毛萼内酯素B衍生物及其药物组合物与抗新冠肺炎的应用

毛萼内酯素b衍生物及其药物组合物与抗新冠肺炎的应用
技术领域
1.本发明属于药物技术领域，具体地，本发明涉及毛萼内酯素b衍生物及其药物组合物，以及它们在制备治疗sars-cov-2感染引起的疾病的药物中的应用。


背景技术：

2.2019新型冠状病毒(2019-ncov)是此前从未在人类中发现的冠状病毒新毒株。2020年2月11日，国际病毒分类委员会(ictv)宣布，2019新型冠状病毒(2019-ncov)的正式分类名为严重急性呼吸综合征冠状病毒2(severe acute respiratory syndrome coronavirus 2，sars-cov-2)。sars-cov-2编码4种结构蛋白和16种非结构蛋白，为识别潜在药物靶点提供了多个靶点。在非结构蛋白中，sars-cov-2 3cl
pro
是病毒生命周期中必不可少的蛋白酶，并且在人体细胞中没有密切相关的同源蛋白，使其成为有吸引力的药物靶点。
3.毛萼内酯素b的14位羟基取代衍生物具有新型骨架类型及全新的结构。现有报道中未见有毛萼内酯素b衍生物的结构及抗sars-cov-2的报道。


技术实现要素：

4.本发明目的是提供毛萼内酯素b衍生物及其药物组合物，和它们在制备治疗sars-cov-2感染引起的疾病的药物中的应用。本发明通过创造性的研究发现毛萼内酯素b衍生物对sars-cov-2 3cl
pro
具有抑制活力及抑制sars-cov-2在细胞中的病毒载量，在治疗sars-cov-2引起的疾病方面具有潜在的治疗效果。
5.为了实现本发明的上述目的，本发明提供了如下的技术方案：
6.如下结构式所示的毛萼内酯素b衍生物，
[0007][0008]
根据所述的毛萼内酯素b衍生物，其中r为以下官能团:
[0009][0010]
本发明同时提供了毛萼内酯素b衍生物的制备方法，该方法以毛萼内酯素b为起始原料，通过酯化反应得到毛萼内酯素b衍生物，
[0011][0012]
该方法包括如下步骤：氩气保护下，将毛萼内酯素b溶解在0.1～0.5ml的二氯甲烷中，加入edci及dmap，将反应置于室温中反应1～12个小时，tlc检测反应完全后，饱和nahco3淬灭，etoac萃取，饱和食盐水洗涤、无水mgso4干燥，过滤，浓缩后柱层析纯化或经过制备纯化，分离得到毛萼内酯素b衍生物。
[0013]
本发明还提供了毛萼内酯素b衍生物在制备sars-cov-2 3cl
pro
抑制剂中的应用，所述的sars-cov-2 3cl
pro
蛋白为新冠病毒3cl蛋白酶(主蛋白酶)；
[0014]
在制备治疗sars-cov-2感染引起的疾病的药物中的应用；
[0015]
在制备治疗单纯性感染、肺炎、急性呼吸道感染、低氧性呼吸衰竭、急性呼吸窘迫综合征、脓毒症、脓毒性休克病的药物中的应用；
[0016]
在制备治疗严重急性呼吸道感染、发热、咳嗽、咽痛的药物中的应用。
[0017]
此外，本发明还提供了一种含有毛萼内酯素b衍生物任其一或任其组合及可药用载体的药物组合物。
[0018]
以及，所述的药物组合物在制备sars-cov-2 3cl
pro
抑制剂中的应用，所述的sars-cov-2 3cl
pro
蛋白为新冠病毒3cl蛋白酶，即主蛋白酶。
[0019]
所述的药物组合物在制备治疗sars-cov-2感染引起的疾病的药物中的应用，所述的疾病为单纯性感染、肺炎、急性呼吸道感染、低氧性呼吸衰竭、急性呼吸窘迫综合征、脓毒症、脓毒性休克病。
[0020]
所述的药物组合物的制备方法，是按上述制备毛萼内酯素b衍生物的方法，先分离得到毛萼内酯素b衍生物，然后再加入可药用载体即可。
[0021]
本发明化合物用作药物时，可以直接使用，或者以药物组合物的形式使用。该药物组合物含有0.1-99％，优选0.5-90％的本发明化合物，其余为药物学上可接受的，对人和动物无毒和惰性的可药用载体和/或赋形剂。
[0022]
所述的药用载体或赋形剂是一种或多种固体、半固体和液体稀释剂、填料以及药物制品辅剂。将本发明的药物组合物以单位体重服用量的形式使用。本发明的药物可经多种形式(液体制剂、固体制剂、注射剂、外用制剂、喷剂、复方制剂)给药。
[0023]
与现有技术相比，本发明具备如下的优益性：
[0024]
1.本发明提供了一类新的毛萼内酯素b衍生物，填补了现有技术的空白。
[0025]
2.本发明提供了制备毛萼内酯素b衍生物的方法，该方法原料易得，易于操作，收率高，适于工业化生产。
[0026]
3.本发明提供了毛萼内酯素b衍生物作为有效成分的药物组合物，为新的抗新冠肺炎药物提供了具有较好药用作用的新的药物。
[0027]
4.本发明的毛萼内酯素b衍生物可在酶水平上抑制sars-cov-2 3cl
pro
的活力，在细胞水平上抑制sars-cov-2病毒复制，减少细胞培养物中sars-cov-2病毒核酸载量。
[0028]
5.毛萼内酯素b衍生物可作为药物用于治疗相关的疾病。可用于制备sars-cov-2 3cl
pro
抑制剂中，所述的sars-cov-2 3cl
pro
蛋白为新冠病毒3cl蛋白酶(主蛋白酶)；用于制备治疗sars-cov-2感染引起的疾病的药物；用于制备治疗包括单纯性感染、肺炎、急性呼吸道感染、低氧性呼吸衰竭、急性呼吸窘迫综合征、脓毒症、脓毒性休克病的药物中；用于制备治疗严重急性呼吸道感染、发热、咳嗽、咽痛的药物。
附图说明
[0029]
图1毛萼内酯素b衍生物在5μm浓度下对sars-cov-2 3cl
pro
的抑制率；
[0030]
图2毛萼内酯素b衍生物结构示意图。
具体实施方式
[0031]
下面结合附图，用本发明的实施例来对本发明作进一步的说明，但不以任何方式对本发明加以限制，基于本发明教导所作的任何变换或改进，均落入本发明的保护范围
[0032]
实施例1
[0033]
1.先制备毛萼内酯素b：
[0034]
制备中间体l：将化合物k溶解在(1～100ml)的丙酮中，在0℃冰浴条件下添加琼斯试剂(10ml)反应15分钟，使用tlc检测反应完全后，使用异丙醇淬灭，加水稀释后etoac萃取，饱和食盐水洗涤，无水mgso4干燥，过滤，浓缩后柱层析纯化，得到无定型固体化合物l，经质谱、一维和二维核磁共振鉴定化合物为中间体l。
[0035][0036]
制备中间体m：将化合物l溶解在(1～100ml)的1，2-二氯乙烷中，在0℃冰浴条件下加入nabh(oac)3(1～50g)后随后缓慢滴加乙酸(0.1～1ml)反应10分钟，使用tlc检测反应完全后，使用丙酮淬灭，加水稀释后etoac萃取，饱和食盐水洗涤，无水mgso4干燥，过滤，浓缩后柱层析纯化，得到无定型固体化合物，经质谱、一维和二维核磁共振鉴定化合物为中间体m。
[0037][0038]
制备中间体n：将化合物m溶解在(1～100ml)的二氯甲烷中，在0℃冰浴条件下依次添加dipea(1～50ml)以及tmscl(1～50ml)，在冰浴条件下反应30分钟后减压浓缩。浓缩后产品溶解于乙腈，后依次添加dbu(1～50ml)和对甲苯磺酰叠氮(1～50ml)反应2小时后滴加tbaf(1～50g)反应30分钟，使用tlc检测反应完全后，加水稀释加etoac萃取，饱和食盐水洗涤，无水mgso4干燥，过滤，浓缩后柱层析纯化，得到无定型固体化合物，经质谱、一维和二维核磁共振鉴定化合物为中间体n。
[0039][0040]
制备中间体o：将化合物n溶解在(1～100ml)的二氯甲烷中，在0℃冰浴条件下依次添加nahco3(1～10g)以及戴斯-马丁试剂(1～10g)，在冰浴条件下反应10分钟，使用tlc检测反应完全后，使用na2o3s2淬灭，加水稀释加etoac萃取，饱和食盐水、洗涤，无水mgso4干燥，过滤，浓缩后柱层析纯化，得到无定型固体化合物，经质谱、一维和二维核磁共振鉴定化合物为中间体o。
[0041][0042]
制备毛萼内酯素b：将化合物o溶解于(1～50ml)甲苯中，放置于110℃加热搅拌下充分反应两个小时。tlc检测反应完全后，将反应挪到室温，待冷却后，直接减压浓缩，浓缩后柱层析纯化，得到无定型白色固体化合物，经质谱、一维和二维核磁共振鉴定化合物为毛萼内酯素b。
[0043][0044]
2.制备毛萼内酯素b衍生物：
[0045]
氩气保护下，将毛萼内酯素b(10～20mg)溶解在(0.1～0.5ml)的二氯甲烷中，加入edci(10～50mg)及dmap(10～50mg)，将反应置于室温中反应1～12个小时，tlc检测反应完全后，饱和nahco3淬灭，etoac萃取，饱和食盐水洗涤、无水mgso4干燥，过滤，浓缩后柱层析纯化或经过制备纯化，得到化合物经质谱、一维共振鉴定的衍生物1-63。
[0046][0047][0048]
实施例2
[0049]
毛萼内酯素b衍生物核磁、质谱数据：
[0050][0051]
data for 1:1h nmr(600mhz,acetone-d6)δ＝6.09(s,1h),5.67(s,1h),5.59(s,1h),4.93(d,j＝10.8,1h),4.87(d,j＝10.8,1h),4.52(dt,j＝14.3,3.6,1h),4.06(d,j＝
3.7,1h),2.88(dd,j＝11.7,3.3,1h),2.49(dd,j＝9.2,2.6,1h),2.48
–
2.41(m,1h),2.23(dd,j＝13.8,11.8,1h),2.20
–
2.15(m,1h),2.11(t,j＝7.4,1h),1.99
–
1.92(m,2h),1.90(dd,j＝13.9,3.3,1h),1.82
–
1.73(m,1h),1.68
–
1.62(m,1h),1.49(q,j＝7.4,2h),1.24(s,3h),1.21(s,3h),0.86(t,j＝7.4,3h)；hresims(m/z):[m na]

