EA043661B1 - Pim KINASE INHIBITOR SALT - Google Patents

Description

Field of technology to which the invention relates

The invention relates to salt forms of the kinase inhibitor Pim N-{(7R)-4-[(3R,4R,5S)-3-amino-4hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro -5H-cyclopenta[b]pyridin-3-yl}-6-(2,6difluorophenyl)-5-fluoropyridine-2-carboxamide, including methods for their preparation and intermediates in their preparation, wherein the compound is useful in the treatment of Pim kinase-related diseases such as cancer.

State of the art

Increased expression of Pim kinases is detected in a wide range of hematological and solid cancer types. Increased expression of various family members has been seen in multiple myeloma, AML, pancreatic cancer and hepatocellular cancer. Claudio et al., Blood 2002, 100, 217586; Amson et al., Proc. Nat. Acad. Sci. USA, 1989, 86, 8857-61; Mizuki et al., Blood, 2003, 101, 3164-73; Li et al., Canc.. Res., 2006, 66, 6741-7; Fujii et al., Int. J. Canc., 2005, 114, 209-18. Increased expression of Pim1 is associated with poor prognosis in mantle cell lymphoma, esophageal, and head and neck cancers. Hsi et al., Leuk. Lymph., 2008, 49, 2081-90; Liu et al., J. Surg. Oncol., 2010,102, 683-88; Peltola et al., Neoplasia, 2009, 11, 629-36. Increased expression of Pim2 is associated with aggressive disease in a subgroup of patients with DLBCL. Gomez-Abad et al., Blood, 2011, 118, 5517-27. Increased expression is often observed when Myc is overexpressed and Pim kinases can confer resistance to traditional chemotherapeutic agents and radiation. Chen et al., Blood, 2009, 114, 4150-57; Isaac et al., Drug Resis. Updates, 2011, 14, 203-11; Hsu et al., Cancer Lett., 2012, 319, 214; Peltola et al., Neoplasia, 2009, 11, 62936. In this regard, these data indicate that inhibition of Pim kinases will be useful in providing therapeutic benefit in patients with cancer.

Pim kinase inhibitors have been described, for example, in US patents. with publication numbers 2014/0200216, 2014/0200227 and 2015/0057265. In particular, the Pim inhibitory compound N-{4-[(3R,4R,5S)-3-amino-4-hydroxy5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b ]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide (including both diastereomers 7R and 7S) is described in US patent publication number 2014/0200227. Accordingly, new forms of Pim-inhibiting molecules are required to facilitate the development of pharmaceutically useful formulations and dosage forms with suitable properties associated, for example, with facilitating the production of safe, effective, high-quality drugs. The present invention described herein is aimed at achieving this goal.

The essence of the invention

This invention is directed to salts of N-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H- cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5fluoropyridin-2-carboxamide.

This invention is further directed to the phosphoric acid salt N-{(7R)-4-[(3R,4R,5S)3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7- Dihydro-5H-cyclopenta[b]pyridin-3-yl}6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide.

This invention is further directed to crystalline forms of the phosphoric acid salt N-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6 ,7-dihydro-5Hcyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide.

This invention is further directed to the two hydrochloric acid salt N{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6 ,7-dihydro-5Hcyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide.

This invention is further directed to the single hydrochloric acid salt N{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6 ,7-dihydro-5Hcyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide.

This invention is further directed to maleic acid salt, adipic acid salt, hydrobromic acid salt, (I)-(-)-mandelic acid salt, salicylic acid salt, benzoic acid salt, pyroglutamic acid salt, methanesulfonic acid salt, (1S)- salt (+)-10camphorsulfonic acid, fumaric acid salt, sulfuric acid salt, L-tartaric acid salt and D-tartaric acid salt N-{(1R)-4-[(3R,4R,5S)-3-amino-4-hydroxy- 5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide.

The present invention is further directed to the crystalline forms of the salts described herein.

The present invention is further directed to pharmaceutical compositions containing a salt or crystalline form described herein and at least one pharmaceutically acceptable carrier.

The present invention is further directed to therapeutic methods of using the salts and crystalline forms described herein.

The present invention is further directed to methods for preparing the ee salt and crystalline forms described herein.

The present invention is further directed to intermediates useful in the preparation of the salts and crystalline forms described herein.

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Brief description of graphic materials

In fig. Figure 1 shows a X-ray powder diffraction (XRD) diffraction pattern of Form I of the phosphoric acid salt of compound 1.

In fig. Figure 2 shows a DSC thermogram of form I of the phosphoric acid salt of compound 1.

In fig. Figure 3 shows a TGA thermogram of form I of the phosphoric acid salt of compound 1.

In fig. Figure 4 shows the X-ray diffraction pattern of the solid form of the salt with two molecules of hydrochloric acid of compound 1.

In fig. Figure 5 shows a DSC thermogram of the solid form of the salt with two molecules of hydrochloric acid of compound 1.

In fig. Figure 6 shows a TGA thermogram of the solid form of the salt with two molecules of hydrochloric acid of compound 1.

In fig. Figure 7 shows the X-ray diffraction pattern of form II of the phosphoric acid salt of compound 1.

In fig. Figure 8 shows the DSC thermogamut of form II of the phosphoric acid salt of compound 1.

In fig. Figure 9 shows a TGA thermogram of form II of the phosphoric acid salt of compound 1.

In fig. Figure 10 shows the X-ray diffraction pattern of form III of the phosphoric acid salt of compound 1.

In fig. Figure 11 shows a DSC thermogram of form III of the phosphoric acid salt of compound 1.

In fig. Figure 12 shows a TGA thermogram of form III of the phosphoric acid salt of compound 1.

In fig. Figure 13 shows the X-ray diffraction pattern of form IV of the phosphoric acid salt of compound 1.

In fig. Figure 14 shows a DSC thermogram of form IV of the phosphoric acid salt of compound 1.

In fig. Figure 15 shows a TGA thermogram of form IV of the phosphoric acid salt of compound 1.

In fig. Figure 16 shows the X-ray diffraction pattern of form V of the phosphoric acid salt of compound 1.

In fig. Figure 17 shows a DSC thermogram of form V of the phosphoric acid salt of compound 1.

In fig. Figure 18 shows a TGA thermogram of form V of the phosphoric acid salt of compound 1.

In fig. Figure 19 shows the X-ray diffraction pattern of form VI of the phosphoric acid salt of compound 1.

In fig. Figure 20 shows a DSC thermogram of form VI of the phosphoric acid salt of compound 1.

In fig. Figure 21 shows a TGA thermogram of form VI of the phosphoric acid salt of compound 1.

In fig. Figure 22 shows the X-ray diffraction pattern of the solid form of the salt with one molecule of hydrochloric acid of compound 1.

In fig. Figure 23 shows a DSC thermogram of the solid form of the salt with one molecule of hydrochloric acid of compound 1.

In fig. Figure 24 shows the X-ray diffraction pattern of the solid form of the maleic acid salt of compound 1.

In fig. Figure 25 shows a DSC thermogram of the solid form of the maleic acid salt of compound 1.

In fig. Figure 26 shows a TGA thermogram of the solid form of the maleic acid salt of compound 1.

In fig. Figure 27 shows the X-ray diffraction pattern of the solid form of the adipic acid salt of compound 1.

In fig. Figure 28 shows a DSC thermogram of the solid form of the adipic acid salt of compound 1.

In fig. Figure 29 shows a TGA thermogram of the solid form of the adipic acid salt of compound 1.

In fig. Figure 30 shows a X-ray diffraction pattern of the solid form of the hydrobromic acid salt of compound 1.

In fig. Figure 31 shows a DSC thermogram of the solid form of the hydrobromic acid salt of compound 1.

In fig. Figure 32 shows a TGA thermogram of the solid form of the hydrobromic acid salt of compound 1.

In fig. Figure 33 shows the X-ray diffraction pattern of the solid form of the R-(-)-mandelic acid salt of compound 1.

In fig. Figure 34 shows a DSC thermogram of the solid form of the R-(-)-mandelic acid salt of compound 1.

In fig. Figure 35 shows a TGA thermogram of the solid form of the R-(-)-mandelic acid salt of compound 1.

In fig. Figure 36 shows the X-ray diffraction pattern of the solid form of the salicylic acid salt of compound 1.

In fig. Figure 37 shows a DSC thermogram of the solid form of the salicylic acid salt of compound 1.

In fig. Figure 38 shows the X-ray diffraction pattern of the solid form of the benzoic acid salt of compound 1.

In fig. Figure 39 shows the X-ray diffraction pattern of the solid form of the benzenesulfonic acid salt of compound 1.

In fig. Figure 40 shows the X-ray diffraction pattern of the solid form of the L-pyroglutamic acid salt of compound 1.

In fig. Figure 41 shows the X-ray diffraction pattern of the solid form of the methanesulfonic acid salt of compound 1.

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In fig. Figure 42 shows the X-ray diffraction pattern of the solid form of the (1S)-(+)-10camphorsulfonic acid salt of compound 1.

In fig. Figure 43 shows the X-ray diffraction pattern of the solid form of the fumaric acid salt of compound 1.

In fig. Figure 44 shows the X-ray diffraction pattern of the solid form of the sulfuric acid salt of compound 1.

In fig. Figure 45 shows the X-ray diffraction pattern of the solid form of the L-tartaric acid salt of compound 1.

In fig. Figure 46 shows the X-ray diffraction pattern of the solid form of the D-tartaric acid salt of compound 1.

Detailed Description of the Invention

This invention is directed to, inter alia, salts of N-{(1R)-4-[(3R,4R,5S)-3-amino-4-hydroxy5-methylpiperidin-1-yl]-7-hydroxy-6,7- Dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)5-fluoropyridine-2-carboxamide (compound 1), the structure of which is presented below.

But

Connection 1

Compound 1 and its salts are Pim kinase inhibitors useful in the treatment of diseases in which, for example, one or more Pim kinases (eg, Pim1, Pim2 and/or Pim3) are up-regulated. In some embodiments, the salt of Compound 1 provided herein is in solid form. In some embodiments, the present invention provides a solid form containing a salt provided herein. In some embodiments, the solid form is crystalline.

In some embodiments, the salt of this invention is a phosphoric acid salt of Compound 1, such as a single molecule phosphoric acid salt form. The salt form of Compound 1 with one molecule of phosphoric acid is referred to herein as the phosphoric acid salt of Compound 1, Phosphoric Acid Compound 1, or Compound 1 phosphate. An alternative name for the salt is N-{(R)-4-[(3R,4R,5S )-3-amino-4-hydroxy-5methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)- 5fluoropicolinamide phosphate.

In some embodiments, the salt of this invention is a hydrochloric acid salt of Compound 1, such as the two-hydrochloric acid salt form. The salt form of Compound 1 with two molecules of hydrochloric acid is referred to herein as the salt of Compound 1 with two molecules of hydrochloric acid, Compound 1 with two molecules of hydrochloric acid, or the dihydrochloride of Compound 1. An alternative name for the salt is N-{(R)-4-[ (3R,4R,5S)3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}6-(2 ,6-difluorophenyl)-5-fluoropicolinamide dihydrochloride.

In some embodiments, the hydrochloric acid salt of Compound 1 is the one-hydrochloric acid salt of Compound 1. The one-hydrochloric acid salt form of Compound 1 is referred to herein as the one-hydrochloric acid salt of Compound 1, the one-hydrochloric acid salt of Compound 1. , or the monohydrochloride of compound 1. An alternative name for the salt is N-{(R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5methylpiperidin-1-yl]-7-hydroxy-6, 7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5fluoropicolinamide monohydrochloride.

In some embodiments, the salt of this invention is the maleic (cis-butenedioic) acid salt of Compound 1. The maleic acid salt form of Compound 1 is referred to herein as Compound 1 maleic acid salt, Compound 1 with maleic acid, or Compound 1 maleate. By its alternative name for salt is N-{(R)-4-[(3R,4R,5S)-3-amino-4-hydroxy5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[ b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropicolinamide maleate.

In some embodiments, the salt of this invention is the adipic acid salt of Compound 1. The adipic acid salt form of Compound 1 is referred to herein as Compound 1 adipic acid salt, Compound 1 adipic acid, or Compound 1 adipate. An alternative name for the salt is N- {(R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin1 -yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3 -yl}-6-(2,6-difluorophenyl)-5-fluoropicolinamide adipate.

In some embodiments, the salt of this invention is the hydrobromic acid salt of Compound 1. In some embodiments, the hydrobromic acid salt of Compound 1 is the two-molecule hydrobromic acid salt form. In some embodiments, the hydrobromic acid salt of Compound 1 is a single molecule hydrobromic acid salt form. The hydrobromic acid salt form of Compound 1 is referred to herein as the hydrobromic acid salt of Compound 1, Compound 1 with hydrobromic acid, or the hydrobromide of Compound 1. An alternative name for the salt is N-{(R)-4-[(3R,4R,5S)- 3-amino-4-hydroxy-5methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5fluoropicolinamide hydrobromide .

In some embodiments, the salt of this invention is the (R)-(-)mandelic acid salt of Compound 1. In some embodiments, the (R)-(-)-mandelic acid salt of Compound 1 is a single molecule salt (R) -(-)-mandelic acid. In some embodiments, the (D)-(-)-mandelic acid salt of Compound 1 is a salt with two molecules of (R)-(-)mandelic acid. The (D)-(-)-mandelic acid salt form of Compound 1 is referred to herein as Compound 1 mandelic acid salt, Compound 1 with mandelic acid, or Compound 1 mandelate. An alternative name for the salt is N-{(R)-4-[ (3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2 ,6-difluorophenyl)-5-fluoropicolinamide mandelate.

In some embodiments, the salt of this invention is the salicylic acid salt of Compound 1. In some embodiments, the salicylic acid salt of Compound 1 is a single molecule salicylic acid salt. In some embodiments, the salicylic acid salt of Compound 1 is a salt with two salicylic acid molecules. The salicylic acid salt form of Compound 1 is referred to herein as Compound 1 salicylic acid salt, Compound 1 with salicylic acid, or Compound 1 salicylate.

An alternative name for the salt is N-{(R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H- Cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5fluoropicolinamide salicylate.

In some embodiments, the salt of this invention is the benzoic acid salt of Compound 1. In some embodiments, the benzoic acid salt of Compound 1 is the single molecule benzoic acid salt. In some embodiments, the benzoic acid salt of Compound 1 is a salt with two benzoic acid molecules. The benzoic acid salt form of Compound 1 is referred to herein as Compound 1 benzoic acid salt, Compound 1 with benzoic acid, or Compound 1 benzoate. An alternative name for the salt is N-{(R)-4-[(3R,4R,5S)- 3amino-4-hydroxy-5-methylpiperidin-1 -yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3 -yl} -6-(2,6-difluorophenyl)-5- fluoropicolinamide benzoate.

In some embodiments, the salt of this invention is the benzenesulfonic acid salt of Compound 1. In some embodiments, the benzenesulfonic acid salt of Compound 1 is a single molecule benzenesulfonic acid salt. In some embodiments, the benzenesulfonic acid salt of Compound 1 is a salt with two benzenesulfonic acid molecules. The benzenesulfonic acid salt form of Compound 1 is referred to herein as the benzenesulfonic acid salt of Compound 1, Compound 1 with benzenesulfonic acid, or besylate of Compound 1. An alternative name for the salt is N-{(R)-4-[(3R,4R,5S)- 3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5Hcyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5- fluoropicolinamide besylate.

In some embodiments, the salt of this invention is the Lpyroglutamic acid salt of Compound 1. In some embodiments, the Lpyroglutamic acid salt of Compound 1 is a single molecule Lpyroglutamic acid salt. In some embodiments, the L-pyroglutamic acid salt of Compound 1 is a salt with two L-pyroglutamic acid molecules. The L-pyroglutamic acid salt form of Compound 1 is referred to herein as Compound 1 L-pyroglutamic acid salt, Compound 1 with L-pyroglutamic acid, or Compound 1 L-pyroglutamate. An alternative name for the salt is N-{(R)-4-[(3R,4R ,5S)-3amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6(2,6-difluorophenyl) -5-fluoropicolinamide L-pyroglutamate.

In some embodiments, the salt of this invention is the methanesulfonic acid salt of Compound 1. In some embodiments, the methanesulfonic acid salt of Compound 1 is a single molecule methanesulfonic acid salt form. In some embodiments, the methanesulfonic acid salt of Compound 1 is a salt form with two methanesulfonic acid molecules. The methanesulfonic acid salt form of Compound 1 is referred to herein as Compound 1 methanesulfonic acid salt, Compound 1 with methanesulfonic acid, or Compound 1 mesylate. An alternative name for the salt is N-{(R)-4-[(3R,4R,5S)- 3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5- fluoropicolinamide mesylate.

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In some embodiments, the salt of this invention is the (1S)(+)-10-camphorsulfonic acid salt of Compound 1. In some embodiments, the (1S)-(+)10-camphorsulfonic acid salt of Compound 1 is a single molecule salt ( 1S)-(+)-10camphorsulfonic acid. In some embodiments, the (1S)-(+)-10camphorsulfonic acid salt of Compound 1 is a salt with two molecules of (1S)-(+)-10camphorsulfonic acid. The (1S)-(+)-10-camphorsulfonic acid salt form of Compound 1 is referred to herein as (1S)-(+)-10-camphorsulfonic acid salt of Compound 1, Compound 1 with (1S)-(+)-10- camphorsulfonic acid or camsylate of compound 1. An alternative name for the salt is N-{(R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7hydroxy-6 ,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropicolinamide camsylate.

In some embodiments, the salt of this invention is the fumaric (trans-butenedioic) acid salt of Compound 1. The fumaric acid salt form of Compound 1 is referred to herein as Compound 1 fumaric acid salt, Compound 1 with fumaric acid, or Compound 1 fumarate. Alternative name for salt is N-{(R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[ b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropicolinamide fumarate.

In some embodiments, the salt of this invention is the sulfuric acid salt of Compound 1. In some embodiments, the sulfuric acid salt of Compound 1 is the single-molecule sulfuric acid salt form. The sulfuric acid salt form of Compound 1 is referred to herein as Compound 1 sulfuric acid salt, Compound 1 with sulfuric acid, or Compound 1 sulfate. An alternative name for the salt is N-{(R)-4-[(3R,4R,5S)- 3-amino-4-hydroxy-5methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5- fluoropicolinamide sulfate.

In some embodiments, the salt of this invention is the tartaric acid salt of Compound 1, such as the salt form of L-tartaric acid. The L-tartaric acid salt form of Compound 1 is referred to herein as Compound 1 L-tartaric acid salt, Compound 1 with L-tartaric acid, or Compound 1 L-tartrate. An alternative name for the salt is N-{(R)-4-[ (3R,4R,5S)-3-amino-4hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2 ,6-difluorophenyl)5-fluoropicolinamide L-tartrate.

In some embodiments, the tartaric acid salt of Compound 1 is the D-tartaric acid salt of Compound 1. The D-tartaric acid salt form of Compound 1 is referred to herein as. compound 1 D-tartaric acid salt, compound 1 with D-tartaric acid, or compound 1 D-tartrate. An alternative name for the salt is N-{(R)-4-[(3R,4R,5S)-3-amino-4 -hydroxy-5-methylpiperigin-1-yl]-7hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropicolinamide D-tartrate.

The salts of this invention may be isolated in one or more solid forms. As used herein, the phrase solid form refers to a salt of this invention in either an amorphous state or a crystalline state (crystalline form or crystalline solid), wherein the salt of this invention in a crystalline state may optionally include a solvent or water within the crystal lattice , for example, to form a solvated or hydrated crystalline form. The term hydrated as used herein is intended to refer to a crystalline form comprising water molecules in a crystal lattice. For example, hydrated crystalline forms include hemihydrates, monohydrates, dihydrates and the like. Other hydrated forms, such as channel hydrates and the like, are also included within the meaning of the term.

The various crystalline forms of the salts of this invention are characterized by X-ray powder diffractometry (XRD), differential scanning calorimetry (DSC) and/or thermogravimetric analysis (TGA). The pattern of X-ray powder diffractometry (XRD) reflections (peaks) is typically considered characteristic of a particular crystalline form. It is well known that the relative intensities of PRD peaks can vary widely depending on, among other things, the sample preparation technique, the crystal size distribution, the various filters used, the sample mounting procedure, and the specific instrument used. In some cases, new peaks may be observed or existing peaks may disappear depending on the instrument type or settings (for example, whether a Ni filter is used or not). As used herein, the term peak or characteristic peak refers to a reflection having a relative height/intensity of at least about 3% of the maximum height/intensity of the peak. Additionally, tool changes and other factors can affect 2theta values.

Therefore, peak assignments such as those given herein may vary by plus or minus about 0.2° (2-theta), and the term essentially or about when used in the context of PDP herein is intended to refer to the above-mentioned variations.

Likewise, temperature readings associated with DSC, TGA or other thermal experiments can vary by approximately ±3°C depending on the instrument, specific settings, sample preparation, etc. Accordingly, a crystalline form reported herein having a DSC thermogram substantially as shown in either figure is intended to correspond to such variation.

The salts and compounds disclosed herein may include all isotopes of the atoms present therein. Isotopes include atoms that have the same atomic number but different mass numbers. For example, isotopes of hydrogen include tritium and deuterium. The salts and compounds of this invention may also include all isotopes of the atoms present in the intermediates or final compounds. Isotopes include atoms that have the same atomic number but different mass numbers. For example, isotopes of hydrogen include tritium and deuterium. One or more of the constituent atoms of the compounds of this invention may be replaced or replaced by isotopes of atoms common or uncommon in nature. In some embodiments, the compound includes at least one deuterium atom. For example, one or more hydrogen atoms in the compound of this invention may be replaced or replaced by deuterium. In some embodiments, the compound contains two or more deuterium atoms. In some embodiments, the compound contains 1, 2, 3, 4, 5, 6, 7, or 8 deuterium atoms. Synthetic methods for incorporating isotopes into organic compounds are known in the art.

In some embodiments, the compounds of this invention or salts thereof are substantially isolated. By essentially isolated is meant that the compound or salt is at least partially or substantially separated from the environment in which it was formed or discovered. Partial separation may include, for example, a composition enriched with the compounds of this invention. The separation may substantially include compositions containing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95% , at least about 97% or at least about 99% by weight of the compounds or salts of this invention.

The phrase pharmaceutically acceptable is used herein to refer to those compounds, materials, compositions and/or dosage forms that are medically suitable for use in contact with tissues of humans and animals without undue toxicity, irritation, allergic response or other problems or complications. commensurate with a reasonable balance of benefit and risk.

As used herein, the term melting point refers to an endothermic phenomenon or an endothermic phenomenon observed, for example, in a DSC experiment. An endothermic phenomenon is a process or reaction in which a sample absorbs energy from its environment in the form of heat, for example, as in a DSC experiment. An exothermic event is a process or reaction in which a sample releases energy. The process of heat absorption and release can be detected using DSC. In some embodiments, the term melting point is used to describe the underlying endothermic phenomenon identified in a particular DSC thermogram.

The term room temperature as used herein is understood in the art and generally refers to a temperature, for example, a reaction temperature, that is approximately equal to the temperature of the room in which the reaction is carried out, for example, a temperature of from about 20°C to about 30°C.

The term elevated temperature as used herein is understood in the art and generally refers to a temperature, for example a reaction temperature, that is above room temperature, for example above 30°C.

Phosphoric acid salt.

This invention is directed to, inter alia, the phosphoric acid salt N-{(1R)-4-[(3R,4R,5S)3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6 ,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide, such as the salt below.

Phosphoric acid salt of Compound 1

The phosphoric acid salt of Compound 1 may be obtained as an amorphous solid, as a crystalline solid, or as a mixture thereof. In some embodiments, the crystalline solid is Form I, which is described below in the Examples. In some embodiments, the crystalline solid having Form I has a characteristic PDP peak of 2-theta at about 4.6°. In some embodiments, crystalline

- 6 043661 a solid having Form I has at least one characteristic PDP peak, expressed in 2-theta, at about 4.6 or about 9.4°. In some embodiments, the crystalline solid having Form I has at least one characteristic PDP peak, expressed in 2-theta, at about 4.6, about 9.4, or about 13.1°. In some embodiments, the crystalline solid of Form I has two or more characteristic PDP peaks expressed in 2-theta selected from about 4.6, about 9.4, about 13.1, about 16.2, about 17.4, about 17.9, about 18.8, about 19.4, about 21.1, about 23.0 and about 24.8. In some embodiments, the crystalline solid of Form I has three or more characteristic PDP peaks expressed at 2 theta selected from about 4.6, about 9.4, about 13.1, about 16.2, about 17.4, about 17 .9, about 18.8, about 19.4, about 21.1, about 23.0, about 24.8 and about 25.2°. In some embodiments, the Form I crystalline solid has four or more characteristic PDP peaks expressed in 2-theta selected from about 4.6, about 9.4, about 13.1, about 16.2, about 17.4, about 17.9, about 18.8, about 19.4, about 21.1, about 23.0, about 24.8, about 25.2°. In some embodiments, the crystalline solid of Form I has a PXRD pattern substantially as shown in FIG. 1. In some embodiments, the Form I crystalline solid has a melting point of about 250°C. In some embodiments, the Form I crystalline solid has an endotherm of about 198°C or about 250°C. In some embodiments, the Form I crystalline solid has a DSC thermogram substantially as shown in FIG. 2. In some embodiments, the crystalline solid of Form I has a TGA thermogram substantially as shown in FIG. 3.

Advantages of the phosphoric acid salt include high crystallinity, high melting point, stable crystalline form (e.g., Form I), and non-hygroscopic properties, all of which facilitate purification, reproducibility, scale-up, production, and formulation of the drug compound.

In some embodiments, the phosphoric acid salt of Compound 1 is a crystalline solid having Form II. In some embodiments, the crystalline solid having Form II has at least one characteristic PDP peak expressed in 2-theta selected from about 4.7, about 9.4, about 18.8, about 21.7, about 24.8 and about 33.3°. In some embodiments, the crystalline solid having Form II has at least 2 characteristic PDP peaks, expressed in 2-theta, selected from about 4.7, about 9.4, about 18.8, about 21.7, about 24.8 and about 33.3°. In some embodiments, the crystalline solid having Form II has at least 3 characteristic PDP peaks, expressed in 2-theta, selected from about 4.7, about 9.4, about 18.8, about 21.7, about 24.8 and about 33.3°. In some embodiments, the crystalline solid having Form II has at least one characteristic PDP peak expressed in 2-theta selected from about 4.7, about 9.4, about 18.8°. In some embodiments, the crystalline solid having Form II has at least two characteristic PDP peaks expressed in 2-theta selected from about 4.7, about 9.4, about 18.8°. In some embodiments, the crystalline solid having Form II has a PXRD pattern shown in FIG. 7. In some embodiments, the crystalline solid having Form II has a melting point of about 249°C. In some embodiments, the crystalline solid having Form II is endothermic at about 249°C. In some embodiments, the crystalline solid having Form II has a DSC thermogram substantially as shown in FIG. 8. In some embodiments, the crystalline solid having Form II has a TGA thermogram substantially as shown in FIG. 9.

