Use of pyridine derivatives

Abstract

This invention discloses the use of a pyridine derivative. Specifically, it discloses the use of substance X in the preparation of drugs for inhibiting nontuberculous mycobacteria, drugs for treating and / or preventing nontuberculous mycobacterial infections, nontuberculous mycobacterial inhibitors, or nontuberculous mycobacterial antibacterial agents, wherein substance X is a compound represented by Formula I or a pharmaceutically acceptable salt thereof. The pyridine derivative of this invention exhibits good inhibitory activity against nontuberculous mycobacteria, and has low cytotoxicity and good safety.

Description

Technical Field

[0001] This invention relates to the use of a pyridine derivative. Background Technology

[0002] The continued increase in cases worldwide has made nontuberculous mycobacterial (NTM) disease a major public health concern. In countries or regions with high prevalence, NTMs can account for 30–50% of all mycobacterial isolates. Nontuberculous mycobacterial (NTM) lung disease is more prevalent in patients with underlying lung diseases such as bronchiectasis and COPD (chronic obstructive pulmonary disease), and is a significant contributor to increased mortality in these patients. More than 200 NTM species or subspecies have been reported, with the Mycobacterium avium complex (MAC, mainly consisting of Mycobacterium avium and intracellular mycobacteria), Mycobacterium abscessus (Mab), and Mycobacterium kansasii being the most common causes of NTM lung disease (NTM-PD). Treatment of NTM infections is often challenging due to the widespread natural resistance of different NTM species to most antibiotics.

[0003] Bedaquiline (BDQ) is a novel antibiotic that targets drug-resistant tuberculosis by inhibiting ATP synthesis. BDQ has demonstrated potent efficacy against multidrug-resistant tuberculosis both in vitro and in vivo, and also exhibits effective activity against many different NTM strains in vitro. However, the numerous serious side effects of BDQ, such as unexplained mortality, QT interval prolongation, hepatotoxicity, and phospholipid deposition, limit its widespread adoption. Therefore, identifying highly active anti-NTM drugs is a primary task in establishing effective treatment regimens for NTM infections. Summary of the Invention

[0004] The technical problem this invention aims to solve is to overcome the scarcity of drugs in the prior art for inhibiting nontuberculous mycobacterial diseases, and to provide a use for a pyridine derivative. The pyridine derivative of this invention exhibits good inhibitory effects against nontuberculous mycobacteria, and also demonstrates low cytotoxicity and good safety.

[0005] This invention provides the use of substance X in the preparation of a drug for inhibiting nontuberculous mycobacteria; said substance X is a compound of formula I or a pharmaceutically acceptable salt thereof;

[0006]

[0007] In one embodiment, the substance X is preferably a compound represented by formula I-1;

[0008]

[0009] In some implementations, substance X may be one of the active ingredients of the drug or the only active ingredient.

[0010] In one embodiment, the nontuberculous mycobacteria may be selected from one or more of rapidly growing nontuberculous mycobacteria and slowly growing nontuberculous mycobacteria.

[0011] In one embodiment, the rapidly growing nontuberculous mycobacteria may be selected from one or more of the following: Mycobacterium abscessus, Mycobacterium tannosum, Mycobacterium Aichii, Mycobacterium aureum, Mycobacterium africanum, Mycobacterium guilloché, Mycobacterium chida, Mycobacterium chub, Mycobacterium cosmetii, Mycobacterium dissecans, Mycobacterium occulta, Mycobacterium auburn, Mycobacterium paraocculta, Mycobacterium septicemia, Mycobacterium smegmatis, Mycobacterium thermophilum, Mycobacterium tsuifolium, Mycobacterium occulta, Mycobacterium occulta, Mycobacterium nigra ...

[0012] In one embodiment, the rapidly growing nontuberculous mycobacterium is preferably Mycobacterium new Orleans.

[0013] In one embodiment, the rapidly growing nontuberculous mycobacteria may be selected from one or more of ATCC19977, ATCC27406, ATCC27280, ATCC23366, ATCC33464, ATCC14472, ATCC19627, ATCC27278, DSM44829, ATCC19340, ATCC6841, DSM44124, ATCC27023, ATCC19686, DSM43271, ATCC35154, ATCC35796, ATCC700731, ATCC19420, ATCC19527, ATCC27282, DSM46621, DSM44679, DSM44177, DSM45103, and DSM44017.

[0014] In one embodiment, the rapidly growing nontuberculous mycobacterium is preferably DSM44679.

[0015] In one embodiment, the slow-growing nontuberculous mycobacteria may be selected from one or more of the following: Mycobacterium Asianum, Mycobacterium avium, Mycobacterium cryptotarum, Mycobacterium chimera, Mycobacterium gastroenterum, Mycobacterium Gordonum, Mycobacterium intracellularis, Mycobacterium Kansas, Mycobacterium vulgatum, Mycobacterium nonchromogenicum, Mycobacterium parasporum, Mycobacterium rhodesiae, Mycobacterium sulgaris, Mycobacterium terrestris, minor mycobacteria, Mycobacterium bufota, Mycobacterium caedes, Mycobacterium aubergine, Mycobacterium kubica, Mycobacterium intermedium, Mycobacterium sphagnum, Mycobacterium schrenckii, and Mycobacterium marinum.

[0016] In one embodiment, the slow-growing nontuberculous mycobacteria may be selected from one or more of ATCC25276, ATCC25291, DSM44243, DSM44622, ATCC15754, ATCC14470, ATCC13950, ATCC12478, ATCC19422, ATCC19530, DSM44648, ATCC27024, ATCC19981, ATCC35799, ATCC15755, ATCC23292, ATCC19250, ATCC27726, DSM45069, DSM44627, DSM44064, ATCC33027, ATCC27962, and ATCC927.

