Phosphates of ptk inhibitor a and uses thereof

Abstract

The disclosure herein relates to the biological activity and preparation of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5-azaspiro[2.4]-heptan-7-ol (Compound A) stable crystalline forms (Compound A-Xln); tablets thereof (Compound A-Xln tablets); its phosphate (Compound A-P) and phosphate stable crystalline forms (Compound A-P-Xln); and its enantiomers (Compound R-A, Compound S-A). In addition, its anticancer activity, stability, chirality, dissolution, and therapeutic indications are also investigated.

Description

[0001] This application is a divisional application of a Chinese Invention Patent application No. 2023800352991, titled “Biological Activity of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5-azaspiro[2.4]-heptan-7-ol (Compound A) Crystals, Phosphate and Its Enantiomers” and filed on June 9, 2023, which claims priority to U.S. Provisional Application No. 63 / 351,205, filed on June 10, 2022. This application claims priority to U.S. Non-Provisional Application No. 18 / 206,506, filed on June 6, 2023.

[0002] This application is a divisional application of a Chinese Invention Patent application No. 2023800352991, titled “Biological Activity of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5-azaspiro[2.4]-heptan-7-ol (Compound A) Crystals, Phosphate and Its Enantiomers” and filed on June 9, 2023, which claims priority to U.S. Provisional Application No. 63 / 351,205, filed on June 10, 2022. This application claims priority to U.S. Non-Provisional Application No. 18 / 206,506, filed on June 6, 2023. TECHNICAL FIELD

[0003] The present disclosure relates to the biological activity and preparation of a stable crystalline form of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5-azaspiro[2.4]-heptan-7-ol (Compound A) (Compound A-Xln); Tablets thereof (Compound A-Xln tablets); Figure 2 its phosphate (Compound A-P) and phosphate stable crystalline form (Compound A-P-Xln); and its enantiomers (Compound R-A, Compound S-A). BACKGROUND

[0004] Protein tyrosine kinases (PTKs) are a family of enzymes that catalyze the phosphorylation process that transfers a phosphate group from a nucleoside triphosphate (usually ATP) to a protein amino acid residue. This phosphorylation process can activate the phosphorylated protein. Thus, PTKs act as “switches” to regulate many cellular functions by controlling the signal cascade from outside the cell through the membrane to the inside cytoplasm and even the nucleus. SUMMARY

[0005] Several embodiments disclosed herein relate to PTK inhibitors with improved stability. These inhibitors can be used to treat PTK-mediated disorders and diseases. For example, PTKs can be classified into epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR), fibroblast growth factor receptor (FGFR), etc. depending on their location on different extracellular domains. A large number of studies have shown that normal cells usually exhibit low or no activity of PTK, while many tumor cells, especially glioma and cancer, exhibit overexpression of PTK. Apparently, abnormal overactivity of PTK is closely related to the processes of tumor cell growth and angiogenesis.

[0006] Inhibition or blockade of PTK activity can significantly inhibit the growth of tumor cells by inhibiting cell signaling. Thus, targeted therapy by inhibition of the expression of PTKs by PTK inhibitors has become a widely accepted therapy.

[0007] The novel PTK inhibitor 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6- methoxyquinolin-7-yloxy)ethyl)-5-azaspiro-[2.4]-heptan-7-ol (Compound A) has the potential to inhibit the activity of a variety of protein tyrosine kinases (PTKs), including but not limited to VEGFr, EGFr, c-kit, PDGF, FGF, SRC, Aurora B, and the like. The structure and synthetic pathway of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5-azaspiro-[2.4]-heptan-7-ol (Compound A) has been disclosed in patent WO2010021918.

[0008] Several embodiments relate to stable crystalline forms of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5-azaspiro[2.4]-heptan-7-ol (Compound A-Xln).

[0009] In several embodiments, the stable crystalline form of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5-azaspiro[2.4]-heptan-7-ol is prepared by a method comprising recrystallization of amorphous form of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5-azaspiro[2.4]-heptan-7-ol.

[0010] In several embodiments, the stable crystalline form of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5-azaspiro[2.4]-heptan-7-ol is prepared by a method comprising two recrystallization steps.

[0011] In several embodiments, the stable crystalline form of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5-azaspiro[2.4]-heptan-7-ol is prepared by a method comprising a recrystallization process using a high boiling point solvent or a mixture of solvents with high boiling points together.

[0012] In several embodiments, the stable crystalline form of 5-(2-(4-(4-fluoro-2-methyl-1H- indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5-azaspiro[2.4]-heptan-7-ol is prepared by a method comprising a recrystallization process using a low boiling point solvent or a mixture of solvents together having a low boiling point.

[0013] In several embodiments, the stable crystalline form of 5-(2-(4-(4-fluoro-2-methyl-1H- indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5-azaspiro[2.4]-heptan-7-ol is prepared by a method comprising a first recrystallization process and a second recrystallization process.

[0014] In several embodiments, the stable crystalline form of 5-(2-(4-(4-fluoro-2-methyl-1H- indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5-azaspiro[2.4]-heptan-7-ol is prepared by a method comprising: a first recrystallization process using a high boiling point solvent or a mixture of solvents together having a high boiling point; and a second recrystallization process using a low boiling point solvent or a mixture of solvents together having a low boiling point. In several embodiments, the high boiling point solvent is DMF and the low boiling point solvent is EtOH.

[0015] Several embodiments are directed to a crystalline form of 5-(2-(4-(4-fluoro-2-methyl-1H- indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5-azaspiro[2.4]-heptan-7-ol that exhibits at least one of the following properties: DSC melting range (Endo): 240-260°C with peak temperature range: 244-254°C; more particularly, DSC melting range (Endo): 247-253°C with peak temperature = 249°C, as shown in the figure Figure 3 TGA thermogram showing undissolved material with weight loss above 250°C, as shown in the figure Figure 4

[0016] Several embodiments are directed to a crystalline form of 5-(2-(4-(4-fluoro-2-methyl-1H- indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5-azaspiro[2.4]-heptan-7-ol characterized by an XRPD pattern as shown in Figure 11

[0017] ​​Certain embodiments are directed to a crystalline form of 5-(2-(4-(4-fluoro-2-methyl-1H- indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5-azaspiro[2.4]-heptan-7-ol characterized by an XRPD pattern comprising 14 characteristic peaks, where greater than 10% of the intensity % is expressed in d-values and angles as follows:

[0018]

[0019] Certain embodiments are directed to a crystalline form of 5-(2-(4-(4-fluoro-2-methyl-1H- indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5-azaspiro[2.4]-heptan-7-ol characterized by an XRPD pattern as shown in Figure 9

[0020] Certain embodiments are directed to a crystalline form of 5-(2-(4-(4-fluoro-2-methyl-1H- indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5-azaspiro[2.4]-heptan-7-ol characterized by an XRPD pattern comprising 43 characteristic peaks, where all of the intensity % is expressed in d-values and angles as follows:

[0021]

[0022]

[0023] In certain embodiments, the crystalline form of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5- yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5-azaspiro[2.4]-heptan-7-ol has better stability and / or solubility than its amorphous form.

[0024] In certain embodiments, the stable crystalline form of 5-(2-(4-(4-fluoro-2-methyl-1H-indol- 5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5-azaspiro[2.4]-heptan-7-ol is a pharmaceutically acceptable salt. In certain embodiments, the stable crystalline form of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5-azaspiro[2.4]-heptan-7-ol is a phosphate salt.

[0025] In certain embodiments, the phosphate salt is prepared by a process comprising the steps of neutralization using a solvent or a mixed solvent of phosphoric acid solution and recrystallization. In certain embodiments, the solvent in which the neutralization and / or recrystallization is performed is EtOH.

[0026] ​In several embodiments, the phosphate salt of 5-(2-(4-(4-fluoro-2-methyl-1H- indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5-azaspiro[2.4]-heptan-7-ol is characterized by one or more of the following properties: DSC melting range (Endo): 221-235 °C with peak temperature = 229 °C, graph as shown in Figure 10 TGA showing a slight weight loss at about 30-60 °C and a significant weight loss above 210 °C, graph as shown in Figure 11

[0027] In several embodiments, the phosphate salt of 5-(2-(4-(4-fluoro-2-methyl-1H- indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5-azaspiro[2.4]-heptan-7-ol includes an XRPD pattern as shown in Figure 1 XRPD results include all characteristic peaks, where all intensity % are expressed in d-value and angle as follows:

[0028]

[0029]

[0030] Several embodiments are directed to a compound having a structure represented by: 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5- azaspiro[2.4]-heptan-7-ol, a stable crystalline form thereof, a salt form thereof, and / or a stable crystalline salt form thereof, for use in inhibiting a protein tyrosine kinase (PTK). In several embodiments, the PTK is selected from, but not limited to, FGFR1(h), FGFR2(h), FGFR3(h), Flt1(h) (VEGFr1), Flt4(h) (VEGFr3), KDR(h) (VEGFr2), Aurora-B PDGFRa(h), PDGFRa(h), and PDGFRp(h). In several embodiments, the salt form is the phosphate salt form.

[0031] ​Several embodiments are directed to a compound having a structure represented by: 5-(2-(4-(4-fluoro-2-methyl-lH-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5- azaspiro[2.4]-heptan-7-ol, a stable crystalline form thereof, a salt form thereof, and / or a stable crystalline salt form thereof, wherein the compound inhibits common cancer cell lines including, but not limited to, PANC-1, NCI-H157, MDA-MB-231, Hela, PC-3, BEL7404, MKN45, Ishikawa, Saos-2, SKOV3, SW579, and HCT116. In several embodiments, the salt form is the phosphate salt form.