calcd for c
24h30
o7na

430.1884,found 430.1892.
[0052]
data for 2:1h nmr(600mhz,acetone-d6)δ＝6.09(s,1h),5.68(d,j＝1.4,1h),5.59(d,j＝1.3,1h),5.49(s,1h),4.96(d,j＝10.8,1h),4.91(d,j＝10.8,1h),4.58(dt,j＝14.2,3.7,1h),4.02(d,j＝3.6,1h),3.33(s,1h),2.88(dd,j＝11.7,3.3,1h),2.48(dd,j＝9.3,2.3,1h),2.47
–
2.41(m,1h),2.24(dd,j＝13.9,11.7,1h),2.05(s,3h),1.99
–
1.95(m,1h),1.92(s,1h),1.90(dd,j＝13.8,3.4,1h),1.87(s,3h),1.78(s,1h),1.66(s,1h),1.25(s,3h),1.21(s,3h)；hresims(m/z):[m na]

calcd for c
25h30
o7na

465.1884,found 465.1889.
[0053][0054]
data for 3:1h nmr(600mhz,acetone-d6)δ＝6.09(s,1h),5.67(d,j＝1.4,1h),5.59(s,1h),4.93(d,j＝10.8,1h),4.87(d,j＝10.8,1h),4.52(dt,j＝14.3,3.7,1h),4.04(d,j＝3.7,1h),2.88(dd,j＝11.7,3.3,1h),2.49(dd,j＝9.1,2.6,1h),2.47
–
2.40(m,1h),2.27
–
2.09(m,3h),1.99
–
1.93(m,2h),1.90(dd,j＝13.9,3.3,1h),1.82
–
1.73(m,1h),1.69
–
1.61(m,1h),1.51
–
1.44(m,2h),1.32
–
1.25(m,8h),1.24(s,3h),1.21(s,3h),0.87(t,j＝7.0,3h)；hresims(m/z):[m na]

calcd for c
28h38
o7na

509.2510,found 509.2516.
[0055]
data for 4:1h nmr(600mhz,acetone-d6)δ＝6.09(s,1h),5.67(s,1h),5.60(s,1h),4.93(d,j＝10.8,1h),4.87(d,j＝10.8,1h),4.52(dt,j＝14.3,3.5,1h),4.06(d,j＝3.6,1h),3.33(s,1h),2.89(dd,j＝11.5,3.1,1h),2.49(dd,j＝9.2,2.5,1h),2.45
–
2.39(m,1h),2.26
–
2.08(m,3h),1.98
–
1.93(m,2h),1.90(dd,j＝13.9,3.2,1h),1.82
–
1.73(m,1h),1.68
–
1.60(m,1h),1.51
–
1.43(m,2h),1.33
–
1.24(m,10h),1.24(s,3h),1.21(s,3h),0.90
–
0.85(m,3h)；hresims(m/z):[m na]

calcd for c
29h40
o7na

523.2666,found 523.2658.
[0056][0057]
data for 5:1h nmr(600mhz,acetone-d6)δ＝6.10(s,1h),5.63(d,j＝1.4,1h),5.61(t,j＝1.0,1h),4.91(d,j＝10.9,1h),4.86(d,j＝10.8,1h),4.52(dd,j＝14.3,3.6,1h),4.11(d,j＝3.5,1h),3.35(s,1h),2.89
–
2.88(m,1h),2.48(dd,j＝9.3,2.4,1h),2.46
–
2.39(m,1h),2.23(dd,j＝13.9,11.8,1h),1.99
–
1.95(m,1h),1.93(d,j＝14.2,1h),1.89(dd,j＝13.8,3.3,1h),1.82
–
1.72(m,1h),1.67
–
1.60(m,1h),1.51
–
1.43(m,1h),1.23(s,
3h),1.20(s,3h),0.86
–
0.79(m,3h),0.76(dt,j＝8.8,4.5,1h)；hresims(m/z):[m k]

calcd for c
24h28
o7k

467.1467,found 467.1463.
[0058]
data for 6:1h nmr(600mhz,acetone-d6)δ＝6.08(s,1h),5.64(d,j＝1.4,1h),5.59(s,1h),4.94(d,j＝10.8,1h),4.88(d,j＝10.8,1h),4.56(dd,j＝14.3,3.7,1h),4.13(dd,j＝3.6,1.0,1h),3.32(s,1h),3.02
–
2.96(m,1h),2.89
–
2.88(m,1h),2.49(dd,j＝9.2,2.5,1h),2.47
–
2.40(m,1h),2.23(dd,j＝13.8,11.7,1h),2.13
–
2.09(m,4h),1.98
–
1.93(m,2h),1.92
–
1.87(m,2h),1.84
–
1.73(m,2h),1.68
–
1.61(m,1h),1.24(s,3h),1.21(s,3h)；hresims(m/z):[m na]

calcd for c
25h30
o7na

465.1884,found 465.1882.
[0059][0060]
data for 7:1h nmr(600mhz,acetone-d6)δ＝6.11
–
6.08(m,1h),5.63(d,j＝1.4,1h),5.59(t,j＝1.0,1h),4.93(d,j＝10.8,1h),4.88(d,j＝10.8,1h),4.54(dd,j＝14.3,3.7,1h),4.06(d,j＝3.7,1h),3.36
–
3.30(m,1h),2.88(dd,j＝11.7,3.3,1h),2.60
–
2.56(m,1h),2.49(dd,j＝9.2,2.5,1h),2.47
–
2.39(m,1h),2.23(dd,j＝13.9,11.7,1h),2.00
–
1.92(m,2h),1.90(dd,j＝13.9,3.3,1h),1.83
–
1.70(m,3h),1.70
–
1.62(m,3h),1.62
–
1.55(m,2h),1.55
–
1.47(m,2h),1.24(s,3h),1.21(s,3h)；hresims(m/z):[m na]

calcd for c
26h32
o7na

479.2040,found 479.2042.
[0061]
data for 8:hresims(m/z):1h nmr(600mhz,acetone-d6)δ＝6.62(q,j＝2.3,1h),6.10(s,1h),5.67(d,j＝1.4,1h),5.60(s,1h),4.97(d,j＝10.8,1h),4.90(d,j＝10.8,1h),4.58(dd,j＝14.2,3.7,1h),4.06(d,j＝3.7,1h),3.38(d,j＝3.9,1h),2.89(dd,j＝11.7,3.4,1h),2.50(dd,j＝9.1,2.4,1h),2.48
–
2.36(m,5h),2.24(dd,j＝13.9,11.7,1h),2.00
–
1.93(m,2h),1.93
–
1.86(m,3h),1.83
–
1.74(m,1h),1.66(s,1h),1.25(s,3h),1.21(s,3h)；hresims(m/z):[m na]

calcd for c
26h30
o7na

477.1884,found 477.1888.
[0062][0063]
data for 9:1h nmr(600mhz,acetone-d6)δ＝6.10(s,1h),5.67(d,j＝1.4,1h),5.61(s,1h),4.93(d,j＝10.8,1h),4.86(d,j＝10.8,1h),4.55(dd,j＝14.3,3.7,1h),4.25(dd,j＝8.7,4.1,1h),4.17(d,j＝3.6,1h),3.82
–
3.75(m,2h),3.38
–
3.32(m,1h),2.89(dd,j＝11.7,3.4,1h),2.51(dd,j＝9.1,2.7,1h),2.48
–
2.40(m,1h),2.24(dd,j＝13.9,11.7,1h),2.13
–
2.09(m,1h),1.99
–
1.93(m,2h),1.93
–
1.84(m,3h),1.84
–
1.74(m,2h),1.69
–
1.63(m,1h),1.24(s,3h),1.22(s,3h)；hresims(m/z):[m na]

calcd for c
25h30
o8na

481.1833,found 481.1842.
[0064]
data for 10:1h nmr(600mhz,acetone-d6)δ＝7.81(d,j＝1.8,1h),7.10(d,j＝3.5,1h),6.65
–
6.59(m,1h),6.14(s,1h),5.85(s,1h),5.65(s,1h),4.98(d,j＝10.8,1h),
4.92(d,j＝10.8,1h),4.62(dd,j＝14.2,3.4,1h),4.14(d,j＝3.6,1h),3.49(d,j＝3.9,1h),2.91(dd,j＝11.7,3.3,1h),2.55(dd,j＝9.1,2.7,1h),2.53
–
2.46(m,1h),2.25(dd,j＝13.9,11.7,1h),2.02
–
1.96(m,2h),1.92(dd,j＝13.9,3.3,1h),1.86
–
1.77(m,1h),1.73
–
1.67(m,1h),1.25(s,3h),1.22(s,3h)；hresims(m/z):[m na]

