Modulators of methyl modifying enzymes, compositions and uses thereof

Abstract

Provided are novel compounds of Formula (I): # imgabs0 # and pharmaceutically acceptable salts thereof, which are useful in the treatment of various diseases, disorders or conditions associated with methyl modifying enzymes. Also provided are pharmaceutical compositions comprising the novel compounds of Formula (I), pharmaceutically acceptable salts thereof, and methods of using the pharmaceutical compositions in the treatment of one or more diseases, disorders or conditions associated with a methyl modifying enzyme.

Description

[0001] This application is a divisional application of a patent application with application number 201980039345.9, application date May 17, 2019, and name “Regulators of methyl modification enzymes, their compositions and uses”.

[0002] Related Applications

[0003] This application claims priority to U.S. Provisional Application No. 62 / 674,141, filed on May 21, 2018, the contents of which are incorporated herein by reference. Background Art

[0004] Eukaryotic chromatin is composed of a macromolecular complex called a nucleosome. A nucleosome has 147 base pairs of DNA wrapped around a protein octamer, which has two subunits of each of histones H2A, H2B, H3 and H4. Histones undergo post-translational modifications, which in turn affect chromatin structure and gene expression. One post-translational modification found on histones is the methylation of lysine and arginine residues. Histone methylation plays a key role in the regulation of gene expression in eukaryotes. Methylation affects chromatin structure and is associated with the activation and inhibition of transcription (Zhang and Reinberg, Genes Dev., 15: 2343-2360, 2001). Enzymes that catalyze the attachment and removal of histones to methyl groups are associated with gene silencing, embryonic development, cell proliferation and other processes.

[0005] A class of histone methyltransferases is characterized by the presence of a SET domain comprising about 130 amino acids. EZH2 is an example of a human SET domain containing a methyltransferase. EZH2 combines with EED (embryonic ectoderm development) and SUZ12 (suppressor of zeste 12 homologs) to form a complex called PRC2 (polyclonal suppressor 2), which has the ability to tri-methylate histone H3 at lysine 27 (Cao and Zhang, "Cell Biology (Mol.Cell)", 15:57-67, 2004). The PRC2 complex can also include RBAP46 subunits and RBAP48 subunits. Another example is the related methyltransferase EZH1.

[0006] The oncogenic activity of EZH2 has been shown in many studies of various cancer types. Approximately 15-20% of GCB-DLBCLs contain gain-of-function mutations in EZH2 (residue Y641), and these cells are highly sensitive to EZH2 inhibition both in vitro and in vivo (McCabe et al., 2012; Bradley et al., 2014). In cell line experiments, overexpression of EZH2 induces cell invasion, growth in soft agar, and motility, while knockout of EZH2 inhibits cell proliferation and cell invasion (Kleer et al., 2003, Proc. Nat. Acad. Sci. USA, 100: 11606-11611; Varambally et al., (2002), "The polycomb group protein EZH2 is involved in progression of prostate cancer", Nature, 419, 624-629). EZH2 has been shown to inhibit the expression of several tumor suppressors, including E-cadherin, DAB2IP, and RUNX3. In xenograft models, EZH2 knockout inhibits tumor growth and metastasis. Downregulation of EZH2 has been shown to block metastasis of prostate cancer in mouse models (Min et al., "An oncogene-tumor suppressor cascade drives metastatic prostate cancer by coordinately activating Ras and nuclear factor-kappaB", Nat Med., 2010 Mar;16(3):286-94). Recently, studies have shown that EZH2 is overexpressed in neuroendocrine tumors and that inhibition of EZH2 in mouse tumors restores androgen dependence (Ku et al., Science, 355, 2017). EZH2 overexpression is associated with the aggressiveness of certain cancers, such as breast cancer (Kleer et al., Proc. Natl. Acad. Sci. USA, 100:11606-11611, 2003).Recent studies have also shown that the prostate cancer-specific oncogenic fusion gene TMPRSS2-ERG induces a repressive epigenetic program by directly activating EZH2 (Yu et al., "An Integrated Network of Androgen Receptor, Polycomb, and TMPRSS2-ERG Gene Fusions in Prostate Cancer Progression," Cancer Cell. 2010 May 18;17(5):443-454).

[0007] Given their roles in regulating a variety of biological processes, methyl-modifying enzymes, particularly EZH2 and its mutants, are attractive targets for regulation. Summary of the invention

[0008] It has been found that the compounds described herein and pharmaceutically acceptable compositions thereof can modulate the activity of EZH2 (see, for example, Table 1). Such compounds include those of structural formula I:

[0009]

[0010] or a pharmaceutically acceptable salt thereof, wherein R 1 , R 2 , R 3 , R 4 , R 5 and R 6 Each of which is as defined herein.

[0011] The disclosed compounds, pharmaceutically acceptable salts and pharmaceutically acceptable compositions can be used to treat various conditions associated with methyl modification enzymes. These conditions include, for example, one or more cancers. DETAILED DESCRIPTION

[0012] 1. Compound Review

[0013] Provided are compounds of Formula I:

[0014]

[0015] or a pharmaceutically acceptable salt thereof, wherein:

[0016] R 1 Is a halogen group, (C 1 -C 4 ) alkyl, halogen (C 1 -C 4) alkyl, (C 1 -C 4 ) alkoxy or halogen (C 1 -C 4 )alkoxy, -S(C 1 -C 4 )alkyl or -S[halogen(C 1 -C 4 )alkyl];

[0017] R 2 is hydrogen, halogen or (C 1 -C 4 )alkyl;

[0018] R 3 Yes (C 1 -C 4 ) alkyl or halogen (C 1 -C 4 )alkyl;

[0019] R 4 Is a halogen group, (C 1 -C 4 ) alkyl or halogen (C 1 -C 4 )alkyl;

[0020] R 5 is hydrogen, (C 1 -C 4 ) alkyl or halogen (C 1 -C 4 )alkyl;

[0021] R 6 Is a halogen group, (C 1 -C 4 ) alkyl or halogen (C 1 -C 4 )alkyl; or

[0022] R 5 and R 6 Together with the nitrogen atom to which it is attached, it forms a substituted alkyl group optionally substituted with 1 to 3 groups selected from halogen, (C 1 -C 4 ) alkyl, halogen (C 1 -C 4 )alkyl and -OR 7 A 4- to 7-membered heterocyclic group substituted with a group;

[0023] R 7 Yes (C 1 -C 4 ) alkyl, halogen (C 1 -C 4 ) alkyl or (C 3 -C7 )cycloalkyl; and

[0024] Dashed lines represent single or double bonds.

[0025] 2. definition

[0026] When used to describe a chemical group that may have multiple points of attachment, the hyphen (-) indicates the point of attachment of the group to the variable that defines it. For example, -S[halo(C 1 -C 4 )alkyl] means that the point of attachment of this group occurs at the sulfur atom.

[0027] As used herein, the terms "halo" and "halogen" refer to an atom selected from fluorine (fluoro, -F), chlorine (chloro, -Cl), bromine (bromo, -Br) and iodine (iodo, -I).

[0028] As used herein, the term "alkyl" refers to a monovalent saturated, straight or branched hydrocarbon radical having 1 to 4 carbon atoms, unless otherwise specified. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, and sec-butyl.

[0029] The term "haloalkyl" includes monohaloalkyl, polyhaloalkyl and perhaloalkyl wherein the halogen is independently selected from fluorine, chlorine, bromine and iodine.

[0030] "Alkoxy" is an alkyl group attached to another moiety through an oxygen linker (-O(alkyl)). Non-limiting examples include methoxy, ethoxy, propoxy, and butoxy.

