具体实施方式

通过下述实例有助于理解本发明，但是不能限制本发明的内容，在下列实例中，所有目标化合物的编号与表1相同。

合成路线：

试剂与条件：(i)3-氨基-2,6-哌啶二酮盐酸盐，乙酸，乙酸钠，120℃，10h；(ii)碳酸钾，碘甲烷，N,N-二甲基甲酰胺，室温，24h；(iii)2-溴-4-氟苯甲醛，乙酰乙酸乙酯，乙酸钠，乙醇，80℃；；(iv)N-溴代丁二酰亚胺，二氯甲烷，40℃；(v)1-叔丁氧羰基-哌嗪，碳酸钾，碘化钾，乙腈，75℃，1h；(vi)三氟乙酸，二氯甲烷，室温，10h；(vii)N-叔丁氧羰基-Linker，HATU，DIPEA，二氯甲烷，0℃，30min，室温，6h；(viii)三氟乙酸，二氯甲烷，室温，10h；(ix)II-2或者II-3，N,N-二甲基甲酰胺，DIPEA，90℃，10h。

实施例1.化合物II-2的制备

将3-氟酞酐(200mg，1.2mmol)、3-氨基-2,6-哌啶二酮盐酸盐(259mg，1.2mmol)乙酸钠(118mg，1.44mmol)溶解在20ml乙酸中，120℃回流10h。反应结束后，旋干溶剂，得大量黑色固体，加入大量甲醇溶解，并加入硅胶拌样上柱，快速柱色谱分离，得到II-2。

白色固体产物，产率58％；¹H NMR(400MHz,DMSO-d₆)δ11.15(s,1H,CONHCO),7.95(q,J＝7.4Hz,1H,Ph-H),7.83–7.66(m,2H,Ph-H),5.16(dd,J＝12.8,5.1Hz,1H,COCHN),2.97–2.82(m,1H,CH₂),2.68–2.52(m,1H,CH₂),2.18–1.96(m,2H,CH₂)；EI-MS:275.06[M-H]^-；C₁₃H₉FN₂O₄[276.05].

实施例2.化合物II-3的制备

将II-2(100.00mg，0.36mmol)和碳酸钾(50mg，0.36mmol)加入溶剂N,N-二甲基甲酰胺5ml，室温搅拌下，逐滴滴加碘甲烷，室温搅拌24h。反应结束后，旋干溶剂，加入水(20ml)和二氯甲烷(20ml*2)萃取，合并有机相，用饱和氯化钠萃取一次，用无水硫酸镁干燥，过滤。有机相加入硅胶拌样，快速柱色谱分离，得到II-3。

白色固体产物，产率46％；¹H NMR(400MHz,DMSO-d₆)δ7.96(tdt,J＝7.4,4.7,2.3Hz,1H,Ph-H),7.83–7.70(m,2H,Ph-H),5.30–5.12(m,1H,COCHN),3.08–2.85(m,3H,CH₃),2.85–2.73(m,1H,CH₂),2.66–2.51(m,1H,CH₂),2.08(tdd,J＝12.7,7.5,4.2Hz,1H,CH₂)；EI-MS:290.25[M-H]^-；C₁₄H₁₁FN₂O₄[290.07].

实施例3.化合物2的制备

称取2-噻唑甲脒盐酸盐(1.0g，6.11mmol)，2-溴-4-氟苯甲醛(1.86g，9.16mmol)和乙酸钠(1.0g，1.22mmol)溶于无水乙醇(100mL)中，室温搅拌下加入乙酰乙酸乙酯(1.2mL，9.20mmol)，80℃下乙醇回流8h；反应结束后过滤，除去盐类。将母液冷却至室温，有黄色晶体(I-2)析出。剩余母液减压除去无水乙醇，加入水(60mL)，用乙酸乙酯萃取(25mL×3)，收集并合并有机相，用饱和氯化钠萃取一次(25mL)，有机相用无水硫酸镁干燥。过滤，加入200目硅胶，拌样，快速柱色谱分离，重结晶得到化合物2。得黄色粉末0.75g，产率：58％；熔点153-156℃。

¹H NMR(400MHz,DMSO-d6)δ9.92(s,1H),7.97(d,J＝2.8Hz,1H),7.89(s,1H),7.59–7.50(m,1H),7.42–7.31(m,1H),7.23(t,J＝8.3Hz,1H),5.98(s,1H),3.94(q,J＝6.9Hz,2H),2.48(s,3H),1.03(t,J＝7.0Hz,3H)；¹³C NMR(100MHz,DMSO-d6)δ166.07,163.13,159.93,147.99,144.78,143.69,141.19,131.14(d,J＝8.7Hz),124.85,122.94(d,J＝9.6Hz),119.98(d,J＝24.2Hz),115.85(d,J＝21.0Hz),97.33,59.57,58.14,17.86,14.46；EI-MS:426.04[M+2+H]⁺,C₁₇H₁₅BrFN₃O₂S[423.01].

实施例4.化合物3的制备

将中间体2(1.86g，4.39mmol)溶于二氯甲烷(50mL)中，室温搅拌下少量多次加入NBS(1.95g，1.10mmol)，二氯甲烷回流1.5h；反应结束后，减压除去二氯甲烷，加入水(50mL)，用乙酸乙酯萃取(20mL×3)，收集并合并有机相，用饱和氯化钠萃取一次(25mL)，有机相用无水硫酸镁干燥。过滤，快速柱色谱分离，重结晶得到化合物3。黄色固体1.30g，收率59％；熔点123-128℃。

¹H NMR(400MHz,CDCl₃)δ7.84(d,J＝3.1Hz,1H),7.52(s,2H),7.44–7.35(m,1H),7.32(dd,J＝8.1,2.6Hz,1H),7.02(t,J＝8.0Hz,1H),6.09(s,1H),4.94(d,J＝8.9Hz,1H),4.61(s,1H),4.09(d,J＝7.0Hz,2H),1.16(t,J＝7.1Hz,3H)；EI-MS:502.2[M+H]⁺；¹³C NMR(100MHz,CDCl₃)δ164.73,163.27,160.76,155.66,150.28,143.87,143.01,137.84,130.60(d,J＝8.6Hz),124.62,123.45,122.10(d,J＝9.2Hz),120.26(d,J＝24.8Hz),115.72(d,J＝20.9Hz),106.39,60.72,51.61,31.79,14.03；EI-MS:499.90[M-H]^-,501.94[M+2-H]^-,503.91[M+4-H]^-,C₁₇H₁₄Br₂FN₃O₂S[500.92].

