Use of epigallocatechin-7-gallate in anti-coronavirus drugs

Abstract

The invention relates to a preparation method of a natural product (‑) gallocatechin‑7‑gallate and its enantiomer (+) gallocatechin‑7‑gallate and their racemates and their use as a medicine for preventing and treating coronavirus infection, and belongs to the pharmaceutical field. The invention is innovative in that: research has found that (‑) gallocatechin‑7‑gallate and its enantiomers and their racemates have the effect of resisting the new coronavirus SARS‑CoV‑2; having the effect of resisting the coronavirus HCoV‑229E; its new coronavirus M pro / 3CL pro The inhibitory effect is an important mechanism for its antiviral effect. It is expected to become a clinical drug for the prevention and treatment of coronavirus infection.

Description

Technical Field

[0001] The present invention relates to a preparation method of (-)-gallocatechin-7-gallate and its chiral enantiomer (+)-gallocatechin-7-gallate and its medicinal use in preventing and treating coronavirus infectious diseases, belonging to the pharmaceutical field. Background Art

[0002] Due to the lack of effective treatments for the prevention and treatment of the new coronavirus (SARS-CoV-2) and common coronaviruses (α-type and β-type coronaviruses), there are challenges in the prevention and treatment of these viruses. Therefore, it is necessary to find drugs with broad-spectrum anti-coronavirus effects. Summary of the invention

[0003] The present invention relates to an activity evaluation method and results of formula I and formula II for preventing and treating coronavirus infection.

[0004] (-)-gallocatechin-7-gallate, referred to herein as compound formula I (codenamed LG0026), has been found to have an anti-SARS-CoV-2 infection effect; an anti-CoV-229E infection effect; and a CoV-2 protease M pro / 3CL pro Inhibition is an important mechanism for its antiviral effect. Its structure is as follows:

[0005]

[0006] (+)-gallocatechin-7-gallate, referred to herein as compound II (codenamed LG0027), has been found to have anti-SARS-CoV-2 infection and anti-coronavirus HCoV-229E infection effects. Its novel coronavirus protease M pro / 3CL pro Inhibition is an important mechanism for its antiviral effect. Its structure is as follows:

[0007]

[0008] The racemate of (-)-gallocatechin-7-gallate and (+)-gallocatechin-7-gallate, i.e., a 1:1 mixture of the two, is referred to as LG0316 or HEFE in this article. Studies have found that LG0316 has an anti-coronavirus HCoV-229E infection effect; its novel coronavirus protease M pro / 3CL pro Inhibition is an important mechanism for its antiviral effect.

[0009] A pharmaceutical composition is used in the preparation of a drug for preventing and / or treating coronavirus infection, wherein the pharmaceutical composition comprises (-)-gallocatechin-7-gallate shown in formula I and (+)-gallocatechin-7-gallate shown in formula II and its racemate or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

[0010] The pharmaceutically acceptable salts include pharmaceutically acceptable organic salts or inorganic salts, wherein the organic salts include sulfonates, carboxylates, amino acid salts and fatty acid salts, and the inorganic salts include hydrochlorides, bromates, iodates, sulfates, hydrogen sulfates, phosphates, hydrogen phosphates, dihydrogen phosphates and nitrates. The sulfonates include alkyl sulfonates containing 1 to 15 carbon atoms, benzene sulfonates, p-toluene sulfonates, o-toluene sulfonates and m-toluene sulfonates; carboxylates include tartrates, maleates, fumarates, citrates, malates, cinnamates, benzoates, malonates, succinates, glutarates, adipates, pamoates and lactates; amino acid salts include glutamates and aspartates; fatty acid salts include long-chain fatty acid salts containing 2 to 18 carbon atoms.

[0011] Beneficial technical effects

[0012] (-)-Epigallocatechin-7-gallate (LG0026), also known as epigallocatechin-7-gallate, is the main chemical component of the traditional Chinese medicine Archidendron clypearia (Jack) ICNielsen. The present invention finds that LG0026 has the effect of preventing and treating infection with the new coronavirus (SARS-CoV-2); and has the effect of preventing and treating infection with the coronavirus HCoV-229E.

