Novel purine derivative and use thereof

Abstract

The present invention relates to a novel purine derivative compound and a composition for enhancing an immune response comprising same as an active ingredient. The compound of the present invention not only has a nanomolar EC50 value for TLR7, which is an intracellular membrane receptor of immune cells, but also has high selectivity for TLR7 compared to TLR8, which has a similar structure and is mainly distributed in the endoplasmic reticulum (ER), thereby being able to induce sustained immune activation. Therefore, the compound of the present invention can be effectively used as an efficient vaccine adjuvant composition against various RNA viruses including influenza virus, SARS-CoV-2, and hepatitis C virus.

Description

Novel purine derivatives and uses thereof

[0001] The present invention relates to a novel purine derivative compound having TLR7 agonist activity and a vaccine adjuvant composition comprising the same as an active ingredient.

[0002]

[0003] Toll-like receptors (TLRs) are transmembrane proteins, a type of pattern recognition receptor (PRR), that play a crucial role in triggering the innate immune response by recognizing pathogen-associated molecular patterns (PAMPs) associated with invading pathogens. Among the ten subtypes (TLR1-10) of the human TLR family, TLR7 and TLR8 are endosomal receptors that bind single-stranded RNA (ssRNA) derived from RNA viruses. The discovery of TLR7-mediated innate immune activation has revealed its potential as a promising target for the treatment of various diseases, including viral infections, autoimmune diseases, and cancer immunotherapy. In particular, research is focusing on developing broad-spectrum, immune-enhancing antiviral agents against various RNA viruses, including influenza virus, severe acute respiratory coronavirus 2 (SARS-CoV-2), and hepatitis C virus, by leveraging the therapeutic effects of TLR7 agonists. Given TLR7's ability to induce interferon (IFN) responses and proinflammatory cytokines and chemokines by activating NF-κB, efforts to apply it as a vaccine adjuvant are gaining increasing attention. This is based on the observation that TLR7 ligands, such as imiquimod, resiquimod, and GS-9620, can efficiently induce cellular and humoral immune stimulation by mediating interactions between CD4+ or CD8+ T cells and antigen-presenting cells (APCs).

[0004] TLR7, an endomembrane receptor that can detect RNA virus genomes, shares a high degree of similarity with TLR8 due to their similar amino acid sequences and tertiary structures. Both receptors regulate innate immunity primarily by activating type I IFN and NF-κB signaling pathways, leading to the induction of proinflammatory cytokines and chemokines and the stimulation of B cells, which are crucial for the adaptive immune response. However, subsequent secondary activation varies depending on factors such as cell type and activating molecules. TLR7 is primarily expressed in the endosomes of immune cells such as dendritic cells, macrophages, and B cells, and TLR7 agonists can induce sustained signaling through persistent complex formation with TLR7, leading to higher levels of type I IFNs or proinflammatory cytokines, which are crucial for antiviral responses. However, TLR8 is primarily distributed in the endosomes of monocytes and myeloid dendritic cells, which is a less favorable environment for sustained immune activation compared to endosomes. Therefore, TLR8-mediated immune activation generally tends to be more transient than TLR7-mediated activation, and in terms of the dynamics of immune activation, TLR7 generally shows more sustained immune activation.

[0005] The development of TLR7-selective agonists is challenging due to the structural similarity between TLR7 and TLR8. Among the best-known TLR7 agonists are small molecules from the imidazoquinolines family, such as imiquimod and resiquimod. However, these have been tested for potential as vaccine adjuvants in infectious diseases, and local vaccination has been hindered due to excessively rapid diffusion from the injection site. Another TLR7 agonist, GS-9620, is a TLR7 EC agonist. 50The value was 290 nM and showed 30-fold selectivity for TLR8. The structures of other TLR7 agonists are based on imidazopyridine, guanosine, oxoadenine, and pyrrolopyrimidine, and several small-molecule compounds have been continuously reported, but most of them do not have high selectivity for TLR7 compared to TLR8, and thus need improvement. Therefore, it is necessary to develop new TLR7 agonists that simultaneously exhibit high efficacy and remarkable selectivity for TLR7.

[0006]

[0007] Numerous papers and patents are referenced and cited throughout this specification. The disclosures of these cited papers and patents are incorporated herein by reference in their entirety to provide a clearer understanding of the state of the art and the scope of the invention.

[0008]

[0009] The present inventors have conducted extensive research to search for excellent immunostimulatory small molecule compounds that can significantly enhance cellular and humoral immune responses by activating Toll-like receptors (TLRs), which play a key role in pathogen invasion recognition and subsequent initiation of the innate immune response. As a result, the present inventors have completed the present invention by discovering that the purine derivative compound of Chemical Formula 1 described below can highly selectively activate TLR7, an immune cell membrane receptor that detects RNA virus genomes, and induce sustained immune stimulation against various RNA viruses such as influenza virus, SARS-CoV-2 (severe acute respiratory coronavirus 2), and hepatitis C virus.

[0010] Therefore, the purpose of the present invention is to provide a novel purine derivative compound and a composition for enhancing immune response containing the same as an active ingredient.

[0011] Other objects and advantages of the present invention will become more apparent from the detailed description, claims and drawings below.

[0012]

[0013] According to one aspect of the present invention, the present invention provides a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof:

[0014] Chemical Formula 1

[0015]

[0016] In the above chemical formula,

[0017] R1 to R4 are each independently hydrogen or C1-C8 alkyl;

[0018] R5 and R6 are each independently hydrogen or C1-C6 alkyl, or R5 and R6 are combined with each other to form a 5- to 7-membered heterocycloalkyl ring which is unsubstituted or substituted with C1-C4 alkyl;

[0019] A is an aryl or heteroaryl of a 6- to 10-membered ring which is unsubstituted or substituted with C1-C4 alkyl or C1-C4 alkoxy;

[0020] m and n are each independently integers from 0 to 3.

[0021] The present inventors have conducted extensive research to identify excellent immunostimulatory small-molecule compounds that can significantly enhance cellular and humoral immune responses by activating Toll-like receptors (TLRs), which play a key role in the innate immune response. As a result, the purine derivative compound represented by Chemical Formula 1 was found to be effective as an immunostimulatory enhancer against various RNA viruses, such as influenza virus, severe acute respiratory coronavirus 2 (SARS-CoV-2), and hepatitis C virus, by specifically activating TLR7 among various TLR subtypes.

[0022] The term “alkyl” as used herein means a straight-chain or branched saturated hydrocarbon group, and includes, for example, methyl, ethyl, propyl, isopropyl, etc. For example, C1-C8 alkyl means an alkyl group having an alkyl unit having 1 to 8 carbon atoms, and when C1-C8 alkyl is substituted, the carbon number of the substituent is not included.

[0023] As used herein, the term “heterocycloalkyl” refers to a saturated carbon ring containing oxygen, sulfur, or nitrogen as a heteroatom within the ring. Specifically, the heteroatom is nitrogen or oxygen, and more specifically, nitrogen. The number of heteroatoms is 1-3, and specifically 1-2. “Heterocycloalkyl having a 5- to 7-membered ring” refers to a ring in which the sum of the carbon and heteroatoms forming the ring is 5 to 7.