[0017] In some implementations, the drug may contain pharmaceutical excipients.

[0018] In some embodiments, the drug may be administered in a manner conventional in the art, such as oral administration.

[0019] In some implementations, the substance X is a therapeutic and / or preventative effective amount.

[0020] The present invention also provides a pharmaceutical composition for inhibiting nontuberculous mycobacteria, comprising: substance X as described above, and pharmaceutical excipients.

[0021] The present invention also provides the use of substance X in the preparation of medicaments for treating and / or preventing nontuberculous mycobacterial infections; said substance X is a compound of formula I or a pharmaceutically acceptable salt thereof;

[0022]

[0023] In one embodiment, the substance X is preferably a compound represented by Formula I-1;

[0024]

[0025] In some implementations, substance X may be one of the active ingredients of the drug or the only active ingredient.

[0026] In one embodiment, the nontuberculous mycobacteria may be selected from one or more of rapidly growing nontuberculous mycobacteria and slowly growing nontuberculous mycobacteria.

[0027] In one embodiment, the rapidly growing nontuberculous mycobacteria may be selected from one or more of the following: Mycobacterium abscessus, Mycobacterium tannosum, Mycobacterium Aichii, Mycobacterium aureum, Mycobacterium africanum, Mycobacterium guilloché, Mycobacterium chida, Mycobacterium chub, Mycobacterium cosmetii, Mycobacterium dissecans, Mycobacterium occulta, Mycobacterium auburn, Mycobacterium paraocculta, Mycobacterium septicemia, Mycobacterium smegmatis, Mycobacterium thermophilum, Mycobacterium tsuifolium, Mycobacterium occulta, Mycobacterium occulta, Mycobacterium nigra ...

[0028] In one embodiment, the rapidly growing nontuberculous mycobacterium is preferably Mycobacterium new Orleans.

[0029] In one embodiment, the rapidly growing nontuberculous mycobacteria may be selected from one or more of ATCC19977, ATCC27406, ATCC27280, ATCC23366, ATCC33464, ATCC14472, ATCC19627, ATCC27278, DSM44829, ATCC19340, ATCC6841, DSM44124, ATCC27023, ATCC19686, DSM43271, ATCC35154, ATCC35796, ATCC700731, ATCC19420, ATCC19527, ATCC27282, DSM46621, DSM44679, DSM44177, DSM45103, and DSM44017.

[0030] In one embodiment, the rapidly growing nontuberculous mycobacterium is preferably DSM44679.

[0031] In one embodiment, the slow-growing nontuberculous mycobacteria may be selected from one or more of the following: Mycobacterium Asianum, Mycobacterium avium, Mycobacterium cryptotarum, Mycobacterium chimera, Mycobacterium gastroenterum, Mycobacterium Gordonum, Mycobacterium intracellularis, Mycobacterium Kansas, Mycobacterium vulgatum, Mycobacterium nonchromogenicum, Mycobacterium parasporum, Mycobacterium rhodesiae, Mycobacterium sulgaris, Mycobacterium terrestris, minor mycobacteria, Mycobacterium bufota, Mycobacterium caedes, Mycobacterium aubergine, Mycobacterium kubica, Mycobacterium intermedium, Mycobacterium sphagnum, Mycobacterium schrenckii, and Mycobacterium marinum.

[0032] In one embodiment, the slow-growing nontuberculous mycobacteria may be selected from one or more of ATCC25276, ATCC25291, DSM44243, DSM44622, ATCC15754, ATCC14470, ATCC13950, ATCC12478, ATCC19422, ATCC19530, DSM44648, ATCC27024, ATCC19981, ATCC35799, ATCC15755, ATCC23292, ATCC19250, ATCC27726, DSM45069, DSM44627, DSM44064, ATCC33027, ATCC27962, and ATCC927.

[0033] In one embodiment, the nontuberculous mycobacterial infection is preferably NTM lung disease and / or disseminated NTM disease.

[0034] In some implementations, the drug may contain pharmaceutical excipients.

[0035] In some embodiments, the drug can be administered in any manner conventional in the art, such as oral administration.

[0036] In one embodiment, the substance X may be a therapeutic and / or preventative effective amount.

[0037] The present invention also provides a pharmaceutical composition for treating and / or preventing nontuberculous mycobacterial infections, comprising: substance X as described above, and pharmaceutical excipients.

[0038] In one embodiment, the nontuberculous mycobacterial infection is preferably NTM lung disease and / or disseminated NTM disease.

[0039] The present invention also provides the use of substance X in the preparation of a nontuberculous mycobacterial inhibitor; wherein substance X is a compound of formula I or a pharmaceutically acceptable salt thereof;

[0040]

[0041] In one embodiment, the substance X is preferably a compound represented by formula I-1;

[0042]

[0043] In some implementations, substance X may be one of the active ingredients of the drug or the only active ingredient.

[0044] In one embodiment, the nontuberculous mycobacteria may be selected from one or more of rapidly growing nontuberculous mycobacteria and slowly growing nontuberculous mycobacteria.

[0045] In one embodiment, the rapidly growing nontuberculous mycobacteria may be selected from one or more of the following: Mycobacterium abscessus, Mycobacterium tannosum, Mycobacterium Aichii, Mycobacterium aureum, Mycobacterium africanum, Mycobacterium guilloché, Mycobacterium chida, Mycobacterium chub, Mycobacterium cosmetii, Mycobacterium dissecans, Mycobacterium occulta, Mycobacterium auburn, Mycobacterium paraocculta, Mycobacterium septicemia, Mycobacterium smegmatis, Mycobacterium thermophilum, Mycobacterium tsuifolium, Mycobacterium occulta, Mycobacterium occulta, Mycobacterium nigra ...

