WO2023019614A1 - Use of p-benzoquinone and/or derivative thereof in preparation of anti novel-coronavirus drug - Google Patents

Use of p-benzoquinone and/or derivative thereof in preparation of anti novel-coronavirus drug Download PDF

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WO2023019614A1
WO2023019614A1 PCT/CN2021/114452 CN2021114452W WO2023019614A1 WO 2023019614 A1 WO2023019614 A1 WO 2023019614A1 CN 2021114452 W CN2021114452 W CN 2021114452W WO 2023019614 A1 WO2023019614 A1 WO 2023019614A1
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benzoquinone
phenyl
halogenated
alkyl
dichloro
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PCT/CN2021/114452
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French (fr)
Chinese (zh)
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颜世强
杨杰
何淑旺
王文笙
程中伟
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山东达因海洋生物制药股份有限公司
北京达因高科儿童药物研究院有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/275—Nitriles; Isonitriles
    • A61K31/277—Nitriles; Isonitriles having a ring, e.g. verapamil
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to the technical field of biomedicine, in particular to the application of p-benzoquinone and/or p-benzoquinone derivatives in the preparation of anti-new coronavirus drugs.
  • Quinone compounds are a class of chemical components with a quinone structure, mainly divided into four types: benzoquinone, naphthoquinone, phenanthrenequinone and anthraquinone.
  • Benzoquinones are divided into two categories: o-benzoquinone and p-benzoquinone.
  • the structure of o-benzoquinone is unstable, so most of the naturally occurring benzoquinones are derivatives of p-benzoquinone.
  • ubiquinone (coenzyme Q class), which exists widely, belongs to p-benzoquinone derivatives, which participate in redox reactions in the body.
  • coenzyme Q10 can be used to treat hypertension, Heart disease and cancer.
  • idebenone which was developed and marketed by Takeda Pharmaceutical Co., Ltd.
  • the object of the present invention is to provide the application of p-benzoquinone and/or p-benzoquinone derivatives in the preparation of anti-new coronavirus drugs, p-benzoquinone and/or p-benzoquinone derivatives can effectively inhibit the 3C-like protease ( 3C-like protease, 3CLP) and papain-like cysteine protease (papain-like cysteine protease, PLP) activity can be used to prepare anti-new coronavirus drugs.
  • 3C-like protease 3C-like protease, 3CLP
  • papain-like cysteine protease papain-like cysteine protease, PLP
  • the invention provides the application of p-benzoquinone and/or p-benzoquinone derivatives in the preparation of anti-new coronavirus drugs.
  • the invention provides the application of p-benzoquinone and/or p-benzoquinone derivatives in the preparation of drugs for inhibiting the proliferation of novel coronavirus.
  • the invention provides the application of p-benzoquinone and/or p-benzoquinone derivatives in the preparation of enzyme inhibitors for novel coronavirus.
  • the present invention provides the application of p-benzoquinone and/or p-benzoquinone derivatives in the preparation of 3C-like protease and/or papain-like cysteine protease inhibitors of novel coronavirus.
  • the R is selected from hydrogen, halogen, hydroxyl, cyano, nitro, C1-12 alkyl, C1-12 alkoxy, phenyl, halogenated C1-6 alkyl, halogenated C1-6 alkane Oxygen, C3-6 cycloalkyl, mono-substituted phenyl or multi-substituted phenyl;
  • the R is selected from hydrogen, halogen, hydroxyl, cyano, nitro, C1-12 alkyl, C1-12 alkoxy, phenyl, halogenated C1-6 alkyl, halogenated C1-6 alkoxy , C3-6 cycloalkyl, mono-substituted phenyl or multi-substituted phenyl;
  • the R is selected from hydrogen, halogen, hydroxyl, cyano, nitro, C1-12 alkyl, C1-12 alkoxy, phenyl, halogenated C1-6 alkyl, halogenated C1-6 alkoxy , C3-6 cycloalkyl, mono-substituted phenyl or multi-substituted phenyl;
  • the R is selected from hydrogen, halogen, hydroxyl, cyano, nitro, C1-12 alkyl, C1-12 alkoxy, phenyl, halogenated C1-6 alkyl, halogenated C1-6 alkoxy , C3-6 cycloalkyl, mono-substituted phenyl or multi-substituted phenyl.
  • the p-benzoquinone derivatives include 2-methyl-1,4-p-benzoquinone, 2-tert-butyl-1,4-p-benzoquinone, 2-phenyl-1,4-p-benzoquinone , 2-chloro-1,4-p-benzoquinone, 2-bromo-1,4-p-benzoquinone, thymoquinone, 2,5-dimethyl-1,4-p-benzoquinone, 2,6-di Methyl-1,4-p-benzoquinone, 2,6-dimethoxy-1,4-p-benzoquinone, 2,5-diphenyl-1,4-p-benzoquinone, 2-chloro-5- Methyl-1,4-p-benzoquinone, 2,5-dichloro-1,4-p-benzoquinone, 2,6-dichloro-1,4-p-benzoquinone, tetrafluoro-1,4-
  • the p-benzoquinone derivatives include 2-methyl-1,4-p-benzoquinone, 2-phenyl-1,4-p-benzoquinone, 2,6-dichloro-1,4-p-benzoquinone One or more of quinone, tetrafluoro-1,4-p-benzoquinone and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone.
  • the p-benzoquinone derivatives include 2-methyl-1,4-p-benzoquinone and/or 2-phenyl-1,4-p-benzoquinone.
  • the invention provides the application of p-benzoquinone and/or p-benzoquinone derivatives in the preparation of anti-new coronavirus drugs.
  • P-benzoquinone and/or p-benzoquinone derivatives can inhibit the activity of 3CLP and PLP of the new coronavirus, thereby inhibiting the activity and proliferation of the new coronavirus, alleviating the immune imbalance in the infected host cells, and then achieving the effect of treating the new coronavirus .
  • the invention provides the application of p-benzoquinone and/or p-benzoquinone derivatives in the preparation of anti-new coronavirus drugs.
  • the present invention also provides the application of p-benzoquinone and/or p-benzoquinone derivatives in the preparation of drugs for inhibiting the proliferation of novel coronavirus.
  • the present invention also provides the application of p-benzoquinone and/or p-benzoquinone derivatives in the preparation of novel coronavirus enzyme inhibitors.
  • the present invention also provides the application of p-benzoquinone and/or p-benzoquinone derivatives in the preparation of 3C-like protease and/or papain-like cysteine protease inhibitors of novel coronavirus.
  • the R is selected from hydrogen, halogen, hydroxyl, cyano, nitro, C1-12 alkyl, C1-12 alkoxy, phenyl, halogenated C1-6 alkyl, halogenated C1-6 Alkoxy, C3-6 cycloalkyl, single-substituted phenyl or multi-substituted phenyl; said R2 is selected from hydrogen, halogen, hydroxyl, cyano, nitro, C1-12 alkyl, C1-12 alkoxy Base, phenyl, halogenated C1-6 alkyl, halogenated C1-6 alkoxy, C3-6 cycloalkyl, monosubstituted phenyl or polysubstituted phenyl; the R3 is selected from hydrogen, halogen, hydroxyl , cyano, nitro, C1-12 alkyl, C1-12 alkoxy, phenyl, halogenated C1-6 alkyl, halogenated C1-6 alkoxy,
  • the p-benzoquinone derivatives preferably include 2-methyl-1,4-p-benzoquinone, 2-tert-butyl-1,4-p-benzoquinone, 2-phenyl-1,4 -p-benzoquinone, 2-chloro-1,4-p-benzoquinone, 2-bromo-1,4-p-benzoquinone, thymoquinone, 2,5-dimethyl-1,4-p-benzoquinone, 2 ,6-Dimethyl-1,4-p-benzoquinone, 2,6-dimethoxy-1,4-p-benzoquinone, 2,5-diphenyl-1,4-p-benzoquinone, 2- Chloro-5-methyl-1,4-p-benzoquinone, 2,5-dichloro-1,4-p-benzoquinone, 2,6-dichloro-1,4-p-benzoquinone, tetrafluoro-1, 4-p-
  • the present invention is to described p-benzoquinone (1,4-p-benzoquinone), 2-methyl-1,4-p-benzoquinone, 2-tert-butyl-1,4-p-benzoquinone, 2-phenyl- 1,4-p-benzoquinone, 2-chloro-1,4-p-benzoquinone, 2-bromo-1,4-p-benzoquinone, thymoquinone, 2,5-dimethyl-1,4-p-benzoquinone Quinone, 2,6-dimethyl-1,4-p-benzoquinone, 2,6-dimethoxy-1,4-p-benzoquinone, 2,5-diphenyl-1,4-p-benzoquinone , 2-chloro-5-methyl-1,4-p-benzoquinone, 2,5-dichloro-1,4-p-benzoquinone, 2,6-dichloro-1,4-p-benzoquinone, tetra
  • Polyproteins (polyproteins, PP) pp1a and pp1b are essential for maintaining the synthesis of coronavirus RNA, and the maturation of polyproteins requires a series of cleavage processes to generate multi-subunit protein complexes (called “viral replication”). Transcriptase”).
  • the main protease (main protease, Mpro) of the multi-subunit protein complex is 3C-like protease (3C-like protease, 3CLP) and papain-like cysteine protease (papain-like cysteine protease, PLP), 3CLP and PLP are highly coordinated Catalytic polyprotein cleavage, loss of 3CLP and PLP activity leads to arrest of the coronavirus life cycle.
  • the p-benzoquinone and/or p-benzoquinone derivatives can inhibit the activity of 3CLP and PLP of the new coronavirus, thereby inhibiting the activity and proliferation of the new coronavirus, and alleviating the immune imbalance in infected host cells.
  • the benzoquinone derivatives exhibiting inhibitory activity against 3CLP and PLP depend on their 1,4-p-benzoquinone core.
  • Design gene-specific primers (3CL-F: gtgccgcgcggcagccatatgtcggcagtgctgcaaagcggtttccgtaagatg, SEQ ID NO: 1; 3CL-R: gtggtggtggtggtggtggtggtgctagggga , SEQ ID NO: 2), wherein the 3CLPro-F primer sequence has an NdeI restriction site, and the 3CLPro-R primer sequence has an XhoI restriction site.
  • the novel coronavirus 3CLPro gene with E. coli codon preference was synthesized by General Bio.
  • the total amplification system was 100 ⁇ L.
