Oxazolidinone compounds, liposomal compositions comprising oxazolidinone compounds, and methods of using the same

Abstract

Disclosed are compositions and methods for the treatment of tuberculosis and other mycobacterial and gram-positive bacterial infections. These compositions comprise highly potent and selective oxazolidinones encapsulated with high efficiency to maximize the potential for low-toxicity drug delivery and are stable in the presence of plasma. The compositions are long-circulating and retain their encapsulated drug while in circulation following intravenous delivery to allow effective accumulation at the site of bacterial or mycobacterial infection. The high doses achievable and the long-circulating nature of the drug combined with the high stability of the formulation allow for a reduction in the frequency of administration compared to the once-daily or twice-daily administration of other drugs commonly used to treat these infections.

Description

[0001] Related Applications

[0002] 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. Utility Patent Application No. 17 / 351,631, filed June 18, 2021, the entire contents of which are hereby incorporated by reference herein. TECHNICAL FIELD

[0003] The present disclosure relates to novel aminoalkyl azolindinones, compositions comprising novel aminoalkyl liposomal compositions of azolindinones, and aminoalkyl Use of azolindinones in the treatment of Mycobacterium tuberculosis and other Gram-positive infections. BACKGROUND

[0004] Mycobacterium is a genus of bacteria that causes tuberculosis (TB). According to the World Health Organization, TB is among the top 10 causes of death and the leading cause of death due to a single infectious agent worldwide. Rifampicin is the most effective first-line drug for treating TB. However, an increasing number of cases of infection with Mycobacterium tuberculosis are 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

[0005] Disclosed are compositions and methods for treating tuberculosis and other mycobacterial and Gram-positive infections.

[0006] One aspect of the present disclosure provides a compound of Formula I, or a pharmaceutically acceptable salt thereof:

[0007]

[0008] Formula I

[0009] wherein R2 is amine (NH2) or acetamide (NHCOCH3), and

[0010] wherein R1 is a tetrazole ring substituted at the 2’ position with an aminoalkyl group.

[0011] In some embodiments, the aminoalkyl group is a dimethylaminoalkyl group. In some embodiments, Aminoalkyl derivatives of azolindinones include those in which an amine or acetamide group at the R2position of the oxazolidinone ring and a dimethylaminoethyl group on the tetrazole ring.

[0012] In some embodiments, a compound of Formula 1a is provided:

[0013]

[0014] In some embodiments, a compound of Formula 1b is provided:

[0015]

[0016] In some embodiments, a compound of Formula 1c or a pharmaceutically acceptable salt thereof is provided:

[0017]

[0018] In some embodiments, a compound of Formula 1d or a pharmaceutically acceptable salt thereof is provided:

[0019]

[0020] In some embodiments, a compound of Formula 1e is provided:

[0021]

[0022] In some embodiments, the compound has a Selectivity Index (SI) of 100 to 1700 for Erd / HepG2 and H37Rv / HepG2.

[0023] In some embodiments, the compound has a SI of 200 to 1700 for Erd / HepG2 and H37Rv / HepG2.

[0024] In some embodiments, the compound has a SI of 300 to 1700 for Erd / HepG2 and H37Rv / HepG2.

[0025] Another aspect of the present disclosure provides a liposomal composition comprising a liposomal vesicle, wherein the liposomal vesicle comprises a compound of Formula I or a pharmaceutically acceptable salt thereof

[0026]

[0027] Formula I

[0028] wherein R2is an amine (NH2) or an acetamide (NHCOCH3), and

[0029] wherein R1is a tetrazole ring substituted at the 2' position with an aminoalkyl group.

[0030] In some embodiments, the aminoalkyl group is a dimethylaminoalkyl group. In some embodiments, Aminoalkyl derivatives of oxazolidinones include an amine or acetamide group at the R2position of the oxazolidinone ring and a dimethylaminoethyl group on the tetrazole ring. Aminoalkyl derivatives of oxazolidinones include an amine or acetamide group at the R2position of the oxazolidinone ring and a dimethylaminoethyl group on the tetrazole ring.

[0031] In some embodiments, a liposomal composition comprising a liposomal vesicle is provided, the liposomal vesicle comprising a compound of Formula la:

[0032]

[0033] In some embodiments, a liposomal composition comprising a liposomal vesicle is provided, the liposomal vesicle comprising a compound of Formula lb:

[0034]

[0035] In some embodiments, a liposomal composition comprising a liposomal vesicle is provided, the liposomal vesicle comprising a compound of Formula lc, or a pharmaceutically acceptable salt thereof:

[0036]

[0037] In some embodiments, a liposomal composition comprising a liposomal vesicle is provided, the liposomal vesicle comprising a compound of Formula Id:

[0038]

[0039] In some embodiments, a liposomal composition comprising a liposomal vesicle is provided, the liposomal vesicle comprising a compound of Formula le:

[0040]

[0041] In some embodiments, the liposomal vesicle is in an aqueous medium.

[0042] In some embodiments, the compound is entrapped in the liposomal vesicle by means of a trapping agent, wherein the trapping agent comprises a polyanion. In some embodiments, the trapping agent is sucrose octasulfate triethylammonium or ammonium sulfate. In some embodiments, the trapping agent is sucrose octasulfate triethylammonium. In some embodiments, the trapping agent is ammonium sulfate.

[0043] In some embodiments, the liposomal composition comprises a salt of the compound, wherein the salt is a sulfate salt, a citrate salt, a sulfoglycoside salt, a salt with phosphorylated or sulfated polyols, or a salt with phosphorylated or sulfated polyanionic polymers. In some embodiments, the liposomal composition comprises a sulfate salt of the compound.

[0044] In some embodiments, the water solubility of the compound in the liposomal vesicle is less than 1 mg / mL. In some embodiments, the water solubility of the compound in the liposomal vesicle is less than 0.1 mg / mL.

[0045] In some embodiments, the liposomal vesicle comprises a membrane comprising phosphatidylcholine and cholesterol. In some embodiments, the liposomal vesicle comprises a membrane comprising phosphatidylcholine and cholesterol, wherein the membrane separates the interior of the liposomal vesicle from an aqueous medium. In some embodiments, the phosphatidylcholine is distearoylphosphatidylcholine (DSPC) or hydrogenated soyphosphatidylcholine (HSPC). In some embodiments, the molar ratio of the phosphatidylcholine to cholesterol is about 60:40 to 35:65. In some embodiments, the molar ratio of the phosphatidylcholine to cholesterol is about 55:45 to about 35:65. In some embodiments, the molar ratio of the phosphatidylcholine to cholesterol is about 50:50 to about 40:60.

[0046] In some embodiments, the molar ratio of the phosphatidylcholine to cholesterol is about 50:50 to about 45:55.

[0047] In some embodiments, the membrane further comprises a lipid conjugated to a polymer.

[0048] In some embodiments, the liposomal vesicle comprises HSPC, cholesterol, and a lipid conjugated to a polymer in a molar ratio of about 55:45:2.75.

[0049] In some embodiments, the lipid conjugated to a polymer is PEG(molecular weight 2,000)-distearoylglycerol (PEG-DSG) or PEG(molecular weight 2,000)-distearoylphosphatidylethanolamine (PEG-DSPE).

[0050] In some embodiments, the Z-average particle size of the liposomes in the liposomal composition is about 80 to about 130 nm.

[0051] In some embodiments, the composition is a liquid pharmaceutical formulation for parenteral administration.

[0052] Further aspects of the present disclosure relate to methods of treating a bacterial infection, comprising administering to a subject in need thereof a therapeutically effective amount of the liposomal composition provided herein.

[0053] In some embodiments, the bacterial infection is a Mycobacterium tuberculosis infection. In some embodiments, the minimum inhibitory concentration (MIC) of the compound in the liposomal vesicle is about 0.01 pg / ml to about 0.25 pg / ml. In some embodiments, the minimum inhibitory concentration (MIC) of the compound in the liposomal vesicle is about 0.01 pg / ml to about 0.1 pg / ml.

[0054] In some embodiments, the liposomal composition is administered parenterally.

[0055] In some embodiments, the method comprises administering one or more additional active agents simultaneously or sequentially. In some embodiments, the one or more active agents comprise bedaquiline, pretomanid, pyrazinamide, moxifloxacin, pharmaceutically acceptable salts thereof, or combinations thereof.

[0056] In some embodiments, the liposomal composition is administered once every week to once every six weeks.

[0057] In some embodiments, the percentage of compound remaining in the blood 6 hours after administration to a subject in need thereof is greater than 20% of the amount administered. In some embodiments, the percentage of compound remaining in the blood is greater than 10% of the amount administered.

[0058] Some aspects of the present disclosure relate to a method of preparing a liposomal composition, comprising the steps of: (i) preparing the liposome comprising a phospholipid, a cholesterol, and a PEG-lipid and having an internal space comprising a trapping agent in a medium substantially free of the trapping agent; (ii) contacting the liposome with the compound of any one of claims 1 to 8 in an aqueous medium to effect encapsulation of the compound in the liposome; (iii) removing unencapsulated compound; and (iv) providing the liposome in a physiologically acceptable medium suitable for parenteral use. BRIEF DESCRIPTION OF DRAWINGS

[0060] Figure 1 is a graph showing the effect of pH on liposomal loading of compounds AKG-3, AKG-5, and AKG-16.

[0061] Figure 2A and Figure 2BThis diagram shows the encapsulation of compounds AKG-3, AKG-5, and AKG-16 into liposomes using the TEA-SOS trapping agent at different drug-to-lipid (DL) ratios. Figure 2A The effect of the added drug / lipid (DL0) ratio (in grams of drug per mole of liposomal phospholipid (PhL)) on the effective load of liposomes is shown, expressed as the drug / lipid ratio (DL) after loading. Figure 2B The effect of the DL0 ratio (drug / lipid input ratio) on liposome loading efficiency is shown, calculated as the percentage of DL relative to DL0 after loading.

[0062] Figure 3A , Figure 3B , Figure 3C ,and Figure 3D This diagram illustrates 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. Figure 3A The effect of the DL0 ratio on the liposome payload of AKG-5 and AKG-16 is shown. Figure 3B The effect of the DL0 ratio on the liposome loading efficiency of AKG-5 and AKG-16 is shown. Figure 3C The effect of the DL0 ratio on the liposome payload of AKG-3 is shown. Figure 3D The effect of the DL0 ratio on the liposome loading efficiency of AKG-3 is shown.

[0063] Figure 4A and Figure 4B The figure shows the encapsulation of AKG-28 and AKG-38 with TEA-SOS and ammonium sulfate as capture agents at different DL0 ratios. Figure 4A The effect of the DL0 ratio on the effective load of liposomes is shown. Figure 4B The effect of the DL0 ratio on load efficiency is shown.

[0064] Figure 5A , Figure 5B , Figure 5C ,and Figure 5D This shows the compound AKG-28 ( Figure 5A , Figure 5C ) and AKG-38 Figure 5B , Figure 5DFigure 6 is a plot showing the dependence of the rapid drug leakage after in vitro contact of liposomes of the present application with mouse (denoted "mouse") or human (denoted "human") plasma, as described in Example 19 below. The liposomes contained 5 mol% PEG(2000)-DSPE (denoted "DSPE") or PEG-DSG (denoted "DSG"). Trapping agent: 0.5 M ammonium sulfate (AS) Figure 5A , Figure 5B , 1 N triethylammonium sucrose octasulfate (TEA-SOS) Figure 5C , Figure 5D .

[0065] Figure 6 The numbered ring structure of the compound of Formula I is shown.

[0066] Figure 7 Figure 7 is a plot showing the profile of plasma concentration versus time of total drug in Sprague-Dawley rats after a single intravenous dose (IVx1) of Ls-AKG28 administered at 10 mg / kg (diamonds), 20 mg / kg (squares), and 40 mg / kg (circles). For comparison, the profile of plasma concentration versus time of 50 mg / kg linezolid in 5% methylcellulose (pH 3 to 4) (single oral dose, POx1) is also included. The mean and SD concentrations at each time point are shown.

[0067] Figure 8 Figure 8 is a plot showing the profile of plasma concentration versus time of total drug in Sprague-Dawley rats after a single intravenous dose (IVx1) of Ls-AKG38 administered at 20 mg / kg (diamonds), 40 mg / kg (squares), and 80 mg / kg (diamonds). For comparison, the profile of plasma concentration versus time of 50 mg / kg linezolid in 5% methylcellulose (pH 3 to 4) (single oral dose, POx1) is also included. The mean and SD concentrations at each time point are shown.

[0068] Figure 9A , Figure 9B , and Figure 9C Figure 9 is a plot showing the profile of plasma concentration versus time of total drug in Sprague-Dawley rats after IVx1 of 10 mg / kg (circles), 20 mg / kg (squares), and 40 mg / kg (diamonds) of Ls-AKG28 administered on day 1 (circles), day 15 (squares), day 29 (diamonds), and day 43 (triangles). Figure 9A Figure 9B Figure 9C ​​Figure 1 is a plot showing the profile of plasma concentration of total drug versus time following IV x 1 administration of Ls-AKG28 at 20 mg / kg (■), 40 mg / kg (♦), and 80 mg / kg (▲) in Sprague-Dawley rats. Mean and SD concentrations are shown for each time point.

[0069] Figure 10A , Figure 10B and Figure 10C Figure 2 is a plot showing the profile of plasma concentration of total drug versus time following IV x 1 administration of Ls-AKG38 at 20 mg / kg (■), 40 mg / kg (♦), and 80 mg / kg (▲) in Sprague-Dawley rats. Mean and SD concentrations are shown for each time point. Figure 10A Figure 10B Figure 10C Figure 3 is a plot showing the profile of plasma concentration of total drug versus time following IV x 1 administration of Ls-AKG38 at 20 mg / kg (■), 40 mg / kg (♦), and 80 mg / kg (▲) in Sprague-Dawley rats. Mean and SD concentrations are shown for each time point.

[0070] Figure 11A , Figure 11B , and Figure 11C Figure 4 is a plot showing the profile of plasma concentration of both lipid (labeled with non-exchangeable DiIC18(3)-DS) and drug following a single IV injection of liposomal AKG-28 (■) and liposomal AKG-38 (♦) in CD-1 mice, and the change in plasma drug / lipid ratio (a measure of the rate of drug release from the liposome) for both Ls-AKG28 and Ls-AKG38 (■) in CD-1 mice. Mean and SD are shown for each time point. Figure 11A Figure 11B Figure 11C Figure 5 is a plot showing the comparison of plasma drug concentration expressed as % of injected dose for Ls-AKG28 and Ls-AKG38 for various formulations of liposomal AKG-28 and liposomal AKG-38 following first and fourth week dosing. Mice were injected once / week for a total of 4 injections with the indicated dose and formulation.

[0071] Figure 12 Figure 6 is a plot showing the effect of Ls-AKG28 dose escalation on body weight of female CD-1 mice over time.

[0072] Figure 13A Figure 7 is a plot showing the effect of Ls-AKG38 dose escalation on body weight of female CD-1 mice over time.

[0073] Figure 13B Figure 8 is a plot showing the effect of Ls-AKG38 dose escalation on body weight of female CD-1 mice over time.

[0074] Figure 13C ​​​​Figure 2 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.

[0075] Figure 13D Figure 3 is a heatmap showing the effect of single treatment of Ls-AKG28 or Ls-AKG38 on histopathology findings in female CD-1 mice.

[0076] Figure 14A Figure 4 is a graph showing the effect of Ls-AKG28 in combination with bedaquiline and premanil (BP) or bedaquiline, premanil and moxifloxacin (BPM) on body weight over time in female CD-1 mice.

[0077] Figure 14B Figure 5 is a graph showing the effect of Ls-AKG38 in combination with BP or BPM on body weight over time in female CD-1 mice.

[0078] Figure 14C Figure 2 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.

[0079] Figure 14D Figure 3 is a heatmap showing the effect of single treatment of Ls-AKG28 or Ls-AKG38 on histopathology findings in female CD-1 mice.

[0080] Figure 15A Figure 6 is a graph showing the change in body weight over time in female CD-1 mice treated with Ls-AKG28 injected at 50 mg / kg twice weekly (2qw) or at 100 mg / kg once weekly (1qw) alone or in combination with BP.

[0081] Figure 15B Figure 7 is a graph showing the change in body weight over time in female CD-1 mice treated with Ls-AKG38 injected at 100 mg / kg 2qw or at 200 mg / kg 1qw alone or in combination with BP.

[0082] Figure 15C Figure 8 is a graph showing hematological and blood biochemistry parameters in female CD-1 mice treated with Ls-AKG28 (at 50 mg / kg 2qw or at 100 mg / kg 1qw) or Ls-AKG38 (at 100 mg / kg 2qw or at 200 mg / kg 1qw) alone or in combination with BP.

[0083] Figure 15Dis a heatmap showing histopathology results in female CD-1 mice treated with Ls-AKG28 (at 50 mg / kg 2qw or at 100 mg / kg 1qw) or Ls-AKG28 (at 100 mg / kg 2qw or at 200 mg / kg 1qw) alone or in combination with BP.

[0084] Figure 16A is a graph showing the effect of Ls-AKG28 on body weight over time in male Sprague-Dawley rats treated for a total of eight weeks.

[0085] Figure 16B is a graph showing the effect of Ls-AKG38 on body weight over time in male Sprague-Dawley rats treated for a total of eight weeks. DETAILED DESCRIPTION

[0087] It is to be understood that both 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.

[0088] Disclosed herein are compounds, compositions, and methods related to the treatment of bacterial infections. As used herein, the terms "compound" and "drug" are used interchangeably. Some aspects of the present disclosure relate to Novel aminoalkyl derivatives of oxazolidinones. Some aspects of the present disclosure relate to methods for synthesizing Novel aminoalkyl derivatives of oxazolidinone compounds. Other aspects relate to compositions comprising Aminoalkyl derivatives of oxazolidinone compounds. Still other aspects of the present disclosure relate to Aminoalkyl derivatives of oxazolidinone compounds or compositions comprising Liposomal compositions of aminoalkyl derivatives of oxazolidinones for use in treating bacterial infections. In some embodiments, the compounds and compositions described herein can be used to treat infections of 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, Streptococcus, vancomycin-resistant enterococci (VRE), methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus pneumoniae, Enterococcus faecium, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus viridans, Listeria monocytogenes, Nocardia, and Corynebacterium.

[0089] In some embodiments, the compounds and compositions described herein exhibit at least 1000-fold unexpectedly high selectivity against Mycobacterium tuberculosis compared to mammalian cells (e.g., kidney or liver mammalian cells). Aminoalkyl derivatives of oxazolidinones exhibit selective activity against Mycobacterium tuberculosis. In some embodiments, the compounds and compositions described herein exhibit at least 1000-fold unexpectedly high selectivity against Mycobacterium tuberculosis compared to mammalian cells (e.g., kidney or liver mammalian cells). Aminoalkyl derivatives of oxazolidinones exhibit selective activity against Mycobacterium tuberculosis. In some embodiments, the compounds and compositions described herein exhibit at least 1000-fold unexpectedly high selectivity against Mycobacterium tuberculosis compared to mammalian cells (e.g., kidney or liver mammalian cells). Aminoalkyl derivatives of oxazolidinones exhibit selective activity against Mycobacterium tuberculosis. In some embodiments, the compounds and compositions described herein exhibit at least 1000-fold unexpectedly high selectivity against Mycobacterium tuberculosis compared to mammalian cells (e.g., kidney or liver mammalian cells). The aminoalkyl derivatives of oxazolidinones exhibit unexpectedly high selectivity against M. tuberculosis of 100 to 6,500 fold, 100 to 6,000 fold, 100 to 5,500 fold, 100 to 5,000 fold, 100 to 4,500 fold, 100 to 4,000 fold, 100 to 3,500 fold, 100 to 3,000 fold, 100 to 2,500 fold, 100 to 2,000 fold, 100 to 1,500 fold, 100 to 1,000 fold, 500 to 6,500 fold, 500 to 6,000 fold, 500 to 5,500 fold, 500 to 5,000 fold, 500 to 4,500 fold, 500 to 4,000 fold, 500 to 3,500 fold, 500 to 3,000 fold, 500 to 2,500 fold, 500 to 2,000 fold, 500 to 1,500 fold, 500 to 1,000 fold, 1,000 to 6,500 fold, 1,000 to 6,000 fold, 1,000 to 5,500 fold, 1,000 to 5,000 fold, 1,000 to 4,500 fold, 1,000 to 4,000 fold, 1,000 to 3,500 fold, 1,000 to 3,000 fold, 1,000 to 2,500 fold, 1,000 to 2,000 fold, 1,000 to 1,500 fold.

[0090] In some embodiments, the compounds and compositions described herein can facilitate selective uptake of mycobacteria-resident macrophages in the liver, spleen, or lung, which helps to provide potent intracellular killing. Macrophages are responsible for clearance of foreign particles, including both foreign infectious agents such as mycobacteria as well as laboratory-derived nanoparticles (e.g., liposomes) by phagocytosis. This results in both having the opportunity to co-reside in the same biological host, effectively concentrating the active agent in an important reservoir of disease.

[0091] Some aspects of the present disclosure relate to compounds that are aminoalkyl derivatives of oxazolidinones (see ) that exhibit unexpectedly high selectivity against M. tuberculosis. In some embodiments, the compounds have the following chemical formula I and pharmaceutically acceptable salts thereof: Figure 6

[0092]

[0093] wherein R2 is an amine (NH2) or an acetamide (NHCOCH3), and

[0094] wherein R1 is a tetrazole ring substituted at the 2' position with an aminoalkyl group.

[0095] In other embodiments, the compounds have the following chemical formula I and pharmaceutically acceptable salts thereof:

[0096]

[0097] ​wherein R2 is an amine (NH2) or an acetamide (NHCOCH3), and

[0098] wherein R1 is a tetrazole ring substituted at the 1' position with an aminoalkyl group.

[0099] In some embodiments, the aminoalkyl group is a dimethylaminoalkyl group. In some embodiments, Aminoalkyl derivatives of azolindinones include those having an amine or acetamide group at the R2 position of the azolindinone ring and a dimethylaminoethyl group on the tetrazole ring.

[0100] The present disclosure shows that the aminoalkyl derivatives of azolindinones described herein very specific structure-activity relationships (SAR) for the aminoalkyl derivatives of azolindinones described Aminoalkyl derivatives of azolindinones include those having an amine or acetamide group at the R2 position of the azolindinone ring and a dimethylaminoethyl group on the tetrazole ring. These compounds (1) have high selectivity for M. tuberculosis when compared to activity in mammalian cells (e.g., human kidney or liver cells), (2) have high activity against M. tuberculosis, and (3) are effectively loaded into liposomes.

[0101] In some embodiments, the aminoalkyl derivatives of azolindinones described herein are loaded in liposomes with 85% or better efficiency. In some embodiments, the loading efficiency of these derivatives is 90% or higher. In some embodiments, the loading of these derivatives is 95% or more, or even quantitative. In some embodiments, methods for loading Aminoalkyl derivatives of azolindinones are loaded in liposomes with 85% or better efficiency. In some embodiments, the loading efficiency of these derivatives is 90% or higher. In some embodiments, the loading of these derivatives is 95% or more, or even quantitative. In some embodiments, methods for loading Aminoalkyl derivatives of azolindinones are loaded in liposomes with 85% or better efficiency. In some embodiments, the loading efficiency of these derivatives is 90% or higher. In some embodiments, the loading of these derivatives is 95% or more, or even quantitative. In some embodiments, methods for loading 2+ , Mn 2+ , Zn 2+ , Mg 2+gradient, 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. 20180369143A1, and International Patent Application Publication No. WO199001405, which are incorporated herein by reference in their entirety. See also Allen et al. (1995) Int J Cancer 62: 199-204. Without being bound by theory, the cations contained inside the liposome play a role in establishing a transmembrane pH gradient, which helps drive the weakly basic drug to accumulate inside the liposome, or directly exchange with the drug molecules. In some embodiments, this results in a quantitative loading of the drug below the total capacity 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).

[0102] Definitions

[0103] For convenience, certain terms used in the specification, examples, and appended claims are collected here. Unless defined otherwise, 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.

[0104] As used herein, the following terms and phrases are intended to have the following meanings:

[0105] A noun, not modified by a number, used here refers to one or more than one (i.e., at least one) grammatical object. For example, “element” means one element or more than one element.

[0106] As used herein, the term “comprising” or “including,” or “having” is used in reference to compositions, methods, and respective components thereof, that are encompassed by the given embodiment, but is also open-ended, including unrecited elements.

[0107] The term “consisting essentially of’ as used herein means those elements required for the given embodiment. This term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic of the embodiment of the disclosure.

[0108] The term “consisting of’ means the compositions, methods, and respective components thereof, as described herein, which do not include any element not recited in the description of the embodiment.

[0109] When the term "comprising" is used in the present specification, it is meant "including", "consisting of" and "consisting essentially of".

[0110] If reference is made in the present specification to "as described above" or "as described

[0111] If reference is made in the present specification to "as described herein", "described herein" or "provided herein", it refers to any of the present disclosure of the present specification in any page of the preceding or following pages.

[0112] As used herein, the term "about" means acceptable variations of 20% or less, 10% or less, and 5% or less of the stated value. In certain embodiments, "about" can mean + / - 1%, 2%, 3%, 4%, 5%, 10%, or 20% variation.

[0113] The term "effective amount" as used herein with respect to a compound or composition means an amount of active compound (also referred to herein as active agent or drug) sufficient to produce a bactericidal or bacteriostatic effect. In one embodiment, an effective amount is a "therapeutically effective amount" meaning an amount of active compound sufficient to reduce the symptoms of the bacterial infection being treated.

[0114] The term "subject" (or, alternatively, "patient") as used herein refers to an animal, preferably a mammal, most preferably a human, who has been the object of treatment, either prophylactic or therapeutic.

[0115] The term "administering" as used herein includes all means of introducing a compound or pharmaceutical composition into a subject in need thereof, including, but not limited to, orally, intravenously, intramuscularly, intraperitoneally, subcutaneously, transdermally, by inhalation, buccally, intraocularly, sublingually, vaginally, rectally, and the like. The administration of the compound or composition is suitably parenteral. For example, the compound or composition can be administered intravenously, but can also be administered intraperitoneally or by inhalation, similar to what is currently used in the clinic for liposomal amikacin against Mycobacterium avium complex (see Shirley et al., Amikacin Liposome Inhalation Suspension: A Review in Mycobacterium avium Complex Lung Disease. Drugs. 2019 Apr; 79(5): 555-562).

[0116] The terms "treatment" and "treating" as used herein refer to therapeutic or prophylactic measures, such as those described herein.

[0117] The term "synergy" as used herein means that the effect achieved by the combined use of the compounds is greater than the sum of the effects produced by the individual compounds, i.e. greater than the effect predicted based on the separate administration of the two active ingredients.

[0118] The term "pharmaceutically acceptable salt" refers to a relatively non-toxic, inorganic or organic acid addition salt of a compound of the present disclosure that possesses the desired pharmacological activity.

[0119] The term "alkyl" means a saturated carbon chain, which can be straight-chained or branched-chained or a combination thereof, unless the carbon chain is otherwise defined. Some examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, and t-butyl, pentyl, hexyl, heptyl, octyl, and the like.

[0120] The term "aminoalkyl" means an alkyl group in which at least one carbon of the alkyl carbon chain forms a bond with an amino group, wherein the amino group is a primary amino group, a mono-alkyl substituted (secondary) amino group, a di-alkyl substituted (tertiary) amino group, or an alkyl substituted amino group in which the amine nitrogen atom forms a heterocyclic ring with the alkyl chain substituted for the hydrogen of the amine.

[0121] The term "liposome" means a vesicle comprised of a bilayer (unilamellar) and / or concentric series of multiple bilayers (multilamellar), separated by an aqueous compartment formed by amphiphilic molecules (e.g., phospholipids) surrounding a central water compartment. In a liposomal drug product, the drug substance is typically contained within the liposome. Typically, water-soluble drugs are contained within the aqueous compartment, while hydrophobic drugs are contained within the lipid bilayer of the liposome. Release of the drug from the liposomal formulation, as well as other characteristics (e.g., liposome clearance and circulation half-life), can be improved by the presence of polyethylene glycol and / or cholesterol or other potential additives in the liposome.

[0122] A "unilamellar liposome", also called a "unilamellar vesicle", is a liposome comprising one lipid bilayer membrane that defines a single enclosed water compartment. The bilayer membrane comprises 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 the external aqueous environment, and their hydrophobic ("tail") portions pointing downward into the interior of the liposome. The inner layer of lipids lies directly beneath the outer layer, with the lipids oriented with their heads facing the aqueous interior of the liposome, and their tails facing the tails of the outer layer.

[0123] A "multilamellar liposome", also called a "multilamellar vesicle", comprises more than one lipid bilayer membrane, which membranes define more than one enclosed water compartment. The membranes are arranged concentrically, such that different membranes are separated by water compartments.

[0124] The terms "encapsulating" and "entrapping" as used herein mean the incorporation of The azolide agent is incorporated into or associated with the liposome.

[0125] 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 in grams of drug per mole of liposomal phospholipid (PhL).

[0126] The term "mol.%" with respect to cholesterol refers to the mole amount of cholesterol expressed in percentage points relative to the sum of the mole amounts of cholesterol and non-poly-PEGylated phospholipids. For example, "55 mol.% cholesterol" in a liposome comprising cholesterol and HSPC refers to a composition of 55 mol. parts cholesterol / 45 mol. parts HSPC.

[0127] The term "mol.%" with respect to PEG-lipid refers to the ratio of the mole amounts of PEG-lipid and non-PEGylated phospholipids expressed in percentage points. For example, "5 mol.% PEG-DSPE" in a liposome comprising HSPC and PEG-DSPE refers to a composition having 5 mol. parts PEG-DSPE / 100 mol. parts HSPC.

[0128] The terms "sucrose octasulfate," "sulfatose," and "sucrose sulfate" refer to the same compound, sucrose octasulfate or its anion, and are used interchangeably herein.

[0129] The symbols "Ac," "Me," and "Et" as seen in chemical formulas refer to acetyl groups (CH3CO), methyl groups (CH3), and ethyl groups (C2H5), respectively.

[0130] Aspects and embodiments are described in further detail in the following sections.

[0131] Compounds

[0132] Oxazolidinones are synthetic antibiotics that exert their function by inhibiting protein synthesis. Linezolid (LZD) is an oxazolidinone that exhibits bacteriostatic activity against Mycobacterium tuberculosis. Oxazolidinone compounds. However, administration of LZD can result in severe side effects, such as anemia, thrombocytopenia, and peripheral neuropathy. Tedizolid is an oxazolidinone that has been shown to inhibit Gram-positive bacteria. Oxazolidinone compounds. While there is limited experience with long-term dosing of phospho-tedizolid (e.g., required for the treatment of tuberculosis) compared to the extensive experience with linezolid, the side effects of phospho-tedizolid are similar but generally less severe than those observed for linezolid.

[0133] Some aspects of the present disclosure relate to Compounds that are aminoalkyl derivatives of oxazolidinones (see Figure 6). In some embodiments, the compound has the following chemical formula I and pharmaceutically acceptable salts thereof:

[0134]

[0135] wherein R2 is an amine (NH2) or an acetamide (NHCOCH3),

[0136] wherein R1 is a tetrazole ring substituted at the 2’ position with an aminoalkyl group.

