Oxazolidinone compounds, liposomal compositions comprising oxazolidinone compounds, and methods of use thereof

Abstract

To provide a composition for treating tuberculosis and other mycobacterial and gram-positive bacterial infections.SOLUTION: There is provided a pharmaceutical composition comprising a compound of formula I or a pharmaceutically acceptable salt thereof for use in the treatment of bacterial infections. Wherein R1 is a tetrazole ring substituted with aminoalkyl at the 2 '-position and R2 is amine or acetamide SELECTED DRAWING: Figure 6

Description

[Technical Field]

[0001] Related Applications This application is a continuation of U.S. Provisional Patent Application No. 63 / 040,810, filed June 18, 2020. No. 17 / 351,631, filed June 18, 2021. Priority and benefit of the present invention are claimed, the entire contents of which are incorporated herein by reference. This shall be done.

[0002] Field The present disclosure relates to novel aminoalkyl oxazolidinone compounds, novel aminoalkyl oxazolidinone compounds, Liposome compositions containing lysinone compounds and Mycobacterium tuberculosis Aminoalcohols in the treatment of tuberculosis and other Gram-positive bacterial infections The present invention relates to the use of hydroxyl oxazolidinone compounds. [Background technology]

[0003] Mycobacteria is a genus of bacteria that causes tuberculosis (TB). According to the World Health Organization For example, TB is one of the top 10 causes of death worldwide and the leading cause of death from a single infectious pathogen. Rifampicin is the most effective first-line drug for treating TB. However, there has been an increase in cases of infection with rifampicin-resistant tuberculosis bacteria. Multidrug-resistant tuberculosis (MDR-TB) is a type of tuberculosis that does not respond to isoniazid and rifampicin. It is a form of TB caused by a bacterium. Summary of the Invention

[0004] Compositions for treating tuberculosis and other mycobacterial and gram-positive bacterial infections - Patent Application 20070122999 Products and methods are disclosed.

[0005] One aspect of the present disclosure is a compound of formula I: [ka] [In the formula, R2 is an amine (NH2) or acetamide (NHCOCH3); R1 is a tetrazole ring substituted at the 2' position with an aminoalkyl. or a pharmaceutically acceptable salt thereof.

[0006] In some embodiments, the aminoalkyl is dimethylaminoalkyl. In the form, the aminoalkyl derivative of the oxazolidinone compound is an R of the oxazolidinone ring. Either an amine or acetamide group at the 2-position and a diamine group on the tetrazole ring Contains a methylaminoethyl group.

[0007] In some embodiments, a compound of Formula 1a: [ka] The compound of formula (I) is provided.

[0008] In some embodiments, Formula 1b: [ka] The compound of formula (I) is provided.

[0009] In some embodiments, the compound of formula 1c: [ka] or a pharmaceutically acceptable salt thereof.

[0010] In some embodiments, the compound of formula 1d: [ka] or a pharmaceutically acceptable salt thereof.

[0011] In some embodiments, the compound of formula 1e: [ka] The compound of formula (I) is provided.

[0012] In some embodiments, the compound is effective in Erd / HepG2 and H37Rv / HepG2. The selectivity index (SI) for these compounds ranges from 100 to 1700.

[0013] In some embodiments, the compound is effective in Erd / HepG2 and H37Rv / HepG2. The corresponding SI ranges from 200 to 1700.

[0014] In some embodiments, the compound is effective in Erd / HepG2 and H37Rv / HepG2. The corresponding SI ranges from 300 to 1700.

[0015] Another aspect of the present disclosure provides a liposome composition comprising liposome vesicles, The cell has formula I: [ka] [In the formula, R2 is an amine (NH2) or acetamide (NHCOCH3); R1 is a tetrazole ring substituted at the 2' position with an aminoalkyl. or a pharmaceutically acceptable salt thereof.

[0016] In some embodiments, the aminoalkyl is dimethylaminoalkyl. In the form, the aminoalkyl derivative of the oxazolidinone compound is an R of the oxazolidinone ring. Either an amine or acetamide group at the 2-position and a diamine group on the tetrazole ring Contains a methylaminoethyl group.

[0017] In some embodiments, the liposome composition comprises a liposome vesicle, , Equation 1a: [ka] This includes compounds of the formula:

[0018] In some embodiments, the liposome composition comprises a liposome vesicle, , Equation 1b: [ka] This includes compounds of the formula:

[0019] In some embodiments, the liposome composition comprises a liposome vesicle, , Equation 1c: [ka] or a pharmaceutically acceptable salt thereof.

[0020] In some embodiments, the liposome composition comprises a liposome vesicle, , Equation 1d: [ka] This includes compounds of the formula:

[0021] In some embodiments, the liposome composition comprises a liposome vesicle, , Equation 1e: [ka] This includes compounds of the formula:

[0022] In some embodiments, the liposome vesicles are in an aqueous medium.

[0023] In some embodiments, the compound is captured in the liposome vesicle with a capturing agent, and the capturing agent is a poly In some embodiments, the scavenger comprises triethylammonium octasulfate. In some embodiments, the scavenger is triethyl octasulfate or ammonium sulfate. In some embodiments, the scavenger is ammonium sulfate. do.

[0024] In some embodiments, the liposome composition comprises a salt of the compound, the salt being a sulfate, a citrate, or a methyl ... salts with phosphates, sucrosophates, salts with phosphated or sulfated polyols, or salts with phosphated polyols In some embodiments, the salt is a salt with a lysine anionic polymer or a sulfated polyanionic polymer. In the present invention, the liposome composition comprises a sulfate salt of the compound.

[0025] In some embodiments, the compound in the liposome vesicle has an aqueous solubility of less than 1 mg / mL. In some embodiments, the compound in the liposome vesicle has an aqueous solubility of less than 0.1 mg / mL. It has a resolution.

[0026] In some embodiments, the liposome vesicles contain phosphatidylcholine and cholesterol. In some embodiments, the liposome vesicle comprises a membrane comprising phosphatidylcholine and The liposomes contain a cholesterol-containing membrane that separates the interior of the vesicle from the aqueous medium. In some embodiments, the phosphatidylcholine is distearoylphosphatidylcholine ( In some embodiments, the phosphate buffered saline (PBSC) is hydrogenated soy phosphatidylcholine (DSPC) or hydrogenated soy phosphatidylcholine (HSPC). The molar ratio of phosphatidylcholine to cholesterol is approximately 60:40 to 35:65. In some embodiments, the molar ratio of phosphatidylcholine to cholesterol is about 55 In some embodiments, the phosphatidylcholine and cholesterol The molar ratio of the alcohol to the alcohol is about 50:50 to about 40:60.

[0027] In some embodiments, the molar ratio of phosphatidylcholine to cholesterol is about 50:5 0 to approximately 45:55.

[0028] In some embodiments, the membrane further comprises a polymer-conjugated lipid.

[0029] In some embodiments, the liposome vesicles comprise HSPCs at a molar ratio of about 55:45:2.75. , cholesterol and polymer-conjugated lipids.

[0030] In some embodiments, the polymer-conjugated lipid is PEG (molecular weight 2,000 0)-distearoylglycerol (PEG-DSG) or PEG (molecular weight 2,000 )-distearoylphosphatidylethanolamine (PEG-DSPE).

[0031] In some embodiments, the liposomes in the liposome composition have a Z of about 80 to about 130 nm. It has an average particle size.

[0032] In some embodiments, the composition is a liquid pharmaceutical formulation for parenteral administration.

[0033] Another aspect of the present disclosure relates to a method of treating a bacterial infection, the method comprising administering to a subject in need thereof The method comprises administering to an animal a therapeutically effective amount of a liposome composition provided herein. nothing.

[0034] In some embodiments, the bacterial infection is a Mycobacterium tuberculosis infection. The compounds in the vesicles have minimum inhibitory concentrations ranging from about 0.01 μg / ml to about 0.25 μg / ml. In some embodiments, the compound in the liposome vesicle has a MIC of about 0.01 It has a minimum inhibitory concentration (MIC) in the range of 0.1 μg / ml to about 0.1 μg / ml.

[0035] In some embodiments, the liposome composition is administered parenterally.

[0036] In some embodiments, the method further comprises administering one or more additional active agents simultaneously or sequentially. In some embodiments, the one or more active agents include bedaquiline, pretomanid, pyrazinamide, moxifloxacin, a pharmaceutically acceptable salt thereof, or This includes combinations thereof.

[0037] In some embodiments, the liposome composition is administered once a week to once every six weeks.

[0038] In some embodiments, the percentage of the compound remaining in the blood after administration to a subject in need thereof In some embodiments, the percentage remaining in the blood exceeds 20% of the administered dose at 6 hours. The percentage of compound is greater than 10% of the administered dose.

[0039] Aspects of the present disclosure relate to methods of making liposome compositions, including: (i) a liposome composition substantially containing an entrapment agent; The phospholipids, cholesterol, and PEG-lipids in a medium containing no sequestering agent were (ii) preparing liposomes having an internal space corresponding to the liposomes; 10. The method of claim 1, wherein the compound of claim 1 is contacted with the liposome in the body. (iii) encapsulating the compound; and (iv) in a physiologically acceptable medium suitable for parenteral use. providing the liposomes. [Brief explanation of the drawings]

[0040] [Figure 1] 1 is a graph showing the effect of pH on liposome loading of compounds AKG-3, AKG-5, and AKG-16. [Figure 2] Figures 2A and 2B are graphs showing the encapsulation of compounds AKG-3, AKG-5, and AKG-16 into liposomes using different drug-to-lipid (DL) ratios of TEA-SOS scavengers. Figure 2A shows the effect of the added drug-to-lipid (DL) ratio on the liposome payload, expressed as the post-loading drug-to-lipid ratio (DL), in grams of drug per mole of liposomal phospholipid (PhL). Figure 2B shows the effect of the DL ratio (input ratio of drug to lipid) on the liposome loading efficiency, calculated as a percentage of the post-loading DL relative to DL. [Figure 3A] 1 is a graph showing the encapsulation of compounds AKG-3, AKG-5, and AKG-16 into liposomes using 0.5 M ammonium sulfate as a trapping agent at different DL ratios. The effect of DL ratio on liposome payload for AKG-5 and AKG-16 is shown. [Figure 3B] 1 is a graph showing the encapsulation of compounds AKG-3, AKG-5, and AKG-16 into liposomes using 0.5 M ammonium sulfate as a trapping agent at different DL ratios. The effect of DL ratio on liposome loading efficiency for AKG-5 and AKG-16 is shown. [Figure 3C] 1 is a graph showing the encapsulation of compounds AKG-3, AKG-5, and AKG-16 into liposomes using 0.5 M ammonium sulfate as a trapping agent at different DL ratios. The effect of DL ratio on liposome payload for AKG-3 is shown. [Figure 3D]1 is a graph showing the encapsulation of compounds AKG-3, AKG-5, and AKG-16 into liposomes using 0.5 M ammonium sulfate as a trapping agent at different DL ratios. The effect of DL ratio on liposome loading efficiency for AKG-3 is shown. [Figure 4] Figures 4A and 4B are graphs showing the encapsulation of AKG-28 and AKG-38 using TEA-SOS and ammonium sulfate as trapping agents at different DL ratios. Figure 4A shows the effect of DL ratio on liposome payload. Figure 4B shows the effect of DL ratio on loading efficiency. [Figure 5] Figures 5A, 5B, 5C, and 5D are graphs showing the dependence of rapid drug leakage from liposomes encapsulating compounds AKG-28 (Figures 5A, 5C) and AKG-38 (Figures 5B, 5D) upon in vitro contact with mouse (labeled "mouse") or human (labeled "human") plasma, as described in Example 19 below. Liposomes contained 5 mol% PEG(2000)-DSPE (labeled "DSPE") or PEG-DSG (labeled "DSG"). Entrapment agents: 0.5 M ammonium sulfate (AS) (Figures 5A, 5B), 1 N triethylammonium sucrose octasulfate (TEA-SOS) (Figures 5C, 5D). [Figure 6] FIG. 1 depicts the numbered ring structures of compounds of Formula I. [Figure 7] Figure 1 shows the plasma concentration versus time profiles of total drug in Sprague-Dawley rats after administration of a single intravenous dose (IVx1) of Ls-AKG28 at 10 mg / kg (diamonds), 20 mg / kg (squares), and 40 mg / kg (circles). The plasma concentration versus time profiles of linezolid at 50 mg / kg (single oral dose, POx1) in 5% methylcellulose (pH 3-4) were also included for comparison. Mean and SD concentrations are presented at each time point. [Figure 8]Figure 1 shows the plasma concentration versus time profiles of total drug in Sprague-Dawley rats after administration of single intravenous doses (IVx1) of Ls-AKG38 at 20 mg / kg (diamonds), 40 mg / kg (squares), and 80 mg / kg (diamonds). The plasma concentration versus time profiles of linezolid at 50 mg / kg (single oral dose, POx1) in 5% methylcellulose (pH 3-4) were also included for comparison. Mean and SD concentrations are presented at each time point. [Figure 9A] 1 is a graph showing plasma concentration versus time profiles of total drug in Sprague-Dawley rats following administration of 10 mg / kg Ls-AKG28, IVx1 on days 1 (circles), 15 (squares), 29 (diamonds), and 43 (triangles). Mean and SD concentrations are presented at each time point. [Figure 9B] 1 is a graph showing plasma concentration versus time profiles of total drug in Sprague-Dawley rats following administration of 20 mg / kg Ls-AKG28, IVx1 on days 1 (circles), 15 (squares), 29 (diamonds), and 43 (triangles). Mean and SD concentrations are presented at each time point. [Figure 9C] 1 is a graph showing plasma concentration versus time profiles of total drug in Sprague-Dawley rats following administration of 40 mg / kg Ls-AKG28, IVx1 on days 1 (circles), 15 (squares), 29 (diamonds), and 43 (triangles). Mean and SD concentrations are presented at each time point. [Figure 10A] 1 is a graph showing plasma concentration versus time profiles of total drug in Sprague-Dawley rats following administration of 20 mg / kg Ls-AKG38, IVx1 on days 1 (circles), 15 (squares), 29 (diamonds), and 43 (triangles). Mean and SD concentrations are presented at each time point. [Figure 10B] 1 is a graph showing plasma concentration versus time profiles of total drug in Sprague-Dawley rats following administration of 40 mg / kg Ls-AKG38, IVx1 on days 1 (circles), 15 (squares), 29 (diamonds), and 43 (triangles). Mean and SD concentrations are presented at each time point. [Figure 10C] 1 is a graph showing plasma concentration versus time profiles of total drug in Sprague-Dawley rats following administration of 80 mg / kg Ls-AKG38, IVx1 on days 1 (circles), 15 (squares), 29 (diamonds), and 43 (triangles). Mean and SD concentrations are presented at each time point. [Figure 11] Figures 11A, 11B, and 11C are graphs showing the plasma concentration versus time profiles of both lipid (non-exchangeable DiIC18(3)-DS labeled) and drug for liposomal AKG-28 (Figure 11A) and liposomal AKG-38 (Figure 11B) in CD-1 mice after a single intravenous injection, as well as the change in plasma drug-to-lipid ratio, a measure of the drug release rate from liposomes, for both Ls-AKG28 and Ls-AKG38 (Figure 11C). Mean and SD concentrations are presented for each time point. [Figure 12] 1 is a graph showing plasma drug concentrations, presented as % injected dose, of compared multiple formulations of liposomal AKG-28 and liposomal AKG-38, Ls-AKG28 and Ls-AKG38, after the first and fourth weekly doses. Mice were injected with the indicated doses and formulations once a week for a total of four injections. [Figure 13A] 1 is a graph showing the effect of increasing doses of Ls-AKG28 on body weight in female CD-1 mice over time. [Figure 13B] 1 is a graph showing the effect of increasing doses of Ls-AKG38 on body weight in female CD-1 mice over time. [Figure 13C] 1 is a graph showing the effect of Ls-AKG28 and Ls-AKG38 in combination with BP or BPM on hematological (RBC, HTC, PLT, WBC) and blood biochemistry (ALT, AST) parameters in female CD-1 mice. [Figure 13D] 1 is a heat map showing the effect of Ls-AKG28 or Ls-AKG38 monotherapy on histopathological findings in female CD-1 mice. [Figure 14A]1 is a graph showing the effect of Ls-AKG28 in combination with bedaquiline and pretomanid (BP) or bedaquiline, pretomanid, and moxifloxacin (BPM) on body weight in female CD-1 mice over time. [Figure 14B] 1 is a graph showing the effect of Ls-AKG38 in combination with BP or BPM on body weight in female CD-1 mice over time. [Figure 14C] 1 is a graph showing the effect of Ls-AKG28 and Ls-AKG38 in combination with BP or BPM on hematological (RBC, HTC, PLT, WBC) and blood biochemistry (ALT, AST) parameters in female CD-1 mice. [Figure 14D] 1 is a heat map showing the effect of Ls-AKG28 and Ls-AKG38 in combination with BP or BPM on histopathological findings in female CD-1 mice. [Figure 15A] Graph showing weight change over time in female CD-1 mice treated with Ls-AKG28 injected at 50 mg / kg twice a week (2qw) or 100 mg / kg once a week (1qw), alone or in combination with BP. [Figure 15B] 1 is a graph showing body weight changes in female CD-1 mice treated with Ls-AKG38 injected 2 qw at 100 mg / kg or 1 qw at 200 mg / kg alone or in combination with BP. [Figure 15C] Graphs showing hematological and blood biochemistry parameters in female CD-1 mice treated with Ls-AKG28 (50 mg / kg 2 qw or 100 mg / kg 1 qw) or Ls-AKG38 (100 mg / kg 2 qw or 200 mg / kg 1 qw) alone or in combination with BP. [Figure 15D] 1 is a heat map showing histopathological results in female CD-1 mice treated with Ls-AKG28 (50 mg / kg 2 qw or 100 mg / kg 1 qw) or Ls-AKG38 (100 mg / kg 2 qw or 200 mg / kg 1 qw) alone or in combination with BP. [Figure 16] Figure 16A is a graph showing the effect of Ls-AKG28 on body weight in male Sprague-Dawley rats chronically treated for a total of 8 weeks over time, and Figure 16B is a graph showing the effect of Ls-AKG38 on body weight in male Sprague-Dawley rats chronically treated for a total of 8 weeks over time. DETAILED DESCRIPTION OF THE INVENTION

[0041] The foregoing general description and the following detailed description are exemplary and explanatory only. Therefore, it should be understood that this is not a limitation on the compositions and methods of the present disclosure.

[0042] Disclosed herein are compounds, compositions and methods for the treatment of bacterial infections. As used herein, the terms "compound" and "drug" are used interchangeably. Some aspects of the present disclosure relate to novel aminoalkyl derivatives of oxazolidinone. This aspect relates to a process for synthesizing novel aminoalkyl derivatives of oxazolidinone compounds. Other embodiments include the use of aminoalkyl derivatives of oxazolidinone compounds in liposomes. Another aspect of the present disclosure relates to compositions containing oxazolidinone compounds in the treatment of bacterial infections. aminoalkyl derivatives of oxazolidinone compounds, In some embodiments, the present invention relates to the use of liposomal compositions containing the compounds described herein. and compositions are used to treat infections from mycobacteria and gram-positive bacteria. In some embodiments, the bacterial infection is Mycobacterium tuberculosis. In this regard, the compounds and compositions described herein are useful for the treatment of mycobacteria and gram-positive bacteria. Inhibits bacterial growth, including but not limited to Mycobacterium tuberculosis, Mycobacterium albicans, Mycobacterium leprae complex ), Mycobacterium gordonae ), Mycobacterium abscess us), Mycobacterium mucogenicum (Mycobacterium mucog enicum), streptococci, vancomycin-resistant enterococci (VRE), methicillin-resistant yellow Staphylococcus aureus (MRSA), Staphylococcus pneumoniae Staphylococcus pneumoniae, Enterococcus pneumoniae Enterococcus faecium, Group B Streptococcus ococcus agalactiae), Streptococcus pneumoniae pneumoniae), Streptococcus pyogen es), viridans group streptococci, Listeria monocytogenes, Nocardia and Corynebacterium Contains rium.

[0043] In some embodiments, the aminoalkyl of the oxazolidinone compounds described herein The derivatives were selective for M. tuberculosis when compared to mammalian cells such as human kidney or hepatocytes. In some embodiments, the oxazolidinone compounds described herein are selectively active. Aminoalkyl derivatives of are compared with mammalian cells, such as kidney or hepatic mammalian cells. When tested, it showed unexpectedly high selectivity of at least 1000-fold against M. tuberculosis. In embodiments, the aminoalkyl derivatives of the oxazolidinone compounds described herein are In some embodiments, the compounds described herein exhibit unexpectedly high selectivity of at least 100-fold. Aminoalkyl derivatives of oxazolidinone compounds are used to treat kidney or liver cells of mammals. Compared to mammalian cells such as cerebrospinal fluid, the antibody against Mycobacterium tuberculosis is 100 to 6,500 times more effective than that against Mycobacterium tuberculosis. ~6,000x, 100~5,500x, 100~5,000x, 100~4,500x , 100~4,000 times, 100~3,500 times, 100~3,000 times, 100~2, 500x, 100~2,000x, 100~1,500x, 100~1,000x, 50 0~6,500x, 500~6,000x, 500~5,500x, 500~5,000 times, 500~4,500 times, 500~4,000 times, 500~3,500 times, 500~3 ,000x, 500~2,500x, 500~2,000x, 500~1,500x, 5 00~1,000x, 1,000~6,500x, 1,000~6,000x, 1,00 0~5,500x, 1,000~5,000x, 1,000~4,500x, 1,000 ~4,000x, 1,000~3,500x, 1,000~3,000x, 1,000~ Unexpectedly high selection of 2,500 times, 1,000-2,000 times, and 1,000-1,500 times It shows selectivity.

[0044] In some embodiments, the compounds and compositions described herein are administered to the liver, spleen, or promotes selective uptake of Mycobacterium by resident macrophages in the lungs, Macrophages can help provide potent intracellular killing of mycobacteria. Both foreign infectious agents, such as cerebrospinal fluids, and laboratory-derived nanoparticles, such as liposomes, involved in the clearance of foreign particles via phagocytosis, including the Co-localization in the reservoir provides an opportunity to effectively concentrate the active agent in a critical reservoir of disease. vinegar.

[0045] Aspects of the present disclosure relate to compounds that are aminoalkyl derivatives of oxazolidinone (Figure 1). 6). In some embodiments, the compound has the following formula I: [ka] [In the formula, R2 is an amine (NH2) or acetamide (NHCOCH3); R1 is a tetrazole ring substituted at the 2' position with an aminoalkyl. and pharmaceutically acceptable salts thereof.

[0046] In other embodiments, the compound of Formula I: [ka] [In the formula, R2 is an amine (NH2) or acetamide (NHCOCH3); R1 is a tetrazole ring substituted at 1' with an aminoalkyl group. and pharmaceutically acceptable salts thereof.

[0047] In some embodiments, the aminoalkyl is dimethylaminoalkyl. In the form, the aminoalkyl derivative of the oxazolidinone compound is an R of the oxazolidinone ring. Either an amine or acetamide group at the 2-position and a diamine group on the tetrazole ring Contains a methylaminoethyl group.

[0048] The present disclosure provides a compound having either an amine group or an acetamide group at the R2 position of the oxazolidinone ring. and any of the compounds described herein containing a dimethylaminoethyl group on the tetrazole ring. Highly specific structure-activity studies of aminoalkyl derivatives of selected oxazolidinone compounds. These compounds exhibit a specific activity correlation (SAR). (2) It is highly selective for Mycobacterium tuberculosis compared to its activity in hepatocytes; It is highly active against sclerotial bacteria, and (3) it is efficiently packed into liposomes.

[0049] In some embodiments, the aminoalkyl derivatives of oxazolidinones described herein used a gradient-based drug loading method to deliver liposomes with an efficiency of 85% or better. In some embodiments, the loading efficiency of these derivatives is 90% or greater. In some embodiments, the loading of these derivatives is 95% or greater, or even quantitative. In some embodiments, the aminoalkyl derivatives of oxazolidinone are loaded into liposomes. In some embodiments, the loading method includes: To efficiently load and subsequently stabilize weakly basic amphiphiles, a transmembrane gradient and The gradient can be (1) a simple pH gradient formed using, for example, a citrate solution. gradient, (2) ammonium ion gradient employing citric acid or ammonium sulfate salts, ( 3) alkyl, dialkyl, or trialkyl ammonium salts, (4) transition metals (Cu 2+ , Mn 2+ , Zn 2+ , Mg 2+ ) gradient, or even (5) transmembrane drug solubility The present invention relates to a method for producing a stoichiometrically controlled ... No. 16,771, No. 5,800,833, No. 8,147,867, No. 7,74 Nos. 4,921, 8,349,360, and 6,110,491, U.S. Pat. Appl. Patent Application Publication No. 2018 / 0369143A1 and International Patent Application Publication No. 199001405 See Allen et al. (1995) Int J Cancer 6 2:199-204. Without being bound by theory, The cations that are released may drive the accumulation of weakly basic drugs inside the liposome or may act as a catalyst for the drug molecules. It plays a role in establishing a pH gradient across the membrane that helps direct exchange of This, in some embodiments, results in quantitative loading of the drug in less than the full volume of the gradient. By forming a stable complex with the drug inside the liposome, can play an important role in stabilizing the formulation against early leakage of ond et al.(2008)J.Pharm Sci 97,4696-4740 (See

[0050] definition For convenience, certain specific examples are employed in the specification, examples, and appended claims. The following terms are collected here. All technical terms used in this specification are used unless otherwise defined. All terms and scientific terms have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. It has.

[0051] As used herein, the following terms and phrases are intended to have the following meanings: will be done.

[0052] The articles "a" and "an" are used herein to refer to one or to more than one (e.g., (i.e., at least one). For example, "element" refers to one element. It means an element or elements.

[0053] As used herein, the terms "comprising" or "including" mean that a given embodiment Compositions, methods, and respective examples that may include elements that are present but not specified Used to refer to its component(s).

[0054] As used herein, the term "consisting essentially of" means that all components required for a given embodiment are This term refers to an element that is fundamental and novel or functional in that embodiment of the disclosure. The present invention allows for the presence of additional elements that do not materially affect the intended characteristic(s).

[0055] The term "consisting of" refers to compositions, methods, and their respective components as described herein. These refer to components of the embodiment excluding any elements not mentioned in that description. .

[0056] The term "comprising" as used herein includes the terms "consisting of" and "including" "essentially consisting of"

[0057] As used herein, "as mentioned above" or "mentioned above" or "the aforementioned" " refers to any of the disclosures made herein in any of the preceding pages. It is often mentioned.

[0058] As used herein, "as referred to herein" and "described herein" , "provided herein," or "as referred to in the text," or When reference is made to "described herein," the present specification includes the preceding or following pages. Reference is made to any of the disclosures made within the specification in any of the following:

[0059] As used herein, the term "about" refers to within 20%, within 10%, and within the stated value. In certain embodiments, "about" refers to an acceptable variation of ±1%, ±2%, ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, ±9%, ±10%, ±11%, ±12%, ±15%, ±16%, ±17%, ±18%, ±19%, ±19%, ±20%, ±21%, ±22%, ±23%, ±24%, ±25%, ±26%, ±27%, ±28%, ±29%, ±29%, ±29%, ±29 It can mean a variation of %, 3%, 4%, 5%, 10% or 20%.

[0060] As used herein, the term "effective amount" in reference to a compound or composition refers to a bactericidal or is sufficient to produce a bacteriostatic effect. In one embodiment, an effective amount refers to an amount sufficient to alleviate the symptoms of the bacterial infection being treated. "Therapeutically effective amount" means the amount of active compound that combines

[0061] As used herein, the term "subject" (or alternatively, "patient") refers to a patient receiving prophylactic or therapeutic The term "animal" refers to an animal, preferably a mammal, most preferably a human, receiving therapeutic treatment.

[0062] As used herein, the term "administration" or "administering" refers to the administration of a compound or drug. The pharmaceutical composition can be administered to a subject in need thereof by any method, including but not limited to, oral, intravenous, intramuscular, intraperitoneal, or intravenous route. This includes all means of administration, such as subcutaneous, transdermal, inhalation, buccal, ocular, sublingual, vaginal, and rectal administration. Alternatively, administration of the composition is suitably parenteral. For example, the compound or composition is preferably Typically, it can be administered intravenously, but it can also be administered intraperitoneally or by inhalation. There is currently clinical use of liposomal amikacin for the treatment of Mycobacterium avium. Amikacin Liposome Inhalation Suspension:A Review in Mycoba cterium avium Complex Lung Disease.Drugs .2019 Apr;79(5):555-562).

[0063] As used herein, "treat," "treating," and "treatment" refer to ent)" refers to therapeutic or prophylactic measures such as those described herein.

[0064] As used herein, the terms "synergistic" and "synergistically" refer to compounds used together. The effect achieved with the compound is greater than the sum of the effects resulting from using the compounds separately. greater than that predicted based on the two active ingredients administered separately also means that it is large.

[0065] The term "pharmaceutically acceptable salt" refers to a relatively unsaturated fatty acid salt of a compound of the present disclosure that possesses the desired pharmacological activity. refers to essentially non-toxic, inorganic or organic acid addition salts.

[0066] The term "alkyl" refers to straight or branched chain carbon atoms, unless the carbon chain is otherwise defined. means a saturated carbon chain which may be a combination thereof. Examples of alkyl groups include methyl, Ethyl, propyl, isopropyl, butyl, sec- and tert-butyl, pentyl , hexyl, heptyl, octyl, and the like.

[0067] The term "aminoalkyl" refers to an alkyl group in which at least one carbon atom of the alkyl chain is bonded to an amino group. The amino group means a primary amino group, a monoalkyl-substituted (Secondary) amino group, dialkyl-substituted (tertiary) amino group, or alkyl-substituted amino group where the amine nitrogen atom and the alkyl chain replacing the amine hydrogen form a heterocyclic ring.

[0068] The term "liposome" refers to a liposome that has a bilayer (unilayer) and / or a central aqueous compartment. by aqueous compartments formed by amphiphilic molecules such as phospholipids that surround the Liposomes are small vesicles made up of multiple concentric bilayers (multilayers) separated from each other. In pharmaceutical products, the active pharmaceutical ingredient is generally contained in liposomes. The hydrophobic drug is contained in the compartment(s) and is absorbed by the lipid bilayer(s) of the liposome. ) are included in the liposome formulation. Other characteristics, such as the circulatory half-life, depend on the polyethylene glycol and / or can be modified by the presence of cholesterol or other potential additives.

[0069] "Unilamellar liposomes," also known as "unilamellar vesicles," are liposomes that contain a single, closed aqueous compartment. Liposomes are liposomes that contain a single lipid bilayer membrane that defines the membrane. The bilayer membrane contains an inner and outer layer. The lipid molecules in the outer layer are hydrophilic ("head group") and hydrophilic ("head group"). ") portions are oriented towards the outer aqueous environment, and their hydrophobic ("tail") portions are oriented towards the liposome. The inner layer of lipids is directly beneath the outer layer and is oriented downwards towards the interior of the smosome. The lipid heads face the aqueous interior of the liposome and the tails point towards the tail of the lipid outer layer.

[0070] "Multilamellar liposomes" are also called "multilamellar vesicles" or "multi-lamellar vesicles" and are composed of more than one These membranes define multiple closed aqueous compartments. The membranes are arranged concentrically, with different membranes separated by aqueous compartments. There are.

[0071] As used herein, the terms "encapsulated" and "entrapped" refer to liposomes. The term "liposome" refers to the incorporation or association of an oxazolidinone pharmaceutical agent in or with a liposome.

[0072] The terms "DL", "DL ratio", "D / L", or "D / L ratio" are used interchangeably; It refers to the ratio of drug to liposomal lipid. Unless otherwise specified, it refers to the ratio of liposomal phospholipid (Ph It is expressed as grams of drug per mole of 0.01 L.

[0073] The term "mol%" in reference to cholesterol is expressed in percentage points. The molar amount of cholesterol relative to the sum of the molar amounts of cholesterol and non-PEGylated phospholipids present For example, in liposomes containing cholesterol and HSPC, "Cholesterol" is a combination of 55 mol of cholesterol per 45 mol of HSPC. Refers to the finished product.

[0074] The term "mol%" in reference to PEG-lipids is expressed in percentage points. This refers to the molar ratio of PEG-lipid to non-PEGylated phospholipid. For example, HSPC and PEG-D "5 mol% PEG-DSPE" in liposomes containing SPE means 100 mol parts This refers to a composition having 5 mol parts PEG-DSPE per 100 mol of HSPC.

[0075] The terms "sucrose octasulfate" and "sucrose" "Sucrose octasulfate" and "sucrose octasulfate" are the same compound, sucrose octasulfate (sucrose octasulfate). This specification refers to a compound of the formula (C1, C2, C3, C4, C5, C6, C7, C8, C9, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C20, C21, C31, C42, C43, C54, C65, C76, C77, C78, ​​C79, C81, C99 are used interchangeably in the literature.

[0076] The symbols "Ac", "Me", and "Et" found in the chemical formula represent acetyl, It refers to the alkyl group (CH3CO), methyl group (CH3), and ethyl group (C2H5).

[0077] Various aspects and embodiments are described in further detail in the following subsections.

[0078] compound Oxazolidinones exert their function by inhibiting protein synthesis Linezolid (LZD) is a synthetic antibiotic that exhibits bacteriostatic activity against Mycobacterium tuberculosis. However, administration of LZD can lead to anemia, thrombocytopenia, and peripheral neuropathy. Tedizolid may cause serious side effects such as peripheral neuropathy. Tedizolid phosphate is an oxazolidinone compound that has been shown to inhibit the side effects of The effects are similar but generally milder than with linezolid. compared with the experience with long-term linezolid use, such as that required for the treatment of tuberculosis. experience is limited.

[0079] Aspects of the present disclosure relate to compounds that are aminoalkyl derivatives of oxazolidinone (Figure 1). 6). In some embodiments, the compound has the following formula I: [ka] [In the formula, R2 is an amine (NH2) or acetamide (NHCOCH3); R1 is a tetrazole ring substituted at the 2' position with an aminoalkyl. and pharmaceutically acceptable salts thereof.

[0080] In some embodiments, the aminoalkyl is dimethylaminoalkyl. In the form, the aminoalkyl derivative of the oxazolidinone compound is an R of the oxazolidinone ring. Either an amine or acetamide group at the 2-position and a diamine group on the tetrazole ring Contains a methylaminoethyl group.

[0081] In other embodiments, the compound of Formula I: [ka] [In the formula, R2 is an amine (NH2) or acetamide (NHCOCH3); R1 is a tetrazole ring substituted at 1' with an aminoalkyl. and pharmaceutically acceptable salts thereof.

[0082] Aminoalkyl derivatives of oxazolidinone compounds having the chemical structures in Table 1 below were used. Synthesized as described in Example 1.

[0083] The compounds of the present disclosure may be in a free form, e.g., as a free base, as a free acid, or as an amphoteric isomer. It can exist as an alkyl group or in the form of a salt. , any salt, organic or inorganic addition salt, or co-crystal commonly used in pharmacy, In particular, it may be any pharmaceutically acceptable organic or inorganic addition salt, or co-crystal. The chemical formula showing the salt or ionic form of a compound of is the undissociated free base (or free acid) form. It is understood that this disclosure also discloses this compound in various forms.

[0084] The present disclosure encompasses all stereoisomeric forms of the compounds. The compound of formula 1 is substantially pure (i.e., at least 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280 0%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, e.g. For example, 100%.

[0085] JPEG2026035569000018.jpg255166JPEG2026035569000019.jpg242170JPEG2026035569000020.jpg248170JPEG2026035569000021.jpg99170

[0086] In some embodiments, the compound has the following chemical formula: [ka] It has.

[0087] In some embodiments, the compound has the following chemical formula: [ka] It has.

[0088] In some embodiments, the compound has the following chemical formula: [ka] It has.

[0089] In some embodiments, the compound has the following chemical formula: [ka] It has.

[0090] In some embodiments, the compound has the following chemical formula: [ka] It has.

[0091] Provided herein are compounds of formula I or their derivatives that are useful in the treatment of mycobacterial infections. Pharmaceutically acceptable salts are disclosed. In some embodiments, the compound is represented by formula 1a, 1b , 1c, 1d, or 1e. In some embodiments, the compound has the formula 1b In some embodiments, the compounds of Formula I have a minimum inhibitory concentration (MIC), e.g., a minimum inhibitory concentration (MIC) of M. tuberculosis. 0.1μg / ml~1μg / ml, 0.25μg / ml~1μg / ml, 0.5μ g / ml~1μg / ml, 0.1μg / ml~0.25μg / ml, 0.1μg / ml~ 0.5μg / ml, 0.25μg / ml~0.5μg / ml, 0.01μg / ml~1μ g / ml, 0.01μg / ml~0.25μg / ml, 0.01μg / ml~0.5μg / ml, and has an MIC in the range of 0.01 μg / ml to 0.1 μg / ml. In this embodiment, the compounds of formula I have a minimum inhibitory concentration (MIC), for example, 1 μg / mL against Mycobacterium tuberculosis. have an MIC of less than 0.1 μg / ml, less than 0.25 μg / ml, or less than 0.1 μg / ml. In some embodiments, the compound of formula I is administered in the range of 0.01 μg / ml to 0.25 μg / ml. In some embodiments, the compound of formula I has an MIC of 0.01 μg / ml to 0.1 μg / ml. The MIC values ​​are given in the range of 0.01 mg / ml depending on the bacterium. It should be understood that the range may be lower than the range specified.