  • the PCR reaction system includes: 1 ⁇ L of cDNA template, 4 ⁇ L of 3CL-R, 4 ⁇ L of 3CL-F, 50 ⁇ L of 2 ⁇ Phanta Master Mix and 41 ⁇ L of ddH 2 O.
  • the specific reaction program is: pre-denaturation at 94°C for 3 minutes, 30 amplification reactions (15s at 94°C, 30s at 65°C, 30s at 72°C) and full extension at 72°C for 5 minutes.
  • pET28a(+) was digested with NdeI and XhoI restriction enzymes, and then the target plasmid fragment was recovered by gel for recombination connection.
  • the specific connection system includes: pET28a(+) plasmid fragment 20 ⁇ g, 3CLPro target gene 40 ⁇ g, 5 ⁇ CE II Buffer 5 ⁇ L, Exnase II 2 ⁇ L, and water to make up 50 ⁇ L.
  • the successfully transformed Escherichia coli strains were pre-cultured overnight in 25 mL of LB liquid medium containing kanamycin (50 ⁇ g/mL) at 37 °C at a 1% (v/v) inoculum size, and then the culture was 1% (v/v) inoculum was inoculated in 1 L of LB liquid medium containing kanamycin (50 ⁇ g/mL) and cultured at 37°C.
  • OD600 reached 0.8
  • IPTG isopropyl-D-thiogalactoside
  • Design gene-specific primers (PLP-F: GTGCCGCGCGCGGCAGCCATATGTCGGCAGTGCTGCAA, SEQ ID NO: 3; PLP-R: GTGCCGCGCGCGGCAGCCATATGTCGGCAGTGCTGCAAGCGGTTCGTACCATCAAG, SEQ ID NO: 4) according to the gene sequence of the new coronavirus PLPro, wherein the PLP-F primer sequence contains NdeI Restriction site, the PLP-R primer sequence has an EcoRI restriction site. Similarly, 5 amino acids of SAVLQ were added to the N-terminus of the protein to construct a 3CLPro self-cleavage site; a stop codon was added to the C-terminus of the protein for later termination of translation.
  • the novel coronavirus PLPro gene with E. coli codon preference was synthesized by Universal Biotechnology.
  • the total amplification system was 100 ⁇ L.
  • the PCR reaction system includes: 1 ⁇ L of cDNA template, 4 ⁇ L of 3CL-R, 4 ⁇ L of 3CL-F, 50 ⁇ L of 2 ⁇ Phanta Master Mix and 41 ⁇ L of ddH 2 O.
  • the specific reaction program is: pre-denaturation at 94° C. for 3 minutes, 30 amplification reactions (94° C. for 15 s, 65° C. for 30 s, and 72° C. for 30 s) and full extension at 72° C. for 5 minutes.
  • the specific connection system includes: 20 ⁇ g of pET28a(+) plasmid fragment, 40 ⁇ g of PLPro target gene, 5 ⁇ L of 5 ⁇ CE II Buffer, 2 ⁇ L of Exnase II, and make up 50 ⁇ L with water.
  • the p-benzoquinone (1,4-p-benzoquinone), 2-methyl-1,4-p-benzoquinone, 2-tert-butyl-1,4-p-benzoquinone, 2-benzoquinone Base-1,4-p-benzoquinone, 2-chloro-1,4-p-benzoquinone, 2-bromo-1,4-p-benzoquinone, thymoquinone, 2,5-dimethyl-1,4- p-benzoquinone, 2,6-dimethyl-1,4-p-benzoquinone, 2,6-dimethoxy-1,4-p-benzoquinone, 2,6-di-tert-butyl-1,4- p-benzoquinone, 2,5-diphenyl-1,4-p-benzoquinone, 2-chloro-5-methyl-1,4-p-benzoquinone, 2,5-dichloro-1,4-p-benzoquinon
  • an emission resonance energy transfer fluorescence spectrophotometer uses an emission resonance energy transfer fluorescence spectrophotometer to detect the Dabcyl fluorescence signal (340nm (excitation)/490nm (emission)) due to the cleavage of the substrate catalyzed by 3CLPro, and determine whether the compound has inhibitory activity by changing the fluorescence signal within 3 minutes .
  • IC50 for compounds with high inhibitory activity screened out through primary screening.
  • the compound was diluted to gradient concentrations (1.5625-100 ⁇ M and 0.15625-10 ⁇ M) using Tris-HCl buffer, and assayed using the same final concentration of the novel coronavirus 3CLPro and fluorescent substrate system above.
  • the IC50 values of compounds were calculated by GraphPad Prism 8.0 software. Each experiment was performed in triplicate, and the final results were expressed as mean ⁇ standard deviation (SD). See Table 3 for the measurement results.
  • Fluorescence resonance energy transfer was used to measure the PLpro activity of the new coronavirus and the inhibitory activity of the compound on the PLpro of the new coronavirus.
  • the assay uses a fluorogenic substrate (CBE-RLRGG ⁇ AMC) with a protein-specific cleavage site (arrow) to initiate the reaction. Proteins and fluorescent substrates were diluted with PLpro buffer (150mM NaCl, 40mM Tris-HCl, 5mM DTT, pH 8.0).
  • 2,6-di-tert-butyl-1,4-p-benzoquinone, idebenone and tetramethyl-1,4-p-benzoquinone can promote the enzymatic activity of 2019-nCoV PLPro.
  • the IC50 values of compounds were calculated by GraphPad Prism 8.0 software. Each experiment was performed in triplicate, and the final results were expressed as mean ⁇ standard deviation (SD). See Table 3 for the measurement results. It can be seen from Table 3 that the compound 2-methyl-1,4-p-benzoquinone and 2-phenyl-1,4-p-benzoquinone have an IC50 value of less than 1.0 ⁇ M for the new coronavirus PLPro, showing that the new Excellent inhibitory activity of coronavirus PLPro.

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Abstract

Use of p-benzoquinone and/or a derivative thereof in the preparation of an anti novel-coronavirus drug, which belongs to the technical field of biomedicine. The p-benzoquinone and/or the derivative thereof can inhibit the activity of 3C-like protease (3CLP) and/or papain-like cysteine protease (PLP) of a novel coronavirus, thereby inhibiting the activity and proliferation of a novel coronavirus and alleviating the immune imbalance in infected host cells, so that the effect of treating a novel coronavirus is achieved.

Description

对苯醌和/或对苯醌衍生物在制备抗新型冠状病毒药物中的应用Application of p-benzoquinone and/or p-benzoquinone derivatives in the preparation of anti-new coronavirus drugs 技术领域technical field
本发明涉及生物医药技术领域，尤其涉及对苯醌和/或对苯醌衍生物在制备抗新型冠状病毒药物中的应用。The invention relates to the technical field of biomedicine, in particular to the application of p-benzoquinone and/or p-benzoquinone derivatives in the preparation of anti-new coronavirus drugs.
背景技术Background technique
醌类化合物是一类具有醌式结构的化学成分，主要分为苯醌、萘醌、菲醌和蒽醌四种类型。苯醌类(benzoquinones)化合物分为邻苯醌和对苯醌两大类，邻苯醌结构不稳定，故天然存在的苯醌化合物多数为对苯醌的衍生物。Quinone compounds are a class of chemical components with a quinone structure, mainly divided into four types: benzoquinone, naphthoquinone, phenanthrenequinone and anthraquinone. Benzoquinones are divided into two categories: o-benzoquinone and p-benzoquinone. The structure of o-benzoquinone is unstable, so most of the naturally occurring benzoquinones are derivatives of p-benzoquinone.
现有技术中公开了苯醌类化合物的多种生物活性，例如广泛存在的泛醌(辅酶Q类)属于对苯醌衍生物，其参与体内氧化还原反应，其中辅酶Q10可用于治疗高血压、心脏病及癌症。再比如，1986年由日本武田药品工业株式会社开发上市的艾地苯醌，亦属于对苯醌衍生物，其对线粒体功能有激活作用；对脑功能代谢和脑功能障碍有改善作用；能提高脑内葡萄糖的利用率，促进ATP生成；能改善脑内神经递质5-羟色胺的代谢，具有较强抗氧化和清除自由基作用。Various biological activities of benzoquinone compounds have been disclosed in the prior art. For example, ubiquinone (coenzyme Q class), which exists widely, belongs to p-benzoquinone derivatives, which participate in redox reactions in the body. Among them, coenzyme Q10 can be used to treat hypertension, Heart disease and cancer. For another example, idebenone, which was developed and marketed by Takeda Pharmaceutical Co., Ltd. in Japan in 1986, also belongs to p-benzoquinone derivatives, which can activate mitochondrial function; improve brain function metabolism and brain dysfunction; The utilization rate of glucose in the brain can promote the generation of ATP; it can improve the metabolism of the neurotransmitter 5-hydroxytryptamine in the brain, and has strong anti-oxidation and free radical scavenging effects.
现有技术中还没有将苯醌类化合物用于抗新型冠状病毒的报道。In the prior art, there is no report that benzoquinone compounds are used for anti-new coronavirus.
发明内容Contents of the invention
本发明的目的在于提供对苯醌和/或对苯醌衍生物在制备抗新型冠状病毒药物中的应用，对苯醌和/或对苯醌衍生物能够有效抑制新型冠状病毒的3C样蛋白酶(3C-like protease，3CLP)和木瓜样半胱氨酸蛋白酶(papain-like cysteine protease，PLP)的活性，能够用于制备抗新型冠状病毒药物。The object of the present invention is to provide the application of p-benzoquinone and/or p-benzoquinone derivatives in the preparation of anti-new coronavirus drugs, p-benzoquinone and/or p-benzoquinone derivatives can effectively inhibit the 3C-like protease ( 3C-like protease, 3CLP) and papain-like cysteine protease (papain-like cysteine protease, PLP) activity can be used to prepare anti-new coronavirus drugs.
为了实现上述发明目的，本发明提供以下技术方案：In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:
本发明提供了对苯醌和/或对苯醌衍生物在制备抗新型冠状病毒药物中的应用。The invention provides the application of p-benzoquinone and/or p-benzoquinone derivatives in the preparation of anti-new coronavirus drugs.
本发明提供了对苯醌和/或对苯醌衍生物在制备抑制新型冠状病毒增殖药物中的应用。The invention provides the application of p-benzoquinone and/or p-benzoquinone derivatives in the preparation of drugs for inhibiting the proliferation of novel coronavirus.