[0013] (+)-gallocatechin-7-gallate (LG0027) and its racemate shown in formula II are both artificially synthesized compounds. Studies have found that they, like LG0026, have anti-coronavirus pharmacological activity and have good clinical application prospects.

[0014] General Description

[0015] As used herein, the term "antiviral drug" refers to a compound or biologic that effectively inhibits the formation and / or replication of a virus in humans, including but not limited to compounds or biologics that interfere with host or viral mechanisms necessary for the formation and / or replication of a virus in humans.

[0016] The "pharmaceutical composition" refers to a composition formed by combining two enantiomeric compounds of the present invention in any ratio, and a pharmaceutical composition prepared by mixing them with excipients or other active ingredients in any ratio.

[0017] Unless otherwise indicated, the term "treating" as used herein refers to reversing, alleviating, inhibiting or preventing the disorder or condition to which the term applies, or one or more symptoms of such disorder or condition. The term "treatment" as used herein refers to the act of treating. In certain embodiments, the term "treatment" refers to a compound or composition according to the invention to reduce or eliminate the symptoms of a viral infection and / or reduce the viral load in a patient.

[0018] "Prevention" or "preventing" refers to any treatment of a disease or condition that results in clinical symptoms of the disease or condition not developing. The term "prevention" also includes the use of a therapeutically effective amount of a compound or composition of the invention before an individual is exposed to a virus (e.g., pre-exposure prophylaxis) to prevent the onset of symptoms of the disease and / or to prevent the virus from reaching detectable levels in the blood.

[0019] As used herein, the term "inhibitor of Coronaviridae virus replication" refers to a compound or biological agent that is capable of reducing or eliminating the ability of Coronaviridae virus to replicate in a host cell (whether in vitro, ex vivo or in vivo).

[0020] The compound names provided above are named according to the literature records, and those skilled in the art will appreciate that other recognized naming systems and symbols can be used to name or identify the compound structure. For example, the compound can be named or identified by a common name, a systematic or non-systematic name. The naming systems and symbols generally recognized in the chemical field include, but are not limited to, Chemical Abstracts Service (CAS) and the International Union of Pure and Applied Chemistry (IUPAC). Therefore, the compound formula I (LG0026) structure provided above is named (S)-5-hydroxy-2-(3,4,5-trihydroxyphenyl)chroman-7-yl 3,4,5-trihydroxybenzoate using ChemBioDraw Ultra, and the compound formula II (LG0027) is named (R)-5-hydroxy-2-(3,4,5-trihydroxyphenyl)chroman-7-yl3,4,5-trihydroxybenzoate using ChemBioDraw Ultra.

[0021] “Composition, racemate” refers to the composition or mixture of Formula I and Formula II mentioned in the embodiments or experimental examples, and the composition in a ratio of 1:1 is also called a racemate. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Formula I, i.e. LG0026 1 H NMR spectrum

[0023] Figure 2 Formula I, i.e. LG0026 13 C NMR spectrum

[0024] Figure 3 Formula I, i.e. H-H_COSY_MOF spectrum of LG0026

[0025] Figure 4 Formula I, i.e., mass spectrum of LG0026

[0026] Figure 5 Formula I, i.e., LG0026 against the novel coronavirus M pro / 3CL pro Dose-effect diagram of

[0027] Figure 6 Formula II, i.e., LG0027 against the novel coronavirus M pro / 3CL pro Dose-effect diagram of

[0028] Figure 7 The racemic body of Formula I and Formula II, i.e., LG0316 against the novel coronavirus M pro / 3CL pro Dose-effect diagram of DETAILED DESCRIPTION

[0029] Example 1 Preparation of (-)-gallocatechin-7-gallate

[0030] 1kg of the medicinal material of the monkey earring was extracted three times with 95% ethanol to obtain 38g of extract, which was dissolved in water. Petroleum ether, ethyl acetate and methanol were selected in sequence and roughly divided into three parts. 5 grams of the ethyl acetate part was separated by silica gel column chromatography, and chloroform-acetone was used for gradient elution. Each 100ml was a fraction, and 256mg of the crude product of Clypearin, i.e. (-)-gallocatechin-7-gallate, was collected and purified by sephadex LH20 column chromatography to obtain 130mg of the pure compound clypearin.