[0032] Several embodiments are directed to a pharmaceutical composition comprising an active ingredient that is a compound described above or elsewhere herein and a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable excipient.

[0033] Several embodiments are directed to a pharmaceutical composition comprising an active ingredient that is a compound described above or elsewhere herein and a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable excipient to form a tablet.

[0034] Several embodiments are directed to a pharmaceutical composition comprising an active ingredient that is a compound selected from a stable crystalline free base form of 5-(2-(4-(4-fluoro-2-methyl-lH-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5- azaspiro[2.4]-heptan-7-ol or a stable crystalline form of a phosphate salt and a pharmaceutically acceptable carrier.

[0035] Several embodiments relate to a method of treating a neoplastic disease, the method comprising administering to a subject in need thereof a compound as defined above or elsewhere herein or a pharmaceutical composition comprising such a compound and a pharmaceutically acceptable excipient. In several embodiments, the method comprises administering an additional agent, which is a chemotherapeutic compound and / or an immunotherapy agent. In several embodiments, the neoplastic disease is a solid tumor selected from lung cancer, renal cancer, colorectal cancer, gastric cancer, melanoma, head and neck cancer, thyroid cancer, pancreatic cancer, liver cancer, prostate cancer, bladder cancer, brain cancer, sarcoma, breast cancer, ovarian cancer, cervical cancer, and endometrial cancer; and a blood cancer selected from ALL, CLL, AML, CML, and multiple myeloma, especially in human clinical studies treating small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), and ovarian cancer. Several embodiments relate to a method of treatment, wherein the combination chemotherapeutic agent is selected from a platinum or a taxane or a topoisomerase 1 inhibitor or an alkaloid or an alkylating agent. In several embodiments, the chemotherapeutic agent is selected from cisplatin, carboplatin, paclitaxel or cisplatin / paclitaxel or carboplatin / paclitaxel or carboplatin / etoposide or topotecan, irinotecan, or lomustine. In several embodiments, the immunotherapy agent is selected from a PD-1 antibody or a PD-L1 antibody including but not limited to nivolumab, pembrolizumab, ipilimumab, blinatumomab, elotuzumab, daratumumab, cemiplimab, avelumab, durvalumab, atezolizumab, toripalimab, sintilimab, camrelizumab, tislelizumab, AK104, Penpulimab, KN035, CS1001, talimogene laherparepvec. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 2 DSC pattern of amorphous (Compound A-Amp) form of 5-(2-(4-(4-fluoro-2-methyl-1H- indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5-azaspiro[2.4]-heptan-7-ol.

[0037] Figure 3 DSC pattern of a crystalline form of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6- methoxyquinolin-7-yloxy)ethyl)-5-azaspiro[2.4]-heptan-7-ol.

[0038] Figure 4 TGA pattern of a crystalline form of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6- methoxyquinolin-7-yloxy)ethyl)-5-azaspiro[2.4]-heptan-7-ol.

[0039] Figure 5 XRPD pattern of a crystalline form of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6- methoxyquinolin-7-yloxy)ethyl)-5-azaspiro[2.4]-heptan-7-ol.

[0040] Figure 6 DSC pattern of a crystalline form of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6- methoxyquinolin-7-yloxy)ethyl)-5-azaspiro[2.4]-heptan-7-ol. 1 H-HMR pattern.

[0041] Figure 7 Stability testing of amorphous and crystalline API of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5- yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5-azaspiro[2.4]-heptan-7-ol.

[0042] Figure 8 Stability testing of amorphous and crystalline tablets of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5- yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5-azaspiro[2.4]-heptan-7-ol.

[0043] Figure 9 Dissolution profile of amorphous and crystalline tablets of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5- yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5-azaspiro[2.4]-heptan-7-ol.

[0044] Figure 10 DSC pattern of a crystalline form of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6- methoxyquinolin-7-yloxy)ethyl)-5-azaspiro[2.4]-heptan-7-ol.

[0045] Figure 11TGA plot of a crystalline form of the phosphate salt of 5-(2-(4-(4-fluoro-2-methyl-1H- indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5-azaspiro[2.4]-heptan-7-ol.

[0046] Figure 12 XRPD plot of a crystalline form of the phosphate salt of 5-(2-(4-(4-fluoro-2-methyl-1H- indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5-azaspiro[2.4]-heptan-7-ol.

[0047] Figure 13 TGA plot of a crystalline form of the phosphate salt of 5-(2-(4-(4-fluoro-2-methyl-1H- indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5-azaspiro[2.4]-heptan-7-ol. 1 H-HMR plot.

[0048] Figure 14 Proliferation inhibition of breast cancer cell line MCF7 by Compound A-Xln, anti-breast cancer agent Anastrozole and Letrozole, combination of Compound A-Xln and Anastrozole, and combination of Compound A-Xln and Letrozole.

[0049] Figure 15 Proliferation inhibition of breast cancer cell line BT474 by Compound A-Xln, anti-breast cancer agent Anastrozole and Letrozole, combination of Compound A-Xln and Anastrozole, and combination of Compound A-Xln and Letrozole.

[0050] Figure 16 Proliferation inhibition of colon cancer cell line HT29 by Compound A-Xln, anti-colon cancer agent 5FU / O, and combination of Compound A-Xln and 5FU / O.

[0051] Figure 17 Proliferation inhibition of colon cancer cell line HCT116 by Compound A-Xln, anti-colon cancer agent 5FU / O, and combination of Compound A-Xln and 5FU / O.

[0052] Figure 18 Proliferation inhibition of colon cancer cell line colo205 by Compound A-Xln, anti-colon cancer agent 5FU / O, and combination of Compound A-Xln and 5FU / O.

[0053] Figure 19 Comparison of mean tumor volume change after administration of Compound A-Xln, an anti-PD1 antibody agent, and combination therapy of Compound A-Xln and anti-PD1 antibody agent to a murine colorectal CT26 cell line xenograft model.

[0054] Figure 20 The relative tumor volume changes after treatment of a small cell lung cancer NCI-H1436 cell line xenograft model with compound A-Xln.

[0055] Figure 21 The relative tumor volume changes after treatment of a non-small cell lung cancer (NSCLC) 95D cell line xenograft model with compound A-Xln.

[0056] Figure 22 The relative tumor volume changes after treatment of an ovarian cancer SKOV3 cell line xenograft model with compound A-Xln.

[0057] Figure 23 The relative tumor volume change after treatment of a renal cell carcinoma 786-O cell line xenograft model with compound A-Xln.

[0058] Figure 24 The relative tumor volume changes after treatment of a Bel-7402 hepatocellular carcinoma cell line xenograft model with compound A-Xln.

[0059] Figure 25 The relative tumor volume changes after treatment of a glioblastoma U87 cell line xenograft model with compound A-Xln.

[0060] Figure 26 Kaplan-Meier progression-free survival (PFS) curves for SCLC patients in group C (≥2 prior lines of therapy, n=30) and group B (=1 prior line of therapy, n=30).

[0061] Compound Kaplan-Mayer overall survival (OS) curves for SCLC patients in group C (≥2 prior lines of therapy, n=30) and group B (=1 prior line of therapy, n=30). Detailed Implementation

[0062] Several embodiments relate to the compound 5-(2-(4-(4-fluoro-2-methyl-1H- indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5-azaspiro[2.4]-heptan-7-ol (Compound A), stable crystalline forms thereof (Compound A-Xln), amorphous (Compound A-Amp), stable crystalline forms of crystallization thereof from phosphates (Compound A-P) (Compound A-P-Xln); and enantiomers of any of the foregoing phosphates or crystallizations (both chiral isomers). Compound A is represented by the following structure:

[0063]

[0064] The following description provides context and examples, but should not be interpreted as limiting the scope of the application encompassed by the claims in this specification or any other application for which this specification has priority. No single component (including method steps) or set of components (e.g., multiple steps) is essential or indispensable. Any feature, structure, component, material, step, or method described and / or illustrated in any embodiment in this specification can be used with or instead of any feature, structure, component, material, step, or method described and / or illustrated in any other embodiment in this specification.

[0065] The terms and phrases used in this application, and variations thereof, unless otherwise expressly stated, should be construed as open ended, rather than limiting. As examples of the foregoing, the term 'including' should be construed as meaning 'including, without limitation;' 'comprising' as meaning 'comprising, without limitation,' 'containing' as meaning 'containing, without limitation,' and 'having' as meaning 'having, without limitation.' The term "consisting of" is to be construed as meaning "consisting of, without limitation," unless otherwise indicated. The term "consisting essentially of" should be construed as meaning "consisting essentially of, without limitation," unless otherwise indicated. The term "example" is used to provide exemplary instances of the items being discussed and does not, unless otherwise stated, imply or require that all such items have the same characteristics. The use of terms "preferably," "preferred," "desired," or "desirable," and words of similar meaning in the detailed description should not be understood as implying that certain features are critical, essential, or even important to the structure or function of the application. Rather, these terms have simply been used to enable the patent to direcdy to the claimed application. Additionally, the term "include" should be interpreted as meaning "include, but not limited to." As used in the process context, the term "include" means that the process includes at least the recited steps, but can include additional steps. As used in the compound, composition, or device context, the term "include" means that the compound, composition, or device includes at least the recited features or components, but can also include additional features or components. Likewise, a group of items linked with the conjunction "and" should not be construed as requiring that each and every one of those items be present in the grouping, unless otherwise expressly specified in the claim. Similarly, a group of items linked with the conjunction "or" should not be construed as requiring mutual exclusivity among that group, unless otherwise expressly specified in the claim.