实施例5.化合物4的制备

取中间体3(100mg，0.2mmol)和1-Boc-哌嗪(37mg，0.2mmol)、碳酸钾(41mg，0.3mmol)、碘化钾(50mg，0.3mmol)，加入10mL乙腈，得黄色混悬液，75℃回流1h。反应结束后，减压除去乙腈，加入水(20mL)，用乙酸乙酯萃取(20mL×3)，收集并合并有机相，用饱和氯化钠萃取一次(25mL)，再用无水硫酸镁干燥，过滤。快速柱色谱分离，重结晶得到化合物4。得到黄色固体产物76.2mg，产率63％。

¹H NMR(400MHz,DMSO-d₆)δ9.69(s,1H,dihydropyrimidine-H),8.00(s,1H,thiazole-H),7.93(s,1H,thiazole-H),7.56(d,J＝8.1Hz,1H,Ph-H),7.37(m,1H,Ph-H),7.22(s,1H,Ph-H),6.02(s,1H,CH),4.08–3.79(m,4H,dihydropyrimidine-CH₂,CH₂ CH₃),3.31(s,4H,CH₂NCH₂),2.50(s,4H,2×BocNCH₂),1.39(s,9H,Boc),1.05(t,J＝6.9Hz,3H,CH₂CH₃)；EI-MS:610.01[M+H]⁺.

实施例6.化合物5的制备

取中间体4(100mg，0.16mmol)溶解于5mL二氯甲烷中，室温搅拌下逐滴加入三氟乙酸0.5mL，室温搅拌过夜。反应结束后，加入饱和碳酸钠溶液(20mL)，产生大量气泡。用二氯甲烷萃取(20mL×3)，收集并合并有机相，用饱和氯化钠萃取一次(25mL)，再用无水硫酸镁干燥，过滤。旋干得粗品5，直接进行下一步反应。

实施例7.化合物6的制备

将不同的N-Boc-Linker“连接臂”(68mg，0.26mmol)和HATU(112mg，0.26mmol)溶解于二氯甲烷中，冰浴30min，加入5(100mg，0.20mmol)和DIPEA(60μL，0.59mmol)，室温搅拌过夜。反应结束后，旋干溶剂，加入水(20ml)和二氯甲烷(20mL×2)萃取，合并有机相，用饱和氯化钠萃取一次，用无水硫酸镁干燥，过滤。快速柱色谱分离，重结晶得到6(a-e)。

所用N-Boc-Linker为N-叔丁氧羰基-8-氨基辛酸，产物6a为黄色固体，产率51.7％，熔点73-75℃；¹H NMR(400MHz,DMSO-d₆)δ9.67(s,1H,dihydropyrimidine-H),8.01(d,J＝3.0Hz,1H,thiazole-H),7.95(d,J＝2.8Hz,1H,thiazole-H),7.57(dd,J＝8.6,2.8Hz,1H,Ph-H),7.44–7.35(m,1H,Ph-H),7.22(t,J＝8.4Hz,1H,Ph-H),6.75(s,1H,NH),6.03(s,1H,dihydropyrimidine-CH),3.95(q,J＝6.7,5.8Hz,2H,CH₂ CH₃),3.89(d,J＝17.2Hz,2H,dihydropyrimidine-CH₂),3.52(s,4H,CH₂ NCOCH₂ ),2.89(q,J＝6.8Hz,2H,COCH₂CH₂CH₂CH₂CH₂CH₂ CH₂ NH),2.69(d,J＝2.1Hz,2H,COCH₂ CH₂CH₂CH₂CH₂CH₂CH₂NH),2.55(s,4H,CH₂ NCH₂ ),2.31(t,J＝7.5Hz,2H,COCH₂ CH₂ CH₂CH₂CH₂CH₂CH₂NH),1.52-1.44(m,2H,COCH₂CH₂CH₂CH₂CH₂ CH₂ CH₂NH),1.36(s,9H,Boc),1.25(d,J＝2.2Hz,6H,COCH₂CH₂ CH₂CH₂CH₂ CH₂CH₂NH),1.05(td,J＝7.1,2.1Hz,3H,CH₂ CH₃ )；EI-MS:848.09[M+H]⁺；C₃₄H₄₆BrFN₆O₅S[848.24].

所用N-Boc-Linker为N-叔丁氧羰基-6-氨基己酸，产物6b为黄色固体，产率76％，熔点53-55℃；¹H NMR(400MHz,DMSO-d₆)δ9.66(s,1H,dihydropyrimidine-H),8.02(s,1H,thiazole-H),7.95(s,1H,thiazole-H),7.58(d,J＝8.5Hz,1H,Ph-H),7.41(s,1H,Ph-H),7.24(s,1H,Ph-H),6.77(s,1H,NH),6.02(s,1H,dihydropyrimidine-CH),3.99–3.93(m,4H,CH₂ CH₃,dihydropyrimidine-CH₂),3.55–3.50(m,4H,CH₂ NCOCH₂ ),2.89(q,J＝6.9Hz,2H,COCH₂CH₂CH₂CH₂ CH₂ NH),2.69(d,J＝2.0Hz,2H,COCH₂ CH₂CH₂CH₂CH₂NH),2.57(s,4H,CH₂ NCH₂ ),2.32(s,2H,COCH₂ CH₂ CH₂CH₂CH₂NH),1.53–1.45(m,2H,COCH₂CH₂CH₂ CH₂ CH₂NH),1.37(s,9H,Boc),1.26(q,J＝7.2Hz,2H,COCH₂CH₂ CH₂ CH₂CH₂NH),1.09–1.02(m,3H,CH₂ CH₃ )；EI-MS:721.19[M+H]⁺；C₃₂H₄₂BrFN₆O₅S[720.21].

