)calculated for C26H35N7NaO2 +: 500.2744,found 500.2747.
实施例26：化合物26的制备
Figure BDA0002838769190000231
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为3-苄氧基苯硼酸，等摩尔代替化合物(1-2)的制备方法中的3，5-二甲氧基苯硼酸，化合物26的核磁测试结果为：
1H NMR(400MHz,CDCl3)δ8.37(s,1H),7.49–7.27(m,8H),7.11(d,J＝8.5 Hz,1H),6.54(dd,J＝16.8,10.4Hz,1H),6.28(d,J＝16.8Hz,1H),5.69(d,J＝10.4 Hz,1H),5.43(s,2H),5.17(s,2H),4.44(t,J＝7.4Hz,2H),3.62(d,J＝49.9Hz,4H), 2.39(d,J＝27.3Hz,6H),2.03–1.93(m,2H),1.38(s,2H).13C NMR(100MHz, CDCl3)δ165.3,159.4,157.7,155.4,154.2,144.1,136.6,134.5,133.3,130.6,128.7, 128.1,127.7,127.5,127.4,120.9,116.0,114.5,98.2,70.0,58.1,53.3,52.7,47.1,45.7, 41.8,29.5,26.2,24.5,21.2.HRMS(ESI)calculated for C30H35N7NaO2 +:548.2744, found 548.2747.
实施例27：化合物27的制备
Figure BDA0002838769190000232
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为2-氯-5-甲氧基苯硼酸，等摩尔代替化合物(1-2)的制备方法中的3，5-二甲氧基苯硼酸，化合物27的核磁测试结果为：
1H NMR(400MHz,CDCl3)δ8.35(d,J＝4.2Hz,1H),7.43(s,1H),6.99(dq,J ＝19.8,3.2Hz,2H),6.53(ddd,J＝15.6,10.7,3.9Hz,1H),6.25(dd,J＝16.9,3.9Hz, 1H),5.80–5.22(m,3H),4.45(t,J＝6.5Hz,2H),3.82(d,J＝4.9Hz,3H),3.68– 3.63(m,2H),3.51(s,2H),2.39(d,J＝5.4Hz,4H),2.31(q,J＝6.2,5.4Hz,2H),1.99 (q,J＝6.8Hz,2H),1.53(q,J＝7.1Hz,2H),1.42–1.28(m,2H).13C NMR(100 MHz,CDCl3)δ165.3,158.6,157.7,155.9,153.8,141.1,132.6,131.1,127.7,127.5, 124.7,116.9,99.9,70.5,58.2,55.7,53.4,52.7,47.1,45.7,41.9,29.5,26.2,24.5. HRMS(ESI)calculated for C24H30ClN7NaO2 +:506.2042,found 506.2044.
实施例28：化合物28的制备
Figure BDA0002838769190000241
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为对氟苯硼酸，等摩尔代替化合物(1-2)的制备方法中的3，5-二甲氧基苯硼酸，化合物28的核磁测试结果为：
1H NMR(400MHz,CDCl3)δ8.31(s,1H),7.25(s,2H),6.54(dd,J＝16.8,10.5 Hz,1H),6.29(dd,J＝16.8,1.7Hz,1H),6.24(s,2H),5.70(d,J＝10.5Hz,1H),4.35 (t,J＝7.0Hz,2H),3.69(t,J＝29.6Hz,4H),2.44(d,J＝31.0Hz,6H),1.92(dd,J＝ 14.9,7.4Hz,2H),1.58(s,2H),1.35–1.30(m,2H).13C NMR(100MHz,CDCl3)δ 165.21,158.54,157.68,155.61,153.68,141.11,132.50,131.04,127.66,127.44, 124.68,116.84,99.80,58.07,55.65,53.26,52.63,47.07,45.59,41.74,29.40,26.09, 24.40.HRMS(ESI)calculatedfor C23H28FN7NaO+:460.2232,found 460.2230.
