A crystalline form of (S)-N-(3-amino-1-(hydroxylamino)-3-methyl-1-oxobutan-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide salt

Abstract

(S)- N‑ A crystalline form of (3-amino-l-(hydroxylamino)-3-methyl-l-oxobutan-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide salt.

Description

Technical Field

[0001] Organic compounds, especially organic salts, more specifically (S)- N- (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide salt. Background Technology

[0002] WO2023118558 has been released (S)- N- Synthesis of (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide dihydrochloride .

[0003] WO2023118557 discloses an injectable pharmaceutical composition for treating respiratory diseases in animals. It discloses a composition comprising (S)- N- Preparations of (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide dihydrochloride. Summary of the Invention

[0004] This invention relates to (S)- N- The crystalline form of (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide salt. Attached Figure Description

[0005] Figure 1 For crystal (S)- N- X-ray powder diffraction pattern of (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide di-p-toluenesulfonate monohydrate.

[0006] Figure 2 For (S)- N- C13 cross-polarized magic angle rotation (CPMAS) NMR spectrum of (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-((((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide di-p-toluenesulfonate monohydrate. Peaks marked with an asterisk correspond to rotation sidebands.

[0007] Figure 3A For (S)- N-Typical DSC thermogram of (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide di-p-toluenesulfonate monohydrate. Figure 3B (S)- was displayed N- Thermogravimetric analysis (TGA) plot of (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide di-p-toluenesulfonate monohydrate.

[0008] Figure 4 It is in crystalline state (S)- N- X-ray powder diffraction pattern of (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide dipropionate.

[0009] Figure 5 It is in crystalline state (S)- N- Carbon-13 cross-polarized magic angle rotation (CPMAS) nuclear magnetic resonance (NMR) spectrum of (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide dipropionate.

[0010] Figure 6 This is a typical DSC thermogram of (S)-4-(hydroxyamino)-3-(4-((4-((((2-methoxyethyl)amine)methyl)phenyl)ethynyl)benzamido)-2-methyl-4-oxobut-2-amine propionate.

[0011] Figure 7 The DVS isotherm plot of di-toluenesulfonate monohydrate is shown.

[0012] Figure 8 The DVS isotherm plot of propionate is shown.

[0013] Figure 9 (S)- was displayed N- The structure of (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide di-p-toluenesulfonate monohydrate, according to ChemDraw® version 21.0.0.28, can also be named as: (S)-4-(hydroxyamino)-3-(4-((4-((((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamido)-2-methyl-4-oxobut-2-amine-4-methylbenzenesulfonate hydrate, or (S)-4-(hydroxyamino)-3-(4-((4-((((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamido)-2-methyl-4-oxobut-2-amine bis-4-methylbenzenesulfonate monohydrate, or Or, based on the name in BIOVIA Draw version 20.1: [4-[2-[4-[[(1S)-2-amino-1-(hydroxycarbamoyl)-2-methyl-propyl]carbamoyl]phenyl]ethynyl]phenyl]methyl-(2-methoxyethyl)amine 4-methylbenzenesulfonate hydrate, or [4-[2-[4-[[(1S)-2-amino-1-(hydroxycarbamoyl)-2-methyl-propyl]carbamoyl]phenyl]ethynyl]phenyl]methyl-(2-methoxyethyl)amine bis(4-methylbenzenesulfonate) hydrate.

[0014] Figure 10 (S)- was displayed N- The structure of (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide dipropionate, according to ChemDraw® version 21.0.0.28, can also be named as: (S)-4-(hydroxyamino)-3-(4-((4-((((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamido)-2-methyl-4-oxobut-2-amine propionate, or (S)-4-(hydroxyamino)-3-(4-((4-((((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamido)-2-methyl-4-oxobut-2-amine dipropionate, or Based on the naming convention of BIOVIA Draw version 20.1: [4-[2-[4-[[(1S)-2-amino-1-(hydroxycarbamoyl)-2-methyl-propyl]carbamoyl]phenyl]ethynyl]phenyl]methyl-(2-methoxyethyl)aminopropionate, or [4-[2-[4-[[(1S)-2-ammonium-1-(hydroxycarbamoyl)-2-methyl-propyl]carbamoyl]phenyl]ethynyl]phenyl]methyl-(2-methoxyethyl)amine dipropionate.

[0015] Figure 11The PXRD pattern of anhydrous di-p-toluenesulfonate is shown.

[0016] Figure 12 The DSC curve of anhydrous di-p-toluenesulfonate is shown.

[0017] Figure 13 The TGA thermogram of anhydrous di-p-toluenesulfonate is shown.

[0018] Figure 14 It shows free base (S)- N- PXRD of (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide.

[0019] Figure 15 It shows free base (S)- N- TGA of (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide.

[0020] Figure 16 It shows free base (S)- N- DSC of (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide.

[0021] Figure 17 Showing hydrochloride (S)- N- PXRD of (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide dihydrochloride.

[0022] Figure 18 Showing hydrochloride (S)- N- TGA of (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide dihydrochloride.

[0023] Figure 19 Showing hydrochloride (S)- N- DSC of (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide dihydrochloride. Detailed Implementation

[0024] Compared to other salts and free bases (S)- N- (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide, crystalline (S)- N- (3-amino-1-(hydroxyamino)-3-methyl-1-oxobutyl-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide di-p-toluenesulfonate monohydrate and (S)- N- (3-amino-1-(hydroxyamino)-3-methyl-1-oxobutyl-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide dipropionate has some advantages N- .

[0025] When selecting salts for pharmaceutical formulations, their stability in both the solid state and the formulation must be considered. Furthermore, the salt's toxicity, bioavailability, and therapeutic efficacy must also be evaluated. In long-term stability studies conducted at room temperature and under accelerated conditions, both di-toluenesulfonate monohydrate and dipropionate were more stable than the hydrochloride and the parent free base. Moreover, di-toluenesulfonate monohydrate exhibited superior stability compared to dipropionate. Di-toluenesulfonate monohydrate is currently the most stable salt known. This is unexpected.

[0026] (S)- N- (3-amino-1-(hydroxyamino)-3-methyl-1-oxobutyl-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide di-p-toluenesulfonate monohydrate (S)- N- (3-amino-1-(hydroxyamino)-3-methyl-1-oxobutyl-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide dipropionate .

[0027] As stated above, WO2023118558 discloses (S)- N- Synthetic methods for (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide dihydrochloride. The methods for preparing di-p-toluenesulfonate monohydrate and dipropionate are summarized below.

[0028] Scheme 1 is to prepare (S)- N-A method for (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide di-p-toluenesulfonate monohydrate. The advantage of this method is that the (S)-(4-(hydroxyamino)-3-(4-((4-((((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide)-2-methyl-4-oxobut-2-yl)carbamate tert-butyl ester intermediate can be deprotected and converted to di-p-toluenesulfonate monohydrate in a single reaction step.

