Small molecule compound, preparation method thereof and application of small molecule compound in preparation of anti-coronavirus drugs

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a small molecule compound, a preparation method thereof and application of the small molecule compound in preparation of anti-coronavirus medicines. The small molecule compound provided by the invention shows broad-spectrum anti-coronavirus activity, can effectively inhibit replication, proliferation and virus protein expression of MHV in cells, and resists fatal MHV infection. The compound can also block the duplication process of PEDV in cells, and shows a remarkable anti-PEDV effect. Due to the fact that the MHV and the SARS-CoV-2 are similar in virus structure and transmission mechanism, the antiviral research results aiming at the MHV and the PEDV can provide important reference for developing effective drugs aiming at the SARS-CoV-2 and other coronaviruses. The compounds have important application prospects in the field of development of drugs for resisting coronaviruses such as MHV, PEDV and SARS-CoV-2.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine. More specifically, it relates to a class of small molecule compounds, a preparation method thereof and an application thereof in preparing anti-coronavirus drugs. BACKGROUND

[0002] Coronaviruses (CoVs) are a class of single-strand RNA viruses with envelope, and the length of their genome is between 26-32 kb, which is the largest RNA virus known so far. Since the first isolation from avian in 1937, more than 30 coronaviruses have been found, which can infect a variety of hosts including humans, livestock and birds. Due to the easy genetic recombination and variation of coronaviruses, their host range is wide and their transmission ability is strong, which poses a serious threat to public health and animal husbandry. In addition, animal coronaviruses such as porcine epidemic diarrhea virus (PEDV) and swine acute diarrhea syndrome coronavirus (SADS-CoV) also have a significant impact on animal husbandry production. Therefore, it is of great practical significance to develop effective therapeutic drugs against coronaviruses.

[0003] The virions of coronaviruses are spherical or oval, with a diameter of about 60-200 nm, and the spike protein (S protein) on the envelope gives the virus a coronal morphology. The viral genome encodes non-structural proteins (such as replicase pp1a / 1b) and structural proteins (S, E, M, N proteins). Among them, the S protein is a key protein for the virus to invade host cells, and the S1 subunit binds to the host receptor, and the S2 subunit mediates membrane fusion to release the viral genome into the cytoplasm. Viral replication depends on RNA-dependent RNA polymerase (RdRp), and structural proteins such as N protein are involved in viral assembly and immune escape. Due to the high variability of coronaviruses, the development of antiviral drugs targeting key steps in their life cycle (such as viral adsorption, replication and assembly) is a key research direction.

[0004] Currently, the prevention and control of coronaviruses mainly relies on vaccines, but vaccines have limitations such as limited protection range, safety issues and poor effect on variant strains. For example, the high mutation rate of RNA viruses can lead to vaccine escape, and the immune effect of some animal coronaviruses (such as PEDV) is not ideal. Antiviral drugs are an important means to make up for the shortcomings of vaccines, and existing research includes interferons, neutralizing antibodies, Chinese herbal medicine extracts and chemically synthesized compounds. However, interferons and antibody drugs have the problems of insufficient specificity or high production cost, and the antiviral mechanisms of natural products (such as bisbenzylisoquinoline alkaloids) and chemical molecular compounds are still in the laboratory stage, and have not yet realized commercial application. In addition, the existing anti-coronavirus drugs have poor broad-spectrum, which is difficult to cover a variety of coronaviruses.

[0005] Therefore, it is urgent to develop more efficient new anti-coronavirus drugs to provide broad-spectrum, significant efficacy and high safety treatment options to meet the clinical needs of different patients. SUMMARY

[0006] The technical problem to be solved by the present application is to overcome the defects and deficiencies of the current limited anti-coronavirus drugs and the poor broad-spectrum of the existing anti-coronavirus drugs, and to provide a class of small molecule compounds.

[0007] The purpose of the present application is to provide a preparation method of the small molecule compound.

[0008] Another purpose of the present application is to provide the application of the small molecule compound.

[0009] Still another purpose of the present application is to provide a pharmaceutical composition.

[0010] The above purposes of the present application are achieved by the following technical solutions: The present application protects a class of small molecule compounds, which have any of the following structures: ; Among them, the single bond represented by the wavy line represents the E configuration or the Z configuration or their mixture in any ratio relative to the stereochemical configuration of the double bond to which it is bonded. The wavy line in the structure of other compounds has the same meaning.

[0011] The small molecule compound obtained by the present application exhibits broad-spectrum anti-coronavirus activity and shows significant antiviral effect on coronaviruses including MHV and PEDV. Since MHV and SARS-CoV-2 have similarities in virus structure and transmission mechanism, the research results of anti-virus against MHV and PEDV can provide an important reference for the development of effective drugs against coronaviruses such as SARS-CoV-2. This class of compounds has important application prospects in the field of developing anti-MHV, PEDV and SARS-CoV-2 coronavirus drugs.