[0024] The term “alkoxy” in this specification means a radical formed by the removal of hydrogen from an alcohol, for example, C1-C4 alkoxy means a radical formed by the removal of hydrogen from an alcohol having 1 to 4 carbon atoms.

[0025] As used herein, the term “aryl” means a monocyclic or polycyclic carbon ring that is wholly or partially unsaturated and has aromaticity.

[0026] As used herein, the term “heteroaryl” refers to a heterocyclic aromatic group containing oxygen, sulfur or nitrogen as a heteroatom within the ring. The number of heteroatoms contained within the ring is 1-3, and specifically 1-2. The term “heteroaryl of a 6- to 10-membered ring” refers to a heteroaryl having 6 to 10 ring atoms including both carbon and heteroatoms, and the ring may be a monocycle or a bicycle.

[0027] As used herein, the term “pharmaceutically acceptable salt” includes salts derived from pharmaceutically acceptable inorganic acids, organic acids, or bases. Examples of suitable acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, trifluoroacetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, and the like. Salts derived from suitable bases may include alkali metals such as sodium, alkaline earth metals such as magnesium, and ammonium.

[0028] According to a specific embodiment of the present invention, R1 and R2 are hydrogen.

[0029] According to a specific embodiment of the present invention, R3 and R4 are each hydrogen and C1-C6 alkyl, more specifically hydrogen and C2-C5 alkyl, and most specifically hydrogen and C4 alkyl, respectively.

[0030] According to a specific embodiment of the present invention, R5 and R6 are each independently C1-C4 alkyl, or R5 and R6 are combined with each other to form a heterocycloalkyl selected from the group consisting of piperazine, piperidine, pyrrolidine, diazepan, and morpholin, which are unsubstituted or substituted with C1-C3 alkyl. More specifically, R5 and R6 are each independently C1-C2 alkyl, or R5 and R6 are combined with each other to form a heterocycloalkyl selected from the group consisting of piperazine, piperidine, pyrrolidine, and diazepane, which are unsubstituted or substituted with C1-C2 alkyl.

[0031] According to a specific embodiment of the present invention, A is an aryl having a 6- to 10-membered ring which is unsubstituted or substituted with C1-C4 alkoxy, and more specifically, an aryl having a 6-membered ring which is unsubstituted or substituted with C1-C2 alkoxy.

[0032] According to a specific embodiment of the present invention, m and n are 1.

[0033] According to a specific embodiment of the present invention, the compound represented by the chemical formula 1 is a compound represented by the following chemical formula 2:

[0034] Chemical Formula 2

[0035]

[0036] In the above chemical formula,

[0037] R1 to R6, m and n are as defined in paragraph 1;

[0038] R7 to R 10 are each independently hydrogen or C1-C3alkoxy.

[0039] More specifically, the above R7 is C1-C2 alkoxy and the above R8 to R 10 is hydrogen.

[0040] According to a specific embodiment of the present invention, the compound represented by the chemical formula 1 is selected from the group consisting of compounds represented by the following chemical formulas 3 to 10:

[0041] Chemical Formula 3 Chemical Formula 4

[0042]

[0043] Chemical Formula 5 Chemical Formula 6

[0044]

[0045] Chemical Formula 7 Chemical Formula 8

[0046]

[0047] Chemical Formula 9 Chemical Formula 10

[0048] .

[0049]

[0050] According to another aspect of the present invention, the present invention provides a composition for enhancing an immune response comprising the compound of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient.

[0051] According to another aspect of the present invention, the present invention provides a method for enhancing an immune response comprising administering to a subject a compound of the present invention or a pharmaceutically acceptable salt thereof.

[0052] As used herein, the term “immune response enhancement” refers to the process of strengthening an antigen-specific immune response within a subject when administered to the subject, and the “immune response enhancement composition” is a substance that promotes an immune response to an antigen during the initial activation process of the immune response, and encompasses all molecules that do not directly act as an immunogen on the host’s immune system but enhance the immune response by enhancing the activity of the immune system. Some antigens have low immunogenicity when administered alone or are toxic at concentrations that induce a significant immune response within the subject, so the immune response enhancement composition can enhance the immunogenicity of the same antigen and reduce the effective amount of the antigen required to induce an immune response sufficient to eliminate the pathogen. Therefore, the “immune response enhancement composition (enhancer)” is used synonymously with “immune adjuvant” and “immune enhancer.”

[0053] The composition for enhancing the immune response used in the present invention may be administered simultaneously with a vaccine composition containing an antigen, or administered sequentially with a time interval between them. When the composition for enhancing the immune response of the present invention is administered simultaneously with a vaccine composition, it may be prepared as a single formulation mixed with the vaccine composition and packaged in a single vial or prefilled syringe, or it may be prepared as a separate formulation and administered individually and simultaneously.

[0054] As used herein, the term “administration” or “administer” refers to directly administering a therapeutically effective amount of the composition of the present invention to a subject so that the same amount is formed in the body of the subject.

[0055] In the present invention, the term “therapeutically effective amount” means the content of a composition in which the pharmacological ingredient in the composition is contained in an amount sufficient to provide a therapeutic or preventive effect to a subject to whom the pharmaceutical composition of the present invention is to be administered, and includes a “prophylactically effective amount”.

[0056] The term “subject” as used herein includes, without limitation, a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, monkey, chimpanzee, baboon, or rhesus macaque. Specifically, the subject of the present invention is a human.

[0057] According to a specific embodiment of the present invention, the composition specifically enhances the activity of TLR7 (Toll-like receptor 7).

[0058] As used herein, the term "specifically enhancing activity" means enhancing the intrinsic biological activity of TLR7 with high selectivity compared to other Toll-like receptor family members, such as TLR8. Although TLR7 and TLR8 are very similar in amino acid sequence and three-dimensional structure, the composition of the present invention is more advantageous in sustained immune activation by specifically activating TLR7.

[0059] The composition of the present invention can be administered parenterally, specifically intravenously, subcutaneously, intraperitoneally, or intranasally. According to the present invention, the composition for enhancing immune responses of the present invention or a vaccine composition comprising the same can be usefully applied as a nasal administration composition that effectively induces an immune response, particularly against respiratory viruses, by inducing a strong mucosal immunity in the body.

[0060] The appropriate dosage of the composition of the present invention can be prescribed in various ways depending on factors such as the formulation method, administration method, patient age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity. The preferred dosage of the composition of the present invention is within the range of 0.001-100 mg / kg for adults.

[0061] The composition of the present invention can be manufactured in the form of a unit dose or can be manufactured by inserting it into a multi-dose container by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains. In this case, the formulation may be in the form of a solution, suspension, syrup or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granule, tablet or capsule, and may additionally include a dispersant or stabilizer.

