Arginase inhibitors and methods of use thereof

Abstract

Disclosed herein include crystalline (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide of Form D and Form E: Formula (I); (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiro[4.7]dodecan-6-one or a pharmaceutically acceptable salt thereof or Crystal Form 1 thereof: Formula (II); and pharmaceutical compositions and methods of using the same. #imgabs0#

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of priority of U.S. Provisional Application No. 62 / 721,113, filed on Aug. 22, 2018, and entitled “Arginase Inhibitors and Methods of Use thereof [Arginase Inhibitors and Their Use Methods]”, the content of which is hereby incorporated by reference in its entirety for all purposes. Background of the Invention

[0003] Arginase is a manganese metalloenzyme that catalyzes the conversion of L - arginine to urea and L - ornithine. There are two isoforms: arginase 1 is a cytoplasmic enzyme mainly present in hepatocytes, where it plays a key role in ammonia removal through urea synthesis, while arginase 2 is a mitochondrial enzyme highly expressed in the kidney, which is involved in the production of ornithine, a precursor of polyamines and proline that are important for cell proliferation and collagen production, respectively.

[0004] Although L - arginine is not an essential amino acid because it can be provided by protein turnover in healthy adults, under various physiological and pathological conditions (e.g., pregnancy, autoimmune diseases, cancer), increased expression and secretion of arginase lead to reduced L - arginine levels. In particular, immune cells are sensitive to reduced L - arginine levels. When faced with a low L - arginine microenvironment, T cells reduce their proliferation rate and the expression of CD3ζ chain, IFNγ, and lytic enzymes, resulting in impaired T - cell reactivity. Dendritic cells respond to low L - arginine conditions by reducing their ability to present antigens, and natural killer cells reduce the proliferation and expression of lytic enzymes.

[0005] Tumors use multiple immunosuppressive mechanisms to evade the immune system. One of them is to reduce L - arginine by increasing the level of circulating arginase, increasing the expression and secretion of arginase in tumor cells, and recruiting myeloid - derived suppressor cells that express and secrete arginase. These mechanisms together lead to reduced L - arginine in the tumor microenvironment and generate an immunosuppressive phenotype. Pharmacological inhibition of arginase activity has been shown to reverse low L - arginine - induced immunosuppression in animal models. Therefore, there is a need for potent and selective arginase inhibitors, either as single agents or in combination with therapies that reverse other immunosuppressive mechanisms, to reverse immunosuppression and re - activate the anti - cancer immunity of patients. Summary of the Invention

[0006] In some embodiments, crystalline (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide in Form D and Form E are disclosed (the chemical structures are shown below):

[0007]

[0008] In some embodiments, a pharmaceutical composition is disclosed that comprises crystalline Form D or Form E of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide, and a pharmaceutically acceptable carrier.

[0009] In some embodiments, a method of treating cancer is disclosed, the method comprising administering to a subject in need thereof crystalline Form D or Form E of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide.

[0010] In some embodiments, a pharmaceutical composition for treating cancer is disclosed, the pharmaceutical composition comprising crystalline Form D or Form E of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide.

[0011] In some embodiments, the use of crystalline Form D or Form E of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide in the manufacture of a medicament for treating cancer is disclosed.

[0012] In some embodiments, crystalline Form D or Form E of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide is disclosed for use in treating cancer.

[0013] In some embodiments, (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiro[4.7]dodecan-6-one (the chemical structure is shown below) or a pharmaceutically acceptable salt thereof is disclosed.

[0014]

[0015] In some embodiments, crystalline (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiro[4.7]dodecan-6-one in Form 1 is disclosed.

[0016] In some embodiments, a pharmaceutical composition is disclosed that comprises (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiro[4.7]dodecan-6-one or a pharmaceutically acceptable salt or crystalline Form 1 thereof and a pharmaceutically acceptable carrier.

[0017] In some embodiments, a method of treating cancer is disclosed that comprises administering to a subject an effective amount of (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiro[4.7]dodecan-6-one or a pharmaceutically acceptable salt or crystalline Form 1 thereof.

[0018] In some embodiments, a pharmaceutical composition for treating cancer is disclosed that comprises (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiro[4.7]dodecan-6-one or a pharmaceutically acceptable salt or crystalline Form 1 thereof.

[0019] In some embodiments, (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiro[4.7]dodecan-6-one or a pharmaceutically acceptable salt or crystalline Form 1 thereof for treating cancer is disclosed.

[0020] In some embodiments, the use of (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiro[4.7]dodecan-6-one or a pharmaceutically acceptable salt or crystalline Form 1 thereof in the manufacture of a medicament for treating cancer is disclosed. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The powder X-ray diffraction pattern of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide Form D is illustrated.

[0022] Figure 2Describes the single crystal structure of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide Form D.

[0023] Figure 3 Describes the differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) traces of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide Form D.

[0024] Figure 4 Describes the gravimetric vapor sorption (GVS) traces of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide Form D.

[0025] Figure 5 Describes the powder X-ray diffraction pattern of (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiro[4.7]dodecan-6-one Form 1.

[0026] Figure 6 Describes the single crystal structure of (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiro[4.7]dodecan-6-one Form 1.

[0027] Figure 7 Describes the differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) traces of (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiro[4.7]dodecan-6-one Form 1.

[0028] Figure 8 Describes the gravimetric vapor sorption (GVS) traces of (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiro[4.7]dodecan-6-one Form 1.

[0029] Figure 9 Describes the powder X-ray diffraction pattern of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide Form E.

[0030] Figure 10 The single crystal structure of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide Form E is described.

[0031] Figure 11 The differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) traces of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide Form E are described.

[0032] Figure 12 The gravimetric vapor sorption (GVS) traces of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide Form E are described. Detailed Description

[0033] In some embodiments, crystalline (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide in Forms D and E are disclosed (chemical structure shown below):

[0034]

[0035] In some embodiments, crystalline form D of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide has an XRPD pattern comprising at least one peak at about 7.8° expressed as 2θ±0.2°. In some embodiments, crystalline form D of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide has an XRPD pattern comprising at least one peak at about 19.2° expressed as 2θ±0.2°. In some embodiments, crystalline form D of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide has an XRPD pattern comprising at least one peak at about 15.0° expressed as 2θ±0.2°. In some embodiments, crystalline form D of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide has an XRPD pattern comprising at least one peak at about 7.8° or at about 19.2° expressed as 2θ±0.2°. In some embodiments, crystalline form D of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide has an XRPD pattern comprising at least one peak at about 7.8° or at about 15.0° expressed as 2θ±0.2°. In some embodiments, crystalline form D of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide has an XRPD pattern comprising at least one peak at about 15.0° or at about 19.2° expressed as 2θ±0.2°. In some embodiments, crystalline form D of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide has an XRPD pattern comprising at least one peak selected from about 7.8°, about 19.2° and about 15.0° expressed as 2θ±0.2°. In some embodiments, crystalline form D of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide has an XRPD pattern comprising at least one peak selected from the peaks listed in Table 1 expressed as 2θ±0.2°.In some embodiments, the crystalline form D of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide has substantially similar to. Figure 1 XRPD pattern.

[0036] In some embodiments, the crystalline form D of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide characterized by X-ray powder diffraction contains peaks having the following 2θ ± 0.2° values: 7.8, 19.2, and 15.0 degrees. In some embodiments, the crystalline form D of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide has an X-ray powder diffraction pattern further comprising peaks at 2θ ± 0.2° of 16.4, 13.1, and 13.7 degrees.

[0037] In some embodiments, crystalline form D of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide, as characterized by X-ray powder diffraction, comprises at least 3 peaks selected from 7.8, 19.2, 15.0, 16.4, 13.1, 13.7, 26.4, 19.8, 17.9, and 22.5 degrees 2θ ± 0.2°. In some embodiments, crystalline form D of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide, as characterized by X-ray powder diffraction, comprises at least 5 peaks selected from 7.8, 19.2, 15.0, 16.4, 13.1, 13.7, 26.4, 19.8, 17.9, and 22.5 degrees 2θ ± 0.2°. In some embodiments, crystalline form D of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide, as characterized by X-ray powder diffraction, comprises at least 7 peaks selected from 7.8, 19.2, 15.0, 16.4, 13.1, 13.7, 26.4, 19.8, 17.9, and 22.5 degrees 2θ ± 0.2°. In some embodiments, crystalline form D of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide, as characterized by X-ray powder diffraction, comprises at least 9 peaks selected from 7.8, 19.2, 15.0, 16.4, 13.1, 13.7, 26.4, 19.8, 17.9, and 22.5 degrees 2θ ± 0.2°.