[0012] Further, the small molecule compound is replaced by a pharmaceutically acceptable salt or a solvate thereof.

[0013] Preferably, the salt is an acetate (also known as acetic acid salt).

[0014] The present application protects a preparation method of the small molecule compound, which comprises the following steps: S1. Compound 1 is dissolved in dichloromethane (DCM), N,N-diisopropylethylamine (DIPEA) and compound 2 are added, and the reaction is carried out at room temperature to obtain a crude product containing compound 3; S2. The crude product containing compound 3 obtained in step S1 is dissolved in dichloromethane, trifluoroacetic acid is added, and the reaction is carried out at room temperature to obtain a crude product containing compound 4; S3. The crude product containing compound 4 obtained in step S2 is dissolved in dichloromethane, N,N-diisopropylethylamine, compound 5 and HATU (2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate, a common condensation reagent) are added, and the reaction is carried out at room temperature, and then purified to obtain compound 6. S4. Compound 6 obtained in step S3 is dissolved in anhydrous ethanol, compound 7 and acetic acid are added, and the reaction is carried out under reflux, and then purified to obtain the target compound. .

[0015] Further, in step S1, the reaction is carried out and then the post-treatment is carried out, which includes stopping the reaction, extraction, washing and drying, and the specific operation is as follows: water is added to the reaction solution obtained after the reaction is carried out, DCM is extracted, the organic phase is combined and washed with saturated brine, anhydrous sodium sulfate is dried, and rotary evaporation is carried out to obtain the crude product containing compound 3.

[0016] Further, in step S2, the reaction is carried out and then the post-treatment is carried out, which includes removing the solvent, and the specific operation is as follows: the reaction solution obtained after the reaction is carried out is rotary evaporated to obtain the crude product containing compound 4.

[0017] Further, in step S3, the purification treatment includes stopping the reaction, extraction, washing, drying, concentration and purification, and the specific operation is as follows: water is added to the reaction solution, DCM is extracted, the organic phase is combined and washed with saturated brine, anhydrous sodium sulfate is dried, and then concentrated under reduced pressure, and then purified by silica gel column chromatography (PE:EA=1:1) to obtain the product as a white or white-like solid, which is compound 6.

[0018] Further, in step S4, the purification treatment includes removing the solvent, dissolving, adjusting the pH, suction filtration and recrystallization, and the specific operation is as follows: the reaction solution is evaporated under reduced pressure to remove the solvent, dissolved in water, and then the pH is adjusted to about 7 with saturated sodium bicarbonate solution, and then a white-like precipitate is obtained in the solution, which is suction filtered to obtain a solid, and then recrystallized to obtain the product as a white-like solid, which is the target compound.

[0019] The application protects the use of the small molecule compound in the preparation of an anti-coronavirus drug.

[0020] Further, the coronavirus includes alpha-coronavirus and / or beta-coronavirus.

[0021] Preferably, the alpha-coronavirus includes PEDV (Porcine Epidemic Diarrhea Virus) or a variant thereof.

[0022] Preferably, the beta-coronavirus includes MHV (Mouse Hepatitis Virus), SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2), or a variant of any of the above viruses.

[0023] Preferably, the cell administration dose of the small molecule compound is ≥ 0.1 μM.

[0024] When the small molecule compound is G003, the cell administration dose of the small molecule compound is preferably 0.1-20 μM, and more preferably 5-20 μM.

[0025] When the small molecule compound is G004, the cell administration dose of the small molecule compound is ≥ 1 μM, preferably 1-20 μM, and more preferably 5-20 μM.

[0026] Preferably, the organism administration dose of the small molecule compound is preferably 10 mg / kg.

[0027] The present application protects a pharmaceutical composition comprising one or two of the small molecule compounds.

[0028] Further, the pharmaceutical composition further contains a pharmaceutically acceptable excipient.

[0029] Still further, the dosage form of the pharmaceutical composition is an injection, a capsule, a tablet, a pill, a granule, a powder or a spray.

[0030] Compared with the prior art, the present application has the following beneficial effects: the small molecule compound provided by the present application exhibits broad-spectrum anti-coronavirus activity, can effectively inhibit the replication, proliferation and viral protein expression of MHV in cells, and can resist fatal MHV infection. Such compounds can also block the replication process of PEDV in cells, and exhibit significant anti-PEDV effect. Since MHV and SARS-CoV-2 have similarities in viral structure and transmission mechanism, the anti-virus research results for MHV and PEDV can provide an important reference for developing effective drugs against SARS-CoV-2 and other coronaviruses. Such compounds have important application prospects in the field of developing drugs against coronaviruses such as MHV, PEDV and SARS-CoV-2. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 CC for G22, G001-G005 and G004-AcOH 50 Data statistics chart.

