Crystalline a-xln of ptk inhibitor a and use 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 Chinese Patent Application No. 2023800352991, filed on June 9, 2023, entitled “5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-7-ol (compound A) crystals, phosphates and the bioactivity of their enantiomers”.

[0002] This application claims the benefit of U.S. Provisional Application 63 / 351,205, filed June 10, 2022. This application claims the benefit of U.S. Non-Provisional Application 18 / 206,506, filed June 6, 2023. Technical Field

[0003] This disclosure relates to the stable crystalline form (compound A-Xln) of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-7-ol (compound A); Its tablets (chemical) Compound A-Xln tablets); Its phosphate (compound AP) and stable crystalline form of phosphate (compound AP-Xln); and the bioactivity and preparation of its enantiomers (compound RA, compound SA). Background Technology

[0004] Protein tyrosine kinases (PTKs) are a series of enzymes that catalyze phosphorylation, a process that transfers phosphate groups from nucleoside triphosphates (usually ATP) to amino acid residues in proteins. This phosphorylation process activates phosphorylated proteins. Therefore, PTKs act as "switches," regulating many cellular functions by controlling a signaling cascade that travels from the extracellular space across the membrane to the 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 conditions and diseases. For example, based on their location on different extracellular domains, 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. Numerous studies have shown that normal cells typically exhibit low or no PTK activity, while many tumor cells, especially gliomas and cancers, exhibit PTK overexpression. Clearly, abnormal overactivity of PTKs is closely related to tumor cell growth and angiogenesis.

[0006] Disruption or blockage of PTK activity can significantly inhibit tumor cell growth by suppressing cell signal transduction. Therefore, targeted therapy that inhibits PTK expression through PTK inhibitors has become a widely accepted treatment.

[0007] The novel PTK inhibitor 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro-[2,4]-hepta-7-ol (compound A) has the potential to inhibit the activity of various 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-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro-[2,4]-hepta-7-ol (compound A) have been disclosed in patent WO2010021918.

[0008] Several examples relate to the stable crystalline form of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-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-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-7-ol was prepared by a method comprising recrystallizing the amorphous form of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-7-ol.

[0010] In several embodiments, the stable crystalline form of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-7-ol was 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-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-7-ol is prepared by a recrystallization process using a high-boiling solvent or a mixture of solvents having 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-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-7-ol is prepared by a method comprising a recrystallization process using a low-boiling solvent or a mixture of solvents having low boiling points together.

[0013] In several embodiments, the stable crystalline form of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-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-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-7-ol is prepared by a method comprising: a first recrystallization process using a high-boiling solvent or a mixture of solvents having high boiling points together; and a second recrystallization process using a low-boiling solvent or a mixture of solvents having low boiling points together. In several embodiments, the high-boiling solvent is DMF and the low-boiling solvent is EtOH.

[0015] Several embodiments relate to the crystalline form of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-7-ol, which exhibits at least one of the following properties: DSC melting range (Endo): 240-260 °C, with a peak temperature range of 244-254 °C; more specifically, DSC melting range (Endo): 247-253 °C, with a peak temperature of 249 °C, as shown in the figure. Figure 2 As shown, or a TGA thermogram, which shows undissolved material exhibiting weight loss above 250°C, as illustrated in the figure. Figure 3 As shown.

[0016] Several embodiments relate to the crystalline form of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-7-ol, characterized as follows Figure 4 The XRPD diagram shown.

[0017] Several embodiments relate to the crystalline form of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-7-ol, characterized by XRPD results comprising 14 characteristic peaks, wherein an intensity % greater than 10% is expressed in d-values ​​and angles as follows:

[0018]

[0019] Several embodiments relate to the crystalline form of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-7-ol, characterized as follows Figure 11 The XRPD diagram shown.

[0020] Several embodiments relate to the crystalline form of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-7-ol, characterized by XRPD results comprising 43 characteristic peaks, wherein all intensity % is expressed in d-values ​​and angles as follows:

[0021]

[0022]

[0023] In several embodiments, the crystalline form of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-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-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-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-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-7-ol is a phosphate.

[0025] In several embodiments, the method for preparing phosphates includes steps of neutralization with a phosphoric acid solution using a solvent or a mixture of solvents, followed by recrystallization. In several embodiments, the solvent used for neutralization and / or recrystallization is EtOH.

[0026] In several embodiments, the phosphate of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-7-ol is characterized by one or more of the following properties: DSC melting range (Endo): 221-235°C, where peak temperature = 229°C, as shown in the figure. Figure 9 As shown; TGA, which is shown as a slight weight loss at approximately 30-60°C and a significant weight loss above 210°C, as illustrated in the figure. Figure 10 As shown.

[0027] In several embodiments, the phosphate of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-7-ol comprises as follows Figure 11 The XRPD plot is shown below. The XRPD results include all characteristic peaks, where all intensity % is expressed in d-values ​​and angles as follows:

[0028]

[0029]

[0030] Several embodiments relate to compounds having a structure represented by 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-7-ol, its stable crystalline form, its salt form, and / or its stable crystalline salt form, for 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 compounds having a structure represented by: 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-7-ol, its stable crystalline form, its salt form, and / or its stable crystalline salt form, wherein the compounds inhibit 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 as a compound described above or elsewhere herein and a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable excipient.

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

[0034] Several embodiments relate to a pharmaceutical composition comprising an active ingredient and a pharmaceutically acceptable carrier as a stable crystalline free base form or a stable crystalline form of a phosphate of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-7-ol.

[0035] Several embodiments relate to a method of treating aplastic diseases, the method comprising administering to a subject of need 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 additional agents, said additional agents being chemotherapeutic compounds and / or immunotherapeutic agents. In several embodiments, the aplastic disease is a solid tumor 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 hematologic malignancies selected from ALL, CLL, AML, CML, and multiple myeloma, particularly in human clinical studies of small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), and ovarian cancer. Several embodiments relate to treatment methods in which said combination chemotherapeutic agents are selected from platinum-based or taxane-based or topoisomerase 1 inhibitors or alkaloids or alkylating agents. 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, but is not limited to, PD-1 or PD-L1 antibodies including: nivolumab, pembrolizumab, ipilimumab, blinatumomab, elotuzumab, daratumumab, cemiplimab, avelumab, durvalumab, atezolizumab, toripalimab, sintilimab, camrelizumab, tislelizumab, AK104, penpulimab, KN035, CS1001, and talimogenelaherparepvec. Attached Figure Description

[0036] Figure 1 DSC diagram of the amorphous (compound A-Amp) form of .5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-7-ol.

