Ezh2 inhibition in combination therapies for the treatment of cancers

Abstract

To provide methods for treating advanced relapsed solid tumors.SOLUTION: Provided is a method for treating advanced relapsed solid tumors using 7-chloro-2-(4-(3-methoxyazetidin-1-yl)cyclohexyl)-2, 4-dimethyl-N-((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)benzo[d][1,3]dioxole-5-carboxamide; or a pharmaceutically acceptable salt thereof. Also provided herein are methods of treating cancers (e.g., solid tumors) using the compound or a pharmaceutically acceptable salt thereof, and a second agent selected from a topoisomerase inhibitor, a DNA alkylating agent, and an androgen receptor signaling inhibitor.SELECTED DRAWING: None

Description

Related Applications

[0001] This application claims priority to U.S. Provisional Application No. 62 / 878,021, filed July 24, 2019, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Enhancer of Zeste Homolog 2 (EZH2) is a histone lysine methyltransferase that is involved in the pathogenesis of both hematological and non-hematological malignancies. EZH2 catalyzes the transfer of one, two, and three methyl groups to lysine 27 of histone 3 (H3K27). EZH2 is a catalytic component of a large multiprotein complex called Polycomb Repressive Complex 2 (PRC2), which generally functions in transcriptional repression (Margueron, R., and Reinberg, D. (2011). The Polycomb complex PRC2 and its mark in life. Nature 469, 343-349). In many cases, transcriptional silencing by PRC2 relies on the catalytic activity of EZH2, but it is clear that the physical association of the PRC2 complex with specific genes is also important for transcriptional repression. Alternatively, the PRC2 complex may contain a closely related homolog of EZH2 known as EZH1. These two catalytic subunits of the PRC2 complex are the only enzymes known to catalyze H3K27 methylation. In addition to their catalytic activity, EZH1 and EZH2 are multidomain proteins that mediate other biological effects through protein-protein and protein-nucleic acid interactions. H3K27 di- and trimethylation (H3K27me2 and H3K27me3) correlate well with transcriptionally repressed genes, whereas H3K27 monomethylation (H3K27me1) is found on transcriptionally active genes (Barski, A., et al. (2007). High-resolution profiling of histone methylations in the human genome. Cell 129, 823-837; Ferrari, KJ, et al. (2014). Polycomb-dependent H3K27me1 and H3K27me2 regulate active transcription and enhancer fidelity. Mol. Cell 53, 49-62). Recent genetic studies suggest that EZH1-containing PRC2 regulates H3K27me1 levels (Hidalgo, I., et al. (2012). Ezh1 is required for hematopoietic stem cell maintenance and prevents senescence-like cell cycle arrest. Cell Stem Cell 11, 649-662; Xie, H., et al. (2014). Polycomb repressive complex 2 regulates normal hematopoietic stem cell function in a developmental-stage-specific manner. Cell Stem Cell 14, 68-80). This is consistent with the putative role of EZH1 in transcription elongation (Mousavi, K., et al. (2012). Polycomb protein Ezh1 promotes RNA polymerase II elongation. Mol. Cell 45, 255-262). Thus, PRC2-dependent H3K27 methyltransferase activity is involved in both transcriptional repression and activation, depending on the composition of the complex.

[0003] EZH2 (but not EZH1) is frequently overexpressed in human cancers. The molecular basis for EZH2 overexpression in cancer includes (1) genomic amplification of the EZH2-encoding gene locus (Tiffen, J., et al. (2016). Somatic Copy Number Amplification and Hyperactivating Somatic Mutations of EZH2 Correlate With DNA Methylation and Drive Epigenetic Silencing of Genes Involved in Tumor Suppression and Immune Responses in Melanoma. Neoplasia 18(2), 121-132., Ding, L., et al.(2006). Identification of EZH2 as a molecular marker for a precancerous state in morphologically normal breast tissues. Cancer Research 66(8), 4095-4099., Saramaki, OR, et al.(2006). The gene for polycomb group protein enhancer of zeste homolog 2 (EZH2) is amplified in late-stage prostate cancer. Genes Chromosomes Cancer 45(7), 639-645.), (2) deletion and epigenetic silencing of microRNAs that attenuate EZH2 expression (Varambally, S., et al.(2008). Genomic loss of microRNA-101 leads to overexpression of histone methyltransferase EZH2 in cancer. Science 322(5908),1695-1699), and (3) dysregulation of gene regulation exerted by the E2F family of transcription factors (Santos, M., et al.(2014).In In vivo disruption of an Rb-E2F-Ezh2 signaling loop causes bladder cancer. Cancer Research 74(22), 6565-6577. Coe, BP, et al. (2013). Genomic deregulation of the E2F / Rb pathway leads to activation of the oncogene EZH2 in small cell lung cancer. PLoS One 8(8), e71670. Bracken, AP, et al. (2003). EZH2 is downstream of the pRB-E2F pathway, essential for proliferation and amplified in cancer. EMBO J 22(20), 5323-5335), including, for example, deletion of the RB1 gene. Thus, there are several recurrent genomic abnormalities in cancer that cause EZH2 overexpression, demonstrating that increased EZH2 levels promote tumor progression. To this end, EZH2 has been implicated in numerous cancer targets, including hematological malignancies and solid tumors. See, for example, WO2014 / 124418.

[0004] An EZH2 inhibitor that has attracted attention due to its antitumor activity and prolonged residence in the PRC2 complex (approximately 101 days) is 7-chloro-2-(4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4-dimethyl-N-((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)benzo[d][1,3]dioxole-5-carboxamide. See, for example, PCT / US2019 / 027932, the contents of which are incorporated herein by reference. Given its therapeutic potential and the prevalence of diseases such as cancer, there is a need for alternative therapeutic uses for this compound, for example, for use in combination-based therapies. Summary of the Invention

[0005] 7-chloro-2-(4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4-dimethyl-N-((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)benzo[d][1,3]dioxole-5-carboxamide Provided herein are methods for treating cancer using a cisplatin, or a pharmaceutically acceptable salt thereof, and a second agent selected from a topoisomerase inhibitor, a DNA alkylating agent, and an androgen receptor signaling inhibitor.

[0006] Also provided herein are methods of treating advanced recurrent solid tumors using 7-chloro-2-(4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4-dimethyl-N-((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)benzo[d][1,3]dioxole-5-carboxamide, or a pharmaceutically acceptable salt thereof, as monotherapy.

