Combination therapy for treating cancer

Abstract

The present disclosure provides compounds of formula (I) and pharmaceutically acceptable salts thereof that inhibit phosphatidylinositol 4, 5-bisphosphate 3-kinase (PI3K) isoform alpha (PI3K alpha) for use in combination with additional therapeutic agents for the treatment of a condition, disease or condition in which increased (e.g., e.g., e.g., e.g., e.g., e.g., e.g., e.g., e.g., e.g., e.g., e.g., e.g., e.g., e.g., e.g., e.g., e.g., e.g., e.g., e.g., e.g., e.g., e.g., e.g., e.g., e.g., e.g., etc. Excessive) PI3K alpha activation contributes to a pathological condition and / or symptom and / or progression of a condition, disease or condition (e.g., cancer) in a subject.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of the filing dates of U.S. Provisional Application No. 63 / 421,082, filed October 31, 2022, U.S. Provisional Application No. 63 / 423,383, filed November 7, 2022, U.S. Provisional Application No. 63 / 488,674, filed March 6, 2023, and U.S. Provisional Application No. 63 / 531,990, filed August 10, 2023. The contents of each application are incorporated herein by reference in their entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing that has been submitted electronically as an XML file named 50006-0099WO1_ST26_SL.XML. The XML file was created on October 30, 2023 and is 2,946 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety. Technical Field

[0005] The present disclosure provides compounds of Formula (I) and pharmaceutically acceptable salts thereof that inhibit phosphatidylinositol 4,5-bisphosphate 3-kinase (PI3K) isoform alpha (PI3Kα) for use in combination with an additional therapeutic agent for treating a condition, disease, or disorder in which increased (e.g., excessive) PI3Kα activation contributes to the pathology and / or symptoms and / or progression of the condition, disease, or disorder (e.g., cancer) in a subject. Background Art

[0006] Phosphatidylinositol 4,5-bisphosphate 3-kinase (PI3K) isoform α (PI3Kα), encoded by the PIK3CA gene, is part of the PI3K / AKT / TOR signaling network and is altered in several human cancers. Several researchers have demonstrated that the role of PI3K / AKT signaling is involved in physiological and pathophysiological functions that drive tumor progression, such as metabolism, cell growth, proliferation, angiogenesis, and metastasis. See, Fruman, D Cell 2017, 170, 605–635 and Janku, F. et al., Nat. Rev. Clin. Oncol. 2018, 15, 273–291. Pharmacological or genetic inhibition of PI3K / AKT / TOR signaling may result in cancer cell death and regression of tumor growth.

[0007] The PI3K pathway can be activated, for example, by point mutations in the PIK3CA gene or by inactivation of the phosphatase and tensin homolog (PTEN) gene. Activation of this pathway occurs in approximately 30–50% of human cancers and contributes to resistance to various anticancer therapies. See, Martini, M. et al., Ann. Med. 2014, 46, 372–383 and Bauer, TM et al., Pharmacol. Ther. 2015, 146, 53–60. Summary of the Invention

[0008] Provided herein are methods for treating cancer in a subject in need thereof, comprising administering to the subject (a) Compound 1, or a pharmaceutically acceptable salt thereof, and (b) one or more additional therapeutic agents.

[0009] Provided herein are methods for treating cancer in a subject in need thereof, comprising administering to the subject (a) Compound 1 or a pharmaceutically acceptable salt thereof, and (b) one or more independently selected additional therapeutic agents selected from the group consisting of selective estrogen receptor modulators (SERMs) / selective estrogen receptor degraders (SERDs), CDK4 / 6 inhibitors, HER2 inhibitors, EGFR inhibitors, immune checkpoint inhibitors, MEK inhibitors, RAS inhibitors and RAF inhibitors, PIM ( Mo Loni's leukemia virus kinase Original Virus all binding sites, such as PIM1, PIM2, and PIM3) inhibitors, or combinations of any of the foregoing.

[0010] Also provided herein are pharmaceutical compositions comprising a compound of Formula (I) or a pharmaceutically acceptable salt thereof, one or more pharmaceutically acceptable excipients, and one or two independently selected additional therapeutic agents.

[0011] The present disclosure also provides a method for inhibiting PI3Kα in mammalian cells, the method comprising contacting mammalian cells with a compound of formula (I) or a pharmaceutically acceptable salt thereof and one or more additional therapeutic agents. In some embodiments, the one or more additional therapeutic agents are selective estrogen receptor modulators (SERMs) / selective estrogen receptor degraders (SERDs), CDK4 / 6 inhibitors, HER2 inhibitors, EGFR inhibitors, immune checkpoint inhibitors, MEK inhibitors, RAS inhibitors, RAF inhibitors, PIM (e.g., PIM1 and PIM3) inhibitors, or any combination thereof.

[0012] Other embodiments include those described in the detailed description and / or claims.

[0013] Additional definition

[0014] In order to promote the understanding of the disclosure as herein described, some other terms are defined below. Generally, the terms used herein and the laboratory procedures in organic chemistry, medicinal chemistry and pharmacology as herein described are those laboratory procedures well-known and commonly used in the art. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as those of ordinary skill in the art to which the present disclosure belongs. The patents, applications, disclosed applications and other publications mentioned throughout the specification and appendix are each incorporated herein by reference in their entirety.

[0015] When referring to a number or a numerical range, the term "about" means that the referenced number or numerical range is approximate, e.g., within experimental variability and / or statistical experimental error, and thus the number or numerical range may vary by up to ±10% of the stated number or numerical range.

[0016] As used herein, the term "acceptable" with respect to a formulation, composition or ingredient means having no persistent detrimental effect on the general health of the subject being treated.

[0017] The phrase "therapeutically effective amount" means an amount of a compound that, when administered to a subject in need of such treatment, is sufficient to (i) treat a PI3Kα protein-related disease or disorder, (ii) alleviate, ameliorate, or eliminate one or more symptoms of a specific disease, condition, or disorder, or (iii) delay the onset of one or more symptoms of a specific disease, condition, or disorder described herein. When used in reference to a treatment using more than one therapeutic agent, each agent can be administered independently in a therapeutically effective amount (e.g., an amount that would be therapeutically effective as a monotherapy), or one or more therapeutic agents can be administered together in a therapeutically effective amount (e.g., a therapeutically effective amount of a combination therapy) for the treatment of the indicated disease or disorder. In other words, the amount of the individual components of the therapeutically effective amount of the combination therapy can be independently administered in a therapeutically effective amount (in the combination) that is lower than when administered as a monotherapy.

[0018] The term "pharmaceutically acceptable excipient" means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, carrier, solvent or encapsulating material. In one embodiment, each component is "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of the pharmaceutical formulation and suitable for use in contact with human and animal tissues or organs without excessive toxicity, irritation, allergic response, immunogenicity or other problems or complications, commensurate with a reasonable benefit / risk ratio. See, e.g., Remington: The Science and Practice of Pharmacy, 21st ed.; Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 6th ed.; Rowe et al., eds.; The Pharmaceutical Press and the American Pharmaceutical Association: 2009; Handbook of Pharmaceutical Additives, 3rd ed.; Ash and Ash, eds.; Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, 2nd ed.; Gibson, ed.; CRC Press LLC: Boca Raton, FL, 2009.

[0019] The term "pharmaceutically acceptable salt" refers to a preparation of a compound that does not cause significant irritation to the organism to which it is applied, nor does it eliminate the biological activity and properties of the compound. In some cases, a pharmaceutically acceptable salt is obtained by reacting a compound described herein with an acid (e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, etc.). In some cases, a pharmaceutically acceptable salt is obtained by reacting a compound having an acidic group as described herein with a base to form a salt, such as an ammonium salt, an alkali metal salt (e.g., sodium salt or potassium salt), an alkaline earth metal salt (e.g., calcium salt or magnesium salt), a salt of an organic base (e.g., dicyclohexylamine, N-methyl-D-glucamine, tris(hydroxymethyl)methylamine), and a salt with an amino acid (e.g., arginine, lysine, etc.), or obtained by other methods previously determined. The pharmacologically acceptable salt is not particularly limited as long as it can be used in a medicament. Examples of salts formed with bases of the compounds described herein include salts thereof with inorganic bases such as sodium, potassium, magnesium, calcium, and aluminum; salts thereof with organic bases such as methylamine, ethylamine, and ethanolamine; salts thereof with basic amino acids such as lysine and ornithine; and ammonium salts. The salts may be acid addition salts, specifically exemplified by acid addition salts with mineral salts such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, and phosphoric acid; organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, methanesulfonic acid, and ethanesulfonic acid; and acidic amino acids such as aspartic acid and glutamic acid.

[0020] As used herein, "subject" refers to any animal, including mammals such as primates (e.g., humans), mice, rats, other rodents, rabbits, dogs, cats, pigs, cattle, sheep, horses, primates, and humans. In some embodiments, the subject is a human. In some embodiments, the subject has experienced and / or exhibits at least one symptom of the cancer to be treated.

[0021] "Treatment" or "therapy" of a subject refers to any type of intervention or process performed on a subject, or the administration of an active agent to a subject, with the purpose of reversing, alleviating, ameliorating, inhibiting, slowing down or preventing the onset, progression, development, severity or recurrence of symptoms, complications, conditions or biochemical indicators associated with a disease. In some embodiments, the disease is cancer. As used herein, when referring to, for example, the treatment of cancer, the terms "treatment" and "treating" are not intended to be absolute terms. For example, as used in a clinical setting, "treatment of cancer" and "treating cancer" are intended to include obtaining beneficial or desired clinical results, and may include improvements in the condition of a subject with cancer. Beneficial or desired clinical results include, but are not limited to, one or more of the following: reduction in proliferation (or destruction) of neoplastic or cancerous cells, inhibition of metastasis of neoplastic cells, reduction in metastases in a subject, reduction in or decrease in the size of a tumor, a change in the growth rate of one or more tumors in a subject, an increase in the duration of remission (partial or complete) in a subject (e.g., as compared to one or more metrics in subjects with similar cancer who have not received treatment or who have received a different treatment, or as compared to one or more metrics in the same subject before treatment), reduction in symptoms due to the disease, increase in the quality of life of those subjects with the disease (e.g., as assessed using FACT-G or EORTC-QLQC30), reduction in the doses of other medications required to treat the disease, delaying the progression of the disease, and / or prolonging the survival of a subject with the disease. "Treatment" may also mean prolonging survival as compared to expected survival if not receiving treatment.

[0022] The term "metastasis" is a term known in the art that refers to the spread of cancer cells from the location where they first formed (primary site) to one or more other sites (secondary sites) in a subject. In metastasis, cancer cells break away from the original (primary) tumor, travel through the blood or lymphatic system, and form new tumors (metastatic tumors) in other organs or tissues of the body. The new metastatic tumors include cancer cells that are identical or similar to the primary tumor. At the secondary site, tumor cells may proliferate and begin the growth or colonization of a secondary tumor at that distant site.

[0023] As used herein, the term "metastatic cancer" (also called "secondary cancer") refers to a type of cancer that originates in one tissue type but then spreads to one or more tissues other than the (primary) cancer's origin. Metastatic brain cancer refers to cancer in the brain, i.e., a cancer that originates in a tissue other than the brain and has metastasized to the brain. In some embodiments described herein, the subject has metastatic brain cancer and / or metastatic spinal cord cancer.

[0024] The term "tumor growth inhibition (TGI) index" refers to a value that indicates the extent to which an agent (e.g., Compound 1 or a pharmaceutically acceptable salt thereof, alone or in combination with one or more additional therapeutic agents as described herein) inhibits tumor growth when compared to an untreated control or a control treated with a monotherapy evaluated as part of a combination therapy. The TGI index is calculated for a specific time point (e.g., a specific day of an experiment or clinical trial) according to the following formula:

[0025]

[0026] Wherein "Tx Day 0" means the first day of administration of treatment (i.e., the first day of administration of the experimental therapy or the control therapy (e.g., vehicle only)), and "Tx Day X" means X days after Day 0. Typically, the mean volume for the treatment and control groups is used. As a non-limiting example, in an experiment in which study day 0 corresponds to "Tx Day 0" and the TGI index is calculated on study day 28 (i.e., "Tx Day 28"), if the mean tumor volume in both groups on study day 0 is 250 mm 3 , and the average tumor volume in the experimental and control groups was 125 mm 3 and 750mm 3 , the TGI index on the 28th day is 125%.

[0027] The compounds provided herein may encompass various stereochemical forms. Compounds also encompass mixtures of enantiomers (e.g., R and S isomers), diastereomers, and enantiomers (e.g., R and S isomers), including racemic mixtures and diastereomeric mixtures, as well as individual enantiomers and diastereomers that occur as a consequence of the structural asymmetry of certain compounds. Unless otherwise stated, when disclosed compounds are named or described by structure without specifying stereochemistry (e.g., "planar" structure) and with one or more chiral centers, it is understood that all possible stereoisomers representing the compound are represented. Similarly, unless otherwise stated, when disclosed compounds are named or described by structure with specified stereochemistry (e.g., structure with "wedge-shaped" and / or "dotted line" bonds) and with one or more chiral centers, it is understood that the stereoisomers representing the indicated compound are represented.

[0028] The compound of "Formula (I)" or a pharmaceutically acceptable salt thereof refers to:

[0029]

[0030] or a pharmaceutically acceptable salt thereof, wherein:

[0031] Z is O or NR x ;

[0032] R x is hydrogen, C1-C6 alkyl or C3-C6 cycloalkyl;

[0033] Each R 1 Independently selected from halogen, hydroxy, cyano, C1-C6 alkyl optionally substituted by hydroxy, and C3-C6 cycloalkyl;

[0034] m is 0, 1, 2, or 3;

[0035] R 2 is halogen, hydroxy, C1-C6 alkyl optionally substituted by hydroxy, C1-C6 haloalkyl, or C3-C6 cycloalkyl optionally substituted by 1 or 2 fluorine groups;

[0036] R 3 is C1-C6 alkyl, C1-C6 haloalkyl, or C3-C6 cycloalkyl optionally substituted by 1 or 2 substituents independently selected from fluorine and C1-C6 alkyl;

[0037] Ring A is a 6-10 membered aryl group, a C3-C8 cycloalkyl group, a 5-10 membered heteroaryl group or a 4-10 membered heterocyclyl group;

[0038] Each R 4 Independently selected from the following:

[0039] (i) halogen,

[0040] (ii) optionally substituted with 1 or 2 hydroxyl groups or -NR A R B Substituted C1-C6 alkyl,

[0041] (iii) C1-C6 alkoxy optionally substituted by 1-2 substituents independently selected from hydroxyl and C3-C6 cycloalkyl,

[0042] (iv) C1-C6 haloalkyl,

[0043] (v) hydroxyl groups,

[0044] (vi) cyano group,

[0045] (vii) -CO2H,

[0046] (viii)-NR A R B ,

[0047] (ix) = NR A2 ,

[0048] (x)-C(=O)NR C R D ,

[0049] (xi)-SO2(NR E R F ),

[0050] (xii) -SO2(C1-C6 alkyl),

[0051] (xiii) -S(=O)(=NH)(C1-C6 alkyl),

[0052] (xiv) -C(=O)(C1-C6 alkyl),

[0053] (xv) -CO2(C1-C6 alkyl),

[0054] (xvi) a 5-6 membered heteroaryl group optionally substituted by a C1-C6 alkyl group,

[0055] (xvii) optionally 1 or 2 independently selected R G substituted 3-9 membered heterocyclyl, and (xvii) optionally substituted by 1 or 2 independently selected R G substituted 3-6 membered cycloalkyl;

[0056] n is 0, 1, or 2;

[0057] Each R A 、R A1 、R B 、R B1 、R C 、R C1 、R D 、R D1 、R E and R F are independently (i) hydrogen,

[0058] (ii) hydroxyl groups,

[0059] (iii) a 4- to 6-membered heterocyclic group,

[0060] (iv) C1-C6 haloalkyl,

[0061] (v) -C(=O)(C1-C6 alkyl),

[0062] (vi) -C(=O)O(C1-C6 alkyl),

[0063] (vi) -SO2(C1-C6 alkyl),

[0064] (vii) a 3-6 membered cycloalkyl group optionally substituted by a hydroxy group, or

[0065] (ix) is optionally independently selected from hydroxy, -C(=O)NR B2 R C2, C1-C6 alkyl substituted with 1-2 substituents selected from the group consisting of -SO2(C1-C6 alkyl), -CO2H and -SO2(NH2); or

[0066] R C and R D , together with the nitrogen atom to which they are attached, form a 4-10 membered heterocyclic group, which is optionally independently selected from hydroxy, halogen, -C(=O)NR B1 R C1 , -SO2(C1-C6 alkyl), -CO2H, 1-2 substituents of C1-C6 alkyl, C1-C6 alkoxy and C1-C6 haloalkoxy optionally substituted by hydroxy;

[0067] Each R A2 、R B2 and R C2 are independently hydrogen or C1-C6 alkyl; and

[0068] Each R G are independently selected from the following: fluoro, cyano, hydroxy, C1-C6 alkyl optionally substituted with hydroxy, C1-C6 alkoxy, -NR A1 R B1 、=NR A2 、-C(=O)NR C1 R D1 , -CO2(C1-C6 alkyl), C1-C6 haloalkyl, C3-C6 cycloalkyl, C1-C6 haloalkoxy, -SO2(C1-C6 alkyl) and -CO2H.

[0069] “Compound 1” or a pharmaceutically acceptable salt thereof refers to (R)-1-(2-aminopyrimidin-5-yl)-3-(1-(5,7-difluoro-3-methylbenzofuran-2-yl)-2,2,2-trifluoroethyl)urea, or a pharmaceutically acceptable salt thereof, which has the following structure:

[0070] or a pharmaceutically acceptable salt thereof.

[0071] The details of one or more embodiments of the present disclosure are set forth in the accompanying drawings and the description below. Other features and advantages of the present disclosure will be apparent from the description, drawings, and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1A An isobologram of fulvestrant relative to compound 1 is shown. Figure 1B An isobologram of lapatinib relative to compound 1 is shown. Figure 1C An isobologram of abemaciclib relative to compound 1 is shown. Figure 1DSchematic representation of potential inhibitors of RAS pathway and MEK1 / 2 pathway genes. Figure 1E Shown are the results of a Profiling Relative Inhibition Simultaneously in Mixtures (PRISM) screen of drugs against wild-type and mutant MAPKs. Figure 1F An isobologram of trametinib relative to compound 1 is shown. Figure 1G Shown is the growth inhibition of MSI-H colon cancer cells when treated with Compound 1 and / or binimetinib.

[0073] Figure 2A Compound 1 was shown to reduce tumor volume in PDX models, and the reduction in tumor volume was more pronounced when compound 1 was administered in combination with fulvestrant. PDX model 1 measures PIK3CA H1047R , ER + Tumor volumes, where circles represent vehicle, diamonds represent fulvestrant 5 mg SC QW, triangles represent Compound 1 at 100 mg / kg PO QD, and squares represent Compound 1 + fulvestrant at 100 mg / kg. Figure 2B Tumor volumes before and after treatment with 100 mg / kg of Compound 1 + Fulvestrant are shown. Figure 2C Shown are tumor volumes before and after treatment with Compound 1 at 100 mg / kg POQD.

[0074] Figures 3A-3B Compound 1 was shown to reduce tumor volume in PDX models, and the reduction in tumor volume was more pronounced when compound 1 was administered in combination with palbociclib. H1047R / R108H , ER + / HER2 + Tumor volume, where circles represent vehicle, triangles represent Compound 1 POQD at 100 mg / kg, green circles represent Palbociclib POQD, and squares represent Compound 1 at 100 mg / kg + Palbociclib at 50 mg / kg. Figure 3B PDX model 3: PIK3CA is shown H1047R , ER + / HER2 + Tumor volume changes, where circles represent vehicle, triangles represent Compound 1 POQD at 100 mg / kg, green circles represent Palbociclib POQD, and squares represent Compound 1 at 100 mg / kg + Palbociclib at 50 mg / kg.

[0075] Figures 4A-4GShown in ER + Compound 1 combination therapy with fulvestrant and / or palbociclib in breast cancer models. Figure 4A Figure 2 shows the establishment of T47D xenograft tumors in female NSG mice. 3 Mice were randomized and treated as indicated. Data represent the percent change in tumor volume of individual tumors at day 20 relative to randomization. Figure 4B and 4C NSG mice bearing T47D tumors were shown to be given a single dose of vehicle or compound as indicated. Tumors were harvested 4 hours after treatment with Compound 1 and 24 hours after treatment with Fulvestrant for (4B) Western blotting of pAKT (S473) and S6 (S240 / 244), and (4C) IHC of AKT and S6, with pS6 and pAKT (S473) groups blotted and analyzed together. All samples for a given protein were run on the same gel (ge). Analysis of vinculin is shown. Images have been cropped to remove other compounds for clarity. 20X IHC images are shown with a 200 μm scale bar. Figure 4D The ST1056 xenograft tumors were established in BALB / c nude mice. 3 Mice were randomized to treatment groups as indicated. Mice were treated for 94 days or until removed from the study due to tumor volume. After 94 days, treatment was terminated and tumor regrowth was monitored. Figure 4E Shows about Figure 4D Percent change in body weight. Figure 4F and 4G Shows the Figure 4A T47D efficacy study (N=9) shown in Figure 2. Percent change in body weight (top) and tumor volume measurements (bottom) over time.

[0076] Figure 5A Inhibition of the enzymatic activities of WT PI3Kα and kinase domain and helical domain mutant proteins by alpelisib and compound 1 is shown, with geometric means and standard deviations shown. Figure 5B Surface plasmon resonance (SPR) sensorgrams showing compound 1 (upper panel) and duvelisib (lower panel) binding to WT, H1047R, and E545K mutant proteins are shown in the table below the sensorgrams. Figure 5C PI3Kα (p110 = purple, p85 = orange) is shown X-ray structure where GDC-0077 (red spheres) is bound in the ATP-binding site and compound 1 (green spheres) is bound in the allosteric site. Figure 5D Arrows indicate alignments between H1047R (cyan = PDB3HHm) and compound 1 binding structures (purple). 3HHM lacks residues 941-952 for direct comparison. Figure 5E A detailed view of bound compound 1 is shown, and residues significantly contributing to compound binding are labeled. Figure 5F The molecular structure of compound 1 is shown. 5G shows that compound 1 has broad kinome selectivity. Above, a table summarizing the kinome profile analysis of compound 1 compared with the published data for apellisin. AurB kinase is the only off-target with IC 50 <10 mM (-1658 nM). Lower panel, dose-dependent inhibition of phosphorylated H3 (Ser10) was observed 1 hour after treatment with the AurB kinase inhibitor balacetib. There was no significant inhibition of AurB observed with Compound 1 treatment.

[0077] Figures 6A-6F Shown is the PI3Kα mutant selectivity profile of compound 1 in a cellular assay. Figure 6A Correlation plots for Apellisin and Compound 1 are shown, comparing pAKTIC at 1 hour across the panel of cell lines. 50 and 72-hour activity (CTGlo) GI 50 . Figure 6B Shown are pAKT inhibition dose response curves (HTRF assay). Figure 6C Shown is a correlation plot comparing the potency of apellisin and Compound 1 (pAKTHTRF assay) in a panel of kinase domain mutant cell lines (orange dots) and WT PI3Kα SKBR3 (black dots). Figure 6D Compound 1 and apellisib AUC across CCLE from the Broad Institute's PRISM screen (theprismlab.org) are shown and grouped by PIK3CA mutation status. Figure 6E Shown are 2-hour glucose uptake in primary human adipocytes as indicated in the bar graph (percent vehicle response). 72-hour viability data for the H1047R-PI3Kα mutant T47D cell line are overlaid in the orange dose response curve for comparison.

[0078] Figures 7A-7D The effects of Compound 1 and apellisin on glucose homeostasis are shown. Figure 7A Shows no history of tumor Female BALB / c mice (n=5 / group) were dosed as indicated for 5 days and subjected to the insulin tolerance test (ITT). On day 5, animals were fasted for 6 hours and dosed as indicated 1 hour before the end of the fast (-1 hour). At T=0, animals were dosed with 0.75 U / kg intraperitoneal insulin. Blood glucose levels were monitored over time, with group means and standard error means shown. Figure 7B Shows the Figure 7A Calculated AUC. The AUC of each treatment group was compared to the vehicle group using ordinary one-way ANOVA and Dunnet's multiple comparison test. Figure 7C Shows something like Figure 7A Oral glucose tolerance test was performed using the ITT, except that at T=0, mice were dosed with 2 g / kg glucose and blood glucose was monitored over time, with group means and standard error of the mean indicated. Figure 7D Shows the Figure 7C The AUC was calculated from the data shown in Figure 7B analysis completed in ).