[0066] Additionally, the phrase "consisting essentially of should be construed as including those elements specifically recited and not excluding additional elements of the technology. The phrase "consisting of should be construed as excluding any element not specified.

[0067] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. A feature disclosed under one heading (such as a composition) can be used in combination with a feature disclosed under a different heading (a method of treatment). Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. It should be noted that the use of particular terms should not be interpreted as implying that the terms are redefined herein to be restricted to only the specific characteristics associated with the term as used in this disclosure.

[0068] When referring to various features, the terms "or" and / or "and" can be used in the alternative, or in the sense of including each possibility. These terms (and variations thereof) are intended to encompass any range of values encompassing or spanning any of the values. For example, with respect to temperature, temperature can be expressed as "equal to or at least about: 40°C, 50°C, 60°C, 70°C, or encompassing and / or spanning ranges of the above values." This language is intended to encompass not only the specific temperatures provided and ranges above the temperature (e.g., equal to or at least about 40°C, equal to or at least about 50°C, equal to or at least about 60°C, and equal to or at least about 70°C), but also temperature ranges spanning these values (e.g., 40°C to 50°C, 40°C to 60°C, 40°C to 70°C, 50°C to 60°C, 50°C to 70°C, or 60°C to 70°C). Similarly, with respect to temperature, temperature can be expressed as "equal to or less than about: 40°C, 50°C, 60°C, 70°C, or encompassing and / or spanning ranges of the above values." This language is intended to encompass not only the specific temperatures provided and ranges below the temperature (e.g., equal to or less than about 40°C, equal to or less than about 50°C, equal to or less than about 60°C, and equal to or less than about 70°C), but also temperature ranges spanning these values (e.g., 40°C to 50°C, 40°C to 60°C, 40°C to 70°C, 50°C to 60°C, 50°C to 70°C, or 60°C to 70°C).

[0069] The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. In addition, various adjuvants can be included as is useful in the art. Considerations for including various components in pharmaceutical compositions are described, for example, in Gilman et al. (eds.) (1990); Goodman and Gilman's: The Pharmacological Basis of Therapeutics, 8th Ed., Pergamon Press, which is incorporated herein by reference in its entirety.

[0070] In some embodiments, a“patient” or“subject” disclosed herein is a human patient, although it is understood that the principles of the presently disclosed subject matter indicate that the presently disclosed subject matter is effective for all vertebrate species, including mammals, intended to be encompassed by the terms“subject” and“patient.” Suitable subjects are generally mammalian subjects. The subject matter described herein can be used in research as well as in veterinary and medical applications. As used herein, the term“mammal” includes, but is not limited to, humans, non-human primates, cows, sheep, goats, pigs, horses, cats, dogs, rabbits, rodents (e.g., rats or mice), monkeys, and the like. Human subjects include neonatal, infant, adolescent, adult, and geriatric subjects.

[0071] As used herein, the term“therapeutically effective amount” refers to an amount that imparts a therapeutic modulation to a subject afflicted with a disorder, disease, or condition, for example, that can be a beneficial effect, including ameliorating the subject’s condition (e.g., modulating one or more symptoms), delaying or minimizing progression of the condition, preventing or delaying onset of the disorder, and / or altering a clinical parameter, disease, or condition, etc. For example, in some embodiments, an effective amount can refer to an amount of a composition, compound, or agent that results in at least a 5% (e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%) improvement in the subject’s condition. Actual dosage levels of active ingredients and agents in an active composition of the disclosed subject matter can be varied so as to administer an amount that is effective to achieve the desired response for a particular subject and / or application. The selected dosage level will depend on a variety of factors including but not limited to the activity of the composition, formulation, route of administration, combination with other drugs or treatments, severity of the condition being treated, and physical condition and prior medical history of the subject being treated. Determination and adjustment of effective dosages are contemplated herein, as well as evaluation of when and how to make such adjustments. The term“therapeutically effective amount” can mean a therapeutic amount sufficient to prevent the spread of cancer or reverse cancer.

[0072] Methods of making crystalline forms

[0073] Several embodiments relate to therapeutic compounds (e.g., therapeutic agents). Several embodiments relate to 5-(2-(4-(4-fluoro-2-methyl-lH-indol-5-yloxy)-6-methoxyquinolin-7- yloxy)ethyl)-5-azaspiro[2.4]-heptan-7-ol (Compound A), a crystalline form (Compound A-Xln), a stable crystalline form of a phosphate salt thereof (Compound A-P-Xln), and an enantiomer of any of the foregoing (e.g., Compound R-A, Compound S-A, Compound R-A-Xln, Compound S-A-Xln, Compound R-A-P-Xln, Compound S-A-P-Xln), each of which is a therapeutic compound. In several embodiments, S in the Compound A name indicates the S configuration, and R in the Compound A name indicates the R configuration. The two enantiomers of Compound A, Compound R-A and Compound S-A, are further represented by the following structures, respectively:

[0074]

[0075] In several embodiments, a crystalline form of Compound A is provided. In several embodiments, the crystalline form of Compound A is a free base form. In several embodiments, the crystalline form is denoted as Compound A-Xln. In several embodiments, the crystalline form is characterized by one or more peaks in an XRPD pattern. In several embodiments, the crystalline form is characterized by an XRPD peak at one or more angles selected from 8.899, 9.927, 11.586, 13.145, 14.938, 15.374, 16.104, 16.558, 17.327, 17.820, 18.492, 18.809, 19.912, 20.445, 21.511, 22.004, 22.499, 23.188, 23.859, 24.314, 24.590, 25.171, 25.952, 26.464, 26.841, 27.217, 27.648, 28.457, 29.310, and 29.856 or a combination of any of the foregoing. In several embodiments, the crystalline form is characterized by an XRPD peak with an intensity exceeding 10% at one or more angles selected from 8.899, 9.927, 14.938, 15.374, 16.104, 16.558, 18.492, 19.912, 20.445, 21.511, 23.859, 24.314, 24.590, and 25.952 or a combination of any of the foregoing. In several embodiments, the crystalline form is characterized by an XRPD peak with an intensity exceeding 20% at one or more angles selected from 9.927, 14.938, 16.558, 20.445, and 21.511 or a combination of any of the foregoing.

[0076] In several embodiments, the DSC melting range of compound A-Xln is characterized by a melting range equal to or less than about 245 °C, 250 °C, 255 °C, or a range including and / or spanning the aforementioned values. In several embodiments, the DSC melting range of compound A-Xln is characterized by a melting point of 249 °C.

[0077] In several embodiments, the TGA peak weight loss range of compound A-Xln is equal to or less than about 245 °C, 250 °C, 255 °C, or a range including and / or spanning the aforementioned values. In several embodiments, the TGA peak weight loss of compound A-Xln occurs at a temperature of 249 °C.

[0078] In several embodiments, a crystalline form of a salt of compound A is provided. In several embodiments, the crystalline form is denoted as compound A-P-Xln. In several embodiments, the crystalline form is characterized by one or more peaks in an XRPD pattern. In several embodiments, the crystalline form is characterized by an XRPD peak at one or more angles selected from 5.268, 7.139, 9.805, 10.455, 11.799, 12.417, 12.669, 13.672, 14.307, 15.546, 16.064, 16.719, 17.495, 18.035, 18.802, 19.530, 21.095, 22.535, 23.426, 25.916, and 26.577 or a combination of any of the foregoing. In several embodiments, the crystalline form is characterized by an XRPD peak with an intensity exceeding 50% at one or more angles selected from 7.139, 12.417, 12.669, 13.672, 16.064, 16.719, 19.530, 21.095, and 23.426 or a combination of any of the foregoing. In several embodiments, the crystalline form is characterized by an XRPD peak with an intensity exceeding 80% at one or more angles selected from 7.139, 13.672, 19.530, and 23.426 or a combination of any of the foregoing.

[0079] In several embodiments, the DSC melting range of compound A-P-Xln is characterized by a melting range equal to or less than about 220 °C, 225 °C, 230 °C, 235 °C, 240 °C, 245 °C, or a range including and / or spanning the aforementioned values. In several embodiments, the DSC melting range of compound A-P-Xln is characterized by a melting point of 229 °C.

[0080] In several embodiments, the TGA peak weight loss of compound A-P-Xln ranges from about equal to or less than: 205 °C, 210 °C, 215 °C, 220 °C, 225 °C, 230 °C, or a range including and / or spanning the above values. In several embodiments, the TGA peak weight loss of compound A-P-Xln, compound, occurs at a temperature of 210 °C.

[0081] Pharmaceutical compositions

[0082] Several embodiments relate to methods of preparing a stable crystalline form of 5-(2-(4-(4-fluoro-2-methyl-lH-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5- azaspiro-[2.4]-heptan-7-ol (Compound A) from an amorphous form of Compound A (Compound A-Amp), such as Compound A-Xln, Compound S-A-Xln, Compound R-A-Xln, Compound A-P-Xln, Compound S-A-P-Xln, or Compound R-A-P-Xln. In several embodiments, the method of preparing a stable crystalline form of Compound A includes one or more crystallization and / or recrystallization steps.