[0137] In some embodiments, the aminoalkyl group is a dimethylaminoalkyl group. In some embodiments, Aminoalkyl derivatives of the oxazolidinone class of compounds include those in which the R2 position of the oxazolidinone ring is an amine or acetamide group and the dimethylaminoethyl group is on the tetrazole ring. Aminoalkyl derivatives of the oxazolidinone class of compounds include those in which the R2 position of the oxazolidinone ring is an amine or acetamide group and the dimethylaminoethyl group is on the tetrazole ring.

[0138] In other embodiments, the compound has the following chemical formula I and pharmaceutically acceptable salts thereof:

[0139]

[0140] wherein R2 is an amine (NH2) or an acetamide (NHCOCH3), and

[0141] wherein R1 is a tetrazole ring substituted at the 2’ position with an aminoalkyl group.

[0142] The compounds of the present disclosure were synthesized as described in Example 1 having the chemical structures of Table 1 below: Aminoalkyl derivatives of the oxazolidinone class of compounds include those in which the R2 position of the oxazolidinone ring is an amine or acetamide group and the dimethylaminoethyl group is on the tetrazole ring.

[0143] The compounds of the present disclosure can exist in free form (e.g., as a free base, or as a free acid, or as a zwitterion) or can exist in a salt form. The salt can be any salt, which is an organic or inorganic addition salt or co-crystal, in particular any pharmaceutically acceptable organic or inorganic addition salt or co-crystal commonly used in the pharmaceutical industry. It is understood that the chemical formula showing a compound in a particular salt form or ionic form also discloses the compound in its undissociated free base (or free acid) form.

[0144] The present disclosure encompasses all stereoisomeric forms of the compounds. In some embodiments, the compounds of 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%).

[0145] Table 1

[0146]

[0147]

[0148]

[0149]

[0150]

[0151] In some embodiments, the compound has the following formula:

[0152]

[0153] In some embodiments, the compound has the following formula:

[0154]

[0155] In some embodiments, the compound has the following formula:

[0156]

[0157] In some embodiments, the compound has the following formula:

[0158]

[0159] In some embodiments, the compound has the following formula:

[0160]

[0161] Disclosed herein are compounds of Formula I or pharmaceutically acceptable salts thereof useful for treating mycobacterial infections. In some embodiments, the compounds have the chemical formula la, lb, lc, Id, or le. In some embodiments, the compounds have the chemical formula lb. In some embodiments, the compounds of Formula I have a minimum inhibitory concentration (MIC) (e.g., against Mycobacterium tuberculosis) 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, 0.01 μg / ml to 0.1 μg / ml. In some embodiments, the minimum inhibitory concentration (MIC) of the compounds of Formula I (e.g., against Mycobacterium tuberculosis) is less than 1 μg / ml, less than 0.25 μg / ml, or less than 0.1 μg / ml. In some embodiments, the MIC of the compounds of Formula I is 0.01 μg / ml to 0.25 μg / ml. In some embodiments, the MIC of the compounds of Formula I is 0.01 μg / ml to 0.1 μg / ml. It is understood that the MIC values can be lower or lower than the ranges provided herein, depending on the bacteria.

[0162] In some embodiments for treating mycobacteria (e.g., Mycobacterium tuberculosis), the MIC of the compound (AKG-28 or AKG-38) is less than 0.1 μg / mL. In some embodiments for treating mycobacteria (e.g., Mycobacterium tuberculosis), the compound has a selectivity index (SI) against killing Mycobacterium tuberculosis relative to human kidney cells (VERO) of at least 1,000. In some embodiments for treating mycobacteria (e.g., Mycobacterium tuberculosis), the MIC of the compound is less than 0.1 μg / mL and the compound has a selectivity index (SI) against killing Mycobacterium tuberculosis relative to human kidney cells (VERO) of at least 1,000. In some embodiments, the compound has the structure of AKG-28 (Formula lb) or AKG-38 (Formula lc). In some embodiments, the MIC is less than 0.05 μg / mL and the selectivity index (SI-MPS) of the MIC in Mycobacterium tuberculosis relative to mitochondrial protein synthesis inhibition is greater than 20, e.g., for AKG-28.

[0163] In some embodiments, the potency of the compounds described herein is increased by a factor of 2 to 20 (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) compared to linezolid for Mycobacterium tuberculosis.

[0164] In some embodiments for treating methicillin-resistant Staphylococcus aureus (MRSA), the compound has an MIC against MRSA strains of less than 2 pg / 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 pg / mL. In some embodiments for treating methicillin-resistant Staphylococcus aureus (MRSA), the compound has an MIC against MRSA strains of less than 2 pg / mL and an IC50 against human VERO kidney cells of greater than 100 pg / mL. In some embodiments, the compound has the structure of AKG-38 (Formula lc), AKG-39 (Formula le), and AKG-40 (Formula Id).

[0165] Water solubility

[0166] In some embodiments, the compound is in a salt form (e.g., hydrochloride or methanesulfonate) and is dissolved in water at greater than 1 mg / ml and preferably greater than 10 mg / ml (and up to 1 g / ml) prior to encapsulation in the liposome. Additional salts prior to encapsulation can include, but are not limited to, benzenesulfonate, bitartrate, carbonate, citrate, ethanesulfonate, gluconate, glutamate, glycolate, lactate, malate, maleate, mandelate, methylsulfate, naphthalenesulfonate, phosphate, propionate, salicylate, succinate, tartrate, and tosylate. In some embodiments, the compound is in a hydrate or solvate or co-crystal form prior to encapsulation in the liposome.

[0167] In some embodiments, the drug is encapsulated inside the liposome in a different salt form with reduced water solubility (e.g., less than 1 mg / mL, and preferably less than 0.1 mg / mL (0.1 to 0.001 mg / mL)). Once encapsulated in the liposome, the salt of the compound includes, but is not limited to, sulfate, citrate, phosphate, sulfoglycolate, or a variety of phosphorylated or sulfated polyols or polyanionic polymers. Some exemplary polyols include, but are not limited to, sucrose, erythritol, mannitol, xylitol, sorbitol, inositol, and combinations thereof. Some exemplary polyanionic polymers include, but are not limited to, polyvinyl sulfonate, polyvinyl sulfate, polyphosphate, copolymers of acrylic acid and vinyl alcohol sulfate, and combinations thereof.

[0168] Working stock of compounds were prepared as follows: 1 to 1.5 equivalents of HC1 as a 1 N aqueous solution were added to an aliquot of the compound (free base) in powder form, and the mixture was vortexed until uniform. To the resulting cake or syrup, typically water was added to a final 10 mg / ml, and complete dissolution was observed. In some cases, 0.95 equivalents of HC1 were added to the drug in free base form, and a 20 mg / ml stock solution was prepared.

[0169] Water solubility of the compounds of the present disclosure is illustrated by the following observations of obtaining visually clear solutions:

[0170]

[0171] These results indicate that the water solubility of the compounds provided herein is higher than the known water solubility of:

[0172] - Linezolid (3 mg / ml) www.drugbank.ca / drugs / DB00601 )

[0173]

[0174] - Sutezolid (0.237 mg / ml) www.drugbank.ca / drugs / DB11905 )

[0175] and

[0176] - Tedizolid (0.382 mg / ml) www.drugbank.ca / drugs / DB14569 )

[0177]

[0178] In some embodiments, the water solubility of the compounds described herein prior to encapsulation into liposomes is at least 5, at least 10, at least 20, at least 30, or at least 40 times higher than the water solubility of: the azolides described above.

[0179] The superior water solubility of the compounds described herein and their amphiphilic weak base properties allow for the efficient use of methods based on transmembrane gradients and liposomal internalization complexation (active loading) to produce liposomal encapsulated forms of these compounds with high drug / carrier (drug / lipid) ratios and pharmacokinetic properties that favor the delivery of the drug to infected tissues following systemic administration of the encapsulated drug. As used herein, the pKa of the amphiphilic weak base is between 7 and 12, and the logP is between 1 and 6.

[0180] Liposomal loading properties and anti-mycobacterial activity.

[0181] One of the important features of the compounds described herein is their amphiphilic weak base character, which facilitates loading of these compounds into liposomes driven by a transmembrane gradient. In some embodiments, the weak base character of the compounds of the present disclosure is characterized by an electrolytic dissociation constant in the range of 7.0 to 12.0, 7.5 to 11.0, 7.8 to 10.5, or 8.0 to 10.0. In some embodiments, the amphiphilic character of the compounds described herein is characterized by a logP parameter in the range of 0.5 to 5.0, 1.0 to 4.0, 1.0 to 3.5, or 1.0 to 3.0. It was unexpectedly discovered that certain embodiments having these favorable characteristics with respect to liposomal loading also have superior anti-mycobacterial activity that rivals or exceeds the activity of similar compounds in the same class of drugs that are less favorably characterized for efficient and stable liposomal encapsulation.

[0182] Liposomal composition

[0183] Disclosed are compositions and uses of compositions for the treatment of tuberculosis and other mycobacterial and gram-positive bacterial infections. The compositions provided herein comprise highly efficient and selective azolindinones, and are stable in the presence of blood plasma. In some embodiments, the compositions are long-circulating and retain their encapsulated drugs in circulation after intravenous administration to allow effective accumulation at the site of bacterial or mycobacterial infection. In some embodiments, high doses can be achieved when combined with the long-circulating character and highly stable retention of the drug when compared to the once-daily or twice-daily administration of other drugs commonly used to treat these infections, allowing for a reduction in the frequency of administration.

[0184] Disclosed herein are pharmaceutical compositions for the treatment of bacterial infections, particularly Mycobacterium tuberculosis infections. In some embodiments, the pharmaceutical compositions are liposomal compositions comprising a polyanion or a polyanion-containing sulfate salt and an aminoalkyl azolindinone.

[0185] In some embodiments, the compositions comprise a liposome in a medium, wherein the liposome interior space comprises an aqueous phase with a polyanion and a compound of Formula I. In some embodiments, the compositions comprise a liposome in a medium, wherein the liposome interior space comprises a polyanion or a polyanion-containing sulfate salt and a compound AKG-16, AKG-28, or AKG-38. In some embodiments, the medium is an aqueous medium, wherein the main components in the medium are the compound of Formula I and the corresponding trapping agent.

[0186] The compound of Formula I can be entrapped in liposomes with a suitable polyanion, such as sucrose octasulfate (e.g., derived from a sucrose octasulfate triethylammonium (TEA-SOS) gradient) or sulfate (e.g., derived from an ammonium sulfate gradient). Additional polyanion trapping agents include, but are not limited to, inositol hexaphosphate, inositol hexasulfate, polyvinylsulfonate, dextran sulfate, citrate, polyphosphate, and suramin.

[0187] The external aqueous medium is typically composed of a suitable buffer and an isotonic agent. Suitable buffers can include histidine, citrate, HEPES, MOPS, MES, TRIS, phosphate, glycine, and imidazole, borate, carbonate, and succinate. Isotonic agents can include salts (e.g., sodium chloride, potassium chloride), sucrose, glycerol, dextrose, or mannitol.

[0188] In some embodiments, the composition comprises a compound of Formula I or Formula la, lb, lc, or Id, or a pharmaceutically acceptable salt thereof, encapsulated with a polyanion in predominantly unilamellar vesicles formed from one or more phospholipids, a sterol, and optionally a lipid conjugated to a hydrophilic polymer (lipid conjugated to polymer). In some embodiments, the composition can comprise a compound of Formula I or Formula la, lb, lc, or Id, or a pharmaceutically acceptable salt thereof, encapsulated with a polyanion in unilamellar and multilamellar vesicles (e.g., having two or three layers). It is understood that multilamellar vesicles clear from circulation more rapidly than unilamellar vesicles. In some embodiments, the phospholipid is hydrogenated soy phosphatidyl choline (HSPC), distearoylphosphatidyl choline (DSPC), or egg sphingomyelin (ESM). The term "phospholipid" as used herein refers to any one or a combination of phospholipids capable of forming liposomes. Neutral phospholipids can include diacylphosphatidyl choline, dialkylphosphatidyl choline, sphingomyelin, and diacylphosphatidyl ethanolamine. Phosphatidyl choline (PC), including those obtained from egg, soy, or other plant sources, or those that are partially or wholly synthetic, or having variable fatty chain length and unsaturation, are suitable for use in the compositions of the present application. Synthetic, semi-synthetic, and natural product phosphatidyl choline, including but not limited to distearoylphosphatidyl choline (DSPC), hydrogenated soy phosphatidyl choline (HSPC), soy PC, egg PC, hydrogenated egg phosphatidyl choline (HEPC), dipalmitoylphosphatidyl choline (DPPC), and dimyristoylphosphatidyl choline (DMPC), are suitable phosphatidyl choline for use in the present disclosure. Charged phospholipids can include phosphatidyl serine, phosphatidic acid, phosphatidyl inositol, phosphatidyl glycerol, cardiolipin, or head group modified lipids such as N-succinyl-phosphatidyl ethanolamine, N-glutaryl-phosphatidyl ethanolamine, and PEG derivatized phosphatidyl ethanolamine.

[0189] Lipids conjugated with polymers may include poly(ethylene glycol) conjugated (polyethylene glycolated) phospholipids (PEG-lipids), such as PEG(molecular weight 2,000) methoxy-poly(ethylene glycol)-1,2-distearyl-sm-glycerol (PEG(2000)-distearylglycerol, PEG-DSG), PEG(molecular weight 2,000) 1,2-distearyl-sm-glycerol-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG(molecular weight 2,000)-distearylphosphatidylethanolamine, PEG-DSPE), or PEG(molecular weight 2,000) N-palmitoyl-sphingosine-1-p-succinyl[methoxy(polyethylene glycol)-2000]} (PEG-ceramide). The molecular weight of the PEG moiety in the PEG-lipid fraction can also be 500 to 10,000 g / mol, 1,500 to 6,000 g / mol, but preferably about 2,000 MW. Other polymers used for conjugation with lipid anchors may include poly(2-methyl-2- (PMOZ), poly(2-ethyl-2-) Poly(phenylene oxaliplatin) (PEOZ), poly-N-vinylpyrrolidone (PVP), polyglycerol, poly(hydroxyethyl L-asparagine) (PHEA), and poly(hydroxyethyl L-glutamine) (PHEG).

[0190] In some embodiments, the sterol is cholesterol. Other exemplary sterols include, but are not limited to, ergosterol, phytosterols (e.g., β-sitosterol), and hopanes. In some embodiments, the phospholipid-to-cholesterol ratio is selected to provide a desired amount of liposome membrane rigidity while maintaining a sufficiently low leakage of Formula I compounds from the liposomes. In some embodiments, optional polymer-conjugated lipids may be added to reduce the tendency of liposome aggregation. The type and amount of polymer-conjugated lipids can be selected to provide desired levels 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 be from about 60:40 to 35:65, from about 50:50 to 35:65, from about 50:50 to about 45:55. Specifically, the liposomes may comprise vesicles composed of HSPC, cholesterol, and polymer-conjugated lipids (PEG-DSG or PEG-DSPE) in a molar ratio of about 55:45:2.75, said molar ratio corresponding to a PEG-lipid concentration of 5 mol% relative to the phospholipid concentration. The concentration of PEG-lipids relative to (non-PEGylated) phospholipids may be 0.5 to 10 mol%, preferably 3 to 10 mol%, and even more preferably 4 to 8 mol%.

[0191] In some embodiments, the liposome composition provides the following: a desired pharmacokinetic property (e.g., extended plasma half-life), measured as the percentage of the injected dose (or amount) retained in the blood 6 or 24 hours after intravenous injection in an immunocompetent mouse; and stable encapsulation of the drug in the plasma within 24 hours after iv administration in mice, determined by the change in the drug-to-lipid ratio (DL ratio). In some embodiments, the percentage of drug retained in the blood at 6 hours is greater than 20%, preferably greater than 30%, and most preferably greater than 40% of the injected dose. The percentage retained in the blood after 24 hours is preferably greater than 10%, more preferably greater than 20% of the injected dose. The DL ratio at 24 hours is greater than 20%, preferably greater than 50%, and most preferably greater than 80% of the liposomal drug initially injected. The desired liposome composition also shows stable encapsulation in vitro in the presence of human plasma using a burst release method, where the liposomes retain greater than 50% of the drug within 20 minutes, greater than 60% of the encapsulated drug within 20 minutes, greater than 70%, preferably greater than 80%, and most preferably greater than 90%.

[0192] Liposomes of the present disclosure can be prepared by any method known in the art. See, e.g., G. Gregoriadis (ed.), Liposome Technology, Vols. 1-3, 1stEd., 1983; 2ndEd., 1993; 3rdEd., 2006; CRC Press, Boca Raton, Fla. Some examples of methods suitable for preparing the liposomal compositions of the present disclosure include membrane extrusion, reverse phase evaporation, sonication, solvent (e.g., ethanol) injection (including microfluidic, Y- junction and T-junction mixing), microfluidization, detergent dialysis, ether injection, and dehydration / rehydration. The size of the liposomes can be controlled by controlling the pore size of the membrane used for extrusion or the pressure used in microfluidization, as well as by any other suitable method. In some embodiments, the desired lipids are first hydrated by thin film hydration or by ethanol injection, and then size classified by extrusion through a membrane of defined pore size (e.g., 50 nm, 80 nm, 100 nm, or 200 nm, or a combination thereof), resulting in liposomes with an average size of 70 to 150 nm, or 80 to 130 nm, and a polydispersity index of 0.1 or less. The pharmaceutical compound to be encapsulated can be added to the liposomal lipids prior to liposome formation, dissolved in an aqueous medium in which the liposomes are formed by the methods described above, whereby the drug is sequestered in the liposomes. In some embodiments, the pharmaceutical compound is encapsulated in the liposomes using a capture agent that incorporates into the interior space of the liposome (see Drummond, D.C., et al. (2006) in Liposome Technology, 3rdEd. (Ed. Gregoriadis, G.) Vol. 2, pp. 149-168).

[0193] In some embodiments, the method of preparing a liposome composition of the disclosure comprises the steps of: (i) preparing liposomes comprising a phospholipid, a cholesterol, and a PEG-lipid, and having an internal space comprising a capture agent, in a medium substantially free of the capture agent; (ii) contacting the liposomes with a compound of the disclosure in an aqueous medium to effect encapsulation of the compound in the liposomes; (iii) removing unencapsulated compound; and (iv) providing the liposomes in a physiologically acceptable medium suitable for parenteral use. In some embodiments, the method of producing liposomes having a compound therein comprises the steps of: (a) preparing liposomes comprising a capture agent consisting of an ammonium or substituted ammonium salt of a polyanion, (b) subsequently removing the capture agent outside the liposomes to form an electrochemical gradient across the membrane, and (c) contacting the liposomes with the compound under conditions effective for the compound to enter the liposomes and allow a corresponding amount of ammonia or substituted ammonia to exit the liposomes, thereby depleting or reducing the pH gradient of the resulting liposomes. Liposome compositions comprising a capture agent inside the liposomes can be prepared by forming the liposomes in a solution of the capture agent. A transmembrane concentration gradient of the capture agent on the liposomes can be formed after liposome formation or prior to drug loading (encapsulation) by removing the capture agent outside the liposomes or diluting the liposomes.

[0194] In some embodiments, the contacting comprises incubation of the liposomes with the drug in an aqueous medium at a temperature above ambient temperature and below the boiling point of water, preferably 30°C to 90°C, 40°C to 80°C, 50°C to 80°C, or 60°C to 75°C. In some embodiments, the incubation is performed at an ionic strength of 50 mM NaCl or less, or more preferably 30 mM NaCl or less. After incubation, a concentrated salt (e.g., NaCl) solution can be added to increase the ionic strength to above 50 mM NaCl or about 100 mM NaCl. The increase in ionic strength after the drug loading incubation step helps to reduce aggregation after liposome loading. The incubation time can vary in the range of a few 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, the liposomes are cooled to 2 to 15°C. In some embodiments, the liposomes are cooled to 4 to 10°C. After the cooling step, a concentrated salt (e.g., NaCl) solution can be added to increase the ionic strength to above 50 mM NaCl or about 100 mM NaCl. The increase in ionic strength after the drug loading incubation step helps to reduce aggregation after liposome loading.

[0195] In some embodiments, the contacting further comprises incubation of the liposome with the drug in an aqueous medium in the presence of an osmotic (tonicity) balancing agent. In some embodiments, the osmotic balancing agent (osmotic agent) is a non-ionic agent. Some 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 osmotic concentration (expressed in osmolality or osmolality concentration) that is equal to the osmotic concentration of the trapping agent solution in the interior space of the liposome prior to drug loading. The osmotic concentration of the trapping agent solution can be measured by any known method prior to the solution being combined with the lipids to form the liposome. In another embodiment, the concentration of the osmotic agent provides an osmotic concentration that is lower than the osmotic concentration of the trapping agent solution, and 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 trapping agent solution. In another embodiment, the concentration of the osmotic agent during the drug loading process is 200 to 400 mmol / kg, preferably 250 to 350 mmol / kg. In another embodiment, the osmotic agent is dextrose and the concentration is 45 g / L. In another embodiment, no osmotic agent is used during the incubation of the liposome with the drug. In another embodiment, the incubation is performed in the presence of an ionic strength adjusting agent. One example of an ionic strength adjusting agent is sodium chloride, for example added to the liposome-drug solution at a concentration of 5 to 50 mM, 10 to 20 mM, or about 10 mM. In contrast to the conventional in the art of liposomes, the compounds of the present disclosure (e.g., AKG-28 and AKG-38) are loaded into the liposomes of the present disclosure in a stable and efficient manner, even during the drug-liposome contacting step, the amount of osmotic agent provides an osmotic concentration that is lower than the osmotic concentration of the trapping agent solution (osmotically imbalanced liposomes), up to no added osmotic agent at all.

[0196] Methods of use

[0197] Disclosed herein are methods for inhibiting the growth of mycobacteria (e.g., Mycobacterium tuberculosis) or gram-positive bacteria (e.g., methicillin-resistant Staphylococcus aureus (MRSA)). Other mycobacteria and gram-positive bacteria include, but are not limited to, the Mycobacterium avium complex, Mycobacterium leprae, Mycobacterium gordonae, Mycobacterium abscessus, Mycobacterium abscessus, Mycobacterium mucogenicum, Streptococcus, vancomycin-resistant enterococci (VRE), Staphylococcus pneumoniae, Enterococcus faecium, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus viridans, Listeria monocytogenes, Nocardia, and Corynebacterium. In some embodiments, the compounds and compositions provided herein inhibit the growth of drug-resistant strains of Mycobacterium tuberculosis. In some embodiments, methods of treating mycobacterial infections are provided. In some embodiments, the compounds and compositions provided herein are useful for treating non-tuberculosis mycobacterial infections. In some embodiments, the method comprises administering to a subject in need thereof a therapeutically effective amount of an aminoalkyl oxazolidinone and / or a pharmaceutically acceptable salt thereof. In some embodiments, the method comprises administering to a subject in need thereof a therapeutically effective amount of a liposomal composition comprising an aminoalkyl oxazolidinone compound and / or a pharmaceutically acceptable salt thereof.

[0198] In some embodiments, the composition is a liquid pharmaceutical formulation for parenteral administration. In some embodiments, the liquid pharmaceutical formulation is a liposomal formulation comprising an appropriate amount of an aminoalkyl oxazolidinone compound, wherein The oxazolidinone compound is encapsulated inside the liposome. In another embodiment, the compound is in salt form inside the liposome with a polyanion (e.g., sulfate, citrate, sucrose octasulfate, inositol hexaphosphate). In some embodiments, the compound is a precipitated or gelled salt with sulfate inside the interior of the liposome composed of a variety of lipid excipients including, but not limited to, phosphatidylcholine, cholesterol, and pegylated phosphatidylethanolamine. The liposomes of the present disclosure show encapsulation efficiencies of over 85%, over 90%, and over 95%. In some embodiments, a retention amount of unencapsulated drug is removed from the liposomal composition. This can be achieved by a variety of methods (e.g., size exclusion chromatography, ion exchange, dialysis, ultrafiltration, tangential flow filtration, adsorption, or precipitation). During or after the unencapsulated drug removal step, the liposomes can be placed in a desired pharmaceutically acceptable carrier (e.g., physiological saline, isotonic dextrose, isotonic sucrose, Ringer's solution, or Hanks' solution). Buffering substances can be added to provide a desired physiologically acceptable pH. The liposomal composition can be adjusted for a desired drug concentration and sterilized, e.g., by sterile filtration through a 0.2 to 0.22 μιη filter. In some embodiments, the compound concentration in the liposomal composition is 1 to 50 mg / ml, 3 to 30 mg / ml, or 5 to 25 mg / ml.

[0199] In some embodiments, the liposomes are mixed with one or more additional excipients for isotonicity or pH control. In some embodiments, the excipients include, but are not limited to, sodium chloride, Hepes buffer, phosphate buffer, and histidine buffer.

[0200] In other embodiments, the composition is an oral formulation. In some embodiments, the composition is a liquid formulation. In some embodiments, the composition is a solid formulation (e.g., a tablet, a capsule, a pill, a dragee, a caplet, etc.). When intended for oral use, for example, tablets, troches, lozenges, aqueous or oil suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups or elixirs can be prepared. 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 include one or more agents including antioxidants, sweetening agents, flavoring agents, coloring agents and preservatives, to provide a palatable or otherwise acceptable preparation. Tablets containing the active ingredient in admixture with non-toxic pharmaceutically acceptable excipient or auxiliary agent which is suitable for tablet manufacture are acceptable. Suitable excipients or auxiliary agents include, but are not limited to, for example, inert diluents, solubilizers, suspending agents, binders, humectants, sweetening agents, flavoring or flavoring agents, isotonic agents, colloid dispersing agents and surface active or wetting agents.

[0201] Tablets, dragees, capsules, pills, granules, suppositories, solutions, suspensions and emulsions, pastes, ointments, gels, creams, lotions, powders and sprays can be suitable pharmaceutical compositions.

[0202] The compound or composition can be administered topically, orally, parenterally, intraperitoneally and / or rectally.

[0203] The dosage regimen is 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.

[0204] The dosage of the compound and / or its pharmaceutically acceptable salt or liposome containing the compound and / or its pharmaceutically acceptable salt can vary within wide limits and will, of course, be adjusted to the individual requirements in each particular case, with the natural fluctuations in the response of the individual and the natural fluctuations of the therapeutic effect to be expected.

[0205] In some embodiments, for use in treating a bacterial infection, a compound or a pharmaceutical liposome composition is administered to a subject in need thereof once every 7 days (i.e., once a week), once every 14 days (i.e., once every two weeks), once every 21 days (i.e., once every three weeks), once every 28 days (i.e., once every four weeks), and once every 42 days (i.e., once every six 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, 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 longer. The particular therapeutically effective amount will depend on a variety of factors including the bacterial infection being treated, the activity of the particular compound being administered, the pharmaceutical composition used, 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 particular compound, and similar factors known to those of ordinary skill in the medical arts. 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 a monotherapy. In some embodiments, the treatment can include the 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 to treat Mycobacterium tuberculosis and other gram-positive bacterial infections. In some embodiments, the treatment can include the simultaneous and / or sequential administration of an effective amount of a compound described herein and an effective amount of two or more (two, three, four, etc.) additional active agents to treat Mycobacterium tuberculosis and other gram-positive bacterial infections. Synergistic antibacterial effect indicates an antibacterial effect greater than the expected complete additive effect of the individual compounds of the combination. When administered simultaneously, the compound and the active agents can be included in the same composition or in different compositions. When administered sequentially, the composition including the compound and the composition including the additional active agents can be administered with a certain time interval (e.g., 20 minutes, 40 minutes, 60 minutes, or longer). In some embodiments, the additional active agents can be administered using a different route of administration or by a different injection.For example, the compound of the present disclosure can be administered intravenously, and one or more additional agents can be administered orally.

[0206] In some embodiments, administration of the compound with one or more (e.g., one, two, three, or four) additional active agents can result in a reduction in the length of the treatment duration. For example, administration of the compound with one or more (e.g., one, two, three, or four) additional active agents can result in a treatment duration that is at least three-fold, at least two-fold, at least 1.5-fold shorter than a treatment with only one active agent. In some embodiments, the additional agent is an antibacterial agent. In some embodiments, the additional active agent can include, but is not limited to, a fluoroquinolone (e.g., moxifloxacin, gatifloxacin, or levofloxacin), bedaquiline and other diarylquinoline analogs (e.g., TBAJ-587 and TBAJ-876), delamanid, pretomanid, isoniazid, rifampin, rifapentine, pyrazinamide, clofazimine, spectinamide, ethambutol, streptomycin, kanamycin, capreomycin, amikacin, Leucyl-tRNA Synthetase (LeuRS) inhibitor GSK 3036656, tryptophanase inhibitor GSK839, DprE1 inhibitor OPC-167832 and Macozinone (PBTZ-169), Telacebec, GSK-656, TBA-7371, and amoxicillin plus clavulanate, each of their pharmaceutically acceptable salts, and any combination thereof. For the treatment of Gram-positive bacterial infections, the additional active agent can include, but is not limited to, vancomycin, gentamycin, daptomycin, teicoplanin, ceftaroline, ceftrobiprole, telavancin, dalbavancin, oritavancin, fluoroquinolones (e.g., delafloxacin), tetracyclines (e.g., eravacycline and omadacycline), sulfonamides (e.g., sulfamethoxazole), and any combination thereof. azithromycin), clarithromycin, doxycycline, minocycline, tetracycline, trimethoprim, lefamulin, and any combination thereof. In some embodiments, the treatment can comprise simultaneous and / or sequential administration of an effective amount of a compound described herein and an effective amount of bedaquiline, pretomanid, sutezolid, moxifloxacin, or a pharmaceutically acceptable salt of each thereof or a combination of the foregoing.

[0207] Actual dosage levels of the active ingredients in the pharmaceutical compositions disclosed herein can be varied in order to obtain an amount of the active ingredient(s) that is effective to achieve the desired therapeutic response for a particular patient, compositions, and modes of administration, without being toxic to the patient.

[0208] As used herein, “parenteral” in the context of administration means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinally, epidural, and intrastemal injection and infusion.

[0209] As used herein, the phrases “parenterally administered” and “administered parenterally” refer to modes of administration other than enteral (i.e., through the digestive tract) and topical administration, usually by injection or infusion, and include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, inhalation, subcapsular, subarachnoid, intraspinally, epidural, and intrastemal injection and infusion. Intravenous injection and infusion are often, but not exclusively, used for liposomal drug administration.

[0210] 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 a week), once every 14 days (i.e., once every two weeks), once every 21 days (i.e., once every three weeks), once every 28 days (i.e., once every four weeks), and once every 42 days (i.e., once every six 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, about 1400 mg to about 1500 mg. The specific therapeutically effective dose will depend on a variety of factors including the bacterial infection being treated, the activity of the specific compound being administered, the pharmaceutical composition used, the age, body 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 concurrently) with the particular compound, and like factors known to one of ordinary skill in the art.

[0211] In some embodiments, for use in treating a bacterial infection, the compound or pharmaceutical composition is administered once or twice daily. The specific therapeutically effective dose will depend on a variety of factors including the bacterial infection being treated, the activity of the specific compound being administered, the pharmaceutical composition used, the age, body 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 concurrently) with the particular compound, and like factors known to one of ordinary skill in the art. Examples

[0212] The following examples, including the experiments performed and results achieved, are provided for illustrative purposes only, and should not be construed as limiting the disclosure.