[0079] Figures 8A-8J Shown are the efficacy and pharmacodynamic profiles of Compound 1 relative to apellisin in CAL33 xenograft tumors. Figures 8A-8B The Cal33 xenograft tumors were established in female BALB / c nude mice. 3 Patients were randomized into treatment groups (n=6) at 4 hr and treated as indicated. Tumors (8A) and BW (8B) were measured twice weekly, with group means and standard error of the mean shown. Figures 8C-8D Cal33 tumor-bearing mice were treated as indicated for 3 days and serum insulin (8C) and blood glucose (8D) were measured, with individual values, group means, and standard error means shown for each treatment group compared to the vehicle group using ordinary one-way ANOVA and Dunnet's multiple comparison test. Figure 8E Figure 2 shows pAKT (S473) measured by Western blot in all Cal33 tumors from mice in a 28-day efficacy study (30- and 100-mg dose groups) and a 3-day PK / PD study (30-, 100-, and 300-mg / kg dose groups). pAKT (S473) and total AKT were measured in Cal33 tumors by Western blot. Normalized pAKT (S473) levels were plotted for unbound concentrations (blue). In vitro pAKT dose responses in Cal33 have been added for reference. Figure 8FShown are pAKT levels in Cal33 tumors, showing only Compound 1 (100 mg / kg) and Apellix (50 mg / kg) over time. Figure 8G The results of Western blot analysis are shown. Figures 8E-8F pAKT (S473) in the gastrocnemius muscles of mice with α-AKT-positive mice was analyzed using a standard one-way ANOVA and Dunnet's multiple comparison test to determine significance. Figure 8H N=4 mice were fasted for 4 hours and then administered with vehicle, Compound 1 (100 mg / kg), or Apellis (50 mg / kg). One hour later, mice were orally administered [U- 13 C]-glucose. After 30 minutes, tissues were collected and analyzed by MS. The abundance of labeled [M+4]succinate (S), fumarate (F), and malate (M) in tumors and gastrocnemius muscle is shown. Two-way ANOVA and Holm- Post hoc tests were performed on individual values, group means, and standard error means compared with the vehicle group. Figure 8I Plasma insulin measured before and 30 minutes after administration of labeled glucose is shown. Figure 8J Shows the Figure 8H Measurements of plasma from mice shown in Figure 2. Isotopic status indicates glucose labeled with U- 13 Animals from the 0-hour group were not given glucose; 13 C]-glucose. Samples collected when glucose was administered to mice were collected 30 min after administration of glucose. Bars represent SD.

[0080] Figure 9 Cal33 tumor-bearing mice were treated for 3 days as indicated and serum insulin was measured, with individual values, group means, and standard error means shown for each treatment group compared to the vehicle group using ordinary one-way ANOVA and Dunnet's multiple comparison test.

[0081] Figure 10 p-AKT and AKT WB analysis in efficacy groups. Tumor samples were analyzed by Western blot for total AKT and p-AKT. For each treatment group analyzed, the corresponding vehicle control samples were processed in parallel and run on the same gel and WB to minimize variation. The p-AKT:AKT ratio for the time-matched vehicle control was defined as 100%, and all treated samples were expressed relative to this and indicated in the corresponding scatter plots with the indicated mean and SEM.

[0082] Figures 11A-11IIt was shown that across PI3Kα mutant tumor xenografts, a metabolically sparing dose of Compound 1 was similarly efficacious as a high dose of apellix. Figures 11A-11C Shown are tumor volumes over time from PDX models (N=3 / group) treated with vehicle, compound 1, or apellis, which have PI3Kα mutations in the kinase domain (ST1056-H1047R; 11C), kinase and helical domains (ST1799-E542K / H1065L; 11B), and helical domains (ST2652-E545K; 11A). Statistical significance was calculated using a two-way ANOVA and Dunnett's multiple comparison test. End-of-study tumors were harvested 4 hours after the last dose and analyzed by Western blot for pAKT (S473). One of three tumors from model ST1799 was used to determine by IsoSeq that the E542K / H1065LPI3Kα mutation was cis. AKT and pAKT (S473) were blotted separately and vinculin analysis was shown. Figure 11D Shown are N=9 for NCI-H1048, Detroit562, and GP2D models; n=6 for model HCC1954. Percent change in body weight in PDX efficacy studies Figures 11A-11C N = 3 in each model; bars indicate SEM. Figure 11E Final percent tumor volume changes are shown for GP2D, Detroit562, NCI-H1048, and HCC 1954CDX tumors. Figures 11F-11I Shows the Figure 11E Tumor volume measurements (left) and percent change in body weight (right) over time in the CDX efficacy study.

[0083] Figure 12 Compound 1 exposure versus time following a single oral dose of 30, 100, or 300 mg / kg of Compound 1 in CD1 mice is shown. Total Exposure and IC 80 Potency was corrected for mouse PPB (fu = 0.033) and assay medium binding (10% FBS fu = 0.26), respectively. DETAILED DESCRIPTION

[0084] The present disclosure provides compounds of Formula (I) and pharmaceutically acceptable salts thereof that inhibit PI3Kα for use in combination with additional therapeutic agents for treating a condition, disease, or disorder in which increased PI3Kα activation contributes to the pathology and / or symptoms and / or progression of the condition, disease, or disorder (e.g., cancer) in a subject.

[0085] As described herein, compound 1 selectively targets the systemic metabolic dysfunction caused by apellix in mutant PI3Kα deficiency. Compared to apellix, compound 1 preserves glucose uptake in human adipocytes and does not induce systemic insulin resistance in vivo, as demonstrated by the lack of insulin spikes following long-term compound 1 administration in six independent CDX studies and OGTTs. Selective targeting of mutant PI3Kα was further demonstrated by the differential effects of compound 1 on pAKT / AKT inhibition in tumors and muscle, and by assessing glucose oxidation in these tissues using an isotope-labeled glucose OGTT. These data suggest a superior metabolic safety profile for compound 1 relative to apellix, which may enhance efficacy by suppressing the counter-regulatory insulin spike, which has limited efficacy in preclinical studies (Hopkins et al. Nature 2018;560(7719):499–503).

[0086] Compound 1 monotherapy was studied in a panel of ten CDX and PDX tumors, primarily breast and HNSCC tumors, with one example each for colon and lung cancer. This panel represents prevalent PI3Kα-mutated cancer types for which there is a significant unmet need for improved therapeutic options. Compound 1 100 mg / kg QD demonstrated robust efficacy similar to or better than high-dose apellisin in a xenograft model, with mouse apellisin exposure approximately twice that of patients (based on AUC using a 50 mg dose). 24h Compound 1 was equally effective in GP2D mutant colon cancer xenografts carrying the H1047L variant (the second most prevalent kinase domain mutation after H1047R). Importantly, treatment was highly effective in ST2652 and ST1799 PDX tumors carrying E545K and E542K mutations (the second and third most common mutation hotspots, respectively). Finally, both NCIH1048 and ST1799 tumors carry secondary PI3Kα mutations, which occur in approximately 10% of primary tumor samples, and both tumors responded favorably to Compound 1 treatment.

[0087] CDK4 / 6 inhibitors and anti-estrogen therapy are used for ER + An important standard of care treatment for breast cancer. The clear advantage of apellisin and fulvestrant combination therapy over fulvestrant monotherapy highlights why it is critical to test PI3Kα inhibitors in combination in preclinical models. + HER2 -In the BrCa CDX model, fulvestrant monotherapy provides a moderate level of tumor growth control, while low-dose compound 1 monotherapy and high-dose apellisib cause tumor stagnation. The combination of fulvestrant and low dose is superior to low-dose compound 1 monotherapy, with the regression in most animals. With or without fulvestrant combination therapy, high-dose compound 1 causes deep tumor regression. In the aggressive BrCaPDX model (ST1056), palbociclib, fulvestrant and compound 1 monotherapy and all combinations, including triple therapy, were studied. Compound 1 monotherapy provides a stable and lasting response in this model, far superior to fulvestrant or palbociclib monotherapy or its combination. However, the combination of fulvestrant and compound 1 provides excellent tumor growth control, wherein regression continues within 90 days of treatment in each animal, and maintains several weeks until the end of the study after stopping dosing. Although palbociclib demonstrated limited efficacy as a monotherapy or in combination with compound 1 in this PDX model, a triple combination therapy with compound 1 and fulvestrant was well tolerated within 90 days of treatment in mice. In contrast, the triple combination of apellisib with ribociclib and fulvestrant resulted in elevated hepatobiliary toxicity, as well as an increased incidence and severity of rash, and was intolerant in patients (Tolaney SM, et al. Clin Cancer Res 2021;27(2):418–28). Of note, the toxicity from the triple combination may be due to interference with drug metabolism rather than intolerance to the mechanism of action of the combination; given that another non-mutant selective PI3Kα inhibitor, inarilicept, appears to be tolerated in triple combinations (Bedard PL, et al. J Clin Oncol 2022;40(16_suppl):1052) and is in progress in a phase 3 study with a CDK4 / 6 inhibitor and fulvestrant (NCT04191499).

[0088] Treatment

[0089] Indications

[0090] Provided herein are methods for treating or preventing a disease or condition associated with dysregulation of the expression or activity or level of a PIK3CA gene, a PI3Ka protein, or any of them (i.e., a PI3Ka-associated disease or condition), such as PIK3CA-associated overgrowth syndrome ((PROS), see, e.g., Venot, et al., Nature, 558, 540-546 (2018)), a brain disorder (e.g., such as macrocephaly-capillary malformation (MCAP) and hemimegalencephaly), a congenital lipoma (e.g., an overgrowth of a vascular malformation), epidermal nevi and skeletal / spinal abnormalities (e.g., CLOVES syndrome) and fibrofatty hyperplasia (FH), or a cancer (e.g., a PI3Ka-associated cancer).

[0091] In some embodiments, provided herein are compounds that can exhibit potent and selective inhibition of PI3Kα. For example, provided herein are compounds that can bind to the helical phosphatidylinositol kinase homology domain catalytic domain of PI3Kα. In some embodiments, provided herein are compounds that can exhibit nanomolar potency against PI3Kα kinases comprising one or more mutations (e.g., mutations in Tables 1 and 2).

[0092] In some embodiments, provided herein are compounds that can show powerful and selective inhibition of mutant PI3K α. For example, provided herein are compounds that can be bound to allosteric sites in the kinase domain. In some embodiments, provided herein are compounds that can show nanomolar efficacy for PI3K α proteins including activating mutations, with minimal activity for related kinases (e.g., wild-type PI3K α). The inhibition of wild-type PI3K α can cause undesirable side effects (e.g., hyperglycemia and rash), which can affect quality of life and compliance. In some cases, the inhibition of wild-type PI3K α can lead to dose-limiting toxicity. See, for example, Hanker, et al., Cancer Disc. 2019, 9, 4, 482-491. Mutant selective inhibitors may reduce such dose-limiting toxicities observed with wild-type PI3K α inhibitors, including the risk of hyperglycemia.

[0093] In some embodiments, Compound 1 or a pharmaceutically acceptable salt thereof selectively targets PI3Kα. For example, Compound 1 or a pharmaceutically acceptable salt thereof selectively targets PI3Kα over another kinase or non-kinase target.

[0094] In some embodiments, compound 1 or a pharmaceutically acceptable salt thereof may exhibit greater inhibition of PI3K α containing one or more mutations as described herein (e.g., one or more mutations as described in Table 1 or Table 2) relative to the inhibition of wild-type PI3K α. In some embodiments, compound 1 or a pharmaceutically acceptable salt thereof may exhibit at least 2-fold, 3-fold, 5-fold, 10-fold, 25-fold, 50-fold, or 100-fold inhibition of PI3K α containing one or more mutations as described herein relative to the inhibition of wild-type PI3K α. In some embodiments, compound 1 or a pharmaceutically acceptable salt thereof may exhibit up to 1000-fold inhibition of PI3K α containing one or more mutations as described herein relative to the inhibition of wild-type PI3K α. In some embodiments, compound 1 or a pharmaceutically acceptable salt thereof may exhibit up to 10,000-fold inhibition of PI3K α with a combination of mutations described herein relative to the inhibition of wild-type PI3K α.

[0095] In some embodiments, Compound 1 or a pharmaceutically acceptable salt thereof selectively targets mutant PI3Kα (e.g., PI3Kα H1047R ) exceeded wild-type PI3Kα.

[0096] Compound 1 or a pharmaceutically acceptable salt thereof can be used to treat diseases and conditions that can be treated with PI3Kα inhibitors, such as PI3Kα-related diseases and conditions, such as PIK3CA-associated overgrowth syndrome (PROS) and proliferative disorders such as cancer, including hematological cancers and solid tumors (e.g., advanced or metastatic solid tumors).

[0097] In some embodiments, the subject has been identified or diagnosed as having a cancer (PI3Kα-associated cancer) with an imbalance of expression or activity or level of a PIK3CA gene, a PI3Kα protein, or any one thereof (e.g., as determined using an assay or kit approved by a regulatory agency, such as an FDA-approved assay). In some embodiments, the subject has a tumor that is positive for an imbalance of expression or activity or level of a PIK3CA gene, a PI3Kα protein, or any one thereof (e.g., as determined using an assay or kit approved by a regulatory agency). For example, the subject has a tumor that is positive for a mutation as described in Table 1 or Table 2. The subject can be a subject having a tumor that is positive for an imbalance of expression or activity or level of a PIK3CA gene, a PI3Kα protein, or any one thereof (e.g., identified as positive using an assay or kit approved by a regulatory agency, such as an FDA-approved assay). The subject can be a subject whose tumor has an imbalance of expression or activity or level of a PIK3CA gene, a PI3Kα protein, or any one thereof (e.g., where the tumor is identified as such using an assay or assay approved by a regulatory agency, such as an FDA-approved assay). In some embodiments, the subject is suspected of having a PI3Kα-associated cancer. In some embodiments, the subject has a clinical record indicating that the subject has a tumor with a dysregulation of the expression or activity or level of the PIK3CA gene, PI3Kα protein, or any of them (and optionally, the clinical record indicates that the subject should be treated with any of the compositions provided herein).

[0098] In some embodiments, the subject is a pediatric subject. See, e.g., Berhman RE, et al., Textbook of Pediatrics, 15th ed. Philadelphia: WB Saunders Company, 1996; Rudolph AM, et al., Rudolph's Pediatrics, 21st ed. New York: McGraw-Hill, 2002; and Avery and First, Pediatric Medicine, 2nd ed. Baltimore: Williams & Wilkins; 1994.

[0099] In certain embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof can be used to prevent diseases and conditions as defined herein (e.g., PIK3CA-associated overgrowth syndrome (PROS) and cancer). As used herein, the term "prevent" means to delay the onset, recurrence or spread of the whole or part of a disease or condition as described herein or its symptoms.

[0100] As used herein, the term "PI3Kα-associated disease or disorder" refers to a disease or disorder associated with or having a dysregulation of the expression or activity or level of a PIK3CA gene, a PI3Kα protein, or any one thereof (e.g., one or more) (e.g., any type of dysregulation of the expression or activity or level of a PIK3CA gene, or a PI3Kα protein, or any one thereof as described herein). Non-limiting examples of PI3Kα-associated diseases or disorders include, for example, PIK3CA-associated overgrowth syndrome (PROS), brain disorders (e.g., such as macrocephaly-capillary malformation (MCAP) and hemimegalencephaly), congenital lipomas (e.g., overgrowth of vascular malformations), epidermal nevi and bone / spinal abnormalities (e.g., CLOVES syndrome) and fibrofatty hyperplasia (FH), or cancer (e.g., PI3Kα-associated cancer).

[0101] As used herein, the term "PI3Kα-associated cancer" refers to a cancer associated with or having a dysregulation of the PIK3CA gene, PI3Kα protein, or the expression or activity or level of any of them. Non-limiting examples of PI3Kα-associated cancers are described herein.

[0102] The phrase "dysregulation of the expression or activity or level of the PIK3CA gene, PI3Kα protein, or any of them" refers to a genetic mutation (e.g., a mutation in the PIK3CA gene that results in the expression of a PI3Kα comprising a deletion of at least one amino acid compared to wild-type PI3Kα, a mutation in the PIK3CA gene that results in the expression of a PI3Kα having one or more point mutations compared to wild-type PI3Kα, a mutation in the PIK3CA gene that results in the expression of a PI3Kα having at least one insertion of an amino acid ... The invention relates to a method for the treatment of PI3Kα in mammalian cells, which comprises the following steps: (1) a gene duplication that causes an increase in the level of PI3Kα in the cell, or a mutation in a regulatory sequence (e.g., a promoter and / or enhancer) that causes an increase in the level of PI3Kα in the cell, (2) a PI3Kα mRNA that results in an alternative splicing form of PI3Kα having at least one amino acid deletion in PI3Kα compared to wild-type PI3Kα, or (3) an increase in the expression (e.g., level increase) of wild-type PI3Kα in mammalian cells due to abnormal cell signaling and / or autocrine / paracrine signaling dysregulation (e.g., compared to control non-cancerous cells). As another example, the PIK3CA gene, PI3Kα protein, or the dysregulation of the expression or activity or level of any one thereof can be a mutation in the PIK3CA gene that encodes a PI3Kα that is constitutively active or has increased activity compared to a protein encoded by a PIK3CA gene that does not include the mutation. Non-limiting examples of PI3Kα point mutations / substitutions / insertions / deletions are described in Tables 1 and 2.

[0103] The term "activating mutation" referring to PI3K α describes a mutation in the PIK3CA gene that results in, for example, when measured under the same conditions, for example, expression of PI3K α with increased kinase activity compared to wild-type PI3K α. For example, an activating mutation can be a mutation in the PIK3CA gene that results in, for example, when measured under the same conditions, for example, expression of PI3K α with increased kinase activity compared to wild-type PI3K α, the PI3K α having one or more (e.g., two, three, four, five, six, seven, eight, nine or ten) amino acid substitutions (e.g., any combination of any amino acid substitutions described herein). In another example, an activating mutation can be a mutation in PIK3CA that results in, for example, when measured under the same conditions, for example, expression of PI3K α with one or more (e.g., two, three, four, five, six, seven, eight, nine or ten) amino acid deletions compared to wild-type PI3K α. In another example, an activating mutation can be a mutation in the PIK3CA gene that results in, for example, expression of a PI3K α having at least one (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 18, or at least 20) amino acid insertion compared to wild-type PI3K α, such as the exemplary wild-type PI3K α described herein, when assayed under the same conditions. Additional examples of activating mutations are known in the art.

[0104] The terms "wild type" or "wild-type" describe a nucleic acid (e.g., PIK3CA gene or PI3Kα mRNA) or protein (e.g., PI3Kα) sequence that is typically found in subjects who do not have a disease or disorder associated with the reference nucleic acid or protein.

[0105] The term "wild type PI3Kα" or "wild-type PI3Kα" describes a normal PI3Kα nucleic acid (e.g., PIK3CA or PI3Kα mRNA) or protein found in a subject who does not have a PI3Kα-associated disease, such as a PI3Kα-associated cancer (and optionally does not also have an increased risk of developing a PI3Kα-associated disease and / or is not suspected of having a PI3Kα-associated disease), or found in cells or tissues from a subject who does not have a PI3Kα-associated disease, such as a PI3Kα-associated cancer (and optionally does not also have an increased risk of developing a PI3Kα-associated disease and / or is not suspected of having a PI3Kα-associated disease).

[0106] Provided herein are methods for treating cancer (e.g., PI3Kα-associated cancer) in a subject in need thereof, comprising administering to the subject a compound of formula (I) or a pharmaceutically acceptable salt thereof (e.g., Compound 1 or a pharmaceutically acceptable salt thereof), or a pharmaceutical composition thereof, and one or more independently selected additional therapeutic agents, as described herein. For example, provided herein are methods for treating PI3Kα-associated cancer in a subject in need thereof, comprising a) detecting a PIK3CA gene, a PI3Kα protein, or an expression or activity or level disorder of any one thereof in a sample from the subject; and b) administering Compound 1 or a pharmaceutically acceptable salt thereof and one or more independently selected additional therapeutic agents. In some embodiments, the PIK3CA gene, the PI3Kα protein, or an expression or activity or level disorder of any one thereof comprises one or more PI3Kα protein substitutions / point mutations / insertions. Non-limiting examples of PI3Kα protein substitutions / insertions / deletions are described in Tables 1 and 2.

[0107] Some embodiments provide methods of treating cancer in a subject in need thereof, comprising administering to the subject Compound 1, or a pharmaceutically acceptable salt thereof, and one or more independently selected additional therapeutic agents.

[0108] Some embodiments provide methods of treating cancer in a subject in need thereof, comprising administering to the subject a combination therapy comprising Compound 1, or a pharmaceutically acceptable salt thereof, and one or more independently selected additional therapeutic agents.

[0109] Some embodiments provide a method of treating cancer in a subject in need thereof, comprising:

[0110] (a) testing or having tested a subject to determine that the subject has a PI3Kα-associated cancer, and

[0111] (b) administering Compound 1, or a pharmaceutically acceptable salt thereof, and one or more independently selected additional therapeutic agents to the subject.

[0112] Some embodiments provide a method of treating cancer in a subject in need thereof, comprising:

[0113] (a) determining that the subject has a PI3Kα-associated cancer; and

[0114] (b) administering Compound 1, or a pharmaceutically acceptable salt thereof, and one or more independently selected additional therapeutic agents to the subject.

[0115] Some embodiments provide a method of treating cancer in a subject in need thereof, comprising:

[0116] (a) determining that the subject has a PI3Kα-associated cancer; and

[0117] (b) administering to the subject a combination therapy comprising Compound 1, or a pharmaceutically acceptable salt thereof, and one or more independently selected additional therapeutic agents.

[0118] Some embodiments provide methods of treating cancer in a subject previously identified as having a PI3Kα-associated cancer, comprising administering to the subject Compound 1, or a pharmaceutically acceptable salt thereof, and one or more independently selected additional therapeutic agents.

[0119] Some embodiments provide methods of treating cancer in a subject previously identified as having a PI3Kα-associated cancer, comprising administering to the subject a combination therapy comprising Compound 1, or a pharmaceutically acceptable salt thereof, and one or more additional therapeutic agents.

[0120] Some embodiments provide a method of treating cancer in a subject in need thereof, comprising administering to the subject

[0121] (a) Compound 1 or a pharmaceutically acceptable salt thereof, and

[0122] (b) one or more independently selected additional therapeutic agents selected from the following: selective estrogen receptor modulators (SERM) / selective estrogen receptor degraders (SERD), CDK4 / 6 inhibitors, HER2 inhibitors, EGFR inhibitors, immune checkpoint inhibitors, MEK inhibitors, RAS inhibitors and RAF inhibitors, PIM (e.g., PIM1 and PIM3) inhibitors or any combination thereof. In some embodiments, compound 1 or a pharmaceutically acceptable salt thereof is administered together with a further therapeutic agent. In some embodiments, compound 1 or a pharmaceutically acceptable salt thereof is administered together with two independently selected further therapeutic agents.

[0123] Some embodiments provide methods of treating cancer in a subject in need thereof, comprising administering to the subject a combination therapy comprising:

[0124] (a) Compound 1 or a pharmaceutically acceptable salt thereof, and

[0125] (b) one or more independently selected additional therapeutic agents selected from the following: selective estrogen receptor modulators (SERM) / selective estrogen receptor degraders (SERD), CDK4 / 6 inhibitors, HER2 inhibitors, EGFR inhibitors, immune checkpoint inhibitors, MEK inhibitors, RAS inhibitors and RAF inhibitors, PIM (e.g., PIM1 and PIM3) inhibitors or any combination thereof. In some embodiments, compound 1 or a pharmaceutically acceptable salt thereof is administered together with a further therapeutic agent. In some embodiments, compound 1 or a pharmaceutically acceptable salt thereof is administered together with two independently selected further therapeutic agents.

[0126] Some embodiments provide a method of treating cancer in a subject in need thereof, comprising:

[0127] (a) testing or having tested a subject to determine that the subject has a PI3Kα-associated cancer, and

[0128] (b) Compound 1 or its pharmaceutically acceptable salt is administered to the subject, and a selective estrogen receptor modulator (SERM) / selective estrogen receptor degrader (SERD), CDK4 / 6 inhibitor, HER2 inhibitor, EGFR inhibitor, immune checkpoint inhibitor, MEK inhibitor, RAS inhibitor, RAF inhibitor, PIM (e.g., PIM1 and PIM3) inhibitor or any combination thereof. In some embodiments, compound 1 or its pharmaceutically acceptable salt is administered together with an additional therapeutic agent. In some embodiments, compound 1 or its pharmaceutically acceptable salt is administered together with two independently selected additional therapeutic agents.

[0129] Some embodiments provide a method of treating cancer in a subject in need thereof, comprising:

[0130] (a) determining that the subject has a PI3Kα-associated cancer; and

[0131] (b) Compound 1 or its pharmaceutically acceptable salt is administered to the subject, and a selective estrogen receptor modulator (SERM) / selective estrogen receptor degrader (SERD), CDK4 / 6 inhibitor, HER2 inhibitor, EGFR inhibitor, immune checkpoint inhibitor, MEK inhibitor, RAS inhibitor, RAF inhibitor, PIM (e.g., PIM1 and PIM3) inhibitor or any combination thereof. In some embodiments, compound 1 or its pharmaceutically acceptable salt is administered together with an additional therapeutic agent. In some embodiments, compound 1 or its pharmaceutically acceptable salt is administered together with two independently selected additional therapeutic agents.

