CN114409643A - 一种二氯苯基多取代哌嗪类化合物及其制备方法与应用 - Google Patents
一种二氯苯基多取代哌嗪类化合物及其制备方法与应用 Download PDFInfo
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- CN114409643A CN114409643A CN202210076418.5A CN202210076418A CN114409643A CN 114409643 A CN114409643 A CN 114409643A CN 202210076418 A CN202210076418 A CN 202210076418A CN 114409643 A CN114409643 A CN 114409643A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
- C07D409/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing three or more hetero rings
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
- C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
- C07D409/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
- C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings
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Abstract
本发明提供了一种二氯苯基多取代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
本发明涉及一种衍生物及其制备方法,具体涉及二氯苯基多取代哌嗪类化合物、包含所述化合物的组合物、制备所述化合物的制备及其在抗冠状病毒药物领域的应用,属于有机合成与医药应用技术领域。
背景技术
新型冠状病毒(Severe Acute Respiratory Syndrome Coronavirus 2,SARS-CoV-2)所致的新型冠状病毒肺炎(Corona Virus Disease 2019,COVID-19)在当前及未来仍是一个重大的公共卫生问题,抗SARS-CoV-2药物是应对当前COVID-19大流行和未来冠状病毒爆发的疫苗的重要补充。在病毒基因组的复制和结构蛋白的合成过程中,3CL蛋白酶(3C-like protease)负责多聚蛋白11个位点的切割,以产生对病毒生存、增殖非常重要的蛋白,因此又被称为主蛋白酶(Mpro)。Mpro作为一种二聚体,在底物P1位置对谷氨酰胺有独特的要求。2020年4月,杨海涛、饶子和团队报道了Mpro的晶体结构,该蛋白酶为同源二聚体,每个单体由三个结构域组成,在结构域Ⅰ和结构域Ⅱ之间形成底物结合口袋,Cys145和His41形成催化二联体,能够结合并切割多聚蛋白(附图1)。另外,Mpro在不同属冠状病毒间高度保守。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。
迄今为止,Mpro抑制剂的种类及结构相对较少,特别是现有共价主蛋白酶抑制剂多以醛基、α,β-不饱和羰基等高活性结构为共价弹头,具有的药物安全隐患。另一方面,非共价抑制剂对主蛋白酶的抑制活性普遍较低,因此,发现活性高、选择性好的SARS-CoV-2主蛋白酶抑制剂是当前抗新冠药物研发的重要方向。
通过高通量筛选发现的嘧啶酮酰基哌嗪类化合物Mcule-5948770040(IC50=4.2μM)是一类骨架新颖的SARS-CoV-2主蛋白酶抑制剂。共晶结构显示Mcule-5948770040与主蛋白酶通过非共价结合来抑制Mpro活性(PDB ID:7LTJ)。然而其活性较低,值得进一步优化。
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二氯苯基多取代哌嗪类化合物、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:
1.二氯苯基多取代哌嗪类化合物
二氯苯基多取代哌嗪类化合物,或其药学上可接受的盐,具有如下通式I所示的结构:
其中,
R为:各类取代磺酰基、取代苯甲酰基、杂环甲酰基、内酰胺甲酰基、取代乙酰基、取代氧代甲酰基、取代丙烯酰基;所述的取代基选自甲基、卤代甲基、硝基、卤素、氰基或硼酸基。
根据本发明优选的,R为取代苯甲磺酰基、取代芳香杂环甲磺酰基、单取代苯甲酰基、多取代苯甲酰基、五元杂环甲酰基、六元取代杂环甲酰基、五元内酰胺甲酰基、嘧啶酮酰基、芳香氧代甲酰基、丙烯酰基、卤代乙酰基、氰乙酰基;所述的取代基选自甲基、卤代甲基、硝基、卤素、氰基或硼酸基;式I所示化合物为S构型。
根据本发明进一步优选的,R为下列取代基I(a)、I(b)、I(c)、I(d)或I(e)中任意一种:
n为选自0、1或2的整数;R1在每次出现时各自独立地为卤素、氰基、硝基;X为=NH-或=CH-。
n为选自0、1或2的整数;R2在每次出现时各自独立地为H、卤素、硼酸基、硝基;X为=NH-或=CH-。
n为选自0或1的整数;X为-S-、-O-或=CH-。
n为选自0、1和2的整数,X和Y各自为-S-、=NH-或=CH-,且至多一个为=CH-;Ar为H、苯环或5-6元芳杂环。
R3、R4、R5在每次出现时各自独立地为H、F、Cl、Br、CN,且至多两个为H。
根据本发明更进一步优选的,二氯苯基多取代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。
2.二氯苯基多取代哌嗪类化合物的制备方法
二氯苯基多取代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(S)-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;然后经氢氧化锂水解甲酯得到中间体4;随后将4溶解在适量的二氯甲烷中,在缩合剂HATU的作用下与噻吩-2-甲胺缩合得到关键中间体5;5再经三氟乙酸脱保护得中间体6;然后6再与取代磺酰氯反应或在HATU的作用下与各类羧酸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)氢氧化锂,甲醇,四氢呋喃,水,室温;(iii)噻吩-2-甲胺,HATU,N,N,-二异丙基乙胺,二氯甲烷,室温;(iv)三氟乙酸,二氯甲烷,室温;(v)各类磺酰氯化合物,N,N,-二异丙基乙胺,二氯甲烷,室温;(vi)各类羧酸化合物,HATU,N,N,-二异丙基乙胺,二氯甲烷,室温。
其中,R6如上述通式I(a)所述结构;R7如上述通式I(b)、I(c)、I(d)、I(e)所述结构;本发明所述的室温为20-30℃。
根据本发明优选的,含有二氯苯基多取代哌嗪类化合物的制备方法,具体步骤如下:
(1)将(S)-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检测反应乙酸乙酯/石油醚=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)中间体3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4,为微黄色粉末状固体;
(3)冰浴下,将中间体4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30min后加入N,N,-二异丙基乙胺和噻吩-2-甲胺,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,以二氯甲烷洗涤所得固体至灰白色,干燥,得中间体5;
