Antiviral composition comprising nucleoside derivative

Abstract

The present invention relates to a novel compound exhibiting antiviral activity and, more particularly, to a compound that is based on a nucleoside derivative structure and exhibits a selective and excellent antiviral effect against respiratory syncytial virus (RSV). The compound of the present invention exhibits a low effective concentration (IC50) against RSV while also showing low cytotoxicity, thereby ensuring safety. It has been confirmed that the compound of the present invention exhibits selective inhibitory activity against RSV on the basis of structural specificity. Accordingly, the compound of the present invention can be effectively utilized as a pharmaceutical composition for the prevention or treatment of RSV infection.

Description

Antiviral composition containing a nucleoside derivative

[0001] The present invention relates to nucleoside derivatives, and more particularly to antiviral compounds against Respiratory Syncytial Virus (RSV) based on an 8-aza purine structure. The compound of the present invention is a compound that simultaneously improves stability and biological activity by modifying and introducing an 8-aza purine backbone, exhibits significant antiviral activity against RSV, and shows low cytotoxicity, thus having great potential for use as a therapeutic agent.

[0002] This invention was carried out with support from the "Seoul National University Institute of Pharmacy" research project (Project No.: 1345364311, Performing Organization: Seoul National University Industry-Academic Cooperation Foundation, Research Period: 2022.06.01 ~ 2025.02.28) of the Basic Research Project in Science and Engineering - University Key Research Institute Support Project supported by the Ministry of Education.

[0003] In addition, the present invention was carried out with support from the research project "Development of nucleoside derivatives with broad-spectrum antiviral activity" (Project No.: 1465040280, Performing Organizations: Seoul National University Industry-Academic Cooperation Foundation, Korea Research Institute of Chemical Technology [Joint Research], Research Period: 2023.04.01 ~ 2025.12.31) of the Antiviral Treatment Development Project for RNA Virus Infections (DiseaseX) supported by the Ministry of Health and Welfare.

[0004] Respiratory Syncytial Virus (RSV) and influenza are representative respiratory viruses that cause significant public health problems worldwide. RSV can cause severe bronchitis, pneumonia, and respiratory impairment, while influenza leads to serious respiratory complications through recurring seasonal outbreaks. Currently, ribavirin is approved as a small molecule treatment for RSV, but its use is limited in some countries. Regarding influenza, four drugs—oseltamivir, zanamivir, peramivir, baloxavir, and marboxil—are approved; however, all of these treatments have limitations, such as the emergence of drug-resistant viruses. Therefore, the development of new therapeutic substances is required to address the annually recurring outbreaks of RSV and influenza infections.

[0005] In particular, ribavirin, used as a treatment for RSV, has a delayed onset of action after administration, making immediate treatment difficult; furthermore, side effects such as dyspnea, respiratory problems, and severe fatigue may occur during the administration process. Additionally, there is a risk of side effects including kidney damage and anemia, requiring caution in patients with impaired renal function or blood disorders. Moreover, there is a limitation in that it is difficult to use in pregnant women as administration during pregnancy may be harmful to the fetus. In the case of influenza, while a preventive vaccine exists, its efficacy is only 40–60%, so the need for effective treatments remains significant. Sesltamivir, a representative treatment, is highly effective only when administered within 48 hours of symptom onset and has limitations in that it exhibits resistance against certain virus variants. Additionally, its use is restricted due to reported side effects such as abdominal pain, vomiting, diarrhea, headache, rash, tachycardia, and fatigue. For these reasons, the discovery of broad-spectrum antivirals effective against RNA virus infections, such as RSV and influenza, is essential.

[0006] Meanwhile, nucleoside drugs are a class of drugs that exhibit various antiviral activities due to their structural similarity to natural nucleosides, and they have long been developed for the treatment of viral diseases. In fact, more than 90 types of antiviral agents have been developed since 1963, and about 50% of them are nucleoside analogs. Ribavirin is a representative nucleoside analog known to be active against various RNA viruses, including Hepatitis C virus and RSV. On the other hand, oseltamivir, zanamivir, peramivir, baloxavir, and marboxil, which are approved as treatments for influenza, do not possess a nucleoside structure. Currently, efforts are underway to develop new antiviral agents through the synthesis of novel lead compounds that possess a nucleoside structure.

[0007] Starting from this need, the present invention provides an 8-aza purine-based nucleoside derivative that mimics the mechanism of ribavirin while improving stability and biological activity through structural modification. The compound of the present invention exhibits significant antiviral activity against RSV and can be utilized as a candidate for a novel RSV treatment due to its low cytotoxicity.

[0008] The present invention aims to overcome the limitations of the existing RSV treatment ribavirin, such as delayed onset of effect, various side effects, and usage restrictions, and to provide a new compound that exhibits specific and stable antiviral activity against RSV infection.

[0009] In particular, the present invention provides an effective treatment for RSV by synthesizing an 8-aza purine-based nucleoside derivative, thereby simultaneously securing low cytotoxicity and high antiviral activity. Accordingly, the compound of the present invention complements the limitations of existing drugs and can be utilized as a new candidate substance for the treatment of RSV infection.

[0010] The present invention will be described in detail below. The advantages and features of the present invention and the embodiments described below for achieving them will become clear. However, the present invention is not limited to the embodiments disclosed below but can be implemented in various different forms. These embodiments are provided merely to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention, and the present invention is defined only by the scope of the claims. Throughout the specification, the same reference numerals refer to the same components.

[0011] Unless otherwise defined, all terms used herein (including technical and scientific terms) may be used in a meaning commonly understood by those skilled in the art to which the present invention pertains. Furthermore, terms defined in commonly used dictionaries are not to be interpreted ideally or excessively unless explicitly and specifically defined otherwise. The terms used herein are for describing embodiments and are not intended to limit the present invention. In this specification, the singular form includes the plural form unless specifically stated otherwise in the text.

[0012] To achieve the above objective, the present invention provides an antiviral composition comprising a compound of Formula 1 below or a pharmaceutically acceptable salt thereof.

[0013] [Chemical Formula 1]

[0014]

[0015] In the above chemical formula 1,

[0016] X is carbon or oxygen, and if X is carbon, it forms a saturated bond with hydrogen or fluorine (F), and

[0017] R1 is non-existent or carbon,

[0018] If R1 is absent, R2 is also absent, a condensation ring is not formed, and the adjacent nitrogen atom exists as an amino group (-NH2), and

[0019] If R1 is a carbon, R2 is hydrogen or an amino group (-NH2), and

[0020] R3 has an oxygen or primary to tertiary amino group structure.

[0021] R3 of the above chemical formula 1 may be an N-methylamino group, an N,N-dimethylamino group, a hydroxyamino group, a cyclopropylamino group, a benzylamino group, or a benzoylamino group, but is not limited thereto.

[0022]

[0023] For example, the above chemical formula 1 may be a compound having the structure of the following chemical formula 2.

[0024] [Chemical Formula 2]

[0025]

[0026] In the above chemical formula 1,

[0027] R1 is non-existent or carbon,

[0028] If R1 is absent, R2 is also absent, a condensation ring is not formed, and the adjacent nitrogen atom exists as an amino group (-NH2), and

[0029] If R1 is a carbon, R2 is hydrogen or an amino group (-NH2), and

[0030] R3 has an oxygen or primary to tertiary amino group structure.

[0031]

[0032] In a preferred embodiment of the present invention, the formula 2 comprises compounds of the following formulas 3 to 13.

