Processes to make crystalline forms of (s)- n-(3-amino-1-(hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4- ((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide salts

Abstract

Processes to make crystalline forms of (S)-N-(3-amino-1-(hydroxyamino)-3- methyl-1-oxobutan-2-yl)-4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide salts.

Description

[0001] PROCESSES TO MAKE CRYSTALLINE FORMS OF (S)- / V-(3-AMINO-1- (HYDROXYAMINO)-3-METHYL-1-OXOBUTAN-2-YL)-4-((4-(((2- METHOXYETHYL)AMINO)METHYL)PHENYL)ETHYNYL)BENZAMIDE SALTS

[0002] TECHNICAL FIELD

[0003] Organic synthesis, specifically processes of making salts, more specifically of making (S)- / V-(3-amino-1-(hydroxyamino)-3-methyl-1 -oxobutan-2-yl)-4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide salts

[0004] BACKGROUND

[0005] WO20231 18558 discloses the synthesis of (S)-A / -(3-amino-1-(hydroxyamino)-3- methyl-1-oxobutan-2-yl)-4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide dihydrochloride

[0006] WO20231 18557 discloses injectable pharmaceutical compositions for the treatment of respiratory diseases in animals. Formulations comprising (S)-A / -(3- amino-1-(hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide dihydrochloride are disclosed.

[0007] SUMMARY OF INVENTION

[0008] The invention concerns processed to make crystalline forms of (S)- / V-(3-amino-1-

[0009] (hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide salts. (DESCRIPTION OF DRAWINGS)

[0010] FIG. 1 is a characteristic X-ray diffraction pattern of the crystalline (S)- / V-(3-amino- 1 -(hydroxyam ino)-3-methyl-1 -oxobutan-2-yl)-4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis tosylate monohydrate salt.

[0011] FIG. 2 is a carbon-13 cross-polarization magic-angle spinning (CPMAS) nuclear magnetic resonance (NMR) spectrum of the crystalline (S)- / V-(3-amino-1- (hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis tosylate monohydrate salt. Peaks labeled with asterisks correspond to spinning sidebands.

[0012] FIG. 3A is a typical DSC thermogram of (S)-A / -(3-amino-1 -(hydroxyamino)-3- methyl-1-oxobutan-2-yl)-4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis tosylate monohydrate salt. FIG. 3B shows the TGA thermogram of (S)W-(3-amino-1 -(hydroxyam ino)-3- methyl-1-oxobutan-2-yl)-4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis tosylate monohydrate salt.

[0013] FIG. 4 is a characteristic X-ray diffraction pattern of the crystalline (S)-4- (hydroxyamino)-3-(4-((4-(((2- methoxyethyl)ammonio)methyl)phenyl)ethynyl)benzamido)-2-methyl-4-oxobutan- 2-aminium propionate

[0014] FIG. 5 is a carbon-13 cross-polarization magic-angle spinning (CPMAS) nuclear magnetic resonance (NMR) spectrum of the crystalline (S)-4-(hydroxyamino)-3-(4- ((4-(((2-methoxyethyl)ammonio)methyl)phenyl)ethynyl)benzamido)-2-methyl-4- oxobutan-2-aminium propionate

[0015] FIG. 6 is a typical DSC thermogram of (S)-4-(hydroxyamino)-3-(4-((4-(((2- methoxyethyl)ammonio)methyl)phenyl)ethynyl)benzamido)-2-methyl-4-oxobutan- 2-aminium propionate. FIG. 7 shows the DVS isotherm plot for the tosylate monohydrate salt.

[0016] FIG. 8 shows the DVS isotherm plot for the propionate salt.

[0017] FIG. 9 shows the structure of the (S)-A / -(3-amino-1-(hydroxyamino)-3-methyl-1- oxobutan-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis tosylate monohydrate salt which might also be named (S)-4-(hydroxyamino)-3-(4-((4-(((2- methoxyethyl)ammonio)methyl)phenyl)ethynyl)benzamido)-2-methyl-4-oxobutan- 2-aminium 4-methylbenzenesulfonate hydrate according to Chem Draw® version 21.0.0.28 or, (S)-4-(hydroxyamino)-3-(4-((4-(((2- methoxyethyl)ammonio)methyl)phenyl)ethynyl)benzamido)-2-methyl-4-oxobutan- 2-aminium bis 4-methylbenzenesulfonate monohydrate or, [4-[2-[4-[[(1 S)-2-azaniumyl-1 -(hydroxycarbamoyl)-2-methyl- propyl]carbamoyl]phenyl]ethynyl]phenyl]methyl-(2-methoxyethyl)ammonium 4- methylbenzenesulfonate hydrate according to BIOVIA Draw version 20.1 or, [4-[2-[4-[[(1 S)-2-azaniumyl-1 -(hydroxycarbamoyl)-2-methyl- propyl]carbamoyl]phenyl]ethynyl]phenyl]methyl-(2-methoxyethyl)ammonium bis 4- methylbenzenesulfonate monohydrate

[0018] FIG. 10 shows the structure of the (S)-N-(3-amino-1 -(hydroxyamino)-3-methyl-1 - oxobutan-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis propionate salt which might also be named (S)-4-(hydroxyamino)-3-(4-((4-(((2- methoxyethyl)ammonio)methyl)phenyl)ethynyl)benzamido)-2-methyl-4-oxobutan- 2-aminium propionate according to ChemDraw® version 21 .0.0.28 or, (S)-4-(hydroxyamino)-3-(4-((4-(((2- methoxyethyl)ammonio)methyl)phenyl)ethynyl)benzamido)-2-methyl-4-oxobutan- 2-aminium bis propionate or, [4-[2-[4-[[(1 S)-2-azaniumyl-1 -(hydroxycarbamoyl)-2-methyl- propyl]carbamoyl]phenyl]ethynyl]phenyl]methyl-(2-methoxyethyl)ammonium propanoate according to BIOVIA Draw version 20.1 or, [4-[2-[4-[[(1 S)-2-azaniumyl-1 -(hydroxycarbamoyl)-2-methyl- propyl]carbamoyl]phenyl]ethynyl]phenyl]methyl-(2-methoxyethyl)ammonium bis propanoate

[0019] FIG. 11 shows the PXRD of bis-tosylate anhydrate.

[0020] FIG. 12 shows the DSC of bis-tosylate anhydrate.

[0021] FIG. 13 shows the TGA of bis-tosylate anhydrate.

[0022] FIG. 14 shows the PXRD of the free base (S)-A / -(3-amino-1-(hydroxyamino)-3- methyl-1-oxobutan-2-yl)-4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide.

[0023] FIG. 15 shows the TGA of the free base (S)- / V-(3-amino-1-(hydroxyamino)-3- methyl-1-oxobutan-2-yl)-4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide.

[0024] FIG. 16 shows of the DSC of the free base(S)-A / -(3-amino-1-(hydroxyamino)-3- methyl-1-oxobutan-2-yl)-4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide.

[0025] FIG. 17 shows the PXRD of the HCI salt (S)- / V-(3-amino-1-(hydroxyamino)-3- methyl-1-oxobutan-2-yl)-4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide dihydrochloride.