[0007] Also provided herein is a pharmaceutical composition comprising 7-chloro-2-(4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4-dimethyl-N-((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)benzo[d][1,3]dioxole-5-carboxamide, or a pharmaceutically acceptable salt thereof, a second agent selected from a topoisomerase inhibitor, a DNA alkylating agent, and an androgen receptor signaling inhibitor, and optionally a pharmaceutically acceptable carrier. [Brief explanation of the drawings]

[0008] [Figure 1A] Figure 1 shows the phenotypic response of cisplatin-sensitive and -resistant A2780 (ovarian cancer) and HT1376 (bladder cancer) cell lines to a single treatment with cisplatin. Data shown are mean cell viability ± standard error of the mean (SEM), n = 2–3, and are representative of independent experiments with replicates. [Figure 1B]Figure 1 shows the phenotypic response of cisplatin-sensitive and -resistant A2780 (ovarian cancer) and HT1376 (bladder cancer) cell lines to a single treatment with compound 1. Data shown are mean cell viability ± standard error of the mean (SEM), n = 2–3, representative of independent experiments with replicates. [Figure 2A] Representative growth curves are shown for cisplatin alone and in combination with a dose titration of Compound 1 in cisplatin-sensitive and -resistant A2780 ovarian cancer cell lines. Representative of replicate independent experiments, mean ± SD is shown. [Figure 2B] Figure 1 shows the sub-GI50 dose for cisplatin in A2780-P and A2780-CR in combination with a sub-GI50 dose of 16 nM Compound 1. Representative of replicate independent experiments, mean ± SD is shown. [Figure 3A] Representative growth curves are shown for cisplatin alone and in combination with dose titrations of Compound 1 in cisplatin-sensitive and -resistant HT1376 bladder cancer cell lines. Shown are representative of replicate independent experiments, mean ± SD. [Figure 3B] Figure 1 shows near-GI50 dose combinations for cisplatin and Compound 1 in HT1376-DMF and HT1376-CR. Representative of replicate independent experiments, mean ± SD is shown. [Figure 4] Figure 1 shows the antitumor effects of Compound 1, cisplatin, and a combination of both on HT1376 tumors in CB17 SCID mice. Data shown are mean tumor size ± SEM, n = 6. PO = oral administration, IV = intravenous administration, QD = once daily, QW = once weekly. [Figure 5] Figure 1 shows the antitumor effects of Compound 1, enzalutamide, and a combination of both in the CTG-2428 patient-derived xenograft (PDX) model of prostate cancer. Data shown are mean tumor size ± SEM, n = 5 per arm. Arrows indicate unscheduled death or end of life due to achievement of maximum tumor volume, and n indicates the remaining animals per arm. PO = oral administration, QD = once daily. [Figure 6] Figure 1 shows the antitumor effects of Compound 1, enzalutamide, and the combination of both in the CTG-2440 PDX model of prostate cancer. Data shown are mean tumor size ± SEM, PO = oral administration, QD = once daily. Arrows indicate the death of animals in the combination arm, causing a reduction in group size, and removal of material from individual animals due to maximum tumor volume; n = remaining animals in the arm. [Figure 7] Figure 1 shows the antitumor effects of Compound 1, enzalutamide, and a combination of both in a CTG-2441 PDX model of prostate cancer. Data shown are mean tumor size ± SEM, n = 5. Arrows indicate animal deaths, causing a reduction in group size; n = remaining animals in the arm. PO = oral administration, QD = once daily. DETAILED DESCRIPTION OF THE INVENTION

[0009] In a first embodiment, a method of treating cancer in a subject is provided, comprising administering to the subject an effective amount of 7-chloro-2-(4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4-dimethyl-N-((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)benzo[d][1,3]dioxole-5-carboxamide, or a pharmaceutically acceptable salt thereof, and an effective amount of a second agent selected from a topoisomerase inhibitor and an androgen receptor signaling inhibitor. Alternatively, as part of the first embodiment, there is provided the use of an effective amount of 7-chloro-2-(4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4-dimethyl-N-((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)benzo[d][1,3]dioxole-5-carboxamide, or a pharmaceutically acceptable salt thereof, and an effective amount of a second agent selected from a topoisomerase inhibitor and an androgen receptor signaling inhibitor, for the manufacture of a medicament for treating cancer in a subject. In another alternative, as part of the first embodiment, there is provided an effective amount of 7-chloro-2-(4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4-dimethyl-N-((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)benzo[d][1,3]dioxole-5-carboxamide, or a pharmaceutically acceptable salt thereof, and an effective amount of a second agent selected from a topoisomerase inhibitor and an androgen receptor signaling inhibitor, for use in treating cancer in a subject.

[0010] 7-Chloro-2-(4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4-dimethyl-N-((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)benzo[d][1,3]dioxole-5-carboxamide has the following chemical formula: [ka] and is disclosed in International Application No. PCT / US2019 / 027932, the contents of which are incorporated herein by reference. "Compound 1" is used interchangeably with 7-chloro-2-(4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4-dimethyl-N-((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)benzo[d][1,3]dioxole-5-carboxamide, each including stereoisomeric and geometric isomeric forms.

[0011] The topoisomerase inhibitor of this method refers to a chemical or biological agent that blocks the action of topoisomerases (including topoisomerase I and II). As an example, the topoisomerase inhibitor of the present method (e.g., as in the first embodiment) includes, but is not limited to, irinotecan, topotecan, camptothecin, lamellarin, etoposide, teniposide, doxorubicin, daunorubicin, mitoxantrone, amsacrine, ellipticine, aurintricarboxylic acid, HU-331, epirubicin, valrubicin, idarubicin, pixantrone, teniposide, belotecan, gimatecan, indotecan, and indimitecan. Alternatively, as part of a second embodiment, the topoisomerase inhibitor of the present method (e.g., as in the first embodiment) is a topoisomerase I inhibitor. In another alternative, as part of a second embodiment, the topoisomerase inhibitor of the present method (e.g., as in the first embodiment) is irinotecan. In another alternative, as part of a second embodiment, the topoisomerase inhibitor of the method (eg, as in the first embodiment) is topotecan.

[0012] The DNA alkylating agent of the present method refers to a chemical or biological agent that acts by preventing DNA strands from joining together as they should. As part of a third embodiment, the DNA alkylating agent of the present method (e.g., as in the first embodiment) is selected from busulfan, cyclophosphamide, bendamustine, carboplatin, chlorambucil, cyclophosphamide, cisplatin, temozolomide, melphalan, carmustine, lomustine, dacarbazine, oxaliplatin, ifosamide, thiotepa, trabectedin, altretamine, mechlorethamine, procarbazine, and streptozocin. Alternatively, as part of a third embodiment, the DNA alkylating agent of the present method (e.g., as in the first embodiment) is cisplatin.

[0013] The androgen receptor signaling inhibitor of this method refers to a chemical or biological agent that blocks androgen receptor (AR) and inhibits or suppresses androgen production.As part of the fourth embodiment, the androgen receptor signaling inhibitor of this method (such as the first embodiment) is selected from bicalutamide, enzalutamide, apalutamide, flutamide, nilutamide, darolutamide, and abiraterone acetate (abiraterone acetate can be included alone or in combination with prednisone).Alternatively, as part of the fourth embodiment, the androgen receptor signaling inhibitor of this method (such as the first embodiment) is enzalutamide.In another alternative example, as part of the fourth embodiment, the androgen receptor signaling inhibitor of this method (such as the first embodiment) is abiraterone acetate (abiraterone acetate can be included alone or in combination with prednisone).

