Process for preparing immunogenic azalides

Abstract

Defined herein is a new process for preparing higher yield and purity immunomodulating macrolides, particularly Formula (1), by using safer non-toxic reagents and reducing or eliminating certain mutagenic impurities.

Description

[0001] ZP000525A

[0002] -1-

[0003] PROCESS FOR PREPARING IMMUNOGENIC AZALIDES

[0004] FIELD OF THE INVENTION

[0005] 5 Defined herein is a new process for preparing immune-modulating azalides. The compounds prepared by the processes defined herein can be used for treating an inflammatory and / or immunological disease or disorder in an animal.

[0006] BACKGROUND OF THE INVENTION

[0007] 10

[0008] Macrolides are known antibacterial compounds that consist of a large macrocyclic lactone ring containing 12 to 16 atoms, which are attached, via glycosidic bonds, to at least one or two deoxy sugars. Azalides are a class of macrolide wherein the lactone ring contains a nitrogen atom. Draxxin® is a semi-synthetic macrolide (azalide) antibiotic that is sold as a ready to use sterile parenteral preparation containing tulathromycin. The preparation consists of an equilibrated mixture of two isomeric forms of tulathromycin in a 9:1 (Tula-A: Tula-B) ratio. Tula-A is a 15-membered lactone ring structure wherein Tula-B is a 13-membered lactone ring structure. Equilibrium is pH and time dependent. Draxxin® is marketed for bovine respiratory disease (BRD) and 20 porcine respiratory disease (SRD).

[0009] Macrolides are known for inhibiting protein synthesis in bacteria (gram positive and gram negative) by reversibly binding to the P site of the 50S unit of the ribosome. They tend to be bacteriostatic and may be bactericidal against some pathogens. Their activity against the Gram-negative pathogens of BRD and ability to concentrate in lung tissue make them an excellent therapeutic. They are the first-line treatment against BRD and are also used to treat respiratory infections in humans.

[0010] Known macrolide antibiotics include, for example, erythromycin, tilmicosin, azithromycin, clarithromycin, gamithromycin, fidaxomicin, roxithromycin, tulathromycin and others. In addition, some macrolides have been shown to have anti-inflammatory 30 and immune-modulating properties. For example, azithromycin, a broad-spectrum antibiotic, suppresses interleukin 12p40 expression in lipopolysaccharide (LPS) and interferon-gamma stimulated macrophages and attenuates LPS induced induction of CXCL8 (IL-8) and GM-CSF from primary bronchial epithelial cells; and in epithelial cells, interactions with phospholipids and Erk1 / 2, are followed by down-regulation of transcription factors AP-1, NFκB, inflammatory cytokines and mucin release following LPS stimulation. US2016-0031925 describes certain azithromycin analogues that are immune-modulating but have been modified to reduce or eliminate the antibiotic effect. Clarithromycin has an immunomodulatory effect on ERJ-mediated inflammation induced by Pseudomonas aeruginosa flagellin. Erythromycin inhibits interleukin-6 and interleukin-8 expression and promotes apoptosis of activated human neutrophils in vitro. Tilmicosin modulates COX-2 and iNOS gene expression and production of cytokines in LPS-stimulated macrophages and monocytes. Roxithromycin down-regulates Th2 chemokine production by keratinocytes and chemokine receptor expression on Th2 cells. Tulathromycin promotes apoptosis and down-regulates pro-inflammatory mediators like leukotriene B4 and CXCL8; and induces production of anti-inflammatory and pro-resolving lipid lipoxin A4. Research findings demonstrate that these antibacterial macrolides modulate certain excessive immune responses which in turn cascade into certain anti-inflammatory benefits.

[0011] Inflammation and pro-inflammatory mediators negatively affect production in the food animal industry by reducing growth, feed and water intake, reproduction, milk production and metabolic health. Increased clinical use of macrolide antibiotics is linked with an increase in pneumococcal macrolide resistance and resistance in BRD pathogens. Recent concerns by global governmental agencies and the general public relative to use of antibiotics in food producing animals (e.g., cattle and swine) has been thought to lead to cross resistance to human pathogens.

[0012] Bovine respiratory disease remains a major problem in modern day cattle production and judicious management is vitally important for both animal welfare and human food safety. In fact, Mannheimia haemolytica is a principal bacterium isolated from respiratory disease in feedlot cattle and is a significant component of enzootic pneumonia in neonatal calves. One of the hallmarks of BRD is a heightened inflammatory response in the host that promotes progression to the full BRD complex. Inhibiting or reversing the inflammation in the host can prevent or control the development of BRD in cattle and other inflammatory diseases or disorders in animals. Porcine respiratory disease, also referred to as enzootic pneumonia, is a common, widely distributed disease that occurs year-round, and is caused by the etiologic agent, Mycoplasma hyopneumoniae. Affected swine have chronic, persistent and nonproductive cough. As pneumonia develops, dyspnea becomes more marked and pulmonary lesions may be present, growth is retarded and feed efficiency decreases with a high probability of secondary infections. Despite methods to eliminate or control M. hyponeumoniae, it remains a significant economic problem.

[0013] Antibiotics may augment management in swine and cattle, however, due to the concerns of resistance and cross-resistance, there is an unmet desire to develop a new anti-inflammatory and immune-modulating agent that lacks the antibacterial effects of the known macrolides. A number of immune-modulating and non-antibacterial compounds and processes to prepare them were recently developed and described in patent application publications WO2021 / 183758, WO2021 / 183754, WO2021 / 183759, WO2021 / 183762 and WO2023 / 038852. As a means of increasing compound yield and purity, reducing and / or eliminating the need for toxic solvents and per-fluorinated catalysts and eliminating Class 2 / 3 mutagenic impurities during the preparation of these compounds, a new process was developed.

[0014] SUMMARY OF THE INVENTION

[0015] In one aspect of the invention, is a process for preparing M9, (2,3S,4,5,8,10R,11R,12S,13S,14R)-2-ethyl-3,4,10-trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-5-((propylamino)methyl)tetrahydro-2H-pyran-2-yl)oxy)-11-(((2S,3R,4S,6R)-3-hydroxy-6-methyl-4-(methylamino)tetrahydro-2H-pyran-2-yl)oxy)-3,5,8, 10,12,14-hexamethyl-1 -oxa-6-azacyclopentadecan-15-one 

[0016]

[0017] y I )oxy )-3, 5, 8, 10,12,14-hexamethyl-1 -oxa-6-azacyclopentadecan-15-one from tulathromycin A, (2R,3S,4R,5R,8R,10R,11 R,12S,13S,14R)-11-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-2-ethyl-3,4,10-trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-5-((propylamino)methyl)tetrahydro-2H-pyran-2-yl)oxy)-3,5,8, 10,12,14-hexamethyl-1 -oxa-6-azacyclopentadecan-15-one, comprising:

[0018] a) reacting tulathromycin A in n-butanol, methyl tert-butyl ether with the catalyst ethyl benzoylformate at a temperature of about 10°C to about 20°C for about 6 hours in the presence of an oxidant;

[0019] b) adding an aqueous solution of acetic acid at less than about 25°C and then discarding the organic phase;

[0020] c) adding toluene, methanol and sodium chloride then adjusting the pH to 9.5 to 10.0 with aqueous ammonium hydroxide and then discarding the aqueous phase; d) adding acetic acid, methanol and anhydrous cupric sulfate and heating to reflux for about 3 hours then cooling to less than about 20°C;

[0021] e) adding aqueous O-methylhydroxylamine hydrochloride and stirring for about 2 hours at about 27°C then discarding the organic phase;

[0022] f) adding water, 2-methyltetrahydrofuran and sodium chloride then adjusting the pH to at least 9.5 with aqueous ammonium hydroxide and then discarding the aqueous phase;

[0023] g) concentrating the organic phase under vacuum then adding acetonitrile to the slurry and then concentrating the slurry under vacuum then heating the resulting slurry to about 65°C while stirring for about 1 hour then cooling to about 5°C for at least 2 hours and holding at about 5°C for an additional hour; and

[0024] h) isolating the resulting M9 solids by filtration, washing the solids with cold acetonitrile at about 10°C then drying the M9 solids under vacuum.

[0025] In another aspect, is a process for preparing M9, (2R,3S,4R,5R,8R,10R,11 R,12S,13S,14R)-2-ethyl-3,4,10-trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-5-((propylamino)methyl)-tetrahydro-2H-pyran-2-yl)oxy)-11-(((2S,3R,4S,6R)-3-hydroxy-6-methyl-4-(methylamino)tetrahydro-2H-pyran-2-y I )oxy )-3, 5, 8, 10,12,14-hexamethyl-1 -oxa-6-azacyclopentadecan-15-one from tulathromycin A, (2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-11-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-2-ethyl-3,4,10-trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-5- ((propylamino)methyl)tetrahydro-2H-pyran-2-yl)oxy)-3,5,8, 10,12,14-hexamethyl-1 -oxa-6-azacyclopentadecan-15-one,

[0026] comprising:

[0027] a) reacting tulathromycin A in n-butanol, methyl tert-butyl ether with the catalyst ethyl benzoylformate at a temperature of about 10°C to about 20°C for about 6 hours in the presence of aqueous 30% H2O2;

[0028] b) adding an aqueous 3-4% solution of acetic acid at less than about 25°C and then discarding the organic phase;

[0029] c) adding toluene, methanol and sodium chloride then adjusting the pH to 9.5 to 10.0 with aqueous 30% ammonium hydroxide and then discarding the aqueous phase;

[0030] d) adding acetic acid, methanol and anhydrous cupric sulfate and heating to reflux for about 3 hours then cooling to less than about 20°C;

[0031] e) adding aqueous 16% O-methylhydroxylamine hydrochloride and stirring for about 2 hours at about 27°C then discarding the organic phase;

[0032] f) adding water, 2-methyltetrahydrofuran and sodium chloride then adjusting the pH to at least 9.5 with aqueous ammonium hydroxide and then discarding the aqueous phase;

[0033] g) concentrating the organic phase under vacuum then adding acetonitrile to the slurry and then concentrating the slurry under vacuum then heating the resulting slurry to about 65°C while stirring for about 1 hour then cooling to about 5°C for at least 2 hours and holding at about 5°C for an additional hour; and

[0034] h) isolating the resulting M9 solids by filtration, washing the solids with cold acetonitrile at about 10°C then drying the M9 solids under vacuum.