[0032] Figure 2 TCID measured after incubating infected cells with G22 and G001-G005 (10 μΜ) 50 Data statistics chart.

[0033] Figure 3 RT-qPCR data statistics chart after pre-treating cells with different concentrations of G22, G001-G005 and G004-AcOH and infecting with MHV.

[0034] Figure 4 IC for G22, G001-G005 and G004-AcOH 50 Data statistics chart.

[0035] Figure 5 RT-qPCR data statistics chart after pre-treating cells with different concentrations of G22 and G001-G005 and infecting with PEDV.

[0036] Figure 6 TCID in extracellular supernatant after treating cells with DMSO and 10 μΜ G004 and infecting with MHV 50 Data statistics chart.

[0037] Figure 7 TCID in intracellular after treating cells with DMSO and 10 μΜ G004 and infecting with MHV 50 Data statistics chart.

[0038] Figure 8 Western Blot chart (left) and gray scale analysis chart (right) for detection of MHV-N protein by different dosages of G004

[0039] Figure 9 Data statistics chart for median lethal dose LD50 challenge dosage in mouse animal experiment.

[0040] Figure 10 Survival curve chart of mice after administration of G004-AcOH (10 mg / kg) in animal experiment.

[0041] Figure 11 Body weight change chart of mice after administration of G004-AcOH (10 mg / kg) in animal experiment. DETAILED DESCRIPTION

[0042] The present application is further described in conjunction with the accompanying drawings and specific examples, which are intended to illustrate but not limit the application. Unless otherwise indicated, the reagents, methods, and apparatuses used in the examples below are of an artisan's choice for use in the methods.

[0043] Unless otherwise indicated, the reagents and materials used in the following examples are commercially available.

[0044] Preparation of G003~G004 The synthetic route of G003~G004 is as follows:

[0045] The preparation of G003 includes the following steps: Step 1: Dissolve N-Boc piperazine (compound 1, 1.0 eq) in DCM, add DIPEA (1.2 eq), and add 2-chlorothiophene-5-sulfonyl chloride (compound 2, 1.0 eq) at room temperature. React overnight at room temperature, add water to the reaction solution, extract with DCM, combine the organic phases and wash with saturated brine, dry over anhydrous sodium sulfate, and spin dry to obtain the product (compound 3 crude product), which is used directly in the next step without purification.

[0046] Step 2: Dissolve the product of the previous step in DCM, add trifluoroacetic acid (10 eq), and react at room temperature for 1 hour. Spin dry the product (compound 4 crude product), which is used directly in the next step without purification.

[0047] Step 3: Dissolve the product of the previous step in DCM, and add DIPEA (5 eq), 3-formylbenzoic acid (compound 5, 1 eq), and HATU (1.2 eq) in sequence. React overnight at room temperature, add water to the reaction solution, extract with DCM, combine the organic phases and wash with saturated brine, dry over anhydrous sodium sulfate, concentrate under reduced pressure, and purify by silica gel column chromatography (PE:EA=1:1) to obtain the product as a white or off-white solid (compound 6).

[0048] Step 4: Dissolve the product of the previous step in anhydrous ethanol, and add tryptamine (compound 7, 1 eq) and acetic acid (10 eq). Reflux overnight, evaporate the solvent under reduced pressure, dissolve in water, adjust the pH to about 7 with saturated sodium bicarbonate solution, and extract with ethyl acetate. Dry the organic phase over anhydrous sodium sulfate, filter, and concentrate under reduced pressure. Purify the residue by recrystallization to obtain the product as an off-white solid (target compound G003, ).

[0049] The NMR data of G003 are as follows: 1H NMR (500 MHz, DMSO-d6) δ 10.48 (s, 1H), 7.49 - 7.30 (m, 7H), 7.22 (d, J = 7.9 Hz, 1H), 7.16 (d, J = 2.0 Hz, 1H), 7.07 - 7.01 (m, 1H), 6.97 (t, J = 7.4 Hz, 1H), 5.11 (s, 1H), 3.80 (s, 1H), 3.62 - 3.44 (m, 3H), 3.30 (t, J = 7.1 Hz, 0H), 3.19 - 2.89 (m, 8H), 2.81 - 2.62 (m, 3H). 13 C NMR (126 MHz, DMSO-d6) δ 169.12, 143.27, 135.88, 135.04, 134.70, 129.89, 128.15, 126.89, 126.72, 125.92, 120.51, 118.10, 117.42, 110.85, 108.54, 59.50, 56.08, 45.36, 41.02, 26.00, 24.62, 24.36. The preparation of G004 comprises the following steps: Step 1: Dissolve N-Boc piperazine (compound 1, 1.2 eq) in DCM, add DIPEA (1.2 eq), add 4-chlorobenzenesulfonyl chloride (compound 2, 1.0 eq) at room temperature, react overnight at room temperature, add water to the reaction solution, extract with DCM, combine the organic phases and wash with saturated brine, dry over anhydrous sodium sulfate, and spin dry to obtain the product (compound 3 crude product), which is used directly in the next step without purification.