[0062]

[0063] According to another aspect of the present invention, the present invention provides a vaccine composition comprising, as active ingredients, one or more antigen proteins or nucleic acid molecules encoding the same; and the compound of the present invention or a pharmaceutically acceptable salt thereof as described above.

[0064] According to another aspect of the present invention, the present invention provides a vaccination method comprising administering to a subject one or more antigen proteins or nucleic acid molecules encoding the same; and a compound of the present invention or a pharmaceutically acceptable salt thereof as described above.

[0065] Since the compound for enhancing the immune response used in the present invention has already been described above, its description is omitted to avoid excessive duplication.

[0066] The term "antigen" as used herein refers to a general term for external foreign substances or internal substances (self antigens) that, upon penetration into a living body, trigger an immune response, induce the production of antibodies, and specifically bind to the produced antibodies. Antigens include polypeptides, microorganisms or fragments thereof (live vaccines, attenuated vaccines, killed vaccines, subunit vaccines, etc.), viruses, etc. that induce an immune response. Antigens that can be used in the present invention may be, for example, antigens derived from influenza viruses, but are not limited thereto.

[0067] The term "vaccine" in this specification refers to a pharmaceutical preparation used to artificially induce, promote, or enhance a protective immune response against tumors, viruses, fungi, bacteria, or other infectious agents or pathogens in a subject. Vaccines are divided into preventive and therapeutic vaccines, and contain as active ingredients cells or antigens that, when administered to a subject, induce an immune response along with the production of antibodies and immune lymphocytes (T cells and B cells).

[0068] The vaccine of the present invention can be used in the form of a DNA vaccine or mRNA vaccine containing a nucleic acid molecule encoding an antigen suitable for the type of disease to be prevented or treated, as well as a protein vaccine.

[0069] In this specification, the term “nucleic acid molecule” and the “nucleotide” constituting the same are deoxyribonucleotides or ribonucleotides existing in single-stranded or double-stranded form, and unless specifically stated otherwise, include analogs of natural nucleotides and mutated bases for reducing immunogenicity (e.g., pseudouridine, N1-methyl pseudouridine, 5-methylcytosine, etc.). When the present invention is used in the form of a DNA vaccine or mRNA vaccine, the antigen encoding gene of the present invention may be included in each gene delivery system and expressed in a subject.

[0070] As used herein, the term “prevention” means inhibiting the development of a disease or condition in a subject who has not been diagnosed with the disease or condition but is susceptible to such disease or condition. As used herein, the term “treatment” means (a) inhibiting the development of a disease, condition, or symptom; (b) alleviating the disease, condition, or symptom; or (c) eliminating the disease, condition, or symptom.

[0071]

[0072] The features and advantages of the present invention are summarized as follows:

[0073] (a) The present invention provides a novel purine derivative compound and a composition for enhancing immune response comprising the same as an active ingredient.

[0074] (b) The compound of the present invention has an EC of nanomolar units for TLR7, an immune cell membrane receptor. 50 Not only does it have value, but it also has a similar structure and has high selectivity for TLR7 compared to TLR8, which is mainly distributed in the ER (endoplasmic reticulum), and can induce continuous immune activation.

[0075] (c) Accordingly, the compound of the present invention can be usefully utilized as an effective vaccine adjuvant composition against various RNA viruses, including influenza virus, SARS-CoV-2, and hepatitis C virus.

[0076]

[0077] Figure 1 is a schematic representation of the predicted binding mode of N6-butyl-7-(4-hydroxymethyl)-2-methoxybenzyl)- 7H-purine-2,6-diamine (represented by cyan bars), a precursor compound of Example 4, at the R848 binding site of monkey TLR7 (PDB ID: 5GMH). Dimerization partners of TLR7 are indicated in pink and blue, and key interactions are indicated by dotted lines.

[0078] Figure 2 is a schematic representation of the predicted binding mode of compound 4 (GM91669, denoted “compound 27b” in the figure below, cyan bar) at the R848 binding site of monkey TLR7 (PDB ID: 5GMH). Dimerization partners of TLR7 are indicated in pink and blue, and key interactions are indicated by dotted lines.

[0079] Figure 3 is a diagram showing the activation of mTLR7 by Example 4 compound (GM91669). Figures 3a and 3b show the results of cell culture-based mTLR7 and mTLR8 reporter assays, in which HEK293 cells stably expressing mTLR7 (Figure 3a) or mTLR8 (Figure 3b) were treated with increasing concentrations of Example 4 compound (GM91669) (light blue circle), with GS-9620 (dark blue circle) and Resiquimod (brown circle) as controls. On the 1st day after treatment, the cell culture supernatant was collected, and the mTLR7- or mTLR8-mediated NF-kB / AP-1 promoter activity was quantified by measuring the SEAP level at 620 nm. The 50% effective concentration (EC) 50 ) and the absorbance for the maximum effective concentration (Emax) are summarized in the tables below each graph in Figs. 3a and 3b. NA indicates no activity at the maximum concentration of 100 μM, not applicable. Figs. 3c to 3f are pictures showing the results of ELISA for quantifying cytokine or IFN levels. RAW264.7 cells were treated with increasing concentrations of Example 4 compound (GM91669), using DMSO as a mock control, and mIL-6 (Fig. 3c), mTNF-α (Fig. 3d), IFN-α1 (Fig. 3e) and mIL-4 (Fig. 3f) were quantified by ELISA using specific antibodies at 8, 12, 24, 36 and 48 hours. Statistical analysis was performed compared to the mock-treated control at each time point, and each graph is expressed as the mean ± standard deviation obtained from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

[0080] Figure 4 is a diagram showing that Example 4 compound (GM91669) enhances the production of IgG and IgA specific to the cognate A / H1N1 virus PR8 in mice. Figure 4a is a schematic diagram summarizing the vaccination and sample collection process. BALB / c mice were administered inactivated PR8 virus alone (0.5 μg per mouse) or together with a TLR7 agonist (Example 4 compound (GM91669) or a positive control, GS-9620), and serum was collected at 3 weeks. Figure 4b is a diagram showing the results of IgG ELISA. Serum was diluted 1,000-fold and loaded onto a plate immobilized with PR8 virus, and cognate virus-specific total IgG (left panel), IgG1 (middle panel), and IgG2a (right panel) were measured. Figure 4c shows the results of calculating the relative ratio of IgG2a / IgG1 compared to the control group (DMSO) that received only the vaccine. Figure 4d shows the results of IgA ELISA. Nasal and bronchial washings were simultaneously sampled from immunized mice, and 10-fold diluted serum (left panel), nasal (middle panel), and tracheal (right panel) fluids were loaded onto PR8 virus-coated plates for comparative titration of IgA. The relative amounts were quantified by measuring absorbance at 450 nm using HRP-conjugated goat anti-mouse IgA antibody, and the data are expressed as the mean ± standard deviation from three mice. ELISA was performed in three independent experiments, and statistical significance was determined using Dunnett's multiple comparison test followed by two-way ANOVA (Figures 4b and 4d) or one-way ANOVA (Figure 4c). ns, not significant; *, P < 0.05; and ****, P < 0.0001.

