CN114213395B - 一种嘧啶酮酰基哌嗪类化合物及其制备方法与应用 - Google Patents
一种嘧啶酮酰基哌嗪类化合物及其制备方法与应用 Download PDFInfo
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Abstract
本发明提供了一种嘧啶酮酰基哌嗪类化合物及其制备方法和应用。所述化合物具有式I所示的结构。本发明还涉及含有式I结构化合物的制备方法,药物组合物以及提供上述化合物在制备抗SARS‑CoV‑2Mpro药物中的应用。
Description
技术领域
本发明属于有机化合物合成与医药应用技术领域,具体涉及一种抑制蛋白酶如新型冠状病毒主蛋白酶的嘧啶酮酰基哌嗪类化合物、包含所述化合物的组合物、制备所述化合物的方法和使用所述化合物治疗由冠状病毒引起的疾病或疾病症状的方法。
背景技术
急性呼吸道综合征冠状病毒2型(Severe Acute Respiratory SyndromeCoronavirus 2,SARS-CoV-2)严重威胁人们的生命健康。SARS-CoV-2为单股正链RNA病毒,属于β冠状病毒属,其复制周期包括吸附、融合、转录、翻译、组装和释放等环节。SARS-CoV-2与以往的冠状病毒相比,其传播性更强、潜伏期更长、无症状感染者更多,这极大地增加了疫情传播扩散的风险,阻碍了对疫情的有效控制。新冠肺炎或将成为全球或局部地区长期流行的传染性疾病。然而现有药物对新型冠状病毒肺炎(Corona Virus Disease 2019,COVID-19)的治疗缺乏针对性,且疗效差。尽管已成功研发出多种新冠疫苗,但接种疫苗后仍存在被感染的风险,加之,新的变异病毒的出现导致了病毒传播力的增加以及一些地方疫情的持续恶化,这给疫情防控带来更大的挑战。因此研发具有临床应用价值的抗SARS-CoV-2药物刻不容缓。
蛋白酶在病毒复制周期的转录和翻译等关键阶段发挥重要作用。SARS-CoV-2主蛋白酶(Main protease,Mpro)是由同源二聚体组成,以半胱氨酸为活性中心的蛋白酶,其活性催化位点由His41和Cys145组成(附图1)。主蛋白酶作为冠状病毒种间相似性极高的共有蛋白酶,催化活性位点高度保守。同时,人体内没有Mpro的同源蛋白酶,为病毒特有,因此该靶点抑制剂选择性好、毒副作用小,这使得Mpro成为一个理想的抗SARS-CoV-2药物靶标。
新冠疫情爆发后,SARS-CoV-2主蛋白酶成为药物设计的重要靶标,现有主蛋白酶抑制剂多为拟肽类共价抑制剂,但共价抑制剂存在选择性较差的问题,不仅抑制病毒主蛋白酶活性,也能抑制人体半胱氨酸组织蛋白酶L和B,具有安全性隐患。非共价抑制剂往往具有较高的选择性,是抗新冠药物研发的重要方向,但是它们的活性较弱,因此发现高活性的SARS-CoV-2主蛋白酶非共价抑制剂是当前的研究热点。
嘧啶酮酰基哌嗪类化合物7(Mcule-5948770040)是经高通量筛选发现的新骨架类型的SARS-CoV-2主蛋白酶抑制剂(IC50=4.2μM),共晶结构显示化合物7与主蛋白酶通过非共价作用结合来抑制Mpro活性(PDB ID:7LTJ)。然而活性较低严重制约该类化合物进一步开发。
发明内容
针对现有技术的不足,本发明提供了一种抑制主蛋白酶的嘧啶酮酰基哌嗪类化合物、包含所述化合物的组合物及其制备方法,本发明还提供上述化合物作为SARS-CoV-2Mpro抑制剂的活性筛选结果及其应用。
本发明的技术方案如下:
1.嘧啶酮酰基哌嗪类化合物
一种嘧啶酮酰基哌嗪类化合物,或其药学上可接受的盐、酯或前药,具有通式I所示的结构:
其中,
R1为:各类取代的脂肪胺、芳香胺、六元杂环甲胺或者五元杂环甲胺;所述的代基选自甲基、卤代甲基、卤素或叔丁氧羰基。
根据本发明优选的,R1为取代苯胺、取代苄胺、取代苯乙胺、吲哚甲胺、吲哚乙胺、联苯甲胺、取代环状甲胺、取代环状乙胺、螺环甲胺、取代哌啶环、取代呋喃甲胺、取代噻吩甲胺、吡咯甲胺、吡啶甲胺、吡唑甲胺、咪唑甲胺、恶唑甲胺、异恶唑甲胺、噻唑甲胺;所述的取代基选自甲基、卤代甲基、卤素或叔丁氧羰基;式I所示化合物构型为消旋体、R或S构型。
根据本发明进一步优选的,R1为下列取代基I(a)、I(b)或I(c)中任意一种:
n为选自0、1或2的整数;
m选自0、1或2的整数,R在每次出现时各自独立地为C1-C2的烷基、苯基、卤素、羟基、氰基、硝基、卤代甲基、卤代苯基;
m和n为选自0、1或2的整数;
R2、R3为H、C1-C2的烷基、卤素、卤代甲基、或环烷基。
n为选自0、1和2的整数,X和Y各自为-S-、-O-、=NH-或=CH-,且至多一个为=CH-;Ar为H、苯环或5-6元芳杂环;R4为H、甲基或卤素。
根据本发明更进一步优选的,嘧啶酮酰基哌嗪类化合物是下列化合物之一:
本发明中所述的“药学上可接受的盐”是指在可靠的医药评价范围内,化合物的盐类适于与人或较低等动物的组织相接触而无不适当的毒性、刺激及过敏反应等,具有相当合理的收益与风险比例,通常是水或油可溶的或可分散的,并可有效地用于其预期的用途。包括药学上可接受的酸加成盐和药学上可接受的碱加成盐,在这里是可做预期的用途并与式I、II化合物的化学性质相容的。适宜的盐的列表参见S.M.Birge等,J.Pharm.Sci.,1977,66,1-19页。
本发明中所述的“前药”是指药学上可接受的衍生物,以便这些衍生物所得的生物转换产物是如式I化合物所定义的活性药物。
2.嘧啶酮酰基哌嗪类化合物的制备方法
嘧啶酮酰基哌嗪类化合物的制备方法,步骤如下:以3,4-二氯苯硼酸1和Boc-哌嗪甲酸酯2为起始原料,二氯甲烷作为反应溶剂,在醋酸铜催化下通过Chan-Lam偶联反应得中间体3;然后将中间体3溶解在适量的二氯甲烷中,经三氟乙酸脱保护得中间体4;随后中间体4在缩合剂HATU的作用下与乳清酸缩合得到关键中间体5;中间体5再经氢氧化锂水解甲酯得到中间体6,然后中间6再在HATU的作用下与各类胺在二氯甲烷中进行酰胺缩合得到各个目标产物。
合成路线如下:
试剂及条件:(i)醋酸酮,氧气,吡啶,二氯甲烷,室温;(ii)三氟乙酸,二氯甲烷,室温;(iii)乳清酸,HATU,N,N,-二异丙基乙胺,二氯甲烷,室温;(iv)氢氧化锂,甲醇,四氢呋喃,水,室温;(v)各种胺类化合物,HATU,N,N,-二异丙基乙胺,二氯甲烷,室温。
其中,R1如上述通式I中所述;本发明所述的室温为20-30℃。
根据本发明优选的,含有嘧啶酮酰基哌嗪类化合物的制备方法,具体步骤如下:
(1)将1-叔丁基-3-甲基哌嗪-1,3二羧酸(1)与3,4-二氯苯硼酸(2)加入二氯甲烷,搅拌中溶解近澄清;在溶液中一次性加入无水醋酸铜与2当量吡啶;混悬均匀后在氧气氛围下反应24h,TLC检测反应EA/PE=1:2,v/v;反应完全后,在体系中加水淬灭反应;分液,以蒸馏水反复洗至有机相无蓝色;有机相经饱和氯化钠溶液洗涤,分出有机相,依次经无水硫酸钠干燥、过滤、减压浓缩,所得粗品经硅胶柱层析分离纯化得中间体3,为无色油状液体;
