CN112457281B - 阻断covid-19棘突状蛋白与人血管紧张素转化酶2结合的小分子抑制剂及其用途 - Google Patents
阻断covid-19棘突状蛋白与人血管紧张素转化酶2结合的小分子抑制剂及其用途 Download PDFInfo
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Abstract
本发明属于生物与医药技术领域,涉及阻断COVID‑19棘突状蛋白与人血管紧张素转化酶2结合的小分子抑制剂及其用途。本发明通过虚拟筛选、阻断COVID‑19棘突状蛋白与人血管紧张素转化酶2结合的实验、抗病毒转录实验,发现了在0.5‑500μM范围内,对COVID‑19棘突状蛋白与人血管紧张素转化酶2的结合有抑制作用、阻断COVID‑19转入细胞的小分子抑制剂,可以用于制备防治新型冠状病毒感染的药物。
Description
技术领域
本发明属于生物与医药技术领域,涉及阻断COVID-19棘突状蛋白与人血管紧张素转化酶2结合的小分子抑制剂及其用途,具体的说,涉及高效特异性防治新型冠状病毒感染的化合物及其应用。
背景技术
新型冠状病毒肺炎,简称“新冠肺炎”,是一类急性传染性疾病,由2019新型冠状病毒(COVID-19)感染引起。人感染了新型冠状病毒以后,其主要症状包括呼吸道症状、发热、咳嗽、气促和呼吸困难等,在较严重病例中,感染可导致肺炎、严重急性呼吸综合征、肾衰竭,甚至死亡。目前针对新型冠状病毒引起的疾病,缺乏针对病原体的有效抗病毒药物。对广大易感人群,目前的防护产品如口罩、含酒精消毒用品等均为广谱式防护,市场上尚无高效、特异性阻隔病毒传染的防护用品。因此,应用现代科学技术,开发作用明确,具有高效、特异性防治新冠病毒感染的产品具有极为重要的现实意义和社会意义。
人冠状病毒棘突状蛋白(COVID-19Spike protein,S蛋白)是一类病毒融合蛋白,S蛋白结合宿主受体是病毒感染细胞的关键步骤,可作为抑制人冠状病毒的药物的靶点[Structural and functional properties of SARS-CoV-2spikeprotein: potentialantivirus drug development for COVID-19,Acta Pharmacologica Sinica,2020]。人新型冠状病毒COVID-19属于β冠状病毒,其S蛋白的S1亚基含有多个结构域A-D,其中结构域A和B组成受体结构域(Receptor binding domain,RBD),与人血管紧张素转化酶2(hACE2)产生相互作用。目前针对该靶点研究较多的是宿主中和抗体和疫苗设计,小分子抑制剂的报道较为稀少,并且大多数只停留在计算模拟层面的活性预测[Computational evaluationof major components from plant essential oils as potent inhibitors of SARS-CoV-2 spike protein,Journal of Molecular Structure,2020;Repurposing approveddrugs as inhibitors of SARS-CoV-2 S-protein from molecular modeling andvirtual screening, Journal of Biomolecular Structure and Dynamics,2020;Truncated human angiotensin converting enzyme 2;a potential inhibitor ofSARS-CoV-2 spike glycoprotein and potent COVID-19 therapeutic agent,Journalof Biomolecular Structure and Dynamics,2020;Targeting SARS-CoV-2spike proteinof COVID-19 with naturally occurring phytochemicals:an in silico study fordrug development, Journal of Biomolecular Structure and Dynamics,2020;Repurposing of the approved small molecule drugs in order to inhibit SARS-CoV-2 S protein and human ACE2 interaction through virtual screeningapproaches,Journal of Biomolecular Structure and Dynamics,2020]。