[0033] [Chemical Formula 3]

[0034] ,

[0035] [Chemical Formula 4]

[0036] ,

[0037] [Chemical Formula 5]

[0038] ,

[0039] [Chemical Formula 6]

[0040] ,

[0041] [Chemical Formula 7]

[0042] ,

[0043] [Chemical Formula 8]

[0044] ,

[0045] [Chemical Formula 9]

[0046] ,

[0047] [Chemical Formula 10]

[0048] ,

[0049] [Chemical Formula 11]

[0050] ,

[0051] [Chemical Formula 12]

[0052] ,

[0053] [Chemical Formula 13]

[0054] .

[0055]

[0056] In a preferred embodiment of the present invention, the formula 1 comprises a compound having the structure of the following formulas 14 and 15.

[0057] [Chemical Formula 14]

[0058] ,

[0059] [Chemical Formula 15]

[0060] .

[0061]

[0062] The present invention also provides a pharmaceutical composition for the prevention, improvement, or treatment of viral diseases comprising the above formula 1 or a pharmaceutically acceptable salt thereof.

[0063] The above composition has an antiviral effect against Respiratory Syncytial Virus. Therefore, the viral disease includes infectious diseases caused by Respiratory Syncytial Virus.

[0064] In the present invention, "antiviral effect" includes inhibition of viral infection, inhibition of viral replication, inhibition of viral infection of host cells, inhibition of viral replication within host cells, and inhibition of host cell functions involved in viral release. In particular, the present invention refers to an inhibitory effect against Respiratory Syncytial Virus (RSV).

[0065] The present invention provides a pharmaceutical composition for the prevention or treatment of RSV infection disease comprising an 8-aza purine-based nucleoside derivative represented by Formula 1 or Formula 2.

[0066] In this specification, the term "prevention" refers to any act of suppressing or delaying the onset of RSV infection by administering the pharmaceutical composition of the present invention. Additionally, "treatment" includes any act of improving symptoms or inhibiting the progression of the disease by administering the pharmaceutical composition of the present invention to a patient with an RSV infection.

[0067] In the present invention, the antiviral composition may be used in combination with a drug already in use, preferably an antiviral agent. The antiviral composition of the present invention may include a pharmaceutically acceptable carrier.

[0068] Carriers used in the composition of the present invention include pharmaceutically acceptable carriers, adjuvants, and vehicles, collectively referred to as "pharmaceutically acceptable carriers." Pharmaceutically acceptable carriers that may be used in the composition of the present invention include, but are not limited to, ion exchange, alumina, aluminum stearate, lecithin, serum proteins (e.g., human serum albumin), buffering materials (e.g., mixtures of various phosphates, glycine, sorbic acid, potassium sorbate, and partial glycerides of saturated vegetable fatty acids), water, salts or electrolytes (e.g., protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, and zinc salts), colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substrates, polyethylene glycol, sodium carboxymethylcellulose, polyarylates, waxes, polyethylene-polyoxypropylene-barrier polymers, polyethylene glycol, and wool paper.

[0069] The composition according to the present invention may be administered orally or parenterally, and parenteral administration may be administered in the form of intranasal, intranasal, oral, intravenous, intramuscular, intra-arterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intradermal, intraperitoneal, intestinal, topical, sublingual, or rectal administration, but is not limited thereto. Preferably, it may be administered orally, intranasally, or intranasally, and particularly when administered intranasally or parenterally, it is more preferable to administer in the form of a spray or aerosol, or by inhalation, but is not limited thereto.

[0070] The above composition for intranasal or intranasal administration is prepared according to techniques well known in the field of pharmaceuticals and can be prepared as a solution in saline using benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons and / or other solubilizing agents or dispersing agents known in the field.

[0071] In another form, the composition according to the present invention may be in the form of a sterile injectable formulation as a sterile aqueous or oily suspension for injection. This suspension may be formulated according to techniques known in the art using a suitable dispersant or wetting agent (e.g., Tween 80) and a suspending agent. The sterile injectable formulation may also be a sterile injectable solution or suspension (e.g., a solution in 1,3-butanediol) in a non-toxic, parenterally acceptable diluent or solvent.

[0072] Vehicles and solvents that may be used permissibly include mannitol, water, Ringer's solution, and isotonic sodium chloride solution. Additionally, sterile nonvolatile oils are typically used as solvents or suspension media. For this purpose, any nonvolatile oil with low irritation, including synthetic mono- or diglycerides, may be used. Fatty acids, such as oleic acid and its glyceride derivatives, are useful in injectable formulations as well as pharmaceutically acceptable natural oils (e.g., olive oil or castor oil), particularly their polyoxyethylated forms.

[0073] The compositions of the present invention may be administered orally in any orally acceptable dosage form, including but not limited to capsules, tablets, and aqueous suspensions and solutions. For oral tablets, commonly used carriers include lactose and corn starch. Lubricants such as magnesium stearate are also typically added.

[0074] Useful diluents for oral administration in capsule form include lactose and dried corn starch. When an aqueous suspension is administered orally, the active ingredient is combined with an emulsifier and a suspending agent. If necessary, sweeteners and / or flavorings and / or colorings may be added.

[0075] The compositions of the present invention may also be administered in the form of suppositories for rectal administration. These compositions may be prepared by mixing the compounds of the present invention with suitable non-irritating excipients that are solid at room temperature but liquid at rectal temperature. Such materials include, but are not limited to, cocoa butter, beeswax, and polyethylene glycol.

[0076] The composition according to the present invention can be formulated as a pill, a coated tablet, a capsule, a liquid, a gel, a syrup, a slurry, or a suspension.

[0077] In a preferred embodiment, a pharmaceutical composition for oral administration may be prepared by mixing an active ingredient with a solid excipient and may be prepared in granular form to be prepared in the form of a tablet or a coated tablet. Suitable excipients may include sugars such as lactose, sucrose, mannitol, and sorbitol; starch from corn, wheat flour, rice, potatoes, or other plants; cellulose such as methylcellulose, hydroxypropylmethylcellulose, or sodium carboxymethylcellulose; carbohydrates such as gums including arabic gum and tagacanthus gum; or protein fillers such as gelatin and collagen. If necessary, disintegrants or solubilizers in the form of their respective salts, such as cross-linked polyvinylpyrrolidone, agar and alginate, or sodium alginate, may be added.

[0078] In addition to the description provided, the pharmaceutical composition of the present invention may be manufactured in various forms using formulation techniques known in the art, and may be prepared as oral tablets, injectables, suspensions, tablets, capsules, syrups, ointments, creams, etc., depending on the purpose of administration. In particular, it is preferable to formulate it in the form of an injectable to achieve a rapid therapeutic effect for RSV infection.

[0079] In this specification, "patient," "subject," and "object" include mammals, and in certain embodiments, the patient is a human. However, in other embodiments, it may be an animal such as a dog, cat, or livestock (e.g., pig, horse, etc.). Accordingly, the pharmaceutical composition of the present invention can be applied to the prevention or treatment of RSV infection in humans as well as various mammals.

[0080] The "therapeutically effective amount" used in this invention refers to an amount sufficient for the prevention or treatment of RSV infection. The effective dose may vary depending on several factors, including the patient's age, weight, gender, degree of RSV infection, and drug activity, and such a dose can be easily determined by a person skilled in the art. The active ingredient of this invention has excellent safety and can be used in doses exceeding the determined dosage.

[0081] The present invention also provides a health functional food composition for the prevention or improvement of viral diseases comprising the above formula 1, formula 2, or a pharmaceutically acceptable salt thereof.

[0082] The health functional food of the present invention can be manufactured, for example, into various food products, candy, chocolate, beverages, gum, tea, vitamin complexes, and health supplements, and can be used in the form of powder, granules, tablets, capsules, or beverages.

