WO2021175123A1 - 1,4-naphthoquinone compound for resisting novel coronavirus and medical use thereof - Google Patents

1,4-naphthoquinone compound for resisting novel coronavirus and medical use thereof Download PDF

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WO2021175123A1
WO2021175123A1 PCT/CN2021/077207 CN2021077207W WO2021175123A1 WO 2021175123 A1 WO2021175123 A1 WO 2021175123A1 CN 2021077207 W CN2021077207 W CN 2021077207W WO 2021175123 A1 WO2021175123 A1 WO 2021175123A1
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compound
naphthoquinone
naphthoquinone compound
hydrogen
methyl
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PCT/CN2021/077207
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French (fr)
Chinese (zh)
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贾金平
崔家华
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上海交通大学
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Priority claimed from CN202010133985.0A external-priority patent/CN111233649B/en
Priority claimed from CN202011544212.8A external-priority patent/CN112645809B/en
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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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C50/00—Quinones
    • C07C50/26—Quinones containing groups having oxygen atoms singly bound to carbon atoms
    • C07C50/32—Quinones containing groups having oxygen atoms singly bound to carbon atoms the quinoid structure being part of a condensed ring system having two rings

Definitions

  • the invention belongs to the field of medicine, and relates to a 1,4-naphthoquinone compound against a new type of coronavirus and its medical use; more specifically, to a 1,4-naphthoquinone compound based on a juglone structure and the same Use in the preparation of drugs against the new coronavirus 2019-nCoV.
  • 2019 Novel Coronavirus is a positive-stranded single-stranded RNA coronavirus with an envelope, which has caused acute viral pneumonia that has recently spread globally (Zhou et al. a new coronavirus of probable bat origin.Nature,579:270-273.).
  • Viruses are non-cellular microorganisms, which are mainly composed of external proteins and internal nucleic acids. They lack an independent metabolic structure and can only parasitize in host cells. They use host cell proteins and nucleic acids as essential materials for their survival and reproduction. Viruses replicate nucleic acids in host cells, synthesize proteins, and assemble them together to form a complete virion. The process of reproduction is called virus replication.
  • RNA of the new coronavirus contains nearly 30,000 bases, and its genome encodes two partially overlapping polyproteins pp1a and pp1ab (Wu et al.2020.A new coronavirus associated with human respiratory disease in China.Nature, 579:265-269).
  • the functional protein necessary for virus replication is derived from the cleavage of polyprotein by the virus 3CL protease (3-Chymotrypsin-like protease, 3CL pro ). There are currently more than 11 known cleavage sites; this protease is also a polyprotein.
  • a part of the protein is released from the polyprotein by cutting its own C-terminal peptide bond (Chen et al.2020.Prediction of the SARS-CoV-2(2019-nCoV)3C-like protease(3CLpro)structure: virtual screening reveals velpatasvir, ledipasvir, and other drug repurposing candidates. F1000Research 9:129). Because the 3CL protease of the new coronavirus plays a vital role in its replication process, and at the same time promotes self-reproduction by damaging host cells, it is an important target for the development of anti-new coronavirus drugs.
  • Natural medicine also known as Chinese herbal medicine in China, is an important part of medicine. Since ancient times, natural medicine has played an extremely important role in the survival and health of the Chinese nation.
  • the material basis for natural medicines to exert their physiological effects is the active ingredients; the separation of active compounds from natural medicines, structural modification and transformation are important means of drug discovery at present.
  • the joint research team of Shanghai Institute of Pharmacy and Shanghai University of Science and Technology will focus on drug screening for listed drugs and self-built “highly patented compound database” and "medicinal plant-derived compound database”.
  • the naphtha structure produces a large amount of reactive oxygen species in the host cell through the redox cycle, and has a Michael addition reaction with biological macromolecules such as proteins and nucleic acids, which has a strong killing effect on normal host cells (Zhang et al. 2018. Advance in Anti-tumor Mechanisms of Shikonin, Alkannin and Their Derivatives. Mini-Rev. Med. Chem., 18:164-172.). This defect limits the clinical application of shikonin as an anti-new coronavirus drug.
  • the traditional Chinese medicine Qinglongyi (Walnut green peel) is the exocarp of the immature fruit of Juglans mandshurica Maxim. It can be used for the treatment of "heat toxins" such as carbuncle swelling and poison (Shandong Chinese Herbal Medicine Handbook Writing Group, Shandong Chinese Herbal Medicine Handbook. First edition; Shandong Provincial People's Publishing House: Jinan, June 1970).
  • Juglone Juglone, 3
  • Juglone, ⁇ -hydrojuglone, and ⁇ -hydrojuglone are the main ingredients in Qinglongyi (Wang Haixiang, research progress on the chemical composition and action mechanism of juglone green peel.
  • juglone as the active ingredient in Qinglongyi has anti-tumor, analgesic, insecticidal, antibacterial and antiviral activities (Strugstad, M, et al., A summary of extraction, properties, and potential uses of juglone: a literature) review.J.Ecosyst.Manage.2012,13,1-16.), its anti-tumor effect is a research hotspot in recent years.
  • the purpose of the present invention is to overcome the above-mentioned shortcomings of the prior art, and provide a 1,4-naphthoquinone novel coronavirus 3CL enzyme inhibitor based on the jugquinone structure, and a preparation method and medical use thereof.
  • the results of pharmacological studies show that the 1,4-naphthoquinone compounds of the present invention have inhibitory activity on the 2019-nCoV virus 3CL protease (3C-like proteinase, 3CL pr o ), and the enzyme inhibitory activity of some compounds is stronger than that of comfrey It has an inhibitory effect on the replication of the virus in the host cell. Compared with shikonin, these compounds have reduced toxicity to normal cells of the host.
  • the present invention relates to a naphthoquinone compound based on a juglone structure.
  • the structure of the naphthoquinone compound is shown in formula (I):
  • R 4 is hydrogen, methyl, ethyl, acetyl or propionyl
  • R 5 is hydrogen, methyl or ethyl
  • R 4 is hydrogen, methyl or ethyl.
  • the R 6 is methyl or ethyl.
  • R 5 is hydrogen, methyl or ethyl.
  • the present invention relates to a naphthoquinone compound based on the menadione structure, characterized in that the structure of the naphthoquinone compound is as shown in formula (V):
  • R is a hydrogen atom, a methyl group, an acetyl group or a hydroxyl group
  • R 1 is a hydrogen, a methoxy group, a benzyloxy group or a benzoyloxy group.
  • R 1 is hydrogen or a methoxy group
  • R 1 is a hydrogen, benzyloxy or benzoyloxy group.
  • R 7 is hydrogen or methoxy.
  • the R 7 is hydrogen or methoxy.
  • R 7 is hydrogen or methoxy.
  • R 7 is hydrogen, benzyloxy or benzoyloxy group.
  • the present invention relates to the use of a naphthoquinone compound as described in the above structure I and structure V in the preparation of anti-new coronavirus 2019-nCoV drugs.
  • the present invention relates to the use of the naphthoquinone compounds described in the above structures II, III, VI, VII, VIII, IX in the preparation of anti-new coronavirus 2019-nCoV drugs.
  • the present invention relates to the use of a naphthoquinone compound as described in the above structure I and structure V in the preparation of a drug for inhibiting the proteolytic enzyme of the novel coronavirus 2019-nCoV 3CL.
  • the present invention relates to the use of the naphthoquinone compounds described in the above structures II, III, VI, VII, VIII, IX in the preparation of drugs for inhibiting the proteolytic enzyme of the novel coronavirus 2019-nCoV 3CL.
  • the present invention takes its 3CL hydrolase as the target point.
  • the chemical components and pharmacological activities of the traditional Chinese medicine Qinglongyi were systematically studied, and juglone was determined as the main active component.
  • Jugquinone and its derivatives are synthesized by chemical methods.
  • the prepared compound is sent to the New Drug Screening Center of Shanghai Institute of Materia Medica for high-throughput screening.
  • the high-throughput screening model is the 3CL proteolytic enzyme of the 2019-nCoV novel coronavirus.
  • the screening results showed that in the in vitro enzyme inhibitory activity experiment, the synthesized 1,4-naphthoquinone compounds showed very strong inhibitory activity on 3CL proteolytic enzymes. Some of the compounds have an inhibitory rate of over 90% against the 2019-nCoV novel coronavirus 3CL proteolytic enzyme at a concentration of 1 ⁇ M.
  • the cell-level toxicity test results show that the 1,4-naphthoquinone compound of the present invention has reduced cytotoxicity compared with shikonin; some compounds can inhibit the replication of 2019-nCoV novel coronavirus in host cells .
  • the 1,4-naphthoquinone compound described in this patent has a clear structure, simple preparation method and high yield. Taking this class of compounds as new drug candidates, the development of highly effective and low-toxic anti-2019-nCoV new coronavirus external preparations, oral preparations, injections and other drugs has good application prospects.
  • the present invention has the following beneficial effects:
  • the 1,4-naphthoquinone compound of the present invention has a clear structure, a simple preparation method, is suitable for industrial production, and has a prospect of further development.
  • Figure 1 shows the chemical structures of shikonin, ebselen and juglone
  • Figure 2 is a schematic diagram of the in vitro anti-new coronavirus activity test results of some compounds of the present invention; among them, (a) is the in vitro anti-new coronavirus activity test results of the compound (VII-1); (b) is the compound (IX-1) ) In vitro anti-new coronavirus activity test results.
  • This embodiment relates to a preparation method of 5-hydroxy-1,4-naphthoquinone (juglone, II-1) with structural formula (II), including the following steps:
  • This embodiment relates to a preparation method of 5-methoxy-1,4-naphthoquinone (Jugtoquinone methyl ether, II-2) with structural formula (II), which includes the following steps:
  • Example 2 The compound II-1 (3g, 17.2mmol) described in Example 1 was dissolved in 90mL of THF-H 2 O (2:1) mixed solution, and tetrabutylammonium bromide (1.6g, 5.2 mmol), sodium hydroxide (11.2g, 64.3mmol) and sodium hydroxide (9.6g, 240mmol). After the addition, under the protection of N 2 , dimethyl sulfate (10 mL, 103 mmol) was added dropwise. After the addition is complete, react at room temperature for 4 hours for post-treatment.
  • the reaction solution was extracted with ethyl acetate (80 mL ⁇ 3), and the organic layers were combined; the organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure.
  • the residue was purified by silica gel column chromatography (mobile phase was ethyl acetate-petroleum ether, volume ratio 1:5) to obtain 3.3 g of white crystalline powder.
  • Dissolve the white crystalline powder in dichloromethane-acetonitrile (3:1, V/V) cool the solution to about 5°C, drop 2.5 times the equivalent of an aqueous solution of cerium ammonium nitrate under stirring, and continue stirring until the reaction materials disappear.
  • This embodiment relates to a preparation method of 5-acetoxy-1,4-naphthoquinone (5-acetyl juglone, III-1) with structural formula (III), which includes the following steps:
  • the compound II-1 (3 g, 17.2 mmol) described in Example 1 was dissolved in 10 mL of acetic anhydride, and 5 drops of concentrated sulfuric acid were added dropwise with stirring. The reaction solution was placed in an ice-water bath and stirred for 2 hours. Afterwards, the reaction solution was suction filtered, washed with ice water and a small amount of methanol successively, to obtain about 3.4 g of compound III-1, which was pale yellow powder crystals, with a yield of 91%.
