WEE1 inhibitors and methods for treating cancer

Abstract

Disclosed herein is a method for determining the sensitivity of a subject to a WEE1 inhibitor, comprising obtaining or obtaining a biological sample from the subject, and performing or performing at least one assay on the biological sample to determine whether the subject has an altered function of CCNE1. Also disclosed is a method for treating cancer with a WEE1 inhibitor, comprising: (a) identifying a subject having cancer and (b) an endogenous or altered function of CCNE1; and administering to the subject an effective amount of a WEE1 inhibitor, or a pharma- ceutically acceptable salt thereof.

Description

[Technical field]

[0001] (Incorporation by reference to priority application) This application claims priority to U.S. Provisional Patent Application No. 63 / 202,770, filed June 23, 2021, which is incorporated by reference in its entirety.

[0002] FIELD OF THEINVENTION This application relates generally to compounds that are WEE1 inhibitors and methods of using them to treat conditions characterized by excessive cell proliferation, such as cancer. The invention also relates to methods of identifying mutations in subjects with cancer and subsequent treatment of those subjects with WEE1 inhibitors. [Background technology]

[0003] DNA is constantly damaged by the environment. Light, chemicals, stress and cell replication result in single or double strand breaks along the DNA backbone. Typically, organisms defend against DNA damage by repair proteins that reconnect or resynthesize damaged DNA. Correct functioning of these proteins is essential for life. Incorrect substitution of nucleotides into DNA can cause mutations (and other genetic alterations, including but not limited to insertions, deletions and frameshifts), genetic diseases and loss of protein function. Complete loss of DNA repair can cause cell death, tumor progression and cancer.

[0004] Cell cycle checkpoints are important for proper DNA repair and ensure that cells do not proceed with cell replication until their genomic integrity is restored. WEE1 is a nuclear kinase involved in arrest at the G2-M cell cycle checkpoint for DNA repair before the onset of mitosis. Normal cells repair damaged DNA during the G1 arrest. Cancer cells often lack a G1-S checkpoint and rely on a functional G2-M checkpoint for DNA repair. WEE1 is overexpressed in a variety of cancer types. A compound known as ZN-c3 is a WEE1 inhibitor with the formula:

[0005] [ka] Summary of the Invention [Means for solving the problem]

[0006] Various embodiments provide a method for determining a subject's susceptibility to compound ZN-c3, or a pharma- ceutically acceptable salt thereof, comprising: obtaining or having obtained a biological sample from a subject; performing, or having performed, at least one assay on the biological sample to determine whether the subject has an altered function of CCNE1.

[0007] Another embodiment is a method of treating cancer, comprising: obtaining or having obtained a biological sample from a subject; performing or having performed at least one assay on the biological sample to determine whether the subject has an altered function of CCNE1; and administering to the subject an effective amount of compound ZN-c3, or a pharma- ceutically acceptable salt thereof, based on the results of the assay.

[0008] Another embodiment is a method of treating cancer, comprising: (a) identifying a subject having cancer and (b) endogenous or altered function of CCNE1; and administering to the subject an effective amount of compound ZN-c3, or a pharma- ceutically acceptable salt thereof.

[0009] Another embodiment is a method of treating cancer, comprising: (a) identifying a subject having cancer and (b) overexpression or altered function of CCNE1; and administering to the subject an effective amount of compound ZN-c3, or a pharma- ceutically acceptable salt thereof.

[0010] Another embodiment is a method of treating cancer in a subject, comprising: determining whether a subject is sensitized to treatment with compound ZN-c3, or a pharma- ceutically acceptable salt thereof, the determining comprising: obtaining or having obtained a biological sample from the subject; and determining whether the subject has an altered function of CCNE1, comprising performing or having performed at least one assay on the biological sample; and selecting a treatment protocol for the subject based on a determination of whether the subject is sensitized to treatment with compound ZN-c3, or a pharma- ceutically acceptable salt thereof.

[0011] These and other embodiments are described in more detail below. [Brief description of the drawings]

[0012] [Figure 1] 1 shows Western blot results confirming CCNE1 overexpression in stable SKOV3 cell lines compared to empty vector control SKOV3 cells ("Control"). [Diagram 2]1 is a plot showing that stable SKOV3 cells overexpressing CCNE1 are more sensitive to ZN-c3 compared to empty vector control SKOV3 cells ("empty vector") as measured by cell viability. [Diagram 3] Figure 2 shows a plot showing the percent cell density of OVCAR-3 ovarian cancer cells after 72 hours of treatment with a 10-point dose response of ZN-c3. OVCAR-3 has CCNE1 amplification (Table 2) and is sensitive to ZN-c3. Viability is calculated using cell titer glow assay. [Figure 4] Figure 1 shows a plot of tumor volume in 10 NOD / SCID mice inoculated with 2x107 OVCAR-3 cells subcutaneously in the right flank and treated daily with 80mg / kg vehicle or ZN-c3 (KP-2638). ZN-c3 treated tumors show significant tumor growth inhibition when compared to vehicle controls. [Diagram 5] Figure 2 shows a plot of tumor volume in 10 BALB / c nude mice subcutaneously inoculated with 1x106 HCC1806 human triple-negative breast cancer cells in the right flank and treated daily with 80mg / kg vehicle or ZN-c3 (KP-2638). HCC1806 cells have high copy numbers of CCNE1 (see Table 2). ZN-c3-treated tumors show significant tumor growth inhibition when compared to vehicle controls. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] WEE1 is a tyrosine kinase that is a key component of the ATR-mediated G2 cell cycle checkpoint control that blocks the entry of mitosis in response to cellular DNA damage. ATR phosphorylates and activates CHK1, which in turn activates WEE1, leading to the selective phosphorylation of cyclin-dependent kinase 1 (CDK1) at Tyr15. WEE1 activation leads to selective phosphorylation of CDK2 at Tyr15, thereby regulating the CDK2-cyclin A / E complex that controls G1 / S phase progression. This process confers a survival advantage by allowing tumor cells time to repair damaged DNA before the onset of mitosis. Inhibition of WEE1 abrogates the G2 checkpoint and promotes cancer cells with DNA damage to enter premature mitosis and undergo cell death by mitotic apoptosis. Thus, inhibition of WEE1 has the potential to increase tumor sensitivity to DNA damaging agents such as cisplatin and induce tumor cell death. In addition, WEE1 activation can also lead to selective phosphorylation of CDK2 at Tyr15, thereby regulating the CDK2-cyclin A / E complex that controls G1 / S phase progression. Inhibition of WEE1 can lead to excessive replication activity, thereby resulting in replication collapse.

