Crystal morphology of omadacycline, its synthesis method, and its use.

Abstract

This invention provides a method for purifying the free base form of omadacycline. [Solution] A method for purifying the free base form of omadacycline represented by formula (1), JPEG2026067882000027.jpg30145 The above method is a method comprising the step of subjecting a solution containing the crude free base form of omadacycline to purification by high-performance liquid chromatography (HPLC), wherein the HPLC includes the use of a modifier selected from the group consisting of weak acids and organic amines, thereby obtaining a solution containing the HPLC-purified free base form of omadacycline.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 037,807, filed on June 11, 2020, the entire content of which is incorporated herein by reference.

Background Art

[0002] Introduction Omadacycline (also known as OMC or PTK 0796) is a 9 - aminomethyltetracycline derivative that is currently in advanced clinical development for the treatment of various bacterial infections. The structure of omadacycline is shown below.

[0003]

Chemical Formula

[0004] The chemical synthesis of omadacycline has been previously described, for example, in U.S. Patent No. 9,434,680, U.S. Patent No. 9,522,872, and U.S. Patent No. 8,383,610, the entire content of each of which is incorporated herein by reference. An exemplary procedure for the synthesis of omadacycline is shown in Scheme 1 below. In this procedure, minocycline (A) is used as the starting material and is subjected to alkylation at the 9 - position with N - (hydroxymethyl) phthalimide in the first step. The product (B) of this conversion is reacted with methylamine, which results in deprotection of the amine group and formation of intermediate (C). In the next step, intermediate (C) is reacted with trimethylacetaldehyde under reductive alkylation conditions to obtain crude omadacycline in the free - base form.

[0005]

Chemical Formula

[0006] After synthesis, the crude omadacycline free base is purified and converted to the tosylate form, which is used to prepare pharmaceutical compositions of omadacycline. Purification of the crude omadacycline free base involves the use of high-performance liquid chromatography (HPLC) and collection of the HPLC fraction containing the purified omadacycline free base. Subsequently, the omadacycline free base in the HPLC fraction is concentrated by extraction with dichloromethane. This purification procedure is time-consuming; for example, producing 1 kilogram of pure omadacycline may require processing times exceeding 70 hours. This exposes the omadacycline to high temperatures for extended periods during solvent evaporation, leading to decomposition. Furthermore, this purification procedure requires a large amount of solvent, thereby generating a considerable amount of waste. Therefore, a simpler and more efficient procedure for purifying omadacycline free base to obtain a high-purity product is desired. Additionally, novel pure forms of omadacycline that can be formulated into pharmaceutical compositions are also needed. [Overview of the Initiative]

[0007] Accordingly, in some embodiments, the present invention provides a method for purifying omadacycline, for example, crude omadacycline free base. In some embodiments, this method includes an improved HPLC procedure that utilizes a modifier, for example acetic acid, and results in a faster and more efficient purification of crude omadacycline free base than previously used methods.

[0008] The method for purifying omadacycline free base according to the present invention also, in some embodiments, includes a step of concentrating the HPLC fraction containing omadacycline free base using nanofiltration before crystallization of omadacycline. The use of nanofiltration replaces the extraction of the HPLC fraction with a solvent, such as dichloromethane, thereby eliminating the need to use large amounts of toxic solvents in the purification process. The use of nanofiltration also avoids prolonged exposure of omadacycline to high temperatures, which can lead to the degradation of omadacycline. If extraction with a solvent, such as dichloromethane, is used after nanofiltration, nanofiltration results in a significant reduction in the amount of solvent required for extraction of omadacycline compared to extraction performed without nanofiltration. The method for purifying omadacycline described herein also, in some embodiments, includes a step of crystallizing the omadacycline free base after, for example, HPLC purification, nanofiltration, and optionally extraction, to obtain extremely pure omadacycline crystalline free base with significantly lower levels of impurities than omadacycline produced using the previously described method.

[0009] The novel purification method of the present invention has led to the first synthesis of omadacycline crystalline free base. Accordingly, in some embodiments, the present invention also provides omadacycline crystalline free base, pharmaceutical compositions comprising omadacycline crystalline free base, and methods for treating bacterial infections using omadacycline crystalline free base.

[0010] The crystalline free base of omadacycline of the present invention can also be used in the synthesis of extremely pure omadacycline tosylate. Therefore, in some embodiments, the present invention also provides a method for preparing omadacycline tosylate from crystalline free base of omadacycline. The present invention also provides omadacycline tosylate prepared by the method of the present invention.

[0011] Therefore, in some embodiments, the present invention provides a crystalline form of the free base of omadacycline represented by formula (1).

[0012]

Chem.

[0013] In a further embodiment, omadacycline is represented by formula (2).

[0014]

Chem.

[0015] In some embodiments, the present invention also provides a peak at approximately 7.25° 2θ, a peak at approximately 7.37° 2θ, a peak at approximately 10.33° 2θ, a peak at approximately 12.58° 2θ, a peak at approximately 12.81° 2θ, a peak at approximately 14.75° 2θ, a peak at approximately 16.44° 2θ, a peak at approximately 17.86° 2θ, a peak at approximately 19.32° 2θ, a peak at approximately 19.44° 2θ, a peak at approximately 19.62° 2θ, a peak at approximately 22.19° 2θ, and a peak at approximately 23.38° 2θ and provides a polymorph of the crystalline form of the free base of omadacycline, characterized by a powder X-ray diffraction pattern comprising at least one peak selected from the group consisting of

[0016] In some embodiments, the polymorph is a peak at approximately 7.25° 2θ, a peak at approximately 7.37° 2θ, a peak at approximately 12.58° 2θ, a peak at approximately 12.81° 2θ, a peak at approximately 16.44° 2θ, and a peak at approximately 17.86° 2θ characterized by a powder X-ray diffraction pattern comprising

[0017] In some embodiments, the polymorph is a peak at approximately 7.25° 2θ, a peak at approximately 7.37° 2θ, a peak at approximately 10.33° 2θ, a peak at approximately 12.58° 2θ, a peak at approximately 12.81° 2θ, a peak at approximately 14.75° 2θ, a peak at approximately 16.44° 2θ, a peak at approximately 17.86° 2θ, a peak at approximately 19.32° 2θ, a peak at approximately 19.44° 2θ, a peak at approximately 19.62° 2θ, a peak at approximately 22.19° 2θ, and a peak at approximately 23.38° 2θ characterized by a powder X-ray diffraction pattern comprising

[0018] In some aspects, the invention also provides a method for preparing the above polymorph, the method comprising crystallizing the free base form of omadacycline from a solvent system comprising an organic solvent and water. In some embodiments, the organic solvent and water are present in the solvent system at an organic solvent:water ratio in the range of about 5:95 v / v to about 95:5 v / v. In further embodiments, the organic solvent is selected from the group consisting of acetonitrile, acetone, isopropyl alcohol, and methyl ethyl ketone. In one particular embodiment, the organic solvent is acetone.

[0019] In some aspects, the invention also provides a polymorph of the crystalline form of the free base of omadacycline prepared by the above method. For example, in some embodiments, the invention provides a polymorph of the crystalline form of the free base of omadacycline prepared by a method comprising crystallizing the free base form of omadacycline from a solvent system comprising an organic solvent and water.

[0020] In some embodiments, the organic solvent and water are present in the solvent system in an organic solvent:water ratio ranging from about 5:95 v / v to about 95:5 v / v. In further embodiments, the organic solvent is selected from the group consisting of acetonitrile, acetone, isopropyl alcohol, and methyl ethyl ketone. In one particular embodiment, the organic solvent is acetone.

[0021] In some embodiments, the polymorph is A peak at approximately 7.25°2θ, The peak is approximately 7.37°2θ. The peak is approximately 10.33°2θ. The peak is approximately 12.58°2θ. The peak is approximately 12.81°2θ. The peak is approximately 14.75°2θ. The peak is approximately 16.44°2θ. The peak is approximately 17.86°2θ. The peak is approximately 19.32°2θ. The peak is approximately 19.44°2θ. The peak is approximately 19.62°2θ. A peak at approximately 22.19°2θ, and The peak is approximately 23.38°2θ. It is characterized by a powder X-ray diffraction pattern that includes at least one peak selected from the group consisting of the following.

[0022] In some embodiments, the polymorph is A peak at approximately 7.25°2θ, The peak is approximately 7.37°2θ. The peak is approximately 12.58°2θ. The peak is approximately 12.81°2θ. A peak at approximately 16.44°2θ, and The peak is approximately 17.86°2θ. It is characterized by a powder X-ray diffraction pattern containing [a specific element].

[0023] In some embodiments, the polymorph is A peak at approximately 7.25°2θ, The peak is approximately 7.37°2θ. The peak is approximately 10.33°2θ. The peak is approximately 12.58°2θ. The peak is approximately 12.81°2θ. The peak is approximately 14.75°2θ. The peak is approximately 16.44°2θ. The peak is approximately 17.86°2θ. The peak is approximately 19.32°2θ. The peak is approximately 19.44°2θ. The peak is approximately 19.62°2θ. A peak at approximately 22.19°2θ, and Approximately 23.38°2θ It is characterized by a powder X-ray diffraction pattern that includes a peak.

[0024] In some embodiments, acetone and water are present in the solvent system in an acetone:water ratio of approximately 50:50 v / v. In certain embodiments, the organic solvent is selected from the group consisting of isopropanol, acetonitrile, and methyl ethyl ketone.

[0025] In some embodiments, the present invention also provides a method for purifying the free base form of omadacycline represented by formula (1),

[0026] [ka] The present invention provides a method comprising the step of subjecting a solution containing the crude free base form of omadacycline to purification by high-performance liquid chromatography (HPLC), wherein the HPLC includes the use of a modifier selected from the group consisting of a strong acid other than methylsulfonic acid (e.g., hydrochloric acid), a weak acid, and an organic amine, thereby obtaining a solution containing the HPLC-purified free base form of omadacycline.

[0027] In some embodiments, the adjusting agent is a weak acid, selected from the group consisting of oxalic acid, methanesulfonic acid, trifluoroacetic acid, sulfite, phosphoric acid, nitrite, hydrofluoric acid, benzoic acid, acetic acid, and formic acid. In some embodiments, the weak acid is selected from the group consisting of oxalic acid, methanesulfonic acid, trifluoroacetic acid, benzoic acid, acetic acid, and formic acid. In certain embodiments, the weak acid is acetic acid. In some embodiments, the adjusting agent is added to the mobile phase during HPLC, or the adjusting agent is added to a solution containing the crude free base form of omadacycline before being introduced into the HPLC column. In some embodiments, the mobile phase comprises elution buffer A and elution buffer B, where elution buffer A contains water and acetonitrile, and / or elution buffer B contains acetonitrile.

[0028] In some embodiments, the amount of beta-epimer impurities of omadacycline in a solution containing the HPLC-purified free base form of omadacycline is less than or equal to one-fifth of the amount of beta-epimer impurities of omadacycline present in a solution containing the crude free base form of omadacycline.

[0029] In some embodiments, the method of the present invention further comprises the step of concentrating a solution containing the HPLC-purified free base form of omadacycline using nanofiltration, wherein the nanofiltration comprises filtering the solution containing the HPLC-purified free base form of omadacycline through a membrane to form a filtrate and a retention solution, the retention solution being the concentrated solution containing the HPLC-purified free base form of omadacycline. In one embodiment, the method further comprises the step of collecting the retention solution.

[0030] In some embodiments, the method of the present invention further includes the step of adding an antioxidant to a solution containing the HPLC-purified free base form of omadacycline prior to nanofiltration. In further embodiments, the antioxidant is added in an amount sufficient to achieve an antioxidant concentration in the solution of about 0.01% to about 0.5% w / v.

[0031] In some embodiments, the membrane has a molecular weight cutoff (MWCO) in the range of about 150 to about 500 daltons. In some embodiments, the concentration of omadacycline in the retention solution is at least about 2 times higher than the concentration of omadacycline in the solution containing the HPLC-purified free base form of omadacycline, for example, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 8 times, or at least 10 times higher.

[0032] In some embodiments, the method of the present invention further includes the step of crystallizing the free base form of omadacycline to obtain the crystalline form of the free base of omadacycline. In some embodiments, the free base form of omadacycline is crystallized from a solvent system comprising an organic solvent and water. In further embodiments, the organic solvent and water are present in the solvent system in an organic solvent:water ratio in the range of about 5:95 v / v to about 95:5 v / v. In some embodiments, the organic solvent is selected from the group consisting of nitriles, alcohols, ketones and ethers. For example, the organic solvent may be selected from the group consisting of acetonitrile, acetone, isopropyl alcohol and methyl ethyl ketone. In certain embodiments, the organic solvent is acetone.

[0033] In some embodiments, acetone and water are present in the solvent system in an acetone:water ratio of approximately 50:50 v / v. In some embodiments, the organic solvent is selected from the group consisting of isopropanol, acetonitrile, and methyl ethyl ketone.

[0034] In some embodiments, the crystalline form of the free base of omadacycline obtained by the method of the present invention is A peak at approximately 7.25°2θ, The peak is approximately 7.37°2θ. The peak is approximately 10.33°2θ. The peak is approximately 12.58°2θ. The peak is approximately 12.81°2θ. The peak is approximately 14.75°2θ. The peak is approximately 16.44°2θ. The peak is approximately 17.86°2θ. The peak is approximately 19.32°2θ. The peak is approximately 19.44°2θ. The peak is approximately 19.62°2θ. A peak at approximately 22.19°2θ, and The peak is approximately 23.38°2θ. It is a polymorph characterized by a powder X-ray diffraction pattern containing at least one peak selected from the group consisting of the following.

[0035] In some embodiments, the present invention also relates to a method for preparing a tosylate of omadacycline represented by formula (1),

[0036] [ka] The steps include: purifying the free base form of omadacycline by the method described above, thereby obtaining the purified free base form of omadacycline; and The step involves reacting the purified free base form of omadacycline in a tosylation reaction to obtain the tosylate of omadacycline. This provides a method that includes this.

[0037] In some embodiments, the present invention also relates to a method for preparing a tosylate of omadacycline represented by formula (1),

[0038] [ka] The present invention provides a method comprising the steps of crystallizing the free base form of omadacycline to obtain a crystalline form of the free base of omadacycline, and reacting the crystalline form of the free base of omadacycline in a tosylation reaction to obtain a tosylate salt of omadacycline.