  • This embodiment relates to a preparation method of 7-methyl-5-hydroxy-1,4-naphthoquinone (7-methyl juglone, IV-1) with structural formula (IV), which includes the following steps:
  • This embodiment relates to a preparation method of 7-methyl-5-benzyloxy-1,4-naphthoquinone (7-methyl juglone benzyl ether, IV-2) of structural formula (IV), including the following step:
  • This embodiment relates to a preparation method of 2-methyl-1,4-naphthoquinone (VI-1) with structural formula (VI), including the following steps:
  • This embodiment relates to a preparation method of 2-acetyl-8-methoxy-1,4-naphthoquinone (VII-1) with structural formula (VII), including the following steps:
  • This embodiment relates to a preparation method of 2-hydroxy-1,4-naphthoquinone (VIII-1) with structural formula (VIII), including the following steps:
  • 1,4-Naphthoquinone (3.16 g, 20 mmol) was dissolved in acetic anhydride (20 mL), and 4 drops of concentrated sulfuric acid were added dropwise. The mixture was stirred and reacted for 8 hours in an ice-water bath. The reaction liquid was suction filtered, and the filter cake was washed with petroleum ether and a small amount of pre-cooled absolute ethanol to obtain about 4.72 g of off-white powder. The powder was dissolved in methanol, 0.5 g of sodium methoxide was added in an ice-water bath, and the mixture was stirred and reacted in an ice-water bath for 4 hours, and then filtered with suction.
  • This embodiment relates to a preparation method of 1,4-naphthoquinone (IX-1) with structural formula (IX), including the following steps:
  • the 3CL hydrolase inhibitory activity was determined using the enzymatic test method established by the Shanghai Institute of Materia Medica and the Institute of Immunochemistry of Shanghai University of Science and Technology to fight against 2019-nCoV virus infection.
  • the 3CL hydrolase expressed by the eukaryotic system Using the 96-channel automatic sample loading system equipped with the high-throughput drug screening platform, the 3CL hydrolase expressed by the eukaryotic system, its characteristic oligopeptide substrate (the oligopeptide substrate is connected to the fluorescent group through an amide bond) and the The test compound is placed in the incubation system.
  • a blank control group without inhibitors was set, and shikonin was used as the positive control drug; the final concentration of each inhibitor in the incubation system was 1 ⁇ mol/L, and 3 replicate wells were set.
  • the inhibition rate was calculated based on the fluorescence intensity of the substrate in each well after hydrolysis.
  • the specific experimental method is to inoculate 293A cells in a 96-well plate at a concentration of 0.5*10 5 cells/well, and carry out plasmid transfection after culturing for 18-20 hours.
  • 293A cells were transfected into 293A cells at the ratio of 3CL protein pellet pcDNA4-3C:pcDNA3-mYFP at 50ng:100ng per well, 4h later, the solvent was replaced with DMSO, 1 ⁇ mol/L of the small molecule compound to be tested, or 10 ⁇ mol/L shikonin.
  • For the culture medium set 3 replicate wells for each concentration. After 72h, discard the culture medium, wash twice with PBS, use PerkinElmer EnVision multi-function microplate reader to detect the fluorescence intensity of each well under excitation wavelength Ex (500nm) and emission wavelength Em (535nm), and record the analysis.
  • Inhibition rate (average fluorescence value of the control group-average fluorescence value of the administration group)/average fluorescence value of the control group. The measurement results are shown in Table 1.
  • the method for determining the 3CL proteolytic enzyme inhibitory activity of the 2019-nCoV novel coronavirus of Example 5 to Example 9 includes the following steps:
  • the incubation system contains 2019-nCoV 3CL protease (0.2 ⁇ M), fluorescently labeled peptide (20 ⁇ M) and a series of concentrations of the test compound (0-20 ⁇ M).
  • the fluorescence intensity of the system when incubated for 2-3 minutes was measured by a microplate reader.
  • the excitation wavelength and detection wavelength were 320nm and 405nm, respectively.
  • the enzyme inhibition rate of the analyte under different concentrations is calculated. All experiments were repeated 3 times, and the IC 50 value of the analyte inhibited enzyme was calculated by Prism5 software.
  • Inhibition rate (average fluorescence value of blank control group-average fluorescence value of administration group)/average fluorescence value of blank control group*100%. The measurement results are shown in Table 1.
  • the test results showed that the positive control substance shikonin at a concentration of 10 ⁇ M, its enzyme inhibition rate was only 51.4%.
  • This example relates to the determination of the growth inhibitory activity of the compounds described in Examples 1-9 on normal host HSF cells.
  • HSF cells in good growth condition, trypsinize, adjust the cell suspension to an appropriate concentration with complete medium, inoculate 5000 cells/well in a 96-well plate, and place it in an incubator for 24 hours, and wait until the cells are attached.
  • Complete medium (100 ⁇ L) containing the compound to be tested was added to each well behind the wall, and three multiple wells were set up in each group.
  • Each 96-well plate is equipped with a zero adjustment hole (only compound and medium, no cells) and a blank control hole (only cell and medium, no compound). After 72 hours of incubation in the incubator, add 20 ⁇ L/well of MTT solution.
  • Example 1-9 The compound which inhibits cell growth IC 50 values were higher than the IC 50 value of the positive control shikonin.
  • This example relates to the in vitro anti-new coronavirus activity determination of the compound (VII-1) described in Example 7 and the compound (IX-1) described in Example 9.
  • the in vitro antiviral activity test of the test compound was performed. Count the well-growing Vero E6 cells, inoculate 1 ⁇ 10 4 cells per well into a 96-well plate, and incubate in a 37°C cell incubator for 24 hours to make the cells adhere to the wall. The cells were pretreated with the candidate drug (10 ⁇ M) for 1 h, and then the 2019-nCoV virus was added (the MOI parameter was set to 0.01), and the cells were infected for 2 h. The supernatant medium containing the virus and the drug to be tested is removed, and Vero E6 cells are further cultured with the newly prepared medium containing the drug to be tested.
  • the positive control, shikonin is a compound that has been reported to have inhibitory activity on the new coronavirus 3CL hydrolase, and may be used for the treatment of the new coronavirus.
  • Clinical studies during the "SARS" period have shown that oral shikonin and its derivatives have a significant effect on patients with early SARS virus infection (Wang, F., Method of treatment of virus infections using shikonin compounds. US Patent, No. 7897640 ).
  • the naphthoquinone compound of the present invention shows stronger 3CL hydrolase inhibitory activity than the positive control shikonin, some compounds have low toxicity to normal human cells, have anti-new coronavirus activity, and have good application prospects.
  • the 1,4-naphthoquinone compound in the present invention has a simple preparation method, easy-to-obtain raw materials and high yield.
  • the in vitro test results show that this type of compound has a very strong inhibitory activity on the 3CL hydrolase of the 2019-nCoV novel coronavirus.
  • the naphthoquinone compounds in the present invention can be used to prepare oral, injection or compound drugs against the new coronavirus.
  • the drug or compound drug can inhibit the 3CL hydrolase of the new coronavirus, thereby inhibiting its replication process in the host cell, and is used for the treatment of the new coronavirus infection.

Abstract

Disclosed are a 1,4-naphthoquinone compound for resisting novel coronavirus and the medical use thereof. The structure of the compound is as shown in formula (X): (X), wherein R1 is hydrogen, hydroxy, methoxy, ethoxy, benzyloxy, acetyloxy, propionyloxy or benzoyloxy; R2 is hydrogen, methyl or ethyl; and R3 is hydrogen, hydroxy, methyl or acetyl. The naphthoquinone compound can inhibit 3CL hydrolase (3C-like proteinase, 3CLpro) of the 2019 novel coronavirus (2019-nCOV), and has an anti-novel coronavirus activity. The 1,4-naphthoquinone compound has a clear structure, simple preparation method, high yield, and is of great significance for developing a novel anti-novel coronavirus drug with a high efficiency and a low toxicity.

Description

一种抗新型冠状病毒的1,4-萘醌类化合物及其医药用途A 1,4-naphthoquinone compound against novel coronavirus and its medical use 技术领域Technical field
本发明属于医药领域，涉及一种抗新型冠状病毒的1,4-萘醌类化合物及其医药用途；更更具体的，涉及一种基于胡桃醌结构的1,4-萘醌类化合物及其在制备抗新型冠状病毒2019-nCoV的药物中的用途。The invention belongs to the field of medicine, and relates to a 1,4-naphthoquinone compound against a new type of coronavirus and its medical use; more specifically, to a 1,4-naphthoquinone compound based on a juglone structure and the same Use in the preparation of drugs against the new coronavirus 2019-nCoV.
背景技术Background technique
2019新型冠状病毒(2019-nCoV)是一种具有包膜的正链单股RNA冠状病毒，它引起了近期在全球范围内流行的急性病毒性肺炎(Zhou et al.2020.A pneumonia outbreak associated with a new coronavirus of probable bat origin.Nature,579:270-273.)。病毒属于非细胞微生物，主要由外部蛋白质和内部核酸组成，缺乏独立的代谢结构，只能寄生在宿主细胞内，利用宿主细胞的蛋白质、核酸作为其生存和繁殖的必须物质。病毒在宿主细胞内复制核酸，合成蛋白质，再装配在一起构成完整病毒体的繁殖过程称为病毒复制。新型冠状病毒的核酸(RNA)包含近3万个碱基，其基因组编码2个部分重叠的多聚蛋白pp1a及pp1ab(Wu et al.2020.A new coronavirus associated with human respiratory disease in China.Nature,579:265‐269)。病毒复制过程中所必须的功能性蛋白源于病毒3CL蛋白酶(3‐Chymotrypsin‐like protease,3CL pro)对多聚蛋白的切割，目前已知的剪切位点超过11处；这个蛋白酶也是多聚蛋白的一部分，通过剪切其自身C端的肽键从多聚蛋白中释放(Chen et al.2020.Prediction of the SARS‐CoV‐2(2019‐nCoV)3C‐like protease(3CLpro)structure:virtual screening reveals velpatasvir,ledipasvir,and other drug repurposing candidates.F1000Research 9:129)。由于新型冠状病毒的3CL蛋白酶在其复制过程中起着至关重要的作用，同时又通过损伤宿主细胞促进自身繁殖，是抗新型冠状病毒药物研发的重要靶标。 2019 Novel Coronavirus (2019-nCoV) is a positive-stranded single-stranded RNA coronavirus with an envelope, which has caused acute viral pneumonia that has recently spread globally (Zhou et al. a new coronavirus of probable bat origin.Nature,579:270-273.). Viruses are non-cellular microorganisms, which are mainly composed of external proteins and internal nucleic acids. They lack an independent metabolic structure and can only parasitize in host cells. They use host cell proteins and nucleic acids as essential materials for their survival and reproduction. Viruses replicate nucleic acids in host cells, synthesize proteins, and assemble them together to form a complete virion. The process of reproduction is called virus replication. The nucleic acid (RNA) of the new coronavirus contains nearly 30,000 bases, and its genome encodes two partially overlapping polyproteins pp1a and pp1ab (Wu et al.2020.A new coronavirus associated with human respiratory disease in China.Nature, 579:265-269). The functional protein necessary for virus replication is derived from the cleavage of polyprotein by the virus 3CL protease (3-Chymotrypsin-like protease, 3CL pro ). There are currently more than 11 known cleavage sites; this protease is also a polyprotein. A part of the protein is released from the polyprotein by cutting its own C-terminal peptide bond (Chen et al.2020.Prediction of the SARS-CoV-2(2019-nCoV)3C-like protease(3CLpro)structure: virtual screening reveals velpatasvir, ledipasvir, and other drug repurposing candidates. F1000Research 9:129). Because the 3CL protease of the new coronavirus plays a vital role in its replication process, and at the same time promotes self-reproduction by damaging host cells, it is an important target for the development of anti-new coronavirus drugs.
