Use of a chrysene diterpenoid compound in the preparation of an antiviral drug

Abstract

This invention belongs to the field of biomedical technology, specifically relating to the application of a chogranane diterpenoid compound in the preparation of antiviral drugs. Studies have shown that the chogranane diterpenoid compound exhibits significant inhibitory effects against Zika virus, with low cytotoxicity and a good selectivity index, making it highly suitable for use in the preparation of antiviral drugs. Furthermore, the chogranane diterpenoid compound described in this invention can be extracted from sea lacquer, a relatively abundant source, resulting in a simple and low-cost preparation method.

Description

Technical Field

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

[0002] The Zika virus was first discovered in 1947 in the Zika Forest of Uganda, Europe. It parasitizes rhesus monkeys and is primarily transmitted through mosquito bites, spreading worldwide. Currently, about 80% of individuals infected with the Zika virus do not show obvious clinical symptoms. A small number of infected individuals who do show clinical symptoms, such as fever, rash, joint pain, muscle pain, headache, vomiting, and conjunctivitis. These clinical symptoms usually last for more than a week and are relatively mild. However, the most worrying aspect is that the Zika virus can be transmitted vertically from the pregnant woman to the fetus across the placental barrier, causing serious consequences for the fetus, such as abnormalities in the fetal brain structure, including incomplete development of the brainstem and cerebellum, delayed myelination, enlarged ventricles, severe calcification of the thin wall tissue of the brain, and a few cases of anejaculation, which are irreversible consequences (Chen Zhangzhou, Li Jin, Xie Duoyuan, et al. Research progress on Zika virus [J]. Chinese Journal of Virology, 2016, 6(05):391-397.DOI:10.16505 / j.2095-0136.2016.05.014.).

[0003] Currently, there is no specific treatment for Zika virus. Prevention primarily relies on mosquito bites (especially in areas where the disease is prevalent) to avoid infection. Besides this, vaccine and antiviral drug development are two promising directions for Zika virus prevention and treatment; however, a Zika virus vaccine has not yet been developed. Regarding antiviral drug development, existing technologies have yielded relevant results. For example, Chinese patent application CN110859833A discloses the application of compound ZP10 in the preparation of an anti-Zika virus drug targeting the Zika virus protease. Studies on its affinity for the Zika virus protease have shown that compound ZP10 exhibits high anti-Zika virus activity, with an IC50 value of [missing information]. 50 2.3 μM, EC 50 The concentration is 7.65 μM. ZP10 exhibits low cytotoxicity, high affinity for Zika virus protease, and inhibits Zika virus replication at both transcriptional and translational levels, thus demonstrating significant anti-Zika virus efficacy. It could be used to prepare drugs for the prevention or treatment of Zika virus. However, this drug is currently only in the research stage, and its clinical efficacy is unknown. Therefore, more anti-Zika virus drugs are still needed for research or clinical use. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the shortcomings and deficiencies of the limited number of existing anti-Zika virus drugs, and to provide an application of chogranditerpenoid compounds in the preparation of antiviral drugs.

[0005] The above-mentioned objective of this invention is achieved through the following technical solution:

[0006] The application of a chogranane diterpenoid compound in the preparation of antiviral drugs, wherein the chogranane diterpenoid compound has the structure of formula I:

[0007]

[0008] Chochiane diterpenoids are a relatively rare class of diterpenoids in nature. The chochiane diterpenoid compound in this invention has the same structure as the compound agallolide M reported in the literature (Bioorg. Chem., 2020, 104, 104206.), but its biological activity has not yet been studied and reported.

[0009] Furthermore, the virus is Zika virus ZIKV SMGC-1 (Zika virus SMGC-1strain).

[0010] The present invention has found that the chocoside diterpenoid compounds have significant inhibitory activity against Zika virus, and exhibit low cytotoxicity and good selectivity.

[0011] The present invention claims the use of the chogranditerpenoid 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 diseases caused by new viruses.

[0012] 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.

[0013] Furthermore, based on the principle of similar pharmaceutical structures and similar effects, the chocoside diterpenoids may also be pharmaceutically acceptable salts, solvates, or stereoisomers.

