WO2023227123A1

WO2023227123A1 - Use of pyridine derivative

Info

Publication number: WO2023227123A1
Application number: PCT/CN2023/096638
Authority: WO
Inventors: 黄海荣; 朱芮; 尚园园; 初乃惠; 聂文娟; 李永国; 李磊
Original assignee: 广州嘉越医药科技有限公司; 首都医科大学附属北京胸科医院; 上海嘉坦医药科技有限公司
Priority date: 2022-05-27
Filing date: 2023-05-26
Publication date: 2023-11-30
Also published as: CN117122595A

Abstract

Disclosed in the present invention is use of a pyridine derivative. Particularly disclosed is use of a substance X in the preparation of a drug for inhibiting non-mycobacterium tuberculosis, a drug for treating and/or preventing non-mycobacterium tuberculosis infection, a non-mycobacterium tuberculosis inhibitor or a non-mycobacterium tuberculosis antibacterial agent. The substance X is a compound represented by formula I, a solvate thereof, a pharmaceutically acceptable salt thereof, or a solvate of a pharmaceutically acceptable salt thereof. The pyridine derivative of the present invention has a good inhibition effect on non-mycobacterium tuberculosis, and is low in cytotoxicity and good in safety.

Description

Uses of pyridine derivatives

This application claims priority to Chinese patent application 2022106003296 with a filing date of 2022/5/27. This application cites the full text of the above-mentioned Chinese patent application.

Technical field

The present invention relates to the use of a pyridine derivative.

Background technique

The continued increase in the number of cases worldwide has made nontuberculous mycobacterial (NTM) disease a major public health problem. In countries or regions with high prevalence, NTM can account for 30-50% of all mycobacterial isolates. Non-tuberculous mycobacterial (NTM) lung disease is more common in patients with underlying lung diseases such as bronchiectasis and COPD (chronic obstructive pulmonary disease) and is an important cause of increased mortality in these patients. More than 200 NTM species or subspecies have been reported, among which Mycobacterium avium complex (MAC, mainly including Mycobacterium avium and Mycobacterium intracellulare), Mycobacterium abscessus (Mab) and Mycobacterium kansasii are The most common bacterial species causing NTM lung disease (NTM-PD). Treatment of NTM infections is often challenging as different NTM species are generally naturally resistant to most antibiotics.

Bedaquiline (BDQ) is a novel antibiotic that targets drug-resistant tuberculosis by inhibiting ATP synthesis. BDQ not only showed potent efficacy against multidrug-resistant tuberculosis in vitro and in vivo, but also showed potent activity against many different NTM strains in vitro. However, the multiple serious side effects of BDQ, such as unexplained mortality, QT interval prolongation, hepatotoxicity, and phospholipidosis, limit the promotion of BDQ. Therefore, finding highly active anti-NTM drugs is the primary task to establish effective NTM infection treatment regimens.

Contents of the invention

The technical problem to be solved by the present invention is to provide the use of a pyridine derivative in order to overcome the lack of drugs for inhibiting non-tuberculous mycobacterial diseases in the prior art. The pyridine derivative of the invention has good inhibitory effect on non-tuberculous mycobacteria, has low cytotoxicity and good safety.

The present invention provides the use of substance X in the preparation of drugs for inhibiting non-tuberculous mycobacteria; the substance Acceptable salt solvates;

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

In some embodiments, the substance X may be one of the active ingredients or the only active ingredient of the drug.

In one embodiment, the non-tuberculous mycobacteria may be selected from one or more of fast-growing non-tuberculous mycobacteria and slow-growing non-tuberculous mycobacteria.

In one embodiment, the fast-growing non-tuberculous mycobacteria can be selected from the group consisting of Mycobacterium abscessus, Mycobacterium field, Mycobacterium aichi, Mycobacterium aureus, Mycobacterium South Africa, Mycobacterium chelonae, Mycobacterium Chida, Mycobacterium trubucus, Mycobacterium cosmetics, Mycobacterium dieli, Mycobacterium fortuitum, Mycobacterium mucogenes, Mycobacterium orbu, Mycobacterium parafortuitum, Mycobacterium exogenous , Mycobacterium dust, Mycobacterium senegal, Mycobacterium septicemia, Mycobacterium smegmatis, Mycobacterium heat-resistant, Mycobacterium eastsea, Mycobacterium fortuitum subgenus fortuitum, Mycobacterium new orleans, Mycobacterium winter bacilli, Mycobacterium abscessus subspecies Marseilles, and Mycobacterium coblenzii.

In one embodiment, the fast-growing non-tuberculous mycobacteria is preferably Mycobacterium New Orleans.

In one embodiment, the rapidly growing non-tuberculous mycobacteria can be selected from the group consisting of ATCC19977, ATCC27406, ATCC27280, ATCC23366, ATCC33464, ATCC14472, ATCC19627, ATCC27278, DSM44829, ATCC19340, ATCC6841, DSM44124, ATCC27023, AT CC19686, DSM43271, ATCC35154 , one or more of ATCC35796, ATCC700731, ATCC19420, ATCC19527, ATCC27282, DSM46621, DSM44679, DSM44177, DSM45103 and DSM44017.