[0031] The physicochemical properties and spectral data of clypearin are as follows:

[0032] 1. Melting point: 182-188℃; (c0.1010, CH3OH);

[0033] 2.FABMS: m / z 443[M+1], 291; EIMS: m / z 290, 164, 139, 125 and other fragment peaks. Molecular formula: C 22 H 18 O 10 ;

[0034] 3. cm -1 :3406,2925,1701,1618,1535,1446,1321,1211,1190,1126, 12028,1007,758;

[0035] 4.UV:λ max 215,280nm;

[0036] 5. 1 H-NMR (500MHz, CD3COCD3): δ4.79(1H,dd,J=9.96,2.0Hz)1.92(1H,m), 2.71(1H,m), 2.64(1H,m),2.71(1H,m),6.16(1H,d,J=2.0Hz), 6.26(1H,d, J=2.0Hz), 6.48(2H,s), 7.20(2H,s);

[0037] 6. 13 C-NMR (125MHz, CD3COCD3): δ78.4(C-2),20.1(C-3),29.9(C-4), 108.0(C-4a),157.3(C-5),101.4(C-6),157.3(C-7),102.3(C-8),156.6(C-8a), 132.2(C-1′),108.0(C-2′6′),151.3(C-3′5′),139.6(C-4′),133.8(C-1″), 110.2(C-2″,6″),146.4(C,3″,5″),146.2(C-4″),165.6(C(C-7″).

[0038] Structural analysis of bisabolin:

[0039] Clypearin, white powder, mp182-188℃, (c0.1010, CH3OH). Infrared spectrum at 1701cm -1 There is a carbonyl absorption peak at 1618,1535cm-1 The structure may contain benzene rings, and the absorption peak at 3406cm-1 indicates that the structure contains hydroxyl groups. FABMS gives a quasi-molecular ion peak of m / z 443, indicating a molecular weight of 442. 1 H-NMR indicated that the structure contained a benzene ring at δ7.20 (2H, s), 6.48 (2H, s), 6.28 (d, J = 2.2 Hz, 1H), 6.16 (d, J = 2.2 Hz, 1H). Five proton signals appeared at δ1.92 (1H, m), 2.12 (1H, m), 2.64 (1H, m), 2.71 (1H, m), 4.79 (1H, dd, J = 9.96, 2.0 Hz). 1 H- 1 These five protons in the H COSY spectrum are coupled in sequence, and based on the coupling relationship, it is inferred that there may be a -O-CH-CH2-CH2- structural fragment in the structure.

[0040] The mass spectrometer gave the following data: FAB + -MS m / z, 443 (M + +1), 291. EIMS showed fragment peaks such as m / z 290, 164, 139, and 125, and the structure was inferred to be a flavanoid compound.

[0041] From the above, the structure of clypearin is inferred to be:

[0042]

[0043] The NMR data of clypearin are as follows:

[0044]

[0045]

[0046] Example 2 Synthesis of Epigallocatechin-7-gallate racemate

[0047] The synthetic route is as follows:

[0048]

[0049] Compound 3

[0050] A two-phase mixture of trihydroxybenzophenone (1, 4.0 g, 21.06 mmol) and galloyl chloride (2, 13.34 g, 56.14 mmol) in toluene (100 mL) and saturated aqueous K2CO3 solution (100 mL) was stirred vigorously at 60°C for 30 minutes. Tetrabutylammonium sulfate (7.3 g, 21.5 mmol) was added and the mixture was stirred at 75°C for another two hours. During this period, the organic layer turned orange and an orange-brown liquid separated at the interface. The toluene layer was separated and the orange-brown liquid was extracted with CHCl3 (50 mL). The toluene and CHCl3 solutions were washed with water (2×90 mL), dried over Na2SO4, filtered and concentrated to give a brown oil (15.2 g). The mixture was redissolved in methanol (60 mL), CH3ONa (2.32 g) was added, and the solution was stirred at room temperature for 30 minutes. Until 2M HCl (aqueous solution, 18 mL) was added to adjust the pH to The resulting yellow solid was isolated by filtration and washed with 40 mL of CHCl3 to afford Compound 3 (2.19 g) as a light yellow solid in 50% yield.

