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

Abstract

Disclosed are compositions and methods for treating tuberculosis and other mycobacterial and gram-positive bacterial infections. These compositions contain highly potent, selective oxazolidinones encapsulated with high efficiency to maximize the dosability of low-toxicity drugs and are stable in the presence of plasma. The compositions are long-circulating and retain their encapsulated drug in the circulation after intravenous administration, allowing for efficient accumulation at the site of bacterial or mycobacterial infection. The high doses that can be achieved, combined with the long-circulating properties and highly stable drug retention, allow for reduced dosing frequency compared to the once-daily or twice-daily administration of other drugs typically used to treat these infections.

Description

[Technical Field]

[0001] Related Applications This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 040,810, filed June 18, 2020, and U.S. Patent Application No. 17 / 351,631, filed June 18, 2021, the entire contents of which are incorporated herein by reference.

[0002] Field The present disclosure relates to novel aminoalkyloxazolidinone compounds, liposomal compositions containing the novel aminoalkyloxazolidinone compounds, and the use of the aminoalkyloxazolidinone compounds in the treatment of Mycobacterium tuberculosis and other gram-positive bacterial infections. [Background technology]

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

[0004] Compositions and methods for treating tuberculosis, as well as other mycobacterial and gram-positive bacterial infections, 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 some embodiments, the aminoalkyl derivative of the oxazolidinone compound contains either an amine group or an acetamide group at the R2 position of the oxazolidinone ring and a dimethylaminoethyl group on the tetrazole ring.

[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 has a selectivity index (SI) in the range of 100-1700 for Erd / HepG2 and H37Rv / HepG2.

[0013] In some embodiments, the compounds have an SI in the range of 200-1700 against Erd / HepG2 and H37Rv / HepG2.

[0014] In some embodiments, the compounds have an SI in the range of 300-1700 against Erd / HepG2 and H37Rv / HepG2.

[0015] Another aspect of the present disclosure provides a liposomal composition comprising a liposomal vesicle, the liposomal vesicle having a structure represented by 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 some embodiments, the aminoalkyl derivative of the oxazolidinone compound contains either an amine group or an acetamide group at the R2 position of the oxazolidinone ring and a dimethylaminoethyl group on the tetrazole ring.

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

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

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

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

[0021] In some embodiments, the liposome composition comprises a liposome vesicle, the liposome vesicle having Formula 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 trapped in the liposome vesicle with a trapping agent, and the trapping agent comprises a polyanion. In some embodiments, the trapping agent is triethylammonium sucrose octasulfate or ammonium sulfate. In some embodiments, the trapping agent is triethylammonium sucrose octasulfate. In some embodiments, the trapping agent is ammonium sulfate.

[0024] In some embodiments, the liposome composition comprises a salt of the compound, the salt being a sulfate, citrate, sucrosophate, a salt with a phosphorylated or sulfated polyol, or a salt with a phosphorylated or sulfated polyanionic polymer. In some embodiments, 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.

[0026] In some embodiments, the liposome vesicle comprises a membrane comprising phosphatidylcholine and cholesterol. In some embodiments, the liposome vesicle comprises a membrane comprising phosphatidylcholine and cholesterol, the membrane separating the interior of the liposome vesicle from the aqueous medium. In some embodiments, the phosphatidylcholine is distearoylphosphatidylcholine (DSPC) or hydrogenated soybean phosphatidylcholine (HSPC). In some embodiments, the molar ratio of phosphatidylcholine to cholesterol is about 60:40 to 35:65. In some embodiments, the molar ratio of phosphatidylcholine to cholesterol is about 55:45 to about 35:65. In some embodiments, the molar ratio of phosphatidylcholine to cholesterol is about 50:50 to about 40:60.

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

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

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

[0030] In some embodiments, the polymer-conjugated lipid is PEG (molecular weight 2,000)-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-average particle size of about 80 to about 130 nm.

[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 a therapeutically effective amount of a liposome composition provided herein.

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

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

[0036] In some embodiments, the method comprises simultaneously or sequentially administering one or more additional active agents, hi some embodiments, the one or more active agents comprise bedaquiline, pretomanide, pyrazinamide, moxifloxacin, pharmaceutically acceptable salts thereof, or 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 is greater than 20% of the administered amount at 6 hours, hi some embodiments, the percentage of the compound remaining in the blood is greater than 10% of the administered amount.

[0039] An embodiment of the present disclosure relates to a method of making a liposome composition, comprising the steps of: (i) preparing liposomes comprising phospholipids, cholesterol, and PEG-lipids in a medium substantially free of the entrapment agent, and having an interior space containing the entrapment agent; (ii) contacting the liposomes with a compound described in any one of claims 1 to 8 in an aqueous medium to encapsulate the compound in the liposomes; (iii) removing any unencapsulated compound; and (iv) providing the liposomes in a physiologically acceptable medium suitable for parenteral use. [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] It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the compositions and methods of the present disclosure.

[0042] Disclosed herein are compounds, compositions, and methods for treating bacterial infections. As used herein, the terms "compound" and "drug" are used interchangeably. Some aspects of the disclosure relate to novel aminoalkyl derivatives of oxazolidinone compounds. Some aspects of the disclosure relate to processes for synthesizing novel aminoalkyl derivatives of oxazolidinone compounds. Other aspects relate to compositions comprising aminoalkyl derivatives of oxazolidinone compounds in liposomes. Other aspects of the disclosure relate to the use of aminoalkyl derivatives of oxazolidinone compounds or liposomal compositions comprising aminoalkyl derivatives of oxazolidinone compounds in the treatment of bacterial infections. In some embodiments, the compounds and compositions described herein can be used to treat infections from mycobacteria and gram-positive bacteria. In some embodiments, the bacterial infection is Mycobacterium tuberculosis. In some embodiments, the compounds and compositions described herein inhibit the growth of mycobacteria and gram-positive bacteria.These include, but are not limited to, Mycobacterium tuberculosis, Mycobacterium avium complex, Mycobacterium leprae, Mycobacterium gordonae, Mycobacterium abscessus, Mycobacterium mucogenicum, streptococci, vancomycin-resistant enterococci (VRE), methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus pneumoniae, Enterococcus faecium, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, These include Streptococcus pyogenes, viridans group streptococci, Listeria monocytogenes, Nocardia, and Corynebacterium.

[0043] In some embodiments, the aminoalkyl derivatives of the oxazolidinone compounds described herein are selectively active against Mycobacterium tuberculosis when compared to mammalian cells, such as human kidney or hepatocytes. In some embodiments, the aminoalkyl derivatives of the oxazolidinone compounds described herein exhibit unexpectedly high selectivity of at least 1000-fold against Mycobacterium tuberculosis when compared to mammalian cells, such as mammalian kidney or hepatocyte cells. In some embodiments, the aminoalkyl derivatives of the oxazolidinone compounds described herein exhibit unexpectedly high selectivity of at least 100-fold. In some embodiments, the aminoalkyl derivatives of the oxazolidinone compounds described herein have an activity against Mycobacterium tuberculosis that is 100-6,500 fold, 100-6,000 fold, 100-5,500 fold, 100-5,000 fold, 100-4,500 fold, 100-4,000 fold, 100-3,500 fold, 100-3,000 fold, 100-2,500 fold, 100-2,000 fold, 100-1,500 fold, 100-1,000 fold, 500-6,500 fold, 500-6,000 fold, 500-5,500 fold, 500-6,000 fold, 500-5,500 fold, 500-7,000 fold, 500-8,000 fold, 500-9,000 fold, 500-10,000 fold, 500-11,000 fold, 500-12,000 fold, 500-13,000 fold, 500-14,000 fold, 500-15,000 fold, 500-16,000 fold, 500-17,000 fold, 500-18,000 fold, 500-19,000 fold, 500-21,000 fold, 500-22,000 fold, 500-23,000 fold, 500-24,000 fold, 500-25,000 fold, 500- ~5,000x, 500~4,500x, 500~4,000x, 500~3,500x, 500~3,000x, 500~2,500x, 500~2,000x, 500~1,500x, 500~1,000x, 1,000~6,500x, 1,000~6,000x, 1,00 The results show unexpectedly high selectivity: 0-5,500-fold, 1,000-5,000-fold, 1,000-4,500-fold, 1,000-4,000-fold, 1,000-3,500-fold, 1,000-3,000-fold, 1,000-2,500-fold, 1,000-2,000-fold, and 1,000-1,500-fold.

[0044] In some embodiments, the compounds and compositions described herein can promote selective uptake of Mycobacterium in resident macrophages in the liver, spleen, or lungs, helping to provide potent intracellular killing. Macrophages are involved in the clearance of foreign particles via phagocytosis, including both foreign infectious pathogens such as Mycobacterium and laboratory-derived nanoparticles such as liposomes. This allows the two to co-localize in the same biological reservoir, effectively concentrating the active agent in a critical reservoir of disease.

[0045] Aspects of the present disclosure relate to compounds that are aminoalkyl derivatives of oxazolidinone (see Figure 6). In some embodiments, compounds have 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 some embodiments, the aminoalkyl derivative of the oxazolidinone compound contains either an amine group or an acetamide group at the R2 position of the oxazolidinone ring and a dimethylaminoethyl group on the tetrazole ring.

[0048] The present disclosure demonstrates highly specific structure-activity relationships (SAR) for aminoalkyl derivatives of the oxazolidinone compounds described herein, which contain either an amine or acetamide group at the R2 position of the oxazolidinone ring and a dimethylaminoethyl group on the tetrazole ring. These compounds are (1) highly selective for Mycobacterium tuberculosis when compared to activity in mammalian cells (e.g., human kidney or hepatocytes), (2) highly active against Mycobacterium tuberculosis, and (3) efficiently loaded into liposomes.

[0049] In some embodiments, the aminoalkyl derivatives of oxazolidinone described herein are loaded into liposomes with efficiencies of 85% or better using gradient-based drug loading methods. 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, methods for loading aminoalkyl derivatives of oxazolidinone into liposomes are described. In some embodiments, the loading method employs a transmembrane gradient and a sequestering agent to efficiently load and subsequently stabilize the weakly basic amphiphilic agent in the internal aqueous space of the liposome. The gradient can be (1) a simple pH gradient formed, for example, using a citrate solution; (2) an ammonium ion gradient employing citrate or ammonium sulfate salts; (3) an alkyl, dialkyl, or trialkyl ammonium salt; or (4) a transition metal (Cu 2+ , Mn 2+ , Zn 2+ , Mg 2+), or even (5) a transmembrane gradient of drug solubility. See U.S. Patent Nos. 5,316,771, 5,800,833, 8,147,867, 7,744,921, 8,349,360, 6,110,491, U.S. Patent Application Publication No. 2018 / 0369143A1, and International Patent Application Publication No. 199001405, all of which are incorporated herein by reference in their entireties. See also Allen et al. (1995) Int J Cancer 62:199-204. Without being bound by theory, cations contained within the liposome interior play a role in establishing a transmembrane pH gradient that drives the accumulation of weakly basic drugs within the liposome interior or helps them directly exchange with drug molecules. This, in some embodiments, results in quantitative loading of the drug at less than the full volume of the gradient. Counterions can play an important role in stabilizing the formulation against premature leakage during circulation or storage by forming stable complexes with the drug inside the liposome (see Drummond et al. (2008) J. Pharm Sci 97, 4696-4740).

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

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

[0052] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0053] As used herein, the terms "comprising" or "comprises" are used to refer to compositions, methods, and their respective component(s) that are present in a given embodiment but may include elements not specified.

[0054] As used herein, the term "consisting essentially of" refers to elements required for a given embodiment. The term allows for the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the present disclosure.

[0055] The term "consisting of" refers to compositions, methods, and each component thereof as described herein, excluding any element not recited in that description of an embodiment.

[0056] The term "comprising" as used herein includes "consisting of" and "consisting essentially of."

[0057] When reference is made herein to "as mentioned above" or "mentioned above" or "the foregoing", reference is made to any of the disclosures made within this specification in any of the preceding pages.

[0058] When reference is made in this specification to "as referred to herein," "described herein," "provided herein," or "as referred to in this text," or "described herein," this specification refers to any of the disclosures made in the specification, either on the preceding or following pages.

[0059] As used herein, the term "about" refers to an acceptable variation of within 20%, 10%, and 5% of the stated value. In certain embodiments, "about" can refer to a variation of ±1%, 2%, 3%, 4%, 5%, 10%, or 20%.

[0060] The term "effective amount," as used herein with respect to a compound or composition, means an amount of an active compound (also referred to herein as an active agent or drug) sufficient to produce a bactericidal or bacteriostatic effect. In one embodiment, the effective amount is a "therapeutically effective amount," meaning the amount of active compound sufficient to alleviate the symptoms of the bacterial infection being treated.

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

[0062] As used herein, the term "administration" or "administering" includes all means of introducing a compound or pharmaceutical composition into a subject in need thereof, including, but not limited to, oral, intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, inhalation, buccal, ocular, sublingual, vaginal, rectal, etc. Administration of the compound or composition is suitably parenteral. For example, the compound or composition can be preferentially administered intravenously, but can also be administered intraperitoneally or by inhalation, etc., currently used clinically for liposomal amikacin in the treatment of Mycobacterium avium Complex Lung Disease. Drugs. 2019 Apr; 79(5):555-562).

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

[0064] As used herein, the terms "synergistic" and "synergistic" mean that the effect achieved with compounds used together is greater than the sum of the effects resulting from using the compounds separately, i.e., greater than the effect that would be predicted based on the two active ingredients administered separately.

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

[0066] The term "alkyl" means saturated carbon chains which may be linear or branched or combinations thereof, unless the carbon chain is defined otherwise. 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" means an alkyl in which at least one carbon of the alkyl carbon chain forms a bond with an amino group, which may be a primary amino group, a monoalkyl-substituted (secondary) amino group, a dialkyl-substituted (tertiary) amino group, or an alkyl-substituted amino group, and the amine nitrogen atom and the alkyl chain replacing the amine hydrogen atom form a heterocyclic ring.

[0068] The term "liposome" refers to a vesicle composed of a bilayer (unilamellar) and / or multiple concentric bilayers (multilamellar) separated by aqueous compartments formed by amphiphilic molecules such as phospholipids that surround a central aqueous compartment. In liposomal drug products, the active pharmaceutical ingredient is generally contained in a liposome. Typically, the water-soluble drug is contained in the aqueous compartment(s), and the hydrophobic drug is contained in the lipid bilayer(s) of the liposome. Drug release from liposomal formulations, particularly liposome clearance and other characteristics such as circulation half-life, can be modified by the presence of polyethylene glycol and / or cholesterol or other potential additives in the liposome.

[0069] A "unilamellar liposome," also known as a "unilamellar vesicle," is a liposome containing a single lipid bilayer membrane that defines a single, closed aqueous compartment. The bilayer membrane contains two layers of lipids: an inner layer and an outer layer (leaflet). The lipid molecules in the outer layer are oriented with their hydrophilic ("head") portions facing toward the outer aqueous environment and their hydrophobic ("tail") portions pointing downward toward the interior of the liposome. The inner lipid layer is located directly beneath the outer lipid layer, with the lipid heads facing the aqueous interior of the liposome and the lipid tails pointing toward the tails of the outer lipid layer.

[0070] "Multilamellar liposomes," also known as "multilamellar vesicles" or "multilamellar vesicles," contain more than one lipid bilayer membrane, which defines multiple closed aqueous compartments. The membranes are arranged concentrically, with different membranes separated by aqueous compartments.

[0071] As used herein, the terms "encapsulated" and "entrapped" refer 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 and refer to the ratio of drug to liposomal lipid. Unless otherwise specified, it is expressed as grams of drug per mole of liposomal phospholipid (PhL).

[0073] The term "mol%" with respect to cholesterol refers to the molar amount of cholesterol relative to the sum of the molar amounts of cholesterol and non-PEGylated phospholipid expressed in percentage points. For example, "55 mol% cholesterol" in a liposome containing cholesterol and HSPC refers to a composition of 55 mol parts cholesterol per 45 mol parts HSPC.

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

[0075] The terms "sucrose octasulfate," "sucrosofate," and "sucrooctasulfate" refer to the same compound, sucrose octasulfuric acid, or its anion, and are used interchangeably herein.

[0076] The symbols "Ac," "Me," and "Et" found in chemical formulas refer to the acetyl group (CH3CO), the methyl group (CH3), and the ethyl group (C2H5), respectively.

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

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

[0079] Aspects of the present disclosure relate to compounds that are aminoalkyl derivatives of oxazolidinone (see Figure 6). In some embodiments, compounds have 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 some embodiments, the aminoalkyl derivative of the oxazolidinone compound contains either an amine group or an acetamide group at the R2 position of the oxazolidinone ring and a dimethylaminoethyl group on the tetrazole ring.

[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 synthesized as described in Example 1.

[0083] The compounds of the present disclosure can exist in free form, for example, as free base, free acid, or zwitterion, or can exist in the form of salt.The salt can be any salt, organic or inorganic addition salt, or cocrystal commonly used in pharmacy, particularly any pharmaceutically acceptable organic or inorganic addition salt, or cocrystal.It is understood that the chemical formula showing a compound in a particular salt form or ion form also discloses this compound in undissociated free base (or free acid) form.

[0084] The present disclosure encompasses all stereoisomeric forms of the compounds. In some embodiments, the compounds in Table 1 below are substantially pure (i.e., at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, e.g., 100%).

[0085] JPEG0007805319000017.jpg255166JPEG0007805319000018.jpg242170JPEG0007805319000019.jpg248170JPEG0007805319000020.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] Disclosed herein are compounds of Formula I, or pharmaceutically acceptable salts thereof, that are useful for treating mycobacterial infections. In some embodiments, the compounds have formula 1a, 1b, 1c, 1d, or 1e. In some embodiments, the compounds have formula 1b: In some embodiments, the compound of formula I has a minimum inhibitory concentration (MIC), e.g., an MIC in the range of 0.1 μg / ml to 1 μg / ml, 0.25 μg / ml to 1 μg / ml, 0.5 μg / ml to 1 μg / ml, 0.1 μg / ml to 0.25 μg / ml, 0.1 μg / ml to 0.5 μg / ml, 0.25 μg / ml to 0.5 μg / ml, 0.01 μg / ml to 1 μg / ml, 0.01 μg / ml to 0.25 μg / ml, 0.01 μg / ml to 0.5 μg / ml, or 0.01 μg / ml to 0.1 μg / ml against Mycobacterium tuberculosis. In some embodiments, the compound of formula I has a minimum inhibitory concentration (MIC), e.g., an MIC of less than 1 μg / ml, less than 0.25 μg / ml, or less than 0.1 μg / ml against Mycobacterium tuberculosis. In some embodiments, the compound of Formula I has an MIC in the range of 0.01 μg / ml to 0.25 μg / ml. In some embodiments, the compound of Formula I has an MIC in the range of 0.01 μg / ml to 0.1 μg / ml. It should be understood that the MIC value may be lower than the range provided herein, depending on the bacterium.

[0092] In some embodiments for treating mycobacteria, e.g., Mycobacterium tuberculosis, the compound (AKG-28 or AKG-38) has an MIC of less than 0.1 μg / mL. In some embodiments for treating mycobacteria, e.g., Mycobacterium tuberculosis, the compound has a selectivity index (SI) for killing Mycobacterium tuberculosis versus human kidney cells (VERO) of at least 1,000. In some embodiments for treating mycobacteria, e.g., Mycobacterium tuberculosis, the compound has an MIC of less than 0.1 μg / mL and a selectivity index (SI) for killing Mycobacterium tuberculosis versus human kidney cells (VERO) of at least 1,000. In some embodiments, the compound has the structure of AKG-28 (Formula 1b) or AKG-38 (Formula 1c). In some embodiments, the MIC is less than 0.05 μg / mL and the selectivity index for MIC compared to mitochondrial protein synthesis inhibition (SI-MPS) for Mycobacterium tuberculosis is greater than 20, such as AKG-28.

[0093] In some embodiments, the compounds described herein have a 2-20 fold increase (about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20) in efficacy-adjusted doses compared to linezolid for Mycobacterium tuberculosis.

[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 methicillin-resistant Staphylococcus aureus (MRSA), the compound has an IC50 against human VERO kidney cells of greater than 100 μg / mL. In some embodiments for treating methicillin-resistant Staphylococcus aureus (MRSA), the compound has an MIC against MRSA strains of less than 2 μg / mL and an IC50 against human VERO kidney cells of greater than 100 μg / mL. In some embodiments, the compound has the structure of AKG-38 (Formula 1c), AKG-39 (Formula 1e), and AKG-40 (Formula 1d).

[0095] Water solubility In some embodiments, the compound is in the form of a salt, such as a hydrochloride or mesylate, and is soluble in water at a concentration of more than 1 mg / ml, preferably more than 10 mg / ml (and up to 1 g / ml) before encapsulation in liposomes. Additional salts before encapsulation may include, but are not limited to, besylate, bitartrate, carbonate, citrate, esylate, gluconate, glutamate, glycolate, lactate, malate, maleate, mandelate, methylsulfate, napsylate, phosphate, propionate, salicylate, succinate, tartrate, and tosylate. In some embodiments, the compound is in the form of a hydrate, solvate, or co-crystal before encapsulation in liposomes.

[0096] In some embodiments, drugs are entrapped inside liposomes in different salt forms with reduced aqueous solubility, e.g., less than 1 mg / mL, preferably less than 0.1 mg / mL (0.1-0.001 mg / mL). Salts of compounds once entrapped in liposomes include, but are not limited to, sulfate, citrate, phosphate, sucrose, or various phosphorylated or sulfated polyols or polyanionic polymers. Exemplary polyols include, but are not limited to, sucrose, erythritol, mannitol, xylitol, sorbitol, inositol, and combinations thereof. Exemplary polyanionic polymers include, but are not limited to, polyvinyl sulfonate, polyvinyl sulfonate, polyphosphate, copolymers of acrylic acid and vinyl alcohol sulfate, and combinations thereof.

[0097] Working stocks of compounds were prepared as follows: To an aliquot of the compound (free base) in powder form, 1 to 1.5 equivalents of HCl in the form of a 1N aqueous solution was added, and the mixture was vortexed until homogeneous. To the resulting cake or syrup, water was added to a final concentration of typically 10 mg / ml, and complete dissolution was observed. In some cases, 0.95 equivalents of HCl were 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 is illustrated by the following observations to obtain a visually clear solution.

[0099] JPEG0007805319000026.jpg67170

[0100] These results demonstrate that the compounds provided herein: -Linezolid (3mg / ml) (www.drugbank.ca / drugs / DB00601) [ka] -Stezolid (0.237mg / ml) (www.drugbank.ca / drugs / DB11905) [ka] and -Tedizolid (0.382 mg / mL) (www.drugbank.ca / drugs / DB14569) [ka] This indicates that the compound has a higher water solubility than the known water solubility of

[0101] In some embodiments, prior to encapsulation in liposomes, the aqueous solubility of the compounds described herein is at least 5 times, at least 10 times, at least 20 times, at least 30 times, or at least 40 times that of the oxazolidinone.

[0102] The excellent water solubility of the compounds described herein, and their amphiphilic weak base properties, enable the efficient use of transmembrane gradient-based and intraliposomal complexation (active loading) approaches to generate liposome-encapsulated forms of these compounds with high drug / carrier (drug / lipid) ratios and pharmacokinetic properties favorable for delivery of the encapsulated drug to infected tissues after systemic drug administration. As used herein, an amphiphilic weak base has a pKa of 7-12 and a logP of 1-6.

[0103] Liposome loading properties and antimycobacterial activity An important feature of the compounds described herein is their weakly amphipathic basic character, which facilitates transmembrane gradient-driven loading of these compounds into liposomes. In some embodiments, the weakly basic character of the compounds disclosed herein is characterized by an electrolytic dissociation constant in the pKa range of 7.0-12.0, 7.5-11.0, 7.8-10.5, or 8.0-10.0. In some embodiments, the amphipathic character of the compounds described herein is characterized by a logP parameter in the range of 0.5-5.0, 1.0-4.0, 1.0-3.5, or 1.0-3.0. It has been unexpectedly discovered that certain embodiments possessing these favorable properties for liposome loading also possess superior activity against mycobacteria, matching or exceeding the activity of similar compounds in the same class of drugs that have unfavorable properties for efficient and stable liposome encapsulation.

[0104] Liposome Composition Disclosed herein are compositions and uses of the compositions for treating tuberculosis and other mycobacterial and gram-positive bacterial infections. These compositions provided herein contain highly potent, selective oxazolidinones that are encapsulated with high efficiency to maximize the drug's potential for low toxicity, and are stable in the presence of plasma. In some embodiments, the compositions are long-circulating and retain their encapsulated drug in the circulation after intravenous administration, allowing for efficient accumulation at the site of bacterial or mycobacterial infection. In some embodiments, the high doses that can be achieved, combined with the long-circulating properties and highly stable drug retention, allow for reduced administration frequency compared to the once-daily or twice-daily administration of other drugs typically used to treat these infections.

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

[0106] In some embodiments, the composition comprises liposomes in a medium, the intraliposomal space comprising an aqueous phase having a polyanion and a compound of Formula I. In some embodiments, the composition comprises liposomes in a medium, the intraliposomal space comprising a polyanion or a sulfate containing a polyanion and the compound AKG-16, AKG-28, or AKG-38. In some embodiments, the medium is an aqueous medium, and the primary composition in the medium is a compound of Formula I and a corresponding sequestering agent.

[0107] The compounds of Formula I can be entrapped within liposomes using a suitable polyanion, such as sucrose octasulfate (e.g., derived from a triethylammonium sucrose octasulfate (TEA-SOS) gradient) or sulfate (e.g., derived from an ammonium sulfate gradient). Additional polyanion entrapment agents include, but are not limited to, inositol hexaphosphate, inositol hexasulfate, polyvinylsulfonic acid, dextran sulfate, citrate, polyphosphate, and suramin.

[0108] The external aqueous medium typically contains an appropriate buffer and isotonicity agent. Suitable buffers include histidine, citrate, HEPES, MOPS, MES, TRIS, phosphate, glycine, imidazole, borate, carbonate, and succinate. Isotonicity agents include salts such as sodium chloride, potassium chloride, sucrose, glycerin, dextrose, or mannitol.

[0109] In some embodiments, the composition comprises a compound of Formula I, or Formula 1a, 1b, 1c, or 1d, or a pharmaceutically acceptable salt thereof, encapsulated with a polyanion in primarily unilamellar vesicles formed from one or more phospholipids, a sterol, and a lipid optionally conjugated to a hydrophilic polymer (polymer-conjugated lipid). In some embodiments, the composition may comprise a compound of Formula I, or Formula 1a, 1b, 1c, or 1d, or a pharmaceutically acceptable salt thereof, encapsulated with a polyanion in unilamellar and multilamellar vesicles (e.g., having two or three layers). It should be understood that multilamellar vesicles may be cleared from the circulation more rapidly than unilamellar vesicles. In some embodiments, the phospholipid is hydrogenated soybean phosphatidylcholine (HSPC), distearoylphosphatidylcholine (DSPC), or egg sphingomyelin (ESM). As used herein, the term "phospholipid" refers to any one or combination of phospholipids capable of forming liposomes. Neutral phospholipids can include diacylphosphatidylcholine, dialkylphosphatidylcholine, sphingomyelin, and diacylphosphatidylethanolamine. Phosphatidylcholine (PC) includes those obtained from egg, soybean, or other plant sources, or partially or completely synthetic, or those with variable lipid chain length and unsaturation, and are suitable for use in the present composition.Synthetic, semi-synthetic, and natural phosphatidylcholine include, but are not limited to, distearoylphosphatidylcholine (DSPC), hydrogenated soybean phosphatidylcholine (HSPC), soybean phosphatidylcholine (soybean PC), egg phosphatidylcholine (egg PC), hydrogenated egg phosphatidylcholine (HEPC), dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC), and are suitable phosphatidylcholine for use in the present disclosure.Charged phospholipids can include phosphatidylserine, phosphatidic acid, phosphatidylinositol, phosphatidylglycerol, cardiolipin, or head group-modified lipids such as N-succinyl-phosphatidylethanolamine, N-glutaryl-phosphatidylethanolamine, and PEG-derivatized phosphatidylethanolamine.