In one embodiment, the rapidly growing non-tuberculous mycobacterium is preferably DSM44679.

In one embodiment, the slow-growing nontuberculous mycobacteria can be selected from the group consisting of Mycobacterium asiatica, Mycobacterium avium, Mycobacterium cryptica, Mycobacterium chimera, Mycobacterium gastricus, Mycobacterium gordonii , Mycobacterium intracellulare, Mycobacterium kansasii, Mycobacterium vole, chromogenic Mycobacterium, Mycobacterium parascrofulaceus, Mycobacterium rhodesia, Mycobacterium scrofula, Mycobacterium sulgaris , Mycobacterium terrestrialis, Mycobacterium minora, Mycobacterium xenoides, Mycobacterium caddis bacilli, Mycobacterium aarhusii, Mycobacterium kubica, Mycobacterium intermedius, Mycobacterium sphagnum, Mycobacterium stutzeri, and Mycobacterium marinum.

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

In some embodiments, the medicament may include pharmaceutical excipients.

In some embodiments, the administration method of the drug can be a conventional administration method in the art, such as oral administration.

In some embodiments, the substance X is a therapeutically and/or prophylactically effective amount.

The present invention also provides a pharmaceutical composition for inhibiting non-tuberculous mycobacteria, which includes: substance X as described above, and pharmaceutical excipients.

The present invention also provides the use of substance X in the preparation of drugs for treating and/or preventing non-tuberculous mycobacterial infections; the substance salts or solvates of pharmaceutically acceptable salts thereof;

In one embodiment, the above-mentioned substance X is preferably a compound represented by formula I-1;

In one embodiment, the rapidly growing non-tuberculous mycobacteria can be selected from the group consisting of Mycobacterium abscessus, Mycobacterium bacilli, Mycobacterium aichi, Mycobacterium aureus, Mycobacterium South Africa, Mycobacterium chelonae, Mycobacterium Chida, Mycobacterium trubucus, Mycobacterium cosmetics, Mycobacterium dieli, Mycobacterium fortuitum , Mycobacterium mucogenes, Mycobacterium obdu, Mycobacterium parafortuitum, Mycobacterium exogenous, Mycobacterium dust, Mycobacterium senegal, Mycobacterium septicemia, Mycobacterium smegmatis, Mycobacterium heat-resistant , Mycobacterium donghaiense, Mycobacterium fortuitum subgenus fortuitum, Mycobacterium new orleans, Mycobacterium winter, Mycobacterium abscessus subspecies Marseille and Mycobacterium koblenz.

In one embodiment, the slow-growing nontuberculous mycobacteria can be selected from the group consisting of Mycobacterium asiatica, Mycobacterium avium, Mycobacterium cryptica, Mycobacterium chimera, Mycobacterium gastricus, Mycobacterium gordonii , Mycobacterium intracellulare, Mycobacterium kansasii, Mycobacterium vole, chromogenic Mycobacterium, Mycobacterium parascrofulaceus, Mycobacterium rhodesia, Mycobacterium scrofula, Mycobacterium sulgaris , Mycobacterium terrestrialis, Mycobacterium minora, Mycobacterium xenoides, Mycobacterium caddis, Mycobacterium aarhusii, Mycobacterium kubica, Mycobacterium intermedius, Mycobacterium sphagnum, One or more species of Mycobacterium marinum and Mycobacterium marinum.

In one embodiment, the non-tuberculous mycobacterial infection is preferably NTM lung disease and/or disseminated NTM disease.

In some embodiments, the medicament may include pharmaceutical excipients.

In some embodiments, the administration method of the drug can be any method conventional in the art, such as oral administration.

In one embodiment, the substance X may be in a therapeutically and/or prophylactically effective amount.

The present invention also provides a pharmaceutical composition for treating and/or preventing non-tuberculous mycobacterial infection, which includes: substance X as described above, and pharmaceutical excipients.

The present invention also provides the use of substance X in the preparation of non-tuberculous mycobacteria inhibitors; the substance Acceptable salt solvates;

In one embodiment, the slow-growing nontuberculous mycobacteria can be selected from the group consisting of Mycobacterium asiatica, Mycobacterium avium bacteria, Mycobacterium cryptica, Mycobacterium chimera, Mycobacterium gastricus, Mycobacterium gordonii, Mycobacterium intracellulare, Mycobacterium kansasii, Mycobacterium vole, Chromogenous Mycobacterium, Parascrofula Mycobacterium, Mycobacterium rhodesia, Mycobacterium scrofula, Mycobacterium sulgaris, Mycobacterium terrestrialis, Mycobacterium minora, Mycobacterium xenoides, Mycobacterium caddis, Aarhus One or more of Mycobacterium spp., Mycobacterium kubica, Mycobacterium intermedius, Mycobacterium sphagnum, Mycobacterium stutzeri, and Mycobacterium marinum.