[0213] Example 1- Synthesis of oxazolidinone derivatives

[0214] Compounds AKG-1, AKG-2, AKG-6, AKG-8, AKG-9 and AKG-19 were synthesized by reacting escitalopram mesylate (escitalopram-MS) with the corresponding amine in N-methyl-2-pyrrolidone (NMP) as solvent at 60 °C (Scheme-1). Escitalopram-MS was obtained by mesylation of the 1° hydroxyl group of escitalopram with methanesulfonyl chloride in the presence of a base at room temperature (RT). Treatment of escitalopram-MS with sodium azide followed by reduction of the resulting azide (AKG-3-A) gave intermediate-1 as a free base or AKG-3 as a hydrochloride salt depending on the eluent chosen for purification. Amide formation of intermediate-1 with the corresponding acid followed by salt formation using HC1 / EtOAc gave compounds AKG-17 and AKG-18. Reaction of escitalopram with the corresponding dialkylamino acid under standard esterification conditions gave compounds AKG-5 and AKG-20. O-alkylation of escitalopram with 2-chloro-N,N-diethylaminoethylamine using sodium hydride as a base gave compound AKG-7.

[0215] Intermediate-2 was synthesized by borylation of a commercially available aryl bromide using bis(pinacolato)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 to AKG-15.

[0216] 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 a 3:1 ratio of intermediates 5 and 6. The structure of these intermediates was deduced from HMBC analysis. Intermediates 7 to 12 were synthesized and the regioisomers were obtained in a similar fashion (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.

[0217] Intermediates-13 were synthesized by mesylation of readily available aryl bromides. Intermediates-15 were obtained by reduction of intermediates-14 with hydrazine (Scheme-5). Boc protection or acetylation of the primary amine in intermediates-15 followed by borylation produced intermediates-18 and 19, respectively. Suzuki coupling (US Patent Application Publication No. 20100022772, PCT International Application Publication No. WO2013044845, incorporated herein by reference in their entirety) of the borylated intermediates with the corresponding aryl bromide intermediates and deprotection of the amine group (where applicable) produced compounds AKG-28 to AKG-31 and AKG-38 to AKG-40.

[0218] Synthetic schemes

[0219] See US Patent Application Publication No. 20100022772, PCT International Application No. 2013044845, incorporated herein by reference in their entirety, for the synthesis of intermediates-19.

[0220] Scheme 1

[0221]

[0222] Scheme 2

[0223]

[0224] Scheme 3

[0225]

[0226] Scheme 4

[0227]

[0228] Scheme 5

[0229]

[0230] Synthesis

[0231] Materials and Methods

[0232] Latanoprost, (R)-3-(4-bromo-3-fluorophenyl)-5-(hydroxymethyl) Zolazepam was purchased from Skychemical and dimethyl-(2-piperidin-4-yl-ethyl)-amine was purchased from Enamine, other reagents and solvents were purchased from Adams and used as such. The chemical structure of the final product was confirmed by nuclear magnetic resonance spectra (1H NMR,13C NMR) determined on a Bruker NMR spectrometer (500 MHz or 400 MHz) and mass spectrometry (LC-MS). 1 H NMR, 13characterized by1H NMR and13C NMR. 13 C NMR spectra were fully decoupled. Chemical shifts are in parts per millions (ppm) using either the deuterated solvent peak or tetramethylsilane (internal) as internal standard. For1H NMR, chemical shifts are reported in parts per millions (ppm), multiplicity (s, singlet; br s, broad singlet; d, doublet; t, triplet; m, multiplet), integration, coupling constant (Hz). For13C NMR, data are reported as chemical shifts (d, ppm). 1 Data for1H NMR are reported as chemical shifts (d, ppm), multiplicity (s, singlet; br s, broad singlet; d, doublet; t, triplet; m, multiplet), integration, coupling constant (Hz). For13C NMR, data are reported as chemical shifts (d, ppm). 13 Data for13C NMR are reported as chemical shifts (d, ppm).

[0233] The purity of the final product was confirmed by analytical HPLC (>95%). Analytical HPLC was performed on an Agilent analytical HPLC system using a Sunfire column 3.5 pm (150 em x 4.6 mm) and a gradient system (water (0.01% TFA) / ACN (0.01% TFA)) with a flow rate of 1 mL / min and detection wavelengths of 254 and 214 nm. Flash Chromatographic (FC) purification was performed with Silica Gel 60 (0.04 to 0.063 pm; 230 to 400 mesh) from Santai Technologies.

[0234] Procedure A. The reaction mixture of Tedizolid-Ms (1.0 eq), R1R2NH (4.0 eq) in NMP (10 mL) was heated to 60 °C for 15 hours in a sealed tube. After completion (LCMS), the reaction was diluted with H2O (40 mL) and extracted with EtOAc (2 x 50 mL). The combined extracts were washed with saturated brine, dried over Na2S04and filtered. The solvent was removed in vacuo and the residue was purified using FC to give the product with purity >95%.

[0235] 1. Synthesis of Tedizolid-Ms

[0236]

[0237] To a solution of Tedizolid (7.00 g, 18.90 mmol) and triethylamine (3.83 g, 37.80 mmol) in CH2Cl2(50 mL) at 0 °C, methanesulfonyl chloride (3.25 g, 28.36 mmol) was added dropwise at 0 °C under argon. After stirring at room temperature for 2 hours, the reaction mixture was poured into water and extracted with CH2Cl2. The organic layer was washed with brine, dried over Na2S04and collected by filtration. The solvent was removed in vacuo to give the pure product Tedizolid-Ms (7.0 g, 82.6% yield) as a yellow solid.

[0238] 1 H NMR (400 MHz, DMSO-d6) δ 8.95 (s, 1H), 8.31 - 8.14 (m, 2H), 7.88 - 7.65 (m, 2H), 7.53 (d, J = 8.6 Hz, 1H), 5.14 - 4.96 (m, 1H), 4.59 - 4.39 (m, 5H), 4.28 (t, J = 9.4 Hz, 1H), 3.92 (dd, J = 9.2, 6.3 Hz, 1H), 3.28 (s, 3H). MS (ESI+) m / z 449.1 ([M + 1] + )

[0239] 2. Synthesis of AKG-1, 2, 6, 8, 9 and 19

[0240]

[0241] AKG-1 was obtained as a white solid (0.5 g, 56.4% yield) using Procedure A from leteridamine-Ms and dimethylamine.

[0242] 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 (8, 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] + )。

[0243]

[0244] AKG-2 was obtained as a white solid (0.52 g, 54.8% yield) using Procedure A from leteridamine-Ms and diethylamine.

[0245] 1H 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] + )

[0246]

[0247] AKG-6 was obtained as a white solid using Procedure A from lumezepam-Ms and N,N-dimethyl-2-(piperidin-4-yl)ethan-l-amine (0.66 g, 58.2% yield).

[0248] 1 H NMR (400 MHz, CDCl3) δ 8.93 (s, 1H), 8.30 (dd, J = 8.1, 2.4 Hz, 1H), 8.05 (d, J = 7.8 Hz, 1H), 7.62 (d, J = 12.9 Hz, 1H), 7.56 - 7.47 (m, 1H), 7.45 - 7.37 (m, 1H), 4.89 - 4.74 (m, 1H), 4.48 (s, 3H), 4.11 (t, J = 8.6 Hz, 1H), 3.86 (t, J = 7.8 Hz, 1H), 2.93 (dd, J = 28.8, 10.9 Hz, 2H), 2.80 - 2.64 (m, 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). 13C NMR (101 MHz, CDC13) 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] + )

[0249]

[0250] Using Procedure A, AKG-8 was obtained as a white solid (0.62 g, 57.6% yield) from tetomilast-Ms and N 1 , N 1 -diethylpropane-1, 3-diamine.

[0251] 1 H 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] + )

[0252]

[0253] Using Procedure A, AKG-9 was obtained as a white solid (0.36 g, 34.4% yield) from tetomilast-Ms and N 1 , N 1 -diethyl ethane-1, 2-diamine.

[0254] 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] + )

[0255] AKG-19 was obtained as a white solid using Procedure A from Diammonium phosphate and ethane-1, 2-diamine (0.60 g, 55% yield).

[0256] 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), 7.59 (d, J = 13.5 Hz, 1H), 7.49 (d, J = 8.7 Hz, 1H), 5.25-5.19 (m, 1H), 4.49 (s, 3H), 4.33 (t, J = 9.2 Hz, 1H), 4.05 (dd, J = 9.1, 6.7 Hz, 1H), 3.52 (s, 2H), 3.43-3.23 (m, 4H). 13 C NMR (101 MHz, DMSO-d6) δ 164.26, 160.94, 158.50, 153.79, 149.84, 145.51, 140.58, 137.78, 132.04, 131.42, 122.61, 119.50, 114.94, 106.46, 69.38, 49.59, 47.87, 45.16, 40.34, 35.58

[0257] 3. Synthesis of AKG-3

[0258]

[0259] To a solution of lurasidone-Ms (1.00 g, 2.23 mmol) in DMF (20 mL) was added NaN3(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 MgSO4, filtered and concentrated in vacuo. The residue was further purified by column chromatography to obtain the title compound AKG-3-1 (0.7 g, 79.4% yield) as a white solid.

[0260]

[0261] A reaction mixture of AKG-3-1 (0.7 g, 1.77 mmol) and Ph3P (1.39 g, 5.31 mmol) in H2O (2 mL) and THF (20 mL) was heated to reflux for 1 h. After completion (LCMS), the reaction was concentrated in vacuo and purified using reverse phase FC. Purification was performed using MeOH 0 to 10% in DCM as eluent and lyophilized to give free base intermediate-1 (2.5 g, 76.5% yield) as a yellow solid, while FC purification was performed using MeCN / 0 to 30% in 0.006 M HC1 in H2O as eluent to give AKG-3 hydrochloride (0.35 g, 48.8% yield) as a yellow solid after lyophilization.

[0262] 1 H NMR (400 MHz, DMSO-d6) δ 8.95 (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] + )

[0263] 4. Synthesis of AKG-17

[0264]

[0265] To a solution of 3-((tert-butoxycarbonyl)amino)propanoic acid (0.62 g, 3.25 mmol, 1.2 eq) and TEA (0.63 g, 6.25 mmol, 2.5 eq) in DMF (10 mL) was added HATU (1.44 g, 3.78 mmol, 1.4 eq) at room temperature under argon. The mixture was stirred for 0.5 h, and then intermediate 1 (1.0 g, 2.70 mmol, 1.0 eq) was added. The whole mixture was stirred at room temperature overnight. LCMS showed the reaction was complete, it was poured into H2O, and the solid was collected by filtration and washed with H2O. The solid was dried under vacuum, and the residue was directly used for the next step by dissolving it in EtOAc and then adding HC1 / EtOAc (4 M, 20 mL). The whole mixture was stirred for 16 h, and the solvent was removed by N2. The residue was purified by reverse phase FC (eluent with 0 to 30% MeCN in H2O with 0.006 M HC1), to give the product AKG-17 (0.5 g, 39.5% yield) as a yellow solid after lyophilization.

[0266] 1 H NMR (500 MHz, DMSO-d6) δ 8.95 (s, 1H), 8.67 (s, 1H), 8.23 (q, J = 8.3 Hz, 2H), 8.14 (s, 3H), 7.77 (t, J = 8.6 Hz, 1H), 7.69 (d, J = 13.5 Hz, 1H), 7.50 (d, J = 8.6 Hz, 1H), 4.87 - 4.78 (m, 1H), 4.49 (s, 3H), 4.22 (t, J = 9.0 Hz, 1H), 3.89 (dd, J = 9.0, 6.5 Hz, 1H), 3.50 (t, J = 5.3 Hz, 2H), 2.98 (dd, J = 12.5, 6.4 Hz, 2H), 2.58 (t, J = 7.1 Hz, 2H) 13 C NMR (101 MHz, DMSO-d6) δ 170.60, 164.22, 160.97, 158.53, 154.42, 149.78, 145.42, 140.89, 137.79, 132.11, 131.41, 122.61, 119.19, 114.72, 106.23, 105.95, 72.13, 47.77, 40.33, 35.58, 32.58

[0267] 5. Synthesis of AKG-18

[0268]

[0269] AKG-18 was obtained as a yellow solid from intermediate-1 and 4-((tert- butoxycarbonyl)amino)butanoic acid using the procedure for AKG-17 (0.5 g, 37.6% yield).

[0270] 1 H NMR (500 MHz, DMSO-d6) δ 8.95 (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

[0271] 6. Synthesis of AKG-5

[0272]

[0273] To a mixture of tafluprost (1.0 g, 2.70 mmol), 4-(dimethylamino)butanoic acid hydrochloride (0.57 g, 3.37 mmol) and TEA (0.27 g, 2.70 mmol), catalytic amount of DMAP in DMF (20 mL) was added DCC (0.84 g, 4.05 mmol) under N2. The mixture was stirred at room temperature for 16 hours. After completion of the reaction (LCMS), it was diluted with H2O (100 mL) and filtered. The filtrate was acidified to pH = 5 to 6 with 0.02 M HC1 and subsequently purified using RP-FC (eluent, 0.5% formic acid in MeCN / H2O) to give the product AKG-5 as formate salt after lyophilization. The product was re-dissolved in H2O and 1 equivalent of aq. HC1 (0.02 M) was added. Lyophilization of the product gave AKG-5 as hydrochloride salt (600 mg, 42.7% yield). 1H NMR (400 MHz, DMSO-d6) δ 10.40 (br, 1H), 8.95 (s, 1H), 8.23 (q, J = 8.5 Hz, 2H), 7.78 (t, J = 8.8 Hz, 1H), 7.71 (dd, J = 13.6, 2.1 Hz, 1H), 7.53 (dd, J = 8.6, 2.1 Hz, 1H), 5.03 (dd, J = 5.6, 3.1 Hz, 1H), 4.48 (s, 3H), 4.36 (qd, J = 12.4, 4.2 Hz, 2H), 4.26 (t, J = 9.3 Hz, 1H), 3.95 (dd, J = 9.2, 6.2 Hz, 1H), 2.99 - 2.86 (m, 2H), 2.64 (s, 6H), 2.45 (t, J = 7.3 Hz, 2H), 1.87 (m, 2H). 13 C NMR (126 MHz, DMSO-d6) δ 172.3, 164.3, 158.8, 154.3, 149.9, 145.6, 140.8, 137.7, 132.0, 131.5, 122.6, 119.4, 114.7, 106.2, 71.1, 64.8, 56.3, 46.7, 40.3, 30.9, 19.9. m / z 469.3 ([M + 1] + ). m / z 484.1 ([M - HC1 + 1] + )

[0274] 7. Synthesis of AKG-7

[0275]

[0276] To a mixture of tafoxanide (1.0 g, 2.70 mmol) in DMF (20 mL) was added NaH (0.13 g, 60%, 5.40 mmol) at room temperature under N2. The mixture was stirred at 0 °C for 0.5 h, and then 2-diethylaminoethyl chloride hydrochloride (930 mg, 5.40 mmol) was added in one portion. The whole mixture was stirred at room temperature for 3 h. LCMS showed the reaction was complete. The reaction was carefully poured into ice / H2O (20 mL) and extracted with DCM (2 x 50 mL). The combined organic extracts were washed with saturated brine, then dried over Na2SO4. The solvent was removed in vacuo, and the residue was purified using FC (eluent with 0 to 15% MeOH in DCM) to give AKG-7 (0.5 g, 39.4% yield) as a white solid.

[0277] 1H NMR (500 MHz, CDC13) 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 CNMR (126 MHz, CDC13) 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] + )。

[0278] 8. Synthesis of AKG-20

[0279]

[0280] To a reaction mixture of tafoxanide (1.0 g, 2.70 mmol), 4-(diethylamino)butanoic acid hydrochloride (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 room temperature under N2. The mixture was stirred at room temperature for 16 hours. After completion (LCMS), the reaction was diluted with H2O (100 mL) and filtered. The filtrate was acidified to pH = 5 to 6 with 0.02 M HC1 and subsequently purified with RP-FC (eluent with MeCN in 0.5% FA / H2O) to give the product as formate salt after lyophilization. The salt was then re-dissolved in H2O and 1 equivalent of HC1 (0.02 M) was added to give the product AKG-20 as hydrochloride salt (0.61 g, 42% yield) after lyophilization.

[0281] 1H NMR (400 MHz, DMSO-d6) δ 8.94 (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 = 7.2 Hz, 6H). 13 CNMR (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)

[0282] 9. Synthesis of Intermediate-3

[0283]

[0284] (R)-3-(4-bromo-3-fluorophenyl)-5-(hydroxymethyl)oxazolidin-2-one (Intermediate-3) was prepared according to the procedure described in Example 1, Step 9, using (R)-3-(4-bromo-3-fluorophenyl)oxazolidin-2-one (Intermediate-2) and sodium borohydride. A mixture of oxazolidine-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 argon for 10 minutes and then (Ph3P)2PdCl2(1.09 g, 1.55 mmol) was added. After purging the mixture with argon again, it was heated to 90 °C for 15 hours. LCMS showed the reaction was complete. It was cooled to room temperature and filtered with Celite to give intermediate-2 as filtrate. To the filtrate was added 5-bromo-2-fluoropyridine (6.55 g, 37.22 mmol), K3PO4(14.47 g, 6.80 mmol) and H2O (20 mL). The mixture was purged with argon for 10 minutes and (dppf)PdCl2(2.27 g, 3.10 mmol) was added. The mixture was purged with argon again. It was then heated to 90 °C for 15 hours. The reaction was monitored by LCMS. After completion, it was concentrated in vacuo and the residue was diluted with H2O (200 mL) and extracted with EtOAc (2 x 200 mL). The combined extracts were washed with saturated brine and then dried over Na2SO4. It was filtered and the solvent was removed in vacuo to give a residue which was purified using FC (eluent with 0 to 15% MeOH in DCM) to give the product intermediate-3 as a yellow solid (6.8 g, 71.6% yield for two steps).

[0285] 1 H NMR (400 MHz, DMSO-d6) δ 8.43 (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] + ).

[0286] 10. Synthesis of AKG-11, 12, 13, 14, 15

[0287] Procedure B. A mixture of intermediate-3 (1.0 eq), R1R2NH (4.0 eq) and catalytic amount of DMAP (10 mL) in NMP was heated to 100 °C in a sealed tube for 16 h. Upon completion of the reaction (LCMS), it was diluted with H2O (50 mL) and extracted with EtOAc (2 x 50 mL). The combined organic extracts were washed with saturated brine, dried over Na2S04and filtered. The solvent was removed under vacuum and the residue was purified using RPFC (eluent with 0 to 40% MeCN in 0.1% NH4HCO3 / H2O, C18) to afford the product.

[0288]

[0289] AKG-11 was obtained as a white solid (0.40 g, 30.1% yield) using Procedure B from intermediate-3 and N,N-dimethyl-2-(piperidin-4-yl)ethan-1-amine.

[0290] 1 H NMR (400 MHz, DMSO-d6) δ 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).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] + )。

[0291]

[0292] Using procedure B, intermediate-3 and N 1 , N 1 -dimethylethane-1,2-diamine afforded AKG-12 as a white solid (0.52 g, 42.6% yield).

[0293] 1 H NMR (400 MHz, DMSO-d6) δ 8.16 (s, 1 H), 7.64 - 7.46 (m, 3H), 7.39 (dd, J = 8.6, 2.2 Hz, 1 H), 6.58 (dd, J = 9.9, 5.6 Hz, 2H), 5.25 (t, J = 5.6 Hz, 1 H), 4.80 - 4.66 (m, 1 H), 4.11 (t, J = 9.0 Hz, 1 H), 3.86 (dd, J = 8.9, 6.2 Hz, 1 H), 3.76 - 3.65 (m, 1 H), 3.61 - 3.49 (m, 1 H), 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] + )

[0294]

[0295] Using procedure B, intermediate-3 and N 1 , N 1 -dichloroethane-1,2-diamine afforded AKG-13 as a white solid (0.68 g, 51.9% yield).

[0296] 1H NMR (400 MHz, DMSO-d6) δ 8.17 (s, 1H), 7.67 - 7.46 (m, 3H), 7.39 (dd, J = 8.6, 2.1 Hz, 1H), 6.63 - 6.44 (m, 2H), 5.25 (s, 1H), 4.74 (dd, J = 9.2, 5.8 Hz, 1H), 4.12 (t, J = 9.0 Hz, 1H), 3.87 (dd, J = 8.9, 6.2 Hz, 1H), 3.76 - 3.65 (m, 1H), 3.63 - 3.52 (m, 1H), 3.34 (dd, J = 13.2, 6.2 Hz, 2H), 2.60 - 2.55 (m, 2H), 2.54 - 2.50 (m, 4H), 0.97 (t, J = 7.1 Hz, 6H). 13 C NMR (101 MHz, DMSO-d6) δ 160.60, 158.53, 158.18, 154.81, 147.62, 138.91, 137.13, 130.20, 121.16, 118.54, 114.29, 108.24, 105.87, 73.80, 62, 10, 52.19, 47.13, 46.45, 39.48, 12.31. MS (ESI+) m / z 417.1 ([M + 1] + )

[0297]

[0298] AKG-14 was obtained as a white solid using Procedure B from Intermediate-3 and N 1 , N 1 -dimethylpropane-1, 3-diamine (0.6 g, 47.3% yield).

[0299] 1 H 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), 6.73 (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). 13C NMR (101 MHz, DMSO-d6) δ 160.50, 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] + )

[0300]

[0301] AKG-15 was obtained as a white solid using Procedure B from Intermediate-3 and N 1 , N 1 -diethylpropane-1, 3-diamine (0.65 g, 48.0% yield).

[0302] 1 H 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 C NMR (101 MHz, DMSO-d6) δ 160.50, 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] + )

[0303] 11. Synthesis of AKG-16

[0304]

[0305] ZnCl2(11.2 g, 81.9 mmol) was added portionwise to pyridine (40 mL) followed by NaN3(8.90 g, 137 mmol) and 5-bromo-2-cyanopyridine (10.0 g, 54.6 mmol) at room temperature and the reaction mixture was heated to reflux at 120 °C for 2 h. After cooling the mixture to room temperature, it was diluted with water (200 mL), stirred at room temperature for 1 h, filtered and washed with water (200 mL). The filtered solid was collected and suspended in HCl (200 mL, 6 M) at room temperature for 2 h. The product was collected by filtration and washed with H2O. Dried under vacuum to give intermediate-4 (10.0 g, 81.3% yield) as a white solid.

[0306] 1 H NMR (400 MHz, DMSO-d6) δ 8.96 (s, 1H), 8.36 (dd, J = 8.4, 2.2 Hz, 1H), 8.18 (d, J = 8.4 Hz, 1H). MS (ESI+) m / z 225.9227.9 ([M+1] + )

[0307]

[0308] A mixture of intermediate-4 (10.0 g, 44.25 mmol) and Ca(OH)2(7.20 g, 97.35 mmol) in H2O (150 mL) and DMF (20 mL) was stirred at room temperature for 0.5 h and then (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. The mixture was diluted with H2O (40 mL) and extracted with EtOAc (2 x 50 mL). The combined extracts were washed with saturated brine, dried over Na2SO4and filtered. The solvent was removed under vacuum and the residue was purified using FC (eluent with 0 to 15% MeOH in DCM) to give the crude product. The crude product was further purified by RPFC (0 to 30% MeCN in 0.1% NH4HCO3 / H2O, C18, eluting intermediate-5 first, then intermediate-6) to give intermediate-5 (0.74 g, 5.6% yield) and intermediate-6 (0.25 g as a light yellow solid) as white solids.

[0309] 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, 0.6Hz, 1H), 4.87 (t, J=6.1Hz, 2H), 2.87 (t, J=6.1Hz, 2H), 2.17 (s, 6H). 13 C NMR (101MHz, DMSO-d6) δ163.74, 151.48, 145.51, 140.81, 124.40, 122.21, 57.73, 51.54, 45.29.MS (ESI+) m / z297.1, 299.1 ([M+1] + Intermediate-6: 1 H NMR (400MHz, DMSO-d6) δ8.98 (s, 1H), 8.38 (dd, J=8.4, 2Hz, 1H), 8.20 (d, J=8.4Hz, 1H), 5 .00 (t, J=6.4Hz, 2H), 2.75 (t, J=6Hz, 2H), 2.10 (s, 6H), MS (ESI+) m / z297.1, 299.1 ([M+1] + ).

[0310]

[0311] Freshly prepared intermediate-2 (1.68 g, 4.98 mmol) (from 1.44 g of (R)-3-(4-bromo-3-fluorophenyl)-5-(hydroxymethyl) using the procedure with intermediate-3) A mixture of oxazolidin-2-one, intermediate-5 (740 mg, 2.49 mmol), and K3PO4 (1.16 g, 5.48 mmol) in dioxane (50 mL) and H2O (5 mL) was purged with argon for 10 min. PdCl2 (182 mg, 0.25 mol) was added. The mixture was purged with argon again. It was then heated to 90 °C for 15 h. LCMS showed the reaction was complete. The mixture was concentrated under vacuum and the residue was diluted with H2O (200 mL) and extracted with EtOAc (2 × 200 mL). The combined extracts were washed with saturated brine, dried over Na2SO4, and filtered. The solvent was removed under vacuum, and the residue was purified using RPFC (eluent with 0 to 40% MeCN in H2O) to give product AKG-16 (520 mg, 49.0% yield) as a white solid.

[0312] 1H NMR (400 MHz, DMSO-d6) δ 8.95 (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] + )

[0313] 12. Synthesis of AKG-21

[0314]

[0315] A solution of Intermediate-2 (1.5 g, 4.5 mmol), Intermediate-6 (0.9 g, 3 mmol), Pd(dppf)Cl2(247 mg, 0.3 mmol) and K3PO4(1.3 g, 6 mmol) in dioxane (30 mL) and H2O (5 mL) was purged with argon for 10 min and heated to 100 °C for 15 h. On completion of the reaction (LCMS), it was concentrated in vacuo and the residue was diluted with H2O (100 mL) and extracted with EtOAc (2 x 50 mL). The combined extracts were washed with saturated brine, dried over Na2SO4and filtered. The solvent was removed in vacuo and the residue was purified using FC (eluent with 0 to 10% MeOH in DCM (10% of NH4OH)) to get AKG-21 (450 mg as white solid) in 35% yield.

[0316] 1H NMR (400 MHz, DMSO-d6) δ 9.01 (s, 1H), 8.36 (d, J = 8.4 Hz, 1H), 8.30 (d, J = 8.4 Hz, 1H), 7.81 (t, J = 8.8 Hz, 1H), 7.74 (dd, J = 13.6, 2.0 Hz, 1H), 7.55 (dd, J = 8.4, 2.0 Hz, 1H), 5.26 (t, J = 5.6 Hz, 1H), 5.08 (t, J = 6.4 Hz, 2H), 4.79 - 4.75 (m, 1H), 4.18 (t, J = 9.2 Hz, 1H), 3.93 - 3.89 (m, 1H), 3.74 - 3.68 (m, 1H), 3.62 - 3.56 (m, 1H), 2.80 (t, J = 6.4 Hz, 2H), 2.13 (s, 6H). 13 C NMR (101 MHz, DMSO-d6): δ 161.09, 158.64, 154.80, 152.10, (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.

[0317] 13. Synthesis of AKG-22

[0318]

[0319] To a mixture of Intermediate-7 (500 mg, 1.354 mmol) in H20 (2 mL) and dioxane (8 mL) was added Intermediate-2 (685 mg, 2.03 mmol), K3P04 (862 mg, 4.06 mmol) and (dppf)PdCI2 (99 mg, 0.135 mmol). The flask was evacuated and backfilled with argon. The mixture was then stirred at 90 °C for 16 h. Water (20 mL) was added and extracted with EtOAc (2 x 20 mL). The organic phase was washed with brine, dried over Na2S04, filtered and concentrated. The residue was purified by silica gel column chromatography (Biotage, 40 g silica gel column @ 30 mL / min, eluting with 0 to 100% EtOAc in petroleum ether) to give the desired product AKG-22-1 (450 mg, yield: 66%) as a grey solid.

[0320]

[0321] To a mixture of AKG-22-1 (450 mg, 0.9 mmol) in DCM (8 mL) was added 4 M HC1 / dioxane (2 mL). The mixture was then stirred at room temperature for 5 hours. The solvent was removed under vacuum to give the desired product AKG-22 as a grey solid (390 mg, yield: 99%).

[0322] 1 H NMR (400 MHz, DMSO-d6) δ 9.03 (s, 1H), 8.39 (d, J = 8.0 Hz, 1H), 8.32 (d, J = 8.0 Hz, 1H) 8.19 (br s, 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 (400 MHz, DMSO-d6) δ (161.07, 158.63), 154.72, 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

[0323] 14. Synthesis of AKG-23

[0324]

[0325] To intermediate-8 (1.0 g, 2.71 mmol) in 20 mL of 1,4-dioxane and 5 mL of H2O was added intermediate-2 (4.86 mmol, 1.63 g), K3PO4 (1.14 g, 5.42 mmol) and (dppf)PdCl2 (0.23 g, 0.27 mmol) and the mixture was stirred at 100 °C for 16 hours. After consumption of starting material, 100 mL of saturated NaHC03was added. The aqueous phase was extracted with EtOAc (3 x 30 mL), the combined organic extracts were washed with H2O, concentrated under vacuum and purified by FC to give the desired compound AKG-23-1 (1.0 g, 70% yield).

[0326]

[0327] To AKG-23-1 (1.0 g, 2 mmol) in 30 mL DCM was added 1 mL of HC1 (4 M in 1,4-dioxane) and the mixture was stirred for 1 hour. After consumption of starting material, the mixture was filtered to give crude product. The crude was stirred in 3 mL MeOH for 1 hour, filtered to give the desired product AKG-23 (0.53 g, 63% yield) as a white solid.

[0328] 1 H NMR (400 MHz, DMSO-d6) δ 8.97 (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). 13 C 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, 149.85, 154.82, [158.57, 161.02], 164.50.

[0329] 15. Synthesis of AKG-24

[0330]

[0331] A mixture of intermediate-2 (1.66 g, 4.98 mmol), intermediate-9 (800 mg, 2.49 mmol) and K3PO4 (1.16 g, 5.48 mmol) in dioxane (50 mL) and H2O (5 mL) was purged with argon for 10 minutes and (dppf)PdCl2 (182 mg, 0.25 mmol) was added. The mixture was purged again with argon. It was then heated to 90 °C for 15 hours. LCMS showed the reaction was complete; concentrated in vacuo and the residue was diluted with H2O (200 mL) and extracted with EtOAc (2 x 200 mL). The combined extracts were washed with saturated brine, then dried over Na2SO4 and filtered. The solvent was removed in vacuo and the residue was purified using RPFC (eluent with 0 to 40% MeCN in H2O) to give the product AKG-24 (440 mg, 39.0% yield) as a white solid.

[0332] 1 H NMR (400 MHz, DMSO-d6) δ 8.95 (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) δ 164.13, 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] + )

[0333] 16. Synthesis of AKG-25

[0334]

[0335] 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. Diluted with H2O (40 mL) and extracted with EtOAc (2 x 100 mL). The combined extracts were washed with saturated brine, then dried over Na2SO4and filtered. The solvent was removed in vacuo and the residue indicated the presence of two regioisomers of N-alkylation. The isomers were separated using FC (eluent with 0 to 15% MeOH in DCM) to give Intermediate-10 (0.98 g, 31.6% yield) as a white solid.

