TWI650120B - Use of a pharmaceutical composition for preparing a medicament for treating or preventing an individual infected with a virus - Google Patents

Abstract

The invention relates to the use of a medicinal composition for preparing a medicament for treating or preventing infection of an individual, wherein the medicinal composition comprises an active ingredient selected from the group consisting of a compound of formula (I) and a compound of formula (II) Group.

Description

A medicinal composition for preparing a treatment or Use of medicine for preventing infection of virus in individuals

An embodiment of the present invention relates to the use of a pharmaceutical composition for preparing a medicament for treating or preventing infection of a virus in an individual.

Influenza virus is easily spread among humans, humans, and vertebrates through droplet infection and contact. Because of its rapid change in antigens, it often spreads on a large or small scale around the world. Patients infected with the virus often cause many concurrent severe illnesses, and these concurrent severe illnesses are usually the main cause of death in influenza patients, the most common of which is lung injury caused by acute lung inflammation caused by influenza. When the virus is recognized by the cell's receptor, it will deactivate macrophages, and macrophages can release some pro-inflammatory cytokines or proteins through the NF-κB pathway, including iNOS, COX-2, IL -6 and so on, and cytokines will activate and start the innate immune response to resist virus invasion, but if the immune response Excessive levels can cause severe inflammation. According to this, the development of a treatment plan with good curative effect is an urgent need at present.

According to one aspect of the present invention, there is provided a medicinal composition for use in preparing a medicament for treating or preventing infection of a virus in an individual, wherein the medicinal composition comprises an active ingredient selected from the group consisting of 1,2,3,4-tetramethoxy -5-toluene, 1,2-dimethoxybenzene, 1,2,3-trimethoxybenzene, 2,3,4-trimethoxy-6-methylphenol, 3,4-dimethoxy -6-Toluene-1,2-diol, 3,4-dimethoxy-5-toluene-1,2-diol, 2,4-dimethoxy-6-toluene-1,3-di Alcohol, 3,6-dimethoxy-4-toluene-1,2-diol, 4,5-dimethoxy-7-toluo [d] [1,3] dioxane, 4, 5-dimethoxy-6-toluo [d] [1,3] dioxe, 4,7-dimethoxy-5-toluo [d] [1,3] dioxe, 5-iodo-4,7-dimethoxy-6-toluo [d] [1,3] dioxane, 1-iodo-2,3,4,5-tetramethoxy-6-toluene 1,2,3,4-tetramethoxy-5-methyl-6- (3-methylbut3-ene-1-yn-1-yl) benzene, 4,7-dimethoxy- 5-methyl-6- (3-methylbut-3-ene-1-yn-1-yl) benzo [d] [1,3] dioxane, 1,2,4-trimethoxy Benzene, 1-iodo-2,4,5-trimethoxybenzene, 3,4-dimethoxyphenol, 4,5-dimethoxy-2-methylphenol, 2,5-dimethoxy Phenol, 2,4-dimethoxyphenol and its The composition of the combined group.

According to some embodiments of the invention, the drug is capable of reducing the infection of cells by the virus.

According to some embodiments of the invention, the drug is Can reduce the amount of virus in the individual's lungs.

According to some embodiments of the invention, the medicament is capable of reducing lung damage caused by the virus in the individual's lungs.

According to some embodiments of the invention, the drug is capable of reducing a cellular inflammatory response.

According to some embodiments of the invention, the medicament is used at a dose of 12.5-37.5 mg per kg of body weight of the individual.

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 virus (NDV), chicken infectious bursal virus (IBDV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), swine fever virus (CSFV), swine Respiratory Coronavirus (PRCV), Porcine Parvovirus (PPV) or Porcine Infectious Gastroenteritis Virus (TGEV).

According to some embodiments of the present invention, the drug can reduce the expression levels of interleukin (IL-6), tumor necrosis factor (TNF) and interleukin-1β (IL-1β).

Another aspect of the present invention provides a method for manufacturing a compound, comprising: reacting a compound having the structure of formula (III) with dichloromethane in an environment protected by argon in the presence of aluminum chloride to form a compound having the structure A compound having the structure of chemical formula (I), wherein the structure of the chemical formula (III) is: In the chemical formula (III), R ₇ and R ₈ are independently selected from the group consisting of hydrogen, methoxy, hydroxyl and methyl. The chemical formula (I) is: In the chemical formula (I), R ₁ , R ₂ , R ₃ and R ₄ are independently selected from the group consisting of hydrogen, methyl, methoxy and hydroxyl.

According to some embodiments of the present invention, the above-mentioned chemical compound having the structure of formula (I), wherein R ₁ is a hydroxyl group, R ₂ is a hydroxyl group, R ₃ is a methoxy group, and R ₄ is hydrogen or a methyl group. .

100‧‧‧ steps

200‧‧‧ steps

300‧‧‧ steps

400‧‧‧ steps

The embodiments of the present invention are read based on the following detailed description with drawings.

Figure 1 depicts compounds 1-22 according to various embodiments. Synthesis flow chart.

FIG. 2 to FIG. 5 are graphs showing the results of cell survival rate in the prevention test according to various embodiments.

Figures 6 to 9 are graphs showing the results of cell survival rates in a therapeutic test according to various embodiments.