[0014] As used herein, the terms "treatment," "treat," and "treating" refer to ameliorating, alleviating, or inhibiting the progression of cancer, or one or more symptoms thereof, as described herein.

[0015] When used to define cancer, the term "advanced," such as "advanced cancer" or "advanced prostate cancer," means that the recited cancer is defined as unresectable, i.e., the cancer cannot be completely removed by surgery, or the cancer is metastatic, or both. In one embodiment, "advanced cancer" means that the cancer is unresectable.

[0016] The cancers described herein may also be "recurrent" cancers. The term "recurrent cancer" refers to cancers that were previously in remission and have returned, or cancer signs and symptoms have returned. Remission includes both partial remission (where some or not all signs and symptoms of cancer have disappeared) and complete remission (where all signs and symptoms of cancer have disappeared, but the cancer may still remain in the body). Thus, "advanced recurrent" cancer means that the cancer was in remission, has returned, and is no longer resectable.

[0017] Exemplary types of cancers treated by the present methods (e.g., as in the first, second, third, or fourth embodiments) include, for example, adrenal gland carcinoma, acinic cell carcinoma, acoustic neuroma, acral lentiginous melanoma, acral hidradenoma, acute eosinophilic leukemia, acute erythroleukemia, acute lymphoblastic leukemia, acute megakaryoblastic leukemia, acute monocytic leukemia, acute promyelocytic leukemia, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenomatous odontogenic tumor, adenosquamous carcinoma, adipose tissue neoplasm, adrenocortical carcinoma, adult T-cell leukemia / lymphoma, aggressive NK-cell leukemia, AIDS-related lymphoma, alveolar rhabdomyosarcoma, alveolar leukemia, and pulmonary leukemia. Focal soft part sarcoma, ameloblastic fibroma, anaplastic large cell lymphoma, anaplastic thyroid carcinoma, angioimmunoblastic T-cell lymphoma, angiomyolipoma, angiosarcoma, astrocytoma, atypical teratoid rhabdoid tumor, B-cell chronic lymphocytic leukemia, B-cell prolymphocytic leukemia, B-cell lymphoma, basal cell carcinoma, biliary tract cancer, bladder cancer, blastoma, bone cancer, Brenner tumor, Brown tumor, Burkitt lymphoma, breast cancer, brain cancer, carcinoma, carcinoma in situ, carcinosarcoma, cartilage tumor, cementoma, myeloid sarcoma, chondroma, chordoma, choriocarcinoma, choroid plexus papilloma, clear cell sarcoma of the kidney, craniopharyngioma , cutaneous T-cell lymphoma, cervical cancer, colorectal cancer, Degos disease, desmoplastic small round cell tumor, diffuse large B-cell lymphoma, dysembryoplastic neuroepithelial tumor, dysgerminoma, embryonal carcinoma, endocrine neoplasms, endodermal sinus tumor, enteropathy-associated T-cell lymphoma, esophageal cancer, inclusion fetal tumor, fibroma, fibrosarcoma, follicular lymphoma, thyroid follicular carcinoma, ganglioneuroma, gastrointestinal cancer, germ cell tumor, gestational choriocarcinoma, giant cell fibroblastoma, giant cell tumor of bone, glial tumor, glioblastoma multiforme, glioma, gliomatosis cerebri, glucagonoma, gonadoblastoma, granulosa cell tumor, male germ cell tumor Cystoma, gallbladder cancer, gastric cancer, hairy cell leukemia, hemangioblastoma, head and neck cancer, hemangiopericytoma, hematologic malignancies, hepatoblastoma, hepatosplenic T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, invasive lobular carcinoma, intestinal cancer, kidney cancer, laryngeal cancer, lentigo maligna, lethal midline carcinoma, leukemia, Leydig cell tumor, liposarcoma, lung cancer, lymphangioma, lymphangiosarcoma, lymphoepithelioma, lymphoma, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, liver cancer, small cell lung cancer, non-small cell lung cancer, MALT lymphoma, malignant fibrous histiocytoma, malignant peripheral nerve sheath tumor, malignant Triton tumor,Mantle cell lymphoma, marginal zone B-cell lymphoma, mast cell leukemia, mediastinal germ cell tumor, medullary carcinoma of the breast, medullary thyroid carcinoma, medulloblastoma, melanoma, meningioma, Merkel cell carcinoma, mesothelioma, metastatic urothelial carcinoma, mixed Müllerian tumor, mucinous tumor, multiple myeloma, muscle tissue neoplasm, mycosis fungoides, myxoid liposarcoma, myxoma, myxosarcoma, nasopharyngeal carcinoma, schwannoma, neuroblastoma, neurofibroma, neuroma, nodular melanoma, ocular Cancer, oligoastrocytoma, oligodendroglioma, oncocytoma, optic nerve sheath meningioma, optic nerve tumor, oral cancer, osteosarcoma, ovarian cancer, Pancoast tumor, papillary thyroid cancer, paraganglioma, pineoblastoma, pineocytoma, pituitary adenoma, pituitary tumor, plasmacytoma, polyembryomatous tumor, precursor T lymphoblastic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, primary peritoneal cancer, prostate cancer, pancreatic cancer, pharyngeal cancer, Pseudomyxoma peritonei, renal cell carcinoma, renal medullary carcinoma, retinoblastoma, rhabdomyoma, rhabdomyosarcoma, Richter transformation, rectal cancer, sarcoma, schwannomatosis, seminoma, Sertoli cell tumor, sex cord-gonadal stromal tumor, signet ring cell carcinoma, skin cancer, small blue round cell tumor, small cell carcinoma, soft tissue sarcoma, somatostatinoma, sooty warts, spinal tumor, splenic marginal zone lymphoma, squamous cell carcinoma, synovial sarcoma, Sézary These include small intestine cancer, squamous cell carcinoma, gastric cancer, T-cell lymphoma, testicular cancer, theca cell carcinoma, thyroid cancer, transitional cell carcinoma, laryngeal cancer, urachal cancer, genitourinary cancer, urothelial carcinoma, uveal melanoma, metastatic castration-resistant prostate cancer, ovarian clear cell carcinoma, uterine cancer, verrucous carcinoma, visual pathway glioma, vulvar cancer, vaginal cancer, Waldenstrom's macroglobulinemia, Warthin's tumor, and Wilms' tumor.