[0031] "Haloalkoxy" is a haloalkyl group attached to another moiety through an oxygen atom, such as but not limited to -OCHCF 2 or -OCF 3 .

[0032] The term "4- to 7-membered heterocyclyl" refers to a 4- to 7-membered (e.g., 4, 5, 6, and 7-membered) saturated or partially unsaturated monocyclic heterocyclic ring containing 1 to 4 heteroatoms independently selected from N, O, and S. The terms "heterocycle," "heterocyclic group," "heterocyclic ring," "heterocyclic group," "heterocyclic moiety," and "heterocyclic radical" are used interchangeably herein. The heterocyclic ring may be attached to its pendant group at any heteroatom or carbon atom that forms a stable structure. Examples of such saturated or partially unsaturated heterocyclyls include, but are not limited to, tetrahydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, pyrrolidinyl, pyridinyl, pyrrolidonyl, piperidinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, morpholinyl, dihydrofuranyl, dihydropyranyl, dihydropyridinyl, tetrahydropyridinyl, dihydropyrimidinyl, oxetanyl, azetidinyl, and tetrahydropyrimidinyl. When specified as optionally substituted or substituted, the substituents on the heterocyclyl (e.g., in the case of an optionally substituted heterocyclyl) may be present at any substitutable position, and include, for example, the position at which the heterocyclyl is attached.

[0033] As used herein, "geometric isomer" refers to isomers that differ in the orientation of substituent atoms relative to the cycloalkyl ring, i.e., cis or trans isomers. When a disclosed compound is named or described by structure without specifying a specific cis or trans geometric isomer form, it is understood that the name or structure encompasses one geometric isomer free of the other geometric isomers, a mixture of geometric isomers, or a mixture enriched in one geometric isomer relative to its corresponding geometric isomer. When a specific geometric isomer (i.e., cis or trans geometric isomer) is described, the isomer described has a purity of at least 60%, 70%, 80%, 90%, 99%, or 99.9% by weight relative to the other geometric isomers.

[0034] Unless otherwise indicated, when the stereochemistry of a disclosed compound is named or depicted by structure, the named or depicted stereoisomer has a purity of at least 60%, 70%, 80%, 90%, 99%, or 99.9% by weight relative to all other stereoisomers. When a single enantiomer is named or depicted by structure, the depicted or named enantiomer has an optical purity of at least 60%, 70%, 80%, 90%, 99%, or 99.9% by weight. The percent optical purity by weight is the ratio of the weight of the enantiomer to the weight of the enantiomer plus the weight of its optical isomers. Enantiomers can be resolved by methods known to those skilled in the art, for example, by the formation of diastereomeric salts that can be separated by, for example, crystallization; the formation of diastereomeric derivatives or complexes that can be separated by, for example, crystallization, supercritical fluid or liquid chromatography; the selective reaction of one enantiomer with an enantiomer-specific reagent, such as enzymatic esterification; or supercritical fluid or liquid chromatography in a chiral environment, such as on a chiral support (e.g., silica with bound chiral ligands) or in the presence of a chiral solvent. Specific enantiomers can also be synthesized by asymmetric synthesis using optically active reagents, substrates, catalysts or solvents, or by converting one enantiomer to the other by asymmetric transformation.

[0035] Unless otherwise indicated, when a disclosed compound is named or depicted by a structure without indication of stereochemistry, and the name or structure encompasses more than one enantiomer and / or more than one geometric isomer, the name or structure encompasses one enantiomer or geometric isomer of the compound free of the corresponding optical or geometric isomer, the racemic mixture of the compound, and mixtures enriched in one enantiomer or geometric isomer relative to its corresponding optical or geometric isomer.

[0036] As used herein, the term "patient" refers to animals, such as mammals, and such as humans. The terms "subject" and "patient" can be used interchangeably.

[0037] The term "pharmaceutically acceptable carrier" refers to a non-toxic carrier, adjuvant or excipient that does not destroy the pharmacological activity of the compound formulated with it. Pharmaceutically acceptable carriers, adjuvants or excipients that can be used in the compositions described herein include, but are not limited to, ion exchangers, alumina, aluminum 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 silicon dioxide, magnesium trisilicate, polyvinyl pyrrolidone, cellulosic substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene glycol-polyoxypropylene block polymers, polyethylene glycol and lanolin.

[0038] The term "treatment" or "treat" or "treating" refers to reversing, alleviating or inhibiting the progression of a disease or condition as described herein or one or more symptoms thereof. Treatment may also be continued after symptoms disappear, for example to delay recurrence.

[0039] Disease, disorder and condition are used interchangeably herein.

[0040] The term "effective amount" or "therapeutically effective amount" refers to the amount of a compound described herein that can elicit a biological or medical response in a subject, such as a dose of 0.01-100 mg / kg body weight / day. The terms "subject" and "patient" are used interchangeably to refer to mammals in need of treatment, such as companion animals (e.g., dogs, cats, etc.), farm animals (e.g., cattle, 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.

[0041] The terms "inhibit," "inhibition," and "inhibiting" include a decrease in the baseline activity of a biological activity or process.

[0042] The compounds described herein may exist in the form of pharmaceutically acceptable salts. For medical purposes, the salts of the compounds described herein refer to non-toxic "pharmaceutically acceptable salts". Pharmaceutically acceptable salt forms include pharmaceutically acceptable acidic / anionic or basic / cationic salts. Suitable pharmaceutically acceptable acid addition salts of the compounds described herein include, for example, salts of inorganic acids (such as hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid and sulfuric acid) and organic acids (such as acetic acid, benzenesulfonic acid, benzoic acid, methanesulfonic acid and p-toluenesulfonic acid).

[0043] 3. Description of Exemplary Compounds

[0044] In a first embodiment, the present disclosure provides a compound of Formula I:

[0045]

[0046] or a pharmaceutically acceptable salt thereof, wherein the variables are as described above for Formula I.

[0047] In a second embodiment, the compound of Formula I has Formula II:

[0048]

[0049] or a pharmaceutically acceptable salt thereof, wherein the variables are as described above for Formula I.

[0050] In a third embodiment, the compound of Formula I or Formula II has Formula III:

[0051]

[0052] or a pharmaceutically acceptable salt thereof, wherein the variables are as described above for Formula I.

[0053] In a fourth embodiment, the compound of Formula I, II or III has Formula IV:

[0054]

[0055] or a pharmaceutically acceptable salt thereof, wherein the variables are as described above for Formula I.

[0056] In a fifth embodiment, R in the compound of Formula I, II, III or IV 1 is a halogen or -S(C 1 -C 4 )alkyl, wherein the remainder of the variables are as described above for Formula I.

[0057] In a sixth embodiment, R in the compound of Formula I, II, III or IV 4 is a halogen or (C 1 -C 4 )alkyl, wherein the remaining variables are as described above for Formula I or the Fifth Embodiment.

[0058] In a seventh embodiment, R in the compound of Formula I, II, III or IV 5 Yes (C 1 -C 4 )alkyl, wherein the remaining variables are as described above for Formula I or the fifth or sixth embodiment.

[0059] In an eighth embodiment, R in the compound of Formula I, II, III or IV 6 Yes (C 1 -C 4 )alkyl, wherein the remaining variables are as described above for Formula I or the fifth or sixth embodiment.

[0060] In a ninth embodiment, R in the compound of Formula I, II, III or IV is 5 and R 6 Each is methyl, wherein the remaining variables are as described above for Formula I or the fifth or sixth embodiment.

