本發明屬藥物合成領域，涉及嘧啶類化合物，尤其涉及一種含有假環的嘧啶類化合物，及其制備方法和應用。本發明的嘧啶類化合物，可以抑制多種腫瘤細胞，尤其能夠選擇性作用于egfrl858r/t790m肺癌細胞，該類化合物能夠克服現有egfr抑制劑耐藥。
背景技術：
據統計顯示，全球由癌癥引起的死亡中，大約有三分之一死于肺癌，這其中80％屬于非小細胞肺癌。外科治療是目前肺癌首選和最主要的治療方法，但是大約有70％的病人會出現繼發性疾病以及轉移。小分子酪氨酸激酶抑制劑(tkis)的發展取得了顯著進展，多種小分子藥物上市。
egfr是erbb酪氨酸激酶受體家族成員之一，該信號通路對細胞的生長、增值和分化等生理過程有重要作用。研究表明，許多腫瘤中egfr高度表達或異常表達。
egfr廣泛分布于哺乳動物的上皮細胞，由細胞為的配體結合區、疏水跨膜結構域和細胞內的激酶區三部分組成。酪氨酸蛋白激酶過度表達時，會阻礙細胞程序死亡，使細胞的生長調控失控，始終處于增生狀態，發展成為惡性腫瘤。臨床研究表明，許多類型的實質性腫瘤如神經膠質細胞瘤、乳腺癌、肺癌、卵巢癌、頭頸部鱗癌、宮頸癌、胃癌等有高水平表達。
現有技術公開了酪氨酸激酶抑制劑是研究最為廣泛的小分子抑制劑之一，其通過競爭性結合胞外配體結合位點，阻斷分子內酪氨酸的自身磷酸化，抑制酪氨酸激酶激活，從而阻斷細胞周期，加速細胞凋亡。
egfrt790m突變是egfr抑制劑最為主要的耐藥機制，目前的egfr抑制劑仍不能完全解決藥物耐藥所導致的臨床局限，而且現有的藥物多以喹唑啉為基本母核的egfr可逆或不可逆抑制劑，其對野生型egfr(egfrwt)選擇性差所導致的毒副作用不可避免。因此迫切需要具有選擇性的新穎骨架化合物來解決egfrt790m導致的耐藥性問題。
技術實現要素：
本發明主要解決的技術問題是提供新的嘧啶類化合物，尤其涉及一種含有假環的嘧啶類化合物，及其制備方法和應用。該化合物能抑制腫瘤細胞生長。
為解決上述技術問題，本發明采用的一個技術方案是：提供一種含有假環的嘧啶類化合物，其特征在于，所述化合物具有的結構為式i：
其中w為nh，r1為含氮五元雜環,r2為含氮堿性基團。
在本發明一個較佳實施例中所述含氮五元雜環的結構為：
在本發明一個較佳實施例中，所述r2為：
在本發明一個較佳實施例中，所述嘧啶類化合物用于制備治療腫瘤的藥物及其藥物組合物中。
在本發明一個較佳實施例中，所述腫瘤為非小細胞肺癌、小細胞肺癌、胰腺癌、鼻咽癌中的任一種。
本發明的有益效果是：
本發明的含有假環的嘧啶類化合物，可以抑制多種腫瘤細胞，尤其能夠選擇性作用于egfrl858r/t790m肺癌細胞，對比野生型細胞，該類化合物的ic50高10-100倍數量級差別，該類化合物是一類新的能夠克服現有egfr抑制劑耐藥并具有選擇性的絡氨酸激酶抑制劑。
具體實施方式
下面將對本發明實施例中的技術方案進行描述，所描述實施例僅是本發明的一部分實施例，而不是全部實施例。基于本發明中的實施例，本領域普通技術人員在沒有做出創造性勞動前提下所獲得的所有其他實施例，都屬于本發明保護的范圍。
下列是本發明中使用的術語定義。除另外指出，本文提供的基團或術語的最初定義單獨或作為其他基團的一部分應用于本說明書中。
“取代雜環”和“取代雜環的”是指雜環或雜環基團中的一個或多個位置被取代，尤其是1-4個取代基，可在任何位置上取代。典型的取代包括但不局限于一個或多個以下取代基：如氫、鹵素、氰基、硝基、三氟甲基、三氟甲氧基、環烷基。
本發明特別優選的通式(i)所示的化合物包括
在一個實施方案中，本發明提供了上述本發明化合物的制備方法。
本發明上述化合物可以采用下述流程中描述的方法和/或本領域普通技術人員已知的其它技術來合成，但不僅限于以下方法。
為方便起見，本發明使用眾所周知的縮寫代表多種化合物，包括但不限于dmf:n,n-二甲基甲酰胺；thf:四氫呋喃；dipea:n,n-二異丙基乙胺反應路線：
反應步驟：
實施例1
步驟1中間體2a的制備
將2-硝基苯乙酮(100mg,0.6mmol)溶于dmf-dma，加熱(100℃)攪拌反應過夜。冷卻，蒸干溶劑，得到中間體1。不經純化直接用于下步反應。220.9[m+h]+
步驟2中間體3a的制備
將中間體1(114mg,0.6mmol)與溶劑量乙醇混合，加入約兩當量水合肼，攪拌反應數小時至中間體1消失。冷卻，蒸干溶劑，得90mg中間體2，收率90％，190[m+h]+。
步驟3中間體4a的制備
將中間體2(90mg,0.5mmol)溶于thf，冰浴條件下加入nah(40mg,1mmol)，攪拌一小時后加入碘甲烷(31ul,0.5mmol)。室溫攪拌反應數小時至中間體2消失，濃縮反應體系，柱層析，得168mg中間體3，收率62％,204[m+h]+。
步驟4中間體5a的制備
將中間體4(168mg,0.8mmol)溶于甲醇，加入pd/c，氫氣加壓，室溫反應過夜。過濾，濃縮，得44mg中間體4，直接用于下步反應,173.9[m+h]+。
實施例2
中間體9a的制備
將中間體5a(60mg,0.3mmol)溶于2-丁醇，加入2,6-二氯嘧啶(44mg,0.3mmol)，dipea(110ul,0.6mmol)，加熱攪拌過夜，冷卻，蒸干溶劑，經柱層析 純化(流動相90:10hexane/etoac)得66mg中間體9a，收率73％,286[m+h]+。
實施例3
將中間體9a(57mg，0.2mmol)與中間體4-氟-2-甲氧基-5-硝基苯胺(50mg，0.2mmol)混合，加入2-戊醇，一次性加入對甲苯磺酸(19mg，0.07mmol)加熱攪拌過夜，冷卻，析出固體，過濾，得26mg中間體10a，不純化直接用于下步反應,436[m+h]+。
實施例4n2-(4-((2-(二甲氨基)乙基)(甲基)氨基)-2-甲氧基-5-硝基苯基)-n4-(2-(1-甲基-1h-吡唑-3-yl)苯基)嘧啶-2,4-二胺(11a)
中間體10a(56mg，0.13mmol)溶于dmf，加入dipea(6ul,0.036mmol)，n,n,n-三甲基乙二胺(13mg，0.13mmol)，加熱攪拌兩小時，蒸干溶劑，經柱層析(流動相1:15甲醇/二氯甲烷)純化得28mg中間體11a.