[0026] FIG. 18 shows the TGA of the HCI salt (S)- / V-(3-amino-1-(hydroxyamino)-3-methyl- 1-oxobutan-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide dihydrochloride.

[0027] FIG. 19 shows the DSC of the HCI salt (S)- / V-(3-amino-1 -(hydroxyamino)-3- methyl-1-oxobutan-2-yl)-4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide dihydrochloride.

[0028] Detailed Description

[0029] There are several advantages of the crystalline (S)-A / -(3-amino-1-(hydroxyamino)- 3-methyl-1 -oxobutan-2-yl)-4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis tosylate monohydrate salt and the (S)-N-(3-amino-1 -(hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-((4- (((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis propionate salt over other salts and the free base (S)-A / -(3-amino-1-(hydroxyamino)-3-methyl-1- oxobutan-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide.

[0030] When selecting a salt for incorporation into a pharmaceutical product, the salt’s stability in both the solid state and in the formulation must be considered. Furthermore, the salt’s toxicity, bioavailability and efficacy must be evaluated. Both the bis tosylate mono hydrate salt and bis propionate salt were more stable than the hydrochloride salt and the parent free base in long term stability studies at ambient and accelerated conditions. Furthermore, the bis tosylate mono hydrate salt was more stable than the bis propionate salt. The bis tosylate mono hydrate salt is the most stable salt identified. This was unexpected.

[0031] (S)-A / -(3-amino-1 -(hydroxyamino)-3-methyl-1 -oxobutan-2-yl)-4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis-tosylate monohydrate salt the (S)-N-(3-amino-1-(hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis propionate salt As indicated above, WO2023118558 discloses the synthesis of (S)-A / -(3-amino-1- (hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide dihydrochloride.

[0032] Processes to prepare the bis tosylate monohydrate and bis propionate salts are outlined below.

[0033] Scheme 1 is a process to prepare (S)-N-(3-amino-1-(hydroxyamino)-3-methyl-1- oxobutan-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis tosylate monohydrate salt. An advantage of this process is that the tert-butyl (S)-(4-(hydroxyamino)-3-(4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamido)-2-methyl-4-oxobutan-2- yl)carbamate intermediate is deprotected and converted to the bis tosylate monohydrate salt in a single reaction.

[0034] Scheme 1

[0035] (S)- / V-(3-amino-1 -(hydroxyamino)-3-methyl-1 -oxobutan-2-yl)-4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis tosylate monohydrate salt can be also prepared directly from the free base as indicated in Scheme 2 Scheme 2

[0036] The intermediate 4-(4-(((2-methoxyethyl)amino)methyl)phenyl)-2-methylbut-3-yn- 2-ol in Scheme 1 can alternatively be prepared as described in Scheme 3.

[0037] Scheme 3 Finally, the (S)-N-(3-amino-1 -(hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis propionate salt can be prepared as described in Scheme 4.

[0038] Scheme 4 Further details of the preparation of these compounds are given below in the Examples

[0039] Bovine respiratory disease (BRD) is the most common and costly disease affecting beef cattle in the world. Bovine respiratory disease (BRD) has a multifactorial etiology and develops as a result of complex interactions between environmental factors, host factors, and pathogens. Environmental factors (e.g., weaning, transport, commingling, crowding, inclement weather, dust, and inadequate ventilation) serve as stressors that adversely affect the immune and nonimmune defense mechanisms of the host. In addition, certain environmental factors (e.g., crowding and inadequate ventilation) can enhance the transmission of infectious agents among animals. It is a complex, bacterial infection that causes Enzootic pneumonia in calves and other bovine animals and can possibly be fatal. The infection is usually a sum of three codependent factors: Stress, an underlying viral infection, and a new bacterial infection. The diagnosis of the disease is complex since there are multiple possible causes.

[0040] The term swine respiratory disease (SRD) was used to describe pneumonia of multiple etiology causing clinical disease and failure to gain weight later in the finishing process (15 to 20 weeks of age).

[0041] The term “substantially as shown” as used herein refers to an X- ray powder diffraction (XRPD) spectrum, carbon-13 cross-polarization magic-angle spinning (CPMAS) nuclear magnetic resonance (NMR) spectrum, or differential scanning calorimetry (DSC) thermogram that is non-identical to those depicted herein, but may fall within the limits of experimental error, when considered by one of ordinary skill in the art. One of ordinary skill in the art would understand that an X-ray powder diffraction spectrum may contain peaks that fall within ±0.2 degrees 20 of the peaks contained in the spectrum of FIG. 1 and a differential scanning calorimetry (DSC) thermogram as depicted in FIG. 3 may contain an endotherm at ±5 °C of what is depicted.

[0042] The term “substantially purified” as used herein refers to a crystalline form of the compound that is at least 90% pure. In an alternate embodiment, “substantially purified” refers to a crystalline form of the compound that is at least 95%, 99%, or 99.9% pure.

[0043] DESCRIPTION OF EMBODIMENTS

[0044] An embodiment of the invention is a crystalline form of (S)- / V-(3-amino-1- (hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis tosylate monohydrate salt having at least one of the following characteristics: an X-ray powder diffraction (XRPD) pattern having at least one peak in terms of d-spacing [A] (±0.2) selected from the group consisting of 14.07, 10.96, 9.46, 7.37, 6.07, 5.48, 5.23, 4.92, 4.55, 4.26 and 3.82 or in terms of °29(±0.2) selected from the group consisting of 6.28, 8.07, 9.35, 12.00, 14.60, 16.16, 16.94, 18.05, 19.51 , 20.84 and 23.28; a carbon-13 cross-polarization magic-angle spinning (CPMAS) nuclear magnetic resonance (NMR) spectrum having at least one peak selected from the group consisting of, 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 a differential scanning calorimetry (DSC) thermogram comprising an endothermic peak at about 68.6 °C ± 5°C.

[0045] Another embodiment of the invention is the crystalline form having an X-ray powder diffraction (XRPD) pattern substantially as shown in Figure 1.

[0046] Another embodiment of the invention is the crystalline form having carbon-13 cross-polarization magic-angle spinning (CPMAS) nuclear magnetic resonance (NMR) spectrum substantially as shown in Figure 2.

[0047] Another embodiment of the invention is the crystalline form having a differential scanning calorimetry (DSC) thermogram substantially as shown in Figure 3. Another embodiment of the invention is the crystalline form having a differential scanning calorimetry (DSC) thermogram comprising an endothermic peak at about 68.6 °C+ / -5°C and an exotherm greater than 200 °C.

[0048] An embodiment of the invention is a crystalline form of (S)- / V-(3-amino-1- (hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis tosylate monohydrate salt having an X-ray powder diffraction (XRPD) pattern having at least one peak in terms of °20(±0.2) selected from the group consisting of 6.28, 8.07 and 9.35.

[0049] An embodiment of the invention is a crystalline form of (S)- / V-(3-amino-1- (hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis tosylate monohydrate salt having an X-ray powder diffraction (XRPD) pattern having at least one peak in terms of °20(±0.2) selected from the group consisting of 12.00, 14.60, 16.16 and 16.94.