In some embodiments, the phosphoric acid salt of Compound 1 is a crystalline solid having Form III. In some embodiments, the crystalline solid having Form III has at least one characteristic PDP peak expressed in 2-theta selected from about 4.6, about 9.4, about 13.3, about 16.3, about 18.9, about 19.2, about 21.2, about 22.5, about 23.1, about 24.9 and about 26.7°. In some embodiments, the crystalline solid having Form III has at least two characteristic PDP peaks expressed in 2-theta selected from about 4.6, about 9.4, about 13.3, about 16.3, about 18.9, about 19.2, about 21.2, about 22.5, about 23.1, about 24.9 and about 26.7°. In some embodiments, the crystalline solid having Form III has at least three characteristic PDP peaks expressed in 2-theta selected from about 4.6, about 9.4, about 13.3, about 16.3, about 18.9, about 19.2, about 21.2, about 22.5, about 23.1, about 24.9 and about 26.7°. In some embodiments, the crystalline solid having Form III has at least one characteristic PDP peak expressed in 2-theta selected from about 4.6, about 18.9, about 19.2, about 22.5, and about 23.1°. In some embodiments, the crystalline solid having Form III has at least two characteristic PDP peaks expressed in 2-theta selected from about 4.6, about 18.9, about 19.2, about 22.5, and about 23.1°. In some embodiments it is possible to

- 7 043661 a crystalline solid having Form III has at least three characteristic PDP peaks expressed in 2-theta selected from about 4.6, about 18.9, about 19.2, about 22.5 and about 23.1°. In some embodiments, the crystalline solid having Form III has a PXRD pattern shown in FIG. 10. In some embodiments, the crystalline solid having Form III has a melting point of about 250°C. In some embodiments, the Form III crystalline solid is endothermic at about 250°C. In some embodiments, the crystalline solid having Form III has a DSC thermogram substantially as shown in FIG. 11. In some embodiments, the crystalline solid having Form III has a TGA thermogram substantially as shown in FIG. 12.

In some embodiments, the phosphoric acid salt of Compound 1 is a crystalline solid having Form IV. In some embodiments, the crystalline solid having Form IV has at least one characteristic PDP peak expressed in 2-theta selected from about 4.1, about 13.3, about 16.4, about 17.7, about 18, 6, about 19.8, about 21.4 and about 23.3°. In some embodiments, the crystalline solid having Form IV has at least two characteristic PDP peaks expressed in 2-theta selected from about 4.1, about 13.3, about 16.4, about 17.7, about 18.6, about 19.8, about 21.4 and about 23.3°. In some embodiments, the crystalline solid having Form IV has at least three characteristic PDP peaks expressed in 2-theta selected from about 4.1, about 13.3, about 16.4, about 17.7, about 18.6, about 19.8, about 21.4 and about 2 3.3°. In some embodiments, the crystalline solid having Form IV has at least one characteristic PDP peak expressed in 2-theta selected from about 4.1, about 13.3, about 16.4, about 18.6, about 19.8 and about 21.4°. In some embodiments, the crystalline solid having Form IV has at least two characteristic PDP peaks expressed in 2-theta selected from about 4.1, about 13.3, about 16.4, about 18.6, about 19.8 and about 21.4°. In some embodiments, the crystalline solid having Form IV has at least three characteristic PDP peaks expressed in 2-theta selected from about 4.1, about 13.3, about 16.4, about 18.6, about 19.8 and about 21.4°. In some embodiments, the crystalline solid having Form IV has a PXRD pattern shown in FIG. 13. In some embodiments, the crystalline solid having Form IV has a melting point of about 245°C. In some embodiments, the crystalline solid having Form IV has a melting point of about 245°C. In some embodiments, the Form IV crystalline solid has a DSC thermogram substantially as shown in FIG. 14. In some embodiments, the Form IV crystalline solid has a TGA thermogram substantially as shown in FIG. 15.

In some embodiments, the phosphoric acid salt of Compound 1 is a crystalline solid having Form V. In some embodiments, the crystalline solid having Form V has at least one characteristic PDP peak expressed in 2-theta selected from about 7 ,3, about 10.9, about 16.4, about 18.5, about 19.8, about 22.6 and about 26.1°. In some embodiments, the crystalline solid having Form V has at least two characteristic PDP peaks expressed in 2-theta selected from about 7.3, about 10.9, about 16.4, about 18.5, about 19.8, about 22.6 and about 2 6.1°. In some embodiments, the crystalline solid having Form V has at least three characteristic PDP peaks expressed in 2-theta selected from about 7.3, about 10.9, about 16.4, about 18.5, about 19.8, about 22.6 and about 26.1°. In some embodiments, the crystalline solid having Form V has at least one characteristic PDP peak expressed in 2-theta selected from about 7.3, about 10.9, about 16.4, about 18.5, and about 19.8°. In some embodiments, the crystalline solid having Form V has at least two characteristic PDP peaks expressed in 2-theta selected from about 7.3, about 10.9, about 16.4, about 18.5, and about 19.8°. In some embodiments, the crystalline solid having Form V has at least three characteristic PDP peaks expressed in 2-theta selected from about 7.3, about 10.9, about 16.4, about 18.5, and about 19.8°. In some embodiments, the crystalline solid having Form V has a PXRD pattern shown in FIG. 16. In some embodiments, the Form V crystalline solid has an endotherm of about 95°C or about 245°C. In some embodiments, the Form V crystalline solid has a DSC thermogram substantially as shown in FIG. 17. In some embodiments, the Form V crystalline solid has a TGA thermogram substantially as shown in FIG. 18.

In some embodiments, the phosphoric acid salt of Compound 1 is a crystalline solid having Form VI. In some embodiments, the crystal

- 8 043661 personal solid having Form VI has at least one characteristic PDP peak expressed in 2-theta selected from about 6.5, about 8.3, about 10.7, about 13.2, about 17 .3 and about 19.1°. In some embodiments, the crystalline solid having Form VI has at least two characteristic PDP peaks expressed in 2-theta selected from about 6.5, about 8.3, about 10.7, about 13.2, about 17.3 and about 19.1°. In some embodiments, the crystalline solid having Form VI has at least three characteristic PDP peaks expressed in 2-theta selected from about 6.5, about 8.3, about 10.7, about 13.2, about 17.3 and about 19.1°. In some embodiments, the crystalline solid having Form VI has at least one characteristic PDP peak expressed in 2-theta selected from about 6.5, about 8.3, and about 10.7°. In some embodiments, the crystalline solid having Form VI has at least two characteristic 2-theta PDP peaks selected from about 6.5, about 8.3, and about 10.7°. In some embodiments, the crystalline solid having Form VI has a PXRD pattern shown in FIG. 19. In some embodiments, the crystalline solid having Form VI has a melting point of about 86°C. In some embodiments, the crystalline solid having Form VI has an endotherm of about 86°C or about 221°C. In some embodiments, the crystalline solid having Form VI has a DSC thermogram substantially as shown in FIG. 20. In some embodiments, the crystalline solid having Form VI has a TGA thermogram substantially as shown in FIG. 21.

In a certain embodiment, the present invention provides a mixture of crystalline solid Form I and one or more solid forms selected from amorphous, Form II, Form III, Form IV, and Form V. In some embodiments, the mixture of crystalline solid Form I is more than about 50% , about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, or about 99% of Form I.

In a certain embodiment, crystalline solid Form I is obtained in a high degree of purity. Purity values indicate the percentage of the amount of sample that is Form I. Purity values can be determined, for example, by HPLC/UV methods. In some embodiments, Form I is greater than about 90% pure, greater than about 95%, greater than about 97%, greater than about 98%, or greater than about 99%. In some embodiments, Form I is substantially free of impurities, such as organic impurities, inorganic impurities, and/or residual solvents. Examples of organic impurities include, for example, starting materials and process intermediates such as

Examples of organic impurities include, for example, process impurities such as

Examples of inorganic impurities include, for example, heavy metals, palladium and ruthenium. Examples of residual solvents include, for example, acetonitrile, dichloromethane, N,Ndimethylformamide, 1,4-dioxane, n-heptane, methanol and 2-propanol.

Salts of hydrochloric acid.

The present invention further provides a two-hydrochloric acid salt of Compound 1. In some embodiments, a solid form of the two-hydrochloric acid salt

- 9 043661 acid has a PRD diffraction pattern shown in FIG. 4. In some embodiments, the solid form of the salt with two molecules of hydrochloric acid has a melting point of about 213°C. In some embodiments, the solid form of the salt with two hydrochloric acid molecules is endothermic at about 213°C. In some embodiments, the solid form of the salt with two hydrochloric acid molecules has a DSC thermogram substantially similar to that shown in FIG. 5. In some embodiments, the solid form of the salt with two hydrochloric acid molecules has a TGA thermogram substantially as shown in FIG. 6.

In some embodiments, the two hydrochloric acid salt of Compound 1 is a crystalline solid. In some embodiments, the crystalline solid of the two-hydrochloric acid salt of Compound 1 has at least one characteristic PDP peak expressed in 2-theta selected from about 8.3, about 18.9, and about 25.0°. In some embodiments, the crystalline solid of the two hydrochloric acid salt of Compound 1 has two or more characteristic PDP peaks expressed in 2-theta selected from about 8.3, about 18.9, and about 25.0°.

The present invention further provides the one-hydrochloric acid salt of Compound 1. In some embodiments, the solid form of the one-hydrochloric acid salt has a PXRD pattern shown in FIG. 22. In some embodiments, the solid form of the salt with one molecule of hydrochloric acid has a melting point of about 209°C. In some embodiments, the solid salt form with one molecule of hydrochloric acid is endothermic at about 209°C. In some embodiments, the solid form of the salt with one molecule of hydrochloric acid has a DSC thermogram substantially similar to that shown in FIG. 23.

In some embodiments, the one molecule hydrochloric acid salt of Compound 1 is a crystalline solid. In some embodiments, the crystalline solid of the salt of Compound 1 with one molecule of hydrochloric acid has at least one characteristic PDP peak expressed in 2-theta selected from about 7.8, about 8.8, about 12.6, about 14, 5, about 17.4, about 23.8 and about 25.2°. In some embodiments, the crystalline solid of the salt of Compound 1 with one molecule of hydrochloric acid has two or more characteristic PDP peaks expressed in 2-theta selected from about 7.8, about 8.8, about 12.6, about 14.5 , about 17.4, about 23.8 and about 25.2°. In some embodiments, the crystalline solid of the salt of Compound 1 with one molecule of hydrochloric acid has three or more characteristic PDP peaks expressed in 2-theta selected from about 7.8, about 8.8, about 12.6, about 14.5 , about 17.4, about 23.8 and about 25.2°.

Salt of maleic acid.

The present invention further provides a maleic acid salt of Compound 1. In some embodiments, the solid form of the maleic acid salt has a PXRD pattern shown in FIG. 24. In some embodiments, the solid form of the maleic acid salt has a melting point of about 202°C. In some embodiments, the solid form of the maleic acid salt is endothermic at about 202°C. In some embodiments, the solid form of the maleic acid salt has a DSC thermogram substantially as shown in FIG. 25. In some embodiments, the maleic acid salt has a TGA thermogram substantially as shown in FIG. 26.

In some embodiments, the maleic acid salt of Compound 1 is a crystalline solid. In some embodiments, the crystalline solid of the maleic acid salt of Compound 1 has at least one characteristic PDP peak expressed in 2-theta selected from about 9.0, about 9.5, about 11.2, about 14.8, about 15.9, about 18.5, about 19.5, about 19.9, about 21.3, about 22.9, about 24.8, about 25.8, about 27.6 and about 30.9°. In some embodiments, the crystalline solid of the maleic acid salt of Compound 1 has at least two characteristic PDP peaks expressed in 2-theta selected from about 9.0, about 9.5, about 11.2, about 14.8, about 15.9, about 18.5, about 19.5, about 19.9, about 21.3, about 22.9, about 24.8, about 25.8, about 27.6 and about 30.9°. In some embodiments, the crystalline solid of the maleic acid salt of Compound 1 has at least three characteristic PDP peaks expressed in 2-theta selected from about 9.0, about 9.5, about 11.2, about 14.8, about 15.9, about 18.5, about 19.5, about 19.9, about 21.3, about 22.9, about 24.8, about 25.8, about 27.6 and about 30.9°.

Adipic acid salt.

The present invention further provides the adipic acid salt of Compound 1. In some embodiments, the solid form of the adipic acid salt has a PXRD pattern shown in FIG. 27. In some embodiments, the solid form of the adipic acid salt has a melting point of about 182°C. In some embodiments, the solid form of the adipic acid salt has an endotherm of about 150°C or about 182°C. In some embodiments, the solid form of the adipic acid salt has a DSC thermogram substantially as shown in FIG. 28. In some embodiments, the adipic acid salt has a TGA thermogram substantially as shown in FIG. 29.

In some embodiments, the adipic acid salt of Compound 1 is a crystalline solid. In some embodiments, the crystalline solid of the adipic acid salt of Compound 1 has at least one characteristic PDP peak expressed in 2-theta selected from about 9.3, about 15.0, about 16.2, about 17.6, about 18.7, about 20.0, about 22.1, about 22.7, about 24.3, about 24.9, about 27.1 and about 28.7°. In some embodiments, the crystalline solid of the adipic acid salt of Compound 1 has at least two characteristic PDP peaks expressed in 2-theta selected from about 9.3, about 15.0, about 16.2, about 17.6, about 18.7, about 20.0, about 22.1, about 22.7, about 24.3, about 24.9, about 27.1 and about 28.7°. In some embodiments, the crystalline solid of the adipic acid salt of Compound 1 has at least three characteristic PDP peaks expressed in 2-theta selected from about 9.3, about 15.0, about 16.2, about 17.6, about 18.7, about 20.0, about 22.1, about 22.7, about 24.3, about 24.9, about 27.1 and about 28.7°.

Salt of hydrobromic acid.

The present invention further provides the hydrobromic acid salt of Compound 1. In some embodiments, the solid form of the hydrobromic acid salt has a PRD diffractogram shown in FIG. 30. In some embodiments, the solid form of the hydrobromic acid salt has a melting point of about 247°C. In some embodiments, the solid form of the hydrobromic acid salt has a DSC thermogram substantially as shown in FIG. 31. In some embodiments, the solid form of the hydrobromic acid salt has a TGA thermogram substantially as shown in FIG. 32.

In some embodiments, the hydrobromic acid salt of Compound 1 is a crystalline solid. In some embodiments, the crystalline solid of the hydrobromic acid salt of Compound 1 has at least one characteristic PDP peak expressed in 2-theta selected from about 6.5, about 9.5, about 12.9, about 16.6, about 17.9, about 19.5, about 21.7, about 22.5, about 23.7, about 24.3, about 26.5, about 27.5 and about 2 8.3°. In some embodiments, the crystalline solid of the hydrobromic acid salt of Compound 1 has at least two characteristic PDP peaks expressed in 2-theta selected from about 6.5, about 9.5, about 12.9, about 16, 6, about 17.9, about 19.5, about 21.7, about 22.5, about 23.7, about 24.3, about 26.5, about 27.5 and about 28.3°. In some embodiments, the crystalline solid of the hydrobromic acid salt of Compound 1 has at least three characteristic PDP peaks expressed in 2-theta selected from about 6.5, about 9.5, about 12.9, about 16.6, about 17.9, about 19.5, about 21.7, about 22.5, about 23.7, about 24.3, about 26.5, about 27.5 and about 28.3°.

Salt of (R)-(-)-mandelic acid.

The present invention further provides the (R)-(-)-mandelic acid salt of Compound 1. In some embodiments, the solid form of the (R)-(-)-mandelic acid salt has a PXRD pattern shown in FIG. 33. In some embodiments, the solid form of the (R)-(-)-mandelic acid salt has a melting point of about 224°C. In some embodiments, the solid form of the (R)-(-)-mandelic acid salt has an endotherm of about 223°C or about 225°C. In some embodiments, the solid form of the (R)-(-)-mandelic acid salt has a DSC thermogram substantially as shown in FIG. 34. In some embodiments, the solid form of the (R)-(-)-mandelic acid salt has a TGA thermogram substantially as shown in FIG. 35.

In some embodiments, the (R)-(-)-mandelic acid salt of Compound 1 is a crystalline solid. In some embodiments, the crystalline solid of the (R)-(-)-mandelic acid salt of Compound 1 has at least one characteristic PDP peak expressed in 2-theta selected from about 11.2, about 13.8, about 18, 6, about 20.6, about 22.5 and about 24.1°. In some embodiments, the crystalline solid of the (R)-(-)-mandelic acid salt of Compound 1 has at least two characteristic PDP peaks expressed in 2-theta selected from about 11.2, about 13.8, about 18, 6, about 20.6, about 22.5 and about 24.1°. In some embodiments, the crystalline solid of the (R)-(-)-mandelic acid salt of Compound 1 has at least three characteristic PDP peaks expressed in 2-theta selected from about 11.2, about 13.8, about 18, 6, about 20.6, about 22.5 and about 24.1°.

Salicylic acid salt.

In some embodiments, the salt of Compound 1 is a salicylic acid salt. In some embodiments, the solid form of the salicylic acid salt has a PXRD pattern shown in FIG. 36. In some embodiments, solid form

- 11 043661 salicylic acid salt has an endothermic phenomenon of about 180°C or about 208°C. In some embodiments, the solid form of the salicylic acid salt has a DSC thermogram substantially as shown in FIG. 37.

In some embodiments, the salicylic acid salt of Compound 1 is a crystalline solid. In some embodiments, the crystalline solid of the salicylic acid salt of Compound 1 has at least one characteristic PDP peak expressed in 2-theta selected from about 21.2 and about 23.5°. In some embodiments, the crystalline solid of the salicylic acid salt of Compound 1 has at least one characteristic PDP peak expressed in 2-theta selected from about 11.8, about 16.7, about 18.7, about 21.2, about 21.9, about 23.0, about 23.5 and about 24.1°. In some embodiments, the crystalline solid of the salicylic acid salt of Compound 1 has at least two characteristic PDP peaks expressed in 2-theta selected from about 11.8, about 16.7, about 18.7, about 21.2, about 21.9, about 23.0, about 23.5 and about 24.1°. In some embodiments, the crystalline solid of the salicylic acid salt of Compound 1 has at least three characteristic PDP peaks expressed in 2-theta selected from about 11.8, about 16.7, about 18.7, about 21.2, about 21.9, about 23.0, about 23.5 and about 24.1°.

Other salts.

The present invention further provides the benzoic acid salt of Compound 1. In some embodiments, the solid form of the benzoic acid salt has a PXRD pattern shown in FIG. 38. In some embodiments, the benzoic acid salt of Compound 1 is a crystalline solid. In some embodiments, the crystalline solid of the benzoic acid salt of Compound 1 has at least one characteristic PDP peak expressed in 2-theta selected from about 11.6, about 14.9, about 16.9, about 18.8, about 21.5, about 23.2, about 23.7 and about 24.9°. In some embodiments, the crystalline solid of the benzoic acid salt of Compound 1 has at least two characteristic PDP peaks expressed in 2-theta selected from about 11.6, about 14.9, about 16.9, about 18.8, about 21.5, about 23.2, about 23.7 and about 24.9°. In some embodiments, the crystalline solid of the benzoic acid salt of Compound 1 has at least three characteristic PDP peaks expressed in 2-theta selected from about 11.6, about 14.9, about 16.9, about 18.8, about 21.5, about 23.2, about 23.7 and about 24.9°.

The present invention further provides the benzenesulfonic acid salt of Compound 1. In some embodiments, the solid form of the benzenesulfonic acid salt has a PXRD pattern shown in FIG. 39. In some embodiments, the benzenesulfonic acid salt of Compound 1 is a crystalline solid. In some embodiments, the crystalline solid of the benzenesulfonic acid salt of Compound 1 has at least one characteristic PDP peak expressed in 2-theta selected from about 6.6, about 9.1, about 12.9, about 13.3, about 14.5, about 18.0, about 23.5 and about 23.9°. In some embodiments, the crystalline solid of the benzenesulfonic acid salt of Compound 1 has at least two characteristic PDP peaks expressed in 2-theta selected from about 6, 6, about 9.1, about 12.9, about 13.3, about 14.5, about 18.0, about 23.5 and about 23.9°. In some embodiments, the crystalline solid of the benzenesulfonic acid salt of Compound 1 has at least three characteristic PDP peaks expressed in 2-theta selected from about 6, 6, about 9.1, about 12.9, about 13.3, about 14.5, about 18.0, about 23.5 and about 23.9°.

The present invention further provides the L-pyroglutamic acid salt of Compound 1. In some embodiments, the solid form of the L-pyroglutamic acid salt has a PXRD pattern shown in FIG. 40. In some embodiments, the Lpyroglutamic acid salt of Compound 1 is a crystalline solid. In some embodiments, the crystalline solid of the L-pyroglutamic acid salt of Compound 1 has at least one characteristic PDP peak expressed in 2-theta selected from about 4.4, about 10.7, about 11.5, about 18.0 , about 20.7, about 21.2 and about 22.9°. In some embodiments, the crystalline solid of the L-pyroglutamic acid salt of Compound 1 has at least two characteristic PDP peaks expressed in 2-theta selected from about 4.4, about 10.7, about 11.5, about 18.0 , about 20.7, about 21.2 and about 22.9°. In some embodiments, the crystalline solid of the L-pyroglutamic acid salt of Compound 1 has at least three characteristic PDP peaks expressed in 2-theta selected from about 4.4, about 10.7, about 11.5, about 18.0 , about 20.7, about 21.2 and about 22.9°.

The present invention further provides the methanesulfonic acid salt of Compound 1. In some embodiments, the solid form of the methanesulfonic acid salt has a PXRD pattern shown in FIG. 41. In some embodiments, the methanesulfonic acid salt of Compound 1 is a crystalline solid. In some embodiments, a crystalline solid of the methanesulfonic acid salt of Compound 1

- 12 043661 has at least one characteristic PDP peak, expressed in 2-theta, selected from about 13.5, about 14.7, about 16.7, about 18.6, about 19.3, about 20.0, about 20.7, about 22.4, about 2 5.7, about 26.8, about 27.2 and about 28.1°. In some embodiments, the crystalline solid of the methanesulfonic acid salt of Compound 1 has at least two characteristic PDP peaks expressed in 2-theta selected from about 13.5, about 14.7, about 16.7, about 18.6, about 19.3, about 20.0, about 20.7, about 22.4, about 25.7, about 26.8, about 27.2 and about 28.1°. In some embodiments, the crystalline solid of the methanesulfonic acid salt of Compound 1 has at least three characteristic PDP peaks expressed in 2-theta selected from about 13.5, about 14.7, about 16.7, about 18.6, about 19.3, about 20.0, about 20.7, about 22.4, about 25.7, about 26.8, about 27.2 and about 28.1°.

The present invention further provides the (1S)-(+)-10-camphorsulfonic acid salt of Compound 1. In some embodiments, the solid form of the (1S)-(+)-10-camphorsulfonic acid salt has a PXRD pattern shown in FIG. 42. In some embodiments, the (1S)-(+)-10-camphorsulfonic acid salt of Compound 1 is a crystalline solid. In some embodiments, the crystalline solid of the (1S)-(+)-10-camphorsulfonic acid salt of Compound 1 has at least one characteristic PDP peak expressed in 2-theta selected from about 7.1, about 10.9, about 13.6, about 16.1, about 17.7, about 18.8, about 19.9 and about 23.2°. In some embodiments, the crystalline solid of the (1S)-(+)-10-camphorsulfonic acid salt of Compound 1 has at least two characteristic PDP peaks expressed in 2-theta selected from about 7.1, about 10.9, about 13.6, about 16.1, about 17.7, about 18.8, about 19.9 and about 23.2°. In some embodiments, the crystalline solid of the (1S)-(+)-10-camphorsulfonic acid salt of Compound 1 has at least three characteristic PDP peaks expressed in 2-theta selected from about 7.1, about 10.9, about 13.6, about 16.1, about 17.7, about 18.8, about 19.9 and about 23.2°.

The present invention further provides a fumaric acid salt. In some embodiments, the solid form of the fumaric acid salt has a PXRD pattern shown in FIG. 43. In some embodiments, the fumaric acid salt is amorphous.

The present invention further provides a sulfuric acid salt of Compound 1. In some embodiments, the solid form of the sulfuric acid salt has a PXRD pattern shown in FIG. 44. In some embodiments, the sulfuric acid salt is amorphous.

The present invention further provides the L-tartaric acid salt of Compound 1. In some embodiments, the L-tartaric acid salt is amorphous. The solid form of Ltartaric acid salt has a PXRD pattern shown in FIG. 45.

The present invention further provides the D-tartaric acid salt of Compound 1. In some embodiments, the D-tartaric acid salt is amorphous. The solid form of Ltartaric acid salt has a PXRD pattern shown in FIG. 46.

Synthetic production of phosphoric acid salts.

In general, the phosphoric acid salts of this invention can be prepared by coupling N-{(1R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7- hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide (referred to herein as Compound 1 or the free base of Compound 1 ) with phosphoric acid. In some embodiments, the phosphoric acid is provided in molar excess relative to the free base of Compound 1. In some embodiments, the combination of the free base of Compound 1 and phosphoric acid is carried out in the presence of a solvent. In some embodiments, the solvent includes water, methanol, 2-propanol, or a mixture thereof. In some embodiments, the couplings can be carried out at an elevated temperature, such as, for example, from about 40 to about 80, from about 50 to about 70, or from about 55 to about 65°C. In some embodiments, the phosphoric acid salt product of Compound 1 obtained from the combination is substantially crystalline. In some embodiments, the crystalline product contains one or more of Forms I, II, III, IV, V, and VI. In some embodiments, the crystalline product contains Form I. In some embodiments, the crystalline product substantially contains Form I. In some embodiments, the phosphoric acid salt product of Compound 1 obtained from the combination of phosphoric acid with Compound 1 is substantially amorphous or contains an amorphous solid substance.

The free base of compound 1, the phosphate salt precursor, can be prepared by coupling the salt N-{(1R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7 -hydroxy-6,7-dihydro5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide with two molecules of hydrochloric acid (referred to herein as the salt of compound 1 with two molecules of hydrochloric acid or dihydrochloride of compound 1) with sodium carbonate. In some embodiments, the sodium carbonate is provided in molar excess with respect to the dihydrochloride of Compound 1. In some embodiments, the combination of Compound 1 with two molecules of hydrochloric acid and

- 13 043661 sodium carbonate is carried out in the presence of a solvent. In some embodiments, the solvent includes water, methylene chloride, or a mixture thereof. In some embodiments, the coupling is carried out at room temperature or at elevated temperature. Examples of reaction temperatures include from about 20 to about 40, from about 20 to about 30, and from about 23 to about 27°C.

The salt of compound 1 with two molecules of hydrochloric acid can be obtained by introducing into the reaction tert-butyl {(3R,4R,5S)-4-{[tert-butyl(dimethyl)silyl]oxy}-1-[(7R)-7-{ [tert-butyl(dimethyl)silyl]oxy}-3-({[6-(2,6-difluorophenyl)-5-fluoropyridin-2-yl]carbonyl}amino)-6,7-dihydro-5H-cyclopenta[ b]pyridin-4-yl]-5-methylpiperidin-3yl}carbamate (25):

with hydrogen chloride. In some embodiments, hydrogen chloride is provided in a molar excess, such as, for example, between about 2 and about 30, between about 5 and about 25, between about 10 and about 20, or about 15 equivalents. with respect to (25). In some embodiments, the reaction with hydrogen chloride is carried out in the presence of a solvent. In some embodiments, the solvent includes 1,4-dioxane, methanol, or a mixture thereof. In some embodiments, the reaction with hydrogen chloride is carried out at room temperature.

Intermediate (25) can be obtained by reacting tert-butyl ((3R,4R,5S)-1-(3-amino-7-{[tert-butyl(dimethyl)silyl]oxy}-6,7-dihydro-5H-cyclonent[b ]pyridin-4-yl)-4-{[tert-butyl(dimethyl)silyl]oxy} 5-methylpiperidin-3-yl)carbamate:

with 6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxylic acid:

In some embodiments, (24) is provided in a slight molar excess (eg, about 1.1 to about 1.5 equivalents, or about 1.2 equivalents) over (23). In some embodiments, the reaction is carried out in the presence of N,N-diisopropylethylamine (DIEA) and N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (HATU). In some embodiments, DIEA is provided in a molar excess of (23) (eg, about 3 to 6 eq., or about 5 eq.). In some embodiments, HATU is provided in a molar excess relative to (23) (eg, about 1.5 to 3.5 eq., or about 2.4 eq.). In additional embodiments, the reaction is carried out in the presence of a solvent. In some embodiments, the solvent includes dimethylformamide (DMF). In some embodiments, the reaction is carried out at about 10 to about 40, or about 15 to about 30°C.