发明内容
本发明的目的在于提供阻断COVID-19棘突状蛋白与人血管紧张素转化酶2 结合的小分子抑制剂及其用途,为高效特异性防治新型冠状病毒感染提供新的策略。
第一方面,本发明提供了一种阻断COVID-19棘突状蛋白与人血管紧张素转化酶2结合的小分子抑制剂,所述的小分子抑制剂为木犀草素二磺化衍生物,其结构式如下式(I)所示:
其中,R1为SO3Na,R2为H;或者R1为H,R2为SO3Na。
第二方面,本发明提供了上述的如式(I)所示的阻断COVID-19棘突状蛋白与人血管紧张素转化酶2结合的小分子抑制剂的制备方法,其包括以下步骤:
1)将浓硫酸加入鸡心瓶中,冰水浴冷却至-10~10℃;
2)在搅拌下,向步骤1)中的浓硫酸中分批加入木犀草素粉末,木犀草素粉末与浓硫酸的配比为1.0g:1.0mL~1.0g:10mL,获得混合物;
3)将步骤2)获得的混合物升温,并在10℃-100℃下反应2h-72h,获得反应液;
4)将步骤3)获得的反应液移入无机盐的水溶液中进行盐析,无机盐溶液的浓度范围为0.5mol/L至该无机盐的饱和浓度,用量为反应液体积的1~10倍,无机盐为常用于盐析的氯化锂、氯化钠、硫酸铵、硫酸镁等,优选氯化钠,反应液与用于盐析的无机盐水溶液配比为体积比1:1~1:50,搅拌至固体完全析出;
5)在步骤4)中固体完全析出后,进行抽滤,抽滤后滤饼采用饱和的盐析水溶液洗涤,并抽滤至干燥,得到土黄色产物;
6)将步骤5)中获得的产物经蒸馏水溶解、调pH值至中性后,加入盐析用无机盐进行盐析、过滤;
7)将步骤6)中过滤后所得固体干燥、以C18反相色谱柱纯化后,采用含有甲醇和水的洗脱液洗脱后,得到木犀草素二磺化衍生物,其结构式如式(I) 所示:
其中,R1为SO3Na,R2为H;或者R1为H,R2为SO3Na。
第三方面,本发明提供了选自以下的化合物在制备用于治疗和/或预防新型冠状病毒感染所引起的疾病的试剂中的用途:木犀草素二磺化衍生物、白藜芦醇类似物、槲皮素二磺化衍生物、雨蛙素、阿卡波糖、阿利吉伦及其组合。
进一步地,所述的新型冠状病毒感染所引起的疾病为新型冠状病毒肺炎。
进一步地,所述的新型冠状病毒肺炎为哺乳动物中的新型冠状病毒肺炎;优选地,所述的哺乳动物为人。
进一步地,所述的试剂通过阻断COVID-19棘突状蛋白与人血管紧张素转化酶2结合以治疗和/或预防新型冠状病毒感染所引起的疾病。
进一步地,所述的预防新型冠状病毒感染所引起的疾病的试剂为洗手液、漱口水或鼻喷雾剂。
第四方面,本发明提供了一种药物组合物,其包含选自以下的化合物及药学上可接受的载体:木犀草素二磺化衍生物、白藜芦醇类似物、槲皮素二磺化衍生物、雨蛙素、阿卡波糖、阿利吉伦及其组合,所述的药物组合物用于治疗和/或预防新型冠状病毒感染所引起的疾病。
第五方面,本发明提供了一种治疗新型冠状病毒感染所引起的疾病的方法,其包括向受试者给予治疗有效量的选自以下的化合物:木犀草素二磺化衍生物、白藜芦醇类似物、槲皮素二磺化衍生物、雨蛙素、阿卡波糖、阿利吉伦及其组合。
第六方面,本发明提供了一种阻断COVID-19棘突状蛋白与人血管紧张素转化酶2结合的方法,其包括向受试者给予治疗有效量的选自以下的化合物:木犀草素二磺化衍生物、白藜芦醇类似物、槲皮素二磺化衍生物、雨蛙素、阿卡波糖、阿利吉伦及其组合。
本发明的效果和益处是:本发明通过虚拟筛选、阻断COVID-19棘突状蛋白与人血管紧张素转化酶2结合的实验、抗病毒转录实验,发现了在10-500μM 范围内,对COVID-19棘突状蛋白与人血管紧张素转化酶2的结合有抑制作用、阻断COVID-19转入细胞的小分子抑制剂,可以用于制备防治新型冠状病毒感染的药物。
附图说明