In some embodiments, the medicament may include pharmaceutical excipients.

The present invention also provides the use of substance X in the preparation of non-tuberculous mycobacterial antibacterial agents; the substance Acceptable salt solvates;

In some embodiments, the medicament may include pharmaceutical excipients.

The present invention also provides a method for treating and/or preventing non-tuberculous mycobacterial infection, which includes: administering a therapeutically effective amount and/or a preventive effective amount of the substance X or the above-mentioned medicine to a subject in need. combination.

Definition and Description

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 in the absence of a particular definition but should instead be given its ordinary meaning. It is understood that when a trade name appears herein, it is intended to refer to its corresponding trade name or its active ingredient.

In the present invention, the medicine for treating and/or preventing related diseases caused by non-tuberculous mycobacteria can be in conventional dosage forms in the art, such as tablets, capsules, intravenous injections, intraperitoneal injections, inhalants, and aerosols. , lyophilized agent, patch, gel, spray or suppository, etc.

The term "pharmaceutical excipients" refers to the excipients and additives used in the production of drugs and preparation of prescriptions. They can be all substances included in pharmaceutical preparations except active ingredients. Please refer to the fourth volume of the Pharmacopoeia of the People's Republic of China (2020 Edition) Or Handbook of Pharmaceutical Excipients (Raymond CRowe, 2009 Sixth Edition).

The term "treatment" refers to therapeutic therapy. When referring to a specific condition, treatment means: (1) alleviating one or more biological manifestations of the disease or condition, (2) interfering with (a) the biological cascade that causes or causes the condition. one or more points or (b) one or more biological manifestations of a condition, (3) amelioration of one or more symptoms, effects, or side effects associated with a condition, or one or more symptoms associated with a condition or its treatment or a variety of symptoms, effects or side effects, or (4) slow the progression of a condition or one or more biological manifestations of a condition.

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

The term "therapeutically effective amount" refers to a compound that is sufficient to effectively treat a disease or condition described herein when administered to a subject The amount of a substance, a "therapeutically effective amount" will vary depending on the compound, the condition and its severity, and the age of the patient to be treated, but can be adjusted as necessary by one skilled in the art.

The term "prophylactically 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 to prevent recurrence of a disease or disorder.

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

On the basis of not violating common sense in the field, the above preferred conditions can be combined arbitrarily to obtain preferred examples of the present invention.

The reagents and raw materials used in the present invention are all commercially available.

The positive and progressive effect of the present invention is that the pyridine derivative of the present invention has strong antibacterial activity against different non-tuberculous mycobacteria, has low cytotoxicity, good safety, and has the ability to treat and/or prevent diseases caused by non-tuberculous mycobacteria. potential for related diseases.

Description of the drawings

Figure 1 shows the cytotoxicity test results of BDQ and the compound represented by formula I-1 in THP-1 cells.

Detailed ways

The present invention will be further described below by way of examples, but the present invention is not limited to the scope of the described examples. Experimental methods without specifying specific conditions in the following examples can be carried out according to conventional methods and conditions, or according to Product manual selection,

Example 1

1. Experimental materials

(1) 51 standard strains of non-tuberculous mycobacteria (NTM), including 26 standard strains of rapidly growing non-tuberculous mycobacteria (RGM) and 25 standard strains of chronically growing non-tuberculous mycobacteria (SGM) From the biological sample bank of Beijing Chest Hospital Affiliated to Capital Medical University, purchased from the American Type Culture Collection (ATCC) or the German Culture Collection of Microorganisms (DSM), see Table 1 and Table 2 below;

Table 1 26 RGM strains

Table 2 Standard strains of 25 SGM strains

(2) 132 NTM clinical isolates, including 40 Mycobacterium abscessus strains, 29 Mycobacterium intracellulare strains, 21 Mycobacterium avium strains, and 42 Mycobacterium kansasii strains. All strains were obtained from clinical samples. After clinical samples are isolated and cultured to obtain positive cultures, they pass the growth test on p-nitrobenzoic acid medium and 16SrRNA (16S ribosomal RNA,), hsp65 (heat shock protein 65), rpoB (RNA polymerase beta subunit) , Sequence alignment of the 16-23SrRNA intergenic region, and the isolates were identified to the species level.