1hnmr(400mhz,chloroform-d)δ10.56(s,1h),9.05(s,1h),8.26(d,j＝8.3hz,1h),8.05(d,j＝5.8hz,1h),7.63(dd,j＝7.8,1.5hz,1h),7.40(d,j＝2.3hz,1h),7.34(s,1h),7.33–7.28(m,1h),7.09(t,j＝7.6hz,1h),6.65(s,1h),6.58(d,j＝2.3hz,1h),6.31(d,j＝5.8hz,1h),4.01(s,3h),3.95(s,3h),3.24(t,j＝7.2hz,2h),2.86(s,3h),2.56(t,j＝7.2hz,2h),2.27(s,6h).13cnmr(150mhz,cdcl3)δ161.8,160.7,159.4,156.9,152.0,143.5,142.2,137.9,136.6,129.8,128.6,124.8,122.5,120.6,118.6,116.3,102.1,56.2,55.9,55.3,55.2,52.7,52.3,52.2,46.1,36.5.hrms(esi)(m/z):[m+h]+calcdforc26h32n9o3518.2550；found518.2548.mp.110-112℃。
實施例5n1-(2-(二甲氨基)乙基)-5-甲氧基-n1-甲基-n4-(4-((2-(1-甲基-1h-吡唑-3-yl)苯基)氨基)嘧啶-2-yl)苯基-1,2,4-三唑(12a)
中間體11a(28mg，0.05mmol)溶于甲醇，加入pd/c，催化氫化得中間體12a。過濾，蒸干溶劑，不經純化直接用于下步反應。
1hnmr(400mhz,chloroform-d)δ10.50(s,1h),8.50(d,j＝8.3hz,1h),8.03(d,j＝5.7hz,1h),8.01(s,1h),7.64(dd,j＝7.7,1.4hz,1h),7.44(s,1h),7.41(d,j＝2.4hz,1h),7.37(t,j＝7.9hz,1h),7.08(t,j＝7.5hz,1h),6.68(s,1h),6.59(d,j＝2.3hz,1h),6.19(d,j＝5.8hz,1h),4.01(s,3h),3.82(s,3h),2.96(t,j＝6.9hz,2h),2.66(s,3h),2.42(t,j＝6.9hz,2h),2.27(s,6h).13cnmr(150mhz,cdcl3)δ160.4,159.7,157.2,141.1,135.2,135.0,135.0,132.4,131.3,129.8,128.2,126.5,124.0,123.4,123.2,107.1,104.1,98.8,62.8,56.9,51.2,41.0,28.6.hrms(esi)(m/z):[m+h]+calcdforc26h34n9o488.2886；found488.2870.。
實施例6n-(2-((2-(二甲氨基)乙基)(甲基)氨基)-4-甲氧基-5-((4-((2-(1-甲基-1h-吡唑-3-yl)苯基)氨基)嘧啶-2-yl)氨基)苯基)丙烯酰胺(13a)
中間體12a(17mg,0.035mmol)溶于二氯甲烷，冰浴，加入dipea(7l,0.038mmol)，丙烯酰氯(3l,0.035mmol)，反應半小時后蒸干溶劑，經柱層析(流動相1:15甲醇/二氯甲烷)純化得目標產物17mg，收率53％。
1hnmr(400mhz,chloroform-d)δ10.37(s,1h),10.07(s,1h),9.49(s,1h),8.41(d,j＝8.3hz,1h),8.11(d,j＝5.7hz,1h),7.72–7.55(m,1h),7.39(s,0h),7.35(s, 1h),7.02(t,j＝7.6hz,1h),6.76(s,1h),6.57(d,j＝2.4hz,1h),6.47–6.26(m,1h),6.22(d,j＝5.8hz,1h),5.66(dd,j＝9.5,2.4hz,1h),3.99(s,3h),3.85(s,3h),2.88(t,j＝5.6hz,2h),2.69(s,3h),2.27(s,6h).13cnmr(150mhz,cdcl3)δ163.24,161.09,159.92,156.61,151.11,145.25,137.22,135.68,132.57,130.95,129.47,128.10,128.05,127.15,125.92,122.20,121.71,121.63,112.36,104.68,104.48,99.48,57.51,56.16,45.56,43.80,39.27,29.84.hrms(esi)(m/z):[m+h]+calcdforc29h36n9o2542.2992；found542.2990.。
實施例7n2-(4-(2-(二甲氨基)乙氧基)-2-甲氧基-5-硝基苯基)-n4-(2-(1-甲基-1h-吡唑-3-yl)苯基)嘧啶-2,4-二胺(11b)
步驟同實施例4。
1hnmr(400mhz,chloroform-d)δ10.53(s,1h),8.51(d,j＝8.3hz,1h),8.02(d,j＝5.8hz,1h),8.00(s,1h),7.64(dd,j＝7.8,1.6hz,1h),7.41(d,j＝2.3hz,1h),7.40–7.32(m,2h),7.08(d,j＝1.2hz,0h),6.59(d,j＝2.3hz,1h),6.54(s,1h),6.17(d,j＝5.7hz,1h),4.09(s,1h),4.00(s,3h),3.81(s,3h),2.75(s,1h),2.38(s,6h).13cnmr(150mhz,cdcl3)δ160.8,159.8,156.4,151.2,141.2,140.6,137.2,131.0,130.7,128.1,127.9,124.4,122.2,121.9,121.4,108.3,104.4,100.3,99.1,68.0,58.6,57.0,45.8,39.3.hrms(esi)(m/z):[m+h]+calcdforc25h36n8o4505.2312；found505.2315.。
實施例8n2-(5-氨基-4-(2-(二甲氨基)乙氧基)-2-甲氧基苯基)-n4-(2-(1-甲基-1h-吡唑-3-苯基)嘧啶-2,4-二胺(12b)
步驟同實施例5
1hnmr(400mhz,chloroform-d)δ10.57(s,1h),9.22(s,1h),8.24(d,j＝8.3hz, 1h),8.06(d,j＝5.8hz,1h),7.64(dd,j＝7.8,1.5hz,1h),7.41(d,j＝2.3hz,1h),7.34(s,1h),7.30(td,j＝8.3,7.9,1.6hz,1h),7.09(td,j＝7.6,1.2hz,1h),6.61(s,1h),6.59(d,j＝2.3hz,1h),6.33(d,j＝5.8hz,1h),4.20(t,j＝5.8hz,2h),4.01(s,3h),3.97(s,3h),2.82(t,j＝5.8hz,2h),2.38(s,6h).13cnmr(150mhz,cdcl3)δ161.0,159.5,156.6,152.7,151.0,148.8,136.7,133.1,131.0,128.3,128.1,123.7,122.7,121.7,121.3,116.2,104.4,99.6,98.4,69.7,58.2,56.4,46.2,39.3.hrms(esi)(m/z):[m+h]+calcdforc25h31n8o2475.2570；found475.2569.。
實施例9n-(2-(2-(2-(二甲氨基)乙氧基)-4-甲氧基-5-((4-((2-(1-甲基-1h-吡唑-3-苯基)氨基)嘧啶-2-基)氨基)苯基)丙烯酰胺(13b)
步驟同實施例6。
1hnmr(400mhz,chloroform-d)δ10.39(s,1h),9.78(s,1h),9.34(s,1h),8.41(d,j＝8.3hz,1h),8.09(d,j＝5.8hz,1h),7.61(dd,j＝7.8,1.6hz,1h),7.40(d,j＝2.4hz,1h),7.24(t,j＝4.0hz,2h),7.02(t,j＝7.6hz,1h),6.61(s,1h),6.57(d,j＝2.3hz,1h),6.42(d,j＝1.8hz,0h),6.38(d,j＝1.8hz,1h),6.29(d,j＝10.1hz,1h),6.24(d,j＝10.0hz,0h),6.21(d,j＝5.8hz,1h),5.68(dd,j＝10.0,1.8hz,1h),4.11(t,j＝5.2hz,2h),4.00(s,3h),3.85(s,3h),2.62–2.50(m,2h),2.36(s,6h).13cnmr(150mhz,cdcl3)δ163.3,161.1,160.0,156.6,151.1,145.7,142.8,137.2,132.1,130.9,128.1,128.0,126.3,125.0,124.40,122.1,121.3,113.9,104.4,101.8,99.4,70.3,58.1,56.2,45.3,39.2,29.8.hrms(esi)(m/z):[m+h]+calcdforc28h31n8o3529.2676；found529.2680。