[0110] Polymer-conjugated lipids include poly(ethylene glycol)-conjugated (PEGylated) phospholipids (PEG-lipids), such as PEG(molecular weight 2,000) methoxy-poly(ethylene glycol)-1,2-distearoyl-sn-glycerol (PEG(2000)-distearoylglycerol, PEG-DSG), PEG(molecular weight 2,000) 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-sphingolipids. The molecular weight of the PEG moiety in the PEG-lipid component can also vary from 500 to 10,000 g / mol, 1,500 to 6,000 g / mol, but is preferably approximately 2,000 MW. Other polymers used for conjugation to lipid anchors include poly(2-methyl-2-oxazoline) (PMOZ), poly(2-ethyl-2-oxazoline) (PEOZ), poly-N-vinylpyrrolidone (PVP), polyglycerol, poly(hydroxyethyl L-asparagine) (PHEA), and poly(hydroxyethyl L-glutamine) (PHEG).

[0111] In some embodiments, the sterol is cholesterol. Other exemplary sterols include, but are not limited to, phytosterols such as ergosterol and β-sitosterol, and hopanoids. In some embodiments, the ratio of phospholipid(s) to cholesterol is selected to provide a desired amount of liposome membrane rigidity while maintaining a sufficiently reduced amount of leakage of the compound of Formula I from the liposomes. In some embodiments, an optional polymer-conjugated lipid can be added to reduce the tendency of the liposomes to aggregate. The type and amount of polymer-conjugated lipid can be selected to provide a desired level of protein binding, liposome stability, and circulation time in the bloodstream. For example, liposome vesicles contain phosphatidylcholine (e.g., DSPC or HSPC) and cholesterol in a molar ratio of about 45:55. The molar ratio of phosphatidylcholine to cholesterol can vary from about 60:40 to 35:65, from about 50:50 to 35:65, or from about 50:50 to about 45:55. In particular, liposomes can contain vesicles composed of HSPC, cholesterol, and polymer-conjugated lipids (PEG-DSG or PEG-DSPE) in a molar ratio of approximately 55:45:2.75, corresponding to a 5 mol% PEG-lipid concentration relative to the phospholipid concentration. The PEG-lipid concentration can vary from 0.5 to 10 mol% relative to the (non-PEGylated) phospholipid, with a preferred ratio being 3 to 10 mol%, and an even more preferred ratio being 4 to 8 mol%.

[0112] In some embodiments, the liposome composition provides desirable pharmacokinetic properties, such as an extended plasma half-life, measured as the percentage of the injected dose (ID) (or injected amount) remaining in the blood 6 or 24 hours after intravenous injection in immunocompetent mice, and allows stable encapsulation of the drug in the plasma over 24 hours, as determined by the change in the drug-to-lipid ratio (DL ratio) after iv administration in mice. In some embodiments, the percentage of drug remaining in the blood is greater than 20%, preferably greater than 30%, and most preferably greater than 40% of the injected dose at 6 hours. The percent retained in the blood after 24 hours is preferably greater than 10%, more preferably greater than 20% of the injected dose. The DL ratio is greater than 20% at 24 hours, preferably greater than 50%, and most preferably greater than 80% of the original injected liposomal drug. Desirable liposome compositions also exhibit stable encapsulation in the presence of human plasma in vitro using a burst release method, with the liposomes retaining greater than 50% of the drug over 20 minutes, and greater than 60%, greater than 70%, preferably greater than 80%, and most preferably greater than 90% of the encapsulated drug over 20 minutes.

[0113] The liposomes of the present disclosure can be prepared by any method known in the art. For example, see G. Gregoriadis (editor), Liposome Technology, vol. st edition,1983; 2 nd edition,1993; 3 rdSee, e.g., "Cellulose Gel," ed., 2006; CRC Press, Boca Raton, Fla. Examples of methods suitable for producing liposome compositions of the present disclosure include membrane extrusion, reverse-phase evaporation, sonication, solvent (e.g., ethanol) injection (including microfluidics, Y-junction, and T-junction mixing), microfluidization, detergent dialysis, ether injection, and dehydration / rehydration. Liposome size can be controlled by controlling the pore size of the membrane used for extrusion, or the pressure and number of passes used in microfluidization or any other suitable method. In some embodiments, the desired lipids are first hydrated by thin-film hydration or ethanol injection, and then sized by extrusion through membranes of defined pore sizes, such as 50 nm, 80 nm, 100 nm, or 200 nm, or a combination thereof, to produce liposomes with an average size in the range of 70-150 nm, or 80-130 nm, and a polydispersity index of 0.1 or less. The drug compound to be encapsulated is added to liposomal lipid before liposome formation, and then dissolved in the aqueous medium that liposome is formed by the above-mentioned method, thereby isolating the drug in liposome.In some embodiments, drug compound is encapsulated in liposome by using a capture agent that is incorporated into the inner space of liposome (see Drummond, DC, et al. (2006) in: Liposome Technology, Third Edition (Ed. Gregoriadis, G.) Volume 2, p.149-168).

[0114] In some embodiments, a method for making a liposome composition of the present disclosure includes (i) preparing liposomes comprising phospholipids, cholesterol, and PEG-lipids, and having an interior space containing a sequestering agent in a medium substantially free of the sequestering agent; (ii) contacting the liposomes with a compound of the present disclosure in an aqueous medium to encapsulate the compound in the liposomes; (iii) removing the unencapsulated compound; and (iv) providing liposomes in a physiologically acceptable medium suitable for parenteral use. In some embodiments, a method for producing liposomes with a compound therein includes (a) preparing liposomes containing a sequestering agent consisting of an ammonium or substituted ammonium salt of a polyanion; (b) subsequently removing the sequestering agent outside the liposomes to form an electrochemical gradient across the membrane; and (c) contacting the liposomes with the compound under conditions effective to allow the compound to enter the liposomes and a corresponding amount of ammonia or substituted ammonia to leave the liposomes (thereby depleting or reducing the pH gradient across the resulting liposomes). Liposome compositions containing an entrapment agent inside the liposome can be prepared by forming liposomes in a solution of the entrapment agent. A transmembrane concentration gradient of the entrapment agent can be created across the liposome by removing the entrapment agent outside the liposome or by diluting the liposome either after liposome formation or before drug loading (entrapment).

[0115] In some embodiments, the contacting step involves incubating the liposomes with the drug in an aqueous medium at a temperature above ambient temperature but below the boiling point of water, preferably between 30°C and 90°C, 40°C and 80°C, 50°C and 80°C, or 60°C and 75°C. In some embodiments, the incubation is performed at an ionic strength less than that corresponding to 50 mM NaCl, or more preferably less than that corresponding to 30 mM NaCl. After incubation, concentrated salt, e.g., NaCl solution, can be added to raise the ionic strength to above 50 mM NaCl, or about 100 mM NaCl. Increasing the ionic strength after the drug-loading incubation step helped reduce post-loading aggregation of the liposomes. Incubation times can range from several minutes to several hours. In some embodiments, the incubation time is 5 to 40 minutes, 10 to 30 minutes, or 15 to 25 minutes. After incubation, the liposomes are cooled and then allowed to reach ambient temperature. In some embodiments, liposomes are cooled to 2-15°C. In some embodiments, liposomes are cooled to 4-10°C. After the cooling step, a concentrated salt, e.g., NaCl, solution can be added to increase the ionic strength to greater than 50 mM NaCl, or about 100 mM NaCl. Increasing the ionic strength after the drug loading incubation step helped reduce aggregation after liposome loading.

[0116] In some embodiments, the contacting also includes incubating the liposome with the drug in an aqueous medium in the presence of an osmotic (isotonic) balancing agent. In some embodiments, the osmotic balancing agent (osmotic agent) is a non-ionic substance. Exemplary non-ionic osmotic agents include, but are not limited to, dextrose (glucose), sucrose, trehalose, lactose, mannitol, sorbitol, and polyvinylpyrrolidone. In some embodiments, the concentration of the osmotic agent has an osmolarity (expressed as osmolarity) equal to the osmolarity of the sequestering agent solution in the interior space of the liposome before drug loading. The osmolarity of the sequestering agent solution can be measured by any known method before combining the solution with lipids to form liposomes. In another embodiment, the concentration of the osmotic agent is lower than the osmotic concentration of the sequestering agent solution, providing an osmotic concentration that is less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, or less than about 10% of the osmotic concentration of the sequestering agent solution. In yet another embodiment, the concentration of the osmotic agent during the drug loading process is in the range of 200-400 mmol / kg, preferably 250-350 mmol / kg. In yet another embodiment, the osmotic agent is dextrose at a concentration of 45 g / L. In yet another embodiment, no osmotic agent is used during the incubation of the liposomes with the drug. In yet another embodiment, the incubation is performed in the presence of an ionic strength adjuster. An example of an ionic strength adjuster is sodium chloride, which is added to the liposome-drug solution at a concentration of, for example, 5-50 mM, 10-20 mM, or about 10 mM. Contrary to convention in the liposome field, compounds of the present disclosure, such as AKG-28 and AKG-38, are stable and loaded into liposomes of the present disclosure in a highly efficient manner, even when the amount of osmotic agent during the drug-liposome contacting step provides an osmotic concentration lower than that of the scavenger solution (osmotically unbalanced liposomes) until the complete absence of added osmotic agent.

[0117] How to use Disclosed herein are methods for inhibiting the growth of mycobacteria, such as Mycobacterium tuberculosis, or Gram-positive bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA). Additional mycobacteria and Gram-positive bacteria include, but are not limited to, Mycobacterium avium complex, Mycobacterium leprae, Mycobacterium gordonae, Mycobacterium abscessus, Mycobacterium mucogenicum, streptococci, vancomycin-resistant enterococci (VRE), Staphylococcus pneumoniae, Enterococcus faecium, Group B Streptococcus, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus viridans, Listeria monocytogenes, Nocardia, and Corynebacterium sp. In some embodiments, the compounds and compositions provided herein inhibit the growth of drug-resistant strains of Mycobacterium tuberculosis. In some embodiments, methods for treating mycobacterial infections are provided. In some embodiments, the compounds and compositions provided herein can be used to treat nontuberculous mycobacterial infections. In some embodiments, the method comprises administering a therapeutically effective amount of an aminoalkyloxazolidinone and / or a pharmaceutically acceptable salt thereof to a subject in need thereof. In some embodiments, the method comprises administering a therapeutically effective amount of a liposome composition comprising an aminoalkyloxazolidinone compound and / or a pharmaceutically acceptable salt thereof to a subject in need thereof. In some embodiments, the composition is a liquid pharmaceutical formulation for parenteral administration. In some embodiments, the liquid pharmaceutical formulation is a liposome formulation containing an appropriate amount of an oxazolidinone compound described herein, wherein the oxazolidinone compound is encapsulated inside the liposome. In another embodiment, the compound is in the form of a salt with a polyanion, such as sulfate, citrate, sucrose octasulfate, or inositol hexaphosphate, inside the liposome. In some embodiments, the compound is a precipitated or gelled salt containing the sulfate salt inside liposomes composed of multiple lipid excipients, including, but not limited to, phosphatidylcholine, cholesterol, and PEGylated phosphatidylethanolamine. Liposomes of the present disclosure exhibit entrapment efficiencies of greater than 85%, greater than 90%, and greater than 95%.In some embodiments, residual unentrapped drug is removed from the liposome composition. This can be accomplished by various means, such as size exclusion chromatography, ion exchange, dialysis, ultrafiltration, tangential flow filtration, adsorption, or precipitation. During or after the unentrapped drug removal step, the liposomes can be incorporated into a desired pharmaceutically acceptable carrier, such as saline, isotonic dextrose, isotonic sucrose, Ringer's solution, or Hank's solution. Buffer substances can be added to achieve a desired physiologically acceptable pH. The liposome composition can be adjusted to the desired drug concentration and sterilized, for example, by sterile filtration through a 0.2-0.22 μm filter. In some embodiments, the compound concentration in the liposome composition ranges from 1 to 50 mg / ml, 3 to 30 mg / ml, or 5 to 25 mg / ml.

[0118] In some embodiments, the liposomes are mixed with one or more additional excipients for tonicity or pH control, including, but not limited to, sodium chloride, 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., tablets, capsules, pills, dragees, caplets, etc.). For oral use, for example, tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups, or elixirs can be prepared (Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, Pa.)). Compositions intended for oral use can be prepared according to any method known in the art for the manufacture of pharmaceutical compositions. The composition can contain one or more agents, such as antioxidants, sweeteners, flavoring agents, coloring agents, and preservatives, to provide a palatable preparation. Tablets containing the active ingredient mixed with non-toxic pharmaceutically acceptable excipients or adjuvants suitable for the manufacture of tablets are acceptable. Suitable excipients or adjuvants include, but are not limited to, for example, inert diluents, solubilizing agents, suspending agents, adjuvants, wetting agents, sweetening agents, flavoring or flavoring substances, isotonic substances, colloidal dispersing agents, and surfactants.

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

[0121] The compounds or compositions may be administered topically, orally, parenterally, intraperitoneally, and / or 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 the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.

[0123] The dosage of the compounds and / or their pharmaceutically acceptable salts, or liposomes containing the compounds and / or their pharmaceutically acceptable salts, can vary within wide limits and will naturally have to be adjusted in each particular case to the individual conditions and the pathogens to be controlled.

[0124] In some embodiments, for use in treating bacterial infections, the compound or pharmaceutical liposomal composition is administered to a subject in need thereof 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 (i.e., once every 4 weeks), or once every 42 days (i.e., once every 6 weeks). In some embodiments, the average weekly dosage is from about 1 mg to about 1500 mg, from about 10 to about 700 mg, from about 25 to about 500 mg, or from about 70 to about 250 mg. In some embodiments, the average weekly dosage is about 1 mg to about 10 mg, about 10 mg to about 25 mg, about 25 mg to about 50 mg, about 50 mg to about 100 mg, about 100 mg to about 200 mg, about 200 mg to about 300 mg, about 300 mg to about 400 mg, about 400 mg to about 500 mg, about 500 mg to about 600 mg, about 600 mg to about 700 mg, about 700 mg to about 800 mg, about 800 mg to about 900 mg, about 900 mg to about 1000 mg, about 1000 mg to about 1100 mg, about 1100 mg to about 1200 mg, about 1200 mg to about 1300 mg, about 1300 mg to about 1400 mg, or about 1400 mg to about 1500 mg. In some embodiments, the compound or composition is administered for up to one month, up to two months, up to three months, up to four months, or more. The specific therapeutically effective amount depends on various factors, such as the bacterial infection being treated, the activity of the specific compound administered, the pharmaceutical composition employed, the age, weight, sex, etc., of the subject, the route of administration, the severity of the bacterial infection, optional drugs / active agents used in combination (sequentially or simultaneously) with the specific compound, and similar factors known to a physician of ordinary skill in the art. In some embodiments, the compound or composition can be used to treat tuberculosis or other mycobacterial infections. In some embodiments, the compound can be used as monotherapy. In some embodiments, treatment can include simultaneous and / or sequential administration of an effective amount of a compound described herein and an effective amount of one or more additional active agents for treating Mycobacterium tuberculosis and other gram-positive bacterial infections.In some embodiments, treatment can involve simultaneous and / or sequential administration of an effective amount of a compound described herein and two or more (2, 3, 4, etc.) additional active agents in effective amounts for treating Mycobacterium tuberculosis and other Gram-positive bacterial infections. A synergistic antibacterial effect refers to an antibacterial effect that is greater than the expected purely additive effect of the individual compounds of the combination. When administered simultaneously, the compound and the additional active agent can be contained in the same composition or in separate compositions. When administered sequentially, the composition containing the compound and the composition containing the additional active agent can be administered with a time separation (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 or by a different injection. For example, a compound of the present disclosure can be administered intravenously, and one or more additional agents can be administered orally.

[0125] In some embodiments, administration of the compound with one or more (e.g., 1, 2, 3, or 4) additional active agents can result in a reduction in the length of the treatment period. For example, administration of the compound with one or more (e.g., 1, 2, 3, or 4) additional active agents can shorten the treatment period by at least 3 times, at least 2 times, or at least 1.5 times compared to treatment with only one active agent. In some embodiments, the additional agent(s) is an antibacterial agent. In some embodiments, the additional active agent includes, but is not limited to, fluoroquinolines such as moxifloxacin, gatifloxacin, or levofloxacin, bedaquiline and other diarylquinoline analogs (e.g., TBAJ-587 and TBAJ-876), delamanid, pretomanid, isoniazid, rifampicin, rifapentine, pyrazinamide, clofazimine, spectinamide, ethambutol, streptomycin, cannabinoids, fluticasone, fluoxetine, fluoxetine, fluoxetine-1, fluoxetine-2, fluoxetine-3, fluoxetine-4, fluoxetine-5, fluoxetine-6, fluoxetine-7, fluoxetine-8, fluoxetine-9, fluoxetine-10, fluoxetine-11, fluoxetine-12, fluoxetine-13, fluoxetine-14, fluoxetine-15, fluoxetine-15, fluoxetine-16, fluoxetine-17, fluoxetine-18, fluoxetine-19, fluoxetine-20, fluoxetine-21, fluoxetine-22, fluoxetine-23, fluoxetine-24, flu The inhibitors can include mycin, capreomycin, amikacin, the leucyl-tRNA synthetase (LeuRS) inhibitor GSK3036656, the tryptophan synthase inhibitor GSK839, the DprE1 inhibitors OPC-167832 and macodinone (PBTZ-169), Telacebec, GSK-656, TBA-7371, and amoxicillin plus clavulanate, pharmaceutically acceptable salts of each thereof, and any combination thereof. For the treatment of Gram-positive bacterial infections, additional active agents may include, but are not limited to, vancomycin, gentamicin, daptomycin, teicoplanin, ceftaroline, ceftrobiprole, telavancin, dalbavancin, oritavancin, fluoroquinolines (e.g., delafloxacin), tetracyclines (e.g., eravacycline and omadacycline), sulfonamides (e.g., sulfamethoxazole), trimethoprim, lefamulin, and any combination thereof. In some embodiments, treatment may involve simultaneous and / or sequential administration of an effective amount of a compound described herein and an effective amount of bedaquiline, pretomanid, pyrazinamide, moxifloxacin, or a pharmaceutically acceptable salt of each thereof, or a combination of the foregoing.

[0126] Actual dosage levels of the active ingredients in the pharmaceutical compositions disclosed herein can be varied to provide an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without causing toxicity to the patient.

[0127] As used herein in the context of administration, "parenteral" means modes of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

[0128] As used herein, the phrases "parenteral administration" and "parenterally administered" refer to modes of administration other than enteral (i.e., via the digestive tract) and topical administration, usually by injection or infusion, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, inhalation, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion. Intravenous injection and infusion are often (but not exclusively) used for liposome drug administration.

[0129] In some embodiments, the liquid composition is administered intravenously. In some embodiments, the compound or pharmaceutical composition is administered to a subject in need thereof 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 (i.e., once every 4 weeks), and once every 42 days (i.e., once every 6 weeks). In some embodiments, the average weekly dose is about 1 mg to about 1500 mg, 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 dose is about 1 mg to about 10 mg, about 10 mg to about 25 mg, about 25 mg to about 50 mg, about 50 mg to about 100 mg, about 100 mg to about 200 mg, about 200 mg to about 300 mg, about 300 mg to about 400 mg, about 400 mg to about 500 mg, about 500 mg to about 600 mg, about 600 mg to about 700 mg, about 700 mg to about 800 mg, about 800 mg to about 900 mg, about 900 mg to about 1000 mg, about 1000 mg to about 1100 mg, about 1100 mg to about 1200 mg, about 1200 mg to about 1300 mg, about 1300 mg to about 1400 mg, or about 1400 mg to about 1500 mg. The specific therapeutically effective amount will depend on a variety of factors, such as the bacterial infection being treated, the activity of the specific compound being administered, the pharmaceutical composition employed, the age, weight, sex, etc. of the subject, the route of administration, the severity of the bacterial infection, any optional drugs / active agents used in combination (sequentially or simultaneously) with the specific compound, and similar factors known to a physician of ordinary skill in the art.

[0130] In some embodiments, compound or pharmaceutical oral compositions is used for treating bacterial infection, administered once or twice a day.Specific therapeutically effective amount depends on various factors, for example, the bacterial infection to be treated, the activity of the specific compound to be administered, the pharmaceutical composition to be used, the age, weight, sex etc. of the subject, the route of administration, the severity of bacterial infection, the optional drug / active agent that is used in combination with specific compound (sequentially or simultaneously), and the similar factors that are known to the physician skilled in the art. [Example]

[0131] The following examples, including the experiments conducted and results achieved, are provided for illustrative purposes only and are not to 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 were synthesized by reacting tedizolid mesylate (tedizolid-MS) with the respective amines in N-methyl-2-pyrrolidone (NMP) as a solvent at 60 °C (Scheme 1). Tedizolid-MS was obtained by mesylation of the 1° hydroxyl group of tedizolid with methanesulfonyl chloride in the presence of a base at room temperature (RT). Treatment of tedizolid-MS with sodium azide followed by reduction of the resulting azide (AKG-3-A) afforded intermediate 1 as the free base or AKG-3 as the hydrochloride salt, depending on the eluent selected for purification. Amidation of intermediate 1 with the corresponding acid followed by hydrochloride salt formation using HCl / EtOAc afforded compounds AKG-17 and AKG-18. Reaction of tedizolid with the corresponding dialkylamino acids under standard esterification conditions gave compounds AKG-5 and AKG-20. O-Alkylation of tedizolid with 2-chloro-N,N-diethylaminoethylamine using sodium hydride as the base gave compound AKG-7.

[0134] Intermediate 2 was synthesized by boronation of commercially available aryl bromides using bis(pinocolato)diboron (Scheme 2). Suzuki coupling of intermediate 2 with readily available 5-bromo-2-fluoropyridine gave intermediate 3, which was heated with the corresponding amine in NMP in a sealed tube to give compounds AKG-11–AKG-15.

[0135] Compounds AKG-16, AKG-21 to AKG-27 were prepared in a convergent synthesis starting from intermediate 4 (Schemes 3 and 4). Click chemistry using sodium azide on 5-bromo-2-cyanopyridine gave intermediate 4. N-Alkylation of the tetrazole in intermediate 4 gave intermediates 5 and 6 in a 3:1 ratio. The structures of these intermediates were deduced from HMBC analysis. Intermediates 7 to 12 were synthesized, and regioisomers were obtained in a similar manner (only the desired isomer is shown in Scheme 4). Suzuki coupling of intermediates 5 to 12 with intermediate 2 and deprotection of the amine group, where applicable, gave compounds AKG-16, AKG-21 to AKG-27.

[0136] Intermediate 13 was synthesized by mesylation of readily available aryl bromides. Intermediate 14 was reduced with hydrazine to give intermediate 15 (Scheme 5). Boc protection or acetylation of the primary amine in intermediate 15, followed by boronation, gave intermediates 18 and 19, respectively. Suzuki coupling of the boronic acid intermediate with the corresponding aryl bromide intermediate (U.S. Patent Application Publication No. 20100022772, PCT International Patent Application Publication No. WO2013044845, which are incorporated by reference in their entireties) and deprotection of the amine group, where applicable, gave compounds AKG-28 to AKG-31 and AKG-38 to AKG-40.

[0137] Synthesis scheme For the synthesis of intermediate 19, see U.S. Patent Application Publication No. 20100022772, PCT International Patent Application Publication No. WO2013044845, which are incorporated by reference in their entireties.

[0138] [ka]

[0139] [ka]

[0140] [ka]

[0141] [ka]

[0142] [ka]

[0143] synthesis material and method Tedizolid, (R)-3-(4-bromo-3-fluorophenyl)-5-(hydroxymethyl)oxazolidin-2-one, was purchased from Skychemical, dimethyl-(2-piperidin-4-yl-ethyl)-amine was purchased from Enamine, and other reagents and solvents were purchased from Adams and used as received. The chemical structure of the final product was confirmed by nuclear magnetic resonance spectroscopy (NMR) determined on a Bruker NMR spectrometer (500 MHz or 400 MHz). 1 H NMR, 13 It was characterized by C NMR. 13 C NMR spectra were fully resolved, and chemical shifts were expressed in parts per million (ppm) using the deuterated solvent peak or tetramethylsilane (internal) as the internal standard. 1 H NMR data are reported as follows: chemical shift (d, ppm), multiplicity (s, singlet; br s, broad singlet; d, doublet; t, triplet; 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. Analytical HPLC was performed on an Agilent analytical HPLC system using a Sunfire column, 3.5 μm (150 cm × 4.6 mm), and a gradient system (water (0.01% TFA) / ACN (0.01% TFA)) with a flow rate of 1 mL / min, with detection at 254 and 214 nm. Flash chromatography (FC) purification was performed using silica gel 60 (0.04–0.063 nm; 230–400 mesh) from Santai Technologies.

[0145] Procedure A. A reaction mixture of tedizolid-MS (1.0 equiv.), R1R2NH (4.0 equiv.) in NMP (10 mL) was heated to 60° C. in a sealed tube for 15 h. Upon completion (LCMS), the reaction was diluted with HO (40 mL) and extracted with EtOAc (2×50 mL). The combined extracts were washed with saturated brine, dried over Na2SO4, and filtered. The solvent was removed under reduced pressure, and the residue was purified using FC to give the product in greater than 95% purity.

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

[0147] To a solution of tedizolid (7.00 g, 18.90 mmol) and triethylamine (3.83 g, 37.80 mmol) in CHCl (50 mL) at 0° C. under Ar was added methanesulfonyl chloride (3.25 g, 28.36 mmol) dropwise at 0° C. After stirring at RT for 2 h, the reaction mixture was poured into water and extracted with CHCl. ​​The organic layer was washed with brine, dried over NaSO, and collected by filtration. The solvent was removed under reduced pressure to give the pure product tedizolid-Ms (7.0 g, 82.6% yield) as a yellow solid. 1H 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, AKG-1 was obtained from tedizolid-Ms and dimethylamine as a white solid (0.5 g, 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, 149.9, 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, AKG-2 was obtained from tedizolid-Ms and dimethylamine as a white solid (0.52 g, 54.8% yield). 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] Using procedure A, AKG-6 was obtained from tedizolid-Ms and N,N-dimethyl-2-(piperidin-4-yl)ethan-1-amine as a white solid (0.66 g, 58.2% yield). 1H 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, 10.9Hz, 2H), 2.80 - 2.64 (m, 2H), 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). 13 C NMR (101 MHz, CDCl3) δ 164.7 , 161.3, 158.8 , 154.3 , 149.9 , 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 509.2 ([M + 1] + ).