[0081] Figure 5 is a diagram showing that Example 4 compound (GM91669) enhances neutralizing antibody production against various influenza A viruses in mice. BALB / c mice (n = 3 per group) were immunized with inactivated PR8 (A / H1N1; DMSO) vaccine alone or with increasing concentrations of Example 4 compound (GM91669) (0.01, 0.1, and 1 μg per mouse) or the positive control GS-9620 (1 μg per mouse). Whole blood samples were collected 3 weeks after vaccination and subjected to HAI assay using 4 HA titers along with the cognate virus PR8 (green) or the heterologous virus HK (A / H3N2; orange). The log2-transformed ratios of HAI titers for each virus are expressed as mean ± standard deviation. ND, not detected. Statistical significance compared to the vaccine-only group (DMSO) was determined by two-way analysis of variance with Dunnett's multiple comparison test. ns, not significant; *, P < 0.05; and ***, P < 0.001.

[0082] Figure 6 is a diagram showing the protective efficacy of a vaccine combined with Example 4 compound (GM91669) against a homologous virus in mice. Figure 6a is a schematic diagram summarizing the vaccination process of influenza A virus H1N1. Female BALB / c mice were intranasally immunized with inactivated PR8 (A / H1N1) alone (0.5 μg per mouse) or increasing concentrations of Example 4 compound (GM91669) (0.01, 0.1, and 1 μg per mouse) or GS-9620 (1 μg per mouse) as a control for mTLR7 stimulation. After 3 weeks, non-immunized (virus only) or immunized mice were challenged with 10 x MLD 50Mice were intranasally infected with PR8 adapted to the dose. Body weight changes (Fig. 6b) and survival rates (Fig. 6c) were recorded daily for 2 weeks after infection. In Fig. 6b, the values ​​are expressed as the mean ± standard deviation from 6 mice in each group. Statistical significance compared to the vaccine-only group in Fig. 6c was determined using Kaplan-Meier curves and log-rank analysis. ns, not significant; **, P < 0.01.

[0083] Figure 7 is a drawing showing the results of confirming the enhancement of heterologous A / H3N2 virus HK-specific IgG and IgA production in mice by Example 4 compound (GM91669). Figure 7a is a schematic diagram summarizing the vaccination and sample collection process. Serum was collected 3 weeks after vaccination of BALB / c mice with inactivated PR8 virus alone (0.5 μg per mouse) or together with a TLR7 agonist (Example 4 compound (GM91669) or a positive control, GS-9620). Figure 7b is a drawing showing the results of IgG ELISA, which measured the homologous virus-specific total IgG (left panel), IgG1 (middle panel), and IgG2a (right panel), respectively, after loading the serum 1,000-fold diluted and HK virus-immobilized plate. Figure 7c is a graph showing the results of calculating the ratio of IgG2a to IgG1 compared to the control group (DMSO) that used the vaccine alone. Figure 7d shows the results of IgA ELISA. After sampling nasal and bronchial washings from immunized mice, 10-fold diluted serum (left panel), nasal (middle panel), and tracheal (right panel) fluids were loaded onto HK virus-coated plates for comparative titration of IgA. To quantify their relative amounts, absorbance was measured at 450 nm using HRP-conjugated goat anti-mouse IgA antibody. Data are expressed as the mean ± standard deviation from three mice. ELISAs were performed in three independent experiments, and statistical significance was determined using Dunnett's multiple comparison test followed by two-way ANOVA (Figures 7b and 7d) or one-way ANOVA (Figure 7c). ns, not significant; *, P < 0.05; and ****, P < 0.0001.

[0084] Figure 8 is a diagram showing the results of evaluating the protective efficacy against a heterologous virus in mice when co-administered with an A / H1N1 influenza virus vaccine with Example 4 compound (GM91669). Figure 8a is a schematic diagram summarizing the infection process of infectious influenza A virus H3N2 following vaccination. Female BALB / c mice were intranasally immunized with inactivated PR8 (A / H1N1) (0.5 μg per mouse) alone, or with increasing concentrations of Example 4 compound (GM91669) at 0.01, 0.1, and 1 μg per mouse, or with 1 μg of GS-9620 as a control for mTLR7 stimulation. After 3 weeks, non-immunized (virus only) or immunized mice were challenged with a mouse-adapted HK strain at a dose of 10 x MLD. 50 The mice were intranasally infected with the amount of . Body weight changes (Fig. 8b) and survival rates (Fig. 8c) were monitored daily for 2 weeks after viral infection. In Fig. 8b, the values ​​are expressed as the mean ± standard deviation from 6 mice in each group. In Fig. 8c, statistical significance compared to the vaccine-only group was determined using Kaplan-Meier curves and log-rank analysis. ns, not significant; **, P < 0.01.

[0085]

[0086] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0087]

[0088] Example

[0089] Manufacturing example

[0090] Example 4 Preparation of compound: N6-butyl-7-(4-((diethylamino)methyl)-2-methoxybenzyl)-

[0091] 7H-purine-2,6-diamine trifluoroacetate

[0092] Step 1: Preparation of methyl 4-((2,6-dichloro-7H-purin-7-yl)methyl)-3-methoxybenzoate

[0093] 2,6-Dichloropurine (3.00 g, 15.873 mmol) was dissolved in tetrahydrofuran (80.0 mL). A 3.0 M methyl magnesium chloride solution in tetrahydrofuran (6.4 mL, 19.04 mmol) was slowly added, and the resulting mixture was stirred at room temperature for 30 minutes. Next, methyl 4-(bromomethyl)-3-methoxybenzoate 5 (4.52 g, 17.460 mmol) was added, and the mixture was stirred at 70°C for an additional 8 hours. After the reaction was completed, the mixture was cooled to room temperature and methanol was added to neutralize the remaining base. The solvent was then evaporated, and the resulting residue was diluted with water and extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate and purified by silica gel chromatography. This process gave methyl 4-((2,6-dichloro-7H-purin-7-yl)methyl)-3-methoxybenzoate (2.50 g, 42%) as a white solid.

[0094]

[0095] Step 2. Preparation of methyl 4-((6-(butylamino)-2-chloro-7H-purin-7-yl)methyl)-3-methoxybenzoate

[0096] Methyl 4-((2,6-dichloro-7H-purin-7-yl)methyl)-3-methoxybenzoate (2.000 g, 5.447 mmol), triethylamine (2.265 mL, 16.340 mmol), and n-butylamine hydrochloride (1.780 g, 16.340 mmol) were dissolved in isopropyl alcohol (54.5 mL). The resulting mixture was stirred at 70°C for 10 h. Upon completion of the reaction, the mixture was cooled to room temperature and the solvent was evaporated. The resulting residue was diluted with water and extracted with ethyl acetate. The organic phase was collected and dried over anhydrous sodium sulfate. The crude mixture was further purified by silica gel chromatography. This process afforded methyl 4-((6-(butylamino)-2-chloro-7H-purin-7-yl)methyl)-3-methoxybenzoate (1.54 g, 70%) as a white solid.