[0083] The antiviral composition of the present invention may be added to food or beverages for the purpose of preventing or improving viral infections. In this case, the amount of the extract in the food or beverage may generally be added in an amount of 0.01 to 50% by weight, preferably 0.1 to 20% by weight, of the total weight of the food for the health functional food composition of the present invention, and in an amount of 0.02 to 10 g, preferably 0.3 to 1 g, based on 100 ml for the health beverage composition, but is not limited thereto.

[0084] The health beverage composition of the present invention has no particular limitations on the liquid components other than containing the extract as an essential component in the indicated proportion, and may contain various flavoring agents or natural carbohydrates as additional components, as in conventional beverages. Examples of the natural carbohydrates mentioned above include monosaccharides, disaccharides such as glucose and fructose, polysaccharides such as maltose and sucrose, conventional sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) may be advantageously used. The proportion of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g, per 100 ml of the composition of the present invention.

[0085] The composition of the present invention may contain various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the compositions of the present invention may contain fruit pulp for the production of natural fruit juices, fruit juice beverages, and vegetable beverages. These ingredients may be used independently or in combination. Although the proportion of these additives is not particularly important, it is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

[0086] The present invention also provides a method for preventing or treating RSV infection, comprising the step of administering a compound of Formula 1 or Formula 2, a derivative thereof, or a pharmaceutically acceptable salt.

[0087] The matters mentioned in the pharmaceutical composition, use, prevention, or treatment method of the present invention shall apply equally unless they are inconsistent with one another.

[0088] The 8-aza purine-based nucleoside derivative according to the present invention exhibits specific and excellent antiviral activity against RSV infection.

[0089] The compound of the present invention demonstrated low cytotoxicity in cell experiments while confirming a potent inhibitory effect against RSV, and can be utilized as a new therapeutic candidate capable of overcoming the limitations of existing ribavirin.

[0090] Therefore, the compound of the present invention can be usefully applied as a composition for the prevention or treatment of RSV infection and has the potential to simultaneously ensure clinical safety and efficacy.

[0091] FIGS. 1a and 1b are schematic images illustrating the synthetic scheme of a compound of Formula 17 according to one embodiment of the present invention.

[0092] FIGS. 2a to 2c show the RSV inhibitory activity (IC) of the compounds of the present invention. 50 ) and cytotoxicity (CC 50 Figure 2a shows the analysis results and includes data evaluating the antiviral activity of each compound at different concentrations via ELISA. In Figure 2a, the inhibitory effect on RSV infection at various concentrations was confirmed through ELISA analysis. As a result, compounds of Formula 3 (LJ-5234), Formula 4 (LJ-5274), Formula 5 (LJ-5275), and Formula 6 (LJ-5276) exhibited concentration-dependent RSV inhibitory activity; in particular, the compound of Formula 3 (LJ-5234) showed a distinct inhibitory effect even at low concentrations, demonstrating excellent IC50. 50 The values ​​were shown. In addition, the evaluation of cell viability revealed that each compound exhibited an RSV inhibitory effect without showing significant cytotoxicity, confirming a favorable Selectivity Index (SI). Figure 2b shows the results of LDH (Lactate Dehydrogenase) analysis performed to evaluate the cytotoxicity of the RSV inhibitor candidate substances, Formula 3 (LJ-5234) and Formula 4 (LJ-5274). According to these results, no cell damage was observed even at concentrations of 100 μM or higher, confirming that the two compounds exhibit low cytotoxicity. Figure 2c shows the cytotoxicity (CC) of the RSV inhibitor candidate substances, Formula 3 (LJ-5234) and Formula 4 (LJ-5274). 50 This shows the results of evaluating ). As a result of the experiment, no cytotoxicity was observed in the two compounds even at concentrations of 100 μg / mL or higher, so CC 50 The value was confirmed to be 300 μM or higher, which corresponds to the CC of the comparative ribavirin. 50It can be seen that it indicates relatively superior cell safety, as it is higher than the value.

[0093] Figure 3 shows the RSV inhibitory effect (IC) of the compound of the present invention. 50 This is a diagram showing graphs and data tables comparing ). In the log scale graph, each compound exhibited concentration-dependent RSV inhibitory activity, and among them, the compound of Chemical Formula 3 (LJ-5234) had the lowest IC50. 50 The excellent inhibitory effect was confirmed by the observed values. In the absorbance measurements at a wavelength of 450 nm, Formula 3 (LJ-5234) also showed a distinct inhibitory effect on RSV infection compared to the control group. According to the table, the IC50 of the compounds of Formula 3 (LJ-5234), Formula 4 (LJ-5274), Formula 5 (LJ-5275), and Formula 6 (LJ-5276) 50 The IC50 values ​​were measured as 0.10 μg / mL (0.37 μM), 1.03 μg / mL (3.81 μM), 1.71 μg / mL (6.05 μM), and 0.34 μg / mL (1.19 μM), respectively, and the IC50 of the existing treatment, ribavirin... 50 They all showed superior antiviral activity compared to (2.02 μg / mL, 8.28 μM).

[0094] FIGS. 4a to 4c show the RSV inhibitory activity (IC) of the compounds of the present invention (Chemical Formula 7 (LJ-5327), Chemical Formula 8 (LJ-5328)). 50 ) and cytotoxicity (CC 50This is a diagram showing the analysis results. Figure 4a shows the results of evaluating the RSV infection inhibitory effect of each compound at different concentrations through ELISA plate analysis and confirming concentration-dependent RSV inhibitory activity. According to the results in the table of Figure 4a, compounds Formula 7 (LJ-5327) and Formula 8 (LJ-5328) both showed enhanced RSV inhibitory effects as the concentration increased, and exhibited significant inhibitory activity even when compared to the positive control. Figure 4b shows the results of LDH analysis evaluating the cytotoxicity of RSV inhibitor candidate substances Formula 7 (LJ-5327) and Formula 8 (LJ-5328). Both compounds showed low cytotoxicity, with no cell damage observed even at concentrations of 100 μM or higher, indicating superior safety compared to the comparison group, ribavirin. Figure 4c shows the results of evaluating the cytotoxicity of RSV inhibitor candidate substances Formula 7 (LJ-5327) and Formula 8 (LJ-5328). The safety of the two compounds was confirmed as almost no cytotoxicity was observed even at concentrations of 100 μM or higher, showing similar or superior results compared to the control, ribavirin.

[0095] FIG. 5 shows the RSV inhibitory effect (IC) of the compounds of the present invention (Chemical Formula 7 (LJ-5327), Chemical Formula 8 (LJ-5328), Chemical Formula 9 (LJ-5329)) and the control ribavirin. 50 This is a graph and data table showing the results. In the log scale graph, compounds of Chemical Formula 7 (LJ-5327), Chemical Formula 8 (LJ-5328), and Chemical Formula 9 (LJ-5329) all exhibited concentration-dependent RSV inhibitory activity; in particular, Chemical Formula 7 (LJ-5327) showed a distinct inhibitory effect even at low concentrations. According to the results in the table, the IC50 of compounds of Chemical Formula 7 (LJ-5327), Chemical Formula 8 (LJ-5328), and Chemical Formula 9 (LJ-5329) 50The values ​​were confirmed to be 0.288 μg / mL (1.11 μM), 1.563 μg / mL (5.50 μM), and 8.440 μg / mL (31.58 μM), respectively, which corresponds to the IC50 of ribavirin. 50 These values ​​are all lower compared to (6.253 μg / mL, 25.61 μM). In particular, Chemical Formula 7 (LJ-5327) showed antiviral activity approximately 6 times superior to ribavirin. The compounds of the present invention also showed a distinct RSV inhibitory effect compared to the negative control group in the 450 nm OD graph.