Figure 10 shows the number of various immune cells contained in the blood of mice 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 the staining results of mouse lung tissue after sectioning and staining with hematoxylin-eosin according to various embodiments.

FIG. 13 is a graph showing the staining results of mouse lung tissues after sectioning and immunofluorescence staining according to various embodiments.

FIG. 14 to FIG. 15 are graphs showing the results of survival rate and weight change of mice after being infected with the virus according to various embodiments.

In order to make the description of this disclosure more detailed and complete, the following presents an illustrative description of the implementation modes and specific embodiments of the present invention; however, this is not the only form of implementing or using the specific embodiments of the present invention. The embodiments disclosed below can be combined or replaced with each other under beneficial circumstances, and other embodiments can be added to an embodiment without further description or description. in In the following description, many specific details will be described in detail to enable the reader to fully understand the following embodiments. However, embodiments of the invention may be practiced without these specific details.

The invention relates to the use of a medicinal composition for preparing a medicament for treating or preventing infection of an individual, wherein the medicinal composition comprises an active ingredient selected from the group consisting of a compound of formula (I) and a compound of formula (II) Group, wherein the chemical formula (I) has the following chemical structure: Wherein R ₁ , R ₂ , R ₃ and R ₄ are independently selected from hydrogen, methyl, iodine, methoxy, hydroxyl and A group consisting of: wherein the chemical formula (II) has the following chemical structure: Where R ₅ and R ₆ are independently selected from hydrogen, methyl, iodine, methoxy, hydroxyl and A group of people.

In one embodiment, the pharmaceutical composition includes an active ingredient selected from the group consisting of 1,2,3,4-tetramethoxy-5-toluene (1,2,3,4-tetramethoxy-5-methylbenzene), 1 2,2-dimethoxybenzene (1,2-dimethoxybenzene), 1,2,3-trimethoxybenzene (1,2,3-trimethoxybenzene), 2,3,4-trimethoxy-6-methyl Phenol (2,3,4-trimethoxy-6-methylphenol), 3,4-dimethoxy-6-toluene-1,2-diol (3,4-dimethoxy-6-methylbenzene-1,2-diol ), 3,4-dimethoxy-5-toluene-1,2-diol (3,4-dimethoxy-5-methylbenzene-1,2-diol), 2,4-dimethoxy-6- Toluene-1,3-diol (2,4-dimethoxy-6-methylbenzene-1,3-diol), 3,6-dimethoxy-4-toluene-1,2-diol (3,6- dimethoxy-4-methylbenzene-1,2- diol), 4,5- dimethoxyethane and toluene -7- [d] [1,3] dioxine (4,5-dimethoxy-7-methylbenzo [d ] [1,3] dioxole), 4,5-dimethoxy-6-toluo [ d ] [1,3] dioxane (4,5-dimethoxy-6-methylbenzo [ d ] [1, 3] dioxole), 4,7-dimethoxy-5-toluo [ d ] [1,3] dioxane (4,7-dimethoxy-5-methylbenzo [ d ] [1,3] dioxole) , 5-iodo-4,7-dimethoxy-6-toluo [ d ] [1,3] dioxane (5-iodo-4,7-dimethoxy-6-met hylbenzo [ d ] [1,3] dioxole), 1-iodo-2,3,4,5-tetramethoxy-6-toluene (1-iodo-2,3,4,5-tetramethoxy-6-methylbenzene ), 1,2,3,4-tetramethoxy-5-methyl-6- (3-methylbut3-ene-1-yn-1-yl) benzene (1,2,3,4- tetramethoxy-5-methyl-6- (3-methylbut-3-en-1-yn-1-yl) benzene), 4,7-dimethoxy-5-methyl-6- (3-methylbutane -3-ene-1-yn-1-yl) benzo [ d ] [1,3] dioxane (4,7-dimethoxy-5-methyl-6- (3-methylbut-3-en-1 -yn-1-yl) benzo [ d ] [1,3] dioxole), 1,2,4-trimethoxybenzene, 1-iodo-2,4,5-trimethyl 1-iodo-2,4,5-trimethoxybenzene, 1,2,4-trimethoxy-5- (3-methylbut-3-ene-1-yn-1-yl) benzene ( 1,2,4-trimethoxy-5- (3-methylbut-3-en-1-yn-1-yl) benzene), 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, an embodiment of the present invention provides a method for producing a compound having the structure of the chemical formula (I), comprising: reacting a compound having the structure of the chemical formula (III) with a monohalogenated methane compound in the presence of a catalyst To form a compound having a structure of formula (I), wherein the structure of formula (III) is: In the chemical formula (III), R ₇ and R ₈ are independently selected from the group consisting of hydrogen, methoxy, hydroxyl and methyl. The chemical formula (I) is: In the chemical formula (I), R ₁ , R ₂ , R ₃ and R ₄ are independently selected from the group consisting of hydrogen, methyl, methoxy and hydroxyl. By the aforementioned method, only one reaction step is required to prepare a compound having the structure of the formula (I). In one embodiment, the compound of formula (I) can be obtained by the above manufacturing method, wherein R ₁ is a hydroxyl group, R ₂ is a hydroxyl group, R ₃ is a methoxy group, and R ₄ is a hydrogen or methyl group. . 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, chloroform, or tribromethane. In addition, in some embodiments, in addition to the halogenated methane compound, the above-mentioned haloalkane compound may include, but is not limited to, a primary haloalkane compound, a secondary haloalkane compound, and a tertiary haloalkane compound.

