Application of composition containing PTK inhibitor A

Abstract

The content disclosed herein 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]-hept-7-ol (Compound A) (Compound A-Xln); a stable crystalline form of 5-(2-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinolin-7-yloxy) ethyl)-5-azaspiro [2.4]-hept-7-ol (Compound A); a tablet thereof (compound A-Xln tablet); a phosphate (compound A-P) and a stable crystalline form of the phosphate (compound A-P-Xln); and enantiomers (compound R-A, compound S-A) thereof. In addition, the anti-cancer activity, stability, chirality, dissolution and treatment indications of the compound are also studied.

Description

[0001] This application is a divisional application of the Chinese invention patent with application number 2023800352991, application date June 9, 2023, and invention name "5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5-azaspiro[2.4]-hept-7-ol (Compound A) crystals, phosphates and biological activities of their enantiomers".

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 351,205, filed on June 10, 2022. This application claims the benefit of U.S. Non-Provisional Application No. 18 / 206,506, filed on June 6, 2023. Technical Field

[0003] The present disclosure relates to a stable crystalline form (Compound A-Xln) 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); Its tablets (chemical Compound A-Xln tablets); Its phosphate salt (Compound AP) and its phosphate stable crystalline form (Compound AP-Xln); and its enantiomers (Compound RA, Compound SA) biological activity and preparation. Background Art

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

[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, according to their positions on different extracellular domains, PTK can be divided into epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR), fibroblast growth factor receptor (FGFR), etc. A large number of studies have shown that normal cells usually show low or inactive PTK, while many tumor cells, especially gliomas and cancers, show overexpression of PTK. Obviously, the abnormal overactivity of PTK is closely related to tumor cell growth and angiogenesis.

[0006] Interrupting or blocking the activity of PTK can significantly inhibit the growth of tumor cells by inhibiting cell signal transduction. Therefore, targeted therapy that inhibits the expression of PTK 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]-heptane-7-ol (Compound A) has the potential to inhibit the activity of multiple protein tyrosine kinases (PTKs), including but not limited to VEGFr, EGFr, c-kit, PDGF, FGF, SRC, Aurora B, etc. The structure and synthetic route of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5-azaspiro-[2.4]-heptane-7-ol (Compound A) have been disclosed in patent WO2010021918.

[0008] Several embodiments relate to 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-XIn).

[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 recrystallizing an 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 the following 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 that together have a high boiling point.

[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 that together have 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 the following method, which includes 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 having high boiling points together; and a second recrystallization process using a low boiling point solvent or a mixture of solvents having low boiling points together. In several embodiments, the high boiling point solvent is DMF and the low boiling point solvent is EtOH.

[0015] Several embodiments relate 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 exhibiting at least one of the following properties: DSC melting range (Endo): 240-260°C, with peak temperature range: 244-254°C; more specifically, DSC melting range (Endo): 247-253°C, with peak temperature = 249°C, as shown in FIG. Figure 2 As shown, or a TGA thermogram showing undissolved material with weight loss above 250°C, as shown Figure 3 shown.

[0016] Several embodiments relate 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: Figure 4 XRPD pattern shown.

[0017] Several embodiments relate 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 XRPD results comprising 14 characteristic peaks wherein the % intensity greater than 10% is expressed in terms of d value and angle as follows:

[0018]

[0019] Several embodiments relate 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: Fig.11 XRPD pattern shown.

[0020] Several embodiments relate 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 XRPD results comprising 43 characteristic peaks wherein all intensity % are expressed in terms of d value and angle as follows:

[0021]

[0022]

[0023] In several 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 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 a pharmaceutically acceptable salt. 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 a phosphate salt.

[0025] In some embodiments, the method comprises the steps of neutralizing a phosphoric acid solution using a solvent or a mixed solvent and recrystallizing the phosphoric acid solution to prepare the phosphate salt. In some embodiments, the solvent for neutralization and / or recrystallization 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, as shown in Figure 2 Fig. 9 TGA, the TGA shows a slight weight loss at about 30-60 ° C and a significant weight loss above 210 ° C, as shown in FIG Fig.10 shown.

[0027] In some 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 Fig.11 The XRPD pattern shown in . The XRPD results include all characteristic peaks, where all intensity % are expressed in terms of d value and angle as follows:

[0028]

[0029]

[0030] Several embodiments relate 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]-hept-7-ol, a stable crystalline form thereof, a salt form thereof and / or a stable crystalline salt form thereof, for use in inhibiting 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 PDGFRα(h), PDGFRα(h) and PDGFRβ(h). In several embodiments, the salt form is the phosphate form.

[0031] Several embodiments relate 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, 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 form.

[0032] Several embodiments relate 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 relate to a pharmaceutical composition comprising an active ingredient selected from a stable crystalline free base form or a stable crystalline form 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 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, the additional agent being 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, particularly in human clinical studies for the treatment of small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), and ovarian cancer. Several embodiments relate to methods of treatment, wherein the combined 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 some embodiments, the immunotherapy agent is selected from a PD-1 antibody or 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, talimogenelaherparepvec. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 .DSC graph of the 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 2 .DSC chart of 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.

[0038] Figure 3 .TGA chart of 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.

[0039] Figure 4 . 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 5 .5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5-azaspiro[2.4]-heptan-7-ol in crystalline form 1 H-HMR spectrum.

[0041] Figure 6 .Stability testing of amorphous API 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 7 .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 8 .Dissolution profiles 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] Fig. 9 .DSC graph of the 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.

[0045] Fig.10.TGA chart of the 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] Fig.11 . XRPD pattern of the 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] Fig.12 .5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5-azaspiro[2.4]-heptan-7-ol phosphate in crystalline form 1 H-HMR spectrum.

[0048] Fig.13 .Compound A-Xln, anti-breast cancer agents anastrozole and letrozole, the combination of compound A-Xln and anastrozole, and the combination of compound A-Xln and letrozole inhibit the proliferation of breast cancer cell line MCF7.

[0049] Fig.14 . Inhibition of proliferation of breast cancer cell line BT474 by compound A-Xln, anti-breast cancer agents anastrozole and letrozole, the combination of compound A-Xln and anastrozole, and the combination of compound A-Xln and letrozole.

[0050] Fig.15 .Inhibition of proliferation of colon cancer cell line HT29 by compound A-Xln, anti-colon cancer agent 5FU / O, and the combination of compound A-Xln and 5FU / O.