[0096] Figure 6 shows the RSV inhibitory effect (IC) of the compound of the present invention (Chemical Formula 9 (LJ-5358)) and the control ribavirin. 50 This is a diagram showing graphs and data tables comparing ). As a result of the analysis, the compound of Chemical Formula 9 (LJ-5358) potently inhibited RSV replication in a concentration-dependent manner, showing a distinct effect even at low concentrations. Ribavirin showed an RSV inhibitory effect, but had a relatively higher IC50 compared to Chemical Formula 9 (LJ-5358). 50 It represented. According to the table, the IC of Chemical Formula 9 (LJ-5358) 50 The IC50 of ribavirin was 0.4846 μg / mL (1.705 μM). 50 It showed excellent antiviral activity, being significantly lower compared to (11.64 μg / mL, 47.66 μM).

[0097] Figure 7 shows the cytotoxicity (CC) of Formula 9 (LJ-5358). 50 This shows the analysis results. As a result of the LDH assay, almost no cytotoxicity was observed for Chemical Formula 9 (LJ-5358) even at concentrations of 100 μM or higher, and it showed low toxicity at a similar level compared to ribavirin. In the graph, cytotoxicity remained at approximately 0% across the entire concentration range, confirming that Chemical Formula 9 (LJ-5358) possesses RSV inhibitory activity while maintaining high cellular safety.

[0098] Figure 8 shows the antiviral activity of various candidate compounds against RSV (IC 50 This shows the results of evaluating ). According to the graph and table, compounds of Chemical Formula 10 (LJ-5363), Chemical Formula 11 (LJ-5364), Chemical Formula 12 (LJ-5365), and Chemical Formula 14 (LJ-5008) have low IC50. 50 It showed a value indicating relatively strong RSV inhibitory activity. In particular, Chemical Formula 12 (IC 50 = 0.8557 μg / mL, 2.388 μM) and Chemical Formula 14 (IC 50 = 1.351 μg / mL, 5.072 μM) is ribavirin (IC2 50 It showed superior activity compared to = 11.64 μg / mL, 47.66 μM).

[0099] Figure 9 shows the cytotoxicity (CC) of compounds of formula 10 (LJ-5363) and formula 11 (LJ-5364). 50 This shows the results of evaluating ). In the graph, the two compounds induced almost no cytotoxicity within the experimental concentration range, and as presented in the table, CC 50 The values ​​were found to be greater than 100 μg / mL (>320 μM, >330 μM), respectively. This indicates that the two compounds have a minimal effect on cell survival and are therefore highly safe.

[0100] Figure 10 shows the cytotoxicity (CC) of Chemical Formula 12 (LJ-5365). 50 This shows the results of evaluating ). In the graph, the compound of Chemical Formula 12 caused almost no cytotoxicity within the test concentration range, and as presented in the table, CC 50 The values ​​were confirmed to be greater than 100 μg / mL (>280 μM), respectively. This means that the compounds can exhibit antiviral activity within a safe range without affecting cell viability.

[0101] FIG. 11 shows the cytotoxicity (CC) of compounds of formula 14 (LJ-5008) and formula 15 (LJ-5074). 50 This represents the analysis results. In the graph, the two compounds caused almost no cytotoxicity within the test concentration range, and as shown in the table, compound Chemical Formula 14 (LJ-5008) CC 50 For values ​​greater than 100 μg / mL (>370 μM), the compound of chemical formula 15 (LJ-5074) is CC 50 The value is confirmed to be greater than 100 μg / mL (>330 μM). This means that the two compounds act stably without affecting cell viability.

[0102] Figure 12 shows the IC50 evaluating the antiviral activity of compound 13 (LJ-5407) against RSV. 50 This presents the analysis results. As a result of the analysis, compound Chemical Formula 13 (LJ-5407) showed particularly excellent inhibitory activity, and IC 50 It was found to be 2.398 μg / mL (5.725 μM). This is compared to the control, ribavirin (IC2). 50 A value lower than 11.64 μg / mL, 47.66 μM), indicating a stronger RSV inhibitory effect.

[0103] Figure 13 shows the CC evaluating the cytotoxicity of compound 13 (LJ-5407) and the comparative ribavirin. 50 This shows the analysis results. As a result of the analysis, compound Chemical Formula 13 (LJ-5407) is CC 50 The value was found to be 100 μg / mL (>270 μM) or higher, so almost no cytotoxicity was observed within the experimental range. This suggests that the compound of chemical formula 13 (LJ-5407) exhibits an RSV inhibitory effect while maintaining excellent safety for cells.

[0104] Various embodiments are presented below to aid in understanding the invention. The following embodiments are provided merely to facilitate a better understanding of the invention and do not limit the scope of protection of the invention to the following embodiments.

[0105] The present invention relates to a composition for antiviral use against RSV (Respiratory Syncytial Virus) comprising a compound of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient. The compound of the present invention exhibits excellent safety with minimal cytotoxicity or side effects, and has the advantage of being stable even during long-term administration. Therefore, the compound of the present invention can be usefully utilized as a pharmaceutical composition for the prevention or treatment of RSV infection.

[0106]

[0107] The following examples are provided to aid in understanding the invention and do not limit the scope of protection of the invention.

[0108]

[0109] [Experimental Preparation and Preparation Example]

[0110] ingredient

[0111] Cells and viruses

[0112] In the embodiments of the present invention, the antiviral activity was evaluated using the Respiratory Syncytial Virus (RSV) A2 strain. The cell line used was HEp-2 cells (human laryngeal cancer-derived cell line, ATCC CCL-23). ​​The virus was obtained by serial culture in the cell line, and infectivity was quantified in plaque-forming units (PFU). Control drugs may include ribavirin (Sigma-Aldrich), etc.

[0113]

[0114] Cell acquisition and culture

[0115] HEp-2 cells were obtained from ATCC. Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin in an incubator at 37°C with 5% CO2. Cells were subcultured 2 to 3 times per week, and both the control and test groups were maintained under identical conditions. Cell seeding for the RSV infection experiment was performed in 96-well plates at a rate of 3 x 10⁻¹⁰ as one example. 4 The procedure was performed after dispensing at a density of cells / well and allowing attachment for 24 hours.

[0116]

[0117] antiviral compounds

[0118] The antiviral compound is prepared according to the conditions provided in the image of each example. In the following examples, the compound indicated by number (n) in the image is referred to as 'compound no. n', and the compound indicated by chemical formula number (n) is referred to as 'compound of chemical formula n (or compound n)'.

[0119]

[0120] 1) Synthesis of the compound of chemical formula 7

[0121]

[0122] First, D-ribose was used as a starting material and reacted at room temperature for 3 hours in the presence of acetone using sulfuric acid (H2SO) as a catalyst to obtain compound 3 with an acetonide protecting group. Subsequently, compound 3 was reacted with trimethylsilyl chloride (TBSCl), triethylamine (Et3N), and 4-dimethylaminopyridine (DMAP) in the solvent of dichloromethane (CH2Cl2) to introduce a TBS protecting group, and then acetylated in the presence of acetic anhydride (Ac2O) and triethylamine to synthesize compound 5 with a yield of 92%.

[0123] Next, compound 5 was reacted at room temperature for 1 hour under azide reaction conditions [TMSN3, SnCl4, CH2Cl2] to obtain compound 6 in 98% yield. Subsequently, compound 6 was reacted in a DMF / water mixed solvent in the presence of cyanamide and cesium carbonate (Cs2CO3) for 16 hours at room temperature to obtain compound 7 in 91% yield by 1,3-dipolar cycloaddition.