[0336] 1 H NMR (400 MHz, CDC13) 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] + ).

[0337]

[0338] A mixture of Intermediate-10 (0.74 g, 2.4 mmol), Intermediate-2 (1.62 g, 4.8 mmol) and K3PO4(1 g, 4.8 mmol) in dioxane (30 mL) and H2O (5 mL) was purged with argon for 10 min and Pd(dppf)Cl2(175 mg, 0.24 mmol) was added. The mixture was purged again with argon and heated to 90 °C for 15 h. Concentrated in vacuo and the residue was diluted with H2O (80 mL) and extracted with EtOAc (2 x 100 mL). The combined extracts were washed with saturated brine, then dried over Na2SO4and filtered. The solvent was removed in vacuo and the residue was purified using FC (eluent with 0 to 15% MeOH in DCM) to give product AKG-25 (0.73 g, 69.5% yield) as a grey solid.

[0339] 1H NMR (400 MHz, DMSO-d6) δ 8.94 (s, 1H), 8.24-8.26 (m, 1H), 8.19-821 (m, 1H), 7.78-7.70 (m, 2H), 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, 12260, 119.12, 118.99, 114.46, 105.98, 105.70, 73.95, 62.07, 55.96, 51.65, 46.47, 45.53, 27.21. MS (ESI+) m / z 442.1 ([M + 1] + )。

[0340] 17. Synthesis of AKG-26

[0341]

[0342] 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 and poured into an ice-water bath and extracted with EA (2 x 200 mL). The combined organic phase was washed with brine (2 x 50 mL) and dried over Na2SO4. After removal of the solvent, the crude product with N-alkylated regioisomers was purified by FC (PE / EA = 1 : 10) to give intermediate-11 (1.70 g, 22.65%) as a white solid.

[0343] 1H NMR (500 MHz, CDC13) 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] + )

[0344]

[0345] 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)Cl2(0.15 g, 0.20 mmol) was suspended in 1,4-dioxane: water (12 mL, 6: 1). The reaction was stirred at reflux for 16 h. The mixture was partitioned between EtOAc (2 x 100 mL) and water, washed with brine, dried over Na2S04and filtered. The residue containing regioisomers was purified using FC eluted with (DCM / MeOH = 20 / 1) until the solvent was removed to give AKG-26 (0.54 g, 56.04%) as a grey solid.

[0346] 1 H NMR (500 MHz, DMSO-d6) δ 8.95 (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). 13C 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 = 3.2 Hz), 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] + )

[0347] 18. Synthesis of AKG-27

[0348]

[0349] To a solution of intermediate-4 (6.3 g, 27.87 mmol) in DMF (42 mL) was added BocNH(CH2)3Br (16.6 g, 69.71 mmol) and K2CO3(11.1 g, 80.02 mmol) at 80 °C for 3 h. The reaction was cooled and poured into an ice-water bath and extracted with EtOAc (2 x 200 mL). The organic phase was washed with brine (2 x 50 mL), dried over Na2SO4and filtered, the solvent was evaporated under reduced pressure. The crude product with N-alkylated regioisomers was purified by FC (PE / EA = 2: 1) to afford intermediate-12 (14 g, 13.1%) as a yellow solid.

[0350] 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, 2H), 3.03 (q, J = 12.4 Hz, 2H), 2.15 - 2.08 (m, 2H), 1.37 (s, 9H) ppm. MS (ESI+) m / z 383.0 ([M + 1] + ).

[0351]

[0352] A solution of intermediate-12 (0.83 g, 2.15 mmol), NaHC03(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). The mixture was bubbled with N2for 5 min, then charged with Pd(dppf)Cl2(0.078 g, 0.095 mmol). The mixture was stirred at 90 °C for 15 h, then cooled to room temperature. The mixture was partitioned between EtOAc (2 x 100 mL) and water. The organic layer was dried over Na2S04, filtered and concentrated. The filtrate was concentrated and purified by silica gel column chromatography on silica gel (DCM / MeOH = 20 / 1) to give AKG-27-1 as a white solid (0.75 g; 66.9%).

[0353] 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 (1n, 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] +) .

[0354]

[0355] To a solution of AKG-27-1 (0.9 g, 1.75 mmol) in anhydrous DCM (16 mL) was added HC1 in dioxane (4.0 mL) at room temperature under N2atmosphere. The reaction mixture was stirred at the same temperature for 6 h and cooled to room temperature. The mixture was evaporated under reduced pressure to give AKG-27 as a light yellow solid (0.65 g, 82.5%).

[0356] 1H 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] + ).

[0357] 19. Synthesis of AKG-28 to 31

[0358] 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 room temperature for 2 h. The mixture was washed with water (2 x 30 mL) and brine (2 x 30 mL), dried over Na2S04, filtered and concentrated to give intermediate-13 (11.4 g, yield 99%). MS (ESI+) m / z 368 ([M + 1] + ).

[0359] To a solution of intermediate-13 (11.4 g, 31 mmol) in DMF (200 mL) was added potassium 1,3-dioxoisoindoline-2-ide (6.02 g, 32 mmol). The mixture was stirred at 90 °C overnight. The mixture was cooled and poured into water (1000 mL), stirred for 0.5 h. The precipitate was collected and dried under vacuum to give intermediate-14 (11 g, yield 85%). MS (ESI+) m / z 419 ([M + 1] + ).

[0360] To a solution of intermediate-14 (11 g, 26.3 mmol) in EtOH (150 mL) was added NH2NH2-H20 (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, yield 100%). MS (ESI+) m / z 289 ([M + 1] + ).

[0361] To a solution of Intermediate-15 (7.6 g, 26.4 mmol) in THF (50 mL) and water (50 mL) was added (Boc)20 (6.9 g, 32 mmol) and K2CO3 (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 x 50 mL). The combined organic extracts were washed with brine (2 x 50 mL), dried over Na2SO4, filtered and concentrated. The residue was purified by FC (Biotage, 80 g silica gel column @ 65 mL / min, eluting with 0 to 60% EtOAc in petroleum ether for 30 min) to give Intermediate-16 (7.8 g, yield 75%). MS (ESI+) m / z 411 ([M+23] + )..

[0362] 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 argon for 10 min, then (Ph3P)2PdCl2 (1.06 g, 1.5 mmol) was added. The mixture was purged again with argon and stirred at 90 °C overnight. The mixture was cooled and diluted with water (300 mL) and extracted with EtOAc (3 x 100 mL). The combined extracts were washed with brine (2 x 50 mL), dried over Na2SO4, filtered and concentrated. The residue was purified by FC (Biotage, 80 g silica gel column @ 65 mL / min, eluting with 0 to 60% EtOAc in petroleum ether for 30 min) to give Intermediate-18 (6.2 g, yield 70%). MS (ESI+) m / z 459 ([M+23] + )..

[0363] Procedure C: A mixture of one of Intermediate-5 / 8 / 9 / 10 / 11 (1.0 eq), one of Intermediate-18 / 19 (1.5 eq), Pd(dppf)Cl2DCM (0.1 eq) and K3PO4 (2.0 eq) in dioxane / H2O (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 Compound AKG-28-1 / AKG-29-1 / AKG-30-1 / AKG-31-1 / AKG-38 / AKG-39 / AKG-40.

[0364] To a solution of one of compounds AKG-28-1 / AKG-29-1 / AKG-30-1 / AKG-31-1 in DCM (1 mL / 100 mg) was added 3N HC1 in EtOAc (20 eq). The mixture was stirred for 2 hours and then filtered. The solid was dried under vacuum or lyophilized to obtain the final compound AKG-28 / AKG-29 / AKG-30 / AKG-31 (35 to 44% yield for the two steps).

[0365]

[0366] This product was obtained as a white solid using procedure C from intermediate-5 and intermediate-18 (0.35 g, 35% yield).

[0367] 1 H NMR (500 MHz, DMSO-d6) δ 10.53 (s, 1H), 8.97 (s, 1H), 8.37 (s, 3H), 829 (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 = 13.6, 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, D20) 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] + )

[0368]

[0369] This product was obtained as a white solid using procedure C from intermediate-5 and intermediate-18 (0.35 g, 35% yield).

[0370] 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) δ 161.52, 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] + )

[0371]

[0372] This product was obtained as a white solid using procedure C from intermediate-10 and intermediate-18 (0.36 g, 40% yield).

[0373] 1H NMR (400 MHz, DMSO-d6) δ 10.94 (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 CNMR (101 MHz, D20) 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), 50.52 (s), 47.68 (s), 42.89 (s), 42.16 (s), 23.74 (s) ppm. MS (ESI+) m / z 441 ([M + 1] + ).

[0374]

[0375] Procedure C was used. This product was obtained as a white solid from intermediate- 11 and intermediate-18 (0.36 g, 42% yield).

[0376] 1HH NMR (400 MHz, DMSO-d6) δ 10.10 (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, D20) δ 163.04 (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] + ).

[0377] 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 eq) followed by acetyl chloride (AcCl, 2.6 g, 2.74 mL, 39.58 mmol, 1.5 eq) and 4-N,N-dimethylaminopyridine (DMAP, 0.028 g, 2.64 mmol, 0.01 eq) at 0-5 °C under N2. The resulting reaction mixture was then stirred at 0-5 °C for 2 h. When TLC and LCMS showed the completion of the reaction, the reaction mixture was quenched with H2O (100 mL). The two layers were separated and the aqueous layer was then extracted with CH2Cl2(2 x 50 mL) and the combined organic extracts were washed with H2O (2 x 100 mL) and saturated aqueous NaCl (100 mL), dried over MgSO4and concentrated in vacuo. 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, yield 75%). MS (ESI+) m / z 332 ([M+1] + ) at 0-5 °C under N2.

[0378] 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) 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 showed the completion of the reaction, the reaction mixture was cooled to room temperature 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 x 50 mL). The combined organic extracts were washed with water (2 x 50 mL) and saturated aqueous NaCl (50 mL), dried over MgSO4and concentrated in vacuo. 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, yield 88.7%). MS (ESI+) m / z 379 ([M+1] + ) at 0-5 °C under N2.

[0379] 20. Synthesis of AKG-38 to 40.

[0380]

[0381] Procedure C was used. This product was obtained as a white solid from Intermediate-5 and Intermediate-19 (0.48 g, 60% yield).

[0382] 1 H 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 = 60 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 = 5.6 Hz, 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, 114.66, 106.18, 105.90, 72.34, 57.71, 51.45, 47.67, 45.25, 41.87, 22.92 ppm. MS (ESI+) m / z 469.2 ([M + 1] + )

[0383]

[0384] Procedure C was used. This product was obtained as a white solid from Intermediate-5 and Intermediate-19 (0.48 g, 60% yield).

[0385] 1 H 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 = 60 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 = 5.6 Hz, 2H), 2.90 (t, J = 6.0 Hz, 2H), 2.19 (s, 6H), 1.85 (s, 3H) ppm. 13C 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] + ).

[0386]

[0387] This product was obtained as a white solid from Intermediate-11 and Intermediate-19 using Procedure C (0.36 g, 50% yield).

[0388] 1 H NMR (400 MHz, DMSO-d6) δ 8.95 (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] + )

[0389] Example 2. Mycobacterium tuberculosis in vitro activity assay

[0390] The broth microdilution MIC method used is described in Collins et al., 1997 and Gruppo et al., 2006. The MIC or minimum inhibitory concentration of the compound after overnight incubation prevents visible growth of the bacteria.

[0391] In brief, MICs were determined by broth microdilution assay with 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 from blue to pink in the presence of mycobacterial growth activity in broth media.

[0392] In brief, 7H9 complete medium was prepared by adding Middlebrook 4.7 g of 7H9 broth powder (Millipore Sigma Cat# M0178), 2 mL of glycerol and 898 purified water in a 1 L flask and mixed until dissolved, and then adding 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 made at a concentration of 10 mg / mL in DMSO and then further diluted with DMSO to 80 pg / mL, or forty-fold to the desired 2 pg / mL starting concentration. A series of nine 1 :2 dilutions were prepared by adding 50 pi of drug solution in the first well to 50 pi of DMSO in the subsequent well in a drug preparation plate, and this process was carried out to the next eight wells. Stocks of M. tuberculosis (M. tb) H34Rv and M. tb Erdman strains were diluted from their 3 to 4 x 10 7 CFU / mL initial concentration to 5 x 105 CFU / mL final concentration, by pipetting up and down with a multichannel pipettor.

[0393] Prepare assay plates by inoculating the media with 100 μΐ of 5 x 10 5 Prepare assay plates by inoculating the media with 100 μΐ of 5 x 10

[0394] Two unique drug susceptible strains of Mtb (M. tb Erdman and M. tb H37Rv) were used for the assay. The MIC assay 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). In general, a 4-fold shift in MIC for two wells is considered significant. For PA-824 (positive control), a 4-fold shift in MIC is expected to occur.

[0395] The MIC was consistent or differed by only one 2-fold dilution by Alamar Blue (MABA) reading or by optical density reading (OD600) measurement. All tested compounds, except one compound AKG-40, showed MIC values consistent for both Mtb Erdman and H37Rv or within one 2-fold dilution range; this showed MIC values (1 to 2 ug / mL) higher for Erdman and MIC value (0.5) higher for H37Rv. The difference can be due to slower growth (lower OD reading) on Erdman plates.

[0396] The expected MIC value for linezolid was shown to be 2 pg / mL, for sitafloxacin 0.25 pg / mL, and for bedaquiline 0.125 pg / mL. These values are consistent with past MIC data and published values (Ruiz et al. Antimicrob. Agents Chemother. 2019, Mar 27;63(4), 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 an MIC of 0.03 to 0.015 pg / mL, significantly more active than sitafloxacin. Molecules containing an acetamide group AKG-39, a oxazolidinone analog, had an MIC of 0.5 pg / mL and AKG-40 had an MIC of 1 to 0.5 pg / mL. AKG-38, with an MIC of 0.06 pg / mL, also showed much greater activity than sitafloxacin.

[0397] In Molecules with an amine or acetamide group at the C5 position of the oxazolidinone were more active (AKG-3 vs. sitafloxacin, AKG-28 or AKG-38 vs. AKG-16, AKG-39 vs. AKG-24, AKG-40 vs. AKG-26), and compounds with aminoalkyl side chains on the tetrazole showed favorable activity. In Substitution of the primary amine at the C5 position of the oxazolidinone with a tert-butyloxycarbonyl amino (Boc-NH) (AKG-28-1 vs. AKG-28) or acetamide (AKG-28-1 vs. AKG-38) resulted in decreased activity. Compounds containing a dimethylaminoalkyl side chain were particularly superior when compared to aminoethyl or diethylaminoethyl analogs (AKG-16 vs. AKG-24, AKG-28 vs. AKG-29, AKG-30 vs. AKG-31). Likewise, 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). Analogues substituted on the 2’ position of the tetrazole were more active than those substituted on the 1’ position (AKG-16 vs. AKG-21, AKG-23 vs. AKG-22).

[0398]

[0399] Table 2

[0400]

[0401]

[0402] Example 3. In vitro cytotoxicity assay against human kidney and human liver cells

[0403] Compounds were tested in vitro in a series of 10 dilutions to determine the IC50 in either African green monkey kidney (Vero; ATCC #CCL81) or human hepatocyte / liver (HepG2; ATCC #HB8065) cells. Since these molecules are generally expected to be non-toxic, a positive control of doxorubicin was included in all studies. Data were reported as full cell viability curves as well as the calculated IC50 value for each compound.

[0404] Adherent cells were grown to approximately 80% confluence. Cells were trypsinized by the addition of 0.25% trypsin-EDTA (Gibco #25200-072) and subsequently spun down and 5 ml of growth media (MEM media; Corning #10010CM) was added to disperse the cells. Cell density was determined using a hemocytometer. Growth media (MEM media containing 10% FBS; Corning #35015CV) was added to the cells to adjust to the appropriate cell concentration. Then, 200 μΐ of cells (5,000 cells / well) were added to 96-well clear bottom plates (Costar #9804) and incubated in the plates for 24 hours at 37°C in a humidified incubator with 5% C02.

[0405] A series of dilutions of the test compounds was prepared using growth media as the solvent (Table 2). These compounds were supplied as a sterile aqueous solution of the hydrochloride salt at a concentration of 5 mg / ml. To prepare the dilutions, each drug stock was warmed to room temperature, vortexed and checked visually for precipitate. If solid drug was present, the stock was heated on a 60°C water bath and subsequently cooled to near room temperature. Based on the treatment concentration, a 20x working stock was prepared by serial dilution. These were further diluted in growth media to 1x to achieve a maximum test drug concentration of 250 μg / ml.

[0406] Compounds were added to wells in a series of 1:2 dilutions, starting at an initial concentration of 250 μg / ml for each compound, by aspirating the old culture medium and replacing it with 200 μl of medium containing the drug. Plates were incubated at 37°C for 72 h in a humidified incubator with 5% CO2. At the end of the compound incubation period, the culture medium in each well was replaced with 100 μl of 1×PrestoBlue cell viability reagent (ThermoFisher catalog #A13261). Plates were incubated at 37°C for 30 min to 2 h in a humidified incubator with 5% CO2. Readings were taken at 30, 60, and 120 min. Fluorescence at 560 nm excitation and 590 nm emission was read using a SpectraMax M5 plate reader (Molecular Devices). Background was corrected by subtracting the RFU of the control wells (containing only culture medium) from all sample readings. The percentage of cytotoxicity was calculated using the following formula:

[0407] %Cytotoxicity = [(RFU)] 培养基 -RFU 处理 ) / RFU. 培养基 ]×100%

[0408] The IC50 is determined using the following formula with GraphPad Prism:

[0409] Y=100 / (1+10^((LogIC50-X)*HillSlope)))

[0410] Table 3

[0411]

[0412]

[0413] Unexpectedly, in Most analogues containing a hydroxyl group on the C5 side chain of the zolidinone ring (which mimic the substituents of terizolamide, the active metabolite of terizolamide phosphate) are the most hepatotoxic, exhibiting single-digit IC50s against the HepG2 hepatocyte line. Terizolamide is currently the most active approved treatment for MRSA. Azoxyl ketones, and structurally similar to compounds in the tetrazolium D-ring, pyridyl C-ring, and aryl B-ring described herein (see [link to article]). Figure 6 However, when compared with a compound having an amino or acetamide group at the same position on the C5 side chain... Toxicity to hepatocytes increases selectivity index relatively low for those with a hydroxyl group on the C5 side chain (AKG-23, AKG-25, AKG-26, and AKG-27) when compared to the oxazolidinones (AKG 28-31, AKG 38-40, and AKG-3).

[0414] Example 4. Determination of Selectivity Index

[0415] The selectivity index (SI) was calculated to determine the relative inhibitory activity of the compounds against two strains of Mycobacterium tuberculosis, Erdman and H37Rv, compared to mammalian cells, either VERO or human hepatocyte-derived (HepG2) cells, as described in Experimental Examples 2 and 3, respectively. A high SI is preferred because it indicates a drug that preferably kills the bacterial strain of tuberculosis at a concentration that is less harmful to normal cells in the body. The selectivity index was calculated using the following formula:

[0416] SI = IC50 50，哺乳动物 / MIC 细菌

[0417] where the bacteria is either the Erdman or H37Rv strain of Mycobacterium tuberculosis and the mammalian cells are either the VERO or HepG2 cell line.

[0418] If the IC50 is greater than the highest value tested against VERO or HepG2 cells, then the SI is shown as greater than (>) the ratio calculated using that highest concentration. Likewise, if the MIC against either the Erdman or H37Rv strain is greater than the highest concentration of drug tested (8 mg / ml), then the SI is shown as less than (<) the ratio calculated using that highest concentration. Calculations where both values are higher than the highest concentration tested are shown as not determined (nd). The results are shown in Table 4. The SI is not directly correlated to the molecular activity in either the mycobacterial strain or the mammalian cell line, and the increase in efficacy in the mycobacterial strain is not directly correlated to an increase in toxicity against the mammalian cell line. For example, AKG-38 exhibits nanomolar MICs against both strains of Mycobacterium tuberculosis, while AKG-38 is relatively inactive against both the VERO and HepG2 cell lines compared to the other molecules in the group, giving AKG-38 a high SI. The same is true for AKG-28. Of note, both molecules, AKG-28 and AKG-38, have a dimethylaminoethyl substituent at the 2' position of the tetrazole ring.

[0419] Table 4

[0420]

[0421]

[0422] In some embodiments, the compound of interest has an SI index against Erd / HepG2 and H37Rv / HepG2 higher than 100, higher than 200, higher than 300, higher than 400, higher than 500, higher than 1000, higher than 1500, higher than 2000, higher than 2500, higher than 3000, higher than 3500, higher than 4000, higher than 4500, higher than 5000, higher than 5500, higher than 6000, higher than 6500, from 100 to 7000, from 100 to 6000, from 100 to 5000, from 100 to 4000, from 100 to 3000, from 100 to 2000, from 100 to 1000, from 100 to 900, from 100 to 800, from 100 to 700, from 100 to 600, from 100 to 500, from 100 to 400, from 100 to 300, from 100 to 200, from 200 to 7000, from 200 to 6000, from 200 to 5000, from 200 to 4000, from 200 to 3000, from 200 to 2000, from 200 to 1000, from 200 to 900, from 200 to 800, from 200 to 700, from 200 to 600, from 200 to 500, from 200 to 400, from 200 to 300, from 300 to 7000, from 300 to 6000, from 300 to 5000, from 300 to 4000, from 300 to 3000, from 300 to 2000, from 300 to 1000, from 300 to 900, from 300 to 800, from 300 to 700, from 300 to 600, from 300 to 500, from 300 to 400. In some embodiments, the compound of interest has an SI index against Erd / HepG2 and H37Rv / HepG2 ranging from 100 to 1700, from 200 to 1700, from 300 to 1700.

[0423] compounds having a hydroxyl group on the C5 side chain compared to Compounds having 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 show relatively higher SI. In addition, the specific tetrazole substitution also improves the SI, with dimethylaminoethyl substitution at the 2' position of the tetrazole ring (AKG-28 and AKG-38) being superior to methyl, diethylaminoethyl, aminoethyl, or dimethylaminopropyl substitution at that same position. Moving the dimethylaminoethyl group to the 1' position of the tetrazole ring (compound AKG-21 vs. AKG-28) unexpectedly results in a significant loss of activity against Mycobacterium tuberculosis.

[0424] Example 5. In vitro activity assay against methicillin-resistant Staphylococcus aureus (MRSA).

[0425] The lead compounds The activity of the oxazolidinone inhibitors was shown to have sufficient potency against the Gram-positive bacteria, methicillin-resistant Staphylococcus aureus (MRSA), such that it was then delivered in the form of a liposome to treat the methicillin-resistant Staphylococcus aureus. In some embodiments, the MIC of two of the three evaluation strains is less than 6 μg / mL. In some embodiments, the MIC of two of the three evaluation strains is less than 2 μg / mL. Less than 2 μg / mL is more preferred.

[0426] Three strains of Staphylococcus aureus were grown overnight at 37°C in ambient atmosphere on tryptic soy agar plates supplemented with 5% sheep blood cells. The cultures were aseptically swabbed and transferred to test tubes of sterile water and the optical density was adjusted to 0.5 at 600 nm. The cultures were then diluted 1 : 100 to deliver approximately 5 x 10 5 After incubation, the MIC of the test article was determined by whether growth was present in each well. The MIC analysis was performed in triplicate.

[0427] 0.5 μg / ml as described in 6,379. Interestingly, all molecules with a primary amine modification at R2 of the oxazolidinone ring (AKG-3, AKG-28, AKG-29, and AKG-30) showed negligible activity (>50 μg / ml) against all three MRSA strains. The molecules with an acetamide group at the same position (AKG-38, AKG-39, and AKG-40) were 3 to 9 times less active against the three MRSA strains than the parent drug, tedizolid.

[0428] Table 5

[0429]

[0430] Example 6: Liposomal Compositions.

[0431] General Protocol

[0432] 1. The lipid components (phospholipid (PhL), cholesterol, and optionally PEG-lipid derivative and / or lipid fluorescent label) are combined in an amount equal to one-tenth of the volume (V) calculated to obtain a lipid suspension with about 60 mM of phospholipid in 100% ethanol and stirred at a temperature of 65 to 68°C until the lipids are completely dissolved.

[0433] The neutral phospholipid can include diacylphosphatidylcholine, dialkylphosphatidylcholine, sphingomyelin, and diacylphosphatidylethanolamine. Hydrogenated soy phosphatidylcholine, distearoylphosphatidylcholine, and egg sphingomyelin are some preferred phospholipids.

[0434] ​The PEG-lipid component can include PEG (molecular weight 2,000)-distearoyl glycerol (PEG-DSG), l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-2000] (PEG-DSPE), or N-palmitoyl-sphingosyl-1- {succinyl[methoxy(polyethylene glycol) 2000]} (PEG-ceramide). The PEG-lipid component can also vary in molecular weight from 1,500 to 6,000 g / mol, but is preferably about 2,000 MW.

[0435] The lipid fluorescent label can include l,l'-dioctadecyl-3,3,3',3'- tetramethylindocarbocyanine-5,5'-disulfonic acid (DiIC18(3)-DS), l,l'-dioctadecyl-3,3,3',3'- tetramethylindodicarbocyanine-5,5'-disulfonic acid (DiIC8(5)-DS), l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-(cyanine 7) (18:0 Cy7 PE), l,2-distearoyl-sn-glycero-3-phosphoethanolamine- N-[amino(polyethylene glycol)-2000]-N-(cyanine 7) (DSPEPEG(2000)-N-Cy7), l,2-distearoyl-sn- glycero-3-phosphoethanolamine-N-(cyanine 5) (18:0 Cy5 PE), l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[amino(polyethylene glycol)-2000]-N-(cyanine 5) (DSPEPEG(2000)-N-Cy5), l- oleoyl-2-[12-[(7-nitro-l,2,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-glycero-3-phosphocholine (18: 1 to 12:0 NBD PC).

[0436] 2. The ethanol lipid solution is combined with a volume V of a capture agent solution (0.25 to 0.5 M ammonium sulfate or IN sucrose octasulfate triethylammonium) while stirring at 65 to 68 °C until a uniform suspension is obtained.

[0437] Potential capture agents can include, but are not limited to, diethylammonium or triethylammonium salts of sucrose octasulfate, ammonium sulfate, ammonium citrate, citric acid, dextran sulfate, polyvinyl sulfonate, or ammonium salts 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. Ammonium salts are generally used and can include ammonium itself, mono-, di-, or trialkyl-ammonium salts.

[0438] ​3. The lipid suspension is extruded 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) at 65 to 68 °C under a pressure of 400 to 450 psi using a thermobarrel extruder (Lipex, Canada) at least three times. When using two 100 nm membranes in a 100 ml Lipex extruder, the extrusion pressure is typically 260 to 300 psi. The resulting liposomes have a Z-average particle size (diameter) Xz of about 80 to about 130 nm and a PDI of less than 0.1.

[0439] 4. The extruded lipid suspension, known to contain unilamellar and / or oligolamellar liposomes, is cooled in a refrigerator (2 to 8 °C) and filtered through a 0.2 micron polyethersulfone (PES) membrane filter under positive pressure.

[0440] 5. An aliquot of the extruded, filtered liposome suspension thus prepared is chromatographed on a gravity-fed Sepharose CL-4B size exclusion column (eluent - Type 1 water) to purify the liposomes from the extraliposomal capture agent. Purified liposomes are collected near the void volume fraction of the column. For scale-up studies, this step is performed using tangential flow filtration (TFF) over a hollow fiber cartridge (Repligen Spectrum MicroKros PS or mPES membrane with a MWCO of 500 KDa), achieving an 8 to 10 volume exchange with Type 1 or USP "water for injection" endotoxin-free water (or until the conductivity of the liposome suspension decreases to less than 200 μ8 / ηηι).

[0441] 6. The lipid concentration in the purified extruded liposome preparation is determined using HPLC with UV detection by measuring the concentration of cholesterol and correcting for the known phospholipid / cholesterol molar ratio. Alternatively, the phospholipid content is directly quantified using the spectrophotometric blue phosphomolybdate method.

[0442] 7. Dissolve drug as hydrochloride salt (e.g., AKG-3 and AKG-5 as mono-hydrochloride salt, AKG-28 and AKG-29 as di-hydrochloride salt) in Type 1 or endotoxin-free purified water at a drug concentration of 5 to 20 mg / ml. Add an equivalent amount of HC1 to drug prepared as free base (e.g., AKG-16, AKG-38). If necessary, adjust the pH of the solution to pH 2.5 to 5.5 using 1 N NaOH, HC1, or Tris base solution, and filter the solution through a 0.2 micron PES filter under positive pressure. If necessary, verify the drug concentration in the stock solution so prepared by HPLC with UV detection at 305 nm.

[0443] 8. Combine the purified liposomes and drug stock solution of Step 5 in the presence of an osmotic agent (typically dextrose) and water in amounts necessary to provide the desired drug to phospholipid (DL) ratio, drug concentration of 1.5 to 3.3 mg / ml, and osmoticum equivalent to the measured osmoticum of the Step 2 capture solution. Optionally, add a buffer at the desired pH (typically pH 4 to pH 7). In some cases, the amount of osmotic agent added (e.g., about 45 g / L dextrose) provides an osmoticum less than the measured osmoticum of the capture solution, and loading is achieved at drug of 6 to 8 mg / ml.

[0444] 9. Incubate the drug-liposome mixture at 65 to 68 °C for about 15 to 20 minutes with constant stirring and quick-chill on ice. After 5 to 10 minutes, bring the mixture to ambient temperature, and adjust to 0.1 M NaCl by adding a calculated amount of 3 M NaCl stock solution.

[0445] 10. Purify the drug-loaded liposomes from unencapsulated drug by size exclusion chromatography (SEC) on a Sepharose CL-4B column by gravity feed, eluent - 10 mM HEPES buffer pH 7.0 in 140 to 144 mM NaCl (HBS-7). Collect liposome fractions near the void volume of the column. For scale-up studies, purify and buffer exchange using 10 volumes of HBS-7 buffer using TFF as described under item 5 above. Typically, about 8 volumes of exchange are used during scale-up. Optionally, concentrate the purified liposomes by continuing the TFF process in the absence of buffer feed. Sterile filter the purified drug-loaded liposomes using a 0.2 micron sterile PES filter under positive pressure, and store in a freezer (2 to 8 °C).

[0446] 11. Drug and lipid concentrations in the purified drug-loaded liposome preparation are determined by HPLC. Alternatively, phospholipids are quantified using spectrophotometry (blue phosphomolybdate) and drug is quantified by UV absorbance (302 to 305 nm) in liposome samples dissolved in 70% isopropanol-0.1 N HC1 in the presence of 6.5 mg / ml sodium dodecyl sulfate. Encapsulation efficiency is determined as follows:

[0447] EE, % = DL / DL0 * 100%

[0448] where DL0 is the ratio of drug to phospholipid in the liposome loading mixture prior to SEC or TFF purification, and DL is the ratio of drug to phospholipid in the drug-loaded liposomes after purification (step 10).

