Compound and pharmaceutical composition comprising the same for treating tuberculosis infection

Abstract

The present invention relates to a compound represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof for treating a tuberculosis infection; and a pharmaceutical composition for treating a tuberculosis infection comprising the same. More specifically, the invention relates to a pharmaceutical composition for treating a tuberculosis infection comprising a compound represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient. [Chemical Formula 1] In the above chemical formula 1, R1 and R2 are independently selected from the group consisting of C3-C9 heteroaryl groups and C6-C12 aryl groups containing nitrogen.

Description

Technology Field

[0001] The present invention relates to a compound for treating tuberculosis-infected diseases and a pharmaceutical composition containing the same. More specifically, it relates to a compound of a novel structure useful for treating tuberculosis-infected diseases and a pharmaceutical composition containing the same as an active ingredient that is effective not only for treating tuberculosis but also for treating granulomas, which are major pathological conditions associated with tuberculosis. Background Technology

[0002] Tuberculosis (TB), which has a long history, remains one of the top ten causes of death. The principle of treatment is a short-term regimen of six months for initial therapy. Generally, a four-drug combination of first-line anti-tuberculosis agents—rifampin (RIF), isoniazid (INH), pyrazinamide (PZA), and ethambutol (EMB)—is administered, followed by a four-month maintenance regimen of three drugs—RIF, INH, and EMB—after two months. Streptomycin (STR) may be used instead of EMB during the initial two months of intensive treatment. In this case, RIF and INH are administered for maintenance therapy.

[0003] For relapsed patients, the principle of retreatment is to use the original medication again, as most patients retain sensitivity to the initial treatment drugs, and the treatment period is extended by 3 months beyond the prescribed initial treatment period. For patients who failed the initial treatment, drug sensitivity testing is performed to exclude all medications used in the initial treatment, and treatment is administered using a combination of at least three, preferably four or more, new, sensitive medications that were not previously used, for a period of at least 18 months in principle.

[0004] The biggest problem with tuberculosis treatment is that there is no specific treatment method when resistance to first-line anti-tuberculosis drugs develops. Although the incidence and mortality of tuberculosis have decreased dramatically due to the development of effective anti-tuberculosis drugs and systematic management, the disease is currently on the rise due to the recent emergence of multidrug-resistant tuberculosis (MDR-TB) and the AIDS epidemic. Recently, drug-resistant tuberculosis, including MDR-TB, has emerged as a serious public health issue. The tuberculosis infection rate in Korea has not decreased in recent years, which is closely related to the fact that the number of drug-resistant tuberculosis patients is not declining. Furthermore, it is known that 50% of drug-resistant tuberculosis patients die. Therefore, a significant portion of deaths from tuberculosis are attributed to drug-resistant tuberculosis. Currently, although several second-line anti-tuberculosis drugs have been developed, none can replace first-line drugs in terms of efficacy. Consequently, the development of new anti-tuberculosis drugs with mechanisms of action different from existing drugs is a very urgent task.

[0005] Meanwhile, granulomas, a major pathological condition associated with tuberculosis (TB), consist of dense aggregates of cellular debris, bacteria, and host immune cells surrounding macrophages infected with Mycobacterium tuberculosis (Mtb). The primary cause of granuloma formation is mycholic acid in the Mtb cell wall, which inhibits phagosome maturation and phagolysosome fusion and promotes inflammatory responses. Granulomas persist in all stages of tuberculosis, including active, latent, and relapsed states; particularly in latent tuberculosis, Mtb enters a quiescent state under nutrient deprivation and hypoxic conditions but can still maintain metabolic activity. Such granulomas are one of the major factors lowering the success rate of tuberculosis treatment. Furthermore, isoniazid, a first-line drug for TB treatment, is ineffective against inactive Mycobacterium tuberculosis within granulomas, which can lead to reduced therapeutic efficacy and the promotion of resistance. Additionally, the delivery of a sub-dose of the drug to bacteria within the granuloma can impair therapeutic effects and induce resistance. Due to these issues, strategies to treat the disease by regulating the host's immune response or physiological pathways, rather than directly targeting the pathogen, are recently gaining attention in tuberculosis treatment from the perspective of host-directed therapy (HDT). The problem to be solved

[0006] One aspect of the present invention is to provide a compound of a new structure useful for treating tuberculosis infections.

[0007] Another aspect of the present invention is to provide a pharmaceutical composition effective not only for tuberculosis but also for the treatment of granulomas, a major pathological condition associated with tuberculosis, by including a compound of a novel structure useful for treating tuberculosis-infected diseases as an active ingredient. means of solving the problem

[0008] According to one aspect of the present invention, a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof is provided for treating tuberculosis-infected diseases.

[0009] According to another aspect of the present invention, a pharmaceutical composition for treating tuberculosis infection is provided, comprising as an active ingredient a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof.

[0010] [Chemical Formula 1]

[0011] In the above Chemical Formula 1, R1 and R2 are nitrogen-containing C3-C9 heteroaryl groups and C6-C 12 It is independently selected from a group composed of aryls. Effects of the invention

[0012] According to the present invention, a novel compound for treating tuberculosis infection is provided, which effectively inhibits inflammatory mediators and also alleviates hypoxic conditions during the infiltration of immune cells within tissues, thereby effectively improving granulomatous lesions. Furthermore, a composition containing the compound of the present invention is expected to be used as a therapeutic agent or adjuvant for the treatment of tuberculosis and the treatment, alleviation, and improvement of granulomatous lesions from the perspective of host-directed therapy (HDT) during tuberculosis treatment. Brief explanation of the drawing

[0013] Figure 1 shows the peaks resulting from the analysis of isoniazid irradiated with 50 kGy of gamma rays using LC-MS. Figure 2 shows the compounds corresponding to each peak identified by the LC-MS analysis results of Figure 1. Figure 3 shows the results of evaluating cell viability after treating with isoniazid (INH) and derivatives (INH-D1 to INH-D5) at different concentrations to confirm cytotoxicity. Figure 4 is a graph showing the production of inflammatory cytokines IL-6 and TNF-α according to the concentrations of INH and INH derivatives (INH-D1 to INH-D5). Figure 5 shows the effect of INH and INH derivatives on the expression of CD80 and CD86 co-stimulatory molecules by TDM stimulation. Figure 6 shows the effects of INH and INH derivatives on the expression of MHC class I and MHC class II induced by TDM stimulation. Figure 7 is a graph showing the inhibitory effect on the production of inflammatory cytokines IL-6 and TNF-α according to the concentrations of INH derivatives (INH-D1 to INH-D10), INH, and Dexa. Figure 8 schematically illustrates the administration and sacrifice plan for an animal experiment using a mouse model to evaluate whether it alleviates pulmonary granuloma lesions. Figure 9 is a graph showing the results of measuring the body weight of mice for 7 days for the lung granuloma induction group (TDM only) and the INH derivative (INH-D1 and INH-D2) administration group. Figure 10 shows a photograph of the lungs extracted after sacrificing mice on day 7 for the lung granuloma induction group (TDM only) and the INH derivative (INH-D1 and INH-D2) administration group (Figure 10 (left)), and a graph showing the results of calculating the lung index (lung weight / body weight x 100) after measuring the weight. Figure 11 shows a photograph (left) of the tissue lesions observed after sacrificing mice and extracting their lungs on day 7 for the lung granuloma induction group (TDM only) and the INH derivative administration group, and a graph (right) showing the area occupied by the granuloma lesions in the lungs. Figure 12 is a graph showing the total number of leukocytes (CD45+ cells) (Figure 12A), whether hypoxic conditions were improved (Figure 12B), the number of interstitial macrophages (Figure 12C), and the number of dendritic cells (Figure 12D) as a result of analyzing the cellular composition of lung tissue using a flow cytometer. Figure 13 shows the results of the analysis of cytokines and chemokines in serum, confirming the cytokine IFN-γ, the chemokine KC which induces neutrophil infiltration, the chemokine MCP-1 which induces macrophage / monocyte infiltration, and RANTES, IL-6, and TNF-α which induce T cell and monocyte infiltration, respectively, on day 3 of pulmonary granuloma induction (Figure 13A) and day 5 of pulmonary granuloma induction (Figure 13B). Figure 14 shows a photograph of the lungs extracted after sacrificing mice on day 7 for the lung granuloma induction group (TDM only) and the INH derivative (INH-D2, INH-D4, INH-D6, and INH-D8) administration group (Figure 14 (left)), and a graph showing the results of calculating the lung index (lung weight / body weight x 100) after measuring the weight. Figure 15 shows a photograph (top) of the tissue lesions observed after sacrificing mice and extracting their lungs on day 7 for the lung granuloma induction group (TDM only) and the INH derivative (INH-D2, INH-D4, INH-D6, and INH-D8) administration group, and a graph (bottom) showing the area occupied by the granuloma lesions in the lungs. Specific details for implementing the invention