(2)在冰水浴下将中间体3溶于二氯甲烷中,逐滴加入三氟乙酸与二氯甲烷的混合溶液;滴加结束后,转至室温反应;约6h后反应液减压浓缩,得到***油状物;加入乙酸乙酯后,析出大量白色固体;过滤,固体用乙酸乙酯/石油醚洗涤,干燥得目标产物中间体4的三氟乙酸盐;
(3)冰浴下,将乳清酸与2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(HATU)加入到二氯甲烷中,活化20min后加入N,N,-二异丙基乙胺和中间体4的三氟乙酸盐,转至室温反应20小时,溶液变为深黄色,析出固体,TLC监测;过滤反应液,以DCM洗涤所得固体至灰白色,干燥,得中间体5;
(4)中间体5溶于甲醇与四氢呋喃混合液中,冰浴下滴加氢氧化锂的水溶液,室温反应,搅拌4h后减压浓缩部分有机溶剂;在冰浴下向剩余的水溶液中滴加1M稀盐酸溶液,将pH调至3以下,有大量乳白色固体析出。过滤、洗涤、干燥得中间体6,为微黄色粉末状固体;
(5)将中间体6与2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(HATU)加入到二氯甲烷中,活化20min后加入N,N,-二异丙基乙胺和各类取代胺,室温反应12h,TLC监测;过滤反应液,以二氯甲烷洗涤固体,得GA系列终产物。
3.嘧啶酮酰基哌嗪类化合物的靶标活性及应用
本发明公开了含嘧啶酮酰基哌嗪类化合物的活性筛选结果及其作为主蛋白酶抑制剂的首次应用。通过实验证明本发明的嘧啶酮酰基哌嗪类化合物可作为主蛋白酶抑制剂用于制备抗冠状病毒药物。本发明还提供上述化合物在制备抗冠状病毒药物中的应用。
目标化合物的抗SARS-CoV-2主蛋白酶活性实验
对按照上述方法合成的一类嘧啶酮酰基哌嗪类化合物进行了SARS-CoV-2主蛋白酶靶标活性测试,它们的抗主蛋白酶活性数据列于附图2中,以文献报道的主蛋白酶抑制剂化合物7及ML188为阳性对照。
本发明新合成的嘧啶酮酰基哌嗪类化合物绝大部分呈现出显著的主蛋白酶抑制活性。例如,化合物GA-13、GA-13S、GA-17S、GA-19、GA-25、GA-27、GA-33其中GA-17S的活性尤为突出(IC50=0.38±0.07μM),其抑制主蛋白酶的活性较先导化合物7提高了将近20倍;另外由GA-13S和GA-28可知,该系列化合物S构型活性优于R构型。因此,嘧啶酮酰基哌嗪类化合物具有进一步研究和开发的价值,可作为抗SARS-CoV-2的先导化合物加以利用。
本发明的一类嘧啶酮酰基哌嗪类化合物可作为SARS-CoV-2主蛋白酶抑制剂应用,具体地说,作为SARS-CoV-2抑制剂用于制备抗新冠病毒药物。
一种抗新冠病毒药物组合物,包括本发明的嘧啶酮酰基哌嗪类化合物和一种或多种药学上可接受载体或赋形剂。
本发明提供了结构全新的嘧啶酮酰基哌嗪类化合物及其制备方法,本发明还提供了化合物抗SARS-CoV-2主蛋白酶活性筛选结果及其在抗病毒领域中的首次应用。本发明的嘧啶酮酰基哌嗪类化合物可作为SARS-CoV-2主蛋白酶抑制剂应用并具有很高的应用价值。具体地说,本发明通过结构优化发现了活性更高、结构新颖的主蛋白酶抑制剂,可作为SARS-CoV-2主蛋白酶抑制剂用于制备抗新冠病毒药物。
附图说明
图1是Mpro三维结构及其活性中心图;
图2是化合物在10μM浓度下对主蛋白酶的抑制率;
图3是以SARS-CoV-2Mpro为靶点的药物筛选原理图。
具体实施方式
通过下述实施例有助于理解本发明,但是不能限制本发明的内容,所述百分比数均为质量百分比。
实施例1:GA-1至GA-34的制备
将1-叔丁基-3-甲基哌嗪-1,3二羧酸(1,5.0g,20.5mmol,1.0eq.)与3,4-二氯苯硼酸(2,7.8g,41mmol,2.0eq.)加入100mL二氯甲烷,搅拌中溶解近澄清。向溶液中一次性加入无水醋酸铜(3.71g,20.5mmol,1.0eq.)与吡啶(3.20g,41mmol,2.0eq.)。混悬均匀后在氧气氛下反应24h,TLC检测反应EA/PE=1:2,v/v。16h反应完全后,在体系中加水淬灭反应。分液,以蒸馏水反复洗至水相无蓝色。有机相经饱和氯化钠溶液洗涤,分出有机相,依次经无水硫酸钠干燥、过滤、减压浓缩,所得粗品经硅胶柱层析(EA/PE梯度洗脱)分离纯化得中间体3,为无色油状液体4.0g,收率51%。ESI-MS:m/z 389.2[M+H]+.C17H22Cl2N2O4(388.1).
(2)在冰水浴下将中间体3(4.0g,10.3mmol,1.0eq.)溶于100mL二氯甲烷中,逐滴加入5mL三氟乙酸与25mL二氯甲烷的混合溶液。滴加结束后,转至室温反应。约6h后TLC检测反应完后减压浓缩反应液,得到***油状物。加入50mL乙酸乙酯后,析出大量白色固体。过滤,固体用30mL乙酸乙酯洗涤2次,真空干燥。得目标产物中间体4的三氟乙酸盐3.9g,收率94.2%。ESI-MS:m/z 289.2[M+H]+.C12H14Cl2N2O2(288.1).
(3)冰水浴下,将乳清酸(1.67g,10.67mmol,1.1eq.)与2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(HATU,5.5g,14.55mmol,1.5eq.)加入到100mL二氯甲烷中,活化20min后加入二异丙基乙胺(DIPEA,3.75g,29.1mmol,3.0eq.)和中间体4的三氟乙酸盐(3.9g,9.7mmol,1.0eq.),转至室温反应20小时,溶液变为深黄色,有固体析出,TLC监测反应完成;抽滤反应液中固体,用二氯甲烷洗涤所得固体,真空干燥,得灰白色中间体5 3.2g,收率78.5%。ESI-MS:m/z 427.2[M+H]+.C17H16Cl2N4O5(426.1).
(4)中间体5(3.2g,7.5mmol,1.0eq.)溶于50mL甲醇与50mL四氢呋喃混合液中,冰浴下滴加氢氧化锂(1.8g,75mmol,10eq.)的水溶液,室温反应,搅拌4h后减压浓缩部分有机溶剂。在冰浴下向剩余的水溶液中滴加1M稀盐酸溶液,将pH调至<3,有大量乳白色固体析出。过滤、100mL水洗多次、真空干燥得中间体6约2.8g,收率90.6%,为微黄色粉末状固体,熔点275-277℃,1H NMR(400MHz,DMSO-d6)δ13.16(s,1H),11.27(d,J=29.1Hz,2H),7.42(d,J=9.0Hz,1H),7.14(d,J=2.9Hz,1H),6.91(d,J=11.9Hz,1H),5.52(s,1H),4.80(d,J=20.8Hz,1H),4.24(dd,J=62.0,12.5Hz,1H),3.95–3.46(m,3H),3.02(d,J=13.4Hz,1H).ESI-MS:m/z 411.1[M-H]-.C16H14Cl2N4O5(412.03).