本发明提供了对苯醌和/或对苯醌衍生物在制备新型冠状病毒的酶抑制剂中的应用。The invention provides the application of p-benzoquinone and/or p-benzoquinone derivatives in the preparation of enzyme inhibitors for novel coronavirus.
本发明提供了对苯醌和/或对苯醌衍生物在制备新型冠状病毒的3C样蛋白酶和/或木瓜样半胱氨酸蛋白酶抑制剂中的应用。The present invention provides the application of p-benzoquinone and/or p-benzoquinone derivatives in the preparation of 3C-like protease and/or papain-like cysteine protease inhibitors of novel coronavirus.
优选的，所述对苯醌的衍生物的化学结构式如式I所示：Preferably, the chemical structural formula of the derivative of p-benzoquinone is as shown in formula I:
Figure PCTCN2021114452-appb-000001
Figure PCTCN2021114452-appb-000001
其中，所述R 1选自氢、卤素、羟基、氰基、硝基、C1-12烷基、C1-12烷氧基、苯基、卤代C1-6烷基、卤代C1-6烷氧基、C3-6环烷基、单取代苯基或多取代苯基； Wherein, the R is selected from hydrogen, halogen, hydroxyl, cyano, nitro, C1-12 alkyl, C1-12 alkoxy, phenyl, halogenated C1-6 alkyl, halogenated C1-6 alkane Oxygen, C3-6 cycloalkyl, mono-substituted phenyl or multi-substituted phenyl;
所述R 2选自氢、卤素、羟基、氰基、硝基、C1-12烷基、C1-12烷氧基、苯基、卤代C1-6烷基、卤代C1-6烷氧基、C3-6环烷基、单取代苯基或多取代苯基； The R is selected from hydrogen, halogen, hydroxyl, cyano, nitro, C1-12 alkyl, C1-12 alkoxy, phenyl, halogenated C1-6 alkyl, halogenated C1-6 alkoxy , C3-6 cycloalkyl, mono-substituted phenyl or multi-substituted phenyl;
所述R 3选自氢、卤素、羟基、氰基、硝基、C1-12烷基、C1-12烷氧基、苯基、卤代C1-6烷基、卤代C1-6烷氧基、C3-6环烷基、单取代苯基或多取代苯基； The R is selected from hydrogen, halogen, hydroxyl, cyano, nitro, C1-12 alkyl, C1-12 alkoxy, phenyl, halogenated C1-6 alkyl, halogenated C1-6 alkoxy , C3-6 cycloalkyl, mono-substituted phenyl or multi-substituted phenyl;
所述R 4选自氢、卤素、羟基、氰基、硝基、C1-12烷基、C1-12烷氧基、苯基、卤代C1-6烷基、卤代C1-6烷氧基、C3-6环烷基、单取代苯基或多取代苯基。 The R is selected from hydrogen, halogen, hydroxyl, cyano, nitro, C1-12 alkyl, C1-12 alkoxy, phenyl, halogenated C1-6 alkyl, halogenated C1-6 alkoxy , C3-6 cycloalkyl, mono-substituted phenyl or multi-substituted phenyl.
优选的，所述对苯醌衍生物包括2-甲基-1,4-对苯醌、2-叔丁基-1,4-对苯醌、2-苯基-1,4-对苯醌、2-氯-1,4-对苯醌、2-溴-1,4-对苯醌、百里醌、2,5-二甲基-1,4-对苯醌、2,6-二甲基-1,4-对苯醌、2,6-二甲氧基-1,4-对苯醌、2,5-二苯基-1,4-对苯醌、2-氯-5-甲基-1,4-对苯醌、2,5-二氯-1,4-对苯醌、2,6-二氯-1,4-对苯醌、四氟-1,4-对苯醌、四氯-1,4-对苯醌、四溴-1,4-对苯醌和2,3-二氯-5,6-二氰基-1,4-苯醌中的一种或几种。Preferably, the p-benzoquinone derivatives include 2-methyl-1,4-p-benzoquinone, 2-tert-butyl-1,4-p-benzoquinone, 2-phenyl-1,4-p-benzoquinone , 2-chloro-1,4-p-benzoquinone, 2-bromo-1,4-p-benzoquinone, thymoquinone, 2,5-dimethyl-1,4-p-benzoquinone, 2,6-di Methyl-1,4-p-benzoquinone, 2,6-dimethoxy-1,4-p-benzoquinone, 2,5-diphenyl-1,4-p-benzoquinone, 2-chloro-5- Methyl-1,4-p-benzoquinone, 2,5-dichloro-1,4-p-benzoquinone, 2,6-dichloro-1,4-p-benzoquinone, tetrafluoro-1,4-p-benzoquinone One of quinone, tetrachloro-1,4-p-benzoquinone, tetrabromo-1,4-p-benzoquinone and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone or Several kinds.
优选的，所述对苯醌衍生物包括2-甲基-1,4-对苯醌、2-苯基-1,4-对苯醌、2,6-二氯-1,4-对苯醌、四氟-1,4-对苯醌和2,3-二氯-5,6-二氰基-1,4-苯 醌中的一种或几种。Preferably, the p-benzoquinone derivatives include 2-methyl-1,4-p-benzoquinone, 2-phenyl-1,4-p-benzoquinone, 2,6-dichloro-1,4-p-benzoquinone One or more of quinone, tetrafluoro-1,4-p-benzoquinone and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone.
优选的，所述对苯醌衍生物包括2-甲基-1,4-对苯醌和/或2-苯基-1,4-对苯醌。Preferably, the p-benzoquinone derivatives include 2-methyl-1,4-p-benzoquinone and/or 2-phenyl-1,4-p-benzoquinone.
本发明提供了对苯醌和/或对苯醌衍生物在制备抗新型冠状病毒药物中的应用。对苯醌和/或对苯醌衍生物能够抑制新型冠状病毒的3CLP和PLP的活性，从而抑制新型冠状病毒的活性和增殖，缓解感染的宿主细胞内免疫失衡，进而达到治疗新型冠状病毒的效果。The invention provides the application of p-benzoquinone and/or p-benzoquinone derivatives in the preparation of anti-new coronavirus drugs. P-benzoquinone and/or p-benzoquinone derivatives can inhibit the activity of 3CLP and PLP of the new coronavirus, thereby inhibiting the activity and proliferation of the new coronavirus, alleviating the immune imbalance in the infected host cells, and then achieving the effect of treating the new coronavirus .
具体实施方式Detailed ways
本发明提供了对苯醌和/或对苯醌衍生物在制备抗新型冠状病毒药物中的应用。The invention provides the application of p-benzoquinone and/or p-benzoquinone derivatives in the preparation of anti-new coronavirus drugs.
本发明还提供了对苯醌和/或对苯醌衍生物在制备抑制新型冠状病毒增殖药物中的应用。The present invention also provides the application of p-benzoquinone and/or p-benzoquinone derivatives in the preparation of drugs for inhibiting the proliferation of novel coronavirus.
本发明还提供了对苯醌和/或对苯醌衍生物在制备新型冠状病毒酶抑制剂中的应用。The present invention also provides the application of p-benzoquinone and/or p-benzoquinone derivatives in the preparation of novel coronavirus enzyme inhibitors.
本发明还提供了对苯醌和/或对苯醌衍生物在制备新型冠状病毒的3C样蛋白酶和/或木瓜样半胱氨酸蛋白酶抑制剂中的应用。The present invention also provides the application of p-benzoquinone and/or p-benzoquinone derivatives in the preparation of 3C-like protease and/or papain-like cysteine protease inhibitors of novel coronavirus.
在本发明中，所述对苯醌的衍生物的化学结构式优选的如式I所示：In the present invention, the chemical structural formula of the derivative of p-benzoquinone is preferably as shown in formula I:
Figure PCTCN2021114452-appb-000002
Figure PCTCN2021114452-appb-000002
其中，所述R 1地选自氢、卤素、羟基、氰基、硝基、C1-12烷基、C1-12烷氧基、苯基、卤代C1-6烷基、卤代C1-6烷氧基、C3-6环烷基、单取代苯基或多取代苯基；所述R 2选自氢、卤素、羟基、氰基、硝基、C1-12烷基、C1-12烷氧基、苯基、卤代C1-6烷基、卤代C1-6烷氧基、C3-6环烷基、单取代苯基或多取代苯基；所述R 3选自氢、卤素、羟基、氰基、硝基、C1-12烷基、C1-12烷氧基、苯基、卤代C1-6烷基、卤代C1-6烷氧基、C3-6环烷基、单取代苯基或多取代苯基；所述R 4选自氢、卤素、羟基、氰基、硝基、C1-12烷基、C1-12烷氧基、苯基、卤代C1-6 烷基、卤代C1-6烷氧基、C3-6环烷基、单取代苯基或多取代苯基。 Wherein , the R is selected from hydrogen, halogen, hydroxyl, cyano, nitro, C1-12 alkyl, C1-12 alkoxy, phenyl, halogenated C1-6 alkyl, halogenated C1-6 Alkoxy, C3-6 cycloalkyl, single-substituted phenyl or multi-substituted phenyl; said R2 is selected from hydrogen, halogen, hydroxyl, cyano, nitro, C1-12 alkyl, C1-12 alkoxy Base, phenyl, halogenated C1-6 alkyl, halogenated C1-6 alkoxy, C3-6 cycloalkyl, monosubstituted phenyl or polysubstituted phenyl; the R3 is selected from hydrogen, halogen, hydroxyl , cyano, nitro, C1-12 alkyl, C1-12 alkoxy, phenyl, halogenated C1-6 alkyl, halogenated C1-6 alkoxy, C3-6 cycloalkyl, monosubstituted benzene or multi-substituted phenyl; said R is selected from hydrogen, halogen, hydroxyl, cyano, nitro, C1-12 alkyl, C1-12 alkoxy, phenyl, halogenated C1-6 alkyl, halogen C1-6 alkoxy, C3-6 cycloalkyl, mono-substituted phenyl or multi-substituted phenyl.