Intermediate (23) can be obtained by mixing tert-butyl ((3R,4R,5S)-1-((7R)-3(aminocarbonyl)-7-{[tert-butyl(dimethyl)silyl]oxy}-6,7- Dihydro-5H-cyclonenta[b]niridin-4-yl)-4{[tert-butyl(dimethyl)silyl]oxy}-5-methylpiperidin-3-yl)carbamate:

with tetra-N-butylammonium bromide (TBAB) in the presence of sodium hydroxide. In some embodiments, TBAB is provided in an amount of about 1 eq. with respect to (22). In some embodiments, sodium hydroxide is provided in a molar excess (e.g., about 1.1 to about 7 equivalents, or about 2 to about 4 equivalents, or about 4.5 equivalents relative to (22). In some In embodiments, mixing is carried out in the presence of 1,3-dibromo-5,5-dimethylhydantoin.

- 14 043661 In some embodiments, the amount of 1,3-dibromo-5,5-dimethylhydantoin provided is less than 1 eq. (eg, about 0.1 to about 0.9 equivalent, or about 0.3 to about 0.8 equivalent, or about 0.7 equivalent) relative to (22). In some embodiments, mixing is carried out in the presence of a solvent. In some embodiments, the solvent includes tetrahydrofuran (THF). In some embodiments, mixing is carried out at a temperature that is below room temperature, such as from about 0 to about 20, from about 0 to about 15, or from about 5 to about 10°C.

Intermediate (22) can be obtained by introducing tert-butyl [(3R,4R,5S)-4-{[tert-butyl(dimethyl)silyl]oxy}-1-((7R)-7-{[tert-butyl(dimethyl )silyl]oxy}-3-cyano-6,7-dihydro-5H-cyclopenta[b]pyridin-4-yl)-5-methylpiperidin-3-yl]carbamate:

OTBS BosHM-A^ ay s N _T BSO ₂₁ with acetaldoxime. In some embodiments, the reaction with acetaldoxime is carried out in the presence of a palladium catalyst such as [1,1'-bis-(diphenylphosphino)ferrocene]dichloropalladium(P) (Pd(dppf) ₂ Cl ₂ complexed with dichloromethane. In some embodiments, acetaldoxime is provided in molar excess (e.g., about 2 to about 20 equivalents, about 5 to about 15 equivalents, or about 10 equivalents). In some embodiments, the total amount of acetaldoxime is added to the reaction mixture in portions. In some embodiments, the reaction with acetaldoxime is carried out in the presence of a solvent. In some embodiments, the solvent includes water, ethanol, or a mixture thereof. In some embodiments, the reaction is carried out at an elevated temperature (for example, from about 50 to about 150, from about 70 to about 100, or about 90°C) .

Intermediate (21) can be obtained by introducing (7R)-7-{[tert-butyl(dimethyl)silyl]oxy}4-chloro-6,7-dihydro-5H-cyclopenta[b]pyridine-3-carbonitrile into the reaction:

C.I.

Ou t N

TBSO with tert-butyl ((3R,4R,5S)-4-{[tert-butyl(dimethyl)silyl]oxy}-5-methylpiperidin-3-yl)carbamate (9), or its hydrochloric acid salt (9 HCl ):

in the presence of K,K-diisopropylethylamine (DIEA). In some embodiments, intermediate (9) is provided in a slight molar excess (eg, 1.05 eq) relative to (20). In some embodiments, DIEA is provided in a molar excess (eg, about 2 to about 6 equivalents, or about 4 equivalents) relative to (20). In some embodiments, the reaction of (20) with (9) is carried out in the presence of a solvent. In some embodiments, the solvent includes dimethyl sulfoxide. In some embodiments, the reaction is carried out at room temperature or at elevated temperature (eg, about 30 to about 150, or about 100°C).

The intermediate product (R)-7-(tert-butyldimethylsilyloxy)-4-chloro-6,7-dihydro-5H-cyclopenta[b]pyridine3-carbonitrile (20) can be obtained by introducing into the reaction (R)-7-(tert-butyldimethylsilyloxy )-4-chloro6,7-dihydro-5H-cyclopenta[b]pyridine-3-carbaldehyde (19):

CI o

TBSO with ammonia and iodine. In some embodiments, the reaction is carried out in the presence of a solvent. In some embodiments, the solvent includes water, THF, or a mixture thereof. In some embodiments, the reaction is carried out at a temperature below room temperature, for example between about 10 and 22°C. In some embodiments, ammonia and iodine are provided in molar excess.

The intermediate (R)-7-(tert-butyldimethylsilyloxy)-4-chloro-6,7-dihydro-5H-cyclopenta[b]pyridine3-carbaldehyde (19) can be prepared by coupling (R)-7-(tert-butyldimethylsilyloxy) -4-chloro-6,7dihydro-5H-cyclopenta[b]pyridine (18):

- 15 043661

with n-butyllithium in the presence of 2,2,6,6-tetramethylpiperidine, followed by the addition of N,Ndimethylformamide (DMF). In some embodiments, the coupling is carried out below room temperature, for example -100 to -10°C. In some embodiments, the coupling is carried out in a solvent. In some embodiments, the solvent includes THF, hexane, or a mixture thereof.

The intermediate product (R)-7-(tert-butyldimethylsilyloxy)-4-chloro-6,7-dihydro-5H-cyclopenta[b]pyridine (18) can be obtained by introducing into the reaction (R)-4-chloro-6,7- Dihydro-5H-cyclopenta[b]pyridin-7-ol (17):

with tert-butyldimethylsilyl chloride and 1H-imidazole. In some embodiments, the reaction is carried out at a temperature below room temperature, such as from about -15 to 15 or from about -15 to 0°C. In some embodiments, the reaction is carried out in the presence of a solvent, such as methylene chloride or other organic solvent. In some embodiments, tert-butyldimethylsilyl chloride is provided in an amount of about 1 eq. in relation to (17). In some embodiments, 1H-imidazole is provided in a molar excess relative to (17).

The intermediate (R)-4-chloro-6,7-dihydro-5H-cyclopenta[b]pyridin-7-ol (17) can be obtained by introducing 4-chloro-5H-cyclopenta[b]pyridin-7(6H) into the reaction -she (16):

with formic acid in the presence of RuCl(p-cymene)[(R,R)-Ts-DPEN] and triethylamine (TEA). In some embodiments, the reaction is carried out in the presence of a solvent, such as methylene chloride or other organic solvent. In some embodiments, the reaction is carried out at a temperature below room temperature. In some embodiments, RuCl(p-cymene)[(R,R)-Ts-DPEN] is provided in a catalytic amount. In some embodiments, TEA is provided in a molar excess relative to (16). In some embodiments, formic acid is provided in a molar excess relative to (16).

The intermediate product 4-chloro-5H-cyclopenta[b]pyridin-7(6H)-one (16) can be obtained by introducing 4-chloro-6,7-dihydro-5H-cyclopenta[b]pyridin-7-ol (15) into the reaction ):

with pyridine-sulfur trioxide in the presence of N,N-diisopropylethylamine. In some embodiments, (15) and N,N-diisopropylethylamine are combined before adding pyridine sulfur trioxide. In some embodiments, the reaction is carried out in the presence of a solvent. In some embodiments, the solvent includes methylene chloride or other organic solvent. In some embodiments, the reaction is carried out below room temperature, for example, about 0°C. In some embodiments, N,N-diisopropylethylamine is provided in a molar excess relative to (15). In some embodiments, pyridine sulfur trioxide is provided in a molar excess relative to (15).

The intermediate product 4-chloro-6,7-dihydro-5H-cyclopenta[b]pyridin-7-ol (15) can be obtained by introducing 4-chloro-6,7-dihydro-5H-cyclopenta[b]pyridin-7- yl acetate (14):

where Ac represents acetyl, with potassium carbonate. In some embodiments, the reaction is carried out in the presence of a solvent, such as methanol, water, another polar solvent, or a mixture thereof. In some embodiments, the reaction is carried out below room temperature, for example, about 0°C. In some embodiments, the potassium carbonate is provided in a molar excess relative to (14).

- 16 043661

The intermediate product 4-chloro-6,7-dihydro-5H-cyclopenta[b]pyridin-7-ylacetate (14) can be obtained by introducing 4-chloro-6,7-dihydro-5H-cyclopenta[b]pyridin-1- into the reaction oxide (13):

with acetic anhydride. In some embodiments, the reaction is carried out in the presence of an organic solvent, where the organic solvent includes toluene or other non-polar solvent. In some embodiments, the reaction is carried out at an elevated temperature, such as from about 50 to about 150, or from about 70 to about 90, or from about 80 to about 85°C. In some embodiments, acetic anhydride is provided in molar excess relative to (13).

The intermediate 4-chloro-6,7-dihydro-5H-cyclopenta[b]pyridine-1-oxide (13) can be obtained by introducing 4-chloro-6,7-dihydro-5H-cyclopenta[b]pyridine (12) into the reaction :

with urea peroxide (UHP) in the presence of a catalyst. In some embodiments, the catalyst is a transition metal catalyst such as methyltrioxorenium (MT). In some embodiments, the catalyst is provided in a catalytic amount (eg, <0.1 equivalent relative to (12)). In some embodiments, the reaction is carried out in the presence of a solvent. In some embodiments, the solvent includes methanol or other polar solvent. In some embodiments, the reaction is carried out at room temperature. In some embodiments, UHP is provided in an amount of about 1 to about 2 equivalents, or in an amount of about 1.5 equivalents. in relation to (12).

The intermediate product 4-chloro-6,7-dihydro-5H-cyclopenta[b]pyridine (12) can be obtained by introducing 6,7-dihydro-5H-cyclopenta[b]pyridine-1-oxide (11) into the reaction:

CO

N ⁺ 0' 11 with phosphorus oxychloride. In some embodiments, phosphorus oxychloride is provided in an amount of about 2-4 eq., or about 3 eq. in relation to (11). In some embodiments, the reaction is carried out in the presence of an organic solvent, where the organic solvent includes, for example, toluene. In some embodiments, the reaction is carried out at an elevated temperature, such as from about 50 to about 100, or from about 70 to about 90, or from about 80 to about 85°C.

The intermediate 6,7-dihydro-5H-cyclopenta[b]pyridine-1-oxide (11) can be obtained by introducing 6,7-dihydro-5H-cyclopenta[b]pyridine (10) into the reaction:

CO N with urea peroxide UHP in the presence of a catalyst. In some embodiments, the catalyst is a transition metal catalyst such as methyltrioxorenium (MT). In some embodiments, the catalyst is provided in a catalytic amount (eg, <0.1 equivalent relative to (10)). In some embodiments, the reaction is carried out at about room temperature. In some embodiments, UHP is provided in an amount of about 2-4 eq. or about 3 eq. in relation to (10). In some embodiments, the reaction is carried out in the presence of a solvent, such as a solvent including methanol or other polar organic solvent. In some embodiments, the present invention provides an intermediate selected from:

where TBS is tert-butyl(dimethyl)silyl.

- 17 043661

In some embodiments, the present invention provides an intermediate selected from:

where TBS is tert-butyl(dimethyl)silyl and Boc is butyloxycarbonyl.

In some embodiments, the intermediate is a tertiary

In some embodiments, the intermediate is

In some embodiments, the intermediate is

where TBS is tert-butyl(dimethyl)silyl.

In some embodiments, the intermediate is

where TBS is tert-butyl(dimethyl)silyl.

In some embodiments, the intermediate is

where TBS is tert-butyl(dimethyl)silyl.

In some embodiments, the intermediate is

where TBS is tert-butyl(dimethyl)silyl and Boc is butyloxycarbonyl.

In some embodiments, the intermediate is a tertiary

where TBS is tert-butyl(dimethyl)silyl and Boc is butyloxycarbonyl.

tert- 18 043661

In some embodiments, the intermediate is

where TBS is tert-butyl(dimethyl)silyl and Boc is butyloxycarbonyl.

In some embodiments, the intermediate is a tertiary

where TBS is tert-butyl(dimethyl)silyl and Boc is tert-butyloxycarbonyl.

In some embodiments, the present invention provides a method for preparing the phosphoric acid salt of Compound 1, comprising:

reacting compound 10 with urea peroxide and methyltrioxorenium (UT) to form compound 11;

reacting compound 11 with phosphorus oxychloride to form compound 12;

reacting compound 12 with urea peroxide and methyltrioxorenium (UT) to form compound 13;

reacting compound 13 with acetic anhydride to form compound 14;

reacting compound 14 with potassium carbonate to form compound 15;

reacting compound 15 with pyridine-sulfur trioxide in the presence of N,Ndiisopropylethylamine to form compound 16;

reacting compound 16 with formic acid in the presence of RuCl(p-cymene)[(R,R)-TsDPEN] and triethylamine (TEA) to form compound 17;

reacting compound 17 with tert-butyldimethylsilyl chloride and 1H-imidazole to form compound 18;

coupling of compound 18 with n-butyllithium in the presence of 2,2,6,6-tetramethylpiperidine followed by the addition of N,N-dimethylformamide (DMF) to form compound 19;

reacting compound 19 with ammonia and iodine to form compound 20;

reacting compound 20 with compound 9 or its hydrochloric acid salt in the presence of N,N-diisopropylethylamine (DIEA) to form compound 21;

reacting compound 21 with acetaldoxime to form compound 22;

mixing compound 22 with tetra-N-butylammonium bromide (TBAB) in the presence of sodium hydroxide to form compound 23;

reaction of compound 23 with compound 24 to form compound 25;

reacting compound 25 with hydrogen chloride to form a salt of compound 1 with two molecules of hydrochloric acid;

combining a salt of compound 1 with two molecules of hydrochloric acid with sodium carbonate to form the free base of compound 1; and combining the free base of Compound 1 with phosphoric acid to form the phosphoric acid salt of Compound 1.

In some embodiments, the present invention provides a method for preparing the phosphoric acid salt of Compound 1, comprising:

reacting compound 19 with ammonia and iodine to form compound 20;

reacting compound 20 with compound 9 or its hydrochloric acid salt in the presence of N,N-diisopropylethylamine (DIEA) to form compound 21;

reacting compound 21 with acetaldoxime to form compound 22;

mixing compound 22 with tetra-N-butylammonium bromide (TBAB) in the presence of sodium hydroxide to form compound 23;

reaction of compound 23 with compound 24 to form compound 25;

reacting compound 25 with hydrogen chloride to form a salt of compound 1 with two molecules of hydrochloric acid;

combining a salt of compound 1 with two molecules of hydrochloric acid with sodium carbonate to form the free base of compound 1; And

- 19 043661 combination of the free base of compound 1 with phosphoric acid to form the phosphoric acid salt of compound 1.

In some embodiments, the present invention provides a method for preparing compound 20, comprising:

reacting compound 10 with urea peroxide and methyltrioxorenium (VI) to form compound 11;

reacting compound 11 with phosphorus oxychloride to form compound 12;

reacting compound 12 with urea peroxide and methyltrioxorenium (VI) to form compound 13;

reacting compound 13 with acetic anhydride to form compound 14;

reacting compound 14 with potassium carbonate to form compound 15;

reacting compound 15 with pyridine-sulfur trioxide in the presence of N,Ndiisopropylethylamine to form compound 16;

reacting compound 16 with formic acid in the presence of RuCl(p-cymene)[(R,R)-TsDPEN] and triethylamine (TEA) to form compound 17;

reacting compound 17 with tert-butyldimethylsilyl chloride and 1H-imidazole to form compound 18;

coupling of compound 18 with n-butyllithium in the presence of 2,2,6,6-tetramethylpiperidine followed by the addition of N,N-dimethylformamide (DMF) to form compound 19; and reacting compound 19 with ammonia and iodine to form compound 20.

As used herein, the term reacting is used as is known in the art and generally refers to combining chemical reactants in such a way as to cause them to interact at the molecular level to achieve a chemical or physical transformation. In some embodiments, the implementation of the reaction includes at least two reactants. In some embodiments, the reaction step of the synthetic process may include, in addition to the reagents, one or more substances, such as a solvent and/or a catalyst. The reaction step of the synthetic method described herein can be carried out at a time and under conditions suitable to obtain a particular product. The terms combination and mixing in relation to the reactants of a chemical reaction are used interchangeably with the term reacting in this document. The term reaction may also be considered interchangeable with reacting, but may be applied to a reaction step involving cross-linking of two organic moieties.

The processes described herein can be monitored in accordance with any suitable method known in the art. For example, product formation can be monitored by spectroscopic means such as nuclear magnetic resonance spectroscopy (eg, 1H or ^13C ), infrared spectroscopy, spectrophotometry (eg, UV-vis), or mass spectrometry; or chromatography such as high performance liquid chromatography (HPLC) or thin layer chromatography. The compounds produced by the reactions can be purified by any suitable method known in the art. For example, chromatography (medium pressure) on a suitable adsorbent (eg, silica gel, alumina and the like), HPLC or preparative thin layer chromatography; distillation; by sublimation, trituration or recrystallization. The purity of compounds is generally determined by physical methods, such as measuring the melting point (in the case of a solid), obtaining an NMR spectrum, or performing HPLC separation. If the melting point decreases, if unwanted signals in the NMR spectrum are reduced, or if extraneous peaks in the HPLC curve are removed, the compound can be said to have been purified. In some embodiments, the compounds are substantially purified.

Preparation of compounds may involve protection and deprotection of various chemical groups. One skilled in the art can readily determine the need for protection and deprotection and the selection of appropriate protecting groups. Protecting group chemistry is described, for example, in Wuts and Greene, Greene's Protective Groups in Organic Synthesis, ^4th Ed., John Wiley & Sons: New York, 2006, which is incorporated herein by reference in its entirety.

The reactions of the methods described herein can be carried out at appropriate temperatures, which can be readily determined by one skilled in the art. Reaction temperatures will depend, for example, on the melting and boiling points of the reactants and the solvent, if present; reaction thermodynamics (for example, intensely exothermic reactions may need to be carried out at lower temperatures); and reaction kinetics (e.g., a high activation energy barrier may require elevated temperatures). Fever refers to temperatures above room temperature (about 22°C).

The reactions of the methods described herein can be carried out in suitable solvents, which can be readily selected by one skilled in the art of organic synthesis. Suitable solvents may be substantially unreactive with respect to the starting materials (reagents), intermediates or products at the temperatures at which the reactions are carried out,

- 20 043661

those. temperatures that can vary from the freezing point of the solvent to the boiling point of the solvent. The reaction can be carried out in one solvent or a mixture of more than one solvent. Depending on the reaction step, the suitable solvent(s) for that particular reaction step can be selected. Suitable solvents include water, alkanes (such as pentanes, hexanes, heptanes, cyclohexane, etc., or a mixture thereof), aromatic solvents (such as benzene, toluene, xylene, etc.), alcohols (such as methanol, ethanol, isopropanol, etc.), ethers (such as dialkyl ethers, methyl tert-butyl ether (MTBE), tetrahydrofuran (THF), dioxane, etc.), esters (such as ethyl acetate, butyl acetate and etc.), halogenated hydrocarbon solvents (such as dichloromethane (DCM), chloroform, dichloroethane, tetrachloroethane), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), acetone, acetonitrile (ACN), hexamethylphosphoramide (HMPA) and N-methylpyrrolidone ( NMP). Such solvents can be used in their aqueous or anhydrous forms.

The separation of racemic mixtures of compounds can be accomplished by any of numerous methods known in the art. A typical method involves fractional recrystallization using a chiral resolving acid, which is an optically active salt-forming organic acid. Suitable separating agents for fractional recrystallization processes are, for example, optically active acids such as the D- and L-forms of tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid or various optically active camphorsulfonic acids. The separation of racemic mixtures can also be accomplished by elution on a column packed with an optically active separating agent (eg dinitrobenzoylphenylglycine). A suitable composition of the elution solvent can be determined by one skilled in the art.

Preparation of crystalline forms I-VI of phosphate salt.

Form I can be prepared, for example, by precipitation of the solid form from a solution containing the phosphoric acid salt N-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1- yl]-7hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide. In some embodiments, the solution contains a solvent including 2-propanol. In other embodiments, the solution contains a solvent including acetonitrile and/or ethanol. The deposition can be carried out at any suitable temperature, such as around room temperature or at elevated temperature. Precipitation can be accomplished by any means that concentrate the solution, such as evaporation, addition of an antisolvent, or cooling.

Form II can be prepared, for example, by precipitation of the solid form from a solution of the phosphoric acid salt N-{(1R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl] -7-hydroxy-6,7-dihydro5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide in a solvent containing dimethylformamide (DMF). Precipitation can be accomplished by any means that concentrate the solution, such as evaporation, addition of an antisolvent, or cooling. In some embodiments, precipitation is accomplished by evaporation in air around room temperature (eg, about 25°C).

Form III can be prepared by precipitation of the solid form from a solution of the phosphoric acid salt N-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7- hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide in a solvent containing dimethylformamide (DMF), whereby precipitation can be carried out at an elevated temperature, such as from about 30°C to about 70°C, from about 40°C to about 60°C, or at about 50°C.

Form IV can be prepared by precipitation of the solid form from an aqueous solution of the phosphoric acid salt N-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7 -hydroxy-6,7-dihydro5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide. In some embodiments, precipitation is accomplished by evaporation in air at an elevated temperature, such as, for example, between about 30°C and about 70°C, between about 40°C and about 60°C, or between about 45°C and about 55 °C.

Form V can be prepared by precipitation of the solid form from an aqueous solution of the phosphoric acid salt N-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7 -hydroxy-6,7-dihydro5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide by cooling the solution. In some embodiments, the aqueous solution is at a temperature of about 30°C to about 40°C, or about 35°C, before cooling. In some embodiments, the solution is cooled to about 4-5°C. In some embodiments, cooling is accomplished by rapid cooling.

Form VI can be prepared by filtering a slurry of the phosphoric acid salt N-{(7R)-4-[(3R,4R,5S)-3amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7 -dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide in water to obtain a filtrate and then cool the filtrate (for example, to below about 10 °C, or up to about 4-5°C).

Synthetic production of other salt forms.

A salt with two hydrochloric acid molecules can be obtained, for example, by combining N-{(7R)-4

- 21 043661

[(3R,4R,5S)-3-αmino-4-hydroxy-5-methylnineridin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopentα[b]pyridin-3yl}-6-( 2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide (free base of compound 1) with hydrochloric acid. In some embodiments, the coupling is carried out in the presence of a solvent, such as a solvent containing 2-propanol and/or 2-propyl acetate. In some embodiments, the hydrochloric acid is provided in molar excess with respect to the free base of Compound 1. In some embodiments, the molar ratio of the free base of Compound 1 to hydrochloric acid is from about 1:2 to about 1:2.5. In some embodiments, the ratio of Compound 1 free base to hydrochloric acid is about 1:2.34.

A salt with one molecule of hydrochloric acid can be obtained, for example, by combining N-{(7R)-4[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy -6,7-dihydro-5Hcyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide (free base of compound 1) with hydrochloric acid. In some embodiments, the coupling is carried out in the presence of a solvent, such as a solvent containing 2-propanol and/or 2-propyl acetate. In some embodiments, the molar ratio of Compound 1 free base to hydrochloric acid is from about 1:1 to about 1:1.5. In some embodiments, the molar ratio of Compound 1 free base to hydrochloric acid is about 1:1.12.

The maleic acid salt can be prepared, for example, by coupling N-{(7R)-4-[(3R,4R,5S)-3amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7 -dihydro-5H-cyclopenta[b]pyridin-3-yl}-6(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide (free base of compound 1) with maleic acid. In some embodiments, the coupling is carried out in the presence of a solvent, such as a solvent containing 2-propanol. In some embodiments, the molar ratio of Compound 1 free base to maleic acid is from about 1:1 to about 1:1.5. In some embodiments, the molar ratio of Compound 1 free base to maleic acid is about 1:1.21. In some embodiments, the method includes adding crystal seeds to initiate deposition.

The adipic acid salt can be prepared, for example, by coupling N-{(7R)-4-[(3R,4R,5S)-3amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7 -dihydro-5H-cyclopenta[b]pyridin-3-yl}-6(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide (free base of compound 1) with adipic acid. In some embodiments, the coupling is carried out in the presence of a solvent, such as a solvent containing 2-propanol and/or heptane. In some embodiments, the molar ratio of Compound 1 free base to adipic acid is from about 1:2 to about 1:3. In some embodiments, the molar ratio of Compound 1 free base to adipic acid is about 1:2.49.

Hydrobromic acid salts can be prepared, for example, by coupling N-{(7R)-4-[(3R,4R,5S)-3amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7 -dihydro-5H-cyclopenta[b]πpyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide (free base of compound 1) with hydrobromic acid. In some embodiments, the coupling is carried out in the presence of a solvent, such as a solvent containing 2-propanol and/or water. In some embodiments, the molar ratio of Compound 1 free base to hydrobromic acid is from about 1:2 to about 1:3 at the time of coupling. In some embodiments, the molar ratio of Compound 1 free base to hydrobromic acid is about 1:2.4. In some embodiments, the hydrobromic acid salt is a salt with two hydrobromic acid molecules. In further embodiments, the hydrobromic acid salt is a salt with one molecule of hydrobromic acid.

The mandelic acid salt can be prepared, for example, by coupling N-{(7R)-4-[(3R,4R,5S)-3amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7 -dihydro-5H-cyclopenta[b]pyridin-3-yl}-6(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide (free base of compound 1) with (R)-(-)mandelic acid. In some embodiments, the coupling is carried out in the presence of a solvent, such as a solvent containing 2-propanol. In some embodiments, the molar ratio of Compound 1 free base to (R)-(-)-mandelic acid is from about 1:1 to about 1:1.5 at the time of coupling. In some embodiments, the molar ratio of Compound 1 free base to (R)-(-)-mandelic acid is about 1:1.06.

The salicylic acid salt can be prepared, for example, by coupling N-{(7R)-4-[(3R,4R,5S)-3amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7 -dihydro-5H-cyclopenta[b]pyridin-3-yl}-6(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide (free base of compound 1) with salicylic acid, optionally in the presence of a solvent. In some embodiments, the solvent includes isopropyl alcohol. In some embodiments, the acid is provided in molar excess relative to the free base of Compound 1. In some embodiments, the molar ratio of Compound 1 free base to salicylic acid is from about 1:1 to about 1:1.5, or from about 1:1 to about 1:1.2. In some embodiments, the molar ratio of Compound 1 free base to salicylic acid is about 1:1.16. In some embodiments, the combination is carried out around room temperature.

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The benzoic acid salt can be prepared, for example, by coupling N-{(7R)-4-[(3R,4R,5S)-3amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7 -dihydro-5H-cyclopenta[b]pyridin-3-yl}-6(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide (free base of compound 1) with benzoic acid, optionally in the presence of a solvent. In some embodiments, the solvent includes isopropyl alcohol. In some embodiments, the acid is provided in molar excess relative to the free base of Compound 1. In some embodiments, the molar ratio of Compound 1 free base to benzoic acid is from about 1:1 to about 1:1.5, or from about 1:1 to about 1:1.2. In some embodiments, the molar ratio of Compound 1 free base to benzoic acid is about 1:1.16. In some embodiments, the combination is carried out around room temperature.