[0037] Figure 2 DSC diagram of the crystalline form of .5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-7-ol.

[0038] Figure 3 TGA diagram of the crystalline form of .5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-7-ol.

[0039] Figure 4 XRPD diagram of the crystalline form of .5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-7-ol.

[0040] Figure 5 Crystalline form of .5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2,4]-hepta-7-ol 1 H-HMR spectrum.

[0041] Figure 6 Stability tests of amorphous and crystalline APIs of .5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-7-ol.

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

[0044] Figure 9 DSC diagram of the crystalline form of phosphate of .5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-7-ol.

[0045] Figure 10TGA diagram of the crystalline form of phosphate of .5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-7-ol.

[0046] Figure 11 XRPD diagram of the crystalline form of phosphate of .5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-7-ol.

[0047] Figure 12 Crystalline form of phosphate of .5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2,4]-hepta-7-ol 1 H-HMR spectrum.

[0048] Figure 13 The proliferation of the breast cancer cell line MCF7 was inhibited 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.

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

[0050] Figure 15 Compound A-Xln, the anti-colon cancer agent 5FU / O, and the combination of compounds A-Xln and 5FU / O inhibited the proliferation of colon cancer cell line HT29.

[0051] Figure 16 Compound A-Xln, the anti-colon cancer agent 5FU / O, and the combination of compounds A-Xln and 5FU / O inhibited the proliferation of colon cancer cell line HCT116.

[0052] Figure 17 Compound A-Xln, the anti-colon cancer agent 5FU / O, and the combination of compounds A-Xln and 5FU / O inhibited the proliferation of colon cancer cell line colo205.

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

[0054] Figure 19 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 20 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 21 The relative tumor volume change after treatment of an ovarian cancer SKOV3 cell line xenograft model with compound A-Xln.

[0057] Figure 22 The relative tumor volume change after treatment of a renal cell carcinoma 786-O cell line xenograft model with compound A-Xln.

[0058] Figure 23 The relative tumor volume changes after treatment of a Bel-7402 hepatocellular carcinoma cell line xenograft model with compound A-Xln.

[0059] Figure 24 The relative tumor volume changes after treatment of a glioblastoma U87 cell line xenograft model with compound A-Xln.

[0060] Figure 25 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] Figure 26 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 compound 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-7-ol (compound A), its stable crystalline form (compound A-Xln), its amorphous form (compound A-Amp), its stable crystalline form crystallized from phosphate (compound AP) (compound AP-Xln); and any enantiomers (two chiral isomers) of the aforementioned phosphate or crystals (e.g., compounds RA, SA, RA-Xln, SA-Xln, RAP-Xln, 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 invention as covered by the appended claims or any other application claiming priority to this specification. No single component (comprising method steps) or collection of components (e.g., multiple steps) is required or essential. Any feature, structure, component, material, step, or method described and / or illustrated in any embodiment of this specification may be used in conjunction with or in lieu of any feature, structure, component, material, step, or method described and / or illustrated in any other embodiment of this specification.

[0065] Unless otherwise expressly stated, the terms and phrases used in this application, and their variations thereof, especially those in the appended claims, should be interpreted as open-ended rather than 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 “comprising” is synonymous with “comprising,” “containing,” or “characterized by,” and is inclusive or open-ended, and does not exclude additional, unreferenced elements or method steps; the term “having” should be interpreted as “having at least”; the term “comprising” should be interpreted as “including but not limited to”; the term “example” is used to provide an exemplary case of the items under discussion, not an exhaustive or restrictive list thereof; and the use of terms such as “preferred,” “ideal,” “desirable,” or “expected,” and words with similar meanings, should not be construed as implying that certain features are critical, necessary, or even important to the structure or function of the invention, but should be understood merely as intended to emphasize alternative or additional features that may or may not be used in particular embodiments of the invention. Additionally, the term "comprising" should be interpreted synonymously with the phrases "having at least" or "containing at least". When used in the context of a process, the term "comprising" means that the process includes at least the listed steps, 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 contains at least the listed features or components, but may also contain additional features or components. Similarly, unless otherwise expressly stated, a group of items connected by the conjunction "and" should not be construed as requiring each of those items to be present in the group, but rather should be construed as "and / or". Likewise, unless otherwise expressly stated, a group of items connected by the conjunction "or" should not be construed as requiring mutual exclusivity in said group, but rather should be construed as "and / or".

[0066] Furthermore, the phrase "consistently composed of..." will be understood to include those elements specifically described and those additional elements that do not substantially affect the fundamental and novel features of the claimed technology. The phrase "consisting of..." excludes any unspecified elements.

[0067] The section headings used herein are for typographical purposes only and should not be construed as limiting the subject matter described. Features disclosed under one heading (such as compositions) may be used in combination with features disclosed under a different heading (treatment methods). 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 in describing certain features or aspects of this disclosure should not be construed as implying that the term is redefined herein to limit itself to any specific characteristic of the feature or aspect of this disclosure associated with that term.