(4)在冰水浴下将中间体5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,得到紫红色油状物;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,干燥得中间体6的三氟乙酸盐;
(5)将N,N,-二异丙基乙胺和中间体6的三氟乙酸盐加入到二氯甲烷中,冰水浴条件下滴加入各类磺酰氯和二氯甲烷的混合液,室温反应6h,TLC监测;反应完全后,在体系中加水淬灭反应;分液,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终产物GC-1至GC-5;
(6)将各类羧酸与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6的三氟乙酸盐,室温反应12h,TLC监测;反应结束后加水,冰水浴下滴加1M的稀盐酸水溶液,分液,二氯甲烷相依次经饱和碳酸氢钠水溶液、饱和氯化钠溶液洗涤,无水硫酸钠干燥,过滤后硅胶柱色谱分离得到目标化合物GC-11至GC-33。
3.二氯苯基多取代哌嗪类化合物的生物活性及应用
本发明公开了含二氯苯基多取代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二氯苯基多取代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二氯苯基多取代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Mcule-5948770040及ML188为阳性对照。
本发明新合成的二氯苯基多取代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GC-14、GC-16、GC-23。其中GC-23的活性尤为突出(IC50=0.17±0.01μM),较先导化合物Mcule-5948770040提高了30倍。因此,二氯苯基多取代哌嗪类化合物可作为抗SARS-CoV-2先导化合物供进一步研发。
本发明的一类二氯苯基多取代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,包括本发明的二氯苯基多取代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二氯苯基多取代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。本发明的二氯苯基多取代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是以SARS-CoV-2Mpro为靶点的药物筛选原理图;
图3是在10μM浓度下化合物对主蛋白酶的抑制率。
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:GC-1至GC-5的制备
(1)将(S)-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4.5g,收率59%。C17H22Cl2N2O4(388.1).1H NMR(400MHz,DMSO-d6)δ7.41(d,J=9.0Hz,1H),7.13(s,1H),6.83(dd,J=47.5,8.8Hz,1H),4.91–4.70(m,1H),4.38(d,J=13.5Hz,1H),3.96(t,J=16.6Hz,1H),3.60(s,3H),3.50(d,J=11.5Hz,1H),3.24–2.89(m,2H),1.40(d,J=1.6Hz,9H).ESI-MS:m/z 389.2[M+H]+.
(2)中间体3(4.0g,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(1.57g,37.5mmol,5.0eq.)的水溶液,室温反应,搅拌12h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2-3,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4约2.5g,收率89.3%,为微黄色粉末状固体,ESI-MS:m/z 373.2[M-H]-.C16H20Cl2N2O4(374.0)。
(3)冰水浴下,将中间体4(2.4g,6.4mmol,1.0eq.)与2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(HATU,3.65g,9.6mmol,1.5eq.)加入到100mL二氯甲烷中,活化30min后加入二异丙基乙胺(DIPEA,2.5g,19.2mmol,3.0eq.)和噻吩-2-甲胺(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 2.4g,收率80.0%。ESI-MS:m/z 470.2[M+H]+.C21H25Cl2N3O3S(469.1).
(4)在冰水浴下将中间体5(2.4g,5.1mmol,1.0eq.)溶于100mL二氯甲烷中,逐滴加入6mL三氟乙酸与30mL二氯甲烷的混合溶液。滴加结束后,转至室温反应。约5h后TLC检测反应完后减压浓缩反应液,得到紫红色油状物。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6的三氟乙酸盐2.1g,收率86.4%。ESI-MS:m/z 370.3[M+H]+.C16H17Cl2N3OS(369.1).1H NMR(400MHz,DMSO-d6)δ9.09(s,1H),8.49(s,1H),7.58–7.37(m,2H),7.22–7.13(m,1H),7.08–6.87(m,3H),4.71–4.53(m,2H),4.48(dd,J=25.7,5.8Hz,1H),3.69(dd,J=53.0,13.3Hz,2H),3.53(d,J=11.9Hz,1H),3.26(d,J=13.1Hz,2H),3.10(d,J=20.9Hz,1H).
(5)将中间体6的三氟乙酸盐(0.1g,0.2mmol,1.0eq.)和N,N,-二异丙基乙胺(51.6mg,0.4mmol,2.0eq.)加入到5mL二氯甲烷中,冰水浴条件下滴加入吡啶磺酰氯(46.2mg,0.26mmol,1.3eq.)和5mL二氯甲烷的混合液,室温反应6h,TLC监测;反应完全后,在体系中加水淬灭反应;分液,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终产物GC-1。
产物为类白色固体,收率55%,熔点71-80℃。
1H NMR(600MHz,DMSO-d6)δ8.94–8.86(m,2H),8.66(t,J=5.9Hz,1H),8.16(dt,J=7.2,1.6Hz,1H),7.69(dd,J=8.1,4.8Hz,1H),7.41–7.31(m,2H),6.98(d,J=2.9Hz,1H),6.93(d,J=5.0Hz,2H),6.78(dd,J=9.1,3.0Hz,1H),4.47(t,J=3.5Hz,1H),4.45–4.36(m,2H),4.03(dt,J=12.0,2.4Hz,1H),3.70–3.56(m,2H),3.56–3.47(m,1H),2.88(dd,J=12.2,4.4Hz,1H),2.68(td,J=11.0,4.4Hz,1H).13C NMR(150MHz,DMSO-d6)δ169.32,156.01,150.67,148.19,143.09,137.42,133.61,131.96,130.85,127.08,125.56,125.36,124.50,120.99,116.47,115.31,57.44,47.25,45.23,44.52,37.51.ESI-MS:m/z 511.2[M+H]+.C21H20Cl2N4O3S2(510.04).