[0035] In another aspect, is a process for preparing M9,

[0036] (2R,3S,4R,5R,8R, 10R, 11 R, 12S, 13S, 14R)-2-ethyl-3,4, 10-trihydroxy-13- (((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-5-((propylamino)methyl)- tetrahydro-2H-pyran-2-yl)oxy)-11-(((2S,3R,4S,6R)-3-hydroxy-6-methyl-4- (m ethy I am i n o)tetrahyd ro-2 H-pyran-2-y I )oxy )-3, 5, 8, 10,12,14-hexamethyl-1 -oxa-6-azacyclopentadecan-15-one from tulathromycin A,

[0037] (2R,3S,4R,5R,8R,10R,11 R,12S,13S,14R)-11-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-2-ethyl-3,4, 10-trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-5- ((propylamino)methyl)tetrahydro-2H-pyran-2-yl)oxy)-3,5,8, 10,12,14-hexamethyl-1 -oxa-6-azacyclopentadecan-15-one, comprising:

[0038] a) reacting tulathromycin A in n-butanol, methyl tert-butyl ether with the catalyst ethyl benzoylformate at a temperature of about 10°C to about 20°C for about 6 hours in the presence of an oxidant;

[0039] b) adding an aqueous solution of acetic acid at less than 25°C and then discarding the organic phase;

[0040] c) adding acetic acid and cupric sulfate pentahydrate and heating to about 55-60°C for about 3 to 4 hours then cooling to less than 20°C;

[0041] d) adding aqueous O-methylhydroxylamine hydrochloride and stirring for about 3 to 4 hours at about 27°C then cooling to less than 20°C;

[0042] e) (i) adding 2-methyltetrahydrofuran and sodium chloride then adjusting the pH to at least 9.5 with aqueous ammonium hydroxide and then discarding the aqueous phase; or (ii) adding acetonitrile and then adjusting the pH to at least 9.5 with aqueous ammonium hydroxide and then adding tetrahydrofuran;

[0043] f) (i) concentrating the organic phase from e(i) under vacuum then adding acetonitrile to the slurry then concentrating the slurry under vacuum then heating the resulting slurry to about 65°C while stirring for about 1 hour then cooling to about 5°C for at least 2 hours and holding at about 5°C for an additional hour; or (ii) heating the resulting slurry from e(ii) to about 50°C while stirring for about 1 hour then cooling to about 5°C over about 2 hours and holding at about 5°C for an hour; and

[0044] g) isolating the M9 solids from f(i) by filtration, washing the solids with cold acetonitrile at about 10°C and then drying the solids under vacuum; or isolating the M9 solids from f(ii) filtration and washing the solids with 5% aqueous ammonium hydroxide, water and acetonitrile at about 10°C then drying the M9 solids under vacuum. In another aspect, is a process for preparing M9,

[0045] (2R,3S,4R,5R,8R, 10R, 11 R, 12S, 13S, 14R)-2-ethyl-3,4, 10-trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-5-((propylamino)methyl)-tetrahydro-2H-pyran-2-yl)oxy)-11-(((2S,3R,4S,6R)-3-hydroxy-6-methyl-4-(methylamino)tetrahydro-2H-pyran-2-yl)oxy)-3,5,8,10,12,14-hexamethyl-1-oxa-6-azacyclopentadecan-15-one from tulathromycin A,

[0046] (2R,3S,4R,5R,8R,10R,11 R,12S,13S,14R)-11-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-2-ethyl-3,4, 10-trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-5- ((propylamino)methyl)tetrahydro-2H-pyran-2-yl)oxy)-3,5,8, 10,12,14-hexamethyl-1 -oxa-6-azacyclopentadecan-15-one, comprising:

[0047] a) reacting tulathromycin A in n-butanol, methyl tert-butyl ether with the catalyst ethyl benzoylformate at a temperature of about 10°C to about 20°C for about 6 hours in the presence of aqueous 30% H2O2;

[0048] b) adding an aqueous 3-4% solution of acetic acid at less than 25°C and then discarding the organic phase;

[0049] c) adding acetic acid and cupric sulfate pentahydrate and heating to about 55-60°C for about 3 to 4 hours then cooling to less than 20°C;

[0050] d) adding aqueous 16% O-methylhydroxylamine hydrochloride and stirring for about 3 to 4 hours at about 27°C then cooling to less than 20°C;

[0051] e) (i) adding 2-methyltetrahydrofuran and sodium chloride then adjusting the pH to at least 9.5 with aqueous 30% ammonium hydroxide and then discarding the aqueous phase; or (ii) adding acetonitrile and then adjusting the pH to at least 9.5 with aqueous 30% ammonium hydroxide and then adding tetrahydrofuran; f) (i) concentrating the organic phase from e(i) under vacuum then adding acetonitrile to the slurry then concentrating the slurry under vacuum then heating the resulting slurry to about 65°C while stirring for about 1 hour then cooling to about 5°C for at least 2 hours and holding at about 5°C for an additional hour; or (ii) heating the resulting slurry from e(ii) to about 50°C while stirring for about 1 hour then cooling to about 5°C over about 2 hours and holding at about 5°C for an hour; and g) isolating the M9 solids from f(i) by filtration, washing the solids with cold acetonitrile at about 10°C and then drying the M9 solids under vacuum; or isolating the M9 solids from f(ii) filtration and washing the solids with 5% aqueous ammonium hydroxide, water and acetonitrile at about 10°C then drying the M9 solids under vacuum.

[0052] E

[0053] o C0 < D ■u c Z3 co " O o <n

[0054]

[0055] 13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-5-((propylamino)methyl)tetrahydro-2H-pyran-2-yl)oxy)-3,5,8,10,12,14-hexamethyl-15-oxo-1 -oxa-6-azacyclopentadecan-11 -yl)oxy)-3-hydroxy-6-methyltetrahydro-2H-pyran-4-yl)-1,3-dimethyl-3-phenylurea from M9, (2R,3S,4R,5R,8R,10R,11 R,12S,13S,14R)-2-ethyl-3,4, 10-trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-5- ((propylamino)methyl)tetrahydro-2H-pyran-2-yl)oxy)-11-(((2S,3R,4S,6R)-3-hydroxy-6-methyl-4-(methylamino)tetrahydro-2H-pyran-2-yl)oxy)-3,5,8, 10,12,14-hexamethyl-1 -oxa-6-azacyclopentadecan-15-one, comprising:

[0056] a) combining M9 with ethyl acetate and triethylamine and cooling to about 10°C; b) adding a solution of N-methyl-N-phenyl-carbamoyl chloride in ethyl acetate while stirring at about 10°C for about 15 hours;

[0057] c) adding water to the reactants and discarding the aqueous phase;

[0058] d) adding water and acetic acid and discarding the organic phase;

[0059] e) washing the aqueous phase with ethyl acetate then adding methyl tert-butyl ether while adjusting the pH to about 8.0 to about 8.5 with 30% aqueous ammonium hydroxide, then discarding the aqueous phase;

[0060] f) washing the organic phase with water then concentrating the organic phase under vacuum, then adding isopropanol then concentrating again under vacuum and cooling the slurry to about 20°C; and

[0061] g) adding isopropanol then water while adding more water over about 90 minutes, then stirring the mixture for about 1 hour at about 20°C; and

[0062] h) filtering the resulting solids, washing the solids with a solution of water and isopropanol, then washing the solids again with water then drying under vacuum to afford the Formula (1) compound as a free base.

[0063] In another aspect, is a process for preparing the Formula (1) compound, 1-((2S,3R,4S,6R)-2-(((2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-2-ethyl-3,4,10-trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-5-((propylamino)methyl)tetrahydro-2H-pyran-2-yl)oxy)-3,5,8,10,12,14-hexamethyl-15-oxo-1 -oxa-6-azacyclopentadecan-11 -yl)oxy)-3-hydroxy-6-methyltetrahydro-2H-pyran-4-yl)-1,3-dimethyl-3-phenylurea from M9, (2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-2-ethyl-3,4, 10-trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-5- ((propylamino)methyl)tetrahydro-2H-pyran-2-yl)oxy)-11-(((2S,3R,4S,6R)-3-hydroxy-6-methyl-4-(methylamino)tetrahydro-2H-pyran-2-yl)oxy)-3,5,8, 10,12,14-hexamethyl-1 -oxa-6-azacyclopentadecan-15-one, comprising:

[0064] a) combining M9 with 2-methyltetrahydrofuran and triethylamine;

[0065] b) adding a solution of N-methyl-N-phenyl-carbamoyl chloride in 2- methyltetrahydrofuran at about 20°C while stirring for about 6 hours;

[0066] c) adding water to the reactants and discarding the aqueous phase;

[0067] d) adding water and acetic acid and discarding the organic phase;

[0068] e) washing the aqueous phase with methyl-tert-butyl ether and discarding the organic phase;

[0069] f) adding isopropanol and adjusting the pH to about 8.0 with aqueous 30% ammonium hydroxide;

[0070] g) adding seed crystals of the Formula (1) compound while holding the reactants for about 30 minutes then adjusting the pH further to about 9.5 with aqueous ammonium hydroxide while stirring the slurry at about 20°C for about one hour; and

[0071] h) collecting the resulting solids by filtration, washing the solids with a solution of water and isopropanol, followed by water then drying under vacuum to afford the Formula (1) compound as a free base.

[0072] In another aspect, is a process for preparing the Formula (1) compound, 1- ((2S,3R,4S,6R)-2-(((2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-2-ethyl-3,4,10- trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-5- ((propylamino)methyl)tetrahydro-2H-pyran-2-yl)oxy)-3,5,8,10,12,14-hexamethyl-15- oxo-1 -oxa-6-azacyclopentadecan-11-yl)oxy)-3-hydroxy-6-methyltetrahydro-2H- pyran-4-yl)-1,3-dimethyl-3-phenylurea from M9,

[0073] (2R,3S,4R,5R,8R, 10R, 11 R,12S, 13S, 14R)-2-ethyl-3,4,10-trihydroxy-13- (((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-5- ((propylamino)methyl)tetrahydro-2H-pyran-2-yl)oxy)-11-(((2S,3R,4S,6R)-3-hydroxy- 6-methyl-4-(methylamino)tetrahydro-2H-pyran-2-yl)oxy)-3,5,8, 10,12,14-hexamethyl- 1-oxa-6-azacyclopentadecan-15-one, comprising:

[0074] a) combining M9 with 2-methyltetrahydrofuran and triethylamine; b) adding a solution of N-methyl-N-phenyl-carbamoyl chloride in 2- methyltetrahydrofuran at about 20°C while stirring for about 6 hours;

[0075] c) adding water to the reactants and discarding the aqueous phase;

[0076] d) adding water and acetic acid and discarding the organic phase;

[0077] e) washing the aqueous phase with methyl-tert-butyl ether and discarding the organic phase;

[0078] f) adding isopropanol and adjusting the pH to about 9.5 with aqueous 30% ammonium hydroxide;

[0079] g) holding the mixture for about 30 minutes then adding water with stirring at about 20°C for about one hour; and

[0080] h) collecting the resulting solids by filtration, washing the solids with a solution of water and isopropanol, followed by water then drying under vacuum to afford the Formula (1) compound as a free base.