[0083] In several embodiments, the preparation of a stable crystalline form of Compound A (e.g., Compound A-Xln, Compound S-A-Xln, Compound R-A-Xln, Compound A-P-Xln, Compound S-A-P-Xln, or Compound R-A-P-Xln) includes at least one recrystallization step. In several embodiments, the method of preparing Compound A-Xln includes more than one recrystallization step (e.g., 2 steps, 3 steps, 4 steps, etc.). In several embodiments, the preparation of a stable crystalline form of Compound A (e.g., Compound A-Xln, Compound S-A-Xln, Compound R-A-Xln, Compound A-P-Xln, Compound S-A-P-Xln, or Compound R-A-P-Xln) includes at least two recrystallization processes (e.g., two recrystallization steps). In other embodiments, the preparation includes only a single recrystallization step.

[0084] In several embodiments, the preparation of a stable crystalline form of Compound A (e.g., Compound A-Xln, Compound A-P-Xln) as disclosed herein can comprise a recrystallization process in a high-boiling solvent. In several embodiments, the high-boiling solvent is a solvent (or mixture of solvents) having a boiling point equal to or at least about: 100°C, 110°C, 120°C, 130°C, 140°C, 150°C, 160°C, 170°C, 180°C, 190°C, 200°C, 250°C, or a range including and / or spanning the aforementioned values. In several embodiments, following this recrystallization step, one or more stable forms of Compound A-Xln are provided. In several embodiments, to provide a stable form of Compound A-P-Xln, Compound S-A-P-Xln, or Compound R-A-P-Xln, an additional recrystallization step is performed.

[0085] In several embodiments, the preparation of a stable crystalline form of Compound A (e.g., Compound A-Xln, Compound S-A-Xln, Compound R-A-Xln, Compound A-P-Xln, Compound S-A-P-Xln, or Compound R-A-P-Xln) as disclosed herein can comprise a recrystallization process in a low-boiling solvent (or mixture of selected solvents). In several embodiments, the low-boiling solvent is a solvent (or mixture of solvents) having a boiling point equal to or less than about: 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, 90°C, <100°C, or a range including and / or spanning the aforementioned values.

[0086] In several embodiments, where at least two crystallization processes are used to prepare a stable crystalline form of Compound A, one process can comprise a recrystallization process in a high-boiling solvent (e.g., a high-boiling solvent or a mixture of solvents together having a high boiling point), and the other process can comprise a recrystallization process in a low-boiling solvent (a low-boiling solvent or a mixture of solvents together having a low boiling point). In several embodiments, where multiple recrystallization processes are used, an initial recrystallization process can be performed using a high-boiling solvent (or a mixture of selected solvents, including a high-boiling solvent and another solvent which can or can not also be a high-boiling solvent). In several embodiments, where two or more recrystallization processes are used, a second recrystallization process can be performed using a low-boiling solvent (or a mixture of selected solvents, including a low-boiling solvent and another solvent which can or can not also be a low-boiling solvent).

[0087] Alternatively, recrystallization in a low-boiling solvent can precede recrystallization in a high-boiling solvent.

[0088] As disclosed elsewhere herein, in cases where a mixture of solvents is used, a mixture of components can still be considered a "high-boiling solvent" when the mixture itself has a high boiling point. Likewise, in cases where a mixture of solvents is used, a mixture of components can still be considered a "low-boiling solvent" when the mixture itself has a low boiling point.

[0089] In several embodiments, to prepare a stable crystalline form of 5-(2-(4-(4-fluoro-2- methyl-lH-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5-azaspiro-[2.4]-heptan-7-ol (Compound A) (Compound A-Xln), the high-boiling solvent (e.g., for the initial recrystallization process) is selected from, but not limited to, DMF, DMA, NMP, a mixture of any of the foregoing selected solvents, or others. In several embodiments, to prepare a stable crystalline form of 5-(2-(4-(4-fluoro-2-methyl-lH-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5-azaspiro-[2.4]-heptan-7-ol (Compound A) (Compound A-Xln), the low-boiling solvent (e.g., for the second recrystallization process) is selected from, but not limited to, MeOH, EtOH, IPA, a mixture of any of the foregoing selected solvents, or others.

[0090] In several embodiments, to prepare a stable crystalline form of 5-(2-(4-(4-fluoro-2- methyl-lH-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5-azaspiro-[2.4]-heptan-7-ol (Compound A), the high-boiling solvent for the initial recrystallization process is DMF, and the low-boiling solvent for the sequential recrystallization process (second recrystallization process) is EtOH.

[0091] Different crystalline forms of a drug have different physical and chemical properties (such as stability, solubility, dissolution rate, bioavailability, etc.), and thus result in differences in the effectiveness, safety, or quality of the drug. In several embodiments, one or more stability tests and / or dissolution tests can be performed. It has been found that the crystalline form of Compound A-Xln has better stability and dissolution rate compared to the amorphous form of Compound A-Amp.

[0092] In several embodiments, as disclosed elsewhere herein, the present disclosure relates to methods of preparing a salt form of 5-(2-(4-(4-fluoro-2-methyl-lH-indol-5-yloxy)-6- methoxyquinolin-7-yloxy)ethyl)-5-azaspiro[2.4]-heptan-7-ol (Compound A), pharmaceutically acceptable salts of compounds comprising Compound A. The concept of "pharmaceutically acceptable salts" includes, but is not limited to, acid addition salts formed with inorganic acids such as hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, and the like; or formed with organic acids such as 1-hydroxy-2-naphthoic acid, 2,2-dichloroacetic acid, 2-hydroxyethanesulfonic acid, 2-oxoglutaric acid, 4-acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid, adipic acid, ascorbic acid (L), aspartic acid (L), benzenesulfonic acid, benzoic acid, camphoric acid (+), camphor-10-sulfonic acid (+), capric acid (decanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfic acid, ethane- 1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galacturonic acid, gentisic acid, glucoheptonic acid (D), gluconic acid (D), glucuronic acid (D), glutamic acid, glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid (DL), lactobionic acid, lauric acid, maleic acid, malic acid (-L), malonic acid, mandelic acid (DL), methanesulfonic acid, naphthalene- 1,5-disulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, propionic acid, pyroglutamic acid (-L), salicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid (+L), thiocyanic acid, toluenesulfonic acid (p-), and undecylenic acid, and the like.

[0093] Several embodiments relate to the preparation of a salt and / or a stable crystalline salt form of 5-(2-(4-(4-fluoro-2-methyl-lH-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5- azaspiro[2.4]-heptan-7-ol. Several embodiments relate to the preparation of a phosphate salt of 5-(2-(4-(4-fluoro-2-methyl-lH-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5- azaspiro[2.4]-heptan-7-ol (Compound A) (Compound A-P) and / or a stable crystalline phosphate salt form (Compound A-P-Xln).

[0094] In several embodiments, to prepare a stable crystalline phosphate salt form of 5-(2-(4-(4-fluoro-2-methyl-lH-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5- azaspiro[2.4]-heptan-7-ol (Compound A) (Compound A-P-Xln), the method comprises a neutralization process of a solution of Compound A and phosphoric acid in a solvent (or mixture of solvents). In several embodiments, the re-crystallization of Compound A-P is performed in a solvent (or mixture of solvents).

[0095] In several embodiments, to prepare a stable crystalline phosphate salt form of 5-(2-(4-(4-fluoro-2-methyl-lH-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5- azaspiro[2.4]-heptan-7-ol (Compound A) (Compound A-P-Xln), the solvent used in the neutralization process and / or the re-crystallization process comprises a solvent selected from, but not limited to, MeOH, EtOH, IPA, a mixture of any of the foregoing solvents, or other solvents.

[0096] In several embodiments, to prepare a stable crystalline phosphate salt form of 5-(2-(4-(4-fluoro-2-methyl-lH-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5- azaspiro[2.4]-heptan-7-ol (Compound A) (Compound A-P-Xln), the solvent used in the neutralization process and / or the re-crystallization process is EtOH.

[0097] In several embodiments, as disclosed elsewhere herein, Compound A-Xln, Compound S-A-Xln, or Compound R-A-Xln can be prepared using a single re-crystallization step (e.g., in a high boiling point solvent or mixture of solvents). In several embodiments, Compound A is heated in a high boiling point solvent. In several embodiments, a portion of the high boiling point solvent is removed (including under vacuum). In several embodiments, the solution is allowed to sit (e.g., cool), and crystals form over this time. In several embodiments, the crystals are collected.

[0098] In several embodiments, Compound A-P-Xln, Compound S-A-P-Xln, or Compound R-A-P-Xln can be prepared using a subsequent recrystallization process. In several embodiments, for example, Compound A-P-Xln, Compound S-A-P-Xln, or Compound R-A-P-Xln is prepared by introducing phosphoric acid (e.g., excess phosphoric acid) into a second recrystallization solvent (e.g., a low boiling solvent). In several embodiments, Compound A is heated in a solvent (e.g., a low boiling solvent). In several embodiments, a portion of the solvent is removed (including under vacuum). In several embodiments, the solution is allowed to sit (e.g., cool), and crystals form over this time. In several embodiments, the crystals are collected. In several embodiments, where excess phosphoric acid is used, some of the phosphoric acid is neutralized.

[0099] Methods of treatment

[0100] In several embodiments, a pharmaceutical composition is provided. In several embodiments, the pharmaceutical composition includes a stable crystalline form of Compound A or a stable crystalline form of a salt of Compound A as disclosed elsewhere herein and a pharmaceutically acceptable carrier. In several embodiments, the pharmaceutical composition includes a stable crystalline form of Compound A or a stable crystalline form of a salt of Compound A as disclosed elsewhere herein and a pharmaceutically acceptable excipient.