[0061] In a tenth embodiment, the group

[0062] and NR in the compound of formula I, II, III or IV 5 R 6 is trans-oriented about the cyclohexyl group, wherein the variables are as described above for Formula I or the fifth, sixth, seventh, eighth or ninth embodiment.

[0063] In an eleventh embodiment, the group

[0064] and NR in the compound of formula I, II, III or IV 5 R 6 cis-oriented about the cyclohexyl group, wherein the variables are as described above for Formula I or the fifth, sixth, seventh, eighth or ninth embodiment.

[0065] In a twelfth embodiment, the stereochemical configuration of the chiral center of the 1,3-dioxolanyl in the compound of Formula I, II, III or IV is R, wherein the variables and remaining features are as described above for Formula I or the fifth, sixth, seventh, eighth, ninth, tenth or eleventh embodiment.

[0066] In a thirteenth embodiment, the stereochemical configuration of the chiral center of the 1,3-dioxolanyl in the compound of Formula I, II, III or IV is S, wherein the variables and remaining features are as described above for Formula I or the fifth, sixth, seventh, eighth, ninth, tenth or eleventh embodiment.

[0067] Specific examples of compounds are provided in the Examples section and are included herein as part of the fourteenth embodiment. Also included are pharmaceutically acceptable salts and neutral forms of these compounds. In one aspect, the present disclosure includes racemic forms of any compound described herein.

[0068] 4. Use, preparation and application:

[0069] In some embodiments, the present disclosure provides a composition comprising a compound described herein or a pharmaceutically acceptable derivative thereof and a pharmaceutically acceptable carrier. The amount of the compound in the provided composition is effective to measurably modulate a histone methyl modifying enzyme or a mutant thereof in a biological sample or a patient.

[0070] In certain embodiments, the compositions described herein are formulated for use in patients who need such compositions. The compositions described herein can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, orally, vaginally, or by an implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques. In certain embodiments, the compositions are administered orally, intraperitoneally, or intravenously. The sterile injection form of the compositions described herein can be an aqueous or oily suspension. These suspensions can be formulated using appropriate dispersants or wetting agents and suspending agents according to techniques known in the art.

[0071] In some embodiments, the composition is administered orally.

[0072] The specific dosage and treatment regimen for any particular patient will depend on various factors, including the activity of the specific compound employed, age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination and the judgment of the treating physician and the severity of the specific disease being treated. The amount of the composition described herein will also depend on the specific compound in the composition.

[0073] The compounds and compositions described herein are generally useful for modulating the activity of one or more enzymes involved in epigenetic regulation, particularly EZH1 and EZH2, more specifically EZH2 and its mutants. In some embodiments, the compounds described herein downregulate or inhibit the activity of EZH2. In some embodiments, the compounds described herein are antagonists of EZH2 activity. In some embodiments, the compounds described herein downregulate or inhibit the activity of EZH1. In some embodiments, the compounds described herein are antagonists of EZH1 activity.

[0074] In some embodiments, the compounds and compositions described herein can be used to treat diseases and / or conditions associated with overexpression of EZH1 or EZH2 and / or expression of mutant forms of EZH2 (e.g., mutant forms that alter EZH2 substrate activity). Studies of EZH2 deletions, missense, and frameshift mutations have shown that EZH2 plays a tumor suppressor role in blood disorders such as myelodysplastic syndrome (MDS) and myeloid malignancies (Ernst et al., Nat Genet., 2010 Aug; 42(8): 722-6; Nikoloski et al., Nat Genet., 2010 Aug; 42(8): 665-7). In some embodiments, the compounds and compositions described herein can be used to treat diseases and / or conditions associated with the presence of EZH2 with Y641N, Y641C, Y641F, Y641H, Y641S, A677G, or A687 mutations. In a specific aspect of this embodiment, EZH2 has a Y641N mutation.

[0075] In some embodiments, the present disclosure provides a method for treating a subject suffering from a disease and / or condition associated with overexpression of EZH1 or EZH2 and / or expression of a mutant form of EZH2, the method comprising the step of administering a compound described herein or a pharmaceutically acceptable salt or composition thereof. In some embodiments, the above method further comprises a preliminary step of determining whether the subject overexpresses EZH2 or expresses a mutant form of EZH2.

[0076] In some embodiments, the disease or condition associated with the presence of a mutant form of EZH2 is human B-cell lymphoma. In some embodiments, the disease and / or condition associated with the presence of Y641N EZH2 is follicular lymphoma or diffuse large B-cell lymphoma. In some embodiments, the compounds or compositions described herein can be used to treat blood disorders such as myelodysplastic syndrome, leukemia, anemia, and cytopenia. Sneeringer et al., "Coordinated activities of wild-type plus mutant EZH2 drive tumor-associated hypertrimethylation of lysine 27 on histone H3 (H3K27) in human B-cell lymphomas," Sneeringer et al., Proc. Natl Acad. Sci., 2010 Dec;109(48):20980-20985.

[0077] In some embodiments, the compounds and compositions described herein can be used to treat diseases and / or conditions associated with cell proliferation. In some embodiments, the compounds and compositions described herein can be used to treat diseases and / or conditions associated with cell cycle or DNA repair disorders. In some embodiments, the compounds and compositions described herein can be used to treat cancer.

[0078] In one aspect, cancers that may be treated by the compounds, compositions, and methods described herein include, but are not limited to: Cardiac sarcomas: (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyosarcoma, fibroma, lipoma, and teratoma; Lung: bronchial lung cancer (squamous cell carcinoma, undifferentiated small cell carcinoma, undifferentiated large cell carcinoma, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondroitinoma, mesothelioma; Gastrointestinal: esophageal (squamous cell =Carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumor, vasodilatory peptide tumor), small intestine (adenocarcinoma, lymphoma, carcinoid tumor, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large intestine (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma); genitourinary tract: kidney (adenocarcinoma, Wilms' tumor =Tumor (Nephroblastoma), Lymphoma, Leukemia), Bladder and Urethra (Squamous Cell Carcinoma, Transitional Cell Carcinoma, Adenocarcinoma), Prostate (Adenocarcinoma, Sarcoma), Testis (Seminoma, Teratoma, Embryonic Carcinoma Cell, Teratocarcinoma, Choriocarcinoma, Sarcoma, Leydig Cell Carcinoma, Fibroma, Fibroadenoma, Adenomatoid Tumor, Lipoma); Liver: Liver Cancer (Hepatocellular Carcinoma), Bile Duct Carcinoma, Hepatoblastoma, Angiosarcoma, Hepatocellular Adenoma, Hemangioma; Bone: Osteogenic Sarcoma (Osteosarcoma), Fibrosarcoma, Malignant Fibrous Histiocytoma, Chondrosarcoma, Ewing's Sarcoma, Malignant Lymphoma (Reticulum Cell Sarcoma), Multiple Myeloma, Malignant Giant Cell Tumor Chordoma, Osteochondroma (Osteochondral Exostosis), Benign Chondroma, Chondroblastoma, Chondromyxoid Fibroma, Osteoid Osteoma, and Giant Cell Tumor; Nervous System: Skull (Osteoma, Hemangioma) , granuloma, xanthomas, osteitis deformans), meninges (meningioma, meningosarcoma, gliosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma, germ cell tumor (pinealoma), glioblastoma multiforme, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors), spinal neurofibroma, meningioma, glioma, sarcoma); Gynecological: uterus (endometrial carcinoma), cervix (cervical cancer, preneoplastic cervical atypical hyperplasia), ovary (ovarian cancer (serous cystadenocarcinoma, mucinous cystadenocarcinoma, clear cell carcinoma, unclassified cancer), granulocyte tumor, Serratia cell tumor, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma), fallopian tube (carcinoma);Hematological: Blood (myeloid leukemia (acute and chronic), acute lymphocytic leukemia, chronic lymphocytic leukemia, myeloproliferative disorders, multiple myeloma, myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin's lymphoma (malignant lymphoma); Skin: malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, dysplastic nevus, lipoma, hemangioma, dermatofibroma, keloid, psoriasis; and Adrenal: neuroblastoma. ;