[0018] In one aspect, as part of the fifth embodiment, the cancer treated by the method (e.g., as in the first, second, third, or fourth embodiment) is a solid tumor. As presented herein, a solid tumor refers to an abnormal mass of tissue that typically does not contain cysts or liquid areas. Solid tumors can be benign or malignant, and the types of cells that form them can vary. Examples of solid tumors include, for example, sarcomas, carcinomas, and lymphomas.

[0019] In one aspect, as part of a sixth embodiment, the cancer treated by the method (e.g., as in the first, second, third, or fourth embodiment) is a solid malignant tumor. Alternatively, as part of a fifth embodiment, the solid tumor treated by the method (e.g., as in the first, second, third, or fourth embodiment) is selected from bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, uterine cancer, kidney cancer, lip cancer, oral cancer, liver cancer, skin cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, and gastric or gastroesophageal cancer. In another alternative, as part of a sixth embodiment, the solid tumor treated by the method (e.g., as in the first, second, or third embodiment) is selected from prostate cancer, small cell lung cancer (SCLC), gastric or gastroesophageal junction (GEJ) adenocarcinoma, and serous ovarian cancer. In another alternative, as part of a sixth embodiment, the solid tumor treated by the method (e.g., as in the first, second, third, or fourth embodiment) is selected from small cell lung cancer (SCLC), gastric or gastroesophageal junction (GEJ) adenocarcinoma, and serous ovarian cancer. In another alternative, as part of a sixth embodiment, the solid tumor treated by the method (e.g., as in the first, second, third, or fourth embodiment) is prostate cancer. In another alternative, as part of a sixth embodiment, the solid tumor treated by the method (e.g., as in the first, second, third, or fourth embodiment) is selected from urothelial carcinoma, ovarian clear cell carcinoma, and endometrial carcinoma.

[0020] In one aspect, as part of a seventh embodiment, the cancer treated by the method (e.g., as in the first through sixth embodiments) is a recurrent cancer. Thus, as part of a sixth embodiment, the cancer treated by the method (e.g., as in the first through sixth embodiments) is a recurrent solid tumor, such as recurrent prostate cancer, recurrent small cell lung cancer (SCLC), recurrent gastric or gastroesophageal junction (GEJ) adenocarcinoma, and recurrent serous ovarian cancer.

[0021] In one aspect, the cancer described herein (e.g., as in the fourth through seventh embodiments) is an advanced cancer, such as advanced prostate cancer, advanced small cell lung cancer (SCLC), advanced gastric or gastroesophageal junction (GEJ) adenocarcinoma, and advanced serous ovarian cancer.

[0022] Unless otherwise indicated, administration as described herein includes administration of 7-chloro-2-(4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4-dimethyl-N-((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)benzo[d][1,3]dioxole-5-carboxamide to treat the recited cancers (e.g., as in the fifth through seventh embodiments) before, concurrently with, or after administration of a topoisomerase inhibitor or androgen receptor signaling inhibitor disclosed herein (e.g., as in the first, second, third, or fourth embodiments). Thus, simultaneous administration is not required for therapeutic purposes. However, in one embodiment, 7-chloro-2-(4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4-dimethyl-N-((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)benzo[d][1,3]dioxole-5-carboxamide is administered simultaneously with a topoisomerase inhibitor or an androgen receptor signaling inhibitor.

[0023] In an eighth embodiment, provided herein is a method of treating advanced recurrent solid tumors using 7-chloro-2-(4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4-dimethyl-N-((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)benzo[d][1,3]dioxole-5-carboxamide, or a pharmaceutically acceptable salt thereof. Alternatively, one of the seventh embodiment In part, there is provided the use of an effective amount of 7-chloro-2-(4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4-dimethyl-N-((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)benzo[d][1,3]dioxole-5-carboxamide, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for treating an advanced recurrent solid tumor in a subject. In another alternative, as part of an eighth embodiment, there is provided an effective amount of 7-chloro-2-(4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4-dimethyl-N-((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)benzo[d][1,3]dioxole-5-carboxamide, or a pharmaceutically acceptable salt thereof, for use in treating an advanced recurrent solid tumor in a subject.

[0024] The advanced recurrent solid tumors described herein (e.g., as in the seventh embodiment) include, but are not limited to, advanced recurrent urothelial carcinoma, advanced recurrent ovarian clear cell carcinoma, and advanced recurrent endometrial carcinoma.

[0025] In a ninth embodiment, provided herein is a pharmaceutical composition comprising an effective amount of 7-chloro-2-(4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4-dimethyl-N-((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)benzo[d][1,3]dioxole-5-carboxamide, or a pharmaceutically acceptable salt thereof, an effective amount of a second agent selected from a topoisomerase inhibitor and an androgen receptor signaling inhibitor, and optionally a pharmaceutically acceptable carrier. Also included is the use of a pharmaceutical composition (e.g., as in the fifth through seventh embodiments) comprising an effective amount of 7-chloro-2-(4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4-dimethyl-N-((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)benzo[d][1,3]dioxole-5-carboxamide, or a pharmaceutically acceptable salt thereof, and an effective amount of a second agent selected from a topoisomerase inhibitor and an androgen receptor signaling inhibitor, and optionally a pharmaceutically acceptable carrier, for treating one or more cancers described herein. Further provided is the use of a pharmaceutical composition comprising 7-chloro-2-(4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4-dimethyl-N-((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)benzo[d][1,3]dioxole-5-carboxamide, or a pharmaceutically acceptable salt thereof, for treating advanced recurrent solid tumors.