實施例10n2-(4-(4-(二甲氨基)哌啶-1-基)-2-甲氧基-5-硝基苯基)-n4-(2-(1-甲基-1h-吡唑-3-基)苯基)嘧啶-2,4-二胺(11c)
步驟同實施例4。
1hnmr(400mhz,chloroform-d)δ10.62(s,1h),9.15(s,1h),8.27(d,j＝8.2hz,1h),8.04(d,j＝5.8hz,1h),7.76(d,j＝8.1hz,1h),7.62(dd,j＝7.8,1.6hz,1h),7.40(d,j＝2.3hz,1h),7.33–7.25(m,1h),7.17(d,j＝7.9hz,1h),7.07(td,j＝7.5,1.2hz,1h),6.57–6.56(m,2h),6.30(d,j＝5.8hz,1h),3.98(s,3h),3.92(s,3h),3.31(d,j＝11.7hz,2h),2.89–2.75(m,3h),2.00(d,j＝11.9hz,2h),1.83-1.81(m,1h).13cnmr(150mhz,chloroform-d)δ160.8,159.2,156.3,151.9,150.8,142.2,141.9,140.1,136.6,136.4,131.0,128.8,128.1,128.0,125.8,124.8,122.5,121.4,121.0,115.9,104.2,102.4,62.4,56.1,51.7,46.1,40.5,39.1,27.3,21.3.hrms(esi)(m/z):[m+h]+calcdforc28h34n9o3544.2779；found544.2786。
實施例11n2-(5-氨基-4-(4-(二甲氨基)哌啶-1-基)-2-甲氧基苯基)-n4-(2-(1-甲基-1h-吡唑-3-基)苯基)嘧啶-2,4-二胺(12c)
步驟同實施例5。
1hnmr(400mhz,chloroform-d)δ10.53(s,1h),8.53(d,j＝8.3hz,1h),8.05(t,j＝2.9hz,2h),7.66(dd,j＝7.8,1.5hz,1h),7.48–7.34(m,3h),7.28(d,j＝0.9hz,1h),7.11(td,j＝7.6,1.2hz,1h),6.67–6.59(m,2h),6.20(d,j＝5.7hz,1h),4.03(s,3h),3.83(s,3h),3.22(d,j＝11.6hz,2h),2.70–2.59(m,2h),2.46(s,6h),2.02(d,j＝12.4hz,2h),1.73(dd,j＝11.8,3.7hz,2h).13cnmr(150mhz,meod)δ163.0,151.3,143.5,141.7,133.0,129.8,129.5,129.1,126.9,125.9,125.2,105.9,105.7,100.2,65.0,57.1,51.2,40.5,39.1,28.3,21.3.hrms(esi)(m/z):[m+h]+calcdforc28h36n9o,514.3037；found514.3040。
實施例12n-(2-(4-(二甲氨基)哌啶-1-基)-4-甲氧基-5-((4-((2-(1-甲基-1h-吡唑-3-基)苯基)氨基)嘧啶-2-基)氨基)苯基)丙烯酰胺(13c)
步驟同實施例6。
1hnmr(400mhz,chloroform-d)δ10.37(s,1h),9.41(s,1h),8.51(s,1h),8.38(d,j＝8.3hz,1h),8.10(d,j＝5.8hz,1h),7.62(dd,j＝7.8,1.6hz,1h),7.40(d,j＝2.3hz,1h),7.32(s,1h),7.04(t,j＝7.6hz,1h),6.72(s,1h),6.58(d,j＝2.3hz,1h),6.41–6.25(m,2h),6.23(d,j＝6.0hz,1h),5.72(d,j＝9.8hz,1h),3.99(s,3h),3.86(s,3h),3.04(d,j＝11.6hz,2h),2.71(t,j＝11.4hz,2h),2.38(s,6h),2.30-2.28(m,2h),2.20-2.15(m,1h).13cnmr(150mhz,meod)δ162.8,161.1,159.8,156.5,151.0,145.2,137.1,135.9,132.3,130.9,128.1,128.0,126.7,126.6,126.4,122.3,121.7,112.1,104.4,103.2,99.5,62.1,56.1,52.4,42.0,39.2,30.0.hrms(esi)(m/z):[m+h]+calcdforc31h38n9o2,568.3134,found568.3137.。
實施例13n2-(2-甲氧基-4-(4-甲基哌啶-1-基)-5-5-硝基苯基)-n4-(2-(1-甲基-1h-吡唑-3-基)苯基)嘧啶-2,4-二胺(11d)
步驟同實施例4。
1hnmr(400mhz,chloroform-d)δ10.59(s,1h),9.20(s,1h),8.25(d,j＝8.3hz,1h),8.06(d,j＝5.8hz,1h),7.64(dd,j＝7.8,1.5hz,1h),7.47–7.38(m,2h),7.35–7.25(m,2h),7.09(td,j＝7.6,1.2hz,1h),6.64(s,1h),6.59(d,j＝2.3hz,1h),6.33(d,j＝5.8hz,1h),3.99(s,3h),3.97(s,3h),3.25(t,j＝4.7hz,4h),2.88(t,j＝4.6hz,4h),2.54(s,3h).13cnmr(150mhz,cdcl3)δ161.8,160.7,159.4,156.9,152.0,143.5,142.2,137.9,136.6,129.8,128.6,124.8,122.5,120.6,118.6, 116.3,102.1,56.2,55.9,55.3,55.2,52.7,52.3,52.2,46.1,36.5.mp.110-113hrms(esi)(m/z):[m+h]+calcdforc26h30n9o3,516.2472,found5516.2468.。
實施例14n2-(5-氨基-2-甲氧基-4-(4-甲基哌啶-1-基)-5-5-硝基苯基)-n4-(2-(1-甲基-1h-吡唑-3-基)苯基)嘧啶-2,4-二胺(12d)
步驟同實施例5。
1hnmr(400mhz,chloroform-d)δ10.55(s,1h),9.17(s,1h),8.24(d,j＝8.2hz,1h),8.05(d,j＝5.8hz,1h),7.63(dd,j＝7.8,1.5hz,1h),7.40(d,j＝2.1hz,2h),7.33–7.28(m,1h),7.13–7.06(m,1h),6.61(s,1h),6.58(d,j＝2.2hz,1h),6.32(d,j＝5.8hz,1h),4.00(s,3h),3.96(s,3h),3.09(t,j＝4.7hz,4h),2.62(t,j＝4.7hz,4h).13cnmr(150mhz,cdcl3)δ162.1,160.7,159.7,156.6,143.3,141.3,138.3,135.3,132.3,129.6,128.5,126.7,122.1,121.7,118.3,107.2,104.3,99.4,56.9,56.0,51.4,46.3,36.5,29.8；hrms(esi)(m/z):[m+na]+calcdforc26h31n9ona,508.2544,found508.2541.。
實施例15n-(4-甲氧基-5-((4-((2-(1-甲基-1h-吡唑-3-基)苯基)氨基)嘧啶-2-基)氨基)苯基)-2-(4-甲基哌啶-1-基)苯基)丙烯酰胺(13d)
步驟同實施例6。
1hnmr(400mhz,chloroform-d)δ10.52(s,1h),9.42(s,1h),8.55(s,1h),8.42(d,j＝8.2hz,1h),8.17(d,j＝5.8hz,1h),7.76(d,j＝8.1hz,1h),7.62(dd,j＝7.8,1.6hz,1h),7.40(d,j＝2.3hz,1h),7.33–7.25(m,1h),7.17(d,j＝7.9hz,1h),7.07(td,j＝7.5,1.2hz,1h),6.57–6.56(m,2h),6.30(d,j＝5.8hz,1h),4.01(s,3h),3.85(s,3h),2.95-2.89(m,4h),2.60-2.58(m,4h),2.38(s,3h).13cnmr(150mhz,cdcl3)δ162.1,160.7,159.8,156.6,143.3,141.3,138.4,135.4,132.2,129.7, 128.5,126.8,122.1,121.7,118.4,107.3,104.3,99.4,56.9,56.0,51.3,46.2,36.5.hrms(esi)(m/z):[m+h]+calcdforc29h34n9o2,540.2830；found540.2835.。