Compounds represented by formula I-1:

2.Experimental method:

(1) Minimum inhibitory concentration (MIC) test

BDQ (bedaquiline, purchased from Congliye Pharmaceutical (Nanjing, China)) and the compound represented by formula I-1 (provided by Shanghai Jiatan Pharmaceutical Technology Co., Ltd.) were dissolved in dimethyl sulfoxide (DMSO). Prepare a stock solution with a concentration of 8 mg/mL under bacterial conditions. Inoculum was prepared from fresh cultures grown on Roche's medium. Broth microdilution procedures were performed in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines. Cation-adjusted Mueller-Hinton broth (CAMHB) enriched with 5% OADC (oleic acid-calf serum albumin-glucose-catalase) was used for SGM, while OADC-free CAMHB was used for RGM. The broth microdilution form is set to a 2-fold concentration gradient dilution, and the concentration range of BDQ and the compound shown in Formula I-1 is 0.0039-2.0 mg/L. Then place the culture plates inoculated with bacteria in a 37°C incubator and incubate them. The growth of RGM can be observed after 3 days, and the growth of SGM can be observed after 7-10 days. Add 70 μL of a solution containing 20 μL of Alamar Blue and 50 μL of Tween 80 (5%) to each well and incubate at 37°C for 24 hours, and then observe the medium color change. A change in the culture medium from blue to pink or purple indicates bacterial growth. The MIC value is defined as the lowest antibiotic drug concentration at which no color change from blue to pink occurs. For the included clinical strains, the MIC50 and MIC90 of the drug against different strains were calculated respectively. MIC50 and MIC90 refer to the MIC required to inhibit the growth of 50% and 90% of the test bacteria in a batch of tests.

(2) Confirm the preliminary epidemiological critical value (ECOFF)

For strains with more than 20 clinical isolates included and for which the compound shown in BDQ/Formula I-1 shows obvious 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 flora for a unimodal MIC distribution curve, while for a bimodal MIC distribution curve, ECOFF is set between the two peaks.

(3) Minimum bactericidal concentration (MBC) determination

The MBC of five NTM species standard strains was determined by counting colony forming units (CFU) in the wells of a 96-well plate after 3 or 7 days of incubation with relevant compounds. According to CLSI guidelines, MBC refers to the drug concentration that achieves a killing rate of 99.9% of the final inoculum. Initial CFU were calculated on the same day the cultures were incubated in 96-well microplates. The drug concentration range of the compound represented by formula I-1 and BDQ is 1×MIC to 128×MIC. Incubate for three days with RGM and 7-10 days with SGM. Aliquots of 100ul of culture per well were placed in cation-conditioned Mueller-Hinton meat. The broth (CAMHB) was cultured with or without 5% OADC. Count CFU 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 CFU that is at least 3 log10 below the initial CFU. When the MBC/MIC ratio ≤ 4, the antibiotic is considered to have a bactericidal effect; otherwise, it is considered to have a bacteriostatic effect.

(4) Cytotoxicity assay

THP-1 cells were used to evaluate the cytotoxicity of BDQ and the compounds shown in formula I-1. The cells were seeded into 96-well plates and induced to differentiate into macrophages with 100 nMPMA. After 48 hours, cells were washed once and cultured in fresh RPMI medium (purchased from Gibco) containing 10% fetal calf serum (RPMI complete medium). Drug solutions were added to the wells at final concentrations ranging from 1 to 16 μg/mL and incubated for an additional 24 h and 48 h. CCK-8 cell proliferation and cytotoxicity assay kit (purchased from Solarbio, Beijing, China) was used to monitor the cytotoxicity of different concentrations of BDQ and the compound represented by Formula I-1. The cell culture supernatant was analyzed, and the absorbance value (Abs) at 450 nm was recorded using a MultiskanGo microplate reader (purchased from ThermoFisher, 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%.

3. Statistical analysis

SPSS23.0 software and GraphPad Prism7.0 software were used to analyze the data, and the Spearman test was used to analyze the correlation between the BDQMIC values of clinical isolates and the MIC values of the compounds represented by Formula I-1. For cytotoxicity assay, two-way analysis of variance (ANOVA) followed by post hoc test was used to determine significant differences between groups. Differences were considered statistically significant for P values <0.05.

4.Results

(1) MIC of BDQ and the compound represented by formula I-1 against NTM standard strain

The MICs of BDQ and the compounds represented by formula I-1 against 51 standard strains are shown in Tables 3 and 4. For the same standard strain, the antibacterial activity of the compound represented by formula I-1 is similar to that of BDQ, or the MIC of some standard strains is doubled. Like BDQ, the compound represented by Formula I-1 showed strong antibacterial activity against almost all tested SGM strains, with MICs usually lower than 0.25 μg/mL. There are only two standard strains of SGM strains, namely Mycobacterium rhodesiae and Mycobacterium celatum, with MIC>2μg/mL. In addition, the compound represented by formula I-1 also showed very effective in vitro activity against the included RGM standard strain. The MIC of all 26 RGM standard strains is ≤0.5μg/ml, and the MIC of 25 SGM standard strains is ≤0.25μg/ml.

Table 3 MIC of 26 RGM standard strains

Table 4 MIC of 25 SGM standard strains

(2) MIC distribution and ECOFF of BDQ and compounds represented by formula I-1 in clinical isolates of different NTM strains

The MIC distributions of the four most common NTM strains to BDQ and the compounds shown in Formula I-1 are shown in Table 5. The sensitivity distribution of clinical isolates to BDQ and the compounds shown in Formula I-1 is consistent with the standard strains, that is Absolutely most SGM isolates had strong antibacterial activity against all included strains. It also has similar activity against Mycobacterium abscessus, but the MIC value is higher than that of SGM.