[0039] In some embodiments, the free base form of omadacycline is crystallized from a solvent system comprising an organic solvent and water. In some embodiments, the organic solvent and water are present in the solvent system in an organic solvent:water ratio ranging from about 5:95 v / v to about 95:5 v / v. In some embodiments, the organic solvent is selected from the group consisting of acetonitrile, acetone, isopropyl alcohol, methyl ethyl ketone, t-butyl methyl ether, ethyl acetate, toluene, and tetrahydrofuran. In one particular embodiment, the organic solvent is acetone.

[0040] In some embodiments, acetone and water are present in the solvent system in an acetone:water ratio of approximately 50:50 v / v. In some embodiments, the organic solvent is selected from the group consisting of isopropanol, acetonitrile, and methyl ethyl ketone.

[0041] In some embodiments, the crystalline form of the free base of omadacycline is, A peak at approximately 7.25°2θ, The peak is approximately 7.37°2θ. The peak is approximately 10.33°2θ. The peak is approximately 12.58°2θ. The peak is approximately 12.81°2θ. The peak is approximately 14.75°2θ. The peak is approximately 16.44°2θ. The peak is approximately 17.86°2θ. The peak is approximately 19.32°2θ. The peak is approximately 19.44°2θ. The peak is approximately 19.62°2θ. A peak at approximately 22.19°2θ, and The peak is approximately 23.38°2θ. It is a polymorph characterized by a powder X-ray diffraction pattern containing at least one peak selected from the group consisting of the following.

[0042] In some embodiments, the method of the present invention comprises the steps of: subjecting a solution containing the crude free base form of omadacycline to purification by high-performance liquid chromatography (HPLC), wherein the HPLC includes the use of a modifier selected from the group consisting of a strong acid other than methylsulfonic acid (e.g., hydrochloric acid), a weak acid, and an organic amine, thereby obtaining a solution containing the HPLC-purified free base form of omadacycline; crystallizing the free base form of omadacycline from the solution containing the HPLC-purified free base form of omadacycline, thereby obtaining a crystalline form of the free base of omadacycline; and reacting the crystalline form of the free base of omadacycline in a tosylation reaction, thereby obtaining a tosylate of omadacycline.

[0043] In some embodiments, the adjusting agent is a weak acid, which is selected from the group consisting of oxalic acid, methanesulfonic acid, trifluoroacetic acid, sulfurous acid, phosphoric acid, nitrite, hydrofluoric acid, benzoic acid, acetic acid, and formic acid. In some embodiments, the weak acid is selected from the group consisting of oxalic acid, methanesulfonic acid, trifluoroacetic acid, benzoic acid, acetic acid, and formic acid. In one particular embodiment, the weak acid is acetic acid.

[0044] In some embodiments, the adjusting agent is added to the mobile phase during HPLC, or the adjusting agent is added to a solution containing the crude free base form of omadacycline before being introduced into the HPLC column. In some embodiments, the mobile phase comprises elution buffer A and elution buffer B, where elution buffer A comprises water and acetonitrile, and / or elution buffer B comprises acetonitrile.

[0045] In some embodiments, the method of the present invention further comprises the step of concentrating a solution containing the HPLC-purified free base form of omadacycline using nanofiltration, wherein the nanofiltration comprises filtering the solution containing the HPLC-purified free base form of omadacycline through a membrane to form a filtrate and a retention solution, the retention solution being the concentrated solution containing the HPLC-purified free base form of omadacycline. In some embodiments, the method of the present invention further comprises the step of collecting the retention solution.

[0046] In some embodiments, the method of the present invention further includes the step of adding an antioxidant to a solution containing the HPLC-purified free base form of omadacycline prior to nanofiltration. In further embodiments, the antioxidant is added in an amount sufficient to achieve an antioxidant concentration in the solution of about 0.01% to about 0.5% w / v. In some embodiments, the membrane used in nanofiltration has a molecular weight cutoff (MWCO) in the range of about 150 to about 500 daltons.

[0047] In some embodiments, the present invention also relates to a method for preparing a tosylate of omadacycline represented by formula (1),

[0048] [ka] A step of subjecting a solution containing the crude free base form of omadacycline to purification by high-performance liquid chromatography (HPLC), wherein the HPLC includes the use of a modifier selected from the group consisting of weak acids and organic amines, thereby obtaining a solution containing the HPLC-purified free base form of omadacycline. A step of concentrating a solution containing the HPLC-purified free base form of omadacycline using nanofiltration, wherein the nanofiltration comprises filtering the solution containing the HPLC-purified free base form of omadacycline through a membrane to form a filtrate and a retaining solution, the retaining solution being the concentrated solution containing the HPLC-purified free base form of omadacycline. The steps include: crystallizing the free base form of omadacycline from a concentrated solution containing the HPLC-purified free base form of omadacycline, thereby obtaining the crystalline form of the free base of omadacycline; and The step involves reacting the crystalline form of the free base of omadacycline in a tosylation reaction to obtain the tosylate of omadacycline. This provides a method that includes this.

[0049] In some embodiments, the present invention also provides a tosylate of omadacycline represented by formula (1) obtained by the method described above.

[0050] [ka]

[0051] In some embodiments, the present invention also provides a crystalline tosylate of omadacycline represented by formula (1) obtained by the method described above.

[0052] [ka] In further embodiments, the present invention also provides polymorphs of crystalline tosylates, for example, polymorph of form 1 or polymorph of form 3.

[0053] In some embodiments, the present invention also provides pharmaceutical compositions comprising the crystalline form of the free base of omadacycline described above and a pharmaceutically acceptable carrier. In some embodiments, the present invention provides pharmaceutical compositions comprising polymorphs of the crystalline form of the free base of omadacycline described above and a pharmaceutically acceptable carrier.

[0054] In some embodiments, the present invention also provides a pharmaceutical composition comprising the above-mentioned omadacycline tosylate and a pharmaceutically acceptable carrier. In some embodiments, the present invention also provides a pharmaceutical composition comprising the above-mentioned crystalline omadacycline tosylate and a pharmaceutically acceptable carrier. In some embodiments, the present invention also provides a pharmaceutical composition comprising a polymorph of the above-mentioned crystalline omadacycline tosylate and a pharmaceutically acceptable carrier.

[0055] In some embodiments, the pharmaceutical composition is in tablet form. In other embodiments, the pharmaceutical composition is an injectable formulation in the form of a lyophilized powder.

[0056] In some embodiments, the present invention also provides a method for treating or preventing a bacterial infection in a subject requiring treatment or prevention of a bacterial infection, comprising the step of administering an effective amount of the above-mentioned free base of omadacycline in crystalline form or pharmaceutical composition to the subject.

[0057] In some embodiments, the present invention also provides a method for treating or preventing a bacterial infection in a subject requiring treatment or prevention of a bacterial infection, comprising the step of administering an effective amount of the above-mentioned polymorph of the crystalline form of the free base of omadacycline to the subject.

[0058] In some embodiments, the present invention also provides a method for treating or preventing a bacterial infection in a subject requiring treatment or prevention of a bacterial infection, comprising the step of administering an effective amount of the above-mentioned omadacycline tosylate or pharmaceutical composition to the subject.

[0059] In some embodiments, the present invention also provides a method for treating or preventing a bacterial infection in a subject requiring treatment or prevention of a bacterial infection, comprising the step of administering an effective amount of the above-mentioned crystalline tosylate of omadacycline or pharmaceutical composition to the subject.

[0060] In some embodiments, the present invention also provides a method for treating or preventing a bacterial infection in a subject requiring treatment or prevention of a bacterial infection, the method comprising the step of administering an effective amount of the above-mentioned polymorph or pharmaceutical composition of omadacycline crystalline tosylate to the subject.

[0061] In some embodiments, the bacterial infection is caused by Gram-positive or Gram-negative bacteria. In some embodiments, the bacterial infection is caused by bacteria that are resistant to other tetracycline compounds. In some embodiments, the bacterial infection is caused by a species of bacteria selected from the group consisting of Klebsiella pneumoniae, Salmonella, Enterococcus hirae, Acinetobacter baumanii, Branhamella catarrhalis, Haemophilus influenzae, Pseudomonas aeruginosa, Enterococcus faecium, Escherichia coli, Staphylococcus aureus, and Enterococcus faecalis.

[0062] In some embodiments, the bacterial infection is acute bacterial cutaneous tissue infection (ABSSSI). In further embodiments, ABSSSI includes Staphylococcus aureus (methicillin-susceptible and resistant isolates), Staphylococcus lugdunensis, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus anginosus group (including S. anginosus, S. intermedius, and S. constellatus), Streptococcus mitis, and Enterococcus faecalis. It is caused by a bacterial species selected from the group consisting of *Faecalis* (vancomycin-sensitive isolate), *Enterobacter cloacae*, *Klebsiella pneumoniae*, *Prevotella melaninogenica*, and *Finegoldia magna*.

[0063] In some embodiments, the bacterial infection is community-acquired bacterial pneumonia (CABP). In further embodiments, CABP is caused by a species of bacteria selected from the group consisting of Streptococcus pneumoniae (penicillin-susceptible and resistant isolates, macrolide-resistant isolates), Staphylococcus aureus (methicillin-susceptible isolates), Haemophilus influenzae (beta-lactamase-negative and positive isolates), Haemophilus parainfluenzae, Klebsiella pneumoniae, Legionella pneumophila, Mycoplasma pneumoniae, and Chlamydophila pneumoniae, including those with bacteremia.

[0064] In some embodiments, the bacterial infection is caused by bacteria of the species Clostridium difficile. In some embodiments, the bacterial infection is caused by mycobacteria. [Brief explanation of the drawing]

[0065] [Figure 1] This figure shows a typical XRPD spectrum of omadacyclin crystalline free base. [Figure 2] This figure shows the XRPD spectrum of omadacyclin free base crystallized from a solvent system containing acetonitrile and water (wet acetonitrile). [Figure 3] This figure shows the XRPD spectrum of omadacyclin free base crystallized from a solvent system containing isopropanol and water (wet isopropanol). [Figure 4] This figure shows the XRPD spectrum of omadacyclin free base crystallized from a solvent system containing 2-butanone and water (wet 2-butanone). [Figure 5] This figure shows the XRPD spectrum of a control crystalline free base of omadacyclin. [Figure 6] This is a schematic diagram of a method for preparing omadacyclin crystalline free base. [Figure 7] This is a schematic diagram of a method for preparing crystalline tosylate of omadacycline, which includes the step of preparing crystalline free base of omadacycline. [Modes for carrying out the invention]

[0066] Omadacycline crystalline free base The present invention provides a crystalline form of omadacycline free base. Omadacycline is a 9-aminomethyltetracycline derivative that has been developed primarily as a first-line agent for the empirical treatment of severe community-acquired infections, such as acute bacterial cutaneous and skin tissue infections (ABSSSI), moderate to severe community-acquired bacterial pneumonia (CABP), and complicated urinary tract infections (cUTI). The name "omadacycline" may be used herein synonymously with the names "OMC," "PTK 0796," or "Compound 1." In some examples, omadacycline may be represented by formula (1).

[0067] [ka]

[0068] In some cases, omadacycline can be represented by equation (2).

[0069] [ka]

[0070] Omadacycline is a yellow amorphous solid that, in its amorphous form, can be particularly unstable to exposure to air, light, and / or moisture. Therefore, omadacycline, in its solid form, must be stored at temperatures below 0°C, with limited exposure to air, light, and moisture. Outside of these limited exposure conditions, omadacycline decomposes to produce decomposition products, such as air decomposition products represented by formulas (3) and (4), and the 4-epiisomer represented by formula (5).

[0071] [ka]

[0072] The free base of omadacycline and certain pharmaceutically acceptable salts are described in U.S. Patent No. 7,553,828, and certain crystalline salts of omadacycline are described in U.S. Patent No. 8,383,610, the full contents of each are incorporated herein by reference. However, prior to this disclosure, the crystalline form of the free base of omadacycline, or polymorphs of its crystalline form, were unknown.

[0073] Accordingly, the present invention provides a crystalline form of the free base of omadacyclin (compound 1). The terms “crystalline” or “crystalline form,” as used herein, refer to a solid form of omadacyclin in which atoms are arranged in a regular, repeating pattern. In some embodiments, the term “crystalline” encompasses, without distinction, polymorphic or non-amorphous forms of omadacyclin.

[0074] As used herein, the terms "amorphous" or "amorphous form" refer to a non-crystalline form of a substance, such as a solid form that does not have a regular arrangement of atoms.

[0075] The terms “polymorph” or “polymorphic form,” as used herein, refer to an organized structure comprising only solute molecules and possessing characteristic crystalline properties. These terms may refer to different crystalline forms of the same molecule. Different polymorphs may have different physical properties, such as melting point, heat of fusion, solubility, dissolution rate, and / or vibrational spectrum, as a result of different arrangements or conformations of molecules within the crystal lattice. Differences in physical properties exhibited by different polymorphs may affect important parameters for pharmaceutical substances, such as storage stability, compressibility and density (important in formulation and product manufacturing), and dissolution rate (an important factor in bioavailability).

[0076] Differences in the stability of different polymorphs can also arise from differences in chemical reactivity (e.g., different susceptibility to oxidation). Therefore, a dosage form containing one polymorph may discolor more rapidly than a dosage form containing different polymorphs of the same substance. Differences in the stability of different polymorphs can also arise from differences in mechanical properties (e.g., if a kinetically favorable polymorph is converted to a thermodynamically more stable polymorph, the tablet may crumble during storage), or from differences in both chemical and mechanical properties (e.g., a tablet of one polymorph is more fragile in high humidity). As a result of differences in solubility / dissolution, in extreme cases, some polymorphic transitions may lead to loss of efficacy, or in other extreme cases, toxicity. Furthermore, the physical properties of the crystals can be important in processing. For example, different polymorphs of the same substance may exhibit differences in their tendency to form solvates, or differences in their particle shape and size distribution, which can affect purification (e.g., one polymorph may be more difficult to filter and wash away impurities than another).