[0101] In several embodiments, a process for preparing a pharmaceutical composition including a stable crystalline form of 5-(2-(4-(4-fluoro-2-methyl-lH-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5- azaspiro[2.4]-heptan-7-ol (Compound A-P-Xln), or a phosphate salt of 5-(2-(4-(4-fluoro-2-methyl-lH-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5-azaspiro[2.4]-heptan-7-ol (Compound A-P) or a stable crystalline phosphate salt form (Compound A-P-Xln) and a pharmaceutically acceptable carrier or excipient is provided. In several embodiments, the excipient or carrier (or both) is mixed with Compound A-Xln or Compound A-P-Xln.

[0102] Abbreviations and definitions

[0103] In several embodiments, a method for treatment is provided. In several embodiments, the method comprises obtaining a pharmaceutical composition comprising a stable crystalline form of 5-(2-(4-(4-fluoro-2-methyl-lH-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5- azaspiro[2.4]-heptan-7-ol (Compound A-P-Xln), or a phosphate salt of 5-(2-(4-(4-fluoro-2-methyl-lH-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5- azaspiro[2.4]-heptan-7-ol (Compound A-P) or a stable crystalline phosphate salt form (Compound A-P-Xln). In several embodiments, the method comprises administering Compound A-Xln, Compound A-P, or Compound A-P-Xln to a patient having a disease. In several embodiments, the disease is a neoplastic disease.

[0104] In several embodiments, Compound A-Xln, Compound A-P, or Compound A-P-Xln comprises a single chiral isomer of one of Compound A-Xln, Compound A-P, or Compound A-P-Xln. In several embodiments, a method of preparing the chiral isomer is performed. In several embodiments, to separate the enantiomers of 5-(2-(4-(4-fluoro-2-methyl-lH-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5- azaspiro[2.4]-heptan-7-ol (Compound A) from each other (two chiral isomers, Compound R-A, Compound S-A), preparative HPLC with a chiral column is used. In several embodiments, the chiral isomers can then be recrystallized (and / or crystallized) as disclosed elsewhere herein.

[0105] Several embodiments relate to a stable crystalline form of Compound A-Xln, or a phosphate salt or a stable crystalline phosphate salt form of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5- azaspiro[2.4]-heptan-7-ol or both chiral isomers (Compound R-A, Compound S-A) for use in a method of treating a neoplastic disease. Several embodiments relate to a method of treating a disease selected from the group consisting of lung cancer, kidney cancer, colorectal cancer, gastric cancer, melanoma, head and neck cancer, thyroid cancer, pancreatic cancer, liver cancer, prostate cancer, bladder cancer, brain cancer, sarcoma, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, in particular small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC) and ovarian cancer; a tumor resulting from any one or more of the foregoing, a blood cancer (selected from ALL, CLL, AML, CML and multiple myeloma) and combinations thereof. In several embodiments, the method of treatment comprises administering a stable crystalline form of Compound A-Xln, or a phosphate salt or a stable crystalline phosphate salt form of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5- azaspiro[2.4]-heptan-7-ol or both chiral isomers (Compound R-A, Compound S-A).

[0106] Several embodiments relate to a stable crystalline form of Compound A-Xln, or a phosphate salt or a stable crystalline phosphate salt form of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5- azaspiro[2.4]-heptan-7-ol or both chiral isomers (Compound R-A, Compound S-A) for use in a method of monotherapy or in combination with an additional chemotherapeutic agent. In several embodiments, the additional chemotherapeutic agent is selected from a platinum or taxane agent.

[0107] In several embodiments, the method of treatment comprises selecting a patient having a selected disease, including a solid tumor. In several embodiments, the method of treatment comprises selecting a patient having a disease selected from the group consisting of lung cancer, renal cancer, colorectal cancer, gastric cancer, melanoma, head and neck cancer, thyroid cancer, pancreatic cancer, liver cancer, prostate cancer, bladder cancer, brain cancer, sarcoma, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, in particular small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), and ovarian cancer; a tumor resulting from any one or more of the foregoing, a blood cancer (selected from ALL, CLL, AML, CML, and multiple myeloma), and combinations thereof. In several embodiments, the method comprises administering to the patient a therapeutic amount of a stable crystalline form of 5-(2-(4-(4-fluoro-2-methyl-lH-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5- azaspiro[2.4]-heptan-7-ol, or a phosphate salt or stable crystalline phosphate salt form of 5-(2-(4-(4-fluoro-2-methyl-lH-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5- azaspiro[2.4]-heptan-7-ol, or both chiral isomers (Compound R-A, Compound S-A).

[0108] In several embodiments, an effective amount of Compound A-Xln, Compound A-P, and / or Compound A-P-Xln is administered to a patient in need of treatment (e.g., a subject having a disease as disclosed herein). In several embodiments, an effective amount of Compound A-Xln, Compound A-P, and / or Compound A-P-Xln is administered to a patient in need of treatment (e.g., a subject having a disease as disclosed herein).

[0109] Several embodiments are directed to or a stable crystalline form of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5- azaspiro[2.4]-heptan-7-ol or a salt or a stable crystalline salt form or both chiral isomers (Compound R-A, Compound S-A) for use in a method of monotherapy or in combination with an immunotherapy agent. In several embodiments, the immunotherapy agent is selected from the group consisting of PD-1, PD-L1, oncolytic virus therapy, bispecific T cell engager (BiTE), and chimeric antigen receptor (CAR) T cell therapy based agents including but not limited to nivolumab, pembrolizumab, ipilimumab, blinatumomab, elotuzumab, daratumumab, cemiplimab, avelumab, durvalumab, atezolizumab, tremelimumab, sintilimab, camrelizumab, tislelizumab, AK104, pidilizumab, KN035, CS1001, talimogene laherparepvec. In several embodiments, the combination is used to treat a disease or disorder as disclosed elsewhere herein. For example, in treating a solid tumor, the solid tumor is selected from the group consisting of lung cancer, renal cancer, colorectal cancer, gastric cancer, melanoma, head and neck cancer, thyroid cancer, pancreatic cancer, liver cancer, prostate cancer, bladder cancer, brain cancer, sarcoma, breast cancer, ovarian cancer, cervical cancer, and endometrial cancer; and a blood cancer selected from the group consisting of ALL, CLL, AML, CML, and multiple myeloma.

[0110] To test the biological activity, the PTK inhibitory activity of Compound A or Compound A-Xln was compared with the market drug Sunitinib. The in vitro tyrosine kinase inhibitory activity can be measured by commercial resources. Some of the tests can also be done with a contract signed with Eurofin or Reaction Biology to complete the screening.

[0111] For the kinase inhibition test, the receptor tyrosine kinases are selected from FGFR1(h), FGFR2(h), FGFR3(h), Fltl(h) (VEGFr1), Flt4(h) (VEGFr3), KDR(h) (VEGFr2), Aurora-B, PDGFRa(h), and PDGFRp(h). Compound A shows inhibition on all the targets with IC50 values ranging from sub-nanomolar to micromolar magnitude. Compared with the market drug Sunitinib, Compound A shows higher inhibitory efficacy.

[0112] The inhibitory effects of compound A-Xln, compound A-P-Xln and two chiral isomers (compound R-A, compound S-A) on cancer cell lines were tested by in vitro MTT assay. The cancer cell lines were selected from PANC-1, NCI-H157, MDA-MB-231, Hela, PC-3, BEL7404, MKN45, Ishikawa, Saos-2, SKOV3, SW579 and HCT116 cell lines. The IC50 values were in micromolar order.

[0113] The following examples further illustrate the present application but should not be construed as limiting the scope of the application in any way.

[0114] Example

[0115] Example 1: Preparation of crystalline form of 5-(2-(4-(4-fluoro-2-methyl-1H- indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5-azaspiro[2.4]-heptan-7-ol (Compound A-Xln)

[0116] For ease of reference, the following abbreviations are used and have the following meanings. EtOH: ethanol, MeOH: methanol, IPA: isopropanol, EtOAc: ethyl acetate, DCM: dichloromethane, DMF: N,N-dimethylformamide, DMA: N,N-dimethylacetamide, NMP: N-methyl-2-pyrrolidone, RT: room temperature, Temp: temperature, eq: equivalent, g: gram, mg: milligram, ml: milliliter, min: minute. API: active pharmaceutical ingredient. DSC: differential scanning calorimetry, TGA: thermogravimetric analysis, XRPD: X-ray powder diffraction, Exo: exothermic, Endo: endothermic. ALL: acute lymphocytic or lymphoblastic leukemia, CLL: chronic lymphocytic or lymphoblastic leukemia, AML: acute myeloid or myeloblastic leukemia, CML: chronic myeloid or myeloblastic leukemia, NSCLC: non-small cell lung cancer, SCLC: small cell lung cancer, ULMS: uterine leiomyosarcoma (sarcoma), OC: ovarian cancer, BT: brain tumor, SD: stable disease, PR: partial response, PFS: progression free survival, IC50: half maximal inhibitory concentration, which is the concentration of an inhibitor in a cell culture medium needed to inhibit the transport activity of a protein by 50%. MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

[0117] Figure 1 Figure 2

[0118] Crude Compound A was prepared similarly according to WO2010021918 (18 g) to give amorphous solid (Compound A-Amp) with a DSC main peak at 245.99 °C for Compound A. DMF (180 ml) was added and the mixture was stirred at 120 °C until the solid dissolved. The hot solution was filtered and the filtrate was concentrated under vacuum to about half the volume and further cooled to RT. The precipitate was filtered and the filter cake was washed with a small amount of DMF followed by some water. The filter cake was then mixed with ethanol (4 1) and heated to dissolve. The solution was concentrated under vacuum to about half the volume and further cooled to RT. The precipitate was filtered and dried in an oven to give the desired product in stable crystals (Compound A-Xln) with only one peak in DSC at 248.79 °C.