[0449] 12. Mean liposome size (Z-average diameter, Xz) and polydispersity index (PDI) are determined by dynamic laser light scattering using the cumulative method on a Zetasizer mu-V, Zetasizer Nano, or zeta sizer Pro (Malvern Panalytical, US).

[0450] Example 7. In vivo stability and blood clearance of liposomes.

[0451] Drug encapsulation stability and blood clearance of liposomes encapsulating compounds of the disclosure are investigated in mice according to the following general protocol. Mice (three per group) of a given laboratory strain (C3H females or CD-1 males) are injected with drug-loaded liposomes at a dose of 9 mg drug / kg body weight via tail vein. At time points 1 and 2, blood samples are taken from the retro-orbital sinus and the animals are sacrificed. Typically, blood sampling time points include 5 minutes, 1 hour, 6 hours, and 24 hours after injection. Plasma is isolated by centrifugation, extracted with acidified isopropanol optionally containing a solubilizer (sodium octane sulfonate), and analyzed for drug and lipid (when liposomes incorporate the lipid label DiIC18(3)-DS) by HPLC. Blood clearance of liposomal drug is expressed as a percentage of the injected dose remaining at a given time point. In vivo stability of drug encapsulation is assessed by the percent change (decrease) in the DL ratio in plasma at a given time point compared to the pre-injection DL value.

[0452] Example 8. Loading of AKG-3, AKG-5, and AKG-16 into liposomes at different pH

[0453] Sucrose octasulfate trimethylammonium capture agent solution was prepared by passing a solution of commercially available sucrose octasulfate potassium heptahydrate (40.2 g in 145 ml water) through a 500 ml Dowex 50W x8 100-200 mesh ion exchange column (in the hydrogen form) and titrating the resulting sucrose octasulfate free acid form to pH 6.2 with neat triethylamine. The concentration of sucrose octasulfate triethylammonium (TEA-SOS) (1 N corresponding to 0.125 M sucrose octasulfate) was assessed from the amount of triethylamine consumed in the titration. Residual potassium was assessed by the addition method using a Horiba LAQUATwin K-11 potassium analyser and was less than 0.1% of the initial potassium amount.

[0454] Liposomes with 1 N sucrose octasulfate trimethylammonium (TEA-SOS) as capture agent consisting of hydrogenated soy phosphatidyl choline (HSPC) (Lipoid, Germany), cholesterol (3:2 molar ratio) and methoxy poly(ethylene glycol) ether of 1,2-distearoyl glycerol (PEG-DSG, PEG molecular weight 2000, NOF, Japan) (0.5 mol.% of HSPC) were prepared essentially as described in the general protocol above. The drug loading step was performed at a DL ratio (DL0) of 500 g / mol PhL in the presence of 16 mM morpholinoethanesulfonic acid (MES)-4 mM sodium citrate buffer (pH range 4.3 to 7.1) and without addition of any buffer substance (pH 5.2 to 5.9). All drugs were encapsulated into liposomes with high efficiency (more than 98%, except AKG-16 which was loaded with 93.3% efficiency at pH 4.38) over the pH range of the study Figure 1 ). Efficient encapsulation did not require the addition of a buffer substance.

[0455] Example 9. Encapsulation of AKG-3, AKG-5 and AKG-16 into liposomes with TEA-SOS capture agent at different DL ratios.

[0456] Liposomes with 1 N TEA-SOS as a trapping agent consisting of HSPC, cholesterol (3:2 molar ratio) and PEG-DSG (0.5 mol.% of HSPC) were prepared essentially as described in the general protocol (Example 6). The drug loading step was performed with DL0 ratios ranging from 750 to 1500 g / mol PhL, without the addition of a buffering substance (pH 4.98 to 6.22). Maximum drug loading of compounds 3, 5 and 16 was observed in the ranges of 900 to 930 g / mol PhL, 982 to 1197 g / mol PhL and 938 to 951 g / mol PhL, respectively, and the loading efficiency at or near the maximum drug loading was at least 97.6%, 96.0% or 85.2%, respectively Figure 2A and Figure 2B ).

[0457] Example 10. Encapsulation of AKG-3, AKG-5 and AKG-16 into liposomes with higher degree of PEGylation or with 0.25 M Ammonium Sulfate (AS) as a trapping agent.

[0458] Liposomes consisting of HSPC with cholesterol (3:2 molar ratio) with different PEG-DSG content and trapping agents were prepared according to the general protocol and loaded with compounds AKG-3, AKG-5 and AKG-16 as in Example 9 with DL0 ratios of 250 or 500 g / mol PhL. As shown below in Table 6, all three compounds were loaded into the liposomes with high efficiency:

[0459] Table 6.

[0460]

[0461]

[0462] *Data in this row are from Example 8, "no buffer added" loading.

[0463] Thus, compounds AKG-3, AKG-5 and AKG-16 were efficiently loaded into phospholipid-cholesterol liposomes with increased levels of PEGylation and with Ammonium Sulfate as an intraliposomal drug trapping agent. However, the loading efficiency of the three compounds decreased when loading was performed with 0.25 M Ammonium Sulfate as a trapping agent at a higher drug to lipid ratio of 500 g drug / mol PhL. The loading efficiency of two of the oxazolidinones (AKG-3 and AKG-16) decreased.

[0464] Example 11. Loading of compounds AKG-3, AKG-5 and AKG-16 into liposomes using 0.5 M AS as a trapping agent.

[0465] Liposomes with 0.5 mol% or 5 mol% PEG-DSG (relative to PhL) and 0.5 M Ammonium Sulfate (AS) as trapping agent consisting of HSPC with Cholesterol (3:2 molar ratio) were prepared according to the general protocol and loaded with compounds AKG-3, AKG-5 and AKG-16 at DL0 ratios in the range of 500 to 1500 g / mol PhL as in Example 8. Results are shown in Figure 3A , Figure 3B , Figure 3C and Figure 3D . All three compounds were loaded in both liposomes with 93 to 100% encapsulation efficiency at DL ratios of 420 to 450 g / mol PhL; maximum drug loading was as follows:

[0466] Table 7

[0467]

[0468] All three test compounds were loadable in 0.5 M Ammonium Sulfate liposomes at more than 500 g drug / mol PhL in formulations with 0.5 mol% PEG-DSG and also for compounds AKG-3 and AKG-16 for formulations containing 5 mol% PEG-DSG. These high levels of loading are important to be able to reach sufficient doses of administered drug for the treatment of diseases. In comparison to Example 10 where lower loading efficiency was observed when using 0.25 M Ammonium Sulfate, the loading is significantly improved, indicating that the higher Ammonium Sulfate concentration of 0.5 M, despite the higher osmotic and potential osmotic burst, is improving the amount of drug loadable per mol of phospholipid and is preferred for anti-infectives where low toxicity and high dosing can lead to improved outcomes.

[0469] Example 12. Loading of compounds AKG-3, AKG-5, AKG-16 and AKG-28 into liposomes containing various combinations of fluorescent lipid markers.

[0470] Liposomes with 0.5 mol% PEG-DSG (relative to PhL), 0.15 mol.% of the lipid fluorescent marker DiIC18(3)-DS (ThermoFisher, USA) and 0.5 M Ammonium Sulfate (AS) or 1 N TEA-SOS as trapping agent consisting of HSPC with Cholesterol (60:40 molar ratio) were prepared according to the general protocol and loaded with compounds AKG-3, AKG-5 and AKG-16 as in Example 11 at pH 4.7 to 5.8 (no buffer substance added). The liposomes had the following characteristics:

[0471] Table 8

[0472]

[0473] All three drugs were effectively loaded into the liposomes. Degradation of AKG-5 during liposome loading was detected as the appearance of a second peak on HPLC.

[0474] Liposomes composed of various phospholipids (HSPC, distearoylphosphatidylcholine (DSPC, Avanti Polar Lipids, USA), or egg sphingomyelin (ESM, Lipoid, Germany) and cholesterol (60:40 molar ratio) with different amounts of PEG-DSG or N-methoxy poly(ethyleneglycol)oxycarbonyl-1,2-distearoylphosphatidylethanolamine (PEG-DSPE, PEG molecular weight 2000, Lipoid, Germany) and the lipid fluorescent label DiIC18(3)-DS (0.15 mol.% relative to PhL) were prepared according to the same general protocol with different trappers and AKG-16 was loaded in a similar way. When indicated, the liposome extrusion step of the general protocol was supplemented with extrusion through two stacked polycarbonate membranes with a pore size of 50 nm. The liposomes had the following characteristics:

[0475] Table 9

[0476]

[0477] Liposomes composed of HSPC with cholesterol (3:2 molar ratio) with 9.2 mol.% PEG-DSPE (relative to PhL), 0.15 mol.% lipid label DiIC18(3)-DS and 0.25 M ammonium sulfate (AS) as trapping agent were prepared according to the general protocol and Example 12 with additional 50 nm extrusion and AKG-28 was loaded at a drug-lipid ratio (DL0) of 150 g / mol PhL. The DL ratio of the liposomes (batch ID 98) was 73.8 g / mol PhL, the Z-average liposome size was 77.8 nm and the size polydispersity index (PDI) was 0.090.

[0478] These studies show that AKG-3, AKG-5, and AKG-16 can be effectively loaded into liposomes with a wide 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, when using 0.25 M AS, the efficiency decreased significantly from about 500 g AKG-16 / mol PhL to 128 g AKG-16 / mol PhL, as compared to 1 N TEA-SOS. Similar 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 concentration of AS can be preferable for loading high concentration of compounds into liposomes.

[0479] Example 13. Blood persistence and in vivo encapsulation stability of liposomes of Example 12 in mice.

[0480] The studies were performed on male CD-1 mice as described in the general protocol above.

[0481] Table 10

[0482]

[0483] These studies show that liposomes composed of different neutral phospholipid components (HSPC, DSPC, or SM) and loaded with AKG-16 using TEA-SOS trapping agent were cleared slowly, with more than 30% of the injected dose remaining in the plasma at 6 hours for most formulations. In addition, except for liposome batch ID 97, which contained AKG-16 loaded using 0.25 M AS, most formulations showed good drug retention, indicating that loading the drug using 0.25 M ammonium sulfate not only resulted in low loading efficiency (as shown in Table 9), but also low DL ratio at 6 hours (3.7%) due to significant leakage of liposomes in this formulation.

[0484] Example 14. Encapsulation of compounds AKG-28 and AKG-38 into liposomes with different trapping agents at different DL ratios.

[0485] Liposomes with 0.5 mol% PEG-DSG (relative to PhL), 0.15 mol.% lipid label DiIC18(3)-DS and 0.5 M Ammonium Sulfate (AS) or 1 N TEA-SOS as a trapping agent consisting of HSPC with Cholesterol (3:2 molar ratio) were prepared according to the general protocol and loaded with compounds AKG-28 and AKG-38 at pH 4.95 to 5.17 (no buffer substance added) and DLO ratios in the range of 300 to 1050 g / mol PhL (AKG-28) or 400 to 1400 g / mol PhL (AKG-38) as in Example 8. Using 0.5 M AS, the maximum drug loading of compounds AKG-28 and AKG-38 was 404 to 424 g / mol PhL and 818 to 842 g / mol PhL, respectively, and the loading efficiency over 95% was 302 g / mol PhL (quantitative loading) and 387 to 764 g / mol PhL (95.5 to 96.7% loading), respectively. Using 1 N TEA-SOS, the maximum drug loading of compounds AKG-28 and AKG-38 was 315 to 328 g / mol PhL and 989 g / mol PhL, respectively, and the maximum loading efficiency was 83.5% at drug loading of 250 g / mol PhL and 400 to 777 g / mol PhL (over 97.2% loading), respectively. Figure 4A and Figure 4B ).

[0486] AKG-38 showed almost quantitative loading between 400 to 800 g AKG-38 / mol PhL, while the resulting drug to lipid ratio for AKG-28 remained flat in the range of 250 to 1000 g AKG-28 / mol PhL, indicating a lower maximum drug loading for AKG-28 than AKG-38. It is understood that the previously indicated higher potency for AKG-28 will allow liposomal formulations of AKG-28 to be effective for the treatment of infectious diseases such as tuberculosis.

[0487] Example 15. Encapsulation of compounds AKG-28 and AKG-38 into liposomes with different phospholipid compositions, degree of PEGylation and trapping agent.

[0488] Liposomes with 0.5 M AS or 1 N TEA-SOS as a trapping agent consisting of phospholipid (PhL) and cholesterol (3:2 molar ratio), PEG-DSG, and DiIC18(3)-DS (0.15 mol.% of PhL) were prepared according to the general protocol and loaded with compounds AKG-28 and AKG-38 at a D / L ratio selected to optimize drug loading and encapsulation efficiency (EE) in the absence of added buffer substances. Results are in Tables 10 and 11 below.

[0489] Table 11. Encapsulation of compound AKG-28.

[0490]

[0491] Table 12. Encapsulation of compound AKG-38.

[0492]

[0493] This example shows that AKG-28 can be efficiently loaded into liposomes consisting of HSPC using 0.5 M AS or 1 N TEA-SOS as a trapping agent with a maximum drug loading of 230 to 275 g AKG-28 / mol PhL. However, formulations including sphingomyelin as the neutral phospholipid for this compound show a relatively lower loading with a maximum of only about 110 g AKG-28 / mol PhL.

[0494] Compound AKG-38 was loaded at a significantly higher D / L ratio of 525 to 600 g / mol using 0.5 M AS or 1 N TEA-SOS 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. In comparison to AKG-28, compound AKG-38 is less sensitive to the presence of sphingomyelin for loading.

[0495] Example 16. Encapsulation of compounds AKG-16, AKG-28, AKG-29, and AKG-38 into liposomes with increased pegylation and 0.5 M ammonium sulfate as a trapping agent.

[0496] Liposomes with 0.5 M Ammonium Sulfate as a trapping agent consisting of HSPC and Cholesterol (3:2 molar ratio), PEG-DSG (5 mol.%) and DiIC18(3)-DS (0.15 mol.%) were prepared according to the general protocol and loaded with compound AKG-16, AKG-28, AKG-29 or AKG-38 at a DL0 ratio selected to optimize drug loading and encapsulation efficiency (EE) in the absence of added buffer substance. Results are in Table 13 below.

[0497] Table 13

[0498]

[0499] This data shows that all compounds containing a dimethylaminoethyl substituent at the 2-position of the tetrazole ring were loaded into liposomes with an efficiency greater than 80%, while AKG-29, which has an aminoethyl substituent at the same position, was loaded into liposomes only at 14.5% efficiency and a final drug loading of 43.6 g AKG-29 / mol PhL. This demonstrates that, despite the presence of a titratable amine in all of the compounds tested, compounds with a substituted ammonium (e.g., N,N-dimethylaminoethyl) on the tetrazole ring unexpectedly allowed more efficient drug loading than those with a primary amine (aminoethyl group) at the same position.

[0500] Example 17. Blood persistence and in vivo encapsulation stability of the liposomes of Examples 15 and 16 in mice.

[0501] Studies were performed on male CD-1 mice as described in the general protocol above.

[0502] Table 14

[0503]

[0504] The data show that drug in liposome batch IDs 128, 132, 142, 144 and 145, all with 0.5 M AS as a trapping agent, lost almost 25 to 60% of encapsulated drug immediately upon contact with blood, as shown by the low DL ratio at 5 minutes and further reduction in DL ratio at 6 hours, particularly significant for AKG-38 and AKG-16 loaded liposomes. Thus, formulations of 0.5 mol% or 5 mol% PEG-DSG and 40 mol% Cholesterol, 0.5 M AS (as a trapping agent) did not retain drug as effectively as formulations using 1 N TEA-SOS (liposome batch IDs 129, 130, 133-135), where the % of initial DL ratio at both 5 minute and 6 hour time points was greater than 80%.

[0505] Example 18. Preparation of AKG-28 and AKG-38 and their loading into PEGylated liposomes with different phospholipid to cholesterol ratios.

[0506] Liposomes containing 5 mol.% PEG-DSG or PEG-DSPE (relative to PhL), 0.15 mol.% of the lipid label DiIC18(3)-DS and 0.5 M ammonium sulfate (AS) or 1 N TEA-SOS as a trapping agent were prepared according to the general protocol and loaded with the compounds AKG-28 and AKG-38 at pH 5.07 to 5.82 (no buffer substance added) as in Example 8.

[0507] In an attempt to stabilize liposomes with 0.5 M AS as a trapping agent for fast drug release upon contact with blood (as described in Example 17), liposomes were prepared using DSPC (generally known to yield more drug-leakage-stable liposomes compared to HSPC) and a reduced proportion of cholesterol (Chol) and loaded with AKG-28 at DL0 250 g / mol PhL or AKG-38 at DL0 500 g / mol PhL (Table 15). Contrary to expectations, reducing the cholesterol content from 40 mol% to 10 mol% cholesterol resulted in a significant decrease in the encapsulation efficiency for both AKG-28 and AKG-38. Lower cholesterol also destabilized the liposomes against aggregation. At 30 mol.% cholesterol (PhL-cholesterol molar ratio 70:30), liposomes containing AKG-28 prepared with 1 N TEA-SOS and 5 mol.% PEG-DSG or PEG-DSPE irreversibly aggregated during drug loading, as did formulations of AKG-38 containing 30 mol% cholesterol, 5 mol% PEG-DSPE, while AKG-38 with 5 mol% PEG-DSG showed a reduced loading efficiency of 77.1% at 30 mol% cholesterol, or 462.4 g / mol PhL.

[0508] Table 15

[0509]

[0510] In contrast, liposomes prepared with HSPC and containing 40 mol% or more cholesterol (up to 65 mol% cholesterol (the maximum investigated)) showed excellent encapsulation efficiencies of more than 87% for both AKG-28 (DL0 250 g / mol PhL) and AKG-38 (DL0 500 g / mol PhL), PEG-lipid (PEG-DSG and PEG-DSPE) and trapping agent (AS or TEA-SOS) and no liposome aggregation (Table 16).

[0511] Additionally, the potential of optimizing the formulation loading of current standard of care drugs from this class, linezolid, in both 0.5 M AS and 1 N TEA-SOS formulations was evaluated. Telithromycin was not soluble enough in water to proceed with transmembrane gradient assisted loading into liposomes following the general protocol of Example 6. Encapsulation efficiency was below 5% in both cases using linezolid, indicating that these liposomal formulations of AKG-28 and AKG-38 are significantly superior in their ability to stably encapsulate drugs compared to linezolid.

[0512] Z-average size (x z ) and polydispersity index (PDI) of liposomes were determined by dynamic light scattering (DLS) cumulants method using a Malvern Zetasizer Pro (Malvern Panalytical) at 173° measurement angle.

[0513] Table 16

[0514]

[0515]

[0516]

[0517] Example 19. In vitro burst release of PEGylated liposomes comprising AKG-28 or AKG-38 and different phospholipid to cholesterol ratios in the presence of plasma.

[0518] Liposomal formulations of AKG-28 and AKG-38 containing 5 mol% PEG-DSPE or PEG-DSG and different ratios of HSPC to cholesterol (40 to 65 mol% cholesterol) were evaluated for in vitro stability in the presence of mouse CD-1 or human pooled plasma (lithium-heparin stabilized from Innovative Research). If necessary, the plasma was thawed, adjusted to pH 7.4 with 1 N HC1, and filtered sequentially through a glass microfiber filter (GF / C), 1 μιη polyether sulfone (PES), and a 0.22 μιη PES filter. Plasma (80 μΐ) was mixed with liposomal drug formulation (20 μΐ) in 0.5 ml Eppendorf tubes. The mixtures were then incubated at 37°C for 20 minutes and then placed in cold water. The mixtures (0.1 mL) were immediately chromatographed on 2 mL Sepharose CL-4B columns, eluted with Hepes buffered saline (pH 7.0), and 0.25 mL liposomal drug was collected in the void volume fraction. The drug and Dil(3)-DS lipid markers were then analyzed by HPLC as described in Example 7, and the percent drug retained encapsulated was determined using the following formula:

[0519] (A d / A I / (A d，0 / A 1，0 )* 100 = percent drug retained encapsulated

[0520] where A d — area of drug peak, A I — area of lipid marker peak, A d，0 — area of drug peak pre-incubated with plasma, and A I，0 — area of lipid marker peak pre-incubated.

[0521] Results are shown in Figure 5A , Figure 5B , Figure 5C and Figure 5D . For liposomes with encapsulated AKG-28 ( Figure 5A ), a burst release phenomenon (rapid decrease in DL ratio indicating drug release from the liposomes) was observed in human plasma for formulations containing 40 mol.% cholesterol, but not for formulations with 45 mol.% or more cholesterol. For liposomes with encapsulated AKG-38 ( Figure 5B ), a burst release phenomenon was observed in both human and mouse plasma for formulations with 40 mol.% and 45 mol.% cholesterol, but not for formulations with 50 mol.% or more cholesterol.

[0522] Example 20. In vitro plasma release and in vivo pharmacokinetics of 5 mol% PEG-lipid liposomes comprising AKG-38 and either 40 mol% or 55 mol% cholesterol

[0523] Three liposome formulations of Example 18 (as described in Example 7) using 0.5 MAS capturer were evaluated in a two-time point pharmacokinetic study in female CD-1 mice, measuring the percent injected dose (%ID) of liposome lipid retained in the blood at both 5 minutes and 6 hours, and drug release from the liposomes by determining the drug to lipid ratio (D / L). Liposomes with either 5 mol% PEG-DSG or 5 mol% PEG-DSPE and containing 55 mol% cholesterol showed pre-injection D / L ratios of >95% at 5 minutes and >85% at 6 hours, while the PEG-DSG formulation containing 40 mol% cholesterol showed significantly reduced D / L 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 is in contrast to previous experience with drug-loaded liposome formulations, as many highly stable liposome drugs approved for clinical use, such as pegylated liposomal doxorubicin and nanoliposomal irinotecan, contain cholesterol at a rate of about 40 mol% (see, e.g., Liposomal Doxorubicin (Caelyx® / Myocet®) Drug Information Package Insert, updated 08 / 2019, and Drummond, D.C., et al. (2006). “Development of a highly active nanoliposomal irinotecan using a novel intraliposomal stabilization strategy.” Cancer Res. 66(6): 3271-3277). Liposomal Doxorubicin (Caelyx® / Myocet®) Drug Information Package Insert, updated 08 / 2019, and Drummond, D.C., et al. (2006). “Development of a highly active nanoliposomal irinotecan using a novel intraliposomal stabilization strategy.” Cancer Res. 66(6): 3271-3277).

[0524] Table 17

[0525]

[0526] Example 21. 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) inhibition over MPS inhibition.

[0527] Colorimetric MitoBiogenesis from AbCam TM Intracellular ELISA kit (cat# ab11021) was used to determine inhibition of mitochondrial protein synthesis according to manufacturer’s instructions. Mitochondrial protein synthesis inhibition was correlated with linezolid and other Significant toxicities associated with oxazolidinones, most notably ocular and peripheral neuropathy and lactic acidosis (Renslo (2010) Expert Rev 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 cultured at 37°C and 5% CO2 in DMEM media with 10% FBS and 1x glutamine. Cells were plated in 384-well plates at a density of 1,500 cells / well in 47.5 μΐ / well. Ten concentrations of each compound (starting at a high concentration of 200 μΜ, including 9 three-fold dilutions and one replicate / condition) were added to 2.5 μΐ of cells and incubated with cells for 5 days at 37°C and 5% CO2. Compounds tested included linezolid and tedizolid controls, as well as AKG-3, AKG-16, AKG-22, AKG-28, AKG-29, AKG-30, AKG-38, AKG-39, and AKG-40.

[0528] MitoBiogenesis intracellular Elisa was then performed according to manufacturer’s instructions (Abcame cat# ab11021), and alkaline phosphatase (AP) developed in a plate reader to detect SDH-1A at 405 nm in a 15 minute kinetic model with 20 second to 1 minute intervals, and HRP developed to detect COX-I at 600 nm in a 15 minute kinetic model with 20 second to 1 minute intervals. COX-1 and SDH-A signals were plotted as a ratio of COX-1 / SDH-A against concentration of each compound, and IC50s were calculated for each of the nine study compounds and two controls.

[0529] The MPS selectivity index (SI-MPS) was determined by dividing the MPS IC50 in ug / ml by the MIC in the drug susceptible H37Rv Mycobacterium tuberculosis strain as determined in Example 2. Both compounds AKG-28 and AKG-29 tested had SI-MPS that were more than 10 times higher than the SI-MPS determined for linezolid and more than 20 times higher than the SI-MPS determined for tedizolid. Both of these compounds were tested in AKG-28 contains a primary amino group at the R2position of the oxazolidinone ring. Because of the high potency (MIC < 0.1) and high selectivity of AKG-28 against Mycobacterium tuberculosis compared to mitochondrial protein synthesis, AKG-28 is an excellent candidate for encapsulation in liposomes and for the treatment of tuberculosis or other mycobacterial diseases.

[0530] Table 18

[0531]

[0532]

[0533] Example 22. Scale-up preparation of liposomal AKG-28 lot 275.

[0534] Batch 267.The general procedure of Example 6 was followed. HSPC (Lipoid AG) 4.95 g (6.30 mmol), cholesterol (Dishman, high purity) 2.98 g (7.71 mmol) and PEG-DSPE (Lipoid AG) 850 mg (0.315 mmol) (HSPC:cholesterol:PEG-DSPE 45:55:2.25 molar ratio) were combined with 9 ml of absolute ethanol (Sigma, E-7023) and heated with stirring on a 68 °C bath until all the lipid was dissolved. In a separate vessel, 93.3 g of 0.5 M aqueous ammonium sulphate (filtered through 0.2 micron) was pre-heated on a 68 °C bath and poured into the hot lipid ethanol solution with stirring. The resulting suspension was stirred on a 68 °C bath for 20 minutes and extruded through two stacked 47 mm 100 nm pore size and one 200 nm pore size polycarbonate track-etched membranes (Whatman Nucleopore) at 260 to 300 psi using a Lipex 100 ml thermobarrel liposome extruder (Northern Lipids, Inc.) heated with circulating 68 °C water. The resulting extruded liposomes were kept in a freezer (2 to 8 °C) overnight and filtered through a 0.22 pm polyethersulfone (PES) filter under positive pressure. The liposome extraneous capture agent (ammonium sulphate) was removed by TFF buffer exchange with endotoxin-free water using a polysulfone hollow fibre cartridge (Spectrum Laboratories) with a MW cut-off of 500 KDa on a KrosFlo TFF system until the residual conductivity fell to less than 200 pS / cm (143 pS / cm after 5.2 volume changes). The phospholipid concentration in the post-TFF liposome suspension was determined by the blue phosphomolybdate method to be 57.4 mM.

[0535] AKG-28 (as dihydrochloride salt) in the form of a 20 mg / ml aqueous stock solution (adjusted to pH 5.03 with NaOH) was combined with a TFF post-liposome suspension to form a loading mixture with a drug to phospholipid (DL) ratio of 250 g / mol in the presence of AKG-28 at a concentration of 45 mg / ml dextrose and 6 mg / ml. The mixture was rapidly heated to 60 to 63 °C with external heating under constant agitation and incubation was continued at 65 °C bath with agitation. After 20 minutes of incubation, the mixture was rapidly cooled to below 10 °C in ice water and held at that temperature for about 10 minutes. After reaching ambient temperature and adjustment 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. The liposomes were pre-concentrated to about 12 mg / ml AKG-28 by diafiltration and purified of any extraliposomal drug by TFF into 10 mM HEPES-Na buffer (pH 7.0 containing 0.144 M NaCl prepared with endotoxin-free water (HBS-7 buffer)) for a total of about 8 volumes of exchange. The proportion of unencapsulated drug prior to purification was estimated by spectrophotometry at 305 nm in the pre-concentrated diafiltrate and found to be about 0.9% (corresponding to a loading efficiency of 99.1%). The concentrated, purified liposomes were sterile filtered through a 0.2 pm sterile filter and the particle size was analyzed by DLS and the drug and phospholipid concentrations were analyzed by spectrophotometry. This procedure was repeated three more times (batches 269, 271, 273). The liposomes obtained had the characteristics shown in Table 19.

[0536] Table 19

[0537]

[0538] These batches were combined to obtain batch 275 with 12.0 mg / ml AKG-28 in the form of liposomes, a particle size Xz of 113.7 nm, and a PDI of 0.0417.

[0539] Example 23. Scale-up preparation of liposomal AKG-38 batch 276.

[0540] Batch 268.Example 22, with the following differences: an aqueous stock solution of AKG-38 (as the free base) was prepared by dissolving the drug in an equal volume of 1 N HCI and adjusting the volume to obtain 20 mg / ml of AG-38 (as the free base) at pH 5.08. The loading mixture contained 1300 mg AKG-38 and was prepared at a DL ratio of 8 mg / mL AKG-38 and 450 g / mol phospholipid, and additionally contained 10 mM NaCI. The post-loading liposomes were pre-concentrated to approximately 22 mg / ml of drug; the proportion of unencapsulated drug prior to purification was estimated by spectrophotometry at 305 nm in the pre-concentration diafiltrate and was found to be approximately 3.2% (corresponding to a loading efficiency of 96.8%). This procedure was repeated three more times (batches 270, 272, 274). The liposomes obtained had the characteristics shown in Table 20.

[0541] Table 20

[0542]

[0543] These batches were combined to obtain batch 276, which had 22.3 mg / ml AKG-38 in the form of liposomes, a particle size Xz of 113.1 nm, and a PDI of 0.0454.

[0544] Example 24. Preparation of "empty liposome" batch 277.

[0545] Except that 0.13 M sodium sulfate was employed instead of 0.5 M ammonium sulfate (sulfate salt of a non-exchanging cation), 2 mmol HSPC, 2.444 mmol cholesterol, and 0.1 mmol PEG-DSPE (HSPC:cholesterol:PEG-DSPE 45:55:2.25 molar ratio) were dissolved in ethanol to form a liposome suspension and extruded through a polycarbonate membrane as described in Example 22. The extruded liposomes were purified from extraliposomal sodium sulfate and brought into HBS-7 buffer by TFF buffer exchange using a polysulfone hollow fiber cartridge with MWCO 500 KDa, for a total of 10 volume exchanges. The purified liposomes had 42.9 mM phospholipid, a particle size Xz of 113.7 nm, and a PDI of 0.0612. They were sterile filtered through a 0.2 pm sterile filter and adjusted to 20 mM phospholipid with sterile HBS-7.

[0546] Example 25. Liposomal AKG-38 batch 279.

[0547] The general procedure of Example 6 was followed. HSPC (Lipoid AG) 13.102 g (16.67 mmol), Cholesterol (Dishman, high purity) 7.877 g (20.37 mmol) and PEG-DSPE (Lipoid AG) 2.250 g (0.833 mmol) (HSPC: Cholesterol: PEG-DSPE 45:55:2.25 molar ratio) were combined with 25 mL absolute ethanol (Sigma, E-7023) and heated with stirring on a 68°C bath until all lipid was dissolved. In a separate vessel, 259.1 g (250 ml) of 0.5 M aqueous ammonium sulphate (filtered through 0.2 micron) was preheated on 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 four portions. Using a Lipex 100 ml thermobarrel liposome extruder (Northern Lipids, Inc.) heated with circulating 70°C water, each portion was extruded five times through a stack of two 47 mm 100 nm pore size and one 200 nm pore size polycarbonate track-etched membranes (Whatman Nucleopore) at 280 psi. The portions of extruded liposomes were combined (Xz 129.7 nm) and extruded together a further five times through the same membrane stack to give liposomes with a size Xz of 115.9 nm and a PDI of 0.0212. The liposomes were left in a freezer (2 to 8°C) overnight and filtered through a 0.2 pm polyethersulfone (PES) filter under positive pressure. The phospholipid concentration was found to be 60.22 ± 0.34 mM. The endotoxin-free water was exchanged for TFF buffer using a polysulfone hollow fibre cartridge (Spectrum Laboratories) with a MW cut-off of 500 KDa on a KrosFlo TFF system until the residual conductivity dropped to 180 pS / cm after 5.1 volume exchanges. The phospholipid concentration in the post-TFF liposome suspension was determined by the blue phosphomolybdate method to be 54.97 ± 0.32 mM.