[0077] Molecular polymorphs can be obtained by several methods known in the art. Such methods may include, but are not limited to, melt recrystallization, melt cooling, solvent recrystallization, desolvation, rapid evaporation, quenching, slow cooling, vapor diffusion, and sublimation. Techniques for characterizing polymorphs may include, but are not limited to, differential scanning calorimetry (DSC), powder X-ray diffraction (XRPD), single-crystal X-ray diffraction, vibrational spectroscopy, e.g., IR and Raman spectroscopy, solid-state NMR, hot-stage optical microscopy, scanning electron microscopy (SEM), electron crystallography and quantitative analysis, particle size analysis (PSA), surface area analysis, solubility testing, and dissolution testing. Specifically, XRPD is a technique used to characterize the crystallographic structure, size, and preferred orientation in polycrystalline or powder solid samples. This diffraction can also be used to characterize heterogeneous solid mixtures to determine the percentage of crystalline compounds present and may provide structural information about unknown materials. In this specification, the term "powder X-ray diffraction pattern," used synonymously with "XRPD pattern," refers to a graphical representation of data collected by XRPD analysis.

[0078] In some embodiments, the present invention provides polymorphs of the crystalline form of omadacycline free base, also referred herein as “polymorphs of omadacycline crystalline free base.” In some embodiments, polymorphs of omadacycline crystalline free base are A peak at approximately 7.25°2θ, The peak is approximately 7.37°2θ. The peak is approximately 10.33°2θ. The peak is approximately 12.58°2θ. The peak is approximately 12.81°2θ. The peak is approximately 14.75°2θ. The peak is approximately 16.44°2θ. The peak is approximately 17.86°2θ. The peak is approximately 19.32°2θ. The peak is approximately 19.44°2θ. The peak is approximately 19.62°2θ. A peak at approximately 22.19°2θ, and The peak is approximately 23.38°2θ. It is characterized by a powder X-ray diffraction pattern (XRPD pattern) that includes at least one peak selected from the group consisting of the following.

[0079] As used herein, the term "peak" refers to a peak in an XRPD pattern that has an intensity at least 20%, e.g., at least 30%, at least 40%, at least 50%, or at least 100% greater than the baseline noise.

[0080] When used herein in relation to peaks in an XRPD pattern, the terms "approximately" or "about" refer to an XRPD pattern in which the peak appears within 0.5°2θ of a given °2θ value, for example, 0.4, 0.3, 0.2, 0.1, 0.05, or 0.01°2θ.

[0081] In some embodiments, the present invention provides polymorphs of omadacyclin crystalline free bases characterized by a powder X-ray diffraction pattern (XRPD pattern) comprising at least two peaks selected from the group of peaks listed above.

[0082] In some embodiments, the present invention provides polymorphs of crystalline forms of omadacycline characterized by a powder X-ray diffraction pattern (XRPD pattern) comprising at least three peaks selected from the group of peaks enumerated above.

[0083] In some embodiments, the present invention provides polymorphs of omadacyclin crystalline free bases characterized by a powder X-ray diffraction pattern (XRPD pattern) comprising at least four peaks selected from the group of peaks listed above.

[0084] In some embodiments, the present invention provides polymorphs of omadacyclin crystalline free bases characterized by a powder X-ray diffraction pattern (XRPD pattern) comprising at least five peaks selected from the group of peaks listed above.

[0085] In some embodiments, the present invention provides polymorphs of omadacyclin crystalline free bases characterized by a powder X-ray diffraction pattern (XRPD pattern) comprising at least six peaks selected from the group of peaks listed above.

[0086] In some embodiments, the present invention provides polymorphs of omadacyclin crystalline free bases characterized by a powder X-ray diffraction pattern (XRPD pattern) comprising at least seven peaks selected from the group of peaks listed above.

[0087] In some embodiments, the present invention provides polymorphs of omadacyclin crystalline free bases characterized by a powder X-ray diffraction pattern (XRPD pattern) comprising at least eight peaks selected from the group of peaks listed above.

[0088] In some embodiments, the present invention provides polymorphs of omadacyclin crystalline free bases characterized by a powder X-ray diffraction pattern (XRPD pattern) comprising at least nine peaks selected from the group of peaks listed above.

[0089] In some embodiments, the present invention provides polymorphs of omadacyclin crystalline free bases characterized by a powder X-ray diffraction pattern (XRPD pattern) comprising at least 10 peaks selected from the group of peaks enumerated above.

[0090] In some embodiments, the present invention provides polymorphs of omadacyclin crystalline free bases characterized by a powder X-ray diffraction pattern (XRPD pattern) comprising at least 11 peaks selected from the group of peaks listed above.

[0091] In some embodiments, the present invention provides polymorphs of omadacyclin crystalline free bases characterized by powder X-ray diffraction patterns (XRPD patterns) that include all of the peaks listed above.

[0092] In some embodiments, the present invention provides polymorphs of omadacyclin crystalline free bases characterized by powder X-ray diffraction patterns (XRPD patterns) shown in any one of Figures 1 to 5.

[0093] In some embodiments, omadacycline crystalline free base, such as the polymorphs of the omadacycline crystalline free base described above, is at least 90% pure, expressed as the weight of the polymorphs of the omadacycline crystalline free base relative to the weight of the composition (w / w%). For example, the polymorphs of the omadacycline crystalline free base are at least 95% pure, at least 96% pure, at least 97% pure, at least 98% pure, at least 99% pure, at least 99.1% pure, at least 99.5% pure, or at least 99% pure. As used herein, “pure” or “purity” refers to compounds that are about 90–100% pure, for example, about 95–100% pure, 98–100% pure, or about 99–100% pure. In some embodiments, the pure omadacycline free base contains less than about 10%, less than about 5%, less than about 2%, or less than 1% impurities. Impurities may include, for example, one or more decomposition products, oxidation products, epimers, solvents, and / or other undesirable impurities.

[0094] One measure of the purity of omadacycline crystalline free base, such as the polymorphs of omadacycline crystalline free base described above, is defined by the 4-epiisomer (β-epimer) represented by formula (5) shown above. Therefore, in some examples, the β-epimer content in omadacycline crystalline free base, such as the polymorphs of omadacycline crystalline free base described above, is less than or equal to 10%, for example, less than or equal to 5%, less than or equal to 2%, less than or equal to 1%, less than or equal to 0.9%, less than or equal to 0.5%, less than or equal to 0.1%, less than or equal to 0.01%, when measured by HPLC (i.e., %). In one particular example, the β-epimer content in omadacycline crystalline free base, such as the polymorphs of omadacycline crystalline free base described above, is less than or equal to 0.9%.

[0095] Method for synthesizing omadacyclin crystalline free base Omadacycline crystalline free bases, such as the polymorphs of the above-mentioned omadacycline crystalline free bases, can be prepared by crystallizing amorphous omadacycline free bases from a solvent system containing organic solvents and water.

[0096] In such solvent systems, organic solvents and water can exist in organic solvent:water ratios ranging from approximately 5:95 v / v to approximately 95:5 v / v. For example, water may be present in the solvent system at approximately 95% v / v (i.e., an organic solvent:water ratio of approximately 5:95 v / v), approximately 90% v / v (i.e., an organic solvent:water ratio of approximately 10:90 v / v), approximately 80% v / v (i.e., an organic solvent:water ratio of approximately 20:80 v / v), approximately 70% v / v (i.e., an organic solvent:water ratio of approximately 30:70 v / v), approximately 60% v / v (i.e., an organic solvent:water ratio of approximately 40:60 v / v), approximately 50% v / v (i.e., an organic solvent:water ratio of approximately 50:50 v / v), and approximately 40% v / v (i.e., approximately In other examples, water may be present in amounts of approximately 1% to 10%, 2% to 15%, 5% to 20%, or 1% to 15% in the solvent system.

[0097] The organic solvent present in the solvent system may be selected from the group consisting of nitriles, alcohols, ketones, and ethers. For example, the organic solvent may be selected from the group consisting of acetonitrile, acetone, isopropyl alcohol, methyl ethyl ketone, t-butyl methyl ether, ethyl acetate, toluene, and tetrahydrofuran. In some examples, the organic solvent may be selected from the group consisting of acetonitrile, acetone, isopropyl alcohol, and methyl ethyl ketone.

[0098] In one example, the solvent system may include acetone and water. Acetone and water may be present in the solvent system in ratios of approximately 1:99 v / v to approximately 99:1 v / v, for example, approximately 10:90 v / v to approximately 90:10 v / v, approximately 20:80 v / v to approximately 80:20 v / v, approximately 30:70 v / v to approximately 70:30, and approximately 40:60 v / v to approximately 60:40 v / v. In one example, acetone and water may be present in the solvent system in an acetone:water ratio of approximately 50 / 50 v / v. In another example, the solvent system may include acetonitrile and water present in the solvent system in a ratio of approximately 95 / 5 v / v acetonitrile:water. In yet another example, the solvent system may include isopropyl alcohol and water present in the solvent system in a ratio of approximately 95 / 5 v / v isopropyl alcohol:water. In another example, the solvent system may include methyl ethyl ketone and water present in the solvent system, for example, in a methyl ethyl ketone:water ratio of approximately 95 / 5v / v.

[0099] In some cases, omadacycline amorphous free base can be purified from a solution containing crude omadacycline amorphous free base by high-performance liquid chromatography (HPLC) and / or nanofiltration, as described elsewhere in this disclosure, prior to crystallization.

[0100] In some embodiments, the present invention provides omadacyclin crystalline free bases prepared by the methods described above.

[0101] Pharmaceutical composition containing omadacycline crystalline free base The present invention also provides pharmaceutical compositions comprising omadacycline crystalline free base. For example, the pharmaceutical compositions of the present invention may comprise an effective amount of omadacycline crystalline free base and optionally a pharmaceutically acceptable carrier. The pharmaceutical compositions of the present invention may be administered to patients who require it for the treatment or prevention of bacterial infections.

[0102] The term "pharmaceutically acceptable carrier" includes substances that can be administered co-administered with omadacycline crystalline free base, which may enable both to perform their intended functions, for example, to treat or prevent bacterial infections. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, saline solutions, alcohols, vegetable oils, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffins, fragrance oils, fatty acid monoglycerides and diglycerides, petroethral fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, etc. The pharmaceutical composition may be sterilized and, if desired, may be mixed with auxiliary agents that do not react adversely with omadacycline free base, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts affecting osmotic pressure, buffers, colorants, fragrances and / or aromatic substances.

[0103] The pharmaceutical compositions of the present invention, comprising omadacycline crystalline free base, may be configured for administration via oral, parenteral, or topical routes. Generally, omadacycline crystalline free base is administered in an effective dose, most preferably depending on the body weight and condition of the subject being treated, as well as the selected specific route of administration. Variations may occur depending on the species of the subject being treated, its individual response to the pharmaceutical, as well as the type of pharmaceutical composition selected, and the duration and intervals during which such administration is performed.

[0104] For oral administration, omadacycline crystalline free base may be administered in the form of tablets or capsules. The tablets or capsules may contain various excipients, selected from the group consisting of, for example, microcrystalline cellulose, sodium citrate, calcium carbonate, dicalcium phosphate, and glycine. The tablets or capsules may also contain disintegrants, such as starch (and preferably corn, potato, or tapioca starch), alginic acid, and certain complex silicates. The tablets or capsules may also contain granulation binders, such as sucrose, gelatin, or acacia. Furthermore, lubricants, such as magnesium stearate, sodium lauryl sulfate, and talc, may also be added to the tablets or capsules for tableting purposes.

[0105] In some cases, oral formulations containing omadacycline crystalline free base include at least one additional component, such as a diluent, stabilizer, lubricant, and disintegrant. In some cases, the diluent may be lactose or microcrystalline cellulose, or a combination of both. In some cases, the stabilizer may be sodium bisulfite. In some cases, the lubricant may be colloidal silicon dioxide. In some cases, the lubricant may be sodium stearyl fumarate or magnesium stearate. In some cases, the disintegrant may be crospovidone.

[0106] In some embodiments, pharmaceutical compositions of the present invention intended for oral administration, such as tablets or capsules, may contain about 10 to about 1000 mg of omadacycline crystalline free base, for example, about 20 to about 750 mg, about 50 to about 500 mg, about 75 to about 400 mg, about 100 to about 300 mg, about 110 to about 250 mg, about 120 to about 240 mg, about 130 to about 210 mg, about 140 to about 170 mg, or about 150 mg of omadacycline crystalline free base.

[0107] For parenteral administration (including intraperitoneal, subcutaneous, intravenous, intradermal, or intramuscular injection), the present invention also provides injectable formulations containing omadacycline crystalline free base. Such injectable formulations may be in the form of a dry, for example, lyophilized powder that is restored to a carrier, for example, an aqueous carrier, for example, with water, before administration. In some embodiments, the injectable formulations containing omadacycline crystalline free base may also contain at least one additional component, for example, a lyoprotectant, an antioxidant, and a pH-adjusting compound.

[0108] In some examples, the freeze-drying protective agent may be a sugar, such as sucrose. In some examples, the antioxidant may be a bisulfite compound, such as sodium bisulfite. In some examples, the pH-adjusting compound may be an acid, such as an inorganic acid, such as phosphoric acid, nitric acid, sulfuric acid, or hydrochloric acid. The pH-adjusting compound may also be a base, such as an inorganic base, such as sodium hydroxide. In some examples, the pH-adjusting compound may include both an acid, such as an inorganic acid, and a base, which are added together to the injectable formulation containing omadacycline crystalline salt to achieve the desired pH. In some embodiments, the desired pH is about 4.0 to about 4.5, for example, 4.2.

[0109] In some embodiments, pharmaceutical compositions of the present invention intended for parenteral administration, such as in lyophilized form or injectable formulations restored with a carrier, may contain about 5 to about 500 mg of omadacycline crystalline free base, for example, about 10 to about 400 mg, about 25 to about 300 mg, about 50 to about 200 mg, about 50 to about 150 mg, about 60 to about 140 mg, about 70 to about 130 mg, about 80 to about 120 mg, about 90 to about 110 mg, or about 100 mg of omadacycline crystalline free base.

[0110] In some embodiments, the pharmaceutical composition containing omadacycline crystalline free base may be in the form of an aerosol pharmaceutical composition. Such an aerosol pharmaceutical composition may be in the form of a solution, suspension, powder formulation or liposome formulation. In some examples, the aerosol pharmaceutical composition containing omadacycline crystalline free base may be contained in an aerosol dispenser, which may also include a metered-dose spray device. In some examples, the aerosol dispenser may be a nebulizer, e.g., a small volume nebulizer (SVN), a pressurized metered-dose inhaler (pMDI), or a dry powder inhaler (DPI). Administration of a tetracycline compound, e.g., omadacycline, or a pharmaceutically acceptable salt thereof, in aerosol in the context of the present invention may be particularly useful in the treatment of pulmonary diseases, e.g., pulmonary diseases associated with bacterial infections, e.g., mycobacterial infections.