[0097]

[0098] Step 3. Preparation of (4-((6-(butylamino)-2-chloro-7H-purin-7-yl)methyl)-3-methoxyphenyl)methanol

[0099] Dissolve lithium aluminum hydride in tetrahydrofuran and mix the mixture at 0 o C. Methyl 4-((6-(butylamino)-2-chloro-7H-purin-7-yl)methyl)-3-methoxybenzoate (3.00 g, 7.428 mmol) was dissolved in tetrahydrofuran (37.00 mL) and the mixture was stirred for 2 hours after adjusting to room temperature. After completion of the reaction, the mixture was cooled to 0 oThe mixture was cooled to C and quenched with cold water (5.00 mL). The mixture was stirred at 0°C for an additional 30 minutes. 1N aqueous sodium hydroxide solution (5.00 mL) was added and stirred for 10 minutes. The mixture was then diluted with ethyl acetate and filtered through a celite cake. The filtrate was concentrated and extracted with ethyl acetate and brine. The organic phase was dried over anhydrous sodium sulfate and purified by silica gel chromatography to afford (4-((6-(butylamino)-2-chloro-7H-purin-7-yl)methyl)-3-methoxyphenyl) methanol (2.40 g, 86%) as a white solid.

[0100]

[0101] Step 4. Preparation of N-butyl-2-chloro-7-(4-(chloromethyl)-2-methoxybenzyl)-7H-purin-6-amine

[0102] (4-((6-(butylamino)-2-chloro-7H-purin-7-yl)methyl)-3-methoxyphenyl)methanol (2.40 g, 6.385 mmol) was dissolved in dichloromethane (31.93 mL), and the mixture was cooled to 0°C. Thionyl chloride (0.56 mL, 7.662 mmol) was added dropwise and stirred for 2 h. The mixture was quenched with saturated sodium bicarbonate solution. The mixture was then extracted with dichloromethane and brine. The organic phase was dried over anhydrous sodium sulfate and purified by silica gel chromatography to give N-butyl-2-chloro-7-(4-(chloromethyl)-2-methoxybenzyl)-7H-purin-6-amine (1.92 g, 76%) as a white solid.

[0103]

[0104] Step 5. Preparation of N-butyl-2-chloro-7-(4-((diethylamino)methyl)-2-methoxybenzyl)-7H-purin-6-amine

[0105] N-Butyl-2-chloro-7-(4-(chloromethyl)-2-methoxybenzyl)-7H-purin-6-amine (300.0 mg, 0.761 mmol), diethylamine (0.47 mL, 4.57 mmol), and cesium carbonate (1.49 g, 4.57 mmol) were dissolved in N,N-dimethylformamide (3.80 mL). The mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was diluted with water and extracted with ethyl acetate. The organic phase was collected and dried over anhydrous sodium sulfate. The mixture was purified by silica gel chromatography to give N-butyl-2-chloro-7-(4-((diethylamino)methyl)-2-methoxybenzyl)-7H-purin-6-amine (182.0 mg, 54%) as a pale yellow solid.

[0106]

[0107] Step 6. Preparation of N6-butyl-7-(4-((diethylamino)methyl)-2-methoxybenzyl)-N2-(2,4-dimethoxybenzyl)-7H-purine-2,6-diamine

[0108] N-Butyl-2-chloro-7-(4-((diethylamino)methyl)-2-methoxybenzyl)-7H-purin-6-amine (182.0 mg, 0.422 mmol) and 2,4-dimethoxybenzylamine (0.573 mL, 3.801 mmol) were dissolved in n-butanol (2.10 mL). The mixture was stirred at 130 o The mixture was stirred at C for 48 hours. After the reaction was completed, the solvent was evaporated under reduced pressure. The residue was diluted with water and extracted with ethyl acetate. The organic phase was collected and dried over anhydrous sodium sulfate. The mixture was purified by silica gel chromatography to obtain N6-butyl-7-(4-((diethylamino)methyl)-2-methoxybenzyl)-N2-(2,4-dimethoxybenzyl)-7H-purine-2,6-diamine (72.0 mg, 30%) as a yellow solid. The compound was used without further purification.

[0109]

[0110] Step 7. Preparation of N6-butyl-7-(4-((diethylamino)methyl)-2-methoxybenzyl)-7H-purine-2,6-diamine trifluoroacetate

[0111] N6-Butyl-7-(4-((diethylamino)methyl)-2-methoxybenzyl)-N2-(2,4-dimethoxybenzyl)-7H-purine-2,6-diamine (72 mg, 0.128 mmol) o C was dissolved in trifluoroacetic acid (3 mL). The mixture was then allowed to reach room temperature and stirred for 5 h. After completion of the reaction, the mixture was concentrated under reduced pressure and co-evaporated with MeOH (10 mL, 3 times) to remove excess trifluoroacetic acid. The resulting crude mixture was purified by reverse-phase column chromatography on a Gilson preparative HPLC system using an ACN / H2O (0.1% TFA) mobile phase. This purification process yielded N6-butyl-7-(4-((diethylamino) methyl)-2-methoxybenzyl)-7H-purine-2,6-diamine trifluoroacetate (19.2 mg, 36%) as a white solid.

[0112]

[0113] Example 3 Preparation of compound: N6-butyl-7-(2-methoxy-4-(piperidin-1-ylmethyl)benzyl)-7H-purine-2,6-diamine trifluoroacetate

[0114] Step 1. Preparation of N-butyl-2-chloro-7-(2-methoxy-4-(piperidin-1-ylmethyl)benzyl)-7H-purin-6-amine

[0115] N-Butyl-2-chloro-7-(4-(chloromethyl)-2-methoxybenzyl)-7H-purin-6-amine (300.0 mg, 0.761 mmol), piperidine (0.300 mL, 3.043 mmol), and cesium carbonate (991.59 mg, 3.043 mmol) were dissolved in N,N-dimethylformamide (3.80 mL). The mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was diluted with water and extracted with ethyl acetate. The organic phase was collected and dried over anhydrous sodium sulfate. The mixture was purified by silica gel chromatography to give N N-butyl-2-chloro-7-(2-methoxy- 4-(piperidin-1-ylmethyl)benzyl)-7H-purin-6-amine (169.0 mg, 50%) as a pale yellow solid.

