CN116854668A - 酞嗪酮类化合物及其药物组合物和应用 - Google Patents
酞嗪酮类化合物及其药物组合物和应用 Download PDFInfo
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- CN116854668A CN116854668A CN202310843892.0A CN202310843892A CN116854668A CN 116854668 A CN116854668 A CN 116854668A CN 202310843892 A CN202310843892 A CN 202310843892A CN 116854668 A CN116854668 A CN 116854668A
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- acid
- pyrazol
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Abstract
本发明公开了一种酞嗪酮类化合物及其药物组合物和应用，该化合物结构如式(I)，还包含其异构体、前药、稳定的同位素衍生物、药学上可接受的盐或它们的混合物；其在酶水平上对PRMT5具有显著的抑制作用，并且在MTA存在下抑制活性更强，纳摩尔浓度水平下的抑制率达到80％以上，同时对MTAP缺失的肿瘤细胞的抑制活性强于野生型，具有选择性；所制备药物在分子水平和细胞水平均可以发挥药效，应用广泛。
Description
技术领域
本发明涉及一种酞嗪酮类化合物及其药物组合物和应用，尤其涉及一种具有PRMT5抑制活性的酞嗪酮类化合物及其药物组合物和应用。
背景技术
表观遗传调控涉及遗传物质的可遗传修饰而不改变其核苷酸序列，并且它通过选择性和可逆地修饰DNA和蛋白质(例如，组蛋白)来控制染色质的构象在转录与非转录活性之间进行转变。这些共价修饰可通过酶来控制，其中许多酶与可导致人类疾病的特定遗传改变相关联。精氨酸甲基化是组蛋白修饰的一种调控类型，是哺乳动物中最常见的翻译后修饰之一。
蛋白精氨酸甲基转移酶(Protein arginine methyltransferase，PRMTs)是一种依赖S-腺苷甲硫氨酸(SAM)的甲基转移酶，能够将甲基从SAM转移到组蛋白和非组蛋白底物精氨酸残基的胍基氮上。基于它们转移的产物特异性，九种哺乳动物PRMTs可以分为三类，分别为I型(PRMT1、2、3、4、6、8)，II型(PRMT5、9)和III型(PRMT7)，其中PRMT5是目前最有前景的II型PRMTs，能催化精氨酸残基的单甲基化和对称二甲基化。
PRMTs可以甲基化组蛋白和非组蛋白底物，在多种细胞功能中发挥重要作用，可以通过组蛋白甲基化影响信号转导、mRNA剪接和DNA修复的基因表达。研究发现，在人类多种恶性肿瘤包括肺癌、卵巢癌、结直肠癌、乳腺癌、黑色素瘤、白血病以及恶性胶质瘤中PRMT5的表达均出现上调，此外，PRMT5作为套细胞淋巴瘤与胶质母细胞瘤的抗癌靶点已被确证，以PRMT5为靶点的抗肿瘤小分子抑制剂研究越来越受到人们的关注。
甲基硫腺苷磷酸化酶(MTAP)在40％的胶质母细胞瘤、25％的黑色素瘤、尿路上皮癌和胰腺癌、15％的非小细胞肺癌中缺失。MTAP缺失的细胞内甲硫腺苷(MTA)浓度增加，MTA是PRMT5的体内抑制剂，相比于野生型细胞，PRMT5小分子抑制剂能够显著抑制MTAP缺失的癌细胞系。
第一代PRMT5抑制剂主要分为SAM非竞争型和SAM竞争型抑制剂，代表性药物有GSK-3326595、JNJ-64619178、PF-06939999、PRT543、PRT811，它们均对PRMT5具有无选择性的抑制活性，还与患者剂量限制性的血小板减少症、贫血、中性粒细胞减少症有关，存在副作用大、治疗指数低的缺陷，这限制了第一代PRMT5抑制剂的应用。第二代PRMT5抑制剂能特异性地作用于MTAP缺失的肿瘤，从而提高对肿瘤的选择性，降低毒副作用。目前第二代的PRMT5抑制剂研究较少，代表性药物有MRTX1719、TNG908和AMG193，它们均处于早期的临床研究阶段。
发明内容
发明目的：本发明的第一目的是提供一种酞嗪酮类化合物，第二目的是提供一种包含所述酞嗪酮类化合物的药物组合物，第三目的是提供一种所述酞嗪酮类化合物及其药物组合物在制备PRMT5抑制剂药物中的应用。
技术方案：本发明所述的酞嗪酮类化合物具有式(I)的结构，还包含其异构体、前药、稳定的同位素衍生物、药学上可接受的盐或它们的混合物：
其中：
R1a、R1b分别独立地选自氢、卤素、C1～C6烷基，或者R1a、R1b与所连的碳原子一起形成C3～C8环烷基或C2～C6杂环烷基；当R1a、R1b不同时，与其相连的碳原子为消旋构型、R构型或S构型，所述C2～C6杂环烷基包含1～3个选自N、O或S的杂原子；
A环选自取代的C6～C10芳基、取代的C5～C9杂芳基、取代的C3～C8环烷基或取代的C2～C6杂环烷基；所述的取代基选自至少一个氢、卤素、氰基、C1～C6卤代烷基、C1～C6烷基、C1～C6烷氧基、羟基、氨基、甲氨基、二甲氨基、乙酰氨基、羧基、甲氧羰基或硝基；所述的C5～C9杂芳基和C2～C6杂环烷基包含1～3个选自N、O或S的杂原子；
R2选自氢、卤素、C1～C6烷基、C1～C6烷氧基、羧基、C1～C3烷氧羰基，与R2相连的双键构型为Z构型、E构型或两者的混合物。
优选，所述结构中：
R1a、R1b分别独立地选自氢、甲基或乙基，或者R1a、R1b与所连的碳原子一起形成环丙烷；
A环选自取代的苯环、哒嗪环、吡啶环、嘧啶环、噻吩环、呋喃环、吡咯环、喹啉环、喹唑林环、萘环；所述的取代基选自至少一个氢、氟、氯、溴、氰基、甲基、乙基、三氟甲基、甲氧基、乙氧基、苯氧基、羟基、氨基、甲氨基、二甲氨基、乙酰氨基、羧基、甲氧羰基或硝基；
R2为氢原子，与R2相连的双键构型为Z构型、E构型或两者的混合物。
优选，所述结构中：
R1a或R1b选自氢；
A环选自取代的苯环、吡啶环、噻吩环、苯并[d][1,3]二恶茂或萘环；所述的取代基选自至少一个氢、氟、氯、溴、氰基、甲基、乙基、三氟甲基、甲氧基、乙氧基、苯氧基、羟基、氨基或硝基；
R2为氢原子，与R2相连的双键构型为Z构型。
优选，所述结构中：
R1a或R1b选自氢；
A环选自
R2为氢原子，与R2相连的双键构型为Z构型。
优选，所述的酞嗪酮类化合物选自以下任一化合物：
进一步地，所述药学上可接受的盐为所述酞嗪酮类化合物与选自以下任一的酸形成的盐：
盐酸、氢溴酸、硫酸、磷酸、碳酸、甲磺酸、苯磺酸、对甲苯磺酸、萘磺酸、柠檬酸、苹果酸、酒石酸、乳酸、丙酮酸、乙酸、马来酸、琥珀酸、富马酸、水杨酸、苯基乙酸、杏仁酸或阿魏酸。
当本发明所述的酞嗪酮类化合物的结构中：R1a、R1b为氢，R2为氢，与R2相连的双键构型为Z构型时，其制备方法如下：
由化合物1与化合物4-溴-1-甲基-1H-吡唑-5-甲醛反应制备化合物2，所用溶剂选自甲醇、乙醇、二氯甲烷、四氢呋喃、甲苯、1,4-二氧六环、乙酸乙酯、丙酮、N,N-二甲基甲酰胺或任意两者组成的混合溶剂，优选乙醇；所用碱选自氢氧化钾、碳酸钾、醋酸钾、碳酸钠、哌啶，优选氢氧化钾和哌啶。
由化合物2与化合物3反应制备化合物4，所用溶剂选自甲苯、1,4-二氧六环、N,N-二甲基甲酰胺、正丁醇、叔戊醇、和水任意两者组成的混合溶剂，优选叔戊醇与水组成的混合溶剂；所用碱选自氢氧化钾、碳酸钾、醋酸钾、碳酸钠、碳酸氢钠，优选碳酸氢钠；所用催化剂选自[1,1'-双(二苯基膦基)二茂铁]二氯化钯(Pd(dppf)Cl2)、四(三苯基膦)钯(Pd(PPh3)4)、双(三苯基磷)二氯化钯(Pd(PPh3)2Cl2)、醋酸钯(Pd(OAc)2)和氯(2-二环己基膦基-2',6'-二甲氧基-1,1'-联苯基)(2'-氨基-1,1'-联苯-2-基)钯(II)(SPhospdG2)，优选SPhospdG2。
由化合物4脱Boc保护制备化合物Ⅰ，所用酸选自HCl/Dioxane、HCl/Ethyl acetate或TFA/DCM，优选HCl/Dioxane。
其中，环A的定义如前所述；
将相应的酸与以上方法制备的酞嗪酮类化合物成盐，即得所述药学上可接受的盐。
本发明所述的药物组合物包含所述的酞嗪酮类化合物以及药学上可接受的载体。具体通过添加香料、甜味剂、液体/固体填料、稀释剂等常用药用辅料，制成常见的药用制剂，如片剂、胶囊、糖浆、悬浮剂或注射剂。
本发明所述的酞嗪酮类化合物或其药物组合物应用于PRMT5抑制剂药物的制备，具体为抗肿瘤药物，更具体为治疗肺癌、卵巢癌、结直肠癌、乳腺癌、黑色素瘤、白血病或恶性胶质瘤的药物。
有益效果：与现有技术相比，本发明具有如下显著优点：
该类化合物结构新颖，在酶水平上可以对PRMT5具有显著的抑制作用，并且在MTA存在下抑制活性更强(100nM浓度水平下的抑制率达到80％以上)；同时对MTAP缺失的HCT116肿瘤细胞的抑制活性(大部分IC50值低于1μM)强于野生型HCT116肿瘤细胞(大部分IC50值高于1μM)，具有选择性。
具体实施方式
实施例1：(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢酞嗪-6-基)-1-甲基-1H-吡唑-5-基)-2-苯基丙烯腈(I-1)的合成
1、(Z)-3-(4-溴-1-甲基-1H-吡唑-5-基)-2-苯基丙烯腈(2-1)的合成
向25mL三颈瓶中依次加入苯乙腈1-1(124mg,1.06mmol)、4-溴-1-甲基-1H-吡唑-5-甲醛(200mg,1.06mmol)、KOH(120mg,2.12mmol)和2mL EtOH，反应体系在25℃下反应12h后，TLC监测(V石油醚:V乙酸乙酯＝10:1)反应完全，减压浓缩，残余物经柱层析纯化(洗脱剂：V石油醚:V乙酸乙酯＝10:1)得白色固体120mg，收率：38.0％。
1H NMR(400MHz,Chloroform-d)δ(ppm):7.77-7.71(m,2H),7.58(s,1H),7.56–7.49(m,3H),7.33(s,1H),3.98(s,3H).
2、(Z)-((7-(5-(2-氰基-2-苯基乙烯基)-1-甲基-1H-吡唑-4-基)-4-氧代-3,4-二氢酞嗪-1-基)甲基)氨基甲酸叔丁酯(4-1)的合成
向25mL三颈瓶中依次加入中间体2-1(120mg,0.416mmol)、(4-氧代-7-(4,4,5,5-四甲基-1,3,2-二氧硼杂环戊烷-2-基)-3,4-二氢二氮杂萘-1-基)甲基)氨基甲酸叔丁酯3-1(200mg,0.499mmol)、SPhospdG2(34.0mg,0.0500mmol)、NaHCO3(70.0mg,0.832mmol)、5mL叔戊醇和1mL水，搅拌溶解，反应体系在N2保护、80℃下反应6h，TLC监测(V二氯甲烷:V甲醇＝20:1)反应完全，反应液减压浓缩，残余物加5mL水稀释，用乙酸乙酯(5mL×3)萃取，合并有机相，有机相用5mL饱和NaCl洗涤，无水Na2SO4干燥，抽滤，滤液减压浓缩，经柱层析纯化(洗脱剂：V石油醚:V乙酸乙酯＝1:1)得白色固体64.0mg，收率31.9％。
1H NMR(300MHz,DMSO-d6)δ(ppm):12.55(s,1H),8.25-8.22(m,2H),8.14(s,1H),8.03(s,1H),7.95-7.88(m,1H).7.85–7.77(m,2H),7.61–7.49(m,3H),7.45(t,J＝5.3Hz,1H),4.39(d,J＝5.5Hz,2H),3.98(s,3H),1.38(s,9H).
3、(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢酞嗪-6-基)-1-甲基-1H-吡唑-5-基)-2-苯基丙烯腈(I-1)的合成
向25mL三颈瓶中依次加入中间体4-1(64.0mg,0.133mmol)和3mL盐酸二氧六环,反应体系在25℃下反应0.5h，TLC监测(V石油醚:V乙酸乙酯＝1:1)反应完全，抽滤，滤饼用2mL正己烷洗涤，真空干燥滤饼得白色固体50.0mg，收率98.9％。
1H NMR(300MHz,DMSO-d6)δ(ppm):12.89(s,1H),8.64(s,3H).8.27(s,1H),8.23(d,J＝8.3Hz,1H),8.18(s,1H),8.06(s,1H),7.94(d,J＝8.3Hz,1H),7.82–7.76(m,2H),7.59–7.48(m,3H),4.41-4.39(m,2H),3.97(s,3H).
13C NMR(75MHz,DMSO-d6)δ(ppm):159.86,139.03,134.93,132.53,131.12,130.95,130.32,129.91,128.87,127.47,126.70,125.58,121.97,121.71,119.55,116.57,66.77,38.11.