[0111] In some embodiments, the pharmaceutical composition containing omadacycline crystalline free base may be in the form of a pharmaceutical composition configured for topical administration. Such a pharmaceutical composition may be in the form of a gel, ointment, lotion, or cream and may contain a tetracycline compound appropriately mixed in a pharmacologically inert topical carrier. The pharmacologically inert topical carrier may include water, glycerol, alcohol, propylene glycol, fatty alcohol, triglycerides, fatty acid esters, or mineral oil. Other possible topical carriers may include liquid petrolatum, isopropyl palmitate, polyethylene glycol, ethanol, polyoxyethylene monolaurate, sodium lauryl sulfate, etc. Furthermore, materials such as antioxidants, humectants, and viscosity stabilizers may also be added as desired.

[0112] Method for treating or preventing bacterial infections using omadacycline crystalline free bases. The present invention also provides a method for treating or preventing a bacterial infection, comprising the step of administering omadacycline crystalline free base or a pharmaceutical composition containing omadacycline crystalline free base to a subject requiring treatment or prevention of a bacterial infection.

[0113] In some embodiments, bacterial infections may be caused by Gram-positive or Gram-negative bacteria. In some embodiments, bacterial infections may be caused by bacteria that are resistant to other tetracycline compounds. In some examples, bacterial infections may be caused by species selected from the group consisting of Klebsiella pneumoniae, Salmonella, Enterococcus hirae, Acinetobacter baumanii, Branhamella catarrhalis, Haemophilus influenzae, Pseudomonas aeruginosa, Enterococcus faecium, Escherichia coli, Staphylococcus aureus, and Enterococcus faecalis.

[0114] In one example, the bacterial infection is acute bacterial cutaneous infection (ABSSSI). ABSSSI includes Gram-positive and Gram-negative bacteria, such as Staphylococcus aureus (methicillin-susceptible and resistant isolates), Staphylococcus lugdunensis, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus anginosus group (including S. anginosus, S. intermedius, and S. constellatus), Streptococcus mitis, and Enterococcus faecalis. It can be caused by bacterial species selected from the group consisting of *Faecalis* (vancomycin-sensitive isolate), *Enterobacter cloacae*, *Klebsiella pneumoniae*, *Prevotella melaninogenica*, and *Finegoldia magna*.

[0115] In another example, a bacterial infection is community-acquired bacterial pneumonia (CABP). CABP can be caused by bacteria of species selected from the group consisting of Gram-positive, Gram-negative, or atypical bacteria, such as Streptococcus pneumoniae (penicillin-susceptible and resistant isolates, macrolide-resistant isolates), Staphylococcus aureus (methicillin-susceptible isolates), Haemophilus influenzae (beta-lactamase-negative and positive isolates), Haemophilus parainfluenzae, Klebsiella pneumoniae, Legionella pneumophila, Mycoplasma pneumoniae, and Chlamydophila pneumoniae, including those with bacteremia.

[0116] In another example, bacterial infections can be caused by Clostridium difficile.

[0117] In another example, a bacterial infection may be caused by mycobacteria, for example, mycobacteria belonging to the mycobacterial species described in U.S. Patent Application No. 62 / 726,738, U.S. Patent Application No. 62 / 731,410, U.S. Patent Application No. 62 / 746,039, and U.S. Patent Application No. 62 / 760,131, the entire contents of which are incorporated herein by reference.

[0118] In yet another example, a bacterial infection is a urinary tract infection (UTI).

[0119] The terms "to treat" or "treatment" refer to the improvement or reduction of one or more symptoms of the disorder being treated, such as a bacterial infection.

[0120] The terms "prophylaxis," "prevent," or "prevention" mean preventing or reducing the risk of bacterial infection.

[0121] The terms "resistance" or "resistant" refer to antibiotic / biological standards as defined by the Clinical and Laboratories Standards Institute (CLSI) and / or the Food and Drug Administration (FDA).

[0122] The term "subject" includes animals exposed to bacterial infections. Examples of subjects include animals, such as livestock (e.g., cattle, pigs, horses, goats, rabbits, sheep, chickens, etc.), laboratory animals (mice, rats, monkeys, chimpanzees, etc.), pets (e.g., dogs, cats, ferrets, hamsters, etc.), birds (e.g., chickens, turkeys, ducks, geese, crows, ravens, sparrows, etc.), primates (e.g., monkeys, gorillas, chimpanzees, bonobos, and humans), and other animals (e.g., squirrels, raccoons, mice, rats, etc.). In one embodiment, the subject is a mouse or a rat. In one embodiment, the subject is a cattle, pig, or chicken. In one embodiment, the subject is a human.

[0123] The term "effective dose" includes the amount of omadacyclin crystalline free base necessary to treat or prevent a bacterial infection. For example, an effective dose represents a potency level sufficient to achieve the desired therapeutic effect by killing bacteria and / or inhibiting bacterial growth. In one embodiment, an effective dose is sufficient to eradicate the bacteria causing the infection.

[0124] Administration of omadacycline crystalline free base Omadacycline crystalline free base can be administered alone or as part of a pharmaceutical composition to subjects requiring it. Any exemplary pharmaceutical composition containing omadacycline crystalline free base may contain an effective amount of omadacycline crystalline free base and optionally a pharmaceutically acceptable carrier.

[0125] The term "pharmaceutically acceptable carrier" includes substances that can be administered co-administered with omadacycline crystalline free base, which may enable both to perform their intended functions, for example, to treat or prevent bacterial infections. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, saline solutions, alcohols, vegetable oils, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffins, fragrance oils, fatty acid monoglycerides and diglycerides, petroleum ether fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, etc. The pharmaceutical composition may be sterilized and, if desired, may be mixed with auxiliary agents that do not react adversely with omadacycline crystalline free base, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts affecting osmotic pressure, buffers, colorants, fragrances and / or aromatic substances.

[0126] The pharmaceutical compositions that can be used in the methods of the present invention may be configured for administration via oral, parenteral, or topical routes. In some examples, the pharmaceutical compositions that can be used in the methods of the present invention may also be configured for aerosol delivery. Generally, omadacycline crystalline free base is administered in an effective dose, most preferably depending on the body weight and condition of the subject being treated, as well as the selected specific route of administration. Variations may occur depending on the species of the subject being treated and its individual response to omadacycline crystalline free base, as well as the type of pharmaceutical composition selected, and the duration and interval over which such administration is performed.

[0127] For oral administration, omadacycline crystalline free base may be administered in the form of tablets or capsules. The tablets or capsules may contain various excipients, selected from the group consisting of, for example, microcrystalline cellulose, sodium citrate, calcium carbonate, dicalcium phosphate, and glycine. The tablets or capsules may also contain disintegrants, such as starch (and preferably corn, potato, or tapioca starch), alginic acid, and certain complex silicates. The tablets or capsules may also contain granulation binders, such as sucrose, gelatin, or acacia. Furthermore, lubricants, such as magnesium stearate, sodium lauryl sulfate, and talc, may also be added to the tablets or capsules for tableting purposes.

[0128] For parenteral administration (including intraperitoneal, subcutaneous, intravenous, intradermal, or intramuscular injection), the present invention also provides injectable formulations comprising omadacycline crystalline free base. Such injectable formulations may be in the form of a dry, for example, lyophilized powder that is restored with a carrier, for example, an aqueous carrier, for example, with water, before administration. In some embodiments, the injectable formulations may also contain at least one additional component, for example, a lyophilized protective agent, an antioxidant, and a pH-adjusting compound.

[0129] Certain pharmaceutical compositions containing omadacycline crystalline free bases that may be suitable for use in the method of the present invention are described, for example, in U.S. Patent No. 9,315,475, the entirety of which is incorporated herein by reference.

[0130] In the method of the present invention, omadacycline crystalline free base may also be administered to a subject by aerosol. The aerosol pharmaceutical composition containing omadacycline crystalline free base may be in the form of a solution, suspension, powder formulation or liposome formulation. In some examples, the aerosol pharmaceutical composition containing omadacycline crystalline free base may be contained in an aerosol dispenser, which may also include a metered-dose spray device. In some examples, the aerosol dispenser may be a nebulizer, such as a small volume nebulizer (SVN), a pressurized metered-dose inhaler (pMDI), or a dry powder inhaler (DPI).

[0131] In the case of topical administration, omadacycline crystalline free base may also be administered to the subject as part of a pharmaceutical composition formulated for topical administration. Such a composition may be in the form of a gel, ointment, lotion, or cream and may contain a tetracycline compound appropriately mixed in a pharmacologically inert topical carrier. The pharmacologically inert topical carrier may contain water, glycerol, alcohol, propylene glycol, fatty alcohol, triglycerides, fatty acid esters, or mineral oil. Other possible topical carriers may include liquid petrolatum, isopropyl palmitate, polyethylene glycol, ethanol, polyoxyethylene monolaurate, sodium lauryl sulfate, etc. Furthermore, materials such as antioxidants, humectants, and viscosity stabilizers may also be added as desired.

[0132] Omadacycline crystalline free base can be administered to a subject in doses of approximately 100-200 mg, 100-300 mg, 100-400 mg, 100-500 mg, 100-600 mg, 200-500 mg, or 300-600 mg, for example, as a daily dose. In further cases, omadacycline crystalline free base can be administered orally. In further cases, omadacycline crystalline free base can be administered intravenously.

[0133] In some embodiments, omadacycline crystalline free base may be administered to a subject in doses of about 50–150 mg, about 50–400 mg, about 50–300 mg, about 50–200 mg, about 100–300 mg, or about 200–300 mg, or about 100 mg. For example, omadacycline crystalline free base may be administered to a subject in doses of about 100 mg, about 150 mg, about 200 mg, about 250 mg, or about 300 mg, for example, as a daily dose. In one embodiment, the dose is an intravenous dose.

[0134] In some embodiments, omadacycline crystalline free base may be administered to a subject in doses of approximately 50–800 mg, approximately 100–700 mg, approximately 250–600 mg, approximately 300–500 mg, approximately 100–400 mg, approximately 100–600 mg, or approximately 300 mg. For example, omadacycline crystalline free base may be administered in doses of approximately 300 mg, approximately 450 mg, or approximately 600 mg. In one embodiment, the dose is an oral dose.

[0135] In one embodiment, omadacycline crystalline free base may be administered intravenously in doses of about 100 mg, about 200 mg, or about 300 mg. In another embodiment, omadacycline crystalline free base may be administered orally in doses of about 300 mg, about 600 mg, or about 900 mg.

[0136] In some cases, omadacycline crystalline free base may be administered as an aerosol dose, for example, by using an aerosol dispenser. In some cases, the aerosol dispenser may contain doses of omadacycline crystalline free base ranging from about 1 to about 2000 mg, for example, about 1 to about 500 mg, about 25 to about 300 mg, about 50 to about 400 mg, about 100 to about 500 mg, about 200 to about 800 mg, about 500 mg to about 1000 mg, about 10 mg to about 200 mg, or about 300 mg to about 700 mg. In some cases, the aerosol dispenser may contain doses of approximately 1 mg, 5 mg, 10 mg, 30 mg, 50 mg, 80 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, or 1000 mg of omadacycline crystalline free base.

[0137] In some cases, omadacycline crystalline free base can be administered topically, for example, by applying a pharmaceutical composition prepared for topical administration containing omadacycline crystalline free base to the affected area. For example, the pharmaceutical composition prepared for topical administration may be in the form of a solution and may contain omadacycline crystalline free base at a concentration of about 0.01% to about 20% w / v, for example, about 0.01% to about 10% w / v, about 0.1% to about 20% w / v, about 0.5% to about 5% w / v, about 1% to about 10% w / v, or about 5% to about 20% w / v based on the volume of the composition. For example, a pharmaceutical composition configured for topical administration may contain omadacycline crystalline free base at concentrations of approximately 0.01% w / v, approximately 0.05% w / v, approximately 0.1% w / v, approximately 0.5% w / v, approximately 1% w / v, approximately 5% w / v, approximately 10% w / v, approximately 15% w / v, or approximately 20% w / v.

[0138] In another example, a pharmaceutical composition prepared for topical administration may contain omadacycline crystalline free base at concentrations of approximately 0.01% to approximately 20% w / w, based on the volume of the composition, for example, approximately 0.01% to approximately 10% w / w, approximately 0.1% to approximately 20% w / w, approximately 0.5% to approximately 5% w / w, approximately 1% to approximately 10% w / w, or approximately 5% to approximately 20% w / w. For example, a pharmaceutical composition prepared for topical administration may contain omadacycline crystalline free base at concentrations of approximately 0.01% w / w, approximately 0.05% w / w, approximately 0.1% w / w, approximately 0.5% w / w, approximately 1% w / w, approximately 5% w / w, approximately 10% w / w, approximately 15% w / w, or approximately 20% w / w.

[0139] In some cases, omadacycline crystalline free base may be administered at least once daily, for example, once, twice, three, or four times daily, in the doses described above. In further cases, omadacycline crystalline free base may be administered to subjects twice daily. In one particular case, omadacycline crystalline free base may be administered orally to subjects twice daily.

[0140] It should be understood that the administration of dose ranges including the doses listed above is also included in the present invention. For example, any of the above doses may be at the bottom or top of the dose range included in the method of the present invention. Furthermore, it should be understood that all enumerations or sets of numbers used throughout this application are also intended to include ranges of numbers in which any of the enumerated numbers may be at the bottom or top of the range. These ranges are intended to be included in the present invention.

[0141] In one embodiment, the oral dose of omadacycline crystalline free base may be three times greater than the intravenous dose of omadacycline crystalline free base.

[0142] For all the listed embodiments, the dose of omadacycline crystalline free base is also understood to be an effective amount of omadacycline crystalline free base.

[0143] In one embodiment, the effective amount of omadacycline crystalline free base, when administered orally, may be about 100 to about 1000 mg, for example, about 200 to about 750 mg, about 100 to about 500 mg, about 200 to about 600 mg, or about 400 to about 600 mg of omadacycline crystalline free base. In further examples, the effective amount of omadacycline crystalline free base, when administered orally, may be about 300 mg, about 450 mg, or about 600 mg of a tetracycline compound.

