Use of heterocyclic compounds

Abstract

This invention relates to the field of pharmaceutical application technology, and more specifically, to the application of heterocyclic compounds. Example I describes the application of the compound shown in Formula I in the preparation of an inhibitor that inhibits the activity of methylenetetrahydrofolate reductase. Furthermore, the compound shown in Formula I can be used alone, in combination with para-aminosalicylic acid, or in combination with a β-lactamase inhibitor, all of which exhibit good antibacterial activity against Mycobacterium tuberculosis, and can treat or prevent diseases caused by Mycobacterium tuberculosis, such as tuberculosis.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical application technology, and more specifically, to the application of heterocyclic compounds. Background Technology

[0002] Tuberculosis (TB) is a disease caused by infection with Mycobacterium tuberculosis (Mtb). It is primarily transmitted through the air, infecting the lungs to form pulmonary TB. A smaller number of cases are extrapulmonary tuberculosis, which can occur in the larynx, lymph nodes, bones, gastrointestinal tract, central nervous system, and genitourinary system. TB is widespread; the WHO estimates that approximately one-quarter of the global population, or 2 billion people, are latently infected with Mycobacterium tuberculosis, and under suitable conditions, this can develop into active TB. According to the latest WHO global TB report, approximately 10.6 million people were infected with TB in 2022, and the global TB death toll was 1.3 million.

[0003] Drug resistance is currently the most serious problem facing tuberculosis treatment. Drug-resistant tuberculosis requires a longer treatment period, accompanied by higher treatment costs and patients having to endure more suffering, yet the success rate of treatment is lower. In 2022, rifampicin-resistant / multidrug-resistant tuberculosis (RR / MDR-TB) accounted for approximately 3.3% of new tuberculosis cases globally, and RR / MDR-TB patients accounted for approximately 17% of relapsed cases. This is a very high proportion, as there were 410,000 new RR / MDR-TB patients in 2022 alone, while the total number of RR-TB patients receiving standardized treatment was only 175,000. According to currently available data, the treatment success rate for sensitive pulmonary tuberculosis was above 80% from 2012 to 2020, but the treatment success rate for drug-resistant tuberculosis during the same period was only 50-60%. Correspondingly, the treatment period is at least 9 months, or even longer (more than 18 months); and the average treatment cost is approximately US$20,000, or tens of thousands of RMB. my country is a country with both a high burden of tuberculosis and a high burden of drug-resistant tuberculosis, urgently requiring the development of new treatment strategies or new alternative drugs for clinical treatment of tuberculosis to meet the country's pressing requirements for tuberculosis control. Therefore, there is an urgent need to develop a new drug that can inhibit Mycobacterium tuberculosis.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide applications of heterocyclic compounds. The compounds having the structure of Formula I provided in the embodiments of this invention not only inhibit methylenetetrahydrofolate reductase activity but also exhibit anti-tuberculosis effects, thereby demonstrating good therapeutic efficacy against diseases caused by Mycobacterium tuberculosis. Furthermore, they can be used in combination with β-lactamase inhibitors or para-aminosalicylic acid to treat tuberculosis.

[0006] This invention is implemented as follows:

[0007] In a first aspect, the present invention provides the use of a compound of Formula I, its pharmaceutical salt, a hydrate of the salt, or a hydrate thereof in the preparation of an inhibitor that inhibits the activity of methylenetetrahydrofolate reductase.

[0008] Wherein, R1 represents any one of H, carboxylic acid and alkyl, and R2 represents any one of heteroatom-containing heteroaryl-substituted alkenyl, heteroatom-containing heteroaryl-substituted thioether, heteroatom-containing heteroaryl-substituted alkyl and heteroatom-containing fused heteroaryl, wherein the heteroatom includes at least one of sulfur, oxygen and nitrogen.

[0009] In a second aspect, the present invention provides the use of a compound of Formula I, a pharmaceutical salt thereof, a hydrate of the salt thereof, or a hydrate thereof in at least one of the following situations;

[0010] (1) Application in the preparation of antibacterial agents that inhibit the activity of Mycobacterium tuberculosis;

[0011] (2) Application in the preparation of bactericides that kill Mycobacterium tuberculosis;

[0012] (3) Application in the preparation of products that inhibit the activity of Mycobacterium tuberculosis;

[0013] (4) Application in the preparation of products for treating Mycobacterium tuberculosis infection;

[0014] (5) Use in the preparation of products for the treatment and / or prevention of diseases caused by Mycobacterium tuberculosis;

[0015] (6) Application in the preparation of drugs for treating tuberculosis;

[0016] Wherein, R1 represents any one of H, carboxylic acid and alkyl, and R2 represents any one of heteroatom-containing heteroaryl-substituted alkenyl, heteroatom-containing heteroaryl-substituted thioether, heteroatom-containing heteroaryl-substituted alkyl and heteroatom-containing fused heteroaryl, wherein the heteroatom includes at least one of sulfur, oxygen and nitrogen.