[0026] In one aspect, 7-chloro-2-(4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4-dimethyl-N-((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)benzo[d][1,3]dioxole-5-carboxamide is in crystalline Form 1 characterized by at least three X-ray powder diffraction peaks at 2θ angles selected from 10.0°, 13.3°, 14.9°, 20.2°, 20.8°, 22.2°, and 22.5°. Alternatively, 7-chloro-2-(4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4-dimethyl-N-((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)benzo[d][1,3]dioxole-5-carboxamide is of crystalline Form 1 characterized by at least four X-ray powder diffraction peaks at 2θ angles selected from 10.0°, 13.3°, 14.9°, 20.2°, 20.8°, 22.2°, and 22.5°. In another alternative, 7-chloro-2-(4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4-dimethyl-N-((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)benzo[d][1,3]dioxole-5-carboxamide is selected from the group consisting of 10.0°, 13.3°, 14.9°, 20.2°, 20.8°, 22.2°, and 22.5°. In another alternative, 7-chloro-2-(4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4-dimethyl-N-((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)benzo[d][1,3]dioxole-5-carboxamide is of crystalline Form 1 characterized by at least six X-ray powder diffraction peaks at 2θ angles selected from 10.0°, 13.3°, 14.9°, 20.2°, 20.8°, 22.2°, and 22.5°. In another alternative, 7-chloro-2-(4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4-dimethyl-N-((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)benzo[d][1,3]dioxole-5-carboxamide is of crystalline Form 1 characterized by X-ray powder diffraction peaks at 2θ angles selected from 10.0°, 13.3°, 14.9°, 20.2°, 20.8°, 22.2°, and 22.5°. In another alternative, 7-chloro-2-(4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4-dimethyl-N-((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)benzo[d][1,3]dioxole-5-carboxamide is of crystalline Form 1 characterized by X-ray powder diffraction peaks at 2θ angles selected from 10.0°, 10.2°, 12.3°, 12.7°, 13.3°, 14.9°, 15.3°, 20.2°, 20.8°, 21.3°, 22.2°, 22.5°, and 23.8°.In another alternative, 7-chloro-2-(4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4-dimethyl-N-((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)benzo[d][1,3]dioxole-5-carboxamide is of crystalline Form 1 characterized by X-ray powder diffraction peaks at 2θ angles selected from 10.0°, 10.2°, 11.0°, 11.4°, 11.8°, 12.3°, 12.7°, 13.3°, 14.9°, 15.3°, 16.1°, 17.4°, 20.2°, 20.8°, 21.3°, 22.2°, 22.5°, and 23.8°. In another alternative, 7-chloro-2-(4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4-dimethyl-N-((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)benzo[d][1,3]dioxole-5-carboxamide is of crystalline Form 1 characterized by X-ray powder diffraction peaks at 2θ angles selected from 14.9°, 20.2°, and 20.8°. In another alternative, 7-chloro-2-(4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4-dimethyl-N-((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)benzo[d][1,3]dioxole-5-carboxamide is of crystalline Form 1 characterized by X-ray powder diffraction peaks at 2θ angles selected from 10.0°, 14.9°, 20.2°, and 20.8°. In another alternative, 7-chloro-2-(4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4-dimethyl-N-((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)benzo[d][1,3]dioxole-5-carboxamide is of crystalline Form 1 characterized by X-ray powder diffraction peaks at 2θ angles selected from 10.0°, 14.9°, 20.2°, 20.8°, and 22.2°.In another alternative, 7-chloro-2-(4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4-dimethyl-N-((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)benzo[d][1,3]dioxole-5-carboxamide is of crystalline Form 1 characterized by X-ray powder diffraction peaks at 2θ angles selected from 10.0°, 13.3°, 14.9°, 20.2°, 20.8°, and 22.2°.

[0027] In one aspect, the 7-chloro-2-(4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4-dimethyl-N-((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)benzo[d][1 ,3]dioxole-5-carboxamide is (2R)-7-chloro-2-(trans-4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4-dimethyl-N-((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)benzo[d][1,3]dioxole-5-carboxamide.

[0028] The term "pharmaceutically acceptable carrier" refers to a non-toxic carrier, adjuvant, or vehicle that does not adversely affect the pharmacological activity of the compound with which it is formulated and that is safe for human use. Pharmaceutically acceptable carriers, adjuvants, or vehicles that may be used in the compositions of the present disclosure include, but are not limited to, ion exchange agents, alumina, aluminum stearate, magnesium stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substances (e.g., microcrystalline cellulose, hydroxypropylmethylcellulose, lactose monohydrate, sodium lauryl sulfate, and croscarmellose sodium), polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wool fat.

[0029] The compounds described herein can exist in the form of pharmaceutically acceptable salts.For pharmaceutical use, the salts of the compounds described herein refer to non-toxic " pharmaceutically acceptable salts ".Pharmaceutically acceptable salt forms include, if possible, pharmaceutically acceptable acidic / anionic or basic / cationic salts.

[0030] The compositions and methods of administration herein may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implanted reservoir. As used herein, the term "parenteral" includes subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques.

[0031] It will also be understood that the specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including age, weight, general health, sex, diet, time of administration, rate of excretion, drug combination, the judgment of the treating physician, and the severity of the particular disease being treated. The amount of compound provided in the composition will also depend on the particular compound in the composition.

[0032] The terms "subject" and "patient" may be used interchangeably and refer to a mammal in need of treatment, such as companion animals (e.g., dogs, cats, etc.), livestock (e.g., cows, pigs, horses, sheep, goats, etc.), and laboratory animals (e.g., rats, mice, guinea pigs, etc.). Typically, the subject is a human in need of treatment.

[0033] The term "effective amount" or "therapeutically effective amount" refers to an amount of a compound described herein that will induce a biological or medical response in a subject, e.g., a dosage of 0.01 to 100 mg / kg body weight / day. In one embodiment, an effective amount of 7-chloro-2-(4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4-dimethyl-N-((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)benzo[d][1,3]dioxole-5-carboxamide, or a pharmaceutically acceptable salt thereof, and an effective amount of a topoisomerase inhibitor or androgen receptor signaling inhibitor described herein are combined to elicit a measurable combined effect for treating one or more cancers described herein.

[0034] Example The invention will now be illustrated by the following non-limiting examples.

[0035] Compound 1 can be prepared as a single enantiomer, a single geometric isomer using the following procedure.

[0036] Intermediate 1: 7-chloro-2,4-dimethyl-2-(4-oxocyclohexyl)benzo[d][1,3]dioxole-5-carboxylate methyl ester [ka]

[0037] Step 1: Synthesis of methyl 5-chloro-3,4-dihydroxy-2-methylbenzoate To a solution of methyl 3,4-dihydroxy-2-methylbenzoate (5.11 g, 27.9 mmol) in tetrahydrofuran (199 mL) was added sulfuryl chloride (2.45 mL, 30.6 mmol) dropwise at -20°C. The reaction mixture was stirred at -20°C for 3 hours and then quenched with a saturated aqueous solution of ammonium chloride (50 mL). The desired product was extracted with ethyl acetate (25 mL x 3). The combined organic layers were washed with brine (25 mL), dried over sodium sulfate, filtered, and concentrated to dryness under reduced pressure. The residue was purified by flash chromatography (silica gel, 0% to 60% ethyl acetate gradient in heptane) to give the title compound (4.117 g, 68% yield) as a beige solid. LCMS [M+H] + m / z: calculated 217.0, found 217.1 (Cl isotope pattern).

[0038] Step 2: Synthesis of methyl 7-chloro-2,4-dimethyl-2-(4-oxocyclohexyl)-2H-1,3-benzodioxole-5-carboxylate A mixture of methyl 5-chloro-3,4-dihydroxy-2-methylbenzoate (1.2 g, 5.53 mmol), triruthenium dodecacarbonyl (176 mg, 276 μmol), and triphenylphosphine (145 mg, 553 μmol) was degassed under vacuum and purged with nitrogen (three cycles). Toluene (8.1 mL) was added, and the reaction mixture was heated to reflux for 30 minutes. A solution of 4-ethynylcyclohexan-1-one (1.34 g, 11.0 mmol) in toluene (17 mL) was then added dropwise, and the reaction was stirred at reflux for 23 hours. Finally, the reaction mixture was cooled to room temperature and concentrated to dryness under reduced pressure. The residue was purified by flash chromatography (silica gel, 0-60% ethyl acetate gradient in heptane) to give the title compound (1.327 g, 70% yield) as a yellow oil. LCMS [M+Na] + m / z: calculated 361.1, found 361.1 (Cl isotope pattern).