(5)将关键中间体6(0.1g,0.24mmol,1.0eq.)与HATU(0.137g,0.36mmol,1.5eq.)加入到10mL二氯甲烷中,活化20min后加入二异丙基乙胺(DIPEA,0.093g,0.72mmol,3.0eq.)和环己基甲胺(0.03g,0.264mmol,1.1eq.),室温反应12h,TLC监测;过滤反应液,以二氯甲烷洗涤固体,得目标化合物N-(环己基甲基)-1-(3,4-二氯苯基)-4-(2,6-二氧代-1,2,3,6-四氢嘧啶-4-甲酰基)哌嗪-2-甲酰胺(GA-1)。或者二氯甲烷(80mL)溶解后水洗(20mL×3),饱和氯化钠溶液(20mL)洗涤,无水硫酸钠干燥,过滤后硅胶柱色谱分离得到目标化合物。
产物为浅黄色固体,收率55%,熔点211-213℃。
1H NMR(600MHz,DMSO-d6)δ11.20(s,2H),8.09(dt,J=28.9,5.9Hz,1H),7.41(d,J=9.0Hz,1H),7.00(d,J=18.2Hz,1H),6.82(d,J=9.1Hz,1H),5.42(d,J=16.2Hz,1H),4.35(d,J=39.6Hz,1H),4.06(dd,J=44.4,13.5Hz,1H),3.86-3.67(m,1H),3.66-3.42(m,3H),3.28-3.15(m,1H),3.01-2.71(m,2H),1.64-1.49(m,5H),1.36-0.98(m,6H),0.77(d,J=8.5Hz,2H).13CNMR(150MHz,DMSO-d6)δ169.88,164.16,161.64,151.48,149.86,147.17,131.98,130.91,120.02,115.86,114.88,99.40,58.07,47.52,45.29,43.18,41.55,37.77,30.78,26.41,25.86.ESI-MS:m/z 507.3[M-H]-.C23H27Cl2N5O4(508.40).
操作同上,所不同的是使用2-苯基乙胺。
产物为白色固体,收率:72%,熔点177-180℃。
1H NMR(600MHz,DMSO-d6)δ11.37–11.11(m,2H),8.20(d,J=16.7Hz,1H),7.40(d,J=9.1Hz,1H),7.27(t,J=7.4Hz,2H),7.22–7.11(m,3H),7.02(d,J=2.9Hz,1H),6.76(td,J=9.7,9.1,2.9Hz,1H),5.52–5.42(m,1H),4.37(d,J=36.6Hz,1H),4.10(d,J=13.4Hz,1H),3.74–3.55(m,2H),3.47(d,J=39.0Hz,2H),3.32(d,J=10.2Hz,1H),3.18(s,1H),2.66(qt,J=14.0,7.4Hz,2H).13C NMR(150MHz,DMSO-d6)δ169.7,164.2,161.7,151.5,149.9,147.2,139.8,132.0,130.9,129.1,128.7,126.5,119.9,115.8,114.7,99.4,58.0,47.5,43.1,41.4,40.8,35.3.ESI-MS:m/z 516.1[M+H]+.C24H23Cl2N5O4(515.11).
操作同上,所不同的是使用吲哚-3-甲胺。
产物为灰白色固体,收率:59%,熔点242-244℃。
1H NMR(600MHz,DMSO-d6)δ11.22(d,J=37.6Hz,2H),10.90(s,1H),8.37(d,J=32.0Hz,1H),7.46–7.30(m,3H),7.19(d,J=28.9Hz,1H),7.07(t,J=7.0Hz,1H),7.00(dd,J=11.9,3.0Hz,1H),6.92(t,J=7.4Hz,1H),6.84–6.73(m,1H),5.47(d,J=11.4Hz,1H),4.56(dd,J=14.6,6.4Hz,1H),4.50–4.29(m,2H),4.23–4.04(m,2H),3.71–3.58(m,2H),3.52–3.41(m,1H),3.27–3.11(m,1H).13C NMR(150MHz,DMSO-d6)δ173.00,170.03,164.22,152.62,149.92,146.95,137.76,136.86,132.04,130.88,126.84,124.23,122.08,121.65,119.04,116.69,115.26,114.87,111.89,100.06,47.75,43.24,41.39,34.89.ESI-MS:m/z563.3[M+Na]+.C25H22Cl2N6O4(540.11).
操作同上,所不同的是使用(4-氟苯基)甲胺。
产物为白色固体,收率:66%,熔点244-246℃。
1H NMR(600MHz,DMSO-d6)δ11.22(s,2H),8.69(d,J=23.7Hz,1H),7.42(d,J=9.1Hz,1H),7.18(dd,J=8.4,5.5Hz,2H),7.13–7.00(m,3H),6.83(d,J=9.2Hz,1H),5.42(d,J=14.2Hz,1H),4.51–4.31(m,2H),4.09(d,J=15.6Hz,1H),3.91–3.70(m,1H),3.66–3.45(m,3H),3.29–3.16(m,1H).13C NMR(150MHz,DMSO-d6)δ170.51,169.99,164.28,162.46,161.83,160.85,151.64,149.85,147.38,135.63,132.03,130.95,129.66,129.60,129.51,120.00,116.24,115.85,115.51,115.37,114.83,99.37,58.26,47.36,43.10,42.14,41.50.ESI-MS:m/z 520.2[M+H]+.C23H20Cl2FN5O4(519.09).
操作同上,所不同的是使用4,4-二氟环己基-1-胺。
产物为白色固体,收率:71%,熔点162-164℃。
1H NMR(600MHz,DMSO-d6)δ11.40–11.07(m,2H),8.11(dd,J=37.5,7.7Hz,1H),7.41(d,J=9.0Hz,1H),7.01(s,1H),6.80(d,J=9.0Hz,1H),5.45(s,1H),4.54–4.11(m,2H),3.90–3.67(m,2H),3.67–3.38(m,3H),3.16(t,J=9.4Hz,1H),1.96(s,2H),1.86(d,J=15.9Hz,2H),1.70(d,J=13.4Hz,2H),1.42(d,J=11.7Hz,2H).13C NMR(150MHz,DMSO-d6)δ169.62,163.34,161.60,151.50,149.91,147.08,132.00,130.95,119.92,115.67,114.65,99.91,57.60,57.10,47.68,45.62,43.68,41.44,31.77,31.61,28.32,27.94.ESI-MS:m/z530.1[M+H]+.C22H23Cl2F2N5O4(529.11).
操作同上,所不同的是使用3,5-二甲基苯胺。
产物为白色固体,收率:73%,熔点196-198℃。
1H NMR(600MHz,DMSO-d6)δ11.24(s,1H),11.21(s,1H),9.97(d,J=126.1Hz,1H),7.43(d,J=8.9Hz,1H),7.16–7.07(m,3H),6.88(dd,J=9.0,2.9Hz,1H),6.69(s,1H),5.40(d,J=73.7Hz,1H),4.67–4.56(m,1H),4.22(dd,J=81.3,13.6Hz,1H),3.95–3.73(m,1H),3.68(d,J=12.7Hz,1H),3.61–3.41(m,2H),3.23–3.17(m,1H),2.21(s,6H).13C NMR(150MHz,DMSO-d6)δ161.76,151.53,149.97,147.30,138.08,133.59,130.16,127.41,126.35,118.53,117.91,116.03,114.89,99.49,98.97,58.51,57.42,49.07,47.50,44.32,43.54,43.19,41.43,21.44.ESI-MS:m/z 538.2[M+Na]+.C24H23Cl2N5O4(515.11).