HRMS(ESI+):m/z calcd for C22H18N6O 383.1620[M+H]+；found:383.1624
实施例2：(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢酞嗪-6-基)-1-甲基-1H-吡唑-5-基)-2-(4-甲苯基)丙烯腈(I-2)的合成
1、(Z)-3-(4-溴-1-甲基-1H-吡唑-5-基)-2-(对甲苯基)丙烯腈(2-2)的合成
以2-(对甲苯基)乙腈1-2(139mg,1.06mmol)和4-溴-1-甲基-1H-吡唑-5-甲醛(200mg,1.06mmol)为原料，操作同2-1，得白色固体120mg，收率：38.5％。
1H NMR(400MHz,Chloroform-d)δ(ppm):7.63(d,J＝8.2Hz,1H),7.58(s,1H),7.32(d,J＝8.0Hz,2H),7.28(s,1H),3.97(s,3H),2.45(s,3H).
2、(Z)-((7-(5-(2-氰基-2-(对甲苯基)乙烯基)-1-甲基-1H-吡唑-4-基)-4-氧代-3,4-二氢酞嗪-1-基)甲基)氨基甲酸叔丁酯(4-2)的合成
以中间体2-2(120mg,0.400mmol)和3-1(191mg,0.480mmol)为原料，操作同4-1，得白色固体60.0mg，收率：30.4％。
1H NMR(300MHz,DMSO-d6)δ(ppm):12.54(s,1H),8.20(d,J＝8.3Hz,1H),8.14(s,1H),8.11(s,1H),8.01(s,1H),7.91-7.84(m,1H),7.69(d,J＝8.1Hz,2H),7.40(t,J＝5.7Hz,2H),7.34(d,J＝8.1Hz,2H),4.36(d,J＝5.1Hz,2H),3.95(s,3H),2.36(s,3H),1.36(s,9H).
(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢酞嗪-6-基)-1-甲基-1H-吡唑-5-基)-2-(对甲苯基)丙烯腈(I-2)的合成
以中间体4-2(60.0mg,0.120mmol)为原料，操作同I-1得白色固体45.0mg，收率：93.9％。
1H NMR(300MHz,DMSO-d6)δ(ppm):12.89(s,1H),8.70(s,3H),8.27-8.15(m,3H),8.10-8.00(m,1H),7.96-7.84(m,1H),7.69(d,J＝7.9Hz,2H),7.34(d,J＝7.9Hz,2H),4.51–4.24(m,2H),3.95(s,3H),2.35(s,3H).
13C NMR(75MHz,DMSO-d6)δ(ppm):159.87,141.04,139.43,139.36,139.26,135.14,131.10,130.56,130.09,129.57,129.07,127.56,126.83,125.78,122.22,119.60,116.83,66.96,38.34,21.50.
HRMS(ESI+):m/z calcd for C23H20N6O 397.1777[M+H]+；found:397.1772.
实施例3：(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢酞嗪-6-基)-1-甲基-1H-吡唑-5-基)-2-(萘-2-基)丙烯腈(I-3)的合成
1、(Z)-3-(4-溴-1-甲基-1H-吡唑-5-基)-2-(萘-2-基)丙烯腈(2-3)的合成
向25ml三颈瓶中依次加入2-(萘-2-基)乙腈1-3(177mg,1.06mmol)、4-溴-1-甲基-1H-吡唑-5-甲醛(200mg,1.06mmol)、KOH(120mg,2.12mmol)和5mL EtOH，搅拌溶解，反应体系在25℃下反应12h，TLC监测(V石油醚:V乙酸乙酯＝10:1)反应完全，减压浓缩，残余物经柱层析纯化(洗脱剂：V石油醚:V乙酸乙酯＝10:1)得白色固体120mg，收率33.5％。
1H NMR(300MHz,Chloroform-d)δ(ppm):8.23(s,1H),7.94(d,J＝8.7Hz,2H),7.92-7.88(m,1H),7.78-7.75(m,1H),7.62–7.55(m,3H),7.44(s,1H),3.99(s,3H).
2、(Z)-((7-(5-(2-氰基-2-(萘-2-基)乙烯基)-1-甲基-1H-吡唑-4-基)-4-氧代-3,4-二氢酞嗪-1-基)甲基)氨基甲酸叔丁酯(4-3)的合成
向25mL三颈瓶中依次加入2-3(120mg,0.355mmol)、中间体3-1(171mg,0.426mmol)、SPhospdG2(26.0mg,0.0360mmol)、NaHCO3(60.0mg,0.710mmol)、10mL叔戊醇和2mL水，搅拌溶解，反应体系在N2保护、80℃下反应12h，TLC监测(V石油醚:V乙酸乙酯＝1:1)反应完全，反应液减压浓缩，残余物加10mL水稀释，用乙酸乙酯(10mL×3)萃取，合并有机相，有机相用10ml饱和NaCl洗涤，无水Na2SO4干燥，抽滤，滤液减压浓缩，柱层析纯化(洗脱剂：V石油醚:V乙酸乙酯＝1:1)得白色固体67.0mg，收率35.4％。
1H NMR(300MHz,DMSO-d6)δ(ppm):12.54(s,1H),8.36(s,1H),8.25(s,1H),8.21(d,J＝8.3Hz,1H),8.15(s,1H),8.12–7.97(m,5H),7.96-7.90(m,1H),7.63–7.58(m,2H),7.39(t,J＝6.0Hz,1H),4.36(d,J＝5.7Hz,2H),4.00(s,3H),1.35(s,9H).
3、(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢酞嗪-6-基)-1-甲基-1H-吡唑-5-基)-2-(萘-2-基)丙烯腈(I-3)的合成
以中间体4-3(67.0mg,0.130mmol)为原料，操作同I-1得白色固体48.0mg，收率：88.2％。
1H NMR(300MHz,DMSO-d6)δ(ppm):12.89(s,1H),8.64(s,3H),8.27(s,1H),8.23(d,J＝8.3Hz,1H),8.18(s,1H),8.06(s,1H),7.97-7.91(m,1H),7.83–7.76(m,2H),7.60–7.47(m,3H),4.44-4.29(m,2H),4.00(s,3H).
13C NMR(75MHz,DMSO-d6)δ(ppm):159.80,139.29,139.24,139.07,134.98,134.91,132.61,131.11,130.93,130.43,130.17,129.91,129.69,129.08,128.90,127.48,126.73,125.64,123.23,122.03,121.71,119.49,116.59,66.79,38.15.
HRMS(ESI+):m/z calcd for C26H20N6O 433.1777[M+H]+；found:433.1772.
实施例4：(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢酞嗪-6-基)-1-甲基-1H-吡唑-5-基)-2-(苯并[d][1,3]二氧杂环戊烯-5-基)丙烯腈(I-4)的合成
1、(Z)-2-(苯并[d][1,3]二氧杂环戊烯-5-基)-3-(4-溴-1-甲基-1H-吡唑-5-基)丙烯腈(2-4)的合成
向25mL三颈瓶中依次加入2-(苯并[d][1，3]二氧杂环戊二烯-5-基)乙腈1-4(172mg,1.06mmol)、4-溴-1-甲基-1H-吡唑-5-甲醛(200mg,1.06mmol)、KOH(120mg,2.12mmol)和5mL EtOH，搅拌溶解，反应体系在25℃下反应12h，TLC监测(V石油醚:V乙酸乙酯＝10:1)反应完全，减压浓缩，残余物经柱层析纯化(洗脱剂：V石油醚:V乙酸乙酯＝10:1)得白色固体120mg，收率35.5％。
1H NMR(300MHz,Chloroform-d)δ(ppm):7.57(s,1H),7.27(d,J＝1.9Hz,1H),7.18(d,J＝1.9Hz,1H),7.16(s,1H),6.93(d,J＝8.2Hz,1H),6.09(s,2H),3.97(s,3H).
2、(Z)-((7-(5-(2-(苯并[d][1,3]二氧杂环戊烯-5-基)-2-氰基乙烯基)-1-甲基-1H-吡唑-4-基)-4-氧代-3,4-二氢酞嗪-1-基)甲基)氨基甲酸叔丁酯(4-4)的合成
向25mL三颈瓶中依次加入2-4(120mg,0.360mmol)、中间体3-1(174mg,0.430mmol)、SPhospdG2(26.0mg,0.0400mmol)、NaHCO3(61.0mg,0.720mmol)、5mL叔戊醇和1mL水，搅拌溶解，反应体系在N2保护、80℃下反应12h，TLC监测(V石油醚:V乙酸乙酯＝1:1)反应完全，反应液减压浓缩，残余物加5mL水稀释，用乙酸乙酯(5mL×3)萃取，合并有机相，有机相用5mL饱和NaCl洗涤，无水Na2SO4干燥，抽滤，滤液减压浓缩，柱层析纯化(洗脱剂：V石油醚:V乙酸乙酯＝1:1)得白色固体63.0mg，收率33.2％。
1H NMR(300MHz,DMSO-d6)δ(ppm):12.53(s,1H),8.20(d,J＝8.3Hz,1H),8.10(s,1H),8.06(s,1H),7.99(s,1H),7.88(d,J＝8.3Hz,1H),7.55(s,1H),7.39(t,J＝5.4Hz,1H),7.17(d,J＝8.2Hz,1H),7.04(d,J＝8.1Hz,1H),6.13(s,2H),4.36(d,J＝5.8Hz,2H),3.93(s,3H),1.36(s,9H).
3、(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢酞嗪-6-基)-1-甲基-1H-吡唑-5-基)-2-(苯并[d][1,3]二氧杂环戊烯-5-基)丙烯腈(I-4)的合成
以中间体4-4(63.0mg,0.120mmol)为原料，操作同I-1得白色固体50.0mg，收率：98.0％。
1H NMR(300MHz,DMSO-d6)δ(ppm):12.90(s,1H),8.68(s,3H),8.22(d,J＝8.3Hz,1H),8.18(s,1H),8.15(s,1H),8.04(s,1H),7.92(d,J＝7.8Hz,1H),7.56(d,J＝2.0Hz,1H,),7.17(dd,J＝8.2,1.9Hz,1H),7.05(d,J＝8.1Hz,1H),6.14(s,2H),4.46-4.28(m,2H),3.95(s,3H).
13C NMR(75MHz,DMSO-d6)δ(ppm):159.84,149.79,148.98,148.50,139.38,139.19,135.08,131.06,129.01,128.52,127.52,126.98,125.72,122.13,121.60,119.08,116.76,109.30,108.82,106.33,102.60,66.92,38.24.
HRMS(ESI+):m/z calcd for C23H18N6O3427.1519[M+H]+；found:427.1508.
实施例5：(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢酞嗪-6-基)-1-甲基-1H-吡唑-5-基)-2-(4-甲氧基苯基)丙烯腈(I-5)的合成
1、(Z)-3-(4-溴-1-甲基-1H-吡唑-5-基)-2-(4-甲氧基苯基)丙烯腈(2-5)的合成
以2-(4-甲氧基苯基)乙腈1-5(156mg,1.06mmol)和4-溴-1-甲基-1H-吡唑-5-甲醛(200mg,1.06mmol)为原料，操作同2-1，得白色固体115mg，收率：32.5％。
1H NMR(300MHz,Chloroform-d)δ(ppm):7.73(d,J＝8.8Hz,2H),7.62(s,1H),7.24(s,1H),7.07(d,J＝8.8Hz,2H),4.02(s,3H),3.95(s,3H).
2、(Z)-((7-(5-(2-氰基-2-(4-甲氧基苯基)乙烯基)-1-甲基-1H-吡唑-4-基)-4-氧代-3,4-二氢酞嗪-1-基)甲基)氨基甲酸叔丁酯(4-5)的合成
以中间体2-5(115mg,0.360mmol)和中间体3-1(174mg,0.430mmol)为原料，操作同4-1，得白色固体90.0mg，收率：48.6％。
1H NMR(300MHz,DMSO-d6)δ(ppm):12.55(s,1H),8.23(d,J＝8.3Hz,1H),8.13(s,1H),8.08(s,1H),8.05(s,1H),7.91(dd,J＝8.3,1.6Hz,1H),7.80–7.74(m,2H),7.41(t,J＝6.0Hz,1H),7.15–7.09(m,2H),4.39(d,J＝5.5Hz,2H),3.97(s,3H),3.85(s,3H),1.39(s,9H).
3、(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢酞嗪-6-基)-1-甲基-1H-吡唑-5-基)-2-(4-甲氧基苯基)丙烯腈(I-5)的合成
以中间体4-5(90.0mg,0.180mmol)为原料，操作同I-1得白色固体70.0mg，收率：96.6％。
1H NMR(300MHz,DMSO-d6)δ(ppm):12.90(s,1H),8.65(s,3H).8.23(d,J＝8.3Hz,1H),8.17(s,1H),8.13(s,1H),8.04(s,1H),7.97-7.91(m,1H),7.74(d,J＝8.5Hz,2H),7.11(d,J＝8.8Hz,2H),4.46-4.32(m,2H),3.95(s,3H),3.82(s,3H).
13C NMR(75MHz,DMSO-d6)δ(ppm):161.40,159.84,139.34,139.28,138.96,135.18,131.06,128.85,128.36,127.61,127.45,125.53,125.03,121.87,121.41,119.25,116.72,115.27,66.78,55.96,38.04.
HRMS(ESI+):m/z calcd for C23H20N6O2413.1726[M+H]+；found:413.1723.
实施例6：(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢酞嗪-6-基)-1-甲基-1H-吡唑-5-基)-2-(间甲苯基)丙烯腈(I-6)的合成
1、(Z)-3-(4-溴-1-甲基-1H-吡唑-5-基)-2-(间甲苯基)丙烯腈(2-6)的合成
以2-(间甲苯基)乙腈1-6(140mg,1.06mmol)和4-溴-1-甲基-1H-吡唑-5-甲醛(200mg,1.06mmol)为原料，操作同2-1，得白色固体106mg，收率：33.2％。
1H NMR(300MHz,Chloroform-d)δ(ppm):7.62(s,1H),7.57(m,2H),7.44(t,J＝7.9Hz,1H).7.36(m,2H),4.01(s,3H),2.50(s,3H).