calcd for c
25h26
o8na

477.1520,found 477.1524.
[0065][0066]
data for 11:1h nmr(600mhz,acetone-d6)δ＝7.86(dd,j＝5.0,1.3,1h),7.66(dd,j＝3.8,1.3,1h),7.18(dd,j＝5.0,3.7,1h),6.15(s,1h),5.85(d,j＝1.4,1h),5.66(s,1h),5.00(d,j＝10.8,1h),4.93(d,j＝10.8,1h),4.64(dd,j＝14.2,3.5,1h),4.13(s,1h),3.51(s,1h),2.91(dd,j＝11.7,3.4,1h),2.56(dd,j＝9.1,2.7,1h),2.54
–
2.46(m,1h),2.25(dd,j＝13.9,11.7,1h),2.03
–
1.97(m,2h),1.92(dd,j＝13.9,3.4,1h),1.87
–
1.77(m,1h),1.73
–
1.67(m,1h),1.26(s,3h),1.22(s,3h)；hresims(m/z):[m na]

calcd for c
25h26
o7sna

493.1291,found 493.1289.
[0067]
data for 12:1h nmr(600mhz,acetone-d6)δ＝7.05(s,1h),6.71(s,1h),6.18(s,1h),6.12(s,1h),5.79(d,j＝1.4,1h),5.61(d,j＝1.0,1h),4.99(d,j＝10.8,1h),4.95(d,j＝10.8,1h),4.64(dd,j＝14.2,3.6,1h),4.07(d,j＝3.6,1.1,1h),3.44(d,j＝3.9,1h),2.90(dd,j＝11.7,3.3,1h),2.52(dd,j＝9.3,2.4,1h),2.50
–
2.44(m,1h),2.25(dd,j＝13.9,11.7,1h),2.02
–
1.93(m,2h),1.91(dd,j＝13.9,3.4,1h),1.85
–
1.77(m,1h),1.70
–
1.65(m,1h),1.25(s,3h),1.22(s,3h)；hresims(m/z):[m na]

calcd for c
25h27
no7na

476.1680,found 476.1677.
[0068][0069]
data for 13:1h nmr(600mhz,acetone-d6)δ＝8.40(d,j＝2.1,1h),7.72(d,j＝2.0,1h),6.16(s,1h),5.92(s,1h),5.67(s,1h),4.97(d,j＝10.8,1h),4.90(d,j＝10.8,1h),4.60(dd,j＝14.2,3.7,1h),4.19(d,j＝3.6,1h),3.52(s,1h),2.92(dd,j＝11.7,3.3,1h),2.58(dd,j＝9.2,3.0,1h),2.56
–
2.48(m,1h),2.25(dd,j＝13.9,11.7,1h),2.00(dd,j＝12.4,8.1,2h),1.93
–
1.90(m,1h),1.86
–
1.78(m,1h),1.75
–
1.68(m,1h),1.25(s,3h),1.22(s,3h)；hresims(m/z):[m na]

calcd for c
24h25
no8na

456.1653,found 456.1654.
[0070]
data for 14:1h nmr(600mhz,acetone-d6)δ＝9.25(s,1h),8.34(s,1h),6.16(s,1h),5.90(s,1h),5.67(s,1h),4.98(d,j＝10.8,1h),4.91(d,j＝10.9,1h),4.62(dd,j＝14.2,3.6,1h),4.20(d,j＝3.7,1h),3.54(d,j＝3.9,1h),2.92(dd,j＝11.7,3.4,1h),2.58(dd,j＝9.0,2.9,1h),2.55
–
2.48(m,1h),2.26(dd,j＝13.8,11.8,1h),2.03
–
1.99(m,2h),1.92(dd,j＝13.8,3.3,1h),1.86
–
1.78(m,1h),1.71(ddt,j＝15.7,6.6,3.0,1h),1.25(s,
3h),1.22(s,3h)；hresims(m/z):[m na]

calcd for c
24h25
no7sna

472.1424,found 472.1428.
[0071][0072]
data for 15:1h nmr(600mhz,acetone-d6)δ＝9.50(s,1h),6.16(s,1h),6.02(d,j＝1.4,1h),5.68(d,j＝1.2,1h),5.00(d,j＝10.8,1h),4.94(d,j＝10.8,1h),4.65(dd,j＝14.2,3.5,1h),4.22(d,j＝3.6,1h),3.60
–
3.58(m,1h),2.93(dd,j＝11.7,3.4,1h),2.62
–
2.60(m,1h),2.59
–
2.52(m,1h),2.26(dd,j＝13.8,11.7,1h),2.04
–
2.01(m,2h),1.93(dd,j＝13.9,3.4,1h),1.88
–
1.81(m,1h),1.77
–
1.71(m,1h),1.26(s,3h),1.23(s,3h)；hresims(m/z):[m na]

calcd for c
23h24
n2o7sna

495.1196,found 495.1198.
[0073]
data for 16:1h nmr(600mhz,acetone-d6)δ＝6.09(s,1h),5.63(s,1h),5.59(s,1h),4.93(d,j＝10.8,1h),4.88(d,j＝10.8,1h),4.53(dd,j＝14.3,3.4,1h),4.08(d,j＝3.6,1h),3.32(d,j＝4.1,1h),2.88(dd,j＝11.7,3.3,1h),2.80(s,1h),2.48(dd,j＝9.2,2.4,1h),2.47
–
2.40(m,1h),2.23(dd,j＝13.9,11.7,1h),2.16
–
2.10(m,1h),1.99
–
1.92(m,2h),1.90(dd,j＝13.9,3.3,1h),1.82
–
1.72(m,3h),1.65(s,3h),1.61
–
1.55(m,1h),1.31
–
1.24(m,3h),1.24(s,3h),1.21(s,3h),1.20
–
1.13(m,1h)；hresims(m/z):[m
–
h]
–
calcd for c
27h33
o7–
469.2232,found 469.2233.
[0074][0075]
data for 17:1h nmr(600mhz,acetone-d6)δ＝6.82
–
6.77(m,1h),6.10(s,1h),5.67(d,j＝1.4,1h),5.60(s,1h),4.97(d,j＝10.8,1h),4.90(d,j＝10.8,1h),4.58(dd,j＝14.3,3.7,1h),4.06(d,j＝3.7,1h),3.38(d,j＝4.1,1h),2.88(dd,j＝11.7,3.4,1h),2.50(dd,j＝9.2,2.3,1h),2.47
–
2.41(m,1h),2.24(dd,j＝13.9,11.7,1h),2.17
–
2.12(m,2h),2.08
–
2.06(m,2h),2.00
–
1.93(m,2h),1.90(dd,j＝13.9,3.4,1h),1.83
–
1.74(m,1h),1.69
–
1.62(m,1h),1.61
–
1.51(m,4h),1.25(s,3h),1.21(s,3h)；hresims(m/z):[m na]

calcd for c
27h32
o7na

491.2040,found 491.2041.
[0076]
data for 18:1h nmr(600mhz,acetone-d6)δ＝7.86
–
7.82(m,2h),7.66
–
7.62(m,1h),7.51
–
7.47(m,2h),6.16(s,1h),5.90(d,j＝1.5,1h),5.66(s,1h),5.02(d,j＝10.8,1h),4.96(d,j＝10.8,1h),4.65(dt,j＝14.3,3.7,1h),4.14(d,j＝3.5,1h),3.53(s,1h),2.92(dd,j＝11.7,3.4,1h),2.57(dd,j＝9.1,2.5,1h),2.55
–
2.48(m,1h),2.26(dd,j＝13.8,11.8,1h),2.00(d,j＝14.3,2h),1.92(dd,j＝13.9,3.3,1h),1.88
–
1.79(m,1h),1.74
–
1.68(m,1h),1.26(s,3h),1.22(s,3h)；hresims(m/z):[m na]

calcd for c
27h28
o7na

478.1727,found 478.1726.
[0077][0078]
data for 19:1h nmr(800mhz,acetone-d6)δ＝8.80
–
8.76(m,2h),7.69
–
7.65(m,2h),6.18(s,1h),5.95(d,j＝1.4,1h),5.68(d,j＝1.0,1h),5.00(d,j＝10.7,1h),4.94(d,j＝10.7,1h),4.63(dd,j＝14.2,3.6,1h),4.21(d,j＝3.6,1h),3.57(dt,j＝4.8,2.3,1h),2.93(dd,j＝11.7,3.4,1h),2.60(dd,j＝9.3,3.0,1h),2.56
–
2.50(m,1h),2.26(dd,j＝13.9,11.8,1h),2.04
–
2.02(m,2h),1.93(dd,j＝13.9,3.4,1h),1.85
–
1.84(m,1h),1.74
–
1.72(m,1h),1.26(s,3h),1.23(s,3h)；hresims(m/z):[m h] calcd for c
26h28
no7 466.1860,found 466.1863.
[0079]
data for 20:1h nmr(800mhz,acetone-d6)δ＝9.01(d,j＝1.5,1h),8.84(d,j＝2.4,1h),8.75(dd,j＝2.3,1.5,1h),6.18(s,1h),6.00(d,j＝1.4,1h),5.69(s,1h),5.00(d,j＝10.7,1h),4.95(d,j＝10.8,1h),4.64(dd,j＝14.2,3.6,1h),4.24(d,j＝3.5,1h),3.58(d,j＝3.1,1h),2.93(dd,j＝11.8,3.4,1h),2.61(d,j＝7.7,1h),2.57
–
2.52(m,1h),2.27(dd,j＝13.9,11.8,1h),2.04
–
2.02(m,2h),1.93(dd,j＝13.9,3.4,1h),1.87
–
1.82(m,1h),1.76
–
1.72(m,1h),1.26(s,3h),1.23(s,3h)；hresims(m/z):[m h]