[0017] Thirdly, the present invention provides a pharmaceutical composition in which the active ingredient comprises a compound of formula I, a pharmaceutically acceptable salt thereof, a hydrate of the salt thereof, or a hydrate thereof, and para-aminosalicylic acid; or, it comprises a compound of formula I, a pharmaceutically acceptable salt thereof, a hydrate of the salt thereof, or a hydrate thereof, and a β-lactamase inhibitor.

[0018] Wherein, R1 represents any one of H, carboxylic acid and alkyl, and R2 represents any one of heteroatom-containing heteroaryl-substituted alkenyl, heteroatom-containing heteroaryl-substituted thioether, heteroatom-containing heteroaryl-substituted alkyl and heteroatom-containing fused heteroaryl, wherein the heteroatom includes at least one of sulfur, oxygen and nitrogen.

[0019] Fourthly, the present invention provides the use of the pharmaceutical composition described in the foregoing embodiments in at least one of the following situations;

[0020] (1) Application in the preparation of antibacterial agents that inhibit the activity of Mycobacterium tuberculosis;

[0021] (2) Application in the preparation of bactericides that kill Mycobacterium tuberculosis;

[0022] (3) Application in the preparation of products that inhibit the activity of Mycobacterium tuberculosis;

[0023] (4) Application in the preparation of products for treating Mycobacterium tuberculosis infection;

[0024] (5) Use in the preparation of products for the treatment and / or prevention of diseases caused by Mycobacterium tuberculosis;

[0025] (6) Prepare inhibitors that inhibit the activity of methylenetetrahydrofolate reductase;

[0026] (7) Application in the preparation of drugs for treating tuberculosis.

[0027] The present invention has the following beneficial effects: The compound shown in Formula I provided in the embodiments of the present invention can inhibit the activity of methylenetetrahydrofolate reductase, exhibiting good antibacterial activity against Mycobacterium tuberculosis, and consequently also having a certain therapeutic effect on diseases caused by Mycobacterium tuberculosis. Simultaneously, this compound, when used in combination with para-aminosalicylic acid or with β-lactamase inhibitors, also exhibits good antibacterial activity against Mycobacterium tuberculosis and has a certain therapeutic effect on diseases caused by Mycobacterium tuberculosis. Furthermore, several compounds having the structure shown in Formula I, such as cefuroxime, have been approved for marketing, demonstrating high safety, and this "drug repurposing" will significantly reduce development costs. Attached Figure Description

[0028] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 A conservation analysis diagram of the homologous protein of mycobacterial methylenetetrahydrofolate reductase provided in the embodiments of the present invention;

[0030] Figure 2 A schematic diagram of the molecular docking of the compound of Formula I with Mycobacterium tuberculosis methylenetetrahydrofolate reductase provided in an embodiment of the present invention;

[0031] Figure 3 A schematic diagram of the molecular docking between cefuroxime and Mycobacterium tuberculosis methylenetetrahydrofolate reductase provided in an embodiment of the present invention;

[0032] Figure 4 A schematic diagram of the molecular docking of cefepime with Mycobacterium tuberculosis methylenetetrahydrofolate reductase provided in an embodiment of the present invention;

[0033] Figure 5 A schematic diagram of the molecular docking between cefoloza and Mycobacterium tuberculosis methylenetetrahydrofolate reductase provided in an embodiment of the present invention;

[0034] Figure 6 A schematic diagram of the molecular docking between cefazolin and Mycobacterium tuberculosis methylenetetrahydrofolate reductase provided in an embodiment of the present invention;

[0035] Figure 7 The half-maximal inhibitory concentration (IC50) of cefuroxime or cefepime provided in this embodiment of the invention against the activity of methylenetetrahydrofolate reductase in Mycobacterium tuberculosis. 50 The results of the measurement are shown in the figure;

[0036] Figure 8 The image shows the results of detecting the bactericidal effect of cefuroxime against Mycobacterium tuberculosis, as provided in an embodiment of the present invention.

[0037] Figure 9 The pharmacokinetic results of subcutaneous injection of 50 mg / kg cefuroxime in mice are shown in the figure provided in the embodiments of the present invention.

[0038] Figure 10 The image shows the results of detecting the anti-tuberculosis activity of cefuroxime in mice, as provided in an embodiment of the present invention.