[0079] In one aspect, the cancer treated by the compounds, compositions and methods described herein is selected from adrenal cancer, acinar cell carcinoma, acoustic neuroma, acrolenticular melanoma, acrohidrosis, acute eosinophilic leukemia, acute erythroid leukemia, acute lymphocytic leukemia, acute megakaryocytic leukemia, acute monocytic leukemia, acute promyelocytic leukemia, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenomatous odontogenic tumor, adenosquamous carcinoma, adipose tissue tumor, adrenocortical carcinoma, adult T-cell leukemia / lymphoma, Aggressive NK-cell leukemia, AIDS-related lymphoma, alveolar rhabdomyosarcoma, alveolar soft tissue sarcoma, ameloblastic fibroma, anaplastic large cell lymphoma, anaplastic thyroid cancer, angioimmunoblastic T-cell lymphoma, angiomyolipoma, angiosarcoma, astrocytoma, atypical teratoid rhabdoid tumor, B-cell chronic lymphocytic leukemia, B-cell proliferative leukemia, B-cell lymphoma, basal cell carcinoma, biliary tract cancer, bladder cancer, blastoma, bone cancer, Brenner tumor, Brown tumor, Burkitt's lymphoma, breast cancer, brain cancer, carcinoma in situ, carcinosarcoma, chondroma, cementoma, medullary sarcoma, brittle bone tumor, chordoma, choriocarcinoma, choroid plexus papilloma, renal clear cell sarcoma, craniopharyngioma, cutaneous T-cell lymphoma, cervical cancer, colorectal cancer, Degos disease disease), desmoplastic small round cell tumor, diffuse large B-cell lymphoma, dysembryoplastic neuroepithelioma, dysgerminoma, embryonal carcinoma, endocrine gland tumor, endodermal sinus tumor, enteropathy-associated T-cell lymphoma, esophageal cancer, parasitic fetus, fibroma, fibrosarcoma, follicular lymphoma, follicular thyroid cancer, ganglioneuroma, gastrointestinal cancer, germ cell tumor, gestational choriocarcinoma, giant cell fibroblastoma, giant cell tumor of bone, glioma, glioblastoma multiforme, glioma, gliomatosis, glucagonoma, gonadal cell tumor, granular cell tumor, gynogenetic, amphotericin B-cell tumor, gallbladder cancer, gastric cancer, hairy cell leukemia, hemangioblastoma, head and neck cancer, hemangiopericytoma, hematological malignancies, hepatoblastoma, hepatosplenic T-cell lymphoma (hepatosplenic T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin'slymphoma), invasive lobular carcinoma, intestinal cancer, kidney cancer, laryngeal cancer, lentigo maligna, fatal midline cancer, leukemia, stromal 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 salamander tumor, mantle cell lymphoma, marginal zone B-cell lymphoma, mast cell leukemia, mediastinal germ cell tumor, medullary carcinoma of the breast, medullary carcinoma of the thyroid, medulloblastoma, melanoma, meningioma, Merkel cell carcinoma, mesothelioma, metastatic urothelial carcinoma, mixed Mullerian tumor tumor), myxoma, multiple myeloma, muscle tissue tumor, mycosis fungoides, myxoid liposarcoma, myxoma, myxosarcoma, nasopharyngeal carcinoma, schwannoma, neuroblastoma, neurofibroma, neuroma, nodular melanoma, eye cancer, oligoastrocytoma, oligodendroglioma, oncocytoma, optic nerve sheath meningioma, optic nerve tumor, oral cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillary thyroid cancer, paraganglioma, pinealoblastoma, pineocytoma, pituitary adenoma, pituitary tumor, plasmacytoma, multiblastoma, precursor 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, rhabdomyosarcoma, Richter's 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 tumors, splenic marginal zone lymphoma, squamous cell carcinoma, synovial sarcoma, Sezary's disease disease), small intestinal cancer, squamous cell carcinoma, gastric cancer, T-cell lymphoma, testicular cancer, theca cell tumor, thyroid cancer, transitional cell carcinoma, laryngeal cancer, urachal cancer, genitourinary cancer, urothelial carcinoma, uveal melanoma, uterine cancer, verrucous carcinoma, optic pathway glioma, vulvar cancer, vaginal cancer, Waldenstrom's macroglobulinemia, Warthin's tumor, Wilms' tumor.

[0080] In one aspect, the cancer treated by the compounds, compositions and methods described herein is selected from the group consisting of adenocarcinoma, adult T-cell leukemia / lymphoma, bladder cancer, blastoma, bone cancer, breast cancer, brain cancer, malignant epithelial tumors, myeloid sarcoma, cervical cancer, colorectal cancer, esophageal cancer, gastrointestinal cancer, glioblastoma multiforme, glioma, gallbladder cancer, gastric cancer, head and neck cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, intestinal cancer, kidney cancer, laryngeal cancer, leukemia, lung cancer, lymphoma, liver cancer, small cell lung cancer, non-small cell lung cancer, Mesothelioma, multiple myeloma, acute myeloid leukemia (AML), diffuse large B-cell lymphoma (DLBCL), eye cancer, optic nerve tumors, oral cancer, ovarian cancer, pituitary tumors, primary central nervous system lymphoma, prostate cancer, pancreatic cancer, pharyngeal cancer, renal cell carcinoma, rectal cancer, sarcoma, skin cancer, spinal tumors, small intestine cancer, stomach cancer, T-cell lymphoma, testicular cancer, thyroid cancer, laryngeal cancer, genitourinary system cancer, urothelial carcinoma, uterine cancer, vaginal cancer and Wilms' tumor.

[0081] In one aspect, the cancer treated by the compounds, compositions and methods described herein is selected from breast cancer, prostate cancer, colon cancer, renal cell carcinoma, glioblastoma multiforme, bladder cancer, melanoma, bronchial carcinoma, lymphoma and liver cancer.

[0082] Also provided is the use of a compound described herein or a pharmaceutically acceptable salt thereof or a composition comprising the disclosed compound or a pharmaceutically acceptable salt thereof for the preparation of a medicament for treating a condition described herein. Also provided is a compound described herein or a pharmaceutically acceptable salt thereof or a composition comprising the disclosed compound or a pharmaceutically acceptable salt thereof for treating a condition described herein.

[0083] example

[0084] The following representative examples are intended to help illustrate the invention and are not intended, nor should they be construed, to limit the scope of the invention.

[0085] Preparation of intermediates

[0086] Intermediate 1: 3-(Aminomethyl)-6-methyl-4-(methylthio)pyridin-2(1H)-one (hydrochloride)

[0087]

[0088] Step 1: Synthesis of sodium 3-oxobut-1-ene-1,1-bis(thioate)

[0089] The mixture of sodium tert-butoxide (16.6g, 172mmol) in toluene (30mL) is degassed under vacuum and purged with nitrogen (3 cycles). Acetone (5.0g, 6.4mL, 86mmol) is then added at 0°C, followed by the slow addition of carbon disulfide (6.6g, 5.24mL, 86mmol). The resulting mixture is stirred at 0°C for 4 hours and then filtered. The filter cake is dried under vacuum to give the title compound (15.4g, crude product) as a yellow solid, which is used in the next step without further purification.