[0092] In some embodiments for treating mycobacteria, e.g., Mycobacterium tuberculosis, the compound ( Mycobacterium akgenii (AKG-28 or AKG-38) has an MIC of less than 0.1 μg / mL. In some embodiments for treating Mycobacterium, e.g., Mycobacterium tuberculosis, the compound comprises at least It has a selectivity index (SI) of 1,000 for killing Mycobacterium tuberculosis versus human kidney cells (VERO). In some embodiments for treating mycobacteria, e.g., Mycobacterium tuberculosis, The compound has an MIC of less than 0.1 μg / mL and an M. tuberculosis to human seroprevalence of at least 1,000. It has a selectivity index (SI) for killing kidney cells (VERO). The compounds have the structure of AKG-28 (Formula 1b) or AKG-38 (Formula 1c). In this embodiment, the MIC is less than 0.05 μg / mL and the mitochondrial protein The selectivity index of MIC for M. tuberculosis compared with SI-MPS was Like over 20.

[0093] In some embodiments, the compounds described herein have a potent inhibitor of Mycobacterium tuberculosis compared to linezolid. At potency-adjusted doses, a 2- to 20-fold increase (approximately 2, approximately 3, approximately 4, approximately 5, approximately 6, approximately 7, Approx. 8, Approx. 9, Approx. 10, Approx. 11, Approx. 12, Approx. 13, Approx. 14, Approx. 15, Approx. 16, Approx. 17, Approx. 1 8, about 19, about 20).

[0094] In some embodiments for treating methicillin-resistant Staphylococcus aureus (MRSA), The compound has an MIC against MRSA strains of less than 2 μg / mL. In some embodiments for treating Staphylococcus aureus (MRSA), the compound is It has an IC50 of over 100 μg / mL against kidney cells. In some embodiments for treating MRSA, the compound is present at a concentration of less than 2 μg / mL. MICs against MRSA strains and human VERO kidney cells were >100 μg / mL In some embodiments, the compound is AKG-38 (Formula 1c), AK G-39 (formula 1e), and AKG-40 (formula 1d).

[0095] Water solubility In some embodiments, the compound is in the form of a salt, e.g., a hydrochloride or mesylate salt. and should be in excess of 1 mg / ml, preferably 10 mg / ml, before encapsulation in liposomes. Soluble in water at more than 1 g / ml (and up to 1 g / ml). Addition before encapsulation Salts of include, but are not limited to, besylate, bitartrate, carbonate, citrate, and esylate. , gluconate, glutamate, glycolate, lactate, malate, maleate , mandelate, methyl sulfate, napsylate, phosphate, propionate, salicylate In some embodiments, compounds may include salts such as succinates, tartrates, and tosylates. The substance may be in the form of a hydrate, solvate or co-crystal prior to encapsulation in liposomes. is.

[0096] In some embodiments, the drug has reduced aqueous solubility, e.g., less than 1 mg / mL, preferably or less than 0.1 mg / mL (0.1 to 0.001 mg / mL) in different salt forms. Once entrapped in the liposome, the salt of the compound may include, but is not limited to, , sulfate, citrate, phosphate, sucrosophate, or various phosphorylated or Exemplary polyols include sulfated polyols or polyanionic polymers. Not specified, but may include sucrose, erythritol, mannitol, xylitol, sorbitol Exemplary polyanionic polymers include thiamin, ... are not limited to, polyvinyl sulfonates, polyvinyl sulfonates, polyphosphates , copolymers of acrylic acid and vinyl alcohol sulfate, and combinations thereof. .

[0097] Working stocks of compounds were prepared as follows: Aliquots of compounds (free base) in powder form. To the coat, add 1 to 1.5 equivalents of HCl in the form of a 1N aqueous solution and stir until the mixture becomes homogeneous. The resulting cake or syrup was vortexed with water, typically to a final concentration of 10 mg / ml of HCl was added until complete dissolution was observed. was added to the free base form of the drug to prepare a 20 mg / ml stock solution.

[0098] The aqueous solubility of the compounds of the present disclosure can be exemplified by the following observations to obtain a visually clear solution: is shown.

[0099] JPEG2026035569000027.jpg67170

[0100] These results demonstrate that the compounds provided herein: -Linezolid (3mg / ml) (www.drugbank.ca / drugs / DB 00601) [ka] -Stezolid (0.237mg / ml) (www.drugbank.ca / drug s / DB11905) [ka] and -Tedizolid (0.382 mg / mL) (www.drugbank.ca / drug s / DB14569) [ka] This indicates that the compound has a higher water solubility than the known water solubility of

[0101] In some embodiments, the compounds described herein may be added to a liposome prior to encapsulation. The water solubility of the oxazolidinone is at least 5 times, at least 10 times, at least 20-fold, at least 30-fold, or at least 40-fold.

[0102] The excellent water solubility of the compounds described herein and their amphiphilic weak base properties The efficient use of transmembrane gradient-based and intraliposomal complexation (active loading) approaches This allows for the use of encapsulated drugs in the infected tissue after systemic drug administration. Liposomal synthesis of these compounds with novel high drug / carrier (drug / lipid) ratios and pharmacokinetic properties As used herein, an amphiphilic weak base is a 7 It has a pKa of ~12 and a logP of 1-6.

[0103] Liposome loading properties and antimycobacterial activity An important feature of the compounds described herein is their weakly amphiphilic basic properties, In some embodiments, this facilitates transmembrane gradient-driven loading of these compounds into liposomes. , the weak base properties of the compounds of the present disclosure are 7.0 to 12.0, 7.5 to 11.0, 7.8 to 1 Characterized by electrolytic dissociation constants in the pKa range of 0.5 or 8.0-10.0 In some embodiments, the amphiphilic properties of the compounds described herein are between 0.5 and 5 logP parameter in the range 0.0, 1.0-4.0, 1.0-3.5, or 1.0-3.0 These favorable properties for liposome loading are Certain embodiments are also preferred for efficient and stable liposomal encapsulation. Match or exceed the activity of similar compounds in the same class of drugs with properties not found in It has been unexpectedly discovered that it has excellent activity against circulating mycobacteria.

[0104] Liposome Composition Compositions for treating tuberculosis and other mycobacterial and gram-positive bacterial infections - Patent Application 20070122999 The compositions provided herein are useful in the treatment of low Highly potent and highly encapsulated to maximize drug delivery potential In some embodiments, the compound contains a selective oxazolidinone and is stable in the presence of plasma. , the composition to allow efficient accumulation at the site of bacterial or mycobacterial infection. , which are long-circulating and retain their encapsulated drug in the circulation after intravenous administration. In some embodiments, the long-circulating properties combined with highly stable retention of the drug The high doses that can be achieved in some cases are comparable to other drugs typically utilized to treat these infections. It may be possible to reduce the frequency of administration compared with once-daily or twice-daily administration of the drug. To perform Noh.

[0105] Disclosed herein are pharmaceutical compositions for treating bacterial infections, particularly Mycobacterium tuberculosis infections. In some embodiments, the pharmaceutical composition comprises a polyanion or a sulfate containing polyanion. and a liposome composition comprising an aminoalkyloxazolidinone compound.

[0106] In some embodiments, the composition comprises liposomes in a vehicle, and the intraliposomal space is filled with liposomes. In some embodiments, the composition comprises an aqueous phase having a lysine anion and a compound of Formula I. The liposomes are contained in the body, and the intraliposomal space contains polyanions or polyanions. Some of the compounds include sulfates and the compounds AKG-16, AKG-28, or AKG-38. In embodiments, the medium is an aqueous medium and the primary composition in the medium comprises a compound of Formula I and are the corresponding scavengers.

[0107] The compounds of formula I may be combined with suitable polyanions, such as sucrose octasulfate (e.g., triethylenediamine). derived from a triammonium sucrose octasulfate (TEA-SOS) gradient) or sulfate Entrapment within liposomes using acid salts (e.g., derived from an ammonium sulfate gradient) Additional polyanion scavengers include, but are not limited to, inositol hexaphosphate. , inositol hexasulfate, polyvinyl sulfonic acid, dextran sulfate, citrate, polylysine Examples include phosphates, and suramin.

[0108] The external aqueous medium is typically composed of an appropriate buffer and an isotonic agent. Agents include histidine, citrate, HEPES, MOPS, MES, TRIS, and phosphate. , glycine, imidazole, borates, carbonates, and succinates. Contains sodium chloride, potassium chloride, sucrose, glycerin, dextrose, or may contain salts such as mannitol.

[0109] In some embodiments, the composition comprises one or more phospholipids, a sterol, and optionally Lipids conjugated to a hydrophilic polymer of choice (polymer-conjugated lipids) encapsulated with polyanions in predominantly unilamellar vesicles formed from comprises a compound of formula 1a, 1b, 1c or 1d, or a pharmaceutically acceptable salt thereof In some embodiments, the compositions include unilamellar vesicles and multilamellar vesicles (e.g., two or three vesicles). a compound of formula I, or a compound of formula 1a, 1b, 1 The compound may comprise a compound of formula 1c or 1d, or a pharmaceutically acceptable salt thereof. It should be understood that vesicles may be cleared from the circulation more rapidly than unilamellar vesicles. In some embodiments, the phospholipid is hydrogenated soy phosphatidylcholine (HSPC), di Stearoylphosphatidylcholine (DSPC), or egg sphingomyelin (ESM As used herein, the term "phospholipid" refers to a lipid that can form liposomes. Neutral phospholipids refer to any single phospholipid or combination of phospholipids that can dialkylphosphatidylcholine, dialkylphosphatidylcholine, sphingomyelin, and and diacylphosphatidylethanolamine. Phosphatidylcholine (P C) includes those derived from eggs, soybeans, or other plant sources, or those derived partly or wholly from These may be synthetic or of variable lipid chain length and unsaturation, and may be used in the present compositions. Synthetic, semi-synthetic and natural phosphatidylcholines are suitable for a wide range of uses. However, distearoylphosphatidylcholine (DSPC), hydrogenated soy phosphatidylcholine Phosphatidylcholine (HSPC), soybean phosphatidylcholine (soybean PC), egg phosphatidylcholine Hydrogenated egg phosphatidylcholine (EGG PC), hydrogenated egg phosphatidylcholine (HEPC), dipalmitoyl phosphatidylcholine dimyristoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) and phosphatidylcholines suitable for use in the present disclosure. Photidylserine, phosphatidic acid, phosphatidylinositol, phosphatidylglycerol Glycerol, cardiolipin, or N-succinyl-phosphatidylethanolamine , N-glutaryl-phosphatidylethanolamine, and PEG-derivatized phosphatidylethanolamine Head group-modified lipids such as diethanolamine may be included.

[0110] The polymer-conjugated lipids are poly(ethylene glycol)-conjugated. PEGylated phospholipids (PEG-lipids), e.g., PEG (molecular weight 2,000) Methoxy-poly(ethylene glycol)-1,2-distearoyl-sn-glycerol (PEG(2000)-distearoylglycerol, PEG-DSG), PEG (molecular 2,000g) 1,2-distearoyl-sn-glycero-3-phosphoethanolamine -N-[methoxy(polyethylene glycol)-2000] (PEG (molecular weight 2,000 )-distearoylphosphatidylethanolamine, PEG-DSPE, or PEG (Molecular weight 2,000) N-palmitoyl-sphingosine-1-{succinyl[methoxy (polyethylene glycol)-2000] (PEG-ceramide). The molecular weight of the PEG moiety in the lipid component is also 500 to 10,000 g / mol, 1,500 It can vary from ~6000 g / mol, but is preferably around 2,000 MW. Other polymers used for conjugation to the anchor are poly(2-methyl-2- Poly(2-ethyl-2-oxazoline) (PMOZ), Poly(2-ethyl-2-oxazoline) (PEOZ), Poly(hydroxyethyl L-)-N-vinylpyrrolidone (PVP), polyglycerol, poly(hydroxyethyl L-) asparagine) (PHEA), and poly(hydroxyethyl L-glutamine) (PHE G).

[0111] In some embodiments, the sterol is cholesterol. Examples include, but are not limited to, phytosterols such as ergosterol and β-sitosterol. In some embodiments, phospholipid(s) and cholesterol The ratio of terol to hydroxybenzoate is adjusted to achieve a sufficient level of hydroxybenzoate while maintaining a sufficiently reduced amount of leakage of the compound of Formula I from the liposomes. In some embodiments, any of the following may be selected to provide a desired amount of liposome membrane rigidity: Addition of optional polymer-conjugated lipids reduces the tendency of liposomes to aggregate. The type and amount of polymer-conjugated lipids can be reduced in the bloodstream. Select to provide the desired level of protein binding, liposome stability and circulation time. For example, the liposome vesicles may be prepared by dissolving phosphatidylcholine in a molar ratio of about 45:55. Contains phosphorus (e.g., DSPC or HSPC) and cholesterol. The molar ratio of acetylcholine to cholesterol is about 60:40 to 35:65, about 50:50 to 35:65 The ratio of the phospholipids in the liposomes can vary from about 50:50 to about 45:55. at a molar ratio of approximately 55:45:2.75, corresponding to a PEG-lipid concentration of 5 mol%. , HSPCs, cholesterol and polymer-conjugated lipids (PEG-DSG or PEG-DSPE). The concentration of PEG-lipid can be The PEG-modified phospholipids can be varied in the range of 0.5 to 10 mol% compared to non-PEGylated phospholipids. A preferred ratio is 3 to 10 mol %, and an even more preferred ratio is 4 to 8 mol %.

[0112] In some embodiments, the liposome composition is administered by intravenous injection in immunocompetent mice. Percentage of injected dose (ID) (or injected amount) remaining in the blood 6 or 24 hours after administration provides desirable pharmacokinetic properties, such as prolonged plasma half-life, measured as plasma as determined by the change in drug-to-lipid ratio (DL ratio) after iv administration in mice In some embodiments, the drug is encapsulated in a stable manner over a 24-hour period. The percentage of drug remaining in the injection is greater than 20% of the injected dose at 6 hours, preferably at 3 More than 0%, most preferably more than 40%. Percentage retained in blood after 24 hours is preferably greater than 10% of the injected dose, more preferably greater than 20%. greater than 20% in 24 hours, preferably greater than 50% of the original injected liposomal drug; Most preferably, the liposome composition is greater than 80%. Desirable liposome compositions also utilize a burst release method. showed stable encapsulation in the presence of human plasma in vitro, and liposomes retained more than 50% of the drug over 20 minutes and more than 60% over 20 minutes More than 70%, preferably more than 80%, and most preferably more than 90% of the capsules Cells retain encapsulated drugs.

[0113] The liposomes of the present disclosure can be made by any method known in the art. For example, see G. Gregoriadis (editor), Liposom e Technology,vol.1-3,1st edition,1983; 2 nd edition,1993; 3 rd edition,2006; CRC P See, e.g., Reess, Boca Raton, Fla. The liposome compositions of the present disclosure may be Examples of suitable methods for preparation include membrane extrusion, reverse phase evaporation, sonication, and solvent (e.g., ethanol) evaporation. Nord) injection (including microfluidics, Y-junction and T-junction mixing), microfluidization, interfaces These include activator dialysis, ether injection, and dehydration / rehydration. The pore size of the membrane used for extraction, or the membrane used for microfluidization or any other suitable method This can be controlled by controlling the pressure and number of passes. The desired lipid is first hydrated by thin film hydration or ethanol injection, then 5 Defined pore sizes such as 0 nm, 80 nm, 100 nm, or 200 nm, or combinations thereof The size can be adjusted by extrusion through a membrane of 70-150 nm or 80-1 Generate liposomes with an average size in the 30 nm range and a polydispersity index below 0.1 The drug compound to be encapsulated is added to the liposomal lipids prior to liposome formation. The liposomes are dissolved in the aqueous medium formed by the above method, thereby In some embodiments, the drug compound is sequestered within the liposome. It is encapsulated in liposomes with a trapping agent incorporated therein (Drummond ,DC,et al.(2006)in:Liposome Technology ,Third Edition(Ed.Gregoriadis,G.)Volume 2, pp. 149-168).

[0114] In some embodiments, the method of making the liposome composition of the present disclosure comprises: (i) a phospholipid; A medium comprising cholesterol and a PEG-lipid, the medium being substantially free of the sequestrant. (ii) preparing a liposome having an interior space containing a compound To encapsulate an object, the liposome is contacted with a compound of the present disclosure in an aqueous medium. (iii) removing any unencapsulated compound; v) providing liposomes in a physiologically acceptable medium suitable for parenteral use; In some embodiments, the method of producing liposomes with compounds therein comprises: a) a lyophilized ammonium salt containing a scavenger consisting of a substituted ammonium salt of a polyanion; (b) preparing a membrane-based vesicle; and (b) subsequently forming an electrochemical gradient across the membrane. (c) removing the scavenger outside the liposomes to allow the compound to enter the liposomes and bind to the target compound; allowing a corresponding amount of ammonia or substituted ammonia to leave the liposome. contacting the liposome with a compound under conditions effective to obtain (which depletes or reduces the pH gradient across the liposome). The liposome composition containing the capture agent is prepared by forming liposomes in a solution of the capture agent. The transmembrane concentration gradient of the entrapment agent can be created after liposome formation or drug loading. Either before loading (entrapment), by removing the entrapment agent outside the liposomes, or by diluting the liposomes. Thus, it can be formed across the liposome.

[0115] In some embodiments, the contacting comprises contacting the liposomes at a temperature above ambient temperature and below the boiling point of water. The temperature is preferably 30°C to 90°C, 40°C to 80°C, 50°C to 80°C, or 60°C to 70°C. In some embodiments, the method comprises incubating the drug in an aqueous medium at a temperature of 5° C. Incubation should be carried out at an ionic strength equivalent to 50 mM NaCl or less, or more preferably Preferably, the reaction is carried out at an ionic strength less than that corresponding to 30 mM NaCl. After incubation, a concentrated salt, e.g., NaCl solution, is added to bring the ionic strength to 50 mM NaCl, or higher than that of about 100 mM NaCl. Increasing the ionic strength after the incubation step reduces aggregation after liposome loading. Incubation times can range from a few minutes to several hours. Incubation times are 5-40 minutes, 10-30 minutes, or 15-25 minutes. After incubation, the liposomes are cooled and then allowed to reach ambient temperature. In some embodiments, the liposomes are cooled to 2-15° C. Cool the mixture to 4-10°C. After the cooling step, add a concentrated salt, e.g., NaCl, solution. and the ionic strength is 50 mM NaCl, or higher than that of about 100 mM NaCl. The increase in ionic strength after the drug loading incubation step can This helped reduce aggregation after loading of the sucrose.

[0116] In some embodiments, the contacting also comprises contacting an aqueous medium in the presence of an osmotic (isotonic) balancing agent. In some embodiments, the method further comprises incubating the liposomes with the drug in a medium. The balancing agent (osmotic agent) is a non-ionic material. Exemplary non-ionic osmotic agents include, but are not limited to: However, dextrose (glucose), sucrose, trehalose, lactose, maize In some embodiments, the hydroxybenzoates include ethanol, sorbitol, and polyvinylpyrrolidone. In this study, the concentration of the permeant was determined by the permeation rate of the scavenger solution in the liposome interior space before drug loading. Osmolarity or osmolality is the same as osmolality. The osmolality of the sequestering agent solution is expressed as the osmolality of the solution combined with the lipid. Before combining them to form liposomes, they can be measured by any known method. In another embodiment, the concentration of the osmotic agent is less than the osmolality of the scavenger solution, Less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50% of the osmotic concentration of an osmolality concentration of less than about 40%, less than about 30%, less than about 20%, or less than about 10% In yet another embodiment, the concentration of the penetrant during the drug loading process is between 200 and 400 mmol / kg, preferably in the range of 250 to 350 mmol / kg. In another embodiment, the osmotic agent is dextrose at a concentration of 45 g / L. In this embodiment, no penetrating agent is used during the incubation of the liposome with the drug. In yet another embodiment, the incubation is carried out in the presence of an ionic strength adjusting agent. An example of the ionic strength adjusting agent is sodium chloride, for example, 5 to 50 mM, 10 It is added to the liposome-drug solution at a concentration of ∼20 mM, or about 10 mM. Contrary to common practice in the field, compounds of the present disclosure, e.g., AKG-28 and AKG-3 8, during the drug-liposome contact step, the amount of penetrant was such that the added penetrant was completely insufficient. until the osmolality is lower than that of the scavenger solution (osmotically unbalanced liposomes). The liposomes of the present disclosure are stable and highly efficient in providing high concentrations. The material is filled into the

[0117] How to use As used herein, mycobacteria, such as Mycobacterium tuberculosis, or methicillin-resistant Staphylococcus aureus Methods for inhibiting the growth of gram-positive bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), are disclosed. and gram-positive bacteria, including but not limited to Mycobacterium avium complex, Mycobacterium leprae , Mycobacterium gordonae, Mycobacterium abscessus, Mycobacterium Lactobacillus mucogenicum, Streptococcus, Vancomycin-resistant Enterococcus (VRE), Staphylococcus pneumoniae Bacteria, Enterococcus faecium, Group B Streptococcus, Streptococcus pneumoniae, Streptococcus pyogenes, Green Streptococcus, Listeria monocytogenes, Nocardia, and Corynebacterium spp. In some embodiments, the compounds and compositions provided herein include In some embodiments, the compound is used to treat mycobacterial infections. In some embodiments, the compounds provided herein and The compositions can be used to treat nontuberculous mycobacterial infections. In an embodiment, the method comprises the step of: to a subject in need thereof. In some embodiments, the method comprises combining an aminoalkyl oxazolidinone compound of the present disclosure and or a pharmaceutically acceptable salt thereof, In some embodiments, the composition is administered to a subject for parenteral administration. In some embodiments, the liquid pharmaceutical formulation is a liquid pharmaceutical formulation. a liposome preparation containing an oxazolidinone compound in an amount sufficient to In another embodiment, the compound is a sulfate salt. Polyanions such as citrate, sucrose octasulfate, and inositol hexaphosphate, and liposomes In some embodiments, the compound may be in the form of a salt within the system, including but not limited to, a phosphatase. phosphatidylcholine, cholesterol, and PEGylated phosphatidylethanolamine, A precipitated or gelled salt containing sulfate salt within a liposome composed of multiple lipid excipients. The liposomes of the present disclosure have greater than 85%, greater than 90%, and greater than 95% capture. In some embodiments, the remaining amount of unentrapped drug is determined by the liposome composition. This can be achieved by size exclusion chromatography, ion exchange, dialysis, ultrafiltration, This can be achieved by a variety of means, such as filtration, tangential flow filtration, adsorption, or precipitation. During or after the unentrapped drug removal step, the liposomes are resuspended in the desired pharmaceutically acceptable carrier. Vehicles that can be used include, for example, saline, isotonic dextrose, isotonic sucrose, Ringer's solution, Alternatively, the solution may be incorporated into Hank's solution. Buffer substances may be added to obtain the desired physiologically acceptable solution. The liposome composition can be adjusted to achieve the desired drug concentration. and can be sterilized, for example, by sterile filtration through a 0.2-0.22 μm filter. In some embodiments, the compound concentration in the liposome composition is 1 to 50 mg / ml, 3 ~30mg / ml, or in the range of 5-25mg / ml.

[0118] In some embodiments, the liposomes contain one or more liposomes containing one or more liposomes for tonicity or pH control. In some embodiments, the excipients include, but are not limited to, sodium chloride. sodium, Hepes buffer, phosphate buffer, and histidine buffer.

[0119] In other embodiments, the composition is an oral formulation. In some embodiments, the composition is a liquid formulation. In some embodiments, the composition is a solid formulation (e.g., a tablet, capsule, pill, For oral use, for example, tablets, troches, lozenges, etc. Zenji, aqueous or oily suspension, dispersible powder or granule, emulsion, hard or soft capsule A solution, syrup, or elixir can be prepared (see Remington's Ph.D. armaceutical Sciences(Mack Publishing Co. ., Easton, Pa.)) Compositions intended for oral use are not intended for the manufacture of pharmaceutical compositions. The composition can be prepared according to any method known in the art. , antioxidants, sweeteners, flavorings, colorings and the like to provide a palatable preparation. It may contain one or more agents, such as a preservative. Tablets containing the active ingredient mixed with toxic pharmaceutically acceptable excipients or adjuvants are acceptable. Suitable excipients or adjuvants include, but are not limited to, inert diluents, soluble Solubilizing agents, suspending agents, adjuvants, wetting agents, sweetening agents, flavoring or flavoring substances, isotonic substances, These include ionic dispersants and surfactants.

[0120] Tablets, dragees, capsules, pills, granules, suppositories, solutions, suspensions and emulsions, pastes, Ointments, gels, creams, lotions, powders and sprays may be suitable pharmaceutical compositions. do.

[0121] The compound or composition may be administered topically, orally, parenterally, intraperitoneally, and / or intravenously. It can be administered rectally.

[0122] Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, one or more doses can be administered over time, or as indicated by the exigencies of the therapeutic situation. Doses may be proportionally reduced or increased as indicated by the formula:

[0123] compounds and / or pharmaceutically acceptable salts thereof, or compounds and / or The dosage of liposomes, including their pharmaceutically acceptable salts, can vary within a wide range. and in each particular case, naturally adapted to the individual conditions and to the pathogen to be controlled. It should be divided.

[0124] In some embodiments, the compound or pharmaceutical liposome is administered for use in treating a bacterial infection. The serosomal composition may be administered once every 7 days (i.e., once every week), once every 14 days (i.e., twice a week), or once every week), once every 21 days (i.e., once every 3 weeks), once every 28 days (i.e., once every 4 weeks), once every 42 days (i.e., once every 6 weeks), or In some embodiments, the average weekly dosage is from about 1 mg to about 1500 mg. g, about 10 to about 700 mg, about 25 to about 500 mg, or about 70 to about 250 mg In some embodiments, the average weekly dosage is from about 1 mg to about 10 mg, from about 10 mg to about 25 mg. g, about 25mg to about 50mg, about 50mg to about 100mg, about 100mg to about 200mg , about 200mg to about 300mg, about 300mg to about 400mg, about 400mg to about 500 mg, about 500mg to about 600mg, about 600mg to about 700mg, about 700mg to about 8 00mg, about 800mg to about 900mg, about 900mg to about 1000mg, about 1000mg g ~ approx. 1100mg, approx. 1100mg ~ approx. 1200mg, approx. 1200mg ~ approx. 1300m g, about 1300 mg to about 1400 mg, and about 1400 mg to about 1500 mg. In embodiments, the compound or composition may be administered for up to 1 month, up to 2 months, up to 3 months, up to 4 months, The particular therapeutically effective amount may be determined based on various factors, including the type of cell being treated. fungal infection, the activity of the particular compound administered, the pharmaceutical composition employed, the age, weight, and sex of the subject The route of administration, the severity of the bacterial infection, and the use in combination with specific compounds (sequentially or simultaneously) optional drugs / active agents (sometimes used), and similar factors known to a physician of ordinary skill in the art. In some embodiments, the compound or composition is In some embodiments, the compounds can be used as monotherapy to treat pulmonary infections. In some embodiments, treatment can be performed in an effective amount as described herein. and an effective amount of one or more of the compounds of formula (I) and (II) to treat Mycobacterium tuberculosis and other gram-positive bacterial infections. and / or sequential administration of multiple additional active agents. In some embodiments, the treatment comprises administering an effective amount of a compound described herein in combination with Mycobacterium tuberculosis and an effective amount of two or more additional active agents (2, 3, 4, etc.) can be administered simultaneously and / or sequentially. The overall antibacterial effect is greater than the predicted purely additive effect of the individual compounds in the combination. When administered simultaneously, the compound and active agent may be administered in the same composition or in separate compositions. When administered sequentially, the composition containing the compound and the additional active ingredient may be included in the composition. The compositions containing the inhibitors are administered at time intervals (e.g., 20 minutes, 40 minutes, 60 minutes or more). In some embodiments, the additional active agent can be administered using a different route of administration. For example, compounds of the present disclosure may be administered intravenously. and one or more additional agents may be administered orally.

[0125] In some embodiments, one or more (e.g., 1, 2, 3, or 4) additional activities Administration of the compound with an agent can result in a reduction in the length of treatment. Administration of a compound with one or more (e.g., 1, 2, 3, or 4) additional active agents may be combined with one The duration of treatment is at least three times, at least two times, or shorter than treatment with the active agent alone. In some embodiments, the additional agent(s) may be an anti- In some embodiments, the additional active agent is a bacterial agent, including but not limited to, a fluoroquinone. Phosphorus, such as moxifloxacin, gatifloxacin, or levofloxacin, Quinoline and other diarylquinoline analogs (e.g., TBAJ-587 and TBAJ -876), delamanid, pretomanid, isoniazid, rifampicin, rifapentine amine, pyrazinamide, clofazimine, spectinamide, ethambutol, strepto mycin, kanamycin, capreomycin, amikacin, leucyl-tRNA synthetase (LeuRS) inhibitor GSK3036656, tryptophan synthase inhibitor GSK8 39, DprE1 inhibitors OPC-167832 and macodinone (PBTZ-169), Telacebec, GSK-656, TBA-7371, and amoxicillin lavranate, their respective pharmaceutically acceptable salts, and any combination thereof. For the treatment of gram-positive bacterial infections, additional active agents include, but are not limited to: However, vancomycin, gentamicin, daptomycin, teicoplanin, ceftaroline ceftrobiprole, telavancin, dalbavancin, oritavancin, fluoroquinolone nitrates (e.g., delafloxacin), tetracyclines (e.g., eravacycline and and omadacycline), sulfonamides (e.g., sulfamethoxazole), trimetanol In some embodiments, troprim, lefamulin, and any combination thereof may be included. In this case, treatment may involve administering an effective amount of a compound described herein and an effective amount of bedaquiline, prednisolone, or benzocaine. Tomanid, pyrazinamide, moxifloxacin, or their respective pharmaceutically acceptable salts or a combination of the foregoing, including simultaneous and / or sequential administration of It can be done.

[0126] The actual dosage levels of the active ingredients in the pharmaceutical compositions disclosed herein may be adjusted to avoid toxicity to the patient. Without limitation, it is not intended to achieve the desired therapeutic response for a particular patient, composition, and mode of administration. The amount of active ingredient can be varied to obtain an amount of active ingredient effective for treating a range of conditions.

[0127] "Parenteral" as used herein in the context of administration refers to enteral and and modes of administration other than topical administration, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, , intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, spider These include intrathecal, intraspinal, epidural and intrasternal injections and infusions.

[0128] As used herein, the phrases "parenteral administration" and "parenterally administered" generally refer to administration modes other than enteral (i.e., through the digestive tract) and topical administration by injection or infusion Formulas include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, Intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, inhalation, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. Liposomal drug administration is often Intravenous injections and infusions (including, but not limited to) are used.

[0129] In some embodiments, the liquid composition is injected intravenously. Alternatively, the pharmaceutical composition may be administered once every 7 days (i.e., once every week), once every 14 days (i.e., , once every 2 weeks), once every 21 days (i.e., once every 3 weeks), once every 28 days ( once every 4 weeks), and once every 42 days (i.e., once every 6 weeks), In some embodiments, the average weekly dose is from about 1 mg to about 100 mg. 1500 mg, about 10 to about 700 mg, about 25 to about 500 mg, or about 70 to about 250 In some embodiments, the average weekly dose is about 1 mg to about 10 mg, about 10 mg ~25mg, approx. 25mg~approx. 50mg, approx. 50mg~approx. 100mg, approx. 100mg~approx. 200mg, about 200mg to about 300mg, about 300mg to about 400mg, about 400mg ~500mg, 500mg~600mg, 600mg~700mg, 700 mg ~ approx. 800 mg, approx. 800 mg ~ approx. 900 mg, approx. 900 mg ~ approx. 1000 mg, approx. 1000mg ~ approx. 1100mg, approx. 1100mg ~ approx. 1200mg, approx. 1200mg ~ approx. 1300mg, about 1300mg to about 1400mg, about 1400mg to about 1500mg The specific therapeutically effective amount will depend on various factors, e.g., the bacterial infection being treated, the specific The activity of the compound, the pharmaceutical composition employed, the age, weight, sex, etc. of the subject, the route of administration, the bacterial infection Severity of infection, optional combinations of specific compounds (sequentially or simultaneously) The dosage will depend on the drug / active agent and similar factors known to a practitioner of ordinary skill in the art.

[0130] In some embodiments, the compound or pharmaceutical oral composition is administered for use in treating a bacterial infection. The composition is administered once or twice daily. The specific therapeutically effective amount will depend on various factors, including the type of The bacterial infection being treated, the activity of the particular compound being administered, the pharmaceutical composition employed, the age and physical condition of the subject The severity of the bacterial infection, the route of administration, the combination with a specific compound (continuous or Optional drugs / active agents used (or simultaneously) and similar drugs / active agents known to a physician of ordinary skill in the art. It depends on the factors. [Example]

[0131] The following examples, including the experiments conducted and results achieved, are provided for illustrative purposes only. , should not be construed as limiting the present disclosure.

[0132] [Example 1]

[0133] Synthesis of oxazolidinone derivatives Compounds AKG-1, AKG-2, AKG-6, AKG-8, AKG-9 and AKG- 19 was dissolved in N-methyl-2-pyrrolidone (NMP) as a solvent at 60 °C. Tedizolid (Tedizolid-MS) was synthesized by reacting with each amine. (Scheme 1) Desiccated with methanesulfonyl chloride in the presence of a base at room temperature (RT). Mesylation of the 1° hydroxyl group of tedizolid gave tedizolid-MS. -MS was treated with sodium azide, and the resulting azide (AKG-3-A) was reduced. Depending on the eluent chosen for purification, intermediate 1 as the free base or hydrochloric acid Amidation of intermediate 1 with the corresponding acid, followed by HCl afforded AKG-3 as a salt. Hydrochloride salt formation using 1 / 4 ethanol afforded compounds AKG-17 and AKG-18. Tedizolid was reacted with the corresponding dialkylamino acid under standard esterification conditions. The reaction gave compounds AKG-5 and AKG-20. Tedizolid with 2-chloro-N,N-diethylaminoethylamine using thorium O-Alkylation of gave compound AKG-7.

[0134] Intermediate 2 can be prepared by the boronation of commercially available aryl bromides using bis(pinocolato)diboron. The compound was synthesized by the chemistry of the readily available 5-bromo-2-fluoropyridinium nitrate (Scheme 2). Suzuki coupling of intermediate 2 with amine affords intermediate 3, which can be converted to the corresponding amine When heated in NMP in a sealed tube with G-15 was obtained.

[0135] Convergent synthesis of compounds AKG-16, AKG-21 to AKG-27 starting from intermediate 4 The azido-2-cyanopyridine was prepared in the following manner (Schemes 3 and 4). Click chemistry using sodium chloride gave intermediate 4. N-Alkylation of trazole gave intermediates 5 and 6 in a 3:1 ratio. The structure of the intermediate was estimated from HMBC analysis. Intermediates 7-12 were synthesized and the positional isomers were In a similar manner (only the desired isomer is shown in Scheme 4), Suzuki coupling of intermediates 5–12 and deprotection of the amine groups, if applicable, afforded the compounds. The compounds AKG-16, AKG-21 to AKG-27 were obtained.

[0136] Intermediate 13 was synthesized by mesylation of readily available aryl bromides. Reduction of 14 with hydrazine gave intermediate 15 (Scheme 5). Boc protection or acetylation of the primary amine in 15 followed by boronation affords it. The corresponding aryl bromide intermediates were used to give intermediates 18 and 19, respectively. Suzuki Coupling of Phosphonic Acid Intermediates (the entire contents of which are incorporated herein by reference) U.S. Patent Application Publication No. 20100022772 and PCT International Patent Application Publication No. WO20 No. 13044845) and, if applicable, deprotection of the amine group to give the compound AKG- 28 to AKG-31 and AKG-38 to AKG-40 were obtained.

[0137] Synthesis scheme The synthesis of intermediate 19 is described in detail in the accompanying drawings, which are incorporated herein by reference in their entirety. U.S. Patent Application Publication No. 20100022772 and PCT International Patent Application Publication No. W Please refer to issue O2013044845.

[0138] [ka]

[0139] [ka]

[0140] [ka]

[0141] [ka]

[0142] [ka]

[0143] synthesis material and method Tedizolid, (R)-3-(4-bromo-3-fluorophenyl)-5-(hydroxy methyl)oxazolidin-2-one was purchased from Skychemical and (2-Piperidin-4-yl-ethyl)-amine was purchased from Enamine and used in other experiments. Drugs and solvents were purchased from Adams and used as received. Chemical structure of the final product , determined on a Bruker NMR spectrometer (500 MHz or 400 MHz) Magnetic resonance spectrum ( 1 H NMR, 13 It was characterized by C NMR. 13 CN The MR spectra were fully resolved. Chemical shifts were determined using the deuterated solvent peak or an internal standard. The concentrations were expressed in parts per million (ppm) using tetramethylsilane (internal) as the standard. 1 H NMR data are reported as follows: chemical shift (d, ppm), multiplicity Severe (s, singlet; br, s, broad singlet; d, doublet; t, tri m, multiplet), integral, coupling constant (Hz). 13 C NMR Data are reported in units of chemical shift (d, ppm).

[0144] The purity of the final product (>95%) was confirmed by analytical HPLC. C was measured using a Sunfire column, 3.5 μm (150 cm × 4.6 mm) and a gradient system. The solvent was water (0.01% TFA) / ACN (0.01% TFA) and the flow rate was 1 mL / min. The chromatograms were run on an Agilent analytical HPLC system using HPLC at 254 and 214 nm. Flash chromatography (FC) purification was performed by Santai Techno Silica gel 60 (0.04-0.063 nm; 230-400 mesh) from Logies The experiment was carried out using a fluoroscopy.

[0145] Procedure A. Tedizolid-MS (1.0 equiv.), R1R2NH(4 The reaction mixture (L 0.0 equiv.) was heated to 60° C. in a sealed tube for 15 hours. CMS), the reaction was diluted with HO (40 mL) and extracted with EtOAc (2x50 mL). The combined extracts were washed with saturated brine, dried over Na2SO4, and filtered. The was removed under reduced pressure and the residue was purified using FC to give a product with a purity of >95%. Something was obtained.