[0068] When referring to various characteristics, the term "or a range that includes and / or spans the values ​​mentioned above" may be used. These terms (and variations thereof) are intended to include any range that includes or spans any of the values ​​mentioned above. For example, with respect to temperature, temperature may be expressed as "equal to or at least about: 40°C, 50°C, 60°C, 70°C, or a range that includes and / or spans the values ​​mentioned above." This language includes not only the specific temperature provided and the range exceeding that 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 the temperature range that spans 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 may be expressed as "equal to or below about: 40°C, 50°C, 60°C, 70°C, or a range that includes and / or spans the values ​​mentioned above." This language includes not only the specific temperature provided and the range below that value (e.g., equal to or below about 40°C, equal to or below about 50°C, equal to or below about 60°C, and equal to or below about 70°C), but also the temperature ranges that span 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 terms “pharmaceutically acceptable carrier” or “pharmaceuticalally acceptable excipient” encompass any solvent, dispersion medium, coating, antibacterial and antifungal agent, isotonic agent, and absorption delay agent, etc. The use of such media and agents for the pharmaceutically active substance is well known in the art. Unless any conventional media or agent is incompatible with the active ingredient, its use in a therapeutic composition is considered. Additionally, 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., (ed.) (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, the “patient” or “subject” disclosed herein refers to a human patient; however, it should be understood that the principles of the currently disclosed subject matter indicate that the subject matter is valid for all vertebrate species (including mammals) intended to be included in the terms “subject” and “patient.” Suitable subjects are typically 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, cattle, sheep, goats, pigs, horses, cats, dogs, rabbits, rodents (e.g., rats or mice), monkeys, etc. Human subjects include newborns, infants, adolescents, adults, and elderly subjects.

[0071] As used herein, the term "therapeutic effective amount" refers to a therapeutic amount that imparts a modulating effect to a subject suffering from a symptom, disease, or ailment, such modulating effect being, for example, a beneficial effect comprising improving the subject's symptoms (e.g., modulating one or more symptoms), delaying or reducing the progression of symptoms, preventing or delaying the onset of symptoms and / or altering clinical parameters, diseases, or ailments. For example, in some embodiments, an effective amount may refer to an amount of composition, compound, or agent that improves the subject's symptoms 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 dose levels of the active ingredient and agent in the active compositions of the disclosed subject matter may vary to administer an amount of active agent that effectively achieves the desired response in a particular subject and / or application. The chosen dose level will depend on a variety of factors, including, but not limited to, the activity of the composition, the formulation, the route of administration, the combination with other drugs or treatments, the severity of the condition being treated, and the physical condition and medical history of the subject being treated. This article considers the determination and adjustment of the effective dose, as well as the assessment of when and how to make such adjustments. The term "therapeutic effective dose" can mean a therapeutic amount sufficient to prevent the spread of cancer or to 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-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-7-ol (compound A), its crystalline form (compound A-Xln), its stable crystalline form of its phosphate (compound AP) (compound AP-Xln), 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 are further represented by the following structures:

[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 represented as compound A-Xln. In several embodiments, the crystalline form is characterized by one or more peaks in an XRPD plot. 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 any combination thereof. In several embodiments, the crystal 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 any combination thereof. In several embodiments, the crystal 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 any combination thereof.

[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, or 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 includes and / or spans the values ​​described above. 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 represented as compound AP-Xln. In several embodiments, the crystalline form is characterized by one or more peaks in an XRPD plot. In several embodiments, the crystalline form is characterized by XRPD peaks 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 any combination thereof. In several embodiments, the crystal 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 any combination thereof. In several embodiments, the crystal 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 any combination thereof.

[0079] 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, or a range including and / or spanning the values ​​described above. In several embodiments, the DSC melting range of compound AP-Xln is characterized by a melting point of 229°C.

[0080] In several embodiments, the TGA peak weight loss of compound AP-Xln ranges from 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 values ​​above. In several embodiments, the TGA peak weight loss of compound AP-Xln occurs at a temperature of 210°C.

[0081] Methods for preparing crystalline forms

[0082] Several embodiments relate to methods for preparing stable crystalline forms of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro-[2,4]-hepta-7-ol (compound A) from the amorphous form of compound A (compound A-Amp), such as compound A-Xln, compound SA-Xln, compound RA-Xln, compound AP-Xln, compound SAP-Xln, or compound RAP-Xln. In several embodiments, the method for preparing the 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 SA-Xln, compound RA-Xln, compound AP-Xln, compound SAP-Xln, or compound RAP-Xln) includes at least one recrystallization step. In several embodiments, the method for preparing compound A-Xln includes more than one recrystallization step (e.g., two steps, three steps, four 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) 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 as disclosed herein (e.g., compound A-Xln, compound AP-Xln) may comprise a recrystallization process in a high-boiling-point solvent. In several embodiments, the high-boiling-point solvent is a solvent (or mixture of solvents) with 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 values ​​described above. In several embodiments, following this recrystallization step, a stable form of one or more compounds A-Xln is provided. In several embodiments, an additional recrystallization step is performed to provide a stable form of compound AP-Xln, compound SAP-Xln, or compound RAP-Xln.

[0085] 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) may comprise a recrystallization process in a low-boiling-point solvent (or a mixture of selected solvents). In several embodiments, a low-boiling-point solvent is a solvent (or mixture of solvents) with 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 values ​​described above.

[0086] In several embodiments, when using at least two crystallization processes to prepare a stable crystalline form of compound A, one process may comprise a recrystallization process in a high-boiling-point solvent (e.g., a high-boiling-point solvent or a mixture of solvents having high boiling points together), and the other process may comprise a recrystallization process in a low-boiling-point solvent (a low-boiling-point solvent or a mixture of solvents having low boiling points together). In several embodiments, when using multiple recrystallization processes, the initial recrystallization process may be performed using a high-boiling-point solvent (or a mixture of selected solvents, comprising a high-boiling-point solvent and another solvent that may or may not be high-boiling-point). In several embodiments, when using two or more recrystallization processes, the second recrystallization process may be performed using a low-boiling-point solvent (or a mixture of selected solvents, comprising a low-boiling-point solvent and another solvent that may or may not be low-boiling-point).

[0087] Alternatively, recrystallization in low-boiling-point solvents may precede recrystallization in high-boiling-point solvents.

[0088] As disclosed elsewhere in this document, in the case of a mixture using solvents, the mixture of components can still be considered a "high-boiling-point solvent" when the mixture itself has a high boiling point. Similarly, in the case of a mixture using solvents, the mixture of components can still be considered a "low-boiling-point solvent" when the mixture itself has a low boiling point.

