Use of dioxymethylene-containing alkaloid compounds in the preparation of a medicament for treating coronavirus infection

Abstract

The application discloses application of a dioxymethylene-containing alkaloid compound in preparation of a medicine for treating coronavirus infection and belongs to the technical field of medicines. The dioxymethylene-containing alkaloid compound is selected from any one of acetyl trihoutenine, stepholidine, decaline, nanlingensine, fargesin, amurine, betonicine, hydrogenated protoophine, 6-propanone-N-methyl dihydrodecaline or campnospermine. The application first discovers and verifies that the compound has significant coronavirus Mpro protease inhibitory activity, wherein the IC 50 value of hydrogenated protoophine to SARS-CoV-2 Mpro is 0.947 muM, and the IC 50 value of 6-propanone-N-methyl dihydrodecaline is 0.518 muM. Cell level experiments show that the compound can effectively inhibit replication of HCoV-OC43 virus, has excellent antiviral activity and good safety. The application provides a new candidate compound and treatment strategy for development of a novel and efficient anti-coronavirus medicine.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of dioxomethylene-containing alkaloid compounds in the preparation of drugs for treating coronavirus infection, and their application in the preparation of coronavirus Mpro inhibitors. Background Technology

[0002] Coronaviruses are a class of enveloped, positive-stranded RNA viruses, classified into four genera according to systematic taxonomy: α, β, γ, and δ. Among them, β coronaviruses pose the greatest threat to human health. β coronaviruses include HCoV-OC43, HCoV-HKU1, SARS-CoV, the 2019 novel coronavirus (SARS-CoV-2 / 2019-nCoV), and MERS-CoV. The COVID-19 pandemic caused by SARS-CoV-2 has resulted in millions of deaths globally, seriously threatening public health and safety.

[0003] The main targets of coronaviruses include Mpro (main protease), RNA-dependent RNA polymerase (RdRp), PLpro, and S protein. Among them, Mpro (also known as 3C-like protease, 3CLpro) is a core regulatory element of coronavirus replication and transcription. Due to its highly conserved sequence and the absence of homologous proteins in the human body, it has become one of the core targets for the development of drugs against COVID-19. Mpro inhibitors can not only inhibit the proliferation of existing coronaviruses but also have the potential to inhibit variants of the novel coronavirus, thus becoming an ideal target for the development of broad-spectrum antiviral drugs.

[0004] With the widespread use of Mpro inhibitors, drug resistance has begun to emerge, while existing drugs suffer from high prices and uneven supply and demand. Therefore, developing novel, highly effective, and broad-spectrum coronavirus Mpro inhibitors from natural products has significant clinical value and practical implications. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide new uses for dioxomethylene alkaloid compounds or their pharmaceutically acceptable salts, namely, their use in the preparation of medicaments for the prevention and / or treatment of coronavirus infection, and their use in the preparation of coronavirus Mpro inhibitors.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides the use of a dioxomethylene alkaloid compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention and / or treatment of coronavirus infection, wherein the dioxomethylene alkaloid compound is selected from any one or at least a combination of two of the following: acetylcephalotaxine, stephavanine, decarine, Nantenine, Chielanthifoline, Amurine, Hippadine, Hydroptotopine, 6-Acetonyl-N-methyldihydrodecarine, or Cassyfiline.

[0007] The chemical structural formula of the dioxomethylene-containing alkaloid compound is as follows:

[0008] Preferably, the coronavirus is a β-coronavirus.

[0009] More preferably, the coronavirus is HCoV-OC43, SARS-CoV, MERS-CoV, or 2019-nCoV.

[0010] Preferably, the drug inhibits coronavirus replication by inhibiting Mpro protease activity.

[0011] Preferably, the drug further includes pharmaceutically acceptable excipients.

[0012] Preferably, the pharmaceutically acceptable excipients include any one or a combination of at least two of the following: carriers, diluents, fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH adjusters, antioxidants, antibacterial agents, or buffers. Examples of combinations of at least two include, for instance, a combination of binders and diluents, a combination of carriers and flavoring agents, or a combination of binders and fillers; other possible combinations are not described in detail here.