[0124] Finally, compound 7 was reacted with a 3 N hydrochloric acid solution in THF solvent at room temperature for 3 hours to remove the acetonide and TBS protecting groups, thereby producing the compound of formula 7 in 67% yield.

[0125]

[0126] 2) Synthesis of Chemical Formulas 3, 5, 8, and 13

[0127]

[0128] 2-1) Preparation of the compound of Chemical Formula 3

[0129] Compound No. 9 was dissolved in THF, and 3N HCl was added and stirred at room temperature for 3 hours. The reaction mixture was concentrated under reduced pressure, and the concentrated product was suspended in an aqueous solution and neutralized. The product was purified by column chromatography to obtain the compound of Formula 3 (yield 71%).

[0130]

[0131] 2-2) Preparation of the compound of chemical formula 13

[0132] After dissolving the compound of Formula 3 in pyridine, trimethylsilyl chloride (TMSCl) was added and the reaction was carried out at room temperature. Subsequently, benzoyl chloride (BzCl) was added dropwise to continue the reaction. After the reaction was completed, the product obtained by concentrating under reduced pressure was purified by column chromatography to obtain the compound of Formula 13.

[0133]

[0134] 2-3) Preparation of the compound of chemical formula 5

[0135] Compound No. 10 was dissolved in THF, and 3N HCl was added and stirred at room temperature for 3 hours. After concentrating the reaction mixture, the concentrated product was suspended in an aqueous solution and neutralized. After concentrating the product, it was purified by column chromatography to obtain the compound of Formula 5 (yield 71%).

[0136]

[0137] 2-4) Preparation of the compound of chemical formula 8

[0138] Compound No. 10 was dissolved in MeOH, NaOH was added, and the mixture was stirred at 80°C for 16 hours. The reaction mixture was neutralized and concentrated, and the concentrated product was purified to obtain Compound No. 11 (yield 64%).

[0139] Next, compound 11 was dissolved in an 80% aqueous carboxylic acid solution and reacted at room temperature for 16 hours. The reaction mixture was concentrated, and the concentrated product was purified to obtain the compound of formula 8 (yield 73%).

[0140]

[0141] 3) Synthesis of compounds of chemical formulas 4, 6, 9, 10, 11, and 12

[0142]

[0143] 3-1) Preparation of the compound of Chemical Formula 4

[0144] Compound No. 12 was synthesized with a yield of 72% by reacting compound No. 7 with dimethyl oxalate [(CH3)2C(O)OCH(OEt)2] at 130°C for 15 hours. Subsequently, compound No. 12 was reacted with a 3 N hydrochloric acid solution in THF solvent at room temperature for 3 hours to remove the protecting group, thereby producing the compound of Chemical Formula 4 with a yield of 64%.

[0145]

[0146] 3-2) Preparation of compounds of chemical formulas 6, 9, 10, 11, and 12

[0147] Compound 12 was reacted with tetrabutylammonium fluoride (TBAF) in THF solvent at room temperature for 2 hours to obtain Compound 13 with an 87% yield. Subsequently, Compound 13 was reacted with a catalytic amount of 4-dimethylaminopyridine (DMAP) in the presence of acetic anhydride (Ac2O) and triethylamine (Et3N) in dichloromethane (CH2Cl2) solvent at room temperature for 30 minutes to synthesize Compound 14 with a 91% yield. Next, Compound 14 was treated with thionyl chloride (SOCl2) in a DMF / CH2Cl2 mixed solvent at 40°C for 16 hours to prepare Compound 15 with a 77% yield.

[0148] Compound 15 was reacted with RNH2 (general formula) at 80°C in the presence of KOH or Et3N in ethanol (EtOH) solvent to obtain corresponding intermediates (16a-16e), respectively (representative yields: 16a, R=NHOH 72%; 16b, R=NHMe 81%; 16c, R=cyclopropyl 74%; 16d, R=dimethyl 77%; 16e, R=benzyl 69%). Subsequently, each intermediate (16a-16e) was i) treated in an NH3 / MeOH solution at room temperature for 8 hours to remove the acetyl protecting group, and ii) reacted in 80% aqueous formic acid (80% aq. HCOOH) at room temperature for 16 hours to remove the remaining protecting group, thereby obtaining the final product. In this case, depending on the type of R, the final products correspond as follows: Chemical formula 6 for R=NHMe, Chemical formula 9 for R=NHOH, Chemical formula 10 for R=cyclopropyl, Chemical formula 11 for R=dimethyl, and Chemical formula 12 for R=benzyl, respectively. Each of the obtained compounds was purified by conventional methods, and their structures were confirmed by NMR and mass spectrometry.

[0149]

[0150] 4) Synthesis of Chemical Formula 14

[0151] 4-1) Step 1

[0152]

[0153] Compound 16 was obtained by dissolving D-ribose in acetone solvent, adding concentrated sulfuric acid (c-H2SO4) as a catalyst, and reacting at room temperature for 3 hours. Subsequently, Compound 16 was dissolved in anhydrous THF, vinyl magnesium bromide (vinylMgBr) was added dropwise at -78°C, and the mixture was heated to 0°C and reacted for 3 hours to prepare Compound 17, which has vinyl groups introduced. Compound 17 was reacted with NaIO4 in the presence of water and oxidized for 40 minutes while heating from 0°C to room temperature to obtain Compound 18. This was then suspended in THF solvent, and NaH, DMSO, and methyltriphenylphosphonium bromide (CH3PPh3Br) were added, and Compound 19 was synthesized by reacting at 0°C to reflux conditions for 15 hours. Finally, compound 19 was reacted in CH2Cl2 solvent at room temperature for 2 days in the presence of Neolyst M2 catalyst, followed by treatment with PDC (pyridinium dichromate) and oxidation at room temperature for 6 hours to prepare compound 20. The total yield of compound 20 obtained through the above steps was 45%, and its structure was confirmed by NMR and mass spectrometry.

[0154]

[0155] 4-2) Step 2

[0156]

[0157] Compound No. 20 was used as a starting material and reacted for 6 hours while increasing the temperature from -78°C to -20°C in THF solvent in the presence of [(CH3)2CH]2S and (CH3)3COCH3 under conditions containing CuBr·SMe2, sec-BuLi, KOt-Bu, and LiBr to obtain Compound No. 21 with a 70% yield. Subsequently, Compound No. 21 was reduced by reacting it with NaBH4 in methanol solvent at 0°C for 1 hour to produce Compound No. 22 with a 96% yield.

[0158] Next, compound 22 was reacted with Tf2O in the presence of pyridine at 0°C for 1 hour to synthesize compound 23 with a 92% yield. Subsequently, compound 23 was reacted with NaN3 in DMF solvent at 80°C for 16 hours to obtain azide-substituted compound 24 with an 85% yield. Finally, compound 24 was reacted with NCCH2CONH2 and KOH in DMF solvent at 0°C for 16 hours to synthesize compound 25, in which a nucleobase precursor was bound, with an 81% yield. The obtained compound 25 was purified by conventional methods, and its structure was confirmed via NMR and mass spectrometry.