[0132] Some embodiments provide a method of treating cancer in a subject in need thereof, comprising:

[0133] (a) determining that the subject has a PI3Kα-associated cancer; and

[0134] (b) administering to the subject a combination therapy comprising: compound 1 or a pharmaceutically acceptable salt thereof, and a selective estrogen receptor modulator (SERM) / selective estrogen receptor degrader (SERD), a CDK4 / 6 inhibitor, a HER2 inhibitor, an EGFR inhibitor, an immune checkpoint inhibitor, a MEK inhibitor, a RAS inhibitor, a RAF inhibitor, a PIM (e.g., PIM1 and PIM3) inhibitor, or any combination thereof. In some embodiments, compound 1 or a pharmaceutically acceptable salt thereof is administered together with an additional therapeutic agent. In some embodiments, compound 1 or a pharmaceutically acceptable salt thereof is administered together with two independently selected additional therapeutic agents.

[0135] Some embodiments provide a method of treating cancer in a subject previously identified as having a PI3Kα-associated cancer comprising administering to the subject

[0136] (a) Compound 1 or a pharmaceutically acceptable salt thereof, and

[0137] (b) one or more independently selected additional therapeutic agents selected from the following: selective estrogen receptor modulators (SERM) / selective estrogen receptor degraders (SERD), CDK4 / 6 inhibitors, HER2 inhibitors, EGFR inhibitors, immune checkpoint inhibitors, MEK inhibitors, RAS inhibitors and RAF inhibitors, PIM (e.g., PIM1 and PIM3) inhibitors or any combination thereof. In some embodiments, compound 1 or a pharmaceutically acceptable salt thereof is administered together with a further therapeutic agent. In some embodiments, compound 1 or a pharmaceutically acceptable salt thereof is administered together with two independently selected further therapeutic agents.

[0138] Some embodiments provide a method for treating a cancer previously determined to be in a subject with a PI3Kα-related cancer, comprising administering to the subject a combination therapy comprising: Compound 1 or a pharmaceutically acceptable salt thereof, and a selective estrogen receptor modulator (SERM) / selective estrogen receptor degrader (SERD), a CDK4 / 6 inhibitor, a HER2 inhibitor, an EGFR inhibitor, an immune checkpoint inhibitor, a MEK inhibitor, a RAS inhibitor, a RAF inhibitor, a PIM (e.g., PIM1 and PIM3) inhibitor, or any combination thereof. In some embodiments, Compound 1 or a pharmaceutically acceptable salt thereof is administered together with an additional therapeutic agent. In some embodiments, Compound 1 or a pharmaceutically acceptable salt thereof is administered together with two independently selected additional therapeutic agents.

[0139] In some embodiments, the one or more PI3Kα protein substitutions / insertions / deletions are selected from E542A, E542G, E542K, E542Q, E542V, E545A, E545D, E545G, E545K, E545Q, M1043I, M1043L, M1043T, M1043V, H1047L, H1047Q, H1047R, H1047Y, G1049R, and combinations thereof. In some embodiments, the PI3Kα protein substitutions / insertions / deletions are H1047X, wherein X is any amino acid. In some embodiments, the PI3Kα protein substitutions / insertions / deletions are H1047R. In some embodiments, the one or more PI3Ka protein substitutions / insertions / deletions are selected from E542A, E542G, E542K, E542Q, E542V, E545A, E545D, E545G, E545K, E545Q, and H1047R.

[0140] In some embodiments, the cancer (eg, a PI3Kα-associated cancer) is selected from a hematological cancer and a solid tumor.

[0141] In some embodiments, the cancer is refractory to one or more prior therapies. In some embodiments, the subject has experienced dose-limiting toxicity to one or more prior therapies. In some embodiments, the one or more prior therapies comprise a PI3K inhibitor.

[0142] In some embodiments, the cancer is unresectable and / or metastatic.

[0143] In some embodiments, the cancer is locally advanced. In some embodiments, the cancer is unresectable. In some embodiments, the cancer is metastatic. In some embodiments, the cancer is metastatic brain cancer, as described herein. In some embodiments, the cancer is metastatic spinal cord cancer, as described herein.

[0144] In some embodiments, the cancer is selected from breast cancer (including HER2 + and HER2 - Breast cancer, ER + breast cancer and triple-negative breast cancer), endometrial cancer, lung cancer (including adenocarcinoma lung cancer and squamous cell lung cancer), esophageal cancer (including esophageal squamous cell carcinoma), ovarian cancer, colorectal cancer, esophageal adenocarcinoma, gastric cancer, bladder cancer, head and neck cancer (including head and neck squamous cell carcinoma, such as oropharyngeal squamous cell carcinoma), thyroid cancer, glioma, cervical cancer, lymphangioma, meningioma, melanoma (including uveal melanoma), prostate cancer, kidney cancer, pancreatic neuroendocrine neoplasm (pNET), gastric cancer, esophageal cancer, acute myeloid leukemia, relapsed and refractory multiple myeloma, and pancreatic cancer.

[0145] In some embodiments, the cancer is selected from breast cancer (including HER2 + and HER2 - Breast cancer, ER + breast cancer and triple-negative breast cancer), colon cancer, rectal cancer, colorectal cancer, ovarian cancer, lymphangioma, meningioma, head and neck squamous cell carcinoma (including oropharyngeal squamous cell carcinoma), melanoma (including uveal melanoma), renal cancer, pancreatic neuroendocrine neoplasm (pNET), gastric cancer, esophageal cancer, acute myeloid leukemia, relapsed and refractory multiple myeloma, pancreatic cancer, lung cancer (including adenocarcinoma lung cancer and squamous cell lung cancer), and endometrial cancer.

[0146] In some embodiments, the cancer is a gynecological cancer. In some embodiments, the gynecological cancer is endometrial cancer, ovarian cancer, or cervical cancer.

[0147] In some embodiments, the cancer is endometrial cancer without DNA mismatch repair (dMMR) deficiency, ie, the endometrial cancer is not dMMR.

[0148] In some embodiments, the cancer is selected from breast cancer, lung cancer, endometrial cancer, esophageal cancer, gastric cancer, ovarian cancer, colorectal cancer, bladder cancer, head and neck cancer, thyroid cancer, prostate cancer, glioma, and cervical cancer.

[0149] In some embodiments, the cancer is breast cancer.

[0150] In some embodiments, the cancer is lung cancer.

[0151] In some embodiments, the cancer is endometrial cancer.

[0152] In some embodiments, the cancer is esophageal cancer.

[0153] In some embodiments, the cancer is gastric cancer.

[0154] In some embodiments, the cancer is ovarian cancer.

[0155] In some embodiments, the cancer is colorectal cancer.

[0156] In some embodiments, the cancer is bladder cancer.

[0157] In some embodiments, the cancer is head and neck cancer.

[0158] In some embodiments, the cancer is thyroid cancer.

[0159] In some embodiments, the cancer is prostate cancer.

[0160] In some embodiments, the cancer is glioma.

[0161] In some embodiments, the cancer is cervical cancer.

[0162] In some embodiments, the cancer described herein is HER2 + Cancer. In some embodiments, the cancer described herein is HER2 - cancer.

[0163] In some embodiments, the cancer described herein is a HER2-low cancer. In some embodiments, the cancer has a HER2 score (e.g., IHC score) of 0, +1, or +2. In some embodiments, the cancer has a HER2 score (e.g., IHC score) of 0 or +1. In some embodiments, the cancer has a HER2 score of 0. In some embodiments, the cancer has a HER2 score of +1. In some embodiments, the cancer has a HER2 score of +2. In some embodiments, the cancer has a HER2 score of +2 or +3. In some embodiments, the cancer has a HER2 score of +3.

[0164] In some embodiments, the cancers described herein are hormone receptor positive (HR + ) cancer. In some embodiments, the cancer described herein is HER2 - and HR + Cancer. In some embodiments, the cancer described herein is ER + In some embodiments, the cancer described herein is PR + In some embodiments, the cancer is HR + / ER - Breast cancer.

[0165] In some embodiments, a cancer described herein is HR+ and has a HER2 score of 0 or + 1. In some embodiments, a cancer described herein is HR+ and has a HER2 score of 0. In some embodiments, a cancer described herein is HR+ and has a HER2 score of +1.

[0166] In some embodiments, the cancers described herein are HR+ and have a HER2 score of +2 or +3. In some embodiments, the cancers described herein are HR+ and have a HER2 score of +2. In some embodiments, the cancers described herein are HR+ and have a HER2 score of +3.

[0167] In some embodiments, the subject has been previously identified or determined to not have an activating mutation in AKT and / or PTEN.

[0168] In some embodiments, the PI3Kα-associated cancer is selected from the cancers described in Table 1 and Table 2.

[0169] Table 1. PI3Kα protein amino acid substitutions / insertions / deletions A

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179]

[0180]

[0181]

[0182]

[0183]

[0184]

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192] A Unless otherwise indicated, the mutations in Table 1 were found in the cBioPortal database derived from Cerami et al. The cBio Cancer Genomics Portal: An Open Platform for Exploring Multidimensional Cancer Genomics Data. Cancer Discovery. May 20122;401; and Gao et al. Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal. Sci. Signal. 6, p11 (2013).

[0193] Velho S, Oliveira C, Ferreira A, Ferreira AC, Suriano G, Schwartz S Jr, Duval A, Carneiro F, Machado JC, Hamelin R, Seruca R. The prevalence of PIK3CA mutations in gastric and colon cancer. Eur J Cancer. 2005 Jul;41(11):1649-54.doi:10.1016 / j.ejca.2005.04.022.PMID:15994075.

[0194] Table 2. Additional PI3Kα protein amino acid substitutions / insertions / deletions A

[0195]

[0196]

[0197]

[0198]

[0199] A Unless otherwise indicated, the mutations in Table 2 were found in the cBioPortal database derived from Cerami et al. The cBio Cancer Genomics Portal: An Open Platform for Exploring Multidimensional Cancer Genomics Data. Cancer Discovery. May 2012 2;401; and Gao et al. Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal. Sci. Signal. 6, p11 (2013).

[0200] Velho S, Oliveira C, Ferreira A, Ferreira AC, Suriano G, Schwartz S Jr, Duval A, Carneiro F, Machado JC, Hamelin R, Seruca R. The prevalence of PIK3CA mutations in gastric and colon cancer. Eur J Cancer. 2005 Jul;41(11):1649-54.doi:10.1016 / j.ejca.2005.04.022.PMID:15994075.

[0201] In some embodiments, dysregulation of the expression or activity or level of the PIK3CA gene, PI3Kα protein, or any thereof comprises a splice variation in the PI3Kα mRNA that results in the expression of a protein that is an alternatively spliced ​​variant of PI3Kα having at least one residue deleted (compared to the wild-type PI3Kα protein) that results in constitutive activity of the PI3Kα protein domain.

[0202] In some embodiments, the PIK3CA gene, PI3Kα protein, or the dysregulation of expression, activity, or level of any one thereof comprises at least one point mutation in the PIK3CA gene, which results in the production of a PI3Kα protein having one or more amino acid substitutions, insertions, or deletions in the PIK3CA gene, which results in the production of a PI3Kα protein having one or more amino acid insertions or deletions compared to the wild-type PI3Kα protein. In some cases, the resulting mutant PI3Kα protein has increased activity compared to the wild-type PI3Kα protein or the PI3Kα protein that does not include the same mutation. In some embodiments, the compounds described herein selectively inhibit the resulting mutant PI3Kα protein relative to the wild-type PI3Kα protein or the PI3Kα protein that does not include the same mutation.

[0203] Exemplary sequence of human phosphatidylinositol 4,5-bisphosphate 3-kinase isoform alpha (UniProtKB entry P42336) (SEQ ID NO: 1)

[0204]

[0205] In some embodiments, compound 1 or a pharmaceutically acceptable compound thereof can be used to treat cancers that have been identified as having one or more PI3Kα mutations. Accordingly, provided herein are methods for treating subjects diagnosed with (or identified as having) cancer, comprising administering compound 1 or a pharmaceutically acceptable salt thereof, and one or more additional therapeutic agents to the subject. In some embodiments, compound 1 or a pharmaceutically acceptable compound thereof is capable of crossing the blood-brain barrier (BBB) ​​and inhibiting mutant PI3Kα in the brain and / or other central nervous system (CNS) structures. In some embodiments, compound 1 or a pharmaceutically acceptable salt thereof is capable of crossing the BBB in a therapeutically effective amount.

[0206] Also provided herein are methods for treating subjects identified or diagnosed as having PI3Kα-associated cancers, comprising administering compound 1 or a pharmaceutically acceptable salt thereof, and one or more additional therapeutic agents to the subject. In some embodiments, the subject has been identified or diagnosed as having PI3Kα-associated cancers by using a regulatory agency-approved, e.g., FDA-approved test or assay for identifying a PIK3CA gene, PI3Kα protein, or disorder of expression or activity or level of any one thereof in a subject or a biopsy sample from a subject, or performing any non-limiting example of an assay described herein. In some embodiments, the test or assay is provided as a kit. In some embodiments, the cancer is a PI3Kα-associated cancer.

[0207] The term "regulatory agency" refers to an agency in a country that approves pharmaceutical agents for medical use in that country. For example, a non-limiting example of a regulatory agency is the US Food and Drug Administration (FDA).

[0208] Also provided is a method for treating cancer in a subject in need thereof, the method comprising: (a) detecting PI3Kα-associated cancer in the subject; and (b) administering Compound 1 or a pharmaceutically acceptable salt thereof, and one or more additional therapeutic agents to the subject. In some embodiments, the subject has previously been treated with another anti-cancer treatment, such as at least partial resection of a tumor or radiotherapy. In some embodiments, the subject is determined to have PI3Kα-associated cancer by: using a regulatory agency-approved, such as an FDA-approved test or assay for identifying a PIK3CA gene, PI3Kα protein, or an expression or activity or level disorder of any one thereof in a biopsy sample from a subject or from a subject, or performing any non-limiting example of an assay described herein. In some embodiments, the test or assay is provided as a kit. In some embodiments, the cancer is a PI3Kα-associated cancer.

[0209] Also provided is a method for treating a subject, comprising performing an assay on a sample obtained from the subject to determine whether the subject has a PIK3CA gene, a PI3Kα protein, or an expression or activity or level disorder of any one thereof, and administering Compound 1 or a pharmaceutically acceptable salt thereof, and one or more additional therapeutic agents (e.g., specifically or selectively) to a subject determined to have a PIK3CA gene, a PI3Kα protein, or an expression or activity or level disorder of any one thereof. In some embodiments of these methods, the subject has previously been treated with another anti-cancer treatment, such as at least partial resection of a tumor or radiotherapy. In some embodiments, the subject is a subject suspected of having a PI3Kα-related cancer, a subject presenting one or more symptoms of a PI3Kα-related cancer, or a subject with an increased risk of developing a PI3Kα-related cancer. In some embodiments, the assay utilizes next-generation sequencing, pyrosequencing, immunohistochemistry, or decomposition FISH analysis. In some embodiments, the assay is an assay approved by a regulatory agency, such as an FDA-approved kit. In some embodiments, the assay is a liquid biopsy. Additional non-limiting assays that may be used in these methods are described herein. Additional assays are also known in the art.

[0210] Also provided are Compound 1, or a pharmaceutically acceptable salt thereof, and one or more additional therapeutic agents for use in treating a PI3Kα-associated cancer in a subject identified or diagnosed as having a PI3Kα-associated cancer by performing an assay (e.g., an in vitro assay) on a sample obtained from the subject to determine whether the subject has a dysregulation of the expression, activity, or level of the PIK3CA gene, the PI3Kα protein, or any of them, wherein the presence of a dysregulation of the expression, activity, or level of the PIK3CA gene, the PI3Kα protein, or any of them identifies the subject as having a PI3Kα-associated cancer. Also provided is the use of compound 1 or its pharmaceutically acceptable salt, and one or more additional therapeutic agents for the manufacture of a medicament for treating PI3Kα-related cancer in a subject identified or diagnosed as having PI3Kα-related cancer by the following steps: performing an assay on a sample obtained from the subject to determine whether the subject has a PIK3CA gene, PI3Kα protein, or an expression or activity or level disorder of any one thereof, wherein the presence of an expression or activity or level disorder of the PIK3CA gene, PI3Kα protein, or any one thereof identifies the subject as having PI3Kα-related cancer. Some embodiments of any method or use described herein further include recording in the subject's clinical record (e.g., a computer-readable medium) that the subject is determined to have a PIK3CA gene, PI3Kα protein, or an expression or activity or level disorder of any one thereof by performing an assay, and compound 1 or its pharmaceutically acceptable salt, and one or more additional therapeutic agents should be administered. In some embodiments, the assay utilizes next-generation sequencing, pyrosequencing, immunohistochemistry, or decomposition FISH analysis. In some embodiments, the assay is an assay approved by a regulatory agency, such as an FDA-approved kit. In some embodiments, the assay is a liquid biopsy.

[0211] Compound 1 or a pharmaceutically acceptable salt thereof, and one or more additional therapeutic agents are also provided for treating a subject in need thereof or for identifying or diagnosing cancer in a subject with PI3Kα-related cancer. Also provided is the use of Compound 1 or a pharmaceutically acceptable salt thereof, and one or more additional therapeutic agents for the manufacture of a medicament for treating cancer in a subject identified or diagnosed as having PI3Kα-related cancer. In some embodiments, the subject is identified or diagnosed as having PI3Kα-related cancer by using a regulatory agency-approved, e.g., FDA-approved, kit for identifying a PIK3CA gene, PI3Kα protein, or an expression or activity or level disorder in a subject or a biopsy sample from a subject. As provided herein, PI3Kα-related cancers include those described herein and known in the art.

[0212] In some embodiments, the subject has been identified or diagnosed as having a cancer with a PIK3CA gene, PI3Kα protein, or a disorder of expression, activity, or level of any one thereof. In some embodiments, the subject has a tumor that is positive for a disorder of expression, activity, or level of PIK3CA gene, PI3Kα protein, or any one thereof. In some embodiments, the subject may be a subject with a tumor that is positive for a disorder of expression, activity, or level of PIK3CA gene, PI3Kα protein, or any one thereof. In some embodiments, the subject may be a subject whose tumor has a disorder of expression, activity, or level of PIK3CA gene, PI3Kα protein, or any one thereof. In some embodiments, the subject is suspected of having a PI3Kα-related cancer. In some embodiments, provided herein are methods for treating a PI3Kα-related cancer in a subject in need of such treatment, the methods comprising a) detecting a disorder of expression, activity, or level of PIK3CA gene, PI3Kα protein, or any one thereof in a sample from the subject; and b) administering Compound 1 or a pharmaceutically acceptable salt thereof, and one or more additional therapeutic agents. In some embodiments, the PIK3CA gene, PI3Kα protein, or the dysregulation of expression, activity, or level of any one thereof comprises one or more PI3Kα protein point mutations / insertions / deletions. Non-limiting examples of PI3Kα protein point mutations / insertions / deletions are described in Tables 1 and 2. In some embodiments, the PI3Kα protein point mutation / insertion / deletion is H1047X, wherein X is any amino acid. In some embodiments, the PI3Kα protein point mutation / insertion / deletion is selected from E542A, E542G, E542K, E542Q, E542V, E545A, E545D, E545G, E545K, E545Q, M1043I, M1043L, M1043T, M1043V, H1047L, H1047Q, H1047R, H1047Y, and G1049R. In some embodiments, a regulatory agency-approved, e.g., FDA-approved assay or kit is used to identify cancers with dysregulation of the expression, activity, or level of the PIK3CA gene, PI3Kα protein, or any thereof. In some embodiments, a regulatory agency-approved, e.g., FDA-approved assay or kit is used to identify tumors with dysregulation of the expression, activity, or level of the PIK3CA gene, PI3Kα protein, or any thereof.

[0213] In some embodiments, the subject has a clinical record indicating that the subject has a tumor with a PIK3CA gene, a PI3Kα protein, or a disorder of the expression, activity, or level of any one thereof. Also provided is a method of treating a subject, comprising administering Compound 1 or a pharmaceutically acceptable salt thereof, and one or more additional therapeutic agents to the subject, wherein the subject has a clinical record indicating that the subject has a PIK3CA gene, a PI3Kα protein, or a disorder of the expression, activity, or level of any one thereof.

[0214] In some embodiments, the methods provided herein include performing an assay on a sample obtained from a subject to determine whether the subject has a PIK3CA gene, a PI3Kα protein, or an expression or activity or level disorder of any one thereof. In some such embodiments, the method further includes administering compound 1 or a pharmaceutically acceptable salt thereof, and one or more additional therapeutic agents to a subject determined to have a PIK3CA gene, a PI3Kα protein, or an expression or activity or level disorder of any one thereof. In some embodiments, the method includes determining via an assay performed on a sample obtained from a subject to determine whether the subject has a PIK3CA gene, a PI3Kα protein, or an expression or activity or level disorder of any one thereof. In such embodiments, the method further includes administering compound 1 or a pharmaceutically acceptable salt thereof, and one or more additional therapeutic agents to the subject. In some embodiments, the PIK3CA gene, the PI3Kα protein, or an expression or activity or level disorder of any one thereof is one or more point mutations in the PIK3CA gene (e.g., any one of one or more PI3Kα point mutations described herein). One or more point mutations in the PIK3CA gene can result in, for example, translation of a PI3Kα protein having one or more of the following amino acid substitutions, deletions, and insertions: E542A, E542G, E542K, E542Q, E542V, E545A, E545D, E545G, E545K, E545Q, M1043I, M1043L, M1043T, M1043V, H1047L, H1047Q, H1047R, H1047Y, and G1049R. One or more mutations in the PIK3CA gene can result in translation of a PI3Kα protein having, for example, one or more of the following amino acid substitutions, deletions, and insertions: 542, 545, 1043, 1047, and 1049. In some embodiments, the PIK3CA gene, PI3Kα protein, or the dysregulation of expression or activity or level of any one thereof is one or more PI3Kα amino acid substitutions (e.g., any PI3Kα amino acid substitutions described herein). Some embodiments of these methods further comprise administering another anticancer agent (e.g., immunotherapy) to the subject.

[0215] In some embodiments, a sample from a subject is used to determine whether the subject has a PIK3CA gene, PI3Kα protein, or any one thereof, expression or activity or level disorder, and the assay can include, for example, next-generation sequencing, immunohistochemistry, fluorescence microscopy, decomposition FISH analysis, Southern blot, Western blot, FACS analysis, Northern blot, and PCR-based amplification (e.g., RT-PCR and quantitative real-time RT-PCR). As is well known in the art, the assay is typically performed, for example, with at least one labeled nucleic acid probe or at least one labeled antibody or antigen-binding fragment thereof. The assay can utilize other detection methods known in the art for detecting PIK3CA gene, PI3Kα protein, or any one thereof, expression or activity or level disorder (see, for example, references cited herein). In some embodiments, the sample is a biological sample or biopsy sample (e.g., paraffin-embedded biopsy sample) from a subject. In some embodiments, the subject is a subject suspected of having PI3Kα-related cancer, a subject with one or more symptoms of PI3Kα-related cancer, and / or a subject with an increased risk of developing PI3Kα-related cancer).

[0216] In some embodiments, liquid biopsy (variously referred to as fluid biopsy or liquid phase biopsy) can be used to identify the PIK3CA gene, PI3Kα protein, or any one thereof expression or activity or level imbalance. See, for example, Karachialiou et al., Ann.Transl.Med., 3(3):36,2016. Liquid biopsy methods can be used to detect the total tumor load and / or PIK3CA gene, PI3Kα protein, or any one thereof expression or activity or level imbalance. Liquid biopsy can be performed on biological samples that are relatively easy to obtain from a subject (e.g., via a simple blood draw), and is generally less invasive than traditional methods for detecting tumor load and / or PIK3CA gene, PI3Kα protein, or any one thereof expression or activity or level imbalance. In some embodiments, liquid biopsy can be used to detect the presence of PIK3CA gene, PI3Kα protein, or any one thereof expression or activity or level imbalance at an earlier stage than traditional methods. In some embodiments, the biological sample to be used in liquid biopsy can include blood, plasma, urine, cerebrospinal fluid, saliva, sputum, bronchoalveolar lavage fluid, bile, lymph, cystic fluid, feces, ascites and combinations thereof. In some embodiments, liquid biopsy can be used to detect circulating tumor cells (CTC). In some embodiments, liquid biopsy can be used to detect cell-free DNA. In some embodiments, the cell-free DNA detected using liquid biopsy is circulating tumor DNA (ctDNA) derived from tumor cells. The analysis of ctDNA (for example, using sensitive detection technology, such as but not limited to next generation sequencing (NGS), traditional PCR, digital PCR or microarray analysis) can be used to identify PIK3CA gene, PI3K α protein or any one thereof expression or activity or level disorder.

[0217] Also provided herein are methods of inhibiting cell proliferation comprising contacting the cells with Compound 1 and one or two independently selected additional therapeutic agents as defined herein.

[0218] Further provided herein are methods of increasing cell death comprising contacting a cell with Compound 1 and one or two independently selected additional therapeutic agents as defined herein.

[0219] In some embodiments, the contacting is in vitro. In some embodiments, the contacting is in vivo. In some embodiments, the contacting is in vivo, wherein the method comprises administering Compound 1 or a pharmaceutically acceptable salt thereof, and one or two independently selected additional therapeutic agents as defined herein to a subject having cells containing abnormal PI3Kα activity. In some embodiments, the cells are cancer cells. In some embodiments, the cancer cells are any cancer as described herein. In some embodiments, the cancer cells are PI3Kα-associated cancer cells.