Table 5 MIC distribution results of four NTM strains against BDQ and the compound shown in formula I-1

BDQ and the compound shown in Formula I-1 showed the strongest activity against Mycobacterium kansasii and Mycobacterium intracellulare, with ECOFF both being 0.0156 μg/mL and MIC50 both being 0.0078 μg/mL. , MIC90 are both 0.0156μg/mL. The vast majority of isolates of both strains included had MICs below 0.008 μg/mL. BDQ and the compound shown in Formula I-1 have good activity against Mycobacterium abscessus, with MIC50 of 0.125 μg/mL and 0.25 μg/mL respectively. The MIC90 of BDQ and the compound shown in Formula I-1 against Mycobacterium abscessus are 0.25 μg/mL and 0.5 μg/mL respectively. The ECOFF of Mycobacterium abscessus for BDQ and the compound represented by Formula I-1 is 0.25 μg/mL or 0.5 μg/mL respectively.

For Mycobacterium abscessus, there is a good correlation between BDQ and the MIC of the compound represented by formula I-1 (Spearman's q=0.722, P=0.0000). The correlation between the BDQMIC of Mycobacterium kansasii and the MIC of the compound represented by Formula I-1 is significant (Spearman's q=1.000, P=0.0000). For Mycobacterium avium and Mycobacterium intracellulare, the correlation between BDQMIC and the MIC of the compound represented by Formula I-1 is also very strong (Spearman's q=0.967, P=0.0000; Spearman's q=0.967, P=0.0000; q=0.784, two-tailed, P=0.0000).

(3)MBC measurement

The MBCs of the compound represented by formula I-1 and BDQ against the five NTM standard strains were both greater than 32×MIC. Therefore, the MBC/MIC ratio of the compound represented by formula I-1 or BDQ against the five NTM standard strains is much higher than 4, indicating that both drugs are effective against Mycobacterium abscessus (Mycobacterium abscessus), Mycobacterium fortuitum (Mycobacterium) fortuitum), Mycobacterium intracellulare, Mycobacterium avium and Mycobacterium kansasii all have bacteriostatic effects (Table 6).

Table 6 MBC/MIC ratio of SGM and RGM

Note: If the MBC/MIC ratio is higher than 4, the effect is considered bacteriostatic, otherwise it is considered bactericidal.

(4) Cytotoxicity determination of BDQ and compounds represented by formula I-1 in THP-1 cells

When the compound of formula I-1 was incubated for 24 hours, the THP-1 cell survival rate reached more than 75% when the concentration was 16 μg/mL, and the THP-1 cell survival rate reached more than 90% when the concentration was 8 μg/mL. The concentration was 4 μg/mL, At 2 μg/mL and 1 μg/mL, the survival rate of THP-1 cells was close to 100%. When incubated for 48 hours, the cell survival rate dropped to less than 75% at the concentration of 16 μg/mL, the cell survival rate reached more than 75% at the concentration of 8 μg/mL, and the cell survival rate reached more than 80% at the concentration of 4 μg/mL and 2 μg/mL. The cell survival rate at 1 μg/mL was close to 100%, and there was no statistical difference between BDQ and the compound represented by Formula I-1 (Figure 1).

in conclusion:

The compounds represented by formula I-1 of the present application all have good antibacterial activity against strains of different NTM species, and it is proved that the tested standard strains and clinical isolates are sensitive to it, and all tested standard strains of RGM or SGM strains The MICs were all below 0.5 μg/mL, and most of them had MICs well below 0.1 μg/mL (Tables 3 and 4). with BDQ In comparison, the activity is similar. The inhibitory activity of the compound represented by formula I-1 against clinical isolates of the four most isolated strains is consistent with the results of standard strains. The MIC50 and MIC90 of Mycobacterium intracellulare, Mycobacterium avium and Mycobacterium kansasii are ≤0.0625 μg respectively. /mL and ≤0.125μg/mL. The MICs of the two drugs were analyzed by Spearman's test to be closely related (Spearman's q ranged from 0.722 to 1). Overall data on RGM and SGM standard strains as well as clinical isolates of the most commonly isolated NTM species support the potential use of compounds represented by Formula I-1 in the treatment of NTM infections as effective as BDQ.

The ECOFF value of the compound represented by formula I-1 of the present application against NTM bacterial species, which is very important for setting breakpoints for drug susceptibility testing in the future, Mycobacterium abscessus, Mycobacterium intracellulare, Mycobacterium avium and Mycobacterium kansasii The ECOFFs of Bacilli are generally low, ranging from 0.0156 μg/mL to 0.5 μg/mL.

