KR20250158694A - Novel Purine Derivatives and Uses Thereof - Google Patents

Abstract

The present invention relates to a novel purine derivative compound and a composition for enhancing an immune response comprising the same as an active ingredient. The compound of the present invention not only has an EC ₅₀ value in the nanomolar range for TLR7, an immune cell endomembrane receptor, but also exhibits high selectivity for TLR7 compared to TLR8, which has a similar structure and is primarily distributed in the endoplasmic reticulum (ER), thereby inducing sustained immune activation. Therefore, 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.

Description

Novel Purine Derivatives and Uses Thereof

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.

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).

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.

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, when tested for potential vaccine adjuvant use in infectious diseases, their rapid diffusion from the injection site hindered local vaccination. Another TLR7 agonist, GS-9620, demonstrated a TLR7 _EC50 value of 290 nM and 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. However, most of them do not exhibit high selectivity for TLR7 compared to TLR8, and thus require improvement. Therefore, the development of new TLR7 agonists that simultaneously exhibit high efficacy and remarkable selectivity for TLR7 is necessary.

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 present invention.

Patent Document 1. Patent Publication No. 10-2017-0029578

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.

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.

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

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:

Chemical Formula 1

In the above chemical formula,

R ₁ to R ₄ are each independently hydrogen or C ₁ -C ₈ alkyl;

R ₅ and R ₆ are each independently hydrogen or C ₁ -C ₆ alkyl, or R ₅ and R ₆ are combined with each other to form a 5- to 7-membered heterocycloalkyl ring which is unsubstituted or substituted with C ₁ -C ₄ alkyl;

A is an aryl or heteroaryl of a 6- to 10-membered ring which is unsubstituted or substituted with C ₁ -C ₄ alkyl or C ₁ -C ₄ alkoxy;

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

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.

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, C ₁ -C ₈ alkyl means an alkyl group having an alkyl unit having 1 to 8 carbon atoms, and when C ₁ -C ₈ alkyl is substituted, the carbon number of the substituent is not included.

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.

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

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

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.

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.

According to a specific embodiment of the present invention, R ₁ and R ₂ are hydrogen.

According to a specific embodiment of the present invention, R ₃ and R ₄ are hydrogen and C ₁ -C ₆ alkyl, respectively, more specifically hydrogen and C ₂ -C ₅ alkyl, respectively, and most specifically hydrogen and C ₄ alkyl, respectively.

According to a specific embodiment of the present invention, R ₅ and R ₆ are each independently C ₁ -C ₄ alkyl, or R ₅ and R ₆ 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 C ₁ -C ₃ alkyl. More specifically, R ₅ and R ₆ are each independently C ₁ -C ₂ alkyl, or R ₅ and R ₆ 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 C ₁ -C ₂ alkyl.

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 C ₁ -C ₄ alkoxy, and more specifically, an aryl having a 6-membered ring which is unsubstituted or substituted with C ₁ -C ₂ alkoxy.

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

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

Chemical Formula 2

In the above chemical formula,

R ₁ to R ₆ , m and n are as defined in paragraph 1;

R ₇ to R ₁₀ are each independently hydrogen or C ₁ -C ₃ alkoxy.

More specifically, R ₇ is C ₁ -C ₂ alkoxy and R ₈ to R ₁₀ are hydrogen.

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:

Chemical Formula 3 Chemical Formula 4

Chemical Formula 5 Chemical Formula 6

Chemical Formula 7 Chemical Formula 8

Chemical Formula 9 Chemical Formula 10

.

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.

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.”

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.

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.

In the present invention, the term “therapeutically effective amount” means the content of a composition containing a pharmacological ingredient in the composition 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”.

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.

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

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.

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.

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.

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.

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.

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.

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.

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).

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, as an active ingredient.

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.

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.

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

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

(b) The compound of the present invention not only has an EC ₅₀ value in the nanomolar range for TLR7, an immune cell endomembrane receptor, but also has high selectivity for TLR7 compared to TLR8, which has a similar structure and is mainly distributed in the ER (endoplasmic reticulum), and thus can induce sustained immune activation.

(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.