[0038] In some embodiments, crystalline form D of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide is characterized by a differential scanning calorimetry (DSC) curve that includes an endotherm at about 214 °C. In some embodiments, crystalline form D of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide has a DSC thermogram that includes a dehydration endotherm that begins at about 213 °C and peaks at about 214 °C. In some embodiments, crystalline form D of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide has a DSC thermogram that is substantially similar to Figure 3 that shown.

[0039] In some embodiments, crystalline form D of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide has a TGA thermogram showing a weight loss of about 0.4% when heated from about 25 °C to about 150 °C and a weight loss of about 3.1% when heated from about 150 °C to about 225 °C. In some embodiments, crystalline form D of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide has a TGA thermogram that is substantially similar to Figure 3 that shown.

[0040] In some embodiments, crystalline form D of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide absorbs less than about 2% water at about 70% relative humidity (RH) and begins to deliquesce after about 80% RH. In some embodiments, crystalline form D of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide has a GVS trace that is substantially similar to Figure 4 that shown.

[0041] In some embodiments, a pharmaceutical composition is disclosed that comprises an effective amount of crystalline form D of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide and a pharmaceutically acceptable carrier. In some embodiments, a pharmaceutical composition is disclosed that comprises an effective amount of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide and a pharmaceutically acceptable carrier, wherein at least about 85% of the (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide is in form D. In some embodiments, a pharmaceutical composition is disclosed that comprises an effective amount of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide and a pharmaceutically acceptable carrier, wherein at least about 90% of the (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide is in form D. In some embodiments, a pharmaceutical composition is disclosed that comprises an effective amount of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide and a pharmaceutically acceptable carrier, wherein at least about 95% of the (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide is in form D. In some embodiments, a pharmaceutical composition is disclosed that comprises an effective amount of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide and a pharmaceutically acceptable carrier, wherein at least about 96% of the (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide is in form D.In some embodiments, a pharmaceutical composition is disclosed that comprises an effective amount of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide and a pharmaceutically acceptable carrier, wherein at least about 97% of the (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide is in Form D. In some embodiments, a pharmaceutical composition is disclosed that comprises an effective amount of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide and a pharmaceutically acceptable carrier, wherein at least about 98% of the (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide is in Form D. In some embodiments, a pharmaceutical composition is disclosed that comprises an effective amount of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide and a pharmaceutically acceptable carrier, wherein at least about 99% of the (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide is in Form D. In some embodiments, a pharmaceutical composition is disclosed that comprises an effective amount of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide and a pharmaceutically acceptable carrier, wherein at least about 99.5% of the (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide is in Form D.

[0042] In some embodiments, the crystalline form E of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide has an XRPD pattern that includes at least one peak at about 12.3° expressed as 2θ±0.2°. In some embodiments, the crystalline form E of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide has an XRPD pattern that includes at least one peak at about 18.8° expressed as 2θ±0.2°. In some embodiments, the crystalline form E of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide has an XRPD pattern that includes at least one peak at about 9.3° expressed as 2θ±0.2°. In some embodiments, the crystalline form E of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide has an XRPD pattern that includes at least one peak at about 12.3° or at about 18.8° expressed as 2θ±0.2°. In some embodiments, the crystalline form E of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide has an XRPD pattern that includes at least one peak at about 12.3° or at about 9.3° expressed as 2θ±0.2°. In some embodiments, the crystalline form E of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide has an XRPD pattern that includes at least one peak at about 18.8° or at about 9.3° expressed as 2θ±0.2°. In some embodiments, the crystalline form E of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide has an XRPD pattern that includes at least one peak selected from about 12.3°, about 18.8° and about 9.3° expressed as 2θ±0.2°. In some embodiments, the crystalline form E of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide has an XRPD pattern that includes at least one peak selected from the peaks listed in Table 1 expressed as 2θ±0.2°.In some embodiments, the crystalline form E of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide is substantially similar to. Figure 9 the XRPD pattern.

[0043] In some embodiments, the crystalline form E of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide characterized by X-ray powder diffraction comprises peaks having the following 2θ ± 0.2° values: 12.3, 18.8, and 9.3 degrees. In some embodiments, the crystalline form E of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide has an X-ray powder diffraction pattern further comprising peaks at 2θ ± 0.2° of 14.2, 14.1, and 19.8 degrees.

[0044] In some embodiments, the crystalline form E of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide characterized by X-ray powder diffraction contains at least 3 peaks selected from 12.3, 18.8, 9.3, 14.2, 14.1, 19.8, 26.2, 17.3, 7.1, and 25.4 degrees 2θ±0.2°. In some embodiments, the crystalline form E of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide characterized by X-ray powder diffraction contains at least 5 peaks selected from 12.3, 18.8, 9.3, 14.2, 14.1, 19.8, 26.2, 17.3, 7.1, and 25.4 degrees 2θ±0.2°. In some embodiments, the crystalline form E of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide characterized by X-ray powder diffraction contains at least 7 peaks selected from 12.3, 18.8, 9.3, 14.2, 14.1, 19.8, 26.2, 17.3, 7.1, and 25.4 degrees 2θ±0.2°. In some embodiments, the crystalline form E of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide characterized by X-ray powder diffraction contains at least 9 peaks selected from 12.3, 18.8, 9.3, 14.2, 14.1, 19.8, 26.2, 17.3, 7.1, and 25.4 degrees 2θ±0.2°.

[0045] In some embodiments, the crystalline form E of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide is characterized by a differential scanning calorimetry (DSC) curve that includes an endothermic peak at about 125°C. In some embodiments, the crystalline form E of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide has a DSC thermogram that includes an endothermic dehydration peak that begins at about 105°C and peaks at about 125°C. In some embodiments, the crystalline form E of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide has a DSC thermogram that is substantially similar to Figure 11 that shown.

[0046] In some embodiments, the (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide Form E has a TGA thermogram showing a weight loss of about 6.0% when heated from about 25 °C to about 135 °C and a weight loss of about 3.0% when heated from about 125 °C to about 225 °C. In some embodiments, the (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide Form E has a TGA thermogram that is substantially similar to Figure 11 that shown.

[0047] In some embodiments, the (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide Form E absorbs less than about 2% water at about 80% relative humidity (RH). In some embodiments, the (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide Form D has a GVS trace that is substantially similar to Figure 12 that shown.

[0048] In some embodiments, a pharmaceutical composition is disclosed that comprises an effective amount of crystalline form E of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide and a pharmaceutically acceptable carrier. In some embodiments, a pharmaceutical composition is disclosed that comprises an effective amount of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide and a pharmaceutically acceptable carrier, wherein at least about 85% of the (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide is in form E. In some embodiments, a pharmaceutical composition is disclosed that comprises an effective amount of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide and a pharmaceutically acceptable carrier, wherein at least about 90% of the (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide is in form E. In some embodiments, a pharmaceutical composition is disclosed that comprises an effective amount of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide and a pharmaceutically acceptable carrier, wherein at least about 95% of the (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide is in form E. In some embodiments, a pharmaceutical composition is disclosed that comprises an effective amount of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide and a pharmaceutically acceptable carrier, wherein at least about 96% of the (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide is in form E.In some embodiments, a pharmaceutical composition is disclosed that comprises an effective amount of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide and a pharmaceutically acceptable carrier, wherein at least about 97% of the (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide is in Form E. In some embodiments, a pharmaceutical composition is disclosed that comprises an effective amount of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide and a pharmaceutically acceptable carrier, wherein at least about 98% of the (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide is in Form E. In some embodiments, a pharmaceutical composition is disclosed that comprises an effective amount of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide and a pharmaceutically acceptable carrier, wherein at least about 99% of the (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide is in Form E. In some embodiments, a pharmaceutical composition is disclosed that comprises an effective amount of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide and a pharmaceutically acceptable carrier, wherein at least about 99.5% of the (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide is in Form E.

[0049] In some embodiments, the compound (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiro[4.7]dodecan-6-one (chemical structure shown below) or a pharmaceutically acceptable salt thereof is disclosed. The compound can be amorphous, crystalline, or a mixture thereof.

[0050]

[0051] In some embodiments, (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiro[4.7]dodecan-6-one Form 1 is disclosed.