[0146] 1. Synthesis of Tedizolid-Ms [ka]

[0147] Tedizolid (7.00 g, 18.90 mmol) in CH2Cl2 (50 mL) at 0 °C and triethylamine (3.83 g, 37.80 mmol) at 0° C. under Ar. Methanesulfonyl chloride (3.25 g, 28.36 mmol) was added dropwise. After stirring for 2 hours, the reaction mixture was poured into water and extracted with CH2Cl2. The extract was washed with water, dried over Na2SO4, and collected by filtration. The solvent was removed under reduced pressure. The pure product Tedizolid-MS (7.0 g, 82.6% yield) was obtained as a yellow solid. rate) was obtained. 1 H NMR (400 MHz, DMSO-d6) δ 8.95 (s, 1H), 8.31 - 8.14 (m, 2H), 7.88 - 7.65 (m, 2H), 7.53 (d, J = 8.6 Hz, 1H), 5.14 - 4.96 (m, 1H), 4.59 - 4.39 (m, 5H), 4.28 (t, J = 9.4 Hz, 1H), 3.92 (dd, J = 9.2, 6.3 Hz, 1H), 3.28 (s, 3H). MS (ESI+) m / z 449.1 ([M + 1] + ).

[0148] 2. Synthesis of AKG-1, 2, 6, 8, 9, and 19 [ka]

[0149] Using procedure A, a white solid (0.5 g) was obtained from tedizolid-Ms and dimethylamine. AKG-1 was obtained as a 56.4% yield. 1 H NMR (400 MHz, DMSO-d6) δ 8.94 (s, 1H), 8.32 - 8.13 (m, 2H), 7.83 - 7.64 (m, 2H), 7.54 (d, J = 7.6 Hz, 1H), 4.87 (s , 1H), 4.49 (s, 3H), 4.21 (t, J = 8.6 Hz, 1H), 3.84 (t, J = 7.4 Hz, 1H), 2.62 (s , 2H), 2.25 (s, 6H). 13 C NMR (101 MHz, DMSO-d6) δ 164.3, 161.0, 158.6, 154.6, 1 49.9, 145.5, 140.9, 137.6, 132.1, 131.4, 122.6, 119.1, 114.6, 106.0, 72.0, 62.1, 48.7, 46.4, 40.2. MS (ESI+) m / z 398.2 ([M + 1] + ).

[0150] [ka]

[0151] Using procedure A, a white solid (0.52%) was obtained from tedizolid-Ms and dimethylamine. g, 54.8% yield) of AKG-2. 1 H NMR (400 MHz, DMSO-d6)δ 8.94 (s, 1H), 8.29 - 8.11 (m, 2H), 7.81 - 7.65 (m, 2H), 7.52 (dd, J = 8.6, 1.8 Hz, 1H), 4.89 - 4.73 (m, 1H), 4.49 (s, 3H), 4.19 (t, J = 8.8 Hz, 1H), 3.82 (dd, J = 8.7, 7.0 Hz, 1H), 2.75 (dd, J = 5.1, 3.7 Hz, 2H), 2.57 (q, J = 6.9 Hz, 4H), 0.97 (t, J = 7.1 Hz, 6H).13 C NMR (101 MHz, DMSO-d6) δ 164.3, 161.0, 158.6, 154.7, 149.9 , 145.5, 141.0, 137.6, 132.1, 131.3, 122.5, 119.1, 114.6, 106.1, 72.6, 56.1, 48. 6, 47.7, 40.3, 12.3. MS (ESI+) m / z 426.3 ([M + 1] + ).

[0152] [ka]

[0153] Procedure A was used to synthesize tedizolid-Ms and N,N-dimethyl-2-(piperidine-4 -yl)ethan-1-amine as a white solid (0.66 g, 58.2% yield) Got G-6. 1 H NMR (400 MHz, CDCl3) δ 8.93 (s, 1H), 8.30 (dd, J = 8.1, 2.4 Hz , 1H), 8.05 (d, J = 7.8 Hz, 1H), 7.62 (d, J = 12.9 Hz, 1H), 7.56 - 7.47 (m, 1H), 7.45 - 7.37 (m, 1H), 4.89 - 4.74 (m, 1H), 4.48 (s, 3H), 4.11 (t, J = 8.6 Hz, 1H ), 3.86 (t, J = 7.8 Hz, 1H), 2.93 (dd, J = 28.8, 10.9 Hz, 2H), 2.80 - 2.64 (m, 2 H), 2.50 - 2.04 (m, 11H), 1.69 (d, J = 10.8 Hz, 2H), 1.48 (d, J = 7.1 Hz, 2H), 1 .37 - 1.19 (m, 4H). 13C NMR (101 MHz, CDCl3) δ 164.7 , 161.3, 158.8 , 154.3 , 14 9.9, 145.4, 140.2, 137.0, 132.3, 130.5, 122.0, 120.0, 113.8, 106.4, 71.5 , 61.4, 57.0, 55.3, 54.3, 48.9, 45.0, 39.7, 33.6, 32.4. MS (ESI+) m / z 50 9.2 ([M + 1] + ).

[0154] [ka]

[0155] Tedizolid-Ms and N using procedure A 1 ,N 1 -Diethylpropane-1,3- AKG-8 was obtained from the diamine as a white solid (0.62 g, 57.6% yield). 1 HN MR (400 MHz, DMSO-d6) δ 8.94 (s, 1H), 8.34 - 8.11 (m, 2H), 7.84 - 7.59 (m, 2H), 7.52 (dd, J = 8.6, 2.0 Hz, 1H), 4.80 (dd, J = 8.3, 5.7 Hz, 1H), 4.49 (s, 3H), 4 .18 (t, J = 8.9 Hz, 1H), 3.90 (dd, J = 8.8, 6.5 Hz, 1H), 2.94 - 2.77 (m, 2H), 2. 66 - 2.53 (m, 7H), 1.65 - 1.51 (m, 2H), 0.99 (t, J = 7.1 Hz, 6H). 13 C NMR (101 M Hz, DMSO-d6) δ 164.3, 161.0, 158.6, 154.6, 149.9, 145.5, 141.0, 137.6, 132.1, 1 31.4, 122.6, 119.1, 114.6, 106.1, 73.2, 52.1, 50.7, 48.2, 48.0, 46.7, 40.3, 26.4 , 11.4. m / z 483.2 ([M + 1] + ).

[0156] [ka]

[0157] Tedizolid-Ms and N using procedure A 1 ,N 1 -Diethylethane-1,2-di AKG-9 was obtained from the amine as a white solid (0.36 g, 34.4% yield). 1 H NMR (500 MHz, DMSO-d6) δ 8.94 (s, 1H), 8.26 - 8.16 (m, 2H), 7.78 - 7.66 (m, 2H), 7 .53 (d, J = 8.5 Hz, 1H), 4.85 - 4.73 (m, 1H), 4.49 (s, 3H), 4.18 (t, J = 8.8 Hz, 1H), 3.90 (t, J = 7.5 Hz, 1H), 2.88 (t, J = 5.4 Hz, 2H), 2.65 (t, J = 6.1 Hz, 2 H), 0.95 (t, J = 7.0 Hz, 6H). 13 C NMR (101 MHz, DMSO-d6) δ 164.3, 161.0, 158.6, 154.7, 149.9, 145.5, 141.0, 137.6, 132.1, 131.4, 122.6, 119.1, 114.6, 106.1, 73 .3, 52.6, 52.2, 48.1, 47.6, 47.1, 40.3, 12.0. m / z 469.3 ([M + 1] + ).

[0158] Using procedure A, a white solid was obtained from tedizolid-Ms and ethane-1,2-diamine. AKG-19 was obtained as (0.60 g, 55% yield). 1 H NMR (500 MHz, DMSO-d6) δ 10.33 (s, 1H), 9.89 (s, 1H), 8.95 (s, 1H), 8.58 (s, 3H), 8.23 ​​(q, J = 8.3 Hz, 2 H), 7.79 (t, J = 8.8 Hz, 1H), 7.69 (d, J = 13.5 Hz, 1H), 7.49 (d, J = 8.7 Hz, 1H ), 5.25 - 5.19 (m, 1H), 4.49 (s, 3H), 4.33 (t, J = 9.2 Hz, 1H), 4.05 (dd, J = 9. 1, 6.7 Hz, 1H), 3.52 (s, 2H), 3.43 - 3.23 (m, 4H). 13 C NMR (101 MHz, DMSO-d6) δ 164.26, 160.94, 158.50, 153.79, 149.84, 145.51, 140.58, 137.78, 132.04, 131.42, 122.61, 119.50, 114.94, 106.46, 69.38, 49.59, 47.87, 45.16, 40.34, 35.58.

[0159] 3. Synthesis of AKG-3

[0160] [ka] To a solution of tedizolid-Ms (1.00 g, 2.23 mmol) in DMF (20 mL) After stirring at 90°C for 3 hours, NaN3 (0.44 g, 6.69 mmol) was added. The reaction mixture was poured into water and extracted with EtOAc. The organic layer was washed with brine and The mixture was dried over MgSO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography. Further purification gave the title compound AKG-3-1 (0.7 g) as a white solid. , 79.4% yield) was obtained.

[0161] [ka]

[0162] AKG-3-1 (0.7 g, 1.77 mL) in HO (2 mL) and THF (20 mL) A reaction mixture of Ph3P (1.39 g, 5.31 mmol) and Ph3P (1.39 g, 5.31 mmol) was heated for 1 h. Heat to reflux. After completion (LCMS), the reaction was concentrated under reduced pressure and purified using reverse phase FC. The product was purified using 0-10% MeOH in DCM as the eluent and lyophilized. The free base of Intermediate 1 (2.5 g, 76.5% yield) was obtained as a yellow solid. However, the eluent used was 0.006M HCl in HO (0-30%). After FC purification, the hydrochloride salt AKG-3 (0. 35g, 48.8% yield) was obtained. 1 H NMR (400 MHz, DMSO-d6) δ8.95 (s, 1H), 8. 61 (s, 3H), 8.28 - 8.18 (m, 2H), 7.79 (t, J = 8.8 Hz, 1H), 7.69 (dd, J= 13.5, 2. 1 Hz, 1H), 7.48 (dd, J = 8.6, 2.1 Hz, 1H), 5.13 - 5.00 (m, 1H), 4.49 (s, 3H), 4. 29 (t, J = 9.2 Hz, 1H), 4.02 (dd, J = 9.3, 6.6 Hz, 1H), 3.34 - 3.23 (m, 2H). 13 C NMR (101 MHz, DMSO-d6) δ 163.8, 160.4, 158.0, 153.4, 149.4, 145.1, 140.2, 137. 2, 131.5, 130.9, 122.1, 118.9, 114.3, 105.9, 69.8, 47.1, 41.4, 39.8. m / z 370.3 ( [M -HCl + 1] + ).

[0163] 4. Synthesis of AKG-17 [ka]

[0164] 3-((tert-butoxycarbonyl)amino)propanoic acid in DMF (10 mL) (0.62 g, 3.25 mmol, 1.2 equiv.) and TEA (0.63 g, 6.25 m mol, 2.5 equiv.) was added to a solution of HATU (1.44 g, 3.78 mmol) at RT under Ar. The mixture was stirred for 0.5 hours, and then intermediate 1 (1.0 g, 2 The whole mixture was stirred at RT overnight. The reaction was shown to be complete and was poured into H2O and the solid was collected by filtration. The solid was dried under reduced pressure and the residue was dissolved in EtOAc. Therefore, after using it in the next step, HCl / EtOAc (4M, 20 mL ) was added. The whole mixture was stirred for 16 h and the solvent was removed by N2. The residue was purified by reverse phase F C (eluent: 0-30% MeCN in 0.006 M HCl in H2O) This was purified to give product AKG-17 (0.5 g, 39%) as a yellow solid after lyophilization. A 0.5% yield was obtained. 1 H NMR (500 MHz, DMSO-d6) δ8.95 (s, 1H), 8.67 (s, 1H), 8.23 (q, J= 8.3 Hz, 2H), 8.14 (s, 3H), 7.77 (t, J= 8.6 Hz, 1H), 7.69 (d, J = 13. 5 Hz, 1H), 7.50 (d, J = 8.6 Hz, 1H), 4.87 - 4.78 (m, 1H), 4.49 (s, 3H), 4.22 (t, J= 9.0 Hz, 1H), 3.89 (dd, J = 9.0, 6.5 Hz, 1H), 3.50 (t, J = 5.3 Hz, 2H), 2.98 (dd, J = 12.5, 6.4 Hz, 2H), 2.58 (t, J = 7.1 Hz, 2H). 13 C NMR (101 MHz, DMSO-d6) δ 170.60, 164.22, 160.97, 158.53, 154.42, 149.78, 145.42, 140.89, 137.79, 132. 11, 131.41, 122.61, 119.19, 114.72, 106.23, 105.95, 72.13, 47.77, 40.33, 35.58, 32.58.

[0165] 5. Synthesis of AKG-18 [ka]

[0166] Using the procedure in AKG-17, intermediate 1 and 4-((tert-butoxycarbonyl) AKG-18 from (( ... obtained. 1H NMR (500 MHz, DMSO-d6) δ8.95 (s, 1H), 8.52 (t, J = 5.7 Hz, 1H), 8.29 - 8.08 (m, 5H), 7.77 (t, J= 8.8 Hz, 1H), 7.69 (d, J = 13.6 Hz, 1H), 7.50 (d, J = 8.7 Hz, 1H), 4.87 - 4.76 (m, 1H), 4.50 (s, 3H), 4.22 (t, J= 9.0 Hz, 1H), 3.93 - 3.83 (m, 1H), 3.49 (t, J = 5.3 Hz, 2H), 2.83 - 2.72 (m, 2H), 2.28 (t, J = 7.2 Hz, 2H), 1.88 - 1.75 (m, 2H). 13 C NMR (101 MHz, DMSO-d6) δ 172.59, 164.23, 160. 96, 158.52, 154.44, 149.00, 145.39, 140.88, 137.78, 132.10, 131.40, 122.61, 119. 17, 114.70, 106.21, 72.17, 47.78, 41.88, 40.37, 38.78, 32.44, 23.60.

[0167] 6. Synthesis of AKG-5 [ka]

[0168] Tedizolid (1.0 g, 2.70 mmol), 4-(dimethylamino)butanoic acid chloride aqueous solution of acetic acid (0.57 g, 3.37 mmol) and TEA (0.27 g, 2.70 mmol) The mixture was treated with a catalytic amount of DMAP, DCC (0.84 g, 4 0.05 mmol) was added. The mixture was stirred at RT for 16 h. Completion of the reaction (LCMS) Upon completion, it was diluted with H2O (100 mL) and filtered. The filtrate was washed with 0.02 M HCl. After acidification to pH = 5-6, RP-FC (using 0.5% formic acid / MeCN in HO) The product AKG-5 was obtained as the formate salt after lyophilization. The product was redissolved in H2O and 1 equivalent of aqueous HCl (0.02 M) was added. The product was lyophilized to give AKG-5 as the HCl salt (600 mg, 42. 7% yield). 1 H NMR (400 MHz, DMSO-d6) δ10.40 (br, 1H), 8.95 (s, 1H), 8.23 ​​(q, J = 8.5 Hz, 2H), 7.78 (t, J = 8.8 Hz, 1H), 7.71 (dd, J = 13.6, 2.1 Hz, 1H), 7.53 ( dd, J = 8.6, 2.1 Hz, 1H), 5.03 (dd, J = 5.6, 3.1 Hz, 1H), 4.48 (s, 3H), 4.36 (qd , J = 12.4, 4.2 Hz, 2H), 4.26 (t, J = 9.3 Hz, 1H), 3.95 (dd, J = 9.2, 6.2 Hz, 1H) ), 2.99 - 2.86 (m, 2H), 2.64 (s, 6H), 2.45 (t, J = 7.3 Hz, 2H), 1.87 (m, 2H). 13 C NMR (126 MHz, DMSO-d6) δ 172.3, 164.3, 158.8, 154.3, 149.9, 145.6, 140.8, 137 .7, 132.0, 131.5, 122.6, 119.4, 114.7, 106.2, 71.1, 64.8, 56.3, 46.7, 40.3, 30.9 , 19.9. m / z 469.3 ([M + 1] + ). m / z 484.1 ([M -HCl + 1]+ ).

[0169] 7. Synthesis of AKG-7 [ka]

[0170] A mixture of tedizolid (1.0 g, 2.70 mmol in DMF (20 mL)) was added to the NaH (0.13 g, 60%, 5.40 mmol) was added at RT. The mixture was cooled to 0°C. After stirring for 0.5 hours, 2-diethylaminoethyl chloride hydrochloride (930 mg, 5. 40 mmol) was added in one portion. The whole mixture was stirred at RT for 3 h. The reaction was then carefully poured into ice / H2O (20 mL) and the DC The combined organic extracts were washed with saturated brine and then extracted with Na The solvent was removed under reduced pressure and the residue was washed with FC (MeOH in DCM 0-15%). The residue was purified using hexane (eluent with AKG-7 was obtained as a result of the above reaction. 1 H NMR (500 MHz, CDCl3) δ 8.93 (s, 1H), 8.30 (d, J= 8.2 Hz, 1H), 8.05 (d, J = 8.2 Hz, 1H), 7.72 (d, J = 12.9 Hz, 1H), 7.53 (t , J = 8.5 Hz, 1H), 7.42 (d, J = 8.5 Hz, 1H), 4.88 (d, J = 3.5 Hz, 1H), 4.48 (s, 3H), 4.34 - 4.26 (m, 1H), 4.18 - 4.08 (m, 2H), 4.00 - 3.93 (m, 1H), 3.87 (qd, J = 10.8, 2.9 Hz, 2H), 3.19 - 3.11 (m, 2H), 3.06 (q, J = 7.1 Hz, 4H), 1.26 (t, J = 7.2 Hz, 6H). 13 C NMR (126 MHz, CDCl3) δ 164.7, 161.1, 159.1, 154.3, 149.8, 145 .5, 140.0, 137.0, 132.2, 130.6, 122.0, 120.1, 113.8, 106.3, 71.3, 71.3, 66.9, 51 .9, 48.2, 46.6, 39.7, 8.9. m / z 470.3([M + 1] + ).

[0171] 8. Synthesis of AKG-20 [ka]

[0172] Tedizolid (1.0 g, 2.70 mmol), 4-(diethyl acrylate) in DMF (20 mL) (amino)butanoic acid hydrochloride (0.61 g, 3.37 mmol) and DMAP (0.05 To the reaction mixture of (g) was added DCC (0.84 g, 4.05 mmol) at RT under N2. The mixture was stirred at RT for 16 h. Upon completion (LCMS), the reaction was diluted with H2O (100 The filtrate was acidified with 0.02M HCl to pH 5-6, and then Purification was performed using RP-FC (eluent: MeCN in 0.5% FA / H2O). After lyophilization, the product was obtained as the formate salt. The salt was then redissolved in HO and One equivalent of HCl (0.02 M) was added and, after lyophilization, product AKG-2 was obtained as the HCl salt. 0 was obtained (0.61 g, 42% yield). 1 H NMR (400 MHz, DMSO-d6) δ8.94 (s, 1H) , 8.28 - 8.14 (m, 2H), 7.82 - 7.66 (m, 2H), 7.53 (d, J = 8.7 Hz, 1H), 5.11 - 4.9 7 (m, 1H), 4.49 (s, 3H), 4.43 - 4.33 (m, 2H), 4.27 (t, J= 9.3 Hz, 1H), 4.01 - 3. 91 (m, 1H), 3.08 - 2.99 (m, 2H), 2.90 - 2.69 (m, 6H), 1.08 (t, J = 7.2 Hz, 6H). 13 C NMR (101 MHz, DMSO-d6) δ 171.00, 164.33, 158.55, 154.32, 149.90, 145.58, 14 0.71, 137.63, 132.02, 131.45, 122.58, 119.33, 114.69, 106.21, 71.01, 65.04, 46.8 6, 46.63, 40.31, 29.99, 9.95(s).

[0173] 9. Synthesis of Intermediate 3 [ka]

[0174] (R)-3-(4-bromo-3-fluorophenyl)- in dioxane (200 mL) 5-(hydroxymethyl)oxazolidin-2-one (9.0 g, 31.02 mmol) , bis(pinacolato)diboron (11.88 g, 46.54 mmol) and KOAc( A mixture of (4.56 g, 46.54 mmol) was purged with Ar for 10 minutes, and then ( PhP)PdCl (1.09 g, 1.55 mmol) was added. The mixture was heated under Ar. After purging again, it was heated to 90° C. for 15 hours. LCMS showed the reaction was complete. It was cooled to room temperature and filtered through Celite to obtain Intermediate 2 as a filtrate. The filtrate was diluted with 5-bromo-2-fluoropyridine (6.55 g, 37.22 mmol). , KPO (14.47 g, 6.80 mmol) and HO (20 mL) were added. The mixture was purged with Ar for 10 min and (dppf)PdCl (2.27 g, 3.10 m mol) was added. The mixture was purged with Ar again. It was then heated to 90°C for 15 h. The reaction was monitored by LCMS. Upon completion, it was concentrated under reduced pressure. The residue was then diluted with H2O (200 mL) and extracted with EtOAc (2 x 200 mL). The combined extracts were washed with saturated brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The solvent was removed by HCl to give a residue which was purified by FC (MeOH in DCM 0-15%). The product, Intermediate 3 (eluent: 6.8 g, 71.6% yield for two steps) was obtained. 1 H NMR (400 MHz, DMSO-d6 ) δ8.43 (s, 1H), 8.23 ​​- 8.14 (m, 1H), 7.72 - 7.61 (m, 2H), 7.49 (dd, J = 8.6, 2 .2 Hz, 1H), 7.32 (dd, J = 8.6, 2.7 Hz, 1H), 5.27 (t, J = 5.6 Hz, 1H), 4.80 - 4.7 1 (m, 1H), 4.15 (t, J = 9.1 Hz, 1H), 3.90 (dd, J = 8.9, 6.1 Hz, 1H), 3.75 - 3.67 (m, 1H), 3.63 - 3.55 (m, 1H). MS (ESI+) m / z 307 ([M + 1] + ).

[0175] 10. Synthesis of AKG-11, 12, 13, 14, and 15 Use procedure B. Mix intermediate 3 (1.0 equiv.), R1R2NH (4. 0 equiv.) and a catalytic amount of DMAP were heated to 100°C for 16 hours in a sealed tube. Upon completion of the reaction (LCMS), it was diluted with HO (50 mL) and EtOAc (2X50 The combined organic extracts were washed with saturated brine and dried over Na2SO4. The solvent was removed under reduced pressure and RPFC (0.1% NH4HCO3 / H2O in The residue was purified using MeCN, 0-40%, eluent with C18, Something was obtained.

[0176] [ka]

[0177] Use procedure B. Intermediate 3 and N,N-dimethyl-2-(piperidin-4-yl)ethoxyethanol AKG-11 was obtained from benzophenone-1-amine as a white solid (0.40 g, 30.1% yield). Ta. 1 H NMR (400 MHz, DMSO-d6) δ8.28 (s, 1H), 7.73 - 7.65 (m, 1H), 7.60 (dd, J = 13.6, 2.1 Hz, 1H), 7.54 (t, J= 8.9 Hz, 1H), 7.41 (dd, J = 8.6, 2.1 Hz, 1H), 6.8 9 (d, J = 9.0 Hz, 1H), 5.25 (t, J = 5.6 Hz, 1H), 4.78 - 4.68 (m, 1H), 4.33 (d, J = 13.0 Hz, 2H), 4.12 (t, J = 9.0 Hz, 1H), 3.87 (dd, J = 8.9, 6.2 Hz, 1H), 3.74 - 3.64 (m, 1H), 3.62 - 3.52 (m, 1H), 2.87 - 2.71 (m, 2H), 2.23 (t, J = 7.3 Hz, 2 H), 2.11 (s, 6H), 1.72 (d, J = 11.5 Hz, 2H), 1.64 - 1.49 (m, 1H), 1.34 (dd, J = 14.3, 7.0 Hz, 2H), 1.18 - 1.04 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 160.68, 15 8.33, 154.81, 147.54, 139.15, 137.82, 130.32, 120.72, 119.11, 114.35, 106.97, 10 6.03, 105.74, 73.82, 62.09, 56.98, 46.45, 45.73, 45.39, 34.29, 34.15, 31.94.MS ( ESI+) m / z 443.1 ([M + 1] + ).

[0178] [ka]

[0179] Use procedure B. Intermediate 3 and N 1 ,N 1 -Dimethylethane-1,2-diamine to white AKG-12 was obtained as a white solid (0.52 g, 42.6% yield). 1 H NMR (400 MHz, DMSO-d6) δ8.16 (s, 1H), 7.64 - 7.46 (m, 3H), 7.39 (dd, J = 8.6, 2.2 Hz, 1H), 6 .58 (dd, J= 9.9, 5.6 Hz, 2H), 5.25 (t, J = 5.6 Hz, 1H), 4.80 - 4.66 (m, 1H), 4.1 1 (t, J= 9.0 Hz, 1H), 3.86 (dd, J = 8.9, 6.2 Hz, 1H), 3.76 - 3.65 (m, 1H), 3.61 - 3.49 (m, 1H), 3.37 (dd, J = 12.3, 6.5 Hz, 2H), 2.42 (t, J = 6.6 Hz, 2H), 2.18 (s, 6H). 13 C NMR (101 MHz, DMSO-d6) δ 160.60, 158.48, 158.19, 154.82, 147.57, 1 38.92, 137.10, 130.22, 121.18, 118.53, 114.30, 108.37, 106.02, 105.74, 73.81, 62 .10, 58.76, 46.45, 45.76, 39.21. MS (ESI+) m / z 375.1 ([M + 1] + ).

[0180] [ka]

[0181] Use procedure B. Intermediate 3 and N 1 ,N 1 -Diethylethane-1,2-diamine to white AKG-13 was obtained as a white solid (0.68 g, 51.9% yield). 1 H NMR (400 MHz, DMSO-d6) δ8.17 (s, 1H), 7.67 - 7.46 (m, 3H), 7.39 (dd, J = 8.6, 2.1 Hz, 1H), 6 .63 - 6.44 (m, 2H), 5.25 (s, 1H), 4.74 (dd, J = 9.2, 5.8 Hz, 1H), 4.12 (t, J = 9 .0 Hz, 1H), 3.87 (dd, J = 8.9, 6.2 Hz, 1H), 3.76 - 3.65 (m, 1H), 3.63 - 3.52 (m, 1H), 3.34 (dd, J= 13.2, 6.2 Hz, 2H), 2.60 - 2.55 (m, 2H), 2.54 - 2.50 (m, 4H), 0.97 (t, J = 7.1 Hz, 6H). 13 C NMR (101 MHz, DMSO-d6) δ 160.60, 158.53, 158.18, 154.81, 147.62, 138.91, 137.13, 130.20, 121.16, 118.54, 114.29, 108.24, 105.87, 73.80, 62.10, 52.19, 47.13, 46.45, 39.48, 12.31. MS (ESI+) m / z 417.1 ([M + 1] + ).

[0182] [ka]

[0183] Use procedure B. Intermediate 3 and N 1 ,N 1 -dimethylpropane-1,3-diamine AKG-14 was obtained as a white solid (0.6 g, 47.3% yield). 1 H NMR (400 MHz, DMSO-d6) δ8.16 (s, 1H), 7.63 - 7.54 (m, 2H), 7.50 (t, J= 8.9 Hz, 1H), 7.39 (dd , J = 8.6, 2.2 Hz, 1H), 6.73 (t, J = 5.6 Hz, 1H), 6.54 (d, J = 8.7 Hz, 1H), 5.25 (t, J = 5.5 Hz, 1H), 4.78 - 4.68 (m, 1H), 4.11 (t, J = 9.0 Hz, 1H), 3.87 (dd, J = 8.9, 6.2 Hz, 1H), 3.75 - 3.66 (m, 1H), 3.64 - 3.53 (m, 1H), 3.33 - 3.23 (m, 2 H), 2.28 (t, J = 7.1 Hz, 2H), 2.13 (s, 6H), 1.72 - 1.62 (m, 2H). 13 C NMR (101 M H z, DMSO-d6) δ 160.60, 158.62, 158.18, 154.81, 147.62, 138.89, 137.08, 130.19, 1 21.21, 118.39, 114.29, 108.10, 106.02, 105.74, 73.81, 62.10, 57.44, 46.45, 45.72 , 39.58, 27.54. MS (ESI+) m / z 389.1 ([M + 1] + ).

[0184] [ka]

[0185] Use procedure B. Intermediate 3 and N 1 ,N 1 -diethylpropane-1,3-diamine AKG-15 was obtained as a white solid (0.65 g, 48.0% yield). 1 H NMR (400 MH z, DMSO-d6) δ8.16 (s, 1H), 7.63 - 7.54 (m, 2H), 7.50 (t, J= 8.9 Hz, 1H), 7.39 ( dd, J = 8.6, 2.2 Hz, 1H), 6.75 (t, J = 5.5 Hz, 1H), 6.54 (d, J = 8.7 Hz, 1H), 5. 25 (t, J = 5.4 Hz, 1H), 4.79 - 4.68 (m, 1H), 4.12 (t, J = 9.0 Hz, 1H), 3.87 (dd, J = 8.9, 6.2 Hz, 1H), 3.75 - 3.66 (m, 1H), 3.63 - 3.54 (m, 1H), 3.32 - 3.23 (m, 2H), 2.49 - 2.40 (m, 6H), 1.70 - 1.61 (m, 2H), 0.95 (t, J = 7.1 Hz, 6H). 13 C NMR (101 MHz, DMSO-d6) δ 160.60, 158.65, 158.18, 154.81, 147.64, 138.89, 137.05, 1 30.18, 121.21, 118.37, 114.29, 108.01, 106.02, 105.74, 73.80, 62.09, 50.81, 46.8 0, 46.45, 40.11, 27.10, 12.23. MS (ESI+) m / z 417.1 ([M + 1] + ).

[0186] 11.Synthesis of AKG-16

[0187] [ka] ZnCl2 (11.2 g, 81.9 mmol) was added portionwise to pyridine (40 mL). After adding NaN3 (8.90 g, 137 mmol) and 5-bromo-2-cyano 10.0 g, 54.6 mmol) was added and the reaction mixture was heated at 120° C. for 2 h. After the mixture was cooled to RT, it was diluted with water (200 mL) and heated to reflux. Stir for 1 hour, filter, and wash with water (200 mL). Collect the filtered solid and add HCl (200 mL, 6 M) at RT for 2 h. The product was collected by filtration and diluted with HO. It was dried under reduced pressure to give Intermediate 4 (10.0 g, The product was obtained in an 81.3% yield. 1H NMR (400 MHz, DMSO-d6) δ8.96 (s, 1H), 8.36 (dd, MS (ESI+) m / z 225.9 227.9 ([M + 1] + ).

[0188] [ka]

[0189] Intermediate 4 (10.0 g, 44.2%) in HO (150 mL) and DMF (20 mL) A mixture of Ca(OH)2 (7.20 g, 97.35 mmol) and Ca(OH)2 (7.20 g, 97.35 mmol) was After stirring at rt for 0.5 h, (2-bromoethyl)dimethylamine hydrobromide (25 0.0 g, 107.3 mmol) was added. The mixture was heated at 80 °C for 24 h. LCM S indicated a 3:1 mixture of intermediates 5 and 6, respectively. The mixture was diluted with HO (4 The mixture was diluted with 100 mL of EtOAc (2×50 mL) and extracted with EtOAc (2×50 mL). The mixture was washed with water, dried over Na2SO4, and filtered. The solvent was removed under reduced pressure and FC(DC The residue was purified using 0-15% MeOH in M ​​eluent to give the crude product The crude product was purified by RPFC (MeCN in 0.1% NH4HCO3 / H2O) 0-30%, C18, intermediate 5 elutes first, followed by intermediate 6) Further purification gave Intermediate 5 (0.74 g, 5.6% yield) as a white solid and Intermediate 6 (0.25 g, as a light yellow solid) was obtained.

[0190] Intermediate 5: 1H NMR (400 MHz, DMSO-d6) δ 8.89 (dd, J = 2.3, 0.6 Hz, 1H), 8.27 (dd, J = 8.4, 2.4 Hz, 1H), 8.10 (dd, J = 8.4, 0.6 Hz, 1H), 4.87 (t, J = 6.1 Hz, 2H), 2.87 (t, J = 6.1 Hz, 2H), 2.17 (s, 6H). 13 C NMR (101 MHz, DMSO-d6) δ 163.74, 1 51.48, 145.51, 140.81, 124.40, 122.21, 57.73, 51.54, 45.29. MS (ESI+) m / z 297.1, 299.1 ([M + 1] + ). Intermediate 6: 1 H NMR (400 MHz, DMSO-d6) δ 8.98 (s, 1H), 8.38 (dd , J= 8.4, 2 Hz, 1H), 8.20 (d, J = 8.4 Hz, 1H), 5.00 (t, J = 6.4 Hz, 2H), 2.75 (t , J = 6 Hz, 2H), 2.10 (s, 6H), MS (ESI+) m / z 297.1, 299.1 ([M + 1] + ).

[0191] [ka]

[0192] Freshly prepared intermediate 2 (1. 68 g, 4.98 mmol) (1.44 g of (R)-3-(4 -bromo-3-fluorophenyl)-5-(hydroxymethyl)oxazolidin-2-ol (from acetone), intermediate 5 (740 mg, 2.49 mmol) and K3PO4 (1.16 g, A mixture of (dppf)PdC (5.48 mmol) was purged with Ar for 10 min. l2 (182 mg, 0.25 mmol) was added. The mixture was purged with Ar again. It was then heated to 90° C. for 15 hours. LCMS showed the reaction was complete. The residue was concentrated under reduced pressure and diluted with HO (200 mL) and EtOAc (2×200 mL The combined extracts were washed with saturated brine, dried over Na2SO4, and filtered. The solvent was removed under reduced pressure and eluted with RPFC (MeCN in HO, 0-40%). The residue was purified using HCl to give product AKG-16 (520 ml) as a white solid. g, 49.0% yield) was obtained. 1 H NMR (400 MHz, DMSO-d6) δ8.95 (s, 1H), 8.23 ​​( dd, J = 18.3, 8.2 Hz, 2H), 7.82 - 7.66 (m, 2H), 7.54 (dd, J= 8.6, 2.1 Hz, 1H), 5 .28 (s, 1H), 4.89 (t, J= 6.1 Hz, 2H), 4.82 - 4.71 (m, 1H), 4.17 (t, J = 9.0 Hz, 1H), 3.98 - 3.87 (m, 1H), 3.77 - 3.65 (m, 1H), 3.64 - 3.53 (m, 1H), 2.90 (t, J = 6.1 Hz, 2H), 2.19 (s, 6H). 13 C NMR (101 MHz, DMSO-d6) δ 164.17, 161.02, 158.58 , 154.81, 149.91, 145.59, 141.03, 137.63, 132.10, 131.39, 122.59, 119.05, 114.47 , 105.97, 105.69, 73.95, 62.07, 57.72, 51.46, 46.46, 45.26. MS (ESI+) m / z 428.1 ([M + 1] + ).

[0193] 12.Synthesis of AKG-21 [ka]

[0194] Intermediate 2 (1.5 g, 4.5 mmHg) in dioxane (30 mL) and HO (5 mL) ol), intermediate 6 (0.9 g, 3 mmol), Pd(dppf)Cl2 (247 mg, 0 A solution of K3PO4 (1.3 g, 6 mmol) and K3PO4 (1.3 g, 6 mmol) was subjected to Ar gas pressure for 10 min. The mixture was heated to 100° C. for 15 hours. Upon completion of the reaction (LCMS), it was concentrated under reduced pressure. The mixture was concentrated and the residue was diluted with H2O (100 mL) and extracted with EtOAc (2X50 mL). The combined extracts were washed with saturated brine, dried over Na2SO4, and filtered. Remove under pressure and use FC (0-10% MeOH in DCM (10% of NH4OH) The residue was purified using 200 mL of 2,000 sachets of AKG-21 (a white solid) in 35% yield. As a result, 450 mg was obtained. 1 H NMR (400 MHz, DMSO-d6) δ9.01 (s, 1H), 8.36 (d, J = 8.4 Hz, 1H), 8.30 (d, J = 8.4 Hz, 1H), 7.81 (t, J = 8.8 Hz, 1H), 7.74 (dd, J = 13.6, 2.0 Hz, 1H), 7.55 (dd, J = 8.4, 2.0 Hz, 1H), 5.26 (t, J = 5.6 Hz, 1H), 5 .08 (t, J = 6.4 Hz, 2H), 4.79 - 4.75 (m, 1H), 4.18 (t, J = 9.2 Hz, 1H), 3.93 - 3 .89 (m, 1H), 3.74 - 3.68 (m, 1H), 3.62 - 3.56 (m, 1H), 2.80 (t, J = 6.4 Hz, 2H), 2.13 (s, 6H). 13 C NMR (101 MHz, DMSO-d6): δ 161.09, 158.64, 154.80, 152.10, (1 49.45, 149.40), 143.46, (141.35, 141.23), (138.19, 138.15), (132.77, 132.76), (1 31.53, 131.48), 124.58, (118.69, 118.56), (114.51, 114.49), (105.97, 105.68), 73 .96, 62.06, 58.38, 47.26, 46.47, 45.42.

[0195] 13.Synthesis of AKG-22 [ka]

[0196] Intermediate 7 (500 mg, 1.354 mL) in HO (2 mL) and dioxane (8 mL) A mixture of Intermediate 2 (685 mg, 2.03 mmol), K3PO4 (86 2 mg, 4.06 mmol) and (dppf)PdCl2 (99 mg, 0.135 mmol) ol) was added. The flask was evacuated and backfilled with Ar. The mixture was then heated at 90 °C for 1 The mixture was stirred for 6 hours. Water (20 mL) was added and the mixture was extracted with EtOAc (2×20 mL). The organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was evaporated on silica gel. Column chromatography (Biotage, 40g silica gel column @ 30mL / min) Purification by HCl (eluted with 0-100% EtOAc in petroleum ether) gave a grey solid. The desired product AKG-22-1 (450 mg, yield: 66%) was obtained as a solid.