[0051] Fig.16 .Inhibition of proliferation of colon cancer cell line HCT116 by compound A-Xln, anti-colon cancer agent 5FU / O, and the combination of compound A-Xln and 5FU / O.

[0052] Fig.17 .Inhibition of proliferation of colon cancer cell line colo205 by compound A-Xln, anti-colon cancer agent 5FU / O, and the combination of compound A-Xln and 5FU / O.

[0053] Fig.18 . Comparison of mean tumor volume changes after administration of Compound A-Xln, an anti-PD1 antibody agent, and a combination therapy of Compound A-Xln and an anti-PD1 antibody agent to a murine colorectal CT26 cell line xenograft model.

[0054] Fig.19 . Changes in relative tumor volume after treatment with compound A-Xln in the small cell lung cancer NCI-H1436 cell line xenograft model.

[0055] Fig. 20 . Changes in relative tumor volume after treatment with compound A-Xln in non-small cell lung cancer (NSCLC) 95D cell line xenograft model.

[0056] Fig.21 . Changes in relative tumor volume after treatment with compound A-Xln in the ovarian cancer SKOV3 cell line xenograft model.

[0057] Fig. 22 . Changes in relative tumor volume after treatment with compound A-Xln in the renal cancer 786-O cell line xenograft model.

[0058] Fig.23 . Changes in relative tumor volume after treatment of Bel-7402 liver cancer cell line xenograft model with compound A-Xln.

[0059] Fig.24 . Changes in relative tumor volume after treatment of glioblastoma U87 cell line xenograft model with compound A-Xln.

[0060] Fig.25 Kaplan-Meier progression-free survival (PFS) analysis curves of SCLC patients in group C (≥2 previous lines, n=30) and group B (=1 previous line, n=30).

[0061] Fig.26 Kaplan-Meier overall survival (OS) analysis curves of SCLC patients in group C (≥2 previous lines, n=30) and group B (=1 previous line, n=30). DETAILED DESCRIPTION

[0062] Several embodiments relate to 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), its stable crystalline form (Compound A-Xln) amorphous (Compound A-Amp), its stable crystalline form crystallized from a phosphate (Compound AP) (Compound AP-Xln); and the enantiomers (two chiral isomers) of any of the aforementioned phosphates or crystals (e.g., Compound RA, Compound SA, Compound RA-Xln, Compound SA-Xln, Compound RAP-Xln, Compound SAP-Xln). Compound A is represented by the following structure:

[0063]

[0064] The following description provides context and examples, but should not be construed as limiting the scope of the present invention as encompassed by the claims in this specification or any other application claiming priority to this specification. No single component (including method steps) or set of components (e.g., multiple steps) is necessary or essential. Any feature, structure, component, material, step or method described and / or shown in any embodiment in this specification may be used together with any feature, structure, component, material, step or method described and / or shown in any other embodiment in this specification or may replace any feature, structure, component, material, step or method described and / or shown in any other embodiment in this specification.

[0065] Unless otherwise expressly stated, the terms and phrases used in this application and their variations, especially in the appended claims, should be interpreted as open-ended and non-restrictive. As an example of the foregoing, the term "comprising" should be understood to mean "including but not limited to", "including but not limited to", etc.; as used herein, the term "including" is synonymous with "including", "containing" or "characterized by", and is inclusive or open-ended and does not exclude additional, uncited elements or method steps; the term "having" should be interpreted as "having at least"; the term "including" should be interpreted as "including but not limited to"; the term "example" is used to provide an exemplary case of the items discussed, rather than an exhaustive or limiting list thereof; and the use of terms such as "preferably", "preferred", "desirable" or "desirable" and words of similar meaning should not be understood to imply that certain features are critical, necessary, or even important to the structure or function of the present invention, but should be understood to be merely intended to emphasize alternative or additional features that may or may not be used in a particular embodiment of the present invention. In addition, the term "comprising" should be interpreted synonymously with the phrase "having at least" or "comprising at least". When used in the context of a process, the term "comprising" means that the process includes at least the steps listed, but may include additional steps. When used in the context of a compound, composition, or device, the term "comprising" means that the compound, composition, or device includes at least the features or components listed, but may also include additional features or components. Similarly, unless otherwise expressly stated, a group of items connected together with the conjunction "and" should not be understood as requiring that each of those items be present in the grouping, but should be understood as "and / or". Similarly, unless otherwise expressly stated, a group of items connected together with the conjunction "or" should not be understood as requiring mutual exclusion in the group, but should be understood as "and / or".

[0066] Additionally, the phrase "consisting essentially of will be understood to include those elements specifically recited and those additional elements that do not materially affect the basic and novel characteristics of the claimed technology. The phrase "consisting of" excludes any elements not specified.

[0067] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. Features disclosed under one heading (such as compositions) can be used in combination with features disclosed under different headings (therapeutic methods). Unless otherwise defined, all technical terms and scientific terms used herein have the same meanings as those generally understood by those of ordinary skill in the art. It should be noted that when describing certain features or aspects of the present disclosure, the use of specific terms should not be considered to imply that the terms are redefined herein to be limited to any specific characteristics of the features or aspects associated with the terms of the present disclosure.

[0068] When referring to various features, the term "or a range including and / or spanning the above values" may be used. These terms (and variations thereof) are intended to include any range containing or spanning any of the above values. For example, with respect to temperature, the temperature may be expressed as "equal to or at least about: 40°C, 50°C, 60°C, 70°C, or a range including and / or spanning the above values". This language includes not only the specific temperature provided and the range exceeding 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 a temperature range 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, the temperature may be expressed as "equal to or lower than about: 40°C, 50°C, 60°C, 70°C, or a range including and / or spanning the above values". This language includes not only the specific temperatures provided and ranges below that value (e.g., at or below about 40°C, at or below about 50°C, at or below about 60°C, and at or below about 70°C), but also includes 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 solvent, dispersion medium, coating, antibacterial and antifungal agent, isotonic agent, and absorption delaying agent, etc. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agents are incompatible with the active ingredient, their use in therapeutic compositions is considered. In addition, various adjuvants as commonly used in the art may be included. 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, "patients" or "subjects" disclosed herein are human patients, but it should be understood that the principles of the subject matter disclosed herein indicate that the subject matter disclosed herein is effective for all vertebrate species (including mammals) intended to be included in the terms "subjects" and "patients". Suitable subjects are typically mammalian subjects. The subject matter described herein can be used for research and veterinary and medical applications. As used herein, the term "mammal" includes but is not limited to humans, non-human primates, cattle, sheep, goats, pigs, horses, cats, dogs, rabbits, rodents (e.g., rats or mice), monkeys, etc. Human subjects include neonates, infants, teenagers, adults, and elderly subjects.