[0029] Option 1 (S)- N- (3-Amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide di-p-toluenesulfonate monohydrate can also be prepared directly from free base as shown in Scheme 2.

[0030] Option 2 The intermediate 4-(4-(((2-methoxyethyl)amino)methyl)phenyl)-2-methylbut-3-yne-2-ol in Scheme 1 can also be prepared according to the method described in Scheme 3.

[0031] Option 3 Finally, (S)- can be prepared as described in Scheme 4. N- (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide dipropionate.

[0032] Option 4 The preparation methods of these compounds will be described in detail below.

[0033] Bovine respiratory disease (BRD) is the most common and economically damaging disease affecting beef cattle worldwide. BRD has a multifactorial etiology, resulting from a complex interaction between environmental factors, host factors, and pathogens. Environmental factors (such as weaning, transport, mixed herding, overcrowding, inclement weather, dust, and poor ventilation) act as stressors, adversely affecting the host's immune and non-immune defense mechanisms. Furthermore, certain environmental factors (such as overcrowding and poor ventilation) can enhance the spread of infectious agents among animals. It is a complex bacterial infection that can cause endemic pneumonia in calves and other bovine animals, and can be fatal. The infection is usually the result of a combination of three interdependent factors: stress, underlying viral infection, and emerging bacterial infection. Diagnosis of this disease is complex due to the multiple possible causes.

[0034] The term Swine Respiratory Disease (SRD) is used to describe pneumonia caused by a variety of etiologies, resulting in clinical disease and poor weight gain in the late fattening stage (15 to 20 weeks of age).

[0035] The term "basically as shown in the figure" as used herein means that the X-ray powder diffraction (XRPD) pattern, carbon-13 cross-polarized magic-angle rotated (CPMAS) nuclear magnetic resonance (NMR) pattern, or differential scanning calorimetry (DSC) thermogram is not exactly identical to the pattern depicted herein, but such differences are likely within the range of experimental error to a person skilled in the art. It should be understood by a person skilled in the art that X-ray powder diffraction patterns may contain variations from the actual patterns depicted herein. Figure 1 The peaks in the spectrum are within a range of ±0.2 degrees 2θ, and the differential scanning calorimetry (DSC) thermogram shown in Figure 3 may contain endothermic peaks within a range of ±5℃ from the temperature depicted.

[0036] As used herein, the term "substantially purified" means that the crystalline form of the compound has a purity of at least 90%. In another embodiment, "substantially purified" means that the crystalline form of the compound has a purity of at least 95%, 99%, or 99.9%.

[0037] Detailed Implementation Plan One embodiment of the present invention is a (S)- N- The crystalline form of (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide di-p-toluenesulfonate monohydrate has at least one of the following characteristics: X-ray powder diffraction (XRPD) patterns having at least one peak selected from 14.07, 10.96, 9.46, 7.37, 6.07, 5.48, 5.23, 4.92, 4.55, 4.26 and 3.82, represented by d-interval [A] (±0.2), or at least one peak selected from 6.28, 8.07, 9.35, 12.00, 14.60, 16.16, 16.94, 18.05, 19.51, 20.84 and 23.28, represented by °2θ (±0.2); Carbon-13 cross-polarized magic angle rotation (CPMAS) nuclear magnetic resonance (NMR) spectra with at least one peak selected from 20.54, 21.14, 22.05, 23.33, 48.56, 50.43, 55.71, 57.7, 59.29, 68.97, 88.52, 90.16, 122.29, 126.94, 129.73, 133.68, 136.67, 141.72, 163.70, and 170.30 ppm; or The differential scanning calorimetry (DSC) thermogram shows an endothermic peak at approximately 68.6℃ + / -5℃.

[0038] Another embodiment of the invention is a crystalline form, which has essentially the following properties: Figure 1 The X-ray powder diffraction (XRPD) pattern shown is shown.

[0039] Another embodiment of the invention has essentially the following characteristics: Figure 2 The crystalline form of the carbon-13 cross-polarized magic angle rotation (CPMAS) nuclear magnetic resonance (NMR) spectrum is shown.

[0040] Another embodiment of the invention is a crystalline form, which has a differential scanning calorimetry (DSC) thermogram substantially as shown in FIG3.

[0041] Another embodiment of the present invention is a crystalline form having a differential scanning calorimetry (DSC) thermogram containing an endothermic peak of about 68.6℃±5℃ and an exothermic peak of greater than 200℃.

[0042] One embodiment of the present invention is (S)- N- The crystalline form of (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide di-p-toluenesulfonate monohydrate, the X-ray powder diffraction (XRPD) pattern of which has at least one peak selected from the group consisting of °2θ (±0.2): 6.28, 8.07 and 9.35.

[0043] One embodiment of the present invention is (S)- N- The crystalline form of (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide di-p-toluenesulfonate monohydrate, the X-ray powder diffraction (XRPD) pattern of which has at least one peak selected from the group consisting of °2θ (±0.2): 12.00, 14.60, 16.16 and 16.94.

[0044] One embodiment of the present invention is (S)- N- The crystalline form of (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide di-p-toluenesulfonate monohydrate, the X-ray powder diffraction (XRPD) pattern of which has at least one peak selected from the group consisting of °2θ (±0.2): 18.05, 19.51, 20.84 and 23.28.

[0045] Another embodiment of the present invention is a pharmaceutical composition comprising any of the above-described crystalline forms and a pharmaceutical excipient.

[0046] Another embodiment of the invention is a pharmaceutical composition wherein the crystalline form is substantially pure.

[0047] One embodiment of the present invention is a (S)- N- The crystalline form of (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide dipropionate, having at least one of the following characteristics: X-ray powder diffraction (XRPD) patterns having at least one peak selected from 3.57, 7.16, 7.59, 10.74, 11.14, 11.32, 12.76, 13.87, 14.35, 14.69, 15.60, 16.15, 18.22, 18.58, 18.94, 22.40, and 22.80, represented by °2θ (±0.2), or at least one peak selected from 24.73, 12.34, 11.65, 8.24, 7.95, 7.75, 6.94, 6.39, 6.17, 6.03, 5.68, 5.49, 4.93, 4.87, 4.78, and 4.69, represented by d-interval [A] (±0.2). Carbon-13 cross-polarized magic angle rotation (CPMAS) nuclear magnetic resonance (NMR) spectra, which have at least one peak selected from 12.27, 24.57, 26.65, 31.96, 32.96, 51.65, 56.12, 57.39, 59.28, 70.31, 179.96, and 183.67 ppm. or The differential scanning calorimetry (DSC) thermogram shows an endothermic peak at approximately 119.1 °C.

[0048] Another embodiment of the invention is a crystalline form, which has essentially the following properties: Figure 4 The X-ray powder diffraction (XRPD) pattern shown is shown.

[0049] Another embodiment of the invention is a crystalline form, which has essentially the following properties: Figure 5 The carbon-13 cross-polarized magic angle rotation (CPMAS) nuclear magnetic resonance (NMR) spectrum is shown.

