Treatment of prostate cancer

Abstract

The present disclosure relates to methods of treating prostate cancer in a subject comprising administering to the subject (a) an androgen receptor inhibitor and (b) an embryonic ectoderm development (EED) inhibitor, including those in which prostate cancer has been determined to be resistant to one or more androgen receptor signaling inhibitors (ARSIs), including (abiraterone).

Description

Cross-references

[0001] This application claims the benefits of U.S. Provisional Application Serial No. 63 / 517,672, filed August 4, 2023; U.S. Provisional Application Serial No. 63 / 603,336, filed November 28, 2023; U.S. Provisional Application Serial No. 63 / 617,895, filed January 5, 2024; and U.S. Provisional Application Serial No. 63 / 670,485, filed July 12, 2024, which are hereby incorporated herein by reference in their entirety. Background Technology

[0002] Prostate cancer is the most common cancer among men in the United States, with approximately one in nine men being diagnosed in their lifetime. Despite the high incidence, mortality rates remain low due to screening methods that allow for early intervention and new, effective treatments. However, even with this low mortality rate, prostate cancer remains one of the leading causes of death among men.

[0003] The new range of therapeutic agents has significantly improved the treatment landscape for both castration-sensitive (localized and metastatic) and castration-resistant prostate cancer. Despite prolonged clinical responses, improved survival, and quality of life, systemic therapy ultimately fails in almost all patients, as indicated by elevated prostate-specific antigen (PSA) levels and / or radiographic indications of disease progression. Following these systemic therapies, chemotherapy remains an option, offering a moderate improvement in overall survival at the cost of significant complications.

[0004] The use of androgen receptor signaling inhibitors (ARSIs), such as the CYP17 inhibitor abiraterone and androgen receptor inhibitors apalutamide, darolutamide, and enzalutamide, has resulted in an expansion of tumor heterogeneity, prominently manifested in the clinical appearance of multiple phenotypic states that bypass ARSIs, including cells with increased plasticity and different differentiations. Enhanced plasticity is a characteristic of the high regenerative capacity of prostate cancer tumors following ARSI therapy. Therefore, there is a need to develop new treatment options to overcome or circumvent the epigenetic plasticity that pervades the treatment of prostate cancer.

[0005] This disclosure relates to a method of treating prostate cancer in a subject, comprising administering to the subject (a) an androgen receptor inhibitor and (b) an embryonic ectodermal development (EED) inhibitor. Summary of the Invention

[0006] This article discloses a method for treating prostate cancer in subjects, including administering (a) androgen receptor inhibitors and (b) embryonic ectodermal development (EED) inhibitors to subjects.

[0007] This article also discloses a method for treating prostate cancer in subjects who have been identified as resistant to one or more first androgen receptor signaling inhibitors (ARSIs), including administering (a) androgen receptor inhibitors and (b) embryonic ectoderm development (EED) inhibitors to the subjects.

[0008] This article further discloses a method for treating prostate cancer in subjects who have been identified as resistant to CYP17 inhibitors, including administering (a) androgen receptor inhibitors and (b) embryonic ectoderm development (EED) inhibitors to the subjects.

[0009] This article also discloses a method for treating prostate cancer in subjects who have been identified as resistant to abiraterone, including administering (a) androgen receptor inhibitors and (b) embryonic ectoderm development (EED) inhibitors to the subjects.

[0010] This article further discloses a method for treating prostate cancer in subjects who have previously received one or more CYP17 inhibitors, including administering (a) an androgen receptor inhibitor and (b) an embryonic ectoderm development (EED) inhibitor to the subjects.

[0011] This article also discloses methods for treating prostate cancer in subjects who (i) have previously received one or more CYP17 inhibitors and (ii) have never received androgen receptor inhibitor treatment, including administering (a) an androgen receptor inhibitor and (b) an embryonic ectoderm development (EED) inhibitor to the subject.

[0012] This article further discloses any of the described methods for treating prostate cancer in subjects, wherein the prostate cancer in the subjects is selected from metastatic prostate cancer, non-metastatic prostate cancer, metastatic castration-resistant prostate cancer, metastatic castration-sensitive prostate cancer, localized high-risk prostate cancer, recurrent prostate cancer, non-metastatic castration-resistant prostate cancer, non-metastatic castration-sensitive prostate cancer, androgen receptor inhibitor-sensitive prostate cancer, androgen receptor inhibitor-resistant prostate cancer, androgen receptor-dependent prostate cancer, androgen receptor-independent prostate cancer, neuroendocrine prostate cancer (NEPC), metastatic neuroendocrine prostate cancer (NEPC), prostate cancer with small cell characteristics, metastatic prostate cancer with small cell characteristics, and invasive variant prostate cancer.

[0013] This document also discloses a method for treating prostate cancer in a subject, comprising administering (a) an androgen receptor inhibitor and (b) an embryonic ectodermal development (EED) inhibitor to the subject. In some embodiments, the subject has previously received one or more CYP17 inhibitors. In some embodiments, the subject has previously received abiraterone or abiraterone acetate. In some embodiments, the prostate cancer in the subject has been identified as resistant to abiraterone. In some embodiments, the subject has not received CYP17 inhibitor treatment prior to administering the androgen receptor inhibitor and the EED inhibitor. In some embodiments, the subject has not received androgen receptor inhibitor treatment prior to administering the androgen receptor inhibitor and the EED inhibitor. In some embodiments, the subject has received one or more prior androgen deprivation therapies prior to administering the androgen receptor inhibitor and the EED inhibitor. In some embodiments, the subject has been administered a gonadotropin-releasing hormone (GnRH) analogue prior to administration of the androgen receptor inhibitor and the embryonic ectodermal development (EED) inhibitor. In some embodiments, the androgen receptor inhibitor and the EED inhibitor are administered to the subject sequentially or simultaneously. In some embodiments, the androgen receptor inhibitor is selected from apalutamide, dalotamide, and enzalutamide. In some embodiments, the EED inhibitor is selected from EED226, A-395, APG-5918, BR-001, BR-002, EEDi-5285, EEDi-1056, FTX-6058, HJM-353, and MAK683. In some implementations, the prostate cancer in the subject is selected from metastatic prostate cancer, non-metastatic prostate cancer, metastatic castration-resistant prostate cancer, metastatic castration-sensitive prostate cancer, localized high-risk prostate cancer, recurrent prostate cancer, non-metastatic castration-resistant prostate cancer, non-metastatic castration-sensitive prostate cancer, androgen receptor inhibitor-sensitive prostate cancer, androgen receptor inhibitor-resistant prostate cancer, androgen receptor-dependent prostate cancer, androgen receptor-independent prostate cancer, neuroendocrine prostate cancer (NEPC), metastatic neuroendocrine prostate cancer (NEPC), prostate cancer with small cell characteristics, metastatic prostate cancer with small cell characteristics, and aggressive variant prostate cancer.

[0014] In some implementations, the prostate cancer in the subjects is metastatic prostate cancer.

[0015] In some implementations, the prostate cancer in the subjects is metastatic castration-resistant prostate cancer. Incorporation

[0016] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication, patent or patent application expressly and individually indicates that it is incorporated by reference. Attached Figure Description

[0017] Figure 1 The antitumor effects of compound 4, dalotamide, as described in Example 4, in intact male BALB / c nude mice in a non-castrated prostate cancer model environment using a 22Rv1 cell line xenograft model were described.

[0018] Figure 2 The antitumor effects of compound 4 and dalotamide as described in Example 5 were depicted in dalotamide-acquired resistant 22Rv1 xenografts from intact male BALB / c nude mice.

[0019] Figure 3 The antitumor effects of compound 4, PF-06821497, and dalotamid, as described in Example 6, in a subcutaneous C4-2 castrated prostate cancer xenograft model in male NPG mice were described.

[0020] Figure 4 The antitumor effects of compound 4, PF-06821497, and dalotamid, as described in Example 7, in a subcutaneous C4-2 intact prostate cancer xenograft model in male NPG mice were depicted.

[0021] Figure 5 The concentration of compound 4 in the plasma of subjects who were administered the compound as a pharmaceutical composition at doses of 100 mg once daily (QD), 200 mg QD, 400 mg QD, 600 mg QD and 900 mg QD as described in Example 9 was depicted over time on day 1 of cycle 2 (C2D1).

[0022] Figure 6 The percentage change (mean ± SEM) of H3K27me3 / H3 in monocytes on day 15 of cycle 1 in subjects orally administered compound 4 in the form of a pharmaceutical composition at doses of 200 mg QD, 400 mg QD, 600 mg QD, 700 mg QD, 800 mg QD or 900 mg QD was depicted in Example 9.

[0023] Figure 7The changes from baseline in the H3K27me3 to histone 3.1 ratio in cell-free nucleosomes were depicted on day 15 of cycle 1 (C1D15) in subjects orally administered compound 4 in the form of a pharmaceutical composition at doses of 100 mg once daily (QD), 200 mg QD, 400 mg QD, 600 mg QD, 700 mg QD, or 900 mg QD, as described in Example 9.

[0024] Figure 8 The changes from baseline in the H3K27me3 to histone 3.1 ratio in cell-free nucleosomes were depicted on day 1 of cycle 2 (C2D1) in subjects orally administered compound 4 in the form of a pharmaceutical composition at doses of 100 mg once daily (QD), 200 mg QD, 400 mg QD, 600 mg QD, 700 mg QD, or 900 mg QD, as described in Example 9.

[0025] Figure 9 The progression-free survival (PFS)-based assessment of male NPG mice treated with compound 4, PF-06821497, or dalotamid in a xenograft subcutaneous C4-2 castrated prostate cancer model as described in Example 6 is depicted.

[0026] Figure 10 The progression-free survival (PFS) of male NPG mice treated with compound 4, PF-06821497, or dalotamide in a xenograft subcutaneous C4-2 intact prostate cancer model, as described in Example 7, was assessed. Detailed Implementation

[0027] As used in the specification and appended claims, unless otherwise specified, the following terms shall have the meanings indicated below.

[0028] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural indicators unless the context clearly specifies otherwise. Thus, for example, reference to “pharmaceutical” includes multiple such pharmaceuticals, and reference to “cell” includes reference to one or more cells and their equivalents known to those skilled in the art, and so on. When the scope is used herein for physical properties (such as molecular weight) or chemical properties (such as chemical formula), it is intended to include all combinations and sub-combinations of the scope, as well as specific embodiments thereof. The term “about” when referring to a number or numerical range means that the number or numerical range mentioned is an approximation within experimental variability (or within statistical experimental error), and therefore in some cases, the number or numerical range will vary between 1% and 15% of said number or numerical range. The term “comprising” (and related terms such as “having” or “including”) is not intended to exclude, in certain other embodiments (e.g., embodiments of any material composition, composition, method, or process described herein), from “consisting of” or “substantially consisting of” said features.

[0029] When used in conjunction with therapeutic agents, including androgen receptor inhibitors and embryonic ectodermal development (EED) inhibitors, "application" means the systemic or local administration of the therapeutic agent, such as direct application to or onto a target tissue, or administration to a subject, whereby the therapeutic agent has a positive effect on the targeted tissue. Therefore, as used herein, the term "application" when used in conjunction with the compositions described herein can include, but is not limited to, providing the composition to or onto a target tissue; or providing the composition systemically to a subject, for example, by oral administration, whereby the therapeutic agent reaches the target tissue or cells. The "application" of the composition can be accomplished by injection, topical application, and oral administration, or by other methods alone or in combination with other known techniques.

[0030] As used herein, the terms “androgen receptor signaling inhibitor,” “ARSI,” “androgen receptor pathway inhibitor,” and “ARPI” refer to agents that, when administered to a subject with prostate cancer, affect androgen signaling pathways in one or more cells containing prostate cancer. Androgen receptor signaling inhibitors include, but are not limited to, agents that inhibit androgen biosynthesis or regulate androgen receptor function, including by inhibiting androgen receptor function, for example, by binding to the receptor and interfering with the binding of androgens to the androgen receptor. Examples of agents that inhibit androgen biosynthesis are known to those skilled in the art and include, but are not limited to, agents that inhibit enzymes responsible for androgen biosynthesis, such as 17α-hydroxylase / C17,20-lyase (CYP17). Examples of CYP17 inhibitors are known to those skilled in the art and include abiraterone and abiraterone prodrugs, including abiraterone acetate.

[0031] As used herein, the term "androgen receptor inhibitor" means an agent that binds to and modulates the function of androgen receptors, including but not limited to agents that inhibit the binding of androgens to androgen receptors, as well as agents that inhibit the nuclear translocation of androgen receptors and their interaction with DNA. Examples of androgen receptor inhibitors are known to those skilled in the art and include, but are not limited to, apalutamide, dalotamide, and enzalutamide.

[0032] As used herein, the term "animal" includes, but is not limited to, human and non-human vertebrates, such as wild animals, domesticated animals, and farm animals. As used herein, the terms "object," "subject," and "individual" are intended to include living organisms in which certain conditions described herein may occur. Examples include humans, monkeys, cows, sheep, goats, dogs, cats, mice, rats, and their transgenic species. In a preferred embodiment, the object is a primate. In some embodiments, the primate or object is a human. In some cases, the human is an adult. In some cases, the human is a child. In other cases, the human is less than 12 years of age. In some cases, the human is an elderly person. In other cases, the human is 60 years of age or older. Other examples of objects include laboratory animals such as mice, rats, dogs, cats, goats, sheep, pigs, and cows. Laboratory animals can be animal models of diseases, such as transgenic mice with hypertensive pathology.

[0033] As used herein, the term "castration-sensitive prostate cancer" is understood by those skilled in the art to describe a condition also known as hormone-sensitive prostate cancer.

[0034] As used herein, terms such as “certain” mean that the following has been established: the preconditions in the subject existed prior to the administration of androgen receptor inhibitors and embryonic ectoderm development (EED) inhibitors, or that a conditional precedent for the subject has been met.

[0035] As used herein, the term "embryonic ectodermal development (EED) inhibitor" refers to a drug that inhibits the function of EED proteins (including by binding to them).

[0036] As used herein, the term "pharmaceutically acceptable" means a carrier, diluent, or excipient that is compatible with other components of the formulation and is harmless to its recipient.

[0037] The term "pharmaceutical composition" means a composition comprising at least one active ingredient, such that the composition is suitable for studying specified effective results in mammals (such as, but not limited to, humans). Those skilled in the art will understand and appreciate techniques suitable for determining whether an active ingredient has the desired effective results, based on the needs of the technician.

[0038] As used herein, the terms “progression,” “recurrence,” “refractory,” and “resistant” refer to prostate cancer in a subject exhibiting regrowth after a period of time or remission, and / or no longer responding to treatment currently being administered to or previously administered to the subject (e.g., prostate cancer in the subject previously treated with a CYP17 inhibitor such as abiraterone). Determining whether the cancer in a subject, or one or more cells containing cancer, has progressed, recurred, or become refractory or resistant to a particular form of treatment such as a CYP17 inhibitor such as abiraterone can be done by methods known to those skilled in the art. For example, depending on the circumstances, the response or non-response of the cancer in a subject, or one or more cells containing cancer, can be assessed by measuring the subject’s clinical symptoms, performing biopsies of one or more relevant tissue types, a reduction in the size and / or number of tumor lesions, duration of response, or progression-free survival. Furthermore, resistance to previous line of therapy in prostate cancer patients can be determined by referring to the criteria set forth in the Prostate Cancer Clinical Trials Working Group 3 (PCWG3) criteria (see, for example, Scher et al. J. Clinical Oncology, 2016, Vol. 34, No. 12, pp. 1402-1418 and included appendices), which include, but are not limited to: (a) elevated prostate-specific antigen (PSA) levels, defined as at least two elevated values ​​obtained at least one week apart, with the most recent result being at least 2.0 ng / mL (or 1.0 ng / mL if PSA elevation is the only indication of progression); (b) confirmation of two new bone lesions when the last systemic therapy was administered; (c) soft tissue progression according to RECIST 1.1; and (d) progression on radiographic imaging.

[0039] As used herein, the term "therapeutic agent" means a medicine used to treat, counteract, alleviate, prevent, or improve an adverse condition or disease of a subject.

[0040] As used herein, “therapeutic effective amount” or “effective amount” means the amount of an active compound or pharmaceutical agent that elicits a biological or pharmaceutical response in a tissue, system, animal, individual, or human, sought by a researcher, veterinarian, physician, or other clinician, and includes one or more of the following: (1) prevention of disease; for example, prevention of disease, condition, or symptom in an individual who may be susceptible to disease, condition, or symptom but has not yet experienced or shown a pathology or symptom; (2) suppression of disease; for example, suppression of disease, condition, or symptom in an individual who is experiencing or showing a pathology or symptom (i.e., prevention of further development of the pathology and / or symptom); and (3) mitigation of disease; for example, mitigation of disease, condition, or symptom in an individual who is experiencing or showing a pathology or symptom (i.e., reversal of the pathology and / or symptom).

[0041] As used herein, the terms “treatment” or “management” refer in some embodiments to therapeutic treatment and in other embodiments to preventive measures or precautions, wherein the aim is to prevent or mitigate (alleviate) an adverse physical condition, symptom, or disease, or to achieve a beneficial or desired clinical outcome. For the purposes described herein, a beneficial or desired clinical outcome includes, but is not limited to, the reduction of symptoms; a reduction in the severity of the condition, symptom, or disease; stabilization (i.e., non-deterioration) of the condition, symptom, or disease status; a delay in the onset or a slowing of the progression of the condition, symptom, or disease; a reduction in the condition, symptom, or disease status; and remission (partial or complete), whether detectable or undetectable, or improvement or aggravation of the condition, symptom, or disease. Treatment includes inducing a clinically significant response without excessive levels of side effects. Treatment also includes prolonged survival compared to expected survival without treatment. The preventive benefits of treatment include preventing the condition, delaying the progression of the condition, stabilizing the condition, or reducing the likelihood of the condition occurring.

[0042] For simplicity, the chemical moiety is defined and refers primarily to a monovalent chemical moiety (e.g., alkyl, aryl, etc.). However, such terms can also be used to express corresponding polyvalent moieties under appropriate structural conditions that are clear to those skilled in the art. For example, while the "alkyl" moiety generally refers to a monovalent group (e.g., CH3-CH2-), in some cases the divalent linking moiety can be "alkyl," in which case those skilled in the art will understand alkyl to be a divalent group (e.g., -CH2-CH2-), which is equivalent to the term "alkylene". (Similarly, where a divalent moiety is required and is stated as "aryl," those skilled in the art will understand that the term "aryl" refers to the corresponding divalent moiety, arylene.) All atoms are understood to have their normal valences for forming bonds (i.e., 4 for carbon, 3 for nitrogen, 2 for oxygen, and 2, 4, or 6 for S, depending on the oxidation state of S).

[0043] As used in this article, the term "amino" refers to -NH2.

[0044] As used in this article, the term "acetyl" refers to "-C(O)CH3".

[0045] As used herein, the term "acyl" refers to an alkyl carbonyl or aryl carbonyl substituent, wherein the alkyl and aryl moieties are as defined herein.

[0046] As used herein, the term "alkyl" refers to a straight-chain and branched aliphatic group containing 1 to 12 carbon atoms. Therefore, "alkyl" encompasses C1, C2, C3, C4, C5, C6, C7, C8, C9, C1 ... 10 C 11 and C 12 Alkyl groups. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, and hexyl.

[0047] As used herein, the term "alkenyl" refers to an unsaturated straight-chain or branched aliphatic group having one or more carbon-carbon double bonds and 2 to 12 carbon atoms. Therefore, "alkenyl" encompasses C2, C3, C4, C5, C6, C7, C8, C9, C16, C16, C17, C18, C19, C16, C18, C19, C19, C19, C10, C11, C12, C13, C14, C15, C16, C17, C18 ... 10 C 11 and C 12 Groups. Examples of alkenyl groups include, but are not limited to, vinyl, propenyl, butenyl, pentenyl, and hexenyl.

[0048] As used herein, the term "alkynyl" refers to an unsaturated straight-chain or branched aliphatic group having one or more carbon-carbon triple bonds and 2 to 12 carbon atoms. Therefore, "alkynyl" encompasses C2, C3, C4, C5, C6, C7, C8, C9, C16, C17, C18, C19, C16, C19, C16, C19, C10, C11, C12, C13, C14, C15, C16, C17, C18 ... 10 C 11 and C 12 Group. Examples of alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, penynyl, and hexynyl.

[0049] As used herein, the terms “alkylene,” “alkenylene,” and “yntynylene” mean an alkyl, alkenyl, or yntynyl group as defined above that is situated between and serves to connect two other chemical groups. Examples of alkylene groups include, but are not limited to, methylene, ethylene, propylene, and butylene. Exemplary alkenyl groups include, but are not limited to, vinylene, propenyl, and butenylene. Exemplary yntynyl groups include, but are not limited to, ethynylene, propynylene, and butynylene.

[0050] As used in this article, the term "alkoxy" refers to -OC1-C6 alkyl.

[0051] As used herein, the term "cycloalkyl" refers to a saturated and partially unsaturated cyclic hydrocarbon group having 3 to 12 carbon atoms. Therefore, "cycloalkyl" includes C3, C4, C5, C6, C7, C8, C9, C16, C17, C18, C19, C18, C19, C19, C10, C11, C12, C13, C14, C15, C16, C17, C18 ... 10 C 11 and C 12 Cyclic hydrocarbon groups. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.

[0052] As used herein, the term "heteroalkyl" refers to an alkyl group as defined above, wherein one or more carbon atoms in the chain are delimited by O, S, or NR. x Independent substitution, where R x It is hydrogen or a C1-C3 alkyl group. Examples of heteroalkyl groups include methoxymethyl, methoxyethyl, and methoxypropyl.

[0053] As used in this article, the term "aryl" refers to a C6-C group containing one to three aromatic rings. 14 The aromatic component. Therefore, "aryl" includes C6, C6, C7, C8, C9 ... 10 C 13 and C 14 Cyclic hydrocarbon groups. An exemplary aryl group is C6-C. 10 Aryl groups. Specific aryl groups include, but are not limited to, phenyl, naphthyl, anthracene, and fluorene.

[0054] As used herein, the terms "aralkyl" and "arylalkyl" refer to an aryl group covalently linked to an alkylene group, wherein the moiety is linked to another group via an alkyl portion. An exemplary aralkyl group is a -(C1-C6)alkyl (C6-C... 10 )Aryl, including but not limited to benzyl, phenethyl and naphthylmethyl.

[0055] As used herein, the terms “heterocyclic group” and “heterocyclic” refer to a single or double ring (fused or spirocyclic) ring structure having 3 to 12 atoms (3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 atoms), such as 4 to 8 atoms, wherein one or more ring atoms are independently -C(O)-, N, NR. 5The ring must contain an O or S atom, and the remaining portion of the ring atom must be a quaternary carbon or a carbonyl carbon. Examples of heterocyclic groups include, but are not limited to, epoxy, oxetane, oxetane, azirne, azirnepropane, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothiophenyl, pyrrolidinyl, piperidinyl, piperazine, imidazoalkyl, thiatanyl, dithiohexane, trithiohexane, azirnezohexane, oxetanehexane, dioxetanepentyl, oxazolidinyl, oxazolidinone, decahydroquinolinyl, piperidinyl, 4-piperidinone, thiomorpholinyl, dimethylmorpholinyl, and morpholinyl. The scope of this term explicitly excludes compounds having an adjacent ring O and / or S.

[0056] As used herein, the term "L-heterocyclic" means a heterocyclic group covalently attached to another group via an alkylene linker L, where L is a C1-C4 alkylene group.

[0057] As used herein, the term "heteroaryl" refers to a group having 5 to 14 ring atoms, preferably 5, 6, 10, 13, or 14 ring atoms, comprising an aromatic heterocycle (e.g., having 6, 10, or 14 π electrons shared in a ring array), and having one to three heteroatoms (each independently N, O, or S) in addition to a carbon atom. "Heteroaryl" also includes fused polycyclic (e.g., bicyclic) ring systems in which one or more fused rings are non-aromatic rings, provided that at least one ring is an aromatic ring and at least one ring contains N, O, or S ring atoms.

[0058] Examples of heteroaryl groups include acridinel, acridinel, benzimidazolyl, benzofuranyl, benzo[d]oxazol-2(3H)-one, 2H-benzo[b][1,4]oxazin-3(4H)-one, benzothiofuranyl, benzothiophenel, benzooxazolyl, benzothiazolyl, benzotriazolyl, benzotetrazolel, benzoisooxazolyl, benzoisothiazolyl, benzimidazolinyl, carbazolel, 4aH-carbazolel, carbolinyl, chromanyl, chromenyl, terolinyl, furanyl, furazanyl, furazanyl Imidazolinyl, imidazolinyl, 1H-indolenyl, indolinyl, indoleazinyl, indoleyl, 3H-indolenyl, isobenzofuranyl, isochoryl, isoindolelinyl, isoindolenyl, isoquinolinyl, isothiazolyl, isoxazolyl, naphridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolylalkyl, oxazolyl, oxazolidine alkyl, pyrimidinyl, phenanthridine, phenanthrolinyl, phenazinyl, phenothiazinyl, phenothiazinyl, phenothiazinyl, phthalazinyl, piperinyl, pteridinyl, purine, pyranyl, pyrazinyl, pyrazolylyl, pyrazolyl, pyrazolyl, pyridazinyl, pyridoxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrrolinyl, 2H-pyrroleyl, pyrroleyl, quinazolinyl, quinolinyl, 4H-quinazinyl, quinoxalinyl, quinoxalinyl The following are listed: nephronyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thienyl, triazinyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl, and xanthonyl.

[0059] As used herein, the terms “L-heteroaryl,” “heteroarylalkyl,” and “heteroarylalkyl” refer to a group comprising a heteroaryl group covalently attached to another group via an alkylene linker. Examples of heteroaryl groups include C1-C6 alkyl groups and heteroaryl groups having 5, 6, 9, or 10 ring atoms. Examples of heteroarylalkyl groups include pyridinylmethyl, pyridinylethyl, pyrrolithylmethyl, pyrrolithylethyl, imidazolylmethyl, imidazolylethyl, thiazolylmethyl, thiazolylethyl, benzimidazolylmethyl, benzimidazolylethylquinazolinylmethyl, quinolinylmethyl, quinolinylethyl, benzofuranylmethyl, indololinylethyl isoquinolinylmethyl, isoinodylmethyl, benzolinylmethyl, and benzothiophenylethyl. The scope of this term explicitly excludes compounds having adjacent ring O and / or S atoms.

[0060] As used herein, the terms “arylene,” “heteroarylene,” and “heterocyclic” refer to the respective divalent aryl, heteroaryl, or heterocyclic group as defined above, which is located between two other chemical groups and serves to connect the two other chemical groups.

[0061] As used herein, when a part (e.g., cycloalkyl, aryl, heteroaryl, heterocyclic, urea, etc.) is described as “optionally substituted” without clearly stating the substituent, it means that the group optionally has one to four, preferably one to three, more preferably one or two non-hydrogen substituents.

[0062] As used in this article, the terms "halogen" and "halogen group" refer to chlorine, bromine, fluorine, or iodine.

[0063] As used herein, the term "haloalkyl" refers to an alkyl chain in which one or more hydrogen atoms are replaced by halogens. Exemplary haloalkyl groups are trifluoromethyl, difluoromethyl, fluorochloromethyl, chloromethyl, and fluoromethyl.

[0064] As used herein, the term "hydroxyalkyl" means an alkyl chain as defined herein, wherein at least one hydrogen atom of the alkyl chain is replaced by a hydroxyl group.

[0065] This document discloses a method for treating prostate cancer in a subject, comprising administering (a) an androgen receptor inhibitor and (b) an embryonic ectodermal development (EED) inhibitor to the subject. In some embodiments, the androgen receptor inhibitor is selected from apalutamide, dalotamide, and enzalutamide. In some embodiments, the androgen receptor inhibitor is apalutamide. In some embodiments, the androgen receptor inhibitor is dalotamide. In some embodiments, the androgen receptor inhibitor is enzalutamide.

[0066] This document also discloses a method for treating prostate cancer in subjects who have been identified as resistant to one or more first androgen receptor signaling inhibitors (ARSIs), including administering (a) an androgen receptor inhibitor and (b) an embryonic ectodermal development (EED) inhibitor to the subject. In some embodiments, the one or more first androgen receptor signaling inhibitors (ARSIs) are selected from one or more first CYP17 inhibitors and one or more first androgen receptor inhibitors. In some embodiments, the one or more first androgen receptor signaling inhibitors (ARSIs) are selected from one or more first CYP17 inhibitors. In some embodiments, the one or more first CYP17 inhibitors are abiraterone acetate. In some embodiments, the one or more first androgen receptor signaling inhibitors (ARSIs) are selected from one or more androgen receptor inhibitors. In some embodiments, the one or more first androgen receptor inhibitors are selected from apalutamide, dalotamide, and enzalutamide. In some embodiments, the one or more first androgen receptor inhibitors are apalutamide. In some embodiments, the one or more first androgen receptor inhibitors are dalotamide. In some embodiments, the one or more first androgen receptor inhibitors are enzalutamide.

[0067] This article also discloses methods for treating prostate cancer in subjects who have been identified as resistant to CYP17 inhibitors, including administering (a) an androgen receptor inhibitor and (b) an embryonic ectodermal development (EED) inhibitor to the subject. In some embodiments, the CYP17 inhibitor is abiraterone acetate. In some embodiments, the androgen receptor inhibitor is selected from apalutamide, dalotamide, and enzalutamide. In some embodiments, the androgen receptor inhibitor is apalutamide. In some embodiments, the androgen receptor inhibitor is dalotamide. In some embodiments, the androgen receptor inhibitor is enzalutamide.