2、(Z)-((7-(5-(2-氰基-2-(间甲苯基)乙烯基)-1-甲基-1H-吡唑-4-基)-4-氧代-3,4-二氢酞嗪-1-基)甲基)氨基甲酸叔丁酯(4-6)的合成
以中间体2-6(106mg,0.350mmol)和中间体3-1(169mg,0.420mmol)为原料，操作同4-1，得白色固体61.0mg，收率：35.5％。
1H NMR(300MHz,DMSO-d6)δ(ppm):12.67(s,1H),8.35(d,J＝8.6Hz,1H),8.32(s,1H),8.25(s,1H),8.15(s,1H),8.07-7.98(m,1H),7.77(s,1H),7.73(d,J＝8.1Hz,1H),7.63–7.40(m,3H),4.52(d,J＝6.0Hz,2H),4.10(s,3H),2.52(s,3H),1.50(s,9H)
3、(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢酞嗪-6-基)-1-甲基-1H-吡唑-5-基)-2-(间甲苯基)丙烯腈(I-6)的合成
以中间体4-6(61.0mg,0.120mmol)为原料，操作同I-1得白色固体45.0mg，收率：92.4％。
1H NMR(300MHz,DMSO-d6)δ(ppm):12.89(s,1H),8.73(s,3H),8.26(s,1H),8.22(d,J＝8.3Hz,1H),8.19(s,1H),8.06(s,1H),7.96-7.88(m,1H),7.62(s,1H),7.59(d,J＝9.2Hz,1H),7.47-7.37(m,1H),7.35-7.27(m,1H),4.46-4.30(m,2H),3.96(s,3H),2.37(s,3H).
13C NMR(75MHz,DMSO-d6)δ(ppm):159.80,139.39,139.28,139.07,134.94,132.55,131.60,131.06,130.18,129.80,128.91,127.45,127.07,125.62,123.93,122.05,121.70,119.57,116.64,66.79,38.14,27.22,21.38.
HRMS(ESI+):m/z calcd for C23H20N6O 397.1777[M+H]+；found:397.1770.
实施例7：(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢酞嗪-6-基)-1-甲基-1H-吡唑-5-基)-2-(邻甲苯基)丙烯腈(I-7)的合成
1、(Z)-3-(4-溴-1-甲基-1H-吡唑-5-基)-2-(邻甲苯基)丙烯腈(2-7)的合成
以2-(邻甲苯基)乙腈1-7(140mg,1.06mmol)和4-溴-1-甲基-1H-吡唑-5-甲醛(200mg,1.06mmol)为原料，操作同2-1，得白色固体224mg，收率：70.1％。
1H NMR(300MHz,Chloroform-d)δ(ppm):7.58(s,1H),7.43–7.35(m,2H),7.34–7.29(m,2H),6.98(s,1H),3.99(s,3H),2.55(s,3H).
2、(Z)-((7-(5-(2-氰基-2-(邻甲苯基)乙烯基)-1-甲基-1H-吡唑-4-基)-4-氧代-3,4-二氢酞嗪-1-基)甲基)氨基甲酸叔丁酯(4-7)的合成
以中间体2-7(224mg,0.740mmol)和中间体3-1(357mg,0.890mmol)为原料，操作同4-1，得白色固体92.0mg，收率：25.5％。
1H NMR(300MHz,DMSO-d6)δ(ppm):12.58(s,1H),8.27(d,J＝8.3Hz,1H),8.13(s,1H),7.98(s,1H),7.96(d,J＝8.7Hz,1H),7.79(s,1H),7.58(d,J＝7.2Hz,1H),7.48–7.30(m,4H),4.45(d,J＝5.9Hz,2H),3.98(s,3H),2.36(s,3H),1.35(s,9H).
3、(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢酞嗪-6-基)-1-甲基-1H-吡唑-5-基)-2-(邻甲苯基)丙烯腈(I-7)的合成
以中间体4-7(92.0mg,0.230mmol)为原料，操作同I-1得白色固体75.0mg，收率：82.6％。
1H NMR(300MHz,DMSO-d6)δ(ppm):12.93(s,1H),8.73(s,3H),8.30(d,J＝8.1Hz,1H),8.17(s,1H),8.09(s,1H),8.01(d,J＝8.4Hz,1H),7.83(s,1H),7.61-7.49(m,1H),7.42–7.30(m,3H),4.54-4.36(m,2H),3.99(s,3H),2.37(s,3H).
13C NMR(75MHz,DMSO-d6)δ(ppm):160.05,139.56,139.51,136.43,135.88,134.72,133.89,131.75,131.65,130.75,130.25,129.24,127.74,127.68,126.04,122.51,122.09,116.94,67.09,20.39.
HRMS(ESI+):m/z calcd for C23H20N6O 397.1777[M+H]+；found:397.1768.
实施例8：(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢二氮杂萘-6-基)-1-甲基-1H-吡唑-5-基)-2-(4-羟基苯基)丙烯腈(I-8)的合成
1、(Z)-3-(4-溴-1-甲基-1H-吡唑-5-基)-2-(4-羟基苯基)丙烯腈(2-8)的合成
以中间体2-(4-羟基苯基)乙腈1-8(142mg,1.06mmol)和4-溴-1-甲基-1H-吡唑-5-甲醛(200mg,1.06mmol)为原料，操作同2-1，得白色固体200mg，收率：62.2％。
1H NMR(300MHz,DMSO-d6)δ(ppm):10.16(s,1H),7.73(s,1H),7.70(s,1H),7.64(d,J＝8.6Hz,2H),6.91(d,J＝8.6Hz,2H),3.87(s,3H).
2、(Z)-((7-(5-(2-氰基-2-(4-羟基苯基)乙烯基)-1-甲基-1H-吡唑-4-基)-4-氧代-3,4-二氢酞嗪-1-基)甲基)氨基甲酸叔丁酯(4-8)的合成
以中间体2-8(200mg,0.660mmol)和中间体3-1(317mg,0.790mmol)为原料，操作同4-1，得白色固体79.0mg，收率：24.5％。
1H NMR(300MHz,DMSO-d6)δ(ppm):12.54(s,1H),10.13(s,1H),8.20(d,J＝8.3Hz,1H),8.11(s,1H),7.99(s,1H),7.97(s,1H),7.87(d,J＝8.4Hz,1H),7.63(d,J＝8.4Hz,2H),7.42(t,J＝5.9Hz,1H),6.89(d,J＝8.6Hz,2H),4.37(d,J＝6.0Hz,2H),3.92(s,3H),1.36(s,9H).
3、(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢二氮杂萘-6-基)-1-甲基-1H-吡唑-5-基)-2-(4-羟基苯基)丙烯腈(I-8)的合成
以中间体4-8(79.0mg,0.160mmol)为原料，操作同I-1得白色固体55.0mg，收率：87.4％。
1H NMR(300MHz,DMSO-d6)δ(ppm):12.90(s,1H),10.27(s,1H),8.55(s,3H),8.24(d,J＝8.2Hz,1H),8.15(s,1H),8.07-7.99(m,2H),7.94(d,J＝8.2Hz,1H),7.62(d,J＝8.4Hz,2H),6.93(d,J＝8.3Hz,2H),4.47-4.35(m,2H),3.94(s,3H).
HRMS(ESI+):m/z calcd for C22H18N6O2399.1569[M+H]+；found:399.1571.
实施例9：(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢二氮杂萘-6-基)-1-甲基-1H-吡唑-5-基)-2-(4-氟苯基)丙烯腈(I-9)的合成
1、(Z)-3-(4-溴-1-甲基-1H-吡唑-5-基)-2-(4-羟基苯基)丙烯腈(2-9)的合成
以中间体2-(4-氟苯基)乙腈1-9(142mg,1.06mmol)和4-溴-1-甲基-1H-吡唑-5-甲醛(200mg,1.06mmol)为原料，操作同2-1，得白色固体230mg，收率：71.1％。
1H NMR(300MHz,Chloroform-d)δ(ppm):7.74–7.65(m,2H),7.56(s,1H),7.24(s,1H),7.19(t,J＝8.5Hz,2H),3.95(s,3H).
2、(Z)-((7-(5-(2-氰基-2-(4-氟苯基)乙烯基)-1-甲基-1H-吡唑-4-基)-4-氧代-3,4-二氢酞嗪-1-基)甲基)氨基甲酸叔丁酯(4-9)的合成
以中间体2-9(230mg,0.750mmol)和中间体3-1(362mg,0.900mmol)为原料，操作同4-1，得白色固体90.0mg，收率：24.5％。
1H NMR(300MHz,DMSO-d6)δ(ppm):12.55(s,1H),8.21(d,J＝8.9Hz,1H),8.18(s,1H),8.10(s,1H),8.01(s,1H),7.94–7.81(m,3H),7.49–7.29(m,3H),4.35(d,J＝6.0Hz),3.96(s,3H),1.35(s,9H).
3、(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢二氮杂萘-6-基)-1-甲基-1H-吡唑-5-基)-2-(4-氟苯基)丙烯腈(I-9)的合成
以中间体4-9(90.0mg,0.180mmol)为原料，操作同I-1得白色固体70.0mg，收率：97.1％。
1H NMR(300MHz,DMSO-d6)δ(ppm):12.94(s,1H),8.71(s,3H),8.31(s,1H),8.29(d,J＝10.1Hz,1H),8.23(s,1H),8.10(s,1H),8.01(d,J＝8.3Hz,1H),7.96–7.87(m,2H),7.51-7.39(m,2H),4.66-4.30(m,2H),4.02(s,3H).
13C NMR(75MHz,DMSO-d6)δ(ppm):159.84,139.32,139.23,139.10,134.88,131.16,130.53,129.34,129.30,129.23,128.93,127.55,125.68,122.02,121.72,118.42,117.10,116.80,116.57,66.85,38.22.
HRMS(ESI+):m/z calcd for C22H17FN6O 401.1526[M+H]+；found:401.1521.
实施例10：(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢二氮杂萘-6-基)-1-甲基-1H-吡唑-5-基)-2-(3,5-二甲基苯基)丙烯腈(I-10)的合成
1、(Z)-3-(4-溴-1-甲基-1H-吡唑-5-基)-2-(3,5-二甲基苯基)丙烯腈(2-10)的合成
以中间体2-(3，5-二甲基苯基)乙腈1-10(153mg,1.06mmol)和4-溴-1-甲基-1H-吡唑-5-甲醛(200mg,1.06mmol)为原料，操作同2-1，得白色固体250mg，收率：74.7％。
1H NMR(300MHz,Chloroform-d)δ(ppm):7.55(s,1H),7.32(s,2H),7.27(s,1H),7.12(s,1H),3.95(s,3H),2.39(s,6H).
2、(Z)-((7-(5-(2-氰基-2-(3,5-二甲基苯基)乙烯基)-1-甲基-1H-吡唑-4-基)-4-氧代-3,4-二氢酞嗪-1-基)甲基)氨基甲酸叔丁酯(4-10)的合成
以中间体2-10(250mg,0.790mmol)和中间体3-1(381mg,0.950mmol)为原料，操作同4-1，得白色固体96.0mg，收率：23.8％。
1H NMR(300MHz,DMSO-d6)δ(ppm):12.55(s,1H),8.21(d,J＝8.3Hz,1H),8.17(s,1H),8.10(s,1H),8.01(s,1H),7.90-7.85(m,1H),7.45-7.36(m,3H),7.15(s,1H),4.38(d,J＝6.1Hz,2H),3.95(s,3H),2.34(s,6H),1.37(s,9H).
3、(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢二氮杂萘-6-基)-1-甲基-1H-吡唑-5-基)-2-(3,5-二甲基苯基)丙烯腈(I-10)的合成
以中间体4-10(96.0mg,0.190mmol)为原料，操作同I-1得白色固体70.0mg，收率：90.7％。
1H NMR(300MHz,DMSO-d6)δ(ppm):13.07(s,1H),8.93(s,3H),8.45-8.33(m,3H),8.23(s,1H),8.06(d,J＝8.2Hz,1H),7.61-7.53(m,2H),7.28(s,1H),4.63-4.52(m,2H),4.12(s,3H),2.48(s,6H).
13C NMR(75MHz,DMSO-d6)δ(ppm):159.86,139.31,139.27,139.04,134.97,132.42,132.39,131.06,129.76,127.42,124.28,121.99,121.68,119.67,116.67,66.78,40.04,21.25.
HRMS(ESI+):m/z calcd for C24H22N6O 411.1933[M+H]+；found:411.1923.
实施例11：(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢二氮杂萘-6-基)-1-甲基-1H-吡唑-5-基)-2-(2-甲氧基苯基)丙烯腈(I-11)的合成
1、(Z)-3-(4-溴-1-甲基-1H-吡唑-5-基)-2-(2-甲氧基苯基)丙烯腈(2-11)的合成
以中间体2-(2-甲氧基苯基)乙腈1-11(156mg,1.06mmol)和4-溴-1-甲基-1H-吡唑-5-甲醛(200mg,1.06mmol)为原料，操作同2-1，得白色固体209mg，收率：60.9％。
1H NMR(300MHz,Chloroform-d)δ(ppm):7.59(dd,J＝7.7,1.6Hz,1H),7.57(s,1H),7.52(s,1H),7.46(td,J＝8.3,1.7Hz,1H),7.09(td,J＝7.6,0.9Hz,1H),7.04(d,J＝8.4Hz,1H),3.98(s,3H),3.97(s,3H).