操作同上,所不同的是使用色胺。
产物为淡黄色固体,收率:68%,熔点143-145℃。
1H NMR(600MHz,DMSO-d6)δ11.19(d,J=11.7Hz,1H),10.79(d,J=6.8Hz,1H),8.24(q,J=6.3,5.7Hz,1H),7.52(d,J=7.9Hz,1H),7.36(d,J=8.8Hz,1H),7.33(d,J=8.1Hz,1H),7.12(d,J=33.6Hz,1H),7.06(t,J=7.5Hz,1H),7.02(d,J=3.0Hz,1H),6.96(t,J=7.4Hz,1H),6.75(dd,J=18.7,7.7Hz,1H),5.47(d,J=12.6Hz,1H),4.37(d,J=39.9Hz,1H),4.16–4.04(m,1H),3.72–3.56(m,2H),3.51(d,J=9.1Hz,1H),3.39(q,J=6.9Hz,2H),3.27–3.15(m,2H),2.76(dd,J=15.9,9.2Hz,2H).13C NMR(150MHz,DMSO-d6)δ169.69,164.22,162.53,151.32,149.88,147.83,136.78,132.00,130.94,127.66,123.11,121.37,119.78,118.69,115.66,114.52,112.12,111.83,99.93,58.20,47.39,42.92,41.55,38.72,25.50.ESI-MS:m/z 553.0[M-H]-.C26H24Cl2N6O4(554.12).
操作同上,所不同的是使用2-环己基乙胺。
产物为白色固体,收率:59%,熔点222-224℃。
1H NMR(600MHz,DMSO-d6)δ11.19(s,2H),8.09–7.98(m,1H),7.41(d,J=9.1Hz,1H),7.00(d,J=10.4Hz,1H),6.81(t,J=9.5Hz,1H),5.43(d,J=25.6Hz,1H),4.33(d,J=32.7Hz,1H),4.08–4.02(m,1H),3.85–3.64(m,1H),3.64–3.54(m,2H),3.46(d,J=12.6Hz,1H),3.26–3.11(m,1H),3.05(tp,J=13.5,6.7Hz,1H),2.92(dq,J=12.8,6.5Hz,1H),1.60(t,J=10.3Hz,5H),1.22(q,J=7.0Hz,2H),1.08(t,J=8.5Hz,4H),0.80(t,J=12.8Hz,2H).13C NMR(150MHz,DMSO-d6)δ169.60,164.15,161.66,151.47,149.89,147.54,147.51,147.16,131.98,130.91,120.31,119.98,116.21,115.84,115.12,114.79,99.40,98.90,58.15,47.47,43.13,41.48,36.82,34.89,33.14,32.85,26.57,26.17,26.10.ESI-MS:m/z521.9[M+H]+.C24H29Cl2N5O4(521.16).
操作同上,所不同的是使用环己基胺。
产物为白色固体,收率:63%,熔点196-198℃。
1H NMR(600MHz,DMSO-d6)δ11.23(s,1H),11.20(s,1H),7.97(dd,J=29.0,7.9Hz,1H),7.41(d,J=9.0Hz,1H),7.00(d,J=9.4Hz,1H),6.79(d,J=9.2Hz,1H),5.44(d,J=1.9Hz,1H),4.45–4.12(m,2H),4.08–3.97(m,1H),3.89–3.68(m,1H),3.67–3.53(m,2H),3.52–3.38(m,2H),1.62(d,J=10.4Hz,4H),1.51(d,J=14.7Hz,1H),1.26–1.18(m,2H),1.10(q,J=11.3,10.6Hz,3H).13C NMR(150MHz,DMSO-d6)δ169.15,164.08,161.60,151.46,149.91,147.04,131.98,130.93,119.86,115.64,114.63,99.53,57.61,48.16,47.75,43.20,41.48,32.81,32.34,25.63,24.84.ESI-MS:m/z 494.2[M+H]+.C22H25Cl2N5O4(493.13)
操作同上,所不同的是使用2-(4-氟苯基)乙胺。
产物为灰白色固体,收率:79%,熔点184-186℃。
1H NMR(600MHz,DMSO-d6)δ11.24(q,J=13.4,12.1Hz,2H),8.23–8.10(m,1H),7.39(d,J=9.1Hz,1H),7.23–7.15(m,2H),7.07(t,J=8.9Hz,2H),7.00(d,J=3.0Hz,1H),6.74(d,J=9.1Hz,1H),5.46(d,J=16.3Hz,1H),4.36(d,J=29.3Hz,1H),4.10(d,J=12.6Hz,1H),3.71–3.54(m,2H),3.53–3.38(m,2H),3.32–3.26(m,1H),3.25–3.10(m,2H),2.70–2.59(m,2H).13C NMR(150MHz,DMSO-d6)δ169.71,164.22,162.12,161.67,160.51,151.52,149.87,147.17,135.89,132.00,130.88,119.87,115.44,115.31,114.67,99.41,98.92,57.97,47.54,43.06,41.36,40.78,34.40.ESI-MS:m/z 534.1[M+H]+.C24H22Cl2FN5O4(533.10).
操作同上,所不同的是使用2-(4-氯苯基)乙胺。
产物为白色固体,收率:77%,熔点214-216℃。
1H NMR(600MHz,DMSO-d6)δ11.26(s,1H),11.22(d,J=10.5Hz,1H),8.17(q,J=5.7Hz,1H),7.39(d,J=9.0Hz,1H),7.30(d,J=8.5Hz,2H),7.18(t,J=9.6Hz,2H),7.00(d,J=2.9Hz,1H),6.73(t,J=10.7Hz,1H),5.46(d,J=20.1Hz,1H),4.35(d,J=27.1Hz,1H),4.10(d,J=13.6Hz,1H),3.70–3.54(m,2H),3.54–3.38(m,2H),3.30(d,J=6.8Hz,1H),3.20–3.09(m,1H),2.72–2.57(m,2H).13C NMR(150MHz,DMSO-d6)δ169.72,164.24,161.70,151.58,149.86,147.26,138.82,132.00,131.24,130.98,130.90,128.63,119.86,115.77,114.64,99.38,57.95,47.54,43.05,41.35,40.54,34.54.ESI-MS:m/z 550.1[M+H]+.C24H22Cl3N5O4(549.07).
操作同上,所不同的是使用2-(3,5-二氯苯基)乙胺。
产物为白色固体,收率:70%,熔点212-214℃。
1H NMR(600MHz,DMSO-d6)δ11.23(t,J=15.0Hz,2H),8.20(dt,J=20.8,5.8Hz,1H),7.45–7.32(m,2H),7.28(d,J=29.8Hz,2H),6.99(s,1H),6.72(t,J=7.0Hz,1H),5.48(d,J=17.2Hz,1H),4.47–4.29(m,1H),4.10(d,J=12.0Hz,1H),3.70–3.55(m,2H),3.53–3.46(m,1H),3.43(d,J=17.9Hz,1H),3.27–3.10(m,2H),2.70(tt,J=14.6,6.7Hz,2H).13CNMR(150MHz,DMSO-d6)δ169.77,164.25,161.67,151.54,149.84,147.18,144.21,134.28,132.03,130.86,128.00,126.32,119.88,115.74,114.55,99.40,57.96,57.11,47.53,43.04,41.32,34.46.ESI-MS:m/z 584.3[M+H]+.C24H21Cl4N5O4(583.03).
