Use of 2-heptyl-4-quinolinones in the preparation of a medicament for the treatment of human adenovirus

Abstract

The application provides application of 2-heptyl-4-quinolinone in preparation of a medicine for resisting human adenovirus, 2-heptyl-4-quinolinone can significantly inhibit human adenovirus infection and replication, and has no great influence on survival of normal cells, and shows good selectivity and safety, and provides a new scheme for treatment of human adenovirus infection; the application also provides a medicine for treating or preventing human adenovirus infection, the medicine comprises 2-heptyl-4-quinolinone or a derivative thereof, and an effective concentration of CYC-116 in the 2-heptyl-4-quinolinone or the derivative thereof is not less than 0.3uM; and the application provides a new medicine for resisting human adenovirus.

Description

Technical Field

[0001] This invention relates to the application of 2-heptayl-4-quinolinone in the preparation of drugs against human adenovirus, and belongs to the field of biomedical technology. Background Technology

[0002] Despite rapid advancements in medical technology, acute respiratory infections (ARIs) remain a leading threat to human health, maintaining a high mortality rate. Among hospitalized patients, approximately 2% of ARI cases are caused by human adenovirus (HAdV) infection, and HAdV is transmissible year-round. Its high transmissibility often leads to community outbreaks, with the warming spring season typically being a high-risk period for adenovirus infection.

[0003] In addition, adenovirus is a multi-organ tropism double-stranded DNA virus that can invade the respiratory, digestive, eye, urinary, heart, brain, liver and other tissues throughout the body. Most immune-normal people experience mild and self-limiting symptoms, but infants, newborns and immunocompromised individuals are extremely prone to severe illness, permanent damage or even death, and it is highly contagious and prone to outbreaks.

[0004] However, there are currently no drugs available that specifically inhibit HAdV infection and replication in clinical practice. In actual clinical treatment, healthcare professionals can only treat complications arising from HAdV infection, and cannot address the root cause of the viral infection. Therefore, developing a drug that can specifically inhibit HAdV infection or replication is of great practical value. Summary of the Invention

[0005] The purpose of this invention is to discover for the first time the application of 2-hepta-4-quinolinone and its derivatives in the preparation of drugs against human adenovirus. 2-hepta-4-quinolinone can significantly inhibit human adenovirus infection and replication, while having little effect on the survival of normal cells, showing good selectivity and safety, and providing a new solution for the treatment of human adenovirus infection.

[0006] To achieve this objective, the present invention provides the following technical solution:

[0007] In a first aspect, the invention provides the use of 2-heptyl-4-quinolinone and its derivatives in the preparation of medicaments against human adenoviruses.

[0008] In this invention, 2-heptayl-4-quinolinone is abbreviated as HHQ, with CAS number 40522-46-1, and its structural formula is as follows:

[0009] .

[0010] Preferably, the anti-human adenovirus is used to inhibit the replication of human adenovirus.

[0011] Preferably, the effective concentration of 2-heptyl-4-quinolinone in the 2-heptyl-4-quinolinone and its derivatives is not less than 0.3 μM.

[0012] Preferably, the 2-heptyl-4-quinolinone and its derivatives are in the form of tautomers, meso compounds, racemates, enantiomers, diastereomers, or mixtures thereof.

[0013] Preferably, the 2-heptyl-4-quinolinone and its derivatives further include pharmaceutically acceptable salts, ethers, esters, prodrugs, metabolites, solvates, or crystals thereof.

[0014] Preferably, the dosage form of the drug includes, but is not limited to, tablets, capsules, oral solutions, pills, granules, powders, aerosols, patches, ointments, liniments, suppositories, and / or injections.

[0015] In a second aspect, the present invention provides a medicament for treating or preventing human adenovirus infection, the medicament comprising 2-heptayl-4-quinolinone or a derivative thereof, wherein the effective concentration of CYC-116 in the 2-heptayl-4-quinolinone or its derivative is not less than 0.3 μM.

[0016] Preferably, the drug further includes pharmaceutically acceptable salts, ethers, esters, prodrugs, metabolites, solvates, or crystals thereof.

[0017] Preferably, the dosage form of the drug includes, but is not limited to, tablets, capsules, oral solutions, pills, granules, powders, aerosols, patches, ointments, liniments, suppositories, and / or injections.