[0052] In some embodiments, the crystalline form 1 of (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiroundecane-6-one has an XRPD pattern comprising at least one peak at about 11.6° represented as 2θ±0.2°. In some embodiments, the crystalline form 1 of (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiroundecane-6-one has an XRPD pattern comprising at least one peak at about 8.2° represented as 2θ±0.2°. In some embodiments, the crystalline form 1 of (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiroundecane-6-one has an XRPD pattern comprising at least one peak at about 13.3° represented as 2θ±0.2°. In some embodiments, the crystalline form 1 of (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiroundecane-6-one has an XRPD pattern comprising at least one peak at about 11.6° or at about 8.2° represented as 2θ±0.2°. In some embodiments, the crystalline form 1 of (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiroundecane-6-one has an XRPD pattern comprising at least one peak at about 11.6° or at about 13.3° represented as 2θ±0.2°. In some embodiments, the crystalline form 1 of (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiroundecane-6-one has an XRPD pattern comprising at least one peak at about 8.2° or at about 13.3° represented as 2θ±0.2°. In some embodiments, the crystalline form 1 of (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiroundecane-6-one has an XRPD pattern comprising at least one peak represented as 2θ±0.2° selected from about 11.6°, about 8.2° and about 13.3°.In some embodiments, the crystalline form 1 of (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiro[4.7]dodecane-6-one has an XRPD pattern comprising at least one peak selected from the peaks listed in Table 1, represented as 2θ ± 0.2°. In some embodiments, the crystalline form 1 of (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiro[4.7]dodecane-6-one is substantially similar to. Figure 5 the XRPD pattern.

[0053] In some embodiments, the crystalline form 1 of (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiro[4.7]dodecane-6-one characterized by X-ray powder diffraction comprises at least 3 peaks selected from 11.6, 8.2, 13.3, 16.4, 12.9, 17.4, 19.5, 16.6, 22.6, and 15.9 degrees 2θ ± 0.2°. In some embodiments, the crystalline form 1 of (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiro[4.7]dodecane-6-one characterized by X-ray powder diffraction comprises at least 5 peaks selected from 11.6, 8.2, 13.3, 16.4, 12.9, 17.4, 19.5, 16.6, 22.6, and 15.9 degrees 2θ ± 0.2°. In some embodiments, the crystalline form 1 of (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiro[4.7]dodecane-6-one characterized by X-ray powder diffraction comprises at least 7 peaks selected from 11.6, 8.2, 13.3, 16.4, 12.9, 17.4, 19.5, 16.6, 22.6, and 15.9 degrees 2θ ± 0.2°. In some embodiments, the crystalline form 1 of (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiro[4.7]dodecane-6-one characterized by X-ray powder diffraction comprises at least 9 peaks selected from 11.6, 8.2, 13.3, 16.4, 12.9, 17.4, 19.5, 16.6, 22.6, and 15.9 degrees 2θ ± 0.2°.

[0054] In some embodiments, the (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiro[4.7]dodecan-6-one Form 1 has a DSC thermogram that includes an endothermic dehydration peak starting at about 82 °C and peaking at about 122 °C. In some embodiments, the (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiro[4.7]dodecan-6-one Form 1 has a DSC thermogram that is substantially similar to Figure 7 that shown.

[0055] In some embodiments, the (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiro[4.7]dodecan-6-one Form 1 has a TGA thermogram that exhibits a mass loss of about 5.5% when heated from about 25 °C to about 150 °C and a mass loss of about 3.2% when heated from about 150 °C to about 225 °C. In some embodiments, the crystalline (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiro[4.7]dodecan-6-one has a TGA thermogram that is substantially similar to Figure 7 that shown.

[0056] In some embodiments, the (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiro[4.7]dodecan-6-one Form 1 absorbs less than about 2% water at about 80% relative humidity (RH). In some embodiments, the (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiro[4.7]dodecan-6-one Form 1 has a GVS trace that is substantially similar to Figure 8 that shown.

[0057] In some embodiments, a pharmaceutical composition is disclosed that comprises an effective amount of (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiroundecane-6-one or a pharmaceutically acceptable salt or crystalline form 1 thereof and a pharmaceutically acceptable carrier. In some embodiments, a pharmaceutical composition is disclosed that comprises an effective amount of (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiroundecane-6-one or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, wherein at least about 85% of the (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiroundecane-6-one is in form 1. In some embodiments, a pharmaceutical composition is disclosed that comprises an effective amount of (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiroundecane-6-one and a pharmaceutically acceptable carrier, wherein at least about 90% of the (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiroundecane-6-one is in form 1. In some embodiments, a pharmaceutical composition is disclosed that comprises an effective amount of (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiroundecane-6-one and a pharmaceutically acceptable carrier, wherein at least about 95% of the (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiroundecane-6-one is in form 1. In some embodiments, a pharmaceutical composition is disclosed that comprises an effective amount of (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiroundecane-6-one and a pharmaceutically acceptable carrier, wherein at least about 96% of the (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiroundecane-6-one is in form 1.In some embodiments, a pharmaceutical composition is disclosed that comprises an effective amount of (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiroundecane-6-one and a pharmaceutically acceptable carrier, wherein at least about 97% of the (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiroundecane-6-one is in Form 1. In some embodiments, a pharmaceutical composition is disclosed that comprises an effective amount of (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiroundecane-6-one and a pharmaceutically acceptable carrier, wherein at least about 98% of the (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiroundecane-6-one is in Form 1. In some embodiments, a pharmaceutical composition is disclosed that comprises an effective amount of (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiroundecane-6-one and a pharmaceutically acceptable carrier, wherein at least about 99% of the (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiroundecane-6-one is in Form 1. In some embodiments, a pharmaceutical composition is disclosed that comprises an effective amount of (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiroundecane-6-one and a pharmaceutically acceptable carrier, wherein at least about 99.5% of the (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiroundecane-6-one is in Form 1.

[0058] The term "pharmaceutically acceptable carrier" includes compounds, materials, compositions, and / or dosage forms that, as determined by one of ordinary skill in the art, are suitable for use in contact with the tissues of humans and animals within the scope of sound medical judgment, without excessive toxicity, irritation, allergic response, or other problems or complications.

[0059] The disclosed compositions can be in a form suitable for oral use (e.g., as tablets, lozenges, hard or soft gelatin capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs), in a form suitable for topical use (e.g., as creams, ointments, gels or aqueous or oily solutions or suspensions), in a form suitable for administration by inhalation (e.g., as a finely divided powder or a liquid aerosol), in a form suitable for administration by insufflation (e.g., as a finely divided powder) or in a form suitable for parenteral administration (e.g., as a sterile aqueous or oily solution for intravenous, subcutaneous, intramuscular or intramuscular injection or as a suppository for rectal administration).

[0060] The amount of active ingredient combined with one or more pharmaceutically acceptable carriers to produce a single dosage form will necessarily vary depending on the host to be treated and the particular route of administration. For further information on routes of administration and dosage regimens, the reader is referred to Chapter 25.3, Volume 5 of Comprehensive Medicinal Chemistry (Editor-in-Chief Corwin Hansch), Pergamon Press 1990.

[0061] In one aspect, a method for treating cancer in a subject in need thereof is disclosed, the method comprising administering to the subject an effective amount of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide in Form D or Form E.

[0062] In one aspect, (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide in Form D or Form E for treating cancer is disclosed.

[0063] In one aspect, the use of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide in Form D or Form E in the manufacture of a medicament for treating cancer is disclosed.

[0064] In one aspect, a pharmaceutical composition for treating cancer comprising (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide in Form D or Form E is disclosed.

[0065] In one aspect, a method for treating cancer in a subject in need thereof is disclosed, the method comprising administering to the subject an effective amount of (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiroundecane-6-one or a pharmaceutically acceptable salt or polymorph 1 thereof.

[0066] In one aspect, (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiroundecane-6-one or a pharmaceutically acceptable salt or polymorph 1 thereof for treating cancer is disclosed.

[0067] In one aspect, the use of (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiroundecane-6-one or a pharmaceutically acceptable salt or polymorph 1 thereof in the manufacture of a medicament for treating cancer is disclosed.

[0068] In one aspect, a pharmaceutical composition for treating cancer comprising (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiroundecane-6-one or a pharmaceutically acceptable salt or polymorph 1 thereof is disclosed.

[0069] The term "cancer" includes, for example, renal cell carcinoma, head and neck squamous cell carcinoma, lung cancer (e.g., small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), mesothelioma), pancreatic cancer, colorectal cancer, breast cancer, acute myeloid leukemia (AML), prostate cancer, gastric cancer, bladder cancer, melanoma, kidney cancer, and ovarian cancer. In some embodiments, the cancer has metastasized. In some embodiments, the cancer is associated with arginase 1 and / or arginase 2 regulation.

[0070] In some embodiments, the cancer is associated with an increased plasma arginase 1 level. In some embodiments, the cancer is associated with a decreased plasma arginine level. In some embodiments, the cancer is associated with both an increased plasma arginase 1 level and a decreased plasma arginine level. In some embodiments, cancers associated with an increased plasma arginase 1 level and / or a decreased plasma arginine level include renal cell carcinoma, head and neck squamous cell carcinoma, lung cancer (e.g., small cell lung cancer (SCLC), non-small cell carcinoma lung cancer (NSCLC), mesothelioma), pancreatic cancer, colorectal cancer, and breast cancer.