[0014] Compound ZN-c3 and its pharmaceutically acceptable salts are WEE1 inhibitors.The chemical structure of compound ZN-c3 is shown above.Compound ZN-c3 and its pharmaceutically acceptable salts can be prepared by various methods.See, for example, WO 2019 / 173082.

[0015] Cyclin E1 (CCNE1) is a cyclin-dependent kinase that regulates the transcription of cyclins, including CDK2. It is involved in cell cycle regulation by binding to CDKs and thereby promoting cell cycle progression.

[0016] definition Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. All patents, applications, published applications and other publications referenced herein are incorporated by reference in their entirety unless otherwise stated. In the event that there are a plurality of definitions for a term herein, the definition in this section prevails unless otherwise stated.

[0017] As used herein, the term "about" has its ordinary meaning as understood by one of ordinary skill in the art, and thus indicates that a value includes the inherent variation of error for the method used to determine the value or the variation that exists among multiple determinations.

[0018] As used herein, "modify" or "alter" means to " or any form thereof means to alter, change, substitute, delete, substitute, remove, change, or transform.

[0019] As used herein, the terms "function" and "functional" have their ordinary meaning as understood by one of ordinary skill in the art and thus refer to a biological function, an enzymatic function, or a therapeutic function.

[0020] As used herein, the term "endogenous" has its ordinary meaning as understood by one of skill in the art, and thus refers to the native or wild-type characteristics of a gene, protein, or cell. In some embodiments, an endogenous gene is any of the above-mentioned genes. In some embodiments, the endogenous protein is a wild-type sequence of said protein. In some embodiments, the endogenous protein function is a wild-type function and activity level of said protein. In some embodiments, the endogenous cell is a wild-type cell.

[0021] The term "mutation" has its ordinary meaning as understood by those of skill in the art and refers to a change in a gene sequence. In some embodiments, the cell has multiple mutations. In some embodiments, the mutations are in a coding region of the genome. The mutations can range in size from a single nucleotide to a large segment of a chromosome containing multiple genes. In some embodiments, at least one mutation is silent and does not significantly affect gene expression or function. In some embodiments, at least one mutation affects gene expression or function, such as gene amplification, overexpression, or increased copy number. In some embodiments, at least one mutation is silent and does not significantly affect protein expression or function. In some embodiments, at least one mutation has a small effect on protein expression or function. In some embodiments, at least one mutation has a moderate effect on protein expression or function. In some embodiments, at least one mutation has a large effect on protein expression or function. In some embodiments, at least one mutation prevents expression or function of a protein. Non-limiting examples of mutations include insertions, deletions, truncations, substitutions, duplications, translocations, and inversions. In some embodiments, the mutation is "somatic", i.e., occurs in somatic cells and is not heritable. In some embodiments, a subset of somatic cells in an organism have at least one mutation that other somatic cells do not have. In some embodiments, the mutation is "germline", i.e., occurs in germ cells and is heritable.

[0022] As disclosed herein, mutations can be monitored through various sequencing, expression, or functional assays.Non-limiting examples include DNA sequencing, RNA sequencing, DNA hybridization, protein sequencing, targeted genome sequencing, whole exome sequencing, whole genome sequencing, ATAC sequencing, Sanger sequencing, PCR, qPCR, RT-PCR, RT-qPCR, next-generation sequencing, protein truncation test, DNA microarray, heteroduplex analysis, denaturing gradient gel electrophoresis, nucleotide sequencing, single-strand conformation polymorphism, restriction enzyme digestion assay, fluorescence in situ hybridization (FISH), comparative genomic hybridization, restriction fragment length polymorphism, amplification refractory mutation system PCR, nested PCR, multiplex ligation-dependent probe amplification, single-strand conformation polymorphism, and oligonucleotide ligation assay. Mutations can also be monitored via a variety of antibody-based assays using biological samples, including, but not limited to, Western blotting, fluorescence-activated cell sorting, immunofluorescence, immunohistochemistry, immunocytochemistry, immunoprecipitation, enzyme-linked immunosorbent assay, radioimmunoassay, and electrochemiluminescence assay.