2、(Z)-((7-(5-(2-氰基-2-(2-甲氧基苯基)乙烯基)-1-甲基-1H-吡唑-4-基)-4-氧代-3,4-二氢酞嗪-1-基)甲基)氨基甲酸叔丁酯(4-11)的合成
以中间体2-11(209mg,0.660mmol)和中间体3-1(316mg,0.790mmol)为原料，操作同4-1，得白色固体87.0mg，收率：25.8％。
1H NMR(300MHz,DMSO-d6)δ(ppm):12.55(s,1H),8.24(d,J＝8.3Hz,1H),8.12(s,1H),8.01(s,1H),7.98-7.93(m,1H),7.92(s,1H),7.63(d,J＝7.6Hz,1H),7.55-7.46(m,1H),7.41(t,J＝5.9Hz,1H),7.20-7.06(m,2H),4.41(d,J＝6.0Hz,2H),3.94(s,3H),3.82(s,3H),1.35(s,9H).
3、(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢二氮杂萘-6-基)-1-甲基-1H-吡唑-5-基)-2-(2-甲氧基苯基)丙烯腈(I-11)的合成
以中间体4-11(87.0mg,0.170mmol)为原料，操作同I-1得白色固体67.0mg，收率：95.6％。
1H NMR(300MHz,DMSO-d6)δ(ppm):12.96(s,1H),8.84(s,3H),8.40(s,1H),8.27(d,J＝8.9Hz,1H),8.26(s,1H),8.14(s,1H),7.97(d,J＝8.0Hz,1H),7.54-7.41(m,2H),7.45(s,1H),7.36(d,J＝7.4Hz,1H),7.13(d,J＝7.7Hz,1H),4.54-4.35(m,2H),4.02(s,3H),3.89(s,3H).
13C NMR(75MHz,DMSO-d6)δ(ppm):160.26,159.82,139.34,139.25,139.12,134.89,133.96,131.09,130.80,128.94,127.45,125.65,122.08,121.78,119.27,119.21,116.67,116.61,111.84,66.81,56.01,38.21.
HRMS(ESI+):m/z calcd for C23H20N6O2413.1726[M+H]+；found:413.1719.
实施例12：(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢二氮杂萘-6-基)-1-甲基-1H-吡唑-5-基)-2-(3-甲氧基苯基)丙烯腈(I-12)的合成
1、(Z)-3-(4-溴-1-甲基-1H-吡唑-5-基)-2-(3-甲氧基苯基)丙烯腈(2-12)的合成
以中间体2-(3-甲氧基苯基)乙腈1-12(156mg,1.06mmol)和4-溴-1-甲基-1H-吡唑-5-甲醛(200mg,1.06mmol)为原料，操作同2-1，得白色固体261mg，收率：77.5％。
1H NMR(300MHz,Chloroform-d)δ(ppm):7.56(s,1H),7.41(t,J＝8.0Hz,1H),7.32-7.27(m,1H),7.21(t,J＝2.2Hz,1H),7.02(dd,J＝8.2,1.9Hz,2H),3.96(s,3H),3.88(s,3H).
2、(Z)-((7-(5-(2-氰基-2-(3-甲氧基苯基)乙烯基)-1-甲基-1H-吡唑-4-基)-4-氧代-3,4-二氢酞嗪-1-基)甲基)氨基甲酸叔丁酯(4-12)的合成
以中间体2-12(261mg,0.820mmol)和中间体3-1(395mg,0.980mmol)为原料，操作同4-1，得白色固体92.0mg，收率：21.9％。
1H NMR(300MHz,DMSO-d6)δ(ppm):12.54(s,1H),8.21(d,J＝7.5Hz,2H),8.11(s,1H),8.01(s,1H),7.93-7.87(m,1H),7.50–7.37(m,3H),7.30(d,J＝7.7Hz,1H),7.13-7.05(m,1H),4.36(d,J＝5.9Hz,2H),3.96(s,3H),3.84(s,3H),1.35(s,9H).
3、(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢二氮杂萘-6-基)-1-甲基-1H-吡唑-5-基)-2-(3-甲氧基苯基)丙烯腈(I-12)的合成
以中间体4-12(90.0mg,0.180mmol)为原料，操作同I-1得白色固体70.0mg，收率：94.8％。
1H NMR(300MHz,DMSO-d6)δ(ppm):12.93(s,1H),8.69(s,3H),8.30(d,J＝8.2Hz,1H),8.19(s,1H),8.07(s,1H),8.03(d,J＝8.3Hz,2H),7.97(s,1H),7.62(d,J＝6.5Hz,1H),7.56-7.46(m,1H),7.22–7.10(m,2H),4.50-4.37(m,2H),3.97(s,3H),3.86(s,3H).
13C NMR(75MHz,DMSO-d6)δ(ppm):159.85,157.13,139.28,139.22,138.91,134.84,134.00,132.37,131.19,130.35,128.81,127.40,125.60,122.38,121.92,121.73,121.56,117.46,116.58,112.65,66.79,56.39,38.04.
HRMS(ESI+):m/z calcd for C23H20N6O2413.1726[M+H]+；found:413.1719.
实施例13：(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢二氮杂萘-6-基)-1-甲基-1H-吡唑-5-基)-2-(4-乙氧基苯基)丙烯腈(I-13)的合成
1、(Z)-3-(4-溴-1-甲基-1H-吡唑-5-基)-2-(4-乙氧基苯基)丙烯腈(2-13)的合成
以中间体2-(4-乙氧基苯基)乙腈1-13(171mg,1.06mmol)和4-溴-1-甲基-1H-吡唑-5-甲醛(200mg,1.06mmol)为原料，操作同2-1，得白色固体330mg，收率：94.6％。
1H NMR(300MHz,Chloroform-d)δ(ppm):7.69–7.62(m,2H),7.57(s,1H),7.18(s,1H),7.03–6.96(m,2H),4.12(q,J＝7.0Hz,2H),3.97(s,3H),1.48(t,J＝7.0Hz,3H).
2、(Z)-((7-(5-(2-氰基-2-(4-乙氧基苯基)乙烯基)-1-甲基-1H-吡唑-4-基)-4-氧代-3,4-二氢酞嗪-1-基)甲基)氨基甲酸叔丁酯(4-13)的合成
以中间体2-13(330mg,0.990mmol)和中间体3-1(478mg,1.19mmol)为原料，操作同4-1，得白色固体146mg，收率：28.5％。
1H NMR(400MHz,DMSO-d6)δ(ppm):12.54(s,1H),8.21(d,J＝8.3Hz,1H),8.11(s,1H),8.05(s,1H),8.01(s,1H),7.92-7.85(m,1H),7.73(d,J＝8.8Hz,2H),7.40(t,J＝6.0Hz,1H),7.08(d,J＝8.9Hz,2H),4.37(d,J＝6.0Hz,2H),4.10(q,J＝7.0Hz,2H),3.94(s,3H),1.37(s,9H),1.36-1.33(m,3H).
3、(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢二氮杂萘-6-基)-1-甲基-1H-吡唑-5-基)-2-(4-乙氧基苯基)丙烯腈(I-13)的合成
以中间体4-13(146mg,0.280mmol)为原料，操作同I-1得白色固体110mg，收率：93.5％。
1H NMR(300MHz,DMSO-d6)δ(ppm):12.92(s,1H),8.69(s,3H),8.26(d,J＝8.3Hz,1H),8.20(s,1H),8.15(s,1H),8.07(s,1H),7.97(d,J＝8.2Hz,1H),7.76(d,J＝8.4Hz,2H),7.12(d,J＝8.4Hz,2H),4.46-4.38(m,2H),4.13(q,J＝6.9Hz,2H),3.98(s,3H),1.37(t,J＝6.9Hz,3H).
13C NMR(75MHz,DMSO-d6)δ(ppm):160.69,139.39,138.92,135.23,131.13,129.60,128.84,128.35,127.47,127.38,125.48,124.82,121.83,121.42,119.34,116.70,115.69,114.85,66.77,64.06,37.99,14.90.
HRMS(ESI+):m/z calcd for C24H22N6O2427.1882[M+H]+；found:427.1877.
实施例14：(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢二氮杂萘-6-基)-1-甲基-1H-吡唑-5-基)-2-(吡啶-3-基)丙烯腈(I-14)的合成
1、(Z)-3-(4-溴-1-甲基-1H-吡唑-5-基)-2-(吡啶-3-基)丙烯腈(2-14)的合成
向25mL三颈瓶中依次加入2-(吡啶-3-基)乙腈1-14(125mg,1.06mmol)、4-溴-1-甲基-1H-吡唑-5-甲醛(200mg,1.06mmol)、哌啶(180mg,2.12mmol)和5mL EtOH，搅拌溶解，反应体系在25℃下反应12h，TLC监测(V石油醚:V乙酸乙酯＝3:1)反应完全，减压浓缩，残余物经柱层析纯化(洗脱剂：V石油醚:V乙酸乙酯＝3:1)得白色固体250mg，收率81.7％。
1H NMR(300MHz,Chloroform-d)δ(ppm):8.99(d,J＝2.4H 1H),8.74(d,J＝4.0Hz,1H),8.06–8.02(m,1H),7.60(s,1H),7.51-7.45(m,1H),7.41(s,1H),4.00(s,3H).
2、(Z)-((7-(5-(2-氰基-2-(吡啶-3-基)乙烯基)-1-甲基-1H-吡唑-4-基)-4-氧代-3,4-二氢酞嗪-1-基)甲基)氨基甲酸叔丁酯(4-14)的合成
向25mL三颈瓶中依次加入2-14(250mg,0.870mmol)、中间体3-1(416mg,1.04mmol)、SPhospdG2(58.0mg,0.0900mmol)、NaHCO3(145mg,1.73mmol)、5mL叔戊醇和1mL水，搅拌溶解，反应体系在N2保护、80℃下反应12h，TLC监测(V石油醚:V乙酸乙酯＝1:1)反应完全，反应液减压浓缩，残余物加10mL水稀释，用乙酸乙酯(10mL×3)萃取，合并有机相，有机相用10mL饱和NaCl洗涤，无水Na2SO4干燥，抽滤，滤液减压浓缩，柱层析纯化(洗脱剂：V石油醚:V乙酸乙酯＝1:1)得白色固体90.0mg，收率21.5％。
1H NMR(300MHz,DMSO-d6)δ(ppm):12.55(s,1H),9.04(s,1H),8.78-8.64(m,1H),8.34(s,1H),8.24(d,J＝8.3Hz,1H),8.21-8.15(m,1H),8.13(s,1H),8.04(s,1H),7.98-7.91(m,1H),7.63-7.54(m,1H),7.41(t,J＝5.5Hz,1H),4.36(d,J＝5.9Hz,2H),4.00(s,3H),1.37(s,9H).
3、(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢二氮杂萘-6-基)-1-甲基-1H-吡唑-5-基)-2-(吡啶-3-基)丙烯腈(I-14)的合成
以中间体4-14(90.0mg,0190mmol)为原料，操作同I-1得白色固体60.0mg，收率：84.1％。
1H NMR(400MHz,DMSO-d6)δ(ppm):12.90(s,1H),9.36(s,3H),8.94(d,J＝5.2Hz,1H),8.72(d,J＝8.6Hz,2H),8.70-8.64(m,3H),8.24(d,J＝8.4Hz,1H),8.22(s,1H),8.11-8.05(m,1H),8.03(d,J＝8.3Hz,1H),4.44-4.26(m,2H),4.02(s,3H).
13C NMR(75MHz,DMSO-d6)δ(ppm):159.71,143.86,142.05,141.67,139.48,138.79,137.98,135.89,134.15,131.94,131.28,129.00,127.59,127.36,125.86,122.45,122.31,115.56,113.38,66.82,38.51.
HRMS(ESI+):m/z calcd for C21H17N7O 384.1573[M+H]+；found:384.1567.
实施例15：(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢二氮杂萘-6-基)-1-甲基-1H-吡唑-5-基)-2-(吡啶-2-基)丙烯腈(I-15)的合成
1、(Z)-3-(4-溴-1-甲基-1H-吡唑-5-基)-2-(吡啶-2-基)丙烯腈(2-15)的合成
向25mL三颈瓶中依次加入2-(吡啶基)乙腈1-15(125mg,1.06mmol)、4-溴-1-甲基-1H-吡唑-5-甲醛(200mg,1.06mmol)、哌啶(180mg,2.12mmol)和5mL EtOH，搅拌溶解，反应体系在25℃下反应12h，TLC监测(V石油醚:V乙酸乙酯＝3:1)反应完全，减压浓缩，残余物经柱层析纯化(洗脱剂：V石油醚:V乙酸乙酯＝3:1)得白色固体210mg，收率68.6％。
1H NMR(300MHz,Chloroform-d)δ(ppm):8.67(d,J＝4.4Hz,1H),8.31(s,1H),7.89-7.78(m,2H),7.58(s,1H),7.41-7.34(m,1H),3.98(s,3H).
2、(Z)-((7-(5-(2-氰基-2-(吡啶-2-基)乙烯基)-1-甲基-1H-吡唑-4-基)-4-氧代-3,4-二氢酞嗪-1-基)甲基)氨基甲酸叔丁酯(4-15)的合成
向25mL三颈瓶中依次加入2-15(210mg,0.730mmol)、中间体3-1(350mg,0.870mmol)、SPhospdG2(49.0mg,0.0700mmol)、NaHCO3(122mg,1.45mmol)、10mL叔戊醇和2mLH2O，搅拌溶解，反应体系在N2保护、80℃下反应12h，TLC监测(V石油醚:V乙酸乙酯＝1:1)反应完全，反应液减压浓缩，残余物加10mL水，用乙酸乙酯(10mL×3)萃取，合并有机相，有机相用10mL饱和NaCl洗涤，无水Na2SO4干燥，抽滤，滤液减压浓缩，柱层析纯化(洗脱剂：V石油醚:V乙酸乙酯＝1:1)得白色固体85.0mg，收率24.2％。
1H NMR(300MHz,DMSO-d6)δ(ppm):12.54(s,1H),8.69(d,J＝4.2Hz,1H),8.55(s,1H),8.21(d,J＝8.3Hz,1H),8.12(s,1H),8.04(s,1H),8.02–7.84(m,3H),7.54–7.49(m,1H),7.36(t,J＝5.9Hz,1H),4.37(d,J＝6.0Hz,2H),3.99(s,3H),1.36(s,9H).