[0039] Step 3: (R)-7-chloro-2,4-dimethyl-2-(4-oxocyclohexyl) ) Separation of methyl benzo[d][1,3]dioxole-5-carboxylate and (S)-7-chloro-2,4-dimethyl-2-(4-oxocyclohexyl)benzo[d][1,3]dioxole-5-carboxylate A racemic mixture of methyl 7-chloro-2,4-dimethyl-2-(4-oxocyclohexyl)benzo[d][1,3]dioxole-5-carboxylate (4.4 g, 13 mmol) was resolved by preparative SFC [Column: Daicel Chemical Industries ChiralPak AY (250 mm × 50 mm, 10 μm i.d.). Mobile phase A: CO / Mobile phase B: 0.1% NHOH in methanol. Isocratic (85% mobile phase A and 15% mobile phase B). Flow rate: 80 mL / min. Column temperature: 40 °C]. Intermediate 1 (Peak 1) (unwanted enantiomer / distomer): Retention time = 6.2 min. Recovery = 1.4 g, 4.05 mmol, 31% yield, 90% ee, 98% purity (yellow solid). 1H NMR (400 MHz, chloroform-d) δ 7.48 (s, 1H), 3.78 (s, 3H), 2.44-2.36 (m, 2H), 2.35-2.25 (m, 6H), 2.19 (tdd, J = 2.8, 5.6, 13.1 Hz, 2H), 1.70-1.57 (m, 5H). Intermediate 1 (peak 2) (desired enantiomer / eutomer): retention time = 7.0 min. Recovery = 1.1 g, 3.08 mmol, 23.75% yield, 99% ee, 95% purity (yellow solid). 1 H NMR (400 MHz, chloroform-d) δ 7.49 (s, 1H), 3.78 (s, 3H), 2.44-2.36 (m, 2H), 2.36-2.25 (m, 6H), 2.20 (tdd, J = 2.8, 5.6, 13.1 Hz, 2H), 1.72-1.59 (m, 5H). SFC analytical method: [Column: ChiralPak AY-3 (150 × 4.6 mm i.d., 3 μm). Mobile phase A: CO₂ / Mobile phase B: 0.05% Et₂NH in iPrOH. Gradient: 5% to 40% Mobile phase B (over 5.5 min). Flow rate: 2.5 mL / min. Column temperature: 40 °C]. Intermediate 1 (Peak 1 - Unwanted Enantiomer / Distomer): Retention time = 2.853 min. Intermediate 1 (Peak 2 - desired enantiomer / eutomer): Retention time = 2.979 min.

[0040] Intermediate 2: 7-chloro-2-(4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4-dimethylbenzo[d][1,3]dioxole-5-carboxylic acid [ka]

[0041] Step 1: Synthesis of methyl 7-chloro-2-(4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4-dimethylbenzo[d][1,3]dioxole-5-carboxylate A solution of 3-methoxyazetidine hydrochloride (8 g, 64.75 mmol) and N,N-diisopropylethylamine (12 mL, 68.9 mmol) in methanol (30 mL) was added to the chamber. After stirring at room temperature for 30 minutes, another solution of methyl 7-chloro-2,4-dimethyl-2-(4-oxocyclohexyl)-1,3-benzodioxole-5-carboxylate (Intermediate 1 - Peak 2) (4.1 g, 12.10 mmol) in tetrahydrofuran (30 mL) was added. The reaction mixture was stirred at room temperature for 1 hour and then cooled to -70 °C. Lithium borohydride (500 mg, 22.96 mmol) was added, and the reaction was stirred at -70 °C for 30 minutes [or until complete consumption of the starting material was observed by TLC, ethyl acetate / methanol 5:1]. The two batches of reaction were then combined and quenched with saturated aqueous ammonium chloride (120 mL) at 0 °C, and the desired product was extracted with dichloromethane (200 mL × 3). The combined organic layers were dried over sodium sulfate, filtered, and concentrated to dryness under reduced pressure. The residue was purified by flash chromatography (silica gel, 0% to 14% gradient of methanol in dichloromethane) to give the title compound (8.05 g, 67% yield, 83% purity) as a pale yellow oil. A sample (50 mg) was further purified by preparative thin layer chromatography (silica gel, ethyl acetate:methanol 15:1). LCMS [M+H] + m / z: calculated 410.2, observed 410.1. 1 H NMR (400MHz, methanol-d4) δ7.39(s,1H),3.95-3.91(m,1H),3.73(s,3H),3.59-3.51(m,2H),3.16(s,3H),2.97(br dd,J=6.4,8.0Hz,2H),2.26(s,3H),2.11-2.02(m,1H),1.91-1.73(m,5H),1.54(s,3H),1.22-1.12(m,2H),0.98-0.86(m,2H).

[0042] Step 2: Synthesis of 7-chloro-2-(4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4-dimethylbenzo[d][1,3]dioxole-5-carboxylic acid To a solution of methyl 7-chloro-2-(4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4-dimethylbenzo[d][1,3]dioxole-5-carboxylate (4 g, 9.75 mmol) in methanol (48 mL) was added a solution of lithium hydroxide hydrate (4.03 g, 96.06 mmol) in water (12 mL). The reaction was stirred at 70 °C for 2 hours, then the two batches were combined and concentrated under reduced pressure. Water (50 mL) was added and the pH was adjusted to 6 with a saturated aqueous solution of citric acid at 0 °C. The desired product was extracted with a 3:1 mixture of dichloromethane and isopropanol (300 mL × 5). The combined organic layers were dried over sodium sulfate, filtered, and concentrated to dryness under reduced pressure to give the title compound (6.1 g, crude) as an off-white solid, which was used in the next step without further purification. LCMS [M+H] + m / z: calculated 396.2, observed 396.1. 1 H NMR (400MHz, methanol-d4) δ7.07(s,1H),4.05-4.10(m,2H),3.76-3.88(m,1H),3.67(br dd,J=10,3.6Hz,2H),3.22(s,3H),2.71-2.81(m,1H),2.19(s,3H),1.91-1.99 (m,4H),1.75-1.85(m,1H),1.52(s,3H),1.18-1.28(m,2H),1.06-1.14(m,2H). compound 1 [ka]

