Use of a sesquiterpene lactone compound in the preparation of an antiviral drug

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to application of a sesquiterpene lactone compound in preparation of an antiviral drug. The sesquiterpene lactone compound has a significant inhibitory effect on bunyaviruses, and the effect is equivalent to that of a positive control, so that the sesquiterpene lactone compound has a good application prospect in preparation of antiviral drugs. In addition, the sesquiterpene lactone compound can be prepared by fermentation, separation and purification of marine microorganisms of the fungus genus Arenariomyces, and the preparation method is simple, the source is rich, and the industrial production preparation is facilitated.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology. More specifically, it relates to the application of a sesquiterpene lactone compound in the preparation of antiviral drugs. Background Technology

[0002] Bunyavirus (BUNV) is a spherical, enveloped, segmented, negative-sense RNA virus, named after Bunyawira, western Uganda, where it was first isolated. With the discovery of various novel Bunyaviruses, the International Committee on Taxonomy of Viruses (ICTV) unified this group of viruses into a single family, Bunyaviridae, in 1975, and updated it to the order Bunyavirales in its tenth report published in 2017. According to the latest classification, the order Bunyavirales is divided into 12 families, 54 genera, and 384 species. Currently, the Bunyavirales order comprises families such as Phenuiviridae, Nairoviridae, Peribunyaviridae, Hantaviridae, Leishbuviridae, Fimoviridae, Mypoviridae, Tospoviridae, Arenaviridae, and Phasmaviridae. As the most diverse group of negative-sense RNA viruses, Bunyaviruses host vertebrates, arthropods (mosquitoes, ticks, sandflies, etc.), and higher plants. Most viruses are transmitted by arthropods and rodents, circulating in nature with blood-sucking arthropods and susceptible vertebrates as hosts, posing a serious threat to public health and economic development.

[0003] In recent years, a novel Bunyavirus belonging to the genus *Bunyavirus* has been found in most cases of severe fever with thrombocytopenia syndrome (SFTS) discovered in China. Preliminary findings suggest a link between these cases and infection with this newly identified virus, classifying it as a novel virus belonging to the genus *Cobandia* of the order *Bunyavirales*, and naming it severe fever with thrombocytopenia syndrome bunyavirus (SFTSV). SFTSV does not produce significant cytopathic effects when infecting Vero cells in vitro, and interferon is undetectable in patient serum, although large amounts of inflammatory chemokines such as IP-10 are found. Therefore, it is believed that SFTSV may replicate continuously in vivo by inhibiting the production of antiviral interferon by target cells. This process induces the body to produce inflammatory chemokines, leading to an excessive immune inflammatory response and thus damaging tissue and organ function. Furthermore, Bunyaviruses exhibit pantropy, invading the blood system and organs such as the heart, liver, kidneys, lungs, and digestive tract, which may be a cause of multiple organ failure in severe SFTS patients. For those with a confirmed diagnosis, etiological treatment should be initiated as early as possible, and underlying diseases and symptomatic supportive treatment should be strengthened.

[0004] Currently, there is no specific treatment for Bunyavirus; only symptomatic treatment is available. To develop drugs against Bunyavirus, Chinese patent application CN108210880A discloses the application of compound PS-341 in the preparation of Bunyavirus inhibitors. Experiments have shown that compound PS-341 can inhibit the degradation of SFTSV NSs-mediated RIG-I ubiquitination pathway, thereby activating the interferon antiviral pathway in host cells during viral infection, significantly inhibiting viral replication and proliferation. However, most effective drugs are still in the early stages of research, and there are very few specific drugs for Bunyavirus; therefore, there is an urgent need to research more anti-Bunyavirus drugs. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the defects and deficiencies of the lack of existing anti-Bunya virus drugs and to provide an application of sesquiterpene lactone compounds in the preparation of antiviral drugs.

[0006] The purpose of this invention is to provide an application of *Curviflora* fungi in the preparation of sesquiterpene lactone compounds or antiviral drugs.

[0007] Another objective of this invention is to provide a method for preparing sesquiterpene lactone compounds from *Curvus* fungi.

[0008] The above-mentioned objective of this invention is achieved through the following technical solution:

[0009] The application of a sesquiterpene lactone compound in the preparation of antiviral drugs, wherein the sesquiterpene lactone compound has the structure of Formula 1:

[0010]

[0011] Further, the virus is a Bunyavirus. Preferably, the virus is a sandfly fibrillvirus (Cobandavirus). More preferably, the virus is a fever-associated thrombocytopenic syndrome Bunyavirus (SFTSV).