操作同上,所不同的是使用3-氯苯磺酰氯。
产物为类白色固体,收率:72%,熔点77-80℃。
1H NMR(600MHz,DMSO-d6)δ8.65(t,J=5.9Hz,1H),7.81(dt,J=7.7,1.8Hz,1H),7.78–7.65(m,3H),7.41–7.32(m,2H),6.98(d,J=3.0Hz,1H),6.93(d,J=4.7Hz,2H),6.78(dd,J=9.0,3.0Hz,1H),4.46(q,J=4.6,4.0Hz,1H),4.42(dd,J=5.9,3.0Hz,2H),3.99(dt,J=12.0,2.4Hz,1H),3.63(dt,J=12.5,3.5Hz,1H),3.60–3.47(m,2H),2.83(dd,J=12.1,4.4Hz,1H),2.63(td,J=11.0,3.7Hz,1H).13C NMR(150MHz,DMSO-d6)δ169.31,148.66,142.70,137.78,134.72,133.85,132.80,131.95,130.85,127.43,127.08,126.68,125.55,124.93,120.27,116.50,114.58,57.42,48.07,45.90,44.55,37.91.ESI-MS:m/z543.9[M+H]+.C22H20Cl3N3O3S2(543.00).
操作同上,所不同的是使用3-氟-4-硝基苯磺酰氯。
产物为黄色固体,收率:69%,熔点85-86℃。
1H NMR(600MHz,DMSO-d6)δ8.66(t,J=5.9Hz,1H),8.42–8.32(m,1H),7.98(dd,J=10.2,1.9Hz,1H),7.79(dd,J=8.5,1.9Hz,1H),7.37(dd,J=4.9,1.5Hz,1H),7.34(d,J=9.0Hz,1H),6.97(d,J=2.9Hz,1H),6.93(dd,J=7.8,2.8Hz,2H),6.77(dd,J=9.1,3.0Hz,1H),4.46(t,J=3.9Hz,1H),4.42(dd,J=5.9,2.6Hz,2H),4.01(dt,J=12.1,2.5Hz,1H),3.66(dt,J=12.7,3.6Hz,1H),3.63–3.58(m,1H),3.54–3.47(m,1H),3.03(dd,J=12.3,4.6Hz,1H),2.83(td,J=11.3,3.7Hz,1H).
13C NMR(150MHz,DMSO-d6)δ169.33,155.88,154.12,150.24,142.68,140.12,134.18,130.85,128.17,127.07,125.56,125.36,124.54,120.22,118.39,118.24,116.29,115.15,57.47,46.89,45.14,43.89,37.89.ESI-MS:m/z 573.1[M+H]+.C22H19Cl2FN4O5S2(572.02).
操作同上,所不同的是使用4-硝基苯磺酰氯。
产物为黄色固体,收率:71%,熔点97-99℃。
1H NMR(600MHz,DMSO-d6)δ8.67(t,J=5.9Hz,1H),8.42(d,J=8.8Hz,2H),8.01(d,J=8.9Hz,2H),7.40–7.29(m,2H),6.96(d,J=3.0Hz,1H),6.94–6.89(m,2H),6.77(dd,J=9.1,2.9Hz,1H),4.45(t,J=3.8Hz,1H),4.43–4.39(m,2H),4.01(dt,J=12.3,2.4Hz,1H),3.65(dt,J=12.8,3.6Hz,1H),3.59(dt,J=12.8,4.0Hz,1H),3.55–3.47(m,1H),2.92(dd,J=12.2,4.5Hz,1H),2.72(td,J=11.2,3.7Hz,1H).13C NMR(150MHz,DMSO-d6)δ170.20,150.57,149.56,142.70,140.85,131.96,130.27,128.95,127.07,125.56,125.35,124.76,120.25,117.37,115.24,57.47,47.06,45.20,43.99,37.41.ESI-MS:m/z 555.0[M+H]+.C22H19Cl2FN4O5S2(554.03).
操作同上,所不同的是使用4-氰基苯磺酰氯。
产物为类白色固体,收率:65%,熔点98-100℃。
1H NMR(600MHz,DMSO-d6)δ8.65(t,J=5.9Hz,1H),8.12(d,J=8.4Hz,2H),7.92(d,J=8.5Hz,2H),7.41–7.32(m,2H),6.97(d,J=3.0Hz,1H),6.96–6.90(m,2H),6.77(dd,J=9.1,3.0Hz,1H),4.45(t,J=3.7Hz,1H),4.41(dd,J=5.9,3.3Hz,2H),3.99(dt,J=12.2,2.3Hz,1H),3.67–3.60(m,1H),3.60–3.54(m,1H),3.50(td,J=12.4,11.6,3.7Hz,1H),2.89(dd,J=12.2,4.4Hz,1H),2.75–2.64(m,1H).13C NMR(150MHz,DMSO-d6)δ169.29,150.69,142.70,140.27,133.45,131.96,130.85,128.64,127.08,125.56,125.36,121.01,118.04,116.85,116.22,115.28,57.46,47.17,45.24,44.09,37.91.ESI-MS:m/z 535.2[M+H]+.C23H20Cl2N4O3S2(534.04).
实施例2:GC-11至GC-33的制备
(1)将(S)-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4.5g,收率59%。ESI-MS:m/z 389.2[M+H]+.C17H22Cl2N2O4(388.1).1H NMR(400MHz,DMSO-d6)δ7.41(d,J=9.0Hz,1H),7.13(s,1H),6.83(dd,J=47.5,8.8Hz,1H),4.91–4.70(m,1H),4.38(d,J=13.5Hz,1H),3.96(t,J=16.6Hz,1H),3.60(s,3H),3.50(d,J=11.5Hz,1H),3.24–2.89(m,2H),1.40(d,J=1.6Hz,9H).
(2)中间体3(4.0g,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(1.57g,37.5mmol,5.0eq.)的水溶液,室温反应,搅拌12h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2-3,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4约2.5g,收率89.3%,为微黄色粉末状固体,ESI-MS:m/z 373.2[M-H]-.C16H20Cl2N2O4(374.0)。
(3)冰水浴下,将中间体4(2.4g,6.4mmol,1.0eq.)与2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(HATU,3.65g,9.6mmol,1.5eq.)加入到100mL二氯甲烷中,活化30min后加入二异丙基乙胺(DIPEA,2.5g,19.2mmol,3.0eq.)和噻吩-2-甲胺(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,得乳白色中间体5 2.4g,收率80.0%。ESI-MS:m/z 470.2[M+H]+.C21H25Cl2N3O3S(469.1).