所用N-Boc-Linker为N-叔丁氧羰基-4-氨基丁酸，产物6c为为黄色固体，产率88.7％，熔点100-108℃；¹H NMR(400MHz,DMSO-d₆)δ9.67(s,1H,dihydropyrimidine-H),8.01(s,1H,thiazole-H),7.95(s,1H,thiazole-H),7.57(d,J＝8.3Hz,1H,Ph-H),7.41(s,1H,Ph-H),7.23(s,1H,Ph-H),6.81(s,1H,NH),6.02(s,1H,dihydropyrimidine-CH),3.96(s,4H,CH₂ CH₃,dihydropyrimidine-CH₂),3.52(s,4H,CH₂ NCOCH₂ ),2.93(q,J＝6.7Hz,2H,COCH₂CH₂ CH₂ NH),2.69(d,J＝1.9Hz,4H,CH₂ NCH₂ ),2.32(s,2H,COCH₂ CH₂CH₂NH),1.62(q,J＝7.2Hz,2H,COCH₂ CH₂ CH₂NH),1.37(s,9H,Boc),1.05(t,J＝7.3Hz,3H,CH₂ CH₃ )；EI-MS:693.25[M+H]⁺；C₃₀H₃₈BrFN₆O₅S[692.18].

所用N-Boc-Linker为N-叔丁氧羰基-2-(2-(2-氨基乙氧基)乙氧基)乙酸，产物6d为黄色固体，产率48％，熔点73-75℃；¹H NMR(400MHz,DMSO-d₆)δ9.67(s,1H,dihydropyrimidine-H),8.01(s,1H,thiazole-H),7.95(s,1H,thiazole-H),7.57(d,J＝8.3Hz,1H,Ph-H),7.41(s,1H,Ph-H),7.22(s,1H,Ph-H),6.76(s,1H,NH),6.03(s,1H,dihydropyrimidine-CH),4.17(s,2H,COCH₂O),4.04–3.86(m,4H,CH₂ CH₃,dihydropyrimidine-CH₂),3.54(d,J＝10.3Hz,8H,CH₂ NCOCH₂ ,OCH₂CH₂OCH₂CH₂ N),3.38(d,J＝6.4Hz,2H,OCH₂ CH₂OCH₂CH₂N),3.07(q,J＝6.3Hz,2H,OCH₂ CH₂ OCH₂CH₂N),1.36(s,9H,Boc),1.05(t,J＝7.3Hz,3H,CH₂ CH₃ )；EI-MS:755.18[M+2+H]⁺；C₃₂H₄₂BrFN₆O₇S[752.20].

所用N-Boc-LinkerN-叔丁氧羰基-2-(2-氨基乙氧基)乙酸，产物6e为黄色固体，产率93％，熔点53-55℃；¹H NMR(400MHz,DMSO-d₆)δ9.66(s,1H,dihydropyrimidine-H),8.04–7.99(m,1H,thiazole-H),7.95(s,1H,thiazole-H),7.57(d,J＝8.5Hz,1H,Ph-H),7.40(s,1H,Ph-H),7.23(s,1H,Ph-H),6.82(s,1H,NH),6.03(s,1H,dihydropyrimidine-CH),4.15(s,2H,COCH₂O),3.98–3.92(m,4H,CH₂ CH₃,dihydropyrimidine-CH₂),3.49-3.43(m,6H,CH₂ NCOCH₂ ,OCH₂ CH₂N),3.09(q,J＝6.3,5.8Hz,2H,OCH₂ CH₂ N),2.69(d,J＝1.9Hz,2H),2.55(s,4H,CH₂ NCH₂ ),1.35(s,9H,Boc),1.05(t,J＝7.1Hz,3H,CH₂ CH₃ )；EI-MS:708.92[M+H]⁺；C₃₀H₃₈BrFN₆O₆S[708.17].

实施例8.化合物7(a-e)的制备

取中间体6(a-e)(400mg，0.56mmol)溶解于20ml二氯甲烷中，室温搅拌下逐滴加入三氟乙酸2mL，室温搅拌过夜。反应结束后，加入饱和碳酸钠溶液(20mL)，产生大量气泡。用二氯甲烷萃取(20mL×3)，收集并合并有机相，用饱和氯化钠萃取一次(25mL)，再用无水硫酸镁干燥，过滤。减压蒸去多余溶剂，剩下2mL溶剂，使用制备TLC分离，重结晶获得目标化合物7(a-e)。

7a为黄色固体，产率61.7％，熔点98-100℃；¹H NMR(400MHz,DMSO-d₆)δ8.00(s,1H,thiazole-H),7.95(s,1H,thiazole-H),7.55(d,J＝8.2Hz,1H,Ph-H),7.41(t,J＝6.9Hz,1H,Ph-H),7.22(t,J＝7.8Hz,1H,Ph-H),6.75(s,1H,NH),6.04(s,1H,dihydropyrimidine-CH),4.05–3.83(m,4H,CH₂ CH₃,dihydropyrimidine-CH₂),3.53(s,4H,CH₂ NCOCH₂ ),2.80–2.70(m,2H,COCH₂CH₂CH₂CH₂CH₂CH₂ CH₂ NH),2.53(d,J＝18.8Hz,4H,CH₂ NCH₂ ),2.31(s,2H,COCH₂ CH₂CH₂CH₂CH₂CH₂CH₂NH),1.53(d,J＝24.3Hz,4H,COCH₂ CH₂ CH₂CH₂CH₂ CH₂ CH₂NH),1.28(s,6H,COCH₂CH₂ CH₂CH₂CH₂ CH₂CH₂NH),1.04(t,J＝6.5Hz,3H,CH₂ CH₃ )；¹³C NMR(100MHz,DMSO-d₆)δ171.20,165.64,162.50,160.04,146.77,144.40,144.08,140.64,131.43,125.24,123.00,120.20,119.96,116.07,115.86,97.73,59.88,58.66,55.84,53.46,53.04,45.63,41.66,32.71,29.10,28.91,28.00,26.32,25.15,14.45；EI-MS:649.10[M+H]⁺；C₂₉H₃₈BrFN₆O₃S[648.19].