[0220] As used herein, the term "contacting" refers to bringing the indicated moieties together in an in vitro system or an in vivo system. For example, "contacting" a PI3Kα protein with a compound provided herein includes administering a compound provided herein to an individual or subject, such as a human, having the PI3Kα protein, and, for example, introducing a compound provided herein into a sample containing cells or a purified preparation containing the PI3Kα protein.

[0221] Also provided herein are methods of increasing tumor cell death in a subject, comprising administering to the subject Compound 1, or a pharmaceutically acceptable salt thereof, and one or two independently selected additional therapeutic agents as defined herein.

[0222] combination

[0223] The methods described herein particularly relate to treating cancer (eg, a cancer described herein, such as breast cancer) with Compound 1, or a pharmaceutically acceptable salt thereof, and one or more additional therapeutic agents.

[0224] Such additional therapeutic agents include additional therapeutic molecules (e.g., small molecules or antibodies), as well as radiotherapy (or radiotherapy) and surgery, such as open surgery or minimally invasive surgery. The compounds of formula (I) or pharmaceutically acceptable salts thereof, such as Compound 1, may therefore also be used as adjuvants for cancer treatment, i.e., they may be used in combination with one or more additional therapies or therapeutic agents (e.g., chemotherapeutic agents that act by the same or different mechanisms of action).

[0225] In some embodiments, the compound of formula (I) or its pharmaceutically acceptable salt (e.g., compound 1 or its pharmaceutically acceptable salt) can be used before administering one or more independently selected additional therapeutic agents or other therapies. For example, one or more doses of a compound of formula (I) or its pharmaceutically acceptable salt (e.g., compound 1 or its pharmaceutically acceptable salt) can be administered to a subject in need thereof for a period of time, and then undergo at least partial resection of the tumor. In some embodiments, before at least partial resection of the tumor, treatment with one or more doses of a compound of formula (I) or its pharmaceutically acceptable salt (e.g., compound 1 or its pharmaceutically acceptable salt) reduces the size of the tumor (e.g., tumor load). In some embodiments, one or more doses of a compound of formula (I) or its pharmaceutically acceptable salt (e.g., compound 1 or its pharmaceutically acceptable salt) can be administered to a subject in need thereof for a period of time, and undergo one or more rounds of radiotherapy. In some embodiments, treatment with one or more doses of a compound of Formula (I), or a pharmaceutically acceptable salt thereof (e.g., Compound 1, or a pharmaceutically acceptable salt thereof), prior to one or more cycles of radiation therapy, reduces the size of the tumor (e.g., tumor burden).

[0226] In some embodiments, the subject has a cancer (e.g., a locally advanced or metastatic tumor) that is resistant or intolerant to standard therapy (e.g., administration of a chemotherapeutic agent, such as a multi-kinase inhibitor, immunotherapy, or radiation (e.g., radioactive iodine)). In some embodiments, the subject has a cancer (e.g., a locally advanced or metastatic tumor) that is resistant or intolerant to previous therapy (e.g., administration of a chemotherapeutic agent, such as a multi-kinase inhibitor, immunotherapy, or radiation (e.g., radioactive iodine)). In some embodiments, the subject has a cancer (e.g., a locally advanced or metastatic tumor) for which there is no standard therapy. In some embodiments, the subject has not received a PI3Kα inhibitor. For example, the subject has not received treatment with a selective PI3Kα inhibitor. In some embodiments, the subject is not resistant to a PI3Kα inhibitor. In some embodiments, the subject is not resistant to a kinase inhibitor. In some embodiments, the subject is not resistant to a kinase inhibitor. In some embodiments, the subject has undergone previous therapy.For example, treatment with a multikinase inhibitor (MKI) or another PI3K inhibitor, such as bupanisib (BKM120), apellisib (BYL719), WX-037, idelalisib, duvelithib, cupanisib, erbulithib, (ALIQOPA™, BAY80-6946), datolisib (NVP-BEZ235, BEZ-235), taselisib (GDC-0032, RG7604), sonolisib, (PX-866), feminostat (CUDC-907), bimiliset (PQR309), ZSTK474, SF1126, AZD8835, inarilisib (GDC-0077), ASN003, pitilisib (GDC-0941), pilaralisib (XL147, SAR245408), gidalisib (PF-05212384, PKI-587), cerelice (TAK-117, MLN1117, INK 1117), BGT-226 (NVP-BGT226), PF-04691502, apitolisib (GDC-0980), omiliset (GSK2126458, GSK458), votalisib (XL756, SAR245409), AMG 511, CH5132799, GSK1059615, paxalisib (GDC-0084, RG7666), VS-5584 (SB2343), PKI-402, wortmannin, LY294002, PI-103, regosacillin (ON-01910 sodium salt), vostatalis (XL-765), LY2023414, SAR260301, KIN-193 (AZD-6428), acalisib (GS-9820), AMG319, or GSK2636771.

[0227] In some embodiments of any of the methods described herein, a compound of Formula (I) (or a pharmaceutically acceptable salt thereof) is administered in combination with a therapeutically effective amount of at least one additional therapeutic agent selected from one or more additional therapies or therapeutic agents (e.g., chemotherapeutic agents).

[0228] Non-limiting examples of additional therapeutic agents include: other PI3Kα targeted therapeutics (i.e., other PI3Kα inhibitors), EGFR inhibitors, VEGFR inhibitors / VEGF inhibitors, HER2 inhibitors, MEK pathway targeted therapeutics (including RAS pathway targeted therapeutics, which include mTOR modulators / inhibitors, as described herein), SHP2 inhibitors, ULK inhibitors, CDK4 / 6 inhibitors, NTRK / ROS inhibitors, ALK inhibitors, RET inhibitors, MET inhibitors, PARP inhibitors, PIM (e.g., PIM1 and PIM3) inhibitors, other kinase inhibitors (e.g., Trk inhibitors or multi-kinase inhibitors) , KAT6A inhibitors, farnesyltransferase inhibitors, aromatase inhibitors, selective estrogen receptor modulators or degraders (SERMs / SERDs, including ERα inhibitors or ERα degraders), vinca alkaloids, antimetabolites, antiandrogens (e.g., androgen receptor (AR) antagonists, AR degraders, AR modulators), alkylating agents, checkpoint inhibitors, apoptosis pathway modulators, cytotoxic chemotherapeutics (also known as anti-neoplastic chemotherapeutics), angiogenesis-targeted therapies (e.g., angiogenesis inhibitors), immune-targeted agents (including immunotherapy), radiotherapy, glucocorticoids, antidiarrheal drugs (e.g., loperamide and diphenoxylate-atropine), antihistamines, and retinoic acid.

[0229] As used herein, a PIM inhibitor is Mo Loni's leukemia virus kinase Original Virus all The zygote (sometimes also called mouse Leukemia virus protein kinase Original Virus InsertAny inhibitor of a kinase (e.g., a kinase that binds to a specific kinase or mitogen-activated protein kinase) such as PIM1, PIM2, and PIM3, and any isoforms thereof (e.g., PIM-1L (molecular weight of 44 kDa) and PIM-1S (molecular weight of 33 kDa). PIM kinases regulate cell proliferation, survival, metabolism, cell trafficking, and signaling, and are overexpressed in a variety of human cancers. Non-limiting examples of PIM1 inhibitors include A47, abemaciclib (Verzenio; NCT03905889), AZD1208 (NCT01588548), AZD1897, ETH-155008, ETP-390101, ETP-45299, ETP-47551, INCB053914 (Uzansertib), JP11646, K00135, K00486, LGB321, LGH447 (PIM447), PIM447, SEL24 / MEN1703 (SEL24-B489), SGI-1776, and TP-3654 (see, Belon and Nicot (2023) Mol. Cancer 22(1)). :18; Mahata S., et al. (2022) Med. Oncol. 39(5):74; Asasti V., et al. (2019) Eur J Med Chem. 172:95-108; Le X., et al. (2016) Cancer Discov. 6(10):1134-47; Keeton EK, et al. (2014) Blood 123:905-13; Garcia P., et al. (2013) ASH 122:1666; Grundler R, et al. (2009) J Exp Med. 206(9):1957-70; Pogacic V., et al., (2007) Cancer Res. 67(14):6916–6924).

[0230] Exemplary SHP2 inhibitors include JAB-3312, SHP099, SHP099 hydrochloride, SHP504, RMC-3943, AS1949490, SHP394, SHP389, and RMC-4630.

[0231] In some embodiments, the SHP2 inhibitor is RMC-4630.

[0232] Exemplary ULK inhibitors include ULK-101, SBP-7455, SBI-0206965, ULK1-IN-2, MRT68921, MRT68921 dihydrochloride, MRT67307, MRT67307 hydrochloride, XST-14, and GW406108X (CW108X).

[0233] Exemplary NTRK / ROS inhibitors include entrectinib (NMS-E628, RXDX-101, ), tadalafil (DS-6051b, AB-106), or ripretinib (TPX-0005).

[0234] Exemplary ALK inhibitors include crizotinib ( PF-02341066), Ceritinib ( LDK-378), Alectinib ( CH5424802, RO5424802, AF802), brigatinib ( AP-26113), Lorlatinib ( PF-06463922), entrectinib (NMS-E628, RXDX-101, ), ASP3026, TSR-011, PF-06463922, ensartinib (X-396), or CEP-37440.

[0235] Exemplary RET inhibitors include septinib ( LOXO-292), zeteletinib (BOS-172738, DS-5010), GSK3179106, amuvatinib hydrochloride (MP470 hydrochloride, HPK56 hydrochloride), TPX-0046, or pralsetinib ( BLU-667).

[0236] Exemplary MET inhibitors include capmatinib ( INC280; INCB28060), tepotinib Tivantinib (ARQ197), savotinib ( Volitinib, HMPL-504, AZD-6094), foretinib (XL880, GSK1363089, GSK089, EXEL-2880), pamufetinib (TAS-115), c-Met-IN-2, PHA-665752, SU11274, SYN1143, or amuvatinib hydrochloride (MP470 hydrochloride, HPK56 hydrochloride).

[0237] Exemplary TRK or multi-targeted kinase inhibitors include altiratinib (DCC-2701), CH7057288, larotrectinib, Entrectinib, ANA-12, ripretinib (TPX-0005), selutinib (MGCD516, MG-516), lestaurinib (CEP-701, KT-5555), tyrphostin AG879 (AG879), and celiac (LOXO-195).

[0238] Exemplary tyrosine kinase inhibitors include axitinib Dasatinib Erlotinib Imatinib (GLIVEC), nilotinib Pazopanib Sunitinib and vemurafenib.

[0239] Exemplary vinca alkaloids include vinorelbine, vinblastine, vincristine, vindesine, and vinflunine.

[0240] Exemplary antimetabolites include methotrexate, 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine, Floxuridine, cytarabine Fludarabine, gemcitabine Hydroxyurea, pemetrexed phototrexate, decitabine, vidaza, DFP-11207 and RX-3117, TAS-114.

[0241] Exemplary alkylating agents include cyclophosphamide, lomustine, carmustine, streptozotocin, bendamustine, chlorambucil, cyclophosphamide, ifosfamide, mechlorethamine, melphalan, chlorambucil, melphalan, busulfan, dacarbazine, temozolomide, altretamine, thiotepa, carboplatin, cisplatin, rubitidine, trabectedin, carmustine, oxaliplatin, and oxaliplatin.

[0242] Exemplary checkpoint inhibitors include nivolumab, pembrolizumab, cemiplizumab, atezolizumab, durvalumab, avelumab, ipilimumab, LAG525 (IMP701), REGN3767 (R3767), BI 754,091, tepolizumab (MGD013), eftilagimod alpha (IMP321), FS118, MBG453, Sym023, TSR-022, MGC018, FPA150, EOS100850, AB928, CPI-006, monalizumab, COM701, CM24, NEO-201, defatinib, PF-04136309, MSC-1, Hu5F9-G4 (5F9), ALX148, TTI-662, RRx- 001, latuzumab (MCS110), LY3022855, SNDX-6352, imiquizumab (RG7155), pecidinib (PLX3397), CAN04, canakinumab (ACZ885), BMS-986253, pebinumab (VX15 / 2503), trebananib, FP-1305, enapotamab vedotin (EnaV), and baviximab.

[0243] Exemplary modulators of apoptosis pathways include oblimersen, ABT-737, navitoclax (ABT-263), venetoclax (ABT-199), Z-VAD-FMK, enlicasin, Q-VD-Oph, Z-VAD(OH)-FMK, benacasin (caspase-1), Z-DEVD-FMK (caspase-3), Q-VD-Oph, Z-IETD-FMK (caspase-8), and Z-VD-FMK. ), PAC-1 (pro-caspase-3), nutlin-3, nutlin-3a, edanulin, HDM201, APR-246, CBL0137, pifluthrin-α, pifluthrin-μ, Z-VAD-FMK, enlicasin, Q-VD-Oph, Z-VAD(OH)-FMK, pomalidomide, lenalidomide, YM155, venetoclax (Bcl-2), S63845 (MCL-1), and A-1331852 (BCL-XL).

[0244] Exemplary cytotoxic chemotherapy drugs include 5-fluorouracil, gemcitabine, methotrexate, NB1011, cyclophosphamide, dacarbazine, melphalan, tracectedin, temozolomide, doxorubicin, daunorubicin, mitoxantrone, vinblastine, paclitaxel, docetaxel, irinotecan, etoposide, and platinum agents such as carboplatin, cisplatin, and oxaliplatin.

[0245] Exemplary topoisomerase inhibitors include etoposide, irinotecan, camptothecin (CPT), topotecan (TPT), irinotecan, belotecan, indenoisoquinoline, phenanthridine and indolocarbazole. Additional examples of topoisomerase inhibitors include aminocamptothecin, CT-2106, crisnatol mesylate, DE-310, elinefand, lucanthone, MLN576 and midudomide.

[0246] Exemplary angiogenesis-targeted therapies include bevacizumab, itraconazole, carboxyamidotriazole, TNP-470, CM101, IFN-α, IL-12, platelet factor-4, suramin, SU5416, thrombospondin, angiostatin, endostatin, 2-methoxyestradiol, tecogalan, tetrathiomolybdate, thalidomide, thrombospondin, prolactin, linomide, ramucirumab, taquimod, ranibizumab, sorafenib, sunitinib, pazopanib, everolimus, lenalidomide (e.g., ) and pomalidomide (e.g., or ), marimastat, 2-methoxyestradiol (PANZEM), SU5415, SU6668, pemaxanib, sunitinib, vandetanib, vitaxin, YM598, ZD6126, and aflibercept.

[0247] In some embodiments, the angiogenesis-targeted therapy is lenalidomide.

[0248] In some embodiments, the angiogenesis-targeted therapy is pomalidomide.

[0249] In some embodiments, the EGFR inhibitor is osimertinib (AZD9291, merelectinib, ), erlotinib Gefitinib Cetuximab Nexituzumab ( IMC-11F8), neratinib (HKI-272, ), Panitumumab (ABX-EGF, ), Vandetanib Rociletinib (CO-1686), Omotinib ( HM61713, BI-1482694), nacatinib (ASP8273), neratinib (EGF816, NVS-816), maverlertinib (PF-06747775), icotinib (BPI-2009H), afatinib (BIBW2992, ), dacomitinib (PF-00299804, PF-804, PF-299, PF-299804), avitinib (AC0010), AC0010MA, EAI045, matuzumab (EMD-7200), nimotuzumab (h-R3, BIOMAb ), zalutumab, MDX447, depatuxizumab (humanized mAb806, ABT-806), depatuxizumab mafodotin (ABT-414), ABT-806, mAb806, canertinib (CI-1033), shikonin, shikonin derivatives (e.g., deoxyshikonin, isobutyrylshikonin, acetylshikonin, β,β-dimethylacryloylshikonin, and acetylshikonin), pocitinib (NOV120101, HM781-36B), AV-412, ibrutinib, WZ4002, brigatinib (AP26113, ), pelitinib (EKB-569), tarloxotinib (TH-4000, PR610), BPI-15086, Hemay022, ZN-e4, tesevatinib (KD019, XL647), lazertinib (YH25448), epitinib (HMPL-813), olafertinib (CK-101, RX518), MM-151, zolitinib (AZD3759), vandetanib (ZD6474), PF-06459988, varlintinib (ASLAN001, ARRY-334543), mobotinib (AP32788, TAK-788), pimurutumab (pimurutumab), ab)(HLX07), befortinib (D-0316), AEE788 (NVP-AEE788), ametinib (formerly amotinib, HS-10296), avitinib, lapatinib (GW572016), pyrotinib (SHR1258), SCT200, CPGJ602, Sym004 (combination of vortuximab and modotuximab), EMD55900 (MAb-425), modotuximab (TAB-H49), vortuximab (992DS), zalutumumab, RO5083945, lapatuximab (IMGN289), ervantuximab (RYBREVANT TM, JNJ-61186372), LY3164530, Pan-HER (Sym013), AMG595, tuxobertinib (BDTX-189), avatinib, disruptin, CL-387785 (EKI-785, WAY-EKI785), autologous T cells equipped with EGFRBi and EGFRCAR-T therapy. In some embodiments, the EGFR targeted therapeutic agent is selected from gefitinib, erlotinib, afatinib, lapatinib, neratinib, osimertinib (AZD-9291, for example ), CL-387785 (EKI-785, WAY-EKI785), locitinib (CO-1686), WZ4002, OMP-305B83, trastuzumab (e.g., TRAZIMERA TM 、 ), RG-7597, and amivantanab.

[0250] In some embodiments, the EGFR inhibitor is lazertinib. In some embodiments, the EGFR inhibitor is ervantumab. In some embodiments, the EGFR inhibitor is trastuzumab.

[0251] Exemplary HER2 inhibitors include trastuzumab (e.g., TRAZIMERA TM 、 ), pertuzumab (e.g., ), emtansine trastuzumab (T-DM1 or ado-emtansine trastuzumab, e.g., ), fam-de trastuzumab Lapatinib, KU004, neratinib (e.g., ), envatinibgovitecan-hziy Dacomitinib (e.g., ), afatinib Tucatinib (irbinitinib, ONT-380, ARRY-380, such as TUKYSA TM ), erlotinib (e.g., ), pyrotinib, pocitinib, CP-724714, CUDC-101, sabutinib (AZD8931), tanspiramycin (17-AAG), IPI-504, dacomitinib (PF299804, PF299), pelitinib, magetuximab, AEE-788 (NVP-AEE788), enroku and dedabrocitinib.

[0252] In some embodiments, the HER2 inhibitor is fam-detrastuzumab. In some embodiments, the HER2 inhibitor is envatinib govitecan-hziy. In some embodiments, the HER2 inhibitor is dedabruzumab.

[0253] Exemplary VEGFR inhibitors / VEGF inhibitors include pazopanib, sunitinib, envatinib, cabozantinib, sorafenib, regorafenib, ponatinib, envatinib axitinib, ziv-aflibercept, vandetanib, tivozanib, vatalanib, AZD-2932, aflibercept, vanucezumab, BI836880, dual antiangiogenic protein (DAAP), and ramucirumab.

[0254] As used herein, "MEK pathway targeted therapeutics" include any compound showing any protein in the MEK pathway, including any compound showing the inactivation activity of any protein in the RAS pathway and RAF pathway (e.g., kinase inhibition, allosteric inhibition, dimerization inhibition and degradation induction). As used herein, "RAS pathway targeted therapeutics" include any compound showing the inactivation activity of any protein in the RAS pathway (e.g., kinase inhibition, allosteric inhibition, dimerization inhibition and degradation induction). Non-limiting examples of proteins in the RAS pathway include any protein in the RAS-RAF-MAPK pathway or PI3K / AKT pathway, such as RAS (e.g., KRAS, HRAS and NRAS), RAF (ARAF, BRAF, CRAF), MEK, ERK, PI3K, AKT and mTOR. In some embodiments, RAS pathway regulators can be selective for proteins in the RAS pathway, for example, RAS pathway regulators can be selective for RAS (also referred to as RAS regulators). In some embodiments, RAS regulators are covalent inhibitors. In some embodiments, the RAS pathway targeted therapeutic agent is a "KRAS pathway modulator". KRAS pathway modulators include any compound that exhibits inactivation activity of any protein in the KRAS pathway (e.g., kinase inhibition, allosteric inhibition, dimerization inhibition, and degradation induction). Non-limiting examples of proteins in the KRAS pathway include any protein in the KRAS-RAF-MAPK pathway or the PI3K / AKT pathway, such as KRAS, RAF, BRAF, MEK, ERK, PI3K (i.e., other PI3K inhibitors as described herein), AKT, and mTOR. In some embodiments, KRAS pathway modulators can be selective for proteins in the RAS pathway, for example, KRAS pathway modulators can be selective for KRAS (also referred to as KRAS modulators). In some embodiments, KRAS modulators are covalent inhibitors.

[0255] Non-limiting examples of RAS-targeted therapeutics include sotolacib (AMG510, ), adagracib (MRTX849), tipifarnib (R115777, zarnestra), cysmethynil, UCM-1336, deltarasin, NHTD, RM007, RM008, gefitinib, apatinib, onclexin-1, vismodegib (GDC-0449), N-(1-acryloylazetidin-3-yl)-2-(5-bromo-3-(5-methoxy-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-1H-indol-1-yl)acetamide, 2-((4-((1-( N,N-dimethylethanaminium (2-(2,4-dichlorophenoxy)acetylpiperidin-4-ylamino)-4-oxobutyl)disulfaneyl), ARS-1620, ARS-853, besentinib (BGB324), ABT-737, selumetinib (AZD6244), datolisin (NVP-BEZ235), PPIN-1, PPIN-2, pan-RAS inhibitor 3144 (RAS-IN-3144), deltarasin, SML-8-73-1, SML-10- 70-1, 1-(2-hydroxyethyl)-4-(2-methyl-3,5-diphenylpyrazolo[1,5-a]pyrimidin-7-yl)piperazin-1-ium, (2R,4aR)-3-acryloyl-11-chloro-9-fluoro-10-(6-fluoro-2-hydroxycyclohexa-2,4-dien-1-yl)-2,6-dimethyl-2,3,4,4a-tetrahydro-1H-pyrazino[1',2':4,5]pyrazino[2,3-c]quinolin-5(6H)-one, NHTD, PD98059, wortmannin, talniflumate, gefitinib, CPD-0857, KY 1022, KYA1797K (ab229170), 0375-0604 (DUN09716), 7773, NSC-658497, JNJ-74699157, PKF115-584 (calphostin C), Kobe0065, Kobe2602, salirasib, 3,3'-(ethylazanediyl)bis(N-phenylpropionamide), ML264, GDC-6036, LY3499446, and D-1553.

[0256] Non-limiting examples of KRAS targeted therapeutics (e.g., KRAS inhibitors) include BI 1701963, sotolacilib (AMG510), ARS-3248 (JNJ-74699157), ARS1620, AZD4785 (ION651987), SML-8-73-1, SML-10-70-1, VSA9, AA12, adagracilib (MRTX-849), LY3499446, ARS853, and siG12DLODER.

[0257] Non-limiting examples of HRAS-targeted therapeutics (eg, HRAS inhibitors) include tipifarnib, Further non-limiting examples of RAS targeted therapeutics include BRAF inhibitors, MEK inhibitors, ERK inhibitors, PI3K inhibitors, AKT inhibitors, and mTOR inhibitors. In some embodiments,

[0258] In some embodiments, the MEK inhibitor is trametinib ( GSK1120212), cobimetinib Bimetinib ( MEK162), selumetinib (AZD6244), mirdametinib (PD0325901), pimasertib (MSC1936369B), SHR7390, TAK-733, RO5126766 (CH5126766), CS3006, WX-554, PD98059, CI1040 (PD184352), paramycin, or a combination thereof.

[0259] In some embodiments, the ERK inhibitor is amerliorex (FRI-20, ON-01060), VTX-11e, 25-OH-D3-3-BE (B3CD, bromoacetoxycalcifediol), FR-180204, AEZ-131 (AEZS-131), AEZS-136, AZ-13767370, BL-EI-001, temuterkib (LY-3214996), rineterkib (LTT- 462), KO-947, MK-8353 (SCH900353), SCH772984, uritinib (BVD-523), CC-90003, ravoxertinib (GDC-0994, RG-7482), ASN007, 5Z-7-oxaenol (FR148083, L783279, LL-Z1640-2), 5-iodotuberculocidin (NSC113939), ONC201 (TIC10), or a combination thereof.