[0197] [ka]

[0198] To a mixture of AKG-22-1 (450 mg, 0.9 mmol) in DCM (8 mL), 4M HCl / dioxane (2 mL) was added, and the mixture was then stirred at RT for 5 h. The solvent was removed under reduced pressure to give the desired product AKG-22 (39%) as a grey solid. 0 mg, yield: 99%). 1 H NMR (400 MHz, DMSO-d6) δ9.03 (s, 1H), 8.39 (d, J = 8.0 Hz, 1H), 8.32 (d, J = 8.0 Hz, 1H), 8.19 (brs, 3H), 7.82 - 7.70 (m, 2H), 7.57 (dd, J= 8.8, 2.0 Hz, 1H), 5.18 (t, J = 5.8 Hz, 2H), 4.81 - 4.74 (m, 1H ), 4.17 (t, J= 9.2 Hz, 1H), 3.93 (dd, J = 9.2, 6.4 Hz, 1H), 3.71 (dd, J = 12.4, 3.2 Hz, 1H), 3.59 (dd, J = 12.4, 4.0 Hz, 1H), 3.53 - 3.47 (m, 2H). 13 C NMR (400M Hz, DMSO-d6) δ (161.07,158.63), 154.82, 152.52, 149.54, 143.25, (141.39,141.28) , 138.24, 124.54, (118.66,118.53), 114.60, (106.01,105.73), 73.97, 62.02, 47.37, 46.48, 38.84.

[0199] 14.Synthesis of AKG-23 [ka]

[0200] Dissolve intermediate 8 (1.0 g, 2.7 mL) in 20 mL of 1,4-dioxane and 5 mL of HO. 1 mmol), intermediate 2 (4.86 mmol, 1.63 g), K3PO4 (1.14 g , 5.42 mmol) and (dppf)PdCl2 (0.23 g, 0.27 mmol) was added and the mixture was stirred at 100° C. for 16 hours. After the starting material was consumed, 100 mL of saturated NaHCO3 was added. The aqueous phase was extracted with EtOAc (3x30 mL) and combined. The organic extract was washed with H2O, concentrated under reduced pressure and purified by FC to give the desired compound. Compound AKG-23-1 (1.0 g, 70% yield) was obtained.

[0201] [ka]

[0202] To AKG-23-1 (1.0 g, 2 mmol) in 30 mL of DCM, 1 mL of HCl (4M in 1,4-dioxane) was added and the mixture was allowed to stir for 1 hour. After the reaction, the mixture was filtered to give the crude product. The crude product was dissolved in 3 mL of MeOH The mixture was stirred in 0.5% CO₂O for 1 hour and filtered to give the desired product AKG-23 (0. 53 g, 63% yield) was obtained. 1H NMR (400 MHz, DMSO-d6) δ8.97 (s, 1H), 8.26 ( m, 5H), 7.83-7.64 (m, 2H), 7.54 (dd, J = 8.6, 1.9 Hz, 1H), 5.09 (s, 2H), 4.77 (m , 1H), 4.16 (t, J = 9.1 Hz, 1H), 3.92 (dd, J = 8.8, 6.2 Hz, 1H), 3.71 (dd, J = 1 2.3, 3.2 Hz, 1H), 3.61-3.57 (dd, J = 12.3, 3.2 Hz, 1H), 3.53 (m, 3H). 13 C NMR (1 25 MHz, DMSO-d6) δ 38.31, 46.49, 50.86, 62.01, 73.96, [105.69, 105.97], 114.46, [118.90, 119.03], 122.73, [131.37, 131.47], 132.21, [137.66, 137.70], [140.99, 141.10], 145.42, 149.86, 154.82, [158.57, 161.02], 164.50.

[0203] 15.Synthesis of AKG-24 [ka]

[0204] Intermediate 2 (1.66 g, 4.98 mL) in dioxane (50 mL) and HO (5 mL) mmol), intermediate 9 (800 mg, 2.49 mmol) and K3PO4 (1.16 g , 5.48 mmol) mixture was purged with Ar for 10 min and (dppf)PdCl2 ( 182 mg, 0.25 mmol) was added. The mixture was purged with Ar again. Then, It was heated to 90° C. for 15 hours. LCMS showed the reaction was complete; The mixture was concentrated under reduced pressure, and the residue was diluted with H2O (200 mL) and extracted with EtOAc (2 x 200 mL). The combined extracts were washed with saturated brine, dried over Na2SO4, and filtered. The solvent was removed under reduced pressure and the eluent was MeCN in RPFC (H2O, 0-40%). The residue was purified using HCl to give the product AKG-24 (440 mg) as a white solid. , 39.0% yield) was obtained. 1 H NMR (400 MHz, DMSO-d6) δ8.95 (s, 1H), 8.28 - 8 .16 (m,2H), 7.81 - 7.67 (m,2H), 7.54 (dd, J = 8.6, 2.1 Hz, 1H), 5.27 (t, J = 5.6 Hz, 1H), 4.93 - 4.70 (m, 3H), 4.17 (t, J = 9.1 Hz, 1H), 3.92 (dd, J = 8.9, 6.1 Hz, 1H), 3.72 (m, 1H), 3.60 (m, 1H), 3.04 (s, 2H), 0.87 (t, J = 6.9 Hz, 6H). 13 C NMR (101 MHz, DMSO-d6) δ164.13, 161.03, 158.59, 154.81, 149.94, 149.90, 145.66 , 141.09, 140.98, 137.66, 137.62, 132.10, 132.08, 131.41, 131.37, 122.54, 119.15 , 119.02, 114.50, 114.47, 105.99, 105.71, 73.95, 62.08, 52.09, 51.60, 46.85, 46. 48, 12.26. MS (ESI+) m / z 456 ([M + H] + ).

[0205] 16.Synthesis of AKG-25 [ka]

[0206] Intermediate 4 (2.25 g, 10 mmol) and KCO (5 To a mixture of 3-chloro-N,N-dimethylpropane-1-amine (0.52 g, 40 mmol), Amine hydrochloride (3.95 g, 25 mmol) was added and the mixture was heated to 80° C. for 4 h. It was diluted with H2O (40 mL) and extracted with EtOAc (2 x 100 mL). The combined extracts were washed with saturated brine, dried over Na2SO4, and filtered. When removed under pressure, the residue showed the presence of two positional isomers of N-alkylation. The compounds were separated using FC (eluent: MeOH in DCM 0-15%). As a result, intermediate 10 (0.98 g, 31.6% yield) was obtained as a white solid. 1 H NMR ( 400 MHz, CDCl3) δ 8.83 (dd, J = 2.4, 0.8 Hz, 1H), 8.16 (dd, J = 8.0, 0.4 Hz, 1H ), 8.01 (dd, J = 8.4, 2.4 Hz, 1H), 4.78 (t, J = 6.8 Hz, 2H), 2.38 (t, J = 3.2 Hz , 2H), 2.27-2.22 (m, 8H).MS (ESI+) m / z 311.1, 313.1 ([M + 1] + ).

[0207] [ka]

[0208] Intermediate 10 (0.74 g, 2.4 mL) in dioxane (30 mL) and HO (5 mL) mmol), intermediate 2 (1.62 g, 4.8 mmol) and K3PO4 (1 g, 4.8 The mixture was purged with Ar for 10 min and treated with Pd(dppf)Cl (175 mg , 0.24 mmol) was added. The mixture was purged again with Ar and heated to 90° C. for 15 h. It was concentrated under reduced pressure and the residue was diluted with H2O (80 mL) and The combined extracts were washed with saturated brine and then extracted with Na2SO4. The solvent was removed under reduced pressure and FC (MeOH in DCM 0-15%) was used. The residue was purified using the eluent (previously used) to give the product AKG-25 ( 0.73 g, 69.5% yield) was obtained. 1 H NMR (400 MHz, DMSO-d6) δ8.94 (s, 1H) , 8.24-8.26 (m, 1H), 8.19-8.21 (m, 1H), 7.78-7.70 (m, 2 H), 7.54 (dd, J = 8.4, 2 .0 Hz, 1H), 5.27 (t, J=5.6 Hz, 1H), 4.80 (t, J = 6.8 Hz, 2H), 4.77-4.75 (m, 1H), 4.16 (t, J = 9.2 Hz, 1H), 3.92 (dd, J=8.8 Hz, 6.0 Hz, 1H), 3.74 - 3.69 (m, 1H), 3.63 - 3.58 (m, 1H), 2.28 (t, J = 7.2 Hz, 2H), 2.10 - 2.17 (m, 8H). 13 C NMR (101 MHz, DMSO-d6) δ 164.26, 161.03, 158.58, 154.81, 149.92, 145.58, 141.09, 137.65 , 132.10, 131.37, 122.60, 119.12, 118.99, 114.46, 105.98, 105.70, 73.95, 62.07, 55.96, 51.65, 46.47, 45.53, 27.21. MS (ESI+) m / z 442.1 ([M + 1] + ).

[0209] 17.Synthesis of AKG-26 [ka]

[0210] To a solution of intermediate 4 (5.0 g, 22.12 mmol) in DMF (30 mL) was added 100 mL of HCl at 80° C. (3-chloropropyl)diethylamine hydrochloride (8.23 g, 55.30 mmHg) was added for 3 hours. ol) and K2CO3 (9.17 g, 66.36 mmol) were added. The reaction was cooled. The combined organic phase was washed with brine ( 2×50 mL) and dried over Na2SO4. Upon removal of the solvent, the N-alkylation The crude product containing the regioisomer was purified by FC (PE / EA = 1:10) to give a white Intermediate 11 (1.70 g, 22.65%) was obtained as a light-colored solid. 1 H NMR (500 MHz, CDCl3) δ 8.34 (d, J = 2.0 Hz, 1H), 8.15 (d, J = 8.0 Hz, 1H), 8.00 (dd, J = 8.0 , 2.0 Hz, 2H), 4.77 (t, J = 7.0 Hz, 2H), 2.53-2.49 (m, 6H), 2.26-2.20 (m, 2H), 0 .99 (t, J = 7.5 Hz, 6H). MS (ESI + ) m / z 339.1, 341.1 ([M + 1] + ).

[0211] [ka]

[0212] Intermediate 11 (0.68 g, 2.00 mmol), Intermediate 2 (1.07 g, 3.99 mmol), ol), tripotassium phosphate (0.85 g, 3.985 mmol) and Pd(dppf) A mixture of Cl2 (0.15 g, 0.20 mmol) in 1,4-dioxane:water (12 ml L, 6:1). The reaction was stirred at reflux for 16 hours. The mixture was diluted with EtOAc ( Partition between 2×100 mL) and water, wash with brine, dry over Na2SO4, and filter. Upon removal of the solvent, the residue containing the regioisomers was purified by distillation with (DCM / MeOH=20 / 1 ) was purified using FC eluting with 0.54 g, 56.04%) was obtained. 1 H NMR (500 MHz, DMSO-d6) δ8.95 (s, 1H), 8.26-8.1 9 (m, 2H), 7.78-7.70 (m, 2H), 7.53 (dd, J = 10.5, 2.5 Hz, 1H), 5.27 (d, J = 7.5 Hz, 1H), 4.83-4.75 (m ,2H), 4.17 (t, J = 11.5 Hz, 1H), 3.92 (dd, J = 11.0, 7.5 H z, 1H), 3.74-3.69 (m, 1H), 3.62-3.58 (m, 1H), 3.51-3.28 (m, 8H), 2.14 (t, J = 8. 0 Hz, 2H), 0.93 (t, J= 8.5 Hz, 6H). 13 C NMR (101 MHz, DMSO-d6) δ 164.24, 161.03 , 158.59, 154.52, 149.91, (d, J = 3.2 Hz),145.59, 141.03 (d, J = 11.8 Hz), 137.6 4 (d, J = 3.2Hz), 132.12, 131.40 (d, J = 4.5 Hz), 122.57, 119.06(d, J = 12.8 Hz) , 114.47 (d, J = 2.8 Hz,), 105.97, 105.70, 73.96, 62.07, 51.70, 49.19, 46.75, 46 .48. MS (ESI + ) m / z 470.1 ([M + 1] + ).

[0213] 18.Synthesis of AKG-27 [ka]

[0214] To a solution of intermediate 4 (6.3 g, 27.87 mmol) in DMF (42 mL) was added 10 mL of HCl at 80° C. for 3 h, BocNH(CH2)3Br (16.6 g, 69.71 mmol) and K2 CO3 (11.1 g, 80.02 mmol) was added. The reaction was cooled and poured into an ice-water bath. The organic phase was extracted with EtOAc (2x200mL). Wash with HCl, dry over Na2SO4, filter and evaporate the solvent under reduced pressure. The crude product containing the positional isomer of methyl group was purified by FC (PE / EA=2:1). This gave Intermediate 12 (14 g, 13.1%) as a yellow solid. 1 H NMR (400 MHz, D MSO-d6) δ 8.89 (d, J = 2.4 Hz, 1H), 8.28 (dd, J = 8.4 Hz, 1H), 8.11 (d, J = 8. 4 Hz, 1H), 6.97 (s, 1H), 4.77 (t, J = 6.8 Hz, 2H), 3.03 (q, J = 12.4 Hz, 2H), 2. 15-2.08 (m, 2H), 1.37 (s, 9H) ppm. MS (ESI+) m / z 383.0 ([M + 1] + ).

[0215] [ka]

[0216] Intermediate 12 (0.83g, 2.15mmol), NaHCO3 (0.36g, 4.31 A solution of 1.4-dioxadiphenylmethane (1.24 g, 3.68 mmol) and Intermediate 2 (1.24 g, 3.68 mmol) was added to 1,4-dioxadiphenylmethane (1.24 g, 3.68 mmol). The mixture was bubbled with N2 for 5 min. After stirring, Pd(dppf)Cl2 (0.078 g, 0.095 mmol) was added. The mixture was stirred at 90° C. for 15 h and then cooled to RT. The mixture was diluted with EtOAc (2×10 The organic layer was dried over Na2SO4, filtered, and concentrated. The filtrate was concentrated and purified by silica gel column chromatography (DCM / Me OH=20 / 1) to give AKG-27-1 (0.7%) as a white solid. 5g (66.9%) was obtained. 1 H NMR (400 MHz, DMSO-d6) δ 8.95 (s, 1H), 8.25 ( d, J = 8.5 Hz, 1H), 8.21 (d, J = 8.4 Hz, 1H), 7.78-7.70 (m, 2H), 7.54 (d, J = 8. 5 Hz, 1H), 6.93 (s, 1H), 5.26 (t, J = 5.0 Hz, 1H), 4.80-4.75 (m, 3H), 4.17 (t, J = 9.0 Hz, 1H), 3.91 (t, J = 8.5 Hz, 1H), 3.71-3.69 (m, 1H), 3.60-3.59 (m, 1H), 3.06-3.03 (m, 2H), 2.15-2.12 (m, 2H), 1.37 (s, 9H) ppm. MS (ESI+) m / z 514.0 ([ M + 1] + ).

[0217] [ka]

[0218] A solution of AKG-27-1 (0.9 g, 1.75 mmol) in dry DCM (16 mL) To the reaction mixture was added HCl in dioxane (4.0 mL) at RT under a N2 atmosphere. The mixture was stirred at the same temperature for 6 hours and cooled to RT. The solvent of the reaction mixture was evaporated under reduced pressure. As a result, AKG-27 (0.65 g, 82.5%) was obtained as a pale yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ 8.95 (s, 1H), 8.26-8.20 (m, 5H), 7.77-7.70 (m, 2H), 7.53 (d, J = 7.6 Hz, 1H), 4.94 (d, J = 6.4 Hz, 2H), 4.77 (s, 1H), 4.51 (s, 2H), 4.16 (t, J = 8.8 Hz, 1H), 3.93 (t, J = 7.0 Hz, 1H), 3.71 (d, J = 12.4 Hz, 1H), 3.60 (d, J = 12.4 Hz, 1H), 2.94 (s, 2H), 2.34 (t, J = 6.8 Hz, 2H) ppm. MS (ESI+) m / z 414.0 ([M + 1] + ).

[0219] 19.Synthesis of AKG-28~31 (R)-3-(4-bromo-3-fluorophenyl)-5- To a solution of (hydroxymethyl)oxazolidin-2-one (9 g, 31 mmol), 0.92g, 34mmol) and TEA (3.76g, 37mmol) were added. The mixture was stirred at RT for 2 h. The mixture was diluted with water (2×30 mL) and brine (2×30 mL ), dried over Na2SO4, filtered and concentrated to give intermediate 13 (11.4 g, 99% yield. MS (ESI+) m / z 368 ([M + 1] + ).

[0220] To a solution of intermediate 13 (11.4 g, 31 mmol) in DMF (200 mL) was added potassium hydroxide. 1,3-dioxoisoindolin-2-indol (6.02 g, 32 mmol) was added. The mixture was stirred at 90° C. overnight. The mixture was cooled and poured into water (1000 mL). The mixture was stirred for 0.5 hours. The precipitate was collected and dried under reduced pressure to give Intermediate 14 (11 g, The yield was 85%. MS (ESI+) m / z 419 ([M + 1] + ).

[0221] To a solution of intermediate 14 (11 g, 26.3 mmol) in EtOH (150 mL) was added NH 2NH2-H2O (85%, 7.7 g, 131 mmol) was added. The mixture was heated at 90 °C. The mixture was stirred overnight. The mixture was filtered and rinsed with EtOH (2×50 mL). The filtrate was concentrated. Intermediate 15 (7.6 g, 100% yield) was obtained. MS (ESI+) m / z 289 ([M + 1] + ).

[0222] Intermediate 15 (7.6 g, 26.4 mmol) in THF (50 mL) and water (50 mL) To a solution of (Boc)2O (6.9 g, 32 mmol) and K2CO3 (7.29 g, 52.8 mmol) was added and the mixture was stirred for 2 hours. The mixture was diluted with water (100 mL) The combined organic extracts were diluted with brine (2 The residue was washed with 50 mL of HCl, dried over Na2SO4, filtered and concentrated. otage, 80g silica gel column @ 65mL / min, 0-60% Et in petroleum ether Purification by elution with 0.5% H OAc for 30 min gave intermediate 16 (7.8 g, 75% yield). ) was obtained. MS (ESI+) m / z 411 ([M + 23] + ).

[0223] Intermediate 16 (7.8 g, 20 mmol), bis(pinaco) (tetrahydro)diboron (6.54 g, 30 mmol) and KOAc (2.94 g, 30 mmol) The mixture of (PhP)PdCl (1.06 g) was purged with Ar for 10 min. , 1.5 mmol) was added. The mixture was purged with Ar again and stirred at 90° C. overnight. The mixture was cooled, diluted with water (300 mL) and extracted with EtOAc (3×100 mL). The combined extracts were washed with brine (2×50 mL), dried over Na 2 SO 4 and filtered. The residue was purified by FC (Biotage, 80 g silica gel column @ 65 mL / min) Purification by elution with 0-60% EtOAc in petroleum ether over 30 min revealed Intermediate 18 (6.2 g, 70% yield) was obtained. MS (ESI+) m / z 459 ([M + 23] + ).

[0224] Procedure C: Intermediates 5 / 8 / 9 / 10 / in dioxane / HO (10:1, 0.06 M) One of 11 (1.0 equiv.), one of intermediates 18 / 19 (1.5 equiv.), Pd(dppf) A mixture of Cl2DCM (0.1 equiv.) and K3PO4 (2.0 equiv.) was percolated with N2. The mixture was diluted with EtOAc and washed with water and brine. The mixture was dried over anhydrous magnesium sulfate, filtered and concentrated. The residue was purified by FC. Compounds AKG-28-1 / AKG-29-1 / AKG-30-1 / AKG-31- 1 / AKG-38 / AKG-39 / AKG-40 was obtained.

[0225] Compounds AKG-28-1 / AKG-29-1 / AK in DCM (1 mL / 100 mg) To one solution of G-30-1 / AKG-31-1, add 3N HCl (20 equivalents) in EtOAc. A quantity of 1,000 ml of HCl was added. The mixture was stirred for 2 hours and then filtered. The solid was dried under reduced pressure or The final compounds AKG-28 / AKG-29 / AKG-30 / AKG-40 were obtained by freeze-drying or lyophilization. One of G-31 was obtained (35-44% yield for two steps).

[0226] [ka]

[0227] Using Procedure C, this product was obtained from Intermediate 5 and Intermediate 18 as a white solid (0.35 g, The compound was obtained as a 35% yield. 1 H NMR (500 MHz, DMSO-d6) δ10.53 (s, 1H), 8.97 (s, 1H), 8.37 (s, 3H), 8.29 (d, J = 8.5 Hz, 1H), 8.24 (d, J= 8.5 Hz, 1H), 7.80 (t, J = 8 .5 Hz, 1H), 7.69 (dd, J = 13.5, 2.0 Hz, 1H), 7.50 (dd, J = 8.5, 2.0 Hz, 1H), 5.3 1 (t, J = 6.0 Hz, 2H), 5.04-4.99 (m, 1H), 4.28 (t, J = 9.0 Hz, 1H), 3.96 (dd, J = 9.0, 6.5 Hz, 1H), 3.84 (s, 2H), 3.28 (s, 2H), 2.87 (s, 6H) ppm. 13 C NMR (126 M Hz, D2O) δ 163.90 (s), 160.30 (s), 158.33 (s), 154.86 (s), 148.55 (s), 142.64 ( s), 138.93 (d, J= 11.0 Hz), 137.74 (s), 132.43 (s), 130.39 (s), 122.46 (s), 119. 03 (s), 114.36 (s), 106.41 (s), 106.18 (s), 70.31 (s), 55.23 (s), 48.07 (s), 47. 69 (s), 43.29 (s), 42.19 (s) ppm. MS (ESI+) m / z 427.1 ([M + 1] + ).

[0228] [ka]

[0229] Using Procedure C, this product was obtained from Intermediate 8 and Intermediate 18 as a white solid (0.4 g, 4 4% yield). 1 H NMR (400 MHz, DMSO-d6) δ 8.97 (s, 1H), 8.57-8.41 m, 6H ),8.29-8.13 (m, 2H), 7.80 (t, J = 9.0 Hz, 1H), 7.69 (dd, J = 13.5, 2.5 Hz, 1H), 7.49 (dd, J = 8.5, 2.0 Hz, 1H), 5.12 - 5.03 (m, 3H), 4.28 (t, J = 9.0 Hz, 1H), 4 .02-3.98 (m, 1H), 3.54-3.51 (m, 2H), 3.33-3.26 (m, 2H). 8.28 (s, 1H), 7.73 - 7.6 5 (m, 1H), 7.60 (dd, J = 13.6, 2.1 Hz, 1H), 7.54 (t, J = 8.9 Hz, 1H), 7.41 (dd, J = 8.6, 2.1 Hz, 1H), 6.89 (d, J = 9.0 Hz, 1H), 5.25 (t, J = 5.6 Hz, 1H), 4.78 - 4.68 (m, 1H), 4.33 (d, J = 13.0 Hz, 2H), 4.12 (t, J = 9.0 Hz, 1H), 3.87 (dd, J = 8.9, 6.2 Hz, 1H), 3.74 - 3.64 (m, 1H), 3.62 - 3.52 (m, 1H), 2.87 - 2.71 (m, 2H ), 2.23 (t, J = 7.3 Hz, 2H), 2.11 (s, 6H), 1.72 (d, J = 11.5 Hz, 2H), 1.64 - 1.4 9 (m, 1H), 1.34 (dd, J = 14.3, 7.0 Hz, 2H), 1.18 - 1.04 (m, 2H) ppm. 13 C NMR (10 1 MHz, D2O) δ161.52, 160.59, 158.12, 154.86, 145.70, 141.05, 140.16, 139.65, 13 3.57, 130.44, 123.64, 117.70, 114.54, 106.50, 106.22, 70.34, 50.81, 47.68 ppm. S (ESI+) m / z 399.2 ([M + 1] + ).

[0230] [ka]

[0231] Using Procedure C, this product was obtained from Intermediate 10 and Intermediate 18 as a white solid (0.36 g , 40% yield). 1 H NMR (400 MHz, DMSO-d6) δ10.94 (s, 1H), 8.96 (s, 1H ), 8.52 (s, 3H), 8.28-8.22 (m, 2H), 7.79 (t, J = 8.8 Hz, 1H), 7.69 (dd, J = 13.6 , 2.0 Hz, 1H), 7.49 (dd, J = 8.8, 2.0 Hz, 1H), 5.08-5.01 (m, 1H), 4.93 (t, J = 6 .8 Hz, 2H), 4.28 (t, J = 9.2 Hz, 1H), 4.00 (dd, J = 9.2, 6.8 Hz, 1H), 3.29 - 3.2 6 (m, 2H), 3.21-3.16 (m, 2H), 2.75 (d, J = 4.8 Hz, 6H), 2.49-2.43 (m, 2H) ppm. 1 3 C NMR (101 MHz, D2O) δ 162.39 (s), 160.58 (s), 158.11 (s), 154.88 (s), 147.20 (s), 141.66 (s), 139.28 (d, J= 11.3 Hz), 132.86 (s), 130.45 (s), 122.90 (s), 118 .44 (d, J = 12.0 Hz), 114.44 (s), 106.46 (s), 106.17 (s), 70.30 (s), 54.56 (s), 50.62 (s), 47.68 (s), 42.89 (s), 42.16 (s), 23.74 (s) ppm. MS (ESI+) m / z 441 ([M + 1] + ).

[0232] [ka]

[0233] Using Procedure C, this product was obtained from Intermediate 11 and Intermediate 18 as a white solid (0.36 g , 42% yield). 1 H NMR (400 MHz, DMSO-d6) δ10.10 (s, 1H), 8.96 (s, 1H ), 8.390- 8.21 (m, 5H), 7.80 (t, J = 8.8 Hz, 1H), 7.69 (dd, J= 13.6, 2.0 Hz, 1H) , 7.50 (dd, J = 8.8, 2.0 Hz, 1H), 5.04-4.97 (m, 1H), 4.94 (t, J = 6.8 Hz, 2H), 4 .28 (t, J= 9.2 Hz, 1H), 3.94 (dd, J = 9.6, 6.4 Hz,, 1H), 3.31-3.26 (m, 2H), 3.21 -3.17 (m, 2H), 3.15-3.12 (m, 4H), 2.46-2.42 (m, 2H), 1.21 (t, J = 7.2 Hz, 6H) pp m. 13 C NMR (101 MHz, D2O) δ163.04 (s), 160.53 (s), 158.07 (s), 154.84 (s), 147. 95 (d, J = 5.4 Hz), 142.36 (s), 139.05 (d, J = 11.3 Hz), 138.32 (s), 132.44 (s), 130.38 (d, J = 4.0 Hz), 122.50 (s), 118.72 (d, J = 12.8 Hz), 114.35 (s), 106.37 (s), 106.09 (s), 70.29 (s), 50.65 (s), 48.55 (s), 47.64 (d, J = 7.4 Hz), 42.17 (s), 23.05 (s), 8.24 (s) ppm. MS (ESI+) m / z 469 ([M + 1] + ).

[0234] To a solution of intermediate 15 (7.6 g, 26.4 mmol) in DCM (150 mL) was added trichloroethylene. Ethylamine (TEA, 4.57 g, 6.27 mL, 52.77 mmol, 2.0 equiv.) , and then acetyl chloride (AcCl, 2.6 g, 2.74 mL, 39.58 mmol, 1 0.5 equivalents) and 4-N,N-dimethylaminopyridine (DMAP, 0.028 g, 2. 64 mmol, 0.01 equiv.) was added under N2 at 0-5 °C. The mixture was stirred at 0-5° C. for 2 hours. TLC and LCMS indicated that the reaction was complete. When indicated, the reaction mixture was quenched with H2O (100 mL). After separation of the two layers, The aqueous layer was extracted with CH2Cl2 (2X50 mL) and the combined organic extracts were washed with H2O (2X1 00 mL) and saturated aqueous NaCl (100 mL), dried over MgSO4, The mixture was concentrated under reduced pressure. The residue was purified by FC (Biotage, 80 g silica gel column @ 65 mL / elution with 0-60% EtOAc in petroleum ether for 30 min. Intermediate 17 (6.5 g, 75% yield) was obtained. MS (ESI+) m / z 332 ([M + 1] + ).

[0235] of intermediate 17 (6.5 g, 19.7 mmol) in 1,4-dioxane (100 mL) The solution was treated with 1,1'-bis(diphenylphosphino)ferrocene-palladium(II) dichloride. Chloride dichloromethane complex (1.61 g, 1.97 mmol), bis(pinacolato)dichloromethane Boron (10 g, 39.39 mmol) and KOAc (4.83 g, 49.24 mmol) l) was added. The resulting reaction was stirred at 90°C for 4 hours. TLC and LCMS showed When the reaction was shown to be complete, the reaction mixture was cooled to RT and then added water (100 mL) and The two layers were separated and the aqueous layer was treated with EtOAc (2 mL). The combined organic extracts were extracted with water (2×50 mL) and saturated aqueous Na The resulting mixture was washed with Cl (50 mL), dried over MgSO4, and concentrated under reduced pressure. The oil was purified by FC (Biotage, 80 g silica gel column @ 60 mL / min) in petroleum ether Purification by elution with 0–100% EtOAc in 30 min gave intermediate 19 (6. 6g, 88.7% yield. MS (ESI+) m / z 379 ([M + 1] + ).

[0236] 20.Synthesis of AKG-38~40 [ka]

[0237] Using Procedure C. This product was obtained from Intermediate 5 and Intermediate 19 as a white solid (0.48 g, (60% yield). 1H NMR (400 MHz, DMSO) δ 8.95 (s, 1H), 8.29-8.19 (m, 3H ), 7.77 (t, J = 8.8 Hz, 1H), 7.69 (dd, J= 13.6, 2.0 Hz, 1H), 7.50 (dd, J = 8.8, 2.0 Hz, 1H), 4.89 (t, J = 6.0 Hz, 2H), 4.81-4.76 (m, 1H), 4.20 (t, J= 9.2 Hz, 1H ), 3.82 (dd, J = 9.2, 6.8 Hz, 1H), 3.45 (t, J = 5.6Hz, 2H), 2.90 (t, J = 6.0 Hz, 2H), 2.19 (s, 6H), 1.85 (s, 3H) ppm. 13 C NMR (101 MHz, DMSO-d6) δ 170.51 ,164. 17, 154.46, 149.94, 145.62, 140.90, 137.63, 132.07, 131.39, 122.59, 119.2 5, 114.66, 106.18, 105.90, 72.34, 57.71, 51.45, 47.67, 45.25, 41.87, 22 .92 ppm. MS (ESI+) m / z 469.2 ([M + 1] + ).

[0238]

change

[0239] Easy to use.この Product を Intermediate 9 および Intermediate 19 から White solid (0.35g, 40% yield) is obtained. 1 H NMR (400 MHz, DMSO) δ 8.95 (s, 1H), 8.29-8.19 (m, 3H ), 7.76 (t, J = 8.8 Hz, 1H), 7.69 (dd, J= 13.6, 2.0 Hz, 1H), 7.50 (dd, J = 8.8, 2.0 Hz, 1H), 4.84-4.76 (m, 3H), 4.21 (t, J = 9.2 Hz, 1H), 3.82 (dd, J= 9.2, 6.4 Hz, 1H), 3.46 (t, J = 5.6 Hz, 2H), 3.04 (t, J = 5.6 Hz, 2H), 2.50-2.47 (m, 4H), 1.85 (s, 3H), 0.87 (t, J= 7.2 Hz, 6H) ppm. 13 C NMR (101 MHz, DMSO-d6) δ 170.50, 164.11, 154.46, 149.91, 145.68, 137.68, 132.04, 131.40, 122.53, 119.27 (d, J = 13.3 Hz), 114.65, 106.18, 105.90, 72.34, 52.11, 51.61, 47.67, 46.83, 41.87, 40.6 3, 40.42, 40.22, 40.01, 39.80, 39.59, 39.38, 22.92, 12.28 ppm. MS (ESI+) m / z 497 ([M + 1] + ).

[0240] [ka]

[0241] Using Procedure C, this product was obtained from Intermediate 11 and Intermediate 19 as a white solid (0.36 g , 50% yield). 1 H NMR (400 MHz, DMSO-d6) δ8.95 (s, 1H), 8.31 - 8.1 8 (m, 3H), 7.77 (t, J= 8.8 Hz, 1H), 7.69 (dd, J = 13.6, 2.0 Hz, 1H), 7.50 (dd, J = 8.8, 2.0 Hz, 1H), 4.88 - 4.71 (m, 3H), 4.20 (t, J= 9.2 Hz, 1H), 3.81 (dd, J = 9.2, 6.4 Hz, 1H), 3.45 (t, J = 5.6 Hz, 2H), 2.45 (s, 6H), 2.14 (s, 2H), 1.85 (s , 3H), 0.93 (s, 6H) ppm. 13 C NMR (101 MHz, DMSO-d6) δ 137.70, 131.45, 114.69, 4 6.79, 41.86, 40.64, 40.43, 40.22, 40.01, 39.80, 39.59, 39.38 ppm. MS (ESI+) m / z 511 ([M + 1] + ).

[0242] [Example 2]

[0243] In vivo studies on Mycobacterium tuberculosis Assay for tro activity The broth microdilution MIC method used was that described by Collins et al., 1997 and Gruppo et al., 2006. After overnight incubation The MIC or minimum inhibitory concentration of a compound that prevents visible growth of bacteria.

[0244] Briefly, Collins et al., 1997 (Collins L, F ranzblau SG(1997).Microplate alamar blue assay versus BACTEC 460 system for high -throughput screening of compounds again st Mycobacterium tuberculosis and Mycoba cterium avium.AAC.41(5):1004-1009) and Gru ppo et al., 2006 (Gruppo V, Johnson CM, Mari etta KS, Scherman H, Zink EE, Crick DC, Adam s LB, Orme IM, Lenaerts AJ. (2006) Rapid mic. robiologic and pharmacologic evaluation of experimental compounds against Mycoba cerium tuberculosis. AAC 50:1245-1250) Microbial assay using Alamar blue endpoint (MABA) as described MICs were determined by broth dilution assay. MABA was measured using the redox indicator Alamydia truncated broth. r blue turns from blue to pink in the presence of mycobacterial growth activity in broth medium It is a 96-well colorimetric assay that replaces the

[0245] Briefly, 4.7 g of Middlebrook's 7H9 Bromine was added to a 1 L flask. powder (Millipore Sigma catalog #M0178), 2 mL of glycerol Add 100 ml of purified water and 898 ml of ethanol with mixing until dissolved, followed by the same 100 ml of ethanol. Add 100 mL of ADC solution (6 g of bovine serum albumin dissolved in 100 mL of water) to a 1 L flask. by adding 200 mg of erythritol, 2 g of albumin, 2 g of dextrose, and 3 mg of catalase. 7H9 complete medium was prepared. Compounds were diluted to a concentration of 10 mg / mL in DMSO and then Further dilute with DMSO to 80 μg / mL, or 40 times the desired starting concentration of 2 μg / mL. 50 μl of drug solution in the first well was diluted with 50 μl of DMSO in the next well. and repeat this process for the next 8 wells in the drug preparation plate. Nine 1:2 dilution series were prepared by carrying out M. tuberculosis Stocks of M.tb H34Rv and M.tb Erdman strains were cultured for 3 days using the medium. ~4x10 7 From their initial concentration of CFU / mL, 5x10 5 Final concentration of CFU / mL Dilute to 100% and thoroughly mix by pipetting up and down using a multichannel pipettor. Mixed into.

[0246] 5x10 5 by transferring 100 μl of medium inoculated with CFU / mL to all wells. Then, the assay plates were prepared. 2.5 μL was transferred to the corresponding wells in the assay plate. Place the samples in a ziplock bag and place it inside the incubator to incubate them at 37°C. Then, on days 3 and 10, the plates were measured at OD600nm on a plate reader. After the 10 day OD600 reading, 10 μl of Alamar Bl was added. On day 12, all assay plates were The images were scanned on a flatbed color scanner. does not produce a visible color change from red to pink, and / or ... The lowest successive antimicrobial concentration (typically 2 2-fold serial dilutions) were considered as the MICs for these compounds.

[0247] Two unique drug-susceptible strains (M.tb Erdman and M.tb H37Rv) The MIC assay was performed using 4% (w / v) human serum albumin. (huSA) (Sigma #A1653) was performed to detect potential protein binding. The MIC of two wells can also be evaluated (serum shift assay). The shift in MIC (4-fold shift in MIC) is considered significant. For the control (gender), a 4-fold shift in MIC is expected.

[0248] MICs were measured by Alamar Blue (MABA) readout or assay Optical densities that were consistent or different only by a single 2-fold dilution, within the limits of (a) All compounds tested were effective in inhibiting Mtb Er The MIC values ​​for both Erdman and H37Rv were consistent, or Higher MIC values ​​of 1–2 μg / mL against n and an M of 0.5 against H37Rv With the exception of one compound, AKG-40, which showed an IC50, it was within one two-fold dilution. The difference is due to slower growth (lower OD reading) on ​​Erdman plates. It is possible that.

[0249] Linezolid is 2 μg / mL, tedizolid is 0.25 μg / mL and bedaquiline is 0. The predicted MIC values ​​were 125 μg / mL. These values ​​were consistent with previous MIC data and and is consistent with published values ​​(Ruiz et al. Antimicrob. Ag ents Chemother.2019,Mar 27;63(4),pii:e01 939-18, Reddy et al. Antimicrob Agents Che mother.2010 Jul;54(7):2840-6, Torrea et a lJ Antimicrob Chemother.2015 Aug;70(8): 2300-5). AKG-28 exhibited an MIC of 0.03 to 0.015 μg / mL. The oxazolidinone acetamide group showed significantly higher activity than dizolidinone. Among the analogs, AKG-39 showed an MIC of 0.5 μg / mL, and AKG-40 showed an MIC of 1 to 1. AKG-38 showed an MIC of 0.06 μg / mL. It also showed several times higher activity than tedizolid.