[0018] Preferably, the 2-heptyl-4-quinolinone and its derivatives are in the form of tautomers, meso compounds, racemates, enantiomers, diastereomers, or mixtures thereof.

[0019] Compared with existing technologies, the beneficial effects and significant advancements of applying the technical solution of this invention are as follows: This invention demonstrates for the first time that 2-heptayl-4-quinolinone (HHQ) can inhibit human adenovirus infection and replication, while clarifying its EC50 value and its lack of significant toxicological effects on cells. This provides a new method for the treatment and research of human adenovirus infection and replication. Attached Figure Description

[0020] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the embodiments of the present invention will be briefly described below.

[0021] Figure 1The graphs are of fluorescence intensity and viral copy number in Example 1, where the left graph is a Gaussian fluorescence intensity graph and the right graph is a viral copy number graph.

[0022] Figure 2 This is a graph showing the effect of different concentrations of HHQ on the replication of HAdV-Gluc in cells, as described in Example 2.

[0023] Figure 3 This is a graph showing the effect of different concentrations of HHQ on cell viability in Example 2. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight. Unless otherwise specified, the experimental materials and reagents used in the following embodiments are commercially available.

[0025] Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. It should be noted that the terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the exemplary embodiments of this application.

[0026] To better understand this invention, the technical terms used in this invention will be explained below.

[0027] HEK-293A cells are human embryonic kidney cells. The original HEK293 was obtained by transforming human embryonic kidney cells with adenovirus type 5 DNA; 293A is a subclone of the 293 parent, A = Adhesion (strong adhesion), and is a monoclonal strain that is specifically selected for its strong adhesion and flattened morphology.

[0028] HAdV-Gluc is a recombinant human adenovirus. HAdV = Human Adenovirus (most commonly the HAdV-C5 / Ad5 replication-defective ΔE1 / ΔE3 vector). Gluc = Gaussian princeps Luciferase, a secretory reporter gene. Full name: Replication-defective human adenovirus type 5 expressing secretory Gaussian luciferase, often abbreviated as Ad-Gluc.

[0029] Infection is the process by which exogenous pathogens such as viruses and bacteria actively invade host cells, utilize intracellular raw materials, enzyme systems, and organelles to complete their own gene replication and protein expression, and simultaneously introduce exogenous genes (such as Gluc reporter genes) into host cells.

[0030] Viral replication is the complete process by which a virus, after invading a host cell, hijacks the host cell's nucleotides, amino acids, enzymes, ribosomes, and other organelles to sequentially complete viral genome replication, viral structural protein synthesis, and the assembly and release of progeny viral particles. Viruses do not have an independent metabolic system and must rely on living host cells to complete replication.

[0031] Effective drug concentration refers to the minimum working concentration range of a drug required to achieve a predetermined biological effect (such as inhibiting viral replication, reducing HAdV-Gluc fluorescence signal, alleviating cytopathic effects (CPE), and inhibiting cell proliferation) in an in vitro cell culture system or in vivo environment.

[0032] Tautomers are two functional group isomers that rapidly interconvert due to the rapid migration of protons within a molecule and the accompanying displacement of double bonds; they belong to structural isomers.

[0033] A meso compound is a single pure compound containing multiple chiral carbon atoms, but the molecule as a whole has a plane of symmetry / center of symmetry. The optical rotations within the molecule cancel each other out, resulting in macroscopically optically inactive compounds.

[0034] Racemic mixture, a mixture of equal amounts of left-handed and right-handed enantiomers.

[0035] Enantiomers are two chiral molecules that are mirror images of each other and cannot be superimposed.

[0036] Diastereomers are stereoisomers of molecules containing two or more chiral centers that are not mirror images of each other and cannot overlap.

[0037] Gaussian luciferase (Gluc) is a secretory luciferase that is released directly into the cell supernatant without cell lysis. Its substrate is coelenterazine, and it catalyzes the emission of light from the substrate under aerobic conditions. The emission intensity is directly proportional to the activity of the Gluc enzyme in the supernatant, and the instrument reads the RLU (Relative Light Unit), or Luc value. This differs from firefly luciferase (Fluc, which requires lysis and a luciferin substrate): Gluc can be detected simply by sampling the supernatant, making it suitable for dynamic time-series sampling.