[0081] In another aspect, is a process for preparing the Formula (1) compound, 1-((2S,3R,4S,6R)-2-(((2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-2-ethyl-3,4,10-trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-5-((propylamino)methyl)tetrahydro-2H-pyran-2-yl)oxy)-3,5,8,10,12,14-hexamethyl-15-oxo-1 -oxa-6-azacyclopentadecan-11 -yl)oxy)-3-hydroxy-6-methyltetrahydro-2H-pyran-4-yl)-1,3-dimethyl-3-phenylurea from M9, (2R,3S,4R,5R,8R,10R,11 R,12S,13S,14R)-2-ethyl-3,4, 1 O-trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-5-((propylamino)methyl)tetrahydro-2H-pyran-2-yl)oxy)-11-(((2S,3R,4S,6R)-3-hydroxy-6-methyl-4-(methylamino)tetrahydro-2H-pyran-2-yl)oxy)-3,5,8, 10,12,14-hexamethyl-1 -oxa-6-azacyclopentadecan-15-one, that is prepared from tulathromycin A, (2R,3S,4R,5R,8R,10R,11 R,12S,13S,14R)-11-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-2-ethyl-3,4,10-trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-5-((propylamino)methyl)tetrahydro-2H-pyran-2-yl)oxy)-3,5,8, 10, 12, 14-hexamethyl-1 -oxa-6-azacyclopentadecan-15-one, comprising:

[0082] a) reacting tulathromycin A in n-butanol, methyl tert-butyl ether with the catalyst ethyl benzoylformate at a temperature of about 10°C to about 20°C for about 6 hours in the presence of an oxidant; b) adding an aqueous solution of acetic acid at less than 25°C then discarding the organic phase;

[0083] c) adding toluene, methanol and sodium chloride to the aqueous phase and then adjusting the pH to 9.5 to 10.0 with aqueous ammonium hydroxide and then discarding the aqueous phase;

[0084] d) adding acetic acid, methanol and anhydrous cupric sulfate and heating to reflux for about 3 hours then cooling to less than about 20°C;

[0085] e) adding aqueous O-methylhydroxylamine hydrochloride and stirring for about 2 hours at about 27°C then discarding the organic phase;

[0086] f) adding water, 2-methyltetrahydrofuran and sodium chloride and adjusting the pH to at least 9.5 with aqueous ammonium hydroxide and then discarding the aqueous phase;

[0087] g) concentrating the organic phase under vacuum then adding acetonitrile to the slurry then concentrating the slurry under vacuum then heating the resulting slurry to about 65°C while stirring for about 1 hour then cooling to about 5°C for at least 2 hours and holding at about 5°C for an additional hour;

[0088] h) isolating the M9 solids by filtration, washing the solids with cold acetonitrile at about 10°C and then drying the solids under vacuum;

[0089] i) combining M9 with ethyl acetate and triethylamine and cooling to about 10°C; j) adding a solution of N-methyl-N-phenyl-carbamoyl chloride in ethyl acetate while stirring at about 10°C for about 15 hours;

[0090] k) adding water to the reactants and discarding the aqueous phase;

[0091] l) adding water and acetic acid and discarding the organic phase;

[0092] m) washing the aqueous phase with ethyl acetate then adding methyl tert-butyl ether while adjusting the pH to about 8.0 to about 8.5 with aqueous ammonium hydroxide then discarding the aqueous phase;

[0093] n) washing the organic phase with water then concentrating the organic phase under vacuum, then adding isopropanol then concentrating again under vacuum and cooling the slurry to about 20°C; and

[0094] o) adding isopropanol then water while adding more water over about 90 minutes, then stirring the mixture for about 1 hour at about 20°C, filtering the solids, washing the solids with a solution of water and isopropanol, then washing the solids again with water before drying under vacuum to afford the Formula (1) compound as a free base.

[0095] In another aspect, is a process for preparing the Formula (1) compound, 1-((2S,3R,4S,6R)-2-(((2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-2-ethyl-3,4,10-trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-5-((propylamino)methyl)tetrahydro-2H-pyran-2-yl)oxy)-3,5,8,10,12,14-hexamethyl-15-oxo-1 -oxa-6-azacyclopentadecan-11 -yl)oxy)-3-hydroxy-6-methyltetrahydro-2H-pyran-4-yl)-1,3-dimethyl-3-phenylurea from M9, (2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-2-ethyl-3,4, 1 O-trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-5-((propylamino)methyl)tetrahydro-2H-pyran-2-yl)oxy)-11-(((2S,3R,4S,6R)-3-hydroxy-6-methyl-4-(methylamino)tetrahydro-2H-pyran-2-yl)oxy)-3,5,8, 10,12,14-hexamethyl-1 -oxa-6-azacyclopentadecan-15-one, that is prepared from tulathromycin A, (2R,3S,4R,5R,8R,10R,11 R,12S,13S,14R)-11-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-2-ethyl-3,4,10-trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-5-((propylamino)methyl)tetrahydro-2H-pyran-2-yl)oxy)-3,5,8, 10, 12, 14-hexamethyl-1 -oxa-6-azacyclopentadecan-15-one, comprising:

[0096] a) reacting tulathromycin A in n-butanol, methyl tert-butyl ether with the catalyst ethyl benzoylformate at a temperature of about 10°C to about 20°C for about 6 hours in the presence of aqueous 30% H2O2;

[0097] b) adding an aqueous 3-4% solution of acetic acid at less than 25°C then discarding the organic phase;

[0098] c) adding toluene, methanol and sodium chloride to the aqueous phase and then adjusting the pH to 9.5 to 10.0 with aqueous 30% ammonium hydroxide and then discarding the aqueous phase;

[0099] d) adding acetic acid, methanol and anhydrous cupric sulfate and heating to reflux for about 3 hours then cooling to less than about 20°C;

[0100] e) adding aqueous 16% O-methylhydroxylamine hydrochloride and stirring for about 2 hours at about 27°C then discarding the organic phase; f) adding water, 2-methyltetrahydrofuran and sodium chloride and adjusting the pH to at least 9.5 with aqueous 30% ammonium hydroxide and then discarding the aqueous phase;

[0101] g) concentrating the organic phase under vacuum then adding acetonitrile to the slurry then concentrating the slurry under vacuum then heating the resulting slurry to about 65°C while stirring for about 1 hour then cooling to about 5°C for at least 2 hours and holding at about 5°C for an additional hour;

[0102] h) isolating the M9 solids by filtration, washing the solids with cold acetonitrile at about 10°C and then drying the solids under vacuum;

[0103] i) combining M9 with ethyl acetate and triethylamine and cooling to about 10°C; j) adding a solution of N-methyl-N-phenyl-carbamoyl chloride in ethyl acetate while stirring at about 10°C for about 15 hours;

[0104] k) adding water to the reactants and discarding the aqueous phase;

[0105] l) adding water and acetic acid and discarding the organic phase;

[0106] m) washing the aqueous phase with ethyl acetate then adding methyl tert-butyl ether while adjusting the pH to about 8.0 to about 8.5 with aqueous 30% ammonium hydroxide then discarding the aqueous phase;

[0107] n) washing the organic phase with water then concentrating the organic phase under vacuum, then adding isopropanol then concentrating again under vacuum and cooling the slurry to about 20°C; and

[0108] o) adding isopropanol then water while adding more water over about 90 minutes, then stirring the mixture for about 1 hour at about 20°C, filtering the solids, washing the solids with a solution of water and isopropanol, then washing the solids again with water before drying under vacuum to afford the Formula (1) compound as a free base.

[0109] In another aspect, is a process for preparing the Formula (1) compound, 1-((2S,3R,4S,6R)-2-(((2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-2-ethyl-3,4,10-trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-5-((propylamino)methyl)tetrahydro-2H-pyran-2-yl)oxy)-3,5,8,10,12,14-hexamethyl-15-oxo-1 -oxa-6-azacyclopentadecan-11-yl)oxy)-3-hydroxy-6-methyltetrahydro-2H-pyran-4-yl)- 1,3-dimethyl-3-phenylurea from M9, (2R,3S,4R,5R,8R, 10R, 11 R,12S, 13S, 14R)-2-ethyl-3,4,10-trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-5- ((propylamino)methyl)tetrahydro-2H-pyran-2-yl)oxy)-11-(((2S,3R,4S,6R)-3-hydroxy-6-methyl-4-(methylamino)tetrahydro-2H-pyran-2-yl)oxy)-3,5,8, 10,12,14-hexamethyl-1-oxa-6-azacyclopentadecan-15-one (M9) that is prepared from tulathromycin A, (2R,3S,4R,5R,8R,10R,11 R,12S,13S,14R)-11-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-2-ethyl-3,4, 10-trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-5- ((propylamino)methyl)tetrahydro-2H-pyran-2-yl)oxy)-3,5,8, 10,12,14-hexamethyl-1 -oxa-6-azacyclopentadecan-15-one, comprising:

[0110] a) reacting tulathromycin A in n-butanol, methyl tert-butyl ether with the catalyst ethyl benzoylformate at a temperature of about 10°C to about 20°C for about 6 hours in the presence of an oxidant;

[0111] b) adding an aqueous solution of acetic acid at less than 25°C then discarding the organic phase;

[0112] c) adding toluene, methanol and sodium chloride to the aqueous phase then adjusting the pH to 9.5 to 10.0 with aqueous ammonium hydroxide and then discarding the aqueous phase;

[0113] d) adding acetic acid, methanol and anhydrous cupric sulfate and heating to reflux for 3 hours then cooling to less than 20°C;

[0114] e) adding aqueous O-methylhydroxylamine hydrochloride and stirring for about 2 hours at about 27°C then discarding the organic phase;

[0115] f) adding water, 2-methyltetrahydrofuran and sodium chloride and adjusting the pH to at least 9.5 with aqueous ammonium hydroxide then discarding the aqueous phase;

[0116] g) concentrating the organic phase under vacuum then adding acetonitrile to the slurry and then concentrating the slurry under vacuum then heating the resulting slurry to about 65°C while stirring for about 1 hour then cooling to about 5°C for about 2 hours and holding at 5°C for an additional hour;

[0117] h) isolating the M9 solids by filtration, washing the solids with cold acetonitrile at about 10°C then drying the solids under vacuum;

[0118] i) combining M9, 2-methyl tetrahydrofuran and triethylamine, j) adding a solution of N-methyl-N-phenyl-carbamoyl chloride in 2-methyl tetrahydrofuran at about 20°C while stirring for about 6 hours;

[0119] k) adding water to the reactants then discarding the aqueous phase;

[0120] l) adding water and acetic acid then discarding the organic phase;

[0121] m) washing the aqueous phase with methyl tert-butyl ether and discarding the organic phase;

[0122] n) adding isopropanol and adjusting the pH to 8.0 with aqueous ammonium hydroxide;

[0123] o) adding seed crystals of Formula (1) while holding the reactants for about 30 minutes, then adjusting the pH to 9.5 with aqueous ammonium hydroxide while stirring the slurry at about 20°C for about 1 hour; and

[0124] p) collecting the resulting solids by filtration, washing with a solution of water and isopropanol, then washing with water and drying the solids under vacuum to afforfd the Formula (1) compound as a free base.

