Crystalline forms of checkpoint kinase 1 (CHK1) inhibitors and uses thereof

Abstract

Provided herein is a pharmaceutical composition (1) comprising a CHK1 inhibitor. (1)

Description

Cross-referencing

[0001] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 493,846, filed April 3, 2023, the entire contents of which are incorporated herein by reference. Background Technology

[0002] Cancer remains the second leading cause of death in the United States (US), with approximately 1,900,000 new diagnoses and 610,000 deaths each year. Globally, cancer is the second leading cause of death, causing nearly 10 million deaths in 2020; nearly one in six deaths are caused by cancer. New cases are projected to increase by 70% over the next 20 years.

[0003] In solid malignancies, metastasis and systemic disease account for approximately 90% of cancer-related deaths. Despite progress made over the past few decades, there is still a need to develop targeted interventions for advanced or metastatic solid tumors.

[0004] Apart from HER2 (or ERBB2), there are no approved targeted therapies for cancer patients with oncogene amplification as standard treatment. These patients typically experience lower survival rates than those with other forms of oncogene mutations or no known oncogene mutations.

[0005] There are currently no approved therapies for treating patients with these other oncogene-amplified tumors, and in particular, there are no oral therapies that include stable drug forms of CHK1 inhibitors. Summary of the Invention

[0006] This article discloses 5-((5-(4-(((1R,3S)-3-aminocyclopentyl)oxy)-2-methoxy-6-methylpyridin-3-yl)-1H-pyrazol-3-yl)amino)pyrazine-2-carboxynitrile: (Compound 1) or its pharmaceutically acceptable salt or solvate in crystalline form.

[0007] This article also discloses the free base 5-((5-(4-(((1R,3S)-3-aminocyclopentyl)oxy)-2-methoxy-6-methylpyridin-3-yl)-1H-pyrazol-3-yl)amino)pyrazine-2-carboxynitrile: (Compound 1) or its pharmaceutically acceptable solvate in crystalline form.

[0008] This article also discloses the anhydrous free base 5-((5-(4-(((1R,3S)-3-aminocyclopentyl)oxy)-2-methoxy-6-methylpyridin-3-yl)-1H-pyrazol-3-yl)amino)pyrazine-2-carboxynitrile: (Compound 1) in crystal form.

[0009] In some embodiments, crystalline compound 1 is in the free base form FB-1, characterized by having at least one of the following properties: (a) and Figure 1 The X-ray powder diffraction (XRPD) patterns shown are essentially the same; (b) X-ray powder diffraction (XRPD) patterns with characteristic peaks at 11.96±0.1° 2θ, 18.81±0.1° 2θ, 19.91±0.1° 2θ, 21.67±0.1° 2θ and 26.75±0.1° 2θ; (c) X-ray powder diffraction (XRPD) patterns with characteristic peaks at 13.87 ±0.1° 2θ and 17.06 ±0.1° 2θ; (d) Differential scanning calorimetry (DSC) thermogram showing endothermic activity with a peak temperature of approximately 216.5 °C (initial temperature); (e) and Figure 2 The thermogravimetric analysis (TGA) chromatograms shown are essentially the same; (f) Thermogravimetric analysis (TGA) chromatogram showing a mass loss of approximately 0.10% from the initial heating temperature to approximately 100.0 °C; or (g) Its combination.

[0010] This article also discloses a pharmaceutical composition comprising the crystalline form disclosed herein and pharmaceutically acceptable excipients.

[0011] This article also discloses a method for treating cancer in a subject in need, which involves applying the crystalline form disclosed herein to the subject.

[0012] In some implementations, cancer includes solid tumors.

[0013] In some implementations, the cancer includes locally advanced or metastatic unresectable solid tumors.

[0014] In some implementations, cancer comprises a tumor or tumor cells carrying oncogene amplification.

[0015] In some implementations, oncogene amplification includes amplification of ABL, AKT1, AKT2, ALK, androgen receptor, BRAF, CCND1, CCND2, CCND3, CCNE1, CDK12, CDK4, CDK6, EGFR, ERBB2, EZH2, FGFR1, FGFR2, FGFR3, FLT3, IDH1 / 2, JAK2, JAK3, KIT, KRAS, MDM2, MDM4, MET, MYC, MYCL, MYCN, NRAS, PDGFRA, TERT, VEGFRA, or any combination thereof.

[0016] In some implementations, oncogene amplification is located on ecDNA.

[0017] In some implementations, oncogene amplification is located at one or more chromosomal loci.

[0018] In some implementations, oncogene amplification is ecDNA-derived amplification.

[0019] In some implementation schemes, the cancer is ovarian cancer.

[0020] In some implementations, ovarian cancer is platinum-resistant high-grade serous ovarian cancer, primary peritoneal cancer, or fallopian tube cancer.

[0021] In some implementations, the cancer is uterine cancer.

[0022] In some implementations, uterine cancer is defined as high-grade endometrial cancer, serous uterine cancer, or carcinosarcoma of the uterus.

[0023] In some implementation schemes, the cancer is colorectal cancer, esophageal cancer, gastric cancer, gastroesophageal junction (GEJ) cancer, head and neck squamous cell carcinoma, liposarcoma, non-small cell lung cancer, or a subtype of squamous cell carcinoma.

[0024] In some implementations, the cancer is neuroblastoma.

[0025] In some implementation schemes, the cancer is breast cancer, bile duct cancer, esophageal cancer, squamous cell carcinoma of the neck, non-small cell lung cancer, gastric cancer, or a subtype of squamous cell carcinoma.

[0026] In some implementation schemes, the cancer is esophageal cancer, non-small cell lung cancer, sarcoma, or stomach cancer.

[0027] In some implementations, the treatment also includes the administration of additional therapeutic agents.

[0028] In some implementations, oncogene amplification includes CDK4, CDK6, EGFR, FGFR1, FGFR2, or FGFR3.

[0029] In some implementations, treatment further includes administration of a CDK4 / 6 inhibitor, an EGFR inhibitor, or an FGFR inhibitor. In some implementations, the EGFR inhibitor is erlotinib. In some implementations, the FGFR inhibitor is pemigatinib. In some implementations, the FGFR inhibitor is futibatinib. In some implementations, the CDK4 / 6 inhibitor is abemaciclib. Incorporation

[0030] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference for the specific purposes identified herein. Attached Figure Description

[0031] The novel features of the invention are specifically set forth in the appended claims. A better understanding of the features and advantages of the invention will be obtained by referring to the following detailed description of exemplary embodiments utilizing the principles of the invention, along with the accompanying drawings, in which: Figure 1 The X-ray powder diffraction (XRPD) pattern of compound 1 in its free base form FB-1 is shown.

[0032] Figure 2 Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) spectra of compound 1 in its free base form FB-1 are shown.

[0033] Figure 3 The X-ray powder diffraction (XRPD) pattern of compound 1 in its free basic form FB-2 is shown.

[0034] Figure 4 The X-ray powder diffraction (XRPD) pattern of compound 1 in its free basic form FB-3 is shown.

[0035] Figure 5 The X-ray powder diffraction (XRPD) pattern of compound 1 in its free base form FB-4 is shown.

[0036] Figure 6 The X-ray powder diffraction (XRPD) pattern of compound 1 in its free basic form FB-5 is shown. Detailed Implementation

[0037] Focal high-copy-number oncogene amplification is frequently observed on extrachromosomal DNA (ecDNA). ecDNA is found in tumor cells and originates from extrachromosomal segments of genomic DNA that typically encode full-length genes and regulatory regions such as promoters. Physically, ecDNA may differ from chromosomes and possess unique properties, including a tendency to contain open chromatin structures and structural variations associated with overtranscription. Furthermore, due to the lack of centromeres, extrachromosomal ecDNA is inherited through non-centromere, non-Mendelian segregation during cell division, resulting in high copy-number gene heterogeneity within tumor cell populations. Because of these properties, ecDNA is a common cellular mechanism for oncogene amplification (e.g., EGFR), and it promotes the overtranscription and overexpression of oncogenes, driving tumor growth and survival. Moreover, these characteristics endow tumor cells activated by oncogene amplification with unparalleled genomic plasticity, promoting tumorigenesis and evading therapeutic stress through rapid genomic evolution. Cancer cells carrying oncogene amplification on ecDNA experience high levels of intrinsic DNA replication stress (RS). Checkpoint kinase 1 (CHK1) plays a crucial role in managing systolic leukemia (RS), making it a potential therapeutic target for cancers with intrinsically elevated RS, including those with oncogenic amplification and ecDNA activation. Consistent with this hypothesis, tumor cells with ecDNA-activated oncogene amplification exhibit enhanced sensitivity to CHK1 inhibition compared to ecDNA-negative, non-amplified cells. Applying targeted therapeutic pressure (e.g., EGFR inhibitors) to the protein products of oncogenes amplified on ecDNA (e.g., EGFR) induces cancer cells to evade this pressure, and these resistance mechanisms further increase RS and dependence on CHK1. Therefore, in ecDNA-activated tumor cells, the combination of targeted therapeutic pressure (e.g., EGFR inhibitors) and CHK1 pressure (i.e., CHK1 inhibitors) can provide synergistic therapeutic effects. Consequently, as recognized and addressed in this paper, there is a need to provide CHK1 inhibitors with the desired clinical and therapeutic properties, in stable pharmaceutical forms containing such CHK1 inhibitors. definition

[0038] In the following description, certain specific details are set forth in order to provide a comprehensive understanding of various embodiments. However, those skilled in the art will understand that the invention can be practiced without these details. In other instances, well-known structures are not shown or described in detail to avoid unnecessarily obscuring the description of the embodiments. Unless the context requires otherwise, the word “comprise” and its variations such as “comprises” and “comprising” should be interpreted in an open, inclusive sense, i.e., as “including but not limited to”, in the specification and subsequent claims. Furthermore, the headings provided herein are for convenience only and do not constitute an explanation of the scope or meaning of the claimed invention.

[0039] References to “some embodiments” or “one embodiment” in this specification mean that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment. Therefore, the phrases “in one embodiment” or “in an embodiment” appearing throughout this specification do not necessarily refer to the same embodiment. Furthermore, a particular feature, structure, or characteristic may be combined in one or more embodiments in any suitable manner. Additionally, unless expressly stated otherwise, the singular forms “a,” “an,” and “the” as used in this specification and the appended claims include plural references. It should also be noted that, unless expressly stated otherwise, the term “or” is generally used to include the meaning of “and / or.”

[0040] As used herein, the term "treatment" refers to therapeutic treatment aimed at preventing or alleviating (reducing) unwanted physical symptoms, conditions, or diseases, or achieving beneficial or desired clinical outcomes. For the purposes described herein, beneficial or desired clinical outcomes include, but are not limited to, symptom relief; reduction in the severity of symptoms, conditions, or diseases; stabilization (i.e., non-deterioration) of the state of symptoms, conditions, or diseases; delay in the onset or slowing of the progression of symptoms, conditions, or diseases; improvement in the state of symptoms, conditions, or diseases; and detectable or undetectable remission (partial or complete) or improvement or cure of symptoms, conditions, or diseases. Treatment includes evoking a clinically significant response without excessive levels of side effects. Treatment also includes extending survival relative to expected survival without treatment. As used herein, the terms "treatment" and words derived from them do not necessarily imply 100% or complete cure. Rather, there are treatments of varying degrees that are recognized by those skilled in the art as having potential benefit or therapeutic effect. In this regard, the disclosed methods can provide treatment for any amount and level of a symptom in mammals. For example, the symptom, including its symptoms or signs, can be reduced by, for example, about 100%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, or about 10%.

[0041] As used herein, the term "effective amount" or "therapeutic effective amount" refers to an adequate amount of the compound disclosed herein administered that will, to a certain extent, alleviate one or more symptoms of the disease or condition being treated, such as cancer or an inflammatory disease. In some embodiments, the result is a reduction and / or mitigation of the signs, symptoms, or cause of the disease, or any other desired alteration of the biological system. For example, an "effective amount" for therapeutic use is, for instance, the amount of a composition comprising the compound disclosed herein required to provide a clinically significant reduction in the symptoms of the disease. In some embodiments, techniques such as dose escalation studies are used to determine the appropriate "effective" amount in any single case.

[0042] As used herein, the term "biological sample" generally refers to a sample derived from or obtained from an object, such as a mammal (e.g., a human). Biological samples considered include, but are not limited to: hair, nails, skin, sweat, tears, eye discharge, nasal swabs or nasopharyngeal washes, sputum, throat swabs, saliva, mucus, blood, serum, plasma, placental fluid, amniotic fluid, umbilical cord blood, emphatic fluid, cavity fluid, earwax, oil, glandular secretions, bile, lymph, pus, microbiota, meconium, breast milk, bone marrow, bone, CNS tissue, cerebrospinal fluid, adipose tissue, synovial fluid, feces, gastric juice, urine, semen, vaginal secretions, stomach, small intestine, large intestine, rectum, pancreas, liver, kidneys, bladder, lungs, and other tissues and fluids derived from or obtained from the object.

[0043] As used herein, the terms “tumor” or “tumor cell” generally refer to a cell that grows and divides beyond its normal growth or division, or a cell that does not die when it should. In some cases, tumor cells present as a solid mass (such as a solid tumor), or in others, they exist in a non-solid form such as a hematopoietic tumor. A tumor or tumor cell may also contain metastatic cells or cells that are metastasizing, in which cancer cells detach from the original (primary) tumor and may form new tumors in other organs or tissues of the body.

[0044] As used herein, the term "ecDNA signature" generally refers to one or more features common to tumors or tumor cells that are ecDNA+. In some cases, ecDNA signatures are selected from gene amplification; p53 loss-of-function mutations; loss of microsatellite instability (MSI-H); low levels of PD-L1 expression; low levels of tumor inflammatory features (TIS); low levels of tumor mutational burden (TMB); increased frequency of allele substitutions, insertions, or deletions (indels); and any combination thereof. In some cases, ecDNA signatures may include copy number increases (gene amplification) and specific structural variations. In some cases, ecDNA signatures may include focal amplification. In some cases, ecDNA signatures involve the detection or identification of ecDNA using imaging techniques. In some cases, ecDNA signatures do not involve any imaging or direct detection of ecDNA. compound

[0045] This article describes a method for treating cancer in subjects who require treatment, which involves administering a CHK1 inhibitor to the subject.