[0119] The DSC plot of the amorphous form of Compound A (Compound A-Amp) is shown in the Figure 3

[0120] The DSC, TGA, XRPD and H-NMR plots of the crystalline form of Compound A (Compound A-Xln) are shown in the 1 Figure 4 Figure 5 Example 2 Example 3: Comparison of stability of amorphous (Compound A-Amp) and crystalline form (Compound A-Xln) API of (2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5-azaspiro[2.4]-heptan-7-ol (Compound A)

[0121] MS: (M+H+) / z 478;

[0122] 1 H-NMR (DMSO-d6) δ ppm: 0.37-0.39 (m, 1 H), 0.50-0.57 (m, 2 H), 0.82-0.85 (m, 1 H) 2.43 (s, 3 H), 2.54-2.59 (d, 2 H), 3.11-3.13 (d, 1 H), 3.26 (s, 2 H), 3.42-3.46 (m, 1 H), 3.82-3.84 (t, 1 H), 3.98 (s, 3 H), 4.39 (t, 2 H), 6.28 (s, 2 H), 6.35-6.36 (d, 1 H), 6.98-7.02 (t, 1 H), 7.22-7.24 (d, 1 H), 7.48 (s, 1 H), 7.62 (s, 1 H), 8.43-8.46 (d, 1 H), 11.42 (s, 1 H).

[0123] DSC melting range (Endo): 247-253 °C with peak temperature = 248.79 °C. TGA showed undissolved material with weight loss above 250 °C. XRPD had the plot including 14 characteristic peaks with intensity % greater than 10% or 43 characteristic peaks where all intensity % are expressed in d values and angles as follows: ​​​​​​

[0124] XRPD data:

[0125]

[0126]

[0127] Figure 6

[0128] Based on the inventors' experience with the preparation of crystalline forms of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5- azaspiro[2.4]-heptan-7-ol (Compound A) in Example 1, the following crystalline forms of Compound A are expected to be prepared. The preparation of crystalline forms of Compound A (Compound A-Xln) should contain at least one recrystallization process, preferably two recrystallization processes. Preferably, the initial recrystallization requires a relatively high boiling point solvent (or mixture of solvents), which includes but is not limited to DMF, DMA, and NMP. The subsequent recrystallization process requires a solvent (or mixture of solvents), which includes but is not limited to MeOH, EtOH, and IPA.

[0129] Example 4: Comparison of stability of amorphous (Compound A-Amp) and crystalline form (Compound A-Xln) of (2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5-azaspiro[2.4]-heptan-7-ol (Compound A) tablets Tablets Figure 7

[0130] Samples of amorphous and crystalline forms of Compound A API were placed in clean containers and the ambient temperature was controlled at 60 °C. The purity of amorphous (Compound A-Amp) and crystalline forms (Compound A-Xln) of Compound A were tested on day 0, day 10, and day 30. Samples of amorphous and crystalline forms of API were measured and an appropriate amount of diluent was added to dissolve by sonication. The concentration was controlled at 0.2 mg / ml and the sample purity was determined by HPLC with a UV detector at 230 nm. The results are listed in the table below and the fitted curves are shown in the Example 5: Comparison of in vitro dissolution profiles of Compound A tablets A-Xln has better stability than A-Amp

[0131] Table 1: Stability comparison of amorphous and crystalline forms of Compound A API

[0132]

[0133] Figure 8 Times / minute AVG

[0134] RSD

[0135] Samples of amorphous and crystalline form tablets (10 mg or 30 mg) of Compound A (A-Amp or A-Xln; referred to as Compound A-Amp tablets or Compound A-Xln tablets) were prepared using the tablet compositions listed below following the procedure below:

[0136] (1) Sieving procedure: Compound A (A-Amp or A-Xln), lactose T80, microcrystalline cellulose (PH101), sodium starch glycolate were sieved separately using 80 mesh sieve.

[0137] (2) Granulation procedure: Sieved Compound A (A-Amp or A-Xln), lactose T80, microcrystalline cellulose, sodium starch glycolate and silicon dioxide were charged into a V-blender for step blend 1. After blend 1 step, the material was fed into roller compactor for dry granulation. The dried granules were sieved through 24 mesh sieve and the resultant powder was sieved through 50 mesh sieve to provide the desired fine powder which constitutes 10% to 13% of the total granule weight. Sieved granules were blended with pre-sieved magnesium stearate for step blend 2 and the content of the final blend was sampled and tested for blend assay (8.9% to 10.3%).

[0138] (3) Tablet compression procedure: The final blend was compressed into tablets after setting the tablet press to compress 10 mg and 30 mg. In-process control (IPC) tests were performed for weight variation and hardness during processing (weight variation of ±6.0% and hardness of 7 to 13 kg / mm 2 ).

[0139] (4) Coating procedure: The coating solution was prepared according to the ratio of Gastrosol film coating premix: purified water = 15:85 (w:w) and stirred for 45 minutes. The coating weight gain was about 2% to 3%.

[0140] (5) Packaging procedure: The collected tablets were weight sorted. The qualified tablets were collected and packed in 7 tablets / plate of aluminum plastic blister.

[0141] Table 2: Composition of Compound A drug product (10 mg tablets)

[0142]

[0143] Chp: Chinese Pharmacopoeia; USP: United States of Pharmacopoeia

[0144] Table 3: Composition of Compound A drug product (30 mg tablets)

[0145]

[0146] ChP: Chinese Pharmacopoeia; USP: United States Pharmacopoeia

[0147] The prepared tablets were tested in a similar protocol as described in Example 3, and the results are listed in Table 4, and the fitted curves are shown in the Times / minute section of the attached drawings.

[0148] Table 4: Stability comparison of amorphous and crystalline forms of Compound A tablets

[0149]

[0150] Table 5: Stability of Compound A-Xln drug product (DP, tablets) stored at 25 °C ± 2 °C / 60% RH ± 5% RH

[0151]

[0152]

[0153]

[0154]

[0155] AVG

[0156] The crystalline forms of a compound can alter its physical and chemical properties, including dissolution rates that can affect the drug's effectiveness. Dissolution rate measurements can be done in dissolution tests. Samples of amorphous (Compound A-Amp) and crystalline (Compound A-Xln) tablets were dissolved in an appropriate solvent (0.1 mol hydrochloric acid solution) and stirred at an appropriate speed to facilitate the dissolution process (ZRC-8D intelligent dissolution apparatus, speed of 50 rpm). The dissolution percentage was determined by HPLC with a UV detector wavelength set at 230 nm. Samples were collected at 5 minutes, 10 minutes, 20 minutes, 30 minutes, and 45 minutes, and the experimental results are listed in the following table, and the dissolution curves can be found in the RSD section of the attached drawings. After comparing the experimental data, it can be observed that the crystalline tablets dissolve faster than the amorphous tablets.

[0157] Table 6: Dissolution curve results for Compound A-Amp tablets 10 mg size

[0158] Example 6: Preparation of phosphate salt and crystalline form of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5-azaspiro[2.4]-heptan-7-ol (Compound A-P-Xln) 1 2 3 4 5 6 Figure 9 Figure 10 5 35.26％ 40.19％ 38.57％ 34.47％ 36.59％ 38.55％ 37.27％ 5.90％ 10 70.22％ 69.35％ 72.56％ 70.48％ 71.69％ 75.44％ 71.62％ 3.05％ 20 83.54％ 82.06％ 86.68％ 91.49％ 86.16％ 88.32％ 86.37％ 3.90％ 30 84.00％ 82.52％ 87.16％ 91.99％ 86.63％ 88.81％ 86.85％ 3.90％ 45 94.43％ 94.34％ 87.43％ 91.14％ 91.42％ 91.22％ 91.66％ 2.81％

[0159] Table 7: Dissolution curve results for Compound A-Xln tablets 10 mg size

[0160] Figure 11 1 2 3 4 5 6 Figure 12 Example 7 5 88.79％ 88.69％ 82.24％ 80.02％ 85.03％ 89.94％ 85.79％ 4.69％ 10 100.99％ 98.14％ 95.45％ 98.40％ 97.32％ 98.87％ 98.19％ 1.86％ 20 98.82％ 95.70％ 98.58％ 97.03％ 95.58％ 97.27％ 97.16％ 1.41％ 30 99.36％ 96.23％ 99.13％ 97.56％ 96.10％ 97.80％ 97.70％ 1.41％ 45 103.53％ 96.06％ 96.95％ 93.98％ 96.03％ 95.91％ 97.08％ 3.41％

[0161] AVG: average; RSD: relative standard deviation.

[0162] Example 9: Kinase inhibition Example 10

[0163] Compound A (250 mg) was dissolved in EtOH (110 ml) under reflux and to the resulting solution was added 1 M H3PO4in EtOH (0.53 ml). The reaction was refluxed for 1 hour and cooled to RT under slow stirring overnight. The solid was filtered and rinsed with EtOH, further dried in an oven at 50 °C for 10 hours to obtain a white solid which is phosphate salt of Compound A with stable crystalline form (Compound A-P-Xln).