3、(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢二氮杂萘-6-基)-1-甲基-1H-吡唑-5-基)-2-(吡啶-2-基)丙烯腈(I-15)的合成
以中间体4-15(85.0mg,0.180mmol)为原料，操作同I-1得白色固体65.0mg，收率：96.4％。
1H NMR(300MHz,DMSO-d6)δ(ppm):12.89(s,1H),8.70(s,3H),8.63(s,1H),8.23(d,J＝8.5Hz,1H),8.20(s,1H),8.04(s,1H),8.01(d,J＝9.9Hz,2H),7.98-7.88(m,2H).7.58-7.48(m,1H),4.45-4.30(m,2H),3.98(s,3H).
13C NMR(75MHz,DMSO-d6)δ(ppm):159.75,149.97,139.28,139.15,139.01,138.91,134.34,133.22,131.17,128.85,127.48,125.66,125.50,122.23,122.12,122.00,118.98,116.05,66.74,38.33.
HRMS(ESI+):m/z calcd for C21H17N7O 384.1573[M+H]+；found:384.1565.
实施例16：(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢二氮杂萘-6-基)-1-甲基-1H-吡唑-5-基)-2-(噻吩-3-基)丙烯腈(I-16)的合成
1、(Z)-3-(4-溴-1-甲基-1H-吡唑-5-基)-2-(噻吩-3-基)丙烯腈(2-16)的合成
向25mL三颈瓶中依次加入2-(噻吩-3-基)乙腈1-16(130mg,1.06mmol)、4-溴-1-甲基-1H-吡唑-5-甲醛(200mg,1.06mmol)、KOH(120mg,2.12mmol)和5mL EtOH，搅拌溶解，反应体系在25℃下反应12h，TLC监测(V石油醚:V乙酸乙酯＝10:1)反应完全，减压浓缩，残余物经柱层析纯化(洗脱剂：V石油醚:V乙酸乙酯＝10:1)得白色固体240mg，收率70.5％。
1H NMR(400MHz,Chloroform-d)δ(ppm):7.76-7.74(m,1H),7.57(s,1H),7.50-7.47(m,1H),7.43-7.40(m,1H),7.21(s,1H),3.97(s,3H).
2、(Z)-((7-(5-(2-氰基-2-(噻吩-3-基)乙烯基)-1-甲基-1H-吡唑-4-基)-4-氧代-3,4-二氢酞嗪-1-基)甲基)氨基甲酸叔丁酯(4-16)的合成
向25mL三颈瓶中依次加入2-16(240mg,0.816mmol)、中间体3-1(393mg,0.979mmol)、SPhospdG2(55.0mg,0.0820mmol)、NaHCO3(137mg,1.63mmol)、10mL叔戊醇和2mLH2O，搅拌溶解，反应体系在N2保护、80℃下反应12h，TLC监测(V石油醚:V乙酸乙酯＝1:1)反应完全，反应液减压浓缩，残余物加10mL水，用乙酸乙酯(10mL×3)萃取，合并有机相，有机相用10mL饱和NaCl洗涤，无水Na2SO4干燥，抽滤，滤液减压浓缩，柱层析纯化(洗脱剂：V石油醚:V乙酸乙酯＝1:1)得白色固体100mg，收率25.5％。
1H NMR(300MHz,DMSO-d6)δ(ppm):12.55(s,1H),8.21(d,J＝8.3Hz,1H),8.14-8.08(m,2H),8.01(s,1H),7.92–7.83(m,2H),7.80-7.74(m,1H),7.74-7.68(m,1H),7.42(t,J＝5.8Hz,1H),4.37(d,J＝6.0Hz,2H),3.95(s,3H),1.36(s,9H).
3、(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢二氮杂萘-6-基)-1-甲基-1H-吡唑-5-基)-2-(噻吩-3-基)丙烯腈(I-16)的合成
以中间体4-16(95.0mg,0.190mmol)为原料，操作同I-1得白色固体69.0mg，收率：91.4％。
1H NMR(300MHz,DMSO-d6)δ(ppm):12.90(s,1H),8.64(s,3H),8.24(d,J＝8.3Hz,1H),8.20(s,1H),8.18(s,1H),8.04(s,1H),7.96-7.86(m,2H),7.88(s,1H),7.83-7.72(m,2H),4.49-4.33(m,2H),3.96(s,3H).
13C NMR(75MHz,DMSO-d6)δ(ppm):159.81,139.29,139.24,139.09,135.06,134.73,131.06,129.52,128.90,128.39,127.48,126.80,125.61,125.47,122.02,121.54,114.62,66.80,38.12.
HRMS(ESI+):m/z calcd for C20H16N6OS 389.1185[M+H]+；found:389.1178.
实施例17：(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢二氮杂萘-6-基)-1-甲基-1H-吡唑-5-基)-2-(5-氟-2-甲基苯基)丙烯腈(I-17)的合成
1、(Z)-3-(4-溴-1-甲基-1H-吡唑-5-基)-2-(5-氟-2-甲基苯基)丙烯腈(2-17)的合成
以中间体2-(5-氟-2-甲基苯基)乙腈1-17(158mg,1.06mmol)和4-溴-1-甲基-1H-吡唑-5-甲醛(200mg,1.06mmol)为原料，操作同2-1，得白色固体210mg，收率：62.5％。
1HNMR(400MHz,Chloroform-d)δ(ppm):7.59(s,1H),7.28–7.20(m,1H),7.13–7.07(m,2H),7.00(s,1H),4.00(s,3H),2.51(s,3H).
2、(Z)-(7-(5-(2-氰基-2-(5-氟-2-甲基苯基)乙烯基)-1-甲基-1H-吡唑-4-基)-4-氧代-3,4-二氢二氮杂萘-1-基)甲基)氨基甲酸叔丁酯(4-17)的合成
以中间体2-17(210mg,0.660mmol)和中间体3-1(315mg,0.790mmol)为原料，操作同4-1，得白色固体80.0mg，收率：23.7％。
1H NMR(300MHz,DMSO-d6)δ(ppm):12.57(s,1H),8.29(d,J＝8.3Hz,1H),8.14(s,1H),7.99(d,J＝1.9Hz,2H),7.88(s,1H),7.59–7.54(m,1H),7.48–7.34(m,2H),7.32-7.25(m,1H),4.46(d,J＝6.0Hz,2H),4.01(s,3H),2.32(s,3H),1.38(s,9H).
3、(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢二氮杂萘-6-基)-1-甲基-1H-吡唑-5-基)-2-(5-氟-2-甲基苯基)丙烯腈(I-17)的合成
以中间体4-17(80.0mg,0.160mmol)为原料，操作同I-1，得白色固体62.0mg，收率：96.2％。
1H NMR(300MHz,DMSO-d6)δ(ppm):12.94(s,1H),8.68(s,3H),8.32(d,J＝8.2Hz,1H),8.18(s,1H),8.13–8.02(m,2H),7.91(s,1H),7.55–7.46(m,1H),7.43–7.35(m,1H),7.32–7.22(m,1H),4.52-4.42(m,1H),4.02(s,3H),2.33(s,3H).
HRMS(ESI+):m/z calcd for C23H19FN6O 415.1683[M+H]+；found:415.1679.
实施例18：(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢酞嗪-6-基)-1-甲基-1H-吡唑-5-基)-2-(4-氯苯基)丙烯腈(I-18)的合成
1、(Z)-3-(4-溴-1-甲基-1H-吡唑-5-基)-2-(4-氯苯基)丙烯腈(2-18)的合成
以中间体2-(4-氯苯基)乙腈1-18(160mg,1.06mmol)和4-溴-1-甲基-1H-吡唑-5-甲醛(200mg,1.06mmol)为原料，操作同2-1，得白色固体220mg，收率：64.5％。
1HNMR(400MHz,Chloroform-d)δ(ppm):7.67–7.62(m,2H),7.56(s,1H),7.49–7.44(m,2H),7.29(s,1H),3.96(s,3H).
2、(Z)-((7-(5-(2-(4-氯苯基)-2-氰基乙烯基)-1-甲基-1H-吡唑-4-基)-4-氧代-3,4-二氢酞嗪-1-基)甲基)氨基甲酸叔丁酯(4-18)的合成
以中间体2-18(220mg,0.680mmol)和中间体3-1(328mg,0.820mmol)为原料，操作同4-1，得白色固体94.0mg，收率：26.7％。
1H NMR(300MHz,DMSO-d6)δ(ppm):12.58(s,1H),8.27(s,1H),8.25(d,J＝8.7Hz,1H),8.13(s,1H),8.05(s,1H),7.97-7.90(m,1H),7.86(d,J＝8.6Hz,1H),7.65(d,J＝8.6Hz,2H),7.42(t,J＝5.9Hz,1H),4.39(d,J＝6.0Hz,2H),4.00(s,3H),1.39(s,9H).
3、(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢酞嗪-6-基)-1-甲基-1H-吡唑-5-基)-2-(4-氯苯基)丙烯腈(I-18)的合成
以中间体4-18(94.0mg,0.180mmol)为原料，操作同I-1，得白色固体68.0mg，收率：89.6％。
1H NMR(300MHz,DMSO-d6)δ(ppm):12.85(s,1H),8.64(s,3H),8.29(s,1H),8.20(d,J＝8.3Hz,1H),8.15(s,1H),8.02(s,1H),7.91(d,J＝8.4Hz,1H),7.79(d,J＝8.6Hz,2H),7.59(d,J＝8.5Hz,2H),4.41-4.28(m,2H),3.94(s,3H).
HRMS(ESI+):m/z calcd for C22H17ClN6O 417.1231[M+H]+；found:417.1220.
实施例19：(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢二氮杂萘-6-基)-1-甲基-1H-吡唑-5-基)-2-(3,5-二氟苯基)丙烯腈(I-19)的合成
1、(Z)-3-(4-溴-1-甲基-1H-吡唑-5-基)-2-(3,5-二氟苯基)丙烯腈(2-19)的合成
以2-(3，5-二氟苯基)乙腈1-19(162mg,1.06mmol)和4-溴-1-甲基-1H-吡唑-5-甲醛(200mg,1.06mmol)为原料，操作同2-1，得白色固体220mg，收率：64.2％。
1H NMR(400MHz,Chloroform-d)δ(ppm):7.60(s,1H),7.36(s,1H),7.28–7.24(m,1H),6.99-6.93(m,1H),3.99(s,3H).
2、(Z)-((7-(5-(2-氰基-2-(3,5-二氟苯基)乙烯基)-1-甲基-1H-吡唑-4-基)-4-氧代-3,4-二氢酞嗪-1-基)甲基)氨基甲酸叔丁酯(4-19)的合成
以中间体2-19(220mg,0.680mmol)和中间体3-1(327mg,0.820mmol)为原料，操作同4-1，得白色固体91.0mg，收率：25.9％。
1H NMR(300MHz,DMSO-d6)δ(ppm):12.60(s,1H),8.41(s,1H),8.28(d,J＝8.3Hz,1H),8.14(s,1H),8.07(s,1H),7.99-7.93(m,1H),7.61(d,J＝2.1Hz,2H),7.54–7.39(m,2H),4.40(d,J＝6.0Hz,2H),4.03(s,3H),1.40(s,9H).
3、(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢二氮杂萘-6-基)-1-甲基-1H-吡唑-5-基)-2-(3,5-二氟苯基)丙烯腈(I-19)的合成
以中间体4-19(91.0mg,0.180mmol)为原料，操作同I-1得白色固体70.0mg，收率：95.6％。
1H NMR(300MHz,DMSO-d6)δ(ppm):12.92(s,1H),8.68(s,3H),8.47(s,1H),8.24(d,J＝8.3Hz,1H),8.20(s,1H),8.07(s,1H),7.94(d,J＝8.2Hz,1H),7.61–7.53(m,2H),7.50–7.41(m,1H),4.49-4.30(m,2H),4.00(s,3H).
HRMS(ESI+):m/z calcd for C22H16F2N6O 419.1432[M+H]+；found:419.1426.
实施例20：(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢二氮杂萘-6-基)-1-甲基-1H-吡唑-5-基)-2-(3,4-二氟苯基)丙烯腈(I-20)的合成
1、(Z)-3-(4-溴-1-甲基-1H-吡唑-5-基)-2-(3,4-二氟苯基)丙烯腈(2-20)的合成
向25mL三颈瓶中依次加入2-(3，4-二氟苯基)乙腈1-20(62.0mg,1.06mmol)、4-溴-1-甲基-1H-吡唑-5-甲醛(200mg,1.06mmol)、哌啶(180mg,2.12mmol)和4mL EtOH，搅拌溶解，反应体系在25℃下反应12h，TLC监测(V石油醚:V乙酸乙酯＝10:1)反应完全，减压浓缩，残余物经柱层析纯化(洗脱剂：V石油醚:V乙酸乙酯＝10:1)得白色固体200mg，收率：58.3％。
1HNMR(300MHz,Chloroform-d)δ(ppm):7.53(s,1H),7.51–7.40(m,2H),7.33–7.23(m,1H),7.22(s,1H),3.93(s,3H).
2、(Z)-((7-(5-(2-氰基-2-(3,4-二氟苯基)乙烯基)-1-甲基-1H-吡唑-4-基)-4-氧代-3,4-二氢酞嗪-1-基)甲基)氨基甲酸叔丁酯(4-20)的合成
以中间体2-20(200mg,0.620mmol)和中间体3-1(297mg,0.740mmol)为原料，操作同4-1，得白色固体67.0mg，收率：20.9％。
1H NMR(300MHz,DMSO-d6)δ(ppm):12.52(s,1H),8.23(s,1H),8.21(d,J＝8.4Hz,1H),8.09–7.98(m,3H),7.93-7.87(m,1H),7.65–7.51(m,2H),7.35(t,J＝5.4Hz,1H),4.33(d,J＝6.3Hz,2H),3.96(s,3H),1.34(s,9H).