calcd for c
25h27
n2o
7
467.1813,found 467.1815.
[0080][0081]
data for 21:1h nmr(800mhz,acetone-d6)δ＝9.35(s,1h),9.06(s,2h),6.19
–
6.17(m,1h),5.98(d,j＝1.4,1h),5.68(d,j＝1.0,1h),4.99(d,j＝10.7,1h),4.93(d,j＝10.7,1h),4.64
–
4.61(m,1h),4.27(s,1h),3.60(s,1h),2.93(dd,j＝11.8,3.4,1h),2.61(d,j＝5.8,1h),2.56
–
2.51(m,1h),2.26(dd,j＝13.9,11.7,1h),2.04
–
2.01(m,2h),1.92(dd,j＝13.9,3.4,1h),1.87
–
1.81(m,1h),1.75
–
1.71(m,1h),1.25(s,3h),1.22(s,3h)；hresims(m/z):[m h]

calcd for c
25h27
n2o
7
467.1813,found 467.1817.
[0082]
data for 22:1h nmr(600mhz,acetone-d6)δ＝7.96
–
7.84(m,2h),7.34
–
7.18(m,2h),6.16(s,1h),5.88(s,1h),5.66(s,1h),5.01(d,j＝10.8,1h),4.95(d,j＝10.7,1h),4.68
–
4.58(m,1h),4.17(d,j＝3.7,1h),3.53(s,1h),2.98(s,1h),2.57(dd,j＝9.2,2.6,1h),2.54
–
2.46(m,1h),2.25(dd,j＝13.9,11.8,1h),2.00(d,j＝14.3,2h),1.91(dd,j＝13.8,3.3,1h),1.88
–
1.76(m,1h),1.75
–
1.66(m,1h),1.25(s,3h),1.22(s,3h)；hresims(m/z):[m na]

calcd for c
27h27
fo7na

505.1633,found 505.1632.
[0083][0084]
data for 23:1h nmr(600mhz,acetone-d6)δ＝7.85
–
7.80(m,2h),7.57
–
7.52(m,2h),6.16(s,1h),5.90(d,j＝1.4,1h),5.66(s,1h),5.01(d,j＝10.8,1h),4.94(d,j＝10.8,1h),4.64(d,j＝14.2,1h),4.19(s,1h),3.53(s,1h),2.94
–
2.92(m,1h),2.57(dd,j＝9.1,2.6,1h),2.54
–
2.48(m,1h),2.25(dd,j＝13.9,11.7,1h),2.03
–
1.98(m,2h),1.91(dd,j＝13.9,3.3,1h),1.87
–
1.79(m,1h),1.74
–
1.68(m,1h),1.25(s,3h),1.22(s,3h)；hresims(m/z):[m
–
h]
–
calcd for c
27h26
clo7–
497.1373,found 497.1372.
[0085]
data for 24:1h nmr(600mhz,acetone-d6)δ＝7.78
–
7.73(m,2h),7.73
–
7.68(m,2h),6.16(s,1h),5.90(s,1h),5.66(s,1h),5.00(d,j＝10.8,1h),4.94(d,j＝10.8,1h),4.63(dd,j＝14.2,3.6,1h),4.16(d,j＝3.6,1h),3.53(s,1h),2.92(dd,j＝11.8,3.3,1h),2.57(dd,j＝9.1,2.6,1h),2.55
–
2.48(m,1h),2.25(dd,j＝13.9,11.8,1h),2.03
–
1.98(m,2h),1.92(dd,j＝13.9,3.3,1h),1.87
–
1.79(m,1h),1.74
–
1.68(m,1h),1.25(s,3h),1.22(s,3h)；hresims(m/z):[m na]

calcd for c
27h27
bro7na

565.0832,found 565.0826.
[0086][0087]
data for 25:1h nmr(600mhz,acetone-d6)δ＝8.02(d,j＝8.1,2h),7.86(d,j＝8.1,2h),6.16(s,1h),5.94(s,1h),5.67(s,1h),5.00(d,j＝10.7,1h),4.94(d,j＝10.8,1h),4.63(dt,j＝14.3,3.5,1h),4.19(d,j＝3.5,1h),3.56(d,j＝3.8,1h),2.92(dd,j＝11.8,3.3,1h),2.58(d,j＝8.6,1h),2.55
–
2.50(m,1h),2.25(dd,j＝13.8,11.8,1h),2.03
–
1.99(m,2h),1.91(dd,j＝14.0,3.3,1h),1.86
–
1.80(m,1h),1.74
–
1.69(m,1h),1.25(s,3h),1.21(s,3h)；hresims(m/z):[m
–
h]
–
calcd for c
27h26
no9–
508.1613,found 508.1613.
[0088]
data for 26:1h nmr(600mhz,acetone-d6)δ＝7.80
–
7.77(m,2h),7.02
–
6.98(m,2h),6.15(s,1h),5.84(d,j＝1.4,1h),5.65(s,1h),5.02(d,j＝10.8,1h),4.96(d,j＝10.8,1h),4.65(dd,j＝14.2,3.3,1h),4.12(d,j＝3.5,1h),3.87(s,3h),3.50(d,j＝3.9,1h),2.91(dd,j＝11.7,3.3,1h),2.55(dd,j＝9.2,2.3,1h),2.53
–
2.47(m,1h),2.25(dd,j＝13.9,11.8,1h),2.03
–
1.96(m,2h),1.91(dd,j＝13.9,3.3,1h),1.86
–
1.78(m,1h),1.72
–
1.67(m,1h),1.26(s,3h),1.22(s,3h)；hresims(m/z):[m
–
h]
–
calcd for c
28h29
o8–
493.1868,found 493.1865.
[0089]
[0090]
data for 27:1h nmr(600mhz,acetone-d6)δ＝7.72(d,j＝7.9,2h),7.30(d,j＝7.9,2h),6.16(s,1h),5.87(s,1h),5.65(d,j＝1.3,1h),5.02(d,j＝10.8,1h),4.96(d,j＝10.8,1h),4.65(dd,j＝14.3,3.3,1h),4.14(d,j＝3.5,1h),3.51(d,j＝3.8,1h),2.92(dd,j＝11.8,3.2,1h),2.56(dd,j＝9.1,2.3,1h),2.54
–
2.48(m,1h),2.39(s,3h),2.25(dd,j＝13.8,11.8,1h),2.03
–
1.97(m,2h),1.92(dd,j＝13.9,3.3,1h),1.86
–
1.80(m,1h),1.73
–
1.68(m,1h),1.26(s,3h),1.22(s,3h)；hresims(m/z):[m
–
h]
–
calcd for c
28h29
o7–
477.1919,found 477.1919.
[0091]
data for 28:1h nmr(600mhz,acetone-d6)δ＝8.38
–
8.34(m,2h),8.10
–
8.05(m,2h),6.18(s,1h),5.97(s,1h),5.69(s,1h),5.01(d,j＝10.8,1h),4.94(d,j＝10.8,1h),4.64(dt,j＝14.2,3.7,1h),4.22(d,j＝3.7,1h),3.59(d,j＝3.8,1h),2.93(dd,j＝11.7,3.3,1h),2.60
–
2.59(m,1h),2.57
–
2.50(m,1h),2.26(dd,j＝13.9,11.8,1h),2.03
–
2.00(m,2h),1.92(dd,j＝13.9,3.3,1h),1.88
–
1.80(m,1h),1.76
–
1.70(m,1h),1.26(s,3h),1.22(s,3h)；hresims(m/z):[m
–
h]
–
calcd for c
28h26
f3o7–
531.1636,found 531.1629.
[0092][0093]
data for 29:1h nmr(800mhz,acetone-d6)δ＝8.43(d,j＝5.1,1h),7.63(dt,j＝5.1,1.5,1h),7.34(s,1h),6.18(s,1h),5.96(d,j＝1.4,1h),5.68(d,j＝1.0,1h),4.99(d,j＝10.8,1h),4.92(d,j＝10.7,1h),4.63
–
4.60(m,1h),4.24(d,j＝3.6,1h),3.58(s,1h),2.93(dd,j＝11.8,3.4,1h),2.62
–
2.60(m,1h),2.55
–
2.51(m,1h),2.26(dd,j＝13.9,11.7,1h),2.04
–
2.00(m,2h),1.92(dd,j＝13.9,3.4,1h),1.87
–
1.81(m,1h),1.76
–
1.71(m,1h),1.25(s,3h),1.22(s,3h)；hresims(m/z):[m
–
h]
–
calcd for c
26h25
fno7–
482.1621,found 482.1623.
[0094]
data for 30:1h nmr(800mhz,acetone-d6)δ＝8.66(d,j＝2.3,1h),8.58(d,j＝4.8,1h),7.62(dd,j＝6.1,4.9,1h),6.15(s,1h),5.99(d,j＝1.5,1h),5.67(d,j＝1.0,1h),4.98(d,j＝10.7,1h),4.91(d,j＝10.7,1h),4.60(dd,j＝14.2,3.6,1h),4.23(d,j＝3.7,1h),3.54(s,1h),2.93(dd,j＝11.7,3.4,1h),2.60
–
2.58(m,1h),2.56
–
2.50(m,1h),2.26(dd,j＝13.9,11.8,1h),2.01(dd,j＝7.5,3.5,2h),1.92(dd,j＝13.9,3.4,1h),1.86
–
1.80(m,1h),1.76
–
1.71(m,1h),1.26(s,3h),1.22(s,3h)；hresims(m/z):[m
–
h]
–
calcd for c
26h25
fno7–
482.1621,found 482.1624.
[0095][0096]
data for 31:1h nmr(600mhz,acetone-d6)δ＝8.60(d,j＝5.0,1h),7.68(dd,j＝6.6,1.5,2h),6.18(s,1h),5.96(d,j＝1.5,1h),5.68(s,1h),5.62(d,j＝1.7,1h),4.98(d,j＝10.8,1h),4.91(d,j＝10.8,1h),4.61(dd,j＝14.2,3.4,1h),4.25(d,j＝3.6,1h),3.58
(t,j＝3.7,1h),2.93(dd,j＝11.7,3.3,1h),2.64
–
2.59(m,1h),2.57
–
2.48(m,1h),2.26(dd,j＝13.9,11.7,1h),2.03
–
1.99(m,1h),1.92(dd,j＝13.8,3.3,1h),1.84(ddt,j＝16.4,8.6,4.5,1h),1.73(ddt,j＝15.8,6.6,3.0,1h),1.25(s,3h),1.22(d,j＝2.7,3h)；hresims(m/z):[m
–
h]
–
calcd for c
26h25
clno7–
498.1325,found 498.1325.
[0097]
data for 32:1h nmr(600mhz,acetone-d6)δ＝8.71(s,1h),8.65(d,j＝4.9,1h),7.57(d,j＝4.9,1h),6.13(s,1h),6.00(s,1h),5.65(s,1h),4.99(d,j＝10.8,1h),4.93(d,j＝10.8,1h),4.61(dd,j＝14.2,3.5,1h),4.30(dt,j＝3.4,1.6,1h),3.54(d,j＝4.1,1h),2.93(dd,j＝11.8,3.3,1h),2.62
–
2.58(m,1h),2.57
–
2.51(m,1h),2.25(dd,j＝13.8,11.7,1h),2.03
–
1.98(m,2h),1.91(dd,j＝13.8,3.3,1h),1.86
–
1.79(m,1h),1.76
–
1.70(m,1h),1.25(s,3h),1.22(s,3h)；hresims(m/z):[m
–
h]
–
calcd for c
26h25
clno7–
498.1325,found 498.1324.
[0098][0099]
data for 33:1h nmr(600mhz,acetone-d6)δ＝8.84(d,j＝2.4,1h),8.68(dd,j＝5.4,2.3,1h),7.55(d,j＝4.7,1h),6.13(s,1h),6.00(s,1h),5.65(d,j＝2.5,1h),4.99(dd,j＝10.8,2.4,1h),4.94(dd,j＝10.8,2.5,1h),4.64
–
4.57(m,1h),4.34(s,1h),3.54(s,1h),2.95
–
2.92(m,1h),2.60(d,j＝7.9,1h),2.57
–
2.51(m,1h),2.29
–
2.21(m,1h),2.02
–
1.97(m,2h),1.91(dd,j＝13.8,3.2,1h),1.86
–
1.77(m,1h),1.75
–
1.69(m,1h),1.25(s,3h),1.22(s,3h)；hresims(m/z):[m k]