[0089] In several embodiments, to prepare a stable crystalline form (compound A-Xln) of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro-[2.4]-hepta-7-ol (compound A), a high-boiling solvent (e.g., for the initial recrystallization process) is selected from, but not limited to, DMF, DMA, NMP, any mixture of the aforementioned selected solvents, or others. In several embodiments, to prepare a stable crystalline form (compound A-Xln) of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro-[2.4]-hepta-7-ol (compound A), a low-boiling solvent (e.g., for the second recrystallization process) is selected from, but not limited to, MeOH, EtOH, IPA, any mixture of the aforementioned selected solvents, or others.

[0090] In several embodiments, in order to prepare a stable crystalline form of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro-[2.4]-hepta-7-ol (compound A), the high-boiling solvent used for the initial recrystallization process is DMF, and the low-boiling solvent used for the sequential recrystallization process (second recrystallization process) is EtOH.

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

[0092] In several embodiments, as disclosed elsewhere herein, this disclosure relates to a method for preparing a salt form of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-7-ol (compound A), comprising a pharmaceutically acceptable salt of the compound A. The concept of "pharmaceutically acceptable salts" includes, but is not limited to, acid addition salts formed from inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid; or acid addition salts formed from organic acids, such as 1-hydroxy-2-naphthoic acid, 2,2-dichloroacetic acid, 2-hydroxyethanesulfonic acid, 2-oxoglutaric acid, 4-acetaminobenzoic 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, caproic acid, and caprylic acid. Acids, carbonic acid, cinnamic acid, citric acid, cyclic amino acids, dodecyl sulfate, ethane-1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactobionic acid, gentian acid, glucoheponic acid (D), gluconic acid (D), glucuronic acid (D), glutamic acid, glutamate, glycerophosphate, 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, dihydroxynaphthalic acid, phosphoric acid, propionic acid, pyroglutamic acid (-L), salicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid (+L), thiocyanate, toluenesulfonic acid (p), and undecanoic acid, etc.

[0093] Several examples 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-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-7-ol. Several examples relate to the preparation of phosphates (compound AP) and / or stable crystalline phosphate forms (compound AP-Xln) of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-7-ol (compound A).

[0094] In several embodiments, to prepare a stable crystalline phosphate form (compound AP-Xln) of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-7-ol (compound A), 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, recrystallization of compound AP is carried out in a solvent (or mixture of solvents).

[0095] In several embodiments, in order to prepare the stable crystalline phosphate form (compound AP-Xln) of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-7-ol (compound A), the solvent used for the neutralization process and / or recrystallization process comprises a solvent selected from, but not limited to, MeOH, EtOH, IPA, mixtures of any of the foregoing solvents, or other solvents.

[0096] In several embodiments, in order to prepare the stable crystalline phosphate form (compound AP-Xln) of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-7-ol (compound A), the solvent used for the neutralization and / or recrystallization processes is EtOH.

[0097] In several embodiments, as disclosed elsewhere herein, compounds A-Xln, SA-Xln, or RA-Xln can be prepared using a single recrystallization step (e.g., in a high-boiling solvent or a mixture of solvents). In several embodiments, compound A is heated in a high-boiling solvent. In several embodiments, a portion of the high-boiling solvent is removed (including under vacuum). In several embodiments, the solution is allowed to stand (e.g., cool), during which time crystals form. In several embodiments, the crystals are collected.

[0098] In several embodiments, compounds AP-Xln, SAP-Xln, or RAP-Xln can be prepared using a subsequent recrystallization process. In several embodiments, for example, compounds AP-Xln, SAP-Xln, or RAP-Xln are 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 stand (e.g., cool), during which time crystals form. In several embodiments, the crystals are collected. In several embodiments, in the case of using excess phosphoric acid, some of the phosphoric acid is neutralized.

[0099] Pharmaceutical Composition

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

[0101] In several embodiments, a method for preparing a pharmaceutical composition comprising a stable crystalline form of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-7-ol (compound AP-Xln), or a phosphate (compound AP) or stable crystalline phosphate (compound AP-Xln) of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-7-ol, 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.

[0102] Treatment

[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-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-7-ol (compound AP-Xln), or a phosphate of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-7-ol (compound AP) or a stable crystalline phosphate form (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.

[0104] 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 for preparing the chiral isomer is performed. In several embodiments, a preparative HPLC with a chiral column is used to separate the enantiomers (two chiral isomers, compound RA and compound SA) of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-7-ol (compound A) from one another. In several embodiments, the chiral isomers may then be recrystallized (and / or crystallized) as disclosed elsewhere herein.

[0105] Several embodiments relate to the stable crystalline form of compound A-Xln of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-7-ol, or the phosphate or stable crystalline phosphate form of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-7-ol, or both chiral isomers (compound RA, compound SA), for the preparation of a medicament for a method of treating aplastic diseases. Several embodiments relate to methods for treating diseases 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, particularly small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), and ovarian cancer; tumors caused by any one or more of the foregoing, hematologic malignancies (selected from ALL, CLL, AML, CML, and multiple myeloma), and combinations thereof. In several embodiments, the treatment method includes administration of compound A-Xln, a stable crystalline form of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-7-ol, or a phosphate or stable crystalline phosphate form of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-7-ol, or both chiral isomers (compound RA, compound SA).

[0106] Several embodiments relate to a stable crystalline form or salt of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-7-ol, or a stable crystalline salt form or both chiral isomers (compound RA, compound SA), for use as monotherapy or in combination with additional chemotherapeutic agents. In several embodiments, the additional chemotherapeutic agent is selected from platinum-based or taxane-based agents.

[0107] In several embodiments, the treatment method includes selecting a patient suffering from a selected disease, including solid tumors. In several embodiments, the treatment method includes selecting a patient suffering from 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, particularly small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), and ovarian cancer; tumors caused by any one or more of the foregoing, hematologic malignancies (selected from ALL, CLL, AML, CML, and multiple myeloma), and combinations thereof. In several embodiments, the method includes administering to a patient a therapeutic amount of a stable crystalline form of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-7-ol, or a phosphate or stable crystalline phosphate form of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-7-ol, or both chiral isomers (compound RA, compound SA).