實施例16n2-(2-甲氧基-4-嗎啉-5-硝基苯基)-n4-(2-(1-甲基-1h-吡唑-3-基)苯基)嘧啶-2,4-二胺(11e)
步驟同實施例4。
1hnmr(400mhz,chloroform-d)δ10.55(s,1h),9.18(s,1h),8.05(s,2h),7.63(dd,j＝7.8,1.6hz,1h),7.43–7.39(m,2h),7.30(td,j＝8.3,7.8,1.6hz,1h),7.09(td,j＝7.6,1.3hz,1h),6.60(s,1h),6.58(d,j＝2.3hz,1h),6.32(d,j＝5.8hz,1h),4.00(s,3h),3.97(s,3h),3.89–3.84(m,4h),3.07–3.02(m,4h).13cnmr(151mhz,meod)δ161.0,159.4,156.5,151.9,151.0,141.7,137.2,136.7,131.0,128.3,128.1,125.3,122.7,121.7,121.3,116.1,104.4,102.1,99.7,67.3,67.2,66.5,56.2,52.9,45.4,40.6,39.0.hrms(esi)(m/z):[m+h]+calcdforc25h27n8o4,503.2150,found503.2155.。
實施例17n2-(5-氨基-2-甲氧基-4-嗎啉苯基)-n4-(2-(1-甲基-1h-吡唑-3-基)苯基)嘧啶-2,4-二胺(12e)
步驟同實施例5。
1hnmr(400mhz,chloroform-d)δ10.53(s,1h),8.52(d,j＝8.3hz,1h),8.07(t,j＝4.2hz,2h),8.04(s,0h),7.66(dd,j＝7.8,1.6hz,1h),7.48(s,1h),7.44(d,j＝2.4hz,1h),7.38(t,j＝8.1hz,1h),7.11(t,j＝7.6hz,1h),6.66(s,1h),6.62(d,j＝2.5hz,1h),6.21(d,j＝5.7hz,1h),4.03(s,3h),3.87(t,j＝4.5hz,4h),3.85(s,3h),2.92(t,j＝4.6hz,4h).13cnmr(151mhz,meod)δ161.0,160.8,159.7,156.4,151.2,141.3,137.2,135.3,132.0,131.0,128.1,127.8,127.0,122.2,122.0, 121.5,107.3,104.5,104.2,99.3,67.9,67.3,66.6,56.9,52.0,45.9,40.7,39.3.hrms(esi)(m/z):[m+h]+calcdforc25h29n8o2,573.2408；found473.2411.。
實施例18n-(4-甲氧基-5-((4-((2-(1-甲基-1h-吡唑-3-基)苯基)氨基)嘧啶-2-基)氨基)-2-嗎啉苯基)丙烯酰胺(13e)
步驟同實施例6。
1hnmr(400mhz,chloroform-d)δ10.40(s,1h),9.46(s,1h),8.53(s,1h),8.39(d,j＝8.3hz,1h),8.10(d,j＝5.7hz,1h),8.06(s,1h),7.64–7.61(m,1h),7.40(d,j＝2.5hz,2h),7.25(d,j＝7.4hz,1h),7.04(t,j＝7.5hz,1h),6.74(s,1h),6.58(d,j＝2.3hz,1h),6.41–6.26(m,2h),6.23(d,j＝5.9hz,1h),5.74(d,j＝9.9hz,1h),4.00(s,3h),3.88-3.85(m,7h),2.88(t,j＝4.5hz,4h).13cnmr(151mhz,meod)δ162.7,161.0,160.9,159.8,156.4,151.0,145.3,137.1,135.0,132.2,130.9,128.1,128.0,127.2,126.8,126.6,122.3,121.6,112.2,104.4,103.8,99.6,67.8,67.3,66.5,56.2,52.9,45.5,40.7,39.2.hrms(esi)(m/z):[m+h]+calcdforc28h31n8o3,527.2514,found527.2516.。
實施例19n2-(4-((2-(甲氨基)乙基)(甲基)氨基)-2-甲氧基-5-硝基苯基)-n4-(2-(1-甲基-1h-1,2,4-三唑-3-yl)苯基)嘧啶-2,4-二胺(11n)
步驟同實施例4。
1hnmr(400mhz,chloroform-d)δ10.72(s,1h),9.12(s,1h),8.22(s,1h),8.20(s,2h),8.09(d,j＝5.8hz,1h),7.41(t,j＝7.9hz,1h),7.36(s,1h),7.12(t,j＝7.6hz,1h),6.66(s,1h),6.42(d,j＝5.8hz,1h),4.04(s,3h),3.96(s,3h),3.29–3.23(m,2h),2.87(s,3h),2.57(t,j＝7.2hz,2h),2.28(s,6h).13cnmr(150mhz,cdcl3)δ162.0,160.8,159.6,157.0,152.2,143.5,142.4,138.0,135.1,129.9,128.7,123.4, 122.5,120.6,118.7,116.5,102.2,99.0,57.1,56.2,54.2,45.9,41.5,36.5.hrms(esi)(m/z):[m+h]+calcdforc25h31n10o3519.2581；found519.2584.。
實施例20n1-(2-(二甲氨基)乙基)-5-甲氧基-n1-甲基-n4-(4-((2-(1-甲基-1h-1,2,4-三唑-3-基)苯基)氨基)嘧啶-2-基)氨基)苯基-1,2,4-三胺(12n)
步驟同實施例5。
1hnmr(400mhz,chloroform-d)δ10.74(s,1h),8.46(d,j＝8.3hz,1h),8.19(dd,j＝7.9,1.6hz,1h),8.15(s,1h),7.99(d,j＝5.9hz,1h),7.90(s,1h),7.48–7.39(m,1h),7.19–7.10(m,1h),6.62(s,1h),6.25(d,j＝5.9hz,1h),4.01(s,3h),3.81(s,3h),3.47(s,2h),3.30(t,j＝6.0hz,2h),3.06(t,j＝6.2hz,2h),2.82(s,6h).13cnmr(150mhz,cdcl3)δ161.9,160.8,158.6,143.4,141.6,137.8,137.3,130.4,129.8,129.7,128.6,127.4,122.8,121.6,118.7,107.7,105.4,99.5,57.0,55.6,51.1,50.8,43.6,43.4,36.6.hrms(esi)(m/z):[m+h]+calcdforc25h33n10o489.2839；found489.2840.。
實施例21n-(2-((2-(二甲氨基)乙基)(甲基)氨基)-4-甲氧基-5-((4-((2-(1-甲基-1h-1,2,4-三唑-3-基)苯基)氨基)嘧啶-2-基)氨基)苯基)丙烯酰胺(13n)
步驟同實施例6。
1hnmr(600mhz,chloroform-d)δ10.52(s,1h),10.03(s,1h),9.45(s,1h),8.44(d,j＝8.3hz,1h),8.16(d,j＝7.8hz,1h),8.13(s,1h),8.13(s,2h),7.54(d,j＝8.6hz,1h),7.37(d,j＝12.3hz,2h),7.33(t,j＝7.8hz,1h),7.14–7.12(m,1h),7.05(t,j＝7.6hz,1h),6.76(s,1h),4.00(s,3h),3.86(s,3h),2.89(t,j＝5.6hz,2h),2.69(s,3h),2.32(t,j＝5.8hz,2h),2.28(s,6h).13cnmr(15mhz,cdcl3)δ 163.3,162.1,160.9,159.9,156.8,145.3,143.4,138.3,135.8,132.6,129.8,129.3,128.4,127.0,125.9,124.8,121.9,121.1,118.3,104.6,99.5,56.1,45.4,43.8,36.5.hrms(esi)(m/z):[m+h]+calcdforc28h35n10o2543.2944；found543.2950.。
實施例22
鄰硝基氯苯(3g,19mmol)溶于dmf，加入吡唑(1.5g,22.8mmol)，碳酸鉀(3.1g,22.8mmol)，cui(36mg,0.19mmol)，110℃反應過夜，過濾，濃縮，得1.9g黃色固體。
實施例23
操作同實施例2.。273[m+h]+。
實施例24
操作同實施例3。422[m+h]+。
實施例25n4-(2-(1h-吡唑-1-基)苯基)-n2-(4-((2-(二甲氨基)乙基)(甲基)氨基)-2-甲氨基-5-硝基苯基)嘧啶-2,4-二胺(11f)