calcd for c
26h26
brno7k

582.0524,found 582.0528.
[0100]
data for 34:1h nmr(600mhz,acetone-d6)δ＝8.58(d,j＝5.0,1h),7.82(d,j＝1.4,1h),7.70(dd,j＝5.0,1.3,1h),6.17(s,1h),5.94(s,1h),5.68(s,1h),4.98(d,j＝10.8,1h),4.91(d,j＝10.8,1h),4.60(dd,j＝14.3,3.2,1h),4.31(d,j＝3.4,1h),3.58
–
3.55(m,1h),2.95
–
2.92(m,1h),2.60
–
2.59(m,1h),2.55
–
2.48(m,1h),2.28
–
2.22(m,1h),2.03
–
1.99(m,2h),1.90(dd,j＝13.9,3.3,1h),1.85
–
1.79(m,1h),1.74
–
1.68(m,1h),1.23(s,3h),1.21(s,3h)；hresims(m/z):[m k]

calcd for c
26h26
brno7k

582.0524,found 582.0527.
[0101][0102]
data for 35:1h nmr(600mhz,acetone-d6)δ＝7.29
–
7.25(m,2h),7.21
–
7.17(m,3h),6.09(s,1h),5.67(d,j＝1.4,1h),5.58(s,1h),4.92(d,j＝10.8,1h),4.86(d,j＝10.8,1h),4.51(dd,j＝14.3,3.5,1h),4.07(d,j＝3.5,1h),3.28(d,j＝4.1,1h),2.91
–
2.88(m,1h),2.82
–
2.79(m,2h),2.57
–
2.45(m,3h),2.45
–
2.40(m,1h),2.23(dd,j＝13.8,11.7,1h),1.98
–
1.92(m,2h),1.90(dd,j＝13.9,3.3,1h),1.81
–
1.73(m,1h),1.67
–
1.62(m,
for c
29h29
clo7na

547.1494,found 547.1499.
[0109]
data for 40:1h nmr(600mhz,acetone-d6)δ＝7.65(d,j＝8.6,2h),7.62(d,j＝8.6,2h),7.55(d,j＝16.0,1h),6.45(d,j＝16.0,1h),6.13(s,1h),5.79(d,j＝1.4,1h),5.64(s,1h),4.97(d,j＝10.8,1h),4.92(d,j＝10.8,1h),4.60(dd,j＝14.3,3.6,1h),4.13(d,j＝3.6,1h),3.42(d,j＝3.9,1h),2.90(dd,j＝11.7,3.4,1h),2.53(dd,j＝9.2,2.6,1h),2.51
–
2.44(m,1h),2.25(dd,j＝13.9,11.7,1h),2.01
–
1.95(m,2h),1.91(dd,j＝13.9,3.3,1h),1.84
–
1.76(m,1h),1.71
–
1.65(m,1h),1.25(s,3h),1.22(s,3h)；hresims(m/z):[m na]

calcd for c
29h29
bro7na

591.0989,found 591.0990.
[0110][0111]
data for 41:1h nmr(600mhz,acetone-d6)δ＝8.31
–
8.26(m,2h),8.01
–
7.97(m,2h),7.68(d,j＝16.1,1h),6.64(d,j＝16.0,1h),6.14(s,1h),5.82(d,j＝1.4,1h),5.65(t,j＝1.0,1h),4.97(d,j＝10.8,1h),4.92(d,j＝10.8,1h),4.60(dd,j＝14.3,3.7,1h),4.14(d,j＝3.7,1h),3.44(s,1h),2.93
–
2.90(m,1h),2.54(dd,j＝9.1,2.7,1h),2.52
–
2.46(m,1h),2.25(dd,j＝13.9,11.7,1h),2.02
–
1.96(m,2h),1.92(dd,j＝13.9,3.3,1h),1.85
–
1.77(m,1h),1.72
–
1.66(m,1h),1.26(s,3h),1.22(s,3h)；hresims(m/z):[m na]

calcd for c
29h29
no9na

558.1735,found 558.1735.
[0112][0113]
data for 42:1h nmr(600mhz,acetone-d6)δ＝7.65
–
7.59(m,2h),7.52(d,j＝15.9,1h),7.00
–
6.96(m,2h),6.23(d,j＝15.9,1h),6.12(s,1h),5.76(d,j＝1.4,1h),5.62(d,j＝1.1,1h),4.96(d,j＝10.8,1h),4.92(d,j＝10.8,1h),4.60(dd,j＝14.3,3.6,1h),4.11(d,j＝3.6,1h),3.84(s,3h),3.39(d,j＝3.9,1h),2.91
–
2.88(m,1h),2.51(dd,j＝9.2,2.6,1h),2.48
–
2.43(m,1h),2.23(dd,j＝13.9,11.8,1h),2.00
–
1.93(m,2h),1.92
–
1.88(m,1h),1.83
–
1.75(m,1h),1.69
–
1.64(m,1h),1.24(s,3h),1.21(s,3h)；hresims(m/z):[m na]

calcd for c
30h32
o8na

543.1995,found 543.1989.
[0114]
data for 43:1h nmr(600mhz,acetone-d6)δ＝7.58
–
7.52(m,3h),7.25(d,j＝7.9,2h),6.34(d,j＝16.0,1h),6.13(s,1h),5.78(d,j＝1.4,1h),5.64(d,j＝1.1,1h),4.97(d,j＝10.8,1h),4.93(d,j＝10.8,1h),4.61(dd,j＝14.3,3.6,1h),4.13(d,j＝3.6,1h),3.43
–
3.40(m,1h),2.92
–
2.89(m,1h),2.52(dd,j＝9.2,2.6,1h),2.50
–
2.44(m,1h),2.36(s,3h),2.25(dd,j＝13.9,11.8,1h),2.01
–
1.95(m,2h),1.91(dd,j＝13.9,3.4,1h),1.83
–
1.77(m,1h),1.70
–
1.65(m,1h),1.25(s,3h),1.22(s,3h)；hresims(m/z):[m na]

calcd for c
30h32
o7na

527.2040,found 527.2035.
[0115][0116]
data for 44:1h nmr(600mhz,acetone-d6)δ＝7.51(d,j＝15.9,1h),7.33(d,j＝2.0,1h),7.17(dd,j＝8.3,2.0,1h),6.98(d,j＝8.3,1h),6.28(d,j＝15.9,1h),6.13(s,1h),5.77(d,j＝1.3,1h),5.64(s,1h),4.95(q,j＝10.8,2h),4.61(dd,j＝14.3,3.6,1h),4.12(d,j＝3.6,1h),3.87(s,3h),3.86(s,3h),3.40(s,1h),2.92
–
2.89(m,1h),2.52(dd,j＝9.1,2.4,1h),2.50
–
2.44(m,1h),2.24(dd,j＝13.9,11.8,1h),2.01
–
1.93(m,2h),1.91(dd,j＝13.9,3.3,1h),1.84
–
1.76(m,1h),1.70
–
1.65(m,1h),1.25(s,3h),1.22(s,3h)；hresims(m/z):[m na]

calcd for c
31h34
o9na

573.2095,found 573.2091.
[0117]
data for 45:1h nmr(600mhz,acetone-d6)δ＝7.99(s,1h),7.64(d,j＝1.9,1h),7.51(d,j＝15.8,1h),6.91(d,j＝1.9,1h),6.16
–
6.11(m,2h),5.76(s,1h),5.64
–
5.62(m,1h),4.99
–
4.90(m,2h),4.59(dd,j＝14.3,3.6,1h),4.11(d,j＝3.4,1h),3.42
–
3.37(m,1h),2.92
–
2.89(m,1h),2.51(dd,j＝9.3,2.6,1h),2.50
–
2.44(m,1h),2.24(dd,j＝13.8,11.7,1h),1.97(dd,j＝17.4,13.0,2h),1.91(dd,j＝13.9,3.3,1h),1.83
–
1.76(m,1h),1.71
–
1.65(m,1h),1.25(s,3h),1.22(s,3h)；hresims(m/z):[m na]