(4)在冰水浴下将中间体5(2.4g,5.1mmol,1.0eq.)溶于100mL二氯甲烷中,逐滴加入6mL三氟乙酸与30mL二氯甲烷的混合溶液。滴加结束后,转至室温反应。约5h后TLC检测反应完后减压浓缩反应液,得到紫红色油状物。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。得中间体6的三氟乙酸盐2.1g,收率86.4%。ESI-MS:m/z 370.3[M+H]+.C16H17Cl2N3OS(369.1).1H NMR(400MHz,DMSO-d6)δ9.09(s,1H),8.49(s,1H),7.58–7.37(m,2H),7.22–7.13(m,1H),7.08–6.87(m,3H),4.71–4.53(m,2H),4.48(dd,J=25.7,5.8Hz,1H),3.69(dd,J=53.0,13.3Hz,2H),3.53(d,J=11.9Hz,1H),3.26(d,J=13.1Hz,2H),3.10(d,J=20.9Hz,1H).
(5)将呋喃甲酸(0.027g,0.24mmol,1.2eq.)与HATU(0.114g,0.3mmol,1.5eq.)加入到10mL二氯甲烷中,活化30min后加入二异丙基乙胺(DIPEA,0.103g,0.8mmol,4.0eq.)和6的三氟乙酸盐(0.1g,0.2mmol,1.0eq.),室温反应12h,TLC监测;反应结束后加水淬灭,冰水浴下滴加1M的稀盐酸水溶液,分液,二氯甲烷相依次经饱和碳酸氢钠水溶液、饱和氯化钠溶液洗涤,无水硫酸钠干燥,过滤后硅胶柱色谱分离得到目标化合物GC-11。
产物为浅黄色固体,收率55%,熔点211-213℃。
1H NMR(600MHz,DMSO-d6)δ8.81(s,1H),7.84(s,1H),7.40(dd,J=9.0,2.3Hz,1H),7.32(d,J=5.0Hz,1H),7.00(s,1H),6.93(s,1H),6.91–6.87(m,1H),6.85(s,1H),6.75(d,J=9.0Hz,1H),6.63(d,J=2.9Hz,1H),4.57(d,J=13.2Hz,1H),4.40(s,1H),4.39–4.33(m,2H),4.05(s,1H),3.62(h,J=4.9Hz,2H),3.30(d,J=2.3Hz,2H).13C NMR(150MHz,DMSO-d6)δ170.61,160.03,149.67,147.39,145.39,142.72,132.98,131.42,127.88,125.90,125.28,118.38,117.04,115.17,113.70,111.82,59.00,43.43,40.63,37.89.
操作同上,所不同的是使用L-焦谷氨酸。
产物为白色固体,收率:57%,熔点121-123℃。
1H NMR(600MHz,DMSO-d6)δ8.84(t,J=6.1Hz,1H),7.67(s,1H),7.38(dd,J=19.1,6.7Hz,2H),6.97–6.86(m,3H),6.76(t,J=6.2Hz,1H),4.48–4.33(m,4H),3.91–3.82(m,1H),3.76–3.49(m,3H),2.10–2.00(m,2H),1.90–1.73(m,1H),1.27(dd,J=12.1,6.7Hz,3H).13C NMR(150MHz,DMSO-d6)δ177.83,171.28,170.48,149.66,142.60,133.12,130.89,127.05,125.74,124.98,118.75,115.28,114.22,59.20,51.63,45.10,42.99,37.92,29.63,24.93,16.20.ESI-MS:m/z 479.1[M-H]-.C23H20Cl2N4O3S2(480.08).
操作同上,所不同的是使用2-吡啶甲酸。
产物为类白色固体,收率:65%,熔点105-107℃。
1H NMR(600MHz,DMSO-d6)δ8.64–8.60(m,1H),7.96–7.89(m,1H),7.52–7.46(m,2H),7.40–7.33(m,2H),6.95–6.90(m,2H),6.83–6.73(m,2H),4.40(ddd,J=21.5,15.4,6.0Hz,1H),4.23–4.18(m,2H),3.70–3.58(m,2H),3.39(ddd,J=12.6,8.3,4.1Hz,1H),1.44(d,J=6.8Hz,1H),1.24(d,J=5.6Hz,1H),1.10(t,J=6.4Hz,1H).13C NMR(150MHz,DMSO-d6)δ169.57,167.51,153.90,149.83,148.51,142.58,137.77,132.02,130.90,127.04,125.53,125.28,124.20,121.35,119.64,114.96,114.32,58.70,47.55,43.10,41.91,38.72.ESI-MS:m/z 475.0[M+H]+.C22H20Cl2N4O2S(474.07).
操作同上,所不同的是使用烟酸。
产物为泡沫状透明固体,收率:75%,熔点101-103℃。
1H NMR(600MHz,DMSO-d6)δ8.66(d,J=3.3Hz,1H),8.54(d,J=2.1Hz,1H),7.72(dt,J=7.9,2.0Hz,1H),7.52–7.44(m,1H),7.40(d,J=9.0Hz,1H),7.37–7.34(m,1H),6.97(d,J=2.9Hz,1H),6.92(dd,J=5.1,3.3Hz,1H),6.86(s,1H),6.78(dd,J=9.0,3.0Hz,1H),4.48(dd,J=15.0,5.9Hz,1H),4.26(s,2H),3.93–3.75(m,1H),3.69–3.50(m,3H),3.39(d,J=10.9Hz,1H),3.20–3.01(m,1H).13C NMR(150MHz,DMSO-d6)δ169.81,166.52,151.41,149.75,147.07,143.40,137.42,132.05,131.94,130.93,127.62,125.68,125.42,123.36,121.27,116.40,114.51,52.85,34.74,18.58,15.55,12.87.ESI-MS:m/z 475.1[M+H]+.C22H20Cl2N4O2S(474.07).
