CN109276563B - Application of pharmaceutical composition in preparing medicine for treating or preventing individual infection virus - Google Patents

Application of pharmaceutical composition in preparing medicine for treating or preventing individual infection virus Download PDF

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CN109276563B
CN109276563B CN201710994334.9A CN201710994334A CN109276563B CN 109276563 B CN109276563 B CN 109276563B CN 201710994334 A CN201710994334 A CN 201710994334A CN 109276563 B CN109276563 B CN 109276563B
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virus
dimethoxy
methyl
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benzenediol
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CN109276563A (en
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李书豪
吴彰哲
李庆国
杨舒涵
郑育淞
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Zhuhai Xinyaolong Investment Development Co ltd
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Zhuhai Xinzhan Biotechnology Co ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/075—Ethers or acetals
    • A61K31/085—Ethers or acetals having an ether linkage to aromatic ring nuclear carbon
    • A61K31/09—Ethers or acetals having an ether linkage to aromatic ring nuclear carbon having two or more such linkages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/357—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having two or more oxygen atoms in the same ring, e.g. crown ethers, guanadrel
    • A61K31/36—Compounds containing methylenedioxyphenyl groups, e.g. sesamin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C41/00—Preparation of ethers; Preparation of compounds having groups, groups or groups
    • C07C41/01—Preparation of ethers
    • C07C41/18—Preparation of ethers by reactions not forming ether-oxygen bonds
    • C07C41/22—Preparation of ethers by reactions not forming ether-oxygen bonds by introduction of halogens; by substitution of halogen atoms by other halogen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C41/00—Preparation of ethers; Preparation of compounds having groups, groups or groups
    • C07C41/01—Preparation of ethers
    • C07C41/18—Preparation of ethers by reactions not forming ether-oxygen bonds
    • C07C41/26—Preparation of ethers by reactions not forming ether-oxygen bonds by introduction of hydroxy or O-metal groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C41/00—Preparation of ethers; Preparation of compounds having groups, groups or groups
    • C07C41/01—Preparation of ethers
    • C07C41/18—Preparation of ethers by reactions not forming ether-oxygen bonds
    • C07C41/30—Preparation of ethers by reactions not forming ether-oxygen bonds by increasing the number of carbon atoms, e.g. by oligomerisation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D317/00—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms
    • C07D317/08—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3
    • C07D317/44—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D317/46—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 ortho- or peri-condensed with carbocyclic rings or ring systems condensed with one six-membered ring
    • C07D317/48—Methylenedioxybenzenes or hydrogenated methylenedioxybenzenes, unsubstituted on the hetero ring
    • C07D317/62—Methylenedioxybenzenes or hydrogenated methylenedioxybenzenes, unsubstituted on the hetero ring with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to atoms of the carbocyclic ring
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Abstract

The invention relates to a use of a pharmaceutical composition for preparing a medicament for treating or preventing an infection of a subject with a virus, wherein the pharmaceutical composition comprises an effective component selected from the group consisting of a compound of formula (I) and a compound of formula (II). Specifically, the pharmaceutical composition prepared by the embodiment of the present invention can prevent viral infection and reduce the amount of virus infection and lung injury caused by the virus infection.

Description

Application of pharmaceutical composition in preparing medicine for treating or preventing individual infection virus
Technical Field
Embodiments of the present invention relate to the use of a pharmaceutical composition for the preparation of a medicament for treating or preventing an infection of an individual with a virus.
Background
Influenza virus (inflenza virus) is very susceptible to spread from person to person, from person to vertebrate, by droplet infection and by contact infection, and is often prevalent in large or small sizes around the world due to its rapidly changing nature of its antigen. Patients infected with viruses often develop many complications, which are often the major cause of death in influenza patients, most commonly lung injury due to acute lung inflammation caused by influenza. When the virus is recognized by the cellular receptor, it deactivates macrophages, which release some proinflammatory cytokines or proteins, including iNOS, COX-2, IL-6, etc., via the NF- κ B pathway, which activate and initiate the innate immune response to resist virus entry, but if the immune response is too great, severe inflammation occurs. Accordingly, the development of a therapeutic regimen with good therapeutic efficacy is an urgent need.
Disclosure of Invention
One aspect of the present invention provides a use of a pharmaceutical composition for preparing a medicament for treating or preventing viral infection in a subject, wherein the pharmaceutical composition comprises an effective ingredient selected from the group consisting of 2,3,4, 5-tetramethoxytoluene, 1,2-dimethoxybenzene, 1,2,3-trimethoxybenzene, 2,3,4-trimethoxy-6-methylphenol, 3, 4-dimethoxy-6-methyl-1, 2-benzenediol, 3, 4-dimethoxy-5-methyl-1, 2-benzenediol, 2, 4-dimethoxy-6-methyl-1, 3-benzenediol, 3, 6-dimethoxy-4-methyl-1, 2-benzenediol, 2, 3-methoxy-methyl-1, 2-benzenediol, and mixtures thereof, 4,5-dimethoxy-7-methylbenzo [ d ] [1,3] dioxolane, 4,5-dimethoxy-6-methylbenzo [ d ] [1,3] dioxolane, 4,7-dimethoxy-5-methylbenzo [ d ] [1,3] dioxolane, 5-iodo-4,7-dimethoxy-6-methylbenzo [ d ] [1,3] dioxolane, 1-iodo-2,3,4, 5-tetramethoxy-6-toluene, 1,2,3,4-tetramethoxy-5-methyl-6- (3-methylbut-3-en-1-yn-1-yl) benzene, 4,7-dimethoxy-5-methyl-6- (3-methylbut-3-en-1-yn-1-yl) -yl) benzo [ d ] [1,3] dioxopentacene, 1,2,4-trimethoxybenzene, 1-iodo-2,4,5-trimethoxybenzene, 3,4-dimethoxyphenol, 4,5-dimethoxy-2-methylphenol, 2,5-dimethoxyphenol, 2,4-dimethoxyphenol, and combinations thereof.