[0046] In one embodiment, the rapidly growing nontuberculous mycobacterium is preferably Mycobacterium new Orleans.

[0047] In one embodiment, the rapidly growing nontuberculous mycobacteria may be selected from one or more of ATCC19977, ATCC27406, ATCC27280, ATCC23366, ATCC33464, ATCC14472, ATCC19627, ATCC27278, DSM44829, ATCC19340, ATCC6841, DSM44124, ATCC27023, ATCC19686, DSM43271, ATCC35154, ATCC35796, ATCC700731, ATCC19420, ATCC19527, ATCC27282, DSM46621, DSM44679, DSM44177, DSM45103, and DSM44017.

[0048] In one embodiment, the rapidly growing nontuberculous mycobacterium is preferably DSM44679.

[0049] In one embodiment, the slow-growing nontuberculous mycobacteria may be selected from one or more of the following: Mycobacterium Asianum, Mycobacterium avium, Mycobacterium cryptotarum, Mycobacterium chimera, Mycobacterium gastroenterum, Mycobacterium Gordonum, Mycobacterium intracellularis, Mycobacterium Kansas, Mycobacterium vulgatum, Mycobacterium nonchromogenicum, Mycobacterium parasporum, Mycobacterium rhodesiae, Mycobacterium sulgaris, Mycobacterium terrestris, minor mycobacteria, Mycobacterium bufota, Mycobacterium caedes, Mycobacterium aubergine, Mycobacterium kubica, Mycobacterium intermedium, Mycobacterium sphagnum, Mycobacterium schrenckii, and Mycobacterium marinum.

[0050] In one embodiment, the slow-growing nontuberculous mycobacteria may be selected from one or more of ATCC25276, ATCC25291, DSM44243, DSM44622, ATCC15754, ATCC14470, ATCC13950, ATCC12478, ATCC19422, ATCC19530, DSM44648, ATCC27024, ATCC19981, ATCC35799, ATCC15755, ATCC23292, ATCC19250, ATCC27726, DSM45069, DSM44627, DSM44064, ATCC33027, ATCC27962, and ATCC927.

[0051] In some implementations, the drug may contain pharmaceutical excipients.

[0052] In some embodiments, the drug may be administered in a manner conventional in the art, such as oral administration.

[0053] In some implementations, the substance X is a therapeutic and / or preventative effective amount.

[0054] The present invention also provides the use of substance X in the preparation of an antibacterial agent for nontuberculous mycobacteria; wherein substance X is a compound represented by formula I or a pharmaceutically acceptable salt thereof;

[0055]

[0056] In one embodiment, the substance X is preferably a compound represented by formula I-1;

[0057]

[0058] In some implementations, substance X may be one of the active ingredients of the drug or the only active ingredient.

[0059] In one embodiment, the nontuberculous mycobacteria may be selected from one or more of rapidly growing nontuberculous mycobacteria and slowly growing nontuberculous mycobacteria.

[0060] In one embodiment, the rapidly growing nontuberculous mycobacteria may be selected from one or more of the following: Mycobacterium abscessus, Mycobacterium tannosum, Mycobacterium Aichii, Mycobacterium aureum, Mycobacterium africanum, Mycobacterium guilloché, Mycobacterium chida, Mycobacterium chub, Mycobacterium cosmetii, Mycobacterium dissecans, Mycobacterium occulta, Mycobacterium auburn, Mycobacterium paraocculta, Mycobacterium septicemia, Mycobacterium smegmatis, Mycobacterium thermophilum, Mycobacterium tsuifolium, Mycobacterium occulta, Mycobacterium occulta, Mycobacterium nigra ...

[0061] In one embodiment, the rapidly growing nontuberculous mycobacterium is preferably Mycobacterium new Orleans.

[0062] In one embodiment, the rapidly growing nontuberculous mycobacteria may be selected from one or more of ATCC19977, ATCC27406, ATCC27280, ATCC23366, ATCC33464, ATCC14472, ATCC19627, ATCC27278, DSM44829, ATCC19340, ATCC6841, DSM44124, ATCC27023, ATCC19686, DSM43271, ATCC35154, ATCC35796, ATCC700731, ATCC19420, ATCC19527, ATCC27282, DSM46621, DSM44679, DSM44177, DSM45103, and DSM44017.

[0063] In one embodiment, the rapidly growing nontuberculous mycobacterium is preferably DSM44679.

[0064] In one embodiment, the slow-growing nontuberculous mycobacteria may be selected from one or more of the following: Mycobacterium Asianum, Mycobacterium avium, Mycobacterium cryptotarum, Mycobacterium chimera, Mycobacterium gastroenterum, Mycobacterium Gordonum, Mycobacterium intracellularis, Mycobacterium Kansas, Mycobacterium vulgatum, Mycobacterium nonchromogenicum, Mycobacterium parasporum, Mycobacterium rhodesiae, Mycobacterium sulgaris, Mycobacterium terrestris, minor mycobacteria, Mycobacterium bufota, Mycobacterium caedes, Mycobacterium aubergine, Mycobacterium kubica, Mycobacterium intermedium, Mycobacterium sphagnum, Mycobacterium schrenckii, and Mycobacterium marinum.