[0090] Step 2: Synthesis of 4,4-bis(methylthio)but-3-en-2-one

[0091] To a solution of 3-oxobut-1-ene-1,1-bis(thioic acid) sodium (15.4 g, 86.4 mmol) in methanol (90 mL) was slowly added iodomethane (24.5 g, 10.7 mL, 173 mmol). The mixture was stirred at 70 ° C for 1 hour and then concentrated to dryness. Water (30 mL) was added and the desired product was extracted with ethyl acetate (60 mL×3). The combined organic layers were dried over sodium sulfate and concentrated under reduced pressure to give the title compound (6.8 g, 42% yield) as a brown oil, which was used in the next step without further purification. LCMS [M+H] + m / z: Calculated: 163.0; Found: 163.0. 1 H NMR (400 MHz, CHLOROFORM-d) δ 6.02 (s, 1H), 2.45 (s, 3H), 2.43 (s, 3H), 2.15 (s, 3H).

[0092] Step 3: Synthesis of 6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridine-3-carbonitrile

[0093] To a solution of 4,4-bis(methylthio)but-3-en-2-one (2.9 g, 18 mmol) and 2-cyanoacetamide (1.5 g, 18 mmol) in tert-butanol (50 mL) was added sodium tert-butoxide (1.9 g, 20 mmol). The mixture was stirred at 80 ° C for 12 hours (two batches of reactions were carried out and combined at this stage). Water (20 mL) was added, and the pH was adjusted to 5-6 with 10% hydrochloric acid. The resulting mixture was filtered, and the filter cake was washed with petroleum ether (20 mL×2), and then the filter cake was dried under vacuum to give the title compound (4.8 g, 74% yield) as an off-white solid, which was used in the next step without further purification. LCMS [M+H] + m / z: Calculated: 181.0; Found: 181.0. 1 H NMR (400 MHz, dimethyl sulfoxide-d 6)δ6.27(s,1H),2.56(s,3H),2.25(s,3H).

[0094] Step 4: Synthesis of 3-(aminomethyl)-6-methyl-4-(methylthio)pyridin-2(1H)-one

[0095] A mixture of 6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridine-3-carbonitrile (3.6 g, 20 mmol) in tetrahydrofuran (50 mL) was degassed under vacuum and purged with nitrogen (3 cycles). Borane dimethyl sulfide complex (10 M, 8.0 mL, 80 mmol) was then slowly added at 0 ° C, and the reaction mixture was then warmed to 70 ° C and stirred for 2 hours. Before the mixture was concentrated under reduced pressure, methanol (15 mL) was slowly added at 0 ° C to quench the reaction to give the title compound (3.8 g, crude) as a light yellow solid, which was used in the next step without further purification. LCMS [M + H] + m / z: calculated: 185.1; found: 185.0.

[0096] Step 5: Synthesis of tert-butyl ((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)carbamate

[0097] To a solution of 3-(aminomethyl)-6-methyl-4-(methylthio)pyridin-2(1H)-one (3.6 g, 20 mmol) in tetrahydrofuran (80 mL) was added triethylamine (5.9 g, 8.1 mL, 59 mmol). The mixture was stirred for 30 minutes, then di-tert-butyl dicarbonate (6.4 g, 29 mmol) was added, and the reactants were stirred for 12 hours at 25 ° C. The reaction mixture was then concentrated to dryness under reduced pressure, water (35 mL) was then added, and the desired product was extracted with a 5:1 mixture of petroleum ether / ethyl acetate (30 mL×3). The combined organic layers were dried over sodium sulfate and concentrated to give the title compound (5.8 g, crude) as a white solid, which was used in the next step without further purification. LCMS[M+H] + m / z: calculated: 285.12; found: 284.9. 1 H NMR (400 MHz, dimethyl sulfoxide-d 6 )δ6.05(s,1H),4.03-4.00(m,2H),2.42(s,3H),2.15(s,3H),1.39(s,9H).

[0098] Step 6: Synthesis of 3-(aminomethyl)-6-methyl-4-(methylthio)pyridin-2(1H)-one (hydrochloride)

[0099] To a solution of hydrogen chloride in 1,4-dioxane (4M, 100 mL, 400 mmol) at 25°C was added tert-butyl ((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)carbamate (5.0 g, 17.6 mmol). The reaction mixture was stirred at 25°C for 2 hours and then concentrated to dryness under reduced pressure. The residue was washed with dichloromethane (30 mL×2) and ethyl acetate (30 mL) to give the title compound (4.5 g, crude, HCl salt) as a yellow solid, which was used in the next step without further purification. LCMS [M+H] + m / z: calculated: 185.1; found: 185.0. 1 H NMR (400 MHz, D 2 O)δ6.31(s,1H),4.03(s,2H),2.41(s,3H),2.18(s,3H).

[0100] Example 1: 9-chloro-2-(trans-4-(dimethylamino)cyclohexyl)-2,4-dimethyl-6-((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)-7,8-dihydro-[1,3-dioxol[4,5-g]isoquinolin-5(6H)-one

[0101]

[0102] Step 1: Synthesis of methyl 6-bromo-7-chloro-2-(trans-4-(dimethylamino)cyclohexyl)-2,4-dimethylbenzo[d][1,3]dioxole-5-carboxylate

[0103] To a solution of 2-(trans-4-((tert-butoxycarbonyl)amino)cyclohexyl)-7-chloro-2,4-dimethylbenzo[d][1,3]dioxole-5-formic acid methyl ester (7g, 15.9mmol) (prepared according to the procedure described in patent application US2017 / 0073335A1) in dichloromethane (80mL) was added potassium carbonate (2.22g, 15.9mmol) at room temperature, followed by dropwise addition of bromine (3.25mL, 63.6mmol). The mixture was stirred at room temperature for 30 minutes (or until the starting material was completely consumed), and then quenched with a saturated solution of sodium thiosulfate. The organic layer was separated, and the aqueous layer was extracted three times with dichloromethane. The combined organic layers were washed with brine, dried over sodium sulfate, filtered and concentrated under reduced pressure to give the title compound (theoretical yield 8g) as a solid, which was used in the next step without further purification. LCMS[M+Na] + m / z: calculated: 540.1; found: 540.2.

[0104] Step 2: Synthesis of methyl 2-(trans-4-aminocyclohexyl)-6-bromo-7-chloro-2,4-dimethylbenzo[d][1,3]dioxole-5-carboxylate trifluoroacetate

[0105] To a solution of methyl 6-bromo-7-chloro-2-(trans-4-(dimethylamino)cyclohexyl)-2,4-dimethylbenzo[d][1,3]dioxole-5-carboxylate (8 g, 15.4 mmol) in dichloromethane (31 mL) was added trifluoroacetic acid (7.70 mL) at room temperature. The mixture was stirred at room temperature for 15 minutes and then concentrated to dryness under reduced pressure to give the title compound as a solid (theoretical yield 8.20 g), which was used in the next step without further purification. LCMS [M+H] + m / z: calculated: 418.0; found: 418.1.