[0144] In another embodiment, the effective amount of omadacycline crystalline free base, when administered intravenously, may be about 50 to about 500 mg, for example, about 50 to about 400 mg, about 100 to about 300 mg, or about 50 to about 200 mg of omadacycline. For example, the effective amount of omadacycline crystalline free base, when administered intravenously, may be about 100 mg, about 150 mg, about 200 mg, about 250 mg, or about 300 mg.

[0145] In some cases, omadacycline crystalline free base may be administered orally, parenterally, systemically, topically, or via aerosol delivery in the context of the present invention. Generally, omadacycline crystalline free base is administered in an effective dose, most preferably depending on the body weight and condition of the subject being treated, as well as the specific route of administration selected. Variations may occur depending on the species of the subject being treated and its individual response to the pharmaceutical, as well as the type of pharmaceutical formulation selected, and the duration and interval over which such administration is performed.

[0146] In some embodiments, omadacycline crystalline free base may be administered for at least 3 days, at least 7 days, at least 14 days, at least 21 days, at least 30 days, at least 60 days, at least 5 weeks, at least 10 weeks, at least 15 weeks, at least 20 weeks, at least 30 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 13 months, at least 14 months, at least 15 months, at least 16 months, at least 17 months, at least 18 months, at least 19 months, at least 20 months, at least 21 months, at least 22 months, at least 23 months, or at least 24 months. For example, administration of omadacycline crystalline free base is recommended for 3-7 days, 3-14 days, 3-21 days, 3-30 days, 3-60 days, 7-14 days, 7-21 days, 7-30 days, 7-60 days, 14-21 days, 14-30 days, 14-60 days, 21-30 days, 21-60 days, 30-60 days, 1-5 weeks, and 3-10 weeks. The period may last for 5 to 20 weeks, 10 to 30 weeks, 20 to 35 weeks, 1 week to 1 month, 2 weeks to 2 months, 1 month to 3 months, 1 month to 6 months, 1 month to 9 months, 3 months to 12 months, 6 months to 12 months, 9 months to 12 months, 9 months to 16 months, 12 months to 18 months, 14 months to 24 months, 12 months to 24 months, or longer than 24 months.

[0147] For example, omadacyclin crystalline free base lasts for 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days, 48 ​​days, 49 days, 50 days, 51 days, 52 days, 53 days, 54 days, 55 days, 56 days, 57 days, 58 days, 59 days, 60 days, 1 week, 2 weeks It may be administered for 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, or 24 months. In other cases, omadacycline crystalline free base may be administered for more than 24 months, for example, 25 months, 26 months, 27 months, 28 months, 29 months, 30 months, 31 months, 32 months, 33 months, 34 months, 35 months, 36 months, 37 months, 38 months, 39 months, 40 months, 41 months, 42 months, 43 months, 44 months, 45 months, 46 months, 47 months, 48 ​​months, or more than 48 months.

[0148] In some embodiments, administration of omadacycline crystalline free base to a subject may include administering one or more loading doses of omadacycline crystalline free base, followed by one or more maintenance doses of omadacycline crystalline free base. In some embodiments, one or more loading doses of omadacycline crystalline free base may be greater than one or more maintenance doses of omadacycline crystalline free base. For example, the loading dose may be a daily dose of about 450 mg, for example, an oral daily dose, while the maintenance dose may be a daily dose of about 300 mg, for example, an oral daily dose. In another example, the loading dose may be a daily dose of about 200 mg, for example, an intravenous daily dose, while the maintenance dose may be a daily dose of about 100 mg, for example, an intravenous daily dose, or a daily dose of 300 mg, for example, an oral daily dose.

[0149] The loading dose and the maintenance dose of omadacycline crystalline free base may be administered via the same or different routes. For example, the loading dose may be administered intravenously, and the maintenance dose may be administered orally. In other embodiments, both the loading dose and the maintenance dose may be administered orally, or both the loading dose and the maintenance dose may be administered intravenously.

[0150] In some cases, the loading dose of omadacycline crystalline free base may be an oral or intravenous dose administered twice daily, and the maintenance dose may be an oral or intravenous dose administered once daily. For example, omadacycline crystalline free base may be administered as an intravenous loading dose of 100 mg twice daily, followed by an intravenous maintenance dose of 100 mg once daily. In another case, omadacycline crystalline free base may be administered as an intravenous loading dose of 100 mg twice daily, followed by an oral maintenance dose of 300 mg once daily. In yet another case, omadacycline crystalline free base may be administered as an oral loading dose of 300 mg twice daily, followed by an oral maintenance dose of 300 mg once daily.

[0151] In other examples, administration of omadacycline crystalline free base may not involve administration of one or more loading doses of omadacycline crystalline free base. Therefore, in some examples, omadacycline crystalline free base may be administered to the subject at the same dose throughout the treatment period. For example, omadacycline crystalline free base may be administered to the subject in an intravenous dose of about 100 mg, about 200 mg, or about 300 mg. The intravenous dose may be administered to the subject once or twice daily throughout the treatment period. In other examples, omadacycline crystalline free base may be administered to the subject in an oral dose of about 300 mg, about 450 mg, or about 600 mg. The oral dose may be administered to the subject once daily throughout the treatment period.

[0152] In some cases, omadacycline crystalline free base may be administered to a subject alone or in combination with at least one additional therapeutic agent. The phrase "in combination with a therapeutic agent" is intended to include simultaneous administration of omadacycline crystalline free base and the therapeutic agent, administration of the therapeutic agent following the initial administration of omadacycline crystalline free base, and administration of omadacycline crystalline free base following the initial administration of the therapeutic agent. In one example, the therapeutic agent is an antibiotic.

[0153] The term "approximately" refers to a range of values ​​that may be 15%, 10%, 8%, 5%, 3%, 2%, 1%, or 0.5% greater or less than a specified value. For example, "approximately 10%" could be between 8.5% and 11.5%. In one embodiment, the term "approximately" refers to a range of values ​​that are 5% greater or less than a specified value. In another embodiment, the term "approximately" refers to a range of values ​​that are 2% greater or less than a specified value. In yet another embodiment, the term "approximately" refers to a range of values ​​that are 1% greater or less than a specified value.

[0154] In this specification, whenever values ​​and ranges are given for, for example, the age of the target population, dosage, duration, etc., it should be understood that all values ​​and ranges encompassed by these values ​​and ranges are intended to be included within the scope of the present invention. Furthermore, all values ​​within these values ​​and ranges may also be upper or lower limits of the range.

[0155] Method for purifying omadacyclin free base The present invention also provides a method for purifying omadacycline free base, for example, crude omadacycline free base. In some examples, the method for purifying omadacycline free base includes the step of subjecting a solution containing crude omadacycline free base to purification by high-performance liquid chromatography (HPLC), for example, preparative HPLC, thereby obtaining a solution containing HPLC-purified omadacycline free base. In some embodiments, the method for purifying omadacycline free base may also include the step of concentrating the solution containing the HPLC-purified omadacycline free base using nanofiltration after HPLC purification, thereby obtaining a concentrated solution containing the HPLC-purified omadacycline free base. In some embodiments, the method for purifying omadacycline free base may also include the step of crystallizing omadacycline free base from the concentrated solution containing the HPLC-purified omadacycline free base after the nanofiltration step, thereby producing crystalline omadacycline free base, for example, polymorphs of the crystalline omadacycline free base described above.

[0156] The methods for purifying omadacycline free base, such as crude omadacycline free base, provided by the present invention can achieve higher omadacycline purification rates and productivity than previous methods for purifying omadacycline (such as those described in U.S. Patent No. 9,434,680). Specifically, in some embodiments, the methods for purifying omadacycline provided by the present invention produce purified omadacycline free base containing lower levels of impurities than previous methods, such as the 4-epiisomer (β-epimer) represented by formula (5) shown above. In some embodiments, the methods for purifying omadacycline provided by the present invention are characterized by significantly shorter processing times than previous omadacycline purification methods. The term "processing time," as used herein, refers to the time required to produce 1 kilogram of crystalline omadacycline free base starting from crude omadacycline free base. In some examples, the term “processing time” refers to the time required to produce one kilogram of omadacycline crystalline free base by following the procedure shown in Figure 6, i.e., the steps of HPLC, nanofiltration, and crystallization. In some examples, the term “processing time” does not include the time required to dry the omadacycline crystalline free base after crystallization.

[0157] For example, compared to previous methods, the present invention makes it possible to reduce processing time to half or less, for example, about one-third or less, about one-fifth or less, about one-sixth or less, about one-seventh or less, about one-eighth or less, about one-ninth or less, about one-tenth or less, about one-eleventh or less, or about one-twelfth or less, compared to the processing time required by previous methods. In one particular embodiment, the present invention makes it possible to reduce processing time to about one-twelfth or less, i.e., from about 73.9 hours achieved by previous methods to about 6.1 hours.

[0158] High-performance liquid chromatography In some examples, a method for purifying omadacycline free base includes the step of subjecting a solution containing crude omadacycline free base to purification by high-performance liquid chromatography (HPLC), for example, preparative HPLC, thereby obtaining a solution containing HPLC-purified omadacycline free base. Purification of crude omadacycline free base by HPLC allows for the removal of impurities, such as the compounds (3), (4) and (5) described above, as well as synthetic by-products. A solution containing crude omadacycline free base can be obtained by any method known in the art for preparing crude omadacycline free base, for example, by the procedure shown in Scheme 1 above, or by the methods of U.S. Patent No. 9,434,680, U.S. Patent No. 9,522,872, or U.S. Patent No. 8,383,610, for example, the methods of which the entire contents of each are incorporated herein by reference.

[0159] In some embodiments, HPLC purification involves the use of a stationary phase, such as a reversed phase (RP). The stationary phase that can be used in the context of the present invention may be any stationary phase that provides adequate purification of crude omadacyclin free base. In some examples, the RP stationary phase may be a C18 stationary phase. In some examples, the RP stationary phase may have a pore size of about 50 Å to about 200 Å, for example, about 50 Å to about 90 Å, about 80 Å to about 150 Å, about 120 Å to about 200 Å, or about 100 Å. In some examples, the RP stationary phase may have a particle size of about 0.5 μm to about 20 μm, for example, about 0.5 μm to about 5 μm, about 2 μm to about 15 μm, about 5 μm to about 20 μm, or about 10 μm. In one example, the RP stationary phase may be a C18 phase having a pore size of about 100 Å and a particle size of about 10 μm. In one embodiment, the RP stationary phase may be Luna® 10 μm PREP C18(2) 100 Å from Phenomenex, LC Column 250 × 21.2 mm, AXIA® Packed, Ea. In another embodiment, the RP stationary phase may be Synergi Polar RP10 10 μm 80 Å.

[0160] In some embodiments, HPLC purification involves the use of a mobile phase, such as elution buffers, such as elution buffer A and elution buffer B. Elution buffer A may contain a mixture of water and an organic solvent, such as methanol, ethanol, methylene chloride, or acetonitrile. In certain embodiments, buffer A contains water and acetonitrile. Elution buffer A may also, in some examples, contain inorganic salts, such as phosphates (e.g., potassium phosphate, dibasic potassium phosphate, sodium phosphate, dibasic sodium phosphate, or ammonium phosphate), acetates (e.g., ammonium acetate, potassium acetate, or sodium acetate), or formates (e.g., sodium formate or potassium formate). Elution buffer B may contain an organic solvent, such as methanol, ethanol, methylene chloride, or acetonitrile. In certain embodiments, elution buffer B contains acetonitrile.

[0161] In some embodiments, elution buffer A and / or elution buffer B may also include a modifier selected from the group consisting of strong acids other than methylsulfonic acid (e.g., hydrochloric acid), weak acids, and organic amines. In other embodiments, a modifier selected from the group consisting of weak acids and organic amines may be added to the solution containing crude omadacycline free base before HPLC purification.

[0162] As used herein, the term “modifier” refers to a chemical that, when added to a solution containing elution buffer A and / or elution buffer B, and / or crude omadacycline free base, facilitates the purification of crude omadacycline free base by HPLC.

[0163] As used herein, the term "weak acid" refers to an acid that does not completely dissociate when dissolved in a solution, such as an aqueous solution. In some embodiments, a weak acid is an acid having a pKa of about 3.0 to about 6.0.

[0164] In some embodiments, the modifier may be a weak acid. Examples of weak acids that can be used as modifiers in the context of the present invention include, but are not limited to, oxalic acid, methanesulfonic acid, trifluoroacetic acid, sulfurous acid, phosphoric acid, nitrite, hydrofluoric acid, benzoic acid, acetic acid, and formic acid. For example, the weak acid may be selected from the group consisting of oxalic acid, methanesulfonic acid, trifluoroacetic acid, benzoic acid, acetic acid, and formic acid. In one particular example, the weak acid may be acetic acid.

[0165] In other embodiments, the modifier may be an organic amine. Examples of organic amines that may be used as modifiers in the context of the present invention include 3-ethanolamine, diethylamine, and trimethylamine.

[0166] In one particular embodiment, the adjusting agent is a weak acid, such as acetic acid. In a further example, the weak acid, such as acetic acid, is added to elution buffer A. In yet another further example, the weak acid, such as acetic acid, is added to elution buffer B. In yet another further example, the weak acid, such as acetic acid, is added to a solution containing crude omadacycline free base before HPLC purification.

[0167] While we do not wish to be bound by any particular theory, it is believed that certain modifiers, such as weak acids, e.g., acetic acid, when added to a solution containing elution buffer A and / or elution buffer B, and / or crude omadacycline free base, before HPLC purification, may enable faster and more efficient purification of omadacycline free base by HPLC compared to previous HPLC-based purification methods used for omadacycline. Previous omadacycline purification methods, such as the method described in U.S. Patent No. 8,946,196, whose entire contents are incorporated herein by reference, utilized strong acids, i.e., methanesulfonic acid, as mobile phase modifiers. While we do not wish to be bound by any particular theory, it is believed that adding a weak acid, e.g., acetic acid, to a solution containing elution buffer A and / or elution buffer B, and / or crude omadacycline free base before HPLC purification may generate a distinct HPLC peak corresponding to the omadacycline free base, e.g., an HPLC peak with a clear boundary. This, on the one hand, makes it possible to collect a single HPLC fraction or several HPLC fractions containing omadacycline free base instead of many HPLC fractions, as required by previous purification methods. On the other hand, this makes it possible to load more material, i.e., crude omadacycline free base, onto the HPLC column while using a significantly smaller amount of HPLC solvent, and to collect HPLC fractions in a much shorter time than with previous purification methods. Furthermore, this also makes it possible to achieve a better purification effect and / or recovery of the product, i.e., omadacycline free base, compared to previous purification methods. Thus, the HPLC purification method of the present invention is particularly suitable for the purification of large quantities of omadacycline free base.