[0043] To a solution of 7-chloro-2-(4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4-dimethylbenzo[d][1,3]dioxole-5-carboxylic acid (Intermediate 2 - single enantiomer and geometric isomer) (5 g, 12.63 mmol) in N,N-dimethylformamide (50 mL) was added O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (5.7 g, 14.99 mmol) and N,N-diisopropylethylamine (11 mL, 63.15 mmol). The mixture was stirred at 20 °C for 30 min, and then 3-(aminomethyl)-6-methyl-4-(methylthio)pyridin-2(1H)-one hydrochloride (Intermediate 1) (4.2 g, 19.03 mmol) was added. The reaction mixture was stirred at room temperature for an additional 1.5 hours and then filtered. The filtrate was purified by preparative HPLC [column: Phenomenex Gemini C18 (250 mm × 50 mm, 10 μm), mobile phase A: water (0.04% ammonium hydroxide v / v and 10 mM ammonium bicarbonate) / mobile phase B: acetonitrile. Gradient (75% to 44% mobile phase A / 25% to 56% mobile phase B, over 23 min). Column temperature: 30 °C] to give the title compound (4.4 g, 60% yield, 96% purity) as a white solid. LCMS [M+H] + m / z: calculated 562.2, observed 562.2. 1 H NMR (400MHz, methanol-d4) δ6.91(s,1H),6.29(s,1H),4.50(s,2H),4.01(sextet,J=6Hz,1H),3.58(dd,J=8.8,6.4Hz,2H),3.26(s,3H),2.92-3.0 2(m,2H),2.54(s,3H),2.31(s,3H),2.21(s,3H),2.01-2.11(m,1H),1 .79-2.00(m,5H),1.62(s,3H),1.19-1.34(m,2H),0.91-1.08(m,2H).

[0044] 1. Primary In Vitro Pharmacology A. Mechanism of Action In biochemical assays, compound 1 inhibited the catalytic activity of wild-type and Y641N mutant EZH2-containing PRC2 complexes, as well as EZH1-containing PRC2 complexes, with half-maximal inhibitory concentration (IC50) values ​​of 0.02 nM and 0.03 nM for wild-type and Y641N mutant EZH2, respectively, and 0.06 nM for EZH1. See, e.g., PCT / US2019 / 027932. The biochemical potency underestimated the true affinity of compound 1, and further characterization of binding by kinetic assays confirmed an inhibition constant of approximately 0.11 pM for EZH2, and approximately 70-fold selectivity for EZH2 over EZH1. Based on kinetic analysis, compound 1 was determined to bind to PRC2 with a long residence time (approximately 101 days). See, e.g., PCT / US2019 / 027932.

[0045] B. Global H3K27me3 intracellular levels and effects on gene expression The ability of Compound 1 to reduce global H3K27me3 intracellular levels was evaluated in a wild-type EZH2-containing cervical cancer cell line (HeLa). After 4 days of treatment, Compound 1 was able to reduce global H3K27me3 levels to an EC50 of 0.40 nM. See, for example, PCT / US2019 / 027932. Compound 1 also showed similar efficacy in other solid tumor cell lines, including bladder cancer (639V and HT1197) and ovarian cancer TOV21G cell lines, with EC50 values ​​of 0.09 nM, 0.14 nM, and 0.26 nM, respectively, on day 3.

[0046] The reduction in H3K27me3 levels leads to changes in gene expression. RNA sequencing of bladder cancer cell lines after 4 days of treatment with Compound 1 revealed significant changes in the expression levels of multiple genes. The predominant change was an increase in gene expression, with very few genes significantly decreased. The increase in gene expression was both dose- and time-dependent, with increased expression observed at higher concentrations of Compound 1 and later time points. This contrasts with the reduction in H3K27me3, as changes in methyl marks were observed 1 day after treatment with Compound 1. Notably, one of the highly up-regulated genes was CDKN1C (also known as p57 or Kip2), a known tumor suppressor and negative regulator of the cell cycle previously reported as an EZH2 target gene. See Yang X, Karuturi RK, Sun F, et al. CDKN1C(p57) is a direct target of EZH2 and suppressed by multiple epigenetic mechanisms in breast cancer cells. PLoS One. 2009;4(4):e5011. Low expression of CDKN1C is seen in advanced bladder and breast cancer and correlates with poor prognosis. See Yang above and Hoffmann MJ, Florl AR, Seifert HH, et al. Multiple mechanisms downregulate CDKN1C in human bladder cancer. Int J Cancer. 2005 Apr 10;114(3):406-13.

[0047] 2. Antiproliferative effects A. Synergistic effect of Compound 1 with cisplatin (a DNA alkylating agent) We evaluated the sensitivity of multiple solid tumor cancer cell lines to the antiproliferative activity of Compound 1, with or without cisplatin. We first found that the cisplatin-resistant forms of the ovarian cancer cell line A2780 (A2780-CR) and bladder cancer cell line (HT1376-CR) were less sensitive to cisplatin than the parental (A2780-P) or age-matched DMF control (HT1376-DMF) cell lines (see Figure 1A), whereas the A2780 and We found that cisplatin-resistant forms of HT1376 and HT1376 remained sensitive to Compound 1 (see Figure 1B). However, combined treatment with Compound 1 and cisplatin resulted in a greater than 50% reduction in growth. See Figures 2A and 2B.

[0048] Similar results were observed in the HT1376 bladder cancer cell line. For example, cisplatin-sensitive (-DMF) and -resistant (-CR) HT1376 cell lines showed enhanced effects on cell growth when cisplatin treatment was combined with compound 1 (see Figures 3A and 3B). In addition, compound 1 alone and in combination with cisplatin were effective in reducing tumor growth (see Figure 4). Collectively, this data demonstrates that compound 1 can be combined with other chemotherapeutic agents to synergistically treat solid tumors, such as bladder and ovarian cancer.

[0049] B. Synergistic effect of Compound 1 with enzalutamide (an androgen receptor signaling inhibitor) The antitumor effects of compound 1 alone and in combination with the androgen receptor signaling inhibitor enzalutamide were evaluated in CTG-2428 PDX tumors in NOG mice. As shown in Figure 5, the combination of compound 1 and enzalutamide significantly reduced absolute tumor volume compared with compound 1 or enzalutamide alone. Similar results were observed in CTG-2440 PDX (Figure 6) and CTG-2441 PDX tumors (Figure 7) in NOG mice. This data establishes that compound 1 can be combined with androgen receptor signaling inhibitors, such as enzalutamide, to treat solid tumor cancers, including prostate cancer.

[0050] 3. Primary in vivo pharmacology (monotherapy and combination therapy) A Phase 1 / 2 study to evaluate the safety, tolerability, and preliminary clinical activity of Compound 1 monotherapy and in combination with irinotecan in six disease-specific dose expansion cohorts will be conducted according to the general procedures outlined below. Phase 1 will consist of a Compound 1 monotherapy dose escalation and combination therapy (Compound 1 + irinotecan) dose escalation period in patients with advanced recurrent solid tumors, and Phase 2 will include a monotherapy dose escalation and combination therapy dose escalation period in six disease-specific dose expansion cohorts.