天然药物，在中国又被称为中草药，是药物的一个重要组成部分。自古以来，天然药物对中华民族的生存和健康起到了极其重要的作用。天然药物发挥生理作用的物质基础是其中的活性成分；从天然药物中分离活性化合物、进行结构修饰和改造是目前药物发现的重要手段。2020年，上海药物所与上海科技大学联合攻关团队重点针对已上市药物以及自建的“高成药性化合物数据库”和“药用植物来源化合物成分数据库”进行药物筛 选，从超过10000个化合物中，发现了包括天然萘醌类化合物紫草素(Shikonin,1)、农药中间体依布硒(Ebselen,2)在内的多个化合物，对新型冠状病毒的3CL酶具有抑制活性(Jin et al.2020.Structure of Mpro from SARS‐CoV‐2 and discovery of its inhibitors.Nature,582:289–293)。由于紫草素分子中具有萘茜结构，可与宿主细胞内的金属离子发生络合。同时，萘茜结构通过氧化还原循环在宿主细胞内产生大量活性氧，并与蛋白质、核酸等生物大分子发生Michael加成反应，对于宿主正常细胞有很强的杀伤作用(Zhang et al.2018.Advance in Anti‐tumor Mechanisms of Shikonin,Alkannin and Their Derivatives.Mini‐Rev.Med.Chem.,18:164‐172.)。这一缺陷，限制了紫草素作为抗新型冠状病毒药物的临床应用。Natural medicine, also known as Chinese herbal medicine in China, is an important part of medicine. Since ancient times, natural medicine has played an extremely important role in the survival and health of the Chinese nation. The material basis for natural medicines to exert their physiological effects is the active ingredients; the separation of active compounds from natural medicines, structural modification and transformation are important means of drug discovery at present. In 2020, the joint research team of Shanghai Institute of Pharmacy and Shanghai University of Science and Technology will focus on drug screening for listed drugs and self-built "highly patented compound database" and "medicinal plant-derived compound database". From more than 10,000 compounds, Several compounds including the natural naphthoquinone compound shikonin (Shikonin, 1) and the pesticide intermediate ebselen (Ebselen, 2) have been discovered, which have inhibitory activity on the 3CL enzyme of the new coronavirus (Jin et al. 2020. Structure of Mpro from SARS-CoV-2 and discovery of its inhibitors. Nature, 582:289–293). Because the shikonin molecule has a naphtha structure, it can complex with the metal ions in the host cell. At the same time, the naphtha structure produces a large amount of reactive oxygen species in the host cell through the redox cycle, and has a Michael addition reaction with biological macromolecules such as proteins and nucleic acids, which has a strong killing effect on normal host cells (Zhang et al. 2018. Advance in Anti-tumor Mechanisms of Shikonin, Alkannin and Their Derivatives. Mini-Rev. Med. Chem., 18:164-172.). This defect limits the clinical application of shikonin as an anti-new coronavirus drug.
中药青龙衣(胡桃青皮)是胡桃楸(Juglans mandshurica Maxim.)未成熟果实的外果皮，可用于疮痈肿毒等“热毒症”的治疗(山东中草药手册编写小组,《山东中草药手册》。第一版；山东省人民出版社:济南,1970年6月)。胡桃醌(Juglone，3)及其衍生物胡桃苷、α-氢化胡桃醌、β-氢化胡桃醌是青龙衣中的主要成份(王海香，胡桃属植物青皮的化学成分及作用机理研究进展.生物质化学工程,2008,42(1):47-52)。其中，胡桃醌作为青龙衣中的活性成分具有抗肿瘤、镇痛、杀虫、抗菌及抗病毒活性(Strugstad,M,et al.,A summary of extraction,properties,and potential uses of juglone:a literature review.J.Ecosyst.Manage.2012,13,1-16.)，其抗肿瘤作用是近年来的研究热点。The traditional Chinese medicine Qinglongyi (Walnut green peel) is the exocarp of the immature fruit of Juglans mandshurica Maxim. It can be used for the treatment of "heat toxins" such as carbuncle swelling and poison (Shandong Chinese Herbal Medicine Handbook Writing Group, Shandong Chinese Herbal Medicine Handbook. First edition; Shandong Provincial People's Publishing House: Jinan, June 1970). Juglone (Juglone, 3) and its derivatives Juglone, α-hydrojuglone, and β-hydrojuglone are the main ingredients in Qinglongyi (Wang Haixiang, research progress on the chemical composition and action mechanism of juglone green peel. Biomass Chemical Engineering, 2008, 42(1): 47-52). Among them, juglone as the active ingredient in Qinglongyi has anti-tumor, analgesic, insecticidal, antibacterial and antiviral activities (Strugstad, M, et al., A summary of extraction, properties, and potential uses of juglone: a literature) review.J.Ecosyst.Manage.2012,13,1-16.), its anti-tumor effect is a research hotspot in recent years.
发明内容Summary of the invention
本发明的目的在于克服上述现有技术的不足，提供一种基于胡桃醌结构的1,4-萘醌类新型冠状病毒3CL酶抑制剂及其制备方法和医药用途。药理学研究结果显示，本发明的1,4-萘醌类化合物对2019-nCoV病毒3CL蛋白水解酶(3C－like proteinase，3CL pr o)具有抑制活性，部分化合物的酶抑制活性强于紫草素，对病毒在宿主细胞内的复制有抑制作用。该类化合物与紫草素相比，对宿主正常细胞的毒性降低。 The purpose of the present invention is to overcome the above-mentioned shortcomings of the prior art, and provide a 1,4-naphthoquinone novel coronavirus 3CL enzyme inhibitor based on the jugquinone structure, and a preparation method and medical use thereof. The results of pharmacological studies show that the 1,4-naphthoquinone compounds of the present invention have inhibitory activity on the 2019-nCoV virus 3CL protease (3C-like proteinase, 3CL pr o ), and the enzyme inhibitory activity of some compounds is stronger than that of comfrey It has an inhibitory effect on the replication of the virus in the host cell. Compared with shikonin, these compounds have reduced toxicity to normal cells of the host.
本发明的目的是通过以下技术方案实现的：The purpose of the present invention is achieved through the following technical solutions:
我们用活性追踪的方法，对中药青龙衣中的化学成分和药理活性进行了系统的研究，确定胡桃醌为其中的主要活性成分。随后以胡桃醌为先导物进行其结构改造，合成了基于胡桃醌结构的1,4-萘醌类化合物；测定了该类化合物抑制新型抗新型冠状病毒3CL酶的活性，测试化合物对宿主正常细胞HSF的毒性及抗病毒活性。We used the method of activity tracking to systematically study the chemical components and pharmacological activities of the traditional Chinese medicine Qinglongyi, and determined that juglone is the main active ingredient. Subsequently, jugquinone was used as the lead for its structural modification, and 1,4-naphthoquinone compounds based on the jugquinone structure were synthesized; the activity of this type of compound in inhibiting the new anti-new coronavirus 3CL enzyme was determined, and the test compound has an effect on normal host cells. The toxicity and antiviral activity of HSF.
第一方面，本发明涉及一种基于胡桃醌结构的萘醌类化合物，所述萘醌类化合物的 结构如式(Ⅰ)所示：In the first aspect, the present invention relates to a naphthoquinone compound based on a juglone structure. The structure of the naphthoquinone compound is shown in formula (I):
Figure PCTCN2021077207-appb-000001
其中，R 4为氢、甲基、乙基、乙酰基或丙酰基；R 5为氢、甲基或乙基。
Figure PCTCN2021077207-appb-000001
Wherein, R 4 is hydrogen, methyl, ethyl, acetyl or propionyl; R 5 is hydrogen, methyl or ethyl.
作为本发明的一个实施方案，所述萘醌类化合物结构如式(Ⅱ)所示：As an embodiment of the present invention, the structure of the naphthoquinone compound is shown in formula (II):
Figure PCTCN2021077207-appb-000002
其中，R 4为氢、甲基或乙基。
Figure PCTCN2021077207-appb-000002
Wherein, R 4 is hydrogen, methyl or ethyl.
作为本发明的又一个实施方案，所述萘醌类化合物结构如式(Ⅲ)所示：As another embodiment of the present invention, the structure of the naphthoquinone compound is shown in formula (III):
Figure PCTCN2021077207-appb-000003
所述R 6为甲基或乙基。
Figure PCTCN2021077207-appb-000003
The R 6 is methyl or ethyl.
作为本发明的又一个实施方案，所述萘醌类化合物结构如式(Ⅳ)所示：As another embodiment of the present invention, the structure of the naphthoquinone compound is shown in formula (IV):
Figure PCTCN2021077207-appb-000004
其中，R 5为氢、甲基或乙基。
Figure PCTCN2021077207-appb-000004
Wherein, R 5 is hydrogen, methyl or ethyl.
第二方面，本发明涉及一种基于甲萘醌结构的萘醌类化合物，其特征在于，所述萘醌类化合物的结构如式(Ⅴ)所示：In the second aspect, the present invention relates to a naphthoquinone compound based on the menadione structure, characterized in that the structure of the naphthoquinone compound is as shown in formula (V):
Figure PCTCN2021077207-appb-000005
其中，R为氢原子、甲基、乙酰基或羟基，R 1为氢、甲氧基、苄氧基或苯甲酰氧基团。优选，R为甲基、乙酰基或羟基时，R 1为氢或甲氧基；R为氢 原子时，R 1为氢、苄氧基或苯甲酰氧基团。
Figure PCTCN2021077207-appb-000005
Wherein, R is a hydrogen atom, a methyl group, an acetyl group or a hydroxyl group, and R 1 is a hydrogen, a methoxy group, a benzyloxy group or a benzoyloxy group. Preferably, when R is a methyl group, an acetyl group or a hydroxyl group, R 1 is hydrogen or a methoxy group; when R is a hydrogen atom, R 1 is a hydrogen, benzyloxy or benzoyloxy group.
作为本发明的一个实施方案，所述萘醌类化合物结构如式(Ⅵ)所示：As an embodiment of the present invention, the structure of the naphthoquinone compound is shown in formula (VI):
Figure PCTCN2021077207-appb-000006
其中，R 7为氢或甲氧基。
Figure PCTCN2021077207-appb-000006
Among them, R 7 is hydrogen or methoxy.
作为本发明的一个实施方案，所述萘醌类化合物结构如式(Ⅶ)所示：As an embodiment of the present invention, the structure of the naphthoquinone compound is shown in formula (VII):
Figure PCTCN2021077207-appb-000007
所述R 7为氢或甲氧基。
Figure PCTCN2021077207-appb-000007
The R 7 is hydrogen or methoxy.