[0068] This article also discloses a method for treating prostate cancer in subjects who have been identified as resistant to abiraterone, comprising administering (a) an androgen receptor inhibitor and (b) an embryonic ectodermal development (EED) inhibitor to the subject. In some embodiments, the androgen receptor inhibitor is selected from apalutamide, dalotamide, and enzalutamide. In some embodiments, the androgen receptor inhibitor is apalutamide. In some embodiments, the androgen receptor inhibitor is dalotamide. In some embodiments, the androgen receptor inhibitor is enzalutamide.

[0069] This document also discloses methods for treating prostate cancer in subjects who have previously received one or more CYP17 inhibitors, including administering (a) an androgen receptor inhibitor and (b) an embryonic ectodermal development (EED) inhibitor to the subject. In some embodiments, the CYP17 inhibitor is abiraterone acetate. In some embodiments, the androgen receptor inhibitor is selected from apalutamide, dalotamide, and enzalutamide. In some embodiments, the androgen receptor inhibitor is apalutamide. In some embodiments, the androgen receptor inhibitor is dalotamide. In some embodiments, the androgen receptor inhibitor is enzalutamide.

[0070] This document further discloses a method for treating prostate cancer in subjects who (i) have received prior treatment with one or more CYP17 inhibitors and (ii) have never received androgen receptor inhibitor treatment, including administering (a) an androgen receptor inhibitor and (b) an embryonic ectodermal development (EED) inhibitor to the subject. In some embodiments, the CYP17 inhibitor is abiraterone acetate. In some embodiments, the androgen receptor inhibitor is selected from apalutamide, dalotamide, and enzalutamide. In some embodiments, the androgen receptor inhibitor is apalutamide. In some embodiments, the androgen receptor inhibitor is dalotamide. In some embodiments, the androgen receptor inhibitor is enzalutamide.

[0071] In another embodiment, any of the methods disclosed herein for treating prostate cancer, wherein the embryonic ectodermal development (EED) inhibitor is a small molecule having a molecular weight of less than or equal to 1000 Daltons. In some embodiments, the embryonic ectoderm development (EED) inhibitor is a small molecule having a molecular weight of less than or equal to 900 Daltons, or less than or equal to 800 Daltons, or less than or equal to 750 Daltons, or less than or equal to 700 Daltons, or less than or equal to 650 Daltons, or less than or equal to 600 Daltons, or less than or equal to 575 Daltons, or less than or equal to 525 Daltons, or less than or equal to 500 Daltons, or less than or equal to 475 Daltons, or less than or equal to 450 Daltons, or less than or equal to 425 Daltons, or less than or equal to 400 Daltons, or less than or equal to 375 Daltons, or less than or equal to 350 Daltons, or less than or equal to 325 Daltons, or less than or equal to 300 Daltons, or less than or equal to 275 Daltons, or less than or equal to 250 Daltons, or less than or equal to 200 Daltons.

[0072] In another embodiment, there is any of the methods disclosed herein for treating prostate cancer, wherein the embryonic ectodermal development (EED) inhibitor is selected from EED226, A-395, APG-5918, BR-001, BR-002, EEDi-5285, EEDi-1056, FTX-6058, HJM-353, and MAK683. In some embodiments, the EED inhibitor is EED226. In some embodiments, the EED inhibitor is A-395. In some embodiments, the EED inhibitor is APG-5918. In some embodiments, the EED inhibitor is BR-001. In some embodiments, the EED inhibitor is BR-002. In some embodiments, the EED inhibitor is EEDi-5285. In some embodiments, the EED inhibitor is EEDi-1056. In some embodiments, the ectodermal development (EED) inhibitor is FTX-6058. In some embodiments, the ectodermal development (EED) inhibitor is HJM-353. In some embodiments, the ectodermal development (EED) inhibitor is MAK683.

[0073] In another embodiment, there is any method of treating prostate cancer disclosed herein, wherein the embryonic ectodermal development (EED) inhibitor is a compound of formula (I).

[0074] Or its pharmaceutically acceptable salt: in: Indicates a single bond or a double bond; Z is either O or S; X represents O and CR. 5 CR 5 OH or C(R) 5 )2, of which: When X is 0 It is a single key; When X is C(R) 5 At 2 o'clock, It is a single key; When X is CR 5 When OH, It is a single key; or When X is CR 5 hour, It is a double bond; R 1 It is aryl, heteroaryl, L-cycloalkyl, -N(R) 5 Heterocyclic or L-heterocyclic, wherein L-cycloalkyl, -N(R)5 The aryl, heteroaryl, or cyclic moiety of the heterocyclic or L-heterocyclic group is optionally surrounded by one or more R... 4 replace; R 2 It is cyano, -COOR 5 -C(O)N(R) 5 )2 or -C(O)N(R 5 )2, where each R 5 Together with the nitrogen atom to which it is attached, it forms an optional structure with one or more R atoms. 4 Replaced 5-8 membered heterocycles; Each R 3 It is independently a C1-C3 alkyl or halogen; Each R 4 Independently, it is oxo, cyano, halogen, -PO3 (C1-C3 alkyl)2, hydroxyl, alkoxy, hydroxyalkyl, heteroalkyl, aralkyl, haloalkyl, -COOR 5 -Y 2 -haloalkyl, -Y 1 -C1-C6 alkyl, -Y 2 -C1-C6 alkyl, -L-cycloalkyl, -L-heteroaryl, -L-heterocyclic, -Y 1 -heterocyclic group, -Y 2 -heterocyclic group, -LN(R) 5 )2、-OLN(R 5 )2、-C(CF3)N(R 5 )2、-Y 1 -N(R 5 )2 or -Y 2 -N(R 5 )2, wherein aralkyl, -L-cycloalkyl, -L-heteroaryl, -L-heterocyclic or -Y 1 -The ring portion of the heterocyclic group is optionally separated by one or more R 7 replace; L is a bond or a C1-C4 alkylene group; Y 1 It is a bond, -C(O)- or -NHC(O)-; Y 2 It is a key, -S-, -SO-, -SO2-, or -NR 5 SO2-, Each R 5 It is hydrogen or C1-C3 alkyl; R 6 It is hydrogen, C1-C3 alkyl, halogen, haloalkyl, hydroxyalkyl, or heteroalkyl; Each R 7It is oxo, cyano, hydroxy, alkoxy, halogen, haloalkyl, hydroxyalkyl, heteroalkyl, cycloalkyl, -LN(R) 5 2. C1-C6 alkyl or -Y 1 - Heterocyclic group; and n is 1 or 2.

[0075] In some embodiments, the embryonic ectodermal development (EED) inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein Z is O. In some embodiments, the embryonic ectodermal development (EED) inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein Z is S.

[0076] In some implementations, the embryonic ectoderm development (EED) inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt thereof, where n is 1.

[0077] In some embodiments, the embryonic ectoderm development (EED) inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R 2 It is a cyano group. In some embodiments, the embryonic ectodermal development (EED) inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R 2 Yes - COOR 5 In some embodiments, the embryonic ectoderm development (EED) inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R 2 It is -C(O)N(R) 5 )2.

[0078] In some embodiments, the embryonic ectoderm development (EED) inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R 3 It is a halogen. In some embodiments, the embryonic ectoderm development (EED) inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R 3 It's fluorine.

[0079] In some embodiments, the embryonic ectoderm development (EED) inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein X is a C(R) compound. 5 )2, and It is a single key.

[0080] In some embodiments, the embryonic ectoderm development (EED) inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein X is a CR 5 and It is a double bond.

[0081] In some embodiments, the embryonic ectoderm development (EED) inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein X is O and It is a single key.

[0082] In some embodiments, the embryonic ectoderm development (EED) inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R 1 It is optionally controlled by one or more R 4 Substituted aryl group. In some embodiments, R 1 It is optionally controlled by one or more R 4 Substituted phenyl groups. In some embodiments, R 1 Is it by one, two, or three Rs? 4 Substituted phenyl groups. In some embodiments, one, two, or three R groups are used. 4 Each of these can be independently a halogen, -PO3 (C1-C3 alkyl)2, hydroxyl, hydroxyalkyl, aralkyl, haloalkyl, or -COOR. 5 -Y 1 -C1-C6 alkyl, Y 2 -C1-C6 alkyl groups, -LN(R) 5 )2、-OLN(R 5 )2、-C(CF3)N(R 5 ) 2、 -Y 1 -N(R 5 )2、-Y 2 -N(R 5 )2、Y 2 -haloalkyl, -L-heteroaryl, -L-heterocyclic or -Y 1 - Heterocyclic group, wherein -L-heterocyclic group or -Y 1 -The heterocyclic moiety of the heterocyclic group is optionally surrounded by one or more R 7 Replacement. In some implementations, R 4 Yes-Y 1 -C1-C6 alkyl and Y 1 It is a bond and the C1-C6 alkyl group is methyl, ethyl, isopropyl, butyl, or pentyl. In some embodiments, R 4 Yes-Y 2 -C1-C6 alkyl and Y 2 It is -SO2- and the C1-C6 alkyl group is methyl. In some embodiments, R 4 Yes-Y 2 - Haloalkyl and Y 2 It is -S- or -SO2- and the haloalkyl group is trifluoromethyl. In some embodiments, R 4 It is -LN(R) 5)2 and L is a key and each R 5 It's hydrogen, each R 5 It is a methyl group, or an R group. 5 It is methyl and has an R 5 It is hydrogen. In some implementations, R 4 It is -LN(R) 5 )2 and L is methylene or ethylene and each R 5 It's hydrogen, each R 5 It is a methyl group, or an R group. 5 It is methyl and has an R 5 It is hydrogen. In some implementations, R 4 Yes-Y 1 -N(R 5 )2, Y 1 It is -C(O)- and each R 5 Independently, each R is hydrogen. 5 It is independently a methyl group, or an R group. 5 It is methyl and has an R 5 It is hydrogen. In some implementations, R 4 Yes-Y 2 -N(R 5 )2, Y 2 It is -SO2- and each R 5 Independently, each R is hydrogen. 5 It is a methyl group, or an R group. 5 It is methyl and has an R 5 It is hydrogen independently. In some implementations, R 4 Yes-Y 1 -heterocyclic group and Y 1 It is -C(O)-, and the heterocyclic moiety of the L-heterocyclic group is piperazinyl or 4-methyl-piperazinyl. In some embodiments, R 4 It is an L-heterocyclic group where L is a bond and the heterocyclic moiety of the L-heterocyclic group is a nitrogen-heterocyclic butyl, oxocyclic butyl, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, piperazineyl, or... -Azabicyclo[3.1.0]hexyl, each optionally separated by one or more R groups selected from oxo, C1-C3 alkyl, alkoxy, hydroxyl and halogen. 7 Replacement. In some implementations, R 4 It is an L-heterocyclic group, wherein L is methylene and the heterocyclic moiety of the L-heterocyclic group is azirrobutyl, oxobutyl, pyrrolidinylpiperidinyl, each optionally separated by one or more R groups selected from C1-C3 alkyl, alkoxy, hydroxyl and halogen. 7 Replacement. In some implementations, R 4 Yes-Y 1 -heterocyclic group and Y 1It is -C(O)- and Y 1 - The heterocyclic moiety of the heterocyclic group is a morpholino group optionally substituted with one or more C1-C3 alkyl groups. In some embodiments, R 4 It is optionally controlled by one or more R 7 Substituted -L-heteroaryl groups. In some embodiments, R 4 It is a tetrazolium group. In some embodiments, R 4 It is -PO3(C1-C3 alkyl)2. In some embodiments, R 4 Yes - COOR 5 In some implementations, R 4 It is a hydroxyalkyl group. In some embodiments, R 4 It is -OLN(R) 5 2. In some implementation schemes, R 4 It is an aralkyl group.

[0083] In some embodiments, the embryonic ectoderm development (EED) inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R 1 It is optionally controlled by one or more R 4 Substituted heteroaryl groups. In some embodiments, R 1 It is pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, triazinyl, pyridinyl, pyridyl-2-one, pyrazinyl, pyridazinyl, pyrimidinyl, isoxazolyl, isoindolinyl, naphridinyl, 1,2,3,4-tetrahydroisoquinolinyl, or 5,6-dihydro-4H-pyrrolo[1,2-b]pyrazolyl, each optionally separated by one or more R 4 Replacement. In some implementations, R 1 by one or more R 4 Replace; where each R 4 Independently, it is cyano, halogen, -Y 1 -C1-C6 alkyl, -Y 2 -C1-C6 alkyl, alkoxy, hydroxyalkyl, heteroalkyl, haloalkyl, -L-cycloalkyl, -LN(R) 5 )2、-Y 1 -N(R 5 2. -L-heteroaryl, -L-heterocyclic, or -Y 1 -Heterocyclic group, wherein the -L-heteroaryl group is a heteroaryl group or an L-heterocyclic group or a Y-heterocyclic group. 1 -The heterocyclic moiety of the heterocyclic group is optionally surrounded by one or more R 7 replace.

[0084] In some embodiments, the embryonic ectoderm development (EED) inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R 1It is arbitrarily assigned to an R 4 The substituted pyrazol group, that one R 4 Independently selected from hydroxyalkyl, heteroalkyl, haloalkyl, -Y 1 -C1-C6 alkyl groups, -LN(R) 5 2. L-heterocyclic or L-heteroaryl, wherein the heteroaryl or heterocyclic portion of the L-heteroaryl is optionally surrounded by one or more R 7 Replacement. In some implementations, R 4 It is an L-heteroaryl group and L is a methylene group, wherein the heteroaryl group is optionally surrounded by one or more R groups. 7 Substituted pyridinyl group. In some embodiments, R 4 It is optionally controlled by one or more R 7 A substituted -L-heterocyclic group, wherein L is a bond and the heterocyclic moiety of the L-heterocyclic group is an oxehezyl, tetrahydrofuranyl, tetrahydropyranyl, piperazineyl, or 4-methylpiperazineyl. In some embodiments, R 4 It is optionally controlled by one or more R 7 The substituted -L-heterocyclic group, wherein L is methylene and the heterocyclic moiety of the L-heterocyclic group is azirrobutyl, oxetane, pyrrolyl, pyrrolidone, tetrahydrofuranyl, tetrahydropyranyl, morpholinyl, piperazineyl, or 4-methylpiperazineyl. In some embodiments, R 4 It is -LN(R) 5 )2, where L is methylene and each R 5 Independently, each R is hydrogen. 5 It is independently a C1-C3 alkyl group or an R 5 It is a C1-C3 alkyl group and has one R 5 It is hydrogen. In some implementations, R 4 Yes-Y 1 -C1-C6 alkyl, wherein Y 1 It is a bond and the C1-C6 alkyl group is methyl, ethyl or isopropyl.

[0085] In some embodiments, the embryonic ectoderm development (EED) inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R 1 It is arbitrarily determined by two Rs 4 The two R groups are substituted with pyrazol groups. 4 Each group is independently selected from hydroxyalkyl, heteroalkyl, haloalkyl, and -Y groups. 1 -C1-C6 alkyl.

[0086] In some embodiments, the embryonic ectoderm development (EED) inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R 1 It is arbitrarily assigned to an R 4Substituted pyridinyl group, that one R 4 Independently selected from cyano, halogen, alkoxy, hydroxyalkyl, heteroalkyl, haloalkyl, -Y 1 -C1-C6 alkyl groups, -LN(R) 5 )2、-Y 1 -N(R 5 )2, -L-cycloalkyl or optionally with one or more R 7 Substituted -L-heterocyclic group.

[0087] In some embodiments, the embryonic ectoderm development (EED) inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R 1 It is optionally controlled by one or more R 4 Substituted -L-cycloalkyl groups.

[0088] In some embodiments, the embryonic ectoderm development (EED) inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R 1 It is optionally controlled by one or more R 4 Substituted -L-heterocyclic group.

[0089] In some embodiments, the embryonic ectoderm development (EED) inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein L is a bond and the heterocyclic group is piperidinyl or tetrahydropyranyl.

[0090] In some implementations, the embryonic ectoderm development (EED) inhibitor is a compound selected from the following: , , , , , , , , , , , , , , , , , , , , , , , , , , , 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , and Or, or a pharmaceutically acceptable salt thereof.

[0091] In some implementations, the embryonic ectoderm development (EED) inhibitor is a compound selected from the following: , , , , , , , , , , and Or, or a pharmaceutically acceptable salt thereof.

[0092] In some implementations, the ectodermal development (EED) inhibitor is: (Compound 1), or a pharmaceutically acceptable salt thereof.

[0093] In some implementations, the ectodermal development (EED) inhibitor is: (Compound 2), or a pharmaceutically acceptable salt thereof.

[0094] In some implementations, the ectodermal development (EED) inhibitor is: (Compound 3), or a pharmaceutically acceptable salt thereof.

[0095] In some implementations, the ectodermal development (EED) inhibitor is: (Compound 4), or a pharmaceutically acceptable salt thereof.

[0096] In some implementations, the ectodermal development (EED) inhibitor is: (Compound 5), or a pharmaceutically acceptable salt thereof.

[0097] In some implementations, the ectodermal development (EED) inhibitor is: (Compound 6), or a pharmaceutically acceptable salt thereof.

[0098] In some implementations, the ectodermal development (EED) inhibitor is: (Compound 7), or a pharmaceutically acceptable salt thereof.

[0099] In some implementations, the ectodermal development (EED) inhibitor is: (Compound 8), or a pharmaceutically acceptable salt thereof.

[0100] In some implementations, the ectodermal development (EED) inhibitor is: (Compound 9), or a pharmaceutically acceptable salt thereof.

[0101] In some implementations, the ectodermal development (EED) inhibitor is: (Compound 10), or a pharmaceutically acceptable salt thereof.

[0102] In some implementations, the ectodermal development (EED) inhibitor is: (Compound 11), or a pharmaceutically acceptable salt thereof.

[0103] In some implementations, the ectodermal development (EED) inhibitor is: (Compound 12), or a pharmaceutically acceptable salt thereof.

[0104] This article also provides a method for treating prostate cancer in the subjects disclosed herein, including administering a therapeutically effective amount of compound 4 to the subjects: (Compound 4) or a pharmaceutically acceptable salt thereof, wherein Compound 4 is in crystalline form. Such methods are also provided herein, wherein the crystalline form of Compound 4 is in anhydrous form. Such methods are also provided herein, wherein the crystalline form of Compound 4 exhibits a peak at 8.1° ± 0.2° 2θ in an X-ray powder diffraction (XRPD) pattern. Such methods are also provided herein, wherein the crystalline form of Compound 4 exhibits further peaks at 9.6° ± 0.2° 2θ in an X-ray powder diffraction (XRPD) pattern. Such methods are also provided herein, wherein the crystalline form of Compound 4 exhibits further peaks at 5.7° ± 0.2° 2θ, 19.7° ± 0.2° 2θ, and 22.0° ± 0.2° 2θ in an X-ray powder diffraction (XRPD) pattern. This paper also provides a method in which the crystalline form of compound 4 exhibits further peaks at 9.8° ± 0.2° 2θ, 15.2° ± 0.2° 2θ, and 17.7° ± 0.2° 2θ in X-ray powder diffraction (XRPD) patterns. This paper also provides a method in which the crystalline form of compound 4 exhibits a peak at approximately 172 °C in differential scanning calorimetry (DSC). This paper also provides a method in which the crystalline form of compound 4 exhibits peaks at approximately 205 °C to approximately 210 °C in DSC patterns. This paper also provides a method in which the crystalline form of compound 4 exhibits peaks at approximately 206 °C to approximately 210 °C, or approximately 207 °C to approximately 210 °C, or approximately 208 °C to approximately 210 °C, or approximately 209 °C to approximately 210 °C in DSC patterns. This paper also provides a method in which the crystalline form of compound 4 exhibits a mass loss of less than about 1% in thermogravimetric analysis when the sample is heated from about 25°C to the pre-melting temperature. This paper also provides a method in which the crystalline form of compound 4 exhibits a mass loss of less than about 1% in thermogravimetric analysis when the sample is heated from about 25°C to about 380°C.

[0105] This paper also provides a method in which the crystalline form of compound 4 exhibits a peak at 7.7° ± 0.2° 2θ in the X-ray powder diffraction (XRPD) pattern. This paper also provides a method in which the crystalline form of compound 4 exhibits further peaks at 13.7° ± 0.2° 2θ and 19.2° ± 0.2° 2θ in the X-ray powder diffraction (XRPD) pattern. This paper also provides a method in which the crystalline form of compound 4 exhibits further peaks at 5.5° ± 0.2° 2θ, 8.6° ± 0.2° 2θ, 15.9° ± 0.2° 2θ, 19.9° ± 0.2° 2θ, and 24.1° ± 0.2° 2θ in the X-ray powder diffraction (XRPD) pattern. This paper also provides a method in which the crystalline form of compound 4 exhibits further peaks at 10.6° ± 0.2° 2θ, 11.0° ± 0.2° 2θ, 15.4° ± 0.2° 2θ, 21.0° ± 0.2° 2θ, and 26.3° ± 0.2° 2θ in X-ray powder diffraction (XRPD) patterns. This paper also provides a method in which the crystalline form of compound 4 exhibits peaks at approximately 203 °C to approximately 210 °C in differential scanning calorimetry (DSC). This paper also provides a method in which the crystalline form of compound 4 exhibits peaks at approximately 203 °C to approximately 208 °C, or approximately 203 °C to approximately 206 °C, or approximately 203 °C to approximately 205 °C in DSC. This paper also provides a method in which the crystalline form of compound 4 exhibits a mass loss of less than approximately 2% in thermogravimetric analysis when the sample is heated from approximately 25 °C to approximately 380 °C. This paper also provides such a method, in which the crystalline form of compound 4 exhibits a mass loss of less than about 2% in thermogravimetric analysis when the sample is heated from about 25°C to about 210°C.

[0106] This paper also provides a method in which the crystalline form of compound 4 exhibits a peak at 7.7° ± 0.2° 2θ in the X-ray powder diffraction (XRPD) pattern. This paper also provides a method in which the crystalline form of compound 4 further exhibits a peak at 15.4° ± 0.2° 2θ in the X-ray powder diffraction (XRPD) pattern. This paper also provides a method in which the crystalline form of compound 4 further exhibits a peak at 19.2° ± 0.2° 2θ in the X-ray powder diffraction (XRPD) pattern. This paper also provides a method in which the crystalline form of compound 4 exhibits a further peak at 13.7° ± 0.2° 2θ in the X-ray powder diffraction (XRPD) pattern. This paper also provides a method in which the crystalline form of compound 4 exhibits further peaks at 5.5° ± 0.2° 2θ, 8.6° ± 0.2° 2θ, 15.9° ± 0.2° 2θ, 19.9° ± 0.2° 2θ, and 24.1° ± 0.2° 2θ in X-ray powder diffraction (XRPD) patterns. This paper also provides a method in which the crystalline form of compound 4 exhibits further peaks at 10.6° ± 0.2° 2θ, 11.0° ± 0.2° 2θ, 21.0° ± 0.2° 2θ, and 26.3° ± 0.2° 2θ in X-ray powder diffraction (XRPD) patterns. This paper also provides a method in which the crystalline form of compound 4 exhibits peaks at approximately 203 °C to approximately 210 °C in differential scanning calorimetry (DSC) patterns. This paper also provides a method in which the crystalline form of compound 4 exhibits a peak at approximately 206 °C to approximately 210 °C in differential scanning calorimetry (DSC). This paper also provides a method in which the crystalline form of compound 4 exhibits a peak at approximately 203 °C to approximately 208 °C, or approximately 203 °C to approximately 206 °C, or approximately 203 °C to approximately 205 °C in DSC. This paper also provides a method in which the crystalline form of compound 4 exhibits a mass loss of less than approximately 2% in thermogravimetric analysis when the sample is heated from approximately 25 °C to approximately 380 °C. This paper also provides a method in which the crystalline form of compound 4 exhibits a mass loss of less than approximately 2% in thermogravimetric analysis when the sample is heated from approximately 25 °C to approximately 210 °C.

[0107] This paper also provides a method in which the crystalline form of compound 4 exhibits peaks at 7.7° ± 0.2° 2θ and 15.4° ± 0.2° 2θ in X-ray powder diffraction (XRPD) patterns. This paper also provides a method in which the crystalline form of compound 4 exhibits a peak at 19.2° ± 0.2° 2θ in X-ray powder diffraction (XRPD) patterns. This paper also provides a method in which the crystalline form of compound 4 exhibits a further peak at 13.7° ± 0.2° 2θ in X-ray powder diffraction (XRPD) patterns. This paper also provides a method in which the crystalline form of compound 4 exhibits further peaks at 5.5° ± 0.2° 2θ, 8.6° ± 0.2° 2θ, 15.9° ± 0.2° 2θ, 19.9° ± 0.2° 2θ, and 24.1° ± 0.2° 2θ in X-ray powder diffraction (XRPD) patterns. This paper also provides a method in which the crystalline form of compound 4 exhibits further peaks at 10.6° ± 0.2° 2θ, 11.0° ± 0.2° 2θ, 21.0° ± 0.2° 2θ, and 26.3° ± 0.2° 2θ in X-ray powder diffraction (XRPD) patterns. This paper also provides a method in which the crystalline form of compound 4 exhibits peaks at approximately 203 °C to approximately 210 °C in differential scanning calorimetry (DSC) patterns. This paper also provides a method in which the crystalline form of compound 4 exhibits a peak at approximately 206 °C to approximately 210 °C in differential scanning calorimetry (DSC). This paper also provides a method in which the crystalline form of compound 4 exhibits a peak at approximately 203 °C to approximately 208 °C, or approximately 203 °C to approximately 206 °C, or approximately 203 °C to approximately 205 °C in DSC. This paper also provides a method in which the crystalline form of compound 4 exhibits a mass loss of less than approximately 2% in thermogravimetric analysis when the sample is heated from approximately 25 °C to approximately 380 °C. This paper also provides a method in which the crystalline form of compound 4 exhibits a mass loss of less than approximately 2% in thermogravimetric analysis when the sample is heated from approximately 25 °C to approximately 210 °C.

[0108] This paper also provides a method in which the crystalline form of compound 4 exhibits peaks at 7.7° ± 0.2° 2θ and 19.2° ± 0.2° 2θ in X-ray powder diffraction (XRPD) patterns. This paper also provides a method in which the crystalline form of compound 4 exhibits a peak at 15.4° ± 0.2° 2θ in X-ray powder diffraction (XRPD) patterns. This paper also provides a method in which the crystalline form of compound 4 exhibits a further peak at 13.7° ± 0.2° 2θ in X-ray powder diffraction (XRPD) patterns. This paper also provides a method in which the crystalline form of compound 4 exhibits further peaks at 5.5° ± 0.2° 2θ, 8.6° ± 0.2° 2θ, 15.9° ± 0.2° 2θ, 19.9° ± 0.2° 2θ, and 24.1° ± 0.2° 2θ in X-ray powder diffraction (XRPD) patterns. This paper also provides a method in which the crystalline form of compound 4 exhibits further peaks at 10.6° ± 0.2° 2θ, 11.0° ± 0.2° 2θ, 21.0° ± 0.2° 2θ, and 26.3° ± 0.2° 2θ in X-ray powder diffraction (XRPD) patterns. This paper also provides a method in which the crystalline form of compound 4 exhibits peaks at approximately 203 °C to approximately 210 °C in differential scanning calorimetry (DSC) patterns. This paper also provides a method in which the crystalline form of compound 4 exhibits a peak at approximately 206 °C to approximately 210 °C in differential scanning calorimetry (DSC). This paper also provides a method in which the crystalline form of compound 4 exhibits a peak at approximately 203 °C to approximately 208 °C, or approximately 203 °C to approximately 206 °C, or approximately 203 °C to approximately 205 °C in DSC. This paper also provides a method in which the crystalline form of compound 4 exhibits a mass loss of less than approximately 2% in thermogravimetric analysis when the sample is heated from approximately 25 °C to approximately 380 °C. This paper also provides a method in which the crystalline form of compound 4 exhibits a mass loss of less than approximately 2% in thermogravimetric analysis when the sample is heated from approximately 25 °C to approximately 210 °C.

[0109] This paper also provides a method in which the crystalline form of compound 4 exhibits less than about 10% degradation when stored at 25°C and 60% relative humidity for at least 7 days. This paper also provides a method in which the crystalline form of compound 4 exhibits less than about 1%, or less than about 2%, or less than about 3%, or less than about 4%, or less than about 5%, or less than about 6%, or less than about 7%, or less than about 8%, or less than about 9% degradation when stored at 25°C and 60% relative humidity for at least 7 days.

[0110] This paper also provides a method in which the crystalline form of compound 4 (a) exhibits a peak at 8.1° ± 0.2° 2θ in an X-ray powder diffraction (XRPD) pattern, and (b) shows less than about 10% degradation when the crystalline form is stored at 25°C and 60% relative humidity for at least 7 days. This paper also provides a method in which the crystalline form of compound 4 (a) exhibits peaks at 9.6° ± 0.2° 2θ, 5.7° ± 0.2° 2θ, 19.7° ± 0.2° 2θ, and 22.0° ± 0.2° 2θ in an X-ray powder diffraction (XRPD) pattern, and (b) shows less than about 10% degradation when the crystalline form is stored at 25°C and 60% relative humidity for at least 7 days. This article also provides such a method, wherein the crystalline form of compound 4 exhibits a degradation of less than about 1%, or less than about 2%, or less than about 3%, or less than about 4%, or less than about 5%, or less than about 6%, or less than about 7%, or less than about 8%, or less than about 9% when the crystalline form is stored at 25°C and 60% relative humidity for at least 7 days.