[0046] Compound 1 In some embodiments, the CHK1 inhibitor is compound 1 or a pharmaceutically acceptable salt thereof. Compound 1 is 5-((5-(4-(((1R,3S)-3-aminocyclopentyl)oxy)-2-methoxy-6-methylpyridin-3-yl)-1H-pyrazol-3-yl)amino)pyrazine-2-carboxynitrile: In some embodiments, compound 1 is a free base. In some embodiments, compound 1 is in the form of a salt.

[0047] Crystalline Compound 1 This article discloses 5-((5-(4-(((1R,3S)-3-aminocyclopentyl)oxy)-2-methoxy-6-methylpyridin-3-yl)-1H-pyrazol-3-yl)amino)pyrazine-2-carboxynitrile: (Compound 1) or its pharmaceutically acceptable salt or solvate in crystalline form.

[0048] This article also discloses the free base 5-((5-(4-(((1R,3S)-3-aminocyclopentyl)oxy)-2-methoxy-6-methylpyridin-3-yl)-1H-pyrazol-3-yl)amino)pyrazine-2-carboxynitrile: (Compound 1) or its pharmaceutically acceptable solvate in crystalline form.

[0049] This article also discloses the anhydrous free base 5-((5-(4-(((1R,3S)-3-aminocyclopentyl)oxy)-2-methoxy-6-methylpyridin-3-yl)-1H-pyrazol-3-yl)amino)pyrazine-2-carboxynitrile: (Compound 1) in crystal form.

[0050] This article also discloses the crystalline form of the HCl salt of 5-((5-(4-(((1R,3S)-3-aminocyclopentyl)oxy)-2-methoxy-6-methylpyridin-3-yl)-1H-pyrazol-3-yl)amino)pyrazine-2-carboxynitrile (compound 1) or its pharmaceutically acceptable solvates.

[0051] This article also discloses the crystalline form of the HCl salt of 5-((5-(4-(((1R,3S)-3-aminocyclopentyl)oxy)-2-methoxy-6-methylpyridin-3-yl)-1H-pyrazol-3-yl)amino)pyrazine-2-carboxynitrile (compound 1).

[0052] This article also discloses the crystalline form of the maleate salt of 5-((5-(4-(((1R,3S)-3-aminocyclopentyl)oxy)-2-methoxy-6-methylpyridin-3-yl)-1H-pyrazol-3-yl)amino)pyrazine-2-carboxynitrile (compound 1) or a pharmaceutically acceptable solvate thereof.

[0053] This article also discloses the crystalline form of the maleate of 5-((5-(4-(((1R,3S)-3-aminocyclopentyl)oxy)-2-methoxy-6-methylpyridin-3-yl)-1H-pyrazol-3-yl)amino)pyrazine-2-carboxynitrile (compound 1).

[0054] This article also discloses the fumarate of 5-((5-(4-(((1R,3S)-3-aminocyclopentyl)oxy)-2-methoxy-6-methylpyridin-3-yl)-1H-pyrazol-3-yl)amino)pyrazine-2-carboxynitrile (compound 1) or its pharmaceutically acceptable solvate in crystalline form.

[0055] This article also discloses the crystalline form of the fumarate of 5-((5-(4-(((1R,3S)-3-aminocyclopentyl)oxy)-2-methoxy-6-methylpyridin-3-yl)-1H-pyrazol-3-yl)amino)pyrazine-2-carboxynitrile (compound 1).

[0056] This article also discloses the crystalline form of the citrate of 5-((5-(4-(((1R,3S)-3-aminocyclopentyl)oxy)-2-methoxy-6-methylpyridin-3-yl)-1H-pyrazol-3-yl)amino)pyrazine-2-carboxynitrile (compound 1) or a pharmaceutically acceptable solvate thereof.

[0057] This article also discloses the crystalline form of the citrate of 5-((5-(4-(((1R,3S)-3-aminocyclopentyl)oxy)-2-methoxy-6-methylpyridin-3-yl)-1H-pyrazol-3-yl)amino)pyrazine-2-carboxynitrile (compound 1).

[0058] This article also discloses the crystalline form of the lactate of 5-((5-(4-(((1R,3S)-3-aminocyclopentyl)oxy)-2-methoxy-6-methylpyridin-3-yl)-1H-pyrazol-3-yl)amino)pyrazine-2-carboxynitrile (compound 1) or a pharmaceutically acceptable solvate thereof.

[0059] This article also discloses the crystalline form of the lactate of 5-((5-(4-(((1R,3S)-3-aminocyclopentyl)oxy)-2-methoxy-6-methylpyridin-3-yl)-1H-pyrazol-3-yl)amino)pyrazine-2-carboxynitrile (compound 1).

[0060] This article also discloses the crystalline form of hippurate or a pharmaceutically acceptable solvate thereof of 5-((5-(4-(((1R,3S)-3-aminocyclopentyl)oxy)-2-methoxy-6-methylpyridin-3-yl)-1H-pyrazol-3-yl)amino)pyrazine-2-carboxynitrile (compound 1).

[0061] This article also discloses the crystalline form of hippuric acid salt of 5-((5-(4-(((1R,3S)-3-aminocyclopentyl)oxy)-2-methoxy-6-methylpyridin-3-yl)-1H-pyrazol-3-yl)amino)pyrazine-2-carboxynitrile (compound 1).

[0062] This article also discloses the crystalline form of the sulfate of 5-((5-(4-(((1R,3S)-3-aminocyclopentyl)oxy)-2-methoxy-6-methylpyridin-3-yl)-1H-pyrazol-3-yl)amino)pyrazine-2-carboxynitrile (compound 1) or a pharmaceutically acceptable solvate thereof.

[0063] This article also discloses the crystalline form of the sulfate of 5-((5-(4-(((1R,3S)-3-aminocyclopentyl)oxy)-2-methoxy-6-methylpyridin-3-yl)-1H-pyrazol-3-yl)amino)pyrazine-2-carboxynitrile (compound 1).

[0064] Free alkali crystalline compound 1 form FB-1 In some embodiments, crystalline compound 1 is in the free base form FB-1, characterized by having at least one of the following properties: (a) and Figure 1 The X-ray powder diffraction (XRPD) patterns shown are essentially the same; (b) X-ray powder diffraction (XRPD) patterns with characteristic peaks at 11.96±0.1° 2θ, 18.81±0.1° 2θ, 19.91±0.1° 2θ, 21.67±0.1° 2θ and 26.75±0.1° 2θ; (c) X-ray powder diffraction (XRPD) patterns with characteristic peaks at 13.87 ±0.1° 2θ and 17.06 ±0.1° 2θ; (d) Differential scanning calorimetry (DSC) thermogram showing endothermic activity with a peak temperature of approximately 216.5 °C (initial temperature); (e) and Figure 2 The thermogravimetric analysis (TGA) chromatograms shown are essentially the same; (f) Thermogravimetric analysis (TGA) chromatogram showing a mass loss of approximately 0.10% from the initial heating temperature to approximately 100.0 °C; or (g) Its combination.

[0065] In some implementations, the crystal form has the same characteristics as... Figure 1 The X-ray powder diffraction (XRPD) patterns shown are essentially the same.

[0066] In some embodiments, the crystalline free alkali compound 1, in form FB-1, has an X-ray powder diffraction (XRPD) pattern with the characteristic peaks shown in Table 1.

[0067] In some embodiments, the X-ray powder diffraction (XRPD) pattern of the crystalline free alkali compound 1, in form FB-1, has characteristic peaks at 11.96 ± 0.1° 2θ, 18.81 ± 0.1° 2θ, 19.91 ± 0.1° 2θ, 21.67 ± 0.1° 2θ, and 26.75 ± 0.1° 2θ.

[0068] In some embodiments, the X-ray powder diffraction (XRPD) pattern of the crystalline free alkali compound 1, in form FB-1, has characteristic peaks at 13.87 ± 0.1° 2θ and 17.06 ± 0.1° 2θ.

[0069] In some embodiments, the X-ray powder diffraction (XRPD) pattern of the crystalline free alkali compound 1, in the form FB-1, has a characteristic peak at 13.87 ± 0.1° 2θ.

[0070] In some embodiments, the X-ray powder diffraction (XRPD) pattern of the crystalline free alkali compound 1, in the form FB-1, has a characteristic peak at 17.06 ± 0.1° 2θ.

[0071] In some implementations, the X-ray powder diffraction (XRPD) pattern also includes a peak at 5.27 ± 0.1° 2θ.

[0072] In some implementations, the X-ray powder diffraction (XRPD) pattern also includes a peak at 13.34 ± 0.1° 2θ.

[0073] In some implementations, the X-ray powder diffraction (XRPD) pattern also includes a peak at 15.66 ± 0.1° 2θ.

[0074] In some implementations, the X-ray powder diffraction (XRPD) pattern also includes a peak at 22.24 ± 0.1° 2θ.

[0075] In some implementations, the X-ray powder diffraction (XRPD) pattern also includes a peak at 27.84 ± 0.1° 2θ.

[0076] In some embodiments, the X-ray powder diffraction (XRPD) pattern of the crystalline free alkali compound 1, in the form FB-1, has a characteristic peak at 5.27 ± 0.1° 2θ.

[0077] In some embodiments, the X-ray powder diffraction (XRPD) pattern of the crystalline free alkali compound 1, in the form FB-1, has a characteristic peak at 11.96 ± 0.1° 2θ.

[0078] In some embodiments, the X-ray powder diffraction (XRPD) pattern of the crystalline free alkali compound 1, in form FB-1, has a characteristic peak at 13.34 ± 0.1° 2θ.

[0079] In some embodiments, the X-ray powder diffraction (XRPD) pattern of the crystalline free alkali compound 1, in the form FB-1, has a characteristic peak at 13.87 ± 0.1° 2θ.

[0080] In some embodiments, the X-ray powder diffraction (XRPD) pattern of the crystalline free alkali compound 1, in the form FB-1, has a characteristic peak at 15.66 ± 0.1° 2θ.

[0081] In some embodiments, the X-ray powder diffraction (XRPD) pattern of the crystalline free alkali compound 1, in the form FB-1, has a characteristic peak at 17.06 ± 0.1° 2θ.

[0082] In some embodiments, the X-ray powder diffraction (XRPD) pattern of the crystalline free alkali compound 1, in the form FB-1, has a characteristic peak at 18.81 ± 0.1° 2θ.

[0083] In some embodiments, the X-ray powder diffraction (XRPD) pattern of the crystalline free alkali compound 1, in form FB-1, has a characteristic peak at 19.91 ± 0.1° 2θ.

[0084] In some embodiments, the X-ray powder diffraction (XRPD) pattern of the crystalline free alkali compound 1, in the form FB-1, has a characteristic peak at 21.67 ± 0.1° 2θ.

[0085] In some embodiments, the X-ray powder diffraction (XRPD) pattern of the crystalline free alkali compound 1, in form FB-1, has a characteristic peak at 22.24 ± 0.1° 2θ.

[0086] In some embodiments, the X-ray powder diffraction (XRPD) pattern of the crystalline free alkali compound 1, in the form FB-1, has a characteristic peak at 26.75 ± 0.1° 2θ.

[0087] In some embodiments, the X-ray powder diffraction (XRPD) pattern of the crystalline free alkali compound 1, in the form FB-1, has a characteristic peak at 27.84 ± 0.1° 2θ.

[0088] In some embodiments, the X-ray powder diffraction (XRPD) pattern of the crystalline free alkali compound 1, in form FB-1, has characteristic peaks at 5.27±0.1° 2θ, 11.96±0.1° 2θ, 13.34±0.1° 2θ, 15.66±0.1° 2θ, 18.81±0.1° 2θ, 19.91±0.1° 2θ, 21.67±0.1° 2θ, 22.24±0.1° 2θ, 26.75±0.1° 2θ, and 27.84±0.1° 2θ.

[0089] In some embodiments, the X-ray powder diffraction (XRPD) pattern of the crystalline free alkali compound 1 in form FB-1 has at least two characteristic peaks selected from the following: 5.27±0.1° 2θ, 11.96±0.1° 2θ, 13.34±0.1° 2θ, 13.87±0.1° 2θ, 15.66±0.1° 2θ, 17.06±0.1° 2θ, 18.81±0.1° 2θ, 19.91±0.1° 2θ, 21.67±0.1° 2θ, 22.24±0.1° 2θ, 26.75±0.1° 2θ, and 27.84±0.1° 2θ.

[0090] In some embodiments, the X-ray powder diffraction (XRPD) pattern of the crystalline free alkali compound 1, in form FB-1, has at least three characteristic peaks selected from the following: 5.27±0.1° 2θ, 11.96±0.1° 2θ, 13.34±0.1° 2θ, 13.87±0.1° 2θ, 15.66±0.1° 2θ, 17.06±0.1° 2θ, 18.81±0.1° 2θ, 19.91±0.1° 2θ, 21.67±0.1° 2θ, 22.24±0.1° 2θ, 26.75±0.1° 2θ, and 27.84±0.1° 2θ.

[0091] In some embodiments, the X-ray powder diffraction (XRPD) pattern of the crystalline free alkali compound 1, in form FB-1, has at least four characteristic peaks selected from the following: 5.27±0.1° 2θ, 11.96±0.1° 2θ, 13.34±0.1° 2θ, 13.87±0.1° 2θ, 15.66±0.1° 2θ, 17.06±0.1° 2θ, 18.81±0.1° 2θ, 19.91±0.1° 2θ, 21.67±0.1° 2θ, 22.24±0.1° 2θ, 26.75±0.1° 2θ, and 27.84±0.1° 2θ.

[0092] In some embodiments, the X-ray powder diffraction (XRPD) pattern of the crystalline free alkali compound 1, in form FB-1, has at least five characteristic peaks selected from the following: 5.27±0.1° 2θ, 11.96±0.1° 2θ, 13.34±0.1° 2θ, 13.87±0.1° 2θ, 15.66±0.1° 2θ, 17.06±0.1° 2θ, 18.81±0.1° 2θ, 19.91±0.1° 2θ, 21.67±0.1° 2θ, 22.24±0.1° 2θ, 26.75±0.1° 2θ, and 27.84±0.1° 2θ.