[0013] Preferably, the dosage form of the drug is a capsule, tablet, granule, gel, sustained-release agent, oral liquid, drop pill, or nano-formulation.

[0014] Secondly, the present invention provides the use of dioxomethylene alkaloid compounds or pharmaceutically acceptable salts thereof in the preparation of coronavirus Mpro inhibitors, wherein the dioxomethylene alkaloid compounds are selected from any one or at least two combinations of acetylcephalotaxine, sennain, decarine, nandina, corydaline, amolinine, amaryllidine, proopioline, 6-acetone-N-methyldihydrodecarine, or genistein.

[0015] Thirdly, the present invention provides the use of dioxomethylene-containing alkaloids or their pharmaceutically acceptable salts in the preparation of reagents for inhibiting the activity of Mpro protein in vitro, wherein the dioxomethylene-containing alkaloids are selected from any one or at least two combinations of acetylcephalotaxine, sennaline, decarine, nandinaline, corydaline, amolinine, amaryllidine, protopine, 6-acetone-N-methyldihydrodecarine, or genistein.

[0016] Fourthly, the present invention provides a pharmaceutical composition comprising the above-mentioned dioxomethylene alkaloid compound or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.

[0017] Preferably, the pharmaceutical composition is a capsule, tablet, granule, gel, sustained-release agent, oral liquid, pellet, or nanoformulation.

[0018] Compared with the prior art, the present invention has the following significant advantages: New use discovery: For the first time, natural alkaloid compounds containing dioxane have been discovered and validated to have anti-coronavirus activity, particularly as Mpro inhibitors.

[0019] Excellent inhibitory activity: The compounds of this invention exhibit significant inhibitory activity against Mpro protease, with protopine showing a high IC50 inhibitory effect on SARS-CoV-2 Mpro. 50 The IC50 value of 6-acetone-N-methyldihydrodecarbin was 0.947 μM. 50 The value is 0.518 μM.

[0020] Cellular-level antiviral activity: The compounds of this invention exhibit good anti-HCoV-OC43 activity at the cellular level, wherein the EC50 of protopine is... 50 EC50 of 6-acetone-N-methyldihydrodecarbin at 0.60 μM 50 The value is 0.37 μM, and it has a high selectivity index and good safety.

[0021] Natural product source: The compounds of this invention are derived from natural products, have good biocompatibility and safety, and can be used as lead compounds for further development. Attached Figure Description

[0022] Figure 1 The graph shows the initial screening inhibition rate of 14 dioxane-containing alkaloids against HCoV-OC43 Mpro at a final concentration of 1 μM.

[0023] Figure 2 IC50 values ​​for the inhibitory effects of proopioridine and 6-acetone-N-methyldihydrodecaline on SARS-CoV-2 Mpro 50 Value graph.

[0024] Figure 3 The results of experiments on the inhibition rate and cytotoxicity of protopine and 6-propionyl-N-methyldihydrodecaline against HCoV-OC43 are presented. Detailed Implementation

[0025] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments, but this does not limit the present invention in any way. Any modifications or improvements made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0026] The processes, conditions, reagents, and experimental methods used in implementing this invention, except as specifically mentioned below, are all common knowledge and general knowledge in the field, and this invention does not have any particular limitations. Experimental methods in the embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer.

[0027] Unless otherwise stated, all technical terms and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. However, in the event of any conflict, the specification containing the definitions shall prevail.

[0028] The dioxomethylene-containing alkaloids (acetylcephalotaxine, sennain, decarine, cephalotaxine, nandina, corydaline, amrinine, amaryllidine, protopine, 6-acetone-N-methyldihydrodecarine, and geniopine) selected in this invention can be obtained from commercially available sources or through chemical synthesis methods known in the art or by extraction and isolation from natural plants. Those skilled in the art can prepare the above compounds according to the methods described in the prior art.