[0111] This paper also provides a method in which the crystalline form of compound 4 (a) exhibits a peak at 7.7° ± 0.2° 2θ in an X-ray powder diffraction (XRPD) pattern, and (b) shows less than about 10% degradation when the crystalline form is stored at 25°C and 60% relative humidity for at least 7 days. This article also provides such a method, wherein the crystalline form of compound 4 exhibits a degradation of less than about 1%, or less than about 2%, or less than about 3%, or less than about 4%, or less than about 5%, or less than about 6%, or less than about 7%, or less than about 8%, or less than about 9% when the crystalline form is stored at 25°C and 60% relative humidity for at least 7 days.

[0112] This paper also provides a method in which the crystalline form of compound 4 exhibits less than about 10% degradation when stored at 40°C and 75% relative humidity for at least 7 days. This paper also provides a method in which the crystalline form of compound 4 exhibits less than about 1%, or less than about 2%, or less than about 3%, or less than about 4%, or less than about 5%, or less than about 6%, or less than about 7%, or less than about 8%, or less than about 9% degradation when stored at 40°C and 75% relative humidity for at least 7 days.

[0113] This paper also provides a method in which the crystalline form of compound 4 (a) exhibits a peak at 8.1° ± 0.2° 2θ in an X-ray powder diffraction (XRPD) pattern, and (b) shows less than about 10% degradation when the crystalline form is stored at 40°C and 75% relative humidity for at least 7 days. This paper also provides a method in which the crystalline form of compound 4 (a) exhibits peaks at 9.6° ± 0.2° 2θ, 5.7° ± 0.2° 2θ, 19.7° ± 0.2° 2θ, and 22.0° ± 0.2° 2θ in an X-ray powder diffraction (XRPD) pattern, and (b) shows less than about 10% degradation when the crystalline form is stored at 40°C and 75% relative humidity for at least 7 days. This article also provides such a method, wherein the crystalline form of compound 4 exhibits a degradation of less than about 1%, or less than about 2%, or less than about 3%, or less than about 4%, or less than about 5%, or less than about 6%, or less than about 7%, or less than about 8%, or less than about 9% when the crystalline form is stored at 40°C and 75% relative humidity for at least 7 days.

[0114] This paper also provides a method in which the crystalline form of compound 4 (a) exhibits a peak at 7.7° ± 0.2° 2θ in an X-ray powder diffraction (XRPD) pattern, and (b) shows less than about 10% degradation when the crystalline form is stored at 40°C and 75% relative humidity for at least 7 days. This article also provides such a method, wherein the crystalline form of compound 4 exhibits a degradation of less than about 1%, or less than about 2%, or less than about 3%, or less than about 4%, or less than about 5%, or less than about 6%, or less than about 7%, or less than about 8%, or less than about 9% when the crystalline form is stored at 40°C and 75% relative humidity for at least 7 days.

[0115] This paper also provides a method in which the crystalline form of compound 4 exhibits less than about 10% degradation when stored at 60°C for at least one week. This paper also provides a method in which the crystalline form of compound 4 exhibits less than about 1%, or less than about 2%, or less than about 3%, or less than about 4%, or less than about 5%, or less than about 6%, or less than about 7%, or less than about 8%, or less than about 9% degradation when stored at 60°C for at least one week.

[0116] This article also discloses a method for treating prostate cancer in subjects, including administering (a) an androgen receptor inhibitor and (b) a... An inhibitor of embryonic ectoderm development (EED) or a pharmaceutically acceptable salt thereof. In some embodiments, the androgen receptor inhibitor is selected from apalutamide, dalotamide, and enzalutamide. In some embodiments, the androgen receptor inhibitor is apalutamide. In some embodiments, the androgen receptor inhibitor is dalotamide. In some embodiments, the androgen receptor inhibitor is enzalutamide.

[0117] This article also discloses a method for treating prostate cancer in subjects who have been identified as resistant to one or more first androgen receptor signaling inhibitors (ARSIs), including administering (a) an androgen receptor inhibitor and (b) a formula The first androgen receptor signaling inhibitor (ARSI) is selected from CYP17 inhibitors and androgen receptor inhibitors. In some embodiments, one or more ARSIs are selected from CYP17 inhibitors. In some embodiments, the CYP17 inhibitor is abiraterone acetate. In some embodiments, one or more ARSIs are selected from one or more androgen receptor inhibitors. In some embodiments, one or more ARSIs are selected from apalutamide, dalotamide, and enzalutamide. In some embodiments, one or more ARSIs are apalutamide. In some embodiments, one or more ARSIs are dalotamide. In some embodiments, one or more ARSIs are enzalutamide.

[0118] This article also discloses a method for treating prostate cancer in subjects whose prostate cancer had been identified as resistant to CYP17 inhibitors, including administration of (a) an androgen receptor inhibitor and (b) a... An inhibitor of embryonic ectoderm development (EED) or a pharmaceutically acceptable salt thereof. In some embodiments, the CYP17 inhibitor is abiraterone acetate. In some embodiments, an androgen receptor inhibitor is selected from apalutamide, dalotamide, and enzalutamide. In some embodiments, the androgen receptor inhibitor is apalutamide. In some embodiments, the androgen receptor inhibitor is dalotamide. In some embodiments, the androgen receptor inhibitor is enzalutamide.

[0119] This article also discloses a method for treating prostate cancer in subjects who have been identified as resistant to abiraterone, including administration of (a) an androgen receptor inhibitor and (b) a combination of these methods. An inhibitor of embryonic ectoderm development (EED) or a pharmaceutically acceptable salt thereof. In some embodiments, the androgen receptor inhibitor is selected from apalutamide, dalotamide, and enzalutamide. In some embodiments, the androgen receptor inhibitor is apalutamide. In some embodiments, the androgen receptor inhibitor is dalotamide. In some embodiments, the androgen receptor inhibitor is enzalutamide.

[0120] This article also discloses a method for treating prostate cancer in subjects who have previously received one or more CYP17 inhibitors, including administering (a) an androgen receptor inhibitor and (b) a... An inhibitor of embryonic ectoderm development (EED) or a pharmaceutically acceptable salt thereof. In some embodiments, the CYP17 inhibitor is abiraterone acetate. In some embodiments, the androgen receptor inhibitor is selected from apalutamide, dalotamide, and enzalutamide. In some embodiments, the androgen receptor inhibitor is apalutamide. In some embodiments, the androgen receptor inhibitor is dalotamide. In some embodiments, the androgen receptor inhibitor is enzalutamide.

[0121] This article also discloses a method for treating prostate cancer in subjects (i) who have previously received one or more CYP17 inhibitors and (ii) who have never received androgen receptor inhibitor treatment, including administering (a) an androgen receptor inhibitor and (b) a [method / treatment]. An inhibitor of embryonic ectoderm development (EED) or a pharmaceutically acceptable salt thereof. In some embodiments, the CYP17 inhibitor is abiraterone acetate. In some embodiments, the androgen receptor inhibitor is selected from apalutamide, dalotamide, and enzalutamide. In some embodiments, the androgen receptor inhibitor is apalutamide. In some embodiments, the androgen receptor inhibitor is dalotamide. In some embodiments, the androgen receptor inhibitor is enzalutamide.

[0122] This document also discloses any of the methods for treating prostate cancer disclosed herein, wherein the subject has received one or more prior chemotherapy treatments prior to the administration of androgen receptor inhibitors and embryonic ectodermal development (EED) inhibitors. In some embodiments, the subject has received at most one chemotherapy treatment prior to the administration of androgen receptor inhibitors and embryonic ectodermal development (EED) inhibitors.

[0123] This article also discloses any methods of treating prostate cancer disclosed herein, wherein the subjects had not previously received CYP17 inhibitor treatment prior to administration of androgen receptor inhibitors and embryonic ectodermal development (EED) inhibitors.

[0124] This article also discloses any methods of treating prostate cancer disclosed herein, wherein the subjects had not previously received androgen receptor inhibitor treatment prior to administration of androgen receptor inhibitors and embryonic ectodermal development (EED) inhibitors.

[0125] This document also discloses any of the methods disclosed herein for treating prostate cancer, wherein the subject has not received a CYP17 inhibitor or an androgen receptor inhibitor prior to administration of the androgen receptor inhibitor and the embryonic ectodermal development (EED) inhibitor. In some embodiments, the subject has not received abiraterone or an androgen receptor inhibitor selected from apalutamide, dalotamide, or enzalutamide prior to administration of the androgen receptor inhibitor and the embryonic ectodermal development (EED) inhibitor. In some embodiments, the subject has not received abiraterone or apalutamide prior to administration of the androgen receptor inhibitor and the embryonic ectodermal development (EED) inhibitor. In some embodiments, the subject has not received abiraterone or enzalutamide prior to treatment prior to administration of the androgen receptor inhibitor and the embryonic ectodermal development (EED) inhibitor. In some embodiments, the embryonic ectodermal development (EED) inhibitor is compound 4.

[0126] This document also discloses any of the methods disclosed herein for treating prostate cancer, wherein the subject has not received abiraterone or an androgen receptor inhibitor selected from apalutamide, dalotamide, or enzalutamide prior to administration of apalutamide and compound 4. In some embodiments, the subject has not received abiraterone or an androgen receptor inhibitor selected from apalutamide, dalotamide, or enzalutamide prior to administration of dalotamide and compound 4. In some embodiments, the subject has not received abiraterone or an androgen receptor inhibitor selected from apalutamide, dalotamide, or enzalutamide prior to administration of enzalutamide and compound 4.

[0127] This document also discloses any of the methods disclosed herein for treating prostate cancer, wherein the subject has not been given abiraterone or apalutamide prior to administration of apalutamide and compound 4. In some embodiments, the subject has not been given abiraterone or dalotamide prior to administration of apalutamide and compound 4. In some embodiments, the subject has not been given abiraterone or enzalutamide prior to administration of apalutamide and compound 4.

[0128] This document also discloses any of the methods disclosed herein for treating prostate cancer, wherein the subject has not been given abiraterone or apalutamide prior to administration of dalotamide and compound 4. In some embodiments, the subject has not been given abiraterone or dalotamide prior to administration of dalotamide and compound 4. In some embodiments, the subject has not been given abiraterone or enzalutamide prior to administration of dalotamide and compound 4.

[0129] This document also discloses any of the methods disclosed herein for treating prostate cancer, wherein the subject has not been given abiraterone or apalutamide prior to administration of enzalutamide and compound 4. In some embodiments, the subject has not been given abiraterone or dalotamide prior to administration of enzalutamide and compound 4. In some embodiments, the subject has not been given abiraterone or enzalutamide prior to administration of enzalutamide and compound 4.

[0130] This document also discloses any of the methods disclosed herein for treating prostate cancer, wherein an androgen receptor inhibitor and an embryonic ectodermal development (EED) inhibitor are administered to the subject sequentially or simultaneously. In some embodiments, the androgen receptor inhibitor and the EED inhibitor are administered to the subject sequentially. In another embodiment, the androgen receptor inhibitor and the EED inhibitor are administered to the subject simultaneously.

[0131] This article also discloses any methods used in the treatment of prostate cancer in subjects disclosed herein, in which androgen receptor inhibitors and embryonic ectodermal development (EED) inhibitors are administered to subjects on the same day.

[0132] This document also discloses any of the methods disclosed herein for treating prostate cancer, wherein an androgen receptor inhibitor and an embryonic ectodermal development (EED) inhibitor are administered to the subject within a 24-hour timeframe. In another embodiment, the androgen receptor inhibitor and EED inhibitor are administered to the subject within a 12-hour timeframe, or a 10-hour timeframe, or an 8-hour timeframe, or a 6-hour timeframe, or a 4-hour timeframe, or a 2-hour timeframe, or at 1-hour intervals between each other.

[0133] This document also discloses any of the methods disclosed herein for treating prostate cancer, wherein an androgen receptor inhibitor and an embryonic ectodermal development (EED) inhibitor are administered to the subject once or twice daily. In some embodiments, an androgen receptor inhibitor and an EED inhibitor are administered to the subject once daily. In some embodiments, an androgen receptor inhibitor and an EED inhibitor are administered to the subject twice daily. In some embodiments, an androgen receptor inhibitor is administered to the subject twice daily, and an EED inhibitor is administered to the subject once daily. In some embodiments, an androgen receptor inhibitor is administered to the subject once daily, and an EED inhibitor is administered to the subject twice daily.

[0134] This document also discloses any of the methods disclosed herein for treating prostate cancer, wherein an androgen receptor inhibitor is administered to the subject with or without food. In one embodiment, the androgen receptor inhibitor is administered to the subject with food. In another embodiment, the androgen receptor inhibitor is administered to the subject without food.

[0135] This article also discloses any of the methods disclosed herein for treating prostate cancer in which the subject has been given one or more prior androgen deprivation therapies prior to administration of androgen receptor inhibitors and embryonic ectodermal development (EED) inhibitors.

[0136] This article also discloses any of the methods disclosed herein for treating prostate cancer in which the subject has been given a gonadotropin-releasing hormone (GnRH) analogue prior to the administration of an androgen receptor inhibitor and an embryonic ectodermal development (EED) inhibitor.

[0137] This document also discloses any of the methods disclosed herein for treating prostate cancer, wherein the subject receives a gonadotropin-releasing hormone (GnRH) analogue concurrently with administration of an androgen receptor inhibitor and an embryonic ectodermal development (EED) inhibitor. In one embodiment, the GnRH analogue is selected from leuprolide, goserelin, histrelin, triptorelin, degarelix, and relugolix. In one embodiment, the GnRH analogue is leuprolide. In one embodiment, the GnRH analogue is goserelin. In one embodiment, the GnRH analogue is histrelin. In one embodiment, the GnRH analogue is triptorelin. In one embodiment, the GnRH analogue is degarelix. In one embodiment, the GnRH analogue is relugolix.

[0138] This article also discloses any of the methods for treating prostate cancer disclosed herein, in which the subject had undergone bilateral orchiectomy prior to administration of androgen receptor inhibitors and embryonic ectodermal development (EED) inhibitors.

[0139] This article also discloses any methods of treating prostate cancer disclosed herein, wherein during the period of administration of androgen receptor inhibitors and embryonic ectodermal development (EED) inhibitors to subjects, compounds that are substrates of CYP3A4, CYP2C19, CYP2C8, CYP2C9, UGT, P-gp, BCRP, or OATP1B1 are not administered to subjects.

[0140] This article also discloses any methods of treating prostate cancer disclosed herein, wherein during the period of administration of androgen receptor inhibitors and embryonic ectodermal development (EED) inhibitors to subjects, compounds that are substrates of CYP3A4, CYP2C9, UGT, P-gp, BCRP, or OATP1B1 are not administered to subjects.

[0141] This article also discloses any methods of treating prostate cancer disclosed herein, wherein during the period of administration of androgen receptor inhibitors and embryonic ectodermal development (EED) inhibitors to subjects, the following compounds are not administered to subjects: (a) CYP2C8 inhibitors, (b) CYP3A4 inducers, or (c) substrates of CYP3A4, CYP2C9, or CYP2C19.

[0142] This article also discloses any of the methods disclosed herein for treating prostate cancer in which the following compounds are not administered to the subject during the period of administration of androgen receptor inhibitors and embryonic ectodermal development (EED) inhibitors: (a) CYP3A4 inducers, (b) PG-p inhibitors, (c) CYP3A4 inhibitors, (d) BCRP substrates, (e) OATP1B1 substrates, or (f) OATP1B3 substrates.

[0143] This document also discloses any methods for treating prostate cancer disclosed herein, wherein the prostate cancer in the subject is selected from metastatic prostate cancer, non-metastatic prostate cancer, metastatic castration-resistant prostate cancer, metastatic castration-sensitive prostate cancer, localized high-risk prostate cancer, recurrent prostate cancer, non-metastatic castration-resistant prostate cancer, non-metastatic castration-sensitive prostate cancer, androgen receptor inhibitor-sensitive prostate cancer, androgen receptor inhibitor-resistant prostate cancer, androgen receptor-dependent prostate cancer, androgen receptor-independent prostate cancer, neuroendocrine prostate cancer (NEPC), metastatic neuroendocrine prostate cancer (NEPC), prostate cancer with small cell characteristics, metastatic prostate cancer with small cell characteristics, and invasive variant prostate cancer. In some embodiments, the prostate cancer in the subject is metastatic prostate cancer. In some embodiments, the prostate cancer in the subject is non-metastatic prostate cancer. In some embodiments, the prostate cancer in the subject is metastatic castration-resistant prostate cancer. In some embodiments, the prostate cancer in the subject is metastatic castration-sensitive prostate cancer. In some embodiments, the prostate cancer in the subject is localized high-risk prostate cancer. In some embodiments, the prostate cancer in the subjects is recurrent prostate cancer. In some embodiments, the prostate cancer in the subjects is non-metastatic castration-resistant prostate cancer. In some embodiments, the prostate cancer in the subjects is non-metastatic castration-sensitive prostate cancer. In some embodiments, the prostate cancer in the subjects is androgen receptor inhibitor-sensitive prostate cancer. In some embodiments, the prostate cancer in the subjects is androgen receptor inhibitor-resistant prostate cancer. In some embodiments, the prostate cancer in the subjects is androgen receptor-dependent prostate cancer. In some embodiments, the prostate cancer in the subjects is androgen receptor-independent prostate cancer. In some embodiments, the prostate cancer in the subjects is neuroendocrine prostate cancer (NEPC). In some embodiments, the prostate cancer in the subjects is metastatic neuroendocrine prostate cancer (NEPC). In some embodiments, the prostate cancer in the subjects is prostate cancer with small cell characteristics. In some embodiments, the prostate cancer in the subjects is metastatic prostate cancer with small cell characteristics. In some embodiments, the prostate cancer in the subjects is aggressive variant prostate cancer.

[0144] In other embodiments of the methods for treating prostate cancer in subjects disclosed herein, subjects with prostate cancer who may benefit from administration of androgen receptor inhibitors and embryonic ectodermal development (EED) inhibitors may include one or more of the following criteria: (a) the subject has undergone bilateral orchiectomy or is willing to continue with GnRH analogs or antagonists to maintain castration levels of testosterone; (b) the subject has progressed after at least one first-line ARSI (abiraterone, enzalutamide, apalutamide, dalotamide) and must not have received more than two chemotherapy regimens in an mCRPC setting; (c) the subject demonstrates evidence of disease progression according to PCWG3 criteria, including elevated PSA, defined as at least two elevated values ​​obtained at least one week apart, with the most recent result being at least 2.0 ng / mL (or 1.0 ng / mL). (d) The subject has a disease that is measurable and / or assessable according to RECIST 1.1; (e) an ECOG performance status of 0 or 1; and (f) adequate organ function.

[0145] In other embodiments, this document provides a method for treating prostate cancer in a subject, wherein a therapeutically effective amount of (a) an androgen receptor inhibitor and (b) an embryonic ectodermal development (EED) inhibitor is administered to the subject, and further administered one or more adjunctive therapeutic agents. In further embodiments, such a method is provided, wherein the one or more adjunctive therapeutic agents are selected from androgen receptor degraders, chemotherapeutic agents, mitotic inhibitors, antimetabolites, platinum-based agents, histone deacetylase (HDAC) inhibitors, CD30-directing antibody-drug conjugates, farnesyltransferase inhibitors, SYK inhibitors, JAK inhibitors, PI3K pathway inhibitors, immunomodulatory agents, AKT inhibitors, radiopharmaceuticals, PARP inhibitors, or combinations thereof. In further embodiments, such a method is provided, wherein the one or more adjunctive therapeutic agents are selected from chemotherapeutic agents.

[0146] Such methods are provided in other embodiments, wherein the androgen receptor degrader is a proteolytically targeted chimera (PROTAC). Androgen receptor (AR) degrader. In a further embodiment, the androgen receptor degrader is bavdegalutamide (ARV-110).

[0147] Such a method is provided in a further embodiment, wherein the chemotherapeutic agent is selected from actinomycin, azacytidine, azathioprine, bendamustine, bleomycin, bortezomib, chlorambucil, cyclophosphamide, daunorubicin, doxifluridine, doxorubicin, epirubicin, etc. n), epothilone, etoposide, idarubicin, irinotecan, lurbinectedin, mechlorethamine, mitoxantrone, teniposide, topotecan, valrubicin, vemurafenib, vinblastine, vincristine, and vindesine. In some embodiments, the chemotherapeutic agent is actinomycin. In some embodiments, the chemotherapeutic agent is azacitidine. In some embodiments, the chemotherapeutic agent is azathioprine. In some embodiments, the chemotherapeutic agent is bleomycin. In some embodiments, the chemotherapeutic agent is bortezomib. In some embodiments, the chemotherapeutic agent is chlorambucil. In some embodiments, the chemotherapeutic agent is cyclophosphamide. In some embodiments, the chemotherapeutic agent is daunorubicin. In some embodiments, the chemotherapy agent is docefluuridine. In some embodiments, the chemotherapy agent is doxorubicin. In some embodiments, the chemotherapy agent is epirubicin. In some embodiments, the chemotherapy agent is epothilone. In some embodiments, the chemotherapy agent is etoposide. In some embodiments, the chemotherapy agent is idarubicin. In some embodiments, the chemotherapy agent is irinotecan. In some embodiments, the chemotherapy agent is rubitidine. In some embodiments, the chemotherapy agent is nitrogen mustard. In some embodiments, the chemotherapy agent is mitoxantrone. In some embodiments, the chemotherapy agent is teniposide. In some embodiments, the chemotherapy agent is topotecan. In some embodiments, the chemotherapy agent is pentorubicin. In some embodiments, the chemotherapy agent is vemurafenib. In some embodiments, the chemotherapy agent is vincristine. In some embodiments, the chemotherapy agent is vincristine. In some embodiments, the chemotherapy agent is vincristine.

[0148] Such methods are provided in further embodiments, wherein one or more additional therapeutic agents are selected from mitotic inhibitors. Such methods are provided in further embodiments, wherein the mitotic inhibitor is selected from paclitaxel, docetaxel, cabazitaxel, tesetaxel, and nab-paclitaxel. In some embodiments, the mitotic inhibitor is paclitaxel. In some embodiments, the mitotic inhibitor is docetaxel. In some embodiments, the mitotic inhibitor is cabazitaxel. In some embodiments, the mitotic inhibitor is tesetaxel. In some embodiments, the mitotic inhibitor is nab-paclitaxel.

[0149] In other embodiments, one or more additional therapeutic agents are selected from antimetabolites. In some embodiments, one or more antimetabolites are selected from azacitidine, 6-mercaptopurine, capecitabine, hydroxyurea, cladribine, pralatrexate, thioguanine, decitabine, clofarabine, nelarabine, fludarabine, 5-fluorouracil, gemcitabine, cytarabine, pemetrexed, methotrexate, Ara-C, fluxuridine, fludarabine, pentostatin, and combinations of trifluridine / tipiracil. In some embodiments, one or more antimetabolites are selected from 6-mercaptopurine, capecitabine, hydroxyurea, cladribine, pralatrexate, thioguanine, decitabine, clofarabine, nerabine, fludarabine, 5-fluorouracil, gemcitabine, cytarabine, pemetrexed, and methotrexate.

[0150] In some embodiments, the antimetabolite is azacitidine. In some embodiments, the antimetabolite is 6-mercaptopurine. In some embodiments, the antimetabolite is capecitabine. In some embodiments, the antimetabolite is hydroxyurea. In some embodiments, the antimetabolite is cladribine. In some embodiments, the antimetabolite is pralatrexate. In some embodiments, the antimetabolite is thioguanine. In some embodiments, the antimetabolite is decitabine. In some embodiments, the antimetabolite is clofarapine. In some embodiments, the antimetabolite is nelabine. In some embodiments, the antimetabolite is fludarabine. In some embodiments, the antimetabolite is 5-fluorouracil. In some embodiments, the antimetabolite is gemcitabine. In some embodiments, the antimetabolite is cytarabine. In some embodiments, the antimetabolite is pemetrexed. In some embodiments, the antimetabolite is methotrexate. In some embodiments, the antimetabolite is cytarabine (Ara-C). In some embodiments, the antimetabolite is fluorouracil. In some embodiments, the antimetabolite is fludarabine. In some embodiments, the antimetabolite is pentostatin. In some embodiments, the antimetabolite is a combination of trifluorouridine and tipyrimidine.

[0151] In some embodiments, one or more additional therapeutic agents are selected from platinum-based agents. In some embodiments, the platinum-based agents are selected from cisplatin, carboplatin, oxaliplatin, nedaplatin, lobaplatin, triplatintetranitrate, pheanthriplatin, picoplatin, and satraplatin. In a further embodiment, the platinum-based agent is cisplatin. In a further embodiment, the platinum-based agent is carboplatin. In a further embodiment, the platinum-based agent is oxaliplatin. In a further embodiment, the platinum-based agent is nedaplatin. In a further embodiment, the platinum-based agent is lobaplatin. In a further embodiment, the platinum-based agent is triplatintetranitrate. In a further embodiment, the platinum-based agent is pheanthriplatin. In a further embodiment, the platinum-based agent is picoplatin. In a further embodiment, the platinum-based agent is satraplatin.

[0152] Such a method is provided in a further embodiment, wherein one or more additional therapeutic agents are selected from histone deacetylase (HDAC) inhibitors. Such a method is provided in a further embodiment, wherein the histone deacetylase (HDAC) inhibitor is selected from vorinostat, romidepsin, belinostat, tucidinostat, panobinostat, mocetinostat, givinostat, resmiostat, abexinostat, ricolinostat, entinostat, tinostamustin, fimepinostat, CXD-101, quisinostat, and chidamide. In a further embodiment, the histone deacetylase (HDAC) inhibitor is vorinostat. In a further embodiment, the histone deacetylase (HDAC) inhibitor is romidepsin. In a further embodiment, the histone deacetylase (HDAC) inhibitor is belinostat. In a further embodiment, the histone deacetylase (HDAC) inhibitor is tonostat. In a further embodiment, the histone deacetylase (HDAC) inhibitor is pabistat. In a further embodiment, the histone deacetylase (HDAC) inhibitor is moxinata. In a further embodiment, the histone deacetylase (HDAC) inhibitor is gemvinata. In a further embodiment, the histone deacetylase (HDAC) inhibitor is resmiostat. In a further embodiment, the histone deacetylase (HDAC) inhibitor is abexistat. In a further embodiment, the histone deacetylase (HDAC) inhibitor is ricolinostat. In a further embodiment, the histone deacetylase (HDAC) inhibitor is entenoxat. In a further embodiment, the histone deacetylase (HDAC) inhibitor is tinostamustin. In a further embodiment, the histone deacetylase (HDAC) inhibitor is femenoxat. In a further embodiment, the histone deacetylase (HDAC) inhibitor is CXD-101. In a further embodiment, the histone deacetylase (HDAC) inhibitor is quinsinostat. In a further embodiment, the histone deacetylase (HDAC) inhibitor is chidamide.

[0153] Such a method is provided in a further embodiment, wherein one or more additional therapeutic agents are selected from CD30-targeting antibody-drug conjugates. Such a method is provided in a further embodiment, wherein the CD30-targeting antibody-drug conjugate is selected from brentuximab vedotin and SGN-CD30C. In a further embodiment, the CD30-targeting antibody-drug conjugate is brentuximab vedotin. In a further embodiment, the CD30-targeting antibody-drug conjugate is SGN-CD30C.

[0154] Such a method is provided in further embodiments, wherein one or more additional therapeutic agents are selected from farnesyltransferase inhibitors. Such a method is provided in further embodiments, wherein the farnesyltransferase inhibitor is selected from antroquinonol, BMS-214662, L778123, L744832, FTI-276, FTI-277, manumycin A, LB-42708, moverastin, PD169541, ABT-100, FTI-2153, tipifarnib, and lonafamib. In further embodiments, the farnesyltransferase inhibitor is antroquinonol. In further embodiments, the farnesyltransferase inhibitor is BMS-214662. In further embodiments, the farnesyltransferase inhibitor is L778123. In further embodiments, the farnesyltransferase inhibitor is L744832. In further embodiments, the farnesyltransferase inhibitor is FTI-276. In a further embodiment, the farnesyltransferase inhibitor is FTI-277. In a further embodiment, the farnesyltransferase inhibitor is cholecystokinin A. In a further embodiment, the farnesyltransferase inhibitor is LB-42708. In a further embodiment, the farnesyltransferase inhibitor is moveerastin. In a further embodiment, the farnesyltransferase inhibitor is PD169541. In a further embodiment, the farnesyltransferase inhibitor is ABT-100. In a further embodiment, the farnesyltransferase inhibitor is FTI-2153. In a further embodiment, the farnesyltransferase inhibitor is tepifenab. In a further embodiment, the farnesyltransferase inhibitor is lonafamib.

[0155] Such methods are provided in further embodiments, wherein one or more additional therapeutic agents are selected from SYK inhibitors. Such methods are provided in further embodiments, wherein the SYK inhibitor is selected from fostamatinib (R788), entoprilinib (GS-9973), cerdulatinib (PRT062070), and TAK-659. In further embodiments, the SYK inhibitor is fostamatinib (R788). In further embodiments, the SYK inhibitor is entoprilinib (GS-9973). In further embodiments, the SYK inhibitor is cerdulatinib (PRT062070). In further embodiments, the SYK inhibitor is TAK-659.