[0125] In another aspect, is a process for preparing the Formula (1) compound, 1-((2S,3R,4S,6R)-2-(((2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-2-ethyl-3,4,10-trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-5-((propylamino)methyl)tetrahydro-2H-pyran-2-yl)oxy)-3,5,8,10,12,14-hexamethyl-15-oxo-1 -oxa-6-azacyclopentadecan-11-yl)oxy)-3-hydroxy-6-methyltetrahydro-2H-pyran-4-yl)-1,3-dimethyl-3-phenylurea from M9,

[0126] (2R,3S,4R,5R,8R, 10R, 11 R,12S, 13S, 14R)-2-ethyl-3,4,10-trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-5-((propylamino)methyl)tetrahydro-2H-pyran-2-yl)oxy)-11-(((2S,3R,4S,6R)-3-hydroxy-6-methyl-4-(methylamino)tetrahydro-2H-pyran-2-yl)oxy)-3,5,8, 10,12,14-hexamethyl-1-oxa-6-azacyclopentadecan-15-one (M9) that is prepared from tulathromycin A, (2R,3S,4R,5R,8R,10R,11 R,12S,13S,14R)-11-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-2-ethyl-3,4, 10-trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-5-((propylamino)methyl)tetrahydro-2H-pyran-2-yl)oxy)-3,5,8, 10,12,14-hexamethyl-1 -oxa-6-azacyclopentadecan-15-one, comprising: a) reacting tulathromycin A in n-butanol, methyl tert-butyl ether with the catalyst ethyl benzoylformate at a temperature of about 10°C to about 20°C for about 6 hours in the presence of aqueous 30% H2O2;

[0127] b) adding an aqueous 3-4% solution of acetic acid at less than 25°C then discarding the organic phase;

[0128] c) adding toluene, methanol and sodium chloride to the aqueous phase then adjusting the pH to 9.5 to 10.0 with aqueous 30% ammonium hydroxide and then discarding the aqueous phase;

[0129] d) adding acetic acid, methanol and anhydrous cupric sulfate and heating to reflux for 3 hours then cooling to less than 20°C;

[0130] e) adding aqueous 16% O-methylhydroxylamine hydrochloride and stirring for about 2 hours at about 27°C then discarding the organic phase;

[0131] f) adding water, 2-methyltetrahydrofuran and sodium chloride and adjusting the pH to at least 9.5 with aqueous ammonium hydroxide then discarding the aqueous phase;

[0132] g) concentrating the organic phase under vacuum then adding acetonitrile to the slurry and then concentrating the slurry under vacuum then heating the resulting slurry to about 65°C while stirring for about 1 hour then cooling to about 5°C for about 2 hours and holding at 5°C for an additional hour;

[0133] h) isolating the M9 solids by filtration, washing the solids with cold acetonitrile at about 10°C then drying the solids under vacuum;

[0134] i) combining M9, 2-methyl tetrahydrofuran and triethylamine,

[0135] j) adding a solution of N-methyl-N-phenyl-carbamoyl chloride in 2-methyl tetrahydrofuran at about 20°C while stirring for about 6 hours;

[0136] k) adding water to the reactants then discarding the aqueous phase;

[0137] l) adding water and acetic acid then discarding the organic phase;

[0138] m) washing the aqueous phase with methyl tert-butyl ether and discarding the organic phase;

[0139] n) adding isopropanol and adjusting the pH to 8.0 with aqueous 30% ammonium hydroxide; o) adding seed crystals of Formula (1) while holding the reactants for about 30 minutes, then adjusting the pH to 9.5 with aqueous 30% ammonium hydroxide while stirring the slurry at about 20°C for about 1 hour; and

[0140] p) collecting the resulting solids by filtration, washing with a solution of water and isopropanol, then washing with water and drying the solids under vacuum to afford the Formula (1) compound as a free base.

[0141] In another aspect, is a process for preparing the Formula (1) compound, 1-((2S,3R,4S,6R)-2-(((2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-2-ethyl-3,4,10-trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-5-((propylamino)methyl)tetrahydro-2H-pyran-2-yl)oxy)-3,5,8,10,12,14-hexamethyl-15-oxo-1 -oxa-6-azacyclopentadecan-11 -yl)oxy)-3-hydroxy-6-methyltetrahydro-2H-pyran-4-yl)-1,3-dimethyl-3-phenylurea from M9, (2R,3S,4R,5R,8R,10R,11 R,12S,13S,14R)-2-ethyl-3,4, 1 O-trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-5-((propylamino)methyl)tetrahydro-2H-pyran-2-yl)oxy)-11-(((2S,3R,4S,6R)-3-hydroxy-6-methyl-4-(methylamino)tetrahydro-2H-pyran-2-yl)oxy)-3,5,8, 10,12,14-hexamethyl-1 -oxa-6-azacyclopentadecan-15-one, that is prepared from tulathromycin A, (2R,3S,4R,5R,8R,10R,11 R,12S,13S,14R)-11-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-2-ethyl-3,4,10-trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-5-((propylamino)methyl)tetrahydro-2H-pyran-2-yl)oxy)-3,5,8, 10, 12, 14-hexamethyl-1 -oxa-6-azacyclopentadecan-15-one, comprising:

[0142] a) reacting tulathromycin A in n-butanol, methyl tert-butyl ether with the catalyst ethyl benzoylformate at a temperature of about 10°C to about 20°C for about 6 hours in the presence of an oxidant;

[0143] b) adding an aqueous solution of acetic acid at less than 25°C and then discarding the organic phase;

[0144] c) adding toluene, methanol and sodium chloride to the aqueous phase and then adjusting the pH of the aqueous phase to 9.5 to 10.0 with aqueous ammonium hydroxide then discarding the aqueous phase;

[0145] d) adding acetic acid, methanol and anhydrous cupric sulfate and heating to reflux for 3 hours then cooling to less than 20°C; e) adding aqueous O-methylhydroxylamine hydrochloride and stirring for about 2 hours at about 27°C then discarding the organic phase;

[0146] f) adding water, 2-methyltetrahydrofuran and sodium chloride and adjusting the pH to at least 9.5 with aqueous ammonium hydroxide and then discarding the aqueous phase;

[0147] g) concentrating the organic phase under vacuum then adding acetonitrile to the slurry and then concentrating the slurry under vacuum then heating the resulting slurry to about 65°C while stirring for about 1 hour then cooling to about 5°C for about 2 hours and holding at about 5°C for an additional hour; and

[0148] h) isolating the M9 solids by filtration, washing the solids with cold acetonitrile at about 10°C and then drying the solids under vacuum;

[0149] i) combining M9, 2-methyl tetrahydrofuran and triethylamine;

[0150] j. adding a solution of N-methyl-N-phenyl-carbamoyl chloride in 2-methyl tetrahydrofuran at about 20°C while stirring for about 6 hours;

[0151] k) adding water to the reactants and discarding the aqueous phase;

[0152] l) adding water acetic acid and discarding the organic phase;

[0153] m) washing the aqueous phase with methyl tert-butyl ether and discarding the organic phase;

[0154] n) adding isopropanol and adjusting the pH to 9.5 with aqueous ammonium hydroxide;

[0155] o) holding the mixture for about 30 minutes, then adding water and stirring the slurry at about 20°C for about 1 hour; and

[0156] p) collecting the resulting solids by filtration, washing with a solution of water and isopropanol, then washing with water and drying the solids under vacuum to afford the Formula (1) compound as a free base.

[0157] In another aspect, is a process for preparing the Formula (1) compound, 1-((2S,3R,4S,6R)-2-(((2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-2-ethyl-3,4,10-trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-5-((propylamino)methyl)tetrahydro-2H-pyran-2-yl)oxy)-3,5,8,10,12,14-hexamethyl-15-oxo-1 -oxa-6-azacyclopentadecan-11 -yl)oxy)-3-hydroxy-6-methyltetrahydro-2H-pyran-4-yl)-1,3-dimethyl-3-phenylurea from M9, (2R,3S,4R,5R,8R, 10R, 11 R, 12S, 13S, 14R)-2-ethyl- 3,4, 10-trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-5-((propylamino)methyl)tetrahydro-2H-pyran-2-yl)oxy)-11-(((2S,3R,4S,6R)-3-hydroxy-6-methyl-4-(methylamino)tetrahydro-2H-pyran-2-yl)oxy)-3,5,8, 10,12,14-hexamethyl-1 -oxa-6-azacyclopentadecan-15-one, that is prepared from tulathromycin A, (2R,3S,4R,5R,8R,10R,11 R,12S,13S,14R)-11-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-2-ethyl-3,4,10-trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-5-((propylamino)methyl)tetrahydro-2H-pyran-2-yl)oxy)-3,5,8, 10, 12, 14-hexamethyl-1 -oxa-6-azacyclopentadecan-15-one, comprising:

[0158] a) reacting tulathromycin A in n-butanol, methyl tert-butyl ether with the catalyst ethyl benzoylformate at a temperature of about 10°C to about 20°C for about 6 hours in the presence of aqueous 30% H2O2;

[0159] b) adding an aqueous 3-4% solution of acetic acid at less than 25°C and then discarding the organic phase;

[0160] c) adding toluene, methanol and sodium chloride to the aqueous phase and then adjusting the pH of the aqueous phase to 9.5 to 10.0 with aqueous 30% ammonium hydroxide then discarding the aqueous phase;

[0161] d) adding acetic acid, methanol and anhydrous cupric sulfate and heating to reflux for 3 hours then cooling to less than 20°C;

[0162] e) adding aqueous 16% O-methylhydroxylamine hydrochloride and stirring for about 2 hours at about 27°C then discarding the organic phase;

[0163] f) adding water, 2-methyltetrahydrofuran and sodium chloride and adjusting the pH to at least 9.5 with aqueous 30% ammonium hydroxide and then discarding the aqueous phase;

[0164] g) concentrating the organic phase under vacuum then adding acetonitrile to the slurry and then concentrating the slurry under vacuum then heating the resulting slurry to about 65°C while stirring for about 1 hour then cooling to about 5°C for about 2 hours and holding at about 5°C for an additional hour; and

[0165] h) isolating the M9 solids by filtration, washing the solids with cold acetonitrile at about 10°C and then drying the solids under vacuum;

[0166] i) combining M9, 2-methyl tetrahydrofuran and triethylamine; j. adding a solution of N-methyl-N-phenyl-carbamoyl chloride in 2-methyl tetrahydrofuran at about 20°C while stirring for about 6 hours;

[0167] k) adding water to the reactants and discarding the aqueous phase;

[0168] l) adding water acetic acid and discarding the organic phase;

[0169] m) washing the aqueous phase with methyl tert-butyl ether and discarding the organic phase;

[0170] n) adding isopropanol and adjusting the pH to 9.5 with aqueous 30% ammonium hydroxide;

[0171] o) holding the mixture for about 30 minutes, then adding water and stirring the slurry at about 20°C for about 1 hour; and

[0172] p) collecting the resulting solids by filtration, washing with a solution of water and isopropanol, then washing with water and drying the solids under vacuum to afford the Formula (1) compound as a free base.

[0173] DISCUSSION

[0174] Unless otherwise defined, scientific and technical terms used in connection with the compounds of the invention defined herein shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures utilized in connection with, and techniques of, chemistry synthesis, macrolides, and immunomodulation defined herein are those that are well known and commonly used in the art. The term “about” as used herein, when used in connection with a measurable numerical variable, refers to the indicated value of the variable and to all values of the variable that are within the experimental error of the indicated value (e.g., within the 95% confidence interval for the mean) or within 10 percent of the indicated value, whichever is greater.

[0175] Other than in operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about”. “About” when used in connection with a measurable numerical variable, refers to the indicated value of the variable and to all values of the variable that are within the experimental error of the indicated value (e.g., within the 95% confidence interval for the mean) or within 10 percent of the indicated value, whichever is greater. In some jurisdictions, the term is considered ambiguous, as such, the term can be readily deleted from the claims thereby ensuring the range as claimed.

[0176] The compounds of the present invention have several asymmetric centers.

[0177] Compounds with asymmetric centers give rise to enantiomers (optical isomers), diastereomers (configurational isomers) or both, and it is intended that all of the possible enantiomers and diastereomers in mixtures and as pure or partially purified compounds are included within the scope of this invention. The present invention is meant to encompass all steric forms of the compounds of the invention. The present invention includes all stereoisomers of compounds of the invention.