[0023] The term "cancer" is used herein in its ordinary biological sense and is understood by those of skill in the art. Cancer therefore includes cancer of any cell type, including, but not limited to, glioblastoma, astrocytoma, meningioma, craniopharyngioma, medulloblastoma, and other brain cancers, leukemia, skin cancer, adrenal gland cancer, anal cancer, biliary tract cancer, bladder cancer, bone cancer, breast cancer, cervical cancer, colorectal cancer, uterine cancer, esophageal cancer, eye cancer, gallbladder cancer, gastrointestinal cancer, Hodgkin's lymphoma, hematologic malignancies, hematologic malignancies, Kaposi's sarcoma, renal cancer, pharyngeal cancer, and leukemia. Head and hypopharyngeal cancer, liver cancer, lung cancer, lymphoma, mesothelioma, melanoma, multiple myeloma, neuroblastoma, nasopharyngeal cancer, ovarian cancer, osteosarcoma, pancreatic cancer, pituitary cancer, retinoblastoma, salivary gland cancer, stomach cancer, small intestine cancer, testicular cancer, thymus cancer, thyroid cancer, uterine cancer, uterine sarcoma, uterine serous adenocarcinoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, Wilms' tumor, solid tumor, or liquid tumor Examples of such tumors include

[0024] As used herein, the term "tumor" has its ordinary meaning as understood by those of skill in the art and refers to an abnormal growth of cells or tissue. In some embodiments, a tumor is benign. In some embodiments, a tumor is malignant. A tumor becomes cancerous when it metastasizes or spreads to other areas of the body. As used herein, the term "solid tumor" has its ordinary meaning as understood by those of skill in the art and refers to an abnormal mass of tissue that does not contain liquid areas or cysts. Non-limiting examples of solid tumors include sarcomas, carcinomas, or lymphomas. Many cancerous tissues can form solid tumors, such as, but not limited to, breast cancer, brain cancer, lung cancer, liver cancer, stomach cancer, spleen cancer, colon cancer, kidney cancer, pancreatic cancer, prostate cancer, uterine cancer, skin cancer, head cancer, neck cancer, sarcoma, neuroblastoma, or ovarian cancer. The terms "cancer" and "tumor" may generally be used interchangeably unless the context clearly indicates that a more specific meaning is intended.

[0025] The term "cell" as used herein has its ordinary meaning as understood by one of skill in the art and can refer to any cell type. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell.

[0026] The terms "individual," "subject," or "patient" as used herein have their ordinary meanings as understood by those of skill in the art, and thus include a human or non-human mammal. The term "mammal" is used in its ordinary biological sense, and thus specifically includes primates, including monkeys (chimpanzees, apes, monkeys) and humans, cows, horses, sheep, goats, swine, rabbits, dogs, cats, rodents, rats, mice, guinea pigs, and the like. In some embodiments, the subject is The subject may be a human. In some embodiments, the subject may be a child and / or infant. In other embodiments, the subject may be an adult.

[0027] The term "cancer therapy" as used herein has its ordinary meaning as understood by those of skill in the art and refers to a therapy (such as surgery and / or radiation) or an anti-cancer agent, such as a small molecule, compound, protein, or other pharmaceutical agent, used to treat, inhibit, or prevent cancer. Non-limiting examples of general classes of anti-cancer agents that can be used with any one or more of the alternatives described herein include alkylating agents, anti-EGFR antibodies, anti-Her-2 antibodies, antimetabolites, vinca alkaloids, platinum-based agents, anthracyclines, topoisomerase inhibitors, taxanes, antibiotics, immunomodulators, immune cell antibodies, interferons, interleukins, HSP90 inhibitors, antiandrogens, antiestrogens, antihypercalcemic agents, apoptosis inducers, aurora kinase inhibitors, Bruton's tyrosine kinase inhibitors, calcitoninib, cyclosporine kinase inhibitors ... Sineurin inhibitors, CaM kinase II inhibitors, CD45 tyrosine phosphatase inhibitors, CDC25 phosphatase inhibitors, CHK kinase inhibitors, cyclooxygenase inhibitors, bRAF kinase inhibitors, cRAF kinase inhibitors, Ras inhibitors, cyclin-dependent kinase inhibitors, cysteine ​​protease inhibitors, DNA intercalators, DNA strand breakers, E3 ligase inhibitors, EGF pathway inhibitors, farnesyltransferase inhibitors, Flk-1 kinase inhibitors, glycogen synthase kinase-3 (GSK3) inhibitors, histone deacetylase (HDAC) inhibitors, I-kappa B-alpha kinase inhibitors, imidazoline Tetrazinone, insulin tyrosine kinase inhibitor, c-Jun-N-terminal kinase (JNK) inhibitor, mitogen-activated protein kinase (MAPK) inhibitor, MDM2 inhibitor, MEK inhibitor, E RK inhibitors, MMP inhibitors, mTor inhibitors, NGFR tyrosine kinase inhibitors, p38 MAP kinase inhibitors, p56 tyrosine kinase inhibitors, PDGF pathway inhibitors, phosphatidylinositol 3-kinase inhibitors, phosphatase inhibitors, protein phosphatase inhibitors p53, p53-p54 ... nicotinic acid, amsacrine, asparaginase, atrasentan, bexarotene, carboquone, demecolcine, efaproxiral, elsamitrucin, etoglucide, hydroxycarbamide, leucovorin, lonidamine, lucantone, masoprocol, methyl aminolevulinate, mitoguazone, mitotane, oblimersen, omacetaxine, pegaspargase, porfimer sodium, prednimustine, sitimagene ceradenovec, talaporfin, temoporfin, trabectedin, or verteporfin.