THP-1 cells have a survival rate of nearly 100% when exposed to compounds of formula I-1 below 4 μg/mL for 24 hours. This concentration is the concentration of the three most commonly isolated bacterial species (Mycobacterium intracellulare, Mycobacterium avium). Mycobacterium and Mycobacterium kansasii) 200-300 times the MIC obtained by testing with standard strains. 4 μg/mL is 16 times the MIC obtained by the standard strain of Mycobacterium abscessus tested in this study. The macrophage assay shows that the compound represented by Formula I-1 is safe at an effective therapeutic concentration.

In summary, both reference strains and clinically isolated NTM tests show that the compound represented by formula I-1 has strong antibacterial activity against different NTM strains and has low cytotoxicity.

Example 2 Evaluation of in vivo antimicrobial activity and prolonged survival against Mycobacterium abscessus

1. Experimental materials

AZM (azithromycin, purchased from Shanghai Modern Pharmaceutical Co., Ltd.), BDQ (bedaquiline, purchased from Congliye Pharmaceutical (Nanjing, China)) and the compound represented by formula I-1 (provided by Shanghai Jiatan Pharmaceutical Technology Co., Ltd.) ;

2. Experimental methods

(1) Wild-type AB zebrafish were selected for natural pair fertilization and cultured in a water temperature of 28°C. Formulated with Middlebrook 7H9 (Becton Dickinson) broth enriched with 10% OADC (oleic acid-calf serum albumin-glucose-catalase) and containing 0.05% Tween 80 (Sigma-Aldrich), it will have a smooth (S ) morphology of Mycobacterium abscessus ATCC19977 was incubated in a 37°C incubator for 5 to 7 days, and then the moderate to logarithmic growth of Mycobacterium abscessus was centrifuged, washed and placed in phosphate buffered saline containing 0.05% Tween 80 ( The bacterial suspension was homogenized and sonicated in PBS), and the bacterial colonies were allowed to settle for 5 to 10 minutes. The bacteria were then enriched in phosphate buffered saline (PBS), and DIO (green fluorescence) was used to label Mycobacterium abscessus. The bacterial cells were transplanted into the wild-type AB zebrafish strain 2 days after fertilization through microvenous injection, and each tail was transplanted with approximately 3.6×10 ³ CFU to establish the Mycobacterium abscessus-zebrafish model.

(2) Under a microscope, select zebrafish 3 days after fertilization and randomly distribute them into six-well plates, with 30 fish in each well. Will DMSO (Shanghai Aladdin Biochemical Technology Co., Ltd., China) stored at -20°C was diluted with water to a 20.0 mg/mL solution, which was used to prepare AZM, BDQ and compounds of formula I-1 respectively. The AZM concentrations were 62.5μg/mL, 125μg/mL, 250μg/mL, 500μg/mL and 1000μg/mL; the BDQ concentrations were 3.91μg/mL, 7.81μg/mL, 15.6μg/mL, 31.2μg/mL and 62.5 μg/mL; the concentrations of the compounds shown in Formula I-1 are 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. After the plate containing zebrafish was kept at 35°C for 48 hours, a blank control group (no injection of Mycobacterium abscessus, no medication) and a negative control group (no injection of Mycobacterium abscessus, no medication) were set up in each well. Capacity: 3mL, measure the MTC (maximum tolerated concentration) of the sample in the zebrafish model.

(3) Under a microscope, zebrafish 3 days after fertilization were randomly distributed into six-well plates, with 30 fish in each well. DMSO (Shanghai Aladdin Biochemical Technology Co., Ltd., China) stored at -20°C was diluted with water to a 20.0 mg/mL solution, which was used to prepare AZM, BDQ and the compound of formula I-1 respectively. The concentration of AZM is 62.5 μg/mL, the concentration of BDQ is 15.6 μg/mL, and the concentration of the compound shown in Formula I-1 is 1.95 μg/mL, 3.91 μg/mL, 7.81 μg/mL, 15.6 μg/mL, and 31.2 μg/mL. mL and 62.5μg/mL. Set up a control group and a negative control group at the same time, with the capacity of each well being 3mL. After 48 hours of treatment at 35°C, 10 zebrafish were randomly selected from each experimental group and photographed under a fluorescence microscope. Image processing software (NIS-Elements D 3.20) was used to analyze and collect the data. The whole-body fluorescence intensity of zebrafish was used as an indicator to evaluate the drug's ability to inhibit zebrafish. The growth of Mycobacterium abscessus in fish, and the inhibitory effect of the test product on the growth of Mycobacterium abscessus. Calculate the abscess based on the fluorescence intensity, and calculate the inhibition rate (%) at each concentration according to the following formula: Inhibition rate (%) = (S _{control group} - S _{drug group} )/S _{control group} × 100%.