[0260] In some embodiments, the antiandrogen is leuprolide Goserelin Triptorelin Leuprorelin mesylate Flutamide Bicalutamide Nilutamide Degarelix Ruilugoli Enzalutamide (MDV3100, ), Abiraterone Flutamide AR inhibitors EPI-506 and apalutamide and darolamide

[0261] In some embodiments, the other PI3K inhibitor is another PI3Kα inhibitor. In some embodiments, the other PI3K inhibitor is a pan-PI3K inhibitor. In some embodiments, the other PI3K inhibitor is selected from bupanixiba (BKM120), apellisib (BYL719, ), Idelalis, Duvilis, Erbulis, WX-037, Kupanisi ( BAY80-6946), datolisib (NVP-BEZ235, BEZ-235), taselisib (GDC-0032, RG7604), sonolisib (PX-866), feminostat (CUDC-907), bimilisib (PQR309), ZSTK474, SF1126, AZD8835, inarilisib (GDC-0077), ASN003, pitilisib (GDC-0941), pilaralisib (XL147, SAR245408), gidalisib (PF-05212384, PKI-587), cerellisib (TAK-117, MLN1117, INK1117), BGT-226 (NVP-BGT226), PF-046 91502, apitolisib (GDC-0980), omilivac (GSK2126458, GSK458), votalisib (XL756, SAR245409), AMG511, CH5132799, GSK1059615, paxalisib (GDC-0084, RG7666), VS-5584 (SB2343), PKI-402, wortmannin, LY294002, PI-103, regosacillin (ON-01910 sodium salt), votalisib (XL-765), LY2023414, SAR260301, KIN-193 (AZD-6428), acalisib (GS-9820), AMG319, GSK2636771, or a combination thereof.

[0262] In some embodiments, the AKT inhibitor is selected from miltefosine Wortmannin, NL-71-101, H-89, GSK690693, CCT128930, capasertib (AZD5363), ipatasertib (GDC-0068, RG7440), A-674563, A-443654, AT7867, AT13148, eupresertib (GSK2141795), afuresertib (GSK2110183), DC120, 2-[4-(2-aminopropyl-2-yl)phenyl]-3-phenylquinoxaline, MK-2206, edelfosine, miltefosine, perifosine (KRX-0401), erucylphophocholine, erufosine, SR13668, OSU-A9, PH-316, PHT-427 (CS-0223), PIT-1, DM-PIT-1, triciribine (triciribine phosphate monohydrate), API-1, N-(4-(5-(3-acetamidophenyl)-2-(2-aminopyridin-3-yl)-3H-imidazo[4,5-b]pyridin-3-yl)benzyl)-3-fluorobenzamide, miransertib (ARQ092), BAY1125976, 3-oxo-tirucallicacid, lactoquinomycin, boc-Phe-vinyl ketone, perifosine (D-21266), TCN, TCN-P, ONC201 (TIC10), and TAS117.

[0263] In some embodiments, the AKT inhibitor is capasitinib.

[0264] In some embodiments, the mTOR inhibitor is a rapamycin analog. Examples of rapamycin analogs include sapacertin (MLN0128), vetosertib (AZD-2014), onatasertib (CC-223), CC-115, everolimus (RAD001), temsirolimus (CCI-779), defostiolimus (AP-23573), sirolimus (rapamycin), defostiolimus (MK-8669), everolimus (RAD001, e.g. or ), umirolumus, zotarolimus and RMC-5552. In some embodiments, the mTOR inhibitor is an ATP-competitive mTOR kinase inhibitor that competes with ATP in the catalytic site of mTOR. Examples of ATP-competitive mTOR kinase inhibitors include torin-1, torin-2 and vestusertib. Types of ATP-competitive mTOR kinase inhibitors can include mTOR / PI3J dual inhibitors and mTORC1 / mTORC2 dual inhibitors (also known as TORCdI). Examples of mTOR / PI3K dual inhibitors include datolis, votarolimus, BGT226, SF1126, PKI-587 and NVPBE235. Examples of mTORC1 / mTORC2 dual inhibitors include sapasetin (codename INK128), AZD8055 and AZD2014.

[0265] Non-limiting examples of farnesyl transferase inhibitors include lonafarnib, tipifarnib, BMS-214662, L778123, L744832, and FTI-277.

[0266] In some embodiments, chemotherapeutic agents include anthracyclines, alkylating agents, taxanes, platinum-based agents, mitomycin, gemcitabine, pemetrexed, eribulin (HALAVEN TM ) or a combination thereof.

[0267] Non-limiting examples of taxanes include paclitaxel, docetaxel, abraxane, and taxotere.

[0268] In some embodiments, the anthracycline is selected from daunorubicin, doxorubicin, epirubicin, and idarubicin.

[0269] In some embodiments, the platinum-based agent is selected from carboplatin, cisplatin, oxaliplatin, nedaplatin, triplatintetranitrate, phenanthriplatin, picoplatin, satraplatin, and lobaplatin. Any platinum-based agent can be conjugated to a nanocarrier, such as a gold nanocluster, gold nanoparticles, or superparamagnetic iron oxide nanoparticles. See, for example, Zhang et al. 2022. Theranostics. 12(5): 2115-2132.

[0270] Non-limiting examples of PARP inhibitors include Lynparza Talazoparib, rucaparib, niraparib, veliparib, BGB-290 (pamiparib), CEP-9883, CEP9722, E7016 (GPI21016), iniparib, cenaparib (IMP4297), venadaparib-idience (NOV1401, IDX-1197), stenoparib (2X-121), ABT-767, atamparib (RBN-2397), talazoparib (BMN673), olaparib (KU-0059436, AZD2281), for example, LYNPARZA TM ), iniparib (BSI-201, SAR240550), rucaparib (AG-014699, PF-01367338), INO-1001, and ameparib (JPI-289).

[0271] Non-limiting examples of aromatase inhibitors include aminoglutethimide, testolactone, anastrozole, letrozole, exemestane, vorozole, formestane, and fadrozole.

[0272] Non-limiting examples of selective estrogen receptor modulators or degraders (SERMs / SERDs) include clomiphene, cyclofenil, anordrin, bromopastriene, nafoxidine, ormeloxifene, raloxifene, toremifene, lasofoxifene, bazedoxifene, ospemifene, afexifene, enclomiphene, serophene, arzoxifene, tamoxifen, etacstil (GW-5638, DPC974), fulvestrant, Blindactone, Elastrant (ORSERDU TM ), giredestrant, amcenestrant (SAR439859), camizestrant (AZD9833), rintodestrant, imlunestrant, LSZ102, LY3484356, ZN-c5, taragarestrant (D-0502), AZD9496, clotrimazole, fenticonazole, SHR9549, and paralastrant (OP-1250).

[0273] In some embodiments, the SERM / SERD is palastatin. In some embodiments, the SERM / SERD is alastatin. In some embodiments, the SERM / SERD is camistrant.

[0274] Non-limiting examples of glucocorticoids include dexamethasone, beclomethasone, betamethasone, budesonide, cortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, and triamcinolone.

[0275] In some embodiments, the glucocorticoid is dexamethasone.

[0276] In some embodiments, the additional therapeutic agent is retinoic acid.

[0277] Non-limiting examples of epigenetic agents include EZH2 inhibitors (e.g., tazemestat, 3-deazaneplanocinA (DZNep or C-c3Ado), EPZ005687, EI1, GSK126 and UNC1999) and HDAC inhibitors (e.g., vorinostat (SAHA) and panobinostat (LBH589)). Non-limiting examples of HDAC inhibitors include hydroxamic acids (or hydroxamates), such as trichostatin A, vorinostat (SAHA), belinostat (PXD101), LAQ824 and panobinostat (LBH589); cyclic tetrapeptides, such as trapoxin B and depsipeptide; benzamides, such as entinostat (MS-275), tacedinaline (CI994) and moxistat (MGCD0103); electrophilic ketones; and fatty acid compounds such as phenylbutyrate and valproic acid.

[0278] In some embodiments, the epigenetic agent is an EZH2 inhibitor. In some embodiments, the epigenetic agent is tazemetostat. In some embodiments, the EZH2 inhibitor is tazemetostat.

[0279] In some embodiments, the epigenetic agent is an HDAC inhibitor. In some embodiments, the epigenetic agent is vorinostat. In some embodiments, the epigenetic agent is panobinostat. In some embodiments, the HDAC inhibitor is vorinostat. In some embodiments, the HDAC inhibitor is panobinostat.

[0280] Non-limiting examples of KAT6A inhibitors include WM-8014, PF-07248144, CTx-648 (PF-9363), and CTX-0124143. In some embodiments, the KAT6A inhibitor is WM-8014. In some embodiments, the KAT6A inhibitor is PF-07248144. In some embodiments, the KAT6A inhibitor is CTx-648. In some embodiments, the KAT6A inhibitor is CTX-0124143.

[0281] Non-limiting examples of immunotherapy include immune checkpoint therapy, atezolizumab Albumin-bound paclitaxel. Non-limiting examples of immune checkpoint therapies include inhibitors targeting CTLA-4, PD-1, PD-L1, BTLA, LAG-3, A2AR, TIM-3, B7-H3, VISTA, IDO, and combinations thereof. In some embodiments, the CTLA-4 inhibitor is ipilimumab. In some embodiments, the PD-1 inhibitor is selected from nivolumab Pembrolizumab Cimiprizumab Atezolizumab Durvalumab Avelumab Dotalizumab Rivulimab Vopalizumab (JTX-4014), spartalizumab (PDR001), camrelizumab (SHR1210), sintilimab (IBI308), tislelizumab (BGB-A317), toripalimab (JS001), INCMGA00012 (MGA012), AMP-224, AMP-514 (MEDI0680) or acrixolimab (YBL-006). In some embodiments, the PD-1 inhibitor is selected from pembrolizumab Nivolumab Cimiprizumab or a combination thereof. In some embodiments, the PD-L1 inhibitor is selected from atezolizumab Avelumab Durvalumab or a combination thereof. In some embodiments, the LAG-3 inhibitor is leramilimab (IMP701, LAG525). In some embodiments, the A2AR inhibitor is ciforadenant (CPI-444). In some embodiments, the TIM-3 inhibitor is sabatolimab (MBG453). In some embodiments, the B7-H3 inhibitor is enoxaparin. In some embodiments, the VISTA inhibitor is ovalimab (JNJ-61610588). In some embodiments, the IDO inhibitor is indomod. See, for example, Marin-Acevedo, et al., Table 1 of J Hematol Oncol. 11:39 (2018), which is incorporated herein in its entirety.

[0282] In some embodiments, the CDK4 / 6 inhibitor is selected from palbociclib ( TQB3616, PD-0332991), Ribociclib Abecilil Volusiclib (P1446A-05), troraciclib, darcillib (SHR6390), roniciclib (BAY1000394), dinaciclib, flavopiridol (alvocilib, L868275, HMR-1275), roscovitine (R-roscovitine, CYC202, seliciclib), riviciclib (P276-00, P276), AT7519, TG02 (SB1317), RGB-286 638, dinaciclib (SCH727965), PHA-793887, ZK-304709, xytocydine, SNS032 (BMS-387032), R547 (R04584820), RGB286147, prvalanol A (NG60), meriolin 3, JNJ7706621, indirubin, AZD-5438, 10Z-hymenialdisine, AGO24322, PF-06873600, and KIN-8741.

[0283] In some embodiments, the CDK4 / 6 inhibitor is KIN-8741. In some embodiments, the CDK4 / 6 inhibitor is palbociclib. In some embodiments, the CDK4 / 6 inhibitor is ribociclib. In some embodiments, the CDK4 / 6 inhibitor is triclasiclib. In some embodiments, the CDK4 / 6 inhibitor is darcil. In some embodiments, the CDK4 / 6 inhibitor is waluxili. In some embodiments, the CDK4 / 6 inhibitor is roniciclib. In some embodiments, the CDK4 / 6 inhibitor is dinaciclib.

[0284] In some embodiments, the additional therapy or therapeutic agent is selected from fulvestrant, capecitabine, trastuzumab, ado-enmetazocine, pertuzumab, paclitaxel, nab-paclitaxel, enzalutamide, olaparib, pegylated liposomal doxorubicin (PLD), trametinib, palbociclib, Bupanib, sorafenib (AEB071), everolimus, exemestane, cisplatin, letrozole, ganizumab (AMG479), LSZ102, ribociclib (LEE011), cetuximab, luminespib (NVP-AUY922, AUY922), infigratinib (BGJ398), bimetinib (MEK162, ARRY-162, ARRY-438162), LJM716, PIM447 (LGH447, LGB321), imatinib, gemcitabine, connefenib (LGX818), and amcenestrant.

[0285] In some embodiments, the additional therapeutic agent is everolimus. In some embodiments, additional therapeutic agents may also be administered to treat potential side effects of specific anticancer therapies and / or as palliative care, such as opioids and corticosteroids. In some embodiments, the additional therapies or therapeutic agents described herein are selected from glucagon-like peptide-1 (GLP-1) receptor agonists, sodium-glucose transporter 2 (SGLT-2) inhibitors, dipeptidyl peptidase 4 (DPP-4) inhibitors, metformin, and combinations thereof.

[0286] Non-limiting examples of GLP-1 receptor agonists include liraglutide (NN2211, e.g. ), dulaglutide (LY2189265, e.g. ), Exenatide (e.g., Exendin-4), taseroglutide, lixisenatide (e.g., ), albiglutide (e.g., ), semaglutide (e.g., ), ZP2929, NNC0113-0987(QBR110395), BPI-3016 and TT401.

[0287] Non-limiting examples of SGLT-2 inhibitors include bepagliflozin, canagliflozin (e.g., ), dapagliflozin (e.g., ), empagliflozin (e.g., ), ergliflozin (e.g., STEGLATRO TM ), ipagliptin (e.g., ), rugagliflozin (e.g., ), repagliflozin, serfliflozin, licofliglozin, sogliatin (e.g., ZYNQUISTA TM ) and topagliflozin.

[0288] Non-limiting examples of DPP-4 inhibitors include sitagliptin (e.g., ), vildagliptin, saxagliptin (e.g., ), linagliptin (e.g., ), gemagliptin, alagliptin, tenegliptin, alogliptin, trelagliptin (e.g., ), omagliptin, ipagliptin, and dulagliptin.

[0289] In some embodiments, the additional therapeutic agent is metformin.In some embodiments, the methods described herein further comprise administering a therapeutically effective amount of metformin to the subject.

[0290] In some embodiments, the method comprises administering Compound 1 or a pharmaceutically acceptable salt thereof and an additional therapeutic agent, such as an aromatase inhibitor, a CDK4 / 6 inhibitor, a SERM / SERD, radiation therapy, an anti-HER2 antibody or its antibody-drug conjugate (ADC), immunotherapy, a checkpoint inhibitor (e.g., an anti-PD-1 or PD-L1 antibody, an anti-CLTA4 antibody), a VEGFR inhibitor / VEGF inhibitor, a KAT6A inhibitor (also known as a MOZ inhibitor or a MYST3 inhibitor), a PI3Kα inhibitor, a MEK pathway targeted therapeutic (including a RAS pathway targeted therapeutic), or a combination thereof. Therapeutic agents, including mTOR inhibitors, as described herein), SHP2 inhibitors, ULK inhibitors, NTRK / ROS inhibitors, ALK inhibitors, RET inhibitors, MET inhibitors, PARP inhibitors, PIM (e.g., PIM1 and PIM3) inhibitors, other kinase inhibitors (e.g., Trk inhibitors or multikinase inhibitors), farnesyl transferase inhibitors, vinca alkaloids, antimetabolites, antiandrogens, alkylating agents, checkpoint inhibitors, apoptosis pathway modulators; cytotoxic chemotherapeutics, angiogenesis targeted therapies, immune targeted agents including immunotherapies or anti-EGFR antibodies.

[0291] In some embodiments, the additional therapeutic agent is an antibody or ADC as described herein. In some embodiments, the antibody is daratumumab (e.g., ).

[0292] In some embodiments, the method comprises administering Compound 1, or a pharmaceutically acceptable salt thereof, and an additional therapeutic agent, such as a HER2 inhibitor, a SERM / SERD, a CDK4 / 6 inhibitor, a MEK inhibitor, a checkpoint inhibitor (e.g., an anti-PD-1 or PD-L1 antibody, an anti-CLTA4 antibody), a multikinase inhibitor, and a PI3K inhibitor.

[0293] In some embodiments, the method comprises administering Compound 1 or a pharmaceutically acceptable salt thereof and an additional therapeutic agent, such as trastuzumab, pertuzumab, enmetazocumab, fam-dertrastuzumab, lapatinib, neratinib, dacomitinib, afatinib, tucatinib, erlotinib, pyrotinib, tanspiramycin, dacomitinib, pelitinib, magetuximab, clomiphene, cyclofenib, bromostriene, ormeloxifene, raloxifene, toremifene, lasofoxifene, bazedoxifene, ospemifene, enclomiphene, selanfen, tamoxifen, fulvestrant, elastrant , camistran, rintodestrant, clotrimazole, fenticonazole, nivolumab, pembrolizumab, cemiplimab, atezolizumab, durvalumab, avelumab, ipilimumab, palbociclib, ribociclib, abemaciclib, triaciclib, darcillib, trametinib, cobimetinib, binimetinib, selumetinib, mirdatinib, pimaticinib, apellis, idelalisib, durvalumab, cupanisib, erbulisib, ribociclib, triaciclib, darcillib, voruximab, and roniciclib.

[0294] In some embodiments, the additional therapeutic agent is fulvestrant.

[0295] In some embodiments, the additional therapeutic agent is lapatinib.

[0296] In some embodiments, the additional therapeutic agent is abemaciclib.

[0297] In some embodiments, the additional therapeutic agent is trametinib.

[0298] In some embodiments, the additional therapeutic agent is binimetinib.

[0299] In some embodiments, the additional therapeutic agent is apellis.

[0300] In some embodiments, the additional therapeutic agent is palbociclib.

[0301] In some embodiments, the additional therapeutic agent is ribociclib.

[0302] In some embodiments, the additional therapeutic agent is triraciclib.

[0303] In some embodiments, the additional therapeutic agent is dacillin.

[0304] In some embodiments, the additional therapeutic agent is waluxib.

[0305] In some embodiments, the additional therapeutic agent is roniciclib.

[0306] In some embodiments, the additional therapeutic agent is dinaciclib.

[0307] In some embodiments, the additional therapeutic agents are palbociclib and clomiphene.

[0308] In some embodiments, the additional therapeutic agents are palbociclib and cyclofenac.

[0309] In some embodiments, the additional therapeutic agents are palbociclib and ardrin.

[0310] In some embodiments, the additional therapeutic agents are palbociclib and bromocriptine.

[0311] In some embodiments, the additional therapeutic agents are palbociclib and nafoxidine.

[0312] In some embodiments, the additional therapeutic agents are palbociclib and ormeloxifene.

[0313] In some embodiments, the additional therapeutic agents are palbociclib and raloxifene.

[0314] In some embodiments, the additional therapeutic agents are palbociclib and toremifene.

[0315] In some embodiments, the additional therapeutic agents are palbociclib and lasofoxifene.

[0316] In some embodiments, the additional therapeutic agents are palbociclib and bazedoxifene.

[0317] In some embodiments, the additional therapeutic agents are palbociclib and ospemifene.

[0318] In some embodiments, the additional therapeutic agents are palbociclib and afoxifene.

[0319] In some embodiments, the additional therapeutic agents are palbociclib and enclomiphene.

[0320] In some embodiments, the additional therapeutic agents are palbociclib and selanphen.

[0321] In some embodiments, the additional therapeutic agents are palbociclib and arzoxifene.

[0322] In some embodiments, the additional therapeutic agents are palbociclib and tamoxifen.

[0323] In some embodiments, the additional therapeutic agents are palbociclib and etacstil.

[0324] In some embodiments, the additional therapeutic agents are palbociclib and fulvestrant.

[0325] In some embodiments, the additional therapeutic agents are palbociclib and brinostrand.

[0326] In some embodiments, the additional therapeutic agents are palbociclib and elastrant.

[0327] In some embodiments, the additional therapeutic agent is palbociclib and giredestrant. In some embodiments, the additional therapeutic agent is palbociclib and amcenestrant.

[0328] In some embodiments, the additional therapeutic agents are palbociclib and camistrant.

[0329] In some embodiments, the additional therapeutic agents are palbociclib and rintodestrant.

[0330] In some embodiments, the additional therapeutic agents are palbociclib and imlunestrant.

[0331] In some embodiments, the additional therapeutic agents are palbociclib and LSZ102.

[0332] In some embodiments, the additional therapeutic agents are palbociclib and LY3484356.

[0333] In some embodiments, the additional therapeutic agents are palbociclib and ZN-c5.

[0334] In some embodiments, the additional therapeutic agents are palbociclib and taragarestrant.

[0335] In some embodiments, the additional therapeutic agents are palbociclib and AZD9496.

[0336] In some embodiments, the additional therapeutic agents are palbociclib and clotrimazole.

[0337] In some embodiments, the additional therapeutic agents are palbociclib and fenticonazole.

[0338] In some embodiments, the additional therapeutic agents are palbociclib and SHR9549.

[0339] In some embodiments, the additional therapeutic agents are palbociclib and palablastrant.

[0340] In some embodiments, the additional therapeutic agents are ribociclib and clomiphene.

[0341] In some embodiments, the additional therapeutic agents are ribociclib and cyclofenac.

[0342] In some embodiments, the additional therapeutic agents are ribociclib and ardrin.

[0343] In some embodiments, the additional therapeutic agent is ribociclib and bromocriptine.

[0344] In some embodiments, the additional therapeutic agents are ribociclib and nafoxidine.

[0345] In some embodiments, the additional therapeutic agents are ribociclib and ormeloxifene.

[0346] In some embodiments, the additional therapeutic agents are ribociclib and raloxifene.

[0347] In some embodiments, the additional therapeutic agents are ribociclib and toremifene.

[0348] In some embodiments, the additional therapeutic agents are ribociclib and lasofoxifene.

[0349] In some embodiments, the additional therapeutic agents are ribociclib and bazedoxifene.

[0350] In some embodiments, the additional therapeutic agents are ribociclib and ospemifene.

[0351] In some embodiments, the additional therapeutic agents are ribociclib and afoxifene.

[0352] In some embodiments, the additional therapeutic agents are ribociclib and enclomid.

[0353] In some embodiments, the additional therapeutic agents are ribociclib and selinexor.

[0354] In some embodiments, the additional therapeutic agents are ribociclib and arzoxifene.

[0355] In some embodiments, the additional therapeutic agents are ribociclib and tamoxifen.

[0356] In some embodiments, the additional therapeutic agents are ribociclib and etacstil.

[0357] In some embodiments, the additional therapeutic agents are ribociclib and fulvestrant.

[0358] In some embodiments, the additional therapeutic agents are ribociclib and brinostrand.

[0359] In some embodiments, the additional therapeutic agents are ribociclib and elastatre.

[0360] In some embodiments, the additional therapeutic agent is ribociclib and giredestrant. In some embodiments, the additional therapeutic agent is ribociclib and amcenestrant.

[0361] In some embodiments, the additional therapeutic agents are ribociclib and camistrant.

[0362] In some embodiments, the additional therapeutic agents are ribociclib and rintodestrant.

[0363] In some embodiments, the additional therapeutic agents are ribociclib and imlunestrant.

[0364] In some embodiments, the additional therapeutic agents are ribociclib and LSZ102.

[0365] In some embodiments, the additional therapeutic agents are ribociclib and LY3484356.

[0366] In some embodiments, the additional therapeutic agents are ribociclib and ZN-c5.

[0367] In some embodiments, the additional therapeutic agents are ribociclib and taragarestrant.

[0368] In some embodiments, the additional therapeutic agents are ribociclib and AZD9496.

[0369] In some embodiments, the additional therapeutic agents are ribociclib and clotrimazole.

[0370] In some embodiments, the additional therapeutic agents are ribociclib and fenticonazole.

[0371] In some embodiments, the additional therapeutic agents are ribociclib and SHR9549.

[0372] In some embodiments, the additional therapeutic agents are ribociclib and palablastrant.

[0373] In some embodiments, the additional therapeutic agents are abemaciclib and clomiphene.

[0374] In some embodiments, the additional therapeutic agents are abemaciclib and cyclofenac.

[0375] In some embodiments, the additional therapeutic agents are abemaciclib and anordrin.

[0376] In some embodiments, the additional therapeutic agents are abemaciclib and bromopastriene.

[0377] In some embodiments, the additional therapeutic agents are abemaciclib and nafoxidine.

[0378] In some embodiments, the additional therapeutic agents are abemaciclib and ormeloxifene.

[0379] In some embodiments, the additional therapeutic agents are abemaciclib and raloxifene.

[0380] In some embodiments, the additional therapeutic agents are abemaciclib and toremifene.

[0381] In some embodiments, the additional therapeutic agents are abemaciclib and lasofoxifene.

[0382] In some embodiments, the additional therapeutic agents are abemaciclib and bazedoxifene.

[0383] In some embodiments, the additional therapeutic agents are abemaciclib and ospemifene.

[0384] In some embodiments, the additional therapeutic agents are abemaciclib and afexifene.

[0385] In some embodiments, the additional therapeutic agents are abemaciclib and enclomiphene.

[0386] In some embodiments, the additional therapeutic agents are abemaciclib and selanphen.

[0387] In some embodiments, the additional therapeutic agents are abemaciclib and arzoxifene.

[0388] In some embodiments, the additional therapeutic agents are abemaciclib and tamoxifen.

[0389] In some embodiments, the additional therapeutic agents are abemaciclib and etacstil.

[0390] In some embodiments, the additional therapeutic agents are abemaciclib and fulvestrant.

[0391] In some embodiments, the additional therapeutic agents are abemaciclib and brinostrand.

[0392] In some embodiments, the additional therapeutic agents are abemaciclib and elastrant.

[0393] In some embodiments, the additional therapeutic agent is abemaciclib and giredestrant. In some embodiments, the additional therapeutic agent is abemaciclib and amcenestrant.