[0038] μM = micromolar, micromoles per liter.

[0039] Example 1: A high-throughput screening system capable of rapidly screening inhibitory drugs was constructed.

[0040] Experimental methods

[0041] By modifying the HAdV virus and inserting a Gaussian luciferase gene into its genome, a high-throughput screening system capable of rapidly screening inhibitory drugs was constructed. The specific method is as follows:

[0042] 1.1 Using molecular biology, the Gaussian luciferase gene with a total length of 555 bp was successfully inserted into the E3 protein gene of the HADV virus genome, and an infectious plasmid containing the complete HADV virus genome was obtained.

[0043] 1.2 Transfect the plasmid into HEK-293A cells to mimic the process of genome injection during viral infection, allowing it to self-assemble into infectious viral particles (HAdV-Gluc) within the cells.

[0044] 1.3. Viral replication was detected by detecting the expression of Gaussia-Lumi.

[0045] Experimental results

[0046] The results are as follows Figure 1 As shown, the constructed virus exhibits good replication ability. This demonstrates that this embodiment successfully constructed a high-throughput screening system capable of rapidly screening inhibitory drugs.

[0047] Example 2: HHQ significantly inhibits the replication of HAdV-Gluc in cells.

[0048] Experimental methods

[0049] 2.1. Dilute HHQ small molecule drug with cell culture medium to concentrations of 100 μM, 20 μM, 4 μM, 0.8 μM, 0.16 μM, 0.032 μM, 0.0064 μM, 0.00128 μM, 0.000256 μM, 0.0000512 μM, and 0 μM (as a control).

[0050] 2.2. Seed virus-free HEK-293A cells (2 × 10⁶ cells per well) into 96-well cell culture plates. 4 After the cells reached 90% confluency, they were infected with the HADV-Gluc virus constructed in Example 1 at an MOI of 0.01 (i.e., 50 μl of diluted virus was added to each well). After 1 hour of infection, HHQ small molecule drug was added (i.e., 50 μl of diluted drug was added to each well), and the cells were cultured in a cell culture incubator.

[0051] 2.3 After 24 hours, remove the 96-well cell culture plate. For the virus-infected cell culture plate, use a Gaussian fluorescein assay kit to detect Luciferase levels. The standard detection procedure (general kit procedure) includes: a) Preparing the substrate working solution: Dilute coelenterate to the working concentration according to the instructions. Protect from light and operate on ice throughout the process, as the substrate is easily inactivated by light; b) Adding samples: Add supernatant to each well of the white luminescent plate; set up wells for blank culture medium, negative cell supernatant, and gradient standards; c) Adding the luminescent substrate: Quickly add an equal volume of substrate working solution to each well, gently pipetting to mix, avoiding air bubbles (air bubbles will significantly lower the RLU value); d) Incubating to stabilize the luminescence signal: Incubate at room temperature in the dark for 2–10 min (the luminescence stability window varies between kits; the incubation time for all samples must be consistent); e) Reading the Luciferase value using a microplate reader: Parameter settings: Chemiluminescence mode, integration time 0.5–2 s / well; reading unit: RLU (Relative Light Units).

[0052] 2.4 Statistical analysis: Plot the curve of HHQ inhibiting HADV-Gluc virus replication and calculate EC50.

[0053] Experimental results

[0054] The results are as follows Figure 2 As shown, HHQ small molecule drug can inhibit the replication of HAdV-Gluc virus, with an EC50 of 0.3 μM.

[0055] Example 3: HHQ showed no significant toxic side effects on cells within its effective concentration range.

[0056] Experimental methods

[0057] 3.1 Dilute HHQ small molecule drug with cell culture medium to concentrations of 0.8 μM, 0.16 μM, 0.032 μM, 0.0064 μM, 0.00128 μM, 0.000256 μM, 0.0000512 μM, and 0 μM (as controls).

[0058] 3.2. Seed uninfected HEK-293A cells (2 × 10⁶ cells per well) into 96-well cell culture plates. 4 Once the cells reached 90% confluence, HHQ small molecule drug was added to the uninfected HEK-293A cell culture plate, i.e., 50 μl of diluted drug was added to each well, and the cells were cultured in a cell incubator.