[0159]

[0160] 4-3) Step 3

[0161]

[0162] Compound 25 was reacted with p-toluenesulfochloride (TsCl) in pyridine solvent at room temperature for 24 hours to obtain sulfonylated compound 27 with an 83% yield. Subsequently, compound 27 was reacted with (i) CH3C(O)OCH(OEt)2 at 90°C for 4 hours, followed by (ii) a reaction in an ammonia / methanol (NH3 / MeOH) solution at room temperature for 24 hours to prepare compound 28 with a transformed ring structure with an 86% yield. Finally, compound 28 was treated in a 65% aqueous solution of trifluoroacetic acid (TFA) at room temperature for 24 hours to synthesize the final product, compound of Formula 14, with the protecting group removed, with a 73% yield.

[0163]

[0164] 5) Synthesis of Chemical Formula 15

[0165] 5-1) Step 1

[0166]

[0167] Compound 21 of Example 4-2 was suspended in THF solvent and treated with LiHMDS at -78°C for 1 hour, after which TESCl was added to introduce a silyl protecting group, thereby obtaining Compound 29 (Step a). Subsequently, Compound 29 was reacted with Selectfluor in DMF at 0°C for 16 hours to perform selective fluorination (including intermediate purification if necessary), followed by sequential processing to synthesize Compound 30 (Step b, total yield of two steps 76%). Next, Compound 30 was treated again under conditions of LiHMDS / TESCl, THF, -78°C, and 1h to obtain Compound 31 (OTES retainer) (Step c), and this was converted to Compound 32 by re-fluorinating with Selectfluor / DMF at 0°C and 16h (Step d, total yield of two steps 70%). Next, compound 32 was reduced with NaBH4 / MeOH at 0°C for 1 hour to produce the alcohol form of compound 33 in 74% yield (step e). Compound 33 was treated with Tf2O / pyridine at 0°C for 1 hour to obtain the trilaterated compound 34 in 89% yield (step f), and then compound 34 was subjected to a substitution reaction with NaN3 / DMF at 80°C for 16 hours to synthesize the azide-substituted compound 35 in 75% yield (step g).

[0168]

[0169] 5-2) Step 2

[0170]

[0171] Compound 36 was prepared by suspending Compound 35 in a methanol solvent and carrying out a reduction reaction by stirring at room temperature for 12 hours in a hydrogen (H2) atmosphere in the presence of a Pd / C catalyst (Step a). Subsequently, Compound 36 was reacted with 5-amino-4,6-dichloropyrimidine and DIPEA in an n-BuOH solvent under microwave conditions (180°C, 7 hours) to induce a substitution reaction and obtain Compound 37, with a total yield of 67% from the two-step synthesis (Step b).

[0172] Compound 38 was synthesized with a yield of 72% by a rearrangement reaction in a 35% aqueous solution of NaNO2 and AcOH at room temperature for 4 hours (step c). Subsequently, Compound 38 was reacted at 110°C for 12 hours in the presence of NH3 in t-BuOH solvent to produce Compound 39 with an introduced amino group in a yield of 81% (step d). Finally, Compound 39 was treated with a 65% aqueous solution of TFA at room temperature for 24 hours to remove the protecting group, thereby synthesizing the final product, Compound of Formula 15, in a yield of 68% (step e). The structure of the obtained Formula 15 was purified according to conventional methods and confirmed by NMR and mass spectrometry.

[0173]

[0174] [Comparative Example]

[0175] 1) Synthesis of Chemical Formula 16

[0176] [Chemical Formula 16]

[0177]

[0178] 1-1) Step 1

[0179]

[0180] Compound 4, into which an isopropylidene protective group was introduced, was prepared by reacting D-ribose as a starting material at room temperature in the presence of acetone and concentrated sulfuric acid. Subsequently, Compound 4 was subjected to a methylation reaction with MsCl and pyridine at 0°C for 4 hours, followed by a reaction at room temperature for 12 hours under KOH / H2O conditions to obtain Compound 5, a cyclic ketal compound (step a).

[0181] Compound 6, into which a silyl protecting group is introduced, was synthesized by reacting Compound 5 in a CHCl2 solvent in the presence of TBDPSCl and imidazole, while increasing the temperature from 0°C to room temperature for 12 hours (Step b). Subsequently, Compound 6 was reduced by reacting it in a NaBH4 / THF·MeOH mixed solvent, while increasing the temperature from 0°C to room temperature for 2 hours, to obtain Compound 7 (Step c). Compound 8, a dimethicated compound, was synthesized by reacting Compound 7 in a CH2Cl2 solvent under MsCl / Et3N conditions, while increasing the temperature from 0°C to room temperature for 2 hours (Step d). Later, in the separation pathway from Compound 4, Compound 8 was reacted in a DMF solvent in the presence of Na2S·9H2O at 90°C for 15 hours to obtain Compound 9, into which a sulfur atom is introduced (Step e, total yield 28% from Compound 4). Compound 9 was reacted with mCPBA in CH2Cl2 solvent at -78°C for 45 minutes to prepare sulfur-oxidized Compound 10 with an 82% yield (step f). Finally, Compound 10 was reacted at 110°C for 4 hours in the presence of acetic anhydride (Ac2O) to synthesize acetylated Compound 11 with a 72% yield (step g).

[0182]

[0183] 1-2) step 2

[0184]

[0185] An acetic acid ester precursor having a TBDPSO protecting group was reacted in CH2Cl2 solvent at 0°C in the presence of TMSN3 and SnCl4 to synthesize an azide-substituted TBDPSO-azide compound with a 56% yield. Subsequently, the said compound was reacted with cyanoacetamide in DMF solvent at room temperature in the presence of Cs2CO3 and water to obtain a TBDPSO-base conjugate with a nucleoside backbone with a 76% yield.

[0186] This compound was reacted with pyridine and POCl3 at room temperature for 1 hour to synthesize a TBDPSO-cyano compound with an introduced cyano group in 54% yield. Subsequently, this was (i) treated with CH3C(O)OCH(OEt)2 at 90°C for 4 hours, and then (ii) reacted in an NH3 / MeOH solution at room temperature for 24 hours to prepare a TBDPSO-nucleoside derivative in 72% yield. Finally, the product was reacted with a 3 N HCl solution in THF solvent at room temperature for 3 hours to synthesize the final product, Formula 16, with the protecting group removed, in 60% yield.

[0187]

[0188] 2) Synthesis of Chemical Formula 17

[0189] [Chemical Formula 17]

[0190]

[0191] Chemical formula 17 was synthesized according to the conditions described in Figures 1a and 1b (Chemical formula 17).

[0192]

[0193] [Example 1]

[0194] 1-1. Antiviral effect against RSV

[0195] Antiviral effect against RSV (ELISA-based IC50 calculation)

[0196] 3 x 10 HEp-2 cells per well in a flat-bottom 96-well plate 4Cells were inoculated. The test compound was prepared by serially diluting it twofold. RSV A2 strain was prepared at 300 PFU per well, and ribavirin (200 μg) was included as the reference drug.

[0197] RSV antigens were detected via ELISA after infection and treatment. A 5H6 clone (500-fold dilution) was used as the primary antibody, and anti-mouse IgG-HRP (2000-fold dilution) was used as the secondary antibody; absorbance was measured at 450 nm after the substrate reaction. An inhibition rate-concentration curve was fitted using the obtained absorbance data to determine the concentration at which RSV inhibition reaches 50% (IC10). 50 ) was produced.

[0198]

[0199] Cytotoxicity (CC50) Evaluation (LDH Release Method)

[0200] 1 x 10 HEp-2 cells per well in a 96-well plate 4 Cells were inoculated one day prior to the experiment. Samples were treated in triplicate and incubated at 37°C. The maximum LDH control was prepared by adding 10 μL of 10X lysis buffer (3 wells each). 50 μL of the culture supernatant was transferred to new 96-wells, and 50 μL of the LDH reaction solution was added to each well. The mixture was incubated for 30 minutes under dark conditions at room temperature, after which 50 μL of stop solution was added. Subsequently, the mixture was centrifuged at 1000 rpm for 2 minutes to remove air bubbles, and absorbance was measured at 490 and 680 nm. The obtained toxicity-concentration curve was fitted to determine the concentration at which cell viability decreased by 50% (CC). 50 Decided on ).