The benzenesulfonic acid salt can be prepared, for example, by coupling N-{(7R)-4-[(3R,4R,5S)-3amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7 -dihydro-5H-cyclopenta[b]πpyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide (free base of compound 1) with benzenesulfonic acid, optionally in the presence of a solvent. In some embodiments, the solvent includes isopropyl alcohol. In some embodiments, the acid is provided in molar excess relative to the free base of Compound 1. In some embodiments, the molar ratio of Compound 1 free base to benzenesulfonic acid is from about 1:1 to about 1:1.5 or about 1:1 to about 1:1,2. In some embodiments, the molar ratio of Compound 1 free base to benzenesulfonic acid is about 1:1.1. In some embodiments, the combination is carried out around room temperature.

L-pyroglutamic acid salt can be prepared, for example, by coupling N-{(7R)-4-[(3R,4R,5S)-3amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6 ,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide (free base of compound 1) with L-pyroglutamic acid, optionally in the presence of a solvent . In some embodiments, the solvent includes isopropyl alcohol. In some embodiments, the solvent includes isopropyl alcohol and heptane. In some embodiments, the acid is provided in molar excess relative to the free base of Compound 1. In some embodiments, the molar ratio of Compound 1 free base to L-pyroglutamic acid is from about 1:1 to about 1:1.5, or from about 1: 1 to about 1:1.2. In some embodiments, the molar ratio of Compound 1 free base to Lpyroglutamic acid is about 1:1.12. In some embodiments, the combination is carried out around room temperature.

The methanesulfonic acid salt can be prepared, for example, by coupling N-{(7R)-4-[(3R,4R,5S)-3amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7 -dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide (free base of compound 1) with methanesulfonic acid, optionally in the presence of a solvent. In some embodiments, the solvent includes isopropyl alcohol. In some embodiments, the solvent includes isopropyl alcohol and ethanol. In some embodiments, the solvent includes isopropyl alcohol, ethanol, and heptane. In some embodiments, the acid is provided in molar excess relative to the free base of Compound 1. In some embodiments, the molar ratio of Compound 1 free base to methanesulfonic acid is from about 1:1 to about 1:1.5, or from about 1:1 to about 1:1.2. In some embodiments, the molar ratio of Compound 1 free base to methanesulfonic acid is about 1:1.1. In some embodiments, the combination is carried out around room temperature.

The (1S)-(+)-10-camphorsulfonic acid salt can be prepared, for example, by coupling N-{(7R)-4[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidine-1 -yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide (free base of compound 1) with ( 1S)-(+)-10 camphorsulfonic acid, optionally in the presence of a solvent. In some embodiments, the solvent includes isopropyl alcohol. In some embodiments, the solvent includes isopropyl alcohol and heptane. In some embodiments, the acid is provided in molar excess with respect to the free base of Compound 1. In some embodiments, the molar ratio of the free base of Compound 1 to (lS)-(+)-10-camphorsulfonic acid is from about 1:1 to about 1: 1.5 or from about 1:1 to about 1:1.2. In some embodiments, the molar ratio of Compound 1 free base to (1S)-(+)-10-camphorsulfonic acid is about 1:1.1. In some embodiments, the combination is carried out around room temperature.

The fumaric acid salt can be prepared, for example, by coupling N-{(7R)-4-[(3R,4R,5S)-3amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7 -dihydro-5H-cyclopenta[b]pyridin-3-yl}-6(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide (free base of compound 1) with fumaric acid, optionally in the presence of a solvent. In some embodiments, the solvent includes isopropyl alcohol. In some embodiments, the solvent includes isopropyl alcohol and heptane. In some embodiments, the acid is provided in molar excess relative to the free base of Compound 1. In some embodiments, the molar ratio of Compound 1 free base to fumaric acid is about

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1:1,16. In some embodiments, the combination is carried out around room temperature.

The sulfuric acid salt can be prepared, for example, by coupling N-{(7R)-4-[(3R,4R,5S)-3-amino4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7 -dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide (free base of compound 1) with sulfuric acid, optionally in the presence of a solvent. In some embodiments, the solvent includes isopropyl alcohol. In some embodiments, the acid is provided in molar excess relative to the free base of Compound 1. In some embodiments, the molar ratio of Compound 1 free base to sulfuric acid is from about 1:1 to about 1:1.5, or from about 1:1 to about 1:1.2. In some embodiments, the molar ratio of Compound 1 free base to sulfuric acid is about 1:1.1. In some embodiments, the combination is carried out around room temperature.

The L-tartaric acid salt can be prepared, for example, by coupling N-{(7R)-4-[(3R,4R,5S)-3amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6 ,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide (free base of compound 1) with L-tartaric acid, optionally in the presence of a solvent. In some embodiments, the solvent includes isopropyl alcohol. In some embodiments, the solvent includes isopropyl alcohol and heptane. In some embodiments, the acid is provided in molar excess relative to the free base of Compound 1. In some embodiments, the molar ratio of Compound 1 free base to L-tartaric acid is from about 1:1 to about 1:1.5, or from about 1: 1 to about 1:1.2. In some embodiments, the molar ratio of Compound 1 free base to L-tartaric acid is about 1:1.16. In some embodiments, the combination is carried out around room temperature.

The D-tartaric acid salt can be prepared, for example, by coupling N-{(7R)-4-[(3R,4R,5S)-3amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6 ,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide (free base of compound 1) with D-tartaric acid, optionally in the presence of a solvent. In some embodiments, the solvent includes isopropyl alcohol. In some embodiments, the solvent includes isopropyl alcohol and heptane. In some embodiments, the acid is provided in molar excess relative to the free base of Compound 1. In some embodiments, the molar ratio of Compound 1 free base to D-tartaric acid is from about 1:1 to about 1:1.5, or from about 1: 1 to about 1:1.2. In some embodiments, the molar ratio of Compound 1 free base to D-tartaric acid is about 1:1.16. In some embodiments, the combination is carried out around room temperature.

Methods of application.

Compound 1 and the salts described herein can inhibit the activity of one or more members of the Pim kinase family and are therefore useful in the treatment of diseases and disorders associated with Pim kinase activity. For example, Compound 1 and its salts can inhibit one or more of Pim1, Pim2 and Pim3. Thus, this disclosure provides methods for treating a Pim kinase-related disease or disorder in an individual (e.g., a patient) by administering to the individual in need of such treatment a therapeutically effective amount or dose of the phosphoric acid salt of Compound 1, or any of its embodiments, or its pharmaceutical composition. This disclosure also provides the phosphoric acid salt of Compound 1, or any of its embodiments, or a pharmaceutical composition thereof, for use in the treatment of a Pim kinase-related disease or disorder. Also contemplated is the use of a phosphoric acid salt of Compound 1, or any of its embodiments, or a pharmaceutical composition thereof, in the manufacture of a medicament for the treatment of a Pim kinase-related disease or disorder.

A Pim kinase-related disease may include any disease, disorder or condition that is directly or indirectly associated with the expression or activity of a Pim kinase, including increased expression and/or abnormal levels of activity. Abnormal levels of activity can be determined by comparing the level of activity in normal, healthy tissue or cells with the level of activity in diseased cells. A Pim kinase-related disease may also include any disease, disorder or condition that can be prevented, mitigated, inhibited or treated by modulating Pim kinase activity. In some embodiments, the disease is characterized by abnormal activity or expression (eg, increased expression) of one or more of Pim1, Pim2, and Pim3. In some embodiments, the disease is characterized by mutant Pim1, Pim2, or Pim3. Pim kinase-related disease may also refer to any disease, disorder or condition in which modulation of the expression or activity of one or more Pim kinases is useful.

Pim kinase-associated diseases that can be treated according to the present invention include cancers, including in particular cancers in which Pim kinases are up-regulated or an oncogene, such as Myc or BCL2, is activated. Pim kinase-related diseases include

- 24 043661 solid tumors, e.g. prostate cancer, colon cancer, esophageal cancer, endometrial cancer, ovarian cancer, uterine cancer, kidney cancer, liver cancer, pancreatic cancer, stomach cancer, breast cancer, lung cancer, head cancer or neck, thyroid cancer, glioblastoma, sarcoma, bladder cancer, etc. Pim kinase-associated diseases also include hematological cancers such as lymphoma, leukemia such as acute lymphoblastic leukemia (ALL), acute myeloblastic leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma, non-Hodgkin lymphoma (including relapsed non-Hodgkin lymphoma, refractory non-Hodgkin lymphoma and relapsed follicular non-Hodgkin lymphoma), Hodgkin lymphoma and multiple myeloma.

Pim kinase-related diseases that can be treated according to the present invention also include myeloproliferative disorders such as polycythemia vera (PV), essential thrombocythemia (ET), chronic myeloid leukemia (CML), and the like. The myeloproliferative disorder may be a myelofibrosis, such as primary myelofibrosis (PMF), polycythemia vera/essential thrombocythemia vera/essential thrombocythemia myelofibrosis (Post-IP/ET MF), essential thrombocythemia vera myelofibrosis (Post-ET MF), or post-Essential thrombocythemia myelofibrosis (Post-ET MF). polycythemia vera (Post-IP MF).

Pim kinase-related diseases that can be treated according to this invention also include immunological disorders such as autoimmune diseases. Immunological disorders include multiple sclerosis, rheumatoid arthritis, allergies, food allergies, asthma, lupus, inflammatory bowel disease and ulcerative colitis.

Pim kinase-related diseases that can be treated according to this invention also include atherosclerosis.

The salts of this invention can also be used to inhibit pathological processes involving Pim kinases, including angiogenesis and tumor metastasis.

Due to the fact that Pim kinases are regulated by the JAK/STAT signaling pathway, the salts of this invention are suitable for the treatment of diseases in which modulation of activation of the JAK/STAT signaling pathway is useful. Therefore, other diseases that can be treated include Crohn's disease, irritable bowel syndrome, pancreatitis, diverticulosis, Graves' disease, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis, ankylosing spondylitis, myasthenia gravis, vasculitis, autoimmune thyroiditis, dermatitis, psoriasis, scleroderma, systemic sclerosis, vitiligo, graft-versus-host disease, Sjögren's syndrome, glomerulonephritis and diabetes mellitus (type I).

The terms person or patient, when used interchangeably, refer to any animal, including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, pigs, cattle, sheep, horses or primates, and most preferably humans.

The phrase therapeutically effective amount refers to the amount of an active compound or pharmaceutical agent that produces in a tissue, system, animal, individual or human the biological or medical response sought by a researcher, veterinarian, doctor or other practitioner.

The term treatment refers to one or more of (1) inhibiting a disease; for example, inhibiting a disease, condition or disorder in an individual who is experiencing or exhibiting pathology or symptoms of the disease, condition or disorder (ie, stopping further development of the pathology and/or symptoms); and (2) disease mitigation; for example, alleviating a disease, condition or disorder in an individual who is experiencing or exhibiting pathology or symptoms of the disease, condition or disorder (ie, regression of pathology and/or symptomatology), such as reducing the severity of the disease.

In some embodiments, the salts of this invention may be useful for preventing or reducing the risk of developing a disease; for example, preventing or reducing the risk of developing a disease, condition or disorder in an individual who may be predisposed to the disease, condition or disorder but is not yet experiencing or exhibiting disease pathology or symptoms.

Complex therapies.

The growth and survival of cancer cells can be influenced by many signaling pathways. Therefore, it is useful to combine different kinase inhibitors that exhibit different preferences in the kinases whose activity they modulate to treat such conditions. Targeting more than one signaling pathway (or more than one biological molecule involved in a given signaling pathway) may reduce the likelihood of drug resistance occurring in a population of cells, and/or reduce the toxicity of treatment.

Accordingly, the Pim inhibitor of this invention can be used in combination with one or more other kinase inhibitors to treat diseases, such as cancer, that are affected by multiple signaling pathways. For example, the combination may include one or more inhibitors of the following kinases for the treatment of cancer: Akt1, Akt2, Akt3, TGF-eR, PKA, PKG, PKC,

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CaM kinase, phosphorylase kinase, MEKK, ERK, MAPK, mTOR, EGFR, HER2, HER3, HER4, INS-R, IGF1R, IR-R, PDGFaR, PDGFeR, CSFIR, KIT, FLK-II, KDR/FLK-1 , FLK-4, flt-1, FGFR1, FGFR2, FGFR3, FGFR4, c-Met, Ron, Sea, TRKA, TRKB, TRKC, FLT3, VEGFR/Flt2, Flt4, EphA1, EphA2, EphA3, EphB2, EphB4, Tie2 , Src, Fyn, Lck, Fgr, Btk, Fak, SYK, FRK, JAK, ABL, ALK and B-Raf. Non-limiting examples of inhibitors that can be combined with a Pim inhibitor provided herein for the treatment of diseases such as cancer include an FGFR inhibitor (FGFR1, FGFR2, FGFR3 or FGFR4), a JAK inhibitor (JAK1 and/or JAK2, e.g. ruxolitinib, baricitinib or INCB39110), IDO inhibitor (eg, epacadostat and NLG919), TDO inhibitor, PI3K delta inhibitor, PI3K gamma inhibitor, CSF1R inhibitor, TAM receptor tyrosine kinase inhibitor (Tyro-3, Axl and Mer), angiogenesis inhibitor, interleukin receptor inhibitor and an adenosine receptor antagonist or a combination thereof. In addition, the Pim inhibitors of the present invention can be combined with inhibitors of kinases associated with the PIK3/Akt/mTOR signaling pathway, such as PI3K, Akt (including Akt1, Akt2 and Akt3) and mTOR kinases.

In some embodiments, the Pim inhibitors of this invention can be combined with inhibitors selective for JAK1 and/or JAK2 (e.g., ruxolitinib, baricitinib, momelotinib, filgotinib, pacritinib, INCB039110, INCB052793, INCB054707, CYT387, ABT494, AZD1480, XL 019, CEP-33779, AZ 960, TG101209 and gandotinib). In some embodiments, the Pim inhibitors of this invention can be combined with inhibitors selective for JAK1 (eg, INCB039110, INCB052793, INCB054707, and ABT494) such as those disclosed, for example, in WO 2010/135650, WO 2011/028685, WO 2011 /112662, WO 2012/068450, WO 2012/068440, WO 2012/177606, WO 2013/036611, WO 2013/026025, WO 2014/138168, WO 2013/173720, WO 2015/0 21153, WO 2014/071031, WO 2014 /106706, WO 2015/131031, WO 2015/168246 and WO 2015/184305. In some embodiments, the Pim inhibitors of this invention can be combined with inhibitors selective for JAK2 (eg, pacritinib, AZD1480, XL019, CEP-33779, AZ 960, TG101209, and gandotinib).

In some embodiments, the Pim inhibitors of this invention can be combined with inhibitors selective for PI3K delta (eg, idelalisib, INCB040093, INCB050465, and TGR 1202), such as those disclosed, for example, in WO 2011/θ0θ8487, WO 2011/075643 , WO 2011/075630, WO 2011/163195, WO 2011/130342, WO 2012/087881, WO 2012/125629, WO 2012/135009, WO 2013/033569, WO2013/151930, W O 2014/134426, WO 2015/191677 and WO 2015/157257.

The Pim inhibitor of the present invention may further be used in combination with other cancer treatments, such as chemotherapy, radiation therapy, tumor-directed therapy, adjuvant therapy, immunotherapy, or surgery. Examples of immunotherapies include cytokine treatment (eg, interferons, GM-CSF, G-CSF, IL-2), CRS-207 immunotherapy, cancer vaccine, monoclonal antibody, adoptive T cell transfer, oncolytic virotherapy, and immunomodulatory small molecules including thalidomide or JAK1/2 inhibitor and the like. For example, the salts of this invention can be administered in combination with one or more anti-cancer drugs, such as chemotherapeutic drugs. Examples of chemotherapy drugs include any of the following: abarelix, aldesleukin, alemtuzumab, alitretinoin, allopurinol, altretamine, anastrozole, arsenic trioxide, asparaginase, azacitidine, bevacizumab, bexarotene, bleomycin, bortezom, bortezomib, intravenous busulfan, oral busulfan, calastone, capecitabine, carboplatin , carmustine, cetuximab, chlorambucil, cisplatin, cladribine, clofarabine, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, dalteparin sodium, dasatinib, daunorubicin, decitabine, denileukin, denileukin-diftitox, dextrazoxane, docetaxel, doxorubicin, dromostanol propi onat, eculizumab, epirubicin, erlotinib , estramustine, etoposide phosphate, etoposide, exemestane, fentanyl citrate, filgrastim, fluxuridine, fludarabine, fluorouracil, fulvestrant, gefitinib, gemcitabine, gemtuzumab ozogamicin, goserelin acetate, histrelin acetate, ibritumomab tiuxetan, idarubicin, ifosfami d, imatinib mesylate, interferon alfa 2a, irinotecan, lapatinib ditosylate, lenalidomide, letrozole, leucovorin, leuprolide acetate, levamisole, lomustine, mechlorethamine, megestrol acetate, melphalan, mercaptopurine, methotrexate, methoxalen, mitomycin C, mitotane, mitoxantrone, nandrolone fenpropionate, nelarabine, but fetumomab, oxaliplatin, paclitaxel, pamidronate, panitumumab , pegaspargase, pegfilgrastim, pemetrexed disodium, pentostatin, pipobromane, plicamycin, procarbazine, quinine, rasburicase, rituximab, ruxolitinib, sorafenib, streptozocin, sunitinib, sunitinib maleate, tamoxifen, temozolomide, teniposide, testolactone, thalidomide, thiogu anine, thiotepa, topotecan, toremifene , tositumomab, trastuzumab, tretinoin, uramustine, valrubicin, vinblastine, vincristine, vinorelbine, vorinostat and zoledronate.

In some embodiments, the Pim inhibitors of this invention can be combined with cytarabine.

Other anticancer agents include antibody therapeutics such as trastuzumab (Herceptin), antibodies to costimulatory molecules such as CTLA-4, 4-1BB, PD-1 and PD-L1, or antibodies to cytokines (IL-10, TGF -β, etc.). The Pim inhibitors provided herein may additionally be used in combination with one or more control inhibitors.

- 26 043661 points (for example, immune checkpoint molecule inhibitors). Examples of immune checkpoint inhibitors include inhibitors against immune checkpoint molecules such as CD27, CD28, CD40, CD122, CD96, CD73, CD47, OX40, GITR, CSF1R, JAK (eg, JAK1 and/or JAK2), PI3K delta, PI3K gamma, TAM, arginase, CD137 (also known as 4-1BB), ICOS, A2AR, B7-NZ, B7H4, BTLA, CTLA-4, LAG3, TIM3, VISTA, PD-1, PD-L1 and PD-L2. In some embodiments, the immune checkpoint molecule is a stimulatory checkpoint molecule selected from CD27, CD28, CD40, ICOS, OX40, GITR, and CD137. In some embodiments, the immune checkpoint molecule is an inhibitory checkpoint molecule selected from A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, TIM3, and VISTA. In some embodiments, the compounds provided herein can be used in combination with one or more agents selected from KIR inhibitors, TIGIT inhibitors, LAIR1 inhibitors, CD160 inhibitors, 2B4 inhibitors and beta TGFR inhibitors.

In some embodiments, immune checkpoint inhibitors include inhibitors against immune checkpoint molecules such as JAK1 and/or JAK2.

In some embodiments, immune checkpoint inhibitors include inhibitors against immune checkpoint molecules such as CD96.

In some embodiments, the immune checkpoint molecule inhibitor is an anti-PD1 antibody, an anti-PD-L1 antibody, or an anti-CTLA-4 antibody.

In some embodiments, the immune checkpoint molecule inhibitor is a PD-1 inhibitor, such as an anti-PD-1 monoclonal antibody. In some embodiments, the anti-PD-1 monoclonal antibody is nivolumab, pembrolizumab (also known as MK-3475), pidilizumab, SHR-1210, or AMP-224. In some embodiments, the anti-PD-1 monoclonal antibody is nivolumab or pembrolizumab. In some embodiments, the anti-PD-1 antibody is pembrolizumab.

In some embodiments, the immune checkpoint molecule inhibitor is a PD-L1 inhibitor, such as an anti-PD-L1 monoclonal antibody. In some embodiments, the anti-PD-L1 monoclonal antibody is BMS-935559, MEDI4736, MPDL3280A (also known as RG7446), or MSB0010718C. In some embodiments, the anti-PD-L1 monoclonal antibody is MPDL3280A or MEDI4736.

In some embodiments, the immune checkpoint molecule inhibitor is a CTLA-4 inhibitor, such as an anti-CTLA-4 antibody. In some embodiments, the anti-CTLA-4 antibody is ipilimumab.

In some embodiments, the immune checkpoint molecule inhibitor is a LAG3 inhibitor, such as an anti-LAG3 antibody. In some embodiments, the anti-LAG3 antibody is BMS-986016 or LAG525.

In some embodiments, the immune checkpoint molecule inhibitor is a GITR inhibitor, such as an anti-GITR antibody. In some embodiments, the anti-GITR antibody is TRX518 or MK-4166. In some embodiments, the anti-GITR antibody is INCAGN01876.

In some embodiments, the immune checkpoint molecule inhibitor is an OX40 inhibitor, such as an anti-OX40 antibody or an OX40L fusion protein. In some embodiments, the anti-OX40 antibody is MEDI0562. In some embodiments, the OX40L fusion protein is MEDI6383. In some embodiments, the anti-OX40 antibody is INCAGN01949.

In some embodiments, the immune checkpoint molecule inhibitor is a TIM3 inhibitor, such as an anti-TIM3 antibody.

In some embodiments, the Pim inhibitors of this invention can be combined with TIGIT inhibitors.

The Pim inhibitors of this invention can be used in combination with one or more other anticancer agents, including BET inhibitors (eg, INCB054329, OTX015 and CPI0610), LSD1 inhibitors (eg, GSK2979552 and INCB059872), HDAC inhibitors (eg, panobinostat, vorinostat and entinostat ), DNA methyltransferase inhibitors (eg, azacitidine and decitabine) and other epigenetic modulators.

In some embodiments, the Pim inhibitors of this invention can be combined with BET inhibitors. In some embodiments, the Pim inhibitors of this invention can be combined with LSD1 inhibitors. In some embodiments, the Pim inhibitors of this invention can be combined with HDAC inhibitors. In some embodiments, the Pim inhibitors of this invention can be combined with DNA methyltransferase inhibitors.

The Pim inhibitors of this invention can be used in combination with one or more agents to treat diseases such as cancer. In some embodiments, the agent is an alkylating agent, a proteasome inhibitor, a corticosteroid, or an immunomodulatory agent. Examples of the alkylating agent include cyclophosphamide (CY), melphalan (MEL)

- 27 043661 and bendamustine. In some embodiments, the proteasome inhibitor is carfilzomib. In some embodiments, the corticosteroid is dexamethasone (DEX). In some embodiments, the immunomodulatory agent is lenalidomide (LEN) or pomalidomide (POM).

One or more anti-inflammatory drugs, steroids, immunosuppressants or therapeutic antibodies can also be combined with the salts of this invention.

When more than one pharmaceutical agent is administered to a patient, they may be administered simultaneously, sequentially, or in combination (eg, for more than two agents).

Composition, dosage forms and administration.

The salts of this invention can be administered in the form of a pharmaceutical composition. Therefore, this disclosure provides a composition containing salts of this invention or any of its embodiments, and at least one pharmaceutically acceptable carrier. These compositions can be prepared by methods well known in the pharmaceutical field and can be administered in various ways depending on whether local or systemic treatment is prescribed and the site to be treated. Administration may be topical (including transdermal, epidermal, ophthalmic and mucosal, including intranasal, vaginal and rectal delivery), pulmonary (eg, inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal or intranasal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal intramuscular injection or infusion; or intracranial, eg intrathecal or intraventricular, administration. Parenteral administration may be as a single dose bolus or may be administered, for example, using a continuous infusion pump. Pharmaceutical and topical compositions may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powdery or oily bases, thickeners and the like may be necessary or desirable.

This invention also includes pharmaceutical compositions containing as an active ingredient a salt of this invention in combination with one or more pharmaceutically acceptable carriers (excipients). In some embodiments, the composition is suitable for topical administration. In preparing the compositions of this invention, the active ingredient is typically mixed with an excipient, diluted with an excipient, or enclosed in such a carrier, for example, a capsule, pouch, paper, or other casing. When the excipient serves as a diluent, it may be a solid, semi-solid or liquid material that acts as a base, carrier or vehicle for the active ingredient. Thus, the compositions may be in the form of tablets, pills, powders, lozenges, sachets, starch capsules, elixirs, suspensions, emulsions, solutions, syrups, aerosols (solid or liquid), ointments containing, for example, up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions and sterile packaged powders.

When formulated, the active compound may be crushed to provide the appropriate particle size before combining with other ingredients. If the active compound is substantially insoluble, it can be ground to a particle size of less than 200 mesh. If the active compound is substantially water soluble, the particle size can be adjusted by grinding to provide substantially uniform distribution within the formulation, for example, about 40 mesh.

The salts of this invention can be ground using known grinding methods, such as wet grinding, to obtain a particle size suitable for the formation of tablets, and for other types of formulations. Finely divided (nanoparticle-containing) preparations of the compounds of this invention can be prepared by methods known in the art, see, for example, WO 2002/000196.

Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup and methylcellulose. The compositions may further include: lubricants such as talc, magnesium stearate and mineral oil; wetting agents; emulsifying and suspending agents; preservatives such as methyl and propyl hydroxybenzoates; and sweeteners and flavorings. The compositions of this invention can be formulated to provide rapid, sustained or sustained release of the active ingredient following administration to a patient using procedures known in the art.

In some embodiments, the pharmaceutical composition comprises silicified microcrystalline cellulose (SMCC) and at least one compound described herein or a pharmaceutically acceptable salt thereof. In some embodiments, the silicified microcrystalline cellulose contains about 98% microcrystalline cellulose and about 2% silica by weight.

- 28 043661

In some embodiments, the composition is a sustained release composition comprising at least a salt as described herein and at least one pharmaceutically acceptable carrier. In some embodiments, the composition contains a salt described herein and at least one component selected from microcrystalline cellulose, lactose monohydrate, hydroxypropyl methylcellulose and polyethylene oxide. In some embodiments, the composition contains at least one compound described herein, or a pharmaceutically acceptable salt thereof, and microcrystalline cellulose, lactose monohydrate, and hydroxypropyl methylcellulose. In some embodiments, the composition contains a salt described herein and microcrystalline cellulose, lactose monohydrate and polyethylene oxide. In some embodiments, the composition further comprises magnesium stearate or silicon dioxide. In some embodiments, the microcrystalline cellulose is Avicel PH102™. In some embodiments, the lactose monohydrate is Fast-flo 316™. In some embodiments, the hydroxypropyl methylcellulose is hydroxypropyl methylcellulose 2208 K4M (eg, Methocel K4 M Premier™) and/or hydroxypropyl methylcellulose 2208 K100LV (eg, Methocel K00LV™). In some embodiments, the polyethylene oxide is polyethylene oxide WSR 1105 (eg, Polyox WSR 1105™).

In some embodiments, a wet granulation method is used to prepare the composition. In some embodiments, a dry granulation method is used to prepare the composition.

The compositions can be formulated into unit dosage form, with each dosage containing from about 5 to about 1000 mg (1 g), more often from about 100 mg to about 500 mg of active ingredient. In some embodiments, each dosage contains about 10 mg of active ingredient. In some embodiments, each dosage contains about 50 mg of active ingredient. In some embodiments, each dosage contains about 25 mg of active ingredient. The term dosage unit forms refers to physically discrete units suitable as unit dosages for human subjects and other mammals, each unit containing a predetermined amount of active material calculated to produce the desired therapeutic effect, in combination with a suitable pharmaceutical excipient. Solid oral dosage forms include, for example, tablets, capsules and pills.