操作同上,所不同的是使用3,5-二氯苯甲酸。
产物为白色固体,收率:63%,熔点169-171℃。
1H NMR(600MHz,DMSO-d6)δ8.76(d,J=77.0Hz,1H),7.74(s,1H),7.40(d,J=9.0Hz,1H),7.36(d,J=5.0Hz,3H),6.98(d,J=2.6Hz,1H),6.92(dd,J=5.1,3.3Hz,1H),6.86(s,1H),6.78(d,J=8.4Hz,1H),4.51(dd,J=15.2,6.2Hz,1H),4.27(s,2H),3.90–3.72(m,1H),3.62(d,J=34.7Hz,3H),3.31(s,2H).13C NMR(150MHz,DMSO-d6)δ170.49,166.87,149.72,142.13,140.26,136.13,133.02,130.93,129.73,127.06,126.22,125.67,125.43,120.34,118.14,116.57,115.62,114.54,57.33,49.31,42.62,41.78,37.01.ESI-MS:m/z542.2[M+H]+.C23H19Cl4N3O2S(541.0).
操作同上,所不同的是使用2-羟基-4-嘧啶甲酸。
产物为白色固体,收率:52%,熔点130-132℃。
1H NMR(600MHz,DMSO-d6)δ12.74(s,1H),8.72(dt,J=61.4,5.9Hz,1H),8.25(s,1H),7.51–7.27(m,2H),6.97(dd,J=6.5,3.0Hz,1H),6.92(q,J=2.9,2.5Hz,1H),6.86(d,J=3.4Hz,1H),6.78(dd,J=9.1,3.2Hz,1H),6.30(d,J=21.5Hz,1H),4.59–4.37(m,2H),4.31–4.20(m,1H),4.16–4.03(m,1H),3.79–3.69(m,1H),3.64(ddd,J=18.9,11.8,5.6Hz,1H),3.61–3.54(m,1H),3.52–3.42(m,1H),3.31–3.27(m,1H).13C NMR(150MHz,DMSO-d6)δ170.44,170.11,166.62,161.44,150.65,149.42,142.59,133.07,130.89,127.08,125.58,125.39,124.44,119.76,115.53,114.49,58.51,47.14,43.05,41.51,37.88.ESI-MS:m/z492.1[M+H]+.C21H19Cl2N5O3S(491.06).
操作同上,所不同的是使用2,6-二氯嘧啶-4-甲酸。
产物为白色固体,收率:58%,熔点178-180℃。
1H NMR(600MHz,DMSO-d6)δ8.70(dt,J=141.7,5.9Hz,1H),7.72(d,J=74.6Hz,1H),7.43–7.31(m,2H),7.00(dd,J=14.9,2.9Hz,1H),6.96–6.90(m,1H),6.85(dd,J=26.2,3.2Hz,1H),6.80(dd,J=11.5,2.9Hz,1H),4.64–4.43(m,2H),4.44–4.36(m,1H),4.22(dd,J=15.2,5.1Hz,1H),4.05(d,J=14.0Hz,1H),3.78–3.68(m,1H),3.59–3.52(m,1H),3.52–3.45(m,1H),3.32(ddd,J=13.5,9.8,4.1Hz,1H).13C NMR(150MHz,DMSO-d6)δ170.36,169.67,165.81,163.98,159.97,150.21,143.57,132.01,130.87,127.09,125.69,125.48,121.04,116.32,114.75,58.93,47.20,43.06,41.77,37.94.ESI-MS:m/z 492.1[M+H]+.C21H19Cl2N5O3S(491.06).ESI-MS:m/z 544.0[M+H]+.C21H17Cl4N5O2S(542.99).
操作同上,所不同的是使用1H-苯并[d][1,2,3]三氮唑-1-乙酸。
产物为白色固体,收率:71%,熔点186-188℃。
1H NMR(600MHz,DMSO-d6)δ8.86(dt,J=46.0,6.0Hz,1H),8.05(d,J=8.3Hz,1H),7.65(dd,J=48.3,8.3Hz,1H),7.52(td,J=7.4,3.2Hz,1H),7.46–7.37(m,2H),7.34(t,J=4.8Hz,1H),7.10(d,J=3.0Hz,1H),6.97(dd,J=26.7,2.6Hz,1H),6.92–6.85(m,2H),5.98–5.83(m,1H),5.69(dd,J=68.6,17.2Hz,1H),4.65–4.45(m,2H),4.43(d,J=11.2Hz,1H),4.36(d,J=15.3Hz,1H),4.01(dd,J=31.8,14.0Hz,1H),3.79–3.72(m,1H),3.67(d,J=12.9Hz,1H),3.53–3.36(m,1H),3.18(ddd,J=13.2,9.5,3.7Hz,1H).13C NMR(150MHz,DMSO-d6)δ170.55,164.69,149.58,145.62,142.68,135.52,132.10,130.94,127.64,127.09,125.76,125.52,125.30,124.21,119.49,116.79,114.90,111.24,58.83,49.14,45.55,43.54,41.52,38.08.C21H19Cl2N5O3S(491.06).ESI-MS:m/z 544.0[M+H]+.C21H17Cl4N5O2S(542.99).
操作同上,所不同的是使用苯甲酰甲酸。
产物为淡黄色油状物,收率:52%。
1H NMR(600MHz,DMSO-d6)δ8.74(dt,J=137.8,6.1Hz,1H),7.95–7.91(m,1H),7.86–7.83(m,1H),7.76(q,J=7.5,6.8Hz,1H),7.64–7.56(m,2H),7.40(d,J=10.0Hz,1H),7.37–7.33(m,1H),6.99(dd,J=26.4,3.0Hz,1H),6.96–6.91(m,1H),6.91–6.80(m,1H),6.79(dd,J=8.5,3.6Hz,1H),4.73–4.55(m,1H),4.49(d,J=5.8Hz,1H),4.36–4.29(m,1H),4.19(dt,J=13.1,4.8Hz,1H),3.90–3.83(m,1H),3.82–3.71(m,1H),3.69–3.62(m,1H),3.55–3.48(m,2H),3.47–3.36(m,1H).13C NMR(150MHz,DMSO-d6)δ191.31,169.81,165.53,150.43,143.26,135.69,135.49,132.09,130.96,130.09,129.92,129.52,127.10,127.04,125.69,125.39,120.22,116.54,114.95,58.16,46.37,43.97,43.07,37.88.ESI-MS:m/z502.2[M+H]+.C24H21Cl2N3O3S(501.07).