[0394] In some embodiments, the additional therapeutic agents are abemaciclib and camistrant.

[0395] In some embodiments, the additional therapeutic agents are abemaciclib and rintodestrant.

[0396] In some embodiments, the additional therapeutic agents are abemaciclib and imlunestrant.

[0397] In some embodiments, the additional therapeutic agents are abemaciclib and LSZ102.

[0398] In some embodiments, the additional therapeutic agents are abemaciclib and LY3484356.

[0399] In some embodiments, the additional therapeutic agents are abemaciclib and ZN-c5.

[0400] In some embodiments, the additional therapeutic agents are abemaciclib and taragarestrant.

[0401] In some embodiments, the additional therapeutic agents are abemaciclib and AZD9496.

[0402] In some embodiments, the additional therapeutic agents are abemaciclib and clotrimazole.

[0403] In some embodiments, the additional therapeutic agents are abemaciclib and fenticonazole.

[0404] In some embodiments, the additional therapeutic agents are abemaciclib and SHR9549.

[0405] In some embodiments, the additional therapeutic agents are abemaciclib and palablastrant.

[0406] , the method comprises administering compound 1 or a pharmaceutically acceptable salt thereof, and two additional independently selected therapeutic agents, such as HER2 inhibitors, SERM / SERDs, CDK4 / 6 inhibitors, MEK inhibitors, checkpoint inhibitors (e.g., anti-PD-1 or PD-L1 antibodies, anti-CLTA4 antibodies), multikinase inhibitors, and PI3K inhibitors.

[0407] In some embodiments, the method comprises administering Compound 1, or a pharmaceutically acceptable salt thereof, and two additional independently selected therapeutic agents, such as trastuzumab, pertuzumab, emtansine trastuzumab, fam-der trastuzumab, lapatinib, neratinib, dacomitinib, afatinib, tucatinib, erlotinib, pyrotinib, tanspiramycin, dacomitinib, pelitinib, magetuximab, clomiphene, cyclofenib, bromocriptine, ormeloxifene, raloxifene, toremifene, lasofoxifene, bazedoxifene, orpemimifene. Fen, enclomiphene, selanfen, tamoxifen, fulvestrant, elastrant, camistrant, rintodestrant, clotrimazole, fenticonazole, nivolumab, pembrolizumab, cemiplimab, atezolizumab, durvalumab, avelumab, ipilimumab, palbociclib, ribociclib, abemaciclib, tracerib, dacillinib, trametinib, cobimetinib, binimetinib, selumetinib, mirdatinib, pimaticinib, apelisib, idelalisib, durvalumab, cupanisib, and erbulisib.

[0408] In some embodiments, the additional therapeutic agents are fulvestrant and lapatinib.

[0409] In some embodiments, the additional therapeutic agents are fulvestrant and trametinib.

[0410] In some embodiments, the additional therapeutic agents are fulvestrant and binimetinib.

[0411] In some embodiments, the additional therapeutic agents are fulvestrant and apellisidone.

[0412] In some embodiments, the additional therapeutic agents are fulvestrant and triraciclib.

[0413] In some embodiments, the additional therapeutic agents are fulvestrant and dacillin.

[0414] In some embodiments, the additional therapeutic agents are fulvestrant and waluxid.

[0415] In some embodiments, the additional therapeutic agents are fulvestrant and roniciclib.

[0416] In some embodiments, the additional therapeutic agents are fulvestrant and dinaciclib.

[0417] In some embodiments, the method comprises administering Compound 1, or a pharmaceutically acceptable salt thereof, and an ERa inhibitor or degrader (eg, a SERM or SERD).

[0418] In some embodiments, the method comprises administering Compound 1, or a pharmaceutically acceptable salt thereof, and a CDK4 / 6 inhibitor.

[0419] In some embodiments, the method comprises administering Compound 1, or a pharmaceutically acceptable salt thereof, and a HER2 inhibitor.

[0420] In some embodiments, the method comprises administering Compound 1, or a pharmaceutically acceptable salt thereof, and apellisor.

[0421] In some embodiments, the method consists essentially of administering Compound 1, or a pharmaceutically acceptable salt thereof, and an ERa inhibitor or degrader.

[0422] In some embodiments, the method consists essentially of administering Compound 1, or a pharmaceutically acceptable salt thereof, and a CDK4 / 6 inhibitor.

[0423] In some embodiments, the method consists essentially of administering Compound 1, or a pharmaceutically acceptable salt thereof, and a HER2 inhibitor.

[0424] In some embodiments, the method consists essentially of administering Compound 1, or a pharmaceutically acceptable salt thereof, and apellisor.

[0425] In some embodiments, the method consists essentially of administering Compound 1, or a pharmaceutically acceptable salt thereof, an ERa inhibitor or degrader, and a CDK4 / 6 inhibitor.

[0426] In some embodiments, the method consists essentially of administering Compound 1, or a pharmaceutically acceptable salt thereof, fulvestrant, and a CDK4 / 6 inhibitor.

[0427] In some embodiments, the method consists essentially of administering Compound 1, or a pharmaceutically acceptable salt thereof, fulvestrant, and palbociclib. In some embodiments, the method consists essentially of administering Compound 1, or a pharmaceutically acceptable salt thereof, fulvestrant, and ribociclib. In some embodiments, the method consists essentially of administering Compound 1, or a pharmaceutically acceptable salt thereof, fulvestrant, and abemaciclib.

[0428] In some embodiments, the methods comprise administering Compound 1, or a pharmaceutically acceptable salt thereof, and Fulvestrant. In some embodiments, the methods comprise administering Compound 1, or a pharmaceutically acceptable salt thereof, and Fulvestrant, wherein Fulvestrant is administered at a dose ranging from about 250 mg to about 500 mg. In some embodiments, the methods comprise administering Compound 1, or a pharmaceutically acceptable salt thereof, and Fulvestrant, wherein Fulvestrant is administered at a dose of about 250 mg. In some embodiments, the methods comprise administering Compound 1, or a pharmaceutically acceptable salt thereof, and Fulvestrant, wherein Fulvestrant is administered at a dose of about 500 mg. In some embodiments, the methods comprise administering Compound 1, or a pharmaceutically acceptable salt thereof, and Fulvestrant, wherein Fulvestrant is administered as two 5 mL injections on days 1, 15, 29, and monthly thereafter, wherein each 5 mL injection comprises 250 mg of Fulvestrant.

[0429] In some embodiments, the method comprises administering compound 1, or a pharmaceutically acceptable salt thereof, and lapatinib. In some embodiments, the method comprises administering compound 1, or a pharmaceutically acceptable salt thereof, and lapatinib, wherein lapatinib is administered at a dose ranging from about 1250 mg to about 1500 mg. In some embodiments, the method comprises administering compound 1, or a pharmaceutically acceptable salt thereof, and lapatinib, wherein lapatinib is administered at a dose of about 1250 mg. In some embodiments, the method comprises administering compound 1, or a pharmaceutically acceptable salt thereof, and lapatinib, wherein lapatinib is administered at a dose of about 1500 mg. In some embodiments, the method comprises administering compound 1, or a pharmaceutically acceptable salt thereof, and lapatinib, wherein lapatinib is administered as 5 tablets once daily, wherein each tablet comprises 250 mg of lapatinib. In some embodiments, the method comprises administering compound 1, or a pharmaceutically acceptable salt thereof, and lapatinib, wherein lapatinib is administered as 6 tablets once daily, wherein each tablet comprises 250 mg of lapatinib.

[0430] In some embodiments, the method comprises administering Compound 1, or a pharmaceutically acceptable salt thereof, and Abemaciclib. In some embodiments, the method comprises administering Compound 1, or a pharmaceutically acceptable salt thereof, and Abemaciclib, wherein Abemaciclib is administered at a dose ranging from about 150 mg to about 400 mg. In some embodiments, the method comprises administering Compound 1, or a pharmaceutically acceptable salt thereof, and Abemaciclib, wherein Abemaciclib is administered at a dose of about 150 mg. In some embodiments, the method comprises administering Compound 1, or a pharmaceutically acceptable salt thereof, and Abemaciclib, wherein Abemaciclib is administered at a dose of about 200 mg. In some embodiments, the method comprises administering Compound 1, or a pharmaceutically acceptable salt thereof, and Abemaciclib, wherein Abemaciclib is administered at a dose of about 300 mg. In some embodiments, the method comprises administering Compound 1, or a pharmaceutically acceptable salt thereof, and Abemaciclib, wherein Abemaciclib is administered at a dose of about 400 mg. In some embodiments, the method comprises administering Compound 1, or a pharmaceutically acceptable salt thereof, and abemaciclib, wherein abemaciclib is administered as a 50 mg, 100 mg, 150 mg, or 200 mg tablet. In some embodiments, the method comprises administering Compound 1, or a pharmaceutically acceptable salt thereof, and abemaciclib, wherein 150 mg of abemaciclib is administered twice daily as a tablet. In some embodiments, the method comprises administering Compound 1, or a pharmaceutically acceptable salt thereof, and abemaciclib, wherein 200 mg of abemaciclib is administered twice daily as a tablet.

[0431] In some embodiments, the method comprises administering Compound 1, or a pharmaceutically acceptable salt thereof, and a MEK inhibitor.

[0432] In some embodiments, the method comprises administering Compound 1, or a pharmaceutically acceptable salt thereof, and trametinib. In some embodiments, the method comprises administering Compound 1, or a pharmaceutically acceptable salt thereof, and trametinib, wherein trametinib is administered at a dose ranging from about 1 mg to about 2 mg. In some embodiments, the method comprises administering Compound 1, or a pharmaceutically acceptable salt thereof, and trametinib, wherein when the subject is a pediatric patient weighing about 26 kg to about 37 kg, trametinib is administered at a dose of about 1 mg. In some embodiments, the method comprises administering Compound 1, or a pharmaceutically acceptable salt thereof, and trametinib, wherein when the subject is a pediatric patient weighing about 38 kg to about 50 kg, trametinib is administered at a dose of about 1.5 mg. In some embodiments, the method comprises administering Compound 1, or a pharmaceutically acceptable salt thereof, and trametinib, wherein when the subject is a pediatric patient weighing about 51 kg or more, trametinib is administered at a dose of about 2 mg. In some embodiments, the method comprises administering Compound 1, or a pharmaceutically acceptable salt thereof, and trametinib, wherein trametinib is administered at a dose of about 2 mg. In some embodiments, the method comprises administering Compound 1, or a pharmaceutically acceptable salt thereof, and trametinib, wherein trametinib is administered as a 0.5 mg or 2 mg tablet. In some embodiments, the method comprises administering Compound 1, or a pharmaceutically acceptable salt thereof, and trametinib, wherein 2 mg of trametinib is administered once daily. In some embodiments, the method comprises administering Compound 1, or a pharmaceutically acceptable salt thereof, and trametinib, wherein 1.5 mg of trametinib is administered once daily. In some embodiments, the method comprises administering Compound 1, or a pharmaceutically acceptable salt thereof, and trametinib, wherein 1 mg of trametinib is administered once daily.

[0433] In some embodiments, the method comprises administering compound 1 or a pharmaceutically acceptable salt thereof and bimetinib. In some embodiments, the method comprises administering compound 1 or a pharmaceutically acceptable salt thereof and bimetinib, wherein bimetinib is administered at a dose ranging from about 30 mg to about 90 mg. In some embodiments, the method comprises administering compound 1 or a pharmaceutically acceptable salt thereof and bimetinib, wherein bimetinib is administered at a dose of about 30 mg. In some embodiments, the method comprises administering compound 1 or a pharmaceutically acceptable salt thereof and bimetinib, wherein bimetinib is administered at a dose of about 45 mg. In some embodiments, the method comprises administering compound 1 or a pharmaceutically acceptable salt thereof and bimetinib, wherein bimetinib is administered at a dose of about 60 mg. In some embodiments, the method comprises administering compound 1 or a pharmaceutically acceptable salt thereof and bimetinib, wherein bimetinib is administered at a dose of about 90 mg. In some embodiments, the method comprises administering compound 1 or a pharmaceutically acceptable salt thereof and bimetinib, wherein bimetinib is administered as a 15 mg tablet. In some embodiments, the method comprises administering Compound 1, or a pharmaceutically acceptable salt thereof, and bimetinib, wherein 45 mg of bimetinib is administered as three 15 mg tablets twice daily. In some embodiments, the method comprises administering Compound 1, or a pharmaceutically acceptable salt thereof, and bimetinib, wherein 30 mg of bimetinib is administered as two 15 mg tablets twice daily.

[0434] In some embodiments, the method comprises administering Compound 1, or a pharmaceutically acceptable salt thereof, and palbociclib. In some embodiments, the method comprises administering Compound 1, or a pharmaceutically acceptable salt thereof, and palbociclib, wherein palbociclib is administered at a dose ranging from about 75 mg to about 125 mg. In some embodiments, the method comprises administering Compound 1, or a pharmaceutically acceptable salt thereof, and palbociclib, wherein palbociclib is administered at a dose of about 75 mg. In some embodiments, the method comprises administering Compound 1, or a pharmaceutically acceptable salt thereof, and palbociclib, wherein palbociclib is administered at a dose of about 100 mg. In some embodiments, the method comprises administering Compound 1, or a pharmaceutically acceptable salt thereof, and palbociclib, wherein palbociclib is administered at a dose of about 125 mg. In some embodiments, the method comprises administering Compound 1, or a pharmaceutically acceptable salt thereof, and palbociclib, wherein palbociclib is administered as a 75 mg, 100 mg, or 125 mg tablet. In some embodiments, the method comprises administering Compound 1, or a pharmaceutically acceptable salt thereof, and palbociclib, wherein 75 mg of palbociclib is administered once daily. In some embodiments, the method comprises administering Compound 1, or a pharmaceutically acceptable salt thereof, and palbociclib, wherein 100 mg of palbociclib is administered once daily. In some embodiments, the method comprises administering Compound 1, or a pharmaceutically acceptable salt thereof, and palbociclib, wherein 125 mg of palbociclib is administered once daily. In some embodiments, the method comprises administering Compound 1, or a pharmaceutically acceptable salt thereof, and palbociclib, wherein 125 mg of palbociclib is administered as a 125 mg tablet once daily.

[0435] Pharmaceutical composition

[0436] Some embodiments provide pharmaceutical compositions comprising a compound of Formula (I) or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.

[0437] Some embodiments provide pharmaceutical compositions comprising Compound 1 or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.

[0438] In some embodiments, the pharmaceutical composition comprises one or two additional therapeutic agents, i.e., as a fixed dose combination. In some embodiments, the pharmaceutical composition comprises one additional therapeutic agent. In some embodiments, the pharmaceutical composition comprises two additional therapeutic agents.

[0439] Drug administration

[0440] In some embodiments, Compound 1, or a pharmaceutically acceptable salt thereof, is administered at a dose ranging from about 1 mg to about 500 mg, for example, from about 1 mg to about 450 mg, from about 1 mg to about 400 mg, from about 1 mg to about 350 mg, from about 1 mg to about 300 mg, from about 1 mg to about 250 mg, from about 1 mg to about 200 mg, from about 1 mg to about 150 mg, from about 1 mg to about 100 mg, from about 1 mg to about 50 mg, from about 1 mg to about 40 mg, from about 1 mg to about 30 mg, from about 1 mg to about 25 mg, from about 1 mg to about 20 mg, from about 1 mg to about 15 mg, from about 1 mg to about 10 mg, or from about 1 mg to about 5 mg.

[0441] In some embodiments, Compound 1, or a pharmaceutically acceptable salt thereof, is administered at a dose in the range of about 1 mg to about 100 mg, for example, about 1 mg to about 80 mg, about 1 mg to about 75 mg, about 1 mg to about 60 mg, about 1 mg to about 50 mg, about 1 mg to about 40 mg, about 1 mg to about 30 mg, or about 1 mg to about 20 mg.

[0442] In some embodiments, Compound 1, or a pharmaceutically acceptable salt thereof, is administered at a dose in the range of about 20 mg to about 100 mg, for example, about 20 mg to about 80 mg, about 30 mg to about 80 mg, about 30 mg to about 70 mg, about 30 mg to about 60 mg, about 20 mg to about 50 mg, about 40 mg to about 70 mg, about 40 mg to about 60 mg, or about 45 mg to about 55 mg.

[0443] In some embodiments, Compound 1, or a pharmaceutically acceptable salt thereof, is administered at a dose in the range of about 1 mg to about 50 mg, e.g., about 1 mg to about 45 mg, about 1 mg to about 40 mg, about 1 mg to about 35 mg, about 1 mg to about 30 mg, about 1 mg to about 25 mg, about 1 mg to about 20 mg, about 1 mg to about 15 mg, about 1 mg to about 10 mg, or about 1 mg to about 5 mg.

[0444] In some embodiments, the total daily dose of Compound 1 or a pharmaceutically acceptable salt thereof is as described herein. In some embodiments, Compound 1 or a pharmaceutically acceptable salt thereof is administered once a day. In some embodiments, Compound 1 or a pharmaceutically acceptable salt thereof is administered twice a day.

[0445] In some embodiments, Compound 1, or a pharmaceutically acceptable salt thereof, is administered as a monotherapy at the doses described herein.

[0446] In some embodiments, Compound 1, or a pharmaceutically acceptable salt thereof, is administered in combination with one or more additional therapeutic agents as described herein, at a dose of Compound 1 as described herein. In some embodiments, the dose of the one or more additional therapeutic agents is a standard dose of the one or more additional therapeutic agents, for example, as indicated on labeling approved by a regulatory agency (e.g., USFDA or EMA).

[0447] Example

[0448] Compound preparation

[0449] Compound 1: (R)-1-(2-aminopyrimidin-5-yl)-3-(1-(5,7-difluoro-3-methylbenzofuran-2-yl)- 2,2,2-trifluoroethyl)urea

[0450]

[0451] Step 1

[0452] To a mixture of pyrimidine-2,5-diamine (3.0 g, 27.2 mmol) and NaHCO 3 (11.4 g, 135.9 mmol) in THF (300 mL) was added phenyl chloroformate (4.5 g, 28.5 mmol) dropwise at 0° C. The mixture was stirred at 25° C. for 4 hours. After the reaction, the reaction mixture was concentrated to give a residue which was purified by silica gel chromatography (0-10% DCM / MeOH) to give phenyl (2-aminopyrimidin-5-yl)carbamate (2.2 g, 34%) as a brown solid. MS (ESI): for C 11 H 10 Mass calculated for N4O2, 230.1, m / z found 231.1 [M+H] + .

[0453] Step 2

[0454] To a solution of 1-(3,5-difluoro-2-hydroxyphenyl)ethan-1-one (20 g, 116.2 mmol) in DMF (200 mL) was added methyl 2-bromoacetate (19.4 g, 127.9 mmol) and K2CO3 (24.1 g, 174.4 mmol) at room temperature. The reaction mixture was stirred at room temperature for 2 hours. After the reaction, the insoluble material was filtered off and DBU (17.7 g, 116.2 mmol) was added to the filtrate, which was again stirred at 80°C for 2 hours. After the reaction, the reaction mixture was concentrated to give a residue which was purified by silica gel chromatography (0-35% PE / DCM) to give methyl 5,7-difluoro-3-methylbenzofuran-2-carboxylate (8.4 g, 32%) as an off-white solid. MS (ESI): for C 11Mass calculated for H8F2O3, 226.0, m / z found 227.0 [M+H] + .

[0455] Step 3

[0456] To a solution of 5,7-difluoro-3-methylbenzofuran-2-carboxylate (8.1 g, 35.8 mmol) in THF (160 mL) was added LiAlH4 (21.5 mL, 21.5 mmol, 1 M in THF) at 0°C. The reaction mixture was stirred at 0°C for 2 hours, then slowly quenched with saturated aqueous potassium carbonate (150 mL) and extracted with EA (200 mL x 3). The combined organic layers were washed with brine and dried over Na2SO4 and concentrated to give a residue which was purified by silica gel chromatography (0-30% PE / EA) to give (5,7-difluoro-3-methylbenzofuran-2-yl)methanol (6.5 g, 91%) as an off-white solid. MS (ESI): for C 10 Mass calculated for H8F2O2, 198.0, m / z found 181.0 [M-H2O+H] + .

[0457] Step 4

[0458] To a solution of (5,7-difluoro-3-methylbenzofuran-2-yl)methanol (6.5 g, 32.6 mmol) in ACN (65 mL) was added IBX (13.7 g, 49.0 mmol) at room temperature. The reaction mixture was stirred at 80°C for 2 hours. The insoluble material was then filtered off and the filtrate was concentrated to give a residue which was purified by silica gel chromatography (0-15% PE / EA) to give 5,7-difluoro-3-methylbenzofuran-2-carbaldehyde (6.1 g, 95%) as an off-white solid. MS (ESI): 40.84 for C 10 Mass calculated for H6F2O2, 196.0, m / z found 197.1 [M+H] + .

[0459] Step 5

[0460] At 0 ℃, trimethyl (trifluoromethyl) silane (8.89g, 62.2mmol) and K are added to the DMF (92mL) solution of 5,7-difluoro-3-methylbenzofuran-2-carboxaldehyde (6.1g, 31.1mmol).The reaction mixture is stirred at room temperature for 0.5 hour, then KCO (4.3g, another of 31.0mmol) is added in batches in the reaction mixture.The mixture is stirred at room temperature for 16 hours, then H is added O (2.8g, 115.5mmol), and the reaction mixture is further stirred for 1 hour at 0 ℃.The mixture is quenched with ice water and extracted with EA (200mLx 3). The combined organic layers were washed with brine, dried over Na2SO4 and concentrated to give a residue which was purified by silica gel chromatography (0-20% PE / EA) to give 1-(5,7-difluoro-3-methylbenzofuran-2-yl)-2,2,2-trifluoroethan-1-ol (6.5 g, 78%) as a pale yellow oil. MS (ESI): 40.8 % for C 11 Mass calculated for H7F5O2, 266.0, m / z found 249.1 [M-H2O+H] + .

[0461] Step 6

[0462] To a solution of 1-(5,7-difluoro-3-methylbenzofuran-2-yl)-2,2,2-trifluoroethan-1-ol (370 mg, 1.39 mmol) in ACN (10 mL) was added IBX (584 mg, 2.08 mmol). The reaction mixture was refluxed for 16 hours. After the reaction, the mixture was filtered and washed with EA. The filtrate was collected and concentrated to give a residue which was purified by silica gel chromatography (0-20% PE / EA) to give 1-(5,7-difluoro-3-methylbenzofuran-2-yl)-2,2,2-trifluoroethan-1-one (320 mg, 87%) as a yellow oil. 1 H NMR (400MHz, DMSO) δ7.82–7.74 (m, 2H), 2.65 (s, 3H).

[0463] Step 7

[0464] A mixture of 1-(5,7-difluoro-3-methylbenzofuran-2-yl)-2,2,2-trifluoroethan-1-one (320 mg, 1.21 mmol), hydroxylamine hydrochloride (585 mg, 8.48 mmol) and NaOAc (992 mg, 12.10 mmol) in EtOH (10 mL) was refluxed for 16 hours. After the reaction, the mixture was concentrated and redissolved in MeOH (10 mL), to which RaneyNi (50 mg) and a drop of ammonia were added. The mixture was stirred at room temperature under H2 for 6 hours. After the reaction, the mixture was filtered and the filtrate was concentrated to give a residue which was purified by silica gel chromatography (0-50% PE / EA) to give 1-(5,7-difluoro-3-methylbenzofuran-2-yl)-2,2,2-trifluoroethan-1-amine (150 mg, 47%) as a light yellow oil. MS (ESI): for C 11 Mass calculated for H8F5NO, 265.0, m / z found 249.1 [M-NH3+H] + .

[0465] Step 8

[0466] To a mixture of 1-(5,7-difluoro-3-methylbenzofuran-2-yl)-2,2,2-trifluoroethane-1-amine (150 mg, 0.57 mmol), DIEA (219 mg, 1.70 mmol) in DMF (10 mL) at 0°C was added phenyl (2-aminopyrimidin-5-yl)carbamate (143 mg, 0.62 mmol). The reaction mixture was then stirred at room temperature for 16 hours before being quenched with water and extracted with EA (50 mL x 3). The combined organic layers were concentrated to give a residue which was purified by silica gel chromatography (0-50% PE / EA) to give (rac)-1-(2-aminopyrimidin-5-yl)-3-(1-(5,7-difluoro-3-methylbenzofuran-2-yl)-2,2,2-trifluoroethyl)urea (150 mg, 66%) as a light yellow oil. MS (ESI): For C 16 H 12 Mass calculated for F5N5O2, 401.1, m / z found 402.1 [M+H] + .

[0467] Step 9

[0468] (Racemic) 1-(2-aminopyrimidin-5-yl)-3-(1-(5,7-difluoro-3-methylbenzofuran-2-yl)-2,2,2-trifluoroethyl)urea (150 mg) was separated by chiral HPLC to give the (S) enantiomer of compound 1 (peak 1, 61 mg, 41%) and compound 1 (peak 2, 58 mg, 39%). MS (ESI): for C16 H 12 Mass calculated for F5N5O2, 401.1, m / z found 402.1 [M+H] + .