[0116]

[0117] Step 2. Preparation of N6-butyl-N2-(2,4-dimethoxybenzyl)-7-(2-methoxy-4-(piperidin-1-ylmethyl) benzyl)-7H-purine-2,6-diamine

[0118] N-Butyl-2-chloro-7-(2-methoxy-4-(piperidin-1-ylmethyl)benzyl)-7H-purin-6-amine (169.0 mg, 0.381 mmol) and 2,4-dimethoxybenzylamine (0.517 mL, 3.433 mmol) were dissolved in n-butanol (5.7 mL). The mixture was stirred at 130 o C for 48 h. After the reaction was completed, the solvent was evaporated under reduced pressure. The residue was diluted with water and extracted with ethyl acetate. The organic phase was collected and dried over anhydrous sodium sulfate. The mixture was purified by silica gel chromatography to obtain N6-butyl-N2-(2,4-dimethoxybenzyl)-7-(2-methoxy-4-(piperidin-1-ylmethyl)benzyl)-7H-purine-2,6-diamine (68.0 mg, 31%) as a yellow solid. The compound was used without further purification.

[0119]

[0120] Step 3. Preparation of N6-butyl-7-(2-methoxy-4-(piperidin-1-ylmethyl)benzyl)-7H-purine-2,6-diamine trifluoroacetate

[0121] N6-Butyl-N2-(2,4-dimethoxybenzyl)-7-(2-methoxy-4-(piperidin-1-ylmethyl)benzyl)-7H-purine-2,6-diamine (68.0 mg, 0.119 mmol) o C was dissolved in trifluoroacetic acid (3 mL). The mixture was then allowed to reach room temperature and stirred for 5 h. After completion of the reaction, the mixture was concentrated under reduced pressure and co-evaporated with MeOH (10 mL, 3 times) to remove excess trifluoroacetic acid. The resulting crude mixture was purified by reverse-phase column chromatography on a Gilson preparative HPLC system using an ACN / H2O (0.1% TFA) mobile phase to give N6-butyl-7-(2-methoxy-4-(piperidin-1-ylmethyl)benzyl)-7H-purine-2,6-diamine trifluoroacetate (21.0 mg, 42%) as a white solid.

[0122] The structures and IUPAC names of the compounds of Examples 1 to 8 manufactured by a method similar to the manufacturing method of the above manufacturing example are summarized in Table 1 below.

[0123] Example Chemical Structure Compound Name 1GM-91649 TFAN6-Butyl-7-(4-((dimethylamino)methyl)-2-methoxybenzyl)-7H-purine-2,6-diamine trifluoroacetate 2GM-91650 TFAN6-Butyl-7-(2-methoxy-4-(pyrrolidin-1-ylmethyl)benzyl)-7H-purine-2,6-diamine trifluoroacetate 3GM-91651 TFAN6-Butyl-7-(2-methoxy-4-(piperidin-1-ylmethyl)benzyl)-7H-purine-2,6-diamine trifluoroacetate 4GM-91669 TFAN6-Butyl-7-(4-((diethylamino)methyl)-2-methoxybenzyl)-7H-purine-2,6-diamine trifluoroacetate 5GM-91671 TFAN6-Butyl-7-(2-methoxy-4-((4-methylpiperazin-1-yl)methyl)benzyl)-7H-purine-2,6-diamine trifluoroacetate 6GM-91699 TFAN6-Butyl-7-(2-methoxy-4-(piperazin-1-ylmethyl)benzyl)-7H-purine-2,6-diamine trifluoroacetate 7GM-91713 TFAN6-Butyl-7-(2-methoxy-4-((4-methyl-1,4-diazepan-1-yl)methyl)benzyl)-7H-purine-2,6-diamine trifluoroacetate 8GM-91714 TFA7-(4-((1,4-diazepan-1-yl)methyl)-2-methoxybenzyl)-N6-butyl-7H-purine-2,6-diamine trifluoroacetate

[0124]

[0125] The results of analyzing the compounds of Examples 1 to 8 of the present invention using nuclear magnetic resonance spectroscopy (NMR) or liquid chromatography-mass spectrometry (LC-MS) are summarized in Table 2 below.

[0126] 실시예LCMS 또는 NMR11H-NMR (400 MHz, METHANOL-D4) δ 8.03 (s, 1H), 7.25 (s, 1H), 7.17 (d, J = 7.9 Hz, 1H), 7.10 (d, J = 7.6 Hz, 1H), 5.64 (s, 2H), 4.32 (s, 2H), 3.94 (s, 3H), 3.60 (t, J = 7.3 Hz, 2H), 2.86 (s, 6H), 1.62-1.54 (m, 2H), 1.33-1.22 (m, 2H), 0.93 (t, J = 7.3 Hz, 3H)21H-NMR (400 MHz, METHANOL-D4) δ 8.02(s, 1H), 7.26(s, 1H), 7.17(d, J=7.9 Hz, 1H), 7.11(dd, J=7.8, 1.1 Hz, 1H), 5.63(s, 2H), 4.38(s, 2H), 3.93(s, 3H), 3.60(t, J=7.2 Hz, 2H), 3.53-3.48(m, 2H), 3.21-3.14(m, 2H), 2.21-2.15(m, 2H), 2.05-1.99(m, 2H), 1.62-1.55(m, 2H), 1.29(td, J=15.0, 7.5 Hz, 2H), 0.93(t, J=7.3 Hz, 3H)31H-NMR (400 MHz, METHANOL-D4) δ 7.99(s, 1H), 7.22(s, 1H), 7.16(d, J=7.6 Hz, 1H), 7.08(d, J=7.6 Hz, 1H), 5.61(s, 2H), 4.27(s, 2H), 3.91(s, 3H), 3.58(t, J=7.1 Hz, 2H), 3.43(t, J=9.7 Hz, 2H), 2.93(t, J=12.0 Hz, 2H), 1.94-1.90(m, 2H), 1.82-1.76 (m, 2H), 1.71(t, J=5.7 Hz, 2H), 1.60-1.53 (m, 2H), 1.27(td, J=15.1, 7.2 Hz, 2H), 0.91(t, J=7.4 Hz, 3H)41H-NMR (400 MHz, METHANOL-D4) δ 8.02(s, 1H), 7.28(s, 1H), 7.18 (d, J = 7.6 Hz, 1H), 7.12 (d, J=7.6 Hz, 1H), 5.64(s, 2H), 4.35 (s, 2H), 3.93 (s, 3H), 3.6 (t, J=7.1 Hz, 2H), 3.25-3.18(m, 4H), 1.63-1.55 (m, 2H), 1.34 (t, J=7.2 Hz, 9H), 0.94(t, J=7.4 Hz, 3H)51H-NMR (400 MHz, METHANOL-D4) δ 8.00(s, 1H), 7.11(d, J=10.3 Hz, 2H), 7.00(d, J=7.6 Hz, 1H), 5.58(s, 2H), 3.89(s, 3H), 3.70(s, 2H), 3.60(t, J=7.1 Hz, 2H), 3.32-3.53(4H), 2.87(s, 3H), 2.87-2.83(m, 4H), 1.62-1.54(m, 2H), 1.29(td, J=14.9, 7.4 Hz, 2H), 0.93(t, J=7.2 Hz, 3H)61H-NMR (400 MHz, METHANOL-D4) δ 8.00 (s, 1H), 7.17 (s, 1H), 7.13 (d, J = 7.6 Hz, 1H), 7.03 (d, J = 7.6 Hz, 1H), 5.59 (s, 2H), 3.90 (s, 3H), 3.86 (s, 2H), 3.61 (t, J = 7.2 Hz, 2H), 3.34-3.33 (m, 4H), 2.94 (t, J = 4.6 Hz, 4H), 1.62-1.55 (m, 2H), 1.29 (td, J = 15.1, 7.4 Hz, 2H), 0.93 (t, J = 7.2 Hz, 3H)71H-NMR (400 MHz, METHANOL-D4) δ 8.01 (s, 1H), 7.31 (s, 1H), 7.17 (d, J = 8.0 Hz, 1H), 7.12 (d, J = 7.6 Hz, 1H), 5.62 (s, 2H), 4.37 (s, 2H), 3.92 (s, 3H), 3.76 (t, J = 3.8 Hz, 2H), 3.67 (t, J = 3.6 Hz, 2H), 3.61 (t, J = 7.2 Hz, 2H), 3.52-3.52 (m, 2H), 3.44 (t, J = 5.1 Hz, 2H), 2.96 (s, 3H), 2.33-2.27 (m, 2H), 1.63-1.56 (m, 2H), 1.30 (td, J = 15.0, 7.5 Hz, 2H), 0.94 (t, J = 7.4 Hz, 3H)8LCMC [M+H] 439.3.