[0028] The term "pharmaceutical acceptable salt" refers to a salt of a compound that does not cause significant irritation to an organism to which it is administered and does not abolish the biological activity and properties of the compound. In some embodiments, the salt is an acid addition salt of the compound. Pharmaceutical salts can be obtained by reacting a compound with an inorganic acid, such as hydrohalic acids (e.g., hydrochloric acid or hydrobromic acid), sulfuric acid, nitric acid, and phosphoric acid (such as 2,3-dihydroxypropyl dihydrogen phosphate). Pharmaceutical salts can also be obtained by reacting a compound with an organic acid, such as an aliphatic or aromatic carboxylic or sulfonic acid, such as formic acid, acetic acid, succinic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, nicotinic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, trifluoroacetic acid, benzoic acid, salicylic acid, 2-oxopentanedioic acid, or naphthalenesulfonic acid. Pharmaceutical salts can also be obtained by reacting a compound with a base to form a salt, for example, an ammonium salt, an alkali metal salt, for example, sodium, potassium, or lithium salt, an alkaline earth metal salt, for example, calcium or magnesium salt, a carbonate salt, a bicarbonate salt, a salt with an organic base, for example, dicyclohexylamine, N-methyl-D-glucamine, tris(hydroxymethyl)methylamine, C1-C7 alkylamines, cyclohexylamine, triethanolamine, ethylenediamine, and amino acids such as arginine and lysine. With respect to the WEE1 inhibitor compounds, one skilled in the art will appreciate that when a salt is formed by protonation of a nitrogen-based group (e.g., NH2), the nitrogen-based group can be associated with a positive charge (e.g., NH2 becomes NH3). + ), and the positive charge can be a negatively charged counterion (Cl - Understand that balance can be achieved by

[0029] Where the compounds disclosed herein have unfilled valences, it is understood that the valences are filled with hydrogen or an isotope thereof, such as hydrogen-1 (protium) and hydrogen-2 (deuterium).

[0030] It is understood that the compounds described herein can be isotopically labeled. Substitution with an isotope such as deuterium can provide certain therapeutic advantages due to greater metabolic stability, such as, for example, increased in vivo half-life or reduced required dosage. Each chemical element represented in a compound structure can include any isotope of that element. For example, in a compound structure, a hydrogen atom can be expressly disclosed or understood as being present in the compound. In any position of a compound where a hydrogen atom can be present, the hydrogen atom can be any isotope of hydrogen, including, but not limited to, hydrogen-1 (protium) and hydrogen-2 (deuterium). Thus, reference to a compound herein encompasses all possible isotopic forms, unless the context clearly indicates otherwise.

[0031] The compounds described herein may be present in crystalline forms (also known as polymorphs, which are compounds in which the same elements of the compound are present). It is understood that the term "solvate" includes crystalline phases, amorphous phases, salts, solvates, and hydrates, including crystal packing arrangements of different compositions. In some embodiments, the compounds described herein exist in solvated forms with pharma- ceutically acceptable solvents, such as water, ethanol, etc. In other embodiments, the compounds described herein exist in unsolvated forms. Solvates contain either stoichiometric or non-stoichiometric amounts of solvent, and may be formed during the crystallization process with pharma-ceutically acceptable solvents, such as water, ethanol, etc. Hydrates are formed when the solvent is water, or alcoholates are formed when the solvent is alcohol. In addition, the compounds provided herein can exist in unsolvated as well as solvated forms. In general, solvated forms are considered equivalent to unsolvated forms for the purposes of the compounds and methods provided herein.

[0032] When a range of values ​​is provided, it is understood that the upper and lower limits, as well as every intervening value between the upper and lower limits of that range, are encompassed within an embodiment.

[0033] Terms and phrases used in this application, and variations thereof, particularly in the appended claims, should be construed as open-ended rather than limiting, unless expressly stated otherwise. As an example above, the term "including" should be construed to mean "including without limitation," "including but not limited to," and the like. As used herein, the term "comprising" is synonymous with "including," "containing," or "featuring," and is inclusive or open-ended and does not exclude additional unrecited elements or method steps. The term "having" should be construed as "having at least." The term "including" should be construed as "including but not limited to." The term "example" is used to provide illustrative examples rather than an exhaustive or exclusive list of the items under discussion. The use of terms such as "preferably," "preferred," "desired," or "desirable," as well as words of similar import, should not be understood as implying that a particular feature is critical, essential, or even important to the structure or function, but rather is intended merely to highlight alternative or additional features that may or may not be utilized in a particular embodiment. Additionally, the term "comprising" is intended to be construed as synonymous with the phrases "having at least" or "including at least." When used in the context of a compound, composition, or device, the term "comprising" means that the compound, composition, or device includes at least the recited features or components, but may also include additional features or components.

[0034] With respect to the use of substantially any plural and / or singular term herein, a person skilled in the art may convert from plural to singular and / or from singular to plural as appropriate depending on the context and / or application. Various singular / plural permutations may be expressly set forth herein for clarity. The indefinite article "a" or "an" does not exclude a plurality. The mere fact that certain means are recited in mutually different dependent claims does not indicate that a combination of these means cannot be used to advantage. Any reference signs in the claims should not be interpreted as limiting the scope thereof.