作为本发明的一个实施方案，所述萘醌类化合物结构如式(Ⅷ)所示：As an embodiment of the present invention, the structure of the naphthoquinone compound is shown in formula (VIII):
Figure PCTCN2021077207-appb-000008
其中，R 7为氢或甲氧基。
Figure PCTCN2021077207-appb-000008
Among them, R 7 is hydrogen or methoxy.
作为本发明的一个实施方案，所述萘醌类化合物结构如式(Ⅸ)所示：As an embodiment of the present invention, the structure of the naphthoquinone compound is shown in formula (IX):
Figure PCTCN2021077207-appb-000009
其中，R 7为氢、苄氧基或苯甲酰氧基团。
Figure PCTCN2021077207-appb-000009
Wherein, R 7 is hydrogen, benzyloxy or benzoyloxy group.
第三方面，本发明涉及一种如上述结构Ⅰ及结构Ⅴ所述的萘醌类化合物，在制备抗新型冠状病毒2019-nCoV药物中的用途。In the third aspect, the present invention relates to the use of a naphthoquinone compound as described in the above structure I and structure V in the preparation of anti-new coronavirus 2019-nCoV drugs.
作为本发明的一个实施方案，涉及上述结构Ⅱ、Ⅲ、Ⅵ、Ⅶ、Ⅷ、Ⅸ所述的萘醌类化合物，在制备抗新型冠状病毒2019-nCoV药物中的用途。As an embodiment of the present invention, it relates to the use of the naphthoquinone compounds described in the above structures II, III, VI, VII, VIII, IX in the preparation of anti-new coronavirus 2019-nCoV drugs.
第四方面，本发明涉及一种如上述结构Ⅰ及结构Ⅴ所述的萘醌类化合物，在制备抑制新型冠状病毒2019-nCoV 3CL蛋白水解酶的药物中的用途。In the fourth aspect, the present invention relates to the use of a naphthoquinone compound as described in the above structure I and structure V in the preparation of a drug for inhibiting the proteolytic enzyme of the novel coronavirus 2019-nCoV 3CL.
作为本发明的一个实施方案，涉及上述结构Ⅱ、Ⅲ、Ⅵ、Ⅶ、Ⅷ、Ⅸ所述的萘醌类化合物，在制备抑制新型冠状病毒2019-nCoV 3CL蛋白水解酶的药物中的用途。As an embodiment of the present invention, it relates to the use of the naphthoquinone compounds described in the above structures II, III, VI, VII, VIII, IX in the preparation of drugs for inhibiting the proteolytic enzyme of the novel coronavirus 2019-nCoV 3CL.
本发明根据目前国内外对2019-nCoV冠状病毒的研究现状，以其3CL水解酶为作用 靶点。同时，用活性追踪的方法，对中药青龙衣中的化学成分和药理活性进行了系统的研究，确定胡桃醌为其中的主要活性成分。通过化学方法合成胡桃醌及其衍生物。所制备的化合物送上海药物研究所新药筛选中心进行高通量筛选，高通量筛选模型为2019-nCoV新型冠状病毒的3CL蛋白水解酶。筛选结果表明：在体外酶抑制活性实验中，所合成的1,4-萘醌类化合物对3CL蛋白水解酶显示了非常强的抑制活性。其中部分化合物在1μM浓度下，对2019-nCoV新型冠状病毒3CL蛋白水解酶的抑制率可达90％以上。此外，细胞水平的毒性测试结果表明，本发明中的1,4-萘醌类化合物与紫草素相比较，其细胞毒性降低；部分化合物可抑制2019-nCoV新型冠状病毒在宿主细胞内的复制。本专利所述的1,4-萘醌类化合物结构明确，其制备方法简便、收率较高。以该类化合物为新药候选物，开发高效低毒的抗2019-nCoV新型冠状病毒的外用制剂、口服制剂及注射剂等药物具有良好的应用前景。According to the current research status of the 2019-nCoV coronavirus at home and abroad, the present invention takes its 3CL hydrolase as the target point. At the same time, using the method of activity tracking, the chemical components and pharmacological activities of the traditional Chinese medicine Qinglongyi were systematically studied, and juglone was determined as the main active component. Jugquinone and its derivatives are synthesized by chemical methods. The prepared compound is sent to the New Drug Screening Center of Shanghai Institute of Materia Medica for high-throughput screening. The high-throughput screening model is the 3CL proteolytic enzyme of the 2019-nCoV novel coronavirus. The screening results showed that in the in vitro enzyme inhibitory activity experiment, the synthesized 1,4-naphthoquinone compounds showed very strong inhibitory activity on 3CL proteolytic enzymes. Some of the compounds have an inhibitory rate of over 90% against the 2019-nCoV novel coronavirus 3CL proteolytic enzyme at a concentration of 1 μM. In addition, the cell-level toxicity test results show that the 1,4-naphthoquinone compound of the present invention has reduced cytotoxicity compared with shikonin; some compounds can inhibit the replication of 2019-nCoV novel coronavirus in host cells . The 1,4-naphthoquinone compound described in this patent has a clear structure, simple preparation method and high yield. Taking this class of compounds as new drug candidates, the development of highly effective and low-toxic anti-2019-nCoV new coronavirus external preparations, oral preparations, injections and other drugs has good application prospects.
与现有技术相比，本发明具有如下的有益效果：Compared with the prior art, the present invention has the following beneficial effects:
1)本发明化合物的制备方法简便，收率较高且原料易得；1) The preparation method of the compound of the present invention is simple, the yield is high, and the raw materials are easily available;
2)体外酶抑制活性实验研究表明，该类化合物对2019-nCoV新型冠状病毒的3CL蛋白水解酶有强抑制活性；2) Experimental studies on enzyme inhibitory activity in vitro show that these compounds have strong inhibitory activity on the 3CL proteolytic enzyme of 2019-nCoV novel coronavirus;
3)体外抗病毒活性实验及细胞水平毒性研究表明，部分化合物对2019-nCoV新型冠状病毒有较强的生长抑制活性，但对宿主细胞的毒性小，具有良好的新药开发前景；3) In vitro antiviral activity experiments and cell-level toxicity studies have shown that some compounds have strong growth inhibitory activity against 2019-nCoV new coronavirus, but have low toxicity to host cells and have a good prospect for new drug development;
4)本发明的1,4-萘醌类化合物，结构明确、制备方法简便、适合工业化生产，有进一步开发的前景。4) The 1,4-naphthoquinone compound of the present invention has a clear structure, a simple preparation method, is suitable for industrial production, and has a prospect of further development.
附图说明Description of the drawings
通过阅读参照以下附图对非限制性实施例所作的详细描述，本发明的其它特征、目的和优点将会变得更明显：By reading the detailed description of the non-limiting embodiments with reference to the following drawings, other features, purposes and advantages of the present invention will become more apparent:
图1为紫草素、依布硒及胡桃醌的化学结构；Figure 1 shows the chemical structures of shikonin, ebselen and juglone;
图2为本发明的部分化合物的体外抗新型冠状病毒活性测试结果示意图；其中，(a)为化合物(Ⅶ-1)的体外抗新型冠状病毒活性测试结果；(b)为化合物(Ⅸ-1)的体外抗新型冠状病毒活性测试结果。Figure 2 is a schematic diagram of the in vitro anti-new coronavirus activity test results of some compounds of the present invention; among them, (a) is the in vitro anti-new coronavirus activity test results of the compound (VII-1); (b) is the compound (Ⅸ-1) ) In vitro anti-new coronavirus activity test results.
具体实施方式Detailed ways
下面结合附图和具体实施例对本发明进行详细说明。以下实施例将有助于本领域的技术人员进一步理解本发明，但不以任何形式限制本发明。应当指出的是，对本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干调整和改进。这些 都属于本发明的保护范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。The present invention will be described in detail below with reference to the drawings and specific embodiments. The following examples will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be pointed out that for those of ordinary skill in the art, several adjustments and improvements can be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention. The experimental methods that do not indicate specific conditions in the following examples usually follow the conventional conditions or the conditions recommended by the manufacturer.
实施例1Example 1
本实施例涉及一种具有结构式(Ⅱ)的5-羟基-1,4-萘醌(胡桃醌，Ⅱ-1)的制备方法，包括以下步骤：This embodiment relates to a preparation method of 5-hydroxy-1,4-naphthoquinone (juglone, Ⅱ-1) with structural formula (II), including the following steps:
Figure PCTCN2021077207-appb-000010
Figure PCTCN2021077207-appb-000010
在250mL三颈瓶内加入75mL 30％H 2O 2，搅拌滴加42mL乙酸酐，滴加中维持反应液温度不超过40℃。滴毕，在40℃搅拌4小时。之后，在剧烈搅拌下向反应液中滴加1,5-二羟基萘(15g,93.7mmol)的100mL甲醇溶液,控制滴加速度使反应溶液温度不超过60℃。滴加完毕后继续搅拌1小时，冷却后倒入冰水中，析出大量棕褐色沉淀，过滤干燥得暗红色固体粉末。采用硅胶柱层析(流动相：乙酸乙酯-石油醚体积比为1：5)进行纯化，得化合物Ⅱ-1约6.5g，呈橙红色针晶，收率40％。 1H-NMR(400MHz,CDCl 3)：δ11.92(1H,OH),7.65(1H,Ar-H),7.63(1H,Ar-H),7.27(1H,Ar-H),6.95(2H,2×Ar-H)。 Add 75 mL of 30% H 2 O 2 into a 250 mL three-necked flask, add 42 mL of acetic anhydride dropwise with stirring, and maintain the temperature of the reaction solution not to exceed 40° C. during the dropwise addition. After dripping, the mixture was stirred at 40°C for 4 hours. Then, under vigorous stirring, a 100 mL methanol solution of 1,5-dihydroxynaphthalene (15 g, 93.7 mmol) was added dropwise to the reaction solution, and the dropping rate was controlled so that the temperature of the reaction solution did not exceed 60°C. After the dripping was completed, the stirring was continued for 1 hour, and then poured into ice water after cooling, a large amount of tan precipitate was precipitated, which was filtered and dried to obtain a dark red solid powder. Purification was carried out by silica gel column chromatography (mobile phase: ethyl acetate-petroleum ether volume ratio 1:5) to obtain about 6.5 g of compound II-1, which was orange-red needle crystals, and the yield was 40%. 1 H-NMR (400MHz, CDCl 3 ): δ11.92 (1H, OH), 7.65 (1H, Ar-H), 7.63 (1H, Ar-H), 7.27 (1H, Ar-H), 6.95 (2H) ,2×Ar-H).