操作同上,所不同的是使用2-噻吩氧代甲酸。
产物为淡黄色油状物,收率:60%。
1H NMR(600MHz,DMSO-d6)δ8.76(dt,J=118.1,5.9Hz,1H),8.28–8.19(m,1H),7.81(dd,J=113.8,3.9Hz,1H),7.40(d,J=9.0Hz,1H),7.37–7.29(m,2H),6.99(dd,J=32.3,3.0Hz,1H),6.94–6.75(m,3H),4.68–4.53(m,1H),4.51–4.41(m,1H),4.34–4.29(m,1H),4.01–3.87(m,1H),3.83–3.68(m,1H),3.67–3.58(m,1H),3.55–3.45(m,2H),3.40–3.33(m,1H).13C NMR(150MHz,DMSO-d6)δ183.37,170.60,164.65,149.69,142.36,139.99,138.72,137.96,132.62,130.96,129.91,127.11,127.06,125.71,125.48,125.40,120.25,116.05,114.92,58.17,46.54,43.98,42.63,37.62.ESI-MS:m/z 508.0[M+H]+.C22H19Cl2N3O3S2(507.02).
操作同上,所不同的是使用2-三氟甲基-4-氟苯甲酸。
产物为白色固体,收率:61%,熔点132-134℃。
1H NMR(600MHz,DMSO-d6)δ8.65(t,J=6.0Hz,1H),7.80–7.55(m,3H),7.39(d,J=9.4Hz,2H),7.03–6.88(m,3H),6.79(d,J=12.8Hz,1H),4.74–4.55(m,1H),4.49(d,J=4.0Hz,1H),4.26(dd,J=15.8,4.6Hz,1H),3.75–3.50(m,3H),3.28(d,J=32.3Hz,3H).13CNMR(150MHz,DMSO-d6)δ169.80,166.19,160.98,151.05,142.23,133.47,133.41,132.05,131.10,130.91,127.09,125.84,125.56,120.04,119.90,115.88,115.64,114.79,114.59,57.36,47.42,42.55,41.05,37.90.ESI-MS:m/z 560.1[M+H]+.C24H19Cl2F4N3O2S(559.05).
操作同上,所不同的是使用2-呋喃氧代甲酸。
产物为橙色固体,收率:53%,熔点102-104℃。
1H NMR(600MHz,DMSO-d6)δ8.76(dt,J=108.3,5.9Hz,1H),8.18(dd,J=14.8,1.7Hz,1H),7.53(d,J=3.6Hz,1H),7.40(dd,J=9.1,1.4Hz,1H),7.36(td,J=4.1,3.5,1.6Hz,1H),6.99(dd,J=27.0,3.0Hz,1H),6.93–6.77(m,4H),4.64–4.52(m,1H),4.45(qd,J=15.4,5.9Hz,1H),4.40–4.31(m,1H),4.08–3.92(m,1H),3.74(ddd,J=30.8,13.4,4.7Hz,1H),3.62(dt,J=12.5,4.4Hz,1H),3.54–3.43(m,2H),3.35(ddd,J=13.2,9.1,4.1Hz,1H).13C NMR(150MHz,DMSO-d6)δ178.61,169.84,163.82,151.08,149.89,149.68,142.47,132.06,129.41,127.06,125.70,125.40,123.90,120.26,116.06,114.94,113.74,58.24,45.59,44.91,42.67,37.83.ESI-MS:m/z 492.1[M+H]+.C22H19Cl2N3O4S(491.05).
操作同上,所不同的是使用氯乙酸。
产物为白色固体,收率:73%,熔点138-140℃。
1H NMR(600MHz,DMSO-d6)δ8.77(d,J=4.7Hz,1H),7.47–7.31(m,2H),6.98(dd,J=46.1,3.0Hz,1H),6.93–6.88(m,2H),6.78(ddd,J=38.3,9.1,3.0Hz,1H),4.49–4.35(m,4H),4.30(t,J=11.1Hz,1H),4.18–4.08(m,1H),3.89(ddt,J=95.6,11.5,5.8Hz,1H),3.68–3.56(m,2H),3.42(dd,J=13.8,4.9Hz,1H),3.25–3.12(m,1H).13C NMR(150MHz,DMSO-d6)δ170.16,165.10,150.90,142.63,132.03,130.89,127.06,125.80,125.47,120.73,117.09,114.64,58.75,46.03,44.14,43.35,42.06,38.35.ESI-MS:m/z 446.1[M+H]+.C18H18Cl3N3O2S(445.02).
操作同上,所不同的是使用氰基乙酸。
产物为白色固体,收率:69%,熔点188-190℃。
1H NMR(600MHz,DMSO-d6)δ8.78(dt,J=24.4,6.0Hz,1H),7.39(dd,J=9.0,5.3Hz,1H),7.37–7.34(m,1H),7.05–6.91(m,2H),6.90(d,J=3.3Hz,1H),6.78(ddd,J=50.4,9.1,3.0Hz,1H),4.47(t,J=3.4Hz,1H),4.42(dd,J=9.6,5.6Hz,2H),4.12–4.03(m,1H),4.02–3.89(m,2H),3.69(d,J=18.9Hz,1H),3.58(ddd,J=18.2,12.8,3.8Hz,2H),3.50–3.40(m,1H),3.38–3.32(m,1H).13C NMR(150MHz,DMSO-d6)δ169.31,161.99,149.53,142.61,132.63,130.89,127.08,125.81,125.57,125.31,119.80,115.90,114.74,58.72,46.08,44.34,41.60,37.97,24.92.ESI-MS:m/z 437.1[M+H]+.C19H18Cl2N4O2S(436.05).