The term "halogen" means fluorine, chlorine, bromine or iodine.

In one embodiment, the reaction between the aforementioned chemical compound having the structure of formula (III) and the halogenated methane compound is based on the reaction containing an inert gas. To provide protection (for example, to prevent unnecessary oxidation reactions), and the inert gas may include, but is not limited to, nitrogen, argon, or helium.

In one embodiment, the virus is influenza virus, chicken infectious anemia virus (CAV), newcastle disease virus (NDV), and chicken infectious bursa disease virus, IBDV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2, PCV2, classical swine fever virus (CSFV), swine Porcine respiratory coronavirus (PRCV), porcine parvovirus (PPV) or transmissible gastroenteritis virus (TGEV). In another embodiment, the virus is an influenza virus, and the influenza virus is an influenza A virus. Influenza viruses can be divided into three types: A, B, and C based on the antigenic proteins produced by nucleoprotein (NP) and matrix protein (MP).

In one embodiment, the drug is used to prevent or treat an individual with infection with influenza A virus. "Influenza A virus infection" as used herein refers to an infection caused by an influenza A virus. In addition, influenza A viruses can be further distinguished by different combinations of viral envelope glycoproteins hemagglutinin (HA) and neuraminidase protein (NA) Into different subtypes. In an embodiment, the influenza A virus used in the test may include, but is not limited to, H1N1, H2N2, H3N2, H5N1, H7N7, H1N2, H9N2, H7N2, H7N3, H10N7, H5N2, H5N3, H5N6, H5N8, H6N1, H7N9 And H10N8. In a preferred embodiment, the influenza A virus used for the test is H1N1.

As used herein, a "subject" refers to an animal including a human being, which is subject to various embodiments of the present invention.

"Prevention" as used herein refers to preventive measures taken to protect or prevent individuals who have not been infected with the virus. "Treatment" as referred to herein means that after an individual is infected with a virus, the infection of the virus can be cured, ameliorated, reduced, mitigated, affected, or changed through various embodiments of the present invention. In a preferred embodiment, the individual receiving the prevention or treatment is a mammal or a bird. The aforementioned mammals may include, but are not limited to, humans, rats, pigs, sheep, cattle, horses, cats, rabbits, deer, monkeys and dogs. The aforementioned birds include, but are not limited to, pigeons, chickens, ducks and geese. In one embodiment, when the individual receiving the prevention or treatment is a human, the individual may be a newborn, adolescent or adult. The above-mentioned newborns are infants within 28 days of birth.

During the cell phase test, in one example, it was unexpectedly discovered that the pharmaceutical composition prepared by using the aforementioned various compounds can be used to reduce the infection of cells with influenza A virus. In some specific embodiments, the above cell line is derived from a cell line including, but not limited to, BHK-21 cells, chicken embryo cells, CHO cells, and CHO-K1 Cell, CHO-K1 cell, NS-1 cell, MRC-5 cell, WI-38 cell, 3T3 cell, 293 cell, Per.C6 cell, BSC cell, HeLa cell, HepG2 cell, LLC-MK cell, CV-1 Cells, RAF cells, or LLCCP cells. For example, in certain embodiments, the cells used in the test are BHK-21 cells. Before or after BHK-21 cells are infected with the aforementioned H1N1 virus, treatment with benzene 2,4-dimethoxy-6-toluene-1,3-diol can effectively improve the survival rate of BHK-21 cells. The concentration of the benzene 2,4-dimethoxy-6-toluene-1,3-diol is about 3.125-100 μg / ml. In some embodiments, the dose of benzene 2,4-dimethoxy-6-toluene-1,3-diol may be, for example, 3.125 μg / ml, 6.25 μg / ml, 12.5 μg / ml, 25 μg / ml, 50 μg / ml or 100 μg / ml.

In addition, lung injury caused by inflammation of the lung after an individual is infected with the influenza virus is a key factor in death due to influenza. In one embodiment, an individual who is treated or prophylactically administered with the drug described above can effectively reduce the amount of influenza A virus infection in the individual's lungs. In one embodiment, the drug can reduce lung damage caused by influenza A virus infection in the individual's lungs, such as infiltration of immune cell spheres in the alveoli, or bronchial swelling due to inflammation. In another embodiment, the drug can also reduce the inflammatory response of cells.

In addition, the inflammatory response caused by a viral infection also causes an increase in inflammation-related cytokines in the individual. These cytokines are immune-related cytokines. When high it can cause inflammation. In one embodiment, the medicament of the present invention can reduce the expression levels of interleukin (IL-6), tumor necrosis factor (TNF) and interleukin-1β (IL-1β) in infected individuals.

