Application of aminopterin and structural analogue thereof in treatment of influenza virus infection

Abstract

The invention provides application of aminopterin and a structural analogue thereof in preparation of a medicine for treating influenza virus infection. The aminopterin and the structural analogue thereof are a compound as shown in a formula I and pharmaceutically acceptable salt thereof. The compound shown in the formula I and the pharmaceutically acceptable salt of the compound have a good inhibition effect on influenza viruses. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to the application of aminopterin and its structural analogs in the preparation of medicines for treating influenza virus infection. Background Art

[0002] Influenza A virus (IAV) is an enveloped, single-stranded, negative-sense RNA virus belonging to the family Orthomyxoviridae. Its genome consists of eight segments encoding at least 12 proteins, including polymerase acid protein (PA), polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2), hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), matrix protein (M), and nonstructural proteins (NS). IAV can be divided into different subtypes based on the antigenicity of HA and NA. So far, 18 HA and 11 NA subtypes have been discovered, of which H1, H5, and H7 subtypes are highly pathogenic, and H1N1 and H3N2 subtypes of IAV have adapted to humans and caused seasonal epidemics.

[0003] IAV hosts are widely distributed, mainly infecting mammals and poultry. They can break through interspecies barriers, have strong variability and high infectivity. Nowadays, influenza viruses can be effectively prevented by using anti-influenza drugs and vaccination. However, due to the characteristics of antigenic drift and antigenic shift, when new variants of influenza viruses appear, the existing influenza vaccines are affected by their "timeliness" and are difficult to effectively inhibit the spread of the virus. The effectiveness of the vaccine will also vary depending on the year, research population and strain. The effectiveness of the vaccine is reduced due to factors such as vaccine mismatch, influenza immunity of the vaccinated population, age, weight, physiological sex and immune status. Therefore, other alternatives are needed to fill the gaps in preventive and therapeutic interventions caused by vaccine-related problems. Anti-influenza A virus drugs are key tools against influenza viruses. M2 ion channel inhibitors and NA inhibitors are the two earliest drugs approved by the FDA to treat influenza viruses, but IAV has a relatively high resistance to M2 ion channel inhibitors. Secondly, different drugs have different toxic side effects for different patients, so there is an urgent need to develop more alternative drugs.

[0004] Aminopterin has anticancer and immunosuppressive activities and is often used to study childhood leukemia. Methotrexate was developed on the basis of aminopterin and has anti-metabolic, anti-tumor and immunosuppressive activities. It is often used in the study of rheumatoid arthritis and various tumors. Pralatrexate is designed based on the structural modification of methotrexate. It can selectively accumulate in tumor cells to block the synthesis of purine and pyrimidine. It has anti-tumor activity and is widely used to treat relapsed or refractory peripheral T-cell lymphoma. Methotrexate and pralatrexate are both aminopterin structural analogs. No studies have shown that this type of compound can be used to treat influenza virus infection. Summary of the invention

[0005] The present invention provides the use of a compound shown in the following formula I and a pharmaceutically acceptable salt thereof in the preparation of an influenza virus inhibitor;

[0006]

[0007] Wherein, X is selected from -NH-, -N(C 1-6 Alkyl)-, -N(C 2-6 Alkynyl)-, -N(C 2-6 alkenyl)-, -CH(C 1-6 Alkyl)-, -CH(C 2-6 Alkynyl)-, -CH(C 2-6 alkenyl)- or -N(CHO)-.

[0008] According to an embodiment of the present invention, X is selected from -NH-, -N(C 1-3 Alkyl)-, -N(C 2-3 Alkynyl)-, -N(C 2-3 alkenyl)-, -CH(C 1-3 Alkyl)-, -CH(C 2-3 Alkynyl)-, -CH(C 2-3 alkenyl)- or -N(CHO)-.

[0009] According to an embodiment of the present invention, X is selected from -NH-, -N(vinyl)-, -N(propenyl)-, -CH(ethynyl)-, -CH(propynyl)-, -N(CHO)-, -N(methyl)-, -N(ethyl)-, -N(propyl)- or -N(2,2-methylpropyl)-.

[0010] According to an embodiment of the present invention, the compound represented by formula I also includes its stereoisomers and racemates.

[0011] According to an embodiment of the present invention, the compound represented by Formula I is selected from aminopterin, methotrexate, pralatrexate, or a compound as shown below:

[0012]

[0013] According to an embodiment of the present invention, the influenza is influenza A or influenza B.