[0050] An embodiment of the invention is a crystalline form of (S)- / V-(3-amino-1- (hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis tosylate monohydrate salt having an X-ray powder diffraction (XRPD) pattern having at least one peak in terms of °20(±0.2) selected from the group consisting of 18.05, 19.51 , 20.84 and 23.28.

[0051] Another embodiment of the invention is a pharmaceutical composition comprising any of the above crystalline forms and a pharmaceutical excipient.

[0052] Another embodiment of the invention is the pharmaceutical composition, wherein the crystalline form is substantially purified.

[0053] An embodiment of the invention is a crystalline form of the (S)-N-(3-amino-1- (hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis propionate salt having at least one of the following characteristics: an X-ray powder diffraction (XRPD) pattern having at least one peak in terms of °20(±0.2) selected from the group consisting of 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 or in terms of d-spacing [A] (±0.2) selected from the group consisting of 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, 4.69, 3.97 and 3.90; a carbon-13 cross-polarization magic-angle spinning (CPMAS) nuclear magnetic resonance (NMR) spectrum having at least one peak selected from the group consisting of 12.27, 24.57, 26.65, 31.96, 32.96, 51.65, 56.12, 57.39, 59.28, 70.31 , 179.96, and 183.67ppm; or a differential scanning calorimetry (DSC) thermogram comprising an endothermic peak at about 119.1 °C ± 5°C.

[0054] Another embodiment of the invention is the crystalline form having an X-ray powder diffraction (XRPD) pattern substantially as shown in Figure 4.

[0055] Another embodiment of the invention is the crystalline form having carbon-13 cross-polarization magic-angle spinning (CPMAS) nuclear magnetic resonance (NMR) spectrum substantially as shown in Figure 5.

[0056] Another embodiment of the invention is the crystalline form having a differential scanning calorimetry (DSC) thermogram substantially as shown in Figure 6.

[0057] An embodiment of the invention is a crystalline form of (S)-N-(3-amino-1- (hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis propionate salt having an X-ray powder diffraction (XRPD) pattern having at least one peak in terms of °20(±O.2) selected from the group consisting of 3.57, 7.16 and 7.59.

[0058] An embodiment of the invention is a crystalline form of (S)-N-(3-amino-1- (hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis propionate salt having an X-ray powder diffraction (XRPD) pattern having at least one peak in terms of °20(±O.2) selected from the group consisting of 10.74, 11.14, 11.32, 12.76, 13.87, 14.35 and 14.69.

[0059] An embodiment of the invention is a crystalline form of (S)-N-(3-amino-1- (hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis propionate salt having an X-ray powder diffraction (XRPD) pattern having at least one peak in terms of °20(±0.2) selected from the group consisting of 15.60, 16.15, 18.22, 18.58, 18.94, 22.40 and 22.80.

[0060] Another embodiment of the invention is a pharmaceutical composition comprising any of the above crystalline forms and a pharmaceutical excipient.

[0061] Another embodiment of the invention is the pharmaceutical composition, wherein the crystalline form is substantially purified.

[0062] Another embodiment of the invention is a method of treating or preventing a respiratory disease in livestock animals comprising administering any of the above pharmaceutical compositions.

[0063] Another embodiment of the invention is the method of treating or preventing a respiratory disease in livestock animals, wherein the respiratory disease is bovine respiratory disease (BRD) and the livestock animals are cattle.

[0064] Another embodiment of the invention is the method of treating or preventing a respiratory disease in livestock animals, wherein the respiratory disease is swine respiratory disease (SRD) and the livestock animals are swine.

[0065] An embodiment of the invention is a method of preparing (S)-A / -(3-amino-1- (hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis tosylate monohydrate salt comprising reacting tert-butyl (S)-(4-(hydroxyamino)-3-(4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamido)-2-methyl-4-oxobutan-2- yl)carbamate with p-toluenesulfonic acid to yield (S)-A / -(3-amino-1 -(hydroxyamino)-3-methyl-1 - oxobutan-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis tosylate monohydrate salt.

[0066] An embodiment of the invention is a method of preparing (S)-A / -(3-amino-1- (hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis tosylate monohydrate salt

[0067] comprising a) reacting 4-bromobenzaldehyde with 2-methoxyethylamine to yield / V-(4- bromobenzyl)-2-methoxyethan-1 -amine b) reacting / V-(4-bromobenzyl)-2-methoxyethan-1 -amine with 2-methyl-3-butyn-2- ol to yield 4-(4-(((2-methoxyethyl)amino)methyl)phenyl)-2-methylbut-3-yn-2-ol c) reacting 4-(4-(((2-methoxyethyl)am ino)methyl)phenyl)-2-methylbut-3-yn-2-ol with potassium hydroxide to yield A / -(4-ethynylbenzyl)-2-methoxyethan-1 -aminium chloride d) reacting / V-(4-ethynylbenzyl)-2-methoxyethan-1-aminium chloride with with tert- butyl (S)-(3-(4-bromobenzamido)-4-(hydroxyamino)-2-methyl-4-oxobutan-2-yl) carbamate to yield tert-butyl (S)-(4-(hydroxyamino)-3-(4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamido)-2-methyl-4-oxobutan-2- yl)carbamate e) reacting tert -butyl (S)-(4-(hydroxyamino)-3-(4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamido)-2-methyl-4-oxobutan-2- yl)carbamate with p-toluenesulfonic acid to yield (S)- / V-(3-amino-1- (hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis tosylate monohydrate salt.

[0068] In an embodiment, the yield (S)- / V-(3-amino-1 -(hydroxyamino)-3-methyl-1- oxobutan-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis tosylate monohydrate salt is recrstalized from a mixture of an alcohol and water. Preferably, the alcohol is 2-propanol.

[0069] In another embodiment, the ratio of alcohol to water from about 1 / 9 to about 1 / 1 (vol. / vol.). Preferably, the ratio of alcohol is about 1 to 1 vol / vol.

[0070] In another embodiment, the reaction of step a) further comprises a reducing agent

[0071] In another embodiment, the reducing agent is NaBH4.

[0072] In another embodiment, the reaction of step b) further comprises a palladium catalyst. In another embodiment, the palladium catalyst is Pd(PPh3)4.

[0073] In another embodiment, the palladium catalyst is PdCl2(PPh3)2.

[0074] In another embodiment, the product of step a) is isolated prior to being carried on to step b).

[0075] In an alternative embodiment, the the product of step a) is not isolated prior to being carried on to step b).

[0076] In another embodiment, the product of step c) is isolated as the HCI salt.

[0077] In another embodiment, the reaction of step d further comprises a solvent.

[0078] In another embodiment, the solvent is dimethyl sulfoxide (DMSO) or cyclopentyl methyl ether (CPME).

[0079] In another embodiment, the reaction of step d further comprises 1 ,8- diazabicyclo(5.4.0)undec-7-ene.

[0080] In another embodiment, the reaction of step e) further comprises water (H2O).