[0250] Molecules with an amine or acetamide group at the C5 position of the oxazolidinone are more active. Higher (AKG-3 vs. tedizolid, AKG-28 or AKG-38 vs. AKG-16, A KG-39 vs. AKG-24, AKG-40 vs. AKG-26), amino acid on tetrazole Compounds bearing alkyl side chains showed favorable activity. Butoxycarbonylamino (Boc-NH) group, primary amine (AKG-28-1 vs. A Substitution of acetamide (AKG-28) or acetamide (AKG-28-1 vs. AKG-38) resulted in increased activity. Compounds containing dimethylaminoalkyl side chains resulted in a decrease in aminoethyl or It was particularly superior when compared to its diethylaminoethyl analogue (AKG-16 vs. A (KG-24, AKG-28 vs. AKG-29, AKG-30 vs. AKG-31). Shorter dialkylaminoalkyl side chains on the trazole ring (e.g., ethylene vs. propylene) showed higher activity (AKG-16 vs. AKG-25, AKG-24 vs. AKG-26 , AKG-28 vs. AKG-30). Analogs with substitutions on the 2' position of the tetrazole are were more active than those with substitutions at the 1' position (AKG-16 vs. AKG-21, AKG -23 vs. AKG-22).

[0251] [ka]

[0252] JPEG2026035569000079.jpg219170

[0253] [Example 3]

[0254] In vitro cytotoxicity assays for human kidney and human hepatocytes African green monkey kidney (Vero; ATCC#CCL81) or human hepatocytes / liver To determine the IC50 in HepG2 (ATCC #HB8065) ​​cells, 10-fold Compounds were tested in vitro across a dilution series. These molecules generally A doxorubicin positive control was included in all studies because it is expected to be non-toxic. Data are presented as whole cell survival curves as well as calculated actual IC50 values ​​for each compound. It is reported as

[0255] Adherent cells were grown to approximately 80% confluence. After trypsinizing the cells by adding PBS (ibco#25200-072), Spin down the cells and add 5 ml of growth medium (MEM medium; Corning #10010C M) was added to disperse the cells. The cell density was determined using a hemocytometer. Growth medium ( MEM medium (Corning #35015CV) containing 10% FBS was added to the cells. Then, 200 μl of cells (5,000 cells / well) were added to the wells. The mixture was added to a 96-well clear flat-bottom plate (Costar #9804) and incubated in a 5% CO2 atmosphere. Incubate in plates for 24 hours at 37°C in a humidified incubator containing Ta.

[0256] Prepare serial dilutions of test compounds using growth medium as the solvent (Table 2). The compounds were provided as sterile aqueous solutions of the HCl salt with a concentration of 5 mg / ml. To prepare the eluates, warm each drug stock to room temperature, vortex, and remove any sediment. If solid drug was present, the stock was heated in a 60°C water bath. Based on the treatment concentration, 20-fold diluted standard stocks were prepared in series. These were further diluted 1-fold in growth medium to give 250 μg / mL ml of the highest drug concentration tested.

[0257] Each sample was then aspirated and replaced with 200 μl of drug-containing medium. Compounds were added to wells in a 1:2 dilution series starting from an initial concentration of 250 μg / ml. The plates were incubated in a humidified incubator containing 5% CO2 at 37°C for 72 hours. At the end of the compound incubation period, Add 100 μl of 1X PrestoBlue Cell Viability Replace with Reagent (ThermoFisher catalog #A13261). Incubate the plate at 37°C in a humidified incubator containing 100% CO2 for 30 minutes to 2 hours. Reads are taken at 30, 60, and 120 minutes. 5. Measure the fluorescence intensity using a plate reader (Molecular Devices) at 560 nm excitation. Fluorescence is read at 590 nm and 590 nm emission. All sample readings are based on the culture medium only. The background control was calculated by subtracting the RFU of the containing control (background control wells). Correct for ground truth. Calculate the percentage of cytotoxicity using the following formula: Cytotoxicity %=[(RFU .培地 -RFU 処理 ) / RFU .培地 ]×100%.

[0258] The IC50 was determined using GraphPad Prism using the following formula: Y=100 / (1+10^((LogIC50-X) * Hill gradient))).

[0259] JPEG2026035569000080.jpg183170

[0260] Surprisingly, the substituents of the active metabolite tedizolid for tedizolid phosphate mimic those of the Many of the analogs contain a hydroxyl group on the C5 side chain of the oxazolidinone ring, It exhibited high hepatotoxicity, with a single-digit IC50 in the HepG2 hepatocyte cell line. Zolid is the most active oxazolidinone currently approved for the treatment of MRSA. and wherein the tetrazole D ring, pyridyl C ring, and aryl B ring are as described herein. It has structural similarity to the compound described above (see Figure 6). The increased toxicity to hepatocytes was due to the presence of an amino or acetamido group at the same position on the C5 side chain. Compared to those with (AKG28-31, AKG38-40, and AKG-3) , tetraoxazolidinones with a hydroxyl group on the C5 side chain (AKG-23, AKG-25 , AKG-26, and AKG-27).

[0261] [Example 4]

[0262] Determination of selectivity index Mammalian cells, i.e., African leukocytes, as described in Examples 2 and 3, respectively. Compared to green monkey kidney (VERO) or human hepatocyte-derived (HepG2) cells The relative inhibitory activity of the compounds against two M. tuberculosis strains, Erdman and H37Rv, was compared. To determine selectivity, the selectivity index (SI) was calculated. A high SI indicates selectivity against normal cells in the body. It is preferred because it shows favorable killing of M. tuberculosis strains at concentrations of drug that are less harmful to the The selectivity index was calculated using the following formula: SI=IC 50,哺乳動物 / MIC 細菌 wherein the bacterium is Mycobacterium tuberculosis, either the Erdman or H37Rv strain; The cells are VERO or HepG2 cell lines).

[0263] If the IC50 was higher than the highest value tested for VERO or HepG2 cells, In this case, the SI is indicated as higher (>) than the ratio calculated using the highest concentration. MICs for Erdman or H37Rv strains were higher at the highest drug concentration tested (8 μg / ml), the SI is lower (<) than the ratio calculated using that highest concentration. If both numbers are above the highest concentration tested, the calculated value is not determined ( The results are shown in Table 4. SI is the percentage of M. tuberculosis strains or mammalian cell lines. The increased potency in M. tuberculosis strains does not directly correlate with the activity of the molecule in either mammalian The increased toxicity of AKG-38 to both Mycobacterium tuberculosis and M. tuberculosis did not directly correlate with the increased toxicity to the cell lines. showed nanomolar MICs against both strains, which was comparable to other molecules in the panel. It was relatively inactive against both VERO and HepG2 cell lines and gave a high SI. This was also observed for the AKG-28. Both molecules had a dimethylaminoethyl substituent at the 2' position of the tetrazole ring. It is worth noting that.

[0264] JPEG2026035569000081.jpg255170JPEG2026035569000082.jpg18170

[0265] In some embodiments, the compound of interest has a saturation of greater than 100, greater than 200, greater than 300, High, above 400, above 500, above 1000, above 1500, 200 Higher than 0, higher than 2500, higher than 3000, higher than 3500, higher than 4000 Higher than 4500, Higher than 5000, Higher than 5500, Higher than 6000, 65 Higher than 00, 100-7000, 100-6000, 100-5000, 100-40 00, 100-3000, 100-2000, 100-1000, 100-900, 10 0~800, 100~700, 100~600, 100~500, 100~400, 10 0~300, 100~200, 200~7000, 200~6000, 200~5000 , 200~4000, 200~3000, 200~2000, 200~1000, 200 ~900, 200~800, 200~700, 200~600, 200~500, 200 ~400, 200~300, 300~7000, 300~6000, 300~5000, 300~4000, 300~3000, 300~2000, 300~1000, 300~ 900, 300-800, 300-700, 300-600, 300-500, 300- with SI indices for Erd / HepG2 and H37Rv / HepG2 of 400. In some embodiments, the compound of interest is 100 to 1700, 200 to 1700, 300 to SI indices for Erd / HepG2 and H37Rv / HepG2 in the range of 1700 Has.

[0266] An amino or acetamido group on the C5 side chain of the oxazolidinone ring and a tetrazole ring Compounds with an aminoalkyl group at the 2' position of Furthermore, certain tetrazole substitutions further increased the SI. Further improvement was achieved by substituting dimethylaminoethyl at the 2' position of the tetrazole ring with methyl at the same position. , diethylaminoethyl, aminoethyl, or dimethylaminopropyl substitutions are preferred. The tetrazole ring contained dimethylamino groups at the 1'-position (AKG-28 and AKG-38). Unexpectedly, when the ethyl group was transferred (compounds AKG-21 vs. AKG-28), the This resulted in a dramatic loss of activity against the fungus.

[0267] [Example 5]

[0268] Methicillin-resistant Staphylococcus aureus (MR In vitro activity assay for SA Measure the activity of lead oxazolidinone inhibitors against the Gram-positive bacteria Methicillin-Resistant Yellow 1 It has shown sufficient efficacy against Staphylococcus aureus (MRSA) and is available in liposomal form for its treatment. In some embodiments, two of the three strains evaluated In some embodiments, the MIC of the three strains evaluated is less than 6 μg / mL. The MIC for two of them is less than 2 μg / mL, and less than 2 μg / mL is more preferable. .

[0269] Three S. aureus strains were cultured on trypticase soy agar plates supplemented with 5% sheep blood cells. The plates were grown overnight at 37°C under ambient atmosphere. The cultures were aseptically swabbed and The culture was then transferred to a tube of sterile water and the optical density was adjusted to 0.5 at 600 nm. Dilute to 1:100 and deliver approximately 5x10 per well in 120µL 5 cells were delivered. After incubation, the MIC of the test substance was determined by the presence / absence of growth in each well. MIC analysis was performed in triplicate.

[0270] Tedizolid is similar to the 0.5 μg / ml described in U.S. Pat. No. 7,816,379. , and showed MICs of 0.206-0.617 μg / ml. All molecules with primary amine modification at R2 of the non-ring (AKG-3, AKG-28, AK G-29, and AKG-30) showed negligible activity against all three MRSA strains. The molecule with an acetamide group at the same position (AKG-3) showed activity (>50 μg / ml). 8, AKG-39, and AKG-40) demonstrated the efficacy of tedizolid itself against three MRSA strains. The activity was one-third to one-ninth of that of the control.

[0271] JPEG2026035569000083.jpg117170

[0272] [Example 6]

[0273] Liposome Composition General Protocol 1. Lipid components (phospholipids (PhL), cholesterol, and, optionally, PEG- Lipid derivatives and / or fluorescently labeled lipids) were added to a lipid suspension containing approximately 60 mM phospholipids. Mix with a volume of 100% ethanol equal to one-tenth the calculated volume (V) to obtain The mixture was stirred at 65 to 68°C until the lipid was completely dissolved.

[0274] Neutral phospholipids include diacylphosphatidylcholine and dialkylphosphatidylcholine. Phosphatidylcholine, sphingomyelin, and diacylphosphatidylethanolamine Hydrogenated soy phosphatidylcholine, distearoylphosphatidylcholine, and egg Sphingomyelins are some of the preferred phospholipids.

[0275] The PEG-lipid component was PEG (molecular weight 2,000)-distearoylglycerol (P EG-DSG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine Poly(ethylene glycol)-N-[methoxy(polyethylene glycol)-2000] (PEG-DSPE) or N-palmitoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)] The molecular weight of the PEG-lipid component may be 2000 (PEG-ceramide). may also vary from 1,500 to 6,000 g / mol, but is preferably about 2,0 00MW.

[0276] Lipid fluorescent labels include 1,1'-dioctadecyl-3,3,3',3'-tetramethyl Ruindocarbocyanine-5,5'-disulfonic acid (DiIC18(3)-DS), 1, 1'-Dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine-5 ,5'-disulfonic acid (DiIC8(5)-DS), 1,2-distearoyl-sn-glucan Lysero-3-phosphoethanolamine-N-(cyanine 7) (18:0 Cy7 PE) , 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino [D-(polyethylene glycol)-2000]-N-(cyanine 7) (DSPE PEG( 2000)-N-Cy7), 1,2-distearoyl-sn-glycero-3-phosphoeta Nolamine-N-(Cyanine 5) (18:0 Cy5 PE), 1,2-distearoyl 1-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)] PEG(2000)-N-Cy5 ), 1-oleoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazo [4-(4-yl)amino]dodecanoyl]-sn-glycero-3-phosphocholine (18:1 -12:0 NBD PC).

[0277] 2. Stir the ethanolic lipid solution at 65-68°C until a uniform suspension is obtained. Then, add a volume V of scavenger solution (0.25-0.5 M ammonium sulfate or 1 N trisulfate). ethylammonium sucrose).

[0278] Potential scavengers include, but are not limited to, 0.1-2 g equivalents / L (0.1 ~2N), preferably 0.2 to 1.5N of distilled sucrose octasulfate Ethylammonium or triethylammonium salts, ammonium sulfate, ammonium citrate ammonium, citrate, dextran sulfate, polyvinyl sulfonic acid, or inositol The ammonium salt of hexaphosphate is typically used. Ammonium itself, monoalkyl-, dialkyl-, or trialkyl-ammonium It may also contain nium salts.

[0279] 3. Thermobarrel (400-450 psi pressure, 65-68°C) A track-etched (tra rel) extrusion device (Lipex, Canada) was used to ck-etched) polycarbonate membrane stack, typically 100 nm thick Two or four membranes with a nominal pore size of 100 nm and one with a nominal pore size of 200 nm at least three times through a membrane (Whatman Nuclepore, USA) The lipid suspension was extruded. Two 100 nm membranes were placed in 100 ml of Lipex extrusion. When used in the apparatus, extrusion pressures were typically 260-300 psi. The liposomes to be produced have a Z-average particle size (diameter) Xz of about 80 to about 130 nm, and a Z-average particle size (diameter) of less than 0.1 It has a PDI of

[0280] 4. Extruded lipid suspension (containing unilamellar and / or oligolamellar liposomes) The sample was cooled in a refrigerator (2-8°C) and then heated under positive pressure with 0.2 μm polyethylene glycol. The mixture was filtered through a polyethersulfone (PES) membrane filter.

[0281] 5. An aliquot of the extruded and filtered liposome suspension so produced is , on a gravity-fed Sepharose CL-4B size exclusion column (eluent - type 1 water) The liposomes were purified from the extraliposomal entrapped agent by chromatography. The purified liposomes were collected near the void volume fraction of the column. Repeat this step 8-10 times with Type 1 or USP "Water for Injection" endotoxin-free water. Volume exchange (or until the conductivity of the liposome suspension drops below 200 μS / cm) Hollow fiber cartridge (Repligen Spectrum MicroKr Tangential flow filtration on a PS or mPES membrane with a MWCO of 500 KDa This was carried out using a TFF filter.

[0282] 6. Compare the lipid concentration in the purified, extruded liposome preparation with the cholesterol concentration and correcting for the known phospholipid / cholesterol molar ratio. Therefore, it was determined using HPLC with UV detection. Alternatively, spectrophotometric phosphomonomers were used. The phospholipid content was directly quantified using the libertine blue method.

[0283] 7. The drug is administered in the form of the hydrochloride salt at a concentration of 5-20 mg / ml of drug to Type 1 or non-endotoxin (e.g., AKG-3 and AKG-5 were used as monohydrochlorides, AKG-28 and AKG-29 were used as the dihydrochloride salts. To the selected drug (e.g., AKG-16, AKG-38), an equal volume of HCl was added. If necessary, adjust the pH of the solution with 1N NaOH, HCl, or tris(hydroxymethyl)amine. The pH was adjusted to 2.5-5.5 using a dichloromethane (Tris) base solution, and the solution was heated under positive pressure until 0°C. The solution was filtered through a 0.2 μm PES filter. If necessary, the solution was filtered through a 0.2 μm PES filter. Drug concentrations in the stock solutions were verified by HPLC with UV detection at 305 nm. .

[0284] 8. Mix the purified liposome and drug stock solutions from step 5 with the desired drug: lipid (DL) ratio, drug concentration ranging from 1.5 to 3.3 mg / ml, scavenger solution in step 2 The amount of osmotic agent (typically 100%) required to provide an osmolality equal to the measured osmolality of Typically, the mixture was mixed in the presence of dextrose and water. If necessary, the desired pH ( Typically, a buffer solution (pH 4 to pH 7) was added. The amount of dextrose (e.g., about 45 g / L) is determined by the measured osmolality of the scavenger solution. This resulted in a lower osmolality and loading was performed at 6-8 mg / ml of drug.

[0285] 9. Incubate the drug-liposome mixture at 65-68°C for approximately 15-20 minutes with constant stirring. After 5-10 minutes, the mixture was allowed to reach ambient temperature and cooled rapidly on ice. Adjust the NaCl concentration to 0.1 M by adding the calculated amount of 3 M NaCl stock solution. Adjusted.

[0286] 10. The drug-loaded liposomes were loaded onto a gravity-fed Sepharose CL-4B column, Syneresis - 10mM HEPES buffer in 140~144mM NaCl, pH 7.0(H Size exclusion chromatography (SEC) on a PBS-7 column revealed that the encapsulated The liposome fraction was collected near the column void volume. For pull-up testing, purification and buffer exchange were performed as described under item 5 above for TFF. The scale was adjusted using a 10-fold volume exchange with HBS-7 buffer. In the piping process, an exchange of approximately 8 volumes was typically used. The purified liposomes were concentrated by continuing the TFF process without buffer supply. The purified drug-loaded liposomes were filtered under positive pressure through a 0.2 μm sterile PES filter. The solution was sterile filtered using a filter and stored in a refrigerator (2-8°C).

[0287] 11. Drug and lipid concentrations in purified drug-loaded liposome preparations were analyzed by HPLC. Alternatively, spectrophotometry (phosphomolybdenum blue) was used for phospholipid quantification. Using the method, 70% isopropanol was added in the presence of 6.5 mg / ml sodium dodecyl sulfate. UV absorption (302-305) in liposome samples solubilized in 0.1N HCl. The drug was quantified by HPLC (nm). The encapsulation efficiency was evaluated by EE, %=DL / DL0 * 100% where DL0 is the ratio of drug and phospholipid in the liposome loading mixture before SEC or TFF purification. DL is the ratio of drug to phospholipid in the drug-loaded liposomes after purification (step 10). (quality ratio) It was decided as follows.

[0288] 12. Average liposome size (Z-average diameter, Xz) and polydispersity index (PDI) were calculated using Z etasizer mu-V, Zetasizer Nano, or Zetasize Cumulant on r Pro (Malvern Panalytical, US) The ion concentration was determined using the dynamic laser scattering method.

[0289] [Example 7]

[0290] In vivo stability and blood clearance of liposomes Drug encapsulation stability and blood concentration of liposomes encapsulating compounds of the present disclosure The clearance rate was tested in mice according to the following general protocol. Groups of mice of a given laboratory strain (C3H female or CD-1 male) were given 100 mg / kg body weight. Drug-loaded liposomes were injected via the tail vein at a dose of 9 mg of drug. Then, blood was collected from the retro-orbital sinus and the animals were sacrificed. Typically, blood was collected at 100 μg / min post-injection. The time intervals included 5 min, 1 h, 6 h, and 24 h. Plasma was separated by centrifugation. Acidified isopropanol, optionally containing a solubilizer (sodium octasulfate). The drug and lipid (liposomes containing lipid label, DiIC18(3)-DS) were extracted with HCl. The blood clearance of the liposomal drug was analyzed by HPLC. The results were expressed as the percentage of the injected dose remaining at the time point. The stability of the drug was assessed by the percent change in the DL ratio in plasma at a given time point compared to the pre-injection DL value. The results were evaluated by (reduction).

[0291] [Example 8]

[0292] Loading of AKG-3, AKG-5, and AKG-16 into liposomes at different pH Dowex 50Wx8 100-200 mesh ion exchange filter in hydrogen form, 500ml The column was loaded with commercially available potassium sucrose octasulfate (40.2 g in 145 ml of water). The resulting free acid form of sucrose octasulfate is passed through the solution and mixed with the original triethylene glycol solution. Trimethylammonium sucrose octanoate was obtained by titration with methylamine to pH 6.2. A sucrose sulfate scavenger solution was prepared. Triethylammonium sucrose octasulfate (TE A-SOS) concentration (1N, corresponding to 0.125M sucrose octasulfate) was estimated from the amount of triethylamine consumed in the titration. Depending on the addition method, a Horiba LAQUATwin K-11 potassium analyzer was used. The estimated potassium content was less than 0.1% of the initial potassium content.

[0293] 1N trimethylammonium sucrose octasulfate (TEA-S) was used as a scavenger. Hydrogenated soy phosphatidylcholine (HSPC) (Lipoid, Germ) was used. any), cholesterol (3:2 molar ratio), and 1,2-distearoylglycerol Methoxypoly(ethylene glycol) ether (PEG-DSG, PEG molecular weight 2 Liposomes (0.5 mol% HSPC) composed of 000, NOF, Japan was prepared essentially as described in the general protocol above. The mixture was diluted with 16 mM morpholinoethanesulfonic acid (MMA) with a pH ranging from 4.3 to 7.1. S) - in the presence of 4 mM sodium citrate buffer and without any buffer substance added (pH 5.2-5.9) at a DL ratio of 500 g / mol pH (DL0). All drugs were encapsulated in liposomes with high efficiency over the entire pH range tested. (>98%, except for AKG-16 at pH 4.38, which was loaded at 93.3% efficiency) (Fig. 1). The addition of buffer substances was not necessary for efficient encapsulation.

[0294] [Example 9]

[0295] AKG-3, AKG- in liposomes with TEA-SOS scavenger at different DL ratios 5, and AKG-16 encapsulation HSPCs with 1N TEA-SOS as a capture agent, cholesterol (3:2 molar ratio) Liposomes composed of PEG-DSG (0.5 mol% of HSPC) The drug-loaded sachets were prepared essentially as described in the general protocol (Example 6). The tape was prepared without adding any buffering substances (pH 4.98-6.22) at 750-1500 g / m The maximum drug concentration for compounds 3, 5, and 16 was 1.25 mg / L. The loadings were 900-930g / mol PhL and 982-1197g / mol, respectively. pHL and 938–951 g / mol pHL, with maximum drug loading observed. The packing efficiency at or near these limits shall be at least 97.6%, 96.0%, and was 85.2% (Figures 2A and 2B).

[0296] [Example 10]

[0297] Liposomal PEGylated liposomes with high PEGylation degree or 0.25M ammonium sulfate (AS) as a capture agent were used. Encapsulation of AKG-3, AKG-5, and AKG-16 in a sphere HSPCs and cholesterol with various PEG-DSG contents (3:2 molar ratio) Liposomes composed of PEG-100 and encapsulating agent were prepared according to the general protocol and or compound AKG-3 as described in Example 9 at a DL0 ratio of 500 g / mol PhL , AKG-5, and AKG-16 were loaded. All three compounds are shown in Table 6 below. The liposomes were loaded with high efficiency so that the

[0298] JPEG2026035569000084.jpg72170

[0299] Thus, compounds AKG-3, AKG-5, and AKG-16 induce high levels of PE Phospholipid-cholesterol with ammonium sulfate as a glyceride and intraliposomal drug entrapment agent Sterols were efficiently loaded into liposomes. However, at 0.25 M Ammonium sulfate was used to achieve a higher drug:lipid ratio of 500 g drug / mol PhL. When loaded, two of the three oxazolidinones (AKG-3 and AKG-16) The packing efficiency decreased.

[0300] [Example 11]

[0301] Compounds AKG-3, AKG-5 in liposomes using 0.5 M AS as a scavenger , and AKG-16 filling HSPC and 0.5 mol% or 5% mol% PEG-DSG (relative to PhL Cholesterol (3:2 molar ratio) with 0.5M ammonium sulfate as a scavenger Liposomes composed of ammonium sulfate (AS) were prepared according to a general protocol. Compounds were prepared as described in Example 8 at DL ratios ranging from 0 to 1500 g / mol PhL. AKG-3, AKG-5, and AKG-16 were loaded. The results are shown in Figures 3A, 3B, and 3C. 3C and 3D. All three compounds were encapsulated in 4-well plates with 93-100% encapsulation efficiency. Maximum drug loaded in both liposomes at pH / DL ratios of 20-450 g / mol The payload was as follows:

[0302] JPEG2026035569000085.jpg48170

[0303] All three compounds tested were effective in formulations containing 0.5 mol% PEG-DSG as well as For compounds AKG-3 and AKG-16, the PEG-DSG content was 5 mol %. In formulations containing more than 500 g drug / mol PhL, 0.5 M ammonium sulfate liposomes These high levels of loading are sufficient for the treatment of diseases. The loading efficiency is important in that a sufficient dose of the drug can be achieved. The loading was significantly improved over Example 10, where the loading was lower using ammonium. is higher than 0.5M, despite the higher osmolality and the possibility of osmotic burst. High ammonium sulfate concentrations limit the amount of drug that can be loaded per mole of phospholipid. and preferably, low toxicity and high doses resulting in improved outcomes. This indicates that improvements can be made in anti-infective agents if this is possible.

[0304] [Example 12]

[0305] Compounds AKG-3, AKG-5, and A were incorporated into liposomes of various compositions containing fluorescent lipid labels. KG-16 and AKG-28 filling Cholesterol with HSPC and 0.5 mol% PEG-DSG (relative to PhL) rol (60:40 molar ratio), 0.15 mol% lipid fluorescent label DiIC18(3)- DS (ThermoFisher, USA), and 0.5 M ammonium sulfate as a scavenger. Liposomes composed of 1N triethanolamine (AS) or 1N triethanolamine-sodium phosphate-sulfonate (TEA-SOS) were prepared using a general protocol. Prepared according to Cole's method, at pH 4.7-5.8 (no buffering substances added), and Compounds AKG-3, AKG-5, and AKG-16 were loaded as described. The team had the following characteristics:

[0306] JPEG2026035569000086.jpg108170

[0307] All three drugs were efficiently loaded into liposomes. Decomposition of 5 was detected as the appearance of a second peak on HPLC.

[0308] Various phospholipids (HSPC, distearoylphosphatidylcholine (DSPC, Ava Anti Polar Lipids, USA), or egg sphingomyelin (ESM, Lipoid, Germany) and various amounts of PEG-DSG or N-methoxy Poly(ethylene glycol)oxycarbonyl-1,2-distearoylphosphatidyl Ethanolamine (PEG-DSPE, PEG molecular weight 2000, Lipoid, Germ cholesterol (60:40 molar ratio) containing any lipid, and fluorescently labeled DiI Liposomes composed of C18(3)-DS (0.15 mol % with respect to PhL) The same general protocol was followed using different capture agents and AKG-1 was detected in a similar manner. Where indicated, the liposome extrusion step of the general protocol included 50 Add extrusion through two stacked polycarbonate membranes with pore sizes of 200 nm The liposomes had the following characteristics:

[0309] JPEG2026035569000087.jpg134170

[0310] Collagen with HSPC and 9.2 mol% PEG-DSPE (relative to PhL) terol (3:2 molar ratio), 0.15 mol% lipid label DiIC18(3)-DS; and 0.25 M ammonium sulfate (AS) as a scavenger. Prepared according to the general protocol and Example 12 with an additional 50 nm extrusion. AKG-28 was loaded at a drug:lipid ratio (DL0) of 150 g / mol PhL. The liposome (batch ID 98) had a DL ratio of 73.8 g / mol PhL, a Z-average liposome size of 73.8 g / mol It has a size of 77.8 nm and a size polydispersity index (PDI) of 0.090.

[0311] These studies compared AKG-3, AKG-5, and AKG-16 with the neutral phospholipid component. HSPC, DSPC, or ESM, or low (0.5 mol%) or high ( Efficiency in liposomes with a range of lipid compositions, including PEG-lipid contents of 5 mol%) However, compared to 1N TEA-SOS, In comparison, when 0.25M AS was used, the efficiency was approximately 500g AKG-16 / mol The pH significantly decreased from 128 g AKG-16 / mol PhL to 128 g AKG-16 / mol PhL. When AS was used to pack AKG-28, a similar low packing efficiency (i.e., 73.8 g / mol PhL) was observed, which indicates that TEA-SOS or higher concentrations of AS This suggests that it may be preferable to load a high concentration of compounds into liposomes. .

[0312] [Example 13]

[0313] Blood Persistence of Liposomes of Example 12 and In Vivo Encapsulation in Mice Encapsulation Stability Tests were performed on male CD-1 mice as described in the general protocol above. did.

[0314] JPEG2026035569000088.jpg91170

[0315] These studies consisted of varying neutral phospholipid components (HSPC, DSPC, or SM). Liposomes constructed and loaded with AKG-16 using TEA-SOS scavenger were used for 3 0% of the injected dose is slowly cleared, with most formulations clearing plasma levels within 6 hours. Furthermore, many formulations were filled using 0.25M AS. All liposomes showed good drug retention, except for liposome batch ID 97, which contained AKG-16. However, drug loading using 0.25M ammonium sulfate was low as shown in Table 9. Not only does this result in improved loading efficiency, but it is also due to significant leakage from the liposomes in this formulation. This suggests that it also results in a low DL ratio (3.7%) at 6 hours.

[0316] [Example 14]

[0317] Compounds AKG-28 and AKG-28 were incorporated into liposomes with different DL ratios and various entrapment agents. G-38 Encapsulation Cholesterol with HSPC and 0.5 mol% PEG-DSG (relative to PhL) role, 0.15 mol% of lipid-labeled DiIC18(3)-DS, and Liposomal solution consisting of 0.5M ammonium sulfate (AS) or 1N TEA-SOS The solution was prepared according to the general protocol and adjusted to pH 4.95-5.17 (with the addition of buffer substances). (not included) and 300-1050 g / mol PhL (AKG-28) or 400-14 00g / mol PhL(AKG-38) as described in Example 8 , compounds AKG-28 and AKG-38 were loaded. When 0.5 M AS was used, The maximum drug loading for compounds AKG-28 and AKG-38 was 404- 424g / mol PhL and 818-842g / mol PhL, and 95 The loading efficiencies of over 302 g / mol PhL (quantitative loading) and 38% were achieved, respectively. The drug loading ranged from 7 to 764 g / mol PhL (95.5 to 96.7% loading). N When TEA-SOS is used, the most recent results for compounds AKG-28 and AKG-38 The high drug loadings were 315–328 g / mol PhL and 989 g / mol The maximum loading efficiencies were 250 g / mol PhL and 4 83.5% for drug loadings between 0.00 and 777 g / mol PhL (>97.2% loading) (Figures 4A and 4B).

[0318] AKG-38 is almost quantitatively charged at 400-800g AKG-38 / mol PhL. The drug-to-lipid ratio obtained was 250–1000 g AKG-28 / mol It remains flat for AKG-28 across the range of PhL, which is comparable to AKG-3 This suggests that the maximum drug loading is lower for AKG-28 than for 8. The higher efficacy previously shown for tuberculosis-like It should be understood that this makes it effective for treating infections.

[0319] [Example 15]

[0320] Compound AK in liposomes using various phospholipid compositions, PEGylation degrees, and entrapment agents G-28 and AKG-38 encapsulation Phospholipids (PhL) with 0.5 M AS or 1 N TEA-SOS as a scavenger ) and cholesterol (3:2 molar ratio), PEG-DSG, and DiIC18 (3 Liposomes composed of )-DS (0.15 mol% of PhL) were prepared according to the general protocol. The formulation was prepared according to the protocol and selected to optimize drug loading and encapsulation efficiency (EE). Compounds AKG-28 and AKG-38 were loaded at the specified DL ratio (added buffer (In the absence of any substance.) The results are shown in Tables 10 and 11 below.

[0321] JPEG2026035569000089.jpg80170

[0322] JPEG2026035569000090.jpg80170

[0323] In this example, AKG-28 was added at a concentration of 230 to 275 g AKG-28 / mol PhL. HS using 0.5M AS or 1N TEA-SOS as a scavenger at high drug loading It was shown that PC could be efficiently loaded into liposomes. However, formulations containing sphingomyelin as the neutral phospholipid for this compound are relatively It showed relatively low loading, with a maximum of only about 110 g AKG-28 / mol PhL. Ta.

[0324] The compound AKG-38 was added at a dose of 600 g AKG-38 / mol PhL. In this case, use 0.5M AS or 1N TEA-SOS and use 525-600g / mo l, or 735g / mol when added at 800g AKG-38 / mol PhL The loading of compound AKG-38 was significantly higher than that of AKG-38. It was less sensitive to the presence of sphingomyelin than G-28.

[0325] [Example 16]

[0326] into liposomes using high PEGylation and 0.5M ammonium sulfate as a trapping agent Encapsulation of compounds AKG-16, AKG-28, AKG-29, and AKG-38 Enter HSPC and cholesterol (3%) using 0.5 M ammonium sulfate as a capture agent :2 molar ratio), PEG-DSG (5 mol%), and DiIC18(3)-DS (0 Liposomes composed of 0.15 mol%) were prepared according to a general protocol and Compounds were prepared with DLO ratios selected to optimize product loading and encapsulation efficiency (EE). Filled with AKG-16, AKG-28, AKG-29, or AKG-38 (added (In the absence of any buffering substance present). The results are shown in Table 13 below.

[0327] JPEG2026035569000091.jpg55170

[0328] This data is consistent with all dimethylaminoethyl substituents at the 2-position of the tetrazole ring. The compound was efficiently loaded into the liposomes to over 80%, but the aminoethyl group was not present at the same position. AKG-29 with a benzoyl substituent showed an efficiency of 14.5% and a yield of 43.6 g AKG-29 / This indicates that only a small amount of drug was loaded into the liposomes at a final drug loading of 100 mol PhL. This is despite the presence of titratable amines in all compounds tested. The benzophenone ring has a substituted ammonium group (e.g., N,N-dimethylaminoethyl group). Compounds with this structure are more efficient than those with a primary amine (aminoethyl group) at the same position. This demonstrates that drug loading is possible.

[0329] [Example 17]

[0330] Blood persistence and in vivo status of liposomes of Examples 15 and 16 in mice Encapsulation stability of Tests were performed on male CD-1 mice as described in the general protocol above. did.

[0331] JPEG2026035569000092.jpg103170

[0332] Data are from liposome batch ID 128, all with 0.5M AS as a scavenger. The drugs in 132, 142, 144, and 145 showed low DL ratios at 5 min and AK The DL at 6 hours was particularly notable for G-38 and AKG-16 loaded liposomes. Almost immediate encapsulation occurs upon contact with blood, as indicated by a further decrease in the ratio. The results showed that 25-60% of the drug was lost. or 5 mol% PEG-DSG and 40 mol% cholesterol, 0.5 M AS The formulation (as a scavenger) showed a % of initial DL ratio of >80% at both 5 min and 6 h. If the formulation used 1N TEA-SOS (liposome batch ID 129, 13 0, 133-135).

[0333] [Example 18]

[0334] AKG in PEGylated liposomes with varying phospholipid to cholesterol ratios Preparation and filling of AKG-28 and AKG-38 5 mol% PEG-DSG or PEG-DSPE (relative to PhL), 0.15 m 0.5 M ammonium sulfate as a capture agent. Liposomes containing 1N triethanolamine (AS) or 1N triethanolamine-sulfonyl ester (TEA-SOS) were prepared according to the general protocol. The solution was prepared according to the procedure described in Example 8 at a pH of 5.07 to 5.82 (without adding any buffer substance). Compounds AKG-28 and AKG-38 were loaded as described.

[0335] Liposomal preparations with 0.5 M AS as a scavenger for rapid drug release upon contact with blood. In an attempt to stabilize the gel (as described in Example 17), DSPC (generally , which are known to result in stable liposomes with higher drug leakage compared to HSPCs) Liposomes using decreasing proportions of cholesterol (Chol) were prepared and DL 0 AKG-28 at 250g / mol PhL or AK at 600g / mol PhL Contrary to expectations, the change from 40 mol% to 10 mol% of cholesterol The reduction in cholesterol content to steroids was observed for both AKG-28 and AKG-38. This resulted in a dramatic decrease in encapsulation efficiency. Lower cholesterol also reduced aggregation. Liposomes were destabilized by 30 mol% cholesterol (PhL-cholesterol). The molar ratio of 1N TEA-SOS and 5 mol% PEG-DSG was 70:30. Liposomes containing AKG-28 prepared using PEG-DSPE or A Contains 30 mol% cholesterol and 5 mol% PEG-DSPE of KG-38 Similar to the formulation, irreversible aggregation occurred during drug loading, but the AK of 30 mol% cholesterol The 5 mol% PEG-DSG formulation of G-38 was 77.1% or 462.4 g / mol It showed a reduced loading efficiency of PhL.

[0336] JPEG2026035569000093.jpg114170

[0337] In contrast, HSPC-prepared celluloses contain more than 40 mol% cholesterol, up to Liposomes containing 65 mol% cholesterol (the maximum value tested) exhibited over 87% AKG-28 (DL0) showed excellent encapsulation efficiency of 250g / mol PhL ) and AKG-38 (DL0 500g / mol PhL), PEG-lipid (P EG-DSG and PEG-DSPE), and scavenger (AS or TEA-SOS) No liposome aggregation was observed (Table 16).

[0338] Furthermore, both the 0.5M AS and 1N TEA-SOS formulations were found to be effective in this class. Evaluate the ability of the optimized formulation to fill the current standard of care, linezolid, derived from Tedizolid was introduced into liposomes via a membrane-spanning gradient according to the general protocol in Example 6. In both cases for linezolid, the drug was not sufficiently soluble in water to perform a distribution support fill. The capsule encapsulation efficiency was less than 5%, which is comparable to that of AKG-28 and AKG-38. Their liposomal formulations stably encapsulate the drug when compared to linezolid. This shows a dramatic improvement in ability.

[0339] The Z-average size of liposomes (x z ) and polydispersity index (PDI) were measured by Malvern Zetasizer Pro (Malvern Panalytical) at 173° The measurement angle was determined by the dynamic light scattering (DLS) cumulant method.