3、(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢二氮杂萘-6-基)-1-甲基-1H-吡唑-5-基)-2-(3,4-二氟苯基)丙烯腈(I-20)的合成
以中间体4-20(67.0mg,0.130mmol)为原料，操作同I-1得白色固体51.0mg，收率：95.5％。
1H NMR(300MHz,DMSO-d6)δ(ppm):12.90(s,1H),8.68(s,3H),8.35(s,1H),8.24(d,J＝8.3Hz,1H),8.19(s,1H),8.12–8.00(m,2H),7.98-7.90(m,1H),7.69–7.54(m,2H),4.43-4.30(m,2H),3.98(s,3H).
13C NMR(75MHz,DMSO-d6)δ(ppm):159.77,139.24,139.05,134.48,131.61,131.12,128.79,127.47,125.55,124.21,121.82,118.96,118.72,117.10,116.08,115.97,115.71,66.68,38.08.
HRMS(ESI+):m/z calcd for C22H16F2N6O 441.1251[M+Na]+；found:441.1241.
实施例21：(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢二氮杂萘-6-基)-1-甲基-1H-吡唑-5-基)-2-(3-甲氧基-4-甲基苯基)丙烯腈(I-21)的合成
1、(Z)-3-(4-溴-1-甲基-1H-吡唑-5-基)-2-(3-甲氧基-4-甲基苯基)丙烯腈(2-21)的合成以2-(3-甲氧基-4-甲基苯基)乙腈1-21(171mg,1.06mmol)和4-溴-1-甲基-1H-吡唑-5-甲醛(200mg,1.06mmol)为原料，操作同2-1，得白色固体155mg，收率：44.1％。
1H NMR(300MHz,Chloroform-d)δ(ppm):7.56(s,1H),7.26(s,1H),7.22(s,2H),7.10(s,1H),3.96(s,3H),3.91(s,3H),2.27(s,3H).
2、(Z)-((7-(5-(2-氰基-2-(3-甲氧基-4-甲基苯基)乙烯基)-1-甲基-1H-吡唑-4-基)-4-氧代-3,4-二氢酞嗪-1-基)甲基)氨基甲酸叔丁酯(4-21)的合成
以中间体2-21(200mg,0.600mmol)和中间体3-1(286mg,0.710mmol)为原料，操作同4-1，得白色固体56.0mg，收率：23.1％。
1H NMR(300MHz,DMSO-d6)δ(ppm):12.55(s,1H),8.25(d,J＝8.3Hz,1H),8.22(s,1H),8.14(s,1H),8.05(s,1H),7.96-7.92(m,1H),7.46(s,1H),7.42(t,J＝5.8Hz,1H),7.31(d,J＝7.8Hz,1H),7.25-.19(m,1H),4.39(d,J＝5.9Hz,2H),3.98(s,3H),3.93(s,3H),2.23(s,3H),1.38(s,9H).
3、(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢二氮杂萘-6-基)-1-甲基-1H-吡唑-5-基)-2-(3-甲氧基-4-甲基苯基)丙烯腈(I-21)的合成
以中间体4-21(56.0mg,0.110mmol)为原料，操作同I-1得白色固体40.0mg，收率：88.2％。
1H NMR(300MHz,DMSO-d6)δ(ppm):12.88(s,1H),8.72(s,3H),8.31(s,1H),8.22(d,J＝8.3Hz,1H),8.18(s,1H),8.07(s,1H),7.93(d,J＝8.3Hz,1H),7.39(s,1H),7.27(d,J＝7.8Hz,1H),7.19(d,J＝7.2Hz,1H),4.43-4.29(m,2H),3.96(s,3H),3.90(s,3H),2.18(s,3H).
13C NMR(75MHz,DMSO-d6)δ(ppm):148.08,139.25,139.07,134.79,131.01,130.74,129.61,128.83,127.43,125.61,121.97,121.59,120.01,119.91,118.45,116.48,111.85,66.74,56.85.
HRMS(ESI+):m/z calcd for C24H22N6O2427.1882[M+H]+；found:427.1874.
实施例22：(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢二氮杂萘-6-基)-1-甲基-1H-吡唑-5-基)-2-(3-氟-5-甲氧基苯基)丙烯腈(I-22)的合成
1、(Z)-3-(4-溴-1-甲基-1H-吡唑-5-基)-2-(3-氟-5-甲氧基苯基)丙烯腈(2-22)的合成
以2-(3-氟-5-甲氧基苯基)乙腈1-22(175mg,1.06mmol)和4-溴-1-甲基-1H-吡唑-5-甲醛(200mg,1.06mmol)为原料，操作同2-1，得白色固体200mg，收率：56.2％。
1H NMR(300MHz,Chloroform-d)δ(ppm):7.57(s,1H),7.31(s,1H),7.05–6.98(m,2H),6.76-6.70(m,1H),3.96(s,3H),3.87(s,3H).
2、(Z)-((7-(5-(2-氰基-2-(3-氟-5-甲氧基苯基)乙烯基)-1-甲基-1H-吡唑-4-基)-4-氧代-3,4-二氢酞嗪-1-基)甲基)氨基甲酸叔丁酯(4-22)的合成
以中间体2-22(155mg,0.470mmol)和中间体3-1(225mg,0.560mmol)为原料，操作同4-1，得白色固体76.0mg，收率：30.9％。
1H NMR(300MHz,DMSO-d6)δ(ppm):12.54(s,1H),8.29(s,1H),8.22(d,J＝8.3Hz,1H),8.10(s,1H),8.01(s,1H),7.90(d,J＝8.3Hz,1H),7.46-7.31(m,1H),7.25(d,J＝9.6Hz,1H),7.17(s,1H),7.02(d,J＝10.7Hz,1H),4.36(d,J＝5.9Hz,2H),3.96(s,3H),3.84(s,3H),1.36(s,9H).
3、(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢二氮杂萘-6-基)-1-甲基-1H-吡唑-5-基)-2-(3-氟-5-甲氧基苯基)丙烯腈(I-22)的合成
以中间体4-22(76.0mg,0.140mmol)为原料，操作同I-1，得白色固体52.0mg，收率：84.4％。
1H NMR(300MHz,DMSO-d6)δ(ppm):8.66(s,1H),8.33(s,3H),8.24(d,J＝8.3Hz,1H),8.18(s,1H),8.06(s,1H),7.96(d,J＝8.9Hz,1H),7.70–7.61(m,1H),7.42–7.33(m,1H),7.31–7.21(m,1H),4.43-4.30(m,1H),3.97(s,3H),3.96(s,3H).
13C NMR(75MHz,DMSO-d6)δ(ppm):161.29,161.13,159.24,138.99,138.86,138.52,133.99,131.98,130.50,128.53,126.96,125.33,121.84,121.14,115.82,108.80,105.46,105.14,66.36,56.14,55.63.
HRMS(ESI+):m/z calcd for C23H19FN6O2431.1632[M+H]+；found:431.1626.
实施例23：(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢二氮杂萘-6-基)-1-甲基-1H-吡唑-5-基)-2-(4-氟-3-甲氧基苯基)丙烯腈(I-23)的合成
1、(Z)-3-(4-溴-1-甲基-1H-吡唑-5-基)-2-(4-氟-3-甲氧基苯基)丙烯腈(2-23)的合成
以中间体2-(4-氟-3-甲氧基苯基)乙腈1-23(175mg,1.06mmol)和4-溴-1-甲基-1H-吡唑-5-甲醛(200mg,1.06mmol)为原料，操作同2-1，得白色固体247mg，收率：69.4％。
1H NMR(300MHz,Chloroform-d)δ(ppm):7.57(s,1H),7.30-7.27(m,1H),7.25-7.22(m,2H),7.22–7.14(m,1H),3.98(s,3H),3.96(s,3H).
2、(Z)-((7-(5-(2-氰基-2-(4-氟-3-甲氧基苯基)乙烯基)-1-甲基-1H-吡唑-4-基)-4-氧代-3,4-二氢酞嗪-1-基)甲基)氨基甲酸叔丁酯(4-23)的合成
以中间体2-23(247mg,0.740mmol)和中间体3-1(354mg,0.880mmol)为原料，操作同4-1，得白色固体80.0mg，收率：20.5％。
1H NMR(300MHz,DMSO-d6)δ(ppm):12.53(s,1H),8.23(d,J＝8.5Hz,1H),8.21(s,1H),8.10(s,1H),8.02(s,1H),7.92(d,J＝9.5Hz,1H),7.68(d,J＝8.0Hz,1H),7.43–7.34(m,2H),7.28–7.24(m,1H),4.35(d,J＝6.0Hz,2H),3.96(s,3H),3.95(s,3H),1.35(s,9H).
3、(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢二氮杂萘-6-基)-1-甲基-1H-吡唑-5-基)-2-(4-氟-3-甲氧基苯基)丙烯腈(I-23)的合成
以中间体4-23(80.0mg,0.150mmol)为原料，操作同I-1，得白色固体56.0mg，收率：86.3％。
1H NMR(300MHz,DMSO-d6)δ(ppm):12.89(s,1H),8.66(s,3H),8.33(s,1H),8.24(d,J＝8.3Hz,1H),8.18(s,1H),8.06(s,1H),7.96(d,J＝8.4Hz,1H),7.70-7.60(m,1H),7.48-7.33(m,1H),7.28–7.24(m,1H),4.46-4.26(m,2H),3.97(s,3H),3.96(s,3H).
13C NMR(75MHz,DMSO-d6)δ(ppm):159.57,151.31,148.14,147.99,139.17,138.99,134.71,130.91,130.70,129.56,127.27,125.50,121.89,121.51,118.34,117.00,116.76,116.39,111.76.
HRMS(ESI+):m/z calcd for C23H19FN6O2431.1632[M+H]+；found:431.1629.
实施例24：(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢二氮杂萘-6-基)-1-甲基-1H-吡唑-5-基)-2-(4-(叔丁基)苯基)丙烯腈(I-24)的合成
1、(Z)-3-(4-溴-1-甲基-1H-吡唑-5-基)-2-(4-(叔丁基)苯基)丙烯腈(2-24)的合成
以2-(4-(叔丁基)苯基)乙腈1-24(183mg,1.06mmol)和4-溴-1-甲基-1H-吡唑-5-甲醛(200mg,1.06mmol)为原料，操作同2-1，得白色固体338mg，收率：92.8％。
1HNMR(300MHz,Chloroform-d)δ(ppm):7.94–7.88(m,2H),7.82(s,1H),7.80–7.73(m,2H),7.54(s,1H),4.21(s,3H),1.62(s,9H).
2、(Z)-((7-(5-(2-(4-(叔丁基)苯基)-2-氰基乙烯基)-1-甲基-1H-吡唑-4-基)-4-氧代-3,4-二氢酞嗪-1-基)甲基)氨基甲酸叔丁酯(4-24)的合成
以中间体2-24(338mg,0.980mmol)和中间体3-1(473mg,1.18mmol)为原料，操作同4-1，得白色固体110mg，收率：20.8％。
1H NMR(300MHz,DMSO-d6)δ(ppm):12.64(s,1H),8.31(d,J＝8.3Hz,1H),8.25(s,1H),8.22(s,1H),8.11(s,1H),8.02-7.97(m,1H),7.83(d,J＝8.4Hz,2H),7.65(d,J＝8.5Hz,2H),7.51(t,J＝6.1Hz,1H),4.49(d,J＝5.9Hz,2H),4.05(s,3H),1.46(s,9H),1.40(s,9H).
3、(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢二氮杂萘-6-基)-1-甲基-1H-吡唑-5-基)-2-(4-(叔丁基)苯基)丙烯腈(I-24)的合成
以中间体4-24(110mg,0.200mmol)为原料，操作同I-1，得白色固体80.0mg，收率：89.3％。
1H NMR(300MHz,DMSO-d6)δ(ppm):12.88(s,1H),8.72(s,3H),8.24–8.21(m,2H),8.08(s,1H),7.96-7.89(m,1H),7.72(d,J＝8.5Hz,2H),7.55(d,J＝8.6Hz,2H),4.47-4.33(m,2H),3.95(s,3H),1.29(s,9H).
13C NMR(75MHz,DMSO-d6)δ(ppm):159.99,154.09,139.46,135.16,130.06,129.57,129.08,127.59,126.87,126.71,121.76,119.60,116.77,66.94,60.59,38.22,35.18,31.44,31.44,14.64.
HRMS(ESI+):m/z calcd for C26H26N6O 439.2246[M+H]+；found:439.2235.
实施例25：(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢二氮杂萘-6-基)-1-甲基-1H-吡唑-5-基)-2-(3-氟苯基)丙烯腈(I-25)的合成
1、(Z)-3-(4-溴-1-甲基-1H-吡唑-5-基)-2-(3-氟-5-甲基苯基)丙烯腈(2-25)的合成
以2-(3-氟-5-甲基苯基)乙腈1-25(125mg,0.840mmol)和4-溴-1-甲基-1H-吡唑-5-甲醛(158mg,0.840mmol)为原料，操作同2-1，得白色固体145mg，收率：54.2％。
1H NMR(300MHz,Chloroform-d)δ(ppm):7.56(s,1H),7.32(s,1H),7.30(s,1H),7.23-7.17(m,1H),7.00(d,J＝9.1Hz,1H),3.96(s,3H),2.44(s,3H).
2、(Z)-((7-(5-(2-氰基-2-(3-氟苯基)乙烯基)-1-甲基-1H-吡唑-4-基)-4-氧代-3,4-二氢酞嗪-1-基)甲基)氨基甲酸叔丁酯(4-25)的合成
以中间体2-25(145mg,0.450mmol)和中间体3-1(218mg,0.540mmol)为原料，操作同4-1，得白色固体50.0mg，收率：21.5％。
1H NMR(300MHz,DMSO-d6)δ(ppm):12.55(s,1H),8.27(s,1H),8.22(d,J＝8.3Hz,1H),8.10(s,1H),8.01(s,1H),7.89(dd,J＝8.3,1.6Hz,1H),7.55(d,J＝9.9Hz,1H),7.42-7.63(m,2H),7.21(d,J＝9.6Hz,1H),4.36(d,J＝5.9Hz,1H),3.96(s,3H),2.38(s,3H),1.34(s,9H).