[0014] Furthermore, the chogranane diterpenoids are extracted from lacquer trees. Mangroves are a transitional marine-terrestrial ecosystem, and their unique ecological environment creates rich structural diversity and unique biological activities in the secondary metabolites of mangrove plants, making them an important treasure trove for the discovery of new drug lead compounds. The chogranane diterpenoids described in this application are extracted from lacquer trees, which are collected from the Qinglan Port mangrove wetland in Wenchang, Hainan. The secondary metabolites of lacquer trees mainly include diterpenes, triterpenes, flavonoids, and tannins, which exhibit antitumor, anti-inflammatory, anti-HIV, and antioxidant activities. The chogranane diterpenoids of this invention are extracted from lacquer trees, which are widely available and low in cost.

[0015] Furthermore, the preparation method of the chocoside diterpenoid specifically includes the following steps:

[0016] S1. Dry and pulverize the sea lacquer, extract it with ethanol, extract the extract with n-hexane and ethyl acetate, and collect the n-hexane extract;

[0017] S2. The hexane extract obtained in step S1 is subjected to normal phase silica gel column chromatography, with hexane-acetone as the eluent for gradient elution, and the fraction with a hexane-acetone volume ratio of 5:1 is collected.

[0018] S3. The fraction obtained in step S2 with a hexane-acetone 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%.

[0019] S4. The acetone fraction of 40% obtained in step S3 is separated and purified by high performance liquid chromatography to obtain chocolate diterpenoids.

[0020] Furthermore, in step S1, the ethanol is extracted 3 to 5 times, and the filtrates are combined after filtration for subsequent extraction reactions.

[0021] Further, in step S2, the gradient elution is performed using hexane-acetone as the eluent at a ratio of 100:0 to 0:100, v / v. Preferably, the elution gradient is 100:0, 100:1, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1, and 0:1.

[0022] Furthermore, in step S3, 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.

[0023] Further, in step S4, the mobile phase of the high-performance liquid chromatography is 55% acetonitrile / water, the flow rate is 1-3 mL / min, and the detection wavelength is 195 nm.

[0024] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0025] Preferably, the excipients include: solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, binding agents, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, flocculants, anti-flocculation agents, filter aids, release inhibitors, etc. The excipients are selected according to the dosage form to be prepared.

[0026] Furthermore, the drug is an oral preparation, an injection, a topical preparation, or an inhaler.

[0027] Preferably, the oral medication includes dosage forms such as tablets, capsules, pills, granules, and oral liquids.

[0028] The present invention has the following beneficial effects:

[0029] This invention provides the application of chogranidine diterpenoids in the preparation of antiviral drugs. Studies have shown that these chogranidine diterpenoids exhibit significant inhibitory effects against Zika virus, with low cytotoxicity and a good selectivity index, making them highly suitable for use in the preparation of antiviral drugs. Furthermore, the chogranidine diterpenoids described in this invention can be extracted from relatively abundant sources such as sea lacquer, resulting in a simple and low-cost preparation method. Attached Figure Description

[0030] Figure 1 This is the proton NMR spectrum of compound I, a diterpenoid obtained in Example 1 of this invention.

[0031] Figure 2 This is the carbon spectrum of compound I, a diterpenoid obtained in Example 1 of the present invention.

[0032] Figure 3 This is the mass spectrum of compound I, a diterpenoid obtained in Example 1 of the present invention. Detailed Implementation

[0033] 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.

[0034] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0035] Example 1: A method for preparing a diterpenoid compound I.

[0036] The preparation method of the chocoside diterpenoid compound I specifically includes the following steps:

[0037] S1. Collection of sea lacquer samples: Sea lacquer stem bark and tender branches were collected (collected from the mangrove wetland of Qinglan Port, Wenchang, Hainan), and dried to a total of 16 kg;

[0038] S2. Sample extraction: The dried sample is crushed and extracted by soaking in industrial ethanol for a total of 5 times to obtain an ethanol extract.

[0039] S3. Column Chromatography Separation: The ethanol extract was extracted with n-hexane and ethyl acetate, respectively, to obtain n-hexane and ethyl acetate extracts. The n-hexane extract was subjected to normal-phase silica gel column chromatography with n-hexane-acetone solvent (100:0–0:100, v / v) as the eluent, using a gradient elution (100:0, 100:1, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1, 0:1), approximately 0.5 L each time. The fractions were collected, and similar fractions were combined by TLC to separate them into 15 fractions, namely fractions Fr.1 to Fr.15. Among them, fraction Fr.8 (24 g) of n-hexane-acetone (volume ratio 5:1) was further subjected to ODS column chromatography with gradient elution using acetone:water = 30:70, 40:60, 55:45, 70:30, 85:15, and 100:0 as solvents to obtain 6 fractions (Fr.8-1 to Fr.8-6).