[0029] All natural dioxomethylene alkaloid compounds used in the following examples were provided by Xili Biotechnology, and their purity was 98% or higher.

[0030] Experimental Example 1: Mpro Enzyme Activity Inhibition Assay This experimental example uses fluorescence resonance energy transfer (FRET) to evaluate the inhibitory activity of the dioxomethylene alkaloid compounds of the present invention on Mpro.

[0031] 1. Experimental Methods Preparation of drug stock solutions: Acetylcephalotaxine, Stephavanine, Decarine, Cephalotaxine, Drupacine, Homoharringtonine, Nantenine, Lycorine, Chielanthifoline, Amurine, Hippadine, Hydrodroprotopine, 6-Acetonyl-N-methyldihydrodecarine, Cassyfiline, the positive control drug Nirmatrelvir, and Baicalein were prepared into initial stock solutions of 1 mg / mL, which were then diluted to a test solution with a concentration of 1 μM.

[0032] The specific screening method is as follows: 5 μL of Mpro (concentration of 4 μmol·L⁻¹) is added... - ¹) with 155 μL buffer (50 mmol·L⁻¹) - ¹ Tris-HCl, pH 7.2, 1 mmol·L - ¹ EDTA) was mixed, and 20 μL of different compounds were added to a final concentration of 1 μM, and the mixture was incubated at 36.9 °C for 10 minutes. Finally, 20 μL (final concentration 20 μmol·L⁻¹) was added. - The reaction was initiated using a fluorescent substrate (¹). Subsequently, fluorescence intensity was measured using a Bio-Tek SynergyH1 flatbed reader at an excitation wavelength of 320 nm and an emission wavelength of 405 nm. The fluorescence intensity was then compared with that of the negative control group to calculate the inhibition rate.

[0033] 2. Experimental Results The results are as follows Figure 1 As shown, at a concentration of 1 μM, the compounds described in this invention all exhibited varying degrees of inhibitory activity against HCoV-OC43 Mpro. Among them, compound 3 (hydroproopioline) and compound 5 (6-acetone-N-methyldihydrodecaline) showed the highest inhibition rates, comparable to the positive control drug nirmatrelvir, and significantly superior to baicalein.

[0034] Test Example 2 IC 50 Measurement This experiment determined the IC50 values ​​of the most active compounds, 3 (hydroproopioline) and 5 (6-acetone-N-methyldihydrodecaline), against SARS-CoV-2 Mpro. 50 value.

[0035] 1. Experimental Methods The target compounds with good activity were serially diluted to prepare a final concentration of 8 μmol·L⁻¹. - ¹, 4 μmol·L - ¹, 2 μmol·L - ¹, 1 μmol·L - ¹, 0.5 μmol·L - ¹, 0.25 μmol·L - ¹, 0.125 μmol·L - ¹, 0.0625 μmol·L - ¹ Eight concentration gradients were used to evaluate the inhibitory effect of SARS-CoV-2 Mpro according to the method in Example 1, and the IC50 was measured. 50 value.

[0036] 2. Experimental Results The results are as follows Figure 2 As shown, protopine hydrochloride has an IC50 value for SARS-CoV-2 Mpro. 50 The IC50 value of 6-acetone-N-methyldihydrodecaline against SARS-CoV-2 Mpro was 0.947 μM. 50 The value was 0.518 μM, and both exhibited strong Mpro inhibitory activity.

[0037] Experimental Example 3: Cellular Level Antiviral Activity Experiment This study validated the antiviral activity and cytotoxicity of the compound against HCoV-OC43 in RD cells (human rhabdomyosarcoma cells) to assess its potential as an anti-coronavirus drug.

[0038] The test compounds were hydrodroprotopine and 6-acetonyl-N-methyldihydrodecarine, respectively. Stock solutions were prepared by dissolving them in DMSO. During the experiment, these stock solutions were diluted with culture medium to the required working concentration, ensuring the final DMSO concentration did not exceed 0.1%.