[0050] Another embodiment of the invention is a crystalline form, which has essentially the following properties: Figure 6 The differential scanning calorimetry (DSC) thermogram shown is shown.

[0051] One embodiment of the present invention is (S)- N- The crystalline form of (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide dipropionate, the X-ray powder diffraction (XRPD) pattern of which has at least one peak selected from the group consisting of °2θ (±0.2): 3.57, 7.16 and 7.59.

[0052] One embodiment of the present invention is (S)- N- The crystalline form of (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide dipropionate, whose X-ray powder diffraction (XRPD) pattern has at least one peak selected from the group consisting of °2θ (±0.2): 10.74, 11.14, 11.32, 12.76, 13.87, 14.35 and 14.69.

[0053] One embodiment of the present invention is (S)- N-The crystalline form of (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide dipropionate, whose X-ray powder diffraction (XRPD) pattern has at least one peak selected from the group consisting of °2θ (±0.2): 15.60, 16.15, 18.22, 18.58, 18.94, 22.40 and 22.80.

[0054] Another embodiment of the present invention is a pharmaceutical composition comprising any of the above-described crystalline forms and a pharmaceutical excipient.

[0055] Another embodiment of the invention is a pharmaceutical composition wherein the crystalline form is substantially pure.

[0056] Another embodiment of the present invention is a method for treating or preventing respiratory diseases in livestock, comprising administering any of the above-described pharmaceutical compositions.

[0057] Another embodiment of the present invention is a method for treating or preventing respiratory diseases in livestock, wherein the respiratory disease is bovine respiratory disease (BRD) and the livestock is a cow.

[0058] Another embodiment of the present invention is a method for treating or preventing respiratory diseases in livestock, wherein the respiratory disease is swine respiratory disease (SRD) and the livestock is a pig.

[0059] One embodiment of the present invention is a preparation ( S )- N- Method for (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide di-p-toluenesulfonate monohydrate: It includes: tert-butyl(S)-(4-(hydroxyamino)-3-(4-((4-((((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamido)-2-methyl-4-oxobutyl-2-yl)carbamate Reaction with p-toluenesulfonic acid to give (S)- N- (3-Amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide di-p-toluenesulfonate monohydrate.

[0060] One embodiment of the present invention is a preparation of (S)- N-Method for (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide di-p-toluenesulfonate monohydrate: It includes: a) Reacting 4-bromobenzaldehyde with 2-methoxyethylamine to obtain N- (4-Bromobenzyl)-2-methoxyethyl-1-amine ; b) make N- The reaction of (4-bromobenzyl)-2-methoxyethyl-1-amine with 2-methyl-3-butyn-2-ol yields 4-(4-(((2-methoxyethyl)amino)methyl)phenyl)-2-methylbutyn-2-ol ; c) Reacting 4-(4-(((2-methoxyethyl)amino)methyl)phenyl)-2-methylbut-3-yne-2-ol with potassium hydroxide to obtain N- (4-ethynylbenzyl)-2-methoxyethyl-1-ammonium hydrochloride ; d) Make N- (4-ethynylbenzyl)-2-methoxyethyl-1-ammonium hydrochloride and tert-butyl( S )-(3-(4-bromobenzamido)-4-(hydroxyamino)-2-methyl-4-oxobut-2-yl)carbamate The reaction yields tert-butyl ( S )-(4-(hydroxyamino)-3-(4-((4-((((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamido)-2-methyl-4-oxobut-2-yl)carbamate ;as well as e) Make tert-butyl ( S )-(4-(hydroxyamino)-3-(4-((4-((((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamido)-2-methyl-4-oxobut-2-yl)carbamate reacts with p-toluenesulfonic acid to give ( S )- N- (3-Amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide di-p-toluenesulfonate monohydrate.

[0061] In one implementation, the generated ( S )- N- (3-Amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide di-p-toluenesulfonate monohydrate recrystallized from a mixture of alcohol and water. Preferably, the alcohol is isopropanol.

[0062] In another embodiment, the volume ratio of alcohol to water is about 1 / 9 to 1 / 1 (volume / volume). Preferably, the volume ratio of alcohol to water is about 1:1.

[0063] In another embodiment, the reaction in step a) further includes a reducing agent.

[0064] In another embodiment, the reducing agent is NaBH4.

[0065] In another embodiment, the reaction in step b) further comprises a palladium catalyst.

[0066] In another embodiment, the palladium catalyst is Pd(PPh3)4.

[0067] In another embodiment, the palladium catalyst is PdCl2(PPh3)2.

[0068] In another embodiment, the product of step a) is separated before proceeding to step b).

[0069] In an alternative implementation, the product of step a) is not separated before step b).

[0070] In another embodiment, the product of step c) is separated in the form of hydrochloride.

[0071] In another embodiment, the reaction in step d) further includes a solvent.

[0072] In another embodiment, the solvent is dimethyl sulfoxide (DMSO) or cyclopentyl methyl ether (CPME).

[0073] In another embodiment, the reaction in step d further includes 1,8-diazabicyclo[5.4.0]undec-7-ene.

[0074] In another embodiment, the reaction in step e) also includes water (H2O).

[0075] In another embodiment, the reaction in step e) further comprises isopropanol.

[0076] In another implementation, isopropanol is essentially water-free.

[0077] One embodiment of the present invention is the preparation of (S)- N- Method for (3-amino-1-(hydroxyamino)-3-methyl-1-oxobutyl-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide di-p-toluenesulfonate monohydrate It includes: making ( S )- N- (3-amino-1-(hydroxyamino)-3-methyl-1-oxobutyl-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide Reacts with p-toluenesulfonic acid to obtain ( S )- N- (3-Amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide di-p-toluenesulfonate monohydrate.

[0078] In another embodiment, the reaction also includes a solvent.

[0079] In another embodiment, the solvent is isopropanol.

[0080] In another embodiment, the reaction also includes water (H2O).

[0081] One embodiment of the present invention is a preparation of (S)- N- Method for (3-amino-1-(hydroxyamino)-3-methyl-1-oxobutyl-2-yl)-4-((4-((((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide dipropionate Including the reaction S)- N- (3-amino-1-(hydroxyamino)-3-methyl-1-oxobutyl-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide It includes: making (S)- N- (3-Amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide.

[0082] One embodiment of the present invention is a method for preparing 4-((4-(((2-methoxyethyl)amino)methyl)phenyl)-2-methylbut-3-yne-2-ol, It includes: a) React 4-bromobenzaldehyde with 2-methoxyethylamine to obtain N- (4-Bromobenzyl)-2-methoxyethyl-1-amine ;as well as b) make N- (4-Bromobenzyl)-2-methoxyeth-1-amine reacts with 2-methyl-3-butyn-2-ol to give 4-(4-(((2-methoxyethyl)amino)methyl)phenyl)-2-methylbutyn-2-ol.