7b为黄色固体，产率76％，熔点95-100℃；¹H NMR(400MHz,DMSO-d₆)δ8.01(d,J＝2.8Hz,1H,thiazole-H),7.95(d,J＝3.5Hz,1H,thiazole-H),7.57(d,J＝8.6Hz,1H,Ph-H),7.39(t,J＝7.1Hz,1H,Ph-H),7.22(t,J＝8.7Hz,1H,Ph-H),6.03(s,1H,dihydropyrimidine-CH),3.95(q,J＝9.0,8.4Hz,4H,CH₂ CH₃,dihydropyrimidine-CH₂),3.52(s,4H,CH₂ NCOCH₂ ),2.69–2.51(m,6H,COCH₂CH₂CH₂CH₂ CH₂ NH,CH₂ NCH₂ ),2.32(t,J＝7.0Hz,2H,COCH₂ CH₂ CH₂CH₂CH₂NH),1.47(dt,J＝24.4,8.0Hz,4H,COCH₂ CH₂ CH₂ CH₂ CH₂NH),1.28(dd,J＝20.1,12.1Hz,2H,COCH₂CH₂ CH₂ CH₂CH₂NH),1.04(t,J＝7.1Hz,3H,CH₂ CH₃ )；¹³C NMR(100MHz,DMSO-d₆)δ171.06,165.64,162.50,160.04,146.78,144.40,144.08,140.64,125.25,120.20,119.96,116.07,115.86,97.72,59.88,58.66,55.84,53.43,53.02,45.62,41.69,32.50,28.05,26.13,24.72,14.45；EI-MS:622.92[M+2+H]⁺；C₂₇H₃₄BrFN₆O₃S[620.16].

7c为黄色固体，产率52.0％，熔点128-135℃；¹H NMR(400MHz,DMSO-d₆)δ7.99(d,J＝24.0Hz,2H,thiazole-H),7.58(d,J＝8.6Hz,1H,Ph-H),7.40(t,J＝7.5Hz,1H,Ph-H),7.23(t,J＝8.7Hz,1H,Ph-H),6.04(s,1H,dihydropyrimidine-CH),3.96(q,J＝8.9Hz,4H,CH₂ CH₃,dihydropyrimidine-CH₂),3.53(s,4H,CH₂ NCOCH₂ ),2.72–2.52(m,6H,COCH₂CH ₂CH₂ NH,CH₂ NCH₂ ),2.39(t,J＝7.5Hz,2H,COCH₂ CH ₂ CH₂NH),1.64(t,J＝7.3Hz,2H,COCH₂ CH₂ CH₂NH),1.05(t,J＝7.2Hz,3H,CH₂ CH₃ )；¹³C NMR(100MHz,DMSO-d₆)δ170.40,167.42,165.64,162.51,160.04,146.75,144.40,144.06,140.65,132.18,131.98,129.13,125.23,123.11,123.01,120.20,119.95,116.05,115.84,97.73,65.49,59.87,58.68,55.82,53.29,52.96,45.47,41.77,30.48,29.79,23.24,19.12,14.45,14.00；EI-MS:592.97[M+H]⁺；C₂₅H₃₀BrFN₆O₃S[592.13].

7d为黄色固体，产率81.7％，熔点60-68℃；¹H NMR(400MHz,DMSO-d₆)δ8.07(d,J＝22.0Hz,2H,thiazole-H),7.66(d,J＝8.6Hz,1H,Ph-H),7.48(t,J＝7.4Hz,1H,Ph-H),7.34–7.26(m,1H,Ph-H),6.11(s,1H,dihydropyrimidine-CH),4.29(s,2H,COCH₂O),4.03(q,J＝7.7Hz,4H,CH₂ CH₃,dihydropyrimidine-CH₂),3.64(d,J＝27.7Hz,10H,CH₂ NCOCH₂ ,OCH₂CH₂ OCH₂ CH₂N),2.98(s,2H,OCH₂CH₂OCH₂ CH₂ N),2.64(s,4H,CH₂ NCH₂ ),1.13(t,J＝7.0Hz,3H,CH₂ CH₃ )；13C NMR(100MHz,DMSO-d₆)δ167.99,165.65,162.51,160.04,146.74,144.39,144.07,140.65,140.61,131.44,131.36,125.26,120.20,119.96,116.08,115.87,97.73,70.25,69.90,69.62,69.19,59.89,58.66,55.82,53.23,52.91,44.90,41.80,14.45；EI-MS:653.18[M+H]⁺；C₂₇H₃₄BrFN₆O₅S[652.15].

7e为黄色固体，产率58.8％，熔点121-129℃；¹H NMR(400MHz,DMSO-d₆)δ8.03–7.94(m,2H,thiazole-H),7.58(d,J＝8.5Hz,1H,Ph-H),7.40(t,J＝7.4Hz,1H,Ph-H),7.23(t,J＝8.7Hz,1H,Ph-H),6.04(s,1H,dihydropyrimidine-CH),4.25(s,2H,COCH₂O),3.96(q,J＝7.3,6.7Hz,4H,CH₂ CH₃,dihydropyrimidine-CH₂),3.61–3.41(m,6H,CH₂ NCOCH₂ ,OCH₂ CH₂N),2.86(t,J＝5.4Hz,2H,OCH₂ CH₂ N),2.69(s,2H,NH₂),2.56(d,J＝13.5Hz,4H,CH₂ NCH₂ ),1.05(t,J＝7.1Hz,3H,CH₂ CH₃ )；¹³C NMR(100MHz,DMSO-d₆)δ168.38,165.64,162.52,160.04,146.69,144.39,144.04,140.64,131.34,125.24,123.11,120.20,119.96,116.05,115.84,97.81,69.08,67.89,67.78,63.28,59.88,58.69,55.80,53.10,44.67,14.45；EI-MS:608.92[M+H]⁺；C₂₅H₃₀BrFN₆O₄S[608.12].

实施例9.化合物8(a-j)的制备

取7(a-e)(123mg，0.16mmol)和2(57mg，0.20mmol)或3(60mg，0.20mmol)溶于N,N-二甲基甲酰胺中，搅拌下加入DIPEA(38μl，0.29mmol)，90℃反应10h。反应结束后，旋干溶剂，加入水(20mL)和EA(20mL*2)萃取，合并有机相，用饱和氯化钠萃取一次，用无水硫酸镁干燥，过滤。有机相加入硅胶拌样，快速柱色谱分离，重结晶获得目标化合物8(a-j)。