实施例2Example 2
本实施例涉及一种具有结构式(Ⅱ)的5-甲氧基-1,4-萘醌(胡桃醌甲醚，Ⅱ-2)的制备方法，包括以下步骤：This embodiment relates to a preparation method of 5-methoxy-1,4-naphthoquinone (Jugtoquinone methyl ether, II-2) with structural formula (II), which includes the following steps:
Figure PCTCN2021077207-appb-000011
Figure PCTCN2021077207-appb-000011
将实施例1所述化合物Ⅱ-1(3g,17.2mmol)溶于90mL的THF-H 2O(2：1)混合溶液中，在搅拌下依次加入四丁基溴化铵(1.6g,5.2mmol)、保险粉(11.2g,64.3mmol)及氢氧化钠(9.6g,240mmol)。加毕，在N 2保护下，滴加硫酸二甲酯(10mL,103mmol)。滴加完毕后，于室温反应4h后处理。反应液用乙酸乙酯萃取(80mL×3)，合并有机层；有机层用饱和食盐水洗涤，无水硫酸钠干燥，减压浓缩。残留物经硅胶柱色谱(流动相为乙酸乙酯-石油醚，体积比1：5)纯化，得白色晶体粉末3.3g。 将该白色晶体粉末于二氯甲烷-乙腈(3:1，V/V)中溶解，溶液冷却至5℃左右，搅拌下滴入2.5倍当量硝酸铈铵的水溶液，继续搅拌至反应原料消失，二氯甲烷萃取。有机层经饱和氯化钠溶液清洗，无水硫酸钠干燥后蒸干，硅胶柱层析纯化。产物Ⅱ-2呈橙色结晶，总收率74％。 1H NMR(400MHz,CDCl 3):δ7.74(dd,J＝7.6,1.2Hz,1H),7.69(dd,J＝7.6,8.2Hz,1H),7.32(dd,J＝8.2,1.2Hz,1H),6.87(s,2H),4.02(s,3H,OCH 3)。 The compound II-1 (3g, 17.2mmol) described in Example 1 was dissolved in 90mL of THF-H 2 O (2:1) mixed solution, and tetrabutylammonium bromide (1.6g, 5.2 mmol), sodium hydroxide (11.2g, 64.3mmol) and sodium hydroxide (9.6g, 240mmol). After the addition, under the protection of N 2 , dimethyl sulfate (10 mL, 103 mmol) was added dropwise. After the addition is complete, react at room temperature for 4 hours for post-treatment. The reaction solution was extracted with ethyl acetate (80 mL×3), and the organic layers were combined; the organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (mobile phase was ethyl acetate-petroleum ether, volume ratio 1:5) to obtain 3.3 g of white crystalline powder. Dissolve the white crystalline powder in dichloromethane-acetonitrile (3:1, V/V), cool the solution to about 5°C, drop 2.5 times the equivalent of an aqueous solution of cerium ammonium nitrate under stirring, and continue stirring until the reaction materials disappear. Dichloromethane extraction. The organic layer was washed with saturated sodium chloride solution, dried with anhydrous sodium sulfate, evaporated to dryness, and purified by silica gel column chromatography. The product II-2 was orange crystals with a total yield of 74%. 1 H NMR(400MHz, CDCl 3 ): δ7.74(dd,J=7.6,1.2Hz,1H), 7.69(dd,J=7.6,8.2Hz,1H), 7.32(dd,J=8.2,1.2Hz , 1H), 6.87 (s, 2H), 4.02 (s, 3H, OCH 3 ).
实施例3Example 3
本实施例涉及一种具有结构式(Ⅲ)的5-乙酰氧基-1,4-萘醌(5-乙酰基胡桃醌，Ⅲ-1)的制备方法，包括以下步骤：This embodiment relates to a preparation method of 5-acetoxy-1,4-naphthoquinone (5-acetyl juglone, Ⅲ-1) with structural formula (Ⅲ), which includes the following steps:
Figure PCTCN2021077207-appb-000012
Figure PCTCN2021077207-appb-000012
将实施例1所述化合物Ⅱ-1(3g,17.2mmol)溶于10mL乙酸酐中，在搅拌下滴加5滴浓硫酸。将反应液置于冰水浴中搅拌反应2小时。后将反应液抽滤，依次用冰水及少量甲醇清洗，得化合物Ⅲ-1约3.4g，呈淡黄色粉末结晶，收率91％。 1H NMR(400MHz,CDCl 3):δ8.05(dd,J＝7.9,1.0Hz,1H),7.77(t,J＝7.9Hz,1H),7.40(dd,J＝7.9,1.0Hz,1H),6.95(d,J＝10.3Hz,1H),6.85(d,J＝10.3Hz,1H),2.45(s,3H,CH 3CO)。 The compound II-1 (3 g, 17.2 mmol) described in Example 1 was dissolved in 10 mL of acetic anhydride, and 5 drops of concentrated sulfuric acid were added dropwise with stirring. The reaction solution was placed in an ice-water bath and stirred for 2 hours. Afterwards, the reaction solution was suction filtered, washed with ice water and a small amount of methanol successively, to obtain about 3.4 g of compound III-1, which was pale yellow powder crystals, with a yield of 91%. 1 H NMR (400MHz, CDCl 3 ): δ8.05 (dd, J = 7.9, 1.0 Hz, 1H), 7.77 (t, J = 7.9 Hz, 1H), 7.40 (dd, J = 7.9, 1.0 Hz, 1H) ), 6.95 (d, J=10.3 Hz, 1H), 6.85 (d, J=10.3 Hz, 1H), 2.45 (s, 3H, CH 3 CO).
实施例4Example 4
本实施例涉及一种具有结构式(Ⅳ)的7-甲基-5-羟基-1,4-萘醌(7-甲基胡桃醌，Ⅳ-1)的制备方法，包括以下步骤：This embodiment relates to a preparation method of 7-methyl-5-hydroxy-1,4-naphthoquinone (7-methyl juglone, IV-1) with structural formula (IV), which includes the following steps:
Figure PCTCN2021077207-appb-000013
Figure PCTCN2021077207-appb-000013
本实施例步骤采用国家发明专利(申请号CN201911006433)的方法制备，以2,5-二甲氧基苯甲醛及丁二酸二乙酯为原料，产物Ⅳ-1呈橙黄色粉末，总收率37％。 1H NMR(400MHz,CDCl 3):δ11.84(s,1H,OH),7.43(s,1H),7.07(s,1H),6.90(s,2H),2.43(s,3H,CH 3)。 The steps in this example are prepared by the method of the national invention patent (application number CN201911006433), using 2,5-dimethoxybenzaldehyde and diethyl succinate as raw materials, the product IV-1 is orange-yellow powder, and the total yield 37%. 1 H NMR (400MHz, CDCl 3 ): δ 11.84 (s, 1H, OH), 7.43 (s, 1H), 7.07 (s, 1H), 6.90 (s, 2H), 2.43 (s, 3H, CH 3 ).
实施例5Example 5
本实施例涉及一种具有结构式(Ⅳ)的7-甲基-5-苄氧基-1,4-萘醌(7-甲基胡桃醌苄基醚，Ⅳ-2)的制备方法，包括以下步骤：This embodiment relates to a preparation method of 7-methyl-5-benzyloxy-1,4-naphthoquinone (7-methyl juglone benzyl ether, IV-2) of structural formula (IV), including the following step:
Figure PCTCN2021077207-appb-000014
Figure PCTCN2021077207-appb-000014
本实施例步骤采用文献(Cui et al.A regioselective synthesis of 7-methyl juglone and its derivatives.Nat.Prod.Res.,2020,DOI:10.1080/14786419.2020.1761356)的方法制备，以2,5-二甲氧基苯甲醛及丁二酸二乙酯为原料，产物Ⅳ-2呈橙色粉末，总收率46％。 1H NMR(400MHz,CDCl 3):δ7.62–7.57(m,2H),7.55–7.52(m,1H),7.46–7.37(m,2H),7.35–7.29(m,1H),7.13(d,J＝1.6Hz,1H),6.83(s,2H),5.25(s,2H),2.43(s,3H)。 The steps in this example were prepared by the method in the literature (Cui et al. A regioselective synthesis of 7-methyl juglone and its derivatives. Nat. Prod. Res., 2020, DOI: 10.1080/14786419.2020.1761356), with 2,5-two Methoxybenzaldehyde and diethyl succinate are used as raw materials, and the product IV-2 is orange powder with a total yield of 46%. 1 H NMR (400MHz, CDCl 3 ): δ7.62–7.57(m,2H), 7.55–7.52(m,1H), 7.46–7.37(m,2H), 7.35–7.29(m,1H), 7.13( d, J = 1.6 Hz, 1H), 6.83 (s, 2H), 5.25 (s, 2H), 2.43 (s, 3H).
实施例6Example 6
本实施例涉及一种具有结构式(Ⅵ)的2-甲基-1,4-萘醌(Ⅵ-1)的制备方法，包括以下步骤：This embodiment relates to a preparation method of 2-methyl-1,4-naphthoquinone (VI-1) with structural formula (VI), including the following steps:
Figure PCTCN2021077207-appb-000015
Figure PCTCN2021077207-appb-000015
在室温下，将14.2g的2-甲基萘(100mmol)溶解于冰醋酸(50mL)中，将该溶液滴加至58.7克铬酐的200毫升冰醋酸溶液中，滴加过程中反应液温度维持在35-40℃。加毕，在40℃保温0.5小时，升温到65℃保温20分钟；将反应物倾入大量冰水中，不断搅拌下析出2-甲基-1,4-萘醌粗品。将粗品过滤，用冰水反复清洗滤饼，至滤液无酸味。将滤饼用二氯甲烷溶解，分取有机层后，加入少量的活性炭脱色。在减压条件下将二氯甲烷蒸除，残留固体经冰醋酸-甲醇重结晶得2-甲基-1,4-萘醌(Ⅵ-1)纯品5.5g，呈淡黄色粉末状结晶，收率32％。 1H NMR(400MHz,Chloroform-d)δ8.13–7.99(m,2H),7.71(dd,J＝5.7,3.3Hz,2H),6.83(q,J＝1.5Hz,1H),2.18(d,J＝1.5Hz,3H). At room temperature, 14.2 g of 2-methylnaphthalene (100 mmol) was dissolved in glacial acetic acid (50 mL), and the solution was added dropwise to 58.7 g of chromic anhydride in 200 ml of glacial acetic acid solution. The temperature of the reaction solution during the addition was Maintain at 35-40°C. After the addition, the temperature was kept at 40°C for 0.5 hours, and the temperature was raised to 65°C for 20 minutes; the reactants were poured into a large amount of ice water, and the crude 2-methyl-1,4-naphthoquinone was precipitated under constant stirring. Filter the crude product and wash the filter cake repeatedly with ice water until the filtrate has no sour taste. Dissolve the filter cake with dichloromethane, separate the organic layer, and add a small amount of activated carbon for decolorization. The dichloromethane was evaporated under reduced pressure, and the remaining solid was recrystallized from glacial acetic acid-methanol to obtain 5.5 g of pure 2-methyl-1,4-naphthoquinone (VI-1), which was a pale yellow powdery crystal. The yield was 32%. 1 H NMR(400MHz, Chloroform-d)δ8.13–7.99(m,2H), 7.71(dd,J=5.7,3.3Hz,2H), 6.83(q,J=1.5Hz,1H), 2.18(d ,J=1.5Hz,3H).