操作同實施例4。
1hnmr(400mhz,chloroform-d)δ9.55(s,1h),8.93(s,1h),8.20(dd,j＝8.2,1.4hz,1h),8.05(d,j＝5.8hz,1h),7.86(d,j＝1.9hz,1h),7.80(d,j＝2.4hz,1h), 7.40–7.32(m,4h),7.16(td,j＝7.7,1.4hz,1h),6.64(s,1h),6.49(t,j＝2.2hz,1h),6.18(d,j＝5.8hz,1h),3.94(s,4h),3.26–3.22(m,2h),2.86(s,4h),2.55(dd,j＝8.0,6.4hz,3h),2.27(s,6h).13cnmr(150mhz,meod)δ160.5,159.4,157.0,152.1,142.3,141.2,135.1,132.5,130.2,130.2,128.1,123.5,123.5,123.2,123.1,116.4,107.1,102.1,99.2,57.0,56.1,54.2,45.8,41.4.。
實施例26n4-(4-((2-(1h-吡唑-1-基)苯基)氨基)嘧啶-2-基)-n1-(2-(二甲氨基)乙基)-5-甲氧基-n1-甲基苯基-1,2,4-三唑(12f)
同實施例5
1hnmr(400mhz,chloroform-d)δ9.35(s,1h),8.36(dd,j＝8.3,1.3hz,1h),8.02(d,j＝5.7hz,1h),7.92(s,1h),7.81(dd,j＝9.8,2.2hz,2h),7.49–7.39(m,2h),7.36(dd,j＝8.0,1.5hz,1h),7.21–7.12(m,1h),6.67(s,1h),6.48(t,j＝2.2hz,1h),6.09(d,j＝5.7hz,1h),3.80(s,3h),2.96(dd,j＝7.6,6.1hz,2h),2.65(s,3h),2.42(dd,j＝7.6,6.1hz,2h),2,27(s,6h).13cnmr(150mhz,meod)δ155.7,155.0,152.3,136.5,136.4,131.8,128.3,127.7,125.7,125.6,123.0,122.0,119.5,118.9,118.5,102.4,102.2,100.4,94.1,53.1,52.2,50.1,41.2,38.1.。
實施例27n-(5-((4-((2-(1h-吡唑-1-基)苯基)氨基)嘧啶-2-基)氨基)-2-((2-(二甲氨基)乙基)(甲基)氨基)-4-甲氧基苯基)丙烯酰胺(13f)
操作同實施例6。
1hnmr(400mhz,chloroform-d)δ10.07(s,1h),9.42(s,1h),9.20(s,1h),8.25(d,j＝8.1hz,1h),8.09(d,j＝5.7hz,1h),7.82(d,j＝1.9hz,1h),7.78(d,j＝2.5hz,1h),7.32(q,j＝3.5,3.0hz,3h),7.13–7.06(m,1h),6.75(s,1h),6.50–6.42(m, 1h),6.42–6.28(m,2h),6.11(d,j＝5.8hz,1h),5.68(dd,j＝9.2,2.6hz,1h),3.84(s,3h),2.88(t,j＝5.6hz,2h),2.69(s,3h),2.29(s,9h).13cnmr(150mhz,cdcl3)δ163.2,160.6,159.8,157.2,145.2,141.2,135.8,132.9,132.5,130.6,130.3,129.3,127.9,126.8,126.0,123.8,123.7,123.2,112.3,107.1,104.6,98.6,57.4,56.4,56.1,45.5,43.7.hrms(esi)(m/z):[m+h]+calcdforc28h34n9o2,528.2830；found528.2828.。
實施例28n4-(2-(1h-吡唑-1-基)-n2-(4-(4-(二甲氨基)哌啶-1-基)-2-甲氧基-5-硝基苯基)嘧啶-2,4-二胺(11g)
操作同實施例4。
1hnmr(400mhz,chloroform-d)δ9.56(s,1h),9.03(s,1h),8.18(d,j＝8.1hz,1h),8.04(d,j＝5.8hz,1h),7.85(d,j＝1.9hz,1h),7.79(d,j＝2.5hz,1h),7.38–7.32(m,3h),7.18–7.12(m,1h),6.56(s,1h),6.47(t,j＝2.3hz,1h),6.18(d,j＝5.8hz,1h),3.93(s,3h),3.32(d,j＝11.7hz,2h),2.79(td,j＝11.7,2.3hz,2h),2.34(s,6h),1.91-1.89(m,2h),1.91-1.77(m,1h).13cnmr(150mhz,cdcl3)δ160.5,159.3,157.0,151.9,142.6,141.3,141.2,136.3,132.5,130.2,130.2,128.0,124.2,123.6,123.4,123.3,116.4,107.1,102.2,99.3,62.0,56.1,52.2,41.6,28.4.hrms(esi)(m/z):[m+h]+calcdforc27h32n9o3,530.2623；found530.2628.。
實施例29n-(5-((4-((2-(1h-吡唑-1-基)苯基)氨基)嘧啶-2-基)氨基)-2-(4-(二甲氨基)哌啶-1-基)-4-甲氧基苯基)丙烯酰胺(12g)
操作同實施例5。
1hnmr(400mhz,chloroform-d)δ9.36(s,1h),8.06–8.00(m,2h),7.94(s,1h),7.81(dd,j＝8.3,2.1hz,2h),7.49–7.32(m,3h),7.21–7.12(m,1h),6.63(s,1h), 6.49(t,j＝2.2hz,1h),6.08(d,j＝5.7hz,1h),4.40(ddd,j＝15.7,4.8,2.3hz,2h),3.80(s,3h),3.69-3.63(m,2h),3.17(d,j＝11.5hz,2h),3.07(td,j＝12.6,3.0hz,2h),2.44-2.36(m,2h),2.37(s,3h),2.34-2.31(m,2h),2.29(s,6h),1.68-1.62(m,3h).13cnmr(150mhz,cdcl3)δ160.5,160.2,159.5,156.8,140.9,140.9,135.0,132.8,130.4,130.1,127.5,126.1,124.1,123.4,123.1,107.0,106.8,103.9,98.5,62.1,62.0,56.7,51.4,44.9,41.7,41.5,38.6,29.3,29.0,27.4.hrms(esi)(m/z):[m+h]+calcdforc27h34n9o,500.2881；found500.2877.。
實施例30n-(5-((4-((2-(1h-吡唑-1-基)苯基)氨基)嘧啶-2-基)氨基)-2-(4-(二甲氨基)哌啶-1-基)-4-甲氧基苯基)丙烯酰胺(13g)
步驟同實施例6。
1hnmr(400mhz,chloroform-d)δ9.36(s,1h),9.25(s,1h),8.44(s,1h),8.24(d,j＝8.2hz,1h),8.08(d,j＝5.8hz,1h),7.82(d,j＝1.8hz,1h),7.78(d,j＝2.4hz,1h),7.36–7.28(m,3h),7.10(t,j＝7.6hz,1h),6.70(s,1h),6.47(t,j＝2.2hz,1h),6.41–6.29(m,2h),6.13(d,j＝5.8hz,1h),5.74(d,j＝9.8hz,1h),5.30(s,1h),3.85(s,3h),3.05(d,j＝11.6hz,2h),2.77–2.66(m,2h),2.46(s,6h),2.08(d,j＝12.4hz,2h),1.78-1.74(m,1h).13cnmr(150mhz,cdcl3)δ160.6,159.8,157.2,141.2,132.9,132.3,130.5,130.3,127.9,126.6,126.5,123.6,123.3,112.2,107.1,103.25,98.7,62.3,56.1,52.3,41.8,29.8,29.6.hrms(esi)(m/z):[m+h]+calcdforc30h36n9o2,554.2986；found554.2981。
實施例31n4-(2-(1h-吡唑-1-基)苯基)-n2-(2-methoxy-4-(4-甲基哌嗪-1-基)-5-硝基苯基)嘧啶-2,4-二胺(11h)
同實施例4。