[0081] In another embodiment, the reaction of step e) further comprises 2-propanol.

[0082] In another embodiment, the 2-propanol is substantially free of water.

[0083] An embodiment of the invention is a method of preparing (S)-A / -(3-amino-1- (hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis tosylate monohydrate salt comprising reacting S)- / V-(3-amino-1 -(hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-

[0084] ((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide with p-toluenesulfonic acid to yield (S)- / V-(3-amino-1 -(hydroxyamino)-3-methyl-1 - oxobutan-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis tosylate monohydrate salt.

[0085] In another embodiment, the reaction further comprises a solvent.

[0086] In another embodiment, the solvent is isopropanol.

[0087] In another embodiment, the reaction further comprises water (H2O).

[0088] An embodiment of the invention is a method of preparing the (S)-N-(3-amino-1- (hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis propionate salt comprising reacting S)- / V-(3-amino-1 -(hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-

[0089] ((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide with propionic acid to yield the (S)-N-(3-amino-1-(hydroxyamino)-3-methyl-1- oxobutan-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis propionate salt.

[0090] An embodiment of the invention is a method of preparing 4-(4-(((2- methoxyethyl)amino)methyl)phenyl)-2-methylbut-3-yn-2-ol comprising a) reacting 4-bromobenzaldehyde with 2-methoxyethylamine to yield / V-(4- bromobenzyl)-2-methoxyethan-1 -amine b) reacting A / -(4-bromobenzyl)-2-methoxyethan-1 -amine with 2-methyl-3-butyn-2- ol to yield 4-(4-(((2-methoxyethyl)amino)methyl)phenyl)-2-methylbut-3-yn-2-ol.

[0091] EXAMPLES

[0092] HPLC methods:

[0093] Method A

[0094] Agilent Technologies UHPLC / MSD Series 1290 composed of Binary pump G7120A included degasser

[0095] Well plate sampler G4226A

[0096] Column oven G7116B

[0097] Diode array detector G7117B

[0098] Mass detector G6130B Quadrupole LC / MS with ESI-source

[0099] Column: Waters XP, 2.1 x 50mm Xbridge BEH C18 2.5 p, T = 40 °C;

[0100] Eluents:

[0101] A: acetonitrile with 0.05 % (vol. / vol.) formic acid.

[0102] B: water with 0.05 % formic acid (vol. / vol.);

[0103] Flow: 0.8 mL / min;

[0104] Gradient: from 2 to 100 % eluent A 1.2 min, 0.5 min 100 % eluent A;

[0105] Run time: 2.2 min;

[0106] Detection: ESI / MS, positive and negative ions scan: 100-650 m / z;

[0107] UV at 254 and 210 nm;

[0108] Method B

[0109] Agilent Technologies UHPLC / MS 1290 Series composed of:

[0110] Binary pump G4220A included degasser

[0111] Well plate sampler G4226A

[0112] Column oven G7116B

[0113] Diode array detector G7117B

[0114] Mass detector G6130B Quadrupole LC / MS with ESI / APCI multi mode-source

[0115] Column: Waters XP, 2.1 x 50mm Xbridge BEH C18 2.5 p, T = 40 °C;

[0116] Eluents:

[0117] A: acetonitrile.

[0118] B: water with 0.10 % ammonia (vol. / vol.); Flow: 0.8 mL / min;

[0119] Gradient: from 2 to 100 % eluent A 1.2 min, 0.5 min 100 % eluent A;

[0120] Run time: 2.2 min;

[0121] Detection: ESI / APCI / MS, positive ions scan: 100-1000 m / z;

[0122] UV at 254 and 210 nm;

[0123] Method C

[0124] Agilent Technologies UHPLC / MS 1260 Series composed of:

[0125] Binary pump G4220A included degasser

[0126] Well plate sampler G4226A

[0127] Column oven G7116B

[0128] Diode array detector G4212A

[0129] Mass detector G6130B Quadrupole LC / MS with ESI / APCI-multi mode source

[0130] Column: Waters XP, 2.1 x 50mm Xbridge BEH C18 2.5 p, T = 40 °C;

[0131] Eluents: A: acetonitrile with 0.05 % (vol. / vol.) formic acid.

[0132] B: water with 0.05 % formic acid (vol. / vol.);

[0133] Flow: 0.8 mL / min;

[0134] Gradient: from 2 to 100 % eluent A 1.2 min, 0.5 min 100 % eluent A;

[0135] Run time: 2.2 min;

[0136] Detection: ESI / MS, positive and negative ions scan: 100-1000 m / z;

[0137] UV at 254 and 210 nm Example 1A: Preparation of N-(4-bromobenzyl)-2-methoxyethan-1 -amine

[0138] A jacketed glass reactor (2 L) was charged with methanol (600 mL), with 4- bromobenzaldehyde (30 g, 162 mmol) and with 2-methoxyethylamine (22.55 mL, 259 mmol). The resulting mixture was a stirred at room temperature for 14 h. The temperature of the reaction mixture was adjusted between 0 and 5 °C and sodium borohydride (6.75 g, 178 mmol) was added in one portion, while the temperature of the reaction mixture was raised to about 15 °C after completion of the addition. The reaction mixture was stirred at this temperature for 40 min. Aqueous 1 N hydrochloric acid (75 mL) was added and the resulting mixture was stirred for 30 min. The volatiles (about 550 mL) were removed under reduced pressure at 40 °C and the obtained residue was acidified to pH 1 by the addition of aqueous 1 N hydrochloric acid. The aqueous layer was extracted with methyl terf-butylether (2 x 15 mL) and was basified to pH 11 by the addition of aqueous 4M sodium hydroxide (about 15 mL). The aqueous layer was extracted with methyl tert- butylether (3 x 20 mL). The combined organic layers were washed with brine (20 mL) and were concentrated under reduced pressure to afford the desired product as a colorless oil (35.2 g, 143 mmol).

[0139] 1H NMR (300 MHz, Methanol-d4) 5 (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)

[0140] LC / MS (Method A): Rt = 0.616 min, m / z 244, 246

[0141] Variation of Example 1A: Preparation of A / -(4-bromobenzyl)-2-methoxyethan-1- amine A jacketed glass reactor (5 L) was charged with methanol (2 L), with 4- bromobenzaldehyde (200 g, 1080 mmol) and with 2-methoxyethylamine (150 mL, 1730 mmol). The resulting mixture was a 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, while the temperature of the reaction mixture was raised to about 15 °C after completion of the addition. The reaction mixture was stirred at this temperature for 40 min. Aqueous 2N hydrochloric acid (500 mL) was added and the resulting mixture was stirred for 30 min. The volatiles were removed under reduced pressure at 40 °C until a turbid mixture was obtained. The pH was adjusted to pH 1 by the addition of aqueous 2 N hydrochloric acid (750 mL). The aqueous layer was extracted with methyl tert- butylether (2 x 500 mL) and was basified to pH 8 by the addition of aqueous 4M sodium hydroxide (300 mL). The aqueous layer was extracted with cyclopentylmethylether (2 x 500 mL). The combined organic layers were washed with brine (500 mL). The desired product (247 g, 1010 mmol) was obtained in solution in cyclopentylmethylether (1.2 L) which can be engaged in the next step.