[0014] Hereinafter, preferred embodiments of the present invention will be described with reference to the attached drawings. However, embodiments of the present invention may be modified in various other forms, and the scope of the present invention is not limited to the embodiments described below.

[0015] The present invention provides a compound for effectively controlling tuberculosis-infected diseases and a pharmaceutical composition containing the same. In this case, tuberculosis-infected diseases include not only tuberculosis caused by Mycobacterium tuberculosis but also diseases or symptoms such as granulomas caused by or associated with tuberculosis.

[0016] In the present invention, the term "treatment" is understood to mean a pharmaceutical composition that may entail not only treatment but also alleviation and / or improvement effects. In the present invention, "improvement," "alleviation," or "treatment" refers to any act in which the symptoms of the said disease are improved or beneficially altered by the administration of the composition.

[0017] In this specification, 'Cx-Cy' means that the number of carbons constituting the substituents is from x to y. For example, 'C6-C 12 ' means that the number of carbons constituting the substituent is 6 to 12, and the range of 'X to Y' includes all values ​​between X and Y, and also includes X and Y. According to the present invention, a new isoniazid derivative compound is provided, and the new isoniazid derivative compound of the present invention can be used for the treatment of tuberculosis.

[0018] More specifically, the compound of the present invention is a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof for treating tuberculosis infection disease.

[0019] [Chemical Formula 1]

[0020] In the above Chemical Formula 1, R1 and R2 are nitrogen-containing C3-C9 heteroaryl groups and C6-C 12 It is independently selected from a group composed of aryls.

[0021] The salt of the isoniazid derivative compound of the present invention may be in the form of a pharmaceutically acceptable salt, and the term "pharmaceutically acceptable salt" as used herein refers to a formulation of the compound that does not cause severe irritation to the organism to which the compound is administered and does not impair the biological activity and physical properties of the compound. The above pharmaceutical salt includes acid addition salts formed by acids that form non-toxic acid addition salts containing pharmaceutically acceptable anions, such as inorganic acids like hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromide, hydroiodide (iodic acid), perchloric acid, tartaric acid, etc., organic carboxylic acids like succinic acid, oxalic acid, tartaric acid, manderic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid, tartaric acid, formic acid, citric acid, acetic acid, trichloroacetic acid, trifluoroacetic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid, salicylic acid, etc., and sulfonic acids like methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, etc. A compound according to the present invention can be converted into its salt by conventional methods.

[0022] The compound of formula (1) of the present invention can be synthesized by any synthesis process widely known in the art, and the synthesis step is not limited, and can be obtained, for example, by electron beam irradiation.

[0023] In this specification, 'aryl' refers to an organic radical derived from an aromatic hydrocarbon by the removal of a single hydrogen, comprising a single or fused ring system. Examples include, but are not limited to, substituents such as phenyl, naphthyl, biphenyl, anthryl, fluorenyl, phenanthryl, triphenylenyl, pyrenyl, perylenyl, chrysenyl, naphthacenyl, and fluoranthenyl. 'Heteroaryl' refers to an aromatic ring derivative having a heterocycle structure and acting as an aromatic radical in a state where one or more hydrogens of the ring have been removed, wherein the heteroatom may be one or more selected from the group consisting of S, N, O, Si, P, and B. Specific examples include pyridinyl groups, pyrimidinyl groups, pyrazinyl groups, etc.

[0024] For example, in the compound of formula (1) of the present invention, the C3-C9 heteroaryl group containing nitrogen may be selected from pyrrolyl, pyrazolyl, imidazolyl, triazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, and naphthyridinyl.

[0025] For example, in the compound of chemical formula (1) of the present invention, C6-C 12 The aryl group may be selected from the phenyl group, naphthyl group, and biphenylyl group.

[0026] The compound of formula (1) of the present invention may, for example, have R1 selected from the group consisting of pyridyl, pyridazinyl, pyrimidinyl, and pyrazinyl, and R2 selected from the group consisting of phenyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, quinolyl, quinoxalinyl, and naphthyl, and preferably R1 is pyridine, and R2 is selected from the group consisting of pyridine, phenyl, pyrazinyl, quinolyl, quinoxalinyl, and naphthyl.

[0027] For example, in the compound of the above chemical formula (1), if R1 and R2 are each pyridyl, the 2nd or 4th position of R1 pyridine may be bonded to the backbone, and the 3rd or 4th position of R2 pyridine may be bonded to the backbone.

[0028] More specifically, the compound represented by the above chemical formula 1 may be at least one compound selected from the group consisting of compounds of the following chemical formulas D1 to D10 or a pharmaceutically acceptable salt thereof.

[0029]

[0030] [Chemical Formula D1] [Chemical Formula D2] [Chemical Formula D3]

[0031]

[0032] [Chemical Formula D4] [Chemical Formula D5]

[0033]

[0034] [Chemical Formula D6] [Chemical Formula D7]

[0035]

[0036] [Chemical Formula D8] [Chemical Formula D9]

[0037]

[0038] [Chemical Formula D10]

[0039] In the present invention, the above chemical formulas D1 to D10 are each used interchangeably with INH-D1 to INH-D10.

[0040] Furthermore, according to another aspect of the present invention, a pharmaceutical composition for treating tuberculosis infection is provided, comprising as an active ingredient a compound represented by the following chemical formula 1 described above or a pharmaceutically acceptable salt thereof.

[0041] [Chemical Formula 1]

[0042] In the above Chemical Formula 1, R1 and R2 are nitrogen-containing C3-C9 heteroaryl groups and C6-C 12 It is independently selected from a group composed of aryls.

[0043] For the compound of the above chemical formula (1), all the contents described in relation to the above compound apply equally.

[0044] More specifically, the compound represented by the above chemical formula 1 may be at least one compound selected from the group consisting of compounds of the above chemical formulas D1 to D10, or a pharmaceutically acceptable salt thereof.

[0045] At this time, the pharmaceutical composition may contain a compound of formula (1) of the present invention or a salt thereof in an amount of 0.00001% to 90% by weight based on the total weight of the pharmaceutical composition, for example, 0.001% to 50% by weight, preferably 0.01% to 10% by weight. However, the effective amount is not particularly limited thereto.

[0046] In the present invention, the tuberculosis may be of a strain of the genus Mycobacterium, for example, the strain of the genus Mycobacterium may be Mycobacterium tuberculosis erdman, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium microti, Mycobacterium fortuitum,

[0047] It may be one or more strains selected from the group consisting of Mycobacterium avium, Mycobacterium xenopi, Mycobacterium smegmatis, Mycobacterium tuberculosis H37Rv and Mycobacterium tuberculosis K, Mycobacterium kansasii, Mycobacterium marinum, and Mycobacterium chelonae.