[0014] According to an embodiment of the present invention, the influenza A includes 18 HA and 11 NA subtypes.

[0015] According to an embodiment of the present invention, the pharmaceutically acceptable salt is selected from the acid addition salt of the nitrogen atom having sufficient basicity in the structure of the compound shown in Formula I, and the acid addition salt includes the compound shown in Formula I and an inorganic acid such as hydrochloric acid, hydrofluoric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, pyrosulfuric acid, phosphoric acid or nitric acid; an organic acid such as formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, hexanoic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2-(4-hydroxybenzoyl)benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, digluconic acid, 3-hydroxy- Salts formed from 2-naphthoic acid, nicotinic acid, pamonic acid, pectinic acid, persulfuric acid, 3-phenylpropionic acid, picric acid, pivalic acid, 2-hydroxyethanesulfonic acid, itaconic acid, sulfamic acid, trifluoromethanesulfonic acid, dodecylsulfuric acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, adipic acid, alginic acid, maleic acid, fumaric acid, D-gluconic acid, mandelic acid, ascorbic acid, glucoheptanoic acid, glycerophosphoric acid, aspartic acid, sulfosalicylic acid, hemisulfuric acid or thiocyanic acid;

[0016] Alternatively, the compound of formula I may have a base addition salt of a carboxyl group with sufficient acidity, such as an alkali metal salt, alkaline earth metal salt or ammonium salt of the compound of formula I.

[0017] According to an embodiment of the present invention, the influenza virus inhibitor can inhibit the activity of influenza virus, thereby achieving the purpose of alleviating or treating influenza virus infection.

[0018] According to an embodiment of the present invention, the activity of inhibiting influenza virus includes: inhibiting the replication of influenza A virus, and / or inhibiting the effect of influenza A virus after entering the cell.

[0019] In the present invention, influenza virus infection includes mild, moderate and severe infections caused by influenza A virus, including but not limited to respiratory tract infection, gastrointestinal tract infection and nervous system damage.

[0020] In some specific embodiments of the present invention, the influenza A virus is an H1N1 influenza A virus in the H1 subtype.

[0021] The present invention also provides a method for alleviating and / or treating influenza virus infection, comprising: administering at least one of the compounds represented by Formula I and pharmaceutically acceptable salts thereof to an individual in need thereof.

[0022] Beneficial Effects

[0023] The compound represented by formula I of the present invention has a good inhibitory effect on influenza virus. For example, different concentrations of aminopterin, methotrexate and pralatrexate inhibit H1N1 infection of MDCK cells in a dose-dependent manner, and their half effective concentration EC 50 They are 32.45nM, 12.03nM, and 39.79nM respectively. SI is the therapeutic index, which is equal to CC 50 At EC 50 The ratio of the three drugs combined CC 50 With IC 50 The results showed that the SI of aminopterin was >308.17, the SI of methotrexate was >2078.14, and the SI of pralatrexate was >100.53.

[0024] In addition, the results that methotrexate inhibited the expression of H1N1 influenza A virus NP and PA proteins indicated that methotrexate could treat influenza A virus infection, which strongly demonstrated the potential anti-influenza virus activity of methotrexate. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The cytotoxicity of aminopterin, methotrexate and pralatrexate at different concentrations on MDCK cells is shown.

[0026] Figure 2 The results show that different concentrations of aminopterin, methotrexate and pralatrexate inhibited H1N1 infection in MDCK cells in a dose-dependent manner.

[0027] Figure 3 It was shown that at the protein level, methotrexate inhibited the expression of NP and PA proteins of H1N1. DETAILED DESCRIPTION

[0028] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope of protection intended by the present invention.

[0029] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0030] Example 1

[0031] 1. Cell virus culture

[0032] Canine kidney epithelial cells (Madin-darby canine kidney, MDCK) were purchased from the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, and cultured in a 37°C, 5% CO2 incubator using DMEM high-glucose medium (Cytiva) containing 10% fetal bovine serum (FBS, PAN) and 1% double antibiotics (penicillin and streptomycin).

[0033] H1N1 influenza A virus was purchased from the American Type Culture Collection (ATCC, VR-1469). H1N1 (A / PR / 8 / 34, ATCC, VR-1469) was propagated in MDCK cells and the TCID 50 Virus titers were determined by the method of All infection experiments were performed in a biosafety level 2 (BLS2) laboratory.