[0178] Oxidation of tertiary amines to form N-oxides can be accomplished with polyfluoroalkyl ketones (2,2,2-trifluoroacetophenone as an Organocatalyst for the Oxidation of Tertiary Amines and Azines to N-Oxides, Limnios, D., et.al., Chem. Eur. J 2014, 20, 559-563). According to the citation, the trifluoroacetophenone was identified as the optimal catalyst for the oxidation of aliphatic tertiary amines that is chemoselective and proceeds to high-to-quantitative yields, particularly in the presence of H2O2, acetonitrile and a buffer. According to the Organocatalyst citation, fluorinated catalysts provided the highest yield of N-oxides. As described in WO2021 / 183759 (p.58), hexafluoroacetone trihydrate was used as the catalyst with methylene chloride used downstream for preparing the M9 intermediate from tulathromycin epoxide and tulathromycin A. In an effort to remove the perfluoroalkyl (PFA) catalyst from the macrolide process described herein, Applicant used the non-fluorinated catalyst ethyl benzoylformate that was described in the citation (Entry 13, Table 1) which only provided a 46% oxidation yield from pyridine after 18 hours. Regardless, Applicant unexpectedly found that the fluorine-free catalyst, ethyl benzoylformate, converted the tertiary amine of tulathromycin to the N-oxide in exceptionally high yield without the need for acetonitrile or a buffer when stirred for only 6 hours at room temperature. Schemes and Experimentals

[0179] Tulathromycin A is a 15-membered (lactone A) closed ring antibacterial macrolide (azalide). The azalide converts to a 13-membered (lactone B) closed ring, tulathromycin B. This conversion is in an equilibrium ratio of about 9:1 (A: B), and is depicted below.

[0180] / -• A '■>

[0181] HO. ' HO

[0182]

[0183] H H \.

[0184] tulathromycin A tulathromycin B

[0185] One of the metabolites of tulathromycin A is the des-methyl azalide, (2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-2-ethyl-3,4,10-trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-5-((propylamino)methyl)-tetrahydro-2H-pyran-2-yl)oxy)-11-(((2S,3R,4S,6R)-3-hydroxy-6-methyl-4-(methylamino)tetrahydro-2H-pyran-2-yl)oxy)-3,5,8, 10,12, 14-hexamethyl-1-oxa-6-azacyclopentadecan-15-one (M9), depicted in the following diagram below:

[0186]

[0187] The des-methyl tulathromycin (M9) analogue can be used as a starting material for preparing macrolide compounds depicted in WO2021 / 183758, WO2021 / 183754, WO2021 / 183759, WO2021 / 183762 and WO2023 / 038852.

[0188] For illustrative purposes, the reaction scheme depicted below demonstrates a potential route for synthesizing M9 from tulathromycin A. For a more detailed description of the individual reaction steps, see the Examples section below. Those skilled in the art will appreciate that other suitable starting materials, reagents, and synthetic routes may be used to synthesize the intermediates and compounds of the invention and a variety of derivatives thereof. Further, many of the compounds prepared by the methods described below can be prepared and / or modified using conventional chemistry.

[0189] The compounds prepared by the processes described herein can be used in its native form or as a salt. In cases where forming a stable nontoxic acid salt is desired, administration of the compound as a pharmaceutically acceptable salt may be appropriate. For the purpose of the present invention, the term "pharmaceutically acceptable salt" refers to those salts which are, within the scope of sound medical evaluation, suitable for use in contact with the tissues and organs of animals without displaying toxicity, irritation, allergic response and the like and are commensurate with a reasonable benefit / risk ratio. Further, the compounds of the invention have a secondary or tertiary amine group, which has basic character and thus can form acid addition salts, which may be pharmaceutically acceptable acids. Therefore, pharmaceutically acceptable salts according to the present invention include those pharmaceutically acceptable acid addition salts formed with organic and inorganic acids and those pharmaceutically acceptable salts formed with optically active acids. Representative acid addition salts include, but are not limited to, acetate, adipate, alginate, ascorbate, citrate, aspartate, benzoate, benzenesulfonate, besylate, bicarbonate / carbonate, bisulfate / sulfate, borate, butyrate, camphorate, camphorsulfonate, camsylate, citrate, digluconate, edisylate, etoglutarate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, glutamate, glycerophosphate, hemisulfate, heptanoate, hexafluorophosphate, hexanoate, hibenzate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethansulfonate, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulphate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, pectinate, persulfate, phosphate / hydrogen phosphate / dihydrogen phosphate, picrate, pivalate, proprionate, saccharate, stearate, succinate, tartrate, thiocyanate, tosylate and trifluoroacetate salts. Scheme 1. Preparation of M9 from Tulathromycin A

[0190]

[0191] Step 1 b demethylation

[0192]

[0193] (0.4 eq)

[0194] Step 1c

[0195] Oxazolidine

[0196] hydrolysis

[0197]

[0198] The conversion of tulathromycin A to M9 involves three steps with one isolation. First, the dimethyl amine is oxidized to give the N-oxide intermediate, followed by a demethylation reaction with Cu(ll) instead of Fe(ll) (which is normally used in a Polonovski reaction) to demethylate and give a mixture of M9 and the N-propyl oxazolidine. Finally, the N-propyl oxazolidine is deprotected using O-methylhydroxyl-amine hydrochloride to give M9.

[0199] Tulathromycin A is dissolved in an alcohol and ether and reacted with hydrogen peroxide in the presence of a non-PFA catalyst to prepare an N-oxide intermediate. A solution of aqueous acetic acid is added, and the N-oxide is extracted into the aqueous phase, discarding the organic phase. To the aqueous phase is added toluene and an alcohol, and the pH adjusted with ammonium hydroxide to >9.5 to bring the N-oxide into the organic phase, discarding the aqueous phase. To the N-oxide is added acetic acid and cupric sulfate to prepare a mixture of M9 and the N-propyl oxazolidine intermediate. The N-propyl oxazolidine is then deprotected with O-methylhydroxylamine hydrochloride to give M9. The toluene phase is removed, an ether solvent is added, and aqueous ammonium hydroxide. The aqueous phase is discarded, and the ether solvent exchanged for acetonitrile, from which M9 is crystallized and isolated.

[0200] Further, the use of O-methylhydroxylamine-HCI instead of hydroxylamine-HCI eliminated three known or potentially mutagenic impurities and one known carcinogen from the reaction. These three suspected or confirmed mutagens included oxime A, Oxime B and formaldoxime; and the carcinogen hydroxylamine hydrochloride. The M9 oximes form in Step 1c, from an impurity that is formed in Step 1a during the oxidation of tulathromycin A ( / .e., an N-oxide on the n-propyl nitrogen). The formaldoxime forms in Step 1c from the formaldehyde by-product in Step 1b. When O-methylhydroxylamine is substituted for hydroxylamine, it eliminates the formation of the oximes and formaldoxime because it makes the O-methyl versions of these impurities which are not considered potential mutagens via an in-silico analysis, and O-methylhydroxylamine is not a carcinogen. These mutagenic / carcinogenic impurities are shown below:

[0201]

[0202] Formaldoxime Hydroxylamine hydrochloride

[0203] EXAMPLES

[0204] The azalides described in each of WO2021 / 183758, WO2021 / 183754, WO2021 / 183759, WO2021 / 183762 and WO2023 / 038852 which were generally prepared from tulathromycin epoxide and the M9 epoxide can be prepared directly from the M9 intermediate as prepared from tulathromycin A using the new chemical processes described herein.

[0205] Preparation of the Formula (1) compound, 1-((2S,3R,4S,6R)-2-(((2R,3S,4R,5R,8R,10R, 11R,12S,13S,14R)-2-ethyl-3,4,10-trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-5-((propylamino)methyl)tetrahydro-2H-pyran-2-yl)oxy)-3,5,8, 10,12,14-hexamethyl-15-oxo-1 -oxa-6-azacyclopentadecan-11-yl)oxy)-3-hydroxy-6-methyltetra-hydro-2H-pyran-4-yl)-1,3-dimethyl-3-phenylurea

[0206] Step 1. Preparation of M9

[0207] To a reactor was charged tulathromycin A (30.0 kg, 37.2 mol), n-butanol (75 L), and methyl terf-butyl ether (45 L) at 10°C. To this was added ethyl benzoylformate (6.72 kg, 37 mol) followed by addition of 30% aqueous hydrogen peroxide (4.5 kg, 43 mol) maintaining an internal temperature less than 20°C. The reaction was stirred at around 20°C for six hours. A solution of acetic acid (5.14 kg, 85.6 mol) in water (150 L) was added at no more than 25°C. The organic phase was discarded. To the aqueous phase was added toluene (162 L), methanol (18 L), and sodium chloride (30 kg). The aqueous phase was adjusted to pH 9.5-10.0 with 30% aqueous ammonium hydroxide (ca. 27 L). The aqueous phase was removed and discarded. To the organic solution was charged acetic acid (2.23 kg, 37 mol), methanol (60 L) and anhydrous cupric sulfate (6.53 kg, 41 mol). The reaction was heated to reflux for three hours and then cooled to less than 20°C. To the reaction was charged a solution of O-methylhydroxylamine hydrochloride (4.76 kg, 56 mol) in water (30 L). After stirring for two hours at 27°C, the toluene phase was removed. Charged water (150 L), 2-methyltetrahydrofuran (180 L), and sodium chloride (30 kg). Charge 30% aqueous ammonium hydroxide (75 L) to adjust the pH to at least 9.5. The lower aqueous phase was discarded. The organics were concentrated under vacuum to a volume of approximately 50L. Acetonitrile (210 L) was charged and then the reaction was concentrated under vacuum to volume of about 180 L. The resulting slurry was heated to 65°C and held the reaction mass under stirring for one hour and then cooled to 5°C over two hours and held for one hour. The product was isolated by filtration, washed with acetonitrile at 10°C (60 L) and then dried under vacuum to afford 23.5 kg of a white solid (80% yield). HRMS M+H = 792.5587.1H NMR (600 MHz, d6-DMSO): NHMe (3H, 2.37 ppm, S) compared to tulathromycin NMe2(6H, 2.26 ppm).

[0208] Within Step 1, to eliminate perfluoroalkyl substances and chlorinated solvents and reduce or eliminate known mutagens, Applicant a) replaced the PFA catalyst hexafluoroacetone hydrate with ethyl benzoylformate; b) replaced dichloromethane in the aqueous phase with toluene, methanol and sodium chloride; c) replaced hydroxylamine-HCI with O-methylhydroxylamine-HCI to deprotect the oxazolidine; and d) further replaced dichloromethane by charging the reaction with water, 2-methyltetrahydrofuran and sodium chloride. In addition, the use of the ethyl benzoylformate catalyst provides an 85-90% yield of the N-oxide (if isolated). This yield was not expected based on the 46% yield described in the Organocatalyst citation.

[0209] Optionally, tulathromycin A is dissolved in an alcohol and ether and reacted with hydrogen peroxide in the presence of a non-PFA catalyst to prepare an N-oxide intermediate. A solution of aqueous acetic acid is added, and the N-oxide is extracted into the aqueous phase, discarding the organic phase. To the aqueous N-oxide is added acetic acid and cupric sulfate to prepare a mixture of M9 and the N-propyl oxazolidine intermediate. The N-propyl oxazolidine is then deprotected with O-methylhydroxylamine hydrochloride to give M9. To the reaction mixture, an ether solvent is added, and aqueous ammonium hydroxide. The aqueous phase is discarded, and the ether solvent exchanged for acetonitrile, from which M9 is crystallized and isolated.