[0083] Example HPLC method: Method A The Agilent Technologies UHPLC / MS 1290 series consists of the following components: Including the high-speed pump G7120A for the degasser G4226A Orifice Plate Sampler Column oven G7116B Diode array detector G7117B G6105B quadrupole LC / MS mass detector equipped with ESI source Column information: Waters XP, 2.1 x 50mm Xbridge BEH C18 2.5μ, T=40℃; Eluent: A: Acetonitrile containing 0.05% (vol. / vol.) formic acid B: Water containing 0.05% (vol. / vol.) formic acid; Flow rate: 0.8 mL / min Gradient: from 2% to 100% eluent A over 1.2 min, then to 100% eluent A over 0.5 min. Runtime: 2.2 min Detection: ESI / MS, positive and negative ion scan: 100-650 m / z; 254 and 210 nm UV; Method B The Agilent Technologies UHPLC / MS 1290 series consists of the following components: Including the high-speed pump G4220A of the degasser G4226A Orifice Plate Sampler Column oven G7116B Diode array detector G7117B G6130B quadrupole LC / MS mass detector equipped with ESI / APCI multi-mode source Column information: Waters XP, 2.1 x 50mm Xbridge BEH C18 2.5μ, T=40℃; Eluent: A: Acetonitrile B: Water containing 0.10% ammonia (vol. / vol.); Flow rate: 0.8 mL / min Gradient: from 2% to 100% eluent A over 1.2 min, then to 100% eluent A over 0.5 min. Runtime: 2.2 min Detection: ESI / APCI / MS, positive ion scan: 100-1000 m / z; 254 and 210 nm UV; Method C The Agilent Technologies UHPLC / MS 1260 series consists of the following components: Including the high-speed pump G4220A of the degasser G4226A Orifice Plate Sampler Column oven G7116B Diode array detector G4212A G6130B quadrupole LC / MS mass detector equipped with ESI / APCI multi-mode source Column information: Waters XP, 2.1 x 50mm Xbridge BEH C18 2.5μ, T=40℃; Eluent: A: Acetonitrile containing 0.05% (vol. / vol.) formic acid B: Water containing 0.05% (vol. / vol.) formic acid; Flow rate: 0.8 mL / min Gradient: from 2% to 100% eluent A over 1.2 min, then to 100% eluent A over 0.5 min. Runtime: 2.2 min Detection: ESI / MS, positive and negative ion scan: 100-1000 m / z; 254 and 210 nm UV; Example 1A: N- Preparation of (4-bromobenzyl)-2-methoxyethyl-1-amine Methanol (600 mL), 4-bromobenzaldehyde (30 g, 162 mmol), and 2-methoxyethylamine (22.55 mL, 259 mmol) were added to a jacketed glass reactor (2 L). The resulting mixture was stirred at room temperature for 14 h. The temperature of the reaction mixture was adjusted to 0–5 °C, and sodium borohydride (6.75 g, 178 mmol) was added in a single batch. After the addition was complete, the temperature of the reaction mixture was raised to approximately 15 °C. The reaction mixture was stirred at this temperature for 40 min. 1 N hydrochloric acid aqueous solution (75 mL) was added, and the resulting mixture was stirred for 30 min. Volatile substances (approximately 550 mL) were removed under reduced pressure at 40 °C, and the resulting residue was acidified to pH 1 by adding 1 N hydrochloric acid aqueous solution. The aqueous layer was extracted with methyl tert-butyl ether (2 × 15 mL), and the pH was adjusted to 11 by adding 4 M sodium hydroxide aqueous solution (approximately 15 mL). The aqueous layer was extracted with methyl tert-butyl ether (3 × 20 mL). The organic layers were combined and washed with brine (20 mL). The product was then concentrated under reduced pressure to give a colorless oily product (35.2 g, 143 mmol).

[0084] 1 H NMR (300 MHz, methanol-d4) δ (ppm): 7.58 – 7.50 (d, J = 8.5 Hz, 2H), 7.32 (d, J = 8.5 Hz, 2H), 3.88 (s, 2H), 3.56 (dd, J = 5.7, 4.9 Hz, 2H), 3.37(s, 3H), 2.88 (dd, J = 5.7, 4.9 Hz, 2H) LC / MS (Method A): Rt = 0.616 min, m / z 244, 246 Variations of Example 1A: N- Preparation of (4-bromobenzyl)-2-methoxyethyl-1-amine Methanol (2 L), 4-bromobenzaldehyde (200 g, 1080 mmol), and 2-methoxyethylamine (150 mL, 1730 mmol) were added to a jacketed glass reactor (5 L). The resulting mixture was stirred at room temperature for 1 h. The temperature of the reaction mixture was adjusted to 0 °C, and sodium borohydride (30.4 g, 840 mmol) was added in three equal portions. After the additions were complete, the temperature of the reaction mixture was raised to approximately 15 °C. The reaction mixture was stirred at this temperature for 40 min. 2N hydrochloric acid aqueous solution (500 mL) was added, and the resulting mixture was stirred for 30 min. Volatile substances were removed under reduced pressure at 40 °C until a turbid mixture was obtained. The pH was adjusted to 1 by adding 2N hydrochloric acid aqueous solution (750 mL). The aqueous layer was extracted with methyl tert-butyl ether (2 × 500 mL), and the pH was adjusted to 8 by adding 4M sodium hydroxide aqueous solution (300 mL). The aqueous layer was extracted with cyclopentyl methyl ether (2 × 500 mL). The organic layers were combined and washed with brine (500 mL). The desired product (247 g, 1010 mmol) was obtained and dissolved in cyclopentyl methyl ether (1.2 L), which could be used for the next reaction.

[0085] Example 1B: Preparation of 4-(4-(((2-methoxyethyl)amino)methyl)phenyl)-2-methylbut-3-yne-2-ol In an inert atmosphere, N- A cyclopentyl methyl ether solution (30 mL) of (4-bromobenzyl)-2-methoxyethyl-1-amine (247 g, 1010 mmol) was added to a round-bottom flask (1 L). Cuprous iodide (578 mg, 3.03 mmol), bis(triphenylphosphine)palladium(II) dichloride (8.56 g, 12.15 mmol), 2-methyl-3-butyn-2-ol (144 mL, 1478 mmol), and 1,8-diazabicyclo[5.4.0]undec-7-ene (229 mL, 1518 mmol) were added, and the reaction mixture was stirred at 75 °C for 3 hours. Heating was stopped, and the reaction mixture was cooled to ambient temperature. 4N hydrochloric acid aqueous solution (800 mL) was added, and the mixture was filtered through a diatomaceous earth (Celite®) filter. The aqueous layer was washed with methyl tert-butyl ether (300 mL). The organic layers were combined and discarded. The aqueous layer was adjusted to pH 9 with 800 mL of 4M sodium hydroxide solution and extracted with cyclopentyl methyl ether (2 × 500 mL and 200 mL). The organic layers were combined and washed successively with 500 mL of 10% ammonia solution and 500 mL of brine. The desired product (235 g, 950 mmol) was obtained, which was dissolved in 1.2 L of cyclopentyl methyl ether and could be used directly in the next reaction. Alternatively, the solution could be concentrated under reduced pressure to obtain a pure, colorless oily product that solidified upon standing.