[0071] In some embodiments, the cancer secretes arginase 2, such as acute myeloid leukemia and prostate cancer.

[0072] In some embodiments, cancer is associated with arginase 1-positive tumor-infiltrating immune cells, such as lung cancer (small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), gastric cancer, bladder cancer, colorectal cancer, melanoma, head and neck squamous cell carcinoma, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, and kidney cancer).

[0073] In one aspect, a method for inhibiting arginase in a subject in need thereof is disclosed, the method comprising administering to the subject an effective amount of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide in Form D or Form E.

[0074] In one aspect, (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide in Form D or Form E for inhibiting arginase is disclosed.

[0075] In one aspect, the use of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide in Form D or Form E in the manufacture of a medicament for inhibiting arginase is disclosed.

[0076] In one aspect, a pharmaceutical composition for inhibiting arginase comprising (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide in Form D or Form E is disclosed.

[0077] In one aspect, a method for inhibiting arginase in a subject in need thereof is disclosed, the method comprising administering to the subject an effective amount of (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiro[4.7]dodecan-6-one or Polymorph 1.

[0078] In one aspect, (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiro[4.7]dodecan-6-one or a pharmaceutically acceptable salt thereof or Polymorph 1 for inhibiting arginase is disclosed.

[0079] In one aspect, there is disclosed the use of (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiro[4.7]dodecan-6-one or a pharmaceutically acceptable salt or crystalline form 1 thereof in the manufacture of a medicament for inhibiting arginase.

[0080] In one aspect, there is disclosed a pharmaceutical composition for inhibiting arginase comprising (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiro[4.7]dodecan-6-one or a pharmaceutically acceptable salt or crystalline form 1 thereof.

[0081] The term "arginase" includes manganese-containing enzymes belonging to the ureohydrolase family that catalyze the fifth and final step in the urea cycle, converting L-arginine to L-ornithine and urea. The term "arginase" includes two isoenzymes of said enzyme, such as arginase 1, which functions in the urea cycle and is mainly located in the cytoplasm of the liver, and arginase 2, which is located in the mitochondria of several tissues of the body and is involved in the regulation of arginine / ornithine concentrations in cells. In some embodiments, form D or form E of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiroundecane-3-yl)-3-methylbutyramide is selective for arginase 1. In some embodiments, form D or form E of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiroundecane-3-yl)-3-methylbutyramide is selective for arginase 2. In some embodiments, form D or form E of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiroundecane-3-yl)-3-methylbutyramide inhibits both arginase 1 and arginase 2. In some embodiments, (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiroundecane-6-one or a pharmaceutically acceptable salt or crystalline form 1 thereof is selective for arginase 1. In some embodiments, (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiroundecane-6-one or a pharmaceutically acceptable salt or crystalline form 1 thereof is selective for arginase 2. In some embodiments, (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiroundecane-6-one or a pharmaceutically acceptable salt or crystalline form 1 thereof inhibits both arginase 1 and arginase 2.

[0082] The term "effective amount" includes an amount of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiroundecane-3-yl)-3-methylbutyramide in Form D or Form E or (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiroundecane-6-one or a pharmaceutically acceptable salt or Polymorph 1 thereof that will cause a biological or medical response in a subject, such as a decrease or inhibition of arginase or cancer-related enzyme or protein activity, an improvement in cancer symptoms, or a slowing or delay of cancer progression. In some embodiments, the term "effective amount" includes an amount of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiroundecane-3-yl)-3-methylbutyramide in Form D or Form E or (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiroundecane-6-one or a pharmaceutically acceptable salt or Polymorph 1 thereof that, when administered to a subject, effectively at least partially alleviates, inhibits, and / or ameliorates cancer or inhibits arginase, and / or reduces or inhibits the growth of a subject's tumor or the proliferation of cancer cells.

[0083] The term "subject" includes warm-blooded mammals such as primates, dogs, cats, rabbits, rats, and mice. In some embodiments, the subject is a primate, such as a human. In some embodiments, the subject has cancer. In some embodiments, the subject is in need of treatment (e.g., the subject will benefit biologically or medically from treatment). In some embodiments, the patient has cancer. In some embodiments, the subject has an increased plasma arginase 1 level. In some embodiments, the subject has a decreased arginine level. In some embodiments, the patient has an increased plasma arginase 1 level and a decreased arginine level. In some embodiments, the subject has a cancer that secretes arginase 2 (e.g., acute myeloid leukemia or prostate cancer). In some embodiments, the subject has arginase 1-positive tumor-infiltrating immune cells.

[0084] The term "inhibit" ("inhibit", "inhibition", or "inhibiting") includes a decrease in the baseline activity of a biological activity or process.

[0085] The term “treatment” includes reducing or inhibiting the activity of an enzyme or protein associated with arginase or in a subject, ameliorating one or more symptoms of cancer, or slowing or delaying the progression of cancer in a subject. The term “treatment” (“treat”, “treating” and “treatment”) also includes reducing or inhibiting the growth of a tumor or the proliferation of cancerous cells in a subject.

[0086] In some embodiments, the compounds of the invention such as (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide and (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiro[4.7]dodecan-6-one exhibit a structural interconversion between free boric acid and borate ester as shown in Scheme 1 below.

[0087] Scheme 1. Interconversion of free boric acid and borate ester of Form D.

[0088]

[0089] In some embodiments, the compounds of the invention such as (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide and (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiro[4.7]dodecan-6-one can exist in a form in which a coordinate covalent bond is formed between the nitrogen atom of the pyrrolidine moiety and the boron atom. When forming a conventional covalent bond, each atom contributes one electron to the bond, while in a coordinate covalent bond (also known as a coordination bond), both electrons come from the same atom. For example, the coordinate covalent bond between the nitrogen and boron atoms in the compounds of the invention is formed by sharing a pair of electrons from the nitrogen atom. The coordinate covalent bonds in (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide and (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiro[4.7]dodecan-6-one can be represented by arrow lines in the following structural formulas respectively:

[0090]

[0091] Examples

[0092] Aspects of the present disclosure may be further defined by reference to the following non-limiting examples, which detail the preparation of certain compounds and intermediates of the present disclosure and methods of using the compounds of the present disclosure. It will be apparent to those of ordinary skill in the art that many modifications can be made to the materials and methods without departing from the scope of the present disclosure.

[0093] Unless otherwise stated:

[0094] (i) Unless otherwise stated, all syntheses are carried out at ambient temperature (i.e., in the range of 17 °C to 25 °C) and under an atmosphere of an inert gas such as nitrogen.

[0095] (ii) Evaporation is carried out in vacuo by rotary evaporation or using a Genevac facility or a Biotage v10 evaporator, and the processing procedures are carried out after removal of residual solids by filtration.

[0096] (iii) On an automated Teledyne Isco Rf or Teledyne Isco using pre-packed RediSep Rf GoldT M silica columns (20 - 40 μm, spherical particles), GraceResolv TM columns ( silica) or Silicycle columns (40 - 63 μm) for flash column chromatography purification.

[0097] (iv) Preparative chromatography is carried out on a Gilson preparative HPLC instrument with UV collection; alternatively, preparative chromatography is carried out on a Waters automated purification HPLC - MS instrument with MS and UV triggered collection.

[0098] (v) Chiral preparative chromatography is carried out on a Gilson instrument (233 injector / fraction collector, 333 and 334 pumps, 155 UV detector) with UV collection or on a Varian Prep Star instrument (2 SD1 pumps, 325 UV detector, 701 fraction collector), with the pumps operating with Gilson 305 injection; alternatively, chiral preparative chromatography is carried out on a Waters Prep 100SFC - MS instrument with MS and UV triggered collection or on a Thar MultiGram III SFC instrument with UV collection.

[0099] (vi) Yields (when present) need not be the maximum achievable.

[0100] (vii) Generally, the structure of the end product having Formula I is confirmed by nuclear magnetic resonance (NMR) spectroscopy; NMR chemical shift values are measured in δ scale [proton magnetic resonance spectra are determined using Bruker Avance 500 (500 MHz), Bruker Avance 400 (400 MHz), Bruker Avance 300 (300 MHz) or Bruker DRX (300 MHz) instruments]; measurements are made at ambient temperature unless otherwise specified; the following abbreviations are used: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; dd, double doublet; ddd, double doublet of doublets; dt, double triplet; bs, broad signal.