[0048] Meanwhile, in the present invention, the tuberculosis may be at least one of extrapulmonary tuberculosis and pulmonary tuberculosis, which are contracted in at least one region among lymph nodes, the gastrointestinal tract, joints, meninges, and the genitourinary system. For example, it may be one or more selected from the group consisting of ocular tuberculosis, cutaneous tuberculosis, renal tuberculosis, lymph node tuberculosis, laryngeal tuberculosis, intestinal tuberculosis, pulmonary tuberculosis, gallbladder tuberculosis, bone tuberculosis, throat tuberculosis, breast tuberculosis, and spinal tuberculosis. In particular, the tuberculosis bacterium infection disease may be at least one selected from the group consisting of ocular tuberculosis, cutaneous tuberculosis, renal tuberculosis, lymph node tuberculosis, laryngeal tuberculosis, intestinal tuberculosis, pulmonary tuberculosis, gallbladder tuberculosis, bone tuberculosis, throat tuberculosis, breast tuberculosis, and spinal tuberculosis.

[0049] More specifically, in the present invention, the pharmaceutical composition for treating tuberculosis-infected diseases may be for treating not only tuberculosis but also related tuberculosis-infected diseases such as tuberculosis-derived inflammation and granulomas, and for example, the tuberculosis-derived inflammation may be at least one selected from the group consisting of chronic inflammation and granulomas.

[0050] More specifically, the isoniazid derivative compound represented by Formula 1 or its salt, which is included as an active ingredient in the pharmaceutical composition of the present invention, may inhibit an inflammatory mediator mediated by TDM (Trehalose-6,6'-dimycolate). In this case, the inflammatory mediator may be at least one of TNF-α and IL-6.

[0051] The above pharmaceutical composition can be formulated into various forms according to the usual method to suit the intended use, such as oral formulations like powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, or sterile injectable solutions, and can be administered orally or through various routes including intravenous, intraperitoneal, subcutaneous, intramuscular, intradural, rectal, and local administration, and preferably is formulated as an injectable preparation administered through a route selected from the group consisting of intravenous, intraperitoneal, subcutaneous, intramuscular, and intradural.

[0052] Examples of suitable carriers, excipients, and diluents that may be included in the above pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, amorphous cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0053] In addition, the above pharmaceutical composition may further include fillers, anticoagulants, lubricants, humectants, fragrances, emulsifiers, preservatives, etc.

[0054] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms can be formulated by mixing at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc., with the above-mentioned therapeutic pharmaceutical composition. In addition, lubricants such as magnesium stearate and talc may also be used in addition to simple excipients. Liquid dosage forms for oral administration include suspensions, liquid formulations, emulsions, syrups, etc., and may include various excipients, such as humectants, sweeteners, flavorings, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin.

[0055] Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. As non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. As bases for injectables, conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, and preservatives may be included.

[0056] The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on the patient's condition and weight, age, degree of disease, drug form, route of administration, and duration, and may be appropriately selected by those skilled in the art.

[0057] However, since the dosage may be increased or decreased depending on the route of administration, severity of the disease, gender, body weight, age, etc., the above dosage does not limit the scope of the present invention in any way.

[0058] The tuberculosis in which the pharmaceutical composition for treating tuberculosis infection of the present invention can exhibit a therapeutic effect may be isoniazid-resistant tuberculosis (INHr-TB).

[0059] The pharmaceutical composition of the present invention may be administered in combination with a known tuberculosis treatment agent; that is, the pharmaceutical composition for treating tuberculosis infection may be used in combination with an anti-tuberculosis agent.

[0060] More specifically, the pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents or treatment methods, and may be administered simultaneously, sequentially, or simultaneously with conventional therapeutic agents. For example, the pharmaceutical composition for treating tuberculosis infection may be used in combination by administration before, after, or in combination with the anti-tuberculosis agent. Furthermore, the pharmaceutical composition of the present invention may be administered as a single or multiple doses. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects, taking all of the above factors into consideration, and this can be easily determined by a person skilled in the art.

[0061] At this time, the above antituberculosis agent as a conventional treatment may be one or more selected from the group consisting of pyrazinamide, isoniazid, streptomycin, para-aminosalicylic acid, neomycin, viomycin, kanamycin, and rifampin.

[0062] According to another aspect of the present invention, a method for treating tuberculosis is provided, comprising the step of administering the pharmaceutical composition of the present invention to a tuberculosis patient.

[0063] The pharmaceutical composition of the present invention can be administered to a tuberculosis patient in a pharmaceutically effective amount.

[0064] In the present invention, "administration" means providing a specific substance to a patient by any appropriate method, and the route of administration of the composition of the present invention may be oral or parenteral through any general route that can reach the target tissue, and preferably orally.

[0065] In the present invention, "patient" refers to a human and all other animals having a disease whose symptoms can be improved by administering the composition of the present invention. The composition according to the present invention may be applied not only to humans (for treatment, suppression, or prevention) but also to other commercially useful animals, for example, animals other than humans.

[0066] In the present invention, "pharmaceuticalally effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the level of the effective amount may be determined according to factors including the type and severity of the patient's disease, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field.

[0067] The tuberculosis treatment composition of the present invention can not only effectively suppress general inflammatory responses, but can also be effectively applied for host-directed therapy to alleviate pulmonary granulomas, particularly during tuberculosis treatment.

[0068] The present invention will be explained in more detail below through specific embodiments. The following embodiments are merely examples to aid in understanding the present invention and do not limit the scope of the present invention.

[0069] Examples

[0070] 1. Irradiation and LC-MS Analysis of Isoniazid

[0071] After dissolving isoniazid in distilled water at a concentration of 5 mg / mL, it was irradiated in a Cobalt-60 gamma irradiation facility (IR-221) at a dose of 10 kGy per hour to achieve a total absorbed dose of 50 kGy. Subsequently, structural changes were confirmed and analyzed using a high-speed liquid chromatography-mass spectrometer (Thermo Ultimate 3000UPLC system, Thermo LTQ-Orbitrap XL). An ACQUITY BEH C18, 1.7 µm, 150 × 2.1 mm column was used, and solvent A was distilled water containing 0.1% formic acid, while solvent B was methanol. The concentration gradient conditions for the analysis were set based on solvent B as follows: 0 min (5%), 1.5 min (5%), 10 min (80%), 10.1 min (100%), 12 min (100%), 12.1 min (5%), and 15 min (5%). The mass spectrometry conditions were set to ESI, positive mode, spray voltage of 4.0 kV, and capillary temperature of 300℃.

[0072] As such, isoniazid irradiated with 50 kGy of gamma rays was analyzed by LC-MS, and five peaks were observed to be generated. As can be seen in Figure 1, the molecular weight of the first peak was 124.04, the second peak was 138.06, the third peak was 123.05, the fourth peak was 243.09, and the fifth peak was 227.09.

[0073] It was confirmed that the molecular weight of each of these compounds is the same as that of isonicotinic acid, isonicozid, isonicotinamide, and N'-(pyridyl-4-carbonyl)-hydrazide, which are the photo-Fenton reaction compounds of isonicozid reported in prior papers, and based on this, it was determined that their structures are the same. Among these, the fourth peak was confirmed to be N'-(pyridyl-4-carbonyl)-hydrazide (hereinafter referred to as 'INH-D1'), and as can be seen in Figure 1, it was confirmed that in addition to isonicozid, isonicotinic acid, isonicotinamide, and one unidentified substance are included.

[0074] Figure 2 shows the compounds corresponding to each peak identified above.