(4) Under a microscope, zebrafish 3 days after fertilization were randomly allocated into 50mL beakers (experimental group), with 60 fish per cup. DMSO (Shanghai Aladdin Biochemical Technology Co., Ltd., China) stored at -20°C was diluted with water to a 20.0 mg/mL solution, which was used to prepare AZM, BDQ and the compound of formula I-1 respectively. The concentration of AZM is 62.5 μg/mL, the concentration of BDQ is 15.6 μg/mL, and the concentration of the compound shown in Formula I-1 is 1.95 μg/mL, 3.91 μg/mL, 7.81 μg/mL, 15.6 μg/mL, and 31.2 μg/mL. mL and 62.5μg/mL. At the same time, a control group and a negative control group were set up. Each cup had a capacity of 20 mL and was treated at 35°C. The number of zebrafish deaths was recorded every day and dead zebrafish were removed. Perform statistical analysis on the data after the experiment to calculate the survival rate of zebrafish in each experimental group.

3.Data analysis

This study used SPSS 26.0 for statistical analysis. Fluorescence intensity is expressed as mean ± SE. If the independent samples are normally distributed, the t or t′ test is used to compare the two groups; if the independent samples are non-normally distributed, the non-parametric test is used. Kaplan-Meier survival analysis was performed using the log-rank test to present the survival of zebrafish with the compound represented by Formula I-1 at different concentrations. CFU counts were analyzed using one-way analysis of variance. A p value <0.05 was considered a statistically significant difference.

4.Results

(1) MTC (maximum tolerated concentration) of different concentrations of AZM, BDQ, and compounds represented by Formula I-1 in zebrafish

Table 7

Table 7 shows the MTC effects of different concentrations of AZM (azithromycin), BDQ, and the compound represented by Formula I-1 in zebrafish. When AZM was administered, when the concentration was 125 μg/mL, the condition of the zebrafish began to deteriorate; when the concentration When the concentration was increased to 500 μg/mL, blood stasis occurred in the heart; when the concentration was increased to 1000 μg/mL, the mortality rate of zebrafish reached 33%. When BDQ was administered and the concentration increased from 3.91 μg/mL to 15.6 μg/mL, there was no significant change compared with the negative control group. When the concentration increased to 31.2 μg/mL, 4 zebrafish cases showed renal edema, 2 zebrafish cases showed pericardial edema, and 1 zebrafish case showed cardiac congestion. When the concentration was 62.5μg/mL, only one zebrafish survived. When the compound represented by Formula I-1 is administered at a concentration of 62.5 μg/mL, the zebrafish is in good condition. When the concentration increased to 125 μg/mL, the symptoms were heart congestion and body bending. At this time, the zebrafish was not dead yet. The experimental data in Table 7 shows that compared with BDQ, the compound represented by Formula I-1 has lower toxicity and better safety.

(2) Evaluation of the inhibitory effects of different concentrations of AZM, BDQ, and compounds represented by formula I-1 on zebrafish model Mycobacterium abscessus

Table 8

Compared with the negative control group, *p＜0.05, **p＜0.01, ***p＜0.001.
Compared with the compound of formula I-1 at 62.5 μg/mL, ^a1 p＜0.001, ^a2 p＜0.05, ^a3 p＜0.01.

The fluorescence intensity in the zebrafish model represents Mycobacterium abscessus infection. Table 8 shows the fluorescence of the MTC concentration of azithromycin, the MTC concentration of bedaquiline and the MTC concentration of the compound shown in Formula I-1 for the whole body and head of zebrafish respectively. strength. The whole body fluorescence intensity distribution of zebrafish in the azithromycin group and the bedaquiline group 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)).

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 higher (433684±11910 vs. 375347±11359, p<0.001; 433684±11910 vs. 375347±11359, p<0.001); in In the analysis of head fluorescence intensity, compared with the 62.5 μg/mL azithromycin group and the 15.6 μg/mL bedaquiline group, the head fluorescence intensity was higher than that of the compound MTC group shown in Formula I-1 (78397±4815 vs. 70976±5726 , p>0.05. 79664±5809 vs. 70976±5726, p>0.05). There was no statistical difference between the compound MTC group shown in I-1 and the 62.5 μg/mL azithromycin group and the 15.6 μg/mL bedaquiline group.

(3) Evaluation of the efficacy of prolonging the survival time of zebrafish model infected with Mycobacterium abscessus

Table 9

Compared with the negative control group, ^a1 p＜0.05, ^b2 p＜0.01, ^b3 p＜0.001.
Compared with the concentration of the compound shown in formula I-1 which is 62.5μg/mL, ^c1 p＜0.05, ^c2 p＜0.001.

Table 9 shows that when the concentration of the compound shown in Formula I-1 is 62.5 μg/mL, the survival rate of zebrafish is 38.33%, and the survival rate of zebrafish 7 days after fertilization dropped from 90% to 38.33%; when Formula I-1 When the concentrations of the indicated compounds and BDQ were 15.6 μg/mL, the survival rates of zebrafish were 48.33% and 75%, respectively. This shows that compared with BDQ, the compound represented by Formula I-1 has a higher survival rate in treating zebrafish infected by Mycobacterium abscessus and has better antibacterial activity against Mycobacterium abscessus in vivo.

Although specific embodiments of the present invention have been described above, those skilled in the art should understand that these are only examples, and various changes or changes can be made to these embodiments without departing from the principles and essence of the present invention. Revise. Accordingly, the scope of the present invention is defined by the appended claims.