[0039] Figure 11The image shows the results of detecting the synergistic anti-tuberculosis effect of cefuroxime and para-aminosalicylic acid provided in an embodiment of the present invention.

[0040] Figure 12 The figure shows the results of detecting the synergistic anti-tuberculosis effect of cefepime and para-aminosalicylic acid provided in the embodiments of the present invention. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0042] Folic acid is an essential nutrient for the life activities of organisms. It provides a one-carbon carrier for the synthesis of purine and thymine deoxynucleotides (dTMP) and participates in the synthesis and conversion of methionine, serine, and glycine. Mammalian cells cannot synthesize folic acid and must obtain this nutrient from food; however, bacteria lack a folic acid transport system and must synthesize folic acid de novo. This characteristic makes the folic acid synthesis and metabolic pathway an ideal target for the development of antibacterial drugs. For example, para-aminosalicylic acid (PAS), currently used to treat drug-resistant tuberculosis, targets bacterial folic acid synthesis.

[0043] Among them, methylenetetrahydrofolate reductase (MTHFR), a key enzyme in folic acid metabolism, catalyzes the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, which is essential for bacterial synthesis of methionine and S-adenosylmethionine, making it an ideal target for antibacterial drug design. In many species, such as Enterobacteriaceae, MTHFR has been identified, annotated, and its crystal structure has been successfully resolved. Furthermore, the reductase activity of MTHFR in these species depends on both reduced coenzyme I (NADH) and the flavoprotein prosthetic group (FAD / FMN). However, MTHFR has not been identified and annotated in the Mycobacterium tuberculosis genome. The inventors discovered that the Rv2172c protein is the MTHFR of Mycobacterium tuberculosis, and confirmed that the reductase activity of Rv2172c depends only on NADH and not on FAD (see: DOI:10.1128 / AAC.01465-21.Epub2021Nov 15.). Meanwhile, the MTHHR protein structures of Mycobacterium smegmatis and Mycobacterium thermophilum were resolved. The primary amino acid sequences and tertiary structures of these two proteins showed high similarity to Rv2172c, and their reductase activities were not dependent on FAD. Therefore, it is believed that the MTHFR of mycobacteria is a special type of methylenetetrahydrofolate reductase with a catalytic mechanism that is NADH-dependent but not FAD-dependent.

[0044] The inventors have discovered the application of the compound represented by Formula I, its pharmaceutical salt, the salt hydrate, or the hydrate thereof in the preparation of inhibitors that inhibit the activity of methylenetetrahydrofolate reductase.

[0045] Wherein, R1 represents any one of H, carboxylic acid and alkyl, and R2 represents any one of heteroatom-containing heteroaryl-substituted alkenyl, heteroatom-containing heteroaryl-substituted thioether, heteroatom-containing heteroaryl-substituted alkyl and heteroatom-containing fused heteroaryl, wherein the heteroatom includes at least one of sulfur, oxygen and nitrogen.

[0046] Preferably, R1 represents any one of H, C1-C7 carboxylic acid, and C1-C8 alkyl, more preferably any one of H, C1-C5 carboxylic acid, and C1-C5 alkyl, more preferably any one of H, C3-C5 carboxylic acid, and C1-C3 alkyl, and even more preferably any one of hydrogen, n-butyric acid, isobutyric acid, methyl, and ethyl; R2 represents any one of oxygen- and nitrogen-containing C3-C10 heteroaryl-substituted C2-C5 alkenyl, sulfur- and nitrogen-containing C3-C10 heteroaryl-substituted thioether, nitrogen-atom-substituted heteroaryl C1-C3 alkyl, and single-atom-containing C4-C8 fused heteroaryl.

[0047] Preferably, R2 represents Any one of them.

[0048] Specifically, the compound represented by Formula I is selected from any one of cefuroxime, cefuroxime ester, cefoprep, cefoprep ester, cefoloza, and cefazolin.

[0049] Among them, ceftaroline (also known as T-91825, PPI-0903M), CAS No. 189345-04-8, molecular formula: C 22 H 20 N8O5S4, structural formula as follows:

[0050]

[0051] Cefadroline fosamil (also known as TAK-599, PPI-0903), a prodrug of cefadroline, is currently marketed in acetate form. Its CAS No. 400827-46-5 has the molecular formula C2. 24 H 25 N8O 10 PS4, the structure is as follows:

[0052] Cefuroxime axetil was developed by Takeda Pharmaceutical Company of Japan and was first approved by the US FDA in October 2010 for the treatment of community-acquired bacterial pneumonia and acute bacterial skin and soft tissue infections in adults. As an N-phosphonoamino water-soluble prodrug of cefuroxime axetil, cefuroxime axetil is rapidly hydrolyzed in the body by phosphatases in the blood to the biologically active compound cefuroxime.