[0075] 2. Synthesis of Isoniazid Derivatives

[0076] Additional isoniazid derivatives having a structure similar to INH-D1, which was identified as a result of radiation treatment of isoniazid in 1. above, were synthesized. Isonicotinic acid and isonicotinamide are substances previously reported as antibiotics, and are known to be ineffective in treating tuberculosis.

[0077] (1) INH-D1 synthesis

[0078] After dissolving isoniazid (137 mg, 1 mmol) in pyrimidine (8 mL), isonicotinoyl chloride hydrochloride (141 mg, 1 mmol) was added to the mixture at 0 °C, and after the addition of all reactants was finished, the mixture was stirred at 60 °C for 12 hours. After the reaction was finished, the temperature of the mixture was cooled to room temperature and washed 5 times with distilled water at 0 °C. After washing, the solid remaining on the filter was collected, the solvent was sufficiently evaporated under reduced pressure, and INH-D1 (80 mg, 33%) was obtained.

[0079]

[0080] [Scheme 1] Synthesis of INH-D1

[0081] (2) INH-D2 synthesis

[0082] Nicotinic hydrazide (480 mg, 3.5 mmol) and K2CO3 (690 mg, 5 mmol) were sufficiently dissolved in DMF (10 mL), and the solution was cooled to 0°C. Then, isonicotinoyl chloride hydrochloride (890 mg, 5 mmol) was added, and the mixture was stirred at 70°C for 12 hours. After the reaction was complete, the mixture was cooled to room temperature and washed 5 times with distilled water at 0°C. After washing, the solid remaining on the filter was collected, the solvent was sufficiently removed under reduced pressure, and INH-D2 (611 mg, 72%) was obtained.

[0083]

[0084] [Scheme 2] Synthesis of INH-D2

[0085] (3) INH-D3 synthesis

[0086] Isoniazid (342 mg, 2.5 mmol) and K2CO3 (553 mg, 4 mmol) were sufficiently dissolved in DMF (10 mL), and the temperature of the solution was cooled to 0 °C. Then, 2-pyridine carbonyl-chloride hydrochloride (553 mg, 3 mmol) was added, and the mixture was stirred at 50 °C for 12 hours. After the reaction was complete, the temperature of the mixture was cooled to room temperature, and it was washed 5 times with distilled water at 0 °C. After washing, the solid remaining on the filter was collected, the solvent was sufficiently evaporated under reduced pressure, and INH-3 (251 mg, 41%) was obtained.

[0087]

[0088] [Scheme 3] Synthesis of INH-D3

[0089] (4) INH-D4 synthesis

[0090] Nicotinic hydrazide (480 mg, 3.5 mmol) and K2CO3 (690 mg, 5 mmol) were sufficiently dissolved in DMF (10 mL), and the temperature of the solution was cooled to 0 °C. Then, 2-pyridine carbonyl-chloride hydrochloride (890 mg, 5 mmol) was added and stirred at room temperature for 12 hours. After the reaction was complete, the mixture was washed 5 times with distilled water at 0 °C. After washing, the solid remaining on the filter was collected, the solvent was sufficiently evaporated under reduced pressure, and INH-4 (500 mg, 59%) was obtained.

[0091]

[0092] [Scheme 4] Synthesis of INH-D4

[0093] (5) INH-D5 synthesis

[0094] Isoniazid (823 mg, 6 mmol) was dissolved in tetrahydrofuran (5 mL), triethylamine (1 mL, 12.3 mmol) was added, and the solution was cooled to 0 °C. Then, benzoyl chloride (348 μL, 3 mmol) dissolved in 1 mL of tetrahydrofuran was added, and the mixture was stirred at 70 °C for 12 hours. After the reaction was complete, the mixture was washed 5 times with distilled water at 0 °C. After washing, the solid remaining on the filter was collected, the solvent was sufficiently evaporated under reduced pressure, and INH-5 (360 mg, 25%) was obtained.

[0095]

[0096] [Scheme 5] Synthesis of INH-D5

[0097] (6) INH-D6 synthesis

[0098] Pyrazinoic acid (148.92 mg, 1.20 mmol) was dissolved in a mixed solvent of tetrahydrofuran (THF, 7 mL) and dichloromethane (DCM, 7 mL), and then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl, 230.04 mg, 1.20 mmol) was added. Subsequently, isoniazid (137.14 mg, 1.00 mmol) and 4-dimethylaminopyridine (DMAP, 36.65 mg, 0.30 mmol) dissolved in THF (1 mL) and DCM (1 mL) were added to the reaction mixture, and the mixture was stirred at room temperature for 72 hours. After the reaction was complete, the mixture was purified using MPLC (DCM / MeOH) to obtain INH-6 (88.3 mg, 36%).

[0099]

[0100] [Scheme 6] Synthesis of INH-D6

[0101] (7) INH-D7 synthesis

[0102] Quinaldic acid (207.80 mg, 1.20 mmol) was dissolved in a mixed solvent of tetrahydrofuran (THF, 5 mL) and dichloromethane (DCM, 5 mL), and then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl, 230.04 mg, 1.20 mmol) was added. Subsequently, isoniazid (137.14 mg, 1.00 mmol) and 4-dimethylaminopyridine (DMAP, 36.65 mg, 0.30 mmol) dissolved in THF (1 mL) and DCM (1 mL) were added to the reaction mixture, and the mixture was stirred at room temperature for 72 hours. After the reaction was complete, the mixture was purified using MPLC (DCM / MeOH) to obtain INH-7 (227 mg, 78%).

[0103]

[0104] [Scheme 7] Synthesis of INH-D7

[0105] (8) INH-D8 synthesis

[0106] 2-Quinoxaline carboxylic acid (174.04 mg, 1.00 mmol) was dissolved in a mixed solvent of tetrahydrofuran (THF, 8 mL) and dichloromethane (DCM, 8 mL), and then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl, 230.04 mg, 1.20 mmol) was added. Subsequently, isoniazid (164.57 mg, 1.20 mmol) and 4-dimethylaminopyridine (DMAP, 36.65 mg, 0.30 mmol) dissolved in THF (2 mL) and DCM (2 mL) were added to the reaction mixture, and the mixture was stirred at room temperature for 72 hours. After the reaction was completed, the mixture was purified using MPLC (DCM / MeOH) to obtain INH-8 (190 mg, 65%).

[0107]

[0108] [Scheme 8] Synthesis of INH-D8

[0109] (9) INH-D9 synthesis

[0110] 6-quinoline carboxylic acid (519.14 mg, 3.00 mmol) was dissolved in a mixed solvent of tetrahydrofuran (THF, 8 mL) and dichloromethane (DCM, 8 mL), and then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl, 766.8 mg, 4.00 mmol) was added. Subsequently, isoniazid (548.56 mg, 4.00 mmol) and 4-dimethylaminopyridine (DMAP, 122.17 mg, 1.00 mmol) dissolved in THF (2 mL) and DCM (2 mL) were added to the reaction mixture, and the mixture was stirred at room temperature for 72 hours. After the reaction was completed, the mixture was purified using MPLC (DCM / MeOH) to obtain INH-9 (55 mg, 6%).

[0111]

[0112] [Scheme 9] Synthesis of INH-D9

[0113] (10) INH-D10 synthesis

[0114] 2-Naphthoic acid (516.54 mg, 3.00 mmol) was dissolved in a mixed solvent of tetrahydrofuran (THF, 8 mL) and dichloromethane (DCM, 8 mL), and then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl, 766.8 mg, 4.00 mmol) was added. Subsequently, isoniazid (548.56 mg, 4.00 mmol) and 4-dimethylaminopyridine (DMAP, 122.17 mg, 1.00 mmol) dissolved in THF (2 mL) and DCM (2 mL) were added to the reaction mixture, and the mixture was stirred at room temperature for 72 hours. After the reaction was complete, the mixture was purified using MPLC (DCM / MeOH) to obtain INH-10 (392.3 mg, 45%).