实施例7Example 7
本实施例涉及一种具有结构式(Ⅶ)的2-乙酰基-8-甲氧基-1,4-萘醌(Ⅶ-1)的制 备方法，包括以下步骤：This embodiment relates to a preparation method of 2-acetyl-8-methoxy-1,4-naphthoquinone (VII-1) with structural formula (VII), including the following steps:
Figure PCTCN2021077207-appb-000016
Figure PCTCN2021077207-appb-000016
采用已报道的方法(Zhang et al.Synthesis of 4,8-dimethoxy-1-naphthol via an acetyl migration.Synth.Commun.,2017,47(6),536-540)，以胡桃醌为原料，合成4,8-二甲氧基-1-萘酚乙酸酯。将4,8-二甲氧基-1-萘酚乙酸酯(300mg，1.22mmol)溶于三氟化硼-乙醚溶液中(5mL，三氟化硼含量为48％)，将反应液升温至60℃并在该温度下搅拌反应30分钟。反应液冷却后用冰水稀释，二氯甲烷萃取，有机层经干燥后减压浓缩至干，残留物经柱层析纯化后得2-乙酰基-4,8-二甲氧基-1-萘酚,呈淡黄色粉末，约237mg，收率：79％。 1H NMR(400MHz,CDCl 3)δ13.65(s,1H),7.81(d,J＝8.1Hz,1H),7.53(t,J＝8.1Hz,1H),6.96(s,1H),6.94(d,J＝8.1Hz,1H),4.04(s,3H),3.95(s,3H),2.70(s,3H)。将中间体2-乙酰基-4,8-二甲氧基-1-萘酚(230mg，0.93mmol)溶解于二氯甲烷-乙腈混合液(V/V,1:3)中，加入2.5倍当量的硝酸铈铵的水溶液(硝酸铈铵在水溶液中的浓度为20％,w/v)，10℃下搅拌0.5h后，乙酸乙酯萃取。有机层经水洗，饱和食盐水洗，无水硫酸钠干燥，减压浓缩，快速柱层析，得到2-乙酰基-8-甲氧基-1,4-萘醌(Ⅶ-1)，呈亮黄色粉末，共182毫克，总收率85％。 1H NMR(400MHz,Chloroform-d)δ7.76–7.69(m,2H),7.37(dd,J＝5.9,3.7Hz,1H),7.02(s,1H),4.04(s,4H),2.61(s,3H). Using the reported method (Zhang et al. Synthesis of 4,8-dimethoxy-1-naphthol via an acetyl migration. Synth. Commun., 2017, 47(6), 536-540), using juglone as the raw material, the synthesis 4,8-Dimethoxy-1-naphthol acetate. 4,8-Dimethoxy-1-naphthol acetate (300mg, 1.22mmol) was dissolved in boron trifluoride-ether solution (5mL, the content of boron trifluoride was 48%), and the reaction solution was heated up The temperature was raised to 60°C and the reaction was stirred at this temperature for 30 minutes. After cooling, the reaction solution was diluted with ice water and extracted with dichloromethane. The organic layer was dried and concentrated to dryness under reduced pressure. The residue was purified by column chromatography to obtain 2-acetyl-4,8-dimethoxy-1- Naphthol, a light yellow powder, about 237mg, yield: 79%. 1 H NMR (400MHz, CDCl 3 ) δ 13.65 (s, 1H), 7.81 (d, J = 8.1 Hz, 1H), 7.53 (t, J = 8.1 Hz, 1H), 6.96 (s, 1H), 6.94 (d, J=8.1 Hz, 1H), 4.04 (s, 3H), 3.95 (s, 3H), 2.70 (s, 3H). Dissolve the intermediate 2-acetyl-4,8-dimethoxy-1-naphthol (230mg, 0.93mmol) in the dichloromethane-acetonitrile mixture (V/V, 1:3) and add 2.5 times An equivalent aqueous solution of cerium ammonium nitrate (the concentration of cerium ammonium nitrate in the aqueous solution is 20%, w/v), stirred at 10° C. for 0.5 h, and then extracted with ethyl acetate. The organic layer was washed with water, saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and flash column chromatography to obtain 2-acetyl-8-methoxy-1,4-naphthoquinone (Ⅶ-1), which was bright Yellow powder, a total of 182 mg, the total yield is 85%. 1 H NMR(400MHz, Chloroform-d)δ7.76–7.69(m,2H), 7.37(dd,J=5.9,3.7Hz,1H), 7.02(s,1H), 4.04(s,4H), 2.61 (s,3H).
实施例8Example 8
本实施例涉及一种具有结构式(Ⅷ)的2-羟基-1,4-萘醌(Ⅷ-1)的制备方法，包括以下步骤：This embodiment relates to a preparation method of 2-hydroxy-1,4-naphthoquinone (Ⅷ-1) with structural formula (VIII), including the following steps:
Figure PCTCN2021077207-appb-000017
Figure PCTCN2021077207-appb-000017
将1，4-萘醌(3.16g，20mmol)溶于乙酸酐(20mL)中，滴加4滴浓硫酸。将混合物在冰水浴下搅拌反应8小时。将反应液抽滤，滤饼经石油醚及少量预冷的无水乙 醇洗涤，得灰白色粉末约4.72克。将该粉末溶于甲醇中，在冰水浴下加入0.5克的甲醇钠，混合物在冰水浴中搅拌反应4小时后，抽滤，收集红色滤饼并用少量甲醇洗涤。将上述滤饼溶于90℃的水中，趁热抽滤；滤液冷却后用浓盐酸酸化，产物析出；抽滤、水洗及减压干燥，得2-羟基-1,4-萘醌(Ⅷ-1)约2.51g，呈淡黄色粉末状结晶，总收率约70％。 1H NMR(400MHz,DMSO-d 6)δ11.69(s,1H),8.03–7.90(m,2H),7.82(dtd,J＝17.6,7.4,1.5Hz,2H),6.17(s,1H). 1,4-Naphthoquinone (3.16 g, 20 mmol) was dissolved in acetic anhydride (20 mL), and 4 drops of concentrated sulfuric acid were added dropwise. The mixture was stirred and reacted for 8 hours in an ice-water bath. The reaction liquid was suction filtered, and the filter cake was washed with petroleum ether and a small amount of pre-cooled absolute ethanol to obtain about 4.72 g of off-white powder. The powder was dissolved in methanol, 0.5 g of sodium methoxide was added in an ice-water bath, and the mixture was stirred and reacted in an ice-water bath for 4 hours, and then filtered with suction. The red filter cake was collected and washed with a small amount of methanol. The filter cake was dissolved in water at 90°C and filtered while it was hot; the filtrate was acidified with concentrated hydrochloric acid after cooling, and the product precipitated; filtered, washed with water and dried under reduced pressure to obtain 2-hydroxy-1,4-naphthoquinone (Ⅷ- 1) Approximately 2.51g, in the form of light yellow powdery crystals, with a total yield of approximately 70%. 1 H NMR (400MHz, DMSO-d 6 ) δ 11.69 (s, 1H), 8.03-7.90 (m, 2H), 7.82 (dtd, J = 17.6, 7.4, 1.5 Hz, 2H), 6.17 (s, 1H) ).
实施例9Example 9
本实施例涉及一种具有结构式(Ⅸ)的1,4-萘醌(Ⅸ-1)的制备方法，包括以下步骤：This embodiment relates to a preparation method of 1,4-naphthoquinone (IX-1) with structural formula (IX), including the following steps:
Figure PCTCN2021077207-appb-000018
Figure PCTCN2021077207-appb-000018
将1-萘胺(3.6g，25mmol)溶于冰乙酸(50mL)中，在低温下滴加质量分数为45％的硫酸(10mL)。加入完毕后，反应液升温至80℃，缓慢滴加浓度分数为5％的H 2O 2溶液(160mL)。滴加完毕后，80℃继续反应3h。反应完毕后，将反应液倒入冰水中，二氯甲烷(50mL*3)萃取。有机层经水、饱和食盐水清洗后，减压浓缩至干。采用硅胶柱层析方法(流动相：乙酸乙酯-石油醚体积比为1：5)进行纯化，得化合物Ⅸ-1约1.5g，呈淡黄色结晶，收率39％。 1H NMR(400MHz,Chloroform-d)δ8.09(ddd,J＝5.8,3.3,0.9Hz,2H),7.76(ddd,J＝5.8,3.3,0.9Hz,2H),6.98(d,J＝0.8Hz,2H)。 1-Naphthylamine (3.6g, 25mmol) was dissolved in glacial acetic acid (50mL), and 45% sulfuric acid (10mL) was added dropwise at low temperature. After the addition, the reaction solution was heated to 80° C., and H 2 O 2 solution (160 mL) with a concentration fraction of 5% was slowly added dropwise. After the addition, the reaction was continued at 80°C for 3 hours. After the completion of the reaction, the reaction solution was poured into ice water and extracted with dichloromethane (50 mL*3). The organic layer was washed with water and saturated brine, and concentrated to dryness under reduced pressure. Purification was carried out by silica gel column chromatography (mobile phase: ethyl acetate-petroleum ether volume ratio 1:5) to obtain about 1.5 g of compound IX-1, which was pale yellow crystals, and the yield was 39%. 1 H NMR (400MHz, Chloroform-d) δ 8.09 (ddd, J = 5.8, 3.3, 0.9 Hz, 2H), 7.76 (ddd, J = 5.8, 3.3, 0.9 Hz, 2H), 6.98 (d, J = 0.8Hz, 2H).
实施例10Example 10
10.1实施例1至实施例4的萘醌类化合物对2019-nCoV冠状病毒的3CL水解酶抑制活性测定。10.1 Determination of the 3CL hydrolase inhibitory activity of the naphthoquinone compounds of Example 1 to Example 4 against 2019-nCoV coronavirus.
实验原理：Experimental principle:
3CL水解酶抑制活性测定，采用上海药物研究所和上海科技大学免疫化学研究所抗2019-nCoV病毒感染联合应急攻关团队所建立的酶学测试方法。利用高通量药物筛选平台配备的96道自动加样系统，将用真核系统表达的3CL水解酶、其特征的寡肽底物(寡肽底物通过酰胺键与荧光基团连接)及待测化合物置于孵育体系中。测定中设置不含有抑制剂的空白对照组，并采用紫草素为阳性对照药物；每个抑制剂在孵育体系中的终浓 度为1μmol/L，并设定3个复孔。测定中根据每孔中底物水解后的荧光强度，计算抑制率。The 3CL hydrolase inhibitory activity was determined using the enzymatic test method established by the Shanghai Institute of Materia Medica and the Institute of Immunochemistry of Shanghai University of Science and Technology to fight against 2019-nCoV virus infection. Using the 96-channel automatic sample loading system equipped with the high-throughput drug screening platform, the 3CL hydrolase expressed by the eukaryotic system, its characteristic oligopeptide substrate (the oligopeptide substrate is connected to the fluorescent group through an amide bond) and the The test compound is placed in the incubation system. In the determination, a blank control group without inhibitors was set, and shikonin was used as the positive control drug; the final concentration of each inhibitor in the incubation system was 1 μmol/L, and 3 replicate wells were set. In the determination, the inhibition rate was calculated based on the fluorescence intensity of the substrate in each well after hydrolysis.