[0210] Option 1: Extractive Work-up Isolation Process for M 9

[0211] To a reactor was charged tulathromycin A (200 g, 0.248 mol), n-butanol (500 mL), and methyl fert-butyl ether (300 mL) at 10°C. To this was added ethyl benzoylformate (45 g, 0.248 mol) followed by addition of 30% aqueous hydrogen peroxide (28.9 g, 0.285 mol) maintaining an internal temperature less than 20°C. The reaction was stirred at around 20°C for six hours. A solution of acetic acid (41.7 g, 0.695 mol) in water (1.0 L) was added at no more than 25°C. The organic phase was discarded. To the aqueous solution was charged acetic acid (168 g, 2.8 mol), and cupric sulfate pentahydrate (6.2 g, 0.0248 mol). The reaction was heated to 55-60°C for three to four hours and then cooled to less than 20°C. To the reaction was charged a solution of O-methylhydroxylamine hydrochloride (31.7 g, 0.372 mol) in water (200 mL). After stirring for 3-4 hours at 27°C, cooled to less than 20°C. Charged 2-methyltetrahydrofuran (1.4 L), and sodium chloride (200 g). Charge 30% aqueous ammonium hydroxide (900 mL) to adjust the pH to at least 9.5. The lower aqueous phase was discarded. The organics were concentrated under vacuum to a volume of approximately 300 mL. Acetonitrile (1.4 L) was charged and then the reaction was concentrated under vacuum to volume of about 1.2 L. The resulting slurry was heated to 65°C and held the reaction mass under stirring for one hour and then cooled to 5°C over two hours and held for one hour. The product was isolated by filtration, washed with acetonitrile (400 mL) at 10°C and then dried under vacuum to afford 154 g of a white solid (80% yield, before assay). HRMS M+H = 792.5587.1H NMR (600 MHz, d6-DMSO): NHMe (3H, 2.37 ppm, S) compared to tulathromycin NMe2(6H, 2.26 ppm).

[0212] 2: Direct Isolation Process for M9 To a reactor was charged tulathromycin A (200 g, 0.248 mol), n-butanol (500 mL), and methyl ferf-butyl ether (300 mL) at 10°C. To this was added ethyl benzoylformate (45 g, 0.248 mol) followed by addition of 30% aqueous hydrogen peroxide (28.9 g, 0.285 mol) maintaining an internal temperature less than 20°C. The reaction was stirred at around 20°C for six hours. A solution of acetic acid (41.7 g, 0.695 mol) in water (1.0 L) was added at no more than 25°C. The organic phase was discarded. To the aqueous solution was charged acetic acid (168 g, 2.8 mol), and cupric sulfate pentahydrate (6.2 g, 0.0248 mol). The reaction was heated to 55-60°C for three to four hours and then cooled to less than 20°C. To the reaction was charged a solution of O-methylhydroxylamine hydrochloride (31.7 g, 0.372 mol) in water (200 mL). After stirring for 3-4 hours at 27°C, cooled to 20°C. Charged acetonitrile (1.2 L). Charge 30% aqueous ammonium hydroxide (900 mL) to adjust the pH to at least 9.5. Charged tetrahydrofuran (400 mL). The resulting slurry was heated to 50°C and held the reaction mass under stirring for one hour and then cooled to 5°C over two hours and held for one hour. The product was isolated by filtration, washed with 5% aqueous ammonium hydroxide solution (1.0 L), water (1.0 L) and acetonitrile (400 mL) at 10°C and then dried under vacuum to afford 127 g of a white solid (64.6% yield, before assay). HRMS M+H = 792.5587.1H NMR (600 MHz, d6-DMSO): NHMe (3H, 2.37 ppm, S) compared to tulathromycin NMe2(6H, 2.26 ppm).

[0213] Step 2: Preparation of Formula (1) 1-((2S,3R,4S,6R)-2-(((2R,3S,4R,5R,8R,10R, 11R,12S,13S,14R)-2-ethyl-3,4,10-trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-5-((propylamino)methyl)tetrahydro-2H-pyran-2-yl)oxy)-3,5,8, 10,12,14-hexamethyl-15-oxo-1 -oxa-6-azacyclopentadecan-11-yl)oxy)-3-hydroxy-6-methyltetrahydro-2H-pyran-4-yl)-1,3-dimethyl-3-phenylurea from M9

[0214]

[0215] Generally, the conversion of M9 to Formula (1) is a urea formation via reaction with N-methylaniline carbamoyl chloride. The material is isolated as the free base in the final crystallization. M9 is reacted in an ester solvent with N-methyl-N-phenyl-carbamoyl chloride and an organic base to prepare the Formula (1) compound. A solution of aqueous acetic acid is added, and the Formula (1) compound is extracted into the aqueous phase, discarding the organic phase. An ether solvent is charged, and the aqueous phase adjusted with ammonium hydroxide to a basic pH to bring the Formula (1) compound into the organic phase, discarding the aqueous phase. The ether solvent is exchanged for an alcohol, from which the Formula (1) compound is crystallized and isolated as the free base.

[0216] To a reactor was charged M9 (10.0 kg, 12.6 mol), ethyl acetate (50 L), and triethylamine (1.5 kg, 15 mol). Aftercooling to 10°C, a solution of N-methyl-N-phenyl-carbamoyl chloride (2.27 kg, 13.3 mol) in ethyl acetate (25 L) was added. The resulting solution was stirred at 10°C for 15 hours and then water (50 L) was added. The lower aqueous phase was discarded. Charged water (50 L), acetic acid (1.7 kg, 29 mol). The phases were separated, and the upper organic phase was discarded. The aqueous phase was washed with ethyl acetate (20 L). To the aqueous phase was charged methyl tert-butyl ether (35 L) and the mixture was adjusted to pH 8.0-8.5 with 30% aqueous ammonium hydroxide (2 L). The aqueous phase was discarded. The organics were washed with water (15 L). The organic solution was concentrated under vacuum to a volume of 20 L. Charged isopropanol (50 L) and concentrated under vacuum to a volume of 40 L. To the resulting slurry at 20°C was added isopropanol (10 L) followed by water (10 L). Additional water (53 L) was added over 90 minutes. After stirring at 20°C for one hour, the product was isolated by filtration, was washed with a solution of water (30 L) and isopropanol (10 L), followed by water (3 x 40 L) and was dried under vacuum to afford 9.1 kg of a white solid as a free base (78% yield). MS M+H = 925. 1 H NMR (400 MHz, DMSO-d6) δ ppm 1 H NMR (400 MHz, DMSO-d6) δ ppm 0.5 -1.5 (m, 42H), 1.5 -2.3 (m, 8H), 2.4-3.0 (m, 10H), 3.05 (s, 3 H), 3.07 (s, 1 H), 3.09 - 3.15 (m, 1 H), 3.27 - 3.33 (m, 1 H), 3.35 (s, 3 H), 3.44 (br d, 1 H), 3.47 - 3.57 (m, 1 H), 3.63 (br s, 1 H), 3.81 (brs, 1 H), 3.88 (brt, 1 H), 4.10 (brs, 1 H), 4.36 (d, 1 H), 4.39 (brd, 1 H), 4.83 (dd, 1 H), 4.87 (d, 1 H), 4.90 (d, 1 H), 7.06 (t, 1 H), 7.15 (dd, 2 H), 7.24 - 7.36 (m, 2 H).

[0217] Optionally, Formula (1) can be prepared by reacting M9 in an ether solvent with N-methyl-N-phenyl-carbamoyl chloride and an organic base to prepare Formula (1). An acid is added, and the Formula (1) compound is extracted into the aqueous phase, discarding the organic phase. An alcohol solvent is charged, and the pH of the aqueous phase is adjusted to a basic using a base. The Formula (1) compound is crystallized and isolated as the free base.

[0218] Optional: Step 2 to Prepare Formula (1) from M9

[0219] To a reactor was charged M9 (40.0 g, 92.4%w / w, 46.6 mmol), 2-methyl tetrahydrofuran (200 mL), and triethylamine (6.14 g, 60.6 mmol). A solution of N-methyl-N-phenyl-carbamoyl chloride (8.70 g, 51.3 mmol) in 2-methyl tetrahydrofuran (100 mL) was added at 20°C. The resulting solution was stirred at 20°C for 6 hours and then water (200 mL) was added. The lower aqueous phase was discarded. Charged water (200 mL), acetic acid (7.0 g, 116.7 mmol). The phases were separated, and the upper organic phase was discarded. The aqueous phase was washed with methyl tert-butyl ether (120 mL) and discarding the organic phase. To the aqueous phase isopropanol was charged (100 mL) and the mixture was adjusted to pH 8.0 with 30% aqueous ammonium hydroxide (4 mL). Seed crystals (0.25-0.5 %w / w) were added, and the mixture was held for 30 minutes. The pH was further adjusted to 9.5 with 30% aqueous ammonium hydroxide (14 mL) and the slurry was stirred at 20°C for 1 hour. The resulting solid was collected by filtration, washed with a solution of water (120 mL) and isopropanol (40 mL), followed by water (240 mL), and dried under vacuum to afford the free base as a white solid (38.7 g, 90% yield). MS M+H = 925. 1H NMR (400 MHz, DMSO-d6) δ ppm 1 H NMR (400 MHz, DMSO-d6) δ ppm 0.5 -1.5 (m, 42H), 1.5 -2.3 (m, 8H), 2.4-3.0 (m, 10H), 3.05 (s, 3 H), 3.07 (s, 1 H), 3.09 - 3.15 (m, 1 H), 3.27 - 3.33 (m, 1 H), 3.35 (s, 3 H), 3.44 (br d, 1 H), 3.47 - 3.57 (m, 1 H), 3.63 (br s, 1 H), 3.81 (br s, 1 H), 3.88 (br t, 1 H), 4.10 (brs, 1 H), 4.36 (d, 1 H), 4.39 (brd, 1 H), 4.83 (dd, 1 H), 4.87 (d, 1 H), 4.90 (d, 1 H), 7.06 (t, 1 H), 7.15 (dd, 2 H), 7.24 - 7.36 (m, 2 H).

[0220] Optional: Step 2 to Prepare Formula (1) from M9

[0221] To a reactor was charged M9 (20.0 g, 92.4 %w / w, 23.3 mmol), 2-methyl tetrahydrofuran (100 mL), and triethylamine (3.07 g, 30.3 mmol). A solution of N-methyl-N-phenyl-carbamoyl chloride (4.35 g, 25.65 mmol) in 2-methyl tetrahydrofuran (50 mL) was added at 20°C. The resulting solution was stirred at 20°C for 6 hours and then water (100 mL) was added. The lower aqueous phase was discarded. Charged water (50 mL), acetic acid (3.5 g, 58.35 mmol). The phases were separated, and the upper organic phase was discarded. The aqueous phase was washed with methyl tert-butyl ether (60 mL). To the aqueous phase isopropanol was charged (80 mL) and the mixture was adjusted to pH 9.5 with 30% aqueous ammonium hydroxide (9 mL). The mixture was held for 30 minutes, added additional water (80 mL) and the slurry was stirred at 20°C for 1 hour. The resulting solid was collected by filtration, washed with a solution of water (60 mL) and isopropanol (20 mL), followed by water (120 mL), and dried under vacuum to afford the free base as a white solid (18.5 g, 86% yield). This procedure provides better control over supersaturation (potential for oiling out).