[0065] In one embodiment, the slow-growing nontuberculous mycobacteria may be selected from one or more of ATCC25276, ATCC25291, DSM44243, DSM44622, ATCC15754, ATCC14470, ATCC13950, ATCC12478, ATCC19422, ATCC19530, DSM44648, ATCC27024, ATCC19981, ATCC35799, ATCC15755, ATCC23292, ATCC19250, ATCC27726, DSM45069, DSM44627, DSM44064, ATCC33027, ATCC27962, and ATCC927.

[0066] In some implementations, the drug may contain pharmaceutical excipients.

[0067] In some embodiments, the drug may be administered in a manner conventional in the art, such as oral administration.

[0068] In some implementations, the substance X is a therapeutic and / or preventative effective amount.

[0069] The present invention also provides a method for treating and / or preventing nontuberculous mycobacterial infection, comprising: administering to a subject in need a therapeutically effective amount and / or a preventively effective amount of the substance X as described above or the pharmaceutical composition described above.

[0070] In one embodiment, the nontuberculous mycobacterial infection is preferably NTM lung disease and / or disseminated NTM disease.

[0071] Definitions and Explanations

[0072] Unless otherwise stated, the following terms and phrases used herein are intended to have the following meanings: a particular term or phrase should not be considered uncertain or unclear unless specifically defined, but should be understood in its ordinary sense; when a trade name appears in this document, it is intended to refer to the corresponding product or its active ingredient.

[0073] In this invention, the drugs for treating and / or preventing diseases caused by nontuberculous mycobacteria can be conventional dosage forms in the art, such as tablets, capsules, intravenous injections, intraperitoneal injections, inhalers, nebulizers, lyophilized agents, patches, gels, sprays, or suppositories.

[0074] The term "pharmaceutical excipients" refers to excipients and additives used in the production of pharmaceuticals and the dispensing of prescriptions. They can be all substances contained in pharmaceutical preparations other than the active ingredient. See the Pharmacopoeia of the People's Republic of China (2020 edition), Volume IV, or the Handbook of Pharmaceutical Excipients (Raymond C. Wayne, 2009 Sixth Edition).

[0075] The term “treatment” refers to a therapeutic approach. When it relates to a specific condition, treatment means: (1) alleviating one or more biological manifestations of the disease or condition; (2) interfering with (a) one or more points in a biological cascade that causes or precipitates the condition or (b) one or more biological manifestations of the condition; (3) improving one or more symptoms, effects or side effects associated with the condition, or one or more symptoms, effects or side effects associated with the condition or its treatment; or (4) slowing the development of the condition or one or more biological manifestations of the condition.

[0076] The term "prevention" refers to the reduction of the risk of acquiring or developing a disease or disorder.

[0077] The term “therapeutic effective amount” means an amount of compound that is sufficient to effectively treat the disease or condition described herein when administered to a subject. The “therapeutic effective amount” will vary depending on the compound, the condition and its severity, and the age of the patient to be treated, but may be adjusted as needed by those skilled in the art.

[0078] The term "preventive effective amount" refers to an amount sufficient to prevent a disease or disorder, or an amount sufficient to prevent one or more symptoms associated with a disease or disorder, or an amount sufficient to prevent a recurrence of a disease or disorder.

[0079] The term "subject" refers to any animal that is about to or has already been administered the compound according to embodiments of the present invention, preferably a mammal, with humans being the most preferred. The term "mammal" includes any mammal, and examples of mammals include, but are not limited to, cattle, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, and humans, with humans being the most preferred.

[0080] Without violating common sense in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0081] The reagents and raw materials used in this invention are all commercially available.

[0082] The positive and progressive effects of this invention are as follows: the pyridine derivatives of this invention have strong antibacterial activity against different nontuberculous mycobacteria, low cytotoxicity, good safety, and have the potential to treat and / or prevent related diseases caused by nontuberculous mycobacteria. Attached Figure Description

[0083] Figure 1 The results of cytotoxicity tests on BDQ and the compounds shown in Formula I-1 in THP-1 cells are shown. Detailed Implementation

[0084] The present invention is further illustrated below by way of embodiments, but this does not limit the invention to the scope of the embodiments described. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0085] Example 1

[0086] 1. Experimental materials

[0087] (1) 51 nontuberculous mycobacterial (NTM) standard strains, including 26 rapid-growing nontuberculous mycobacterial (RGM) standard strains and 25 chronic-growing nontuberculous mycobacterial (SGM) standard strains, were obtained from the biobank of Beijing Chest Hospital affiliated to Capital Medical University and purchased from the American Type Culture Collection (ATCC) or the German Culture Collection (DSM), as shown in Tables 1 and 2 below;

[0088] Table 1 26 RGM strains

[0089]

[0090]

[0091] Table 2 Standard strains of 25 SGM strains

[0092]

[0093]

[0094] (2) 132 NTM clinical isolates, including 40 Mycobacterium abscessus, 29 Mycobacterium intracellularis, 21 Mycobacterium avium, and 42 Mycobacterium kansas. All strains were derived from clinical samples. After obtaining positive cultures from the clinical samples, the isolates were identified to the species level by growth assays on p-nitrobenzoic acid medium and sequence alignment of 16S rRNA (16S ribosomal RNA), hsp65 (heat shock protein 65), rpoB (RNA polymerase β subunit), and the 16-23S rRNA intergenic region.