[0108] In several embodiments, effective amounts of compound A-Xln, compound AP, and / or compound AP-Xln are administered to patients requiring treatment (e.g., subjects suffering from diseases as disclosed herein).

[0109] Several embodiments relate to a stable crystalline form or salt of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-7-ol, or a stable crystalline salt form or both chiral isomers (compound RA, compound SA), for use as monotherapy or in combination with immunotherapy agents. In several embodiments, the immunotherapy agents are selected from PD-1, PD-L1, oncolytic virus therapy, bispecific T-cell adaptors (BiTE), and chimeric antigen receptor (CAR) T-cell therapy agents, including but not limited to nivolumab, pembrolizumab, ipilimumab, bonnetumab, erlotuzumab, daratumumab, cimiprimab, avelumab, duvalumab, atezolizumab, toripalimab, sintilimab, camrelizumab, tislelizumab, AK104, penexalilimab, KN035, CS1001, and talirapive. In several embodiments, the combination is used to treat diseases or conditions 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, the blood cancers are selected from ALL, CLL, AML, CML and multiple myeloma.

[0110] To test bioactivity, the PTK inhibitory activity of compound A or compound A-Xln was compared with that of the commercially available drug sunitinib. In vitro tyrosine kinase inhibitory activity can be measured using commercial resources. Some of these tests can also be screened under contract with Eurofin or Reaction Biology.

[0111] For the kinase inhibition assay, the receptor tyrosine kinases selected were 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 inhibitory activity against all targets, with IC50 values ​​ranging from sub-nanomolar to micromolar. Compared to the marketed drug sunitinib, compound A showed higher inhibitory efficacy.

[0112] The inhibitory effects of compounds A-Xln, AP-Xln, and two chiral isomers (compounds RA and SA) on cancer cell lines were tested using in vitro MTT assays. Cancer cell lines selected from PANC-1, NCI-H157, MDA-MB-231, HeLa, PC-3, BEL7404, MKN45, Ishikawa, Saos-2, SKOV3, SW579, and HCT116 were used. IC50 values ​​were in the micromolar range.

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

[0114] Example

[0115] Abbreviations and Definitions

[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 lymphoblastic or lymphocytic leukemia; CLL: Chronic lymphoblastic 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 cell culture medium required to inhibit protein transport activity by 50%; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

[0117] Example 1: Crystalline form 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7- Preparation of (2,4)-hepta-7-ol (compound A-Xln)

[0118] Crude compound A (18 g) was prepared similarly according to WO2010021918, yielding an amorphous solid (compound A-Amp) with a DSC main peak at 245.99 °C. 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 its volume under vacuum and further cooled to RT. The precipitate was filtered, and the filter cake was washed with a small amount of DMF, followed by washing with some water. The filter cake was then mixed with ethanol (4 l) and heated to dissolve. The solution was concentrated to about half its volume under vacuum and further cooled to RT. The precipitate was filtered and dried in an oven to obtain stable crystals of the desired product (compound A-Xln), which exhibited only one DSC peak at 248.79 °C.

[0119] The DSC chromatogram of the amorphous form of compound A (compound A-Amp) is shown in the attached figure. Figure 1 What is represented.

[0120] DSC, TGA, XRPD and other methods were used to analyze the crystalline form of compound A (compound A-Xln). 1 The H-NMR spectra are shown in the attached figures. Figure 2 , Figure 3 , Figure 4 and Figure 5 What is represented.

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

[0122] 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).

[0123] DSC melting range (Endo): 247-253℃, with a peak temperature of 248.79℃. TGA shows undissolved material with weight loss above 250℃. XRPD has the aforementioned graph including 14 characteristic peaks with an intensity % greater than 10%, or 43 characteristic peaks where all intensity % is expressed in d-values ​​and angles as follows:

[0124] XRPD data:

[0125]

[0126]

[0127] Example 2

[0128] Based on the inventors' research experience in preparing the crystalline form of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-7-ol (compound A) in Example 1, the following preparation of the crystalline form of compound A is anticipated. The preparation of the crystalline form of compound A (compound A-Xln) should involve at least one recrystallization process, preferably two recrystallization processes. Preferably, the initial recrystallization requires a relatively high-boiling 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 3: (2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl The amorphous (compound A-Amp) and crystalline (compound A-) forms of 5-azaspiro[2.4]-hepta-7-ol (compound A-) Stability comparison of Xln API

[0130] Samples of the 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 the amorphous (compound A-Amp) and crystalline (compound A-Xln) forms of compound A was tested on days 0, 10, and 30. Samples of both amorphous and crystalline API were measured, and appropriate amounts of diluent were added for dissolution by sonication. The concentration was controlled at 0.2 mg / ml, and sample purity was determined by HPLC using a 230 nm UV detector. The results are listed in the table below, and the fitted curves are shown in the attached figure. Figure 6 As shown in the figure, A-Xln has better stability than A-Amp.

[0131] Table 1: Comparison of the stability of amorphous and crystalline forms of compound A (API)

[0132]

[0133] Example 4: (2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl The amorphous (compound A-Amp) and crystalline (compound A-) forms of 5-azaspiro[2.4]-hepta-7-ol (compound A-) Xln)

[0134] Comparison of tablet stability

[0135] Samples of amorphous and crystalline 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 according to the following procedure:

[0136] (1) Screening process: Compound A (A-Amp or A-Xln), lactose T80, microcrystalline cellulose (PH101), and sodium carboxymethyl starch were screened through an 80-mesh sieve.

[0137] (2) Granulation process: The sieved compound A (A-Amp or A-Xln), lactose T80, microcrystalline cellulose, sodium carboxymethyl starch, and silica are loaded into a V-shaped stirrer for step blending 1. After blending 1, 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%).

[0138] (3) Tableting process: After being compressed into 10mg and 30mg tablets using a tablet press, the final blend was compressed into tablets. During processing, in-process control (IPC) tests were performed on weight change and hardness (weight change ±6.0%, and hardness 7 to 13 kg / mm²). 2 ).