[0051] 1 H NMR(500MHz,DMSO-d6)δ12.89(s,1H),10.88(s,1H),7.37(s,2H),7.10 (s,1H),6.61(d,J=2.1Hz,1H),6.25(d,J=2.1Hz,1H),3.94(s,6H),3.78(s,3H). 13 C NMR (100MHz, DMSO-d6) δ182.4,164.8,163.5,161.9,157.9,153.7,141.2,126.5, 105.5,104.6,104.3,99.4,94.8,60.7,56.8,56.5,19.0.HRMS-ESI(m / z):[M+Na + ]Calcd for C 18 H 16 NaO7367.0788; Found 367.0781.

[0052] Compound 4

[0053] To a solution of compound 3 (2.15 g, 6.24 mmol) in 50 mL of dry DMF, aromatic chloride 2 (4.32 g, 18.73 mmol) and anhydrous K2CO3 (1.73 g, 12.51 mmol) were added, and the mixture was stirred for 30 minutes. The pH was adjusted to 5 with 2M HCl (aqueous solution). The solid was precipitated, filtered, washed with DMF and water, and then dried at 50°C to give compound 4 (3.03 g) as a brown solid in 91% yield.

[0054] 1 H NMR (500MHz, CDCl3) δ12.78 (s, 1H), 7.44 (s, 2H), 7.10 (s, 2H), 7.01 (s, 1H), 6.70 (d, J = 3.7Hz, 2H), 3.95 (d, J = 6.6Hz, 18H). 13 C NMR (101MHz, CDCl3) δ182.8,164.6,164.0,162.0,156.7,156.3,153.7,153.2,141.7,126.1,123.5,1 08.9,107.6,105.9,105.7,103.8,101.3,61.1,61.1,56.4.HRMS-ESI(m / z):[M+Na + ]Calcd for C 28 H 26 NaO 11 561.1367; Found 561.1373.

[0055] Compound 5

[0056] To a solution of compound 4 (2.03 g, 3.77 mmol) in 150 mL of CH2Cl2, palladium on carbon (5%, 1.00 g) was added, and the mixture was stirred at room temperature for 72 h under H2 at 0.40 MPa pressure. The solution was filtered, concentrated, and then purified by silica gel column chromatography to give compound 5 (1.23 g) as a white foam solid in a yield of 62%.

[0057] 1H NMR(500MHz, CDCl3)δ7.47(s,2H),6.69(s,2H),6.45(s,1H),6.31(s, 1H),4.98(d,J=10.5Hz,1H),3.97(s,6H),3.92(s,6H),3.90(s,3H),2.87(d,J=16.0 Hz,1H),2.82–2.70(m,2H),2.29(d,J=13.1Hz,1H),2.15–2.03(m,2H). 13 C NMR (100MHz, CDCl3) δ165.2,156.6,154.7,153.4,153.1,149.8,142.9,137.6, 137.0,124.3,107.5,103.1,102.8,101.3,78.0,61.0,60.9,56.4,56.2,29.5,19.6.HRMS-ESI(m / z):[M+Na + ]Calcd for C 28 H 30 NaO 10 549.1731; Found 549.1758.

[0058] Compound 6

[0059] Dimethyl sulfate (238 μL, 2.51 mmol) and anhydrous KCO (472 mg, 3.42 mmol) were added to a solution of compound 5 (600 mg, 1.14 mmol) in 5 mL of anhydrous acetone, and the mixture was stirred for 5 h at 56 ° C. The solution was concentrated, redissolved in EtOAc, washed with water, dried with NaSO, filtered, and then concentrated to give a brown oil. After purification by flash silica gel column chromatography, compound 6 (0.62 g) was obtained as a white solid with a yield of 99.0%.