According to some embodiments of the present invention, it has been unexpectedly discovered in animal experiments that the drug system can improve the survival rate of influenza A virus infection in a desired individual. In one embodiment, the above-mentioned drug is administered at a dose of 12.5-37.5 mg per kg of body weight of the individual. In one embodiment, the above-mentioned drug is administered at a dose of 15 mg, 17.5 mg, 20 mg, 22.5 mg, 25 mg, 27.5 mg, 30 mg, 32.5 mg, or 35 mg per kg of body weight of the individual. For example, in certain embodiments, the individual receiving prophylaxis or treatment is a mammal or avian. Before the mammal or avian is infected with the H1N1 virus or after the virus, if the medicine of the present invention is administered at 25 mg per kilogram of body weight, the survival rate can be effectively improved. In a preferred embodiment, the medicament to be administered comprises benzene 2,4-dimethoxy-6-toluene-1,3-diol, and the subject to be prevented or treated is a mammal or avian, and is preferably administered The dose was 25 mg per kg of body weight of the individual. Administration of the medicament of the present invention is not harmful to the individual, which means that there are no side effects. In addition, in some specific embodiments, the medicament of the present invention is administered to the individual immediately before the infection or to the individual immediately after the infection. In one embodiment, the above medicine is used to prepare a medicine for treating or preventing an individual's infection with influenza A virus, and it can be formulated into various dosage forms, such as lozenges, liquids, granules, capsules, powders, or a combination thereof according to the manner of administration.

Embodiments of the invention may further comprise a pharmaceutically acceptable diluent, excipient or carrier. It should be understood that the above-mentioned pharmaceutically acceptable diluents, excipients or carriers are compatible with the active ingredients and are not harmful to the individual being 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 a combination thereof. The above-mentioned diluent can be used to adjust the osmotic pressure, pH value, decrease / increase consistency or solubility of the drug of the present invention.

In one embodiment, the excipient may be an antioxidant, a sweetener, a flavoring agent, a colorant, a preservative, or a combination thereof.

In one embodiment, the carrier may include, but is not limited to, various emulsifiers, alcoholic liquids, polysorbates, glycerol, or a combination thereof. The alcohol liquid may be, but is not limited to, a monohydric alcohol or a polyhydric alcohol. For example, the 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 can also be used together with other supplements or prepared into other compositions. The aforementioned supplements include, but are not limited to, polysaccharides, adenosine, vitamins, triterpenoids, sterols, lignin, or a combination thereof.

In one embodiment, the medicament of the present invention may further include antibodies, immunoglobulins or other known immune factor therapeutic agents, and the specificity of the target (such as a virus) by immunotherapy is used to enhance the effect of the medicament of the present invention.

In order to confirm that the embodiment of the present invention can prepare a drug for treating or preventing influenza virus, it will be described through the following tests. It should be noted that the following embodiments are for exemplary purposes only, and not for limiting the present invention.

Preparation of compounds

The compound 1, compound 2, compound 3 and compound 16 used in Example 1, Example 2, Example 3 and Example 16 were purchased from Sigma Company in the United States, and were 1,2,3,4-tetramethoxy- 5-Toluene, 1,2-dimethoxybenzene, 1,2,3-trimethoxybenzene and 1,2,4-trimethoxybenzene. Compounds 1 and 16 are used as starting materials for compound synthesis in the examples.

Next, please refer to FIG. 1 for a flowchart of a method for preparing the compound in the example, and the following steps 100-400 are used to synthesize the compound in the example.

Step 100: At room temperature, add 4.43 1,2,3,4-tetramethoxy-5-toluene (compound 1) to a 25 ml round-bottomed flask, followed by 6 ml ( mL) of dichloromethane (DCM), and 0.65 g of aluminum chloride (AlCl ₃ ) was added, followed by argon. After heating to 40 ° C for 16 hours, tracking was performed by thin layer chromatorgraphy (TLC). The reaction was then cooled to room temperature, and 30 ml of ice water and dichloromethane were added for extraction. Finally, column chromatography was used to purify with different ratios of hexane and ethyl acetate (EA) as eluents to obtain 2,3,4-trimethoxy-6-methylphenol. , 2,4-dimethoxy-6-toluene-1,3-diol, 3,4-dimethoxy-6-toluene-1,2-diol, 3,4-dimethoxy- 5-Toluene-1,2-diol and 3,6-dimethoxy-4-toluene-1,2-diol are the compounds 4-8 of Example 4-8, respectively.

If the starting material (compound 1) of the aforementioned step 100 is replaced with compound 16, that is, 1,2,3,4-tetramethoxy-5-toluene is replaced with 1,2,4-trimethoxybenzene, the same procedure can be used. 3,4-dimethoxyphenol, 4,5-dimethoxy-2-methylphenol, 2,5-dimethoxyphenol, and 2,4-dimethoxyphenol were prepared, respectively Compounds 19-22 of Examples 19-22.

Step 200: At room temperature, add 1.49 mmol of 3,4-dimethoxy-6-toluene-1,2-diol (compound 6) to a 25 ml round-bottomed flask, followed by 250 mg of hydrogen Lithium oxide (LiOH) was filled with argon, and 3 ml of dimethylformamide (DMF) was added. Then, 0.42 ml of dibromomethane was slowly added, and the reaction was heated to 80 ° C to continue the reaction overnight. After that, it was tracked by thin layer chromatography. After the reaction 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 saline was added. Finally, column chromatography was used to purify n-hexane and ethyl acetate in different ratios as the eluent to obtain 4,5-dimethoxy-7-toluo [ d ] [1,3] dioxane. The ring is the compound 9 of Example 9.