The components used to prepare the pharmaceutical compositions are of high purity and substantially free of potentially harmful contaminants (e.g., at least nationally food grade, typically at least analytical grade, and more typically at least pharmaceutical grade cleanliness). In particular, for human consumption, the composition is preferably manufactured or formulated according to Good Manufacturing Practice standards as defined in applicable regulations of the US Food and Drug Administration. For example, suitable formulations may be sterile and/or substantially isotonic and/or in full compliance with all US Food and Drug Administration Good Manufacturing Practices.

The active compound can be effective over a wide range of dosages and is typically administered in a therapeutically effective amount. However, it should be understood that the amount of compound actually administered will generally be determined by the physician in accordance with relevant circumstances, including the condition being treated, the route of administration selected, the actual compound administered, the age, weight and response of the individual patient, the severity of the patient's symptoms, and the like.

The therapeutic dosage of a compound of this invention may be varied according to, for example, the specific use being treated, the route of administration of the compound, the health and condition of the patient, and the judgment of the prescriber. The proportion or concentration of a compound of this invention in a pharmaceutical composition may vary depending on a number of factors, including dosage, chemical characteristics (eg, hydrophobicity), and route of administration. For example, the compounds of this invention can be provided in an aqueous physiological buffer solution containing from about 0.1 to about 10% w/v of the compound for parenteral administration. Some typical dose ranges are from about 1 mcg/kg to about 1 g/kg body weight per day. In some embodiments, the dosage range is from about 0.01 mg/kg to about 100 mg/kg body weight per day. Dosage will likely depend on variables such as the type and degree of progression of the disease or disorder, the general health of the individual patient, the relative biological effectiveness of the selected compound, the composition of the excipient, and the route of administration. Effective doses can be extrapolated from dose-response curves obtained from in vitro test systems or animal models.

For the preparation of solid compositions such as tablets, capsules, pills or other oral dosage forms, the main active ingredient is mixed with a pharmaceutical excipient to form a solid preformulation containing a homogeneous mixture of compounds

- 29 043661 research on this invention. When these pre-formulations are referred to as homogeneous, the active ingredient is typically dispersed uniformly throughout the composition so that the composition can be readily divided into equally effective unit dosage forms such as tablets, pills and capsules. The solid preformulation is then divided into unit dosage forms of the type described above, containing from, for example, about 0.1 to about 1000 mg of the active ingredient of this invention.

The tablets or pills of this invention may be coated or otherwise combined to provide a dosage form that provides the benefit of long-lasting action. For example, a tablet or pill may include an internal dose component and an external dose component, the latter being in the form of a coating over the former. The two components may be separated by an enteric layer, which serves to resist breakdown in the stomach and allows the internal component to pass intact into the duodenum or be delayed in release. A variety of materials can be used for such enteric layers or coatings, and such materials include a number of polymer acids and mixtures of polymer acids with materials such as shellac, cetyl alcohol and cellulose acetate.

Liquid forms into which the compounds and compositions of this invention can be administered orally or by injection include aqueous solutions, suitably flavored syrups, aqueous or oily suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil nut or peanut butter, as well as elixirs and similar pharmaceutical bases.

Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents or mixtures thereof, and powders. Liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described above. In some embodiments, the compositions are administered by the oral or nasal route for local or systemic effect. The compositions can be nebulized using inert gases. Nebulized solutions can be inhaled directly from the nebulizing device, or the nebulizing device can be connected to a face mask, tent, or intermittent positive pressure breathing apparatus. Compositions based on solution, suspension or powder can be administered orally or nasally from devices that deliver the drug in an appropriate manner.

Topical formulations may contain one or more conventional carriers. In some embodiments, ointments may contain water and one or more hydrophobic carriers selected from, for example, liquid paraffin, polyoxyethylene alkyl ether, propylene glycol, Vaseline® white petrolatum (petroleum jelly), and the like. Cream carrier compositions may be based on water in combination with glycerin and one or more other components, for example, glycerol monostearate, PEGglycerol monostearate and cetyl stearyl alcohol. Gels can be prepared using isopropyl alcohol and water, suitably in combination with other components such as, for example, glycerin, hydroxyethylcellulose and the like. In some embodiments, topical formulations contain at least about 0.1, at least about 0.25, at least about 0.5, at least about 1, at least about 2, or at least about 5 wt. .% of the compound of this invention. Topical formulations may suitably be packaged in tubes of, for example, 100 g, which are optionally associated with instructions for treating a selected symptom, for example, psoriasis or other skin condition.

The amount of compound or composition administered to a patient will vary depending on what is being administered, the purpose of administration, such as prophylaxis or therapy, the condition of the patient, the route of administration, and the like. In therapeutic applications, the compositions can be administered to a patient already suffering from a disease in an amount sufficient to treat or at least partially arrest the symptoms of the disease and its complications. Effective doses will depend on the disease state being treated as well as the judgment of the attending physician based on factors such as the severity of the disease, age, weight and general condition of the patient, and the like.

Compositions administered to a patient may be in the form of pharmaceutical compositions described above. These compositions may be sterilized by conventional sterilization methods or may be subjected to sterilizing filtration. Aqueous solutions may be packaged for use as is, or lyophilized, wherein the lyophilized preparation is combined with a sterile aqueous vehicle prior to administration. The pH of compound formulations will typically be between 3 and 11, more preferably 5 to 9, and most preferably 7 to 8. It will be understood that the use of some of the above excipients, carriers or stabilizers will result in the formation of pharmaceutical salts.

The therapeutic dosage of a compound of this invention may be varied according to, for example, the particular use being treated, the route of administration of the compound, the health and condition of the patient, and the judgment of the prescribing physician. The proportion or concentration of a compound of this invention in a pharmaceutical composition may vary depending on the range of

- 30 043661 factors, including dosage, chemical characteristics (eg, hydrophobicity), and route of administration. For example, the compounds of this invention can be provided in an aqueous physiological buffer solution containing from about 0.1 to about 10% w/v of the compound for parenteral administration. Some typical dose ranges are from about 1 mcg/kg to about 1 g/kg body weight per day. In some embodiments, the dosage range is from about 0.01 mg/kg to about 100 mg/kg body weight per day. Dosage will likely depend on variables such as the type and degree of progression of the disease or disorder, the general health of the individual patient, the relative biological effectiveness of the selected compound, the composition of the excipient, and the route of administration. Effective doses can be extrapolated from dose-response curves obtained from in vitro test systems or animal models.

It is understood that certain features of the invention, which for clarity are described in the context of individual embodiments, may also be provided in combination in a single embodiment (while it is intended that the embodiments be combined as if written in multiple dependent form). Conversely, various features of the invention, which for brevity are described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.

The invention will be described in more detail by means of specific examples. The following Examples are provided for illustrative purposes and are not intended to limit the invention in any way. Those skilled in the art will readily recognize many non-critical parameters that can be changed or modified to obtain substantially the same results.

Examples

Example 1. Asymmetric synthesis of phosphoric acid salt N-{(1R)-4-[(3R,4R,5S)-3-amino-4-hydroxy5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro -5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridin-2-carboxamide

but but

Free base of Compound 1 Phosphoric acid salt of Compound 1

Step 1. Synthesis of tert-butyl [(3R,4R,5S)-4-{[tert-butyl(dimethyl)silyl]oxy}-1-((7R)-7-{[tert-butyl(dimethyl)silyl]oxy} -3-cyano-6,7-dihydro-5H-cyclopenta[b]pyridin-4-yl)-5-methylpiperidin-3yl]carbamate (21).

To a stirred solution of (7R)-7-{[tert-butyl(dimethyl)silyl]oxy}-4-chloro-6,7-dihydro-5Hcyclopenta[b]pyridine-3-carbonitrile (20) (see example 5, 54.0 g, 173 mmol) (99.2% HPLC purity) in anhydrous dimethyl sulfoxide (DMSO, 162 mL) add tert-butyl ((3R,4R,5S)-4-{[tert-butyl (dimethyl)silyl]oxy}-5-methylpiperidin-3-yl)carbamate hydrochloride (9 (HCl)) (see US patent publication number 2014/0200227, para [0769], 70.0 g, 182.0 mmol , 1.05 eq.) and N,N-diisopropylethylamine (DIEA, 121 ml, 694 mmol, 4 eq.) at room temperature. The resulting reaction mixture was heated at 100°C (oil bath) for 6 hours. When LCMS and HPLC indicated completion of the reaction (>98.5% conversion), the reaction mixture was cooled to room temperature using a water bath, diluted with water (400 ml ), extracted with tert-butyl methyl ether (TBME) twice (700 and 400 ml). The organic layers were washed with brine (500 ml), dried over MgSO ₄ , filtered to remove the drying agent and concentrated in vacuo. The residue was purified by filtration chromatography (330 g silica gel column; eluting with 5% EtOAc/hexanes for 4 minutes, then 25% EtOAc/hexanes for 6 minutes to give the title product, tert-butyl [(3R,4R,5S) -4-{[tert-butyl(dimethyl)silyl]oxy}-1-((7R)-7-{[tert-butyl(dimethyl)silyl]oxy}-3-cyano-6,7-dihydro-5H-cyclopenta[ b]pyridin-4-yl)-5-methylpiperidin-3yl]carbamate (21) (100.6 g, 98.1% HPLC purity, 92% yield) as a white to yellowish solid. 1H NMR (400 MHz, DMSO ₆ ) δ 8.48 (s, 1H), 6.75 (d, J=9.4 Hz, 1H), 5.01 (dd, J=6.9, 5.2 Hz, 1H) , 3.61 (d, J=12.6 Hz, 1H), 3.52 (d, J=12.4 Hz, 1H), 3.48-3.36 (m, 1H), 3.23 ( t, J=9.5 Hz, 1H), 3.02 (t, J=11.6 Hz, 2H), 2.84 (t, J=12.5 Hz, 1H), 2.74 (dt, J=15.2, 7.6 Hz, 1H), 2.32 (td, J=12.9, 8.0 Hz, 1H), 1.82 (ddd,

- 31 043661

J=13.3, 8.1, 4.3 Hz, 1H), 1.75-1.60 (m, 1H), 1.36 (s, 9H), 0.94 (d, J=6, 5 Hz, 3H), 0.87 (s, 9H), 0.84 (s, 9H), 0.13 (s, 3H), 0.08 (s, 6H), 0.06 (s, 3H) ppm; LCMS (EI) m/z 617.4 (C32H ₅ 7^O^ ₂ , (M+H)+).

Step 2. Synthesis of tert-butyl ((3R,4R,5S)-1-((7R)-3-(aminocarbonyl)-7-{[tert-butyl(dimethyl)silyl]oxy}-6,7dihydro-5H- cyclopenta[b]pyridin-4-yl)-4-{[tert-butyl(dimethyl)silyl]oxy}-5-methylpiperidin-3-yl)carbamate (22).

To the stirred solution tert-butyl [(3R,4R,5S)-4-{[tert-butyl(dimethyl)silyl]oxy}-1-((7R)-7{[tert-butyl(dimethyl)silyl]oxy} -3-cyano-6,7-dihydro-5H-cyclopenta[b]pyridin-4-yl)-5-methylpiperidin-3yl]carbamate (21) (65.0 g, 105 mmol) in ethanol (195 ml) in [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(P) (Pd(dppf) ₂ Cl ₂ in complex with dichloromethane (1:1); 1.721 g, 2.107 mmol, 0.02 eq. .), acetaldoxime (32.8 ml, 527 mmol, 5.0 eq.), and water (65 ml) at room temperature. The resulting reaction mixture was degassed three times and refluxed with N2, then refluxed (oil bath; about 90°C) for 6 hours. Additional acetaldoxime (32.8 mL, 527 mmol, 5.0 eq.) was added. The reaction mixture was then refluxed (oil bath; temperature approximately 90°C) for an additional 16 hours. When LCMS and HPLC indicated completion of the reaction (>96.5% conversion), the hot reaction mixture was exposed to water (390 mL). followed by gradual cooling to room temperature. The resulting slurry was stirred at room temperature for an additional 30 minutes before isolation by filtration. The wet filter cake was washed with water (3x100 ml) followed by redispersion in a mixture of acetonitrile and water (400 ml; acetonitrile to water: 1 to 3 by volume). The solid was collected by filtration, washed with water (3x100 ml) and dried in a household vacuum at room temperature overnight to give the target product, tert-butyl ((3R,4R,5 S)-1 -((1 R)-3 -(aminocarbonyl)-7-{[tert-butyl(dimethyl)silyl]oxy}-6,7-dihydro-5Hcyclopenta[b]pyridin-4-yl)-4-{[tert-butyl(dimethyl)silyl]oxy }-5-methylpiperidin-3-yl)carbamate (22), in the form of a light brown powder, which was immediately used in the subsequent reaction without further purification. 1H NMR (400 MHz, DMSO6) δ 8.16 (s, 1H), 7.84 (s, 1H), 7.46 (s, 1H), 6.66 (d, J=9.6 Hz, 1H ), 5.01 (dd, J=6.8, 4.7 Hz, 1H), 3.48-3.36 (m, 1H), 3.23-3.07 (m, 3H), 3, 01-2.88 (m, 1H), 2.82 (t, J=11.7 Hz, 1H), 2.74-2.58 (m, 2H), 2.35 (dt, J=12, 3, 6.0 Hz, 1H), 1.84 (dt, J=13.1, 6.6 Hz, 1H), 1.72-1.56 (m, 1H), 1.35 (s, 9H ), 0.90 (d, J=6.6 Hz, 3H), 0.87 (s, 9H), 0.84 (s, 9H), 0.14 (s, 3H), 0.08 (s , 3H), 0.07 (s, 3H), 0.06 (s, 3H); LCMS (EI) m/z 635.4 ^^N^SR (M+H)+).

Step 3. Synthesis of tert-butyl ((3R,4R,S)-1-(3-amino-1-{[tert-butyl(dimethyl)silyl]oxy}-6,7-dihydro-5Hcyclopenta[b]pyridinium- 4-yl)-4-{[tert-butyl(dimethyl)silyl]oxy}-5-methylpiperidin-3-yl)carbamate (23).

To a stirred solution of tert-butyl ((3R,4R,5S)-1-((7R)-3-(aminocarbonyl)-7-{[tert-butyl(dimethyl)silyl]oxy}-6,7-dihydro-5H-cyclopent [b]pyridin-4-yl)-4-{[tert-butyl(dimethyl)silyl]oxy}-5methylpiperidin-3-yl)carbamate (22) (66.9 g, 105 mmol) in tetrahydrofuran (THF, 470 .0 ml) in a 2 L three-neck round-bottom flask at 0°C, add tetra-N-bugylammonium bromide (34.30 g, 105.4 mmol, 1.0 eq.) and a solution of 3.0 M sodium hydroxide in water ( 158.0 mL, 474.1 mmol, 4.5 eq.), then add 1,3-dibromo-5,5-dimethylhydantoin (21.52 g, 73.75 mmol, 0.7 eq.) portionwise over 44 min. with reaction temperature control from 5 to 10°C. The resulting dark brown solution was stirred at 5 to 10°C for an additional 20 minutes. When LCMS indicated completion of the reaction, the reaction mixture was diluted with MTBE (100 ml), quenched with a 10% aqueous solution of Na ₂ S ₂ O ₃ (300 ml) and water (200 ml). The two phases were separated and the aqueous phase was extracted with MTBE (2x600 ml). The combined organic layers were washed with water (500 ml), dried over MgSO ₄ , filtered to remove drying agent and concentrated in vacuo to give the crude product as a brown soap solid (63.7 g). The technical product was purified by filtration chromatography (330 g silica gel column; eluting with 20% EtOAc/hexanes for 6 minutes, then 45% EtOAc/hexanes for 8 minutes, followed by recrystallization from heptane (180 ml) to obtain the desired product, tert-butyl ((3R,4R,5S)-1-(3-amino-7-{[tert-butyl(dimethyl)silyl]oxy}-6,7-dihydro-5H-cyclopenta[b]pyridine-4- yl)-4-{[tert-butyl(dimethyl)silyl]oxy}-5-methylpiperidin-3-yl)carbamate (23) (46.2 g, 100.0% HPLC purity, 72% yield over two steps ) in the form of a white solid with a yellowish tint.

1H NMR (400 MHz, DMSO6) δ 7.80 (s, 1H), 6.61 (d, J=9.7 Hz, 1H), 4.88 (dd, J=6.8, 3.6 Hz , 1H), 4.81 (s, 2H), 3.52 (td, J=9.8, 4.6 Hz, 1H), 3.14 (t, J=9.5 Hz, 1H), 3 .04-2.84 (m, 3H), 2.85-2.68 (m, 2H), 2.48-2.42 (m, 1H), 2.23 (dq, J=13.2, 6.9 Hz, 1H), 1.79 (dq, J=12.6, 4.4 Hz, 2H), 1.35 (s, 9H), 0.89 (d, J=6.6 Hz, 3H), 0.85 (s, 18H), 0.10 (s, 3H), 0.08 (s, 3H), 0.06 (s, 3H), -0.00 (s, 3H); LCMS (EI) m/z 607.4 (C ₃₁ H ₅₉ N ₄ O ₄ Si ₂ , (M+H)+).

Step 4. Synthesis of tert-butyl {(3R,4R,5S)-4-{[tert-butyl(dimethyl)silyl]oxy}-1-[(7R)-7-{[tert-butyl(dimethyl)silyl]oxy} -3-({[6-(2,6-difluorophenyl)-5-fluoropyridin-2-yl]carbonyl}amino)-6,7-dihydro-5H-cyclopenta[b]pyridin-4-yl]-5- methylpiperidin-3-yl}carbamate (25).

To a stirred solution tert-butyl ((3R,4R,5S)-1-(3-amino-7-{[tert-butyl(dimethyl)silyl]oxy}-6,7dihydro-5H-cyclopenta[b]pyridine-4 -yl)-4-{[tert-butyl(dimethyl)silyl]oxy}-5-methylpiperidin-3-yl)carbamate (23) (100.0 g, 164.8 mmol) and 6-(2,6- difluorophenyl)-5-fluoropyridine-2-carboxylic acid (24) (see US patent number 2014/0200227, para [0625] 50.05 g, 197.7 mmol, 1.2 eq.) in anhydrous N,N- dimethylformamide (DMF, 320.0 ml) was added N,N-diisopropylethylamine (DIEA, 137.8 ml, 790.8 mmol, 4.8 eq.) at room temperature. After stirring at room temperature for 10 min, the reaction mixture was treated with N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (NATi, 150.3 g, 395.4 mmol , 2.4 eq.) in portions with temperature control from 15 to 30°C. Then the reaction mixture was stirred at room temperature for 30 minutes. When LCMS and HPLC indicated completion of the reaction, the reaction mixture was filtered into water (1300 ml) with stirring. The resulting slurry was stirred at room temperature for an additional 30 minutes. The solid was collected by filtration and washed with a mixture of 50% acetonitrile and water (50% v/v; 2x200 ml). The wet solid was then exposed to acetonitrile (420 ml) and the resulting slurry was heated at 70° C. until a clear solution formed. Water (320 ml) was slowly added to the solution at 70°C, and the resulting mixture was gradually cooled to room temperature and stirred at room temperature for 30 minutes. The solid was collected by filtration, washed with a mixture of acetonitrile and water (50% by volume; 320 ml) and dried in a vacuum oven at 50°C overnight to obtain the target product, tert-butyl {(3R,4R,5S)-4- {[tert-butyl(dimethyl)silyl]oxy}-1-[(7R)-7-{[tert-butyl(dimethyl)silyl]oxy}-3-({[6-(2,6-difluorophenyl)-5- fluoropyridin-2-yl]carbonyl}amino)-6,7dihydro-5H-cyclopenta[b]pyridin-4-yl]-5-methylpiperidin-3-yl}carbamate (25) (131.8 g, 99.7% HPLC purity, yield 95%) in the form of a white powder.

¹ H NMR (400 MHz, DMSO6) δ 10.39 (s, 1H), 8.95 (s, 1H), 8.36 (dd, J=8.7, 4.0 Hz, 1H), 8, 19 (t, J=8.9 Hz, 1H), 7.67-7.55 (m, 1H), 7.27 (t, J=8.5 Hz, 3H), 6.46 (d, J =9.6 Hz, 1H), 5.00 (dd, J=6.8, 4.2 Hz, 1H), 3.40 (dd, J=9.3, 5.9 Hz, 1H), 3 .05 (t, J=9.4 Hz, 2H), 2.93 (t, J=10.8 Hz, 4H), 2.82 (dt, J=15.8, 8.1 Hz, 1H) , 2.56 (t, J=11.7 Hz, 1H), 2.40-2.26 (m, 1H), 1.92-1.79 (m, 1H), 1.54 (s, 0H ), 1.32 (s, 9H), 0.87 (s, 11H), 0.84 (s, 9H), 0.71 (d, J=6.5 Hz, 4H), 0.14 (s , 4H), 0.07 (s, 3H), 0.04 (s, 4H), 0.01 (s, 3H); LCMS (EI) m/z 842.4 (C ₄₃ H ₆₃ F ₃ N ₅ O ₅ Si ₂ , (M+H)+).

Step 5. Synthesis of N-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy6,7-dihydro-5H-cyclopent [b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide with two molecules of hydrochloric acid (salt of compound 1 with two molecules of hydrochloric acid).

A solution of 4.0 M hydrogen chloride in 1,4-dioxane (13.9 L, 55600 mmol, 15.0 eq.) was added to the tert-butyl {(3R,4R,5S)-4-{[tert-butyl( dimethyl)silyl]oxy}-1-[(7R)-7-{[tert-butyl(dimethyl)silyl]oxy}-3-({[6-(2,6-difluorophenyl)-5-fluoropyridine-2- ul]carbonyl}amino)-6,7-dihydro-5H-cyclopenta[b]pyridin-4-yl]-5-methylpiperidin-3yl}carbamate (25) (3121.0 g, 3706.0 mmol) in methanol ( 19.5 l) at room temperature. During the addition of the HCl solution in 1,4-dioxane, the internal temperature increased from 17.3 to 36.8°C. The resulting light yellow solution was stirred at room temperature for 22 hours and a solid (2 HCl salt) began to precipitate from solution within 2 hours. When LCMS and HPLC indicated completion of the reaction, the solid was collected by filtration, washed with heptane (5 L), and filter dried under vacuum overnight to obtain the target product, N-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5methylpiperidin-1-yl]-7-hydroxy -6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5fluoropyridine-2-carboxamide dihydrochloride (salt of compound 1 with two molecules of hydrochloric acid) (2150.0 g, 99.1% HPLC purity, 98% yield) as a white powder. 1H NMR (400 MHz, DMSO6) δ 10.57 (s, 1H), 8.57 (s, 3H), 8.36 (dd, J=8.7, 4.1 Hz, 1H), 8.22 (t, J=8.8 Hz, 1H), 7.69 (ddd, J=15.1, 8.5, 6.7 Hz, 1H), 7.34 (t, J=8.1 Hz, 2H), 5.27 (t, J=7.0 Hz, 1H), 4.02 (d, J=11.1 Hz, 1H), 3.44 (d, J=11.3 Hz, 1H) , 3.29 (t, J=12.2 Hz, 1H), 3.13 (q, J=8.5, 7.4 Hz, 2H), 3.09-2.97 (m, 2H), 2.77 (t, J=12.3 Hz, 1H), 2.56-2.50 (m, 1H), 1.95 (dq, J=14.9, 7.7, 7.2 Hz, 1H), 1.66-1.50 (m, 1H), 0.70 (d, J=6.5 Hz, 3H); LCMS (EI) m/z 514.2 (C _m H^s^Oz, (M+H)+).

Step 6. Synthesis of N-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy6,7-dihydro-5H-cyclopent [b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridin-2-carboxamide (free base of compound 1).

A 200 L glass reactor was assembled with an overhead stirrer, reflux condenser, thermocouple, dropping funnel and nitrogen inlet, and the apparatus was purged with nitrogen. Sodium carbonate (18.264 g) and drinking water (86.2 L) were charged into the reactor and stirred for about 50 minutes until a solution was obtained. Methylene chloride (107.8 L) was charged into the reactor. N-{(1R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5methylniperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[ b]niridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide dihydrochloride (hydrochloric acid salt of compound 1) (4300 g) for about 27 min, maintaining the temperature at about 27°C and the reaction mixture was stirred at about 23°C for about 22 hours until a clear solution was obtained. The phases were separated and the aqueous phase was extracted with methylene chloride (26.9 L). The combined organic phases were washed twice with potable water (26.9 L per wash) and concentrated under reduced pressure at about 54°C to give the desired product, N-{(7R)-4-[(3R,4R,5S)-3 -amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine -2-carboxamide (compound 1 free base, 3838 g) as a light yellow solid, which was immediately used in the subsequent reaction without further purification. LCMS (EI) m/z 514.2 (C ₂₆ H ₂₇ F ₃ N ₅ O ₃ , (M+H)+).

Step 7. Synthesis of N-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy6,7-dihydro-5H-cyclopent [b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide with phosphoric acid (phosphoric acid salts of compound 1).

A 100 L glass reactor with overhead stirrer, reflux condenser, thermocouple, dropping funnel and nitrogen inlet was assembled and the apparatus was purged with nitrogen. Prepared separately

- 33 043661 prepared a solution of phosphoric acid by thoroughly mixing an aqueous solution of 85% H ₃ PO ₄ (980 g) and 2 propanol (IPA, 5.7 l) at room temperature. The phosphoric acid solution was filtered through a built-in cleaning filter. Separately, methanol (19.0 L), USP purified water (1.9 L) and 2-propanol were filtered through a built-in cleaning filter. Filtered methanol (19.0 L), N-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7hydroxy-6,7- Dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide (free base of compound 1) (3800 g), filtered USP purified water (1.9 L), and filtered 2-propanol (15.2 L) were sequentially loaded into the reactor. The reaction mixture was heated to about 56°C. The filtered phosphoric acid solution (5.8 L) was charged into the reactor over about 45 minutes, maintaining the temperature at about 59°C. The container was rinsed with filtered 2-propanol (5.8 L) into the reaction mixture, maintaining the temperature at approximately 62°C. Filtered 2-propanol (15.2 L) was charged, maintaining the temperature at about 58°C, and the reaction mixture was stirred at about 57°C for about 1.5 hours. The reaction mixture was cooled to about 26°C and stirred at about 17°C C for about 3.5 hours. The reaction mixture was filtered and the filter cake was washed successively with filtered 2-propanol (22.8 L) and filtered heptane (obtained separately by filtering 22.8 L of heptane through an in-line cleaning filter). The product was filter dried and then dried under reduced pressure at 20-54°C to obtain the target product, phosphoric acid salt N-{(1R)-4-[(3R,4R,5S)-3-amino-4-hydroxy- 5methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5fluoropyridin-2-carboxamide (phosphoric acid salt of the compound 1) (3,952 g, >99.0% HPLC purity, 87.3% yield) as a white to off-white crystalline solid.