[0095] The compound shown in Formula I-1:

[0096]

[0097] 2. Experimental Methods:

[0098] (1) Minimum inhibitory concentration (MIC) test

[0099] BDQ (bedaquiline, purchased from Liye Pharmaceutical (Nanjing, China)) and the compound shown in Formula I-1 (provided by Shanghai Jiatan Pharmaceutical Technology Co., Ltd.) were dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution with a concentration of 8 mg / mL under aseptic conditions. Inoculum was prepared using fresh cultures passaged and grown on Roche medium. The broth microdilution method was performed according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. Cationic-regulated Mueller-Hinton broth (CAMHB) enriched with 5% OADC (oleic acid-fetal serum albumin-glucose-catalase) was used for SGM, while OADC-free CAMHB was used for RGM. The broth microdilution was set as a 2-fold serial dilution, with BDQ and the compound shown in Formula I-1 ranging from 0.0039 to 2.0 mg / L. The inoculated culture plates were then incubated at 37°C. RGM growth was observed after 3 days, and SGM growth was observed after 7-10 days. A 70 μL solution containing 20 μL Alamar Blue and 50 μL Tween 80 (5%) was added to each well and incubated at 37°C for 24 hours. The color change of the medium was then observed. A change from blue to pink or purple indicated bacterial growth. The MIC value was defined as the lowest antibiotic concentration at which no color change from blue to pink occurred. For clinically observed bacterial species, the MIC50 and MIC90 were calculated separately for each species. MIC50 and MIC90 refer to the MICs required to inhibit the growth of 50% and 90% of the test bacteria in a batch of experiments, respectively.

[0100] (2) Confirm the preliminary epidemiological threshold (ECOFF)

[0101] For bacterial species with more than 20 clinical isolates included, and for which the compound shown in BDQ / Formula I-1 exhibits significant antibacterial activity, ECOFF is determined based on the distribution characteristics of MIC values. ECOFF is defined as the concentration that can inhibit >95% of bacterial populations for a single-peak MIC distribution curve, while for a bimodal MIC distribution curve, ECOFF is set between the two peaks.

[0102] (3) Minimum bactericidal concentration (MBC) determination

[0103] After incubation with the relevant compound for 3 or 7 days, the minimum effective concentration (MBC) of five NTM standard strains was determined by counting colony-forming units (CFU) in the wells of a 96-well plate. According to CLSI guidelines, MBC is the drug concentration that achieves 99.9% kill of the final inoculum. Initial CFU was calculated on the same day the culture was incubated in the 96-well microplate. The drug concentrations of the compound shown in Formula I-1 and BDQ ranged from 1 × MIC to 128 × MIC. RGM was performed for 3 days, and SGM for 7–10 days. Aliquots of 100 μL of culture per well were placed in cationic-regulated Mueller-Hinton broth (CAMHB) with or without 5% OADC and incubated. CFU were counted after 7–10 days of RGM or 28 days of SGM. According to CLSI guidelines, MBC is defined as the lowest effective drug concentration in CFUs that is at least 3 log10 lower than the initial CFU. When the MBC / MIC ratio is ≤4, the antibiotic is considered to have bactericidal effect; otherwise, it is considered to have bacteriostatic effect.

[0104] (4) Cytotoxicity assay

[0105] The cytotoxicity of BDQ and the compound shown in Formula I-1 was assessed using THP-1 cells. Cells were seeded in 96-well plates and induced to differentiate into macrophages with 100 nMPMA. After 48 hours, the cells were washed once and cultured in fresh RPMI medium (Gibco) containing 10% fetal bovine serum (RPMI complete medium). Drug solutions were added to the wells at final concentrations ranging from 1 to 16 μg / mL, and incubated for 24 and 48 hours. The cytotoxicity of different concentrations of BDQ and the compound shown in Formula I-1 was monitored using the CCK-8 Cell Proliferation and Cytotoxicity Assay Kit (Solarbio, Beijing, China). Cell culture supernatants were analyzed, and absorbance values ​​(Abs) at 450 nm were recorded using a MultiskanGo microplate reader (Thermo Fisher, USA). Cell viability (%) at each concentration was determined as follows: Cell viability = ((Abs450 of treated cells / Abs450 of control cells) / (Abs450 of untreated cells / Abs450 of control cells)) × 100%.

[0106] 3. Statistical Analysis

[0107] Data were analyzed using SPSS 23.0 and GraphPad Prism 7.0 software. Spearman's test was used to analyze the correlation between the BDQMIC values ​​of clinical isolates and the MIC values ​​of the compounds shown in Formula I-1. For cytotoxicity assays, two-way ANOVA was used, followed by post-hoc tests to determine significant differences between groups. A p-value < 0.05 was considered statistically significant.

[0108] 4. Results

[0109] (1) MIC of BDQ and the compound shown in Formula I-1 against NTM standard strain

[0110] The MICs of BDQ and the compound shown in Formula I-1 against 51 standard strains are shown in Tables 3 and 4. For the same standard strains, the antibacterial activity of the compound shown in Formula I-1 was similar to that of BDQ, or the MIC of some standard strains was increased by 1-fold. Similar to BDQ, the compound shown in Formula I-1 showed strong antibacterial activity against almost all tested SGM strains, with MICs generally below 0.25 μg / mL. Only two SGM strain standards, namely *Mycobacterium rhodesiae* and *Mycobacterium celatum*, had MICs >2 μg / mL. Furthermore, the compound shown in Formula I-1 also showed very effective in vitro activity against the included RGM standard strains. The MICs of all 26 RGM strain standards were ≤0.5 μg / mL, and the MICs of the 25 SGM strain standards were ≤0.25 μg / mL.

[0111] Table 3. MIC of 26 RGM standard strains

[0112]

[0113]

[0114] Table 4. MIC of 25 SGM standard strains

[0115]

[0116]

[0117] (2) MIC distribution and ECOFF of BDQ and the compounds shown in Formula I-1 in clinical isolates from different NTM strains

[0118] Table 5 shows the MIC distributions of the four most common NTM strains against BDQ and the compounds shown in Formula I-1. The sensitivity distributions of clinical isolates to BDQ and the compounds shown in Formula I-1 are consistent with those of the standard strains, indicating strong antibacterial activity. Clinical isolates also exhibit strong antibacterial activity against the vast majority of SGM isolates containing all included strains. Similar activity is also observed against Mycobacterium abscessus, but the MIC values ​​are higher than those against SGM.