[0093] In some embodiments, the X-ray powder diffraction (XRPD) pattern of the crystalline free alkali compound 1, in form FB-1, has at least six characteristic peaks selected from the following: 5.27±0.1° 2θ, 11.96±0.1° 2θ, 13.34±0.1° 2θ, 13.87±0.1° 2θ, 15.66±0.1° 2θ, 17.06±0.1° 2θ, 18.81±0.1° 2θ, 19.91±0.1° 2θ, 21.67±0.1° 2θ, 22.24±0.1° 2θ, 26.75±0.1° 2θ, and 27.84±0.1° 2θ.

[0094] In some embodiments, the X-ray powder diffraction (XRPD) pattern of the crystalline free alkali compound 1, in form FB-1, has at least seven characteristic peaks selected from the following: 5.27±0.1° 2θ, 11.96±0.1° 2θ, 13.34±0.1° 2θ, 13.87±0.1° 2θ, 15.66±0.1° 2θ, 17.06±0.1° 2θ, 18.81±0.1° 2θ, 19.91±0.1° 2θ, 21.67±0.1° 2θ, 22.24±0.1° 2θ, 26.75±0.1° 2θ, and 27.84±0.1° 2θ.

[0095] In some embodiments, the X-ray powder diffraction (XRPD) pattern of the crystalline free alkali compound 1, in form FB-1, has at least eight characteristic peaks selected from the following: 5.27±0.1° 2θ, 11.96±0.1° 2θ, 13.34±0.1° 2θ, 13.87±0.1° 2θ, 15.66±0.1° 2θ, 17.06±0.1° 2θ, 18.81±0.1° 2θ, 19.91±0.1° 2θ, 21.67±0.1° 2θ, 22.24±0.1° 2θ, 26.75±0.1° 2θ, and 27.84±0.1° 2θ.

[0096] In some embodiments, the X-ray powder diffraction (XRPD) pattern of the crystalline free alkali compound 1, in form FB-1, has at least nine characteristic peaks selected from the following: 5.27±0.1° 2θ, 11.96±0.1° 2θ, 13.34±0.1° 2θ, 13.87±0.1° 2θ, 15.66±0.1° 2θ, 17.06±0.1° 2θ, 18.81±0.1° 2θ, 19.91±0.1° 2θ, 21.67±0.1° 2θ, 22.24±0.1° 2θ, 26.75±0.1° 2θ, and 27.84±0.1° 2θ.

[0097] In some embodiments, the X-ray powder diffraction (XRPD) pattern of the crystalline free alkali compound 1, in form FB-1, has at least ten characteristic peaks selected from the following: 5.27±0.1° 2θ, 11.96±0.1° 2θ, 13.34±0.1° 2θ, 13.87±0.1° 2θ, 15.66±0.1° 2θ, 17.06±0.1° 2θ, 18.81±0.1° 2θ, 19.91±0.1° 2θ, 21.67±0.1° 2θ, 22.24±0.1° 2θ, 26.75±0.1° 2θ, and 27.84±0.1° 2θ.

[0098] In some embodiments, the X-ray powder diffraction (XRPD) pattern of the crystalline free alkali compound 1, in form FB-1, has at least 11 characteristic peaks selected from the following: 5.27±0.1° 2θ, 11.96±0.1° 2θ, 13.34±0.1° 2θ, 13.87±0.1° 2θ, 15.66±0.1° 2θ, 17.06±0.1° 2θ, 18.81±0.1° 2θ, 19.91±0.1° 2θ, 21.67±0.1° 2θ, 22.24±0.1° 2θ, 26.75±0.1° 2θ, and 27.84±0.1° 2θ.

[0099] In some embodiments, the differential scanning calorimetry (DSC) thermogram of the crystalline free alkali compound 1, in form FB-1, has an endothermic peak temperature of about 216.5 °C (initial temperature).

[0100] In some embodiments, the crystalline free alkali compound 1, in form FB-1, has the same properties as... Figure 2 The thermogravimetric analysis (TGA) chromatograms shown are essentially the same.

[0101] In some embodiments, the crystalline free alkali compound 1, in form FB-1, has a thermogravimetric analysis (TGA) chromatogram showing a mass loss of about 0.10% as the temperature rises from the initial heating temperature to about 100.0 °C.

[0102] In some implementations, the crystalline free alkali compound 1, in the form FB-1, is physically and chemically stable.

[0103] In some implementations, the crystalline free alkali compound 1, in the form FB-1, is chemically stable.

[0104] In some implementations, based on a four-week stability study conducted in an open-closed dish at 25 °C / 60%RH, the crystalline free alkali compound 1, in form FB-1, is chemically and physically stable.

[0105] In some implementations, based on a four-week stability study conducted in an open-closed dish at 40 °C / 75%RH, the crystalline free alkali compound 1, in form FB-1, is chemically and physically stable.

[0106] In some implementations, based on a four-week stability study conducted in an open-closed dish at 60 °C, the crystalline free alkali compound 1, in form FB-1, is chemically and physically stable.

[0107] In some embodiments, the crystalline free alkali compound 1, in the form FB-1, is highly crystalline.

[0108] In some embodiments, the crystalline free alkali compound 1, in the form FB-1, has a high melting point.

[0109] In some implementations, the crystalline free alkali compound 1, in the form FB-1, is non-hygroscopic.

[0110] In some implementations, the crystalline free alkali compound 1, in the form FB-1, is anhydrous.

[0111] Table 1: XRPD peaks of compound 1, form FB-1

[0112] Free alkali crystal compound 1 form FB-2 In some embodiments, crystalline compound 1 is in the free base form FB-2, characterized by having at least one of the following properties: (a) and Figure 3 The X-ray powder diffraction (XRPD) patterns shown are essentially the same. (b) X-ray powder diffraction (XRPD) pattern with characteristic peaks shown in Table 2; or (c) Its combination.

[0113] In some implementations, the crystal form has the same characteristics as... Figure 3 The X-ray powder diffraction (XRPD) patterns shown are essentially the same.

[0114] In some embodiments, the crystalline free alkali compound 1, in form FB-2, has an X-ray powder diffraction (XRPD) pattern with the characteristic peaks shown in Table 2.

[0115] Table 2: XRPD peaks of compound 1, form FB-2

[0116] Free alkali crystalline compound 1 form FB-3 In some embodiments, crystalline compound 1 is in the free base form FB-3, characterized by having at least one of the following properties: (a) and Figure 4 The X-ray powder diffraction (XRPD) patterns shown are essentially the same. (b) X-ray powder diffraction (XRPD) pattern with characteristic peaks shown in Table 3; or (c) Its combination.

[0117] In some implementations, the crystal form has the same characteristics as... Figure 4 The X-ray powder diffraction (XRPD) patterns shown are essentially the same.

[0118] In some embodiments, the crystalline free alkali compound 1, in form FB-3, has an X-ray powder diffraction (XRPD) pattern with the characteristic peaks shown in Table 3.

[0119] Table 3: XRPD peaks of compound 1, form FB-3

[0120] Free alkali crystalline compound 1 form FB-4 In some embodiments, crystalline compound 1 is in the free base form FB-4, characterized by having at least one of the following properties: (d) and Figure 5 The X-ray powder diffraction (XRPD) patterns shown are essentially the same. (e) X-ray powder diffraction (XRPD) pattern with characteristic peaks shown in Table 4; or (f) Its combination.

[0121] In some implementations, the crystal form has the same characteristics as... Figure 5 The X-ray powder diffraction (XRPD) patterns shown are essentially the same.

[0122] In some embodiments, the crystalline free alkali compound 1, in form FB-4, has an X-ray powder diffraction (XRPD) pattern with the characteristic peaks shown in Table 4.

[0123] Table 4: XRPD peaks of compound 1, form FB-4

[0124] Free alkali crystalline compound 1 form FB-5 In some embodiments, crystalline compound 1 is in the free base form FB-5, characterized by having at least one of the following properties: (g) and Figure 6 The X-ray powder diffraction (XRPD) patterns shown are essentially the same. (h) X-ray powder diffraction (XRPD) patterns with characteristic peaks shown in Table 5; or (i) Its combination.

[0125] In some implementations, the crystal form has the same characteristics as... Figure 6 The X-ray powder diffraction (XRPD) patterns shown are essentially the same.

[0126] In some embodiments, the crystalline free alkali compound 1, in form FB-5, has an X-ray powder diffraction (XRPD) pattern with the characteristic peaks shown in Table 5.

[0127] Table 5: XRPD peaks of compound 1, form FB-5 Pharmaceutical Composition

[0128] In some embodiments, the compounds described herein are administered in the form of pure chemicals. In some embodiments, the compounds described herein are combined with pharmaceutically suitable or acceptable carriers (also referred to herein as pharmaceutically suitable (or acceptable) excipients, physiologically suitable (or acceptable) excipients, or physiologically suitable (or acceptable) carriers), which are selected based on the chosen route of administration and standard pharmaceutical practice, as described, for example, in Remington: The Science and Practice of Pharmacy (Gennaro, 21st edition MackPub. Co., Easton, PA (2005)).

[0129] Therefore, this article provides a pharmaceutical composition comprising the compound described herein or a pharmaceutically acceptable salt, solvate, tautomer or stereoisomer thereof, and a pharmaceutically acceptable excipient.

[0130] Pharmaceutical compositions are administered in a manner suitable for the treatment (or prevention) of a disease. The appropriate dosage, as well as the appropriate duration and frequency of administration, will be determined by factors such as the patient's condition, the type and severity of the patient's disease, the specific form of the active ingredient, and the method of administration. Generally, an appropriate dosage and treatment regimen is provided in an amount sufficient to provide therapeutic and / or preventive benefits, such as improved clinical outcomes, such as increased overall response rate, increased duration of response, more frequent complete or partial remission, or longer disease-free and / or overall survival, or reduction in symptom severity. Optimal dosage is typically determined using experimental models and / or clinical trials. The optimal dosage depends on the patient's body mass, weight, or blood volume.

[0131] In some embodiments, the pharmaceutical composition is formulated for oral, topical (including buccal and sublingual), rectal, vaginal, percutaneous, parenteral, intrapulmonary, intradermal, intrathecal, epidural, or intranasal administration. Parenteral administration includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In some embodiments, the pharmaceutical composition is formulated for intravenous injection, oral administration, inhalation, nasal administration, topical administration, or ocular administration. In some embodiments, the pharmaceutical composition is formulated for oral administration. In some embodiments, the pharmaceutical composition is formulated for intravenous injection. In some embodiments, the pharmaceutical composition is formulated as tablets, pills, capsules, liquids, inhalers, nasal spray solutions, suppositories, suspensions, gels, colloids, dispersions, solutions, emulsions, ointments, lotions, eye drops, or ear drops. In some embodiments, the pharmaceutical composition is formulated as tablets.

[0132] The appropriate dosage and dosage regimen are determined using conventional range-finding techniques known to those skilled in the art. Typically, treatment is initiated with a smaller dose than the optimal dose of the compounds disclosed herein. Thereafter, the dose is increased in small increments until the optimal effect for the given situation is achieved. method

[0133] This document discloses a method for treating cancer in a subject of need, comprising administering to the subject a pharmaceutical composition comprising compound 1 or a pharmaceutically acceptable salt thereof. In some embodiments, the CHK1 inhibitor is compound 1 or a pharmaceutically acceptable salt thereof.

[0134] In some implementations of the methods disclosed herein, the object undergoes a therapeutic response.

[0135] In some embodiments of the methods disclosed herein, the treatment response comprises a reduction in the level of oncogene amplification in the tumor or tumor cells after treatment, compared to the level of oncogene amplification in the tumor or tumor cells before treatment.

[0136] In some embodiments of the methods disclosed herein, the treatment response includes a reduction in one or more of tumor growth, tumor size, number of tumor cells, or tumor metastasis compared to before treatment.

[0137] In some embodiments of the methods disclosed herein, the treatment response includes a treatment benefit. In some embodiments of the methods disclosed herein, the treatment benefit is stable disease (SD). In some embodiments of the methods disclosed herein, the treatment benefit is a partial response (PR). In some embodiments of the methods disclosed herein, the treatment benefit is a complete response (CR). In some embodiments of the methods disclosed herein, a complete response is determined by RECISTv1.1 (or RANO of GBM). In some embodiments of the methods disclosed herein, the treatment benefit is duration of response (DOR). In some embodiments of the methods disclosed herein, the treatment benefit is progression-free survival (PFS). In some embodiments of the methods disclosed herein, the treatment benefit is overall survival (OS).

[0138] In some implementations of the methods disclosed herein, the cancer includes solid tumors.

[0139] In some embodiments of the methods disclosed herein, the cancer comprises a locally advanced or metastatic unresectable solid tumor.

[0140] In some embodiments of the methods disclosed herein, the cancer comprises a tumor or tumor cells carrying oncogene amplification.

[0141] In some embodiments of the methods disclosed herein, oncogene amplification includes amplification of ABL, AKT1, AKT2, ALK, androgen receptor, BRAF, CCND1, CCND2, CCND3, CCNE1, CDK12, CDK4, CDK6, EGFR, ERBB2, EZH2, FGFR1, FGFR2, FGFR3, FLT3, IDH1 / 2, JAK2, JAK3, KIT, KRAS, MDM2, MDM4, MET, MYC, MYCL, MYCN, NRAS, PDGFRA, TERT, VEGFRA, or any combination thereof.

[0142] In some embodiments of the methods disclosed herein, the oncogene amplification comprises the amplification of FGFR1, FGFR2, FGFR3, or combinations thereof. In some embodiments of the methods disclosed herein, the oncogene amplification comprises the amplification of FGFR1, FGFR2, FGFR3, or combinations thereof, and the treatment further comprises the administration of an FGFR inhibitor.

[0143] In some embodiments of the methods disclosed herein, oncogene amplification includes CDK4, CDK6, EGFR, FGFR1, FGFR2, or FGFR3.

[0144] In some embodiments of the methods disclosed herein, the oncogene amplification comprises EGFR amplification. In some embodiments of the methods disclosed herein, the oncogene amplification comprises EGFR amplification, and the treatment further comprises administration of an EGFR inhibitor.

[0145] In some embodiments of the methods disclosed herein, the oncogene amplification comprises amplification of CDK4, CDK6, or a combination thereof. In some embodiments of the methods disclosed herein, the oncogene amplification comprises amplification of CDK4, CDK6, or a combination thereof, and the treatment further comprises administration of a CDK4 / 6 inhibitor.

[0146] In some embodiments of the methods disclosed herein, oncogene amplification is located on ecDNA.