[0548] AKG-38 (free base) was mixed with 0.95 equivalents of 1 N HC1 and formulated with endotoxin-free water to obtain a 20 mg / ml aqueous stock solution (pH 5.16). The solution was passed through a 0.2 pm filter, and the filtered volume containing 3958 mg of drug was combined with a TFF post-liposome suspension in the presence of 44.5 mg / ml dextrose, 10 mM NaCl, and AKG-38 at a concentration of 8 mg / ml (pH 5.54) to form a loading mixture with a drug to phospholipid (DL) ratio of 450 g / mol. The mixture was heated to 61 °C over a 5 minute period with external heating under constant agitation, and incubation was continued at 65 °C bath for another 22 minutes with agitation. The mixture was then transferred to an ice water bath, stirred for 7 minutes to reduce the temperature to 10 °C, and kept in the ice water bath for another 8 minutes. After removal from the ice bath, reaching ambient temperature, and adjustment to 0.1 M NaCl by the addition of a 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 by diafiltration to about 22 mg / ml of AKG-38, and purified from any extraliposomal drug by TFF into HBS-7 buffer, with a total of 8 volume changes. The concentrated, purified liposomes were sterile filtered through a 0.2 pm PES high flow sterile filter, and the particle size was analyzed by DLS and the drug and phospholipid concentrations were analyzed by spectrophotometry. The liposomes had the following characteristics: AKG-38 was 21.1 ± 0.19 mg / ml, the DL ratio was 454 ± 4.7 g / mol phospholipid, Xz was 116.4 nm, and PDI was 0.0231. The yield of the drug formulation was 3834 mg (96.9%).

[0549] Example 26. Liposome AKG-28 Batch 281.

[0550] The general procedure of Example 6 was followed. Extruded liposomes containing 0.5 M ammonium sulfate were prepared as described in Example 25, composed of HSPC, cholesterol, and PEG-DSPE in a molar ratio of 45:55:2.25. The extraliposomal capture agent (ammonium sulfate) was removed by TFF exchange of endotoxin-free water on a KrosFlo TFF system using a polyether sulfone hollow fiber cartridge (Spectrum Laboratories) with a MW cutoff of 500 KDa until the residual conductivity was reduced to 150 pS / cm (4.1 volume changes). The phospholipid concentration in the post-TFF liposome suspension was determined to be 55.4 mM by the blue phosphomolybdate method.

[0551] AKG-28 (as dihydrochloride) at 20 mg / mL aqueous stock solution (adjusted to pH 5.24 with NaOH) was combined with the TFFed liposome suspension at a drug to phospholipid (DL) ratio of 250 g / mol to form a loading mixture in the presence of 44.5 mg / mL dextrose and 6 mg / mL concentration of AKG-28. The mixture was heated to 65.4°C over 2.5 minutes with constant stirring by external heating and continued incubation with stirring on a 65°C bath. After 20 minutes of incubation, the mixture was cooled to 9.3°C in ice water over 2.75 minutes and allowed to remain in the ice water bath for about 10 minutes. The mixture was then allowed to reach ambient temperature and adjusted to 0.1 M NaCl; pH 6.43. The loading mixture, 133.4 g, was purified by TFF using a polysulfone hollow fiber cartridge with a molecular weight cutoff of 500 KD. The liposomes were pre-concentrated by diafiltration to about 12 mg / ml of AKG-28 and purified from any extraliposomal drug by TFF exchange into HBS-7 buffer for a total of 8.1 volume exchanges. The proportion of unencapsulated drug prior to purification was estimated by spectrophotometry at 302 nm in the pre-concentrated diafiltrate and found to be about 0.7% (corresponding to a loading efficiency of 99.3%). The concentrated, purified liposomes were sterile filtered through a 0.2 μιη sterile filter and the particle size was analyzed by DLS and the drug and phospholipid concentrations were analyzed by spectrophotometry. The liposomes had the following characteristics: AKG-28 was 13.26 ± 0.21 mg / ml, the DL ratio was 258.2 ± 3.7 g / mol phospholipid, Xz was 117.3 nm, and PDI was 0.0421.

[0552] Example 27. Liposomal AKG-38 Batch 285.

[0553] The general procedure of Example 6 was followed. Extruded liposomes containing 0.5 M ammonium sulfate were prepared essentially as described in Example 25 and consisted of HSPC, cholesterol, and PEG-DSPE in a molar ratio of 45:55:2.25. The extraliposomal capture agent (ammonium sulfate) was removed by TFF exchange into endotoxin-free water on a KrosFlo TFF system using a polyethersulfone hollow fiber cartridge (Spectrum Laboratories) with a MW cutoff of 500 KDa until the residual conductivity dropped to 138 μ8 / ηηι (5.6 volume exchanges). The phospholipid concentration in the post-TFF liposome suspension was determined to be 53.1 mM by the blue phosphomolybdate method.

[0554] AKG-38 (free base) was mixed with 0.95 equivalents of 1 N HC1 and formulated 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 pm filter, and a quantity of the filtrate containing 1400 mg of drug was combined with a TFF post-liposome suspension in the presence of 44.5 mg / ml dextrose, 10 mM NaCl, and AKG-38 at a concentration of 8 mg / ml (pH 5.58) to form a loading mixture with a drug to phospholipid (DL) ratio of 450 g / mol. The mixture was heated to 63 °C over a period of 2.25 minutes with constant agitation by external heating, and incubation was continued at 65 °C bath with agitation for a total of 21 minutes. The mixture was then transferred to an ice water bath, stirred for 3 minutes to reduce the temperature to 10.3 °C, and allowed to remain in the ice water bath for an additional 7 minutes. After removal from the ice bath, reaching ambient temperature, and adjustment to 0.1 M NaCl by addition of a 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 by diafiltration to approximately 22 mg / ml of AKG-38, and purified from any extraliposomal drug by TFF into HBS-7 buffer, for a total of 7.7 volume changes. The AKG-38 concentration of the concentrated, purified liposomes was 23.1 mg / mL. The drug concentration was adjusted to 20 mg / ml with HBS-7 buffer, the liposomes were sterile filtered through a 0.2 pm PES high flow sterile filter, and the particle size was analyzed by DLS and the drug and phospholipid concentrations were analyzed by spectrophotometry. The liposomes had the following characteristics: AKG-38 was 20.35 ± 0.26 mg / ml, the DL ratio was 437.8 ± 6.5 g / mol phospholipid, Xz was 121.1 nm, and the PDI was 0.0200. The yield of the drug formulation was 1355 mg (96.8%).

[0555] Example 28. Liposome AKG-28 Batch 286.

[0556] Extruded liposomes (HSPC:cholesterol:PEG-DSPE 45:55:2.25 molar ratio) containing 0.5 M ammonium sulfate free of extraliposomal capture agent were obtained as in Example 27.

[0557] AKG-28 (as dihydrochloride salt) at 20 mg / mL aqueous stock solution (adjusted to pH 5.18 with NaOH) was combined with TFF post-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 concentration of AKG-28. The mixture was placed on a 65 °C water bath with stirring and reached 60 °C in 4.5 minutes. Incubation was continued with stirring for a total of 20 minutes, the mixture was cooled in ice water to 10.0 °C in 2 minutes, and it was allowed to remain in an ice water bath for about 10 minutes. The mixture was then brought to ambient temperature and adjusted to 0.1 M NaCl; pH 6.23. The 104.6 g loading mixture was purified by TFF using a polysulfone hollow fiber cartridge with a molecular weight cutoff of 500 KD. The liposomes were pre-concentrated by diafiltration to about 12 mg / ml of AKG-28, and purified from any extraliposomal drug by TFF exchange into HBS-7 buffer, for a total of 8.3 volume exchanges. The concentrated, purified liposomes were sterile filtered through a 0.2 pm sterile filter (chased with HBS-7 buffer), and the particle size was analyzed by DLS and the drug and phospholipid concentrations were analyzed by spectrophotometry. The liposomes had the following characteristics: AKG-28 was 12.05 ± 0.13 mg / ml, the DL ratio was 239.4 g / mol phospholipid, Xz was 120.1 nm, and the PDI was 0.0294. The yield of the formulated drug was 555.5 mg (92.6%).

[0558] Example 29. Liposome AKG-38 Batch 292.

[0559] Batch 288.The general procedure of Example 6 was followed. HSPC (Lipoid AG) 9.17 g (11.67 mmol), cholesterol (Dishman, high purity) 5.51 g (14.26 mmol) and PEG-DSPE (Lipoid AG) 1.575 g (0.583 mmol) were combined with 17.5 mL of absolute ethanol (Sigma, E-7023) and heated with stirring on a 69 to 70 °C bath until all lipid was dissolved. In a separate vessel, 181.4 g (175 ml) of 0.5 M aqueous ammonium sulphate (filtered through 0.2 micron) 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 three portions. Using a Lipex 100 ml thermobarrel liposome extruder (Northern Lipids, Inc.) heated with circulating 70 °C water, each portion was extruded five times through a stack of two 47 mm 100 nm pore size and one 200 nm pore size polycarbonate track-etched membranes (Whatman Nucleopore) at 280 psi. The extruded liposome portions were combined (Xz 126.7 nm) and extruded together a further four times through the same membrane stack to give liposomes with a size Xz of 119.2 nm and a PDI of 0.0385. The liposomes were left in the freezer (2 to 8 °C) overnight and filtered through a 0.2 pm polyethersulfone (PES) filter under positive pressure. The phospholipid concentration was found to be 59.08 ± 0.44 mM. The endotoxin-free water was exchanged for TFF buffer using a polysulfone hollow fibre cartridge (Spectrum Laboratories) with a MW cut-off of 500 KDa on a KrosFlo TFF system until the residual conductivity dropped to 152 pS / cm after 5.4 volume exchanges. The phospholipid concentration in the post-TFF liposome suspension was determined by the blue phosphomolybdate method to be 57.76 ± 0.53 mM.

[0560] AKG-38 (free base) was mixed with 0.95 equivalents of 1 N HC1 and formulated with endotoxin-free water to obtain an aqueous stock solution of 19.7 mg / ml (pH 5.11). The solution was passed through a 0.2 pm filter, and a quantity of 3509 mg of drug in the filtrate was combined with a TFF post-liposome suspension in the presence of 44.5 mg / ml dextrose, 10 mM NaCl, and AKG-38 at a concentration of 8 mg / ml (pH 5.50) to form a loading mixture with a drug to phospholipid (DL) ratio of 450 g / mol. The mixture was heated to 61.6 °C over a period of 5 minutes with constant agitation by external heating and incubated for an additional 20 minutes at 65 °C bath with agitation. The mixture was then transferred to an ice water bath, stirred for 7 minutes to reduce the temperature to 10 °C, and allowed to remain in the ice water bath for an additional 8 minutes. After removal from the ice bath, reaching ambient temperature, and adjusting to 0.1 M NaCl by the addition of a 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 by diafiltration to approximately 22 mg / ml of AKG-38 and purified from any extraliposomal drug by TFF into HBS-7 buffer with a total of 7.8 volume changes. The concentrated, purified liposomes were sterile filtered through a 0.2 pm PES high flow sterile filter, and the particle size was analyzed by DLS and the drug and phospholipid concentrations were analyzed by spectrophotometry. The liposomes had the following characteristics: AKG-38 was 22.47 ± 0.38 mg / ml, the DL ratio was 441.6 g / mol phospholipid, Xz was 121.3 nm, and PDI was 0.0465. The drug formulation yield was 3375 mg (96.2%).

[0561] Batch 289.The procedure of Ls-288 was repeated using 1506 mg of AKG-38 (as a similarly prepared 20.0 mg / mL aqueous stock solution, pH 5.15). The solution was combined with the same TFF post-extruded 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 AKG-38 at a concentration of 8 mg / ml (pH 5.53). The mixture was heated to 64.3 °C over a 2 minute period with constant agitation by external heating and incubated with agitation for a further 20 minutes on a 65 °C bath. The mixture was then transferred to an ice water bath, stirred for 2.75 minutes to reduce the temperature to 9.6 °C and left in the ice water bath for a further 14 minutes. After removal from the ice bath, the loaded mixture was allowed to reach ambient temperature and adjusted to 0.1 M NaCl with a 3 M NaCl stock; pH 6.54. The drug loaded liposomes were purified by TFF using a polysulfone hollow fiber cartridge with a molecular weight cut-off of 500 KD. The liposomes were pre-concentrated by diafiltration to approximately 22 mg / ml of AKG-38 and purified from any extraliposomal drug by TFF into HBS-7 buffer, a total of 8.1 volume changes. The concentrated, purified liposomes were sterile filtered through a 0.2 pm PES high flow sterile filter and the particle size was analyzed by DLS and the drug and phospholipid concentrations by spectrophotometry. The liposomes had the following characteristics: AKG-38 was 22.84 ± 0.41 mg / ml, the DL ratio was 452.7 g / mol phospholipid, Xz was 120.3 nm and the PDI was 0.0522. The yield of drug formulation was 1407 mg (93.4%).

[0562] Batch 290.The general procedure of Example 6 was followed. HSPC (Lipoid AG) 7.86 g (10.00 mmol), Cholesterol (Dishman, high purity) 4.73 g (12.22 mmol) and PEG-DSPE (Lipoid AG) 1.35 g (0.50 mmol) (HSPC: Cholesterol: PEG-DSPE 45:55:2.25 molar ratio) were combined with 15 mL absolute ethanol (Sigma, E-7023) and heated with stirring on a 69 to 70 °C bath until all lipid was dissolved. In a separate vessel, 155.5 g (150 ml) of 0.5 M aqueous ammonium sulphate (filtered through 0.2 micron) 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 two portions. Using a Lipex 100 ml thermobarrel liposome extruder (Northern Lipids, Inc.) heated with circulating 70 °C water, each portion was extruded four times through a stack of two 47 mm 100 nm pore size and one 200 nm pore size polycarbonate track-etched membranes (Whatman Nucleopore) at 280 psi. The extruded liposome portions were combined (Xz 131.5 nm) and extruded together a further four times through the same membrane stack to give liposomes with a size Xz of 122.7 nm and a PDI of 0.0215. The liposomes were left to stand in a refrigerator (2 to 8 °C) overnight and filtered through a 0.2 pm polyethersulfone (PES) filter under positive pressure. The phospholipid concentration was found to be 58.99 ± 0.22 mM. The endotoxin-free water was exchanged for TFF buffer using a polysulfone hollow fibre cartridge (Spectrum Laboratories) with a MW cut-off of 500 KDa on a KrosFlo TFF system until the residual conductivity dropped to 146 pS / cm after 5.5 volume changes. The phospholipid concentration in the post-TFF liposome suspension was determined by the blue phosphomolybdate method to be 56.94 ± 0.41 mM.

[0563] AKG-38 (free base) was mixed with 0.95 equivalents of 1 N HC1 and formulated with endotoxin-free water to obtain a 20 mg / mL aqueous stock solution (pH 5.15). The solution was passed through a 0.2 pm filter, and a quantity of the filtrate containing 2315 mg of drug was combined with a TFF post-liposome suspension in the presence of 44.5 mg / ml dextrose, 10 mM NaCl, and AKG-38 at a concentration of 8.02 mg / ml (pH 5.52) to form a loading mixture with a drug to phospholipid (DL) ratio of 450 g / mol. The mixture was heated to 64.4°C over a period of 3.25 minutes with constant agitation by external heating and continued incubation with agitation for an additional 17 minutes on a 65°C bath. The mixture was then transferred to an ice water bath, agitated to reduce the temperature to less than 10°C, allowed to remain in the ice water bath for a total of 10 minutes, allowed to reach ambient temperature, and adjusted to 0.1 M NaCl with a 3 M NaCl stock; pH 6.63. 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 by diafiltration to approximately 22 mg / ml of AKG-38 and purified from any extraliposomal drug by TFF into HBS-7 buffer, a total of 8.0 volume changes. The concentrated, purified liposomes were sterile filtered through a 0.2 pm PES high flow sterile filter and analyzed for particle size by DLS and drug and phospholipid concentrations by spectrophotometry. The liposomes had the following characteristics: AKG-38 was 22.07 ± 0.23 mg / ml, the DL ratio was 441.6 g / mol phospholipid, Xz was 120.4 nm, and PDI was 0.0395. The yield of drug formulation was 2141 mg (92.5%).

[0564] Batch 292. Batches 288 (150.3 g), 289 (61.2 g), and 290 (19.5 g) were combined to give batch 292 of 278.4 g of 22.5 mg / mL liposome-formulated AKG-38. All liposome formulations were stored at 2 to 8°C.

[0565] Example 30. Preparation of Liposomal AKG-28 Batch 235.

[0566] The general procedure of Example 6 was followed. HSPC (Lipoid AG) 940 mg (1.20 mmol), cholesterol (Dishman, high purity) 568 mg (1.47 mmol), PEG-DSPE (Lipoid AG) 163 mg (0.06 mmol), and 0.0018 mmol of the lipophilic fluorescent label DiIC 18(3)-DS (AAT Bioquest, USA) (HSPC: Cholesterol: PEG-DSPE: DiIC 18 (3)-DS 45:55:2.25:0.0675 molar ratio, 0.15 mol% DiI 3-DS relative to HSPC) were combined in 2 ml of absolute ethanol (Sigma, E-7023) and heated with stirring on a 68°C bath until all the lipids were dissolved. In a separate vessel, 20 ml of 0.5 M aqueous ammonium sulfate (filtered through 0.2 micron) was preheated on a 68°C bath and poured into the hot lipid ethanol solution with stirring. The resulting suspension was stirred on a 68°C bath for 20 minutes and extruded through two stacked 47 mm 100 nm pore size and one 200 nm pore size polycarbonate track-etched membranes (Whatman Nucleopore) at 300 psi using a Lipex 100 ml thermobarrel liposome extruder (Northern Lipids, Inc.) heated with circulating 68°C water. The resulting extruded liposomes were kept in a refrigerator (2 to 8°C) overnight and filtered through a 0.2 pm polyethersulfone (PES) filter under positive pressure. The liposome extraneous capture agent (ammonium sulfate) was removed by TFF buffer exchange with endotoxin-free water using a polysulfone hollow fiber cartridge (Spectrum Laboratories) with a MW cutoff of 500 KDa on a KrosFlo TFF system until the conductivity of the retentate dropped to 60 pS / cm (10 volumes exchange). The phospholipid concentration in the liposome suspension after TFF was determined by the blue phosphomolybdate method to be 37.56 ± 0.62 mM.

[0567] AKG-28 (as dihydrochloride salt) at 50 mg in a 20 mg / ml aqueous stock solution (adjusted to pH 4.99 with NaOH) was combined with the TFF post-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 concentration. The mixture (pH 5.53) was incubated with stirring on a 65 °C bath for 20 minutes, rapidly cooled in ice water and held in an ice water bath for about 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 500 KD molecular weight cutoff. The liposomes were pre-concentrated by diafiltration to about 5 mg / ml AKG-28 and purified from any extraliposomal drug by TFF into HBS-7 buffer, a total of about 10 volume changes. The purified liposomes were further concentrated by TFF by two-fold using a syringe operated small 500 KD hollow fiber cartridge (MicroKros, Spectrum). The concentrated, purified liposomes were sterile filtered through a 0.2 pm sterile filter and analyzed for particle size by DLS and drug and phospholipid concentrations by spectrophotometry. The liposomes had the following characteristics: AKG-28 was 8.22 ± 0.16 mg / ml, DL ratio was 257.3 ± 10.3 g / mol phospholipid, liposome size Xz was 118.2 nm, PDI was 0.0188. The yield of drug formulation was 41.4 mg (82.8%).

[0568] Example 31. Preparation of Liposomal AKG-38 Batch 236.

[0569] Post-TFF extruded liposomes were used from Example 30 containing 0.5 M ammonium sulfate. AKG-38 (free base) was mixed with 0.95 equivalents of 1 N HC1 and formulated with endotoxin-free water to obtain an aqueous stock solution of 20 mg / mL (pH 5.11). The solution was passed through a 0.2 μιη filter, and a volume of filtrate containing 70 mg of drug was combined with the post-TFF liposome suspension (Example 30) in the presence of 140 mg / ml dextrose and 3 mg / ml AKG-38 concentration to form a loading mixture with a drug to phospholipid (DL) ratio of 450 g / mol. The mixture was incubated with stirring on a 65 °C bath for 20 minutes, rapidly cooled in ice water and held in an ice water bath for about 10 minutes. After reaching ambient temperature and adjusting to 0.1 M NaCl with 3 M NaCl stock solution, the pH was 6.33. 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 by diafiltration to about 6 mg / ml of AKG-38 and purified from any extraliposomal drug by TFF into HBS-7 buffer, a total of about 10 volume changes. The purified liposomes were further concentrated by TFF using a syringe-operated small 500 KD hollow fiber cartridge (MicroKros, Spectrum) by a factor of two. The concentrated, purified liposomes were sterile filtered through a 0.2 μιη sterile filter and analyzed by DLS for particle size and by spectrophotometry for drug and phospholipid concentrations. The liposomes had the following characteristics: AKG-38 9.04 ± 0.16 mg / ml, DL ratio of 463.9 ± 19.8 g / mol phospholipid, liposome size Xz of 119.3 nm, PDI of 0.0267. The drug formulation yield was 56 mg (80%).

[0570] Example 32. Retention of drug encapsulation in liposomes of lots 235 and 236 in the presence of plasma in vitro.

[0571] Retention of drug encapsulation in liposomes in the presence of 80% mouse or human plasma at 37 °C was determined as described in Example 19 herein. Incubation time was 20 minutes.

[0572] Table 21.

[0573] Liposomal batch ID 235 236 Mouse plasma 100.2±4.3 91.0±2.5 Human plasma 99.6±3.9 94.8±2.6

[0574] These liposomes are stable against sudden release of drug upon contact with plasma.

[0575] Example 33. Preparation of liposome AKG-28 and AKG-38 lots 231, 232 (HSPC: cholesterol: PEG-DSPE 45:55:2.25 molar ratio, with 0.5 M ammonium sulfate as the trapping agent).

[0576] The general procedure of Example 6 was followed. HSPC (Lipoid AG) 4.255 g (5.41 mmol), cholesterol (Dishman, high purity) 2.56 g (6.62 mmol) and PEG-DSPE (Lipoid AG) 729 mg (0.27 mmol) (HSPC: Cholesterol: PEG-DSPE 45:55:2.25 molar ratio) were combined in 9 ml of absolute ethanol (Sigma, E-7023) and heated with stirring on a 70 °C bath until all the lipid was dissolved. In a separate vessel, 90 ml of 0.5 M aqueous ammonium sulphate (filtered through 0.2 micron) 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 25 minutes and extruded through two 47 mm 100 nm pore size and one 200 nm pore size polycarbonate track-etched membrane (Whatman Nucleopore) stack at 260 psi using a Lipex 100 ml thermobarrel liposome extruder (Northern Lipids, Inc.) heated with circulating 70 °C water. The resulting extruded liposomes were kept in a freezer (2 to 8 °C) overnight and filtered through a 0.2 pm polyethersulfone (PES) filter under positive pressure. The endotoxin-free water was exchanged by TFF buffer using a polysulfone hollow fibre cartridge (Spectrum Laboratories) with a MW cut-off of 500 KDa on a KrosFlo TFF system until the conductivity of the retentate dropped to 60 pS / cm (10 volumes exchange). The concentration of phospholipids in the liposome suspension after TFF was determined by blue phosphomolybdate spectrophotometry to be 46.97 ± 0.80 mM.

[0577] Batch 231.AKG-28 (as dihydrochloride salt) at 20 mg / mL aqueous stock solution adjusted to pH 5.02 with sodium hydroxide) was combined with a TFF post-liposome suspension to form a loading mixture with a drug to phospholipid (DL) ratio of 250 g / mol in the presence of AKG-28 at a concentration of 137.6 mg / mL dextrose and 2.53 mg / mL. The mixture (pH 5.60) was incubated with stirring on 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 a 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. The liposomes were pre-concentrated by diafiltration to approximately 9 mg / ml of AKG-28 and purified from any extraliposomal drug by TFF exchange into HBS-7 buffer, for a total of 10.9 volume exchanges. The purified liposomes were further concentrated to approximately 12 mg / ml of drug by continued TFF diafiltration in the absence of buffer feed. The concentrated, purified liposomes were sterile filtered through a 0.2 pm sterile filter and analyzed for particle size by DLS and drug and phospholipid concentrations by spectrophotometry. The liposomes had the following characteristics: AKG-28 was 11.42 ± 0.09 mg / ml, the DL ratio was 247.7 ± 7.1 g / mol phospholipid, the liposome size Xz was 116.5 nm, and the PDI was 0.0511. The yield of the drug formulation was 322.7 mg (92.2%).

[0578] Batch 232.AKG-38 (free base) was mixed with 0.95 equivalents of 1 N HC1 and formulated with endotoxin-free water to obtain an aqueous stock solution of 20 mg / mL (pH 5.09). The solution was passed through a 0.2 μιη filter, and a quantity of the filtrate containing 580 mg of drug was combined with the TFF post-liposome suspension of this example to form a loading mixture with a drug to phospholipid (DL) ratio of 500 g / mol in the presence of 137.6 mg / ml dextrose, AKG-38 concentration of 2.53 mg / ml, pH 5.72. The mixture was incubated with stirring on a 65 °C bath for 20 minutes, rapidly cooled in ice water and held in an ice water bath for about 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. The liposomes were pre-concentrated by diafiltration to about 12 mg / ml of AKG-38 and purified from any extraliposomal drug by TFF exchange into HBS-7 buffer, for a total of 8.5 volume exchanges. The purified liposomes were further concentrated two-fold by continued diafiltration TFF in the absence of buffer feed. The concentrated, purified liposomes were sterile filtered through a 0.2 μιη sterile filter, and the particle size was analyzed by DLS and the drug and phospholipid concentrations were analyzed by spectrophotometry. The liposomes had the following characteristics: AKG-38 was 16.03 ± 0.07 mg / ml, the DL ratio was 487.3 ± 13.9 g / mol phospholipid, the liposome size Xz was 120.0 nm, and the PDI was 0.0069. The drug yield was 538.9 mg (92.9%)

[0579] Example 34. Preparation of Liposomal AKG-28 Batch 233 (HSPC:Cholesterol:PEG-DSG 60:40:3 molar ratio, with 1 N sucrose octasulfate triethylammonium as a trapping agent)

[0580] The general procedure of Example 6 was followed. HSPC (Lipoid AG) 1.88 g (2.4 mmol), cholesterol (Dishman, high purity) 619 mg (1.6 mmol) and PEG-DSG (Sunbright GS-020, NOF, Japan) 312 mg (0.12 mmol) were combined in 3 ml of absolute ethanol and heated with stirring on a 67 °C bath until all the lipid was dissolved. In a separate vessel, 31.5 g (30 ml) of 1 N aqueous triethylammonium octasaccharide octasulfate (filtered through 0.2 micron, pH 6.20, see Example 8) was preheated on a 65 °C bath and poured into the hot lipid ethanol solution with stirring. The resulting suspension was stirred on a 65 °C bath for 5 minutes and extruded through four 47 mm 100 nm pore size and one 200 nm pore size polycarbonate track-etched membrane (Whatman Nucleopore) stack at 400 psi using a Lipex 100 ml thermobarrel liposome extruder (Northern Lipids, Inc.) heated with circulating 65 °C water. The resulting extruded liposomes were kept in a freezer (2 to 8 °C) overnight and filtered through a 0.2 pm polyethersulfone (PES) filter under positive pressure. 9.2 g of extruded liposomes were purified from the liposome extracapsular agent (TEA-SOS) by TFF buffer exchange into endotoxin-free water using a polysulfone hollow fiber cartridge (Spectrum Laboratories) with a MW cutoff of 500 KDa on a KrosFlo TFF system until the conductivity of the retentate dropped to 21 pS / cm (14.5 volume changes). The phospholipid concentration in the liposome suspension after TFF was determined by blue phosphomolybdate spectrophotometry to be 31.32 ± 0.85 mM.

[0581] AKG-28 (as dihydrochloride) at 140 mg in a 20 mg / ml aqueous stock solution (adjusted to pH 5.02 with NaOH) was combined with the TFF post-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 AKG-28 at a concentration of 2.52 mg / ml. The mixture (pH 5.43) was incubated with stirring on a 65 °C bath for 20 minutes, rapidly cooled in ice water and held in an ice water bath for about 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. The liposomes were pre-concentrated by diafiltration to about 9 mg / ml of AKG-28 and purified from any extraliposomal drug by TFF exchange into HBS-7 buffer, for a total of 10.9 volume exchanges. The purified liposomes were further concentrated to about 12 mg / ml of drug by continued TFF diafiltration in the absence of buffer feed. The concentrated, purified liposomes were sterile filtered through a 0.2 pm sterile filter (traced with HBS-7 buffer) and analyzed for particle size by DLS and drug and phospholipid concentrations by spectrophotometry. The liposomes had the following characteristics: AKG-28 at 10.64 ± 0.20 mg / ml, DL ratio of 246.8 ± 11.7 g / mol phospholipid, liposome size Xz of 116.3 nm, PDI of 0.0022. The yield of drug formulation was 118.2 mg (84.4%).

[0582] Example 35. Preparation of Liposomal AKG-38 Batch 234 (HSPC:Cholesterol:PEG- DSPE 45:55:2.25 molar ratio, with 1 N sucrose octasulfate triethylammonium as a trapping agent)

[0583] The general procedure of Example 6 was followed. HSPC (Lipoid AG) 3.30 g (4.20 mmol), cholesterol (Dishman, high purity) 1.985 g (5.13 mmol) and PEG-DSPE (Lipoid AG) 567 mg (0.21 mmol) (HSPC: Cholesterol: PEG-DSPE 45:55:2.25 molar ratio) were combined in 7 mL of absolute ethanol (Sigma, E-7023) and stirred heated on a 70 μ bath until all the lipid was dissolved. In a separate vessel, 10 ml of 1 N sucrose octasulfate triethylammonium (TEA-SOS) aqueous 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 on a 70 °C bath for 10 minutes and extruded through two 47 mm 100 nm pore size and one 200 nm pore size polycarbonate track-etched membranes (Whatman Nucleopore) stacked in a Lipex 100 ml thermobarrel liposome extruder (Northern Lipids, Inc.) heated with a circulating 70 °C water bath at 260 psi for eight passes. The resulting extruded liposomes were kept in a freezer (2 to 8 °C) overnight and filtered through a 0.2 μm polyethersulfone (PES) filter under positive pressure. The phospholipid concentration was 54.6 mM. 11.33 g of extruded liposomes were purified from the liposome-externalizing agent (TEA-SOS) by TFF buffer exchange into endotoxin-free water using a polysulfone hollow fiber cartridge (Spectrum Laboratories) with a MW cutoff of 500 KDa on a KrosFlo TFF system until the conductivity of the retentate dropped to 64 μS / cm (13.8 volume changes). The phospholipid concentration in the liposome suspension after TFF was determined by blue phosphomolybdate spectrophotometry to be 28.67 ± 1.01 mM.