[0119] Table 5. MIC distribution results of four NTM strains against BDQ and the compound shown in Formula I-1.

[0120]

[0121] BDQ and the compound shown in Formula I-1 exhibited the strongest activity against *Mycobacterium kansasii* and *Mycobacterium intracellulare*, with ECOFF of 0.0156 μg / mL, MIC50 of 0.0078 μg / mL, and MIC90 of 0.0156 μg / mL for both. The vast majority of isolates from these two strains had MICs below 0.008 μg / mL. BDQ and the compound shown in Formula I-1 showed good activity against *Mycobacterium abscessus*, with MIC50s of 0.125 μg / mL and 0.25 μg / mL, respectively. The MIC90s of BDQ and the compound shown in Formula I-1 against *Mycobacterium abscessus* were 0.25 μg / mL and 0.5 μg / mL, respectively. The ECOFF concentrations of BDQ and the compound shown in Formula I-1 for Mycobacterium abscessus were 0.25 μg / mL and 0.5 μg / mL, respectively.

[0122] For *Mycobacterium abscessus*, BDQ and the compound MIC shown in Formula I-1 showed a good correlation (Spearman's q = 0.722, P = 0.0000). For *Mycobacterium kansasii*, the correlation between BDQMIC and the compound MIC shown in Formula I-1 was significant (Spearman's q = 1.000, P = 0.0000). For *Mycobacterium avium* and *Mycobacterium intracellulare*, the correlation between BDQMIC and the compound MIC shown in Formula I-1 was also very strong (Spearman's q = 0.967, P = 0.0000; Spearman's q = 0.784, two-tailed, P = 0.0000).

[0123] (3) MBC measurement

[0124] The MBC of the compound shown in Formula I-1 and BDQ against the five NTM standard strains was greater than 32 × MIC. Therefore, the MBC / MIC ratios of the compound shown in Formula I-1 or BDQ against the five NTM standard strains were much higher than 4, indicating that both drugs have antibacterial activity against Mycobacterium abscessus, Mycobacterium fortuitum, Mycobacterium intracellulare, Mycobacterium avium, and Mycobacterium kansasii (Table 6).

[0125] Table 6. MBC / MIC Ratios of SGM and RGM

[0126]

[0127] Note: If the MBC / MIC ratio is higher than 4, the effect is considered to be antibacterial; otherwise, it is considered to be bactericidal.

[0128] (4) Cytotoxicity assay of BDQ and the compound shown in Formula I-1 in THP-1 cells

[0129] After 24 hours of incubation with compound I-1, the survival rate of THP-1 cells reached over 75% at a concentration of 16 μg / mL, and over 90% at a concentration of 8 μg / mL. At concentrations of 4 μg / mL, 2 μg / mL, and 1 μg / mL, the survival rate of THP-1 cells approached 100%. After 48 hours of incubation, the cell survival rate decreased to below 75% at a concentration of 16 μg / mL, remained above 75% at a concentration of 8 μg / mL, exceeded 80% at concentrations of 4 μg / mL and 2 μg / mL, and approached 100% at a concentration of 1 μg / mL. There was no statistically significant difference between BDQ and the compound shown in Formula I-1. Figure 1 ).

[0130] in conclusion:

[0131] The compound shown in Formula I-1 of this application exhibits good antibacterial activity against strains of various NTM species, demonstrating that both the tested standard strains and clinical isolates are susceptible to it. The MICs of all tested standard strains of RGM or SGM strains are below 0.5 μg / mL, with most MICs well below 0.1 μg / mL (Tables 3 and 4). The activity is similar to BDQ. The inhibitory activity of the compound shown in Formula I-1 against clinical isolates of the four most frequently isolated NTM species is consistent with the results for the standard strains; the MIC50 and MIC90 for *Mycobacterium intracellulare*, *Mycobacterium avium*, and *Mycobacterium kansasum* are ≤0.0625 μg / mL and ≤0.125 μg / mL, respectively. Spearman's test analysis showed a close correlation between the MICs of the two drugs (Spearman's q range is 0.722 to 1). Overall data from RGM and SGM standard strains and clinical isolates of the most frequently isolated NTM species support the potential application of the compound shown in Formula I-1 in the treatment of NTM infections, making it as effective as BDQ.

[0132] The ECOFF values ​​of the compounds shown in Formula I-1 for NTM species are important for setting breakpoints in future drug susceptibility tests. ECOFFs for Mycobacterium abscessus, Mycobacterium intracellularis, Mycobacterium avium, and Mycobacterium kansasus are typically low, ranging from 0.0156 μg / mL to 0.5 μg / mL.

[0133] THP-1 cells exhibited nearly 100% survival after 24 hours of exposure to compounds of Formula I-1 at concentrations below 4 μg / mL. This concentration is 200–300 times the MIC obtained from tests on standard strains of the three most commonly isolated bacterial species (Mycobacterium intracellulare, Mycobacterium avium, and Mycobacterium kansasense). 4 μg / mL is 16 times the MIC obtained from tests on standard strains of Mycobacterium abscessus in this study, and this macrophage assay indicates that the compound of Formula I-1 is safe at effective therapeutic concentrations.

[0134] In summary, both the reference strain and clinically isolated NTM assays demonstrate that the compound represented by Formula I-1 exhibits strong antibacterial activity against different NTM strains and low cytotoxicity.