操作同上,所不同的是使用丙烯酸。
产物为透明油状物,收率:65%。
1H NMR(600MHz,DMSO-d6)δ8.76(d,J=35.2Hz,1H),7.49–7.29(m,2H),7.05–6.85(m,3H),6.76(dd,J=33.2,9.1Hz,1H),6.70–6.59(m,1H),6.09(dd,J=37.9,16.7Hz,1H),5.67(dd,J=39.4,10.5Hz,1H),4.39(s,2H),4.37–4.29(m,1H),4.11–3.90(m,1H),3.72–3.64(m,1H),3.59(s,2H),3.45(d,J=18.5Hz,1H),3.23(d,J=10.5Hz,1H).13C NMR(150MHz,DMSO-d6)δ170.36,165.79,149.89,142.77,132.01,130.88,128.37,127.68,127.04,125.69,125.06,120.38,115.91,114.40,59.34,46.53,43.44,41.23,37.41.ESI-MS:m/z 424.2[M+H]+.C19H19Cl2N3O2S(423.06).
操作同上,所不同的是使用苯并噻唑-2-羧酸。
产物为白色固体,收率:60%,熔点112-114℃。
1H NMR(600MHz,DMSO-d6)δ8.84(dt,J=62.1,5.8Hz,1H),8.22(d,J=9.3Hz,1H),8.14(dd,J=20.0,8.4Hz,1H),7.67–7.56(m,2H),7.41(d,J=9.0Hz,1H),7.17(dd,J=74.3,6.4Hz,1H),6.98(dd,J=7.7,2.9Hz,1H),6.92–6.81(m,1H),6.79(dd,J=9.1,2.7Hz,1H),6.76–6.65(m,1H),4.78(ddd,J=81.1,12.0,3.1Hz,1H),4.55–4.44(m,1H),4.44–4.36(m,1H),4.24(qd,J=15.4,5.8Hz,2H),4.10(dd,J=14.0,4.4Hz,1H),3.76–3.43(m,3H).13CNMR(150MHz,DMSO-d6)δ170.48,164.84,159.90,152.99,149.71,141.21,136.45,133.07,130.95,127.45,127.41,126.86,125.33,125.10,124.80,122.27,119.77,115.39,114.31,58.56,46.91,45.65,43.25,37.71.ESI-MS:m/z 553.2[M+Na]+.C24H20Cl2N4O2S2(530.04).
操作同上,所不同的是使用3-二羟硼基-5-硝基苯甲酸。
产物为白色固体,收率:42%,熔点105-110℃。
1H NMR(600MHz,DMSO-d6)δ8.76(d,J=76.0Hz,1H),8.68–8.43(m,2H),8.32–7.97(m,2H),7.47–7.16(m,2H),7.03–6.62(m,4H),4.49(d,J=33.4Hz,2H),4.18(d,J=74.0Hz,2H),3.85(s,1H),3.68–3.49(m,3H),3.10(s,1H).13C NMR(150MHz,DMSO-d6)δ173.33,166.98,152.90,149.07,147.50,141.55,136.27,131.70,130.87,129.13,127.39,126.93,125.54,125.21,121.72,119.36,115.58,113.61,62.83,53.97,42.29,40.62,37.90,18.49.ESI-MS:m/z 563.1[M+H]+.C23H21BCl2N4O6S(562.07).
操作同上,所不同的是使用二氯乙酸。
产物为白色固体,收率:66%,熔点186-188℃。
1H NMR(600MHz,DMSO-d6)δ8.77(dt,J=24.6,6.0Hz,1H),7.39(dd,J=9.0,2.2Hz,1H),7.35(ddd,J=6.5,5.0,1.3Hz,1H),7.13(d,J=80.5Hz,1H),6.98(dd,J=20.2,3.0Hz,1H),6.91(dt,J=5.2,3.0Hz,1H),6.89(s,1H),6.78(ddd,J=18.4,9.0,3.0Hz,1H),4.51–4.44(m,1H),4.42–4.37(m,2H),4.38–4.22(m,1H),3.97–3.87(m,1H),3.66–3.57(m,2H),3.48(dd,J=13.7,4.8Hz,1H),3.42–3.33(m,1H).13C NMR(150MHz,DMSO-d6)δ170.81,161.70,150.64,143.21,132.03,130.90,127.97,125.71,125.53,121.15,115.46,113.87,66.52,58.21,45.97,44.06,42.74,37.35.ESI-MS:m/z 480.0[M+H]+.C18H17Cl4N3O2S(478.98).
操作同上,所不同的是使用二氟一氯乙酸。
产物为白色固体,收率:70%,熔点118-120℃。
1H NMR(600MHz,DMSO-d6)δ8.86(dt,J=44.9,5.9Hz,1H),7.42(d,J=2.1Hz,2H),7.01–6.86(m,3H),6.77(ddd,J=29.5,9.1,3.0Hz,1H),4.56–4.41(m,2H),4.38–4.34(m,1H),3.99–3.93(m,1H),3.69–3.54(m,3H),3.54–3.35(m,1H).13C NMR(150MHz,DMSO-d6)δ169.72,159.72,153.29,149.33,142.56,132.83,130.97,127.48,125.83,125.52,124.54,115.65,114.54,58.40,51.83,44.77,43.49,37.83.ESI-MS:m/z 480.2[M-H]-.C18H16Cl3F2N3O2S(481.00).
操作同上,所不同的是使用噻唑-2-甲酸。
产物为乳白色固体,收率:79%,熔点118-120℃。
1H NMR(600MHz,DMSO-d6)δ8.81(dt,J=50.5,5.8Hz,1H),8.07–7.95(m,2H),7.40(d,J=9.1Hz,1H),7.29(dd,J=13.5,5.0Hz,1H),6.96(d,J=3.0Hz,1H),6.82(ddd,J=57.3,8.9,3.2Hz,3H),4.78(d,J=117.1Hz,1H),4.44(d,J=14.9Hz,1H),4.37(d,J=25.5Hz,1H),4.26(qd,J=15.3,5.7Hz,1H),4.21–4.09(m,1H),4.08–3.98(m,1H),3.73–3.44(m,3H).13C NMR(150MHz,DMSO-d6)δ170.59,164.69,160.40,151.80,143.90,142.54,132.05,131.37,126.98,125.85,125.43,125.33,119.67,116.22,114.21,59.42,47.29,43.88,43.10,37.76.ESI-MS:m/z 481.0[M+H]+.C20H18Cl2N4O2S2(480.02).