Claims

The use of a substance Characteristically, the substance X is a compound represented by formula I, a solvate thereof, a pharmaceutically acceptable salt thereof, or a solvate of a pharmaceutically acceptable salt thereof;
The use according to claim 1, wherein the substance X is a compound represented by formula I-1;
The use according to claim 1 or 2, characterized in that it meets one or more of the following conditions:

1) The substance X is one of the active ingredients or the only active ingredient of the drug;

2) The medicine contains pharmaceutical excipients;

3) The drug is administered orally;

4) The substance X is a therapeutic and/or preventive effective amount.
The use according to claim 1 or 2, characterized in that it meets one or more of the following conditions:

1) The non-tuberculous mycobacterial infection is NTM lung disease and/or disseminated NTM disease;

2) The non-tuberculous mycobacteria are selected from one or more types of rapidly growing non-tuberculous mycobacteria and slow-growing non-tuberculous mycobacteria.
The use according to claim 4, characterized in that it satisfies one or both of the following conditions:

1) The rapidly growing non-tuberculous mycobacteria are selected from the group consisting of Mycobacterium abscessus, Mycobacterium field, Mycobacterium aichi, Mycobacterium aureus, Mycobacterium South Africa, Mycobacterium chelonae, Mycobacterium Chida, Mycobacterium trubucus, Mycobacterium cosmeticum, Mycobacterium dieli, Mycobacterium fortuitum, Mycobacterium mucogenes, Mycobacterium obdu, Mycobacterium parafortunate, Mycobacterium exogenous, Mycobacterium dust , Mycobacterium senegal, Mycobacterium septicemia, Mycobacterium smegmatis, Mycobacterium thermoresistant, Mycobacterium donghaiense, Mycobacterium fortuitum Mycobacterium fortuitum, Mycobacterium new Orleans, Mycobacterium winter, Mycobacterium abscessus subsp. Marseilles and Mycobacterium coblenz; the fast-growing non-tuberculous mycobacteria are preferably for Mycobacterium New Orleans;

2) The slow-growing non-tuberculous mycobacteria are selected from the group consisting of Mycobacterium asiatica, Mycobacterium avium, Mycobacterium cryptica, Mycobacterium chimera, Mycobacterium gastric, Mycobacterium gordonii, Mycobacterium intracellulare bacilli, Mycobacterium kansasii, Mycobacterium vole, Mycobacterium chromogenes, Mycobacterium parascrofulaceus, Mycobacterium rhodesia, Mycobacterium scrofula, Mycobacterium sulgaris, Mycobacterium terrestrialis , Mycobacterium minora, Mycobacterium xenoides, Mycobacterium caddis, Mycobacterium aarhusii, Mycobacterium kubica, Mycobacterium intermedius, Mycobacterium sphagnum, Mycobacterium stutzeri and One or more species of Mycobacterium marinum.
The use according to claim 5, characterized in that it satisfies one or both of the following conditions:

1) The rapidly growing non-tuberculous mycobacteria are selected from the group consisting of ATCC19977, ATCC27406, ATCC27280, ATCC23366, ATCC33464, ATCC14472, ATCC19627, ATCC27278, DSM44829, ATCC19340, ATCC6841, DSM44124, ATCC27023, ATCC19 686, DSM43271, ATCC35154, ATCC35796, ATCC700731, One or more of ATCC19420, ATCC19527, ATCC27282, DSM46621, DSM44679, DSM44177, DSM45103 and DSM44017; the fast-growing non-tuberculous mycobacterium is preferably DSM44679;

2) The slow-growing non-tuberculous mycobacteria are selected from ATCC25276, ATCC25291, DSM44243, DSM44622, ATCC15754, ATCC14470, ATCC13950, ATCC12478, ATCC19422, ATCC19530, DSM44648, ATCC27024, ATCC19981, ATCC3 5799, ATCC15755, ATCC23292, ATCC19250, ATCC27726, One or more of DSM45069, DSM44627, DSM44064, ATCC33027, ATCC27962 and ATCC927.
A pharmaceutical composition for treating and/or preventing non-tuberculous mycobacteria infection, or inhibiting non-tuberculous mycobacteria, characterized in that it includes: substance X according to any one of claims 1-2, and pharmaceutical excipients.
The pharmaceutical composition according to claim 7, characterized in that it meets one or both of the following conditions:

(1) The substance X has the use as described in any one of claims 1-6;

(2) The non-tuberculous mycobacterial infection is NTM lung disease and/or disseminated NTM disease.
A method for treating and/or preventing non-tuberculous mycobacterial infection, which includes: administering to a patient in need a therapeutically effective amount and/or a prophylactically effective amount of substance X according to any one of claims 1-2 or as The pharmaceutical composition of claim 7 or 8; the substance X has the use as described in any one of claims 1-6.
The method of claim 9, wherein the non-tuberculous mycobacterial infection is NTM lung disease and/or disseminated NTM disease.