[0139] (4) Coating process: Prepare a coating solution according to the ratio of gastric-soluble film coating premix: purified water = 15:85 (w:w) and stir continuously for 45 minutes. The coating weight increases by approximately 2% to 3%.

[0140] (5) Packaging process: The collected tablets are sorted by weight. The qualified tablets are collected and packaged in aluminum-plastic blister packs of 7 tablets / blister.

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

[0142]

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

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

[0145]

[0146] Chp: Chinese Pharmacopoeia; USP: United States Pharmacopeia

[0147] The prepared tablets were tested in a similar manner to that described in Example 3, and the results are listed in Table 4, with the fitting curves shown in the attached figure. Figure 7 As shown in the image.

[0148] Table 4: Comparison of stability of amorphous and crystalline tablets of compound A

[0149]

[0150] Table 5: Stability of Compound A-Xln Pharmaceutical Product (DP, Tablet) at 25℃±2℃ / 60%RH±5%RH

[0151]

[0152]

[0153]

[0154]

[0155] Example 5: Comparison of in vitro dissolution curves of Compound A tablets

[0156] The crystalline form of a compound can alter its physical and chemical properties, including dissolution rate, which can affect drug efficacy. Dissolution rate measurements can be performed in a dissolution test. Samples of amorphous (compound A-Amp) and crystalline (compound A-Xln) tablets were dissolved in a suitable solvent (0.1 mol hydrochloric acid solution) and stirred at an appropriate rate to promote the dissolution process (ZRC-8D intelligent dissolution apparatus, speed 50 rpm). The dissolution percentage was determined by HPLC with the UV detector wavelength set to 230 nm. Samples were collected at 5, 10, 20, 30, and 45 minutes. The experimental results are listed in the table below, and the dissolution curves can be found in the attached figure. Figure 8 Found. After comparing the experimental data, it was observed that crystalline tablets dissolved faster than amorphous tablets.

[0157] Table 6: Dissolution curve results of compound A-Amp tablets (10mg specification)

[0158] Number of 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％

[0159] Table 7: Dissolution curve results of compound A-Xln tablets (10mg specification)

[0160] Number of 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％

[0161] AVG: Mean; RSD: Relative Standard Deviation.

[0162] Example 6: 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl Preparation of phosphate and crystalline form of (compound AP-Xln) of 5-azaspiro[2,4]-hepta-7-ol

[0163] 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 washed with EtOH, and further dried in an oven at 50 °C for 10 hours to obtain a white solid, which is the phosphate of compound A in a stable crystalline form (compound AP-Xln).

[0164] DSC, TGA, XRPD and other methods were used to analyze phosphate crystals (compound AP-Xln). 1 H-NMR spectra are respectively from Figure 9 , Figure 10 , Figure 11 and Figure 12 express.

[0165] 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).

[0166] DSC melting range (Endo): 221-235℃, with a peak temperature of 229℃. TGA shows a weight loss of 1.84% at approximately 30-60℃, and significant weight loss above 210℃. The XRPD graph includes 21 characteristic peaks, where all intensity % is expressed in d-values ​​and angles as follows:

[0167] Table 8: XRPD data for the phosphate of compound A (compound AP-Xln) with stable crystalline form:

[0168]

[0169] Example 7

[0170] Based on the inventors' research experience in preparing a stable crystalline phosphate form (compound AP-Xln) of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-7-ol (compound A), the following preparation of a stable crystalline phosphate form of compound A is anticipated. The stable crystalline phosphate compound (compound AP-Xln) is prepared by reacting compound A with phosphoric acid in a solution of some type of solvent (or solvent mixture) and by recrystallization in some type of solvent (or solvent mixture). Preferably, this stable crystalline phosphate form of (compound AP-Xln) should be obtainable by recrystallization in a solvent (or solvent mixture) containing, 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 at a flow rate of 5 mL / min into a chiral HPLC system equipped with a chiral pre-formed column (CHIRALCEL OD 500 × 50 mm). The mobile phase at UV 240 nm was hexane:IPA:diethylamine (85:15:0.1). The injections were repeated a total of five times, and the collected fractions were combined and further evaporated to give compound RA (optical rotation: -23.49, 1.3 mg) and compound SA (optical rotation: +22.51, 1.6 mg) as two enantiomers.

[0173] 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).

[0174] 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).

[0175] Example 9: Kinase Inhibition

[0176] To test the bioactivity of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-7-ol (compound A-Xln), inhibition of kinases FGFR1(h), FGFR2(h), FGFR3(h), Flt1(h)(VEGFr1), Flt4(h)(VEGFr3), KDR(h)(VEGFr2), Aurora-B PDGFRα(h), PDGFRα(h), and PDGFRβ(h) was tested, and the IC50 values ​​are listed in the table below. Furthermore, the kinase inhibitory effect of the commercially available drug sunitinib was tested, and the results were compared with those of compound A-Xln.

[0177] Table 9: Kinase inhibition of compound A compared to sunitinib

[0178]

[0179]

[0180] - or blank: No inhibition or compound activity cannot fit the IC50 curve.

[0181] Example 10

[0182] The ion chromatography analysis and water solubility results of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-7-ol phosphate (compound AP) are listed in the table below.

[0183] Table 10. Ion chromatography analysis and water solubility study of compound AP

[0184]

[0185] Example 11

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

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

[0188]

[0189]

[0190] Table 12. In vitro inhibitory activities of compounds A and AP on tumor cell lines.

[0191]

[0192] Example 12: Compound A-Xln in vitro and in vivo with chemotherapy or immunotherapy targeting tumor cell lines and targeting... Combined efficacy of various tumor cell lines in vivo xenograft models

[0193] The MTT assay results of compound A-Xln combination therapy were studied. 100 μl of a 5 × 10⁻⁶ m³ solution was used. 4Cell suspension of 10 cells / ml was added to each well of a 96-well plate and incubated at 37°C in 5% CO2. After 24 hours, compound A-Xln sample solution was added according to Table 13, 10 μl / well for double-sample preparations. After incubation at 37°C and 5% CO2 for 72 hours, 20 μl of 5 mg / ml MTT solution was added to each well, followed by 100 μl / well of dissolution solution after 4 hours. The samples were placed in an incubator and the OD value was measured at 570 nm using a full-wavelength multimicroplate reader after dissolution. Figure 13 and 14 The proliferation inhibition of breast cancer cell lines MCF7 and BT474 by compound A-Xln, the anti-breast cancer agents anastrozole and letrozole, the combination of compound A-Xln and anastrozole, and the combination of compound A-Xln and letrozole were demonstrated, respectively.