[0156] Such methods are provided in further embodiments, wherein one or more additional therapeutic agents are selected from JAK inhibitors. Such methods are provided in further embodiments, wherein the JAK inhibitor is selected from tofacitinib, baricitinib, ruxolitinib, upadacitinib, fedratinib, abrocitinib, and ruxolitinib. In further embodiments, the JAK inhibitor is tofacitinib. In further embodiments, the JAK inhibitor is baricitinib. In further embodiments, the JAK inhibitor is ruxolitinib. In further embodiments, the JAK inhibitor is upadatinib. In further embodiments, the JAK inhibitor is fedratinib. In further embodiments, the JAK inhibitor is abuxitinib. In further embodiments, the JAK inhibitor is ruxolitinib.

[0157] Such a method is provided in a further embodiment, wherein one or more additional therapeutic agents are selected from PI3K pathway inhibitors. Such methods are provided in further implementation schemes, wherein the PI3K pathway inhibitors are selected from taselisib (GDC-0032), GDC-0077, perifosine, idelalisib, buparlisib (BKM120), duvelisib (IPI-145), copanlisib (BAY80-6946), PX-866, dactolisib, CUDC-907, voxtalisib (SAR245409, XL765), ME-401, IPI-549, SF1126, RP6530, INK1117, pictilisib (GDC-0941), XL147 (SAR245408), palomid 529, GSK1059615, ZSTK474, and PWT33597. In a further embodiment, the PI3K pathway inhibitor is taselisib (GDC-0032). In a further embodiment, the PI3K pathway inhibitor is GDC-0077. In a further embodiment, the PI3K pathway inhibitor is perifoxine. In a further embodiment, the PI3K pathway inhibitor is ederaliximab. In a further embodiment, the PI3K pathway inhibitor is bupaniximab (BKM120). In a further embodiment, the PI3K pathway inhibitor is duveliximab. In a further embodiment, the PI3K pathway inhibitor is (IPI-145). In a further embodiment, the PI3K pathway inhibitor is cupanixine (BAY 80-6946). In a further embodiment, the PI3K pathway inhibitor is PX-866. In a further embodiment, the PI3K pathway inhibitor is datolis. In a further embodiment, the PI3K pathway inhibitor is CUDC-907. In a further embodiment, the PI3K pathway inhibitor is vortalolixalate (SAR245409, XL765). In a further embodiment, the PI3K pathway inhibitor is ME-401. In a further embodiment, the PI3K pathway inhibitor is IPI-549. In a further embodiment, the PI3K pathway inhibitor is SF1126. In a further embodiment, the PI3K pathway inhibitor is RP6530. In a further embodiment, the PI3K pathway inhibitor is INK1117. In a further embodiment, the PI3K pathway inhibitor is pitigliflozin (GDC-0941). In a further embodiment, the PI3K pathway inhibitor is XL147 (SAR245408).In a further embodiment, the PI3K pathway inhibitor is palomid 529. In a further embodiment, the PI3K pathway inhibitor is GSK1059615. In a further embodiment, the PI3K pathway inhibitor is ZSTK474. In a further embodiment, the PI3K pathway inhibitor is PWT33597.

[0158] Such methods are provided in further embodiments, wherein one or more additional therapeutic agents are selected from immunomodulatory agents. Such methods are provided in further embodiments, wherein the immunomodulatory agents are selected from lenalidomide, PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 agents, T-cell immunoglobulins and ITIM domain (TIGIT) agents, TIM-3 inhibitors, and LAG-3 inhibitors.

[0159] Such methods are provided in other embodiments, wherein one or more additional therapeutic agents are selected from PD-1 inhibitors. In some embodiments, the PD-1 inhibitor is selected from pembrolizumab, nivolumab, cemiplimab, JTX-4014, spartalizumab (PDR001), camrelizumab (SHR1210), sintilimab (IBI308), tislelizumab (BGB-A317), toripalimab (JS 001), dostarlimab (TSR-042, WBP-285), INCMGA00012 (MGA012), AMP-224, and AMP-514 (MEDI0680).

[0160] In some embodiments, the PD-1 inhibitor is pembrolizumab. In some embodiments, the PD-1 inhibitor is nivolumab. In some embodiments, the PD-1 inhibitor is cimiprimab. In some embodiments, the PD-1 inhibitor is spartazolizumab (PDR001). In some embodiments, the PD-1 inhibitor is camrelizumab (SHR1210). In some embodiments, the PD-1 inhibitor is sintilimab (IBI308). In some embodiments, the PD-1 inhibitor is tislelizumab (BGB-A317). In some embodiments, the PD-1 inhibitor is toreplemab (JS 001). In some embodiments, the PD-1 inhibitor is dostalimab (TSR-042, WBP-285). In some embodiments, the PD-1 inhibitor is INCMGA00012 (MGA012). In some embodiments, the PD-1 inhibitor is AMP-224. In some implementations, the PD-1 inhibitor is AMP-514 (MEDI0680).

[0161] Further embodiments of the methods disclosed herein are provided, wherein one or more additional therapeutic agents are selected from PD-L1 inhibitors. In some embodiments, the PD-L1 inhibitor is selected from atezolizumab, avelumab, durvalumab, MPDL3280A (RG7446), MDX-1105 (BMS-936559), BMS-935559, MSB0010718C, and MEDI4736.

[0162] In some implementations, the PD-L1 inhibitor is selected from atezolizumab, avelumab, durvalumab, KN035, CK-301, AUNP12, CA-170, and BMS-986189.

[0163] In some embodiments, the PD-L1 inhibitor is atezolizumab. In some embodiments, the PD-L1 inhibitor is avelumab. In some embodiments, the PD-L1 inhibitor is durvalumab. In some embodiments, the PD-L1 inhibitor is MPDL3280A (RG7446). In some embodiments, the PD-L1 inhibitor is MDX-1105 (BMS-936559). In some embodiments, the PD-L1 inhibitor is BMS-935559. In some embodiments, the PD-L1 inhibitor is MSB0010718C. In some embodiments, the PD-L1 inhibitor is MEDI4736.

[0164] Such a method is provided in a further embodiment, wherein the CTLA-4 inhibitor is selected from ipilimumab and tremelimumab. In a further embodiment, the CTLA-4 inhibitor is ipilimumab. In a further embodiment, the CTLA-4 inhibitor is tremelimumab.

[0165] In a further embodiment, such a method is provided, wherein the T-cell immunoglobulin and ITIM domain (TIGIT) agent is selected from BMS-986207, BGB-A1217, tiragolumab, AB154, ASP8374, MK-7684, CD112RCOM701, and LY3435151. In a further embodiment, the TIGIT agent is BMS-986207. In a further embodiment, the TIGIT agent is BGB-A1217. In a further embodiment, the TIGIT agent is tiragolumab. In a further embodiment, the TIGIT agent is AB154. In a further embodiment, the TIGIT agent is ASP8374. In a further embodiment, the TIGIT agent is MK-7684. In a further embodiment, the TIGIT agent is CD112RCOM701. In a further embodiment, the TIGIT agent is LY3435151.

[0166] In further embodiments, such methods are provided, wherein the TIM-3 inhibitor is selected from Sym023, INCAGN02390, LY331367, Sym021, MBG453, BGB-A425, TSR-022, RO7121661, and LU3415244. In further embodiments, the TIM-3 inhibitor is Sym023. In further embodiments, the TIM-3 inhibitor is INCAGN02390. In further embodiments, the TIM-3 inhibitor is LY331367. In further embodiments, the TIM-3 inhibitor is Sym021. In further embodiments, the TIM-3 inhibitor is MBG453. In further embodiments, the TIM-3 inhibitor is BGB-A425. In further embodiments, the TIM-3 inhibitor is TSR-022. In further embodiments, the TIM-3 inhibitor is RO7121661. In further embodiments, the TIM-3 inhibitor is LU3415244.

[0167] Such methods are provided in further embodiments, wherein the LAG-3 inhibitor is selected from relatlimab, tebotelimab, chlorogenic acid, RO-7247669, favezelimab, INCAGN-2385, IBI-110, eftilagimod alpha, Sym-022, LBL-007, ABL-501, HLX26, IBI-323, ieramilimab, FS 118, EMB-02, and finalimab. In further embodiments, the LAG-3 inhibitor is relatlimab. In further embodiments, the LAG-3 inhibitor is tebotelimab. In further embodiments, the LAG-3 inhibitor is chlorogenic acid. In further embodiments, the LAG-3 inhibitor is RO-7247669. In further embodiments, the LAG-3 inhibitor is favezelimab. In a further embodiment, the LAG-3 inhibitor is INCAGN-2385. In a further embodiment, the LAG-3 inhibitor is IBI-110. In a further embodiment, the LAG-3 inhibitor is eftilagimod alpha. In a further embodiment, the LAG-3 inhibitor is Sym-022. In a further embodiment, the LAG-3 inhibitor is LBL-007. In a further embodiment, the LAG-3 inhibitor is ABL-501. In a further embodiment, the LAG-3 inhibitor is HLX 26. In a further embodiment, the LAG-3 inhibitor is IBI-323. In a further embodiment, the LAG-3 inhibitor is eralimab. In a further embodiment, the LAG-3 inhibitor is FS118. In a further embodiment, the LAG-3 inhibitor is EMB-02. In a further embodiment, the LAG-3 inhibitor is finalimab.

[0168] Such methods are provided in further embodiments, wherein the AKT inhibitor is ipatasertib (GDC-0068), capivasertib (AZD5363), MK2206, afuresertib (GSK2110183), uprosertib (GSK2141795), perifosin (KRX-0401), PHT-427 (CS-0223), or Akti-1 / 2. In some embodiments, the AKT inhibitor is ipatasertib (GDC-0068). In some implementations, the PI3K inhibitors are taselisib (GDC-0032), GDC-0077, perifoxine, ederaliximab, bupaniximab (BKM120), duveliximab (IPI-145), cupanixine (BAY 80-6946), PX-866, datoliximab, CUDC-907, vortaliximab (SAR245409, XL765), ME-401, IPI-549, SF1126, RP6530, INK1117, pitiliximab (GDC-0941), XL147 (SAR245408), palomid 529, GSK1059615, ZSTK474, or PWT33597.

[0169] Such methods are provided in further embodiments, wherein the radiopharmaceutical is selected from radioligand therapy agents. In some embodiments, the radiopharmaceutical is lutetium Lu 177 vipivotide tetraxetan.

[0170] Such a method is provided in a further embodiment, wherein the PARP inhibitor is selected from olaparib, rucaparib, niraparib, veliparib, fuzolaparib, CEP 9722, E7016, talazoparib, veliparib, pamiparib, AZD5305, AZD5135, AZD9574, IMP1734, DM5167, KU-0059436 (AZD2281), NMS-293, SNV-001, compounds disclosed in WO 2022 / 225934, compounds disclosed in WO 2023 / 056039, compounds disclosed in WO2022 / 247816, and compounds disclosed in CN 115677688 A.

[0171] Compounds of formula (I) or pharmaceutically acceptable salts thereof may be prepared using commercially available reagents and intermediates in the synthetic methods and reaction schemes described herein, in the synthetic methods and reaction schemes described in U.S. Patent No. 11,091,495, or using other reagents and conventional methods well known to those skilled in the art. The contents of U.S. Patent No. 11,091,495 are hereby incorporated by reference for this purpose.

[0172] For example, intermediates of compounds and compounds of formula (I) of the present invention can be prepared according to general reaction schemes I or II: General Reaction Scheme I

[0173] In general reaction scheme I, the R2-ester-substituted imidazo[1,2-c]pyrimidine A is coupled to the R3-optionally substituted intermediate amine B via nucleophilic substitution to produce intermediate C. The boric acid derivative (Y)-R1 D is coupled to the halogen-substituted intermediate C via a Suzuki reaction in the presence of a suitable base (e.g., sodium carbonate), and the R2 ester is converted to an acid by saponification with NaOH to generate intermediate acid E. This acid is converted to the corresponding amide, which dehydrates to give the topic compound nitrile G.

[0174] General Reaction Scheme II

[0175] In General Reaction Scheme II, a halogenated intermediate C containing a suitable reactant R2 (e.g., an ester) is converted to an acid intermediate by saponification in the presence of a suitable base, and then treated with NH4Cl in the presence of HATU to form an amide, which is subsequently dehydrated to form a nitrile intermediate H. R1 is coupled to intermediate H via a Suzuki reaction using a boric acid derivative (Y) in the presence of a base. The nitrile group of intermediate G containing R1 is hydrolyzed in the presence of acid and water to give the topic compound amide F.

[0176] Some embodiments provide methods for treating prostate cancer in a subject, including administering a pharmaceutically acceptable salt of a compound of formula (I) to the subject. The desired salt can be prepared by any suitable method in the art, for example, with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc., or with organic acids such as acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, pyranosidyl acid such as glucuronic acid or galacturonic acid, α-hydroxy acids such as citric acid or tartaric acid, amino acids such as aspartic acid or glutamic acid, aromatic acids such as benzoic acid or cinnamic acid, sulfonic acids such as p-toluenesulfonic acid or ethanesulfonic acid, etc. In particular, references to compounds of formula (I) herein also refer to pharmaceutically acceptable salts of compounds of formula (I) in alternative embodiments.

[0177] If the compound of formula (I) or its pharmaceutically acceptable salt is a solid, those skilled in the art will understand that the compound or its salt may exist in different crystalline or polymorphic forms, all of which are intended to be within the scope of the present invention and the prescribed formula.

[0178] This document also provides isotopically labeled compounds of formula (I) or their pharmaceutically acceptable use, wherein one or more atoms are replaced by atoms having the same atomic number but with an atomic mass or mass number different from those commonly found in nature. Examples of isotopes suitable for inclusion in compounds of the present invention include the following isotopes: hydrogen, such as 2 H and 3 H; carbon, such as 11 C 13 C and 14 C; chlorine, such as 36 Cl; fluorine, such as 18 F; iodine, such as 123 I and 125 I; nitrogen, such as 13 N and 15 N; oxygen, such as 15 O、 17 O and 18 O; phosphorus, such as 32 P; and sulfur, such as 35 S. Certain isotope-labeled compounds of the present invention, such as those containing radioactive isotopes, are useful in studies of drug and / or substrate tissue distribution. Radioactive isotope tritium ( 3 H) and carbon-14 ( 14 C) They are particularly useful for this purpose due to the ease of their incorporation and the well-established detection methods. Using heavier isotopes (such as deuterium, 2Substituting H) can yield certain therapeutic advantages from greater metabolic stability, such as increased in vivo half-life or reduced dose requirements, and may therefore be preferred in some cases. Using positron-emitting isotopes (such as...) 11 C 18 F, 15 O and 13 Substitution of N) can be useful in positron emission tomography (PET) studies to examine substrate receptor occupancy. Isotope-labeled compounds of formula (I) or pharmaceutically acceptable salts thereof can generally be prepared by conventional techniques known to those skilled in the art or method analogs of the methods described herein, using appropriate isotope-labeled reagents instead of the unlabeled reagents that would otherwise be used.

[0179] In one aspect, compositions described herein comprising compounds of formula (I) or pharmaceutically acceptable salts thereof are used for the treatment of prostate cancer in the subject. Such compositions can be prepared into pharmaceutically acceptable dosage forms for administration to the subject. Pharmaceutically acceptable dosage forms include, for example, liquids, suspensions, powders for reconstitution, tablets, pills, sachets, or hard or soft gelatin capsules (see, for example, ...). Remington: The Science and Practice of Pharmacy (Gennaro, 21st edition Mack Pub. Co., Easton, PA (2005)). Compounds of formula (I) or pharmaceutically acceptable salts thereof can be formulated into pharmaceutical compositions as described below in any pharmaceutical form that a person skilled in the art would deem suitable. Pharmaceutical compositions of the present invention comprise a therapeutically effective amount of at least one compound of formula (I) or a pharmaceutically acceptable salt thereof, and an inert, pharmaceutically acceptable carrier or diluent.

[0180] The drug carrier used can be solid or liquid. Exemplary solid carriers are lactose, sucrose, talc, gelatin, agar, pectin, gum arabic, magnesium stearate, stearic acid, etc. Exemplary liquid carriers are syrup, peanut oil, olive oil, water, etc. Similarly, the composition may contain time-delayed or time-release materials known in the art, such as glyceryl monostearate or glyceryl distearate alone, or together with waxes, ethyl cellulose, hydroxypropyl methylcellulose, methyl methacrylate. Further additives or excipients may be added to achieve the desired formulation properties. For example, bioavailability enhancers such as Labrasol, Gelucire, etc., or formulations such as CMC (carboxymethyl cellulose), PG (propylene glycol), or PEG (polyethylene glycol) may be added. For example, in the preparation of capsule formulations, Gelucire, a semi-solid mediator that protects the active ingredient from light, moisture, and oxidation, may be added.

[0181] If a solid carrier is used, the preparation can be formulated into tablets, powders, or granules encapsulated in hard gelatin capsules, or into lozenges or tablets. The amount of the solid carrier can vary, but will typically be from about 25 mg to about 1 g. If a liquid carrier is used, the preparation can be in the form of a sterile injectable solution or suspension in syrup, emulsion, soft gelatin capsules, ampoules, or vials, or a non-aqueous liquid suspension. If a semi-solid carrier is used, the preparation can be in the form of hard or soft gelatin capsule formulations. The compositions of the present invention are prepared in unit dosage forms suitable for administration (e.g., parenteral or oral administration).

[0182] To obtain a stable water-soluble dosage form, the compound of formula (I) or a pharmaceutically acceptable salt thereof may be dissolved in an aqueous solution of an organic or inorganic acid, such as a 0.3 M solution of succinic acid or citric acid. If the soluble salt form is unavailable, the compound or a pharmaceutically acceptable salt thereof may be dissolved in a suitable co-solvent or combination of co-solvents. Examples of suitable co-solvents include ethanol, propylene glycol, polyethylene glycol 300, polysorbate 80, glycerol, etc., in concentrations ranging from 0% to 60% by total volume. In one exemplary embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is dissolved in DMSO and diluted with water. The composition may also be in the form of a salt of the active ingredient in a solution in a suitable aqueous medium such as water or isotonic saline or glucose solution.

[0183] A suitable formulation depends on the chosen route of administration. For injection, the compound of formula (I) or a pharmaceutically acceptable salt thereof can be formulated into an aqueous solution, preferably in a physiologically compatible buffer such as Hanks' solution, Ringer's solution, or physiological saline buffer. For administration via mucosa, a penetrant suitable for the barrier to be penetrated is used in the formulation. Such penetrants are generally known in the art.

[0184] For oral administration, compounds can be formulated by combining the active compound with a pharmaceutically acceptable carrier known in the art. Such carriers enable the compounds of the present invention to be formulated into tablets, pills, sugar-coated pills, capsules, liquids, gels, syrups, slurries, suspensions, etc., for oral ingestion by a subject. Pharmaceutical preparations for oral use can be obtained by mixing a solid excipient with the active ingredient (the pharmaceutical preparation), optionally grinding the resulting mixture, and, if desired, processing the granular mixture after adding suitable adjuvants to obtain tablets or sugar-coated tablet cores. Suitable excipients include: fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; and cellulose preparations, such as corn starch, wheat starch, rice starch, potato starch, gelatin, gum, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, or polyvinylpyrrolidone (PVP). If desired, disintegrants, such as croscarmellose, agar, or alginate or salts thereof, such as sodium alginate, can be added.

[0185] The sugar-coated core is provided with a suitable coating. For this purpose, a concentrated sugar solution may be used, optionally containing gum arabic, polyvinylpyrrolidone, Carbopol gel, polyethylene glycol and / or titanium dioxide, lacquer solution, and a suitable organic solvent or solvent mixture. Dyes or pigments may be added to the coating of the tablet or sugar-coated core to identify or characterize different combinations of active pharmaceutical ingredients.

[0186] Orally applicable pharmaceutical preparations include push-in capsules made of gelatin, and closed soft capsules made of gelatin and plasticizers such as glycerin or sorbitol. Push-in capsules may contain the active ingredient mixed with fillers such as lactose, binders such as starch, and / or lubricants such as talc or magnesium stearate, and optionally, stabilizers. In soft capsules, the active pharmaceutical ingredient may be dissolved or suspended in a suitable liquid, such as fatty oil, liquid paraffin, or liquid polyethylene glycol. Additionally, stabilizers may be added. All formulations intended for oral administration should be in doses suitable for such administration. For buccal administration, the composition may be in tablets or lozenges formulated in a conventional manner.

[0187] For intranasal or inhalation administration of compounds of formula (I) or their pharmaceutically acceptable salts, a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas, can be conveniently delivered as an aerosol spray from a pressurized package or nebulizer. In the case of pressurized aerosols, the dosage unit can be determined by providing a valve to deliver the measured amount. Gelatin capsules and cartridges for inhalers or blowpipes, etc., can be formulated into a powder mixture containing the compound and a suitable powder matrix such as lactose or starch.

[0188] These compounds can be formulated for parenteral administration by injection, for example, by bolus or continuous infusion. Formulations for injection may be in unit dose form, for example, in ampoules or multi-dose containers, with added preservatives. These compositions may be in such forms as suspensions, solutions, or emulsions in oily or aqueous media, and may contain formulations such as suspending agents, stabilizers, and / or dispersants.

[0189] Pharmaceutical formulations intended for parenteral administration include aqueous solutions of the active compound in a water-soluble form. Additionally, suspensions of the active pharmaceutical agent can be prepared as suitable oily injectable suspensions. Suitable lipophilic solvents or mediators include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. Aqueous injectable suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that improve the solubility of the compound to allow for the preparation of high-concentration solutions.

[0190] Alternatively, the active ingredient can be in powder form for use in combination with a suitable medium, such as sterile, pyrogen-free water, prior to use.

[0191] In addition to the formulations described above, compounds of formula (I) or pharmaceutically acceptable salts thereof can be formulated into depot preparations. Such long-acting formulations can be administered via implantation (e.g., subcutaneously or intramuscularly) or intramuscular injection. Therefore, for example, these compounds can be formulated with suitable polymers or hydrophobic materials (e.g., as emulsions in acceptable oils) or ion exchange resins, or as slightly soluble derivatives, for example, as slightly soluble salts. The drug carrier for the hydrophobic compound is a cosolvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. The cosolvent system can be a VPD cosolvent system. VPD is a solution of 3% w / v benzyl alcohol, 8% w / v nonpolar surfactant polysorbate 80, and 65% w / v polyethylene glycol 300, diluted to volume with pure ethanol. A VPD cosolvent system (VPD:5W) contains VPD diluted 1:1 with a 5% glucose aqueous solution. This cosolvent system dissolves the hydrophobic compound well and itself produces low toxicity when administered systemically. The proportions of the cosolvent system can be varied appropriately without compromising its solubility and toxicity characteristics. Furthermore, the identity of the cosolvent components can be changed: for example, other low-toxicity nonpolar surfactants can be used instead of polysorbate 80; the fraction size of polyethylene glycol can be varied; other biocompatible polymers can replace polyethylene glycol, such as polyvinylpyrrolidone; and other sugars or polysaccharides can be used instead of glucose.

[0192] Alternatively, other delivery systems for hydrophobic drug compounds can be employed. Liposomes and emulsions are known examples of delivery media or carriers for hydrophobic drugs. Certain organic solvents, such as dimethyl sulfoxide (DMSO), can also be used, although this is generally at the cost of higher toxicity due to the toxic nature of DMSO. Additionally, sustained-release systems can be used to deliver compounds, such as semi-permeable matrices containing solid hydrophobic polymers of therapeutic agents. Various sustained-release materials have been developed and are well known to those skilled in the art. Sustained-release capsules can release compounds for weeks to over 100 days, depending on their chemical properties. Additional strategies for protein stabilization can be employed, depending on the chemical properties and biostability of the therapeutic agent.

[0193] Pharmaceutical compositions may also contain suitable solid-phase or gel-phase carriers or excipients. These carriers and excipients can provide a significant improvement in the bioavailability of poorly soluble drugs. Examples of such carriers or excipients include calcium carbonate, calcium phosphate, sugars, starch, cellulose derivatives, gelatin, and polymers such as polyethylene glycol.

[0194] Furthermore, the pharmaceutical composition can be incorporated into a skin patch for direct delivery of the drug to the skin.

[0195] Additionally, a pharmaceutically acceptable formulation of a compound of formula (I) or a pharmaceutically acceptable salt thereof, which may be used to practice the methods disclosed herein, may contain a compound of formula (I) or a pharmaceutically acceptable salt thereof in an amount from about 0.5 w / w% to about 95 w / w%, or from about 1 w / w% to about 95 w / w%, or from about 1 w / w% to about 75 w / w%, or from about 5 w / w% to about 75 w / w%, or from about 10 w / w% to about 75 w / w%, or from about 10 w / w% to about 50 w / w%.

[0196] It should be understood that the actual dose of the compound of formula (I) or a pharmaceutically acceptable salt thereof to be administered to a subject in need will vary depending on the specific agent used, the specific composition formulated, the mode of administration, and the specific site, host, and disease being treated. Given experimental data on a given compound, using conventional dosing determination tests, those skilled in the art can determine the optimal dose for a given set of conditions. For oral administration, exemplary daily doses typically used will be from about 0.001 to about 1000 mg / kg body weight, wherein courses of treatment are repeated at appropriate intervals. In some embodiments, the method provided herein is used to administer the compound of formula (I) or a pharmaceutically acceptable salt thereof to the subject in an amount between about 0.01 mg / kg daily and about 300 mg / kg daily. In other embodiments, the method disclosed herein is used to administer the compound of formula (I) or a pharmaceutically acceptable salt thereof to the subject in an amount between about 0.1 mg / kg daily and about 100 mg / kg daily. In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in an amount between about 10 mg and about 500 mg daily. In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered daily at an amount between about 100 mg and about 400 mg. In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered daily at an amount between about 150 mg and about 350 mg. In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered daily at an amount between about 150 mg and about 300 mg. In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered daily at an amount between about 160 mg and about 300 mg. In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered daily at an amount between about 160 mg. In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered daily at an amount between about 200 mg. In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered daily at an amount between about 240 mg. In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered daily at an amount between about 280 mg. In some implementations, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered at a dose of about 320 mg per day.

[0197] Some embodiments provide methods disclosed herein in which a compound of formula (I) or a pharmaceutically acceptable salt thereof is administered to a subject in an amount ranging from about 100 mg to about 1000 mg once daily, from about 100 mg to about 900 mg once daily, from about 100 mg to about 850 mg once daily, from about 100 mg to about 800 mg once daily, from about 100 mg to about 750 mg once daily, from about 100 mg to about 700 mg once daily, from about 100 mg to about 650 mg once daily, from about 100 mg to about 600 mg once daily, from about 100 mg to about 550 mg once daily, or from about 100 mg to about 500 mg once daily.

[0198] Some embodiments provide the methods disclosed herein, wherein the dosage is approximately 100 mg once daily, approximately 150 mg once daily, approximately 200 mg once daily, approximately 300 mg once daily, approximately 225 mg once daily, approximately 275 mg once daily, approximately 300 mg once daily, approximately 325 mg once daily, approximately 350 mg once daily, approximately 375 mg once daily, approximately 400 mg once daily, approximately 425 mg once daily, approximately 450 mg once daily, approximately 475 mg once daily, approximately 500 mg once daily, approximately 525 mg once daily, approximately 550 mg once daily, approximately 575 mg once daily, approximately 600 mg once daily, approximately 625 mg once daily, approximately 650 mg once daily, approximately 675 mg once daily, approximately 700 mg once daily, approximately 725 mg once daily, approximately 750 mg once daily, approximately 775 mg once daily, approximately 800 mg once daily, approximately 825 mg once daily, approximately 850 mg once daily, approximately 875 mg once daily. The compound of formula (I) or a pharmaceutically acceptable salt thereof is administered to the subject in an amount of approximately 900 mg once daily, approximately 925 mg once daily, approximately 950 mg once daily, approximately 975 mg once daily, or approximately 1000 mg once daily.

[0199] Some embodiments provide methods disclosed herein in which compound 4 is administered to a subject in amounts ranging from about 100 mg to about 1000 mg once daily, from about 100 mg to about 900 mg once daily, from about 100 mg to about 850 mg once daily, from about 100 mg to about 800 mg once daily, from about 100 mg to about 750 mg once daily, from about 100 mg to about 700 mg once daily, from about 100 mg to about 650 mg once daily, from about 100 mg to about 600 mg once daily, from about 100 mg to about 550 mg once daily, or from about 100 mg to about 500 mg once daily.

[0200] Some embodiments provide the methods disclosed herein, wherein the dosage is approximately 100 mg once daily, approximately 150 mg once daily, approximately 200 mg once daily, approximately 300 mg once daily, approximately 225 mg once daily, approximately 275 mg once daily, approximately 300 mg once daily, approximately 325 mg once daily, approximately 350 mg once daily, approximately 375 mg once daily, approximately 400 mg once daily, approximately 425 mg once daily, approximately 450 mg once daily, approximately 475 mg once daily, approximately 500 mg once daily, approximately 525 mg once daily, approximately 550 mg once daily, approximately 575 mg once daily, approximately 600 mg once daily, approximately 625 mg once daily, approximately 650 mg once daily, approximately 675 mg once daily, approximately 700 mg once daily, approximately 725 mg once daily, approximately 750 mg once daily, approximately 775 mg once daily, approximately 800 mg once daily, approximately 825 mg once daily, approximately 850 mg once daily, approximately 875 mg once daily. Compound 4 was administered to the subject in doses of approximately 900 mg once daily, approximately 925 mg once daily, approximately 950 mg once daily, approximately 975 mg once daily, or approximately 1000 mg once daily.