实施例29：化合物29的制备
Figure BDA0002838769190000251
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为3-氟-4-羟基苯硼酸，等摩尔代替化合物(1-2)的制备方法中的3，5-二甲氧基苯硼酸，化合物29的核磁测试结果为：
1H NMR(400MHz,CDCl3)δ8.34(s,1H),7.72–7.62(m,2H),7.26–7.23(m, 1H),6.55(dd,J＝16.8,10.5Hz,1H),6.28(dd,J＝16.8,1.9Hz,1H),5.69(dd,J＝ 10.5,1.9Hz,1H),4.44(t,J＝7.2Hz,2H),3.68(s,2H),3.51(d,J＝21.5Hz,2H), 2.47–2.39(m,4H),2.36–2.31(m,2H),1.99(dt,J＝15.0,7.5Hz,2H),1.55(dd,J＝ 15.1,7.6Hz,2H),1.42–1.33(m,2H).13C NMR(100MHz,CDCl3)δ165.30, 162.94,161.55,157.73,157.26,156.54,154.10,119.85,117.25,100.41,100.29, 99.99,61.49,57.97,53.20,52.53,47.28,29.69,29.65,29.40,29.26,24.72,24.24.. HRMS(ESI)calculated for C23H28FN7NaO2+:476.2181,found476.2180.
实施例30：化合物30的制备
Figure BDA0002838769190000252
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为6-甲氧基萘-2-硼酸，等摩尔代替化合物(1-2)的制备方法中的3，5-二甲氧基苯硼酸，化合物30的核磁测试结果为：
1H NMR(400MHz,CDCl3)δ8.37(d,J＝5.6Hz,1H),8.08(s,1H),7.91(d,J＝ 8.4Hz,1H),7.83(d,J＝8.9Hz,1H),7.77(dd,J＝8.4,1.6Hz,1H),7.26–7.19(m, 2H),6.51(dd,J＝16.8,10.5Hz,1H),6.31–6.16(m,1H),5.77(d,J＝66.8Hz,2H), 5.67(dd,J＝10.5,1.9Hz,1H),4.47(t,J＝7.2Hz,2H),3.97(s,3H),3.69(s,2H), 3.54(s,2H),2.38(dd,J＝21.6,14.2Hz,6H),2.02(dt,J＝14.8,7.4Hz,2H),1.63– 1.53(m,2H),1.41(dt,J＝15.1,7.5Hz,2H).13C NMR(100MHz,CDCl3)δ165.27, 158.50,157.93,155.54,154.31,144.47,134.66,129.73,128.92,128.23,128.12, 127.81,127.54,127.43,126.28,119.94,105.79,98.49,58.13,55.42,53.27,52.70, 47.05,45.55,41.71,29.57,26.08,24.51.HRMS(ESI)calculated for C28H33N7NaO2 +: 522.2588,found 522.2587.
实施例31：化合物31的制备
Figure BDA0002838769190000261
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为对硝基苯硼酸，等摩尔代替化合物(1-2)的制备方法中的3，5-二甲氧基苯硼酸，化合物31的核磁测试结果为：
1H NMR(400MHz,CDCl3)δ8.45–8.36(m,3H),7.92(d,J＝8.8Hz,2H), 6.54(dd,J＝16.8,10.5Hz,1H),6.27(dd,J＝16.9,2.0Hz,1H),5.90–5.63(m,3H), 4.48(t,J＝7.2Hz,2H),3.67(s,2H),3.59–3.50(m,2H),2.42(t,J＝5.1Hz,4H), 2.35(dd,J＝8.5,6.5Hz,2H),2.01(p,J＝7.6Hz,2H),1.63–1.55(m,2H),1.41(dt, J＝11.4,6.8Hz,2H).13C NMR(100MHz,CDCl3)δ165.3,157.6,155.9,154.7, 148.0,141.7,139.6,129.2,127.8,127.5,124.6,98.4,77.2,58.1,53.4,52.7,47.4,45.7, 41.8,29.7,29.5,26.2,24.5.HRMS(ESI)calculated for C23H28N8NaO3 +:487.2177, found 487.2179.