[0101] (viii) Generally, the end product having the formula I is further characterized by mass spectrometry (LCMS or UPLC) after liquid chromatography; UPLC is carried out as follows: Using Waters UPLC equipped with a Waters SQ mass spectrometer (column temperature 40 °C, UV = 220 - 300 nm or 190 - 400 nm, mass spectrometry = ESI with positive / negative switching) at a flow rate of 1 mL / min, using a solvent system of 97% A + 3% B to 3% A + 97% B over 1.50 min (total run time including equilibration back to starting conditions is 1.70 min), where A = 0.1% formic acid or 0.05% trifluoroacetic acid in water (for acid treatment) or 0.1% ammonium hydroxide in water (for base treatment), and B = acetonitrile. For acid analysis, the column used is Waters Acquity HSS T3 (1.8 μm, 2.1×50 mm); for base analysis, the column used is Waters Acquity BEH C18 (1.7 μm, 2.1×50 mm). Alternatively, UPLC is carried out as follows: Using Waters UPLC equipped with a Waters SQ mass spectrometer (column temperature 30 °C, UV = 210 - 400 nm, mass spectrometry = ESI with positive / negative switching) at a flow rate of 1 mL / min using a solvent gradient of 2% to 98% B for 1.5 min (total run time including equilibration back to starting conditions is 2 min), where A = 0.1% formic acid in water and B = 0.1% formic acid in acetonitrile (for acid work) or A = 0.1% ammonium hydroxide in water and B = acetonitrile (for base work). For acid analysis, the column used is Waters Acquity HSS T3 (1.8 μm, 2.1×30 mm), for base analysis, the column used is Waters Acquity BEH C18 (1.7 μm, 2.1×30 mm); Using Waters Alliance HT (2795) equipped with a Waters ZQ ESCi mass spectrometer and Phenomenex Gemini-NX C18 (5 μm, 110A, 2.1×50 mm column), at a flow rate of 1.1 mL / min, carrying out LCMS from 95% A to 95% B over 4 min, holding for 0.5 min, where A = 0.1% formic acid and B = acetonitrile solution of 0.1% formic acid (for acid treatment) or A = 0.1% aqueous ammonium hydroxide and B = acetonitrile (for base treatment).In addition, LCMS was performed using a Shimadzu UFLC: The Shimadzu UFLC was equipped with a Shimadzu LCMS-2020 mass spectrometer and a Waters HSS C18 (1.8 μm, 2.1×50 mm) or Shim-pack XR-ODS (2.2 μm, 3.0×50 mm) or Phenomenex Gemini-NX C18 (3 μm, 3.0×50 mm) column, at a flow rate of 0.7 mL / min (for Waters HSS C18 column), 1.0 mL / min (for Shim-pack XR-ODS column) or 1.2 mL / min (for Phenomenex Gemini-NX C18), from 95% A to 95% B over 2.2 min, with a hold time of 0.6 min, where A = 0.1% formic acid or 0.05% trifluoroacetic acid in water (for acid treatment) or 0.1% ammonium hydroxide or 6.5 mM ammonium carbonate in water (for base treatment) and B = acetonitrile. Unless otherwise specified, the reported molecular ion corresponds to [M+H]+; for molecules with multiple isotope patterns (Br, Cl, etc.), unless otherwise stated, the reported value is the value obtained for the lowest isotope mass.

[0102] (ix) Ion exchange purification was generally performed using an SCX-2 (Biotage) column.

[0103] (x) The purity of the intermediate was evaluated by thin layer chromatography, mass spectrometry, LCMS, UPLC / MS, HPLC (high performance liquid chromatography) and / or NMR analysis;

[0104] (xi) The following abbreviations were used: -

[0105] EtOAc: Ethyl acetate

[0106] DMSO: Dimethyl sulfoxide

[0107] KHMDS: Potassium hexamethyldisilazide

[0108] MeOH: Methanol

[0109] MeCN: Acetonitrile

[0110] LCMS: Liquid chromatography - mass spectrometry

[0111] rt or RT: Room temperature

[0112] aq: Aqueous

[0113] THF: Tetrahydrofuran

[0114] DCM: Dichloromethane

[0115] DMF: Dimethylformamide

[0116] HATU: (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate)

[0117] HEPES: (4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid)

[0118] XRPD: X-Ray Powder Diffraction

[0119] DSC: Differential Scanning Calorimetry

[0120] TGA: Thermogravimetric Analysis

[0121] GVS: Gravimetric Vapor Sorption

[0122] Example 1: Synthesis of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide Form D Intermediate 1: (2S,4R)-4-azidopyrrolidine-1,2-dicarboxylic acid 1-tert-butyl 2-methyl ester

[0123] Intermediate 2: (2S,4R)-4-azidopyrrolidine-1,2-dicarboxylic acid 2-benzyl 1-tert-butyl ester

[0124]

[0125] At 0 °C, methanesulfonyl chloride (2.86 mL, 36.7 mmol) was added dropwise to a solution of (2S,4S)-4-hydroxypyrrolidine-1,2-dicarboxylic acid 1-tert-butyl 2-methyl ester (7.50 g, 30.6 mmol) and triethylamine (5.11 mL, 36.7 mmol) in DCM (38 mL). The reaction mixture was stirred at 0 °C for 1 h and then warmed to room temperature with stirring for an additional 1 h. The reaction mixture was diluted with dichloromethane and washed with water. The organic layer was dried over Na2SO4, filtered, and concentrated to dryness to give (2S,4S)-4-((methylsulfonyl)oxy)pyrrolidine-1,2-dicarboxylic acid 1-(tert-butyl) 2-methyl ester (9.9 g, 100% yield), which was used without further purification. m / z (ES + ) [M+NH4] + = 341.

[0126] Sodium azide (5.96 g, 91.7 mmol) was added to a solution of (2S,4S)-4-((methylsulfonyl)oxy)pyrrolidine-1,2-dicarboxylic acid 1-(tert-butyl) 2-methyl ester (9.89 g, 30.6 mmol) in DMF (30 mL). The reaction mixture was heated to 50 °C and stirred overnight. The reaction mixture was cooled to room temperature and concentrated. The resulting residue was diluted with EtOAc and washed with water. The organic layer was dried over Na2SO4, filtered and concentrated to dryness. The crude material was purified by silica gel chromatography (hexane / EtOAc) to give the product as a mixture of rotamers (Intermediate 1, 5.95 g, 72% yield). 1 H NMR (300 MHz, DMSO-d6) δ 1.33 and 1.40 (9H, s x2) rotamers, 2.08 - 2.22 (1H, m), 2.26 - 2.41 (1H, m), 3.41 (1H, dt), 3.48 - 3.61 (1H, m), 3.65 and 3.68 (3H, s x2) rotamers, 4.22 (1H, dd), 4.30 - 4.43 (1H, m); m / z (ES + ) [M+H] + = 271.

[0127] Intermediate 3: (4R)-4-azido-2-(but-2-enyl)pyrrolidine-1,2-dicarboxylic acid 2-benzyl 1-tert-butyl ester

[0128]

[0129] At 0 °C, a solution of sodium hydroxide (5.28 g, 132 mmol) in water (22 mL) was added dropwise to a solution of (2S,4R)-4-azidopyrrolidine-1,2-dicarboxylic acid 1-tert-butyl 2-methyl ester (Intermediate 1, 5.95 g, 22.0 mmol) in THF (44 mL) and MeOH (22 mL). The reaction mixture was stirred overnight while slowly warming to room temperature. Volatiles were removed in vacuo and the aqueous layer was acidified to pH ~ 3 with 5M HCl and extracted with DCM. The combined organics were dried over Na2SO4, filtered and concentrated to dryness to give (2S,4R)-4-azido-1-(tert-butoxycarbonyl)pyrrolidine-2-carboxylic acid as a mixture of rotamers (5.64 g, 100% yield), which was used without further purification. 1 H NMR (300 MHz, DMSO-d 6 ) δ 1.35 and 1.40 (9H, s x2) rotamers, 2.07 - 2.18 (1H, m), 2.26 - 2.38 (1H, m), 3.34 - 3.44 (1H, m), 3.48 - 3.63 (1H, m), 4.09 - 4.17 (1H, m), 4.30 - 4.37 (1H, m); m / z (ES- )[M+HCOO] - = 301.

[0130] Benzyl bromide (2.83 mL, 23.8 mmol) was added dropwise to a solution of (2S,4R)-4-azido-1-(tert-butoxycarbonyl)pyrrolidine-2-carboxylic acid (5.19 g, 19.9 mmol) and triethylamine (3.46 mL, 24.8 mmol) in DMF (60 mL), and the reaction mixture was stirred overnight at room temperature. Volatiles were removed in vacuo, and the resulting residue was dissolved in EtOAc and washed with water. The organic layer was dried over Na2SO4, filtered, and concentrated to dryness. The crude material was purified by silica gel chromatography (hexanes / EtOAc) to afford the product (Intermediate 2, 5.09 g, 74% yield). 1 1H NMR (300 MHz, DMSO-d6) δ 1.26 and 1.39 (9H, s x2) rotamers, 2.11 - 2.23 (1H, m), 2.31 - 2.43 (1H, m), 3.43 (1H, ddd), 3.50 - 3.59 (1H, m), 4.25 - 4.40 (2H, m), 5.07 - 5.22 (2H, m), 7.31 - 7.40 (5H, m); m / z (ES + )[M+H] + = 347.