[0147] In some embodiments of the methods disclosed herein, oncogene amplification is located at one or more chromosomal loci.

[0148] In some embodiments of the methods disclosed herein, oncogene amplification is ecDNA-derived amplification.

[0149] In some embodiments of the methods disclosed herein, the oncogene amplification has a copy number of at least 6, at least 8, at least 10, at least 15, at least 20, or more than 20 copies of the oncogene or a portion thereof.

[0150] In some embodiments of the methods disclosed herein, the cancer comprises a malignant tumor whose size may be reduced, whose growth or spread may be slowed or stopped, or whose symptoms are in remission or alleviated, reduced, and / or completely cured by the absence or inhibition of CHK1 function. Target malignant tumors are, but are not limited to, head and neck cancer, gastrointestinal cancer (esophageal cancer, gastric cancer, duodenal cancer, liver cancer, biliary tract cancer (gallbladder cancer, bile duct cancer, etc.), pancreatic cancer, colorectal cancer (colon cancer, rectal cancer, etc.), lung cancer (non-small cell lung cancer, small cell lung cancer, squamous cell lung cancer, lung adenocarcinoma, mesothelioma, etc.), breast cancer, genital cancer (ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, etc.), urinary tract cancer (kidney cancer, bladder cancer, prostate cancer, testicular tumors, etc.), hematopoietic system tumors (leukemia, malignant lymphoma, multiple myeloma, etc.), bone and soft tissue tumors (e.g., soft tissue sarcoma, liposarcoma, and osteosarcoma), skin cancer, and brain tumors (e.g., glioblastoma).

[0151] In some embodiments of the methods disclosed herein, the term cancer is used in accordance with its ordinary meaning and refers to all types of cancer, growths, or malignant tumors found in mammals, including leukemia, lymphoma, melanoma, neuroendocrine tumors, epithelial carcinoma, and sarcoma. Exemplary cancers that can be treated with the compounds disclosed herein or their pharmaceutically acceptable salts, solvates, tautomers, or stereoisomers, or pharmaceutical compositions include acute myeloid leukemia, adrenocortical carcinoma, adrenal carcinoma, bladder cancer, bone cancer, brain cancer, breast cancer (e.g., ductal carcinoma, lobular carcinoma, primary, metastatic), breast cancer, endocrine system cancers, hepatic stellate cell carcinoma, pancreatic stellate cell carcinoma, cervical cancer, colon cancer, colorectal cancer, ductal carcinoma, endometrial cancer, esophageal cancer, gastric cancer, genitourinary tract cancer, glioblastoma, glioma, head and neck cancer, hepatocellular carcinoma, and Hodgkin's disease. Diseases including: kidney cancer, leukemia (e.g., lymphoblastic leukemia, chronic lymphocytic leukemia, hairy cell leukemia), liver cancer (e.g., hepatocellular carcinoma), lobular carcinoma, lung cancer (e.g., non-small cell lung cancer, squamous cell lung cancer, adenocarcinoma, large cell lung cancer, small cell lung cancer, carcinoid, sarcoma), liposarcoma, lymph node carcinoma, lymphoma (e.g., mantle cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, marginal zone lymphoma, Burkitt's lymphoma, non-Hodgkin's lymphoma), malignant carcinoid, malignant hypercalcemia, malignant pancreatic insulinoma, medullary thyroid carcinoma, medulloblastoma, melanoma, mesothelioma, multiple myeloma, myocardial carcinoma, endocrine or exocrine pancreatic vegetations, neuroblastoma, ovarian cancer, and Paget's disease of the nipple. The cancers selected include: pancreatic cancer, papillary thyroid cancer, phyllodes tumor, premalignant skin lesions, primary thrombocytosis, prostate cancer (e.g., castration-resistant prostate cancer), kidney cancer, rhabdomyosarcoma, salivary gland cancer, sarcoma, soft tissue sarcoma, squamous cell carcinoma (e.g., head, neck, or esophagus), gastric cancer, testicular cancer, thyroid cancer, bladder cancer, or uterine cancer. In the implementation scheme, the cancers are selected from bladder cancer, breast cancer, colon cancer, esophageal cancer, glioblastoma, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, salivary gland cancer, soft tissue sarcoma, squamous cell lung cancer, gastric cancer, and uterine cancer.

[0152] In some embodiments of the methods disclosed herein, the cancer is ovarian cancer. In some embodiments of the methods disclosed herein, the ovarian cancer is platinum-resistant high-grade serous ovarian cancer, primary peritoneal cancer, or fallopian tube cancer.

[0153] In some embodiments of the methods disclosed herein, the cancer is uterine cancer. In some embodiments of the methods disclosed herein, the uterine cancer is high-grade endometrial cancer, serous uterine carcinoma, or carcinosarcoma of the uterus.

[0154] In some embodiments of the methods disclosed herein, the cancer is glioblastoma or neuroblastoma.

[0155] In some embodiments of the methods disclosed herein, the cancer is colorectal cancer, esophageal cancer, gastric cancer, gastroesophageal junction (GEJ) cancer, head and neck squamous cell carcinoma, liposarcoma, non-small cell lung cancer, or a subtype of squamous cell carcinoma.

[0156] In some embodiments of the methods disclosed herein, the cancer is breast cancer or head, esophageal cancer, cervical squamous cell carcinoma, non-small cell lung cancer, gastric cancer, or a subtype of squamous cell carcinoma.

[0157] In some embodiments of the methods disclosed herein, the cancer is esophageal cancer, non-small cell lung cancer, sarcoma, or gastric cancer. In some embodiments of the methods disclosed herein, the cancer is esophageal cancer. In some embodiments of the methods disclosed herein, the cancer is non-small cell lung cancer. In some embodiments of the methods disclosed herein, the cancer is sarcoma. In some embodiments of the methods disclosed herein, the cancer is gastric cancer.

[0158] In some implementations of the methods disclosed herein, the subject has received one or more prior treatments.

[0159] In some implementations of the methods disclosed herein, the subject is unresponsive to one or more prior therapies.

[0160] In some implementations of the methods disclosed herein, the subject has developed resistance to one or more prior therapies.

[0161] In some embodiments of the methods disclosed herein, one or more prior therapies are chemotherapy.

[0162] In some embodiments of the methods disclosed herein, one or more prior therapies are PD1 antibodies.

[0163] In some embodiments of the methods disclosed herein, one or more prior therapies are PD-L1 antibodies.

[0164] In some embodiments of the methods disclosed herein, one or more prior therapies are CTLA4 checkpoint inhibitors.

[0165] In some embodiments of the methods disclosed herein, one or more prior therapies are VEGF-targeted therapies (e.g., bevacizumab for ovarian cancer).

[0166] This article discloses a method for treating cancer in a subject of need, comprising administering to the subject a pharmaceutical composition comprising compound 1 or a pharmaceutically acceptable salt thereof, wherein the method further comprises obtaining a diagnostic indicator of oncogene amplification from a biological sample of the subject.

[0167] In some embodiments of the methods disclosed herein, the diagnostic indicator is obtained prior to the first administration of compound 1 or a pharmaceutically acceptable salt thereof.

[0168] In some embodiments of the methods disclosed herein, the diagnostic indicator is obtained after the first administration of compound 1 or a pharmaceutically acceptable salt thereof.

[0169] In some embodiments of the methods disclosed herein, the diagnostic indicator is obtained after multiple administrations of compound 1 or a pharmaceutically acceptable salt thereof.

[0170] In some embodiments of the methods disclosed herein, diagnostic indicators are derived from next-generation sequencing (NGS)-based assays.

[0171] In some embodiments of the methods disclosed herein, the diagnostic indicator is derived from fluorescence in situ hybridization (FISH) assay.

[0172] In some embodiments of the methods disclosed herein, the diagnostic indicator comprises an indicator for ecDNA-derived oncogene amplification.

[0173] In some embodiments of the methods disclosed herein, the diagnostic indicator is obtained from a tumor or liquid biopsy.

[0174] In some embodiments of the method disclosed herein, the method further includes assessing the presence or level of one or more of the following features in a sample from a subject: gene amplification, focal gene amplification, ecDNA, HSR, or ecDNA.

[0175] In some embodiments of the method disclosed herein, the method further includes obtaining information on the presence or level of one or more of the following in the tumor or tumor cells of the subject, before, during, or after the administration of compound 1 or a pharmaceutically acceptable salt thereof: gene amplification, focal gene amplification, ecDNA, HSR, or ecDNA characteristics.

[0176] Oncogene amplification Oncogene amplification-associated tumors represent a highly unmet need within the cancer population. Patients with cancers carrying high-copy-count oncogene amplifications have significantly reduced survival rates compared to the broader cancer population. Pan-cancer analyses of oncogene amplification tumors, cross-referenced with data from the Surveillance, Epidemiology, and End Results projects, indicate that in the United States alone, this population represents more than 400,000 newly diagnosed cancer patients annually, encompassing a wide range of tumor types.

[0177] While precision medicine and targeted therapies offer significant health benefits and survival improvements for cancer patients, unfortunately, most of these therapies have proven ineffective in oncogene amplification (APA) populations. Furthermore, immune checkpoint inhibitors (such as pembrolizumab) may perform poorly in APA-affected cancer populations, and excessive progression is associated with the tumor environment of APA.

[0178] To date, HER2 inhibitors (such as trastuzumab) targeting HER2-overexpressing breast cancer, gastroesophageal junction cancer, and gastric cancer are the only approved targeted therapies in the oncogene amplification (or overexpression) cancer population, with breast cancer being the only single-agent approved. Targeted therapies that have shown efficacy in patients whose cancers are driven by oncogene point mutations, gene fusions, or skipping deletions often fail to demonstrate strong efficacy in patients whose tumors are driven by oncogene amplification. Despite extensive clinical trials of targeted therapies in the oncogene amplification cancer population, including EGFR inhibitors in EGFR-amplified glioblastoma multiforme, FGFR inhibitors in FGFR-amplified cancers, and CDK4 / 6 inhibitors in CDK4-amplified liposarcoma, no approved therapies remain. These clinical data have led to the misconception that oncogene amplification may not be a relevant cancer driver. This erroneous conclusion persists despite substantial data contradicting this. Recurrent focal copy number amplification and overexpression of established oncogene drivers (which could otherwise be activated by mutations and / or gene fusions), along with the antitumor efficacy of targeted inhibition (genetic and pharmacological) in short-term preclinical cancer models, suggest that amplification is a driving factor, even though targeted therapies often fail to translate into long-term clinical benefits. However, this disconnect indicates that cancers driven by oncogene amplification are biologically distinct from other tumors and require novel treatment modalities. Therefore, it is necessary to improve our understanding of the biology of oncogene amplification, with the goal of advancing new therapies, drug targets, and novel molecular entities for this highly unmet patient population.

[0179] Role of extrachromosomal DNA in oncogene amplification Chromosomal instability and tumor heterogeneity are considered reasons for the failure of many targeted therapies. Consistent with this hypothesis, oncogene amplification is a result of prior or persistent chromosomal instability caused by numerical and / or structural alterations in chromosomes, and can generate ecDNA. It has long been recognized that oncogenes can amplify not only on chromosomes but also on ecDNA (originally called "double microsomes"). However, until recently, the frequency, importance, and specific role of ecDNA in cancer biology have only been fully understood.

[0180] Some of the most common driver oncogenes are encoded on ecDNA and confer selective advantage on cancer cells. These oncogenes amplified on ecDNA have several characteristics that distinguish them from oncogene amplifications located on chromosomes: 1. Because ecDNA lacks a centromere, it segregates unevenly into daughter cells during cell division, contrary to its amplified state of chromosomal localization. This characteristic supports a non-Mendelian inheritance pattern, enabling extreme gene copy number changes in relatively few cell divisions and leading to widespread copy number heterogeneity, thereby driving adaptation and tumor evolution.

[0181] 2. ecDNA is epigenetically dysregulated and contains accessible chromatin and supertranscribed gene regions, which are typically more actively expressed than genes located on chromosomes.

[0182] These characteristics distinguish ecDNA from other forms of oncogene amplification and promote genomic plasticity and adaptive levels beyond chromosomal amplification, enabling tumors to evade environmental damage, including the pressure of targeted therapy. Novel therapies that interfere with ecDNA function are necessary to overcome ecDNA-mediated adaptation in tumors with oncogene amplification.

[0183] ecDNA-activated oncogene amplification is a major driver of tumorigenesis, playing a crucial role in driving tumor heterogeneity and enabling cancer cells to rapidly develop resistance to targeted oncogene therapies. ecDNA-activated oncogene amplification has been observed in nearly half of all human cancer types, but is almost never found in normal cells. For example, ecDNA-activated oncogene amplification is found in approximately 14% of primary cancer samples, and more than half of high copy number oncogene amplifications (i.e., copy number > 8) are located on ecDNA. Furthermore, many of the most aggressive tumor types contain the highest incidence of ecDNA, including approximately 60% of glioblastoma multiforme and slightly less than 50% of sarcomas.

[0184] Even when tumor type is controlled, patients whose cancer carries ecDNA experience significantly shorter survival than those whose cancers are driven by other molecular pathologies. These data strongly suggest that a new treatment modality is needed to address this significant unmet need for patients with cancers that have ecDNA activation.

[0185] Role of extrachromosomal DNA in therapy resistance The unique properties of ecDNA, coupled with the significant genomic plasticity activated by ecDNA in tumors, contribute to tumor invasiveness and the ability to evade treatment pressure through rapid genomic evolution. The first evidence of ecDNA-driven treatment resistance was found in mouse cancer cell lines where methotrexate treatment led to high amplification of dihydrofolate reductase (DHFR) on ecDNA, which disappeared upon removal of methotrexate. Similar instances of DHFR ecDNA amplification have been reproduced in various human cancer cell lines. Furthermore, amplification of drug efflux pump genes (including the ABC transporter family) on ecDNA has been observed to mediate resistance to various chemotherapy regimens. The equivalent role of ecDNA in providing resistance to more current targeted therapies has also been well-established. Rapid loss of the EGFRvIII amplified population on ecDNA in patient-derived glioblastoma multiforme cells facilitated evasion of the EGFR inhibitor erlotinib, while a new cell population containing MDM2 amplification appeared on the ecDNA; this observed effect is consistent with the lack of equivalent response to EGFR inhibitors in patients. Preclinical studies in gastric cancer cell lines containing FGFR2 amplification on ecDNA suggest that cell resistance to the pan-FGFR inhibitor infigratinib may be driven by an oncogene-dependent conversion from FGFR2 amplification on ecDNA to rapid amplification of novel EGFR on ecDNA. Surprisingly, this dependence reverses back to FGFR2 amplification on ecDNA under EGFR-inhibiting pressure via erlotinib. In each case, the initial cell population is sensitive to the corresponding targeted therapy, resulting in a transient antiproliferative effect lasting several weeks. Resistance to targeted therapy and regeneration occur simultaneously with the conversion of oncogenes amplified on ecDNA. The rapid rate of amplification change is unique to ecDNA and helps explain the inherent targeted therapy resistance in cancers with newly amplified oncogenes.