[0154] [ka]

[0155] Tedizolid-Ms and N using procedure A 1 ,N 1 -Diethylpropane-1,3-diamine gave AKG-8 as a white solid (0.62 g, 57.6% yield). 1H NMR (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 MHz, DMSO-d6) δ 164.3, 161.0, 158.6, 154.6, 149.9, 145.5, 141.0, 137.6, 132.1, 131.4, 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-diamine to give AKG-9 as a white solid (0.36 g, 34.4% yield). 1H 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, 2H), 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, AKG-19 was obtained from tedizolid-Ms and ethane-1,2-diamine as a white solid (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, 2H), 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). 13C 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) was added NaN (0.44 g, 6.69 mmol). After stirring at 90 °C for 3 h, the reaction mixture was poured into water and extracted with EtOAc. The organic layer was washed with brine, dried over anhydrous MgSO, filtered, and concentrated under reduced pressure. The residue was further purified by column chromatography to give the title compound AKG-3-1 (0.7 g, 79.4% yield) as a white solid.

[0161] [ka]

[0162] A reaction mixture of AKG-3-1 (0.7 g, 1.77 mmol) and PhP (1.39 g, 5.31 mmol) in HO (2 mL) and THF (20 mL) was heated to reflux for 1 h. After completion (LCMS), the reaction was concentrated under reduced pressure and purified using reverse-phase FC. Purification using 0-10% MeOH in DCM as the eluent and lyophilization afforded the free base Intermediate 1 (2.5 g, 76.5% yield) as a yellow solid. FC purification using 0-30% MeCN in 0.006 M HCl in HO as the eluent afforded the hydrochloride salt AKG-3 (0.35 g, 48.8% yield) as a yellow solid after lyophilization. 1H 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

change

[0164] To a solution of 3-((tert-butoxycarbonyl)amino)propanoic acid (0.62 g, 3.25 mmol, 1.2 equiv.) and TEA (0.63 g, 6.25 mmol, 2.5 equiv.) in DMF (10 mL) was added HATU (1.44 g, 3.78 mmol, 1.4 equiv.) under Ar at RT. The mixture was stirred for 0.5 h, and then Intermediate 1 (1.0 g, 2.70 mmol, 1.0 equiv.) was added. The whole mixture was stirred at RT overnight. LCMS showed that the reaction was complete, and it was poured into HO, and the solid was collected by filtration and washed with HO. The solid was dried under reduced pressure, and the residue was used in the next step by dissolving it in EtOAc, followed by the addition of HCl / EtOAc (4 M, 20 mL). The whole mixture was stirred for 16 h, and the solvent was removed by N. The residue was purified by reversed-phase FC (eluent with 0-30% MeCN in 0.006 M HCl in H2O) to give the product AKG-17 (0.5 g, 39.5% yield) as a yellow solid after lyophilization. 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). 13C 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 for AKG-17, AKG-18 was obtained from intermediate 1 and 4-((tert-butoxycarbonyl)amino)butanoic acid as a yellow solid (0.5 g, 37.6% yield). 1 H 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] To a mixture of tedizolid (1.0 g, 2.70 mmol), 4-(dimethylamino)butanoic acid hydrogen chloride (0.57 g, 3.37 mmol), and TEA (0.27 g, 2.70 mmol) under N was added a catalytic amount of DMAP, DCC (0.84 g, 4.05 mmol) in DMF (20 mL). The mixture was stirred at RT for 16 h. Upon completion of the reaction (LCMS), it was diluted with HO (100 mL) and filtered. The filtrate was acidified to pH = 5-6 with 0.02 M HCl and then purified using RP-FC (eluent: MeCN in 0.5% formic acid / HO) to give the product AKG-5 as the formate salt after lyophilization. The product was redissolved in HO, 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). 13C 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] To a mixture of tedizolid (1.0 g, 2.70 mmol) in DMF (20 mL) was added NaH (0.13 g, 60%, 5.40 mmol) at RT under N. The mixture was stirred at 0 °C for 0.5 h, after which 2-diethylaminoethyl chloride hydrochloride (930 mg, 5.40 mmol) was added in one portion. The entire mixture was stirred at RT for 3 h. LCMS showed the reaction was complete. The reaction was carefully poured into ice / HO (20 mL) and extracted with DCM (2 × 50 mL). The combined organic extracts were washed with saturated brine and then dried over NaSO. The solvent was removed under reduced pressure, and the residue was purified using FC (eluent with MeOH in DCM 0-15%) to give AKG-7 as a white solid (0.5 g, 39.4% yield). 11H 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 13C 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

Chemical Structure

[0172] To a reaction mixture of tedizolid (1.0 g, 2.70 mmol), 4-(diethylamino)butanoic acid hydrogen chloride (0.61 g, 3.37 mmol), and DMAP (0.05 g) in DMF (20 mL) was added DCC (0.84 g, 4.05 mmol) at RT under N. The mixture was stirred at RT for 16 h. Upon completion (LCMS), the reaction was diluted with HO (100 mL) and filtered. The filtrate was acidified to pH = 5-6 with 0.02 M HCl and then purified using RP-FC (eluent: MeCN in 0.5% FA / HO) to give the product as the formate salt after lyophilization. The salt was then redissolved in HO, and 1 equivalent of HCl (0.02 M) was added. After lyophilization, the product AKG-20 was obtained as the HCl salt (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 - 4.97 (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 = 7.2Hz, 6H). 13 C NMR (101 MHz, DMSO-d6) δ 171.00, 164.33, 158.55, 154.32, 149.90, 145.58, 140.71, 137.63, 132.02, 131.45, 122.58, 119.33, 114.69, 106.21, 71.01, 65.04, 46.86, 46.63, 40.31, 29.99, 9.95(s).

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

[0174] A mixture of (R)-3-(4-bromo-3-fluorophenyl)-5-(hydroxymethyl)oxazolidin-2-one (9.0 g, 31.02 mmol), bis(pinacolato)diboron (11.88 g, 46.54 mmol), and KOAc (4.56 g, 46.54 mmol) in dioxane (200 mL) was purged with Ar for 10 minutes, and then (PhP)PdCl (1.09 g, 1.55 mmol) was added. The mixture was purged with Ar again, and then heated to 90 °C for 15 hours. LCMS showed the reaction was complete. It was cooled to room temperature and filtered over Celite to give intermediate 2 as the filtrate. To the filtrate was added 5-bromo-2-fluoropyridine (6.55 g, 37.22 mmol), K3PO4 (14.47 g, 6.80 mmol), and HO (20 mL). The mixture was purged with Ar for 10 minutes, and (dppf)PdCl2 (2.27 g, 3.10 mmol) was added. The mixture was purged with Ar again. It was then heated to 90 °C for 15 hours. The reaction was monitored by LCMS. Upon completion, it was concentrated under reduced pressure, and the residue was diluted with HO (200 mL) and extracted with EtOAc (2 x 200 mL). The combined extracts were washed with saturated brine and then dried over Na2SO4. Filtration and removal of the solvent under reduced pressure gave a residue which was purified using FC (eluent with MeOH in DCM 0-15%) to give the product intermediate 3 (6.8 g, 71.6% yield for two steps) as a yellow solid. 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 - 4.71 (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 Procedure B was used. Intermediate 3 (1.0 equiv.), R1R2NH (4.0 equiv.), and a catalytic amount of DMAP in NMP (10 mL) were heated to 100 °C in a sealed tube for 16 h. Upon completion of the reaction (LCMS), it was diluted with HO (50 mL) and extracted with EtOAc (2 x 50 mL). The combined organic extracts were washed with saturated brine, dried over Na2SO4, and filtered. The solvent was removed under reduced pressure, and the residue was purified using RPFC (MeCN in 0.1% NH4HCO3 / HO, 0-40%, eluent with C18) to give the product.

[0176] [ka]

[0177] Using procedure B, AKG-11 was obtained from intermediate 3 and N,N-dimethyl-2-(piperidin-4-yl)ethan-1-amine as a white solid (0.40 g, 30.1% yield). 1H 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), 6.89 (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 13C NMR (101 MHz, DMSO-d6) δ 160.68, 158.33, 154.81, 147.54, 139.15, 137.82, 130.32, 120.72, 119.11, 114.35, 106.97, 106.03, 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 give AKG-12 as a white solid (0.52 g, 42.6% yield). 1H 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), 4.11 (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, 138.92, 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 gave AKG-13 as a white solid (0.68 g, 51.9% yield). 1H 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, 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 to give AKG-14 as a white solid (0.6 g, 47.3% yield). 1H 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, 2H), 2.28 (t, J = 7.1 Hz, 2H), 2.13 (s, 6H), 1.72 - 1.62 (m, 2H). 13 C NMR (101 MHz, DMSO-d6) δ 160.60, 158.62, 158.18, 154.81, 147.62, 138.89, 137.08, 130.19, 121.21, 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 gave AKG-15 as a white solid (0.65 g, 48.0% yield). 11H NMR (400 MHz, DMSO-d6) δ 8.16 (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 13C NMR (101 MHz, DMSO-d6) δ 160.60, 158.65, 158.18, 154.81, 147.64, 138.89, 137.05, 130.18, 121.21, 118.37, 114.29, 108.01, 106.02, 105.74, 73.80, 62.09, 50.81, 46.80, 46.45, 40.11, 27.10, 12.23. MS (ESI+) m / z 417.1 ([M + 1] + ).

[0186] 11. Synthesis of AKG-16

[0187]

Chemical Structure

[0188] [ka]

[0189] A mixture of intermediate 4 (10.0 g, 44.25 mmol) and Ca(OH) (7.20 g, 97.35 mmol) in HO (150 mL) and DMF (20 mL) was stirred at rt for 0.5 h, after which (2-bromoethyl)dimethylamine hydrobromide (25.0 g, 107.3 mmol) was added. The mixture was heated at 80 °C for 24 h. LCMS showed a 3:1 mixture of intermediates 5 and 6, respectively. The mixture was diluted with HO (40 mL) and extracted with EtOAc (2 × 50 mL). The combined extracts were washed with saturated brine, dried over NaSO, and filtered. The solvent was removed under reduced pressure, and the residue was purified using FC (eluent with MeOH in DCM 0-15%) to give the crude product. The crude product was further purified by RPFC (MeCN 0-30% in 0.1% NH4HCO3 / HO, C18, intermediate 5 eluted first, then intermediate 6) to give intermediate 5 (0.74 g, 5.6% yield) as a white solid and intermediate 6 (0.25 g, as a light yellow solid).

[0190] Intermediate 5: 1 H 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, 151.48, 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: 1H 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] A mixture of freshly prepared Intermediate 2 (1.68 g, 4.98 mmol) (from 1.44 g of (R)-3-(4-bromo-3-fluorophenyl)-5-(hydroxymethyl)oxazolidin-2-one using the procedure for Intermediate 3), Intermediate 5 (740 mg, 2.49 mmol), and KPO (1.16 g, 5.48 mmol) in dioxane (50 mL) and HO (5 mL) was purged with Ar for 10 min. To this was added (dppf)PdCl (182 mg, 0.25 mmol). The mixture was purged with Ar again. It was then heated to 90 °C for 15 h. LCMS showed the reaction was complete. It was concentrated under reduced pressure, and the residue was diluted with HO (200 mL) and extracted with EtOAc (2 × 200 mL). The combined extracts were washed with saturated brine, dried over NaSO, and filtered. The solvent was removed under reduced pressure, and the residue was purified using RPFC (eluent with MeCN in HO, 0-40%) to give the product AKG-16 (520 mg, 49.0% yield) as a white solid. 1H 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

change

[0194] A solution of intermediate 2 (1.5 g, 4.5 mmol), intermediate 6 (0.9 g, 3 mmol), Pd(dppf)Cl (247 mg, 0.3 mmol), and KPO (1.3 g, 6 mmol) in dioxane (30 mL) and HO (5 mL) was purged with Ar for 10 min and heated to 100 °C for 15 h. Upon completion of the reaction (LCMS), it was concentrated under reduced pressure, and the residue was diluted with HO (100 mL) and extracted with EtOAc (2 × 50 mL). The combined extracts were washed with saturated brine, dried over NaSO, and filtered. The solvent was removed under reduced pressure, and the residue was purified using FC (eluent with 0–10% MeOH in DCM (10% NHOH)) to give AKG-21 (450 mg as a white solid) in 35% yield. 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). 13C NMR (101 MHz, DMSO-d6): δ 161.09, 158.64, 154.80, 152.10, (149.45, 149.40), 143.46, (141.35, 141.23), (138.19, 138.15), (132.77, 132.76), (131.53, 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] To a mixture of intermediate 7 (500 mg, 1.354 mmol) in HO (2 mL) and dioxane (8 mL) was added intermediate 2 (685 mg, 2.03 mmol), KPO (862 mg, 4.06 mmol), and (dppf)PdCl (99 mg, 0.135 mmol). The flask was evacuated and backfilled with Ar. The mixture was then stirred at 90 °C for 16 h. Water (20 mL) was added and extracted with EtOAc (2 × 20 mL). The organic layer was washed with brine, dried over NaSO, filtered, and concentrated. The residue was purified by silica gel column chromatography (Biotage, 40 g silica gel column @ 30 mL / min, eluted with 0–100% EtOAc in petroleum ether) to give the desired product AKG-22-1 (450 mg, yield: 66%) as a gray solid.

[0197] [ka]

[0198] To a mixture of AKG-22-1 (450 mg, 0.9 mmol) in DCM (8 mL) was added 4 M HCl / dioxane (2 mL). The mixture was then stirred at RT for 5 h. The solvent was removed under reduced pressure to give the desired product AKG-22 (390 mg, yield: 99%) as a gray solid. 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 (400MHz, 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] To intermediate 8 (1.0 g, 2.71 mmol) in 20 mL of 1,4-dioxane and 5 mL of HO was added intermediate 2 (4.86 mmol, 1.63 g), KPO (1.14 g, 5.42 mmol), and (dppf)PdCl (0.23 g, 0.27 mmol), and the mixture was stirred at 100 °C for 16 h. After the starting material was consumed, 100 mL of saturated NaHCO was added. The aqueous phase was extracted with EtOAc (3 × 30 mL), and the combined organic extracts were washed with HO, concentrated under reduced pressure, and purified by FC to give the desired compound AKG-23-1 (1.0 g, 70% yield).

[0201] [ka]

[0202] To AKG-23-1 (1.0 g, 2 mmol) in 30 mL of DCM was added 1 mL of HCl (4 M in 1,4-dioxane), and the mixture was stirred for 1 hour. After the starting material was consumed, the mixture was filtered to obtain the crude product. The crude product was stirred in 3 mL of MeOH for 1 hour and filtered to obtain the desired product AKG-23 (0.53 g, 63% yield) as a white solid. 1 H 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 = 12.3, 3.2 Hz, 1H), 3.61-3.57 (dd, J = 12.3, 3.2 Hz, 1H), 3.53 (m, 3H). 13C NMR (125 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] A mixture of intermediate 2 (1.66 g, 4.98 mmol), intermediate 9 (800 mg, 2.49 mmol), and KPO (1.16 g, 5.48 mmol) in dioxane (50 mL) and HO (5 mL) was purged with Ar for 10 minutes, and (dppf)PdCl (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; it was concentrated under reduced pressure, and the residue was diluted with HO (200 mL) and extracted with EtOAc (2 × 200 mL). The combined extracts were washed with saturated brine, then dried over NaSO, and filtered. The solvent was removed under reduced pressure and the residue was purified using RPFC (eluent with MeCN in HO, 0-40%) to give the product AKG-24 (440 mg, 39.0% yield) as a white solid. 1H 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

change

[0206] To a mixture of intermediate 4 (2.25 g, 10 mmol) and K2CO3 (5.52 g, 40 mmol) in DMF (20 mL) was added 3-chloro-N,N-dimethylpropan-1-amine hydrochloride (3.95 g, 25 mmol), and the mixture was heated to 80 °C for 4 h. It was diluted with HO (40 mL) and extracted with EtOAc (2 x 100 mL). The combined extracts were washed with saturated brine, then dried over Na2SO4 and filtered. The solvent was removed under reduced pressure, and the residue showed the presence of two regioisomers of N-alkylation. The isomers were separated using FC (eluent with MeOH in DCM 0-15%) to give intermediate 10 (0.98 g, 31.6% yield) 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] A mixture of intermediate 10 (0.74 g, 2.4 mmol), intermediate 2 (1.62 g, 4.8 mmol), and KPO (1 g, 4.8 mmol) in dioxane (30 mL) and HO (5 mL) was purged with Ar for 10 min, and Pd(dppf)Cl (175 mg, 0.24 mmol) was added. The mixture was purged with Ar again and heated to 90 °C for 15 h. It was concentrated under reduced pressure, and the residue was diluted with HO (80 mL) and extracted with EtOAc (2 × 100 mL). The combined extracts were washed with saturated brine, then dried over NaSO, and filtered. The solvent was removed under reduced pressure, and the residue was purified using FC (eluent with MeOH in DCM 0–15%) to give the product AKG-25 (0.73 g, 69.5% yield) as a gray solid. 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. + ).

[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 (3-chloropropyl)diethylamine hydrochloride (8.23 g, 55.30 mmol) and K2CO3 (9.17 g, 66.36 mmol) at 80 °C for 3 h. The reaction was cooled, poured into an ice-water bath, and extracted with EA (2 × 200 mL). The combined organic phases were washed with brine (2 × 50 mL) and dried over Na2SO4. Upon removal of the solvent, the crude product containing the N-alkylated regioisomer was purified by FC (PE / EA = 1:10) to give intermediate 11 (1.70 g, 22.65%) as a white 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] A mixture of Intermediate 11 (0.68 g, 2.00 mmol), Intermediate 2 (1.07 g, 3.99 mmol), potassium phosphate tribasic (0.85 g, 3.985 mmol), and Pd(dppf)Cl (0.15 g, 0.20 mmol) was suspended in 1,4-dioxane:water (12 mL, 6:1). The reaction was stirred under reflux for 16 h. The mixture was partitioned between EtOAc (2×100 mL) and water, washed with brine, dried over NaSO, and filtered. Upon removal of the solvent, the residue containing the regioisomers was purified using FC eluting with (DCM / MeOH=20 / 1) to give AKG-26 (0.54 g, 56.04%) as a gray solid. 1 H NMR (500 MHz, DMSO-d6) δ8.95 (s, 1H), 8.26-8.19 (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 Hz, 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), 137.64 (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 BocNH(CH)Br (16.6 g, 69.71 mmol) and KCO (11.1 g, 80.02 mmol) at 80 °C for 3 h. The reaction was cooled, poured into an ice-water bath, and extracted with EtOAc (2 × 200 mL). The organic phase was washed with brine (2 × 50 mL), dried over NaSO, filtered, and the solvent was evaporated under reduced pressure. The crude product, containing the N-alkylated regioisomer, was purified by FC (PE / EA = 2:1) to give intermediate 12 (14 g, 13.1%) as a yellow solid. 1 H NMR (400 MHz, DMSO-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, MS (ESI+) m / z 383.0 ([M + 1] + ).

[0215] [ka]

[0216] A solution of intermediate 12 (0.83 g, 2.15 mmol), NaHCO (0.36 g, 4.31 mmol), and intermediate 2 (1.24 g, 3.68 mmol) was suspended in 1,4-dioxane (32 mL) and water (8 mL). N was bubbled through the mixture for 5 minutes, and then Pd(dppf)Cl (0.078 g, 0.095 mmol) was added. The mixture was stirred at 90 °C for 15 hours and then cooled to RT. The mixture was partitioned between EtOAc (2 × 100 mL) and water. The organic layer was dried over NaSO, filtered, and concentrated. The filtrate was concentrated and purified by silica gel column chromatography (DCM / MeOH = 20 / 1) on silica gel to give AKG-27-1 (0.75 g; 66.9%) as a white solid. 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] To a solution of AKG-27-1 (0.9 g, 1.75 mmol) in dry DCM (16 mL) was added HCl in dioxane (4.0 mL) at RT under a N atmosphere. The reaction mixture was stirred at the same temperature for 6 h and cooled to RT. The solvent of the reaction mixture was evaporated under reduced pressure to give AKG-27 (0.65 g, 82.5%) 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 To a solution of (R)-3-(4-bromo-3-fluorophenyl)-5-(hydroxymethyl)oxazolidin-2-one (9 g, 31 mmol) in DCM (100 mL) was added (3.92 g, 34 mmol) and TEA (3.76 g, 37 mmol). The mixture was stirred at RT for 2 h. The mixture was washed 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 1,3-dioxoisoindolin-2-ide (6.02 g, 32 mmol). The mixture was stirred at 90° C. overnight. The mixture was cooled, poured into water (1000 mL), and stirred for 0.5 h. The precipitate was collected and dried under reduced pressure to give intermediate 14 (11 g, 85% yield). 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 NHNH-H0 (85%, 7.7 g, 131 mmol). The mixture was stirred at 90 °C overnight. The mixture was filtered and rinsed with EtOH (2 x 50 mL). The filtrate was concentrated to give intermediate 15 (7.6 g, 100% yield). MS (ESI+) m / z 289 ([M + 1] + ).

[0222] To a solution of intermediate 15 (7.6 g, 26.4 mmol) in THF (50 mL) and water (50 mL) was added (Boc)O (6.9 g, 32 mmol) and KCO (7.29 g, 52.8 mmol), and the mixture was stirred for 2 h. The mixture was diluted with water (100 mL) and extracted with EtOAc (3×50 mL). The combined organic extracts were washed with brine (2×50 mL), dried over NaSO, filtered, and concentrated. The residue was purified by FC (Biotage, 80 g silica gel column @ 65 mL / min, eluting with 0–60% EtOAc in petroleum ether for 30 min) to give intermediate 16 (7.8 g, 75% yield). MS (ESI+) m / z 411 ([M + 23] + ).

[0223] A mixture of intermediate 16 (7.8 g, 20 mmol), bis(pinacolato)diboron (6.54 g, 30 mmol), and KOAc (2.94 g, 30 mmol) in dioxane (100 mL) was purged with Ar for 10 min, followed by the addition of (PhP)PdCl (1.06 g, 1.5 mmol). 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 NaSO, filtered, and concentrated. The residue was purified by FC (Biotage, 80 g silica gel column @ 65 mL / min, eluting with 0–60% EtOAc in petroleum ether for 30 min) to give intermediate 18 (6.2 g, 70% yield). MS (ESI+) m / z 459 ([M + 23] + ).

[0224] Procedure C: A mixture of one of Intermediates 5 / 8 / 9 / 10 / 11 (1.0 equiv.), one of Intermediates 18 / 19 (1.5 equiv.), Pd(dppf)Cl2DCM (0.1 equiv.), and K3PO4 (2.0 equiv.) in dioxane / HO (10:1, 0.06 M) was purged with N2 and stirred at 90 °C overnight. The mixture was diluted with EtOAc, washed with water and brine, dried over anhydrous magnesium sulfate, filtered, and concentrated. The residue was purified by FC to give one of compounds AKG-28-1 / AKG-29-1 / AKG-30-1 / AKG-31-1 / AKG-38 / AKG-39 / AKG-40.

[0225] To a solution of one of compounds AKG-28-1, AKG-29-1, AKG-30-1, or AKG-31-1 in DCM (1 mL / 100 mg) was added 3N HCl (20 equiv.) in EtOAc. The mixture was stirred for 2 hours and then filtered. The solid was dried under reduced pressure or lyophilized to give one of the final compounds AKG-28, AKG-29, AKG-30, or AKG-31 (35-44% yield for the two steps).

[0226] [ka]

[0227] Use procedure C. The product was obtained from Intermediate 5 and Intermediate 18 as a white solid (0.35 g, 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), 5.31 (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 MHz, 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] Use procedure C. The product was obtained from Intermediate 8 and Intermediate 18 as a white solid (0.4 g, 44% 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 - 7.65 (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), 6.89 (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 - 1.49 (m, 1H), 1.34 (dd, J = 14.3, 7.0 Hz, 2H), 1.18 - 1.04 (m, 2H) ppm. 13 C NMR (101 MHz, D2O) δ161.52, 160.59, 158.12, 154.86, 145.70, 141.05, 140.16, 139.65, 133.57, 130.44, 123.64, 117.70, 114.54, 106.50, 106.22, 70.34, 50.81, 47.68 ppm. MS (ESI+) m / z 399.2 ([M + 1]+ ).

[0230]

change

[0231] Easy to use. The resulting product, Intermediate 10, and Intermediate 18, were obtained 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 - 3.26 (m, 2H), 3.21-3.16 (m, 2H), 2.75 (d, J = 4.8 Hz, 6H), 2.49-2.43 (m, 2H) ppm. 13 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]

change

[0233] Easy to use. The resulting product, Intermediate 11, and Intermediate 18, were obtained 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) ppm. 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 triethylamine (TEA, 4.57 g, 6.27 mL, 52.77 mmol, 2.0 equiv), followed by acetyl chloride (AcCl, 2.6 g, 2.74 mL, 39.58 mmol, 1.5 equiv) and 4-N,N-dimethylaminopyridine (DMAP, 0.028 g, 2.64 mmol, 0.01 equiv) under N at 0-5 °C. The resulting reaction mixture was then stirred at 0-5 °C for 2 h. When TLC and LCMS showed the reaction was complete, the reaction mixture was quenched with HO (100 mL). After separation of the two layers, the aqueous layer was extracted with CHCl (2×50 mL), and the combined organic extracts were washed with HO (2×100 mL) and saturated aqueous NaCl (100 mL), dried over MgSO, and concentrated under reduced pressure. The residue was purified by FC (Biotage, 80 g silica gel column @ 65 mL / min, eluting with 0-60% EtOAc in petroleum ether for 30 min) to give intermediate 17 (6.5 g, 75% yield). MS (ESI+) m / z 332 ([M + 1] + ).

[0235] To a solution of intermediate 17 (6.5 g, 19.7 mmol) in 1,4-dioxane (100 mL) was added 1,1'-bis(diphenylphosphino)ferrocene-palladium(II) dichloride dichloromethane complex (1.61 g, 1.97 mmol), bis(pinacolato)diboron (10 g, 39.39 mmol), and KOAc (4.83 g, 49.24 mmol). The resulting reaction was stirred at 90 °C for 4 h. When TLC and LCMS indicated the reaction was complete, the reaction mixture was cooled to RT and then treated with water (100 mL) and EtOAc (100 mL). The two layers were separated, and the aqueous layer was extracted with EtOAc (2 × 50 mL). The combined organic extracts were washed with water (2 × 50 mL) and saturated aqueous NaCl (50 mL), dried over MgSO4, and concentrated under reduced pressure. The residual brown oil was purified by FC (Biotage, 80 g silica gel column @ 60 mL / min, eluting with 0-100% EtOAc in petroleum ether for 30 min) to give intermediate 19 (6.6 g, 88.7% yield). MS (ESI+) m / z 379 ([M + 1] + ).

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

[0237] Use procedure C. The 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 , 119.25 , MS (ESI+) m / z 469.2 ([M + 1] + ).

[0238] [ka]

[0239] Use procedure C. The product was obtained from Intermediate 9 and Intermediate 19 as a white solid (0.35 g, 40% yield). 1H 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, 40.63, 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] Use procedure C. The product was obtained from Intermediate 11 and Intermediate 19 as a white solid (0.36 g, 50% yield). 1H NMR (400 MHz, DMSO-d6) δ8.95 (s, 1H), 8.31 - 8.18 (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, 46.79, 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] Assay for in vitro activity against Mycobacterium tuberculosis The broth microdilution MIC method used is described in Collins et al., 1997 and Gruppo et al., 2006. MIC or minimum inhibitory concentration of a compound that prevents visible growth of bacteria after overnight incubation.