1hnmr(400mhz,chloroform-d)δ9.58(s,1h),9.07(s,1h),8.23–8.17(m,1h),8.07(d,j＝5.8hz,1h),7.87(d,j＝1.9hz,1h),7.82(d,j＝2.5hz,1h),7.41–7.35(m,2h),7.18(td,j＝7.6,1.3hz,1h),6.62(s,1h),6.50(t,j＝2.2hz,1h),6.21(d,j＝5.8hz,1h),3.97(s,3h),3.11(t,j＝4.8hz,4h),2.66(t,j＝4.7hz,4h),2.40(s,3h).13cnmr(150mhz,meod)δ160.5,159.3,157.0,151.89,141.9,141.2,136.9,132.5,130.2,130.2,128.0,124.7,123.6,123.5,123.3,116.2,107.1,102.2,99.4,56.2,55.2,52.3,46.1.hrms(esi)(m/z):[m+h]+calcdforc25h28n9o3,502.2310；
found502.2306.。
實施例32n4-(2-(1h-吡唑-1-基)苯基)-n2-(5-氨基-2-甲氧基-4-(4-甲基哌嗪-1-基)苯基)嘧啶-2,4-二胺(12h)
同實施例5。
1hnmr(400mhz,chloroform-d)δ9.35(s,1h),8.37(dd,j＝8.3,1.4hz,1h),8.03(t,j＝2.8hz,2h),7.95(s,1h),7.82(d,j＝1.9hz,1h),7.80(d,j＝2.5hz,1h),7.47–7.40(m,2h),7.36(dd,j＝8.0,1.5hz,1h),7.16(td,j＝7.7,1.4hz,1h),6.66(s,1h),6.49(t,j＝2.2hz,1h),6.09(d,j＝5.7hz,1h),3.80(s,3h),3.43–3.36(m,4h),2.44–2.39(m,4h),2.32(s,6h).13cnmr(150mhz,meod)δ160.8,160.4,159.6,157.0,141.2,135.2,133.0,132.3,130.4,130.3,127.7,126.6,124.2,123.5,123.2,107.1,107.0,104.2,98.8,56.8,56.0,55.5,54.3,51.5,46.3,46.2,45.6,39.99.hrms(esi)(m/z):[m+h]+calcdforc25h30n9o,472.2568；found472.2565.。
實施例33n-(5-((4-((2-(1h-吡唑-1-基)苯基)氨基)嘧啶-2-基)氨基)-4-甲氧基-2-(4-甲基哌嗪-1-基)苯基)丙烯酰胺(13h)
同實施例6。
1hnmr(400mhz,chloroform-d)δ10.55(s,1h),9.45(s,1h),8.60–8.55(m,1h),8.45(d,j＝8.4hz,1h),8.25–8.12(m,4h),8.04(s,0h),7.37(d,j＝8.0hz,2h),7.08(t,j＝7.4hz,1h),6.79(s,1h),6.39–6.29(m,2h),5.76(d,j＝9.7hz,1h),4.03(s,3h),3.88(s,3h),2.99-2.94(m,4h),2.63-2.61(m,4h),2.41(s,3h).13cnmr(151mhz,cdcl3)δ162.7,160.6,159.8,157.2,145.1,141.2,135.3,132.9,132.3,130.5,130.2,127.9,126.8,126.7,126.4,123.6,123.3,111.9,107.1,107.1,103.5,98.7,56.1,56.1,52.5,46.2.hrms(esi)(m/z):[m+h]+calcdforc28h32n9o2,526.2673；found526.2669.。
實施例34n4-(2-(1h-吡唑-1-基)苯基)-n2-(2-甲氧基-4-嗎啉-5-硝基苯基)嘧啶-2,4-二胺(11i)
步驟同實施例4。
1hnmr(400mhz,chloroform-d)δ9.58(s,1h),9.07(s,1h),8.18(d,j＝8.2hz,1h),8.06(d,j＝5.8hz,1h),7.86(d,j＝1.9hz,1h),7.80(d,j＝2.5hz,1h),7.37(dd,j＝15.1,7.5hz,3h),7.17(td,j＝7.6,1.4hz,1h),6.59(s,1h),6.49(t,j＝2.2hz,1h),6.21(d,j＝5.8hz,1h),3.96(s,3h),3.91–3.83(m,4h),3.07–3.00(m,4h).13cnmr(151mhz,cdcl3)δ160.5,159.3,157.0,151.8,141.7,141.2,137.2,132.5,130.3,130.2,128.0,125.1,123.6,123.5,123.3,116.1,107.1,102.1,99.4,67.1,56.2,52.9.hrms(esi)(m/z):[m+h]+calcdforc24h25n8o4,489.1993；found489.1987.。
實施例35n4-(2-(1h-吡唑-1-基)苯基)-n2-(5-氨基-2-甲氧基-4-嗎啉苯基)嘧啶-2,4-二胺(12i)
步驟同實施例5。
1hnmr(400mhz,chloroform-d)δ9.38(s,1h),8.38(d,j＝8.4hz,1h),8.03(d,j＝5.7hz,1h),7.97(s,1h),7.83(d,j＝1.9hz,1h),7.80(d,j＝2.4hz,1h),7.49(s,1h),7.46–7.40(m,1h),7.36(dd,j＝8.1,1.5hz,1h),7.26(d,j＝0.9hz,3h),7.17(t,j＝7.8hz,1h),6.64(s,1h),6.50(t,j＝2.2hz,1h),6.10(d,j＝5.7hz,1h),3.85(t,j＝4.5hz,4h),3.82(s,3h),2.90(t,j＝4.5hz,4h).13cnmr(151mhz,cdcl3)δ160.4,159.6,156.9,141.3,141.2,135.3,133.1,132.1,130.5,130.3,127.7,126.8,124.2,123.5,123.2,107.2,107.1,104.2,98.9,67.9,56.9,52.0.。
實施例36n-(5-((4-((吡唑-1-基)苯基)氨基)嘧啶-2基)氨基)-4-甲氧基-2-嗎啉苯基)丙烯酰胺(13i)
步驟同實施例6。
1hnmr(400mhz,chloroform-d)δ9.41(s,1h),9.27(s,1h),8.50(s,1h),8.23(d,j＝8.2hz,1h),8.09(d,j＝5.8hz,1h),7.82(d,j＝1.9hz,1h),7.78(d,j＝2.4hz,1h),7.38–7.27(m,3h),7.10(t,j＝7.6hz,1h),6.72(s,1h),6.46(t,j＝2.2hz,1h),6.42–6.24(m,2h),6.14(d,j＝5.8hz,1h),5.80–5.71(m,1h),3.86-3.79(m,7h),2.86(t,j＝4.5hz,4h).13cnmr(150mhz,cdcl3)δ162.7,160.6,159.7,157.1,145.1,141.2,135.0,132.8,132.2,130.5,130.2,127.8,126.9,126.7,126.5,123.6,123.3,112.1,107.1,103.4,98.8,67.8,56.1,52.9.。
實施例37
操作同實施例3。422.9[m+h]+。
實施例38n4-(2-(2h-1,2,3-三唑-2-基)苯基)-n2-(4-((2-(二甲氨基)乙基)(甲基)氨基)-2-甲氧基-5-硝基苯基)嘧啶-2,4-二胺(11j)
步驟同實施例4。
1hnmr(400mhz,chloroform-d)δ9.82(s,1h),9.01(s,1h),8.16(d,j＝8.3hz,1h),8.08(d,j＝5.8hz,1h),7.99(dd,j＝8.4,1.7hz,1h),7.93(s,2h),7.43–7.35(m,2h),7.23–7.17(m,1h),6.65(s,1h),6.31(d,j＝5.8hz,1h),3.94(s,4h),3.26(t,j＝7.2hz,2h),2.86(s,3h),2.58(t,j＝7.2hz,2h).13cnmr(150mhz,cdcl3)δ160.5,159.4,157.3,152.2,142.5,135.1,135.0,135.0,131.0,129.7,128.5,123.5,123.4,123.1,122.8,116.5,102.1,98.7,57.0,56.1,54.0,45.8,41.4.hrms(esi)(m/z):[m+h]+calcdforc24h29n10o3,505.2419；found505.2418.。
實施例39n4-(4-((2-(2h-1,2,3-三唑-2-基)苯基)氨基)嘧啶-2-基)-n1-(2-(二甲氨基)乙基)-5-甲氧基-n1-甲基苯基-1,2,4-三胺(12j)
步驟同實施例5。