[0127]

[0128] Experimental example

[0129] Activation of TLR7 by the compound of the present invention

[0130] Based on the results of the HTLR7 and 8 reporter assays, the compound of Example 4 (GM91669), which was the most active compound, was selected as a representative small molecule compound for further biological activity evaluation on the in vivo immune response. First, it was investigated whether the agonistic activity of the compound of Example 4 (GM91669) was also implemented in mouse TLR7 (mTLR7) and TLR8 (mTLR8). Secreted embryonic alkaline phosphatase (SEAP) is expressed under the NF-kB / AP-1-induced promoter by the mTLR7 or mTLR8 signaling pathway in HEK293 cells. The reporter assay results showed that the compound of Example 4 (27b, GM91669) (EC 50 <46 nM) is a TLR7 selective agonist and TLR7 / 8 dual agonist, GS-9620 (EC 50 , 5,286 nM) and resiquimod (EC 50 , 312 nM) was found to be a more potent agonist than the compound of Example 4 (27b, GM91669) (Fig. 3a). The maximum efficacy (E ) of the compound of Example 4 (27b, GM91669) measured at 33 μM max ) was 0.816, which was significantly higher than that of the GS-9620 treatment group (0.695 at 100 μM) and the resiquimod treatment group (0.778 at 3.7 μM), and especially in the case of Example 4 compound (GM91669) and resiquimod, E maxAt these concentrations, mTLR7 activity was reduced, which may be related to excessive stimulation of mTLR7 activity and subsequent immunotoxicity. For comparison, the same reporter assay was performed using HEK293 cells expressing mTLR8, and neither compound showed any mTLR8 agonistic effect (Figure 3b), suggesting that compound 4 can activate both hTLR7 and mTLR7 without species specificity.

[0131] To directly evaluate the immunostimulatory effect of Example 4 compound (GM91669), increasing concentrations were applied to the mouse macrophage cell line RAW264.7. Cell culture supernatants were collected at 8, 12, 24, 36, and 48 hours after treatment, and mouse (pro)inflammatory cytokines or IFNs, including mouse IL-6 (mIL-6), tumor necrosis factor-α (mTNF-α), interferon-α1 (mIFN-α1), and mIL-4, were measured. ELISA results showed that all tested cytokines and type I IFNs significantly increased in a concentration-dependent manner (Figures 3c to 3f). However, their concentrations peaked at different time points: mIL-6 at 36 hours, mTNF-α at 48 hours, mIFN-α1 at 8 hours, and mIL-4 at 24 hours. Time-course immunoassays revealed that compound 4 (GM91669) stimulated not only a Th1 immune response based on the secretion of mIL-6, mTNF-α, and mIFN-α1 via TLR7 in immune cells, but also a Th2 immune response based on the secretion of mIL-6 and mIL-4. Therefore, when administered together with a vaccine in the body, activation of both innate and adaptive immunity can be expected.

[0132] We investigated whether activation of the mTLR7-mediated innate immune response by compound 4 (GM91669) could enhance antigen-specific immunity in vivo. To this end, BALB / c mice without immunosuppression were intranasally administered an inactivated vaccine of H1N1 influenza A virus (A / Puerto Rico / 8 / 1934; PR8) at a dose of 0.5 μg, alone or in combination with various doses of compound 4 (GM91669) (0.01, 0.1, and 1 μg) or GS-9620 (1 μg) (Fig. 4a). Three weeks after immunization, serum and nasal and bronchial lavage fluids were collected, and changes in IgG and IgA levels were monitored using ELISA. Total IgG levels against the homologous virus significantly increased in a dose-dependent manner (Fig. 4b, left panel). In particular, IgG2a showed a more prominent change (Fig. 4b, right panel) compared to IgG1 (Fig. 4b, middle panel). Comparative analysis at the same dose (1 μg of Example 4 compound (GM91669) or GS-9620 per mouse) showed that the IgG2a level was increased 5.2-fold by Example 4 compound, whereas it was increased 1.6-fold by GS-9620, indicating that the increase in the IgG2a to IgG1 ratio induced by Example 4 compound induced a Th1-biased immune response more efficiently than GS-9620, thereby strongly inducing vaccine strain-matched antibody production (Fig. 4c).

[0133] Unlike traditional intramuscular vaccines, intranasal administration offers several advantages, including providing sufficient mucosal immunity, particularly at the injection site, and matching the natural route of respiratory viruses. To evaluate these advantages, we analyzed PR8-specific IgA levels in serum, nasal passages, and bronchial passages after vaccination. We confirmed that intranasal vaccination increased PR8-specific IgA production not only in serum but also in the upper and lower respiratory tract (Figure 4d). Compared to GS-9620, compound 4 more strongly increased IgA levels in nasal passages and bronchial passages, but not in serum. Furthermore, to verify whether secreted PR8-specific antibodies could neutralize infectious virus particles, we performed a hemagglutination inhibition (HAI) assay, and confirmed that 1 μg of compound 4 or GS-9620 was sufficient to increase neutralizing antibody production after vaccination with an inactivated virus vaccine (Figure 5, green bars). In summary, immunoglobulin identification and quantitative analysis revealed that compound 4 stimulated the production of neutralizing antibodies within 3 weeks after a single intranasal vaccine administration, inducing robust systemic and mucosal immunity in the body.