[0244] Briefly, MICs were determined by a broth microdilution assay using the Alamar blue endpoint (MABA) as described by Collins et al., 1997 (Collins L, Franzblau SG (1997). Microplate alamar blue assay versus BACTEC 460 system for high-throughput screening of compounds against Mycobacterium tuberculosis and Mycobacterium avium. AAC. 41(5):1004-1009) and Gruppo et al., 2006 (Gruppo V, Johnson CM, Marietta KS, Scherman H, Zink EE, Crick DC, Adams LB, Orme IM, Lenaerts AJ. (2006) Rapid microbiologic and pharmacologic evaluation of experimental compounds against Mycobacterium tuberculosis. AAC 50:1245-1250). MABA is a 96-well colorimetric assay in which the redox indicator Alamar blue changes color from blue to pink in the presence of mycobacterial growth activity in broth medium.

[0245] Briefly, 7H9 complete medium was prepared by adding 4.7 g of Middlebrook's 7H9 broth powder (Millipore Sigma catalog #M0178), 2 mL of glycerol, and 898 mL of purified water to a 1 L flask with mixing until dissolved, followed by the addition of 100 mL of ADC solution (6 g of bovine serum albumin, 2 g of dextrose, and 3 mg of catalase dissolved in 100 mL of water) to the same 1 L flask. Compounds were brought to a concentration of 10 mg / mL in DMSO and then further diluted with DMSO to 80 μg / mL, or 40-fold the desired starting concentration of 2 μg / mL. Nine 1:2 dilution series were prepared by adding 50 μl of drug solution in the first well to 50 μl of DMSO in the next well and carrying this process forward for the next eight wells in the drug preparation plate. Stocks of M. tuberculosis (M.tb) H34Rv and M.tb Erdman strains were cultured at 3–4 x 10 7 From their initial concentration of CFU / mL, 5x10 5 Dilutions were made to a final concentration of CFU / mL and mixed thoroughly by pipetting up and down using a multichannel pipettor.

[0246] 5x10 5Assay plates were prepared by transferring 100 μl of medium inoculated with CFU / mL to all wells. Subsequently, 2.5 μL of each drug dilution from the drug preparation plate was transferred to the corresponding well in the assay plate. The assay plates were then placed in ziplock bags and placed inside an incubator, where they were incubated at 37°C. The plates were then read at OD600nm on a plate reader on days 3 and 10. After the OD600 reading on day 10, 10 μl of Alamar Blue dye was added to each assay well. On day 12, all assay plates were scanned on a flatbed color scanner. The lowest serial antimicrobial concentrations (typically 2-fold serial dilutions) that did not produce a visible color change from blue to pink with Alamar Blue and / or showed an 80% or greater reduction in OD600 relative to drug-free control wells were considered the MICs for these compounds.

[0247] Assays were performed using two unique drug-susceptible strains (M.tb Erdman and M.tb H37Rv). MIC assays can also be performed in the presence of 4% (w / v) human serum albumin (huSA) (Sigma #A1653) to assess potential protein binding (serum shift assay). Generally, a shift in the MIC between two wells (a four-fold shift in the MIC) is considered significant. For PA-824 (positive control), a four-fold shift in the MIC is expected.

[0248] MICs were measured by the Alamar Blue (MABA) readout or by the optical density readout (OD600), which matched or differed by only one two-fold dilution, within the limits of the assay. All compounds tested showed consistent MIC values ​​against both Mtb Erdman and H37Rv, or were within one two-fold dilution, except for one compound, AKG-40, which showed higher MIC values ​​of 1–2 μg / mL against Erdman and an MIC of 0.5 against H37Rv. This difference may be due to slower growth (lower OD reading) on ​​Erdman plates.

[0249] Linezolid showed predicted MIC values ​​of 2 μg / mL, tedizolid showed 0.25 μg / mL, and bedaquiline showed 0.125 μg / mL. These values ​​are consistent with previous MIC data and published values ​​(Ruiz et al. Antimicrob. Agents Chemother. 2019, Mar 27;63(4),pii:e01939-18; Reddy et al. Antimicrob Agents Chemother. 2010 Jul;54(7):2840-6; Torrea et al. J Antimicrob Chemother. 2015 Aug;70(8):2300-5). AKG-28 showed significantly greater activity than tedizolid, with MICs ranging from 0.03 to 0.015 μg / mL. Among oxazolidinone analogs containing an acetamide group, AKG-39 showed an MIC of 0.5 μg / mL, AKG-40 showed MICs ranging from 1 to 0.5 μg / mL, and AKG-38, with an MIC of 0.06 μg / mL, also showed several-fold higher activity than tedizolid.

[0250] Molecules bearing an amine or acetamide group at the C5 position of the oxazolidinone were more active (AKG-3 vs. tedizolid, AKG-28 or AKG-38 vs. AKG-16, AKG-39 vs. AKG-24, AKG-40 vs. AKG-26), while compounds bearing an aminoalkyl side chain on the tetrazole showed favorable activity. Substitution of the t-butoxycarbonylamino (Boc-NH) group at the C5 position of the oxazolidinone with a primary amine (AKG-28-1 vs. AKG-28) or acetamide (AKG-28-1 vs. AKG-38) resulted in decreased activity. Compounds containing dimethylaminoalkyl side chains were particularly superior when compared with aminoethyl or diethylaminoethyl analogs (AKG-16 vs. AKG-24, AKG-28 vs. AKG-29, AKG-30 vs. AKG-31). Similarly, shorter dialkylaminoalkyl side chains on the tetrazole ring (e.g., ethylene vs. propylene) showed greater 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 were more active than those with substitutions at the 1'-position (AKG-16 vs. AKG-21, AKG-23 vs. AKG-22).

[0251] [ka]

[0252] JPEG0007805319000078.jpg219170

[0253] [Example 3]

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

[0255] Adherent cells were grown to approximately 80% confluence. After trypsinization by adding 0.25% trypsin-EDTA (Gibco #25200-072), the cells were spun down, and 5 ml of growth medium (MEM medium; Corning #10010CM) was added to disperse the cells. Cell density was determined using a hemocytometer. Growth medium (MEM medium containing 10% FBS; Corning #35015CV) was added to the cells to adjust the appropriate cell concentration. Then, 200 μl of cells (5,000 cells / well) were added to a 96-well clear flat-bottom plate (Costar #9804) and incubated in the plate at 37°C in a humidified incubator with 5% CO2 for 24 hours.

[0256] Serial dilutions of test compounds were prepared using growth medium as the solvent (Table 2). These compounds were provided as sterile aqueous solutions of the HCl salt with a concentration of 5 mg / ml. To make the dilutions, each drug stock was warmed to room temperature, vortexed, and visually inspected for sediment. If solid drug was present, the stock was heated in a 60°C water bath and then allowed to cool to near room temperature. Based on the treatment concentration, 20-fold diluted standard stocks were made by serial dilution. These were further diluted 1-fold in growth medium, and the highest drug concentration tested was 250 μg / ml.

[0257] Compounds were added to wells in a 1:2 dilution series starting from an initial concentration of 250 μg / ml for each compound by aspirating the old medium and replacing it with 200 μl of drug-containing medium. Plates were incubated for 72 hours at 37°C in a humidified incubator with 5% CO2. At the end of the compound incubation period, the medium in each well was replaced with 100 μl of 1X PrestoBlue Cell Viability Reagent (ThermoFisher catalog #A13261). Plates were incubated for 30 minutes to 2 hours at 37°C in a humidified incubator with 5% CO2. Readings were taken at 30, 60, and 120 minutes. Fluorescence was read at 560 nm excitation and 590 nm emission using a SpectraMax M5 plate reader (Molecular Devices). Background was corrected by subtracting the RFU of a control containing culture medium only (background control well) from all sample readings. 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] JPEG0007805319000079.jpg183170

[0260] Surprisingly, many of the analogs containing a hydroxyl group on the C5 side chain of the oxazolidinone ring, which mimics the substituent of the active metabolite tedizolid for tedizolid phosphate, exhibited the highest hepatotoxicity, exhibiting single-digit IC50s in the HepG2 hepatocyte cell line. Tedizolid is the most active oxazolidinone currently approved for the treatment of MRSA and shares structural similarity with the compounds described herein in the tetrazole D ring, pyridyl C ring, and aryl B ring (see Figure 6). However, increased toxicity to hepatocytes results in a relatively low selectivity index for teoxazolidinones with a hydroxyl on the C5 side chain (AKG-23, AKG-25, AKG-26, and AKG-27) compared with those with an amino or acetamido group at the same position on the C5 side chain (AKG28-31, AKG38-40, and AKG-3).

[0261] [Example 4]

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

[0263] If the IC50 was higher than the highest value tested for VERO or HepG2 cells, the SI was indicated as higher (>) than the ratio calculated using that highest concentration. Similarly, if the MIC for the Erdman or H37Rv strains was higher than the highest concentration of drug tested (8 μg / ml), the SI was indicated as lower (<) than the ratio calculated using that highest concentration. If both numbers were above the highest concentration tested, the calculated value was indicated as not determined (nd). The results are shown in Table 4. The SI did not directly correlate with the activity of the molecule in either the M. tuberculosis strains or the mammalian cell lines, and increased potency in M. tuberculosis strains did not directly correlate with increased toxicity to the mammalian cell lines. For example, AKG-38 exhibited nanomolar MICs against both M. tuberculosis strains, but it was relatively inactive against both VERO and HepG2 cell lines compared to the other molecules in the panel, giving a high SI. This was also observed for AKG-28. It is noteworthy that both the AKG-28 and AKG-38 molecules had a dimethylaminoethyl substituent at the 2' position of the tetrazole ring.

[0264] JPEG0007805319000080.jpg255170JPEG0007805319000081.jpg18170

[0265] In some embodiments, the compound of interest has a saturation level of greater than 100, greater than 200, greater than 300, greater than 400, greater than 500, greater than 1000, greater than 1500, greater than 2000, greater than 2500, greater than 3000, greater than 3500, greater than 4000, greater than 4500, greater than 5000, greater than 5500, greater than 6000, greater than 6500, 100-7000, 100-6000, 100-5000, 100-4000, 100-3000, 100-2000, 100-1000, 100-900, 100-800, 100-700, 100-600, 100-500, 100-400, 100-400, 100-500, 100-400, 100-500, 100-400, 100-6500, 100-7000, 100-800, 100-700, 100-600, 100-500, 100-400, 100-500, 100-400, 100-6500, 100-7000, 100-700, 100-600, 100-500, 100-400, 100-6500, 100-7000, 100-800, 100-800, 100-900, 100-900, 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, 300~700 The compound of interest has an SI index with respect to Erd / HepG2 and H37Rv / HepG2 of 0, 300-6000, 300-5000, 300-4000, 300-3000, 300-2000, 300-1000, 300-900, 300-800, 300-700, 300-600, 300-500, or 300-400. In some embodiments, the compound of interest has an SI index with respect to Erd / HepG2 and H37Rv / HepG2 in the range of 100-1700, 200-1700, or 300-1700.

[0266] Compounds bearing an amino or acetamido group on the C5 side chain of the oxazolidinone ring and an aminoalkyl group at the 2' position of the tetrazole ring exhibited relatively high SI compared with those bearing a hydroxyl group on the C5 side chain. Furthermore, specific tetrazole substitutions further improved SI, with dimethylaminoethyl substitution at the 2' position of the tetrazole ring being preferred over methyl, diethylaminoethyl, aminoethyl, or dimethylaminopropyl substitutions at the same position (AKG-28 and AKG-38). Moving the dimethylaminoethyl group to the 1' position of the tetrazole ring (compounds AKG-21 vs. AKG-28) unexpectedly resulted in a dramatic loss of activity against M. tuberculosis.

[0267] [Example 5]

[0268] Assay for in vitro activity against methicillin-resistant Staphylococcus aureus (MRSA) The activity of lead oxazolidinone inhibitors was measured and showed sufficient potency against the Gram-positive bacterium methicillin-resistant Staphylococcus aureus (MRSA) to justify subsequent delivery in liposomal form for its treatment. In some embodiments, the MIC for two of the three strains evaluated is less than 6 μg / mL. In some embodiments, the MIC for two of the three strains evaluated is less than 2 μg / mL, with less than 2 μg / mL being more preferred.

[0269] Three S. aureus strains were grown overnight at 37°C under ambient atmosphere on trypticase soy agar plates supplemented with 5% sheep blood cells. Cultures were aseptically swabbed and transferred to tubes of sterile water, and the optical density was adjusted to 0.5 at 600 nm. Cultures were then diluted 1:100 to yield approximately 5 x 10 per well in 120 μL. 5 cells were delivered. After incubation, the MIC of the test article was determined by the presence / absence of growth in each well. MIC analysis was performed in triplicate.

[0270] Tedizolid demonstrated MICs ranging from 0.206 to 0.617 μg / ml, similar to the 0.5 μg / ml described in U.S. Patent No. 7,816,379. Interestingly, all molecules with a primary amine modification at R2 of the oxazolidinone ring (AKG-3, AKG-28, AKG-29, and AKG-30) exhibited negligible activity (>50 μg / ml) against all three MRSA strains. Molecules with an acetamide group at the same position (AKG-38, AKG-39, and AKG-40) were three to nine times less active than tedizolid itself against the three MRSA strains.

[0271] JPEG0007805319000082.jpg117170

[0272] [Example 6]

[0273] Liposome Composition General Protocol 1. The lipid components (phospholipids (PhL), cholesterol, and, if necessary, PEG-lipid derivatives and / or fluorescently labeled lipids) were mixed with a volume of 100% ethanol equal to one-tenth of the calculated volume (V) to obtain a lipid suspension containing approximately 60 mM phospholipids, and stirred at a temperature of 65-68 °C until complete dissolution of the lipids.

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

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

[0276] Lipid fluorescent labels include 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-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-glycero-3-phosphoethanolamine-N-(cyanine7) (18:0 Cy7 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000]-N-(cyanine7) (DSPE PEG(2000)-N-Cy7), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(cyanine5) (18:0 Cy5 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000]-N-(cyanine 5) (DSPE PEG(2000)-N-Cy5), 1-oleoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-glycero-3-phosphocholine (18:1-12:0 NBD PC).

[0277] 2. The ethanolic lipid solution was mixed with volume V of scavenger solution (0.25-0.5 M ammonium sulfate or 1 N triethylammonium sucrose octasulfate) with stirring at 65-68 °C until a homogenous suspension was obtained.

[0278] Potential scavengers include, but are not limited to, diethylammonium or triethylammonium salts of sucrose octasulfate, ammonium sulfate, ammonium citrate, citric acid, dextran sulfate, polyvinyl sulfonic acid, or the ammonium salt of inositol hexaphosphate, at a concentration of 0.1 to 2 g equivalents / L (0.1 to 2 N), preferably 0.2 to 1.5 N. Typically, an ammonium salt is used, which may include ammonium itself, monoalkyl-, dialkyl-, or trialkyl-ammonium salts.

[0279] The lipid suspension was extruded at least three times through a stack of track-etched polycarbonate membranes, typically two or four membranes with a nominal pore size of 100 nm and one membrane with a nominal pore size of 200 nm (Whatman Nuclepore, USA), using a thermobarrel extrusion apparatus (Lipex, Canada) at 400-450 psi and 65-68°C. When two 100 nm membranes were used in a 100 ml Lipex extrusion apparatus, the extrusion pressure was typically 260-300 psi. The resulting liposomes had a Z-average particle size (diameter) Xz of approximately 80 to approximately 130 nm and a PDI of less than 0.1.

[0280] 4. The extruded lipid suspension (known to contain unilamellar and / or oligolamellar liposomes) was chilled in a refrigerator (2-8°C) and filtered under positive pressure through a 0.2 μm polyethersulfone (PES) membrane filter.

[0281] 5. An aliquot of the extruded, filtered liposome suspension so produced was chromatographed on a gravity-fed Sepharose CL-4B size-exclusion column (eluent—Type 1 water) to purify the liposomes from extraliposomal entrapment agents. The purified liposomes were collected near the void volume fraction of the column. For scale-up studies, this step was performed using tangential flow filtration (TFF) on a hollow fiber cartridge (Repligen Spectrum MicroKros PS or mPES membrane with a 500 kDa MWCO) with 8-10 volume exchanges with Type 1 or USP "Water for Injection" endotoxin-free water (or until the conductivity of the liposome suspension fell below 200 μS / cm).

[0282] 6. Lipid concentrations in purified, extruded liposome preparations were determined using HPLC with UV detection by measuring cholesterol concentrations and correcting for known phospholipid / cholesterol molar ratios. Alternatively, phospholipid content was directly quantified using a spectrophotometric phosphomolybdenum blue method.

[0283] 7. Drugs were dissolved in type 1 or endotoxin-free purified water in the form of hydrochloride salts at concentrations of 5–20 mg / ml (e.g., AKG-3 and AKG-5 were used as monohydrochlorides, and AKG-28 and AKG-29 were used as dihydrochlorides). To drugs prepared in free base form (e.g., AKG-16, AKG-38), an equal volume of HCl was added. If necessary, the pH of the solution was adjusted to 2.5–5.5 using 1N NaOH, HCl, or tris(hydroxymethyl)aminomethane (Tris) base solution, and the solution was filtered through a 0.2 μm PES filter under positive pressure. Where necessary, the drug concentration in the stock solution was verified by HPLC with UV detection at 305 nm.

[0284] 8. The purified liposomes and drug stock solution from step 5 were mixed in the presence of the amount of osmotic agent (typically dextrose) and water necessary to provide the desired drug:phospholipid (DL) ratio, a drug concentration ranging from 1.5 to 3.3 mg / ml, and an osmolality equal to the measured osmolality of the sequestering agent solution from step 2. If necessary, a buffer solution of the desired pH (typically, pH 4 to pH 7) was added. In some cases, the amount of osmotic agent added (e.g., approximately 45 g / L of dextrose) resulted in an osmolality lower than the measured osmolality of the sequestering agent solution, and loading was performed with 6 to 8 mg / ml of drug.

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

[0286] 10. Drug-loaded liposomes were purified from unencapsulated drug by size-exclusion chromatography (SEC) on a gravity-fed Sepharose CL-4B column, eluting with 10 mM HEPES buffer, pH 7.0 (HBS-7) in 140–144 mM NaCl. The liposome fraction was collected near the column void volume. For scale-up studies, purification and buffer exchange were performed using TFF as described under item 5 above, with a 10-fold volume exchange with HBS-7 buffer. In scale-up processes, approximately 8-fold volume exchanges were typically used. If necessary, purified liposomes were concentrated by continuing the TFF process without buffer feeding. The purified, drug-loaded liposomes were aseptically filtered using a 0.2 μm sterile PES filter under positive pressure and stored in a refrigerator (2–8°C).

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

[0288] 12. The mean liposome size (Z-average diameter, Xz) and polydispersity index (PDI) were determined using dynamic laser scattering by the method of cumulants on a Zetasizer mu-V, Zetasizer Nano, or Zetasizer Pro (Malvern Panalytical, US).

[0289] [Example 7]

[0290] In vivo stability and blood clearance of liposomes The drug encapsulation stability and blood clearance rate of liposomes encapsulating compounds of the present disclosure were tested in mice according to the following general protocol. Three groups of mice (C3H female or CD-1 male) of a given laboratory strain were injected with drug-loaded liposomes via the tail vein at a dose of 9 mg of drug per kg of body weight. At time points 1 and 2, blood was collected from the retro-orbital sinus and the animals were sacrificed. Typically, blood collection time points included 5 minutes, 1 hour, 6 hours, and 24 hours after injection. Plasma was separated by centrifugation, extracted with acidified isopropanol containing a solubilizing agent (sodium octasulfate) as needed, and analyzed for drug and lipids (when the liposomes contained the lipid label, DiIC18(3)-DS) by HPLC. Blood clearance of the liposomal drug was expressed as the percentage of the injected dose remaining at a given time point. The in vivo stability of drug encapsulation was assessed by the percent change (decrease) in the DL ratio in plasma at a given time point compared to the DL value before injection.

[0291] [Example 8]

[0292] Loading of AKG-3, AKG-5, and AKG-16 into liposomes at different pH A trimethylammonium sucrose octasulfate scavenger solution was prepared by passing a solution of commercially available potassium sucrose octasulfate (40.2 g in 145 ml of water) through a 500 ml ion exchange column of hydrogen-form Dowex 50Wx8 100-200 mesh and titrating the resulting free acid form of sucrose octasulfate with neat triethylamine to pH 6.2. The concentration of triethylammonium sucrose octasulfate (TEA-SOS) (1 N, corresponding to 0.125 M sucrose octasulfate) was estimated from the amount of triethylamine consumed in the titration. The remaining potassium was estimated using a Horiba LAQUATwin K-11 potassium analyzer by the addition method and was found to be less than 0.1% of the initial potassium amount.

[0293] Liposomes (0.5 mol% HSPC) composed of hydrogenated soybean phosphatidylcholine (HSPC) (Lipoid, Germany), cholesterol (3:2 molar ratio), and methoxypoly(ethylene glycol) ether of 1,2-distearoylglycerol (PEG-DSG, PEG molecular weight 2000, NOF, Japan) with 1N trimethylammonium sucrose octasulfate (TEA-SOS) as a sequestering agent were prepared essentially as described in the general protocol above. The drug loading step was carried out at a pH of 500 mg / mol DL (DL0) in the presence of 16 mM morpholinoethanesulfonic acid (MES)-4 mM sodium citrate buffer with a pH ranging from 4.3 to 7.1, as well as without the addition of any buffering substance (pH 5.2-5.9). All drugs were encapsulated in liposomes with high efficiency over the entire pH range tested (except for AKG-16 at pH 4.38, which was loaded with 93.3% efficiency, exceeding 98%) (Figure 1). Addition of buffering substances was not necessary for efficient encapsulation.

[0294] [Example 9]

[0295] Encapsulation of AKG-3, AKG-5, and AKG-16 in liposomes with TEA-SOS scavengers at different DL ratios Liposomes composed of HSPC, cholesterol (3:2 molar ratio), and PEG-DSG (0.5 mol% of HSPC) with 1N TEA-SOS as the sequestering agent were prepared essentially as described in the general protocol (Example 6). The drug loading step was carried out without added buffer (pH 4.98-6.22) at DL ratios ranging from 750 to 1500 g / mol pHL. Maximum drug loadings for compounds 3, 5, and 16 were observed in the ranges of 900-930 g / mol pHL, 982-1197 g / mol pHL, and 938-951 g / mol pHL, respectively, with loading efficiencies at or near maximum drug loading of at least 97.6%, 96.0%, or 85.2%, respectively (Figures 2A and 2B).

[0296] [Example 10]

[0297] Encapsulation of AKG-3, AKG-5, and AKG-16 into liposomes using a high degree of PEGylation or 0.25 M ammonium sulfate (AS) as an entrapment agent Liposomes composed of HSPC and cholesterol with various PEG-DSG contents (3:2 molar ratio) and entrapment agents were prepared according to the general protocol and loaded with compounds AKG-3, AKG-5, and AKG-16 at DL ratios of 250 or 500 g / mol PhL as described in Example 9. All three compounds were loaded into liposomes with high efficiency, as shown in Table 6 below.

[0298] JPEG0007805319000083.jpg72170

[0299] Thus, compounds AKG-3, AKG-5, and AKG-16 were efficiently loaded into phospholipid-cholesterol liposomes with high levels of PEGylation and ammonium sulfate as the intraliposomal drug entrapment agent. However, loading efficiency decreased for two of the three oxazolidinones (AKG-3 and AKG-16) when loaded at a higher drug:lipid ratio of 500 g drug / mol PhL using 0.25 M ammonium sulfate as the entrapment agent.

[0300] [Example 11]

[0301] Loading of compounds AKG-3, AKG-5, and AKG-16 into liposomes using 0.5 M AS as a scavenger Liposomes composed of HSPC and cholesterol (3:2 molar ratio) with 0.5 mol% or 5% mol% PEG-DSG (relative to PhL) and 0.5 M ammonium sulfate (AS) as a sequestering agent were prepared according to the general protocol and loaded with compounds AKG-3, AKG-5, and AKG-16 as described in Example 8 at DL ratios ranging from 500 to 1500 g / mol PhL. The results are shown in Figures 3A, 3B, 3C, and 3D. All three compounds were loaded into both liposomes at DL ratios of 420 to 450 g / mol PhL with encapsulation efficiencies of 93 to 100%; the maximum drug payloads were as follows:

[0302] JPEG0007805319000084.jpg48170

[0303] All three tested compounds were able to be loaded into 0.5M ammonium sulfate liposomes at more than 500 g of drug / mol PhL in the preparation containing 0.5 mol% PEG-DSG, and compounds AKG-3 and AKG-16 were able to be loaded at 5 mol% PEG-DSG. These high loading levels are important in achieving sufficient doses of drug administered for disease treatment. Although loading efficiency was significantly improved compared to Example 10, where loading efficiency was lower using 0.25M ammonium sulfate, this indicates that, despite higher osmolality and the possibility of osmotic burst, higher ammonium sulfate concentrations of 0.5M are an improvement in terms of the amount of drug that can be loaded per mole of phospholipid, and preferably for anti-infective agents, where low toxicity and high doses can lead to improved outcomes.

[0304] [Example 12]

[0305] Loading of compounds AKG-3, AKG-5, AKG-16, and AKG-28 into liposomes of various compositions containing fluorescent lipid labels Liposomes composed of HSPC and cholesterol (60:40 molar ratio) with 0.5 mol% PEG-DSG (relative to PhL), 0.15 mol% lipid fluorescently labeled DiIC18(3)-DS (ThermoFisher, USA), and 0.5 M ammonium sulfate (AS) or 1 N TEA-SOS as a scavenger were prepared according to the general protocol and loaded with compounds AKG-3, AKG-5, and AKG-16 at pH 4.7-5.8 (no buffer substances added) as described in Example 11. The liposomes had the following characteristics:

[0306] JPEG0007805319000085.jpg108170

[0307] All three drugs were efficiently loaded into liposomes. Degradation of AKG-5 during liposome loading was detected as the appearance of a second peak on HPLC.

[0308] Liposomes composed of various phospholipids (HSPC, distearoylphosphatidylcholine (DSPC, Avanti Polar Lipids, USA), or egg sphingomyelin (ESM, Lipoid, Germany)) and cholesterol (60:40 molar ratio) containing various amounts of PEG-DSG or N-methoxypoly(ethylene glycol)oxycarbonyl-1,2-distearoylphosphatidylethanolamine (PEG-DSPE, PEG molecular weight 2000, Lipoid, Germany), and the lipid fluorescently labeled DiIC18(3)-DS (0.15 mol% with respect to PhL) were prepared according to the same general protocol using different trapping agents and loaded with AKG-16 in a similar manner. Where indicated, the liposome extrusion step of the general protocol was supplemented with extrusion through two stacked polycarbonate membranes with 50 nm pore sizes. The liposomes had the following characteristics:

[0309] JPEG0007805319000086.jpg134170

[0310] Liposomes composed of HSPC and cholesterol (3:2 molar ratio) with 9.2 mol% PEG-DSPE (relative to PhL), 0.15 mol% lipid-labeled DiIC18(3)-DS, and 0.25 M ammonium sulfate (AS) as a trapping agent were prepared according to the general protocol and Example 12 using an additional 50 nm extrusion and loaded with AKG-28 at a drug:lipid ratio (DL) of 150 g / mol PhL. Liposomes (batch ID 98) have a DL ratio of 73.8 g / mol PhL, a Z-average liposome size of 77.8 nm, and a size polydispersity index (PDI) of 0.090.