[0131] Intermediate 4: (4R)-4-azido-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylic acid 2-benzyl 1-tert-butyl ester

[0132]

[0133] (2S,4R)-4-Azidopyrrolidine-1,2-dicarboxylic acid 2-benzyl 1-tert-butyl ester (Intermediate 2, 5.09 g, 14.7 mmol) and crotyl bromide (2.27 mL, 22.0 mmol) were dissolved in THF (100 mL), and the solution was cooled to -78 °C under a N2 atmosphere. The solution was treated by dropwise addition of a KHMDS solution (0.5 M in toluene, 44.1 mL, 22.0 mmol). The reaction mixture was slowly warmed to room temperature and stirred for 3 h. The crude reaction mixture was quenched with water, and volatiles were removed in vacuo. The crude mixture was diluted in DCM, and the layers were separated. The organic layer was washed with water, dried over Na2SO4, filtered, and concentrated to dryness. The crude material was purified by silica gel chromatography (hexanes / EtOAc) to afford the product as a mixture of rotamers and E / Z olefins (Intermediate 3, 4.6 g, 78% yield). 11H NMR (300 MHz, DMSO-d6) δ 1.26 - 1.43 (9H, m), 1.59 - 1.66 (3H, m), 2.07 - 2.17 (1H, m), 2.32 - 2.48 (2H, m), 2.57 - 3.12 (2H, m), 3.35 - 3.82 (1H, m), 4.20 - 4.38 (1H, m), 5.02 - 5.22 (2H, m), 5.24 - 5.41 (1H, m), 5.46 - 5.68 (1H, m), 7.28 - 7.42 (5H, m); m / z (ES + ) [M+H] + = 401.

[0134] Intermediate 5: (2S,4R)-4-azido-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylic acid 2-benzyl 1-tert-butyl ester and Intermediate 6: (2R,4R)-4-azido-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylic acid 2-benzyl 1-tert-butyl ester Intermediate 7: (2R,4R)-4-amino-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylic acid 2-benzyl 1-tert-butyl ester

[0135]

[0136] Bis(1,5-cyclooctadiene)iridium(I) dichloride (772 mg, 1.15 mmol) and bis(diphenylphosphino)methane (883 mg, 2.30 mmol) were added to an oven-dried round-bottom flask. The flask was sealed and purged with N2. The solid was dissolved in DCM (66 mL), and 4,4,5,5-tetramethyl-1,3,2-dioxaborolane (3.67 mL, 25.3 mmol) was slowly added to the solution. The reaction was stirred at room temperature for 10 min. (4R)-4-Azido-2-(but-2-enyl)pyrrolidine-1,2-dicarboxylic acid 2-benzyl 1-tert-butyl ester (Intermediate 3, 4.60 g, 11.5 mmol) was added to the reaction as a solution in DCM (44 mL), and the reaction mixture was stirred overnight. The reaction mixture was diluted with DCM and quenched with water. The layers were separated, and the aqueous layer was extracted with DCM. The combined organic layers were dried over Na2SO4, filtered, and concentrated to dryness. The crude material was purified by silica gel chromatography (hexane / EtOAc) to give (4R)-4-azido-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylic acid 2-benzyl 1-tert-butyl ester (Intermediate 4, 2.7 g, 44% yield).

[0137] Intermediate 8: (2R,4R)-4-((R)-2-(tert-butoxycarbonylamino)-3-methylbutyramido)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylic acid 2-benzyl 1-tert-butyl ester Intermediate 9: (2R,4R)-4-((S)-2-amino-3-methylbutyramido)-2-(4-dihydroxyboronylbutyl)pyrrolidine-2-carboxylic acid group Example 1: (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide Form D Angle (2θ ± 0.2°)

[0138]

[0139] Subject the purified material obtained from the synthesis of intermediate 4 to chiral SFC (Chiralpak IG column, 21.2 x 250 mm, 5 μm, temperature = 23 °C, mobile phase = 0%-7% MeOH (w / 0.2% NH4OH): CO2, UV detection @ 220 nm, sample loading = 16.8 mg / inj, concentration = 112.5 ng / mL (in MeOH), flow rate = 70 mL / min, outlet pressure = 100 bar), and two diastereomers are obtained. The stereochemistry of the major diastereomer intermediate 6 is designated as the trans addition product, and the minor diastereomer intermediate 5 is designated as the cis addition product.

[0140] Intermediate 5 (436 mg): (2S,4R)-4-azido-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylic acid 2-benzyl 1-tert-butyl ester. 1 H NMR (400 MHz, DMSO-d6) δ 0.58 - 0.70 (2H, m), 1.17 (12H, s), 1.25 - 1.40 (13H, m), 1.74 - 1.83 (1H, s), 2.00 - 2.11 (2H, m), 2.38 - 2.47 (1H, m), 3.07 - 3.16 (1H, m), 3.81 (1H, m), 4.29 - 4.34 (1H, m), 5.04 - 5.17 (2H, m), 7.34 - 7.39 (m, 5H); m / z (ES + ) [M+H] + = 529.

[0141] Intermediate 6 (1.60 g): (2R,4R)-4-azido-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylic acid 2-benzyl 1-tert-butyl ester. 1 H NMR (400 MHz, DMSO-d6) δ 0.56 - 0.73 (2H, m), 0.98 - 1.13 (1H, m), 1.17 (12H, s), 1.26 - 1.37 (13H, m), 1.66 - 1.79 (1H, m), 2.01 - 2.22 (2H, m), 2.34 - 2.47 (1H, m), 3.60 (1H, br dd), 4.29 - 4.35 (1H, m), 5.04 - 5.18 (2H, m), 7.31 - 7.40 (5H, m); m / z (ES + ) [M+H] + = 529.

[0142] Intensity (%) Figure 2

[0143]

[0144] (2R,4R)-4-Azido-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylic acid 2-benzyl 1-tert-butyl ester (Intermediate 6, 688 mg, 1.30 mmol) was dissolved in ethyl acetate (13 mL) and methanol (4 mL), and treated with Pd / C (10% wt, 346 mg, 0.325 mmol). The flask was equipped with an H2 balloon, and the suspension was stirred overnight at room temperature. The reaction mixture was filtered through Celite and rinsed with methanol. The filtrate was concentrated under reduced pressure to give the product (Intermediate 7, 500 mg, 93% yield), which was used without further purification. 1 1H NMR (300 MHz, DMSO-d6) δ 0.67 (2H, t), 0.94 - 1.00 (1H, m), 1.17 (12H, s), 1.22 - 1.38 (11H, m), 1.43 - 1.53 (1H, m), 1.85 (1H, d), 2.00 - 2.15 (2H, m), 3.23 (2H, dd), 3.58 - 3.61 (1H, m), 3.80 - 3.88 (1H, m), 8.96 (2H, m); m / z (ES + ) [M+H] + = 413.

[0145] Figure 3 Figure 4

[0146]

[0147] Triethylamine (0.21 mL, 1.5 mmol) and HATU (254 mg, 0.668 mmol) were added successively to a solution of Boc-Val-OH (145 mg, 0.668 mmol) in DMF (2.9 mL), and the reaction was stirred at room temperature for 30 min. (2R,4R)-4-Amino-1-(tert-butoxycarbonyl)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-2-carboxylic acid (Intermediate 7, 250 mg, 0.606 mmol) was added as a solution in DMF (2.9 mL) to the reaction mixture. The reaction was stirred overnight at room temperature. The crude reaction mixture was concentrated and purified directly by silica gel chromatography (hexane / EtOAc) to give the product as a mixture of rotamers (Intermediate 8, 250 mg, 67% yield). 11H NMR (300 MHz, DMSO-d6) δ 0.64 - 0.73 (2H, m), 0.73 - 0.85 (6H, m), 1.13 - 1.14 (1H, m), 1.17 (12H, s), 1.22 - 1.42 (22H, m), 1.56 - 1.75 (1H, m), 1.79 - 1.97 (1H, m), 2.00 - 2.26 (2H, m), 3.08 - 3.24 (1H, m), 3.54 - 3.77 (2H, m), 4.12 - 4.36 (1H, m), 6.58 (1H, t), 7.96 - 8.03 (2H, m); m / z (ES + ) [M + H] + = 584。

[0148] Example 2: (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiro[4.7]dodecan-6-one Form 1 (acetone adduct) Form 1 Figure 5

[0149]