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

[0195]

[0196]

[0197] Based on the samples in Table 14, the proliferation inhibitors of three colon cancer lines HT29, HCT116, and colo205 were also tested using compound A-Xln, the anti-colon cancer agent 5FU / O, and combinations of compounds A-Xln and 5FU / O. The results are as follows: Figure 15-17 As shown.

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

[0199]

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

[0201] Nude mice were inoculated with the mouse colorectal CT26 cell line for 7 days and randomly divided into groups of 6 mice each. The mice were then treated with either a mediator, compound A-Xln once daily in two treatment groups, anti-mouse PD-1 (PD-1) 200 μg / mouse once every 3 days in one treatment group, or a combination of compound A-Xln and anti-PD-1 in both treatment groups. Table 15 shows the changes in tumor size after administration of the corresponding compounds. Figure 18 The study presents a comparison of the change in mean tumor volume following administration of compound A-Xln and an anti-PD-1 antibody, as well as a combination therapy of compound A-Xln and an anti-PD-1 antibody.

[0202] Table 15: List of drug administration methods for CT26 cells in mouse colorectal region

[0203]

[0204] D0: First administration time. IP: Intraperitoneal injection. ig: Gastrointestinal administration. Qd: Once daily. Q3d: Once every three days. PD-1 (anti-PD-1): Anti-mouse PD-1 antibody.

[0205] The in vivo antitumor efficacy of compound A-Xln against various tumor cell lines has been tested in xenograft models.

[0206] Animal models of in vivo antitumor xenograft activity using various tumor cell lines (SCLC NCI-H1436, NSCLC 95-D, ovarian SKVO3, kidney 786-O, liver Bel-7420, brain U87) are as follows: Tumor tissue from well-grown tumor cell lines is cut into 3 mm pieces, and one piece is subcutaneously inoculated into the right flank of each nude mouse. Animals are grouped and administered the pre-designed dose. When the tumor size reaches 100 mm after 10-14 days... 3 Treatment begins at the above time. Animals with excessively large or small tumors are culled based on tumor size, and animals with similar average tumor volumes are grouped together. Animals are then orally administered the medication daily for 14-21 days. Thirteen days after inoculation, the large diameter a (mm) and small diameter b (mm) are measured twice weekly using calipers. The volume of each tumor is calculated using the following formula: TV = ab 2 / 2 and the relative tumor volume was calculated as: RTV = Vt / Vo, where Vo represents the tumor volume on the first day of treatment; Vt represents the tumor volume on each measurement day. Animals were sacrificed, and tumors were extracted by dissection 20-30 days post-inoculation. Individual body weight and tumor weight were determined and calculated. The percentage of tumor inhibition 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. Experimental drug administration and efficacy results are shown in the attached figure. Figure 19-24 As shown. The optimal tumor inhibition percentage range is >70% to 100%.

[0207] The results demonstrated that the combination of compound A-Xln with anti-breast cancer agents such as aromatase inhibitors anastrozole and letrozole increased the inhibition of human cancer cell proliferation (such as breast cancer cell lines MCF7 and BT474).

[0208] The study demonstrated that the combination of compound A-Xln with chemotherapeutic agents (such as anti-colon cancer 5FU / O) increased the inhibition of proliferation of human cancer cells (such as cancer cell lines HT29, HCT116, and colo205). The inhibitory activity also showed some differences in sequential dosing, with higher activity observed in regimens where the chemotherapeutic agent was administered first, followed by compound A.

[0209] Mouse xenograft models have demonstrated combined inhibitory effects on cancer cell proliferation, such as the proliferation of the mouse colorectal cancer cell line CT26. The combination of compound A-Xln with an anti-PD1 antibody provides strong evidence for enhanced efficacy in in vivo animal models for treating cancer cell proliferation.

[0210] 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, the following antibodies, can produce synergistic clinical efficacy: nivolumab, pembrolizumab, ipilimumab, bonnetumab, erlotuzumab, daratumumab, cimiprimab, avelumab, duvalumab, atezolizumab, toripalimab, sintilimab, camrelizumab, tislelizumab, AK104, AK105, penprimab, KN035, CS1001, and talirapive.

[0211] Various human cancer cell lines and animal xenograft models have demonstrated that compound A-Xln has significant tumor-suppressive activity, particularly in SCLC, NSCLC, ovarian cancer, renal cancer, liver cancer, and brain cancer.

[0212] Example 13: Compound A-Xln tablets are effective against solid tumors such as NSCLC, SCLC, ovarian cancer, endometrial cancer, and uterine cancer. Clinical trials in people with cervical cancer, uterine leiomyosarcoma, and peritoneal mesothelioma.

[0213] A: Overview

[0214] Compound A-Xln tablets are administered orally at 90 mg, 80 mg, 70 mg, 60 mg, or 40 mg once daily for 28 days as one cycle for different cancer patients, until intolerance or disease progression (PD) is assessed by RECISIT 1.1. In cases of observed intolerance, the daily dose of 90 mg can be reduced to 80 mg, 70 mg, 60 mg, 50 mg, 40 mg, 30 mg, or 20 mg daily in 10 mg intervals. Treatment results (as of March 2022) in a small number of representative patients with significant and unexpected antitumor activity are listed below. The very positive objective response rate (ORR) for SCLC ≥3 lines (≥2 lines) of treatment was >20%, and the very positive objective response rate for platinum-resistant ovarian cancer ≥3 lines (≥2 prior lines) of treatment was approximately 20%. Some efficacy was observed in SCLC patients who received maintenance therapy with compound A-Xln tablets after completing a first-line standard of care (SOC) cycle. It has some efficacy for SCLC patients who have already been treated with compound A-Xln tablets as a second-line (previously first-line) treatment.