[0201] In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject at an amount of about 100 mg once daily. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject at an amount of about 200 mg once daily. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject at an amount of about 400 mg once daily. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject at an amount of about 500 mg once daily. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject at an amount of about 600 mg once daily. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject at an amount of about 700 mg once daily. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject at an amount of about 800 mg once daily. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject at an amount of about 900 mg once daily.

[0202] In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 100 nM in the subject's plasma for at least 4 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 100 nM in the subject's plasma for at least 8 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 100 nM in the subject's plasma for at least 12 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 100 nM in the subject's plasma for at least 16 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 100 nM in the subject's plasma for at least 20 hours after administration. Some embodiments provide a method disclosed herein in which compound 4 is administered to the subject in an amount that provides a concentration of compound 4 equal to or greater than 100 nM in the subject's plasma for at least 24 hours after administration.

[0203] In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 200 nM in the subject's plasma for at least 4 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 200 nM in the subject's plasma for at least 8 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 200 nM in the subject's plasma for at least 12 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 200 nM in the subject's plasma for at least 20 hours after administration. Some embodiments provide a method disclosed herein in which compound 4 is administered to the subject in an amount that provides a concentration of compound 4 equal to or greater than 200 nM in the subject's plasma for at least 24 hours after administration.

[0204] In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 250 nM in the subject's plasma for at least 4 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 250 nM in the subject's plasma for at least 8 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 250 nM in the subject's plasma for at least 12 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 250 nM in the subject's plasma for at least 16 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 250 nM in the subject's plasma for at least 20 hours after administration. Some embodiments provide a method disclosed herein in which compound 4 is administered to the subject in an amount that provides a concentration of compound 4 equal to or greater than 250 nM in the subject's plasma for at least 24 hours after administration.

[0205] In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 275 nM in the subject's plasma for at least 4 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 275 nM in the subject's plasma for at least 8 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 275 nM in the subject's plasma for at least 12 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 275 nM in the subject's plasma for at least 16 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 275 nM in the subject's plasma for at least 20 hours after administration. Some embodiments provide a method disclosed herein in which compound 4 is administered to the subject in an amount that provides a concentration of compound 4 equal to or greater than 275 nM in the subject's plasma for at least 24 hours after administration.

[0206] In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 300 nM in the subject's plasma for at least 4 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 300 nM in the subject's plasma for at least 8 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 300 nM in the subject's plasma for at least 12 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 300 nM in the subject's plasma for at least 16 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 300 nM in the subject's plasma for at least 20 hours after administration. Some embodiments provide a method disclosed herein in which compound 4 is administered to the subject in an amount that provides a concentration of compound 4 equal to or greater than 300 nM in the subject's plasma for at least 24 hours after administration.

[0207] In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 400 nM in the subject's plasma for at least 4 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 400 nM in the subject's plasma for at least 8 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 400 nM in the subject's plasma for at least 12 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 400 nM in the subject's plasma for at least 16 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 400 nM in the subject's plasma for at least 20 hours after administration. Some embodiments provide a method disclosed herein in which compound 4 is administered to the subject in an amount that provides a concentration of compound 4 equal to or greater than 400 nM in the subject's plasma for at least 24 hours after administration.

[0208] In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 500 nM in the subject's plasma for at least 4 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 500 nM in the subject's plasma for at least 8 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 500 nM in the subject's plasma for at least 12 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 500 nM in the subject's plasma for at least 16 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 500 nM in the subject's plasma for at least 20 hours after administration. Some embodiments provide a method disclosed herein in which compound 4 is administered to the subject in an amount that provides a concentration of compound 4 equal to or greater than 500 nM in the subject's plasma for at least 24 hours after administration.

[0209] In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 600 nM in the subject's plasma for at least 4 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 600 nM in the subject's plasma for at least 8 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 600 nM in the subject's plasma for at least 12 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 600 nM in the subject's plasma for at least 16 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 600 nM in the subject's plasma for at least 20 hours after administration. Some embodiments provide a method disclosed herein in which compound 4 is administered to the subject in an amount that provides a concentration of compound 4 equal to or greater than 600 nM in the subject's plasma for at least 24 hours after administration.

[0210] In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 625 nM in the subject's plasma for at least 4 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 625 nM in the subject's plasma for at least 8 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 625 nM in the subject's plasma for at least 12 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 625 nM in the subject's plasma for at least 16 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 625 nM in the subject's plasma for at least 20 hours after administration. Some embodiments provide a method disclosed herein in which compound 4 is administered to the subject in an amount that provides a concentration of compound 4 equal to or greater than 625 nM in the subject's plasma for at least 24 hours after administration.

[0211] In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 650 nM in the subject's plasma for at least 4 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 650 nM in the subject's plasma for at least 8 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 650 nM in the subject's plasma for at least 12 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 650 nM in the subject's plasma for at least 16 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 650 nM in the subject's plasma for at least 20 hours after administration. Some embodiments provide the method disclosed herein, wherein compound 4 is administered to the subject in an amount that provides a concentration of compound 4 equal to or greater than 650 nM in the subject's plasma for at least 24 hours after administration.

[0212] In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 675 nM in the subject's plasma for at least 4 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 675 nM in the subject's plasma for at least 8 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 675 nM in the subject's plasma for at least 12 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 675 nM in the subject's plasma for at least 16 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 675 nM in the subject's plasma for at least 20 hours after administration. Some embodiments provide a method disclosed herein in which compound 4 is administered to the subject in an amount that provides a concentration of compound 4 equal to or greater than 675 nM in the subject's plasma for at least 24 hours after administration.

[0213] In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 700 nM in the subject's plasma for at least 4 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 700 nM in the subject's plasma for at least 8 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 700 nM in the subject's plasma for at least 12 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 700 nM in the subject's plasma for at least 16 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 700 nM in the subject's plasma for at least 20 hours after administration. Some embodiments provide a method disclosed herein in which compound 4 is administered to the subject in an amount that provides a concentration of compound 4 equal to or greater than 700 nM in the subject's plasma for at least 24 hours after administration.

[0214] In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 725 nM in the subject's plasma for at least 4 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 725 nM in the subject's plasma for at least 8 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 725 nM in the subject's plasma for at least 12 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 725 nM in the subject's plasma for at least 16 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 725 nM in the subject's plasma for at least 20 hours after administration. Some embodiments provide the method disclosed herein, wherein compound 4 is administered to the subject in an amount that provides a concentration of compound 4 equal to or greater than 725 nM in the subject's plasma for at least 24 hours after administration.

[0215] In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 750 nM in the subject's plasma for at least 4 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 750 nM in the subject's plasma for at least 8 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 750 nM in the subject's plasma for at least 12 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 750 nM in the subject's plasma for at least 16 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 750 nM in the subject's plasma for at least 20 hours after administration. Some embodiments provide a method disclosed herein in which compound 4 is administered to the subject in an amount that provides a concentration of compound 4 equal to or greater than 750 nM in the subject's plasma for at least 24 hours after administration.

[0216] In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 775 nM in the subject's plasma for at least 4 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 775 nM in the subject's plasma for at least 8 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 775 nM in the subject's plasma for at least 12 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 775 nM in the subject's plasma for at least 16 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 775 nM in the subject's plasma for at least 20 hours after administration. Some embodiments provide the method disclosed herein, wherein compound 4 is administered to the subject in an amount that provides a concentration of compound 4 equal to or greater than 775 nM in the subject's plasma for at least 24 hours after administration.

[0217] In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 800 nM in the subject's plasma for at least 4 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 800 nM in the subject's plasma for at least 8 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 800 nM in the subject's plasma for at least 12 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 800 nM in the subject's plasma for at least 16 hours after administration. In some embodiments, the method disclosed herein is provided, wherein compound 4 is administered to a subject in an amount that provides a concentration of compound 4 equal to or greater than 800 nM in the subject's plasma for at least 20 hours after administration. Some embodiments provide a method disclosed herein in which compound 4 is administered to the subject in an amount that provides a concentration of compound 4 equal to or greater than 800 nM in the subject's plasma for at least 24 hours after administration.

[0218] Furthermore, a pharmaceutically acceptable formulation of a compound of formula (I) or a pharmaceutically acceptable salt thereof that can be used to practice the methods disclosed herein may contain a compound of formula (I) or a pharmaceutically acceptable salt thereof in an amount of about 10 mg to about 2000 mg, or about 10 mg to about 1500 mg, or about 10 mg to about 1000 mg, or about 10 mg to about 750 mg, or about 10 mg to about 500 mg, or about 25 mg to about 500 mg, or about 50 mg to about 500 mg, or about 100 mg to about 500 mg.

[0219] In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered to the subject of need once daily. In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered to the subject of need twice daily. In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered to the subject of need three times daily.

[0220] In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered to the subject of need in 28-day cycles. In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered to the subject of need in multiple 28-day cycles. In some embodiments, the administration of the compound of formula (I) or a pharmaceutically acceptable salt thereof to the subject of need is sustained for at least one 28-day cycle. In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered to the subject of need daily within each 28-day cycle.

[0221] In some cases, the methods described herein involve combining compositions and formulations comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof with one or more additional therapeutic agents to administer to a subject or subject in need multiple cycles repeated on a regular schedule with rest periods between each cycle. For example, in some cases, a treatment cycle consists of one week of treatment followed by a three-week rest period. The length of a treatment cycle depends on the treatment administered. In some embodiments, the length of a treatment cycle ranges from two to six weeks. In some embodiments, the length of a treatment cycle ranges from three to six weeks. In some embodiments, the length of a treatment cycle ranges from three to four weeks. In some embodiments, the length of a treatment cycle is three weeks (or 21 days). In some embodiments, the length of a treatment cycle is four weeks (28 days). In some embodiments, the length of a treatment cycle is 56 days. In some embodiments, a treatment cycle lasts one, two, three, or four weeks. In some embodiments, a treatment cycle lasts three weeks. In some embodiments, a treatment cycle lasts four weeks. The number of treatment doses scheduled within each cycle also varies depending on the medication administered.

[0222] The dosage of the compositions described herein can be determined by any suitable method. When administered to a subject, the maximum tolerated dose (MTD) and maximum response dose (MRD) of the compound of formula (I) or a pharmaceutically acceptable salt thereof, along with any additional therapeutic agent, can be determined via established animal and human experimental procedures and in the examples described herein. For example, the toxicity and therapeutic efficacy of the compound of formula (I) or a pharmaceutically acceptable salt thereof, along with any additional therapeutic agent, can be determined by standard pharmaceutical procedures in cell culture or laboratory animals, including but not limited to those used to determine LD50. 50 (The dose that is lethal to 50% of the population) and ED 50 (The dose effective for 50% of the population). The dose ratio between toxicity and therapeutic effect is the therapeutic index, and it can be expressed as LD50. 50 With ED 50 The ratio between these values. Data obtained from cell culture assays and animal studies can be used to formulate dosage ranges for human use. The dosage of such compounds is preferably within the range containing the lowest ED (extra toxicity). 50Within the periodic concentration range. Dosage can vary within this range depending on the dosage form and route of administration used. Additional relative doses (expressed as a percentage of the maximum response or maximum tolerated dose) can be readily obtained via the protocol.

[0223] In some implementations, the amount of the compound of formula (I) or its pharmaceutically acceptable salt, and / or the pharmaceutical formulation containing such amounts, varies depending on factors such as the specific salt or form, the disease condition and its severity, the identity of the person or host requiring treatment (e.g., age, weight, sex), but may still be determined based on the specific circumstances surrounding the case, such as the specific pharmaceutical agent administered, the type of liquid formulation, the condition being treated, and the person or host being treated.

[0224] Methods for detecting biomarkers ARSIs, administered in combination with EED inhibitors to subjects with prostate cancer, can be used in amounts known to those skilled in the art regarding these agents. For example, abiraterone acetate, apalutamide, dalotamide, and enzalutamide can be administered to subjects according to the methods disclosed herein, in amounts and conditions approved by multiple regulatory agencies (e.g., the United States Food and Drug Administration) for the treatment of various types of prostate cancer. In one embodiment, abiraterone acetate can be administered to a subject with metastatic castration-resistant prostate cancer at a dose of 1000 mg orally once daily and prednisone at a dose of 5 mg orally twice daily, according to the methods disclosed herein. In another embodiment, abiraterone acetate can be administered to a subject with metastatic castration-sensitive prostate cancer at a dose of 1000 mg orally once daily and prednisone at a dose of 5 mg orally once daily, according to the methods disclosed herein. In another embodiment, apalutamide may be administered orally at a dose of 240 mg once daily, with or without food, according to the methods disclosed herein, to subjects with metastatic castration-sensitive prostate cancer or non-metastatic castration-resistant prostate cancer. In another embodiment, darostamide may be administered orally at a dose of 600 mg twice daily, with food, according to the methods disclosed herein, to subjects with non-metastatic castration-resistant prostate cancer or metastatic hormone-sensitive prostate cancer (in combination with docetaxel), and for subjects with hormone-sensitive prostate cancer treated in combination with docetaxel, the first cycle of docetaxel is administered to the subject within 6 weeks of the start of darostamide treatment. In another embodiment, enzalutamide may be administered orally at a dose of 160 mg once daily, according to the methods disclosed herein, to subjects with castration-resistant prostate cancer or metastatic castration-sensitive prostate cancer.

[0225] Some embodiments provide methods disclosed herein in which the measurement of one or more biomarkers is used to determine whether a subject will benefit from administration of a compound of formula (I) or a pharmaceutically acceptable salt thereof, or whether a subject responds to administration of a compound of formula (I) or a pharmaceutically acceptable salt thereof. Biomarkers that can be used according to the methods disclosed herein are those known to those skilled in the art, including, but not limited to, those disclosed in the fifth edition of World Health Organization Classification of Haematolymphoid Tumours: Lymphoid Neoplasms, Leukemia, July 2022, Vol. 36, No. 7, pp. 1720-1748.

[0226] This document discloses a method comprising: (a) providing a biological sample obtained from a subject with prostate cancer; (b) determining the presence or absence of a biomarker in the biological sample obtained from the subject; (c) detecting the presence or absence of the biomarker in the biological sample using the method described herein; and (d) if the biomarker is present in the biological sample, administering to the subject a therapeutically effective amount of a compound of an androgen receptor inhibitor and an embryonic ectodermal development (EED) inhibitor. In some embodiments, the presence of one or more of the biomarkers described herein indicates that the prostate cancer in the subject is resistant to one or more prior therapies, such as the cancer being resistant to abiraterone or one or more androgen receptor inhibitors, such as the prostate cancer being resistant to apalutamide and / or dalotamide and / or enzalutamide.

[0227] The presence, absence, or level of such biomarkers can be measured collectively or individually in biological samples obtained from the subject, such as solid tumor samples (e.g., prostate cancer samples), or from relevant biological fluid samples (e.g., blood samples). In some cases, one or more biomarkers are detected in plasma or serum derived from blood samples obtained from the subject. In some cases, the detection methods disclosed herein are used to predict treatment response to the therapies described herein (e.g., administration of androgen receptor inhibitors and embryonic ectodermal development (EED) inhibitors to the subject), to monitor proliferative diseases or conditions in subjects treated with the therapy, and in subjects described herein treated with the therapy.

[0228] In some embodiments, the expression of biomarkers in biological samples from subjects is measured using immunohistochemistry (IHC) assays. Such immunohistochemical (IHC) assays are commercially available or can be developed and utilized according to methods known to those skilled in the art.

[0229] Immunohistochemistry techniques typically utilize antibodies to detect and visualize cellular antigens in situ using colorimetric or fluorescent methods. In such techniques, expression is detected using antibodies or antisera, polyclonal antisera, or monoclonal antibodies specific to each marker. Antibodies can be detected by directly labeling the antibody itself, for example, using radiolabeled markers, fluorescent markers, hapten markers such as biotin, or enzymes such as horseradish peroxidase or alkaline phosphatase. Alternatively, unlabeled primary antibodies can be used in combination with labeled secondary antibodies, including antisera, polyclonal antisera, or monoclonal antibodies specific to the primary antibody. Immunohistochemical protocols and kits are well known in the art and are commercially available.

[0230] Two common methods of IHC are generally available: direct and indirect assays. In a first assay, the binding of an antibody to the target antigen is directly determined. This direct assay uses labeled reagents, such as fluorescent tags or enzyme-labeled primary antibodies, which can be visualized without further antibody-antibody interactions. In a typical indirect assay, an unconjugated primary antibody binds to the antigen, and then a labeled secondary antibody binds to the primary antibody. In the case of enzyme-labeled secondary antibodies, a chromogenic or fluorescent substrate is added to provide visualization of the antigen. Signal amplification occurs because several secondary antibodies can react with different epitopes on the primary antibody. Primary and / or secondary antibodies used in immunohistochemistry are typically labeled with detectable portions. A variety of labels are available, which are generally categorized as follows. First, there are radioactive isotopes, such as… 35 S, 14 C 125 I, 3 H and 131 I. Antibodies can be labeled with radioisotopes using techniques described in Current Protocols in Immunology, Volumes 1 & 2, Coligen et al., eds., Wiley-Interscience, New York, NY, Pubs. (1991), and the radioactivity can be measured using scintillation counting. Next, colloidal gold particles. Third, fluorescent labeling, including but not limited to rare earth chelates (europium chelates), Texas red, rhodamine, fluorescein, dansyl sulfonyl, lissamine, umbelliferone, phycocrytherin, phycocyanin, or commercially available fluorophores such as SPECTRUM ORANGE. and SPECTRUM GREEN And / or one or more derivatives thereof. For example, fluorescent labels can be conjugated to antibodies using techniques disclosed in Current Protocols in Immunology (ibid.). Fluorescence can be quantified using a fluorometer. Fourth, a variety of enzyme-substrate labels are available, and U.S. Patent No. 4,275,149 provides a review of some of these. Enzymes typically catalyze chemical changes in chromogenic substrates, which can be measured using a variety of techniques. For example, an enzyme can catalyze a color change in a substrate, which can be measured spectrophotometrically. Alternatively, an enzyme can alter the fluorescence or chemiluminescence of a substrate. Techniques for quantifying changes in fluorescence have been described above. Chemiluminescent substrates become electronically excited through a chemical reaction and can then emit light, which can be measured (e.g., using a chemiluminometer) or contribute energy to a fluorescent acceptor. Examples of enzyme-labeled enzymes include luciferases (e.g., firefly luciferase and bacterial luciferase; U.S. Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, malate dehydrogenase, urease, peroxidases such as horseradish peroxidase (HRPO), alkaline phosphatase, β-galactosidase, glucosylamylase, lysozyme, sugar oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, etc. Techniques for conjugating enzymes to antibodies are described in O'Sullivan et al., Methods for the preparation of Enzyme-Antibody conjugates for use in Enzyme Immunoassay, in Methods in Enzym. (edited by J. Langone & H. Van Vunakis), Academic press, New York, 73:147-166 (1981). Examples of enzyme-substrate combinations include, for example: (i) horseradish peroxidase (HRPO) with catalase as a substrate, wherein catalase oxidizes a dye precursor [e.g., o-phenylenediamine (OPD) or 3,3',5,5'-tetramethylbenzidine hydrochloride (TMB)]. 3,3-diaminobenzidine (DAB) can also be used to visualize HRP-labeled antibodies; (ii) alkaline phosphatase (AP) with p-nitrophenyl phosphate as a chromogenic substrate; and (iii) β-D-galactosidase (β-D-Gal) having a chromogenic substrate (e.g., p-nitrophenyl-β-D-galactosidase) or a fluorescent substrate (e.g., 4-methylumbelliferyl-β-D-galactosidase). Many other enzyme-substrate combinations are available to those skilled in the art. These methods are generally described in U.S. Patent Nos. 4,275,149 and 4,318,980. Sometimes, the label is indirectly conjugated to the antibody.Those skilled in the art will understand the various techniques for achieving this. For example, antibodies can be conjugated with biotin, and any of the four classes of markers mentioned above can be conjugated with avidin, or vice versa. Biotin binds selectively to avidin, and therefore, the marker can be conjugated with an antibody in this indirect manner. Alternatively, to achieve indirect conjugation of the marker with the antibody, the antibody can be conjugated with a small hapten, and one of the different types of markers mentioned above can be conjugated with an anti-hapten antibody. Thus, indirect conjugation of the marker with the antibody can be achieved.

[0231] Biological samples (including tissue samples) obtained from an object can be prepared according to procedures commonly used in the art. Typically, paraffin-embedded sections of cells or tissues are obtained by: (1) preserving the tissue in a fixative; (2) dehydrating the fixed tissue; (3) infiltrating the tissue with a fixative; (4) orienting the tissue so that the cut surface accurately represents the tissue; (5) embedding the tissue in paraffin (forming a paraffin block); (6) cutting the paraffin block of tissue into 4-5 picometer sections using a microtome; and (7) mounting the sections onto a glass slide. Pathologists, etc., can then read the slides to assess the presence or absence of biomarkers, the presence or absence of abnormal or normal cells, or the presence or absence of a specific cell type, and to determine the location of the cell type of interest. Thus, for example, pathologists, etc., will examine the slides and identify normal and abnormal cells (such as aberrant or tumor cells). Any method for defining the location of the cell of interest (e.g., coordinates on the XY axis) can be used.

[0232] In addition to the sample preparation procedures discussed above, further processing of tissue sections may be required before, during, or after IHC. For example, epitope retrieval methods, such as heating the tissue sample in a citrate buffer, can be performed [see, e.g., Leong et al. Appl. Immunohistochem. 4(3):201(1996)]. Following an optional blocking step, the tissue sections are exposed to the primary antibody for a sufficient period of time, and under suitable conditions, the primary antibody binds to the target protein antigen in the tissue sample. Suitable conditions for achieving this can be determined through routine experiments.

[0233] The degree of antibody binding to the sample is determined using any of the detectable markers discussed above. For example, the marker is an enzyme marker (e.g., HRPO), which catalyzes a chemical change in a chromogenic substrate such as 3,3'-diaminobenzidine chromogen. Preferably, the enzyme marker is conjugated to an antibody that specifically binds to the primary antibody (e.g., the primary antibody is a rabbit polyclonal antibody, and the secondary antibody is a goat anti-rabbit antibody). The sample thus prepared can be mounted and covered with a coverslip. The slide is then evaluated, for example, using a microscope.

[0234] IHC can be combined with morphological staining before or after treatment. After dewaxing, sections mounted on a slide can be stained with morphological stains for evaluation. The morphological stains used provide an accurate morphological assessment of the tissue sections. Sections can be stained with one or more dyes, each staining different cellular components. In one embodiment, hematoxylin is used to stain cell nuclei in the slide. Hematoxylin is widely available. A suitable hematoxylin is hematoxylin II (Ventana). When a lighter blue color is required for cell nuclei, a bluing agent can be used after hematoxylin staining. Those skilled in the art will understand that staining can be optimized for a given tissue by increasing or decreasing the length of time the slide is retained in the dye.

[0235] Automated systems for slide preparation and IHC processing are commercially available. Ventana The BenchMark XT system is an example of such an automated system.

[0236] After staining, tissue sections can be analyzed using standard microscopy techniques. Typically, pathologists assess tissue for the presence of abnormal cells, normal cells, or specific cell types, and identify the location of the cell type of interest. Thus, for example, pathologists will examine slides and identify normal and abnormal cells (such as aberrant or tumor cells). Any method can be used to determine the location of the cells of interest (e.g., coordinates on the X and Y axes).

[0237] In some embodiments, the presence, absence, and / or expression level of biomarkers in samples obtained from a subject are detected by analyzing genetic material in the sample. In some embodiments, the genetic material is obtained from blood, serum, plasma, sweat, hair, tears, urine, and other techniques known to those skilled in the art. In some embodiments, the sample contains circulating tumor RNA (ctRNA). In some embodiments, the sample contains peripheral blood mononuclear cells (PBMCs). In some cases, the sample contains circulating tumor cells (CTCs). In some cases, the genetic material is obtained from tumor biopsy or liquid biopsy. In some embodiments, tumor biopsy includes formalin-fixed paraffin-embedded biopsy, fresh-frozen biopsy, fresh biopsy, or frozen biopsy. In some embodiments, liquid biopsy includes PBMCs, circulating tumor RNA, cell-free plasma RNA, or circulating tumor cells (CTCs). Tumor and liquid biopsies may undergo additional analytical processing for sample dissociation, cell sorting, and enrichment of cell populations of interest.

[0238] In some implementations, methods for detecting the presence, absence, or level of biomarkers in biological samples obtained from a subject involve detecting nucleic acid sequences. In some cases, the nucleic acid sequence comprises deoxyribonucleic acid (DNA), such as in the case of detecting complementary DNA (cDNA) of mRNA transcripts. In some cases, the nucleic acid sequence comprises denatured DNA molecules or fragments thereof. In some cases, the nucleic acid sequence comprises DNA selected from genomic DNA, viral DNA, mitochondrial DNA, plasmid DNA, amplified DNA, circular DNA, circulating DNA, cell-free DNA, or foreign body DNA. In some cases, the DNA is single-stranded DNA (ssDNA), double-stranded DNA, denatured double-stranded DNA, synthetic DNA, and combinations thereof. Circular DNA may be cleaved or fragmented. In some cases, the nucleic acid sequence includes ribonucleic acid (RNA). In some cases, the nucleic acid sequence comprises fragmented RNA. In some cases, the nucleic acid sequence comprises partially degraded RNA. In some cases, the nucleic acid sequence comprises microRNA or portions thereof. In some cases, nucleic acid sequences contain RNA molecules or fragmented RNA molecules (RNA fragments) selected from the following: microRNA (miRNA), premiRNA, primary miRNA, mRNA, premRNA, viral RNA, viroid RNA, viral RNA, circular RNA (circRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), pretRNA, long noncoding RNA (lncRNA), small nuclear RNA (snRNA), circulating RNA, cell-free RNA, exogenous RNA, vector-expressed RNA, RNA transcripts, synthetic RNA, and combinations thereof.

[0239] In some embodiments disclosed herein, biomarkers are detected by performing nucleic acid-based assays on samples obtained from an object. In some cases, nucleic acid-based assays include quantitative polymerase chain reaction (qPCR), reverse transcription PCT (RT-qPCR), gel electrophoresis (including, for example, Northern or Southern blotting), immunohistochemistry (IHC), immunofluorescence (IF), in situ hybridization (ISH) (such as fluorescence in situ hybridization (FISH)), cytochemistry, microarrays, or sequencing. In some embodiments, the sequencing technology includes next-generation sequencing. In some embodiments, the method involves hybridization assays, such as fluorescent qPCR (e.g., TaqMan). TMSYBR Green, SYBR Green I, SYBR Green II, SYBR Gold, ethidium bromide, methylene blue, Pyronin Y, DAPI, acridine orange, Blue View, or phycoerythrin) are used in qPCR assays involving nucleic acid amplification reactions with specific primer pairs and hybridization of nucleic acid probes containing amplified detectable portions or molecules specific to the target nucleic acid sequence. In some cases, the number of amplification cycles used to detect the target nucleic acid in qPCR assays is approximately 5 to approximately 30 cycles. In some cases, the number of amplification cycles used to detect the target nucleic acid is at least approximately 5 cycles. In some cases, the number of amplification cycles used to detect the target nucleic acid is at most approximately 30 cycles. In some cases, the number of amplification cycles used to detect the target nucleic acid is approximately 5 to 10, approximately 5 to 15, approximately 5 to 20, approximately 5 to 25, approximately 5 to 30, approximately 10 to 15, approximately 10 to 20, approximately 10 to 25, approximately 10 to 30, approximately 15 to 20, approximately 15 to 25, approximately 15 to 30, approximately 20 to 25, approximately 20 to 30, or approximately 25 to 30 cycles. For TaqMan... TM The method involves using a hydrolyzable probe containing a fluorophore and a quencher, which is hydrolyzed by DNA polymerase upon hybridization with the target nucleic acid. In some cases, the presence of the target nucleic acid is determined when the number of amplification cycles required to reach the threshold is less than 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, or 20 cycles. In some cases, hybridization can occur at standard hybridization temperatures, such as between approximately 35°C and approximately 65°C in standard PCR buffer.

[0240] Additional exemplary nucleic acid-based assays include the use of nucleic acid probes conjugated or otherwise immobilized on beads, multi-well plates, or other substrates, wherein the nucleic acid probes are configured to hybridize with a target nucleic acid sequence. In some cases, the nucleic acid probes are specific for one or more polynucleotide sequences encoding the relevant biomarkers disclosed herein. In some cases, biomarker-specific nucleic acid probes comprise a nucleic acid probe sequence that is sufficiently complementary to the polynucleotide sequence encoding the relevant biomarker protein. In some cases, the probe comprises a transcribed polynucleotide sequence (e.g., RNA, cDNA). In some embodiments, the nucleic acid probe may be, for example, full-length cDNA or a portion thereof, such as an oligonucleotide of at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides in length and sufficient to specifically hybridize with the target nucleic acid sequence under standard hybridization conditions. In some embodiments, the target nucleic acid sequence is immobilized on a solid surface and in contact with the probe, such as by running the isolated target nucleic acid sequence on an agarose gel and transferring the target nucleic acid sequence from the gel to a membrane, such as nitrocellulose. In some implementations, the probe is immobilized on a solid surface, such as in an Affymetrix gene chip array, and the probe is in contact with the target nucleic acid sequence.