操作同上,所不同的是使用呋喃-2-甲胺。
产物为灰白色固体,收率:63%,熔点216-214℃。
1H NMR(600MHz,DMSO-d6)δ9.56(d,J=47.6Hz,2H),7.52(d,J=25.0Hz,1H),7.38(t,J=9.7Hz,1H),6.97(d,J=2.9Hz,1H),6.79(dd,J=9.1,2.9Hz,1H),6.34(d,J=15.1Hz,1H),6.14(dd,J=56.3,3.1Hz,1H),5.07(d,J=40.8Hz,1H),4.56–4.34(m,2H),4.34–4.14(m,2H),4.10(d,J=13.1Hz,1H),3.66–3.57(m,1H),3.57–3.50(m,1H),3.47(d,J=12.0Hz,1H),3.17–2.98(m,1H).13C NMR(150MHz,DMSO-d6)δ170.23,168.36,167.13,159.18,153.00,150.42,142.19,131.86,130.16,119.28,115.55,114.58,110.36,106.75,95.23,93.85,58.45,46.92,43.14,41.18,36.24.ESI-MS:m/z 492.2[M+H]+.C21H19Cl2N5O5(491.08).
操作同上,所不同的是使用4-苯基苄胺。
产物为灰白色固体,收率:68%,熔点266-268℃。
1H NMR(600MHz,DMSO-d6)δ11.24(d,J=24.7Hz,2H),8.76(d,J=47.1Hz,1H),7.63(d,J=7.6Hz,2H),7.60–7.51(m,2H),7.45(q,J=9.4,8.6Hz,3H),7.35(t,J=7.4Hz,1H),7.23(d,J=8.0Hz,2H),7.06(dd,J=16.0,3.0Hz,1H),6.87(t,J=9.7Hz,1H),5.45(s,1H),4.49(d,J=19.7Hz,1H),4.37–4.21(m,1H),4.19–4.08(m,2H),3.64(t,J=12.7Hz,1H),3.58(p,J=6.7Hz,2H),3.49(d,J=8.4Hz,1H),3.25(t,J=9.2Hz,1H).13C NMR(150MHz,DMSO-d6)δ170.08,164.25,161.57,151.54,149.89,147.24,140.46,139.31,138.67,132.06,130.99,129.40,128.57,128.25,128.08,127.80,127.05,115.88,114.87,99.45,58.20,53.76,47.50,42.03,12.71.ESI-MS:m/z 578.2[M+H]+.C29H25Cl2N5O4(577.13).
操作同上,所不同的是使用2-叔丁氧羰基-2-氮杂螺[3.3]庚烷-6-胺。
产物为白色固体,收率:78%,熔点268-270℃。
1H NMR(600MHz,DMSO-d6)δ11.20(d,J=39.4Hz,2H),8.39(dd,J=36.3,7.1Hz,1H),7.40(d,J=8.8Hz,1H),7.01(d,J=3.0Hz,1H),6.79(dd,J=9.0,3.1Hz,1H),5.41(s,1H),4.52–4.24(m,1H),4.11(t,J=13.3Hz,1H),3.95(hept,J=7.8Hz,1H),3.77(d,J=64.6Hz,4H),3.68–3.62(m,1H),3.56(d,J=9.6Hz,1H),3.46(t,J=9.3Hz,2H),3.12(t,J=9.9Hz,1H),2.43–2.30(m,2H),2.11–1.92(m,2H),1.35(s,9H).13C NMR(150MHz,DMSO-d6)δ169.08,164.25,161.83,155.86,151.75,150.01,131.96,130.92,119.83,115.81,114.78,99.43,78.86,57.77,57.08,47.58,45.51,43.44,43.13,41.34,40.76,31.78,28.54.ESI-MS:m/z 607.2[M+H]+.C27H32Cl2N6O6(606.18).
操作同上,所不同的是使用4,4-二氟哌啶盐酸盐。
产物为白色固体,收率:78%,熔点218-220℃。
1H NMR(600MHz,DMSO-d6)δ11.29(s,1H),11.22(d,J=10.9Hz,1H),7.48–7.35(m,1H),7.10(s,1H),6.88(d,J=9.0Hz,1H),5.37(d,J=70.1Hz,1H),5.21(d,J=60.1Hz,1H),4.40(dd,J=115.4,13.1Hz,1H),3.97–3.85(m,1H),3.81–3.55(m,4H),3.45(d,J=10.3Hz,1H),3.06(t,J=14.5Hz,1H),1.90(q,J=57.1,52.9Hz,4H).13C NMR(150MHz,DMSO-d6)δ169.29,164.08,161.75,151.45,149.69,147.24,132.11,131.18,120.39,116.24,115.10,98.88,53.92,53.50,47.21,45.80,44.35,43.72,43.17,42.11,40.94.ESI-MS:m/z 516.1[M+H]+.C21H21Cl2F2N5O4(515.09).
操作同上,所不同的是使用噻吩-2-甲胺。
产物为白色固体,收率:78%,熔点176-178℃。
1H NMR(600MHz,DMSO-d6)δ11.21(d,J=16.6Hz,2H),8.85–8.70(m,1H),7.40(d,J=9.0Hz,1H),7.36(d,J=4.8Hz,1H),7.00(d,J=3.0Hz,1H),6.94–6.91(m,1H),6.89(s,1H),6.83–6.78(m,1H),5.42(d,J=15.6Hz,1H),4.54(dd,J=15.4,6.6Hz,1H),4.46–4.36(m,2H),4.26(dd,J=15.4,5.1Hz,1H),4.15–4.05(m,1H),3.82–3.59(m,2H),3.60–3.44(m,2H).13CNMR(150MHz,DMSO-d6)δ169.32,164.23,161.71,151.08,149.34,147.14,142.56,132.84,130.91,128.30,125.68,125.45,119.91,115.75,114.29,99.91,58.24,47.30,43.07,41.47,37.94.ESI-MS:m/z 508.2[M+H]+.C21H19Cl2N5O4S(507.05).
操作同上,所不同的是使用3,5-二氟苯胺。
产物为白色固体,收率:69%,熔点174-176℃。
1H NMR(600MHz,DMSO-d6)δ11.17(d,J=44.7Hz,2H),10.55(d,J=179.4Hz,1H),7.43(d,J=8.8Hz,1H),7.26(d,J=9.1Hz,2H),7.15(d,J=12.1Hz,1H),6.90(dq,J=18.4,10.0,9.3Hz,2H),5.36(d,J=126.9Hz,1H),4.68(t,J=25.8Hz,1H),4.28(dd,J=80.4,13.6Hz,1H),3.96–3.74(m,1H),3.73–3.54(m,2H),3.54–3.40(m,2H),3.13(dd,J=16.0,9.5Hz,1H).13CNMR(150MHz,DMSO-d6)δ170.06,169.26,164.31,163.89,163.67,163.30,162.05,161.66,151.47,150.31,147.38,147.10,132.09,131.02,120.77,119.89,116.23,115.05,103.21,103.04,99.34,99.29,58.53,53.90,47.52,43.36.ESI-MS:m/z 524.4[M+H]+.C22H17Cl2F2N5O4(523.06)
操作同上,所不同的是使用4-三氟甲基苯胺。
产物为淡黄色固体,收率:78%,熔点220-222℃。
1H NMR(600MHz,DMSO-d6)δ11.27(d,J=53.2Hz,1H),11.17(d,J=52.1Hz,1H),10.47(d,J=137.1Hz,1H),7.73(d,J=8.4Hz,2H),7.64(dd,J=35.4,8.5Hz,2H),7.44(dd,J=9.3,4.0Hz,1H),7.15(d,J=9.2Hz,1H),6.91(s,1H),5.39(d,J=101.8Hz,1H),4.67(s,1H),4.27(dd,J=86.9,13.8Hz,1H),3.86(dd,J=72.4,11.5Hz,1H),3.69(dd,J=38.8,12.0Hz,1H),3.62–3.44(m,2H),3.17(td,J=17.6,15.8,9.0Hz,1H).13C NMR(150MHz,DMSO-d6)δ169.25,164.17,163.93,161.77,151.52,149.91,146.75,142.30,132.09,131.03,126.33,124.39,120.38,116.21,115.03,99.40,58.59,47.43,43.36,41.29.ESI-MS:m/z554.1[M-H]-.C23H18Cl2F3N5O4(555.07).