实施例32：化合物32的制备
Figure BDA0002838769190000271
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为3-氟-5-甲氧基苯硼酸，等摩尔代替化合物(1-2)的制备方法中的3，5-二甲氧基苯硼酸，化合物31的核磁测试结果为：
1H NMR(400MHz,CDCl3)δ8.38(s,1H),7.24(d,J＝2.1Hz,2H),6.99–6.87 (m,1H),6.54(dd,J＝16.8,10.6Hz,1H),6.27(dd,J＝16.8,1.9Hz,1H),5.80–5.63 (m,3H),4.44(t,J＝7.2Hz,2H),3.92(s,3H),3.66(d,J＝12.5Hz,2H),3.54(s,2H), 2.45–2.33(m,6H),2.02–1.96(m,2H),1.55(dd,J＝15.0,7.5Hz,2H),1.37(dd,J ＝15.2,7.2Hz,2H).13C NMR(100MHz,CDCl3)δ165.31,164.85,164.72,162.35, 162.23,157.51,155.78,154.50,127.78,127.48,111.59,111.33,104.53,104.28, 98.15,58.13,53.38,52.69,47.23,45.66,41.82,29.49,26.17,24.51.HRMS(ESI) calculated for C24H30FN7NaO2 +:490.2337,found 490.2335.
实施例33：化合物33的制备
Figure BDA0002838769190000272
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为3,5-二氟苯硼酸，等摩尔代替化合物(1-2)的制备方法中的3，5-二甲氧基苯硼酸，化合物33的核磁测试结果为：
1H NMR(400MHz,CDCl3)δ8.36(s,1H),7.01(dd,J＝8.4,2.2Hz,2H),6.73 (dt,J＝10.5,2.3Hz,1H),6.55(dd,J＝16.8,10.6Hz,1H),6.28(dd,J＝16.8,1.9Hz, 1H),5.83(s,2H),5.69(dd,J＝10.5,1.9Hz,1H),4.44(t,J＝7.2Hz,2H),3.66(m,J ＝16.1Hz,2H),3.55(m,2H),2.37(dd,J＝23.0,15.7Hz,6H),1.98(dd,J＝14.9,7.5 Hz,2H),1.56(dd,J＝14.8,7.4Hz,2H),1.45–1.36(m,2H).13C NMR(100MHz, CDCl3)δ165.28,162.81,161.77,161.65,157.58,155.96,154.41,127.74,127.49, 110.07,110.04,107.79,107.57,102.38,102.13,98.25,58.15,55.81,53.35,52.74, 47.12,29.51,24.50.HRMS(ESI)calculatedfor C23H27F2N7NaO+:478.2137,found 478.2139.
实施例34：化合物34的制备
Figure BDA0002838769190000281
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为3,5-二三氟甲基苯硼酸，等摩尔代替化合物(1-2)的制备方法中的3，5-二甲氧基苯硼酸，化合物34的核磁测试结果为：
1H NMR(400MHz,CDCl3)δ8.44(s,1H),8.20(s,2H),7.99(s,1H),6.61– 6.51(m,1H),6.28(d,J＝17.0Hz,1H),5.69(d,J＝10.6Hz,1H),4.48(t,J＝7.0Hz, 2H),3.64(s,4H),2.39(d,J＝26.1Hz,6H),2.05–1.99(m,2H),1.78(s,2H),1.38 (dd,J＝23.0,12.9Hz,2H).13C NMR(100MHz,CDCl3)δ165.3,157.5,156.0,154.8, 140.9,135.5,133.2,132.9,132.6,128.5,127.8,127.5,124.4,122.5,121.7,98.4,58.1, 53.4,52.7,47.35 45.7,41.8,29.5,26.2,24.5.HRMS(ESI)calculated for C25H27F6N7NaO+:578.2073,found578.2075.