[0086] 1 H NMR (600 MHz, methanol-d4) δ (ppm): 7.38 (d, J = 8.2 Hz, 2H), 7.32 (d,J = 7.9 Hz, 2H), 3.79 (s, 2H), 3.52 (t, J = 5.3 Hz, 2H), 3.36 (s, 3H), 2.76(m, 2H), 1.57(s, 6H) LC / MS (Method B): Rt = 0.845 min, m / z 248 Example 1C: N- Preparation of (4-ethynylbenzyl)-2-methoxyethane-1-amine chloride Potassium hydroxide (42.6 g, 760 mmol) was added to a cyclopentyl methyl ether solution (1215 mL) of 4-(4-((((2-methoxyethyl)amino)methyl)phenyl)-2-methylbut-3-yn-2-ol (235 g, 950 mmol). The resulting mixture was stirred at reflux temperature for 5 h until the reaction was complete. Heating was stopped, and the reaction mixture was cooled to room temperature. The reaction mixture was filtered through a diatomaceous earth mat, which was then rinsed with cyclopentyl methyl ether (100 mL). The filtrate was extracted with water (2 × 500 mL), and the organic layer was collected. The organic layer was washed with water (100 mL) and dried over brine (500 mL). A 4M hydrochloric acid solution in isopropanol (285 mL, 1140 mmol) was added to the organic layer, and the resulting mixture was stirred for 30 min. The filtered suspension was washed with cyclopentyl methyl ether (1000 mL), and the solid obtained was dried under reduced pressure to obtain the desired product, which was a grayish-white solid (182 g, 780 mmol).

[0087] 1 ¹H NMR (600 MHz, methanol-d⁴) δ (ppm): 7.58 (d, J = 8.3 Hz, 2H), 7.51 (d, J = 8.3 Hz, 2H), 4.26 (s, 2H), 3.69 – 3.66 (m, 2H), 3.65 (s, 1H), 3.43 (s, 3H), 3.24 (t, J = 5.1 Hz, 2H) LC / MS (Method B): retention time = 0.910 min, m / z 190 LC / MS (Method B): Rt = 0.910 min, m / z 190 Example 1D: Preparation of (S)-(4-(hydroxyamino)-3-(4-((4-((((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamido)-2-methyl-4-oxobut-2-yl)tert-butyl carbamate (S)-(3-(4-bromobenzamido)-4-(hydroxyamino)-2-methyl-4-oxobut-2-yl) tert-butyl carbamate (20.5 g, 47.6 mmol) was added under an inert atmosphere. N- (4-ethynylbenzyl)-2-methoxyethane-1-amine chloride (9.68 g, 42.9 mmol), tetrakis(triphenylphosphine)palladium(0) (2.75 g, 2.382 mmol), and cuprous iodide(I) (0.907 g, 4.76 mmol) were added to a jacketed glass reactor (500 mL). Dry and degassed dimethyl sulfoxide (210 mL) and 1,8-diazabicyclo[5.4.0]undec-7-ene (25.1 mL, 167 mmol) were added, and the reaction mixture was heated to 70 °C. After reacting for 1 hour, the reaction mixture was cooled to room temperature. Ethyl acetate (800 mL) and 0.5 N hydrochloric acid aqueous solution (800 mL) were added, and the resulting mixture was stirred for 15 minutes. The mixture was allowed to stand to separate into layers; the aqueous layer was collected, and the organic layer was extracted with 0.5 N hydrochloric acid aqueous solution (400 mL). The combined aqueous layers were washed with ethyl acetate (200 mL), the pH was adjusted to 6 with saturated sodium bicarbonate solution, and then extracted with ethyl acetate (2 × 600 mL). The combined organic layers were filtered through a diatomaceous earth filter, washed with saturated sodium bicarbonate solution (200 mL), dried with brine (200 mL), and concentrated under reduced pressure to give the desired product as a light gray solid (20.20 g, 37.1 mmol).

[0088] 1 H NMR (300 MHz, methanol-d4) δ (ppm): 7.92 (d, J = 7.9 Hz, 2H), 7.64 (d,J = 8.0 Hz, 2H), 7.55 (d, J = 7.7 Hz, 2H), 7.41 (d, J = 7.8 Hz, 2H), 4.72 (s,1H), 3.86 (bs, 2H), 3.55 (bs, 2H), 3.37 (s, 3H), 2.83 (bs, 2H), 1.50 (s, 3H),1.47 (s, 9H), 1.44 (s, 3H) LC / MS (Method B): Rt = 0.743 min, m / z 539 Variation of Example 1D: Preparation of (S)-(4-(hydroxyamino)-3-(4-((4-((((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamido)-2-methyl-4-oxobut-2-yl)tert-butyl carbamate A cyclopentyl methyl ether solution (530 mL) of (S)-(3-(4-bromobenzamido)-4-(hydroxyamino)-2-methyl-4-oxobut-2-yl)carbamate tert-butyl ester (95 g, 77 wt.%, 170 mmol) was added to a 3 L jacketed glass reactor placed under an inert atmosphere, and the temperature was adjusted to 40 °C. N- A solution of (4-ethynylbenzyl)-2-methoxyethyl-1-amine chloride (43 g, 90 wt.%, 170 mmol) in dimethyl sulfoxide (370 mL) was followed by the addition of bis(triphenylphosphine)palladium(II) dichloride (2.4 g, 3.4 mmol), cuprous iodide (I) (0.012 g, 0.63 mmol), and 1,8-diazabicyclo[5.4.0]undec-7-ene (77 mL, 510 mmol). The reaction temperature was adjusted to 70 °C, and the reaction was carried out for 15 hours. Heating was stopped, and the reactants were allowed to cool to ambient temperature. Ethyl acetate (180 mL) and 1N hydrochloric acid aqueous solution (900 mL) were added, and the resulting mixture was stirred for 15 minutes. The mixture was allowed to stand and separate into layers, and the aqueous layer was collected. The aqueous layer was washed with ethyl acetate (270 mL), adjusted to pH 8 by adding saturated sodium carbonate aqueous solution, and then extracted with ethyl acetate (2 × 500 mL). The organic layers were combined, washed with saturated sodium bicarbonate aqueous solution (200 mL), and dried over brine (2 × 200 mL). The desired product (81.8 g, 152 mmol) was obtained as an ethyl acetate solution and could be used for the next reaction.