[0050] Step 2: Dissolve the product of the previous step in DCM, add trifluoroacetic acid (10 eq), and react at room temperature for 1 hour, then spin dry the product (compound 4 crude product), which is used directly in the next step without purification.

[0051] Step 3: Dissolve the product of the previous step in DCM, add DIPEA (5 eq), 3- formylbenzoic acid (compound 5, 1 eq), and HATU (1.2 eq) in sequence, react overnight at room temperature, add water to the reaction solution, extract with DCM, combine the organic phases and wash with saturated brine, dry over anhydrous sodium sulfate, concentrate under reduced pressure, and purify by silica gel column chromatography (PE:EA = 1:1) to obtain the product as a white or off-white solid (compound 6).

[0052] Step 4: The product of the previous step was dissolved in absolute ethanol, tryptamine (1 eq) and acetic acid (10 eq) were added, and the reaction was refluxed overnight. The solvent was evaporated under reduced pressure, and the solution was dissolved in water and adjusted to pH 7 with saturated sodium bicarbonate solution. A white precipitate appeared in the solution, which was filtered to obtain a solid. The product was obtained as a white solid after recrystallization (target compound G004, ).

[0053] The NMR data of G004 are as follows: 1 H NMR (500 MHz, DMSO-d6) δ 10.46 (s, 1H), 7.72 - 7.66 (m, 2H), 7.61 (d, J = 8.5 Hz, 2H), 7.45 (d, J = 7.7 Hz, 1H), 7.43 - 7.37 (m, 2H), 7.29 (dt, J = 6.5, 2.0 Hz, 1H), 7.22 (d, J = 7.9 Hz, 1H), 7.12 (s, 1H), 7.07 - 7.01 (m, 1H), 6.99 (t, J = 7.4 Hz, 1H), 5.10 (s, 1H), 3.74 (s, 0H), 3.50 (t, J = 9.8 Hz, 2H), 3.31 (d, J = 12.4 Hz, 1H), 2.98 (ddt, J = 49.8, 12.3, 5.6 Hz, 2H), 2.74 - 2.57 (m, 2H). 13 C NMR (126 MHz, DMSO-d6) δ 169.11, 143.32, 135.90, 135.77, 134.79, 134.73, 133.33, 132.91, 130.03, 128.59, 128.14, 126.79 (d, J = 12.7 Hz), 125.98, 120.55, 118.14, 117.47, 110.85, 108.54, 56.17, 45.51, 41.16, 39.95, 22.09. Preparation of G004-AcOH: In order to further increase the solubility of G004, G004 and acetic acid were mixed in a 1:1 molar ratio and then lyophilized to obtain the acetate salt of G004, referred to as G004-AcOH.

[0054] Example 2 Preparation of G001, G002, G005, G22 G001, G002, G005, G22 have the following structural formula:

[0055] G001, G002, G005, G22 were prepared according to the method of Reference Example 1.