1hnmr(400mhz,chloroform-d)δ9.63(s,1h),8.40(d,j＝8.3hz,1h),8.07(d,j＝5.7hz,1h),8.01(s,1h),7.95–7.88(m,5h),7.52–7.43(m,2h),7.22(t,j＝7.7hz,1h),6.67(s,1h),6.18(d,j＝5.7hz,1h),3.81(s,3h),2.96(t,j＝6.6hz,2h),2.66(s,3h),2.42(dd,j＝8.6,5.1hz,2h),2.29(s,3h),2.28(s,3h).13cnmr(150mhz,meod)δ160.4,159.7,157.2,141.1,135.2,135.0,135.0,132.4,131.3,129.8,128.2,126.5,124.0,123.4,123.2,107.1,104.1,98.8,62.8,56.9,51.2,41.0,28.6.hrms(esi)(m/z):[m+h]+calcdforc24h31n10o,475.2677；found475.2674.。
實施例40n-(5-((4-((2-(2h-1,2,3-三唑-2-基)苯基)氨基)嘧啶-2-基)氨基)-2-((2-(二甲氨基)乙基)(甲基)氨基)-4-甲氧基苯基)丙烯酰胺(13j)
步驟同實施例6。
1hnmr(400mhz,chloroform-d)δ10.09(s,1h),9.67(s,1h),9.48(s,1h),8.24(d,j＝8.2hz,1h),8.13(d,j＝5.6hz,1h),7.95(dd,j＝8.2,1.6hz,1h),7.91(s,2h),7.39–7.31(m,2h),7.17–7.11(m,1h),6.76(s,1h),6.26(d,j＝5.8hz,1h),5.71–5.66(m,1h),3.85(s,3h),2.88(t,j＝5.6hz,2h),2.69(s,3h),2.33–2.26(m,9h).13cnmr(150mhz,cdcl3)δ163.2,160.6,159.9,157.5,145.1,135.8,135.0,134.9,132.6,131.4,129.9,129.4,128.4,126.8,125.9,123.3,123.3,123.1,112.3,104.6,98.4,57.5,56.1,45.5,43.7.hrms(esi)(m/z):[m+h]+calcdforc27h33n10o2,529.2782；found529.2780。
實施例41n4-(2-(2h-1,2,3-三唑-2-基)苯基)-n2-(4-(4-(二甲氨基)哌啶-1-基)-2-甲氧基-5硝基苯基)嘧啶-2,4-二胺(11k)
步驟同實施例4。
1hnmr(400mhz,chloroform-d)δ9.82(s,1h),9.11(s,1h),8.15(d,j＝8.1hz,1h),8.08(d,j＝5.8hz,1h),8.01–7.97(m,2h),7.93(s,2h),7.43–7.36(m,2h),7.21(ddd,j＝8.5,7.3,1.4hz,1h),6.56(s,1h),6.31(d,j＝5.8hz,1h),3.94(s,3h),3.34(d,j＝11.6hz,2h),2.80(m,2h),2.40-2.36(m,2h),2.34(s,6h),1.90-1.88(m,2h),1.78(m,1h).13cnmr(150mhz,meod)δ160.5,159.3,157.3,152.1,142.9,135.9,135.2,135.1,135.0,130.9,129.7,128.5,124.0,123.6,123.4,122.8,116.5,102.0,98.7,61.9,56.2,56.1,52.2,41.6,41.5,41.4,28.3.hrms(esi)(m/z):[m+h]+calcdforc26h31n10o3,531.2575；found531.2570.。
實施例42n4-(2-(2h-1,2,3-三唑-2-基)苯基)-n2-(5-氨基-4-(4-(二甲氨基)哌啶-1- 基)-2-甲氧基苯基)嘧啶-2,4-二胺(12k)
步驟同實施例5。
1hnmr(400mhz,chloroform-d)δ9.62(s,1h),8.38(d,j＝8.0hz,1h),8.05(d,j＝5.7hz,1h),8.00(d,j＝7.1hz,1h),7.92(s,1h),7.90(s,2h),7.49–7.43(m,3h),7.21(t,j＝7.7hz,1h),6.60(s,1h),6.17(d,j＝5.7hz,1h),3.80(s,3h),3.2-3.18(m,2h),2.65-2.59(m,2h),2.50(s,6h),2.07-2.04(m,2h),1.76(m,2h).13cnmr(150mhz,meod)δ160.4,159.7,157.2,141.1,135.2,135.0,135.0,132.4,131.3,129.8,128.2,126.5,124.0,123.4,123.2,107.1,104.1,98.8,62.8,56.9,51.2,41.0,28.6.hrms(esi)(m/z):[m+h]+calcdforc26h33n10o,501.2833；found501.2835.。
實施例43n-(5-((4-((2-(2h-1,2,3-三唑-2-基)苯基)氨基)嘧啶-2-基)氨基)-2-(4-(二甲氨基)哌啶-1-基)-4-甲氧基苯基)丙烯酰胺(13k)
步驟同實施例6。
1hnmr(400mhz,chloroform-d)δ9.67(s,1h),9.41(s,1h),8.49(s,1h),8.22(d,j＝8.2hz,1h),8.12(d,j＝5.7hz,1h),7.96(d,j＝9.3hz,1h),7.91(s,2h),7.38–7.31(m,2h),7.16(t,j＝7.7hz,1h),6.71(s,1h),6.39–6.29(m,1h),6.27(d,j＝5.7hz,1h),5.77–5.72(m,1h),3.85(s,3h),3.05(d,j＝11.4hz,2h),2.78–2.68(m,2h),2.43(s,6h),2.39-2.35(m,2h),2.09-2.01(m,2h),1.79–1.65(m,2h).13cnmr(150mhz,cdcl3)δ162.8,160.6,159.9,157.4,135.1,132.3,131.4,129.9,128.2,126.7,126.4,123.4,123.3,112.0,103.2,98.4,62.2,56.2,52.3,41.8,29.8.hrms(esi)(m/z):[m+h]+calcdforc29h35n10o2,555.2939；found555.2928.。
實施例44n4-(2-(2h-1,2,3-三唑-2-基)苯基)-n2-(2-甲氧基-4-(4-甲基哌啶-1-基)-5-硝基苯基)嘧啶-2,4-二胺(11l)
步驟同實施例4。
1hnmr(400mhz,chloroform-d)δ9.82(s,1h),9.13(s,1h),8.15(d,j＝8.3hz,1h),8.10(d,j＝6.2hz,1h),8.02–7.98(m,1h),7.93(s,1h),7.43–7.36(m,2h),7.22(t,j＝7.8hz,1h),6.60(s,1h),6.32(d,j＝5.8hz,1h),3.96(s,3h),3.10(t,j＝4.7hz,5h),2.63(t,j＝4.6hz,5h),2.38(s,3h).13cnmr(150mhz,cdcl3)δ160.5,159.4,157.3,152.0,142.2,136.7,135.1,135.1,130.9,129.8,128.5,124.6,123.6,123.5,122.9,116.3,102.1,98.9,56.2,55.2,52.3,46.1.hrms(esi)(m/z):[m+h]+calcdforc24h27n10o3,503.2262；found503.2263.。
實施例45n4-(2-(2h-1,2,3-三唑-2-基)苯基)-n2-(5-氨基-2-甲氧基-4-(4-甲基哌啶-1-基)苯基)嘧啶-2,4-二胺(12l)
步驟同實施例5。
1hnmr(400mhz,chloroform-d)δ9.63(s,1h),8.39(dd,j＝8.4,1.4hz,1h),8.07(d,j＝5.7hz,1h),8.01(dd,j＝8.1,1.6hz,1h),7.95(s,1h),7.91(s,2h),7.50(s,1h),7.46(td,j＝8.4,7.8,1.6hz,1h),7.22(ddd,j＝8.5,7.3,1.3hz,1h),6.66(s,1h),6.19(d,j＝5.7hz,1h),3.82(s,3h),3.07(t,j＝4.9hz,4h),2.54(s,3h).13cnmr(151mhz,cdcl3)δ160.5,159.7,157.1,141.3,135.1,135.08,131.4,129.9,128.2,127.0,124.0,123.4,123.2,107.1,104.3,98.9,56.9,55.6,50.4,45.5.hrms(esi)(m/z):[m+h]+calcdforc24h29n10o,473.2520；found573.2524.。