[0035] Pharmaceutical Compositions Some embodiments described herein relate to pharmaceutical compositions that can include an effective amount of one or more compounds described herein (e.g., compound ZN-c3, or a pharma- ceutically acceptable salt thereof) and a pharma- ceutically acceptable carrier, diluent, excipient, or combination thereof.

[0036] The term "pharmaceutical composition" refers to a mixture of one or more compounds and / or salts disclosed herein with other chemical components, such as diluents or carriers. A pharmaceutical composition is a mixture of a compound or compounds administered to an organism. The pharmaceutical compositions may also be obtained by reacting the compound with an inorganic or organic acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, and salicylic acid. Pharmaceutical compositions are generally tailored to the particular intended route of administration.

[0037] The term "physiologically acceptable" refers to a carrier, diluent, or excipient that does not neutralize the biological activity and properties of the compound or cause substantial damage or injury to the animal to which the composition is intended to be delivered.

[0038] As used herein, "carrier" refers to a compound that facilitates the incorporation of a compound into cells or tissues. For example, and without limitation, dimethyl sulfoxide (DMSO) is a commonly used carrier that facilitates the uptake of many organic compounds into cells or tissues of a subject.

[0039] As used herein, "diluent" refers to an ingredient in a pharmaceutical composition that has no apparent pharmacological activity, but may be pharma- ceutically necessary or desirable. For example, a diluent may be used to bulk a potent drug whose mass is too small for manufacture and / or administration. It may also be a liquid for dissolving a drug to be administered by injection, ingestion, or inhalation. A common form of diluent in the art is a buffered aqueous solution, such as, without limitation, phosphate buffered saline, which mimics the pH and isotonicity of human blood.

[0040] As used herein, "excipient" refers to an essentially inert substance added to a pharmaceutical composition to provide the composition with, but not limited to, bulk, consistency, stability, binding ability, lubrication, disintegration ability, etc. For example, stabilizers such as antioxidants and metal chelators are excipients. In one embodiment, the pharmaceutical composition includes an antioxidant and / or a metal chelator. A "diluent" is a type of excipient.

[0041] The pharmaceutical compositions described herein can be administered to human patients by themselves or in pharmaceutical compositions in which they are mixed with other active ingredients, such as in combination therapy, or with carriers, diluents, excipients, or combinations thereof.The appropriate formulation depends on the route of administration selected.Techniques for formulation and administration of the compounds described herein are known to those skilled in the art.

[0042] The pharmaceutical compositions disclosed herein can be prepared in a manner known per se, for example, by conventional mixing, dissolving, granulating, dragee-making, elutriating, emulsifying, encapsulating, entrapping or tabletting processes. In addition, the active ingredient is contained in an amount effective to achieve its intended purpose. Many of the compounds used in the pharmaceutical combinations disclosed herein may be provided as salts with pharma-ceutically compatible counterions.

[0043] There are multiple techniques in the art for administering WEE1 inhibitor compounds, salts, and / or compositions, including, but not limited to, oral, rectal, intrapulmonary, topical, aerosol, injection, infusion, and parenteral delivery, including intramuscular, subcutaneous, intravenous, intramedullary injection, intrathecal, direct intraventricular, intraperitoneal, intranasal, and intraocular injection. In some embodiments, the WEE1 inhibitor compound, or a pharma- ceutically acceptable salt thereof, may be administered orally.

[0044] The WEE1 inhibitor compounds, salts, and / or compositions may also be administered in a local rather than systemic manner, for example, by injecting the compound directly into the affected area, often as a depot or sustained release formulation, or by implantation. Additionally, the compounds can be administered in targeted drug delivery systems, for example, in liposomes coated with tissue-specific antibodies. Liposomes can be targeted to and taken up selectively by organs. For example, intranasal or intrapulmonary delivery may be desirable to target a respiratory disease or condition.

[0045] The composition may be provided in a pack or dispenser device, which may contain one or more unit dosage forms containing the active ingredient, if desired. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accompanied by a notice associated with the container, in a format prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice reflects the approval by the agency of the form of the drug for human or animal administration. Such notice may, for example, be a label approved by the U.S. Food and Drug Administration for prescription drugs, or an approved product insert. The composition may comprise the compound and / or salt described herein, formulated in a compatible pharmaceutical carrier, and may also be prepared, placed in a suitable container, and labeled for the treatment of the indicated condition.

[0046] method Various embodiments provide a method for determining a subject's susceptibility to compound ZN-c3, or a pharma- ceutically acceptable salt thereof, comprising: obtaining or having obtained a biological sample from a subject; performing, or having performed, at least one assay on the biological sample to determine whether the subject has an altered function of CCNE1.

[0047] Another embodiment is a method of treating cancer, comprising: obtaining or having obtained a biological sample from a subject; performing or having performed at least one assay on the biological sample to determine whether the subject has an altered function of CCNE1; and administering to the subject an effective amount of compound ZN-c3, or a pharma- ceutically acceptable salt thereof, based on the results of the assay.

[0048] Another embodiment is a method of treating cancer, comprising: (a) identifying a subject having cancer and (b) endogenous or altered function of CCNE1; and administering to the subject an effective amount of compound ZN-c3, or a pharma- ceutically acceptable salt thereof.

[0049] Another embodiment is a method of treating cancer, comprising: (a) identifying a subject having cancer and (b) overexpression or altered function of CCNE1; and administering to the subject an effective amount of compound ZN-c3, or a pharma- ceutically acceptable salt thereof.