[0222] Historically, Formula (1) was prepared by combining M9 with N-methyl-N-phenyl-carbamoyl chloride and triethylamine in dichloromethane at room temperature for 16 hours (WO2021 / 183759). As described herein, Applicant removed the dichloromethane from the reaction, thereby further eliminating the use of chlorinated solvents in the preparation of the macrolide and also eliminated certain mutagenic and / or carcinogenic intermediates / impurities with the updated and novel processes.

Claims

CLAIMSWe Claim:

1. A process for preparing M9, (2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-2-ethyl-3,4, 10-trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-5- ((propylamino)methyl)-tetrahydro-2H-pyran-2-yl)oxy)-11-(((2S,3R,4S,6R)-3-hydroxy-6-methyl-4-(methylamino)tetrahydro-2H-pyran-2-yl)oxy)-3,5,8, 10,12,14-hexamethyl-1 -oxa-6-azacyclopentadecan-15-one from tulathromycin A, (2R,3S,4R,5R,8R,10R,11 R,12S,13S,14R)-11-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-2-ethyl-3,4,10-trihydroxy-13- (((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-5-((propylamino)methyl)tetrahydro-2H-pyran-2-yl)oxy)-3,5,8, 10, 12, 14-hexamethyl-1 -oxa-6-azacyclopentadecan-15-one, comprising:a) reacting tulathromycin A in n-butanol, methyl tert-butyl ether with the catalyst ethyl benzoylformate at a temperature of about 10°C to about 20°C for about 6 hours in the presence of an oxidant;b) adding an aqueous solution of acetic acid at less than about 25°C and then discarding the organic phase;c) adding toluene, methanol and sodium chloride then adjusting the pH to 9.5 to 10.0 with aqueous ammonium hydroxide and then discarding the aqueous phase; d) adding acetic acid, methanol and anhydrous cupric sulfate and heating to reflux for about 3 hours then cooling to less than about 20°C;e) adding aqueous O-methylhydroxylamine hydrochloride and stirring for about 2 hours at about 27°C then discarding the organic phase;f) adding water, 2-methyltetrahydrofuran and sodium chloride then adjusting the pH to at least 9.5 with aqueous ammonium hydroxide and then discarding the aqueous phase;g) concentrating the organic phase under vacuum then adding acetonitrile to the slurry and then concentrating the slurry under vacuum then heating the resulting slurry to about 65°C while stirring for about 1 hour then cooling to about 5°C for at least 2 hours and holding at about 5°C for an additional hour; andh) isolating the resulting M9 solids by filtration, washing the solids with cold acetonitrile at about 10°C then drying the M9 solids under vacuum.

2. A process for preparing M9, (2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-2-ethyl-3,4, 10-trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-5-((propylamino)methyl)-tetrahydro-2H-pyran-2-yl)oxy)-11-(((2S,3R,4S,6R)-3-hydroxy-6-methyl-4-(methylamino)tetrahydro-2H-pyran-2-yl)oxy)-3,5,8, 10,12,14-hexamethyl-1 -oxa-6-azacyclopentadecan-15-one from tulathromycin A, (2R,3S,4R,5R,8R,10R,11 R,12S,13S,14R)-11-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-2-ethyl-3,4,10-trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-5-((propylamino)methyl)tetrahydro-2H-pyran-2-yl)oxy)-3,5,8, 10, 12, 14-hexamethyl-1 -oxa-6-azacyclopentadecan-15-one, comprising:a) reacting tulathromycin A in n-butanol, methyl tert-butyl ether with the catalyst ethyl benzoylformate at a temperature of about 10°C to about 20°C for about 6 hours in the presence of an oxidant;b) adding an aqueous solution of acetic acid at less than 25°C and then discarding the organic phase;c) adding acetic acid and cupric sulfate pentahydrate and heating to about 55-60°C for about 3 to 4 hours then cooling to less than 20°C;d) adding aqueous O-methylhydroxylamine hydrochloride and stirring for about 3 to 4 hours at about 27°C then cooling to less than 20°C;e) (i) adding 2-methyltetrahydrofuran and sodium chloride then adjusting the pH to at least 9.5 with aqueous ammonium hydroxide and then discarding the aqueous phase; or (ii) adding acetonitrile and then adjusting the pH to at least 9.5 with aqueous ammonium hydroxide and then adding tetrahydrofuran;f) (i) concentrating the organic phase from e(i) under vacuum then adding acetonitrile to the slurry then concentrating the slurry under vacuum then heating the resulting slurry to about 65°C while stirring for about 1 hour then cooling to about 5°C for at least 2 hours and holding at about 5°C for an additional hour; or (ii) heating the resulting slurry from e(ii) to about 50°C while stirring for about 1hour then cooling to about 5°C over about 2 hours and holding at about 5°C for an hour; andg) isolating the M9 solids from f(i) by filtration, washing the solids with cold acetonitrile at about 10°C and then drying the solids under vacuum; or isolating the M9 solids from f(ii) filtration and washing the solids with 5% aqueous ammonium hydroxide, water and acetonitrile at about 10°C then drying the M9 solids under vacuum.

3. The process of claim 1 or 2, wherein the oxidant is aqueous H2O2.

4. The process of claim 1 or 2, wherein the aqueous ammonium hydroxide is about 30% ammonium hydroxide and the aqueous H2O2is about 30% H2O2.

5. The process of claim 1 or 2, wherein the O-methylhydroxylamine is about 16% O-methylhydroxylamine.

6. The process of claim 1 or 2, wherein the aqueous acetic acid is about 3-4% acetic acid.

7. A process for preparing the Formula (1) compound, 1-((2S,3R,4S,6R)-2-(((2R,3S,4R,5R,8R, 10R, 11 R, 12S, 13S, 14R)-2-ethyl-3,4, 10-trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-5-((propylamino)methyl)tetrahydro-2H-pyran-2-yl)oxy)-3,5,8, 10, 12, 14-hexamethyl-15-oxo-1 -oxa-6-azacyclopentadecan-11 -yl)oxy)-3-hydroxy-6-methyltetrahydro-2H-pyran-4-yl)-1,3-dimethyl-3-phenylurea from M9, (2R,3S,4R,5R,8R, 10R, 11, 12S, 13S, 14R)-2-ethyl-3,4, 10-trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-5-((propylamino)methyl)tetrahydro-2H-pyran-2-yl)oxy)-11-(((2S,3R,4S,6R)-3-hydroxy-6-methyl-4-(methylamino)tetrahydro-2H-pyran-2-yl)oxy)-3,5,8, 10, 12, 14-hexamethyl-1 -oxa-6-azacyclopentadecan-15-one, comprising:a) combining M9 with ethyl acetate and triethylamine and cooling to about 10°C; b) adding a solution of N-methyl-N-phenyl-carbamoyl chloride in ethyl acetate while stirring at about 10°C for about 15 hours;c) adding water to the reactants and discarding the aqueous phase;d) adding water and acetic acid and discarding the organic phase;e) washing the aqueous phase with ethyl acetate then adding methyl tert-butyl ether while adjusting the pH to about 8.0 to about 8.5 with aqueous ammonium hydroxide, then discarding the aqueous phase;f) washing the organic phase with water then concentrating the organic phase under vacuum, then adding isopropanol then concentrating again under vacuum and cooling the slurry to about 20°C; andg) adding isopropanol then water while adding more water over about 90 minutes, then stirring the mixture for about 1 hour at about 20°C; andh) filtering the resulting solids, washing the solids with a solution of water and isopropanol, then washing the solids again with water then drying under vacuum to afford the Formula (1) compound as a free base.

8. The process of claim 7, wherein the aqueous ammonium hydroxide is about 30% ammonium hydroxide.

9. A process for preparing the Formula (1) compound, 1-((2S,3R,4S,6R)-2-(((2R,3S,4R,5R,8R, 10R, 11 R,12S, 13S, 14R)-2-ethyl-3,4,10-trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-5-((propylamino)methyl)tetrahydro-2H-pyran-2-yl)oxy)-3,5,8, 10, 12, 14-hexamethyl-15-oxo-1 -oxa-6-azacyclopentadecan-11 -yl)oxy)-3-hydroxy-6-methyltetrahydro-2H-pyran-4-yl)-1,3-dimethyl-3-phenylurea from M9, (2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-2-ethyl-3,4,10-trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-5-((propylamino)methyl)tetrahydro-2H-pyran-2-yl)oxy)-11-(((2S,3R,4S,6R)-3-hydroxy-6-methyl-4-(methylamino)tetrahydro-2H-pyran-2-yl)oxy)-3,5,8, 10, 12, 14-hexamethyl-1 -oxa-6-azacyclopentadecan-15-one, comprising:a) combining M9 with 2-methyltetrahydrofuran and triethylamine;b) adding a solution of N-methyl-N-phenyl-carbamoyl chloride in 2- methyltetrahydrofuran at about 20°C while stirring for about 6 hours;c) adding water to the reactants and discarding the aqueous phase;d) adding water and acetic acid and discarding the organic phase;e) washing the aqueous phase with methyl-tert-butyl ether and discarding the organic phase;f) adding isopropanol and adjusting the pH to about 8.0 with aqueous ammonium hydroxide;g) adding seed crystals of the Formula (1) compound while holding the reactants for about 30 minutes then adjusting the pH further to about 9.5 with aqueous ammonium hydroxide while stirring the slurry at about 20°C for about one hour; andh) collecting the resulting solids by filtration, washing the solids with a solution of water and isopropanol, followed by water then drying under vacuum to afford the Formula (1) compound as a free base.

10. The process of claim 9, wherein the aqueous ammonium hydroxide is about 30% ammonium hydroxide.

11. A process for preparing the Formula (1) compound, 1-((2S,3R,4S,6R)-2-(((2R,3S,4R,5R,8R, 10R, 11 R, 12S, 13S, 14R)-2-ethyl-3,4, 10-trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-5-((propylamino)methyl)tetrahydro-2H-pyran-2-yl)oxy)-3,5,8, 10, 12, 14-hexamethyl-15-oxo-1 -oxa-6-azacyclopentadecan-11 -yl)oxy)-3-hydroxy-6-methyltetrahydro-2H-pyran-4-yl)-1,3-dimethyl-3-phenylurea from M9, (2R,3S,4R,5R,8R, 10R, 11, 12S, 13S, 14R)-2-ethyl-3,4, 10-trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-5-((propylamino)methyl)tetrahydro-2H-pyran-2-yl)oxy)-11-(((2S,3R,4S,6R)-3-hydroxy-6-methyl-4-(methylamino)tetrahydro-2H-pyran-2-yl)oxy)-3,5,8, 10, 12, 14-hexamethyl-1 -oxa-6-azacyclopentadecan-15-one, comprising:a) combining M9 with 2-methyltetrahydrofuran and triethylamine;b) adding a solution of N-methyl-N-phenyl-carbamoyl chloride in 2- methyltetrahydrofuran at about 20°C while stirring for about 6 hours;c) adding water to the reactants and discarding the aqueous phase;d) adding water and acetic acid and discarding the organic phase;e) washing the aqueous phase with methyl-tert-butyl ether and discarding the organic phase;f) adding isopropanol and adjusting the pH to about 9.5 with aqueous ammonium hydroxide;g) holding the mixture for about 30 minutes then adding water with stirring at about 20°C for about one hour; andh) collecting the resulting solids by filtration, washing the solids with a solution of water and isopropanol, followed by water then drying under vacuum to afford the Formula (1) compound as a free base.