操作同上,所不同的是使用(5-甲基噻吩)-2-甲胺。
产物为淡黄色固体,收率:68%,熔点208-210℃。
1H NMR(600MHz,DMSO-d6)δ11.21(d,J=17.2Hz,2H),8.71(d,J=22.0Hz,1H),7.40(d,J=9.0Hz,1H),6.99(d,J=8.4Hz,1H),6.81(d,J=9.1Hz,1H),6.65(d,J=3.3Hz,1H),6.57(s,1H),5.41(d,J=15.3Hz,1H),4.44–4.33(m,2H),4.21(ddd,J=60.6,15.3,5.3Hz,1H),4.08(t,J=12.5Hz,1H),3.81–3.69(m,1H),3.64(d,J=12.6Hz,1H),3.56–3.42(m,2H),3.28–3.17(m,1H),2.36(s,3H).13C NMR(150MHz,DMSO-d6)δ170.20,169.11,164.23,161.69,151.52,149.79,147.16,140.09,138.09,132.03,130.91,125.52,125.08,115.82,114.76,99.45,58.59,47.35,42.63,41.88,38.08,15.37.ESI-MS:m/z 522.2[M+H]+.C22H21Cl2N5O4S(521.07).
操作同上,所不同的是使用环丁基甲胺。
产物为白色固体,收率:55%,熔点222-224℃。
1H NMR(600MHz,DMSO-d6)δ11.21(d,J=9.7Hz,2H),8.10(s,1H),7.41(s,1H),6.98(s,1H),6.80(s,1H),5.42(s,1H),4.34(d,J=46.5Hz,1H),4.06(dd,J=23.3,13.4Hz,1H),3.90–3.67(m,1H),3.66–3.39(m,3H),3.28–2.86(m,3H),2.34(p,J=7.5Hz,1H),1.86(qt,J=7.7,3.3Hz,2H),1.82–1.65(m,2H),1.61–1.48(m,2H).13C NMR(150MHz,DMSO-d6)δ169.98,163.54,161.64,150.89,148.70,146.04,131.99,130.92,119.89,115.66,114.64,99.42,58.11,47.48,44.05,43.05,41.52,35.04,25.43,18.14.ESI-MS:m/z 502.1[M+Na]+.C21H23Cl2N5O4(479.11).
操作同上,所不同的是使用环丁胺。
产物为白色固体,收率:64%,熔点188-190℃。
1H NMR(600MHz,DMSO-d6)δ10.86(s,2H),8.37(dd,J=41.2,7.5Hz,1H),7.36(dd,J=58.1,9.0Hz,1H),7.01(d,J=2.9Hz,1H),6.79(dd,J=9.1,2.9Hz,1H),5.41(s,1H),4.37–4.25(m,1H),4.19–4.05(m,2H),3.83–3.57(m,2H),3.43(d,J=71.3Hz,2H),3.12(t,J=11.8Hz,1H),2.10(t,J=8.2Hz,2H),1.91–1.74(m,2H),1.69–1.49(m,2H).13C NMR(150MHz,DMSO-d6)δ168.74,164.17,161.65,151.55,150.02,147.20,131.97,130.93,119.83,115.80,114.77,99.51,57.65,47.74,44.53,43.16,41.36,30.13,27.86,15.13.ESI-MS:m/z 466.3[M+H]+.C20H21Cl2N5O4(465.10).
操作同上,所不同的是使用环戊胺。
产物为白色固体,收率:71%,熔点176-178℃。
1H NMR(600MHz,DMSO-d6)δ11.21(s,2H),8.05(dd,J=28.8,7.2Hz,1H),7.41(d,J=9.0Hz,1H),7.10–6.91(m,1H),6.80(t,J=11.3Hz,1H),5.44(s,1H),4.49–4.13(m,2H),3.92(q,J=6.8Hz,1H),3.87–3.67(m,1H),3.63(d,J=12.3Hz,1H),3.59–3.38(m,2H),1.72(dp,J=13.1,7.1,6.3Hz,2H),1.58(d,J=16.8Hz,2H),1.54–1.39(m,3H),1.39–1.23(m,2H).13CNMR(150MHz,DMSO-d6)δ169.42,164.10,161.58,151.49,150.01,147.09,131.98,130.93,115.71,114.70,100.68,99.53,57.49,50.90,43.24,41.46,33.03,32.32,23.90.ESI-MS:m/z 480.1[M+H]+.C21H23Cl2N5O4(479.11).
操作同上,所不同的是使用(5-甲基呋喃)-2-甲胺。
产物为白色固体,收率:73%,熔点188-190℃。
1H NMR(600MHz,DMSO-d6)δ11.21(d,J=9.3Hz,2H),8.58(d,J=30.2Hz,1H),7.41(d,J=9.0Hz,1H),7.00(t,J=3.8Hz,1H),6.81(dt,J=8.9,4.2Hz,1H),6.01(d,J=3.0Hz,1H),5.94(s,1H),5.42(s,1H),4.43(d,J=34.5Hz,1H),4.31(dd,J=15.7,6.2Hz,1H),4.15–3.96(m,2H),3.85–3.67(m,1H),3.67–3.40(m,3H),3.27–3.04(m,1H),2.20(s,3H).13CNMR(150MHz,DMSO-d6)δ169.81,164.24,161.68,151.53,151.19,150.43,149.86,147.17,131.99,130.91,119.90,115.74,114.70,108.00,106.76,99.39,58.09,47.48,43.06,41.45,36.25,13.66.ESI-MS:m/z 506.1[M+H]+.C22H21Cl2N5O5(505.09).
操作同上,所不同的是使用吡咯-2-甲胺。
产物为淡黄色固体,收率:73%,熔点152-154℃。
1H NMR(600MHz,DMSO-d6)δ11.21(d,J=16.0Hz,1H),10.48(d,J=36.5Hz,1H),8.44(d,J=37.8Hz,1H),7.40(t,J=8.0Hz,1H),7.03(d,J=3.1Hz,1H),6.82(dd,J=9.2,2.9Hz,1H),6.62(d,J=6.5Hz,1H),5.94–5.86(m,1H),5.82(d,J=11.4Hz,1H),5.46(s,1H),4.43(d,J=29.7Hz,1H),4.33–4.15(m,2H),4.15–4.04(m,2H),3.72–3.46(m,4H).13CNMR(150MHz,DMSO-d6)δ169.65,164.35,161.79,151.63,149.88,147.39,132.59,130.91,127.21,118.68,117.58,115.84,114.02,107.72,106.42,99.37,57.94,47.50,43.10,41.80,41.36.ESI-MS:m/z489.0[M-H]-.C21H20Cl2N6O4(490.09).
操作同上,所不同的是使用苯胺。
产物为淡黄色固体,收率:83%,熔点179-180℃。
1H NMR(600MHz,DMSO-d6)δ11.28(d,J=42.4Hz,1H),11.19(d,J=36.5Hz,1H),10.10(d,J=117.8Hz,1H),7.48(d,J=8.0Hz,2H),7.44(dd,J=9.2,3.2Hz,1H),7.28(dt,J=20.6,7.7Hz,2H),7.13(d,J=7.8Hz,1H),7.06(t,J=7.4Hz,1H),6.89(d,J=9.1Hz,1H),5.42(d,J=71.3Hz,1H),4.62(d,J=32.5Hz,1H),4.22(dd,J=85.8,13.5Hz,1H),3.97–3.76(m,1H),3.69(t,J=13.7Hz,1H),3.62–3.43(m,2H),3.22(t,J=9.6Hz,1H).13C NMR(150MHz,DMSO-d6)δ168.64,164.57,161.77,152.01,150.35,148.24,146.12,137.63,131.02,129.29,129.05,125.17,124.34,120.76,120.32,116.72,114.94,101.33,58.63,47.84,45.66,41.37.ESI-MS:m/z488.0[M+H]+.C22H19Cl2N5O4(487.08).