图1为100倍镜下100μM化合物DUT202001对荧光标记的COVID-19棘突状蛋白和表达荧光标记人血管紧张素转化酶2细胞结合的抑制情况。
图2A至图2C为基于新冠病毒S蛋白的类病毒细胞检测体系的结果图,其中图2A为化合物DUT202001的检测结果,图2B为化合物DUT202003的检测结果,图2C为化合物DUT202004的检测结果。
具体实施方式
以下结合附图,通过实施例进一步说明本发明,但不作为对本发明的限制。以下提供了本发明实施方案中所使用的具体材料及其来源。但是,应当理解的是,这些仅仅是示例性的,并不意图限制本发明,与如下试剂和仪器的类型、型号、品质、性质或功能相同或相似的材料均可以用于实施本发明。下述实施例中所使用的实验方法如无特殊说明,均为常规方法。下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
术语(定义)
为了更容易理解本公开,以下具体定义了某些技术和科学术语。除非在本文中另有明确定义,本文使用的所有其它技术和科学术语都具有本公开所属领域的一般技术人员通常理解的含义。
术语“抑制”或“阻断”可互换使用,并涵盖部分和完全抑制/阻断这两者。配体的抑制/阻断优选地降低或改变无抑制或阻断的情况下发生配体结合时出现活性的正常水平或类型。
“治疗”意指给予患者内用或外用治疗剂,诸如包含本发明的任一种化合物的组合物,所述患者具有一种或多种疾病症状,而已知所述治疗剂对这些症状具有治疗作用。通常,在受治疗患者或群体中以有效缓解一种或多种疾病症状的量给予治疗剂,无论是通过诱导这类症状退化还是抑制这类症状发展到任何临床测量的程度。有效缓解任何具体疾病症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化,例如患者的疾病状态、年龄和体重,以及药物在患者产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法,可评价疾病症状是否已被减轻。尽本发明的实施方案(例如治疗方法或制品)在缓解每个患都有的目标疾病症状方面可能无效,但是根据本领域已知的任何统计学检验方法如Student t 检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定,其在统计学显著数目的患者中应当减轻目标疾病症状。
“药物组合物”表示含有一种或多种本公开所述化合物或其生理学上/可药用的盐或前体药物与其他化学组分的混合物,所述其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药,利于活性成分的吸收进而发挥生物活性。
“有效量”、“有效剂量”是指获得任一种或多种有益的或所需的治疗结果所必需的药物、化合物或药物组合物的量。对于预防用途,有益的或所需的结果包括消除或降低风险、减轻严重性或延迟病症的发作,包括病症、其并发症和在病症的发展过程中呈现的中间病理表型的生物化学、组织学和/或行为症状。对于治疗应用,有益的或所需的结果包括临床结果,诸如减少各种本公开新型冠状病毒感染所引起的疾病的发病率或改善所述疾病的一个或多个症状,减少治疗病症所需的其它药剂的剂量,增强另一种药剂的疗效,和/或延缓患者的新型冠状病毒感染所引起的疾病的进展。
术语“药学上可接受的载体”指适合用于制剂中用于递送抗体或抗原结合片段的任何无活性物质。载体可以是抗粘附剂、粘合剂、包衣、崩解剂、充填剂或稀释剂、防腐剂(如抗氧化剂、抗菌剂或抗真菌剂)、增甜剂、吸收延迟剂、润湿剂、乳化剂、缓冲剂等。合适的药学上可接受的载体的实例包括水、乙醇、多元醇(例如甘油、丙二醇、聚乙二醇等)右旋糖、植物油(例如橄榄油)、盐水、缓冲液、缓冲的盐水和等渗剂例如糖、多元醇、山梨糖醇和氯化钠。
“有效量”、“有效剂量”是指获得任一种或多种有益的或所需的治疗结果所必需的药物、化合物或药物组合物的量。对于预防用途,有益的或所需的结果包括消除或降低风险、减轻严重性或延迟病症的发作,包括病症、其并发症和在病症的发展过程中呈现的中间病理表型的生物化学、组织学和/或行为症状。对于治疗应用,有益的或所需的结果包括临床结果,诸如减少各种本公开靶抗原相关病症的发病率或改善所述病症的一个或多个症状,减少治疗病症所需的其它药剂的剂量,增强另一种药剂的疗效,和/或延缓患者的本公开靶抗原相关病症的进展。