[0053] Ceftobiprole (also known as Ro 63-9141, BAL 9141; also called: cefopiprole, cefopiro, cefotopro), CAS No. 209467-52-7, molecular formula: C 20 H 22 N8O6S2, structural formula as follows:

[0054]

[0055] Ceftobiprole medocaril (also known as BAL5788; also called cefopiprole, cefopirox, cefotorox), a prodrug of cefpirome, CAS No. 376653-43-9, molecular formula: C 26 H 26 N8O 11 S2, the structural formula is as follows:

[0056] Because the free form of cefpirome is unstable, the more common form on the market is the stable sodium salt form, ceftobiprole medocaril sodium (CAS No.: 252188-71-9), which has the same biological activity. Developed by Basilier AG of Switzerland, it was first approved for marketing in Canada in March 2018 for the treatment of community-acquired and hospital-acquired pneumonia. Cefpirome is rapidly broken down into its active form, cefpirome, by plasma esterases in plasma.

[0057] Ceftolozane (also known as FR-264205, CXA-101), CAS No. 689293-68-3, molecular formula: C 23 H 30 N 12 O8S2; the structural formula is as follows:

[0058] Cefoloza was developed by Astellas Pharma and Urahide Pharmaceutical Co., Ltd. of Japan, and a patent was applied for in 2003. The drug is currently undergoing phase 2 / 3 clinical trials for the treatment of pyelonephritis, urinary tract infections, and abdominal infections, and has not yet been approved for marketing.

[0059] Cefozopran (also known as SCE-2787), CAS No. 113359-04-9, molecular formula: C 19 H 17 N9O5S2, structural formula as follows:

[0060] The most common form of cefazolin on the market is cefazolin hydrochloride, which has the same biological activity. It was developed by Takeda Pharmaceutical Company of Japan and was first approved for marketing in Japan in August 1995 for the treatment of diseases such as cholangitis, cholecystitis, and cystitis.

[0061] The methylenetetrahydrofolate reductase activity was selected from mycobacterial methylenetetrahydrofolate reductase.

[0062] In a second aspect, embodiments of the present invention provide the use of the compound shown in Formula I above, its pharmaceutical salt, salt hydrate or hydrate thereof in at least one of the following situations;

[0063] (1) Application in the preparation of antibacterial agents that inhibit the activity of Mycobacterium tuberculosis;

[0064] (2) Application in the preparation of bactericides that kill Mycobacterium tuberculosis;

[0065] (3) Application in the preparation of products that inhibit the activity of Mycobacterium tuberculosis;

[0066] (4) Application in the preparation of products for treating Mycobacterium tuberculosis infection;

[0067] (5) Use in the preparation of products for the treatment and / or prevention of diseases caused by Mycobacterium tuberculosis;

[0068] (6) Application in the preparation of drugs for treating tuberculosis.

[0069] The compounds shown in Formula I are identical to those defined above. Cefuroxime and cefepime, among other compounds, belong to the cephalosporin family. Most cephalosporins have almost no antibacterial activity against Mycobacterium tuberculosis. The biggest difference between cefuroxime, cefepime, and other cephalosporins is that they all possess the core structure shown in Formula I. Embodiments of this invention have found that compounds with the core structure shown in Formula I, such as cefuroxime and cefepime, possess antibacterial activity against Mycobacterium tuberculosis, exhibiting both antibacterial and bactericidal effects, thereby providing therapeutic or preventative effects against Mycobacterium tuberculosis infection or diseases caused by it, such as pulmonary tuberculosis. Simultaneously, they can also inhibit the activity of methylenetetrahydrofolate reductase, and can be used as products that inhibit methylenetetrahydrofolate reductase activity.

[0070] Thirdly, embodiments of the present invention provide a pharmaceutical composition whose active ingredient is composed of a compound of formula I, its pharmaceutical salt, a hydrate of the salt or a hydrate thereof, and para-aminosalicylic acid, or composed of a compound of formula I, its pharmaceutical salt, a hydrate of the salt or a hydrate thereof, and a β-lactamase inhibitor.

[0071] Wherein, the mass ratio of the compound shown in Formula I to the para-aminosalicylic acid is (0.01-100):(0.001-100), preferably (0.02-32):(0.0025-0.32);

[0072] The mass ratio of the compound shown in Formula I to the β-lactamase inhibitor is (0.02-20):(0.02-200), preferably (0.2-2):(0.2-32);

[0073] The β-lactamase inhibitor is selected from oxadiazine β-lactamase inhibitors and / or penicillin sulfone β-lactamase inhibitors.