[0039] 1. Cytotoxicity assay (CC) 50 (Measurement) The cytotoxicity of the test compound to RD cells was detected using the CCK-8 assay.

[0040] Cell plating: RD cells in logarithmic growth phase were resuspended in DMEM medium containing 10% FBS and the cell density was adjusted to 1×10⁶ cells / year. 5 Cells / mL. Seed the cell suspension into 96-well plates at 100 µL per well (i.e., 1 × 10⁶ cells / mL). 4 (10 cells) were cultured in a 37°C, 5% CO2 incubator for 24 hours to allow the cells to adhere to the incubator.

[0041] Drug treatment: Discard the old culture medium and add 100 µL of fresh culture medium (DMEM containing 2% FBS) containing different concentration gradients of the test compound to each well. Concentration settings: Starting at 100 µM, perform 3-fold serial dilutions to set up a total of 6 concentrations (200 μM, 100 μM, 50 μM, 25 μM, 12.5 μM, and 6.25 μM, with 3 replicates for each concentration). The following control group was also set up: Blank control group: only 100 µL of DMEM medium containing 2% FBS was added (cell-free, drug-free).

[0042] Negative control group: 100 µL of DMEM medium containing 2% FBS (containing cells, no drugs) was added.

[0043] Incubation: Place the 96-well plate in a 37°C, 5% CO2 incubator and continue incubation for 48 hours.

[0044] CCK-8 assay: After incubating the experimental plate in an incubator for 48 h, dilute 10× CCK-8 to 1× using CCK-8 dilution medium, vortex to mix, and place in the dark. Discard the supernatant on the experimental plate, add 100 μL / well of 1× CCK-8 assay solution, wrap the plate with aluminum foil, and incubate in the dark for 30-50 min. Absorbance measurement: The absorbance (OD value) of each well was measured at a wavelength of 450 nm using an ELISA reader.

[0045] Data processing: Calculate the average OD value of each group of replicates.

[0046] Cell viability (%) = [(OD value of drug-treated group - OD value of blank control group) / (OD value of negative control group - OD value of blank control group)] × 100%.

[0047] A dose-response curve was plotted with the logarithm of drug concentration on the x-axis and cell viability (%) on the y-axis. Nonlinear regression analysis was performed using GraphPad Prism software to calculate the median cytotoxic concentration (CC). 50 value).

[0048] 2. Antiviral activity assay (EC) 50 (Measurement) The effect of the test compound on the replication of HCoV-OC43 in RD cells was detected by quantitative RT-PCR.

[0049] Cell plating and drug pretreatment: RD cells in logarithmic growth phase were resuspended in DMEM medium containing 10% FBS and the cell density was adjusted to 5 × 10⁶ cells / year. 5 Cells / mL. Seed the cell suspension into 48-well plates at 200 µL per well (i.e., 1 × 10⁶ cells / mL). 5 Cells were cultured at 37°C in a 5% CO2 incubator for 12 hours. The old medium was discarded, and each well was replaced with 200 µL of DMEM (containing 2% FBS) with different concentration gradients of the test compounds, and incubated for another 2 hours. Concentration settings: according to CC... 50 The test results showed that six concentration gradients were set within the non-toxic concentration range (40 μM, 8 μM, 1.6 μM, 0.32 μM, 0.064 μM, and 0.0128 μM, respectively).

[0050] Viral infection: 2 hours later, add HCoV-OC43 virus solution to each well at a multiplicity of infection (MOI) of 0.1 and mix gently. Incubate the 48-well plate at 37°C in a 5% CO2 incubator for another 48 hours.

[0051] Viral RNA extraction and quantification: 48 hours post-infection, supernatants were collected from each well. Viral RNA was extracted from the supernatants using an RNA extraction kit. RNA was reverse transcribed into cDNA using a PrimeScript RT kit (containing a gDNA Eraser). Absolute quantitative RT-PCR was performed using TB Green® Premix Ex Taq™ II (primers for qRT-PCR: M-qF1: 5′-ggcttatgtggccccttact-3′; M-qR1: 5′-ggcaaatctgcccaagaata-3′) to determine viral copy number. A viral control group (infected with virus only, without drugs) and a cell control group (not infected with virus, without drugs) were also set up.