If the starting material (compound 6) of the aforementioned step 200 is replaced with compound 7, a 4,5-dimethoxy-6-toluo [ d ] [1,3] dioxecyclic ring can be prepared in the same procedure. Compound 10 of Example 10.

If the starting material (compound 6) of the foregoing step 200 is replaced with compound 8, 4,7-dimethoxy-5-toluo [ d ] [1,3] dioxane can be prepared in the same procedure. Compound 11 of Example 11.

Step 300: At room temperature, add 4.43 millimoles of 4,7-dimethoxy-5-toluo [ d ] [1,3] dioxane (Compound 11) to a 25 ml round-bottomed flask. Then, add 2 mg of acetonitrile, 1.1 millimolar N-iodosuccinimide (NIS), and then add 0.3 ml of trifluoroacetic acid (CF ₃ COOH), and cover with tin foil . After heating to 50 ° C for 5 hours, the reaction was tracked by thin layer chromatography. After the reaction was cooled to room temperature, 5 ml of sodium dithionite was added. It is then extracted with 6 ml of dichloromethane, and 10 ml of water is added to extract to obtain 5-iodo-4,7-dimethoxy-6-toluo [ d ] [1,3] dioxane. Compound 12 of Example 12.

If the starting material (compound 11) of the aforementioned step 300 is replaced with compound 1, 1-iodo-2,3,4,5-tetramethoxy-6-toluene can be prepared in the same procedure, which is the compound 13 of Example 13. .

If the starting material (compound 11) of the aforementioned step 300 is replaced with compound 16, 1-iodo-2,4,5-trimethoxybenzene can be prepared in the same procedure as Compound 17 of Example 17.

Step 400: Add 10 millimolar 5-iodo-4,7-dimethoxy-6-toluo [ d ] [1,3] dioxane to a 25 ml round-bottomed bottle at room temperature. (Compound 12), 130 mg of cuprous iodide (CuI), 130 mg, and palladium acetate (Pd (OAc) ₂ ), followed by adding 1.0 ml of trimethylamine (Et ₃ N), and heating to 60 ° C. for reaction, During the reaction, 192 mg of 2-methylbut-1-en-3-yne (2-methylbut-1-en-3-yne) was slowly added and reacted for 36 hours, followed by tracking by thin layer chromatography. After the above reaction was cooled to room temperature, 15 ml of ethyl acetate was added for extraction, and then filtered, impurities were washed with 6 ml of water, concentrated and dried under vacuum. Finally, column chromatography was used to produce n-hexane in different proportions. 4,7-Dimethoxy-5-methyl-6- (3-methylbut-3-ene-1-yn-1-yl) benzo [ d ] [1,3] Dioxane, Compound 15 of Example 15.

If the starting material (compound 12) of the aforementioned step 400 is replaced by compound 13, 1,2,3,4-tetramethoxy-5-methyl-6- (3-methylbut 3- Ene-1-yn-1-yl) benzene, compound 14 of Example 14.

If the starting material (compound 12) of the aforementioned step 400 is replaced with compound 17, 1,2,4-trimethoxy-5- (3-methylbut-3-ene-1-yne-1) can be prepared in the same procedure. -Yl) benzene, which is the compound 18 of Example 18.

As described in Table 1 below, compounds 1-22 were used to prepare solutions of different concentrations for subsequent tests.

Cell culture

The cells used in the test in this embodiment are baby hamster kidney cells BHK-21 (ATCC CCL-10, baby hamster kidney cells). Subculture was performed when the cells grew to eighth full on the medium. First, aspirate the cell culture solution, wash the residual serum and cell metabolites with phosphate buffered saline (PBS), add 1 ml of trypsin-EDTA (typsin-EDTA), and place at 37 ° C for 3 hours. -5 minutes . Then the effect of trypsin-EDTA was stopped with 0.5 ml of fetal bovine serum (FBS), and the cells were washed with RPMI-1640 medium and collected. Aliquot into a 50 ml centrifuge tube, centrifuge at 500G for 5 minutes at 4 ° C, remove the supernatant, resuspend the cells in RPMI-1640 medium, count the cells, and finally cultivate them in 10% fetal bovine Cell culture fluid of serum.

Virus culture

Inject 100 microliters (μl) of Influenza A virus (H1N1) into the allantoic membrane of the egg embryo, and then seal the cavity with wax to reproduce. Incubate in the incubator for 1 to 2 days, and then place at 4 ° C to wait for coagulation. Finally, the liquid in the allantoic membrane was withdrawn and stored at -80 ° C.

Virus titer

During subculture, BHK-21 cells were seeded in 6-well culture plates and placed in an incubator containing 5% CO ₂ . When the cells are cultured to 6 to 7 minutes, the cell culture solution is aspirated, the residual cell solution is washed with PBS, and virus suspensions of different dilutions are added. After the infection in the incubator for 1 hour, the virus suspension was aspirated, and the RPMI-1640 culture solution containing 2% FBS and 2% agarose gel was added. The solution was left to stand for solidification and cultured in the incubator. 2 to 3 days. Next, 2 ml of 10% formalin was added, and the gel was removed after standing at room temperature for 1 hour, and stained with 1% crystal violet for 1 hour. Finally, the residual crystal violet was washed away with water, the number of virus classes was calculated, and the virus titer was determined. The unit is PFU / ml (plaque forming unit).