實施例46n-(5-((4-((2-(2h-1,2,3-三唑-2-基)苯基)氨基)嘧啶-2-基)氨基)-4-甲氧基-2-(4-甲基哌啶-1-基)苯基)嘧啶-2,4-二胺(13l)
步驟同實施例6。
1hnmr(400mhz,chloroform-d)δ9.69(s,1h),9.43(s,1h),8.50(s,1h),8.22(d,j＝8.3hz,1h),8.12(d,j＝5.8hz,1h),7.96(dd,j＝8.1,1.5hz,1h),7.91(s,2h),7.45(s,1h),7.34(t,j＝7.8hz,1h),7.16(t,j＝7.7hz,1h),6.76(s,1h),6.37(d,j＝1.7hz,1h),6.32(d,j＝9.9hz,1h),6.27(d,j＝5.8hz,1h),5.75(d,j＝10.0hz,1h),3.85(s,3h),2.95-2.90(m,4h),2.74-2.63(m,4h)2.46(s,3h).13cnmr(151mhz,cdcl3)δ162.7,160.7,159.7,157.1,135.1,132.3,131.3,129.9,128.4,126.8,126.5,123.4,123.3,112.0,103.6,98.5,56.1,55.9,52.2,45.9.hrms(esi)(m/z):[m+h]+calcdforc27h31n10o2,527.2626；found527.2626.。
實施例47n4-(2-(2h-1,2,3-三唑-2-基)苯基)-n2-(2-甲氧基-4-嗎啉-5-硝基苯基)嘧啶-2,4-二胺(11m)
步驟同實施例4。
1hnmr(400mhz,chloroform-d)δ9.82(s,1h),9.16(s,1h),8.15(d,j＝8.3hz,1h),8.10(d,j＝5.8hz,1h),8.00(dd,j＝8.2,1.5hz,1h),7.94(s,2h),7.44–7.37(m,2h),7.22(ddd,j＝8.4,7.4,1.3hz,1h),6.60(s,1h),6.34(d,j＝5.8hz,1h),5.30(s,0h),3.98(s,3h),3.92–3.82(m,4h),3.13–3.01(m,4h).13cnmr(151mhz,cdcl3)δ160.6,159.4,157.4,151.9,141.9,137.0,135.1,130.9,129.9,128.5,125.0,123.7,123.5,122.9,116.3,102.1,99.0,67.2,56.2,52.9.hrms(esi)(m/z):[m+h]+calcdforc23h24n9o4,490.1946；found490.1943。
實施例48n4-(2-(2h-1,2,3-三唑-2-基)苯基)-n2-(5-氨基-2-甲氧基-4-(4-甲基哌啶-1-基)苯基)嘧啶-2,4-二胺(12m)
步驟同實施例5。
1hnmr(400mhz,chloroform-d)δ9.64(s,1h),8.41(d,j＝8.3hz,1h),8.07(d,j＝5.7hz,1h),8.01(dd,j＝8.2,1.4hz,1h),7.96(s,1h),7.91(s,1h),7.52–7.42(m,2h),7.22(t,j＝8.0hz,1h),6.64(s,1h),6.19(d,j＝5.7hz,1h),3.86(t,j＝4.5hz,4h),3.83(s,3h),2.94–2.87(m,4h).13cnmr(151mhz,cdcl3)δ160.5,159.7,157.2,141.3,135.4,135.0,132.2,131.4,129.9,128.2,126.7,124.0,123.4,123.2,107.1,104.2,98.9,67.9,56.9,52.0.hrms(esi)(m/z):[m+h]+calcdforc23h26n9o2,460.2204；found460.2206.。
實施例49n-(5-((4-((2-(2h-1,2,3-三唑-2-基)苯基)氨基)嘧啶-2-基)氨基)-4-甲氧基-2-嗎啉苯基)丙烯酰胺(13m)
步驟同實施例6。
1hnmr(400mhz,chloroform-d)δ9.69(s,1h),9.47(s,1h),8.53(s,1h),8.21(d,j＝8.4hz,11h),8.13(d,j＝5.8hz,11h),7.97(dd,j＝8.1,1.4hz,10h),7.92(d,j＝0.9hz,19h),7.34(t,j＝7.6hz,9h),7.16(t,j＝7.7hz,11h),6.74(s,11h),6.43–6.29(m,18h),6.28(d,j＝6.0hz,14h),5.76(d,j＝10.0hz,10h),5.30(s,10h),2.88(t,j＝4.5hz,47h).13cnmr(151mhz,cdcl3)δ160.7,135.1,132.3,128.4,126.5,123.4,103.5,98.5,67.8,56.2,53.5,53.0,29.8,29.4.hrms(esi)(m/z):[m+h]+calcdforc26h28n9o3,514.2310；found514.2314.。
實施例50藥學實驗
1)嘧啶衍生物的激酶抑制活性測試
檢測本發明化合物對激酶的抑制活性，采用方法為酶聯免疫吸附法 (enzyme-linkedimmunosorbentassay,elisa)檢測激酶磷酸化底物的能力，計算化合物對激酶活性的抑制作用。采用bac-to-bactm桿狀病毒表達系統(invitrogen,carlsbad,ca,usa)進行野生型egfr和egfrt790m/l858r突變的激酶區表達，經鎳柱(qiageninc.,valencia,ca,usa)純化。
elisa主要步驟如下：酶反應底物poly(glu,tyr)4:1用無鉀離子的pbs(10mm磷酸鈉緩沖液，150mmnacl,ph7.2-7.4)稀釋成20μg/ml，于37℃反應12-16h包被酶標板，然后用含0.1％tween-20的pbs(t-pbs)洗板，每孔加入用反應緩沖液(50mmhepesph7.4,50mmmgcl2,0.5mmmncl2,0.2mmna3vo4,1mmdtt)稀釋的atp(終濃度5μm)溶液，加入不同濃度的待測化合物或溶劑對照，然后分別加入相應激酶啟動反應，37℃搖床反應1h。t-pbs洗板三次，加入抗體py99100μl/孔(用含bsa的t-pbs稀釋)于37℃搖床反應0.5h。t-pbs洗板后，加入辣根過氧化物酶標記的羊抗鼠的igg100μl/孔(用含bsa的t-pbs稀釋)，37℃搖床反應0.5h，再次洗板后，加入2mg/ml的opd顯色液100μl/孔，25℃避光反應1-10min。加入2mh2so4終止反應，用可調波長式微孔板酶標儀spectramax190讀數，波長為492nm。ic50值由抑制曲線得到。
表1受試化合物對激酶的抑制活性(ic50,nm)
實驗結果表明，受試化合物對egfrt790m/l858r具有較強的抑制活性，對野生型egfr抑制活性較弱，表現出較好的選擇性，其中化合物13a，13f和13n對egfrt790m/l858r的激酶抑制活性稍強于對照化合物azd9291，且選擇性優于azd9291；
2)嘧啶衍生物的體外人腫瘤細胞增值抑制實驗
檢測本發明化合物對體外人腫瘤細胞增殖抑制活性，采用的方法為磺酰羅丹明b(sulforodamineb，srb)法。
選用含野生型egfr人表皮細胞癌a431細胞株和含egfrt790m/l858r突變人非小細胞肺癌nci-h1975細胞株(均購自atcc)進行檢測。細胞以一定密度接種于96孔板，貼壁過夜，加入不同濃度化合物作用72h，用預冷的10％三氯乙酸于4℃固定1h，蒸餾水洗去固定液后烘干，使用4mg/ml的srb溶液(溶于1％醋酸)室溫染色15分鐘，洗去多余染色液，室溫烘干，在使用酶標儀檢測前加入10mmol/ltris100μl每孔，約15分鐘后，srb完全溶解，使用多功能酶標儀(versamax,moleculardevices)測定515nm的吸收波長，抑制率計算公為：[1–(a515treated/a515control)]×100％。
表2受試化合物對細胞的增值抑制活性(ic50,μm)
結果表明，受試化合物13a，13f和13n對含egfrt790m/l858r突變的nci-h1975細胞的體外生長具有很強的抑制活性，與對照化合物azd9291活性相當，且化合物13a和13f對含野生型egfr的a431細胞的增殖抑制活性弱，其選擇性優于azd9291；其他化合物對其也具有較強的抑制活性。
以上所述實施例僅表達了本發明的幾種實施方式，其描述較為具體和詳細，但并不能因此而理解為對本發明專利范圍的限制。應當指出的是，對于本領域的普通技術人員來說，在不脫離本發明構思的前提下，還可以做出若干變形和改進，這些都屬于本發明的保護范圍。因此，本發明專利的保護范圍應以所附權利要求為準。