According to some embodiments of the invention, the agent is capable of reducing infection of the cell by a virus.
According to some embodiments of the invention, the medicament is capable of reducing the amount of virus infecting the lungs of the subject.
According to some embodiments of the invention, the medicament is capable of reducing lung damage caused by infection of the lungs of the subject with the virus.
According to some embodiments of the invention, the agent is capable of reducing an inflammatory response in a cell.
According to some embodiments of the invention, the medicament is administered in a dose of 12.5-37.5 mg per kg body weight of the subject.
According to some embodiments of the invention, the virus is H1N1, H2N2, H3N2, H5N1, H7N7, H1N2, H9N2, H7N2, H7N3, H10N7, H5N2, H5N3, H5N6, H5N8, H6N1, H7N9, H10N8, chicken infectious anemia virus (CAV), Newcastle Disease Virus (NDV), chicken Infectious Bursal Disease Virus (IBDV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), porcine circovirus type ii (PCV2), swine fever virus (CSFV), Porcine Respiratory Coronavirus (PRCV), Porcine Parvovirus (PPV) or porcine infectious enterogastritis virus (TGEV).
According to some embodiments of the invention, the agent is capable of reducing the expression of interleukin (IL-6), Tumor Necrosis Factor (TNF), and interleukin-1 β (IL-1 β).
Another aspect of the present invention is to provide a method for producing a compound, comprising: reacting a compound having a structure of formula (III) with dichloromethane in an argon-protected environment in the presence of aluminum chloride to form a compound having a structure of formula (I),
wherein the structure of formula (III) is:
Figure GDA0002964276130000021
in the chemical formula (III), R7And R8Is independently selected from the group consisting of hydrogen, methoxy, hydroxy and methyl;
the chemical formula (I) is:
Figure GDA0002964276130000031
in the chemical formula (I),R1、R2、R3and R4Is independently selected from the group consisting of hydrogen, methyl, methoxy and hydroxyl.
According to some embodiments of the invention, the chemical compound having the structure of formula (I) above, wherein R is1Is a hydroxyl group, R2Is a hydroxyl group, R3Is methoxy, and R4Is hydrogen or methyl.
Drawings
Embodiments of the present invention are read in light of the following detailed description with accompanying drawings.
FIG. 1 depicts a flow diagram of the synthesis of compounds 1-22, according to various embodiments;
FIGS. 2-5 are graphs showing the results of preventing cell survival in assays, according to various embodiments;
FIGS. 6-9 are graphs depicting the results of cell viability in a therapeutic assay, according to various embodiments;
FIG. 10 depicts various immune cell numbers contained in the blood of a mouse, according to various embodiments;
FIG. 11 is a graph showing the results of H1N1 nucleoprotein gene expression in mouse lung tissue, according to various embodiments;
FIG. 12 is a graph showing staining results after hematoxylin-eosin staining of mouse lung tissue after being sectioned, according to various embodiments;
FIG. 13 is a graph showing staining results after immunofluorescence staining of mouse lung tissue after being sectioned, according to various embodiments;
FIGS. 14-15 are graphs showing the survival rate and weight change of mice infected with virus according to various embodiments;
wherein, the notation:
100 step 200 step 300 step 400.
Detailed Description
In order to make the disclosure more complete and complete, illustrative descriptions are provided below for aspects and embodiments of the invention; it is not intended to be the only form in which the embodiments of the invention may be practiced or utilized. The various embodiments disclosed below may be combined with or substituted for one another where appropriate, and additional embodiments may be added to one embodiment without further recitation or description. In the following description, numerous specific details are set forth to provide a thorough understanding of the following embodiments. However, embodiments of the invention may be practiced without these specific details.
The present invention relates to the use of a pharmaceutical composition for the preparation of a medicament for treating or preventing a viral infection in a subject, wherein the pharmaceutical composition comprises an active ingredient selected from the group consisting of a compound of formula (I) and a compound of formula (II), wherein formula (I) has the following chemical structure:
Figure GDA0002964276130000041
wherein R is1、R2、R3And R4Is independently selected from hydrogen, methyl, iodine, methoxy, hydroxyl and
Figure GDA0002964276130000042
the group consisting of; wherein the chemical formula (II) has the following chemical structure:
Figure GDA0002964276130000043
wherein R is5And R6Is independently selected from hydrogen, methyl, iodine, methoxy, hydroxyl and
Figure GDA0002964276130000044
the group consisting of.