[0012] This invention has found that sesquiterpene lactone compound 1 has a significant inhibitory effect on Bunyavirus, providing a wider range of options for subsequent drug research and screening and clinical application against Bunyavirus.

[0013] The present invention claims the use of the sesquiterpene lactone compounds in the preparation of antiviral drugs, but is not limited to, the use of administering an effective amount of the compounds of the present invention to a patient to prepare a medicine for the prevention or treatment of viral diseases, the relief of symptoms of viral diseases, or the delay of the development or onset of new viral diseases.

[0014] The novel applications of the compounds claimed in this invention, in addition to their therapeutic benefits for humans, can also be used in veterinary treatment of pets, introduced breeds of animals, and farm animals, including mammals, rodents, birds, etc. Other examples of animals include horses, dogs, cats, and pigs.

[0015] Furthermore, the sesquiterpene lactones may also be pharmaceutically acceptable salts, solvates, or stereoisomers.

[0016] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0017] Furthermore, the drug is an oral preparation, an injection, a topical preparation, or an inhaler. Preferably, the oral preparation includes dosage forms such as tablets, capsules, pills, granules, and oral liquids.

[0018] Additionally, this invention provides the application of *Cyclocarya* fungi in the preparation of sesquiterpene lactone compounds, wherein the sesquiterpene lactone compounds have the structure of Formula 1:

[0019]

[0020] Microbial secondary metabolites are an important source of drug lead compounds. The unique natural environment of the ocean, characterized by high salinity, high pressure, and lack of light, allows marine microorganisms to exhibit significant novelty in terms of biological species and active metabolites, and they are becoming an important source of drug lead compounds. The *Eutypella* sp. F0219 fungus of this invention was isolated from seabed sediments at a depth of 75 m in the South China Sea (GPS 114.6609°E, 21.5942°N), and belongs to the genus *Eutypella*. The fungus is classified as *Eutypella* sp. F0219, provided by Hainan Normal University, and has been disclosed in existing technology (Yi Jiling, Shi Kangqi, Wu Binglin, et al. Study on secondary metabolites of marine fungus *Eutypella* sp. F0219 [J]. Organic Chemistry, 2023, 43(1):4.).

[0021] This invention has discovered that the secondary metabolites of the fungus Eutypella sp. F0219 can be isolated and purified to obtain a sesquiterpene lactone compound 1 with significant anti-Bunya virus activity.

[0022] Therefore, the present invention also provides the application of the aforementioned *Curvularia* fungi in the preparation of antiviral drugs.

[0023] This invention further provides a method for preparing sesquiterpene lactones from *Curvularia* fungi, wherein the sesquiterpene lactones are prepared by fermentation, separation, and purification of the *Curvularia* fungi.

[0024] Furthermore, the method for preparing sesquiterpene lactone compounds from *Curvularia* fungi specifically includes the following steps:

[0025] S1. The fungus *Curvularia* is activated and cultured on a large scale to obtain a ferment.

[0026] S2. The fermentation product obtained in step S1 is extracted by soaking in ethyl acetate. The crude extract is then extracted with n-hexane and ethyl acetate to obtain n-hexane extract and ethyl acetate extract, respectively.

[0027] S3. The hexane extract obtained in step S2 is subjected to normal phase silica gel column chromatography, with petroleum ether-ethyl acetate as the eluent for gradient elution, and the fraction with a volume ratio of petroleum ether-ethyl acetate of 5:1 is collected.

[0028] S4. The fraction obtained in step S3 with a petroleum ether-ethyl acetate volume ratio of 5:1 was subjected to ODS column chromatography with gradient elution using acetone-water as the eluent to obtain a fraction with an acetone volume fraction of 40%.

[0029] S5. The acetone fraction of 40% obtained in step S4 is separated and purified by high performance liquid chromatography to obtain sesquiterpene lactone compounds.

[0030] Furthermore, in step S2, the activated ethyl acetate is soaked and extracted 2 to 3 times, and then concentrated under reduced pressure to obtain a crude extract.

[0031] Further, in step S3, the gradient elution is performed using petroleum ether-ethyl acetate as the eluent at a ratio of 100:0 to 0:100, v / v. Preferably, the elution gradient is sequentially 100:0, 100:1, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1, 1:2, and 0:1.

[0032] Furthermore, in step S4, acetone-water is used as the eluent, and gradient elution is performed sequentially using acetone:water = 30:70, 40:60, 55:45, 70:30, 85:15, and 100:0 as solvents.

[0033] Furthermore, in step S5, the mobile phase of the high-performance liquid chromatography (HPLC) is 75% methanol / water, the flow rate is 1-3 mL / min, and the detection wavelength is 285 nm.