[0201]

[0202] Selectivity Index

[0203] The selectivity index (SI) is CC 50 / IC 50 It was defined as.

[0204]

[0205] 1-2. Antiviral effect against influenza virus

[0206] MDCK cells were seeded into 96-well plates (3 x 10 cells per well) and infected with individual influenza viruses in serum-free MEM under conditions of MOI 0.001 at 35°C or 37°C for 1 hour. After washing with phosphate-buffered saline (PBS), cells were treated with each compound (compound of Formula 3 and control (OSV-C (Oseltamivir))) diluted in MEM containing 2 µg / ml of TPCK-trypsin. After incubation for 3 days at the same temperature for viral infection, inhibition of influenza virus-induced CPE was measured by adding 2.5 mg / ml of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT). The dose-response to the compound according to the present invention or the control (reference antiviral compound) was measured by treating mock- or virus-infected cells with serial dilutions of the test compound. CC 50 and EC 50 The values ​​were calculated using GraphPadPrism 6 software (GraphPad, La Jolla, CA).

[0207]

[0208] 1-3. Antiviral effects against dengue virus

[0209] DENV RNA replication efficiency was determined using BHK-21 cells encoding the luciferase-expressing DENV-2 replicon (BHK-D2-Rluc). Briefly, BHK-21 replicon cells were seeded into 96-well plates and incubated with various concentrations of the compound of Formula 3 (or control (Ribavirin)) at 37°C for 24 hours at the indicated time points. After incubation, antiviral activity was measured using the Renilla Luciferase Assay (Promega, Madison, WI, USA) on a microplate luminometer (Tecan, Mannedorf, Switzerland).

[0210]

[0211] 1-4. Antiviral effects on other compounds

[0212] The procedure was carried out in the same manner as in Examples 1-1 to 1-3 above, except that compounds of Formulas 4 to 18 were used instead of Formula 3. Viability was 50% (CC 50 Cytotoxicity was determined by estimating the compound concentration that reduces it by ), and the selectivity index was CC 50 EC 50 It is the ratio of.

[0213]

[0214] [Example 2]

[0215] Experimental results

[0216] 2-1. Confirmation of antiviral effect against RSV

[0217] The antiviral activity of each compound against RSV was analyzed according to the method described in Example 1-1 above, and the results are shown in Table 1 below.

[0218] Compound RSVIC 50 (uM)CC 50(uM) Chemical Formula 30.37 > 300 Chemical Formula 43.81 > 300 Chemical Formula 56.05 > 300 Chemical Formula 61.19 > 300 Chemical Formula 70.35 > 400 Chemical Formula 81.78 > 400 Chemical Formula 90.3 > 350 Chemical Formula 102.98 > 320 Chemical Formula 113.5 > 330 Chemical Formula 120.41 > 320 Chemical Formula 131 > 320 Chemical Formula 140.88 > 100 Chemical Formula 1510.91 > 100 Control Group (Ribavirin) 8.28 > 300 Chemical Formula 16BA Chemical Formula 17 > 100 > 100

[0219] In Table 1 above, BA means 'no effect'.

[0220] According to the results in Table 1 above, compounds of Chemical Formulas 3 to 15 all exhibited significant antiviral activity against RSV. These compounds are IC 50 Values ​​were confirmed in the range of a few μM or less, and CC indicating cytotoxicity 50 All values ​​were observed at concentrations of several hundred μM or higher, confirming excellent selectivity along with low cytotoxicity. In particular, the compounds of Formulas 3, 7, 9, and 12 exhibited very low IC50 levels of 0.3–0.4 μM. 50 The value is shown as the IC50 of ribavirin used as a control. 50 It showed significantly superior antiviral activity compared to the value (8.28 μM) (see Figs. 2a, 3, 4a, 5, 6, 8, and 12).

[0221] On the other hand, no inhibitory activity against RSV was observed for the compounds of Formula 16 and Formula 17, and IC 50 The value was found to be 100 μM or higher, confirming that it was ineffective unlike other compounds of the present invention. Therefore, the compounds of Formula 3 to Formula 15 of the present invention have excellent RSV inhibitory activity and low cytotoxicity, proving to be promising as antiviral candidate substances.

[0222]

[0223] 2-2. Confirmation of antiviral effect against influenza virus

[0224] The antiviral activity of each compound against the influenza virus was analyzed according to the method described in Examples 1-2 above, and the results are shown in Table 2 below.

[0225] Compound InfluenzaIC 50 (uM)CC 50 (uM) Chemical Formula 3>100>100 Chemical Formula 7>100>100 Chemical Formula 8>100>100 Chemical Formula 10>100>100 Chemical Formula 11>100>100 Chemical Formula 12>100>100 Chemical Formula 13>100>100 Chemical Formula 14 12.6 12.6 Chemical Formula 15>100>100 Control Group (Oseltamivir) 0.3>100

[0226] Compounds of Formulas 3, 7, 8, 10 to 15 have low ICs for RSV. 50 They showed distinct antiviral activity by exhibiting specific values, but against the influenza virus, all IC5s 50 The value was confirmed to be 100 μM or higher, indicating no substantial inhibitory activity (see Table 2). These results indicate that the compounds of the present invention exhibit selective antiviral activity against RSV rather than influenza virus.

[0227]

[0228] 2-3. Confirmation of antiviral effect against dengue virus

[0229] The antiviral activity of each compound against the dengue virus was analyzed according to the method described in Examples 1-3 above, and the results are shown in Table 3 below.

[0230] Compound Dengueic 50 (uM)CC 50(uM) Formula 4 > 8.34 Formula 5 > 3.70 < 3.70 Formula 6 > 3.70 < 3.70 Formula 7 > 100 > 100 Formula 10 > 6.39 Formula 11 > 10.59 Formula 12 > 100 > 100 Formula 15 > 100 > 100 Control Group (Ribavirin) 13.7 > 100

[0231] Referring to Table 3, some compounds (e.g., Formula 4, Formula 5, Formula 6, Formula 10, Formula 11, etc.) are IC 50 Although the values ​​were measured in the range of a few μM, the CC of these compounds 50 The values ​​were also at similar levels, so cytotoxicity and antiviral activity could not be distinguished. In other words, the observed inhibitory effect of these compounds can be interpreted as a non-specific inhibition phenomenon caused by cytotoxicity rather than a dengue virus-specific effect.

[0232] Meanwhile, other compounds such as Chemical Formula 7, Chemical Formula 12, and Chemical Formula 15 are all IC 50 The value was confirmed to be 100 μM or higher, so it did not show significant inhibitory activity against dengue virus.

[0233] Therefore, the compounds of the present invention showed high selectivity and potent inhibitory activity against RSV, but no notable antiviral activity against dengue virus was observed. These results support the possibility that the compounds of the present invention can be developed as RSV-specific antiviral agents and suggest that they possess a differential and selective inhibitory mechanism against respiratory viruses such as RSV.

[0234]

[0235] 2-4. Cytotoxicity Evaluation Results

[0236] To evaluate the cellular safety of the compound of the present invention, LDH (Lactate Dehydrogenase) analysis and CC 50Experiments were conducted to calculate the (Cytotoxic Concentration 50) value. The correlation between antiviral effect and cell safety was verified by measuring cytotoxicity within the concentration range where RSV inhibitory activity was confirmed for each compound.