In one embodiment, the active ingredient contained in the pharmaceutical composition is selected from the group consisting of 2,3,4, 5-tetramethoxytoluene (1,2,3,4-tetramethoxy-5-methylbenzene), 1,2-dimethoxybenzene (1,2-dimethoxybenzene), 1,2,3-trimethoxybenzene (1,2, 3-trimethylbenzene), 2,3,4-trimethoxy-6-methylphenol (2,3,4-trimethoxy-6-methylphenol), 3, 4-dimethoxy-6-methyl-1, 2-benzenediol (3,4-dimethoxy-6-methylbenzene-1, 2-benzenediol), 3, 4-dimethoxy-5-methyl-1, 2-benzenediol (3,4-dimethoxy-5-methylbenzene-1,2-diol), 2, 4-dimethoxy-6-methyl-1, 3-benzenediol (2, 4-dimethoxy-6-methylbenezene-1, 3-diol), 3, 6-dimethoxy-4-methyl-1, 2-benzenediol (3, 6-dimethoxy-4-methylbenezene-1, 2-diol), 4,5-dimethoxy-7-methylbenzo [ d ] [1,3] dioxan (4, 5-dimethoxy-7-methylbenezo [ d ] [1,3] dioxan), 4,5-dimethoxy-6-methylbenzo [ d ] [1,3] dioxan (4,5-dimethoxy-6-methylbenzo [ d ] [1,3] dioxan), 4,7-dimethoxy-5-methylbenzo [ d ] [1,3] dioxan), 3] dioxypentacyclic (4, 7-dimethy-5-methybenzo [ d ] [1,3] dioxole), 5-iodo-4,7-dimethoxy-6-methylbenzo [ d ] [1,3] dioxypentacyclic (5-iododo-4, 7-dimethy-6-methybenzo [ d ] [1,3] dioxole), 1-iodo-2,3,4, 5-tetramethoxy-6-toluene (1-iododo-2, 3,4, 5-tetramethoxy-6-methybene), 1,2,3,4-tetramethoxy-5-methyl-6- (3-methylbut-3-en-1-yn-1-yl) benzene (1,2,3, 4-tetramethoxy-5-methy-6- (3-methylbut-3-en-1-yl) benzene (1,2,3, 4-tetramethoxy-5-methy-6- (3-methyben-1-yl) ) 4,7-dimethoxy-5-methyl-6- (3-methylbut-3-en-1-yn-1-yl) benzo [ d ] [1,3] dioxopentacyclic (4,7-dimethoxy-5-methyl-6- (3-methyllbut-3-en-1-yn-1-yl) benzole [ d ] [1,3] dioxole), 1,2,4-trimethoxy benzene (1,2, 4-trimethoxbene), 1-iodo-2,4,5-trimethoxy benzene (1-iodo-2,4, 5-trimethoxbene), 1,2,4-trimethoxy-5- (3-methylbut-3-en-1-yn-1-yl) benzene (1,2,4-trimethoxy-5- (3-methybout-3-en-1-yn-1-yl) bezene), 3,4-dimethoxyphenol (3,4-dimethoxyphenol), 4,5-dimethoxy-2-methylphenol (4,5-dimethoxy-2-methylphenol), 2,5-dimethoxyphenol (2,5-dimethoxyphenol), 2,4-dimethoxyphenol (2,4-dimethoxyphenol), and combinations thereof.
In one embodiment, the present invention provides a method for producing a compound having the structure of formula (I), comprising: reacting a compound having the structure of formula (III) with a methane halide compound in the presence of a catalyst to form a compound having the structure of formula (I), wherein the structure of formula (III) is:
Figure GDA0002964276130000051
in the chemical formula (III), R7And R8Is independently selected from the group consisting of hydrogen, methoxy, hydroxy and methyl, the formula (I) is:
Figure GDA0002964276130000052
in the formula (I), R1、R2、R3And R4Is independently selected from the group consisting of hydrogen, methyl, methoxy and hydroxyl. By the above method, the compound having the structure of formula (I) can be prepared by only one reaction step. In one embodiment, compounds of formula (I) wherein R is1Is a hydroxyl group, R2Is a hydroxyl group, R3Is methoxy and R4Is hydrogen or methyl. In one embodiment, the catalyst may include, but is not limited to, aluminum chloride, iron oxide, zinc chloride, tin chloride, or aluminum bromide.
According to some embodiments, the halogenated methane compound may include, but is not limited to, methylene chloride, dibromomethane, trichloromethane, or tribromomethane. In addition, in some embodiments, the haloalkanes may include, but are not limited to, primary haloalkanes, secondary haloalkanes, and tertiary haloalkanes in addition to the methyl halide.
The term "halo" means fluoro, chloro, bromo, or iodo.
In one embodiment, the reaction of the compound of formula (III) with the halogenated methane compound is performed in an environment containing an inert gas, such as nitrogen, argon or helium, to provide protection (e.g., to prevent unwanted oxidation).
In one embodiment, the virus is influenza virus, chicken infectious anemia virus (CAV), Newcastle Disease Virus (NDV), chicken Infectious Bursal Disease Virus (IBDV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), second type Porcine Circovirus (PCV) type 2, PCV2), swine fever virus (CSFV), porcine respiratory virus (PRCV), Porcine Parvovirus (PPV), or porcine transmissible gastroenteritis virus (tgv). In another embodiment, the virus is an influenza virus, and the influenza virus is an influenza a virus. Influenza viruses can be distinguished A, B, C types based on antigenic proteins produced by the Nucleoprotein (NP) and Matrix Protein (MP).
In one embodiment, the medicament is for preventing or treating infection of a subject with influenza a virus. As used herein, "infection with influenza A virus" refers to infection by influenza A virus. Furthermore, influenza a viruses can be further divided into different subtypes according to different combinations of the envelope glycoproteins Hemagglutinin (HA) and neuraminidase protein (NA). In one embodiment, the influenza a viruses used for the assay may include, but are not limited to, H1N1, H2N2, H3N2, H5N1, H7N7, H1N2, H9N2, H7N2, H7N3, H10N7, H5N2, H5N3, H5N6, H5N8, H6N1, H7N9, and H10N 8. In a preferred embodiment, the influenza a virus used in the assay is H1N 1.
As used herein, "subject" refers to an animal, including a human, that is subject to various embodiments of the present invention.
As used herein, "prevention" refers to prophylactic measures taken to protect or prevent a subject not already infected with a virus. As used herein, "treating" refers to curing, ameliorating, alleviating, palliating, affecting or modifying an infection by a virus in a subject following infection by the virus by various embodiments of the present invention. In a preferred embodiment, the subject to be prevented or treated is a mammal or a bird. The aforementioned mammals include, but are not limited to, humans, mice, pigs, sheep, cattle, horses, cats, rabbits, deer, monkeys, and dogs. The aforementioned birds may include, but are not limited to, pigeons, chickens, ducks and geese. In one embodiment, when the subject to be prevented or treated is a human, the subject may be a neonate, an adolescent, or an adult. The newborn refers to the infant within 28 days (including 28 days) of birth.