[0115]

[0116] [Scheme 10] Synthesis of INH-D10

[0117] 3. Confirmation of the structure of INH-D1 to INH-D10

[0118] To confirm the structure of each of the synthesized INH-D1 to INH-D10 compounds, nuclear magnetic resonance spectroscopy (NMR) analysis was performed after synthesis to confirm the structure.

[0119] As a result, for each compound, the following 1 H NMR and 13 We were able to verify the C NMR data.

[0120] Analytical data for INH-1: 1H NMR (500 MHz, DMSO-d6) δ 10.99 (s, 2H), 8.81 (d, J=5.5 Hz, 4H), 7.84 (d, J=5.6 Hz, 4H); 13 C NMR (125 MHz, DMSO-d6) δ 164.3, 150.5, 139.3, 121.4.

[0121] Analytical data for INH-2: 1 H NMR (500 MHz, DMSO-d6) δ 10.90 (s, 2H), 8.81 (m, 3H), 8.27 (d, J=7.62 Hz, 1H), 7.84 (d, J=4.61 Hz, 2H), 7.58 (dd, J=4.98, 2.15 Hz, 1H); 13 C NMR (125 MHz, DMSO-d6) δ 164.9, 164.8, 153.1, 151.0, 148.9, 139.9, 165.7, 128.5, 124.2, 121.8.

[0122] Analytical data for INH-3: 1 H NMR (500 MHz, DMSO-d6) δ 10.86 (s, 1H), 10.77 (s, 1H), 8.79 (dd, J= 6.05, 1.65 Hz, 2H), 8.72-8.71 (m, 1H), 8.08-8.03 (m, 2H), 7.82 (dd, J=6.05, 1.6 Hz, 2H), 7.69-7.66 (m, 1H); 13 C NMR (125 MHz, DMSO-d6) δ 163.9, 163.2, 150.4, 149.1, 148.7, 139.5, 137.9, 127.1, 122.4, 121.3.

[0123] Analytical data for INH-4: 1H NMR (500 MHz, DMSO-d6) δ 10.77 (br s, 1H), 10.73 (br s, 1H), 9.07 (d, J = 1.6, 1H), 8.78 (m, 1H), 8.72 (d, J = 4.55, 1H), 8.27-8.25 (m, 1H), 8.08-8.03 (m, 2H), 7.69-7.66 (m, 1H), 7.59-7.56 (m, 1H); 13 C NMR (500 MHz, DMSO-d6) δ 164.1, 163.3, 152.5, 149.1, 148.7, 148.5, 137.9, 135.3, 128.2, 127.1, 123.7, 122.5.

[0124] Analytical data for INH-5: 1 H NMR (500 MHz, DMSO-d6) δ 10.73 (br s, 2H), 8.80 (dd, J = 4.25, 1.15 Hz, 2H), 7.93 (m, 2H), 7.83 (dd, J=4.3, 1.15 Hz, 2H), 7.61 (m, 1H), 7.53 (m, 2H); 13 C NMR (500 MHz, DMSO-d6) δ 165.8, 164.4, 150.6, 150.5, 137.5, 132.4, 132.0, 128.6, 128.5, 127.5, 121.4.

[0125] Analytical data for INH-6: 1 H NMR (500 MHz, DMSO-d6) δ 10.95 (d, J = 24.45, 2H), 9.22 (d, J=1.45, 1H), 8.95 (d, J=2.5, 1H), 8.81-8.82 (m, 1H), 8.80 (m, 2H), 7.82-7.83 (m, 2H); 13 C NMR (500 MHz, DMSO-d6) δ 163.99, 162.30, 150.50, 148.14, 144.07, 143.77, 143.69, 139.41, 121.34.

[0126] Analytical data for INH-7: 1 H NMR (500 MHz, DMSO-d6) δ 10.96 (s, 2H), 8.81-8.82 (m, 2H), 8.62 (d, J=8.3 Hz, 1H), 8.16 (t, J=15.5 Hz, 1H), 8.11 (d, J=0.8 Hz, 1H), 7.90-7.93 (m, 1H), 7.85-7.86 (m, 2H), 7.75-7.78 (m, 1H); 13 C NMR (500 MHz, DMSO-d6) δ 164.11, 163.44, 150.51, 149.17, 146.09, 139.54, 138.04, 130.75, 129.31, 129.00, 128.44, 128.20, 121.39, 118.87.

[0127] Analytical data for INH-8: 1 H NMR (500 MHz, DMSO-d6) δ 11.10 (d, J=58.9, 2H), 9.49 (s, 1H), 8.81-8.82 (m, 2H), 8.22-8.26 (m, 2H), 8.00-8.06 (m, 2H), 7.85-7.86 (m, 2H); 13 C NMR (500 MHz, DMSO- d 6)δ 164.14, 162.57, 150.53, 143.56, 143.52, 143.16, 139.84, 139.42, 132.35, 131.56, 129.58, 129.20, 121.39.

[0128] Analytical data for INH-9: 1H NMR (500 MHz, DMSO-d6) δ 10.95 (s, 1H), 9.02 (s, 1H), 8.82 (d, J=3.6, 2H), 8.65 (d, J=14.15 , 1H), 8.53 (d, J=7.45, 1H), 8.25 (d, J=8.5, 1H), 8.16 (d, J=8.45, 1H), 7.87 (d, J=3.65, 2H), 7.63 (d, J=3.6, 1H);13 1C NMR (500 MHz, DMSO- d 6)δ 165.46, 164.42, 152.48, 150.55, 148.92, 139.52, 137.30, 130.21, 129.41, 128.68, 127.61, 127.17, 122.41, 121.40.

[0129] Analytical data for INH-10: 1 H NMR (500 MHz, DMSO-d6): δ 10.84 (s, 2H), 8.82-8.80 (m, 2H), 8.56 (s, 1H), 8.09-8.06 (m, 2H), 8.02 (d, J=8, 1H), 8.00-7.98 (m, 1H), 7.85 (m, 2H), 7.65(m, 2H); 13 1C NMR (500 MHz, DMSO- d 6)δ 165.84, 164.46, 150.52, 139.55, 134.45, 132.08, 129.69, 129.00, 128.23, 128.12, 128.02, 127.71, 126.97, 123.99, 121.35.

[0130] As a result, the synthesized INH-D1 to INH-D10 compounds were each identified as the following compounds.

[0131] INH-D1: N -Isonicotinoyl-Isonicotinohydrazide ( N '-isonicotinoyl-isonicotinohydrazide)

[0132] INH-D2: N '-Isonicotinoyl-nicotinohydrazide ( N '-isonicotinoyl-nicotinohydrazide)

[0133] INH-D3: N '-Isonicotinoyl-Piccolinohydrazide ( N '-isonicotinoyl-picolinohydrazide

[0134] INH-D4: N '-Nicotinoyl-Piccolinohydrazide ( N '-nicotinoyl-picolinohydrazide)

[0135] INH-D5: N -benzoyl-isonicotinohydrazide ( N '-benzoy-lisonicotinohydrazide)

[0136] INH-D6: N'-isonicotinoylpyrazine-2-carbohydrazide

[0137] INH-D7: N'-isonicotinoylquinoxaline-2-carbohydrazide

[0138] INH-D8: N'-isonicotinoylquinoline-3-carbohydrazide

[0139] INH-D9: N'-isonicotinoylquinoline-6-carbohydrazide

[0140] INH-D10: N'-isonicotinoyl-2-naphthohydrazide

[0141] 4. Evaluation of Cytotoxicity of Isoniazid Derivatives

[0142] Mouse macrophage cell line (RAW264.7) was cultured in high-glucose DMEM (Dulbecco's modified Eagle's medium; WelGene) medium with 10% FBS (fetal bovine serum, Gibco BRL) and 100 U / mL penicillin / streptomycin (Invitrogen) in an incubator containing 37°C and 5% CO2.