[0168] One measure of the purity of omadacycline crystalline free base, such as the polymorphs of omadacycline crystalline free base described above, is defined by the content of the 4-epiisomer (β-epimer) represented by formula (5) shown above. In some cases, purification of crude omadacycline free base by HPLC, such as preparative HPLC, results in the removal of a considerable amount of β-epimer. For example, the amount of β-epimers present in a solution containing HPLC-purified omadacycline free base is at least 50%, for example, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500%, at least 600%, at least 650%, at least 700%, at least 750%, at least 800%, at least 850%, at least 900%, at least 950%, or at least 1000% lower than the amount of β-epimers present in a solution containing crude omadacycline free base, as measured by the area under the % curve of the HPLC trace. For example, in Example 1 and Table 2 described herein, the % content of β-epimers was reduced from approximately 11.13% β-epimers present in the solution of crude omadacycline free base to approximately 1.71% β-epimers present in the solution of HPLC-purified omadacycline free base.

[0169] Nanofiltration In some embodiments, the HPLC fraction containing HPLC-purified omadacycline free base can be concentrated using nanofiltration to produce a concentrated solution of HPLC-purified omadacycline free base. In some embodiments, after nanofiltration, the concentrated solution of HPLC-purified omadacycline free base may be extracted with a solvent, such as dichloromethane (DCM). In other embodiments, the concentrated solution of HPLC-purified omadacycline free base is not extracted with a solvent, such as DCM, and is used directly for crystallization of omadacycline free base as described later.

[0170] In contrast, previous methods for purifying omadacycline, such as the method described in U.S. Patent No. 8,946,196, did not utilize nanofiltration and relied solely on extraction with a solvent, such as DCM. The nanofiltration of the HPLC fraction containing HPLC-purified omadacycline free base by the method of the present invention allows for a significant reduction in the amount of solvent required for extraction after the nanofiltration step. For example, as described in Example 3 of this specification, using nanofiltration before DCM extraction required 892 mL of DCM to produce a concentrated solution containing 71.04 grams of HPLC-purified omadacycline free base. In comparison, producing the same amount of concentrated solution containing HPLC-purified omadacycline free base without nanofiltration would require approximately 43.97 L of DCM, or about 49 times more DCM. Accordingly, in exemplary embodiments of the present invention, including extraction of HPLC-purified omadacyclin free bases by DCM, nanofiltration makes it possible to reduce the amount of solvent required for extraction, such as DCM, to about 1 / 5, about 1 / 10, about 1 / 15, about 1 / 20, about 1 / 25, about 1 / 30, about 1 / 35, about 1 / 40, about 1 / 45, or about 1 / 50. In some embodiments, nanofiltration makes it possible to reduce the amount of solvent required for extraction of HPLC-purified omadacyclin free bases, such as DCM, to about 5 to about 1 / 10, about 10 to about 1 / 20, about 15 to about 1 / 30, about 20 to about 1 / 40, or about 25 to about 1 / 50.

[0171] In some examples, nanofiltration for concentrating the HPLC fraction containing HPLC-purified omadacycline free base is performed without subsequent extraction with a solvent, such as DCM. In these examples, the concentrated solution containing HPLC-purified omadacycline free base obtained after nanofiltration can be used directly for crystallization of omadacycline free base, as described later. Therefore, in these examples, the use of nanofiltration eliminates the need for a solvent.

[0172] Reducing the volume of solvent, such as DCM, used to concentrate HPLC-purified omadacycline free base, or eliminating the use of solvent, such as DCM, reduces the cost of the purification procedure and eliminates the need to dispose of large amounts of chlorinated waste. Eliminating the use of solvent, such as DCM, for concentrating HPLC-purified omadacycline free base also eliminates the need for an evaporation step to remove DCM from the solution containing HPLC-purified omadacycline free base. The evaporation step can cause degradation of omadacycline free base, and its elimination leads to an increased recovery rate of crystalline omadacycline free base.

[0173] Nanofiltration involves filtering a solution containing HPLC-purified omadacycline free base through a membrane. During nanofiltration, a portion of the filtered solution passes through the membrane to form a filtrate, while the solution remaining on the membrane is the retention solution. In the method of the present invention, the majority of the omadacycline free base remains in the retention solution, forming a concentrated solution containing HPLC-purified omadacycline free base. Therefore, in some embodiments of the present invention, the nanofiltration step may further include collecting the retention solution.

[0174] Suitable membranes for nanofiltration by the method steps of the present invention may include any membrane that allows the retention of omadacyclin free base while allowing the solvent to pass through the membrane. In some examples, the nanofiltration membrane may have a molecular weight cutoff (MWCO) in the range of about 150 to about 500 daltons, for example, about 150 to about 300 daltons, about 200 to about 400 daltons, or about 300 to 500 daltons.

[0175] In some embodiments, the method of the present invention may further include the step of adding an antioxidant to the solution containing HPLC-purified omadacycline free base before nanofiltration. For example, the antioxidant may be added in an amount sufficient to achieve a concentration of about 0.01% to about 0.5% w / v of antioxidant in the solution containing HPLC-purified omadacycline free base.

[0176] Antioxidants that may be added to a solution containing HPLC-purified omadacycline free base can be selected from the group consisting of ascorbic acid (vitamin C), glutathione, lipoic acid, carotene, α-tocopherol (vitamin E), ubiquinol (coenzyme Q), deferoxamine, and bisulfites, such as sodium bisulfite.

[0177] The nanofiltration process yields a retention solution, which is a concentrated solution containing HPLC-purified omadacycline free base. In some examples, the concentration of omadacycline in the retention solution is at least about twice as high, e.g., at least about four times higher, at least about five times higher, at least about eight times higher, at least about ten times higher, at least about twenty times higher, or at least about 50 times higher, than the concentration of omadacycline in the solution containing HPLC-purified omadacycline free base, i.e., in the fraction collected as a result of HPLC purification.

[0178] crystallization In some embodiments, the method for purifying omadacycline free base may also include the step of crystallizing the omadacycline free base after nanofiltration and solvent extraction, or after nanofiltration without solvent extraction, thereby producing crystalline omadacycline free base. For example, crystallization of omadacycline free base to produce the polymorphs of the crystalline omadacycline free base described above may be carried out according to the methods described herein.

[0179] In embodiments in which omadacyclin free base is crystallized from a concentrated solution after nanofiltration without extraction by a solvent, such as DCM, the solvent that can be used for crystallization may be selected from the group consisting of a mixture of acetone and water, a mixture of acetonitrile and water, and a mixture of isopropanol and water. In one example, the solvent that can be used for crystallization may be a mixture of acetone and water.

[0180] In embodiments in which omadacyclin free bases are precipitated from a concentrated solution after nanofiltration and extraction with a solvent, such as DCM, heptane and methyl tert-butyl ether (MTBE) may be used to precipitate amorphous free bases from the DCM concentrate.

[0181] In some embodiments, the omadacycline crystalline free base produced as a result of crystallization, such as the polymorphs of the omadacycline crystalline free base described above, are at least 90% pure, e.g., at least 95% pure, at least 96% pure, at least 97% pure, at least 98% pure, at least 99% pure, at least 99.1% pure, at least 99.5% pure, or at least 99% pure, when measured in %w / w. As used herein, “pure” or “purity” refers to a compound that is about 90–100% pure, e.g., about 95–100% pure, 98–100% pure, or about 99–100% pure, when measured in %w / w. In some embodiments, the pure omadacycline free base contains impurities of less than about 10%, less than about 5%, less than about 2%, or less than 1%. The impurities may include, for example, one or more decomposition products, oxidation products, epimers, solvents, and / or other undesirable impurities.

[0182] Crystallization of omadacycline free base from a concentrated solution containing HPLC-purified omadacycline free base results in further purification of the omadacycline free base, as measured, for example, by the β-epimer content. For instance, the β-epimer content in crystalline omadacycline free base can be reduced by at least about 50%, for example, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, compared to the β-epimer content in a concentrated solution containing HPLC-purified omadacycline free base. The % β-epimer content is measured as the % total peak area corresponding to the β-epimer relative to the total area under the curve of the HPLC trace.

[0183] In some cases, the β-epimer content in omadacycline crystalline free base, such as the polymorphs of the omadacycline crystalline free base described above, is 10% or less, e.g., 5% or less, 2% or less, 1% or less, 0.9% or less, 0.5% or less, 0.1% or less, 0.05% or less, or 0.01% or less. In one particular case, the β-epimer content in omadacycline crystalline free base, such as the polymorphs of the omadacycline crystalline free base described above, is 0.9% or less. The % β-epimer content is measured as the % total peak area corresponding to the β-epimer relative to the total area under the curve of the HPLC trace.

[0184] The method for purifying omadacycline free base provided by the present invention offers advantages over previous methods for purifying omadacycline (such as those described in U.S. Patent No. 9,434,680). The method for purifying omadacycline free base provided by the present invention can achieve higher omadacycline purification rates and productivity compared to previous methods.

[0185] Table 1 below shows a comparison between the previous method for omiadacyclin purification and the method of the present invention. Specifically, Table 1 compares the amount of material fed into the HPLC column, the calculated % recovery rate, and the total processing time between the previous method and the method of the present invention.

[0186] [Table 1]

[0187] The amount of crude omadacycline material added to the HPLC column, as indicated in parentheses, is an assay-corrected amount, i.e., an assay-corrected amount, that is, an assay-corrected amount, that is, an assay-corrected amount, that is, that that is correct for the actual amount of omadacycline present in the added material. This figure reflects the actual amount of omadacycline present in the crude omadacycline material added to the HPLC column. The assay-corrected amount of crude omadacycline material can be determined by comparing the HPLC signal generated by a known amount of crude omadacycline material with a calibration curve generated using a known high-purity omadacycline standard.

[0188] The calculated percentage recovery can be determined by dividing the amount of crystalline omadacycline free base obtained after purification, corrected for the assay, by the amount of crude omadacycline introduced into the assay-corrected HPLC column, and multiplying by 100%.

[0189] Table 1 demonstrates that the present invention's method for purifying omadacycline free base allows for the loading of significantly larger amounts of crude omadacycline onto the HPLC column, thereby drastically reducing the amount of resin required for HPLC purification. The present invention's method for purifying omadacycline free base also results in a higher calculated recovery rate, drastically reducing the processing time required to produce 1 kilogram of pure omadacycline free base from approximately 73.9 hours to approximately 6.1 hours. While offering the above benefits compared to previous methods for purifying omadacycline, the present invention's method for purifying omadacycline free base provides omadacycline free base that is at least as pure as omadacycline obtained using previous methods. In some embodiments, the method for purifying omadacycline according to the present invention provides omadacycline free base that is at least 90% pure, for example, at least 95% pure, at least 96% pure, at least 97% pure, at least 98% pure, at least 99% pure, at least 99.1% pure, at least 99.5% pure, or at least 99% pure, when measured in %w / w.

[0190] These characteristics make the HPLC purification method of the present invention particularly suitable for purifying large quantities of omadacyclin free base.

[0191] Method for synthesizing omadacycline tosylate The present invention also provides a method for preparing omadacycline tosylate from omadacycline crystalline free base. For example, according to the method of the present invention, omadacycline tosylate can be prepared by reacting omadacycline crystalline free base in a tosylation reaction to obtain omadacycline tosylate. In some examples, omadacycline tosylate is omadacycline crystalline tosylate, for example, polymorphs of form 1, form 2, or form 3 of omadacycline crystalline tosylate described in U.S. Patent No. 8,383,610, the entire contents of which are incorporated herein by reference.

[0192] Methods for synthesizing omadacycline tosylates, such as crystalline tosylates, such as polymorphs of form 1, form 2, or form 3, from free omadacycline base are known in the art and are described, for example, in U.S. Patent No. 8,383,610, the entirety of which is incorporated herein by reference.

[0193] In some cases, the method for preparing omadacycline tosylate from a solution containing crude omadacycline free base is as follows: The step of subjecting a solution containing crude omadacycline free base to purification by HPLC, thereby obtaining a solution containing HPLC-purified omadacycline free base. The steps involve concentrating a solution containing HPLC-purified omadacycline free base using nanofiltration to obtain a concentrated solution containing HPLC-purified omadacycline free base, The steps include: crystallizing omadacycline free base from a concentrated solution containing HPLC-purified omadacycline free base to produce crystalline omadacycline free base; and The step involves reacting omadacycline crystalline free base in a tosylation reaction to obtain omadacycline tosylate. Includes.

[0194] Figure 7 shows a scheme illustrating the procedure for preparing the omadacycline tosylate described above.

[0195] The step of purifying a solution containing crude omadacycline free base by HPLC to obtain a solution containing HPLC-purified omadacycline free base can be carried out as described in the preceding section of this specification.

[0196] The step of concentrating a solution containing HPLC-purified omadacycline free base using nanofiltration to obtain a concentrated solution containing HPLC-purified omadacycline free base can be carried out as described in the preceding section herein.

[0197] The step of crystallizing omadacycline free base from a concentrated solution containing HPLC-purified omadacycline free base, thereby producing crystalline omadacycline free base, can be carried out as described in the preceding section herein.

[0198] The present invention also provides omadacycline tosylates obtained by the methods described above. In some examples, the omadacycline tosylate is a crystalline omadacycline tosylate, for example, a polymorph of form 1, form 2, or form 3 of the crystalline omadacycline tosylate described in U.S. Patent No. 8,383,610.

[0199] In some embodiments, the present invention also provides pharmaceutical compositions comprising omadacycline tosylate, the omadacycline tosylate being obtained by the method described above. The omadacycline tosylate may be crystalline omadacycline tosylate, for example, a polymorph of form 1, form 2, or form 3 of crystalline omadacycline tosylate. Exemplary pharmaceutical compositions comprising omadacycline tosylate are described, for example, in U.S. Patent No. 9,314,475, the entirety of which is incorporated herein by reference.