In cell-phase assays, in one embodiment, it was unexpectedly found that pharmaceutical compositions prepared using the various compounds described above can be used to reduce infection of cells by influenza a virus. In some embodiments, the cells are derived from cell lines including, but not limited to, BHK-21 cells, chicken embryo cells, CHO-K1 cells, CHO-K1 cells, NS-1 cells, MRC-5 cells, WI-38 cells, 3T3 cells, 293 cells, Per.C6 cells, BSC cells, HeLa cells, HepG2 cells, LLC-MK cells, CV-1 cells, RAF cells, or LLCPK cells. For example, in certain embodiments, the cell used in the assay is a BHK-21 cell. Before or after the BHK-21 cells are infected by the H1N1 virus, if the BHK-21 cells are treated by the benzene 2, 4-dimethoxy-6-methyl-1, 3-benzenediol, the survival rate of the BHK-21 cells can be effectively improved. The concentration of the benzene 2, 4-dimethoxy-6-methyl-1, 3-benzenediol is about 3.125-100. mu.g/ml. In some embodiments, benzene 2, 4-dimethoxy-6-methyl-1, 3-benzenediol may be used in a dose of, for example, 3.125. mu.g/ml, 6.25. mu.g/ml, 12.5. mu.g/ml, 25. mu.g/ml, 50. mu.g/ml, or 100. mu.g/ml.
In addition, lung injury (lung injury) caused by inflammation of the lungs after infection with influenza virus in an individual is a key factor in death due to influenza. In one embodiment, the amount of influenza a virus in the lungs of a subject infected with the aforementioned drug is effectively reduced by administering the drug therapeutically or prophylactically to the subject. In one embodiment, the medicament reduces lung damage in the lungs of the subject caused by infection with influenza a virus, such as immune cell sphere infiltration in the alveoli, or bronchial swelling caused by inflammation. In another embodiment, the drug also reduces inflammatory response.
In addition, the inflammatory response caused by viral infection also causes an increase in cytokines associated with inflammation in the subject. These cytokines are immune-related cytokines that cause inflammation when the immune response is too high. In one embodiment, the agents of the invention reduce the expression of interleukin (IL-6), Tumor Necrosis Factor (TNF), and interleukin-1 β (IL-1 β) in an infected individual.
According to some embodiments of the invention, the medicament is unexpectedly found to enhance survival of a subject in need thereof infected with influenza a virus in an animal assay. In one embodiment, the above-mentioned drugs are administered in an amount of 12.5-37.5 mg/kg body weight of the subject. In one embodiment, the above-mentioned drugs are administered in an amount of 15 mg, 17.5 mg, 20mg, 22.5 mg, 25mg, 27.5 mg, 30 mg, 32.5 mg or 35 mg per kg of body weight of the subject. For example, in certain embodiments, the subject receiving the prophylaxis or treatment is a mammal or avian. Before or after the mammals or birds are infected with the H1N1 virus, if the medicine of the present invention is administered in an amount of 25mg/kg body weight, the survival rate of the mammals or birds can be effectively improved. In a preferred embodiment, the administered drug comprises benzene 2, 4-dimethoxy-6-methyl-1, 3-benzenediol, and the subject to be prevented or treated is a mammal or a bird, and the preferred dose is 25mg per kg body weight of the subject. The administration of the medicament of the invention is not harmful to the subject, i.e. no side effects occur. In some embodiments, the medicament of the invention is administered to the subject immediately prior to or immediately after infection with the virus. In one embodiment, the above medicament is used for preparing a medicament for treating or preventing infection of a subject with influenza a virus, and can be formulated into various dosage forms such as tablets, liquids, granules, capsules, powders or combinations thereof according to administration.
Embodiments of the invention may further comprise a pharmaceutically acceptable diluent, excipient or carrier. It will be appreciated that the above pharmaceutically acceptable diluents, excipients or carriers are compatible with the active ingredient and are not deleterious to the subject to which they are administered. In one embodiment, the diluent may include, but is not limited to, buffered saline, potassium chloride, potassium hydrogen phosphate, potassium dihydrogen phosphate, sodium sulfate, sodium chloride, potassium hydroxide, or combinations thereof. The above diluents may be used to adjust the osmotic pressure, the pH, decrease/increase the consistency or solubility of the drug of the invention.
In one embodiment, the excipient may be an antioxidant, sweetener, flavoring agent, coloring agent, preservative, or a combination thereof.
In one embodiment, the carrier may include, but is not limited to, various emulsifiers, alcoholic liquids, polysorbates, glycerin, or combinations thereof. The above alcohol liquid may be, but is not limited to, monohydric alcohol or polyhydric alcohol, for example, monohydric alcohol includes methanol, ethanol, n-propanol, isopropanol, n-butanol, second butanol, third butanol, isobutanol, n-hexanol, n-heptanol, n-octanol or n-decanol. In one embodiment, the polysorbate may be, for example, polysorbate 20(Tween 20), polysorbate 40(Tween 40), polysorbate 60(Tween 60), polysorbate 80(Tween 80), or a combination thereof.
According to some embodiments, the medicament of the present invention may be used or prepared as other compositions in conjunction with other supplements. Such supplements include, but are not limited to, polysaccharides, adenosine, vitamins, triterpenes, sterols, lignins, or combinations thereof.
In one embodiment, the pharmaceutical composition of the present invention may further comprise an antibody, immunoglobulin or other known immune factor therapeutic agent, which enhances the effect of the pharmaceutical composition of the present invention by utilizing the specificity of immunotherapy for a target (e.g., a virus).
To confirm that embodiments of the present invention can prepare a drug having the function of treating or preventing influenza virus, the following experiments are given for illustrative purposes only and not for limiting the present invention.
Preparation of the Compounds
The compound 1, compound 2, compound 3 and compound 16 used in example 1, example 2, example 3 and example 16 were all purchased from Sigma company, usa and were 2,3,4, 5-tetramethoxytoluene, 1,2-dimethoxybenzene, 1,2,3-trimethoxybenzene and 1,2,4-trimethoxybenzene, respectively. Wherein compound 1 and compound 16 are useful as starting materials for the synthesis of the compounds of the examples.