操作同上,所不同的是使用(4-三氟甲基环己基)胺。
产物为白色固体,收率:78%,熔点168-170℃。
1H NMR(600MHz,DMSO-d6)δ11.21(d,J=12.4Hz,2H),8.09(dd,J=59.5,6.9Hz,1H),7.41(d,J=9.1Hz,1H),7.07–6.96(m,1H),6.89–6.80(m,1H),5.43(d,J=24.0Hz,1H),4.48(d,J=63.0Hz,1H),4.16(dd,J=137.5,13.8Hz,2H),3.93–3.69(m,2H),3.69–3.55(m,2H),3.45(dd,J=11.8,6.6Hz,1H),3.30(s,1H),3.20–3.04(m,1H),1.60(s,4H),1.54–1.36(m,4H).13CNMR(150MHz,DMSO-d6)δ169.89,164.03,161.41,151.47,149.94,147.09,132.04,131.37,115.58,114.66,99.37,98.80,56.60,55.35,48.24,43.95,43.40,41.44,28.30,28.06,19.89.ESI-MS:m/z 562.2[M+H]+.C23H24Cl2F3N5O4(561.12).
操作同上,所不同的是使用(1H-吡唑-5-基)甲胺。
产物为白色固体,收率:58%,熔点172-174℃。
1H NMR(600MHz,DMSO-d6)δ12.56(s,1H),11.27(d,J=20.7Hz,2H),8.49(s,1H),7.41(d,J=13.8Hz,1H),7.01(s,1H),6.83(d,J=9.4Hz,1H),6.01(d,J=24.9Hz,1H),5.46(s,1H),4.54–4.32(m,2H),4.10(q,J=13.3,12.4Hz,2H),3.88–3.42(m,4H),3.20(d,J=9.5Hz,1H).13C NMR(150MHz,DMSO-d6)δ171.06,166.06,162.62,152.44,152.31,149.78,142.05,132.00,130.91,129.30,119.69,115.44,114.69,104.21,99.31,56.25,47.39,45.45,43.08,41.13.ESI-MS:m/z 492.3[M+H]+.C20H19Cl2N7O4(491.09).
操作同上,所不同的是使用噻唑-5-甲胺。
产物为白色固体,收率:68%,熔点218-220℃。
1H NMR(600MHz,DMSO-d6)δ10.99(s,2H),9.00(s,1H),8.87(s,1H),7.63(s,1H),7.32(d,J=9.0Hz,1H),6.92(d,J=10.4Hz,1H),6.72(d,J=9.0Hz,1H),5.34(s,1H),4.52(dd,J=15.3,6.2Hz,1H),4.38(d,J=25.6Hz,2H),4.26(dd,J=15.3,5.1Hz,1H),4.11–3.95(m,1H),3.68–3.49(m,2H),3.38(dd,J=12.4,8.8Hz,2H).13C NMR(150MHz,DMSO-d6)δ170.04,165.42,161.86,154.91,148.91,146.42,141.63,137.73,132.62,130.30,125.98,118.51,116.32,114.65,99.46,58.64,47.12,43.01,41.44,35.33.ESI-MS:m/z 509.3[M+H]+.C20H18Cl2N6O4S(508.05).
操作同上,所不同的是使用噻唑-2-甲胺。
产物为白色固体,收率:68%,熔点161-163℃。
1H NMR(600MHz,DMSO-d6)δ11.31–11.19(m,2H),9.08(t,J=5.7Hz,1H),7.70(d,J=3.3Hz,1H),7.61(d,J=3.2Hz,1H),7.43(d,J=9.0Hz,1H),7.03(d,J=3.0Hz,1H),6.84(dd,J=9.1,3.0Hz,1H),5.46(s,1H),4.69(dd,J=16.2,6.6Hz,1H),4.62–4.45(m,2H),4.37–4.14(m,1H),3.90–3.64(m,2H),3.60–3.45(m,2H),3.30–3.22(m,1H).13C NMR(150MHz,DMSO-d6)δ170.43,169.44,164.25,161.72,152.28,149.74,147.13,143.26,132.04,130.91,120.44,119.53,115.75,114.67,100.62,58.38,49.07,47.22,43.01,41.02.ESI-MS:m/z 509.1[M+H]+.C20H18Cl2N6O4S(508.05).
操作同上,所不同的是使用恶唑-5-甲胺。
产物为淡黄色固体,收率:73%,熔点188-190℃。
1H NMR(600MHz,DMSO-d6)δ11.16(s,2H),8.85–8.66(m,1H),8.16(s,1H),7.34(d,J=9.0Hz,1H),6.94(s,1H),6.83(d,J=6.6Hz,1H),6.73(d,J=5.8Hz,1H),5.33(d,J=9.1Hz,1H),4.45–4.31(m,2H),4.15–3.97(m,2H),3.78–3.54(m,2H),3.52–3.34(m,3H),3.12(t,J=11.5Hz,1H).13C NMR(150MHz,DMSO-d6)δ169.31,165.26,162.29,152.00,151.53,149.88,149.56,133.55,130.53,123.64,123.05,119.86,115.72,114.34,99.03,58.19,47.30,43.04,41.41,34.65.ESI-MS:m/z 493.2[M+H+].C20H18Cl2N6O5(492.07).
实施例2:(S)-1-(3,4-二氯苯基)-4-(2,6-二氧基-1,2,3,6-四氢嘧啶-4-羰基)-N-(呋喃-2-基甲基)哌嗪-2-甲酰胺(GA-13S)的制备
合成路线如下:
试剂及条件:(i)醋酸酮,吡啶,氧气,二氯甲烷,室温;(ii)三氟乙酸,二氯甲烷,室温;(iii)乳清酸,HATU,二异丙基乙胺,二氯甲烷,室温;(iv)氢氧化锂,甲醇,四氢呋喃,水,室温;(v)呋喃甲胺或噻吩甲胺,HATU,二异丙基乙胺,二氯甲烷,室温。
操作步骤同实施例1,所不同的是在步骤1中使用的是(S)-1-叔丁基-3-甲基哌嗪-1,3二羧酸;步骤5中使用的为呋喃甲胺;
产物为白色固体,收率:67%,熔点216-218℃。
1H NMR(600MHz,DMSO-d6)δ9.74–9.36(m,2H),7.52(d,J=26.3Hz,1H),7.37(dd,J=11.5,9.0Hz,1H),6.97(d,J=2.9Hz,1H),6.79(dd,J=9.1,3.1Hz,1H),6.43–6.28(m,1H),6.14(dd,J=57.7,3.2Hz,1H),5.06(d,J=44.5Hz,1H),4.55–4.36(m,2H),4.33–4.04(m,3H),3.66–3.48(m,2H),3.49–3.38(m,1H),3.15–3.02(m,1H).13C NMR(150MHz,DMSO-d6)δ171.29,170.20,167.12,153.04,152.56,150.46,142.38,142.17,131.85,130.04,120.10,119.26,115.16,114.59,110.89,106.88,106.73,95.20,58.43,47.73,43.14,41.19,36.24.ESI-MS:m/z515.1[M+Na]+.C21H19Cl2N5O5(491.08)