1H NMR (400 MHz, DMSO6) δ 10.43 (s, 1H), 9.25 (s, 1H), 8.38 (dd, J=8.7, 4.1 Hz, 1H), 8.20 (t, J=8.8 Hz, 1H), 7.66 (ddd, J=15.2, 8.4, 6.7 Hz, 1H), 7.32 (t, J=8.6 Hz, 2H), 7.19 (s, 2H), 4.82 (dd, J=6.9, 4.5 Hz, 1H), 3.22 (d, J=6.9 Hz, 1H), 3, 18-2.99 (m, 3H), 2.87 (d, J=8.0 Hz, 2H), 2.79-2.67 (m, 1H), 2.59 (t, J=11, 5 Hz, 1H), 2.27 (dt, J=13.5, 6.6 Hz, 1H), 1.92-1.74 (m, 1H), 1.58-1.39 (m, 1H ), 0.70 (d, J=6.5 Hz, 3H) ppm; ¹³ C NMR (100 MHz, DMSO6) δ 163.1, 160.5, 159.5 (dd, Jcf=249.4, 6.6 Hz), 159.0 (d, Jcf=261.8 Hz), 146.0 (d, Jcf=4.2), 144.6, 136.1 (Jcf=18.6), 140.8, 132.8 (t, Jcf=10.6 Hz), 130.6, 128.5, 126.2 (d, Jcf=20.5), 125.4 (d, Jcf=6.4), 112.4 (d, _Jcf =20.8 Hz), 110.7 (td , J _CF =19.5, 3.6 Hz), 74.9, 72.8, 55.7, 54.0, 52.4, 37.7, 33.1, 26.9, 14.4 m .d.; ¹⁹ F NMR (376 MHz, DMSO6) δ -117.26 (m, 1F), -113.77 (m, 2F) ppm; LCMS (EI) m/z 514.2 (C26H27F3N5O3, (M+H)+).

The results of quantitative elemental microanalysis for carbon, hydrogen and nitrogen of the phosphoric acid salt of compound 1 correspond to the expected gross formula (C2 ₆ H ₂₉ F ₃ N ₅ O ₇ P). Anal. Calculated for C ₂₆ H ₂₉ F ₃ N ₅ O ₇ P: C, 51.07; N, 4.78; N, 11.45. Found: C, 51.16; N, 4.70; N, 11.56.

Example 2. X-ray powder diffractometry (XRD) of the phosphoric acid salt form I of compound 1.

Form I crystalline salt of phosphoric acid N-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H -cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5fluoropyridine-2-carboxamide (see example 1, step 7) was characterized by PRD. X-ray powder diffractometry (XRD) was obtained from a Rigaku MiniFlex X-ray Powder Diffractometer (XRD). The general experimental procedures for PRD were as follows: (1) X-ray radiation from copper at 1.054056 A with a K^ filter; (2) X-ray power supply 30 kV, 15 mA; and (3) the sample powder was dispersed on a sample holder with zero background. The general measurement conditions for the PRD were as follows: initial angle 3°; end angle 45°; step 0.02° and scanning speed 2°/min. The XRD diffraction pattern is shown in Fig. 1, and the PRD data are given in table. 1.

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Example 3 Differential scanning calorimetry (DSC) of the phosphoric acid salt of compound 1 (Form I).

Crystalline solid, phosphoric acid salt N-{(1R)-4-[(3R,4R,5S)-3-amino-4hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro- 5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide, (see example 1, step 7) was characterized by DSC. DSC was obtained from a TA Instruments differential scanning calorimetry instrument, model Q200, with an autosampler. The DSC instrument conditions were as follows: 30–350°C at 10°C/min; Tzero type aluminum sample cup and lid and nitrogen gas flow at 50 ml/min. The DSC thermogram is shown in Fig. 2. The DSC thermogram showed a major endotherm (considered to be melting/decomposition) at an initial temperature of 238.8°C with a peak temperature of 247.1°C, and a minor endotherm at an initial temperature of 193.1°C with a peak temperature at 198.4 °C. Several portions of Form I were characterized by DSC, each having a thermogram with the main endothermic peak occurring in the range from 249.7 to 254.7°C.

Example 4 Thermogravimetric analysis (TGA) of the phosphoric acid salt of compound 1 (Form I).

Crystalline solid, phosphoric acid salt N-{(1R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5methylpiperidin-1-ul]-7-hydroxy-6,7-dihydro- 5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide, (see example 1, step 7) was characterized by TGA. TGA was obtained from a TA Instrument Thermogravimetric Analyzer, model Q500. The general experimental conditions for TGA were: ramp from 20°C to 600°C at 20°C/min; nitrogen purge, gas flow 40 ml/min with subsequent balance of purge flow; sample purge flow at 60 ml/min; platinum sample cup. The TGA thermogram is shown in Fig. 3. A mass loss of about 2% was observed below 200°C and is believed to be due to loss of moisture and residual solvents. Karl Fischer analysis was performed on various synthetic portions of Form I, with each experiment showing water content in the range of 1.40-1.50%, indicating that Form I may be a hydrate, such as a hemihydrate.

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Example 5. Asymmetric synthesis of (1R)-1-{[tert-butyl(dimethyl)silyl]oxy}-4-chloro-6,7-dihydro5H-cyclopenta[b]pyridine-3-carbonitrile (20).

Step 1. 6,7-Dihydro-5H-cyclopenta[b]pyridine-1-oxide (11)

To a mixture of 6,7-dihydro-5H-cyclopenta[b]pyridine (10) (10.0 g, 83.9 mmol) in methanol (50 ml) was added urea peroxide adduct (UHP, 24 g, 250 mmol, 3. 0 equiv.) and methyltrioxorenium (UP) (80 mg, 0.3 mmol, 0.0036 equiv.) at room temperature. The resulting mixture was stirred at room temperature overnight, followed by concentration under reduced pressure to remove methanol. After distillation ceased to be observed, methylene chloride (100 ml) was added and concentration continued. The resulting residue was treated with methylene chloride (100 ml) and stirred at room temperature for 10 minutes. The solid was filtered and extracted with methylene chloride 3 times (3x50 ml). The combined filtrates were dried with anhydrous sodium sulfate (Na ₂ SO ₄ ) and sodium bisulfite (NaHSO ₃ ), followed by filtration through a pad of silica gel (SiO ₂ ). The layer was washed with 10% MeOH in CH ₂ CH ₂ (100 ml) 3 times. The combined filtrates were concentrated to give the title product, 6,7-dihydro-5H-cyclopenta[b]pyridine-1-oxide (11, 11.5 g, 99% yield) as a white to yellowish solid, which was used in the following reactions without further purification. For 11: 1H NMR (300 MHz, CDC^) δ 8.05 (d, J=6.0 Hz, 1H), 7.08 (m, 2H), 3.15. (t, J=6.0 Hz, 2H), 3.00. (t, J=6.0 Hz, 2H), 2.16 (m, 2H) ppm; LCMS (EI) m/z 136 (C ₈ H ₉ NO, (M+H)+).

Step 2. 4-Chloro-6,7-dihydro-5H-cyclopenta[b]pyridine (12)

Cl

6,7-Dihydro-5H-cyclopenta[b]pyridine-1-oxide (11, 12.0 g, 84.3 mmol) was slowly added to phosphorus oxychloride ( _POCl3 , 24 ml, 250 mmol, 3.0 eq. ) and toluene (48 ml) at 80-85°C (internal temperature) with an amount of about 0.2 g per portion over 2 hours. After addition was completed, the reaction mixture was continued to stir at 80-85°C (internal temperature) in for 3 hours. Excessive amounts of POCl ₃ and toluene were removed under reduced pressure at 60°C. The resulting residue was then poured into a cooled mixture of ice (50 g) and saturated aqueous sodium carbonate solution (Na ₂ CO ₃ , 50 ml). The pH of the mixture was adjusted to 8 with 25% aqueous sodium hydroxide (NaOH, about 30 ml). The mixture was then extracted with methylene chloride (3x100 ml). The combined organic extracts were dried over anhydrous Na ₂ SO ₄ and filtered through a layer of silica gel (SiO ₂ , 36 g) and carbon (6 g, on top of silica gel). The layer was washed with ethyl acetate (4x100 ml) until no more desired product emerged from the silica gel layer. The combined filtrates were then concentrated under reduced pressure to give the technical title product (12, 10.93 g). To a mixture of technical 4-chloro-6,7dihydro-5H-cyclopenta[b]pyridine (12, 10.7 g, 69.6 mmol) in THF (50 ml) was added 3-chlorobenzoic acid (11 g, 73 mmol) at room temperature. The mixture was then stirred at room temperature until the 3-chlorobenzoic acid dissolved. The mixture was concentrated under reduced pressure to form a technical salt as a solid, which was treated with hexanes (32 ml). The resulting suspension was stirred at room temperature for 10 minutes. The solid was collected by filtration, washed with hexanes (2x32 ml). Additional solids were collected from the filtrates. The combined wet solid (about 18 g) was dissolved in methylene chloride (100 ml) and exposed to saturated aqueous Na ₂ CO ₃ (2 x 2 0 ml). The aqueous layers were again extracted with methylene chloride (50 ml). The combined organic extracts were dried over anhydrous Na ₂ SO ₄ , filtered and concentrated to give the purified title product, 4chloro-6,7-dihydro-5H-cyclopenta[b]pyridine (12, 7.1 g) as a dark oil. For 12: 1H NMR (300 MHz, CDCl3) δ 8.20 (d, J=3.0 Hz, 1H), 7.02 (d, J=3.0 Hz, 1H), 3.06. (t, J=6.0 Hz, 2H), 2.98. (t, J=6.0 Hz, 2H), 2.12 (m, 2H) ppm; LCMS (EI) m/z 154/156 (C ₈ H ₈ ClN, (M+H) ⁺ ).

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Step 3. 4-Chloro-6,7-dihydro-5H-cyclopenta[b]pyridine-1-oxide (13)

Cl С0 o

To a mixture of 4-chloro-6,7-dihydro-5H-cyclopenta[b]pyridine (12, 100.0 g, 618.4 mmol) in methanol (500 ml) was added urea peroxide adduct (UHP, 1.5 eq. , 87.3 g, 928 mmol, 1.5 eq.) and methyltrioxorenium(VII) (925 mg, 3.71 mmol, 0.006 eq.) at room temperature. The resulting mixture was stirred at room temperature for 3 hours. The reaction mixture was concentrated under reduced pressure to remove methanol. Methylene chloride (500 ml) was added and concentration continued. The resulting residue was treated with methylene chloride (500 ml) and the resulting mixture was stirred for 30 minutes. The solid was filtered and extracted with methylene chloride (4x500 ml). The combined filtrates were dried with anhydrous sodium sulfate (Na ₂ SO ₄ ) and sodium bisulfite (NaHSO ₃ ). The mixture was then filtered through a layer of silica gel (SiO ₂ , 180 g) and activated carbon (10 g). The layer was washed with 10% MeOH in CH2CH2 (500 ml) 3 times. The combined filtrates were concentrated under reduced pressure to give the title product, 4-chloro-6,7-dihydro-5Hcyclopenta[b]pyridine-1-oxide (13, 104 g) as a white to yellowish solid. For 13: 1H NMR (300 MHz, DMSO-d ₆ ) δ 8.04 (d, J=6.0 Hz, 1H), 7.34 (d, J=6.0 Hz, 1H), 2.98. (m, 4H), 2.08 (m, 2H) ppm; LCMS (EI) m/z 170/172 (C ₈ H ₈ ClNO, (M+H)+).

Step 4. 4-Chloro-6,7-dihydro-5H-cyclopenta[b]pyridin-7-ylacetate (14) ci

AcO

4-Chloro-6,7-dihydro-5H-cyclopenta[b]pyridine-1-oxide (13) (65.0 g, 340 mmol) was added to a mixture of acetic anhydride (Ac ₂ O, 98 ml, 1000 mmol) and toluene (325 ml) in portions (6 g per portion) at 80-85°C for 1 hour. After addition was complete, the resulting mixture was stirred at 80-85°C for 3 hours. When the completion of the reaction was indicated by LCMS and/ or HPLC, the reaction mixture was concentrated under reduced pressure to remove acetic anhydride and toluene. The resulting technical target product, 4-chloro-6,7-dihydro-5H-cyclopenta[b]pyridin-7-ylacetate (14), was used immediately in the subsequent reaction without additional purification. For 14: 1H NMR (300 MHz, DMSO-d ₆ ) δ 8.38 (d, J=6.0 Hz, 1H), 7.21 (d, J=6.0 Hz, 1H), 6.14 . (m, 1H), 3.08 (m, 1H), 2.93 (m, 1H), 2.64 (m, 1H), 2.10 (s, 3H), 2.07 (m, 1H) ppm; LCMS (EI) m/z 212/214 (C _w HioC1N0 ₂ , (M+H)+).

Step 5. 4-Chloro-6,7-dihydro-5H-cyclopenta[b]pyridin-7-ol (15)

To a mixture of 4-chloro-6,7-dihydro-5H-cyclopenta[b]pyridin-7-ylacetate (14.73 g, 340 mmol, obtained from the previous step) in MeOH (130 ml) and water (130 ml) potassium carbonate (K2CO3, 130 g, 920 mmol) at 0°C. The resulting mixture was gradually warmed to room temperature and stirred at room temperature for 4 hours. The reaction mixture was then concentrated under reduced pressure to remove methanol. The precipitated solid was collected by filtration, washed with water (3x100 ml), and dried to obtain the first batch of technical product (15). The aqueous layers were extracted with DCM (3x300 ml). The combined organic extracts were dried over anhydrous Na ₂ SO ₄ , filtered and concentrated to obtain a second portion of the technical product (15). Technical target product, 4-chloro-6,7-dihydro-5H-cyclopenta[b]pyridin-7-ol (15, 61.9 g, 93% yield over 2 steps), obtained from two portions as dark brown oils were immediately used in the subsequent reaction without further purification. For 15: 1H NMR (300 MHz, DMSO-d ₆ ) δ 8.34 (d, J=3.0 Hz, 1H), 7.18 (d, J=3.0 Hz, 1H), 5.26 . (t, J=3.0 Hz, 1H), 3.08 (m, 1H), 2.86 (m, 1H), 2.60 (m, 1H), 2.09 (m, 1H) m. d.; LCMS (EI) m/z 170/172 (C8H8C1NO, (M+H)+).

Step 6. 4-Chloro-5H-cyclopenta[b]pyridin-7(6H)-one (16)

N,N-Diisopropylethylamine (339 .5 g, 2627.0 mmol) at room temperature. After cooling the mixture to 0°C, pyridine-sulfur trioxide (1:1) (200.0 g, 1256.6 mmol) in dimethyl sulfoxide (DMSO, 800 g) was added dropwise over 35 min and the resulting mixture was stirred at 0°C for 1 hour. The reaction progress was monitored by HPLC and about 30% of starting material 15 remained. Additional amounts of pyridine sulfur trioxide (1:1) (138 g, 866.8 mmol) and N,N were added

- 37 043661 diisopropylethylamine (79.8 g, 617 mmol), and the reaction mixture was stirred in an ice bath for an additional 2 hours. Water (1000 ml) was added to the reaction mixture, and the resulting mixture was concentrated under reduced pressure to remove methylene chloride. The resulting residue was carefully poured into saturated aqueous NaHCO ₃ (2000 ml). The solid was collected by filtration, washed with water (2x200 ml) and dried to obtain the first portion of the technical target product (16). The aqueous filtrate was extracted with methylene chloride (2x200ml). The combined organic extracts were dried over anhydrous Na ₂ SO ₄ , filtered and concentrated to obtain a second portion of the technical target product (16). The combined technical target product was purified by flash chromatography on silica gel (SiO ₂ , eluting with 0 to 50% ethyl acetate/hexane) to give the target product, 4chloro-5H-cyclopenta[b]pyridin-7(6H)-one (16, 87 g, 80% yield), as a yellow to brown oil that solidified when placed under vacuum. For 16: 1H NMR (300 MHz, DMSO-d ₆ ) δ 8.68 (d, J=6.0 Hz, 1H), 7.48 (d, J=6.0 Hz, 1H), 3.18 (m, 2H), 2.81 (m, 2H) ppm; LCMS (EI) m/z 168/170 (C8H6C1NO, (M+H)+).

Step 7. (R)-4-Chloro-6,7-dihydro-5H-cyclopenta[b]pyridin-7-ol (11)

To a solution of 4-chloro-5H-cyclopenta[b]pyridin-7(6H)-one (16, 138.0 g, 823.4 mmol) and triethylamine (TEA, 573.8 ml, 4117 mmol) in methylene chloride (1100 ml) added RuCl(p-cymene)[(R,R)-Ts-DPEN] (1.31 g, 2.06 mmol) at room temperature. The resulting mixture was degassed, cooled to 5-10°C and stirred at 5-10°C in a nitrogen atmosphere. Formic acid (155.3 mL, 4117 mmol) was then slowly added to the reaction mixture at 7-14° C. (internal temperature). After addition, the reaction mixture was gradually warmed to room temperature and stirred at room temperature for 22 hours. When LC/MS and HPLC indicated completion of the reaction, the reaction mixture was diluted with saturated aqueous sodium bicarbonate (NaHCO ₃ ) (1000 ml) and water (1500 ml ). The two layers were separated and the aqueous layer was extracted with methylene chloride (3x500 ml). The organic layers were combined, dried over anhydrous Na ₂ SO ₄ , filtered and concentrated to give the technical target product, (R)-4-chloro-6,7-dihydro-5H-cyclopenta[b]pyridin-7-ol (17, expected yield 100%) in the form of a yellow to brown oil, which was immediately used for the subsequent reaction without further purification. For 17: 1H NMR (300 MHz, DMSO6) δ 8.33 (d, J=6.0 Hz, 1H), 7.19 (d, J=6.0 Hz, 1H), 5.27 (m, 1H), 3.08 (m, 1H), 2.86 (m, 1H), 2.57 (m, 1H), 2.10 (m, 1H) ppm; LCMS (EI) m/z 170/172 (C8H8C1NO, (M+H)+).

Step 8. (R)-7-(tert-Butyldimethylsilyloxy)-4-chloro-6,7-dihydro-5H-cyclopenta[b]pyridine (18)

Tert. -butyldimethylsilyl chloride (172.25 g, 1015.6 mmol) and 1H-imidazole (101.3 g, 1472 mmol) at 0-5°C. The resulting reaction mixture was stirred at room temperature overnight. When LC/MS and HPLC indicated completion of the reaction, the reaction mixture was diluted with water (1000 ml). The two layers were separated and the aqueous layer was extracted with methylene chloride (3x500 ml). The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated to give the technical title product (18). Purification of the technical product by chromatography on silica gel (SiO2, elution with 0-10% ethyl acetate in hexanes) gave the target product, (R)-7-(tert-butyldimethylsilyloxy)-4-chloro-6,7dihydro-5H-cyclopenta[b] pyridine (18, 253.0 g, 87.8% yield over 2 steps) as a yellow to brown oil. For 18: 1H NMR (300 MHz, DMSO-d ₆ ) δ 8.36 (d, J=3.0 Hz, 1H), 7.13 (d, J=3.0 Hz, 1H), 5.21 (m, 1H), 3.08 (m, 1H), 2.81 (m, 1H), 2.43 (m, 1H), 2.05 (m, 1H), 0.93 (s, 9H) , 0.22 (s, 3H), 0.16 (s, 3H) ppm; LCMS (EI) m/z 284/286 (C^ClNOSi, (M+H)+).

Step 9. (R)-7-(tert-Butyldimethylsilyloxy)-4-chloro-6,7-dihydro-5H-cyclopenta[b]pyridine-3carbaldehyde (19)

A solution of 2.5 M n -butyllithium in hexane (180 ml, 450 mmol). The resulting mixture was heated to > -32°C and stirred at -32°C to 0°C for 30 minutes and then cooled to -78°C (external bath). To the mixture, cooled as described above, was added dropwise (R)-7-(tert-butyldimethylsilyloxy)-4-chloro-6,7-dihydro-5H-cyclopenta[b]pyridine

- 38 043661 (18, 106.0 g, 373 mmol) in anhydrous tetrahydrofuran (380 ml) at -74°C to -76°C (internal temperature). After stirring the resulting dark brown solution at -74°C (internal temperature) for 90 minutes, anhydrous N,N-dimethylformamide (DMF, 130 g, 1129 mmol) was added and the internal temperature was maintained at -76°C during addition. up to -70°C. The resulting reaction mixture was continued to stir at -74°C to -72°C for 2 hours, followed by dilution with 1 N aqueous HCl (500 ml) and water (500 ml). The two phases were separated and the aqueous phase was extracted with MTBE (2x250 ml). The combined organic phases were washed with brine (2x250 ml) and concentrated under reduced pressure to give technical product (19). Purification of the technical product by chromatography on silica gel (SiO ₂ , eluting with 0-8% ethyl acetate in hexane) gave the target product, (R)-7-(tert-butyldimethylsilyloxy)-4-chloro-6,7-dihydro-5Hcyclopenta[b ]pyridine-3-carbaldehyde (19, 110.7 g, 94% yield), as a yellow to brown oil. For 19: ^1H NMR (300 MHz, _CDCl3 ) δ 10.4 (s, 1H), 8.91 (s, 1H), 5.22 (m, 1H), 3.10 (m, 1H), 2.87 (m, 1H), 2.48 (m, 1H), 2.09 (m, 1H), 0.92 (s, 9H), 0.21 (s, 3H), 0.16 (s , 3H) ppm; LCMS (EI) m/z 312/314 (C15H22ClNO2Si, (M+H)+).

Step 10. (R)-7-(tert-butyldimethylsilyloxy)-4-chloro-6,7-dihydro-5H-cyclopenta[b]pyridine-3carbonitrile (20)

N

TBSO

To a stirred solution of (R)-7-(tert-butyldimethylsilyloxy)-4-chloro-6,7-dihydro-5Hcyclopenta[b]pyridine-3-carbaldehyde (19, 91.3 g, 292 mmol) in tetrahydrofuran (THF, 700 ml) at room temperature, add a solution of ammonia in water 14.8 M (350 ml, 5950 mmol) and iodine (I ₂ , 80.0 g, 315 mmol) in an ice-water bath (internal temperature controlled at 16 ° C to 22°C). The resulting reaction mixture was stirred at room temperature for 3 hours, followed by dilution with a 10% aqueous solution of Na ₂ S ₂ O ₃ (200 ml). The mixture was extracted with ethyl acetate (2500 ml). The combined organic extracts were washed with water (100 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give the title product, (R)-7(tert-butyldimethylsilyloxy)-4-chloro-6,7-dihydro- 5H-cyclopenta[b]pyridine-3-carbonitrile (20, 89.9 g, 98% yield) as a yellow to brown oil. For 20: 1H NMR (300 MHz, CDCl ₃ ) δ 8.68 (s, 1H), 5.22 (m, 1H), 3.09 (m, 1H), 2.84 (m, 1H), 2 .49 (m, 1H), 2.07 (m, 1H), 0.92 (s, 9H), 0.20 (s, 3H), 0.18 (s, 3H) ppm; LCMS (EI) m/z 309/311 (C ₁₅ H ₂₁ ClN ₂ OSi, (M+H) ⁺ ).

Example 6. Study of the stability of the phosphoric acid salt form I of compound 1.

Phosphoric acid salt form I of compound 1 was exposed to various environmental conditions to evaluate stability. The results are presented in table. 2 below. As can be seen from the data, Form I is highly stable even in the presence of heat and moisture. Only heating at 210°C showed possible conversion to another solid form, but the transformation was reversible when exposed to 40°C/75% relative humidity for 4 days.

table 2

Conditions Crystal Form (PRD) Suspension in 0.5% methylcellulose in water Form I 40 °C/75% r.h. within 1 week Form I vacuum drying at 50 °C for 1 day Form I vacuum drying at 100 °C for 1 day Form I vacuum drying at 210 °C for 20 min Not Form I vacuum drying at 210 °C and then 40 °C/75% r.h. within 4 weeks Form I

Example 8. Preparation of Phosphoric Acid Salt Form I of Compound 1.

Procedure A-isopropanol.

To a solution of compound 1 (25.68 mg, 0.05 mmol) in isopropanol (0.5 ml) was added 0.056 ml of a 0.1 M solution of phosphoric acid (0.12 mmol, 1.12 eq.) in isopropanol. The reaction mixture was stirred overnight. The resulting precipitate was collected by filtration and the filter cake was air dried to obtain Form I.

Procedure B-acetonitrile.

Compound 1 (50.35 mg, 0.216 mmol, 1 eq.) was combined with 0.1 ml of acetonitrile and the mixture was stirred for 2 minutes to obtain a clear solution. To the resulting solution was added 0.108 ml of a 1 M solution of phosphoric acid (0.108 mmol, 1.1 eq.) in isopropanol to obtain a viscous suspension. The reaction mixture was heated to 78°C and stirred for 2 hours (note: slurry), after which the reaction mixture was cooled to room temperature and stirred overnight. The resulting precipitate was collected by filtration, and the filter cake was dried to obtain Form I.

Karl Fischer titration showed that the salt of compound 1, obtained from acetonitrile, contains about 1.419% water.

- 39 043661

Procedure C-ethanol.

Compound 1 (50.30 mg, 0.216 mmol, 1 eq.) was combined with 1.2 ml ethanol and the mixture was stirred to obtain a clear solution. To the resulting solution was added 0.108 ml of a 1 M solution of phosphoric acid (0.108 mmol, 1.1 eq.) in isopropanol to obtain a suspension. The resulting reaction mixture was heated to 79°C and stirred for up to 2 hours, after which the reaction mixture was stirred at 81-83°C for 2 hours (note: slurry). The reaction mixture was cooled to room temperature and stirred for 2 hours. The resulting precipitate was separated by filtration and the filter cake was dried to give Form I (55 mg, 91.8%).

Procedure D-methanol.

Compound 1 (50 mg, 0.216 mmol, 1 eq.) was combined with 0.5 ml methanol to obtain a clear solution. To the resulting solution was added 0.95 ml of a 1 M solution of phosphoric acid (0.95 mmol, 1.25 eq.) in isopropanol to obtain a viscous suspension. Methanol (0.5 ml) was added and the mixture was stirred for 1 hour, after which the reaction mixture was heated to 78°C and stirred at this temperature for 2 hours (note: slurry). The resulting precipitate was collected by filtration and the filter cake was dried to give Form I (42.6 mg, 91.7%).

The stoichiometric ratio between the free base of compound 1 and phosphoric acid was determined by elemental analysis to be 1:1. Elemental analysis: Calculated for C ₂₆ H ₂₉ F ₃ N ₅ O ₇ P: C, 51.07; N, 4.78; N, 11.45; R, 5.07. Found: C, 49.23; N, 4.57; N, 10.85; R, 5.28.

Example 9. Study of the stability of form I of the phosphoric acid salt of compound 1 under phase equilibrium conditions at 25°C and 50°C.

Phase equilibrium studies were designed to obtain information on the predominant crystal form for phase identification. The phosphoric acid salt of compound 1 (Form I) was equilibrated in a representative group of solvents at 25±1°C (chloroform, DMF, 1,4dioxane, methanol and methanol/20% water, 2-methoxyethanol and 2-methoxyethanol/20% water , MIBK, THF and THF/20% water, acetone, n-BuOH and h-BuOH/20% water, EtOH and EtOH/20% water, isobutyl acetate, 1-propanol and 1-propanol/20% water, isopropanol, water and MEK ) and 50±1°C (chloroform, DMF, 1,4-dioxane, methanol and methanol/20% water, 2-methoxyethanol and 2-methoxyethanol/20% water, MIBK, THF and THF/20% water, acetone, n-BuOH and h-BuOH/20% water, EtOH and EtOH/20% water, EtOAc, ethyl formate, 1-propanol and 1propanol/20% water, isopropanol, IPA/MeOH/water (1.73/0.79/ 0.08), IPA/water (3/2), water) under the control of the IKA® ETS-D5 temperature controller and IKA® RCT basic safety control.

Compound 1 (Form I) phosphate was added to 3 ml of solvent or solvent mixture (selected from a list for appropriate temperatures) until a cloudy solution was obtained, then about 30 mg of Compound 1 (Form I) phosphate was added to the cloudy solution. The mixture was stirred at 25±1°C or 50±1°C, respectively, for 2-3 days. The solid was filtered and analyzed by PRD. The material was Form I for phase equilibrium at 25±1°C and 50±1°C in all solvents and solvent mixtures tested, which is the same as the phosphate parent material of Compound 1 (Form I).