“给予”和“处理”当应用于动物、人、实验受试者、细胞、组织、器官或生物流体时,是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触。“给予”和“处理”可以指例如治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触,以及试剂与流体的接触,其中所述流体与细胞接触。“给予”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理例如细胞。“处理”当应用于人、兽医学或研究受试者时,是指治疗处理、预防或预防性措施,研究和诊断应用。
实施例1:化合物的初筛
以实验室自有145个分子数据库及1913个已上市小分子药物为筛选对象进行虚拟筛选。本实施例所用到的虚拟筛选软件为Discovery Studio。据虚拟筛选得分进行排序并结合分子结构特点选出有潜力的分子列表。在本实施例中选择以下表1所示化合物,其中,木犀草素二磺化衍生物MXCS-diSO3Na (DUT202001、DUT202002)由下述实施例2合成,白藜芦醇类似物 (DUT202003)、槲皮素二磺化衍生物(DUT202004)为实验室自有,雨蛙素DUT202005、阿卡波糖DUT202006、阿利吉伦(DUT202007)及实施例中所用木犀草素均为商业购买。
表1化合物信息
实施例2:DUT202001[2-(4,5-二羟基苯基)-5,7-二羟基-苯并-γ-吡喃酮 -3′,6-二磺酸钠(又名:木犀草素-3′,6-二磺酸钠)]和DUT202002[2-(4, 5-二羟基苯基)-5,7-二羟基-苯并-γ-吡喃酮-3′,8-二磺酸钠(又名:木犀草素-3′, 8-二磺酸钠)]的合成
量取4mL浓硫酸于50mL鸡心瓶中,冰水浴冷却至0℃,在搅拌下,分批加入1.0g木犀草素粉末,加料完毕后升温,在30℃下反应72h后,将反应液移入50mL饱和氯化钠水溶液搅拌,有大量固体析出,静置待固体完全析出,减压抽滤,滤饼用饱和氯化钠溶液(5mL×3)洗涤,抽滤至干,得土黄色产物。产物加 40mL去离子水溶解,碳酸氢钠调pH值=6~7,加2.0g氯化钠盐析,过滤析出的固体、干燥、以C18反相色谱柱纯化,洗脱液甲醇:水(1:5),得0.6g目标产物DUT202001,即2-(4,5-二羟基苯基)-5,7-二羟基-苯并-γ-吡喃酮-3′,6- 二磺酸钠(又名:木犀草素-3′,6-二磺酸钠),产率35%;得0.15g目标产物 DUT202002,即2-(4,5-二羟基苯基)-5,7-二羟基-苯并-γ-吡喃酮-3′,6-二磺酸钠(又名:木犀草素-3′,6-二磺酸钠),产率8.7%。
DUT202001:1H NMR(600MHz,DMSO-d6)δ:6.66(1H,s,H-3),6.48(1H,s, H-8),7.61(1H,d,J=2.0Hz,H-2′),7.40(1H,d,J=2.0Hz,H-6′);13C NMR(150MHz, DMSO-d6)δ:163.6(C-2),103.5(C-3),182.2(C-4),160.1(C-5),114.5(C-6),160.9(C-7), 94.6(C-8),157.1(C-9),103.4(C-10),120.6(C-1′),116.5(C-2′),131.7(C-3′), 146.5(C-4′),146.6(C-5′),114.3(C-6′);目标分子酸式质谱数据:m/z 222[M-2H]2-, 467[M-2H+Na]-。
DUT202002:1H NMR(600MHz,DMSO-d6)δ:6.71(1H,s,H-3),6.18(1H,s, H-6),7.79(1H,s,H-2′),7.69(1H,s,H-6′);13C NMR(150MHz,DMSO-d6)δ: 164.2(C-2),103.2(C-3),181.9(C-4),161.7(C-5),99.1(C-6),160.9(C-7),110.5(C-8), 154.3(C-9),104.0(C-10),120.8(C-1′),115.9(C-2′),131.7(C-3′),146.3(C-4′), 146.4(C-5′),116.9(C-6′);目标分子酸式质谱数据:m/z 222[M-2H]2-, 467[M-2H+Na]-。