[0311] These studies demonstrate that AKG-3, AKG-5, and AKG-16 can be efficiently loaded into liposomes with a range of lipid compositions, including HSPC, DSPC, or ESM as the neutral phospholipid component, or low (0.5 mol%) or high (5 mol%) PEG-lipid content. However, compared to 1 N TEA-SOS, when 0.25 M AS was used, the efficiency significantly decreased from approximately 500 g AKG-16 / mol PhL to 128 g AKG-16 / mol PhL. A similarly low loading efficiency (i.e., 73.8 g / mol PhL) was observed when AKG-28 was loaded with 0.25 M AS. This suggests that TEA-SOS or higher concentrations of AS may be preferred for loading higher concentrations of compounds into liposomes.

[0312] [Example 13]

[0313] Blood persistence and in vivo encapsulation stability of liposomes of Example 12 in mice Tests were performed on male CD-1 mice as described in the general protocol above.

[0314] JPEG0007805319000087.jpg91170

[0315] These studies show that liposomes composed of varying neutral phospholipid components (HSPC, DSPC, or SM) and loaded with AKG-16 using a TEA-SOS scavenger were slowly cleared, with over 30% of the injected dose remaining in plasma at 6 hours for most formulations. Furthermore, most formulations showed good drug retention, with the exception of liposome batch ID 97, which contained AKG-16 loaded using 0.25 M AS. However, the results suggest that loading the drug using 0.25 M ammonium sulfate not only resulted in low loading efficiency, as shown in Table 9, but also in a low DL ratio (3.7%) at 6 hours due to significant leakage from liposomes in this formulation.

[0316] [Example 14]

[0317] Encapsulation of compounds AKG-28 and AKG-38 into liposomes with different DL ratios and various entrapment agents Liposomes composed of HSPC and cholesterol with 0.5 mol% PEG-DSG (relative to PhL), 0.15 mol% lipid-labeled DiIC18(3)-DS, and 0.5 M ammonium sulfate (AS) or 1 N TEA-SOS as a scavenger were prepared according to the general protocol and loaded with compounds AKG-28 and AKG-38 at pH 4.95–5.17 (no buffer added) and DL ratios ranging from 300–1050 g / mol PhL (AKG-28) or 400–1400 g / mol PhL (AKG-38), as described in Example 8. When using 0.5 M AS, the maximum drug loadings for compounds AKG-28 and AKG-38 ranged from 404 to 424 g / mol PhL and 818 to 842 g / mol PhL, respectively, with loading efficiencies above 95% at drug loadings of 302 g / mol PhL (quantitative loading) and 387 to 764 g / mol PhL (95.5 to 96.7% loading), respectively. When using 1 N TEA-SOS, the maximum drug loadings for compounds AKG-28 and AKG-38 ranged from 315 to 328 g / mol PhL and 989 g / mol PhL, respectively, with loading efficiencies of 83.5% at drug loadings of 250 g / mol PhL and 400 to 777 g / mol PhL (above 97.2% loading), respectively (Figures 4A and 4B).

[0318] While AKG-38 demonstrated near-quantitative loading of 400-800 g AKG-38 / mol PhL, the resulting drug-to-lipid ratio remained flat for AKG-28 over the range of 250-1000 g AKG-28 / mol PhL, suggesting a lower maximum drug loading for AKG-28 than for AKG-38. It should be appreciated that the higher potency previously demonstrated for AKG-28 makes liposomal formulations of AKG-28 effective for the treatment of infectious diseases such as tuberculosis.

[0319] [Example 15]

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

[0321] JPEG0007805319000088.jpg80170

[0322] JPEG0007805319000089.jpg80170

[0323] This example demonstrates that AKG-28 can be efficiently loaded into liposomes composed of HSPCs using 0.5 M AS or 1 N TEA-SOS as the entrapment agent with a maximum drug loading of 230-275 g AKG-28 / mol PhL. However, formulations containing sphingomyelin as the neutral phospholipid for this compound showed relatively low loading, with a maximum of only about 110 g AKG-28 / mol PhL.

[0324] Compound AKG-38 was loaded at significantly higher D / L ratios of 525-600 g / mol when the drug was added at 600 g AKG-38 / mol PhL, or greater than 735 g / mol when added at 800 g AKG-38 / mol PhL, using 0.5 M AS or 1 N TEA-SOS. The loading of compound AKG-38 was less sensitive to the presence of sphingomyelin than AKG-28.

[0325] [Example 16]

[0326] Encapsulation of compounds AKG-16, AKG-28, AKG-29, and AKG-38 in liposomes using high PEGylation and 0.5 M ammonium sulfate as a trapping agent Liposomes composed of HSPC and cholesterol (3:2 molar ratio) with 0.5 M ammonium sulfate as a scavenger, PEG-DSG (5 mol%), and DiIC18(3)-DS (0.15 mol%) were prepared according to the general protocol and loaded with compounds AKG-16, AKG-28, AKG-29, or AKG-38 (in the absence of added buffer) at DL ratios selected to optimize drug loading and encapsulation efficiency (EE). The results are shown in Table 13 below.

[0327] JPEG0007805319000090.jpg55170

[0328] The data show that all compounds containing a dimethylaminoethyl substituent at the 2-position of the tetrazole ring were efficiently loaded into liposomes at over 80% efficiency, whereas AKG-29, which has an aminoethyl substituent at the same position, was poorly loaded into liposomes with an efficiency of 14.5% and a final drug loading of 43.6 g AKG-29 / mol PhL. This indicates that despite the presence of titratable amines in all compounds tested, compounds with an ammonium group substituted on the tetrazole ring (e.g., an N,N-dimethylaminoethyl group) allowed for more efficient drug loading than those with a primary amine (aminoethyl group) at the same position.

[0329] [Example 17]

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

[0331] JPEG0007805319000091.jpg103170

[0332] The data showed that the drug in liposome batches IDs 128, 132, 142, 144, and 145, all with 0.5 M AS as a sequestering agent, lost 25–60% of the encapsulated drug almost immediately upon contact with blood, as indicated by the low DL ratio at 5 min and a further decline in the DL ratio at 6 h, particularly pronounced for liposomes loaded with AKG-38 and AKG-16. Thus, formulations with 0.5 mol% or 5 mol% PEG-DSG and 40 mol% cholesterol, 0.5 M AS (as a sequestering agent), were unable to retain drug as efficiently as formulations with 1 N TEA-SOS (liposome batches IDs 129, 130, 133–135), when the % initial DL ratio at both 5 min and 6 h was above 80%.

[0333] [Example 18]

[0334] Preparation and loading of AKG-28 and AKG-38 in PEGylated liposomes with varying phospholipid to cholesterol ratios Liposomes containing 5 mol% PEG-DSG or PEG-DSPE (relative to PhL), 0.15 mol% lipid-labeled DiIC18(3)-DS, and 0.5 M ammonium sulfate (AS) or 1 N TEA-SOS as a scavenger were prepared according to the general protocol and loaded with compounds AKG-28 and AKG-38 at pH 5.07–5.82 (no buffer substances added) as described in Example 8.

[0335] In an attempt to stabilize liposomes with 0.5 M AS as a sequestering agent against rapid drug release upon contact with blood (as described in Example 17), liposomes using DSPC (generally known to result in stable liposomes with higher drug leakage compared to HSPC) and decreasing percentages of cholesterol (Chol) were prepared and loaded with AKG-28 at a DLO of 250 g / mol PhL or AKG-38 at 600 g / mol PhL (Table 15). Contrary to expectations, reducing the cholesterol content from 40 mol% to 10 mol% cholesterol resulted in a dramatic decrease in encapsulation efficiency for both AKG-28 and AKG-38. Lower cholesterol also destabilized the liposomes against aggregation. Liposomes containing AKG-28 prepared with 1N TEA-SOS and 5 mol% PEG-DSG or PEG-DSPE at 30 mol% cholesterol (70:30 PhL-cholesterol molar ratio) irreversibly aggregated during drug loading, as did formulations containing AKG-38, 30 mol% cholesterol, 5 mol% PEG-DSPE, whereas formulations of AKG-38, 30 mol% cholesterol, 5 mol% PEG-DSG showed reduced loading efficiencies of 77.1% or 462.4 g / mol PhL.

[0336] JPEG0007805319000092.jpg114170

[0337] In contrast, liposomes prepared using HSPC and containing 40 mol% or more cholesterol, up to 65 mol% cholesterol (the maximum value tested), showed excellent encapsulation efficiencies of over 87% and no liposome aggregation for both AKG-28 (DL0 250 g / mol PhL) and AKG-38 (DL0 500 g / mol PhL), PEG-lipids (PEG-DSG and PEG-DSPE), and entrapment agents (AS or TEA-SOS) (Table 16).

[0338] Additionally, the ability of the optimized formulations to load linezolid, a current standard of care from this class, was evaluated in both 0.5 M AS and 1 N TEA-SOS formulations. Tedizolid was not sufficiently soluble in water to perform transmembrane gradient-assisted loading into liposomes according to the general protocol in Example 6. In both cases for linezolid, encapsulation efficiency was less than 5%, indicating that these liposomal formulations of AKG-28 and AKG-38 were dramatically superior in their ability to stably encapsulate the drug when compared to linezolid.

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

[0340] JPEG0007805319000093.jpg255169JPEG0007805319000094.jpg207170

[0341] [Example 19]

[0342] In vitro burst release of PEGylated liposomes containing AKG-28 or AKG-38 and varying ratios of phospholipid to cholesterol in the presence of plasma The in vitro stability of liposomal formulations of AKG-28 and AKG-38 containing 5 mol% PEG-DSPE or PEG-DSG and varying ratios of HSPC to Chol (50–65 mol% Chol) was evaluated in the presence of mouse CD-1 or human pooled plasma (lithium-heparin stabilized from Innovative Research). Plasma was thawed, adjusted to pH 7.4 with 1 N HCl as needed, and filtered sequentially through glass microfiber filters (GF / C), 1 μm polyethersulfone (PES), and 0.22 μm PES filters. Plasma (80 μl) was mixed with the liposomal drug formulation (20 μl) in a 0.5 ml Eppendorf tube. The mixture was then incubated at 37°C for 20 min before being placed in cold water. The mixture (0.1 mL) was chromatographed without delay on a 2 mL Sepharose CL-4B column and eluted with Hepes-buffered saline (pH 7.0), and 0.25 mL of 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, and the remaining encapsulated drug percentage was calculated using 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,0 is the area of ​​the drug peak before incubation with plasma, and A I,0 is that of the lipid-labeled peak before incubation) was determined using

[0343] The results are shown in Figures 5A, 5B, 5C, and 5D. For liposomes encapsulating AKG-28 (Figure 5A), a burst release phenomenon (a rapid decrease in the DL ratio, indicating drug release from liposomes) was observed in human plasma for formulations containing 40 mol% cholesterol, but not for formulations containing 45 mol% or more cholesterol. For liposomes encapsulating AKG-38 (Figure 5B), a burst release phenomenon was observed in both human and mouse plasma for formulations containing 40 mol% and 45 mol% cholesterol, but not for formulations containing 50 mol% or more cholesterol.

[0344] [Example 20]

[0345] In vitro plasma release and in vivo pharmacokinetics of 5 mol% PEG-lipid liposomes containing AKG-38 and 40 or 55 mol% cholesterol The three liposomal formulations of Example 18 using 0.5M AS scavenger were evaluated in a two-time point pharmacokinetic study in female CD-1 mice as described in Example 7, measuring the percent of injected dose of liposomal lipid remaining in the blood (%ID) at both 5 minutes and 6 hours and measuring drug release from liposomes by determining the drug-to-lipid ratio (DL). Liposomes with 5 mol% PEG-DSG or 5 mol% PEG-DSPE containing 55 mol% Chol showed pre-injection D / L of greater than 95% at 5 minutes and greater than 85% at 6 hours, whereas the PEG-DSG formulation containing 40 mol% Chol showed dramatically reduced DL ratios at both 5 minutes and 6 hours, consistent with the in vitro drug leakage data in the presence of plasma (Table 17). This finding contrasts with previous experience with drug-loaded liposomal formulations, as several highly stable liposomal drugs approved for clinical use, such as PEGylated liposomal doxorubicin and nanoliposomal irinotecan, contain cholesterol at approximately 50 mol% (see, e.g., Doxil® drug information package insert, updated August 2019, and Drummond, DC, et al. (2006). "Development of a highly active nanoliposomal irinotecan using a novel intraliposomal stabilization strategy," Cancer Res. 66(6):3271-3277).

[0346] JPEG0007805319000095.jpg95170

[0347] [Example 21]

[0348] Inhibition of mitochondrial protein synthesis (MPS) by AKG-3, AKG-16, AKG-22, AKG-28, AKG-29, AKG-30, AKG-38, AKG-39, and AKG-40 and selectivity for Mycobacterium tuberculosis (H37Rv) compared with MPS inhibition Inhibition of mitochondrial protein synthesis was determined using a colorimetric MitoBiogenesis™ intracellular ELISA kit (catalog #ab11021) from AbCam according to the manufacturer's instructions. Mitochondrial protein synthesis inhibition has been correlated with significant toxicities associated with linezolid and other oxazolidinones, most notably ocular and peripheral neuropathy and lactic acidosis (Renslo (2010) Expert Review Anti Infect Ther 8(5)565-574; Flanagan et al. (2015) Antimicrob Agents Chemother 59(1)178-185; Santini et al. (2017) Expert Opin Drug Saf 16(7)833-843). Levels of two mitochondrial proteins were measured simultaneously, including the mitochondrial DNA-encoded subunit I of complex IV (COX-1) and the nuclear DNA-encoded 70 kDa subunit of complex II (SDH-A). The H9C2 rat BDIX cardiomyoblast cell line was used in these studies in a 384-well plate assay format. Cells were grown in DMEM medium containing 10% FBS and 1x glutamine at 37°C and 5% CO2. Cells were seeded at a density of 1,500 cells / well in 384-well plates in 47.5 μl / well. One replicate per concentration and condition of each compound, including nine 3-fold dilutions starting at a high concentration of 200 μM, was added to the cells in 2.5 μl and incubated with the cells for 5 days at 37°C and 5% CO2. Compounds tested included tedizolid and linezolid controls, as well as AKG-3, AKG-16, AKG-22, AKG-28, AKG-29, AKG-30, AKG-38, AKG-39, and AKG-40.

[0349] MitoBiogenesis In-Cell Elisa was then performed according to the manufacturer's instructions (Abcame catalog #ab11021) in a plate reader, with alkaline phosphatase (AP) developed for detection of SDH-1A at 405 nm in a kinetic mode for 15 minutes (20 seconds to 1 minute intervals) and HRP developed for detection of COX-I at 500 nm in a kinetic mode for 15 minutes (20 seconds to 1 minute intervals). COX-I and SDH-A signals were plotted as the ratio of COX-1 / SDH-A against the concentration of each compound, and IC50 values ​​were calculated for each of the nine investigated compounds and two controls.

[0350] The MPS selectivity index (SI-MPS) was determined by dividing the MPS IC in μg / ml by the MIC in the drug-susceptible H37Rv M. tuberculosis strain, determined as described in Example 2. The two compounds tested, AKG-28 and AKG-29, had SI-MPS values ​​more than 10-fold higher than those determined for linezolid and more than 20-fold higher than those determined for tedizolid. Both of these compounds contained a primary amino group at the R2 position of the oxazolidinone ring. Because of its high potency (MIC<0.1) and high selectivity for M. tuberculosis compared with mitochondrial protein synthesis, AKG-28 is an excellent candidate for encapsulation in liposomes and treatment of tuberculosis or other mycobacterial diseases.

[0351] JPEG0007805319000096.jpg108170

[0352] [Example 22]

[0353] Scale-up preparation of liposomal AKG-28 Lot 275 Lot 267.The general procedure of Example 6 was followed. 4.95 g (6.30 mmol) of HSPC (Lipoid AG), 2.98 g (7.71 mmol) of cholesterol (Dishman, High purity), and 850 mg (0.315 mmol) of PEG-DSPE (Lipoid AG) (molar ratio of HSPC:Chol:PEG-DSPE 45:55:2.25) were mixed with 9 ml of absolute ethanol (Sigma, E-7023) and heated with stirring on a 68°C bath until all lipids were dissolved. In a separate container, 93.3 g of 0.5 M aqueous ammonium sulfate (0.2 μm filtered) was preheated on a 68°C bath and poured into the hot lipid ethanol solution with stirring. The resulting suspension was stirred for 20 minutes in a 68°C bath and then extruded eight times at 260-300 psi through a stack of two 47 mm 100 nm pore-size and one 200 nm pore-size polycarbonate track-etched membranes (Whatman Nucleopore) using a Lipex 100 ml thermobarrel liposome extrusion apparatus (Northern Lipids, Inc.) heated with circulating water at 68°C. The resulting extruded liposomes were kept overnight in a refrigerator (2-8°C) and then filtered under positive pressure through a 0.2 μm polyethersulfone (PES) filter. The extraliposomal scavenger (ammonium sulfate) was removed by TFF buffer exchange into endotoxin-free water on a KrosFlo TFF system using polysulfone hollow fiber cartridges with a MW cutoff of 500 KDa (Spectrum Laboratories) until the residual conductivity fell to less than 200 μS / cm (143 μS / cm after a 5.2-fold volume exchange). The phospholipid concentration in the post-TFF liposome suspension was determined to be 57.4 mM by the phosphomolybdenum blue method.

[0354] 720 mg of AKG-28 (as the dihydrochloride salt) in the form of a 20 mg / ml aqueous stock solution (adjusted to pH 5.03 with NaOH) was mixed with the post-TFF liposome suspension to form a loading mixture with a drug-to-phospholipid (DL) ratio of 250 g / mol in the presence of 45 mg / ml dextrose and 6 mg / ml AKG-28. The mixture was rapidly heated to 60-63 °C by external heating with constant stirring, and the incubation continued with stirring in a 65 °C bath. After a 20-minute incubation, the mixture was rapidly cooled to below 10 °C in ice water and held at this temperature for approximately 10 minutes. After reaching ambient temperature and adjusting to 0.1 M NaCl, the drug-loaded liposomes were purified by TFF using a polysulfone hollow fiber cartridge with a molecular weight cutoff of 500 kD. Liposomes were pre-concentrated to approximately 12 mg / ml AKG-28 by diafiltration and purified from extraliposomal drug by TFF exchange in 10 mM HEPES-Na buffer, pH 7.0, containing 0.144 M NaCl, made with endotoxin-free water (HBS-7 buffer) over a total of eight volume exchanges. The percentage of unencapsulated drug before purification was estimated spectrophotometrically at 305 nm in the pre-concentrated diafiltrate and found to be approximately 0.9% (corresponding to a loading efficiency of 99.1%). The concentrated, purified liposomes were aseptically passed through a 0.2 μm sterile filter 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 273). The resulting liposomes had the characteristics shown in Table 19.

[0355] JPEG0007805319000097.jpg56170

[0356] These lots were mixed to obtain Lot 275, which had 12.0 mg / ml of AKG-28 in liposomal form, particle size Xz 113.7 nm, and PDI 0.0417.

[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: A stock aqueous solution of AKG-38 (as free base) was prepared by dissolving the drug in an equal volume of 1N HCl and adjusting the volume to obtain 20 mg / ml AKG-38 (as free base), pH 5.08. The loading mixture contained 1300 mg of AKG-38, prepared at a DL ratio of 8 mg / ml AKG-38 and 450 g / mol phospholipid, and further contained 10 mM NaCl. The loaded liposomes were pre-concentrated to approximately 22 mg / ml of drug; the percentage of unencapsulated drug before purification was estimated spectrophotometrically at 305 nm in the diafiltrate before concentration and found to be approximately 3.2% (corresponding to a loading efficiency of 96.8%). This process was repeated three more times (Lots 270, 272, and 274). The resulting liposomes had the characteristics shown in Table 20.

[0359] JPEG0007805319000098.jpg56170

[0360] These lots were mixed to obtain Lot 276, which had 22.3 mg / ml AKG-38 in liposomal form, particle size Xz 113.1 nm, and PDI 0.0454.

[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 (45:55:2.25 molar ratio of HSPC:Chol:PEG-DSPE) were dissolved in ethanol to form a liposome suspension, which was then extruded through a polycarbonate membrane as described in Example 22, except that the non-exchangeable cation sulfate, 0.13 M sodium sulfate, was used instead of 0.5 M ammonium sulfate. The extruded liposomes were purified from extraliposomal sodium sulfate and loaded into HBS-7 buffer by TFF buffer exchange using a polysulfone hollow fiber cartridge with a MWCO of 500 kDa over a total of 10-fold exchange. The purified liposomes had 42.9 mM phospholipid, a particle size of Xz 113.7 nm, and a PDI of 0.0612. They were aseptically passed through a 0.2 μm sterile filter and adjusted to 20 mM phospholipid with sterile HBS-7.

[0363] [Example 25]

[0364] Liposomal AKG-38 Lot 279 The general procedure of Example 6 was followed. 13.102 g (16.67 mmol) of HSPC (Lipoid AG), 7.877 g (20.37 mmol) of cholesterol (Dishman, High purity), and 2.250 g (0.833 mmol) of PEG-DSPE (Lipoid AG) (molar ratio of HSPC:Chol:PEG-DSPE 45:55:2.25) were mixed with 25 ml of absolute ethanol (Sigma, E-7023) and heated with stirring on a 68 °C bath until all lipids were dissolved. In a separate container, 259.1 g (250 ml) of 0.5 M aqueous ammonium sulfate (0.2 μm filtered) was preheated on a 70 °C bath and poured into the hot lipid ethanol solution with stirring. The resulting suspension was stirred on a 70 °C bath for at least 20 minutes and divided into four portions. Each portion was extruded five times at 280 psi through a stack of two 47 mm 100 nm pore-size and one 200 nm pore-size polycarbonate track-etched membranes (Whatman Nucleopore) using a Lipex 100 ml thermobarrel liposome extrusion apparatus (Northern Lipids, Inc.) heated with circulating water at 70°C. These partially extruded liposome portions were mixed (Xz 129.7 nm) and extruded together five more times through the same membrane stack, yielding liposomes with a size of Xz 115.9 nm and a PDI of 0.0212. The liposomes were kept overnight in a refrigerator (2–8°C) and then filtered through a 0.2 μm polyethersulfone (PES) filter under positive pressure. The phospholipid concentration was found to be 60.22 ± 0.34 mM. The extraliposomal scavenger (ammonium sulfate) was removed by TFF buffer exchange into endotoxin-free water on a KrosFlo TFF system using polysulfone hollow fiber cartridges with a MW cutoff of 500 kDa (Spectrum Laboratories) until the residual conductivity dropped to 180 μS / cm after a 5.1-volume exchange. The phospholipid concentration in the post-TFF liposome suspension was determined by the phosphomolybdenum blue method to be 54.97 ± 0.32 mM.

[0365] AKG-38 (free base) was mixed with 0.95 equivalents of 1N HCl and made up with endotoxin-free water to obtain a 20 mg / ml aqueous stock solution (pH 5.16). The solution was passed through a 0.2 μm filter, and an amount of the filtrate containing 3958 mg of drug was mixed with the post-TFF liposome suspension to form a loading mixture with a drug-to-phospholipid (DL) ratio of 450 g / mol in the presence of 44.5 mg / ml dextrose, 10 mM NaCl, and 8 mg / ml AKG-38 at pH 5.54. The mixture was heated to 61°C by external heating with constant stirring for 5 minutes, and incubation continued in a 65°C bath for an additional 22 minutes with stirring. The mixture was then transferred to an ice-water bath, stirred for 7 minutes, the temperature was lowered to 10°C, and the mixture was kept in the ice-water bath for an additional 8 minutes. After removal from the ice bath, allowing to reach ambient temperature, and adjusting to 0.1 M NaCl by adding 3 M NaCl stock, the drug-loaded liposomes (pH 6.53) were purified by TFF using a polysulfone hollow fiber cartridge with a molecular weight cutoff of 500 kD. The liposomes were pre-concentrated to approximately 22 mg / ml AKG-38 by diafiltration and purified from extraliposomal drug by TFF exchange in HBS-7 buffer over a total of eight volume exchanges. The concentrated, purified liposomes were aseptically passed through a 0.2 μm PES high-flow sterile filter and analyzed for particle size by DLS 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 ± 4.7 g / mol phospholipid, Xz 116.4 nm, and PDI 0.0231. 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. Extruded liposomes composed of HSPC, cholesterol, and PEG-DSPE in a molar ratio of 45:55:2.25 containing 0.5 M ammonium sulfate were prepared as described in Example 25. The extraliposomal sequestering agent (ammonium sulfate) was removed by TFF buffer exchange into endotoxin-free water on a KrosFlo TFF system using a polyethersulfone hollow fiber cartridge with a MW cutoff of 500 KDa (Spectrum Laboratories) until the residual conductivity was reduced to 150 μS / cm (4.1 volume exchanges). The phospholipid concentration in the post-TFF liposome suspension was determined to be 55.4 mM by the phosphomolybdenum blue method.

[0368] 969.5 mg of AKG-28 (as dihydrochloride salt) in the form of a 20 mg / ml aqueous stock solution (adjusted to pH 5.24 with NaOH) was mixed with the post-TFF liposome suspension to form a loading mixture with a drug-to-phospholipid (DL) ratio of 250 g / mol in the presence of 44.5 mg / ml dextrose and 6 mg / ml AKG-28. The mixture was heated to 65.4°C in 2.5 minutes by external heating with constant stirring, and incubation continued with stirring in a 65°C bath. After 20 minutes of incubation, the mixture was cooled to 9.3°C in 2.75 minutes in ice water and held in ice water for approximately 10 minutes. The mixture was then allowed to reach ambient temperature and adjusted to 0.1 M NaCl; pH 6.43. 133.4 g of the loading mixture was subjected to purification by TFF using a polysulfone hollow fiber cartridge with a molecular weight cutoff of 500 kD. Liposomes were pre-concentrated to approximately 12 mg / ml AKG-28 by diafiltration and purified from extraliposomal drug by TFF exchange in HBS-7 buffer over a total of 8.1 volume exchanges. The percentage of unencapsulated drug in the pre-concentrated diafiltrate was estimated spectrophotometrically at 302 nm and found to be approximately 0.7% (corresponding to a loading efficiency of 99.3%). The concentrated, purified liposomes were aseptically passed through a 0.2 μm sterile filter and analyzed for particle size by DLS and for drug and phospholipid concentrations by spectroscopy. The liposomes had the following characteristics: AKG-28 13.26 ± 0.21 mg / ml, DL ratio 258.2 ± 3.7 g / mol phospholipid, Xz 117.3 nm, and PDI 0.0421.

[0369] [Example 27]

[0370] Liposomal AKG-38 Lot 285 The general procedure of Example 6 was followed. Extruded liposomes composed of HSPC, cholesterol, and PEG-DSPE in a molar ratio of 45:55:2.25 containing 0.5 M ammonium sulfate were prepared essentially as described in Example 25. The extraliposomal sequestering agent (ammonium sulfate) was removed by TFF buffer exchange into endotoxin-free water on a KrosFlo TFF system using a polyethersulfone hollow fiber cartridge with a MW cutoff of 500 KDa (Spectrum Laboratories) until the residual conductivity was reduced to 138 μS / cm (5.6-fold exchange). The phospholipid concentration in the post-TFF liposome suspension was determined to be 53.1 mM by the phosphomolybdenum blue method.