[0134]

[0135] Protective effect of the compound of the present invention on mice against homologous viruses

[0136] Example 4 It was investigated whether the compound could protect vaccinated mice from influenza virus infection by enhancing antigen-specific mucosal and humoral immune responses. Three weeks after vaccination, mice were challenged with the same virus strain, mouse-adapted PR8 (maPR8, 10 x 50% mouse lethal dose [MLD], in the absence or presence of the compound. 50]) were intranasally infected with a lethal dose of 100 μg of the compound of Example 4 (Fig. 6a). Body weight changes and mortality were monitored daily for the following two weeks. In the non-immunized group (Virus only), body weight gradually decreased until the 8th day, and all mice died on the 9th day (Figs. 6b and 6c). The group vaccinated with the inactivated virus also lost body weight over time, and only one of the six mice recovered to the normal range and survived (survival rate, 16.7%). This indicates that the vaccine dose is limited in protecting mice exposed to a lethal dose of the virus. In contrast, when the inactivated virus was vaccinated together with 1 μg of the compound of Example 4 per mouse, body weight loss was significantly alleviated, and the survival rate of the maPR8-infected mice was completely restored (survival rate, 100%) (Figs. 6b and 6c). This revealed that compound 4, a TLR7 selective agonist, could provide complete protection from the same viral infection by enhancing innate mucosal immunity and antigen-mediated humoral immune responses (Figs. 4 and 5).

[0137]

[0138] Effect of the compound of the present invention on the production of heterologous A / H3N2 virus HK-specific IgG and IgA

[0139] Among influenza A viruses, the H3N2 subtype, along with H1N1, is known to frequently cause severe human infections, sometimes leading to pandemics. Therefore, to evaluate the cross-protective effect of a vaccine adjuvanted with compound Example 4 against these heterologous viruses, the same vaccination regimen used for the A / H1N1-specific immune response assay was followed (Fig. 4a). Serum and nasal and bronchial lavage fluids were collected to quantify the production of broadly neutralizing antibodies against the heterologous virus HK (A / H3N2) (Fig. 7a). ELISA data showed that compound Example 4 significantly increased the production of HK-specific total IgG, IgG1, and IgG2a in serum in a dose-dependent manner (Fig. 7b). Interestingly, 1 μg of Example 4 compound stimulated IgG production at a similar level as the same dose of GS-9620, but unlike the homotypic vaccination (Fig. 4c), the IgG2a / IgG1 ratios of all TLR7 agonist-treated samples were not statistically significant compared to the vaccine-only control (Fig. 7c). These data suggest that Example 4 compound, as an adjuvant in an A / H1N1-based vaccine, stimulated IgG antibodies recognizing H3N2 subtype influenza A virus, and activated them in a Th1 / Th2 balanced manner, favoring IgG2a.

[0140] In an IgA detection ELISA, HK-specific IgA accumulated significantly more in the nasal and bronchial fluids than in the serum after vaccination in the presence of compound 4 (Fig. 7d). The enhancement of mucosal immunity induced by compound 4 was distinct from that induced by GS-9620. Consistent with these results, an HAI assay confirmed that antibodies, including HK-specific IgG and IgA, successfully neutralized infectious HK virus (Fig. 5, orange bars). These data suggest that compound 4, when used in combination with an inactivated vaccine, can expand antigenicity by accelerating serum IgG and mucosal IgA production.

[0141]

[0142] Protective effect against heterologous viruses when co-administered with the compound of the present invention and the A / H1N1 influenza virus vaccine

[0143] To determine the immunostimulatory potential of compound 4 for the development of a universal vaccine, mice immunized with a PR8-derived vaccine were challenged with a lethal dose (10 x MLD) of another subtype virus, mouse-adapted HK (maHK). 50 ) were infected (Fig. 8a). As a result of monitoring body weight changes, all mice vaccinated with the inactivated PR8 virus together with the Example 4 compound or GS-9620 recovered their body weight (Fig. 8b). In contrast, the non-vaccinated group (Virus only) and the group vaccinated with the non-adjuvanted vaccine (Vaccine) did not recover their body weight after HK virus infection. More importantly, the mortality analysis showed that 16.7%, 33.3%, and 66.7% of mice survived after vaccination with 0.01, 0.1, and 1 μg of the Example 4 compound per mouse, respectively (Fig. 8c). Body weight loss was more alleviated in the 1 μg GS-9620 adjuvanted group compared to the same dose of the Example 4 compound group (Fig. 8b), but the survival rate was the same at the endpoint of 14 days after infection (Fig. 8c). In summary, in vivo infection experiments using heterologous viruses demonstrated that compound 4 broadly enhanced neutralizing IgG and IgA, thereby extending the protective spectrum of the vaccine and inducing mouse protection against other subtypes of influenza A viruses.

[0144]

[0145] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof: Chemical Formula 1 In the above chemical formula, R1 to R4 are each independently hydrogen or C1-C8 alkyl; R5 and R6 are each independently hydrogen or C1-C6 alkyl, or R5 and R6 are combined with each other to form a 5- to 7-membered heterocycloalkyl ring which is unsubstituted or substituted with C1-C4 alkyl; A is an aryl or heteroaryl of a 6- to 10-membered ring which is unsubstituted or substituted with C1-C4 alkyl or C1-C4 alkoxy; m and n are each independently integers from 0 to 3.

2. A compound or a pharmaceutically acceptable salt thereof, characterized in that in claim 1, R1 and R2 are hydrogen.

3. A compound or a pharmaceutically acceptable salt thereof, characterized in that in paragraph 1, R3 and R4 are hydrogen and C1-C6 alkyl, respectively.

4. A compound or a pharmaceutically acceptable salt thereof, characterized in that in paragraph 1, R5 and R6 are each independently C1-C4 alkyl, or R5 and R6 are combined with each other to form a heterocycloalkyl selected from the group consisting of piperazine, piperidine, pyrrolidine, diazepan, and morpholin, which are unsubstituted or substituted with C1-C3 alkyl.

5. A compound or a pharmaceutically acceptable salt thereof, characterized in that in paragraph 1, A is an aryl having a 6- to 10-membered ring which is unsubstituted or substituted with C1-C4 alkoxy.

6. A compound or a pharmaceutically acceptable salt thereof, characterized in that in paragraph 1, m and n are 1.

7. In the first paragraph, the compound represented by the formula 1 is a compound represented by the following formula 2, or a pharmaceutically acceptable salt thereof: Chemical Formula 2 In the above chemical formula, R1 to R6, m and n are as defined in paragraph 1; R7 to R 10 are each independently hydrogen or C1-C3alkoxy.

8. In the first paragraph, the compound represented by the chemical formula 1 is a compound characterized in that it is selected from the group consisting of compounds represented by the following chemical formulas 3 to 10, or a pharmaceutically acceptable salt thereof: Chemical Formula 3 Chemical Formula 4 Chemical Formula 5 Chemical Formula 6 Chemical Formula 7 Chemical Formula 8 Chemical Formula 9 Chemical Formula 10 .

9. A composition for enhancing an antigen-specific immune response, comprising a compound of any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof as an active ingredient.

10. A composition according to claim 9, characterized in that the composition specifically enhances the activity of TLR7 (Toll-like receptor 7). 11.(a) A vaccine composition comprising as an active ingredient one or more antigen proteins or nucleic acid molecules encoding the same; and (b) a compound of any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof.

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