[0150] Trifluoroacetic acid (0.63 mL, 8.2 mmol) was added to a solution of (2R,4R)-1-(tert-butoxycarbonyl)-4-((S)-2-(tert-butoxycarbonylamino)-3-methylbutanamido)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-2-carboxylic acid (Intermediate 8, 250 mg, 0.409 mmol) in DCM (4 mL). The resulting solution was stirred at room temperature for 1 h and then concentrated in vacuo. The crude amino acid was dissolved in Et2O (2 mL) and 1 M aqueous HCl (2 mL). Phenylboronic acid (99 mg, 0.81 mmol) was added, and the clear two-phase solution was stirred at room temperature for 1 h. The reaction mixture was diluted with water and washed with Et2O. The aqueous layer was lyophilized and purified by ion-exchange chromatography (PoraPak Rxn CX 60 cc column). The desired product was eluted from the column using 2 M ammonia / methanol. The material obtained was further purified by reverse-phase chromatography (RediSep Rf C18Aq, 0% to 10% acetonitrile in water) to give (2R,4R)-4-((S)-2-amino-3-methylbutanamido)-2-(4-dihydroxyborinobutyl)pyrrolidine-2-carboxylic acid (Intermediate 9, 28 mg, 20% yield) as a mixture of white solid and rotamers. 11H NMR (300 MHz, D2O) δ 0.66 - 0.76 (2H, m), 0.85 (6H, dd), 1.07 - 1.43 (4H, m), 1.55 - 1.68 (1H, m), 1.77 - 1.97 (2H, m), 2.13 - 2.33 (2H, m), 3.07 (1H, d), 3.08 - 3.16 (1H, m), 3.37 - 3.48 (1H, m), 4.27 - 4.40 (1H, m); m / z (ES + ) [M + H] + = 330.

[0151] Angle (2θ ± 0.2°) Intensity (%)

[0152]

[0153] A 20.5 mg quantity of Intermediate 9 was suspended in 0.50 mL of MeCN. The suspension was heated to 75 °C and stirred at 75 °C for 1 hour. The suspension was then cooled to ambient temperature, and the solid was filtered and dried in air. A crystalline material with needle / rod-shaped crystals was obtained and named Form D of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutanamide.

[0154] Example 1 was analyzed by XRPD and the results were tabulated in the following table (Table 1) and shown in Figure 6 .

[0155] Table 1. XRPD Peaks of Form D

[0156] Figure 7 Figure 8 7.8 100.0 19.2 33.1 15.0 27.7 16.4 18.0 13.1 18.0 13.7 12.5 26.4 6.0 19.8 5.6 17.9 4.9 22.5 4.1

[0157] Example 1 is characterized by providing at least one of the following 2θ ± 0.2° values measured using CuKα radiation: 7.8, 19.2, and 15.0°.

[0158] Single crystals of Example 1 were obtained by slow evaporation of an acetonitrile solution, and single crystal structure analysis confirmed that Example 1 is the anhydrous form. The molecular structure of Example 1 is shown in Example 3: (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide Form D . Crystallographic data: Space group orthorhombic P212121, unit cell dimensions:

[0159] Example 1 was analyzed by thermal techniques. DSC analysis showed that Form D had an endothermic dehydration event starting at approximately 213 °C and peaking at approximately 214 °C. TGA showed that Example 1 exhibited a mass loss of approximately 0.4% when heated from approximately 25 °C to approximately 150 °C, and a mass loss of approximately 3.1% when heated from approximately 150 °C to approximately 225 °C. Representative DSC / TGA thermograms of Example 1 are shown in ​ in.

[0160] Example 1 was analyzed by gravimetric vapor sorption. GVS analysis showed that Example 1 absorbed less than 2% water at 70% relative humidity (RH) and began to deliquesce after 80% RH. Representative GVS thermograms of Example 1 are shown in ​ in.

[0161] ​ ​

[0162]

[0163] A 20.3 mg quantity of Intermediate 9 was suspended in 2.0 ml of acetone. After adding 200 μl of H2O, the solid dissolved. The solvent was removed by evaporation under ambient conditions. The resulting solid was suspended in 1.0 ml of acetone and stirred for 3 days at ambient temperature. After drying the solvent in air, a crystalline material (rod-shaped crystals) of the acetone adduct was obtained and named (3R,5R)-8-hydroxy-3-((S)-4-isopropyl-2,2-dimethyl-5-oxoimidazolidin-1-yl)-7-oxa-1-aza-8-boraspiro[4.7]dodecan-6-one.

[0164] Example 2 was analyzed by XRPD and the results are tabulated below (Table 2) and shown in ​ in.

[0165] Table 2. XRPD Peaks of the Acetone Adduct

[0166] ​ ​ 11.6 100.0 8.2 84.7 13.3 70.0 16.4 52.3 12.9 41.7 17.4 41.2 19.5 29.6 16.6 28.5 22.6 27.4 15.9 27.3

[0167] Example 2 is characterized by providing at least one of the following 2θ ± 0.2° values measured using CuKα radiation: 11.6, 8.2, and 13.3°.

[0168] Single crystals of Example 2 were obtained by slow evaporation of an acetone / H2O solution. Single crystal structure analysis confirmed that acetone was a monohydrate. The molecular structure of the acetone adduct is shown in ​ in. Crystallographic data: Space group orthorhombic P212121, unit cell dimensions:

[0169] Example 2 was analyzed by thermal techniques. DSC analysis showed that the acetone adduct had an endothermic dehydration event starting at 82 °C and peaking at 122 °C. Another endothermic dehydration event was also identified as starting at 177 °C and peaking at 182 °C. TGA showed that the acetone adduct exhibited a mass loss of approximately 5.5% when heated from approximately 25 °C to approximately 150 °C and a mass loss of approximately 3.2% when heated from approximately 150 °C to approximately 225 °C. Representative DSC / TGA thermograms of acetone are shown in ​ in.

[0170] Example 2 was analyzed by gravimetric vapor sorption. GVS analysis showed that the acetone adduct absorbed less than 2% water at 80% relative humidity (RH). Representative GVS thermograms of the acetone adduct are shown in ​ in.

[0171] ​ Dodecan-3-yl)-3-methylbutanamide Form E

[0172] A 30 mg quantity of Intermediate 9 was suspended in 0.50 mL of ethyl acetate pre-saturated with water. The suspension was heated to 75 °C and stirred at 75 °C for 1 hour. The suspension was then cooled to ambient temperature, and the solid was filtered and dried in air. A crystalline material with needle / rod crystals was obtained and named as form E of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutanamide.

[0173] Example 3 was analyzed by XRPD and the results were tabulated in Table 3 below and shown in Figure 9 in.

[0174] Table 3. XRPD Peaks of Form E

[0175] Angle (2θ ± 0.2°) Intensity (%) 12.3 100.0 18.8 64.1 9.3 46.9 14.2 35.9 14.1 35.2 19.8 26.1 26.2 19.5 17.3 17.5 7.1 14.0 25.4 13.4

[0176] Example 3 is characterized by providing at least one of the following 2θ ± 0.2° values measured using CuKα radiation: 12.3, 18.8, and 9.3°.

[0177] Single crystals of Example 3 were obtained by slow evaporation of a clear solution of a mixed solvent of acetonitrile / water (ratio 20:1), and single crystal structure analysis confirmed that Example 3 was in the monohydrate form. The molecular structure of Example 3 is shown in Figure 10 in. Crystallographic data: Space group hexagonal P61, unit cell dimensions:

[0178] Example 3 was analyzed by thermal techniques. DSC analysis showed that Form E had an endothermic dehydration event starting at about 105 °C and peaking at about 125 °C. Another endothermic dehydration event was also identified in the broad range from about 125 °C to about 225 °C. TGA showed that Example 3 exhibited a mass loss of about 6.0% when heated from about 25 °C to about 135 °C and a mass loss of about 3.0% when heated from about 125 °C to about 225 °C. Representative DSC / TGA thermograms of acetone are shown in Figure 11 in.

[0179] Example 3 was analyzed by gravimetric vapor sorption. GVS analysis showed that Example 3 absorbed less than 2% water at 80% relative humidity (RH). Representative GVS thermograms of Example 3 are shown in Figure 12 in.

[0180] Example 4: Biological Activity

[0181] Crystalline material Forms D (Example 1), 1 (Example 2) and E (Example 3) are converted into the same active chemical moiety when dissolved in an aqueous medium, e.g., under physiological conditions. The inhibition of human arginase 1 and arginase 2 activities by Example 1 was quantified by measuring the formation of thiol groups from L-thiocitrulline using recombinant arginase 1 or arginase 2 produced by Escherichia coli (E. coli.). Thiol groups were detected with the Ellman's reagent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). DTNB reacts with thiols to produce a mixed disulfide and 2-nitro-5-thiobenzoic acid (TNB), the latter being quantified by the absorbance of the anion (TNB 2- ) at 412 nm.

[0182] Assays were performed in clear 384-well plates (Greiner catalog number: 781101). Different concentrations of Example 1 in 300 nL DMSO were dispensed into the assay plates using an Echo acoustic dispenser, and then the plates were immediately sealed and centrifuged.