[0060] 1 H NMR (400MHz, CDCl3) δ7.48 (s, 2H), 6.70 (s, 2H), 6.49 (d, J = 2.2Hz, 1H),6.36(d,J=2.2Hz,1H),4.98(dd,J=10.8,2.1Hz,1H),3.98(d,J=1.0Hz,9H) ,3.92(s,6H),3.89(s,3H),3.87(s,3H),2.97–2.82(m,1H),2.82–2.67(m,2H), 2.34–2.21(m,1H),2.08–1.99(m,2H). 13C NMR (100MHz, CDCl3) δ165.0, 158.4,156.1,153.4,153.1,150.2,142.8,137.6,137.2,137.2,124.5,108.7,107.4,103.2, 103.1,96.9,78.0,61.0,60.9,60.4,56.4,56.2,55.7,29.7,29.6,19.8,14.2.HRMS-ESI (m / z):[M+Na + ]Calcd for C 29 H 32 NaO 11 563.1888; Found 563.1892.

[0061] Epigallocatechin-7-gallate racemate (LG0316)

[0062] Compound 6 (0.48 g, 0.88 mmol) was dissolved in anhydrous CH2Cl2 (20 mL), and then BBr3 (15.4 mL, 1.0 M in CH2Cl2) was added dropwise at -40 °C. The resulting reddish brown solution was heated to room temperature. Stirred for 12 hours under nitrogen. After completion, the reaction contents were quenched by adding ice water (20 mL), CH2Cl2 was removed, and the aqueous layer was extracted twice with ethyl acetate (50 mL). The combined organic extracts were then washed with water (25 mL) and brine (15 mL), dried over Na2SO4, filtered and concentrated. The residue was purified by Sephadex LH-20 gel to obtain (±) gallocatechin-7-gallate (0.36 g) as a brown solid with a yield of 91% and a purity of 98% (from HPLC-MS).

[0063] 1 H NMR (500MHz, CD3OD) δ7.21(s,2H),6.47(s,2H),6.22(d,J=11.9Hz, 2H),4.84(s,1H),2.79(d,J=17.2Hz,1H),2.74–2.65(m,1H),2.27–2.15(m, 1H),2.09–1.95(m,1H). 13 C NMR(100MHz,CD3OD)δ167.0,157.7,157.2, 151.4,146.8,146.5,140.3,133.9,133.6,120.7,110.4,108.5,106.1,102.3,101.4,79.0,30.4,20.3.HRMS-ESI(m / z):[MH -]Calcdfor C 22 H 19 O 10 441.0827; Found 441.0825.

[0064] Example 3 Separation of enantiomers of epigallocatechin-7-gallate

[0065] The above-mentioned catechin gallate-7-gallate racemate was separated by HPLC using a chiral preparative column and the corresponding components were collected. The solvent was removed by rotary evaporation to obtain a pure product of optical isomers. A total of 300 mg of the racemate was separated to obtain 100 mg of (-)-catechin gallate-7-gallate (LG0026), ee = 98.4%, and 100 mg of (+)-catechin gallate-7-gallate (LG0027), ee = 98.0%.

[0066] Experimental Example 1 Evaluation of the Anti-Novel Coronavirus (SARS-CoV-2) Effect of Compounds

[0067] Cells: Vero cells.

[0068] Virus strain: C-Tan-nCoV Wuhan strain (SARS-CoV-2). Virus infection is equivalent to 100TCID5.

[0069] Experimental Materials and Methods

[0070] (1) Sample treatment: The sample concentration prepared by DMSO is 10 mg / ml as the stock solution. Before using the sample, dilute the drug with virus culture maintenance solution, the first concentration is 100 ug / ml, and then dilute it 5 times with culture solution, 5 dilutions each. The positive drug is Remdesivir.

[0071] (2) Therapeutic administration was adopted. The virus was first incubated with cells for 2 hours, and then the virus solution was discarded, and the drug diluted in the maintenance solution was added, and the culture was continued for 48 hours. At the same time, a drug control group and a virus control group were set up.

[0072] (3) Viral nucleic acid determination: 48 h after drug treatment, 100 μl of cell culture supernatant was taken to extract nucleic acid and perform rRT-PCR experiment (ORF1ab) to determine the Ct value of the novel coronavirus nucleic acid in the cell culture supernatant and calculate the virus inhibition rate.

[0073] (4) Calculate the EC of the sample 50 .

[0074] The experimental results are shown in Table 1. The results show that (-)-gallocatechin-7-gallate has a significant anti-new coronavirus effect, EC50 =0.98ug / ml, which is equivalent to the positive drug Remdesivir. This study will provide a new solution for the clinical treatment of new coronary pneumonia.