实验方法：experimental method:
具体实验方法是将293A细胞按0.5*10 5个/孔浓度接种于96孔板，并于培养18～20h后进行质粒转染。每孔按3CL蛋白质粒pcDNA4-3C∶pcDNA3-mYFP为50ng∶100ng的比例转染293A细胞，4h后更换分别含溶剂DMSO、1μmol/L的待测小分子化合物或10μmol/L紫草素的新鲜培养液，每个浓度设定3个复孔。72h后倒弃培养基，用PBS洗2次，应用PerkinElmer EnVision多功能酶标仪，在激发波长Ex(500nm)、发射波长Em(535nm)下检测每孔的荧光强度，并记录分析。 The specific experimental method is to inoculate 293A cells in a 96-well plate at a concentration of 0.5*10 5 cells/well, and carry out plasmid transfection after culturing for 18-20 hours. 293A cells were transfected into 293A cells at the ratio of 3CL protein pellet pcDNA4-3C:pcDNA3-mYFP at 50ng:100ng per well, 4h later, the solvent was replaced with DMSO, 1μmol/L of the small molecule compound to be tested, or 10μmol/L shikonin. For the culture medium, set 3 replicate wells for each concentration. After 72h, discard the culture medium, wash twice with PBS, use PerkinElmer EnVision multi-function microplate reader to detect the fluorescence intensity of each well under excitation wavelength Ex (500nm) and emission wavelength Em (535nm), and record the analysis.
细胞抑制率计算：Calculation of cell inhibition rate:
抑制率＝(对照组平均荧光值－给药组平均荧光值)/对照组平均荧光值，测定结果如表1所示。Inhibition rate = (average fluorescence value of the control group-average fluorescence value of the administration group)/average fluorescence value of the control group. The measurement results are shown in Table 1.
10.2实施例5到实施例9的2019-nCoV新型冠状病毒的3CL蛋白水解酶抑制活性测定方法，包括以下步骤：10.2 The method for determining the 3CL proteolytic enzyme inhibitory activity of the 2019-nCoV novel coronavirus of Example 5 to Example 9 includes the following steps:
采用文献报道的荧光共振能量转移方法(Jin et al.2020.Structure of Mpro from SARS-CoV-2 and discovery of its inhibitors.Nature,582:289–293)，进行化合物的酶抑制活性测定。以市售荧光标记的多肽MCA-AVLQSGFR-Lys(Dnp)-Lys-NH 2为底物(GLBiochem,Shanghai)，通过酶动力学方法测定3CL酶的催化活性及初始速度。在化合物的酶抑制活性测定中，孵育体系含有2019-nCoV的3CL蛋白酶(0.2μM)，荧光标记多肽(20μM)及系列浓度的待测化合物(0-20μM)。通过酶标仪测定孵育2-3分钟时体系的荧光强度，激发波长及检测波长分别为320nm及405nm。根据加入抑制剂后酶催化底物水解的初始速度变化率，计算不同浓度下待测物的酶抑制率。所有实验均重复3次，并通过Prism5软件计算待测物抑制酶的IC 50值。我们测定了实施例5-9所述化合物对新型冠状病毒3CL蛋白酶的抑制活性。在酶抑制活性实验中，我们选用紫草素作为阳性对照药物。测定结果如表1所示。 Using the fluorescence resonance energy transfer method reported in the literature (Jin et al. 2020. Structure of Mpro from SARS-CoV-2 and discovery of its inhibitors. Nature, 582:289-293), the enzyme inhibitory activity of the compound was determined. Using the commercially available fluorescently labeled polypeptide MCA-AVLQSGFR-Lys(Dnp)-Lys-NH 2 as the substrate (GLBiochem, Shanghai), the catalytic activity and initial speed of the 3CL enzyme were determined by the enzyme kinetic method. In the determination of the enzyme inhibitory activity of the compound, the incubation system contains 2019-nCoV 3CL protease (0.2μM), fluorescently labeled peptide (20μM) and a series of concentrations of the test compound (0-20μM). The fluorescence intensity of the system when incubated for 2-3 minutes was measured by a microplate reader. The excitation wavelength and detection wavelength were 320nm and 405nm, respectively. According to the initial rate change rate of the enzyme-catalyzed substrate hydrolysis after the inhibitor is added, the enzyme inhibition rate of the analyte under different concentrations is calculated. All experiments were repeated 3 times, and the IC 50 value of the analyte inhibited enzyme was calculated by Prism5 software. We determined the inhibitory activity of the compounds described in Examples 5-9 on the new coronavirus 3CL protease. In the enzyme inhibitory activity experiment, we chose shikonin as the positive control drug. The measurement results are shown in Table 1.
酶抑制率计算：Enzyme inhibition rate calculation:
抑制率＝(空白对照组平均荧光值－给药组平均荧光值)/空白对照组平均荧光值*100％，测定结果如表1所示。Inhibition rate=(average fluorescence value of blank control group-average fluorescence value of administration group)/average fluorescence value of blank control group*100%. The measurement results are shown in Table 1.
表1 1,4‐萘醌类化合物对3CL酶的抑制活性测试结果Table 1 Test results of 1,4-naphthoquinone compound's inhibitory activity on 3CL enzyme
Figure PCTCN2021077207-appb-000019
Figure PCTCN2021077207-appb-000019
*注：酶抑制率超过100％是由于孵育2‐3分钟时体系的荧光值低于初始的荧光值，由机器的读数误差造成。*Note: The enzyme inhibition rate exceeds 100% because the fluorescence value of the system is lower than the initial fluorescence value when incubating for 2 to 3 minutes, which is caused by the reading error of the machine.
测定结果显示，阳性对照物紫草素在10μM的浓度下，其酶抑制率仅为51.4％。在1μM的浓度下，化合物胡桃醌(Ⅱ-1)、胡桃醌甲醚(Ⅱ-2)、乙酰基胡桃醌(Ⅲ-1)、7‐甲基胡桃醌(Ⅳ-1)、7‐甲基胡桃醌苄基醚(Ⅳ-2)、2‐乙酰基‐8‐甲氧基‐1,4‐萘醌(Ⅶ-1)及1,4‐萘醌(Ⅸ-1)的酶抑制率均达到90％以上，抑制活性远高于紫草素。The test results showed that the positive control substance shikonin at a concentration of 10 μM, its enzyme inhibition rate was only 51.4%. At a concentration of 1μM, the compounds juglone (Ⅱ-1), juglone methyl ether (Ⅱ-2), acetyl juglone (Ⅲ-1), 7-methyl juglone (Ⅳ-1), 7-methyl Enzyme inhibition rate of benzyl juglone (IV-2), 2-acetyl-8-methoxy-1,4-naphthoquinone (Ⅶ-1) and 1,4-naphthoquinone (Ⅸ-1) Both reached more than 90%, and the inhibitory activity was much higher than shikonin.
实施例11Example 11
本实施例涉及实施例1-9所述化合物对宿主正常细胞HSF细胞的生长抑制活性测定。This example relates to the determination of the growth inhibitory activity of the compounds described in Examples 1-9 on normal host HSF cells.
取生长状态良好的HSF细胞，胰酶消化后用完全培养基调整细胞悬液至合适的浓度，以5000个细胞/孔接种于96孔板内，静置于培养箱中培养24h，待细胞贴壁后每孔加入含待测化合物的完全培养基(100μL)，各组均设三个复孔。每块96孔板上设置调零孔(只加化合物与培养基，不含细胞)与空白对照孔(只含有细胞与培养基，不加化合物)。在培养箱中培养72小时后，加入MTT溶液20μL/孔，避光培养4h后终止培养，吸去孔内液体，加入100μL/孔的二甲基亚砜，于摇床上低速振荡10min，待孔内结晶物充分溶解后，于490nm波长处检测各孔的吸光度(OD值)，按下列公式计算细胞生长抑制率：生长抑制率(％)＝(1–给药组OD值/对照组OD值)×100％。根据不同浓度下的细胞生长抑制率，使用Prism5软件计算IC 50值。 Take HSF cells in good growth condition, trypsinize, adjust the cell suspension to an appropriate concentration with complete medium, inoculate 5000 cells/well in a 96-well plate, and place it in an incubator for 24 hours, and wait until the cells are attached. Complete medium (100 μL) containing the compound to be tested was added to each well behind the wall, and three multiple wells were set up in each group. Each 96-well plate is equipped with a zero adjustment hole (only compound and medium, no cells) and a blank control hole (only cell and medium, no compound). After 72 hours of incubation in the incubator, add 20μL/well of MTT solution. After 4 hours of incubation in the dark, stop the culture, aspirate the liquid in the well, add 100μL/well of dimethyl sulfoxide, shake on a shaker at low speed for 10 minutes, and wait for the well. After the inner crystals are fully dissolved, detect the absorbance (OD value) of each well at a wavelength of 490nm, and calculate the cell growth inhibition rate according to the following formula: Growth inhibition rate (%) = (1-administration group OD value / control group OD value )×100%. The cell growth inhibition at different concentrations of using software to calculate the 50 value Prism5 IC.
表2 1,4-萘醌类化合物对HSF细胞的细胞毒作用Table 2 Cytotoxic effects of 1,4-naphthoquinone compounds on HSF cells
Figure PCTCN2021077207-appb-000020
Figure PCTCN2021077207-appb-000020
Figure PCTCN2021077207-appb-000021
Figure PCTCN2021077207-appb-000021
测定结果表明,天然萘醌类化合物紫草素对人正常细胞HSF具有极强的生长抑制作用(IC 50<2μM)。实施例1-9所述化合物，其抑制细胞生长的IC 50值均高于阳性对照药物紫草素的IC 50值。在所测定的化合物中，化合物Ⅳ-2、Ⅵ-1、Ⅶ-1、Ⅷ-1及Ⅸ-1对宿主正常细胞HSF细胞的毒性显著降低(IC 50>25μM)。 The results of the determination showed that shikonin, a natural naphthoquinone compound, has a strong growth inhibitory effect on normal human HSF cells (IC 50 <2μM). Example 1-9 The compound which inhibits cell growth IC 50 values were higher than the IC 50 value of the positive control shikonin. Among the tested compounds, the toxicity of compounds IV-2, VI-1, VII-1, VIII-1 and IX-1 to the host normal HSF cells was significantly reduced (IC 50 > 25 μM).
实施例12Example 12
本实施例涉及实施例7所述化合物(Ⅶ-1)及实施例9所述化合物(Ⅸ-1)的体外抗新型冠状病毒活性测定。This example relates to the in vitro anti-new coronavirus activity determination of the compound (VII-1) described in Example 7 and the compound (IX-1) described in Example 9.