[0106] Step 3: Synthesis of methyl 6-bromo-7-chloro-2-(trans-4-(dimethylamino)cyclohexyl)-2,4-dimethylbenzo[d][1,3]dioxole-5-carboxylate

[0107] To a solution of 2-(trans-4-aminocyclohexyl)-6-bromo-7-chloro-2,4-dimethylbenzo[d][1,3]dioxole-5-formic acid methyl ester trifluoroacetate (8.20g, 15.4mmol) in dichloromethane (77mL) was added formaldehyde (37% by weight in water) (12.3mL, 154mmol) at room temperature, followed by sodium triacetoxyborohydride (16.3g, 77.0mmol). The reactant was stirred at room temperature for 15 minutes and then quenched with a saturated solution of sodium bicarbonate. The organic layer was separated and the aqueous layer was extracted three times with dichloromethane. The combined organic layers were washed with brine, dried over sodium sulfate, filtered and concentrated to dryness under reduced pressure. The residue was purified by flash chromatography (normal phase KP-NH column, gradient of heptane containing 0 to 100% ethyl acetate) to give the title compound in the form of a light yellow oil (5.8g, 78% yield over three steps). LCMS [M+H] + m / z: calculated: 446.1; found: 446.3.

[0108] Step 4: Synthesis of methyl 7-chloro-2-(trans-4-(dimethylamino)cyclohexyl)-6-(2-ethoxyvinyl)-2,4-dimethylbenzo[d][1,3]dioxole-5-carboxylate

[0109] A mixture of methyl 6-bromo-7-chloro-2-(trans-4-(dimethylamino)cyclohexyl)-2,4-dimethylbenzo[d][1,3]dioxole-5-carboxylate (4.27 g, 9.55 mmol), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex with dichloromethane (1.55 g, 1.91 mmol), cesium carbonate (9.31 g, 28.6 mmol), 1,4-dioxane (35 mL) and water (5 mL) was degassed (four vacuum / nitrogen refill cycles) and then a solution of 2-[(E)-2-ethoxyvinyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (3.78 g, 19.1 mmol) in 1,4-dioxane (15 mL) was added. The resulting black reaction mixture was heated at 100°C for 24 hours, then cooled to room temperature, diluted with ethyl acetate and filtered. The filtrate was washed with water, and the aqueous layer was extracted three times with ethyl acetate. The combined organic layers were washed with brine, dried over sodium sulfate, filtered and concentrated to dryness under reduced pressure. The residue was purified by flash chromatography (normal phase KP-NH column, gradient of heptane containing 0 to 40% ethyl acetate) to give the title compound (3.5 g, 84% yield) as a thick slightly yellow oil. LCMS [M + H] + m / z: calculated: 438.2; found: 438.3.

[0110] Step 5: Synthesis of methyl 7-chloro-2-(trans-4-(dimethylamino)cyclohexyl)-2,4-dimethyl-6-(2-oxoethyl)benzo[d][1,3]dioxole-5-carboxylate

[0111] To a solution of 7-chloro-2-(trans-4-(dimethylamino)cyclohexyl)-6-((E)-2-ethoxyvinyl)-2,4-dimethylbenzo[d][1,3]dioxole-5-carboxylic acid methyl ester (4.4 g, 10.0 mmol) in acetonitrile (63 mL) was added cerium chloride heptahydrate (18.6 g, 50.0 mmol), sodium iodide (7.49 g, 50.0 mmol) and water (100 μL). The reaction mixture was stirred at 80 ° C for 3 hours and then filtered through a plug of celite. The filtrate was used directly in the next step. LCMS [M+H] + m / z: calculated: 410.17; found: 410.3.

[0112] Step 6: Synthesis of methyl 7-chloro-2-(trans-4-(dimethylamino)cyclohexyl)-2,4-dimethyl-6-(2-oxoethyl)benzo[d][1,3]dioxole-5-carboxylate

[0113] To a crude solution of 7-chloro-2-(trans-4-(dimethylamino)cyclohexyl)-2,4-dimethyl-6-(2-oxoethyl)benzo[d][1,3]dioxole-5-carboxylic acid methyl ester in acetonitrile (from the previous step) was added 3-(aminomethyl)-6-methyl-4-(methylsulfanyl)-1,2-dihydropyridin-2-one (free base) (3.68 g, 20.0 mmol) and triethylamine (3 mL, 21.5 mmol). The reaction mixture was stirred at room temperature for 5 minutes, then sodium cyanoborohydride (3.14 g, 50.0 mmol) was added. The reactants were stirred at room temperature for 5 minutes (or until the starting material was completely consumed), then quenched with a saturated solution of sodium bicarbonate. Next, the desired product was extracted three times with dichloromethane. The combined organic layers were then washed with brine, dried over sodium sulfate, filtered and concentrated to dryness under reduced pressure to give the title compound as a solid, which was used in the next step without further purification. LCMS [M+H] + m / z: calculated: 578.24; found: 578.4.

[0114] Step 7: Synthesis of 9-chloro-2-(trans-4-(dimethylamino)cyclohexyl)-2,4-dimethyl-6-((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)-7,8-dihydro-[1,3]-dioxol[4,5-g]isoquinolin-5(6H)-one

[0115] Potassium carbonate (1.38 g, 10.0 mmol) was added to a crude solution of 7-chloro-2-(trans-4-(dimethylamino)cyclohexyl)-2,4-dimethyl-6-(2-oxoethyl)benzo[d][1,3]dioxole-5-carboxylic acid methyl ester in dimethyl sulfoxide (50 mL). The reaction mixture was stirred at room temperature for 30 minutes (or until the starting material was completely consumed) and then quenched with a saturated solution of sodium bicarbonate. Next, the desired product was extracted three times with dichloromethane. The combined organic layers were then washed with brine, dried over sodium sulfate, filtered, and concentrated to dryness under reduced pressure. The residue was purified by reverse phase C18 column (gradient of 30% to 70% acetonitrile in water with 0.1% trifluoroacetic acid) to give the title compound as a white solid as a racemic mixture) (1600 mg, 29% yield over three steps).

[0116] Step 8: Separation of (R)-9-chloro-2-(trans-4-(dimethylamino)cyclohexyl)-2,4-dimethyl-6-((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)-7,8-dihydro-[1,3]-dioxol[4,5-g]isoquinolin-5(6H)-one and (S)-9-chloro-2-(trans-4-(dimethylamino)cyclohexyl)-2,4-dimethyl-6-((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)-7,8-dihydro-[1,3]-dioxol[4,5-g]isoquinolin-5(6H)-one

[0117] (R)-9-chloro-2-(trans-4-(dimethylamino)cyclohexyl)-2,4-dimethyl-6-((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)-7,8-dihydro-[1,3]-dioxol[4,5-g]isoquinoline-5(6H) was resolved by preparative SFC [column: Chiralpak IC (inner diameter 250×21 mm, 10 μm) -one and (S)-9-chloro-2-(trans-4-(dimethylamino)cyclohexyl)-2,4-dimethyl-6-((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)-7,8-dihydro-[1,3]-dioxol[4,5-g]isoquinolin-5(6H)-one (1.6 g mixture). Mobile phase A: CO 2 / Mobile phase B: methanol / dichloromethane 1:1 mixture with 0.25% isopropylamine. Isocratic (70% mobile phase A and 30% mobile phase B). Flow rate: 70 g / min. Column temperature: 25°C]. Example 1 Peak 1 (enantiomer): Retention time = 4.69 minutes. Recovery = 264 mg, 33% yield, 99.7% ee, >99% purity (tan solid). LCMS [M+H] + m / z: calculated: 546.2; found: 546.3. 1 H NMR (400 MHz, chloroform-d) δ 12.34 (br.s., 1H), 6.00 (s, 1H), 4.88 (s, 2H), 3.38-3.28 (m, 2H), 2.83 (d, J = 5.9 Hz, 2H), 2.54 (s, 3H), 2.46-2.25 (m, 12H), 2.03 (br.s., 4H), 1.83 (br.s., 1H), 1.62 (s, 3H), 1.27 (d, J = 8.3 Hz, 5H). Example 1 Peak 2 (minor enantiomer): retention time = 5.33 minutes. 73% ee, 97.5% purity (tan solid). LCMS [M+H] +m / z: calculated: 546.2; found: 546.3. 1 H NMR (400 MHz, CHLOROFORM-d) δ 12.86-12.59 (m, 1H), 6.00 (s, 1H), 4.87 (s, 2H), 3.36-3.28 (m, 2H), 2.81 (t, J = 6.4 Hz, 2H), 2.53 (s, 3H), 2.45 (s, 9H), 2.33 (s, 3H), 2.10 (br. s., 2H), 2.04 (dd, J = 2.9, 8.8 Hz, 2H), 1.90-1.81 (m, 1H), 1.62 (s, 3H), 1.41-1.22 (m, 5H).