[0584] AKG-38 (free base) was mixed with 0.95 equivalents of 1 N HC1 and formulated with endotoxin-free water to obtain an aqueous stock solution of 20 mg / mL (pH 5.09). The solution was passed through a 0.2 μιη filter, and a quantity of the filtrate containing 250 mg of drug was combined with the TFF post-liposome suspension of this example in the presence of 116.4 mg / ml dextrose, AKG-38 concentration of 2.53 mg / ml, pH 5.24, to form a loading mixture with a drug to phospholipid (DL) ratio of 500 g / mol. The mixture was incubated with stirring on a 65 °C bath for 20 minutes, rapidly cooled in ice water and held in an ice water bath for about 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. The liposomes were pre-concentrated by diafiltration to about 10 mg / ml of 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 about two-fold by continued TFF diafiltration in the absence of buffer feed. The concentrated, purified liposomes were sterile filtered through a 0.2 μιη sterile filter, and the particle size was analyzed by DLS and the drug and phospholipid concentrations were analyzed by spectrophotometry. The liposomes had the following characteristics: AKG-38 was 15.71 ± 0.33 mg / ml, the DL ratio was 518.6 ± 18.4 g / mol phospholipid, the liposome size Xz was 114.3 nm, and the PDI was 0.0284. The drug was formulated with a yield of 235.7 mg (94.3%).

[0585] Example 36. Effect of osmotic agent concentration on loading efficiency of AKG-28 and AKG-38 in liposomes and drug retention in liposomes in plasma.

[0586] 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 ammonium sulfate outside the liposomes by TFF exchange of endotoxin-free “water for injection” (WFI) - quality water (Hyclone) using a MicroKros polysulfone hollow fiber cartridge (MWCO 500 KDa, Spectrum Laboratories) operated with a syringe (13.8x volume change, residual conductivity 88 μ8 / cm, phospholipid concentration 55.4 mM). Liposomes were loaded with AKG-28 or AKG-38 by incubating drug (prepared as a 20 mg / ml aqueous stock as described in Examples 30 and 31) with purified extruded liposomes in aqueous solution in a 65 °C water bath for 20 minutes in the presence of different concentrations of osmotic agent (dextrose) 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). Unencapsulated drug was removed by size exclusion chromatography on Sepharose CL-4B (elution buffer HBS-7 buffer) and loading (encapsulation) efficiency was determined from the results of drug and phospholipid analysis. Osmotic agent concentrations are expressed both in absolute terms and as a percentage of a dextrose concentration of 168 mg / ml, which was determined to be isotonic with the 0.5 M ammonium sulfate solution used to form the liposomes. Contrary to expectations based on general consensus in the liposome field, drug was efficiently loaded into the liposomes of the present disclosure (encapsulation efficiency greater than 85%, and most greater than 90%) even under low-osmotic conditions (i.e., when the osmotic strength of the solution outside the liposomes is lower than the osmotic strength of the trapping agent solution inside the liposomes) and even in the complete absence of added osmotic balancing agent (dextrose) (Table 22). Furthermore, drug encapsulation in liposomes loaded with the lowest concentration of osmotic agent was at least as stable upon exposure to plasma under the conditions of the in vitro plasma release assay described in Example 19 as that in those loaded with near-complete (86.3%) osmotic balancing.

[0587] The results show that liposomes with 55 mol% cholesterol, 45 mol% PC, 5 mol% PEG-DSPE of HSPC, and 0.5 M AS trapping agent loaded AKG-28 (Table 22) and AKG-38 (Table 23) at 250 or 500 g / mol PhL with efficiencies >85%, most >90% under low-osmotic conditions with dextrose up to 0%, and that liposomes loaded under low-osmotic conditions effectively retain drug in the presence of plasma.

[0588] Table 22

[0589]

[0590] Table 23

[0591]

[0592] Example 37. Single-dose pharmacokinetic study of Ls-AKG28 and Ls-AKG38 total form (encapsulated + released drug) in rats

[0593] This study was performed to evaluate the PK of AKG28 and AKG38 administered as single doses of Ls-AKG28 and Ls-AKG38 in rats. The study was performed on male Sprague-Dawley rats using IV administration of liposomal AKG-38 (Ls-AKG38) at 20, 40, or 80 mg / kg body weight or liposomal AKG-28 (Ls-AKG28) at 10, 20, and 40 mg / kg body weight. Ls-AKG28 (batch 275) and Ls-AKG38 (batch 276) were prepared as described in Examples 22 and 23, respectively. For comparison, linazolid was administered orally as a gavage at 50 mg / kg body weight as a suspension with 0.5% methylcellulose formulated and acidified to pH 3 to 4 (Sigma M0430) at a concentration of 20 mg / mL. For plasma drug measurements, 0.5 ml of blood was collected in lithium heparin tubes at 5 minutes, 15 minutes, 1 hour, 3 hours, 6 hours, 24 hours, 48 hours, and 72 hours. The samples were centrifuged, and the resulting plasma was isolated and transferred to duplicate clear polypropylene tubes, frozen immediately on dry ice, and stored at -80 °C until analysis. Rat plasma concentrations 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 estimate the plasma maximum concentration (Cmax), the plasma maximum concentration divided by the dose (Cmax / dose), the time of Cmax (Tmax), the last measured concentration (Clast), the time of Clast (Tlast), the area under the plasma concentration versus time curve from 0 hours to the last time point (AUC0-t), and from 0 hours to infinity (AUC0-inf), AUC0-t divided by the dose (AUC0-t / dose), and AUC0-inf divided by the dose (AUC0-inf / dose). max max max last last 0-last 0-inf 0-last 0-last ​​​​​​​​apparent clearance (CL / F) and apparent volume of distribution (Vd / F). For linezolid, the PK software was used to estimate the same PK parameters, except for apparent clearance (CL / F) and apparent volume of distribution (Vd / F).

[0594] Plasma concentration versus time profiles for total drug following administration of Ls-AKG28 at 10, 20, and 40 mg / kg single IV dose (IVx1) are shown in FIG. 6. Figure 7 A summary of plasma PK parameters for total drug following administration of Ls-AKG28 at 10, 20, and 40 mg / kg IVx1 is shown in Table 24.

[0595] Plasma concentration versus time profiles for Ls-AKG28 were detectable from 5 minutes to 72 hours at all doses. Based on C max Based on the results of Cmax / dose and AUC / dose, plasma PK of Ls-AKG28 was linear (proportional to dose) following administration of 10, 20, and 40 mg / kg. Plasma clearance (CL) of Ls-AKG38 (about 2.59 mL / hour / kg) was higher than that of Ls-AKG28 (about 1.67 mL / hour / kg) at all doses. Vd of Ls-AKG28 was greater than that of Ls-AKG38 at the same dose (20 or 40 mg / kg).

[0596] Table 24. Summary of plasma PK parameters for total drug following administration of Ls-AKG28 at 10, 20, and 40 mg / kg IV.

[0597]

[0598] Plasma concentration versus time profiles for total drug following administration of Ls-AKG38 at 20, 40, and 80 mg / kg IVx1 are shown in FIG. 7. Figure 8

[0599] A summary of plasma PK parameters for total drug following administration of Ls-AKG38 at 20, 40, and 80 mg / kg IVx1 is shown in Table 25.

[0600] Plasma concentration versus time profiles for Ls-AKG38 were detectable from 5 minutes to 72 hours at all doses. Based on C max ​ / Dose and AUC / Following administration of 20, 40, and 80 mg / kg, plasma PK of Ls-AKG38 was linear (dose-proportional). At all doses, the plasma clearance (CL) of Ls-AKG38 (approximately 2.59 mL / h / kg) was higher than that of Ls-AKG28 (approximately 1.67 mL / h / kg). At the same doses (20 or 40 mg / kg), the volume depletion (Vd) of Ls-AKG28 was greater than that of Ls-AKG38.

[0601] Table 25. Summary of plasma PK parameters for total drug after administration of Ls-AKG38 at 20, 40 and 80 mg / kg IV.

[0602]

[0603] Following administration of Ls-AKG28 at doses of 10, 20, and 40 mg / kg IV×1 and Ls-AKG38 at doses of 20, 40, and 80 mg / kg IV×1, the plasma concentration profiles of the total drug relative to time were as follows: Figure 7 and Figure 8 The data is shown in the figure. In the single IV dose study, plasma concentrations of Ls-AKG28 and Ls-AKG38 were detectable over time from 5 minutes to 72 hours at all doses. Plasma PK of Ls-AKG28 was linear (dose-proportional) after administration of 10, 20, and 40 mg / kg. 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 higher 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 exposures of Ls-AKG28 (40 mg / kg) and Ls-AKG38 were approximately 73 times and 110 times higher than that of linezolid, respectively (using AUC from 0 to the last).

[0604] Compared to linezolid, both liposomal formulations exhibit significantly higher plasma AUC and greater persistence of the drug in circulation. The p-values ​​of both liposomal formulations show a linear dose-dependent relationship, as observed by the remarkably similar AUC / dose values ​​for each of Ls-AKG28 and Ls-AKG38.

[0605] Example 38. Plasma pharmacokinetics (PK) of total forms (encapsulated + released drug) Ls-AKG28 and Ls-AKG38 after multiple IV dosings in Sprague-Dawley rats.

[0606] This study was conducted to evaluate the PK of AKG28 and AKG 38 in rats administered at progressively increasing doses of Ls-AKG28 and Ls-AKG38 weekly for eight weeks. The study was conducted on Sprague-Dawley rats using IV administration. Ls-AKG28 (batch 275) and Ls-AKG38 (batch 276) were prepared as described in Examples 22 and 23, respectively. Plasma concentrations in rats were determined by HPLC. Following IV x 1 administration of Ls-AKG28 at 10, 20, and 40 mg / kg on days 1, 15, 29, and 43, the profiles of plasma concentrations versus time for total drug are shown in Tables 26 and Figure 9A , Figure 9B and Figure 9C Following IV x 1 administration of Ls-AKG28 at 10, 20, and 40 mg / kg on days 1, 15, 29, and 43, a summary of plasma PK parameters for total drug is shown in Table 26. Figure 9A , Figure 9B and Figure 9C All data in Tables 26, 27, 28, and 29 were used to generate the PK parameter results in Tables 30, 31, 32, and 33, respectively.

[0607] The plasma disposition of Ls-AKG28 was similar following the first dose and multiple doses. For Ls-AKG28 at 10 mg / kg, plasma Cmax and AUC were similar on days 1, 15, 29, and 43. For Ls-AKG28 at 20 mg / kg, plasma Cmax and AUC were increased on days 29 and 43. For Ls-AKG28 at 40 mg / kg, plasma Cmax and AUC were increased following dosing on days 1 to 43.

[0608] Following IV x 1 administration of Ls-AKG38 at 20, 40, and 80 mg / kg on days 1, 15, 29, and 43, the profiles of plasma concentrations versus time for total drug are shown in Tables 27 and Figure 10A , Figure 10B and Figure 10CSummary of plasma PK parameters for total drug after Ls-AKG38 administration at 20, 40, and 80 mg / kg IV x 1 on days 1, 15, 29, and 43 is shown in Table 27. Plasma disposition of Ls-AKG38 was similar in single and multiple dose PK after the first dose. Plasma Cmax and AUC increased from day 1 to day 43 for Ls-AKG38 at 20, 40, and 80 mg / kg. Given concerns of accelerated blood clearance (ABC) for pegylated liposomes containing non-cytotoxic drug payloads, the lack of increase in clearance rate in subsequent cycles was unexpected and suggests that liposomes containing AKG-28 or AKG-38 can be dosed chronically in mammals.

[0609] Table 26. Summary of plasma PK parameters for total drug after Ls-AKG28 administration at 10, 20, and 40 mg / kg IV x 1 on days 1, 15, 29, and 43.

[0610]

[0611] Table 27. Summary of plasma PK parameters for total drug after Ls-AKG38 administration at 20, 40, and 80 mg / kg IV x 1 on days 1, 15, 29, and 43.

[0612]

[0613] Example 39. Pharmacokinetic studies of Ls-AKG28 and Ls-AKG38 drug and liposome lipid in CD-1 mice.

[0614] This study was designed to determine the blood pharmacokinetic parameters of both drug and liposome lipid, as well as the stability of drug retention in liposomes of AKG-28 and AKG-38 liposome formulations in plasma in vivo. The study was performed on male CD-1 (20 to 22 g) mice (5 mice / time point) in the general protocol described in Example 7 above. Ls-AKG28 (batch 235) and Ls-AKG38 (batch 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 sampled at 0.083, 1, 3, 6, 24, and 48 hours post-injection. AKG-28, AKG-38, and the fluorescent liposome lipid marker (DiIC 18(3) Plasma concentration of liposomal phospholipids (DS). Using liposome batches 235 and 236 as standards, plasma concentrations of liposomal phospholipids were calculated from fluorescently labeled quantification. Because unencapsulated liposomal phospholipids were not expected to be... The tissue affinity of azolidinone drugs is many times higher than that of liposome-encapsulated drugs (e.g., in rats, the Vd of unencapsulated AKG-28 was 33.27 to 43.74 mL / kg compared to liposome-encapsulated AKG-28). Supported by the Vd of 2,291.26 mL / kg for the zolidinone linezolid (see Example 37), plasma drug concentrations are primarily attributable to the liposomal associated drug, and the plasma drug-liposomal lipid (DL) ratio normalized relative to the original (pre-injection) DL value is used as a measure of liposomal drug retention. Non-compartmental PK analysis was performed using Summit Research Services, PK Solutions 2.0. For Ls-AKG28 and Ls-AKG38, this PK software was used to estimate the maximum plasma concentration (C0). max ), the maximum plasma concentration divided by the dose (C max / dose), time to Cmax (T) max ), and the final measured concentration (C) last ), the time of the final concentration measurement (T) last From 0 hours to the last time point (AUC0-) last ) and 0 hours to infinity (AUC) 0-inf The plasma concentration relative to the area under the time curve, AUC 0-last Divide by dose (AUC) 0-last / dose), clearance rate (CL), volume of distribution (Vd), and clearance half-life.

[0615] The application of Ls-AKG28 ( Figure 11A ) and Ls-AKG38 ( Figure 11B Following this, the plasma concentration spectrum of the drug versus time was analyzed. A summary of the plasma PK parameters for Ls-AKG28 and Ls-AKG38 is shown in Table 28, and a summary of the plasma PK parameters for liposomal phospholipids is shown in Table 29. The kinetics of the DL ratio, indicating the stability of drug encapsulation in vivo, were also analyzed. Figure 11C As shown in Table 30.

[0616] Ls-AKG28 exhibits near-perfect in vivo stability, with no drug loss detected in mice up to 48 hours after intravenous injection. Using a single exponential equation (R0...) 2 =0.822), the drug release half-life of Ls-AKG28 is 866.3 hours. Ls-AKG28 has a faster drug release rate. Using a single exponential equation (R² = 0.822), 2=0.950), the drug release half-life of Ls-AKG38 is 22.9 hours.

[0617] Table 28. Summary of plasma PK parameters of the drugs after administration of Ls-AKG28 and Ls-AKG38.

[0618]

[0619]

[0620] Table 29. Summary of plasma PK parameters for liposomal phospholipids after administration of Ls-AKG28 and Ls-AKG38.

[0621]

[0622]

[0623] Table 30. Plasma drug to liposomal phospholipid ratios of Ls-AKG28 and Ls-AKG38 after IV administration in mice.

[0624]

[0625] Example 40. Pharmacokinetic study of Ls-AKG28 and Ls-AKG38 in mice after multiple administrations of liposomes in the presence of the ABC effect.

[0626] It has been shown that, following repeated injections, the production of anti-PEG antibodies leads to faster clearance of liposomes containing PEG-lipid conjugates (polyethylene glycol-modified liposomes) (Ishida et al. Journal of Controlled Release 105 (2005) 305-317; Laverman et al. JPET 298 (2001) 607-612), a phenomenon known as the accelerated blood clearance (ABC) effect. This study was conducted to determine whether the ABC effect existed after repeated administration of different doses of Ls-AKG28 and Ls-AKG38 with different compositions. Liposomes were prepared according to Examples 33 to 35, batches 231, 232, 233, and 234. The study was conducted on male CD1 mice, generally as described in Example 7. Five mice were used in each group. Plasma concentrations of AKG-28 and AKG-38 in mice were determined by HPLC. Mice were injected with the specified doses and formulations once per week for a total of four injections. Plasma drug levels were measured at 6-hour intervals following the first and fourth doses. Figure 12). None of the test groups had a significantly accelerated clearance at the 4th injection (two-tailed, unequal variance t-test all p values > 0.05). This data establishes that these liposomes Azolides can be administered chronically over multiple weeks without a significant negative impact on drug exposure.

[0627] Table 31. Plasma drug concentrations of Ls-AKG28 and Ls-AKG38 following administration of Ls-AKG28 and Ls-AKG38. Abbreviations: SOS, IN TEA-SOS; AS, 0.5 M ammonium sulfate; Chol, cholesterol content expressed as mol% of the sum of cholesterol and HSPC; DL, drug to lipid ratio, g / mol of liposome phospholipid; %ID - percent of the injected dose averaged per group; SD - standard deviation.

[0628]

[0629] This data indicates that there was no increase in blood clearance of the liposomal AKG-28 or liposomal AKG-38 of the disclosure after four cycles of treatment, which is in contrast to other pegylated liposomes that do not contain a cytotoxic drug associated with the liposome that have been previously reported.

[0630] Example 41. Dose-dependent tolerability of liposomal AKG-28 and liposomal AKG-38 in CD-1 mice

[0631] The objective of this study was to evaluate the tolerability of Ls-AKG28 and Ls-AKG38 injected as a single agent at different doses in mice. Ls-AKG28 (50, 65, 90, or 100 mg / kg / dose) or Ls-AKG38 (50, 90, 120, or 200 mg / kg / dose) was administered once a week by intravenous injection (tail vein) to 20- to 22-gram female CD-1 mice (5 / group) for 4 weeks. Liposomal formulations were prepared as previously described in Example 33 (Ls-AKG28 lot 231 and Ls-AKG38 lot 232). Control groups were injected with an equal volume of HEPES-buffered saline (HBS, pH 7) once a week for 4 weeks. Body weights were measured 3 times a week throughout the study, and the data are expressed as percent change in body weight relative to the body weight measured on day 0.

[0632] At the end of the study (72 hours after the last treatment), animals were humanely euthanized using CO2 inhalation. Blood samples were collected by cardiac puncture and transferred to EDTA pre-filled microvials for hematological analysis (Homological ADVIA 120 / 2120i analyzer) and to lithium heparin pre-filled microvials for plasma isolation. Plasma was isolated from the cellular fraction by centrifugation at 10000 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 pre-filled with 10% buffered formalin, replaced with 70% ethanol after 24 hours. Tissues were embedded in paraffin, sectioned, stained with hematoxylin and eosin (H&E) and histopathological evaluation was performed by a board-certified veterinary pathologist.

[0633] As shown in Figures 6A and 6B, no significant effect on mouse body weight was observed for both Ls-AKG28 and Ls-AKG38 when treated with up to 90 mg / kg (for Ls-AKG28) and 200 mg / kg (for Ls-AKG38) for a total of four weekly doses, relative to the saline control group. Figure 13A and Figure 13B As shown in Figures 6A and 6B, no significant effect on mouse body weight was observed for both Ls-AKG28 and Ls-AKG38 when treated with up to 90 mg / kg (for Ls-AKG28) and 200 mg / kg (for Ls-AKG38) for a total of four weekly doses, relative to the saline control group.

[0634] Relative to the control group, red blood cell count and hematocrit were not significantly reduced in mice treated with high doses of Ls-AKG38 (90, 120 and 200 mg / kg) relative to the saline control group ( Figure 13C ). No such effect was observed in mice treated with Ls-AKG28. Platelet count was found to be significantly reduced relative to the control group ( Figure 13C ) in mice treated with the highest dose of 90 mg / kg Ls-AKG28, but still less than 25% reduced compared to the saline control. Treatment with Ls-AKG28 or Ls-AKG38 did not significantly affect white blood cell (WBC) count or blood liver enzymes (ALT and AST) count.

[0635] Histopathological analysis showed no findings related to the test samples in animals receiving 50 and 65 mg / kg Ls-AKG28 ( Figure 13D). In the liver, spleen, and kidney of animals receiving 90 mg / kg LS-AKG28, there were test article-related findings consisting of minimal vacuolation of macrophages, including Kupffer cells. Treatment with LS-AKG38 at doses of 90 mg / kg and 120 mg / kg was associated with test article-related findings in the liver and spleen. In the liver, Kupffer cells had mild to moderate vacuolation and hypertrophy at 50 and 90 mg / kg, moderate vacuolation and hypertrophy at 50 and 120 mg / kg, and mild multifocal aggregation of vacuolated macrophages at 90 mg / kg and 120 mg / kg.

[0636] Treatment with the highest dose of LS-AKG38 (200 mg / kg) was associated with minimal to mild multifocal mixed cell infiltrates in the liver and spleen, and minimal individual hepatocyte necrosis and minimal focal hepatocyte necrosis in the liver Figure 13D ) in the liver. Since these microscopic findings occur frequently as background findings in this species, they were not considered test article-related.

[0637] Overall, both LS-AKG28 and LS-AKG38 monotherapy showed good in vivo tolerability in mice, even when the liposomal drugs were injected at the highest evaluated dose (90 and 200 mg / kg for LS-AKG28 and LS-AKG38, respectively).

[0638] Example 42. In vivo tolerability of LS-AKG28 and LS-AKG38 in combination with BDQ / PMD or BDQ / PMD / MOX in mice.

[0639] In this example, liposomal In vivo tolerability of oxazolidinones. When used to treat multidrug-resistant tuberculosis, the three-drug regimen of bedaquiline, pretomanid, and linezolid (BDQ / PMD / LNZ or BPL) or bedaquiline, pretomanid, and moxifloxacin (BDQ / PMD / MOXI or BPM) have shown strong activity in the clinic (Conradie et al (2020) N Engl J Med 382(10) 893-902 and Tweed et al. (2019) Lancet Respir Med 7(12) 1048-1058), but the BPL regimen has been limited by major toxicity associated with the addition of linezolid (Conradie et al (2020) N Engl J Med 382(10) 893-902). Here, the safety and tolerability of two liposomal oxazolidinones, Ls-AKG28 and Ls-AKG38, were evaluated when used as part of both the BPL and BPM regimens. CD-1 mice (5 / 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. In addition, mice were treated with the triple combination of BDQ, PMD, and moxifloxacin (MOXI) and liposomal oxazolidinones Ls-AKG28 (Lot 231) and Ls-AKG38 (Lot 232) as part of the BPL and BPM regimens. In vivo tolerability of oxazolidinones. When used to treat multidrug-resistant tuberculosis, the three-drug regimen of bedaquiline, pretomanid, and linezolid (BDQ / PMD / LNZ or BPL) or bedaquiline, pretomanid, and moxifloxacin (BDQ / PMD / MOXI or BPM) have shown strong activity in the clinic (Conradie et al (2020) N Engl J Med 382(10) 893-902 and Tweed et al. (2019) Lancet Respir Med 7(12) 1048-1058), but the BPL regimen has been limited by major toxicity associated with the addition of linezolid (Conradie et al (2020) N Engl J Med 382(10) 893-902). Here, the safety and tolerability of two liposomal oxazolidinones, Ls-AKG28 and Ls-AKG38, were evaluated when used as part of both the BPL and BPM regimens. CD-1 mice (5 / 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. In addition, mice were treated with the triple combination of BDQ, PMD, and moxifloxacin (MOXI) and liposomal oxazolidinones Ls-AKG28 (Lot 231) and Ls-AKG38 (Lot 232) as part of the BPL and BPM regimens. Co-treatment of mice with oxazolidinones.

[0640] Ls-AKG28 (50 mg / kg / dose) and Ls-AKG38 (90 mg / kg / dose) were administered intravenously via tail vein injection once per week for 4 weeks. The combination of BDQ, PMD, and MOXI (25 / 100 / 100 mg / kg / dose, respectively) was given daily via oral gavage, 5 times per week for 4 weeks. As additional controls, mice were treated with BDQ / PMD / MOXI or BDQ / PMD (25 / 100 mg / kg / dose, respectively) plus linezolid (LNZ) (100 mg / kg / dose daily, p.o.) 5 times per week for 4 weeks. Body weight measurements, tissue collection, and analysis were performed as previously described in Example 41.

[0641] As Figure 14A and Figure 14BAs shown in Table 6, no significant effects on body weight of mice were observed with Ls-AKG28 or Ls-AKG38 co-treatment with BDQ / PMD (BP) or BDQ / PMD / MOX (BPM) combinations during the study period. Both Ls-AKG28 and LsAKG38 in combination with BDQ / PMD or BDQ / PMD / MOX showed good tolerability and did not affect hematology or blood biochemistry in treated mice Figure 14C

[0642] Histopathology data Figure 14D ) showed no treatment-related changes in the case of Ls-AKG28 in combination with BDQ / PMD. Ls-AKG28 + BDQ / PMD / MOX combination had minimal events related to mixed cell and mononuclear cell infiltrates in the lung and heart. Treatment with Ls-AKG38 as a single treatment was associated with minimal test article-related findings in the liver (inflammatory infiltrates and hepatocyte necrosis). Administration of Ls-AKG38 + BDQ / PMD did not show any treatment-related findings, and the combination of Ls-AKG38 + BDQ / PMD / MOX was associated with minimal mixed cell infiltrates in the lung. Animals treated with the BDQ / PMD / LNZ combination were associated with treatment-related findings of inflammatory infiltrates, minimal hepatocyte necrosis in the liver, and an infiltrate of vacuolated macrophages in the lung. Thus, both Ls-AKG28 (50 mg / kg / dose) and Ls-AKG38 (90 mg / kg / dose) administered once weekly for 4 weeks in combination with BDQ / PMD or BDQ / PMD / MOX showed good tolerability in mice.

[0643] Example 43. Effect of dose scheduling on tolerability of Ls-AKG28 and Ls-AKG38 in combination with BDQ / PMD in mice.

[0644] ​In this study, the in vivo tolerability of Ls-AKG28 (50 mg / kg / dose) or Ls-AKG38 (100 mg / kg / dose) administered twice weekly was compared to Ls-AKG28 (100 mg / kg / dose) or Ls-AKG38 (200 mg / kg / dose) given once weekly. Liposomes were prepared according to Example 25 (Ls-AKG38, lot 279) and Example 26 (Ls-AKG28, lot 281). The two liposomal drugs were injected into CD-1 female mice (5 animals / group) alone or in combination with BDQ / PMD (BP). BDQ / PMD (25 and 100 mg / kg / dose, respectively) were given by daily oral gavage, 5 times per week for 4 weeks. The control group was injected with HEPES-buffered saline (HBS, pH 7) once weekly for 4 weeks. Blood and tissue samples were collected and analyzed as described above in Examples 41 and 42.

[0645] Neither the twice weekly nor the once weekly administration of Ls-AKG28 or Ls-AKG38 at the higher doses affected body weight Figure 15A and Figure 15B ) or blood cell counts and biochemistry Figure 15C ) in mice in both single and combination therapy.

[0646] Histopathological analysis of the collected tissues Figure 15D ) showed minimal interstitial mixed cell infiltrates composed of macrophages and neutrophils in 2 of 5 mice receiving Ls-AKG28 at 50 mg / kg (2 qw) and mild interstitial mixed cell infiltrates in 1 of 5 animals receiving Ls-AKG28 at 100 mg / kg (1 qw).

[0647] In the lungs of mice receiving Ls-AKG28 + BP at 50 mg / kg (1 qw), there were minimal interstitial infiltrates composed of macrophages (1 of 5 animals) or mixed (macrophages and neutrophils) inflammatory cells (3 of 5 mice). In mice receiving Ls-AKG28 + BP at 100 mg / kg (1 qw), 4 of 5 animals had minimal interstitial mixed cell infiltrates. Additionally, 2 of 5 animals receiving Ls-AKG28 + BP at 100 mg / kg (1 qw) had minimal multifocal foreign body granulomas associated with pale basophilic foreign bodies in the lung. Since these microscopic findings are frequently present as background findings in this species, they were not considered to be related to the test article, including minimal multifocal mixed cell infiltrates and minimal individual hepatocyte necrosis in the liver of 1 of 5 animals receiving Ls-AKG28 + BP at 50 mg / kg (2 qw).

[0648] Similar microscopic findings associated with Ls-AKG38 treatment (alone or in combination) were not considered test article-related, including minimal to mild increase in extramedullary hematopoiesis (EMH) in the liver and spleen, minimal to mild multifocal mixed cell infiltrates in the liver and minimal single hepatocyte necrosis in the liver, minimal focal hepatocyte necrosis, minimal focal foreign body granulomas in the lung (associated with pale basophilic foreign bodies), and minimal mixed cell infiltrates in the lung. These findings were not considered treatment-related due to their mild nature, sporadic incidence, presence in the saline control group, and occurrence as a common background finding in this species.

[0649] Accordingly, both Ls-AKG28 and Ls-AKG38 (alone or in combination with BDQ / PMD) were well-tolerated in mice administered at doses of 50 mg / kg and 100 mg / kg twice weekly or at double doses of 100 mg / kg and 200 mg / kg once weekly, and did not affect body weight, hematology, or histopathology of the treated animals.

[0650] Example 44. In vivo tolerability of Ls-AKG28 and Ls-AKG38 in rats.

[0651] The objective 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 Ls-AKG-38 (20, 40, or 80 mg / kg / dose) once weekly by intravenous injection (tail vein) for 8 weeks. Control groups were injected with an equivalent volume of HEPES-buffered saline (HBS, pH 7) once weekly for 8 weeks. Prior to the study endpoint, animals were humanely euthanized by exsanguination from the abdominal aorta following isoflurane anesthesia. Blood and tissue samples were collected for evaluation of clinical pathology parameters. Representative samples of tissues were collected and stored in 10% neutral buffered formalin, embedded in paraffin, sectioned, mounted on slides, stained with hematoxylin and eosin, and histopathology evaluation was performed by a board-certified veterinary pathologist. Hematology analysis of blood was performed using the Homological ADVIA 120 / 2120i analyzer and blood biochemistry was analyzed using the Cobas 6000 analyzer.

[0652] The following parameters and endpoints were also evaluated: mortality and moribundity checks, clinical observations, body weight, food consumption, nerve conduction velocity (NCV) and muscle action potential (MAP), functional observation battery (FOB).

[0653] Nerve conduction velocity (NCV) and muscle action potential (MAP) were performed at week 8. During the recording time, animals were anesthetized with isoflurane. Tail nerve NCV measures the conduction velocity in the tail nerve, which runs along the center bone of the tail. This nerve is about 50% longer than any other nerve in the rat and is particularly susceptible to length-dependent distal axonopathy. NCV is measured over a distance of 50 mm, and is sensitive to nodal current and transmembrane current, 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 synchronicity of activated fibers. Data were recorded with a recording electrode located about 10 mm below the hairline (determined visually) on the tail and a stimulating cathode located 50 mm further. The amplitude and onset latency of the recorded signal were noted, and the velocity was calculated by dividing the distance between the stimulating cathode and the recording electrode by the absolute onset latency of the initial depolarizing current.