[0034] The present invention has the following beneficial effects:

[0035] This invention provides the application of sesquiterpene lactones in the preparation of antiviral drugs. The sesquiterpene lactones exhibit significant inhibitory effects against Bunyavirus, with efficacy comparable to the positive control, demonstrating promising application prospects in the preparation of antiviral drugs. Furthermore, the sesquiterpene lactones can be prepared through fermentation, separation, and purification by marine microorganisms of the genus *Curvus*, a simple preparation method with abundant sources, which is conducive to industrial-scale production. Attached Figure Description

[0036] Figure 1 This is the hydrogen spectrum of sesquiterpene lactone compound 1 obtained in Example 1 of the present invention.

[0037] Figure 2 This is the carbon spectrum of sesquiterpene lactone compound 1 obtained in Example 1 of the present invention.

[0038] Figure 3 This is the mass spectrum of sesquiterpene lactone compound 1 obtained in Example 1 of the present invention. Detailed Implementation

[0039] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0040] The endophytic fungus Eutypella sp. F0219 used in the fermentation was isolated from seabed sediments at a depth of 75 m in the South China Sea (GPS 114.6609°E, 21.5942°N), and belongs to the genus Eutypella. The fungus Eutypella sp. F0219 was provided by Hainan Normal University and has been disclosed in existing technology (Yi Jiling, Shi Kangqi, Wu Binglin, et al. Study on secondary metabolites of marine fungus Eutypella sp. F0219 [J]. Organic Chemistry, 2023, 43(1):4.).

[0041] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0042] Example 1: A method for preparing a sesquiterpene lactone compound

[0043] The preparation method of the sesquiterpene lactone compound specifically includes the following steps:

[0044] S1. Activation of the strain: Take out the preserved endophytic fungus Eutypella sp. F0219 and place it in an incubator for pre-culture for 24 hours. Then transfer it to a sterile PDA plate for activation and incubate in an incubator for 2 to 4 days.

[0045] S2. Seed culture preparation: Take an activated endophytic fungus Eutypella sp. F0219 cell the size of a broad bean after activation, inoculate it into sterile PDB liquid medium, and culture it in a constant temperature shaker at 28℃ for 2-3 days to obtain seed culture.

[0046] S3. Extract preparation: The cultured seed culture was transferred to sterile PDB liquid culture medium using a pipette in a sterile laminar flow hood and fermented at a constant temperature of 28°C for 30-40 days to obtain the fermentation product; the fermentation product was extracted 2-3 times by soaking in ethyl acetate solvent, and concentrated under reduced pressure to obtain a crude extract; the crude extract was extracted with n-hexane and ethyl acetate respectively to obtain n-hexane extract and ethyl acetate extract respectively;

[0047] S4. Column Chromatography Separation: The hexane extract was subjected to normal-phase silica gel column chromatography with petroleum ether-ethyl acetate solvents (100:0, 100:1, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1, 1:2, 0:1, v / v) for gradient elution. Fractions were collected in approximately 0.5 L increments, and similar fractions were combined by TLC analysis to separate into 20 fractions, Fr.1 to Fr.20. Fraction Fr.11 (petroleum ether-ethyl acetate volume ratio 5:1) was further subjected to ODS column chromatography with acetone:water = 30:70, 40:60, 55:45, 70:30, 85:15, 100:0 for gradient elution, yielding 6 fractions (Fr.11-1 to Fr.11-6).

[0048] S5. Preparation of monomeric compounds: Component Fr.11-2 (acetone:water = 40:60) was purified by high performance liquid chromatography (HPLC) with 75% methanol / water as the mobile phase, a flow rate of 3 mL / min, a detection wavelength of 285 nm, and a retention time of 12.4 min to obtain compound 1.

[0049] The structure of compound 1 is shown below:

[0050]

[0051] Example 2: Structural confirmation of sesquiterpene lactone compound 1

[0052] The compounds obtained in Example 1 were subjected to proton, carbon, and mass spectrometry analyses. The results are shown in [reference needed]. Figures 1-3 The results show that the proton, carbon, and mass spectra of the obtained compound correspond one-to-one with its structural formula, confirming that its structure is the structure of formula 1.

[0053] Example 3: Anti-Bunya virus activity test of sesquiterpene lactone compound 1

[0054] 1. Cell culture and cell lines

[0055] The human liver cancer cells (Huh7) used in the experiment were provided and preserved by the Institute of Toxicology and Pharmacology, Academy of Military Medical Sciences, China, and their source and passage number were clearly defined.