[0371] AKG-38 (free base) was mixed with 0.95 equivalents of 1N HCl and made up with endotoxin-free water to obtain a 19.9 mg / ml aqueous stock solution (pH 5.13). The solution was passed through a 0.2 μm filter, and an amount of the filtrate containing 1400 mg of drug was mixed with the post-TFF liposome suspension to form a loading mixture with a drug-to-phospholipid (DL) ratio of 450 g / mol in the presence of 44.5 mg / ml dextrose, 10 mM NaCl, and 8 mg / ml AKG-38 at pH 5.58. The mixture was heated to 63°C by external heating with constant stirring over 2.25 minutes, and incubation continued in the 65°C bath with stirring for a total of 21 minutes. The mixture was then transferred to an ice-water bath, stirred for 3 minutes, the temperature was lowered to 10.3°C, and the mixture was maintained in the ice-water bath for an additional 7 minutes. After removal from the ice bath, allowing to reach ambient temperature, and adjusting to 0.1 M NaCl by adding 3 M NaCl stock, the drug-loaded liposomes (pH 6.70) were purified by TFF using a polysulfone hollow fiber cartridge with a molecular weight cutoff of 500 KD. The liposomes were pre-concentrated to approximately 22 mg / ml AKG-38 by diafiltration and purified from extraliposomal drug by TFF exchange in HBS-7 buffer over a total of 7.7 volume exchanges. The concentrated, purified liposomes had an AKG-38 concentration of 23.1 mg / ml. The drug concentration was adjusted to 20 mg / ml with HBS-7 buffer, and the liposomes were aseptically passed through a 0.2 μm PES high-flow sterile filter and analyzed for particle size by DLS and for drug and phospholipid concentrations by spectroscopy. The liposomes had the following characteristics: AKG-38 20.35±0.26 mg / ml, DL ratio 437.8±6.5 g / mol phospholipid, Xz 121.1 nm, PDI 0.0200. The yield of formulated drug was 1355 mg (96.8%).

[0372] [Example 28]

[0373] Liposomal AKG-28 Lot 286 Extruded liposomes (HSPC:Chol:PEG-DSPE 45:55:2.25 molar ratio) containing 0.5 M ammonium sulfate and no extraliposomal entrapped agent were obtained as described in Example 27. 600 mg of AKG-28 (as dihydrochloride salt) in the form of a 20 mg / ml aqueous stock solution (adjusted to pH 5.18 with NaOH) was mixed with the post-TFF liposome suspension to form a loading mixture with a drug-to-phospholipid (DL) ratio of 250 g / mol in the presence of 44.5 mg / ml dextrose and 6 mg / ml AKG-28. The mixture was placed in a 65°C water bath with stirring and allowed to reach 60°C in 4.5 minutes. Incubation was continued with stirring for a total of 20 minutes, and the mixture was cooled to 10.0°C in 2 minutes in ice water and kept in the ice-water bath for approximately 10 minutes. The mixture was then allowed to reach ambient temperature and adjusted to 0.1 M NaCl; pH 6.23. 104.6 g of the loaded mixture was subjected to purification by TFF using a polysulfone hollow fiber cartridge with a molecular weight cutoff of 500 kD. The liposomes were pre-concentrated to approximately 12 mg / ml AKG-28 by diafiltration and purified from extraliposomal drug by TFF exchange in HBS-7 buffer over a total of 8.3 volume exchanges. The concentrated, purified liposomes were aseptically passed through a 0.2 μm sterile filter (chased with HBS-7 buffer) and analyzed for particle size by DLS and for drug and phospholipid concentrations by spectroscopy. The liposomes had the following characteristics: AKG-28 12.05 ± 0.13 mg / ml, DL ratio 239.4 g / mol phospholipid, Xz 120.1 nm, 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. 9.17 g (11.67 mmol) of HSPC (Lipoid AG), 5.51 g (14.26 mmol) of cholesterol (Dishman, High purity), and 1.575 g (0.583 mmol) of PEG-DSPE (Lipoid AG) were mixed with 17.5 ml of absolute ethanol (Sigma, E-7023) and heated with stirring in a 69-70°C bath until all lipids were dissolved. In a separate container, 181.4 g (175 ml) of 0.5 M aqueous ammonium sulfate (0.2 μm filtered) was preheated in a 70°C bath and poured into the hot lipid-ethanol solution with stirring. The resulting suspension was stirred in a 70°C bath for at least 20 minutes and divided into three portions. Each portion was extruded five times at 280 psi through a stack of two 47 mm 100 nm pore-size and one 200 nm pore-size polycarbonate track-etched membranes (Whatman Nucleopore) using a Lipex 100 ml thermobarrel liposome extrusion apparatus (Northern Lipids, Inc.) heated with circulating water at 70°C. These partially extruded liposome portions were mixed (Xz 126.7 nm) and extruded together four more times through the same membrane stack, yielding liposomes with a size of Xz 119.2 nm and a PDI of 0.0385. The liposomes were kept overnight in a refrigerator (2–8°C) and then filtered through a 0.2 μm polyethersulfone (PES) filter under positive pressure. The phospholipid concentration was found to be 59.08 ± 0.44 mM. The extraliposomal scavenger (ammonium sulfate) was removed by TFF buffer exchange into endotoxin-free water on a KrosFlo TFF system using polysulfone hollow fiber cartridges with a MW cutoff of 500 kDa (Spectrum Laboratories) until the residual conductivity fell to 152 μS / cm after a 5.4-fold volume exchange. The phospholipid concentration in the liposome suspension after TFF was determined by the phosphomolybdenum blue method to be 57.76 ± 0.53 mM.

[0376] AKG-38 (free base) was mixed with 0.95 equivalents of 1N HCl and made up with endotoxin-free water to obtain a 19.7 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 3509 mg of drug was mixed with the post-TFF liposome suspension to form a loading mixture with a drug-to-phospholipid (DL) ratio of 450 g / mol in the presence of 44.5 mg / ml dextrose, 10 mM NaCl, and 8 mg / ml AKG-38 at pH 5.50. The mixture was heated to 61.6°C by external heating with constant stirring over 5 minutes, and incubation was continued in the 65°C bath with stirring for an additional 20 minutes. The mixture was then transferred to an ice-water bath, stirred for 7 minutes, the temperature was lowered to 10°C, and the mixture was kept in the ice-water bath for an additional 8 minutes. After removal from the ice bath, allowing to reach ambient temperature, and adjusting to 0.1 M NaCl by adding 3 M NaCl stock, the drug-loaded liposomes were purified by TFF using a polysulfone hollow fiber cartridge with a molecular weight cutoff of 500 KD. The liposomes were pre-concentrated to approximately 22 mg / ml AKG-38 by diafiltration and purified from extraliposomal drug by TFF exchange in HBS-7 buffer over a total of 7.8 volume exchanges. The concentrated, purified liposomes were aseptically passed through a 0.2 μm PES high-flow sterile filter and analyzed for particle size by DLS and for drug and phospholipid concentrations by spectroscopy. The liposomes had the following characteristics: AKG-38 22.47 ± 0.38 mg / ml, DL ratio 441.6 g / mol phospholipid, Xz 121.3 nm, and PDI 0.0465. The yield of formulated drug was 3375 mg (96.2%).

[0377] Lot 289.The Ls-288 process was repeated using 1506 mg of AKG-38 (a similarly prepared 20.0 mg / ml aqueous stock solution, pH 5.15). This solution was mixed with the same post-TFF extruded liposome suspension to form a loading mixture with a drug-to-phospholipid (DL) ratio of 450 g / ml in the presence of 44.5 mg / ml dextrose, 10 mM NaCl, and 8 mg / ml AKG-38 at pH 5.53. The mixture was heated to 64.3°C by external heating with constant stirring over 2 minutes, and incubation continued in the 65°C bath with stirring for an additional 20 minutes. The mixture was then transferred to an ice-water bath, stirred for 2.75 minutes, the temperature reduced to 9.6°C, and held in the ice-water bath for an additional 14 minutes. After removal from the ice bath, the loading mixture was allowed to reach ambient temperature and adjusted to 0.1 M NaCl using 3 M NaCl stock; pH 6.54. Drug-loaded liposomes were purified by TFF using polysulfone hollow fiber cartridges with a molecular weight cutoff of 500 kD. Liposomes were pre-concentrated to approximately 22 mg / ml AKG-38 by diafiltration and purified from extraliposomal drug by TFF exchange in HBS-7 buffer over a total of 8.1 volume exchanges. The concentrated, purified liposomes were aseptically passed through a 0.2 μm PES high-flow sterilizing filter and analyzed for particle size by DLS and for drug and phospholipid concentrations by spectroscopy. The liposomes had the following characteristics: AKG-38 22.84 ± 0.41 mg / ml, DL ratio 452.7 g / mol phospholipid, Xz 120.3 nm, and PDI 0.0522. The yield of formulated drug was 1407 mg (93.4%).

[0378] Lot 290.The general procedure of Example 6 was followed. 7.86 g (10.00 mmol) of HSPC (Lipoid AG), 4.73 g (12.22 mmol) of cholesterol (Dishman, High Purity), and 1.35 g (0.50 mmol) of PEG-DSPE (Lipoid AG) (45:55:2.25 molar ratio of HSPC:Chol:PEG-DSPE) were mixed with 15 ml of absolute ethanol (Sigma, E-7023) and heated with stirring in a 69-70 °C bath until all lipids were dissolved. In a separate container, 155.5 g (150 ml) of 0.5 M aqueous ammonium sulfate (0.2 μm filtered) was preheated in a 70 °C bath and poured into the hot lipid-ethanol solution with stirring. The resulting suspension was stirred in the 70 °C bath for at least 20 min and then divided into two portions. Each portion was extruded four times at 280 psi through a stack of two 47 mm 100 nm pore-size and one 200 nm pore-size polycarbonate track-etched membranes (Whatman Nucleopore) using a Lipex 100 ml thermobarrel liposome extrusion apparatus (Northern Lipids, Inc.) heated with circulating water at 70°C. These partially extruded liposome portions were mixed (Xz 131.5 nm) and extruded together four more times through the same membrane stack, yielding liposomes with a size of Xz 122.7 nm and a PDI of 0.0215. The liposomes were kept overnight in a refrigerator (2–8°C) and then filtered through a 0.2 μm polyethersulfone (PES) filter under positive pressure. The phospholipid concentration was found to be 58.99 ± 0.22 mM. The extraliposomal scavenger (ammonium sulfate) was removed by TFF buffer exchange into endotoxin-free water on a KrosFlo TFF system using polysulfone hollow fiber cartridges with a MW cutoff of 500 kDa (Spectrum Laboratories) until the residual conductivity fell to 146 μS / cm after a 5.5-fold volume exchange. The phospholipid concentration in the post-TFF liposome suspension was determined by the phosphomolybdenum blue method to be 56.94 ± 0.41 mM.

[0379] AKG-38 (free base) was mixed with 0.95 equivalents of 1N HCl and made up with endotoxin-free water to obtain a 20 mg / ml aqueous stock solution (pH 5.15). The solution was passed through a 0.2 μm filter, and an amount of the filtrate containing 2315 mg of drug was mixed with the post-TFF liposome suspension to form a loading mixture with a drug-to-phospholipid (DL) ratio of 450 g / mol in the presence of 44.5 mg / ml dextrose, 10 mM NaCl, and an AKG-38 concentration of 8.02 mg / ml, pH 5.52. The mixture was heated to 64.4°C by external heating with constant stirring over 3.25 minutes, and incubation continued in a 65°C bath with stirring for an additional 17 minutes. The mixture was then transferred to an ice-water bath, stirred to reduce the temperature to below 10°C, and held in the ice-water bath for a total of 10 minutes, allowed to reach ambient temperature, and adjusted to 0.1 M NaCl using 3 M NaCl stock; pH 6.63. Drug-loaded liposomes were purified by TFF using a polysulfone hollow fiber cartridge with a molecular weight cutoff of 500 KD. Liposomes were pre-concentrated to approximately 22 mg / ml AKG-38 by diafiltration and purified from extraliposomal drug by TFF exchange in HBS-7 buffer over a total of 8.0 volume exchanges. The concentrated, purified liposomes were aseptically passed through a 0.2 μm PES high-flow sterile filter and analyzed for particle size by DLS and for drug and phospholipid concentrations by spectroscopy. The liposomes had the following characteristics: AKG-38 22.07±0.23 mg / ml, DL ratio 441.6 g / mol phospholipid, Xz 120.4 nm, PDI 0.0395. The yield of formulated drug was 2141 mg (92.5%).

[0380] Lot 292. Lots 288 (150.3 g), 289 (61.2 g), and 290 (19.5 g) were combined to yield 278.4 g of Lot 292 at 22.5 mg / ml liposomally formulated AKG-38. All liposomal 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: 940 mg (1.20 mmol) HSPC (Lipoid AG), 568 mg (1.47 mmol) cholesterol (Dishman, High purity), 163 mg (0.06 mmol) PEG-DSPE (Lipoid AG), and 0.0018 mmol of lipophilic fluorescently labeled DiIC 18 (3)-DS(AAT Bioquest, USA)(HSPC:Chol:PEG-DSPE:DiIC 18 (3)-DS (45:55:2.25:0.0675 molar ratio, 0.15 mol% DiI3-DS relative to HSPC) was mixed with 2 ml of absolute ethanol (Sigma, E-7023) and heated with stirring in a 68 °C bath until all lipids were dissolved. In a separate container, 20 ml of 0.5 M aqueous ammonium sulfate solution (0.2 μm filtered) was preheated in a 68 °C bath and poured into the hot lipid-ethanol solution with stirring. The resulting suspension was stirred in a 68 °C bath for 20 min and extruded eight times at 300 psi through a stack of two 47 mm 100 nm pore-size and one 200 nm pore-size polycarbonate track-etched membranes (Whatman Nucleopore) using a Lipex 100 ml Thermobarrel liposome extrusion apparatus (Northern Lipids, Inc.) heated with circulating water at 68 °C. The resulting extruded liposomes were kept overnight in a refrigerator (2-8°C) and then filtered under positive pressure through a 0.2 μm polyethersulfone (PES) filter. The extraliposomal scavenger (ammonium sulfate) was removed by TFF buffer exchange into endotoxin-free water on a KrosFlo TFF system using a polysulfone hollow fiber cartridge with a MW cutoff of 500 kDa (Spectrum Laboratories) until the residual conductivity was reduced to 60 μS / cm (10-fold volume exchange). The phospholipid concentration in the post-TFF liposome suspension was determined to be 37.56 ± 0.62 mM by the phosphomolybdenum blue method.

[0383] 50 mg of AKG-28 (as dihydrochloride salt) in the form of a 20 mg / ml aqueous stock solution (adjusted to pH 4.99 with NaOH) was mixed with the post-TFF liposome suspension to form a loading mixture with a drug-to-phospholipid (DL) ratio of 250 g / mol in the presence of 140 mg / ml dextrose and 3 mg / ml AKG-28. The mixture (pH 5.53) was incubated with stirring in a 65°C bath for 20 minutes, rapidly cooled in ice water, and held in an ice-water bath for approximately 10 minutes. After reaching ambient temperature and adjusting to 0.1 M NaCl with 3 M NaCl stock solution, the pH was 5.80. The drug-loaded liposomes were purified by TFF using a polysulfone hollow fiber cartridge with a molecular weight cutoff of 500 KD. Liposomes were pre-concentrated to approximately 5 mg / ml AKG-28 by diafiltration and purified from extraliposomal drug by TFF exchange in HBS-7 buffer over a total of 10 volume exchanges. The purified liposomes were further concentrated 2-fold by TFF using a small, syringe-operated 500 KD hollow fiber cartridge (MicroKros, Spectrum). The concentrated, purified liposomes were aseptically passed through a 0.2 μm sterile filter and analyzed for particle size by DLS and for drug and phospholipid concentrations by spectroscopy. The liposomes had the following characteristics: AKG-28 8.22 ± 0.16 mg / ml, DL ratio 257.3 ± 10.3 g / mol phospholipid, Xz 118.2 nm, PDI 0.0188. The yield of formulated drug was 41.4 mg (82.8%).

[0384] [Example 31]

[0385] Preparation of liposomal AKG-38 Lot 236 The extruded liposomes after TFF containing 0.5 M ammonium sulfate from Example 30 were used. AKG-38 (free base) was mixed with 0.95 equivalents of 1 N HCl and made up with endotoxin-free water to obtain 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 mixed with the post-TFF liposome suspension (Example 30) to form a loading mixture with a drug-to-phospholipid (DL) ratio of 450 g / mol in the presence of 140 mg / ml dextrose and 3 mg / ml AKG-38. The mixture was incubated with stirring in a 65°C bath for 20 minutes, rapidly cooled in ice water, and held in an ice-water bath for approximately 10 minutes. After reaching ambient temperature and adjusting to 0.1 M NaCl with 3 M NaCl stock solution, the pH was 6.33. Drug-loaded liposomes were purified by TFF using a polysulfone hollow fiber cartridge with a molecular weight cutoff of 500 kD. Liposomes were pre-concentrated to approximately 6 mg / ml AKG-38 by diafiltration and purified from extraliposomal drug by TFF exchange in HBS-7 buffer over a total of approximately 10 volume exchanges. The purified liposomes were further concentrated 2-fold by TFF using a small, syringe-operated 500 kD hollow fiber cartridge (MicroKros, Spectrum). The concentrated, purified liposomes were aseptically passed through a 0.2 μm sterile filter and analyzed for particle size by DLS and for drug and phospholipid concentrations by spectroscopy. The liposomes had the following characteristics: AKG-38 9.04 ± 0.16 mg / ml, DL ratio 463.9 ± 19.8 g / mol phospholipid, Xz 119.3 nm, and PDI 0.0267. The yield of formulated drug was 56 mg (80%).

[0386] [Example 32]

[0387] Retention of encapsulated drug in liposomes of Lots 235 and 236 in vitro in the presence of plasma The retention of encapsulated drug in liposomes in the presence of 80% mouse human plasma at 37° C. was determined as described herein in Example 19. The incubation time was 20 minutes.

[0388] JPEG0007805319000099.jpg33170

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

[0390] [Example 33]

[0391] Preparation of liposomes AKG-28 and AKG-38 lots 231 and 232 (HSPC:cholesterol:PEG-DSPE 45:55:2.25 molar ratio, sequestering agent 0.5 M ammonium sulfate) The general procedure of Example 6 was followed. 4.255 g (5.41 mmol) of HSPC (Lipoid AG), 2.56 g (6.62 mmol) of cholesterol (Dishman, High purity), and 729 mg (0.27 mmol) of PEG-DSPE (Lipoid AG) (molar ratio of HSPC:Chol:PEG-DSPE 45:55:2.25) were mixed with 9 ml of absolute ethanol (Sigma, E-7023) and heated with stirring on a 70°C bath until all lipids were dissolved. In a separate container, 90 ml of 0.5 M aqueous ammonium sulfate solution (0.2 μm filtered) was preheated on a 70°C bath and poured into the hot lipid ethanol solution with stirring. The resulting suspension was stirred for 25 min in a 70°C bath and then extruded eight times at 260 psi through a stack of two 47 mm 100 nm pore-size and one 200 nm pore-size polycarbonate track-etched membranes (Whatman Nucleopore) using a Lipex 100 ml thermobarrel liposome extrusion apparatus (Northern Lipids, Inc.) heated with circulating 70°C water. The resulting extruded liposomes were kept overnight in a refrigerator (2-8°C) and then filtered under positive pressure through a 0.2 μm polyethersulfone (PES) filter. The extraliposomal scavenger (ammonium sulfate) was removed by TFF buffer exchange into endotoxin-free water on a KrosFlo TFF system using a polysulfone hollow fiber cartridge with a 500 kDa MW cutoff (Spectrum Laboratories) until the residual conductivity was reduced to 60 μS / cm (10-fold exchange). The phospholipid concentration in the liposome suspension after TFF was determined by the phosphomolybdenum blue method to be 46.97±0.80 mM.

[0392] Lot 231.350 mg of AKG-28 (as dihydrochloride salt) in the form of a 20 mg / ml aqueous stock solution (adjusted to pH 5.02 with NaOH) was mixed with the post-TFF liposome suspension to form a loading mixture with a drug-to-phospholipid (DL) ratio of 250 g / mol in the presence of 137.6 mg / ml dextrose and 2.53 mg / ml AKG-28. The mixture (pH 5.60) was incubated with stirring in a 65°C bath for 20 minutes, rapidly cooled in ice water, and held in an ice-water bath for approximately 10 minutes. After reaching ambient temperature and adjusting to 0.1 M NaCl with 3 M NaCl stock solution, the pH was 5.68. The drug-loaded liposomes were purified by TFF using a polysulfone hollow fiber cartridge with a molecular weight cutoff of 500 kD. Liposomes were pre-concentrated to approximately 9 mg / ml AKG-28 by diafiltration and purified from extraliposomal drug by TFF exchange in HBS-7 buffer over a total of 10.9 volume exchanges. The purified liposomes were further concentrated to approximately 12 mg / ml drug by continuing TFF diafiltration without buffer feed. The concentrated, purified liposomes were aseptically passed through a 0.2 μm sterile filter and analyzed for particle size by DLS and for drug and phospholipid concentrations by spectroscopy. The liposomes had the following characteristics: AKG-28 11.42 ± 0.09 mg / ml, DL ratio 247.7 ± 7.1 g / mol phospholipid, Xz 116.5 nm, PDI 0.0511. The yield of formulated drug was 322.7 mg (92.2%).

[0393] Lot 232.AKG-38 (free base) was mixed with 0.95 equivalents of 1N HCl and made up with endotoxin-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 the filtrate containing 580 mg of drug was mixed with the post-TFF liposome suspension of this example to form a loading mixture at a drug-to-phospholipid (DL) ratio of 500 g / mol in the presence of 137.6 mg / ml dextrose, 2.53 mg / ml AKG-38, and pH 5.72. The mixture was incubated with stirring in a 65°C bath for 20 minutes, rapidly cooled in ice water, and held in an ice-water bath for approximately 10 minutes. After reaching ambient temperature and adjusting to 0.1 M NaCl with 3 M NaCl stock solution, the pH was 6.40. The drug-loaded liposomes were purified by TFF using a polysulfone hollow fiber cartridge with a molecular weight cutoff of 500 KD. Liposomes were pre-concentrated to approximately 12 mg / ml AKG-38 by diafiltration and purified from extraliposomal drug by TFF exchange in HBS-7 buffer over a total of 8.5 volume exchanges. The purified liposomes were further concentrated 2-fold by continuing nTFF diafiltration without buffer feeding. The concentrated, purified liposomes were aseptically passed through a 0.2 μm sterile filter and analyzed for particle size by DLS and for drug and phospholipid concentrations by spectroscopy. The liposomes had the following characteristics: AKG-38 16.03 ± 0.07 mg / ml, DL ratio 487.3 ± 13.9 g / mol phospholipid, Xz 120.0 nm, PDI 0.0069. The yield of formulated drug was 538.9 mg (92.9%).

[0394] [Example 34]

[0395] Preparation of liposome AKG-28 lot 233 (HSPC:cholesterol:PEG-DSG 60:40:3 molar ratio, entrapment agent 1N triethylammonium sucrose octasulfate) The general procedure of Example 6 was followed. 1.88 g (2.4 mmol) of HSPC (Lipoid AG), 619 mg (1.6 mmol) of cholesterol (Dishman, High purity), and 312 mg (0.12 mmol) of PEG-DSG (Sunbright GS-020, NOF, Japan) were mixed in 3 ml of absolute ethanol and heated with stirring in a 67°C bath until all lipids were dissolved. In a separate container, 31.5 g (30 ml) of a 1 N aqueous triethylammonium octasulfate sucrose solution (0.2 μm filtered, pH 6.20, see Example 8) was preheated in a 65°C bath and poured into the hot lipid ethanol solution with stirring. The resulting suspension was stirred for 5 minutes in a 65°C bath and then extruded three times at 400 psi through a stack of four 47 mm 100 nm pore-size and one 200 nm pore-size polycarbonate track-etched membranes (Whatman Nucleopore) using a Lipex 100 ml thermobarrel liposome extrusion apparatus (Northern Lipids, Inc.) heated with circulating water at 65°C. The resulting extruded liposomes were kept overnight in a refrigerator (2-8°C) and then filtered under positive pressure through a 0.2 μm polyethersulfone (PES) filter. 9.2 g of extruded liposomes were purified from the extraliposomal entrapment agent (TEA-SOS) by TFF buffer exchange into endotoxin-free water on a KrosFlo TFF system using polysulfone hollow fiber cartridges with a 500 kDa MV cutoff (Spectrum Laboratories) until the retentate conductivity fell to 21 μS / cm (14.5-fold volume exchange). The phospholipid concentration in the post-TFF liposome suspension was determined by phosphomolybdenum blue spectrophotometry to be 31.32 ± 0.85 mM.

[0396] 140 mg of AKG-28 (as dihydrochloride salt) in the form of a 20 mg / ml aqueous stock solution (adjusted to pH 5.02 with NaOH) was mixed with the post-TFF liposome suspension to form a loading mixture with a drug-to-phospholipid (DL) ratio of 250 g / mol in the presence of 116.1 mg / ml dextrose and 2.52 mg / ml AKG-28. The mixture (pH 5.43) was incubated with stirring in a 65°C bath for 20 minutes, rapidly cooled in ice water, and held in an ice-water bath for approximately 10 minutes. After reaching ambient temperature and adjusting to 0.1 M NaCl with 3 M NaCl stock solution, the pH was 5.80. The drug-loaded liposomes were purified by TFF using polysulfone hollow fiber cartridges with a molecular weight cutoff of 500 kD. Liposomes were pre-concentrated to approximately 9 mg / ml AKG-28 by diafiltration and purified from extraliposomal drug by TFF exchange in HBS-7 buffer over a total of 10.9 volume exchanges. The purified liposomes were further concentrated to approximately 12 mg / ml drug by continuing TFF diafiltration without buffer feed. The concentrated, purified liposomes were aseptically passed through a 0.2 μm sterile filter (chased with HBS-7 buffer) and analyzed for particle size by DLS and for drug and phospholipid concentrations by spectroscopy. The liposomes had the following characteristics: AKG-28 10.64 ± 0.20 mg / ml, DL ratio 246.8 ± 11.7 g / mol phospholipid, Xz 116.3 nm, PDI 0.0022. The yield of 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, sequestering agent 1N triethylammonium sucrooctasulfate) The general procedure of Example 6 was followed. 3.30 g (4.20 mmol) of HSPC (Lipoid AG), 1.985 g (5.13 mmol) of cholesterol (Dishman, high purity), and 567 mg (0.21 mmol) of PEG-DSPE (Lipoid AG) (45:55:2.25 molar ratio of HSPC:cholesterol:PEG-DSPE) were mixed in 7 ml of absolute ethanol (Sigma, E-7023) and heated with stirring on a 70°C bath until all lipids were dissolved. In a separate container, 10 ml of 1 N aqueous triethylammonium sucrose octasulfate (TEA-SOS) solution (0.2 micron filtered) was preheated on a 70°C bath and poured into the hot lipid ethanol solution with stirring. The resulting suspension was stirred for 10 min in a 70°C bath and then extruded eight times at 260 psi through a stack of two 47 mm 100 nm pore-size and one 200 nm pore-size polycarbonate track-etched membranes (Whatman Nucleopore) using a Lipex 100 ml thermobarrel liposome extruder (Northern Lipids, Inc.) heated with circulating 70°C water. The resulting extruded liposomes were kept overnight in a refrigerator (2-8°C) and then filtered under positive pressure through a 0.2 μm polyethersulfone (PES) filter. The phospholipid concentration was 54.6 mM. 11.33 g of extruded liposomes were purified from the extraliposomal scavenger (TEA-SOS) by TFF buffer exchange for endotoxin-free water on a KrosFlo TFF system using a polysulfone hollow fiber cartridge with a MW cutoff of 500 kDa (Spectrum Laboratories) until the conductivity of the retentate decreased to 64 μS / cm (13.8 volume exchanges). The phospholipid concentration in the post-TFF liposome suspension was determined to be 28.67 ± 1.01 mM by blue phosphomolybdate spectrophotometry.