实施例3:阻断COVID-19棘突状蛋白的受体结合域(RBD)与人血管紧张素转化酶2(hACE2)结合的活性测试
实验材料:化合物DUT202001和DUT202002(实施例2中合成)、DUT202003 和DUT202004(实验室自有);化合物DUT202005、DUT202006和DUT202007 (购自上海阿拉丁生化科技股份有限公司);SARS-CoV-2S、pAX2、pHB和 ACE2-pcDNA3(中科普瑞昇科技有限公司);293T细胞(上海合源生物科技有限公司)。pCMV-SNAP-hACE2质粒、SNAP-561荧光染料和RBD541-Halo-640dye 来自于中国科学院大连化学物理研究所徐兆超研究组。
具体地,测试步骤如下:
1)选取实施例1中虚拟筛选归一化FitValue打分值为0.3966的木犀草素二磺化衍生物MXCS-diSO3Na(DUT202001)、归一化FitValue打分值为0.446的木犀草素二磺化衍生物MXCS-diSO3Na(DUT202002)、归一化FitValue打分值为0.400的白藜芦醇类似物(DUT202003)、归一化FitValue打分值为0.4240的槲皮素二磺化衍生物(DUT202004)、归一化FitValue打分值为0.4898的雨蛙素 (DUT202005)、归一化FitValue打分值为0.4881的阿卡波糖(DUT202006)、归一化FitValue打分值为0.4460的阿利吉伦(DUT202007),以各自适宜浓度为起始浓度;
2)阻断COVID-19棘突状蛋白的受体结合域(RBD)与人血管紧张素转化酶2(hACE2)结合的实验
将Hela细胞传于2个共聚焦成像皿,24小时后用Lipo2000试剂瞬时转入 pCMV-SNAP-hACE2质粒,37℃ 5%CO2培养箱中培养48小时。将SNAP-561 荧光染料溶于DMEM高糖培养基中,终浓度为0.2μM。用该探针溶液孵育细胞 10min,用DMEM一遍。对照组加入只含有20nM RBD541-Halo-640dye的1mL DMEM培养基,实验组加入同时含有120μM待测药物分子和20nM RBD541-Halo-640dye的1mL DMEM培养基。在37℃ 5%CO2培养箱中孵育60 min。用荧光共聚焦显微镜在10倍镜下成像,如图1所示。
图1中第一行为640nm激发的RBD541-Halo蛋白成像通道,第二行为561 nm激发的SNAP-ACE2蛋白的成像通道,其中,图1中的(a)为只加入20nM RBD541-Halo-640dye的细胞成像;图1中的(b)为同时加入20nM RBD541-Halo-640dye和120μM待测药物分子DUT202001的细胞成像,其红色通道的细胞荧光微弱,说明该分子有抑制SARS-CoV-2的RBD与hACE2结合的作用。
另外,我们统计了20个细胞的平均荧光强度和以及成像图片背景的平均荧光强度得到和 以图1 中的(a)的IF640/IF561作为100%的RBD相对活性(RA),图1中的(b)与图1中的(a)作对比,得到120μM该分子能够抑制85%的RBD活性。
所选取的化合物测试结果见表2。
表2抑制COVID-19棘突状蛋白类与人血管紧张素转化酶2结合的结果
按照上述实验方法,以上所选取的化合物的活性检测结果如表2和图1所示,候选化合物对COVID-19棘突状蛋白RBD与表达hACE2的Hela细胞具有不同程度的抑制效果,尤其化合物DUT202001在7.6μM的浓度时,达到了51.8%的抑制效果。
实施例4:基于荧光素酶报告体系为基础的针对新冠病毒S蛋白的类病毒细胞检测体系进行评价
本实施例中,新冠病毒S蛋白的类病毒细胞检测由合肥中科普瑞昇生物医药科技有限公司完成。简要地,包括以下步骤:
1)新冠病毒S蛋白的类病毒制备及收集:假病毒用三种假病毒质粒 SARS-CoV-2S、pAX2和pHB瞬时转染293T细胞,S蛋白作为包膜蛋白,荧光素酶基因被包装在病毒内部。6-8h后更换新鲜培养基,37℃,5%CO2培养48h。从60mm培养皿中收集病毒上清液,病毒上清液用0.45um滤膜过滤,立即使用病毒或在-80℃保存。