[0469] (S) enantiomer of compound 1 (peak 1):

[0470] 1 HNMR (400MHz, DMSO-d6) δ8.21(s,2H),8.20(s,1H),7.81(d,J=8.0Hz,1H),7.46-7.39(m,2H),6.40(s,2H),6.09–5.98(m,1H),2.29(s,3H).

[0471] Compound 1 (peak 2):

[0472] 1 HNMR (400MHz, DMSO-d6) δ8.19(s,2H),8.18(s,1H),7.79(d,J=8.0Hz,1H),7.46–7.34(m,2H),6.37(s,2H),6.03-5.97(m,1H),2.27(s,3H).

[0473] Determination

[0474] Homogeneous time-resolved fluorescence (HTRF)-pAKT-T47D

[0475] Compound 1 was measured using homogeneous time-resolved fluorescence (HTRF).

[0476] Materials, reagents, and equipment

[0477] Gibco RPMI1640 Medium, without phenol red; Gibco RPMI1640 Medium; Gibco Trypsin-EDTA (0.5%), without phenol red; Gibco DPBS; Trypan blue solution 0.4% (Corning); Avantor Seradigm Premium Grade Fetal Bovine Serum (FBS); Greiner 7840 80-384 well TC treated white plates; pAKT (Ser473) HTRF; Gibco Insulin, human recombinant, zinc solution; Gibco Recovery Cell Culture Freezing Medium; Countess II FL Automated Cell Counter (ThermoFisher); Countess II Slides (ThermoFisher); microscope; and PHERAstar FSX Microplate Reader (BMG LABTECH, Inc.).

[0478] program

[0479] The scinamic cell line ID is T47D.1, the HTRF detection is pAKT (S473), there is PI3KαH1047R mutation, the seeding density is 5000, the time point is 1 hour, and the culture medium used is RPMI+10% FBS (without phenol red)+0.2 units / ml bovine insulin.

[0480] Cell culture maintenance:

[0481] • Cell density was not allowed to reach 100% confluence. When cells reached -80% confluence, they were split 1:5.

[0482] o Cells were split twice a week (Monday and Friday).

[0483] o Cells older than passage 18 (~2 months of maintenance) were not used.

[0484] ○ No antibiotics were used for tissue culture maintenance or assays.

[0485] For frozen cells:

[0486] 1. Collect and count trypsinized cells. Pellet the cells at 1000 rpm for 5 minutes and aspirate the supernatant.

[0487] 2. Gently resuspend the cell pellet at 3e6 cells / 1 mL of Gibco Freezing Medium. For example, if there are 9e6 total cells, resuspend the cell pellet in 3 mL of freezing medium.

[0488] 3. Measure 1 mL aliquots of resuspended cells per cryovial. Freeze cells at -80°C in an appropriate cell freezing container (ie, Mr. Frosty or Corning CoolCell Freezing System).

[0489] 4. Transfer cells to a Liquid Nitrogen Cryotank for long-term storage.

[0490] For thawing cells:

[0491] 1. Remove the cells from the liquid nitrogen tank. Thaw the cryovials in a 37°C water bath until small "ice pellets" remain. Spray them with 70% ethanol before moving to the TC / BSC hood.

[0492] 2. Add 9 ml of fresh culture medium to a 15 ml conical tube. Add 10 ml of fresh culture medium to the T75 TC-treated flask.

[0493] 3. Gently transfer 1 mL of cells in freezing medium from the cryovial to a 15 mL conical tube containing culture medium.

[0494] 4. Centrifuge at 1000 rpm for 5 minutes to pellet the cells.

[0495] 5. Aspirate the culture medium / freezing medium.

[0496] 6. Gently resuspend the cell pellet in 5 mL of fresh medium and transfer to a T75 flask with 10 mL of fresh medium. Place the flask in a 37°C incubator with 5% CO2.

[0497] plan

[0498] Day 1

[0499] The procedure is as follows:

[0500] 1. Prepare ARP:

[0501] a. Stamp 12.5 nL from the 10 mM source plate to the destination plate using Echo. If the plate is not used that day, seal it immediately and freeze it at -20°C.

[0502] b. If using frozen ARP, thaw the plate and spin at 1000 rpm x 1 minute.

[0503] 2. Preparation of cells (adherent):

[0504] a. Aspirate the culture medium from the cells. Wash the cells with sterile 1X PBS. Aspirate the PBS and add an appropriate amount of trypsin.

[0505] b. Once the cells are completely trypsinized, add appropriate culture medium to resuspend the cells. Transfer the cells to a 15 mL or 50 mL conical tube.

[0506] c. Count cells on a Countess II Cell Counter.

[0507] 3. Cell plating:

[0508] a. Prepare cells at the appropriate plating density. Using a Multidrop Combi, dispense 12 μL of diluted cells / well of a Greiner 784080–384-well TC-treated white plate into columns 1–23. Add 12 μL of the appropriate phenol-free medium to column 24 only.

[0509] b. Place the plate in a 37°C tissue culture incubator for the appropriate treatment time (see "Assay" table).

[0510] 4. Preparation of HTRF Lysis Buffer

[0511] a. Calculate the amount of HTRF lysis buffer master mix needed to perform the desired experiment, plus any additional dead volume necessary for dispensing (4 μL per well is required). Dilute the blocking reagent into 4X lysis buffer at a ratio of 1:25 (i.e., 0.1 mL of blocking reagent solution plus 2.4 mL of 4X lysis buffer).

[0512] b. Add 4 uL of lysis buffer master mix to all wells with sample or DMSO. Centrifuge the plate at 1000 rpm for 1 minute.

[0513] c. Incubate at room temperature for 30 minutes.

[0514] 5. Preparation of HTRF Antibodies

[0515] a. Calculate the amount of HTRF antibody master mix needed to perform the desired experiment, plus any additional dead volume necessary for dispensing (4 mL per well is required). Add the Eu cryptate antibody and the d2 antibody to the assay buffer at a 1:40 ratio (i.e., 100 μL Eu cryptate + 100 μL d2 cryptate + 3800 μL assay buffer).

[0516] b. Add 4 μL of the antibody master mix to each well, including column 24, which contains only medium.

[0517] c. Centrifuge the plate at 1000 rpm for 1 minute. Cover and create a "humidity chamber" by placing the plate in a sealed bag with a wet paper towel or similar and incubate overnight at room temperature in the dark.

[0518] Day 2

[0519] 6. Measure on PHERAstar / Envision using the HTRF protocol. When reading the plate, read all wells.

[0520] IC of compound 1 in T47D pAKT assay 50 (nM) between 31 and 42 nM.

[0521] Surface plasmon resonance (SPR)

[0522] PI3K binding can also be determined by SPR. SPR experiments were performed on a Biacore 8K instrument. Biotinylated recombinant PI3KαH1047R protein was used, which contains the full-length p110-α subunit carrying the H1047R mutation, with an N-terminal AviTag, complexed with a truncated p85-α subunit (amino acid residues 322-694). The protein was first incubated with 1 μM wortmannin for 30 minutes at RT to covalently block the ATP binding site and then immobilized on a streptavidin sensor chip by flowing the protein through the sensor chip at a typical concentration of 20 μg / mL and a flow rate of 2 μL / min for 1200 seconds. Compound binding affinities were measured in multi-cycle kinetic mode at a flow rate of 90 μL / min, with an association time of 90 seconds and a dissociation time of 240 seconds. The running buffer contained 50 mM Tris, pH 7.5, 150 mM NaCl, 0.01% Brij35, 1 mM DTT, 1 mM MgCl2, 0.05% Tween-20, and 2% DMSO. The temperature was maintained at 25°C during the experiment, and the data were fitted to a 1:1 binding model.

[0523] crystallization

[0524] Crystals of PI3Kα (H1047R) / p85α heterodimers in complex with GDC-0077 and compound 1 were obtained at a concentration of 10 mg / ml (20 mM Tris / HCl, 150 mM NaCl, 1 mM TCEP, pH 8.0) and pre-incubated for 1 hour with 1.5 excess GDC-0077 and compound 1 (150 mM dimethyl sulfoxide (DMSO) solution); 0.8 μl of protein solution was then mixed with 0.8 μl of stock solution (0.1 M MES pH 6.8, 0.5 M NaCl, 5% (w / v) polyethylene glycol 3350) and equilibrated at 293 K in 60 μl of stock solution. After 5 days, well-diffracting crystals were selected for data collection. Crystals were cryopreserved in stock solution supplemented with 30% ethylene glycol and quickly frozen in liquid nitrogen before data collection. The complete crystals of PI3Kα(H1047R) / p85α / GDC-0077 / Compound 1 were collected at the European Synchotron Radiation Facility (ESRF, Grenoble, FR, beamline ID30a1). The data sets were integrated, analyzed, and scaled using the programs XDS, Pointless, and STARANISO from the autoPROC pipeline, respectively.

[0525] Structural analysis and refinement.

[0526] The isostructural reference model of PI3Kα in complex with p85α and GDC-0077 was used as a starting model for refinement using the constraints of REFMAC5 for the H1047R data set. The final model was obtained using the program REFMAC5 and then several rounds of refinement using BUSTER. The atomic displacement factors were modeled using a single isotropic B factor / atom and a single TLS group / chain. Non-crystallographic symmetry constraints were used. Constraints for compound GDC-0077 and compound 1 were generated using GRADE from GlobalPhasing using the large plane option. The final model consists of two heterodimers in the asymmetric unit, both of which are bound to compound 1 and GDC-0077. Chain A and B heterodimers are better resolved than the other heterodimer and are therefore used in the analysis of compound 1 binding. Statistics on the crystal structure are reported in Table 13. Images were generated using PyMOL (www.pymol.org).

[0527] Table 13. X-ray crystallographic statistics of compound 1

[0528]

[0529]

[0530] The values ​​in brackets represent the highest resolution shell. RMS, root mean square.

[0531] PI3Kα ATPase assay

[0532] Full-length WT, M1043X, H1047X or G1049R enzyme (1-10nM) is incubated at room temperature for 1 hour with vehicle or compound, followed by the addition of ATP (final 90 μM) to initiate the enzyme reaction. The assay buffer contains 50mMTris, 150mMNaCl, 0.01%Brij35, 15mMMgCl2, 0.05%Tween-20 and 1mM DTT. ADP-Glo ​​test kit (Promega #V9102) is used to measure ADP production after a 100-minute incubation at room temperature.

[0533] Kinome selectivity profiling

[0534] Compound 1 was evaluated in vitro by kinase profiling across 373 kinases (KinaseProfiler TM , IC 50 Profiler TM ; Eurofins Cerep, Le Bois l'évêque, France).

[0535] Cell assay

[0536] See Table 14 for cell line details.

[0537] Table 14. Cell lines and plating densities used for HTRF (pAKT) and viability (CTGLO).

[0538]

[0539]

[0540] pAKT homogeneous time-resolved fluorescence (HTRF)

[0541] A 384-well phosphorylated AKT (S473) HTRF (PerkinElmer #64AKSPEH) assay was used for targeted engagement. Each well was seeded with the number of cells indicated in Supplementary Table S3 in a volume of 12.5 uL in phenol red-free medium and then treated at 37°C for 1 hour before reading on a PHERAstar plate reader.

[0542] Cell proliferation

[0543] Cell viability was measured using CellTiterGlo (Promega #G9243). The indicated cell lines were seeded in 50 uL of culture medium in 384-well plates according to Supplementary Table S3 and treated for 72 hours at 37°C with 5% CO2.

[0544] Compound 1 was submitted to the Broad Institute PRISM high-throughput cell viability screen (www.theprismlab.org / ).

[0545] human adipocytes 3 H-2-deoxyglucose uptake

[0546] Primary human subcutaneous adipocytes were treated with test compounds or vehicle for 1 hour, followed by the addition of 10 nM insulin and 3 H-2-deoxyglucose (ZenBio Durham, NCUSA, assay #CA-25, batch #SL0071). Cytochalasin B treatment was used to control for nonspecific glucose uptake. Corrected counts per minute were determined using a scintillation counter.

[0547] Animal studies

[0548] All animal handling and treatment procedures were performed in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care guidance and in accordance with approved Institutional Animal Care and Use Committee guidelines. Cell line xenografts were performed in BALB / c nude mice, except for the T47D model using NSG mice implanted with 17-β estradiol tablets (0.5 mg, 90-day release). PK / PD studies with xenograft tumors were established using standard protocols. PDX models were performed by XenoSTART (San Antonio, TX). Compound 1 and apellis were formulated in 30% 2-hydroxypropyl-β-cyclodextrin pH 8. Tissue Western blotting used standard protocols using snap-frozen tissue in radioimmunoprecipitation assay buffer with protease inhibitors. Primary antibodies pAKT (S473) (AB_2315049), AKT (AB_1147620), and vinculin (AB_2728768) from Cell Signaling Technologies were used, along with secondary antibodies from LiCOR: (IRDye 680CW goat anti-mouse IgG, AB_10956588) and IRDye 800CW goat anti-rabbit IgG (AB_621843). IHC samples were fixed in 10% NBF for 24 hours, transferred to 70% ethanol, embedded, sectioned, stained, and tumor pAKT quantified (Cell Signaling #4060). Plasma and tissue bioanalysis of Compound 1 or apellisin was measured using liquid chromatography followed by tandem mass spectrometry after protein precipitation. All methods and limits of quantification were sufficient in terms of specificity and sensitivity to support PK analysis.

[0549] OGTT and ITT studies in BALB / c nude mice were performed after 5 days of treatment. Fast for 5 hours, administer the drug, and then administer 2g / kg oral glucose (OGTT) or 0.75U / kg intraperitoneal insulin (ITT) (Lilly Inc, France, #H1079) 1 hour later. After tail vein collection or terminal blood sampling, blood glucose was measured at the indicated time (One touch Glucose Meter, Roche, ACCU-CHEK Performa #06454038). Insulin was measured by ELISA (Crystal Chem, #90082). Metabolic profile analysis in mice bearing CAL33 tumors was performed after 4 hours of fasting, followed by drug administration for 1 hour, followed by 300-mg 13C-labeled glucose (Cambridge Isotope Labs #CLM-1396-0) was administered orally. Tissues were collected immediately before (0 hours) or 30 minutes after administration of labeled glucose, snap-frozen, and analyzed (NYU Metabolism Core using the Hybrid Metabolomics protocol, RRID: SCR_017935).

[0550] Example 1: Isobolograms as a measure of combination benefit

[0551] Determination of constant enzyme activity as determined by varying substrate and inhibitor concentrations.

[0552] Three models for measuring synergy include:

[0553] 1. Loewe additivity model Additivity can be defined as a drug combining with itself, where no interaction occurs. The Loewe additivity approach is widely used but assumes that the compounds have the same Ymax and hill slope.

[0554] 2. The Bliss Independence model is based on the concept of pharmacological independence.

[0555] 3. The HSA model defines additivity as the maximum effect of the most potent compound in the combination. See Di Veroli, et al., Bioinformatics. 2016. Sep 15; 32(18): 2866-8.

[0556] Synergy was quantified and plotted as follows. Both the combination index and the isobologram were derived from the Loewe additivity method. Using IC 50 The combination index (CI) is calculated to quantify synergy. See, Altenburger, et al., Handbook of Hazardous Materials. 1993, p. 15-27. A CI less than 0.75 indicates synergy, while a CI greater than 1.25 indicates antagonism. A CI between 0.75 and 1.25 indicates additivity. The linear concentrations of the two drugs are located on different axes, and the IC of the combination is generally plotted. 50 Linear isobol lines representing Loewe additivity run between concentrations of equal value. Figures 1A-1E .

[0557] Example 2: Compound 1 in combination with fulvestrant supports clinical benefit

[0558] Strong tumor growth inhibition and stasis were observed with 100 mg / kg Compound 1. There was little to no growth inhibition with Fulvestrant alone. Only sustained tumor regression was observed with the combination of Compound 1 and Fulvestrant. Figures 2A-2C and Table 3.

[0559] Table 3

[0560]

[0561] Example 3: Compound 1 in combination with palbociclib supports clinical benefit

[0562] Strong tumor growth inhibition and stasis were observed with 100 mg / kg Compound 1 and Palbociclib alone. Only tumor regression was observed with the combination of Compound 1 and Palbociclib. Figures 3A-3B C and Table 4.

[0563] Table 4

[0564]

[0565] Example 4: CDX Model Studies Using GP2D Colon Adenocarcinoma Cells and Detroit 562 Head and Neck Squamous Cell Carcinoma (HNSCC) Cells

[0566] GP2D and Detroit562 tumor cell lines were cultured and transplanted into immunodeficient BALB / c nude mice. Mice were provided with the treatments indicated in Tables 5 and 6.

[0567] Table 5

[0568]

[0569]

[0570] Table 6

[0571] Detroit562 MAPK-driven HNSCC treat dose vehicle POQD Cetuximab 5 mg / kg QW to 10 mg / kg Compound 1 30 mg / kg Compound 1 + cetuximab Same as single agent Compound 1 100mg / kg Compound 1 + cetuximab Same as single agent

[0572] Example 5: PDX model studies using ST1056 breast cancer cells and ST433 head and neck squamous cell carcinoma (HNSCC) cells

[0573] ST1056 tumor cell line was biopsied and transplanted into athymic, immunodeficient nude mice. Table 7 shows the study details. Mice were given the treatments indicated in Table 8.

[0574] Table 7

[0575]

[0576]

[0577] Table 8

[0578]

[0579] *Administered as a fixed-volume dose.

[0580] ST433 tumor cell line was biopsied and transplanted onto FoxChase SCID (CB17 / Icr-Prkdcscid / IcrIcoCrl). Table 9 shows the study details and mice were provided with treatments as indicated in Table 10.

[0581] Table 9

[0582]

[0583]

[0584] Table 10

[0585]

[0586] Example 6: Evaluation of the anti-tumor effect of Compound 1 in combination with Fulvestrant in the T-47D human breast cancer xenograft model in immunodeficient mice

[0587] The cell line T-47D was used for this ER + HER2 - CDX breast cancer model, T-47D (PI3Kα H1047R ) were transplanted into immunodeficient mice.

[0588] Compound 1 (100 mg / kg) monotherapy caused stable tumor regression (141% TGI). Compound 1 in combination with fulvestrant (5 mg QW) was well tolerated, with more consistent and deeper tumor regression. Table 11 shows that fulvestrant monotherapy (G2) or compound 1 at suboptimal doses (50 mg / kg, G3) each produced tumor growth inhibition (52% and 92%, respectively), but collectively (G5), the combination demonstrated benefits exceeding either monotherapy alone, with 16% tumor regression.

[0589] Table 11 shows the antitumor activity in T-47D xenografts. After 20 days of treatment, the mean tumor volume (TV) of the vehicle control group reached 413 mm 3Fulvestrant at 50 mg / kg QD and 5 mg / mouse sc, respectively, showed antitumor activity relative to vehicle control with a TGI of 52% (P≤0.01) when analyzed by two-way RM ANOVA followed by Tukey post hoc comparison of means.

[0590] Compound 1 at 50 mg / kg QD and 100 mg / kg QD showed dose-dependent antitumor activity, with TGI values ​​of 92% (P≤0.0001) and 141% (P≤0.0001), respectively. Compound 1 at 50 mg / kg QD in combination with Fulvestrant 5 mg / mouse sc showed strong antitumor activity, with a TGI value of 116% (P≤0.0001). Compound 1 at 100 mg / kg QD in combination with Fulvestrant 5 mg / mouse sc showed strong antitumor activity, with a TGI value of 155% (P≤0.0001). Table 11 shows that G2 or G3 demonstrated therapeutic synergy relative to G5, with tumor regression seen only in combination.

[0591] Table 11

[0592]

[0593] A: mean ± SEM; B: TGI = (1-T / C) x 100%, T / C = 100% x (TV at the end of treatment - TV at the beginning of treatment) / (TV at the end of vehicle - TV at the beginning of vehicle); C: relative to vehicle control; NS = not significant, by two-way RMANOVA followed by Tukey post hoc comparison of means, *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.

[0594] Example 7: Evaluation of Compound 1 as Monotherapy and in Combination with Other Antineoplastic Agents in Participants with Advanced Solid Tumors

[0595] This clinical trial is a multi-part, open-label, Phase 1 / 2 study that evaluated the safety, tolerability, pharmacokinetics (PK), and preliminary anti-tumor activity of Compound 1 in participants with advanced solid tumors harboring specific mutations.

[0596] Part 1 will evaluate Compound 1 as monotherapy in participants with breast cancer and other solid tumor types; Part 2 will evaluate Compound 1 as combination therapy with fulvestrant in participants with breast cancer.

[0597] Each study part will include a 28-day screening period followed by treatment with Compound 1 monotherapy or combination therapy. Participants will remain in the study part in which they were initially enrolled throughout their study participation (ie, they will not move to other study parts).

[0598] Table 12. Arms and Interventions

[0599]

[0600]

[0601] Main outcome measures

[0602] Part 1.1 (Dose Escalation):

[0603] 1. MTD: number and proportion of subjects who experienced at least 1 DLT during the first 28 days of treatment.

[0604] 2. OBD: PK, pharmacodynamics, ORR, TEAE / SAE ≥ Grade 2. Type, frequency, and severity of TEAEs according to the CTCAE v5.0 criteria.

[0605] Section 1.2 (Dose Selection) and Section 2.1:

[0606] 1. PK, pharmacodynamics, ORR, and safety parameters

[0607] Part 1.2 (Dose Expansion), Part 1.3, and Part 2.2:

[0608] 1. ORR is defined as the percentage of participants with PR or CR based on RECIST 1.1 Inclusion criteria for selection

[0609] 1. Patients with advanced or refractory solid tumor malignancies that are metastatic or locally advanced and unresectable

[0610] 2. Have a new or recent tumor biopsy (collected at screening, if feasible) or archived tumor sample within 12 months prior to screening

[0611] 3. Patients with a tumor with a documented PI3Kα mutation as described herein, obtained from a tumor or plasma sample, as determined by a PCR- or NGS-based assay such as an FDA-approved test in the United States, or obtained as part of routine clinical care in a CLIA-certified or similarly certified laboratory.

[0612] 4. Have at least one measurable tumor lesion according to RECIST 1.1

[0613] 5. ≥18 years old when signing the ICF

[0614] 6. Having an ECOG performance status score of 0 or 1 at screening

[0615] Selected exclusion criteria

[0616] 1. History of solid tumors or hematologic malignancies histologically different from the cancer to be studied (≤2 years before screening)

[0617] 2. With symptomatic brain or spinal metastasis

[0618] 3. Patients with tumors that have mutations / deletions in PTEN and activating mutations in AKT or mTOR confirmed by a CLIA-certified or similarly certified laboratory, or patients with tumors that have mutations / deletions in PTEN and activating mutations in AKT (e.g., E17K) confirmed by a CLIA-certified or similarly certified laboratory.

[0619] 4. Have a confirmed diagnosis of type 1 diabetes or uncontrolled type 2 diabetes requiring anti-glycemic medication

[0620] 5. Cohorts A0, A1, A2, A3, A4, and B: Prior treatment with PI3K / AKT / mTOR inhibitors, except in certain cases

[0621] 6. Treatment with any local or systemic antineoplastic therapy or investigational anticancer agent within 14 days or 4 half-lives (whichever is longer) before the start of study treatment and until a maximum washout period of 28 days

[0622] 7. Toxicity from previous anticancer therapy that has not recovered to baseline levels or CTCAE grade ≤ 1, except for alopecia and peripheral neuropathy

[0623] 8. Has received radiotherapy within 14 days before the start of study treatment

[0624] Example 8. Compound 1 as a mutant-selective allosteric PI3Kα inhibitor

[0625] Compound 1 was characterized by surface plasmon resonance (SPR; Figure 5B ) was confirmed as a potent binder of H1047RPI3Kα. In addition to its low nanomolar binding affinity for the H1047R mutant (equilibrium dissociation constant, K D ~2.9nM), compound 1 has a binding affinity of 1 / 20 for WT PI3Kα (K D~56nM). In contrast, duvelithide (a non-selective orthosteric PI3Kα inhibitor) showed roughly equal binding affinity for mutant and WT PI3Kα. Without being bound by theory, the selectivity of compound 1 for the H1047R mutant may be largely due to the faster binding constant (k) for H1047R relative to WT PI3Kα. on ) and a slightly slower dissociation constant (k off )drive( Figure 5B ), suggesting that the allosteric site occupied by compound 1 may be more accessible in the H1047R mutant form of the enzyme.

[0626] The biochemical potency and mutant selectivity of compound 1 were compared with those of apellix in a panel of common oncogenic mutant PI3Kα forms ( Figure 5A Compound 1 was found to be effective against all kinase domain mutant PI3Kα forms found in cancer, including the most common variant, H1047R (IC 50 ~9.4nM) of potent and selective inhibitors with 14-fold selectivity for WT PI3Kα (IC 50 Under these same assay conditions, compound 1 had limited selectivity for the hotspot helical domain mutant (E542K / E545K) PI3Kα, whereas apellix did not show mutant selectivity as previously reported (Fritsch C, et al. Mol Cancer Ther 2014; 13(5): 1117–29).

[0627] Compound 1 also demonstrated excellent broad kinome selectivity ( Figure 5G A biochemical screen using 373 kinases representing approximately 70% of the human kinome, including PI3Kβ, PI3Kδ, and PI3Kγ isoforms, showed that only AurB kinase was inhibited by >50% (IC 50 = 1.6 μM). Follow-up confirmed that compound 1 showed limited AurB inhibition in cells at concentrations up to 10 μM.