[0052] Data processing: Calculate the average number of virus copies in each group.

[0053] Viral inhibition rate (%) = [1 - (viral copy number in drug-treated group / viral copy number in virus control group)] × 100%.

[0054] A dose-response curve was plotted with the logarithm of drug concentration on the x-axis and the viral inhibition rate (%) on the y-axis. Nonlinear regression analysis was performed using GraphPad Prism software to calculate the median effective concentration (EC50). 50 value).

[0055] Selectivity Index (SI) Calculation: SI = CC 50 / EC 50 A higher SI value indicates better selectivity and higher safety of the compound.

[0056] 3. Experimental Results The results are as follows Figure 3 As shown, protopine hydrochloride has an effect on the ECG of HCoV-OC43. 50 The value is 0.60 μM, CC 50 The concentration was approximately 6.25 μM, with a selectivity index (SI) of approximately 10.42; 6-acetone-N-methyldihydrodecaline was effective against ECGs of HCoV-OC43. 50 The value is 0.37 μM, CC 50 The concentration was 15.40 μM, and the SI was 41.62. These results indicate that the compounds of this invention exhibit significant anti-HCoV-OC43 activity at the cellular level and demonstrate good safety. 6-Propane-N-methyldihydrodecalin, in particular, exhibits superior selectivity and safety, showing promising potential for the development of anti-coronavirus drugs.

[0057] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The use of dioxomethylene alkaloid compounds or pharmaceutically acceptable salts thereof in the preparation of medicaments for the prevention and / or treatment of coronavirus infection, characterized in that, The dioxomethylene-containing alkaloids are selected from any one or at least two combinations of the following: protopine, 6-propiono-N-methyldihydrodecarline, acetylcephalotaxine, sennain, decarline, nandina, corydaline, amolinine, amaryllidine, or geniocarline.

2. The application according to claim 1, characterized in that, The coronavirus in question is a beta coronavirus.

3. The application according to claim 2, characterized in that, The coronaviruses mentioned are HCoV-OC43, SARS-CoV, MERS-CoV, or 2019-nCoV.

4. The application according to claim 1, characterized in that, The drug inhibits coronavirus replication by suppressing the activity of the Mpro protease.

5. The application according to any one of claims 1-4, characterized in that, The drug also includes pharmaceutically acceptable excipients.

6. The application according to claim 5, characterized in that, The pharmaceutically acceptable excipients include any one or a combination of at least two of the following: carriers, diluents, fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH adjusters, antioxidants, antibacterial agents, or buffers.

7. The application according to claim 1, characterized in that, The dosage form of the drug is capsule, tablet, granule, gel, sustained-release agent, oral liquid, drop pill or nano-formulation.

8. The use of dioxomethylene alkaloids or their pharmaceutically acceptable salts in the preparation of coronavirus Mpro protease inhibitors, characterized in that, The dioxomethylene-containing alkaloids are selected from any one or at least two combinations of acetylcephalotaxine, sennain, decarine, nandina, corydaline, amolinine, amaryllidine, proopiopinine, 6-acetone-N-methyldihydrodecarine, or genistein.

9. The use of dioxomethylene alkaloids or their pharmaceutically acceptable salts in the preparation of reagents for inhibiting Mpro protease activity in vitro, characterized in that, The dioxomethylene-containing alkaloids are selected from any one or at least two combinations of acetylcephalotaxine, sennain, decarine, nandina, corydaline, amolinine, amaryllidine, proopiopinine, 6-acetone-N-methyldihydrodecarine, or genistein.

10. A pharmaceutical composition, characterized in that, It comprises a dioxomethylene alkaloid compound as described in claim 1 or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.

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