[0056] Huh7 cells were cultured in a humidity-saturated cell culture incubator at 37°C and 5% CO2. They were passaged at a ratio of 1:3 to 1:6, with the medium changed every 48 hours during the culture process. After about 2 to 5 days (when the cells reached a monolayer), the cells were passaged again after digestion with 0.25% EDTA trypsin for 2 minutes. The complete culture medium used for cell growth was DMEM high-glucose medium supplemented with 10% FBS and penicillin-streptomycin antibiotics, and the maintenance medium was DMEM high-glucose medium supplemented with 2% FBS and penicillin-streptomycin antibiotics.

[0057] 2. Cell viability assay

[0058] Cell viability using CellTiter Luminescent Cell Viability Assay.

[0059] The specific experimental design is as follows:

[0060] Cells covering the bottom of the flask were digested with 0.25% EDTA trypsin and resuspended in complete culture medium to prepare a single-cell suspension. After counting, the cells were seeded at a density of 5000 cells per well in 96-well transdermal plates and cultured at 37°C with 5% CO2 saturation for 24 hours. The SFTSV stock solution was diluted with maintenance medium and added to the 96-well transdermal plates to achieve a final concentration of 100 TCID. 50 Simultaneously, the test compounds were serially diluted with maintenance medium in a 3-fold gradient and added to 96-well plates, resulting in 8 concentrations, starting at 100 μM. Favipiravir (T-705) purchased from Merck was used as a positive compound, and cell and virus control groups were established. After 6 days of treatment, the supernatant was discarded, and CellTiter diluted 2-fold with PBS buffer was added to each well. Luminescent Cell Viability assay solution was subjected to light-protected shaking lysis for 5 min, allowed to stand for 3 min, and finally the fluorescence signal intensity was measured using MolecularDevices M5.

[0061] The cytotoxicity assay for the compound and the experimental conditions are the same as above, except that no virus is added.

[0062] The formula for calculating cell viability is:

[0063] Cell Viability (%) = (Average value of drug-treated group / Cell control group) × 100%, used to calculate CC. 50 value.

[0064] Inhibition rate of CPE (%) = (mean value of drug-treated group - mean value of virus control group) / (mean value of virus control group - mean value of cell control group) × 100%, used to calculate EC 50 value.

[0065] 3. Using Origin 8.0 software, an S-shaped curve was fitted to the inhibition rate-concentration ratio, and the EC50 of the test compound was calculated. 50 Value. Calculate CC using the same method. 50 Value, and according to EC 50 and CC 50 According to the formula SI=CC 50 / EC 50Calculate the Selection Index (SI).

[0066] 4. Experimental Results:

[0067] EC of sesquiterpene lactone compound 1 of the present invention 50 =6.39±2.505μM, CC 50 =30.05±2.64μM, SI=4.70; EC50 of positive drug T-705 50 =6.53±0.44μM, CC 50 >200 μM, SI>30.63. It is evident that sesquiterpene lactone compound 1 of this invention exhibits significant inhibitory activity against Bunyavirus, and its efficacy is close to that of positive control drugs.

[0068] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Use of a sesquiterpene lactone compound in the preparation of a drug for treating fever with thrombocytopenia syndrome bunyavirus, characterized in that, The sesquiterpene lactone compound has a structure of formula 1: 。 2. Use according to claim 1, characterized in that, The sesquiterpene lactone compound can also be a pharmaceutically acceptable salt thereof.

3. Use according to claim 1, characterized in that, The drug is an oral agent, an injection, a topical agent or an inhalant.

4. A method for preparing a sesquiterpene lactone compound, characterized by, The sesquiterpene lactone compound has a structure of formula 1: ； The sesquiterpene lactones were produced by fungi of the genus *Curvus* ( Eutypella The preparation of sp.) F0219 involves fermentation, separation, and purification, specifically including the following steps: S1, activating and expanding the culture of the Neopeckia fungus to obtain a fermentation product; S2, soaking and extracting the fermentation product obtained in step S1 with ethyl acetate, and extracting the obtained crude extract with n-hexane and ethyl acetate respectively to obtain n-hexane extract and ethyl acetate extract; S3, performing normal phase silica gel column chromatography on the n-hexane extract obtained in step S2, and performing gradient elution with petroleum ether-ethyl acetate as an eluent to collect a fraction with a petroleum ether-ethyl acetate volume ratio of 5:1; S4, performing ODS column chromatography on the fraction with a petroleum ether-ethyl acetate volume ratio of 5:1 obtained in step S3, and performing gradient elution with acetone-water as an eluent to obtain a component with an acetone volume fraction of 40%; S5, performing separation and purification on the component with an acetone volume fraction of 40% obtained in step S4 by high performance liquid to obtain a sesquiterpene lactone compound.

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