[0118] Example 2: 9-chloro-2-(trans-4-(dimethylamino)cyclohexyl)-2,4-dimethyl-6-((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)-[1,3]-dioxol[4,5-g]isoquinoline

[0119] -5(6H)-Keto

[0120]

[0121] To a solution of 7-chloro-2-(trans-4-(dimethylamino)cyclohexyl)-6-(2-ethoxyvinyl)-2,4-dimethylbenzo[d][1,3]dioxole-5-carboxylic acid methyl ester (5.1 g, 11.6 mmol) (prepared according to the procedure described in Example 1, Step 4) in acetonitrile (110 mL) was added cerium chloride heptahydrate (21.5 g, 57.9 mmol), sodium iodide (8.67 g, 57.9 mmol) and water (110 μL). The reaction mixture was stirred at 80 °C for 3 hours and then filtered through a plug of celite. The resulting filtrate was concentrated under vacuum and used directly in the next step. The crude mixture was dissolved in 100 mL of methanol, and 3-(aminomethyl)-6-methyl-4-(methylsulfanyl)-1,2-dihydropyridin-2-one hydrochloride (5.96 g, 23.2 mmol) and triethylamine (6.43 mL, 46.4 mmol) were added. The mixture was stirred at room temperature for 2 hours (the title compound could be observed by LCMS at this stage), and then sodium cyanoborohydride (3.63 g, 57.9 mmol) was added to the reactants. The mixture was stirred for 1 hour and then filtered through celite. The solid was rinsed with methanol (100 mL), and the filtrate was concentrated under reduced pressure to remove the methanol. Ethyl acetate (100 mL) and a saturated solution of sodium bicarbonate were added to the residue, and the desired product was extracted three times with ethyl acetate. The combined organic layers were then washed with brine, dried over sodium sulfate, filtered, and concentrated to dryness under reduced pressure to give a mixture containing the title compound. This crude mixture was dissolved in dimethyl sulfoxide (50 mL), and potassium carbonate (3.2 g, 23.2 mmol) was added. The reaction mixture was stirred at room temperature for 30 minutes, and then a saturated solution of sodium bicarbonate was added. The desired product was extracted three times with dichloromethane, and then the combined organic layer was washed with brine, dried over sodium sulfate, filtered, and concentrated to dryness under reduced pressure. The residue was purified by a reverse phase C18 column (gradient of 30 to 70% acetonitrile in water and 0.1% trifluoroacetic acid) to give the title compound (Example 2-racemic mixture) as a tan solid (356 mg, 5% yield). LCMS [M + H] + m / z: calculated: 544.2; found: 544.3. 1 H NMR (400 MHz, CHLOROFORM-d) δ 13.26-13.11 (m, 1H), 7.11 (d, J=7.8 Hz, 1H), 6.62 (s, 1H), 6.00 (s, 1H), 5.18 (s, 2H), 2.76 (s, 3H), 2.42 (s, 3H), 2.34-2.28 (m, 9H), 2.24 (br. s., 1H), 2.01 (br. s., 3H), 1.89-1.82 (m, 1H), 1.66 (s, 3H), 1.33-1.21 (m, 5H).

[0122] By SFC[Chiralpak The racemic mixture was separated into (R)-9-chloro-2-((1r,4R)-4-(dimethylamino)cyclohexyl)-2,4-dimethyl-6-((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)-[1,3]-dioxol[4,5-g]isoquinolin-5(6H)-one and (S)-9-chloro-2-((1r,4S)-4-(dimethylamino)cyclohexyl)-2,4-dimethyl-6-((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)-[1,3]-dioxol[4,5-g]isoquinolin-5(6H)-one using an AD-H column (inner diameter 250×30 mm, 5 μm). Mobile phase A: CO2 / mobile phase B: 2:5 ethanol / water with 0.1% ammonium hydroxide. Isocratic (85% mobile phase A and 15% mobile phase B). Flow rate: 50 mL / min. Column temperature: 40°C]. Retention time: Peak 1 (minor enantiomer): 4.8 minutes. Peak 2 (euantiomer): 6.5 minutes. Peak 1: 53.8 mg, 96.90 umol, 52.72% yield, 98% purity, >99% ee, as a white solid. LCMS (M+H+) calculated value: 544.1; found value: 544. 1H NMR (400 MHz, CD3OD) δ 6.91 (d, J = 7.8 Hz, 1H), 6.61 (d, J = 7.5 Hz, 1H), 6.20 (s, 1H), 5.18-4.86 (m, 2H), 2.61-2.55 (m, 3H), 2.40-2.31 (m, 3H), 2.23 (s, 3H), 2.21 (s, 7H), 1.92 (br d, J = 6.5 Hz, 4H), 1.87-1.76 (m, 1H), 1.58 (s, 3H), 1.21 (br s, 4H). Peak 2: 37.6 mg, 68.41 umol, 37.22% yield, 99% purity, 97.6% ee, in the form of a white solid. LCMS (M+H+) calculated value: 544.2; found value: 544. 1H NMR (400 MHz, CD3OD) δ 6.91 (d, J = 7.5 Hz, 1H), 6.61 (d, J = 7.5 Hz, 1H), 6.19 (s, 1H), 4.98 (s, 2H), 2.59 (s, 3H), 2.38 (s, 3H), 2.22 (s, 10H), 1.93 (br d, J = 6.5 Hz, 4H), 1.82 (br s, 1H), 1.58 (s, 3H), 1.25-1.16 (m, 4H).

[0123] EZH2 assay

[0124] Measuring IC of inhibitors using EZH2 50

[0125] EZH2 biochemical assay (IC 50 ): By 3 The potency of the compounds was assessed by incorporation of H-SAM into biotinylated H3 peptide. Specifically, 30 pM PRC2 containing wt EZH2 (pentameric complex prepared in-house) was incubated with 450 nM SAM, 450 nM 3 H-SAM, 2 μM H3K27me3 activating peptide (H 2 N-RKQLATKAAR(Kme3)SAPATGGVKKP-amide) and compounds (same as 10-point replicate dose response titration in DMSO, final assay 0.8% DMSO (v / v)) were pre-incubated for 3-5 hours in a total volume of 12.5 μl of 50 mM Tris (pH 8.5), 1 mM DTT, 0.07 mM Brij-35, 0.1% BSA, 0.8% DMSO. Biotinylated H3 substrate peptide (H 2 The reaction was initiated with 2 μM stock solution of N-RKQLATKAAR(Kme1)SAPATGGVKKP-NTPEGBiot) in 12.5 μl buffer and allowed to react for 18-22 hours at room temperature. Quenching was completed by adding 20 μl STOP solution (50 mM Tris (pH 8.5), 200 mM EDTA, 2 mM SAH). 35 μl of the quenched solution was transferred to a streptavidin-coated FlashPlate (PerkinElmer), incubated for 1-2 hours, washed, and read in a TopCount Reader (PerkinElmer). IC was calculated in a Genedata Screener using a nonlinear least squares four-parameter fit 50 , where the four parameters are IC 50 , Hill slope, pre-conversion baseline (0% INH), and post-conversion baseline (100% INH).