[0135] Example 2: Evaluation of in vivo antibacterial activity and prolonged survival time against Mycobacterium abscessus.

[0136] 1. Experimental materials

[0137] AZM (azithromycin, purchased from Shanghai Modern Pharmaceutical Co., Ltd.), BDQ (bedaquiline, purchased from Liye Pharmaceutical (Nanjing, China)) and the compound shown in Formula I-1 (provided by Shanghai Jiatan Pharmaceutical Technology Co., Ltd.);

[0138] 2. Experimental Methods

[0139] (1) Wild-type AB zebrafish were naturally paired and fertilized, and cultured in water at 28℃. Middlebrook 7H9 (Becton Dickinson) broth rich in 10% OADC (oleic acid-fetal serum albumin-glucose-catalase) and 0.05% Tween 80 (Sigma-Aldrich) was prepared. Smooth (S) morphological Mycobacterium abscessus ATCC19977 was incubated at 37℃ for 5 to 7 days. The moderately to logarithmically growing Mycobacterium abscessus was then centrifuged, washed, and placed in phosphate-buffered saline (PBS) containing 0.05% Tween 80. The bacterial suspension was homogenized and sonicated. After colonies settled for 5 to 10 minutes, the bacteria were enriched in PBS and labeled with DIO (green fluorescent dye). The bacterial cells were then intravenously injected into wild-type AB zebrafish strains 2 days after fertilization, with approximately 3.6 × 10⁶ cells per fish. 3 CFU was used to establish a zebrafish model of Mycobacterium abscessus.

[0140] (2) Under a microscope, zebrafish 3 days after fertilization were randomly assigned to six-well plates, with 30 fish per well. DMSO (Shanghai Aladdin Biochemical Technology Co., Ltd., China) stored at -20℃ was diluted with water to a solution of 20.0 mg / mL, which was used to prepare AZM, BDQ, and the compound shown in Formula I-1. The concentrations of AZM were 62.5 μg / mL, 125 μg / mL, 250 μg / mL, 500 μg / mL, and 1000 μg / mL, respectively; the concentrations of BDQ were 3.91 μg / mL, 7.81 μg / mL, 15.6 μg / mL, 31.2 μg / mL, and 62.5 μg / mL, respectively; and the concentrations of the compound shown in Formula I-1 were 15.7 μg / mL, 31.3 μg / mL, 62.5 μg / mL, 125 μg / mL, 250 μg / mL, 500 μg / mL, and 1000 μg / mL, respectively. After incubating zebrafish plates at 35°C for 48 hours, a blank control group (no Mycobacterium abscessus injection, no drug administration) and a negative control group (Mycobacterium abscessus injection, no drug administration) were set up. The volume of each well was 3 mL, and the MTC (maximum tolerated concentration) of the sample in the zebrafish model was measured.

[0141] (3) Under a microscope, zebrafish 3 days after fertilization were randomly assigned to six-well plates, with 30 fish per well. DMSO (Shanghai Aladdin Biochemical Technology Co., Ltd., China) stored at -20℃ was diluted with water to a solution of 20.0 mg / mL, which was used to prepare AZM, BDQ, and the compound shown in Formula I-1. The concentrations of AZM were 62.5 μg / mL, BDQ was 15.6 μg / mL, and the concentrations of the compound shown in Formula I-1 were 1.95 μg / mL, 3.91 μg / mL, 7.81 μg / mL, 15.6 μg / mL, 31.2 μg / mL, and 62.5 μg / mL, respectively. A control group and a negative control group were also set up, with a volume of 3 mL per well. After treatment at 35℃ for 48 hours, 10 zebrafish were randomly selected from each experimental group and photographed under a fluorescence microscope. Data were analyzed using image processing software (NIS-Elements D 3.20). The overall fluorescence intensity of the zebrafish was used as an indicator to evaluate the drug's inhibitory effect on the growth of *Mycobacterium abscessus*. The inhibitory effect of the test sample on *Mycobacterium abscessus* was measured. The abscess size was calculated based on fluorescence intensity, and the inhibition rate (%) at each concentration was calculated using the following formula: Inhibition rate (%) = (S... 对照组 -S 药物组 ) / S 对照组 ×100%.

[0142] (4) Under a microscope, zebrafish 3 days after fertilization were randomly assigned to 50 mL beakers (experimental group), with 60 zebrafish per beaker. DMSO (Shanghai Aladdin Biochemical Technology Co., Ltd., China) stored at -20℃ was diluted with water to a solution of 20.0 mg / mL, which was used to prepare AZM, BDQ, and the compound shown in Formula I-1. The concentrations of AZM were 62.5 μg / mL, BDQ was 15.6 μg / mL, and the concentrations of the compound shown in Formula I-1 were 1.95 μg / mL, 3.91 μg / mL, 7.81 μg / mL, 15.6 μg / mL, 31.2 μg / mL, and 62.5 μg / mL, respectively. A control group and a negative control group were also set up, with each beaker containing 20 mL. The beakers were treated at 35℃, and the number of zebrafish deaths was recorded daily, after which dead zebrafish were removed. The data after the experiment were statistically analyzed, and the survival rate of zebrafish in each experimental group was calculated.

[0143] 3. Data Analysis

[0144] Statistical analysis was performed using SPSS 26.0. Fluorescence intensity is expressed as mean ± SE. If the independent samples were normally distributed, t-tests or t' tests were used for comparisons between groups; if they were not normally distributed, nonparametric tests were used. Kaplan-Meier survival analysis was performed using the log-rank test to present the survival of zebrafish at different concentrations of the compound shown in Formula I-1. One-way ANOVA was used to analyze CFU counts. A p-value < 0.05 was considered statistically significant.