[0074] Furthermore, the above-mentioned pharmaceutical composition may also incorporate one or more pharmaceutically acceptable carriers; said carriers include diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorbents, lubricants, etc., which are conventional in the pharmaceutical field.

[0075] The above-mentioned pharmaceutical compositions can be prepared in various forms such as injections, tablets, powders, granules, capsules, oral liquids, ointments, and creams; all of the above dosage forms can be prepared according to conventional methods in the pharmaceutical field.

[0076] The above-mentioned drug composition can be introduced into the body, such as into muscles, intradermal tissues, subcutaneous tissues, veins, or mucous membranes, by means of injection, spray, nasal drops, eye drops, penetration, absorption, or physical or chemical mediation; or it can be introduced into the body after being mixed or encapsulated with other substances.

[0077] When using this pharmaceutical composition, the individual compounds (drugs) may be used simultaneously or sequentially, at their respective effective doses, and administered according to the standard administration method using appropriate routes of administration. Alternatively, the effective doses of each compound may be mixed to form a mixture for use.

[0078] The compound shown in Formula I is identical to the above-described definition. Embodiments of the present invention have found that the compound shown in Formula I, when used in combination with para-aminosalicylic acid or with a β-lactamase inhibitor, exhibits good antibacterial activity against Mycobacterium tuberculosis and can be used for the treatment of tuberculosis. Therefore, embodiments of the present invention provide the application of the above-described pharmaceutical composition in at least one of the following situations;

[0079] (1) Application in the preparation of antibacterial agents that inhibit the activity of Mycobacterium tuberculosis;

[0080] (2) Application in the preparation of bactericides that kill Mycobacterium tuberculosis;

[0081] (3) Application in the preparation of products that inhibit the activity of Mycobacterium tuberculosis;

[0082] (4) Application in the preparation of products for treating Mycobacterium tuberculosis infection;

[0083] (5) Use in the preparation of products for the treatment and / or prevention of diseases caused by Mycobacterium tuberculosis;

[0084] (6) Prepare inhibitors that inhibit the activity of methylenetetrahydrofolate reductase;

[0085] (7) Application in the preparation of drugs for treating tuberculosis.

[0086] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0087] Example 1 Molecular docking

[0088] Molecular docking plays a crucial role in drug discovery and development, building upon the discovery of novel drug targets. It is used to identify potential drug candidates and understand their binding interactions with target proteins. Virtual screening is one of the main applications of molecular docking in drug discovery, involving the rapid screening of large libraries of compounds to identify potential lead compounds that may bind to specific target proteins. Docking algorithms can predict the binding affinity and binding mode of small molecules to target proteins, enabling the selection of promising candidates for further experimental evaluation. Simultaneously, molecular docking can also be used for drug repurposing, i.e., screening existing drugs for new targets. Docking helps identify potential therapeutic effects beyond the original indication by predicting its binding interactions with other target proteins, reducing costs and time in the drug development process.

[0089] Therefore, in this embodiment of the invention, molecular docking was used to study the inhibitory effect of the compound shown in Formula I on the activity of methylenetetrahydrofolate reductase.

[0090] Mycobacterial methylenetetrahydrofolate reductases are a unique class of reductases that function solely in relation to NADH and do not depend on FAD. The structures of the resolved Mycobacterium tuberculosis methylenetetrahydrofolate reductase Rv2172c, Mycobacterium smegmatis methylenetetrahydrofolate reductase MSMEG_6649, and Mycobacterium thermophilum methylenetetrahydrofolate reductase MHAS_04356 are extremely similar in their three-dimensional protein structures, and the amino acid sequences of methylenetetrahydrofolate reductase homologs are highly conserved among mycobacteria (see [link to article]). Figure 1Using the protein structure of Mycobacterium tuberculosis Rv2172c resolved by the inventors, molecular docking was performed to search for small molecule compounds that may inhibit the activity of mycobacterial methylenetetrahydrofolate reductase. Through molecular docking, we found that the compound structure shown in Formula I may bind to the active site of Rv2172c, and in the two highest-scoring docking poses, the compound structure shown in Formula I interacts with several key amino acids remaining in Rv2172c enzyme activity-dependent states, including Val117, Met121, Arg153, and Arg159 (see [link to relevant documentation]). Figure 2 This may inhibit the binding of the enzyme to its substrate or coenzyme NADH, thereby suppressing enzyme activity. A search was conducted in all available compound libraries from MCE Corporation for compounds with the structure shown in Formula I. The results, as shown in Table 1, primarily included ceftaroline, ceftobiprole, ceftolozane, cefozopran, and their pharmaceutical salts or hydrates.