[0186] Similarly, mutated oncogenes (such as BRAFV600E and KRASG12C) can amplify on ecDNA as a resistance mechanism to corresponding targeted therapies (such as BRAF / MEK or KRAS inhibitors). For example, mutated BRAFV600E melanoma cell lines exhibited ecDNA-activated BRAFV600E amplification after exposure to dual BRAF / MEK inhibition. This phenomenon has also been documented in clinical cases. Relatedly, several putative acquired resistance mechanisms to the KRASG12C inhibitor adagrasib have been reported, among which high-level focal amplification of KRASG12C on ecDNA, confirmed in vitro and in vivo, confers resistance to two clinically validated KRASG12C inhibitors, adagrasib and sotorasib.

[0187] In summary, these studies highlight remarkable genomic plasticity and a dramatic increase in ecDNA-activated oncogene amplification, enabling cancer cells to rapidly adapt to therapeutic stress. Ultimately, cancers driven by ecDNA-activated oncogene amplification are biologically distinct from other oncogene-activated tumors and require novel therapeutic modalities. Dosage / Administration

[0188] This document discloses a method for treating cancer in a subject, comprising administering to the subject a pharmaceutical composition comprising compound 1 or a pharmaceutically acceptable salt thereof. In some embodiments, the pharmaceutical composition comprising compound 1 comprises a crystalline form of compound 1, a crystalline free base form of compound 1, or a crystalline anhydrous free base form of compound 1. In some cases, such forms of compound 1 have X-ray powder diffraction (XRPD) patterns, differential scanning calorimetry (DSC) thermography, thermogravimetric analysis (TGA) thermography, or any combination thereof, as described herein.

[0189] The principle of the initial human dose of compound 1 GLP-compliant repeated-dose toxicology studies were conducted in rats and dogs with oral intensive Q2D administration for up to 29 days. The major effects observed in both species were associated with myelosuppression / depletion and gastrointestinal toxicity and were considered to be on-target pharmacological effects of compound 1 directly related to CHK1 inhibition. Partial or complete reversibility of the major changes associated with compound 1 were confirmed in all tissues, and no major unexpected toxicities were identified.

[0190] In some embodiments of the methods disclosed herein, compound 1 is administered at a dose of about 10 mg to about 800 mg.

[0191] In some embodiments of the methods disclosed herein, the dosage of compound 1 is from about 10 mg to about 400 mg.

[0192] In some embodiments of the methods disclosed herein, compound 1 is administered at a dose of about 10 mg to about 400 mg, thereby causing the subject to experience a therapeutic response.

[0193] In some embodiments of the method disclosed herein, the dosage of compound 1 is between about 10 mg to about 20 mg, about 20 mg to about 40 mg, about 40 mg to about 80 mg, about 80 mg to about 120 mg, about 120 mg to about 160 mg, about 160 mg to about 200 mg, and about 200 mg to about 400 mg.

[0194] In some embodiments of the methods disclosed herein, the dose of compound 1 is between about 20 to about 40 mg, about 40 to about 80 mg, about 80 to about 120 mg, about 120 mg to about 160 mg, or about 160 mg to about 200 mg.

[0195] In some embodiments of the methods disclosed herein, the dose of compound 1 is between about 10 mg to about 20 mg, about 20 mg to about 40 mg, about 40 mg to about 80 mg, or about 80 mg to about 120 mg.

[0196] In some embodiments of the methods disclosed herein, the dose of compound 1 is between about 10 mg and about 20 mg. In some embodiments of the methods disclosed herein, the dose of compound 1 is between about 20 mg and about 40 mg. In some embodiments of the methods disclosed herein, the dose of compound 1 is between about 40 mg and about 80 mg. In some embodiments of the methods disclosed herein, the dose of compound 1 is between about 80 mg and about 120 mg. In some embodiments of the methods disclosed herein, the dose of compound 1 is between about 120 mg and about 160 mg. In some embodiments of the methods disclosed herein, the dose of compound 1 is between about 160 mg and about 200 mg. In some embodiments of the methods disclosed herein, the dose of compound 1 is between about 200 mg and about 400 mg.

[0197] In some embodiments of the methods disclosed herein, the dose of compound 1 is about 10 mg, about 20 mg, about 40 mg, about 80 mg, about 120 mg, about 160 mg, or about 200 mg. In some embodiments of the methods disclosed herein, the dose of compound 1 is about 10 mg. In some embodiments of the methods disclosed herein, the dose of compound 1 is about 15 mg. In some embodiments of the methods disclosed herein, the dose of compound 1 is about 20 mg. In some embodiments of the methods disclosed herein, the dose of compound 1 is about 25 mg. In some embodiments of the methods disclosed herein, the dose of compound 1 is about 30 mg. In some embodiments of the methods disclosed herein, the dose of compound 1 is about 35 mg. In some embodiments of the methods disclosed herein, the dose of compound 1 is about 40 mg. In some embodiments of the methods disclosed herein, the dose of compound 1 is about 45 mg. In some embodiments of the methods disclosed herein, the dose of compound 1 is about 50 mg. In some embodiments of the methods disclosed herein, the dose of compound 1 is about 55 mg. In some embodiments of the methods disclosed herein, the dose of compound 1 is about 60 mg. In some embodiments of the methods disclosed herein, the dose of compound 1 is about 65 mg. In some embodiments of the methods disclosed herein, the dose of compound 1 is about 70 mg. In some embodiments of the methods disclosed herein, the dose of compound 1 is about 75 mg. In some embodiments of the methods disclosed herein, the dose of compound 1 is about 80 mg. In some embodiments of the methods disclosed herein, the dose of compound 1 is about 85 mg. In some embodiments of the methods disclosed herein, the dose of compound 1 is about 90 mg. In some embodiments of the methods disclosed herein, the dose of compound 1 is about 95 mg. In some embodiments of the methods disclosed herein, the dose of compound 1 is about 100 mg. In some embodiments of the methods disclosed herein, the dose of compound 1 is about 105 mg. In some embodiments of the methods disclosed herein, the dose of compound 1 is about 110 mg. In some embodiments of the methods disclosed herein, the dose of compound 1 is about 115 mg. In some embodiments of the methods disclosed herein, the dose of compound 1 is about 120 mg. In some embodiments of the methods disclosed herein, the dose of compound 1 is about 125 mg. In some embodiments of the methods disclosed herein, the dose of compound 1 is about 130 mg. In some embodiments of the methods disclosed herein, the dose of compound 1 is about 135 mg. In some embodiments of the methods disclosed herein, the dose of compound 1 is about 140 mg. In some embodiments of the methods disclosed herein, the dose of compound 1 is about 145 mg.In some embodiments of the methods disclosed herein, the dose of compound 1 is about 150 mg. In some embodiments of the methods disclosed herein, the dose of compound 1 is about 155 mg. In some embodiments of the methods disclosed herein, the dose of compound 1 is about 160 mg. In some embodiments of the methods disclosed herein, the dose of compound 1 is about 165 mg. In some embodiments of the methods disclosed herein, the dose of compound 1 is about 170 mg. In some embodiments of the methods disclosed herein, the dose of compound 1 is about 175 mg. In some embodiments of the methods disclosed herein, the dose of compound 1 is about 180 mg. In some embodiments of the methods disclosed herein, the dose of compound 1 is about 185 mg. In some embodiments of the methods disclosed herein, the dose of compound 1 is about 190 mg. In some embodiments of the methods disclosed herein, the dose of compound 1 is about 195 mg. In some embodiments of the methods disclosed herein, the dose of compound 1 is about 200 mg.

[0198] In some embodiments of the methods disclosed herein, the composition is administered orally.

[0199] In some embodiments of the methods disclosed herein, the composition is administered parenterally.

[0200] In some embodiments of the method disclosed herein, the composition is applied daily.

[0201] In some embodiments of the method disclosed herein, the composition is applied every other day.

[0202] In some embodiments of the method disclosed herein, the composition is administered on a cycle of day 1 and day 3, followed by a 4-day dosing break.

[0203] In some embodiments of the method disclosed herein, the composition is applied once every 3 days or weekly.

[0204] In some embodiments of the method disclosed herein, the composition is applied once every 3 days.

[0205] In some embodiments of the method disclosed herein, the composition is applied once a week.

[0206] In some embodiments of the method disclosed herein, the composition is administered with a dosing holiday of 4 days, 4-7 days, 7 days, or 14 days. combination

[0207] This document discloses a method for treating cancer in a subject of need, comprising administering to the subject a pharmaceutical composition comprising compound 1 or a pharmaceutically acceptable salt thereof and an additional therapeutic agent. In some embodiments, the CHK1 inhibitor is compound 1 or a pharmaceutically acceptable salt thereof. In some embodiments, the pharmaceutical composition comprising compound 1 comprises a crystalline form of compound 1, a crystalline free base form of compound 1, or a crystalline anhydrous free base form of compound 1. In some cases, such forms of compound 1 have X-ray powder diffraction (XRPD) patterns, differential scanning calorimetry (DSC) thermography, thermogravimetric analysis (TGA) thermography, or any combination thereof, as described herein.

[0208] In some embodiments of the methods disclosed herein, the additional therapeutic agent is an EGFR inhibitor.

[0209] In some embodiments of the methods disclosed herein, the EGFR inhibitor is selected from 602, 705, 707, abivertinib, ABX-900, afatinib, agerafenib (RXDX-105), alflutinib mesylate, amivantamab, APL-1898, ASK-120067, aumoletinib / almonertinib, BBT-176, BDTX-1535, BDTX-189, BEBT-109, and befortinib mesylate. mesylate), beitatini, BLU-701, BLU-945, BPI-361175, BPI-7711, BPI-D0316, C-005, CDP1, cetuximab, CH-7233163, CK-101, CMAB-017, dacomitinib, depatuxizumab, martin-depatuxizumab Mafodotin (ABT-414), DFP-17729, Dositinib, DS-2087, DZD-9008, E01001, E-10C, Epertinib, Epitinib (HMPL-813), Erlotinib, ES-072, FCN-411, FHND-9041, Furmonertinib, FWD-1509, GB-263, GC-1118A, Gefitinib GMA-204, GR-1401, Hemay-022, HLX-07, HS-627, I-010, icotinib, imgatuzumab, IN-A008, JMT-101, JRF-103, JS-111, JS-113, JZB-28, KN-023, KN-026, KP-673, lapatinib, larotinib, lazertinib, LL-191, LYN 205, M1231, maihuatinib, marizomib, mobocertinib, MP-0274, MRG003, naputinib (tosylate)tosilate, nazartinib, necitumumab, neptinib, nimotuzumab, NRC-2694-A, NT-004, OBX1-012, olafertinib, olmutinib, ORIC-114, oritinib, osimertinib, panitumumab, pirotinib, poziotinib, PRB-0 01. Pyrotinib, QL-1203, SCT-200, serclutamab, SHR-A1307, SIM-200, SPH-1188, SSGJ-612, SYN-004, TAD-011, tarloxotinib, TAS-6417, TGRX-360, theliatinib (HMPL-309), TPC-064, TQB-3804, TY-9591, WJ-13404, WSD-0922, XZP-5809, yinlitinib maleate, YK-029A, YZJ-0318, zorifertinib, and ZSP-0391.

[0210] In some embodiments of the methods disclosed herein, the EGFR inhibitor is erlotinib. Erlotinib (TARCEVA) Ò Erlotinib is an oral small molecule inhibitor of the receptor tyrosine kinase EGFR. Early data showed that erlotinib has anticancer activity in a variety of tumors, including lung and pancreatic cancer, and it was approved by the FDA in 2013 for EGFR-mutant NSCLC with EGFR exon 19 deletion or exon 21 substitution mutations. Notably, erlotinib can inhibit wild-type EGFR and is not selective only for mutant EGFR.

[0211] The planned dose of erlotinib is 150 mg orally once daily, administered >1 hour before or 2 hours after a meal. This is the dosage of erlotinib for the treatment of NSCLC according to the Tarceva® United States Prescribing Information (USPI).

[0212] In some embodiments of the methods disclosed herein, erlotinib is administered to the subject at a dose of 150 mg PO, 100 mg PO, or 50 mg PO per day.

[0213] In some embodiments of the methods disclosed herein, the additional therapeutic agent is an FGFR inhibitor.

[0214] In some embodiments of the methods disclosed herein, the FGFR inhibitor is selected from 3D-185, ABSK-011, ABSK-012, ABSK-061, ABSK-091, aldafermin, alofanib, AST-56100, AZD-4547, bemarituzumab, BFKB-8488A, BGS-2219, BIO-1262, BPI-17509, BPI-43487, CPL-304-110, derazantinib, E-7090, erdafitinib, EVER-4010001, and EVT-601. FGF-401, fisogatinib, FPI-1966, fabatinib, gunagratinib, H3B-6527, HH-185, HMPL-453, HS-236, ICP-105, ICP-192, Infiglioli, JAB-6000, KIN-3248, M-6123, MAX-40279, OM-RCA-001, pemitinib, RLY-4008, rogaratinib, SAR-439115, SAR-442501, SC-0011, SY-4798, TT-00434, zoligratinib (FF-284), and WXSH-0011.

[0215] In some embodiments of the methods disclosed herein, the FGFR inhibitor is pemetinib. Pemetinib (PEMAZYRE) ® Pemitinib is an oral small molecule inhibitor of the receptor tyrosine kinase FGFR. It was first approved by the FDA in 2020 for previously treated, unresectable locally advanced or metastatic cholangiocarcinoma with FGFR2 fusions or other rearrangements as detected by FDA-approved tests. Notably, pemitinib inhibits wild-type FGFR1, FGFR2, and FGFR3 receptors.