[0164] DSC, TGA, XRPD and 1 H-NMR patterns are represented by Example 11 , Example 12: Combination efficacy of Compound A-Xln in vitro, in vivo with chemotherapy or immunotherapy against tumor cell lines and in vivo xenograft models against various tumor cell lines , Figure 13 and Figures 15-17 respectively.

[0165] 1 H NMR (DMSO-d6) δ ppm 0.37-0.39 (m, 1 H), 0.50-0.57 (m, 2 H), 0.82-0.85 (m, 1 H) 2.43 (s, 3 H), 2.51-2.56 (m, 1 H), 2.58-2.60 (d, 1 H), 2.72-2.74 (d, 1 H), 2.90 (t, 2 H), 3.14-3.18 (m, 1 H), 3.77-3.82 (m, 1 H), 3.97 (s, 3 H), 4.24 (t, 2 H), 4.60 (s, 1 H), 6.28 (s, 1 H), 6.33-6.34 (d, 1 H), 7.00 (t, 1 H), 7.22-7.24 (d, 1 H), 7.42 (s, 1 H), 7.60 (s, 1 H), 8.42-8.43 (d, 1 H), 11.42 (s, 1 H).

[0166] DSC melting range (Endo): 221-235 °C with peak temperature = 229 °C. TGA shows 1.84% weight loss at about 30-60 °C and significant weight loss above 210 °C. XRPD with figure includes 21 characteristic peaks with all intensity % expressed in d values and angles as follows:

[0167] Table 8: XRPD data of phosphate salt of Compound A with stable crystalline form (Compound A-P-Xln):

[0168]

[0169] Figure 18

[0170] Based on the research experience of the inventors on the preparation of stable crystalline phosphate salt forms of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5-azaspiro[2.4]-heptan-7-ol (Compound A), the following stable crystalline phosphate salt forms of Compound A were expected to be prepared. The stable crystalline phosphate salt forms of Compound A (Compound A-P-Xln) were prepared by the process of Compound A in solution of phosphoric acid in some type of solvent (or mixture of solvents) and the process of recrystallization in some types of solvents (or mixture of solvents). Preferably, this stable crystalline phosphate salt form of (Compound A-P-Xln) should be able to be obtained by recrystallization from solvents (or mixture of solvents) including but not limited to MeOH, EtOH and IPA.

[0171] Example 8: Preparation of enantiomers (chiral isomers)

[0172] Compound A (10 mg) was dissolved in IPA (10 ml) and 2 ml of the solution was injected into a chiral HPLC machine with a chiral pre-packed column CHIRALCEL OD 500 x 50 mm at a flow rate of 5 ml / min with a mobile phase of hexane:IPA:diethylamine (85:15:0.1) at UV 240 nm. The injection was repeated a total of five times and the collected fractions were combined and further evaporated to obtain Compound R-A (optical rotation: -23.49, 1.3 mg) and Compound S-A (optical rotation: +22.51, 1.6 mg) as two enantiomers.

[0173] R-A: 1 H-NMR (DMSO-d6) δ ppm: 0.34-0.40 (m, 1H), 0.48-0.60 (m, 2H), 0.80-0.87 (m, 1H), 2.42 (s, 3H), 2.59-2.62 (d, 1H), 2.73-2.75 (d, 1H), 2.87-2.92 (m, 1H), 2.89 (t, 2H), 3.14-3.19 (m, 1H), 3.74-3.79 (m, 1H), 3.96 (s, 3H), 4.24 (t, 2H), 6.28 (s, 1H), 6.32-6.33 (d, 1H), 6.99 (t, 1H), 7.20-7.23 (d, 1H), 7.41 (s, 1H), 7.59 (s, 1H), 8.41-8.43 (d, 2H), 11.42 (s, 1H).

[0174] S-A: 1H-NMR (DMSO-d6) δ ppm: 0.34-0.41 (m, 1H), 0.49-0.59 (m, 2H), 0.80-0.85 (m, 1H), 2.42 (s, 3H), 2.59-2.62 (d, 1H), 2.73-2.76 (d, 1H), 2.87-2.92 (m, 1H), 2.89 (t, 2H), 3.14-3.19 (m, 1H), 3.74-3.79 (m, 1H), 3.96 (s, 3H), 4.24 (t, 2H), 6.28 (s, 1H), 6.32-6.33 (d, 1H), 6.99 (t, 1H), 7.20-7.23 (d, 1H), 7.41 (s, 1H), 7.59 (s, 1H), 8.41-8.43 (d, 2H), 11.42 (s, 1H).

[0175] Figures 19-24

[0176] To test the biological activity of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6- methoxyquinolin-7-yloxy)ethyl)-5-azaspiro[2.4]-heptan-7-ol (Compound A-Xln), the inhibition of the kinases FGFR1(h), FGFR2(h), FGFR3(h), Flt1(h) (VEGFr1), Flt4(h) (VEGFr3), KDR(h) (VEGFr2), Aurora-B PDGFRa(h), PDGFRa(h), PDGFRb(h) was tested and the IC50 values are listed in the table below. In addition, the kinase inhibition of the marketed drug Sunitinib was tested and the results were compared to Compound A-Xln.

[0177] Table 9: Kinase inhibition of Compound A compared to Sunitinib

[0178]

[0179]

[0180] - or blank: no inhibition or compound activity could not be fitted to IC50 curve.

[0181] Example 13: Human clinical trials of Compound A-Xln tablets against solid tumors, such as NSCLC, SCLC, ovarian cancer, endometrial cancer, cervical cancer, uterine leiomyosarcoma, and peritoneal mesothelioma.

[0182] The results of the ion chromatography analysis and the aqueous solubility of the phosphate salt of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinolin-7- yloxy)ethyl)-5-azaspiro[2.4]-heptan-7-ol (Compound A-P) are listed in the table below.

[0183] Table 10. Ion Chromatography analysis and water solubility studies of compounds A-P

[0184]

[0185] Figure 25

[0186] In vitro MTT (proliferation) assays were performed with compounds from the above examples to give the following inhibition results:

[0187] Table 11: Inhibition results of compounds A-Xln

[0188]

[0189]

[0190] Table 12. In vitro inhibition activity of compounds A and compounds A-P on tumor cell lines

[0191]

[0192] Figure 26 ​

[0193] Combination therapy MTT assay results were investigated for compounds A-Xln. 100 μΐ of cell suspension at a concentration of 5 x 10 4 cells / ml was added to each well of a 96-well plate and placed in a 37 °C, 5% C02 incubator. After 24 hours, compound A-Xln sample solutions were added according to Table 13, 10 μΐ / well for double one-fold duplicate wells. After 72 hours of incubation at 37 °C and 5% C02, 20 μΐ of 5 mg / ml MTT solution was added to each well, followed by 100 μΐ / well of lysis solution after 4 hours. The samples were placed in the incubator and a full wavelength multi-function microplate reader was used after lysis. OD values were measured at 570 nm. ​ and 14 show the proliferation inhibition of breast cancer cell lines MCF7 and BT474 by compounds A-Xln, the anti-breast cancer agents anastrozole and letrozole, the combination of compounds A-Xln and anastrozole, and the combination of compounds A-Xln and letrozole, respectively.

[0194] Table 13: Dosing list for two breast cancer cell lines MCF-7 and BT474

[0195]

[0196]

[0197] The samples according to Table 14 were also tested for the proliferation inhibitor of three colon cancer lines HT29, HCT116 and colo205 by Compound A-Xln, the anti-colon cancer agent 5FU / O and the combination of Compound A-Xln and 5FU / O. The results are shown in Table 16. ​

[0198] Table 14: Dosing list of three colon cancer cell lines HT29, HCT116 and colo205

[0199]

[0200] The efficacy of Compound A-Xln in combination with immunotherapy, such as anti-mouse PD-1 antibody, was also tested.

[0201] Nude mice were inoculated with murine colorectal CT26 cell line for 7 days and randomized into groups of 6 nude mice and then treated with vehicle, Compound A-Xln once daily in two dosing groups, with anti-mouse PD-1 (PD-1) once every 3 days at 200 pg / mouse in one group, or with the combination of Compound A-Xln and anti-PD-1 in two dosing groups. Table 15 shows the change in tumor size after administration of the respective compounds. ​ A comparison of the average tumor volume change after administration of Compound A-Xln and anti-PD1 antibody agent and the combination therapy of Compound A-Xln and anti-PD-1 antibody agent is shown.

[0202] Table 15: Dosing list of murine colorectal CT26 cells

[0203]

[0204] D0: Time of first drug administration. IP: Intraperitoneal injection. i.g.: Gastrointestinal administration. Qd: Once a day. Q3d: Once every three days. PD-1 (anti-PD-1): Anti-mouse PD-1 antibody

[0205] Compound A-Xln has been tested for in vivo anti-tumor efficacy against various tumor cell lines on xenograft models.