[0071] As used herein, the term "therapeutically effective amount" refers to a therapeutic amount that imparts a modulating effect to a subject suffering from a condition, disease or illness, which modulating effect, for example, can be a beneficial effect, including improving the subject's condition (e.g., regulating one or more symptoms), delaying or reducing the progression of the condition, preventing or delaying the onset of the condition and / or changing clinical parameters, diseases or illnesses, etc. For example, in some embodiments, an effective amount can refer to an amount of a composition, compound or medicament that improves the subject's condition by at least 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%). The actual dosage levels of active ingredients and medicaments in the active compositions of the disclosed subject matter can be varied so as to administer an amount of active agent 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 the physical condition and previous medical history of the subject being treated. The determination and adjustment of effective doses, as well as the assessment of when and how to make such adjustments are contemplated herein. The term "therapeutically effective amount" may mean a therapeutic amount sufficient to prevent the spread of cancer or reverse cancer.

[0072] Compound

[0073] Several embodiments relate to therapeutic compounds (e.g., therapeutic agents). Several embodiments relate to 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), a crystalline form (Compound A-Xln), a stable crystalline form (Compound AP-Xln) of its phosphate salt (Compound AP), and any of the foregoing enantiomers (e.g., Compound RA, Compound SA, Compound RA-Xln, Compound SA-Xln, Compound RAP-Xln, Compound SAP-Xln), each of which is a therapeutic compound. In several embodiments, the S in the name of Compound A indicates the S configuration, and the R in the name of Compound A indicates the R configuration. The two enantiomers of Compound A, Compound RA, and Compound SA, respectively, are further represented by the following structures:

[0074]

[0075] (S)-5-(2-((4-((4-fluoro-2-methyl-1H-indol-5-yl)oxy(R)-5-(2-((4-((4-fluoro-2-methyl-1H-indol-5-yl)oxy)-6-methoxyquinolin-7-yl)oxy)ethyl)-5-azaspiroyl)-6-methoxyquinolin-7-yl)oxy)ethyl)-5-azaspiroyl

[0076] [2.4]Hept-7-ol [2.4]Hept-7-ol.

[0077] 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 represented 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 XRPD peaks 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 greater than 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 greater than 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.

[0078] 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 above values. In several embodiments, the DSC melting range of Compound A-Xln is characterized by a melting point of 249°C.

[0079] 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 above values. In several embodiments, the TGA peak weight loss Compound A-Xln appears at a temperature of 249°C.

[0080] In several embodiments, a crystalline form of a salt of Compound A is provided. In several embodiments, the crystalline form is represented as Compound AP-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 XRPD peaks at one or more angles selected from the group consisting of 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 XRPD peaks with intensities 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 XRPD peaks with intensities 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.

[0081] In several embodiments, the DSC melting range of compound AP-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 above values. In several embodiments, the DSC melting range of compound AP-Xln is characterized by a melting point of 229°C.

[0082] In several embodiments, the TGA peak weight loss range of Compound AP-Xln is equal to or less than about: 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 AP-Xln, Compound occurs at a temperature of 210°C.

[0083] Process for preparing the crystalline form

[0084] Several embodiments relate to methods for preparing 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), such as Compound A-Xln, Compound SA-Xln, Compound RA-Xln, Compound AP-Xln, Compound SAP-Xln or Compound RAP-Xln, from an amorphous form of Compound A (Compound A-Amp). In several embodiments, the method for preparing a stable crystalline form of Compound A comprises one or more crystallization and / or recrystallization steps.

[0085] In several embodiments, the preparation of a stable crystalline form of Compound A (e.g., Compound A-Xln, Compound SA-Xln, Compound RA-Xln, Compound AP-Xln, Compound SAP-Xln, or Compound RAP-Xln) comprises at least one recrystallization step. In several embodiments, the method for preparing Compound A-Xln comprises 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 SA-Xln, Compound RA-Xln, Compound AP-Xln, Compound SAP-Xln, or Compound RAP-Xln) comprises at least two recrystallization processes (e.g., two recrystallization steps). In other embodiments, the preparation comprises only a single recrystallization step.

[0086] In several embodiments, the preparation of a stable crystalline form of Compound A (e.g., Compound A-Xln, Compound AP-Xln) as disclosed herein may include a recrystallization process in a high boiling point solvent. In several embodiments, the high boiling point 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 above values. In several embodiments, after this recrystallization step, a stable form of one or more Compounds A-Xln is provided. In several embodiments, in order to provide a stable form of Compound AP-Xln, Compound SAP-Xln, or Compound RAP-Xln, an additional recrystallization step is performed.

[0087] In several embodiments, the preparation of a stable crystalline form of Compound A as disclosed herein (e.g., Compound A-Xln, Compound SA-Xln, Compound RA-Xln, Compound AP-Xln, Compound SAP-Xln, or Compound RAP-Xln) can include a recrystallization process in a low boiling point solvent (or a mixture of selected solvents). In several embodiments, the low boiling point 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 above values.

[0088] In several embodiments, when at least two crystallization processes are used to prepare a stable crystalline form of compound A, one process may include a recrystallization process in a high boiling point solvent (e.g., a high boiling point solvent or a mixture of solvents having a high boiling point together), and another process may include a recrystallization process in a low boiling point solvent (a mixture of low boiling point solvents or solvents having a low boiling point together). In several embodiments, when multiple recrystallization processes are used, a high boiling point solvent (or a mixture of selected solvents, including a high boiling point solvent and another solvent that may or may not be a high boiling point) may be used for the initial recrystallization process. In several embodiments, when two or more recrystallization processes are used, a second recrystallization process may be performed using a low boiling point solvent (or a mixture of selected solvents, including a low boiling point solvent and another solvent that may or may not be a low boiling point).

[0089] Alternatively, recrystallization in a low boiling point solvent may precede recrystallization in a high boiling point solvent.

[0090] As disclosed elsewhere herein, in the case where a mixture of solvents is used, when the mixture itself has a high boiling point, the mixture of components can still be considered a "high boiling point solvent." Similarly, in the case where a mixture of solvents is used, when the mixture itself has a low boiling point, the mixture of components can still be considered a "low boiling point solvent."