[0398] AKG-38 (free base) was mixed with 0.95 equivalents of 1N HCl and prepared in endotoxin-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 the filtrate containing 250 mg of drug was combined with the post-TFF liposome suspension of this example to form a loading mixture at a drug-to-phospholipid (DL) ratio of 500 g / mol in the presence of 116.4 mg / ml dextrose, an AKG-38 concentration of 2.53 mg / ml, and a pH of 5.24. The mixture was incubated with stirring in a 65°C bath for 20 minutes, rapidly cooled in ice water, and held in an ice-water bath for approximately 10 minutes. After reaching ambient temperature and adjusting to 0.1 M NaCl with 3 M NaCl stock solution, the pH was 6.60. The drug-loaded liposomes were purified by TFF using a polysulfone hollow fiber cartridge with a molecular weight cutoff of 500 KD. Liposomes were pre-concentrated by diafiltration into approximately 10 mg / ml AKG-38 and purified from any extraliposomal drug by TFF exchange into HBS-7 buffer for a total of 8.0 volume exchanges. The purified liposomes were further concentrated approximately 2-fold by continuing TFF diafiltration without a buffer feed. The concentrated purified liposomes were aseptically passed through a 0.2 μm sterile filter and analyzed for particle size by DLS and drug and phospholipid concentrations by spectrophotometry. The liposomes were characterized as follows: AKG-38 15.71 ± 0.33 mg / ml, DL ratio 518.6 ± 18.4 g / mol phospholipid, liposome size Xz 114.3 nm, and PDI 0.0284. The yield of formulated drug was 235.7 mg (94.3%).

[0399] [Example 36] Effect of osmotic agent concentration on the loading efficiency of AKG-28 and AKG-38 into liposomes and drug retention by liposomes in plasma The general protocol of Example 6 was followed. Extruded liposomes containing 0.5 M ammonium sulfate and a lipid composition of HPSC, cholesterol, PEG-DSPE, and DiIC18(3)-DS (fluorescent lipid label) in a molar ratio of 45:55:2.25:0.0675 were prepared as described in Example 30. Liposomes were purified from extraliposomal ammonium sulfate by TFF exchange with endotoxin-free water (Hyclone) for water for injection (WFI) using a syringe-actuated microcloth polysulfone hollow fiber cartridge (MWCO 500 KDa, Spectrum Laboratories) (13.8 volume exchange, residual conductivity 88 μS / cm, phospholipid concentration 55.4 mM). Liposomes were loaded with AKG-28 or AKG-38 by incubating the drug (prepared as an aqueous 20 mg / ml stock as described in Examples 30 and 31) with purified extruded liposomes in aqueous solution in a 65°C water bath for 20 minutes at a drug concentration of 2.22 mg / ml and a DL ratio of 250 g / mol phospholipid (AKG-28) or 450 g / mol phospholipid (AKG-38) in the presence of various concentrations of osmotic agent (dextrose). Unencapsulated drug was removed by size-exclusion chromatography using Sepharose CL-4B, eluting with HBS-7 buffer, and loading (encapsulation) efficiency was determined from the results of drug and phospholipid analysis. Osmotic agent concentrations were expressed in absolute terms and as a percentage of the 168 mg / ml dextrose concentration, which was determined to be isoosmotic to the 0.5 M ammonium sulfate solution used to form the liposomes. Contrary to expectations from the general consensus in the liposome field, drugs were effectively loaded into the disclosed liposomes (with encapsulation efficiencies greater than 85%, mostly greater than 90%) even under hypotonic conditions (i.e., at an osmotic pressure of the solution outside the liposomes lower than that of the intraliposomal entrapped agent solution) and in the complete absence of added osmotic balancing agent (dextrose) (Table 22).Furthermore, when exposed to plasma under the conditions of the in vitro plasma release assay described in Example 19, drug encapsulation in liposomes loaded with the lowest concentration of osmotic agent was at least as stable as those loaded at near perfect (86.3%) osmotic equilibrium.

[0400] The results showed that liposomes loaded with 55 mol% Chol, 45 mol% PC, 5 mol% HSPC with PEG-DSPE, and 0.5 MAS scavenger both AKG-28 (Table 22) and AKG-38 (Table 23) at 250 or 500 g / mol PhL with efficiencies >85%, mostly >90%, and under hypoosmotic conditions down to zero percent dextrose, liposomes loaded under hypoosmotic conditions efficiently retained the drug in the presence of plasma.

[0401] JPEG0007805319000100.jpg88170

[0402] JPEG0007805319000101.jpg90170

[0403] [Example 37] Single-dose pharmacokinetic study of all forms (encapsulated + released drug) of Ls-AKG28 and Ls-AKG38 in rats This study was conducted to evaluate the pharmacokinetics of Ls-AKG28 and Ls-AKG38 administered as a single dose in rats. The study was conducted in male Sprague-Dawley rats using IV administration of 20, 40, or 80 mg / kg body weight of liposomal AKG-38 (Ls-AKG38) or 10, 20, or 40 mg / kg body weight of liposomal AKG-28 (Ls-AKG28). Ls-AKG28 (Lot 275) and Ls-AKG38 (Lot 276) were prepared as described in Examples 22 and 23, respectively. For comparison, linezolid 50 mg / kg body weight formulated in 0.5% methylcellulose and acidified to pH 3-4 (Sigma M0430) at a concentration of 20 mg / mL was administered by oral gavage. For plasma drug measurements, 0.5 ml of blood was collected in lithium heparin tubes at 5 min, 15 min, 1 h, 3 h, 6 h, 24 h, 48 h, and 72 h. Samples were centrifuged, and the resulting plasma was separated, transferred to double-sided clear polypropylene tubes, immediately frozen on dry ice, and stored at -80°C until analysis. Plasma concentrations in rats were determined by HPLC. Non-compartmental PK analysis was performed using Phoenix WinNonlin (version 7.0). For Ls-AKG28 and Ls-AKG38, this PK software was used to calculate the maximum plasma 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-last ) and 0h to infinity (AUC 0-inf ), AUC 0-last divided by the dose (AUC 0-last The apparent clearance (CL / F), volume of distribution (Vd), and elimination half-life (T1 / 2) were estimated for linezolid using this PK software, except for the apparent clearance (CL / F) and apparent volume of distribution (Vd / F).

[0404] The plasma concentration versus time profiles of all drugs after administration of Ls-AKG28 at a single intravenous dose of 10, 20, and 40 mg / kg (IV x 1) are shown in Figure 7. A summary of the plasma PK parameters of all drugs after administration of Ls-AKG28 at 10, 20, 40 mg / kg IV x 1 is shown in Table 24.

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

[0406] JPEG0007805319000102.jpg134170

[0407] The plasma concentration versus time profiles of all drugs following administration of Ls-AKG38 at 20, 40, and 80 mg / kg IV×1 are shown in FIG.

[0408] A summary of plasma PK parameters for all drugs following 20, 40, and 80 mg / kg IV x 1 dose of Ls-AKG38 is shown in Table 25.

[0409] At all doses, the plasma concentration versus time profile of Ls-AKG38 was detectable from 5 minutes to 72 hours. max Based on the / dose and AUC / dose results, the plasma PK of Ls-AKG38 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 greater than that of Ls-AKG28 (approximately 1.67 mL / h / kg). At the same dose (20 or 40 mg / kg), the Vd of Ls-AKG28 was greater than that of Ls-AKG38.

[0410] JPEG0007805319000103.jpg135170

[0411] The plasma concentration versus time profiles of all drugs 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 are shown in Figures 7 and 8, respectively. In single IV dose studies, the plasma concentration versus time profiles of Ls-AKG28 and Ls-AKG38 were detectable from 5 minutes to 72 hours at all doses. The plasma PK of Ls-AKG28 was linear (dose-proportional) after administration of 10, 20, and 40 mg / kg. The plasma PK of Ls-AKG38 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 greater than that of Ls-AKG28 (approximately 1.67 mL / h / kg). At the same dose (20 or 40 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 at 40 mg / kg was approximately 73-fold and 110-fold higher than the plasma PK of linezolid (using AUC from 0 to end).

[0412] The plasma AUC and drug persistence in the circulation were much greater for both liposomal formulations compared to linezolid. The PK of both liposomal formulations, with a linear dose-dependence as seen by the AUC / dose values, was very similar for Ls-AKG28 and Ls-AKG38, respectively.

[0413] [Example 38] Plasma pharmacokinetics (PK) of Ls-AKG28 and Ls-AKG38 in all forms (encapsulated + released) after repeated IV dosing in Sprague-Dawley rats This study was conducted to evaluate the PK of Ls-AKG28 and Ls-AKG38 in rats administered once weekly at increasing doses for a total of 8 weeks. The study was conducted in Sprague-Dawley rats via IV administration. Ls-AKG28 (Lot 275) and Ls-AKG38 (Lot 276) were prepared as described in Examples 22 and 23, respectively, and plasma concentrations in rats were determined by HPLC. The plasma concentration versus time profiles of total drug after administration of Ls-AKG28 at 10, 20, and 40 mg / kg IV × 1 on days 1, 15, 29, and 43 are shown in Table 26 and Figures 9A, 9B, and 9C. A summary of plasma PK parameters for all drugs following administration of Ls-AKG28 at 10, 20, 40 mg / kg IV on days 1, 1, 15, 29, and 43 is shown in Table 26. All data from Figures 9A, 9B, and 9C were 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 similar after the first dose. At 10 mg / kg Ls-AKG28, plasma C max The plasma C and AUC were similar on days 1, 15, 29, and 43. At 20 mg / kg of Ls-AKG28, max and AUC increased on days 29 and 43. At 40 mg / kg Ls-AKG28, plasma C max and AUC increased after dosing on days 1-43.

[0415] The plasma concentration versus time profiles of all drugs after administration of Ls-AKG38 at 20, 40, and 80 mg / kg IV × 1 on Days 1, 15, 29, and 43 are shown in Table 27 and Figures 10A, 10B, and 10C. A summary of plasma PK parameters of all drugs after administration of Ls-AKG38 at 20, 40, and 80 mg / kg IV × 1 on Days 1, 1, 15, 29, and 43 is shown in Table 27. In single- and multiple-dose PK studies, the plasma kinetics of Ls-AKG38 were similar after the first dose. At 20, 40, and 80 mg / kg, Ls-AKG38 showed plasma C maxand AUC increased from day 1 to day 43. Given concerns about accelerated blood clearance (ABC) for PEGylated liposomes containing non-cytotoxic drug payloads, the lack of increased clearance in later cycles was surprising and suggests that liposomes containing AKG-28 or AKG-38 can be chronically dosed in mammals.

[0416] JPEG0007805319000104.jpg137170

[0417] JPEG0007805319000105.jpg137170

[0418] [Example 39] Pharmacokinetic study of drugs and liposomal lipids of Ls-AKG28 and Ls-AKG38 in CD-1 mice This study was designed to determine the in vivo plasma pharmacokinetic parameters of drug and liposomal lipids, as well as the stability of drug retention in liposomes for liposomal formulations of AKG-28 and AKG-38. The study was conducted in male CD-1 (20-22 g) mice (five mice per time point) as described in the general protocol in Example 7 above. Ls-AKG28 (Lot 235) and Ls-AKG38 (Lot 236) were prepared as described in Examples 30 and 31, respectively. Liposomes at a dose of 50 mg / kg (Ls-AKG28) or 90 mg / kg (Ls-AKG38) were injected into the lateral tail vein at time 0, and blood was collected at 0.083, 1, 3, 6, 24, and 48 h post-injection. AKG-28, AKG-38, and fluorescent liposomal lipid label (DiIC) were used. 18Plasma concentrations of (3)-DS were determined by HPLC. Using liposome lots 235 and 236 as standards, plasma concentrations of liposomal phospholipids were calculated from fluorescently labeled quantification. Because the tissue affinity of unencapsulated oxazolidinone drugs is expected to be several times higher than that of liposome-encapsulated drugs (e.g., as supported by the Vd of 2,291.26 mL / kg for unencapsulated oxazolidinone, linezolid, compared with Vd of 33.27-43.74 mL / kg for liposome-encapsulated AKG-28 in rats; see Example 37), plasma drug concentrations were considered to be primarily attributable to liposome-associated drug. The plasma drug-to-liposomal lipid (DL) ratio, normalized to the original (pre-injection) DL value, was employed as a measure of drug retention by liposomes. Noncompartmental PK analysis was performed using Summit Research Services' PK Solutions 2.0. For Ls-AKG28 and Ls-AKG38, the PK software was used to calculate the maximum plasma 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-last ) and 0h to infinity (AUC 0-inf ), AUC 0-last divided by the dose (AUC 0-last / dose), clearance (CL), volume of distribution (Vd), and elimination half-life were estimated.

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

[0420] Ls-AKG28 has almost complete in vivo stability, with undetectable loss of drug up to 48 hours after IV injection in mice. The half-life of drug release for Ls-AKG28 follows a monoexponential equation (R 2 = 0.822), the half-life of Ls-AKG28 is 866.3 hours. Ls-AKG38 has a fast drug release rate. The half-life of Ls-AKG38 drug release is calculated using a monoexponential equation (R 2 =0.950), the result is 22.9 hours.

[0421] JPEG0007805319000106.jpg143170

[0422] JPEG0007805319000107.jpg140170

[0423] JPEG0007805319000108.jpg75170

[0424] [Example 40] Pharmacokinetic study of Ls-AKG28 and Ls-AKG38 drugs in mice after repeated administration of liposomes, presence of ABC effect The generation of anti-PEG antibodies has been shown to cause faster clearance of liposomes containing PEG-lipid conjugates (PEGylated liposomes) after repeated injection, a phenomenon known as accelerated blood clearance (ABC) (Ishida et al. Journal of Controlled Release 105 (2005) 305-317; Laverman et al. JPET 298 (2001) 607-612). This study was conducted to determine whether there was an ABC effect after repeated administration of various doses of Ls-AKG28 and Ls-AKG38 with different compositions. Liposomes were prepared according to Examples 33-35, lots 231, 232, 233, and 234. This study was conducted in male CD-1 mice as generally described in Example 7. Groups of five mice were used. Plasma concentrations of AKG-28 and AKG-38 in mice were determined by HPLC. Mice were injected four times weekly with the indicated doses and formulations. Drug was measured in plasma at 6-hour time points after the first and fourth doses (FIG. 12). None of the tested groups showed significantly accelerated clearance after the fourth injection (two-tailed t-test, all p values ​​>0.05). This data confirms that these liposomal oxazolidinones can be chronically dosed over multiple weekly cycles without significantly negatively impacting drug exposure.

[0425] JPEG0007805319000109.jpg89170

[0426] This data shows that after four cycles of treatment, the blood clearance rate of liposomal AKG-28 or liposomal AKG-38 of the present disclosure was not increased, in contrast to what was previously reported for other PEGylated liposomes that do not contain a liposome-associated cytotoxic drug.

[0427] [Example 41] Dose-dependent tolerability of liposomal AKG-28 and liposomal AKG-38 in CD-1 mice The purpose of this study was to evaluate the tolerability of Ls-AKG28 and Ls-AKG38 injected into mice as single agents at different doses. Female CD-1 mice weighing 20–22 g (five mice per group) received Ls-AKG28 (50, 65, 90, or 100 mg / kg / dose) or Ls-AKG38 (50, 90, 120, or 200 mg / kg / dose) intravenously (tail vein) once weekly for 4 weeks. Liposomal formulations (Ls-AKG28 Lot 231 and Ls-AKG38 Lot 232) were prepared as previously described in Example 33. The control group received an equal volume of HEPES-buffered saline (HBS, pH 7) injection once weekly for 4 weeks. Body weight was measured three times weekly throughout the study, and data are presented as a percentage of weight change relative to the weight measured on day 0.

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

[0429] As shown in Figures 13A and 13B, compared to the saline control group, there was no significant effect on mouse body weight observed for both Ls-AKG28 and Ls-AKG38 when treated at doses up to 90 mg / kg for Ls-AKG28 and up to 200 mg / kg for Ls-AKG38 for a total of 4 weeks.

[0430] Compared with the control group, mice treated with high doses of Ls-AKG38 (90, 120, and 200 mg / kg) did not show a significant decrease in red blood cell count and hematocrit (Figure 13C) compared with the saline control group. No such effect was observed in mice administered Ls-AKG28. A significant decrease in platelet count was observed in mice treated with Ls-AKG28 at the highest dose of 90 mg / kg (Figure 13C) compared with the control group, but the decrease was still less than 25% compared with the saline control. Treatment with either Ls-AKG28 or Ls-AKG38 did not significantly affect white blood cell (WBC) count or blood liver enzymes (ALT and AST).

[0431] Histopathological analysis revealed no test article-related findings in animals administered 50 and 65 mg / kg of Ls-AKG28 (Figure 13D). Test article-related findings, consisting of minimal vacuolation of macrophages (including Kupffer cells), were observed in the liver, spleen, and kidneys of animals administered 90 mg / kg of LS-AKG28. Treatment with Ls-AKG38 was associated with test article-related findings in the liver and spleen at doses of 90 mg / kg and 120 mg / kg. In the liver, mild to mild vacuolation and Kupffer cell hypertrophy were observed at 50 and 90 mg / kg, moderate Kupffer cell vacuolation and hypertrophy at 50 and 120 mg / kg, and minimal multifocal aggregation of vacuolated macrophages at 90 mg / kg and 120 mg / kg.

[0432] Treatment with the highest dose of Ls-AKG38 (200 mg / kg) was associated with a slight increase in extramedullary hematopoiesis (EMH) in the liver and spleen, minimal to mild multifocal mixed cell infiltrates, and minimal individual and focal hepatocyte necrosis in the liver (Fig. 13D). These microscopic findings were not considered test article related, as they were common background findings in this species.

[0433] Overall, both Ls-AKG28 and Ls-AKG38 monotherapy were well tolerated in vivo in mice, even when injected at the highest evaluated doses of liposomal drug, 90 and 200 mg / kg for Ls-AKG28 and Ls-AKG38, respectively.

[0434] [Example 42] In vivo tolerability of Ls-AKG28 and Ls-AKG38 in combination with BDQ / PMD or BDQ / PMD / MOX in mice In this example, the in vivo tolerability of liposomal oxazolidinone was evaluated in combination with therapeutically relevant anti-TB drugs. Three-drug regimens of bedaquiline, pretomanide, and linezolid (BDQ / PMD / LNZ or BPL) or bedaquiline, pretomanide, and moxifloxacin (BDQ / PMD / MOXI or BPM) have demonstrated strong clinical activity in treating multidrug-resistant tuberculosis (Conradie et al. (2020) N Engl J Med 382(10)893-902 and Tweed et al. (2019) Lancet Respir Med 7(12)1048-1058). However, the BPL regimen is limited primarily by toxicity associated with the addition of linezolid (Conradie et al. (2020) N Engl J Med 382(10)893-902). Here, we evaluated the safety and tolerability of two liposomal oxazolidinones, Ls-AKG28 and Ls-AKG38, when used as part of either the BPL regimen (substituting for linezolid) or the BPM regimen (addition). CD-1 mice (5 per group) were treated with Ls-AKG28 (Lot 231) or Ls-AKG38 (Lot 232) alone or in combination with bedaquiline (BDQ) and pretomanid (PMD). Ls-AKG28 (Lot 231) and Ls-AKG38 (Lot 232) were prepared as described in Example 33. Additionally, mice were co-treated with a triple combination of BDQ, PMD, and moxifloxacin (MOXI) and liposomal oxazolidinone.

[0435] Ls-AKG28 (50 mg / kg / dose) and Ls-AKG38 (90 mg / kg / dose) were intravenously injected via the tail vein once a week for 4 weeks. A combination of BDQ, PMD, and MOXI (25 / 100 / 100 mg / kg / dose, respectively) was administered by oral gavage once daily, 5 times a week for 4 weeks. As an additional control, mice were treated with BDQ / PMD / MOX or BDQ / PMD (25 / 100 mg / kg / dose, respectively) plus linezolid (LNZ), given orally at 100 mg / kg / dose once daily, 5 times a week for 4 weeks. Body weight measurements, tissue collection, and analysis were performed as described above in Example 41.

[0436] As shown in Figures 14A and 14B, no significant effect of Ls-AKG28 or Ls-AKG38 on mouse body weight was observed during the study when co-treated with BDQ / PMD (BP) or BDQ / PMD / MOX (BPM). Both Ls-AKG28 and LsAKG38 were well tolerated in combination with BDQ / PMD or BDQ / PMD / MOX and did not affect hematology or blood biochemistry in treated mice (Figure 14C).

[0437] Histopathological data (Figure 14D) showed no treatment-related changes in the case of Ls-AKG28 in combination with BDQ / PMD. The combination of Ls-AKG28 + BDQ / PMD / MOX had minimal events associated with mixed cell and mononuclear cell infiltration in the lungs and heart. Treatment with Ls-AKG38 as monotherapy was associated with minimal test article-related findings in the liver (inflammatory infiltration and hepatocellular necrosis). Administration of Ls-AKG38 + BDQ / PMD showed no treatment-related findings, and the combination of Ls-AKG38 + BDQ / PMD / MOX was associated with minimal mixed cell infiltration in the lungs. Animals treated with the BDQ / PMD / LNZ combination were associated with treatment-related findings of inflammatory infiltration in the liver, minimal hepatocellular necrosis, and vacuolated macrophage infiltration in the lungs. Thus, both Ls-AKG28 (50 mg / kg / dose) and Ls-AKG38 (90 mg / kg / dose) administered once weekly for 4 weeks were well tolerated in mice in combination with BDQ / PMD or BDQ / PMD / MOX.

[0438] [Example 43] Effect of dose scheduling on the tolerability of Ls-AKG28 and Ls-AKG38 in combination with BDQ / PMD in mice In this study, the in vivo tolerability of twice-weekly administered Ls-AKG28 (50 mg / kg / dose) or Ls-AKG38 (100 mg / kg / dose) was compared with once-weekly administered Ls-AKG28 (100 mg / kg / dose) or Ls-AKG38 (200 mg / kg / dose). Liposomes were prepared according to Example 25 (Ls-AKG38, lot 279) and Example 26 (Ls-AKG28, lot 281). Both liposomal drugs were injected alone or in combination with BDQ / PMD (BP) into CD-1 female mice (five mice per group). BDQ / PMD (25 and 100 mg / kg / dose, respectively) was administered by oral gavage once daily, five times a week for four weeks. The control group received weekly injections of HEPES-buffered saline (HBS, pH 7) for four weeks. Blood and tissue samples were collected and analyzed as described in Examples 41 and 42.

[0439] Neither monotherapy nor combination therapy of mice treated with Ls-AKG28 or Ls-AKG38 twice weekly or once weekly at higher doses affected body weight (Figures 15A and 15B) or blood counts and biochemistry (Figure 15C).

[0440] Histopathological analysis of the recovered tissues (Fig. 15D) revealed a minimal interstitial mixed cell infiltrate consisting of macrophages and neutrophils in two of five mice receiving 50 mg / kg (2 qw) Ls-AKG28 and a mild interstitial mixed cell infiltrate in one of five animals receiving 100 mg / kg (1 qw) Ls-AKG28.

[0441] Lungs from mice receiving Ls-AKG28+BP at 50 mg / kg (1 qw) showed a minimal interstitial infiltrate consisting of macrophages (1 of 5) or mixed (macrophages and neutrophils) inflammatory cells (3 of 5). Four of 5 mice receiving 100 mg / kg (1 qw) of Ls-AKG28+BP showed a minimal interstitial mixed cell infiltrate. Furthermore, lungs from 2 of 5 animals receiving 100 mg / kg (1 qw) of Ls-AKG28+BP showed minor multifocal foreign-body granulomas associated with pale basophilic foreign bodies. These microscopic findings were not considered test article related because of the occurrence of minor multifocal mixed cell infiltrates and minimal individual hepatocellular necrosis in the liver of one of five animals receiving Ls-AKG28 + BP 50 mg / kg (2 qw), a common background finding in this species.

[0442] Similar microscopic findings associated with Ls-AKG38 treatment (alone or in combination) were not considered test article related and included a slight increase in extramedullary hematopoiesis (EMH) in the liver and spleen, slight to mild multifocal mixed cell infiltrates and minimal individual hepatocyte necrosis in the liver, minimal focal hepatocyte necrosis in the lung, minimal focal foreign body granulomas (associated with pale basophilic foreign body), and minimal mixed cell infiltrates in the lung. These findings were not considered treatment-related due to their minimal to mild nature, sporadic incidence, presence in the saline control group, and occurrence as a common background finding in this species.

[0443] Thus, Ls-AKG28 and Ls-AKG38 (alone or in combination with BDQ / PMD) administered twice weekly at doses of 50 mg / kg and 100 mg / kg, respectively, or once weekly at doubling doses of 100 mg / kg and 200 mg / kg, were both well tolerated in mice and did not affect the body weight, hematology, or histopathology of treated animals.

[0444] [Example 44] In vivo tolerability of Ls-AKG28 and Ls-AKG38 in rats The purpose of this study was to determine the potential toxicity of Ls-AKG28 (Lot 275) and Ls-AKG38 (Lot 276) in rats. Ls-AKG28 (Lot 275) and Ls-AKG38 (Lot 276) were prepared 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 80 mg / kg / dose) intravenously (tail vein) once weekly for 8 weeks. A control group received an equal volume of HEPES-buffered saline (HBS, pH 7) once weekly for 8 weeks. Prior to the study endpoint, animals were euthanized by exsanguination via the abdominal aorta after isoflurane anesthesia. Blood and tissue samples were collected, and clinical pathological parameters were evaluated. Representative samples of tissues were collected, preserved in 10% neutral buffer, embedded in paraffin, sectioned, mounted on glass slides, stained with hematoxylin and eosin, and evaluated for histopathology by a board-certified veterinary pathologist. Hematological analyses were performed using a Homology ADVIA 120 / 2120i Analyzer, and blood biochemistry analyses were performed using a Cobas 6000 Analyzer.

[0445] Mortality and moribund checks, clinical observations, body weight, food intake, nerve conduction velocity (NCV) and muscle action potential (MAP), parameters and endpoints of the Functional Observation Battery (FOB) were also assessed.

[0446] Nerve conduction velocity (NCV) and muscle action potential (MAP) measurements were performed at 8 weeks. Animals were anesthetized with isoflurane during recording sessions. The tail nerve NCV measures the conduction velocity of the tail nerve, which runs along the central bone of the tail. This nerve is approximately 50% longer than any other nerve in the rat, making it particularly susceptible to length-dependent distal axonopathy. NCV is measured over a distance of 50 mm and is sensitive to nodal and transmembrane currents, the structure and mean cross-sectional diameter of the responding axon, and the integrity of the associated myelin sheath. The amplitude of the evoked response reflects the number and synchrony of activated fibers. Data were recorded with an active recording electrode (visually determined) positioned approximately 10 mm below the hairline of the tail and a stimulating cathode 50 mm further distal. Signal amplitude and onset latency were recorded, and velocity was calculated by dividing the distance between the stimulating cathode and active electrode by the absolute onset latency of the initial depolarizing current.