[0216] In some embodiments of the methods disclosed herein, pemitinib is administered to subjects at doses of 13.5 mg PO, 9 mg PO, and 4.5 mg PO daily, once daily for 14 days, followed by 7 days without pemitinib administration.

[0217] In some implementations of the methods disclosed herein, the FGFR inhibitor is fobatinib.

[0218] Fabatinib (LYTGOBI®) is indicated for the treatment of adults with previously treated, unresectable, locally advanced, or metastatic intrahepatic cholangiocarcinoma carrying fibroblast growth factor receptor 2 (FGFR2) gene fusions or other rearrangements. Fabatinib was approved for medical use in the United States in September 2022.

[0219] In some embodiments of the methods disclosed herein, fobatinib is administered to the subject at a daily dose of 20 mg PO.

[0220] In some implementations of the methods disclosed herein, the additional therapeutic agent is a CDK4 / 6 inhibitor.

[0221] In some embodiments of the methods disclosed herein, the CDK4 / 6 inhibitor is selected from abexicillin, AG-122275, AM-5992, AT-7519, AU2-94, auceliciclib, BEBT-209, BPI-1178, BPI-16350, CS-3002, fascaplysin, FCN-437, FN-1501, GLR-2007, GW-491619, HEC-80797, HS-10342, IIIM-290, IIIM-985, lerociclib, and milciclib maleate. maleate), MM-D37K, MS-140, NP-102, NUV-422, ON-123300, palbociclib, PF-06842874, PF-06873600, PF-07220060, QHRD-110, R-547, RGB-286199, RGT-419B, ribociclib, riviciclib, RO-0505124, SHR-6390, THR-53, THR-79, TQB-3303, TQB-3616, trilaciclib, TY-302, TY-302, voruciclib, VS2-370, WXWH-0240, XH-30002 and XZP-3287.

[0222] In some embodiments of the methods disclosed herein, the CDK4 / 6 inhibitor is abecicilline. Abecicilline (VERZENIO) Ò Abecib is an oral small molecule inhibitor of CDK4 / 6. It was first approved by the FDA in 2017 for hormone receptor-positive and HER-2-negative advanced or metastatic breast cancer. Notably, abecib inhibits both wild-type CDK4 and CDK6 receptors.

[0223] In some embodiments of the methods disclosed herein, abexilide is administered to the subject at a dose of about 50 mg twice daily, about 100 mg twice daily, or 150 mg twice daily.

[0224] In some embodiments of the methods disclosed herein, the additional therapeutic agent is a BRAF inhibitor. In some embodiments of the methods disclosed herein, the BRAF inhibitor is ABM-1310, agorafenib (RXDX-105), ARQ-736, ASN-003, AZ-304, AZ-628, BAL-3833, bevacirafenib, BGB-3245, BI-882370, dabrafenib, DAY101, DP-2874, EBI-907, EBI-945. Encorafenib, GDC-0879, lifirafenib, LUT-014, LYN204, NMS-P285, NMS-P730, PF-04880594, PF-07284890, PLX-8394, RX-208, TL-241, UAI-201, UB-941, vemurafenib, VS-6766, or XL-281.

[0225] In some embodiments of the methods disclosed herein, the additional therapeutic agent is an MDM2 or MDM4 inhibitor.

[0226] In some embodiments of the methods disclosed herein, the MDM2 inhibitors are AD-021.32, ALRN-6924, APG-115, ASTX-295, ATSP-7041, BI-907828, CGM-097, CYC700, DS-5272, idasanutlin, KRT-232 (AMG-232), MD-224, MI-1061, MI-219, MI-43, and MI-77301 (SAR405838, SAR299155). MK-8242, NU-8231, NVP-CGM097, OM-301, PXN-527, RAIN-32 (milademetan), RG7112 (RO5045337), RG7388 (RG7775), Rigel-3, RO-2468, RO-5353, RO-5963, serdemetan (JNJ-26854165), SIL-43, siremadlin, or UBX-0101. In some embodiments of the methods disclosed herein, the MDM4 inhibitor is 17AAG, 489-PXN, ALRN-6924, APG-115, ATSP-7041, BI-907828, CTX1, FL-118, inulanolide A, K-178, or SAH-p53-8.

[0227] In some embodiments of the methods disclosed herein, the additional therapeutic agent is a MET inhibitor.

[0228] In some embodiments of the methods disclosed herein, the MET inhibitors are ABP-1130, BPI-1831, BPI-2021, BYON-3521, CG-203306, CX-1003, Debio-1144, EMD-94283, EMT-100, EMT-101, HE-003, LMV-12, LS-177, NX-125, OMO-2, PF-4254644, PR X-MET, PTX-2173, QBH-196, RP-1400, SAB-Y14, SAR-125844, SGX-126, SYD-3521, WXSH-0011, X-379 and XL-265, as well as anti-MET antibodies (such as ABX-900, GB-263, FS-101, LY-3164530, LY-3343544, PMC-002 or SAIT-301). In some embodiments of the methods disclosed herein, the MET inhibitors are ABN-401, ABT-700, AMG-208, AMG-337, ARGX-111, BAY-85-3474, BMS-817378, bozitinib, BPI-9016M, glumetinib, and golvatinib tartrate. tartrate), GST-HG161, HQP-8361, I-020, JNJ-38877605, kanitinib, merestinib, MK-2461, MK-8033, OMO-1, pamufetinib, S-49076, savolitinib, SPH-3348, tivantinib, SAR-125844, SCR-1515 and TPX-0022, or anti-MET antibodies (such as APL-101, CKD-702, EMB-01, EMI-137, etc.). The MET inhibitor may be ficlatuzumab, HLX-55, HS-10241, MCLA-129, MT-8633, NOV-1105, RC-108, REGN-5093, SHR-A1403, Sym-015, or telisotuzumab vedotin. In some embodiments of the methods disclosed herein, the MET inhibitor is ervantuzumab, capmatinib, crizotinib, or tepotinib.

[0229] In some embodiments of the methods disclosed herein, the additional therapeutic agent is a KRAS inhibitor. In some embodiments of the methods disclosed herein, the KRAS inhibitor is ABREV01, ARS-1620, APG-1842, ATG-012, BBP-454, BEPT-607, BI-2852, BI-1823911, BPI-421286, BTX-2541, COTI-219, IMM-1811900, JAB-21000, JAB-22000, JAB-23000, JAB-BX300, JP-002, KR-12, LYN202, MRTX-1133, RAS-F, RMC-6236, RMC-6291, SDGR5, STX-301, and YL-15293, or an anti-KRAS antibody (such as SBT-100, SBT-102, or SBT-300). In some embodiments of the methods disclosed herein, the KRAS inhibitor is adagrasibu, ARS-3248, D-1553, GDC-6036, JDQ-443, LY3537982, sotorazib (AMG510), or BI1701963. Example

[0230] Example 1: Solid-state characterization of compound 1 in form FB-1 The XRPD pattern of the crystal formed by compound 1 in form FB-1 can be seen in Figure 1 DSC analysis showed that at 216.6 ℃ (T 峰 Heat is absorbed at ) and TGA loss is 0.11 wt% between room temperature and 100 °C. Figure 2 ).

[0231] Table 6. Solid-state characterization of compound 1, form FB-1

[0232] pKa measurement The material used in this study was FB-1. The dissociation constant of the free base of compound 1 was measured using potentiometric acid-base titration. The experimental procedure is described below. Measurements were performed in triplicate, showing a pKa of 8.99 ± 0.6 (SD). The experimental pKa value was similar to the predicted value.

[0233] program: 1) Weigh approximately 10 mg of the starting material into 10 ml of MeOH. 2) Add 25 mL of 0.5M NaCl 3) Adjust the pH to around 3-4 using 0.1 M HCl. 4) Titrate the above solution with 0.5M NaOH solution with pH 3-12. Table 7: pKa of compound 1 form FB-1

[0234] Water activity determination The material used in this study was FB-1. The water activity of the free base of compound 1 was measured using an Aqualab TDL-2. The experimental procedure is as follows. Measurements were performed in duplicate, and the material showed a water activity of 0.47.

[0235] program: 1) Take approximately 3-5 mL of the reference standard into the sample dish. 2) Set the temperature of Aqualab TDL-2 to 25℃. 3) Close the sample chamber and begin measurement. 4) Confirm that the reference solution value is within the standard limit. 5) Take approximately 50-100 mg of API into a new sample pan and begin the measurement. Table 8: Water activity of compound 1 form FB-1

[0236] The distribution coefficients were determined by the shake-flask method. The Log P value was measured using the shake-flask method. The Log P value in DI water was 2.01 ± 0.309, lasting for 1 day (24 hours). Since the product of compound 1 has low solubility in DI water, the experiment was continued by slow stirring. The Log P value in DI water was 1.64 ± 0.259, lasting for 5 days (120 hours).

[0237] Log P was determined using both a standard shake-flask Log P program and a slow-stirring Log P program. The average Log P result was obtained from three replicate tests using HPLC quantification.

[0238] program: 1) The partition coefficient was determined under 1-octanol / aqueous phase conditions. (The aqueous phase was deionized water, and the ratio of 1-octanol to aqueous phase was 50:50.) 2) Before determining the partition coefficient, the two solvents are mutually saturated at the experimental temperature. Transfer 500 mL of 1-octanol and 500 mL of aqueous phase to a 1 L flask, shake well for 24 hours, and let stand for at least 24 hours to allow the two phases to separate.

[0239] 3) Test conditions - Dissolve approximately 10 mg of sample in 10 mL of saturated 1-octanol, and transfer 9 mL of the solution to a 20 mL glass sample vial containing 9 mL of aqueous phase. Prepare three copies for each sample.

[0240] 4) Vigorously shake a 20 mL glass sample vial at 200 rpm for 24 hours using a track-type shaker (BT Lab Systems, model BT909, MO, USA), then let the vial stand for 2 hours to allow the system to equilibrate. This is the sample for 1 day (24 hours). Continue shaking at 200 rpm for 96 hours, then let the vial stand for 2 hours to allow the system to equilibrate. This is the sample for 5 days (120 hours). Compound 1 has too low a solubility in water to be defined using HPLC; therefore, the results from the 1-octanol layer were used for Log P calculation.

[0241] Table 9: Results of Compound 1 Log P 1 day (24 hours)

[0242] Note: 1. The concentration of compound 1 in the aqueous layer is defined by the concentration of the 1-octanol layer sample over 1 day (24 hours) at T0. 2. The concentration of compound 1 in the 1-octanol layer is defined by the calibration curve of compound 1.

[0243] Table 10: Results of Compound 1 Log P over 5 days (120 hours)

[0244] Note: 1. The concentration of compound 1 in the 1-octanol layer is defined by the calibration curve of compound 1, with an additional point of 0.068 mg / mL.

[0245] Reference: 1. "Partition coefficient (n-octanol / water): shake flask method", OECD Guideline 107, 07 / 27 / 1995.

[0246] 2. "Partition coefficient (1-octanol / water): slow stirring method", OECD Guideline 123, 03 / 23 / 2006.

[0247] Example 2: Screening of Polymorphs FB-1 solubility study The approximate solubility of crystalline compound 1 in 20 solvents was evaluated, and the status / results are reported in Table 11.

[0248] program: 1) Weigh approximately 5 mg of the starting material into a 4.0 mL vial. Add 25 µL of solvent at room temperature. 2) Shake and stir the solution. If a clear solution is not obtained, add another 25 µL and repeat until 0.75 mL of solvent is added.

[0249] 3) Then, add solvent in increments of 0.25 mL until a final volume of 3.0 mL is reached. 4) Stir the solution overnight. Table 11. Approximate solubility of compound 1

[0250] CL- clear; TSL- thin slurry; * after overnight stirring Screening of polymorphs of compound 1, form FB-1 Based on the approximate solubility of the starting material, a total of 80 polymorphs were screened using different techniques, including antisolvent addition, solid vapor diffusion, liquid vapor diffusion, room temperature slurrying, 50 °C slurrying, slow evaporation, polymer-induced crystallization, grinding, heating-cooling-heating, and hydration experiments. The material used in this study was free alkali compound 1 in the form FB-1.

[0251] program 1. Approximately 30 mg was plasma-processed at room temperature and 50°C. Approximately 30 mg of API was slurried in 0.5–1.0 mL of different solvents in 4.0 mL glass vials using a magnetic stirrer at room temperature. After 5–7 days, the solids in the slurry were characterized by XRPD. 2. Hydration at room temperature and 50°C Approximately 30 mg of API was slurried in 0.5–1.0 mL of different solvents in 4.0 mL glass vials using a magnetic stirrer at room temperature. After 5–7 days, the solids in the slurry were characterized by XRPD. 3. Solid vapor diffusion Approximately 30 mg of API was stored in a 4.0 mL glass vial; this vial was then placed inside a 20 mL glass vial containing solvent. The solid was characterized by XRPD after 7–10 days. 4. Addition of antisolvent Approximately 30 mg of API was dissolved in a solvent to form a saturated solution, and an antisolvent was added until a volume ratio of 10 was reached. The resulting solid was characterized by XRPD. 5. Liquid vapor diffusion Approximately 30 mg of API was dissolved in a solvent to form a saturated solution in a 4.0 mL glass vial, which was then placed into a 20 mL glass vial containing the antisolvent. After 7–10 days, the resulting solid was characterized by XRPD. 6. Polymer-induced crystallization Approximately 30 mg of API and 2.0 mg of the listed polymer were added to the solvent and stirred continuously at room temperature. After 7–10 days, the solids in the slurry or the precipitated solids in the solution were characterized by XRPD. 7. Grinding: Approximately 30 mg of API was ground in a mortar and pestle for several minutes. The ground material was then analyzed by XPRD.

[0252] 8. Compaction: Approximately 100 mg of API was compressed at 20 kN using a STYL'One Nano and Type B instrument. The tablets were then crushed, and the powder material was analyzed by XPRD.