Statistical Analysis

The data of the test results are expressed as mean ± standard error (SEM), and Student's t-test is used to compare the differences between the treatment groups. The asterisk "*" indicates that there is a significant difference, * indicates p <0.05; ** indicates p <0.01; *** indicates p <0.001.

Cytotoxicity test

The cytotoxicity test was performed using MTS assay (MTS assay). BHK-21 cells were seeded at 1 × 10 ⁴ cells / well in a 96-well culture dish, cultured in an incubator containing 5% CO ₂ at 37 ° C for 24 hours, and then the cells were covered and added separately. Different concentrations of test samples and cells were incubated in the incubator for 48 hours. Finally, the MTS reagent was added to the culture medium for 1 hour to prevent light from the reaction, and its absorbance at 490 nm was measured to detect the cell survival rate.

The MTS test can be used to determine the preventive or therapeutic effects of various compounds (compound 1-22) of this embodiment on influenza virus. In Example 1-22, each compound was prepared into six different concentrations for testing, which were 3.125 μg / ml, 6.25 μg / ml, 12.5 μg / ml, 25 μg / ml, 50 μg / ml and 100 μg / ml. The cells without any treatment are the control group, and the relative survival rate of other treatment groups can be calculated with the survival rate of the control group being 100%. The group infected only with the virus without any preventive and therapeutic treatment was the virus group. In addition, since Compound 1-22 uses DMSO as a solvent, the DMSO group refers to a control group treated with DMSO only after being infected with the virus. Tamiflu is a commercially available antiviral preparation, and the positive control group test concentrations were 25 μg / ml, 50 μg / ml, 100 μg / ml, 200 μg / ml, 400 μg / ml, and 800 μg / ml, respectively. Based on the above, this test is divided into two groups according to the time point of test compound 1-22 addition:

(1) Prophylaxis test group: BHK-21 cells were cultured in a 96-well culture plate, and compounds 1-22 of different concentrations were added to the incubator for 1 hour. Subsequently, H1N1 influenza virus with a multiplicities of infection (MOI) of 0.1 was added for 1 hour. Then, the culture medium was removed, and PRMI-1640 medium containing 2% FBS was added, and cultured in an incubator containing 5% CO ₂ at 37 ° C. for 48 hours. The cell survival rate was measured by the MTS test.

(2) Treatment test group: BHK-21 cells were cultured in 96-well culture plates, and H1N1 influenza virus with MOI of 0.1 was added for 1 hour. The virus was then removed, and compounds 1-22 at different concentrations were added to the incubator for 1 hour. Finally, the culture medium was removed, PRMI-1640 medium containing 2% FBS was added, and the cells were cultured in an incubator at 37 ° C containing 5% CO ₂ for 48 hours. The cell survival rate was measured by the MTS test.

Referring to Figures 2 to 5, the cell survival rate of the prevention test group is shown. The results showed that the survival rate of the virus group without any treatment after infection was only 35.27%. Compared with the virus group, the compound 1-22 of Example 1-22 can effectively prevent virus infection after treatment, and has significant differences. In Example 20, the survival rate can reach more than 80% at a dose of 12.5 μg / ml. However, Example 4, Example 5, and Example 9 had the best effects, with survival rates close to 80% and showing dose-dependent.

Continuing to refer to Figures 6 to 9, the cell survival rate of the treatment test group is shown. The results showed that the survival rate of the virus group without any treatment after infection was only 36.52%. Compared with the virus group, after treatment with the compound 1-22 of Example 1-22, the virus infection can be effectively treated with significant differences. In addition, the Tamiflu group showed survival rates of 57.60 ± 4.73%, 55.51 ± 4.07%, and 66.39 ± 4.68% at concentrations of 25 μg / ml, 50 μg / ml, 100 μg / ml, 200 μg / ml, 400 μg / ml, and 800 μg / ml, respectively. , 61.39 ± 3.72%, 55.57 ± 2.76%, 54.18 ± 4.79%. Except for the dose-relatedness in the treatment stage, Example 5 was close to 80% at the test concentrations of 50 μg / ml and 100 μg / ml, and the effect was higher than that of Tamiflu in the positive control group. Based on the above, Compound 5 of Example 5 was selected for further animal testing.

Animal model establishment

In this experiment, mice of the BALB / c strain purchased from the National Laboratory Animal Center were used, and all mice were housed in the Animal Room of the National Ocean University of Taiwan.

This test divides animal test modes into four groups:

(1) Control group: Mice were fed PBS before challenge, and PBS was inhaled into the nasal cavity during challenge.

(2) Virus group: Mice were fed PBS before challenge, and 500PFU / mouse H1N1 virus was inhaled into the nasal cavity during challenge.

(3) Tamiflu group: mice were given tube PBS before challenge, and 500PFU / mouse H1N1 virus was inhaled into the nasal cavity for one hour after challenge, and Tamiflu (20mg / kg / day) was about 20 mg / kg body weight per day. Perform tube feeding.