[0050] Another embodiment is a method of treating cancer in a subject, comprising: determining whether a subject is sensitized to treatment with compound ZN-c3, or a pharma- ceutically acceptable salt thereof, said determining comprising: obtaining or having obtained a biological sample from the subject; and determining whether the subject has an altered function of CCNE1, comprising performing or having performed at least one assay on the biological sample; The subject is sensitized to treatment with the compound ZN-c3, or a pharma- ceutically acceptable salt thereof. and selecting a treatment protocol for the subject based on the determination of whether or not the subject is a candidate for the disease.

[0051] Details of various embodiments and implementations of the methods described above are described in more detail elsewhere herein, and all such embodiments and implementations will be understood to apply to all of the methods described above unless the context clearly indicates otherwise.

[0052] In various embodiments of the above-mentioned method, the altered function of CCNE1 is CCNE1 gene amplification. In other embodiments, the altered function of CCNE1 is CCNE1 protein overexpression. In some embodiments, the altered function of CCNE1 results from CCNE1 gene mutation.

[0053] In various embodiments of the above-mentioned methods, the cancer comprises one or more of the types of cancer (including tumors) described herein. In one embodiment, the cancer is a solid tumor. In one embodiment, the cancer is a hemolytic malignancy. In some embodiments, the cancer is endometrial cancer, gallbladder cancer, or ovarian cancer. Examples of ovarian cancer include epithelial ovarian cancer, germ cell cancer, and stromal cancer. In one embodiment, the epithelial ovarian cancer is high-grade serous ovarian cancer. In various embodiments of the above-mentioned methods, the subject is a human.

[0054] A "functional assay" is a method for detecting the activity of a gene, protein, or cell in response to a stimulus or injury. The particular functional assay performed depends on the particular mutation or mutations incorporated into the genome of the cell. For example, in various embodiments, the methods described herein may include performing or having performed at least one assay on a biological sample to determine whether a subject has an altered function of CCNE1 (or does not have an altered function of CCNE1). Functional assays include, but are not limited to, kinase assays, transcription assays, e.g., using reporter constructs, proliferation assays, apoptosis assays, migration / chemotaxis assays, nutrient sensitivity assays, drug (e.g., drugs, chemotherapeutic agents, mutagens) or radiation sensitivity assays, nucleic acid binding assays, or protein binding assays, all of which are within the capabilities of one of ordinary skill in the art.

[0055] As used herein, the terms "treat," "treating," "treatment," "therapeutic," and "therapy" do not necessarily mean a complete cure or elimination of a disease or condition. Any alleviation of any undesirable signs or symptoms of a disease or condition, to any degree, may be considered treatment and / or therapy. Additionally, treatment may include actions that may worsen a subject's overall feeling of health or appearance.

[0056] The terms "administration" or "administering" as used herein have their usual meaning as understood by those skilled in the art, and refer to providing or giving a pharmaceutical agent, such as compound ZN-c3, or a pharma-ceutically acceptable salt thereof, to a subject by any effective route. Exemplary administration routes include, but are not limited to, oral, injection (including intracranial, subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), sublingual, rectal, transdermal, intranasal, vaginal, intraocular, or inhalation routes.

[0057] The terms "therapeutically effective amount" and "effective amount" are used to indicate an amount of an active compound or pharmaceutical agent that induces a described biological or medical response. For example, a therapeutically effective amount of a compound, salt or composition may be the amount necessary to prevent, alleviate, or ameliorate the symptoms of a disease or condition, or to prolong the survival of the subject being treated. This response may occur in a tissue, system, animal, or human, and may include alleviation of signs or symptoms of the disease or condition being treated. Determination of an effective amount is well within the capabilities of one of ordinary skill in the art in light of the disclosure provided herein. The therapeutically effective amount of the ZN-c3 derivatives disclosed herein required as a dose is The dosage of the compound, and its salts, will depend on the route of administration, the type of animal, including humans, being treated, and the physical characteristics of the particular animal under consideration. Dosages can be adjusted to achieve the desired effect, but will depend on factors such as body weight, diet, concurrent medications, and other factors that one of ordinary skill in the medical arts will recognize.

[0058] For example, an effective amount of a compound or radiation is an amount that results in (a) the reduction, alleviation, or elimination of one or more symptoms caused by cancer, (b) a reduction in tumor size, (c) the elimination of tumor, and / or (d) long-term disease stabilization (growth arrest) of tumor. In the treatment of lung cancer (such as non-small cell lung cancer), a therapeutically effective amount is an amount that reduces or eliminates cough, shortness of breath, and / or pain. As another example, an effective amount, i.e., a therapeutically effective amount, of a WEE1 inhibitor is an amount that results in a reduction in WEE1 activity and / or phosphorylation (e.g., phosphorylation of CDC2, also known as CDK1). The reduction in activity of WEE1 is known to those skilled in the art and can be determined by analysis of WEE1 intrinsic kinase activity and phosphorylation of downstream substrates.

[0059] The amount of compound ZN-c3, or its pharmaceutically acceptable salt, required for use in treatment will vary depending on the particular compound or salt selected, the route of administration, the nature and / or symptoms of the disease or condition being treated, and the age and condition of the patient, and will ultimately be at the discretion of the attending physician or clinician. In the case of administration of a pharmaceutically acceptable salt, the dosage may be calculated as a free base. As will be understood by those skilled in the art, in certain circumstances, it may be necessary to administer the compounds disclosed herein in amounts that exceed or even far exceed the dosage ranges described herein in order to effectively and aggressively treat, particularly progressive, diseases or conditions.