[0237] LDH analysis of compounds of Formula 3 (LJ-5234) and Formula 4 (LJ-5274) showed that both compounds exhibited low cytotoxicity, with no cell damage observed even at concentrations of 100 μM or higher (see Fig. 2b). Furthermore, the CC of Formula 3 (LJ-5234) and Formula 4 (LJ-5274) 50 Analysis results showed that both compounds were CC 50 The value was confirmed to be 100 μg / mL or higher (>300 μM). This corresponds to the CC of ribavirin, the subject of comparison. 50 It can be seen that the compound of the present invention exhibits relatively superior cell safety with a value higher than the value (Fig. 2c).

[0238] LDH analysis of Chemical Formula 7 (LJ-5327) and Chemical Formula 8 (LJ-5328) revealed that for both compounds, almost no cell damage was observed even at concentrations of 100 μM or higher, confirming low cytotoxicity (Fig. 4b). CC of the above Chemical Formula 7 (LJ-5327) and Chemical Formula 8 (LJ-5328) 50 The evaluation results also showed that both compounds were CC 50 The value was found to be 100 μg / mL or higher, confirming high cell safety and almost no toxicity (see Fig. 4c).

[0239] The cytotoxicity of Chemical Formula 9 (LJ-5358) was analyzed, and LDH analysis and CC 50 As a result of the measurements, no cytotoxicity was observed in the compound even at concentrations of 100 μM or higher, and cell viability was maintained stably across the entire concentration range (see Fig. 7).

[0240] CC of Chemical Formula 10 (LJ-5363) and Chemical Formula 11 (LJ-5364)50 As a result of the evaluation, both compounds were CC 50 The value was found to be greater than 100 μg / mL (>320 μM, >330 μM), indicating that the effect on cell viability is negligible (see Fig. 9).

[0241] As a result of the cytotoxicity analysis of Chemical Formula 12 (LJ-5365), the compound caused almost no cell damage within the experimental concentration range, and CC 50 The value was found to be 100 μg / mL or higher (>280 μM), confirming excellent safety (see Fig. 10).

[0242] As a result of the cytotoxicity analysis of Chemical Formula 14 (LJ-5008) and Chemical Formula 15 (LJ-5074), both compounds were CC 50 The values ​​were found to be greater than 100 μg / mL (>370 μM, >330 μM), which means that the two compounds have RSV inhibitory activity without exhibiting cytotoxicity (see Fig. 11).

[0243] As a result of the cytotoxicity analysis of Chemical Formula 13 (LJ-5407), the present compound is CC 50 The value was confirmed to be greater than 100 μg / mL (>270 μM), indicating that despite having RSV inhibitory activity, the toxicity to cells is low (see Fig. 13).

[0244] That is, the compounds represented by Formulas 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, and 15 of the present invention all exhibited antiviral activity against RSV, while showing almost no cytotoxicity within the experimental range. Therefore, the compounds of the present invention are evaluated as having secured not only excellent antiviral activity but also high cellular safety.

[0245]

[0246] Collectively, the compounds represented by Chemical Formulas 3 to 15 exhibited significant antiviral activity against RSV, particularly with low IC50s. 50 A high inhibitory effect was confirmed through the values. On the other hand, although Chemical Formulas 16 and 17 have a structural framework similar to Chemical Formulas 3 to 15, they did not show substantial inhibitory activity against RSV. This result supports the fact that the compounds of the present invention exert a selective inhibitory effect against RSV based on structural specificity.

[0247] Therefore, the compound of the present invention is a novel compound exhibiting antiviral activity specific to RSV, and a pharmaceutical composition containing it as an active ingredient can be a useful means for the prevention or treatment of RSV infection. Furthermore, since the compound of the present invention ensures safety due to its low cytotoxicity, it has the advantage of being stably applicable even in clinical situations requiring long-term administration.

[0248]

[0249] The present invention has been described above with reference to its preferred embodiments. Those skilled in the art will understand that the present invention may be embodied in modified forms without departing from the essential characteristics of the invention. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the invention is defined by the claims, not by the foregoing description, and all variations within the scope of the claims should be interpreted as being included in the invention.

Claims

1. An antiviral composition comprising the following chemical formula 1 or a pharmaceutically acceptable salt thereof: [Chemical Formula 1] In the above chemical formula 1, X is carbon or oxygen, and if X is carbon, it forms a saturated bond with hydrogen or fluorine (F), and R1 is non-existent or carbon, If R1 is absent, R2 is also absent, a condensation ring is not formed, and the adjacent nitrogen atom exists as an amino group (-NH2), and If R1 is a carbon, R2 is hydrogen or an amino group (-NH2), and R3 has an oxygen or primary to tertiary amino group structure.

2. In Paragraph 1, An antiviral composition in which R3 of the above chemical formula 1 is an N-methylamino group, an N,N-dimethylamino group, a hydroxyamino group, a cyclopropylamino group, a benzylamino group, or a benzoylamino group.

3. In Paragraph 1, An antiviral composition, wherein the above chemical formula 1 is a compound having the structure of the following chemical formula 2: [Chemical Formula 2] In the above chemical formula 1, R1 is non-existent or carbon, If R1 is absent, R2 is also absent, a condensation ring is not formed, and the adjacent nitrogen atom exists as an amino group (-NH2), and If R1 is a carbon, R2 is hydrogen or an amino group (-NH2), and R3 has an oxygen or primary to tertiary amino group structure.

4. In Paragraph 3, An antiviral composition wherein the compound of Chemical Formula 2 above is a compound having any one of the structures of Chemical Formulas 3 to 13 below: [Chemical Formula 3] ,, [Chemical Formula 4] ,, [Chemical Formula 5] ,, [Chemical Formula 6] , [Chemical Formula 7] , [Chemical Formula 8] , [Chemical Formula 9] , [Chemical Formula 10] , [Chemical Formula 11] , [Chemical Formula 12] , [Chemical Formula 13] .

5. In Paragraph 2, An antiviral composition wherein the above chemical formula 1 is a compound having the structure of any one of the following chemical formulas 14 and 15: [Chemical Formula 14] , [Chemical Formula 15] .

6. In Paragraph 1, The above composition is an antiviral composition having an antiviral effect against Respiratory Syncytial Virus.

7. A pharmaceutical composition for the prevention or treatment of viral diseases comprising the following chemical formula 1 or a pharmaceutically acceptable salt thereof: [Chemical Formula 1] In the above chemical formula 1, R1 is non-existent or carbon, If R1 is absent, R2 is also absent, a condensation ring is not formed, and the adjacent nitrogen atom exists as an amino group (-NH2), and If R1 is a carbon, R2 is hydrogen or an amino group (-NH2), and R3 has an oxygen or primary to tertiary amino group structure.

8. In Paragraph 7, The above composition is a pharmaceutical composition having an antiviral effect against Respiratory Syncytial Virus.

9. A health functional food composition for the prevention or improvement of viral diseases comprising the following chemical formula 1 or a pharmaceutically acceptable salt thereof: [Chemical Formula 1] In the above chemical formula 1, R1 is non-existent or carbon, If R1 is absent, R2 is also absent, a condensation ring is not formed, and the adjacent nitrogen atom exists as an amino group (-NH2), and If R1 is a carbon, R2 is hydrogen or an amino group (-NH2), and R3 has an oxygen or primary to tertiary amino group structure.

10. In Paragraph 9, The above composition is a health functional food composition having an antiviral effect against Respiratory Syncytial Virus.

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