实施例35：化合物35的制备
Figure BDA0002838769190000291
具体制备方法为：与化合物1的制备方法类似，不同的是所使用的原料之一为3-氟-5-三氟甲基苯硼酸，等摩尔代替化合物(1-2)的制备方法中的3，5-二甲氧基苯硼酸，化合物35的核磁测试结果为：
1H NMR(400MHz,CDCl3)δ8.35(s,1H),7.73(s,1H),7.58(dd,J＝8.7,2.0 Hz,1H),7.40–7.37(m,1H),6.48(dd,J＝16.8,10.5Hz,1H),6.20(dd,J＝16.8,1.9 Hz,1H),5.61(dd,J＝10.5,1.9Hz,1H),4.39(t,J＝7.2Hz,2H),3.60(s,2H),3.47(s, 2H),2.34(t,J＝5.1Hz,4H),2.30–2.24(m,2H),1.96–1.90(m,2H),1.52–1.47(m, 2H),1.32(t,J＝7.6Hz,2H).13C NMR(100MHz,CDCl3)δ162.0,161.5,157.8, 155.6,154.1,144.2,135.0,129.5,127.8,127.7,106.4,101.0,98.2,58.0,55.6,53.3, 52.5,47.0,46.0,42.2,29.7,29.5,26.2,24.5.HRMS(ESI)calculated for C24H27F4N7NaO+:528.2105,found 528.2107.
实施例36：化合物36的制备
Figure BDA0002838769190000292
具体制备方法为：与化合物10的制备方法类似，不同的是所使用的原料之一为4,4,4-三氟丁烯酸，等摩尔代替化合物(1-3)的制备方法中的丙烯酰氯，化合物36的核磁测试结果为：
1H NMR(400MHz,CDCl3)δ8.32(s,1H),6.92(dt,J＝15.4,2.1Hz,1H),6.78 (d,J＝2.3Hz,2H),6.73–6.64(m,1H),6.52(t,J＝2.3Hz,1H),4.40(t,J＝7.2Hz, 2H),3.82(s,6H),3.65(t,J＝5.2Hz,2H),3.50(t,J＝5.0Hz,2H),2.40(q,J＝4.7Hz, 4H),2.32(t,J＝7.5Hz,2H),1.96(p,J＝7.4Hz,2H),1.52(q,J＝7.6Hz,2H),1.36 (q,J＝8.2Hz,2H).13C NMR(100MHz,CDCl3)δ162.3,161.8,158.1,155.8,154.3, 144.4,135.3,129.7,129.4,128.0,127.9,121.4,106.6,101.3,98.5,58.3,55.8,53.5, 52.7,47.3,46.2,42.5,29.9,29.8,26.4,24.7.HRMS(ESI)calculated for C26H32F3N7NaO3 +:570.2411,found 570.2413.
实施例37：化合物37的制备
Figure BDA0002838769190000301
具体制备方法为：与化合物34的制备方法类似，不同的是所使用的原料之一为4,4,4-三氟丁烯酸，等摩尔代替化合物(1-3)的制备方法中的丙烯酰氯，化合物37的核磁测试结果为：
1H NMR(400MHz,CDCl3)δ8.43(d,J＝2.3Hz,1H),8.20(s,2H),7.99(s,1H), 6.94(d,J＝15.5Hz,1H),6.72(dt,J＝14.9,6.8Hz,1H),4.48(t,J＝7.0Hz,2H), 3.69(s,2H),3.54(s,2H),2.41(d,J＝31.2Hz,6H),2.02(q,J＝7.8Hz,2H),1.59(s, 2H),1.44–1.26(m,2H).13C NMR(100MHz,CDCl3)δ164.1,162.1,161.6,157.4, 155.9,154.7,141.3,136.6,127.7,121.0,118.9,118.7,113.4,113.2,98.2,58.0,53.3, 52.5,47.2,45.9,42.2,29.7,29.5,26.1,24.5,14.1.HRMS(ESI)calculated for C26H26F9N7NaO+:646.1947,found646.1947.
