Application of atesane diterpenoid compound in preparation of antiviral drugs

Abstract

The invention discloses an application of an atestin diterpenoid compound Agallochaol Q. The atestin diterpenoid compound Agallochaol Q has remarkable inhibitory activity on enteroviruses (especially EV-D68), so that the atestin diterpenoid compound has a relatively great development prospect and a relatively great research value in the aspect of resisting the EV-D68, and the atestin diterpenoid compound Agallochaol Q has a relatively good application prospect in resisting the enteroviruses (especially the EV-D68). The compound is expected to be further designed and developed into a novel anti-EV-D68 medicine in the future.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to use of an athisanane diterpenoid compound in the preparation of antiviral drugs. Background Art

[0002] Enterovirus D68 (EV-D68) is an RNA virus that primarily infects children and young adults. It belongs to the genus Enterovirus in the family Picornaviridae. Unlike other enteroviruses, EV-D68 is primarily transmitted through the respiratory tract, rather than the traditional fecal-oral route. This is because EV-D68 is sensitive to acidic environments and cannot survive in the gastrointestinal tract. In addition, EV-D68 is more stable at lower temperatures (33°C), making it closer to rhinoviruses than to other enteroviruses. EV-D68 infection mostly presents with mild respiratory symptoms such as cough, runny nose, and fever. However, in a few cases, the virus can invade the central nervous system, leading to serious neurological complications such as acute flaccid myelitis (AFM). The clinical manifestations of AFM are acute onset of limb paralysis, often accompanied by lesions in the gray matter of the spinal cord.

[0003] In recent years, EV-D68 infection has increased significantly among children and young people worldwide.

[0004] EV-D68 enters cells by binding to sialic acid receptors on the surface of host cells. After the virus enters the cell, its genomic RNA is released and translated and replicated. Viral nonstructural proteins (such as 2C, 3C, and 2A) play a key role in viral replication and evasion of the host immune response. Existing antiviral drugs include Pleconaril (capsid inhibitor) and Telaprevir (2A protease inhibitor), but these drugs have shown certain limitations in clinical trials. Pleconaril showed good antiviral activity in in vitro experiments, but its effect in animal models was limited. Although Telaprevir has an inhibitory effect on the 2A protease of EV-D68, it failed to significantly improve the neurological functional outcomes of patients with AFM in clinical trials. Currently, the treatment of EV-D68 infection mainly relies on supportive care, and there are no specific antiviral drugs or vaccines. Therefore, there is an urgent need to find highly effective anti-EV-D68 therapeutic drugs. Summary of the Invention

[0005] The present invention aims to solve at least one of the above-mentioned technical problems existing in the prior art. To this end, the present invention aims to provide a method for preparing an antiviral drug comprising using an athisanane diterpenoid compound.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] In one aspect, the present invention provides the use of a compound of formula I, a geometric isomer thereof, a pharmaceutically acceptable salt thereof, a solvate thereof, or a hydrate thereof in the preparation of a medicament for use in one or more of the following:

[0008] 1) Prevent or treat enterovirus infection or diseases caused by enterovirus infection;

[0009] 2) Inhibit the replication or reproduction of enterovirus in cells;

[0010]

[0011] The compound of formula I is an atisenane diterpenoid compound Agallochaol Q obtained by extraction and separation from the stems and branches of Acris pumila.

[0012] In some embodiments, the enterovirus is selected from poliovirus (PV), Coxsackie A Virus (CV-A), Coxsackie B Virus (CV-B), Echovirus, Enterovirus 71 (EV71), Enterovirus D68 (EV-D68), or any combination thereof.

[0013] In some embodiments, the enterovirus is selected from EV-D68.

[0014] In some embodiments, the disease caused by enterovirus infection is selected from hand, foot and mouth disease, respiratory system infection, central nervous system disease, acute flaccid paralysis, nonspecific febrile illness, pneumonia, bronchiolitis, encephalitis and myocarditis, acute flaccid myelitis or any combination thereof.

[0015] In some embodiments, the disease caused by enterovirus infection is selected from hand, foot and mouth disease, respiratory system infection, central nervous system disease, acute flaccid paralysis or any combination thereof.

[0016] In some embodiments, the cell is a mammalian cell.

[0017] In some embodiments, the mammal is selected from the group consisting of bovine, equine, ovine, porcine, canine, feline, rodent, and primate.

[0018] In some embodiments, the mammal is a human, a cat, a pig, or a dog.

[0019] In some embodiments, the mammal is a human.

[0020] In some embodiments, the drug contains the compound of formula I, its geometric isomers, pharmaceutically acceptable salts, solvates or hydrates thereof as the sole active pharmaceutical ingredient.

[0021] In some embodiments, the drug further contains other anti-enteroviral active ingredients.