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.
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.
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, cell culture supernatants were collected, and SEAP levels were measured at 620 nm to quantify mTLR7- or mTLR8-mediated NF-kB/AP-1 promoter activity. The absorbance for the 50% effective concentration (EC ₅₀ ) and maximum effective concentration (E max ) are summarized in the tables below each graph in Figures 3a and 3b . NA indicates no activity at the maximum concentration of 100 μM, not applicable. Figures 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 (Figure 3c), mTNF-α (Figure 3d), IFN-α1 (Figure 3e), and mIL-4 (Figure 3f) were quantified by ELISA using specific antibodies at 8, 12, 24, 36, and 48 hours. Statistical analysis was performed compared with the simulated control group 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.
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.
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.
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 with 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 intranasally challenged with mouse-adapted PR8 at a dose of 10 x MLD _50. Body weight changes (Figure 6b) and survival rates (Figure 6c) were recorded daily for 2 weeks after infection. In Figure 6b, values are expressed as the mean ± standard deviation from six mice in each group. Statistical significance compared to the vaccine-only group in Figure 6c was determined using Kaplan-Meier curves and log-rank analysis. ns, not significant; **, P < 0.01.
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.
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 together 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) alone (0.5 μg per mouse) or with increasing concentrations of Example 4 compound (GM91669) at 0.01, 0.1, and 1 μg per mouse, or together with 1 μg of GS-9620 as a control for mTLR7 stimulation. After 3 weeks, non-immunized (virus only) or immunized mice were intranasally challenged with a mouse-adapted HK strain at a dose of 10 x MLD ₅₀ . Body weight changes (Fig. 8b) and survival rates (Fig. 8c) were monitored daily for two weeks after virus infection. In Fig. 8b, the values are expressed as the mean ± standard deviation from six mice in each group. Statistical significance compared to the vaccine-only group in Fig. 8c was determined using Kaplan-Meier curves and log-rank analysis. ns, not significant; **, P < 0.01.

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.

Example

Manufacturing example

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

7H-purine-2,6-diamine trifluoroacetate

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

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.

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

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.

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

Lithium aluminum hydride was dissolved in tetrahydrofuran, and the mixture was cooled to 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 the temperature of the resulting mixture. After completion of the reaction, the mixture was cooled to 0 ^o C and quenched with cold water (5.00 mL). The mixture was stirred for an additional 30 minutes at 0 ° C. 1 N 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.

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

(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.

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

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.

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

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 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 give 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.

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

N6-Butyl-7-(4-((diethylamino)methyl)-2-methoxybenzyl)-N2-(2,4-dimethoxybenzyl)-7H-purine-2,6-diamine (72 mg, 0.128 mmol) was dissolved in trifluoroacetic acid (3 mL) at 0 ^o C. 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 the excess trifluoroacetic acid. The resulting crude mixture was purified by reverse-phase column chromatography on a Gilson preparative HPLC system using ACN/H ₂ O (0.1% TFA) as a mobile phase. This purification process gave N6-butyl-7-(4-((diethylamino) methyl)-2-methoxybenzyl)-7H-purine-2,6-diamine trifluoroacetate (19.2 mg, 36%) as a white solid.

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

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

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.

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

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 give 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.

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

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) was dissolved in trifluoroacetic acid (3 mL) at 0 ^o C. 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 ACN/H ₂ O (0.1% TFA) as a 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.

Table 1 below summarizes the structures and IUPAC names of the compounds of Examples 1 to 8, which were prepared using methods similar to those of the above-mentioned manufacturing examples.

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

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.

Example LCMS or NMR 1 1H-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) 2 1H-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) 3 1H-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) 4 1H-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) 5 1H-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) 6 1H-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) 7 1H-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) 8 LCMC [M+H] 439.3

Experimental example

Activation of TLR7 by the compound of the present invention

Based on the results of the HTLR7 and 8 reporter assays, the most active compound, Example 4 (GM91669), was selected as a representative small-molecule compound for further biological activity evaluation on the in vivo immune response. First, we investigated whether the agonistic activity of Example 4 (GM91669) was also demonstrated at 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. Reporter assay results showed that compound 4 (27b, GM91669) (EC ₅₀ <46 nM) was a TLR7 selective agonist and a more potent agonist than GS-9620 (EC ₅₀ , 5,286 nM) and resiquimod (EC ₅₀ , 312 nM), which are TLR7/8 dual agonists (Fig. 3a). The maximum potency (E _max ) of the Example 4 compound (27b, GM91669) measured at 33 μM 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). In particular, the mTLR7 activity was reduced at the E _max concentration for the Example 4 compound (GM91669) and resiquimod, 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 both compounds did not show mTLR8 agonist effect (Fig. 3b), suggesting that the Example 4 compound can activate not only hTLR7 but also mTLR7 without species specificity.