Referring to FIG. 1, a flow chart of a method for preparing the compound of the embodiment is shown, wherein the compound of the embodiment is synthesized by the following steps 100-400.
Step 100: to a 25 mL round bottom flask was added 4.43 millimoles (mmol) of 2,3,4, 5-tetramethoxytoluene (compound 1) at room temperature, followed by 6 mL (mL) of dichloromethane (dichromethane,DCM) was added, 0.65 g of aluminum chloride (AlCl)3) Then argon gas is filled. The reaction was heated to 40 ℃ for 16 hours and followed by Thin Layer Chromatography (TLC). The reaction was then allowed to cool to room temperature and extracted with 30 ml of ice water and dichloromethane. Finally, by using a column chromatography, n-hexane (hexane) and Ethyl Acetate (EA) in different proportions as eluent, 2,3,4-trimethoxy-6-methylphenol, 2, 4-dimethoxy-6-methyl-1, 3-benzenediol, 3, 4-dimethoxy-6-methyl-1, 2-benzenediol, 3, 4-dimethoxy-5-methyl-1, 2-benzenediol and 3, 6-dimethoxy-4-methyl-1, 2-benzenediol, which are respectively the compounds 4 to 8 of examples 4 to 8, can be obtained by purification.
When the starting material (compound 1) in the aforementioned step 100 is replaced with compound 16, that is, 2,3,4, 5-tetramethoxytoluene is replaced with 1,2,4-trimethoxybenzene, 3,4-dimethoxyphenol, 4,5-dimethoxy-2-methylphenol, 2,5-dimethoxyphenol and 2,4-dimethoxyphenol, which are compounds 19 to 22 of examples 19 to 22, respectively, can be obtained in the same manner.
Step 200: to a 25 ml round-bottomed flask was added 1.49 mmol of 3, 4-dimethoxy-6-methyl-1, 2-benzenediol (compound 6) at room temperature, followed by addition of 250 mg of lithium hydroxide (LiOH) and then argon filling, followed by addition of 3 ml of Dimethylformamide (DMF). Then 0.42 ml of dibromomethane (dibromomethane) was added slowly and heated to 80 ℃ for overnight reaction (overhead) followed by thin layer chromatography. After the reaction mixture was cooled to room temperature, 15 ml of ice water and 15 ml of ethyl acetate were added for extraction, and an equal volume of saturated brine was added. Finally, by using a column chromatography, the 4,5-dimethoxy-7-methylbenzo [ d ] [1,3] dioxopentacene, which is the compound 9 of example 9, can be obtained by purifying with n-hexane and ethyl acetate in different proportions as an eluent.
When the starting material (Compound 6) in the aforementioned step 200 is replaced with Compound 7, 4,5-dimethoxy-6-methylbenzo [ d ] [1,3] dioxopentacene, which is Compound 10 of example 10, can be obtained in the same manner.
When the starting material (Compound 6) in the aforementioned step 200 is replaced with Compound 8, 4,7-dimethoxy-5-methylbenzo [ d ] [1,3] dioxopentacene, which is Compound 11 of example 11, can be obtained in the same manner.
Step 300: to a 25 ml round-bottomed flask was added 4.43 mmol of 4,7-dimethoxy-5-methylbenzo [ d ] at room temperature][1,3]Dioxopentacene (Compound 11), followed by 2 mg acetonitrile (Acetonitrile), 1.1 mmol N-iodosuccinimide (NIS), and 0.3 mL trifluoroacetic acid (CF)3COOH) and wrapped in tinfoil. The reaction was continued for 5 hours by heating to 50 ℃ and followed by thin layer chromatography. After the reaction mixture was cooled to room temperature, 5 ml of sodium dithionite (sodium dithionite) was added. Then extracting with 6 ml dichloromethane, then adding 10 ml water to extract to obtain 5-iodo-4,7-dimethoxy-6-methylbenzo [ d][1,3]Dioxolane, compound 12 of example 12.
When the starting material (compound 11) in the aforementioned step 300 is replaced with compound 1, 1-iodo-2,3,4, 5-tetramethoxy-6-toluene, which is compound 13 of example 13, can be obtained in the same procedure.
When the starting material (compound 11) in the aforementioned step 300 is replaced with compound 16, 1-iodo-2,4,5-trimethoxybenzene, which is compound 17 of example 17, can be obtained in the same procedure.
Step 400, add 10 mmol of 5-iodo-4,7-dimethoxy-6-methylbenzo [ d ] to a 25 mL round-bottomed flask at room temperature][1,3]Dioxolane (Compound 12), 130 mg of copper iodide (CuI), 130 mg of palladium acetate (Pd (OAc))2) Followed by the addition of 1.0 ml of triethylamine (trimethyamine, Et)3N), heated to 60 ℃ to react, and 192 mg of 2-methylbut-1-en-3-yne (2-methylbut-1-en-3-yne) were slowly added in the middle of the reaction and reacted for 36 hours, followed by thin layer chromatography. After the reactants are cooled to room temperature, 15 ml of ethyl acetate is added for extraction, then filtration is carried out, 6 ml of water is used for washing out impurities, concentration and vacuum-pumping drying are carried out, and finally, 4,7-dimethoxy-5-methyl-6- (3-methylbut-3-en-1-yn-1-yl) benzo [ d ] can be obtained by utilizing a column chromatography and using n-hexane and ethyl acetate with different proportions as extracting agents for purification][1,3]Dioxolane, compound 15 of example 15.
When the starting material (compound 12) in the aforementioned step 400 is replaced with compound 13, 1,2,3,4-tetramethoxy-5-methyl-6- (3-methylbut-3-en-1-yn-1-yl) benzene, which is compound 14 of example 14, can be obtained in the same manner.