8a为黄色固体，产率41.7％，熔点119-120℃；¹H NMR(400MHz,DMSO-d₆)δ11.10(s,1H,CONHCO),9.67(s,1H,dihydropyrimidine-H),8.01(s,1H,thiazole-H),7.95(s,1H,thiazole-H),7.58(t,J＝7.8Hz,2H,Ph-H,Pomalidomide-Ph-H),7.44–7.35(m,1H,Ph-H),7.22(t,J＝8.4Hz,1H,Ph-H),7.10(d,J＝8.6Hz,1H,Pomalidomide-Ph-H),7.02(d,J＝7.0Hz,1H,Pomalidomide-Ph-H),6.53(s,1H,NH),6.03(s,2H,dihydropyrimidine-CH),5.05(dd,J＝12.7,4.8Hz,1H,Pomalidomide-CH),3.95(q,J＝9.4,8.4Hz,4H,CH₂ CH₃,dihydropyrimidine-CH₂),3.52(s,4H,CH₂ NCOCH₂ ),3.30(d,J＝6.4Hz,2H,Pomalidomide-CH₂),2.95–2.84(m,1H,Pomalidomide-CH₂),2.64–2.53(m,4H,CH₂ NCOCH₂ ),2.31(t,J＝7.1Hz,2H,COCH₂ CH₂ CH₂CH₂CH₂CH₂CH₂NH),2.07–1.99(m,1H,Pomalidomide-CH₂),1.58(s,2H,COCH₂ CH₂ CH₂CH₂CH₂CH₂CH₂NH),1.50(s,2H,COCH₂CH₂CH₂CH₂CH₂ CH₂ CH₂NH),1.28(d,J＝34.5Hz,6H,COCH₂CH₂ CH₂CH₂CH₂ CH₂CH₂NH),1.04(t,J＝6.9Hz,3H,CH₂ CH₃ )；¹³C NMR(100MHz,DMSO-d₆)δ174.13,173.76,171.78,169.15,166.82,166.78,162.39,160.37,152.23,146.11,143.15,142.71,138.89,138.87,138.65,131.84,129.35,129.28,126.57,126.51,125.44,124.96,122.52,121.71,120.94,120.78,113.30,113.14,112.47,100.34,65.57,61.37,60.21,52.75,52.71,45.07,41.58,36.13,30.69,30.12,29.30,28.98,26.77,26.02,24.30,14.46；EI-MS:906.97[M+2+H]⁺；C₄₂H₄₆BrFN₈O₇S[904.24].

8b为黄色固体，产率20.6％，熔点114-117℃；¹H NMR(400MHz,DMSO-d₆)δ11.11(s,1H,CONHCO),9.73(s,1H,dihydropyrimidine-H),8.09(s,2H,thiazole-H),7.66–7.55(m,2H,Ph-H,Pomalidomide-Ph-H),7.51(s,1H,Ph-H),7.29(s,1H,Ph-H),7.11(d,J＝8.4Hz,1H,Pomalidomide-Ph-H),7.04(d,J＝6.9Hz,1H,Ph-H),6.55(s,1H,NH),6.02(s,1H,dihydropyrimidine-CH),5.06(dd,J＝13.0,5.0Hz,1H,Pomalidomide-CH),4.23(t,J＝6.4Hz,2H,dihydropyrimidine-CH₂),4.02(d,J＝6.7Hz,2H,CH₂ CH₃),3.32(d,J＝5.7Hz,4H,CH₂ NCOCH₂ ),2.89(t,J＝13.0Hz,1H,Pomalidomide-CH₂),2.60(d,J＝17.6Hz,2H,COCH₂CH₂CH₂CH₂ CH₂ NH),2.39(s,2H,COCH₂ CH₂CH₂CH₂CH₂NH),2.03(d,J＝7.7Hz,1H,Pomalidomide-CH₂),1.69–1.52(m,4H,COCH₂ CH₂ CH₂ CH₂ CH₂NH),1.39(s,2H,Pomalidomide-CH₂),1.24(s,2H,COCH₂CH₂ CH₂ CH₂CH₂NH),1.10(d,J＝6.3Hz,3H,CH₂ CH₃ )；¹³C NMR(100MHz,DMSO-d₆)δ174.13,173.76,171.78,169.15,166.82,166.78,162.39,160.37,152.23,146.11,143.15,142.71,138.89,138.87,138.65,131.84,129.35,129.28,126.57,126.51,125.44,124.96,122.52,121.71,120.94,120.78,113.30,113.14,112.47,100.34,65.57,61.37,60.21,52.75,52.71,45.07,41.58,36.13,30.69,28.98,26.02,24.80,24.30,14.46；EI-MS:878.95[M+2+H]⁺；C₄₀H₄₂BrFN₈O₇S[876.21].

8c为黄色固体，产率26.6％，熔点76-82℃；¹H NMR(400MHz,DMSO-d₆)δ11.11(s,1H,CONHCO),9.69(s,1H,dihydropyrimidine-H),8.08(s,2H,thiazole-H),7.61(t,J＝7.5Hz,2H,Ph-H,Pomalidomide-Ph-H),7.50(s,1H,Ph-H),7.29(s,1H,Ph-H),7.19(d,J＝8.5Hz,2H,Ph-H,Pomalidomide-Ph-H),7.04(d,J＝6.8Hz,1H,Pomalidomide-Ph-H),6.69(s,1H,NH),6.02(s,1H,dihydropyrimidine-CH),5.06(dd,J＝13.1,5.0Hz,1H,Pomalidomide-CH),4.58(s,2H),4.23(t,J＝6.4Hz,4H,dihydropyrimidine-CH₂),4.01(d,J＝6.3Hz,2H,CH₂ CH₃),3.35(d,J＝6.1Hz,4H,CH₂ NCOCH₂ ),2.96–2.57(m,3H,Pomalidomide-CH₂,COCH₂CH₂ CH₂ NH),2.48(s,2H,COCH₂ CH₂CH₂NH),2.07–2.00(m,1H,Pomalidomide-CH₂),1.88–1.76(m,2H,COCH₂ CH₂ CH₂NH),1.09(t,J＝6.4Hz,3H,CH₂ CH₃ )；13C NMR(100MHz,DMSO-d₆)δ174.13,173.76,171.78,169.15,166.82,166.78,162.39,160.37,152.23,146.11,143.15,142.71,138.89,138.87,138.65,131.84,129.35,129.28,126.57,126.51,125.44,124.96,122.52,121.71,120.94,120.78,113.30,113.14,112.47,100.34,65.57,61.37,60.21,52.75,52.71,45.07,41.56,34.75,30.69,25.15,24.30,14.46；EI-MS:848.93[M+H]⁺；C₃₈H₃₈BrFN₈O₇S[848.18].