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.

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).

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 three weeks after a single intranasal vaccine administration, inducing robust systemic and mucosal immunity in the body.

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

Example 4: We investigated whether compound 4 could enhance antigen-specific mucosal and humoral immune responses, thereby protecting vaccinated mice from influenza virus infection. Three weeks after vaccination, mice were intranasally challenged with a lethal dose of the same virus strain, mouse-adapted PR8 (maPR8, 10 × 50% mouse lethal dose [MLD ₅₀ ]), in the absence or presence of compound 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 day 8, and all mice died by day 9 (Figs. 6b and 6c). The group vaccinated with inactivated virus also lost weight over time, and only one of six mice regained weight within 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 virus. In contrast, when inactivated viruses were inoculated with 1 μg of Example 4 compound per mouse, weight loss was significantly alleviated, and the survival rate of maPR8-infected mice was completely restored (survival rate, 100%) (Figs. 6b and 6c). This suggests that Example 4 compound, a TLR7-selective agonist, can provide complete protection against infection with the same virus by enhancing innate mucosal immunity and antigen-mediated humoral immune responses (Figs. 4 and 5).

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

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.

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.

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

To confirm 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 ₅₀ ) of a different subtype of virus, the mouse-adapted HK (maHK) virus (Fig. 8a). Monitoring of body weight changes revealed that all mice vaccinated with the inactivated PR8 virus together with Compound 4 or GS-9620 regained their weight (Fig. 8b). In contrast, the unvaccinated group (Virus only) and the group vaccinated with the non-adjuvanted vaccine (Vaccine) did not regain their weight after HK virus infection. More importantly, mortality analysis showed that 16.7%, 33.3%, and 66.7% of mice survived after vaccination with Compound 4 at doses of 0.01, 0.1, and 1 μg per mouse, respectively (Fig. 8c). Body weight loss was more moderate in the 1 μg GS-9620 adjuvant group compared to the same dose of Example 4 compound group (Figure 8b), but survival rates remained the same at the 14-day post-infection endpoint (Figure 8c). In summary, in vivo infection experiments using heterologous viruses confirmed that Example 4 compound enhanced broadly neutralizing IgG and IgA, extending the vaccine's protective spectrum and inducing mouse protection against other subtypes of influenza A viruses.

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 (11)

A compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof:
Chemical Formula 1

In the above chemical formula,
R ₁ to R ₄ are each independently hydrogen or C ₁ -C ₈ alkyl;
R ₅ and R ₆ are each independently hydrogen or C ₁ -C ₆ alkyl, or R ₅ and R ₆ are combined with each other to form a 5- to 7-membered heterocycloalkyl ring which is unsubstituted or substituted with C ₁ -C ₄ alkyl;
A is an aryl or heteroaryl of a 6- to 10-membered ring which is unsubstituted or substituted with C ₁ -C ₄ alkyl or C ₁ -C ₄ alkoxy;
m and n are each independently integers from 0 to 3.
A compound or a pharmaceutically acceptable salt thereof, characterized in that in claim 1, R ₁ and R ₂ are hydrogen.
A compound or a pharmaceutically acceptable salt thereof, characterized in that in claim 1, R ₃ and R ₄ are hydrogen and C ₁ -C ₆ alkyl, respectively.
A compound or a pharmaceutically acceptable salt thereof _{, characterized} in that in claim 1, R ₅ and R ₆ are each independently C ₁ -C ₄ alkyl, or R ₅ and R ₆ 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 C 1 -C 3 alkyl.
A compound or a pharmaceutically acceptable salt thereof, characterized in that in claim 1, A is an aryl having a 6- to 10-membered ring which is unsubstituted or substituted with C ₁ -C ₄ alkoxy.
A compound or a pharmaceutically acceptable salt thereof, characterized in that in claim 1, m and n are 1.
In claim 1, the compound represented by the chemical formula 1 is a compound represented by the following chemical formula 2, or a pharmaceutically acceptable salt thereof:
Chemical Formula 2

In the above chemical formula,
R ₁ to R ₆ , m and n are as defined in paragraph 1;
R ₇ to R ₁₀ are each independently hydrogen or C ₁ -C ₃ alkoxy.
In claim 1, 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
.
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.
In claim 9, the composition is characterized in that it specifically enhances the activity of TLR7 (Toll-like receptor 7).
A vaccine composition comprising as an active ingredient (a) 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.