[0091] In some embodiments, to prepare 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), a high boiling point solvent (e.g., for the initial recrystallization process) is selected from, but not limited to, DMF, DMA, NMP, a mixture of any of the aforementioned selected solvents, or others. In some embodiments, to prepare 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), a low boiling point solvent (e.g., for the second recrystallization process) is selected from, but not limited to, MeOH, EtOH, IPA, a mixture of any of the aforementioned selected solvents, or others.

[0092] In several embodiments, in order to prepare 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), the high boiling point solvent used in the initial recrystallization process is DMF, and the low boiling point solvent used in the sequential recrystallization process (second recrystallization process) is EtOH.

[0093] Different crystalline forms of a drug have different physical and chemical properties (such as stability, solubility, dissolution rate, bioavailability, etc.), and thus lead to differences in the effectiveness, safety or quality of the drug. In several embodiments, one or more stability tests and / or dissolution tests may be performed. It has been found that the crystalline form of compound A-Xln has better stability and dissolution rate than the amorphous form of compound A-Amp.

[0094] In several embodiments, as disclosed elsewhere herein, the present disclosure relates to methods for preparing 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), including pharmaceutically acceptable salts of compounds of Compound A. The concept of "pharmaceutically acceptable salt" includes but is not limited to acid addition salts formed by inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc.; or acid addition salts formed by 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), acid), carbonic acid, cinnamic acid, citric acid, cycloamine, dodecyl sulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactonic 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, etc.

[0095] Several embodiments relate to the preparation of salts and / or stable crystalline 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. Several embodiments relate to the preparation of phosphate salts (Compound AP) and / or stable crystalline phosphate salt forms (Compound AP-Xln) 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).

[0096] In some embodiments, to prepare a stable crystalline phosphate 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 AP-Xln), the method comprises neutralizing a solution of Compound A and phosphoric acid in a solvent (or a mixture of solvents). In some embodiments, the recrystallization of Compound AP is carried out in a solvent (or a mixture of solvents).

[0097] In some embodiments, in order to prepare a stable crystalline phosphate form (Compound AP-Xln) 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 solvent used in the neutralization process and / or the recrystallization process includes a solvent selected from but not limited to the following: MeOH, EtOH, IPA, a mixture of any of the foregoing solvents, or other solvents.

[0098] In certain embodiments, in order to prepare a stable crystalline phosphate form (Compound AP-Xln) 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 solvent used in the neutralization process and / or the recrystallization process is EtOH.

[0099] In several embodiments, as disclosed elsewhere herein, Compound A-Xln, Compound SA-Xln, or Compound RA-Xln can be prepared using a single recrystallization step (e.g., in a high boiling point solvent or a 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 stand (e.g., cool), during which time crystals are formed. In several embodiments, the crystals are collected.

[0100] In several embodiments, a subsequent recrystallization process can be used to prepare Compound AP-Xln, Compound SAP-Xln, or Compound RAP-Xln. In several embodiments, for example, Compound AP-Xln, Compound SAP-Xln, or Compound RAP-Xln is prepared by introducing phosphoric acid (e.g., excess phosphoric acid) into a second recrystallization solvent (e.g., a low boiling point solvent). In several embodiments, Compound A is heated in a solvent (e.g., a low boiling point solvent). In several embodiments, a portion of the solvent is removed (including under vacuum). In several embodiments, the solution is allowed to stand (e.g., cool), during which time crystals are formed. In several embodiments, the crystals are collected. In several embodiments, where excess phosphoric acid is used, some of the phosphoric acid is neutralized.

[0101] Pharmaceutical composition

[0102] In several embodiments, a pharmaceutical composition is provided. In several embodiments, the pharmaceutical composition comprises 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 comprises 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.

[0103] In several embodiments, a method for preparing a pharmaceutical composition is provided, the pharmaceutical composition comprising 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 AP-Xln), or 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 (Compound AP) or a stable crystalline phosphate salt form (Compound AP-Xln) and a pharmaceutically acceptable carrier or excipient. In several embodiments, the excipient or carrier (or both) is mixed with Compound A-Xln or Compound AP-Xln.

[0104] Treatment

[0105] 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-1H-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5-azaspiro[2.4]-hept-7-ol (Compound AP-Xln), or a phosphate salt (Compound AP) or a stable crystalline phosphate salt form (Compound AP-Xln) of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5-azaspiro[2.4]-hept-7-ol (Compound AP-Xln). In several embodiments, the method comprises administering Compound A-Xln, Compound AP, or Compound AP-Xln to a patient suffering from a disease. In several embodiments, the disease is a neoplastic disease.

[0106] In several embodiments, Compound A-Xln, Compound AP, or Compound AP-Xln comprises a single chiral isomer of one of Compound A-Xln, Compound AP, or Compound AP-Xln. In several embodiments, a method of preparing chiral isomers is performed. In several embodiments, in order to separate the enantiomers (two chiral isomers, Compound RA, Compound SA) of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5-azaspiro[2.4]-hept-7-ol (Compound A) from each other, 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.

[0107] Several embodiments relate to 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-Xln, or a phosphate salt or a stable crystalline phosphate form or two chiral isomers 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 RA, Compound SA), for use in the preparation of a medicament for use in a method for treating a neoplastic disease. Several embodiments relate to methods 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; tumors caused by any one or more of the foregoing, blood cancer (selected from ALL, CLL, AML, CML and multiple myeloma) and combinations thereof. In certain embodiments, the method of treatment comprises administering 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-Xln, or a phosphate salt or a stable crystalline phosphate salt form or two chiral isomers 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 RA, Compound SA).

[0108] Several embodiments relate to a stable crystalline form or salt or a stable crystalline salt form or two chiral isomers 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 RA, Compound SA) 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.

[0109] In some embodiments, the method of treatment comprises selecting a patient with a selected disease, the disease comprising a solid tumor. In some embodiments, the method of treatment comprises selecting a patient with a disease, the disease is 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; tumors caused by any one or more of the foregoing, 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-1H-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5-azaspiro[2.4]-heptan-7-ol, or a phosphate salt or a stable crystalline phosphate salt form or two chiral isomers 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 RA, Compound SA).

[0110] In several embodiments, an effective amount of Compound A-Xln, Compound AP and / or Compound AP-Xln is administered to a patient in need of treatment (e.g., a subject suffering from a disease as disclosed herein). In several embodiments, an effective amount of Compound A-Xln, Compound AP and / or Compound AP-Xln is administered to a patient in need of treatment (e.g., a subject suffering from a disease as disclosed herein).