[0241] In some implementations, the term "probe" in relation to nucleic acids refers to any nucleic acid molecule capable of selectively binding to a specific intended target nucleic acid sequence. In some cases, probes are specifically designed to be labeled with, for example, radiolabels, fluorescent labels, enzymes, chemiluminescent tags, colorimetric tags, or other labels or tags known in the art. In some cases, the fluorescent label includes a fluorophore. In some cases, the fluorophore is an aromatic or heteroaromatic compound. In some cases, the fluorophore is pyrene, anthracene, naphthalene, acridine, zirconia, benzoxazole, indole, benzoindole, oxazole, thiazole, benzothiazole, canine, carbocyanine, salicylates, anthranilates, xanthones, or coumarins. Exemplary xanthones include, for example, fluorescein and rhodamine dyes. Fluorescein and rhodamine dyes include, but are not limited to, 6-carboxyfluorescein (FAM), 2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), tetrachlorofluorescein (TET), 6-carboxyrhodamine (R6G), N,N,N,N'-tetramethyl-6-carboxyrhodamine (TAMRA), and 6-carboxy-X-rhodamine (ROX). Suitable fluorescent probes also include naphthylamine dyes having an amino group at the α or β position. For example, naphthylamine compounds include 1-dimethylaminonaphthyl-5-sulfonate / salt, 1-anilino-8-naphthylsulfonate / salt, and 2-p-toluidine-6-naphthylsulfonate / salt, and 5-(2'-aminoethyl)aminonaphthyl-1-sulfonic acid (EDANS). Exemplary coumarins include, for example, 3-phenyl-7-isocyanate coumarin; acridines, such as 9-isothiocyanate coumarin and acridine orange; N-(p-(2-benzoxazolyl)phenyl)maleimide; cyanines, such as, for example, indole-dicarbocyanine 3 (Cy3), indole-dicarbocyanine 5 (Cy5), indole-dicarbocyanine 5.5 (Cy5.5), 3-(-carboxy-pentyl)-3'-ethyl-5,5'-dimethyloxacarbocyanine (CyA); 1H,5H,11H,15H-xanthondo[2,3,4-ij: 5,6,7-i'j']diquinazine-18-onium, 9-[2(or 4)-[[6-[2,5-dioxo-1-pyrrolyl]oxy]-6-oxohexyl]amino]sulfonyl]-4(or 2)-sulfonylphenyl]-2,3,6,7,12,13,16,17-octahydro-internal salt (TR or Texas Red); or BODIPY™ dye. In some cases, the probe contains FAM as a dye marker.

[0242] In some implementations, detecting one or more biomarkers involves sequencing genetic material obtained from a sample of the subject. Sequencing can be performed using any suitable sequencing technology, including but not limited to single-molecule real-time (SMRT) sequencing, Polony sequencing, ligation sequencing, reversible terminator sequencing, proton detection sequencing, ion semiconductor sequencing, nanopore sequencing, electron sequencing, pyrosequencing, Maxam-Gilbert sequencing, chain termination (such as Sanger) sequencing, +S sequencing, or synthetic sequencing. Sequencing methods also include next-generation sequencing, such as modern sequencing technologies, such as Illumina sequencing (such as Solexa), Roche 454 sequencing, Ion Torrent sequencing, and SOLiD sequencing. In some cases, next-generation sequencing involves high-throughput sequencing methods. Additional sequencing methods available to those skilled in the art may also be used.

[0243] In some cases, the number of nucleotides sequenced is at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 300, 400, 500, 2000, 4000, 6000, 8000, 10000, 20000, 50000, 100000, or more than 100000 nucleotides. In some cases, the number of nucleotides sequenced ranges from approximately 1 to 100,000 nucleotides, approximately 1 to 10,000 nucleotides, approximately 1 to 1,000 nucleotides, approximately 1 to 500 nucleotides, approximately 1 to 300 nucleotides, approximately 1 to 200 nucleotides, approximately 1 to 100 nucleotides, approximately 5 to 100,000 nucleotides, approximately 5 to 10,000 nucleotides, approximately 5 to 1,000 nucleotides, approximately 5 to 500 nucleotides, approximately 5 to 300 nucleotides, approximately 5 to 200 nucleotides, approximately 5 to 100 nucleotides, approximately 10 to 100,000 nucleotides, approximately 10 to 10,000 nucleotides, approximately 10 to 1,000 nucleotides, approximately 10 to 500 nucleotides, approximately 10 to 300 nucleotides, approximately 10 to 200 nucleotides, approximately 10 to 100 nucleotides. The range of approximately 20 to approximately 100,000 nucleotides, approximately 20 to approximately 10,000 nucleotides, approximately 20 to approximately 1,000 nucleotides, approximately 20 to approximately 500 nucleotides, approximately 20 to approximately 300 nucleotides, approximately 20 to approximately 200 nucleotides, approximately 20 to approximately 100 nucleotides, approximately 30 to approximately 100,000 nucleotides, approximately 30 to approximately 1,000 nucleotides, approximately 30 to approximately 1,000 nucleotides, approximately 30 to approximately 500 nucleotides, approximately 30 to approximately 300 nucleotides, approximately 30 to approximately 200 nucleotides, approximately 30 to approximately 100 nucleotides, approximately 50 to approximately 100,000 nucleotides, approximately 50 to approximately 10,000 nucleotides, approximately 50 to approximately 1,000 nucleotides, approximately 50 to approximately 500 nucleotides, approximately 50 to approximately 300 nucleotides, approximately 50 to approximately 200 nucleotides, or approximately 50 to approximately 100 nucleotides.

[0244] In some cases, hybridization assays (such as those described herein) are used to detect mRNA encoding biomarkers in a sample. Exemplary probe sequences that can hybridize with a target nucleic acid sequence contain at least 10 but no more than 100 consecutive nucleotides containing the relevant sequence. In some cases, RNA sequencing (RNA-seq) is used to detect mRNA encoding proteins encoding the relevant biomarker.

[0245] In some cases, mRNA detection involves amplifying the nucleic acid of the target protein via polymerase chain reaction (PCR). In some embodiments, the PCR assay involves using a pair of primers capable of amplifying at least about 10 consecutive nucleobases in a nucleic acid sequence, thereby amplifying one or more gene products of a biomarker. In quantitative real-time PCR, quantification is based on the amount of fluorescence signal (TaqMan and SYBR green). In some embodiments, the nucleic acid probe is conjugated to a detectable molecule. The detectable molecule may be a fluorophore. The nucleic acid probe may also be conjugated to a quencher.

[0246] In some embodiments, the assay for detecting the presence or absence of mRNA encoding the relevant biomarker includes reverse transcription of the relevant mRNA molecule to produce a corresponding complementary DNA (cDNA) molecule. In some embodiments, the assay further includes contacting the cDNA molecule with a nucleic acid probe containing a nucleic acid sequence complementary to the nucleic acid sequence of the cDNA molecule. In some embodiments, the assay includes detecting a double-stranded hybridization product between the nucleic acid probe and the cDNA molecule. In some embodiments, a pair of primers is used to further amplify the hybridization product. In some embodiments, the primers comprise a first primer having a nucleic acid sequence containing at least 10 but no more than 50 consecutive nucleic acids within a relevant nucleic acid sequence that binds to the top strand of the double-stranded hybridization product; and a second primer having a nucleic acid sequence containing at least 10 but no more than 50 consecutive nucleic acids within a nucleic acid sequence that is reverse complementary to the relevant nucleic acid sequence that binds to the bottom strand of the double-stranded hybridization product.

[0247] In some embodiments, methods for preparing complementary DNA (cDNA) libraries are disclosed herein. In some embodiments, the cDNA library is sequenced using suitable sequencing methods disclosed herein. In some embodiments, the cDNA library is labeled to generate multiple nucleic acid probes and immobilized onto a fixation surface (such as a microarray). In some embodiments, the multiple nucleic acid probes are capable of hybridizing with at least about 10 consecutive nucleotides of two or more genes in a sample obtained from a subject. In some embodiments, detecting the presence or absence of a biomarker includes detecting high or low expression levels of one or more genes compared to reference levels.

[0248] In some embodiments, this document discloses the extraction of genetic material from biological samples (e.g., blood, serum, or tissue samples) obtained from a subject. In some embodiments of nucleic acid extraction, any technique that does not interfere with subsequent analysis is used to extract the nucleic acids. In some embodiments, the technique uses alcohol precipitation utilizing ethanol, methanol, or isopropanol. In some embodiments, the technique uses phenol, chloroform, or any combination thereof. In some embodiments, the technique uses cesium chloride. In some embodiments, the technique uses sodium acetate, potassium acetate, or ammonium acetate, or any other salt commonly used to precipitate DNA. In some embodiments, the technique utilizes column- or resin-based nucleic acid purification protocols, such as those commonly commercially available; a non-limiting example is the GenElute Bacterial Genomic DNA Kit available from Sigma Aldrich. In some embodiments, after extraction and before subsequent analysis, the nucleic acids are stored in water, Tris buffer, or Tris-EDTA buffer. In one exemplary embodiment, the nucleic acid material is extracted in water. In some cases, extraction does not include nucleic acid purification. In some implementations, RNA can be extracted from cells using RNA extraction techniques, including techniques such as using acidic phenol / guanidine isothiocyanate extraction (RNAzol B; Biogenesis), RNeasy RNA preparation kit (Qiagen), or PAXgene (PreAnalytix, Switzerland).

[0249] In some respects, circulating tumor RNA (ctRNA) is used to assess the expression levels of RNA molecules released from tumors into the bloodstream. In some implementations, ctRNA detection is useful, for example, for detecting and diagnosing tumors. Because tumor DNA and RNA have acquired multiple genetic mutations leading to tumor development, ctRNAs do not perfectly match an individual's DNA and RNA, respectively. Identifying genetically differentiated DNA and RNA aids in tumor detection. Using ctRNA to diagnose the type of tumor can reduce the need for obtaining samples of tumor tissue (tumor biopsies, which can be challenging when tumors are difficult to access, such as those in the brain or lungs).

[0250] In some embodiments, a reduction in the amount of ctRNA indicates that the solid tumor is shrinking and that treatment with the compound of formula (I) or a pharmaceutically acceptable salt thereof is effective. In some embodiments, a lack of ctRNA in the bloodstream indicates that the cancer has not recurred after treatment with the compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0251] This article describes a method for assessing genetic alterations using ctRNA profiling. In some embodiments, genomic profiling is performed after each treatment cycle with the compound of formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, the genetic alteration indicates that the cancer has become resistant to treatment with the compound of formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, the absence of the genetic alteration indicates that the cancer has not become resistant to treatment with the compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0252] This article describes a method for assessing genetic alterations (including, but not limited to, mutations in certain genes and / or copy number alterations in certain genes) through circulating tumor DNA (ctDNA) and / or cell-free DNA (cfDNA) mapping. In some embodiments, genomic mapping is performed after each treatment cycle with the compound of formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, the genetic alteration indicates that the cancer has become resistant to treatment with the compound of formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, the absence of the genetic alteration indicates that the cancer has not become resistant to treatment with the compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0253] In some embodiments, biomarker expression is measured by immunofluorescence (IF) assay. In some embodiments, biomarker expression is measured by in situ hybridization (ISH) assay. In some embodiments, biomarker transcript expression is measured using assays such as quantitative polymerase chain reaction (qPCR), microarrays, and RNA sequencing, or commercially available assays from companies such as Fluidigm and Nanostring.

[0254] This document discloses a method for treating a subject with prostate cancer, comprising: (a) providing a biological sample obtained from the subject with prostate cancer; (b) determining to detect the presence or absence of a biomarker in the biological sample obtained from the subject; (c) detecting the presence or absence of the biomarker in the biological sample using the method described herein; and (d) if the biomarker is present in the biological sample, administering to the subject a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, the expression of the biomarker is based on the deviation of the biomarker's expression level from a reference expression level. In some embodiments, the expression level is high relative to a reference expression level. In some embodiments, the expression level is low relative to a reference expression level. In some embodiments, the reference expression level is derived from an individual or group of individuals without cancer. In some embodiments, the reference expression level is derived from an individual or group of individuals with cancer that has no therapeutic response to a compound of formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, the expression level deviates from the reference expression level by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0255] In some embodiments, the determination of biomarker expression or presence is based on the percentage of cells that stain weakly, moderately, or strongly for the relevant biomarker, where a threshold defines the minimum percentage of cells required for staining positivity at various intensity levels (≥a% of tumor cells staining weakly, ≥b% of tumor cells staining moderately, ≥c% of tumor cells staining strongly, or a combination thereof). In some embodiments, the determination is made when ≥10%, ≥15%, ≥20%, ≥25%, ≥30%, ≥35%, ≥40%, ≥45%, ≥50%, ≥55%, ≥60%, ≥65%, ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, or ≥95% of tumor cells stain weakly for the biomarker; or when ≥10%, ≥15%, ≥20%, ≥25%, ≥30%, ≥35%, ≥40%, ≥45%, ≥50%, ≥55%, ≥60%, or ≥65% of tumor cells stain weakly for the biomarker. When ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, or ≥95% of tumor cells are stained with the biomarker, or when ≥10%, ≥15%, ≥20%, ≥25%, ≥30%, ≥35%, ≥40%, ≥45%, ≥50%, ≥55%, ≥60%, ≥65%, ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, or ≥95% of tumor cells are strongly stained with the biomarker, or when any combination thereof, one or more cells, including cancer cells, are identified as expressing the biomarker.

[0256] reagent kits and products In some embodiments, kits and articles thereof are disclosed for use with one or more methods and compositions described herein. Such kits include carriers, packages, or containers that are divided to contain one or more containers, such as vials, tubes, etc., each containing one of the independent elements to be used in the methods described herein. Suitable containers include bottles, vials, syringes, and test tubes. In one embodiment, the container is formed of a variety of materials, such as glass or plastic.

[0257] Kits typically include a label listing the contents and / or instructions for use, as well as a package insert with instructions for use. A set of instructions will also usually be included.

[0258] In one embodiment, the label is on or associated with the container. In one embodiment, the label is on the container when the letters, numbers, or other characters forming the label are attached, molded, or etched into the container itself, and is associated with the container when the label is present in a receiver or carrier that also holds the container, such as as a packaging insert. In one embodiment, the label is used to indicate that the contents will be used for a specific therapeutic application. The label also indicates instructions for use of the contents, such as those described herein.

[0259] In some embodiments, the pharmaceutical composition is contained in a packaging or dispenser device that holds one or more unit dosage forms containing the compounds provided herein. For example, the packaging may contain metal or plastic foil, such as blister packs. In one embodiment, the packaging or dispenser device includes instructions for use. In one embodiment, the packaging or dispenser also includes a notification relating to the container, in the form prescribed by a government agency regulating the manufacture, use, or sale of the drug, reflecting that agency’s approval of the form of the drug for human or veterinary use. For example, such a notification is a drug label or approved product insert approved by the U.S. Food and Drug Administration. In one embodiment, a composition containing the compounds provided herein, formulated in a compatible drug carrier, placed in a suitable container, and labeled for the treatment of a specified condition is also prepared.

[0260] This document discloses a kit comprising (a) an androgen receptor inhibitor and (b) an embryonic ectodermal development (EED) inhibitor for treating prostate cancer in a subject of need, and a packaging insert containing instructions for determining when to administer the kit to a subject with prostate cancer, measuring the expression of the biomarker described herein in one or more cells including prostate cancer, and the use of (a) the androgen receptor inhibitor and (b) the embryonic ectodermal development (EED) inhibitor if it has been determined that one or more cells including prostate cancer express the biomarker.

[0261] Implementation Plan Implementation Scheme 1: A method for treating prostate cancer in a subject, comprising administering to the subject (a) an androgen receptor inhibitor and (b) an embryonic ectodermal development (EED) inhibitor.

[0262] Implementation Scheme 2: A method for treating prostate cancer in a subject, wherein the prostate cancer in the subject has been identified as resistant to one or more first androgen receptor signaling inhibitors (ARSIs), including administering (a) an androgen receptor inhibitor and (b) an embryonic ectoderm development (EED) inhibitor to the subject.

[0263] Implementation Scheme 3: According to the method of Implementation Scheme 2, one or more first androgen receptor signaling inhibitors (ARSIs) are selected from one or more first CYP17 inhibitors and one or more first androgen receptor inhibitors.

[0264] Implementation Scheme 4: The method according to Implementation Scheme 3, wherein one or more first androgen receptor signaling inhibitors (ARSIs) are selected from one or more first CYP17 inhibitors.

[0265] Implementation Scheme 5: According to the method of Implementation Scheme 4, one or more of the first CYP17 inhibitors are abiraterone acetate.

[0266] Implementation Scheme 6: The method according to Implementation Scheme 2, wherein one or more first androgen receptor signaling inhibitors (ARSIs) are selected from one or more androgen receptor inhibitors.

[0267] Implementation Scheme 7: The method according to Implementation Scheme 6, wherein one or more first androgen receptor inhibitors are selected from apalutamide, darotamide, and enzalutamide.

[0268] Implementation Scheme 8: A method for treating prostate cancer in a subject, wherein the prostate cancer in the subject has been identified as resistant to CYP17 inhibitors, comprising administering to the subject (a) an androgen receptor inhibitor and (b) an embryonic ectoderm development (EED) inhibitor.

[0269] Implementation Scheme 9: The method according to Implementation Scheme 8, wherein the CYP17 inhibitor is abiraterone acetate.

[0270] Implementation Scheme 10: A method for treating prostate cancer in a subject, wherein the prostate cancer in the subject has been identified as resistant to abiraterone, comprising administering to the subject (a) an androgen receptor inhibitor and (b) an embryonic ectodermal development (EED) inhibitor.

[0271] Implementation Scheme 11: A method for treating prostate cancer in a subject, wherein the subject has previously received one or more CYP17 inhibitors, including administering to the subject (a) an androgen receptor inhibitor and (b) an embryonic ectoderm development (EED) inhibitor.

[0272] Implementation Scheme 12: A method for treating prostate cancer in a subject, wherein the subject (i) has received prior administration of one or more CYP17 inhibitors and (ii) has never received androgen receptor inhibitor treatment, comprising administering to the subject (a) an androgen receptor inhibitor and (b) an embryonic ectoderm development (EED) inhibitor.

[0273] Implementation Scheme 13: The method according to any one of Implementation Schemes 1 to 12, wherein the androgen receptor inhibitor is selected from apalutamide, darotamide, and enzalutamide.

[0274] Implementation Scheme 14: The method according to any one of Implementation Schemes 1 to 13, wherein the embryonic ectoderm development (EED) inhibitor is a small molecule having a molecular weight of less than or equal to 1000 Daltons.

[0275] Implementation Scheme 15: The method according to any one of Implementation Schemes 1 to 14, wherein the embryonic ectoderm development (EED) inhibitor is selected from EED226, A-395, APG-5918, BR-001, BR-002, EEDi-5285, EEDi-1056, FTX-6058, HJM-353 and MAK683.

[0276] Implementation Scheme 16: The method according to any one of Implementation Schemes 1 to 13, wherein the embryonic ectoderm development (EED) inhibitor is a compound of formula (I).

[0277] Or its pharmaceutically acceptable salt: in: Indicates a single bond or a double bond; Z is either O or S; X represents O and CR. 5 CR 5 OH or C(R) 5 )2, of which: When X is 0 It is a single key; When X is C(R) 5 At 2 o'clock, It is a single key; When X is CR 5 When OH, It is a single key; or When X is CR5 hour, It is a double bond; R 1 It is aryl, heteroaryl, L-cycloalkyl, -N(R) 5 Heterocyclic or L-heterocyclic, wherein L-cycloalkyl, -N(R) 5 The aryl, heteroaryl, or cyclic moiety of the heterocyclic or L-heterocyclic group is optionally surrounded by one or more R... 4 replace; R 2 It is cyano, -COOR 5 -C(O)N(R) 5 )2 or -C(O)N(R 5 )2, where each R 5 Together with the nitrogen atom to which it is attached, it forms an optional structure with one or more R atoms. 4 Replaced 5-8 membered heterocycles; Each R 3 It is independently a C1-C3 alkyl or halogen; Each R 4 Independently, it is oxo, cyano, halogen, -PO3 (C1-C3 alkyl)2, hydroxyl, alkoxy, hydroxyalkyl, heteroalkyl, aralkyl, haloalkyl, -COOR 5 -Y 2 -haloalkyl, -Y 1 -C1-C6 alkyl, -Y 2 -C1-C6 alkyl, -L-cycloalkyl, -L-heteroaryl, -L-heterocyclic, -Y 1 -heterocyclic group, -Y 2 -heterocyclic group, -LN(R) 5 )2、-OLN(R 5 )2、-C(CF3)N(R 5 )2、-Y 1 -N(R 5 )2 or -Y 2 -N(R 5 )2, wherein aralkyl, -L-cycloalkyl, -L-heteroaryl, -L-heterocyclic or -Y 1 -The ring portion of the heterocyclic group is optionally separated by one or more R 7 replace; L is a bond or a C1-C4 alkylene group; Y 1 It is a bond, -C(O)- or -NHC(O)-; Y 2 It is a key, -S-, -SO-, -SO2-, or -NR 5 SO2-, Each R 5It is hydrogen or C1-C3 alkyl; R 6 It is hydrogen, C1-C3 alkyl, halogen, haloalkyl, hydroxyalkyl, or heteroalkyl; Each R 7 It is oxo, cyano, hydroxy, alkoxy, halogen, haloalkyl, hydroxyalkyl, heteroalkyl, cycloalkyl, -LN(R) 5 2. C1-C6 alkyl or -Y 1 - Heterocyclic group; and n is 1 or 2.

[0278] Implementation Scheme 17: The method according to Implementation Scheme 16, wherein Z is O in the compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0279] Implementation Scheme 18: The method according to Implementation Scheme 17, wherein Z is S in the compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0280] Implementation Scheme 19: The method according to any one of Implementation Schemes 16 to 18, wherein n is 1 in the compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0281] Implementation Scheme 20: The method according to any one of Implementation Schemes 16 to 19, wherein R is in the compound of formula (I) or a pharmaceutically acceptable salt thereof. 2 It is a cyano group.

[0282] Implementation Scheme 21: The method according to any one of Implementation Schemes 16 to 19, wherein R is in the compound of formula (I) or a pharmaceutically acceptable salt thereof. 2 Yes - COOR 5 .

[0283] Implementation Scheme 22: The method according to any one of Implementation Schemes 16 to 19, wherein R is in the compound of formula (I) or a pharmaceutically acceptable salt thereof. 2 It is -C(O)N(R) 5 )2.

[0284] Implementation Scheme 23: The method according to any one of Implementation Schemes 16 to 22, wherein R is in the compound of formula (I) or a pharmaceutically acceptable salt thereof. 3 It is halogen.

[0285] Implementation Scheme 24: The method according to Implementation Scheme 23, wherein R is in the compound of formula (I) or a pharmaceutically acceptable salt thereof. 3 It's fluorine.

[0286] Implementation Scheme 25: The method according to any one of Implementation Schemes 16 to 24, wherein X is C(R) in the compound of formula (I) or a pharmaceutically acceptable salt thereof. 5 )2 and It is a single key.

[0287] Implementation Scheme 26: The method according to any one of Implementation Schemes 16 to 24, wherein X is a CR in the compound of formula (I) or a pharmaceutically acceptable salt thereof. 5 and It is a double bond.

[0288] Implementation Scheme 27: The method according to any one of Implementation Schemes 16 to 24, wherein X is O in the compound of formula (I) or a pharmaceutically acceptable salt thereof and It is a single key.

[0289] Implementation Scheme 28: The method according to any one of Implementation Schemes 16 to 27, wherein R is in the compound of formula (I) or a pharmaceutically acceptable salt thereof. 1 It is optionally controlled by one or more R 4 Substituted aryl groups.

[0290] Implementation Scheme 29: The method according to Implementation Scheme 28, wherein R is in the compound of formula (I) or a pharmaceutically acceptable salt thereof. 1 It is optionally controlled by one or more R 4 Substituted phenyl groups.

[0291] Implementation Scheme 30: The method according to Implementation Scheme 29, wherein R is in the compound of formula (I) or a pharmaceutically acceptable salt thereof. 1 Is it by one, two, or three Rs? 4 Substituted phenyl groups.

[0292] Implementation Scheme 31: The method according to Implementation Scheme 30, wherein in the compound of formula (I) or a pharmaceutically acceptable salt thereof, one, two or three R 4 Each of these can be independently a halogen, -PO3 (C1-C3 alkyl)2, hydroxyl, hydroxyalkyl, aralkyl, haloalkyl, or -COOR. 5 -Y 1 -C1-C6 alkyl, Y 2 -C1-C6 alkyl groups, -LN(R) 5 )2、-OLN(R 5 )2、-C(CF3)N(R 5 )2、-Y 1 -N(R 5 )2、-Y 2 -N(R 5 )2、Y2 -haloalkyl, -L-heteroaryl, -L-heterocyclic or -Y 1 - Heterocyclic group, wherein -L-heterocyclic group or -Y 1 -The heterocyclic moiety of the heterocyclic group is optionally surrounded by one or more R 7 replace.

[0293] Implementation Scheme 32: The method according to Implementation Scheme 31, wherein R is in the compound of formula (I) or a pharmaceutically acceptable salt thereof. 4 Yes-Y 1 -C1-C6 alkyl, and Y 1 It is a bond, and the C1-C6 alkyl group is methyl, ethyl, isopropyl, butyl, or pentyl.

[0294] Implementation Scheme 33: The method according to Implementation Scheme 31, wherein R is in the compound of formula (I) or a pharmaceutically acceptable salt thereof. 4 Yes-Y 2 -C1-C6 alkyl, and Y 2 It is -SO2-, and the C1-C6 alkyl group is methyl.

[0295] Implementation Scheme 34: The method according to Implementation Scheme 31, wherein R is in the compound of formula (I) or a pharmaceutically acceptable salt thereof. 4 Yes-Y 2 - Haloalkyl, and Y 2 It is -S- or -SO2-, and the haloalkyl group is trifluoromethyl.

[0296] Implementation Scheme 35: The method according to Implementation Scheme 31, wherein R is in the compound of formula (I) or a pharmaceutically acceptable salt thereof. 4 It is -LN(R) 5 )2, and L is the key, and each R 5 It's hydrogen, each R 5 It is a methyl group, or an R group. 5 It is methyl and has an R 5 It is hydrogen.

[0297] Implementation Scheme 36: The method according to Implementation Scheme 31, wherein R is in the compound of formula (I) or a pharmaceutically acceptable salt thereof. 4 It is -LN(R) 5 )2, and L is methylene or ethylene, and each R 5 It's hydrogen, each R 5 It is a methyl group, or an R group. 5 It is methyl and has an R 5 It is hydrogen.

[0298] Implementation Scheme 37: The method according to Implementation Scheme 31, wherein R is in the compound of formula (I) or a pharmaceutically acceptable salt thereof. 4 Yes-Y 1 -N(R 5 )2, Y 1 It is -C(O)-, and each R 5 Independently, each R is hydrogen. 5 It is independently a methyl group, or an R group. 5 It is methyl and has an R 5 It is hydrogen.

[0299] Implementation Scheme 38: The method according to Implementation Scheme 31, wherein R is in the compound of formula (I) or a pharmaceutically acceptable salt thereof. 4 Yes-Y 2 -N(R 5 )2, Y 2 It is -SO2-, and each R 5 Independently, each R is hydrogen. 5 It is a methyl group, or an R group. 5 It is methyl and has an R 5 It is hydrogen on its own.

[0300] Implementation Scheme 39: The method according to Implementation Scheme 31, wherein R is in the compound of formula (I) or a pharmaceutically acceptable salt thereof. 4 Yes-Y 1 - Heterocyclic group, and Y 1 It is -C(O)-, and the heterocyclic portion of the L-heterocyclic group is piperazinyl or 4-methyl-piperazinyl.

[0301] Implementation Scheme 40: The method according to Implementation Scheme 31, wherein R is in the compound of formula (I) or a pharmaceutically acceptable salt thereof. 4 It is an L-heterocyclic group, and L is a bond, and the heterocyclic moiety of the L-heterocyclic group is a nitrogen-heterocyclic butyl, oxocyclic butyl, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, piperazineyl, or... -Azabicyclo[3.1.0]hexyl, each optionally selected from one or more R groups selected from oxo, C1-C3 alkyl, alkoxy, hydroxyl and halogen. 7 replace.

[0302] Implementation Scheme 41: The method according to Implementation Scheme 31, wherein R is in the compound of formula (I) or a pharmaceutically acceptable salt thereof. 4 It is an -L-heterocyclic group, wherein L is methylene, and the heterocyclic moiety of the L-heterocyclic group is azirrobutyl, oxobutyl, pyrrolidinylpiperidinyl, each optionally selected from one or more R groups selected from C1-C3 alkyl, alkoxy, hydroxyl, and halogen. 7 replace.

[0303] Implementation Scheme 42: The method according to Implementation Scheme 31, wherein R is in the compound of formula (I) or a pharmaceutically acceptable salt thereof. 4 Yes-Y 1 - Heterocyclic group, and Y 1 It is -C(O)-, and Y 1 - The heterocyclic moiety of the heterocyclic group is a morpholino group optionally substituted with one or more C1-C3 alkyl groups.