[0022] In some embodiments, the compound of formula I, its geometric isomers, pharmaceutically acceptable salts, solvates or hydrates thereof are used in combination with other anti-enteroviral active ingredients.

[0023] In some embodiments, the compound of formula I, its geometric isomers, pharmaceutically acceptable salts, solvates or hydrates thereof are in the same dosage unit with other anti-enteroviral active ingredients.

[0024] In some embodiments, the compound of formula I, its geometric isomers, pharmaceutically acceptable salts, solvates or hydrates thereof and other anti-enteroviral active ingredients are in different formulation units.

[0025] In some embodiments, the compound of formula I, its geometric isomers, pharmaceutically acceptable salts, solvates or hydrates thereof are administered simultaneously, separately or sequentially with other anti-enteroviral active ingredients.

[0026] In some embodiments, the other anti-enteroviral active ingredients are selected from one or more of Pleconaril, Vapendavir, Pirodavir, Pocapavir, R856932, OBR-5-340, DAS181, Suramin, Guanidine, Fluoxetine, Pirlindole mesylate, Formoterol, Zuclopenthixol, Dibucaine, Telaprevir, Rupintrivir, AG7404, Emetine, Azvudine, GPC-N114, Enviroxime, Itraconazole, and NITD008.

[0027] In some embodiments, the drug may further comprise a pharmaceutically acceptable carrier and / or excipient.

[0028] In some embodiments, the drug is prepared into a dosage form selected from the group consisting of tablets, capsules, pills, oral liquid preparations, granules, powders, and injections.

[0029] In some embodiments, the drug is administered by a route selected from the group consisting of oral administration, injection, implantation, topical application, spraying, and inhalation.

[0030] definition

[0031] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. At the same time, in order to better understand the present invention, the definitions and explanations of relevant terms are provided below.

[0032] In the present invention, "geometric isomers" refer to stereoisomers in molecules with double bonds or ring structures, which are produced by different spatial arrangements of atoms or atomic groups connected to the double bonds or rings in the molecules due to the hindrance of free rotation, such as cis / trans isomers.

[0033] In the present invention, "pharmaceutically acceptable salts" include salts formed between the compound of Formula I and a pharmaceutically acceptable inorganic or organic acid, or a pharmaceutically acceptable inorganic or organic base. Examples of suitable acid addition salts include salts formed with hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, perchloric acid, fumaric acid, acetic acid, propionic acid, succinic acid, glycolic acid, formic acid, lactic acid, maleic acid, tartaric acid, citric acid, pamoic acid, malonic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, hydroxynaphthoic acid, hydroiodic acid, malic acid, or tannic acid. Examples of suitable base addition salts include salts formed with sodium, lithium, potassium, magnesium, aluminum, calcium, zinc, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine, or procaine. Methods for preparing pharmaceutically acceptable salts of the compounds of the present invention are known to those skilled in the art.

[0034] As used herein, "solvate" refers to a substance formed by the association of a compound of Formula I or a pharmaceutically acceptable salt thereof with organic solvent molecules, including but not limited to methanol, ethanol, propanol, acetonitrile, and the like. The compound of Formula I or a pharmaceutically acceptable salt thereof may form a hydrate with water.

[0035] In the present invention, "the pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), including but not limited to binders, diluents, fillers, disintegrants, wetting agents, lubricants, colorants, flavorings, solubilizers, osmotic pressure regulators or other conventional additives. Typical pharmaceutically acceptable carriers and / or excipients include, for example, microcrystalline cellulose, starch, cross-linked polyvinylpyrrolidone, povidone, polyvinylpyrrolidone, maltitol, citric acid, sodium lauryl sulfate or magnesium stearate.

[0036] The beneficial effects of the present invention are:

[0037] The present invention provides use of an artisanane diterpenoid compound, Agallochaol Q, in anti-enterovirus treatment. The artisanane diterpenoid compound, Agallochaol Q, exhibits significant inhibitory activity against enteroviruses (particularly EV-D68). Therefore, the artisanane diterpenoid compound has great development prospects and research value in anti-EV-D68 treatment and is expected to be further designed and developed into a new anti-EV-D68 drug in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 1H NMR spectrum of the atissenane diterpenoid compound Agallochaol Q obtained in Example.

[0039] Figure 2 This is the 13C NMR spectrum of the atissenane diterpenoid compound Agallochaol Q obtained in Example.

[0040] Figure 3 This is the HRESIMS spectrum of the atissenane diterpenoid compound Agallochaol Q obtained in Example.