[0253] Table 12. Results of polymorph screening by slurry conditioning at room temperature

[0254] CL- clear liquid; SL- slurry; TSL- thin slurry; LT- temperature between 2-8 °C; SE- solvent evaporated at room temperature SL- slurry Table 13. Results of polymorph screening by slurry conditioning at 50 °C

[0255] SL- slurry Table 14. Results of polymorph screening by hydration at room temperature

[0256] SL- slurry Table 15. Results of polymorph screening by hydration at 50 °C

[0257] CL- clear liquid; SL- slurry; LT- temperature between 2-8 °C; SE- solvent evaporated at room temperature Table 16. Results of polymorph screening by antisolvent addition at room temperature

[0258] CL- clear liquid; TSL- thin slurry; LT- temperature between 2-8 °C; SE- solvent evaporated at room temperature Table 17. Results of polymorph screening by solid vapor diffusion at room temperature

[0259] Table 18. Results of polymorph screening by liquid vapor diffusion at room temperature

[0260] CL- clear liquid; SL- slurry; LT- temperature between 2-8 °C; SE- solvent evaporated at room temperature Table 19. Results of polymorph screening by polymer-induced crystallization at room temperature

[0261] i.e. The polymorphic form FB-4 was discovered under two testing conditions. Equilibrium solubility study of Form FB-1 Slurry conditioning and solid vapor diffusion were performed using acetone as the solvent system. DSC showed two endothermic events at 120.8 and 206.7 °C (T peak), with TGA loss of 4.4 wt% between room temperature and 175 °C. Thermal analysis data suggest that form FB-4 may be a hydrated form of the free base.

[0262] Polymorphic form FB-5 was discovered in the DMSO / toluene solvent system. Due to the limited amount of material obtained under the test conditions, only DSC analysis was performed, which showed two endothermic events at 132.0 and 221.1 °C (T peak), indicating that it is most likely a solvated or hydrated form of the free base.

[0263] Form FB-1 was tested under milling conditions to understand its potential for solid-to-solid phase transition. However, XRPD data indicated that no phase transition occurred under milling conditions, and the material was stable. The potential for form FB-1 to form a hydrated form using a water-organic solvent system was also tested. XRPD analysis of the final solids showed that form FB-1 was stable under these test conditions.

[0264] The pyrolysis of compound 1 hydrochloride yielded a new polymorphic form, termed form FB-3. However, the final solids from multiple batches of salt pyrolysis showed some variation at lower angles (2θ) in the XRPD diffraction pattern. Analysis of the solids by DSC-TGA indicated that the XRPD variation was likely due to differences in solvate content. Analysis of milled and compacted materials of form FB-1 by XRPD showed no solid-to-solid morphological change.

[0265] ND- not detected The experimental procedures and results are summarized in Table 20. The buffer medium was prepared according to USP 35-NF 30 (page 5774), and the biorelevant media were prepared according to the provided instructions (biorelevant.com). Approximately 40.0 mg of solid was added to 2.0 mL of medium to obtain a slurry. The system was set at 37 °C for approximately 18 h. The solubility of the solution was determined by HPLC and pH testing.

[0266] Calibration curves were generated using compound 1 for concentrations ranging from 0.05 to 0.51 mg / mL. The regression value was found to be >0.999. The solubility concentration was further determined using the calibration curve equation.

[0267] Table 20. Equilibrium solubility data of compound 1 form FB-1 from biologically relevant media.

[0268] CL- clear; TSL- thin slurry; * after overnight stirring Table 21. Equilibrium solubility data of compound FB-1 from pH medium (form 1)

[0269] Solid-state stability of compound 1 in form FB-1 The material used in this study was compound 1, FB-1. According to solid-state analysis and HPLC analysis, compound 1, FB-1, was stable for four weeks at 25 °C / 60%RH, 40 °C / 75%RH, and 60 °C. The experimental results are summarized in Table 22.

[0270] Table 22. Results of solid-state stability study of compound 1 / state

[0271] Example 2: Salt Screening Solubility Study The approximate solubility of crystalline compound 1, FB-1, in 20 solvents was evaluated, and the status / results are reported in Table 23.

[0272] program: 1) Weigh approximately 5 mg of the starting material into a 4.0 mL vial. Add 25 µL of solvent at room temperature. 2) Shake and stir the solution. If a clear solution is not obtained, add another 25 µL and repeat until 0.75 mL of solvent has been added.

[0273] 3) Then, add solvent in increments of 0.25 mL until a final volume of 3.0 mL is reached. 4) Stir the solution overnight. Table 23. Approximate solubility of compound 1 in form FB-1

[0274] * not tested; Salt screening of compound 1 Salt screening of crystalline compound 1 was conducted at room temperature in eight acids and five solvents. The experimental procedures are described below. The experimental results are summarized in Table 24. Detailed solid-state characterization data can be found in Appendix II.

[0275] FB + Acidic CI Salt screening was performed using eight counterions in five solvent systems. Approximately 25 mg of free base was weighed into a 2.0 mL vial. The counterion (CI) was added to the solid at a 1:1 molar ratio (API:CI), followed by approximately 0.25 mL of solvent. The vial was then kept stirred at room temperature. After stirring at room temperature for 3–5 days, any solid-state changes in the sample were analyzed by PXRD.

[0276] Table 24: Screening results for salts of compound 1

[0277] Table 25: Solid-state characterization results of potential salt-attached compounds of compound 1

[0278] Not in plan; NA- not applicable + Scale-up and characterization of salts SL- slurry; CL- clear liquid; RT- room temperature Hippuric acid-2 The scale-up of crystalline compound 1 hippurate was tested at the sub-mg scale. The results of the salt-scale experiments are summarized in Tables 26 and 27. The results show good reproducibility. Hippuric acid-2 was analyzed by DVS, and the data showed an increase of 0.74% wt in H2O between 0-80% RH and an increase of 3.8% wt in H2O between 0-90% RH. However, the solid XRPD changed after DVS, resulting in a mixture of hippuric acid-2 and hippuric acid-4.

[0279] Table 26: Salt Amplification Settings

[0280] * recovered by solid Table 27: Salt Magnification Results

[0281] SL- slurry; RT- room temperature Maleic acid-1 The maleate salt of crystalline compound 1 was tested at a sub-mg scale. The salt amplification results are summarized in Tables 28 and 29. The results show good reproducibility in the formation of maleic acid-1 salt. Maleic acid-1 exhibits a 1.03% wt H₂O increase between 0 and 90% RH and is slightly hygroscopic. The solid after DVS remains maleic acid-1.

[0282] Table 28: Salt Amplification Settings

[0283] * recovered by solid Table 29: Salt Magnification Results

[0284] Assuming it is a THF solvate † SL- slurry; CL- clear liquid; RT- room temperature Lactic Acid-1 and Lactic Acid-2 A test run was performed using 25 mg FB-1 to replicate lactate-1. The results and status of the salt amplification are summarized below. The test run successfully replicated lactate-1.

[0285] Table 30: Reproducibility test of lactate-1 in IPA

[0286] SL- slurry; CL- clear liquid; RT- room temperature Table 31: Results of the reproducibility study of lactate-1

[0287] The crystalline lactate of compound 1 was scaled up at a scale of 550 mg at room temperature. The results and states of the salt scale-up are summarized in Tables 32 and 33. At a scale of 500 mg, the reproducibility of crystalline lactate-1 was poor, and lactate-2 was obtained at the end of the study, not lactate-1.

[0288] Table 32: Lactate Amplification Settings

[0289] * recovered by solid (IPA contains 0.23wt% H2O) Table 33: Magnification results of lactate in IPA

[0290] * ML (equivalent to FB); DVS analysis of lactate-2 salt showed an increase of 2.98% wt between 0-80% RH, classifying it as a moderately hygroscopic material. Between 0-90% RH, the material showed an increase of 3.66% wt, and the solid XRPD after DVS matched that of lactate-2.

[0291] Slurry conditioning in H2O Free alkali and selected 3-lead salts were slurried in water and stirred at room temperature for 5 days. After 5 days of stirring, the final solids were analyzed by XRPD. In summary, hippurtae, maleate, and lactate showed a tendency to transform into different solid forms, indicating that these selected salt forms are unstable when the slurry is conditioned in water.

[0292] Table 34: XRPD results of slurry conditioning in H2O

[0293] Equilibrium solubility study of FB-1, hippuric acid-2 and lactic acid-2 The experimental procedures and results are summarized in Tables 35, 36, and 37. Buffer media were prepared according to USP 35-NF 30 (page 5774), and biorelevant media were prepared according to the provided instructions (biorelevant.com). Approximate amounts of solids were added to 0.5–1.0 mL of the medium to obtain a slurry. The system was set at room temperature for 24 hours. Afterward, the solids were filtered using a centrifugal filter.

[0294] The solubility of the solution was determined by HPLC and pH testing. Due to the low solubility of the sample concentration, the injection volume was adjusted from 0.3 µL to 3 µL. The final solids were analyzed by XRPD.

[0295] Table 35. Results of Free Alkali Solubility Test

[0296] Final solid; SL- slurry ‡ * ML (equivalent to FB); Table 36. Results of hippuric acid-2 salt solubility test

[0297] Final solid; SL- slurry ‡ * ML (equivalent to FB); Table 37. Results of Lactate-2 Salt Solubility Test

[0298] Final solid; SL- slurry ‡ * ML (equivalent to FB); Example 3: Salt pyrolysis Salt cleavage was performed at a scale of 4.2 g. The experimental results are summarized in Table 38.

[0299] Table 38. Salt Crack

[0300] HPLC method development Various methods have been tested in the salt screening, polymorph screening, and excipient compatibility studies. For the solubility studies reported in this report, the concentration of compound 1 was analyzed using the following HPLC methods (Table 39). The HPLC methods reported in Table 40 were used for Log P and solid-state stability studies.

[0301] Table 39. HPLC Method Information (for equilibrium solubility studies)

[0302] Table 40. HPLC Method Information (for Log P and Solid State Stability Studies)

[0303] X-ray powder diffraction (XRPD) Instrument: Panalytical Empyrean Parameters: X-ray tube Cu (Kα radiation); Power: 45 kV x 40 mA Scan range: 2 to 40 2θ (degrees) Step size: 0.01 degrees Scanning speed: 6.33 degrees (2θ) / minute Thermogravimetric analysis (TGA) Instrument: TA Instruments Discovery TGA Parameters: Temperature rise of 10 °C per minute, 25 to 300 °C, N2 purging at 50 mL / min Differential scanning calorimetry (DSC) Instrument: TA Instruments Discovery DSC Parameters: Temperature increases by 10°C per minute, up to 300°C. Polarized light microscope (PLM) Instrument: Nikon Eclipse Ci POL Camera: Nikon DS-Fi3 Software: Nikon NIS Elements 1 H NMR Instrument: Bruker 400 MHz Ultrashield Solvent: DMSO d 6 Water activity meter Instrument: Aqualab TDL-2 Titrator Instrument: Mettler Toledo G20S Compact Potentiometric Titrator Dynamic vapor adsorption (DVS) Instrument: DVS Intrinsic, Surface Measurement System Parameters: 25 ℃, 30-90-0-90-0% RH, 2 cycles. High-performance liquid chromatography (HPLC)

[0304] The embodiments and implementations described herein are for illustrative purposes only, and in some implementations, various modifications or changes are included within the scope of this disclosure and the appended claims.

Claims

1. A 5-((5-(4-(((1R,3S)-3-aminocyclopentyl)oxy)-2-methoxy-6-methylpyridin-3-yl)-1H-pyrazol-3-yl)amino)pyrazine-2-carboxynitrile: (Compound 1) or its pharmaceutically acceptable salt or solvate in crystalline form.

2. A free base 5-((5-(4-(((1R,3S)-3-aminocyclopentyl)oxy)-2-methoxy-6-methylpyridin-3-yl)-1H-pyrazol-3-yl)amino)pyrazine-2-carboxynitrile: (Compound 1) or its pharmaceutically acceptable solvate in crystalline form.

3. An anhydrous free base 5-((5-(4-(((1R,3S)-3-aminocyclopentyl)oxy)-2-methoxy-6-methylpyridin-3-yl)-1H-pyrazol-3-yl)amino)pyrazine-2-carboxynitrile: (Compound 1) in crystal form.

4. The crystalline form according to claim 1, wherein the crystalline compound 1 is in the free base form FB-1, characterized in that... It has at least one of the following properties: (a) An X-ray powder diffraction (XRPD) pattern that is essentially the same as that shown in Figure 1; (b) X-ray powder diffraction (XRPD) patterns with characteristic peaks at 11.96±0.1° 2θ, 18.81±0.1° 2θ, 19.91±0.1° 2θ, 21.67±0.1° 2θ and 26.75±0.1° 2θ; (c) X-ray powder diffraction (XRPD) patterns with characteristic peaks at 13.87 ±0.1° 2θ and 17.06 ±0.1° 2θ; (d) Differential scanning calorimetry (DSC) thermogram showing endothermic activity with a peak temperature of approximately 216.5 °C (initial temperature); (e) Thermogravimetric analysis (TGA) chromatograms that are essentially the same as those shown in Figure 2; (f) Thermogravimetric analysis (TGA) chromatogram showing a mass loss of approximately 0.10% from the initial heating temperature to approximately 100.0 °C; or (g) Its combination.

5. The crystal form according to any one of claims 1-4, wherein the crystal form has an X-ray powder diffraction (XRPD) pattern substantially the same as that shown in FIG1.

6. The crystalline form according to any one of claims 1-5, wherein the crystalline free alkali compound 1, form FB-1, has an X-ray powder diffraction (XRPD) pattern having the characteristic peaks shown in Table 1.

7. The crystalline form according to any one of claims 1-6, wherein the crystalline free alkali compound 1, form FB-1, has an X-ray powder diffraction (XRPD) pattern with characteristic peaks at 11.96±0.1° 2θ, 18.81±0.1° 2θ, 19.91±0.1° 2θ, 21.67±0.1° 2θ, and 26.75±0.1° 2θ.

8. The crystalline form according to any one of claims 1-6, wherein the crystalline free alkali compound 1, form FB-1, has an X-ray powder diffraction (XRPD) pattern with characteristic peaks at 13.87±0.1° 2θ and 17.06±0.1° 2θ.

9. The crystalline form according to any one of claims 1-6, wherein the crystalline free alkali compound 1, form FB-1, has an X-ray powder diffraction (XRPD) pattern with a characteristic peak at 13.87 ± 0.1° 2θ.

10. The crystalline form according to any one of claims 1-6, wherein the crystalline free alkali compound 1, form FB-1, has an X-ray powder diffraction (XRPD) pattern with a characteristic peak at 17.06 ± 0.1° 2θ.

11. The crystal form according to any one of claims 1-10, wherein the X-ray powder diffraction (XRPD) pattern further includes a peak at 5.27 ± 0.1° 2θ.