在本发明中，所述对苯醌衍生物优选的包括2-甲基-1,4-对苯醌、2-叔丁基-1,4-对苯醌、2-苯基-1,4-对苯醌、2-氯-1,4-对苯醌、2-溴-1,4-对苯醌、百里醌、2,5-二甲基-1,4-对苯醌、2,6-二甲基-1,4-对苯醌、2,6-二甲氧基-1,4-对苯醌、2,5-二苯基-1,4-对苯醌、2-氯-5-甲基-1,4-对苯醌、2,5-二氯-1,4-对苯醌、2,6-二氯-1,4-对苯醌、四氟-1,4-对苯醌、四氯-1,4-对苯醌、四溴-1,4-对苯醌和2,3-二氯-5,6-二氰基-1,4-苯醌(DDQ)中的一种或几种；更优选的，所述对苯醌衍生物包括2-甲基-1,4-对苯醌、2-苯基-1,4-对苯醌、2,6-二氯-1,4-对苯醌、四氟-1,4-对苯醌和2,3-二氯-5,6-二氰基-1,4-苯醌中的一种或几种；最优选的，所述对苯醌衍生物包括2-甲基-1,4-对苯醌和/或2-苯基-1,4-对苯醌。In the present invention, the p-benzoquinone derivatives preferably include 2-methyl-1,4-p-benzoquinone, 2-tert-butyl-1,4-p-benzoquinone, 2-phenyl-1,4 -p-benzoquinone, 2-chloro-1,4-p-benzoquinone, 2-bromo-1,4-p-benzoquinone, thymoquinone, 2,5-dimethyl-1,4-p-benzoquinone, 2 ,6-Dimethyl-1,4-p-benzoquinone, 2,6-dimethoxy-1,4-p-benzoquinone, 2,5-diphenyl-1,4-p-benzoquinone, 2- Chloro-5-methyl-1,4-p-benzoquinone, 2,5-dichloro-1,4-p-benzoquinone, 2,6-dichloro-1,4-p-benzoquinone, tetrafluoro-1, 4-p-benzoquinone, tetrachloro-1,4-p-benzoquinone, tetrabromo-1,4-p-benzoquinone and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone ( DDQ) in one or more; more preferably, the p-benzoquinone derivatives include 2-methyl-1,4-p-benzoquinone, 2-phenyl-1,4-p-benzoquinone, 2, One of 6-dichloro-1,4-p-benzoquinone, tetrafluoro-1,4-p-benzoquinone and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone or several; most preferably, the p-benzoquinone derivatives include 2-methyl-1,4-p-benzoquinone and/or 2-phenyl-1,4-p-benzoquinone.
本发明对所述对苯醌(1,4-对苯醌)、2-甲基-1,4-对苯醌、2-叔丁基-1,4-对苯醌、2-苯基-1,4-对苯醌、2-氯-1,4-对苯醌、2-溴-1,4-对苯醌、百里醌、2,5-二甲基-1,4-对苯醌、2,6-二甲基-1,4-对苯醌、2,6-二甲氧基-1,4-对苯醌、2,5-二苯基-1,4-对苯醌、2-氯-5-甲基-1,4-对苯醌、2,5-二氯-1,4-对苯醌、2,6-二氯-1,4-对苯醌、四氟-1,4-对苯醌、四氯-1,4-对苯醌、四溴-1,4-对苯醌和2,3-二氯-5,6-二氰基-1,4-苯醌的来源没有特殊限制，来源于常规市售。The present invention is to described p-benzoquinone (1,4-p-benzoquinone), 2-methyl-1,4-p-benzoquinone, 2-tert-butyl-1,4-p-benzoquinone, 2-phenyl- 1,4-p-benzoquinone, 2-chloro-1,4-p-benzoquinone, 2-bromo-1,4-p-benzoquinone, thymoquinone, 2,5-dimethyl-1,4-p-benzoquinone Quinone, 2,6-dimethyl-1,4-p-benzoquinone, 2,6-dimethoxy-1,4-p-benzoquinone, 2,5-diphenyl-1,4-p-benzoquinone , 2-chloro-5-methyl-1,4-p-benzoquinone, 2,5-dichloro-1,4-p-benzoquinone, 2,6-dichloro-1,4-p-benzoquinone, tetrafluoro -1,4-p-benzoquinone, tetrachloro-1,4-p-benzoquinone, tetrabromo-1,4-p-benzoquinone and 2,3-dichloro-5,6-dicyano-1,4- The source of benzoquinone is not particularly limited, and it is obtained from conventional commercial products.
多聚蛋白(polyproteins，PP)pp1a和pp1b对于维持冠状病毒RNA的合成至关重要，而多聚蛋白的成熟需要经历一系列裂解过程，才能生成多亚基蛋白质复合物(被称为“病毒复制转录酶”)。多亚基蛋白质复合物的主要蛋白酶(main protease，Mpro)为3C样蛋白酶(3C-like protease，3CLP)和木瓜样半胱氨酸蛋白酶(papain-like cysteine protease，PLP)，3CLP和PLP高度协调催化多聚蛋白的裂解，3CLP和PLP活性缺失会导致冠状病毒生命周期停止。Polyproteins (polyproteins, PP) pp1a and pp1b are essential for maintaining the synthesis of coronavirus RNA, and the maturation of polyproteins requires a series of cleavage processes to generate multi-subunit protein complexes (called "viral replication"). Transcriptase"). The main protease (main protease, Mpro) of the multi-subunit protein complex is 3C-like protease (3C-like protease, 3CLP) and papain-like cysteine protease (papain-like cysteine protease, PLP), 3CLP and PLP are highly coordinated Catalytic polyprotein cleavage, loss of 3CLP and PLP activity leads to arrest of the coronavirus life cycle.
在本发明中，所述对苯醌和/或对苯醌衍生物能够抑制新型冠状病毒的3CLP和PLP的活性，从而抑制新型冠状病毒的活性和增殖，缓解感染的宿主细胞内免疫失衡。在本发明中，对3CLP和PLP发挥抑制的活性的苯醌衍生物依赖于其1,4-对苯醌母核。In the present invention, the p-benzoquinone and/or p-benzoquinone derivatives can inhibit the activity of 3CLP and PLP of the new coronavirus, thereby inhibiting the activity and proliferation of the new coronavirus, and alleviating the immune imbalance in infected host cells. In the present invention, the benzoquinone derivatives exhibiting inhibitory activity against 3CLP and PLP depend on their 1,4-p-benzoquinone core.
下面将结合本发明中的实施例，对本发明中的技术方案进行清楚、完整地描述。显然，所描述的实施例仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。The technical solutions in the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Apparently, the described embodiments are only some of the embodiments of the present invention, but not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.
实施例1Example 1
1、新型冠状病毒3CLPro蛋白的表达与纯化1. Expression and purification of novel coronavirus 3CLPro protein
1.1引物设计1.1 Primer design
根据新型冠状病毒3CLPro的基因(ORF1ab的蛋白残基3264-3569，GenBank检索号：MN908947.3)为模板设计基因特异性引物(3CL-F：gtgccgcgcggcagccatatgtcggcagtgctgcaaagcggtttccgtaagatg，SEQ ID NO：1；3CL-R：gtggtggtggtggtgctcgaggggcccttgaaaggtcacaccgctgc，SEQ ID NO：2)，其中3CLPro-F引物序列带有NdeI酶切位点，3CLPro-R引物序列带有XhoI酶切位点。在蛋白N端加入SAVLQ5个氨基酸，构建3CLPro自身酶切位点；在蛋白C端的VTFQ氨基酸后加入GP两个氨基酸，构建Precission蛋白酶酶切位点，促进目的基因与His标签融合表达。Design gene-specific primers (3CL-F: gtgccgcgcggcagccatatgtcggcagtgctgcaaagcggtttccgtaagatg, SEQ ID NO: 1; 3CL-R: gtggtggtggtggtggtggtgctagggga , SEQ ID NO: 2), wherein the 3CLPro-F primer sequence has an NdeI restriction site, and the 3CLPro-R primer sequence has an XhoI restriction site. Add 5 amino acids of SAVLQ to the N-terminus of the protein to construct 3CLPro's own enzyme cleavage site; add two amino acids of GP after the VTFQ amino acid to the C-terminus of the protein to construct a Precision protease cleavage site to promote the fusion expression of the target gene and His tag.
1.2新型冠状病毒3CLPro基因扩增1.2 Amplification of 3CLPro gene of novel coronavirus
首先由通用生物合成具有大肠杆菌密码子偏爱性的新型冠状病毒3CLPro基因。以合成的新型冠状病毒3CLPro的基因作为模板，扩增总体系为100μL。PCR反应体系包括：cDNA模板1μL、3CL-R 4μL、3CL-F 4μL、2×Phanta Master Mix 50μL和ddH 2O 41μL。反应程序具体为：94℃3min的预变性，30次扩增反应(94℃15s，65℃30s，72℃30s)及72℃充分延伸5min。PCR产物取9μL，加入1μL 6×Gel Loading Dye Purple混合均匀后上样，其中第一个胶孔中加入2μL DNA Marker，以供样品分离出的条带进行对照(电压150V，40min)。在紫外灯下观察PCR结果，并用切胶刀从凝胶上切下PCR正确片段的条带，放入1.5mL EP管中，称重并记录。使用SanPrep柱式DNA胶回收试剂盒回收PCR产物，使用Nano Drop测定回收的目的基因浓度与纯度。 First, the novel coronavirus 3CLPro gene with E. coli codon preference was synthesized by General Bio. Using the synthetic 3CLPro gene of the novel coronavirus as a template, the total amplification system was 100 μL. The PCR reaction system includes: 1 μL of cDNA template, 4 μL of 3CL-R, 4 μL of 3CL-F, 50 μL of 2×Phanta Master Mix and 41 μL of ddH 2 O. The specific reaction program is: pre-denaturation at 94°C for 3 minutes, 30 amplification reactions (15s at 94°C, 30s at 65°C, 30s at 72°C) and full extension at 72°C for 5 minutes. Take 9 μL of the PCR product, add 1 μL of 6×Gel Loading Dye Purple, mix well and load the sample, and add 2 μL of DNA Marker to the first gel hole for comparison of the bands isolated from the sample (voltage 150V, 40min). Observe the PCR results under ultraviolet light, and use a gel knife to cut out the band of the correct PCR fragment from the gel, put it into a 1.5mL EP tube, weigh and record. The PCR product was recovered using the SanPrep Column DNA Gel Recovery Kit, and the concentration and purity of the recovered target gene was determined using Nano Drop.
1.3新型冠状病毒3CLPro表达载体的构建1.3 Construction of novel coronavirus 3CLPro expression vector
使用NdeI和XhoI限制性内切酶对pET28a(+)进行酶切，然后胶 回收目的质粒片段用于重组连接。具体连接体系包括：pET28a(+)质粒片段20μg、3CLPro目的基因40μg、5×CE Ⅱ Buffer 5μL、Exnase Ⅱ 2μL，并用水补足50μL。pET28a(+) was digested with NdeI and XhoI restriction enzymes, and then the target plasmid fragment was recovered by gel for recombination connection. The specific connection system includes: pET28a(+) plasmid fragment 20 μg, 3CLPro target gene 40 μg, 5×CE Ⅱ Buffer 5 μL, Exnase Ⅱ 2 μL, and water to make up 50 μL.