2)表面表达hACE2的293T细胞的制备及种板:用ACE2-pcDNA3细胞质粒转染瞬时转染293T细胞,6-8h后更换新鲜培养基,37℃,5%CO2培养48h。 48h后,将细胞旋转至生长培养基中,然后用细胞计数器计数,将细胞悬浮液稀释至所需密度,取50μl细胞悬液至96孔板,每孔的细胞数为25000个。37℃、 5%CO2培养12h,培养12h后细胞完全贴壁于96孔板上。
3)加药孵育:根据实验设计,在96孔板上每孔加入10μL配制好浓度的待测化合物溶液,37℃,5%CO2孵育1h。
4)加假病毒:96孔板上每孔加40μL病毒上清液。37℃,5%CO2培养24h。 24h后更换新鲜培养基,100μL/孔。37℃,5%CO2培养24h。
5)测量:测量前将孔板平衡至室温,每孔加入30uL肾素荧光素酶试剂;在摇床上混合2分钟,诱导细胞溶解;在室温下孵育5分钟以稳定发光信号;记录酶标仪上的荧光数据。
使用以下公式计算与DMSO处理的对照与加药的孔之间的相对荧光强度 (%):
相对荧光强度(%)=加药孔荧光值/对照孔荧光值*100%
化合物对于COVID-19S蛋白假病毒侵染细胞的抑制率为100%-相对荧光强度(%)。
表3抑制COVID-19S蛋白类病毒侵染实验分组及测试浓度
表4抑制COVID-19S蛋白类病毒侵染结果
按照上述实验方法,以上所选取的化合物的活性检测结果如表4和图2A至图2C所示,候选化合物对COVID-19S蛋白假病毒侵染表达hACE2的293T细胞具有良好的抑制效果,在化合物的浓度大于250μM时,对于假病毒侵染的抑制率可达到75%以上。
以上示例性实施方式所呈现的描述仅用以说明本发明的技术方案,并不想要成为毫无遗漏的,也不想要把本发明限制为所描述的精确形式。显然,本领域的普通技术人员根据上述教导做出很多改变和变化都是可能的。选择示例性实施方式并进行描述是为了解释本发明的特定原理及其实际应用,从而使得本领域的其它技术人员便于理解、实现并利用本发明的各种示例性实施方式及其各种选择形式和修改形式。本发明的保护范围意在由所附权利要求书及其等效形式所限定。
Claims (9)
2.如权利要求1所述的小分子抑制剂的制备方法,其特征在于,所述的制备方法包括以下步骤:
1)将浓硫酸加入鸡心瓶中,冷却至-10~10℃;
2)在搅拌下,向步骤1)中的浓硫酸中分批加入木犀草素粉末,木犀草素粉末与浓硫酸的配比为1.0g:1.0mL~1.0g:10mL,获得混合物;
3)将步骤2)获得的混合物升温,并在10℃-100℃下反应2h-72h,获得反应液;
4)将步骤3)获得的反应液移入无机盐的水溶液中进行盐析,无机盐溶液的浓度范围为0.5mol/L至该无机盐的饱和浓度,用量为反应液体积的1~10倍,无机盐为常用于盐析的氯化锂、氯化钠、硫酸铵、硫酸镁,反应液与用于盐析的无机盐水溶液配比为体积比1:1~1:50,搅拌至固体完全析出;
5)在步骤4)中固体完全析出后,进行抽滤,抽滤后滤饼采用饱和盐析水溶液洗涤,并抽滤至干燥,得到土黄色产物;
6)将步骤5)中获得的产物经蒸馏水溶解、调pH值至中性后,加入盐析用的无机盐进行盐析、过滤;
7)将步骤6)中过滤后所得固体干燥、以C18反相色谱柱纯化后,采用含有甲醇和水的洗脱液洗脱后,得到所述的小分子抑制剂。
3.如权利要求2所述的制备方法,所述的无机盐为常用于盐析的氯化钠。
4.如权利要求1所述的小分子抑制剂在制备用于治疗和/或预防新型冠状病毒感染所引起的疾病的试剂中的用途,所述的试剂通过阻断COVID-19棘突状蛋白与人血管紧张素转化酶2结合以治疗和/或预防新型冠状病毒感染所引起的疾病。
5.根据权利要求4所述的用途,其中,所述的新型冠状病毒感染所引起的疾病为新型冠状病毒肺炎。
6.根据权利要求5所述的用途,其中,所述的新型冠状病毒肺炎为哺乳动物中的新型冠状病毒肺炎。
7.根据权利要求6所述的用途,所述的哺乳动物为人。
8.根据权利要求4-7中任一项所述的用途,所述试剂为洗手液、漱口水或鼻喷雾剂。
9.一种药物组合物,其包含权利要求1所述的小分子抑制剂及药学上可接受的载体。
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