[0075] Table 1 Inhibitory effect of (-)-gallocatechin-7-gallate on the cytopathic effect induced by the novel coronavirus C-Tan-nCoV Wuhan strain (SARS-CoV-2) in vitro on Vero cells

[0076]

[0077] Experimental Example 2 Evaluation of the activity of compounds against coronavirus HCoV-229E

[0078] Huh7 cells were used as virus hosts to measure the degree of inhibition of virus-induced cytopathic effect (CPE) by the samples. Virus strain: Coronavirus HCoV-229E, stored at -80°C. Compound samples were prepared into a stock solution with DMSO, the sample concentration was 10 mg / ml, and then diluted 3 times with culture medium, with 8 dilutions each. Positive control drug: Ribavirin (RBV), Hubei Tianyao Pharmaceutical Co., Ltd. (Batch No. 31712252).

[0079] Test method: Huh7 cells were inoculated into 96-well culture plates and placed in a 5% CO2 atmosphere. Culture. After 24 hours, the infection was about 100 TCID 50 Coronavirus, add maintenance solution containing samples of different dilutions and positive control drugs, set up cell control wells and virus control wells, 5% CO2, When the cytopathic effect (CPE) of the virus control group reached 4+, the cytopathic effect (CPE) of each group was observed, and the half toxic concentration (TC) of the samples to cells was calculated by the Reed-Muench method. 50 ) and the median effective concentration (EC 50 The experimental results are shown in Table 2.

[0080] The results showed that (-)-gallocatechin-7-gallate (LG0026) and (+)-gallocatechin-7-gallate (LG0027) had similar activities against coronavirus HCoV-229E, indicating that the two enantiomers have similar effects against coronavirus HCoV-229E infection, and their activities are also similar to those of the positive drug ribavirin.

[0081] Table 2 Inhibitory effect of epigallocatechin-7-gallate on cytopathic effect induced by coronavirus HCoV-229E in Huh7 cells in vitro

[0082]

[0083] Experimental Example 3 Novel Coronavirus M pro / 3CL pro Evaluation of inhibitory activity

[0084] 1. Experimental Principle

[0085] The fluorescence resonance energy transfer (FRET) method is used. The detection principle is as follows: Edans is a fluorescent donor, Dabcyl is a fluorescent acceptor or quencher. The absorption spectra of these two fluorescent groups overlap to a certain extent. When the distance between the two fluorescent groups is appropriate (generally 7-10nm), the fluorescent energy is transferred from the donor to the acceptor, resulting in the attenuation of the fluorescence intensity of the donor fluorescent molecule itself. Edans and Dabcyl are connected to the 2019-nCoV M pro / 3CL pro The two ends of the natural substrate of the protease, namely Dabcyl-KTSAVLQSGFRKME-Edans. pro / 3CL pro When the protease does not cleave the substrate, the two groups are close enough to cause fluorescence resonance energy transfer, that is, Dabcyl can quench the fluorescence of Edans and cause no fluorescence to be detected; when the substrate is 2019-nCoV M pro / 3CL pro After protease cleavage, the two ends of the polypeptide are separated, and the two groups are separated. The fluorescence of Edans is no longer quenched by Dabcyl, and the fluorescence of Edans can be detected. In this way, 2019-nCoVM can be detected very sensitively through fluorescence detection. pro / 3CL pro The enzymatic activity of protease. If 2019-nCoVM is added to the reaction system pro / 3CL pro The generation of fluorescence will be inhibited by the inhibitor, and the fluorescence intensity is inversely proportional to the inhibitory effect of the inhibitor, so that 2019-nCoV M can be detected. pro / 3CL pro The inhibitory effect of protease inhibitors. The maximum excitation wavelength of Edans is 340nm and the maximum emission wavelength is 490nm.

[0086] 2. Experimental methods

[0087] (1) Preparation of samples and positive drugs

[0088] Take appropriate amount of inhibitor sample to be tested and positive drug Ebselen, prepare a solution with concentration of 10 mg / ml with DMSO, and dilute it for activity evaluation. The positive drug is Ebselen.