[0051] A. Efficacy of single agents Patients enrolled in the following cohorts will receive oral Compound 1 monotherapy: ·Monotherapy dose-escalation cohort in patients with advanced recurrent solid tumors. Dose expansion cohort 1 in patients with urothelial carcinoma. Dose expansion cohort 2 in patients with ovarian clear cell carcinoma. Dose expansion cohort 3 in patients with endometrial carcinoma.

[0052] This study will enroll evaluable patients with advanced solid tumors over two phases. Eligibility includes specific criteria, such as relapse after or progression during standard therapy. Phase 1 is intended to determine the maximum tolerated dose (MTD) and / or recommended phase 2 dose (RP2D) of Compound 1 as monotherapy in patients with advanced solid tumors. Secondary objectives include the safety and tolerability of Compound 1, its pharmacokinetic (PK) and pharmacodynamic (PD) profiles, and preliminary clinical activity of Compound 1. Phase 2 is designed to evaluate the antitumor activity of Compound 1 as monotherapy in patients with selected solid tumors (e.g., urothelial carcinoma, ovarian clear cell carcinoma, and endometrial carcinoma).

[0053] Patients enrolled in the monotherapy dose-escalation portion of this study will receive Compound 1 once daily (QD) orally (PO) in consecutive 4-week (28-day) cycles. The starting dose is 50 mg. The Compound 1 dose will be titrated by no more than 100% until at least one Grade 2 study drug-related adverse event (excluding anemia or lymphopenia) is reported, after which the Compound 1 dose may be titrated by no more than 40%. Intermediate or additional dose levels may be evaluated if recommended based on review of new safety, PK, or PD data. Compound 1 dose levels above 300 mg QD will be titrated by no more than 25%.

[0054] B. Effectiveness of Combination Therapy Patients enrolled in the following cohorts will receive oral Compound 1 monotherapy: Combination therapy dose-escalation cohort in patients with advanced recurrent solid tumors Dose expansion cohort 4 in patients with small cell lung cancer (SCLC). Dose expansion cohort 5 in patients with gastric or gastroesophageal junction (GEJ) adenocarcinoma. Dose expansion cohort 6 in patients with serous ovarian cancer.

[0055] This trial will enroll evaluable patients with advanced solid tumors over two phases identical to the monotherapy dose, except the selected solid tumors are small cell lung cancer, gastric or gastroesophageal junction, and serous ovarian cancer. Eligibility includes specific criteria, such as recurrence after or progression during standard therapy.

[0056] While several embodiments of the present disclosure have been described, it will be apparent that the inventors' basic examples may be modified to provide other embodiments that utilize the compounds and methods of the present disclosure. It will therefore be understood that the scope of the present disclosure is defined by the appended claims rather than by the specific embodiments that have been represented by example.

[0057] The contents of all references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are expressly incorporated herein by reference in their entirety. Unless otherwise defined, all technical and scientific terms used herein are to be given the meaning commonly known to those skilled in the art.

Claims

1. 1. A method of treating a solid tumor in a subject, comprising administering to the subject an effective amount of a compound having the formula: 【Chemistry 1】 or a pharmaceutically acceptable salt thereof and an effective amount of a second agent selected from a topoisomerase inhibitor, a DNA alkylating agent, and an androgen receptor signaling inhibitor.

2. 10. The method of claim 1, wherein the second agent is an androgen receptor signaling inhibitor.

3. 3. The method of claim 1 or 2, wherein the second drug is an androgen receptor signaling inhibitor selected from bicalutamide, enzalutamide, apalutamide, flutamide, nilutamide, darolutamide, and abiraterone acetate.

4. The method of any one of claims 1 to 3, wherein the second drug is enzalutamide.

5. The method of claim 1 , wherein the second agent is a DNA alkylating agent.

6. 6. The method of claim 1 or 5, wherein the DNA alkylating agent is selected from busulfan, cyclophosphamide, bendamustine, carboplatin, chlorambucil, cyclophosphamide, cisplatin, temozolomide, melphalan, carmustine, lomustine, dacarbazine, oxaliplatin, ifosamide, thiotepa, trabectedin, altretamine, mechlorethamine, procarbazine, and streptozocin.

7. 10. The method of claim 1 or 5, wherein the DNA alkylating agent is cisplatin.

8. The method of claim 1 , wherein the second agent is a topoisomerase inhibitor.

9. The method of claim 1 or 8, wherein the topoisomerase inhibitor is a topoisomerase I inhibitor.

10. 9. The method of claim 1 or 8, wherein the topoisomerase inhibitor is selected from irinotecan, topotecan, camptothecin, lamellarin, etoposide, teniposide, doxorubicin, daunorubicin, mitoxantrone, amsacrine, ellipticine, aurintricarboxylic acid, HU-331, epirubicin, valrubicin, idarubicin, pixantrone, teniposide, belotecan, gimatecan, indotecan, and indimitecan.

11. 10. The method of any one of claims 1, 8, and 9, wherein the topoisomerase inhibitor is irinotecan.

12. 12. The method of any one of claims 1 to 11, wherein the solid tumor is selected from prostate cancer, small cell lung cancer (SCLC), gastric or gastroesophageal junction (GEJ) adenocarcinoma, and serous ovarian cancer.

13. 13. The method of any one of claims 1 to 12, wherein the solid tumor is selected from small cell lung cancer (SCLC), gastric or gastroesophageal junction (GEJ) adenocarcinoma, and serous ovarian cancer.

14. The method of any one of claims 1 to 12, wherein the solid tumor is prostate cancer.

15. The method of any one of claims 1 to 14, wherein the solid tumor is characterized as an advanced tumor.

16. The method of any one of claims 1 to 15, wherein the solid tumor is characterized as a recurrent solid tumor.

17. The method of any one of claims 1 to 16, wherein the compound is administered simultaneously with the second agent.

18. The compound has the formula: 【Chemistry 2】 18. The method of any one of claims 1 to 17, wherein the compound is a compound having the formula:

19. A method of treating advanced recurrent solid tumors using 7-chloro-2-(4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4-dimethyl-N-((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)benzo[d][1,3]dioxole-5-carboxamide, or a pharmaceutically acceptable salt thereof.

20. 20. The method of claim 19, wherein the advanced recurrent solid tumor is advanced recurrent urothelial carcinoma, advanced recurrent ovarian clear cell carcinoma, or advanced recurrent endometrial carcinoma.

21. The method of claim 19 or 20, wherein the 7-chloro-2-(4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4-dimethyl-N-((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)benzo[d][1,3]dioxole-5-carboxamide is (2R)-7-chloro-2-(trans-4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4-dimethyl-N-((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)benzo[d][1,3]dioxole-5-carboxamide.

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