8d为黄色固体，产率13.0％，熔点122-129℃；¹H NMR(400MHz,DMSO-d₆)δ11.10(s,1H,CONHCO),9.65(s,1H,dihydropyrimidine-H),8.00(t,J＝2.7Hz,1H,thiazole-H),7.93(t,J＝2.8Hz,1H,thiazole-H),7.57(ddd,J＝8.7,5.9,2.5Hz,2H,Ph-H,Pomalidomide-Ph-H),7.39(dd,J＝8.8,6.3Hz,1H,Ph-H),7.25–7.18(m,1H,Ph-H),7.13(d,J＝8.6Hz,1H,Pomalidomide-Ph-H),7.03(dd,J＝7.1,2.1Hz,1H,Pomalidomide-Ph-H),6.62(d,J＝6.3Hz,1H,NH),6.02(s,1H,dihydropyrimidine-CH),5.05(dd,J＝13.0,5.4Hz,1H,Pomalidomide-CH),4.17(s,2H,COCH₂O),4.00–3.88(m,4H,CH₂ CH₃,dihydropyrimidine-CH₂),3.65–3.44(m,12H,CH₂ NCOCH₂ ,OCH₂CH₂ OCH₂CH₂ N),2.93–2.82(m,1H,Pomalidomide-CH₂),2.69–2.52(m,6H,Pomalidomide-CH₂,CH₂ NCH₂ ),2.08–1.90(m,1H,Pomalidomide-CH₂),1.04(td,J＝7.1,2.1Hz,3H)；¹³C NMR(100MHz,DMSO-d₆)δ173.20,170.49,169.42,167.82,167.74,165.62,162.52,160.03,146.89,146.68,144.37,144.03,140.64,136.68,132.58,131.43,125.17,123.10,123.01,120.19,119.95,117.87,116.03,115.82,111.15,109.78,97.75,70.31,70.05,69.96,69.33,59.86,58.69,55.83,49.07,42.24,41.80,31.47,22.63,14.44；EI-MS:909.10[M+H]⁺；C₄₀H₄₂BrFN₈O₉S[908.20].

8e为黄色固体，产率4.3％，熔点62-68℃；¹H NMR(400MHz,DMSO-d₆)δ11.10(s,1H,CONHCO),9.66(s,1H,dihydropyrimidine-H),8.05–7.92(m,2H,thiazole-H),7.66–7.50(m,2H,Ph-H,Pomalidomide-Ph-H),7.40(t,J＝7.6Hz,1H,Ph-H),7.23(d,J＝8.5Hz,1H,Ph-H),7.18(t,J＝9.5Hz,1H,Pomalidomide-Ph-H),7.04(d,J＝7.1Hz,1H,Pomalidomide-Ph-H),6.70(d,J＝6.2Hz,1H,NH),6.03(s,1H,dihydropyrimidine-CH),5.05(dd,J＝12.9,5.5Hz,1H,Pomalidomide-CH),4.24(s,2H,COCH₂O),4.00–3.81(m,4H,CH₂ CH₃,dihydropyrimidine-CH₂),3.72–3.63(m,2H,OCH₂ CH₂N),3.56–3.46(m,4H,CH₂ NCOCH₂ ),2.92–2.81(m,1H,Pomalidomide-CH₂),2.56(d,J＝17.2Hz,6H,CH₂ NCH₂ ,OCH₂ CH₂ N),2.01(d,J＝11.7Hz,1H,Pomalidomide-CH₂),1.12–0.97(m,3H,CH₂ CH₃ )；¹³C NMR(100MHz,DMSO-d₆)δ173.22,170.51,169.36,167.79,167.75,165.63,162.50,160.04,146.87,146.73,144.39,144.05,140.65,136.68,132.61,131.36,125.20,123.11,123.01,120.20,119.96,117.90,116.05,115.84,111.15,109.75,97.72,69.66,69.54,67.77,63.27,59.87,58.68,55.82,53.23,49.05,44.91,42.24,41.80,41.02,31.46,22.64,14.44；EI-MS:865.10[M+H]⁺；C₃₈H₃₈BrFN₈O₈S[864.17].

8f为黄色固体，产率8％，熔点85-90℃；¹HNMR(400MHz,DMSO-d₆)δ9.67(s,1H,dihydropyrimidine-H),8.00(t,J＝2.7Hz,1H,thiazole-H),7.94(d,J＝2.7Hz,1H,thiazole-H),7.64–7.49(m,2H,Ph-H,Pomalidomide-Ph-H),7.39(t,J＝7.3Hz,1H,Ph-H),7.22(t,J＝8.7Hz,1H,Ph-H),7.10(d,J＝8.5Hz,1H,Pomalidomide-Ph-H),7.02(d,J＝7.0Hz,1H,Pomalidomide-Ph-H),6.54(d,J＝5.6Hz,1H,NH),6.03(s,1H,dihydropyrimidine-CH),5.32(dd,J＝12.7,4.8Hz,1H,Pomalidomide-CH),4.03–3.84(m,4H,CH₂ CH₃,dihydropyrimidine-CH₂),3.52(s,4H,CH₂ NCOCH₂ ),3.30(d,J＝6.4Hz,2H,Pomalidomide-CH₂),3.01(d,J＝2.1Hz,3H,Pomalidomide-CH₃),2.93–2.63(m,2H,Pomalidomide-CH₂),2.53(d,J＝13.6Hz,4H,CH₂ NCH₂ ),2.31(t,J＝7.5Hz,2H,COCH₂ CH₂ CH₂CH₂CH₂CH₂CH₂NH),2.01(dt,J＝13.9,9.6Hz,2H,COCH₂ CH₂ CH₂CH₂CH₂ CH₂ CH₂NH),1.53(d,J＝34.6Hz,4H,COCH₂ CH₂ CH₂CH₂CH₂ CH₂ CH₂NH),1.30(d,J＝19.0Hz,6H,COCH₂CH₂ CH₂CH₂CH₂ CH₂CH₂NH),1.09–1.00(m,3H,CH₂ CH₃ )；¹³CNMR(100MHz,DMSO-d₆)δ172.68,171.78,169.15,166.82,166.78,166.64,162.39,160.37,152.23,146.11,143.15,142.71,138.89,138.87,138.65,131.84,129.35,129.28,126.57,126.51,125.44,124.96,122.52,121.71,120.94,120.78,113.30,113.14,112.47,100.34,65.57,61.37,60.21,52.75,51.73,45.07,41.58,36.13,30.50,30.12,29.30,28.98,28.93,26.77,26.02,24.21,14.46；EI-MS:920.99[M+2+H]⁺；C₄₃H₄₈BrFN₈O₇S[918.25].