实施例3:(S)-1-(3,4-二氯苯基)-4-(2,6-二氧基-1,2,3,6-四氢嘧啶-4-羰基)-N-(噻吩-2-基甲基)哌嗪-2-甲酰胺(GA-17S)的制备
操作同上,所不同的是使用噻吩甲胺。
产物为浅黄色固体,收率:69%,熔点176-178℃。
1H NMR(600MHz,DMSO-d6)δ11.29–11.16(m,2H),8.78(dt,J=19.5,5.8Hz,1H),7.43–7.28(m,2H),7.00(t,J=3.8Hz,1H),6.96–6.87(m,2H),6.81(dd,J=9.0,3.3Hz,1H),5.42(d,J=15.3Hz,1H),4.54(dd,J=15.4,6.6Hz,1H),4.47–4.37(m,2H),4.26(dd,J=15.4,5.1Hz,1H),4.10(t,J=13.8Hz,1H),3.81–3.42(m,4H),3.27–3.17(m,1H).13C NMR(150MHz,DMSO-d6)δ168.94,164.24,161.70,152.35,149.77,147.14,142.56,132.03,130.22,127.11,125.68,124.38,119.28,115.74,114.00,100.84,58.22,48.07,43.06,41.47,37.86.ESI-MS:m/z508.1[M+H]+.C21H19Cl2N5O4S(507.05)
实施例4:(R)-1-(3,4-二氯苯基)-4-(2,6-二氧基-1,2,3,6-四氢嘧啶-4-羰基)-N-(呋喃-2-基甲基)哌嗪-2-甲酰胺(GA-28)的制备
操作步骤同实施例1,所不同的是在步骤1中使用的是(R)-1-叔丁基-3-甲基哌嗪-1,3二羧酸;步骤5中使用的为呋喃甲胺;
产物为白色固体,收率:78%,熔点248-251℃。
1H NMR(600MHz,DMSO-d6)δ11.22(d,J=10.3Hz,1H),9.58(s,1H),8.71(dt,J=54.8,5.7Hz,1H),7.53(s,1H),7.41(d,J=9.0Hz,1H),7.00(t,J=3.4Hz,1H),6.82(td,J=9.0,8.2,2.9Hz,1H),6.38–6.34(m,1H),6.14(d,J=3.1Hz,1H),5.42(s,1H),4.54–4.44(m,1H),4.36(dd,J=15.6,6.3Hz,1H),4.24(qd,J=15.8,5.7Hz,1H),4.15–4.02(m,2H),3.71(dd,J=13.8,4.3Hz,1H),3.58(h,J=6.9Hz,4H),3.54–3.41(m,2H),3.20(ddd,J=13.5,9.7,4.1Hz,1H).13CNMR(150MHz,DMSO-d6)δ169.96,164.24,161.68,151.51,149.86,147.19,142.58,132.01,130.92,119.89,115.72,114.69,110.86,107.17,99.38,98.63,53.72,41.99,18.44,17.22,12.59.ESI-MS:m/z 492.0[M+H]+.C21H19Cl2N5O5(491.08)
实施例5:目标化合物的对主蛋白酶(Mpro)的抑制实验
实验原理:
采用的荧光共振能量转移(fluorescence resonance energy transfer,FRET)法,底物结构为:MCA-AVLQSGFR-Lys(Dnp)-Lys-NH2,其中MCA是荧光供体,Dnp是荧光受体或称为荧光淬灭基团,完整的序列即含有荧光基团,又含有荧光淬灭基团,由于两个基团空间距离较近,淬灭基团的抑制作用使得荧光基团不会产生荧光。当加入SARS-CoV-2主蛋白酶Mpro后,由于主蛋白酶能够在氨基酸Q和S之间进行切割,使得荧光基团远离淬灭基团,在激发光为320nm下发射波长为405nm的荧光,通过测定荧光来检测Mpro的活性,进而间接反应化合物的抑制活性(见附图3)(Dai Wenhao,et al.,Science.368(6497):1331-1335,2020.Qiao Jingxin,et al.,Science.371(6536):1374-1378,2021.)。
实验方法:
使用荧光共振能量转移法,测试了目标化合物的对主蛋白酶的抑制活性。使用MCA-AVLQSGFR-Lys(Dnp)-Lys-NH2为反应底物。在避光条件下,将1.5μM的SARS-CoV-2Mpro,500μM的底物和10μM的化合物加入96孔板中进行初筛,37℃下孵育10分钟,使用多功能酶标仪检测每组的荧光强度,激发波长为320nm,发射波长为405nm,每隔10s测一次,测10min,获取荧光强度。首先根据标曲将荧光强度值换算成单位时间内荧光强度的增加量。取用第一分钟的数据来获取速率用反应初速率的变化来表示抑制剂对酶活力的抑制程度是研究酶抑制作用,空白对照的反应初速率为V0,添加抑制剂后为Vi,则酶活力的抑制程度可用如下方程式:i%=(1-Vi)/V0×100%。实验分为空白对照组,阳性对照组和实验组。以化合物7作为实验阳性对照组,将化合物在10μM浓度下,抑制率超过阳性对照的化合物进行复筛。实验初筛结果见附图2。
复筛:选取1.5μM的SARS-CoV-2 Mpro,500μM的底物和四个浓度梯度(0.5μM,1μM,5μM,10μM)测试化合物的IC50。每组设置3个复孔,37℃下孵育10分钟,使用多功能酶标仪检测每组的荧光强度,激发波长为320nm,发射波长405nm,每隔10s测一次,测10min,获取荧光强度。最后根据不同浓度下的抑制率利用GraphPad Prism 5进行IC50的计算。实验结果如表1所示。
表1.目标化合物(嘧啶酮酰基哌嗪类化合物)抑制SARS-CoV-2主蛋白酶的复筛结果
aIC50(μM):对酶的抑制达到50%时,所需化合物浓度,即半数抑制浓度;ML188:已报道的一种SARS-CoV-2主蛋白酶抑制剂,作为阳性对照。
实验结论分析:
本发明新合成的嘧啶酮酰基哌嗪类化合物绝大部分呈现出显著的主蛋白酶抑制活性。化合物GA-13、GA-13S、GA-17S、GA-19、GA-25、GA-27,GA-33。活性均优于先导化合物7和另一类已知的SARS-CoV-2主蛋白酶抑制剂ML188,其中GA-17S的活性尤为突出(IC50=0.38±0.07μM),其抑制主蛋白酶的IC50值较先导化合物7提高了将近20倍;另外由GA-13S和GA-28可知,该系列化合物S构型活性优于R构型。因此,嘧啶酮酰基哌嗪类化合物具有进一步研发的价值。
Claims (6)
3.如权利要求1所述的嘧啶酮酰基哌嗪类化合物的制备方法,步骤如下:
以3,4-二氯苯硼酸1和Boc-哌嗪甲酸酯2为起始原料,二氯甲烷作为反应溶剂,在醋酸铜催化下通过Chan-Lam偶联反应得中间体3;然后将中间体3溶解在适量的二氯甲烷中,经三氟乙酸脱保护得中间体4;随后中间体4在缩合剂HATU的作用下与乳清酸缩合得到关键中间体5;中间体5再经氢氧化锂水解甲酯得到中间体6,然后中间6再在HATU的作用下与各类胺在二氯甲烷中进行酰胺缩合得到各个目标产物;
合成路线如下:
试剂及条件:(i)醋酸酮,氧气,吡啶,二氯甲烷,室温;(ii)三氟乙酸,二氯甲烷,室温;(iii)乳清酸,HATU,N,N,-二异丙基乙胺,二氯甲烷,室温;(iv)氢氧化锂,甲醇,四氢呋喃,水,室温;(v)R1-NH2,HATU,N,N,-二异丙基乙胺,二氯甲烷,室温;
其中,R1如上述权利要求1中通式I中所述;所述的室温为20-30℃。
4.权利要求1或2所述的嘧啶酮酰基哌嗪类化合物在制备抗SARS-CoV-2Mpro的药物中的应用。
5.一种抗冠状病毒药物组合物,包含权利要求1或2所述嘧啶酮酰基哌嗪类化合物和一种或多种药学上可接受载体或赋形剂。
6.如权利要求5所述的冠状病毒为SARS-CoV-2。
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