[0111] Several embodiments relate to a stable crystalline form or salt or a stable crystalline salt form or two chiral isomers 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 RA, Compound SA) for use in monotherapy or in combination with an immunotherapy agent. In some embodiments, the immunotherapy agent is selected from 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, toripalimab, sintilimab, carrelizumab, tislelizumab, AK104, penampalimumab, KN035, CS1001, talilav. In some embodiments, the combination is used to treat a disease or condition as disclosed elsewhere herein. For example, in the treatment of solid tumors, the solid tumors are selected from 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 and endometrial cancer; and blood cancers are selected from ALL, CLL, AML, CML and multiple myeloma.

[0112] To test biological activity, the PTK inhibitory activity of Compound A or Compound A-Xln was compared to the marketed drug Sunitinib. In vitro tyrosine kinase inhibitory activity can be measured by commercial sources. Some of these tests can also be screened under contract with Eurofin or Reaction Biology.

[0113] For the kinase inhibition test, the receptor tyrosine kinases were selected from FGFR1(h), FGFR2(h), FGFR3(h), Flt1(h)(VEGFr1), Flt4(h)(VEGFr3), KDR(h)(VEGFr2), Aurora-B, PDGFRα(h) and PDGFRβ(h). Compound A showed inhibition of all targets, and the IC50 values ​​ranged from subnanomolar to micromolar. Compared with the marketed drug sunitinib, Compound A showed higher inhibitory efficacy.

[0114] The inhibitory effects of compound A-Xln, compound AP-Xln and two chiral isomers (compound RA, compound SA) 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 value was in the micromolar range.

[0115] The following examples further illustrate the present invention but should not be construed as limiting the scope of the present invention in any way.

[0116] Examples

[0117] Abbreviations and definitions

[0118] 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 lymphoblastic or lymphocytic leukemia, CLL: chronic lymphocytic or lymphocytic leukemia, AML: acute myeloid or myeloid leukemia, CML: chronic myeloid or myeloid 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 inhibitor in the cell culture medium required to inhibit the transport activity of a protein by 50%. MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

[0119] Example 1: Crystalline Form 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7- Preparation of 5-azaspiro[2.4]-heptane-7-ol (Compound A-Xln)

[0120] Crude compound A (18 g) was prepared similarly according to WO2010021918 to obtain an amorphous solid (Compound A-Amp) with a DSC main peak of 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 to about half volume under vacuum and further cooled to RT. The precipitate was filtered and the filter cake was washed with a small amount of DMF and then washed with some water. The filter cake was then mixed with ethanol (4 l) and heated to dissolve. The solution was concentrated to about half volume in a vacuum and further cooled to RT. The precipitate was filtered and dried in an oven to obtain stable crystals (Compound A-Xln) of the desired product, wherein there was only one DSC peak at 248.79°C.

[0121] The DSC diagram of the amorphous form of Compound A (Compound A-Amp) is shown in the attached figure. Figure 1 Indicated.

[0122] DSC, TGA, XRPD and 1 The H-NMR diagrams are shown in the attached drawings. Figure 2 , Figure 3 , Figure 4 and Figure 5 Indicated.

[0123] MS: (M+H + ) / z 478;

[0124] 1 H-NMR(DMSO-d6)δppm:0.37-0.39(m,1H),0.50-0.57(m,2H),0.82-0.85(m,1H)2.43 (s,3H),2.54-2.59(d,2H),3.11-3.13(d,1H),3.26(s,2H),3.42-3.46(m,1H),3.82- 3.84(t,1H),3.98(s,3H),4.39(t,2H),6.28(s,2H),6.35-6.36(d,1H),6.98-7.02(t ,1H),7.22-7.24(d,1H),7.48(s,1H),7.62(s,1H),8.43-8.46(d,1H),11.42(s,1H).

[0125] 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 pattern including 14 characteristic peaks with intensity % greater than 10% or 43 characteristic peaks where all intensity % are expressed as d value and angle as follows:

[0126] XRPD data:

[0127]

[0128]

[0129] Example 2

[0130] Based on the inventor's research experience on the preparation of the crystalline form of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5-azaspiro[2.4]-heptane-7-ol (Compound A) in Example 1, the following crystalline form preparation of Compound A is expected. The preparation of the crystalline form 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 a mixture of solvents), which includes but is not limited to DMF, DMA and NMP. The subsequent recrystallization process requires a solvent (or a mixture of solvents), which includes but is not limited to MeOH, EtOH and IPA.

[0131] Example 3: (2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl The amorphous form (Compound A-Amp) and crystalline form (Compound A- Xln) API Stability Comparison

[0132] Samples of the amorphous and crystalline forms of Compound A API were placed in a clean container and the ambient temperature was controlled at 60°C. The purity of the amorphous (Compound A-Amp) and crystalline forms (Compound A-Xln) of Compound A was tested on Day 0, Day 10, and Day 30. Samples of the amorphous and crystalline forms of API were measured and a suitable amount of diluent was added to dissolve by ultrasonic treatment. 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 curve is shown in the attached figure section. Figure 6 A-Xln has better stability than A-Amp

[0133] Table 1: Comparison of the stability of the amorphous and crystalline forms of Compound A API

[0134]

[0135] Example 4: (2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl The amorphous form (Compound A-Amp) and crystalline form (Compound A- Xln)

[0136] Comparison of tablet stability

[0137] Samples of amorphous and crystalline forms of Compound A (A-Amp or A-Xln; referred to as Compound A-Amp tablets or Compound A-Xln tablets) tablets (10 mg or 30 mg) were prepared using the tablet compositions listed below by the following process:

[0138] (1) Screening process: Compound A (A-Amp or A-Xln), lactose T80, microcrystalline cellulose (PH101), and sodium carboxymethyl starch were sieved through 80 mesh sieves respectively.

[0139] (2) Granulation process: The sieved compound A (A-Amp or A-Xln), lactose T80, microcrystalline cellulose, sodium carboxymethyl starch and silicon dioxide are charged into a V-shaped blender for step blending 1. After the blending 1 step, the material is fed into a roller compactor for dry granulation. The dried granules are sieved through a 24 mesh sieve, and the resulting powder is sieved through a 50 mesh sieve to provide the desired fine powder, which accounts for 10% to 13% of the total granule weight. The sieved granules are blended with pre-sieved magnesium stearate for step blending 2, and the content of the final blend is sampled and tested for blend determination (8.9% to 10.3%).

[0140] (3) Tablet Compression Process: After setting the tablet press to compress to 10 mg and 30 mg, the final blend was compressed into tablets. During processing, in-process control (IPC) tests were conducted on weight variation and hardness (weight variation was ± 6.0%, and hardness was 7 to 13 kg / mm 2 ).