[0304] Implementation Scheme 43: The method according to Implementation Scheme 31, wherein R is in the compound of formula (I) or a pharmaceutically acceptable salt thereof. 4 It is optionally controlled by one or more R 7 Substituted -L-heteroaryl.

[0305] Implementation Scheme 44: The method according to Implementation Scheme 31, wherein R is in the compound of formula (I) or a pharmaceutically acceptable salt thereof. 4 It is a tetrazolium group.

[0306] Implementation Scheme 45: The method according to Implementation Scheme 31, wherein R is in the compound of formula (I) or a pharmaceutically acceptable salt thereof. 4 It is -PO3(C1-C3 alkyl)2.

[0307] Implementation Scheme 46: The method according to Implementation Scheme 31, wherein R is in the compound of formula (I) or a pharmaceutically acceptable salt thereof. 4 Yes - COOR 5 .

[0308] Implementation Scheme 47: The method according to Implementation Scheme 31, wherein R is in the compound of formula (I) or a pharmaceutically acceptable salt thereof. 4 It is a hydroxyalkyl group.

[0309] Implementation Scheme 48: The method according to Implementation Scheme 31, wherein R is in the compound of formula (I) or a pharmaceutically acceptable salt thereof. 4 It is -OLN(R) 5 )2.

[0310] Implementation Scheme 49: The method according to Implementation Scheme 31, wherein R is in the compound of formula (I) or a pharmaceutically acceptable salt thereof. 4 It is an aralkyl group.

[0311] Implementation Scheme 50: The method according to any one of Implementation Schemes 15 to 26, wherein R is in the compound of formula (I) or a pharmaceutically acceptable salt thereof. 1 It is optionally controlled by one or more R 4 Substituted heteroaryl groups.

[0312] Implementation Scheme 51: The method according to Implementation Scheme 50, wherein R is in the compound of formula (I) or a pharmaceutically acceptable salt thereof. 1 It is pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, triazinyl, pyridinyl, pyridyl-2-one, pyrazinyl, pyridazinyl, pyrimidinyl, isoxazolyl, isoindolinyl, naphridinyl, 1,2,3,4-tetrahydroisoquinolinyl, or 5,6-dihydro-4H-pyrrolo[1,2-b]pyrazolyl, each optionally separated by one or more R 4 replace.

[0313] Implementation Scheme 52: The method according to Implementation Scheme 50, wherein R is in the compound of formula (I) or a pharmaceutically acceptable salt thereof. 1 by one or more R 4 Replace; where each R 4 Independently, it is cyano, halogen, -Y 1 -C1-C6 alkyl, -Y 2 -C1-C6 alkyl, alkoxy, hydroxyalkyl, heteroalkyl, haloalkyl, -L-cycloalkyl, -LN(R) 5 )2、-Y 1 -N(R 5 2. -L-heteroaryl, -L-heterocyclic, or -Y 1 -Heterocyclic group, wherein the -L-heteroaryl group is a heteroaryl group or an L-heterocyclic group or a Y-heterocyclic group. 1 -The heterocyclic moiety of the heterocyclic group is optionally surrounded by one or more R 7 replace.

[0314] Implementation Scheme 53: The method according to Implementation Scheme 50, wherein R is in the compound of formula (I) or a pharmaceutically acceptable salt thereof. 1 It is arbitrarily assigned to an R 4 The substituted pyrazol group, that one R 4 Independently selected from hydroxyalkyl, heteroalkyl, haloalkyl, -Y 1 -C1-C6 alkyl groups, -LN(R) 5 2. L-heterocyclic or L-heteroaryl, wherein the heteroaryl or heterocyclic portion of the L-heteroaryl is optionally surrounded by one or more R 7 replace.

[0315] Implementation Scheme 54: The method according to Implementation Scheme 53, wherein R is in the compound of formula (I) or a pharmaceutically acceptable salt thereof. 4 It is -L-heteroaryl, and L is methylene, wherein the heteroaryl group is optionally surrounded by one or more R groups. 7 Substituted pyridinyl group.

[0316] Implementation Scheme 55: The method according to Implementation Scheme 53, wherein R is in the compound of formula (I) or a pharmaceutically acceptable salt thereof. 4 Is -L- optionally controlled by one or more R 7 The substituted heterocyclic group, wherein L is a bond, and the heterocyclic portion of the L-heterocyclic group is oxobutyryl, tetrahydrofuranyl, tetrahydropyranyl, piperazineyl, or 4-methylpiperazineyl.

[0317] Implementation Scheme 56: The method according to Implementation Scheme 53, wherein R is in the compound of formula (I) or a pharmaceutically acceptable salt thereof. 4 It is optionally controlled by one or more R 7 The substituted -L-heterocyclic group, wherein L is methylene, and the heterocyclic moiety of the L-heterocyclic group is azirrobutyl, oxobutyl, pyrrolyl, pyrrolidone, tetrahydrofuranyl, tetrahydropyranyl, morpholinyl, piperazineyl, or 4-methylpiperazineyl.

[0318] Implementation Scheme 57: The method according to Implementation Scheme 53, wherein R is in the compound of formula (I) or a pharmaceutically acceptable salt thereof. 4 It is -LN(R) 5 )2, where L is methylene, and each R 5 Independently, each R is hydrogen. 5 It is independently a C1-C3 alkyl group, or an R 5 It is a C1-C3 alkyl group and has one R 5 It is hydrogen.

[0319] Implementation Scheme 58: The method according to Implementation Scheme 53, wherein R is in the compound of formula (I) or a pharmaceutically acceptable salt thereof. 4 Yes-Y 1 -C1-C6 alkyl, wherein Y 1 It is a bond, and the C1-C6 alkyl group is methyl, ethyl, or isopropyl.

[0320] Implementation Scheme 59: The method according to Implementation Scheme 50, wherein R is in the compound of formula (I) or a pharmaceutically acceptable salt thereof. 1 It is arbitrarily determined by two Rs 4 The two R groups are substituted with pyrazol groups. 4 Each group is independently selected from hydroxyalkyl, heteroalkyl, haloalkyl, and -Y groups. 1 -C1-C6 alkyl.

[0321] Implementation Scheme 60: The method according to Implementation Scheme 50, wherein R is in the compound of formula (I) or a pharmaceutically acceptable salt thereof. 1 It is arbitrarily assigned to an R 4 Substituted pyridinyl group, that one R 4Independently selected from cyano, halogen, alkoxy, hydroxyalkyl, heteroalkyl, haloalkyl, -Y 1 -C1-C6 alkyl groups, -LN(R) 5 )2、-Y 1 -N(R 5 )2, -L-cycloalkyl or optionally with one or more R 7 Substituted -L-heterocyclic group.

[0322] Implementation Scheme 61: The method according to any one of Implementation Schemes 16 to 27, wherein R is in the compound of formula (I) or a pharmaceutically acceptable salt thereof. 1 It is optionally controlled by one or more R 4 Substituted -L-cycloalkyl groups.

[0323] Implementation Scheme 62: The method according to any one of Implementation Schemes 16 to 27, wherein R is in the compound of formula (I) or a pharmaceutically acceptable salt thereof. 1 It is optionally controlled by one or more R 4 Substituted -L-heterocyclic group.

[0324] Implementation Scheme 63: The method according to Implementation Scheme 62, wherein in the compound of formula (I) or a pharmaceutically acceptable salt thereof, L is a bond and the heterocyclic group is piperidinyl or tetrahydropyranyl.

[0325] Implementation Scheme 64: The method according to any one of Implementation Schemes 1 to 13, wherein the embryonic ectoderm development (EED) inhibitor is a compound selected from the following: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , and Or, or a pharmaceutically acceptable salt thereof.

[0326] Implementation Scheme 65: The method according to any one of Implementation Schemes 1 to 13, wherein the embryonic ectoderm development (EED) inhibitor is a compound selected from the following: , , , , , , , , , , and Or, or a pharmaceutically acceptable salt thereof.

[0327] Implementation Scheme 66: The method according to Implementation Scheme 65, wherein the embryonic ectoderm development (EED) inhibitor is: (Compound 1), or a pharmaceutically acceptable salt thereof.

[0328] Implementation Scheme 67: The method according to Implementation Scheme 65, wherein the embryonic ectoderm development (EED) inhibitor is: (Compound 2), or a pharmaceutically acceptable salt thereof.

[0329] Implementation Scheme 68: The method according to Implementation Scheme 65, wherein the embryonic ectoderm development (EED) inhibitor is: (Compound 3), or a pharmaceutically acceptable salt thereof.

[0330] Implementation Scheme 69: The method according to Implementation Scheme 65, wherein the embryonic ectoderm development (EED) inhibitor is: (Compound 4), or a pharmaceutically acceptable salt thereof.

[0331] Implementation Scheme 70: The method according to Implementation Scheme 65, wherein the embryonic ectoderm development (EED) inhibitor is: (Compound 5), or a pharmaceutically acceptable salt thereof.

[0332] Implementation Scheme 71: The method according to Implementation Scheme 65, wherein the embryonic ectoderm development (EED) inhibitor is: (Compound 6), or a pharmaceutically acceptable salt thereof.

[0333] Implementation Scheme 72: The method according to Implementation Scheme 65, wherein the embryonic ectoderm development (EED) inhibitor is: (Compound 7), or a pharmaceutically acceptable salt thereof.

[0334] Implementation Scheme 73: The method according to Implementation Scheme 65, wherein the embryonic ectoderm development (EED) inhibitor is: (Compound 8), or a pharmaceutically acceptable salt thereof.

[0335] Implementation Scheme 74: The method according to Implementation Scheme 65, wherein the embryonic ectoderm development (EED) inhibitor is: (Compound 9), or a pharmaceutically acceptable salt thereof.

[0336] Implementation Scheme 75: The method according to Implementation Scheme 65, wherein the embryonic ectoderm development (EED) inhibitor is: (Compound 10), or a pharmaceutically acceptable salt thereof.

[0337] Implementation Scheme 76: The method according to Implementation Scheme 65, wherein the embryonic ectoderm development (EED) inhibitor is: (Compound 11), or a pharmaceutically acceptable salt thereof.

[0338] Implementation Scheme 77: The method according to Implementation Scheme 65, wherein the embryonic ectoderm development (EED) inhibitor is: (Compound 12), or a pharmaceutically acceptable salt thereof.

[0339] Implementation Scheme 78: A method for treating prostate cancer in a subject, comprising administering to the subject (a) an androgen receptor inhibitor and (b) a... Inhibitors of embryonic ectoderm development (EED) or pharmaceutically acceptable salts thereof.

[0340] Implementation Scheme 79: A method for treating prostate cancer in a subject, wherein the prostate cancer in the subject has been identified as resistant to one or more first androgen receptor signaling inhibitors (ARSIs), comprising administering to the subject (a) an androgen receptor inhibitor and (b) a... Inhibitors of embryonic ectoderm development (EED) or pharmaceutically acceptable salts thereof.

[0341] Implementation Scheme 80: The method according to Implementation Scheme 78, wherein one or more first androgen receptor signaling inhibitors (ARSIs) are selected from CYP17 inhibitors and androgen receptor inhibitors.

[0342] Implementation Scheme 81: The method according to Implementation Scheme 80, wherein one or more first androgen receptor signaling inhibitors (ARSIs) are selected from CYP17 inhibitors.

[0343] Implementation Scheme 82: The method according to Implementation Scheme 81, wherein the CYP17 inhibitor is abiraterone acetate.

[0344] Implementation Scheme 83: The method according to Implementation Scheme 80, wherein one or more first androgen receptor signaling inhibitors (ARSIs) are selected from one or more androgen receptor inhibitors.

[0345] Implementation Scheme 84: The method according to Implementation Scheme 81, wherein one or more first androgen receptor inhibitors are selected from apalutamide, darotamide, and enzalutamide.

[0346] Implementation Scheme 85: A method for treating prostate cancer in a subject, wherein the prostate cancer in the subject has been identified as resistant to CYP17 inhibitors, comprising administering to the subject (a) an androgen receptor inhibitor and (b) a CYP17 inhibitor. Inhibitors of embryonic ectoderm development (EED) or pharmaceutically acceptable salts thereof.

[0347] Implementation Scheme 86: The method according to Implementation Scheme 85, wherein the CYP17 inhibitor is abiraterone acetate.

[0348] Implementation Scheme 87: A method for treating prostate cancer in a subject, wherein the prostate cancer in the subject has been identified as resistant to abiraterone, comprising administering to the subject (a) an androgen receptor inhibitor and (b) a... Inhibitors of embryonic ectoderm development (EED) or pharmaceutically acceptable salts thereof.

[0349] Implementation Scheme 88: A method for treating prostate cancer in a subject, wherein the subject has previously received one or more CYP17 inhibitors, comprising administering to the subject (a) an androgen receptor inhibitor and (b) a CYP17 inhibitor. Inhibitors of embryonic ectoderm development (EED) or pharmaceutically acceptable salts thereof.

[0350] Implementation Scheme 89: The method according to any one of Implementation Schemes 1 to 88, wherein the androgen receptor inhibitor is selected from apalutamide, darotamide, and enzalutamide.

[0351] Implementation Scheme 90: The method according to Implementation Scheme 89, wherein the androgen receptor inhibitor is apalutamide.

[0352] Implementation Scheme 91: The method according to Implementation Scheme 89, wherein the androgen receptor inhibitor is dalotamide.

[0353] Implementation Scheme 92: The method according to Implementation Scheme 89, wherein the androgen receptor inhibitor is enzalutamide.

[0354] Implementation Scheme 93: A method for treating prostate cancer in a subject, wherein the subject (i) has previously received one or more CYP17 inhibitors and (ii) has never received androgen receptor inhibitor treatment, comprising administering to the subject (a) an androgen receptor inhibitor and (b) a [method / formula missing]. Inhibitors of embryonic ectoderm development (EED) or pharmaceutically acceptable salts thereof.

[0355] Implementation Scheme 94: The method according to any one of Implementation Schemes 1 to 93, wherein the subject has received one or more prior chemotherapy treatments prior to the administration of the androgen receptor inhibitor and the embryonic ectoderm development (EED) inhibitor.

[0356] Implementation Scheme 95: The method according to any one of Implementation Schemes 1 to 93, wherein the subject has received at most one prior chemotherapy treatment prior to the administration of the androgen receptor inhibitor and the embryonic ectoderm development (EED) inhibitor.

[0357] Implementation Scheme 96: The method described in any one of Implementation Schemes 1 to 95, wherein the subject has not received CYP17 inhibitor treatment prior to administration of the androgen receptor inhibitor and the embryonic ectoderm development (EED) inhibitor.

[0358] Implementation Scheme 97: The method according to any one of Implementation Schemes 1 to 96, wherein the subject has not received androgen receptor inhibitor treatment prior to administration of the androgen receptor inhibitor and the embryonic ectoderm development (EED) inhibitor.

[0359] Implementation Scheme 98: The method described in any one of Implementation Schemes 1 to 97, wherein an androgen receptor inhibitor and an embryonic ectoderm development (EED) inhibitor are administered to the subject sequentially or simultaneously.

[0360] Implementation Scheme 99: The method described in Implementation Scheme 98, wherein an androgen receptor inhibitor and an embryonic ectoderm development (EED) inhibitor are administered sequentially to the subject.

[0361] Implementation scheme 100: The method described in any one of implementation schemes 1 to 99, wherein an androgen receptor inhibitor and an embryonic ectoderm development (EED) inhibitor are administered to the subject on the same day.

[0362] Implementation Scheme 101: The method described in any one of Implementation Schemes 1 to 100, wherein an androgen receptor inhibitor and an embryonic ectoderm development (EED) inhibitor are administered to the subject over a 24-hour period.

[0363] Implementation Scheme 102: The method according to Implementation Scheme 98, wherein an androgen receptor inhibitor and an embryonic ectoderm development (EED) inhibitor are administered to the subject simultaneously.

[0364] Implementation Scheme 103: The method described in any one of Implementation Schemes 1 to 102, wherein the androgen receptor inhibitor and the embryonic ectoderm development (EED) inhibitor are administered to the subject once or twice daily.

[0365] Implementation Scheme 104: The method described in Implementation Scheme 103, wherein an androgen receptor inhibitor and an embryonic ectoderm development (EED) inhibitor are administered to the subject once daily.

[0366] Implementation Scheme 105: The method described in Implementation Scheme 103, wherein an androgen receptor inhibitor and an embryonic ectoderm development (EED) inhibitor are administered to the subject twice daily.

[0367] Implementation Scheme 106: The method described in any one of Implementation Schemes 1 to 102, wherein the subject is given an androgen receptor inhibitor twice daily and an embryonic ectoderm development (EED) inhibitor once daily.

[0368] Implementation Scheme 107: The method described in any one of Implementation Schemes 1 to 102, wherein an androgen receptor inhibitor is administered to the subject once daily and an embryonic ectoderm development (EED) inhibitor is administered to the subject twice daily.

[0369] Implementation Scheme 108: The method described in any one of Implementation Schemes 1 to 107, wherein an androgen receptor inhibitor is administered to the subject with or without food.

[0370] Implementation Scheme 109: The method according to Implementation Scheme 108, wherein an androgen receptor inhibitor is administered to the subject along with food.

[0371] Implementation Scheme 110: The method according to Implementation Scheme 108, wherein the androgen receptor inhibitor is not administered to the subject along with food.

[0372] Implementation Scheme 111: The method according to any one of Implementation Schemes 1 to 110, wherein the subject has been given one or more prior androgen deprivation therapies before administering the androgen receptor inhibitor and the embryonic ectoderm development (EED) inhibitor to the subject.

[0373] Implementation Scheme 112: The method according to any one of Implementation Schemes 1 to 110, wherein the subject has been administered a gonadotropin-releasing hormone (GnRH) analogue prior to administration of the androgen receptor inhibitor and the embryonic ectoderm development (EED) inhibitor to the subject.

[0374] Implementation Scheme 113: The method according to any one of Implementation Schemes 1 to 110, wherein the subject receives a gonadotropin-releasing hormone (GnRH) analogue while being administered an androgen receptor inhibitor and an embryonic ectoderm development (EED) inhibitor.

[0375] Implementation Scheme 114: The method according to any one of Implementation Schemes 1 to 110, wherein the subject has undergone bilateral orchiectomy prior to administration of androgen receptor inhibitors and embryonic ectodermal development (EED) inhibitors to the subject.

[0376] Implementation Scheme 115: The method according to any one of Implementation Schemes 1 to 114, wherein during the period of administration of androgen receptor inhibitors and embryonic ectoderm development (EED) inhibitors to the subject, no compound that is a substrate of CYP3A4, CYP2C19, CYP2C8, CYP2C9, UGT, P-gp, BCRP, or OATP1B1 is administered to the subject.

[0377] Implementation Scheme 116: The method according to any one of Implementation Schemes 1 to 114, wherein during the period of administration of androgen receptor inhibitors and embryonic ectoderm development (EED) inhibitors to the subject, no compound that is a substrate of CYP3A4, CYP2C9, UGT, P-gp, BCRP, or OATP1B1 is administered to the subject.

[0378] Implementation Scheme 117: The method according to any one of Implementation Schemes 1 to 114, wherein during the period of administration of androgen receptor inhibitors and embryonic ectoderm development (EED) inhibitors to the subject, the following compounds are not administered to the subject: (a) CYP2C8 inhibitors, (b) CYP3A4 inducers, or (c) substrates of CYP3A4, CYP2C9, or CYP2C19.

[0379] Implementation Scheme 118: The method according to any one of Implementation Schemes 1 to 114, wherein during the period of administration of the androgen receptor inhibitor and the embryonic ectoderm development (EED) inhibitor to the subject, the following compounds are not administered to the subject: (a) CYP3A4 inducer, (b) PG-p inhibitor, (c) CYP3A4 inhibitor, (d) BCRP substrate, (e) OATP1B1 substrate, or (f) OATP1B3 substrate.

[0380] Implementation Scheme 119: The method described in any one of Implementation Schemes 1 to 118, wherein the prostate cancer in the subject is selected from metastatic prostate cancer, non-metastatic prostate cancer, metastatic castration-resistant prostate cancer, metastatic castration-sensitive prostate cancer, localized high-risk prostate cancer, recurrent prostate cancer, non-metastatic castration-resistant prostate cancer, non-metastatic castration-sensitive prostate cancer, androgen receptor inhibitor-sensitive prostate cancer, androgen receptor inhibitor-resistant prostate cancer, androgen receptor-dependent prostate cancer, androgen receptor-independent prostate cancer, neuroendocrine prostate cancer (NEPC), metastatic neuroendocrine prostate cancer (NEPC), prostate cancer with small cell characteristics, metastatic prostate cancer with small cell characteristics, and invasive variant prostate cancer.

[0381] Implementation Scheme 120: The method described in Implementation Scheme 119, wherein the prostate cancer in the subject is metastatic prostate cancer.

[0382] Implementation Scheme 121: The method described in Implementation Scheme 119, wherein the prostate cancer in the subject is non-metastatic prostate cancer.

[0383] Implementation Scheme 122: The method described according to Implementation Scheme 119, wherein the prostate cancer in the subject is metastatic castration-resistant prostate cancer.

[0384] Implementation Scheme 123: The method according to Implementation Scheme 119, wherein the prostate cancer in the subject is metastatic castration-sensitive prostate cancer.

[0385] Implementation Scheme 124: The method described in Implementation Scheme 119, wherein the prostate cancer in the subject is localized high-risk prostate cancer.

[0386] Implementation Scheme 125: The method described in Implementation Scheme 119, wherein the prostate cancer in the subject is recurrent prostate cancer.

[0387] Implementation Scheme 126: The method according to Implementation Scheme 119, wherein the prostate cancer in the subject is non-metastatic castration-resistant prostate cancer.

[0388] Implementation Scheme 127: The method according to Implementation Scheme 119, wherein the prostate cancer in the subject is non-metastatic castration-sensitive prostate cancer.

[0389] Implementation Scheme 128: The method according to Implementation Scheme 119, wherein the prostate cancer in the subject is androgen receptor inhibitor sensitive prostate cancer.

[0390] Implementation Scheme 129: The method according to Implementation Scheme 119, wherein the prostate cancer in the subject is androgen receptor inhibitor resistant prostate cancer.

[0391] Implementation Scheme 130: The method described in Implementation Scheme 119, wherein the prostate cancer in the subject is androgen receptor-dependent prostate cancer.

[0392] Implementation Scheme 131: The method described in Implementation Scheme 119, wherein the prostate cancer in the subject is androgen receptor-independent prostate cancer.

[0393] Implementation Scheme 132: The method according to Implementation Scheme 119, wherein the prostate cancer in the subject is neuroendocrine prostate cancer (NEPC).

[0394] Implementation Scheme 133: The method according to Implementation Scheme 119, wherein the prostate cancer in the subject is metastatic neuroendocrine prostate cancer (NEPC).

[0395] Implementation Scheme 134: The method according to Implementation Scheme 119, wherein the prostate cancer in the subject is prostate cancer with small cell characteristics.

[0396] Implementation Scheme 135: The method according to Implementation Scheme 119, wherein the prostate cancer in the subject is metastatic prostate cancer with small cell characteristics.

[0397] Implementation Scheme 136: The method described according to Implementation Scheme 119, wherein the prostate cancer in the subject is an aggressive variant of prostate cancer.

[0398] Example Example 1: Preparation of Crystallization Form 1 of Compound 4 150 µL of methanol was added to 50 mg of the free base of compound 4, and the resulting slurry was stirred at room temperature for one day. The resulting solid was filtered under vacuum and dried overnight under ambient conditions to give compound 4 in form 1.

[0399] Example 2A: Preparation of Crystallization Form 2 of Compound 4 400 mg of the free base of compound 4 was dissolved in 1.5 mL of 2-methyltetrahydrofuran at 50 °C, and 1.5 mL of n-heptane was added at about 47 °C. The resulting mixture was then cooled to 10 °C. The resulting solid was filtered under vacuum and allowed to air dry overnight under ambient conditions to give compound 4 in form 2.

[0400] Example 2B: Preparation of Crystallization Form 2 of Compound 4 A certain amount of the free base of compound 4 was dissolved in 2-methyltetrahydrofuran (10 volumes) and then distilled down to 3 volumes. The temperature of the solution was adjusted to approximately 25°C, and the resulting slurry was stirred for more than 30 minutes. After 2 hours, n-heptane (7 volumes) was added to the slurry, and the resulting mixture was stirred for more than 4 hours. The resulting solid was filtered, the filter cake was washed with 30% 2-methyltetrahydrofuran / heptane (2 volumes), and dried in a vacuum oven to provide compound 4 in form 2.

[0401] Example 3A: X-ray powder diffraction (XRPD) analysis of forms 1 and 2 of compound 4 Using Panalytical X'pert 3 XRPD analysis of the crystalline polymorph of compound 4 was performed using X-ray powder diffraction. The sample was deposited in the center of a zero-background Si substrate. The 2θ position was calibrated according to the Panalytical Si reference disk. The parameters used in the analysis are described in Table 1.

[0402] Table 1

[0403] Polymorphic form 1 of compound 4, as described above, was analyzed by XRPD and the peaks are shown in Table 2. The error associated with each °2θ position was determined to be ± 0.2° θ.

[0404] Table 2

[0405] Polymorphic form 2 of compound 4, as described above, was analyzed by XRPD and the peaks are shown in Table 3. The error associated with each °2θ position was determined to be ± 0.2°-θ.

[0406] Table 3

[0407] Example 3B: Thermogravimetric analysis and differential scanning calorimetry of compound 4 in forms 1 and 2 Thermogravimetric analysis (TGA) data were collected using a TA Discovery TGA550 TGA from TA Instruments, and differential scanning calorimetry (DSC) analysis was performed using a TA Q2000 DSC from TA Instruments with the parameters described in Table 4.

[0408] Table 4

[0409] When performed under the conditions described in Table 4, thermogravimetric analysis (TGA) of the sample of form 1 of compound 4 showed a weight loss of approximately 1% when the sample was heated from room temperature to approximately the onset of melting (approximately 207 °C). Differential scanning calorimetry (DSC) analysis of form 1 of compound 4, when performed under the conditions described in Table 4, showed peaks between approximately 170 °C and 172 °C and between approximately 207 °C and 208 °C.

[0410] When performed under the conditions described in Table 4, thermogravimetric analysis (TGA) of the sample of form 2 of compound 4 showed approximately 2% weight loss as the sample was heated from room temperature to approximately the point of melting (approximately 204 °C). Differential scanning calorimetry (DSC) analysis of form 2 of compound 4 showed a peak between approximately 203 °C and 204 °C when performed under the conditions described in Table 4.

[0411] Example 3C: Preparation of Crystallization Form 2 of Compound 4 The reactor was purged and purged with nitrogen to atmospheric pressure. A solution of compound 4 (approximately 2.41 kg, as determined by solution analysis using HPLC) in 2-methyltetrahydrofuran (2-MeTHF, 36 kg, 15 volumes) was then added to the reactor, and this batch was concentrated by distillation under reduced pressure to approximately 5 L (approximately 2 volumes). The resulting solution was adjusted to approximately 25°C, and then n-heptane (0.4 kg, 0.2 volumes) was added in portions over a period of approximately 3 hours. The resulting solution was then inoculated with form 2 of compound 4 (9 g, 0.4 wt%), and the resulting mixture was stirred for approximately 1.3 hours, followed by the addition of additional n-heptane (24 kg, 10 volumes) over approximately 6 hours. The resulting slurry was stirred at 25°C for approximately 4.25 hours and then filtered. The reactor was then flushed with n-heptane (5.8 kg, 2.5 V), and the mixture was flushed into a filter cake, which was dehydrated and the solids were dried under reduced pressure at 40°C and 50°C for 19 hours to provide 2.48 kg of compound 4 in form 2.

[0412] Example 4: Study of compound 4 and dalotamide in 22Rv1 of intact BALB / c nude mice (low hormone). Compound 4, alone and in combination with dalotamide, was evaluated in intact male mice with low hormone levels in 22Rv1 (an enzalutamide-resistant prostate cancer cell line xenograft model). Intact male BALB / c nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Animals were housed in individually ventilated cages in a specific pathogen-free (SPF) environment of an animal husbandry facility and acclimatized to their new environment for at least 7 days prior to the start of any experiments. Mice were approximately 6–8 weeks old at the time of tumor cell inoculation. The human prostate cancer cell line 22Rv1 was purchased from the American Type Culture Collection (ATCC). CRL-2505 TM22Rv1 cells were cultured in RPMI 1640 medium containing L-glutamine and 10% fetal bovine serum (FBS) at 37°C in a 5% CO2 atmosphere. The medium was changed every 2 to 3 days. Cells were passaged with trypsin-EDTA when confluence reached 80-90%. Cells growing in the exponential growth phase were harvested and counted for inoculation in mice. The drug delivery medium for compound 4 was prepared using medium 2, which consisted of 0.5% methylcellulose (MC) in 50 mM phosphate buffer at pH 6.8, and stored at 4°C. Preparation of medium 2: Equal volumes of 50 mM Na2HPO4 (Sigma, catalog number S5136) and 50 mM NaH2PO4 (Sigma, catalog number 5011) were mixed, and the pH of the resulting mixture was adjusted to pH 6.8 using a 50 mM H3PO4 (Sigma, catalog number 79622) solution. Methylcellulose (MC; Sigma, catalog number M0262) powder was added to prepare a 0.5% MC 50 mM phosphate buffer, and the final pH of the 20 mg / mL dosing solution of compound 4 was approximately pH 6.8. The 20 mg / mL suspension of compound 4 was prepared by adding 2 mL of the medium (0.5% methylcellulose in 50 mM phosphate buffer, pH 6.8) to 40 mg of compound 4, and placing the resulting mixture in a heated water bath (approximately 50–60°C) and an ultrasonic bath, and sonicating for 15 minutes to provide a suspension of compound 4, which was found to be stable for 7 days when stored at 4°C between uses. When used for dosing, the vial was removed from storage at 4°C approximately 15 to 20 minutes before use, and the suspension was mixed between loaded syringes to ensure mixing.