When the starting material (compound 12) in the aforementioned step 400 is replaced with compound 17, 1,2,4-trimethoxy-5- (3-methylbut-3-en-1-yn-1-yl) benzene, compound 18 of example 18, can be obtained in the same manner.
Solutions of varying concentrations were prepared using compounds 1-22 as described in the table one below for subsequent experiments.
Watch 1
Figure GDA0002964276130000111
Figure GDA0002964276130000121
Cell culture
The cell used in the test of this embodiment was baby hamster kidney cell BHK-21(ATCC CCL-10, baby hamster kidney cells). Subculture was performed when the cells grew to eighty-percent full on the medium. First, the cell culture solution was aspirated, the residual serum and cell metabolites were washed with Phosphate Buffered Saline (PBS), 1 ml of trypsin-EDTA (typsin-EDTA) was added, and the mixture was allowed to stand at 37 ℃ for 3 to 5 minutes. Then, the action of trypsin-EDTA was stopped with 0.5 ml of Fetal Bovine Serum (FBS), and the cells were washed and collected with RPMI-1640 culture medium. Subpackaging in a 50 ml centrifuge tube, centrifuging at 4 deg.C and 500G for 5 min, removing supernatant, suspending cells in RPMI-1640 culture medium, counting cells, and culturing in cell culture medium containing 10% fetal calf serum.
Virus culture
Injecting 100 μ l (μ l) influenza A virus (H1N1) into allantoic membrane of egg embryo, sealing the opening with wax, culturing in incubator for 1-2 days, and standing at 4 deg.C for coagulation. Finally, the liquid in the allantoic membrane is extracted and stored at-80 ℃.
Determination of viral titres
In the subculture, BHK-21 cells were inoculated into 6-well culture plates and placed in a medium containing 5% CO2The incubator of (1). When the cells are cultured to 6 to 7 minutes, the cell culture solution is sucked up, the residual cell solution is washed by PBS, and virus suspensions with different dilution times are added. After being placed in an incubator and infected for 1 hour, the virus suspension was aspirated, and RPMI-1640 medium containing 2% FBS and 2% agarose gel (agarose gel) was added thereto, and the mixture was left to stand until it was coagulated and then cultured in the incubator for 2 to 3 days. Then 2 ml of 10% formalin (formalin) was added, and after standing at room temperature for 1 hour, the gel was removed and stained with 1% crystal violet (crystal violet) for 1 hour. Finally, washing off residual crystal violet by using clear water, calculating the number of virus classes and determining the virus titer. The unit is PFU/ml (queue forming unit).
Statistical analysis
Data for experimental results are presented as mean (means) ± Standard Error (SEM) and Student's t-test was used to compare the differences between treatment groups. Significant differences are indicated by asterisks, indicating p < 0.05; denotes p < 0.01; denotes p < 0.001.
Cytotoxicity test
Cytotoxicity assays were performed using the MTS assay (MTS assay). At 1 × 104The amount of BHK-21 cells per well was plated in a 96-well plate at 37 ℃ with 5% CO2After culturing for 24 hours in the incubator, the cells are pasted, and samples to be detected with different concentrations and the cells are respectively added to be cultured for 48 hours in the incubator. And finally, adding an MTS reagent into the culture solution, and carrying out a light-shielding reaction for 1 hour, and then measuring the light absorption value of the MTS reagent at the wavelength of 490nm to detect the cell survival rate.
The prophylactic or therapeutic effects of the various compounds (compounds 1 to 22) of the present embodiment on influenza viruses were measured by the MTS assay. In examples 1-22, each compound was prepared at 6 different concentrations for testing, 3.125. mu.g/ml, 6.25. mu.g/ml, 12.5. mu.g/ml, 25. mu.g/ml, 50. mu.g/ml and 100. mu.g/ml. The cells without any treatment were used as control groups (control), and relative survival rates of other treatment groups were calculated based on the survival rate of the control groups as 100%. The group that only infects viruses but has not undergone any prophylactic or therapeutic treatment is the virus group. In addition, since compounds 1-22 all used DMSO as a solvent, the DMSO group refers to a control group treated with DMSO only after infection with virus. Tamiflu is a commercially available antiviral preparation and tested as a positive control at concentrations of 25. mu.g/ml, 50. mu.g/ml, 100. mu.g/ml, 200. mu.g/ml, 400. mu.g/ml and 800. mu.g/ml, respectively. The assay was divided into two groups according to the time points of addition of test compounds 1-22 as described above:
(1) prevention (prophyxias) test group: BHK-21 cells were cultured in 96-well plates and first added with different concentrations of compounds 1-22 for 1 hour in an incubator. Subsequently, an additional H1N1 influenza virus was added at a virus infection dose (MOI) of 0.1 for 1 hour. Then removing the culture medium, adding 2% FBS-containing PRMI-1640 culture medium, and standing at 37 deg.C with 5% CO2Was cultured in the incubator of (1) for 48 hours, and the cell viability was measured by the MTS assay.
(2) Treatment (treatment) trial group: BHK-21 cells were cultured in a 96-well plate and first treated with H1N1 influenza virus at an MOI of 0.1 for 1 hour. The virus was then removed and compounds 1-22 were added at various concentrations for 1 hour in the incubator. Finally removing the culture medium, adding 2% FBS-containing PRMI-1640 culture medium, and standing at 37 deg.C with 5% CO2Was cultured in the incubator of (1) for 48 hours, and the cell viability was measured by the MTS assay.
Referring to fig. 2 to 5, cell viability in the prevention test group is shown. The results showed that the survival rate of the virus group without any treatment after infection with virus was only 35.27%. The compounds 1-22 of examples 1-22 were all effective in preventing viral infection, and were significantly different from the virus group. Example 20 the survival rate was more than 80% at a dose of 12.5. mu.g/ml. Whereas examples 4,5 and 9 were the best, with survival rates approaching 80% and were dose-dependent.