[0091] The compounds shown in Formula I in Table 1

[0092]

[0093] Note: Ceftobiprole; Ceftaroline; Ceftolozane; Cefozopran.

[0094] Molecular docking revealed that compounds with the structure shown in Formula I, such as cefuroxime or cefepime, can bind to the active site of Rv2172c. Furthermore, in their two highest-scoring docking poses, these compounds interact with multiple key amino acid residues dependent on Rv2172c enzyme activity, such as Glu9, Thr86, Pro118, Arg119, Met121, Arg153, and Arg159 (see [link to documentation]). Figure 3 , Figure 4 , Figure 5 and Figure 6 ).

[0095] Further enzyme kinetic experiments were conducted as follows: The inhibitory effects of cefuroxime and cefepime on the methylenetetrahydrofolate reductase activity of Rv2172c were detected at room temperature using 100 mM phosphate buffer (pH 8.0) and a substrate concentration of 100 μM. The concentrations of cefuroxime and cefepime ranged from 0.1 to 50 μM, and the concentrations of cefepime ranged from 0.01 to 3 μM. After incubating 1 μM of Rv2172c protein with cefuroxime and cefepime at room temperature for 20 min, the substrate was added, and the reaction began. Subsequently, the absorbance of NADH was measured at 340 nm and 25 °C.

[0096] See the results of the enzyme kinetics experiment. Figure 7 The results showed that both cefuroxime and cefepime could inhibit the activity of methylenetetrahydrofolate reductase of Rv2172c, with an IC50 value of [missing value]. 50 The concentrations of 1.75 μM and 0.37 μM, respectively, demonstrate that compounds with the structure shown in Formula I can inhibit the enzymatic catalytic activity of methylenetetrahydrofolate reductase, a mycobacterial specific enzyme.

[0097] Example 2: Detection of the antibacterial activity of the compound shown in Formula I against Mycobacterium tuberculosis.

[0098] Mycobacterium tuberculosis H37Ra and H37Rv were cultured in 7H9+OADC liquid medium until the logarithmic growth phase, and the drug-containing bacterial suspension was diluted to 1×10⁻⁶ using the microplate two-fold dilution method. 5 The minimum inhibitory concentration (MIC) of cefuroxime and cefepime against Mycobacterium tuberculosis was determined using CFU / mL. The MICs of different cephalosporins against Mycobacterium tuberculosis were also measured as controls. Detailed information on all tested cephalosporins is shown in Table 2.

[0099] Table 2 Cephalosporin Types

[0100]

[0101] The test results are shown in Table 3.

[0102] Table 3. MICs of different cephalosporins against Mycobacterium tuberculosis

[0103]

[0104] Note: Cephalexin; Cefazolin; Cefoxitin; Cefaclor; Ceftazidime; Cefoperazone; Cefixime; Ceftriaxone; Ceftobiprole; Ceftaroline.

[0105] Table 3 shows that cefuroxime or cefepime exhibits good antibacterial activity against Mycobacterium tuberculosis even at low concentrations (1-2 μg / mL). Other cephalosporins tested showed no good antibacterial activity against Mycobacterium tuberculosis (MIC > 16 μg / mL), indicating that only compounds with the core structure shown in Formula I can inhibit the activity of Mycobacterium tuberculosis.

[0106] Since cefuroxime and cefepime exhibit completely identical antibacterial activity against Mycobacterium tuberculosis, cefuroxime was selected as a representative ingredient in subsequent embodiments of this invention to further investigate the bactericidal effect of compounds with the core structure shown in Formula I against Mycobacterium tuberculosis.

[0107] Example 3: Test of the bactericidal effect of cefuroxime against Mycobacterium tuberculosis

[0108] Mycobacterium tuberculosis H37Ra was cultured in 7H9+OADC liquid medium until the logarithmic growth phase, and then the bacterial suspension was diluted to 1×10⁻⁶. 6 CFU / mL was added to the bacterial culture at concentrations of 1, 2, and 5 times the MIC (1, 2, and 5 μg / mL). The bacterial culture was incubated at 37°C after drug addition, and the culture was diluted and plated at regular intervals for viable cell count. Each experiment was performed in triplicate (biological replicates) and twice (technical replicates).

[0109] See results Figure 8 ,according to Figure 8 It is known that cefuroxime has a very strong bactericidal effect against Mycobacterium tuberculosis at a concentration of 1 μg / mL, and 1 times the MIC is the minimum bactericidal concentration (MBC).