8g为黄色固体，产率14.5％，熔点128-130℃；¹HNMR(400MHz,DMSO-d₆)δ9.67(s,1H,dihydropyrimidine-H),7.98(d,J＝24.6Hz,2H,thiazole-H),7.58(s,2H,Ph-H,Pomalidomide-Ph-H),7.40(s,1H,Ph-H),7.22(s,1H,Ph-H),7.11(s,1H,Ph-H),7.03(s,1H,Ph-H),6.55(s,1H,NH),6.03(s,1H,dihydropyrimidine-CH),5.13(s,1H,Pomalidomide-CH),3.94(s,4H,CH₂ CH₃,dihydropyrimidine-CH₂),3.53(s,4H,CH₂ NCOCH₂ ),3.02(s,3H,Pomalidomide-CH₃),2.95(s,1H,Pomalidomide-CH₂),2.76(d,J＝16.1Hz,1H,Pomalidomide-CH₂),2.54(s,4H,CH₂ NCH₂ ),2.34(s,2H,COCH₂CH₂CH₂CH₂ CH₂ NH),2.05(s,2H,COCH₂ CH₂CH₂CH₂CH₂NH),1.58(s,4H,Pomalidomide-CH₂,COCH₂ CH₂ CH₂ CH₂ CH₂NH),1.38(s,2H,COCH₂CH₂CH₂ CH₂ CH₂NH),1.24(s,2H,COCH₂CH₂ CH₂ CH₂CH₂NH),1.05(s,3H,CH₂ CH₃ )；¹³CNMR(100MHz,DMSO-d₆)δ172.26,171.11,170.30,169.40,167.74,165.63,162.50,160.03,146.93,146.79,144.38,144.07,140.66,140.62,136.77,132.66,131.43,125.21,123.11,123.01,120.20,119.96,117.68,116.05,115.84,110.86,109.44,97.69,59.87,58.66,55.85,53.43,53.03,49.58,45.61,42.26,41.67,32.63,31.60,29.03,27.06,26.58,24.98,21.86,14.44；EI-MS:890.94[M+H]⁺；C₄₁H₄₄BrFN₈O₇S[890.22].

8h为黄色固体，产率26.8％，熔点108-114℃；¹H NMR(400MHz,DMSO-d₆)δ9.67(s,1H,dihydropyrimidine-H),8.01(s,1H,thiazole-H),7.95(s,1H,thiazole-H),7.69–7.48(m,2H,Ph-H,Pomalidomide-Ph-H),7.40(t,J＝7.2Hz,1H,Ph-H),7.29–7.12(m,2H,Ph-H,Pomalidomide-Ph-H),7.03(d,J＝7.0Hz,1H,Pomalidomide-Ph-H),6.69(s,1H,NH),6.03(s,1H,dihydropyrimidine-CH),5.13(dd,J＝12.7,4.7Hz,1H,Pomalidomide-CH),3.95(q,J＝7.2Hz,4H,CH₂ CH₃,dihydropyrimidine-CH₂),3.52(s,4H,CH₂ NCOCH₂ ),3.01(s,3H,Pomalidomide-CH₃),2.94–2.62(m,2H,Pomalidomide-CH₂),2.55(s,4H,CH₂ NCOCH₂ ),2.40(d,J＝23.7Hz,2H,COCH₂CH₂ CH₂ NH),2.04(d,J＝11.7Hz,2H,Pomalidomide-CH₂),1.77(d,J＝32.1Hz,2H,COCH₂ CH₂CH₂NH),1.24(s,2H,COCH₂ CH₂ CH₂NH),1.05(t,J＝6.9Hz,3H,CH₂ CH₃ )；¹³C NMR(100MHz,DMSO-d₆)δ172.68,171.78,169.15,166.82,166.78,166.64,162.39,160.37,152.23,146.11,143.15,142.71,138.89,138.87,138.65,131.84,129.35,129.28,126.57,126.51,125.44,124.96,122.52,121.71,120.94,120.78,113.30,113.14,112.47,100.34,65.57,61.37,60.21,52.75,51.73,45.07,41.56,34.75,30.50,28.93,25.15,24.21,14.46；EI-MS:862.90[M+H]⁺；C₃₉H₄₀BrFN₈O₇S[862.19].

8i为黄色固体，产率4.8％，熔点90-104℃；¹H NMR(400MHz,DMSO-d₆)δ9.66(s,1H,dihydropyrimidine-H),8.00(s,1H,thiazole-H),7.94(s,1H,thiazole-H),7.57(s,2H,Ph-H,Pomalidomide-Ph-H),7.40(s,1H,Ph-H),7.22(s,1H,Ph-H),7.14(s,1H,Pomalidomide-Ph-H),7.04(s,1H,Pomalidomide-Ph-H),6.62(s,1H,NH),6.03(s,1H,dihydropyrimidine-CH),5.12(s,1H,Pomalidomide-CH),4.17(s,1H,COCH₂O),3.94(s,4H,CH₂ CH₃,dihydropyrimidine-CH₂),3.64(s,8H,OCH₂CH₂OCH₂CH₂N)3.34(s,4H,CH₂ NCOCH₂ ),3.01(s,3H,Pomalidomide-CH₃),2.76(s,1H,Pomalidomide-CH₂),2.02(s,2H,Pomalidomide-CH₂),1.04(s,3H,CH₂ CH₃ )；¹³C NMR(100MHz,DMSO-d₆)δ172.23,170.26,169.41,167.82,167.71,165.62,162.51,160.03,146.90,146.68,144.36,144.02,140.67,136.71,132.55,131.43,125.18,123.10,120.18,119.94,117.89,116.02,115.82,111.17,109.72,97.72,70.30,70.06,69.96,69.32,59.86,58.68,55.83,53.23,52.92,49.62,44.99,42.23,41.79,31.61,27.05,21.84,14.43；EI-MS:924.95[M+2+H]⁺；C₄₁H₄₄BrFN₈O₉S[922.21].