实施例38：化合物38的制备
Figure BDA0002838769190000311
具体制备方法为：与化合物35的制备方法类似，不同的是所使用的原料之一为4,4,4-三氟丁烯酸，等摩尔代替化合物(1-3)的制备方法中的丙烯酰氯，化合物38的核磁测试结果为：
1H NMR(400MHz,CDCl3)δ8.43(d,J＝2.3Hz,1H),8.20(s,2H),7.99(s,1H), 6.94(d,J＝15.5Hz,1H),6.71(dq,J＝14.2,6.7Hz,1H),4.48(t,J＝7.0Hz,2H), 3.69(s,2H),3.54(s,2H),2.41(d,J＝31.2Hz,6H),2.01(t,J＝7.6Hz,2H),1.59(s, 2H),1.48–1.27(m,2H).13C NMR(100MHz,CDCl3)δ162.0,155.2,154.6,142.9, 133.2,132.1,132.1,129.4,129.0,128.6,128.5,127.9,121.3,120.8,111.6,105.6, 58.1,53.3,52.6,46.9,46.0,42.3,29.5,26.3,24.5,22.1.HRMS(ESI)calculated for C26H26F9N7NaO+:646.1947,found646.1947.
实施例39：化合物39的制备
Figure BDA0002838769190000312
具体制备方法为：与化合物10的制备方法类似，不同的是所使用的原料之一为反式-4-二甲基氨基巴豆酸，等摩尔代替化合物(1-3)的制备方法中的丙烯酰氯，化合物39的核磁测试结果为：
1H NMR(400MHz,CDCl3)δ8.38(s,1H),6.81(d,J＝2.3Hz,2H),6.56(t,J＝ 2.3Hz,1H),5.60(s,2H),4.43(t,J＝7.2Hz,2H),3.86(s,6H),3.67(t,J＝5.1Hz, 2H),3.59(t,J＝5.1Hz,2H),3.49(s,1H),2.45(t,J＝5.1Hz,4H),2.33(t,J＝7.4Hz, 2H),1.96(d,J＝7.5Hz,2H),1.50–1.35(m,4H).13C NMR(100MHz,CDCl3)δ 161.5,161.5,157.7,155.9,144.0,135.1,106.4,101.0,100.0 98.3,58.1,55.6,53.1, 52.4,52.3,49.4,47.1,45.8,43.4,43.3,43.2,37.4,29.6,26.9,26.5.HRMS(ESI) calculated for C25H31N7NaO3 +:500.2831,found 500.2833.
实施例40：化合物40的制备
Figure BDA0002838769190000321
具体制备方法为：与化合物10的制备方法类似，不同的是所使用的原料之一为巴豆酸，等摩尔代替化合物(1-3)的制备方法中的丙烯酰氯，化合物40的核磁测试结果为：
1H NMR(400MHz,CDCl3)δ8.31(s,1H),6.88–6.73(m,3H),6.52(t,J＝2.3 Hz,1H),6.20(dq,J＝15.0,1.7Hz,1H),4.39(t,J＝7.2Hz,2H),3.82(s,6H),3.62(d, J＝8.1Hz,2H),3.56–3.43(m,2H),2.36(t,J＝5.1Hz,4H),2.29(dd,J＝8.7,6.4 Hz,2H),2.01–1.91(m,2H),1.83(dd,J＝6.9,1.7Hz,3H),1.52(t,J＝7.6Hz,2H), 1.36(ddd,J＝15.3,9.1,5.4Hz,2H).13C NMR(100MHz,CDCl3)δ165.5,161.5, 158.0,155.7,154.1,144.1,141.5,135.1,121.4,119.9,106.4,101.1,98.2,60.4,58.2, 55.5,53.5,53.4,52.8,47.0,45.5,41.8,29.6,26.2,24.5,18.2.HRMS(ESI)calculated for C26H35N7NaO3 +:516.2694,found516.2696.