[0126] EC of inhibitors in HeLa cell assays 50 Measurement

[0127] H3K27me3α Hella Assay (AlphaLISA). Ten different doses of each test compound (in a series of 3-fold dilutions) were plated in duplicate 384-well tissue culture treated plates (Catalog No. 6007680; Perkin Elmer, Waltham, MA). HeLa cells grown in culture were trypsinized and lysed using Cells were counted using a cell counter (Catalog No. C10281; Life Technologies, Grand Island, NY). Cells were diluted to 67,000 cells / mL in 10% DMEM (Catalog No. 10569-010, Life Technologies, Grand Island, NY) and plated using a Biotek MicroFlo TM 15 μL (1,000 cells) was seeded into each well using a dispenser (BioTek Instruments, Inc. Vermont, USA). The plate was incubated at 37°C / 5% CO 2 Incubate for 72 hours at 4 °C. One replicate plate was processed for HeLa assay, and another replicate plate was processed for viability. 5 μL of cell-histone lysis buffer (1X) (Catalog No.: AL009F1, Perkin Elmer, Waltham, Massachusetts) per well was added to the plate processed for AlphaLISA, and this plate was incubated at low speed for 30 minutes on a plate shaker at RT (Model: 4625-Q, Thermo Scientific, Waltham, Massachusetts; Waltham, MA). Then, 10 μL of histone extraction buffer (Catalog No.: AL009F2; Perkin Elmer, Waltham, Massachusetts) was added to each well, and the plate was incubated at low speed for 20 minutes on a plate shaker at RT. Next, 10 μL of a 5X mixture of anti-K27me3 acceptor microbeads and biotinylated anti-histone H3 (C-ter) antibody (diluted to 3 nM final concentration) (Catalog No. AL118; PerkinElmer, Waltham, MA) was added to each well. Dilution of acceptor microbeads and anti-histone H3 was done in 1X Histone Assay Buffer (Catalog No. AL009F3; PerkinElmer, Waltham, MA), which was prepared by diluting the provided 10X stock solution. The plate was sealed with aluminum plate sealant and incubated at 23°C for 60 minutes. Next, 10 μL of a 5X solution of Streptavidin Donor beads (Catalog No. 6760002, PerkinElmer, Waltham, MA) (final concentration of 20 μg / mL in 1X Histone Assay Buffer) was added, = the plate was sealed with aluminum plate sealant and incubated at 23°C for 30 minutes. The plate was then read using an EnVision-Alpha Reader (Model: 2104, PerkinElmer, Waltham, MA).

[0128] Cell viability was determined by adding 15 μL of Cell Titer Glo ((Catalog No.: G9241, Promega Madison, WI)) to each well containing cells containing culture medium. The plate was incubated at low speed on a plate shaker at RT for 15-20 minutes. The plate was then read using an EnVision-Alpha plate reader (Model: 2104, PerkinElmer, Waltham, Massachusetts).

[0129] GI of inhibitors in Karpas-422 viability assay 50 Measurement

[0130] The Karpas-422 cell line was obtained from DSMZ (Braunschweig, Germany) and grown in RPMI-1640 medium. All media contained 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (Invitrogen). 20K cells per well were seeded on 96-well compound-coated plates. Every 4 days, cells were split and seeded at the original seeding density (based on DMSO well counts) into plates containing fresh EZH2 inhibitors. On day 8, relative cell numbers were assessed by Cell Titer-Glo fluorescent cell viability assay (Promega). Curve fitting was performed using GraphPad Prism5, and GI values ​​are reported. 50 The data are shown in Table 1.

[0131] Table 1.

[0132]

Claims

1. A compound of the formula: or a pharmaceutically acceptable salt thereof, wherein R 1 Is a halogen group, (C 1 -C 4 ) alkyl, halogen (C 1 -C 4 )alkyl, -O(C 1 -C 4 )alkyl or -O[halogen(C 1 -C 4 )alkyl], -S(C 1 -C 4 )alkyl or -S[halogen(C 1 -C 4 )alkyl]; R 2 is hydrogen, halogen or (C 1 -C 4 )alkyl; R 3 Yes (C 1 -C 4 ) alkyl or halogen (C 1 -C 4 )alkyl; R 4 Is a halogen group, (C 1 -C 4 ) alkyl or halogen (C 1 -C 4 )alkyl; R 5 is hydrogen, (C 1 -C 4 ) alkyl or halogen (C 1 -C 4 )alkyl; R 6 Is a halogen group, (C 1 -C 4 ) alkyl or halogen (C 1 -C 4 )alkyl; or R 5 and R 6 Together with the nitrogen atom to which it is attached, it forms a substituted alkyl group optionally substituted with 1 to 3 groups selected from halogen, (C 1 -C 4 ) alkyl, halogen (C 1 -C 4 )alkyl and -OR 7 A 4- to 7-membered heterocyclic group substituted with a group; R 7 Yes (C 1 -C 4 ) alkyl, halogen (C 1 -C 4 ) alkyl or (C 3 -C 7 )cycloalkyl; and Dashed lines represent single or double bonds.

2. The compound according to claim 1, wherein the compound has the following formula: or a pharmaceutically acceptable salt thereof.

3. The compound according to claim 1 or 2, wherein the compound has the following formula: or a pharmaceutically acceptable salt thereof.

4. The compound according to any one of claims 1 to 3, wherein the compound has the following formula: or a pharmaceutically acceptable salt thereof.

5. A compound according to any one of claims 1 to 4, wherein R 1 is a halogen or -S(C 1 -C 4 )alkyl.

6. A compound according to any one of claims 1 to 5, wherein R 4 is a halogen or (C 1 -C 4 )alkyl.

7. A compound according to any one of claims 1 to 6, wherein R 5 Yes (C 1 -C 4 )alkyl.

8. A compound according to any one of claims 1 to 7, wherein R 6 Yes (C 1 -C 4 )alkyl.

9. A compound according to any one of claims 1 to 8, wherein R 5 and R 6 Each is a methyl group.

10. A compound according to any one of claims 1 to 9, wherein the group and NR 5 R 6 Trans oriented about the cyclohexyl group.

11. A compound according to any one of claims 1 to 9, wherein the group and NR 5 R 6 Cis-oriented around the cyclohexyl group.

12. The compound according to any one of claims 1 to 11, wherein the stereochemical configuration of the chiral center of the 1,3-dioxolanyl group is R.

13. The compound according to any one of claims 1 to 11, wherein the stereochemical configuration of the chiral center of the 1,3-dioxolanyl group is S.

14. The compound of claim 1, wherein the compound has the formula: or a pharmaceutically acceptable salt thereof.

15. The compound of claim 1, wherein the compound has the formula: or a pharmaceutically acceptable salt thereof. 16 . A pharmaceutical composition comprising the compound according to claim 1 or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier.

17. A method for treating cancer in a subject, the method comprising administering to the subject an effective amount of a compound according to any one of claims 1 to 15 or a pharmaceutically acceptable salt thereof, or a composition according to claim 16.

18. The method of claim 17, wherein the cancer is selected from the group consisting of breast cancer, prostate cancer, colon cancer, renal cell carcinoma, glioblastoma multiforme, bladder cancer, melanoma, bronchial carcinoma, lymphoma, leukemia, bile duct cancer, sarcoma, multiple myeloma, lung cancer, ovarian cancer, gastric cancer, adenoid cystic carcinoma, and liver cancer.

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