calcd for c
27h28
o8na

503.1686,found 503.1676.
[0118][0119]
data for 46:1h nmr(600mhz,acetone-d6)δ＝8.65(d,j＝5.2,2h),7.64
–
7.59(m,2h),7.53(d,j＝16.1,1h),6.65(d,j＝16.0,1h),6.14(s,1h),5.81(d,j＝1.4,1h),5.65(t,j＝1.0,1h),4.97(d,j＝10.8,1h),4.92(d,j＝10.8,1h),4.60(dd,j＝14.3,3.6,1h),4.23
–
4.18(m,1h),3.44(s,1h),2.94
–
2.90(m,1h),2.54(dd,j＝9.1,2.7,1h),2.53
–
2.44(m,1h),2.25(dd,j＝13.9,11.7,1h),2.03
–
1.95(m,2h),1.91(dd,j＝13.9,3.3,1h),1.86
–
1.75(m,1h),1.72
–
1.64(m,1h),1.25(s,3h),1.22(s,4h)；hresims(m/z):[m h]

calcd for c
27h30
no
7
492.2018,found 492.2017.
[0120]
data for 47:1h nmr(600mhz,acetone-d6)δ＝7.22
–
7.15(m,2h),7.15
–
7.09(m,2h),6.11(s,1h),5.70(s,1h),5.61(s,1h),4.98
–
4.91(m,1h),4.91
–
4.85(m,1h),4.59
–
4.52(m,1h),4.18
–
4.13(m,1h),3.37(s,1h),3.25
–
3.18(m,1h),3.12
–
3.04(m,4h),2.92
–
2.88(m,1h),2.54
–
2.49(m,1h),2.49
–
2.41(m,1h),2.28
–
2.20(m,1h),2.00
–
1.93(m,2h),1.92
–
1.88(m,1h),1.84
–
1.74(m,1h),1.69
–
1.63(m,1h),1.23(s,3h),1.21(s,3h)；hresims(m/z):[m na]

calcd for c
30h32
o7na

537.1884,found 537.1893.
[0121][0122]
data for 48:1h nmr(600mhz,acetone-d6)δ＝7.86
–
7.79(m,1h),7.63(d,j＝8.4,1h),7.57
–
7.50(m,2h),7.38(t,j＝7.4,1h),6.18(s,1h),5.95(s,1h),5.73
–
5.67(m,1h),5.01(d,j＝10.8,1h),4.95(d,j＝10.7,1h),4.65(dd,j＝14.2,3.5,1h),4.23(d,j＝3.6,1h),3.56(d,j＝3.8,1h),2.93(dd,j＝11.8,3.3,1h),2.60
–
2.57(m,1h),2.56
–
2.50(m,1h),2.26(dd,j＝13.8,11.8,1h),2.04
–
1.98(m,2h),1.92(dd,j＝14.0,3.3,1h),1.88
–
1.79(m,1h),1.75
–
1.69(m,1h),1.26(s,3h),1.22(s,3h)；hresims(m/z):[m k]

calcd for c
29h28
o8k

543.1416,found 543.1426.
[0123]
data for 49:1h nmr(600mhz,acetone-d6)δ＝7.69(d,j＝8.1,1h),7.48(d,j＝8.3,1h),7.31
–
7.27(m,1h),7.13
–
7.08(m,1h),7.06(d,j＝2.2,1h),6.17(s,1h),5.92(s,1h),5.67(s,1h),5.01(d,j＝10.8,1h),4.97(d,j＝10.8,1h),4.65(dd,j＝14.2,3.6,1h),4.14(dd,j＝3.6,1.5,1h),3.55
–
3.51(m,1h),2.92(dd,j＝11.7,3.3,1h),2.56(dd,j＝9.2,2.6,1h),2.54
–
2.48(m,1h),2.26(dd,j＝13.9,11.8,1h),2.03
–
1.97(m,2h),1.92(dd,j＝13.9,3.3,1h),1.87
–
1.80(m,1h),1.73
–
1.68(m,1h),1.26(s,3h),1.22(s,3h)；hresims(m/z):[m na]

calcd for c
29h29
no7na

526.1836,found 526.1839.
[0124][0125]
data for 50:1h nmr(600mhz,acetone-d6)δ＝8.03(d,j＝8.1,1.3,2h),8.00(s,1h),7.57
–
7.52(m,1h),7.51
–
7.46(m,1h),6.19(s,1h),5.92(s,1h),5.69(s,1h),5.02(d,j＝10.8,1h),4.94(d,j＝10.8,1h),4.66(dd,j＝14.2,3.4,1h),4.21(d,j＝3.4,1h),3.56(s,1h),2.95
–
2.92(m,1h),2.58(dd,j＝9.2,2.7,1h),2.56
–
2.49(m,1h),2.26(dd,j＝13.9,11.8,1h),2.04
–
1.98(m,2h),1.92(dd,j＝13.9,3.3,1h),1.87
–
1.79(m,1h),1.75
–
1.69(m,1h),1.26(s,3h),1.22(s,3h)；hresims(m/z):[m na]

calcd for c
29h28
o7sna

543.1448,found 543.1449.
[0126]
data for 51:1h nmr(800mhz,acetone-d6)δ＝8.56(d,j＝6.9,1h),8.22(s,1h),7.56(d,j＝9.2,1h),7.38
–
7.31(m,1h),6.99(t,j＝6.7,1h),6.16(s,1h),5.90(s,1h),5.66(s,1h),5.02(d,j＝10.8,1h),4.98(d,j＝10.8,1h),4.67(dd,j＝14.2,3.5,1h),4.15(d,j＝3.6,1h),3.51(s,1h),2.93
–
2.91(m,1h),2.56(dd,j＝9.1,2.5,1h),2.54
–
2.50(m,1h),2.25(dd,j＝13.9,11.8,1h),1.99
–
1.96(m,2h),1.92(dd,j＝13.9,3.4,1h),1.86
–
1.80(m,1h),1.73
–
1.69(m,1h),1.26(s,3h),1.22(s,3h)；hresims(m/z):[m na]

calcd for c
28h28
n2o7na

527.1792,found 527.1789.
[0127][0128]
data for 52:1h nmr(600mhz,acetone-d6)δ＝9.08(dd,j＝7.0,1.8,1h),8.80(dd,j＝4.1,1.8,1h),8.31(s,1h),7.30(dd,j＝7.0,4.1,1h),6.16(s,1h),5.88(s,1h),5.65(s,1h),5.04(d,j＝10.8,1h),4.99(d,j＝10.8,1h),4.70(dd,j＝14.3,3.6,1h),4.10(d,j＝3.4,1h),3.52(s,1h),2.93
–
2.92(m,1h),2.55(dd,j＝9.3,2.5,1h),2.53
–
2.48(m,1h),2.26(dd,j＝13.8,11.8,1h),2.03
–
1.96(m,2h),1.92(dd,j＝13.8,3.4,1h),1.87
–
1.79(m,1h),1.74
–
1.68(m,1h),1.27(s,3h),1.22(s,3h)；hresims(m/z):[m na]

calcd for c
27h27
n3o7na

528.1741,found 528.1738.
[0129]
data for 53:1h nmr(600mhz,acetone-d6)δ＝8.56(s,1h),6.15(s,1h),6.01(d,j＝1.4,1h),5.67(s,1h),5.01(d,j＝10.8,1h),4.95(d,j＝10.8,1h),4.66(dd,j＝14.2,3.9,1h),4.09(d,j＝3.9,1h),3.98(s,3h),3.53
–
3.50(m,1h),2.95
–
2.92(m,1h),2.57(dd,j＝9.2,3.2,1h),2.56
–
2.51(m,1h),2.26(dd,j＝13.8,11.8,1h),2.03
–
1.98(m,2h),1.93(dd,j＝13.9,3.4,1h),1.88
–
1.80(m,1h),1.76
–
1.70(m,1h),1.28(s,3h),1.22(s,3h)；hresims(m/z):[m na]

calcd for c
26h27
n5o8na

560.1752,found 560.1744.
[0130][0131]
data for 54:1h nmr(600mhz,acetone-d6)δ＝8.48
–
8.45(m,1h),8.05(d,j＝8.2,1h),7.99(dd,j＝8.5,4.3,2h),7.85(dd,j＝8.6,1.7,1h),7.68(ddd,j＝8.2,6.8,1.3,1h),7.62(ddd,j＝8.1,6.8,1.3,1h),6.20(s,1h),5.96(s,1h),5.69(t,j＝1.0,1h),5.05(d,j＝10.8,1h),5.00(d,j＝10.8,1h),4.70(dd,j＝14.3,3.6,1h),4.20(d,j＝3.5,1h),3.58(d,j＝3.9,1h),2.94(dd,j＝11.7,3.3,1h),2.60
–
2.58(m,1h),2.57
–
2.51(m,1h),2.27(dd,j＝13.8,11.8,1h),2.04
–
1.99(m,2h),1.93(dd,j＝13.9,3.4,1h),1.89
–
1.81(m,1h),1.76
–
1.70(m,1h),1.27(s,3h),1.22(s,3h)；hresims(m/z):[m na]