Example 10. Preparation and characterization of the phosphoric acid salt form II of compound 1.

A saturated solution of phosphate of compound 1 form I (obtained in example 1, 20 ml) in DMF was evaporated in air without stirring at 25±1°C to obtain a solid, which was characterized by PRD, DSC and TGA as form II.

The experimental parameters for obtaining PXR data are the same as those described in example 2. The PXD diffraction pattern is shown in FIG. 7, and the PRD data are given in table. 3.

- 40 043661

The experimental parameters for obtaining DSC data are the same as those described in Example 3. The DSC thermogram is shown in FIG. 8 and displays an endothermic phenomenon at about 249°C.

The experimental parameters for obtaining TGA data are the same as those described in Example 4. The TGA thermogram is shown in FIG. 9.

Example 11. Preparation and characterization of the phosphoric acid salt form III of compound 1.

ml of a saturated solution of phosphate of compound 1 (form I) in DMF was evaporated in air without stirring at 50 ± 1°C to obtain a solid, which was characterized by PRD, DSC and TGA as form III.

The experimental parameters for obtaining PXR data are the same as those described in example 2. The PXD diffraction pattern is shown in FIG. 10, and the PRD data are given in table. 4.

Table 4

PRD form III data

2-theta (°) Height N% 4, 6 425 85, 1 7.1 50 9, 9 9, 4 129 25, 9 13.3 166 33.3 15, 7 89 17.8 16, 3 173 34.7 18.9 500 100 19, 2 289 57.9 21.2 290 58 22.5 220 44.1 23, 1 216 43.2 24.3 88 17.7 24.9 142 28.4 25, 6 65 13, 1 26, 7 165 33, 1 27.7 89 17.7 29, 1 61 12.1 30.4 62 12.4 33.4 41 8.2 34.2 66 13.2 35, 0 72 14.3 38.3 46 9, 3 38.8 57 11.5 43.4 43 8.7

The experimental parameters for obtaining DSC data are the same as those described in Example 3. The DSC thermogram is shown in FIG. 11 and shows an endothermic phenomenon at about 250°C.

The experimental parameters for obtaining TGA data are the same as those described in Example 4. The TGA thermogram is shown in FIG. 12.

Example 12.

Preparation and characterization of phosphoric acid salt form IV of compound 1.

ml of a saturated solution of phosphate compound 1 form I in water was evaporated in air without stirring at 50±1°C to obtain a solid, which was characterized by PRD, DSC and TGA as form IV.

The experimental parameters for obtaining PXR data are the same as those described in example 2. The PXD diffraction pattern is shown in FIG. 13, and the PRD data are given in table. 5.

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Table 5

PRD form IV data

2-theta (°) Height N% 4, 1 81 62.1 4.9 52 39, 9 6, 9 45 34.7 7.4 63 48.3 8.0 37 28, 6 11.0 44 34 12.8 38 29 13.3 96 73, 9 16, 4 130 100 17.7 78 59, 4 18.1 88 67.1 18, 6 102 77.8 19, 0 55 42 19, 8 118 90, 1 20, 6 42 32.3 21.4 114 87 22, 6 46 35.5 23.3 81 62 25, 0 61 46, 8 26, 7 46 35.5 35, 7 26 20.2 38.4 27 20.3

The experimental parameters for obtaining DSC data are the same as those described in Example 3. The DSC thermogram is shown in FIG. 14 and shows an endothermic phenomenon at about 245°C.

The experimental parameters for obtaining TGA data are the same as those described in Example 4. The TGA thermogram is shown in FIG. 15.

Example 13. Preparation and characterization of the phosphoric acid salt form V of compound 1.

100 ml of a saturated solution of compound 1 phosphate in water, obtained at 35°C, was quickly cooled to 4-5°C and kept at this temperature for 1 hour to obtain a fine suspension, which was filtered and air-dried for 1 hour. Solid the substance was identified as the V phosphate form of compound 1 and characterized by PRD, DSC and TGA.

The experimental parameters for obtaining PXR data are the same as those described in example 2. The PXD diffraction pattern is shown in FIG. 16, and the PRD data are given in table. 6.

Table 6

Form V PRD data

2-theta (°) Height N% 4.2 50 8.5 5, 5 53 9 7.3 301 51.4 9, 3 75 12.8 10.2 46 7, 8 10, 9 316 53, 9 11.9 50 8.5 12.7 72 12.4 13, 1 148 25, 2 14.7 84 14.4 16, 4 479 81.8 17.5 159 27.2 18.1 232 39, 6 18.5 586 100 19, 8 531 90, 6 20, 6 88 15 21.2 102 17.3 22, 6 244 41, 6 23, 1 91 15.5 23, 8 81 13, 9 24.7 120 20, 6 26, 1 252 43, 1 26, 7 161 27.5 30.5 116 19, 7 30, 8 61 10.5 31, 8 69 11.8 35, 1 86 14, 6 35.5 65 11.2 37.3 104 17.7 37.7 65 11.1 39, 7 53 9, 1 44.2 51 8.7

The experimental parameters for obtaining DSC data are the same as those described in Example 3. The DSC thermogram is shown in FIG. 17.

The experimental parameters for obtaining TGA data are the same as those described in Example 4. The TGA thermogram is shown in FIG. 18.

Example 14. Preparation and characterization of Form VI of the phosphoric acid salt of compound 1.

The phosphate of compound 1 was added to 150 ml of water to form a slurry, which was stirred for 2 hours to form a suspension. The suspension was filtered and the filtrate was cooled to 4-5°C and kept

-42043661 at 4-5°C for 3 days. The suspension was filtered to isolate a crystalline solid, which was characterized by PRD, DSC and TGA as Form VI.

The experimental parameters for obtaining PXR data are the same as those described in example 2. The PXD diffraction pattern is shown in FIG. 19, and the PRD data are given in table. 7.

Table 7

DRP Form VI data

2-theta (°) Height N% 3.7 133 21.2 4.1 222 35, 4 6, 5 629 100 8.3 437 69.5 10, 7 216 34.4 13, 2 230 36, 6 14, 6 171 27.3 16, 1 61 9, 7 17.3 263 41.8 18.3 59 9, 4 19, 1 444 70.7 20, 1 171 27.2 20, 8 77 12.2 21.5 130 20.7 21.8 74 11.7 22.8 59 9, 4 24.1 85 13.5 25, 1 114 18.1 26, 6 108 17.2 27.1 74 11.7 27.8 83 13.2 30, 8 93 14.8 34.5 47 7.5 36, 2 80 12.7 38.5 60 9, 5 40, 2 54 8.7 40, 8 49 7.8 44, 6 45 7.2

The experimental parameters for obtaining DSC data are the same as those described in Example 3. The DSC thermogram is shown in FIG. 20.

The experimental parameters for obtaining TGA data are the same as those described in Example 4. The TGA thermogram is shown in FIG. 21.

Example 15 Investigation of phosphate polymorphism of compound 1 by addition of antisolvent.

Prepare saturated solutions of compound 1 phosphate by adding compound 1 phosphate (Form I) to DMF, MeOH, MeOH/20% water and DMSO, respectively. An antisolvent was added to initiate precipitation. MTBE, IP Ac, EtOAc, MeCN, MIBK, MEK and toluene were chosen as antisolvents. Experiments that did not produce any particulate matter upon addition of the antisolvent were not examined further.

Upon addition of antisolvent (see Table 8), Form I was identified from MeOH/IPAc, MeOH/EtOAc, MeOH/MEK, aq. MeOH/MTBE, DMSO/MeCN, DMSO/IPAc and DMSO/MEK. Form II was identified from MeOH/MeCN, aq. MeOH/MeCN and water. MeOH/IPAc. The amorphous form was found from MeOH/TBME and MeOH/MIBK.

The X-ray diffraction pattern for amorphous phosphate of compound 1 is shown in Fig. 23.

Table 8

Solvent (ml) Antisolvent (ml) Solid state form DMF (2 ml) IPAc (8 ml) n/a DMF (2 ml) MTBE (8 ml) n/a MeOH (2 ml) MTBE (8 ml) amorphous MeOH (2 ml) IPAc (8 ml) Form I MeOH (2 ml) EtOAc (8 ml) Form I MeOH (2 ml) CH ₂ C1 ₂ (8 ml) Clear solution MeOH (2 ml) MeCN (8 ml) Form II MeOH (2 ml) MIBK (8 ml) amorphous MeOH (2 ml) MEK (8 ml) Form I MeOH/20% water (2 ml) MeCN (8 ml) Form II MeOH/20% water (2 ml) MTBE (8 ml) Form I MeOH/20% water (2 ml) IPAc (8 ml) Form II DMSO (0.5 ml) MeCN (5 ml) Form I DMSO (0.5 ml) IPAc (5 ml) Form I DMSO (0.5 ml) MEK (5.5 ml) Form I DMSO (0.5 ml) Acetone (8 ml) n/a DMSO (0.5 ml) Toluene (6 ml) n/a

Example 16: Investigation of Compound 1 Phosphate Polymorphism by Reverse Addition.

We prepared saturated solutions of phosphate of compound 1 in DMF, MeOH, MeOH/20% water and DMSO, shown in table. 9, and added to a larger volume of miscible antisolvent (ie, MTBE, IP Ac, EtOAc, MeCN). Experiments that did not produce any particulate matter when added to the antisolvent were not examined further. From most solvents, Form I was identified, except that Form II was identified from MeOH/MeCN, aq. MeOH/MeCN and aq. MeOH/IPAc. The amorphous solid was identified from MeOH/MTBE, MeOH/EtOAc and

MeOH/MIBK.

Table 9

Solvent (ml) Antisolvent (ml) Solid state form DMF (2 ml) IPAc (8 ml) n/a DMF (2 ml) MTBE (8 ml) n/a MeOH (2 ml) MTBE (8 ml) Amorphous MeOH (2 ml) IPAc (8 ml) Form I MeOH (2 ml) EtOAc (8 ml) amorphous MeOH (2 ml) CH ₂ C1 ₂ (8 ml) n/a MeOH (2 ml) MeCN (8 ml) Form II MeOH (2 ml) MIBK (8 ml) Amorphous MeOH (2 ml) MEK (8 ml) Form I MeOH/20% water (2 ml) MeCN (8 ml) Form II MeOH/20% water (2 ml) MTBE (8 ml) Form I MeOH/20% water (2 ml) IPAc (8 ml) Form II DMSO (0.5 ml) IPAc (5 ml) Form I DMSO (0.5 ml) MeCN (5 ml) Form I DMSO (0.5 ml) MEK (5.5 ml) Form I DMSO (0.5 ml) Acetone (8 ml) n/a DMSO (0.5 ml) Toluene (6 ml) n/a

Example 17 Phosphate Test of Compound 1 by Rapid Cooling.

Saturated phosphate solutions of Compound 1 prepared at 35°C were rapidly cooled to about -20°C to -25°C to initiate precipitation of higher energy crystalline forms. Representative solvents were selected based on solubility data measured at 25°C and 50°C (see example 7). The investigated solvents and crystalline forms obtained from the experiments are given in Table. 10. Form I was identified from aq. THF and aq. EtOH. Form V and Form VI were identified from water.

Table 10

Solvent (ml) Solid state form DMF Clear solution MeOH Clear solution MeOH/20% water Clear solution 2-methoxyethanol Clear solution 2-methoxyethanol/20% water Clear solution THF/20% water Form I EtOH/20% water Form I 1-RgOH/20% water Clear solution 2-RgOH/20% water Clear solution Water (chilled to 4-5 °C) Form V Water (chilled to 4-5 °C) Form VI

Example 18 Study of phosphate polymorphism of compound 1 using heating and cooling cycles.

This experiment was designed to search for stable crystalline forms. Prepare saturated solutions at 50°C and cool them slowly in a bath using a programmable circulating bath to obtain a clear solution for all solvents. About 10 mg of Compound 1 Form I phosphate was added to the clear solution to form a slurry. The resulting slurry was then heated to 50°C over 2 hours and then cooled to 5°C over 2 hours. This process was repeated for 3 days and the solid was filtered for further analysis.

The results are presented in table. 11. Form I was identified for all samples.

Table 11

Solvent (mp) Solid state form DMF Form I Methanol Form I Methanol/20% water Form I 2-Methoxyethanol Form I 2-Methoxyethanol/20% water Form I THF/20% water Form I n-ViON/10% water Form I EtOH/20% water Form I n-propanol/20% water Form I IPA/30% water Form I Water Form I

Example 19 Phosphate polymorphism study of compound 1 using evaporation.

Evaporation studies were conducted to identify the predominant crystalline form during uncontrolled precipitation. Experiments that did not result in any particulate matter (i.e., clear thin films and oils) were not further investigated (n/a). To characterize the morphology of the solid state of the crystalline forms of the samples obtained by evaporation at 25±1°C and 50±1°C under the control of the IKA® ETS-D5 temperature controller and IKA® RCT basic safety control, PRD was used.

The results are presented in table. 12 (25±1°С) and table. 13 (50±1 °C). Evaporation at 25+1 °C (Table 12) resulted in polymorphic form II (DMF). Evaporation at 50±1°C (Table 13) resulted in two polymorphic forms, including Form III (DMF) and Form IV (water).

-44043661

Table 12

Evaporation at 25±1°С

Solvent (ml) Solid state form MeCN n/a Chloroform n/a Dichloromethane n/a DMF Form II 1,4-Dioxane/5% water n/a 1,4-Dioxane n/a Methanol n/a Methanol/20/water Form I 2-Methoxyethanol Form I MIBK n/a Toluene n/a Hexane n/a THF n/a Acetone n/a n-ViON n/a MTBE n/a DMSO Form I EtOH n/a EtOAc n/a Ethyl formate n/a Heptane n/a Isobutyl acetate n/a IPAc n/a 1-Propanol n/a IPS n/a Water n/a MEK n/a

Table 13

Evaporation 50±1°С

Solvent (ml) Solid state form MeCN n/a Chloroform n/a DMF Form III 1,4-Dioxane n/a Methanol Form I MeOH/20% water Form I 2-Methoxyethanol Form I MIBK n/a Toluene n/a Hexane n/a THF n/a Acetone n/a n-ViON n/a MTBE n/a DMSO Form I EtOH n/a EtOAc n/a Ethyl formate n/a Heptane n/a Isobutyl acetate n/a IPAc n/a 1-Propanol n/a IPS n/a Water Form IV MEK n/a

Example 20 Comparative stability study of the crystalline phosphate solid of Compound 1 in IPA/MeOH/water/DMSO.

To evaluate the transformation of solid forms of phosphate of compound 1, comparative experiments were carried out with suspensions as follows: to a saturated solution (1.5 ml) of phosphate of compound 1, form I, in the solvent given in table. 14, Form I (6 mg) was added followed by stirring to obtain a cloudy solution, then 6 mg of each of Form II to Form VI was added to the mixture. The suspension was stirred for 2 days at room temperature and analyzed by PRD. The results shown in table. 14 showed that the phosphate form I of compound 1 was the most stable form in each of the IPA/methanol/water/DMSO mixtures.

Table 14

Solvent (ml) Solid state form IPA/MeOH/water/DMSO (61.8/28.2/2.9/7.1) Form I IPA/MeOH/water/DMSO (56.6/28.3/3.0/12.1) Form I

Example 22 Karl Fischer titration of crystalline solid phosphate forms of compound 1.

The results of Karl Fischer titration of polymorphic forms I-VI of phosphate of compound 1 are presented in table. 15.

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Table 15

Solid state form % Water Notes Form I 1.5 Dried according to Good Manufacturing Practices Form II 1.4 Sample sealed in ampoule Form III 1.4 Sample sealed in ampoule Form IV thirty Sample sealed in ampoule Form V 2.4 Sample sealed in ampoule Form VI 15, 9 Almost fresh sample

Karl Fischer analysis of the phosphate forms I-VI of compound 1 was carried out. Each experiment showed water content ranging from 1.40-15.9%, which shows that each of the forms I-VI can be hydrated.

Example 23. Preparation and determination of the characteristics of the salt of compound 1 with two molecules of hydrochloric acid.

Compound 1 (55.2 mg, 0.107 mmol) was combined with 0.7 ml isopropyl alcohol (IPA) and stirred for 2 min to obtain a clear solution. A solution of hydrochloric acid (0.25 ml, 0.25 mmol, 2.34 eq; 1 M HCl in IPA/IPAc from 3.7 M HCl in IPAc) was added to form a slurry, which was heated to 50 °C and stirred for 15 min. The resulting mixture was cooled to room temperature, stirred overnight, filtered and dried under vacuum (6 hours) to obtain the final product (61.8 mg, 98%).

The stoichiometric ratio between compound 1 and hydrochloric acid was determined by elemental analysis to be 1:2. 1H NMR showed the salt to contain 7.8% isopropanol, and Karl Fischer titration showed a water content of about 0.586%. Elemental analysis: Calculated for C29H3sCl2F3NsO4: C, 53.67; N, 5.35; N, 10.80; Cl, 10.95. Found: C, 53.26; N, 5.21; N, 10.57; Cl, 10.83.

The salt of compound 1 with two hydrochloric acid molecules was characterized by PRD, DSC, and TGA. The experimental parameters for obtaining PXR data are the same as those described in example 2. The PXD diffraction pattern is shown in FIG. 4. The experimental parameters for obtaining DSC data are the same as described in example 3. The DSC thermogram is shown in Fig. 5. The DSC thermogram showed a major endothermic peak at about 213°C. The experimental parameters for obtaining TGA data are the same as those described in Example 4. The TGA thermogram is shown in FIG. 6.

PDP data are presented in table. 17.

Example 24. Preparation and determination of the characteristics of the salt of compound 1 with one molecule of hydrochloric acid.

To a solution of the free base of compound 1 (see example 1, step 6; 0.05 mmol, 25.68 mg) in isopropanol (0.5 ml, 0.1 M) was added 0.056 ml of hydrochloric acid (0.056 mmol, 1.12 eq., 1.0 M solution in IPA/IPAc, prepared from 3.7 M HCl in IPAc (isopropyl acetate)). The reaction mixture was stirred overnight. The resulting precipitate was removed by filtration, the filter cake was washed with MTBE, and the solid was dried under vacuum overnight to give the title salt.

The salt of compound 1 with one molecule of hydrochloric acid was characterized by PRD and DSC. The XRD diffraction pattern is shown in Fig. 22. The experimental parameters for obtaining DSC data are the same as described in example 3. The DSC thermogram is shown in FIG. 23.

PDP data are presented in table. 18.

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Table 18

2-theta (°) Height N% 3.5 102 27.3 4.0 105 28 7.8 126 33, 6 8.8 134 35, 7 11.9 57 15, 3 12, 6 121 32.2 14.5 138 36, 8 15, 0 47 12.5 15, 9 70 18.8 16, 6 44 11, 6 17.4 113 30.2 19, 7 74 19, 8 20.4 59 15, 8 20, 6 54 14.4 22.3 74 19, 9 23, 8 186 49, 6 24, 6 84 22.3 25, 2 375 100 26, 3 80 21.4 26.5 82 21.8 27.4 38 10.2 28.8 90 24 29, 2 72 19.2 31.1 75 19, 9 31.5 44 11.7 31.7 42 11.3 34.5 69 18.3 37, 6 46 12.4 40, 9 43 11.4 41.3 32 8, 6 41.8 34 8.9

Example 25. Preparation and characterization of the maleic acid salt of compound 1.

A volume of 1.0 mL of isopropanol was added to the free base of Compound 1 (50.30 mg, 0.216 mmol, 1 eq.). The resulting mixture was stirred to obtain a clear solution. Maleic acid (14.2 mg, 0.122 mmol, 1.21 eq.) was added to this solution and the resulting reaction mixture was stirred to obtain a clear solution. Stirring was continued for 1 hour. To this solution was added 1 mg of crystals (seeds) obtained from IPA/heptane, and the resulting mixture was stirred to form a slurry. The slurry was stirred continuously for 3 hours. The precipitate was removed by filtration, and the filter cake was washed with MTBE and dried under vacuum overnight to obtain the title salt (56.8 mg, 89.2%).

The stoichiometric salt ratio between the free base of compound 1 and maleic acid was determined by Ή NMR to be 1:1. The crystallinity of the maleate of compound 1 was confirmed by PRD, DSC and TGA.

The experimental parameters for obtaining PXR data are the same as those described in example 2. The PXD diffraction pattern is shown in FIG. 24. The experimental parameters for obtaining DSC data are the same as described in example 3. The DSC thermogram is shown in FIG. 25. The experimental parameters for obtaining TGA data are the same as described in example 4. The TGA thermogram is presented in FIG. 26.

PDP data are presented in table. 19.

-47043661

Table 19

2-theta (°) Height N% 5.2 108 3, 9 9, 0 762 27.4 9, 5 851 30, 6 10.4 504 18.1 11.2 1255 45, 1 12.8 204 7.3 14.8 632 22.7 15, 9 748 26, 9 17.0 323 11.6 17.8 460 16.5 18.5 2781 100 19, 5 687 24.7 19, 9 1036 37.2 20, 9 1007 36.2 21.3 1421 51.1 22.9 2122 76, 3 23, 6 538 19, 3 24.4 575 20.7 24.8 1904 68.5 25, 8 1638 58.9 27, 6 1166 41.9 29, 2 492 17.7 30, 9 1025 36, 9 32.5 268 9, 7 33.4 97 3.5 34.2 429 15, 4 35, 6 252 9 36, 0 152 5, 5 36, 7 200 7.2 37, 6 115 4.2 38.2 61 2.2 38, 6 77 2.8 39, 9 123 4.4 40.7 220 7.9 41, 6 251 9 42.3 471 16.9 43.2 328 11.8 43, 6 161 5, 8

Example 26. Preparation and characterization of the adipic acid salt of compound 1.

A volume equal to 0.6 ml of isopropanol was added to the free base of compound 1 (37.8 mg, 0.216 mmol 1 eq) and the resulting mixture was stirred for 1 min to obtain a clear solution. Adipic acid (26.8 mg, 0.183 mmol, 2.49 eq) was added to the solution and the resulting slurry was stirred at room temperature for 5 minutes. The reaction mixture was heated to 50°C and stirred at this temperature for 15 minutes. (note: clear solution). The reaction mixture was cooled to room temperature and stirred for 3 hours. Heptane (0.2 ml) was added and the reaction mixture was stirred to form a slurry, which was stirred continuously overnight. The precipitate was removed by filtration and the filter cake was washed with MTBE, collected and dried under vacuum overnight to give the title salt (36.5 mg, 84.5% yield).

The stoichiometric salt ratio between the free base of compound 1 and adipic acid was determined by Ή NMR to be 1:1. The crystallinity of the adipate of compound 1 was confirmed by PRD, DSC and TGA. The experimental parameters for obtaining PXR data are the same as those described in example 2. The PXD diffraction pattern is shown in FIG. 27. The experimental parameters for obtaining DSC data are the same as described in example 3. The DSC thermogram is presented in Fig. 28. The experimental parameters for obtaining TGA data are the same as those described in Example 4. The TGA thermogram is shown in FIG. 29.

PDP data are presented in table. 20.

Table 20

2-theta (°) Height N% 3, 8 169 10 9, 0 443 26, 3 9, 3 1025 60.8 10, 1 235 14 12.0 267 15, 8 12.5 62 3.7

-48043661

13.3 110 6, 5 15, 0 754 44.7 16, 2 464 27.5 17, 6 368 21.8 18.7 792 46, 9 20, 0 1687 100 21.2 147 8.7 22.1 708 42 22.7 415 24.6 24.3 1154 68.4 24.9 766 45, 4 26, 9 254 15, 1 27.1 425 25.2 28.7 337 20 30.2 75 4.5 31.7 124 7.3 33.7 140 8.3 35, 0 95 5, 6 35, 6 58 3.4 36, 3 71 4.2 36, 7 77 4, 6 38.2 119 7 40.5 62 3.7 41.8 168 9, 9 43.2 100 5, 9

Example 27. Preparation and characterization of the hydrobromic acid salt of compound 1.

To a 0.1 M solution of the free base of compound 1 (0.05 mmol, 25.68 mg) in isopropanol (0.5 ml) was added 0.12 ml hydrobromic acid (0.12 mmol, 2.4 eq., 1. 0 M solution in isopropanol/water). The resulting mixture was stirred overnight to obtain a slurry. The precipitate was removed by filtration and the filter cake was washed with MTBE and dried under vacuum overnight to obtain the desired product.

The experimental parameters for obtaining PXR data are the same as those described in example 2. The PXD diffraction pattern is shown in Fig. 30. The experimental parameters for obtaining DSC data are the same as described in example 3. The DSC thermogram is shown in FIG. 31. The experimental parameters for obtaining TGA data are the same as those described in Example 4. The TGA thermogram is shown in FIG. 32.

PDP data are presented in table. 21.

Table 21

2-theta (°) Height N% 3, 8 171 55, 4 4.2 142 46.2 6, 5 99 32.3 9, 5 93 30.3 10, 1 41 13.2 10, 9 47 15, 1 12.9 189 61.3 13.2 102 33.2 13, 9 64 20, 6 15, 6 69 22.4 16, 6 263 85, 3 17.9 143 46, 6 18.9 48 15, 6 19, 5 201 65.2 19, 9 77 24.9 21.7 101 33 22.5 308 100 23, 0 111 35, 9 23.7 179 58.2 24.3 188 61 26.5 256 83.1 27.5 239 77.5 28.3 135 43.8 30, 6 48 15.5 32.5 49 15, 9 33, 6 77 25, 1 33, 9 86 27.8 35, 3 50 16, 3

Example 28. Preparation and characterization of the mandelic acid salt of compound 1.

To a 0.1 M solution of the free base of compound 1 (0.05 mmol, 25.68 mg) in IPA (0.5 ml) was added mandelic acid (8.1 mg, 0.053 mmol, 1.06 eq). The mixture was stirred overnight. The suspension was filtered and washed with MTBE to obtain the mandelate salt of compound 1, which was analyzed by PRD, DSC and TGA.

The experimental parameters for obtaining PXR data are the same as those described in example 2. The PXD diffraction pattern is shown in FIG. 33. The experimental parameters for obtaining DSC data are the same as described in example 3. The DSC thermogram is presented in Fig. 34. The experimental parameters for obtaining TGA data are the same as those described in Example 4. The TGA thermogram is shown in FIG. 35.

PDP data are presented in table. 22.

- 49 043661

Table 22

2-theta (°) Height N% 4.0 190 9, 7 4.8 54 2.7 8.7 89 4.5 11.2 753 38.3 12.0 286 14.5 13, 8 207 10, 6 14.9 359 18.3 15, 7 142 7.2 16, 7 206 10.5 17.3 222 11.3 18, 6 788 40.1 20.2 107 5, 4 20, 6 379 19, 3 22.5 537 27.3 24.1 1965 100 25, 3 338 17.2 26, 6 221 11.3 27.9 118 6 28, 6 162 8.2 29, 4 124 6, 3 30.2 60 3 31.0 138 7 31.5 59 3 32.7 91 4, 6 33.5 110 5, 6 35, 7 125 6, 4 37.5 103 5, 2 38, 6 129 6, 6 39, 3 126 6, 4 40, 9 112 5, 7 42.3 85 4.4 44.0 113 5, 7

Example 29. Salicylic acid salt of compound 1.

To a 0.1 M solution of the free base of compound 1 (0.05 mmol, 25.68 mg) in isopropyl alcohol (IPA) (0.5 ml) was added salicylic acid (0.058 mmol, 8.01 mg, 1.16 eq. ). The resulting reaction mixture was stirred overnight to form a slurry. The precipitate was removed by filtration, and the filter cake was washed with methyl tert-butyl ether (MTBE) to obtain the salicylic acid salt of compound 1, which was characterized by PRD and DSC.