8j为黄色固体，产率19.8％，熔点78-80℃；¹H NMR(400MHz,DMSO-d₆)δ9.67(s,1H,dihydropyrimidine-H),8.00(s,2H,thiazole-H),7.94(s,1H,Ph-H),7.67–7.52(m,2H,Ph-H,Pomalidomide-Ph-H),7.40(t,J＝6.9Hz,1H,Ph-H),7.28–7.13(m,1H,Pomalidomide-Ph-H),7.05(d,J＝6.7Hz,1H,Pomalidomide-Ph-H),6.69(s,1H,NH),6.03(s,1H,dihydropyrimidine-CH),5.12(dd,J＝12.7,4.6Hz,3H,Pomalidomide-CH),4.24(s,2H,COCH₂O),3.93(dd,J＝15.1,11.3Hz,5H,CH₂ CH₃,dihydropyrimidine-CH₂),3.70(d,J＝17.1Hz,2H,OCH₂ CH₂N),3.50(d,J＝20.7Hz,6H,OCH₂ CH₂ N,CH₂ NCOCH₂ ),3.00(s,2H,Pomalidomide-CH₃),2.73(d,J＝16.8Hz,1H,Pomalidomide-CH₂),2.53(d,J＝8.3Hz,4H,CH₂ NCH₂ ),2.02(s,1H,Pomalidomide-CH₂),1.05(t,J＝6.8Hz,3H,CH₂ CH₃ )；13C NMR(100MHz,DMSO-d₆)δ172.22,170.27,169.34,167.75,167.71,165.62,162.50,160.04,146.88,146.76,144.38,144.05,140.66,136.71,132.59,131.43,131.34,125.22,123.11,123.01,120.20,119.96,117.94,116.05,115.84,111.17,109.69,97.65,69.72,69.53,67.77,63.27,59.86,58.66,55.82,53.22,52.87,49.60,44.94,42.21,41.81,41.01,31.60,27.05,21.84,14.44；EI-MS:878.91[M+H]⁺；C₃₉H₄₀BrFN₈O₈S[878.19].

实施例10.目标化合物的体外抗HBV活性实验(HepDES19细胞)

测试原理：HepDES19细胞是在四环素(可抑制启动子)的控制下，稳定转染了HBV D型基因组基因的HepG2(人肝母细胞瘤)细胞系衍生物¹。在不存在四环素的情况下，诱导HepDES19细胞HBV复制，添加化合物，并将细胞孵育3天,细胞表达的HBV DNA含量和细胞的生存状况会有所变化²。通过定量聚合酶链反应(qPCR)分析HBV DNA含量也就是化合物抑制HBV复制的有效性，得到HBV DNA降低至一半所需要的化合物浓度，即为半数有效浓度(EC₅₀)，表示化合物的抗HBV活性。通过MTS法测试化合物对细胞的毒性大小，得到化合物杀死半数细胞所需浓度，即为半数致死浓度(CC₅₀)，表示化合物的细胞毒性。(Guo H,Jiang D,Zhou T,et al.Journal of virology 2007；81(22):12472-12484；Edwards TC,Lomonosova E,Patel JA,et al.Antiviral research 2017；(143):205-217.)

实验方法：

(1)细胞培养。将细胞保存在Dulbecco改良的Eagle培养基(Dulbecco’s modifiedEagle’smedium，DMEM)/F12培养基中，该培养基补充了10％的胎牛血清(FBS)和1％的青霉素/链霉素(P/S)和1μg/mL的四环素。通过从培养基中去除四环素来诱导HBV pgRNA的同步表达。

(2)细胞含药培养。在不存在四环素的情况下，将HepDES19细胞以每孔4x 10⁴个细胞的密度接种在96孔板中。诱导HBV复制48小时后，添加最终DMSO浓度为1％的化合物溶液，与细胞共同孵育3天。

(3)细胞活性(EC₅₀)测试方法。细胞在200μL磷酸盐缓冲盐水(PBS)中洗涤，并在150μL核心裂解缓冲液(10mM Tris pH 7.4，1％Tween20，150mM NaCl)中裂解。将细胞在20-23℃的定轨摇床上以350rpm孵育40分钟。将细胞裂解液转移至96孔PCR板中，并以3300×g离心5分钟。将50μL上清液转移至另一个96孔PCR板中，并与20单位微球菌核酸酶和100μMCaCl₂混合。将裂解液在37℃孵育1小时，然后将核酸酶在70℃灭活10分钟。将Qiagen蛋白酶(0.005Anson单位)加入到裂解物中，并将混合物孵育过夜，然后在95℃使蛋白酶失活10分钟。

将裂解物用作链优先定量聚合酶链反应(qPCR)分析的模板。用40个循环在95℃下进行15s，在60℃下进行1min进行定量PCR。使用Kappa Probe Force通用PCR预混液。正极性DNA链的引物和探针为5′CATGAACAAGAGATGTGTAGTAGGCAGAG3′，5′GGAGGCTGTAGGCATAAATTGG3′和5′/56-FAM/CTGCGCACC/ZEN/AGCACCATGCA/3IABkFQ。负极性DNA链的引物和探针是5'GCAGATGAGAAGGCACAGA3'，5'CTTCTCCGTCTGCCGTT3'和5'/56-FAM/AGTCCGCGT/ZEN/AAAGAGAGGTGCG/3IABkFQ。使用GraphPad Prism和三参数变量响应对数(抑制剂)-反应算法(variable-response log(inhibitor)-versus-response algorithm)将最低值设置为零，计算正链DNA的EC₅₀值。

(4)细胞毒性(CC₅₀)测试方法。使用CellTiter 96^TMAQueous非放射性细胞增殖测定法(MTS)在HepDES19细胞中测量存在化合物时的细胞生存力。在不存在四环素的情况下，将细胞以每孔1x 10⁴个细胞的密度接种到96孔板中，两天后使用化合物，并将细胞孵育3天。使用GraphPad Prism和三参数变量响应对数(抑制剂)-反应算法(底值设置为零)计算出50％的细胞毒性浓度(CC₅₀)值。

表2.目标化合物(二氢嘧啶-泊马度胺缀合物)抑制HBV DNA复制及细胞毒性的活性评价

结果显示，本发明新合成的二氢嘧啶-泊马度胺缀合物呈现出显著的抗HBV活性。所有二氢嘧啶-PROTAC类似物都在低微摩尔浓度水平显示出抗HBV活性，EC₅₀值在0.43-3.77μM范围内。三个化合物的活性尽管低于GLS4(EC₅₀＝0.046μM)，但是接近阳性药物3TC(EC₅₀＝0.40μM)，特别是8c(EC₅₀＝0.48μM)、8i(EC₅₀＝0.46μM)和8j(EC₅₀＝0.43μM)，具有进一步研究的价值。