[0040] S4. Preparation of monomeric compounds: Component Fr.8-2 was purified by high performance liquid chromatography (HPLC) with 55% acetonitrile / water as the mobile phase, a flow rate of 3 mL / min, a detection wavelength of 195 nm, and a retention time of 25.5 min to obtain compound I.

[0041] The structure of compound I is shown below:

[0042]

[0043] Example 2: Structural confirmation of chochiane diterpenoid compound I

[0044] 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 I.

[0045] Example 3: Test of anti-Zeka virus activity of chocoside diterpenoid compound I

[0046] 1. Cells and Viruses:

[0047] BHK (Baby Hamster Syrian Kidney) cells, Zika virus ZIKV SMGC-1 (Zika virus SMGC-1 strain), were provided and preserved by the Institute of Toxicology and Pharmacology, Academy of Military Medical Sciences.

[0048] 2. Preparation of main reagents:

[0049] 2-1. DMEM high glucose medium containing 10% fetal bovine serum: Mix 50 ml fetal bovine serum (FBS), antibiotics (containing 100 U / ml penicillin and 100 μg / ml streptomycin) thoroughly with 500 ml DMEM medium to obtain the medium.

[0050] 2-2. DMEM high glucose medium (maintenance medium) containing 2% fetal bovine serum: Mix 10 ml fetal bovine serum (FBS), antibiotics (containing 100 U / ml penicillin and 100 μg / ml streptomycin) thoroughly with 500 ml DMEM medium to obtain the medium.

[0051] 2-3. Preparation of working solution for cell viability assay kit (Promega, G7572): Dilute the assay solution with PBS at a ratio of 1:1 and mix well.

[0052] 3. Virus titer (TCID) 50 ) Measurement

[0053] S1. BHK cells were prepared at a ratio of 5 × 10 3 / wells were inoculated into white-walled, transparent 96-well plates and cultured overnight in an adherent manner;

[0054] S2. Take the virus to be titrated and dilute it sequentially with 2% maintenance medium to a concentration of 10. -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 10 -8 Store at 4℃ until use;

[0055] S3. Take out the 96-well plate that was pre-inoculated in step S1, discard the supernatant, add 150 μl of 2% maintenance medium to each well of the virus titration group, and add 200 μl of 2% maintenance medium to each well of the control group.

[0056] S4. Take out the diluted virus from step S2 and sequentially inoculate it into the 96-well plate in step S3 from low concentration to high concentration, 50 μl per well, and set 8 replicates for each concentration.

[0057] S5. Place the virus-inoculated 96-well plate back into the incubator for further incubation. Observe the viral lesions in the wells daily, recording the number and severity of lesions. Observe continuously for 7 days. The concentration at which 50% of the wells show complete viral lesions is TCID50. 50 (50% tissue culture infectious dose).

[0058] 4. In vitro evaluation of the compound's anti-Zika virus activity

[0059] 4-1. Cell Counting and Plating

[0060] (1) 75cm of cells covered with BHK cells 2 Add 2 ml of trypsin to the culture flask, digest at 37°C for 2 min, add 5 ml of 10% DMEM and disperse the cells, centrifuge at 2000 rpm for 5 min;

[0061] (2) Discard the supernatant, resuspend the cells in 5 ml of 10% DMEM, mix well, and then aspirate 20 μl onto a cell counting plate and count the cells using a cell counter.

[0062] (3) Calculate the required number of cells, dilute with 10% DMEM, then mix the cell dilution using a pipette, and spray at 5 × 10⁻⁶. 3 100 μl of cells per well was added to a 96-well plate with a white-walled bottom. The cell seeding plate was placed in a humidified chamber and incubated overnight in a CO2 incubator. After 24 hours, the diluted virus and the test compound were inoculated.

[0063] 4.2 Compound Dilution

[0064] The stock solution of the compound was vortexed and mixed. A certain volume of the stock solution was transferred to 2% DMEM. Then, the compound to be tested was serially diluted in a 3-fold dilution gradient using a multi-channel pipette to obtain a total of 8 concentrations, with an initial concentration of 100 μM. NITD008 purchased from Selleck Biotechnology was used as the positive compound.