[0142] Example 1 B Preparation of 4-(4-(((2-methoxyethyl)amino)methyl)phenyl)-2- methylbut-3-yn-2-ol

[0143] A round bottom flask (1 L) was charged with the solution of A / -(4-bromobenzyl)-2- methoxyethan-1 -amine (247 g, 1010 mmol) in cyclopentylmethylether (30 mL) under inert atmosphere. Copper (I) iodide (578 mg, 3.03 mmol), bis(triphenylphosphine)palladium (II) chloride (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 h. Heating was stopped and the reaction mixture was allowed to reach ambient temperature. Aqueous 4N hydrochloric acid was added (800 mL) and the mixture was filtered over a thin pad of Celite®. The aqueous layer was washed with methyl ferf-butylether (300 mL). The combined organic layers were discarded. The pH of the aqueous layer was adjusted to pH 9 by the addition of aqueous 4M sodium hydroxide (800 mL) and was extracted with cyclopentylmethylether (2 * 500 mL and 200 mL). The combined organic layers were washed with aqueous 10% ammonia solution (500 mL) and with brine (500 mL). The desired product (235 g, 950 mmol) was obtained in solution in cyclopentylmethylether (1.2 L) which can be directly engaged in the next step. Alternatively the solution can be concentrated under reduced pressure to afford the pure desired product as a colorless oil which solidifies upon standing.

[0144] 1H NMR (600 MHz, Methanol-d4) 5 (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)

[0145] LC / MS (Method B): Rt = 0.845 min, m / z 248

[0146] Example 1 C: Preparation of / V-(4-ethynylbenzyl)-2-methoxyethan-1 -aminium chloride

[0147] Potassium hydroxide (42.6 g, 760 mmol) was added to a solution of 4-(4-(((2- methoxyethyl)amino)methyl)phenyl)-2-methylbut-3-yn-2-ol (235 g, 950 mmol) in cyclopentylmethylether (1215 mL) the resulting mixture was stirred at reflux for 5 h until complete conversion was observed. Heating was stopped and reaction mixture was allowed to cool to ambient temperature. The reaction mixture was filtered over a pad of celite which was rinsed with cyclopentylmethylether (100 mL). The filtrate was extracted with water (2 x 500 mL) and the organic layer was collected. The organic layer was washed with water (100 mL) and dried with brine (500 mL). A 4M solution of hydrochloric acid in 2-propanol (285 mL, 1140 mmol) was added to the organic layer and the resulting mixture was stirred for 30 min. The suspension formed was filtered, the filter cake was wshed with cyclopentylmethylether (1000 mL) and the isolated solid was dried under reduced pressure to afford the desired product as a off white solid (182 g, 780 mmol).1H NMR (600 MHz, Methanol-d4) 5 (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, 1 H), 3.43 (s, 3H), 3.24 (t, J =

[0148] 5.1 Hz, 2H)

[0149] LC / MS (Method B): Rt = 0.910 min, m / z 190

[0150] Example 1 D: Preparation of tert-butyl (S)-(4-(hydroxyamino)-3-(4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamido)-2-methyl-4-oxobutan-2- yl)carbamate

[0151] A jacketed glass reactor (500 mL) placed under inert atmosphere was charged with fert-butyl (S)-(3-(4-bromobenzamido)-4-(hydroxyamino)-2-methyl-4-oxobutan- 2-yl)carbamate (20.5 g, 47.6 mmol), / V-(4-ethynylbenzyl)-2-methoxyethan-1 - aminium chloride (9.68 g, 42.9 mmol), tetrakis(triphenylphosphine)palladium (0) (2.75 g, 2.382 mmol) and copper (I) iodide (0.907 g, 4.76 mmol). Dry and degassed dimethylsulfoxide (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 1 h reaction time, the reaction mixture was allowed to reach ambient temperature. Ethyl acetate (800 mL) and aqueous 0.5N hydrochloric acid (800 mL) were added and the resulting mixture was stirred for 15 min. The layers were allowed to settle, the aqueous layer was collected, and the organic layer was extracted with aqueous 0.5N hydrochloric acid (400 mL). The combined aqueous layers were washed with ethyl acetate (200 mL), the pH was adjusted to pH 6 by the addition of aqueous saturated sodium bicarbonate and the aqueous layers were extracted ethyl acetate (2 x 600 mL). The combined organic layers were filtered over a celite pad, extracted with aqueous saturated sodium hydrogencarbonate (200 mL), dried with brine (200 mL), and concentrated under reduced pressure to afford the desired product as a slightly grey solid (20.20 g, 37.1 mmol).qH NMR (300 MHz, Methanol-ck) 6 (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, 1 H), 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)

[0152] LC / MS (Method B): Rt = 0.743 min, m / z 539

[0153] Variation of Example 1 D: Preparation of tert-butyl (S)-(4-(hydroxyamino)-3-(4-((4- (((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamido)-2-methyl-4-oxobutan- 2-yl)carbamate

[0154] A jacketed glass reactor (3 L) placed under inert atmosphere was charged with a solution of te / t-butyl (S)-(3-(4-bromobenzamido)-4-(hydroxyamino)-2-methyl-4- oxobutan-2-yl)carbamate (95 g, 77 wt.%, 170 mmol) in cyclopentylmethylether (530 mL) and the temperature was adjusted to 40°C. A solution of A / -(4- ethynylbenzyl)-2-methoxyethan-1-aminium chloride (43 g, 90 wt.%, 170 mmol) in dimethylsulfoxide (370 mL) was added followed by bis(triphenylphosphine) palladium (II) chloride (2.4 g, 3.4 mmol), copper (I) iodide (0.012 g, 0.63 mmol) and 1 ,8-diazabicyclo(5.4.0)undec-7-ene (77 mL, 510 mmol). The temperature of the contents was adjusted to 70 °C to react for 15h. Heating was stopped and the contents was allowed to reach ambient temperature. Ethyl acetate (180 mL) and aqueous 1 N hydrochloric acid (900 mL) were added and the resulting mixture was stirred for 15 min. The layers were allowed to settle and the aqueous layer was collected. The aqueous layer was washed with ethyl acetate (270 mL), the pH was adjusted to pH 8 by the addition of aqueous saturated sodium carbonate and the aqueous layer was extracted ethyl acetate (2 x 500 mL). The combined organic layers were washed with aqueous saturated sodium hydrogencarbonate (200 mL) and were dried with brine (2 * 200 mL). The desired product (81.8 g, 152 mmol) was obtained as a solution in ethyl acetate which can be engaged in the next step.