1.4新型冠状病毒3CLPro的诱导表达1.4 Induced expression of novel coronavirus 3CLPro
将转化成功的大肠杆菌菌种在37℃下按1％(v/v)接种量接种于25mL含有卡那霉素(50μg/mL)的LB液体培养基中预培养过夜，然后将培养物按1％(v/v)接种量接种于1L含有卡那霉素(50μg/mL)的LB液体培养基中37℃培养。当OD600达到0.8时，添加0.5mM异丙基-D-硫代半乳糖苷(IPTG)在25℃下诱导12h，使3CLPro蛋白表达。The successfully transformed Escherichia coli strains were pre-cultured overnight in 25 mL of LB liquid medium containing kanamycin (50 μg/mL) at 37 °C at a 1% (v/v) inoculum size, and then the culture was 1% (v/v) inoculum was inoculated in 1 L of LB liquid medium containing kanamycin (50 μg/mL) and cultured at 37°C. When OD600 reached 0.8, 0.5mM isopropyl-D-thiogalactoside (IPTG) was added to induce 12h at 25°C to express 3CLPro protein.
1.5新型冠状病毒3CLPro的纯化1.5 Purification of novel coronavirus 3CLPro
1)取1L菌液在5000×g条件下离心10min，弃去上清液。1) Take 1L of bacterial liquid and centrifuge at 5000×g for 10 min, discard the supernatant.
2)将沉淀重悬于40mL Lysis Buffer中(所有缓冲液的pH在4℃下调节)，然后通过高压均质仪裂解3～5min。2) Resuspend the pellet in 40mL Lysis Buffer (the pH of all buffers is adjusted at 4°C), and then lyse for 3-5 minutes with a high-pressure homogenizer.
3)使用冷冻低温离心机将裂解液在4℃下超速离心(15000×g)30min，取上清再次超速离心(15000×g)30min。3) The lysate was ultracentrifuged (15000×g) for 30 minutes at 4° C. using a refrigerated low-temperature centrifuge, and the supernatant was taken and ultracentrifuged again (15000×g) for 30 minutes.
4)使用Ni-NTA柱进行蛋白纯化前，先用Lysis Buffer平衡Ni-NTA柱。4) Before using the Ni-NTA column for protein purification, equilibrate the Ni-NTA column with Lysis Buffer.
5)将已离心2～3次的裂解液上清分批次加入至Ni-NTA柱流穿，流穿结束后，为除去非特异性结合蛋白，加入100mL左右的Lysis Buffer洗涤Ni-NTA柱，洗涤结束后使用25mL Wash Buffer及35mL Elution Buffer洗脱收集3CLPro。5) Add the supernatant of the lysate that has been centrifuged for 2 to 3 times in batches to flow through the Ni-NTA column. After the flow-through is completed, in order to remove non-specific binding proteins, add about 100 mL of Lysis Buffer to wash the Ni-NTA column. After washing, 3CLPro was collected by elution with 25mL Wash Buffer and 35mL Elution Buffer.
6)将裂解液、流穿液、洗涤液、Wash Buffer及Elution Buffer各收集20μL。6) Collect 20 μL each of lysate, flow-through, washing buffer, Wash Buffer and Elution Buffer.
7)每个样品加入5μL 5×SDS-PAGE Protein Loading Buffer吹打混匀，在95℃条件下加热10min使蛋白样品变性，每管样品取10μL进行SDS-PAGE电泳，其中第一个胶孔中加入2μL Prestained Protein Ladder，以供样品分离出的条带进行对照(电压220V，45min)。7) Add 5 μL of 5×SDS-PAGE Protein Loading Buffer to each sample, pipette and mix well, heat at 95°C for 10 minutes to denature the protein sample, take 10 μL of each tube of sample for SDS-PAGE electrophoresis, and add 2μL Prestained Protein Ladder, for the band isolated from the sample for control (voltage 220V, 45min).
8)根据SDS-PAGE电泳结果浓缩洗脱液至2mL，使用替换液A(20 mM HEPES，400mM NaCl，pH 7.5)替换洗脱液3次，最终得到4mL 3CLPro浓缩液，使用Nano Drop测定蛋白浓度。在浓缩液中加入13mg/mL Precission蛋白酶(PPE)100μL酶切过夜。8) Concentrate the eluent to 2mL according to the results of SDS-PAGE electrophoresis, replace the eluent 3 times with replacement solution A (20 mM HEPES, 400mM NaCl, pH 7.5), and finally obtain 4mL 3CLPro concentrated solution, use Nano Drop to determine the protein concentration . Add 100 μL of 13mg/mL Precision Protease (PPE) to the concentrated solution to digest overnight.
9)使用GFC Buffer(100mM NaCl，20mM HEPES，pH 7.5)平衡凝胶过滤分子筛层析柱(HiLoad 16/600 Superdex 200pg)。9) Use GFC Buffer (100mM NaCl, 20mM HEPES, pH 7.5) to equilibrate the gel filtration molecular sieve chromatography column (HiLoad 16/600 Superdex 200pg).
10)将4mL酶切处理后的3CLPro浓缩液均分至3个1.5mL EP管中，4℃下超速离心12000×g，10min。10) Divide 4mL of the 3CLPro concentrate after enzyme digestion into three 1.5mL EP tubes, and ultracentrifuge at 12000×g for 10min at 4°C.
11)去除沉淀后，使用螺旋注射器将蛋白样品推入5mL上样环中，通过AKTA蛋白纯化仪进行蛋白纯化。11) After removing the precipitate, use a spiral syringe to push the protein sample into a 5mL sample loop, and perform protein purification through the AKTA protein purification instrument.
12)浓缩从凝胶过滤分子筛层析柱洗脱下来的含有高纯度3CLPro部分，浓缩液依次使用替换液B(20mM HEPES，200mM NaCl，pH 7.5)和替换液C(20mM HEPES，100mM NaCl，pH7.5)替换3次，使用Nano Drop测定蛋白浓度。将3CLPro蛋白按50μL/管进行分装，液氮速冻后放入-80℃保存。12) Concentrate the part containing high-purity 3CLPro eluted from the gel filtration molecular sieve chromatography column, and use the replacement solution B (20mM HEPES, 200mM NaCl, pH 7.5) and the replacement solution C (20mM HEPES, 100mM NaCl, pH7. .5) Replace 3 times, use Nano Drop to measure protein concentration. The 3CLPro protein was divided into 50 μL/tubes, quickly frozen in liquid nitrogen and stored at -80°C.
2、新型冠状病毒PLpro的表达纯化2. Expression and purification of novel coronavirus PLpro
2.1引物设计2.1 Primer design
根据新型冠状病毒PLPro的基因序列为模板设计基因特异性引物(PLP-F：GTGCCGCGCGGCAGCCATATGTCGGCAGTGCTGCAA，SEQ ID NO：3；PLP-R：GTGCCGCGCGGCAGCCATATGTCGGCAGTGCTGCAAGCGGTTCGTACCATCAAG，SEQ ID NO：4)，其中PLP-F引物序列带有NdeI酶切位点，PLP-R引物序列带有EcoR I酶切位点。同样，在蛋白N端加入SAVLQ 5个氨基酸，构建3CLPro自身酶切位点；在蛋白C端的加入终止密码子以备后期终止翻译。Design gene-specific primers (PLP-F: GTGCCGCGCGGCAGCCATATGTCGGCAGTGCTGCAA, SEQ ID NO: 3; PLP-R: GTGCCGCGCGGCAGCCATATGTCGGCAGTGCTGCAAGCGGTTCGTACCATCAAG, SEQ ID NO: 4) according to the gene sequence of the new coronavirus PLPro, wherein the PLP-F primer sequence contains NdeI Restriction site, the PLP-R primer sequence has an EcoRI restriction site. Similarly, 5 amino acids of SAVLQ were added to the N-terminus of the protein to construct a 3CLPro self-cleavage site; a stop codon was added to the C-terminus of the protein for later termination of translation.
2.2新型冠状病毒PLPro基因扩增2.2 New coronavirus PLPro gene amplification
首先由通用生物合成具有大肠杆菌密码子偏爱性的新型冠状病毒PLPro基因。以合成的新型冠状病毒PLPro的基因作为模板，扩增总体系为100μL。PCR反应体系包括：cDNA模板1μL、3CL-R 4μL、3CL-F 4μL、2×Phanta Master Mix 50μL和ddH 2O 41μL。反应程序具体为：94℃3min的预变性，30次扩增反应(94℃15s，65℃30s，72℃30s)及72℃ 充分延伸5min。PCR产物取9μL，加入1μL 6×Gel Loading Dye Purple混合均匀后上样，其中第一个胶孔中加入2μL DNA Marker，以供样品分离出的条带进行对照(电压150V，40min)。在紫外灯下观察PCR结果，并用切胶刀从凝胶上切下PCR正确片段的条带，放入1.5mL EP管中，称重并记录。使用SanPrep柱式DNA胶回收试剂盒回收PCR产物，使用Nano Drop测定回收的目的基因浓度与纯度。 First, the novel coronavirus PLPro gene with E. coli codon preference was synthesized by Universal Biotechnology. Using the synthetic novel coronavirus PLPro gene as a template, the total amplification system was 100 μL. The PCR reaction system includes: 1 μL of cDNA template, 4 μL of 3CL-R, 4 μL of 3CL-F, 50 μL of 2×Phanta Master Mix and 41 μL of ddH 2 O. The specific reaction program is: pre-denaturation at 94° C. for 3 minutes, 30 amplification reactions (94° C. for 15 s, 65° C. for 30 s, and 72° C. for 30 s) and full extension at 72° C. for 5 minutes. Take 9 μL of the PCR product, add 1 μL of 6×Gel Loading Dye Purple, mix well and load the sample, and add 2 μL of DNA Marker to the first gel hole for comparison of the bands isolated from the sample (voltage 150V, 40min). Observe the PCR results under ultraviolet light, and use a gel knife to cut out the band of the correct PCR fragment from the gel, put it into a 1.5mL EP tube, weigh it and record it. The PCR product was recovered using the SanPrep Column DNA Gel Recovery Kit, and the concentration and purity of the recovered target gene was determined using Nano Drop.