[0143] To evaluate cytotoxicity, cells were treated with isoniazid (INH) and derivatives (INH-D1 to INH-D5) at different concentrations, and after 24 hours, cell viability was evaluated by staining with 0.5 mg / mL of MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) reagent.

[0144] As a result of cell viability experiments, INH, INH-D1, and INH-3 showed no cytotoxicity at concentrations of 200 μM or lower, while INH-D2, INH-4, and INH-D5 showed no cytotoxicity up to a concentration of 400 μM (Fig. 3).

[0145] Based on this, it was confirmed that when each derivative was treated with TDM (Trehalose-6'6-dimycolate) derived from Mycobacterium tuberculosis, there was no decrease in cell viability for both INH and INH derivatives at concentrations of 200 μM or less.

[0146] 5. Evaluation of the Inhibitory Efficacy of Isoniazid Derivatives on Pulmonary Granuloma-Related Inflammatory Factors (In-vitro)

[0147] The efficacy of inhibiting inflammatory mediators associated with the formation of lung granulomas was evaluated using an ELISA assay to measure cytokine content and surface molecule expression analysis using a flow cytometer.

[0148] (1) Measurement of cytokines according to the concentration of INH and INH-D1 to INH-D5 derivatives

[0149] 50 μg of TDM (Trehalose-6'6-dimycolate) derived from Mycobacterium tuberculosis was dissolved in 1 mL of a mixture of hexane and ethanol (volume ratio 9:1), dispensed into a 48-well plate at a concentration of 1 μg per well, and coated by evaporating the organic solvent in a clean bench. RAW264.7 cells were placed in the TDM-coated 48-well plate at a concentration of 1 × 10⁶ 5 Inoculated at a cell / well density and cultured for 2 hours.

[0150] Subsequently, INH, INH-D1, INH-D2, INH-3, INH-4, and INH-5 were treated at concentrations of 50, 100, and 200 μM, respectively. Cells were cultured for an additional 18 hours at 37°C in a 5% CO2 environment. After culture, the supernatant was extracted, and the concentrations of TNF-α (Cat.555268), IL-6 (Cat.555540), and IL-12p70 (Cat.555256) cytokines were measured using an ELISA assay kit. All subsequent procedures were performed according to the instructions of the manufacturer, BD Biosciences.

[0151] TNF-α plays a crucial role in the formation and maintenance of pulmonary granulomas, and contributes to the isolation and elimination of Mycobacterium tuberculosis by recruiting and activating immune cells. IL-6 amplifies the inflammatory response and activates immune cells to strengthen the immune response against Mycobacterium tuberculosis. However, this amplification of the inflammatory response leads to tissue damage and functional impairment; in particular, the formation of pulmonary granulomas causes Mycobacterium tuberculosis to enter a dormant state, which leads to resistance and recurrent tuberculosis.

[0152] It was confirmed that the compound of the present invention reduces the production of inflammatory cytokines in a concentration-dependent manner at all concentrations (50, 100, 200 μM).

[0153] Compared to INH at the same concentration, INH-D1 to INH-D5 showed superior IL-6 and TNF-α inhibitory efficacy compared to INH (Fig. 4).

[0154] (2) Measurement of cytokines according to the concentration of INH and INH-D1 to INH-D10 derivatives

[0155] 50 μg of TDM (Trehalose-6'6-dimycolate) derived from Mycobacterium tuberculosis was dissolved in 1 mL of a mixture of hexane and ethanol (volume ratio 9:1), dispensed into a 48-well plate at a concentration of 1 μg per well, and coated by evaporating the organic solvent in a clean bench. RAW264.7 cells were placed in the TDM-coated 48-well plate at a concentration of 1 × 10⁶ 5 Cells were inoculated at a cell / well density and cultured for 2 hours. Afterward, INH derivatives (INH-D1 to INH-D10), INH, and Dexa were treated at various concentrations, and cultured for 18 hours in a 37°C, 5% CO2 environment. After 18 hours of culture, the cell supernatant was collected, and the cytokine levels in the supernatant were measured using an ELISA kit, and the results are shown in Figure 7.

[0156] (3) Analysis of surface molecules

[0157] RAW264.7 cells were seeded into TDM-coated 48-well plates at a density of 1×10^5 cells / well and cultured for 2 hours. Subsequently, INH, INH-D1, INH-D2, INH-3, INH-4, and INH-5 were treated at concentrations of 50, 100, and 200 μM, respectively. The cells were cultured for an additional 18 hours at 37°C in a 5% CO2 environment. After culture, the cells were harvested (125 xg, 5 min) and washed with PBS. The cells were then cultured with surface molecular antibodies in the dark for 15 minutes. The antibodies used were FITC-labeled anti-CD80 (Cat.561954, BD Biosciences), PE-labeled anti-CD86 (Cat.553692, BD Biosciences), APC-labeled anti-MHC-I (Cat.17-5958-82, Invitrogen, Waltham, MA, USA), PE-Cyanine7 (Cy7)-labeled anti-MHC-II (Cat.25-5321-82, Invitrogen), and BV510-Live / Dead (Cat.L34966, Invitrogen). After washing with PBS, cells were analyzed. Cell analysis was performed using a MACSQuant VYB flow cytometer.

[0158] CD80 and CD86 are co-stimulatory molecules expressed on the surface of antigen-presenting cells (APCs) that play a role in regulating and amplifying inflammatory responses by inducing the full activation of T cells. MHC-I presents intracellular antigens to CD8+ T cells (cytotoxic T cells), enabling them to recognize and eliminate infected or damaged cells; this enhances the cytotoxic response at the site of inflammation, contributing to the rapid elimination of infectious agents. MHC-II is primarily expressed on antigen-presenting cells and regulates and amplifies inflammatory responses by presenting extracellular antigens to CD4+ T cells (helper T cells).

[0159] The effects of isoniazid derivatives on the expression of co-stimulatory molecules and MHC induced by TDM stimulation were evaluated. Significant inhibitory effects were observed on CD80 and CD86 in the INH-D2 200 μM treatment group (Fig. 5). For MHC-I, INH was inhibited at 100 μM and 200 μM treatments, INH-D1 was significantly reduced at a concentration of 200 μM, and INH-D2 was significantly reduced at all concentrations. For MHC-II, INH showed no reducing effect, while INH-D1 was reduced at 100 μM and INH-D2 was reduced at 50 μM and 200 μM (Fig. 6).

[0160] 6. Evaluation of efficacy in alleviating pulmonary granuloma in a mouse model

[0161] Experiments using a mouse model were conducted to evaluate whether isoniazid derivatives have an inhibitory effect on TDM-induced inflammatory mediators and alleviate actual pulmonary granuloma lesions. The administration and sacrifice plan for the animal experiment is shown in Figure 8.

[0162] 1 mg of TDM (Cord factor, GB61684, GLPBIO) was mixed with 90 μL of mineral oil (cat. 330779, Sigma-Aldrich Co., St. Louis, MO, USA) and then homogenized with 910 μL of PBS containing Tween-80. 100 μL of the emulsion containing 100 μg of TDM was injected into the tail vein of 9-week-old mice (100 μg / mouse). An emulsion solution without TDM was injected into the control group. INH (25 mg / kg), INH-D1 (5 and 25 mg / kg), and INH-D2 (5, 25 mg / kg) were administered orally daily for 5 days, starting 1 day after the intravenous injection of TDM. As a positive control, 0.5 mg / kg of dexamethasone was administered orally on days 1, 3, and 5. Mice were euthanized after 7 days.