[0155] Example 1 E: Preparation of (S)-4-(hydroxyamino)-3-(4-((4-(((2- methoxyethyl)ammonio)methyl)phenyl)ethynyl)benzamido)-2-methyl-4-oxobutan- 2-aminium 4-toluenesulfonate hydrate tert-butyl (S)-(4-(hydroxyamino)-3-(4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamido)-2-methyl-4-oxobutan-2- yl)carbamate (4.881 g, 9.06 mmol) was dissolved in 2-propanol (25 mL), p- toluenesulfonic acid (6.89 g, 36.2 mmol) was added and the mixture heated to 50 °C and was stirred at this temperature for 5 h. Heating was stopped and the reaction mixture was allowed to reach ambient temperature. The obtained suspension was filtered, and content of the filter was rinsed with 2-propanol (20 mL) and dried under reduced pressure at 35 °C. The obtained solid was slurred in 2-propanol (25 mL) at 50 °C. After 40 min, the slurry was cooled to ambient temperature, was filtered off, and dried under reduced pressure at 40 °C to deliver the desired compound as an off white solid (5.23 g, 6.52 mmol).

[0156] 1H NMR (300 MHz, Methanol-d4) 5 (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, 1 H), 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)

[0157] LC / MS (Method A): Rt = 0.744 min, m / z 439 Example 1 F: Preparation of (S)-4-(hydroxyamino)-3-(4-((4-(((2- methoxyethyl)ammonio)methyl)phenyl)ethynyl)benzamido)-2-methyl-4-oxobutan-

[0158] 2-aminium 4-toluenesulfonate hydrate

[0159] (S)-A / -(3-amino-1 -(hydroxyamino)-3-methyl-1 -oxobutan-2-yl)-4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide (50 g, 114 mmol) was dissolved in a mixture of 2-propanol (335 mL) and of water (88 mL). The solution was filtered and transferred into a three-necked flask (1 L) equipped with an overhead stirrer. A solution of p-toluenesulfonic acid (49.1 g, 285 mmol) in 2- propanol (230 mL) was added dropwise under stirring. After addition of half of this solution, seeds of the desired product were added to the mixture to induce precipitation. The reaction mixture was further stirred for 16h to complete precipitation. The obtained suspension was filtered and the obtained solid was dried under reduced pressure at 40 °C to afford the desired product as an off-white solid (77 g, 96 mmol).

[0160] LC / MS (Method B): Rt = 0.715 min, m / z 439

[0161] Example 1 G: Purification / Recrystallization of (S)-4-(hydroxyamino)-3-(4-((4-(((2- methoxyethyl)ammonio)methyl)phenyl)ethynyl)benzamido)-2-methyl-4-oxobutan- 2-aminium 4-toluenesulfonate hydrate

[0162] (S)-4-(hydroxyamino)-3-(4-((4-(((2-methoxyethyl)ammonio)methyl)phenyl) ethynyl)benzamido)-2-methyl-4-oxobutan-2-aminium 4-toluenesulfonate hydrate (46.65 g, 58.24 mmol) was added in portions to a stirring mixture of water (60 mL) and 2-propanol (60 mL) at 60 °C. Heating was stopped after 1 h and the mixture was allowed to reach 30 °C over 90 min. The temperature was lowered to 5 °C and the obtained thick suspension was filtered. The wet cake was rinsed with 2- propanol (50 ml), sucked dry and the wet cake was finally dried under reduced pressure at 40 °C to deliver the desired product as an off-white solid (39.55 g, 49.38 mmol).

[0163] Example 2 - Preparation of the (S)-N-(3-amino-1 -(hydroxyamino)-3-methyl-1 - oxobutan-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis propionate salt

[0164] Ethanol (6.75 mL) was added to (S)- / V-(3-amino-1-(hydroxyamino)-3-methyl-1- oxobutan-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide (0.5 g, 1.140 mmol) and the resulting suspension was stirred at ambient temperature. Heptane (4.5 mL) was added and the resulting mixture was heated to about 30 °C to obtain a clear solution. The solution was cooled to ambient temperature and a solution of propionic acid (0.2 mL, 2.67 mmol) in heptane (4.5 mL) was added dropwise. Additional heptane (14.8 mL) was slowly added and the mixture was stirred at room temperature for 16 h. The obtained fine suspension was filtered under a nitrogen stream over a Nalgene filter paper pore size 0.2 pm and the obtained solid was dried at 40 °C under reduced pressure to deliver the desired product as an off-white solid (544 mg, 0.93 mmol).

[0165] 1H NMR (300 MHz, d3-Dimethylsulfoxide) 5 (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, 1 H), 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,).

[0166] Example 3 - Characterization Description of Physical Characterization Methods

[0167] The crystalline (S)-A / -(3-amino-1 -(hydroxyamino)-3-methyl-1 -oxobutan-2-yl)-4-((4- (((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis tosylate monohydrate salt was characterized by X-ray powder diffraction (XRPD), carbon- 13 solid state NMR (ssNMR), and Differential Scanning Calorimetry (DSC).

[0168] The X-ray powder diffraction (XRPD)

[0169] X-ray powder diffraction studies are widely used to characterize molecular structures, crystallinity, and polymorphism. The X-ray powder diffraction pattern of (S)- / V-(3-amino-1 -(hydroxyamino)-3-methyl-1 -oxobutan-2-yl)-4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis tosylate monohydrate salt was generated on Bruker AXS D8 Advance with a LYNXEYE XE-T detector in reflection mode. The instrument was configured in the Bragg-Brentano configuration and equipped with a Cu radiation source with monochromatization to Ka achieved using a Nickel filter. A fixed slit optical configuration was employed for data acquisition. Data were acquired between 2 and 40° 20. Samples were prepared by gently pressing powdered sample onto a shallow cavity zero background silicon holder.

[0170] Solid State NMR

[0171] In addition to the X-ray powder diffraction pattern described above, (S)-A / -(3- amino-1-(hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis tosylate monohydrate salt was further characterized based on its carbon-13 solid-state nuclear magnetic resonance (NMR) spectrum. A (S)- / V-(3-amino-1-(hydroxyamino)-3-methyl-1- oxobutan-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis tosylate monohydrate salt sample was packed into a Zirconia 4-mm solid-state NMR rotor to capacity and the rotor sealed with a Kel-F® drive tip.13C CPMAS spectrum of the sample was collected on a Bruker AV400 III NMR spectrometer operating at a1H Larmor frequency of 400.120 MHz using a Bruker 4 mm H / F / X BB double resonance MAS probe. An MAS rate of 13 kHz and a pulse delay of 1 .8 s were utilized. During CP, a 100 kHz1H p / 2 excitation pulses was applied, followed by1H lock pulse for which the power was linearly increased from 41 .7 kHz to 83.3 kHz (50% to 100%) over 3 ms. The pulse power for the corresponding13C square pulse was matched to the respective1H ramp to produce maximum signal. High-power TPPM1H decoupling at 100 kHz was applied during data acquisition, while collecting 23200 scans for signal averaging. A Lorentzian line broadening of 30 Hz, zero filling to 32k data points, and standard baselinecorrection methods were applied during processing. Throughout the CP experiment, the sample temperature was controlled with a Bruker VT control unit at 275 K. Based on temperature calibration experiments, a set temperature of 275 K at 13 kHz MAS corresponds to an actual sample temperature of 300 K. The13C CP spectrum was externally referenced using a sample of a-glycine with the carboxyl carbon set to 176.70 ppm. Chemical shifts and the corresponding relative peak intensities were measured with standard peak-picking routines as part of the instrument software.