[0041] Figure 4 This is an experiment to evaluate the in vitro anti-EV-D68 activity of the athysenane diterpenoid compound Agallochaol Q based on cytopathic effect. DETAILED DESCRIPTION

[0042] The present invention is further described in detail below by way of specific examples. Unless otherwise specified, the raw materials, reagents, or devices used in the examples are all available from conventional commercial sources or can be obtained by existing methods. Unless otherwise specified, all experiments or testing methods are conventional methods in the art.

[0043] Example 1 Preparation of Agallochaol Q, a diterpenoid compound of athysenane

[0044] The preparation of the atissenane diterpenoid compound Agallochaol Q specifically comprises the following steps:

[0045] S1. Raw material processing: drying and crushing the stems and branches of Aconitum carmichaelii collected from Wenchang City, Hainan Province;

[0046] S2. Preparation and extraction of extract: The obtained lacquer sample was soaked and stirred in 95% ethanol (ethanol: water = 95:5, v / v), and the extract was concentrated under reduced pressure to obtain the lacquer extract; the obtained extract was suspended in water (extract: water = 1:2, v / v), and extracted with n-hexane (n-hexane: water = 3:1, v / v) to obtain the n-hexane layer extract;

[0047] S3. Column chromatography separation: The n-hexane layer extract of the lacquer was separated by a normal phase silica gel column, and the elution system was selected as petroleum ether-ethyl acetate. The petroleum ether-ethyl acetate volume ratio was 100:0, 100:1, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1, and 0:100, and one fraction was collected per 1 L. A total of 314 fractions were collected, namely Fr.1 to Fr.314; subsequently, the extract was eluted by high performance liquid chromatography Based on the TLC analysis results, similar fractions Fr.90 to Fr.100 were combined and separated and purified by ODS (octadecylsilane bonded silica gel) column chromatography. Acetone-water was selected as the elution system, and gradient elution was performed with acetone-water solution at volume fractions of 40%, 55%, 65%, 75%, 85%, and 100%, respectively. A total of 51 subfractions (Fr.90-100-1 to Fr.90-100-51) were obtained.

[0048] S4. Preparation of monomeric compounds: Component Fr.90-100-14 (65% acetone aqueous solution) was separated and purified by semi-preparative high performance liquid chromatography with a mobile phase of 60% acetonitrile / water at a flow rate of 3 mL / min, a detection wavelength of 195 nm, and a retention time of 59 min to obtain the atissenane diterpenoid compound Agallochaol Q represented by formula (I).

[0049]

[0050] 2. Structural confirmation of athisanane diterpenoids

[0051] The preparation of the obtained athysenane diterpenoid compound Agallochaol Q was carried out. 1 H NMR spectrum, 13 C NMR spectrum and HR-ESIMS spectrum were measured, and the corresponding spectra were Figures 1 to 3 As can be seen from the results, a series of spectral data and NMR data of the obtained compound all correspond to its structural formula, and it can be confirmed that its structure is the structure shown in formula (I).

[0052] Example 2 Test of the Anti-Enterovirus 68 Activity of Agallochaol Q, a Diterpenoid Compound of Athisanane

[0053] 1. Cell culture and cell lines

[0054] Human rhabdomyosarcoma cell line (RD cells) was purchased from the American Type Culture Collection (Cat. No. CCL-136).

[0055] Enterovirus: Enterovirus type 68 (EV-D68), preserved in the applicant's laboratory.

[0056] The cells were cultured in a 37°C, 5% CO2 incubator. The complete growth medium used was DMEM high-glucose medium (Gibco, Catalog No. 2367374) supplemented with 10% FBS (Gibco, Catalog No. 16000044) and penicillin-streptomycin (Gibco, Catalog No. 2321152). The maintenance medium used for cell culture was DMEM high-glucose medium supplemented with 2% FBS and penicillin-streptomycin.

[0057] 2. Cell Viability Detection

[0058] PBS and Mix the luminescent detection reagents to prepare the detection solution. Discard the supernatant from the plate and add 100 μL of the detection solution. Gently shake the plate on a horizontal shaker for 5 minutes and then let it rest for 3 minutes, protecting it from light throughout the process to ensure even distribution of the detection solution. Finally, measure the luminescence intensity using a multi-function microplate reader to assess cell viability.

[0059] 3. The experimental plan is as follows:

[0060] In the experiment, RD cells in T75 culture flasks were first digested and cultured at a rate of 1×10 4Cells were seeded at a density of 100 μL / well into a transparent white-bottomed 96-well plate. 100 μL of cell suspension was added to each well, and the plate was then placed in an incubator for overnight incubation to ensure uniform cell distribution. After incubation, the supernatant was discarded and replaced with 100 μL of 2% DMEM medium. 100 mM DMSO-prepared stock solutions of Agallochaol Q and 10 mM NITD008 (CAS No. 1044589-82-3) were diluted with 2% DMEM to 4 times the test concentration of Agallochaol Q and NITD008 solutions.