3、(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢二氮杂萘-6-基)-1-甲基-1H-吡唑-5-基)-2-(3-氟苯基)丙烯腈(I-25)的合成
以中间体4-25(50.0mg,0.100mmol)为原料，操作同I-1得白色固体35.0mg，收率：86.9％。
1H NMR(300MHz,DMSO-d6)δ(ppm):12.91(s,1H),8.74(s,3H),8.37(s,1H),8.22(d,J＝8.3Hz,1H),8.20(s,1H),8.07(s,1H),7.91(d,J＝8.3Hz,1H),7.54(d,J＝9.9Hz,1H),7.39(s,1H),7.19(d,J＝8.8Hz,1H),4.45-4.32(m,2H),3.97(s,3H),2.37(s,3H).
13C NMR(75MHz,DMSO-d6)δ(ppm):165.52,162.28,160.83,143.45,143.34,140.36,140.22,135.64,135.50,132.67,132.16,129.97,128.52,126.72,124.63,123.14,122.99,119.06,117.37,111.71,111.40,67.83,39.25,22.30.
HRMS(ESI+):m/z calcd for C23H19FN6O 415.1683[M+H]+；found:415.1679.
实施例26：(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢酞嗪-6-基)-1-甲基-1H-吡唑-5-基)-2-(4-氯-3-氟苯基)丙烯腈(I-26)的合成
1、(Z)-3-(4-溴-1-甲基-1H-吡唑-5-基)-2-(4-氯-3-氟苯基)丙烯腈(2-26)的合成
以2-(4-氯-3-氟苯基)乙腈1-26(180mg,1.06mmol)和4-溴-1-甲基-1H-吡唑-5-甲醛(200mg,1.06mmol)为原料，操作同2-1，得白色固体280mg，收率：77.7％。
1HNMR(300MHz,Chloroform-d)δ(ppm):7.60(s,1H),7.58–7.54(m,1H),7.53–7.45(m,2H),7.33(s,1H),3.99(s,3H).
2、(Z)-((7-(5-(2-(4-氯-3-氟苯基)-2-氰基乙烯基)-1-甲基-1H-吡唑-4-基)-4-氧代-3,4-二氢酞嗪-1-基)甲基)氨基甲酸叔丁酯(4-26)的合成
以中间体2-26(115mg,0.360mmol)和中间体3-1(174mg,0.430mmol)为原料，操作同4-1，得白色固体96.0mg，收率：21.8％。
1H NMR(300MHz,DMSO-d6)δ(ppm):12.56(s,1H),8.33(s,1H),8.24(d,J＝8.3Hz,1H),8.11(s,1H)),8.04(s,1H),8.01(d,J＝8.6Hz,1H),7.93(d,J＝8.3Hz,1H),7.79-7.75(m,1H),7.58(d,J＝8.9Hz,1H),7.38(t,J＝6.8Hz,1H),4.36(d,J＝6.1Hz,2H),3.99(s,3H),1.37(s,9H).
3、(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢酞嗪-6-基)-1-甲基-1H-吡唑-5-基)-2-(4-氯-3-氟苯基)丙烯腈(I-26)的合成
以中间体4-26(96.0mg,0.180mmol)为原料，操作同I-1，得白色固体72.0mg，收率：92.3％。
1H NMR(400MHz,DMSO-d6)δ(ppm):12.91(s,1H),8.66(s,3H),8.42(s,1H),8.24(d,J＝8.3Hz,1H),8.19(s,1H),8.06(s,1H),8.03-7.99(m,1H),7.97-7.93(m,1H),7.78-7.74(m,1H),7.60-7.54(m,1H),4.42-4.32(m,2H),3.99(s,3H).
13C NMR(75MHz,DMSO-d6)δ(ppm):159.60(d,J＝17.8Hz),156.20,139.24,139.11,139.00,134.39,133.62(d,J＝7.8Hz),132.32,131.90,131.10,128.81,127.47,125.61,124.35–123.85(m),121.96,121.78,116.87(d,J＝2.4Hz),116.00,115.04,114.72,66.71,38.17.
HRMS(ESI+):m/z calcd for C22H16ClFN6O 435.1136[M+H]+；found:435.1132.
实施例27：(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢酞嗪-6-基)-1-甲基-1H-吡唑-5-基)-2-(3-氯-5-氟苯基)丙烯腈(I-27)的合成
1、(Z)-3-(4-溴-1-甲基-1H-吡唑-5-基)-2-(3-氯-5-氟苯基)丙烯腈(2-27)的合成
以2-(2-氯-4-氟苯基)乙腈1-27(125mg,0.740mmol)和4-溴-1-甲基-1H-吡唑-5-甲醛(139mg,0.740mmol)为原料，操作同2-1，得白色固体210mg，收率：83.7％。
1H NMR(300MHz,Chloroform-d)δ(ppm):7.60(s,1H),7.55-7.52(m,1H),7.38-7.33(m,2H),7.27-7.22(m,1H),3.99(s,3H).
2、(Z)-((7-(5-(2-(3-氯-5-氟苯基)-2-氰基乙烯基)-1-甲基-1H-吡唑-4-基)-4-氧代-3,4-二氢酞嗪-1-基)甲基)氨基甲酸叔丁酯(4-27)的合成
以中间体2-27(297mg,0.740mmol)和中间体3-1(210mg,0.620mmol)为原料，操作同4-1，得白色固体69.0mg，收率：20.9％。
1H NMR(300MHz,DMSO-d6)δ(ppm):12.54(s,1H),8.37(s,1H),8.23(d,J＝8.3Hz,1H),8.10(s,1H),8.02(s,1H),7.92(d,J＝8.4Hz,1H),7.71(s,1H),7.69–7.59(m,2H),7.41–7.34(m,1H),4.35(d,J＝5.8Hz,2H),3.98(s,3H),1.35(s,9H).
3、(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢酞嗪-6-基)-1-甲基-1H-吡唑-5-基)-2-(3-氯-5-氟苯基)丙烯腈(I-27)的合成
以中间体4-27(69.0mg,0.130mmol)为原料，操作同I-1得白色固体45.0mg，收率：80.2％。
1H NMR(400MHz,DMSO-d6)δ(ppm):12.91(s,1H),8.67(s,3H),8.47(s,1H),8.24(d,J＝8.3Hz,1H),8.19(s,1H),8.07(s,1H),7.97-7.92(m,1H),7.73-7.67(m,2H),7.65-7.60(m,1H),4.45-4.35(m,2H),3.99(s,3H).
13C NMR(75MHz,DMSO-d6)δ(ppm):162.78(d,J＝248.6Hz),159.74,139.33,139.28,139.02,136.22(d,J＝9.4Hz),135.47(d,J＝11.3Hz),134.27,133.33,131.17,128.93,127.50,125.73,123.00,122.19,118.17,117.83,116.16(d,J＝27.8Hz),112.79(d,J＝24.1Hz),66.77,38.29.HRMS(ESI+):m/z calcd for C22H16ClFN6O 435.1136[M+H]+；found:435.1129.
实施例28：(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢酞嗪-6-基)-1-甲基-1H-吡唑-5-基)-2-(3-氯苯基)丙烯腈(I-28)的合成
1、(Z)-3-(4-溴-1-甲基-1H-吡唑-5-基)-2-(3-氯苯基)丙烯腈(2-28)的合成
以中间体2-(3-氯苯基)乙腈1-28(160mg,1.06mmol)和4-溴-1-甲基-1H-吡唑-5-甲醛(200mg,1.06mmol)为原料，操作同2-1，得白色固体175mg，收率：51.3％。
1H NMR(300MHz,Chloroform-d)δ(ppm):7.62–7.58(m,1H),7.51(dt,J＝6.4,2.0Hz,1H),7.48(s,1H),7.40–7.33(m,2H),7.23(s,1H),3.87(s,3H).
2、(Z)-((7-(5-(2-(3-氯苯基)-2-氰基乙烯基)-1-甲基-1H-吡唑-4-基)-4-氧代-3,4-二氢酞嗪-1-基)甲基)氨基甲酸叔丁酯(4-28)的合成
以中间体2-28(175mg,0.540mmol)和中间体3-1(260mg,0.650mmol)为原料，操作同4-1，得白色固体72.0mg，收率：26.6％。
1H NMR(300MHz,DMSO-d6)δ(ppm):12.55(s,1H),8.31(s,1H),8.22(d,J＝8.3Hz,1H),8.02(s,1H),7.98(s,1H),7.94-7.88(m,1H),7.71–7.66(m,1H),7.61–7.52(m,2H),7.41(t,J＝6.0Hz,1H),4.37(d,J＝5.9Hz,2H),3.98(s,3H),1.36(s,9H).
3、(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢酞嗪-6-基)-1-甲基-1H-吡唑-5-基)-2-(3-氯苯基)丙烯腈(I-28)的合成
以中间体4-28(72.0mg,0.140mmol)为原料，操作同I-1，得白色固体51.0mg，收率：87.8％。
1H NMR(300MHz,DMSO-d6)δ(ppm):12.91(s,1H),8.74(s,3H),8.40(s,1H),8.22(d,J＝10.1Hz,2H),8.08(s,1H),7.96-7.90(m,2H),7.73–7.67(m,1H),7.59-7.53(m,2H),4.46-4.35(m,2H),3.98(s,3H).
13C NMR(75MHz,DMSO-d6)δ(ppm):159.67,139.17,139.01,134.43,131.75,131.54,131.00,130.41,128.76,127.31,126.08,125.49,125.38,121.91,121.83,117.56,116.11,66.61,38.05.
HRMS(ESI+):m/z calcd for C22H17ClN6O 417.1231[M+H]+；found:417.1223.
实施例29：(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢二氮杂萘-6-基)-1-甲基-1H-吡唑-5-基)-2-(3-氟苯基)丙烯腈(I-29)的合成
1、(Z)-3-(4-溴-1-甲基-1H-吡唑-5-基)-2-(3-氟苯基)丙烯腈(2-29)的合成
以2-(3-氟苯基)乙腈1-29(142mg,1.06mmol)和4-溴-1-甲基-1H-吡唑-5-甲醛(200mg,1.06mmol)为原料，操作同2-1，得白色固体254mg，收率：78.4％。
1HNMR(300MHz,Chloroform-d)δ(ppm):7.57(s,1H),7.54–7.46(m,2H),7.44–7.38(m,2H),7.33(s,1H),7.23–7.15(m,1H),3.97(s,3H).
2、(Z)-(7-(5-(2-氰基-2-(3-氟苯基)乙烯基)-1-甲基-1H-吡唑-4-基)-4-氧代-3,4-二氢二氮杂萘-1-基)甲基)氨基甲酸叔丁酯(4-29)的合成
以中间体2-29(254mg,0.830mmol)和中间体3-1(400mg,0.990mmol)为原料，操作同4-1，得白色固体120mg，收率：28.9％。
1H NMR(300MHz,DMSO-d6)δ(ppm):12.54(s,1H),8.29(s,1H),8.22(d,J＝8.3Hz,1H),8.11(s,1H),8.02(s,1H),7.93-7.87(m,1H),7.81–7.71(m,1H),7.62–7.52(m,2H),7.43–7.31(m,2H),4.36(d,J＝6.0Hz,2H),3.97(s,3H),1.35(s,9H).
3、(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢二氮杂萘-6-基)-1-甲基-1H-吡唑-5-基)-2-(3-氟苯基)丙烯腈(I-29)的合成
以中间体4-29(120mg,0.120mmol)为原料，操作同I-1，得白色固体72.0mg，收率：75.5％。
1H NMR(300MHz,DMSO-d6)δ(ppm):12.91(s,1H),8.67(s,3H),8.38(s,1H),8.24(d,J＝8.3Hz,1H),8.20(s,1H),8.07(s,1H),7.94(d,J＝8.3Hz,1H),7.77(d,J＝10.1Hz,1H),7.66–7.53(m,2H),7.42–7.32(m,1H),4.46-4.32(m,2H),3.99(s,3H).
13C NMR(75MHz,DMSO-d6)δ(ppm):162.92(d,J＝244.8Hz),159.81,139.31,139.17,134.93(d,J＝8.5Hz),134.59,132.02(d,J＝8.5Hz),131.85,131.18,128.92,127.52,125.69,123.16(d,J＝1.9Hz),122.08,121.97,117.94(d,J＝2.6Hz),117.73(d,J＝22.4Hz),116.30,113.41(d,J＝24.0Hz),66.80,38.22.
HRMS(ESI+):m/z calcd for C22H17FN6O 401.1526[M+H]+；found:401.1519.
实施例30：(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢二氮杂萘-6-基)-1-甲基-1H-吡唑-5-基)-2-(3-苯氧基苯基)丙烯腈(I-30)的合成
1、(Z)-3-(4-溴-1-甲基-1H-吡唑-5-基)-2-(3-苯氧基苯基)丙烯腈(2-30)的合成
以2-(3-苯氧基苯基)乙腈1-30(221mg,1.06mmol)和4-溴-1-甲基-1H-吡唑-5-甲醛(200mg,1.06mmol)为原料，操作同2-1，得白色固体170mg，收率：42.2％。
1H NMR(300MHz,Chloroform-d)δ(ppm):7.52(s,1H),7.44–7.32(m,4H),7.24(s,1H),7.23(s,1H),7.13(t,J＝7.4Hz,1H),7.09–7.02(m,2H),7.01(s,1H),3.91(s,3H).
2、(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢二氮杂萘-6-基)-1-甲基-1H-吡唑-5-基)-2-(3-苯氧基苯基)丙烯腈(4-30)的合成
以中间体2-30(145mg,0.450mmol)和中间体3-1(218mg,0.540mmol)为原料，操作同4-1，得白色固体50.0mg，收率：21.5％。
1H NMR(300MHz,DMSO-d6)δ(ppm):12.56(s,1H),8.24(s,1H),8.22(d,J＝8.4Hz,1H),8.10(s,1H),8.00(s,1H),7.93–7.84(m,1H),7.56–7.50(m,3H),7.46-7.38(m,3H),7.17(t,J＝7.4Hz,1H),7.13-7.03(m,3H),4.34(d,J＝5.6Hz,2H),3.96(s,3H),1.34(s,9H).