[0145] 4. Results

[0146] (1) MTC (maximum tolerated concentration) of different concentrations of AZM, BDQ, and the compound shown in Formula I-1 in zebrafish

[0147] Table 7

[0148]

[0149] Table 7 shows the MTC effects of different concentrations of AZM (azithromycin), BDQ, and the compound shown in Formula I-1 in zebrafish. With AZM, the condition of zebrafish began to deteriorate at a concentration of 125 μg / mL; cardiac congestion occurred at a concentration of 500 μg / mL; and the mortality rate reached 33% at a concentration of 1000 μg / mL. With BDQ, increasing the concentration from 3.91 μg / mL to 15.6 μg / mL showed no significant change compared to the negative control group. At a concentration of 31.2 μg / mL, four zebrafish developed renal edema, two developed pericardial edema, and one developed cardiac congestion. Only one zebrafish survived at a concentration of 62.5 μg / mL. With the compound shown in Formula I-1, the zebrafish were in good condition at a concentration of 62.5 μg / mL. When the concentration was increased to 125 μg / mL, the zebrafish exhibited cardiac congestion and body contortion, but were not yet dead. The experimental data in Table 7 demonstrate that, compared to BDQ, the compound represented by Formula I-1 has lower toxicity and better safety.

[0150] (2) Evaluation of the inhibitory effects of different concentrations of AZM, BDQ, and the compound shown in Formula I-1 on Mycobacterium abscessus in zebrafish model

[0151] Table 8

[0152]

[0153] Compared with the negative control group, *p<0.05, **p<0.01, ***p<0.001.

[0154] Compared with the compound shown in Formula I-1 at 62.5 μg / mL, a1p<0.001, a2 p<0.05, a3 p<0.01.

[0155] In the zebrafish model, fluorescence intensity represented Mycobacterium abscessus infection. Table 8 shows the fluorescence intensity of azithromycin MTC concentration, bedaquiline MTC concentration, and the MTC concentration of the compound shown in Formula I-1 at the whole body and head of zebrafish, respectively. The fluorescence intensity distribution of the whole body of zebrafish in the azithromycin and bedaquiline groups was lower than that in the negative control group (433684±11910 vs. 671089±22305, p<0.01; 438648±8280 vs. 671089±22305, p<0.01).

[0156] In whole-body fluorescence intensity analysis, the whole-body fluorescence intensity of the 62.5 μg / mL azithromycin group and the 15.6 μg / mL bedaquiline group was significantly higher than that of the MTC group (433684±11910 vs. 375347±11359, p<0.001; 433684±11910 vs. 375347±11359, p<0.001). In head fluorescence intensity analysis, the head fluorescence intensity of the 62.5 μg / mL azithromycin group and the 15.6 μg / mL bedaquiline group was significantly higher than that of the MTC group shown in Formula I-1 (78397±4815 vs. 70976±5726, p>0.05; 79664±5809 vs. 70976±5726, p>0.05). The MTC group shown in I-1 showed no statistically significant difference compared to the 62.5 μg / mL azithromycin group and the 15.6 μg / mL bedaquiline group.

[0157] (3) Evaluation of the efficacy of prolonging the survival time of a zebrafish model infected with Mycobacterium abscessus.

[0158] Table 9

[0159]

[0160]

[0161] Compared with the negative control group, b1 p<0.05, b2 p<0.01, b3 p<0.001.

[0162] Compared to the concentration of the compound shown in Formula I-1, which is 62.5 μg / mL, c1 p<0.05, c2 p<0.001.

[0163] Table 9 shows that when the concentration of the compound shown in Formula I-1 was 62.5 μg / mL, the survival rate of zebrafish was 38.33%, and the survival rate of zebrafish decreased from 90% to 38.33% 7 days after fertilization. When the concentrations of the compound shown in Formula I-1 and BDQ were 15.6 μg / mL, the survival rates of zebrafish were 48.33% and 75%, respectively. This indicates that the compound shown in Formula I-1, compared with BDQ, resulted in a higher survival rate in zebrafish treated with Mycobacterium abscessus infection and exhibited better antibacterial activity against Mycobacterium abscessus in vivo.

[0164] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. Use of a substance X for the manufacture of a medicament for inhibiting non-tuberculous mycobacteria, a medicament for the treatment and / or prevention of a non-tuberculous mycobacterial infection, a non-tuberculous mycobacterial inhibitor, or a non-tuberculous mycobacterial antimicrobial, characterized in that, The substance X is a compound represented by Formula I or a pharmaceutically acceptable salt thereof; the nontuberculous mycobacterium is a rapidly growing nontuberculous mycobacterium, and the rapidly growing nontuberculous mycobacterium is Mycobacterium abscessus; I。 2. The use as described in claim 1, characterized in that, The substance X is a compound represented by formula I-1; I-1。 3. The use as described in claim 1 or 2, characterized in that, The rapidly growing nontuberculous mycobacterium mentioned is Mycobacterium abscessus subsp. Marseilles.

4. The use as described in claim 1 or 2, characterized in that, It meets one or more of the following conditions: 1) Substance X is one or the only effective ingredient of the drug; 2) The drug contains pharmaceutical excipients; 3) The medication is administered orally; 4) The substance X is in a therapeutic and / or preventative effective amount.

5. The use as described in claim 1 or 2, characterized in that, The nontuberculous mycobacterial infection is NTM lung disease and / or disseminated NTM disease.

6. The use as described in claim 1, characterized in that, The rapidly growing nontuberculous mycobacteria are ATCC19977 or DSM45103.

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