[0163] (1) Analysis of lung tissue pathology

[0164] The left lung lobe was fixed in 10% formalin for histopathological examination. Lung tissue was dehydrated, progressively immersed in alcohol and xylene, and embedded in paraffin. Deparaffinized tissue sections (5 μm) stained with hematoxylin and eosin (H&E, #ab245880) at Abcam (Cambridge, UK) were used. Histopathological analysis of lung inflammation was performed using Motic Digital Slide Assistant software version 1.0.7.44 (Kowloon Bay, Kowloon, Hong Kong) (PMID: 31734231). High-resolution images of H&E-stained slides were scanned. Inflammation was measured in two stages using ImageJ software version 1.52 (NIH, Bethesda, MD; https: / / imagej.nih.gov / ij). The maximum threshold for all analyzed digital transparency in the lung region was set to 200. The minimum and maximum values ​​of hue, saturation, and brightness of the images were set to 120, 255; 0, 255; 0, 255, respectively. The total cell area was determined by modifying the procedure described in the online ImageJ stained section examples directory (NIH, https: / / imagej.nih.gov / ij / docs / examples). Pulmonary inflammation was calculated using the ratio of the total area occupied by the cell area.

[0165] As a result of measuring the body weight of mice for 7 days, significant body weight recovery was observed in the INH-D2 25 mg / kg administration group compared to the pulmonary granuloma induction group (TDM only) (Fig. 9).

[0166] On day 7, mice were sacrificed, their lungs were extracted and weighed, and the lung index (lung weight / body weight x 100) was calculated. As a result, there was no significant difference between the TDM only group and the INH 25 mg / kg group. However, the lung index decreased in the INH-D1 25 mg / kg and INH-D2 25 mg / kg groups administered high concentrations of derivatives. The lung index also decreased significantly in the positive control group administered dexamethasone. Figure 10(a) shows a photograph of the extracted lung, and Figure 10(b) shows a graph of the lung index.

[0167] Furthermore, histopathological analysis revealed clear granulomatous lesions in the TDM-only group compared to the vehicle group, and computer analysis of the tissue lesions showed a significant increase in the area occupied by the lesions in the lungs. This alleviation of the lesions was observed in the INH-D2 25 mg / kg administration group and the positive control group administered dexamethasone. Figure 11(a) shows a photograph of the tissue lesions, and Figure 11(b) shows a graph of the area occupied by the granulomatous lesions in the lungs.

[0168] (2) Analysis of lung immune cells

[0169] Pimonidazole was prepared at a concentration of 6 mg / ml and administered via intraperitoneal injection 90 minutes prior to euthanasia of the mice. After euthanizing the mice, the lungs were collected and weighed. The lungs were cut using scissors on a clean workbench, and each lung was immersed in 0.5 mL of digestion buffer. The digestion buffer consisted of RPMI1640 medium containing 1 mg / mL of collagenase IV (Cat.C1639, Sigma-Aldrich Co.) and 0.1 mg / mL of DNase I (Cat.10104159001, Sigma-Aldrich Co.). After incubation at 37°C for 30 minutes, the samples were passed through a 40 μm mesh. Cells were obtained by centrifugation at 1800 rpm for 3 minutes and lysed in erythrocyte lysis buffer (Cat.R7757, Sigma-Aldrich Co.). The cells were neutralized by adding RPMI (containing 10% FBS and 1% penicillin) and passed through a 40 μm mesh again. After centrifugation at 1800 rpm for 3 minutes, the supernatant was removed and the cells were immersed in PBS. 100 μL of lung single cells were stained in a dark room for 15 minutes with BV421-labeled anti-CD45 (Cat.563890, BD Biosciences), BV510-labeled anti-Ly6G (Cat.740157, BD Biosciences), PE-Cy7-labeled anti-MHC-II (Cat.25-5321-82, ebioscience), PE-labeled anti-CD11b (Cat. 101208, Biolegend), Alexa fluor 647-labeled anti-CD64(FcγRI) (Cat.558539, BD Biosciences), PE-CF594-labeled anti-CD24 (Cat.562477, BD Biosciences), and FITC-hypoxyprobe antibodies. Cells were collected at 1800 rpm for 3 minutes and then washed with PBS. After collecting the cells again, 400 μL of PBS was added.30 μL of cell suspension was analyzed using a MACSQuant VYB flow cytometer.

[0170] Immune cells such as macrophages, lymphocytes, neutrophils, and dendritic cells, as well as fibroblasts and plasma cells, are involved in the formation of pulmonary granulomas during tuberculosis infection. To confirm whether the alleviating effect of the compound of the present invention on granuloma lesions was substantially associated with cell infiltration within lung tissue, the cellular composition of lung tissue was analyzed using a flow cytometer. Total leukocytes (CD45+ cells) decreased in the INH-D2 25 mg / kg and dexamethasone administration groups (Fig. 12A).

[0171] When these immune cells infiltrate, hypoxic conditions occur, causing Mycobacterium tuberculosis to enter a dormant phase, making drug treatment difficult. As a result of measuring whether hypoxic conditions improved using a Hypoxyprobe, a significant improvement in hypoxic conditions was observed in the INH-D2 25 mg / kg administration group (Fig. 12B). Subsequently, the number of stromal macrophages and dendritic cells also significantly improved in the INH-D2 25 mg / kg and dexamethasone administration groups, respectively (Figs. 12C and 12D).

[0172] (3) Analysis of cytokines and chemokines in serum

[0173] Levels of chemokines involved in the migration of immune cells and cytokines mediating inflammatory responses were analyzed in serum on days 3 and 5 of pulmonary granuloma induction.

[0174] Blood obtained by orbital blood collection on days 3 and 5 of TDM administration, respectively, was centrifuged at 12,000 rpm for 15 minutes to separate serum. A cytometric bead array kit (Cat.558266, BD Biosciences) was used to analyze the levels of cytokines and chemokines in mouse serum, including IFN-γ (#558296), IL-2 (#558297), KC (558340), IL-6 (#558301), MCP-1 (558342), IL-10 (#558300), TNF-α (#558299), IL-12p70 (#558303), RANTES (#558345), and IL-1β (#560232).

[0175] 25 μL of capture beads coated with specific capture antibodies for each cytokine and chemokine (0.5 μL diluted in 25 μL of phosphate buffered saline containing 0.5% BSA, 1 mM EDTA, and 0.05% Tween-20) were mixed with 50 μL of serum and incubated in the dark for 1 hour. 25 μL of PE-detection beads (0.5 μL diluted in 25 μL of phosphate buffered saline containing 0.5% BSA and 5% polyethylene glycol) were added, and the mixture was stained in the dark for an additional 1 hour. After adding wash buffer, the sample was centrifuged at 200 × g for 5 minutes. After washing, cytokines and chemokines in the serum were analyzed using a MACSQuant VYB flow cytometer (Miltenyi Biotec).

[0176] On day 3 of pulmonary granuloma induction, compared to the TDM-only group, cytokine IFN-γ decreased in the INH-D1 25 mg / kg, INH-D2 5 mg / kg, and INH-D2 25 mg / kg groups. IL-6 and TNF-α were significantly reduced in the INH-D2 25 mg / kg administration group. Chemokine KC, which induces neutrophil infiltration, and chemokine MCP-1, which induces macrophage / monocyte infiltration, were also significantly reduced in the INH-D2 25 mg / kg administration group, while RANTES, which induces T-cell and monocyte infiltration, showed no significant difference across all drug administration groups (Fig. 13A).