[0172] Differential Scanning Calorimetry (DSC)

[0173] DSC data was acquired using TA Instruments DSC Q2000 or equivalent instrumentation. A sample with a weight between 1 and 6 mg is weighed into a open pan. This pan was placed in the sample position in the calorimeter cell. An empty pan was placed in the reference position. The calorimeter cell was closed and a flow of nitrogen was passed through the cell. The heating program was set to heat the sample at a heating rate of 10 °C / min to a temperature of approximately 250 °C. When the run was complete, the data were analyzed using the DSC analysis program in the system software. The observed endo- and exotherms were integrated between baseline temperature points that were above and below the temperature range over which the endotherm or exotherm was observed. The data reported included the onset temperature, peak temperature and enthalpy.

[0174] Thermogravimetric analysis was carried out on a TA Q50 Thermogravimetric Analyzer (TA Instrument). Samples (2-15 mg) in open pans were heated from 25 to 300°C at 10°C / min, with a nitrogen purge of 100 mL / min. When the run was complete, the data were analyzed using the TGA analysis program in the system software. The observed weight loss up to a specific temperature was reported. Physical Characterization of (S)-A / -(3-amino-1 -(hydroxyamino)-3-methyl-1 - oxobutan-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis-tosylate monohydrate salt

[0175] FIG. 1 shows the X-ray powder diffraction pattern of (S)- / V-(3-amino-1- (hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis tosylate monohydrate salt. (S)- / V-(3-amino-1-(hydroxyamino)-3-methyl-1 -oxobutan-2-yl)-4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis tosylate monohydrate salt exhibited characteristic diffraction peaks corresponding to d-spacings of 14.07, 10.96 and 9.46 angstroms. (S)- / V-(3-amino-1-(hydroxyamino)-3-methyl-1- oxobutan-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis tosylate monohydrate salt was further characterized by the d-spacings of 7.37, 6.07, 5.48 and 5.23 angstroms. (S)- / V-(3-amino-1-(hydroxyamino)-3-methyl-1- oxobutan-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis tosylate monohydrate salt was even further characterized by the d-spacings of 4.92, 4.55, 4.62 and 3.82 angstroms.

[0176] Table 1. Characteristic Peak Position and Corresponding d-Spacing for (S)- / V-(3- amino-1-(hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis tosylate monohydrate salt

[0177] FIG. 2 is a carbon-13 cross-polarization magic-angle spinning (CPMAS) nuclear magnetic resonance (NMR) spectrum of the crystalline (S)-A / -(3-amino-1- (hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis tosylate monohydrate salt. Peaks labeled with asterisks correspond to spinning sidebands.

[0178] Table 2. Chemical shifts and Relative peak intensities of (S)-A / -(3-amino-1- (hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis tosylate monohydrate salt

[0179] FIG. 3A is a typical DSC curve of (S)- / V-(3-amino-1-(hydroxyamino)-3-methyl-1- oxobutan-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis tosylate monohydrate salt. The DSC curve is characterized by a dehydration endotherm with an extrapolated onset temperature of 47.5°C, a peak temperature of 68.6°C and enthalpy of 51 ,7 / g.

[0180] FIG. 3B is thermal gravimetric analysis thermogram for (S)- / V-(3-amino-1 - (hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis tosylate monohydrate salt. The thermogram showed a weight loss of 2.24% which is close to that expected for the loss of water from theoretical monohydrate (theoretical 2.25%). Significant weight loss was observed above 200°C, indicating decomposition. .

[0181] Physical Characterization of (S)-N-(3-amino-1 -(hydroxyamino)-3-methyl-1 - oxobutan-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis propionate salt. The below analysis were conducted on the Form B polymorph of the (S)-N-(3-amino-1 -(hydroxyamino)-3-methyl-1 -oxobutan-2-yl)-4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis propionate salt.

[0182] FIG. 4 shows the X-ray powder diffraction pattern of (S)-N-(3-amino-1- (hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis propionate salt.

[0183] Table 3. Characteristic Peak Position and Corresponding d-Spacing for (S)-N-(3- amino-1-(hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis propionate salt. (S)-N- (3-amino-1-(hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis propionate salt exhibited characteristic diffraction peaks corresponding to d-spacings of 24.73, 12.34, 11.65. The (S)-N-(3-amino-1-(hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4- ((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis propionate salt was further characterized by d-spacings 8.24, 7.95, 7.75, 6.94, 6.39, 6.17 and 6.03. The (S)-N-(3-amino-1-(hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis propionate salt was even further characterized by d-spacings 5.68, 5.49, 4.93, 4.87, 4.78, 4.69, 3.97, and 3.90

[0184] (S)-N-(3-amino-1-(hydroxyamino)-3-methyl-1 -oxobutan-2-yl)-4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis propionate salt was further characterized based on its carbon-13 solid-state nuclear magnetic resonance (NMR) spectrum. The (S)-N-(3-amino-1-(hydroxyamino)-3-methyl-1- oxobutan-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis propionate salt sample was packed into a Zirconia 4-mm solid-state NMR rotor to capacity and the the rotor sealed with a Kel-F® drive tip.13C CPMAS spectrum of the sample was collected on a Bruker AV400 III NMR spectrometer operating at a1H Larmor frequency of 400.120 MHz using a Bruker 4 mm H / F / X BB double resonance MAS probe. An MAS rate of 13 kHz and a pulse delay of 1 .2 s were utilized. During CP, a 100 kHz1H / 2 excitation pulses was applied, followed by1H lock pulse for which the power was linearly increased from 41 .7 kHz to 83.3 kHz (50% to 100%) over 3 ms. The pulse power for the corresponding13C square pulse was matched to the respective1H ramp to produce maximum signal. High- power TPPM1H decoupling at 100 kHz was applied during data acquisition, while collecting 100000 scans for signal averaging. A Lorentzian line broadening of 30 Hz, zero filling to 32k data points, and standard baseline-correction methods were applied during processing. Throughout the CP experiment, the sample temperature was controlled with a Bruker VT control unit at 270 K. Based on temperature calibration experiments, a set temperature of 270 K at 13 kHz MAS corresponds to an actual sample temperature of 290 K. The13C CP spectrum was externally referenced using a sample of a-glycine with the carboxyl carbon set to 176.70 ppm. Characterisatic13C chemical shifts and the corresponding relative peak intenisties were measured with standard peak-picking routines as part of the instrument software.

[0185] FIG. 5 is a carbon-13 cross-polarization magic-angle spinning (CPMAS) nuclear magnetic resonance (NMR) spectrum of the crystalline of (S)-N-(3-amino-1- (hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis propionate salt. Peaks labeled with asterisks correspond to spinning sidebands.

[0186] Table 4. Chemical shifts and Relative peak intensities of the (S)-N-(3-amino-1- (hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis propionate salt. FIG. 6 is a typical DSC curve of the (S)-N-(3-amino-1-(hydroxyamino)-3-methyl-1 - oxobutan-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis propionate salt. The DSC curve is characterized by an endotherm with an extrapolated onset temperature of 107.8°C, a peak temperature of 119.1 °C and enthalpy of 75.2J / g.