12. A process for preparing the Formula (1) compound, 1-((2S,3R,4S,6R)-2- (((2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-2-ethyl-3,4,10-trihydroxy-13- (((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-5- ((propylamino)methyl)tetrahydro-2H-pyran-2-yl)oxy)-3,5,8, 10,12,14-hexamethyl-15- oxo-1 -oxa-6-azacyclopentadecan-11-yl)oxy)-3-hydroxy-6-methyltetrahydro-2H- pyran-4-yl)-1,3-dimethyl-3-phenylurea from M9,(2R,3S,4R,5R,8R, 10R, 11 R,12S, 13S, 14R)-2-ethyl-3,4,10-trihydroxy-13- (((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-5- ((propylamino)methyl)tetrahydro-2H-pyran-2-yl)oxy)-11-(((2S,3R,4S,6R)-3-hydroxy- 6-methyl-4-(methylamino)tetrahydro-2H-pyran-2-yl)oxy)-3,5,8, 10,12,14-hexamethyl- 1-oxa-6-azacyclopentadecan-15-one, that is prepared from tulathromycin A, (2R,3S,4R,5R,8R,10R,11 R,12S,13S,14R)-11-(((2S,3R,4S,6R)-4-(dimethylamino)-3- hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-2-ethyl-3,4, 10-trihydroxy-13- (((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-5- ((propylamino)methyl)tetrahydro-2H-pyran-2-yl)oxy)-3,5,8, 10,12,14-hexamethyl-1 - oxa-6-azacyclopentadecan-15-one, comprising:a) reacting tulathromycin A in n-butanol, methyl tert-butyl ether with the catalyst ethyl benzoylformate at a temperature of about 10°C to about 20°C for about 6 hours in the presence of an oxidant;b) adding an aqueous solution of acetic acid at less than 25°C then discarding the organic phase;c) adding toluene, methanol and sodium chloride to the aqueous phase and then adjusting the pH to 9.5 to 10.0 with aqueous ammonium hydroxide and then discarding the aqueous phase;d) adding acetic acid, methanol and anhydrous cupric sulfate and heating to reflux for about 3 hours then cooling to less than about 20°C;e) adding aqueous O-methylhydroxylamine hydrochloride and stirring for about 2 hours at about 27°C then discarding the organic phase;f) adding water, 2-methyltetrahydrofuran and sodium chloride and adjusting the pH to at least 9.5 with aqueous ammonium hydroxide and then discarding the aqueous phase;g) concentrating the organic phase under vacuum then adding acetonitrile to the slurry then concentrating the slurry under vacuum then heating the resulting slurry to about 65°C while stirring for about 1 hour then cooling to about 5°C for at least 2 hours and holding at about 5°C for an additional hour;h) isolating the M9 solids by filtration, washing the solids with cold acetonitrile at about 10°C and then drying the solids under vacuum;i) combining M9 with ethyl acetate and triethylamine and cooling to about 10°C; j) adding a solution of N-methyl-N-phenyl-carbamoyl chloride in ethyl acetate while stirring at about 10°C for about 15 hours;k) adding water to the reactants and discarding the aqueous phase;l) adding water and acetic acid and discarding the organic phase;m) washing the aqueous phase with ethyl acetate then adding methyl tert-butyl ether while adjusting the pH to about 8.0 to about 8.5 with aqueous ammonium hydroxide then discarding the aqueous phase;n) washing the organic phase with water then concentrating the organic phase under vacuum, then adding isopropanol then concentrating again under vacuum and cooling the slurry to about 20°C; ando) adding isopropanol then water while adding more water over about 90 minutes, then stirring the mixture for about 1 hour at about 20°C, filtering the solids, washing the solids with a solution of water and isopropanol, then washing the solids again with water before drying under vacuum to afforf the Formula (1) compound as a free base.

13. A process for preparing the Formula (1 ) compound, 1 -((2S,3R,4S,6R)-2- (((2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-2-ethyl-3,4,10-trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-5- ((propylamino)methyl)tetrahydro-2H-pyran-2-yl)oxy)-3,5,8, 10,12,14-hexamethyl-15-oxo-1 -oxa-6-azacyclopentadecan-11-yl)oxy)-3-hydroxy-6-methyltetrahydro-2H-pyran-4-yl)-1,3-dimethyl-3-phenylurea from M9,(2R,3S,4R,5R,8R, 10R, 11 R,12S, 13S, 14R)-2-ethyl-3,4,10-trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-5- ((propylamino)methyl)tetrahydro-2H-pyran-2-yl)oxy)-11-(((2S,3R,4S,6R)-3-hydroxy-6-methyl-4-(methylamino)tetrahydro-2H-pyran-2-yl)oxy)-3,5,8, 10,12,14-hexamethyl-1-oxa-6-azacyclopentadecan-15-one, that is prepared from tulathromycin A, (2R,3S,4R,5R,8R,10R,11 R,12S,13S,14R)-11-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-2-ethyl-3,4, 10-trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-5- ((propylamino)methyl)tetrahydro-2H-pyran-2-yl)oxy)-3,5,8, 10,12,14-hexamethyl-1 -oxa-6-azacyclopentadecan-15-one, comprising:a) reacting tulathromycin A in n-butanol, methyl tert-butyl ether with the catalyst ethyl benzoylformate at a temperature of about 10°C to about 20°C for about 6 hours in the presence of an oxidant;b) adding an aqueous solution of acetic acid at less than 25°C then discarding the organic phase;c) adding toluene, methanol and sodium chloride to the aqueous phase then adjusting the pH to 9.5 to 10.0 with aqueous ammonium hydroxide and then discarding the aqueous phase;d) adding acetic acid, methanol and anhydrous cupric sulfate and heating to reflux for 3 hours then cooling to less than 20°C;e) adding aqueous O-methylhydroxylamine hydrochloride and stirring for about 2 hours at about 27°C then discarding the organic phase;f) adding water, 2-methyltetrahydrofuran and sodium chloride and adjusting the pH to at least 9.5 with aqueous ammonium hydroxide then discarding the aqueous phase;g) concentrating the organic phase under vacuum then adding acetonitrile to the slurry and then concentrating the slurry under vacuum then heating the resultingslurry to about 65°C while stirring for about 1 hour then cooling to about 5°C for about 2 hours and holding at 5°C for an additional hour;h) isolating the M9 solids by filtration, washing the solids with cold acetonitrile at about 10°C then drying the solids under vacuum;i) combining M9, 2-methyl tetrahydrofuran and triethylamine,j) adding a solution of N-methyl-N-phenyl-carbamoyl chloride in 2-methyl tetrahydrofuran at about 20°C while stirring for about 6 hours;k) adding water to the reactants then discarding the aqueous phase;l) adding water and acetic acid then discarding the organic phase;m) washing the aqueous phase with methyl tert-butyl ether and discarding the organic phase;n) adding isopropanol and adjusting the pH to 8.0 with aqueous ammonium hydroxide;o) adding seed crystals of Formula (1) while holding the reactants for about 30 minutes, then adjusting the pH to 9.5 with aqueous ammonium hydroxide while stirring the slurry at about 20°C for about 1 hour; andp) collecting the resulting solids by filtration, washing with a solution of water and isopropanol, then washing with water and drying the solids under vacuum to afford the Formula (1) compound as a free base.

14. A process for preparing the Formula (1) compound, 1-((2S,3R,4S,6R)-2-(((2R,3S,4R,5R,8R, 10R, 11 R, 12S, 13S, 14R)-2-ethyl-3,4, 10-trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-5-((propylamino)methyl)tetrahydro-2H-pyran-2-yl)oxy)-3,5,8, 10, 12, 14-hexamethyl-15-oxo-1 -oxa-6-azacyclopentadecan-11 -yl)oxy)-3-hydroxy-6-methyltetrahydro-2H-pyran-4-yl)-1,3-dimethyl-3-phenylurea from M9, (2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-2-ethyl-3,4,10-trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-5-((propylamino)methyl)tetrahydro-2H-pyran-2-yl)oxy)-11-(((2S,3R,4S,6R)-3-hydroxy-6-methyl-4-(methylamino)tetrahydro-2H-pyran-2-yl)oxy)-3,5,8, 10, 12, 14-hexamethyl-1 -oxa-6-azacyclopentadecan-15-one, that is prepared from tulathromycin A, (2R,3S,4R,5R,8R,10R,11 R, 12S, 13S, 14R)-11-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-2-ethyl-3,4,10-trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-5-((propylamino)methyl)tetrahydro-2H-pyran-2-yl)oxy)-3,5,8, 10,12,14-hexamethyl-1 -oxa-6-azacyclopentadecan-15-one, comprising:a) reacting tulathromycin A in n-butanol, methyl tert-butyl ether with the catalyst ethyl benzoylformate at a temperature of about 10°C to about 20°C for about 6 hours in the presence of an oxidant;b) adding an aqueous solution of acetic acid at less than 25°C and then discarding the organic phase;c) adding toluene, methanol and sodium chloride to the aqueous phase and then adjusting the pH of the aqueous phase to 9.5 to 10.0 with aqueous ammonium hydroxide then discarding the aqueous phase;d) adding acetic acid, methanol and anhydrous cupric sulfate and heating to reflux for 3 hours then cooling to less than 20°C;e) adding aqueous O-methylhydroxylamine hydrochloride and stirring for about 2 hours at about 27°C then discarding the organic phase;f) adding water, 2-methyltetrahydrofuran and sodium chloride and adjusting the pH to at least 9.5 with aqueous ammonium hydroxide and then discarding the aqueous phase;g) concentrating the organic phase under vacuum then adding acetonitrile to the slurry and then concentrating the slurry under vacuum then heating the resulting slurry to about 65°C while stirring for about 1 hour then cooling to about 5°C for about 2 hours and holding at about 5°C for an additional hour; andh) isolating the M9 solids by filtration, washing the solids with cold acetonitrile at about 10°C and then drying the solids under vacuum;i) combining M9, 2-methyl tetrahydrofuran and triethylamine;j) adding a solution of N-methyl-N-phenyl-carbamoyl chloride in 2-methyl tetrahydrofuran at about 20°C while stirring for about 6 hours;k) adding water to the reactants and discarding the aqueous phase;l) adding water acetic acid and discarding the organic phase;m) washing the aqueous phase with methyl tert-butyl ether and discarding the organic phase;n) adding isopropanol and adjusting the pH to 9.5 with aqueous ammoniumhydroxide;o) holding the mixture for about 30 minutes, then adding water and stirring the slurry at about 20°C for about 1 hour; andp) collecting the resulting solids by filtration, washing with a solution of water and isopropanol, then washing with water and drying the solids under vacuum to afford the Formula (1) compound as a free base.

15. The process of claim 12, 13 or 14, wherein the aqueous oxidant is about 30% H2O2, the aqueous ammonium hydroxide is about 30% ammonium hydroxide, the aqueous O-methylhydroxylamine is about 16% O-methylhydroxylamine and the aqueous acetic acid is about 3-4% acetic acid.

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