[0628] Compared with the published WT- and H1047R-PI3Kα x-ray crystal structures, the cocrystal structure of compound 1 reveals that compound 1 occupies a novel allosteric site ( Figure 5C , Table 13), which results from a major conformational transition in residues 936–940, together with other smaller local rearrangements ( Figure 5D). Specifically, residues F937 and L938 occupy positions that directly conflict with compound 1. Rearrangement of the side chain and main chain atoms of these residues results in the repositioning of F937 and L938 to create space for the allosteric site. In addition, the activation loop is better resolved in the compound 1 co-structure relative to the existing H1047R structure (3hhm and 3hiz; Mandelker D, et al. Proc Natl Acad Sci USA 2009; 106(40): 16996–7001). Compound 1 makes several specific protein contacts within the allosteric site ( Figure 5E ).

[0629] Example 9. Compound 1 selectively targets PI3Kα activity and cell viability in PI3Kα mutant cells

[0630] Compound 1 was studied in a panel of 10 human tumor cell lines harboring PI3Kα kinase domain mutations and compared to apellisin (Table 15). In addition to H1047R, the panel also included three cell lines harboring the second most common kinase domain mutation, H1047L (EFM19, GP2D, and OAW42 cell lines), as well as three double mutant forms (BT20, CAL148, and NCIH1048 cell lines). SKBR3 breast cancer cells rely on amplified WT PI3Kα activity for growth and were included as a comparator for WT selectivity (Cerami E, et al. Cancer Discov 2012; 2(5): 401–4; Gao J, et al. Sci Signal 2013; 6(269): p11).

[0631] Target engagement was assessed using phosphorylated (serine 473) AKT (pAKT) as a marker of PI3Kα / AKT pathway activity. Figure 6B Compound 1 and apellisin dose response curves are shown for PI3Kα kinase domain mutants T47D and Cal33 cells, as well as WTSKBR3 cells. Compound 1 potently inhibited mutant PI3Kα activity across the panel of cell lines, with IC ranging from ∼15 to ∼319 nM. 50 values, which are consistent with the apellisin IC values ​​of ∼28 to ∼268 nM. 50 Compound 1 was more potent than apellisin in 9 of the 11 cell lines, with the only exception being WT-PI3Kα–SKBR3 cells, which was expected based on the mutant selectivity of compound 1 (Figure 15). Figure 6C , Table 15). In ER + HER2 -In the breast cancer benchmark T47D (H1047RPI3Kα) cell line, compound 1 was 9-fold more selective than in the SKBR3 (WTPI3Kα) cell line. In contrast, apellis did not show differences in selectivity between mutant and WT driven cell lines. When cell viability was studied in the same cell lines, there was a strong correlation between target engagement (pAKT) and cell viability (Pearson correlation coefficient = 0.8 [log scale]) ( Figure 6A These results confirm that pAKT is a relevant translational biomarker for mutant-selective inhibitors such as Compound 1.

[0632] Table 15. Compound 1 Target engagement and cell viability activity in a panel of human tumor cell lines.

[0633]

[0634]

[0635] GI 50 , the concentration of compound that reduces total cell growth by 50%; GMean, geometric mean; RRID, Research Resource Identifier.

[0636] Compound 1 activity was then evaluated in a high-throughput cell viability panel of approximately 900 tumor cell lines to identify markers of sensitivity. Consistent with the selectivity profiles described above, cell lines harboring PI3Kα kinase domain mutations were significantly more sensitive than WT cell lines ( Figure 6D Unexpectedly, tumor cells with mutations in the PI3K α-helical domain had almost the same level of growth sensitivity to compound 1, despite earlier results using recombinant proteins ( Figure 5E The increased sensitivity of the helical domain mutant cell lines compared to the WT PI3Kα cell lines may be explained by the dependence of the mutant cell lines on PI3Kα for proliferation, whereas most WT cell lines are not. PI3Kα mutations located in the hotspot helical and kinase domains appear to be sensitive to compound 1 ( Figure 6D ). PTEN inactivating mutations conferred resistance to PI3Kα inhibition in this study (P < 0.0081), consistent with previous reports (Razavi P, et al. Nat Cancer 2020; 1(4): 382–93).

[0637] The effect of compound 1 on WT PI3Kα was evaluated by assessing insulin-mediated glucose uptake in primary human adipocytes (Hauner H, Int J Obes Relat Metab Disord 1998; 22(5): 448–53). Differentiated primary human subcutaneous adipocytes were pretreated with apellis or compound 1 and then supplemented with [ 3 H]-2-deoxyglucose and 10 nM insulin. Apellisin inhibited glucose uptake at concentrations as low as 100 nM, with almost complete inhibition at 10 μM, while 50% inhibition (EC 50 ) The required concentration of compound 1 is ≥10 μM ( Figure 6E The maximum effect of compound 1 on glucose uptake (E max ) was 38%, while apellisin caused a deeper inhibition with an E of 88%. max . Overlay of cell viability dose-response curves obtained in T47D cells ( Figure 6A ) shows the potentially improved therapeutic index of Compound 1 relative to apellisin in a relevant human-cell system.

[0638] Example 10. Compound 1 treatment produces robust antitumor efficacy in PI3Kα mutant tumors in mice without metabolic dysregulation

[0639] In vivo pharmacological characterization of compound 1 was designed to determine the metabolic safety and antitumor efficacy profile of compound 1 compared to apellix. A 50 mg / kg once daily (QD) dose of apellix was selected because it was effective in a published mouse xenograft model, albeit with significant glycemic dysregulation (Fritsch C, et al. (2014) Mol Cancer Ther. 13(5):1117–29). At this dose, apellix plasma exposure (AUC) in mice (~75,000 ng*hr / mL) was approximately twice the exposure at the maximum approved human dose (~33,000 ng*hr / mL) (Juric D, et al. J Clin Oncol 2018;36(13):1291–9). A lower dose (20 mg / kg) was also included. Based on the pharmacokinetic (PK) profile of compound 1, the 30- and 100-mg / kg QD doses were expected to achieve 80% inhibitory concentrations (IC 80 ), while the 300 mg / kg QD dose exceeded these levels ( Figure 12 ).

[0640] With regard to metabolic control, the major consequence of WT PI3Kα inhibition is blockade of insulin action (e.g., insulin resistance), impaired glucose disposal and leading to hyperglycemia (Fruman DA, Chiu H, Hopkins BD, Bagrodia S, Cantley LC, Abraham RT. The PI3K pathway in human disease. Cell 2017; 170(4): 605–35; James DE, Stockli J, Birnbaum MJ. The aetiology and molecular landscape of insulin resistance. Nat Rev Mol Cell Biol 2021; 22(11): 751–7).

[0641] The effects of apellisin and Compound 1 doses on insulin sensitivity were analyzed using the insulin tolerance test (ITT) and oral glucose tolerance test (OGTT) following 5 days of repeated dosing in non-tumor-bearing female BALB / c nude mice (the sex / strain commonly used in xenograft studies). Apellisin treatment resulted in a dose-dependent decrease in glucose disposal consistent with insulin resistance in both the ITT and OGTT (100 mg / dL, 0.1 mmol / l, and 0.2 mmol / l, respectively). Figure 7A / 7B and 7C / 7D). In contrast, Compound 1 treatment was not associated with significant changes in glucose AUC, although there was a non-statistically significant increase in the ITT at the 300 mg / kg dose. Notably, Compound 1 had no effect on body weight or fasting glucose after 5 days of treatment. These studies established that repeated dosing of Compound 1 at 100 mg / kg QD was well tolerated without metabolic derangements, whereas apellix caused significant insulin resistance at the 50 mg / kg dose level.

[0642] Example 11. Benchmarking Compound 1 in the Cal33 (H1047R PI3Kα) Human HNSCC Xenograft Model

[0643] The Cal33 (H1047R PI3Kα) head and neck squamous cell carcinoma (HNSCC) cell model was chosen to benchmark in vivo therapeutic activity and pharmacodynamic biomarkers because this cell line demonstrates moderate sensitivity to PI3Kα inhibition in culture ( Figure 6A The study consisted of three arms: an efficacy arm in which animals received the test article for 28 days; a PK / pharmacodynamic (PK / PD) arm in which animals received either Compound 1 or apellisin for 3 days; and a tumor-bearing arm in which animals received [U- 13C]-glucose bolus to evaluate the effects of apellisin and compound 1 on glucose uptake and glucose oxidation in target tissues.

[0644] In the efficacy arm, there was a dose-dependent reduction in tumor volume for both compounds; Compound 1 at 30 mg / kg showed similar efficacy to Apellix at 20 mg / kg, while Compound 1 at 100 mg / kg showed similar efficacy to Apellix at 50 mg / kg ( Figure 8A Both compounds were well tolerated at all doses with no changes in body weight ( Figure 8B Apellix treatment increased serum insulin 1 hour after the dose on day 28 (P = 0.0585, Figure 8C and Figure 9 ), with a similar trend in glucose (P < 0.083, Figure 8D In all subsequent cell line-derived xenograft (CDX) studies, apellisib 50 mg / kg, but not Compound 1 at 100 mg / kg, caused a significant increase in serum insulin 1 hour post-dose (Table 16).

[0645] Table 16. Summary of CDX Study: Tumor Growth Inhibition and Insulin Levels (1 hour post-dose).

[0646]

[0647] Tumor growth inhibition relative to the first day of administration or a Regression (negative TGI).

[0648] b 1 hour after dose.

[0649] P≤0.05*, 0.01**, 0.001***, 0.0001****.

[0650] Negative TGI values ​​indicate % regression. NS, not significant; NSG, NOD scidγ.

[0651] PD biomarkers of target engagement (pAKT / AKT ratio) and Compound 1 tumor drug levels were measured 1, 4, and 12 hours post-dose on Day 3 (PK / PD group), and 1 and 6 hours after the last dose (Day 28). Figure 8E The curve fit relationship between tumor-drug concentration and pAKT / AKT levels in Cal33 tumor xenografts and in vitro is shown in Figure 2. The calculated IC from this curve fit was 0.01 when corrected for matrix binding, compared to 0.018 nM from cell culture. 50The 82% tumor growth inhibition (TGI) in the compound 1100 mg / kg QD dose group was associated with a 57% mean pAKT inhibition (AUC of treatment / vehicle). 1-12h ); and for the apellisib 50 mg / kg QD group, 79% TGI was associated with a 66% mean pAKT inhibition ( Figure 8F Unlike apellisin, compound 1 did not reduce skeletal muscle pAKT / AKT ( Figure 8G ).

[0652] Inhibition of PI3Kα is known to suppress glucose metabolism in tumors and host tissues (Hopkins BD, et al. Nature 2018;560(7719):499–503; Juric D, et al. (2018) J Clin Oncol 36(13):1291–9; DockxY, et al. Mol Imaging 2021;2021:5594514; Sarker D, et al. Clin Cancer Res 2015;21(1):77–86).

[0653] To evaluate the effects of apellisin and compound 1 on this process, Cal33 tumor-bearing mice were pretreated with drug or vehicle and then given U- 13 C-glucose. After 30 minutes, tumor and skeletal muscle metabolites were extracted and quantified using liquid chromatography-mass spectrometry (Lopes M, et al. Cell Rep 2021;37(2):109833). Oral bolus administration resulted in robust labeling of more than 70% of circulating glucose carbons in all groups ( Figure 8J In tumors, both apellisin and compound 1 significantly reduced 13 C was incorporated into TCA intermediates, a marker of glucose oxidation (Figure 4H). However, only mice treated with apellisib demonstrated decreased glucose oxidation in skeletal muscle ( Figure 8H This decrease occurred despite higher levels of circulating insulin in this group (30 min apellisin vs. vehicle), suggesting insulin resistance in this tissue ( Figure 8I These data support that compound 1 selectively inhibits mutant PI3Kα but not the WT enzyme found in host tissues.

[0654] Example 12. Compound 1 is effective across PI3Kα mutant CDX and PDX tumors without evidence of insulin resistance

[0655] This detailed metabolic characterization of Compound 1 and its activity in the Cal33 xenograft model identified the optimal Compound 1 dose as 100 mg / kg QD and confirmed that Compound 1 was more effective than the clinically matched dose of apellix (20 mg / kg). Therefore, Compound 1 was administered at 100 mg / kg to a panel of PI3Kα mutant CDX and patient-derived xenograft (PDX) models representing several cancers. Models included colon cancer (GP2D), lung cancer (NCIH1048), HNSCC (Detroit562), and HR - HER2 + Breast cancer (HCC1954). High-dose apellisin (50 mg / kg QD) was included as a benchmark, and all studies were performed in BALB / c nude mice.

[0656] The key efficacy endpoint was TGI or regression, and tolerability measures included body weight at the end of the study and insulin levels 1 hour after the dose. No treatment-specific effects on body weight were noted for either Compound 1 or Apellisib ( Figures 11F-11I The TGI of compound 1 treated with 100 mg / kg QD was similar to or better than that of apellisin treated with 50 mg / kg QD ( Figure 11E , Table 17). Compound 1 demonstrated robust efficacy in GP2D tumor xenografts expressing H1047LPI3Kα, the second most prevalent kinase domain mutation. The waterfall plot shows tumor regression in half of the animals treated with Compound 1, whereas no regression was seen with apellisin ( Figure 11E In the Detroit562 HNSCC model, robust efficacy was seen for both compounds, including tumor regression in 5 of 9 study animals. Similarly, Compound 1 and Apellix treated NCIH1048 lung cancer cells harboring dual (H1047R / K411R) PI3Kα mutations, as well as HCC1954 HR + HER2 + Provided comparable tumor growth control in breast cancer models ( Figure 11E In each of the above studies, a significant increase in serum insulin was observed in animals dosed with apellisin 50 mg / kg at 1 hour post-dose, whereas Compound 1 at 100 mg / kg was not associated with an increase in insulin (Table 17). Although both apellisin and Compound 1 treatment resulted in significant TGI, HCC1954 cells showed the lowest response of all cell lines tested in the CDX panel. HCC1954 cells were also the weakest responders to both agents in cell culture ( Figure 6A ), and may reflect the efficacy of PI3Kα inhibitor monotherapy in HER2 +Reduced efficacy in cancer, which is thought to be related to compensatory HER3 activation (Serra V, et al. Oncogene 2011; 30(22):2547–57; Chandarlapaty S, et al. Cancer Cell 2011; 19(1):58–71).

[0657] Table 17. Summary of CDX Study: Tumor Growth Inhibition and Insulin Levels (1 hour post-dose).

[0658]

[0659]

[0660] Tumor growth inhibition relative to the first day of administration or a Regression (negative TGI).

[0661] b 1 hour after dose.

[0662] P≤0.05*, 0.01**, 0.001***, 0.0001****.

[0663] Negative TGI values ​​indicate % regression. NS, not significant; NSG, NOD scidγ.

[0664] Compound 1 and apellix monotherapy were evaluated in two breast cancer PDX models: ST1056 (kinase domain mutant [H1047R]) and ST1799 (double mutant, E542K / H1065L). A third HNSCC xenograft model (ST2652) carrying only the helical domain mutation (E542K) was also evaluated. Compound 1 was highly effective in all three models including the (E542K) helical domain mutant and generally similar to apellix ( Figures 11A-11C For both Compound 1 and Apellix, a decrease in pAKT / AKT was seen 4 hours post-dose in all models ( Figures 11A-11C ).

[0665] Example 13. Safety and Efficacy of Clinically Relevant Combination Therapies

[0666] Compound 1 and fulvestrant were evaluated as monotherapy and in combination in a T47D cell xenograft model. + HER2 - In PDX models, compound 1, fulvestrant, and palbociclib monotherapy as well as paired and triple combinations of these compounds were evaluated.

[0667] The T47D cell line represents an important benchmark for mutant PI3Kα mechanisms and is a well-established estrogen-dependent ER cell line previously characterized with apellix treatment. + HER2 - Breast cancer xenograft model (Fritsch C, et al. (2014) Mol Cancer Ther 13 (5): 1117–29). Compound 1 monotherapy demonstrated a dose-dependent TGI, with a 50 mg / kg QD dose achieving a TGI similar to that of high-dose apellisib (50 mg / kg QD), while a 100 mg / kg QD dose of compound 1 produced significant tumor regression in every animal ( Figure 4A Fulvestrant monotherapy provided only ~50% TGI, and the addition of Compound 1 at doses of 50 or 100 mg / kg resulted in regression of the majority of xenografts (20% and 70% regression, respectively). Fulvestrant monotherapy and Compound 1 combination were well tolerated based on body weight ( Figures 4F-4G Single-dose PK / PD measurements were performed in tumors collected 4 hours after administration of 100 mg / kg of Compound 1 and 24 hours after fulvestrant. Phosphorylated AKT and pS6 (biomarkers of PI3Kα pathway activity) were modestly reduced by fulvestrant, while Compound 1 resulted in significant inhibition, as assessed by Western blotting and IHC (respectively). Figure 4B and 4C ).

[0668] Combination studies including a triple combination of compound 1 with palbociclib and fulvestrant were extended to aggressive ER + HER2 - Breast cancer PDX model. ST1056 tumors grew rapidly in the vehicle, palbociclib monotherapy, fulvestrant monotherapy, and fulvestrant and palbociclib combination groups, requiring animals to be removed from the study at day 17 ( Figure 4D Compound 1 resulted in durable tumor growth inhibition for 49 days or more in most animals. Adding palbociclib to Compound 1 did not provide additional efficacy but was well tolerated until day 71 when the group was terminated. The most significant responses occurred for the combination of Compound 1 and Fulvestrant, and the triple combination of Compound 1, Fulvestrant, and palbociclib. Tumor inhibition persisted between days 28 and 94 in each animal in both groups. Treatment was stopped on day 94, and modest regrowth was observed only one month after treatment was stopped at the end of the study ( Figure 4D Compound 1 monotherapy, Compound 1 in combination with fulvestrant, and the triple combination including palbociclib were all well tolerated based on body weight changes ( Figure 4E ).

Claims

1. A method of treating cancer in a subject in need thereof, comprising administering to the subject: (a) Compound 1 or a pharmaceutically acceptable salt thereof, and (b) one or more independently selected additional therapeutic agents selected from the group consisting of selective estrogen receptor modulators (SERMs), selective estrogen receptor degraders (SERDs), CDK4 / 6 inhibitors, HER2 inhibitors, EGFR inhibitors, immune checkpoint inhibitors, MEK inhibitors, RAS inhibitors and RAF inhibitors, PIM (e.g., PIM1, PIM2 and PIM3) inhibitors, or a combination of any of the foregoing.

2. The method of claim 1, wherein the one or more independently selected additional therapeutic agents is one additional therapeutic agent.

3. The method of claim 1 or 2, wherein the additional therapeutic agent is a SERM / SERD.

4. The method of claim 1 or 2, wherein the additional therapeutic agent is a CDK4 / 6 inhibitor.

5. The method of claim 1 or 2, wherein the additional therapeutic agent is a HER2 inhibitor.

6. The method of claim 1 or 2, wherein the additional therapeutic agent is an EGFR inhibitor.

7. The method of claim 1 or 2, wherein the additional therapeutic agent is an immune checkpoint inhibitor.

8. The method of claim 1 or 2, wherein the additional therapeutic agent is a MEK inhibitor.

9. The method of claim 1 or 2, wherein the additional therapeutic agent is a RAS inhibitor.

10. The method of claim 1 or 2, wherein the additional therapeutic agent is a RAF inhibitor.

11. The method of claim 1, wherein the one or more independently selected additional therapeutic agents are two independently selected additional therapeutic agents.

12. The method of claim 1 or 11, wherein one of the additional therapeutic agents is a SERM / SERD and the other additional therapeutic agent is a HER2 inhibitor, a CDK4 / 6 inhibitor or a MEK inhibitor.

13. The method of claims 1 or 11-12, wherein one of the additional therapeutic agents is a SERM / SERD and the other additional therapeutic agent is a HER2 inhibitor.

14. The method of claims 1 or 11-12, wherein one of the additional therapeutic agents is a SERM / SERD and the other additional therapeutic agent is a CDK4 / 6 inhibitor.

15. The method of claims 1 or 11-12, wherein one of the additional therapeutic agents is a SERM / SERD and the other additional therapeutic agent is a MEK inhibitor.

16. The method of any one of claims 1-3 or 11-15, wherein the SERM / SERD is clomiphene, cyclofenil, bromopastriene, ormeloxifene, raloxifene, toremifene, lasofoxifene, bazedoxifene, ospemifene, enclomiphene, selanfen, tamoxifen, fulvestrant, elastrant, camistrant, rintodestrant, clotrimazole, parastrant, or fenticonazole.

17. The method of any one of claims 1-3 or 11-16, wherein the SERM / SERD is fulvestrant.

18. The method of any one of claims 1, 2, 4, 11, 12, 14, or 17, wherein the CDK4 / 6 inhibitor is palbociclib, ribociclib, abemaciclib, or tricaciclib.

19. The method of any one of claims 1, 2, 4, 11, 12, 14, 17, or 18, wherein the CDK4 / 6 inhibitor is palbociclib.

20. The method of any one of claims 1, 2, 4, 11, 12, 14, 17, or 18, wherein the CDK4 / 6 inhibitor is abemaciclib.

21. The method of any one of claims 1, 2, 5, 11, 12, or 14, wherein the HER2 inhibitor is trastuzumab, pertuzumab, emtansine trastuzumab, fam-detrastuzumab, lapatinib, neratinib, dacomitinib, afatinib, tucatinib, erlotinib, pyrotinib, tanspiramycin, dacomitinib, pelitinib, or magetuximab.

22. The method of any one of claims 1, 2, 5, 11, 12, 14, or 21, wherein the HER2 inhibitor is lapatinib.

23. The method of any one of claims 1, 2, 6, or 11, wherein the EGFR inhibitor is gefitinib, erlotinib, afatinib, neratinib, osimertinib, vandetanib, cetuximab, necituzumab, lazertinib, ervantumab, or panitumumab.

24. The method of any one of claims 1, 2, 7, or 11, wherein the immune checkpoint inhibitor is nivolumab, pembrolizumab, cemiplimab, atezolizumab, durvalumab, avelumab, or ipilimumab.

25. The method of any one of claims 1, 2, 4, 11, 12, 14, or 15, wherein the MEK inhibitor is dacillinib, trametinib, cobimetinib, bimetinib, selumetinib, mirdatinib, or pimatitinib.

26. The method of any one of claims 1, 2, 4, 11, 12, 14, 15, or 25, wherein the MEK inhibitor is trametinib.

27. The method of any one of claims 1, 2, 4, 11, 12, 14, 15, or 25, wherein the MEK inhibitor is bimetinib.

28. The method of any one of claims 1, 2, 9, or 11, wherein the RAS inhibitor is sotolacib.

29. The method of any one of claims 1, 2, 10, or 11, wherein the RAF inhibitor is vemurafenib, dabrafenib, conefantfenib, sorafenib, belvarafenib, or naporafenib.

30. The method of claim 1, wherein the one or more additional therapeutic agents is fulvestrant.

31. The method of claim 1, wherein the one or more additional therapeutic agents are fulvestrant and lapatinib.

32. The method of claim 1, wherein the one or more additional therapeutic agents are fulvestrant and abemaciclib.

33. The method of claim 1, wherein the one or more additional therapeutic agents are fulvestrant and palbociclib.

34. The method of claim 1, wherein the one or more additional therapeutic agents are fulvestrant and trametinib.

35. The method of claim 1, wherein the one or more additional therapeutic agents are fulvestrant and binimetinib.

36. The method of any one of claims 1-35, wherein the cancer is selected from breast cancer, lung cancer, endometrial cancer, esophageal cancer, gastric cancer, ovarian cancer, colorectal cancer, bladder cancer, head and neck cancer, thyroid cancer, prostate cancer, glioma, and cervical cancer.

37. The method of any one of claims 1-36, wherein the cancer is breast cancer.

38. The method of any one of claims 1-37, wherein the breast cancer is HER2 + Breast cancer.

39. The method of any one of claims 1-37, wherein the breast cancer is HER2 - Breast cancer.

40. The method of any one of claims 1-37, wherein the breast cancer is ER + Breast cancer.

41. The method of any one of claims 1-37, wherein the breast cancer is triple-negative breast cancer.

42. The method of any one of claims 1-36, wherein the cancer is lung cancer.

43. The method of any one of claims 1-36, wherein the cancer is endometrial cancer.

44. The method of any one of claims 1-36, wherein the cancer is esophageal cancer.

45. The method of any one of claims 1-36, wherein the cancer is gastric cancer.

46. ​​The method of any one of claims 1-36, wherein the cancer is ovarian cancer.

47. The method of any one of claims 1-36, wherein the cancer is colorectal cancer.

48. The method of any one of claims 1-36, wherein the cancer is bladder cancer.

49. The method of any one of claims 1-36, wherein the cancer is head and neck cancer.

50. The method of any one of claims 1-36, wherein the cancer is thyroid cancer.

51. The method of any one of claims 1-36, wherein the cancer is prostate cancer.

52. The method of any one of claims 1-36, wherein the cancer is glioma.

53. The method of any one of claims 1-36, wherein the cancer is cervical cancer.

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