[0089] Example 1E: Preparation of (S)-4-(hydroxyamino)-3-(4-((4-((((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamido)-2-methyl-4-oxobut-2-amine-4-toluenesulfonate hydrate (S)-(4-(hydroxyamino)-3-(4-((4-(((((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamido)-2-methyl-4-oxobut-2-yl)carbamate tert-butyl ester (4.881 g, 9.06 mmol) was dissolved in isopropanol (25 mL), and p-toluenesulfonic acid (6.89 g, 36.2 mmol) was added. The mixture was heated to 50 °C and stirred at this temperature for 5 h. Heating was stopped, and the reaction mixture was cooled to ambient temperature. The resulting suspension was filtered, the filtrate was washed with isopropanol (20 mL), and dried under reduced pressure at 35 °C. The resulting solid was slurried with isopropanol (25 mL) at 50 °C. After 40 minutes, the slurry was cooled to room temperature, filtered, and dried under reduced pressure at 40 °C to give the desired compound as a grayish-white solid (5.23 g, 6.52 mmol).

[0090] 1 H NMR (300 MHz, methanol-d4) δ (ppm): 7.96 (d, J = 8.5 Hz, 2H), 7.72 (d,J = 8.2 Hz, 4H), 7.66 (dd, J = 8.5, 1.6 Hz, 4H), 7.56 (d, J = 8.3 Hz, 2H),7.29 – 7.19 (m, 4H), 4.83 (s, 1H), 4.28 (s, 2H), 3.71 – 3.64 (m, 2H), 3.43(s, 3H), 3.30 – 3.21 (m, 2H), 2.38 (s, 6H), 1.50 (s, 3H), 1.46 (s, 3H) LC / MS (Method A): Rt = 0.744 min, m / z 439 Example 1F: Preparation of (S)-4-(hydroxyamino)-3-(4-((4-((((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamido)-2-methyl-4-oxobut-2-amine-4-toluenesulfonate hydrate (S)- N-(3-Amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-((((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide (50 g, 114 mmol)) was dissolved in a mixture of 335 mL isopropanol and 88 mL water. The solution was filtered and transferred to a three-necked flask (1 L) equipped with a top stirrer. While stirring, a solution of p-toluenesulfonic acid (49.1 g, 285 mmol) in 230 mL of isopropanol was added dropwise. After half the solution was added, seed crystals of the desired product were added to the mixture to induce precipitation. The reaction mixture was stirred for 16 hours until complete precipitation. The resulting suspension was filtered, and the resulting solid was dried under reduced pressure at 40 °C to give the desired product as a grayish-white solid (77 g, 96 mmol).

[0091] LC / MS (Method B): Rt = 0.715 min, m / z 439 Example 1G: Purification / recrystallization of (S)-4-(hydroxyamino)-3-(4-((4-((((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamido)-2-methyl-4-oxobut-2-amine-4-toluenesulfonate hydrate (S)-4-(hydroxyamino)-3-(4-((4-((((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamido)-2-methyl-4-oxobut-2-amine-4-toluenesulfonate hydrate (46.65 g, 58.24 mmol) was added in portions at 60 °C to a mixture of water (60 mL) and isopropanol (60 mL) under stirring. Heating was stopped after 1 hour, and the mixture was cooled to 30 °C over 90 minutes. The temperature was lowered to 5 °C, and the resulting thick suspension was filtered. The wet filter cake was washed with isopropanol (50 mL), dried under vacuum at 40 °C, and the desired product was obtained as a grayish-white solid (39.55 g, 49.38 mmol).

[0092] Example 2: (S)- N- Preparation of (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide dipropionate Add 6.75 mL of ethanol to (S)- N-(3-Amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-((((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide (0.5 g, 1.140 mmol) was added, and the resulting suspension was stirred at ambient temperature. Heptane (4.5 mL) was added, and the resulting mixture was heated to approximately 30 °C to obtain a clear solution. The solution was cooled to room temperature, and a heptane solution of propionic acid (0.2 mL, 2.67 mmol) was added dropwise (4.5 mL). Additional heptane (14.8 mL) was slowly added, and the mixture was stirred at room temperature for 16 hours. The resulting fine suspension was filtered through Nalgene filter paper with a pore size of 0.2 µm under a nitrogen stream, and the resulting solid was dried under reduced pressure at 40 °C to obtain the desired product as a grayish-white solid (544 mg, 0.93 mmol).

[0093] 1 ¹H NMR (300 MHz, d³-dimethyl sulfoxide) δ (ppm): 7.94 (d, J = 8.5 Hz, 2H), 7.66 (d, J = 8.5 Hz, 2H), 7.61 (d, J = 8.3 Hz, 2H), 7.49 (d, J = 8.3 Hz, 2H), 4.66 (s, 1H), 4.09 (s, 2H), 3.66 – 3.59 (m, 2H), 3.41 (s, 3H), 3.09 – 3.00 (m, 2H), 2.24 (q, J = 7.6 Hz, 4H), 1.40 (s, 3H), 1.33 (s, 3H), 1.11 (t, J = 7.6 Hz, 6H) Example 3: Characterization Description of physical characterization methods X-ray powder diffraction (XRPD), carbon-13 solid-state NMR (ssNMR), and differential scanning calorimetry (DSC) were used to analyze the crystallized (S)- N- Characterization of (3-amino-1-(hydroxyamino)-3-methyl-1-oxobutyl-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide di-p-toluenesulfonate monohydrate N- .

[0094] X-ray powder diffraction (XRPD) X-ray powder diffraction is widely used to characterize molecular structure, crystallinity, and polymorphism. (S)- N-The X-ray powder diffraction pattern of (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-((((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide di-p-toluenesulfonate monohydrate was acquired in reflection mode on a Bruker AXS D8 Advance diffractometer using a LYNXEYE XE-T detector. The instrument employs a Bragg-Brentano geometry, equipped with a Cu radiation source, and Kα monochromaticity is achieved through a nickel filter. Data acquisition was performed using a fixed slit optics configuration. The data acquisition range was 2° to 40° (2θ). Sample preparation involved gently pressing the powdered sample onto a shallow-cavity zero-background silicon sample holder.

[0095] Solid-state NMR In addition to the X-ray powder diffraction pattern mentioned above, (S)- N- (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide di-p-toluenesulfonate monohydrate was further characterized by its carbon-13 solid-state nuclear magnetic resonance (NMR) spectrum. N- (3-Amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide di-p-toluenesulfonate monohydrate sample was filled to full capacity into a 4 mm zirconia solid-state NMR rotor and the rotor tip was sealed using a Kel-F® driven rotor. 13 The C2C PMA-S spectra were acquired using a Bruker AV400 III NMR spectrometer. 1 The H-Ramohr frequency was operated at 400.120 MHz using a Bruker 4 mm H / F / X BB dual resonant MAS probe. The MAS rate was 13 kHz, and the pulse delay was 1.8 s. During CP, a 100 kHz pulse was applied. 1 H p / 2 excitation pulse, followed by application 1 The H-lock pulse, whose power linearly increases from 41.7 kHz to 83.3 kHz (from 50% to 100%) within 3 ms. Correspondingly... 13 The pulse power matching of the C-square pulse 1An H-slope was used to generate the maximum signal. During data acquisition, a high-power TPPM 1H decoupling at 100 kHz was applied, and 23,200 scans were acquired simultaneously for signal averaging. During data processing, 30 Hz Lorentz linewidth broadening, zero-fill to 32k data points, and standard baseline correction were applied. Throughout the CP experiments, the sample temperature was controlled at 275 K by a Bruker VT control unit. According to temperature calibration experiments, setting the temperature to 275 K at 13 kHz MAS corresponds to an actual sample temperature of 300 K. 13 The CCP spectra used an α-glycine sample as an external standard reference, with its carboxyl carbon set at 176.70 ppm. Chemical shifts and corresponding relative peak intensities were measured using the standard peak picking procedure in the instrument software.