calcd for c
31h30
o7na

537.1884,found 537.1893.
[0132]
data for 55:1h nmr(600mhz,acetone-d6)δ＝7.34
–
7.32(m,1h),7.31
–
7.25(m,2h),6.99
–
6.95(m,1h),6.83(d,j＝8.1,1h),6.14(s,1h),5.79(s,1h),5.64(s,1h),4.98(d,j＝10.8,1h),4.91(d,j＝10.8,1h),4.81(t,j＝1.7,2h),4.60(dd,j＝14.2,3.3,1h),4.21(d,j＝3.4,1h),3.47(d,j＝3.9,1h),2.90(dd,j＝11.7,3.3,1h),2.54(dd,j＝9.1,2.6,1h),2.51
–
2.45(m,1h),2.25(dd,j＝13.9,11.7,1h),2.02
–
1.97(m,2h),1.91(dd,j＝13.9,3.4,1h),1.84
–
1.78(m,1h),1.71
–
1.66(m,1h),1.25(s,3h),1.22(s,3h)；hresims(m/z):[m h]

calcd for c
30h31o8
519.2013,found 519.2017.
[0133][0134]
data for 56:1h nmr(600mhz,acetone-d6)δ＝8.53
–
8.48(m,1h),8.16
–
8.11(m,1h),8.08
–
8.04(m,1h),7.95
–
7.91(m,1h),7.90
–
7.84(m,1h),7.78
–
7.72(m,1h),6.19(s,1h),6.03(s,1h),5.70(s,1h),5.08
–
4.97(m,2h),4.73
–
4.66(m,1h),4.20(s,1h),3.61(s,1h),2.97
–
2.92(m,1h),2.60
–
2.54(m,2h),2.31
–
2.23(m,1h),2.10
–
2.08(m,2h),1.96
–
1.91(m,1h),1.90
–
1.83(m,1h),1.79
–
1.72(m,1h),1.28(s,3h),1.23(s,3h)；hresims(m/z):[m na]

calcd for c
30h29
no7na

538.1836,found 538.1842.
[0135]
data for 57:1h nmr(600mhz,acetone-d6)δ＝9.25(s,1h),8.21
–
8.17(m,2h),8.05
–
8.01(m,1h),8.00
–
7.96(m,1h),6.21(s,1h),6.09(s,1h),5.72(s,1h),5.03(d,1h),4.99(d,j＝10.8,1h),4.68(dd,j＝14.2,3.4,1h),4.29(d,j＝3.5,1h),3.65(d,j＝3.9,1h),2.95(dd,j＝11.8,3.3,1h),2.63(dd,j＝9.1,3.1,1h),2.60
–
2.55(m,1h),2.28(dd,j＝13.8,11.8,1h),2.09
–
2.06(m,2h),1.95
–
1.92(m,1h),1.88
–
1.83(m,1h),1.79
–
1.74(m,1h),1.28(s,3h),1.23(s,3h)；hresims(m/z):[m na]

calcd for c
29h28
n2o7na

539.1789,found 539.1794.
[0136][0137]
data for 58:1h nmr(600mhz,acetone-d6)δ＝7.57(dt,j＝7.7,1.3,1h),7.49(t,j＝8.0,1h),7.46
–
7.42(m,2h),7.41(dd,j＝2.6,1.5,1h),7.25(ddd,j＝8.2,2.6,1.1,1h),7.23
–
7.20(m,1h),7.07
–
7.04(m,2h),6.13(s,1h),5.87(s,1h),5.63(s,1h),5.00(d,j＝10.8,1h),4.93(d,j＝10.8,1h),4.63(dd,j＝14.3,3.6,1h),4.16(d,j＝3.6,1h),3.51(s,1h),2.94
–
2.91(m,1h),2.56(dd,j＝9.2,2.6,1h),2.52
–
2.46(m,1h),2.25(dd,j＝13.8,11.7,1h),2.03
–
1.97(m,2h),1.91(dd,j＝13.9,3.3,1h),1.85
–
1.79(m,1h),1.72
–
1.67(m,1h),1.25(s,3h),1.21(s,3h)；hresims(m/z):[m na]

calcd for c
33h32
o8na

579.1989,found 579.1985.
[0138]
data for 59:1h nmr(600mhz,acetone-d6)δ＝8.11(d,j＝5.1,1h),7.94(d,j＝7.8,1h),7.82(d,j＝7.7,1h),7.67(d,j＝7.7,2h),7.59(t,j＝7.8,1h),7.51(t,j＝7.7,2h),7.42(t,j＝7.4,1h),6.16(s,1h),5.92(s,1h),5.67(s,1h),5.03(d,j＝10.6,1h),5.01
–
4.95(m,1h),4.73
–
4.63(m,1h),4.22(s,1h),3.60
–
3.54(m,1h),2.96
–
2.92(m,1h),2.61
–
2.57(m,1h),2.56
–
2.50(m,1h),2.29
–
2.22(m,1h),2.03
–
1.98(m,2h),1.95
–
1.91(m,1h),1.88
–
1.80(m,1h),1.75
–
1.69(m,1h),1.26(s,3h),1.22(s,3h)；hresims(m/z):[m
–
h]
–
calcd for c
33h31
o7–
539.2075,found 539.2084.
[0139][0140]
data for 60:1h nmr(600mhz,acetone-d6)δ＝8.69(d,j＝4.8,2h),7.99
–
7.95(m,2h),7.92
–
7.88(m,2h),7.74
–
7.69(m,2h),6.18(s,1h),5.94(s,1h),5.68(s,1h),5.03(d,j＝10.8,1h),4.97(d,j＝10.8,1h),4.67(dd,j＝14.2,3.6,1h),4.18(d,j＝3.6,1h),3.57(d,j＝3.9,1h),2.93(dd,j＝11.7,3.3,1h),2.59(dd,j＝9.5,2.9,1h),2.56
–
2.50(m,1h),2.27(dd,j＝13.9,11.8,1h),2.04
–
2.00(m,2h),1.93(dd,j＝13.9,3.4,1h),1.89
–
1.81(m,1h),1.76
–
1.69(m,1h),1.27(s,3h),1.23(s,3h)；hresims(m/z):[m h]

calcd for c
32h32
no
7
542.2173,found 542.2180.
[0141]
data for 61:1h nmr(600mhz,acetone-d6)δ＝7.79
–
7.74(m,2h),7.27(t,j＝7.5,2h),7.25
–
7.21(m,2h),7.19
–
7.14(m,1h),7.01
–
6.96(m,2h),6.15(s,1h),5.84(s,1h),5.64(s,1h),5.01(d,j＝10.8,1h),4.96(d,j＝10.8,1h),4.65(dd,j＝14.3,3.6,1h),4.15
–
4.06(m,3h),3.50(s,1h),2.91(dd,j＝11.8,3.4,1h),2.70(t,j＝7.1,2h),2.56
–
2.53(m,1h),2.53
–
2.46(m,1h),2.25(dd,j＝13.9,11.7,1h),2.03
–
1.96(m,2h),1.94
–
1.89(m,1h),1.86
–
1.77(m,5h),1.72
–
1.67(m,1h),1.26(s,3h),1.22(s,3h)；hresims(m/z):[m k]

calcd for c
37h40
o8k

651.2355,found 651.2352.
[0142][0143]
data for 62:1h nmr(600mhz,acetone-d6)δ＝9.19(s,1h),9.10
–
9.07(m,2h),8.18(d,j＝2.1,1h),8.08
–
8.05(m,1h),7.96
–
7.91(m,1h),7.69(t,j＝7.8,1h),6.17(s,1h),5.93(s,1h),5.67(s,1h),5.03(d,j＝10.7,1h),4.97(d,j＝10.8,1h),4.68(dd,j＝14.3,3.6,1h),4.19(dd,j＝3.7,1.4,1h),3.59(s,1h),2.93(dd,j＝11.7,3.3,1h),2.59(d,j＝9.3,1h),2.57
–
2.50(m,1h),2.27(dd,j＝13.9,11.8,1h),2.04
–
2.00(m,2h),1.93(dd,j＝13.9,3.3,1h),1.89
–
1.81(m,1h),1.76
–
1.69(m,1h),1.26(s,3h),1.23(s,3h)；hresims(m/z):[m na]

calcd for c
31h30
n2o7na

565.1945,found 565.1936.
[0144]
data for 63:1h nmr(600mhz,acetone-d6)δ＝9.13(d,j＝2.6,1h),8.80(s,1h),8.61(dd,j＝4.7,1.5,1h),8.26(ddd,j＝8.4,2.7,1.4,1h),7.98(s,1h),7.56(dd,j＝8.3,4.7,1h),6.15(s,1h),5.86(s,1h),5.65(s,1h),5.00(d,j＝10.8,1h),4.95(d,j＝10.8,1h),4.65(dd,j＝14.3,3.2,1h),4.14(d,j＝3.5,1h),3.50(s,1h),2.91(dd,j＝11.7,3.4,1h),2.56(dd,j＝9.2,2.5,1h),2.54
–
2.47(m,1h),2.26(dd,j＝13.9,11.8,1h),2.04
–
1.97(m,2h),1.92(dd,j＝13.9,3.4,1h),1.87
–
1.78(m,1h),1.73
–
1.67(m,1h),1.26(s,3h),1.22(s,3h)；hresims(m/z):[m h]