[0141] (4) Coating process: Prepare a coating solution according to the ratio of gastric film coating premix: purified water = 15:85 (w:w), and continue stirring for 45 minutes. The coating weight increase is about 2% to 3%.

[0142] (5) Packaging process: The collected tablets are sorted by weight. The qualified tablets are collected and packaged in aluminum-plastic blisters with 7 tablets / plate.

[0143] Table 2: Composition of Compound A drug product (10 mg tablets)

[0144]

[0145] Chp: Chinese Pharmacopoeia; USP: United States Pharmacopoeia

[0146] Table 3: Composition of Compound A drug product (30 mg tablets)

[0147]

[0148] Chp: Chinese Pharmacopoeia; USP: United States Pharmacopoeia

[0149] 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 accompanying drawings. Figure 7 as shown in .

[0150] Table 4: Comparison of the stability of amorphous and crystalline tablets of Compound A

[0151]

[0152] Table 5: Stability of Compound A-Xln Drug Product (DP, Tablets) Storage at 25°C ± 2°C / 60% RH ± 5% RH

[0153]

[0154]

[0155]

[0156]

[0157] Example 5: Comparison of in vitro dissolution profiles of Compound A tablets

[0158] The crystalline form of a compound can change its physical and chemical properties, including the dissolution rate that can affect the effectiveness of the drug. Dissolution rate measurements can be completed in a dissolution test. Samples of amorphous (Compound A-Amp) tablets 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 promote the dissolution process (ZRC-8D intelligent dissolution equipment, speed of 50 rpm). The dissolution percentage was determined by HPLC, with the UV detector wavelength set to 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 curve can be found in the accompanying drawings. Figure 8 After comparing the experimental data, it can be observed that the crystalline tablets dissolve faster than the amorphous tablets.

[0159] Table 6: Dissolution curve results of Compound A-Amp tablets 10 mg

[0160] Times / minute 1 2 3 4 5 6 AVG RSD 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％

[0161] Table 7: Dissolution curve results of Compound A-Xln tablets 10 mg

[0162] Times / minute 1 2 3 4 5 6 AVG RSD 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％

[0163] AVG: average value; RSD: relative standard deviation.

[0164] Example 6: 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl Preparation of the phosphate salt and crystalline form of 5-azaspiro[2.4]-heptan-7-ol (compound AP-Xln)

[0165] Compound A (250 mg) was dissolved in EtOH (110 ml) under reflux, and 1 M H3PO4 (0.53 ml) in EtOH was added to the resulting solution. The reaction was refluxed for 1 hour and cooled to RT overnight with slow stirring. The solid was filtered and rinsed with EtOH and further dried in an oven at 50° C. for 10 hours to obtain a white solid, which is the phosphate salt of compound A (Compound AP-Xln) in a stable crystalline form.

[0166] DSC, TGA, XRPD and 1 The H-NMR spectra were Fig. 9 , Fig.10 , Fig.11 and Fig.12 express.

[0167] 1 H NMR(DMSO-d6)δppm: 0.37-0.39(m,1H),0.50-0.57(m,2H),0.82-0.85(m,1H)2.43(s,3H), 2.51-2.56(m,1H),2.58-2.60(d,1H),2.72-2.74(d,1H),2.90(t,2H),3.14-3.18(m,1H),3 .77-3.82(m,1H),3.97(s,3H),4.24(t,2H),4.60(s,1H),6.28(s,1H),6.33-6.34(d,1H),7 .00(t,1H),7.22-7.24(d,1H),7.42(s,1H),7.60(s,1H),8.42-8.43(d,1H),11.42(s,1H).

[0168] 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 pattern includes 21 characteristic peaks with all intensity % expressed as d value and angle as follows:

[0169] Table 8: XRPD data of the phosphate salt of Compound A (Compound AP-Xln) having a stable crystalline form:

[0170]

[0171] Example 7

[0172] Based on the inventors' research experience on the preparation of a stable crystalline phosphate 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), the following stable crystalline phosphate form of Compound A is expected to be prepared. The stable crystalline phosphate compound (Compound AP-Xln) is prepared by a process of neutralizing Compound A with a solution of phosphoric acid in a certain type of solvent (or solvent mixture) and a process of recrystallizing in some type of solvent (or solvent mixture). Preferably, this stable crystalline phosphate form of (Compound AP-Xln) should be obtainable by recrystallization from a solvent (or solvent mixture) including but not limited to MeOH, EtOH and IPA.

[0173] Example 8: Preparation of enantiomers (chiral isomers)

[0174] 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 preformed column CHIRALCEL OD 500×50 mm at a flow rate of 5 ml / min, wherein the mobile phase at UV 240 nm was hexane: IPA: diethylamine (85: 15: 0.1). The injection was repeated five times in total, and the collected fractions were combined and further evaporated to obtain compound RA (optical rotation: −23.49, 1.3 mg) and compound SA (optical rotation: +22.51, 1.6 mg) as two enantiomers.

[0175] RA: 1H-NMR(DMSO-d6)δppm: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),6.9 9(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).

[0176] SA: 1 H-NMR(DMSO-d6)δppm: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),6.9 9(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).

[0177] Example 9: Kinase inhibition

[0178] 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 kinases FGFR1(h), FGFR2(h), FGFR3(h), Flt1(h)(VEGFr1), Flt4(h)(VEGFr3), KDR(h)(VEGFr2), Aurora-B PDGFRα(h), PDGFRα(h), PDGFRβ(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 with those of Compound A-Xln.

[0179] Table 9: Kinase inhibition of Compound A compared to Sunitinib

[0180]

[0181]

[0182] - or Blank: No inhibition or compound activity cannot fit the IC50 curve.

[0183] Example 10

[0184] The ion chromatography analysis and water solubility results 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 AP) are listed in the table below.