[0056] The NMR data of G001 are as follows: 1 H NMR (500 MHz, DMSO-d6) δ 10.46 (s, 1H), 7.51 - 7.37 (m, 7H), 7.28 (dt, J = 6.4, 2.0 Hz, 1H), 7.22 (d, J = 7.9 Hz, 1H), 7.10 (s, 1H), 7.04 (t, J = 7.3 Hz, 1H), 6.99 (t, J = 7.4 Hz, 1H), 5.11 (s, 1H), 3.74 (s, 1H), 3.53 - 3.45 (m, 2H), 3.31 (s, 0H), 3.10 - 2.99 (m, 1H), 2.93 (dt, J = 12.2, 5.9 Hz, 1H), 2.78 - 2.55 (m, 2H), 2.41 (s, 3H). 13 C NMR (126 MHz, DMSO-d6) δ 169.05, 143.60, 142.97, 135.90, 134.73, 134.54, 132.05, 130.06, 129.78, 128.17, 127.36, 126.86, 126.68, 126.06, 120.59, 118.16, 117.45, 110.85, 108.49, 56.14, 45.58, 41.15, 39.95, 21.93, 20.90. The NMR data of G002 are as follows: 1H NMR (500 MHz, DMSO-d6) δ 10.50 (s, 1H), 7.48 - 7.40 (m, 3H), 7.38 - 7.32 (m, 1H), 7.25 - 7.18 (m, 2H), 7.01 (t, J = 7.5 Hz, 1H), 6.96 (t, J = 7.4 Hz, 1H), 5.14 (s, 1H), 3.71 (s, 1H), 3.54 - 3.37 (m, 2H), 3.24 - 3.17 (m, 2H), 3.08 - 2.91 (m, 4H), 2.78 - 2.63 (m, 2H), 1.93 (t, J = 10.5 Hz, 3H), 1.75 (dt, J = 13.2, 3.4 Hz, 3H), 1.61 (d, J = 12.7 Hz, 1H), 1.38 - 1.04 (m, 6H). 13 C NMR (126 MHz, DMSO-d6) δ 169.13, 143.27, 135.88, 135.05, 134.70, 129.90, 128.16, 126.90, 126.73, 125.93, 120.52, 118.11, 117.43, 110.86, 108.55, 59.49, 56.09, 45.37, 41.02, 39.95, 26.03 (d, J = 4.9 Hz), 24.63, 24.36, 22.06. NMR data for G005 is as follows: 1H NMR (500 MHz, DMSO-d6) δ 10.46 (s, 1H), 7.72 (d, J = 8.5 Hz, 1H), 7.60 (d, J = 2.3 Hz, 1H), 7.48 (dd, J = 8.6, 2.3 Hz, 1H), 7.41 (dd, J = 8.2, 6.2 Hz, 3H), 7.32 (td, J = 4.2, 3.8, 1.5 Hz, 1H), 7.20 (d, J = 8.0 Hz, 2H), 7.01 (t, J = 7.4 Hz, 1H), 6.95 (t, J = 7.4 Hz, 1H), 5.11 (s, 1H), 3.69 (s, 1H), 3.61 - 3.41 (m, 0H), 3.30 (s, 0H), 3.20 - 2.91 (m, 5H), 2.68 (td, J = 15.2, 13.9, 7.9 Hz, 2H), 2.54 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ 169.04, 143.17, 138.19, 135.87, 134.72, 133.94, 130.01, 129.48, 129.20, 128.12, 126.83, 126.71, 125.97, 120.54, 118.13, 117.42, 110.83, 108.51, 56.15, 45.47, 41.15, 40.11, 39.94, 22.02. NMR data for G22 are as follows: 1 H NMR (500 MHz, DMSO-d6) δ 10.51 (s, 1H), 7.78 (td, J = 8.5, 6.3 Hz, 1 H), 7.59 (ddd, J = 11.3, 9.2, 2.6 Hz, 1H), 7.44–7.39 (m, 3H), 7.35–7.31 (m, 2H), 7.25–7.18 (m, 2H), 7.07–7.00 (m, 1H), 7.01–6.94 (m, 1H), 5.24 (s, 1H), 3.69–3.37 (m, 4H), 3.20–3.04 (m, 4H), 3.05–2.90 (m, 2H), 2.83–2.65 (m, 2H). 13 C NMR (126 MHz, DMSO-d6) δ 169.08, 165.35 (dd, J = 254.7, 12.1 Hz), 159.25 (dd, J = 256.5, 13.8 Hz), 135.99, 135.00, 132.96 (d, J = 10.7 Hz), 130.27, 128.32, 127.33 (x2), 126.59, 126.45, 120.87, 120.59 (dd, J = 14.6, 3.7 Hz), 118.36 (x2), 117.68, 112.68 (dd, J = 22.1, 3.1 Hz), 111.05, 108.40, 106.47 (t, J = 26.6 Hz), 56.03, 45.29, 45.25, 40.98, 21.55. Example 3 CCK8 detection of drug cytotoxicity and CC50 calculation First, 17Cl-1 was plated in a 96-well plate (8000 cells / well) and incubated at 37 °C with 5% CO2 for 12 h. First, the drug was dissolved in DMSO to 100 mM, and then 100 mM drug was used as the substrate to dilute to 0.01, 0.1, 1, 5, 20, 40, 80 mM with DMSO, and finally mM level of drug was diluted to 0.01, 0.1, 1, 5, 20, 40, 80, 120, 150 μΜ with 10% FBS DMEM. After 12 h of cell culture, to prevent evaporation error, 60 wells of the 96-well plate except the outermost circle of cell wells were all discarded, and then the drugs of various concentrations were added from left to right, one column for each concentration (6 repeated wells), 60 μL of the corresponding concentration of drug was added to each well, at the same time, a 96-well plate was prepared, one column of 10% FBS DMEM was added to each well (60 μL) as a background control group. After adding the drug to the cell plate, it was incubated at 37 °C with 5% CO2 for 24 h. CCK8 detection reagent (GENVIEW) was prepared, and the amount used per well was 10 μL CCK-8 solution + 30 μL 10% FBS DMEM. After 24 h of drug incubation with cells, CCK8 detection reagent was added in the dark at 37 °C with 5% CO2 for 2 h, and then the OD450 nM absorbance was detected using an enzyme marker. The half cytotoxicity concentration CC50 was calculated by GraphPad software. 50 and plotted.