采用qRT‐PCR方法，以Vero E6细胞作为宿主细胞，进行待测化合物的体外抗病毒活性测试。将生长状态良好的Vero E6细胞计数，以1×10 4个每孔接种到96孔板中，并在37℃细胞培养箱中培养24小时，使细胞贴壁。用候选药物(10μM)预处理细胞1h，随后加入2019‐nCoV病毒(MOI参数设定为0.01)，以细胞感染2h。除去含有病毒及待测药物的上清培养基，用新配制的含待测药物的培养基进一步培养Vero E6细胞。在细胞感染病毒72小时后，各孔收集上清液，使用试剂盒(Qiagen,Germany)从上清 培养液中提取病毒RNA，通过qRT‐PCR分析检测上清液中的病毒拷贝数，采用标准曲线法计算抑制率。使用Prism5软件计算半数有效浓度(EC 50)。结果表明(图2)，化合物Ⅸ-1及Ⅸ-1均显示了较强的体外抗病毒活性，其中化合物Ⅶ-1在体外抗新型冠状病毒的EC 50值仅为4.55μM，与文献(Jin et al.2020.Structure of Mpro from SARS-CoV-2 and discovery of its inhibitors.Nature,582:289–293)所报道的农药中间体依布硒(图1，化合物2)的EC 50值接近(EC 50＝4.67μM)。化合物Ⅸ-1也显示了较强的抗新型冠状病毒活性，其EC 50值接近10μM。阳性对照物紫草素，为已报道的对新型冠状病毒3CL水解酶具有抑制活性的化合物，可能用于新型冠状病毒的治疗。“非典”时期的临床研究表明，口服紫草素及其衍生物，对SARS病毒感染早期的患者有显著疗效(Wang,F.,Method of treatment of virus infections using shikonin compounds.US Patent,No.7897640)。本发明所述萘醌类化合物较阳性对照物紫草素显示了更强的3CL水解酶抑制活性，部分化合物对人正常细胞的毒性低，具有抗新型冠状病毒活性，有良好的应用前景。 Using the qRT-PCR method, with Vero E6 cells as the host cell, the in vitro antiviral activity test of the test compound was performed. Count the well-growing Vero E6 cells, inoculate 1×10 4 cells per well into a 96-well plate, and incubate in a 37°C cell incubator for 24 hours to make the cells adhere to the wall. The cells were pretreated with the candidate drug (10 μM) for 1 h, and then the 2019-nCoV virus was added (the MOI parameter was set to 0.01), and the cells were infected for 2 h. The supernatant medium containing the virus and the drug to be tested is removed, and Vero E6 cells are further cultured with the newly prepared medium containing the drug to be tested. 72 hours after the cells were infected with the virus, the supernatant was collected in each well, and the viral RNA was extracted from the supernatant culture using a kit (Qiagen, Germany), and the virus copy number in the supernatant was detected by qRT-PCR analysis. The curve method calculates the inhibition rate. Prism5 software was used to calculate the median effective concentration (EC 50 ). The results showed (Figure 2) that both compounds Ⅸ-1 and Ⅸ-1 showed strong in vitro antiviral activity. Among them, the EC 50 value of compound Ⅶ-1 against the new coronavirus in vitro was only 4.55μM. Compared with the literature (Jin et al. 2020. Structure of Mpro from SARS-CoV-2 and discovery of its inhibitors. Nature, 582: 289–293) reported that the EC 50 value of the pesticide intermediate ebselen (Figure 1, compound 2) is close to ( EC 50 =4.67 μM). Compound IX-1 also showed strong anti-new coronavirus activity, and its EC 50 value was close to 10 μM. The positive control, shikonin, is a compound that has been reported to have inhibitory activity on the new coronavirus 3CL hydrolase, and may be used for the treatment of the new coronavirus. Clinical studies during the "SARS" period have shown that oral shikonin and its derivatives have a significant effect on patients with early SARS virus infection (Wang, F., Method of treatment of virus infections using shikonin compounds. US Patent, No. 7897640 ). The naphthoquinone compound of the present invention shows stronger 3CL hydrolase inhibitory activity than the positive control shikonin, some compounds have low toxicity to normal human cells, have anti-new coronavirus activity, and have good application prospects.
综上所述，本发明中的1,4‐萘醌类化合物，其制备方法简便、原料易得且收率较高。体外测定结果表明，该类化合物对2019-nCoV新型冠状病毒的3CL水解酶有非常强的抑制活性。可使用本发明中的萘醌类化合物，制备抗新型冠状病毒的口服、注射药物或复方药物。该药物或复方药物可抑制新型冠状病毒的3CL水解酶，进而抑制其在宿主细胞内的复制过程，用于新型冠状病毒感染的治疗。In summary, the 1,4-naphthoquinone compound in the present invention has a simple preparation method, easy-to-obtain raw materials and high yield. The in vitro test results show that this type of compound has a very strong inhibitory activity on the 3CL hydrolase of the 2019-nCoV novel coronavirus. The naphthoquinone compounds in the present invention can be used to prepare oral, injection or compound drugs against the new coronavirus. The drug or compound drug can inhibit the 3CL hydrolase of the new coronavirus, thereby inhibiting its replication process in the host cell, and is used for the treatment of the new coronavirus infection.
以上对本发明的具体实施例进行了描述。需要理解的是，本发明并不局限于上述特定实施方式，本领域技术人员可以在权利要求的范围内做出各种变形或修改，这并不影响本发明的实质内容。The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various deformations or modifications within the scope of the claims, which does not affect the essence of the present invention.

Claims (16)

  1. 一种基于胡桃醌结构的萘醌类化合物，其特征在于，所述萘醌类化合物的结构如式(Ⅰ)所示：A naphthoquinone compound based on a juglone structure, characterized in that the structure of the naphthoquinone compound is as shown in formula (I):
    Figure PCTCN2021077207-appb-100001
    其中，R 4为氢、甲基、乙基、乙酰基或丙酰基；R 5为氢、甲基或乙基。
    Figure PCTCN2021077207-appb-100001
    Wherein, R 4 is hydrogen, methyl, ethyl, acetyl or propionyl; R 5 is hydrogen, methyl or ethyl.
  2. 一种如权利要求1所述的基于胡桃醌结构的萘醌类化合物，在制备抑制新型冠状病毒2019-nCoV 3CL水解酶的药物中的用途。A use of the naphthoquinone compound based on the juglone structure of claim 1 in the preparation of a drug for inhibiting the hydrolase of the novel coronavirus 2019-nCoV 3CL.
  3. 如权利要求2所述的用途，其特征在于，所述萘醌类化合物结构如式(Ⅱ)所示：The use according to claim 2, wherein the structure of the naphthoquinone compound is as shown in formula (II):
    Figure PCTCN2021077207-appb-100002
    其中，R 4为氢、甲基或乙基。
    Figure PCTCN2021077207-appb-100002
    Wherein, R 4 is hydrogen, methyl or ethyl.
  4. 如权利要求2所述的用途，其特征在于，所述萘醌类化合物结构如式(Ⅲ)所示：The use according to claim 2, wherein the structure of the naphthoquinone compound is as shown in formula (III):
    Figure PCTCN2021077207-appb-100003
    所述R 6为甲基或乙基。
    Figure PCTCN2021077207-appb-100003
    The R 6 is methyl or ethyl.
  5. 如权利要求2所述的用途，其特征在于，所述萘醌类化合物结构如式(Ⅵ)所示：The use according to claim 2, wherein the structure of the naphthoquinone compound is as shown in formula (VI):
    Figure PCTCN2021077207-appb-100004
    其中，R 5为氢、甲基或乙基。
    Figure PCTCN2021077207-appb-100004
    Wherein, R 5 is hydrogen, methyl or ethyl.
  6. 一种如权利要求1所述的基于胡桃醌结构的萘醌类化合物，在制备抗新型冠状病毒2019-nCoV药物中的用途。A use of the naphthoquinone compound based on the juglone structure as claimed in claim 1 in the preparation of an anti-new coronavirus 2019-nCoV drug.
  7. 如权利要求6所述的用途，其特征在于，所述萘醌类化合物结构如式(Ⅱ)所示：The use according to claim 6, wherein the structure of the naphthoquinone compound is as shown in formula (II):
    Figure PCTCN2021077207-appb-100005
    其中，R 4为氢、甲基或乙基。
    Figure PCTCN2021077207-appb-100005
    Wherein, R 4 is hydrogen, methyl or ethyl.
  8. 如权利要求6所述的用途，其特征在于，所述萘醌类化合物结构如式(Ⅲ)所示：The use according to claim 6, wherein the structure of the naphthoquinone compound is as shown in formula (III):
    Figure PCTCN2021077207-appb-100006
    所述R 6为甲基或乙基。
    Figure PCTCN2021077207-appb-100006
    The R 6 is methyl or ethyl.
  9. 如权利要求6所述的用途，其特征在于，所述萘醌类化合物结构如式(Ⅳ)所示：The use according to claim 6, wherein the structure of the naphthoquinone compound is as shown in formula (IV):
    Figure PCTCN2021077207-appb-100007
    其中，R 5为氢、甲基或乙基。
    Figure PCTCN2021077207-appb-100007
    Wherein, R 5 is hydrogen, methyl or ethyl.
  10. 一种基于甲萘醌结构的萘醌类化合物，其特征在于，所述萘醌类化合物的结构如式(Ⅴ)所示：A naphthoquinone compound based on the menadione structure, characterized in that the structure of the naphthoquinone compound is as shown in formula (V):
    Figure PCTCN2021077207-appb-100008
    其中，R为氢原子、甲基、乙酰基或羟基，R 1为氢、甲氧基、苄氧基或苯甲酰氧基团。
    Figure PCTCN2021077207-appb-100008
    Wherein, R is a hydrogen atom, a methyl group, an acetyl group or a hydroxyl group, and R 1 is a hydrogen, a methoxy group, a benzyloxy group or a benzoyloxy group.
  11. 如权利要求10所述的化合物，其特征在于，所述萘醌类化合物结构如式(Ⅵ) 所示：The compound of claim 10, wherein the structure of the naphthoquinone compound is as shown in formula (VI):
    Figure PCTCN2021077207-appb-100009
    其中，R 7为氢或甲氧基。
    Figure PCTCN2021077207-appb-100009
    Among them, R 7 is hydrogen or methoxy.
  12. 如权利要求10所述的化合物，其特征在于，所述萘醌类化合物结构如式(Ⅶ)所示：The compound of claim 10, wherein the structure of the naphthoquinone compound is as shown in formula (VII):
    Figure PCTCN2021077207-appb-100010
    所述R 7为氢或甲氧基。
    Figure PCTCN2021077207-appb-100010
    The R 7 is hydrogen or methoxy.
  13. 如权利要求10所述的化合物，其特征在于，所述萘醌类化合物结构如式(Ⅷ)所示：The compound of claim 10, wherein the structure of the naphthoquinone compound is as shown in formula (VIII):
    Figure PCTCN2021077207-appb-100011
    其中，R 7为氢或甲氧基。
    Figure PCTCN2021077207-appb-100011
    Among them, R 7 is hydrogen or methoxy.
  14. 如权利要求10所述的化合物，其特征在于，所述萘醌类化合物结构如式(Ⅸ)所示：The compound of claim 10, wherein the structure of the naphthoquinone compound is as shown in formula (IX):
    Figure PCTCN2021077207-appb-100012
    其中，R 7为氢、苄氧基或苯甲酰氧基团。
    Figure PCTCN2021077207-appb-100012
    Wherein, R 7 is hydrogen, benzyloxy or benzoyloxy group.
  15. 一种如权利要求10所述的基于甲萘醌结构的萘醌类化合物，在制备抑制新型冠状病毒2019-nCoV 3CL蛋白水解酶的药物中的用途。A use of the naphthoquinone compound based on the menadione structure of claim 10 in the preparation of a drug for inhibiting the proteolytic enzyme of the novel coronavirus 2019-nCoV 3CL.
  16. 一种如权利要求10所述的基于甲萘醌结构的萘醌类化合物，在制备抗新型冠状病毒2019-nCoV药物中的用途。A use of the naphthoquinone compound based on the menadione structure as claimed in claim 10 in the preparation of anti-new coronavirus 2019-nCoV drugs.
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