[0187] Example 4 - Stability of crystalline (S)-A / -(3-amino-1 -(hydroxyamino)-3-methyl-1- oxobutan-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis tosylate monohydrate salt and the (S)-N-(3-amino-1-(hydroxyamino)-3-methyl- 1-oxobutan-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis propionate salt comparing to other forms

[0188] Samples of (S)- / V-(3-amino-1 -(hydroxyamino)-3-methyl-1 -oxobutan-2-yl)-4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis tosylate monohydrate salt (listed below as bis-tosylate mono hydrate), the (S)-N-(3-amino-1 - (hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis propionate salt (listed below as the bis propionate), (S)-A / -(3-amino-1 -(hydroxyamino)-3-methyl-1- oxobutan-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide dihydrochloride salt (listed below as the bis HCI salt) and (S)- / V-(3-amino-1 - (hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide (listed below as the free base) were evaluated for stability. The conditions were 40°C and 75% relative humidity (RH). Chemical stability was evaluated by monitoring the %Area of API peak in stability samples. The separation of API main peak from impurity and degradation peaks was achieved on UPLC instrument with C18 column and UV detector.

[0189] The bis-tosylate mono hydrate has better chemical and physical stability comparing to other forms as shown in Tables 6 and 6A.

[0190] Table 6

[0191] Table 6A - Solid state stability study

[0192] Solid state stability studies were performed to assess the physicochemical stability of the free base, bis-tosylate anhydrate, bis-tosylate monohydrate and bis propionate Type B under three stability conditions (25 °C / 60%RH, 40 °C / 75 %RH and 50C or 60 °C, open). Assay purity values are presented in Table 6A.

[0193] For bis tosylate monohydrate:

[0194] 1. Chemically and physically stable at 25 °C / 60%RH, 40 °C / 75%RH and 50 °Cfor 6 months.

[0195] For bis propionate Type B:

[0196] 1. Chemically stable at 25 °C / 60%RH for two weeks

[0197] 2. Decrease in %A purity by -13% at 40 °C / 75%RH in two weeks and loss of crystallinity

[0198] 3. Decrease in %A purity by -5% at 60 °C open conditions in two weeks and physically stable

[0199] Table 6A

[0200] Hygroscopicity was evaluated by dynamic vapor sorption (DVS). The water sorption-desorption isotherms were obtained using a DVS system (Surface Measurement Systems, DVS Adventure). At 25°C, one RH cycle was performed for the sample. In the cycle, RH was raised by 10% per step, from 0% or 30% to 90% and then back to 0% or 30%RH. A rate of change in mass per time unit (dm / dt) of 0.002 wt% / min was set as the equilibrium criteria. Once the criteria was met, the system would hold the set parameters for 10 minutes. The maximum equilibration time was 180 minutes. Figure 7 shows the DVS isotherm plot for the tosylate monohydrate salt. The adsorption phase of the first cycle started at 30%RH and ended at 90% with < 0.3% water uptake. The desorption phase of the first cycle showed water loss at below 10%RH, The second cycle followed the same trace as the first cycle, indicating no form change. Figure 8 shows the DVS isotherm plot for the propionate salt. A water uptake of >14% at 80%RH indicated the salt was hygroscopic. Figure 9 shows the DVS isotherm plot for the anhydrous tosylate salt. In the adsorption phase, the anhydrate adsorbed ~ 2.4% water in one step and at the desorption phase it retained the water at RH above 10%. The large hysterisis effect observed indicated the formation of the hydrate after the adsorption phase.

Claims

CLAIMS1 . A method of preparing (S)-A / -(3-amino-1-(hydroxyamino)-3-methyl-1- oxobutan-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis tosylate monohydrate saltcomprising reacting tert-butyl (S)-(4-(hydroxyamino)-3-(4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamido)-2-methyl-4-oxobutan-2- yl)carbamatewith p-toluenesulfonic acid to yield (S)-A / -(3-amino-1 -(hydroxyamino)-3-methyl-1 - oxobutan-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis tosylate monohydrate salt.

2. A method of preparing (S)- / V-(3-amino-1-(hydroxyamino)-3-methyl-1- oxobutan-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis tosylate monohydrate saltcomprising a) reacting 4-bromobenzaldehyde with 2-methoxyethylamine to yield / V-(4- bromobenzyl)-2-methoxyethan-1 -amineb) reacting / V-(4-bromobenzyl)-2-methoxyethan-1 -amine with 2-methyl-3-butyn-2- ol to yield 4-(4-(((2-methoxyethyl)amino)methyl)phenyl)-2-methylbut-3-yn-2-olc) reacting 4-(4-(((2-methoxyethyl)am ino)methyl)phenyl)-2-methylbut-3-yn-2-ol with potassium hydroxide to yield A / -(4-ethynylbenzyl)-2-methoxyethan-1 -aminium chlorided) reacting / V-(4-ethynylbenzyl)-2-methoxyethan-1-aminium chloride with with tert- butyl (S)-(3-(4-bromobenzamido)-4-(hydroxyamino)-2-methyl-4-oxobutan-2-yl) carbamateto yield tert-butyl (S)-(4-(hydroxyamino)-3-(4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamido)-2-methyl-4-oxobutan-2- yl)carbamatee) reacting tert -butyl (S)-(4-(hydroxyamino)-3-(4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamido)-2-methyl-4-oxobutan-2- yl)carbamate with p-toluenesulfonic acid to yield (S)-A / -(3-amino-1- (hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-((4-(((2- methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis tosylate monohydrate salt.

3. A method of preparing (S)- / V-(3-amino-1-(hydroxyamino)-3-methyl-1-oxobutan- 2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis tosylate monohydrate saltcomprising reacting S)- / V-(3-amino-1 -(hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamidewith p-toluenesulfonic acid to yield (S)- / \ / -(3-amino-1 -(hydroxyamino)-3-methyl-1 - oxobutan-2-yl)-4-((4-(((2-methoxyethyl)arnino)methyl)phenyl)ethynyl)benzamide bis tosylate monohydrate salt.

4. A method of preparing the (S)-N-(3-amino-1-(hydroxyamino)-3-methyl-1- oxobutan-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis propionate saltcomprising reacting S)- / V-(3-amino-1 -(hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4- ((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamidewith propionic acid to yield the (S)-N-(3-amino-1-(hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-((4-(((2-methoxyethyl)amino)methyl)phenyl)ethynyl)benzamide bis propionate salt.

5. A method of preparing 4-(4-(((2-methoxyethyl)amino)methyl)phenyl)-2- methylbut-3-yn-2-olcomprising a) reacting 4-bromobenzaldehyde with 2-methoxyethylamine to yield N-(4- bromobenzyl)-2-methoxyethan-1 -amineb) reacting A / -(4-bromobenzyl)-2-methoxyethan-1 -amine with 2-methyl-3-butyn-2- ol to yield 4-(4-(((2-methoxyethyl)amino)methyl)phenyl)-2-methylbut-3-yn-2-ol.