[0096] Differential scanning calorimetry (DSC) DSC data were acquired using a TA Instruments DSC Q2000 or equivalent instrument. Samples weighing between 1 and 6 mg were placed in an open aluminum pan. This pan was placed at the sample position in the calorimeter. An empty pan was placed at the reference position. The calorimeter was closed, and a nitrogen flow was introduced through the cell. The heating program was set to heat the sample to approximately 250 °C at a heating rate of 10 °C / min. After the run, the data were analyzed using the DSC analysis program in the system software. Observed endothermic and exothermic peaks were integrated between baseline temperatures above and below the temperature range of the thermal event. Reported data include the onset temperature, peak temperature, and enthalpy change.

[0097] Thermogravimetric analysis (TGA) was performed on a TA Q50 thermogravimetric analyzer (TA Instruments). Samples (2–15 mg) were placed in open pans and heated from 25 °C to 300 °C at a heating rate of 10 °C / min, with nitrogen purging at a flow rate of 100 mL / min. After the run, the data were analyzed using the TGA analysis program in the system software. The observed weight loss at the specific temperatures was reported.

[0098] (S)- N- Physical characterization of (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide di-p-toluenesulfonate monohydrate Figure 1 (S)- was displayed N- X-ray powder diffraction pattern of (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide di-p-toluenesulfonate monohydrate. (S)- N-(3-Amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide di-p-toluenesulfonate monohydrate exhibits characteristic diffraction peaks with corresponding d-intervals of 14.07, 10.96, and 9.46 Å. (S)- N- (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide di-p-toluenesulfonate monohydrate was further characterized with d-intervals of 7.37, 6.07, 5.48, and 5.23 Å. (S)- N- The d-intervals of (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide di-p-toluenesulfonate monohydrate are 4.92, 4.55, 4.62 and 3.82 Å.

[0099] Table 1. (S)- N- Characteristic peak positions and corresponding d-spacings of (3-amino-1-(hydroxyamino)-3-methyl-1-oxobutyl-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide di-p-toluenesulfonate monohydrate

[0100] Figure 2 It is (S)- N- Carbon-13 cross-polarized magic angle rotation (CPMAS) nuclear magnetic resonance (NMR) spectrum of (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-((((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide di-p-toluenesulfonate monohydrate. Peaks marked with an asterisk correspond to rotation sidebands.

[0101] Table 2: (S)- N- Chemical shift and relative peak intensity of (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide di-p-toluenesulfonate monohydrate

[0102] Figure 3A For (S)- N-Typical DSC curve of (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide di-p-toluenesulfonate monohydrate. This DSC curve is characterized by an endothermic dehydration peak, with an extrapolated onset temperature of 47.5 °C, a peak temperature of 68.6 °C, and an enthalpy of 51.7 g / g.

[0103] Figure 3B For (S)- N- Thermogravimetric analysis (TGA) curves of the monohydrate salt of (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide di-p-toluenesulfonate. The TGA curves show a weight loss of 2.24%, which is close to the theoretical value of water loss for the monohydrate (2.25%). Significant weight loss was observed above 200°C, indicating decomposition.

[0104] (S)- N- Physical characterization of (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide dipropionate. The following analysis focuses on (S)- N- The B-type polymorph of (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide dipropionate was obtained.

[0105] Figure 4 (S)- was displayed N- X-ray powder diffraction pattern of (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide dipropionate.

[0106] Table 3. (S)- N- Characteristic peak positions and corresponding d-spacings of (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide dipropionate. (S)- N- The characteristic diffraction peaks of (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide dipropionate correspond to d-intervals of 24.73, 12.34, and 11.65. (S)- N-The d-intervals of (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide dipropionate were 8.24, 7.95, 7.75, 6.94, 6.39, 6.17, and 6.03, respectively. (S)- N- The d-spacing of (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide dipropionate was further characterized as 5.68, 5.49, 4.93, 4.87, 4.78, 4.69, 3.97 and 3.90.

[0107] (S)- N- (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide dipropionate was further characterized by its carbon-13 solid-state nuclear magnetic resonance (NMR) spectrum. N- (3-Amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide dipropionate sample was filled to full capacity into a 4 mm zirconia solid-state NMR rotor and the rotor tip was sealed using a Kel-F® driven rotor. 13 The C2C PMA-S spectra were acquired using a Bruker AV400 III NMR spectrometer. 1 The H-Ramohr frequency was operated at 400.120 MHz using a Bruker 4 mm H / F / XBB dual resonant MAS probe. The MAS rate was 13 kHz, and the pulse delay was 1.2 s. During CP, a 100 kHz pulse was applied. 1 An H π / 2 excitation pulse is applied, followed by a 1H lock-in pulse, the power of which linearly increases from 41.7 kHz to 83.3 kHz (from 50% to 100%) within 3 ms. The pulse power of the corresponding 13C square pulse is matched to its respective... 1 An H-slope is applied to generate the maximum signal. A high-power TPPM of 100 kHz is applied during data acquisition. 1H-decoupling was used, and 100,000 scans were acquired simultaneously for signal averaging. During data processing, 30 Hz Lorentz linewidth broadening, zero-fill to 32k data points, and standard baseline correction were applied. Throughout the CP experiments, the sample temperature was controlled at 270 K by a Bruker VT control unit. According to temperature calibration experiments, setting the temperature to 270 K at 13 kHz MAS corresponds to an actual sample temperature of 290 K. 13 CCP spectra used an α-glycine sample as an external standard reference, with its carboxyl carbon set at 176.70 ppm. Characteristic 13 The C chemical shift and the corresponding relative peak intensity were measured using the standard peak picking program in the instrument software.

[0108] Figure 5 It is in crystalline state (S)- N- Carbon-13 cross-polarized magic angle rotation (CPMAS) nuclear magnetic resonance (NMR) spectrum of (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-((((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide dipropionate. Peaks marked with asterisks correspond to rotation sidebands.

[0109] Table 4. (S)- N- Chemical shift and relative peak intensity of (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide dipropionate.

[0110] Figure 6 For (S)- N- Typical DSC curve of (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide dipropionate. This DSC curve is characterized by an endothermic peak, with an extrapolated onset temperature of 107.8 °C, a peak temperature of 119.1 °C, and an enthalpy of 75.2 J / g.