[0200] The present invention also provides a method for treating or preventing a bacterial infection in a subject requiring treatment or prevention of a bacterial infection, comprising the step of administering to the subject an omadacycline tosylate prepared according to the method of the present invention. Furthermore, the present invention also provides a method for treating or preventing a bacterial infection in a subject requiring treatment or prevention of a bacterial infection, comprising the step of administering to the subject a pharmaceutical composition comprising an omadacycline tosylate prepared using the method of the present invention.

[0201] In some cases, bacterial infections may be caused by Gram-positive or Gram-negative bacteria. In some cases, bacterial infections may be caused by bacteria that are resistant to other tetracycline compounds. In some cases, bacterial infections may be caused by species selected from the group consisting of Klebsiella pneumoniae, Salmonella, Enterococcus hirae, Acinetobacter baumanii, Branhamella catarrhalis, Haemophilus influenzae, Pseudomonas aeruginosa, Enterococcus faecium, Escherichia coli, Staphylococcus aureus, and Enterococcus faecalis.

[0202] In one example, the bacterial infection is acute bacterial cutaneous infection (ABSSSI). In some embodiments, ABSSSI includes Gram-positive or Gram-negative bacteria, such as Staphylococcus aureus (methicillin-susceptible and resistant isolates), Staphylococcus lugdunensis, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus anginosus group (including S. anginosus, S. intermedius, and S. constellatus), Streptococcus mitis, and Enterococcus faecalis. It can be caused by bacterial species selected from the group consisting of *Faecalis* (vancomycin-sensitive isolate), *Enterobacter cloacae*, *Klebsiella pneumoniae*, *Prevotella melaninogenica*, and *Finegoldia magna*.

[0203] In another example, the bacterial infection may be community-acquired bacterial pneumonia (CABP). In some embodiments, CABP may be caused by bacteria of species selected from the group consisting of Gram-positive, Gram-negative, or atypical bacteria, such as Streptococcus pneumoniae (penicillin-susceptible and resistant isolates, macrolide-resistant isolates), Staphylococcus aureus (methicillin-susceptible isolates), Haemophilus influenzae (beta-lactamase-negative and positive isolates), Haemophilus parainfluenzae, Klebsiella pneumoniae, Legionella pneumophila, Mycoplasma pneumoniae, and Chlamydophila pneumoniae, including those associated with bacteremia.

[0204] In another example, the bacterial infection is a Clostridium difficile infection.

[0205] In yet another example, the bacterial infection may be a urinary tract infection (UTI).

[0206] Examples of the invention [Example 1] Crystallization of omadacyclin free base from acetone This example illustrates a typical procedure for crystallizing the free base of omadacycline from acetone and characterizing the crystalline product. The free base of omadacycline was crystallized from a holding solution obtained after nanofiltration. The holding solution, having a concentration of 80-100 mg / mL, was diluted with an equal volume of acetone, a seed crystal was added, and the mixture was stirred to induce crystallization of the free base of omadacycline. The product was filtered and dried.

[0207] The crystalline free base of omadacycline was characterized by XRPD analysis. The obtained XRPD spectrum is shown in Figure 1. The prominent peaks (°2θ) present in the XRPD spectrum of Figure 1 are listed in Table 2 below.

[0208] [Table 2]

[0209] Crystallization improved the overall purity of omadacyclin free base, particularly with respect to the removal of β-epimers, as is evident from the data shown in Table 3.

[0210] [Table 3]

[0211] [Example 2] Further identification of solvents for crystallizing omadacyclin free base. The purpose of this experiment is to identify additional solvents other than acetone that can be used to crystallize omadacyclin free base.

[0212] Materials and methods The starting material used was amorphous free base prepared by dissolving purified free base from different batches of omadacycline in dilute hydrochloric acid. The base was extracted with dichloromethane (DCM) at neutral pH, and the extract was concentrated and dried. The resulting material was an orange powder, which, as determined by HPLC, had a purity of 98.22% by area percent and a β-epimer level of 1.44%.

[0213] The initial crystallization attempts were made with 0.5-gram scale omadacycline, using a typical solvent volume of 10 mL. Forty-one different solvents or solvent combinations were tested for the crystallization of amorphous omadacycline. In the case of two-component solvent combinations, the content of the second solvent almost never exceeded 5%. The omadacycline solution mixture was stirred overnight at room temperature. Any solid formed was filtered, washed, and dried. In each case, the solid and filtrate samples were analyzed by HPLC and compared to the starting material. Conclusions regarding the crystallinity of the product were based on the HPLC results. Crystalline products should be purer than the starting material.

[0214] Based on these initial HPLC results, three solvents were selected for crystallization and used for further crystallization on a 1-gram scale. These three solvents were isopropanol, acetonitrile, and 2-butanone (methyl ethyl ketone), all containing 5% water. The crystallinity of the products from these three crystallizations was then confirmed by XRPD analysis and compared to the control crystalline free base of omadacycline.

[0215] result Table 4 below includes a comparison of HPLC purity between the starting materials and the three products. Production yields are also shown. All solvents used for crystallization contained 5% water.

[0216] [Table 4]

[0217] The crystallinity of the crystallization products listed in Table 4 was confirmed by XRPD analysis. Figure 2 shows the XRPD spectrum of omadacycline free base crystallized from wet acetonitrile. Figure 3 shows the XRPD spectrum of omadacycline free base crystallized from wet isopropanol. Figure 4 shows the XRPD spectrum of omadacycline free base crystallized from wet 2-butanone. Figure 5 shows the XRPD spectrum of a control crystalline free base of omadacycline. As is clear from comparing Figures 1-3 with Figure 4, the crystallization products from wet acetonitrile, wet isopropanol, and wet 2-butanone were crystalline.

[0218] [Example 3] Use of nanofiltration for concentration of omadacycline aqueous solutions The objective of this experiment was to demonstrate that nanofiltration can be effectively used to concentrate an aqueous solution of omadacycline free base after purification by preparative HPLC. In a typical experiment, 150 grams of crude omadacycline free base (67.4% w / w purity, adjusted to 101.1 grams for the assay) was purified by HPLC. For further processing, a fraction containing 80.34 g of omadacycline (concentration 19.74 mg / mL) in a 93:7 mixture of water / acetonitrile was selected. This solution was divided into two parts, 2000 mL and 2070 mL, and concentrated by nanofiltration. As a product, two retention solutions with concentrations of 80.67 mg / mL and 52.46 mg / mL were obtained, containing a total of 76.09 grams of product. Each retention solution was subjected to extraction by DCM to obtain omadacycline as a dry powder, which was corrected to 71.04 grams for the assay. A total of 892 mL of DCM was used for extraction and recovery. In comparison, a process utilizing DCM extraction to concentrate an aqueous solution of omadacycline requires approximately 43.97 L of DCM for the same amount of product. Further reduction in DCM volume would be possible if the second portion of nanofiltration were concentrated to the same level as the first portion (i.e., approximately 80 mg / mL).

[0219] It should be noted that subjecting the retained solution obtained as a result of nanofiltration to extraction with DCM is optional. As described in Example 4 below, the free base of omadacycline may be directly crystallized from the retained solution to produce crystalline omadacycline free base.

[0220] In the next step, the omadacycline free base was subjected to a tosylation / crystallization procedure to obtain 82.85 grams (adjusted to 59.17 grams with respect to the assay) of tosylate. The purity of the product was within the specification range.

[0221] [Example 4] Production of Crystalline Tosylate of Omadacycline via Crystalline Free Base The purpose of this experiment was to produce crystalline tosylate of omadacycline via crystalline free base. Crystallization of the free base of omadacycline was carried out using the retained solution obtained after purification of the crude free base by preparative HPLC and nanofiltration, which contained 26.4 kg of crude omadacycline. The retained solution was transferred to a reactor, an equal amount of acetone was added thereto, and the solution was heated to 20 - 25°C. Using acetic acid / trimethylamine (AcOH / TEA), the pH of the solution was adjusted to 7.8 - 8.0, and seed crystals in an acetone / water mixture were added. The reaction mixture was stirred at 20 - 25°C for 8 - 12 hours and filtered. The yellow solid was washed with an acetone - water mixture and dried. The corrected yield of crystalline free base of omadacycline was 10.62 kg (41.8%).

[0222] To prepare the crystalline tosylate of omadacycline, a solution of p-toluenesulfonic acid in acetone / water was prepared in a glass reactor. In parallel, a solution containing 9.76 kg of the crystalline free base of omadacycline in dry acetone was prepared in a second reactor at a temperature of 25 - 30 °C. Then, 20% of the volume of the p-toluenesulfonic acid solution was added to the second reactor at a temperature of 25 - 30 °C. Seed crystals were added to the reaction mixture and the remainder of the p-toluenesulfonic acid solution was transferred over a period of 2 - 3 hours. The resulting suspension was stirred at 10 - 15 °C for 1 - 3 hours, filtered, washed and dried. The yield of the crystalline tosylate of omadacycline was 11.78 kg (92.2%).

[0223] Equivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments and methods described herein. Such equivalents are intended to be encompassed by the scope of the present invention. All patents, patent applications and references cited herein are hereby expressly incorporated by reference into this specification.

Claims

1. Crystal morphology of the free base of omadacycline represented by formula (1): 【Chemistry 1】 。

2. The crystalline form of omadacycline according to claim 1, represented by formula (2): 【Chemistry 2】 。

3. A peak at approximately 7.25°2θ, The peak is approximately 7.37°2θ. The peak is approximately 10.33°2θ. The peak is approximately 12.58°2θ. The peak is approximately 12.81°2θ. The peak is approximately 14.75°2θ. The peak is approximately 16.44°2θ. The peak is approximately 17.86°2θ. The peak is approximately 19.32°2θ. The peak is approximately 19.44°2θ. The peak is approximately 19.62°2θ. A peak at approximately 22.19°2θ, and The peak is approximately 23.38°2θ. A polymorph of a crystalline form according to claim 1 or 2, characterized by a powder X-ray diffraction pattern including at least one peak selected from the group consisting of the following.

4. A peak at approximately 7.25°2θ, The peak is approximately 7.37°2θ. The peak is approximately 12.58°2θ. The peak is approximately 12.81°2θ. A peak at approximately 16.44°2θ, and The peak is approximately 17.86°2θ. The polymorph according to claim 3, characterized by a powder X-ray diffraction pattern containing the following.

5. A peak at approximately 7.25°2θ, The peak is approximately 7.37°2θ. The peak is approximately 10.33°2θ. The peak is approximately 12.58°2θ. The peak is approximately 12.81°2θ. The peak is approximately 14.75°2θ. The peak is approximately 16.44°2θ. The peak is approximately 17.86°2θ. The peak is approximately 19.32°2θ. The peak is approximately 19.44°2θ. The peak is approximately 19.62°2θ. A peak at approximately 22.19°2θ, and The peak is approximately 23.38°2θ. The polymorph according to claim 3, characterized by a powder X-ray diffraction pattern containing the following.

6. A method for preparing the polymorph according to any one of claims 3 to 5, comprising the step of crystallizing the free base form of omadacycline from a solvent system comprising an organic solvent and water.

7. The method according to claim 6, wherein an organic solvent and water are present in the solvent system in an organic solvent:water ratio in the range of about 5:95 v / v to about 95:5 v / v.

8. The method according to claim 7, wherein the organic solvent is selected from the group consisting of acetonitrile, acetone, isopropyl alcohol, and methyl ethyl ketone.

9. The method according to claim 8, wherein the organic solvent is acetone.

10. The method according to claim 9, wherein acetone and water are present in the solvent system in an acetone:water ratio of approximately 50:50 v / v.

11. The method according to claim 8, wherein the organic solvent is selected from the group consisting of isopropanol, acetonitrile, and methyl ethyl ketone.

12. Polymorphs of the crystalline form of the free base of omadacycline represented by formula (1), prepared by a method comprising the step of crystallizing the free base form of omadacycline from a solvent system containing an organic solvent and water: 【Transformation 3】 。

13. The polymorph according to claim 12, wherein an organic solvent and water are present in the solvent system in an organic solvent:water ratio in the range of approximately 5:95 v / v to approximately 95:5 v / v.

14. The polymorph according to claim 13, wherein the organic solvent is selected from the group consisting of acetonitrile, acetone, isopropyl alcohol, and methyl ethyl ketone.

15. The polymorph according to claim 14, wherein the organic solvent is acetone.

16. The polymorph according to claim 15, wherein acetone and water are present in the solvent system in an acetone:water ratio of approximately 50:50 v / v.

17. The polymorph according to claim 14, wherein the organic solvent is selected from the group consisting of isopropanol, acetonitrile, and methyl ethyl ketone.

18. A peak at approximately 7.25°2θ, The peak is approximately 7.37°2θ. The peak is approximately 10.33°2θ. The peak is approximately 12.58°2θ. The peak is approximately 12.81°2θ. The peak is approximately 14.75°2θ. The peak is approximately 16.44°2θ. The peak is approximately 17.86°2θ. The peak is approximately 19.32°2θ. The peak is approximately 19.44°2θ. The peak is approximately 19.62°2θ. A peak at approximately 22.19°2θ, and The peak is approximately 23.38°2θ. A polymorph according to any one of claims 12 to 17, characterized by a powder X-ray diffraction pattern having at least one peak selected from the group consisting of .

19. A peak at approximately 7.25°2θ, The peak is approximately 7.37°2θ. The peak is approximately 12.58°2θ. The peak is approximately 12.81°2θ. A peak at approximately 16.44°2θ, and The peak is approximately 17.86°2θ. The polymorph according to claim 18, characterized by a powder X-ray diffraction pattern containing the following.

20. A peak at approximately 7.25°2θ, The peak is approximately 7.37°2θ. The peak is approximately 10.33°2θ. The peak is approximately 12.58°2θ. The peak is approximately 12.81°2θ. The peak is approximately 14.75°2θ. The peak is approximately 16.44°2θ. The peak is approximately 17.86°2θ. The peak is approximately 19.32°2θ. The peak is approximately 19.44°2θ. The peak is approximately 19.62°2θ. A peak at approximately 22.19°2θ, and The peak is approximately 23.38°2θ. The polymorph according to claim 18, characterized by a powder X-ray diffraction pattern containing the following.

21. A method for purifying the free base form of omadacycline represented by formula (1), 【Chemistry 4】 The method described above is A step of purifying a solution containing the crude free base form of omadacycline by high-performance liquid chromatography (HPLC), The HPLC procedure includes the use of a modifier selected from the group consisting of weak acids and organic amines, thereby obtaining a solution containing the HPLC-purified free base form of omadacycline. A method that includes this.