[0654] Toe nerve NCV measures the conduction velocity of the sensory toe nerve. The toe nerve is the distal portion of the sciatic nerve that innervates the dorsal surface of the hind paw. Nerve conduction velocity is sensitive to nodal current and transmembrane current, structure and mean cross-sectional diameter of the responding axon, and the integrity of the associated myelin sheath. Data were recorded with a recording electrode located at the lateral malleolus of the hind paw and a stimulating cathode located at the base of the second toe of the hind paw. The amplitude and onset latency of the recorded signal were noted, and the velocity was calculated by dividing the distance between the stimulating cathode and the recording electrode by the absolute onset latency of the initial depolarizing current.

[0655] Tibial motor conduction (Onset Latency) measures the response characteristics of the rat hind paw intrinsic muscles following stimulation of motor fibers at the distal portion of the tibial nerve. Data were recorded with a recording electrode located in the lateral dorsalis muscle of the hind paw (equivalent to the extensor digitorum brevis in humans) and a stimulating cathode located at the lateral malleolus of the hind paw, near the ankle joint. Nerve conduction velocity in motor axons was estimated from the onset latency of the evoked compound muscle action potential (CMAP). The amplitude of the CMAP was determined at the peak of the response following supramaximal stimulation of the relevant nerve.

[0656] There were no Ls-AKG28, Ls-AKG38 related unexpected deaths, clinical observations, or effects on body weight ( Figure 16A andFigure 16B ), NCV and MAP (Table 34), FOB (Table 35), food consumption, coagulation parameters, organ weights, or macroscopic findings (data not shown). Although potency adjustment of Ls-AKG28 or Ls-AKG38 (based on free drug potency against M. tuberculosis Erdmann strain in Example 2) was increased by 16.5-fold compared to linezolid, the maximum decreased nerve conduction velocity of the tail or left toe nerve was less than 5% in any of the liposomal treatment groups.

[0657] Administration of Ls-AKG28 and Ls-AKG38 at doses > 20 mg / kg resulted in statistically significant decreases in platelet counts (up to 20% difference compared to control groups). No additional effects on other hematological and blood biochemical parameters were observed (Tables 32, 33).

[0658] Administration of Ls-AKG28 by intravenous injection at doses > 10 mg / kg / dose, once weekly for 8 weeks, to male Sprague-Dawley rats resulted in microscopic findings in the spleen, kidney, and liver (Table 36). The spleen had minimal to moderate macrophage vacuolization (with basophilic granulation) and minimal to mild basophilic material accumulation in rats given 20 or 40 mg / kg / dose. Glomerular mesangial cell vacuolization was minimal in the kidney of rats given 40 mg / kg / dose. The liver had minimal centrilobular single cell necrosis and minimal to mild centrilobular hepatocyte degeneration at all doses.

[0659] Table 32 Effect of liposomal AKG-28 and AKG-38 on liver enzyme levels in blood after eight weeks of dosing in male Sprague Dawley rats.

[0660]

[0661] Table 33 Effect of liposomal AKG-28 and AKG-38 on blood cell counts and hematocrit (HCT) after eight weeks of dosing in male Sprague Dawley rats.

[0662]

[0663]

[0664] Table 34 Effect of liposomal AKG-28 and AKG-38 on nerve conduction after eight weeks of dosing in male Sprague Dawley rats.

[0665]

[0666] Table 35. Effect of liposomal AKG-28 and AKG-38 on neurological observations groups in male Sprague Dawley rats after eight weeks of dosing.

[0667]

[0668] Table 36. Summary of microscopic findings in tissues in male Sprague Dawley rats after eight weeks of dosing with liposomal AKG-28 and AKG-38.

[0669]

[0670] a The number in parentheses represents the number of animals with that finding.

[0671] Ls-AKG38 administered at doses > 20 mg / kg / dose resulted in minimal centrilobular single cell necrosis and minimal to mild centrilobular hepatocyte degeneration in the liver microscopic findings at all doses.

[0672] In contrast, rats dosed with Ls-AKG28 and Ls-AKG38 had increased incidence of liver single cell necrosis at all doses compared to rats dosed with linezolid. Rats dosed with Ls-AKG28 and Ls-AKG38 had similar incidence and severity of centrilobular hepatocyte degeneration in the liver at all doses. Rats dosed with Ls-AKG28 also had vacuolated macrophages and basophilic material in the spleen at 20 or 40 mg / kg / dose and glomerular mesangial cell vacuolation in the kidney at 40 mg / kg / dose.

[0673] In summary, Ls-AKG28 administered at levels of 10, 20, and 40 mg / kg / dose by multiple intravenous injections over 8 weeks was well tolerated in rats. Ls-AKG38 administered at levels of 20, 40, and 80 mg / kg / dose by multiple intravenous injections over 8 weeks was well tolerated in rats. Example 2 shows that AKG-28 was 33 times more potent than linezolid in killing Mycobacterium tuberculosis (Erdmann strain) in vitro, while AKG-38 was 17 times more potent than linezolid. Thus, when potency is corrected, rats did not show significant neuropathy (change in nerve conduction velocity), liver enzyme elevation, decrease in red blood cell count or hematocrit, or decrease in body weight at linezolid equivalent doses of 1320 to 1336 mg / kg, which is 16.5 times higher than the clinically relevant dose of linezolid, 80 mg / kg.

[0674] Example 45. Efficacy of liposomal AKG-28 and AKG-38 in combination with bedaquiline and pretomanid or in combination with bedaquiline (B), pretomanid (Pa) and moxifloxacin (M) in Kramnik (C3HeB / FeJ mice model of M. tuberculosis infection).

[0675] The C3HeB / FeJ (Kramnik) mouse model of infection exhibits late hypoxic caseous granulomas in the lung after TB infection (Driver E., et al., Antimicrobial Agents and Chemotherapy, 2012, vol. 56, p. 3181-3195). The lung pathology observed in C3HeB / FeJ mice more closely resembles the lesion pathology and heterogeneity in bacterial population as seen in TB patients and was used to evaluate the efficacy of liposomal AKG-28 and AKG-38 at intermediate weekly doses of 50 and 90 mg / kg. Ls-AKG28 (batch 275) and Ls-AKG38 (batch 276) were prepared as described in Examples 22 and 23, respectively. Lung pathology of C3HeB / FeJ mice showed three different types of lesions classified as caseous necrotic lesions delineated by a collagen rim (type I), fulminant neutrophilic alveolitis (type II) and cellular lesions (type III) (see Irwin et al. (2015) Dis Model Mech 8, 591-602). 8- to 10- week-old C3HeB / FeJ female mice were infected with LDA (low dose aerosol infection). Mice were infected at a target of about 50 to 75 bacilli / mouse (Erdman strain) using a Glas-Col inhalation exposure system. At day 1 post-infection, 5 mice per round of aerosol run were sacrificed to determine bacterial uptake.

[0676] Eight weeks post-infection, eight mice were sacrificed to determine bacterial load in the lungs and spleen at the start of treatment. Mice were weighed prior to sacrifice. Gross pathology observations were made on the lungs and spleen. The entire lung and spleen were removed and frozen at -80°C. Previously frozen tissues were recovered and homogenized in lx PBS using a Precellys homogenizer. Lung and spleen homogenates were plated on 7H11 agar square plates. CFU counts were performed after 3-5 weeks of incubation at 37°C in a dry air incubator. Treatments were administered by oral gavage or intraperitoneal (i.p.) injection, starting 8 weeks post-infection and continuing for 4-6 consecutive weeks (M-F gavage, i.p. injection once per week). Bedaquiline (B), pretomanid (Pa), moxifloxacin (M), and linezolid (L) were given by gavage at 200 μL / dose, 5 days per week for 4 or 6 weeks. Bedaquiline (25 mg / kg) was given first, followed by pretomanid (100 mg / kg) no less than one hour later. Moxifloxacin (100 mg / kg) or linezolid (100 mg / kg) were given 4 hours after pretomanid administration. Liposomal formulations were given once per week for 4 or 6 weeks.

[0677] Mice were observed daily at the time of dosing and weighed at least once per week. Sacrifice was completed two weeks after the end of four or six weeks of treatment. Eight mice per treatment group were weighed prior to sacrifice. The entire lung and spleen were aseptically harvested for all treatment groups. Gross pathology observations of the lung and spleen were tabulated. Lungs were photographed for gross lesion analysis. The entire lung and spleen were frozen at -80°C. Previously frozen tissues were recovered and homogenized in lx PBS or 10% bovine serum albumin (BSA) in lx PBS (to avoid drug carryover, *see explanation below) using a Precellys homogenizer. Lung and spleen homogenates were plated on 7H11 agar or 7H11 square plates containing charcoal and serially diluted in lx PBS or 10% BSA. CFU counts were performed after 5 weeks of incubation at 37°C in a dry air incubator.

[0678] The addition of Ls-AKG28 to the BPaM treatment resulted in a further 0.64 logio CFU reduction in the lung vs. BPaM after 4 weeks of treatment, while BPaM + Ls-AKG38 treatment resulted in a further 0.25 logio CFU reduction vs. BPaM. In the NIX (BPaL) regimen, Ls-AKG28 or Ls-AKG38 replacement of linezolid (L) resulted in improved efficacy relative to BPaL over six weeks of treatment. Ls-AKG38 replacement of linezolid in the BPaL regimen did result in a significant improvement in efficacy compared to the BPaL treated group at 6 weeks of treatment. Specifically, BPaL treatment for 6 weeks resulted in a 4.18 logio CFU reduction with one of 8 animals having no CFU in the plate. BPa + Ls-AKG28 treatment for 6 weeks resulted in a 4.68 logio CFU reduction, which was not statistically different from BPaL. BPa + Ls-AKG38 treatment for 6 weeks resulted in a 5.26 logio CFU reduction with 2 of 8 animals having no CFU in the plate. This was a statistically significant reduction vs. BPaL (p = 0.04, Dunnett’s test).

[0679] In the spleen at 6 weeks of treatment, replacement of linezolid in the NIX regimen with either liposomal formulation resulted in a slight improvement in lung efficacy relative to the BPaL treated group. Treatment with the NIX regimen for 6 weeks resulted in a 3.56 logio CFU reduction with 3 of 8 animals showing no CFU in the plate. Treatment with BPa + Ls-AKG28 for 6 weeks resulted in a 4.18 logio CFU reduction with 5 of 8 mice showing no CFU in the plate. Treatment with BPa + Ls-AKG38 for 6 weeks resulted in a 4.38 logio CFU reduction with 6 of 8 mice showing no CFU in the plate. CFU burden in the spleen of mice treated with drugs for 6 weeks was low and close to the lower limit of detection of 0.66 logio CFU.

[0680] Table 37 - Lung Log CFU

[0681]

[0682] * 2 of 8 mice had no measurable CFU; all mice were listed at the limit of detection of 0.96 log CFU.

[0683] Table 38 - Spleen Log CFU

[0684]

[0685] * 3 of 8 mice had no measurable CFU

[0686] ** 5 of 8 mice had no measurable CFU;

[0687] ***6 of 8 mice had no measurable CFU; all mice are listed at the limit of detection of 0.66 log CFU.

[0688] All mice with no measurable CFU are listed at the limit of detection of 0.66 log CFU. This indicates that Ls-AKG38 and Ls-AKG28 are more effective than linezolid when combined with bedaquiline and preravaprevir (both drugs at moderately and highly tolerable doses) after only six weeks of treatment. As shown in Examples 42 and 43, both Ls-AKG28 and Ls-AKG38 can be safely dosed in this combination, at least up to twice the doses used in this study. It also indicates that when Ls-AKG28 is added to the BPaM regimen, CFU are further reduced in both the lung and spleen at this same highly tolerable dose.

[0689] Example 46. Efficacy of single treatment with liposomal AKG-28 in a Balb / c model of M. tuberculosis infection.

[0690] The regimen and dose-dependent efficacy of Ls-AKG28 was determined in comparison to free linezolid at clinically relevant doses of 50 and 100 mg / kg in a chronic Balb / c tuberculosis model. In the chronic Balb / c mouse model, bacterial load in the lung reaches steady state 4 to 5 weeks after M. tuberculosis infection (Lenaerts et al. (2005) AAC 49(6) 2294-2301). Ls-AKG28 (batch 286) was prepared as described in Example 28. 6 to 8 week old Balb / c female mice were obtained from Jackson Laboratories and mice were infected with LDA (low dose aerosol infection), i.e. using a Glas-Col inhalation exposure system to infect mice with approximately 50 to 100 bacilli / mouse of M. tuberculosis Erdman.

[0691] Mice (n=3) were sacrificed on day 1 post-infection to determine bacterial uptake. Whole lungs were aseptically harvested in Precellys tubes (Bertin cat# KT03961-1-396.7) and homogenized in 4 ml 1xPBS using a Precellys tissue homogenizer. Undiluted homogenate was transferred to two large 7H11 agar plates (150x15mm) and the plates were incubated in a dry air incubator at 37°C in sealed zip top bags for at least 21 days until colonies could be counted. Mice (n=5) were sacrificed on day 28 post-aerosol infection to determine bacterial load in lungs and spleen at the start of treatment. Mice were weighed prior to sacrifice. Lungs and spleen were grossly pathologically observed. Lungs (divided into left lobe and right upper [cranial] lobe + accessory lobe) and spleen were aseptically harvested and frozen at -80°C. The right lower lobe of lung [caudal] was collected in 4% paraformaldehyde (PFA) for histology. Previously frozen tissues were recovered and homogenized in 1xPBS using a Precellys homogenizer. Lung and spleen homogenate was plated on 7H11 agar square plates. CFU counts were performed after 3 to 5 weeks incubation at 37°C in a dry air incubator.

[0692] Linazolid was administered by oral gavage (200 uL / mouse) in 5% PEG-200 (Sigma P3015, batch MKBW3119V) / 95% (0.5%) methylcellulose (Sigma M0430, batch 031M0051) starting on day 28 (Mon) post-aerosol infection and continuing for 2 to 8 weeks, 5 days out of 7 per week. Ls-AKG28 was administered by i.p. injection once or twice per week at a dose of 50 or 100 mg / kg. Final sacrifice was performed 3 days after the last dosing day for mice treated for 2, 4 or 8 weeks with the drug. Mice were weighed prior to sacrifice. Lungs and spleen were grossly pathologically observed. Lungs (divided into left lobe, right upper lobe + accessory lobe) and spleen were aseptically harvested and frozen at -80°C. The right lower lobe of lung was collected in 4% PFA for histology. Previously frozen tissues were recovered and homogenized in 10% bovine serum albumin (BSA) in 1xPBS to avoid drug carryover. After homogenization, lung and spleen homogenate was serially diluted in 1xPBS and 10% BSA and then plated on 7H11 agar or 7H11 square plates containing charcoal. CFU counts were performed after 3 to 5 weeks incubation at 37°C in a dry air incubator.

[0693] The reduction in lung CFU counts is shown in Table 39, and the reduction in spleen CFU counts is shown in Table 40. Treatment with 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 two weeks, compared to a reduction of less than 0.15 Log10 CFU with linezolid at 100 mg / kg (q1×5). This reduction in the spleen with Ls-AKG28 was close to 3 Log10 CFU at 2 weeks. At eight weeks, all mice treated with Ls-AKG28 were completely sterile (or below the detection limits of 1.13 in the lungs and 0.66 in the spleen) compared to 2.45 Log10 CFU in the lungs and 3.15 Log10 CFU in the spleen with linezolid at a higher dose of 100 mg / kg. This single therapeutic activity is significant for mice lacking active combination partners such as bedaquiline. For zolidinones, this is surprising, and within a relatively short period of only eight weeks. For example, in an eight-week treatment, linezolid monotherapy showed log10 CFU counts in the range of 4 to 6 in Balb / c and C3HeB / FeJ mice (Lanoix et al. (2015) Dis Models Mech. 8, 603-610), and even at regimens of 3 to 14 doses / week up to 1000 mg / kg / week, the activity remained moderate (Bigelow et al. (2021) J Infec. Dis. 223 (11) 1855-1864).

[0694] Table 39 - Lung Log CFU

[0695]

[0696] *No measurable CFU was found in 6 / 6 mice; all mice are listed below the detection limit of 1.13 log CFU.

[0697] Table 40 - Spleen Log CFU

[0698]

[0699] *No measurable CFU was found in 4 / 5 mice.

[0700] **1 / 6 of the mice had no measurable CFU.

[0701] ***No measurable CFU was found in 6 / 6 mice; all mice are listed below the detection limit of 0.66 log CFU.

[0702] At a detection limit of 0.66 log CFU, all mice with no measurable CFU were listed.

[0703] Example 47. Efficacy of liposome AKG-38 in a rabbit endocarditis model of methicillin-resistant Staphylococcus aureus (MRSA).

[0704] Staphylococcus aureus infections, particularly those involving the intravascular system (e.g., IE; heart and hemodialysis device infections, etc.) are prevalent and associated with unacceptably high morbidity, mortality, and post-treatment recurrence rates. This is particularly true when such infections are caused by MRSA strains that are resistant to multiple drugs. Moreover, even when MRSA strains have minimal inhibitory concentrations (MICs) for vancomycin ("workhouse" anti-MRSA agent) within the "susceptible" range of the recognized Clinical Standards Laboratory Institute (CLSI), i.e., < 2 ug / ml, the clinical outcome is suboptimal.

[0705] A typical high-inoculum intravascular biofilm MRSA infection model, left-sided aortic valve rabbit IE, was used in 6-month-old and 2.2 to 2.5 kg female New Zealand white rabbits. Rabbits were anesthetized systemically with intramuscular injections of xylazine and ketamine. The fur over their right carotid artery was then clipped to expose the skin. The cut-down site over the right carotid artery was locally anesthetized with 1% lidocaine. A cut-down was then performed to expose the right carotid artery. It was isolated, proximally ligated, and then retrograde cannulated with a polyethylene catheter through the aortic valve into the left ventricle, where it was then secured in place and left in place for the duration of the study. For left-sided IE at 48 hours after catheter placement (to induce an aseptic aortic valve and ventricular vegetations), animals were challenged with approximately 2 x 10 5 IE was induced by IV challenge with the cfu MW2 strain. The MRSA strain MW-2 (USA 400-Clone Complex [CC] 1) used: i) was clinically derived; ii) was genome sequenced; iii) represented a common hospital-acquired MRSA clonotype; iv) was virulent in an experimental IE model; and v) was in vitro susceptible to daptomycin (DAP). Infection spread from the heart valve infected vegetations to the kidneys and spleen.

[0706] In different animal groups, liposomal AKG-38 (Ls-AKG38) was given at 40 mg / kg / dose once (in combination with DAP treatment) or twice (once in combination with DAP treatment; then a second infusion at the time of sacrifice after DAP treatment in the "relapse animal group" that did not receive further DAP treatment). Ls-AKG38 (batch 292) was prepared as described in Example 29. The first Ls-AKG38 infusion would be performed approximately 1 hour after the first DAP iv administration. DAP was given at a sub-lethal dose of 2 mg / kg daily for 4 days, alone or in combination with Ls-AKG38.

[0707] Animals were humanely euthanized on day 6 (DAP + single dose of Ls-AKG38 or DAP alone) or day 12 (two doses of Ls-AKG38 + DAP on day 1 and day 6) and key target organs were aseptically removed and quantitatively cultured (blood, cardiac vegetations; kidney and spleen for left IE). Quantitative target tissue cultures were performed by standard preparation of aseptically removed organs by weighing, homogenization, serial dilution and plating. Serial dilution and quantitative culture of blood was similarly performed. Data from blood cultures and each target organ for different treatment groups were calculated as mean and median log 10 cfu / ml or log 10 cfu / gm (± SD) for each tissue, respectively.

[0708] Preliminary data from the MRSA left ventricular endocarditis model in rabbits are shown in Table 41 below. Daptomycin alone or daptomycin plus a single dose of Ls-AKG38 showed no significant efficacy at day 6 post-inoculation. Surprisingly, a second injection of Ls-AKG38 produced significant efficacy at day 12, including sterilization in all five tissues in 4 / 5 rabbits and CFU reduction in multiple organs by more than 6 Log. This data indicates that Ls-AKG38 can be effective in treating endocarditis after cessation of daily daptomycin.

[0709] Table 41

[0710]

[0711] Example 48. In vitro activity of AKG-28 and AKG-38 against multiple non-tuberculous mycobacteria.

[0712] MIC testing was performed by using the microbroth dilution method of Mueller Hinton (MH) broth (cationically adjusted) (Obregon-Henao et al. (2015) Antimicrobial Agents Chemother 59, 6904-6912) to achieve the calcium and magnesium ion concentrations recommended in the CLSI standard M7-A7 (Becton Dickinson). MIC testing was also performed using the microbroth dilution method using 7H9 broth (Shang et al. (2011) PLoS One 6, e24726; Chan et al. (2010) Am J Respir Cell Mol Biol 43, 287-393) with the goal of optimizing the ability to detect more compounds with anti-NTM activity by using different broths in the microbroth dilution method. Sigma-Aldrich NTM were incubated on 7H11 agar plates (Becton Dickinson) at 35 to 37 °C in ambient air for 3 to 25 days (depending on the bacterial strain). CFU were removed from the agar plates and placed in MH broth with 0.05% Tween 80 and incubated at 35 to 37 °C in ambient air until the optical density (OD) absorbance was 0.08 to 0.1 (0.5 McFarland standard) after 7 days of growth. The bacterial cell suspension was then prepared by making a bacterial cell suspension in saline, matching the (OD) to 0.08 to 0.1 (0.5 McFarland standard). Sigma-Aldrich

[0713] Broth (MH) 180 μΐ was added to the first column in a 96-well plate. Then 100 μΐ of broth (MH) was added to the other columns in the 96-well plate. Compounds were prepared in DMSO using 1.28 mg / mL and immediately used for a test range of 64 to 0.062 μg / ml and 20 μΐ of compound was added to the first column well and serially diluted 100 μΐ. Finally, 100 μΐ of NTM cell suspension was added to all wells except the control wells of just media. QC reagents specific to each organism 1) bacterial only negative control 2) media only negative control 3) or clofazimine positive drug control 4) optional E. coli control.

[0714] ​On day 3, the OD of RGM was determined. Thereafter, the plate was assayed by using the Resazurin Microtiter Assay Plate 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 employs the addition of resazurin (7-hydroxy-3H-phen xazine-3-ketone 10-oxide) to the MIC 96-well plate. Resazurin is a blue dye that has a weak fluorescence until it is irreversibly reduced to a pink and highly red fluorescent Thymol resorufin. Resazurin is used as a redox indicator in bacterial cell viability MIC assays.

[0715] The results show that both AKG-28 and AKG-38 are generally more effective than terizidone across a range of different NTM species and strains. This includes M. avium, M. chelonae, M. abscessus and M. kansasii. Terizidone was more active in only one of the three strains evaluated, M. massiliense.

[0716] Table 42

[0717]

[0718] Example 49. Activity of selected compounds against drug resistant strains of M. tuberculosis in vitro.

[0719] The compounds of the disclosure that showed activity against drug susceptible strains of M. tuberculosis were further evaluated for activity against several multi-drug resistant (MDR) clinical isolates M70, M28, M94, M14 (Cheng A.F., et al., 2004, Antimicrob. Agents Chemother. v. 48, p. 596-601) and TN5904 (Palanisamy G.S., et al., 2008, Tuberculosis (Edinb.) vol. 88 p. 295-306). These strains are characterized by the following resistance profiles:

[0720] Table 43. Drug resistance profile of M. tuberculosis MDR strains used in the study. (Abbreviations: R - resistant; S - susceptible; STR - streptomycin, INH - isoniazid; RIF - rifampin, EMB - ethambutol, PZA - pyrazinamide.

[0721] Table 43

[0722] Strain / drug STR INH RIF EMB PZA M70 R R R S R M28 S R R R R M14 R R S S R M94 R R S S S TN5904 R R R S R

[0723] MICs of test compounds, including comparators / resistance controls (RIF, INH, STR, moxifloxacin (MOX) and linezolid (LNZ)), were determined using broth microdilution method with Alamar Blue endpoint (MABA) essentially as described in Example 2 with the following modifications. Test compounds and comparators serially diluted in DMSO at two-fold factor were added to wells of 96-well assay plates containing 100 μL of 7H9-glycerol medium supplemented with ADC. Compounds were diluted in DMSO so as to keep the compound concentration within the desired range and to keep the final DMSO concentration in the wells at 2% (M70, M28, M94) or 2.5% (M14, TN5904), except for STR which was serially diluted and added as an aqueous solution due to low solubility in DMSO. Bacterial stocks of MDR strains and susceptible H37Rv strain (positive control) were removed from cold storage, thawed and diluted with 7H9-ADC-glycerol medium to provide a bacterial density of 10 6 CFU / mL (H37Rv, TN5904), 2 x 10 6 CFU / mL (M70, M14) or 3 x 10 6 CFU / mL (M28, M94) and 50 μL of the 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. Plates were sealed in Ziplock bags, incubated at 37°C and bacterial growth was monitored by periodic reading of optical density at 600 nm (OD600). On day 14 (if OD600 reached or exceeded 0.40) or day 17, 15 μL of Alamar Blue solution was added to the wells, incubation was continued and color of the incubation mixture was recorded after three days (seven days in the case of the slowly growing M28 strain). The lowest continuous antimicrobial concentration at which two-fold serial dilutions did not produce a visible color change of Alamar Blue relative to the drug-free control well was considered the MIC of these compounds. MIC determinations based on OD600 reduction > 80% relative to the no drug control well were consistent with the MABA results. A two-fold change in MIC for two wells was considered significant. Results are summarized in Table 44 below.

[0724] Table 44. Minimum inhibitory concentrations (MIC) in vitro (MABA assay) of various compounds in drug sensitive and drug resistant strains of M. tuberculosis.

[0725]

[0726] The comparator / control compounds RIF, IHN, MOX and STR show the expected in vitro activity against DR-TB / MDR-TB strains as well as H37Rv. All the test compounds of the present disclosure show at least the same activity against MDR-TB strains as against the drug sensitive strain H37Rv, within the range of variation of the typical MIC assay. The highest activity is shown by AKG-28, followed by AKG-38 and AKG-3. Compounds AKG-28 and AKG-38 stand out as the most active compounds, even compared to their structurally similar analogues.

[0727] Aspects of the present disclosure can be used alone, in combination, or in multiple arrangements not specifically discussed in the foregoing implementations, and thus application is not limited to the details of the components or arrangements set forth in the foregoing description or shown in the drawings. For example, aspects described in one implementation can be combined with aspects described in other implementations in any manner.

[0728] While specific embodiments of the subject disclosure have been discussed, the above specification is illustrative and not restrictive. Many variations of the disclosure will become apparent to those skilled in the art upon review of the specification. The scope of the disclosure should be determined, not with reference to the above description, but instead with reference to the appended claims, along with their full scope of equivalents, and the specification. The disclosure is further defined by the following claims.

[0729] INCORPORATED BY REFERENCE

[0730] All publications, patents, and patent applications cited in this specification are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated by reference for all purposes.

Claims

1. A compound of formula I or a pharmaceutically acceptable salt thereof: in R1 is selected from groups A to N: And R2 is an amine or acetamide.

2. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R2 is -NHCOCH3.

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

4. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is a compound of formula 1d or a pharmaceutically acceptable salt thereof:

5. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is a compound of formula 1e or a pharmaceutically acceptable salt thereof:

6. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the pharmaceutically acceptable salt of said compound is a compound of formula 1b or a pharmaceutically acceptable salt thereof:

7. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the pharmaceutically acceptable salt of said compound is:

8. A liposome composition comprising the compound of any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof.

9. The liposome composition of claim 8, comprising liposome vesicles, said liposome vesicles comprising a compound of formula I or a pharmaceutically acceptable salt thereof.

10. The liposome composition of claim 8, wherein the liposome composition comprises a compound of formula I or a pharmaceutically acceptable salt thereof within a liposome vesicle, the liposome vesicle comprising a polyanion scavenger, phospholipids and cholesterol, and the liposome vesicle is in an aqueous medium.

11. The liposome composition of claim 10, wherein the polyanion scavenger is sucrose octasulfate triethylammonium or ammonium sulfate.

12. The liposome composition of claim 10, wherein the pharmaceutically acceptable salt of the compound of formula I is a sulfate encapsulated within the liposome vesicles.

13. The liposome composition of claim 10, wherein the phospholipid is phosphatidylcholine.

14. The liposome composition of claim 13, wherein the phosphatidylcholine is distearate phosphatidylcholine or hydrogenated soybean phosphatidylcholine.

15. The liposome composition of claim 13, wherein the molar ratio of phosphatidylcholine to cholesterol in the liposome composition is from 60:40 to 35:

65.

16. The liposome composition of claim 13, wherein the molar ratio of phosphatidylcholine to cholesterol is 45:

55.

17. The liposome composition of claim 9, wherein the liposome composition further comprises lipids conjugated to a polymer.

18. The liposome composition of claim 17, wherein the lipid conjugated with the polymer is PEG 2000-distearylglycerol or PEG 2000-distearylphosphatidylethanolamine.

19. The liposome composition of claim 13, wherein the liposome vesicles comprise: a. A sulfate of compound of formula I encapsulated within the liposome vesicles; b. Phosphatidylcholine and cholesterol in a molar ratio of 60:40 to 35:65 in the liposome composition; and c. Lipids conjugated with polymers.

20. The liposome composition of claim 19, wherein the liposome composition comprises liposome vesicles of a sulfate of a compound of formula I, and the liposome vesicles comprise phosphatidylcholine HSPC, cholesterol, and a polymer-conjugated lipid in a molar ratio of 45:55:2.25, wherein the polymer-conjugated lipid is PEG-distearate phosphatidylethanolamine.

21. The liposome composition of claim 19, wherein the lipid conjugated with the polymer is a PEG-lipid in an amount of 4 to 8 mol% relative to the phosphatidylcholine.

22. The liposome composition of claim 21, wherein the liposome composition comprises: aZ-average particle size is 80nm to 130nm; and b. The polydispersity index is less than 0.

1.

23. Use of the liposome composition according to any one of claims 8 to 22 in the preparation of a medicament for treating bacterial infections.

24. A method for preparing a liposome composition, comprising the following steps: (i) Prepare liposomes containing phospholipids, cholesterol and PEG-lipids and having an internal space containing a trapping agent in a medium; (ii) Contact the liposomes with the compound or pharmaceutically acceptable salt thereof as described in any one of claims 1 to 7 in an aqueous medium to encapsulate the compound or pharmaceutically acceptable salt thereof in the liposomes; (iii) Remove unencapsulated compounds; as well as (iv) The liposomes are provided in a physiologically acceptable medium suitable for parenteral use.

25. The method of claim 24, wherein the trapping agent in step (i) is ammonium sulfate, and in step (ii) the concentration of the trapping agent in the internal space of the liposome is 0.25 M to 0.5 M before drug loading.

26. The method of claim 25, wherein the concentration of the trapping agent in the internal space of the liposome in step (i) is 0.5 M.

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