[0065] 4.3 Change of fluid and administration of medication

[0066] (1) Discard the culture medium in the 96-well cell seeding plate and add 100 μl of 2% DMEM to each well;

[0067] (2) The diluted compounds were added to the cells in 96-well plates in ascending order of concentration, 50 μl per well. An equal volume of 2% DMEM was added to both the cell control group and the virus control group, resulting in a volume of 150 μl per well.

[0068] (3) Remove the test virus from the -80℃ freezer and allow it to equilibrate to room temperature;

[0069] (4) Dilute the test virus to 100 TCID using 2% DMEM.50 Add 50 μl of the above 96-well plate cells to each well; add an equal volume of 2% DMEM to the cell control group, so that the final volume of each well is 200 μl.

[0070] (5) Place the 96-well plate in a humidified box and incubate it at 37°C and 5% CO2. Observe the cell pathogenesis every day.

[0071] 4.4 Cell viability assay

[0072] (1) The buffer and substrate of the chemiluminescent cell viability assay reagent are mixed in the dark to prepare the working solution, which is then stored at -80°C. Before detection, the working solution is taken out, thawed in the dark, and mixed with PBS at a 1:1 ratio for later use.

[0073] (2) When the virus control wells are completely infected (8 days for BHK), the culture medium in the 96-well plate is discarded, 100 μl of the detection reagent is added to each well, and the 96-well plate is shaken for 5 min using an orbital oscillator to induce cell lysis. After the signal is stabilized in the dark for 2 min, the chemiluminescence units are measured and the plate is read using a microplate reader with the CellTiter-Glo preset program.

[0074] 5. Results Analysis

[0075] (1) Calculate the inhibition rate of the drug at each dilution using the following formula:

[0076]

[0077] Among them, μSample, μvirus, and μcell represent the average cell viability of the drug administration group, the virus control group, and the cell control group, respectively.

[0078] (2) 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 50 Calculate the selection index (SI).

[0079] (3) Experimental results:

[0080] EC of compound I 50 =19.98±14.85μM, CC 50 >100μM, SI>5.01; EC50 of positive compound NITD008 50 =1.86±1.48μM, CC 50>10μM, SI>5.38.

[0081] It is evident that the diterpenoid compound I of this invention has a significant inhibitory effect on Zika virus and exhibits good safety.

[0082] 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. The application of a diterpenoid compound in the preparation of antiviral drugs, characterized in that, The chocoside diterpenoid compound has the structure of Formula I: The virus in question is the Zika virus.

2. The application according to claim 1, characterized in that, The chocolate diterpenoids can also be pharmaceutically acceptable salts.

3. The application according to claim 1, characterized in that, The diterpenoid compound was obtained from sea lacquer.

4. The application according to claim 3, characterized in that, The preparation method of the chocoside diterpenoid specifically includes the following steps: S1. Dry and pulverize the sea lacquer, extract it with ethanol, extract the extract with n-hexane and ethyl acetate, and collect the n-hexane extract; S2. The hexane extract obtained in step S1 is subjected to normal phase silica gel column chromatography, with hexane-acetone as the eluent for gradient elution, and the fraction with a hexane-acetone volume ratio of 5:1 is collected. S3. The fraction obtained in step S2 with a hexane-acetone 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%. S4. The acetone fraction of 40% obtained in step S3 is separated and purified by high performance liquid chromatography to obtain chocolate diterpenoids.

5. The application according to claim 4, characterized in that, In step S2, the gradient elution is performed using hexane-acetone as the eluent at a ratio of 100:0 to 0:100, v / v.

6. The application according to claim 4, characterized in that, In step S3, acetone-water is used as the eluent, and gradient elution is performed sequentially using acetone:water volume ratios of 30:70, 40:60, 55:45, 70:30, 85:15, and 100:

0.

7. The application according to claim 4, characterized in that, In step S4, the mobile phase of the high-performance liquid chromatography (HPLC) is 55% acetonitrile / water, the flow rate is 1-3 mL / min, and the detection wavelength is 195 nm.

8. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable excipients.

9. The application according to claim 1, characterized in that, The drug is an oral, injectable, topical, or inhaled preparation.

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