[0034] 2. Cytotoxicity of different concentrations of aminopterin, methotrexate and pralatrexate on MDCK cells

[0035] Cell Counting Kit-8 (CCK-8) was used to detect the cytotoxicity of aminopterin, methotrexate and pralatrexate.

[0036] (1) MDCK cells were inoculated into a 96-well cell culture plate. When the cell density reached 80% to 90%, the cells were washed twice with PBS buffer;

[0037] (2) Use PBS + 0.2% BSA as a diluent to dilute the drugs ten-fold or two-fold, and dilute aminopterin, methotrexate, and pralatrexate to the required concentrations. Dilute aminopterin ten-fold, eight dilutions, with a concentration range of 0.01nM to 100μM. Dilute methotrexate two-fold, thirteen dilutions, with a concentration range of 0.006μM to 25μM. Dilute pralatrexate two-fold, nine dilutions, with a concentration range of 15.625nM to 4μM. Inoculate 2 wells for each concentration;

[0038] (3) A cell control group without drug and a blank control group without drug and cells were set up. An equal volume of diluent was added to the cell control group and incubated for 1 h;

[0039] (4) Prepare the growth medium, which is DMEM high-glucose medium containing 1% double antibody (penicillin and streptomycin), 2 μg / mL TPCK-trypsin, and 0.2% BSA. After the incubation, wash the cells once with PBS buffer;

[0040] (5) Add drug-containing growth solution diluted with growth solution to the wells at the same concentration as during incubation, and add an equal volume of growth solution to the cell control group;

[0041] (6) Place the 96-well cell culture plate in an incubator at 37°C and 5% CO2 for 24 h;

[0042] (7) After the incubation, the liquid in the well plate was discarded, and the plate was washed with PBS buffer. DMEM high-glucose medium containing 10% CCK-8 solution was added, and the 96-well plate was placed in an incubator in the dark for 1.5 h.

[0043] (8) Detect the luminescence intensity at 450 nm and calculate the inhibition rate. The formula is:

[0044] Inhibition rate (%) = [OD (0 drug addition) - OD (drug addition)] ÷ [OD (0 drug addition) - OD (blank)] × 100%.

[0045] Finally, GraphPad-Prism 8 software was used for data analysis and CC calculation. 50 .

[0046] The results are as follows Figure 1 As shown in Figure 2, aminopterin below 10 μM, methotrexate below 25 μM, and pralatrexate below 4 μM had no significant cytotoxicity to MDCK cells, all less than 50%. Therefore, the cell half-toxic concentrations of the three drugs were CC. 50 CC 50 >10μM、CC 50 >25μM、CC 50 >4μM.

[0047] Example 2

[0048] Aminopterin, methotrexate and pralatrexate inhibit EC of H1N1 influenza A virus 50 Determination

[0049] (1) 2.5×10 4 MDCK cells were inoculated into a sterile 96-well plate and cultured in a 37°C, 5% CO2 incubator for 18-24 h;

[0050] (2) Dissolve the three compounds in DMSO to make 10 mM stock solutions and store them in a -80°C ultra-low temperature refrigerator for later use;

[0051] (3) When the cell density reaches 80% to 90%, discard the culture medium in the 96-well plate and wash each well twice with PBS buffer;

[0052] (4) Use PBS + 0.2% BSA as diluent to dilute the three drugs to twice the concentration required in Example 1.

[0053] (5) Dilute the H1N1 influenza A virus solution to the desired concentration (1000 TCID 50 / mL), mix the drugs of different concentrations with the diluted virus solution in equal volumes, and set up a virus control group with only virus added and a cell control group without virus and drugs added;

[0054] (6) Add 50 μL of the corresponding prepared virus-drug mixture, and add an equal volume of the virus control group to a final concentration of 1000 TCID 50 / mL virus solution, and the cell control group was added with an equal volume of PBS + 0.2% BSA diluent. Finally, the 96-well plate was placed in an incubator at 37°C and 5% CO2 for 1 hour;

[0055] (7) After the incubation, the drug was diluted to the final concentration required in Example 1 using the growth medium in Example 1, the 96-well plate was washed once with PBS buffer, and then the growth medium containing only the drug was added. The control group was added with only an equal volume of growth medium, and the cells were cultured in an incubator at 37° C. and 5% CO 2 .

[0056] (8) Observe the cell lesions under a microscope 24 hours after virus infection. Collect the supernatant samples when obvious lesions appear in the virus control group;

[0057] (9) Extract viral RNA from the supernatant and perform quantitative analysis using RT-PCR and qRT-PCR. The inhibition rate is calculated as follows:

[0058] Inhibition rate (%) = (virus control copy number - sample copy number) ÷ virus control copy number × 100%

[0059] (10) GraphPad-Prism 8 software was used for data analysis and calculation of EC 50 .