[0061] 3.1 Antiviral efficacy

[0062] During drug treatment, 50 μL of 100 TCID 50 Virus solution and 50 μL of Agallochaol Q solution of athysenane diterpenoid compound. For the positive control group, 50 μL of 100 TCID 50 Virus solution and 50 μL NITD008 solution. Add 50 μL of virus solution and 50 μL of 2% DMEM medium to the virus control group, and 100 μL of 2% DMEM medium to the cell control group. Place the 96-well plate in a humidified chamber and continue incubation at 37°C, 5% CO2, ensuring appropriate humidity until the virus-treated cells develop pathological changes.

[0063] To detect cell viability, PBS and The luminescent detection reagents were mixed to prepare the detection solution. After discarding the supernatant on the well plate, 100 μL of detection solution was added. The well plate was then gently shaken on a horizontal shaker for 5 minutes and then allowed to stand for 3 minutes. The whole process was protected from light to ensure that the detection solution was evenly distributed. Finally, the luminescence intensity was measured by a multifunctional microplate reader to evaluate cell viability and calculate the half effective concentration (EC50) of the drug. 50 ) to evaluate the antiviral efficacy of drugs.

[0064] The formula for calculating the virus inhibition rate of each concentration of the athysenane diterpenoid compound Agallochaol Q is:

[0065]

[0066] 3.2 Cytotoxicity

[0067] At the beginning of the experiment, 100 μL of cell suspension (1×10 4Cells / well) were seeded into a transparent white bottom 96-well plate, 100 μL of cell suspension was added to each well, and incubated overnight in an incubator. After incubation, the culture medium in the well plate was discarded and 150 μL of 2% DMEM medium was added. For the drug treatment group, 50 μL of different concentrations of the adipsinane diterpenoid compound Agallochaol Q solution was added to each well; for the cell control group, 50 μL of 2% DMEM medium was added. The 96-well plate was placed in a humidified box and placed in a 37°C, 5% CO2 incubator for continued culture. After 3 days of culture, the cells were used Luminescent cell viability assay was used to assess cell viability and calculate the half cytotoxic concentration (CC 50 ).

[0068] The inhibition rate of each concentration of the athysenane diterpenoid compound Agallochaol Q on cells can be calculated by the following formula:

[0069]

[0070] The selectivity index (SI) is calculated as follows:

[0071] SI=CC 50 / EC 50

[0072] 4. Test results:

[0073] EC of Agallochaol Q, a diterpenoid compound of artisan 50 =4.15±0.08μM, CC 50 =36.02±2.90μM, SI=8.68( Figure 4 ); EC of positive drug NITD008 50 =0.34±0.003μM, CC 50 >10 μM, SI>29.41. It can be seen that the atissenane diterpenoid compound Agallochaol Q of the present invention has a significant inhibitory effect on EV-D68 enterovirus, and the effect is close to that of positive drugs.

[0074] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. Use of a compound of formula I, its geometric isomers, its pharmaceutically acceptable salts, its solvates or its hydrates in the preparation of a medicament for one or more of the following: 1) Prevent or treat enterovirus infection or diseases caused by enterovirus infection; 2) Inhibit the replication or reproduction of enterovirus in cells; 2. The use according to claim 1, characterized in that The enterovirus is selected from poliovirus, coxsackie A virus, coxsackie B virus, echovirus, enterovirus 71, enterovirus D68 or any combination thereof.

3. The use according to claim 1, characterized in that The enterovirus is selected from enterovirus D68.

4. The use according to claim 1, characterized in that The disease caused by enterovirus infection is selected from hand, foot and mouth disease, respiratory system infection, central nervous system disease, acute flaccid paralysis, nonspecific febrile disease, pneumonia, bronchiolitis, encephalitis and myocarditis, acute flaccid myelitis or any combination thereof.

5. The use according to claim 4, characterized in that The disease caused by enterovirus infection is selected from hand, foot and mouth disease, respiratory system infection, central nervous system disease, acute flaccid paralysis or any combination thereof.

6. The use according to any one of claims 1 to 5, characterized in that The cells are mammalian cells.

7. The use according to any one of claims 1 to 5, characterized in that The drug contains the compound of formula I, its geometric isomers, its pharmaceutically acceptable salts, its solvates or its hydrates as the sole active pharmaceutical ingredient.

8. The use according to any one of claims 1 to 5, characterized in that The medicine further contains other anti-enterovirus active ingredients.

9. The use according to any one of claims 1 to 5, characterized in that The medicament may further comprise a pharmaceutically acceptable carrier and / or excipient.

10. The use according to any one of claims 1 to 5, characterized in that The drug is prepared into a dosage form selected from the following: tablets, capsules, pills, oral liquid preparations, granules, powders, and injections.

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