[0059] 3.3 After 24 hours, remove the 96-well cell culture plates. Cell viability of the virus-free cell culture plates was assessed using the CCK8 cell viability assay kit. Standard operating procedures included: a) Plate seeding, cell digestion and resuspending, adjusting density, adding 100 μL of cell suspension to each of the 96 wells; adding PBS to the edge wells to prevent evaporation. Incubate overnight; b) Drug treatment, discarding the old culture medium and adding complete culture medium containing gradient concentrations of HHQ; adding an equal volume of solvent-containing culture medium to the control group (solvent control, excluding toxicity such as DMSO); adding only culture medium to the blank wells. c) Incubate for 24 / 48 / 72 h according to the experimental design; d) Add CCK-8 working solution, directly add 10 μL of CCK-8 solution (medium:CCK8 = 10:1) to each well, gently shake to mix, avoid generating air bubbles, and do not vigorously blow or pipette to prevent cell detachment; operate in the dark throughout the process; e) Incubate for color development at 37 ℃ in the dark for 1–4 h (adjust according to the cell proliferation rate); for a large number of cells and high viability, color development is faster, so it is recommended to uniformly incubate for the same duration; f) Read the values ​​using a microplate reader, with a detection wavelength of 450 nm and a reference wavelength of 630 nm; record the OD of each well. 450 .

[0060] 3.4 Statistical analysis: The results of statistical analysis on the effects of different concentrations of HHQ on cell viability.

[0061] Experimental results

[0062] The results are as follows Figure 3 As shown, HHQ has no obvious toxic side effects on cells within the effective concentration range.

[0063] The applicant declares that, in the process of describing the above-mentioned specification:

[0064] The terms "this embodiment," "an embodiment of the present invention," "as shown," "further," and "further improved technical solutions," etc., indicate that the specific features, structures, materials, or characteristics described in the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms are not necessarily directed at the same embodiment or example, and the specific features, structures, materials, or characteristics described can be combined or combined in any suitable manner in one or more embodiments or examples. Furthermore, without causing contradiction, those skilled in the art can combine or combine the different embodiments or examples described in this specification and the features of the different embodiments or examples.

[0065] Finally, it should be noted that:

[0066] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them;

[0067] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention. Non-essential improvements, adjustments or substitutions made by those skilled in the art based on the content of this specification are all within the scope of protection claimed by the present invention.

Claims

1. 2-Heptyl-4-quinolinone and its derivatives in the preparation of drugs against human adenovirus.

2. The application according to claim 1, characterized in that, The anti-human adenovirus is used to inhibit the replication of human adenovirus.

3. The application according to claim 1, characterized in that, The effective concentration of 2-heptyl-4-quinolinone in the 2-heptyl-4-quinolinone and its derivatives is not less than 0.3 μM.

4. The application according to claim 1, characterized in that, The 2-heptyl-4-quinolinone and its derivatives are in the form of tautomers, meso compounds, racemates, enantiomers, diastereomers, or mixtures thereof.

5. The application according to claim 1 or 5, characterized in that, The 2-heptyl-4-quinolinone and its derivatives also include pharmaceutically acceptable salts, ethers, esters, prodrugs, metabolites, solvates, or crystals thereof.

6. The application according to claim 1, characterized in that, The dosage forms of the drug include, but are not limited to, tablets, capsules, oral solutions, pills, granules, powders, aerosols, patches, ointments, liniments, suppositories, and / or injections.

7. A drug for treating or preventing human adenovirus infection, characterized in that, The drug contains 2-heptayl-4-quinolinone or a derivative thereof, and the effective concentration of CYC-116 in the 2-heptayl-4-quinolinone or its derivative is not less than 0.3 μM.

8. The medicament for treating or preventing human adenovirus infection according to claim 7, characterized in that, The drug also includes its pharmaceutically acceptable salts, ethers, esters, prodrugs, metabolites, solvates or crystals thereof.

9. The medicament for treating or preventing human adenovirus infection according to claim 7, characterized in that, The dosage forms of the drug include, but are not limited to, tablets, capsules, oral solutions, pills, granules, powders, aerosols, patches, ointments, liniments, suppositories, and / or injections.

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