Continuing with fig. 6-9, cell viability for the treatment trial groups is shown. The results showed that the survival rate of the virus group without any treatment after infection with virus was only 36.52%. The treatment with compounds 1-22 of examples 1-22 was effective in treating viral infections and was significantly different compared to the virogroup. Furthermore, the Tamiflu group showed survival rates of 57.60. + -. 4.73%, 55.51. + -. 4.07%, 66.39. + -. 4.68%, 61.39. + -. 3.72%, 55.57. + -. 2.76%, 54.18. + -. 4.79% at concentrations of 25. mu.g/ml, 50. mu.g/ml, 100. mu.g/ml, 200. mu.g/ml, 400. mu.g/ml and 800. mu.g/ml, respectively. Example 5 in addition to presenting dose-related relationship during the treatment period, the effect was close to 80% at both the test concentrations of 50. mu.g/ml and 100. mu.g/ml, which was also higher than that of Tamiflu in the positive control group. Compound 5 of example 5 was selected for further animal testing according to the above.
Animal model establishment
BALB/c mice were used in this experiment, and all mice were housed in the animal house of the institute of Life sciences, Taiwan university of oceanic sciences, China.
The test divides animal test modes into four groups:
(1) control group: the mice were fed with PBS before challenge, and PBS was inhaled through the nasal cavity during challenge.
(2) And (3) virus group: the mice were fed with PBS before challenge, and 500 PFU/mouse H1N1 virus was inhaled through the nasal cavity during challenge.
(3) Tamiflu group: mice were tube-fed with PBS prior to challenge, and were tube-fed with approximately 20mg of Tamiflu (20mg/kg/day) per kg body weight per day one hour after nasal inhalation of 500 PFU/mouse H1N1 virus.
(4) Example 5: mice were tube-fed with PBS prior to challenge, and were tube-fed with approximately 25mg of compound 5 per kg of body weight per day (25mg/kg/day) one hour after nasal inhalation of 500 PFU/mouse H1N1 virus.
Blood assay immunization
Mice administered CO on day nine2After sacrifice, whole blood was collected from the mice as a cardiac blood sample. Blood analysis was performed using a Hematology analyzer (Exigo Veterinary Hematology analyzer, Medonic, Stockholm, Sweden).
As shown in FIG. 10, the cell numbers of white blood cells (leukocytes), lymphocytes (lymphocytes), intermediate cells (intermediate cells) and neutrophils (neutrophiles) after treatment of the above four different groups are shown. The mice treated in example 5 had no significant differences in leukocytes, lymphocytes, intermediate cells and neutrophils compared to the control group. It can be seen that example 5 did not affect the number of immune cells in mice.
Real-time quantitative polymerase chain reaction (Real-time PCR) detection of virus expression
Since the H1N1 virus is an RNA virus, it is necessary to extract its RNA and then reverse transcribe it into cDNA to perform real-time quantitative polymerase chain reaction.
First, a lung tissue mass was weighed and ground with a grinding bar, total RNA extraction was performed using commercially available RNA extraction kit RNeasy (QIAGEN, Germantown, USA), 2. mu.l of RNA was diluted 10-fold with DEPC (diethyl pyrocarbonate) water, and absorbance values at wavelengths of 260nm and 280nm were measured and converted into RNA concentration and purity. Then, quantitative RNA was put into a PCR tube, DEPC water was added to make up 12.5. mu.l, 1. mu.l of Oligo (dT) primer was added thereto, and after reacting at 72 ℃ for 2 minutes, the mixture was put into an ice bath, 4. mu.l of 5-fold buffer, 1. mu.l of 10mM dNTP mixture, 1. mu. l M-MLV reverse transcriptase and 0.5. mu.l of RNase inhibitor were sequentially added thereto, and after uniform mixing, the mixture was reacted at 42 ℃ for 1 hour, followed by reaction at 95 ℃ for 5 minutes to remove the activity of the reverse transcriptase, whereby cDNA of the above-mentioned RNA could be obtained.
Next, 5. mu.l of the 100-fold diluted cDNA was taken, followed by addition of 12.5. mu.l of iQTM SYBR
Figure GDA0002964276130000161
Supermix (Bio-Rad, Hercules, USA) and 10. mu.M double primers each in an amount of 0.5. mu.l, and finally sterile water was added to make the total volume 25. mu.l. Using a PCR reactor (iCycler)
Figure GDA0002964276130000162
Multicolor Real-Time PCR Detection System, BIO-RAD) performs polymerase chain reaction by using the optimal adhesion temperature of each primer, and the expression quantity of the H1N1 nucleoprotein gene can be obtained after the reaction is finished.
Referring to FIG. 11, the expression level of the H1N1 nucleoprotein gene is shown after treatment of the four different groups. Both Tamiflu and example 5 groups were reduced by more than about 5-fold compared to the untreated virus group after challenge and were significantly different. Further, the decrease degree of example 5 was similar to that of the Tamiflu group, and it was found that example 5 can decrease the virus infectivity.
Mouse lung pathological tissue paraffin embedded section
Mouse lung tissue was soaked in 10% formalin, left to stand at 4 ℃ for one day, and then dehydrated with 70%, 90%, and 100% ethanol and xylene in this order. Tissue sections were performed after paraffin embedding. The slides were placed in a 50 ℃ oven for baking and then stored at 4 ℃ or tissue stained.
Hematoxylin-eosin staining (Hematoxylin and eosin stain, H & E stain)
Before staining, paraffin of the coated tissue was dewaxed with xylene and then the slide was immersed in hematoxylin and stained for 10 seconds, and then washed with running water for 15 minutes. Then staining with eosin for 1 minute, washing with running water, drying the slide, sequentially soaking 70%, 90% and 100% alcohol and xylene for 5 minutes for dehydration, and sealing and storing after the slide is dried.