[0185] Table 10. Ion chromatography analysis and water solubility study of compound AP

[0186]

[0187] Example 11

[0188] In vitro MTT (proliferation) assays were performed with compounds from the above examples to give the following inhibition results:

[0189] Table 11: Inhibition results of compound A-Xln

[0190]

[0191]

[0192] Table 12. In vitro inhibitory activity of compound A and compound AP on tumor cell lines

[0193]

[0194] Example 12: Compound A-Xln in vitro and in vivo with chemotherapy or immunotherapy against tumor cell lines and against Combination efficacy in in vivo xenograft models of various tumor cell lines

[0195] The results of MTT assay for the combination therapy of compound A-Xln were studied. 100 μl of 5×10 4Cell suspension of 10 cells / ml was added to each well of a 96-well plate and placed in a 37°C, 5% CO2 incubator. After 24 hours, compound A-Xln sample solution was added according to Table 13, 10 μl / well for double duplicate wells. After incubation for 72 hours at 37°C and 5% CO2, 20 μl of 5 mg / ml MTT solution was added to each well, and then 100 μl / well of dissolution solution was added after 4 hours. The sample was placed in an incubator and a full-wavelength multifunctional microplate reader was used after dissolution. The OD value at 570 nm was measured. Fig.13 and 14 Proliferation inhibition of breast cancer cell lines MCF7 and BT474 by Compound A-Xln, anti-breast cancer agents anastrozole and letrozole, the combination of Compound A-Xln and anastrozole, and the combination of Compound A-Xln and letrozole are shown, respectively.

[0196] Table 13: Dosing schedule for two breast cancer cell lines MCF-7 and BT474

[0197]

[0198]

[0199] The samples according to Table 14 were also tested for proliferation inhibitors of three colon cancer lines, HT29, HCT116 and colo205, by compound A-Xln, anti-colon cancer agent 5FU / O and a combination of compound A-Xln and 5FU / O. The results are shown in Figure 15-17 shown.

[0200] Table 14: Dosing schedule for three colon cancer cell lines: HT29, HCT116 and colo205

[0201]

[0202] The efficacy of combination treatment with Compound A-Xln plus immunotherapy, such as anti-mouse PD-1 antibody, was also tested.

[0203] Nude mice were inoculated with murine colorectal CT26 cell line for 7 days and randomly divided into groups of 6 nude mice, and then treated with vehicle, compound A-Xln once a day in two dosing groups, anti-mouse PD-1 (PD-1) once every 3 days in one group at 200 μg / mouse, or a combination of compound A-Xln and anti-PD-1 in two dosing groups. Table 15 shows the changes in tumor size after administration of the corresponding compounds. Fig.18 Comparison of mean tumor volume changes after administration of Compound A-Xln and anti-PD1 antibody agent and combination therapy of Compound A-Xln and anti-PD-1 antibody agent is shown.

[0204] Table 15: Dosing schedule for murine colorectal CT26 cells

[0205]

[0206] D0: First drug administration time. IP: Intraperitoneal injection. ig: Gastrointestinal administration. Qd: Once a day. Q3d: Once every three days. PD-1 (anti-PD-1): Anti-mouse PD-1 antibody

[0207] The in vivo antitumor efficacy of Compound A-Xln has been tested on xenograft models against various tumor cell lines.

[0208] The xenograft model of antitumor activity in animals with various tumor cell lines (SCLC NCI-H1436, NSCLC 95-D, ovarian SKVO3, kidney 786-O, liver Bel-7420, brain U87) was as follows: Tumor tissue of well-growing tumor cell lines was cut into 3 mm pieces and one piece was subcutaneously inoculated into the right flank of each nude mouse. The animals were divided into groups and administered according to the pre-designed dose. When the tumor size reached 100 mm after 10-14 days, 3 Treatment was started at 1:10 pm. Animals with tumors that were too large or too small were excluded according to the size of the tumor, and animals with similar average tumor volumes were grouped. The animals were then orally administered daily for 14-21 consecutive days. After 13 days of inoculation, the large diameter a (mm) and the small diameter b (mm) were measured twice a week with a caliper. The volume of each tumor was calculated by the following formula: TV = ab 2 / 2 and the relative tumor volume was calculated as: RTV = Vt / Vo, Vo represents the tumor volume on the first day of treatment; Vt represents the tumor volume on each measurement day. The animals were sacrificed and the tumors were extracted by dissection 20-30 days after inoculation. The individual body weight and tumor weight were determined and calculated. The tumor inhibition percentage was calculated as [1-((TWt) / (TWc)]×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. The experimental dosing and efficacy results are shown in the attached figure Figure 19-24 Optimal tumor inhibition percentage ranged from >70% to 100%.

[0209] The results showed that the combination of compound A-Xln and anti-breast cancer agents such as aromatase inhibitors anastrozole and letrozole increased the proliferation inhibition of human cancer cells (such as breast cancer cell lines MCF7 and BT474).

[0210] Compound A-Xln was demonstrated to increase the proliferation inhibition of human cancer cells (such as cancer cell lines HT29, HCT116 and colo205) in combination with chemotherapeutic agents (such as anti-colon cancer 5FU / O). The inhibitory activity also showed some differences in the sequential administration, with higher activity in the regimen of first administering the chemotherapeutic agent and then administering compound A.

[0211] Murine xenograft models have shown combined inhibitory effects on cancer cell proliferation, such as mouse colorectal cancer cell line CT26 proliferation. The combination of Compound A-Xln and anti-PD1 antibody agents in the treatment of cancer cell proliferation provides strong evidence of improved efficacy in in vivo animal models.

[0212] Combination therapy of Compound A-Xln with an immunotherapeutic agent, such as a PD-1 or PD-L1 antibody, such as those selected from but not limited to the following antibodies can produce synergistic clinical efficacy: nivolumab, pembrolizumab, ipilimumab, blinatumomab, elotuzumab, daratumumab, cemiplimab, avelumab, durvalumab, atezolizumab, toripalimab, sintilimab, carrelizumab, tislelizumab, AK104, AK105, penampalimumab, KN035, CS1001, talilavin.

[0213] Various human cancer cell line animal xenograft models have demonstrated that compound A-Xln has significant tumor inhibitory activity, especially in SCLC, NSCLC, ovarian cancer, renal cancer, liver cancer and brain cancer.

[0214] Example 13: Compound A-Xln tablets for solid tumors, such as NSCLC, SCLC, ovarian cancer, endometrial cancer, uterine cancer Human clinical trials in cervical cancer, uterine leiomyosarcoma, and peritoneal mesothelioma.