2.3新型冠状病毒PLPro表达载体的构建2.3 Construction of novel coronavirus PLPro expression vector
使用NdeI和EcoR I限制性内切酶对pET28a(+)进行酶切，然后胶回收目的质粒片段用于重组连接。具体连接体系包括：pET28a(+)质粒片段20μg、PLPro目的基因40μg、5×CE Ⅱ Buffer 5μL、Exnase Ⅱ 2μL，并用水补足50μL。pET28a(+) was digested with NdeI and EcoR I restriction enzymes, and then the target plasmid fragment was recovered by gel for recombination connection. The specific connection system includes: 20 μg of pET28a(+) plasmid fragment, 40 μg of PLPro target gene, 5 μL of 5×CE Ⅱ Buffer, 2 μL of Exnase Ⅱ, and make up 50 μL with water.
2.4新型冠状病毒PLPro的诱导表达2.4 Induced expression of novel coronavirus PLPro
将构建成功的大肠杆菌转接到装有100mL LB液体培养基锥形瓶中，按照0.1％培养基体积加入菌体对应的50mg/mL氨苄青霉素抗生素，放入37℃摇床中恒温培养，2～3h左右，待菌液OD600值达到0.8时，添加1μM ZnCl2，按0.5mM终浓度加入诱导剂IPTG，25℃过夜诱导，使蛋白表达。Transfer the successfully constructed Escherichia coli to a Erlenmeyer flask containing 100mL LB liquid medium, add 50mg/mL ampicillin antibiotic corresponding to the bacterium according to the volume of 0.1% medium, and put it into a shaker at 37°C for constant temperature cultivation, 2 ~3h or so, when the OD600 value of the bacterial solution reaches 0.8, add 1μM ZnCl2, add the inducer IPTG at a final concentration of 0.5mM, and induce overnight at 25°C to express the protein.
2.5新型冠状病毒PLPro的纯化2.5 Purification of novel coronavirus PLPro
(1)取菌液于4000×g条件下离心15min，弃去上清液。将沉淀以1L菌20mL Lysis Buffer的比例进行重悬(Lysis Buffer中含终浓度为5mMβ-巯基乙醇)。然后通过高压均质仪在70～80MPa条件下裂解10min。使用冷冻低温离心机将裂解液在4℃下超速离心(18000×g)30min，重复该操作。(1) Take the bacterial liquid and centrifuge it at 4000×g for 15 min, discard the supernatant. Resuspend the pellet at a ratio of 1L bacteria to 20mL Lysis Buffer (the final concentration in Lysis Buffer is 5mM β-mercaptoethanol). Then, it was cracked for 10 minutes under the condition of 70-80 MPa by a high-pressure homogenizer. The lysate was ultracentrifuged (18000×g) at 4° C. for 30 min using a refrigerated low-temperature centrifuge, and the operation was repeated.
(2)使用Lysis Buffer平衡Ni-NTA柱后，将两次离心的裂解液上清经膜过滤后，加入至Ni-NTA柱流穿，后加入Lysis Buffer洗涤Ni-NTA柱以除去非特异性结合的蛋白，洗涤结束后使用Elution Buffer洗脱收集PLpro。收集上述实验中的裂解液、流穿液、高盐洗脱液、洗涤液及Elution Buffer 20μL，并各自加入5μL 5×SDS-PAGE ProteinLoading Buffer。取10μL样品进行SDS-PAGE电泳，电泳条件为220V，45min，电泳结束后对蛋白胶进行染色、脱色，检测蛋白是否表达。(2) After using Lysis Buffer to equilibrate the Ni-NTA column, filter the supernatant of the lysate obtained by two centrifugations through membrane filtration, add to the Ni-NTA column to flow through, and then add Lysis Buffer to wash the Ni-NTA column to remove non-specific binding After washing, use Elution Buffer to elute and collect PLpro. Collect 20 μL of the lysate, flow-through, high-salt eluate, washing solution and Elution Buffer in the above experiments, and add 5 μL of 5×SDS-PAGE Protein Loading Buffer to each. Take 10 μL sample for SDS-PAGE electrophoresis, the electrophoresis condition is 220V, 45min, after the electrophoresis, the protein gel is stained and decolorized to detect whether the protein is expressed.
(3)根据从镍柱洗脱的蛋白浓度，对蛋白进行浓缩。使用GFC Buffer (20mM HEPES，100mM NaCl，pH 7.5)替换洗脱液3次，最终获得PLpro浓缩液，使用Nano Drop测定蛋白浓度。在浓缩液中加入1M的TCEP 5μL和51mg/mL 3CLpro蛋白酶3μL酶切过夜。(3) Concentrate the protein according to the concentration of the protein eluted from the nickel column. Use GFC Buffer (20mM HEPES, 100mM NaCl, pH 7.5) to replace the eluent 3 times, and finally obtain the PLpro concentrate, and use Nano Drop to determine the protein concentration. Add 5 μL of 1M TCEP and 3 μL of 51 mg/mL 3CLpro protease to the concentrated solution to digest overnight.
(4)使用GFC Buffer平衡凝胶过滤分子筛层析柱(HiLoad 16/600 Superdex 200pg)。将经酶切处理后的PLpro浓缩液超速离心(12000×g)10min。使用螺旋注射器将蛋白样品加入上样环中，注意不要吸入沉淀。使用AKTA对蛋白进行二次纯化。浓缩从凝胶过滤分子筛层析柱洗脱下来的含有高纯度PLpro的部分。使用Nano Drop测定蛋白浓度。将PLpro按后续实验需求进行分装，分装后使用液氮速冻，放入-80℃保存。(4) Use GFC Buffer to equilibrate the gel filtration molecular sieve chromatography column (HiLoad 16/600 Superdex 200pg). Ultracentrifuge (12000×g) the PLpro concentrate after enzyme digestion for 10 min. Add the protein sample to the sample loop using a spiral syringe, taking care not to aspirate the pellet. The protein was purified a second time using AKTA. Concentrate the fractions containing high-purity PLpro eluted from the gel filtration molecular sieve chromatography column. Protein concentration was determined using Nano Drop. The PLpro was subpackaged according to the requirements of the follow-up experiments. After subpackaging, it was quickly frozen with liquid nitrogen and stored at -80°C.
3、活性测试3. Activity test
化合物：1,4-对苯醌、2-甲基-1,4-对苯醌、2-叔丁基-1,4-对苯醌、2-苯基-1,4-对苯醌、2-氯-1,4-对苯醌、2-溴-1,4-对苯醌、百里醌、2,5-二甲基-1,4-对苯醌、2,6-二甲基-1,4-对苯醌、2,6-二甲氧基-1,4-对苯醌、2,6-二叔丁基-1,4-对苯醌、2,5-二苯基-1,4-对苯醌、2-氯-5-甲基-1,4-对苯醌、2,5-二氯-1,4-对苯醌、2,6-二氯-1,4-对苯醌、四氟-1,4-对苯醌、四氯-1,4-对苯醌、四溴-1,4-对苯醌、2,3-二氯-5,6-二氰基-1,4-苯醌、艾地苯醌和四甲基-1,4-对苯醌；上述所述化合物购自于成都吉安特医药科技有限公司。Compounds: 1,4-p-benzoquinone, 2-methyl-1,4-p-benzoquinone, 2-tert-butyl-1,4-p-benzoquinone, 2-phenyl-1,4-p-benzoquinone, 2-Chloro-1,4-p-benzoquinone, 2-bromo-1,4-p-benzoquinone, thymoquinone, 2,5-dimethyl-1,4-p-benzoquinone, 2,6-dimethyl Base-1,4-p-benzoquinone, 2,6-dimethoxy-1,4-p-benzoquinone, 2,6-di-tert-butyl-1,4-p-benzoquinone, 2,5-diphenyl Base-1,4-p-benzoquinone, 2-chloro-5-methyl-1,4-p-benzoquinone, 2,5-dichloro-1,4-p-benzoquinone, 2,6-dichloro-1 ,4-p-benzoquinone, tetrafluoro-1,4-p-benzoquinone, tetrachloro-1,4-p-benzoquinone, tetrabromo-1,4-p-benzoquinone, 2,3-dichloro-5,6 -Dicyano-1,4-benzoquinone, idebenone and tetramethyl-1,4-p-benzoquinone; the above-mentioned compounds were purchased from Chengdu Jiante Pharmaceutical Technology Co., Ltd.
在本发明中，所述对苯醌(1,4-对苯醌)、2-甲基-1,4-对苯醌、2-叔丁基-1,4-对苯醌、2-苯基-1,4-对苯醌、2-氯-1,4-对苯醌、2-溴-1,4-对苯醌、百里醌、2,5-二甲基-1,4-对苯醌、2,6-二甲基-1,4-对苯醌、2,6-二甲氧基-1,4-对苯醌、2,6-二叔丁基-1,4-对苯醌、2,5-二苯基-1,4-对苯醌、2-氯-5-甲基-1,4-对苯醌、2,5-二氯-1,4-对苯醌、2,6-二氯-1,4-对苯醌、四氟-1,4-对苯醌、四氯-1,4-对苯醌、四溴-1,4-对苯醌、2,3-二氯-5,6-二氰基-1,4-苯醌、艾地苯醌和四甲基-1,4-对苯醌的化学结构式如表1所示。In the present invention, the p-benzoquinone (1,4-p-benzoquinone), 2-methyl-1,4-p-benzoquinone, 2-tert-butyl-1,4-p-benzoquinone, 2-benzoquinone Base-1,4-p-benzoquinone, 2-chloro-1,4-p-benzoquinone, 2-bromo-1,4-p-benzoquinone, thymoquinone, 2,5-dimethyl-1,4- p-benzoquinone, 2,6-dimethyl-1,4-p-benzoquinone, 2,6-dimethoxy-1,4-p-benzoquinone, 2,6-di-tert-butyl-1,4- p-benzoquinone, 2,5-diphenyl-1,4-p-benzoquinone, 2-chloro-5-methyl-1,4-p-benzoquinone, 2,5-dichloro-1,4-p-benzoquinone quinone, 2,6-dichloro-1,4-p-benzoquinone, tetrafluoro-1,4-p-benzoquinone, tetrachloro-1,4-p-benzoquinone, tetrabromo-1,4-p-benzoquinone, The chemical structural formulas of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone, idebenone and tetramethyl-1,4-p-benzoquinone are shown in Table 1.