The experimental parameters for obtaining PXR data are the same as those described in example 2. The PXD diffraction pattern is shown in FIG. 36. The experimental parameters for obtaining DSC data are the same as described in example 3. The DSC thermogram is shown in FIG. 37.

PDP data are presented in table. 23.

- 50 043661

Table 23

2-theta (°) Height N% 8.7 58 2.7 9, 7 77 3, 5 10, 9 234 10.7 11.3 406 18.7 11.8 609 28 13.2 80 3.7 13, 6 48 2.2 14.5 163 7.5 14.8 366 16, 8 15, 6 231 10, 6 16, 7 288 13.2 18.3 143 6, 6 18.7 422 19, 4 19, 9 161 7.4 20, 6 58 2.7 21.2 735 33.8 21.9 1536 70, 6 23, 0 1123 51.6 23.4 2176 100 24.1 716 32.9 25, 1 501 23 25, 8 93 4.3 26, 3 410 18.8 27.1 57 2, 6 27.7 151 6, 9 28.1 142 6, 5 29, 4 243 11.1 29, 9 63 2.9 31.3 131 6 32.9 110 5 35, 7 84 3, 9 36.5 120 5, 5 36, 8 231 10, 6 37.9 170 7.8 39, 4 57 2, 6 42, 0 185 8.5 42, 6 139 6, 4 44.0 66 3 44.4 62 2.9

Example 30. Benzoic acid salt of compound 1.

To a 0.1 M solution of the free base of compound 1 (0.05 mmol, 25.68 mg) in isopropyl alcohol (IPA) (0.5 ml) was added benzoic acid (7.05 mg, 0.0577 mmol, 1.16 eq.). The resulting reaction mixture was stirred overnight to form a slurry. The precipitate was removed by filtration, and the filter cake was washed with methyl tert-butyl ether (MTBE) to obtain the benzoic acid salt of compound 1, which was analyzed by PRD.

The experimental parameters for obtaining PXR data are the same as those described in example 2. The PXD diffraction pattern is shown in FIG. 38.

PDP data are presented in table. 24.

Example 31. Benzenesulfonic acid salt of compound 1.

To a 0.1 M solution of the free base of compound 1 (0.05 mmol, 25.68 mg) in isopropyl alcohol (IPA) (0.5 ml) was added benzenesulfonic acid (0.11 ml 0.5 M in isopropyl alcohol,

- 51 043661

0.055 mmol, 1.1 eq.). The resulting reaction mixture was stirred overnight to obtain a slurry. The precipitate was removed by filtration to obtain the besylate salt of compound 1, which was characterized by PRD.

The experimental parameters for obtaining PXR data are the same as those described in example 2. The PXD diffraction pattern is shown in FIG. 39.

PDP data are presented in table. 25.

Table 25

2-theta (°) Height N% 3, 9 156 20 6, 6 406 52.1 9, 1 188 24.1 12.0 109 14 12.9 306 39.2 13.3 267 34.3 14.5 237 30.4 15, 7 56 7.1 16, 2 162 20.7 18.0 780 100 19, 2 197 25.2 19, 8 109 14 20.5 94 12 21.2 125 16, 1 21.8 182 23.3 23.5 356 45, 7 23, 9 522 66, 9 25, 2 56 7.1 26, 1 79 10.1 28.4 89 11.4 29, 7 148 18.9 31, 6 65 8.4 32.9 46 5, 8 35, 9 45 5, 7 36, 4 69 8.8 39, 8 47 6 42.8 71 9, 1 43.7 52 6, 6

Example 32. L-pyroglutamic acid salt of compound 1.

To a 0.1 M solution of the free base of compound 1 (0.05 mmol, 25.68 mg) in isopropyl alcohol (IPA) (0.5 ml) was added L-pyroglutamic acid (7.25 mg, 0.056 mmol, 1.12 eq.). The resulting reaction mixture was stirred for 5 hours, after which heptane (0.3 ml) was added. The reaction mixture was stirred continuously overnight to form a slurry. The precipitate was removed by filtration, and the filter cake was washed with methyl tert-butyl ether (MTBE) to obtain the L-pyroglutamic acid salt of compound 1, which was characterized by PRD.

The experimental parameters for obtaining PXR data are the same as those described in example 2. The PXD diffraction pattern is shown in FIG. 40.

PDP data are presented in table. 26.

Table 26

2-theta (°) Height N% 4.4 344 32.7 8.3 49 4, 6 9, 2 86 8.2 10.7 343 32.6 11.5 1052 100 12.5 74 7.1 13.5 176 16, 7 15, 0 102 9, 7 15, 4 202 19.2 16, 1 100 9, 5 17.5 73 6, 9 18.0 365 34.7 19, 0 145 13.8 19, 8 72 6, 8 20.7 576 54.7 21.2 706 67.1 22.9 539 51.2 23.7 66 6, 3 24.5 139 13.2 25, 2 98 9, 3 26, 4 124 11.8 29.2 158 15 32.4 85 8.1 33, 0 47 4.4 34.0 49 4, 6 41.3 51 4.9 41.9 46 4.4

Example 33. Methanesulfonic acid salt of compound 1.

To a 0.1 M solution of the free base of compound 1 (0.05 mmol, 25.68 mg) in isopropyl alcohol (IPA) (0.5 ml) was added methanesulfonic acid (0.055 mmol, 1.1 eq., 0.055 ml 1, 0 M solution in EtOH). The resulting reaction mixture was stirred for 5 hours, after which heptane (0.3 ml) was added to the reaction mixture. The reaction mixture was stirred continuously for 24 hours to obtain a slurry. The precipitate was removed by filtration to obtain the mesylate salt of compound 1, which was analyzed by PRD.

The experimental parameters for obtaining PXR data are the same as those described in example 2. The PXD diffraction pattern is shown in Fig. 41.

PDP data are presented in table. 27.

Table 27

2-theta (°) Height N% 3, 6 143 18.6 6, 4 60 7.8 9, 6 55 7.1 10.4 44 5, 7 12.7 222 28.9 13.5 650 84.6 14.7 528 68.7 15, 1 135 17, 6 16, 7 598 77.8 18, 6 425 55, 3 19, 3 697 90.7 20, 0 697 90.8 20.7 434 56.5 21, 6 81 10, 6 22.1 214 27.9 22.4 146 19 23, 0 295 38.5 24.1 447 58.2 24.4 768 100 24.8 234 30.4 25, 7 354 46.1 26, 8 434 56.5 27.2 715 93.1 28.1 364 47.3 29, 4 240 31.3 30, 1 236 30.7 31.5 138 17.9 32.1 140 18.2 34.1 236 30.8 35, 0 94 12.2 35, 4 71 9, 2 36, 2 120 15, 6 37.4 196 25.5 38.0 73 9, 5 38.8 183 23.8 39, 8 90 11.7 40, 1 61 8 40.4 59 7.7 41.0 76 9, 9 41.4 54 7 41.9 70 9, 1 42.1 49 6, 4 42.9 67 8.7 43.4 108 14

Example 34. (18)-(+)-10-camphorsulfonic acid salt of compound 1.

To a 0.1 M solution of the free base of compound 1 (0.05 mmol, 25.68 mg) in isopropyl alcohol (IPA) (0.5 ml) was added (lS)-(+)-10-camphorsulfonic acid (0.055 mmol, 1.1 equiv., 0.11 mL 0.5 M solution in IPA) (CAS registration number 3144-16-9; Aldrich catalog number C2107-500G). The resulting reaction mixture was stirred for 5 hours, after which heptane (0.3 ml) was added to the reaction mixture. The reaction mixture was stirred continuously overnight to obtain a slurry. The precipitate was removed by filtration to obtain the camsylate salt of compound 1, which was analyzed by PRD.

The experimental parameters for obtaining PXR data are the same as those described in example 2. The PXD diffraction pattern is shown in FIG. 42.

PDP data are presented in table. 28.

-53 043661

Table 28

2-theta (°) Height N% 3.7 140 25, 9 6, 6 232 43 7.1 539 100 10, 9 249 46, 1 13, 6 249 46.2 16, 1 292 54.1 17.7 372 69 18.8 188 34.8 19, 9 500 92.7 21.3 70 12.9 23.2 321 59, 6 25, 6 90 16, 7 28.4 124 22.9 29, 6 62 11.5 31.7 60 11.1 35, 1 99 18.4 43.5 64 11.8

Example 35. Fumaric acid salt of compound 1.

To a 0.1 M solution of the free base of compound 1 (0.05 mmol, 25.68 mg) in isopropyl alcohol (IPA) (0.5 ml) was added maleic acid (6.69 mg, 0.058 mmol, 1.16 eq. ). The reaction mixture was stirred for 5 hours, after which heptane (0.3 ml) was added to the reaction mixture. The reaction mixture was stirred continuously overnight to obtain a slurry. The precipitate was removed by filtration to obtain the fumarate salt of compound 1, which was analyzed by PRD.

The fumaric acid salt of compound 1 was characterized by PRD. The experimental parameters for obtaining PXR data are the same as those described in example 2. The PXD diffraction pattern is shown in FIG. 43.

PDP data are presented in table. 29.

Table 29

2-theta (°) Height N% 3, 8 397 77.6 5, 2 120 23.4 7.3 511 100 10.4 89 17.4 11, 6 74 14.5 13.2 145 28.4 16.2 68 13.3 17.1 126 24.6 20, 8 239 46, 7 24.3 216 42.2 25, 6 81 15, 9 27.8 78 15, 3 31.1 71 13.9 41.9 52 10.1

Example 36. Sulfuric acid salt of compound 1.

To a 0.1 M solution of the free base of compound 1 (0.05 mmol, 25.68 mg) in IPA (0.5 ml) was added sulfuric acid (0.055 mmol, 1.1 eq., 0.055 ml of a 1.0 M solution in IPS). The reaction mixture was stirred overnight to obtain a slurry. The precipitate was removed by filtration to obtain the sulfate salt of compound 1, which was characterized by PRD.

The sulfuric acid salt of compound 1 was characterized by PRD. The experimental parameters for obtaining PXR data are the same as those described in example 2. The PXD diffraction pattern is shown in FIG. 44.

PDP data are presented in table. 16.

Table 16

2-theta (°) Height N% 3, 6 185 100 6, 3 169 91.8 8.7 89 48.3 10, 8 54 29, 1 12.8 51 27.6 13.7 57 31.1 15, 4 132 71.7 19, 0 152 82.1 20.2 114 62 21.2 85 46 21, 6 114 61.9 23, 1 76 40.9 24.0 62 33.4 24.8 68 36, 6 25, 4 59 31.8 27.1 110 59, 6 27.2 111 60.4

Example 37. L-tartaric acid salt of compound 1.

To a 0.1 M solution of the free base of compound 1 (0.05 mmol, 25.68 mg) in isopropyl alcohol (IPA) (0.5 ml) was added L-tartaric acid (8.71 mg, 0.058 mmol, 1.16 eq.). Reaction mixture

-54043661 was stirred for 5 hours, after which heptane (0.3 ml) was added to the reaction mixture. The reaction mixture was stirred continuously overnight to obtain a slurry. The precipitate was removed by filtration to obtain the L-tartrate salt of compound 1, which was analyzed by PRD.

The L-tartaric acid salt of compound 1 was obtained as an amorphous solid. The experimental parameters for obtaining PXR data are the same as those described in example 2. The PXD diffraction pattern is shown in FIG. 45.

Example 38. D-tartaric acid salt of compound 1.

To a 0.1 M solution of the free base of compound 1 (0.05 mmol, 25.68 mg) in IPA (0.5 ml) was added D-tartaric acid (8.64 mg, 0.058 mmol, 1.16 eq). The reaction mixture was stirred for 5 hours, after which heptane (0.3 ml) was added to the reaction mixture. The reaction mixture was stirred continuously overnight to obtain a slurry. The precipitate was removed by filtration to obtain the D-tartrate salt of compound 1, which was analyzed by PRD.

The D-tartaric acid salt of compound 1 was obtained as an amorphous solid (PRD diffraction pattern is shown in Fig. 46).

Example A: Pim Enzyme Assays.

Pim-1 and Pim-3 kinase assays - 20 µl reactions can be performed in white 384-well polystyrene plates dotted with 0.8 µl compound/DMSO in assay buffer (50 mM Tris, pH 7.5, 0.01% Tween-20, 5 mM MgCl ₂ , 0.01% BSA, 5 mM DTT) containing 0.05 μM biotin-labeled peptide substrate BAD (AnaSpec 62269), 1 mM ATP, and 2.5 pM enzyme ( Pim-1, Invitrogen PV3503) or 1.25 pM (Pim-3, Millipore 14-738) for 1 hour at 25°C. Reactions are stopped by adding 10 μl of stopping buffer (150 mM Tris, pH = 7.5, 150 mM NaCl, 75 mM EDTA, 0.01% Tween-20, 0.3% BSA) supplemented with Phospho-Bad antibody (Serll2) ( Cell Signaling 9291) diluted 666-fold, and streptavidin donor beads (PerkinElmer 6760002) along with Protein-A acceptor beads (PerkinElmer 6760137) at 15 μg/ml each. Addition of beads to the stopping buffer and stopping of reactions is carried out under dim light. Before stopping the reactions, the stopping buffer with beads is preliminarily kept for 1 hour in the dark at room temperature. After the reactions have stopped, the plates are kept for 1 h in the dark at room temperature before reading on a PHERAstar FS plate reader (BMG Labtech) under dim light.

Pim-2 kinase assays - 20 µl reactions can be performed in white 384-well polystyrene plates labeled with 0.8 µl compound/DMSO in assay buffer (50 mM Tris, pH 7.5, 0.01% Tween -20.5 mM MgCl ₂ , 0.01% BSA, 5 mM DTT) containing 0.05 μM fluorescein-labeled peptide substrate CREBtide (Invitrogen PV3508), 1 mM ATP, and 1 nM enzyme (Invitrogen PV3649) for 2 h at 25°C. Reactions were stopped by adding 10 μl of TR-FRET dilution buffer (Invitrogen PV3574) with 30 mM EDTA and 1.5 nM LanthaScreen Tb-CREB pSer133 antibody (Invitrogen PV3566). After 30 min of incubation at room temperature, the plates were read on a PHERAstar FS plate reader (BMG Labtech).

Compounds or salts of this invention having an IC50 of 2 μmol or less when tested for Pim kinase activity under the assay conditions described above are considered active. Compound 1 was tested according to this assay and found to have an IC ₅₀ <100 nmol. The phosphate salt of compound 1 and the two-hydrochloric acid salt of compound 1 were tested according to this analysis, and the data are presented below in table. 16.

Although the above in vitro assays are performed at 1 mM ATP, compounds can also be assessed for potency and in vitro activity against PIM targets using Km conditions, in which the ATP concentration is set to the _Km value and the assay is more sensitive to PIM inhibitory activity.

Example B: Analysis of Pim cells and whole blood.

Cell growth assay with Pim.

KMS12BM and MOLM16 cells were purchased from DSMZ (Germany) and stored according to the supplier's recommendations. To measure the antiproliferative activity of test compounds, cells were seeded in their respective culture medium (2x103 cells/200 μl/well) in 96-well ultra-low binding plates (Corning), with or without test compound(s). After 3-4 days, [3H]-thymidine (1 µCi/well) (PerkinElmer) in PBS (10 µl) was added to the cell culture for an additional 12 hours, after which the incorporated radioactivity was separated by filtering with water through GF/B filters (Packard Bioscience ) and measured by liquid scintillation counting using TopCount (Packard Bioscience).

Pim pBAD signaling pathway assays.

To measure the effect of test compounds on pBAD levels in KMS12BM cells (DSMZ, Germany) were seeded with RPMI solution and 10% FBS (4x105 cells/well/100 μl) in 96-well v-bottom polypropylene plates (Greiner) in the presence of or absence of 5 µl of test compound(s) in the concentration range. After 2.5 h at 37°C and 5% CO2, cells were disrupted in 100 μl cell extraction buffer (Cell Signaling Technology) containing PMSF, HALT

-

Claims (54)

and protease inhibitors (Thermo, EMD Calbiochem). The amount of pBAD protein in cell lysates was assessed using the Human pBAD S112 ELISA Kit (Cell Signaling Technology). The determination of IC ₅₀ was carried out by plotting the percentage of inhibition versus the logarithm of the inhibitor concentration using GraphPad Prism 5.0 software. To measure the effect of test compounds on pBAD levels in cells in the presence of human whole blood, heparinized human whole blood (Biological Specialty Corp, Colmar PA) was prepared and added at 350 μl/well to a 96-well 2 ml block plate (Costar 3961) in presence or absence of 20 µl of test compound(s) over a range of concentrations. KMS12BM cells (1x10 ⁶ ) or MOLM-16 cells (5x105) (DSMZ, Germany) in 25 μl RPMI and 10% FBS (GIBCO) were added to each well. After 2.5 h at 37°C and 5% CO ₂ , red blood cells were disrupted with erythrocyte lysis buffer (Qiagen) and the remaining cells were centrifuged at 1200 rpm. The resulting pellets were disrupted using 100 μl of cell extraction buffer (Cell Signaling Technology) containing Halt, PMSF and protease inhibitors (Thermo, Calbiochem, Sigma). The amount of pBAD protein in cell lysates was then assessed using the commercial Human pBAD S112 ELISA Kit (Cell Signaling Technology). Determination of IC ₅₀ was carried out by plotting the percentage of inhibition versus the logarithm of inhibitor concentration using GraphPad Prism 5.0 software. Example C. Analysis data. The phosphoric acid salt of Compound 1 and the two-hydrochloric acid salt of Compound 1 were tested in the analyzes of Examples A and B described above. The data are shown in Table 1 below. thirty. Table 30 Type of analysis (Example No., cell type) 1C ₅ 0 (pm) Compound 1 2-HC1 salt 1C ₅ 0 (pm) Compound 1 salt NZPO4 Enzyme PIM1 (Note A) < 35 < 35 Enzyme PIM2 (Note A) < 35 < 35 Enzyme PIM3 (Note A) < 35 < 35 Tumor cell growth (Note B, KMS12BM cells) < 100 < 100 Tumor cell growth (Note B, MOLM16 cells) < 35 < 35 pBAD of KMS12BM cells (Note B) < 35 < 35 pBAD whole blood (Note B, KMS12BM cells) < 200 <200 pBAD whole blood (Ex. B, MOLM16 cells) < 100 < 100 Various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications should also come within the scope of the appended claims. Each reference, including, without limitation, all patents, patent applications and publications cited in this application, is incorporated herein by reference in its entirety. CLAIM 1. Solid form salt formula where the solid form is crystalline. 2. Solid form according to claim 1, characterized in that it is hydrated. 3. Solid form according to claim 2, characterized in that it is a hemihydrate. 4. The solid form of claim 1, characterized in that it is Form I having three or more characteristic X-ray powder diffraction (XPD) peaks expressed in degrees 2-theta selected from about 4.6, 9.4, 13, 1, 16.2, 17.4, 17.9, 18.8, 19.4, 21.1, 23.0, 24.8 and 25.2°. 5. The solid form according to claim 4, having a XRD pattern essentially the same as shown in FIG. 1. 6. Solid form according to claim 4, having a melting point of about 250°C. 7. The solid form of claim 4, having a differential scanning calorimetry (DSC) thermogram substantially as shown in FIG. 2. 8. The solid form of claim 4, having a thermogravimetric analysis (TGA) thermogram substantially as shown in FIG. 3. 9. The solid form of claim 1, having Form II, having at least three characteristic PDP peaks expressed in 2-theta selected from about 4.7, 9.4, 18.8, 21.7, 24.8 and 33.3°. 10. The solid form of claim 9, having a PXRD pattern substantially the same as shown in FIG. 7. - 56 043661 11. The solid form of claim 1, having Form III, having at least three characteristic PDP peaks expressed in 2-theta selected from about 4.6, 9.4, 13.3, 16.3, 18.9 , 19.2, 21.2, 22.5, 23.1, 24.9 and 26.7°. 12. The solid form of claim 11, having a XRD pattern substantially the same as shown in FIG. 10. 13. The solid form of claim 1, having Form IV, having at least three characteristic PDP peaks expressed in 2-theta selected from about 4.1, 13.3, 16.4, 17.7, 18.6 , 19.8, 21.4 and 23.3°. 14. The solid form according to claim 3, having a PXRD pattern essentially the same as shown in FIG. 13. 15. The solid form of claim 1, having a V shape having at least three characteristic PDP peaks expressed in 2-theta selected from about 7.3, 10.9, 16.4, 18.5, 19.8 , 22.6 and 26.1°. 16. The solid form of claim 15, having a PXRD pattern substantially the same as shown in FIG. 16. 17. The solid form of claim 2, having Form VI, having at least three characteristic PDP peaks expressed in 2-theta selected from about 6.5, 8.3, 10.7, 13.2, 17.3 and 19.1°. 18. The solid form of claim 17, having a PXRD pattern substantially the same as shown in FIG. 19. 19. Salt, which is a salt of hydrochloric acid N-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5methylpiperudin-1-yl]-7-hydroxy-6,7 -dihydro-5H-cyclopenta[b]pyrudin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide. 20. Salt according to claim 19, characterized in that it is a salt with two molecules of hydrochloric acid. 21. Solid form of hydrochloric acid salt N-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro- 5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5fluoropyridine-2-carboxamide, characterized in that it is crystalline and has a PRD diffraction pattern essentially the same as shown in FIG. 4. 22. Solid form according to claim 21, characterized in that it has a melting point of 213°C (±3°C). 23. Salt according to claim 19, characterized in that it is a salt with one molecule of hydrochloric acid. 24. Solid form of hydrochloric acid salt N-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro- 5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5fluoropyridine-2-carboxamide, characterized in that it is crystalline and has a PRD diffraction pattern essentially the same as shown in FIG. 22. 25. Solid form according to claim 24, characterized in that it has a melting point of 209°C (±3°C). 26. Salt, which is a salt of maleic acid N-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5methylpiperudin-1-yl]-7-hydroxy-6,7 -dihydro-5H-cyclopenta[b]pyrudin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide. 27. Salt, which is a salt of adipic acid N-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5methylpiperudin-1-yl]-7-hydroxy-6,7 -dihydro-5H-cyclopenta[b]pyrudin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide. 28. Salt which is the hydrobromic acid salt N-{(7R)-4-[(3R,4R,5S)-3amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7 -dihydro-5H-cyclopenta[b]pyridin-3-yl}-6(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide. 29. Salt which is a salt of (R)-(-)-mandelic acid N-{(7R)-4-[(3R,4R,5S)-3amino-4-hydroxy-5-methylpiperidin-1-yl] -7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide. 30. Salt which is a salt of salicylic acid N-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy5-methylpiperidin-1-yl]-7-hydroxy-6,7 -dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide. 31. Salt, which is a salt of benzoic acid N-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5methylpiperudin-1-yl]-7-hydroxy-6,7 -dihydro-5H-cyclopenta[b]pyrudin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide. 32. Salt which is benzenesulfonic acid salt N-{(7R)-4-[(3R,4R,5S)-3amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7 -dihydro-5H-cyclopenta[b]pyridin-3-yl}-6(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide. 33. Salt which is L-pyroglutamic acid salt N-{(7R)-4-[(3R,4R,5S)-3amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6 ,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide. 34. Salt which is the methanesulfonic acid salt N-{(7R)-4-[(3R,4R,5S)-3amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7 -dihydro-5H-cyclopenta[b]pyridin-3-yl}-6(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide. - 57 043661 35. Salt, which is a salt of (1S)-(+)-10-camphorsulfonic acid N-{(7R)-4-[(3R,4R,5S)-3amino-4-hydroxy-5-methylpiperidine-1 - yl]-7-hydroxy-6,7-gihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide. 36. Salt, which is a fumaric acid salt! N-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5methylnineridin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridine -3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridin-2-carboxamide. 37. Salt, which is a salt of sulfuric acid! N-{(7R)-4-[(3R,4R,5S)-3-αmino-4-hydroxy-5methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridine -3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridin-2-carboxamide. 38. Salt, which is L-tartaric acid salt! N-{(7R)-4-[(3R,4R,5S)-3-αmino-4-hydroxy-5methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridine -3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridin-2-carboxamide. 39. Salt, which is a salt of D-tartaric acid! N-{(7R)-4-[(3R,4R,5S)-3-αmino-4-hydroxy-5methylpineridin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridine -3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridin-2-carboxamide. 40. A pharmaceutical composition comprising an effective amount of a solid form according to any one of claims 1 to 18 or an effective amount of a salt according to any one of claims 19 to 39 and at least one pharmaceutically acceptable carrier for inhibiting Pim kinase. 41. Solid oral dosage form containing the pharmaceutical composition according to claim 40. 42. A method of inhibiting Pim kinase, comprising contacting Pim kinase with an effective amount of a solid form according to any one of claims 1 to 18 or an effective amount of a salt according to any one of claims 19 to 39. 43. The method according to claim 42, characterized in that Pim is a Pim1 kinase. 44. The method according to claim 42, characterized in that Pim is Pim 2 kinase. 45. The method according to claim 42, characterized in that Pim is Pim 3 kinase. 46. A method of treating cancer, comprising administering to a patient in need thereof an effective amount of a solid form according to any one of claims 1 to 18 or an effective amount of a salt according to any one of claims 1939, characterized in that the cancer is prostate cancer, colon cancer colon, esophageal cancer, endometrial cancer, ovarian cancer, uterine cancer, kidney cancer, liver cancer, pancreatic cancer, stomach cancer, breast cancer, lung cancer, head or neck cancer, thyroid cancer, glioblastoma, sarcoma, bladder cancer , lymphoma, leukemia, acute lymphoblastic leukemia, acute myeloblastic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, diffuse large cell lymphoma, mantle cell lymphoma, non-Hodgkin lymphoma, Hodgkin lymphoma or multiple myeloma. 47. The method of claim 42, wherein the activity of at least one of Pim1, Pim2 and Pim3 is up-regulated in cancer. 48. A method of treating a myeloproliferative disorder associated with Pim kinase activity, comprising administering to a patient in need thereof an effective amount of a solid form according to any one of claims 1 to 18 or an effective amount of a salt according to any one of claims 19 to 39. 49. A method of treating an immune disorder associated with Pim kinase activity, comprising administering to a patient in need thereof an effective amount of a solid form according to any one of claims 1 to 18 or an effective amount of a salt according to any one of claims 19 to 39. 50. The method according to claim 49, characterized in that the immune disorder is an autoimmune disease. 51. A method of treating atherosclerosis associated with Pim kinase activity, comprising administering to a patient in need thereof an effective amount of a solid form according to any one of claims 1 to 18 or an effective amount of a salt according to any one of claims 19 to 39. 52. A method of reducing angiogenesis or metastasis of tumors, comprising administering to a patient in need thereof an effective amount of a solid form according to any one of claims 1 to 18 or an effective amount of a salt according to any one of claims 19 to 39. 53. A method for obtaining the solid form of phosphoric acid salt! N-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5methylnineridin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridine -3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide according to claim 1, including the combination N-{(7R)-4-[(3R,4R,5S)-3- amino-4-hydroxy-5-methylniperidin1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridin- 2-carboxamide (the free base of compound 1) with phosphoric acid, where the coupling is carried out in the presence of methanol and 2-propanol. 54. A method for producing solid form 1 according to any one of claims 4 and 5, including precipitation of the solid form from a solution containing a phosphoric acid salt N-{(7R)-4-[(3R,4R,5S)-3-amino-4 -hydroxy-5methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5fluoropyridine-2-carboxamide, characterized in that the solution contains a solvent that contains a) 2-propanol or b) acetonitrile, ethanol or both. -