[0183] Two premixes were prepared from reagents thawed immediately prior to addition to the assay plates. Premix 1 contained human arginase 1 or human arginase 2 at a final concentration of 5 nM and 0.5 mM DTNB in the following assay buffer: 45 mM HEPES pH 7.5, brij 35 (0.045% (w / v)) and 100 μM MnCl2. Premix 2 contained freshly thawed 0.5 mM L-thiocitrulline in the assay buffer. 15 μL of Premix 1 was dispensed into the assay plates containing Example 1, centrifuged and incubated at room temperature for 30 minutes, then 15 μL of Premix 2 was added.

[0184] Centrifuge the assay plate and then read the absorbance at 412 nm in a Pherastar multimode microplate reader to collect data at time point 0 (T0). Incubate the plate at room temperature for 60 min and then read again to collect data at time point 1 (T1). Obtain the data by subtracting the A412 signal measured at T0 (time point 0) from the A412 signal measured at T1 (time point 1). Convert the data to % activity using the following equation:

[0185] % Activity of compound = 100 * [(X - minimum) / (maximum - minimum)]

[0186] where X represents the normalized value of the compound based on the minimum (vehicle) and maximum (reference compound) inhibition controls.

[0187] Calculate the concentration of Example 1 that inhibits activity by 50% (i.e., IC 50 ) by plotting % activity versus the test compound concentration and fitting the data using the Genedata Screener Smart fitting algorithm. The IC 50 of Example 1 for arginase 1 is 0.222 μM and for arginase 2 is 0.282.

Claims

1. (S)-2-Amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide Polymorph D, characterized in that, The X-ray powder diffraction pattern contains peaks having the following 2θ±0.2° values: 7.8, 19.2, 15.0, 16.4, 13.1, and 13.7 degrees.

2. (S)-2-Amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide polymorph D according to claim 1, wherein, The X-ray powder diffraction pattern is substantially as shown in Figure 1.

3. (S)-2-Amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide Crystal Form D, characterized in that, The X-ray powder diffraction pattern contains at least 7 peaks selected from 7.8, 19.2, 15.0, 16.4, 13.1, 13.7, 26.4, 19.8, 17.9, and 22.5 degrees 2θ±0.2°.

4. (S)-2-Amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide polymorph D, characterized in that, The X-ray powder diffraction pattern contains at least 9 peaks selected from 7.8, 19.2, 15.0, 16.4, 13.1, 13.7, 26.4, 19.8, 17.9, and 22.5 degrees 2θ±0.2°.

5. The crystalline form D of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide according to any one of claims 1 to 4, further characterized by a differential scanning calorimetry (DSC) curve including an endothermic peak at 214 °C.

6. (S)-2-Amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide polymorph D according to claim 5, wherein, The DSC curve is substantially as shown in Figure 3.

7. The crystalline form D of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide according to any one of claims 1 to 4, wherein a coordinate covalent bond is formed between the nitrogen atom and the boron atom in the pyrrolidine moiety, as represented by the arrow line in the following structural formula:

8. A pharmaceutical composition comprising the crystalline form D of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide according to any one of claims 1 to 7 and a pharmaceutically acceptable carrier.

9. A pharmaceutical composition comprising an effective amount of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide and a pharmaceutically acceptable carrier, wherein at least 85% of the (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide is the crystalline form D according to any one of claims 1 to 7.

10. The pharmaceutical composition according to claim 9, wherein at least 90% of the (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide is the crystalline form D according to any one of claims 1 to 7.

11. The pharmaceutical composition according to claim 9, wherein at least 95% of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide is crystalline form D as described in any one of claims 1 to 7.

12. The pharmaceutical composition according to claim 9, wherein at least 96% of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide is crystalline form D as described in any one of claims 1 to 7.

13. The pharmaceutical composition according to claim 9, wherein at least 97% of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide is crystalline form D as described in any one of claims 1 to 7.

14. The pharmaceutical composition according to claim 9, wherein at least 98% of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide is crystalline form D as described in any one of claims 1 to 7.

15. The pharmaceutical composition according to claim 9, wherein at least 99% of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide is crystalline form D as described in any one of claims 1 to 7.

16. The pharmaceutical composition according to claim 9, wherein at least 99.5% of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide is crystalline form D as described in any one of claims 1 to 7.

17. Use of the pharmaceutical composition for the manufacture of a medicament for the treatment of cancer, said pharmaceutical composition comprising an effective amount of crystalline form D of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide as described in any one of claims 1 to 7, wherein the cancer is selected from lung cancer, pancreatic cancer, colorectal cancer, breast cancer, acute myeloid leukemia (AML), prostate cancer, bladder cancer, melanoma and ovarian cancer.

18. Use of crystalline form D of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiroundecane-3-yl)-3-methylbutyramide according to any one of claims 1 to 7 for the manufacture of a medicament for the treatment of cancer, wherein the cancer is selected from lung cancer, pancreatic cancer, colorectal cancer, breast cancer, acute myeloid leukemia (AML), prostate cancer, bladder cancer, melanoma and ovarian cancer.

19. (S)-2-Amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide Crystal Form E, characterized in that, The X-ray powder diffraction pattern comprises peaks having the following 2θ ± 0.2° values: 12.3, 18.8, 9.3, 14.2, 14.1 and 19.8 degrees.

20. (S)-2-Amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutanamide polymorph E according to claim 19, wherein, The X-ray powder diffraction pattern is substantially as shown in Figure 9.

21. (S)-2-Amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide crystal form E, characterized in that, The X-ray powder diffraction pattern comprises at least 7 peaks selected from 12.3, 18.8, 9.3, 14.2, 14.1, 19.8, 26.2, 17.3, 7.1 and 25.4 degrees 2θ ± 0.2°.

22. (S)-2-Amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide Crystal Form E, characterized in that, The X-ray powder diffraction pattern comprises at least 9 peaks selected from 12.3, 18.8, 9.3, 14.2, 14.1, 19.8, 26.2, 17.3, 7.1 and 25.4 degrees 2θ ± 0.2°.

23. Crystalline form E of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiroundecane-3-yl)-3-methylbutyramide according to any one of claims 19 to 22, further characterized in that it comprises a differential scanning calorimetry (DSC) curve with an endothermic peak at 125 °C.

24. (S)-2-Amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide crystal form E according to claim 23, wherein, The DSC curve is substantially as shown in Figure 11.

25. Crystalline form E of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiroundecane-3-yl)-3-methylbutyramide according to any one of claims 19 to 22, wherein a coordination covalent bond is formed between the nitrogen atom of the pyrrolidine moiety and the boron atom, as represented by the arrow line in the following structural formula:

26. A pharmaceutical composition comprising crystalline form E of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiroundecane-3-yl)-3-methylbutyramide according to any one of claims 19 to 25 and a pharmaceutically acceptable carrier.

27. A pharmaceutical composition comprising an effective amount of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiroundecane-3-yl)-3-methylbutyramide and a pharmaceutically acceptable carrier, wherein at least 85% of the (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiroundecane-3-yl)-3-methylbutyramide is crystalline form E according to any one of claims 19 to 25.

28. The pharmaceutical composition according to claim 27, wherein at least 90% of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide is crystalline form E as described in any one of claims 19 to 25.

29. The pharmaceutical composition according to claim 27, wherein at least 95% of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide is crystalline form E as described in any one of claims 19 to 25.

30. The pharmaceutical composition according to claim 27, wherein at least 96% of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide is crystalline form E as described in any one of claims 19 to 25.

31. The pharmaceutical composition according to claim 27, wherein at least 97% of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide is crystalline form E as described in any one of claims 19 to 25.

32. The pharmaceutical composition according to claim 27, wherein at least 98% of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide is crystalline form E as described in any one of claims 19 to 25.

33. The pharmaceutical composition according to claim 27, wherein at least 99% of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide is crystalline form E as described in any one of claims 19 to 25.

34. The pharmaceutical composition according to claim 27, wherein at least 99.5% of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide is crystalline form E as described in any one of claims 19 to 25. Use of a pharmaceutical composition for the manufacture of a medicament for the treatment of cancer, said pharmaceutical composition comprising an effective amount of crystalline form E of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide according to any one of claims 19 to 25, wherein the cancer is selected from lung cancer, pancreatic cancer, colorectal cancer, breast cancer, acute myeloid leukemia (AML), prostate cancer, bladder cancer, melanoma and ovarian cancer.

36. Use of crystalline form E of (S)-2-amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutyramide according to any one of claims 19 to 25 for the manufacture of a medicament for the treatment of cancer, wherein the cancer is selected from lung cancer, pancreatic cancer, colorectal cancer, breast cancer, acute myeloid leukemia (AML), prostate cancer, bladder cancer, melanoma and ovarian cancer.

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