3、(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢二氮杂萘-6-基)-1-甲基-1H-吡唑-5-基)-2-(3-苯氧基苯基)丙烯腈(I-30)的合成
以中间体4-30(59.0mg,0.0400mmol)为原料，操作同I-1，得白色固体17.0mg，收率：90.5％。
1H NMR(300MHz,DMSO-d6)δ(ppm):12.92(s,1H),8.72(s,3H),8.33(s,1H),8.22(d,J＝8.5Hz,1H),8.19(s,1H),8.07(s,1H),7.91(d,J＝8.5Hz,1H),7.60-7.52(m,2H),7.49(s,1H),7.45-7.37(m,2H),7.21-7.12(m,1H),7.11-7.03(m,3H),4.48-4.29(m,2H),3.96(s,3H).
13C NMR(75MHz,DMSO-d6)δ(ppm):159.84,157.88,156.30,139.36,139.22,134.72,134.51,131.68,131.21,130.76,128.93,127.46,125.63,124.62,122.11,121.89,121.70,120.36,119.45,118.53,116.51,116.44,66.78,38.18.
HRMS(ESI+):m/z calcd for C28H22N6O2475.1882[M+H]+；found:475.1871.
实施例31：(Z)-3-(4-溴-1-甲基-1H-吡唑-5-基)-2-(2,2-二氟苯并[d][1,3]二氧杂环戊烯-5-基)丙烯腈(Ⅰ-31)的合成(I-31)的合成
1、(Z)-3-(4-溴-1-甲基-1H-吡唑-5-基)-2-(2,2-二氟苯并[d][1,3]二氧杂环戊烯-5-基)丙烯腈(2-31)的合成
向25mL三颈瓶中依次加入2-(2，2-二氟苯并[d][1,3]二氧杂环戊烯-5-基)乙腈1-31(209mg,1.06mmol)、4-溴-1-甲基-1H-吡唑-5-甲醛(200mg,1.06mmol)、KOH(120mg,2.12mmol)和5mL EtOH，搅拌溶解，反应体系在25℃下反应12h，TLC监测(V石油醚:V乙酸乙酯＝10:1)反应完全，减压浓缩，残余物经柱层析纯化(洗脱剂：V石油醚:V乙酸乙酯＝10:1)得白色固体270mg，收率69.4％。
1H NMR(300MHz,Chloroform-d)δ(ppm):7.56(s,1H),7.48(d,J＝8.4Hz,1H),7.41(s,1H),7.23(s,1H),7.18(d,J＝8.4Hz,1H),3.95(s,3H).
2、(Z)-((7-(5-(2-氰基-2-(2,2-二氟苯并[d][1,3]二氧杂环戊烯-5-基)乙烯基)-1-甲基-1H-吡唑-4-基)-4-氧代-3,4-二氢二氮杂萘-1-基)甲基)氨基甲酸叔丁酯(4-31)的合成
向25mL三颈瓶中依次加入2-31(270mg,0.730mmol)、中间体3-1(354mg,0.880mmol)、SPhospdG2(49.0mg,0.0700mmol)、NaHCO3(123mg,1.47mmol)、10mL叔戊醇和2mL水，搅拌溶解，反应体系在N2保护、80℃下反应12h，TLC监测(V石油醚:V乙酸乙酯＝1:1)反应完全，反应液减压浓缩，残余物加5mL水稀释，用乙酸乙酯(5mL×3)萃取，合并有机相，有机相用5mL饱和NaCl洗涤，无水Na2SO4干燥，抽滤，滤液减压浓缩，柱层析纯化(洗脱剂：V石油醚:V乙酸乙酯＝1:1)得白色固体107mg，收率26.5％。
1H NMR(300MHz,DMSO-d6)δ(ppm):12.56(s,1H),8.22(s,1H),8.21(d,J＝8.1Hz,1H),8.09(s,1H),8.04-7.99(m,2H),7.94–7.89(m,1H),7.59–7.50(m,2H),7.40(t,J＝5.7Hz,1H),4.34(d,J＝5.8Hz,2H),3.96(s,3H),1.34(s,9H).
3、(Z)-3-(4-(4-(氨甲基)-1-氧代-1,2-二氢酞嗪-6-基)-1-甲基-1H-吡唑-5-基)-2-(2,2-二氟苯并[d][1,3]二氧杂环戊二烯-5-基)丙烯腈(I-31)的合成
以中间体4-31(107mg,0.190mmol)为原料，操作同I-1，得白色固体85.0mg，收率：97.5％。
1H NMR(300MHz,DMSO-d6)δ(ppm):12.90(s,1H),8.70(s,3H),8.34(s,1H),8.21(d,J＝8.6Hz,1H),8.19(s,1H),8.11-8.04(m,2H),7.98-7.89(m,1H),7.60-7.50(m,1H),4.45-4.31(m,2H),3.97(s,3H).
13C NMR(75MHz,DMSO-d6)δ(ppm):159.61,144.21,143.87,139.20,139.04,138.98,134.49,131.54,131.22,130.92,129.57,128.78,127.34,125.53,123.96,121.68,117.68,116.21,111.03,108.33,66.65,38.08.
HRMS(ESI+):m/z calcd for C23H16F2N6O3463.1330[M+H]+；found:463.1324.
实施例32：本发明部分化合物的PRMT5抑制活性研究
1、实验试剂
PRMT5(Active Motif,Cat.No.31921)[3H]-SAM(PerkinElmer,Cat.No.NET155V001MC)SAM(Sigma,Cat.No.A7007-100MG)SAH(Sigma,Cat.No.A9384-25MG)MTA(Sigma,D5011-100MG)OptiPlate(384-well,white)(Perkin Elmer,Cat.No.6007299)384-well Flashplate(Perkin Elmer,Cat.No.SMP410A001PK)
2、实验方法
(1)准备1x检测缓冲液(改良的Tris缓冲液)；
(2)化合物梯度稀释：在100％DMSO中通过Echo将化合物转移到测试板，DMSO的最终浓度分数是1％；
(3)PRMT5：在1x检测缓冲液中制备酶和[3H]-SAM混合溶液(PRMT5+MTA：在1x检测缓冲液中制备酶、[3H]-SAM和MTA的混合溶液)；
(4)在1x检测缓冲液中制备底物溶液；
(5)PRMT5：将15μL酶和[3H]-SAM混合溶液转移到测试板上(PRMT5+MTA：将15μL酶、[3H]-SAM和MTA混合溶液转移至测试板)；
(6)向每个孔中添加10μL底物溶液以开始反应，将10μL 1x检测缓冲液作为空白对照；
(7)室温孵育90min；
(8)在1x检测缓冲液中加入冷SAM，制成终止液；
(9)每孔加入5μL终止液，停止反应；
(10)每孔转移25μL至Flashplate；
(11)室温下孵育至少1h；
(12)通过Microbeta检测Flashplate；
(13)处理数据：将数据在Excel中拟合，用公式(1)：inh％＝(Max-Signal)/(Max-Min)*100得到抑制值；用公式(2)：Y＝Bottom+(Top-Bottom)/(1+(IC50/X)*HillSlope)得到IC50值，其中Y是抑制率而X是化合物浓度。
3、实验结果
a：A：Inhibitionrate％@100nM≥90％，B：80％≤Inhibitionrate％@100nM＜90％，C：Inhibitionrate％@100nM＜80％。
实施例33：本发明部分化合物的体外细胞SDMA活性评价
1、实验试剂
蛋白酶抑制剂购自MCE公司；磷酸酶抑制剂II购自MCE公司；BCA试剂盒购自碧云天有限公司；PVDF膜购自美国Millipore公司；ECL购自南京诺维赞生物科技有限公司；SDMA抗体购自CST公司，货号13222S；β-Tubulin购自CST公司，货号2146S；HRP偶联二抗购自CST公司；PRMT5抗体购自Abcam公司，货号ab109451。
2、实验方法
在MV-4-11细胞系中，以预期浓度和预期时间给药化合物。用PBS洗涤两次后，用RIPA缓冲液、蛋白酶抑制剂、磷酸酶抑制剂II裂解细胞1h。混合物在12000rpm下离心15min。总蛋白含量由BCA试剂盒定量，稀释蛋白至统一浓度，加入6×Loading Buffer，于100℃变性蛋白，后转移至-80℃保存至分析。等效蛋白通过8～10％的SDS-PAGE电泳，然后转移到PVDF膜。随后，用5％(w/v)脱脂牛奶将膜封闭1～2h。用一抗检测蛋白，然后用HRP偶联的二抗，用ECL作为HRP底物进行可视化。目标印迹用化学发光系统检测。用Image J软件进行灰度分析。
3、实验结果
a：A：IC50＜100nM，B：100nM≤IC50＜1μM，C：IC50≥1.0μM；
b：D：IC50≥10μM，E：1μM≤IC50＜10μM，F：IC50＜1μM。
Claims (10)
1.一种酞嗪酮类化合物，其特征在于，具有式(I)的结构，还包含其异构体、前药、稳定的同位素衍生物、药学上可接受的盐或它们的混合物：
其中：
R1a、R1b分别独立地选自氢、卤素、C1～C6烷基，或者R1a、R1b与所连的碳原子一起形成C3～C8环烷基或C2～C6杂环烷基；当R1a、R1b不同时，与其相连的碳原子为消旋构型、R构型或S构型，所述C2～C6杂环烷基包含1～3个选自N、O或S的杂原子；
A环选自取代的C6～C10芳基、取代的C5～C9杂芳基、取代的C3～C8环烷基或取代的C2～C6杂环烷基；所述的取代基选自至少一个氢、卤素、氰基、C1～C6卤代烷基、C1～C6烷基、C1～C6烷氧基、羟基、氨基、甲氨基、二甲氨基、乙酰氨基、羧基、甲氧羰基或硝基；所述的C5～C9杂芳基和C2～C6杂环烷基包含1～3个选自N、O或S的杂原子；
R2选自氢、卤素、C1～C6烷基、C1～C6烷氧基、羧基、C1～C3烷氧羰基，与R2相连的双键构型为Z构型、E构型或两者的混合物。
2.根据权利要求1所述的酞嗪酮类化合物，其特征在于，所述结构中：
R1a、R1b分别独立地选自氢、甲基或乙基，或者R1a、R1b与所连的碳原子一起形成环丙烷；
A环选自取代的苯环、哒嗪环、吡啶环、嘧啶环、噻吩环、呋喃环、吡咯环、喹啉环、喹唑林环、萘环；所述的取代基选自至少一个氢、氟、氯、溴、氰基、甲基、乙基、三氟甲基、甲氧基、乙氧基、苯氧基、羟基、氨基、甲氨基、二甲氨基、乙酰氨基、羧基、甲氧羰基或硝基；
R2为氢原子，与R2相连的双键构型为Z构型、E构型或两者的混合物。
3.根据权利要求1所述的酞嗪酮类化合物，其特征在于，所述结构中：
R1a或R1b选自氢；
A环选自取代的苯环、吡啶环、噻吩环、苯并[d][1,3]二恶茂或萘环；所述的取代基选自至少一个氢、氟、氯、溴、氰基、甲基、乙基、三氟甲基、甲氧基、乙氧基、苯氧基、羟基、氨基或硝基；
R2为氢原子，与R2相连的双键构型为Z构型。
4.根据权利要求1所述的酞嗪酮类化合物，其特征在于，所述结构中：
R1a或R1b选自氢；
A环选自
R2为氢原子，与R2相连的双键构型为Z构型。
5.根据权利要求1所述的酞嗪酮类化合物，其特征在于，选自以下任一化合物：
6.根据权利要求1所述的酞嗪酮类化合物，其特征在于，所述药学上可接受的盐为所述化合物与选自以下任一的酸形成的盐：
盐酸、氢溴酸、硫酸、磷酸、碳酸、甲磺酸、苯磺酸、对甲苯磺酸、萘磺酸、柠檬酸、苹果酸、酒石酸、乳酸、丙酮酸、乙酸、马来酸、琥珀酸、富马酸、水杨酸、苯基乙酸、杏仁酸或阿魏酸。
7.一种药物组合物，其特征在于，包含权利要求1所述的酞嗪酮类化合物以及药学上可接受的载体。
8.一种权利要求1所述的酞嗪酮类化合物或权利要求7所述的药物组合物在制备PRMT5抑制剂药物中的应用。
9.根据权利要求8所述的应用，其特征在于，所述药物抗肿瘤药物。
10.根据权利要求9所述的应用，其特征在于，所述药物为治疗肺癌、卵巢癌、结直肠癌、乳腺癌、黑色素瘤、白血病或恶性胶质瘤的药物。
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| WO2023059798A1 (en) * | 2021-10-06 | 2023-04-13 | Mirati Therapeutics, Inc. | Methods for separation of enantiomers |
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| WO2023098439A1 (zh) * | 2021-11-30 | 2023-06-08 | 上海和誉生物医药科技有限公司 | 一种吡唑衍生物，其制备方法和在药学上的应用 |
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| WO2023059798A1 (en) * | 2021-10-06 | 2023-04-13 | Mirati Therapeutics, Inc. | Methods for separation of enantiomers |
| WO2023064880A1 (en) * | 2021-10-13 | 2023-04-20 | Remix Therapeutics Inc. | Compounds and methods for modulating nucleic acid splicing |
| WO2023098439A1 (zh) * | 2021-11-30 | 2023-06-08 | 上海和誉生物医药科技有限公司 | 一种吡唑衍生物，其制备方法和在药学上的应用 |
| CN116178347A (zh) * | 2022-06-29 | 2023-05-30 | 苏州浦合医药科技有限公司 | Prmt5-mta抑制剂 |
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