[0060] The results are as follows Figure 2 As shown in the results of the half effective concentration experiment, different concentrations of aminopterin, methotrexate and pralatrexate showed a dose-dependent effect on the inhibition of H1N1 infection in MDCK cells. 50 They are 32.45nM, 12.03nM, and 39.79nM respectively. SI is the therapeutic index, which is equal to CC 50 At EC 50 The ratio of the three drugs combined CC 50 With IC 50 The results show that the SI of aminopterin is greater than 308.17, the SI of methotrexate is greater than 2078.14, and the SI of pralatrexate is greater than 100.53. The test results show that the compounds aminopterin, methotrexate and pralatrexate have the effect of resisting influenza A virus infection.

[0061] CC 50 It is the cell half-toxic concentration, which is the drug concentration that causes 50% of the cells to change in morphology. The higher the value, the lower the toxicity of the drug to cells.

[0062] EC 50 It is the half effective concentration, which means the drug concentration that reduces the virus infection rate by 50%. The smaller the value, the better the drug's inhibitory effect on the virus.

[0063] SI is the therapeutic index, which is equal to CC 50 With EC 50 The ratio is used to evaluate the therapeutic potential of a drug. The larger the value, the higher the possibility of drug development and the stronger the antiviral effect of the drug.

[0064] Example 3

[0065] Experimental study on the inhibition of expression of NP and PA proteins of H1N1 influenza A virus by methotrexate

[0066] (1) 1.5×10 5 MDCK cells were cultured for 18 to 24 hours, and then the 24-well plate was washed twice with PBS buffer; the experiment was divided into cell control wells (CC wells), virus control wells (VC wells), positive drug baloxavir (Baloxavir, BXA) control wells (VC+BXA wells) and two drug-treated wells (VC+methotrexate wells). The final drug concentrations in the drug-treated wells were 1μM and 5μM, respectively, the final concentration of baloxavir in the positive drug control wells was 0.1μM, and the virus infection dose was 200TCID 50 / mL. The difference between the cell control wells and the drug control wells is that no virus and drug solution are added, and the difference between the virus control wells and the drug control wells is that no drug is added.

[0067] (2) Place the 24-well plate in an incubator and incubate for 1 hour. After 1 hour, wash the 24-well plate with PBS buffer, add 500 μL of growth solution to the CC well and VC well, and add growth solution containing the corresponding drug concentration to the remaining drug wells;

[0068] (3) After 24 hours, cells were collected for western blot. The growth medium was aspirated, and each well was washed with 500 μL PBS buffer, and then the buffer was aspirated; 200 μL lysis buffer (180 μL RIPA lysis buffer + 10 μL DMSF + 10 μL Protease inhibitor cocktail) was added to each well, and lysed at 4°C for 10 minutes, shaking every 2 minutes to allow all cells to detach from the cell culture plate.

[0069] (4) Aspirate the cell lysate and centrifuge at 4°C, 12,000 rpm for 10 min.

[0070] (5) Take 120 μL of the supernatant and prepare the sample for SDS PAGE gel (electrophoresis conditions: 150 V, 40-50 min. Sample loading: add 5 μL of protein marker to the first well, and add other samples to the subsequent wells in sequence).

[0071] (6) After electrophoresis is complete, remove the gel, cut off the portion above the protein marker, and place in pure water.

[0072] (7) Transfer: Place the electrotransfer tank in an ice-water mixture for precooling. Cut the membrane and activate it with methanol for 30 seconds. Place the nitrocellulose membrane and gel in the electrotransfer solution and then transfer the membrane (transfer clip with white side down → a sponge → two filter papers → nitrocellulose membrane → gel on the front → two filter papers → a sponge → transfer clip), constant current 200 mA, transfer for 1.5 hours;

[0073] (8) After the transfer, the nitrocellulose membrane was washed briefly with TBST and then blocked with 5% skim milk on a shaker at room temperature for 0.5 h.