表1对苯醌和对苯醌的衍生物的结构式The structural formula of table 1 p-benzoquinone and derivatives of p-benzoquinone
Figure PCTCN2021114452-appb-000003
Figure PCTCN2021114452-appb-000003
Figure PCTCN2021114452-appb-000004
Figure PCTCN2021114452-appb-000004
Figure PCTCN2021114452-appb-000005
Figure PCTCN2021114452-appb-000005
Figure PCTCN2021114452-appb-000006
Figure PCTCN2021114452-appb-000006
1)化合物对新型冠状病毒3CLPro的抑制活性测试1) The inhibitory activity test of the compound against the novel coronavirus 3CLPro
(1)通过荧光共振能量转移技术(FRET)测定新型冠状病毒3CLPro活性及化合物对新型冠状病毒3CLPro的抑制活性。测定试验中使用带有新型冠状病毒3CLPro切割位点(箭头所示)的荧光底物(Dabcyl-KTSAVLQ↓SGFRKM-E(Edans)-NH 2)和Tris-HCl缓冲液(50mM Tris-HCl，1mM EDTA，pH 7.3)。 (1) The activity of 2019-nCoV 3CLPro and the inhibitory activity of compounds on 2019-nCoV 3CLPro were determined by fluorescence resonance energy transfer (FRET). Fluorescent substrate (Dabcyl-KTSAVLQ↓SGFRKM-E(Edans)-NH 2 ) and Tris-HCl buffer (50mM Tris-HCl, 1mM EDTA, pH 7.3).
(2)化合物由100％DMSO溶解。取上述10μL化合物分别与40μL新型冠状病毒3CLPro(终浓度0.1μM)在Tris-HCl缓冲液中孵育1h，通过添加50μL荧光底物(Dabcyl-KTSAVLQ↓SGFRKM-E(Edans)-NH 2、终 浓度为20μM)引发反应。使用放射共振能量转移荧光分光光度计检测由于3CLPro催化的底物裂解而产生的Dabcyl荧光信号(340nm(激发)/490nm(发射))，通过3min内荧光信号的变化，从而判定化合物是否具有抑制活性。 (2) Compounds were dissolved in 100% DMSO. Take 10 μL of the above compounds and incubate with 40 μL of novel coronavirus 3CLPro (final concentration 0.1 μM) in Tris-HCl buffer for 1 h, and add 50 μL of fluorescent substrate (Dabcyl-KTSAVLQ↓SGFRKM-E(Edans)-NH 2 , final concentration 20 μM) to initiate the reaction. Use an emission resonance energy transfer fluorescence spectrophotometer to detect the Dabcyl fluorescence signal (340nm (excitation)/490nm (emission)) due to the cleavage of the substrate catalyzed by 3CLPro, and determine whether the compound has inhibitory activity by changing the fluorescence signal within 3 minutes .
(3)新型冠状病毒3CLPro动力学常数通过将数据拟合到MichaelisMenten方程中得出。通过初筛筛选出高抑制活性化合物(选择终浓度为50μM时抑制率大于0.9的化合物)，加入紫草素(已知高活性抑制剂)作为阳性对照，对比抑制率。筛选结果参见表2。由表2可以看出化合物：1,4-对苯醌、2-甲基-1,4-对苯醌、2-叔丁基-1,4-对苯醌、2-苯基-1,4-对苯醌、2-氯-1,4-对苯醌、2-溴-1,4-对苯醌、百里醌、2,5-二甲基-1,4-对苯醌、2,6-二甲基-1,4-对苯醌、2,6-二甲氧基-1,4-对苯醌、2-氯-5-甲基-1,4-对苯醌、2,5-二氯-1,4-对苯醌、2,6-二氯-1,4-对苯醌、四氟-1,4-对苯醌、四氯-1,4-对苯醌、四溴-1,4-对苯醌以及2,3-二氯-5,6-二氰基-1,4-苯醌对新型冠状病毒3CLPro的酶抑制活性优于阳性对照药紫草素。(3) The kinetic constants of the 2019-nCoV 3CLPro were obtained by fitting the data to the MichaelisMenten equation. Compounds with high inhibitory activity were screened out through primary screening (compounds with an inhibitory rate greater than 0.9 at a final concentration of 50 μM were selected), and shikonin (a known highly active inhibitor) was added as a positive control to compare the inhibitory rate. See Table 2 for screening results. It can be seen from Table 2 that the compounds: 1,4-p-benzoquinone, 2-methyl-1,4-p-benzoquinone, 2-tert-butyl-1,4-p-benzoquinone, 2-phenyl-1, 4-p-benzoquinone, 2-chloro-1,4-p-benzoquinone, 2-bromo-1,4-p-benzoquinone, thymoquinone, 2,5-dimethyl-1,4-p-benzoquinone, 2,6-Dimethyl-1,4-p-benzoquinone, 2,6-dimethoxy-1,4-p-benzoquinone, 2-chloro-5-methyl-1,4-p-benzoquinone, 2,5-dichloro-1,4-p-benzoquinone, 2,6-dichloro-1,4-p-benzoquinone, tetrafluoro-1,4-p-benzoquinone, tetrachloro-1,4-p-benzoquinone Quinone, tetrabromo-1,4-p-benzoquinone and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone have better enzyme inhibitory activity against novel coronavirus 3CLPro than the positive control drug, comfrey white.
(4)对通过初筛筛选出的高抑制活性化合物进行IC50的测定。使用Tris-HCl缓冲液将化合物稀释至梯度浓度(1.5625-100μM和0.15625-10μM)，并使用上述相同终浓度的新型冠状病毒3CLPro和荧光底物体系进行测定。化合物的IC50值由GraphPad Prism 8.0软件计算。每次实验三个平行，最终结果用平均值±标准差(SD)表示。测定结果参见表3。由表3可以看出，化合物：1,4-对苯醌、2-甲基-1,4-对苯醌、2-苯基-1,4-对苯醌、2-氯-1,4-对苯醌、2-溴-1,4-对苯醌、百里醌、2,5-二甲基-1,4-对苯醌、2,6-二甲基-1,4-对苯醌、2-氯-5-甲基-1,4-对苯醌、2,6-二氯-1,4-对苯醌、四溴-1,4-对苯醌以及2,3-二氯-5,6-二氰基-1,4-苯醌对新型冠状病毒3CLPro的IC50值低于1.0μM，显示出对新型冠状病毒3CLPro的优异抑制活性。(4) Determination of IC50 for compounds with high inhibitory activity screened out through primary screening. The compound was diluted to gradient concentrations (1.5625-100 μM and 0.15625-10 μM) using Tris-HCl buffer, and assayed using the same final concentration of the novel coronavirus 3CLPro and fluorescent substrate system above. The IC50 values of compounds were calculated by GraphPad Prism 8.0 software. Each experiment was performed in triplicate, and the final results were expressed as mean ± standard deviation (SD). See Table 3 for the measurement results. As can be seen from Table 3, compounds: 1,4-p-benzoquinone, 2-methyl-1,4-p-benzoquinone, 2-phenyl-1,4-p-benzoquinone, 2-chloro-1,4 -p-benzoquinone, 2-bromo-1,4-p-benzoquinone, thymoquinone, 2,5-dimethyl-1,4-p-benzoquinone, 2,6-dimethyl-1,4-p- Benzoquinone, 2-chloro-5-methyl-1,4-p-benzoquinone, 2,6-dichloro-1,4-p-benzoquinone, tetrabromo-1,4-p-benzoquinone and 2,3- Dichloro-5,6-dicyano-1,4-benzoquinone has an IC50 value of less than 1.0 μM against 2019-nCoV 3CLPro, showing excellent inhibitory activity against 2019-nCoV 3CLPro.
2)化合物对新型冠状病毒PLpro的抑制活性测试2) The inhibitory activity test of the compound against the novel coronavirus PLpro
(1)使用荧光共振能量转移技术(FRET)测定新型冠状病毒PLpro活性及化合物对新型冠状病毒PLpro的抑制活性。测定试验使用带有蛋白特异性切割位点(箭头所示)的荧光底物(CBE-RLRGG↓AMC)以启动反应。蛋白及荧光底物由PLpro缓冲液(150mM NaCl、40mM Tris-HCl、 5mM DTT，pH 8.0)进行稀释。(1) Fluorescence resonance energy transfer (FRET) was used to measure the PLpro activity of the new coronavirus and the inhibitory activity of the compound on the PLpro of the new coronavirus. The assay uses a fluorogenic substrate (CBE-RLRGG↓AMC) with a protein-specific cleavage site (arrow) to initiate the reaction. Proteins and fluorescent substrates were diluted with PLpro buffer (150mM NaCl, 40mM Tris-HCl, 5mM DTT, pH 8.0).
(2)化合物由100％DMSO溶解。取上述10μL化合物分别与40μL新型冠状病毒PLpro(终浓度0.3μM)在PLpro缓冲液中孵育10min，通过添加50μL荧光底物(终浓度为20μM)引发反应。PLpro将底物裂解产生AMC荧光信号(340nm(激发)/490nm(发射))，通过3min内荧光信号的变化，从而判定化合物是否具有抑制活性。(2) Compounds were dissolved in 100% DMSO. 10 μL of the above compounds were incubated with 40 μL of 2019-nCoV PLpro (final concentration 0.3 μM) in PLpro buffer for 10 min, and the reaction was initiated by adding 50 μL of fluorescent substrate (final concentration 20 μM). PLpro cleaves the substrate to generate AMC fluorescence signal (340nm (excitation)/490nm (emission)), and through the change of fluorescence signal within 3min, it is determined whether the compound has inhibitory activity.