[0206] The animal in vivo anti-tumor activity xenograft models with various tumor cell lines (SCLC NCI-H1436, NSCLC 95-D, ovarian SKVO3, renal 786-O, hepatic Bel-7420, brain U87) were as follows: Tumor tissue of well-grown tumor cell lines was cut into 3 mm pieces and one piece was inoculated to the right flank subcutaneously for each nude mouse. The animals were grouped and administered according to the pre-designed dose. When the tumor size reached 100 mm 3 ​Treatment was initiated on day 0. Animals with tumors that were too large or too small were culled according to tumor size, and animals with similar average tumor volumes were grouped. Animals were then dosed orally daily for 14-21 consecutive days. The large diameter a (mm) and small diameter b (mm) were measured twice per week with calipers 13 days after inoculation. The volume of each tumor was calculated by the formula: TV = ab 2 / 2 and relative tumor volume was calculated as: RTV = Vt / Vo, Vo representing the tumor volume on the first day of treatment; Vt representing the tumor volume on each measurement day. Animals were sacrificed and tumors were extracted by dissection 20-30 days after inoculation. Individual body weights and tumor weights were determined and calculated. The percent tumor inhibition was calculated as [1 - ((TWt) / (TWc)] x 100%, where TWt represents the average tumor weight of the treated group on the last day of the experiment, and TWc represents the average tumor weight of the control group on the last day of the experiment. Experimental dosing and efficacy results are shown in the ​

[0207] Results demonstrate that the combination of Compound A-Xln with anti-breast cancer agents such as aromatase inhibitors anastrozole and letrozole increases the inhibition of proliferation of human cancer cells such as breast cancer cell lines MCF7 and BT474.

[0208] The combination of Compound A-Xln with chemotherapeutic agents such as anti-colon cancer 5FU / O increases the inhibition of proliferation of human cancer cells such as cancer cell lines HT29, HCT116 and colo205. Inhibitory activity also shows some differences in sequential dosing, with higher activity in the regimen where the chemotherapeutic agent is dosed first followed by Compound A.

[0209] Murine xenograft models show combined inhibition of cancer cell proliferation such as mouse colon cancer cell line CT26 proliferation. The combination of Compound A-Xln with anti-PD1 antibody agents to treat cancer cell proliferation provides strong evidence of improved efficacy in in vivo animal models.

[0210] The combination therapy of Compound A-Xln with immunotherapeutic agents such as PD-1 or PD-L1 antibodies such as those selected from, but not limited to, nivolumab, pembrolizumab, ipilimumab, bintrafusp alfa, elotuzumab, durvalumab, cemiplimab, avelumab, durvalumab, atezolizumab, tremelimumab, sintilimab, camrelizumab, tislelizumab, AK104, AK105, pidilizumab, KN035, CS1001, talimogene laherparepvec.

[0211] ​Various human cancer cell line animal xenograft models have demonstrated significant tumor inhibitory activity of Compound A-Xln, particularly in SCLC, NSCLC, ovarian cancer, renal cancer, liver cancer, and brain cancer.

[0212] ​ ​

[0213] A: Overview

[0214] Compound A-Xln tablets are administered orally at 90 mg or 80 mg or 70 mg or 60 mg or 40 mg once daily for 28 days as one cycle for different cancer patients until intolerance or progressive disease (PD) is assessed by RECISIT 1.1. In case of intolerance observed, 90 mg per day can be reduced to 80 mg or 70 mg or 60 or 50 mg or 40 mg or 30 mg or 20 mg per day with 10 mg interval. The treatment results of a few representative patients with significant unexpected anti-tumor activity are listed below (as of March 2022). The very positive objective response rate (ORR) for SCLC >3 line (>2 line) treatment is >20%, and the very positive objective response rate for platinum-resistant ovarian cancer >3 line (>2 line) treatment is about 20%. There is some efficacy for SCLC patients using Compound A-Xln tablets maintenance therapy after the end of 1st line standard of care (SOC) therapy cycle. There is some efficacy for SCLC patients who have been treated with Compound A-Xln tablets as a 2nd line (prior 1st line) treatment regimen.

[0215] Efficacy has also been observed in sarcoma and brain tumors.

[0216]

[0217]

[0218] *: Non-progression disease time / still in study. Mnt: maintenance therapy

[0219] In addition, a completed SCLC clinical study (with thirty patients of prior 1st line (Group B) and 30 patients of > prior 2nd line (Group C)) has demonstrated positive clinical efficacy for B: 3.68 months and C: 3.62 months of progression-free survival (PFS) and for B: 10.39 months and for C: > 15 months of overall survival (OS). They are much better than the currently reported available treatment regimens for prior 1st line or > 2nd line treatment. Kaplan-Meier curves are shown in the attached figures ​ and ​ .

[0220] B: Representative subject examples (each using 10 mg tablets)

[0221] (1) : Representative subject S01013 in Phase 1b

[0222] Subject S01013 with NSCLC (prior lines = 3) was administered Compound A-Xln tablets 60 mg (10 mg x 6) once daily in 28-day cycles. The dose was reduced to 40 mg and 30 mg at the second cycle, and similar treatment regimen described above was used in subsequent cycles. The subject’s best treatment response was PR (54.70% reduction compared to baseline target lesions).

[0223] (2) : Representative subject S01014 in Phase 1b

[0224] Subject S01014 with NSCLC (prior lines = 3) was administered Compound A-Xln tablets 60 mg once daily in 28-day cycles. The dose was reduced to 40 mg at the third cycle, and similar treatment regimen described above was used in subsequent cycles. The subject’s best treatment response was PR (56.95% reduction compared to baseline target lesions).

[0225] (3) : Representative subject S05002 in Phase 1b

[0226] Subject S05002 with NSCLC (prior lines = 2) was administered Compound A-Xln tablets 60 mg once daily in 28-day cycles. Similar treatment regimen described above was used in subsequent cycles. The subject’s best treatment response was PR (38.42% reduction compared to baseline target lesions).

[0227] (4) : Representative subject 020207 in Phase 1

[0228] Subject 020207 with peritoneal mesothelioma (prior lines = 3) was administered Compound A-Xln tablets 40 mg in combination with tremelimumab injection (240 mg, every 3 weeks) once daily in 28-day cycles. Similar treatment regimen described above was used in subsequent cycles. The subject’s best treatment response was PR (36.4% reduction compared to baseline target lesions).

[0229] (5) : Representative subject 020202 in Phase 1

[0230] Subject 020202 with small cell lung cancer (prior lines = 1) was administered Compound A-Xln tablets 40 mg in combination with tremelimumab injection (240 mg, every 3 weeks) once daily in 28-day cycles. Similar treatment regimen described above was used in subsequent cycles. The subject’s best treatment response was PR (38.22% reduction compared to baseline target lesions).

Claims

1. A crystalline Form A-P-Xln of a phosphate salt of 5-(2-(4-(4-fluoro-2-methyl-1H- indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5-azaspiro[2.4]-heptan-7-ol, characterized by, The XRPD pattern of said crystalline Form A-P-Xln comprises the following characteristic peaks: 。 2. The crystalline Form A-P-Xln of claim 1, characterized in that, The XRPD pattern of said crystalline Form A-P-Xln is shown in Figure 11.

3. The crystalline form AP-Xln according to claim 1, characterized in that, DSC melting range (Endo): 221-235 °C, with a peak temperature of 229 °C.

4. Process for the preparation of the crystalline Form A-P-Xln according to any one of claims 1 to 3, characterized in that, 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5- azaspiro[2.4]-heptan-7-ol was neutralized with phosphoric acid in a single solvent or a multi-solvent mixture, and then crystallized.

5. The production method according to claim 4, characterized by, The solvent used for neutralization was EtOH.

6. Use of the crystalline Form A-P-Xln according to any one of claims 1 to 3 in the preparation of a protein tyrosine kinase inhibitor.

7. Use according to claim 6, characterized in that, The protein tyrosine kinase is selected from the group consisting of human FGFR1, human FGFR2, human FGFR3, human Fltl, i.e. VEGFr1, human Flt4, i.e. VEGFr3, human KDR, i.e. VEGFr2, human Aurora-B PDGFRa, human PDGFRa and human PDGFRb.

8. Use of the crystalline Form A-P-Xln according to any one of claims 1 to 3 in the preparation of a medicament for inhibiting pancreatic cancer, prostate cancer, sarcoma, thyroid cancer, colon cancer, ovarian cancer, hematological cancer, breast cancer, brain cancer, non-small cell lung cancer, small cell lung cancer, liver cancer, kidney cancer, cervical cancer and gastric cancer.

9. Use of the crystalline Form A-P-Xln according to any one of claims 1 to 3 in the preparation of a medicament for inhibiting cancer cells selected from the group consisting of PANC-1, NCI-H157, MDA-MB-231, Hela, PC-3, BEL7404, MKN45, Ishikawa, Saos-2, SKOV3, SW579, NCI-H1436, Bel-7402, U87 and HCT116.

10. Use of the crystalline Form A-P-Xln according to any one of claims 1 to 3 in the preparation of a medicament for treating a neoplastic disease, wherein the neoplastic disease is a solid tumor.

11. Use according to claim 10, characterized in that, The solid tumor is selected from the group consisting of lung cancer, mesothelioma, renal cancer, colorectal cancer, gastric cancer, melanoma, head and neck cancer, thyroid cancer, pancreatic cancer, liver cancer, prostate cancer, bladder cancer, brain cancer, sarcoma, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, and blood cancer selected from the group consisting of acute lymphoblastic leukemia, chronic lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia and multiple myeloma.

12. Use according to claim 11, characterized in that, The lung cancer is non-small cell lung cancer or small cell lung cancer.

13. The use according to claim 11, characterized in that, The solid tumor is selected from the group consisting of small cell lung cancer, non-small cell lung cancer, mesothelioma, ovarian cancer, brain tumor and sarcoma.

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