[0111] Example 4: Crystallization (S) - N- (3-amino-1-(hydroxyamino)-3-methyl-1-oxobutyl-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide di-p-toluenesulfonate monohydrate and (S)- N- Stability of (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide dipropionate compared to other crystal forms (S)- N- (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide di-p-toluenesulfonate monohydrate sample (hereinafter referred to as di-p-toluenesulfonate monohydrate), (S)- N- (3-amino-1-(hydroxyamino)-3-methyl-1-oxobutyl-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide dipropionate (hereinafter referred to as dipropionate), (S)- N- (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide dihydrochloride (hereinafter referred to as dihydrochloride) and (S)- N- Stability evaluation was performed on samples containing (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide (listed hereinafter as a free base). Evaluation conditions were 40 °C and 75% relative humidity (RH). Chemical stability was assessed by monitoring the percentage area of ​​the API peak in the stable samples. Separation of the API main peak from impurity and degradation peaks was achieved using a UPLC instrument equipped with a C18 column and a UV detector.

[0112] As shown in Tables 6 and 6A, di-toluenesulfonate monohydrate exhibits better chemical and physical stability compared to other forms.

[0113] Table 6

[0114] Table 6A – Solid-state stability study To evaluate the physicochemical stability of the free base, anhydrous di-p-toluenesulfonate, di-p-toluenesulfonate monohydrate, and type B dipropionate under three stability conditions (25℃ / 60%RH, 40℃ / 75%RH, and 50℃ or 60℃, open), solid-state stability studies were conducted. The determined purity values ​​are shown in Table 6A.

[0115] For di-p-toluenesulfonate monohydrate: 1. Chemical and physical properties are stable for 6 months at 25℃ / 60%RH, 40℃ / 75%RH and 50℃.

[0116] For type B dipropionate: 1. The chemical properties are stable for two weeks under conditions of 25℃ / 60%RH.

[0117] 2. Under conditions of 40℃ / 75%RH, the purity of %A decreases by about 13% within two weeks, and it loses its crystallinity.

[0118] 3. Under open conditions at 60℃, the purity of A decreases by about 5% within two weeks, while its physical properties remain stable.

[0119] Table 6A

[0120] Hygroscopicity was assessed using dynamic vapor adsorption (DVS). Water adsorption-desorption isotherms were obtained using a DVS system (Surface Measurement Systems, DVS Adventure). The sample was subjected to one RH cycle at 25°C. During the cycle, RH was increased from 0% or 30% to 90% in 10% increments per step, and then decreased back to 0% or 30% RH. An equilibrium criterion was set at a rate of change of mass per unit time (dm / dt) of 0.002 wt% / min. Once this criterion was reached, the system was maintained at the set parameters for 10 minutes. The maximum equilibrium time was 180 minutes. Figure 7 The DVS isotherm plot of di-toluenesulfonate monohydrate is shown. The adsorption phase of the first cycle started at 30% RH and ended at 90% RH, with water absorption <0.3%. The desorption phase of the first cycle occurred at below 10% RH, resulting in water loss. The trajectory of the second cycle was the same as that of the first cycle, indicating that no crystal form transformation occurred. Figure 8 The DVS isotherm plot of propionate is shown. A water absorption of >14% at 80% RH indicates that the salt is hygroscopic. Figure 9 The DVS isotherm plot of anhydrous di-p-toluenesulfonate is shown. During the adsorption phase, the anhydrous product adsorbed approximately 2.4% water in one step, and during the desorption phase, water was still retained at RH above 10%. The observed large hysteresis effect indicates that hydrates formed after the adsorption phase.

Claims

1. A (S)- N- The crystalline form of (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide di-p-toluenesulfonate monohydrate has at least one of the following characteristics: X-ray powder diffraction (XRPD) pattern having at least one peak selected from 6.28, 8.07, 9.35, 12.00, 14.60, 16.16, 16.94, 18.05, 19.51, 20.84 and 23.28, expressed in °2θ (±0.2); Carbon-13 cross-polarized magic angle rotation (CPMAS) nuclear magnetic resonance (NMR) spectra with at least one peak selected from 20.54, 21.14, 22.05, 23.33, 48.56, 50.43, 55.71, 57.7, 59.29, 68.97, 88.52, 90.16, 122.29, 126.94, 129.73, 133.68, 136.67, 141.72, 163.70, and 170.30 ppm; or The differential scanning calorimetry (DSC) thermogram shows an endothermic peak at approximately 68.6℃ + / -5℃.

2. The crystalline form according to claim 1, having an X-ray powder diffraction (XRPD) pattern substantially as shown in Figure 1.

3. The crystalline form according to claim 1, having a carbon-13 cross-polarized magic angle rotation (CPMAS) nuclear magnetic resonance (NMR) spectrum substantially as shown in Figure 2.

4. The crystalline form according to claim 1, having a differential scanning calorimetry (DSC) thermogram substantially as shown in FIG3.

5. A pharmaceutical composition comprising a crystalline form according to any one of claims 1-4 and a pharmaceutical excipient.

6. The pharmaceutical composition according to claim 5, wherein the crystalline form is substantially pure.

7. A (S)- N- The crystalline form of (3-amino-1-(hydroxyamino)-3-methyl-1-oxobut-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide dipropionate, having at least one of the following characteristics: X-ray powder diffraction (XRPD) pattern having at least one peak selected from 3.57, 7.16, 7.59, 10.74, 11.14, 11.32, 12.76, 13.87, 14.35, 14.69, 15.60, 16.15, 18.22, 18.58, 18.94, 22.40 and 22.80, expressed in °2θ (±0.2); Carbon-13 cross-polarized magic angle rotation (CPMAS) nuclear magnetic resonance (NMR) spectra with at least one peak selected from 12.24, 24.57, 26.65, 31.96, 32.96, 51.65, 56.12, 57.39, 59.28, 70.31, 179.96, and 183.67 ppm; or The differential scanning calorimetry (DSC) thermogram shows an endothermic peak at approximately 119.1 °C.

8. The crystalline form according to claim 7, having an X-ray powder diffraction (XRPD) pattern substantially as shown in FIG4.

9. The crystalline form according to claim 7, having a carbon-13 cross-polarized magic angle rotation (CPMAS) nuclear magnetic resonance (NMR) spectrum substantially as shown in FIG5.

10. The crystalline form according to claim 7, having a differential scanning calorimetry (DSC) thermogram substantially as shown in FIG6.

11. A pharmaceutical composition comprising a crystalline form according to any one of claims 7-10 and a pharmaceutical excipient.

12. The pharmaceutical composition according to claim 11, wherein the crystalline form is substantially pure.

13. A method for treating or preventing respiratory diseases in livestock, comprising administering a pharmaceutical composition according to any one of claims 5-6 or 11-12.

14. The method of claim 13, wherein the respiratory disease is bovine respiratory disease (BRD) and the livestock is a cattle.

15. The method of claim 13, wherein the respiratory disease is swine respiratory disease (SRD) and the livestock is a pig.

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