22. The method according to claim 21, wherein the adjusting agent is a weak acid, and the weak acid is selected from the group consisting of oxalic acid, methanesulfonic acid, trifluoroacetic acid, sulfurous acid, phosphoric acid, nitrite, hydrofluoric acid, benzoic acid, acetic acid, and formic acid.

23. The method according to claim 22, wherein the weak acid is acetic acid.

24. The method according to any one of claims 21 to 23, wherein the adjusting agent is added to the mobile phase during HPLC, or the adjusting agent is added to a solution containing the crude free base form of omadacycline before being introduced into the HPLC column.

25. The method according to any one of claims 21 to 24, wherein the mobile phase comprises elution buffer A and elution buffer B, wherein elution buffer A comprises water and acetonitrile, and / or elution buffer B comprises acetonitrile.

26. The method according to any one of claims 21 to 25, wherein the amount of beta-epimer impurities of omadacycline in a solution containing the HPLC-purified free base form of omadacycline is one-fifth or less of the amount of beta-epimer impurities of omadacycline present in a solution containing the crude free base form of omadacycline.

27. A step of concentrating a solution containing the HPLC-purified free base form of omadacycline using nanofiltration, Nanofiltration involves filtering a solution containing the HPLC-purified free base form of omadacycline through a membrane to form a filtrate and a retaining solution, the retaining solution being a concentrated solution containing the HPLC-purified free base form of omadacycline. The method according to any one of claims 21 to 26, further comprising:

28. The method according to claim 27, further comprising the step of collecting the retaining liquid.

29. The method according to claim 28, further comprising the step of adding an antioxidant to a solution containing the HPLC-purified free base form of omadacycline before nanofiltration.

30. The method according to claim 29, wherein the antioxidant is added in an amount sufficient to achieve a concentration of the antioxidant in the solution of about 0.01% to about 0.5% w / v.

31. The method according to any one of claims 27 to 30, wherein the membrane has a molecular weight cutoff (MWCO) in the range of about 150 to about 500 daltons.

32. The method according to any one of claims 27 to 31, wherein the concentration of omadacycline in the holding solution is at least about twice as high as the concentration of omadacycline in a solution containing the HPLC-purified free base form of omadacycline.

33. The method according to any one of claims 21 to 32, further comprising the step of crystallizing the free base form of omadacycline to obtain a crystalline form of the free base of omadacycline.

34. The method according to claim 33, wherein the free base form of omadacycline is crystallized from a solvent system containing an organic solvent and water.

35. The method according to claim 34, wherein an organic solvent and water are present in the solvent system in an organic solvent:water ratio in the range of about 5:95 v / v to about 95:5 v / v.

36. The method according to claim 35, wherein the organic solvent is selected from the group consisting of acetonitrile, acetone, isopropyl alcohol, and methyl ethyl ketone.

37. The method according to claim 36, wherein the organic solvent is acetone.

38. The method according to claim 37, wherein acetone and water are present in the solvent system in an acetone:water ratio of approximately 50:50 v / v.

39. The method according to claim 36, wherein the organic solvent is selected from the group consisting of isopropanol, acetonitrile, and methyl ethyl ketone.

40. The crystalline form of the free base of omadacycline is A peak at approximately 7.25°2θ, The peak is approximately 7.37°2θ. The peak is approximately 10.33°2θ. The peak is approximately 12.58°2θ. The peak is approximately 12.81°2θ. The peak is approximately 14.75°2θ. The peak is approximately 16.44°2θ. The peak is approximately 17.86°2θ. The peak is approximately 19.32°2θ. The peak is approximately 19.44°2θ. The peak is approximately 19.62°2θ. A peak at approximately 22.19°2θ, and The peak is approximately 23.38°2θ. The method according to any one of claims 33 to 39, characterized by a polymorph having a powder X-ray diffraction pattern including at least one peak selected from the group consisting of .

41. A method for preparing the tosylate of omadacycline represented by formula (1), 【Transformation 5】 The method described above is A step of purifying the free base form of omadacycline by the method described in any one of claims 21 to 40, thereby obtaining the purified free base form of omadacycline, and The purified free base form of omadacycline is reacted in a tosylation reaction to obtain the tosylate of omadacycline. A method that includes this.

42. A method for preparing the tosylate of omadacycline represented by formula (1), 【Transformation 6】 The method described above is The steps include: crystallizing the free base form of omadacycline to obtain the crystalline form of the free base of omadacycline; and The step of reacting the crystalline form of the free base of omadacycline in a tosylation reaction to obtain the tosylate of omadacycline. A method that includes this.

43. The method according to claim 42, wherein the free base form of omadacycline is crystallized from a solvent system containing an organic solvent and water.

44. The method according to claim 43, wherein an organic solvent and water are present in the solvent system in an organic solvent:water ratio in the range of about 5:95 v / v to about 95:5 v / v.

45. The method according to claim 44, wherein the organic solvent is selected from the group consisting of acetonitrile, acetone, isopropyl alcohol, methyl ethyl ketone, t-butyl methyl ether, ethyl acetate, toluene, and tetrahydrofuran.

46. The method according to claim 45, wherein the organic solvent is acetone.

47. The method according to claim 46, wherein acetone and water are present in the solvent system in an acetone:water ratio of approximately 50:50 v / v.

48. The method according to claim 45, wherein the organic solvent is selected from the group consisting of isopropanol, acetonitrile, and methyl ethyl ketone.

49. The crystalline form of the free base of omadacycline is A peak at approximately 7.25°2θ, The peak is approximately 7.37°2θ. The peak is approximately 10.33°2θ. The peak is approximately 12.58°2θ. The peak is approximately 12.81°2θ. The peak is approximately 14.75°2θ. The peak is approximately 16.44°2θ. The peak is approximately 17.86°2θ. The peak is approximately 19.32°2θ. The peak is approximately 19.44°2θ. The peak is approximately 19.62°2θ. A peak at approximately 22.19°2θ, and The peak is approximately 23.38°2θ. The method according to any one of claims 42 to 48, characterized by a polymorph having a powder X-ray diffraction pattern including at least one peak selected from the group consisting of .

50. A step of purifying a solution containing the crude free base form of omadacycline by high-performance liquid chromatography (HPLC), The HPLC process involves the use of a modifier selected from the group consisting of weak acids and organic amines, thereby obtaining a solution containing the HPLC-purified free base form of omadacycline. The steps include: crystallizing the free base form of omadacycline from a solution containing the HPLC-purified free base form of omadacycline, thereby obtaining the crystalline form of the free base of omadacycline; and The step of reacting the crystalline form of the free base of omadacycline in a tosylation reaction to obtain the tosylate of omadacycline. The method according to any one of claims 42 to 49, including the method described in any one of claims 42 to 49.

51. The method according to claim 50, wherein the adjusting agent is a weak acid, and the weak acid is selected from the group consisting of oxalic acid, methanesulfonic acid, trifluoroacetic acid, sulfurous acid, phosphoric acid, nitrite, hydrofluoric acid, benzoic acid, acetic acid, and formic acid.

52. The method according to claim 51, wherein the weak acid is acetic acid.

53. The method according to any one of claims 50 to 52, wherein the adjusting agent is added to the mobile phase during HPLC, or the adjusting agent is added to a solution containing the crude free base form of omadacycline before being introduced into the HPLC column.

54. The method according to any one of claims 50 to 53, wherein the mobile phase comprises elution buffer A and elution buffer B, wherein elution buffer A comprises water and acetonitrile, and / or elution buffer B comprises acetonitrile.

55. A step of concentrating a solution containing the HPLC-purified free base form of omadacycline using nanofiltration, Nanofiltration involves filtering a solution containing the HPLC-purified free base form of omadacycline through a membrane to form a filtrate and a retaining solution, the retaining solution being a concentrated solution containing the HPLC-purified free base form of omadacycline. The method according to any one of claims 50 to 54, further comprising:

56. The method according to claim 55, further comprising the step of collecting the retaining liquid.

57. The method according to claim 56, further comprising the step of adding an antioxidant to a solution containing the HPLC-purified free base form of omadacycline before nanofiltration.

58. The method according to claim 57, wherein the antioxidant is added in an amount sufficient to achieve a concentration of the antioxidant in the solution of about 0.01% to about 0.5% w / v.

59. The method according to any one of claims 56 to 58, wherein the membrane has a molecular weight cutoff (MWCO) in the range of about 150 to about 500 daltons.

60. A method for preparing the tosylate of omadacycline represented by formula (1), 【Transformation 7】 The method described above is A step of purifying a solution containing the crude free base form of omadacycline by high-performance liquid chromatography (HPLC), The HPLC process involves the use of a modifier selected from the group consisting of weak acids and organic amines, thereby obtaining a solution containing the HPLC-purified free base form of omadacycline. A step of concentrating a solution containing the HPLC-purified free base form of omadacycline using nanofiltration, Nanofiltration involves filtering a solution containing the HPLC-purified free base form of omadacycline through a membrane to form a filtrate and a retaining solution, wherein the retaining solution is a concentrated solution containing the HPLC-purified free base form of omadacycline. The steps include: crystallizing the free base form of omadacycline from a concentrated solution containing the HPLC-purified free base form of omadacycline, thereby obtaining the crystalline form of the free base of omadacycline; and The step of reacting the crystalline form of the free base of omadacycline in a tosylation reaction to obtain the tosylate of omadacycline. A method that includes this.

61. Tosylate of omadacycline represented by formula (1), obtained by the method described in any one of claims 41 to 60: 【Transformation 8】 。

62. A crystalline tosylate of omadacycline represented by formula (1), obtained by the method described in any one of claims 50 to 60: 【Chemistry 9】 。

63. A polymorph of the crystalline tosylate according to claim 62.

64. A polymorph of a crystalline tosylate according to claim 62, which is a polymorph of form 1 or a polymorph of form 3.

65. A pharmaceutical composition comprising the crystalline form of the free base of omadacycline according to claim 1 or 2 and a pharmaceutically acceptable carrier.

66. A pharmaceutical composition comprising a polymorph of the crystalline form of the free base of omadacycline according to any one of claims 3 to 5 or 12 to 20 and a pharmaceutically acceptable carrier.

67. A pharmaceutical composition comprising the tosylate of omadacycline according to claim 61 and a pharmaceutically acceptable carrier.

68. A pharmaceutical composition comprising the crystalline tosylate of omadacycline according to claim 62 and a pharmaceutically acceptable carrier.

69. A pharmaceutical composition comprising a polymorph of crystalline tosylate of omadacycline according to claim 63 or 64 and a pharmaceutically acceptable carrier.

70. A pharmaceutical composition according to any one of claims 65 to 69, which is in tablet form.

71. A pharmaceutical composition according to any one of claims 65 to 69, which is an injectable formulation in the form of a freeze-dried powder.

72. A method for treating or preventing a bacterial infection in a subject requiring treatment or prevention of a bacterial infection, comprising the step of administering to the subject an effective amount of a crystalline form of the free base of omadacycline according to claim 1 or 2, or the pharmaceutical composition according to claim 65.

73. A method for treating or preventing a bacterial infection in a subject requiring treatment or prevention of a bacterial infection, comprising the step of administering to the subject an effective amount of a polymorph of the crystalline form of the free base of omadacycline according to any one of claims 3 to 5, or the pharmaceutical composition according to claim 66.

74. A method for treating or preventing a bacterial infection in a subject requiring treatment or prevention of a bacterial infection, comprising the step of administering to the subject an effective amount of omadacycline tosylate according to claim 61, or the pharmaceutical composition according to claim 67.

75. A method for treating or preventing a bacterial infection in a subject requiring treatment or prevention of a bacterial infection, comprising the step of administering to the subject an effective amount of crystalline tosylate of omadacycline according to claim 62, or the pharmaceutical composition according to claim 68.

76. A method for treating or preventing a bacterial infection in a subject requiring treatment or prevention of a bacterial infection, comprising the step of administering to the subject an effective amount of a polymorph of crystalline tosylate of omadacycline according to claim 63 or 64, or the pharmaceutical composition according to claim 69.

77. The method according to any one of claims 72 to 76, wherein the bacterial infection is caused by Gram-positive or Gram-negative bacteria.

78. The method according to any one of claims 72 to 76, wherein the bacterial infection is caused by bacteria that are resistant to other tetracycline compounds.

79. The method according to any one of claims 72 to 76, wherein the bacterial infection is caused by a bacterium of a species selected from the group consisting of Klebsiella pneumoniae, Salmonella, Enterococcus hirae, Acinetobacter baumanii, Branhamella catarrhalis, Haemophilus influenzae, Pseudomonas aeruginosa, Enterococcus faecium, Escherichia coli, Staphylococcus aureus, and Enterococcus faecalis.

80. The method according to any one of claims 72 to 76, wherein the bacterial infection is acute bacterial cutaneous tissue infection (ABSSSI).

81. ABSSSI includes Staphylococcus aureus (methicillin-susceptible and resistant isolates), Staphylococcus lugdunensis, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus anginosus group (including Streptococcus anginosus, Streptococcus intermedius, and Streptococcus constellatus), Streptococcus mitis, and Enterococcus faecalis. The method according to claim 80, caused by a bacterium of a species selected from the group consisting of *Faecalis* (vancomycin-sensitive isolate), *Enterobacter cloacae*, *Klebsiella pneumoniae*, *Prevotella melaninogenica*, and *Finegoldia magna*.

82. The method according to any one of claims 72 to 76, wherein the bacterial infection is community-acquired bacterial pneumonia (CABP).

83. The method according to claim 82, wherein CABP is caused by a bacterium of a species selected from the group consisting of Streptococcus pneumoniae (penicillin-susceptible and resistant isolates, macrolide-resistant isolates), Staphylococcus aureus (methicillin-susceptible isolates), Haemophilus influenzae (beta-lactamase-negative and positive isolates), Haemophilus parainfluenzae, Klebsiella pneumoniae, Legionella pneumophila, Mycoplasma pneumoniae, and Chlamydophila pneumoniae, including cases with bacteremia.

84. The method according to any one of claims 72 to 76, wherein the bacterial infection is caused by a bacterium of the species Clostridium difficile.

85. The method according to any one of claims 72 to 76, wherein the bacterial infection is caused by mycobacteria.

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