[0215] Its efficacy has also been observed in sarcomas and brain tumors.

[0216]

[0217]

[0218] *: Non-progressive disease time / still under investigation. Mnt: Maintenance therapy.

[0219] Furthermore, a completed SCLC clinical trial (with 30 patients who had received first-line therapy (Group B) and 30 patients who had received ≥ second-line therapy (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 first-line or ≥ second-line therapy, these are significantly better than currently reported treatment options. The Kaplan-Mayer curve is shown in the attached figure. Figure 25 and Figure 26 As shown.

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

[0221] (1): Representative subjects in phase 1b, S01013

[0222] Subject S01013, with NSCLC (previous line >3), received 60 mg of compound A-Xln tablets orally once daily (10 mg x 6) for a 28-day cycle. At the second cycle, the dose was reduced to 40 mg and 30 mg, and a similar treatment regimen was used in subsequent cycles. The subject's best treatment response was PR (a 54.70% reduction in target lesions compared to baseline).

[0223] (2): Representative subjects in phase 1b, S01014

[0224] Subject S01014 (previous line = 3) with NSCLC received 60 mg of compound A-Xln tablets orally once daily for 28 days. The dose was reduced to 40 mg at the third cycle and continued in subsequent cycles with a similar treatment regimen. The best treatment response in the subject was PR (a 56.95% reduction in target lesions compared to baseline).

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

[0226] Subject S05002 (previous baseline = 2) with NSCLC received 60 mg of compound A-Xln tablets orally once daily for 28 days. A similar treatment regimen was used in subsequent cycles. The subject's best treatment response was PR (a 38.42% reduction in target lesions compared to baseline).

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

[0228] Subject 020207 (previous baseline = 3) with peritoneal mesothelioma received a combination of compound A-Xln tablets 40 mg orally once daily for 28 days, along with toripalimab injection (240 mg, every 3 weeks). A similar treatment regimen was used in subsequent cycles. The subject's best treatment response was PR (a 36.4% reduction in target lesion compared to baseline).

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

[0230] Subject 020202 (previous baseline = 1) with small cell lung cancer received a combination of compound A-Xln tablets 40 mg orally once daily for 28 days, along with toripalimab injection (240 mg, once every 3 weeks). Similar treatment regimens were used in subsequent cycles. The subject's best treatment response was PR (a 38.22% reduction in target lesions compared to baseline).

Claims

1. 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-7-ol crystals A-Xln, characterized in that, 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. Crystals of 2,5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2,4]-hepta-7-ol A-Xln, characterized in that, The intensity percentage greater than 10% in the XRPD of the crystal A-Xln is expressed as follows, using d-values ​​and angles: 。 3. The crystal A-Xln according to claim 2, characterized in that, The XRPD characteristic peaks of the crystal A-Xln, with all intensities (%) expressed in terms of d-values ​​and angles, are as follows: 。 4. The crystal A-Xln according to claim 1, characterized in that, The XRPD of the crystal A-Xln is shown in Figure 4.

5. The crystal A-Xln according to claim 1, characterized in that, The DSC melting range of the crystal A-Xln is 240-260℃, with a peak temperature range of 244-254℃.

6. The crystal A-Xln according to claim 5, characterized in that, The exact melting range of the crystal, A-Xln, is 247-253°C, with a peak temperature of 249°C.

7. The crystal A-Xln according to claim 1, characterized in that, The TGA thermogravimetric analysis of the crystal A-Xln showed that the substance was a solvate-free compound and exhibited weight loss above 250 °C.

8. The method for preparing crystal A-Xln according to any one of claims 1 to 7, characterized in that, The amorphous form of 5-(2-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinoline-7-yloxy)ethyl)-5-azaspiro[2.4]-hepta-7-ol is subjected to recrystallization, the recrystallization being first performed using a high-boiling solvent and then using a low-boiling solvent; the high-boiling solvent is selected from at least one of DMF, DMA or NMP; and the low-boiling solvent is selected from at least one of MeOH, EtOH or IPA.

9. The method for preparing crystal A-Xln according to claim 8, characterized in that, The high-boiling-point solvent is DMF.

10. The method for preparing crystal A-Xln according to claim 8, characterized in that, The low-boiling-point solvent is EtOH.

11. The use of crystal A-Xln according to any one of claims 1 to 7 in the preparation of protein tyrosine kinase inhibitors.

12. The application according to claim 11, characterized in that, The protein tyrosine kinase is selected from human FGFR1, human FGFR2, human FGFR3, human Flt1 (VEGFr1), human Flt4 (VEGFr3), human KDR (VEGFr2), Aurora-B-human PDGFRα, human PDGFRα, or human PDGFRβ.

13. The use of crystal A-Xln according to any one of claims 1 to 7 in the preparation of a medicament for inhibiting cancer cell lines of the pancreas, prostate, sarcoma, thyroid, colon, ovary, blood, breast, brain, NSCLC, SCLC, liver, kidney, rectum, cervix or stomach.

14. The application according to claim 13, characterized in that, The cancer cells are selected from cancer cell lines such as PANC-1, NCI-H157, MDA-MB-231, HeLa, PC-3, BEL7404, MKN45, Ishikawa, Saos-2, SKOV3, SW579, NCI-H1436, Bel-7402, U87, or HCT116.

15. The use of crystal A-Xln according to any one of claims 1 to 7 in the preparation of a medicament for treating aplastic diseases, wherein the aplastic disease is a solid tumor or leukemia, and the solid tumor is selected from: 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, or endometrial cancer.

16. The application according to claim 15, characterized in that, The lung cancer is selected from NSCLC or SCLC.

17. The application according to claim 15, characterized in that, The blood cancers mentioned are selected from: ALL, CLL, AML, CML or multiple myeloma.

18. The application according to claim 15, characterized in that, The neoplastic diseases mentioned are small cell lung cancer, non-small cell lung cancer, mesothelioma, ovarian cancer, brain tumors, or sarcomas.

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