实施例41酶活测试
本发明采用均相时间分辨荧光(HIRF)技术评价化合物对FGFR1,2,3和4 四个亚型的抑制作用。首先构建FGFR1,2,3和4原核表达载体，鉴定正确后，大肠杆菌体系大批量表达纯化FGFR蛋白，测定蛋白浓度，分装保存。酶活测试开始前，选用cisbio HTRF KinEA(62TK0PEB)预先将酶活体系中需要的各种组分稀释到所需要的工作浓(使用酶活缓冲液进行稀释)。准备384孔板 (66PL384025)，每孔依次加入5μL蛋白溶液、1μL化合物、2μL底物，最后加入2μL ATP启动反应。每个浓度设置3个平行实验，空白对照组将1μL化合物换成1μLDMSO，阳性对照组为阳性药AZD4547，其他条件保持不变。密封 384孔板，室温下孵育1h左右。孵育完成后，向每孔中加入5μL澜系元素标记的磷酸化底物的抗体(供体)和5μL链酶亲和素标记的XL665(受体)终止反应，避光，室温下继续孵育1h左右。最后使用TECANinfinite M1000PRO多功能酶标仪检测荧光信号，计算抑制率，用GraphPad Prism 7.0拟合IC50值。
结果显示，化合物1-40均可不同程度的抑制FGFR1/2/3/4的蛋白活性。
表1.基于酶活结果的构效关系研究
Figure BDA0002838769190000331
Figure BDA0002838769190000341
Figure BDA0002838769190000351
实施例42细胞增殖试验
本发明选取化合物10和36，采用MTT比色法评价该化合物10和36对多种肿瘤细胞的增殖抑制作用。胰酶消化收集对数生长期的肿瘤细胞，用1ml新鲜培养基重悬，然后取少量进行稀释，用血球计数板计算细胞数量。根据细胞生长速度不同，按照3000-5000个/孔的密度将肿瘤细胞接种到96孔板中，然后放置细胞培养箱中培养24h。随后用化合物10和36处理肿瘤细胞，设置8个浓度，每个浓度设置三个复孔，孵育96h。孵育完成后，每孔加入10μLMTT溶液(5 mg/ml)，拍打混匀后继续放入细胞培养箱中培养3-4h。孵育完成后，小心地吸空孔板中液体，随后每孔加入100μL DMSO溶液。设置酶标仪程序，分别在 490nm和570nm处的测定吸光光度值，根据公式计算每个浓度的增殖抑制率，最后用Graphpad Prism 7软件进行数据处理，计算IC50。
结果显示(表1)，化合物10和36显著抑制人肺鳞癌细胞NCI-H1581(FGFR1 扩增)和人胃癌细胞SNU-16(FGFR2扩增)的增殖，对人肝癌细胞SK-hep-1 (FGGR4扩增)具有一定程度的增殖抑制作用，而对FGFR表达正常的肿瘤细胞无明显抑制作用，表明化合物10和36具有很强的选择性。
表2.化合物36对特定癌细胞系的抗增殖活性
Figure BDA0002838769190000352
Figure BDA0002838769190000361
实施例43化合物10和36抑制FGF/FGFR及其下游信号传导途径
为了进一步阐明化合物10和36对FGF/FGFR信号的影响，通过蛋白质免疫印迹实验验证其对FGFR和下游信号蛋白磷酸化的影响。胰酶消化收集处于对数生长期的人肺鳞癌细胞NCI-H1581和人胃癌细胞SNU-16，用1ml新鲜培养基重悬，取少量体积进行稀释，用血球计数板计算细胞数量。按照3x105/个每孔的密度，取相应体积的肿瘤细胞悬浮液接种到6孔板中，放置细胞培养箱中培养24h。待细胞完全贴壁后，吸去旧培养基，加入含有不同浓度的化合物10和 36的新鲜培养基，细胞培养箱中共孵育12h。