As shown in fig. 12, in the virus group that was not treated after challenge, the extent of infiltration of the surrounding alveoli with the immune cell globules was denser than that of the control group, indicating that the lung injury was severe. The extent of infiltration was less in Tamiflu and example 5 treated mice compared to the virus group. From this, it is understood that example 5 can reduce lung injury caused by influenza virus.
Immunofluorescent staining (Immunofluorescence stain)
The slides were prepared by embedding the sections in paraffin as described above, and after 5 minutes of oven action at 65 ℃, the slides were dewaxed with xylene, then washed three times with PBST (PBS-Tween 20), and then filled (blocking) with 5% Bovine Serum Albumin (BSA) for 1 hour. After completion of the priming, PBST was washed three times, and Mouse anti-influenza A/WSN/33M protein antibody (Mouse anti-influenza A/WSN/33M-protein) was added and left to react overnight at 4 ℃ (overnight). The following day, the tissue was washed three times with PBST, treated with a fluorescent isothiocyanate-goat anti-mouse antibody (FITC-goat anti mouse IgG) for 1 hour, washed three times with PBST, stained nuclei with 1. mu.g/ml of 4',6-Diamidino-2-phenylindole (4',6-Diamidino-2-phenylindole dihydrate, DAPI), and observed for the expression of tissue fluorescence signals using a fluorescence microscope.
Referring to FIG. 13, the fluorescence staining results show that the virus group not treated after challenge has a large amount of virus. In contrast, example 5 showed much reduced fluorescence signal and similar results to those of Tamiflu. Example 5 was again shown to be effective in reducing the amount of viral infection in lung tissue.
Survival rate and weight change in mice
The experimental groups were control, virus, Tamiflu and example 5, as established in the animal model described above. However, mice were not sacrificed, but their survival rate after infection with virus was observed and their weight changes were recorded.
Referring to fig. 14 and 15, the survival rate and weight change of the mice after the four groups are treated for 19 days are shown. The survival rate of mice was 100% until 19 days when the control group was not infected with virus; the virus group only infected the virus alone and did not have any treatment, so death began to occur on day 9, and only 25% survival rate remained in mice by day 19; the Tamiflu group died beginning on day 14 with a 40% survival rate of mice by day 19; the group treated with compound 5 of example 5 began to die at day 14, the survival rate of the mice by day 19 reached 80%, and the body weight gradually increased back to the original weight compared to the other groups.
In summary, the embodiment of the present invention utilizes the 22 compounds, and the prepared drugs can be used for treating or preventing influenza a virus. Specifically, the medicament prepared by the embodiment of the invention can reduce the infection amount of the influenza A virus and the lung injury caused by the influenza A virus, and can improve the survival rate of mice and reduce the percentage of weight loss in animal experiments.
The foregoing outlines features of several embodiments so that those skilled in the art may better understand the aspects of the present disclosure. Those skilled in the art should appreciate that the present disclosure may be readily utilized as a basis for designing or modifying other methods or processes for carrying out the same purposes and/or achieving the same advantages of the embodiments introduced herein. Those skilled in the art should also realize that such equivalents do not depart from the spirit and scope of the present disclosure, and that they may make various changes, substitutions, and alterations herein without departing from the spirit and scope of the present disclosure.

Claims (7)

1. Use of a pharmaceutical composition for the preparation of a medicament for treating or preventing infection of a subject with influenza a virus, wherein the pharmaceutical composition comprises an active ingredient selected from the group consisting of 2,3,4, 5-tetramethoxytoluene, 1,2-dimethoxybenzene, 1,2,3-trimethoxybenzene, 2,3,4-trimethoxy-6-methylphenol, 3, 4-dimethoxy-6-methyl-1, 2-benzenediol, 3, 4-dimethoxy-5-methyl-1, 2-benzenediol, 2, 4-dimethoxy-6-methyl-1, 3-benzenediol, 3, 6-dimethoxy-4-methyl-1, 2-benzenediol, 2, 3-dimethyl-6-methyl-1, 2-benzenediol, 2, N-dimethyl-2-benzenediol, N-methyl-phenyl-1, N-dimethyl-ol, 4,5-dimethoxy-7-methylbenzo [ d ] [1,3] dioxolane, 4,5-dimethoxy-6-methylbenzo [ d ] [1,3] dioxolane, 4,7-dimethoxy-5-methylbenzo [ d ] [1,3] dioxolane, 5-iodo-4,7-dimethoxy-6-methylbenzo [ d ] [1,3] dioxolane, 1-iodo-2,3,4, 5-tetramethoxy-6-toluene, 1,2,3,4-tetramethoxy-5-methyl-6- (3-methylbut-3-en-1-yn-1-yl) benzene, 4,7-dimethoxy-5-methyl-6- (3-methylbut-3-en-1-yn-1-yl) -yl) benzo [ d ] [1,3] dioxopentacene, 1,2,4-trimethoxybenzene, 1-iodo-2,4,5-trimethoxybenzene, 3,4-dimethoxyphenol, 4,5-dimethoxy-2-methylphenol, 2,5-dimethoxyphenol, 2,4-dimethoxyphenol, and combinations thereof.
2. The use of claim 1, wherein the medicament is capable of reducing infection of a cell by a virus.
3. The use of claim 1, wherein the medicament is capable of reducing the number of pulmonary infections of the subject.
4. The use of claim 1, wherein the medicament is capable of reducing lung damage caused by a pulmonary infection of the subject with the virus.
5. The use of claim 1, wherein the medicament is capable of reducing an inflammatory response in a cell.
6. The use of claim 1, wherein the medicament is administered in an amount of 12.5-37.5 mg/kg body weight of the subject.
7. The use of claim 1, wherein the influenza a virus is H1N1, H2N2, H3N2, H5N1, H7N7, H1N2, H9N2, H7N2, H7N3, H10N7, H5N2, H5N3, H5N6, H5N8, H6N1, H7N9, or H10N 8.
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