[0110] Example 4: Detection of anti-tuberculosis activity of cefuroxime in mice

[0111] Balb / c mice were injected with a safe dose of 50 mg / kg of cefuroxime for pharmacokinetic analysis.

[0112] The results are as follows Figure 9As shown, cefuroxime can reach a blood concentration of 40 μg / mL 0.5 hours after injection, but is rapidly metabolized to below 1 μg / mL within 2 hours after injection.

[0113] The anti-tuberculosis activity of cefuroxime in mice was assessed using a mouse model of non-selective autonomous luminescent Mycobacterium tuberculosis H37Ra (UA1Ra). The specific procedure was as follows: UA1Ra was transferred to fresh 7H9 medium containing OADC and cultured to the logarithmic growth phase (OD600 ≈ 0.5–1.0). Mice with a relative luminescent unit (RLU) value ≥ 2 million / 200 μl of bacterial suspension were used for infection. 200 μl of UAlRa bacterial suspension was injected intravenously into 4–6 week old female BALB / c mice. On day 2 post-infection (day 0 of treatment), the RLU value of the mice was measured. Mice were anesthetized with isoflurane, then placed in a luminescence detector with their chests pressed against the photometer's detection aperture. The light production was measured twice for 3 seconds, and the RLU value was recorded. Mice with excessively low RLU values ​​were culled, and mice with acceptable RLU values ​​were randomly divided into groups of 5 mice each and marked. PBS was administered as a negative control, and rifampicin (RIF, 10 mg / kg) was administered as a positive control. The cefuroxime treatment groups received doses of 20 mg / kg and 50 mg / kg. Except for the positive control, which was administered via gavage, both the treatment and negative control groups received subcutaneous injections once daily for four consecutive days. RLU values ​​were measured on days 2, 4, and 5 of treatment. Mice were euthanized on the second day after treatment (day 5), and lung tissue was harvested and homogenized in 2 mL of sterile PBS using a tissue homogenizer. The RLU values ​​of the mouse lung homogenate were then measured using a chemiluminescence immunoassay analyzer. The RLU values ​​were used to assess the number of viable bacteria in the system and evaluate the antibacterial activity of the compound.

[0114] See results Figure 10 ,according to Figure 10 It is known that cefuroxime has anti-tuberculosis activity in mice, with the most significant effect observed at a dose of 50 mg / kg, but slightly less effective than rifampin, a first-line anti-tuberculosis drug. However, based on pharmacokinetic data, this compound at a dose of 50 mg / kg in mice maintains an effective bactericidal concentration of 1 μg / mL for less than 2 hours per day. In contrast, 600 mg (for a patient weighing 60 kg, the dose is 10 mg / kg) of cefuroxime proxetil maintains a serum drug concentration above 1 μg / mL for approximately 8 hours in humans.

[0115] It is evident that compounds with Formula I have broad prospects for development and application as anti-tuberculosis drugs.

[0116] Example 5: Detection of the enhanced antibacterial activity of β-lactamase inhibitors against Mycobacterium tuberculosis.

[0117] It is generally believed that β-lactamase inhibitors do not have antibacterial activity against Mycobacterium tuberculosis. The inventors have indeed detected that β-lactamase inhibitors, such as Avibactam or Relebactam, have a MIC greater than 64 μg / mL against Mycobacterium tuberculosis.

[0118] To test whether β-lactamase inhibitors enhance the antibacterial activity of compounds having Formula I against Mycobacterium tuberculosis, all available β-lactamase inhibitors were purchased, details of which are shown in Table 4.

[0119] Table 4 shows the β-lactamase inhibitors detected.

[0120]

[0121] Mycobacterium tuberculosis H37Ra was cultured in 7H9+OADC liquid medium until the logarithmic growth phase, and the drug-containing bacterial suspension was diluted to 1×10⁻⁶ using the microplate two-fold dilution method. 5 The MIC of cefuroxime against Mycobacterium tuberculosis was determined by adding 0.5 μg / mL of different β-lactamase inhibitors. The experiment was repeated three times, and the results are shown in Table 5.

[0122] As shown in Table 5, various β-lactamase inhibitors can increase the antibacterial activity of cefuroxime against Mycobacterium tuberculosis, such as avibactam, relebactam, taniborbactam, and clavulanate. Avibactam and relebactam were selected as representative β-lactamase inhibitors for further research.

[0123] Table 5. MICs of cefuroxime against Mycobacterium tuberculosis when used in combination with different β-lactamase inhibitors.