[0340] JPEG2026035569000094.jpg255169JPEG2026035569000095.jpg207170

[0341] [Example 19]

[0342] AKG-28 or AKG-38 in the presence of plasma and varying ratios of phospholipids to cholesterol In vitro burst release of pegylated liposomes containing terols. 5 mol% PEG-DSPE or PEG-DSG and varying ratios of HSPC vs. C AKG-28 and AKG-38 containing hol (50-65 mol% Chol) The in vitro stability of the liposomal formulation was evaluated using mouse CD-1 or human pooled Plasma (lithium-heparin stabilized from Innovative Research) Plasma was thawed and, if necessary, diluted to 1N. The pH was adjusted to 7.4 with HCl, and the solution was filtered through a glass microfiber filter (GF / C), 1 μ m polyethersulfone (PES), and through a 0.22 μm PES filter. Plasma (80 μl) was added to a 0.5 ml Eppendorf tube containing liposomes. The mixture was then mixed with the somatic drug formulation (20 μl). The mixture was then incubated at 37° C. for 20 minutes. The mixture (0.1 mL) was added to 2 mL of Sepharose gel and then placed in cold water. Chromatography was performed without delay on a CL-4B column in Hepes-buffered saline ( The liposomal drug was eluted at pH 7.0, and 0.25 mL of the liposomal drug was collected in the void volume fraction. The drug and DiI(3)-DS lipid label were then analyzed by HPLC as described in Example 7. The % remaining encapsulated drug was calculated according to the following formula: (A d / A I / (A d,0 / A I,0 ) * 100 = % encapsulated drug remaining (In the formula, A d is that of the drug peak, and A I is the area of ​​the lipid-labeled peak, and A d, is the area of ​​the drug peak before incubation with plasma, and A I,0 is Inc. (This is the lipid-labeled peak before incubation) was determined using

[0343] The results are shown in Figures 5A, 5B, 5C and 5D. For liposomes (Figure 5A), a burst release phenomenon (DL) was observed, indicating drug release from liposomes. A rapid decrease in the ratio of cholesterol to cholesterol in human plasma was observed for a formulation containing 40 mol% cholesterol. However, this was not observed in formulations containing 45 mol% or more cholesterol. For AKG-38-encapsulated liposomes (Fig. 5B), the burst release phenomenon was For formulations containing 40 mol% and 45 mol% cholesterol, It was observed in both mouse and rat plasma, but not at cholesterol levels above 50 mol%. It wasn't done.

[0344] [Example 20]

[0345] AKG-38 and 5 mol% P containing 40 or 55 mol% cholesterol In vitro plasma release and in vivo pharmacokinetics of EGTA-lipid liposomes status The three liposome formulations of Example 18 using 0.5M AS scavenger were incubated for 5 minutes and 6 hours. The percent injected dose (%ID) of liposomal lipid remaining in the blood was measured in both Drug release from liposomes is measured by determining the drug to lipid ratio (DL), see Example 7. The drug was evaluated in a two-time point pharmacokinetic study in female CD-1 mice as described. PEG-DSG or 5 mol% PEG-DSPE, 55 mol% Chol Liposomes containing DMSO retained more than 95% of their pre-injection D at 5 minutes and more than 85% at 6 hours. / L, but the PEG-DSG formulation containing 40 mol% Chol showed The DL ratio was dramatically reduced both during and after the treatment with α-glucan in the presence of plasma in vitro. This finding was consistent with the drug leakage data from the PEGylated liposomal drug approved for clinical use, such as xorubicin and nanoliposomal irinotecan Some very stable liposomal drugs contain cholesterol at a ratio of about 50 mol%. This finding contrasts with previous experience with drug-loaded liposomal formulations, as (For example, the Doxil (C) Drug Information Package Insert, updated in August 2019) , and Drummond, DC, et al. (2006). "Developm ent of a highly active nanoliposomal iri notecan using a novel intraliposomal sta bilization strategy”, Cancer Res.66(6):32 71-3277).

[0346] JPEG2026035569000096.jpg95170

[0347] [Example 21]

[0348] AKG-3, AKG-16, AKG-22, AKG-28, AKG-29, AKG-30 Mitochondrial protein synthesis by AKG-38, AKG-39, and AKG-40 Inhibition of MPS formation and selectivity for Mycobacterium tuberculosis (H37Rv) compared with MPS inhibition Colorimetric MitoBiogenesis from AbCam according to the manufacturer's instructions Use the ® Intracellular ELISA Kit (catalog #ab11021) to detect mitochondrial Inhibition of mitochondrial protein synthesis was determined. Important toxicities associated with oxazolidinone and other oxazolidinones, most notably to the eyes and peripheral nerves, have been reported. It was correlated with pulmonary hypertension and lactic acidosis (Renslo (2010) Exp ert Reve Anti Infect Ther 8(5)565-574;Fl anagan et al. (2015) Antimicrob Agents Che mother 59(1)178-185;Santini et al.(2017) Expert Opin Drug Saf 16(7)833-843). Complex IV Subunit I (COX-1) and complex encoded by mitochondrial DNA of Two, including a 70 kDa subunit (SDH-A) encoded by nuclear DNA of II The levels of mitochondrial proteins in H9C2 rat BDIX myocardial buds were simultaneously measured. The cell lines were used in these studies in a 384-well plate assay format. in DMEM medium containing 10% FBS and 1x glutamine at 37°C and 5% CO Cells were grown at 1,500 cells / well in 384-well plates at 47.5 μl / well. Each well contained nine 3-fold dilutions, starting at a high concentration of 200 μM. Compound concentrations and one replicate per condition were added to the cells in 2.5 μl and incubated at 37°C and The cells were incubated at 4°C for 5 days at 5% CO2. and linezolid control, as well as AKG-3, AKG-16, AKG-22, and AKG- 28, AKG-29, AKG-30, AKG-38, AKG-39, and AKG-40 It included:

[0349] MitoBiogenesis In-Cell Elisa was then performed according to the manufacturer's instructions. Follow the instructions (Abcame catalog #ab11021) and use a plate reader. During this period, alkaline phosphatase (AP) was measured in a dynamic model for 15 minutes (20 seconds to 1 minute intervals). The plate was then developed for detection of SDH-1A at 405 nm, and HRP was added for 15 minutes (20 seconds). The system was developed for detection of COX-I at 500 nm in kinetic mode (~1 min intervals). COX-1 and SDH-A signals were measured against the concentration of each compound. The values ​​were plotted as a ratio of 0.01 to 0.1, and IC50 values ​​were calculated for each of the nine investigated compounds and two controls. I put it out.

[0350] MPS IC50 in μg / ml was calculated using drug-sensitive H The MPS selectivity index (SI-M) was calculated by dividing by the MIC for the 37Rv M. tuberculosis strain. The two compounds tested, AKG-28 and AKG-29, were was more than 10-fold higher than that determined for tedizolid. Both of these compounds had SI-MPS greater than 20 times higher than that of It contained a primary amino group at the R2 position of the oxazolidinone ring. Mitochondrial protein Due to its high potency (MIC<0.1) and high selectivity against Mycobacterium tuberculosis compared to synthetic Therefore, AKG-28 has been shown to be useful for encapsulation in liposomes and for the treatment of tuberculosis or other mycobacterial diseases. are excellent candidates for treatment of

[0351] JPEG2026035569000097.jpg108170

[0352] [Example 22]

[0353] Scale-up preparation of liposomal AKG-28 Lot 275 Lot 267. The general procedure of Example 6 was followed. HSPC (Lipoid AG) 4. 95g (6.30mmol), cholesterol (Dishman, High Purit y) 2.98 g (7.71 mmol), and PEG-DSPE (Lipoid AG) 850mg(0.315mmol)(HSPC:Chol:PEG-DSPE 45:5 5:2.25 molar ratio) was mixed with 9 ml of absolute ethanol (Sigma, E-7023). The mixture was combined and heated in a 68°C bath with stirring until all the lipids were dissolved. 3.3 g of 0.5 M aqueous ammonium sulfate (0.2 μm filtered) was preheated in a 68°C bath. The resulting suspension was heated to 100°C and poured into the hot lipid ethanol solution with stirring. Lipex 100ml sample was heated in a 68°C bath for 20 minutes with stirring and circulating water at 68°C. A Mobarel liposome extrusion device (Northern Lipids, Inc.) was used. Two 47 mm 100 nm pore diameter and one 200 nm pore diameter polycarbonate tubes were used. Through a stack of rack-etched membranes (Whatman Nucleopore) The resulting extruded liposomes were stored in a refrigerator for 8 hours. (2-8°C) overnight, and then filtered under positive pressure with a 0.2 μm polyethersulfone (PES) filter. The remaining conductivity was less than 200 μS / cm (5.2 volumes of replacement). The ion exchange rate was then increased by 100 kJ / s, until the ion exchange rate was reduced to 143 μs / cm (after 143 μs / cm). Use a Hollow Fiber Cartridge (Spectrum Laboratories) On the KrosFlo TFF system, TFF buffer exchange into endotoxin-free water resulted in The scavenger (ammonium sulfate) outside the liposomes was removed. The phospholipid concentration was determined by the phosphomolybdenum blue method to be 57.4 mM. It was.

[0354] 720 ml in the form of a 20 mg / ml aqueous stock solution (adjusted to pH 5.03 with NaOH) g of AKG-28 (as dihydrochloride salt) was mixed with the liposome suspension after TFF and In the presence of 250 mg / ml dextrose and 6 mg / ml AKG-28 A loading mixture was formed with a drug to phospholipid (DL) ratio of 1000 mg / mol. The mixture was then rapidly heated to 60-63°C by external heating and placed on a 65°C bath with stirring. After 20 minutes of incubation, the mixture was placed in ice water for 10 minutes. The mixture was cooled rapidly to below 0.1 MN and held at this temperature for approximately 10 minutes. After adjusting to aCl, a polysulfone hollow fiber cartridge with a molecular weight cut-off of 500 kD was used. The drug-loaded liposomes were purified by TFF using a ridge. The liposomes were purified by diafiltration. The AKG-28 was pre-concentrated to approximately 12 mg / ml by filtration and then mixed over a total of 8 volume exchanges. Contains 0.144M NaCl made with endotoxin-free water (HBS-7 buffer) Liposomes by TFF exchange in 10 mM HEPES-Na buffer, pH 7.0 The proportion of unencapsulated drug before purification was determined by 305 nm filtration in the diafiltrate before concentration. The spectrophotometric estimation was about 0.9% (corresponding to a packing efficiency of 99.1%). The concentrated and purified liposomes were then sterile filtered through a 0.2 μm sterilizing filter. The particles were passed through the tubes and analyzed for particle size by DLS and for drug and phospholipid concentrations by spectroscopy. This procedure was repeated three more times (Lots 269, 271, and 272). 3) The obtained liposomes had the characteristics shown in Table 19.

[0355] JPEG2026035569000098.jpg56170

[0356] These lots were mixed to produce liposomes with a particle size of Xz 113.7nm and a PDI of 0. Lot 275 was obtained with 0417 having 12.0 mg / ml AKG-28.

[0357] [Example 23]

[0358] Scale-up preparation of liposomal AKG-38 Lot 276 Lot 268. The protocol of Example 22 was used with the following differences: an equal volume of 1N HCl The drug was dissolved in 20 mg / ml AKG-38 (as free base), pH 5.08 Stock AKG-38 (as free base) by adjusting the volume to obtain An aqueous solution was prepared. The fill mixture contained 1300 mg of AKG-38. was prepared at a DL ratio of 8 mg / ml AKG-38 and 450 g / mol phospholipids, The liposomes after loading were diluted to approximately 22 mg / ml. The percentage of unencapsulated drug before purification was determined by measuring the concentration at 305 nm in the diafiltrate before concentration. Spectrophotometrically estimated to be approximately 3.2% (corresponding to a packing efficiency of 96.8%) This process was repeated three more times (Lots 270, 272, and 273). 4) The resulting liposomes had the characteristics shown in Table 20.

[0359] JPEG2026035569000099.jpg56170

[0360] These lots were mixed to produce liposomes with a particle size of Xz 113.1 nm and a PDI of 0. Lot 276 was obtained with 22.3 mg / ml AKG-38 of 0454.

[0361] [Example 24]

[0362] Preparation of "empty liposomes" lot 277 2 mmol of HSPC, 2.444 mmol of cholesterol and 0.1 mmol of PEG-DSPE (HSPC:Chol:PEG-DSPE 45:55:2.25) The liposomes were dissolved in ethanol and formed into a liposome suspension. The only difference was that the non-exchangeable cation sulfate, 0.13M sodium sulfate, was used instead of ammonium sulfate. The extruded material was extruded through a polycarbonate membrane as described in Example 22. The liposomes were purified from extraliposomal sodium sulfate and subjected to a total of 10-fold volume exchange. TFF using polysulfone hollow fiber cartridges with MWCO 500 kDa The purified liposomes were then buffer exchanged into HBS-7 buffer. The phospholipid had a particle size of Xz 113.7 nm and a PDI of 0.0612. The mixture was aseptically passed through a 0.2 μm sterile filter and diluted to 20 mM with sterile HBS-7. The phospholipids were prepared as follows.

[0363] [Example 25]

[0364] Liposomal AKG-38 Lot 279 The general procedure of Example 6 was followed. HSPC (Lipoid AG) 13.102 g (1 6.67mmol), cholesterol (Dishman, High purity) 7. 877 g (20.37 mmol), and PEG-DSPE (Lipoid AG)2. 250g(0.833mmol)(HSPC:Chol:PEG-DSPE 45:55 : 2.25 molar ratio) was mixed with 25 ml of absolute ethanol (Sigma, E-7023). The mixture was combined and heated in a 68°C bath with stirring until all the lipids were dissolved. 59.1 g (250 ml) of 0.5 M aqueous ammonium sulfate (0.2 μm filtered) The mixture was preheated on a 70°C bath and poured into the hot lipid ethanol solution while stirring. The resulting suspension was stirred in a 70°C bath for at least 20 minutes and then divided into four portions. Lipex 100ml Thermobarrel heated with 70℃ water circulating in the area. Two 4-well plates were extruded using a liposome extrusion device (Northern Lipids, Inc.). 7mm polycarbonate track etch with 100nm hole diameter and one 200nm hole diameter 280ps through a stack of membranes (Whatman Nucleopore) These partially extruded liposome fractions were mixed (Xz 1 29.7 nm), extruded together five more times through the same membrane stack. Liposomes with a size of Xz 115.9 nm and a PDI of 0.0212 were obtained. The film was kept in a refrigerator (2-8°C) overnight and then coated with 0.2 μm polyethersulfone under positive pressure. The phospholipid concentration was 60.22 ± 0.34 mM. The residual conductivity was found to be 180 μS / cm after 5.1 times the volume of the exchange. Polysulfone hollow fiber cartridges (S) with a MW cutoff of 500 KDa were used until KrosFlo TFF system using the Spectrum Laboratories On the other hand, TFF buffer exchange into endotoxin-free water allowed the extraliposomal entrapment agent (e.g., ammonium sulfate) to be removed. The phospholipid concentration in the liposome suspension after TFF was determined by the phosphomolybdenum (PMO) method. It was determined by the Denbleu method to be 54.97±0.32 mM.

[0365] AKG-38 (free base) was mixed with 0.95 equivalents of 1N HCl and diluted with endotoxin-free water. The solution was diluted to 0.05 ml and made up to a 20 mg / ml aqueous stock solution (pH 5.16). The filtrate containing 3958 mg of drug was passed through a 0.2 μm filter and used as the post-TFF lyophilizer. Mix with the liposome suspension and use 44.5 mg / ml dextrose, 10 mM NaCl , and 450 g / mol in the presence of 8 mg / ml AKG-38 concentration, pH 5.54 The drug and phospholipid (DL) ratio was adjusted to form a loading mixture. The mixture was heated to 61°C and then stirred in a 65°C bath for an additional 22 minutes. The mixture was then transferred to an ice-water bath and stirred for 7 minutes to bring the temperature to 1 The temperature was lowered to 0°C and kept in the ice-water bath for an additional 8 minutes. The mixture was then removed from the ice bath and allowed to reach ambient temperature. After adjusting to 0.1M NaCl by adding 3M NaCl stock The drug-loaded liposomes (pH 6.53) were prepared using polysulfone with a molecular weight cut-off of 500 kD. Liposomes were purified by TFF using hollow fiber cartridges. AKG-38 was pre-concentrated to approximately 22 mg / ml by HCl and diluted with HBS over a total of 8 volume exchanges. The drug was purified from the extraliposomes by TFF exchange in HCl-7 buffer. The liposomes were aseptically passed through a 0.2 μm PES high-flow sterilizing filter and analyzed by DLS. The particles were analyzed for particle size by HPLC and for drug and phospholipid concentrations by spectroscopy. The liposomes had the following characteristics: AKG-38 21.1±0.19 mg / ml , DL ratio 454±4.7g / mol phospholipid, Xz 116.4nm, PDI 0.02 31. The yield of the formulated drug was 3834 mg (96.9%).

[0366] [Example 26]

[0367] Liposomal AKG-28 Lot 281 The general procedure of Example 6 was followed. 45:55:2 with 0.5M ammonium sulfate The extrusion was composed of HSPC, cholesterol, and PEG-DSPE at a molar ratio of 0.25. The extruded liposomes were prepared as described in Example 25. The remaining conductivity was 150 with a MW cutoff of 500 KDa until the concentration drops to 4.1 μS / cm (4.1 volume exchange). Polyethersulfone hollow fiber cartridge (Spectrum Laboratories) TFF buffer in endotoxin-free water on a KrosFlo TFF system using The sequestering agent (ammonium sulfate) was removed from the liposomes by exchange. The phospholipid concentration in the phosphosome suspension was determined by the phosphomolybdenum blue method. The concentration was 55.4 mM.

[0368] 969 in the form of a 20 mg / ml aqueous stock solution (adjusted to pH 5.24 with NaOH). 5 mg of AKG-28 (as dihydrochloride salt) was mixed with the liposome suspension after TFF. In the presence of 44.5 mg / ml dextrose and AKG-28 at a concentration of 6 mg / ml A loading mixture of 250 g / mol of drug to phospholipid (DL) was formed. The mixture was heated to 65.4°C in 2.5 minutes with external heating while stirring, and then placed in a 65°C bath. After 20 minutes of incubation, the mixture was transferred to ice water. The mixture was cooled to 9.3°C in 2.75 minutes and then kept in ice water for about 10 minutes. After reaching ambient temperature, the solution was adjusted to 0.1 M NaCl; pH 6.43. 133.4 g The packed mixture was then loaded onto a polysulfone hollow fiber cartridge with a molecular weight cut-off of 500 KD. The liposomes were purified by TFF using a diafiltration system. ml of AKG-28 pre-concentrated in HBS-7 buffer over a total of 8.1 volume exchanges The drug outside the liposomes was purified by TFF exchange at 1000 rpm. The percentage of unencapsulated drug before purification was In the diafiltrate before concentration, the concentration was estimated spectrophotometrically at 302 nm to be approximately 0.7% (99 The concentrated and purified liposomes were then Aseptically passed through a 0.2 μm sterile filter and analyzed for particle size by DLS and The drug and phospholipid concentrations were analyzed by spectroscopy. The liposomes were characterized as follows: AKG-28 13.26±0.21mg / ml, DL ratio 258.2±3.7 g / mol phospholipid, Xz 117.3nm, PDI 0.0421.

[0369] [Example 27]

[0370] Liposomal AKG-38 Lot 285 The general procedure of Example 6 was followed. 45:55:2 with 0.5M ammonium sulfate The extrusion was composed of HSPC, cholesterol, and PEG-DSPE at a molar ratio of 0.25. The extruded liposomes were prepared essentially as described in Example 25. MW cutoff 500KD until the temperature drops to 138μS / cm (5.6 times the exchange rate) Polyethersulfone hollow fiber cartridges (Spectrum Laboratories) with a TFF to endotoxin-free water on a KrosFlo TFF system using tethers The extraliposomal sequestering agent (ammonium sulfate) was removed by FF buffer exchange. The phospholipid concentration in the liposome suspension after F was determined by the phosphomolybdenum blue method. The result was 53.1 mM.

[0371] AKG-38 (free base) was mixed with 0.95 equivalents of 1N HCl and diluted with endotoxin-free water. The solution was then diluted to give a 19.9 mg / ml aqueous stock solution (pH 5.13). After TFF, the filtrate containing 1400 mg of drug was passed through a 0.2 μm filter. and mixed with the liposome suspension of 44.5 mg / ml dextrose, 10 mM Na Cl, and 450 g / mo in the presence of 8 mg / ml AKG-38 concentration, pH 5.58 The loading mixture was formed at a drug to phospholipid (DL) ratio of 1.001. With constant stirring, the mixture was heated to 63°C by external heating and placed in a 65°C bath for a total of 21 minutes. The incubation was continued with stirring. The mixture was then transferred to an ice-water bath and incubated for 3 minutes. With stirring, the temperature was reduced to 10.3°C and kept in the ice-water bath for an additional 7 minutes. Remove from bath, allow to reach ambient temperature, and add 3M NaCl stock After adjusting to 0.1 M NaCl, the drug-loaded liposomes (pH 6.70) were diluted with 1000 sachets of ... Purification was performed by TFF using a 500 kD polysulfone hollow fiber cartridge. The liposomes were pre-concentrated by diafiltration to approximately 22 mg / ml of AKG-38. Liposomes were exfoliated by TFF exchange in HBS-7 buffer over a total of 7.7 volume exchanges. The concentrated and purified liposomes contained 23.1 mg / ml of AKG-3. The drug concentration was adjusted to 20 mg / ml with HBS-7 buffer, and the liposomes were The mixture was aseptically passed through a 0.2 μm PES high-flow sterile filter and analyzed by DLS. The particles were analyzed for particle size and for drug and phospholipid concentrations by spectroscopy. The formulation had the following characteristics: AKG-38 20.35±0.26 mg / ml, D L ratio 437.8±6.5g / mol phospholipid, Xz 121.1nm, PDI 0.02 00. The yield of the formulated drug was 1355 mg (96.8%).

[0372] [Example 28]

[0373] Liposomal AKG-28 Lot 286 Extruded liposomes containing 0.5M ammonium sulfate and no extraliposomal entrapment agent Liposomes (HSPC:Chol:PEG-DSPE 45:55:2.25 molar ratio) was obtained as described in Example 27. A 20 mg / ml aqueous stock solution (with NaOH) 600 mg of AKG-28 (as dihydrochloride salt) in the form of TFF (adjusted to pH 5.18) was added to the The resulting liposome suspension was mixed with 44.5 mg / ml dextrose and 6 mg / In the presence of AKG-28 at a concentration of 1 ml, a drug to phospholipid (DL) ratio of 250 g / mol was used. The filling mixture was formed. The mixture was placed in a 65°C water bath with stirring and heated to 60°C for 4.5 minutes. The incubation was continued with stirring for a total of 20 minutes, and the mixture was then placed on ice. The mixture was cooled to 10.0°C in 2 minutes and kept in an ice-water bath for about 10 minutes. After reaching ambient temperature, the mixture was adjusted to pH 6.23 with 0.1 M NaCl. The packed mixture was placed in a polysulfone hollow fiber cartridge with a molecular weight cut-off of 500 kD. The liposomes were purified by TFF using 1000 mg / ml of 10 ... 1 pre-concentrated in AKG-28 and HBS-7 buffer over a total of 8.3 volume exchanges The liposomes were purified from the extraliposomal drug by TFF exchange. , aseptically passed through a 0.2 μm sterile filter (expelled with HBS-7 buffer), Analyzed for particle size by DLS and for drug and phospholipid concentrations by spectroscopy The liposomes had the following characteristics: AKG-28 12.05±0.13m g / ml, DL ratio 239.4g / mol phospholipid, Xz 120.1nm, PDI 0. 0294. The yield of formulated drug was 555.5 mg (92.6%).

[0374] [Example 29]

[0375] Liposomal AKG-38 Lot 292 Lot 288. The general procedure of Example 6 was followed. HSPC (Lipoid AG) 9. 17g (11.67mmol), cholesterol (Dishman, High purity ty) 5.51 g (14.26 mmol), and PEG-DSPE (Lipoid A 1.575 g (0.583 mmol) of Sigma-Aldrich (Sigma-Aldrich) in 17.5 ml of absolute ethanol a, E-7023) and stir in a 69-70°C bath until all lipids are dissolved. In a separate container, 181.4 g (175 ml) of 0.5 M aqueous ammonium sulfate was added. The lipids (0.2 μm filtered) were preheated on a 70°C bath and added to the hot lipid ethanol solution. The resulting suspension was stirred in a 70°C bath for at least 20 minutes. The mixture was divided into three parts. Each part was heated in a Lip ex 100ml Thermobarrel liposome extrusion device (Northern Lipids, Inc.) to fabricate two 47mm 100nm pore diameter and one 200nm pore diameter Polycarbonate track-etched membrane (Whatman Nucleopore These partially extruded resins were extruded five times at 280 psi through a stack of The liposome portions were mixed (Xz 126.7 nm) and further passed through the same membrane stack. When extruded four times, the size was Xz 119.2 nm and the PDI was 0.0385. The liposomes were kept in a refrigerator (2-8°C) overnight and then heated under positive pressure. The solution was filtered through a 0.2 μm polyethersulfone (PES) filter. The residual conductivity was found to be 59.08 ± 0.44 mM. After exchange, the antibody was purified by a polysaccharide with a MW cutoff of 500 KDa until the antibody level dropped to 152 μS / cm. A fluorocarbon hollow fiber cartridge (Spectrum Laboratories) was used. TFF buffer exchange into endotoxin-free water on a KrosFlo TFF system The scavenger (ammonium sulfate) outside the liposomes was removed. The phospholipid concentration of the sucrose was determined by the phosphomolybdenum blue method and was found to be 57.76±0 The concentration was 0.53mM.

[0376] AKG-38 (free base) was mixed with 0.95 equivalents of 1N HCl and diluted with endotoxin-free water. The solution was then diluted to give a 19.7 mg / ml aqueous stock solution (pH 5.11). After TFF, the filtrate containing 3509 mg of drug was filtered through a 0.2 μm filter. and mixed with the liposome suspension of 44.5 mg / ml dextrose, 10 mM Na Cl, and 450 g / mo in the presence of 8 mg / ml AKG-38 concentration, pH 5.50 The loading mixture was formed at a drug to phospholipid (DL) ratio of 1.0. The mixture was stirred constantly for 5 minutes. The mixture was heated to 61.6°C with external heating while stirring and then placed in a 65°C bath for an additional 20 minutes. The incubation was continued with stirring. The mixture was then transferred to an ice-water bath and incubated for 7 minutes. With stirring, the temperature was lowered to 10°C and kept in the ice-water bath for an additional 8 minutes. , allowed to reach ambient temperature, and diluted to 0.1 M NaCl by adding 3 M NaCl stock. After preparation in aCl, the drug-loaded liposomes were then injected into a polysulfone-containing PBS containing 500 kDa of molecular weight cut-off. Liposomes were purified by TFF using a PEG hollow fiber cartridge. Therefore, AKG-38 was pre-concentrated to approximately 22 mg / ml, and a total of 7.8 volumes were exchanged. The drug was purified from outside the liposomes by TFF exchange in HBS-7 buffer. The prepared liposomes were aseptically passed through a 0.2 μm PES high-flow sterilizing filter. Analyzed for particle size by DLS and for drug and phospholipid concentrations by spectroscopy The liposomes had the following characteristics: AKG-38 22.47±0.38m g / ml, DL ratio 441.6g / mol phospholipids, Xz 121.3nm, PDI 0. 0465. The yield of formulated drug was 3375 mg (96.2%).

[0377] Lot 289. The process for Ls-288 was repeated using 1506 mg of AKG-38. Repeat (similarly prepared 20.0 mg / ml aqueous stock solution, pH 5.15). The solution was mixed with the same extruded liposome suspension after TFF to give a 44.5 mg / ml dextrose, 10 mM NaCl, and 8 mg / ml AKG-38 concentration, pH A loading mixture was formed in the presence of 5.53 at a drug to phospholipid (DL) ratio of 450 g / ml. The mixture was heated to 64.3°C by external heating with constant stirring over a period of 2 minutes. The mixture was then incubated in a 65°C bath for an additional 20 minutes with stirring. The mixture was transferred to an ice-water bath and stirred for 2.75 minutes, allowing the temperature to drop to 9.6°C. After removal from the ice bath, the filling mixture was allowed to reach ambient temperature and 3M NaCl stock; pH 6.54 adjusted to 0.1M NaCl. The loaded liposomes were then loaded onto a polysulfone hollow fiber cartridge with a molecular weight cut-off of 500 KD. Liposomes were purified by TFF using diafiltration to an A of approximately 22 mg / ml. TF pre-concentrated in KG-38 and in HBS-7 buffer over a total of 8.1 volume exchanges The drug outside the liposomes was purified by F exchange. The concentrated and purified liposomes were then diluted with 0. The particles were aseptically passed through a 2 μm PES high-flow sterile filter and analyzed for particle size by DLS. The liposomes were analyzed for drug and phospholipid concentrations by HPLC and spectroscopy. The characteristics were: AKG-38 22.84±0.41mg / ml, DL ratio 452.7 g / mol phospholipid, Xz 120.3nm, PDI 0.0522. Formulated drug The yield was 1407 mg (93.4%).

[0378] Lot 290. The general procedure of Example 6 was followed. HSPC (Lipoid AG) 7. 86g (10.00mmol), cholesterol (Dishman, High puri ty) 4.73 g (12.22 mmol), and PEG-DSPE (Lipoid A G)1.35g(0.50mmol)(HSPC:Chol:PEG-DSPE 45: 55:2.25 molar ratio) in 15 ml of absolute ethanol (Sigma, E-7023) The mixture was mixed with the above and heated in a bath at 69-70°C with stirring until all the lipids were dissolved. In a container, 155.5 g (150 ml) of 0.5 M aqueous ammonium sulfate (0.2 μm filter) The filtered lipids were preheated in a 70°C bath and poured into the hot lipid ethanol solution while stirring. The resulting suspension was stirred in a 70°C bath for at least 20 minutes and then divided into two portions. Each part was placed in a 100ml container of Lipex heated with circulating water at 70°C. A Mobarel liposome extrusion device (Northern Lipids, Inc.) was used. Two 47mm 100nm pore size and one 200nm pore size polycarbonate tubes were used. Through a stack of rack-etched membranes (Whatman Nucleopore) These partially extruded liposome fractions were mixed and extruded four times at 280 psi. (Xz 131.5 nm) and push together four more times through the same membrane stack. When extracted, liposomes with a size of Xz 122.7 nm and a PDI of 0.0215 were obtained. The liposomes were kept in a refrigerator (2-8°C) overnight and then filtered under positive pressure with a 0.2 μm polyethylene terephthalate (PEP). The phospholipid concentration was 58.99 ± 0.01. The residual conductivity was found to be 146 μS after a 5.5-fold exchange. Polysulfone hollow fiber cartridge with a MW cutoff of 500 KDa was used until the molecular weight was reduced to 1000 kJ / cm. KrosFlo using a cartridge (Spectrum Laboratories) On the TFF system, extraliposomal entrapment was achieved by exchanging the TFF buffer into endotoxin-free water. The agent (ammonium sulfate) was removed. The phospholipid concentration in the liposome suspension after TFF was It was 56.94±0.41mM as determined by the phosphomolybdenum blue method. .

[0379] AKG-38 (free base) was mixed with 0.95 equivalents of 1N HCl and diluted with endotoxin-free water. The solution was diluted to 0.05 mL to obtain a 20 mg / ml aqueous stock solution (pH 5.15). The filtrate containing 2315 mg of drug was passed through a 0.2 μm filter and the resulting solution was used as the post-TFF lyophilizer. Mix with the liposome suspension and use 44.5 mg / ml dextrose, 10 mM NaCl , and in the presence of AKG-38 concentration of 8.02 mg / ml, pH 5.52, 450 g / m The loading mixture was formed at a drug to phospholipid (DL) ratio of 0.5% over 3.25 minutes. With constant stirring, the mixture was heated to 64.4°C by external heating, then placed in a 65°C bath. Incubation was continued for 17 minutes with stirring. The mixture was then transferred to an ice-water bath. , stirring, reducing the temperature to below 10°C, and holding in an ice-water bath for a total of 10 minutes; Allow to reach ambient temperature and 0.1M NaCl using 3M NaCl stock; pH 6.63 The drug-loaded liposomes were prepared using polysulfone hollow capsules with a molecular weight cut-off of 500 KD. Liposomes were purified by TFF using a fiber cartridge. Pre-concentrated to approximately 22 mg / ml AKG-38 and diluted with HBS over a total of 8.0 volume exchanges The drug was purified from the extraliposomes by TFF exchange in HCl-7 buffer. The liposomes were aseptically passed through a 0.2 μm PES high-flow sterilizing filter and analyzed by DLS. The particles were analyzed for particle size by HPLC and for drug and phospholipid concentrations by spectroscopy. The liposomes had the following characteristics: AKG-38 22.07±0.23 mg / m l, DL ratio 441.6g / mol phospholipid, Xz 120.4nm, PDI 0.039 5. The yield of the formulated drug was 2141 mg (92.5%).

[0380] Lot 292. Lots 288 (150.3g), 289 (61.2g), and 290 (19.5 g) were mixed to give 22.5 mg / ml of liposomally formulated AKG-38 This yielded 278.4 g of lot 292. All liposome formulations were stored at 2-8°C.

[0381] [Example 30]

[0382] Preparation of liposomal AKG-28 Lot 235 The general procedure of Example 6 was followed. HSPC (Lipoid AG) 940 mg (1.2 0mmol), Cholesterol (Dishman, High purity) 568mg (1.47mmol), PEG-DSPE (Lipoid AG) 163mg (0.06 mmol), and 0.0018 mmol of lipophilic fluorescent label DiIC 18 (3)-DS( AAT Bioquest, USA)(HSPC:Chol:PEG-DSPE:DiI C 18 (3)-DS 45:55:2.25:0.0675 molar ratio relative to HSPC 0.15 mol% DiI3-DS) in 2 ml of absolute ethanol (Sigma, E-7 023) and heated in a 68°C bath with stirring until all lipids were dissolved. In a vessel, 20 ml of 0.5 M aqueous ammonium sulfate solution (0.2 μm filtered) The mixture was preheated in a 68°C bath and poured into the hot lipid ethanol solution while stirring. The resulting suspension was stirred for 20 minutes in a 68°C bath, and then heated with Lipex while circulating water at 68°C. 100ml Thermobarrel liposome extrusion device (Northern Lipids, In c.) Two 47 mm 100 nm pore diameter and one 200 nm pore diameter polystyrene were used. Carbonate track-etched membrane (Whatman Nucleopore) The resulting extruded liposomes were extruded through the stack eight times at 300 psi. The tubes were kept in a refrigerator (2-8°C) overnight and then heated under positive pressure with 0.2 μm polyethersulfone (PE S) filter. The remaining conductivity was 60 μS / cm (10-fold exchange). Polysulfone hollow fiber cartridges with a MW cutoff of 500 KDa were used until the KrosFlo TFF using PEG (Spectrum Laboratories) In the system, the TFF buffer exchange into endotoxin-free water removes the extraliposomal entrapment agent (sulfur). The phospholipid concentration in the liposome suspension after TFF was measured using the phospholipid It was determined by the molybdenum blue method to be 37.56±0.62 mM.

[0383] 50 mg in the form of a 20 mg / ml aqueous stock solution (adjusted to pH 4.99 with NaOH) of AKG-28 (as dihydrochloride salt) was mixed with the liposome suspension after TFF. In the presence of 250 mg / ml dextrose and 3 mg / ml AKG-28 The loading mixture was formed at a drug to phospholipid (DL) ratio of g / mol. The mixture (pH 5. 53) was incubated in a 65°C bath with stirring for 20 minutes, then rapidly cooled in ice water. The solution was then placed in an ice-water bath for approximately 10 minutes. After reaching ambient temperature, the solution was diluted with 3M NaCl stock solution. After adjusting to 0.1 M NaCl, the pH was 5.80. by TFF using polysulfone hollow fiber cartridges with a volume cutoff of 500KD. Liposomes were pre-concentrated to approximately 5 mg / ml AKG-28 by diafiltration. Liposomes by TFF exchange in HBS-7 buffer over a total of 10 volume exchanges The purified liposomes were purified from foreign substances. The purified liposomes were then injected into a small syringe-operated 500KD TFF using hollow fiber cartridges (MicroKros, Spectrum) The concentrated and purified liposomes were then filtered through a 0.2 μm sterile filter. The sample was passed aseptically and analyzed for particle size by DLS and for drug and linker activity by spectroscopy. Liposomes were analyzed for lipid concentration. The liposomes had the following characteristics: AKG-28 8.22±0.16mg / ml, DL ratio 257.3±10.3g / mol phospholipid, Xz 118.2 nm, PDI 0.0188. The yield of the formulated drug was 41.4 mg ( 82.8%).

[0384] [Example 31]

[0385] Preparation of liposomal AKG-38 Lot 236 Extruded liposomes after TFF containing 0.5M ammonium sulfate in Example 30 AKG-38 (free base) was mixed with 0.95 equivalents of 1N HCl to remove endotoxin. Compensated with free water to give a 20 mg / ml aqueous stock solution (pH 5.11). The solution was passed through a 0.2 μm filter, and an amount of the filtrate containing 70 mg of drug was used for TFF. Mixed with the subsequent liposome suspension (Example 30) and 140 mg / ml dextrose and In the presence of AKG-38 at a concentration of 3 mg / ml, 450 g / mol of drug and phospholipid (D The mixture was stirred in a 65°C bath for 20 minutes to form a filling mixture. The mixture was incubated, rapidly cooled in ice water, and kept in an ice water bath for approximately 10 minutes. After adjusting to 0.1M NaCl with a 3M NaCl stock solution, the pH was 6.33. Drug-loaded liposomes were then loaded onto polysulfone hollow fiber capsules with a molecular weight cut-off of 500 kD. Liposomes were purified by TFF using a diafiltration cartridge. Pre-concentrated to 100 μg / ml AKG-38 and HBS-7 buffer over a total of approximately 10 volume exchanges The liposomes were purified from the drug outside the liposomes by TFF exchange in a liquid. , a syringe-operated miniature 500KD hollow fiber cartridge (MicroKros, The concentrated, purified protein was further concentrated two-fold by TFF using a Spectrum™ HPLC system. The liposomes were aseptically passed through a 0.2 μm sterile filter and analyzed for particle size by DLS. The liposomes were analyzed for drug and phospholipid concentrations by HPLC and spectroscopy. The following characteristics were observed: AKG-38 9.04±0.16mg / ml, DL ratio 463. 9±19.8 g / mol phospholipid, Xz 119.3 nm, PDI 0.0267. Formulation The yield of the purified drug was 56 mg (80%).