孵育完成后，吸去上清，用预冷的1XPBS溶液洗涤三次，彻底吸除残液，每孔加入60μL RIPA裂解液，用细胞刮子将细胞轻轻地刮下来，将裂解液转移至1.5ml EP管中，冰浴10min， 14000rpm离心10min，吸取上清至新的1.5ml EP管中，吸取少量进行蛋白含量测定，剩余加相应体积的5X上样缓冲液，涡旋混匀，100℃金属浴10min。根据测定的蛋白含量，按每孔30μg蛋白的上样量，计算相应的上样量。利用 8％的SDS-PAGE凝胶电泳分离蛋白，将分离好的蛋白转至PDVF膜上，丽春红染色，根据目的蛋白的分子量裁剪含有蛋白的PDVF膜，1X TBST溶液洗净丽春红，5％的脱脂奶粉封闭30min。1X TBST溶液洗涤三次，将含有目标蛋白条带的PDVF 膜放入孵育盒中，加入相应的一抗孵育液，4℃过夜孵育。一抗孵育完成后， 1X TBST溶液洗涤三次，加入相应的二抗孵育液，常温孵育1.5h。孵育完成后， 1X TBST溶液洗涤三次，用超敏HRP化学发光试剂盒显色。
如图1和2结果显示，化合物10和36不仅可以显著抑制NCI-H1581细胞中FGFR1和SNU-16细胞中FGFR2的磷酸化，而且可以剂量依赖的方式抑制下游PLCγ，AKT和ERK的激活。总的来说，化合物10和36可以在体外强烈抑制FGF/FGFR和下游信号传导途径。
实施例44化合物10和36明显抑制小鼠体内肿瘤生长
为了评估化合物10和36在体内的抗肿瘤功效，用FGFR1高表达的人肺鳞癌细胞NCI-H1581建立了小鼠皮下移植瘤模型。胰酶消化收集处于对数生长期的细胞，1X PBS洗涤三次，细胞沉淀用重悬液(1640培养基：Matrigel＝1:1)稀释至1x108/mL，按5x106个/只的数量将细胞接种至5-6周龄的雌性BALB/c Nude 小鼠的前肢腋窝下端。待肿瘤体积长至50-100mm3时，随机将小鼠分为三组(对照组，50mg/kg,100mg/kg),每组6只小鼠，化合物10每天口服给药，化合物 36每天腹腔注射给药，并量取肿瘤体积和小鼠体重，给药26天后处理小鼠，剖取肿瘤组织，固定于福尔马林溶液中备用。
图3和4结果显示，化合物10和36剂量依赖性地抑制NCI-H1581细胞在小鼠体内的增殖，高剂量组的抑制率达72％，抑制肿瘤组织内FGFR1的磷酸化激活，且没有明显的体重减轻现象，表明化合物10和36在体内对所有剂量均具有良好的耐受性。
总之，本发明优化并发现了2H-吡唑并[3,4-d]嘧啶的新型衍生物化合物10 和36，作为对FGFR1，FGFR2和FGFR3的有效和选择性抑制剂，表征了对靶蛋白的不可逆结合作用。该先导化合物不仅可以在低浓度下抑制FGF/FGFR 及其下游信号通路，而且在体内外均显示出对NCI-H1581癌细胞系的显著抗增殖作用。而且，化合物10和36显示出低毒性并且具有良好的PK特性，目前正被确认为潜在的候选药物。
以上所述仅为本发明创造的较佳实施例而已，并不用以限制本发明创造，凡在本发明创造的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明创造的保护范围之内。
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Claims (3)

1.一种结构如下所示的化合物或其在药学上可接受的盐：
Figure FDA0003489593650000011
2.一种含有权利要求1所述的化合物或其药学上可接受的盐的药物组合物，其中，包含一种或多种药学上可接受的赋形剂，该药物组合物的剂型为药学上可接受的任一剂型。
3.权利要求1所述的化合物或其药学上可接受的盐或权利要求2所述的药物组合物在用于制备作为不可逆的泛成纤维细胞生长因子受体FGFR1，FGFR2和FGFR3抑制剂的用途。
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