[0413] The dosing mixture containing 10 mg / mL dalolatadine was prepared using media 1, which consisted of 50% PEG 400, 30% propylene glycol (PG), and 20% D5W. The dosing mixture was prepared by dissolving 100 mg dalolatadine in 5 mL of PEG 400 and vortexing and sonicating, adding 3 mL of PG and vortexing and sonicating, and adding 2 mL of D5W and vortexing and sonicating to obtain a suspension. The preparation was stored at 4°C and protected from light before use.

[0414] The dosing mixture containing 3 mg / mL enzalutamide was prepared using a medium consisting of 5% DMSO, 0.25% carboxymethyl cellulose (CMC), and 0.2% Tween 80 in water for injection (WFI) and stored at 4°C. The dosing mixture containing enzalutamide was prepared as follows: 99 mg of enzalutamide was added to a suitable glass vial, followed by 1.65 mL of DMSO (equivalent to 5% of the final total volume), and the mixture was vortexed and sonicated for 10 minutes to obtain a clear solution. Then, 31.35 mL of diluent (0.25% CMC / 0.2% Tween 80) was added, and the resulting mixture was vortexed to obtain a mixture with a total volume of 33 mL, sonicated in a water bath for 2 hours, and continuously stirred until a homogeneous suspension was formed. The dosing formulation was prepared one day prior to administration and weekly, and stored at room temperature and protected from light before use.

[0415] The design of this study is illustrated in Table 5 below (where n / a = not applicable; PO = oral gavage; QD = once daily starting from day 0; BID = twice daily starting from day 0).

[0416] Table 5

[0417] 22Rv1 tumor cells were inoculated into the right ventral region of intact male BALB / c nude mice. Each mouse received 4 × 10⁻⁶ cells mixed 1:1 with Matrigel in a total volume of 100 µL. 6 Individual cells. Monitor and measure tumor growth when the tumor is detectable. When the tumor size reaches approximately 100-175 mm... 3 Forty tumor-bearing mice were randomly divided into five groups of eight for treatment as described in Table 5. Treatment began on the day of randomization. The start date of treatment is designated as Day 0. Mice were administered the following medications orally for 21 days (days 0 to 20): a carrier solution, compound 4 at a dose of 100 mg / kg QD, dalotamide at a dose of 50 mg / kg BID, or enzalutamide at a dose of 30 mg / kg QD, according to the groups listed in Table 5. BIDs were administered at 7–8 hour intervals. Except for group 5, which was administered at 10 mL / kg, the volume of each formulation was 5 mL / kg based on recent body weight. Subcutaneous tumor volume was measured twice weekly using calipers with the following formula: Tumor volume (TV) = (length × width) 2) / 2. Mouse weight was measured daily using a weighing device. Animal health and clinical signs of adverse events were monitored through daily observation of overall morphology and necropsy of animals euthanized at the study endpoint. The study was terminated after 21 days of treatment as defined in the study protocol. Tumor growth inhibition (TGI) and regression were calculated using the following formula: TGI = [1 - (TVtf - TVt0) / (TVcf - TVc0)] × 100%, where: TVtf is the mean tumor volume (TV) of the treatment group on the last or most recent treatment day; TVt0 is the mean TV of the treatment group on day 0 of treatment; TVcf is the mean TV of the control group on the last or most recent treatment day; and TVc0 is the mean TV of the control group on day 0 of treatment. Mean tumor volume, SD, and SEM were calculated using a standard statistical tool (GraphPad PRISM 9.5). One-way ANOVA was used to compare tumor volume differences between groups at the end of the study (EOS). Dunnett's multiple comparison test was then used to compare the treated tumor volume against the overall tumor growth, and Tukey's multiple comparison test was used to compare the treated tumor volume at the end of the study across all treatment groups. A p-value less than 0.05 was considered statistically significant.

[0418] Results: Compound 4, as a monotherapy and in combination with dalolactam, showed tumor growth inhibition, as shown in Table 6. Compared with the mediator control, compound 4 administered orally at a dose of 100 mg / kg QD for 21 days and its combination with dalolactam at a dose of 50 mg / kg twice daily (BID) inhibited tumor growth by 46% and 62%, respectively. Figure 1 (Table 6). At the end of the study (EOS), both the compound 4 monotherapy group and the compound 4 plus dalolactam combination therapy group had significantly smaller tumor volumes compared to the mediator group (p < 0.005). No statistically significant difference was observed between dalolactam monotherapy at 50 mg / kg BID and enzalutamide monotherapy at 30 mg / kg QD. In summary, in a 22Rv1 prostate cancer xenograft model established under hypohormonal conditions, compound 4 as monotherapy demonstrated tumor growth inhibition and a significant reduction in EOS tumor volume, and its combination with dalolactam showed further improved tumor growth inhibition.

[0419] Table 6

[0420] Example 5: Study of Compound 4 and Darlotamine in a Darlotamine Acquired Resistance 22Rv1 Model Using Intact BALB / c Nude Mice Compound 4 alone and in combination with dallotamine were evaluated in a dallotamine-acquired resistance 22Rv1 model using intact BALB / c nude mice. Intact male BALB / c nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Animals were housed in individually ventilated cages in a specific pathogen-free (SPF) environment at an animal husbandry facility and acclimatized to their new environment for at least 7 days prior to the start of any experiments. Mice were approximately 6–8 weeks old when tumor cells were inoculated. 22Rv1 cells were cultured in RPMI 1640 medium containing L-glutamine and 10% fetal bovine serum (FBS) at 37°C in a 5% CO2 atmosphere. The medium was changed every 2–3 days. Cells were passaged with trypsin-EDTA when confluence reached 80–90%. Cells in the exponential growth phase were harvested and counted for inoculation in mice. The administration medium for compound 4 was prepared using medium 2, which consisted of 0.5% methylcellulose (MC) in a 50 mM phosphate buffer at pH 6.8, and stored at 4°C. Preparation of medium 2: Equal volumes of 50 mM Na₂HPO₄ (Sigma, catalog number S5136) and 50 mM NaH₂PO₄ (Sigma, catalog number 5011) were mixed, and the pH of the resulting mixture was adjusted to pH 6.8 using a 50 mM H₃PO₄ (Sigma, catalog number 79622) solution. Methylcellulose (MC; Sigma, catalog number M0262) powder was added to prepare a 0.5% MC 50 mM phosphate buffer, and the final pH of the 20 mg / mL administration solution of compound 4 was approximately pH 6.8. A 20 mg / mL suspension of compound 4 was prepared by adding 2 mL of the medium (0.5% methylcellulose in 50 mM phosphate buffer, pH 6.8) to 40 mg of compound 4, and placing the resulting mixture in a heated water bath (approximately 50–60°C) and an ultrasonic bath for 15 minutes to provide a suspension of compound 4. It was found to be stable for 7 days between use when stored at 4°C. When intended for administration, the vial was removed from storage at 4°C approximately 15–20 minutes prior to use, and the suspension was mixed between loaded syringes to ensure proper mixing.

[0421] The dosing mixture containing 10 mg / mL dalolatadine was prepared using media 1, which consisted of 50% PEG 400, 30% propylene glycol (PG), and 20% D5W. The dosing mixture was prepared by dissolving 100 mg dalolatadine in 5 mL of PEG 400 and vortexing and sonicating, adding 3 mL of PG and vortexing and sonicating, and adding 2 mL of D5W and vortexing and sonicating to obtain a suspension. The preparation was stored at 4°C and protected from light before use.

[0422] The design of the study (Phase I and Phase II) is illustrated in Tables 7 and 8 below (where n / a = not applicable; PO = oral gavage; QD = once daily starting from day 0; BID = twice daily starting from day 0).

[0423] Table 7: Stage I processing before randomization

[0424] Table 8: Stage II processing after randomization

[0425] During Phase I, 64 intact male BALB / c nude mice were treated with dalolactam at 50 mg / kg BID for 28 days, as described in Table 7. On day 14 of dalolactam treatment in Phase I, 22Rv1 tumor cells were inoculated into the right ventral region of the intact male BALB / c nude mice. Each mouse received 4 × 10⁻⁶ cells mixed 1:1 with Matrigel in a total volume of 100 µL. 6 Individual cells. Tumor growth was monitored and measured when the tumor became perceptible. Two weeks after dalotamide treatment in Phase I, the tumor size reached approximately 130–140 mm. 3 Thirty-two tumor-bearing mice were randomly assigned to four groups of eight mice each for treatment as described in Table 8. Phase II treatment began on the day of randomization. The start date of Phase II treatment is designated as Day 0 of treatment. Mice were administered oral mediator solution, compound 4 at a QD of 100 mg / kg, and dallotamine at a BID of 50 mg / kg according to the groups listed in Table 8 for 28 days (days 0 to 27). BID administration was performed at 7–8 hour intervals. The administration volume for each formulation was 5 mL / kg based on recent body weight. Subcutaneous tumor volume was measured twice weekly using calipers with the following formula: Tumor volume (TV) = (length × width) 2) / 2. Mouse weight was measured daily using a weighing device. Animal health and clinical signs of adverse events were monitored through daily observation of overall morphology and necropsy of animals euthanized at the study endpoint. The study was terminated after 28 days of treatment as defined in the study protocol. Tumor growth inhibition (TGI) was calculated using the following formula: TGI = [1 - (TVtf - TVt0) / (TVcf - TVc0)] × 100%, where TVtf is the mean tumor volume (TV) of the treatment group on the last or most recent treatment day, TVt0 is the mean TV of the treatment group on day 0 of treatment, TVcf is the mean TV of the control group on the last or most recent treatment day, and TVc0 is the mean TV of the control group on day 0 of treatment. Mean tumor volume, SD, and SEM were calculated using a standard statistical tool (GraphPad PRISM 9.5). One-way ANOVA was used to compare tumor volume differences between groups at the end of the study (EOS). Dunnett's multiple comparison test was then used to compare the treated tumor volume against the overall tumor growth, and Tukey's multiple comparison test was used to compare the treated tumor volume at the end of the study across all treatment groups. A p-value less than 0.05 was considered statistically significant.

[0426] Results: Compared with the mediator control group, compound 4 administered orally at a dose of 100 mg / kg QD for 28 days and its combination with dalolactam at a dose of 50 mg / kg BID inhibited tumor growth by 22% and 59%, respectively, while dalolactam monotherapy resulted in 23% tumor growth inhibition. At the end of the study (EOS), the tumor volume in the compound 4 and dalolactam combination group was significantly smaller than that in the mediator group (p < 0.04) (Table 9 and 2000). Figure 2 However, no statistically significant differences were observed in the remaining single-treatment groups.

[0427] Table 9

[0428] Example 6: Study of compound 4, PF-06821497 and dalotamid in a subcutaneous C4-2 castrated prostate cancer xenograft model in male NPG mice. Compound 4 and PF-06821497 (an EZH2 inhibitor) alone, as well as their combination with dallotamide, were evaluated in a subcutaneous C4-2 castrated prostate cancer xenograft model in male NPG mice. The administration medium for compound 4 was prepared using medium 2, consisting of 0.5% methylcellulose (MC) in 50 mM phosphate buffer at pH 6.8, and stored at 4°C. Preparation of medium 2: Equal volumes of 50 mM Na2HPO4 (Sigma, catalog number S5136) and 50 mM NaH2PO4 (Sigma, catalog number 5011) were mixed, and the pH of the resulting mixture was adjusted to pH 6.8 using a 50 mM H3PO4 (Sigma, catalog number 79622) solution. Methylcellulose (MC; Sigma, catalog number M0262) powder was added to prepare a 0.5% MC 50 mM phosphate buffer, and the final pH of the 20 mg / mL dosing solution of compound 4 was approximately pH 6.8. The 20 mg / mL suspension of compound 4 was prepared by adding 2 mL of the medium (0.5% methylcellulose in 50 mM phosphate buffer, pH 6.8) to 40 mg of compound 4, placing the resulting mixture in a heated water bath (approximately 50–60°C), and then sonicating the mixture in an ultrasonic bath for 15 minutes to provide a suspension of compound 4. It was found to be stable for 7 days between uses when stored at 4°C. When used for dosing, the vial was removed from storage at 4°C approximately 15–20 minutes before use, and the suspension was mixed between loaded syringes to ensure mixing.

[0429] The dosing mixture containing 10 mg / mL dalolatadine was prepared using media 1, which consisted of 50% PEG 400, 30% propylene glycol (PG), and 20% D5W. The dosing mixture was prepared by dissolving 100 mg dalolatadine in 5 mL of PEG 400 and vortexing and sonicating; adding 3 mL of PG and vortexing and sonicating; and adding 2 mL of D5W and vortexing and sonicating to obtain a suspension. The preparation was stored at 4°C and protected from light before use.

[0430] The dosing mixture containing 20 mg / mL of PF-06821497 was prepared in a mixture of 0.5% NaCMC and 0.1% Tween 80, with a final pH of 4.5.

[0431] C4-2 prostate cancer cells were cultured in DMEM / Ham's F12K (4:1) + insulin + T3 + transferrin + d-biotin + adenine supplemented with 10% fetal bovine serum and maintained in vitro at 37°C in a 5% CO2 atmosphere. Cells in the exponential growth phase were harvested prior to tumor inoculation and quantified using a cell counter. Each mouse was subcutaneously inoculated with 0.1 ml of C4-2 tumor cells (5 × 10⁶ cells) in PBS mixed with Matrigel (1:1) in the right upper quadrant. 6 (One) is used for tumor development. When the average tumor size reaches approximately 200 mm. 3 Castration was performed on all mice. The average tumor size reached approximately 239 mm. 3 Randomization was initiated at that time. This study included 60 mice. All animals were randomly assigned to 6 study groups, with 10 mice in each group, as described in Table 10. Randomization was performed using the matched distribution method (StudyDirector™ software, version 3.1.399.19).

[0432] Table 10

[0433] Results: TGI data were based on 28 days of administration per group, which was the number of days animals in the vector group remained surviving. In this model, darostamid (50 mg / kg twice daily (BID)) provided significant antitumor efficacy relative to the vector control group, resulting in a 68% TGI (Table 10). Figure 3 Compound 4 (100 mg / kg, QD) provided 69% tumor growth inhibition as a single agent, and exhibited even more robust inhibition in combination with dalolactone, reaching a TGI of 83%. PF-06821497 also provided antitumor growth inhibition as a single agent (100 mg / kg, BID) and in combination with dalolactone, with TGIs of 62% and 68%, respectively. All treatment groups resulted in significantly smaller EOS tumors relative to the mediator treatment groups, with no differences between groups. Comparison of the dalolactone combination groups revealed that, in the C4-2 castration model, the compound 4 combination had the most robust antitumor activity relative to the PF-06821497 combination, with TGIs of 83% and 68%, respectively.

[0434] Based on progression-free survival (PFS) (defined as tumor volume greater than 800 mm) 3Treatments (or those resulting in death) were assessed to determine the increased PFS relative to the vehicle treatment in all groups. PFS data were based on mice in each group receiving the drug for a QD of 31 days, excluding the vehicle treatment. Darolantadine prolonged median PFS by 1.5 days relative to the vehicle treatment. PF-06821497, as a single agent or in combination with darolantadine, also provided increased PFS, resulting in median PFS of 15.5 days and 24.5 days, respectively. Compound 4 treatment, as a single agent or in combination with darolantadine, provided the greatest benefit for median PFS prolongation, reaching 27 days for the single-agent group and not reaching the median PFS at the end of the study (Table 10). Figure 9 ).

[0435] Example 7: Study of compound 4, PF-06821497 and dalotamid in a subcutaneous C4-2 prostate cancer xenograft model in intact male NPG mice. Compound 4 and PF-06821497 (an EZH2 inhibitor) alone, as well as their combination with dallotamide, were evaluated in a subcutaneous C4-2 prostate cancer xenograft model in intact male NPG mice. The study was conducted according to the general method described in Example 6, and the dosage and frequency of administration for each of compound 4, PF-06821497, and dallotamide are illustrated in Table 11. In this model, dallotamide (50 mg / kg twice daily (BID)) provided a TGI of -10.33% (Table 11, ...). Figure 4 Compound 4 (100 mg / kg QD) provided approximately 31% tumor growth inhibition when used as a single agent, while the combination of compound 4 (100 mg / kg QD) with dalolactam (50 mg / kg BID) provided approximately 72% TGI (Table 11). PF-06821497 provided approximately 55% TGI when used as a single agent (100 mg / kg BID), while the combination of PF-06821497 (100 mg / kg BID) with dalolactam (50 mg / kg BID) provided approximately 58% TGI (Table 11).

[0436] Based on progression-free survival (PFS) (defined as tumor volume greater than 800 mm) 3 Treatments (or death) were evaluated across all groups to result in increased PFS compared to the median treatment. Darlotamine prolonged median PFS by 4 days relative to the median treatment. PF-06821497, as a monotherapy or in combination with darlotamine, also provided increased PFS, resulting in median PFS of 15.5 days and 17 days, respectively. Compound 4 treatment as a monotherapy provided a median PFS extension benefit to 14 days. Compound 4 in combination with darlotamine provided a further extension of median PFS, reaching 24 days before the end of the study (Table 11). Figure 10 ).

[0437] Table 11

[0438] Example 8: In vitro study of compound 4 in combination with an androgen receptor signaling inhibitor in C4-2 and LNCaP prostate cancer cell lines.

[0439] Cell-based studies were performed in the C4-2 and LNCaP prostate cancer cell lines to evaluate the in vitro synergistic effects of compound 4 with an androgen receptor signaling inhibitor. Cell viability was assessed by quantitatively measuring ATP (a measure of metabolically active cells) following treatment with compound 4, an androgen receptor signaling inhibitor, and a combination of compound 4 and an androgen receptor signaling inhibitor. Synergistic effects (Bliss, Loewe, and HSA) were assessed using quantitative methods, and synergistic response data were analyzed and visualized, revealing the synergistic effects of compound 4 with the androgen receptor signaling inhibitor in multiple prostate cancer cell lines.

[0440] The test samples were as follows: (a) compound 4, (b) PF-06821497 (an EZH2 inhibitor), (c) enzalutamide, and (d) dalotamide. Each test sample was dissolved in DMSO to prepare a 10 mM stock solution. The dosing solution for each test sample was prepared by serial dilution in DMSO followed by dilution in complete culture medium, resulting in 0.4% DMSO for the assay. The stock formulation was stored at -80°C, and the dosing solution was prepared immediately before use for each experiment. The efficacy of the test samples was based on the effect of CellTiter-Globe assay on C4-2 and LNCaP cells treated with cells containing the test samples for 14 days. The activity is measured by (CTG) assay.

[0441] The sources and preparation of the cells, reagents, and working solutions used in this study are as follows. C4-2 and LNCaP cell line samples were obtained from the American Type Culture Collection (ATCC). The culture media for both cell lines were: RPMI with 10% fetal bovine serum (FBS) plus 2 mM L-Glut and 0.5 µg / mM penicillin-streptomycin; RPMI 1640 containing phenol red (Corning, catalog number 15-040-CV); FBS (Omega Scientific, catalog number FB-11); L-glutamine (Corning, catalog number 25-005-CI); and penicillin-streptomycin (Gibco, catalog number 15140122); the assay medium was the same as the culture medium; CellTiter-Glo 2.0 Cell viability assay (Promega, catalog number G9243); and DMSO (Sigma, catalog number D2660). The following materials and equipment were used to perform the study: Bravo liquid handling workstation (Agilent, catalog number G5523BA); 96-well non-sterile polypropylene V-shaped base plate (Corning, catalog number 3363); 384-well sterile white flat-bottomed transparent plate (Greiner, catalog number 781098); and TopSeal-A Plus plate seal (PerkinElmer, catalog number 6050185).

[0442] Prior to each assay, C4-2 and LNCaP cells were cultured in complete culture medium to the exponential growth phase for at least one week. Different cell suspensions were counted using a Bravo liquid handling workstation and seeded into 384-well sterile white flat-bottomed clear plates at a concentration of 500 cells per well in 56 µL of medium. To prepare dilutions of the compounds from 10 mM stock solutions, dilution plates were prepared using a Bravo liquid handling workstation by performing 10-point 1 / 3-fold serial dilutions in DMSO (for compounds 4 and PF-06821497) or 6-point 1 / 3-fold serial dilutions in DMSO (for dalolamide and enzalutamide) to a final concentration 500 times the initial concentration in the final assay plate. The compound dilutions in the medium were added to the cells by dispensing 2 μL to cells placed in 56 μL of culture medium on the same day. The plates were incubated at 37°C in a 5% CO2 incubator. After 7 days of incubation, the cell culture medium and inhibitors were replaced and incubated for an additional 7 days. Before viability assay, the plate was allowed to equilibrate at room temperature, and then CTG 2.0 was added at a 1:1 ratio using a Bravo liquid handling workstation. The plate was sealed using a PerkinElmer TopSeal-A Plus plate sealer and incubated at room temperature for 10 minutes, after which the luminescence was read on a Tecan Spark microplate reader. The measured luminescence is a direct readout of the presence of ATP in the cells, used to measure cell viability. Cell viability was measured with CTG in four replicates.

[0443] Data Analysis: Data from the Tecan SPARK microplate reader was imported into a Microsoft Excel file and then into GraphPad Prism. Curves were fitted using a four-parameter model, and percentage inhibition was calculated using GraphPad Prism. DMSO wells were used to define the upper limit (zero percentage inhibition). The degree of synergistic, neutralizing, or antagonistic effects of the drug combinations was quantified by comparing the observed drug combination responses with the expected responses calculated using a reference model assuming no interaction between the drugs from the SynergyFinder website (Ianevski 2022). These models used different algorithms to quantify the degree of synergistic effects as a multiplicative effect of the single drugs as if they acted independently (Bliss), corresponding to an additive effect as the expected response of the single drugs being the same compound (Loewe), or exceeding the maximum single-drug response (HSA).

[0444] Results: Compound 4, PF-06821497, dalolamide, and enzalutamide, when used as single agents (not in combination), showed nanomolar cell titers in C4-2 and LNCaP prostate cancer cells grown in complete medium with full FBS and no additional hormones. The following combinations (a) Compound 4 with enzalutamide; and (b) Compound 4 with dalolamide, respectively, showed synergistic effects in reducing cell viability and altering cell growth inhibition curves in both C4-2 and LNCaP cell lines, as shown in the data in Table 12. As also shown in Table 12, the combination of PF-06821497 and enzalutamide achieved a synergistic effect in the C4-3 cell line.

[0445] Table 12

[0446] Example 9: Clinical evaluation of compound 4 in human subjects with cancer A clinical study was conducted on the use of compound 4 in human subjects with cancer. Compound 4 was administered orally to human subjects in the form of a pharmaceutical composition. Enrolled subjects were assigned to groups where they were administered compound 4 for 28 days at doses of 100 mg once daily (QD), 200 mg QD, 400 mg QD, 600 mg QD, 700 mg QD, 800 mg QD, or 900 mg QD. The number of subjects enrolled and administered compound 4 at each dose level was as follows: 100 mg QD (subjects), 200 mg QD (4 subjects), 400 mg QD (3 subjects), 600 mg QD (3 subjects), and 900 mg QD (6 subjects).

[0447] The plasma concentrations of compound 4 in subjects who received the drug composition orally on day 1 of cycle 2 (C2D1) were determined over time, and the available C2D1 data (100 mg QD (2 subjects), 200 mg QD (3 subjects), 400 mg QD (3 subjects), 600 mg QD (3 subjects), and 900 mg QD (3 subjects)) were determined and plotted as follows: Figure 5 As shown. In C2D1, the concentration of compound 4 in the plasma of subjects generally showed increasing exposure with increasing dose level, low intra-patient variability, low intra-group variability, and a half-life greater than 12 hours (t). 1 / 2 ).

[0448] Peripheral blood mononuclear cell (PBMC) and plasma samples were collected from subjects before administration of compound 4 on day 1 of cycle 1 (C1D1), and after oral administration of compound 4 as a pharmaceutical composition on day 15 of cycle 1 (C1D15) and day 1 of cycle 2 (C2D1) to assess target binding in monocytes and cell-free nucleosomes, respectively. H3K27me3 and total histone H3 levels were measured in monocytes to assess changes in H3K27me3 intensity normalized to H3 during treatment. Dying tumor cells release nucleosomes into circulation, so changes in H3K27me3 levels in cell-free nucleosomes are expected to reflect target binding in the tumor microenvironment. Pharmacodynamic changes were thus captured by quantifying treatment-induced changes in H3K27me3 levels in cell-free nucleosomes (normalized to histone variant H3.1 as shown).

[0449] On day 15 of cycle 1, the percentage change (mean ± SEM) of H3K27me3 / H3 in monocytes of subjects orally administered compound 4 at doses of 200 mg QD, 400 mg QD, 600 mg QD, 700 mg QD, 800 mg QD, or 900 mg QD was determined from baseline, and plotted as follows: Figure 6 As shown. Figure 6 As shown, H3K27me3 in the mononuclear cells of the subjects was reduced by more than 75% at each dose level.

[0450] On day 15 of cycle 1 (C1D15), the change from baseline in the H3K27me3 to histone 3.1 (H3.1) ratio in cell-free nucleosomes of subjects orally administered compound 4 in the form of the pharmaceutical composition at doses of 100 mg once daily (QD), 200 mg QD, 400 mg QD, 600 mg QD, 700 mg QD, or 900 mg QD was determined. Figure 7The description.

[0451] On day 1 of cycle 2 (C2D1), the change from baseline in the H3K27me3 to histone 3.1 (H3.1) ratio in cell-free nucleosomes of subjects orally administered compound 4 in the form of the pharmaceutical composition at doses of 100 mg once daily (QD), 200 mg QD, 400 mg QD, 600 mg QD, 700 mg QD, or 900 mg QD was determined. Figure 8 The description.

Claims

1. A method of treating prostate cancer in a subject, comprising administering to the subject (a) an androgen receptor inhibitor and (b) an embryonic ectodermal development (EED) inhibitor.

2. The method of claim 1, wherein the subject has previously received one or more CYP17 inhibitors.

3. The method of claim 2, wherein the subject has previously received abiraterone or abiraterone acetate.

4. The method according to any one of claims 1 to 3, wherein the prostate cancer in the subject has been determined to be resistant to abiraterone.

5. The method of claim 1, wherein the subject has not received CYP17 inhibitor treatment prior to administering the androgen receptor inhibitor and the embryonic ectoderm development (EED) inhibitor to the subject.

6. The method according to any one of claims 1 to 5, wherein the subject has not received androgen receptor inhibitor treatment prior to administering the androgen receptor inhibitor and the embryonic ectoderm development (EED) inhibitor to the subject.

7. The method according to any one of claims 1 to 6, wherein the subject has been given one or more prior androgen deprivation therapies before administering the androgen receptor inhibitor and the embryonic ectoderm development (EED) inhibitor to the subject.

8. The method according to any one of claims 1 to 6, wherein the subject has been administered a gonadotropin-releasing hormone (GnRH) analogue prior to administration of the androgen receptor inhibitor and the embryonic ectoderm development (EED) inhibitor to the subject.

9. The method according to any one of claims 1 to 8, wherein the androgen receptor inhibitor and the embryonic ectoderm development (EED) inhibitor are administered to the subject sequentially or simultaneously.

10. The method according to any one of claims 1 to 9, wherein the androgen receptor inhibitor is selected from apalutamide, darotamide, and enzalutamide.

11. The method according to any one of claims 1 to 10, wherein the embryonic ectoderm development (EED) inhibitor is selected from EED226, A-395, APG-5918, BR-001, BR-002, EEDi-5285, EEDi-1056, FTX-6058, HJM-353 and MAK683.

12. The method according to any one of claims 1 to 10, wherein the embryonic ectoderm development (EED) inhibitor is a compound selected from the group consisting of: , , , , , , , , , , and Or, or a pharmaceutically acceptable salt thereof.

13. The method according to any one of claims 1 to 12, wherein the prostate cancer in the object is selected from metastatic prostate cancer, non-metastatic prostate cancer, metastatic castration-resistant prostate cancer, metastatic castration-sensitive prostate cancer, localized high-risk prostate cancer, recurrent prostate cancer, non-metastatic castration-resistant prostate cancer, non-metastatic castration-sensitive prostate cancer, androgen receptor inhibitor-sensitive prostate cancer, androgen receptor inhibitor-resistant prostate cancer, androgen receptor-dependent prostate cancer, androgen receptor-independent prostate cancer, neuroendocrine prostate cancer (NEPC), metastatic neuroendocrine prostate cancer (NEPC), prostate cancer with small cell characteristics, metastatic prostate cancer with small cell characteristics, and invasive variant prostate cancer.

14. The method of claim 13, wherein the prostate cancer in the subject is metastatic prostate cancer.

15. The method of claim 14, wherein the prostate cancer in the subject is metastatic castration-resistant prostate cancer.

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