[0386] [Example 32]

[0387] In vitro liposomal encapsulation of Lots 235 and 236 in the presence of plasma Possession of contaminated drugs Retention of Encapsulated Drug in Liposomes in the Presence of 80% Mouse Human Plasma at 37°C was determined as described in Example 19 herein. The incubation time was 20 minutes. there were.

[0388] JPEG2026035569000100.jpg33170

[0389] These liposomes were stable to burst release of drug upon contact with plasma.

[0390] [Example 33]

[0391] Liposomal AKG-28 and AKG-38 Lots 231, 232 (HSPC: cholesterol) Molar ratio of PEG-DSPE: 45:55:2.25, scavenger: 0.5M ammonium sulfate Preparation of ammonium The general procedure of Example 6 was followed. HSPC (Lipoid AG) 4.255 g (5. 41mmol), cholesterol (Dishman, High purity) 2.56 g (6.62 mmol), and PEG-DSPE (Lipoid AG) 729 mg ( 0.27 mmol) (HSPC:Chol:PEG-DSPE 45:55:2.25 (molar ratio) was mixed with 9 ml of absolute ethanol (Sigma, E-7023) to remove all fats. Heat with stirring in a 70°C bath until the solids are dissolved. Aqueous ammonium sulfate solution (0.2 μm filtered) was preheated on a 70°C bath and heated The resulting suspension was poured into the lipid ethanol solution while stirring. Stir for 1 minute, then heat the Lipex 100ml thermobarrel with 70℃ water in circulation. Two 4-well plates were extruded using a liposome extrusion device (Northern Lipids, Inc.). Polycarbonate track etch with 7 mm of 100 nm hole diameter and one 200 nm hole diameter 260ps through a stack of membranes (Whatman Nucleopore) The resulting extruded liposomes were stored in a refrigerator (2-8°C) overnight. Retain and filter under positive pressure through a 0.2 μm polyethersulfone (PES) filter The MW cut was continued until the residual conductivity decreased to 60 μS / cm (10 times the amount of replacement). Polysulfone hollow fiber cartridges (Spectrum Laser) with a capacity of 500 kDa Endotoxin-free water on the KrosFlo TFF system using The extraliposomal sequestering agent (ammonium sulfate) was removed by TFF buffer exchange to The phospholipid concentration in the liposome suspension after TFF was measured by the phosphomolybdenum blue method. The concentration was determined to be 46.97±0.80 mM.

[0392] Lot 231. of a 20 mg / ml aqueous stock solution (adjusted to pH 5.02 with NaOH) 350 mg of AKG-28 (as dihydrochloride salt) was added to the liposome suspension after TFF. Mixed to obtain 137.6 mg / ml dextrose and 2.53 mg / ml A In the presence of KG-28, a loading mixture of 250 g / mol drug to phospholipid (DL) was formed. The mixture (pH 5.60) was incubated in a 65°C bath with stirring for 20 minutes. The mixture was cooled rapidly in ice water and kept in an ice water bath for approximately 10 minutes. After adjusting to 0.1 M NaCl with NaCl stock solution, the pH was 5.68. Drug-loaded liposomes were loaded into polysulfone hollow fiber cartridges with a molecular weight cut-off of 500 KD. Liposomes were purified by TFF using a diafiltration system. Liposomes were purified to approximately 9 mg / ml by diafiltration. AKG-28 pre-concentrated in HBS-7 buffer over a total of 10.9 volume exchanges The purified liposomes were purified from the extraliposomal drug by TFF exchange in 1000 mL of PBS. Continuing TFF diafiltration without fluid delivery further increased the drug concentration to approximately 12 mg / ml. The concentrated and purified liposomes were aseptically passed through a 0.2 μm sterile filter. The particle size was measured by DLS and the drug and phospholipid concentrations by spectroscopy. The liposomes had the following characteristics: AKG-28 11.42±0. 09mg / ml, DL ratio 247.7±7.1g / mol phospholipid, Xz 116.5nm , PDI 0.0511. The yield of the formulated drug was 322.7 mg (92.2%). there were.

[0393] Lot 232. AKG-38 (free base) was mixed with 0.95 equivalents of 1N HCl. Make up with toxin-free water to obtain a 20 mg / ml aqueous stock solution (pH 5.09). The solution was passed through a 0.2 μm filter, and an amount of filtrate containing 580 mg of drug was , and mixed with the post-TFF liposome suspension of this example to obtain a 137.6 mg / ml dextromethorphan 500 g / m in the presence of 2.53 mg / ml AKG-38 concentration, pH 5.72 The loading mixture was formed with a drug to phospholipid (DL) ratio of 0.5. The mixture was stirred in a 65°C bath. Incubate for 20 minutes with stirring, then rapidly cool in ice water and keep in an ice-water bath for approximately 10 minutes. Allow to reach ambient temperature and adjust to 0.1M NaCl with 3M NaCl stock solution. The pH was 6.40 after the procedure. The drug-loaded liposomes were prepared using a 500KD molecular weight cutoff. Liposomes were purified by TFF using a polysulfone hollow fiber cartridge. The AKG-38 was pre-concentrated by diafiltration to approximately 12 mg / ml, and a total of 8.5 volumes were exchanged. The drug was purified from extraliposomal drugs by TFF exchange in HBS-7 buffer over the entire exchange period. The purified liposomes were purified by continuing nTFF diafiltration without buffer supply. The concentrated and purified liposomes were then passed through a 0.2 μm sterile filter. Passed aseptically and analyzed for particle size by DLS and for drug and phospholipid spectroscopy. Liposomes were analyzed for their quality and solubility. The liposomes had the following characteristics: AKG-38 16 .03±0.07mg / ml, DL ratio 487.3±13.9g / mol phospholipid, Xz 120.0 nm, PDI 0.0069. The yield of the formulated drug was 538.9 mg ( 92.9%).

[0394] [Example 34]

[0395] Liposomal AKG-28 Lot 233 (HSPC:Cholesterol:PEG-DSG 6 Preparation of sucrose (scavenger 1N triethylammonium octasulfate, 0:40:3 molar ratio) The general procedure of Example 6 was followed. HSPC (Lipoid AG) 1.88 g (2.4 mmol), cholesterol (Dishman, High purity) 619 mg ( 1.6 mmol), and PEG-DSG (Sunbright GS-020, NOF 312 mg (0.12 mmol) of benzophenone-3, benzodiazepine (B1), benzodiazepine (B2), benzodiazepine (B3), benzodiazepine (B4), benzodiazepine (B5), benzodiazepine (B6), benzodiazepine (B7), benzodiazepine (B8), benzodiazepine (B9), benzodiazepine (B10), benzodiazepine (B11), benzodiazepine ( The mixture was heated in a 67°C bath with stirring until all the lipids were dissolved. 5 g (30 ml) of 1 N aqueous triethylammonium octasulfate sucrose solution (0.2 μm Filtered, pH 6.20, see Example 8) was preheated on a 65°C bath and added to hot lipid ethanol. The resulting suspension was stirred in a 65°C bath for 5 minutes and then poured into the ethanol solution. Lipex 100ml thermo-barrel liposome extrusion device heated with circulating water at ℃ (Northern Lipids, Inc.) was used to prepare four 47mm 100n one 200 nm pore size polycarbonate track-etched membrane (W Extruded three times at 400 psi through a stack of (Hatman Nucleopore) The extruded liposomes were stored overnight in a refrigerator (2-8°C) and then heated under positive pressure. The retentate was filtered through a 0.2 μm polyethersulfone (PES) filter. MV cutoff 500k until conductivity drops to 21 μS / cm (14.5 times exchange volume) Polysulfone hollow fiber cartridges (Spectrum Laboratories) with Da TFF to endotoxin-free water on the KrosFlo TFF system using ries Buffer exchange resulted in the extrusion of 9.2 g of the entrapped agent (TEA-SOS) from outside the liposomes. The liposomes were purified. The phospholipid concentration in the liposome suspension after TFF was measured using the phosphomolybdenum (PMP) method. It was determined by Buden blue spectrophotometry to be 31.32±0.85 mM.

[0396] 140 ml in the form of a 20 mg / ml aqueous stock solution (adjusted to pH 5.02 with NaOH) g of AKG-28 (as dihydrochloride salt) was mixed with the liposome suspension after TFF. In the presence of 6.1 mg / ml dextrose and 2.52 mg / ml AKG-28 Under this condition, a loading mixture was formed with a drug to phospholipid (DL) ratio of 250 g / mol. The mixture (pH 5.43) was incubated in a 65°C bath with stirring for 20 minutes, and then rapidly soaked in ice water. The mixture was cooled rapidly and kept in an ice-water bath for approximately 10 minutes. After adjusting to 0.1 M NaCl with the drug-loaded liposomes, the pH was 5.80. The RT-PCR was performed using a polysulfone hollow fiber cartridge with a molecular weight cut-off of 500 KD. The liposomes were purified by diafiltration to approximately 9 mg / ml of AKG-28. and subjected to TFF exchange in HBS-7 buffer over a total of 10.9 volume exchanges. The purified liposomes were purified from the drug outside the liposomes without supplying a buffer. Continuing TFF diafiltration further concentrated the drug to approximately 12 mg / ml. The purified liposomes were aseptically passed through a 0.2 μm sterile filter (HBS- 7 buffer), particle size was analyzed by DLS, and drug and Liposomes were analyzed for phospholipid concentration and had the following characteristics: AKG-28 10.64±0.20mg / ml, DL ratio 246.8±11.7g / mol phospholipids; Xz 116.3nm, PDI 0.0022. The yield of the formulated drug was 118.2 mg (84.4%).

[0397] [Example 35] Preparation of liposomal AKG-38 Lot 234 (HSPC:cholesterol:PEG- DSPE 45:55:2.25 molar ratio, scavenger 1N triethylammonium sucrose sulfate) The general procedure of Example 6 was followed. HSPC (Lipoid AG) 3.30 g (4. 20 mmol), cholesterol (Dishman, high purity) 1.985 g (5.13 m mol), and PEG-DSPE (Lipoid AG) 567 mg (0.21 mmol) l) (HSPC:cholesterol:PEG-DSPE 45:55:2.25 molar ratio) Mix in 7 ml of absolute ethanol (Sigma, E-7023) until all lipids are dissolved. The mixture was heated with stirring in a 70°C bath until the mixture was dissolved. 10 ml of tetraethyl ammonium sucrose octasulfate (TEA-SOS) aqueous solution (0.2 micron filtered) The suspension was preheated in a 70°C bath and poured into the hot lipid ethanol solution while stirring. The mixture was stirred in a 70°C bath for 10 minutes, and then 100 ml of Lipex solution heated with circulating 70°C water was added. Using a Mobarrel liposome extruder (Northern Lipids, Inc.), Two 47mm 100nm pore size and one 200nm pore size polycarbonate trabeculae 260p through a stack of black-etched membranes (Whatman Nucleopore) The extruded liposomes were stored overnight in a refrigerator (2-8°C). The solution was filtered under positive pressure through a 0.2 μm polyethersulfone (PES) filter. The lipid concentration was 54.6 mM. 11.33 g of extruded liposomes were diluted with 100% ethanol at a MW cutoff of 1000 mg / ml. 500 KDa (Spectrum Laboratories) polysulfone hollow fiber For endotoxin-free water on the KrosFlo TFF system using cartridges The TFF buffer exchange was used to purify the extraliposomal entrapped agent (TEA-SOS) and remove the residual The conductivity was maintained until it decreased to 64 μS / cm (13.8 volume exchanges). The phospholipid concentration in the gel suspension was 28.67 ± 100 μg / ml by blue phosphorus molybdate spectrophotometry. It was measured to be 1.01 mM.

[0398] AKG-38 (free base) was mixed with 0.95 equivalents of 1N HCl to obtain an endotoxin-free The solution was diluted with water to give a 20 mg / ml aqueous stock solution (pH 5.09). The amount of filtrate containing 250 mg of drug was used as the post-TFF liposome of this example. Combined with the dextrose suspension, 116.4 mg / ml dextrose and 2.53 mg / ml AK G-38 concentration, at a drug-phospholipid (DL) ratio of 500 g / mol in the presence of pH 5.24 The filling mixture was formed. The mixture was incubated in a 65°C bath for 20 minutes with stirring. The mixture was cooled rapidly in ice water and kept in an ice-water bath for approximately 10 minutes. After adjusting to 0.1 M NaCl with NaCl stock solution, the pH was 6.60. The loaded liposomes were placed in a polysulfone hollow fiber cartridge with a molecular weight cut-off of 500 kD. The liposomes were purified by TFF using approximately 10 mg / ml of AKG-38. Preconcentrated by precipitation filtration and TFF exchange into HBS-7 buffer for a total of 8.0 volume exchange. The purified liposomes were purified from any extraliposomal drug by conversion. By continuing TFF diafiltration without feeding, the concentration was further increased by approximately 2-fold. The somes were aseptically passed through a 0.2 μm sterile filter and analyzed for particle size by DLS and spectrophotometric analysis. The drug and phospholipid concentrations were analyzed by fluorometric method. The liposomes were AKG-38 15.7 1±0.33mg / ml, DL ratio 518.6±18.4g / mol phospholipid, liposome The particle size was 114.3 nm and the PDI was 0.0284. 235.7 mg (94.3%).

[0399] [Example 36] Loading efficiency of AKG-28 and AKG-38 into liposomes and liposomes in plasma Effect of osmotic agent concentration on drug retention by The general protocol of Example 6 was followed. 0.5M ammonium sulfate and 45:5 HPSC, cholesterol, PEG-DSPE and Extruded liposomes containing the lipid composition of DiIC18(3)-DS (fluorescent lipid label) The liposomes were prepared as described in Example 30. TFF exchange with endotoxin-free water (Hyclone) allows for syringe-operated microclasts. Polysulfone hollow fiber cartridge (MWCO 500KDa, Spectrum L aboratories) (13.8 volume exchange, residual conductivity 88 μS / cm, phospholipid concentration The liposomes were purified from extraliposomal ammonium sulfate using various concentrations of ammonium sulfate (55.4 mM). In the presence of osmotic agent (dextrose), the drug concentration was 2.22 mg / ml and the DL ratio was 250 g / mol phospholipid (AKG-28) or 450 g / mol phospholipid (AKG-38) The liposomes were loaded with the drug (20 mg / ml aqueous solution as described in Examples 30 and 31). stock) in a water bath at 65°C for 20 minutes. AKG-28 or AKG-38 was incubated with the Sepharose CL-4B was loaded and eluted by size exclusion using HBS-7 buffer. Unencapsulated drug is removed by dechromatography, and drug and phospholipid are The loading (encapsulation) efficiency was measured from the results of the quality analysis. The penetrant concentration was measured in absolute terms. , and for the 0.5 M ammonium sulfate solution used to form the liposomes Expressed as a percentage of the 168 mg / ml dextrose concentration determined to be isoosmotic Contrary to expectations from the general consensus in the liposome field, the drug exhibited low permeability. Under osmotic conditions (i.e., the osmotic pressure of the solution outside the liposomes is lower than that of the solution of the entrapped agent inside the liposomes), even in the complete absence of added osmotic balance agent (dextrose). were effectively loaded into the disclosed liposomes (with encapsulation efficiencies exceeding 85%, with the majority exceeding 90%). Furthermore, in vitro plasma release assays described in Example 19 When exposed to plasma under the conditions of (a), the drug encapsulation in liposomes loaded with the lowest concentration of osmotic agent was Cell encapsulation was at least as good as that of the filled cells with nearly perfect (86.3%) osmotic equilibrium. It was stable.

[0400] The results showed that liposomes were 55 mol% Chol, 45 mol% PC, and 5 mol% HSPC. PEG-DSPE at 0.5 MAS scavenger, and AKG-28 (Table 22) and AKG-3 8 (Table 23) at 250 or 500 g / mol P with efficiencies >85%, most >90%. Filled with 0.05% dextrose and under hypoosmolar conditions up to zero percent showed that liposomes loaded with α-glucan efficiently retained the drug in the presence of plasma.

[0401] JPEG2026035569000101.jpg88170

[0402] JPEG2026035569000102.jpg90170

[0403] [Example 37] All forms (encapsulation + release) of Ls-AKG28 and Ls-AKG38 in rats Single-dose pharmacokinetic study of the drug This study was conducted in rats using single-dose Ls-AKG28 and Ls-AKG38. This study was conducted to evaluate the PK of AKG28 and AKG38 in male Sprag In Iowa-Dawley rats, 20, 40, and 80 mg of liposomes per kg of body weight were administered. AKG-38 (Ls-AKG38), or 10, 20, or 40 mg per kg of body weight This was performed using IV administration of liposomal AKG-28 (Ls-AKG28). G28 (Lot 275) and Ls-AKG38 (Lot 276) were used in Example 2, respectively. 2 and 23. For comparison, 0.5% methylcellulose was used in the formulation. The rats were given 50 mg / kg of ionized linezolid by oral gavage at a concentration of 20 mg / mL, pH 3. Acidified to 4°C (Sigma M0430). For plasma drug determination, 0.5 ml of blood was Lithium heparin tubes are tested for 5 minutes, 15 minutes, 1 hour, 3 hours, 6 hours, 24 hours, and 48 hours. The samples were collected at 1 hour, 2 hours, and 72 hours. The samples were centrifuged and the resulting plasma was separated and double-clarified. Transfer to polypropylene tubes, freeze immediately on dry ice, and store at -80°C until analysis. The plasma concentrations in rats were determined by HPLC. Non-compartmental PK analysis was performed using lin (version 7.0). For G28 and Ls-AKG38, the plasma maximum Concentration (C max ), the maximum plasma concentration divided by the dose (C max / dose), C max Time (T max ), the last measured concentration (C last ), the time of the last measured concentration (T last ), area under the plasma concentration versus time curve from 0 h to the last time point (AUC 0-l ast ) and 0h to infinity (AUC 0-inf ), AUC 0-last Divide by the dose The value (AUC 0-last / dose), clearance (CL), volume of distribution (Vd), and The elimination half-life (T1 / 2) was estimated for linezolid using this PK software. The same parameters were obtained except for the apparent clearance (CL / F) and apparent volume of distribution (Vd / F). The same PK parameters were estimated.

[0404] Ls-AKG2 at 10, 20, and 40 mg / kg single intravenous doses (IV × 1) The plasma concentration versus time profiles of all drugs after administration of 8 are shown in Figure 7. Summary of plasma PK parameters for all drugs after administration of 0, 20, and 40 mg / kg IV × 1 is shown in Table 24.

[0405] At all doses, the plasma concentration versus time profile of Ls-AKG28 was observed from 5 minutes to 72 hours. It was possible to extract. C max Based on the results of / dose and AUC / dose, Ls-AKG28 The plasma PK of 10, 20, and 40 mg / kg was linear (dose proportional). At this dose, the plasma clearance (CL) of Ls-AKG38 (approximately 2.59 mL / h / kg) was This was greater than that of Ls-AKG28 (approximately 1.67 mL / h / kg). 40 mg / kg), the Vd of Ls-AKG28 was greater than that of Ls-AKG38.

[0406] JPEG2026035569000103.jpg134170

[0407] Total drugs after administration of Ls-AKG38 at 20, 40, and 80 mg / kg IV × 1 The plasma concentration versus time profile of is shown in FIG.

[0408] Plasma PK of all drugs after single IV administration of Ls-AKG38 at 20, 40, and 80 mg / kg The parameters are outlined in Table 25.

[0409] At all doses, the plasma concentration versus time profile of Ls-AKG38 was observed from 5 minutes to 72 hours. It was possible to extract. C max Based on the results of / dose and AUC / dose, Ls-AKG3 The plasma PK of 8 was linear (dose-proportional) after administration of 20, 40, and 80 mg / kg. At all doses, the plasma clearance (CL) of Ls-AKG38 (approximately 2.59 mL / h / kg) was ) was greater than that of Ls-AKG28 (approximately 1.67 mL / h / kg). At the same doses (40 mg / kg or 40 mg / kg), the Vd of Ls-AKG28 was greater than that of Ls-AKG38.

[0410] JPEG2026035569000104.jpg135170

[0411] After administration of Ls-AKG28 at 10, 20, and 40 mg / kg IV × 1, and Ls-AKG38 at 20, 40, and 80 mg / kg IV × 1 The plasma concentration versus time profiles of the drugs are shown in Figures 7 and 8, respectively. Single IV Dose Study The plasma concentration versus time profiles of Ls-AKG28 and Ls-AKG38 were measured at all doses. The plasma PK of Ls-AKG28 was 1 The blood levels of Ls-AKG38 were linear (dose proportional) after administration of 0, 20, and 40 mg / kg. Plasma PK was linear (dose proportional) after administration of 20, 40, and 80 mg / kg. The plasma clearance (CL) of Ls-AKG38 (approximately 2.59 mL / h / kg) is The results were higher than those of s-AKG28 (approximately 1.67 mL / h / kg). 0 mg / kg), the Vd of Ls-AKG28 was greater than that of Ls-AKG38. The total plasma PK exposure of Ls-AKG28 and Ls-AKG38 in g / kg was The plasma PK (using AUC from 0 to end) was approximately 73-fold and 110-fold higher.

[0412] The plasma AUC and drug duration in circulation were significantly higher for both liposomal formulations compared with linezolid. The AUC / dose values ​​showed a linear dose-dependence. The PK of both liposomal formulations was significantly higher for Ls-AKG28 and Ls-AKG38, respectively. is similar to

[0413] [Example 38] Whole Form (Encapsulated) After Repeated IV Dosing in Sprague-Dawley Rats Plasma pharmacokinetics (PK) of Ls-AKG28 and Ls-AKG38 (+ release) This study was conducted in rats using increasing doses of Ls-AKG28 and Ls-AKG38 for a total of 8 This study was conducted to evaluate the PK of AKG28 and AKG38 administered once weekly for 12 weeks. The study was conducted in Sprague-Dawley rats by IV administration. (Lot 275) and Ls-AKG38 (Lot 276) were used in Examples 22 and 23. Each was prepared as described, and plasma concentrations in rats were determined by HPLC. AKG28 at 10, 20, and 40 mg / kg IV x 1 on days 1, 15, 2 The plasma concentration versus time profiles of all drugs after administration on Days 9 and 43 are shown in Table 26. 9A, 9B, and 9C. / kg IV × total drug after administration on days 1, 15, 29, and 43 A summary of plasma PK parameters is shown in Table 26. All data in Figures 9A, 9B, and 9C The data was used to generate the PK parameter results in Table 26.

[0414] In single- and multiple-dose PK studies, the plasma kinetics of Ls-AKG28 were At 10 mg / kg, Ls-AKG28 increased plasma C max and AUC is first, The results were similar on days 15, 29, and 43. At 20 mg / kg of Ls-AKG28, blood Plasma C max and AUC increased on days 29 and 43. In 8, plasma C max and AUC increased after dosing on days 1-43.

[0415] On days 1, 15, 29, and 43, Ls-AKG38 was administered at 20, 40, and 60 mg / mL. The plasma concentration versus time profiles of all drugs after administration of 80 mg / kg IV × 1 are shown in Table 2. 7 and Figures 10A, 10B, and 10C. Total after administration of 80mg / kg intravenous injection on days 1, 15, 29, and 43 A summary of the plasma PK parameters of the drug is shown in Table 27. In single and multiple dose PK studies: The plasma kinetics of Ls-AKG38 was similar after the first dose. g of Ls-AKG38, plasma C max and AUC increased from day 1 to day 43. Blood clearance (ABC) of pegylated liposomes containing cytotoxic drug payloads Given the concerns about acceleration of It is expected that liposomes containing AKG-28 or AKG-38 will be effective in preventing chronic inflammation in mammals. It is suggested that the drug can be administered sexually.

[0416] JPEG2026035569000105.jpg137170

[0417] JPEG2026035569000106.jpg137170

[0418] [Example 39] Drug and liposomal analysis of Ls-AKG28 and Ls-AKG38 in CD-1 mice Pharmacokinetic studies of lipids This study aims to investigate the blood pharmacokinetic parameters of the drug and liposomal lipids in vivo in plasma. and liposomal formulations of AKG-28 and AKG-38. The study was designed to determine the stability of the product retention. The study was performed on male CD-1 (20–22 g) mice as described in the protocol (time (5 mice per time point). Ls-AKG28 (Lot 235) and Ls-AKG38 (Lot 236) were prepared as described in Examples 30 and 31, respectively. Lipo at a dose of 90 mg / kg (Ls-AKG28) or 90 mg / kg (Ls-AKG38) The thrombin was injected into the lateral tail vein at time 0, and blood was collected at 0.083, 1, 3, 6, 24, and 30 min post-injection. AKG-28, AKG-38, and fluorescent liposomal lipid labels (D iIC 18 The plasma concentration of (3)-DS) was determined by HPLC. Liposome Lot 23 Using 5 and 236 as standards, the plasma concentration of liposomal phospholipids was calculated from the fluorescence-labeled quantification. The tissue affinity of the non-encapsulated oxazolidinone drug was significantly higher than that of the liposome-encapsulated drug. It is expected that the ATP concentration will be several times higher than that of liposome capsules in rats (for example, Vd of 33.27-43.74 mL / kg for cell-encapsulated AKG-28 compared with non-encapsulated Supported by a Vd of 2,291.26 mL / kg of encapsulated oxazolidinone, linezolid (See Example 37 for an example), plasma drug concentrations were primarily attributable to liposome-associated drug. The plasma drug-liposomal lipid normalized to the original (pre-injection) DL value was The (DL) ratio was taken as a measure of drug retention by liposomes. K analysis is provided by Summit Research Services, PK Solutions For Ls-AKG28 and Ls-AKG38, This PK software can be used to determine the maximum plasma concentration (C max ), the maximum plasma concentration is divided by the dose. The value (C max / dose), Cmax Time (T max ), the last measured concentration (C l ast ), the time of the last measured concentration (T last ), the time from 0h to the last time point Area under the plasma concentration curve (AUC 0-last ) and 0h to infinity (AUC 0- inf ), AUC 0-last divided by the dose (AUC 0-last / dose), clear The lance (CL), volume of distribution (Vd), and elimination half-life were estimated.

[0419] of drugs after administration of Ls-AKG28 (Fig. 11A) and Ls-AKG38 (Fig. 11B) Plasma concentration versus time profiles are shown. Ls-AKG28 and Ls-AKG38 in plasma A summary of the plasma PK parameters of the drug is shown in Table 28, and a summary of the liposomal phospholipids is shown in Table 29. The dynamics of the DL ratio, which indicates the stability of drug encapsulation in vivo, are shown in Figures 11C and 11D. Shown in Table 30.

[0420] Ls-AKG28 has almost complete in vivo stability and was shown to be effective in mice after intravenous administration. With undetectable loss of drug up to 48 hours post-injection. The period is a monoexponential equation (R 2 = 0.822), the result is 866.3 hours. 8 has a fast drug release rate. The half-life of drug release from Ls-AKG38 follows a monoexponential equation (R 2 =0 Using the time constant (.950), the result is 22.9 hours.

[0421] JPEG2026035569000107.jpg143170

[0422] JPEG2026035569000108.jpg140170

[0423] JPEG2026035569000109.jpg75170

[0424] [Example 40] Ls-AKG28 and Ls-AKG38 drugs in mice after repeated administration of liposomes Pharmacokinetic studies of substances, existence of ABC effect The generation of anti-PEG antibodies is a phenomenon known as accelerated blood clearance. Faster clearing of liposomes containing EG-lipid conjugates (PEGylated liposomes) It has been shown to induce the ABC effect after repeated injections (Ishida et al. Journal of Controlled Release 105( 2005)305-317; Laverman et al.JPET 298(20 01)607-612). This study investigated the effects of Ls-AKG28 and Ls -To determine whether there is an ABC effect after repeated administration of various doses of AKG38 The liposomes were prepared in Examples 33 to 35, Lots 231, 232, 233 and 234. This study was carried out using male CD-1 mice as generally described in Example 7. Five mice were used in each group. Plasma concentrations of G-38 were determined by HPLC. Mice received the indicated doses and formulations. The injections were given once a week for a total of four times. The drug was measured in the blood at 6-hour time points after the first and fourth doses. None of the groups tested showed a significant accelerated clonogenic effect after the fourth injection (Figure 12). No clearance was observed (two-tailed t-test, all p values ​​>0.05). These liposomal oxazolidinones were shown to be effective without significantly negatively impacting drug exposure. , confirming that the drug can be administered chronically over multiple weekly cycles.

[0425] JPEG2026035569000110.jpg89170

[0426] This data shows that after four cycles of treatment, liposomal AKG-28 or liposomal AKG-28 of the present disclosure The blood clearance rate of the liposome-associated AKG-38 was significantly higher than that of the liposome-associated cytotoxic drug. In contrast to what has been previously reported for other PEGylated liposomes that do not This indicates that

[0427] [Example 41] Doses of liposomal AKG-28 and liposomal AKG-38 in CD-1 mice dependent tolerance The purpose of this study was to compare the efficacy of Ls-AKG28 and Ls-AKG28 injected as single agents at different doses in mice. The objective of this study was to evaluate the tolerability of s-AKG38. Mice were treated with Ls-AKG28 (50, 65, 90 or 100 mg / kg / dose) or Ls-AKG38 (50, 90, 120 or 200 mg / kg / dose) was administered once weekly for 4 The liposome formulation (Ls-AKG28 Lot 2) was administered intravenously (tail vein) for 2 weeks. 31 and Ls-AKG38 Lot 232) were prepared as previously described in Example 33. The control group received the same amount of HEPES-buffered saline (HBS, pH 7) once a week for 4 weeks. Body weights were measured three times per week during the study period, and data were expressed relative to the body weight measured on day 0. Presented as percentage of weight change.

[0428] At the end of the study (72 hours after the final treatment), animals were euthanized using CO2 inhalation. Fluid samples were collected by cardiac puncture and prefilled with EDTA for hematological analysis. Microtainer (Homology ADVIA 120 / 2120i Analyzer) and and transferred to microtainers prefilled with lithium heparin for plasma separation. Plasma was separated from the cellular fraction by centrifugation at 10,000 rpm for 5 min and used for biochemical analysis. The Cobas 6000 Analyzer was used. Tissue samples (liver, spleen, kidney, lung) The tissue (e.g., heart, small intestine, and column) was placed in a 50 ml tube prefilled with 10% buffered formalin. The tissue was collected in a tube and replaced with 70% ethanol after 24 hours. The tissue was embedded in paraffin and Sectioned and stained with hematoxylin and eosin (H&E) by a board-certified veterinarian. Histopathological examination was performed by a medical pathologist.

[0429] As shown in Figures 13A and 13B, compared with the saline control group, Ls-AK at doses up to 90 mg / kg for G28 and 20 mg / kg for Ls-AKG38 When treated for a total of 4 weeks at doses up to 0 mg / kg, Ls-AKG28 and Ls No significant effect on mouse body weight was observed for either α-AKG38 or α-AKG38.

[0430] Compared with the control group, high doses of Ls-AKG38 (90, 120 and 200 mg / kg )-treated mice showed significantly increased red blood cell counts and hematocrit compared to saline controls. There was no significant decrease in the IL-1 receptor agonist (Fig. 13C). No such effect was observed. At the highest dose of 90 mg / kg (Fig. 13C), Ls-AKG2 A significant decrease in platelet count was observed in mice treated with 8 compared with the control group. The reduction was still less than 25% compared to the saline control. Treatment with either Ls-AKG38 or Ls-AKG38 did not significantly increase white blood cell (WBC) counts or blood liver enzymes (A However, the effect of thiamin on the LT and AST levels was not significant.

[0431] Histopathological analysis showed that animals administered 50 and 65 mg / kg Ls-AKG28 No test article-related findings were observed in the subjects (Figure 13D). In the liver, spleen, and kidney of animals administered S-AKG28, macrophages ( Test article-related findings consisted of minimal vacuolization of the spleen (including Kupffer cells). Treatment with Ls-AKG38 at doses of 90 mg / kg and 120 mg / kg significantly reduced liver and spleen. In the liver, 50 and 90 mg / kg, mild to mild vacuolization and Kupffer cell hypertrophy, 50 and 120 mg / kg, moderate Kupffer cell vacuolization and hypertrophy, 90 mg / kg and 120 mg / kg Minimal multifocal aggregation of vacuolated macrophages was noted in g.

[0432] Treatment with the highest dose of Ls-AKG38 (200 mg / kg) resulted in increased activity in the liver and spleen. There was a slight increase in extramedullary hematopoiesis (EMH), minimal to mild multifocal mixed cell infiltrates, and liver associated with minimal individual hepatocellular necrosis in the liver and minimal focal hepatocellular necrosis. These microscopic findings were not considered to be related to the test article. This was due to the common background finding in this species. do.

[0433] Overall, both Ls-AKG28 and Ls-AKG38 monotherapy were and Ls-AKG38 were administered at 90 and 200 mg / kg liposomal drug, respectively. Even when injected at the highest evaluated dose, it was well tolerated in vivo in mice. did.

[0434] [Example 42] Ls-A in combination with BDQ / PMD or BDQ / PMD / MOX in mice In vivo tolerability of KG28 and Ls-AKG38 In this example, the in vivo tolerability of liposomal oxazolidinone was evaluated in a therapeutically relevant It was evaluated in combination with related anti-TB drugs: bedaquiline, pretomannide, and linezolid. (BDQ / PMD / LNZ or BPL), or bedaquiline, pretomanid, and mogamulizumab The three-drug regimen of xifloxacin (BDQ / PMD / MOXI or BPM) It has shown strong clinical activity in treating multidrug-resistant tuberculosis (Conradie et al., 2004). t al(2020)N Engl J Med 382(10)893-902 an d Tweed et al. (2019) Lancet Respir Med 7( 12)1048-1058), the BPL regimen has been associated with toxicity primarily related to the addition of linezolid. Limited by (Conradie et al. (2020) N Engl J Med 382(10)893-902). Here, two liposomal oxazolidinyl The safety and tolerability of Zinone, Ls-AKG28, and Ls-AKG38 were evaluated in the BPL register. Either substituting linezolid in the BPM regimen or adding it to the BPM regimen CD-1 mice (5 mice per group) were treated with Ls-AK G28 (Lot 231) or Ls-AKG38 (Lot 232) alone, or Bedaki The mice were treated with either BDQ or pretomanid in combination. 28 (Lot 231) and Ls-AKG38 (Lot 232) were described in Example 33. Furthermore, mice were treated with BDQ, PMD, and moxifloxacin (MOXI). ) and liposomal oxazolidinone triple combination.

[0435] Ls-AKG28 (50 mg / kg / dose) and Ls-AKG38 (90 mg / kg The BDQ, PMD, and M OXI combination (25 / 100 / 100 mg / kg / dose, respectively) once daily, 5 times a week, 4 As an additional control, mice were given 100 mg / kg / dose once daily by oral gavage. , given orally, 5 times a week for 4 weeks, BDQ / PMD / MOX or BDQ / PMD (25 / 100 mg / kg / dose, respectively) + linezolid (LNZ). Measurements, tissue collection and analysis were performed as described above in Example 41.

[0436] As shown in Figures 14A and 14B, the effect of Ls-AKG28 or Both Ls-AKG38 and BDQ / PMD(BP) or BDQ / PMD / MOX When co-treated in combination with (BPM), no significant effects were observed during the study. Both Ls-AKG28 and LsAKG38 are BDQ / PMD or BDQ / PMD / M The combination with OX was well tolerated and did not result in any significant changes in hematology or blood biochemistry in treated mice. chemistry (Fig. 14C).

[0437] Histopathological data (Fig. 14D) showed that Ls-AKG28 in combination with BDQ / PMD In the case of Ls-AKG28+BDQ / PMD / MO, no treatment-related changes were observed. The X combination was associated with minimal mixed cell and mononuclear cell infiltrates in the lungs and heart. Treatment with Ls-AKG38 as monotherapy caused minimal damage to the liver. Ls-AKG3 was associated with test article-related findings (inflammatory infiltrates and hepatocellular necrosis). Administration of 8+BDQ / PMD showed no treatment-related findings, whereas Ls-AKG38+BDQ The / PMD / MOX combination was associated with minimal mixed cell infiltrates in the lung. Animals treated with the D / LNZ combination had liver inflammatory infiltrates, minimal hepatocellular necrosis, and This was associated with treatment-related findings of vacuolated macrophage infiltration in the lungs. Both G28 (50 mg / kg / dose) and Ls-AKG38 (90 mg / kg / dose) When administered once weekly for 4 weeks, it is effective in combination with BDQ / PMD or BDQ / PMD / MOX. It was well tolerated in mice.

[0438] [Example 43] Ls-AKG28 and Ls-AKG3 in combination with BDQ / PMD in mice Effect of dose scheduling on tolerability of 8 In this study, Ls-AKG28 (50 mg / kg / dose) administered twice weekly or Ls -The in vivo tolerability of AKG38 (100 mg / kg / dose) administered once weekly in L s-AKG28 (100 mg / kg / dose) or Ls-AKG38 (200 mg / kg Liposomes were compared with those of Example 25 (Ls-AKG38, lot 279) and Both liposomal drugs were prepared according to Example 26 (Ls-AKG28, Lot 281). were administered alone or in combination with BDQ / PMD(BP) to female CD-1 mice (5 mice per group). BDQ / PMD (25 and 100 mg / kg / dose, respectively) were injected once daily. The mice were given HEPES-buffered saline (HBS) by oral gavage five times a week for four weeks. H7) was injected once a week for 4 weeks. Blood and tissue samples were collected and used in Examples 41 and 42. were analyzed as described.