[0057] The results are shown in Figure 1As shown, the cytotoxicity of the drugs after incubation with different concentrations of drugs was within a reasonable range, among which the CC 50 of G003, G004 and G004-AcOH were 22.525 μM, 23.352 μM and 17.174 μM, respectively. The above results showed that these compounds had certain safety.

[0058] Example 4 Verification of the anti-MHV antiviral activity and IC 50 of G22 and G004 in vitro For example, in the study of MHV, 17Cl-1 was first plated in a 24-well plate (2 x 10 5 cells / well) and cultured at 37 ˚C with 5% CO2 for 12 h. The drugs were dissolved in DMSO to 100 mM, and then diluted to 0.01, 0.1, 1, 5, 20 mM with DMSO using 100 mM drug as the substrate. Finally, the mM-level drugs were diluted to 0.01, 0.1, 1, 5, 10, 20 μM with 10% FBS DMEM. When the plated cells were incubated for 12 h, 400 μL / well of each drug (2 replicates for each drug) was added, and a DMSO control group without drug (3 replicates) and a MOCK group without infection were set. After the drugs were incubated with the cells in a 37 ˚C cell culture box with 5% CO2 for 3 h, the cells were directly infected with virus MHV (100 μL / well, 10% FBS DMEM dilution) without discarding the liquid. After 2 h of infection, the cells were washed with PBS for 3 times, and then 400 μL / well of drug and 100 μL / well of 10% FBS DMEM were added. After 24 h of continuous culture at 37 ˚C with 5% CO2, the cell culture solution was discarded, the cells were washed with PBS for 3 times, and the total RNA of the cells was extracted according to the instructions of the RNAfast200 total RNA fast extraction kit (Feijie Biology). Reverse transcription was performed using the HiScript II Q RT SuperMix for qPCR (+gDNAwiper) (Novozyme) reagent, and the qPCR reaction system was configured using the ChamQ SYBR qPCR Master Mix (Novozyme) reagent. The relative fluorescence quantitative detection of viral mRNA level was performed on an ABI 7500 real-time fluorescence quantitative PCR instrument to show the antiviral activity of each drug. The half-inhibitory concentration IC 50 was calculated by GraphPad software, and the drug selection index SI (SI = CC 50 / IC 50 ) was calculated in combination with the CC 50 data.

[0059] The results are shown in Table 1. Figure 2As shown, after different drugs (10 μM) incubated with virus infected cells, TCID 50 The virus titers of virus particles showed that the tested compounds had anti-MHV viral activity in vitro.

[0060] The results are shown in Table 1. Figure 3 As shown, after different drugs (10 μM) incubated with virus infected cells, TCID

[0061] The results are shown in Table 1. Figure 4 As shown, the IC 50 of G003, G004 were 143.7 nM, 266.6 nM (the IC 50 of G004 solution prepared by acetic acid was 154.6 nM). According to the CC 50 / IC 50 , the selectivity index SI of G003, G004, G004-AcOH were 156.75, 87.59 and 111.09 respectively, which further indicated that the compounds had high SI index and good safety.

[0062] Example 5 Verification of anti-PEDV antiviral activity of G22, G004 drugs in vitro Vero cells were plated in 24-well plates (2×10 5The cells were incubated at 37 °C with 5% CO2 for 12 h. The drugs were dissolved in DMSO to 100 mM, and then diluted to 1, 5, 15 mM with DMSO, and finally diluted to 1, 5, 15 μM with DMEM (containing 0.4% trypsin). When the cells were incubated for 12 h, 400 μL / well of each drug (3 repeats for each drug) was added, and a DMSO control group (3 repeats) without drug and a MOCK group without virus were set. After the cells were incubated with the drugs at 37 °C with 5% CO2 for 3 h, the virus PEDV was added at MOI=0.001 without discarding the liquid. After 17 h of continuous culture at 37 °C with 5% CO2, the cell culture solution was discarded, the cells were washed with PBS for 3 times, and the total RNA was extracted according to the instructions of the RNAfast200 total RNA fast extraction kit (Feijie Biology). The reverse transcription was performed using the HiScript II Q RT SuperMix for qPCR (+gDNA wiper) (Novozyme) reagent, the qPCR reaction system was configured using the ChamQ SYBR qPCR Master Mix (Novozyme) reagent, and the relative fluorescence quantitative detection of the virus mRNA level was performed on the ABI 7500 real-time fluorescence quantitative PCR instrument to show the antiviral activity of each drug. The antiviral activity of the drug was analyzed by GraphPad software.

[0063] The results are shown in Figure 5 G001, G002, G005 and G22 have no anti-viral activity against PEDV, while G003 and G004 have significant anti-PEDV viral activity. Since G004 has good antiviral effect on both MHV and PEDV, and especially the anti-viral effect on PEDV is better than that of G003, G004 drug is selected for subsequent related research.