[0177] On day 5 of pulmonary granuloma induction, IL-6 was suppressed in the INH-D1 5 mg / kg, INH-D2 5 mg / kg, INH-D2 25 mg / kg, and dexamethasone administration groups compared to the TDM-only group. TNF-α was reduced in the INH 25 mg / kg, INH-D1 25 mg / kg, INH-D2 5 mg / kg, INH-D2 25 mg / kg, and dexamethasone administration groups, while there were no significant differences in other cytokines and chemokines (Fig. 13B).

[0178] 7. Comparative experiment of isoniazid dimers INH-D2, D4, D6, and D8

[0179] 1 mg of TDM (Cord factor, GB61684, GLPBIO) was mixed with 90 μL of mineral oil (cat. 330779, Sigma-Aldrich Co., St. Louis, MO, USA), and then homogenized with 910 μL of PBS containing Tween-80. 100 μL of the emulsion containing 100 μg of TDM was injected into the tail vein of 9-week-old mice (100 μg / mouse). An emulsion solution without TDM was injected into the control group. INH-D2, INH-D4, INH-D6, and INH-D8 were all administered orally at a dose of 30 mg / kg daily for 5 days, starting 1 day after the intravenous injection of TDM. As a positive control, 0.5 mg / kg of dexamethasone was administered intraperitoneally on days 1, 3, and 5. The mouse was euthanized after 7 days.

[0180] (1) Lung index analysis

[0181] On day 7, mice were sacrificed, their lungs were extracted and weighed, and the lung index (lung weight / body weight x 100) was calculated. Compared to the TDM only group, the lung index was significantly reduced in the groups administered INH-D2 30 mg / kg, INH-D8 30 mg / kg, and Dexa 0.5 mg / kg. Figure 14 shows a photograph of the lungs extracted after mice were sacrificed on day 7 for the lung granuloma induction group (TDM only) and the INH derivative (INH-D2, INH-D4, INH-D6, and INH-D8) administration groups (Figure 14 (left)), and a graph showing the results of calculating the lung index (lung weight / body weight x 100) after weighing.

[0182] (2) Analysis of lung histopathology

[0183] The left lung lobe was fixed in 10% formalin for histopathological examination. Lung tissue was dehydrated, progressively immersed in alcohol and xylene, and embedded in paraffin. Deparaffinized tissue sections (5 μm) stained with hematoxylin and eosin (H&E, #ab245880) at Abcam (Cambridge, UK) were used. Histopathological analysis of lung inflammation was performed using Motic Digital Slide Assistant software version 1.0.7.44 (Kowloon Bay, Kowloon, Hong Kong) (PMID: 31734231). High-resolution images of H&E-stained slides were scanned. Inflammation was measured in two stages using ImageJ software version 1.52 (NIH, Bethesda, MD; https: / / imagej.nih.gov / ij). The maximum threshold for all analyzed digital transparency in the lung region was set to 200. The minimum and maximum values ​​of hue, saturation, and brightness of the images were set to 120, 255; 0, 255; and 0, 255, respectively. The total cell area was determined by modifying the procedure described in the online ImageJ stained section examples directory (NIH, https: / / imagej.nih.gov / ij / docs / examples). Pulmonary inflammation was calculated using the ratio of the total area occupied by the cell area. Analysis results showed that the pulmonary granuloma lesion index was significantly attenuated in the INH-D2, INH-D8, and Dexa groups compared to the TDM group.

[0184] Figure 15 shows a photograph (top) of the tissue lesions observed after sacrificing mice and extracting their lungs on day 7 for the lung granuloma induction group (TDM only) and the INH derivative (INH-D2, INH-D4, INH-D6, and INH-D8) administration group, and a graph (bottom) showing the area occupied by the granuloma lesions in the lungs.

[0185] 8. Statistical Analysis

[0186] Statistical comparisons were performed using one-way analysis of variance (ANOVA). Significance levels were determined using Tukey's multiple comparison test distribution with GraphPad Prism statistical software (version 8; San Diego, CA). P < 0.05, P < 0.01, and P < 0.001 were considered statistically significant.

[0187] As a result of the experiment described above, it was confirmed that the isoniazid derivative of the present invention effectively inhibits pulmonary granuloma lesions and inflammatory mediators. On the other hand, it was confirmed that isoniazid, which is used as a tuberculosis treatment, did not exhibit effects in alleviating inflammatory mediators or pulmonary granuloma lesions. In conclusion, it can be seen that the isoniazid derivative of the present invention can be used to alleviate pulmonary granuloma lesions in terms of HDT during tuberculosis treatment.

[0188] Although embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and it will be obvious to those skilled in the art that various modifications and variations are possible within the scope of the technical concept of the present invention as described in the claims.

Claims

Claim 1 A compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof for the treatment of tuberculosis infection: [Chemical Formula 1] In the above Chemical Formula 1, R1 and R2 are nitrogen-containing C3-C9 heteroaryl groups and C6-C 12 It is independently selected from a group composed of aryls. Claim 2 A compound or a pharmaceutically acceptable salt thereof, wherein R1 is selected from the group consisting of pyridine, pyridizine, pyrimidine, and pyrazine, and R2 is selected from the group consisting of pyridine, pyridizine, pyrimidine, pyrazine, and phenyl. Claim 3 A compound or a pharmaceutically acceptable salt thereof, wherein, in the case where R1 is pyridine, the 2nd or 4th position of the pyridine is bound to the backbone, and in the case where R2 is pyridine, the 3rd or 4th position of the pyridine is bound to the backbone. Claim 4 In claim 1, the compound represented by Formula 1 is at least one selected from the group consisting of compounds of Formulas D1 to D5, or a pharmaceutically acceptable salt thereof: [Chemical Formula D1] [Chemical Formula D2] [Chemical Formula D3] [Chemical Formula D4] [Chemical Formula D5] Claim 5 A pharmaceutical composition for treating tuberculosis infection, comprising as an active ingredient a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof: [Chemical Formula 1] In the above Chemical Formula 1, R1 and R2 are nitrogen-containing C3-C9 heteroaryl groups and C6-C 12 It is independently selected from a group composed of aryls. Claim 6 A pharmaceutical composition for treating tuberculosis infection, wherein in claim 5, R1 is selected from the group consisting of pyridine, pyridizine, pyrimidine, and pyrazine, and R2 is selected from the group consisting of pyridine, pyridizine, pyrimidine, pyrazine, and phenyl. Claim 7 A pharmaceutical composition for treating tuberculosis infection, wherein, in claim 5, when R1 is pyridine, the 2nd or 4th position of the pyridine is bound to the backbone, and when R2 is pyridine, the 3rd or 4th position of the pyridine is bound to the backbone. Claim 8 A pharmaceutical composition for treating tuberculosis infection, wherein the compound represented by Chemical Formula 1 is at least one selected from the group consisting of compounds of Chemical Formulas D1 to D5 below: [Chemical Formula D1] [Chemical Formula D2] [Chemical Formula D3] [Chemical Formula D4] [Chemical Formula D5] Claim 9 A pharmaceutical composition for treating a tuberculosis infection disease according to claim 5, wherein the tuberculosis infection disease is at least one selected from the group consisting of ocular tuberculosis, cutaneous tuberculosis, kidney tuberculosis, lymph node tuberculosis, laryngeal tuberculosis, intestinal tuberculosis, pulmonary tuberculosis, gallbladder tuberculosis, bone tuberculosis, throat tuberculosis, breast tuberculosis, and spinal tuberculosis. Claim 10 A pharmaceutical composition for treating a tuberculosis infection, wherein, in paragraph 5, the tuberculosis infection is a granuloma. Claim 11 A pharmaceutical composition for treating a tuberculosis infection disease, wherein, in claim 5, the tuberculosis is isoniazid-resistant tuberculosis (INHr-TB).

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