[0060] Generally, however, suitable doses will often be within the range of about 0.05 mg / kg to about 10 mg / kg. For example, suitable doses may be within the range of about 0.10 mg to about 7.5 mg per kg of body weight per day, such as about 0.15 mg to about 5.0 mg per kg of recipient body weight per day, about 0.2 mg to 4.0 mg per kg of recipient body weight per day, or any amount therebetween. The compound may be administered in a unit dosage form, which may contain, for example, 1 to 500 mg, 10 to 100 mg, 5 to 50 mg, or any amount therebetween of active ingredient per unit dosage form.

[0061] The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more partial doses per day. The partial dose itself may be further divided, for example, into several discrete loosely spaced administrations.

[0062] As will be readily apparent to those skilled in the art, the useful in vivo dosages and the specific administration methods will vary depending on the age, weight, severity of the affliction, the mammalian species being treated, the specific compound being used, and the specific application for which these compounds are being used. The determination of effective dosage levels, i.e., the dosage levels required to achieve the desired results, can be accomplished by those skilled in the art using routine methods, such as human clinical trials, in vivo studies, and in vitro studies. For example, the useful dosages of compound ZN-c3, or its pharma-ceutically acceptable salts, can be determined by comparing their in vitro activity and in vivo activity in animal models. Such comparisons can be made by comparison with established drugs, such as cisplatin and / or gemcitabine).

[0063] Dosage amount and interval are adjusted individually to provide plasma concentrations of the active moiety sufficient to maintain the modulating effect or minimal effective concentration (MEC). The MEC may vary for each compound but can be estimated from in vivo and / or in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and administration. The effective local concentration of the drug will depend on the route of administration. However, HPLC assays or bioassays can be used to determine plasma concentrations. Dosage intervals can also be determined using the MEC value. Compositions should be administered using a regimen that maintains plasma levels above the MEC for 10-90% of the time, preferably 30-90% and most preferably 50-90%. In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to the plasma concentration.

[0064] It should be noted that the attending physician would know how and when to terminate, interrupt, or adjust administration due to toxicity or organ dysfunction. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response is not adequate (precluding toxicity). The magnitude of the dose administered in the management of the disease of interest will vary with the severity of the disease or condition to be treated and with the route of administration. The severity of the disease or condition can, for example, be assessed, in part, by standard prognostic evaluation methods. Furthermore, the dose and perhaps the frequency of administration will also vary with the age, weight, and response of the individual patient. Programs comparable to those discussed above can be used in veterinary medicine.

[0065] Compound ZN-c3, its pharma- ceutically acceptable salts and compositions disclosed herein can be evaluated for efficacy and toxicity using known methods. For example, the toxicology of a particular compound or a subset of compounds sharing a certain chemical moiety can be established by evaluating in vitro toxicity on cell lines, such as mammalian cell lines, preferably human cell lines. The results of such studies are often predictive of toxicity in animals, such as mammals, or especially humans. Alternatively, the toxicity of a particular compound in an animal model, such as mice, rats, rabbits, dogs or monkeys, can be determined using known methods. The efficacy of a particular compound can be established using several recognized methods, such as in vitro methods, animal models, or human clinical trials. When selecting a model to determine efficacy, a person skilled in the art can be guided by the state of the art in selecting the appropriate model, dose, route of administration and / or regimen. EXAMPLES

[0066] Example 1 - Cell culture analysis shows that CCNE1 overexpression correlates with the effectiveness of WEE1 inhibitors in arresting cell proliferation. Cyclin E1 (CCNE1) participates in cell cycle regulation by binding to cyclin-dependent kinases (CDKs), including CDK2, thereby promoting cell cycle progression. The ovarian cancer cell line SKOV3, which has low endogenous expression levels of CCNE1, was transduced with lentiviral vectors carrying CCNE1 or an empty vector control. Stable SKOV3 cell lines were established by puromycin selection. CCNE1 overexpression in stable SKOV3 cell lines compared to empty vector control cells was confirmed by Western blotting (Figure 1). These cells were then treated with increasing concentrations of ZN-c3 for 6 days and monitored for cell survival (Figure 2). Compared to control cells, cells overexpressing CCNE1 function were 3.1-fold more sensitive to WEE1 inhibition (Table 1). These results indicate that CCNE1 amplification enhances the efficacy of WEE1 inhibitors in cancer cells.

[0067] [Table 1]

[0068] Table 1 shows the statistical analysis of the results of the cell culture experiments shown in Figure 2 (IC 50 , μM) This is what was done.

[0069] Example 2 - Tumor cell growth analysis shows that cell lines with high copy numbers of CCNE1 correlate with the efficacy of WEE1 inhibitors in arresting cell proliferation. OVCAR-3 human ovarian cancer cell line has an amplified copy number of CCNE1 (Table 2), which correlates with high expression of CCNE1 protein. OVCAR-3 cells were treated with increasing concentrations of ZN-c3 for 72 hours and monitored for cell survival (Figure 3). ZN-c3-treated cells showed IC50 sensitivity of 395 nM (Table 3). These results indicate that high copy number of CCNE1 confers sensitivity to ZN-c3 treatment.

[0070] [Table 2]

[0071] Table 2 summarizes the copy numbers of CCNE1 in the cell lines used in Figures 3, 4, and 5. Data was obtained from the Catalogue Of Somatic Mutations In Cancer (COSMIC) database.