[0064] Example 6 TCID 50 Detection of antiviral effect of drug For example, the research on MHV: 17Cl-1 was first plated in a 12-well plate (3×10 5The cells were incubated at 37 °C with 5% CO2for 12 h. The drugs were dissolved in DMSO to 100 mM, and then diluted to 20 mM with DMSO. Finally, the mM-level drugs were diluted to 10 μM with 10% FBS DMEM. When the cells were incubated for 12 h, 400 μL / well of each drug (3 repeats for each drug) was added, and a DMSO control group without the drug was set. After the drug was incubated with the cells at 37 °C with 5% CO2for 3 h, the virus MHV was added at a MOI of 0.01 (600 μL / well, 10% FBS DMEM dilution) without discarding the liquid. After 2 h of infection, the cells were washed with PBS for 3 times, and then 400 μL / well of the drug and 600 μL / well of 10% FBS DMEM were added. After 24 h of continuous culture at 37 °C with 5% CO2, the cell culture fluid (extracellular virus) was collected and frozen-thawed once at -80 °C, and 1 mL / well of 10% FBS DMEM was added and frozen-thawed once at -80 °C to collect the cell culture fluid (intracellular virus).

[0065] The collected intracellular and extracellular viruses were gradiently diluted, i.e., 100 μL of virus solution + 900 μL of 2% FBS DMEM, for a total of 10 gradients. After 17Cl-1 was incubated at 37 °C with 5% CO2for 12 h, the cell culture fluid was discarded, and 100 μL / well of each dilution of the virus solution was added from left to right, one dilution per column, with a total of 8 repeats. The cells were continuously incubated at 37 °C with 5% CO2for 2-5 days, and the TCID50was calculated by observing the cytopathic effect (cell fusion). 50 The change in virus titer was detected.

[0066] The results are shown in Table 1. Figures 6-7 Compared with the DMSO group, the G004 drug group showed a significant inhibitory effect on the multiplication of MHV both in the extracellular (Fig. 1) and intracellular (Fig. 2) spaces. Figure 6 Figure 7

[0067] Example 7: Western Blot detection of the antiviral effect of the drug For example, the MHV was used: 17Cl-1 was first plated in a 12-well plate (3×10 5 ​​Cells were cultured at 37 ˚C with 5% CO2 for 12 h. The drug was dissolved in DMSO to 100 mM, and then diluted to 20 mM with DMSO using 100 mM drug as substrate. Finally, the mM-level drug was diluted to 10 μM with 10% FBS DMEM. When the seeded cells had been incubated for 12 h, 400 μL / well of G004 was added, and a DMSO control group without drug was set up. After the cells were incubated in a cell culture incubator at 37 ˚C with 5% CO2 for 3 h, the medium was not discarded, and the virus MHV (600 μL / well, diluted in 10% FBSDMEM) was directly inoculated at MOI=0.01, MOI=0.05, and MOI=0.1. Two h after inoculation, the cells were washed three times with PBS, and 400 μL / well of drug and 600 μL / well of 10% FBSDMEM were added again. After culturing at 37°C with 5% CO2 for 24 h, the culture medium was discarded, the cell culture was collected, total protein was extracted, and the expression level of MHV-N protein was detected by Western blotting.

[0068] Experimental results are as follows Figure 8 As shown, at the same exposure dose, compared with the DMSO group, the MHV-N band in the drug-treated group (G004 group) was significantly weakened, indicating that G004 showed a significant inhibitory effect on MHV-N protein at different exposure doses. Example 8: The effect of the drug on inhibiting MHV infection in a mouse model (1) LD 50 Determination: Taking the MHV study as an example: 10 PFU group (5 animals), 50 PFU group (5 animals), and 10 PFU group were set up. 2 PFU group (5 animals), 200 PFU group (5 animals), 10 3 PFU group (5 animals), 10 4 PFU group (5 animals), 10 5 The PFU group (n=5) and the control group (n=5) were challenged with nasal drops of PFU according to the set dosage. The weight changes and mortality of the mice were recorded for 14 consecutive days to calculate the median lethal dose (LD50). 50 This will provide reference data for the challenge dosage in subsequent animal experiments. Figure 9 As shown, all animals in the 10 PFU group died, 4 animals in the 50 PFU group died, and 10 PFU group... 2 Three animals died in the PFU group, all of the 200 PFU group survived, and 10... 3 All PFU group members survived; 10 4 All PFU group members survived; 10 5PFU group all survived and the non-toxic control group all survived, according to the data results, the LD 50 = 93.33 PFU.