操作同上,所不同的是使用一溴一氯乙酸。
产物为白色固体,收率:82%,熔点162-164℃。
1H NMR(600MHz,DMSO-d6)δ8.77(dq,J=29.6,6.1Hz,1H),7.39(d,J=7.3Hz,1H),7.38–7.32(m,1H),7.19–7.00(m,1H),6.99–6.93(m,1H),6.93–6.86(m,2H),6.83–6.73(m,1H),4.52–4.45(m,1H),4.45–4.34(m,2H),4.29–4.20(m,1H),3.98–3.92(m,1H),3.72–3.59(m,2H),3.56–3.37(m,3H).13C NMR(150MHz,DMSO-d6)δ170.24,163.04,152.64,150.20,144.81,133.75,130.89,127.06,125.47,119.81,115.81,113.90,58.58,52.38,46.26,44.48,43.27,37.38.ESI-MS:m/z 523.9[M+H]+.C18H17BrCl3N3O2S(522.93).
操作同上,所不同的是使用溴乙酸。
产物为白色固体,收率:69%,熔点152-154℃。
1H NMR(600MHz,DMSO-d6)δ8.90–8.74(m,1H),7.45–7.27(m,2H),7.02(dd,J=43.1,2.8Hz,1H),6.96–6.89(m,2H),6.88–6.71(m,1H),4.63–4.21(m,4H),4.19–3.77(m,3H),3.73–3.48(m,2H),3.48–3.36(m,1H),3.34–3.07(m,1H).13C NMR(150MHz,DMSO-d6)δ172.96,170.30,150.19,143.30,132.01,130.88,127.05,125.70,125.41,119.65,115.63,114.54,60.33,58.66,55.37,54.29,43.86,37.88.ESI-MS:m/z 490.0[M+H]+.C18H18BrCl2N3O2S(488.97).
操作同上,所不同的是使用二溴乙酸。
产物为白色固体,收率:81%,熔点158-160℃。
1H NMR(600MHz,DMSO-d6)δ8.76(dt,J=33.7,6.0Hz,1H),7.39(d,J=9.0Hz,1H),7.37–7.31(m,1H),7.09–6.97(m,1H),6.95(d,J=2.9Hz,1H),6.94–6.85(m,2H),6.77(ddd,J=20.0,9.1,3.0Hz,1H),4.51–4.39(m,3H),4.38–4.34(m,1H),3.98–3.86(m,1H),3.66–3.55(m,2H),3.50–3.42(m,1H).13C NMR(150MHz,DMSO-d6)δ170.73,164.92,150.19,142.74,132.02,130.88,127.71,125.69,125.52,125.28,119.75,115.41,113.51,58.42,55.72,44.12,38.35,37.94.ESI-MS:m/z 568.1[M+H]+.C18H17Br2Cl2N3O2S(566.88).
实施例3: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法(fluorescence resonance energy transfer,FRET),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,进而间接反映化合物的抑制活性(见附图2)(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-2 Mpro,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Mcule-5948770040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3。
复筛:选取1.5μM的SARS-CoV-2 Mpro,500μM的底物和四个浓度梯度(0.1μM,0.5μM,1μM,5μM)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 8进行IC50的计算。实验结果如表2所示。
表2.二氯苯基多取代哌嗪类目标化合物抑制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:
本发明新合成的二氯苯基多取代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GC-14、GC-16、GC-23和GC-32活性均优于先导化合物Mcule-5948770040和另一类已知的SARS-CoV-2主蛋白酶抑制剂ML188,其中GC-23的活性尤为突出(IC50=0.17±0.011μM),其抑制主蛋白酶的活性较先导化合物Mcule-5948770040提高了30倍;因此,二氯苯基多取代哌嗪类化合物具有进一步研发的价值。
Claims (8)
2.如权利要求1所述的二氯苯基多取代哌嗪类化合物,其特征在于,R为取代苯甲磺酰基、取代芳香杂环甲磺酰基、单取代苯甲酰基、多取代苯甲酰基、五元杂环甲酰基、六元取代杂环甲酰基、五元内酰胺甲酰基、嘧啶酮酰基、芳香氧代甲酰基、丙烯酰基、卤代乙酰基、氰乙酰基;所述的取代基选自甲基、卤代甲基、硝基、卤素、氰基或硼酸基;式I所示化合物为S构型。
5.如权利要求1-3所述的二氯苯基多取代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(S)-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;然后经氢氧化锂水解甲酯得到中间体4;随后将4溶解在适量的二氯甲烷中,在缩合剂HATU的作用下与噻吩-2-甲胺缩合得到关键中间体5;5再经三氟乙酸脱保护得中间体6;然后6再与取代磺酰氯反应或在HATU的作用下与各类羧酸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)氢氧化锂,甲醇,四氢呋喃,水,室温;(iii)噻吩-2-甲胺,HATU,N,N,-二异丙基乙胺,二氯甲烷,室温;(iv)三氟乙酸,二氯甲烷,室温;(v)各类磺酰氯化合物,N,N,-二异丙基乙胺,二氯甲烷,室温;(vi)各类羧酸化合物,HATU,N,N,-二异丙基乙胺,二氯甲烷,室温;
其中,R6如上述通式I(a)所述结构;R7如上述通式I(b)、I(c)、I(d)、I(e)所述结构。
6.权利要求1-4任一项所述的二氯苯基多取代哌嗪类化合物在制备抗SARS-CoV-2Mpro的药物中的应用。
7.一种抗冠状病毒药物组合物,包含权利要求1-4任一项所述二氯苯基多取代哌嗪类化合物和一种或多种药学上可接受载体或赋形剂。
8.如权利要求7所述的冠状病毒为SARS-CoV-2。
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