[0072] [Table 3]

[0073] Table 3 shows the statistical analysis of the results of the cell culture experiments shown in Figure 3 (IC 50 , μM).

[0074] Example 3 - In vivo tumor growth inhibition analysis shows that cell lines with high copy numbers of CCNE1 correlate with the efficacy of WEE1 inhibitors in halting tumor growth. Twenty million OVCAR-3 cells were inoculated into the right flank of ten 6- to 8-week-old female NOD / SCID mice. Animals were randomized and the mean tumor volume was 111 mm. 3 Treatment was initiated when tumor volume reached 100 μg / kg. Animals were treated daily with vehicle (20% HP-β-CD) or 80 mg / kg ZN-c3, and tumor volume and body weight were measured twice weekly. OVCAR-3 has an amplified copy number of CCNE1 (Table 2). Compared to vehicle-treated mice, ZN-c3-treated mice show a significant reduction in tumor growth after 28 days (Figure 4). These results indicate that high copy number of CCNE1 confers sensitivity to ZN-c3 treatment.

[0075] 1×10 6 HCC1806 cells were inoculated into the right flank of 10 6-8 week-old female BALB / c nude mice. The animals were randomized to obtain 1000 mice with a mean tumor volume of 155 mm. 3Treatment was initiated when the tumor volume reached 100 mg / kg. Animals were treated daily with vehicle (20% HP-β-CD) or 80 mg / kg ZN-c3, and tumor volume and body weight were measured twice weekly. HCC1806 human triple-negative breast cancer cell line has an amplified copy number of CCNE1 (Table 2). Compared to vehicle-treated mice, ZN-c3-treated mice show a significant reduction in tumor growth after 28 days (Figure 5). These results suggest that high copy numbers of CCNE1 are essential for the induction of tumor growth in ZN-c3-treated mice. 3 shows that it confers sensitivity to treatment.

[0076] Example 4 - Clinical trial demonstrates the superior ability of WEE1 inhibitors to treat cancer in subjects with cyclin E1 (CCNE1) gene amplification. The efficacy of WEE1 inhibitors in human subjects with CCNE1 gene amplification was evaluated by clinical trial.Based on existing genomic reports showing CCNE1 gene amplification, a human subject (Patient 1) with stage IVB gallbladder cancer who had received four prior therapies was enrolled in clinical trial ZN-c3-005.

[0077] Patient 1 began treatment with 300 mg QD of ZN-c3. Patient 1 was evaluated for response to ZN-c3 approximately 6 weeks later. Tumor progression was monitored using tumor imaging analysis, which showed a 38% reduction in measurable tumors. These results demonstrate the enhanced efficacy of WEE1 inhibition in human subjects with CCNE1 gene amplification in a clinical setting.

Claims

1. A method for determining the sensitivity of a biological sample obtained from a subject to a compound or a pharmaceutically acceptable salt thereof, comprising: performing or having performed at least one assay on the biological sample to determine whether the biological sample obtained from the subject has an altered function of CCNE1; wherein the compound is as follows: 【Chemical 1】 A method, which is a compound represented by.

2. A pharmaceutical composition for treating cancer, comprising an effective amount of a compound or a pharmaceutically acceptable salt thereof, which is administered to a subject having an altered function of CCNE1, wherein the subject is determined based on performing or having performed at least one assay on a biological sample obtained from the subject; wherein the compound is as follows: 【Chemical 2】 A pharmaceutical composition, which is a compound represented by.

3. A pharmaceutical composition for treating cancer, comprising an effective amount of a compound or a pharmaceutically acceptable salt thereof, which is administered to a subject having (a) cancer and (b) an endogenous or altered function of CCNE1, wherein the compound is as follows: A pharmaceutical composition, which is a compound represented by. 【Chemical Formula 3】

4. A pharmaceutical composition for treating cancer, comprising an effective amount of a compound or a pharmaceutically acceptable salt thereof, which is administered to a subject having (a) cancer and (b) overexpression or an altered function of CCNE1, wherein the compound is as follows: A pharmaceutical composition, which is a compound represented by.

5. 【Chemical Formula 4】 The method according to claim 1, wherein the altered function of CCNE1 is CCNE1 gene amplification.

6. The method according to claim 1, wherein the altered function of CCNE1 is CCNE1 protein overexpression.

7. The method according to claim 1, wherein the altered function of CCNE1 results from a CCNE1 gene mutation.

8. The pharmaceutical composition according to any one of claims 2 to 4, wherein the altered function of CCNE1 is CCNE1 gene amplification.

9. The pharmaceutical composition according to any one of claims 2 to 4, wherein the altered function of CCNE1 is CCNE1 protein overexpression.

10. The pharmaceutical composition according to any one of claims 2 to 4, wherein the altered function of CCNE1 results from a CCNE1 gene mutation.

11. The pharmaceutical composition according to any one of claims 2 to 4, wherein the cancer is a solid tumor or a hematological malignancy.

12. The pharmaceutical composition according to any one of claims 2 to 4, wherein the cancer is endometrial cancer, gallbladder cancer, or ovarian cancer. ​ ​

13. The pharmaceutical composition according to claim 12, wherein the ovarian cancer is epithelial ovarian cancer, germ cell cancer, or stromal cancer.

14. The pharmaceutical composition according to claim 13, wherein the epithelial ovarian cancer is high-grade serous ovarian cancer.

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