[0069] (2) Animal experiment G004 was dissolved in DMSO to 100 mM, and then 100 mM G004 was used as a substrate to dilute to 20 mM with DMSO. Finally, mM G004 was used for subsequent experiments. In the animal experiment, it was found that mM G004 was not fully dissolved in the subsequent solvent (normal saline). Therefore, the drug was introduced into the group modification to improve the solubility of G004 in the solvent. The designed G004-AcOH enhanced the solubility of the drug, and the addition of acetate group did not affect the antiviral effect of the drug. Therefore, G004-AcOH was used for subsequent animal experiments. Similarly, LD 50 =93.33 PFU was used to determine the challenge dose, and different dosages were used for grouping. The drug administration group: intraperitoneal injection of drug at 21:00, 10 mg / kg, intranasal challenge at 10:00 the next morning, 150 PFU per mouse, intraperitoneal injection of drug 6 hours later, 10 mg / kg, and then continuous drug administration (twice a day: 9:00 and 16:00) for 1-5 days. Seven mice were continuously weighed to record the mortality rate, and three mice were dissected on the fourth day to measure the virus titer. The DMSO group: intraperitoneal injection of drug (solvent) at 21:00, 10 mg / kg, intranasal challenge at 10:00 the next morning, 150 PFU per mouse, intraperitoneal injection of drug (solvent) 6 hours later, 10 mg / kg, and then continuous drug administration (solvent, twice a day: 9:00 and 16:00) for 1-5 days. Seven mice were continuously weighed to record the mortality rate, and three mice were dissected on the fourth day to measure the virus titer. The MOCK group: except for no challenge, the rest of the conditions were the same as the DMSO group. Two mice were continuously weighed to record the mortality rate, and three mice were dissected on the fourth day to measure the virus titer.

[0070] The results are shown in Figure 9 , 10 The survival rate of the drug administration group: one mouse died on the seventh day, and the rest survived. The survival rate of the DMSO group: one mouse died on the seventh day, three mice died on the eighth day, and the rest survived. Body weight detection found that the body weight of mice in the drug administration group fluctuated less and recovered faster ( Figure 11 ). Therefore, G004-AcOH (10 mg / kg) can effectively protect mice from lethal attack by MHV, with higher survival rate and better weight recovery.

[0071] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications, etc. made without departing from the spirit and principles of the present application should be equivalent replacement manners and should be included in the protection scope of the present application.

Claims

1. A class of small molecule compounds characterized in that, The small molecule compound has any one of the following structures: ； The single bond represented by the wavy line indicates that the stereochemical configuration of the double bond to which it is bonded is E configuration or Z configuration or a mixture thereof in any ratio.

2. The small molecule compound of claim 1, wherein, The small molecule compound is replaced by a pharmaceutically acceptable salt thereof or a solvate thereof.

3. A method of producing the small molecule compound of claim 1, characterized by, The method comprises the following steps: S1. Compound 1 is dissolved in dichloromethane, N,N-diisopropylethylamine and compound 2 are added, and the reaction is carried out at room temperature to obtain a crude product containing compound 3; S2. The crude product containing compound 3 obtained in step S1 is dissolved in dichloromethane, and trifluoroacetic acid is added, and the reaction is carried out at room temperature to obtain a crude product containing compound 4; S3. The crude product containing compound 4 obtained in step S2 is dissolved in dichloromethane, N,N-diisopropylethylamine, compound 5 and HATU are added, and the reaction is carried out at room temperature, and then purification treatment is carried out to obtain compound 6; S4. Compound 6 obtained in step S3 is dissolved in anhydrous ethanol, compound 7 and acetic acid are added, and the reaction is carried out under reflux, and after the reaction is completed, purification treatment is carried out to obtain the target compound: 。 4. The small molecule compound of claim 1 or 2 for use in the preparation of a drug for resisting coronavirus.

5. The use according to claim 1, characterized in that, The coronavirus includes alpha-coronavirus and / or beta-coronavirus.

6. Use according to claim 4, characterized in that, The alpha-coronavirus includes PEDV or a variant thereof.

7. Use according to claim 4, characterized in that, The beta-coronavirus includes MHV, SARS-CoV-2 or a variant of any of the above viruses.

8. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises one or both of the small molecule compounds of claim 1 or claim 2.

9. The pharmaceutical composition according to claim 8, wherein The pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

10. The pharmaceutical composition of claim 8, wherein, The dosage form of the pharmaceutical composition is injection, capsule, tablet, pill, granule, powder or spray. The coronavirus includes alpha-coronavirus and / or beta-coronavirus. The alpha-coronavirus includes PEDV or a variant thereof. The beta-coronavirus includes MHV, SARS-CoV-2 or a variant of any of the above viruses. The pharmaceutical composition comprises one or both of the small molecule compounds of claim 1 or claim 2. The pharmaceutical composition further comprises a pharmaceutically acceptable excipient. The dosage form of the pharmaceutical composition is injection, capsule, tablet, pill, granule, powder or spray.

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