[0074] (9) After blocking, discard the milk, wash with TBST, add the primary antibody (NP antibody or PA antibody), and incubate on a shaker at room temperature for 2 h;

[0075] (10) After the primary antibody incubation, the membrane was placed in a small box and washed three times with TBST, each time for 5 minutes. Then, the secondary antibody was added in the same way as the primary antibody incubation, and incubated at room temperature for 1 hour;

[0076] (11) Color development: Wash with TBST three times, 5 min each time. Then place the membrane in a dark folder and add the freshly prepared luminescent solution (mix solution A and solution B in a volume ratio of 1:1, and prepare 1.2 mL of luminescent solution for each membrane). Be careful to add the solution evenly, use luminescent imaging, and perform color development in a dark room.

[0077] The results are as follows Figure 3 As shown, methotrexate effectively inhibited the expression of NP and PA proteins of H1N1. This indicates that methotrexate can treat influenza A virus infection, which strongly proves the potential anti-influenza virus activity of methotrexate.

[0078] The above is a detailed introduction to the technical solution provided by the present application. The description of the above embodiments is only used to help understand the method and core idea of ​​the present application; at the same time, for those skilled in the art, according to the idea of ​​the present application, there will be changes in the specific implementation method and application scope. In summary, the content of this specification should not be understood as limiting the present application.

Claims

1. Use of the compound represented by formula I and its pharmaceutically acceptable salt in the preparation of influenza virus inhibitors; in, X is selected from -NH-, -N(C 1-6 Alkyl)-, -N(C 2-6 Alkynyl)-, -N(C 2-6 alkenyl)-, -CH(C 1-6 Alkyl)-, -CH(C 2-6 Alkynyl)-, -CH(C 2-6 alkenyl)- or -N(CHO)-.

2. The use according to claim 1, wherein X is selected from -NH-, -N(C 1-3 Alkyl)-, -N(C 2-3 Alkynyl)-, -N(C 2-3 alkenyl)-, -CH(C 1-3 Alkyl)-, -CH(C 2-3 Alkynyl)-, -CH(C 2-3 alkenyl)- or -N(CHO)-; Preferably, X is selected from -NH-, -N(vinyl)-, -N(propenyl)-, -CH(ethynyl)-, -CH(propynyl)-, -N(CHO)-, -N(methyl)-, -N(ethyl)-, -N(propyl)- or -N(2,2-methylpropyl)-.

3. The use according to claim 1 or 2, wherein The compound represented by formula I is selected from aminopterin, methotrexate, pralatrexate, or the compound shown below:

4. The use according to any one of claims 1 to 3, wherein The influenza is influenza A or influenza B.

5. The use according to any one of claims 1 to 4, wherein The influenza A includes 18 HA and 11 NA subtypes.

6. The use according to any one of claims 1 to 5, wherein The pharmaceutically acceptable salt is selected from the acid addition salt of the nitrogen atom with sufficient basicity in the structure of the compound shown in Formula I, and the acid addition salt includes the compound shown in Formula I and an inorganic acid such as hydrochloric acid, hydrofluoric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, pyrosulfuric acid, phosphoric acid or nitric acid; an organic acid such as formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, hexanoic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2-(4-hydroxybenzoyl)benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, digluconic acid, 3-hydroxy-2-naphthoic acid, Salts formed from nicotinic acid, pamoic acid, pectinic acid, persulfuric acid, 3-phenylpropionic acid, picric acid, pivalic acid, 2-hydroxyethanesulfonic acid, itaconic acid, sulfamic acid, trifluoromethanesulfonic acid, dodecylsulfuric acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, adipic acid, alginic acid, maleic acid, fumaric acid, D-gluconic acid, mandelic acid, ascorbic acid, glucoheptanoic acid, glycerophosphoric acid, aspartic acid, sulfosalicylic acid, hemisulfuric acid or thiocyanic acid; Alternatively, the compound of formula I may have a base addition salt of a carboxyl group with sufficient acidity, such as an alkali metal salt, alkaline earth metal salt or ammonium salt of the compound of formula I.

7. The use according to any one of claims 1 to 6, wherein The influenza virus inhibitor can inhibit the activity of influenza virus, thereby achieving the purpose of alleviating or treating influenza virus infection.

8. The use according to any one of claims 1 to 7, wherein The activity of inhibiting influenza virus includes: inhibiting the replication of influenza A virus, and / or inhibiting the effect of influenza A virus after entering the cell.

9. The use according to any one of claims 1 to 8, wherein Influenza virus infection includes mild, moderate and severe infections caused by influenza A virus, including but not limited to respiratory tract infections, gastrointestinal infections and nervous system damage.

10. The use according to any one of claims 1 to 9, wherein The influenza A virus is an H1N1 influenza A virus in the H1 subtype.

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