calcd for c
29h30
n3o
7
532.2078,found 532.2086.
[0145]
实施例3
[0146]
sars-cov-2 3cl
pro
抑制剂筛选流程：
[0147]
1.样品的准备。取适量待测定的抑制剂样品，用实验缓冲液(assay buffer)或dmso等适当的溶剂配制成适宜浓度的溶液，如果有必要可配制成适当的浓度梯度待用。
[0148]
2.样品测定。
[0149]
a.根据样品数量(含相关对照)，参考下表配制适量的assay reagent。注：由于2019-ncov m
pro
/3cl
pro
的甘油含量较高，微量吸取时要注意避免吸头吸附损失并吹打完全，加入2019-ncov m
pro
/3cl
pro
后需要注意充分混匀。
[0150] 1个样品5个样品10个样品20个样品assay buffer92μl460μl920μl1.84ml2019-ncov 3cl
pro
1μl5μl10μl20μl
[0151]
b.参考下表，使用96孔黑板设置各组别，并按照下表依次加入检测试剂和样品。加入待测样品后，混匀。为获得更加可靠的检测结果，建议每个样品设置2个重复孔的检测。
[0152][0153]
注：样品溶剂是指配制和稀释待测抑制剂所用的溶剂。
[0154]
c.各孔快速加入substrate 2μl，混匀。注：加入substrate后反应会立即开始，如果孔数较多的情况下，建议在低温或使用排枪操作以减小各孔间加入substrate的时间差而导致的误差，混匀操作可在培养板振荡器上进行。
[0155]
d.37℃避光孵育10分钟后信号即趋于稳定，可在5-20分钟内使用多功能酶标进行荧光测定。最大激发波长为325nm，最大发射波长为393nm。当荧光读数偏低时，也可适当延长孵育时间至20-30分钟。
[0156]
3.计算:a.计算每个样品孔和空白对照孔的平均荧光值，可分别记录为rfu
空白对照
、rfu
100％酶活性对照
、rfu
阳性对照
和rfu
样品
。b.计算每个样品的抑制百分率。计算公式如下：抑制率(％)＝(rfu
100％酶活性对照-rfu
样品
)/(rfu
100％酶活性对照-rfu
空白对照
)
×
100％；c.对于检测发现有效的抑制剂，通过检测该抑制剂的剂量效应就可以计算出该抑制剂的ic
50
。
[0157]
4.结果:如图1所示，所有化合物的初筛浓度为5μm，筛选结果显示不同衍生物的抑制活力从10％到99％不等。共有16个化合物的抑制率超过了80％，这些对sars-cov-2 3cl
pro
具有高抑制率的化合物具有含溴或含有2-卤代异烟碱的特征。对以上16个化合物测定了ic
50
值。结果如表1所示，化合物12、24、30和47具有良好的抑制sars-cov-2 3cl
pro
的能力，以上4个化合物的ic
50
值小于1.0μm。
[0158]
表1化合物对sars-cov-2 3cl
pro
的半数有效抑制浓度(ic
50
)
[0159]
no.ic
50
[μm]no.ic
50
[μm]71.794
±
0.099271.662
±
0.20081.253
±
0.158300.523
±
0.093
120.463
±
0.013321.115
±
0.001161.128
±
0.322342.427
±
0.065171.932
±
0.146361.731
±
0.197181.535
±
0.310402.304
±
0.457240.477
±
0.012470.824
±
0.121251.498
±
0.144551.093
±
0.021
[0160]
实施例4
[0161]
抗sars-cov-2实验流程：
[0162]
1.测定药物和化合物
[0163]
待测样品溶解于dmso中。阳性对照为瑞德西韦，购自aa blocks公司，溶解于dmso中。待测样品贮存液浓度均为10mm，储存条件为：4℃保存。
[0164]
2.试剂和溶液
[0165]
(1)试剂
[0166]
cck-8(cell counting kit-8)为碧云天公司产品。dmem高糖培养基、胎牛血清、100
×
penicillin-streptomycin(10,000units/ml青霉素,10000μg/ml链霉素)为gibco公司产品。mtt(3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide)、dmf(n,n
’‑
dimethyl formamine)购自sigma公司。easypure viral rna kit为全式金生物公司产品。probe one-step qrt-pcr kit为toyobo公司产品。引物和探针购自宝日医生物技术有限公司，n-f：5’ggggaacttctcctgctagaat 3’、n-r：5’cagacattttgctctcaagctg 3’、probe:5’ttgctgctgcttgacagatt 3’。
[0167]
(2)培养基
[0168]
dmem高糖培养基，含10％胎牛血清、100units/ml青霉素、100μg/ml链霉素。
[0169]
3.细胞和病毒
[0170]
非洲绿猴肾上皮细胞系vero e6、人肺泡上皮细胞系hpa epic购自atcc，sars-cov-2病毒107株，来自广东cdc。所有细胞和病毒均以含10％胎牛血清的dmem完全培养基进行培养。按常规方法制备sars-cov-2，滴定并计算出病毒的tcid
50
。病毒贮存液分装后，置
–
80℃保存。细胞和病毒按常规方法冻存和复苏。
[0171]
4.sars-cov-2感染性滴定
[0172]
p3实验室内以4
×
104个/孔接种vero e6细胞到96孔板，37℃，5％co2培养过夜，待单层细胞长至70％左右时，转移至p3实验室待用。将sars-cov-2贮存液用含3％胎牛血清的培养基进行10倍梯度稀释，共10个梯度。吸去细胞培养板中的培养基，每孔加入100μl病毒稀释液，每个梯度10个重复孔，同时设置对照孔10孔。37℃、5％co2培养箱培养，次日开始显微镜下观察细胞病变效应，记录每个梯度阳性孔数，待病变孔数不再增加时(3～7天)，统计细胞病变孔数，按reed&muench方法计算病毒的tcid
50
(50％tissue culture infection dose)。
[0173]
5.对hpa epic细胞的毒性实验
[0174]8×
105/ml将hpa epic细胞以100μl体积接种至96孔板中，37℃，5％co2培养过夜。待单层细胞长至70％左右时，加入不同浓度的待测药物，100μl/孔，设3个重复孔。同时设置不含药物的对照孔,37℃,5％co2培养3天。采用mtt比色法检测细胞毒性。elx800酶标仪测
定od值，测定波长为570nm，参考波长为630nm。计算得到cc
50
值(50％cytotoxic concentration)，即对50％的正常人肺泡上皮细胞系hpa epic产生毒性时的药物浓度。
[0175]
6.对sars-cov-2在hpa epic细胞中复制的抑制实验
[0176]
将8
×
105/ml的hpa epic细胞200μl/孔接种到48孔细胞培养板上，37℃，5％co2培养过夜。待单层细胞长至70％左右时，转移至p3实验室待用。
[0177]
吸去培养液后，以100μl/孔加入梯度稀释的药物后，加入moi＝1的sars-cov-2，100μl/孔。同时设置不含药物的正常细胞对照孔和病毒感染孔。瑞德西韦为阳性药物对照。37℃，5％co2孵育1h后，吸去上清pbs清洗，随后加入含同等浓度药物的培养基。37℃，5％co2培养48h后使用easypure viral rna kit试剂盒对上清中病毒rna进行提取，probe one-step qrt-pcr kit试剂进行rt-qpcr定量，graphpad作图计算得到ec
50
，ec
50
(50％effective concentration)为抑制细胞病变效应形成50％时的药物浓度。
[0178]
7.对vero e6细胞的毒性实验
[0179]4×
105/ml将vero e6细胞以100μl体积接种至96孔板中，37℃，5％co2培养过夜。待单层细胞长至70％左右时，加入不同浓度的待测药物，100μl/孔，设3个重复孔。同时设置不含药物的对照孔,37℃,5％co2培养3天。采用mtt比色法检测细胞毒性。elx800酶标仪测定od值，测定波长为570nm，参考波长为630nm。计算得到cc
50
值(50％cytotoxic concentration)，即对50％的正常非洲绿猴肾上皮细胞系vero e6产生毒性时的药物浓度。
[0180]
8.对sars-cov-2在vero e6细胞中复制的抑制实验
[0181]
将4
×
105/ml的vero e6细胞200μl/孔接种到48孔细胞培养板上，37℃，5％co2培养过夜。待单层细胞长至70％左右时，转移至p3实验室待用。
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吸去培养液后，以100μl/孔加入梯度稀释的药物后，加入moi＝0.1的sars-cov-2，100μl/孔。同时设置不含药物的正常细胞对照孔和病毒感染孔。瑞德西韦为阳性药物对照。37℃，5％co2孵育1h后，吸去上清pbs清洗，随后加入含同等浓度药物的培养基。37℃，5％co2培养48h后使用easypure viral rna kit试剂盒对上清中病毒rna进行提取，probe one-step qrt-pcr kit试剂进行rt-qpcr定量，graphpad作图计算得到ec
50
，ec
50
(50％effective concentration)为抑制细胞病变效应形成50％时的药物浓度。
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9.结果
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如表2所示，从ec
50
值上来说，化合物32、47都强于阳性对照药瑞德西韦，其中化合物47，无论从ec
50
值上或治疗指数值甚至都强于瑞德西韦，ec
50
值达到了19
±
1nm，ti值大于1000。以上结果显示该类结构修饰物可作为抗新冠病毒分子进行下一步的开发。
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表2对sars-cov-2病毒感染抑制实验(hpa细胞，vero e6细胞)
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[0187][0188]
制剂实施例
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在以下制剂实施例中，选择常规试剂，并按照现有常规方法进行制剂制备，本应用例仅体现本发明所述毛萼内酯素b衍生物制备成不同的制剂，对具体试剂和操作不作具体限定。
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1.将毛萼内酯素b衍生物任其一或任其组合用dmso溶解后，按常规方法加注射用水，精滤，灌封灭菌制成注射液，所述注射液的浓度为0.5～5mg/ml。
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2.将毛萼内酯素b衍生物任其一或任其组合用dmso溶解后，将其溶于无菌注射用水中，搅拌使其溶解，用无菌抽滤漏斗过滤，再无菌精滤，分装于安瓿中，低温冷冻干燥后无菌熔封，得粉针剂。
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3.将毛萼内酯素b衍生物任其一或任其组合按其与赋形剂质量比为9:1的比例加入赋形剂，制成粉剂。
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4.将毛萼内酯素b衍生物任其一或任其组合按其与赋形剂质量比为5:1的比例加入赋形剂，制粒压片。
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5.将毛萼内酯素b衍生物任其一或任其组合按常规口服液制备方法制成口服液。
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6.将毛萼内酯素b衍生物任其一或任其组合按其与赋形剂质量比为5:1的比例加入赋形剂，制成胶囊。
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7.将毛萼内酯素b衍生物任其一或任其组合按其与赋形剂质量比为5:1的比例加入赋形剂，制成颗粒剂。
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以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。