[0447] The digital nerve NCV measures conduction velocity in the sensory digital nerve. The digital nerve is the distal end of the sciatic nerve, innervating the dorsal surface of the hind paw. Nerve conduction velocity is sensitive to nodal and transmembrane currents, the structure and mean cross-sectional diameter of the responding axon, and the integrity of the associated myelin sheath. Data were recorded with an active recording electrode positioned at the ankle posterior to the lateral malleolus and a stimulating cathode located at the base of the second toe of the hind paw. Signal amplitude and onset latency were recorded, and velocity was calculated by dividing the distance between the stimulating cathode and the active electrode by the absolute onset latency of the initial depolarizing current.

[0448] Tibial motor conduction (onset latency) measures the response characteristics of intrinsic muscles in the rat hindpaw following stimulation of motor fibers in the distal tibial nerve. Data were recorded with the active electrode positioned over the lateral dorsi muscle of the hindpaw (equivalent to the extensor digitorum brevis in humans) and the stimulating cathode positioned proximal to the ankle joint, behind the lateral malleolus. Nerve conduction velocity in motor axons was estimated from the onset latency of the induced compound muscle action potential (CMAP). CMAP amplitude was measured at the peak of the response following supramaximal stimulation of the relevant nerve.

[0449] There were no Ls-AKG28- or Ls-AKG38-related unscheduled deaths, clinical observations, effects on body weight (Figures 16A and 16B), NCV and MAP (Table 34), FOB (Table 35), food consumption, coagulation parameters, organ weights, or gross findings (data not shown). Despite a 16.5-fold increase in the efficacy-adjusted dose (based on free drug potency against M. tuberculosis Erdmann in Example 2) for Ls-AKG28 or Ls-AKG38 compared with linezolid in the liposome-treated groups, the lowest decrease in nerve conductance velocity in the caudal or left digital nerve was less than 5% in either liposome-treated group.

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

[0451] Administration of Ls-AKG28 to male Sprague-Dawley rats by intravenous injection once weekly for 8 weeks at doses of 10 mg / kg / dose or higher resulted in microscopic findings in the spleen, kidneys, and liver (Table 36). Spleens showed minimal to moderate macrophage vacuolation with basophilic granules and minimal to mild accumulation of basophilic material in rats administered 20 or 40 mg / kg / dose. Kidneys of rats given 40 mg / kg / dose showed microvacuolation of glomerular mesangial cells. Livers showed minimal centrilobular single-cell necrosis and minimal to mild centrilobular hepatocyte degeneration at all doses.

[0452] JPEG0007805319000110.jpg73170

[0453] JPEG0007805319000111.jpg73170

[0454] JPEG0007805319000112.jpg80170

[0455] JPEG0007805319000113.jpg89170

[0456] JPEG0007805319000114.jpg146170

[0457] Administration of Ls-AKG38 at doses of 20 mg / kg / dose and above resulted in hepatic microscopic findings of centrilobular minimal single-cell necrosis and centrilobular minimal to mild hepatocellular degeneration at all doses.

[0458] In contrast, rats treated with Ls-AKG28 and Ls-AKG38 showed increased incidence of hepatic single-cell necrosis at all doses compared with rats treated with linezolid. The incidence and severity of centrilobular hepatocellular degeneration were similar in the livers of rats treated with Ls-AKG28 and Ls-AKG38 at all doses. Furthermore, rats treated with Ls-AKG28 showed vacuolated macrophages and basophilic material in the spleen at 20 or 40 mg / kg / dose, and vacuolated glomerular mesangial cells in the kidney at 40 mg / kg / dose.

[0459] In conclusion, repeated intravenous injections of Ls-AKG28 over 8 weeks were well tolerated in rats at doses of 10, 20, and 40 mg / kg. Repeated intravenous injections of Ls-AKG38 over 8 weeks were well tolerated in rats at doses of 20, 40, and 80 mg / kg. Example 2 demonstrated that AKG-28 was 33-fold more potent than linezolid, and AKG-38 was 17-fold more potent than linezolid in killing M. tuberculosis (Erdmann strain) in vitro. Thus, corrected for efficacy, rats did not experience significant neuropathy (changes in nerve conduction velocity), elevated liver enzymes, decreases in red blood cell count or hematocrit, or weight loss at linezolid-equivalent doses of 1320-1336 mg / kg, which is 16.5 times the clinically relevant dose of linezolid 80 mg / kg.

[0460] [Example 45] Efficacy of liposomal AKG-28 and AKG-38 in combination with bedaquiline and pretomannide, or bedaquiline (B), pretomannide (Pa), and moxifloxacin (M) in Kramnik (C3HeB / FeJ mouse model of pulmonary Mycobacterium tuberculosis infection) The C3HeB / FeJ (Kramnik) mouse infection model exhibits advanced hypoxic caseous granulomas in the lungs after TB infection (Driver E., et al., Antimicrobial Agents and Chemotherapy, 2012, vol. 56, pp. 3181-3195). The lung pathology observed in C3HeB / FeJ mice more closely resembles the lesion pathology and heterogeneity in bacterial populations seen in TB patients and was used to evaluate the efficacy of liposomal AKG-28 and AKG-38 at moderate weekly doses of 50 and 90 mg / kg. Ls-AKG28 (Lot 275) and Ls-AKG38 (Lot 276) were prepared as described in Examples 22 and 23, respectively. Lung pathology in C3HeB / FeJ mice shows three distinct types of lesions, classified as caseous necrotic lesions delineated by collagen rims (type I), fulminant neutrophilic alveolitis (type II), and cellular lesions (type III) (see Irwin et al. (2015) Dis Model Mech 8, 591-602). Eight- to ten-week-old C3HeB / FeJ female mice were infected with LDA (low-dose aerosol infection). Using the Glas-Col inhalation challenge system, mice were infected with a target of approximately 50-75 bacilli / mouse (Erdman strain). Five mice per aerosol challenge were sacrificed 1 day after infection to determine bacterial uptake.

[0461] Eight weeks after infection, eight mice were sacrificed to measure the bacterial burden in the lungs and spleens at the start of treatment. Mice were weighed before sacrifice. Gross pathological examination of the lungs and spleens was performed. Whole lungs and spleens were extracted and frozen at -80°C. Previously frozen tissues were 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 3–5 weeks of incubation at 37°C in a dry-air incubator. Treatment was initiated 8 weeks after infection and continued for 4–6 consecutive weeks by oral gavage or intraperitoneal (ip) injection (MF for oral gavage, weekly for ip injection). Bedaquiline (B), pretomanid (Pa), moxifloxacin (M), and linezolid (L) were administered orally by gavage at a total volume of 200 μL per dose, 5 days per week for 4 or 6 weeks. Bedaquiline (25 mg / kg) was administered first, followed at least 1 hour later by pretomanid (100 mg / kg). Moxifloxacin (100 mg / kg) or linezolid (100 mg / kg) was administered 4 hours after pretomanid. The liposomal formulations were administered once per week for a total of 4 or 6 weeks.

[0462] Mice were observed daily during dosing and weighed at least weekly. Two weeks after completing the 4-6 week treatment, mice were sacrificed. Eight mice per treatment group were weighed prior to sacrifice. Whole lungs and spleens were aseptically collected for all treatment groups. Gross pathology of the lungs and spleens is shown graphically. Lungs were photographed for gross lesion analysis. Whole lungs and spleens were frozen at -80°C, and previously frozen tissues were collected and homogenized in either 1x PBS or 10% bovine serum albumin (BSA) in 1x PBS using a Precellys homogenizer (to avoid drug carryover; see below for clarification). After homogenization, lung and spleen homogenates were plated on 7H11 agar or charcoal containing 7H11 quad plates and serially diluted in 1x PBS or 10% BSA. CFU counts were performed after 5 weeks of incubation at 37°C in a dry air incubator.

[0463] The addition of Ls-AKG28 to BPaM treatment resulted in a further 0.64 log CFU reduction in BPaM in the lung, whereas BPaM + Ls-AKG38 treatment resulted in a further 0.25 log CFU reduction in BPaM after 4 weeks of treatment. Substituting either Ls-AKG28 or Ls-AKG38 for linezolid (L) in the NIX (BPaL) regimen improved efficacy over BPaL at 6 weeks of treatment. Substituting Ls-AKG38 for linezolid in the BPaL regimen significantly improved efficacy compared to the BPaL-treated group at 6 weeks of treatment. Specifically, 6 weeks of BPaL administration resulted in a 4.18 log CFU reduction, with no CFU observed in one of eight mice. Six weeks of treatment with BPa+Ls-AKG28 resulted in a 4.68 log10 CFU reduction, but this was not statistically significant compared to BPaL. Six weeks of treatment with BPa+Ls-AKG38 resulted in a 5.26 log10 CFU reduction, with no CFU observed on plates from two of eight mice. This reduction was statistically significant compared to BPAL (p=0.04, Dunnett's test).

[0464] In the spleens after 6 weeks of treatment, replacing the liposomal formulation of linezolid with the NIX regimen slightly improved lung efficacy compared with the BPaL-treated group. Six weeks of treatment with the NIX regimen resulted in a 3.56 log10 CFU reduction, with plates from 3 of 8 mice showing no CFU. Six weeks of treatment with BPa+Ls-AKG28 resulted in a 4.18 log10 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 log10 CFU reduction, with plates from 6 of 8 mice showing no CFU. The CFU burden in the spleens of mice treated with the drugs was low, approaching the lower limit of detection of 0.66 log10 CFU.

[0465] JPEG0007805319000115.jpg83170

[0466] JPEG0007805319000116.jpg93170

[0467] All mice without measurable CFUs were enumerated at a detection limit of 0.66 log CFU. This indicates that Ls-AKG38 and Ls-AKG28, when combined with bedaquiline and pretomanid, are more active than linezolid at moderate and highly tolerated doses of both drugs after only 6 weeks of treatment. As shown in Examples 42 and 43, both Ls-AKG28 and Ls-AKG38 can be safely administered in this combination at doses at least twice those used in this study. Furthermore, the addition of Ls-AKG28 to the BPaM regimen further reduced CFUs in both the lungs and spleen at these same highly tolerated doses.

[0468] [Example 46] Efficacy of liposomal AKG-28 monotherapy in a Balb / c model of pulmonary Mycobacterium tuberculosis infection The schedule- and dose-dependent efficacy of Ls-AKG28 was determined compared to free linezolid at clinically relevant doses of 50 and 100 mg / kg in a chronic Balb / c model of tuberculosis. In the chronic Balb / c mouse model, lung bacterial burden reaches a steady state 4–5 weeks after M. tuberculosis infection (Lenaerts et al. (2005) AAC 49(6)2294–2301). Ls-AKG28 (Lot 286) was prepared as described in Example 28. Six- to eight-week-old Balb / c female mice were obtained from Jackson Laboratories and infected with approximately 50–100 bacilli of M. tuberculosis Erdman per mouse using a low-dose aerosol (LDA) challenge system.

[0469] Mice (n = 3) were sacrificed 1 day postinfection to determine bacterial uptake. Whole lungs were aseptically collected in Precelly tubes (Bertin cat# KT03961-1-396.7) and homogenized in 4 ml of 1x PBS using a Precellys tissue homogenizer. The undiluted homogenate was transferred to two large 7H11 agar plates (150 x 15 mm), and the plates were incubated in sealed zip-top bags in a dry-air incubator at 37°C for at least 21 days until colonies could be counted. On day 28 postaerosol infection, mice (n = 5) were sacrificed to determine the bacterial burden in the lungs and spleens at the start of treatment. Mice were weighed before sacrifice. Gross pathological examination of the lungs and spleens was performed. Lungs (divided into left and upper right [cranial] lobes + accessory lobes) and spleens were aseptically collected and frozen at -80°C. The lower right lung lobe [caudal] was collected for histological examination in 4% paraformaldehyde (PFA). Previously frozen tissue 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 3-5 weeks of incubation at 37°C in a dry air incubator.

[0470] Linezolid in 5% PEG-200 (Sigma P3015, lot MKBW3119V) / 95% (0.5%) methylcellulose (Sigma M0430, lot 031M00051) was administered by oral gavage (200 μL per mouse) starting on day 28 (month) after aerosol infection and continued 5 days out of 7 days per week for 2–8 weeks. Ls-AKG28 was administered by intraperitoneal injection at a dose of 50 or 100 mg / kg once or twice weekly. Terminal sacrifice occurred 3 days after the last administration of the drug in mice treated for 2, 4, or 8 weeks. Mice were weighed before sacrifice. Gross pathological examination of the lungs and spleens was performed. Lungs (divided into left lobe, right upper lobe, and accessory lobe) and spleens were aseptically collected and frozen at -80°C, and the right lower lobe was collected in 4% PFA for histological examination. To avoid drug carryover, previously frozen tissues were collected and homogenized in 10% bovine serum albumin (BSA) in 1x PBS. After homogenization, lung and spleen homogenates were serially diluted in 1x PBS and 10% BSA and then plated on charcoal-containing 7H11 agar or 7H11 quad plates. CFU counts were performed after 3–5 weeks of incubation at 37°C in a dry air incubator.

[0471] Reductions in lung CFU counts are shown in Table 39, and spleen CFU counts are shown in Table 40. Treatment with Ls-AKG28 at 50 mg / kg twice weekly or 100 mg / kg once weekly resulted in a 1.5 Log10 CFU reduction in the lungs after only 2 weeks, whereas linezolid at 100 mg / kg resulted in a 0.15 Log10 CFU reduction (q1 x 5). This reduction was approximately 3 Log10 CFU in the spleen at 2 weeks for Ls-AKG28. At 8 weeks, all mice treated with Ls-AKG28 were completely germ-free (below the detection limit of 1.13 in the lungs and 0.66 in the spleen) at 8 weeks, compared with 2.45 log10 CFU in the lungs and 3.15 log10 CFU in the spleen for the high dose of linezolid at 100 mg / kg. This monotherapy activity is surprising for an oxazolidinone without an active combination partner like bedaquiline and with a relatively short duration of only 8 weeks. For example, at 8 weeks of treatment, linezolid monotherapy demonstrated log CFU counts in the range of 4–6 in Balb / c and C3HeB / FeJ mice (Lanoix et al. (2015) Dis Models Mech. 8, 603–610), and activity remained modest even at doses up to 1000 mg / kg / week on schedules ranging from 3–14 doses / week (Bigelow et al. (2021) J Infec. Dis. 223(11) 1855–1864).

[0472] JPEG0007805319000117.jpg84170

[0473] JPEG0007805319000118.jpg104170

[0474] All mice without measurable CFU were enumerated at a detection limit of 0.66 log CFU.

[0475] [Example 47] Efficacy of liposomal AKG-38 in a rabbit methicillin-resistant Staphylococcus aureus (MRSA) endocarditis model Staphylococcus aureus infections, particularly those involving the endovascular system (e.g., IE; cardiac and hemodialysis machine infections, etc.), are widespread and associated with unacceptably high morbidity, mortality, and posttreatment recurrence rates. This is particularly true when such infections are caused by multidrug-resistant strains of MRSA. Furthermore, even when MRSA strains have a minimum inhibitory concentration (MIC) for vancomycin (the "workhouse" anti-MRSA drug) within the accepted Clinical Standards Institute (CLSI) "susceptibility" range (i.e., ≤2 μg / ml), clinical outcomes remain suboptimal.

[0476] A typical high-inoculum intravascular biofilm MRSA infection model, left-sided aortic valve IE, was performed in female New Zealand White rabbits aged 6 months and weighing 2.2–2.5 kg. The rabbits were anesthetized with intramuscular injections of xylazine and ketamine. The fur was then clipped over the right carotid artery to expose the skin. The cut site on the right carotid artery was locally anesthetized with 1% lidocaine. An incision was then made to expose the right carotid artery. This was isolated, proximally ligated, and then retrogradely cannulated across the aortic valve into the left ventricle using a polyethylene catheter, which remained fixed and indwelled in the left ventricle for the duration of the study. For left-sided IE 48 hours after catheter placement (to induce sterile aortic valve and ventricular vegetations), the animals received approximately 2 × 10 of the MW2 strain. 5 IE was induced by intravenous challenge with 100 cfu of MRSA strain MW-2 (USA400-clonal complex [CC]1). The MRSA strain used was i) clinically derived, ii) genome sequenced, iii) represents a common nosocomial MRSA clonal type, iv) virulent in an experimental IE model, and v) susceptible to daptomycin (DAP) in vitro. Infection spread from infected warts on heart valves to the kidneys and spleen.

[0477] Liposomal AKG-38 (Ls-AKG38) was given in separate groups of animals at a dose of 40 mg / kg / dose either once (in combination with DAP) or twice (once in combination with DAP; then a second injection at sacrifice after DAP treatment in a "relapse group" of animals that did not receive further DAP therapy). Ls-AKG38 (Lot 292) was prepared as described in Example 29. The first Ls-AKG38 injection was administered approximately 1 hour after the initial iv dose of DAP. DAP was administered either alone or in combination with Ls-AKG38 at a sublethal dose of 2 mg / kg daily for 4 days.

[0478] Animals were euthanized, and major target organs were sterilely removed and quantitatively cultured (blood, cardiac vein for left-sided IE; kidney and spleen) on either day 6 (DAP alone or DAP + single dose of Ls-AKG38) or day 12 (DAP + two doses of Ls-AKG38 on days 1 and 6). Quantitative target tissue cultures were performed by standard preparation of sterilely removed organs by weighing, homogenization, serial dilution, and plating. Serial dilutions and quantitative cultures of blood were performed similarly. Data for blood cultures and target organs of different treatment groups are shown in logarithmic scale, respectively. 10 cfu / ml or log 10 Calculated as mean and median cfu / gm tissue (±SD).

[0479] Preliminary data from a left ventricular endocarditis model of MRSA in rabbits are shown in Table 41 below. A single dose of daptomycin alone or daptomycin plus Ls-AKG38 showed no significant effect on day 6 post-inoculation. Surprisingly, a second injection of Ls-AKG38 resulted in significant efficacy on day 12, including sterilization in 4 / 5 rabbits in all five tissues and a greater than 6-log reduction in CFU in multiple organs. This data suggests that endocarditis can be effectively treated with Ls-AKG38 after discontinuation of daily daptomycin.

[0480] JPEG0007805319000119.jpg113170

[0481] [Example 48] In vitro activity of AKG-28 and AKG-38 against various nontuberculous mycobacteria MIC tests were performed by the microbroth dilution method (Obregon-Henao et al. (2015) Antimicrobial Agents Chemother 59, 6904-6912) using Mueller Hinton (MH) broth (cation-adjusted) to the calcium and magnesium ion concentrations recommended in CLSI standard M7-A7 (Becton Dickinson). MIC tests were also performed using 7H9 broth ( Sigma-Aldrich The microbroth dilution method was performed using 7H11 agar plates (Shang et al. (2011) PLoS One 6, e24726; Chan et al. (2010) Am J Respir Cell Mol Biol 43, 287-393). The goal was to optimize the ability to detect more compounds active against NTM by using different broths in our microbroth dilution method. NTM was detected by 7H11 agar plates ( Sigma-Aldrich CFU were grown on agar plates at 35-37°C for 3-25 days (depending on the bacterial strain) in ambient air. CFU were harvested from the agar plates, placed in either MH broth containing 0.05% Tween 80, and grown at 35-37°C in ambient air until the optical density (OD) absorbance, harvested after 7 days of growth, reached 0.08-0.1 (0.5 McFarland Standard). Bacterial cell suspensions were then confirmed to match 0.08-0.1 (0.5 McFarland Standard) by preparing them in saline.

[0482] 180 μl of broth (MH) was added to the first column of a 96-well plate. Next, 100 μl of broth (MH) was added to the other columns of the 96-well plate. Compounds were prepared using 1.28 mg / mL in DMSO and used immediately for the test range of 64 to 0.062 μg / mL. 20 μl of compound was added to the first column of wells, and 100 μl was serially diluted. Finally, 100 μl of NTM cell suspension was added to all wells except for the media-only control well. QC agents specific to each microorganism were: 1) Bacteria only negative control; 2) Media only negative control; 3) Tedizolid positive drug control; and 4) Optional E. coli control.

[0483] RGM was assayed for OD on day 3. The plates were then assayed using the resazurin microtiter assay plate method recommended by the Clinical and Laboratories Standards Institute (Brown-Elliott et al. (2012) Clin Microbiol Rev vol. 25(3), p. 545-582). Briefly, this method used the addition of resazurin (7-hydroxy-3H-phenoxazin-3-one 10-oxide) to MIC 96-well plates. Resazurin is a blue dye that itself exhibits weak fluorescence and a pink, very red fluorescence. Resorufin It is irreversibly reduced to and used as a redox indicator in bacterial cell viability MIC assays.

[0484] Results indicate that both AKG-28 and AKG-38 are generally more potent than tedizolid across different NTM species and strains, including Mycobacterium avium, Microbacterium chelonae, Microbacterium abscessus, and Microbacterium kansasii. Only in Microbacterium massiliense was tedizolid more active than in all three strains evaluated.

[0485] JPEG0007805319000120.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 were further evaluated for activity against several multidrug-resistant (MDR) clinical isolates M70, M28, M94, M14 (Cheng AF, et al., 2004, Antimicrob. Agents Chemother. v. 48, pp. 596-601) and TN5904 (Palanisamay GS, et al., 2008, Tuberculosis (Edinb.) vol. 88 pp. 295-306). These strains are characterized by the following resistance characteristics:

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

[0488] JPEG0007805319000121.jpg62170

[0489] MICs of test compounds, including comparator / resistant controls (RIF, INH, STR, moxifloxacin (MOX), and linezolid (LNZ)), were determined using the broth microdilution method with Alamar Blue endpoint (MABA) essentially as described in Example 2, with the following modifications. Serially diluted test compounds and comparators in 2-fold increments in DMSO were added to wells of a 96-well assay plate containing 7H9-glycerol medium supplemented with 100 μL of ADC. Compounds were diluted in DMSO to maintain compound concentrations in the desired range, with final DMSO concentrations maintained at 2% (M70, M28, M94) or 2.5% (M14, TN5904) in the wells. However, due to low solubility in DMSO, STRs were serially diluted and added as aqueous solutions. Bacterial stocks of MDR strains and the susceptible H37Rv strain (positive control) were removed from the refrigerator, thawed, and diluted in 7H9-ADC-glycerol medium to obtain 100 μL of STR. 6 CFU / mL (H37Rv, TN5904), 2×10 6 CFU / mL (M70, M14), or 3 × 10 6 Bacterial densities of CFU / mL (M28, M94) were obtained, and 50 μL of diluted bacterial stock was added to the compound-containing medium in the wells. The range of final drug concentrations in the wells is shown in the table below. The plates were sealed in Ziploc bags and incubated at 37°C. Bacterial growth was monitored by periodic optical density measurements at 600 nm (OD600). On day 14 (when OD600 reached or exceeded 0.40) or day 17, 15 μL of Alamar Blue solution was added to the wells, and incubation was continued. The color of the incubation mixture was recorded after 3 days (7 days for the slow-growing M28 strain). The lowest serial antimicrobial concentration of the 2-fold serial dilutions that did not produce a visible color change in Alamar Blue compared to drug-free control wells was considered the MIC for these compounds. OD600-based MIC determinations (≥80% reduction in OD600 compared to drug-free control wells) were consistent with the MABA results. The shift in MIC for two wells (4-fold) was considered significant. The results are summarized in Table 44 below.

[0490] JPEG0007805319000122.jpg145170

[0491] The comparative / control compounds RIF, IHN, MOX, and STR demonstrated expected in vitro activity against DR-TB / MDR-TB strains and H37Rv. Within the variance of typical MIC assays, all tested compounds of the present disclosure were at least as active against MDR-TB strains as they were against the drug-susceptible strain H37Rv. AKG-28 was the most active, followed by AKG-38 and AKG-3. Compounds AKG-28 and AKG-38 were also the most active compared to their structurally close analogs.

[0492] Various aspects of the present disclosure may be used alone, in combination, or in various arrangements not specifically described in the foregoing embodiments, and therefore are not limited in their application to the details and arrangements of components set forth in the foregoing description or illustrated in the drawings. For example, aspects described in one embodiment may be combined in any manner with aspects described in other embodiments.

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

[0494] Incorporation by Reference All publications, patents, and patent applications cited herein are incorporated by reference for all purposes to the same extent as if each individual publication, patent, or patent application was specifically indicated to be incorporated by reference.

Claims

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

2. R ２ is -NHCOCH ３ 2. The compound of claim 1, wherein:

3. The compound has formula 1c: 【Chemistry 2】 2. The compound of claim 1, wherein the compound is: or a pharmaceutically acceptable salt thereof.

4. The compound has formula 1d: 【Transformation 3】 2. The compound of claim 1, wherein the compound is: or a pharmaceutically acceptable salt thereof.

5. The compound has the formula 1e: 【Chemistry 4】 2. The compound of claim 1, wherein the compound is: or a pharmaceutically acceptable salt thereof.

6. R ２ The compound of claim 1 , wherein is an amine.

7. The compound is compound AKG-28: 【Transformation 5】 2. The compound of claim 1, wherein:

8. The compound is compound AKG-29: 【Transformation 6】 2. The compound of claim 1, wherein:

9. The compound is compound AKG-30: 【Transformation 7】 2. The compound of claim 1, wherein:

10. The compound is compound AKG-31: 【Transformation 8】 2. The compound of claim 1, wherein:

11. A liposome composition comprising the compound according to any one of claims 1 to 10.

12. 12. The liposomal composition of claim 11, comprising a liposomal vesicle, wherein the liposomal vesicle comprises the compound of Formula I or a pharmaceutically acceptable salt thereof.

13. 12. The liposome composition of claim 11, wherein the liposome composition comprises a physiologically acceptable medium containing liposome vesicles, the liposome vesicles comprising (i) a membrane comprising phospholipids and cholesterol, and (ii) an interior space comprising a polyanion trapping agent and the compound of Formula I or a pharmaceutically acceptable salt thereof.

14. 14. The liposome composition of claim 13, wherein the polyanion scavenger is triethylammonium sucrose octasulfate (TEA-SOS) or ammonium sulfate (AS).

15. 11. A liposome composition comprising a physiologically acceptable medium containing liposome vesicles, the liposome vesicles comprising (i) a membrane comprising phospholipids and cholesterol, and (ii) a polyanion sequestering agent and the compound of Formula I or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 10, wherein the pharmaceutically acceptable salt of the compound of Formula I is a sulfate salt encapsulated in the liposome vesicles.

16. The liposome composition of claim 13 , wherein the phospholipid is a phosphatidylcholine.

17. 17. The liposome composition of claim 16, wherein the phosphatidylcholine is distearoylphosphatidylcholine (DSPC) or hydrogenated soy phosphatidylcholine (HSPC).

18. The liposome composition according to claim 16, wherein the amount of cholesterol in the liposome composition is 50 mol % to 65 mol % relative to the total amount of phosphatidylcholine and cholesterol in the liposome composition.

19. 17. The liposome composition of claim 16, wherein the molar ratio of phosphatidylcholine to cholesterol is 45:

55.

20. The liposome composition of claim 12 , wherein the liposome composition further comprises a polymer-conjugated lipid.

21. 21. The liposome composition of claim 20, wherein the polymer-conjugated lipid is PEG (molecular weight 2,000)-distearoylglycerol (PEG-DSG) or PEG (molecular weight 2,000)-distearoylphosphatidylethanolamine (PEG-DSPE).

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