(4) Example 5: Mice were fed with PBS before challenge, and 500 PFU / mouse of H1N1 virus was inhaled into the nose for one hour after challenge, and about 25 mg of compound 5 (25 mg / kg / day) Perform tube feeding.

Blood analysis

After the mice were sacrificed with CO ₂ on the ninth day, whole blood was collected by heart blood collection. Blood analysis was performed using an Exigo Veterinary Hematology analyzer (Medonic, Stockholm, Sweden).

As shown in Figure 10, the numbers of leukocytes, lymphocytes, intermediate cells, and neutrophile granulocytes after treatment with the four different groups are shown. Compared with the control group, the white blood cells, lymphocytes, intermediate cells, and neutrophils in the blood of the mice treated in Example 5 were not significantly different. It can be seen that Example 5 does not affect the number of immune cells in mice.

Real-time PCR to detect viral expression

Because H1N1 virus is an RNA virus, its RNA needs to be extracted first and then reverse transcribed into cDNA for real-time quantitative polymerase chain reaction.

First, weigh the lung tissue and grind it with a grinding rod. Use a commercially available RNA extraction kit RNeasy (QIAGEN, Germantown, USA) for total RNA extraction. Dilute 2 μl of RNA with DEPC (diethylpyrocarbonate) water for 10 times and measure it. The absorbance values at the wavelengths of 260nm and 280nm were converted into RNA concentration and purity. Then take the quantified RNA in a PCR tube, add DEPC water to make up to 12.5 μl, and then add 1 μl Oligo (dT) primers. After reacting at 72 ° C for 2 minutes, immediately put in an ice bath, and then add 4 μl 5 times buffer RNA, 1 μl of 10 mM dNTP mixture, 1 μl of M-MLV reverse transcriptase, and 0.5 μl of RNase inhibitor, react at 42 ° C for 1 hour, and then react at 95 ° C for 5 minutes to remove the reverse transcriptase activity. Of cDNA.

Next, by taking 5μl cDNA 100-fold diluted, followed by addition of ^{12.5μl iQ TM SYBR Green ® Supermix (} Bio-Rad, Hercules, USA) 0.5μl of 10μM bidirectional and each primer, and finally sterile water was added to bring the total reaction volume of 25μl. A PCR reactor (iCycler iQ ^® Multicolor Real-Time PCR Detection System, BIO-RAD) was used to perform the polymerase chain reaction using the optimal adhesion temperature of each primer. After the reaction was completed, the expression of H1N1 nucleoprotein gene was obtained.

Referring to Fig. 11, the expression level of H1N1 nucleoprotein gene after the treatment of the aforementioned four different groups is shown. Compared with the untreated virus group after the challenge, the groups of Tamiflu and Example 5 both decreased by about 5 times and there was a significant difference. In addition, the degree of decline in Example 5 was similar to that of the Tamiflu group. From this, it can be seen that Example 5 can reduce the virus infectivity.

Paraffin-embedded sections of mouse lung tissue

The mouse lung tissue was immersed in a 10% formalin solution, and left at 4 ° C for one day, and then dehydrated with 70%, 90%, and 100% alcohol and xylene in order. After paraffin embedding, tissue sections were performed. Place the slides in a 50 ° C oven and bake the slides, then store at 4 ° C or perform tissue staining.

Hematoxylin and eosin stain (H & E stain)

Before staining, the paraffin coated tissue was dewaxed with xylene and covered with water, and then the slide was soaked in hematoxylin for 10 seconds, and then washed with running water for 15 minutes. After staining with eosin for 1 minute, rinse with running water. After the slides have dried, soak in 70%, 90%, and 100% alcohol and xylene for 5 minutes in order to dehydrate. After the slides are dried, they can be sealed and stored.

As shown in Figure 12, in the virus group that was not treated after the challenge, the range of infiltration of immune cell spheres in the surrounding alveoli was denser than that in the control group, indicating that the lung damage caused by it was more serious. Compared with the virus group, the infiltration range of the mice treated with Tamiflu and Example 5 was milder. From this, it can be seen that Example 5 can reduce lung damage caused by influenza virus.

Immunofluorescence stain

Slides were prepared using the paraffin-embedded sections, and after 5 minutes in an oven at 65 ° C, the slides were dewaxed with xylene, then washed three times with PBST (PBS-Tween 20), and then 5% bovine serum protein ( bovine serum albumin (BSA) for 1 hour. After the filling effect was completed, the cells were washed three times with PBST, and mouse anti-influenza A / WSN / 33 M protein was added, and the reaction was left at 4 ° C overnight. Wash three times the next day with PBST and add fluorescence Isothiocyanate-goat anti mouse IgG (FITC-goat anti mouse IgG) was treated for 1 hour, and then washed three times with PBST, and 1'g / ml of 4 ', 6-diamidino-2-phenylindole ( 4 ', 6-Diamidino-2-phenylindole dihydrochloride (DAPI) was used to stain the nucleus, and then the fluorescence signal of the tissue was observed with a fluorescence microscope.