12. The crystal form according to any one of claims 1-11, wherein the X-ray powder diffraction (XRPD) pattern further includes a peak at 13.34 ± 0.1° 2θ.

13. The crystal form according to any one of claims 1-12, wherein the X-ray powder diffraction (XRPD) pattern further includes a peak at 15.66 ± 0.1° 2θ.

14. The crystal form according to any one of claims 1-13, wherein the X-ray powder diffraction (XRPD) pattern further includes a peak at 22.24 ± 0.1° 2θ.

15. The crystal form according to any one of claims 1-14, wherein the X-ray powder diffraction (XRPD) pattern further includes a peak at 27.84 ± 0.1° 2θ.

16. The crystalline form according to any one of claims 1-5, wherein the crystalline free alkali compound 1, form FB-1, has an X-ray powder diffraction (XRPD) pattern with a characteristic peak at 5.27 ± 0.1° 2θ.

17. The crystalline form according to any one of claims 1-5, wherein the crystalline free alkali compound 1, form FB-1, has an X-ray powder diffraction (XRPD) pattern with a characteristic peak at 11.96 ± 0.1° 2θ.

18. The crystalline form according to any one of claims 1-5, wherein the crystalline free alkali compound 1, form FB-1, has an X-ray powder diffraction (XRPD) pattern with a characteristic peak at 13.34 ± 0.1° 2θ.

19. The crystalline form according to any one of claims 1-5, wherein the crystalline free alkali compound 1, form FB-1, has an X-ray powder diffraction (XRPD) pattern with a characteristic peak at 13.87 ± 0.1° 2θ.

20. The crystalline form according to any one of claims 1-5, wherein the crystalline free alkali compound 1, form FB-1, has an X-ray powder diffraction (XRPD) pattern with a characteristic peak at 15.66 ± 0.1° 2θ.

21. The crystalline form according to any one of claims 1-5, wherein the crystalline free alkali compound 1, form FB-1, has an X-ray powder diffraction (XRPD) pattern with a characteristic peak at 17.06 ± 0.1° 2θ.

22. The crystalline form according to any one of claims 1-5, wherein the crystalline free alkali compound 1, form FB-1, has an X-ray powder diffraction (XRPD) pattern with a characteristic peak at 18.81 ± 0.1° 2θ.

23. The crystalline form according to any one of claims 1-5, wherein the crystalline free alkali compound 1, form FB-1, has an X-ray powder diffraction (XRPD) pattern with a characteristic peak at 19.91 ± 0.1° 2θ.

24. The crystalline form according to any one of claims 1-5, wherein the crystalline free alkali compound 1, form FB-1, has an X-ray powder diffraction (XRPD) pattern with a characteristic peak at 21.67 ± 0.1° 2θ.

25. The crystalline form according to any one of claims 1-5, wherein the crystalline free alkali compound 1, form FB-1, has an X-ray powder diffraction (XRPD) pattern with a characteristic peak at 22.24 ± 0.1° 2θ.

26. The crystalline form according to any one of claims 1-5, wherein the crystalline free alkali compound 1, form FB-1, has an X-ray powder diffraction (XRPD) pattern with a characteristic peak at 26.75 ± 0.1° 2θ.

27. The crystalline form according to any one of claims 1-5, wherein the crystalline free alkali compound 1, form FB-1, has an X-ray powder diffraction (XRPD) pattern with a characteristic peak at 27.84 ± 0.1° 2θ.

28. The crystalline form according to any one of claims 1-5, wherein the crystalline free alkali compound 1, form FB-1, has an X-ray powder diffraction (XRPD) pattern with characteristic peaks at 5.27 ± 0.1° 2θ, 11.96 ± 0.1° 2θ, 13.34 ± 0.1° 2θ, 15.66 ± 0.1° 2θ, 18.81 ± 0.1° 2θ, 19.91 ± 0.1° 2θ, 21.67 ± 0.1° 2θ, 22.24 ± 0.1° 2θ, 26.75 ± 0.1° 2θ, and 27.84 ± 0.1° 2θ.

29. The crystalline form according to any one of claims 1-5, wherein the crystalline free alkali compound 1, form FB-1, has an X-ray powder diffraction (XRPD) pattern having at least two characteristic peaks selected from 5.27±0.1° 2θ, 11.96±0.1° 2θ, 13.34±0.1° 2θ, 13.87±0.1° 2θ, 15.66±0.1° 2θ, 17.06±0.1° 2θ, 18.81±0.1° 2θ, 19.91±0.1° 2θ, 21.67±0.1° 2θ, 22.24±0.1° 2θ, 26.75±0.1° 2θ, and 27.84±0.1° 2θ.

30. The crystalline form according to any one of claims 1-5, wherein the crystalline free alkali compound 1, form FB-1, has an X-ray powder diffraction (XRPD) pattern having at least three characteristic peaks selected from 5.27±0.1° 2θ, 11.96±0.1° 2θ, 13.34±0.1° 2θ, 13.87±0.1° 2θ, 15.66±0.1° 2θ, 17.06±0.1° 2θ, 18.81±0.1° 2θ, 19.91±0.1° 2θ, 21.67±0.1° 2θ, 22.24±0.1° 2θ, 26.75±0.1° 2θ, and 27.84±0.1° 2θ.

31. The crystalline form according to any one of claims 1-5, wherein the crystalline free alkali compound 1, form FB-1, has an X-ray powder diffraction (XRPD) pattern having at least four characteristic peaks selected from 5.27±0.1° 2θ, 11.96±0.1° 2θ, 13.34±0.1° 2θ, 13.87±0.1° 2θ, 15.66±0.1° 2θ, 17.06±0.1° 2θ, 18.81±0.1° 2θ, 19.91±0.1° 2θ, 21.67±0.1° 2θ, 22.24±0.1° 2θ, 26.75±0.1° 2θ, and 27.84±0.1° 2θ.

32. The crystalline form according to any one of claims 1-5, wherein the crystalline free alkali compound 1, form FB-1, has an X-ray powder diffraction (XRPD) pattern having at least five characteristic peaks selected from 5.27±0.1° 2θ, 11.96±0.1° 2θ, 13.34±0.1° 2θ, 13.87±0.1° 2θ, 15.66±0.1° 2θ, 17.06±0.1° 2θ, 18.81±0.1° 2θ, 19.91±0.1° 2θ, 21.67±0.1° 2θ, 22.24±0.1° 2θ, 26.75±0.1° 2θ, and 27.84±0.1° 2θ.

33. The crystalline form according to any one of claims 1-5, wherein the crystalline free alkali compound 1, form FB-1, has an X-ray powder diffraction (XRPD) pattern having at least six characteristic peaks selected from 5.27±0.1° 2θ, 11.96±0.1° 2θ, 13.34±0.1° 2θ, 13.87±0.1° 2θ, 15.66±0.1° 2θ, 17.06±0.1° 2θ, 18.81±0.1° 2θ, 19.91±0.1° 2θ, 21.67±0.1° 2θ, 22.24±0.1° 2θ, 26.75±0.1° 2θ, and 27.84±0.1° 2θ.

34. The crystalline form according to any one of claims 1-5, wherein the crystalline free alkali compound 1, form FB-1, has an X-ray powder diffraction (XRPD) pattern having at least seven characteristic peaks selected from 5.27±0.1° 2θ, 11.96±0.1° 2θ, 13.34±0.1° 2θ, 13.87±0.1° 2θ, 15.66±0.1° 2θ, 17.06±0.1° 2θ, 18.81±0.1° 2θ, 19.91±0.1° 2θ, 21.67±0.1° 2θ, 22.24±0.1° 2θ, 26.75±0.1° 2θ, and 27.84±0.1° 2θ.

35. The crystalline form according to any one of claims 1-5, wherein the crystalline free alkali compound 1, form FB-1, has an X-ray powder diffraction (XRPD) pattern having at least eight characteristic peaks selected from 5.27±0.1° 2θ, 11.96±0.1° 2θ, 13.34±0.1° 2θ, 13.87±0.1° 2θ, 15.66±0.1° 2θ, 17.06±0.1° 2θ, 18.81±0.1° 2θ, 19.91±0.1° 2θ, 21.67±0.1° 2θ, 22.24±0.1° 2θ, 26.75±0.1° 2θ, and 27.84±0.1° 2θ.

36. The crystalline form according to any one of claims 1-5, wherein the crystalline free alkali compound 1, form FB-1, has an X-ray powder diffraction (XRPD) pattern having at least nine characteristic peaks selected from 5.27±0.1° 2θ, 11.96±0.1° 2θ, 13.34±0.1° 2θ, 13.87±0.1° 2θ, 15.66±0.1° 2θ, 17.06±0.1° 2θ, 18.81±0.1° 2θ, 19.91±0.1° 2θ, 21.67±0.1° 2θ, 22.24±0.1° 2θ, 26.75±0.1° 2θ, and 27.84±0.1° 2θ.

37. The crystalline form according to any one of claims 1-5, wherein the crystalline free alkali compound 1, form FB-1, has an X-ray powder diffraction (XRPD) pattern having at least ten characteristic peaks selected from 5.27±0.1° 2θ, 11.96±0.1° 2θ, 13.34±0.1° 2θ, 13.87±0.1° 2θ, 15.66±0.1° 2θ, 17.06±0.1° 2θ, 18.81±0.1° 2θ, 19.91±0.1° 2θ, 21.67±0.1° 2θ, 22.24±0.1° 2θ, 26.75±0.1° 2θ, and 27.84±0.1° 2θ.

38. The crystalline form according to any one of claims 1-5, wherein the crystalline free alkali compound 1, form FB-1, has an X-ray powder diffraction (XRPD) pattern having at least 11 characteristic peaks selected from 5.27±0.1° 2θ, 11.96±0.1° 2θ, 13.34±0.1° 2θ, 13.87±0.1° 2θ, 15.66±0.1° 2θ, 17.06±0.1° 2θ, 18.81±0.1° 2θ, 19.91±0.1° 2θ, 21.67±0.1° 2θ, 22.24±0.1° 2θ, 26.75±0.1° 2θ, and 27.84±0.1° 2θ.

39. The crystalline form according to any one of claims 1-38, wherein the crystalline free alkali compound 1, form FB-1, has an endothermic differential scanning calorimetry (DSC) thermogram with a peak temperature of about 216.5 °C (initial temperature).

40. The crystalline form according to any one of claims 1-39, wherein the crystalline free alkali compound 1, form FB-1, has a thermogravimetric analysis (TGA) chromatogram substantially the same as that shown in FIG2.

41. The crystalline form according to any one of claims 1-40, wherein the crystalline free alkali compound 1, form FB-1, has a thermogravimetric analysis (TGA) chromatogram showing a mass loss of about 0.10% as it rises from the initial heating temperature to about 100.0 °C.

42. The crystalline form according to any one of claims 1-41, wherein the crystalline free alkali compound 1, in form FB-1, is physically and chemically stable.

43. The crystalline form according to any one of claims 1-42, wherein the crystalline free alkali compound 1, in form FB-1, is chemically stable.

44. A pharmaceutical composition comprising a crystalline form according to any one of claims 1-43 and a pharmaceutically acceptable excipient.

45. A method of treating cancer in a subject of need, the method comprising administering to the subject a crystalline form according to any one of claims 1-43.

46. ​​The method of claim 45, wherein the cancer comprises a solid tumor.

47. The method of claim 45, wherein the cancer comprises a locally advanced or metastatic unresectable solid tumor.

48. The method according to any one of claims 45-47, wherein the cancer comprises a tumor or tumor cells carrying oncogene amplification.

49. The method of claim 48, wherein the oncogene amplification comprises amplification of ABL, AKT1, AKT2, ALK, androgen receptor, BRAF, CCND1, CCND2, CCND3, CCNE1, CDK12, CDK4, CDK6, EGFR, ERBB2, EZH2, FGFR1, FGFR2, FGFR3, FLT3, IDH1 / 2, JAK2, JAK3, KIT, KRAS, MDM2, MDM4, MET, MYC, MYCL, MYCN, NRAS, PDGFRA, TERT, VEGFRA, or any combination thereof.

50. The method of claim 48 or claim 49, wherein the oncogene amplification is located on ecDNA.

51. The method of claim 48 or claim 49, wherein the oncogene amplification is located at one or more chromosomal loci.

52. The method of claim 48 or claim 49, wherein the oncogene amplification is an ecDNA-derived amplification.

53. The method according to any one of claims 45-52, wherein the cancer is ovarian cancer.

54. The method according to claim 53, wherein the ovarian cancer is platinum-resistant high-grade serous ovarian cancer, primary peritoneal cancer, or fallopian tube cancer.

55. The method according to any one of claims 45-52, wherein the cancer is uterine cancer.

56. The method of claim 55, wherein the uterine cancer is high-grade endometrial cancer, serous uterine carcinoma, or carcinosarcoma of the uterus.

57. The method according to any one of claims 45-52, wherein the cancer is colorectal cancer, esophageal cancer, gastric cancer, gastroesophageal junction (GEJ) cancer, head and neck squamous cell carcinoma, liposarcoma, non-small cell lung cancer, or a subtype of squamous cell carcinoma.

58. The method according to any one of claims 45-52, wherein the cancer is neuroblastoma.

59. The method according to any one of claims 45-52, wherein the cancer is breast cancer, bile duct cancer, esophageal cancer, cervical squamous cell carcinoma, non-small cell lung cancer, gastric cancer, or a subtype of squamous cell carcinoma.

60. The method according to any one of claims 45-52, wherein the cancer is esophageal cancer, non-small cell lung cancer, sarcoma, or gastric cancer.

61. The method according to any one of claims 45-60, wherein the treatment further comprises administering an additional therapeutic agent.

62. The method of claim 61, wherein the oncogene amplification comprises CDK4, CDK6, EGFR, FGFR1, FGFR2 or FGFR3.

63. The method of claim 62, wherein the treatment further comprises administering a CDK4 / 6 inhibitor, an EGFR inhibitor, or an FGFR inhibitor.

64. The method of claim 63, wherein the EGFR inhibitor is erlotinib.

65. The method of claim 63, wherein the FGFR inhibitor is pemitinib.

66. The method of claim 63, wherein the FGFR inhibitor is fobatinib.

67. The method of claim 63, wherein the CDK4 / 6 inhibitor is abexilide.

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