[0124]

[0125] Note: Ceftaroline

[0126] Mycobacterium tuberculosis H37Rv was cultured in 7H9+OADC liquid medium until the logarithmic growth phase, and the drug-containing bacterial suspension was diluted to 1×10⁻⁶ using the microplate two-fold dilution method. 5 The MIC of cefuroxime against Mycobacterium tuberculosis was determined by adding different concentrations of the β-lactamase inhibitors avibactam or retelebactam. The experiment was repeated three times, and the results are shown in Table 6.

[0127] Table 6. MIC of cefuroxime against Mycobacterium tuberculosis when different concentrations of β-lactamase inhibitors are added.

[0128]

[0129] Note: Avibactam, Ceftaroline, Relebactam

[0130] As can be seen from Table 6, Avibactam or Relebactam can increase the anti - tuberculosis activity of Ceftaroline, and the effect of Avibactam is slightly better than that of Relebactam. That is to say, β - lactamase inhibitors can cooperate with the compound shown in Formula I to have antibacterial activity against Mycobacterium tuberculosis.

[0131] Example 6 Detection of the Synergistic Anti - tuberculosis Effect of Ceftaroline or Ceftobiprole and Para - aminosalicylic Acid

[0132] Para - aminosalicylic acid (PAS), as an anti - tuberculosis drug used clinically, inhibits Mycobacterium tuberculosis by inhibiting dihydrofolate reductase. PAS is mainly used for the treatment of drug - resistant tuberculosis and has good anti - tuberculosis activity (MIC is 0.02 - 0.04 μg / mL).

[0133] To detect its synergistic anti - tuberculosis activity with the compound shown in Formula I, Mycobacterium tuberculosis H37Rv was cultured to the logarithmic phase and then serially diluted to 10 5 CFU / mL. Each 500 μl was aliquoted into a 48 - well plate and increasing concentrations of Ceftaroline or Ceftobiprole and PAS were added. Calculate the FICI of the combination (FICI = MIC of A when used in combination / MIC of A when used alone+MIC of B when used in combination / MIC of B when used alone). FICI ≤ 0.5 indicates synergistic effect, 0.5 < FICI ≤ 4 indicates no interaction, and FICI > 4 indicates antagonistic effect.

[0134] The results are as Figure 11 and Figure 12 shown. According to Figure 11 and Figure 12 it can be seen that Ceftaroline or Ceftobiprole and the clinical anti - tuberculosis drug PAS have good synergistic anti - tuberculosis effects. That is, adding a very small amount of PAS can significantly reduce the MIC of Ceftaroline or Ceftobiprole against Mycobacterium tuberculosis, and adding a very small amount of Ceftaroline or Ceftobiprole can also significantly reduce the MIC of PAS against Mycobacterium tuberculosis. It shows that the compound shown in Formula I can produce a synergistic effect with PAS, has antibacterial effect on Mycobacterium tuberculosis, and has a therapeutic effect on the diseases caused by Mycobacterium tuberculosis.

[0135] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Application of cefepime or its pharmaceutical salt as the sole active ingredient in the preparation of anti-tuberculosis drugs.

2. The application of cefepime or its pharmaceutically acceptable salt as the sole active ingredient in the preparation of anti-tuberculosis drugs, characterized in that, The English name of the ceftobiprole is ceftobiprole medocaril.

3. The use of cefepime or its pharmaceutical salt as the sole active ingredient in the preparation of drugs for the treatment or prevention of tuberculosis caused by Mycobacterium tuberculosis.

4. The use of cefepime or its pharmaceutically acceptable salt as the sole active ingredient in the preparation of a medicament for the treatment or prevention of tuberculosis caused by Mycobacterium tuberculosis, characterized in that, The English name of the ceftobiprole is ceftobiprole medocaril.

5. The use of the combination of cefuroxime and rabezantamine in the preparation of an anti-tuberculosis drug; characterized in that, The concentration of the relebactam is 4-32 µg / mL.

6. The use of a combination of cefuroxime and relebactam in the preparation of a medicament for the treatment or prevention of tuberculosis caused by Mycobacterium tuberculosis; characterized in that, The concentration of the relebactam is 4-32 µg / mL.

7. The use of a combination of cefepime and para-aminosalicylic acid or a combination of cefuroxime and para-aminosalicylic acid in the preparation of anti-tuberculosis drugs.

8. The use of a combination of cefepime and para-aminosalicylic acid or a combination of cefuroxime and para-aminosalicylic acid in the preparation of a medicine for the treatment or prevention of tuberculosis caused by Mycobacterium tuberculosis.

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