[0215] A: Overview

[0216] Compound A-Xln tablets are orally administered at 90mg or 80mg or 70mg or 60mg or 40mg, once a day, for 28 days as a cycle for different cancer patients, until intolerance or progressive disease (PD) is assessed by RECISIT 1.1. In the case of observed intolerance, 90mg per day can be reduced to 80mg or 70mg or 60 or 50mg or 40mg or 30mg or 20mg per day within a 10mg interval. The treatment results of a few representative patients with significant and unexpected anti-tumor activity are listed below (cutoff time, March 2022). The very positive objective response rate (ORR) for SCLC ≥ 3 lines (≥ 2 lines) treatment is > 20%, and the very positive objective response rate for platinum-resistant ovarian cancer ≥ 3 lines (≥ 2 previous lines) treatment is about 20%. SCLC patients who use compound A-Xln tablets for maintenance therapy after the end of the 1-line standard of care (SOC) therapy cycle have some efficacy. There was some efficacy in SCLC patients who had been treated with Compound A-Xln tablets as a 2nd line (previously 1st line) treatment regimen.

[0217] Efficacy has also been observed in sarcomas and brain tumors.

[0218]

[0219]

[0220] *: Non-progressive disease time / still under investigation. Mnt: maintenance therapy

[0221] In addition, a completed SCLC clinical study (with thirty patients with 1 previous line (Group B) and 30 patients with ≥ 2 previous lines (Group C)) has demonstrated positive clinical efficacy in progression-free survival (PFS) of 3.68 months for B and 3.62 months for C, and overall survival (OS) of 10.39 months for B and > 15 months for C. For patients with 1 or ≥ 2 previous lines of treatment, they are much better than the currently available treatment options reported historically. The Kaplan-Meier curve is shown in the attached figure Fig.25 and Fig.26 shown.

[0222] B: Representative subject examples (each using 10 mg tablets)

[0223] (1): Representative subject S01013 in Phase 1b

[0224] Subject S01013 (previous line> 3) with NSCLC was orally administered Compound A-Xln tablets 60 mg (10 mg×6) once a day in a 28-day cycle. The dose was reduced to 40 mg and 30 mg at the second cycle, and a similar above-mentioned treatment regimen was used in subsequent cycles. The best treatment response of the subject was PR (54.70% reduction compared to baseline target lesions).

[0225] (2): Representative subject S01014 in Phase 1b

[0226] Subject S01014 (previous line = 3) with NSCLC was orally administered Compound A-Xln tablets 60 mg once a day in a 28-day cycle. The dose was reduced to 40 mg at the third cycle, and a similar above-mentioned treatment regimen was used in subsequent cycles. The subject's best treatment response was PR (56.95% reduction compared to baseline target lesions).

[0227] (3): Representative subject S05002 in Phase 1b

[0228] Subject S05002 (previous line = 2) with NSCLC was orally administered Compound A-Xln tablets 60 mg once daily in 28-day cycles. Similar above treatment regimens were used in subsequent cycles. The subject's best treatment response was PR (38.42% reduction compared to baseline target lesions).

[0229] (4): Representative subjects of Phase 1 020207

[0230] Subject 020207 (previous line = 3) with peritoneal mesothelioma was orally administered a combination of Compound A-Xln tablets 40 mg and Teplizumab injection (240 mg, once every 3 weeks) once daily in a 28-day cycle. A similar above-mentioned treatment regimen was used in subsequent cycles. The subject's best treatment response was PR (36.4% reduction compared to baseline target lesions).

[0231] (5): Representative subjects of Phase 1 020202

[0232] Subject 020202 (previous line = 1) with small cell lung cancer was orally administered a combination of Compound A-Xln tablets 40 mg and Teplizumab injection (240 mg, once every 3 weeks) once a day in a 28-day cycle. A similar treatment regimen as described above was used in subsequent cycles. The best treatment response of the subject was PR (reduced by 38.22% compared to baseline target lesions).

Claims

1. Use of crystalline A-Xln of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)ethyl)-5-azaspiro[2.4]-hept-7-ol and a second therapeutic agent in the preparation of a drug for treating a neoplastic disease, wherein: The second therapeutic agent is a chemotherapeutic compound and / or an immunotherapy agent; the XRPD of the crystal A-Xln includes 14 characteristic peaks with an intensity % greater than 10% at the following locations: 8.9, 9.9, 14.9, 15.4, 16.1, 16.6, 18.5, 19.9, 20.4, 21.5, 23.9, 24.3, 24.6 and 26.0 degrees 2θ.

2. The use according to claim 1, characterized in that: The intensity % greater than 10% in the XRPD of the crystal A-Xln is expressed as d value and angle as follows:

3. The use according to claim 1, characterized in that: All intensities of the XRPD characteristic peaks of the crystal A-Xln are expressed as d values ​​and angles as follows:

4. The use according to claim 1, characterized in that: The XRPD of the crystal A-Xln is shown in FIG4 .

5. The use according to claim 1, characterized in that: The DSC melting range of the crystal A-Xln is 240-260°C, wherein the peak temperature range is 244-254°C.

6. The use according to claim 1, characterized in that: The melting range of the crystal A-Xln is 247-253°C, with a peak temperature of 249°C.

7. The use according to claim 1, characterized in that: The TGA thermogram of the crystalline A-Xln showed undissolved material with weight loss above 250°C.

8. The use according to claim 1, characterized in that: The chemotherapeutic compound is selected from but not limited to the group consisting of platinums or taxanes or topoisomerase 1 inhibitors or alkaloids or alkylating agents: cisplatin, carboplatin, paclitaxel or cisplatin / paclitaxel or carboplatin / paclitaxel or carboplatin / etoposide or topotecan, irinotecan or lomustine.

9. The use according to claim 1, characterized in that: The immunotherapy agent is selected from but not limited to the group of PD-1 antibodies or PD-L1 antibodies consisting of: nivolumab, pembrolizumab, ipilimumab, blinatumomab, elotuzumab, daratumumab, cemiplimab, avelumab, durvalumab, atezolizumab, toripalimab, sintilimab, camrelizumab, tislelizumab, AK104, AK105, penpulimab, KN035, CS1001, talimogenelaherparepvec.

10. The use according to claim 1, characterized in that: The neoplastic disease is a solid tumor selected from but not limited to the following group: lung cancer, NSCLC, SCLC, 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 and endometrial cancer; and a blood cancer selected from but not limited to the following group: ALL, CLL, AML, CML and multiple myeloma; preferably, the neoplastic disease is small cell lung cancer, non-small cell lung cancer, mesothelioma, ovarian cancer, brain tumor and sarcoma.

11. The method of claim 1, wherein the treatment is using Compound A-Xln for 1st-line patient maintenance therapy or for 2nd-line or ≥3rd-line patient treatment.

12. The method of claim 1, wherein the therapeutic dose is 90 mg, or 80 mg, or 70 mg, or 60 mg, or 50 mg, or 40 mg, or 30 mg or 20 mg per day.

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