[0439] Ls-AKG28 or Ls-AKG38 was administered twice weekly or once weekly at higher doses. Neither monotherapy nor combination therapy significantly affected the body weight (Figures 15A and 15B) or There was no effect on blood counts and biochemistry (Figure 15C).

[0440] Histopathological analysis of the recovered tissues (Figure 15D) showed that 50 mg / kg (2 qw) of L Two out of five mice given s-AKG28 developed microscopic cells consisting of macrophages and neutrophils. Interstitial mixed cell infiltrate, and 100 mg / kg (1qw) Ls-AKG28 One of five animals had a mild interstitial mixed cell infiltrate.

[0441] In the lungs of mice administered Ls-AKG28+BP at 50 mg / kg (1qw), macrophages (1 out of 5) or mixed (macrophage and neutrophil) inflammatory cells (5 Slight interstitial infiltration consisting of 100 mg / kg (1qw) Ls - Four out of five mice receiving AKG28+BP had minimal interstitial mixed cell infiltrates. Furthermore, Ls-AKG28+BP was administered at 100 mg / kg (1qw). Two of the five animals examined had minor multifocal foreign body granulomas associated with pale basophilic foreign bodies in the lungs. These microscopic findings were consistent with those observed in the Ls-AKG28+BP50mg / kg (2qw) In the liver of one of five animals receiving Because of the occurrence of small individual hepatocellular necrosis as a common background finding in this species, were not considered to be related to the test substance.

[0442] Similar microscopic findings associated with Ls-AKG38 treatment (alone or in combination) were observed with the test substance. There appears to be no association with the development of leukemia, and a slight increase in extramedullary hematopoiesis (EMH) in the liver and spleen. In addition, there was minimal to mild multifocal mixed cell infiltrate in the liver and very few individual hepatocytes. Hepatocellular necrosis, minimal focal hepatocellular necrosis in the lungs, minimal focal foreign body granuloma (faintly These findings included a small amount of pulmonary embolism (associated with a basic foreign body) and a minimal mixed cell infiltrate in the lung. are due to its subtle to mild nature, sporadic incidence, presence in saline control groups, and Not considered treatment-related due to occurrence as a common background finding in the species I couldn't.

[0443] Therefore, Ls-AKG28 and Ls-AKG38 (alone or with BDQ / PMD) combination) at doses of 50 mg / kg and 100 mg / kg twice weekly, or In mice treated weekly with doubling doses of 0 mg / kg and 200 mg / kg, It was well tolerated and had no effect on the body weight, hematology, or histopathology of treated animals. It didn't affect me.

[0444] [Example 44] In vivo tolerability of Ls-AKG28 and Ls-AKG38 in rats The purpose of this study was to evaluate the efficacy of Ls-AKG28 (Lot 275) and Ls-AKG3 in rats. The objective of this study was to determine the potential toxicity of Ls-AKG28 (Lot 276). 75) and Ls-AKG38 (Lot 276) were used as described in Examples 22 and 23, respectively. Male Sprague-Dawley rats were administered Ls-AKG28( 10, 20 or 40 mg / kg / dose) or LsAKG-38 (20, 40 or 8 The control group received a single dose of 0 mg / kg / dose via intravenous injection (tail vein) once a week for 8 weeks. The mice were injected with a single dose of HEPES-buffered saline (HBS, pH 7) once a week for 8 weeks. Before endpoint, animals were euthanized by exsanguination from the abdominal aorta after isoflurane anesthesia. Blood and tissue samples were collected and clinical pathological parameters were evaluated. The material was collected, stored in 10% neutral buffer, embedded in paraffin, sectioned, and then inserted into glass slides. The specimens were mounted on plates, stained with hematoxylin and eosin, and examined by a board-certified veterinary pathologist. Histopathology was evaluated by a human veterinarian. Hematological analysis was performed by the Homology ADVIA 120 / Blood biochemistry analysis was performed using a Cobas 600 analyzer. The test was performed using a 0 Analyzer.

[0445] Mortality and moribund checks, clinical observations, body weight, food intake, nerve conduction velocity (NCV) and Muscle action potential (MAP), functional observation battery (FOB) parameters and endpoints The study also evaluated the

[0446] Nerve conduction velocity (NCV) and muscle action potential (MAP) were measured at week 8. During the procedure, animals were anesthetized with isoflurane. The caudal nerve (NCV) runs along the central bone of the tail. This nerve is approximately 50% longer than any other nerve in the rat, and Susceptible to length-dependent distal axonopathy. NCV is measured over a distance of 50 mm. , nodal and transmembrane currents, the structure and mean cross-sectional diameter of responding axons, and associated The amplitude of the evoked response is sensitive to the integrity of the myelin sheath. The data reflect the activity and synchronization of the stimuli. The signals were recorded at the stimulating cathode (as determined visually) and 50 mm further distal to the stimulating cathode. The amplitude and onset latency were recorded, and the distance between the stimulating cathode and the active electrode was adjusted to reflect the initial depolarizing current. Velocity was calculated by dividing by the paired onset latency.

[0447] Digital nerve NCV measures the conduction velocity in the sensory digital nerves. The digital nerves support the dorsal surface of the hind paw. The nerve conduction velocity is determined by the nodal and transmembrane currents, the responsive axon, and the sciatic nerve. It is sensitive to the structure and average cross-sectional diameter of the cords, as well as the integrity of the associated myelin sheath. The data was collected using an active recording electrode located at the ankle posterior to the lateral malleolus and a stimulating electrode located at the base of the second toe of the hind foot. The amplitude and onset latency of the signal were recorded and the stimulation cathode and the activation potential were measured. Velocity was calculated by dividing the distance between the poles by the absolute onset latency of the initial depolarizing current.

[0448] Tibial motor conduction (onset latency) was measured in the intrinsic muscles of the rat hindpaw after stimulation of the motor fibers of the distal tibial nerve. The response characteristics of the hind paw are measured. The data are obtained by positioning the lateral dorsi muscles (equivalent to the extensor digitorum brevis muscle in humans) in the hind paw. Recordings were made with the active electrode positioned in the ankle joint and the stimulating cathode positioned proximal to the lateral malleolus. Nerve conduction velocity in axons is estimated from the onset latency of the induced compound muscle action potential (CMAP). The amplitude of the CMAP was measured at the peak of the response after supramaximal stimulation of the relevant nerve.

[0449] Ls-AKG28- and Ls-AKG38-related unplanned deaths, clinical observations, and effects on body weight (Fig. 16A and 16B), NCV and MAP (Table 34), FOB (Table 35), food intake, There was no effect on coagulation parameters, organ weights, or macroscopic findings (data not shown). In the liposome-treated group, the efficacy of Ls-AKG28 or Ls-AKG38 was investigated. The adjusted dose (based on the free drug efficacy against M. tuberculosis strain Erdmann in Example 2) was Despite a 16.5-fold increase compared to the control group, the tail and The largest decrease in nerve conductance velocity in the left or right digital nerve was less than 5%.

[0450] Administration of Ls-AKG28 and Ls-AKG38 at doses of 20 mg / kg or higher resulted in blood This resulted in a statistically significant reduction in platelet count (up to 20% difference compared to the control group). No additional effects were observed on hematological and blood chemistry parameters (Table 32, Table 33).

[0451] Male Sprague-Dawley rats were randomly assigned to receive 10 mg / kg / dose or more once a week. Administration of Ls-AKG28 by intravenous injection once a day for 8 weeks resulted in microscopic changes in the spleen, kidney, and liver. The spleen was treated with 20 or 40 mg / kg / dose, resulting in microscopic findings (Table 36). In rats treated with IFN-γ, very mild to moderate macrophage vacuolation and basophilic granules were observed. There was very slight to mild accumulation of basophilic material. In the kidneys of rats treated with IFN-γ, microvacuolation of glomerular mesangial cells was observed. Minimal centrilobular single-cell necrosis and minimal to mild centrilobular hepatocellular degeneration were observed at all doses. I was criticized.

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[0457] Administration of Ls-AKG38 at doses of 20 mg / kg / dose or higher resulted in centrilobular vasculopathy at all doses. Hepatic microscopic findings of minimal single-cell necrosis and minimal to mild centrilobular hepatocellular degeneration brought about.

[0458] In contrast, rats treated with Ls-AKG28 and Ls-AKG38 showed no significant changes compared with those treated with linezolid. The incidence of hepatic single-cell necrosis was increased at all doses compared to the control rats. In the livers of rats treated with 8 and Ls-AKG38, the incidence and severity of centrilobular hepatocellular degeneration were significantly increased. The severity of the lesions was similar across all doses. At 0 or 40 mg / kg / dose, vacuolated macrophages and basophilic material were observed in the spleen. At 40 mg / kg / dose, vacuolation of glomerular mesangial cells was observed in the kidney. .

[0459] In conclusion, administration of Ls-AKG28 by repeated intravenous injections over 8 weeks was associated with the following clinical outcomes: Ls-AKG was well tolerated in rats at 20 and 40 mg / kg / dose. Repeated intravenous injections of 38 over an 8-week period were administered at 20, 40, and 80 mg / kg / day. Example 2 demonstrated that AKG-28 is more potent than linezolid. AKG is 33 times more effective at killing tuberculosis bacteria in vitro (Erdmann strain) -38 was 17-fold higher. Therefore, when corrected for potency, rats were clinically The linezolid equivalent dose of 1 mg / kg is 16.5 times that of 80 mg / kg, which is a reasonable dose for At 320-1336 mg / kg, significant neuropathy (changes in nerve conduction velocity) and elevated liver enzymes were observed. No elevation in blood glucose level, decrease in red blood cell count or hematocrit, or weight loss was observed.

[0460] [Example 45] Liposomes in Kramnik (C3HeB / FeJ mouse model of pulmonary Mycobacterium tuberculosis infection) AKG-28 and AKG-38 in combination with bedaquiline and pretomannide, or Efficacy of Daquiline (B), Pretomannide (Pa), and Moxifloxacin (M) The C3HeB / FeJ (Kramnik) mouse infection model demonstrates the role of TB in lung function after infection. Showing advanced hypoxic caseous granulomas (Driver E., et al., Anti Microbial Agents and Chemotherapy,2012,v ol.56, pp.3181-3195). Lung pathology is characterized by heterogeneity in lesion pathology and bacterial populations seen in TB patients. More similar to liposomal AK at moderate doses of 50 and 90 mg / kg once weekly This was used to evaluate the efficacy of Ls-AKG28 and AKG-38. Ls-AKG38 (Lot 275) and Ls-AKG38 (Lot 276) were used in Examples 22 and 23, respectively. Lung pathology in C3HeB / FeJ mice was characterized by collagen-rimmed lungs. (Type I), fulminant neutrophilic alveolitis (Type II), and cellular lesions (Type III) Three different types of lesions classified as caseous necrotic lesions are shown (Irwin et al., 2011). See et al. (2015) Dis Model Mech 8, 591-602 Eight to ten week old C3HeB / FeJ female mice were administered LDA (low dose aerosol challenge). The Glas-Col inhalation exposure system was used to infect the mice with approximately 50-75 bacilli / mammary. Mice were infected with the target strain of Erdman. Five mice were infected per aerosol dose. Mice were sacrificed 1 day after infection and bacterial uptake was determined.

[0461] Eight weeks after infection, eight mice were sacrificed to measure the bacterial counts in the lungs and spleens at the start of treatment. The burden was measured. Mice were weighed before sacrifice. Gross pathology of lungs and spleen Whole lungs and spleens were extracted and frozen at -80°C. was collected and homogenized in 1x PBS using a Precellys homogenizer. Lung and spleen homogenates were plated on 7H11 agar quad plates. CFU Counts were performed after incubation at 37°C in a dry air incubator for 3 to 5 weeks. Treatment was administered by oral gavage or intraperitoneal (ip) injection 8 weeks after infection. The treatment was started between 10 and 12 weeks and continued for 4 to 6 consecutive weeks (MF for oral gavage, i.p. injection). Once a week for flu-like symptoms). Bedaquiline (B), pretomanid (Pa), moxifloxacin (M ), and linezolid (L) were orally administered at a total dose of 200 μL / dose, 5 days a week for 4 or 6 weeks. Bedaquiline (25 mg / kg) was administered first, followed by a 25 mg / kg dose for at least 1 hour. Later, pretomannide (100 mg / kg) was administered. Moxifloxacin (100 mg / kg) or linezolid (100 mg / kg) was administered 4 hours after pretomanid administration. The liposomal formulation was administered once a week for a total of 4 or 6 weeks.

[0462] Mice were observed daily during dosing and weighed at least once a week. Eight mice per treatment group were weighed before sacrifice. Lungs and spleens were collected aseptically from all treated groups. Gross pathology of the lungs and spleens was The findings are shown in the figure. Lungs were photographed for gross lesion analysis. Whole lungs and spleens were stored at -80°C. Freeze and collect previously frozen tissue and homogenize it to 1x P using a Precellys homogenizer. Homogenize in either 10% bovine serum albumin (BSA) in 1x PBS or 10% bovine serum albumin (BSA) in 1x PBS. (To avoid drug carryover. *See below for clarification. After homogenization, the lung and spleen homogenates were transferred to a 7-well containing 7H11 quad plate. Plate on H11 agar or charcoal and serially dilute in 1x PBS or 10% BSA CFU counts were performed after incubation at 37°C for 5 weeks in a dry air incubator. This was done after the test.

[0463] The addition of Ls-AKG28 to BPaM treatment further reduced BPaM in the lung by 0. BPaM+Ls-AKG38 treatment resulted in a 64 log CFU reduction, but After treatment with 100 mg of ... Either Ls-AKG28 or Ls-AKG38 was used as the NIX (BPaL) register. Substitution of linezolid (L) in men showed improved efficacy over BPaL after 6 weeks of treatment Substituting Ls-AKG38 for linezolid in a BPaL regimen for 6 weeks of treatment The efficacy of BPaL was significantly improved when compared with the BPaL treatment group. Treatment resulted in a 4.18 log CFU reduction, with CF in 1 of 8 plates. After 6 weeks of BPa+Ls-AKG28 administration, the Although a log10 CFU reduction was observed, there was no statistically significant difference compared to BPaL. Treatment with BPa+Ls-AKG38 for 6 weeks resulted in a 5.26 log10 CFU reduction. However, no CFU was observed on the plates of 2 out of 8 mice. This is statistically significant for BPAL. The decrease was significantly greater than that observed in the control group (p=0.04, Dunnett's test).

[0464] In the spleen 6 weeks after treatment, the NIX regimen replaces the liposomal formulation of linezolid The NIX regimen resulted in slightly improved pulmonary efficacy compared with the BPaL treatment group. Weekly treatment resulted in a 3.56 log CFU reduction, with 3 of 8 plates showing CF Six weeks of treatment with BPa+Ls-AKG28 showed a 4.18 log 0 CFU reduction, with plates from 5 of 8 mice showing no CFU. Six weeks of treatment with BPa+Ls-AKG38 resulted in a 4.38 log CFU reduction. The plates of 6 out of 8 mice showed no CFU. The CFU burden in the spleens of mice in this study was low, approaching the lower limit of detection of 0.66 log10 CFU. It was.

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[0467] All mice without measurable CFU were enumerated with a detection limit of 0.66 log CFU. This is because Ls-AKG38 and Ls-AKG28 contain bedaquiline and pretomanid in combination. When used with linezolid, after only 6 weeks of treatment, moderate and highly tolerated doses of both drugs As shown in Examples 42 and 43, Ls-AKG28 and Both Ls-AKG38 and Ls-AKG38 were effective in this combination at the doses used in this study. Both drugs can be safely administered at double the dose. When added to Zymen, this same highly tolerated dose further reduced CFU in both the lung and spleen. It has also been shown to lower

[0468] [Example 46] Efficacy of liposomal AKG-28 monotherapy in a Balb / c model of pulmonary Mycobacterium tuberculosis infection Schedule- and dose-dependent efficacy of Ls-AKG28 in chronic Balb / tuberculosis c model at clinically relevant doses of 50 and 100 mg / kg compared with free linezolid In the chronic Balb / c mouse model, the lung bacterial burden was determined by comparing the A steady state is reached within 4-5 weeks (Lenaerts et al. (2005) AAC 49(6)2294-2301). Ls-AKG28 (Lot 286) was used in Example 28. Balb / c female mice, 6–8 weeks old, were cultured at Jackson Laboratory and prepared as described. Mice were obtained from the Glas-Col inhalation exposure system. , LDA (low dose aerosol infection) was used to infect the tuberculosis bacillus Erdman with approximately 50-100 Bacillus / mice were infected.

[0469] Mice (n=3) were sacrificed 1 day after infection and bacterial uptake was determined. Whole lungs were placed in a ly tube (Bertin cat# KT03961-1-396.7). Aseptically collect and pre-dissolve in 4 ml of 1x PBS using a Precellys tissue homogenizer The undiluted homogenate was spread onto two large 7H11 agar plates (150× 15 mm) and place in a dry air incubator at 37°C until colonies can be counted. Plates were incubated in sealed zip-top bags for at least 21 days. On day 28 after infection with the rosol, mice (n=5) were sacrificed and the lungs and spleens at the start of treatment were analyzed. The bacterial load in the lungs and spleen was determined. Mice were weighed before sacrifice. Pathological observation was performed on the lungs (divided into left lobe and upper right [cranial] lobe + accessory lobe) and spleen. The lower right lung lobe (caudal) was collected aseptically and frozen at -80°C. The tissues were then collected for histological examination in Perfluorooctadecane (PFA). The lung and spleen homogenates were homogenized in 1x PBS using a Ellys homogenizer. The genates were plated onto 7H11 agar quad plates. CFU counts were performed in a dry air This is done after incubating at 37°C for 3 to 5 weeks.

[0470] 5% PEG-200 (Sigma P3015, lot MKBW3119V) / 95% (0.5%) methylcellulose (Sigma M0430, lot 031M00051 ) was started by oral gavage on the 28th day (Mon) after aerosol infection ( The Ls-AK was administered in a single dose of 200 μL per mouse, 5 days out of 7 days per week for 2 to 8 weeks. G28 was administered by intraperitoneal injection at a dose of 50 or 100 mg / kg once or twice a week. Terminal sacrifice was performed 3 days after the last administration of drugs in mice treated for 2, 4, or 8 weeks. Before sacrificing the mice, their body weights were measured. Macroscopic pathological observations of the lungs and spleens were performed. The lungs (divided into left lobe, right upper lobe and accessory lobe) and spleen were collected aseptically and stored at -80°C. The right lower lobe was frozen at 4°C and collected in 4% PFA for histological examination. To avoid burns, retrieve previously frozen tissue and resuspend it in 10% bovine serum albumin in 1x PBS. After homogenization, the lung and spleen homogenates were homogenized in 1×PB. Serial dilutions were made in 10% BSA and then plated on 7H11 agar or 7H11 quad plates. CFU counts were performed in a dry air incubator. This was done after incubation at 37°C for 3 to 5 weeks.

[0471] The reduction in lung CFU counts is shown in Table 39, and the reduction in spleen CFU counts is shown in Table 40. Treatment with 100 mg / kg twice weekly or 100 mg / kg once weekly resulted in a significant improvement in lung function after just 2 weeks. A 1.5 Log10 CFU reduction was observed with linezolid 100 mg / kg, but 0. A 15Log10 CFU reduction was not observed (q1×5). This reduction was due to the At 2 weeks, the spleen count for G28 was approximately 3 Log10 CFU. At 8 weeks, the spleen count for Ls All mice treated with AKG28 were completely germ-free at 8 weeks (1.13 in the lungs). and spleen (less than the detection limit of 0.66), whereas the lungs had 2.45 log10 CFU and the lineage At the high dose of 100 mg / kg, the spleen count was 3.15 log10 CFU. Monotherapy activity is limited to only 8% in the absence of an active combination partner such as bedaquiline. The relatively short duration of the oxazolidinone treatment, i.e., 8 weeks, is surprising. Linezolid monotherapy improved the survival of Balb / c and C3HeB / FeJ mice (Lan oix et al.(2015)Dis Models Mech.8,603-61 0) showed a log10 CFU count in the range of 4-6, and activity was observed at doses of 3-14 / week. The effects remain moderate even on a range of schedules up to 1000 mg / kg / week (Bige low et al(2021)J Infec.Dis.223(11)1855-1 864).

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[0474] All mice with no measurable CFU were enumerated at a detection limit of 0.66 log CFU. did.

[0475] [Example 47] Liposomal analysis of methicillin-resistant Staphylococcus aureus (MRSA) in a rabbit endocarditis model The effectiveness of AKG-38 Staphylococcus aureus infections, especially those affecting the vascular system (e.g., IE; cardiac and hemodialysis machine infections, etc.) ) are widespread and are associated with unacceptably high morbidity, mortality, and post-treatment recurrence rates This is because such infections are caused by multidrug-resistant strains of MRSA. Furthermore, if the MRSA strain is identified by a recognized Clinical Standards Institute (CLSI) ) within the "susceptible" range (i.e., 2ug / ml or less) of vancomycin ("Workhouse"). Even when anti-MRSA drugs have minimal inhibitory concentrations (MICs), clinical outcomes are minimal. It remains unsuitable.

[0476] A typical high-inoculum intravascular biofilm MRSA infection model, left-sided aortic valve IE in rabbits Female New Zealand White rabbits, 6 months old and weighing 2.2-2.5 kg, were used. General anesthesia was administered to the rat by intramuscular injection of xylazine and ketamine. The cut site on the right carotid artery was locally anesthetized with 1% lidocaine. A cut was then made to expose the right carotid artery, which was isolated and proximally ligated. Then, cannulate retrogradely across the aortic valve and into the left ventricle using a polyethylene catheter. The catheter was inserted and then secured to the left ventricle and left in place for the duration of the study. For left-sided IE (to induce sterile aortic valve and ventricular vegetations), animals were cultured at approximately 100°C for 12 min at 12°C. 2×10 5 IE was induced by intravenous challenge with 1000 cfu of MRSA strain MW. -2 (USA400-clonal conjugate [CC]1) is i) clinically derived and ii) germline. iii) identify common hospital-acquired MRSA clonal types; and iv) experimental I E model and v) sensitive to daptomycin (DAP) in vitro The infection spreads from infected warts on the heart valves to the kidneys and spleen.

[0477] Liposomal AKG-38 (Ls-AKG38) was administered at 40 mg / kg / dose, once (in combination with DAP) or twice (in combination with DAP) 1 time; then after DAP treatment in the "relapse group of animals" that did not receive further DAP therapy Ls-AKG38 (Lot 292) was given either as a single injection or as a second injection at the time of sacrifice. The first Ls-AKG38 injection was performed as described in Example 29. The dose is administered approximately 1 hour after the initial intravenous administration of DAP. The dose was administered for 4 days either alone or in combination with Ls-AKG38.

[0478] Animals were euthanized and major target organs were examined on day 6 (L of DAP alone or DAP + single dose). s-AKG38) or 12 days (DAP + 2 doses of Ls-AKG38 on days 1 and 6) The blood was collected from either the left or right IE, and the remaining blood was collected from the heart and cultured. verruca; kidney and spleen). Sterile isolation by weighing, homogenization, serial dilution and plating. Quantitative target tissue culture was performed by standard preparation of the excised organs. Digestion and quantitative cultures were performed similarly. Blood culture and target organ data for the different treatment groups were , respectively. 10 cfu / ml or log 10 of cfu / gm tissue (±SD) The mean and median values ​​were calculated.

[0479] Preliminary data from the MRSA left ventricular endocarditis model in rabbits are presented in Table 41 below. A single dose of daptomycin alone or daptomycin plus Ls-AKG38 was There was no significant effect on day 6. Surprisingly, the second injection of Ls-AKG38 showed significant efficacy at 12 days, killing 4 / 5 rabbits in all 5 tissues, and a 6-log or greater reduction in CFU in multiple organs. These results suggest that Ls-AKG38 can be effectively treated after discontinuation of daily daptomycin. do.

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[0481] [Example 48] In vitro studies of AKG-28 and AKG-28 in various nontuberculous mycobacteria and AKG-38 activity MIC testing is recommended by CLSI standard M7-A7 (Becton Dickinson). Mueller H for the calcium and magnesium ion concentrations Microbroth dilution method (Obre) was used with cation-adjusted MH broth. gon-Henao et al. (2015) Antimicrobial Agen MIC test was performed by Chemother 59, 6904-6912. Also, 7H9 Broth ( Sigma-Aldrich ) using the microbroth dilution method ( Shang et al.(2011)PLoS One 6,e24726; Cha n et al. (2010) Am J Respir Cell Mol Biol 43, 287-393). The goal was to By using different broths in The objective of this study was to optimize the ability to detect NTM on 7H11 agar plates ( Sigm a-Aldrich ) at 35-37°C for 3-25 days (depending on the bacterial strain) in air. CFU were picked from the agar plates and plated in MH containing 0.05% Tween 80. The optical density (OD) absorbance was measured after 7 days of growth in either broth. in ambient air until the temperature reaches 0.08 to 0.1 (0.5 McFarland Standard). The bacterial cell suspension was then prepared in saline and grown at 35-37°C. By doing so, the (OD) is 0.08 to 0.1 (0.5 McFarland Standard d) was confirmed to be consistent with

[0482] 180 μl of broth (MH) was added to the first column of a 96-well plate. 0 μl of broth (MH) was added to another column of the 96-well plate. Made using 1.28 mg / mL in SO, test range 64 to 0.062 μg / mL For immediate use, 20 μl of compound is added to the first column of wells, and 100 μl Finally, 100 μl of the NTM cell suspension is added to the medium-only control wells. QC agents specific to each microorganism were added to all wells except: 1) negative control bacteria only 2) Media only negative control 3) Tedizolid positive drug control 4) Optional E. coli control.

[0483] RGM was assayed for OD on day 3. Clinical and Laboratories Standards Institute(Brown-E lliott et al. (2012) Clin Microbiol Rev vo Resazurin microtiter plate recommended by I.25(3), p.545-582 The plates were assayed using the assay plate method. This method involves the MIC analysis of resazurin (7-hydroxy-3H-phenanthrin) in a 96-well plate. Resazurin is a blue pigment and , which itself shows weak fluorescence, pink and very red fluorescence Resorufin irreversibly reduced to It was used as a redox indicator in bacterial cell viability MIC assays.

[0484] The results showed that both AKG-28 and AKG-38 were effective in different NTM species and strains. It is generally more potent than tedizolid. Microbacterium chelonae, Microbacterium abscessus, and Microbacterium Only Microbacterium massiliense was included in all three strains evaluated. Tedizolid was more active in this setting.

[0485] JPEG2026035569000121.jpg228170

[0486] [Example 49] In vitro activity of selected compounds against drug-resistant strains of Mycobacterium tuberculosis. Compounds of the present disclosure that exhibit activity against drug-susceptible strains of M. tuberculosis include some multidrug-resistant (M DR) Clinical isolates M70, M28, M94, M14 (Cheng AF, et al .,2004,Antimicrob.Agents Chemother.v.48, p.596-601)and TN5904(Palanisamay GS,et al.,2008,Tuberculosis(Edinb.)vol.88 p.2 95-306). These strains are characterized by the following resistance characteristics:

[0487] Table 43. Drug resistance characteristics of M. tuberculosis MDR strains used in this study. (Abbreviation: R-resistant; S - susceptible; STR - streptomycin; INH - isoniazid; RIF - rifampin EMB-ethambutol, PZA-pyrazineamide.

[0488] JPEG2026035569000122.jpg62170

[0489] Comparison / resistant controls (RIF, INH, STR, moxifloxacin (MOX), and MICs of test compounds, including nezolid (LNZ), were determined essentially as described in Example 2. The following was performed using the liquid microdilution method with Alamar Blue endpoint (MABA): The test compounds were serially diluted in 2-fold in DMSO and the ratios were determined with the modification of The control was a 96-well plate containing 7H9-glycerol medium supplemented with 100 μL of ADC. Compounds were diluted in DMSO and added to wells of a 1000-well assay plate. Maintain the pH in the desired range and keep the final DMSO concentration in the wells at 2% (M70, M28, M94). or 2.5% (M14, TN5904), but due to its low solubility in DMSO STR was serially diluted and added as an aqueous solution. Remove the bacterial stock (sexual control) from the refrigerator, thaw, and incubate in 7H9-ADC-glycerol. Dilute with medium to 10 6 CFU / mL (H37Rv, TN5904), 2×10 6 CFU / mL (M70, M14), or 3 × 10 6 Bacterial density in CFU / mL (M28, M94) After obtaining the desired concentration, 50 μL of the diluted bacterial stock in compound-containing medium was added to the wells. The range of final drug concentrations in the plates is shown in the table below. Incubate at 7°C and monitor bacterial growth by periodic optical density measurements at 600 nm (OD600). Growth was monitored. On day 14 (when OD600 reached or exceeded 0.40), On day 17 or if the number of days exceeded 100, 15 μL of Alamar Blue solution was added to the wells. Continue incubation and after 3 days (7 days for the slow-growing M28 strain), The color of the reaction mixture was recorded. The lowest serial two-fold dilutions of these compounds were used to determine the antimicrobial concentrations that did not produce a visible color change. MIC determination based on OD600 (compared to control wells without drug) was considered as MIC. The results of the MABA assay were consistent with those of the two wells. The shift in MIC (4-fold) was considered significant. The results are summarized in Table 44 below.

[0490] JPEG2026035569000123.jpg145170

[0491] The comparative / control compounds RIF, IHN, MOX, and STR were showed promising in vitro activity against TB strains H37Rv and H37Rv. Within the variance of the MIC assay, all tested compounds of the present disclosure inhibited the drug-susceptible strain H It was at least as active against MDR-TB strains as it was against 37Rv. The most active compound was AKG-28, followed by AKG-38 and AKG-3. G-28 and AKG-38 were the most active compared to their structurally close analogues. It was a good compound.

[0492] Various aspects of the present disclosure may be used alone, in combination, or as specifically illustrated in the foregoing embodiments. can be used in various arrangements not described in The details and arrangement of components set forth in the foregoing description or illustrated in the drawings are not intended to be limiting. For example, aspects described in one embodiment may be interchangeable with aspects described in other embodiments. They can be combined in various ways.

[0493] While specific embodiments of the present disclosure have been described, the above specification is illustrative and not restrictive. Many variations of this disclosure will become apparent to those skilled in the art upon review of this specification. The full scope of the disclosure extends to the claims, their full scope of equivalents, and such variations. The nature and shape of the composition should be determined by reference to the present specification.

[0494] Incorporation by Reference All publications, patents, and patent applications cited herein are to be construed as wholly-owned subsidiaries of each individual publication, If a patent or patent application is specifically incorporated herein by reference, Portions of this specification are incorporated by reference for all purposes to the same extent as if expressly set forth herein. This shall be done.

Claims

1. Formula I: 【Chemistry 1】 [In the formula, R ２ is an amine (NH ２ ) or acetamide (NHCOCH ３ ) and R １ is a tetrazole ring substituted at the 2' position with an aminoalkyl. or a pharmaceutically acceptable salt thereof.

2. The compound of claim 1 , wherein the aminoalkyl is dimethylaminoethyl.

3. Formula 1b: 【Chemistry 2】 2. The compound of claim 1 having the formula:

4. Formula 1c: 【Transformation 3】 2. The compound of claim 1, wherein:

5. Formula 1d or Formula 1e: 【Chemistry 4】 2. The compound of claim 1 having the formula:

6. Selectivity index (SI) for Erd / HepG2 and H37Rv / HepG2 is 10 6. The compound according to any one of claims 1 to 5, wherein the molecular weight is in the range of 0 to 1700.

7. SI to Erd / HepG2 and H37Rv / HepG2 is 200-1700 6. The compound of claim 1, wherein

8. SI for Erd / HepG2 and H37Rv / HepG2 is 300-1700 6. The compound of claim 1, wherein

9. 1. A liposomal composition comprising a liposomal vesicle, the liposomal vesicle comprising a compound of Formula I: 【Transformation 5】 [In the formula, R ２ is an amine (NH ２ ) or acetamide (NHCOCH ３ ) and R １ is a tetrazole ring substituted at the 2' position with an aminoalkyl. or a pharmaceutically acceptable salt thereof.

10. 10. The liposome composition of claim 9, wherein the aminoalkyl is dimethylaminoethyl. thing.

11. The liposome vesicle has formula 1b: 【Transformation 6】 The liposome composition of claim 9 , comprising the compound:

12. The liposome vesicle has formula 1c: 【Transformation 7】 The liposome composition of claim 9 , comprising the compound:

13. The liposome vesicle is represented by Formula 1d or Formula 1e: 【Transformation 8】 The liposome composition of claim 9 , comprising the compound:

14. 14. The liposome vesicle of claim 9, wherein the liposome vesicle is in an aqueous medium. osmosome composition.

15. The compound is entrapped in the liposome vesicle with a trapping agent, the trapping agent trapping a polyanion. The liposome composition according to any one of claims 9 to 13, comprising:

16. The scavenger is triethylammonium sucrose octasulfate or ammonium sulfate. The liposome composition according to claim 15 .

17. 16. The method of claim 15, wherein the scavenger is triethylammonium sucrose octasulfate. osmosome composition.

18. The liposome composition of claim 15, wherein the sequestering agent is ammonium sulfate.

19. and salts of the compounds, the salts being sulfate, citrate, sucrosophate, phosphate, or salts with sulfated polyols, or phosphated or sulfated polyanionic polymers The liposome composition according to any one of claims 9 to 13, which is a salt of

20. 14. The method of claim 9, further comprising the step of: providing a salt of said compound, said salt being a sulfate. The liposome composition described above.

21. 10. The compound in the liposome vesicle of claim 9, wherein the compound has an aqueous solubility of less than 1 mg / mL.

14. The liposome composition according to any one of claims 1 to 13.

22. The compound in the liposome vesicle has an aqueous solubility of less than 0.1 mg / mL. Item 14. The composition according to any one of items 9 to 13.

23. the liposome vesicle comprises a membrane comprising phosphatidylcholine and cholesterol; The liposome composition according to any one of claims 9 to 13.

24. 24. The method of claim 23, wherein the membrane separates the interior of the liposome vesicle from the aqueous medium. A liposome composition comprising:

25. The phosphatidylcholine is distearoylphosphatidylcholine (DSPC) or The liposome of claim 23, wherein the phospholipid is hydrogenated soy phosphatidylcholine (HSPC). composition.

26. The molar ratio of phosphatidylcholine to cholesterol is about 60:40 to about 35:65 24. The liposome composition of claim 23, wherein:

27. The molar ratio of the phosphatidylcholine to cholesterol is about 55:45 to about 35:65 24. The liposome composition of claim 23, wherein:

28. The molar ratio of phosphatidylcholine to cholesterol is about 50:50 to about 45:55 24. The liposome composition of claim 23, wherein:

29. The molar ratio of the phosphatidylcholine to cholesterol is about 50:50 to about 40:60 24. The liposome composition of claim 23, wherein:

30. 30. Any of claims 23 to 29, wherein the membrane further comprises a polymer-conjugated lipid. The liposome composition according to any one of claims 1 to 10.

31. The liposome vesicles comprise HSPC, cholesterol in a molar ratio of about 55:45:2.

75. and a polymer-conjugated lipid. The liposome composition described above.

32. The polymer-conjugated lipid is PEG (molecular weight 2,000)-distearoyl PEG-DSG or PEG (molecular weight 2,000)-distearoyl 32. The method according to claim 30 or 31, wherein the compound is phosphatidylethanolamine (PEG-DSPE). The liposome composition described.

33. 14. The liposome of claim 9, which is a liquid pharmaceutical formulation for parenteral administration. Room composition.

34. 10. The liposomes according to claim 9, wherein the liposomes have a Z-average particle size ranging from about 80 to about 130 nm.

14. The liposome composition according to any one of claims 13.

35. A method for treating a bacterial infection, comprising administering to a subject in need thereof a therapeutically effective amount of any one of claims 9 to 10.

14. A method comprising administering the liposome composition described in any one of claims 13 to 14.

36. 36. The method of claim 35, wherein the bacterial infection is a Mycobacterium tuberculosis infection.

37. The compound in the liposome vesicles is about 0.01 μg / ml to about 0.25 μg / ml 37. The method of claim 35 or 36, having a minimum inhibitory concentration (MIC) in the range of

38. The compound in the liposome vesicles is about 0.01 μg / ml to about 0.1 μg / ml 37. The method of claim 35 or 36, wherein the antibody has an MIC in the range

39. 39. Any one of claims 35 to 38, comprising parenterally administering the liposome composition. The method described in paragraph .

40. 40. The method of claim 39, comprising administering one or more active agents simultaneously or sequentially. How to post.

41. The one or more active agents are bedaquiline, pretomanid, pyrazinamide, moxibustion 41. The method of claim 40, comprising administering to a subject a composition comprising floxacin, a pharmaceutically acceptable salt thereof, or a combination thereof. The method described.

42. 40. The method of claim 39, wherein the liposome composition is administered once a week to once every six weeks.

43. The percentage of compound remaining in the blood after administration to a subject in need thereof is 40. The method of claim 39, wherein the dose is greater than 20%.

44. The percentage of a compound remaining in the blood after administration to a subject in need thereof is the dose.

40. The method of claim 39, wherein the concentration of the ion exchange medium is greater than 10%.

45. 1. A method of making a liposome composition, comprising: (i) phospholipids, cholesterol, and PEG- in a medium that is substantially free of sequestering agents. preparing a liposome comprising lipids and having an interior space containing an entrapped agent; (ii) mixing the liposome with a compound according to any one of claims 1 to 8 in an aqueous medium; contacting to encapsulate the compound in the liposome; (iii) removing any unencapsulated compound; (iv) providing the liposomes in a physiologically acceptable medium suitable for parenteral use; Tep and A method comprising:

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