(3)新型冠状病毒PLpro动力学常数通过将数据拟合到MichaelisMenten方程中得出。通过初筛筛选出高抑制活性化合物(选择终浓度为50μM时抑制率大于0.9的化合物)，加入紫草素(已知高活性抑制剂)作为阳性对照，对比抑制率。筛选结果参见表2。由表2可以看出化合物：2-甲基-1,4-对苯醌、百里醌、2,5-二甲基-1,4-对苯醌、2,6-二甲基-1,4-对苯醌、2,6-二甲氧基-1,4-对苯醌、2,5-二苯基-1,4-对苯醌、2,5-二氯-1,4-对苯醌、2,6-二氯-1,4-对苯醌、四氟-1,4-对苯醌、四氯-1,4-对苯醌以及2,3-二氯-5,6-二氰基-1,4-苯醌对新型冠状病毒PLPro的酶抑制活性优于阳性对照药紫草素。但是2,6-二叔丁基-1,4-对苯醌、艾地苯醌和四甲基-1,4-对苯醌对新型冠状病毒PLPro的酶活性为促进作用。(3) The PLpro kinetic constants of the novel coronavirus were obtained by fitting the data to the MichaelisMenten equation. Compounds with high inhibitory activity were screened out through primary screening (compounds with an inhibitory rate greater than 0.9 at a final concentration of 50 μM were selected), and shikonin (a known highly active inhibitor) was added as a positive control to compare the inhibitory rate. See Table 2 for screening results. It can be seen from Table 2 that the compounds: 2-methyl-1,4-p-benzoquinone, thymoquinone, 2,5-dimethyl-1,4-p-benzoquinone, 2,6-dimethyl-1 ,4-p-benzoquinone, 2,6-dimethoxy-1,4-p-benzoquinone, 2,5-diphenyl-1,4-p-benzoquinone, 2,5-dichloro-1,4 -p-benzoquinone, 2,6-dichloro-1,4-p-benzoquinone, tetrafluoro-1,4-p-benzoquinone, tetrachloro-1,4-p-benzoquinone and 2,3-dichloro-5 , 6-dicyano-1,4-benzoquinone has better enzyme inhibitory activity against the new coronavirus PLPro than the positive control drug shikonin. However, 2,6-di-tert-butyl-1,4-p-benzoquinone, idebenone and tetramethyl-1,4-p-benzoquinone can promote the enzymatic activity of 2019-nCoV PLPro.
(4)对通过初筛筛选出的高抑制活性化合物进行IC50的测定。使用PLpro缓冲液将化合物稀释至梯度浓度，并使用上述相同终浓度的SARS-CoV-2 PLpro和荧光底物体系进行测定。(4) Determination of IC50 for compounds with high inhibitory activity screened out through primary screening. Compounds were diluted to gradient concentrations using PLpro buffer and assayed using the same final concentrations of SARS-CoV-2 PLpro and fluorogenic substrate system as above.
化合物的IC50值由GraphPad Prism 8.0软件计算。每次实验三个平行，最终结果用平均值±标准差(SD)表示。测定结果参见表3。由表3可以看出，化合物2-甲基-1,4-对苯醌以及2-苯基-1,4-对苯醌对新型冠状病毒PLPro的IC50值低于1.0μM，显示出对新型冠状病毒PLPro的优异抑制活性。The IC50 values of compounds were calculated by GraphPad Prism 8.0 software. Each experiment was performed in triplicate, and the final results were expressed as mean ± standard deviation (SD). See Table 3 for the measurement results. It can be seen from Table 3 that the compound 2-methyl-1,4-p-benzoquinone and 2-phenyl-1,4-p-benzoquinone have an IC50 value of less than 1.0 μM for the new coronavirus PLPro, showing that the new Excellent inhibitory activity of coronavirus PLPro.
表2对苯醌或对苯醌的衍生物抗新冠活性50μm初筛抑制率Table 2 Inhibition rate of anti-coronavirus activity of p-benzoquinone or derivatives of p-benzoquinone at 50 μm primary screening
Figure PCTCN2021114452-appb-000007
Figure PCTCN2021114452-appb-000007
Figure PCTCN2021114452-appb-000008
Figure PCTCN2021114452-appb-000008
表3对苯醌或对苯醌的衍生物抗新冠活性IC50测定结果Table 3 Determination results of p-benzoquinone or derivatives of p-benzoquinone anti-coronavirus activity IC50
Figure PCTCN2021114452-appb-000009
Figure PCTCN2021114452-appb-000009
Figure PCTCN2021114452-appb-000010
Figure PCTCN2021114452-appb-000010
注：“—”表示表2中初筛活性相对较低的化合物，没有进行IC50测定；“*”表示差异大，不可求。Note: "—" means that the compounds with relatively low initial screening activity in Table 2 have not been tested for IC50; "*" means that the difference is large and cannot be determined.
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。The above is only a preferred embodiment of the present invention, it should be pointed out that for those skilled in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications are also It should be regarded as the protection scope of the present invention.

Claims (8)

  1. 对苯醌和/或对苯醌衍生物在制备抗新型冠状病毒药物中的应用。Application of p-benzoquinone and/or p-benzoquinone derivatives in the preparation of anti-new coronavirus drugs.
  2. 对苯醌和/或对苯醌衍生物在制备抑制新型冠状病毒增殖药物中的应用。Application of p-benzoquinone and/or p-benzoquinone derivatives in the preparation of drugs for inhibiting the proliferation of novel coronavirus.
  3. 对苯醌和/或对苯醌衍生物在制备新型冠状病毒的酶抑制剂中的应用。Application of p-benzoquinone and/or p-benzoquinone derivatives in the preparation of enzyme inhibitors for novel coronaviruses.
  4. 对苯醌和/或对苯醌衍生物在制备新型冠状病毒的3C样蛋白酶和/或木瓜样半胱氨酸蛋白酶抑制剂中的应用。Application of p-benzoquinone and/or p-benzoquinone derivatives in the preparation of 3C-like protease and/or papain-like cysteine protease inhibitors of novel coronavirus.
  5. 根据权利要求1～4任意一项所述的应用，其特征在于，所述对苯醌衍生物的化学结构式如式I所示：According to the application described in any one of claims 1 to 4, it is characterized in that the chemical structural formula of the p-benzoquinone derivative is as shown in formula I:
    Figure PCTCN2021114452-appb-100001
    Figure PCTCN2021114452-appb-100001
    其中，所述R 1选自氢、卤素、羟基、氰基、硝基、C1-12烷基、C1-12烷氧基、苯基、卤代C1-6烷基、卤代C1-6烷氧基、C3-6环烷基、单取代苯基或多取代苯基； Wherein, the R is selected from hydrogen, halogen, hydroxyl, cyano, nitro, C1-12 alkyl, C1-12 alkoxy, phenyl, halogenated C1-6 alkyl, halogenated C1-6 alkane Oxygen, C3-6 cycloalkyl, mono-substituted phenyl or multi-substituted phenyl;
    所述R 2选自氢、卤素、羟基、氰基、硝基、C1-12烷基、C1-12烷氧基、苯基、卤代C1-6烷基、卤代C1-6烷氧基、C3-6环烷基、单取代苯基或多取代苯基； The R is selected from hydrogen, halogen, hydroxyl, cyano, nitro, C1-12 alkyl, C1-12 alkoxy, phenyl, halogenated C1-6 alkyl, halogenated C1-6 alkoxy , C3-6 cycloalkyl, mono-substituted phenyl or multi-substituted phenyl;
    所述R 3选自氢、卤素、羟基、氰基、硝基、C1-12烷基、C1-12烷氧基、苯基、卤代C1-6烷基、卤代C1-6烷氧基、C3-6环烷基、单取代苯基或多取代苯基； The R is selected from hydrogen, halogen, hydroxyl, cyano, nitro, C1-12 alkyl, C1-12 alkoxy, phenyl, halogenated C1-6 alkyl, halogenated C1-6 alkoxy , C3-6 cycloalkyl, mono-substituted phenyl or multi-substituted phenyl;
    所述R 4选自氢、卤素、羟基、氰基、硝基、C1-12烷基、C1-12烷氧基、苯基、卤代C1-6烷基、卤代C1-6烷氧基、C3-6环烷基、单取代苯基或多取代苯基。 The R is selected from hydrogen, halogen, hydroxyl, cyano, nitro, C1-12 alkyl, C1-12 alkoxy, phenyl, halogenated C1-6 alkyl, halogenated C1-6 alkoxy , C3-6 cycloalkyl, mono-substituted phenyl or multi-substituted phenyl.
  6. 根据权利要求5所述的应用，其特征在于，所述对苯醌衍生物包括2-甲基-1,4-对苯醌、2-叔丁基-1,4-对苯醌、2-苯基-1,4-对苯醌、2-氯-1,4-对苯醌、2-溴-1,4-对苯醌、百里醌、2,5-二甲基-1,4-对苯醌、2,6-二甲基-1,4- 对苯醌、2,6-二甲氧基-1,4-对苯醌、2,5-二苯基-1,4-对苯醌、2-氯-5-甲基-1,4-对苯醌、2,5-二氯-1,4-对苯醌、2,6-二氯-1,4-对苯醌、四氟-1,4-对苯醌、四氯-1,4-对苯醌、四溴-1,4-对苯醌和2,3-二氯-5,6-二氰基-1,4-苯醌中的一种或几种。The application according to claim 5, wherein the p-benzoquinone derivatives include 2-methyl-1,4-p-benzoquinone, 2-tert-butyl-1,4-p-benzoquinone, 2- Phenyl-1,4-p-benzoquinone, 2-chloro-1,4-p-benzoquinone, 2-bromo-1,4-p-benzoquinone, thymoquinone, 2,5-dimethyl-1,4 -p-benzoquinone, 2,6-dimethyl-1,4-p-benzoquinone, 2,6-dimethoxy-1,4-p-benzoquinone, 2,5-diphenyl-1,4- p-benzoquinone, 2-chloro-5-methyl-1,4-p-benzoquinone, 2,5-dichloro-1,4-p-benzoquinone, 2,6-dichloro-1,4-p-benzoquinone , tetrafluoro-1,4-p-benzoquinone, tetrachloro-1,4-p-benzoquinone, tetrabromo-1,4-p-benzoquinone and 2,3-dichloro-5,6-dicyano-1 , One or more of 4-benzoquinones.
  7. 根据权利要求6所述的应用，其特征在于，所述对苯醌衍生物包括2-甲基-1,4-对苯醌、2-苯基-1,4-对苯醌、2,6-二氯-1,4-对苯醌、四氟-1,4-对苯醌和2,3-二氯-5,6-二氰基-1,4-苯醌中的一种或几种。The application according to claim 6, wherein the p-benzoquinone derivatives include 2-methyl-1,4-p-benzoquinone, 2-phenyl-1,4-p-benzoquinone, 2,6 One or more of -dichloro-1,4-p-benzoquinone, tetrafluoro-1,4-p-benzoquinone and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone kind.
  8. 根据权利要求7所述的应用，其特征在于，所述对苯醌衍生物包括2-甲基-1,4-对苯醌和/或2-苯基-1,4-对苯醌。The application according to claim 7, characterized in that the p-benzoquinone derivatives include 2-methyl-1,4-p-benzoquinone and/or 2-phenyl-1,4-p-benzoquinone.
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