Referring to FIG. 13, according to the fluorescent staining results, a large number of viruses were present in the virus group that had not been treated after the challenge. On the contrary, the fluorescent signal of Example 5 was reduced a lot, and the result was similar to that of Tamiflu. It was proved again that Example 5 can effectively reduce the amount of viral infection in lung tissues.

Mouse survival rate and weight change

The animal model was established as described above, and the test group was the control group, the virus group, the Tamiflu group, and Example 5. However, the mice were not sacrificed, but their survival rate after virus infection was observed and their body weight changes were recorded.

Referring to Figure 14 and Figure 15, the mice's survival rate and weight change during 19 days after treatment in the four groups are shown. The control group was not infected with the virus until the 19th day, and the mouse survival rate was 100%; the virus group was infected with the virus alone without any treatment, so death began to occur on the 9th day, and by the 19th day, the mouse survival rate remained only 25%; Tamiflu group began to die on day 14 and the survival rate of mice by day 19 was 40%; the group treated with compound 5 of Example 5 began to die on day 14 and reached day 19 Mouse The survival rate can reach 80%, and compared with other groups, the weight gradually returns to the original weight.

In summary, the embodiment of the present invention uses the aforementioned 22 compounds, and the medicines prepared by them can be used to treat or prevent influenza A virus. Specifically, the medicine prepared by using the embodiments of the present invention can not only reduce the amount of influenza A virus infection and the lung damage caused by it, but also 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 can better understand the aspects of the present disclosure. Those skilled in the art will appreciate that this disclosure can be readily used as a basis for designing or modifying other methods or processes for achieving the same purpose and / or achieving the same advantages of the embodiments introduced herein. Those skilled in the art should also realize that such equivalents do not violate the spirit and scope of this disclosure, and can make various changes, substitutions and alterations without departing from the spirit and scope of this disclosure.

Claims (10)

A medicinal composition for use in preparing a medicament for treating or preventing infection of an influenza A virus in an individual, wherein the medicinal composition comprises an active ingredient selected from the group consisting of 1,2,3,4-tetramethoxy-5-toluene, 1,2-dimethoxybenzene, 1,2,3-trimethoxybenzene, 2,3,4-trimethoxy-6-methylphenol, 3,4-dimethoxy-6-toluene- 1,2-diol, 3,4-dimethoxy-5-toluene-1,2-diol, 2,4-dimethoxy-6-toluene-1,3-diol, 3,6 -Dimethoxy-4-toluene-1,2-diol, 4,5-dimethoxy-7-toluo [ d ] [1,3] dioxane, 4,5-dimethoxy -6-tolyl [ d ] [1,3] dioxe, 4,7-dimethoxy-5-tolyl [ d ] [1,3] dioxe, 5-iodo-4 , 7-dimethoxy-6-toluo [ d ] [1,3] dioxane, 1-iodo-2,3,4,5-tetramethoxy-6-toluene, 1,2, 3,4-tetramethoxy-5-methyl-6- (3-methylbut3-ene-1-yn-1-yl) benzene, 4,7-dimethoxy-5-methyl- 6- (3-methylbut-3-ene-1-yn-1-yl) benzo [ d ] [1,3] dioxane, 1,2,4-trimethoxybenzene, 1-iodine -2,4,5-trimethoxybenzene, 3,4-dimethoxyphenol, 4,5-dimethoxy-2-methylphenol, 2,5-dimethoxyphenol, 2,4 -Dimethoxyphenol and its combination Into the group. The use according to item 1 of the scope of patent application, wherein the drug is capable of reducing the infection of cells with influenza A virus. The use according to item 1 of the scope of patent application, wherein the drug is capable of reducing the amount of influenza A virus in the individual's lungs. The use according to item 1 of the scope of the patent application, wherein the drug is capable of reducing lung damage caused by influenza A virus in the individual's lungs. The use according to item 1 of the scope of patent application, wherein the drug is capable of reducing the inflammatory response of cells. The use as described in item 1 of the scope of patent application, wherein the dosage of the drug is 12.5-37.5 mg per kg of body weight of the individual. The use as described in item 1 of the patent application scope, wherein the influenza A virus is H1N1, H2N2, H3N2, H5N1, H7N7, H1N2, H9N2, H7N2, H7N3, H10N7, H5N2, H5N3, H5N6, H5N8, H6N1, H7N9 Or H10N8. The use as described in item 1 of the scope of patent application, wherein the drug is capable of reducing the expression levels of interleukin (IL-6), tumor necrosis factor (TNF) and interleukin-1β (IL-1β). A method for producing a benzene compound, comprising: reacting a compound having a structure of formula (III) with dichloromethane in an environment protected by argon in the presence of aluminum chloride to form a structure having formula (I) The compound of formula (III) is: In the chemical formula (III), R ₇ and R ₈ are independently selected from the group consisting of hydrogen, methoxy, hydroxyl and methyl. The chemical formula (I) is: In this formula _{(I), R 1, R} 2, R 3 and R ₄ is independently selected from the group consisting of hydrogen Department, methyl, methoxy and hydroxyl group thereof. The method as described in item 9 of the scope of patent application, wherein R ₁ is a hydroxyl group, R ₂ is a hydroxyl group, R ₃ is a methoxy group, and R ₄ is a hydrogen or a methyl group.