[0089] (2) Preparation of Assay Reagent

[0090] Prepare an appropriate amount of Assay Reagent according to the number of samples (including relevant controls). pro / 3CL pro 92μl Assay Buffer is needed to prepare Assay Reagent for testing one sample.

[0091] (3) Sample testing

[0092] The samples were tested in a 96-well black plate. 93μl Assay Reagent and 5μl sample were added to each sample well, 93μl Assay Reagent and 5μl DMSO were added to the model well, and 93μl Assay Buffer and 5μl DMSO were added to the blank control. Oscillate for 1 minute on an oscillator and mix thoroughly. 2μl Substrate was quickly added to each well, oscillated for 1 minute on an oscillator and mixed thoroughly. After incubation at 37°C in the dark for 15-20 minutes, fluorescence was determined using a multifunctional enzyme label. The excitation wavelength was 340nm and the emission wavelength was 490nm.

[0093] 3. Activity Results

[0094] The initial screening evaluated the 2019-nCoV M in the reaction system at a sample detection concentration of 50ug / ml. pro / 3CL pro Protease inhibitory activity. Compounds with an inhibition rate greater than 50% were screened three times, and their IC 50 The experimental results are shown in Table 3. All three samples showed good inhibitory activity.

[0095] Table 3 Effects of epigallocatechin-7-gallate on novel coronavirus M pro / 3CL pro The inhibitory effect

[0096]

[0097] Novel CoronavirusM pro / 3CL proThe inhibitory activity results showed that all epigallocatechin-7-gallate compounds had significant activity, among which LG0026 had the strongest activity, and the activities of LG0027 and the racemic isomer LG0316 were comparable.

[0098] In summary, (-)-gallocatechin-7-gallate (LG0026) has obvious anti-new coronavirus SARS-CoV-2 effects and anti-coronavirus HCoV-229E effects; it is expected to become a broad-spectrum anti-coronavirus infection drug, laying a scientific experimental foundation for further research and development and clinical application.

[0099] (+)-Gallocatechin-7-gallate (LG0027) and its racemate (LG0316) have similar anti-coronavirus activity as (LG0026), and are slightly weaker than LG0026.

[0100] Novel CoronavirusM pro / 3CL pro The inhibitory effect is the important mechanism of the anti-coronavirus effects of (-)-gallocatechin-7-gallate (LG0026), (+)-gallocatechin-7-gallate (LG0027) and their racemate (LG0316), and has good application prospects as anti-coronavirus drugs.

Claims

1. Use of (-)-gallocatechin-7-gallate or a pharmaceutically acceptable salt thereof as shown in formula I in the preparation of a drug for preventing and / or treating coronavirus infection, 2. The use according to claim 1, characterized in that The coronavirus is selected from the new coronavirus SARS-CoV-2.

3. The use according to claim 1, characterized in that The coronavirus is selected from HCoV-229E coronavirus.

4. Use of a pharmaceutical composition in the preparation of a drug for preventing and / or treating coronavirus infection, characterized in that: The pharmaceutical composition comprises (-)-gallocatechin-7-gallate or a pharmaceutically acceptable carrier thereof as shown in Formula I, 5. The use according to claim 1, characterized in that The pharmaceutically acceptable salt is selected from pharmaceutically acceptable organic salts or inorganic salts, wherein the organic salt is selected from sulfonates, carboxylates, amino acid salts and fatty acid salts, and the inorganic salt is selected from hydrochlorides, bromates, iodates, sulfates, hydrogen sulfates, phosphates, hydrogen phosphates, dihydrogen phosphates and nitrates.

6. Use according to claim 5, characterized in that The sulfonate is selected from alkyl sulfonates containing 1 to 15 carbon atoms, benzene sulfonates, p-toluene sulfonates, o-toluene sulfonates, and m-toluene sulfonates; the carboxylates are selected from tartrate, maleate, fumarate, citrate, malate, cinnamate, benzoate, malonate, succinate, glutarate, adipate, pamoate, and lactate; the amino acid salt is selected from glutamate and aspartate; and the fatty acid salt is selected from long-chain fatty acid salts containing 2 to 18 carbon atoms.

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